CN118510788A - Lipid amine - Google Patents

Abstract

Provided are lipid amine compounds that can be used to prepare lipid nanoparticle compositions for delivering therapeutic or prophylactic payloads into cells.

Description

Lipid amine

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing dates of U.S. Provisional Application No. 63/273,421, filed on October 29, 2021, U.S. Provisional Application No. 63/308,180, filed on February 9, 2022, and U.S. Provisional Application No. 63/395,243, filed on August 4, 2022, the contents of which are incorporated herein by reference in their entirety.

Technical Field

Provided are lipid amine compounds that can be used to prepare lipid nanoparticle compositions for delivering therapeutic or prophylactic payloads into cells.

Background Art

Delivery of biologically active payloads (e.g., nucleic acids and proteins) to cells has the potential to be used to treat a variety of diseases and/or disorders. However, effective targeted delivery of such payloads is a continuing medical challenge. In particular, delivery of nucleic acids to cells becomes difficult due to the relative instability and low cell permeability of such substances.

Lipid nanoparticles provide an effective transport vehicle for payloads into cells and intracellular compartments, but there is still a need to improve safety, efficacy and specificity. Therefore, it is necessary to develop lipid nanoparticle compositions to help deliver therapeutic and prophylactic agents (e.g., nucleic acids) into cells.

Summary of the invention

Provided herein is a lipid amine having a structure of Formula A1:

or a salt thereof, wherein the constituent members are as defined herein.

Also provided herein is a lipid nanoparticle composition comprising a lipid amine of formula A1 or a salt thereof.

Also provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a lipid nanoparticle composition comprising a lipid amine of Formula A1 or a salt thereof.

Also provided herein is a method of delivering a payload into a cell, the method comprising contacting the cell with a lipid nanoparticle composition described herein.

Also provided herein is a method of delivering a therapeutic or prophylactic payload to a patient, the method comprising administering to the patient a lipid nanoparticle composition described herein.

Also provided herein is a method for preparing a lipid nanoparticle composition, the method comprising contacting a lipid nanoparticle core with a lipid amine compound of formula A1 or a salt thereof.

Also provided herein are the products of any of the methods described herein.

Each limitation may encompass multiple embodiments. Therefore, it is contemplated that each limitation involving any element or combination of elements may be included in each of the aspects described. The application of the present invention is not limited to the construction details and component arrangements set forth in the following description or illustrated in the accompanying drawings. Other embodiments that may be practiced or implemented in various ways are possible.

DETAILED DESCRIPTION

Provided herein is a lipid amine having a structure of Formula A1:

or a salt thereof, wherein:

Z is N or CH;

^R1 is _C1-14 alkyl, _C1-14 alkenyl or _C1-14 hydroxyalkyl;

^R2 and ^R3 are each _C2-20 alkyl, wherein:

(i) the C _2-20 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) 1, 2, 3 or 4 non-terminal carbons of the C _2-20 alkyl group are optionally replaced by O;

(iii) 1, 2, 3 or 4 non-terminal carbons of the C _2-20 alkyl group are optionally replaced by NR ¹⁰ ;

(iv) 1, 2, 3 or 4 non-terminal carbons of the _C2-20 alkyl group are optionally replaced by C(=O); and

(v) 1, 2, 3 or 4 non-terminal carbons of the _C2-20 alkyl group are optionally replaced ^{by CRaRb} , wherein ^Ra and ^Rb together with the C atom to which they are attached form a _C3-6 cycloalkyl group;

wherein ^R2 and ^R3 are the same or different;

or R ² and R ³ together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group comprising 1, 2 or 3 ring-forming NR ¹⁰ groups, wherein the 7-18 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo;

or R ² , R ³ and R ⁶ together with the atoms to which they are attached and any intervening atoms form a 7-18 membered bridged heterocycloalkyl group optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo;

R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently selected from H, halo and C _1-4 alkyl;

or ^R4 and ^R5 together with the carbon atom to which they are attached form a _C3-7 cycloalkyl group;

or R ⁶ and R ⁷ together with the carbon atom to which they are attached form a C _3-7 cycloalkyl group;

R ⁸ , R ⁹ and R ¹⁰ are each independently selected from H and C _1-4 alkyl;

j is 0 or 1;

k is 0, 1, 2, 3, 4, 5 or 6;

l is 0 or 1;

m is 0, 1, 2, 3, 4, 5 or 6; and

n is 0 or 1;

When j is 0, l is 1.

Where j and l are not both 0.

Provided herein is a lipid amine having a structure of Formula A1:

or a salt thereof, wherein:

Z is N or CH;

^R1 is _C1-14 alkyl, _C1-14 alkenyl or _C1-14 hydroxyalkyl;

^R2 and ^R3 are _C2-20 alkyl groups, wherein:

(i) the C _2-20 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) 1, 2, 3 or 4 non-terminal carbons of the _C2-20 alkyl group are optionally replaced by O; and

(iii) 1, 2, 3 or 4 non-terminal carbons of the C _2-20 alkyl group are optionally replaced by NR ¹⁰ ;

wherein ^R2 and ^R3 are the same or different;

or R ² and R ³ together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group comprising 1, 2 or 3 ring-forming NR ¹⁰ groups, wherein the 7-18 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ₈ R ₉ , OH and halo;

or R ² , R ³ and R ⁶ together with the atoms to which they are attached and any intervening atoms form a 7-18 membered bridged heterocycloalkyl group optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ₈ R ₉ , OH and halo;

R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently selected from H, halo and C _1-4 alkyl;

or ^R4 and ^R5 together with the carbon atom to which they are attached form a _C3-7 cycloalkyl group;

or R ⁶ and R ⁷ together with the carbon atom to which they are attached form a C _3-7 cycloalkyl group;

R ⁸ , R ⁹ and R ¹⁰ are each independently selected from H and C _1-4 alkyl;

j is 0 or 1;

k is 0, 1, 2, 3, 4, 5 or 6;

l is 0 or 1;

m is 0, 1, 2, 3, 4, 5 or 6; and

n is 0 or 1;

When j is 0, l is 1.

Where j and l are not both 0.

In some embodiments, the compound is not:

In some embodiments, Z is N. In some embodiments, Z is CH.

In some embodiments, R ¹ is C _1-14 alkyl. In some embodiments, R ¹ is C _3-12 alkyl. In some embodiments, R ¹ is C _6-12 alkyl. In some embodiments, R ¹ is C _8-10 alkyl. In some embodiments, R ¹ is C ₈ alkyl. In some embodiments, R ¹ is C ₁₀ alkyl.

In some embodiments, R ¹ is C _1-14 hydroxyalkyl. In some embodiments, R ¹ is C _3-12 hydroxyalkyl. In some embodiments, R ¹ is C _6-12 hydroxyalkyl. In some embodiments, R ¹ is C _8-10 hydroxyalkyl. In some embodiments, R ¹ is C ₈ hydroxyalkyl. In some embodiments, R ¹ is C ₁₀ hydroxyalkyl.

In some embodiments, R ¹ is C _1-14 alkenyl. In some embodiments, R ¹ is C _3-12 alkenyl. In some embodiments, R ¹ is C _6-12 alkenyl. In some embodiments, R ¹ is C _8-10 alkenyl. In some embodiments, R ¹ is C ₈ alkenyl. In some embodiments, R ¹ is C ₁₀ alkenyl.

In some embodiments, R ¹ is

In some embodiments, ^R1 is

In some embodiments, R ¹ is

In some embodiments, ^R1 is

In some embodiments, when j is 1, then l is 0.

In some embodiments, when j is 0, then l is 1.

In some embodiments, when one of j and l is 1, the other is 0.

In some embodiments, j is 0. In some embodiments, j is 1.

In some embodiments, k is 0, 1, 2, 3, or 4. In some embodiments, k is 0, 2, 3, or 4. In some embodiments, k is 0. In some embodiments, k is 1. In some embodiments, k is 2. In some embodiments, k is 3. In some embodiments, k is 4. In some embodiments, k is 5. In some embodiments, k is 6.

In some embodiments, l is 0. In some embodiments, l is 1.

In some embodiments, m is 0, 1, 2, or 4. In some embodiments, m is 0. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

In some embodiments, n is 0. In some embodiments, n is 1.

In some embodiments, j is 0, k is 0, l is 1, m is 1, and n is 1. In some embodiments, j is 0, k is 0, l is 1, m is 2, and n is 1. In some embodiments, j is 0, k is 0, l is 1, m is 4, and n is 1. In some embodiments, j is 1, k is 0, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 1, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 1, l is 0, m is 2, and n is 0. In some embodiments, j is 1, k is 1, l is 1, m is 1, and n is 1. In some embodiments, j is 1, k is 2, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 2, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 3, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 4, l is 0, m is 0, and n is 1.

In some embodiments, k is 1 and both R ⁴ and R ⁵ are H. In some embodiments, k is 1 and one of R ⁴ and R ⁵ is C _1-4 alkyl and the other of R ⁴ and R ⁵ is H. In some embodiments, k is 1 and one of R ⁴ and R ⁵ is methyl and the other of R ⁴ and R ⁵ is H. In some embodiments, k is 2 and each of R ⁴ and R ⁵ is H. In some embodiments, k is 2 and one R ⁴ is C _1-4 alkyl and the remaining R ⁴ and R ⁵ substituents are H. In some embodiments, k is 2 and one R ⁴ is methyl and the remaining R ⁴ and R ⁵ substituents are H. In some embodiments, k is 3 and each of R ⁴ and R ⁵ is H. In some embodiments, k is 4 and each of R ⁴ and R ⁵ is H.

In some embodiments, m is 1 and ^R6 and ^R7 are both H. In some embodiments, m is 2 and each ^R6 and ^R7 is H. In some embodiments, m is 4 and each ^R6 and ^R7 is H. In some embodiments, m is 2, one ^R6 together with ^R2 and ^R3 together with the atoms to which they are attached and any intervening atoms form a 7-18 membered bridged heterocycloalkyl, and the other ^R6 is H, and ^R7 are both H.

In some embodiments, j is 0, k is 0, l is 1, m is 1, R ⁶ and R ⁷ are both H, and n is 1. In some embodiments, j is 0, k is 0, l is 1, m is 2, each R ⁶ and R ⁷ is H, and n is 1. In some embodiments, j is 0, k is 0, l is 1, m is 4, each R ⁶ and R ⁷ is H, and n is 1. In some embodiments, j is 1, k is 1, each R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, one of R ⁴ and R ⁵ is C _1-4 alkyl and the other of R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, each R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 1, one of R ⁴ and R ⁵ is C _1-4 alkyl and the other of R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 2, each of R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 2, one R ⁴ is C _1-4 alkyl and the remaining R ⁴ and R ⁵ substituents are H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 2, each of R ^{4 and R 5 is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 2, each of R 4} and R ⁵ is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 3, each of R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 4, each R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 1, each R ⁴ and R ⁵ is H, l is 1, m is 1, R ⁶ and R ⁷ are both H, and n is 1. In some embodiments, j is 1, k is 1, each R ⁴ and R ⁵ is H, l is 0, m is 2, one R ⁶ and R ² and R ³ together with the atoms to which they are attached and any intervening atoms form a 7-18 membered bridged heterocycloalkyl, and the other R ⁶ is H, R ⁷ are both H, and n is 0.

In some embodiments, j is 1, k is 1, one of R ⁴ and R ⁵ is methyl and the other of R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 0. In some embodiments, j is 1, k is 1, one of R ⁴ and R ⁵ is methyl and the other of R ⁴ and R ⁵ is H, l is 0, m is 0, and n is 1. In some embodiments, j is 1, k is 2, one R ⁴ is methyl and the remaining R ⁴ and R ⁵ substituents are H, l is 0, m is 0, and n is 0.

In some embodiments, R ² and R ³ are each independently selected from C _2-10 alkyl, wherein the C _2-10 alkyl is substituted with 1, 2, 3, 4, or 5 substituents independently selected from -NR ⁸ R ⁹ , OH, and halo, wherein at least one substituent is -NR ⁸ R ⁹ .

In some embodiments, ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein:

(i) the C _2-10 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; and

(ii) 1, 2, 3 or 4 non-terminal carbons of the C _2-10 alkyl group are optionally replaced by O.

In some embodiments, ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein:

(i) the C _2-10 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; and

(iii) 1, 2, 3 or 4 non-terminal carbons of the C _2-10 alkyl group are optionally replaced by NR ¹⁰ .

In some embodiments, ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein:

(i) the C _2-10 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; and

(iv) 1, 2, 3 or 4 non-terminal carbons of the C _2-10 alkyl group are optionally replaced by C(═O).

In some embodiments, ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein:

(i) the C _2-20 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; and

(v) 1, 2, 3 or 4 non-terminal carbons of the C _2-20 alkyl group are optionally replaced by CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl group.

In some embodiments, ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein:

(i) the C _2-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) one or two non-terminal carbons of the C _2-10 alkyl group are optionally replaced by O;

(iii) one or two non-terminal carbons of the C _2-10 alkyl group are optionally replaced by NR ¹⁰ ;

(iv) 1 or 2 non-terminal carbons of the C _2-10 alkyl group are optionally replaced by C(═O); and

(v) One or two non-terminal carbons of the _C2-10 alkyl group are optionally replaced ^{by CRaRb} , wherein ^Ra and ^Rb together with the C atom to which they are attached form a _C3-6 cycloalkyl group.

In some embodiments, ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein:

(i) the C _2-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; and

(ii) One or two non-terminal carbon atoms of the C _2-10 alkyl group are optionally replaced by O.

In some embodiments, ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein:

(i) the C _2-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; and

(iii) 1 or 2 non-terminal carbons of the C _2-10 alkyl group are optionally replaced by NR ^10. In some embodiments, R ² and R ³ are each independently selected from C _2-10 alkyl groups, wherein:

(i) the C _2-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; and

(iv) One or two non-terminal carbons of the C _2-10 alkyl group are optionally replaced by C(═O). In some embodiments, R ² and R ³ are each independently selected from C _2-10 alkyl groups, wherein:

(i) the C _2-20 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; and

(v) One or two non-terminal carbons of the _C2-10 alkyl group are optionally replaced ^{by CRaRb} , wherein ^Ra and ^Rb together with the C atom to which they are attached form a _C3-6 cycloalkyl group.

In some embodiments, ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein:

(i) the C _2-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) 1 or 2 non-terminal carbons of the C _2-10 alkyl group are optionally replaced by O; and

(iii) 1 or 2 non-terminal carbons of the C _2-10 alkyl group are optionally replaced by NR ^10. In some embodiments, R ² and R ³ are each independently selected from C _4-10 alkyl groups, wherein:

(i) the C _4-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) one or two non-terminal carbons of the C _4-10 alkyl group are optionally replaced by O;

(iii) one or two non-terminal carbons of the C _4-10 alkyl group are optionally replaced by NR ¹⁰ ;

(iv) one or two non-terminal carbons of the C _4-10 alkyl group are optionally replaced by C(═O); and

(v) One or two non-terminal carbons of the C _4-10 alkyl group are optionally replaced by CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl group.

In some embodiments, ^R2 and ^R3 are each independently selected from _C4-10 alkyl, wherein:

(i) the C _4-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) 1 or 2 non-terminal carbons of the C _4-10 alkyl group are optionally replaced by O; and

(iii) One or two non-terminal carbons of the C _4-10 alkyl group are optionally replaced by NR ¹⁰ .

In some embodiments, one of R ² and R ³ is C _2-5 alkyl, wherein:

The C _2-5 alkyl group is substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

And wherein the other of R ² and R ³ is a C _7-10 alkyl group, wherein:

(i) the C _7-10 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) 1, 2, 3 or 4 non-terminal carbons of the C _7-10 alkyl group are optionally replaced by O;

(iii) 1, 2, 3 or 4 non-terminal carbons of the C _7-10 alkyl group are optionally replaced by NR ¹⁰ ;

(iv) 1, 2, 3 or 4 non-terminal carbons of the _C7-10 alkyl group are optionally replaced by C(=O); and

(v) 1, 2, 3 or 4 non-terminal carbons of the C _7-10 alkyl group are optionally replaced by CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl group.

In some embodiments, one of R ² and R ³ is C _2-5 alkyl, wherein:

(i) the C _2-5 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

And wherein the other of R ² and R ³ is a C _7-10 alkyl group, wherein:

(i) the C _7-10 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) 1, 2, 3 or 4 non-terminal carbons of the _C7-10 alkyl group are optionally replaced by O; and

(iii) 1, 2, 3 or 4 non-terminal carbons of the C _7-10 alkyl group are optionally replaced by NR ¹⁰ .

In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted by 1 -NR ⁸ R ^{9. In some embodiments, one of R 2 and R 3 is a C 2-20 alkyl substituted by 1 -NR 8 R 9 and 1} _non ^{- terminal} carbon of the C 2-20 alkyl is replaced by NR ^10. In some embodiments, one of R ² and R ³ is _a C _2-20 alkyl substituted by 1 -NR ⁸ R ⁹ and 1 non-terminal carbon of the C _2-20 alkyl is replaced by O. In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted by 1 -NR ⁸ R ⁹ and 2 halogen groups and 1 non-terminal carbon of the C _2-20 alkyl is replaced by NR ¹⁰ . In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 -F, and 1 non-terminal carbon of the C _2-20 alkyl is replaced with NR ^10. In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 halo. In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 -F. In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 halo, wherein 1 non-terminal carbon of the C _2-20 alkyl is replaced with NR ¹⁰ . In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 -F, wherein 1 non-terminal carbon of the C _2-20 alkyl is replaced with NR ^10. In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 halo. In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 -F. In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 OH.

In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted by 1 -NR ⁸ R ⁹ and 1 non-terminal carbon of the C _2-20 alkyl is replaced by C(=O). In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted by 2 -NR ⁸ R ⁹ and 1 non-terminal carbon of the C _2-20 alkyl is replaced by C(=O). In some embodiments, one of R ² and R ³ is a C _2-20 alkyl substituted by 1 -NR ⁸ R ⁹ , 1 non-terminal carbon of the C _2-20 alkyl is replaced by NR ¹⁰ , and 1 non-terminal carbon of the C _2-20 alkyl is replaced by CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl. In some embodiments, one of R ² and R ³ is C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ , and 1 non-terminal carbon of the C _2-20 alkyl is replaced with CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl.

In some embodiments, one of R ² and R ³ is selected from:

C _2-20 alkyl substituted by one -NR ⁸ R ⁹ ,

A C _2-20 alkyl group substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-20 alkyl group is replaced by NR ¹⁰ ,

A C _2-20 alkyl group substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-20 alkyl group is replaced by O,

A C _2-20 alkyl group substituted by 1 -NR ⁸ R ⁹ and 2 halogen groups, wherein 1 non-terminal carbon of the C _2-20 alkyl group is replaced by NR ¹⁰ ,

A C _2-20 alkyl group substituted by 1 -NR ⁸ R ⁹ and 1 halogen group, wherein 1 non-terminal carbon of the C _2-20 alkyl group is replaced by NR ¹⁰ ,

A C _2-20 alkyl group substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-20 alkyl group is replaced by C(═O),

C _2-20 alkyl substituted by 2 -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-20 alkyl is replaced by C(═O), and

_C2-20 alkyl substituted by ^{1 -NR8R9} , wherein 1 non-terminal carbon of the _C2-20 alkyl is replaced ^{by NR10} and 1 non-terminal carbon of the _C2-20 alkyl is replaced by CRaRb, wherein R ^{a and} R ^b together with the C atom to which they are attached form ^a _C3-6 cycloalkyl, and

The other of ^R2 and ^R3 is selected from:

C _2-20 alkyl substituted by one -NR ⁸ R ⁹ ,

A C _2-20 alkyl group substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-20 alkyl group is replaced by NR ¹⁰ ,

C _2-20 alkyl substituted by 1 -NR ⁸ R ⁹ and 2 halogen groups,

C _2-20 alkyl substituted by 1 -NR ⁸ R ⁹ and 1 halogen,

C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 OH, and

C _2-20 alkyl substituted with one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-20 alkyl is replaced with CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl.

In some embodiments, one of R ² and R ³ is selected from C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ ; C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _2-20 alkyl is replaced with NR ¹⁰ ; C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _2-20 alkyl is replaced with O; C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 halo, wherein 1 non-terminal carbon of the C _2-20 alkyl is replaced with NR ¹⁰ ; and C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 halo, wherein 1 non-terminal carbon of the C 2-20 alkyl is replaced with NR ¹⁰ , and the other of R ² and R ³ is selected from C _2-20 alkyl substituted with 1 -NR ⁸ R ⁹ ; C _2-20 alkyl substituted with 1 -NR ⁸ R 9; C _2-20 alkyl substituted with -NR 8 R ⁹ , wherein one non-terminal carbon of the C _2-20 alkyl is replaced with NR ¹⁰ ; C _2-20 alkyl substituted with one -NR ⁸ R ⁹ and two halogen groups; C _2-20 alkyl substituted with one -NR ⁸ R ⁹ and one halogen group; and C _2-20 alkyl substituted with one -NR ⁸ R ⁹ and one OH group.

In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ^{8 R 9. In some embodiments, one of R 2 and R 3 is a C 2-10 alkyl substituted with 1 -NR 8 R 9 , and} ₁ ^{non -} terminal carbon of the C _2-10 alkyl is replaced with NR ^10. In some embodiments, one of R ² and R ³ is a C 2-10 alkyl substituted with 1 -NR ⁸ R ⁹ , and 1 non-terminal carbon of the C _2-10 alkyl is replaced with O. In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 halogen groups, and 1 non _- terminal carbon of the C _2-10 alkyl is replaced with NR ¹⁰ . In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 -F, and 1 non-terminal carbon of the C _2-10 alkyl is replaced by NR ^10. In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 halo. In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 -F. In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 halo, wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced by NR ¹⁰ . In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 -F, wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced with NR ^10. In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 halo. In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 -F. In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 OH.

In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted by 1 -NR ⁸ R ⁹ , and 1 non-terminal carbon of the C _2-10 alkyl is replaced by C(=O). In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted by 2 -NR ⁸ R ⁹ , and 1 non-terminal carbon of the C _2-10 alkyl is replaced by C(=O). In some embodiments, one of R ² and R ³ is a C _2-10 alkyl substituted by 1 -NR ⁸ R ⁹ , 1 non-terminal carbon of the C _2-10 alkyl is replaced by NR ¹⁰ , and 1 non-terminal carbon of the C _2-10 alkyl is replaced by CR ^a R ^b , wherein ^Ra and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl. In some embodiments, one of R ² and R ³ is C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ , and 1 non-terminal carbon of the C _2-10 alkyl is replaced with CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl.

In some embodiments, one of R ² and R ³ is selected from:

C _2-10 alkyl substituted by one -NR ⁸ R ⁹ ,

C _2-10 alkyl substituted by 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced by NR ¹⁰ ,

A C _2-10 alkyl group substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-10 alkyl group is replaced by O,

C _2-10 alkyl substituted by 1 -NR ⁸ R ⁹ and 2 halogen groups, wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced by NR ¹⁰ ,

C _2-10 alkyl substituted by 1 -NR ⁸ R ⁹ and 1 halogen, wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced by NR ¹⁰ ,

A C _2-10 alkyl group substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-10 alkyl group is replaced by C(═O),

C _2-10 alkyl substituted by 2 -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-10 alkyl is replaced by C(=O), and

_C2-10 alkyl substituted by ^{1 -NR8R9} , wherein 1 non-terminal carbon of the _C2-10 alkyl is replaced ^{by NR10} and 1 non-terminal carbon of the _C2-10 alkyl is replaced by ^CRaRb , wherein R ^a and R ^b together with the C atom to which they are attached form ^a _C3-6 cycloalkyl, and

The other of ^R2 and ^R3 is selected from:

C _2-10 alkyl substituted by one -NR ⁸ R ⁹ ,

C _2-10 alkyl substituted by 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced by NR ¹⁰ ,

C _2-10 alkyl substituted by 1 -NR ⁸ R ⁹ and 2 halogen groups,

C _2-10 alkyl substituted by 1 -NR ⁸ R ⁹ and 1 halogen,

C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 OH, and

C _2-10 alkyl substituted with one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-10 alkyl is replaced with CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl.

In some embodiments, one of R ² and R ³ is selected from C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ ; C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced with NR ¹⁰ ; C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced with O; C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 2 halo, wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced with NR ¹⁰ ; and C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 halo, wherein 1 non-terminal carbon of the C _2-10 alkyl is replaced with NR ¹⁰ , and the other of R ² and R ³ is selected from C _2-10 alkyl substituted with 1 -NR ⁸ R ⁹ ; C 2-10 alkyl substituted with 1 -NR ⁸ R 9; C _2-10 alkyl substituted with -NR 8 R ⁹ , wherein one non-terminal carbon of the C _2-10 alkyl is replaced with NR ¹⁰ ; C _2-10 alkyl substituted with one -NR ⁸ R ⁹ and two halogen groups; C _2-10 alkyl substituted with one -NR ⁸ R ⁹ and one halogen group; and C _2-10 alkyl substituted with one -NR ⁸ R ⁹ and one OH group.

In some embodiments, one of R ² and R ³ is selected from:

C _5-10 alkyl substituted by one -NR ⁸ R ⁹ ,

A C _5-10 alkyl group substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _5-10 alkyl group is replaced by NR ¹⁰ ,

A C _5-10 alkyl group substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _5-10 alkyl group is replaced by O,

A C _5-10 alkyl group substituted by 1 -NR ⁸ R ⁹ and 2 halogen groups, wherein 1 non-terminal carbon of the C _5-10 alkyl group is replaced by NR ¹⁰ ,

_C5-10 alkyl substituted by ^{1 -NR8R9} and 1 halogen, wherein 1 non-terminal carbon of the _C5-10 alkyl is replaced by ^NR10 , and

_C5-10 alkyl substituted by ^{1 -NR8R9} , wherein 1 non-terminal carbon of the _C2-10 ^alkyl is replaced ^{by NR10} and 1 non-terminal carbon of the _C2-10 alkyl is replaced by ^CRaRb , wherein R ^a and R ^b together with the C atom to which they are attached form a _C3-6 cycloalkyl,

and the other of ^R2 and ^R3 is selected from:

C _3-6 alkyl substituted by one -NR ⁸ R ⁹ ,

C _3-6 alkyl substituted by 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _3-6 alkyl is replaced by NR ¹⁰ ,

C _3-6 alkyl substituted by 1 -NR ⁸ R ⁹ and 2 halogen groups,

C _3-6 alkyl substituted by 1 -NR ⁸ R ⁹ and 1 halogen,

C _3-6 alkyl substituted by 1 -NR ⁸ R ⁹ and 1 OH,

C _3-6 alkyl substituted by one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-10 alkyl is replaced by C(=O),

C _3-6 alkyl substituted by 2 -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-10 alkyl is replaced by C(=O), and

C _3-6 alkyl substituted with one -NR ⁸ R ⁹ , wherein one non-terminal carbon of the C _2-10 alkyl is replaced with CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl.

In some embodiments, one of R ² and R ³ is selected from C _5-10 alkyl substituted with 1 -NR ⁸ R ⁹ ; C _5-10 alkyl substituted with 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _5-10 alkyl is replaced with NR ¹⁰ ; C _5-10 alkyl substituted with 1 -NR ⁸ R ⁹ , wherein 1 non-terminal carbon of the C _{5-10 alkyl is replaced with O; C 5-10 alkyl} substituted with 1 -NR ⁸ R ⁹ and 2 halo, wherein 1 non-terminal carbon of the C _5-10 alkyl is replaced with NR ¹⁰ ; and C _5-10 alkyl substituted with 1 -NR ⁸ R ⁹ and 1 halo, wherein 1 non-terminal carbon of the C _5-10 alkyl is replaced with NR ¹⁰ , and the other of R ² and R ³ is selected from C 3-6 alkyl substituted with 1 -NR ⁸ R ⁹ ; C _3-6 alkyl substituted with 1 -NR ⁸ R 9; ⁹ -substituted C _3-6 alkyl, wherein one non-terminal carbon of the C _3-6 alkyl is replaced by NR ¹⁰ ; C _3-6 alkyl substituted by one -NR ⁸ R ⁹ and two halogen groups; C _3-6 alkyl substituted by one -NR ⁸ R ⁹ and one halogen group; and C _3-6 alkyl substituted by one -NR ⁸ R ⁹ and one OH group.

In some embodiments, one of R ² and R ³ is C ₃ alkyl, which is substituted with at least one -NR ⁸ R ⁹ group and further optionally substituted with one or two groups selected from OH and halo.

In some embodiments, one of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

and the other of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

and the other of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

and the other of ^R2 and ^R3 is selected from

In some embodiments, one of ^R2 and ^R3 is selected from

and the other of ^R2 and ^R3 is selected from

In some embodiments, R ² and R ³ together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group comprising 1, 2 or 3 ring-forming NR ¹⁰ groups, wherein the 7-18 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo.

In some embodiments, R ² and R ³ together with the N atom to which they are attached form a 7-12 membered heterocycloalkyl group comprising 1, 2 or 3 ring-forming NR ¹⁰ groups, wherein the 7-12 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo.

In some embodiments, R ² and R ³ together with the N atom to which they are attached form an 8-10 membered heterocycloalkyl group comprising 1, 2 or 3 ring-forming NR ¹⁰ groups, wherein the 8-10 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo.

In some embodiments, R ² and R ³ together with the N atom to which they are attached form an 8-10 membered heterocycloalkyl containing 1, 2, or 3 annular NCH ₃ or NH groups, wherein the 8-10 membered heterocycloalkyl is optionally substituted with 1, 2, or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH, and halo.

In some embodiments, R ² and R ³ together with the N atom to which they are attached form an 8-10 membered heterocycloalkyl group containing 1, 2, or 3 annular NCH ₃ or NH groups.

In some embodiments, R ² and R ³ together with the N atom to which they are attached form a heterocycloalkyl of the formula:

In some embodiments, ^R2 , ^R3 , and ^R6 , together with the atoms to which they are attached and any intervening atoms, form a 7-18 membered bridged heterocycloalkyl optionally substituted with 1, 2, or 3 substituents _{independently} selected from _C1-4 alkyl, _-NR8R9 , OH, and halo.

In some embodiments, ^R2 , ^R3 , and ^R6 , together with the atoms to which they are attached and any intervening atoms, form a 7-13 membered bridged heterocycloalkyl optionally substituted with 1, 2, or 3 substituents _{independently} selected from _C1-4 alkyl, _-NR8R9 , OH, and halo.

In some embodiments, ^R2 , ^R3 , and ^R6 , together with the atoms to which they are attached and any intervening atoms, form a 7-10 membered bridged heterocycloalkyl optionally substituted with 1, 2, or 3 substituents _{independently} selected from _C1-4 alkyl, _-NR8R9 , OH, and halo.

In some embodiments, ^R2 , ^R3 , and ^R6, taken together with the atoms to which they are attached and any intervening atoms, form a 7-10 membered bridged heterocycloalkyl.

In some embodiments, R ² , R ³ , and R ⁶ , together with the atoms to which they are attached and any intervening atoms, form a 7-18 membered bridged heterocycloalkyl having the formula:

In some embodiments, R ⁴ and R ⁵ are each independently H or C _1-4 alkyl. In some embodiments, R ⁴ and R ⁵ are each independently H or methyl. In some embodiments, R ⁴ and R ⁵ are both H. In some embodiments, R ⁴ and R ⁵ are both C _1-4 alkyl. In some embodiments, R ⁴ and R ⁵ are both methyl. In some embodiments, one of R ⁴ and R ⁵ is H and the other of R ⁴ and R ⁵ is C _1-4 alkyl. In some embodiments, one of R ⁴ and R ⁵ is H and the other of R ⁴ and R ⁵ is methyl.

In some embodiments, R ⁶ and R ⁷ are each independently H or C _1-4 alkyl. In some embodiments, R ⁶ and R ⁷ are each independently H or methyl. In some embodiments, R ⁶ and R ⁷ are both H. In some embodiments, R ⁶ and R ⁷ are both C _1-4 alkyl. In some embodiments, R ⁶ and R ⁷ are both methyl. In some embodiments, one of R ⁶ and R ⁷ is H and the other of R ⁶ and R ⁷ is C _1-4 alkyl. In some embodiments, one of R ⁶ and R ⁷ is H and the other of R ⁶ and R ⁷ is methyl.

In some embodiments, R ⁸ , R ⁹ and R ¹⁰ are each independently selected from H and methyl. In some embodiments, R ⁸ and R ⁹ are both H. In some embodiments, R ⁸ and R ⁹ are both C _1-4 alkyl. In some embodiments, R ⁸ and R ⁹ are both methyl. In some embodiments, one of R ⁸ and R ⁹ is H and the other of R ⁸ and R ⁹ is C _1-4 alkyl. In some embodiments, one of R ⁸ and R ⁹ is H and the other of R ⁸ and R ⁹ is methyl. In some embodiments, R ¹⁰ is H or methyl. In some embodiments, R ¹⁰ is H. In some embodiments, R ¹⁰ is methyl.

In some embodiments, ^Ra and ^R form a _C3 cycloalkyl together with the C atom to which they are attached, for example, cyclopropyl. In some embodiments, ^Ra and ^R form a C4 cycloalkyl together with the C atom to which they are attached, for example, cyclobutyl. In some embodiments, ^Ra and ^R form a _C5 cycloalkyl together with the C atom to which they are attached, for example, cyclopentyl. In some embodiments, ^Ra and ^R form _{a C6} cycloalkyl together with the C atom to which they are attached, for example, cyclopentyl.

In some embodiments:

Z is N or CH;

^R1 is _C1-14 alkyl, _C1-14 alkenyl or _C1-14 hydroxyalkyl;

^R2 and ^R3 are each _C2-20 alkyl, wherein:

(i) the C _2-20 alkyl group is substituted by 1 or 2 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) one non-terminal carbon of the C _2-20 alkyl group is optionally replaced by O;

(iii) one non-terminal carbon of the C _2-20 alkyl group is optionally replaced by NR ¹⁰ ;

(iv) one non-terminal carbon of the _C2-20 alkyl group is optionally replaced by C(=O); and

(v) one non-terminal carbon of the _C2-20 alkyl group is optionally replaced by CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl group;

wherein ^R2 and ^R3 are the same or different;

or ^R2 and ^R3 together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group containing two ring-forming ^NR10 groups;

R ⁴ is selected from H and C _1-4 alkyl;

R ⁵ , R ⁶ and R ⁷ are each H;

R ⁸ , R ⁹ and R ¹⁰ are each independently selected from H and C _1-4 alkyl;

j is 0 or 1;

k is 0, 1, 2, 3, or 4;

l is 0 or 1;

m is 0, 1, 2 or 4; and

n is 0 or 1;

When j is 0, l is 1.

Where j and l are not both 0.

In some embodiments:

Z is N or CH;

^R1 is _C1-14 alkyl, _C1-14 alkenyl or _C1-14 hydroxyalkyl;

^R2 and ^R3 are each _C2-20 alkyl, wherein:

(i) the C _2-20 alkyl group is substituted by 1 or 2 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) one non-terminal carbon of the _C2-20 alkyl group is optionally replaced by O; and

(iii) one non-terminal carbon of the C _2-20 alkyl group is optionally replaced by NR ¹⁰ ;

wherein ^R2 and ^R3 are the same or different;

or ^R2 and ^R3 together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group comprising two ring-forming ^NR10 groups;

R ⁴ is selected from H and C _1-4 alkyl;

R ⁵ , R ⁶ and R ⁷ are each H;

R ⁸ , R ⁹ and R ¹⁰ are each independently selected from H and C _1-4 alkyl;

j is 0 or 1;

k is 0, 1, 2, 3, or 4;

l is 0 or 1;

m is 0, 1, 2 or 4; and

n is 0 or 1;

Where j and l are not both 0,

When j is 0, l is 1.

In some embodiments,

Z is N;

^R1 is _C1-14 alkyl, _C1-14 alkenyl or _C1-14 hydroxyalkyl;

^R2 and ^R3 are each _C2-20 alkyl, wherein:

(i) the C _2-20 alkyl group is substituted by 1 or 2 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ;

(ii) one non-terminal carbon of the _C2-20 alkyl group is optionally replaced by O; and

(iii) one non-terminal carbon of the C _2-20 alkyl group is optionally replaced by NR ¹⁰ ;

wherein ^R2 and ^R3 are the same or different;

or ^R2 and ^R3 together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group comprising two ring-forming ^NR10 groups;

R ⁴ is selected from H and C _1-4 alkyl;

R ⁵ , R ⁶ and R ⁷ are each H;

R ⁸ , R ⁹ and R ¹⁰ are each independently selected from H and C _1-4 alkyl;

j is 0 or 1;

k is 0, 1, 2, 3, or 4;

l is 0 or 1;

m is 0, 1 or 4; and

n is 0 or 1;

Where j and l are not both 0, and when j is 0, l is 1.

In some embodiments, the compound of formula A1 is a compound of formula A2:

or a salt thereof.

In some embodiments, the compound of formula A1 is selected from compounds of formula A3-A15:

or a salt thereof. In some embodiments, the compound of formula A1 is selected from:

or a salt of any one of the above mentioned.

In some embodiments, the compound of formula A1 is selected from:

or a salt of any one of the above mentioned.

In some embodiments, the compound of formula A1 is selected from:

or a salt of any one of the above mentioned.

It should be understood that, for clarity, certain features described herein that are set forth in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for simplicity, various features described herein that are set forth in the context of a single embodiment may also be provided separately or in any suitable sub-combination.

Lipid nanoparticle compositions

Further provided is a lipid nanoparticle (LNP) composition comprising a lipid amine disclosed herein, such as a lipid amine of formula A1. In some embodiments, in addition to the lipid amine, the lipid nanoparticle composition further comprises at least one of an ionizable lipid, a phospholipid, a structural lipid, and a PEG-lipid. In some embodiments, the lipid nanoparticles of the lipid nanoparticle composition are loaded with an effective load. In some embodiments, the lipid amine is primarily disposed on the outer surface of the lipid nanoparticles of the lipid nanoparticle composition. In some embodiments, the lipid nanoparticle composition has a zeta potential greater than neutral at physiological pH.

In some embodiments, the lipid nanoparticle composition comprises:

(i) ionizable lipids,

(ii) phospholipids,

(iii) structural lipids,

(iv) optionally a PEG-lipid,

(v) optionally a payload for delivery into the cell, and

(vi) a lipid amine as disclosed herein, for example a lipid amine of formula A1.

The lipid nanoparticle composition may also include other components, including but not limited to helper lipids, stabilizers, salts, buffers and solvents. The helper lipid is a non-cationic lipid. The helper lipid may include at least one fatty acid chain of at least 8 carbons and at least one polar head group portion. In some embodiments, the lipid nanoparticle core has a neutral charge at neutral pH.

In some embodiments, the lipid nanoparticle composition has a weight ratio of lipid amine to payload of about 0.1:1 to about 15:1, about 0.2:1 to about 10:1, about 1:1 to about 10:1, about 1:1 to about 8:1, about 1:1 to about 7:1, about 1:1 to about 6:1, about 1:1 to about 5:1, about 1:1 to about 4:1, or about 1.25:1 to about 3.75:1. In some embodiments, the weight ratio of lipid amine to payload is about 1.25:1, about 2.5:1, or about 3.75:1. In some embodiments, the molar ratio of lipid amine to payload is about 0.1:1 to about 20:1, about 1.5:1 to about 10:1, about 1.5:1 to about 9:1, about 1.5:1 to about 8:1, about 1.5:1 to about 7:1, about 1.5:1 to about 6:1, or about 1.5:1 to about 5:1. In some embodiments, the molar ratio of lipid amine to payload is about 1.5:1, about 2:1, about 3:1, about 4:1, or about 5:1.

In some embodiments, the lipid nanoparticle composition is characterized as having a zeta potential of about 5mV to about 20mV. In some embodiments, the lipid nanoparticle composition has a zeta potential of about 5mV to about 15mV. In some embodiments, the lipid nanoparticle composition has a zeta potential of about 5mV to about 10mV. The zeta potential measures the surface charge of a colloidal dispersion. The magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent similarly charged particles in the dispersion. The zeta potential can be measured on a Wyatt Technologies Mobius zeta potential instrument. This instrument characterizes mobility and zeta potential by the principle of "Massively Parallel Phase Analysis Light Scattering" or MP-PALS. Compared with ISO method 13099-1:2012, which uses only one detection angle and requires a higher operating voltage, this measurement is more sensitive and induces less stress. In some embodiments, an instrument using the MP-PALS principle is used to measure the zeta potential of the lipids of the empty lipid nanoparticle composition described herein. Zeta potential can be measured on a Malvern Zetasizer (NanoZS).

In some embodiments, greater than about 80%, greater than about 90%, or greater than about 95% of the lipid amines are on the surface of lipid nanoparticles of the lipid nanoparticle composition.

In some embodiments, the lipid nanoparticle composition has a polydispersity value of less than about 0.4, less than about 0.3, or less than about 0.2. In some embodiments, the LNP has a polydispersity value of about 0.1 to about 1, about 0.1 to about 0.5, or about 0.1 to about 0.3.

In some embodiments, the lipid nanoparticles of the lipid nanoparticle composition have an average diameter of about 40 nm to about 150 nm, about 50 nm to about 100 nm, about 60 nm to about 120 nm, about 60 nm to about 100 nm, or about 60 nm to about 80 nm.

In some embodiments, the lipid nanoparticles of the lipid nanoparticle composition have an overall polarization of laurdan greater than or equal to about 0.6. In some embodiments, the LNP has a d-spacing greater than about 6 nm or greater than about 7 nm.

In some embodiments, at least about 50%, at least about 75%, at least about 90%, at least about 95% of the lipid nanoparticles of the lipid nanoparticle composition have a surface fluidity value greater than a threshold polarization level.

Ionizable lipids

As used herein, the term "ionizable lipid" has its common meaning in the art and may refer to a lipid comprising one or more charged moieties. In some embodiments, an ionizable lipid may be positively or negatively charged. For example, an ionizable lipid may be positively charged at a relatively low pH, in which case it may be referred to as a "cationic lipid". In certain embodiments, an ionizable lipid molecule may comprise an amine group, and may be referred to as an ionizable amino lipid. As used herein, a "charged moiety" is a chemical moiety that carries a formal charge (e.g., monovalent (+1 or -1), divalent (+2 or -2), trivalent (+3 or -3) etc.). The charged moiety may be anionic (i.e., negatively charged) or cationic (i.e., positively charged). Examples of positively charged moieties include amine groups (e.g., primary amines, secondary amines and/or tertiary amines), ammonium groups, pyridinium groups, guanidinium groups and imidazolium groups. In a specific embodiment, the charged moiety comprises an amine group. Examples of negatively charged groups or their precursors include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, etc. In some cases, the charge of the charged moiety can vary with environmental conditions, for example, changes in pH can change the charge of the moiety, and/or cause the moiety to become charged or uncharged. In general, the charge density of the molecule can be selected as desired.

It should be understood that the terms "charged" or "charged moiety" do not refer to a "partial negative charge" or "partial positive charge" on a molecule. The terms "partial negative charge" and "partial positive charge" are given their ordinary meanings in the art. A "partial negative charge" can be created when a functional group contains a bond that becomes polarized so that electron density is pulled toward one atom of the bond, thereby creating a partial negative charge on the atom. In general, one of ordinary skill in the art will recognize bonds that can become polarized in this manner.

In some embodiments, the LNP comprises about 30 mol% to about 60 mol%, about 35 mol% to about 55 mol%, about 40 mol% to about 50 mol%, or about 45 mol% to about 50 mol% ionizable lipid.

In some embodiments, the ionizable lipid is an ionizable amino lipid. In one embodiment, the ionizable amino lipid may have a positively charged hydrophilic head and a hydrophobic tail connected via a linker structure.

In some embodiments, the ionizable lipid is a compound of formula (I):

or an N-oxide or salt thereof, wherein:

^R1 is in Indicates a connection point;

^Raα , ^Raβ , ^Raγ and ^Raδ are each independently selected from H, _C2-12 alkyl and _C2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

R ⁴ is selected from -(CH ₂ ) _n OH and

wherein n is selected from 1, 2, 3, 4 and 5;

in Represents a connection point,

wherein R ¹⁰ is N(R) ₂ ;

wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H;

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Each R ⁵ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H;

Each R ⁶ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H;

M and M' are each independently selected from -C(O)O- and -OC(O)-;

R' is a C _1-12 alkyl group or a C _2-12 alkenyl group;

l is selected from 1, 2, 3, 4 and 5; and

m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein:

^R1 is in Indicates a connection point;

^Raα , ^Raβ , ^Raγ and ^Raδ are each H;

^R2 and ^R3 are each _C1-14 alkyl;

R ⁴ is -(CH ₂ ) _n OH;

n is 2;

Each R ⁵ is H;

Each R ⁶ is H;

M and M' are each -C(O)O-;

R' is a C _1-12 alkyl group;

l is 5; and

m is 7.

In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein:

^R1 is in Indicates a connection point;

^Raα , ^Raβ , ^Raγ and ^Raδ are each H;

^R2 and ^R3 are each _C1-14 alkyl;

R ⁴ is -(CH ₂ ) _n OH;

n is 2;

Each R ⁵ is H;

Each R ⁶ is H;

M and M' are each -C(O)O-;

R' is a C _1-12 alkyl group;

l is 3; and

m is 7.

In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein:

^R1 is in Indicates a connection point;

R ^aα is C _2-12 alkyl;

^Raβ , ^Raγ and ^Raδ are each H;

^R2 and ^R3 are each _C1-14 alkyl;

^R4 is

^R10 is -NH( _C1-6 alkyl);

n2 is 2;

Each R ⁵ is H;

Each R ⁶ is H;

M and M' are each -C(O)O-;

R' is a C _1-12 alkyl group;

l is 5; and

m is 7.

In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein:

^R1 is in Indicates a connection point;

^Raα , ^Raβ and ^Raδ are each H;

^Raγ is a _C2-12 alkyl group;

^R2 and ^R3 are each _C1-14 alkyl;

R ⁴ is -(CH ₂ ) _n OH;

n is 2;

Each R ⁵ is H;

Each R ⁶ is H;

M and M' are each -C(O)O-;

R' is a C _1-12 alkyl group;

l is 5; and

m is 7.

In some embodiments, the ionizable lipid is selected from:

or an N-oxide or salt thereof.

In some embodiments, the ionizable lipid is a compound:

or an N-oxide or salt thereof.

In some embodiments, the ionizable lipid is a compound:

or an N-oxide or salt thereof.

In some embodiments, the ionizable lipid is a compound:

or an N-oxide or salt thereof.

In some embodiments, the ionizable lipid is a compound:

or an N-oxide or salt thereof.

In some embodiments, the ionizable lipid is a compound of formula (I):

or an N-oxide or salt thereof, wherein:

^R1 is: in Indicates a connection point;

^Raβ , ^Raγ and ^Raδ are each independently selected from H, _C2-12 alkyl and _C2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

R ⁴ is selected from -(CH ₂ ) _n OH and

in Indicates a connection point;

wherein n is selected from 1, 2, 3, 4 and 5;

wherein R ¹⁰ is N(R) ₂ ;

wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H;

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Each R ⁵ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H;

Each R ⁶ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H;

M and M' are each independently selected from -C(O)O- and -OC(O)-;

R' is a C _1-12 alkyl group or a C _2-12 alkenyl group;

l is selected from 1, 2, 3, 4 and 5; and

m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In some embodiments, the ionizable lipid is a compound of formula (I):

or an N-oxide or salt thereof, wherein:

^R1 is: in Indicates a connection point;

^Raα , ^Raβ , ^Raγ and ^Raδ are each independently selected from H, _C2-12 alkyl and _C2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

R ⁴ is -(CH ₂ ) _n OH, wherein n is selected from 1, 2, 3, 4 and 5;

Each R ⁵ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H;

Each R ⁶ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H;

M and M' are each independently selected from -C(O)O- and -OC(O)-;

R' is a C _1-12 alkyl group or a C _2-12 alkenyl group;

l is selected from 1, 2, 3, 4 and 5; and

m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein:

^R1 is in Indicates a connection point;

^Raβ , ^Raγ and ^Raδ are each H;

^R2 and ^R3 are each _C1-14 alkyl;

R ⁴ is -(CH ₂ ) _n OH;

n is 2;

Each R ⁵ is H;

Each R ⁶ is H;

M and M' are each -C(O)O-;

R' is a C _1-12 alkyl group;

l is 5; and

m is 7.

In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein:

^R1 is in Indicates a connection point;

^Raβ , ^Raγ and ^Raδ are each H;

^R2 and ^R3 are each _C1-14 alkyl;

R ⁴ is -(CH ₂ ) _n OH;

n is 2;

Each R ⁵ is H;

Each R ⁶ is H;

M and M' are each -C(O)O-;

R' is a C _1-12 alkyl group;

l is 3; and

m is 7.

In some embodiments, the ionizable lipid is a compound of formula (I) or an N-oxide or salt thereof, wherein:

^R1 is in Indicates a connection point;

R ^aβ and R ^aδ are each H;

^Raγ is a _C2-12 alkyl group;

^R2 and ^R3 are each _C1-14 alkyl;

R ⁴ is -(CH ₂ ) _n OH;

n is 2;

Each R ⁵ is H;

Each R ⁶ is H;

M and M' are each -C(O)O-;

R' is a C _1-12 alkyl group;

l is 5; and

m is 7.

In some embodiments, the ionizable lipid is a compound of formula (I):

or an N-oxide or salt thereof, wherein:

^R1 is: in Indicates a connection point;

^Raα , ^Raβ , ^Raγ and ^Raδ are each independently selected from H, _C2-12 alkyl and _C2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

^R4 is

in Indicates a connection point;

wherein R ¹⁰ is N(R) ₂ ;

wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H;

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Each R ⁵ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H;

Each R ⁶ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H;

M and M' are each independently selected from -C(O)O- and -OC(O)-;

R' is a C _1-12 alkyl group or a C _2-12 alkenyl group;

l is selected from 1, 2, 3, 4 and 5; and

m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13.

In some embodiments:

^R1 is in Indicates a connection point;

^Raβ , ^Raγ and ^Raδ are each H;

R ^aα is C _2-12 alkyl;

^R2 and ^R3 are each _C1-14 alkyl;

^R4 is

in Indicates a connection point;

Wherein R ¹⁰ is NH(C _1-6 alkyl);

Where n2 is 2;

Each R ⁵ is H;

Each R ⁶ is H;

M and M' are each -C(O)O-;

R' is a C _1-12 alkyl group;

l is 5; and

m is 7.

In some embodiments, the ionizable lipid of formula (I) is:

or an N-oxide or salt thereof.

In some embodiments, the ionizable lipid is a compound of formula (II):

or an N-oxide or salt thereof, wherein:

^R'a is R' ^branched or R'^cyclic;

The R' ^branch is: And the R' ^ring is: and

^R'b is: in Indicates a connection point;

^Raγ and ^Raδ are each independently selected from H, C _1-12 alkyl and C _2-12 alkenyl, wherein at least one of ^Raγ and ^Raδ is selected from C _1-12 alkyl and C _2-12 alkenyl;

R ^bγ and R ^bδ are each independently selected from H, C _1-12 alkyl and C _2-12 alkenyl, wherein at least one of R ^bγ and R ^bδ is selected from C _1-12 alkyl and C _2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

R ⁴ is selected from -(CH ₂ ) _n OH and

in Indicates a connection point;

wherein n is selected from 1, 2, 3, 4 and 5;

wherein R ¹⁰ is N(R) ₂ ;

wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H;

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Each R' is independently a C _1-12 alkyl or a C _2-12 alkenyl;

Y ^a is a C _3-6 carbon ring;

R*" ^a is selected from _C1-15 alkyl and _C2-15 alkenyl;

s is 2 or 3;

m is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; and

l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

In some embodiments, the ionizable lipid is a compound of formula (II):

or an N-oxide or salt thereof, wherein:

^R'a is R' ^branched or R'^cyclic;

The R' ^branch is: And ^R'b is:

in Indicates a connection point;

^Raγ and ^Raδ are each independently selected from H, C _1-12 alkyl and C _2-12 alkenyl, wherein at least one of ^Raγ and ^Raδ is selected from C _1-12 alkyl and C _2-12 alkenyl;

R ^bγ and R ^bδ are each independently selected from H, C _1-12 alkyl and C _2-12 alkenyl, wherein at least one of R ^bγ and R ^bδ is selected from C _1-12 alkyl and C _2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

R ⁴ is selected from -(CH ₂ ) _n OH and

in Indicates a connection point;

wherein n is selected from 1, 2, 3, 4 and 5;

wherein R ¹⁰ is N(R) ₂ ;

wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H;

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Each R' is independently a C _1-12 alkyl or a C _2-12 alkenyl;

m is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; and

l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

In some embodiments, the ionizable lipid is a compound of formula (II):

or an N-oxide or salt thereof, wherein:

^R'a is R' ^branched or R'^cyclic;

The R' ^branch is: And ^R'b is:

in Indicates a connection point;

^Raγ and ^Rbγ are each independently selected from _C1-12 alkyl and _C2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

R ⁴ is selected from -(CH ₂ ) _n OH and

in Indicates a connection point;

wherein n is selected from 1, 2, 3, 4 and 5;

wherein R ¹⁰ is N(R) ₂ ;

wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H; and wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Each R' is independently a C _1-12 alkyl or a C _2-12 alkenyl;

m is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; and

l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

In some embodiments, the ionizable lipid is a compound of formula (II):

or an N-oxide or salt thereof, wherein:

^R'a is R' ^branched or R'^cyclic;

The R' ^branch is: And ^R'b is:

in Indicates a connection point;

R ^aγ is selected from C _1-12 alkyl and C _2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

R ⁴ is selected from -(CH ₂ ) _n OH and

in Indicates a connection point;

wherein n is selected from 1, 2, 3, 4 and 5;

wherein R ¹⁰ is N(R) ₂ ;

wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H;

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

R' is a C _1-12 alkyl group or a C _2-12 alkenyl group;

m is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; and

l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

In some embodiments, the ionizable lipid is a compound of formula (II):

or an N-oxide or salt thereof, wherein:

^R'a is R' ^branched or R'^cyclic;

The R' ^branch is: And ^R'b is:

in Indicates a connection point;

^Raγ and ^Rbγ are each independently selected from _C1-12 alkyl and _C2-12 alkenyl;

R ⁴ is selected from -(CH ₂ ) _n OH and

in Indicates a connection point;

wherein n is selected from 1, 2, 3, 4 and 5;

wherein R ¹⁰ is N(R) ₂ ;

wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H;

wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;

Each R' is independently a C _1-12 alkyl or a C _2-12 alkenyl;

m is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8 and 9; and l is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8 and 9.

In some embodiments, the ionizable lipid is a compound of formula (II):

or an N-oxide or salt thereof, wherein:

^R'a is R' ^branched or R'^cyclic;

The R' ^branch is: And ^R'b is: in Indicates a connection point;

R ^aγ is selected from C _1-12 alkyl and C _2-12 alkenyl;

^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl;

R ⁴ is -(CH ₂ ) _n OH, wherein n is selected from 1, 2, 3, 4 and 5;

R' is a C _1-12 alkyl group or a C _2-12 alkenyl group;

m is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9; and

l is selected from 1, 2, 3, 4, 5, 6, 7, 8 and 9.

In some embodiments, m and l are each independently selected from 4, 5, and 6. In some embodiments, m and l are each 5.

In some embodiments, each R' is independently C _1-12 alkyl. In some embodiments, each R' is independently C _2-5 alkyl.

In some embodiments, ^R'b is: And R ² and R ³ are each independently a C _1-14 alkyl group.

In some embodiments, ^R'b is: And R ² and R ³ are each independently a C _6-10 alkyl group.

In some embodiments, ^R'b is: And ^R2 and ^R3 are each a _C8 alkyl group.

In some embodiments, the R' ^branch is: And ^R'b is: ^Raγ is a _C1-12 alkyl group, and ^R2 and ^R3 are each independently a _C6-10 alkyl group.

In some embodiments, the R' ^branch is: And ^R'b is: ^Raγ is _C2-6 alkyl, and ^R2 and ^R3 are each independently _C6-10 alkyl. In some embodiments, the R' ^branch is: And ^R'b is: ^Raγ is a _C2-6 alkyl group, and ^R2 and ^R3 are each a _C8 alkyl group.

In some embodiments, the R' ^branch is: ^R'b is: And R ^aγ and R ^bγ are each a C _1-12 alkyl group.

In some embodiments, the R' ^branch is: ^R'b is: And R ^aγ and R ^bγ are each a C _2-6 alkyl group.

In some embodiments, m and l are each independently selected from 4, 5, and 6 and each R' is independently C _1-12 alkyl. In some embodiments, m and l are each 5 and each R' is independently C _2-5 alkyl.

In some embodiments, the R' ^branch is: ^R'b is: m and l are each independently selected from 4, 5 and 6, each R' is independently a C _1-12 alkyl group, and ^Raγ and ^Rbγ are each a C _1-12 alkyl group.

In some embodiments, the R' ^branch is: ^R'b is: m and l are each 5, each R' is independently a _C2-5 alkyl group, and ^Raγ and ^Rbγ are each a _C2-6 alkyl group.

In some embodiments, the R' ^branch is: And ^R'b is: m and l are each independently selected from 4, 5 and 6, R' is a C _1-12 alkyl group, ^Raγ is a C _1-12 alkyl group, and R ² and R ³ are each independently a C _6-10 alkyl group.

In some embodiments, the R' ^branch is: And ^R'b is: m and l are each 5, R' is a _C2-5 alkyl group, ^Raγ is a _C2-6 alkyl group, and ^R2 and ^R3 are each a _C8 alkyl group.

In some embodiments, ^R4 is wherein R ¹⁰ is NH(C _1-6 alkyl) and n2 is 2.

In some embodiments, ^R4 is wherein R ¹⁰ is NH(CH ₃ ) and n2 is 2.

In some embodiments, the R' ^branch is: ^R'b is: m and l are each independently selected from 4, 5 and 6; each R' is independently C _1-12 alkyl; ^Raγ and ^Rbγ are each C _1-12 alkyl; and ^R4 is wherein R ¹⁰ is NH(C _1-6 alkyl), and n2 is 2.

In some embodiments, the R' ^branch is: ^R'b is: m and l are each 5, each R' is independently _C2-5 alkyl, ^Raγ and ^Rbγ are each _C2-6 alkyl and ^R4 is wherein R ¹⁰ is NH(CH ₃ ) and n2 is 2.

In some embodiments, the R' ^branch is: And ^R'b is: m and l are each independently selected from 4, 5 and 6, R' is C _1-12 alkyl, R ² and R ³ are each independently C _6-10 alkyl, ^Raγ is C _1-12 alkyl and R ⁴ is wherein R ¹⁰ is NH(C _1-6 alkyl) and n2 is 2.

In some embodiments, the R' ^branch is: And ^R'b is: m and l are each 5, R' is a C _2-5 alkyl group, ^Raγ is a C _2-6 alkyl group, R ² and R ³ are each a C ₈ alkyl group and R ⁴ is wherein R ¹⁰ is NH(CH ₃ ) and n2 is 2.

In some embodiments, ^R4 is -( _CH2 ) _nOH and n is 2, 3, or 4. In some embodiments, ^R4 is -( _CH2 ) _nOH and n is 2.

In some embodiments, the R' ^branch is: ^R'b is: m and l are each independently selected from 4, 5 and 6, each R′ is independently C _1-12 alkyl, ^Raγ and ^Rbγ are each C _1-12 alkyl, R ⁴ is —(CH ₂ ) _n OH, and n is 2, 3 or 4.

In some embodiments, the R' ^branch is: ^R'b is: m and l are each 5, each R′ is independently a C _2-5 alkyl group, ^Raγ and R ^bγ are each a C _2-6 alkyl group, R ⁴ is —(CH ₂ ) _n OH, and n is 2.

In some embodiments, the ionizable lipid is a compound of formula (II):

or an N-oxide or salt thereof, wherein:

^R'a is R' ^branched or R'^cyclic;

The R' ^branch is: And ^R'b is:

in Indicates a connection point;

^Raγ is a _C1-12 alkyl group;

^R2 and ^R3 are each independently _C1-14 alkyl;

R ⁴ is -(CH ₂ ) _n OH, wherein n is selected from 1, 2, 3, 4 and 5;

R' is a C _1-12 alkyl group;

m is selected from 4, 5 and 6; and

l is selected from 4, 5 and 6.

In some embodiments, m and l are each 5, and n is 2, 3, or 4.

In some embodiments, R' is _C2-5 alkyl, ^Raγ is _C2-6 alkyl, and ^R2 and ^R3 are each _C6-10 alkyl.

In some embodiments, m and l are each 5, n is 2, 3 or 4, R' is _C2-5 alkyl, ^Raγ is _C2-6 alkyl, and ^R2 and ^R3 are each _C6-10 alkyl.

In some embodiments, the ionizable lipid is a compound of formula (II-g):

or an N-oxide or salt thereof, wherein:

^Raγ is a _C2-6 alkyl group;

R' is _C2-5 alkyl; and

R ⁴ is selected from -(CH ₂ ) _n OH and

in Represents a connection point,

wherein n is selected from 3, 4 and 5; and

wherein R ¹⁰ is NH(C _1-6 alkyl); and

wherein n2 is selected from 1, 2 and 3.

In some embodiments, the ionizable lipid is a compound of formula (II-h):

or an N-oxide or salt thereof, wherein:

R ^aγ and R ^bγ are each independently C _2-6 alkyl;

Each R' is independently _C2-5 alkyl; and

R ⁴ is selected from -(CH ₂ ) _n OH and

in Represents a connection point,

wherein n is selected from 3, 4 and 5;

wherein R ¹⁰ is NH(C _1-6 alkyl); and

wherein n2 is selected from 1, 2 and 3.

In some embodiments, ^R4 is

wherein R ¹⁰ is NH(CH ₃ ) and n2 is 2.

In some embodiments, R ⁴ is -(CH ₂ ) ₂ OH.

In some embodiments, the ionizable lipid is a compound having formula (III):

or an N-oxide or salt thereof, wherein:

R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are independently selected from C _5-20 alkyl, C _5-20 alkenyl, -R"MR', -R*YR", -YR" and -R*OR";

each M is independently selected from -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) _2- , aryl, and heteroaryl;

X ¹ , X ² , and X ³ are each independently selected from a bond, -CH ₂ -, -(CH ₂ ) ₂ -, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -C(O)-CH ₂ -, -CH ₂ -C(O)-, -C(O)O-CH ₂ -, -OC(O)-CH ₂ -, -CH ₂ -C(O)O-, -CH ₂ -OC(O)-, -CH(OH)-, -C(S)-, and -CH(SH)-;

Each Y is independently a C _3-6 carbocyclic ring;

Each R* is independently selected from C _1-12 alkyl and C _2-12 alkenyl;

Each R is independently selected from C _1-3 alkyl and C _3-6 carbocycle;

Each R' is independently selected from C _1-12 alkyl, C _2-12 alkenyl, and H; and

Each R" is independently selected from _C3-12 alkyl and _C3-12 alkenyl, and wherein:

i) at least one of X ¹ , X ² and X ³ is not -CH ₂ -; and/or

ii) At least one of R ₁ , R ₂ , R ₃ , R ₄ and R ₅ is -R"MR'.

In some embodiments, R ₁ , R ₂ , R ₃ , R ₄ , and R ₅ are each C _5-20 alkyl; X ¹ is -CH ₂ -; and X ² and X ³ are each -C(O)-.

In some embodiments, the compound of formula (III) is:

Phospholipids

Phospholipids as defined herein are any lipids comprising a phosphate group. Phospholipids are a subgroup of non-cationic lipids. The LNP core may include one or more phospholipids, such as one or more (poly) unsaturated lipids. Phospholipids can be assembled into one or more lipid bilayers. Generally speaking, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. The phospholipid moiety may be selected from the non-limiting group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine and sphingomyelin. The fatty acid moiety may be selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid and docosahexaenoic acid. Non-natural substances are also contemplated, including natural substances with modification and substitution (including branching, oxidation, cyclization and alkynes). For example, phospholipids can be functionalized or cross-linked with one or more alkynes (e.g., alkenyl groups in which one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, the alkyne groups can undergo copper-catalyzed cycloadditions upon exposure to azides. Such reactions can be used to functionalize the lipid bilayer of nanoparticle compositions to facilitate membrane penetration or cell recognition or to conjugate nanoparticle compositions to useful components, such as targeting or imaging moieties (e.g., dyes).

In some embodiments, the LNP comprises about 5 mol% to about 15 mol%, about 8 mol% to about 13 mol%, or about 10 mol% to about 12 mol% phospholipid.

In some embodiments, the phospholipid is a compound of formula (IV):

or a salt thereof, wherein:

Each R ¹ is independently H or an optionally substituted alkyl; or optionally two R ¹ are joined together with an intermediate atom to form an optionally substituted monocyclic cycloalkyl or an optionally substituted monocyclic heterocyclyl; or optionally three R ¹ are joined together with an intermediate atom to form an optionally substituted bicyclic cycloalkyl or an optionally substituted bicyclic heterocyclyl;

n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

A has the following formula:

^each instance of L is independently a bond or optionally substituted Ci _-6 alkylene, wherein one methylene unit of the optionally substituted Ci _-6 alkylene is optionally replaced by -O-, -N( ^RN )-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C( ^O )O-, -OC(O)-, -OC(O)O-, -OC(O)N( ^RN )-, -NRNC ⁽ O)O-, or ^{-NRNC (} O)N( ^RN )-;

each instance ^of R is independently optionally substituted _C1-30 alkyl, optionally substituted _C1-30 alkenyl, or optionally substituted _C1-30 alkynyl; optionally wherein one or more methylene units of ^R are independently replaced by optionally substituted cycloalkylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N( ^RN )-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C( ^O )O-, -OC(O)-, -OC(O) ^O- , -OC( ^O )N( ^RN )-, -NRNC ⁽ O)O-, ^-C (O)S-, -SC(O)-, -C(═NRN ⁾ -, -C(═NRN ⁾ -, or )N(R ^N )-, -NR ^N C(＝NR ^N )-, -NR ^N C(＝NR ^N )N(R ^N )-, -C(S)-, -C(S)N(R ^N )-, -NR ^N C(S)-, -NR ^N C(S)N(R ^N )-, -S(O)-, -OS(O)-, -OS(O)O-, -OS(O) ₂ -, _{-S(O) 2 O-, -OS(O) 2 O-} , -N(R ^N )S(O)-, -S(O)N(R ^N )-, -N(R ^N )S(O)N(R ^N )-, -OS(O)N(R ^N )-, -N(R ^N )S(O)O-, -S(O) ₂ -, -N(R ^N )S(O) ₂ -, -S(O) ₂ N(R ^N )-, -N(R ^N )S(O) ₂ N(R ^N )-, -OS(O) ₂ N(R ^N )- or -N(R ^N )S(O) ₂ O-;

Each instance of ^RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

Ring B is optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl; and

p is 1 or 2;

Provided that the compound does not have the formula:

wherein each instance of R ² is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

In some embodiments, the phospholipid is selected from:

1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC),

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),

1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),

1,2-Dimyristoyl-sn-glycero-phosphocholine (DMPC),

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC),

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),

1,2-diundecanoyl-sn-glycerophosphocholine (DUPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),

1,2-di-O-octadecene-sn-glycero-3-phosphocholine (18:0 diether PC),

1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC),

1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),

1,2-Dilinolenoyl-sn-glycero-3-phosphocholine,

1,2-Diarachidonoyl-sn-glycero-3-phosphocholine,

1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine,

1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE),

1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0PC),

1,2-Diphytanoyl-sn-glycero-3-phospho-(1'-racemic-glycerol) (sodium salt) (4ME16:0PG),

1,2-Diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0PS),

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine,

1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine,

1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine,

1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine,

1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine,

1,2-Dioleoyl-sn-glycero-3-phospho-racemic-(1-glycerol) sodium salt (DOPG) and

Sphingomyelin.

In some embodiments, the phospholipid is DSPC, DOPE, or a combination thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the phospholipid is DOPE. In some embodiments, the phospholipid is 4ME 16:0PE, 4ME 16:0PC, 4ME 16:0PG, 4ME 16:0PS, or a combination thereof.

In some embodiments, the phospholipid is N-lauroyl-D-erythro-sphingylphosphocholine.

Alternative lipids

In certain embodiments, alternative lipids are used instead of phospholipids. Non-limiting examples of such alternative lipids include the following:

Structural lipids

The LNP core may include one or more structural lipids. Incorporation of structural lipids into lipid nanoparticles may help reduce the aggregation of other lipids in the particle. The structural lipids may be selected from the group including, but not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine glycosides, ursolic acid, alpha-tocopherol, hopanes, plant sterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, "sterol" is a subgroup of the steroid consisting of steroid alcohols.

In some embodiments, the structural lipid is alpha-tocopherol. In some embodiments, the structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid is an analog of cholesterol. In some embodiments, the structural lipid is beta-sitosterol. In some embodiments, the structural lipid is hemisuccinate cholesteryl ester. Hemisuccinate cholesteryl ester has the following structure:

In some embodiments, the LNP comprises about 20mol% to about 60mol%, about 30mol% to about 50mol%, or about 35mol% to about 40mol% of structural lipids. In some embodiments, the LNP comprises about 35mol% of structural lipids. In some embodiments, the LNP comprises about 40mol% of structural lipids.

PEG and PEGylated lipids

The LNP core can include one or more molecules comprising polyethylene glycol (PEG), such as PEG-modified lipids. Such substances may alternatively be referred to as PEGylated lipids. PEG-lipids are lipids modified with polyethylene glycol. PEG-lipids can be selected from the following non-limiting groups: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

In some embodiments, the PEG lipid is a compound of formula (V):

or a salt thereof, wherein:

^R3 is ^-ORO ;

R ^O is hydrogen, optionally substituted alkyl or an oxygen protecting group;

r is an integer between 1 and 100, inclusive;

^L is optionally substituted C _1-10 alkylene, wherein at least one methylene group of the optionally substituted C _1-10 alkylene is independently replaced by optionally substituted cycloalkylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -O-, -N( ^RN )-, -S-, -C(O) ^- , -C(O)N(RN)-, -NRNC( ^O )-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)N( ^RN )-, -NRNC ⁽ O)O-, or ^-NRNC (O)N( ^RN )-;

D is a moiety obtained by click chemistry or a moiety that can be cleaved under physiological conditions;

m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

A has the following formula:

^each instance of L is independently a bond or optionally substituted Ci _-6 alkylene, wherein one methylene unit of the optionally substituted Ci _-6 alkylene is optionally replaced by -O-, -N( ^RN )-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C( ^O )O-, -OC(O)-, -OC(O)O-, -OC(O)N( ^RN )-, -NRNC ⁽ O)O-, or ^{-NRNC (} O)N( ^RN )-;

each instance ^of R is independently optionally substituted _C1-30 alkyl, optionally substituted _C1-30 alkenyl, or optionally substituted _C1-30 alkynyl; optionally wherein one or more methylene units of ^R are independently replaced by optionally substituted cycloalkylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N( ^RN )-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C( ^O )O-, -OC(O)-, -OC(O) ^O- , -OC( ^O )N( ^RN )-, -NRNC ⁽ O)O-, ^-C (O)S-, -SC(O)-, -C(═NRN ⁾ -, -C(═NRN ⁾ -, or )N(R ^N )-, -NR ^N C(＝NR ^N )-, -NR ^N C(＝NR ^N )N(R ^N )-, -C(S)-, -C(S)N(R ^N )-, -NR ^N C(S)-, -NR ^N C(S)N(R ^N )-, -S(O)-, -OS(O)-, -OS(O)O-, -OS(O) ₂ -, _{-S(O) 2 O-, -OS(O) 2 O-} , -N(R ^N )S(O)-, -S(O)N(R ^N )-, -N(R ^N )S(O)N(R ^N )-, -OS(O)N(R ^N )-, -N(R ^N )S(O)O-, -S(O) ₂ -, -N(R ^N )S(O) ₂ -, -S(O) ₂ N(R ^N )-, -N(R ^N )S(O) ₂ N(R ^N )-, -OS(O) ₂ N(R ^N )- or -N(R ^N )S(O) ₂ O-;

Each instance of ^RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

Ring B is optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl; and

p is 1 or 2.

In some embodiments, the PEG lipid is PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE. In some embodiments, the PEG lipid is PEG-DMG. In some embodiments, the PEG lipid is PEG-DMG 2k. In some embodiments, the PEG lipid has the following structure:

DMG-PEG 2k has the following structure:

In some embodiments, the PEG-modified lipid is a modified form of PEG-DMG.

In one embodiment, the PEG-lipid may be a PEGylated lipid as described in International Publication No. WO2012099755, the contents of which are herein incorporated by reference in their entirety.

Any PEG-lipid described herein can be modified to include one or more hydroxyl groups on the PEG chain (OH-PEG-lipid) or one or more hydroxyl groups on the lipid (PEG-lipid-OH). In some embodiments, the PEG-lipid is an OH-PEG-lipid. In some embodiments, the OH-PEG-lipid comprises a hydroxyl group at the end of the PEG chain. In some embodiments, the PEG-lipid described herein can be modified to include one or more alkyl groups on the PEG chain (alkyl-PEG-lipid). In some embodiments, the alkyl-PEG-lipid is a methoxy-PEG-lipid.

In some embodiments, the LNP comprises about 0.1 mol% to about 5.0 mol%, about 0.5 mol% to about 5.0 mol%, about 1.0 mol% to about 5.0 mol%, about 1.0 mol% to about 2.5 mol%, about 0.5 mol% to about 2.0 mol%, or about 1.0 mol% to about 1.5 mol% of PEG-lipid. In some embodiments, the LNP comprises about 1.5 mol% or about 3.0 mol% of PEG-lipid.

Certain LNPs provided herein contain no or low levels of PEG-lipid. Some LNPs contain less than 0.5 mol% PEG-lipid.

In some embodiments, PEG is used as a stabilizer. In some embodiments, the PEG stabilizer is PEG-lipid. In some embodiments, the LNP comprises less than 0.5 mol% of a PEG stabilizer.

Payload molecule

Lipid nanoparticle compositions of the present disclosure can be used for delivering numerous different payloads to cells. The payload can be a therapeutic agent or preventive agent that can mediate (e.g., directly mediate or via a bystander effect) the therapeutic or preventive effects in such cells. Typically, the payload delivered by the composition is a nucleic acid, but the disclosure also encompasses non-nucleic acid agents, such as small molecules, chemotherapeutic drugs, peptides, polypeptides, and other biomolecules. Deliverable nucleic acids include molecules based on DNA (i.e., comprising deoxyribonucleotides) and molecules based on RNA (i.e., comprising ribonucleotides). In addition, nucleic acids can be naturally occurring forms of molecules or chemically modified forms of molecules (e.g., comprising one or more modified nucleotides).

In one embodiment, the therapeutic or preventive agent is an agent that enhances (i.e., increases, stimulates, upregulates) protein expression. Non-limiting examples of the types of therapeutic or preventive agents that can be used to enhance protein expression include RNA, mRNA, dsRNA, CRISPR/Cas9 technology, ssDNA, and DNA (e.g., expression vectors).

In one embodiment, the therapeutic or preventive agent is an agent that reduces (i.e., reduces, inhibits, downregulates) protein expression. Non-limiting examples of the types of therapeutic or preventive agents that can be used to reduce protein expression include mRNAs that incorporate microRNA binding sites (miR binding sites), microRNAs (miRNAs), antagomirs, small (short) interfering RNAs (siRNAs) (including short polymers and Dicer substrate RNAs), RNA interference (RNAi) molecules, antisense RNAs, ribozymes, small hairpin RNAs (shRNAs), locked nucleic acids (LNAs), and CRISPR/Cas9 technology.

In one embodiment, the therapeutic or prophylactic agent is a peptide therapeutic agent. In one embodiment, the therapeutic or prophylactic agent is a polypeptide therapeutic agent. In some embodiments, the therapeutic or prophylactic agent includes an mRNA encoding a secreted protein; a membrane-bound protein; or an intercellular protein, or a peptide, polypeptide, or a biologically active fragment thereof.

In some embodiments, at least about 50%, at least about 75%, at least about 90%, or at least about 95% of the effective load is encapsulated in the lipid nanoparticles. In some embodiments, about 50% to about 99%, about 65% to about 99%, about 75% to about 95%, or about 80% to about 95% of the effective load is encapsulated in the lipid nanoparticles.

cell

LNP can be used to deliver payload molecules to cell colonies. In some embodiments, LNP is contacted with cell colonies. In some embodiments, when LNP is contacted with cell colonies, about 10% or more, 15% or more, 20% or more, or 30% or more of the cell colonies have accumulated LNP. In some embodiments, when LNP is contacted with cell colonies, about 1% to about 75%, about 5% to about 50%, about 10% to about 40%, or about 15% to about 25% of the cell colonies have accumulated LNP.

In some embodiments, when the LNP is contacted with a cell population, about 5% or more, about 10% or more, or about 20% or more of the cells in the cell population express the effective load. In some embodiments, when the LNP is contacted with a cell population, about 0.5% to about 50%, about 1% to about 40%, about 3% to about 20%, or about 5% to about 15% of the cells in the cell population express the effective load.

In some embodiments, the cell population is an epithelial cell population. In some embodiments, the cell population is a respiratory epithelial cell population. In some embodiments, the respiratory epithelial cell population is a lung cell population. In some embodiments, the respiratory epithelial cell population is a nasal cell population. In some embodiments, the respiratory epithelial cell population is an alveolar epithelial cell population. In some embodiments, the respiratory epithelial cell population is a bronchial epithelial cell population. In some embodiments, the respiratory epithelial cell population is a HBE population. In some embodiments, the cell population is a HeLa population.

Pharmaceutical compositions and preparations

The present disclosure provides pharmaceutical compositions and formulations comprising any of the LNPs described herein.

The pharmaceutical composition or formulation may optionally include one or more other active substances, such as therapeutic and/or prophylactic active substances. The pharmaceutical composition or formulation of the present disclosure may be sterile and/or pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents can be found in, for example, Remington: The Science and Practice of Pharmacy 21st edition, Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference in its entirety). In some embodiments, the composition is administered to humans, human patients, or subjects. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to nanoparticles containing a polynucleotide or polypeptide payload to be delivered as described herein.

The formulations and pharmaceutical compositions described herein can be prepared by any method known or later developed in the art of pharmacology. In general, such preparation methods include the steps of associating the nanoparticles with excipients and/or one or more other auxiliary ingredients, and then dividing, shaping and/or packaging the product into desired single or multiple dose units when necessary and/or desired.

Pharmaceutical compositions or formulations according to the present disclosure can be prepared, packaged and/or sold in bulk, in a single unit dose and/or in multiple single unit doses. As used herein, "unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and/or a suitable fraction of this dose, such as half or one-third of this dose.

The relative amounts of active ingredient, pharmaceutically acceptable excipient and/or any other ingredients in a pharmaceutical composition according to the present disclosure may vary depending on the identity, size and/or condition of the subject being treated and further depending on the route by which the composition is to be administered.

Although the descriptions of pharmaceutical compositions and formulations provided herein are primarily directed to pharmaceutical compositions and formulations suitable for administration to humans, one skilled in the art will appreciate that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals.

As used herein, pharmaceutically acceptable excipients include, but are not limited to, any and all solvents, dispersion media or other liquid vehicles, dispersion or suspension aids, diluents, granulation and/or dispersants, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, adhesives, lubricants or oils, colorants, sweeteners or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmotic pressure regulators, pH regulators, buffers, chelating agents, cryoprotectants and/or bulking agents suitable for the desired specific dosage form. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see Remington: The Science and Practice of Pharmacy, 21st ed., A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; which is incorporated herein by reference in its entirety).

Exemplary diluents include, but are not limited to, calcium or sodium carbonate, calcium phosphate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, the like, and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are not limited to, starch, pregelatinized starch or microcrystalline starch, alginic acid, guar gum, agar, poly(vinyl-pyrrolidone), (povidone), cross-linked poly(vinyl-pyrrolidone) (crospovidone), cellulose, methylcellulose, carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), magnesium aluminum silicate, Sodium lauryl sulfate, and the like and/or combinations thereof.

Exemplary surfactants and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [ 80], Sorbitan monopalmitate[ 40], monooleylglycerol, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g. ), polyoxyethylene ethers (e.g. polyoxyethylene lauryl ether [ 30]), F 68, 188, etc. and/or combinations thereof.

Exemplary binders include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., gum arabic, sodium alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, the like, and combinations thereof.

Oxidation is a potential degradation pathway for mRNA, especially liquid mRNA formulations. To prevent oxidation, antioxidants can be added to the formulation. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, and the like, and combinations thereof.

Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, trisodium edetate, the like, and combinations thereof.

Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, the like, and combinations thereof.

Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisole, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), the like, and combinations thereof.

In some embodiments, the pH of the polynucleotide solution is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers for controlling pH may include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, the like, and/or combinations thereof.

Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, and the like, and combinations thereof.

The pharmaceutical compositions described herein may contain cryoprotectants to stabilize the polynucleotides described herein during freezing. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and the like, and combinations thereof.

The pharmaceutical compositions described herein may contain bulking agents in the lyophilized polynucleotide formulations to produce a "pharmaceutically elegant" cake that stabilizes the lyophilized polynucleotides during long-term (e.g., 36 months) storage. Exemplary bulking agents of the present disclosure may include, but are not limited to, sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof.

The composition may be in liquid form or solid form. In some embodiments, the composition or formulation is in liquid form. In some embodiments, the composition is suitable for inhalation. The composition may be applied to the lungs. Aerosolized drug formulations may be delivered to the lungs, preferably using a variety of commercially available devices.

The composition can be administered to the respiratory tract by a suitable method (e.g., intranasal instillation, intratracheal instillation, and intratracheal injection). In some embodiments, the composition or nanoparticles are administered by intranasal, intrabronchial, or pulmonary administration. For example, the composition and nanoparticles are administered by a nebulizer or inhaler.

In some embodiments, the composition is delivered to the lungs by inhalation of an aerosolized pharmaceutical formulation. Inhalation can be performed via the nose and/or mouth of the subject. Administration can be performed by self-administering the formulation during inhalation or administering the formulation to the subject on the ventilator via a ventilator. Exemplary devices for delivering the formulation to the lungs include, but are not limited to, dry powder inhalers, pressurized metered dose inhalers, nebulizers, and electrohydrodynamic aerosol devices.

A pressurized metered dose inhaler (pMDI) can be used to apply a liquid formulation to a patient's lungs. A pMDI typically includes at least two components: a canister, in which the liquid formulation is held together under pressure in combination with one or more propellants, and a container for holding and starting the canister. The canister may contain a single or multiple doses of the formulation. The canister may include a valve, typically a metering valve, from which the contents of the canister may be discharged. Aerosolized drugs are dispensed from a pMDI by the following method: a force is applied to the canister to push it into the container, thereby opening the valve and allowing drug particles to be transported from the valve through the container outlet. After discharge from the canister, the liquid formulation is atomized to form an aerosol. A pMDI typically uses one or more propellants to pressurize the contents of the canister, and pushes the liquid formulation out of the container outlet to form an aerosol. Any suitable propellant may be used. The propellant may take a variety of forms. For example, the propellant may be a compressed gas or a liquefied gas.

Liquid preparations can also be applied using a nebulizer. A nebulizer is a liquid aerosol generator that converts liquid preparations into mist or cloud of small droplets, preferably a mist or cloud with a diameter less than 5 micrometers of mass median aerodynamic diameter, which can be inhaled into the lower respiratory tract. This process is referred to as atomization. When an aerosol cloud is inhaled, the droplets carry one or more active agents to the nose, upper respiratory tract or deep lung. Any type of nebulizer can be used to apply preparations to patients, including, but not limited to, pneumatic (jet) nebulizers and electromechanical nebulizers. Pneumatic (jet) nebulizers use pressurized gas supply as the driving force for atomization of liquid preparations. Compressed gas is delivered via a nozzle or jet machine to produce a low-pressure field, which entrains the surrounding liquid preparation and shears it into a film or filament. The film or filament is unstable and can be decomposed into small droplets, which are carried by compressed airflow to the breath of inhalation. The baffle inserted in the droplet plume screens out larger droplets and returns them to the total liquid storage tank. Electromechanical atomizers use electromechanical forces to atomize liquid preparations. Electromechanical driving forces can be applied, for example, by vibrating the liquid preparation at ultrasonic frequencies or by forcing a large amount of liquid through small holes in a film. The force generates a thin film of liquid or a stream of filaments that breaks up into small droplets to form a slowly moving aerosol stream that can be entrained in the inhaled stream. Liquid preparations can also be administered using an electrohydrodynamic (EHD) aerosol device. An EHD aerosol device uses electrical energy to aerosolize a liquid drug solution or suspension.

Dry powder inhalers (DPIs) typically use a mechanism such as gas burst to produce a cloud of dry powder in a container that can then be inhaled by a subject. In a DPI, the dose to be administered is stored in the form of a non-pressurized dry powder, and when the inhaler is activated, the subject inhales particles of the powder. In some cases, compressed gas (i.e., propellant) can be used to dispense the powder, similar to a pressurized metered dose inhaler (pMDI). In some cases, a DPI can be breathably activated, meaning that an aerosol is produced in precise response to inhalation. Typically, a dry powder inhaler administers a dose less than tens of milligrams at each inhalation to avoid causing a cough. Examples of DPIs include Inhaler (Astrazeneca, Wilmington, Del.), Inhaler (Innovata, Ruddington, Nottingham, UKL), Inhaler (Glaxo, Greenford, Middlesex, UK), (Orion,Expo,FI), Inhaler (Pfizer, New York, NY), Inhaler (Microdose, Monmouth Junction, NJ) and Inhaler (Dura, San Diego, Calif.).

The pharmaceutical composition is administered in an effective amount to cause a desired biological effect, such as a therapeutic or preventive effect, for example, due to the expression of a normal gene product to supplement or replace a defective protein or reduce the expression of an undesirable protein, as measured in some embodiments by alleviation of one or more symptoms. The formulation can be administered in an effective amount to deliver the LNP to, for example, the apical membrane of respiratory and non-respiratory epithelial cells to deliver the payload.

How to use

Lipid amine compounds can be used to prepare lipid nanoparticle compositions that can be loaded with a payload and administered to cells, such as cells of a patient for treating a disease. Thus, provided herein are methods of delivering a payload to a cell, such as by contacting the cell with a lipid nanoparticle composition disclosed herein.

In some embodiments, the cell is an epithelial cell. In some embodiments, the cell is an airway epithelial cell. In some embodiments, the cell is a respiratory epithelial cell. The respiratory epithelial cell can be, for example, a lung cell, a nasal cell, an alveolar epithelial cell, or a bronchial epithelial cell. In some embodiments, the cell is an HBE cell or a HeLa population. In some embodiments, the cell is in a patient.

In some embodiments, the payload is a polynucleotide or a polypeptide. The polynucleotide includes, but is not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribose configuration, α-LNA with α-L-ribose configuration (diastereomers of LNA), 2′-amino-LNA with 2′-amino functionalization, and 2′-amino-α-LNA with 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), or a hybrid or combination thereof. In some embodiments, the polynucleotide is mRNA, rRNA, or tRNA. In some embodiments, the polynucleotide is mRNA.

The lipid nanoparticle composition can be administered to a patient via intranasal, intrabronchial or pulmonary administration. For example, the composition and nanoparticles can be administered via a nebulizer or inhaler.

As will be appreciated by those skilled in the art, the lipid amines disclosed herein have additional uses. For example, lipid amines can be used to treat inflammatory diseases. Lipid amines can also be used as antimicrobial agents.

Kits and Devices

The present disclosure provides a variety of kits for suitably and/or effectively using the claimed nanoparticles of the present disclosure. Typically, the kit will contain sufficient amounts and/or sufficient numbers of components to allow the user to perform multiple treatments on a subject and/or perform multiple experiments.

In one aspect, the present disclosure provides a kit comprising a nanoparticle of the present disclosure.

The kit may also include packaging and instructions and/or a delivery agent to form a formulation composition. The delivery agent may include saline, a buffer solution, a lipid, or any delivery agent disclosed herein. In one embodiment, the kit also includes an administration device, such as a nebulizer or an inhaler.

Method for preparing LNP

The present disclosure also provides a method of preparing a lipid nanoparticle composition, the method comprising contacting a lipid nanoparticle core disclosed herein with a lipid amine disclosed herein.

In some embodiments, a method of preparing a lipid nanoparticle composition comprises:

(a) mixing a nucleic acid payload with a lipid solution, the lipid solution comprising:

(1) Ionizable lipids,

(2) phospholipids,

(3) structural lipids, and

(4) Optionally PEG-lipid

producing a filled lipid nanoparticle (fLNP) core; and

(c) contacting the fLNP core with a lipid amine.

In some embodiments, a method of preparing nanoparticles comprises:

(a) a mixed lipid solution comprising:

(1) Ionizable lipids,

(2) phospholipids,

(3) structural lipids, and

(4) Optionally PEG-lipid

Generation of empty lipid nanoparticle (eLNP) cores;

(b) contacting the eLNP core with a nucleic acid payload to form an fLNP; and

(c) contacting the fLNP core with a lipid amine.

In some embodiments, the mixing further comprises an aqueous buffer solution. In some embodiments, the aqueous buffer solution has a pH of about 3.5 to about 4.5. In other embodiments, the aqueous buffer solution has a pH of about 4. In some embodiments, the aqueous buffer solution has a pH of about 4.6 to about 6.5. In some embodiments, the aqueous buffer solution has a pH of about 5.

In some embodiments, the aqueous buffer solution may include acetate buffer, citrate buffer, phosphate buffer or Tris buffer. In some embodiments, the aqueous buffer solution includes acetate buffer or citrate buffer. In other embodiments, the aqueous buffer solution is an acetate buffer, such as sodium acetate buffer.

In some embodiments, the aqueous buffer solution has a buffer concentration greater than about 30mM. In some embodiments, the aqueous buffer solution has a buffer concentration greater than about 40mM. In some embodiments, the aqueous buffer solution has a buffer concentration of about 30mM to about 100mM. In some embodiments, the aqueous buffer solution has a buffer concentration of about 40mM to about 75mM. In other embodiments, the aqueous buffer solution has a buffer concentration of about 33mM, about 37.5mM, or about 45mM.

In some embodiments, the aqueous buffer solution may have an ionic strength of about 15 mM or less, about 10 mM or less, or about 5 mM or less. In some embodiments, the aqueous buffer solution has an ionic strength of about 0.1 mM to about 15 mM, about 0.1 mM to about 10 mM, or about 0.1 mM to about 5 mM.

In some embodiments, the lipid solution has a lipid concentration of about 5 mg/mL to about 100 mg/mL, about 15 mg/mL to about 35 mg/mL, about 20 mg/mL to about 30 mg/mL, or about 24 mg/mL.

The lipid solution may also include an organic solvent, such as an alcohol, such as ethanol. The organic solvent may be present in an amount of about 1% by volume to about 50% by volume, about 5% by volume to about 40% by volume, or about 10% by volume to about 33% by volume. In other embodiments, the solvent is 100% by volume ethanol or greater than 95% by volume ethanol.

In some embodiments, the lipid solution comprises about 30mol% to about 60mol%, about 35mol% to about 55mol%, or about 40mol% to about 50mol% of ionizable lipids relative to total lipids. In some embodiments, the lipid solution comprises about 5mol% to about 15mol%, about 8mol% to about 13mol%, or about 10mol% to about 12mol% of phospholipids relative to total lipids. In some embodiments, the lipid solution comprises about 30mol% to about 50mol%, about 35mol% to about 45mol%, or about 37mol% to about 42mol% of structural lipids relative to total lipids. In some embodiments, the lipid solution comprises about 0.1mol% to about 2mol%, about 0.1mol% to about 1mol%, or about 0.25mol% to about 0.75mol% of PEG-lipids relative to total lipids.

In some embodiments, the lipid solution comprises:

about 40 mol % to about 50 mol % ionizable lipid;

about 10 mol % to about 12 mol % of phospholipids;

about 37 mol % to about 42 mol % structural lipids; and

About 0.25 mol % to about 0.75 mol % PEG-lipid; each relative to total lipids.

In some embodiments, the lipid solution comprises:

about 49 mol% ionizable lipids;

about 11 mol % to about 12 mol % phospholipids;

about 39 mol % structural lipids; and

About 0.5 mol % PEG-lipid; each relative to total lipids.

Mixing lipid solution and buffer solution can precipitate lipid nanoparticles and prepare empty lipid nanoparticle compositions described herein. Precipitation can be carried out by ethanol drop precipitation using, for example, a high-energy mixer (e.g., T-junction, confined impact jet, microfluidic mixer, vortex mixer) to introduce lipids (in ethanol) into a suitable anti-solvent (i.e., water) in a controllable manner, thereby driving the liquid to be supersaturated and spontaneously precipitated into lipid particles. In some embodiments, mixing is implemented with a multi-inlet vortex mixer. In some embodiments, mixing is implemented with a microfluidic mixer (e.g., described in WO 2014/172045). The mixing step can be implemented at ambient temperature or, for example, at a temperature of less than about 30°C, less than about 28°C, less than about 26°C, less than about 25°C, less than about 24°C, less than about 22°C, or less than about 20°C.

In some embodiments, the mixing includes nanoprecipitation. Nanoprecipitation is a unit operation in which nanoparticles are self-assembled from their individual lipid components by means of kinetic mixing and subsequent maturation and serial dilution. This unit operation includes three individual steps: mixing water and organic input, maturing the nanoparticles, and diluting after a controlled residence time. Due to the continuity of these steps, it is considered as one unit operation. The unit operation includes a continuous online combination of three liquid streams and an online maturation step: mixing an aqueous buffer with a lipid stock solution, maturing via a controlled residence time, and diluting the nanoparticles. The nanoprecipitation itself occurs in a mixer of appropriate size, which is designed to allow continuous high-energy combinations of an aqueous solution with a lipid stock solution dissolved in ethanol. Throughout this operation, the aqueous solution and the lipid stock solution all flow continuously into the mixing hardware at the same time. The ethanol content that keeps the lipids dissolved is suddenly reduced and the lipids are all precipitated from each other. Therefore, the particles self-assemble in the mixing chamber. One goal of the unit operation is to exchange the solution into a complete aqueous buffer without ethanol and reach a target concentration of nanoparticles. This can be achieved by first reaching the target treatment concentration, then using diafiltration, and then (if necessary) a final concentration step once the ethanol has been completely removed.

In some embodiments, the lipid nanoparticle core in contact with the lipid amine comprises a PEG-lipid. In some embodiments, the lipid nanoparticle core in contact with the lipid amine is substantially free of a PEG-lipid. In some embodiments, the PEG-lipid is added to the lipid nanoparticle together with the lipid amine before or after contact with the lipid amine. In some embodiments, the PEG-lipid is used as a stabilizer.

In some embodiments, the contacting of step (b) is performed at a pH of about 3.5 to about 6.5. In some embodiments, the combining is performed at a pH of about 5. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 4.5 to about 5.5 prior to combining the empty lipid nanoparticle composition with the payload. In some embodiments, the pH of the empty lipid nanoparticle composition is adjusted to about 5 prior to combining the empty lipid nanoparticle composition with the payload.

The nucleic acid payload can be provided as a nucleic acid solution comprising (i) a nucleic acid, such as DNA or RNA (e.g., mRNA), and (ii) a buffer capable of maintaining an acidic pH (e.g., a pH of about 3 to about 6, about 4 to about 6, or about 5 to about 6). In some embodiments, the pH of the nucleic acid solution is about 5.

In some embodiments, the buffer of the nucleic acid solution is an acetate buffer, a citrate buffer, a phosphate buffer or a tris buffer. In some embodiments, the buffer is an acetate buffer or a citrate buffer. In other embodiments, the buffer is an acetate buffer, such as a sodium acetate buffer. The buffer concentration of the nucleic acid solution may be about 5mM to about 140mM. In some embodiments, the buffer concentration is about 20mM to about 100mM, about 30mM to about 70mM, or about 40mM to about 50mM. In some embodiments, the buffer concentration is about 42.5mM.

The nucleic acid solution may include nucleic acids at a concentration of about 0.05 mg/mL to about 5.0 mg/mL, 0.05 mg/mL to about 2.0 mg/mL, about 0.05 mg/mL to about 1.0 mg/mL, about 0.1 mg/mL to about 0.5 mg/mL, or about 0.2 mg/mL to about 0.3 mg/mL. In some embodiments, the nucleic acid concentration is about 0.25 mg/mL.

A high energy mixer (e.g., T-junction, confined impact jet, microfluidic mixer, vortex mixer) can be used for the contacting of step (b). In some embodiments, the combination is implemented with a multi-inlet vortex mixer. In some embodiments, the combination is implemented with a microfluidic mixer (e.g., described in WO 2014/172045). The combination step can be implemented at ambient temperature or, for example, at a temperature of less than about 30°C, less than about 28°C, less than about 26°C, less than about 25°C, less than about 24°C, less than about 22°C, or less than about 20°C.

In some embodiments, contacting the LNP core with the lipid amine comprises dissolving the lipid amine in a non-ionic excipient. In some embodiments, the non-ionic excipient is selected from macrogol 15-hydroxystearate (HS15), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG-2K), PL1, polyoxyethylene sorbitan monooleate [ 80] and d-α-tocopheryl polyethylene glycol succinate (TPGS). In some embodiments, the non-ionic excipient is macrogol 15-hydroxystearate (HS 15).

In some embodiments, contacting the lipid nanoparticle core with a lipid amine includes dissolving the lipid amine in a buffer solution. In some embodiments, the buffer is an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the buffer solution is a phosphate buffered saline (PBS). In some embodiments, the buffer solution is a Tris-based buffer. In some embodiments, the buffer solution concentration is about 5mM to about 100mM, about 5mM to about 50mM, about 10mM to about 30mM, or about 20mM.

In some embodiments, the lipid amine solution has a pH of about 7 to about 8, or about 7.5. In some embodiments, the lipid amine solution has a concentration of about 0.1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 10 mg/mL, or about 2 mg/mL to about 3 mg/mL.

In some embodiments, the lipid nanoparticle composition is subjected to maturation via a controlled residence time after loading and before neutralization. In some embodiments, the residence time is about 5 to about 120 seconds, about 10 to about 90 seconds, about 20 to about 70 seconds, about 30 to about 60 seconds, about 30 seconds, about 45 seconds, or about 60 seconds.

In some embodiments, the lipid nanoparticle composition is subjected to maturation via a controlled residence time after neutralization and before the addition of a cationic agent. In some embodiments, the residence time is about 1 to about 30 seconds, about 2 to about 20 seconds, about 5 to about 15 seconds, about 7 to about 12 seconds, or about 10 seconds.

In some embodiments, the method of preparing a lipid nanoparticle composition further comprises one or more additional steps selected from the following:

diluting the composition with dilution buffer;

adjusting the pH of the composition;

adding one or more surfactants to the composition;

filtering the composition;

Concentrated compositions;

Buffer exchange composition;

adding a cryoprotectant to the composition; and

An osmotic pressure regulator is added to the composition.

In some embodiments, the method for preparing a lipid nanoparticle composition may also include 1, 2, 3, 4, 5, 6, 7 or all of the steps listed above. Some steps may be repeated. The steps may be implemented in the order listed but not necessarily. Each step refers to an action associated with a composition produced by a previously formulated step. For example, if the method includes a step of adding one or more surfactants to the composition, the surfactant is added to the composition produced by the previous step, wherein the previous step may be any one of the steps listed above.

In some embodiments, one or more additional steps is adjusting the pH of the composition to a pH of about 7 to about 8. In some embodiments, the pH is adjusted to a pH of about 7.5.

In some embodiments, one or more additional steps are to add another surfactant to the filled lipid nanoparticles (e.g., in addition to the lipid amine). The surfactant can be disposed within the nanoparticles and/or disposed on their surface (e.g., by coating, adsorption, covalent bonding or other methods). Surfactants may include, but are not limited to, PEG derivatives (e.g., PEG-DMG), lipid amines (e.g., sterolamines and related lipid amines), anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., acetylcysteine, mugwort, bromelain, papaya, scabious peony, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4, dornase α In some embodiments, the other surfactant is a PEG lipid, such as PEG-DMG. In some embodiments, the other surfactant is provided with the lipid amine. In some embodiments, the other surfactant is present in the lipid amine solution with the lipid amine. In some embodiments, the other surfactant is a PEG-lipid at a concentration of about 0.1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 3 mg/mL.

In some embodiments, one or more additional steps are to add an osmotic pressure regulator to the composition. The osmotic pressure regulator may be a salt or a sugar. In some embodiments, the osmotic pressure regulator is a sugar. The sugar may be selected from (but not limited to) glucose, fructose, galactose, sucrose, lactose, maltose and dextrose. In some embodiments, the osmotic pressure regulator is a salt. The salt may be an inorganic salt, such as sodium chloride, potassium chloride, calcium chloride or magnesium chloride. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the salt is 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt. The salt may be provided as a salt solution having a salt concentration of about 100mM to about 500mM, about 200mM to about 400mM, about 250mM to about 350mM, or about 300mM. The pH value of the salt solution may be about 7 to about 8. The salt solution may also include a buffer, such as an acetate buffer, a citrate buffer, a phosphate buffer or a tris buffer. The buffer concentration can be, for example, about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM.

The cryoprotectant can be added to the filled nanoparticle composition by adding a cryoprotectant aqueous solution, which may include an aqueous buffer solution having a buffer concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 90 mM, about 1.0 mM to about 80 mM, about 2 mM to about 70 mM, about 3 mM to about 60 mM, about 4 mM to about 50 mM, about 5 mM to about 40 mM, about 6 mM to about 30 mM, about 7 mM to about 20 mM, about 8 mM to about 15 mM, or about 9 mM to about 12 mM. In some embodiments, the buffer concentration is about 1 mM to 20 mM, about 1 mM to about 10 mM, or about 5 mM. In some embodiments, the buffer in the cryoprotectant solution includes an acetate buffer, a citrate buffer, a phosphate buffer, or a tris buffer. In some embodiments, the buffer is an acetate buffer or a citrate buffer. In other embodiments, the buffer is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the cryoprotectant solution is about 7 to about 8, such as about 7.5. In some embodiments, the cryoprotectant solution comprises about 40% to about 90% by weight, about 50% to about 85% by weight, about 60% to about 80% by weight, or about 70% by weight sucrose.

In some embodiments, the method further comprises the step of diluting the composition with a dilution buffer. The dilution buffer may be an aqueous buffer solution, wherein the buffer concentration is about 0.1mM to about 100mM, about 0.5mM to about 90mM, about 1.0mM to about 80mM, about 2mM to about 70mM, about 3mM to about 60mM, about 4mM to about 50mM, about 5mM to about 40mM, about 6mM to about 30mM, about 7mM to about 20mM, about 8mM to about 15mM or about 9mM to about 12mM. In some embodiments, the buffer concentration is about 30mM to about 75mM, about 30mM to about 60mM or about 30mM to about 50mM. In some embodiments, the dilution buffer comprises acetate buffer, citrate buffer, phosphate buffer or tris buffer. In some embodiments, the dilution buffer comprises acetate buffer or citrate buffer. In other embodiments, the dilution buffer is an acetate buffer, such as sodium acetate. In some embodiments, the pH of the dilution buffer is about 3 to about 7, about 3 to about 6, about 3 to about 5, about 4, about 5, about 5.5, or about 6. In some embodiments, the dilution buffer comprises the same buffer as the aqueous buffer solution used during combining the empty lipid nanoparticle composition and the nucleic acid solution.

In some embodiments, the method further comprises any one or more of the following steps: filtering the composition; concentrating the composition; and exchanging the buffer of the composition. The filtering, concentrating, and buffer exchanging steps can be implemented together with tangential flow filtration (TFF). Residual organic solvent can be removed by the filtering step.

In some embodiments, buffer exchange can change the composition of the filled lipid nanoparticle composition by increasing or decreasing the buffer concentration, changing the buffer composition, or changing the pH.

In some embodiments, the concentrating step can increase the concentration of filled lipid nanoparticles in the composition.

In some embodiments, the method of preparing a filled lipid nanoparticle composition further comprises at least the following steps: adjusting the pH of the composition to a pH of about 7 to about 8 (e.g., about pH 7.5); and adding an osmotic pressure regulator (e.g., an inorganic salt) to the composition.

In some embodiments, the method of preparing a filled lipid nanoparticle composition further comprises at least the following steps: adjusting the pH of the composition to a pH of about 7 to about 8 (e.g., about pH 7.5); adding a surfactant to the composition; and adding an osmotic pressure regulator (e.g., an inorganic salt) to the composition.

In some embodiments, the method of preparing a lipid nanoparticle composition may further comprise:

(i) adjusting the pH of the composition to a pH of about 7 to about 8;

(ii) adding one or more surfactants to the composition;

(iii) concentrated compositions;

(iv) adding an inorganic salt to the composition; and

(v) diluting the composition.

synthesis

As will be appreciated by those skilled in the art, the compounds provided herein, including salts and stereoisomers thereof, can be prepared using known organic synthesis techniques and can be synthesized according to any of a variety of possible synthetic routes, such as those provided in the schemes below.

The reactions for preparing the compounds described herein can be carried out in suitable solvents that can be easily selected by those skilled in the art of organic synthesis. Suitable solvents may not substantially react with starting materials (reactants), intermediates or products at the temperature at which the reaction is carried out (e.g., a temperature that can range from the solvent's freezing point to the solvent's boiling point). A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, a solvent suitable for a particular reaction step can be selected by a skilled person.

The expressions "ambient temperature", "room temperature" and "rt" as used herein are understood in the art to generally refer to a temperature, such as reaction temperature, which is approximately the temperature of the room in which the reaction is carried out, such as a temperature of about 20°C to about 30°C.

The preparation of the compounds described herein may involve the protection and deprotection of various chemical groups. One skilled in the art can readily determine the need for protection and deprotection and the selection of appropriate protecting groups. The chemistry of protecting groups can be found in, for example, T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, Wiley & Sons, Inc., New York (1999).

The reaction can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means (e.g., nuclear magnetic resonance spectroscopy (e.g., ¹ H or ¹³ C), infrared spectroscopy, spectrophotometry (e.g., UV-visible light), mass spectrometry) or by chromatographic methods (e.g., high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LCMS) or thin layer chromatography (TLC)). Compounds can be purified by a variety of methods (including high performance liquid chromatography (HPLC) and normal phase silica chromatography) by those skilled in the art.

Compounds of formula A1 can be prepared, for example, using the methods illustrated in the scheme below:

Solution 1

Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 1. Appropriate reaction between cholesteryl chloroformate and an amine can be carried out under suitable conditions to produce a precursor of a compound of formula A1 or a compound of formula A1.

Solution 2

Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 2. An appropriate reaction between cholesterol or a cholesterol derivative (e.g., stigmasterol) and 4-nitrophenyl chloroformate can be carried out under suitable conditions (e.g., using triethylamine and 4-dimethylaminopyridine). The product of the reaction can be reacted with an amine under suitable conditions (e.g., using triethylamine) to produce a precursor of a compound of formula A1 or a compound of formula A1.

Solution 3

Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 3. An appropriate reaction between cholesterol hemisuccinate or cholesterol derivative hemisuccinate and an activating agent can be carried out under suitable conditions. The product of the reaction can be reacted with an amine under suitable conditions to produce a precursor of a compound of formula A1 or a compound of formula A1.

Solution 4

Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 4. Appropriate reaction between compounds of formula A1, HCHO, NaBH ₃ CN and AcONa can be carried out under suitable conditions to generate compounds of formula A1.

Solution 5

Precursors of compounds of formula A1 can be prepared via the synthetic pathways outlined in Scheme 5. Appropriate reactions between cholesterol or a cholesterol derivative (e.g., stigmasterol) and can be carried out under suitable conditions (e.g., using triethylamine and 4-dimethylaminopyridine). The product of the reaction can be reacted with an amine under suitable conditions (e.g., using triethylamine) to obtain a precursor of a compound of formula A1.

Solution 6

Precursors of compounds of formula A1 can be prepared via the synthetic pathway outlined in Scheme 5. An appropriate reaction between cholesterol or a cholesterol derivative (e.g., stigmasterol) and a boc-half ester can be carried out under suitable conditions. The product of the reaction can be reacted under suitable conditions to obtain a precursor of a compound of formula A1.

Solution 7

Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic routes outlined in Scheme 7. Appropriate reactions between spermidine or spermine and (E)-N-((tert-butoxycarbonyl)oxy)benzylimidocyanide (BOC-ON) can be carried out under suitable conditions to give intermediates for the synthesis of compounds of formula A1.

Solution 8

Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic routes outlined in Scheme 8. Appropriate reactions between intermediate 1 and acrylonitrile can be carried out under suitable conditions to give intermediate 2. Intermediate 2 can be reacted with benzyl bromide under suitable conditions (e.g. K ₂ CO ₃ and KI) to give intermediate 3. Intermediate 3 can be reacted with Boc ₂ O under suitable conditions (e.g. NaBH ₄ and NiCl ₂ ) to give intermediate 4. The benzyl group of intermediate 4 can be removed under suitable conditions (e.g. H ₂ and Pd/C) to give intermediate 5.

Solution 9

Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic routes outlined in Scheme 9. Appropriate reaction between 1,4-butanediol and acrylonitrile can be carried out under suitable conditions (e.g. Triton B) to give intermediate 6. Intermediate 6 can be reacted with methanesulfonyl chloride under suitable conditions (e.g. triethylamine) to give intermediate 7. Intermediate 7 can be reacted with N-Boc-1,3-diaminopropane under suitable conditions to give intermediate 8. Intermediate 8 can be reacted with benzyl bromide under suitable conditions (e.g. K ₂ CO ₃ and KI) to give intermediate 9. Intermediate 9 can be reacted with Boc ₂ O under suitable conditions (e.g. NaBH ₄ and NiCl ₂ ) to give intermediate 10. The benzyl group of intermediate 10 can be removed under suitable conditions (e.g. H ₂ and Pd/C) to give intermediate 11.

Solution 10

Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic routes outlined in Scheme 10. Appropriate reaction between N-Boc-1,3-diaminopropane and 2-nitrobenzenesulfonyl chloride under suitable conditions (e.g., triethylamine) affords intermediate 12. Intermediate 12 can be reacted with tert-butyl N-(6-bromohexyl)carbamate under suitable conditions (e.g., K ₂ CO ₃ and KI) to afford intermediate 13. The 2-nitrobenzenesulfonyl group can be removed under suitable conditions (e.g., K ₂ CO ₃ and thiophenol) to afford intermediate 14.

Solution 11

Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic routes outlined in Scheme 11. Appropriate reaction between thiocholesterol and 2,2'-bipyridyl disulfide under suitable conditions affords intermediate 15. Intermediate 15 can be reacted with methyl trifluoromethanesulfonate (methyl triflate) under suitable conditions to afford intermediate 16. Intermediate 16 can be reacted with an appropriate mercaptocarboxylic acid to provide intermediate 17.

Solution 12

Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 12. Appropriate reaction between intermediate 17 and an amine can be carried out under suitable conditions (eg using a coupling agent) to produce a precursor of a compound of formula A1 or a compound of formula A1.

Solution 13

Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic routes outlined in Scheme 13. Appropriate reaction between _benzylamine and alkyl halide under suitable conditions (e.g. _K2CO3 and KI) affords intermediate 18. Removal of the benzyl group of intermediate 18 under suitable conditions (e.g. _H2 and Pd/C) affords intermediate 19.

Solution 14

Compounds of formula A1 or precursors for synthesizing compounds of formula A1 can be prepared via the synthetic routes outlined in Scheme 14. Appropriate reaction between cholesterol _{chloroacetate} and amines under suitable conditions (e.g., using, for example, _K2CO3 and KI) affords intermediate 20. Intermediate 20 can be reacted with an appropriate carboxylic acid under suitable conditions to produce a precursor of a compound of formula A1 or a compound of formula A1. In some embodiments, R ^Y is

Solution 15

Precursors of compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 15. Appropriate reaction between intermediate 21 and nitrobenzenesulfonyl chloride can be carried out under suitable conditions (e.g., triethylamine) to give intermediate 22. Intermediate 22 can be reacted with an alkyl bromide under suitable conditions (e.g., K ₂ CO ₃ and KI) to give intermediate 23. In some embodiments, R ^Z is

Scheme 16

Precursors of compounds of formula A1 can be prepared via the synthetic pathways outlined in Scheme 16. An appropriate reaction between cholesterol and a carboxylic acid can be carried out in the presence of a coupling agent under suitable conditions. The product of the reaction can be reacted under suitable conditions to obtain a compound of formula A1 or a precursor of a compound of formula A1. In some embodiments, R ^X is

Solution 17

Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic routes outlined in Scheme 17. Appropriate reactions between 8-bromooctanoic acid, oxalyl chloride and N,O-dimethylhydroxylamine can be carried out under suitable conditions (e.g. catalytic DMF in DCM) to give intermediate 24. Intermediate 24 can be reacted with methylmagnesium bromide and HCl under suitable conditions to give intermediate 25. Intermediate 25 can be reacted with ammonium acetate and sodium cyanoborohydride under suitable conditions to give intermediate 26. Intermediate 26 can be reacted with BOC anhydride under suitable conditions (e.g. triethylamine (TEA)) to give intermediate 27. Intermediate 27 can be reacted with tert-butyl (4-((2-nitrophenyl)sulfonamido)butan-2-yl)carbamate (prepared in a similar manner to intermediate 12) under suitable conditions (e.g. K ₂ CO ₃ , BnBr and thiophenol) to give intermediate 28.

Scheme 18

Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic routes outlined in Scheme 18. Appropriate reaction between intermediate 29 and intermediate 30 under suitable conditions (eg sodium triacetoxyborohydride in anhydrous MeOH) affords intermediate 31.

Solution 19

Intermediates for the synthesis of compounds of formula A1 can be prepared via the synthetic routes outlined in Scheme 19. Appropriate reaction between intermediate 29 and 2-nitrobenzenesulfonyl chloride can be carried out under suitable conditions (e.g. triethylamine in DCM) to give intermediate 32. Intermediate 30A can be reacted with p-toluenesulfonyl chloride under suitable conditions to give intermediate 33. Intermediate 32 can be reacted with intermediate 33 under suitable conditions (e.g. _{K2CO3 ,} BnBr and thiophenol) to give intermediate 31.

Scheme 20

Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic routes outlined in Scheme 20. Appropriate reaction between cholesterol or a cholesterol derivative (e.g., sitosterol) and thionyl chloride under suitable conditions provides intermediate 34. Intermediate 34 can be reacted with sodium thiocyanate under suitable conditions to give intermediate 35. Intermediate 35 can be reacted with lithium aluminum hydride under suitable conditions to give thiocholesterol or a thiocholesterol derivative (e.g., thiositosterol).

Solution 21

Intermediates for the synthesis of compounds of Formula A1 can be prepared via the synthetic routes outlined in Scheme 21. Appropriate reaction between thiocholesterol or a thiocholesterol derivative (e.g., thiositosterol) and 2,2'-dipyridyl disulfide under suitable conditions affords intermediate 15A. Intermediate 15A can be reacted with methyl trifluoromethanesulfonate (methyl triflate) under suitable conditions to afford intermediate 16A. Intermediate 16A can be reacted with an appropriate mercaptocarboxylic acid to provide intermediate 17A.

Solution 22

Compounds of formula A1 can be prepared via the synthetic route outlined in Scheme 22. Appropriate reaction between intermediate 17A and an amine can be carried out under suitable conditions (eg using a coupling agent) to generate a precursor of a compound of formula A1.

definition

In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning described below. Other definitions are set forth throughout this application.

In this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. The term "a" (or "a") and the terms "one or more" and "at least one" are used interchangeably herein. In some aspects, the term "a" or "an" means "single". In other aspects, the term "a" or "an" includes "two or more" or "more".

The term "and/or" should be taken as specifically disclosing each of the two specified features or components, with or without the other. Thus, the term "and/or" as used in phrases herein (e.g., "A and/or B") is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in phrases (e.g., "A, B, and/or C") is intended to cover each of the following: A, B, and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology Revised Edition, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure to those skilled in the art.

Whenever the language "comprising" is used herein to recite an aspect, other similar aspects recited according to "consisting of" and/or "consisting essentially of" are also provided.

Units, prefixes and symbols are expressed in the form accepted by the International System of Units (SI). Numerical ranges include the numbers that define the range. When a range of values is listed, it is understood that each intermediate integer value between the listed upper and lower limits of the range and each fraction thereof and each subrange between such values are also specifically disclosed. The upper and lower limits of any range may be independently included or not included in the range, and each range including any limit, an infinite value or two limits is also included in the present disclosure. When values are explicitly listed, it is understood that values of the number or amount approximately the same as the listed values are also within the scope of the present disclosure. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the present disclosure. On the contrary, when different elements or groups of elements are disclosed individually, their combinations are also disclosed. When any element of the present disclosure is disclosed as having multiple alternatives, examples of the present disclosure are also disclosed herein, wherein each alternative is excluded singly or in any combination with other alternatives; more than one element of the present disclosure may have such exclusions, and all combinations of elements with such exclusions are disclosed herein.

The term "about" as used in conjunction with numerical values throughout the specification and claims indicates an accuracy interval that is familiar and acceptable to those skilled in the art. This accuracy interval is, for example, ±10%.

As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, non-human animals are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, animals are transgenic animals, genetically engineered animals, or clones.

As used herein, the term "compound" is intended to include all stereoisomers and isotopes of the depicted structure. As used herein, the term "stereoisomer" means any geometric isomer (e.g., cis and trans isomers), enantiomers, or diastereomers of a compound. The present disclosure encompasses any and all stereoisomers of the compounds described herein, including stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) as well as enantiomers and stereoisomer mixtures (e.g., racemates). Enantiomers and stereoisomer mixtures of compounds and the manner in which they are separated into their component enantiomers or stereoisomers are well known. "Isotopes" refer to atoms having the same atomic number but having different mass numbers due to different numbers of neutrons in the nucleus. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compounds, salts, or complexes of the present disclosure can be prepared by conventional methods in combination with solvents or water molecules to form solvates and hydrates.

As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a cell (e.g., a mammalian cell) with a nanoparticle composition means that the cell and the nanoparticle share a physical connection. Methods for contacting cells with external entities in vivo and in vitro are well known in the biological field. For example, contacting a nanoparticle composition with a cell disposed in a mammal can be implemented by different routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve different amounts of nanoparticle compositions. In addition, more than one cell can be contacted with a nanoparticle composition.

Another example of contact is between nanoparticles and lipid amines. Contacting nanoparticles (e.g., filled with effective load or empty) and lipid amines can mean that the surface of the nanoparticles is physically linked to the lipid amine so that the lipid amine can form an interaction with the nanoparticles. In some embodiments, contacting the nanoparticles with lipid amines allows the lipid amine to be inserted into the nanoparticles, e.g., starting at the surface of the nanoparticles. In some embodiments, the terms "layering", "coating", "post-addition", and "addition" can be used to mean "contacting" with respect to contacting the nanoparticles with lipid amines.

As used herein, the term "delivery" means providing an entity to a destination. For example, delivering a polynucleotide to a subject may involve administering a nanoparticle composition comprising the polynucleotide to the subject (e.g., via an intravenous, intramuscular, intradermal, or subcutaneous route). Administering a nanoparticle composition to a mammal or mammalian cell may involve contacting one or more cells with the nanoparticle composition.

As used herein, "delivery agent" refers to any substance that at least partially facilitates the delivery of a polynucleotide to a targeted cell in vivo, in vitro, or ex vivo.

As used herein, the term "diastereoisomers" means stereoisomers that are not mirror images of each other and are not superimposable on each other.

As used herein, the "effective amount" of the term agent is an amount sufficient to achieve a beneficial or desired result (e.g., clinical result), and thus the "effective amount" depends on the context in which it is applied. For example, in the context of administering an agent for treating a protein deficiency, the effective amount of an agent is, for example, the amount of mRNA expressing a protein sufficient to improve, alleviate, eliminate or prevent signs and symptoms associated with a protein deficiency compared to the severity of the symptoms observed without the agent. The term "effective amount" can be used interchangeably with "effective dose," "therapeutically effective dose," or "therapeutically effective dose."

As used herein, the term "enantiomer" means each individual optically active form of a disclosed compound having an optical purity or enantiomeric excess of at least 80% (i.e., at least 90% of one enantiomer and up to 10% of the other enantiomer), at least 90% or at least 98% (as determined by standard methods in the art).

As used herein, the term "encapsulate" means to enclose, surround, incorporate, or contain.

As used herein, "encapsulation efficiency" refers to the amount of polynucleotides that become part of a nanoparticle composition relative to the initial total amount of polynucleotides used to prepare the nanoparticle composition. For example, if 97 mg of polynucleotides are encapsulated in the nanoparticle composition out of a total of 100 mg of polynucleotides initially provided to the composition, the encapsulation efficiency can be given as 97%. As used herein, "encapsulation" can refer to complete, substantial or partial encapsulation, restriction, surrounding or enclosing.

As used herein, "epithelial cells" include cells derived from epithelium. Example epithelial cells are respiratory epithelial cells, nasal epithelial cells, alveolar epithelial cells, lung epithelial cells, or bronchial epithelial cells. In some embodiments, the epithelial cells are human bronchial epithelial (HBE) cells. In some embodiments, the epithelial cells are in vitro cells. In some embodiments, the epithelial cells are in vivo cells.

As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an mRNA template from the DNA sequence (e.g., by transcription); (2) processing of the mRNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of the mRNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

As used herein, the term "ex vivo" refers to an event that occurs outside of an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof). An ex vivo event can occur in an environment that is minimally altered from the natural (eg, in vivo) environment.

As used herein, the term "helper lipid" refers to a compound or molecule comprising a lipid portion (for insertion into a lipid layer (e.g., a lipid bilayer)) and a polar portion (for interaction with the physiological solution on the lipid layer surface). Typically, the helper lipid is a phospholipid. The function of the helper lipid is to "supplement" the amino lipid and increase the fusogenicity of the bilayer, and/or to help promote, for example, the intracellular escape of a nucleic acid delivered to a cell. Helper lipids are also considered to be key structural components on the LNP surface.

As used herein, the term "in vitro" refers to events that occur in an artificial environment (eg, in a test tube or reaction vessel, in cell culture, in a Petri dish, etc.) rather than within an organism (eg, an animal, plant, or microorganism).

As used herein, the term "in vivo" refers to events that occur within an organism (eg, an animal, plant, or microorganism, or a cell or tissue thereof).

The term "ionizable amino lipid" includes, for example, those lipids described herein throughout that exhibit one, two, three or more fatty acid or fatty alkyl chains and at least one pH titratable amino head group (e.g., alkylamino or dialkylamino head group). Ionizable amino lipids are typically protonated (i.e., positively charged) at a pH below the pKa of the amino head group and are substantially uncharged at a pH above the pKa. Such ionizable amino lipids include, but are not limited to, DLin-MC3-DMA (MC3) and (13Z, 165Z)-N,N-dimethyl-3-nonyldocosa-13-16-dien-1-amine (L608).

As used herein, the term "isomer" means any tautomer, stereoisomer, enantiomer or diastereomer of any compound of the present disclosure. It should be recognized that the compounds of the present disclosure may have one or more chiral centers and/or double bonds, and therefore exist as stereoisomers (e.g., double bond isomers, i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis/trans isomers). According to the present disclosure, the chemical structures depicted herein and therefore the compounds of the present disclosure encompass all corresponding stereoisomers, i.e., stereoisomerically pure forms (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) as well as enantiomeric and stereoisomeric mixtures (e.g., racemates). Enantiomeric and stereoisomeric mixtures of the disclosed compounds can generally be resolved into their component enantiomers or stereoisomers by well-known methods such as chiral phase gas chromatography, chiral phase high performance liquid chromatography, crystallization of the compound as a chiral salt complex, or crystallization of the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereoisomerically pure or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

As used herein, a "lipid nanoparticle core" is a lipid nanoparticle to which a post-addition layer of additional components (e.g., lipid amine and/or PEG-lipid or other lipid) can be added. In some embodiments, the lipid nanoparticle core comprises: (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) optionally a PEG-lipid. In other embodiments, the lipid nanoparticle core comprises: (i) an ionizable lipid, (ii) a phospholipid, (iii) a structural lipid, and (iv) a PEG-lipid. In some embodiments, the lipid nanoparticle core may contain a payload.

As used herein, "linker" or "linker structure" refers to a group of atoms, such as 10-1,000 atoms, and may include, for example, but not limited to, the following atoms or groups: carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imide. The linker can be connected to a modified nucleoside or nucleotide on a nucleobase or sugar moiety at the first end and a payload (e.g., a detectable agent or therapeutic agent) at the second end. The linker can be of a length sufficient not to interfere with incorporation into a nucleic acid sequence. The linker can be used for any useful purpose, such as forming a polynucleotide polymer (e.g., via linkage of two or more chimeric polynucleotide molecules or IVT polynucleotides) or a polynucleotide conjugate, and administering a payload, as described herein. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amide, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene glycol or propylene glycol monomer units, such as diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers and derivatives thereof. Other examples include, but are not limited to, cleavable moieties within the linker, such as disulfide bonds (-S-S-) or azo bonds (-N=N-), which can be cleaved using reducing agents or photolysis. Non-limiting examples of selectively cleavable bonds include amide bonds, which can be cleaved, for example, by using tris(2-carboxyethyl)phosphine (TCEP) or other reducing agents and/or photolysis, and ester bonds, which can be cleaved, for example, by acidic or basic hydrolysis.

As used herein, "lung cells" include cells derived from the lung. Lung cells can be, for example, lung epithelial cells, airway basal cells, bronchiolar exocrine cells, pulmonary neuroendocrine cells, alveolar cells, or airway epithelial cells. In some embodiments, the lung cells are in vitro cells. In some embodiments, the lung cells are in vivo cells.

The term "nucleic acid" in its broadest sense includes any compound and/or substance comprising a nucleotide polymer. These polymers are generally referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present disclosure include, but are not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA, including LNA with a β-D-ribose configuration, α-LNA with an α-L-ribose configuration (diastereomers of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-α-LNA with 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA), or hybrids or combinations thereof.

As used herein, "patient" refers to a subject who may seek or need treatment, requires treatment, is receiving treatment, will receive treatment, or is under the care of a professional trained for a particular disease or condition.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and are commensurate with a reasonable benefit/risk ratio.

As used herein, the phrase "pharmaceutically acceptable excipient" refers to any ingredient other than the compounds described herein that is substantially non-toxic and non-inflammatory in patients (e.g., a vehicle capable of suspending or dissolving the active compound). Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), softeners, emulsifiers, fillers (diluents), film formers or coatings, flavoring agents, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration.

The "pharmaceutically acceptable salts" of the present disclosure include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or in an organic solvent or in a mixture of the two; generally, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is used. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

The term "solvate" as used herein means a compound of the present disclosure in which molecules of a suitable solvent are incorporated into the crystal lattice. The suitable solvent is physiologically tolerable at the dosage administered. For example, a solvate can be prepared by crystallization, recrystallization or precipitation from a solution comprising an organic solvent, water or a mixture thereof. When water is the solvent, the solvate is referred to as a "hydrate."

The term "polynucleotide" as used herein refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acids ("DNA") and triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes modified (e.g., by alkylation and/or by end-capping) and unmodified forms of the polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose, including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced), any other type of polynucleotide that is an N-glycoside or C-glycoside of a purine or pyrimidine base, and other polymers containing an orthonucleotide backbone (e.g., polyamides (e.g., peptide nucleic acids "PNA") and polymorpholino polymers) and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration that allows base pairing and base stacking, such as found in DNA and RNA. In specific aspects, the polynucleotide comprises mRNA. In other aspects, the mRNA is a synthetic mRNA. In some aspects, the synthetic mRNA comprises at least one non-natural nucleobase. In some aspects, all nucleobases of a certain class have been replaced by non-natural nucleobases (e.g., all uridines in the polynucleotides disclosed herein can be replaced by non-natural nucleobases, such as 5-methoxyuridine). In some aspects, a polynucleotide (e.g., synthetic RNA or synthetic DNA) comprises only natural nucleobases, i.e., A (adenosine), G (guanosine), C (cytidine), and T (thymidine) in the case of synthetic DNA, or A, C, G, and U (uridine) in the case of synthetic RNA.

The skilled artisan will appreciate that the T bases in the codon maps disclosed herein are present in DNA, and in the corresponding RNA, the T bases will be replaced by U bases. For example, the codon-nucleotide sequences disclosed herein in the form of DNA (e.g., vectors or in vitro translation (IVT) templates) will have their T bases transcribed as U bases in their corresponding transcribed mRNAs. In this regard, codon-optimized DNA sequences (comprising T) and their corresponding mRNA sequences (comprising U) are both considered to be codon-optimized nucleotide sequences of the present disclosure. The skilled artisan will also appreciate that equivalent codon maps can be generated by replacing one or more bases with non-natural bases. Thus, for example, a TTC codon (DNA map) will correspond to a UUC codon (RNA map), which in turn will correspond to a ΨΨC codon (RNA map in which U has been replaced by pseudouridine).

Standard A-T and G-C base pairs are formed under conditions that allow hydrogen bonding between the N3-H and C4-oxy groups of thymidine and the N1 and C6-NH2 of adenosine, respectively, and between the C2-oxy group, N3 and C4-NH2 of cytidine and the C2-NH2, N'-H and C6-oxy groups of guanosine, respectively. Thus, for example, guanosine (2-amino-6-oxy-9-β-D-ribofuranosyl-purine) can be modified to form isoguanosine (2-oxy-6-amino-9-β-D-ribofuranosyl-purine). Such modifications produce a nucleobase that will no longer effectively form a standard base pair with cytosine. However, modification of cytosine (1-β-D-ribofuranosyl-2-oxy-4-amino-pyrimidine) to form isocytosine (1-β-D-ribofuranosyl-2-amino-4-oxy-pyrimidine-) produces a modified nucleotide that will not base pair effectively with guanosine, but will form base pairs with isoguanosine (U.S. Pat. No. 5,681,702 to Collins et al.). Isocytosine can be purchased from Sigma Chemical Co. (St. Louis, Mo.); isocytidine can be prepared by the methods described by Switzer et al. (1993) Biochemistry 32:10489-10496 and references cited therein; 2'-deoxy-5-methyl-isocytidine can be prepared by the methods of Tor et al., 1993, J. Am. Chem. Soc. 115:4461-4467 and references cited therein; and isoguanine nucleotides can be prepared using the methods described by Switzer et al., 1993, supra, and Mantsch et al., 1993, Biochem. 14:5593-5601 or by the methods described in U.S. Pat. No. 5,780,610 to Collins et al. Other unnatural base pairs can be synthesized by the method described in Piccirilli et al., 1990, Nature 343:33-37 for the synthesis of 2,6-diaminopyrimidine and its complement (1-methylpyrazolo[4,3]pyrimidine-5,7-(4H,6H)-dione. Other such modified nucleotide units that form unique base pairs are known, such as those described in Leach et al. (1992) J. Am. Chem. Soc. 114:3675-3683 and Switzer et al. (supra).

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may comprise modified amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more analogs of amino acids, including, for example, unnatural amino acids, such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine, as well as other modifications known in the art.

As used herein, the term refers to proteins, polypeptides and peptides of any size, structure or function. Polypeptides include encoded polynucleotide products, naturally occurring polypeptides, synthetic polypeptides, the aforementioned homologues, heterologous homologues, homologous homologues, fragments and other equivalents, variants and analogs. Polypeptides may be monomers or may be multimolecular complexes, such as dimers, trimers or tetramers. They may also include single-chain or multi-chain polypeptides. The most common disulfide bonds are found in multi-chain polypeptides. The term polypeptide may also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. In some embodiments, a "peptide" may be less than or equal to 50 amino acids long, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids long.

As used herein, the term "prevent," "prevent," "prevent," "prevent," "preventing ...

As used herein, "prevention" refers to treatment or a course of action intended to prevent the spread of disease.

The term "salt" includes any anionic and cationic complexes. Pharmaceutically acceptable salts are a subset of the non-toxic salts described above.

"Subject" or "individual" or "animal" or "patient" or "mammal" means any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include, but are not limited to, humans, livestock, farm animals, zoo animals, sports animals, pets, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates, such as apes, monkeys, orangutans, and chimpanzees; canines, such as dogs and wolves; felines, such as cats, lions, and tigers; equines, such as horses, donkeys, and zebras; bears, food animals, such as cows, pigs, and sheep; ungulates, such as deer and giraffes; rodents, such as mice, rats, hamsters, and guinea pigs; and the like. In certain embodiments, the mammal is a human subject. In other embodiments, the subject is a human patient. In a specific embodiment, the subject is a human patient in need of treatment.

As used herein, the term "substantially" refers to the qualitative condition of showing the full or nearly full range or degree of a characteristic or property of interest. It will be understood by those of ordinary skill in the biological field that biological and chemical features rarely, if ever, proceed to completion and/or progress to completion or achieve or avoid an absolute result. Therefore, the term "substantially" is used herein to capture the potential incompleteness inherent in many biological and chemical features.

An individual "suffering from" a disease, disorder, and/or condition has been diagnosed with or displays one or more signs and symptoms of the disease, disorder, and/or condition.

An individual who is "susceptible" to a disease, disorder, and/or condition has not been diagnosed with and/or is unable to display signs and symptoms of the disease, disorder, and/or condition, but has a predisposition to develop the disease or its signs and symptoms. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (e.g., cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with the development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of proteins and/or nucleic acids associated with the disease, disorder, and/or condition; (4) habits and/or lifestyle associated with the development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microorganism associated with the development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.

The term "synthetic" means produced, prepared and/or manufactured by human hand. The synthesis of polynucleotides or other molecules of the present disclosure may be chemical synthesis or enzymatic synthesis.

The term "therapeutic or prophylactic agent" refers to an agent that has a therapeutic, diagnostic and/or prophylactic effect and/or induces a desired biological and/or pharmacological effect when administered to a subject. For example, an mRNA encoding a polypeptide may be a therapeutic or prophylactic agent.

As used herein, the term "therapeutically effective amount" means the amount of an agent to be delivered (e.g., a nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient to treat, ameliorate the signs and symptoms of, diagnose, prevent and/or delay the onset of an infection, disease, disorder and/or condition when administered to a subject suffering from or susceptible to an infection, disease, disorder and/or condition.

Treating, treatment, therapy: As used herein, the term "treating" or "treatment" or "therapy" refers to partially or completely alleviating, ameliorating, improving, alleviating one or more signs and symptoms or characteristics of a disease, delaying its onset, inhibiting its progression, reducing its severity and/or reducing its incidence. For example, "treating" a disease may refer to reducing the signs and symptoms associated with the disease, prolonging the life of the patient (increasing the survival rate), reducing the severity of the disease, preventing or delaying the onset of the disease, etc. Treatment can be applied to subjects who do not show signs of a disease, disorder, and/or condition and/or subjects who only show early signs of a disease, disorder, and/or condition for the purpose of reducing the risk of developing pathologies associated with the disease, disorder, and/or condition.

The term "n-membered" (where n is an integer) generally describes the number of ring-forming atoms in a moiety, where the number of ring-forming atoms is n. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridinyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl.

As used herein, the phrase "optionally substituted" means unsubstituted or substituted. Substituents are independently selected, and substitutions may be in any chemically accessible position. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced by a substituent. A single divalent substituent (e.g., oxo) may replace two hydrogen atoms. It is understood that substitution at a given atom is limited by valence.

Throughout the definitions, the term "C _nm " indicates a range including an endpoint, where n and m are integers and indicate the number of carbons. Examples include C _1-4 , C _1-6 , etc.

As used herein, the term "C _nm alkyl" used alone or in combination with other terms refers to a saturated hydrocarbon group having n to m carbons, which may be linear or branched. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl; higher homologues such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, etc. In some embodiments, the alkyl group contains 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

As used herein, "C _nm alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds and having n to m carbons. Example alkenyl groups include, but are not limited to, vinyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, etc. In some embodiments, the alkenyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, "C _nm alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds and having n to m carbons. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety contains 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

As used herein, the term "C _nm alkylene" used alone or in combination with other terms refers to a divalent alkyl linking group having n to m carbons. Examples of alkylene include, but are not limited to, second-1,1-diyl, second-1,2-diyl, third-1,1,-diyl, third-1,3-diyl, third-1,2-diyl, fourth-1,4-diyl, fourth-1,3-diyl, fourth-1,2-diyl, 2-methyl-third-1,3-diyl, etc. In some embodiments, the alkylene moiety contains 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

As used herein, the term "C _nm alkoxy" used alone or in combination with other terms refers to a radical of the formula -O-alkyl, wherein the alkyl has n to m carbons. Example alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like. In some embodiments, the alkyl has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

As used herein, the term "C _nm hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group (-OH).

As used herein, the term "C _nm alkylamino" refers to a group of the formula -NH(alkyl), wherein the alkyl group has n to m carbon atoms. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylamino groups include, but are not limited to, N-methylamino, N-ethylamino, N-propylamino (e.g., N-(n-propyl)amino and N-isopropylamino), N-butylamino (e.g., N-(n-butyl)amino and N-(tert-butyl)amino), and the like.

As used herein, the term "amino" refers to a group of formula _-NH2 .

As used herein, the term "aryl" used alone or in combination with other terms refers to an aromatic hydrocarbon group, which may be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). The term "C _nm aryl" refers to an aryl group having n to m ring carbon atoms. Aryl includes, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, etc. In some embodiments, aryl has 6 to 10 carbon atoms. In some embodiments, aryl is phenyl or naphthyl.

As used herein, "halo" refers to F, Cl, Br, or I. In some embodiments, halo is F, Cl, or Br. In some embodiments, halo is F.

As used herein, "cycloalkyl" refers to non-aromatic cyclic hydrocarbons, including cyclized alkyl and/or alkenyl. Cycloalkyl may include monocyclic or polycyclic (e.g., with 2, 3 or 4 fused rings) groups and spirocycles. The ring-forming carbon atoms of cycloalkyl may be optionally substituted by oxo or sulfenyl (e.g., C(O) or C(S)). The definition of cycloalkyl also includes a portion having one or more aromatic rings fused to a cycloalkyl ring (i.e., with a shared bond), such as benzo or thienyl derivatives of cyclopentane, cyclohexane, etc. Cycloalkyl containing fused aromatic rings may be connected via any ring-forming atom (including the ring-forming atom of the fused aromatic ring). Cycloalkyl may have 3, 4, 5, 6, 7, 8, 9 or 10 ring-forming carbons (C _3-10 ). In some embodiments, cycloalkyl is a C _3-10 monocyclic or bicyclic cycloalkyl. In some embodiments, cycloalkyl is a C _3-7 monocyclic cycloalkyl. Example cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarbyl, etc. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic heterocyclic ring having at least one heteroatom ring member selected from sulfur, oxygen and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety may be an N-oxide. In some embodiments, the heteroaryl is a 5-10-membered monocyclic or bicyclic heteroaryl having 1, 2, 3 or 4 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a 5-6 monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur and oxygen. In some embodiments, the heteroaryl is a 5- or 6-membered heteroaryl ring. A five-membered heteroaryl ring is a heteroaryl having five ring atoms, wherein one or more (e.g., 1, 2 or 3) ring atoms are independently selected from N, O and S. Exemplary five-membered ring heteroaryls are thienyl, furanyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl. Six-membered heteroaryl rings are heteroaryl rings having six ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary six-membered ring heteroaryls are pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

As used herein, "heterocycloalkyl" refers to a non-aromatic monocyclic or polycyclic heterocycle having one or more ring-forming heteroatoms selected from O, N or S. Heterocycloalkyl includes monocyclic 4-, 5-, 6-, 7-, 8-, 9- or 10-membered heterocycloalkyls. Heterocycloalkyl may also include spirocycles. Example heterocycloalkyls include pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydropyran, oxetanyl, azetidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazepine, etc. The ring-forming carbon atoms and heteroatoms of heterocycloalkyl can be optionally substituted by oxo or sulfide ion groups (e.g., C(O), S(O), C(S) or S(O) ₂ , etc.). Heterocycloalkyl can be connected via ring-forming carbon atoms or ring-forming heteroatoms. In some embodiments, heterocycloalkyl contains 0 to 3 double bonds. In some embodiments, heterocycloalkyl contains 0 to 2 double bonds. The definition of heterocycloalkyl also includes a portion having one or more aromatic rings fused to a cycloalkyl ring (i.e., having a shared bond therewith), such as benzo or thienyl derivatives of piperidine, morpholine, aza-phenanthene, etc. Heterocycloalkyl containing a fused aromatic ring can be connected via any ring-forming atom (including the ring-forming atom of the fused aromatic ring). In some embodiments, heterocycloalkyl is a monocyclic 4-6 membered heterocycloalkyl having 1 or 2 heteroatoms independently selected from nitrogen, oxygen or sulfur and having one or more oxidized ring members. In some embodiments, heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxygenated ring members.

In some places, the definition or embodiment refers to a specific ring (e.g., azetidine ring, pyridine ring, etc.). Unless otherwise indicated, these rings may be connected to any ring member, provided that the valence of the atom is not exceeded. For example, the azetidine ring may be connected to any position of the ring, while the pyridine-3-yl ring is connected at the 3 position.

As used herein, a "bridged ring" or "bridged ring group" is a ring system having at least two linked rings that share three or more atoms. The bridged ring can be a carbocyclic ring or a heterocycloalkyl ring. Example bridged rings include

When a range is given, the endpoints are included. In addition, it should be understood that unless otherwise indicated or otherwise obvious from the context and the understanding of one of ordinary skill in the art, in various embodiments of the present disclosure, the values expressed as ranges can be assumed to be any specific value or sub-range within the range, unless the context clearly indicates otherwise, otherwise accurate to one-tenth of the unit of the lower limit of the range.

In addition, it should be understood that any specific embodiment of the present disclosure that is within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not explicitly stated herein. Any specific embodiment of the composition of the present disclosure (e.g., any nucleic acid or protein encoded thereby; any production method; any method of use, etc.) may be excluded from any one or more claims for any reason, whether or not related to the existence of the prior art.

All reference sources (e.g., references, publications, databases, database entries, and techniques cited herein) are incorporated herein by reference, even if not explicitly stated in the reference. If a reference source conflicts with the statements of the present application, the statements in the present application shall prevail.

Section and table headings are not intended to be limiting.

Example

abbreviation:

ACN: Acetonitrile

Aq.: Aqueous solution

Boc ₂ O: di-tert-butyl pyrocarbonate

DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

BnBr: benzyl bromide

DCC: N,N′-dicyclohexylcarbodiimide

DCM: dichloromethane

DMAP: 4-dimethylaminopyridine

DMF: dimethylformamide

EtOAc: Ethyl acetate

h: hour/hour

LCMS: Liquid chromatography-mass spectrometry

MTBE: Methyl tert-butyl ether

PMA: phosphomolybdic acid

Soln.: solution

rt: room temperature

THF: Tetrahydrofuran

TLC: Thin layer chromatography

Example 1

Synthesis of compounds according to formula A1

Intermediate A: tert-butyl (5-((3-((tert-butoxycarbonyl)amino)butyl)amino)pentan-2-yl)carbamate

Step 1: tert-Butyl (5-hydroxypentan-2-yl)carbamate

To a solution of di-tert-butyl dicarbonate (2.45 mL, 10.66 mmol) in anhydrous DCM (30 mL) stirred at 0°C under nitrogen was added 4-aminopentan-1-ol (1.00 g, 9.69 mmol) dropwise over 10 minutes. The solution was gradually warmed to room temperature and stirred for 3 days. The solvent was then removed under vacuum, and the resulting residue was dissolved in DCM and purified on silica with a 0-100% EtOAc gradient in hexane. The fractions containing the product were pooled and concentrated in vacuo to give tert-butyl (5-hydroxypentan-2-yl)carbamate (0.86 g, 4.23 mmol, 43.6%) as an oil. UPLC/ELSD: RT=0.28 min. MS (ES): m/z (MH ⁺ ) 204.3 for C ₁₀ H ₂₁ NO ₃ . ¹ H NMR (300MHz, CDCl ₃ ) δ 4.51 (br.s, 1H), 3.62 (t, 3H), 2.59 (br.s, 1H), 1.53 (m, 4H), 1.42 (s, 9H), 1.12 (d, 3H, J = 6Hz).

Step 2: 4-((tert-Butoxycarbonyl)amino)pentyl 4-methylbenzenesulfonate

To a solution of tert-butyl (5-hydroxypentan-2-yl)carbamate (0.86g, 4.23mmol) in anhydrous DCM (30mL) stirred under nitrogen, triethylamine (2.95mL, 21.15mmol), dimethylaminopyridine (0.10g, 0.85mmol) and p-toluenesulfonyl chloride (1.61g, 8.46mmol) were added. The solution was stirred at room temperature overnight, during which it became dark red. The mixture was then further diluted with DCM, washed with water (1x30 mL), saturated sodium bicarbonate aqueous solution (1x30mL) and brine (1x30 mL), dried over sodium sulfate, filtered, and concentrated into a dark brown oil. The oil was dissolved in DCM and purified with a 0-50% EtOAc gradient in hexane on silica. Fractions containing product were pooled and concentrated to give 4-((tert-butoxycarbonyl)amino)pentyl 4-methylbenzenesulfonate (0.97 g, 2.72 mmol, 64.3%) as a light brown oil. UPLC/ELSD: RT: 1.02 min _. ^MS (ES): m/z (MH ⁺ ) 358.4 _for Ci7H27NO5S. _1H NMR (300 MHz, CDCl3) δ 7.77 (d, 2H, J = 6 Hz), 7.36 (d, 2H, J = 6 Hz), 4.32 (br _. s, 1H), 4.03 (t, 2H), 3.55 (br. s, 1H), 2.45 (s, 3H), 1.69 (br. m, 2H), 1.41 (s, 11H), 1.06 (d, 3H, J = 6 Hz).

Step 3: tert-Butyl (5-((3-((tert-butoxycarbonyl)amino)butyl)amino)pentan-2-yl)carbamate

To a solution of N-[4-(2-nitrobenzenesulfonamido)butyl-2-yl]carbamic acid tert-butyl ester (0.97g, 2.61mmol) in anhydrous DMF (20mL) stirred under nitrogen at room temperature, 4-methylbenzenesulfonic acid 4-((tert-butoxycarbonyl)amino)pentyl ester (1.03g, 2.87mmol) and potassium carbonate (1.08g, 7.82mmol) were added. The solution was warmed to 100 ° C and stirred for 48 hours. Then, the reaction was quenched with 30mL water and diluted with 50mL EtOAc. The organic layer was separated, and the aqueous layer was extracted with EtOAc (3x30 mL). The organic layers were all combined, washed with water (1x50 mL) and brine (1x50mL), dried over sodium sulfate, filtered, and concentrated into a yellow oil. The oil was dissolved in DCM and purified with 0-100% EtOAc gradient in hexane on silica. The fractions containing product are collected and concentrated into an oil. The oil is dissolved in 20mL DMF, thiophenol (1.03mL, 10.03mmol) and potassium carbonate (1.08g, 7.82mmol) are added thereto. The solution is stirred at room temperature overnight. The next morning, salt is removed from the mixture by centrifugation and the supernatant is concentrated into residue. The residue is dissolved in 40mL DCM, washed with water (2x10 mL) and saline (2x10 mL), dried over potassium carbonate, filtered, and concentrated into an oil. The oil is dissolved in DCM and purified by 0-50% (50:45:5DCM/MeOH/NH ₄ OH) gradient in DCM on silica. Fractions containing product were pooled and concentrated to give tert-butyl (5-((3-((tert-butoxycarbonyl)amino)butyl)amino)pentan-2-yl)carbamate (0.34 g, 0.91 mmol, 34.93%) as a colorless oil. UPLC/ELSD: RT: 0.28 min _{. MS} (ES): m/z (MH ⁺ ) _{for C19H39N3O4} 374.4. ¹ H NMR (300MHz, CDCl ₃ ) δ4.91(br.m,1H),4.67(br.m,1H),3.70(br.m,2H),3.40(s,2H),2.72(t,1H),2.58(br.m,3H),2.41(br.s,4H),1.50(br.m,5H),1.41 (s,19H),1.10(d,6H,J=6Hz).

A. Compound SA50: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate dihydrochloride

Step 1: Tert-butyl malonate ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.95 g, 15.21 mmol) was added to a solution of cholesterol (4.00 g, 10.14 mmol) and 3-(tert-butoxy)-3-oxopropanoic acid (2.39 mL, 15.21 mmol) in dichloromethane (20 mL) stirred under nitrogen. The reaction mixture was then cooled to 0 ° C, and diisopropylethylamine (5.36 mL, 30.41 mmol) was added dropwise over 20 minutes. The resulting mixture was gradually warmed to room temperature and left overnight. The mixture was then diluted to 150 mL with dichloromethane, washed with water (1x70 mL), saturated sodium bicarbonate aqueous solution (2x70 mL) and brine (1x70 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give a yellow oil. The oil was dissolved in dichloromethane and purified on silica with a 0-25% ethyl acetate in hexanes gradient to afford tert-butyl malonate ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) as an oil (4.99 g, 9.44 mmol, 93.1% ₎ . UPLC/ELSD: RT: 3.36 _{min .} MS (ES): m/z (MH ⁺ ) 529.8 for C34H56O4. ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.41(m,1H),4.67(m,1H),3.27(s,2H),2.38(d,2H),1.91(br.m,10H),1.49(s,12H),1.35(br.m,6H),1.04(br.m,17H),0.9 1 (d, 3H, J = 3Hz), 0.87 (d, 3H, J = 3Hz), 0.70 (s, 3H).

Step 2: 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-3-oxopropanoic acid

To a solution of tert-butyl malonate ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (4.99 g, 9.44 mmol) in dichloromethane (50 mL) stirred at 0°C under nitrogen was added trifluoroacetic acid (10.85 mL, 141.63 mmol) dropwise over 20 minutes. The clear light yellow reaction mixture was allowed to gradually warm to room temperature overnight. The next morning, the reaction was quenched at 0°C with 20 mL of 5% aqueous sodium bicarbonate. The organics were separated, washed again with 10 mL of 5% aqueous sodium bicarbonate, dried over sodium sulfate, filtered, and concentrated to give a white solid. The solid was dissolved in dichloromethane and purified on silica with a gradient of 0-60% ethyl acetate in hexanes to give 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-3-oxopropanoic acid (3.12 g, 6.61 mmol, 70.0%) as a white solid. UPLC/ELSD: RT: 2.97 min. MS (ES): m/z (MH ⁺ ) 473.7 for C ₃₀ H ₄₈ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 10.99 (br.s, 1H), 5.42 (m, 1H), 4.73 (m, 1H), 3.45 (s, 2H), 2.37 (d, 2H, J = 9Hz), 1.89 (br.m, 5H), 1.35 (br.m, 18H), 1.05 (s, 5H) ,0.94(d,4H,J＝2Hz),0.89(d,6H,J＝2Hz),0.70(s,3H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate

To a solution of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-oxopropanoic acid (3.12 g, 6.61 mmol) in dichloromethane (60 mL) stirred under nitrogen was added tetramethyldipropylenetriamine (2.30 mL, 9.81 mmol), dimethylaminopyridine (0.08 g, 0.65 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.90 g, 9.81 mmol). The reaction mixture was cooled to 0 °C and diisopropylethylamine (3.46 mL, 19.62 mmol) was added dropwise over 20 minutes. The mixture was gradually warmed to room temperature and left overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution (1x50 mL) and brine (1x50 mL), dried over sodium sulfate, filtered, and concentrated into an oil. The oil was dissolved in dichloromethane and purified on silica with a 0-60% (9:1 methanol/concentrated aqueous ammonium hydroxide) gradient in dichloromethane to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate as a yellow oil (1.82 g, 2.84 mmol, 43.4%). UPLC/ELSD: RT: 1.85 _{min .} MS (ES): m/z (MH ⁺ ) 643.0 _{for C40H71N3O3} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.39 (m, 1H), 4.67 (m, 1H), 3.54 (s, 2H), 3.35 (br.m, 4H), 2.37 (br.m, 6H), 2.22 (d, 12H, J = 3Hz), 1.50 (br.m, 28H), 1.02 (br.s, 5 H), 0.92 (d, 4H, J = 6Hz), 0.88 (d, 6H, J = 9Hz), 0.68 (s, 3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate (0.22 g, 0.32 mmol) in diethyl ether (4.3 mL) and isopropanol (0.22 mL) was added hydrochloric acid (5.5 M in isopropanol, 0.37 mL, 1.85 mmol) dropwise. The mixture was cooled to 0°C and stirred vigorously for 30 minutes, after which the white precipitate was filtered off via vacuum filtration and washed repeatedly with cold ether. The residue was dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-(bis(3-(dimethylamino)propyl)amino)-3-oxopropanoate dihydrochloride (0.11 g, 0.15 mmol, 46.6% ₎ as a white waxy solid. UPLC/ELSD: RT: _1.81 min _. MS (ES): m/z (MH ⁺ ) 627.99 _{for C40H73Cl2N3O3} . ¹ H NMR (300MHz, CD ₃ OD) δ: ppm 5.41 (br.s, 1H), 4.64 (br.m, 1H), 3.57 (br.m, 7H), 3.33 (br.s, 2H), 3.20 (br.m, 5H), 2.93 (d, 15H, J = 6Hz), 2.40 (d, 2H, J = 9Hz), 2. 05(br.m,12H),1.55(br.m,14H),1.20(br.m,13H),1.07(s,7H),0.98(d,5H,J＝6Hz),0.91(d,7H,J＝6Hz),0.74(s,3H).

B. Compound SA51: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate dihydrochloride

Step 1: 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid

Glutaric anhydride (2.63g, 22.81mmol) and triethylamine (3.21mL, 22.81mmol) are added to the solution of cholesterol (5.00g, 12.67mmol) in acetone (50mL) stirred under nitrogen. The reaction mixture is refluxed at 56°C, and becomes a colorless clear solution from a white slurry, and is carried out under reflux for 3 days. Then, the solution is cooled to room temperature, concentrated under vacuum, and dissolved in 150mL dichloromethane. Then this is washed with 0.5M HCl (1x100 mL) and saturated aqueous ammonium chloride solution (1x100mL), dried over sodium sulfate, filtered, and concentrated to give a white solid. The solid was dissolved in dichloromethane and purified on silica with a 0-50% ethyl acetate in hexanes gradient to afford 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (6.011 g, 12.00 mmol, 94.7%) as a white solid. UPLC/ELSD: RT: 2.96 _{min .} MS (ES): m/z (MH ⁺ ) 501.7 for _C32H52O4 . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.40 (m, 1H), 4.66 (m, 1H), 2.45 (br.m, 5H), 2.01 (br.m, 3H), 1.85 (br.m, 3H), 1.34 (br.m, 22H), 0.94 (d, 3H, J = 6Hz), 0.88 (d, 6H, J =9Hz),0.70(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate

To a solution of 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (6.01 g, 11.88 mmol) in stirred dichloromethane (100 mL) under nitrogen was added tetramethyldipropylenetriamine (4.19 mL, 17.82 mmol), dimethylaminopyridine (0.15 g, 1.19 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.45 g, 17.83 mmol). The solution was cooled to 0 ° C, and then diisopropylethylamine (6.29 mL, 35.65 mmol) was added dropwise. The reaction mixture was gradually warmed to room temperature and overnight. The solution was further diluted with dichloromethane, washed with saturated sodium bicarbonate aqueous solution (1x100 mL) and brine (1x100 mL), dried over sodium sulfate, filtered, and concentrated to give an oil. The material was dissolved in dichloromethane and purified on silica in a 0-60% (9:1 methanol:aq. ammonium hydroxide) gradient in dichloromethane to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene- ₃ -yl _5- (bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate as an oil (2.30 g, 11.88 mmol, 28.9%). UPLC/ELSD: RT: 2.02 min _. MS (ES): m/z (MH ⁺ ) 671.1 for _C42H75N3O3 . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.36 (m, 1H), 4.60 (m, 1H), 3.33 (br.m, 5H), 2.39 (br.m, 12H), 2.23 (d, 11H, J = 6Hz), 1.99 (br.m, 4H), 1.84 (br.m, 3H), 1.71 (br.m ,5H),1.33(br.m,11H),1.14(br.m,7H),1.02(s,6H),0.92(d,3H,J＝6Hz),0.87(d,5H,J＝9Hz),0.68(s,3H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate (0.23 g, 0.33 mmol) in diethyl ether (4.6 mL) and isopropanol (0.23 mL) was added hydrochloric acid (5.5 M in isopropanol, 0.37 mL, 1.85 mmol) dropwise. The mixture was cooled to 0°C and stirred vigorously for 30 minutes, after which the white precipitate was filtered off via vacuum filtration and washed repeatedly with cold ether. The residue was dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-(bis(3-(dimethylamino)propyl)amino)-5-oxopentanoate dihydrochloride (0.12 g, 0.16 mmol, 49.5% ₎ as a white waxy solid. UPLC/ELSD: RT: 1.86 min _. MS (ES): m/z (MH ⁺ ) _{671.81 for C42H77Cl2N3O3} . ¹ H NMR(300MHz,CD ₃ OD)δ:ppm 5.41(br.s,1H),4.55(br.m,1H),3.54(t,5H,J＝6Hz),3.24(br.m,6H),2.94(d,14H,J＝6Hz),2.55(t,2H,J＝6Hz),2.43(t,2H,J＝6Hz),2.35(d,2H,J＝9Hz),2.05(br.m,6H),1.90(br.m,6H),1.55(br.m,12H),1.19(br.m,10H),1.07(s,7H),0.98(d,4H,J＝6Hz),0.90(d,7H,J＝6Hz),0.74(s,3H)。

C. Compound SA56: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-oxo-4-(1,4,7-triazacyclononan-1-yl)butanoate

Step 1: Di-tert-butyl 7-(4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoyl)-1,4,7-triazacyclononane-1,4-dicarboxylate

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.060 g, 0.31 mmol) was added to a stirred solution of cholesteryl hemisuccinate (100 mg, 0.205 mmol), 1,4,7-triazacyclononane-1,4-dicarboxylic acid 1,4-di-tert-butyl ester (Enamine, Monmouth Junction, NJ) (0.068 g, 0.20 mmol) and DMAP (catalyst) in DCM (1.4 mL). The reaction mixture was stirred at room temperature and monitored by TLC. Water (1.5 mL) was added at 21.5 h. After stirring for 16 h, water (10 mL) was added. The mixture was then extracted with DCM (2 x 15 mL). The combined organics were passed through a hydrophobic glass frit, dried over Na ₂ SO ₄ , and then concentrated. The crude material was purified via silica gel chromatography (0-4% MeOH in DCM) to afford di-tert-butyl 7-(4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoyl)-1,4,7-triazacyclononane-1,4-dicarboxylate (130 mg, 0.163 mmol, 79.3%) as a clear oil. UPLC/ELSD: RT = 3.41 min. MS (ES): m/z of C ₄₇ H ₇₉ N ₃ O ₇ = 1619.2 [2M+Na] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.33-5.39 (m, 1H), 4.52-4.68 (m, 1H), 3.18-3.79 (br.m, 12H), 2.49-2.71 (m, 4H) ,2.24-2.39(m,2H),1.74-2.06(br.m,5H),0.93-1.71(br.m,39H),1.01(s,3H),0.91(d,3H,J＝6.5Hz),0.87(d,3H,J＝6.6Hz),0.67 (s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-oxo-4-(1,4,7-triazacyclononan-1-yl)butanoate dihydrochloride

To a solution of di-tert-butyl 7-(4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoyl)-1,4,7-triazacyclononane-1,4-dicarboxylate (123 mg, 0.154 mmol) in iPrOH (2.0 mL) was added 5-6 N HCl in iPrOH (0.18 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17 h, more iPrOH (2.0 mL) and 5-6 N HCl in iPrOH (0.06 mL) were added. At 41h, the reaction mixture was cooled to room temperature and ACN (4mL) was added. The solid was collected by vacuum filtration and rinsed with ACN to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) 4-oxo-4-(1,4,7-triazacyclononane-1-yl)butanoate dihydrochloride (0.082g, 0.11mmol, 73.8%) as a white solid. UPLC/ELSD: RT = 2.30min. MS (ES): m/z of C ₃₇ H ₆₃ N ₃ O ₃ = 598.1 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ 10.36 (br.s, 2H), 10.11 (br.s, 2H), 5.34-5.43 (m, 1H), 4.50-4.65 (m, 1H), 3.97-4. 29(m,4H),3.64-3.95(m,6H),3.41-3.61(m,2H),2.66-2.84(m,2H),2.45-2.65( m,2H),2.21-2.40(m,2H),1.75-2.08(br.m,5H),0.94-1.70(br.m,21H),1.01(s,3H),0.91(d,3H,J＝6.4Hz),0.87(d,3H,J＝6.5Hz),0.86(d,3H,J＝6.6Hz) ,0.68(s,3H).

Step 3: 4-oxo-4-(1,4,7-triazacyclononan-1-yl)butanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-oxo-4-(1,4,7-triazacyclononan-1-yl)butanoate dihydrochloride (0.054 g, 0.075 mmol) was suspended in 5% NaHCO ₃ aqueous solution (10 mL) and then extracted with DCM (3 x 10 mL). K ₂ CO ₃ (about 100 mg) was added to the aqueous layer. The aqueous layer was extracted with DCM (2 x 10 mL). The combined organics were passed through a hydrophobic frit, dried over _Na2SO4 , and concentrated to afford (3S, _8S ,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-oxo-4-(1,4,7-triazacyclononan-1-yl)butanoate (0.023 g, 0.037 mmol, 50.0%) as a white solid. ¹ H NMR (300MHz, CDCl ₃ ): δ5.33-5.40(m,1H),4.54-4.70(m,1H),3.41-3.57(m,4H),2.99-3.15(m,4H),2.72-2.84(m,4H),2.58-2.72(m,4H),2.24-2.39 (m,2H),1.74-2.19(br.m,7H),0.94-1.70(br.m,21H),1.01(s,3H),0.91(d,3H,J＝6.5Hz),0.87(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.67(s,3H). UPLC/ELSD: RT=2.39min. MS (ES): m/z of C ₃₇ H ₆₃ N ₃ O ₃ = 598.6 [M+H] ⁺ .

D. Compound SA57: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 6-(bis(3-(dimethylamino)propyl)amino)-6-oxohexanoate

Step 1: 6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-6-oxohexanoic acid

Adipic anhydride (1.66 g, 12.93 mmol) was added to a solution of cholesterol (5.00 g, 12.93 mmol) in dichloromethane (50 mL) stirred under nitrogen. Pyridine (3.97 mL, 28.45 mmol) was then added dropwise over 10 minutes. The reaction mixture was heated to reflux at 40 ° C and allowed to stand overnight. The mixture was then cooled to room temperature and concentrated to a yellow oil. The oil was dissolved in dichloromethane and purified on silica without additional workup in a 0-30% ethyl acetate gradient in hexanes to afford 6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-6-oxohexanoic acid (1.97 g, 3.83 mmol, 29.6%) as a white solid. UPLC/ELSD: RT: 3.09 _min . MS (ES): m/z (MH ⁺ ) 515.7 _{for C33H54O4} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 12.15(br.s,1H),5.39(m,1H),4.63(br.m,1H),2.40(br.m,6H),2.00(br.m,2H),1.85(br.m,3H),1.70(br.m,4H),1.34(br.m,1 9H), 1.03 (s, 6H), 0.93 (d, 4H, J = 6Hz), 0.88 (d, 6H, J = 6Hz), 0.69 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 6-(bis(3-(dimethylamino)propyl)amino)-6-oxohexanoate

To a solution of 6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-6-oxohexanoic acid (1.00 g, 1.92 mmol) in stirred dichloromethane (25 mL) under nitrogen was added tetramethyldipropylenetriamine (0.68 mL, 2.89 mmol), dimethylaminopyridine (0.02 g, 0.19 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.56 g, 2.89 mmol). The resulting solution was cooled to 0 ° C and diisopropylethylamine (1.02 mL, 5.77 mmol) was added dropwise. The mixture was gradually warmed to room temperature and left overnight. The solution was then diluted with dichloromethane, washed with saturated sodium bicarbonate aqueous solution (1x10 mL) and brine (1x10 mL), dried over sodium sulfate, filtered, and concentrated into an oil. The oil was dissolved in DCM and purified on silica with a gradient of 0-60% (8:2:0.1 dichloromethane/methanol/concentrated aqueous ammonium hydroxide solution) in dichloromethane, according to ¹ H NMR afforded (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 6-(bis(3-(dimethylamino)propyl)amino)-6-oxohexanoate as a yellow oil, so 0-25% (8:2:0.1 dichloromethane/methanol/conc.) was used on silica. The material was repurified with an aqueous ammonium hydroxide (aqueous) gradient to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 6-(bis(3-(dimethylamino)propyl)amino)-6-oxohexanoate as a light yellow oil (0.38 g, 0.54 mmol, _28.2 %). UPLC/ELSD: RT: 2.11 _min . MS (ES): m/z (MH ⁺ ₎ 685.1 for _C43H77N3O3 . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.40 (m, 3H), 5.20 (m, 1H), 4.40 (br.m, 1H), 3.22 (m, 4.48), 2.58 (s, 3H), 2.38 (t, 2H, J = 9Hz), 2.26 (s, 6H), 2.20 (d, 3H, J = 9Hz) ), 2.14 (br.s, 9H), 1.49 (br.m, 24H), 0.95 (br.m, 7H), 0.85 (s, 5H), 0.75 (d, 4H, J = 6Hz), 0.70 (d, 5H, J = 9Hz), 0.51 (s, 3H).

E. Compound SA58: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

Step 1: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a stirred solution of 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.76 g, 1.49 mmol) in dichloromethane under nitrogen was added 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.76 g, 1.49 mmol) in a stirred solution of dichloromethane under nitrogen was added 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.76 g, 1.4 To a solution in 1% paraffin (20 mL) was added tert-butyl (3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.75 g, 1.49 mmol), dimethylaminopyridine (0.02 g, 0.15 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.43 g, 2.24 mmol). The resulting solution was cooled to 0 °C and diisopropylethylamine (0.79 mL, 4.48 mmol) was added dropwise. The mixture was gradually warmed to room temperature and allowed to proceed overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution (1 x 10 mL) and brine (1 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica with a gradient of 0-60% ethyl acetate in hexanes to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate as a light yellow oil (0.71 g, 0.72 mmol, 48.2%). UPLC/ELSD: RT: 3.37 min. MS (ES): m/z (MH ⁺ ) 986.4 for C ₅₇ H ₁₀₀ N ₄ O ₉ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm5.39(m,2H),4.64(br.m,1H),3.27(br.m,11H),2.38(br.m,6H),1.86(br.m,13H),1.46(br.d,32H),1.15(br.m,11H),1.03(s,5 H), 0.94 (d, 3H, J = 9Hz), 0.88 (d, 5H, J = 9Hz), 0.70 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-olate (0.71 g, 0.72 mmol) in 2-propanol (10 mL) stirred under nitrogen was added hydrochloric acid (5.5 M in 2-propanol, 1.44 mL, 7.20 mmol) dropwise. The mixture is heated to 45 DEG C and stirred overnight.Then, the solution is cooled to room temperature, and acetonitrile (5mL) is added to the mixture.Then it is sonicated to remove the precipitated solid from the flask side.After stirring for 30 minutes after sonication, solid is filtered out by vacuum filtration, repeatedly washed with acetonitrile, and dried in a vacuum to obtain 5-((3-aminopropyl) (4-((3-aminopropyl) amino) butyl) amino)-5-oxopentanoic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester trihydrochloride (0.39g, 0.47mmol, 65.6%) as light purple solid. UPLC/ELSD: RT: 1.68min. MS (ES): m/z (MH ⁺ ) 686.1 for C ₄₂ H ₇₉ Cl ₃ N ₄ O ₃ . ¹ H NMR (300MHz, CD ₃ OD) δ: ppm 5.40 (s, 1H), 4.90 (br.s, 9H), 4.55 (br.s, 1H), 3.33 (br.m, 12H), 2.32 (br.6H), 2.16 (br.m, 2H), 2.05 (s, 5H), 1.91 (br.m, 10H), 1.54 (br.m, 7H), 1.39 (br.m, 4H), 1.17 (d, 8H, J = 6Hz), 1.06 (s, 5H), 0.97 (d, 4H, J = 6Hz), 0.90 (d, 6H, J = 6Hz), 0.73 (s, 3H).

F. Compound SA59: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-3-oxopropanoate trihydrochloride

Step 1: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a stirred solution of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-3-oxopropanoic acid (0.70 g, 1.47 mmol) in dichloromethane under nitrogen was added 1% ethanol; To a solution in 20 mL of 4-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.74 g, 1.47 mmol), dimethylaminopyridine (0.02 g, 0.15 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.43 g, 2.20 mmol) were added. The resulting solution was cooled to 0° C. and diisopropylethylamine (0.78 mL, 4.40 mmol) was added dropwise. The mixture was gradually warmed to room temperature and allowed to remain overnight. The solution was then diluted with dichloromethane, washed with water (3×20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica with a gradient of 0-60% ethyl acetate in hexanes to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oate (1.13 g, 1.18 mmol, 80.4%) as a light yellow oil. UPLC/ELSD: RT: 3.29 min. MS (ES): m/z (MH ⁺ ) 958.4 for C ₅₅ H ₉₆ N ₄ O ₉ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.39 (m, 2H). 4.70(br.m,2H),3.26(br.m,13H),2.37(d,2H,J＝6Hz),1.86(br.m,16H),1.45(br.s,28H),1.23(br.m,12H),1.03(s,4H),0.94(d,3H,J＝6Hz),0.88(d,5H, J＝6Hz),0.69(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-3-oxopropanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-olate (1.13 g, 1.18 mmol) in 2-propanol (15 mL) stirred under nitrogen was added hydrochloric acid (5.5 M in 2-propanol, 2.36 mL, 11.79 mmol) dropwise. The mixture is heated to 40 DEG C and carried out overnight.Then, acetonitrile (5mL) is added, and the solution is sonicated until all solids are displaced from the flask side.After stirring for 30 minutes after sonication, solid is filtered out by vacuum filtration and repeatedly washed with acetonitrile and dried under vacuum, 3-((3-aminopropyl) (4-((3-aminopropyl) amino) butyl) amino)-3-oxopropionic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester trihydrochloride (0.52g, 0.64mmol, 54.4%) is obtained as light purple solid. UPLC/ELSD: RT: 1.57min. MS (ES): m/z (MH ⁺ ) 657.2 for C ₄₀ H ₇₅ N ₄ O ₃ . ¹ H NMR (300MHz, CD ₃ OD) δ: ppm 5.42 (m, 1H), 4.88 (br.s, 11H), 4.60 (m, 1H), 3.33 (br.m, 16H), 2.39 (d, 2H, J = 3Hz), 1.55 (br.m, 40H), 0.96 (d, 4H, J = 6Hz), 0.9 0(d,6H,J=6Hz),0.74(s,3H).

G. Compound SA60: (8-aminooctyl)(3-aminopropyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester dihydrochloride

Step 1: tert-Butyl N-{8-[(2-cyanoethyl)amino]octyl}carbamate

Acrylonitrile (1.00mL, 15.3mmol) is added to the foil-covered stirred suspension of N-(8-aminooctyl) tert-butyl carbamate (2.50g, 10.2mmol) in water (100mL). The suspension is stirred at room temperature and monitored by TLC. At 26h, the reaction mixture is diluted with 5% _NaHCO3 aqueous solution (200mL) and then extracted with EtOAc ( _3x 100mL). The combined organic matter is washed with brine, dried over _Na2SO4 , and concentrated. The crude material is purified via silica gel chromatography (0-10% MeOH in DCM) to obtain N-{8-[(2-cyanoethyl)amino]octyl} tert-butyl carbamate (1.833g, 6.163mmol, 60.2%) as a yellow oil. UPLC/ELSD: RT=0.28min. MS (ES): m/z=197.9 [(M+H)-(CH ₃ ) ₂ C=CH ₂ -CO ₂ ] ⁺ C ₁₆ H ₃₁ N ₃ O ₂ ; ¹ H NMR (300MHz, CDCl ₃ ): δ4.50 (br.s, 1H), 3.09 (dt, 2H, 6.6, 6.5Hz), 2.92 (t, 2H, J=6.6Hz ), 2.62 (t, 2H, J = 7.1Hz), 2.51 (t, 2H, J = 6.7Hz), 1.18-1.58 (m, 13H), 1.44 (s, 9H).

Step 2: tert-Butyl N-{8-[Benzyl(2-cyanoethyl)amino]octyl}carbamate

A mixture of N-{8-[(2-cyanoethyl)amino]octyl}t-butyl carbamate (0.870g, 2.92mmol), potassium carbonate (0.808g, 5.85mmol), benzyl bromide (0.40mL, 3.4mmol) and potassium iodide (0.097g, 0.58mmol) in ACN (17.5mL) was stirred at 65°C. The reaction was monitored by LCMS. At 3h, the reaction mixture was cooled to room temperature, filtered through a celite pad, rinsed with MTBE, and concentrated. The residue was dissolved in 5% _NaHCO3 aqueous solution (50mL) and then extracted with MTBE ( _3x 30mL). The combined organic matter was washed with brine, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-40% EtOAc in hexanes) to give tert-butyl N-{8-[benzyl(2-cyanoethyl)amino]octyl}carbamate (0.783 g, 2.02 mmol, 69.1%) as a clear oil. UPLC/ELSD: RT = 0.44 _min . MS (ES): m/ _z for _C23H37N3O2 = 331.9 [(M+H) _- (CH3 _)2C = _CH2 ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ7.21-7.39 (m, 5H), 4.49 (br.s, 1H), 3.60 (s, 2H), 3.09 (dt, 2H, J = 6.5, 6.2Hz), 2.78 (t, 2H, J = 6.9Hz), 2.48 (t, 2H, J = 7.4Hz), 2.39 (t,2H,J=7.0Hz),1.38-1.54(m,4H),1.44(s,9H),1.19-1.36(m,8H).

Step 3: tert-Butyl N-{3-[benzyl({8-[(tert-butoxycarbonyl)amino]octyl})amino]propyl}carbamate

To a stirred solution of tert-butyl N-{8-[benzyl(2-cyanoethyl)amino]octyl}carbamate (0.492 g, 1.27 mmol) in MeOH (8.8 mL) was added di-tert-butyl dicarbonate (0.693 g, 3.17 mmol) and nickel(II) chloride hexahydrate (0.030 g, 0.13 mmol). The reaction mixture was cooled to 0 °C in an ice bath and then _NaBH4 (0.336 g, 8.89 mmol) was added portionwise over 30 min to give a black suspension (warning: vigorous gas evolution occurred during addition). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17.3 h, diethylenetriamine (0.15 mL, 1.4 mmol) was added dropwise, and the reaction mixture was stirred at room temperature. After 30 min, diethylenetriamine (0.15 mL) was added. After 1.5 h, the reaction mixture was concentrated, dissolved in 5% _NaHCO3 aqueous solution and extracted with EtOAc ( _3x ). The combined organics were washed with 5% _NaHCO3 aqueous solution and brine, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-65% EtOAc in hexane) to give tert-butyl N-{3-[benzyl({8-[(tert-butoxycarbonyl)amino]octyl})amino]propyl}carbamate (0.512 g, 1.04 mmol, 82.0%) as a clear oil. UPLC/ELSD: RT=0.92 min. MS (ES): m/z of C ₂₈ H ₄₉ N ₃ O ₄ = 492.5 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ7.19-7.35 (m, 5H), 5.52 (br.s, 1H), 3.51 (s, 2H), 3.00-3.22 (m, 4H) ,2.46(t,2H,J＝6.2Hz),2.36(t,2H,J＝7.4Hz),1.56-1.68(m,2H),1.36-1.55(m,22H),1.18-1.33(m,8H).

Step 4: tert-Butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate

A solution of tert _- butyl N-{3-[benzyl({8-[(tert-butoxycarbonyl)amino]octyl})amino]propyl}carbamate (0.496 g, 1.01 mmol) and 10% Pd/C (0.429 g, 0.202 mmol) in ethanol (10 mL) was stirred under H2 balloon. The reaction was monitored by TLC. At 3 h, the reaction mixture was diluted with EtOAc (20 mL), filtered through a diatomaceous earth pad, and rinsed with EtOAc. The filtrate was concentrated, dissolved in EtOAc, and filtered using a 0.45 μm syringe filter. The filtered organic matter was concentrated to give tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.323 g, 0.805 mmol, 79.8%) as an off-white solid. UPLC/ELSD: RT=0.59 min. MS (ES): m/z of C ₂₁ H ₄₃ N ₃ O ₄ = 402.0 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.17 (br.s, 1H), 4.50 (br.s, 1H), 3.20 (dt, 2H, J = 6.0, 6.0Hz), 3.09 (dt, 2H, J = 6.5, 6. 4Hz), 2.67 (t, 2H, J = 6.6Hz), 2.58 (t, 2H, J = 7.1Hz), 1.89 (br.s, 1H), 1.59-1.74 (m, 2H), 1.37-1.55 (m, 22H), 1.21-1.37 (m, 8H).

Step 5: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate

Cholesteryl carbonate 4-nitrophenyl ester (0.300 g, 0.544 mmol), tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.240 g, 0.598 mmol) and triethylamine (0.12 mL, 0.85 mmol) were combined in CHCl ₃ (4.8 mL). The reaction mixture was stirred at 50 °C and monitored by TLC. At 20.25 h, tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (77 mg) and triethylamine (0.04 mL) were added. The reaction mixture was stirred at 60 °C. At 95 h, the reaction mixture was cooled to room temperature, diluted with DCM (20 mL), and washed with water (25 mL). The aqueous layer was extracted with DCM (2 x 20 mL). The combined organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-30% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.398 g, 0.489 mmol, 89.9%) as a clear oil. UPLC/ELSD: RT = 3.47 min. MS (ES): m/z of C ₄₉ H ₈₇ N ₃ O ₆ = 836.5 [M+Na] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.22-5.43 (m, 2H), 4.40-4.84 (m, 2H), 3.00-3.39 (br.m, 8H), 2.21-2.44 (m, 2H), 1. 73-2.07(br.m,5H),0.93-1.71(br.m,53H),1.02(s,3H),0.91(d,3H,J＝6.4Hz),0.86(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.67(s,3H).

Step 6: (8-aminooctyl)(3-aminopropyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.395 g, 0.485 mmol) in iPrOH (2.5 mL) was added 5-6N HCl in iPrOH (0.7 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17.5 h, the reaction mixture was cooled to room temperature. ACN (5 mL) was added, the suspension was stirred for 15 min, and the solid was collected by vacuum filtration, rinsed with 2:1 ACN:iPrOH to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(8-aminooctyl)(3-aminopropyl)carbamate dihydrochloride (0.249 g, 0.356 mmol, 73.4%) as a white solid. UPLC/ELSD: RT = 1.97 min. MS (ES): m/z of C ₃₉ H ₇₃ Cl ₂ N ₃ O ₂ = 614.4 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ8.00-8.64 (br.m, 6H), 5.33-5.44 (m, 1H), 4.39-4.56 (m, 1H), 2.93-3.54 (br.m, 8 H),2.20-2.43(m,2H),1.69-2.16(br.m,10H),0.93-1.66(br.m,30H),1.02(s,3H),0.91(d,3H,J＝6.3Hz),0.87(d,3H,J＝6.5Hz),0.86(d,3H,J＝6.5Hz),0 .68(s,3H).

H. Compound SA61: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-(3-aminopropoxy)butyl)(3-aminopropyl)carbamate dihydrochloride

Step 1: 3-(4-Hydroxybutoxy)propionitrile

Acrylonitrile (3.0 mL, 46 mmol) was added to a solution of 1,4-butanediol (8.0 mL, 91 mmol) and benzyltrimethylammonium hydroxide (0.20 mL, 1.3 mmol, 40 wt% in water). The reaction mixture was stirred at room temperature while covered with foil and monitored by TLC. At 2 h, the reaction mixture was diluted with water (150 mL) and extracted with 1:1 hexane/MTBE (50 mL) and EtOAc (3 x 50 mL). The combined organic matter was washed with water and brine, dried over MgSO ₄ , and concentrated. The crude material was purified via silica gel chromatography (50%-100% EtOAc in hexane) to obtain 3- (4-hydroxybutoxy) propionitrile (1.374 g, 9.596 mmol, 21.0%) as a yellow oil. UPLC/ELSD: RT = 0.20 min. ¹ H NMR (300MHz, CDCl ₃ ): δ3.67 (t, 2H, J = 6.0Hz), 3.66 (t, 2H, J = 6.4Hz), 3.50-3.57 (m, 2H), 2.60 (t, 2H, 6.4Hz), 1.60-1.76 (m, 5H).

Step 2: 4-(2-cyanoethoxy)butyl methanesulfonate

3- (4- hydroxybutoxy) propionitrile (1.00g, 6.98mmol) and triethylamine (1.5mL, 11mmol) in DCM (10mL) stirring solution is cooled to 0 DEG C in an ice bath, and then methanesulfonyl chloride (0.60mL, 7.8mmol) is added dropwise. The reaction is monitored by TLC. The reaction mixture is slowly warmed to room temperature. At 2h, the reaction mixture is cooled to 0 DEG C in an ice bath, and methanesulfonyl chloride (0.06mL) is added again. At 2h 10min, water (10mL) is added, and the reaction mixture is stirred at room temperature for 5min. Thereafter, 5% _NaHCO3 aqueous solution (50mL) is added, and then the reaction mixture is extracted with DCM (3x 30mL). The combined organic matter is washed with water and brine, dried over _MgSO4 , and concentrated to obtain methanesulfonic acid 4- (2- cyanoethoxy) butyl ester (1.556g, 7.032mmol, quantitative) in a yellow oily state. The material was carried forward to the next step without further ^{purification.1H} NMR (300 MHz, _CDCl3 ): δ 4.28 (t, 2H, J = 6.4 Hz), 3.64 (t, 2H, J = 6.2 Hz), 3.54 (t, 2H, J = 5.9 Hz), 3.01 (s, 3H), 2.59 (t, 2H, J = 6.2 Hz), 1.81-1.93 (m, 2H), 1.66-1.78 (m, 2H).

Step 3: tert-Butyl N-(3-{[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate

A solution of tert-butyl N-(3-aminopropyl)carbamate (4.272 g, 24.52 mmol), 4-(2-cyanoethoxy)butyl methanesulfonate (1.550 g, 7.005 mmol) and EtOH (16 mL) was stirred at 65 °C. The reaction was monitored by TLC. At 4 h, the reaction mixture was cooled to room temperature. At 21.5 h, the reaction mixture was concentrated and then dissolved in a mixture of EtOAc (75 mL) and water (75 mL). The layers were separated, and the aqueous solution was extracted with EtOAc (50 mL). The combined organic matter was washed with water (3x) and brine, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-20% (5% concentrated aqueous _NH4OH in MeOH) in DCM) to afford tert-butyl N-(3-{[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate (1.396 g, 4.662 mmol, 66.6%) as a yellow oil. UPLC/ELSD: RT = 0.22 min. MS (ES): m/z of C ₁₅ H ₂₉ N ₃ O ₃ = 243.8 [(M+H)-t-Bu] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.19 (br.s, 1H), 3.64 (t, 2H, J = 6.3Hz), 3.50 (t, 2H, J = 6.0Hz), 3.20 (dt, 2H, J = 6.2, 5.9Hz), 2.67 (t, 2H, J = 6.6Hz), 2.61 (t, 2H, J = 6.7Hz), 2.59 (t, 2H, J = 6.4Hz), 1.48-1.70 (m, 6H), 1.44 (s, 9H), 1.10 (br.s, 1H).

Step 4: tert-Butyl N-(3-{benzyl[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate

To a mixture of tert-butyl N-(3-{[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate (1.380 g, 4.609 mmol), potassium carbonate (1.274 g, 9.218 mmol) and potassium iodide (0.150 g, 0.904 mmol) in ACN (20 mL), benzyl bromide (0.63 mL, 5.3 mmol) was added. The reaction mixture was stirred at 65 ° C and monitored by TLC. At 2.5 h, the reaction mixture was cooled to room temperature and filtered through a celite pad rinsed with ACN, and the filtrate was concentrated. The residue was dissolved in 5% NaHCO aqueous solution (about ₅₀ mL), then extracted with MTBE (2x 25 mL) and EtOAc (25 mL). The combined organics were washed with brine, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (30%-70% EtOAc in hexanes) to give tert-butyl N-(3-{benzyl[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate (1.372 g, 3.522 mmol, 76.4%) as a yellow oil. UPLC/ELSD: RT = 0.29 min. MS (ES): m/z = 390.0 [M+H] ⁺ for C _{2 2} H _{3 5} N ₃ O ₃ . ¹ H NMR (300MHz, CDCl ₃ ): δ7.20-7.39 (m, 5H), 5.41 (br.s, 1H), 3.60 (t, 2H, J = 6.4Hz), 3.51 (s, 2H), 3.38-3.47 (m, 2H), 3.15 (dt, 2H, J = 5.8, 5.6Hz), 2.56 (t ,2H,J＝6.4Hz),2.47(t,2H,J＝6.3Hz),2.35-2.43(m,2H),1.51-1.69(m,6H),1.44(s,9H).

Step 5: tert-Butyl N-{3-[benzyl(4-{3-[(tert-butoxycarbonyl)amino]propoxy}butyl)amino]propyl}carbamate

To a stirred solution of tert-butyl N-(3-{benzyl[4-(2-cyanoethoxy)butyl]amino}propyl)carbamate (1.357 g, 3.484 mmol) in MeOH (23 mL) was added di-tert-butyl dicarbonate (1.901 g, 8.709 mmol) and nickel(II) chloride hexahydrate (0.083 g, 0.35 mmol). The reaction mixture was cooled to 0°C in an ice bath, and then _NaBH4 (0.923 g, 24.4 mmol) was added portionwise over 40 min (caution: vigorous gas evolution occurred during addition). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17.25 h, the reaction mixture was cooled to 0°C in an ice bath, and then _NaBH4 (500 mg) was added portionwise over 30 min. The reaction mixture was stirred at room temperature. At 18.5 h, the reaction mixture was cooled to 0°C in an ice bath, and then _NaBH4 (100 mg) was added. The reaction mixture was stirred at 0 ° C. At 19.5h, NaBH ₄ (101mg) was added. At 20.5h, NaBH ₄ (102mg) was added. At 21.5h, Boc ₂ O (850mg) and NaBH ₄ (103mg) were added. The reaction mixture was slowly warmed to room temperature. At 40.5h, diethylenetriamine (0.55mL, 5.1mmol) was added dropwise, and the reaction mixture was stirred at room temperature for 1h. Thereafter, the reaction mixture was concentrated, dissolved in 5% NaHCO ₃ aqueous solution, and extracted with DCM (3x). The biphasic mixture was concentrated to remove volatile organics, and the mixture was then extracted with MTBE (3x). The combined organics were washed with brine, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-7% (5% concentrated aqueous _NH4OH in MeOH) in DCM) to afford tert-butyl N-{3-[benzyl(4-{3-[(tert-butoxycarbonyl)amino]propoxy}butyl)amino]propyl}carbamate (0.836 g, 1.69 mmol, 48.6%) as a light yellow oil. UPLC/ELSD: RT = 0.65 min. MS (ES): m/z of C ₂₇ H ₄₇ N ₃ O ₅ = 494.5 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ7.19-7.38 (m, 5H), 5.43 (br.s, 1H), 3.51 (s, 2H), 3.43 (t, 2H, J = 5.9 Hz), 3.31-3.39 (m, 2H), 3.06-3.26 (m, 4H), 2.46 (t, 2H, J = 6.2Hz), 2.35-2.43 (m, 2H), 1.49-1.79 (br.m, 8H), 1.44 (s, 18H).

Step 6: tert-Butyl N-{3-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butoxy]propyl}carbamate

Under _H2 balloon, stir N-{3-[benzyl (4-{3-[(tert-butoxycarbonyl) amino] propoxy } butyl) amino] propyl } t-butyl carbamate (0.825g, 1.67mmol) and 10% Pd/C (0.711g, 0.334mmol) in EtOH (10mL) solution. Monitor the reaction by TLC. At 18h, dilute the reaction mixture with EtOAc (40mL) and then filter via the diatomaceous earth pad rinsed with EtOAc. The filtrate is concentrated, dissolved in EtOAc, and filtered using a 0.45 μm syringe filter. Concentrate the filtered organic matter, obtain N-{3-[4-({3-[(tert-butoxycarbonyl) amino] propyl } amino) butoxy] propyl } t-butyl carbamate (0.636g, 1.58mmol, 94.3%) as a yellow oil. UPLC/ELSD: RT=0.40min. MS (ES): m/z of C ₂₀ H ₄₁ N ₃ O ₅ = 404.5 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.20 (br.s, 1H), 4.91 (br.s, 1H), 3.47 (t, 2H, J = 5.9Hz), 3.41 (t, 2H, J = 6.1Hz), 3.13-3 .23(m,4H),2.67(t,2H,J＝6.6Hz),2.61(t,2H,J＝6.6Hz),1.48-1.80(br.m,9H),1.44(s,18H).

Step 7: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-(3-((tert-butoxycarbonyl)amino)propoxy)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate

A solution of 4-nitrophenyl cholesterol carbonate (0.663 g, 1.20 mmol), tert-butyl N-{3-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butoxy]propyl}carbamate (0.630 g, 1.56 mmol) and triethylamine (0.50 mL, 3.6 mmol) in PhMe (10 mL) was stirred at 90 °C. The reaction was monitored by LCMS. At 18 h, the reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in DCM (50 mL) and then washed with water (3 x 30 mL). The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (20%-60% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-(3-((tert-butoxycarbonyl)amino)propoxy)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate as a sticky white foam (0.825 g, 1.01 mmol, 84.2%). UPLC/ELSD: RT = 3.39 min. MS (ES): m/z of C ₄₈ H ₈₅ N ₃ O ₇ = 839.2 [M+Na] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.34-5.43(m,1H),5.30(br.s,1H),4.73-5.00(m,1H),4.41-4.59(m,1H),3.46(t,2H,J＝5.9Hz),3.41(t,2H,J＝5.9Hz),3.00-3.36(br.m,8H),2.20 -2.43(m,2H),0.93-2.09(br.m,34H),1.43(s,18H),1.02(s,3H),0.91(d,3H,J＝6.4Hz),0.86(d,6H,J＝6.5Hz),0.67(s,3H).

Step 8: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-(3-aminopropoxy)butyl)(3-aminopropyl)carbamate dihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-(3-((tert-butoxycarbonyl)amino)propoxy)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.809 g, 0.991 mmol) in iPrOH (6.0 mL) was added 5-6N HCl in iPrOH (1.4 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 15.5 h, the reaction mixture was cooled to room temperature. ACN (18 mL) was added to the reaction mixture, and the suspension was stirred at room temperature for 10 min. After this time, the solid was collected by vacuum filtration and rinsed with 3:1 ACN/iPrOH to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-(3-aminopropoxy)butyl)(3-aminopropyl)carbamate dihydrochloride (0.609 g, 0.828 mmol, 83.6%) as a white solid. UPLC/ELSD: RT = 2.00 min. MS (ES): m/z = 617.0 [M+H] ⁺ for C ₃₈ H ₆₉ N ₃ O ₃ ; ¹ H NMR (300 MHz, CDCl ₃ ): δ8.51-8.82(m,3H),8.05(br.s,3H),5.33-5.42(m,1H),4.42-4.57(m,1H),3.63(t,2H,J＝5.4Hz),2.97-3.58(br.m,10H),2.19-2.43(m,2H),0.93- 2.13 (br.m, 34H), 1.02 (s, 3H), 0.91 (d, 3H, J = 6.4Hz), 0.86 (d, 3H, J = 6.5Hz), 0.86 (d, 3H, J = 6.5Hz), 0.67 (s, 3H).

I. Compound SA62: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-methyl-3-oxopropanoate trihydrochloride

Step 1: 1-(tert-butyl) 2-methylmalonate 3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.36 g, 7.03 mmol) was added to a solution of cholesterol (1.85 g, 4.69 mmol) and 3-(tert-butoxy)-2-methyl-3-oxopropanoic acid (0.96 mL, 5.63 mmol) in dichloromethane (50 mL) stirred under nitrogen. The reaction mixture was then cooled to 0 ° C. and diisopropylethylamine (2.48 mL, 14.07 mmol) was added dropwise over 20 minutes. The resulting mixture was gradually warmed to room temperature and left overnight. The mixture was then diluted to 150 mL with dichloromethane, washed with water (1 x 70 mL), saturated sodium bicarbonate aqueous solution (2 x 70 mL) and brine (1 x 70 mL), dried over sodium sulfate, filtered, and concentrated in vacuo to give a yellow oil. The oil was dissolved in dichloromethane and purified on silica with a gradient of 0-25% ethyl acetate in hexanes to give 1-(tert-butyl)2-methylmalonate 3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)2-methylmalonate (1.62 g, 2.99 mmol, 63.7%) as an oil. UPLC/ELSD: RT: 3.41 _min . MS (ES): m/z (MH ⁺ ) 543.8 _{for C35H58O4} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.31 (m, 1H), 4.58 (br.m, 1H), 3.21 (q, 1H, J = 6Hz), 2.27 (d, 2H, J = 9Hz), 1.87 (br.m, 6H), 1.50 (br.m, 6H), 1.39 (s, 12H), 1.28 (br .m,12H),1.07(br.m,8H),0.95(s,4H),0.91(d,2H,J＝6Hz),0.86(d,4H,J＝6Hz),0.80(d,8H,J＝6Hz),0.61(s,3H).

Step 2: 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-methyl-3-oxopropanoic acid

A solution of 1-(tert-butyl) 2-methylmalonate 3-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (1.62 g, 2.99 mmol) in dichloromethane (50 mL) was cooled to 0°C. Trifluoroacetic acid (3.43 mL, 44.79 mmol) was added dropwise to the solution over 20 minutes. The reaction mixture was gradually warmed to room temperature for 5 hours, slowly turning light pink. The crude reaction mixture was concentrated in vacuo to a pink solid, dissolved in DCM, and purified on silica with a 0-40% ethyl acetate in hexanes gradient to afford 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-methyl-3-oxopropanoic acid (1.05 g, 2.16 mmol, 72.2%) _as a white solid. UPLC/ELSD: RT: 3.02 min. MS (ES): m/z (MH ⁺ ) 487.7 _{for C31H50O4} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 11.03 (br.s, 1H), 5.40 (br.d, 1H), 4.72 (br.m, 1H), 3.49 (q, 1H, J = 6Hz), 2.38 (d, 2H, J = 9Hz), 2.01 (br.m, 5H), 1.61 (br.m, 5H), 1. 50 (d, 5H, J = 6Hz), 1.27 (br.m, 12H), 1.04 (s, 5H), 0.95 (d, 4H, J = 6Hz), 0.90 (d, 6H, J = 6Hz), 0.70 (s, 3H).

Step 3: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2,16-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a stirred solution of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-methyl-3-oxopropanoic acid (0.50 g, 1.02 mmol) was added to a stirred solution of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-methyl-3-oxopropanoic acid (0.50 g, 1.02 mmol) under nitrogen atmosphere was added. To a solution in dichloromethane (10 mL) was added tert-butyl (3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.72 g, 1.42 mmol), dimethylaminopyridine (0.01 g, 0.10 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.30 g, 1.53 mmol). The resulting solution was cooled to 0 °C and diisopropylethylamine (0.54 mL, 3.05 mmol) was added dropwise. The mixture was gradually warmed to room temperature and allowed to remain overnight. The solution was then diluted with dichloromethane, washed with water (3 x 10 mL), dried over sodium sulfate, filtered, and concentrated to give an oil. The oil was dissolved in DCM and purified on silica with a gradient of 0-60% ethyl acetate in hexanes to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,16-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-oate (0.18 g, 0.19 mmol, 18.2%) as a light yellow oil. UPLC/ELSD: RT: 3.26 min. MS (ES): m/z (MH ⁺ ) 972.4 for C ₅₆ H ₉₈ N ₄ O ₉ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.36 (br.s, 1H), 4.58 (br.m, 1H), 4.10 (q, 1H, J = 6Hz), 3.36 (br.m, 13H), 2.26 (br.m, 3H), 2.01 (s, 4H), 1.80 (br.m, 10H), 1.43 (br .m,47H),1.23(t,4H,J＝9Hz),1.08(br.m,7H),0.97(s,8H),0.90(d,4H,J＝9Hz),0.84(d,6H,J＝6Hz),0.65(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-methyl-3-oxopropanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,16-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaheptadecan-17-olate (0.18 g, 0.19 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5.5 M in isopropanol, 0.37 mL, 1.85 mmol) dropwise. The mixture was heated to 45°C and stirred overnight. The solution was then cooled to room temperature and acetonitrile (5 mL) was added to the mixture. It was then sonicated to remove the precipitated solid from the side of the flask. After stirring for 30 minutes after sonication, the solid was filtered off by vacuum filtration, washed repeatedly with acetonitrile, and dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 3-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-methyl-3-oxopropanoate trihydrochloride (0.06 g, 0.07 mmol, 38.7%) as a white solid. UPLC/ELSD: RT: 1.70 min. MS (ES): m/z (MH ⁺ ) 672.1 for C ₄₁ H ₇₇ Cl ₃ N ₄ O ₃ . ¹ H NMR (300MHz, CD ₃ OD) δ: ppm 5.41 (s, 1H), 4.88 (br.m, 1H), 4.58 (br.m, 1H), 3.92 (br.m, 1H), 3.33 (s, 3H), 3.10 (br.m, 8H), 2.34 (br.m, 2H), 2.05(br.m,15H),1.54(br.m,8H),1.38(br.m,8H),1.17(d,9H,J＝6Hz),1.06(s,6H),0.97(d,4H,J＝6Hz),0.90(d,6H,J＝6Hz),0.73(s,3H).

J. Compound SA63: (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-(dimethylamino)propyl)amino)-4-oxobutanoate

Step 1: (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-ol

The mixture of magnesium chips (2.21g, 90.87mmol) and iodine (1.54g, 6.06mmol) was purged twice with vacuum and nitrogen and then maintained under nitrogen. Anhydrous tetrahydrofuran (50mL) was added to this mixture and stirred under nitrogen. 1-bromo-4-methylpentane (8.82mL, 60.58mmol) was added dropwise to this mixture over 10 minutes, and then the reaction was allowed to proceed at room temperature for 1 hour. Then, the reaction mixture was refluxed at 66°C for 3 hours, during which the gray reaction slurry became a clear colorless solution containing some undissolved magnesium. The reactant was then cooled to 0°C, and the solution became turbid again. At 0°C, a solution of pregnenolone (5.75g, 18.17mmol) in anhydrous tetrahydrofuran (25mL) was added dropwise over 1 hour, during which the reaction mixture solidified. The product of 4-nitro-1-yl pyridine-2 ... The resulting residue was dissolved in DCM and purified on silica with a 0-50% ethyl acetate in hexanes gradient to afford (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-ol (2.68 g, 6.64 mmol, 36.6%) as a white solid. UPLC/ELSD: RT: 2.13 min _. MS (ES): m/z (MH ⁺ ) 403.7 _{for C27H46O2} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.36 (br.d, 1H, J=6Hz), 3.54 (br.m, 1H), 2.29 (br.m, 2H), 2.06 (br.m, 2H), 1.85 (br.m, 16H), 1.29 (s, 6H), 1.17 (br.m, 6H), 1.03 ( s, 6H), 0.88 (d, 10H, J = 6Hz).

Step 2: 4-(((3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)butanoic acid

To a solution of (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-ol (0.50 g, 1.24 mmol) in dichloromethane (10 mL) stirred under nitrogen was added succinic anhydride (0.12 g, 1.24 mmol). Pyridine (0.17 mL, 1.24 mmol) was then added dropwise at room temperature and the mixture was refluxed at 40° C. overnight, at which point all solids became a solution. The next day, TLC revealed incomplete conversion, and dimethylaminopyridine (0.05 g, 0.41 mmol) and succinic anhydride (0.03 g, 0.25 mmol) were added and the reaction mixture was refluxed again overnight at 40° C. The next morning, the mixture was concentrated in vacuo to a yellow oil. The yellow oil was dissolved in dichloromethane and purified on silica with a 0-30% ethyl acetate in hexanes gradient to afford 4-(((3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)butanoic acid (0.27 g, 0.54 mmol, 43.3%) as a white solid. UPLC/ELSD: RT: 2.20 _min . MS (ES): m/z (MH ⁺ ) 503.8 _{for C31H50O5} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 6.60 (br.s, 1H), 5.38 (br.s, 1H), 4.64 (br.m, 1H), 4.13 (q, 1H, J = 6Hz), 2.66 (dd, 4H, J = 6Hz), 2.33 (d, 2H, J = 6Hz), 2.05 (br.m, 2H), 1.84(br.m,3H),1.51(br.m,12H),1.28(br.m,8H),1.13(br.m,5H),1.02(s,4H),0.86(d,10H,J=6Hz).

Step 3: (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-(dimethylamino)propyl)amino)-4-oxobutanoate

To a solution of 4-(((3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)butanoic acid (0.35 g, 0.68 mmol) in stirred dichloromethane (10 mL) under nitrogen was added tetramethyldipropylenetriamine (0.24 mL, 1.02 mmol), dimethylaminopyridine (0.01 g, 0.07 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.20 g, 1.02 mmol). The resulting solution was cooled to 0 ° C, and diisopropylethylamine (0.36 mL, 2.04 mmol) was added dropwise. The mixture was gradually warmed to room temperature and left overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution (1 x 10 mL) and brine (1 x 10 mL), dried over sodium sulfate, filtered, and concentrated to give an oil. The oil was dissolved in DCM and purified on silica with a 0-60% (80:19:1 DCM/MeOH/ _NH4OH ) gradient in DCM to afford (3S,8S,9S,10R,13S,14S,17S)-17-(2-hydroxy-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-(dimethylamino)propyl)amino)-4-oxobutanoate as a light yellow oil (0.07 g, 0.09 mmol, 13.6%). UPLC/ELSD: RT: 1.25 min. MS (ES): m/z (MH ⁺ ) 673.0 for C ₄₁ H ₇₃ N ₃ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.37 (br.s, 1H), 4.64 (br.m, 1H), 3.35 (br.t, 4H, J = 9Hz), 2.64 (s, 4H), 2.28 (br.m, 6H), 2.22 (s, 12H), 1.83 (br.m, 4H), 1.60 (br. m,15H),1.28(br.s,7H),1.13(br.m,5H),1.02(s,4H),0.89(d,9H,J=6Hz).

K. Compound SA64: (8-(dimethylamino)octyl)(3-(dimethylamino)propyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)

At room temperature to (8-aminooctyl) (3-aminopropyl) carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R) -10,13- dimethyl -17- ((R) -6- methyl hept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetrahydro -1H- cyclopenta [a] phenanthrene -3- base ester (105mg, 0.15mmol) and sodium acetate trihydrate (208mg, 1.53mmol) in 6mL methanol solution add formaldehyde (0.12mL, 37wt% in water, 1.53mmol) and sodium cyanoborohydride (96.1mg, 1.53mmol).The solution was stirred at room temperature for 16 hours, after which no starting aminosterol remained according to LCMS.The mixture was diluted with 2M NaOH aqueous solution and extracted three times with DCM. The organics were combined, washed once with brine, dried ( _MgSO4 ), filtered, and concentrated. The residue was purified by silica gel chromatography (0-50% (mixture of 1% concentrated aqueous _NH4OH and 20% MeOH in DCM) in DCM) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (8-(dimethylamino)octyl)(3-(dimethylamino)propyl)carbamate (63.2 mg, 0.091 mmol, 60%). UPLC/ELSD: RT = 2.14 min. MS (ES): m/z (MH ⁺ ) 671.2 for C ₄₃ H ₈₀ N ₃ O ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.35 (d, 1H, J = 5Hz); 4.48 (septet, 1H, J = 5Hz); 3.19 (s, 4H); 2.39-2.27 (m, 12H); 2.25 (s, 6H); 2.22 (s, 6H); 2.03-1.63 (m, 8H); 1 .58-1.04 (m, 23H); 1.00 (s, 6H); 0.90 (d, 3H, J = 6Hz); 0.86 (d, 3H, J = 1Hz); 0.84 (d, 3H, J = 1Hz); 0.66 (s, 3H).

L. Compound SA65: N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)glycine ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester tetrahydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloroacetate

Cholesterol (2 g, 5.17 mmol), chloroacetic acid (573 mg, 5.69 mmol), DMAP (63 mg, 0.52 mmol) and DCC (1.17 g, 5.69 mmol) were dissolved in 10 mL DCM. The solution was stirred at room temperature for 17 hours. The mixture was filtered and the filtrate was washed with ethyl acetate. The filtered solution was concentrated and dissolved in ethyl acetate. The organic layer was washed once with water and brine, dried (MgSO ₄ ), filtered, and concentrated. The residue was purified by silica gel chromatography (0-40% ethyl acetate in hexanes) to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloroacetate (0.71 g, 1.53 mmol, 30%) as a white solid. UPLC/ELSD: RT = 3.43 min. ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.40 (d, 1H, J = 5Hz); 4.48 (septet, 1H, J = 4Hz); 4.03 (s, 2H); 2.36 (d, 2H, J = 8Hz); 2.06-1.77 (m, 5H); 1.64-1.05 (m, 21H); 1.02 (s , 3H); 0.91 (d, 3H, J = 6Hz); 0.88 (s, 3H); 0.86 (s, 3H); 0.68 (s, 3H).

Step 2: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

At room temperature, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloroacetate (350 mg, 0.75 mmol) and NaI (1 To a solution of tert-butyl N-{3-[(tert-butoxycarbonyl)amino]propyl}-N-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butyl]carbamate (378 mg, 0.75 mmol) and N,N-diisopropylethylamine (0.2 mL, 1.13 mmol) in 7.5 mL acetonitrile was added. The solution was stirred at 60 °C for 18 hours. The mixture was diluted with ethyl acetate, washed once with water and brine, dried over MgSO ₄ , filtered, and concentrated. The residue was purified by silica gel chromatography (0-100% (1% _NH4OH , 20% MeOH in DCM) in DCM) to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oate as a colorless oil (590 mg, 0.63 mmol, 84%). UPLC/ELSD: RT=2.94min. MS (ES): m/z (MH ⁺ ) 930.0 for C ₅₄ H ₉₇ N ₄ O ₈ . ^1. ₃₁ (d, 2H, J = 8Hz); 2.06-1.03 (m, 64H); 1.00 (s, 3H); 0.91 (d, 3H, J = 6Hz); 0.87 (s, 3H); 0.85 (s, 3H); 0.67 (s, 3H).

Step 3: N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester tetrahydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oate (590 mg, 0.64 mmol) in isopropanol (15 mL) was added a 5 M HCl solution in isopropanol (15 mL, 6.4 mmol). The solution was stirred at 40 °C for 41 hours. The mixture was cooled to room temperature and diluted with acetonitrile (15 mL). The resulting solid was precipitated by centrifugation (5000 g, 5 min). The supernatant was removed and the pellet was dried under vacuum to give N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6 _- methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (350 mg, 0.45 mmol, 71%) _as a _white powder. UPLC/ELSD: RT = 1.83 min. MS (ES): m/z ([M-3HCl-Cl ^- ] ⁺ ) 629.6 for _C39H73N4O2 . ¹ H NMR (300MHz, CD ₃ OD) δ: ppm 5.43 (d, 1H, J = 4Hz); 4.80-4.66 (m, 1H); 4.28 (s, 2H); 3.49-3.33 (m, 4H); 3.22-3.02 (m, 8H); 2.43 (d, 2H, J = 7Hz); 2.28-1.0 9 (m, 29H); 1.06 (s, 3H); 0.95 (d, 3H, J = 6Hz); 0.89 (s, 3H); 0.87 (s, 3H); 0.73 (s, 3H).

M. Compound SA66: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-oxoacetate trihydrochloride

Step 1: 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-oxoacetic acid

To a stirred solution of cholesterol (0.500 g, 1.29 mmol) in a mixture of _Et2O (6.5 mL) and DCM (2.0 mL) cooled to 0°C in an ice bath was slowly added oxalyl chloride (0.23 mL, 2.7 mmol). The reaction mixture was slowly warmed to room temperature and monitored by TLC. At 24 h, the reaction mixture was cooled to 0°C in an ice bath, and then water (3.0 mL) was added dropwise (caution: vigorous gas evolution occurred during addition). The mixture was stirred at room temperature for 1 h, and then the layers were separated. The aqueous layer was extracted with _Et2O (3x). The combined organics were washed with _brine , dried over _Na2SO4 , and concentrated to give 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-oxoacetic acid (0.534 g, 1.16 mmol, 90.0%) as a white solid. UPLC/ELSD: RT = 2.95 min. ¹ H NMR (300MHz, CDCl ₃ ): δ5.68(br.s,1H),5.38-5.46(m,1H),4.75-4.89(m,1H),2.35-2.61(m,2H),1.70-2.11(br.m,6H),0.93-1.65(br.m,20H),1.04( s, 3H), 0.92 (d, 3H, J = 6.5Hz), 0.87 (d, 3H, J = 6.6Hz), 0.86 (d, 3H, J = 6.5Hz), 0.68 (s, 3H).

Step 2: 2-Chloro-2-oxoacetate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a solution of 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-oxoacetic acid (0.100 g, 0.218 mmol) and DMF (catalyst) in DCM (2 mL) was slowly added dropwise oxalyl chloride (0.03 mL, 0.4 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 40 min, the reaction mixture was concentrated and then re-concentrated from PhMe to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloro-2-oxoacetate as a yellow solid. The material was carried forward without further purification, assuming quantitative yield.

Step 3: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazahexadecane-16-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 2-chloro-2-oxoacetate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.104 g, 0.218 mmol) was added. ) and triethylamine (0.10 mL, 0.71 mmol) in toluene (2.0 mL) cooled to 0 ° C in an ice bath was added dropwise tert-butyl N-{3-[(tert-butoxycarbonyl)amino]propyl}-N-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butyl]carbamate (0.150 g, 0.298 mmol) in toluene (0.75 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 30 min, the reaction mixture was stirred at 50 ° C. At 17 h, the reaction mixture was cooled to room temperature and then concentrated. The residue was dissolved in DCM and washed with 5% NaHCO ₃ aqueous solution. The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazahexadecane-16-oate (0.138 g, 0.146 mmol, 67.1%) as a yellow oil. UPLC/ELSD: RT = 3.33 min. MS (ES): m/z of C ₅₄ H ₉₄ N ₄ O ₉ = 844.4 [(M+H)-(CH ₃ ) ₂ C = CH ₂ -CO ₂ ] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.38-5.45(m,1H),5.21(br.s,1H),4.65-4.87(m,2H),3.32-3.47(m,2H),3.02-3.31(br.m,10H),2.35-2.53(m,2H),0.94-2.08(br.m,34H),1.46 (s,9H),1.44(s,18H),1.02(s,3H),0.92(d,3H,J＝6.4Hz),0.87(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.5Hz),0.68(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-oxoacetate trihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazahexadecane-16-oate (0.132 g, 0.140 mmol) in iPrOH (1.3 mL) was added 5-6 N HCl in iPrOH (0.28 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 18h, the reaction mixture was cooled to room temperature. ACN (3mL) was added to the reaction mixture, and the suspension was stirred at room temperature for 1h. After that, solids were collected by vacuum filtration to obtain 2- ((3-aminopropyl) (4- ((3-aminopropyl) amino) butyl) amino) -2- oxoacetic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R) -10, 13- dimethyl -17- ((R) -6- methyl hept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetrahydro -1H- cyclopenta [a] phenanthrene -3- base ester trihydrochloride (0.085g, 0.10mmol, 73.2%) as a white solid. UPLC/ELSD: RT = 1.70min. MS (ES): m/z of C ₃₉ H ₇₀ N ₄ O ₃ = 643.8 [M+H] ⁺ ; ¹ H NMR (300MHz, CD ₃ OD): δ5.42-5.51 (m, 1H), 4.72-4.85 (m, 1H), 3.34-3.61 (br.m, 4H), 3.04-3.19 (br.m, 6H) ,2.92-3.01(m,2H),2.37-2.54(m,2H),0.98-2.19(br.m,34H),1.08(s,3H) ,0.96(d,3H,J＝6.4Hz),0.89(d,3H,J＝6.6Hz),0.89(d,3H,J＝6.6Hz),0.74(s ,3H).

N. Compound SA67: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-(3-(dimethylamino)propoxy)butyl)(3-(dimethylamino)propyl)carbamate

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-(3-aminopropoxy)butyl)(3-aminopropyl)carbamate (100 mg, 0.15 mmol) and sodium acetate trihydrate (197.5 mg, 1.45 mmol) in 5.8 mL of methanol at room temperature was added formaldehyde (0.11 mL, 37 wt% in water, 1.45 mmol) and sodium cyanoborohydride (91.2 mg, 1.45 mmol). The solution was stirred at room temperature for 6 hours after which no starting aminosterol remained according to LCMS. The mixture was diluted with 2M NaOH aqueous solution and extracted three times with DCM. The organics were combined, washed once with brine, dried ( _MgSO ), filtered, and concentrated. The residue was purified by silica gel chromatography (0-20% (1% NH ₄ OH and 20% MeOH in DCM) in DCM) to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10, 13-dimethyl-17-((R)-6-methylhept-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (35.6 mg, 0.051 mmol, 35%) of (4-(3-(dimethylamino)propoxy)butyl)(3-(dimethylamino)propyl)carbamate as a colorless oil. UPLC/ELSD: RT=2.01min. MS (ES): m/z (MH ⁺ ) 673.0 for C ₄₂ H ₇₈ N ₃ O ₃ . ² _.04-1.66 (m,10H); 1.64-1.04(m,15H); 1.01(s,6H); 0.91(d,3H,J＝6Hz); 0.87(d,3H,J＝1Hz); 0.85(d,3H,J＝1Hz); 0.67(s,3H).

O. Compound SA68: (3-(dimethylamino)propyl)(4-((3-(dimethylamino)propyl)(methyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-aminopropyl)(4-((3-aminopropyl)amino)butyl)carbamate trihydrochloride (144 mg, 0.2 mmol) and sodium acetate trihydrate (162.3 mg, 1.19 mmol) in 2 mL of methanol at room temperature was added formaldehyde (0.094 mL, 37 wt% in water, 1.19 mmol) and sodium cyanoborohydride (75 mg, 1.19 mmol). The solution was stirred at room temperature for 17 hours after which no starting aminosterol remained according to LCMS. The mixture was diluted with 2M NaOH aqueous solution and extracted three times with DCM. The organics were combined, washed once with brine, dried (MgSO ₄ ), filtered and concentrated. The residue was purified by silica gel chromatography (0-20% (mixture of 2% concentrated NH ₄ OH aqueous solution and 20% MeOH in DCM) in DCM) to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10, 13-dimethyl-17-((R)-6-methylhept-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (31.7 mg, 0.044 mmol, 22%) as a colorless oil. UPLC/ELSD: RT = 1.71 min. MS (ES): m/z (MH ⁺ ) for C ₄₃ H ₈₁ N ₄ O ₂ 685.6. ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.36 (d, 1H, J = 5Hz); 4.49 (sevent, 1H, J = 5Hz); 3.47 (s, 4H); 3.22 (s, 4H); 2.38-2.24 (m, 12H); 2.22 (s, 6H); 2.21 (s, 6H); 2.20 (s, 3H); 2.05-1.95 (m, 2H); 1.72-1.06 (m, 23H); 1.01 (s, 6H); 0.91 (d, 3H, J = 6Hz); 0.87 (d, 3H, J = 1Hz); 0.85 (d, 3H, J = 1Hz); 0.67 (s, 3H).

P. Compound SA69: (8-aminooctyl)(3-aminopropyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate

4-Nitrophenyl β-sitosterol carbonate (0.300 g, 0.517 mmol), tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.260 g, 0.647 mmol), and triethylamine (0.22 mL, 1.6 mmol) were combined in PhMe (4.5 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 18.25 h, the reaction mixture was cooled to room temperature and concentrated. The residue was dissolved in DCM (20 mL) and washed with water (3x). The organic layer was passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.327 g, 0.388 mmol, 75.0%). UPLC/ELSD: RT = 3.74 min. MS (ES): m/z of C ₅₁ H ₉₁ N ₃ O ₆ = 842.9 [M+H] ⁺ ; ¹ HNMR (300MHz, CDCl ₃ ): δ 5.15-5.47 (m, 2H), 4.40-4.86 (m, 2H), 2.98-3.41 (br.m, 8H). 2.20-2.45(m,2H),1.76-2.12(br.m,5H),0.89-1.75(br.m,54H),1.02(s,3H),0.92(d,3H,J=6.4Hz),0.77-0.88(m,9H),0.68(s,3H).

Step 2: (8-aminooctyl)(3-aminopropyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.315 g, 0.374 mmol) in iPrOH (4.0 mL) was added 5-6N HCl in iPrOH (0.53 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 18 h, the reaction mixture was cooled to room temperature and ACN (12 mL) was added. The solid was collected via vacuum filtration and rinsed with 3:1 ACN/iPrOH to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(8-aminooctyl)(3-aminopropyl)carbamate dihydrochloride (0.236 g, 0.309 mmol, 82.5%) as a white solid. UPLC/ELSD: RT = 3.54 min. MS (ES): m/z of C ₄₁ H ₇₇ Cl ₂ N ₃ O ₂ = 342.6 [M+2Na] ²⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ8.33 (br.s, 3H), 8.22 (br.s, 3H), 5.31-5.42 (m, 1H), 4.38-4.53 (m, 1H), 2.92 -3.53(br.m,8H),2.20-2.42(m,2H),1.72-2.17(br.m,10H),0.94-1.71(br.m,31H),1.02(s,3H),0.92(d,3H,J＝6.3Hz),0.77-0.89(m,9H),0.68(s,3H) .

Q. Compound SA70

SA70 (3-aminopropyl)(4-((3-aminopropyl)amino)butyl)carbamate ((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester analogue from hydroxycholesterol olefin elimination byproduct)

R. Compound SA71: N-(8-aminooctyl)-N-(3-aminopropyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: N-(8-((tert-Butoxycarbonyl)amino)octyl)-N-(3-((tert-Butoxycarbonyl)amino)propyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

Cholesteryl chloroacetate (0.227 g, 0.490 mmol), tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.236 g, 0.589 mmol), potassium carbonate (0.136 g, 0.980 mmol), and potassium iodide (0.008 g, 0.05 mmol) were combined in THF (3.5 mL). The reaction mixture was stirred at 65 °C and monitored by LCMS. At 4 h, the reaction mixture was stirred at 60 °C. At 93 h, the reaction mixture was cooled to room temperature. The reaction mixture was concentrated and then dissolved in DCM. The organics were washed with water, passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (20%-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl N-(8-((tert-butoxycarbonyl)amino)octyl)-N-(3-((tert-butoxycarbonyl)amino)propyl)glycine as a clear oil (0.318 g, 0.384 mmol, 78.3%). UPLC/ELSD: RT = 3.62 min. MS (ES): m/z of C ₅₀ H ₈₉ N ₃ O ₆ = 829.0 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.45 (br.s, 1H), 5.35-5.41 (m, 1H), 4.58-4.72 (m, 1H), 4.50 (br.s, 1H), 3.25 (s, 2H) ,3.20(dt,2H,J＝5.7，6.0Hz),3.09(dt,2H,J＝6.4，5.8Hz),2.59(t,2H,J＝6.4Hz),2.50(t ,2H,J＝7.5Hz),2.28-2.36(m,2H),1.75-2.08(br.m,5H),0.94-1.70(br.m,53H),1.02(s,3H),0.91(d,3H,J＝6.5Hz),0.87(d,3H,J＝6.5Hz),0.86(d,3H ,J＝6.6Hz),0.68(s,3H).

Step 2: N-(8-aminooctyl)-N-(3-aminopropyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl N-(8-((tert-butoxycarbonyl)amino)octyl)-N-(3-((tert-butoxycarbonyl)amino)propyl)glycine (0.310 g, 0.374 mmol) in iPrOH (4.0 mL) was added 5-6N HCl in iPrOH (0.53 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 21.75 h, the reaction mixture was cooled to room temperature and ACN (12 mL) was added. The solid was collected via vacuum filtration and rinsed with 3:1 ACN/iPrOH to give N-(8-aminooctyl)-N-(3-aminopropyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.208 g, 0.248 mmol, 66.4%) as a white solid. UPLC/ELSD: RT = 1.83 min. MS (ES): m/z for C ₄₀ H ₇₃ N ₃ O ₂ = 335.4 [M+2Na] ²⁺ ; ¹ H NMR (300 MHz, CDCl ₃ ): δ10.72(br.s,1H),8.41(br.s,3H),8.27(br.s,3H),5.38-5.48(m,1H),4.59-4.82(m,1H),2.91-4.42(br.m,10H),2.22-2.72(br.m,4H),1.72-2.18( br.m,10H),0.93-1.70(br.m,28H),1.01(s,3H),0.91(d,3H,J＝5.5Hz),0.86(d,6H,J＝6.5Hz),0.67(s,3H).

S. Compound SA72: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((8-aminooctyl)(3-aminopropyl)amino)-4-oxobutanoate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-4-oxobutanoate

To a stirred solution of (-)-cholesterol NHS succinate (0.300 g, 0.514 mmol) in THF (3.0 mL) was added tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.258 g, 0.642 mmol) in THF (1.0 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 h, the reaction mixture was stirred at 50 °C. At 23 h, the reaction mixture was cooled to room temperature and then concentrated. The residue was dissolved in DCM and washed with water. The organics were passed through a _hydrophobic frit, dried over _Na2S04 , and concentrated. The crude material was purified via silica gel chromatography (20%-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-4-oxobutanoate as a white foam (0.315 g, 0.362 mmol, 70.4%). UPLC/ELSD: RT = 3.96 min. MS(ES)：C ₅₂ H ₉₁ N ₃ O ₇的m/z＝871.0[M+H] ⁺ ； ¹ H NMR(300MHz,CDCl ₃ )：δ5.27-5.46(m,2H),4.39-4.76(m,2H),2.95-3.48(br.m,8H),2.53-2.72(m,4H),2.24-2.39(m,2H),1.75-2.06(br.m,5H),0.93-1.70(br.m,53H),1.01(s,3H),0.91(d,3H,J＝6.4Hz),0.86(d,3H,J＝6.5Hz),0.86(d,3H,J＝6.6Hz),0.67(s,3H)。

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((8-aminooctyl)(3-aminopropyl)amino)-4-oxobutanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-4-oxobutanoate (0.307 g, 0.347 mmol) in iPrOH (4 mL) was added 5-6N HCl in iPrOH (0.49 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 21.75 h, the reaction mixture was cooled to room temperature and then ACN (16 mL) was added. The solids were collected via vacuum filtration and rinsed with 4:1 ACN/iPrOH to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((8-aminooctyl)(3-aminopropyl)amino)-4-oxobutanoate dihydrochloride (0.169 g, 0.214 mmol, 61.8%) as a white solid. UPLC/ELSD: RT = 2.15 min. MS (ES): m/z of C ₄₂ H ₇₅ N ₃ O ₃ = 336.0 [M+2H] ²⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ8.01-8.61 (m, 6H), 5.31-5.42 (m, 1H), 4.51-4.69 (m, 1H), 2.92-3.68 (br.m, 8H) ,2.62(s,4H),2.21-2.39(m,2H),1.71-2.20(br.m,10H),0.94-1.70(br.m, 30H),1.01(s,3H),0.91(d,3H,J＝6.4Hz),0.86(d,6H,J＝6.5Hz),0.67(s,3H) .

T. Compound SA73: N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)alanine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oic acid

To a solution of tert-butyl (3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.83 g, 1.65 mmol) and potassium hydroxide (0.37 g, 6.60 mmol) in methanol (10 mL) stirred under nitrogen was added 2-bromopropionic acid (0.30 mL, 3.30 mmol) dropwise at room temperature. The resulting solution was heated to 60° C. overnight. The next day, the solution was concentrated to an oil. The oil was dissolved in dichloromethane and purified on silica with a gradient of 0-60% ethyl acetate in hexanes to give 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oic acid (0.13 g, 0.22 mmol, 13.2%) as an oil. UPLC/ELSD: RT: _2.73 min _. MS (ES): m/z (MH ⁺ ) 575.8 _{for C28H54N4O8} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 7.31 (br.s, 1H), 5.72 (br.s, 1H), 3.17 (br.m, 13H), 1.90 (br.m, 2H), 1.64 (br.m, 7H), 1.40 (s, 26H).

Step 2: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a solution of cholesterol (0.10 g, 0.26 mmol) and 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oic acid (0.13 g, 0.22 mmol) in dichloromethane (10 mL) stirred under nitrogen was added dimethylaminopyridine (0.01 g, 0.04 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.06 g, 0.33 mmol). The resulting solution was cooled to 0°C and diisopropylethylamine (0.12 mL, 0.65 mmol) was added dropwise. The mixture was gradually warmed to room temperature and allowed to proceed overnight. The solution was then diluted with dichloromethane, washed with water (1 x 10 mL), saturated aqueous sodium bicarbonate (1 x 10 mL), and brine (1 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica with a gradient of 0-25% ethyl acetate in hexanes to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oate as a colorless oil (0.05 g, 0.05 mmol, 21.9%). UPLC/ELSD: RT: 2.83 min. MS (ES): m/z (MH ⁺ ) 944.4 for C ₅₅ H ₉₈ N ₄ O _8. The compound was not analyzed by H-NMR in order to avoid loss of previous material required for subsequent reaction.

Step 3: N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)alanine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oate (0.05 g, 0.05 mmol) in 2-propanol (5 mL) stirred under nitrogen was added hydrochloric acid (5.5 M in 2-propanol, 0.10 mL, 0.48 mmol) dropwise. The mixture was heated to 45° C. and stirred overnight. Then, the solution is cooled to room temperature, and acetonitrile (3mL) is added to the mixture. Then it is sonicated to remove the precipitated solid from the flask side. After stirring for 30 minutes after sonication, solid is filtered out by vacuum filtration, repeatedly washed with acetonitrile, and dried in a vacuum to obtain N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)alanine (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester trihydrochloride (0.03g, 0.03mmol, 62.8%) as a white solid. UPLC/ELSD: RT: 1.51min. MS (ES): m/z (MH ⁺ ) 644.1 for C ₄₀ H ₇₇ Cl ₃ N ₄ O ₂ . ¹ H NMR (300MHz, CD ₃ OD) δ: ppm 5.54(br.s,1H),4.50(br.m,1H),3.33(br.d,8H),3.12(br.m,9H),2.45(br.m,2H),2.00(br.m,15H),1.55(br.m,17H),1.19(br .m,14H),0.96(d,4H,J＝6Hz),0.90(d,7H,J＝6Hz),0.74(s,3H).

U. Compound SA74: (4-aminobutan-2-yl)(4-((4-aminobutan-2-yl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (3-((4-nitrophenyl)sulfonamido)butyl)carbamate

To a solution of tert-butyl (3-aminobutyl)carbamate (1.00 g, 5.31 mmol) in anhydrous DCM (15 mL) stirred under nitrogen was added triethylamine (0.89 mL, 6.37 mmol). The solution was cooled to 0°C and then a solution of 4-nitrobenzenesulfonyl chloride (1.30 g, 5.84 mmol) in 5 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with another 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate solution (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid solution (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (3-((4-nitrophenyl)sulfonamido)butyl)carbamate (1.95 _g , 5.22 mmol, 98.3%) as a white solid. UPLC/ELSD: RT = 0.54 min _. MS (ES): m/z (MH ⁺ ) _{for Ci5H23N3O6S} 374.4. ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 8.07(m,1H),7.78(m,1H),7.68(m,1H),5.23(m,1H),4.81(br.s,1H),3.52(m,1H),3.19(m,1H),3.05(m,1H),1.63(m,2H) ,1.37(s,9H),0.98(d,3H,J=6Hz).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-3,1-diyl))dicarbamate

To a solution of tert-butyl (3-((4-nitrophenyl)sulfonamido)butyl)carbamate (1.95 g, 5.22 mmol) in anhydrous DMF (20 mL) stirred under nitrogen was added potassium carbonate (2.10 g, 15.17 mmol) and 1,4-diiodobutane (0.33 mL, 2.49 mmol). The solution was heated to 40 °C overnight. The next morning, benzyl bromide (0.25 mL, 2.06 mmol) was added and the reaction was allowed to proceed at room temperature for 24 h. Then, thiophenol (0.98 mL, 9.57 mmol), potassium carbonate (1.03 g, 7.46 mmol) and an additional 5 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight. The next morning, the salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to give an oil, which was dissolved in 40 mL DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-60% (70:20:10 DCM/MeOH/concentrated aqueous ammonium hydroxide solution) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-3,1-diyl))dicarbamate (0.76 g, 1.77 mmol, 71.0%) as a colorless oil. UPLC/ELSD: RT=0.42 min. MS (ES): m/z (MH ⁺ ) 431.6 for C ₂₂ H ₄₆ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.47 (m, 2H), 3.24 (br.m, 4H), 2.74 (br.m, 4H), 2.55 (m, 2H), 1.53 (m, 10H), 1.44 (s, 18H), 1.09 (d, 6H, J = 6Hz).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)amino)butan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-3,1-diyl))dicarbamate (0.49 g, 1.15 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.48 mL, 3.43 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.63 g, 1.15 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, and the solution was washed with water (3 x 10 mL), dried over sodium sulfate, filtered, and concentrated to give an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (80:19:1 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)amino)butan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate as a colorless _oil (0.78 g, 0.92 mmol, 80.6%). UPLC/ELSD: RT = 2.62 _{min .} MS (ES): m/z (MH ⁺ ) 844.3 for _C50H90N4O6 . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.28(m,1H),3.14(br.m,5H),2.59(m,4H),2.25(m,3H),1.90(br.m,7H),1.46(br.m,22H),1.34(s,23H),1.09(br.m,28H),0. 83 (d, 5H, J = 6Hz), 0.79 (d, 7H, J = 6Hz), 0.59 (s, 3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-aminobutan-2-yl)(4-((4-aminobutan-2-yl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)amino)butan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate (0.78 g, 0.92 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 1.85 mL, 9.23 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give SA74 (4-aminobutan-2-yl)(4-((4-aminobutan-2-yl)amino)butyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.57 g, 0.73 mmol, 78.8%) as a white solid. UPLC/ELSD: RT = 1.67 min. MS (ES): m/z (MH ⁺ ) 753.4 for C ₄₀ H ₇₇ Cl ₃ N ₄ O ₂ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.42(m,1H),4.49(br.m,1H),4.12(br.m,1H),3.45(br.m,1H),3.33(s,2H),3.24(br.m,2H),3.13(br.m,4H),2.90(br.m,2H),2. 41(d,2H,J＝3Hz),2.32(br.m,1H),1.93(br.m,19H),1.43(d,6H,J＝6Hz),1.31 (d,5H,J＝6Hz),1.08(br.m,12H),0.97(d,4H,J＝6Hz),0.90(d,6H,J＝6Hz),0.75 (s,3H).

V. Compound SA75: (3-amino-2-methylpropyl)(4-((3-amino-2-methylpropyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (2-methyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate

To a solution of tert-butyl (3-amino-2-methylpropyl)carbamate (1.00 g, 5.31 mmol) in anhydrous DCM (15 mL) stirred under nitrogen was added triethylamine (0.89 mL, 6.37 mmol). The solution was cooled to 0°C and then a solution of 4-nitrobenzenesulfonyl chloride (1.30 g, 5.84 mmol) in 5 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with another 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate solution (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid solution (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (2-methyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate (2.20 _g , 5.90 mmol, quantitative) as a white solid. UPLC/ELSD: RT = 0.58 min _. MS (ES): m/z (MH ⁺ ) 374.4 _{for Ci5H23N3O6S} . ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 8.12(m,1H),7.84(m,1H),7.74(m,1H),6.20(br.s,1H),4.82(br.s,1H),3.19(m,1H),3.04(m,3H),1.84(m,1H),1.41(s,9H) ,0.91(d,3H,J=6Hz).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylpropane-3,1-diyl))dicarbamate

To a solution of tert-butyl (2-methyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate (2.20 g, 5.90 mmol) in anhydrous DMF (20 mL) stirred under nitrogen was added potassium carbonate (2.37 g, 17.14 mmol) and 1,4-diiodobutane (0.37 mL, 2.81 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.28 mL, 2.33 mmol) was added and the reaction was allowed to proceed at room temperature for 24 h. Then, thiophenol (1.11 mL, 10.82 mmol), potassium carbonate (1.17 g, 8.43 mmol) and an additional 5 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a gradient of 0-60% (70:20:10 DCM/MeOH/concentrated aqueous ammonium hydroxide solution). The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylpropane-3,1-diyl))dicarbamate (0.85 g, 1.97 mmol, 70.0%) as a colorless oil. UPLC/ELSD: RT=0.43 min. MS (ES): m/z (MH ⁺ ) 431.6 for C ₂₂ H ₄₆ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.76 (m, 1H), 2.93 (m, 2H), 2.73 (m, 2H), 2.28 (m, 8H), 1.56 (m, 4H), 1.28 (s, 4H), 1.18 (s, 17H), 0.66 (d, 6H, J = 6Hz).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-((tert-butoxycarbonyl)amino)-2-methylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-methylpropyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylpropane-3,1-diyl))dicarbamate (1.06 g, 2.45 mmol) in anhydrous toluene (20 mL) stirred under nitrogen was added triethylamine (0.86 mL, 6.13 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (1.13 g, 2.04 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, and the solution was washed with water (3 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (80:19:1 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2-methylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-methylpropyl)amino)butyl)carbamate as a colorless oil (1.08 g, 1.28 mmol, 62.8%). UPLC/ELSD: RT = 2.52 _{min .} MS (ES): m/z (MH ⁺ ) 844.3 _{for C50H90N4O6} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.29(m,1H),4.42(br.m,1H),3.07(br.m,5H),2.87(m,3H),2.51(m,4H),2.25(br.m,2H),1.79(br.m,7H),1.46(m,8H),1.34 (s,18H),1.05(br.m,10H),0.94(s,5H),0.82(m,14H),0.59(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-amino-2-methylpropyl)(4-((3-amino-2-methylpropyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2-methylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-methylpropyl)amino)butyl)carbamate (1.08 g, 1.28 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 2.57 mL, 12.83 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give SA75 (3-amino-2-methylpropyl)(4-((3-amino-2-methylpropyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.66 g, 0.85 mmol, 66.1%) as a white solid. UPLC/ELSD: RT=1.59 min. MS (ES): m/z (MH ⁺ ) 753.4 for C ₄₀ H ₇₇ Cl ₃ N ₄ O ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.42 (m, 1H), 4.46 (br.m, 1H), 3.33 (br.m, 4H), 3.12 (br.m, 5H), 2.95 (m, 4H), 2.40 (d, 3H, J = 9Hz), 1.75 (br.m, 19H), 1.20 (br.m, 9H), 1.08 (m, 8H), 0.97 (d, 4H, J = 6Hz), 0.89 (d, 6H, J = 6Hz), 0.75 (s, 3H).

W. Compound SA76: (3-aminobutyl)(4-((3-aminobutyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate

To a solution of tert-butyl (4-aminobutan-2-yl)carbamate (1.00 g, 5.31 mmol) in anhydrous DCM (15 mL) stirred under nitrogen was added triethylamine (0.89 mL, 6.37 mmol). The solution was cooled to 0°C and then a solution of 4-nitrobenzenesulfonyl chloride (1.30 g, 5.84 mmol) in 5 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with another 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate solution (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid solution (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate (1.47 _g , 3.94 mmol, 74.1%) as a white solid. UPLC/ELSD: RT = 0.61 min _. MS (ES): m/z (MH ⁺ ) 374.4 _{for Ci5H23N3O6S} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 8.00(m,1H),7.81(m,2H),7.73(m,2H),6.15(br.s,1H),4.27(br.s,1H),3.64(br.s,1H),3.19(br.s,1H),2.95(br.s,1H),1. 64(m,1H),1.30(s,10H),1.00(d,3H,J=6Hz).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-4,2-diyl))dicarbamate

To a solution of tert-butyl (4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate (1.47 g, 3.94 mmol) in anhydrous DMF (20 mL) stirred under nitrogen was added potassium carbonate (1.58 g, 11.44 mmol) and 1,4-diiodobutane (0.25 mL, 1.88 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.19 mL, 1.56 mmol) was added and the reaction was allowed to proceed at room temperature for 24 h. Then, thiophenol (0.74 mL, 7.22 mmol), potassium carbonate (0.78 g, 5.62 mmol) and an additional 5 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight again. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-60% (70:20:10 DCM/MeOH/concentrated aqueous ammonium hydroxide solution) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-4,2-diyl))dicarbamate (0.17 g, 0.40 mmol, 21.2%) as a colorless oil. UPLC/ELSD: RT=0.45 min. MS (ES): m/z (MH ⁺ ) 431.6 for C ₂₂ H ₄₆ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 4.94 (m, 1H), 3.66 (m, 5H), 2.66 (m, 8H), 1.66 (m, 8H), 1.39 (s, 18H), 1.10 (d, 6H, J=9Hz).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(4-((3-((tert-butoxycarbonyl)amino)butyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-4,2-diyl))dicarbamate (0.17 g, 0.40 mmol) in anhydrous toluene (5 mL) stirred under nitrogen was added triethylamine (0.15 mL, 1.08 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.20 g, 0.36 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, and the solution was washed with water (3 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (80:19:1 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(4-((3-((tert-butoxycarbonyl)amino)butyl)amino)butyl)carbamate as a colorless oil (0.13 g, 0.16 mmol, 43.0%). UPLC/ELSD: RT = 2.77 _{min .} MS (ES): m/z (MH ⁺ ) 844.3 _{for C50H90N4O6} . ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 5.36(m,1H),4.89(m,1H),4.50(m,2H),3.67(br.m,2H),3.22(br.m,4H),2.59(m,4H),2.33(m,2H),1.98(br.m,6H),1.54(br.m ,15H),1.43(s,22H),1.34(m,5H),1.14(m,14H),1.02(s,7H),0.82(m,14H),0.91(d,4H,J＝6Hz),0.86(d,6H,J＝6Hz),0.67(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-aminobutyl)(4-((3-aminobutyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(4-((3-((tert-butoxycarbonyl)amino)butyl)amino)butyl)carbamate (0.13 g, 0.16 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.31 mL, 1.55 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give SA76 (3-aminobutyl) (4-((3-aminobutyl)amino)butyl)carbamate (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.06 g, 0.07 mmol, 47.9%) as a white solid. UPLC/ELSD: RT = 1.49 min. MS (ES): m/z (MH ⁺ ) 753.4 for C ₄₀ H ₇₇ Cl ₃ N ₄ O ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.42 (m, 1H), 4.48 (br.m, 1H), 3.47 (br.m, 2H), 3.33 (br.m, 7H), 3.17 (m, 4H), 2.40 (d, 2H, J = 9Hz), 2.05 (br.m, 8H), 1.62 (br.m, 1 0H), 1.39 (d, 8H, J = 9Hz), 1.16 (d, 7H, J = 6Hz), 1.08 (br.m, 5H), 0.98 (d, 3H, J = 6Hz), 0.91 (d, 5H, J = 6Hz), 0.74 (s, 3H).

X. Compound SA77: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-amino-2,2-dimethylpropyl)(4-((3-amino-2,2-dimethylpropyl)amino)butyl)carbamate trihydrochloride

Step 1: tert-Butyl (2,2-dimethyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate

To a solution of tert-butyl (3-amino-2,2-dimethylpropyl)carbamate (1.00 g, 4.94 mmol) in anhydrous DCM (15 mL) stirred under nitrogen was added triethylamine (0.83 mL, 5.93 mmol). The solution was cooled to 0°C and then a solution of 4-nitrobenzenesulfonyl chloride (1.20 g, 5.44 mmol) in 5 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with another 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate solution (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid solution (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (2,2-dimethyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate (2.00 _g , 5.15 mmol, 104.1%) as a white solid. UPLC/ELSD: RT = 0.85 min _. MS (ES): m/z (MH ⁺ ) 388.4 _{for Ci6H25N3O6S} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 8.10 (m, 1H), 7.82 (m, 1H), 7.73 (m, 2H), 6.54 (br.s, 1H), 4.86 (br.s, 1H), 2.99 (d, 2H, J = 6Hz), 2.81 (d, 2H, J = 9Hz), 1.41 (s, 9H), 0.90(s,6H).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2,2-dimethylpropane-3,1-diyl))dicarbamate

To a solution of tert-butyl (2,2-dimethyl-3-((4-nitrophenyl)sulfonamido)propyl)carbamate (2.00 g, 5.15 mmol) in anhydrous DMF (20 mL) stirred under nitrogen was added potassium carbonate (2.07 g, 14.96 mmol) and 1,4-diiodobutane (0.32 mL, 2.45 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.24 mL, 2.04 mmol) was added and the reaction was allowed to proceed at room temperature for 24 h. Then, thiophenol (0.97 mL, 9.44 mmol), potassium carbonate (1.02 g, 7.36 mmol) and an additional 5 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight again. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to give an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-60% (70:20:10 DCM/MeOH/concentrated aqueous ammonium hydroxide solution) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2,2-dimethylpropane-3,1-diyl))dicarbamate (0.63 g, 1.38 mmol, 56.3%) as a colorless oil. UPLC/ELSD: RT=0.46 min. MS (ES): m/z (MH ⁺ ) 459.7 for C ₂₄ H ₅₀ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.84 (m, 2H), 2.82 (d, 4H, J = 6Hz), 2.39 (br.s, 4H), 2.22 (s, 4H), 1.33 (br.m, 4H), 1.24 (s, 18H), 0.70 (s, 12H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2,2-dimethylpropane-3,1-diyl))dicarbamate (0.66 g, 1.43 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.55 mL, 3.90 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.72 g, 1.30 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, and the solution was washed with water (3 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (80:19:1 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropyl)amino)butyl)carbamate as a colorless oil (0.39 g, 0.45 mmol, 34.7%). UPLC/ELSD: RT = 2.77 min. MS (ES): m/z (MH ⁺ ) 872.3 for C ₅₂ H ₉₄ N ₄ O ₆ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.38(m,1H),5.09(m,1H),4.23(m,1H),2.92(br.m,2H),2.72(m,6H),2.27(m,2H),2.10(m,4H),1.70(m,5H),1.28(br.m,8H ),1.14(s,21H),1.05(m,4H),0.85(m,8H),0.74(s,7H),0.60(br.m,22H),0.39(s,4H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-2,2-dimethylpropyl)(4-((3-amino-2,2-dimethylpropyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-dimethylpropyl)amino)butyl)carbamate (0.39 g, 0.45 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.90 mL, 4.51 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give SA77 (3-amino-2,2-dimethylpropyl) (4-((3-amino-2,2-dimethylpropyl)amino)butyl)carbamate (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.21 g, 0.26 mmol, 58.0%) as a white solid. UPLC/ELSD: RT = 1.49 min. MS (ES): m/z (MH ⁺ ) 781.5 for C ₄₂ H ₈₁ Cl ₃ N ₄ O ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.42 (m, 1H), 4.51 (br.m, 1H), 3.33 (br.m, 5H), 3.08 (d, 6H, J = 9Hz), 2.73 (m, 2H), 2.42 (d, 2H, J = 6Hz), 1.75 (br.m, 16H), 1.39 (br .m,4H),1.22(br.m,11H),1.09(br.m,11H),0.97(d,4H,J＝6Hz),0.90(d,6H,J＝6Hz),0.74(s,3H).

Y. Compound SA78: (3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (2-methyl-4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate

To a solution of tert-butyl (4-amino-2-methylbutan-2-yl)carbamate (1.00 g, 4.94 mmol) in anhydrous DCM (15 mL) stirred under nitrogen was added triethylamine (0.83 mL, 5.93 mmol). The solution was cooled to 0°C and then a solution of 4-nitrobenzenesulfonyl chloride (1.20 g, 5.44 mmol) in 5 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with another 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate solution (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid solution (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (2-methyl-4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate (1.86 g, 4.79 mmol, 96.9%) _as a white solid. UPLC/ELSD: RT = 0.69 min _. MS (ES): m/z (MH ⁺ ) 388.4 _{for Ci6H25N3O6S} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 8.10 (m, 1H), 7.84 (m, 1H), 7.75 (m, 2H), 5.46 (br.s, 1H), 4.47 (s, 1H), 3.15 (q, 2H), 1.94 (t, 2H), 1.39 (s, 9H), 1.23 (s, 6H).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylbutane-4,2-diyl))dicarbamate

To a solution of tert-butyl (2-methyl-4-((4-nitrophenyl)sulfonamido)butan-2-yl)carbamate (1.86 g, 4.79 mmol) in anhydrous DMF (20 mL) stirred under nitrogen was added potassium carbonate (1.92 g, 13.92 mmol) and 1,4-diiodobutane (0.30 mL, 2.28 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.23 mL, 1.89 mmol) was added and the reaction was allowed to proceed at room temperature for 24 h. Then, thiophenol (0.90 mL, 8.78 mmol), potassium carbonate (0.95 g, 6.84 mmol) and an additional 5 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight again. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a gradient of 0-60% (70:20:10 DCM/MeOH/concentrated aqueous ammonium hydroxide solution). The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylbutane-4,2-diyl))dicarbamate (0.57 g, 1.24 mmol, 54.3%) as a colorless oil. UPLC/ELSD: RT=0.46 min. MS (ES): m/z (MH ⁺ ) 459.7 for C ₂₄ H ₅₀ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.94 (m, 2H), 2.61 (m, 8H), 1.92 (br.m, 2H), 1.61 (m, 4H), 1.45 (br.m, 4H), 1.32 (s, 18H), 1.21 (s, 12H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylbutane-4,2-diyl))dicarbamate (0.68 g, 1.48 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.57 mL, 4.03 mmol). Then (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.74 g, 1.34 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, and the solution was washed with water (3 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (80:19:1 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)butyl)carbamate as a colorless _oil (0.57 g, 0.65 mmol, 48.3%). UPLC/ELSD: RT = 2.65 _{min .} MS (ES): m/z (MH ⁺ ) 872.3 for _C52H94N4O6 . ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 5.99(m,1H),5.27(m,1H),4.40(m,2H),3.11(br.m,4H),2.59(t,2H),2.50(t,2H),2.25(m,2H),1.77(m,7H),1.58(m,2H),1 .44(br.m,12H),1.32(s,18H),1.20(d,16H,J＝9Hz),1.01(m,9H),0.92(s,6H),0.82(d,4H,J＝6Hz),0.75(d,6H,J＝9Hz),0.57(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)butyl)carbamate (0.57 g, 0.65 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 1.30 mL, 6.50 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give SA78 (3-amino-3-methylbutyl) (4-((3-amino-3-methylbutyl)amino)butyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.35 g, 0.44 mmol, 67.2%) as a white solid. UPLC/ELSD: RT = 1.50 min. MS (ES): m/z (MH ⁺ ) 781.5 for C ₄₂ H ₈₁ Cl ₃ N ₄ O ₂ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.43(m,1H),4.44(br.m,1H),3.33(br.m,5H),3.15(br.m,3H),2.38(m,2H),2.16(br.m,8H),1.74(br.m,10H),1.43(br.m,14H ), 1.17 (d, 9H, J = 6Hz), 1.08 (br.m, 5H), 0.98 (d, 4H, J = 6Hz), 0.90 (d, 6H, J = 6Hz), 0.74 (s, 3H).

Z. Compound SA79: (3-amino-2,2-difluoropropyl)(4-((3-amino-2,2-difluoropropyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (2,2-difluoro-3-((4-nitrophenyl)sulfonamido)propyl)carbamate

To a solution of tert-butyl (3-amino-2,2-difluoropropyl)carbamate (0.95 g, 4.52 mmol) in anhydrous DCM (15 mL) stirred under nitrogen was added triethylamine (0.76 mL, 5.42 mmol). The solution was cooled to 0°C, and then a solution of 4-nitrobenzenesulfonyl chloride (1.10 g, 4.97 mmol) in 5 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour, and then at room temperature for another 3 hours. The mixture was then diluted with another 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate solution (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid solution (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (2,2-difluoro-3-((4-nitrophenyl)sulfonamido)propyl)carbamate ( _{1.66 g} , 4.19 mmol, 92.6%) as a white solid. UPLC/ELSD: RT = 0.61 min. MS (ES): m/z (MH ⁺ ) _{396.4 for C14H19F2N3O6S} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 8.15 (m, 1H), 7.87 (m, 1H), 7.73 (m, 2H), 6.68 (br.s, 1H), 5.02 (br.s, 1H), 3.55 (br.m, 4H), 1.45 (s, 9H).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2,2-difluoropropane-3,1-diyl))dicarbamate

To a solution of tert-butyl (2,2-difluoro-3-((4-nitrophenyl)sulfonamido)propyl)carbamate (1.65 g, 4.18 mmol) in anhydrous DMF (20 mL) stirred under nitrogen was added potassium carbonate (1.68 g, 12.14 mmol) and 1,4-diiodobutane (0.26 mL, 1.99 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.20 mL, 1.65 mmol) was added and the reaction was allowed to proceed at room temperature for 24 h. Thiophenol (0.78 mL, 7.67 mmol), potassium carbonate (0.83 g, 5.97 mmol) and an additional 5 mL of anhydrous DMF were then added and the reaction was allowed to proceed overnight. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-60% (70:20:10 DCM/MeOH/concentrated aqueous ammonium hydroxide solution) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2,2-difluoropropane-3,1-diyl))dicarbamate (0.50 g, 1.07 mmol, 53.3%) as a colorless oil. UPLC/ELSD: RT=0.39 min. MS (ES): m/z (MH ⁺ ) 475.5 for C ₂₀ H ₃₈ F ₄ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.14 (m, 2H), 3.25 (m, 4H), 2.62 (m, 4H), 2.33 (br.m, 4H), 1.18 (br.m, 4H), 1.12 (s, 18H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2,2-difluoropropane-3,1-diyl))dicarbamate (0.64 g, 1.35 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.52 mL, 3.68 mmol). Then (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.68 g, 1.23 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, and the solution was washed with water (3 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (80:19:1 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)amino)butyl)carbamate as a colorless oil (0.12 g, 0.13 mmol, 10.7%). UPLC/ELSD: RT = 2.63 min. MS (ES): m/z (MH ⁺ ) 888.2 for C ₄₈ H ₈₂ F ₄ N ₄ O ₆ . ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 5.61(m,1H),5.31(m,1H),4.98(m,1H),4.55(br.m,1H),3.63(br.m,5H),3.32(m,2H),2.97(t,2H),2.69(t,2H),2.36(m,2H) ,2.05(br.m,5H),1.60(br.m,5H),1.46(s,21H),1.15(m,6H),1.04(s,5H),0.93(d,3H,J＝6Hz),0.89(d,5H,J＝6Hz),0.69(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-2,2-difluoropropyl)(4-((3-amino-2,2-difluoropropyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)amino)butyl)carbamate (0.12 g, 0.13 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.26 mL, 1.31 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give SA79 (3-amino-2,2-difluoropropyl) (4-((3-amino-2,2-difluoropropyl) amino) butyl) carbamate (3S, 8S, 9S, 10R, 13R, 14S, 17R) -10,13-dimethyl-17-((R) -6-methylhept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthrene-3-yl ester trihydrochloride (0.05 g, 0.06 mmol, 42.8%) as a white solid. UPLC/ELSD: RT = 1.52 min. MS (ES): m/z (MH ⁺ ) 797.4 for C ₃₈ H ₆₉ Cl ₃ F ₄ N ₄ O ₂ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.43(m,1H),4.49(br.m,1H),3.94(br.m,7H),3.77(br.m,4H),3.33(m,3H),3.22(m,2H),2.42(m,2H),1.76(br.m,23H),1.1 8(d,12H,J=6Hz),1.08(br.m,6H),0.98(d,4H,J=9Hz),0.91(d,6H,J=6Hz),0.75(s,3H).

AA. Compound SA81: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-aminohexyl)carbamate dihydrochloride

Step 1: tert-Butyl N-{6-[benzyl({6-[(tert-butoxycarbonyl)amino]hexyl})amino]hexyl}carbamate

Benzylamine (0.50mL, 4.6mmol) is added to a suspension of tert-butyl N-(6-bromohexyl)carbamate (2.694g, 9.613mmol), potassium carbonate (1.898g, 13.73mmol) and potassium iodide (0.152g, 0.915mmol) in dimethylformamide (7.5mL). The reaction mixture is stirred at 50°C and monitored by LCMS. At 23.5h, the reaction mixture is cooled to room temperature and then diluted with methyl tert-butyl ether (150mL). The diluted mixture is washed with water ( _4x ) and brine, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-70% methyl tert-butyl ether in hexanes) to give tert-butyl N-{6-[benzyl({6-[(tert-butoxycarbonyl)amino]hexyl})amino]hexyl}carbamate (2.144 g, 4.239 mmol, 92.6%) as a clear oil. UPLC/ELSD: RT = 0.87 min. MS (ES): m/z of C ₂₉ H ₅₁ N ₃ O ₄ = 506.70 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ7.17-7.37 (m, 5H), 4.50 (br.s, 2H), 3.52 (s, 2H), 3.08 (dt, 4H, J = 6.5, 6.2Hz), 2. 37(t,4H,J=7.2Hz),1.37-1.51(m,8H),1.44(s,18H),1.22-1.33(m,8H).

Step 2: tert-Butyl N-[6-({6-[(tert-butoxycarbonyl)amino]hexyl}amino)hexyl]carbamate

By N-{6-[benzyl({6-[(tert-butoxycarbonyl)amino]hexyl})amino]hexyl}t-butyl carbamate (2.12g, 4.192mmol) and 10% Pd/C (0.892g, 0.419mmol) are combined in ethanol (35mL) and then stirred at room temperature under _H2 balloon. The reaction is monitored by TLC. At 19h, the reaction mixture is filtered through a diatomaceous earth pad rinsed with ethyl acetate. The filtrate is concentrated, dissolved in ethyl acetate, filtered through a 0.45 μm PTFE glass frit, and concentrated to give N-[6-({6-[(tert-butoxycarbonyl)amino]hexyl}amino)hexyl]t-butyl carbamate (1.537g, 3.698mmol, 88.2%) as an off-white solid. UPLC/ELSD: RT=0.62min. MS (ES): m/z of C ₂₂ H ₄₅ N ₃ O ₄ = 416.60 [M+H] ⁺ ; ¹ HNMR (300MHz, CDCl ₃ ): δ4.51 (br.s, 2H), 3.10 (dt, 4H, J = 6.4, 6.4Hz), 2.57 (t, 4H, J = 7.1Hz), 1.13-1.57 (br.m ,17H),1.44(s,18H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-((tert-butoxycarbonyl)amino)hexyl)carbamate

Cholesteryl carbonate 4-nitrophenyl ester (0.200 g, 0.362 mmol), tert-butyl N-[6-({6-[(tert-butoxycarbonyl)amino]hexyl}amino)hexyl]carbamate (0.188 g, 0.453 mmol), and triethylamine (0.15 mL, 1.08 mmol) were combined in toluene (3.5 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature, diluted with dichloromethane (30 mL), and washed with 5% NaHCO ₃ aqueous solution (3 x 25 mL). The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-50% ethyl acetate in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-((tert-butoxycarbonyl)amino)hexyl)carbamate (0.286 g, 0.345 mmol, 95.3%) as a clear oil. UPLC/ELSD: RT = 3.53 min. MS (ES): m/z of C ₅₀ H ₈₉ N ₃ O ₆ = 728.94 [(M+H)-(CH ₃ ) ₂ C = CH ₂ -CO ₂ ] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.33-5.42(m,1H),4.41-4.64(m,3H),2.99-3.28(m,8H),2.20-2.41(m,2H),1.75-2.08(m,5H),0.93-1.67(br.m,37H),1.44(s,18H),1.02(s,3 H), 0.91 (d, 3H, J = 6.4Hz), 0.87 (d, 3H, J = 6.6Hz), 0.86 (d, 3H, J = 6.5Hz), 0.68 (s, 3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-aminohexyl)carbamate dihydrochloride

To a solution of (3S, 8S, 9S, 10R, 13R, 14S, 17R) -10, 13- dimethyl -17- ((R) -6- methylhept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetrahydro -1H- cyclopenta [a] phenanthrene -3-yl ester (0.280 g, 0.338 mmol) in isopropanol (3.5 mL) was added 5-6N HCl (0.52 mL) in isopropanol. The reaction mixture was stirred at 40 ° C and monitored by LCMS. At 18 h, the reaction mixture was cooled to room temperature and then acetonitrile (10.5 mL) was added. The suspension was cooled to 0 ° C in an ice bath. The solid was then collected by vacuum filtration and rinsed with cold 3:1 acetonitrile/isopropanol to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-aminohexyl)carbamate dihydrochloride (0.179 g, 0.251 mmol, 74.4%) as a white solid. UPLC/ELSD: RT = 2.09 min. MS (ES): m/z for C ₄₀ H ₇₃ N ₃ O ₂ = 335.49 [(M+2H)+CH ₃ CN] ²⁺ ; ¹ H NMR (300 MHz, CDCl ₃ ): δ5.36-5.44(m,1H),4.33-4.48(m,1H),3.25(t,4H,J＝7.2Hz),2.92(t,4H,J＝7.6Hz),2.24-2.39(m,2H),1.79-2.12(m,5H),0.97-1.73(br.m,37H),1 .05 (s, 3H), 0.95 (d, 3H, J = 6.4Hz), 0.88 (d, 3H, J = 6.5Hz), 0.88 (d, 3H, J = 6.6Hz), 0.73 (s, 3H).

AB. Compound SA82: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-aminohexyl)carbamate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-((tert-butoxycarbonyl)amino)hexyl)carbamate

4-Nitrophenyl beta-sitosterol carbonate (0.200 g, 0.345 mmol), tert-butyl N-[6-({6-[(tert-butoxycarbonyl)amino]hexyl}amino)hexyl]carbamate (0.179 g, 0.431 mmol), triethylamine (0.15 mL, 1.1 mmol) were combined in toluene (3.5 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature, diluted with dichloromethane (about 30 mL) and washed with 5% aqueous NaHCO ₃ (3 x 25 mL). The combined washings were extracted with dichloromethane (25 mL). The combined organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-40% ethyl acetate in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-((tert-butoxycarbonyl)amino)hexyl)carbamate (0.259 g, 0.302 mmol, 87.7%) as a clear oil. UPLC/ELSD: RT = 3.63 min. MS (ES): m/z of C ₅₂ H ₉₃ N ₃ O ₆ = 857.26 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.33-5.42 (m, 1H), 4.41-4.61 (m, 3H), 3.02-3.28 (m, 8H), 2.19-2.42 (m, 2H), 1.7 6-2.08(m,5H),0.88-1.73(br.m,38H),1.44(s,18H),1.02(s,3H),0.92(d,3H,J＝6.4Hz),0.79-0.89(m,9H),0.68(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-aminohexyl)carbamate dihydrochloride

To a solution of (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)bis(6-((tert-butoxycarbonyl)amino)hexyl)carbamate (0.256 g, 0.299 mmol) in isopropanol (3.2 mL) was added 5-6N HCl in isopropanol (0.45 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 18 h, the reaction mixture was cooled to room temperature and then acetonitrile (9.6 mL) was added. The suspension was cooled to 0 °C in an ice bath and the solids were collected by vacuum filtration, rinsing with cold 3:1 acetonitrile/isopropanol to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(6-aminohexyl)carbamate dihydrochloride (0.167 g, 0.216 mmol, 72.1%) as a white solid. UPLC/ELSD: RT = 2.23 min. MS (ES): m/z of C ₄₂ H ₇₇ N ₃ O ₂ = 349.94 [(M+2H) + CH ₃ CN] ²⁺ ; ¹ H NMR (300MHz, CD ₃ OD): δ 5.36-5.54 (m, 1H), 4.33-4.48 (m, 1H), 3.25 (t, 4H, J = 7.1Hz), 2.92 ( t,4H,J＝7.6Hz),2.25-2.39(m,2H),1.79-2.13(m,5H),0.91-1.76(br.m,38H),1.05(s,3H),0.96(d,3H,J＝6.4Hz),0.81-0.91(m,9H),0.73(s,3H).

AC. Compound SA83: (6-aminohexyl)(3-aminopropyl)carbamic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: tert-Butyl N-[3-(2-nitrobenzenesulfonamido)propyl]carbamate

To a stirred solution of tert-butyl N-(3-aminopropyl)carbamate (1.50 g, 8.35 mmol) and triethylamine (1.50 mL, 10.7 mmol) in dichloromethane (40 mL) cooled to 0 ° C in an ice bath was added a solution of 2-nitrobenzenesulfonyl chloride (2.00 g, 8.75 mmol) in dichloromethane (10 mL) dropwise via a charging funnel over 15 min. Thereafter, the reaction mixture was slowly warmed to room temperature while stirring. The reaction was monitored by TLC. At 23 h, the reaction mixture was diluted with dichloromethane (50 mL) and then washed with 5% aqueous citric acid. The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (20%-50% ethyl acetate in hexanes) to give tert-butyl N-[3-(2-nitrobenzenesulfonamido)propyl]carbamate (2.611 g, 7.265 mmol, 87.0%) as a viscous light yellow oil. UPLC/ELSD: RT = 0.54 min. MS (ES): m/z of C ₁₄ H ₂₁ N ₃ O ₆ S=304.14[(M+H)-(CH ₃ ) ₂ C=CH ₂ ] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ8.09-8.17(m,1H),7.81-7.89(m,1H),7.68-7.77(m,2H),5.8 6(br.s,1H),4.66(br.s,1H),3.21(dt,2H,J=6.2,6.2Hz),3.15(dt,2H,J=6.4,6.4Hz),1.63-1.76(m,2H),1.42(s,9H).

Step 2: tert-Butyl N-[3-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}2-nitrobenzenesulfonamido)propyl]carbamate

To a stirred mixture of N-[3-(2-nitrobenzenesulfonamido)propyl]carbamic acid tert-butyl ester (1.000 g, 2.782 mmol), potassium carbonate (0.769 g, 5.56 mmol) and potassium iodide (0.046 g, 0.28 mmol) in dimethylformamide (15 mL) was added a solution of N-(6-bromohexyl)carbamic acid tert-butyl ester (0.858 g, 3.06 mmol) in dimethylformamide (1.0 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 20.3 h, the reaction mixture was heated to 50 ° C. At 25 h, the reaction mixture was cooled to room temperature. The reaction mixture was diluted with methyl tert-butyl ether and water. The layers were separated, and the organic matter was washed with water (4x) and brine, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (10%-85% methyl tert-butyl ether in hexanes) to give tert-butyl N-[3-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}2-nitrobenzenesulfonamido)propyl]carbamate (1.399 g, 2.504 _mmol , _90.0 %) as a clear oil. UPLC/ _{ELSD: RT = 1.43 min. MS (ES): m/z for C25H42N4O8S =} 403.26 [(M+H)-2[( _CH3 ) _2C = _CH2 ] _-CO2 ] ⁺ ; ^1H NMR (300 MHz, _CDCl3 ): δ7.96-8.04(m,1H),7.58-7.73(m,3H),4.84(br.s,1H),4.50(br.s,1H),3.35(t,2H,J＝7.0Hz),3.25(t,2H,J＝7.6Hz),3.15(td,2H,J＝6.3,6.2Hz),3. 06(td,2H,J=6.7,6.5Hz),1.68-1.79(m,2H),1.34-1.60(m,4H),1.44(s,18H),1.20-1.33(m,4H).

Step 3: tert-Butyl N-[3-({6-[(tert-butoxycarbonyl)amino]hexyl}amino)propyl]carbamate

Tert-butyl N-[3-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}2-nitrobenzenesulfonamido)propyl]carbamate (1.391 g, 2.490 mmol), potassium carbonate (1.032 g, 7.469 mmol) and thiophenol (0.39 mL, 3.82 mmol) were combined in dimethylformamide (20 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 18 h, the reaction mixture was filtered through a pad of celite, rinsed with methyl tert-butyl ether. The filtrate was washed with saturated aqueous NaHCO ₃ solution, water (3x) and brine. The organics were dried over Na ₂ SO ₄ and concentrated. The crude material was purified via silica gel chromatography (0-20% (5% concentrated aqueous _NH4OH in methanol) in dichloromethane) to afford tert-butyl N-[3-({6-[(tert-butoxycarbonyl)amino]hexyl}amino)propyl]carbamate (0.889 g, 2.38 mmol, 95.6%) as a white solid. UPLC/ELSD: RT = 0.42 min. MS (ES): m/z of C ₁₉ H ₃₉ N ₃ O ₄ = 374.38 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.15 (br.s, 1H), 4.52 (br.s, 1H), 3.19 (dt, 2H, J = 5.9, 5.9Hz), 3.10 (dt, 2H, J = 6.4, 6 .4Hz), 2.66 (t, 2H, J = 6.6Hz), 2.57 (t, 2H, J = 7.0Hz), 1.59-1.71 (m, 2H), 1.24-1.55 (m, 9H), 1.44 (s, 18H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (6-((tert-butoxycarbonyl)amino)hexyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate

Cholesteryl carbonate 4-nitrophenyl ester (0.200 g, 0.362 mmol), tert-butyl N-[3-({6-[(tert-butoxycarbonyl)amino]hexyl}amino)propyl]carbamate (0.169 g, 0.453 mmol), and triethylamine (0.15 mL, 1.1 mmol) were combined in toluene (3.5 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature, diluted with dichloromethane (30 mL), and washed with 5% NaHCO ₃ aqueous solution (3 x 30 mL). The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (20%-50% ethyl acetate in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (6-((tert-butoxycarbonyl)amino)hexyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.240 g, 0.305 mmol, 84.2%). UPLC/ELSD: RT = 3.43 min. MS (ES): m/z of C ₄₇ H ₈₃ N ₃ O ₆ = 687.36 [(M+H)-(CH ₃ ) ₂ C = CH ₂ -CO ₂ ] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.17-5.44(m,2H),4.40-4.86(m,2H),2.98-3.40(br.m,8H),2.19-2.44(m,2H),1.74-2.10(m,5H),0.93-1.73(br.m,31H),1.44(s,18H),1.02(s ,3H),0.91(d,3H,J＝6.4Hz),0.86(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.68(s,3H).

Step 5: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (6-aminohexyl)(3-aminopropyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (6-((tert-butoxycarbonyl)amino)hexyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.234 g, 0.298 mmol) in isopropanol (2.8 mL) was added 5-6N HCl in isopropanol (0.42 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature and then acetonitrile (8.4 mL) was added. The suspension was stirred at room temperature for 1 h and the solid was then collected by vacuum filtration, rinsed with cold 3:1 acetonitrile/isopropanol to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(6-aminohexyl)(3-aminopropyl)carbamate dihydrochloride (0.177 g, 0.264 mmol, 88.8%) as a white solid. UPLC/ELSD: RT = 1.98 min. MS (ES): m/z of C ₃₇ H ₆₇ N ₃ O ₂ = 314.63 [(M+2H) + CH ₃ CN] ²⁺ ; ¹ H NMR (300MHz, CD ₃ OD): δ5.35-5.44(m,1H),4.36-4.52(m,1H),3.37(t,2H,J＝6.8Hz),3.28(t,2H,J＝7.6Hz),2.88-2.99(m,4H),2.26-2.46(m,2H),1.78-2.14(m,7H),0. 98-1.75(br.m,29H),1.06(s,3H),0.95(d,3H,J＝6.4Hz),0.88(d,3H,J＝6.6Hz),0.88(d,3H,J＝6.6Hz),0.73(s,3H).

AD. Compound SA84: (6-aminohexyl)(3-aminopropyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (6-((tert-butoxycarbonyl)amino)hexyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate

4-Nitrophenyl beta-sitosterol carbonate (0.200 g, 0.345 mmol), tert-butyl N-[3-({6-[(tert-butoxycarbonyl)amino]hexyl}amino)propyl]carbamate (0.161 g, 0.431 mmol), and triethylamine (0.15 mL, 1.1 mmol) were combined in toluene (3.5 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature, diluted with dichloromethane (30 mL), and washed with 5% aqueous NaHCO ₃ solution (3 x 30 mL). The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (20%-50% ethyl acetate in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (6-((tert-butoxycarbonyl)amino)hexyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.218 g, 0.268 mmol, 77.6%). UPLC/ELSD: RT = 3.55 min. MS (ES): m/z of C ₄₉ H ₈₇ N ₃ O ₆ = 715.12 [(M+H)-(CH ₃ ) ₂ C = CH ₂ -CO ₂ ] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ 5.13-5.44 (m, 2H), 4.35-4.88 (m, 2H), 2.98-3.40 (br.m, 8H ),2.20-2.45(m,2H),1.76-2.09(m,5H),0.88-1.75(br.m,32H),1.44(s,18H),1.02(s,3H),0.92(d,3H,J=6.4Hz),0.78-0.89(m,9H),0.68(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(6-aminohexyl)(3-aminopropyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (6-((tert-butoxycarbonyl)amino)hexyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.213 g, 0.262 mmol) in isopropanol (2.6 mL) was added 5-6N HCl in isopropanol (0.38 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature, and then acetonitrile (7.8 mL) was added. The suspension was stirred at room temperature for 1 h, and the solid was then collected by vacuum filtration, rinsed with cold 3:1 acetonitrile/isopropanol to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(6-aminohexyl)(3-aminopropyl)carbamate dihydrochloride (0.169 g, 0.232 mmol, 88.8%) as a white solid. UPLC/ELSD: RT = 2.17 min. MS(ES)：C ₃₉ H ₇₁ N ₃ O ₂的m/z＝328.70[(M+2H)+CH ₃ CN] ²⁺ ； ¹ H NMR(300MHz,CD ₃ OD)：δ5.36-5.45(m,1H),4.36-5.53(m,1H),3.37(t,2H,J＝6.6Hz),3.28(t,2H,J＝7.5Hz),2.88-2.98(m,4H),2.26-2.43(m,2H),1.80-2.12(m,7H),0.91-1.77(br.m,30H),1.06(s,3H),0.96(d,3H,J＝6.4Hz),0.82-0.91(m,9H),0.73(s,3H)。

AE. Compound SA87: (3-amino-2-fluoropropyl)(4-((3-amino-2-fluoropropyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (2-fluoro-3-((2-nitrophenyl)sulfonamido)propyl)carbamate

To a solution of tert-butyl (3-amino-2-fluoropropyl)carbamate (1.00 g, 5.20 mmol) in anhydrous DCM (15 mL) stirred under nitrogen was added triethylamine (0.87 mL, 6.24 mmol). The solution was cooled to 0°C and then a solution of 2-nitrobenzenesulfonyl chloride (1.27 g, 5.72 mmol) in 5 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate (2 x 15 mL), water (1 x 15 mL), 10% aqueous citric acid (2 x 15 mL), water (1 x 15 mL), and brine (2 x 15 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (2-fluoro-3-((2-nitrophenyl)sulfonamido)propyl)carbamate (1.93 _g , 5.14 mmol, 98.7%) as a white solid. UPLC/ELSD: RT = 1.76 min. MS (ES): m/z (MH ⁺ ) _{378.4 for C14H20FN3O6S} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 8.15 (m, 1H), 7.87 (m, 1H), 7.77 (m, 2H), 6.14 (br.s, 1H), 5.31 (m, 1H), 4.71 (m, 1H), 4.55 (m, 1H), 3.39 (m, 4H), 1.44 (s, 9H).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-fluoropropane-3,1-diyl))dicarbamate

To a solution of tert-butyl (2-fluoro-3-((2-nitrophenyl)sulfonamido)propyl)carbamate (1.94 g, 5.14 mmol) in anhydrous DMF (20 mL) stirred under nitrogen was added potassium carbonate (2.06 g, 14.92 mmol) and 1,4-diiodobutane (0.32 mL, 2.45 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.24 mL, 2.03 mmol) was added and the reaction was allowed to proceed for 8 h at room temperature. Then, thiophenol (0.96 mL, 9.42 mmol), potassium carbonate (1.01 g, 7.34 mmol) and an additional 5 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight again. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatants were concentrated in vacuo to give an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-80% (75:20:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-fluoropropane-3,1-diyl))dicarbamate (0.77 g, 1.76 mmol, 72.0%) as a colorless oil. UPLC/ELSD: RT=0.34 min. MS (ES): m/z (MH ⁺ ) 439.6 for C ₂₀ H ₄₀ F ₂ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.11 (m, 2H), 4.73 (br.s, 1H), 4.56 (br.s, 1H), 3.36 (m, 3H), 2.75 (br.m, 9H), 1.51 (m, 5H), 1.43 (s, 18H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2-fluoropropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-fluoropropyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-fluoropropane-3,1-diyl))dicarbamate (0.73 g, 1.67 mmol) in anhydrous toluene (20 mL) stirred under nitrogen was added triethylamine (0.59 mL, 4.18 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.77 g, 1.39 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, diluted with toluene, washed with water (3x15 mL), dried over sodium sulfate, filtered, and concentrated to give an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (80:19:1 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2-fluoropropyl)( ₄ -((3-((tert-butoxycarbonyl)amino)-2-fluoropropyl)amino)butyl)carbamate as a colorless oil (0.76 g, 0.89 mmol, _64.1 %). UPLC/ELSD: RT = 2.62 _min . _MS (ES): m/z (MH ⁺ ) 852.3 for _C48H84F2N4O6 . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.89 (m, 1H), 4.98 (br.m, 1H), 4.76 (br.m, 1H), 4.60 (m, 2H), 3.42 (br.m, 9H), 2.86 (d, 1H, J = 6Hz), 2.78 (m, 1H), 2.65 (m, 3H), 2.3 5(m,2H),1.90(br.m,6H),1.58(br.m,10H),1.46(s,25H),1.35(m,4H),1.15(br.m,10H),1.04(s,6H),0.94(d,4H,J＝6Hz),0.78(d,6H,J＝6Hz),0.70(s,3H ).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-2-fluoropropyl)(4-((3-amino-2-fluoropropyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2-fluoropropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-fluoropropyl)amino)butyl)carbamate (0.76 g, 0.89 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5N in isopropanol, 1.79 mL, 8.93 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.54 g, 0.67 mmol, 75.4%) as a white solid. UPLC/ELSD: RT = 1.65 min. MS (ES): m/z (MH ⁺ ) 651.7 for C ₃₈ H ₇₁ Cl ₃ F ₂ N ₄ O ₂ . ^1. 75(br.m,17H),1.16(br.m,13H),0.95(d,4H,J=6Hz),0.91(d,6H,J=6Hz),0.74(s,3H).

AF. Compound SA88: (3-amino-2-hydroxypropyl)(4-((3-amino-2-hydroxypropyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (2-hydroxy-3-((2-nitrophenyl)sulfonamido)propyl)carbamate

To a solution of tert-butyl (3-amino-2-hydroxypropyl)carbamate (10.51 g, 55.27 mmol) in anhydrous DCM (200 mL) stirred under nitrogen was added triethylamine (9.24 mL, 66.37 mmol). The solution was cooled to 0°C and then a solution of 2-nitrobenzenesulfonyl chloride (12.25 g, 55.27 mmol) in 100 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate (2 x 100 mL), water (1 x 100 mL), 10% aqueous citric acid (2 x 100 mL), water (1 x 100 mL), and brine (2 x 100 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (2-hydroxy-3-((2-nitrophenyl)sulfonamido)propyl)carbamate (17.54 _g , 46.73 mmol, 84.5%) as a white solid. UPLC/ELSD: RT = 1.23 min _. MS (ES): m/z (MH ⁺ ) 376.4 _{for C14H21N3O7S} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 8.13 (m, 1H), 7.88 (m, 1H), 7.78 (m, 2H), 6.01 (br.s, 1H), 5.01 (m, 1H), 3.86 (m, 1H), 3.29 (m, 4H), 3.12 (m, 1H), 1.45 (s, 9H).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-hydroxypropane-3,1-diyl))dicarbamate

To a solution of tert-butyl (2-hydroxy-3-((2-nitrophenyl)sulfonamido)propyl)carbamate (4.00 g, 10.66 mmol) in anhydrous DMF (40 mL) stirred under nitrogen was added potassium carbonate (4.28 g, 30.95 mmol) and 1,4-diiodobutane (0.67 mL, 5.07 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.50 mL, 4.21 mmol) was added and the reaction was allowed to proceed at room temperature for 16 h. Then, thiophenol (2.00 mL, 19.54 mmol), potassium carbonate (2.10 g, 15.22 mmol) and an additional 5 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight again. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to give an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a gradient of 0-60% (70:20:10 DCM/MeOH/concentrated aqueous ammonium hydroxide solution). The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-hydroxypropane-3,1-diyl))dicarbamate (1.12 g, 2.58 mmol, 50.8%) as a colorless oil. UPLC/ELSD: RT=0.20 min. MS (ES): m/z (MH ⁺ ) 435.6 for C ₂₀ H ₄₂ N ₄ O ₆ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm5.47(m,2H),4.72(br.s,1H),3.76(br.s,2H),3.47(m,9H),3.22(m,3H),3.05(m,3H),2.62(br.m,8H),1.53(m,5H),1.42(s,18 H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2-hydroxypropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-hydroxypropyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-hydroxypropane-3,1-diyl))dicarbamate (1.12 g, 2.58 mmol) in anhydrous toluene (20 mL) stirred under nitrogen was added triethylamine (0.91 mL, 6.44 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (1.19 g, 2.15 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, diluted with toluene, washed with water (3x15 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (80:19:1 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2-hydroxypropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-hydroxypropyl)amino)butyl)carbamate as a colorless oil (0.08 g, 0.09 mmol, 4.1 _% ). UPLC/ELSD: RT = 2.45 _{min .} MS (ES): m/z (MH ⁺ ) 848.3 for _C48H86N4O8 . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.32(m,2H),5.07(br.m,1H),4.43(br.m,1H),3.77(m,2H),3.25(br.m,6H),2.95(m,4H),2.56(m,4H),2.26(m,2H),1.90(m ,5H),1.47(m,9H),1.37(s,18H),1.07(m,11H),0.95(s,6H),0.86(d,4H,J＝6Hz),0.78(d,5H,J＝6Hz),0.61(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-amino-2-hydroxypropyl)(4-((3-amino-2-hydroxypropyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2-hydroxypropyl)(4-((3-((tert-butoxycarbonyl)amino)-2-hydroxypropyl)amino)butyl)carbamate (0.08 g, 0.09 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.18 mL, 0.89 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (6 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3-amino-2-hydroxypropyl)(4-((3-amino-2-hydroxypropyl)amino)butyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.04 g, 0.04 mmol, 49.3%) as a white solid. UPLC/ELSD: RT=1.53 min. MS (ES): m/z (MH ⁺ ) 648.2 for C ₃₈ H ₇₃ Cl ₃ N ₄ O ₄ . ¹ H NMR (300MHz, MeOD) δ: ppm 5.42 (m, 1H), 4.45 (br.m, 1H), 4.26 (br.m, 1H), 4.08 (br.m, 1H), 3.44 (m, 3H), 3.13 (m, 9H), 2.41 (d, 2H, J = 3Hz), 2.05 (s, 3H), 1.9 2(m,3H),1.55(br.m,13H),1.16(br.m,11H),0.97(d,3H,J＝6Hz),0.91(d,5H,J＝6Hz),0.74(s,3H).

AG. Compound SA89: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 6-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-6-oxohexanoate trihydrochloride

Step 1: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaeicosane-20-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a stirred anhydrous D-2-ol of 6-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-6-oxohexanoic acid (0.40 g, 0.77 mmol) was added 0.2% ethanol; To a solution in CM (10 mL) was added tert-butyl (3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.43 g, 0.85 mmol), dimethylaminopyridine (0.02 g, 0.15 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.22 g, 1.15 mmol). The resulting solution was cooled to 0 °C, and diisopropylethylamine (0.41 mL, 2.31 mmol) was added dropwise. The mixture was gradually warmed to room temperature and allowed to proceed overnight. The solution was then diluted with dichloromethane, washed with saturated sodium bicarbonate (1 x 10 mL) and brine (1 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica with a gradient of 0-80% ethyl acetate in hexanes. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaeicosane-20-oic acid as a light yellow oil (0.59 g, 0.59 mmol, 77.0%). UPLC/ELSD: RT: 3.40 min. MS (ES): m/z (MH ⁺ ) 1000.4 for C ₅₈ H ₁₀₂ N ₄ O ₉ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.25 (m, 1H). 4.47(br.m,1H),4.00,(q,1H),3.12(br.m,12H),2.20(br.m,6H),1.91(br.m,8H),1.54(br.m,16H),1.31(br.s,33H),1.13(br.m,13H),0.90(s,6H),0.8 1 (d, 4H, J = 6Hz), 0.73 (d, 6H, J = 6Hz), 0.56 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 6-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-6-oxohexanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaeicosane-20-oate (0.59 g, 0.59 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 1.19 mL, 5.92 mmol) dropwise. The solution was heated to 40° C. overnight. The next morning, anhydrous acetonitrile (15 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.36 g, 0.43 mmol, 71.7%) as a white solid. UPLC/ELSD: RT=1.75 min. MS (ES): m/z (MH ⁺ ) 700.1 for C ₄₃ H ₈₁ Cl ₃ N ₄ O ₃ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.40(m,1H),4.54(br.m,1H),3.96(m,1H),3.52(br.m,4H),3.33(s,1H),3.12(m,9H),2.49(br.m,2H),2.36(br.m,5H),2.17(m, 3H), 2.06 (s, 3H), 1.67 (br.m, 30H), 1.16 (d, 14H, J = 6Hz), 1.07 (s, 6H), 0.98 (d, 5H, J = 6Hz), 0.89 (d, 7H, J = 6Hz), 0.74 (s, 3H).

AH. Compound SA90 6-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-6-oxohexanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 6-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-6-oxohexanoic acid

To a solution of sitosterol (0.44 g, 1.01 mmol) in anhydrous DCM (10 mL) stirred under nitrogen was added oxepane-2,7-dione (0.13 g, 1.01 mmol), followed by dropwise addition of pyridine (0.31 mL, 2.22 mmol). The solution was then refluxed at 40 ° C overnight, during which all solids became a solution. The next day, the mixture was cooled to room temperature, concentrated to a yellow oil, dissolved in DCM, and purified on silica without further treatment. A silica column was run with a 0-30% EtOAc gradient in hexane. Fractions containing product were pooled and concentrated to give 6-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-6-oxohexanoic acid (0.12 g, 0.21 mmol, 21.0%) as a white solid. UPLC/ELSD: RT: 3.23 _min . MS (ES): m/z (MH ⁺ ) 543.8 _{for C35H58O4} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.36 (m, 1H), 3.54 (br.m, 1H), 2.28, (m, 2H), 2.04 (br.m, 3H), 1.86 (br.m, 3H), 1.49 (br.m, 19H), 1.02 (s, 6H), 0.94 (d, 5H, J＝6 Hz),0.86(q,10H),0.69(s,3H).

Step 2: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaeicosane-20-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 6-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-6-oxohexanoic acid (0.12 g, 0.21 mmol) was stirred under nitrogen. To a solution in anhydrous DCM (10 mL) of 10% 4- ((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamic acid tert-butyl ester (0.13 g, 0.25 mmol), dimethylaminopyridine (0.01 g, 0.04 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.08 g, 0.42 mmol) were added. The resulting solution was cooled to 0 ° C and diisopropylethylamine (0.11 mL, 0.64 mmol) was added dropwise. The mixture was gradually warmed to room temperature and left overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution (1×10 mL) and brine (1×10 mL), dried over sodium sulfate, filtered, and concentrated to give an oil. The oil was dissolved in DCM and purified on silica with a 0-80% ethyl acetate in hexanes gradient to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaeicosane-20-oic acid ester (0.15 g, 0.14 mmol, 67.5%). UPLC/ELSD: RT: 3.91min. MS (ES): m/z (MH ⁺ ) 1028.5 for C ₆₀ H ₁₀₆ N ₄ O ₉ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.38(m,2H),4.59(br.m,2H),4.14(m,1H),3.24(br.m,11H),2.32(br.m,6H),1.67(br.m,17H),1.47(s,32H),1.25(br.m,11H ), 1.03 (s, 6H), 0.94 (d, 4H, J = 9Hz), 0.86 (q, 8H, J = 9Hz), 0.69 (s, 3H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 6-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-6-oxohexanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazaeicosane-20-olate (0.15 g, 0.14 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.29 mL, 1.43 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (10 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile and dried in vacuo to give ((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-6-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-6-oxohexanoate trihydrochloride (0.06 g, 0.06 mmol, 43.9%) as a white solid. UPLC/ELSD: RT = 1.97 min. MS (ES): m/z (MH ⁺ 40.4) for C ₄₅ H ₈₅ Cl ₃ N ₄ O ₃ )728.1. ¹ H NMR(300MHz,MeOD)δ:ppm 5.41(m,1H),4.87(br.m,9H),4.55(br.m,1H),3.46(m,3H),3.33(s,1H),3.10(m,6H),2.35(br.m,6H),2.04(s,5H),1.68(br.m ,15H),1.21(m,9H),1.06(s,4H),0.97(d,4H,J＝6Hz),0.86(q,7H,J＝6Hz),0.74(s,3H).

AI. Compound SA95: (10-aminodecyl)(3-aminopropyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: tert-Butyl N-{10-[(2-cyanoethyl)amino]decyl}carbamate

To the suspension of N-(10-aminodecyl) tert-butyl carbamate (1.500g, 5.506mmol), water (15mL) and ethylene glycol dimethyl ether (15mL), triton B (catalyst) is added. The suspension is stirred at 50 ℃, and then acrylonitrile (0.40mL, 6.1mmol) is added. The reaction mixture is stirred at 50 ℃ and monitored by LCMS. At 16h, the reaction mixture is cooled to room temperature. The reaction mixture is concentrated and diluted with dichloromethane (100mL) and 5% NaHCO ₃ aqueous solution (100mL). The aqueous layer is extracted with dichloromethane (3x 30mL). The organic matter merged is passed through a hydrophobic glass frit, through Na ₂ SO ₄ drying, and concentrated. The crude material was purified via silica gel chromatography (0-10% methanol in dichloromethane) to afford tert-butyl N-{10-[(2-cyanoethyl)amino]decyl}carbamate (1.328 g, 3.150 mmol, 57.2%) as a white solid. UPLC/ELSD: RT = 0.53 min. MS (ES): m/z of C ₁₈ H ₃₅ N ₃ O ₂ = 326.48 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ4.49 (br.s, 1H), 3.09 (dt, 2H, J = 6.5, 6.3Hz), 2.93 (t, 2H, J = 6.6Hz), 2.62 (t, 2H, J = 7 .1Hz), 2.52 (t, 2H, J = 6.6Hz), 1.22-1.54 (m, 17H), 1.44 (s, 9H).

Step 2: tert-Butyl N-{10-[benzyl(2-cyanoethyl)amino]decyl}carbamate

Benzyl bromide (0.43 mL, 3.6 mmol) was added to a stirred suspension of N-{10-[(2-cyanoethyl)amino]decyl}carbamic acid tert-butyl ester (1.209 g, 2.867 mmol) and potassium carbonate (0.793 g, 5.74 mmol) in acetonitrile (18 mL). The reaction mixture was stirred at 70 ° C and monitored by LCMS. At 16 h, the reaction mixture was cooled to room temperature and then filtered through a celite pad, rinsed with acetonitrile. The filtrate was concentrated and then purified via silica gel chromatography (0-50% ethyl acetate in hexane) to give N-{10-[benzyl(2-cyanoethyl)amino]decyl}carbamic acid tert-butyl ester (1.283 g, quantitative) as a clear oil. UPLC/ELSD: RT=0.74 min. MS (ES): m/z of C ₂₅ H ₄₁ N ₃ O ₂ = 416.47 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ7.20-7.42 (m, 5H), 4.48 (br.s, 1H), 3.61 (s, 2H), 3.10 (td, 2H, J = 6.6, 6.3Hz), 2.78 (t,2H,J=7.0Hz),2.48(t,2H,J=7.2Hz),2.39(t,2H,J=7.0Hz),1.39-1.58(m,4H),1.44(s,9H),1.21-1.35(m,12H).

Step 3: tert-Butyl N-{3-[benzyl({10-[(tert-butoxycarbonyl)amino]decyl})amino]propyl}carbamate

By N-{10-[benzyl(2-cyanoethyl)amino]decyl}t-butyl carbamate (0.050g, 0.12mmol), di-tert-butyl dicarbonate (0.053g, 0.24mmol) and nickel(II) chloride (0.016g, 0.12mmol) are combined in ethanol (1.0mL). The stirred reaction mixture is cooled to 0 ° C in an ice bath, and then sodium borohydride (0.014g, 0.36mmol) is added. The reaction mixture is warmed to room temperature and monitored by LCMS. At 21h, diethylenetriamine (0.03mL, 0.3mmol) is added. The reaction mixture is stirred at room temperature for 2h and then filtered through a diatomaceous earth pad. The filtrate is concentrated, suspended in 5% _NaHCO3 aqueous solution (25mL), and extracted with ethyl acetate (3x 15mL). The combined organic matter is washed with _brine , dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-7% methanol in dichloromethane) to give tert-butyl N-{3-[benzyl({10-[(tert-butoxycarbonyl)amino]decyl})amino]propyl}carbamate (0.040 g, 0.077 mmol, 64.0%) as a clear oil. UPLC/ELSD: RT = 1.25 min. MS (ES): m/z of C ₃₀ H ₅₃ N ₃ O ₄ = 520.77 [M+H] ⁺ ; ¹ HNMR (300MHz, CDCl ₃ ): δ7.19-7.39 (m, 5H), 5.52 (br.s, 1H), 3.51 (s, 2H), 3.01-3.22 (m, 4H ), 2.46 (t, 2H, J = 6.1Hz), 2.37 (t, 2H, J = 7.1Hz), 1.37-1.68 (m, 6H), 1.44 (s, 9H), 1.44 (s, 9H), 1.15-1.35 (m, 12H).

Step 4: tert-Butyl N-[3-({10-[(tert-butoxycarbonyl)amino]decyl}amino)propyl]carbamate

By N-{3-[benzyl ({10-[(tert-butoxycarbonyl) amino] decyl}) amino] propyl} t-butyl carbamate (0.340g, 0.654mmol) and 10% Pd/C (0.139g, 0.065mmol) combination in ethanol (5.1mL) and then under _H2 balloon, stir at room temperature. Monitor the reaction by LCMS. At 18h, dilute the reaction mixture with ethyl acetate (10mL) and then filter through diatomaceous earth pad, rinse with ethyl acetate. The filtrate is concentrated, dissolved in ethyl acetate, filtered through 0.45μmPTFE syringe filter, and concentrated to obtain t-butyl N-[3-({10-[(tert-butoxycarbonyl) amino] decyl} amino) propyl] carbamate (0.238g, 0.554mmol, 84.7%) as a white solid. UPLC/ELSD: RT=0.97min. MS (ES): m/z of C ₂₃ H ₄₇ N ₃ O ₄ = 430.42 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.17 (br.s, 1H), 4.49 (br.s, 1H), 3.20 (dt, 2H, J = 6.1, 6.0Hz), 3.09 (dt, 2H, J = 6.6, 6 .4Hz), 2.67 (t, 2H, J = 6.6Hz), 2.58 (t, 2H, J = 7.1Hz), 1.17-1.76 (br.m, 19H), 1.44 (s, 18H).

Step 5: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(10-((tert-butoxycarbonyl)amino)decyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate

Cholesteryl carbonate 4-nitrophenyl ester (0.112 g, 0.203 mmol), tert-butyl N-[3-({10-[(tert-butoxycarbonyl)amino]decyl}amino)propyl]carbamate (0.103 g, 0.240 mmol), and triethylamine (0.088 mL, 0.63 mmol) were combined in toluene (3.0 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 17 h, the reaction mixture was heated at 100 °C. At 20 h, DMAP (catalyst) was added. At 41 h, the reaction mixture was cooled to room temperature, diluted with dichloromethane (20 mL), and then washed with 5% NaHCO ₃ in water (3x). The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (10%-30% ethyl acetate in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (10-((tert-butoxycarbonyl)amino)decyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.157 g, 0.186 mmol, 91.9%) as a clear oil. UPLC/ELSD: RT = 3.72 min. MS (ES): m/z of C ₅₁ H ₉₁ N ₃ O ₆ = 743.62 [(M+H)-(CH ₃ ) ₂ C = CH ₂ -CO ₂ ] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.20-5.43(m,2H),4.40-4.83(m,2H),3.02-3.37(m,8H),2.21-2.43(m,2H),1.75-2.10(m,5H),0.93-1.75(br.m,39H),1.44(s,9H),1.44(s,9H) ), 1.02 (s, 3H), 0.92 (d, 3H, J = 6.4Hz), 0.87 (d, 3H, J = 6.5Hz), 0.86 (d, 3H, J = 6.5Hz), 0.68 (s, 3H).

Step 6: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (10-aminodecyl)(3-aminopropyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (10-((tert-butoxycarbonyl)amino)decyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.154 g, 0.183 mmol) in isopropanol (2.5 mL) was added 5-6N HCl in isopropanol (0.28 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature and then acetonitrile (7.5 mL) was added. The solid was collected by vacuum filtration and rinsed with cold 3:1 acetonitrile/isopropanol to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(10-aminodecyl)(3-aminopropyl)carbamate dihydrochloride (0.080 g, 0.11 mmol, 59.2%) as a white solid. UPLC/ELSD: RT = 2.23 min. MS (ES): m/z of C ₄₁ H ₇₅ N ₃ O ₂ = 342.41 [(M+2H) + CH ₃ CN] ²⁺ ; ¹ HNMR (300MHz, CD ₃ OD): δ5.35-5.45(m,1H),4.36-4.51(m,1H),3.36(t,2H,J＝6.9Hz),3.26(t,2H,J＝7.4Hz),2.87-2.98(m,4H),2.27-2.43(m,2H),1.78-2.12(m,7H),0. 97-1.73(br.m,37H),1.06(s,3H),0.95(d,3H,J＝6.4Hz),0.88(d,3H,J＝6.6Hz),0.88(d,3H,J＝6.6Hz),0.73(s,3H).

AJ. Compound SA96: (10-aminodecyl)(3-aminopropyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(10-((tert-butoxycarbonyl)amino)decyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate

4-Nitrophenyl beta-sitosterol carbonate (0.140 g, 0.241 mmol), tert-butyl N-[3-({10-[(tert-butoxycarbonyl)amino]decyl}amino)propyl]carbamate (0.119 g, 0.277 mmol), and triethylamine (0.10 mL, 0.75 mmol) were combined in toluene (3.5 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 17 h, the reaction mixture was heated to 100 °C. At 20 h, DMAP (catalyst) was added. At 41 h, the reaction mixture was cooled to room temperature, diluted with dichloromethane (20 mL), and then washed with 5% NaHCO ₃ aqueous solution (3x). The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (10%-30% ethyl acetate in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (10-((tert-butoxycarbonyl)amino)decyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.187 g, 0.215 mmol, 89.0%). UPLC/ELSD: RT = 3.80 min. MS (ES): m/z of C ₅₃ H ₉₅ N ₃ O ₆ = 770.03 [(M+H)-(CH ₃ ) ₂ C = CH ₂ -CO ₂ ] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.18-5.43 (m, 2H), 4.40-4.83 (m, 2H), 2.97-3.41 (m, 8H), 2.21-2.44(m,2H),1.76-2.13(m,5H),0.87-1.74(br.m,40H),1.44(s,9H),1.44(s,9H),1.02(s,3H),0.92(d,3H,J=6.4Hz),0.78-0.87(m,9H),0.68( s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(10-aminodecyl)(3-aminopropyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (10-((tert-butoxycarbonyl)amino)decyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.182 g, 0.209 mmol) in isopropanol (2.5 mL) was added 5-6N HCl (0.28 mL) in isopropanol. The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature and then acetonitrile (8.25 mL) was added. The mixture was concentrated. Add methyl tert-butyl ether (about 10mL). Concentrate the mixture. Dissolve the residue in isopropanol (1.5mL) and then add dropwise to acetonitrile (10mL). Concentrate the mixture. Add acetonitrile/methyl tert-butyl ether/isopropanol (85:10:5, about 10mL). Concentrate the mixture. Dissolve the residue in isopropanol (1.5mL) and then add dropwise to 3:1 hexane/ethyl acetate (10mL). Concentrate the mixture. Dissolve the residue in isopropanol (1.5mL) and then add 9:1 acetonitrile/ethanol (10mL). Then add acetonitrile (10mL). The supernatant was decanted and the solid was dried under vacuum to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)(10-aminodecyl)(3-aminopropyl)carbamate dihydrochloride (0.075 g, 0.089 mmol, 42.7%) as a white solid. UPLC/ELSD: RT = 2.34 min. MS(ES)：C ₄₃ H ₇₉ N ₃ O ₂的m/z＝356.73[(M+2H)+CH ₃ CN] ²⁺ ； ¹ H NMR(300MHz,CD ₃ OD)：δ5.36-5.46(m,1H),4.36-4.52(m,1H),3.36(t,2H,J＝6.8Hz),3.26(t,2H,J＝7.3Hz),2.87-2.98(m,4H),2.27-2.44(m,2H),1.80-2.12(m,7H),0.91-1.77(br.m,38H),1.06(s,3H),0.96(d,3H,J＝6.4Hz),0.79-0.91(m,9H),0.73(s,3H)。

AK. Compound SA97: N-(8-aminooctyl)-N-(3-aminopropyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pyridine

By thiocholesterol (2.000g, 4.966mmol) and 2,2'-bipyridyl disulfide (1.204g, 5.463mmol) combination in chloroform (12.5mL).The reaction mixture is stirred at room temperature and monitored by LCMS.At 20h, the reaction mixture is concentrated, and then methanol (35mL) is added.The resulting mixture is left to stand for 2h. Afterwards, the solid was ground with methanol into a slurry by mortar and pestle, and then the solid was collected by vacuum filtration to give 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pyridine (1.812 g, 3.540 mmol, 71.3%) as a light brown solid. UPLC/ELSD: RT = 3.45 min. MS (ES): m/z of C ₃₂ H ₄₉ NS ₂ = 512.62 [M+H] ⁺ ; ¹ HNMR (300MHz, CDCl ₃ ): δ 8.39-8.49 (m, 1H), 7.72-7.83 (m, 1H), 7.57-7.69 (m, 1H), 7.01-7.12 (m, 1H), 5.27-5 .43(m,1H),2.70-2.88(m,1H),2.20-2.47(m,2H),0.78-2.11(br.m,38H),0.66(s,3H).

Step 2: 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)-1-methylpyridin-1-ium trifluoromethanesulfonate

To a solution of 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pyridine (1.807 g, 3.530 mmol) in dichloromethane (3.5 mL) and heptane (35 mL) was added methyl triflate (0.40 mL, 3.5 mmol) dropwise over 10 min. The reaction mixture was stirred at room temperature and monitored by TLC. At 4 h, triflate (0.08 mL) was added dropwise. At 4 h 40 min, the solid was collected via vacuum filtration, rinsing with heptane to give 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)-1-methylpyridin-1-ium trifluoromethanesulfonate (2.011 g, 2.975 mmol, 84.3%) as an off-white solid. ¹ H NMR (300MHz, CD ₃ CN): δ8.51-8.63(m,2H),8.30-8.41(m,1H),7.66-7.75(m,1H),5.34-5.45(m,1H),4.19(s,3H),2.87-3.06(m,1H),2.33-2.49(m ,2H),0.78-2.08(br.m,38H),0.69(s,3H).

Step 3: 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid

To a mixture of 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)-1-methylpyridin-1-ium trifluoromethanesulfonate (1.000 g, 1.479 mmol) in dimethylformamide (6.5 mL) was added 3-mercaptopropionic acid (0.14 mL, 1.6 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 21 h, more 3-mercaptopropionic acid (0.02 mL) was added. At 24 h, water (8 mL) was added, and the reaction mixture was stirred at room temperature for 30 min and then sonicated. The solid was collected by vacuum filtration and rinsed with water. The wet solid was then dissolved in dichloromethane and passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. Acetonitrile (40 mL) was added to the residue, which was then sonicated. The solid was collected by vacuum filtration to give 3-(((3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.541 g, 1.07 mmol, 72.2%) as a white solid. UPLC/ELSD: RT＝3.26min.; ¹ H NMR (300MHz, CDCl ₃ ): δ10.32(br.s,1H),5.31-5.40(m,1H),2.86-2.95(m,2H),2.75-2.84(m,2H),2.59-2.73(m,1H),2.23-2.41(m ,2H),1.74-2.08(m,5H),0.93-1.70(br.m,21H),1.00(s,3H),0.92(d,3H,J＝6.5Hz),0.87(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.68(s,3H).

Step 4: tert-Butyl (3-(N-(8-((tert-butoxycarbonyl)amino)octyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)propyl)carbamate

To a mixture of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)propanoic acid (0.200 g, 0.395 mmol), tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.222 g, 0.552 mmol) and N-hydroxysuccinimide (0.068 g, 0.59 mmol) in dichloromethane (6.0 mL) was added dicyclohexylcarbodiimide (0.138 g, 0.671 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17h, N-hydroxysuccinimide (15mg) and dicyclohexylcarbodiimide (35mg) were added. At 5 days, the reaction mixture was filtered through a diatomaceous earth pad and rinsed with dichloromethane. The filtrate was concentrated, dissolved in 9:1 hexane/ethyl acetate (10mL), filtered, and concentrated. The crude material was purified via silica gel chromatography (10%-50% ethyl acetate in hexanes) to afford tert-butyl (3-(N-(8-((tert-butoxycarbonyl)amino)octyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)propyl)carbamate (0.203 g, 0.228 mmol, 57.8%) as a clear oil. UPLC/ELSD: RT = 3.61 min. MS (ES): m/z of C ₅₁ H ₉₁ N ₃ O ₅ S ₂ = 790.32 [(M+H)-(CH ₃ ) ₂ C = CH ₂ -CO ₂ ] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.24-5.42(m,2H),4.42-4.67(m,1H),2.91-3.48(br.m,10H),2.57-2.78(m,3H),2.27-2.38(m,2H),0.93-2.09(br.m,40H),1.44(s,9H),1.43(s, 9H), 1.00 (s, 3H), 0.91 (d, 3H, J = 6.4Hz), 0.87 (d, 3H, J = 6.5Hz), 0.86 (d, 3H, J = 6.6Hz), 0.68 (s, 3H).

Step 5: N-(8-aminooctyl)-N-(3-aminopropyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a mixture of tert-butyl (3-(N-(8-((tert-butoxycarbonyl)amino)octyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)propyl)carbamate (0.200 g, 0.225 mmol) in isopropanol (3.0 mL) was added 5-6N HCl in isopropanol (0.32 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 17 h, the reaction mixture was cooled to room temperature and then acetonitrile (9 mL) was added. The material was concentrated and then dissolved in 4:1 acetonitrile/methanol (10 mL). The suspension was filtered and rinsed with methanol. The filtrate was concentrated, ground with 19:1 acetonitrile/ethanol (10 mL), dissolved in methanol, and concentrated to give N-(8-aminooctyl)-N-(3-aminopropyl)-3-(((3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.126 g, 0.154 mmol, 68.4%) as a white solid. UPLC/ELSD: RT=2.28 min. MS (ES): m/z _{for C41H75N3OS2} = 366.60 [(M+ _2H )+CH3CN] ₂₊ ^.

AL. Compound SA98 2-((2-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-oxoethyl)disulfanyl)acetate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 2-((2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-oxoethyl)disulfanyl)acetic acid

To the solution of cholesterol (5.00g, 12.93mmol) in anhydrous DCM (100mL) stirred under nitrogen, dithiodiglycolic acid (4.53mL, 25.86mmol) is added. The solution is then cooled to 0°C and dimethylaminopyridine (0.32g, 2.59mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (4.96g, 25.86mmol) are added, followed by dripping triethylamine (4.52mL, 25.86mmol). The solution is gradually warmed to room temperature and stirred overnight. The next day, the solution is washed with saturated sodium bicarbonate (1x25 mL) and water (1x25 mL), dried over sodium sulfate, filtered, and concentrated into a brown oil. The oil is dissolved in DCM and purified with 0-100% EtOAc gradient in hexane on silica. Fractions containing product were pooled and concentrated to give 2-((2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-oxoethyl)disulfanyl)acetic acid (3.76 g, 6.82 mmol, 52.7% ₎ as a dark brown solid. UPLC/ELSD: RT: 3.11 _{min .} MS (ES): m/z (MH ⁺ ) 551.8 _for C31H50O4S2. ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 9.04 (br.s, 1H), 5.41 (m, 1H), 4.69 (br.m, 1H), 3.65 (s, 2H), 3.60 (s, 1H), 2.39 (d, 2H, J = 9Hz), 2.01 (br.m, 5H), 1.52 (br.m, 11H) ), 1.16 (br.m, 6H), 1.04 (s, 6H), 0.95 (d, 3H, J = 6Hz), 0.86 (d, 6H, J = 6Hz), 0.70 (s, 3H).

Step 2: 12-(tert-Butoxycarbonyl)-7-(3-((tert-Butoxycarbonyl)amino)propyl)-19,19-dimethyl-6,17-dioxo-18-oxa-3,4-dithia-7,12,16-triazaeicosanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester

To 2-((2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-2-oxoethyl)disulfanyl)acetic acid (0.30 g, 0.55 mmol) was added under nitrogen. To a stirred solution in anhydrous DCM (5 mL) was added tert-butyl (3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.41 g, 0.82 mmol), dimethylaminopyridine (0.03 g, 0.27 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.26 g, 1.36 mmol). The reaction was allowed to proceed overnight at room temperature. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 x 10 mL) and brine (1 x 10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica with a 0-80% ethyl acetate in hexanes gradient to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 12-(tert-butoxycarbonyl)-7-(3-((tert-butoxycarbonyl)amino)propyl)-19,19-dimethyl-6,17-dioxo-18-oxa-3,4-dithia-7,12,16-triazaeicosanoate (0.33 g, 0.31 mmol, 57.6%). UPLC/ELSD: RT: 3.46min. MS (ES): m/z (MH ⁺ ) 1036.5 for C ₅₆ H ₉₈ N ₄ O ₉ S ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.33 (m, 2H), 4.61 (br.m, 1H), 3.72 (s, 2H), 3.51 (s, 2H), 3.29 (br.m, 11H), 2.28 (d, 2H, J = 6Hz), 1.81 (br.m, 6H), 1.50 (s, 26H) ,1.20(br.m,11H),0.97(s,5H),0.88(d,3H,J＝6Hz),0.82(d,5H,J＝6Hz),0.63(s,3H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 2-((2-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-2-oxoethyl)disulfanyl)acetate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 12-(tert-butoxycarbonyl)-7-(3-((tert-butoxycarbonyl)amino)propyl)-19,19-dimethyl-6,17-dioxo-18-oxa-3,4-dithia-7,12,16-triazaeicosanoate (0.33 g, 0.31 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.63 mL, 3.14 mmol) dropwise. The solution was heated to 40 DEG C and overnight. In the morning of the second day, the solution was cooled to room temperature, and anhydrous acetonitrile (10mL) was added to the mixture, the mixture was sonicated and stirred for another 1 hour. Then the white solid was filtered out from the solution, washed repeatedly with acetonitrile, and dried in a vacuum to obtain 2-((2-((3-aminopropyl))(4-((3-aminopropyl)amino)butyl)amino)-2-oxoethyl)disulfanyl)acetic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylheptyl-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester trihydrochloride (0.21g, 0.22mmol, 70.0%) as a white solid. UPLC/ELSD: RT=2.00min. MS (ES): m/z (MH ⁺ ) 735.7 for C ₄₁ H ₇₇ Cl ₃ N ₄ O ₃ S ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.43(m,1H),4.60(br.m,1H),3.90(m,2H),3.67(s,2H),3.53(m,5H),3.33(s,2H),3.11(m,9H),2.39(m,2H),1.98(br.m,10H ),1.55(br.m,13H),1.39(m,7H),1.18(br.m,6H),1.08(s,6H),0.98(d,4H,J＝6Hz),0.91(d,6H,J＝6Hz),0.75(s,3H).

AM. Compound SA110: N-(8-aminooctyl)-N-(3-aminopropyl)-5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pentanamide dihydrochloride

Step 1: 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pentanoic acid

To a stirred mixture of 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)-1-methylpyridin-1-ium trifluoromethanesulfonate (1.000 g, 1.479 mmol) in dimethylformamide (4.5 mL) was added 5-thiopentanoic acid (0.208 g, 1.55 mmol) in dimethylformamide (2.0 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 15 h, more 5-thiopentanoic acid (60 mg) in dimethylformamide (0.5 mL) was added. Water (20mL) is added at 40h, and the reaction mixture is stirred at room temperature for 15min and then sonicated. Solid is collected by vacuum filtration and rinsed with water. Solid is dissolved in dichloromethane, passed through hydrophobic glass frit, dried over Na ₂ SO ₄ , and concentrated. Acetonitrile (25mL) is added to residue, and suspension is sonicated. Solid is collected by vacuum filtration, rinsed in small amounts with cold acetonitrile, 5- (((3S, 8S, 9S, 10R, 13R, 14S, 17R) -10,13- dimethyl -17- ((R) -6- methyl hept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetrahydro -1H- cyclopenta [a] phenanthrene -3- bases) disulfanyl) valeric acid (0.604g, 1.13mmol, 76.3%) is obtained as white solid. UPLC/ELSD: RT=3.47min; ¹ H NMR (300MHz, CDCl ₃ ): δ10.10 (br.s, 1H), 5.30-5.48 (m, 1H), 2.57-2.77 (m, 3H), 2.22-2.46 (m, 4H), 0.94-2.08 (br.m, 30H), 1.01 (s, 3H), 0.92 (d, 3H, J = 6.5Hz), 0.87 (d, 3H, J = 6.6Hz), 0.86 (d, 3H, J = 6.6Hz), 0.68 (s, 3H).

Step 2: tert-Butyl (8-(N-(3-((tert-butoxycarbonyl)amino)propyl)-5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pentanamido)octyl)carbamate

To a mixture of 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)pentanoic acid (0.250 g, 0.467 mmol), tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.263 g, 0.654 mmol) and N-hydroxysuccinimide (0.081 g, 0.70 mmol) in dichloromethane (7.5 mL) was added dicyclohexylcarbodiimide (0.164 g, 0.795 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 50 h, N-hydroxysuccinimide (34 mg) and dicyclohexylcarbodiimide (72 mg) were added. Hexane (38 mL) was added at 92 h, and the reaction mixture was then filtered through a celite pad, rinsed with 5:1 hexane/dichloromethane. The filtrate was concentrated and then purified via silica gel chromatography (10%-50% ethyl acetate in hexanes) to give tert-butyl (8-(N-(3-((tert-butoxycarbonyl)amino)propyl)-5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pentanamido)octyl)carbamate (0.210 g, 0.229 mmol, 48.9%) as a clear oil. UPLC/ELSD: RT = 3.53 min. MS (ES): m/z of C ₅₃ H ₉₅ N ₃ O ₅ S ₂ = 919.93 [M+H] ⁺ ; ¹ H NMR (300MHz, CDCl ₃ ): δ5.23-5.48 (m, 2H), 4.38-4.67 (m, 1H), 2.99-3.45 (br.m, 8H), 2.56-2.76 (m, 3H , 0.68(s,3H).

Step 3: N-(8-aminooctyl)-N-(3-aminopropyl)-5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pentanamide dihydrochloride

To a mixture of tert-butyl (8-(N-(3-((tert-butoxycarbonyl)amino)propyl)-5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pentanamido)octyl)carbamate (0.208 g, 0.226 mmol) in isopropanol (3.0 mL) was added 5-6N HCl in isopropanol (0.32 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 24 h, the reaction mixture was diluted with methanol (3 mL) and filtered, rinsing with methanol. The filtrate was concentrated and the residue was then triturated with 19:1 acetonitrile/ethanol (2 x 3 mL). The residue was dissolved in methanol and then concentrated to give N-(8-aminooctyl)-N-(3-aminopropyl)-5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pentanamide dihydrochloride (0.111 g, 0.140 mmol, 62.0%) as a white foam. UPLC/ELSD: RT = 2.28 min. MS (ES): m/z of C ₄₃ H ₇₉ N ₃ OS ₂ = 359.81 [M+2H] ²⁺ .

¹ H NMR (300 MHz, DMSO, reported as visible in the spectrum): δ 7.67-8.29 (m, 8.78H), 5.25-5.43 (m, 1H), 3.14-3.43 (m, 7.91H), 2.58-2.86 (m, 10.97H), 2.17-2.39 (m, 5.35H), 0.79-2.07 (brm, 84.51H), 0.61-0.70 (m, 3.34H).

AN. Compound SA111 (3-aminobutyl)(4-((3-aminobutyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(4-((3-((tert-butoxycarbonyl)amino)butyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-4,2-diyl))dicarbamate (0.33 g, 0.77 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.32 mL, 2.30 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.44 g, 0.77 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, diluted with toluene, washed with water (3x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-70% (70:25:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(4-((3-((tert-butoxycarbonyl)amino)butyl)amino)butyl)carbamate as a light yellow oil (0.41 g, 0.48 mmol, 62.0%). UPLC/ELSD: RT = 2.76 _{min .} MS (ES): m/z (MH ⁺ ) 872.3 _{for C52H94N4O6} . ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 5.08(m,1H),4.67(br.m,1H),4.19(br.m,2H),3.42(m,2H),3.13(s,3H),2.90(br.m,4H),2.29(m,4H),2.05(m,4H),1.69(m,6 H), 1.35 (br.m, 14H), 1.14 (br.s, 17H), 0.99 (br.m, 6H), 0.86 (d, 9H, J = 6Hz), 0.73 (s, 5H), 0.64 (d, 5H, J = 6Hz), 0.55 (q, 8H, J = 6Hz), 0.38 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-aminobutyl)(4-((3-aminobutyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(4-((3-((tert-butoxycarbonyl)amino)butyl)amino)butyl)carbamate (0.41 g, 0.48 mmol) in isopropanol (7 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.95 mL, 4.75 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (10 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.30 g, 0.36 mmol, 74.8%) as a white solid. UPLC/ELSD: RT = 1.50 min. MS (ES): m/z (MH ⁺ ) 672.3 for C ₄₂ H ₈₁ Cl ₃ N ₄ O ₂ . ¹ H NMR (300MHz, MeOD) δ: ppm 5.41 (m, 1H), 4.48 (br.m, 1H), 3.48 (br.m, 2H), 3.33 (s, 7H), 3.17 (m, 3H), 2.39 (d, 2H, J = 3Hz), 1.92 (br.m, 8H), 1.73 (br.m, 10H), 1.37 (br.m, 9H), 1.17 (d, 4H, J = 6Hz), 1.07 (s, 5H), 0.98 (d, 5H, J = 6Hz), 0.86 (q, 8H, J = 6Hz), 0.74 (s, 3H).

AO. Compound SA113: (4-aminobutan-2-yl)(4-((4-aminobutan-2-yl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-3,1-diyl))dicarbamate (0.19 g, 0.43 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.18 mL, 1.31 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (4-nitrophenyl)carbonate (0.25 g, 0.44 mmol) was added. The solution was heated to 90° C. for 48 h. The reaction mixture was then cooled to room temperature, diluted with toluene, washed with water (3x20 mL), dried over sodium sulfate, filtered, and concentrated to give an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-80% (70:25:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (4-((tert-butoxycarbonyl)amino)butan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate as a light yellow oil (0.16 g, 0.18 mmol, 41.0%). UPLC/ELSD: RT = 2.50 min. MS (ES): m/z (MH ⁺ ) 872.3 for C ₅₂ H ₉₄ N ₄ O ₆ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm5.34(br.m,2H),4.48(br.m,1H),4.17(br.m,1H),3.15(br.m,6H),2.48(br.m,7H),1.95(br.m,7H),1.51(br.m,15H),1.39(s,25H ), 1.11 (br.m, 15H), 1.04 (d, 5H, J = 6Hz), 0.98 (s, 6H), 0.89 (d, 6H, J = 6Hz), 0.78 (q, 10H, J = 6Hz), 0.64 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(4-aminobutan-2-yl)(4-((4-aminobutan-2-yl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)amino)butan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)butan-2-yl)amino)butyl)carbamate (0.16 g, 0.18 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.36 mL, 1.80 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (10 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.11 g, 0.13 mmol, 72.0%) as a white solid. UPLC/ELSD: RT = 1.69 min. MS (ES): m/z (MH ⁺ ) 673.2 for C ₄₂ H ₈₁ Cl ₃ N ₄ O ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.43(m,1H),4.47(br.m,1H),4.12(m,1H),3.43(br.m,1H),3.33(s,4H),3.24(br.m,2H),3.13(m,5H),2.91(br.m,2H),2.41(d,2 H,J＝3Hz),1.99(br.m,10H),1.76(br.m,12H),1.43(d,6H,J＝6Hz),1.31(d,6H,J＝6Hz),1.18(br.m,6H),1.08(s,6H),0.99(d,5H,J＝6Hz),0.89(q,9H,J＝6Hz), 0.75(s,3H).

AP. Compound SA114 (3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2-methylbutane-4,2-diyl))dicarbamate (0.30 g, 0.65 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.32 mL, 2.30 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.38 g, 0.65 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, diluted with toluene, washed with water (3x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-70% (70:25:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)butyl)carbamate as a light yellow oil (0.36 g, 0.40 mmol, 60.5%). UPLC/ELSD: RT = 2.86 min. MS (ES): m/z (MH ⁺ ) 900.3 for C ₅₄ H ₉₈ N ₄ O ₆ . ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 5.63(m,1H),5.09(m,1H),5.01(s,1H),4.22(br.m,2H),2.93(br.m,4H),2.40(t,2H),2.32(t,2H),2.05(br.m,2H),1.59(br.m ,7H),1.26(br.m,13H),1.14(s,20H),1.02(d,16H,J＝9Hz),0.84(br.m,9H),0.73(s,6H),0.65(d,6H,J＝6Hz),0.56(q,10H,J＝6Hz),0.39(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)butyl)carbamate (0.36 g, 0.40 mmol) in isopropanol (7 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.79 mL, 3.96 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (10 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.24 g, 0.27 mmol, 69.3%) as a white solid. UPLC/ELSD: RT = 1.50 min. MS (ES): m/z (MH ⁺ ) 700.3 for C ₄₄ H ₈₅ Cl ₃ N ₄ O ₂ . ¹ H NMR (300MHz, MeOD) δ: ppm 5.42 (m, 1H), 4.44 (br.m, 1H), 3.94 (m, 1H), 3.48 (br.m, 2H), 3.33 (br.m, 8H), 3.15 (m, 4H), 2.40 (d, 2H, J = 3Hz), 2.12 (br.m, 10H), 1.74 (br.m, 12H), 1.42 (d, 16H, J = 6Hz), 1.18 (d, 11H, J = 6Hz), 1.08 (s, 6H), 0.98 (d, 5H, J = 6Hz), 0.87 (q, 9H, J = 6Hz), 0.75 (s, 3H).

AQ. Compound SA116: (3-amino-2,2-difluoropropyl)(4-((3-amino-2,2-difluoropropyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl(3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(2,2-difluoropropane-3,1-diyl))dicarbamate (0.37 g, 0.78 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.33 mL, 2.33 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (4-nitrophenyl)carbonate (0.45 g, 0.78 mmol) was added. The solution was heated to 90° C. for 48 h. The reaction mixture was then cooled to room temperature, diluted with toluene, washed with water (3 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-80% (70:25:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)amino)butyl)carbamate as a light yellow oil (0.05 g, 0.06 mmol, 7.3%). UPLC/ELSD: RT = 2.77 min. MS (ES): m/z (MH+) of C ₅₀ H ₈₆ F ₄ N ₄ O ₆ 916.3. 1H NMR (300MHz, CDCl3) δ: ppm 5.61(br.m,1H),5.41(br.m,1H),5.00(br.m,1H),4.55(br.m,1H),3.65(br.m,4H),3.33(br.m,2H),2.97(t,2H),2.69(t,1H),2.36(br.m,2H),1.87(br.m ,4H),1.59(br.m,7H),1.46(s,17H),1.14(br.m,14H),1.04(s,5H),0.95(d,4H,J＝6Hz),0.86(q,8H,J＝6Hz),0.70(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-2,2-difluoropropyl)(4-((3-amino-2,2-difluoropropyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)(4-((3-((tert-butoxycarbonyl)amino)-2,2-difluoropropyl)amino)butyl)carbamate (0.05 g, 0.06 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.11 mL, 0.57 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, anhydrous acetonitrile (10 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3-amino-2,2-difluoropropyl)(4-((3-amino-2,2-difluoropropyl)amino)butyl)carbamic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.04 g, 0.04 mmol, 77.1%) as a white solid. UPLC/ELSD: RT=1.63 min. MS (ES): m/z (MH ⁺ ) 716.1 for C ₄₀ H ₇₃ Cl ₃ F ₄ N ₄ O ₂ . ¹ H NMR (300MHz, MeOD) δ: ppm 5.32 (m, 1H), 4.38 (br.m, 1H), 3.81 (br.m, 6H), 3.33 (br.m, 4H), 3.22 (s, 5H), 3.08 (br.m, 2H), 2.28 (d, 2H, J = 3Hz), 1.93 (br.m, 5H) ,1.54(br.m,9H),1.26(br.m,6H),1.06(d,6H,J＝6Hz),0.97(s,5H),0.87(d,4H,J＝6Hz),0.77(q,7H,J＝6Hz),0.63(s,3H).

AR. Compound SA117: 5-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid

To a solution of sitosterol (2.50 g, 5.71 mmol) in acetone (30 mL) stirred under nitrogen, glutaric anhydride (1.17 g, 10.28 mmol) and triethylamine (1.43 mL, 10.28 mmol) were added. The reaction mixture was refluxed at 56 ° C, from a white slurry to a colorless clear solution, and was refluxed for 3 days. The solution was then cooled to room temperature, concentrated under vacuum, and dissolved in 150 mL of dichloromethane. This was then washed with 0.5 M HCl (1x100 mL), saturated aqueous ammonium chloride solution (1x100 mL), and water (1x100 mL), dried over sodium sulfate, filtered, and concentrated to a white solid. The solid was dissolved in dichloromethane and purified on silica with a 0-80% ethyl acetate in hexanes gradient to afford 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (2.33 g, 4.40 mmol, 77.1%) as a white solid. UPLC/ELSD: RT: 3.30 _min . MS (ES): m/z (MH ⁺ ) 529.8 _{for C34H56O4} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.38(m,1H),3.53(m,1H),2.31(br.m,3H),2.07(br.m,3H),1.98(br.m,3H),1.50(br.m,7H),1.26(br.m,12H),1.03(s,5H),0. 93 (d, 6H, J = 6Hz), 0.85 (q, 10H, J = 6Hz), 0.70 (s, 3H).

Step 2: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (1.50 g, 2.81 mmol) was stirred under nitrogen. To a solution in anhydrous DCM (25 mL) of 1% ethyl acetate (1.41 g, 2.81 mmol), dimethylaminopyridine (0.04 g, 0.28 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.09 g, 5.62 mmol) were added tert-butyl (3-(tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (1.41 g, 2.81 mmol), dimethylaminopyridine (0.04 g, 0.28 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.09 g, 5.62 mmol). The resulting solution was cooled to 0 °C and diisopropylethylamine (1.49 mL, 8.42 mmol) was added dropwise. The mixture was gradually warmed to room temperature for 48 h. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica with a 0-80% (70:25:5 DCM/MeOH/ _NH4OH ) gradient in DCM. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate as a light yellow oil (0.41 g, 0.40 mmol, 14.3%). UPLC/ELSD: RT: 3.29 min. MS (ES): m/z (MH ⁺ ) 1014.5 for C ₄₉ H ₁₀₄ N ₄ O ₉ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.41 (m, 2H), 4.52 (br.m, 1H), 4.14 (m, 1H), 3.16 (br.m, 13H), 2.29 (br.m, 7H), 1.75 (br.m, 21H), 1.35 (d, 33H, J = 6Hz), 1.09 (br.m ,12H),0.93(s,7H),0.85(d,5H,J＝6Hz),0.76(q,9H,J＝6Hz),0.59(s,3H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminopropyl)(4-((3-aminopropyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-olate (0.41 g, 0.40 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.80 mL, 4.01 mmol) dropwise. The solution was heated to 40 ° C and overnight. In the morning of the next day, anhydrous acetonitrile (15mL) was added to the mixture, the mixture was sonicated and stirred for another 1 hour. Then the white solid was filtered out from the solution, washed repeatedly with acetonitrile, and dried in a vacuum to obtain 5-((3-aminopropyl) (4-((3-aminopropyl) amino) butyl) amino)-5-oxopentanoic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylheptyl-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester trihydrochloride (0.24g, 0.27mmol, 66.1%) as a white solid. UPLC/ELSD: RT=1.64min. MS (ES): m/z (MH ⁺ ) 714.3 for C ₄₄ H ₈₃ Cl ₃ N ₄ O ₃ . ¹ H NMR (300MHz, MeOD) δ: ppm5.40(m,1H),4.56(br.m,1H),3.95(m,1H),3.52(br.m,3H),3.33(s,3H),3.15(br.m,6H),2.42(br.m,5H),1.91(br.m,10H),1.54 (br.m,7H),1.32(br.m,7H),1.17(d,4H,J＝6Hz),1.06(s,4H),0.97(d,4H,J＝6Hz),0.88(q,7H,J＝6Hz),0.74(s,3H).

AS. Compound SA119: N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)glycine (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester tetrahydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloroacetate

To the solution of sitosterol (2.00g, 2.57mmol) in anhydrous dichloroethane (22.84mL), DBU (2.05mL, 13.70mmol) is added. The reactant is cooled to 0°C, and a solution of chloroacetyl chloride (0.73mL, 9.13mmol) in 5mL dichloroethane is added dropwise to the reaction mixture so that a turbid dark brown mixture is changed from a clear colorless solution. The mixture is gradually warmed to room temperature and stirred overnight. The next morning, TLC showed that the reaction was not complete, so the mixture was cooled to 0°C again and 0.50mL DBU and 0.20mL chloroacetyl chloride were added again. The mixture was warmed to room temperature, and the reaction was completed according to TLC after 2 hours. The mixture was cooled to 0°C again, and 30mL water was added. After being warmed to room temperature, the water layer was separated and washed with DCM (3x30 mL), and all organic layers were combined, dried over sodium sulfate, filtered, and concentrated to obtain a brown oil. The oil was dissolved in DCM and purified on silica with a 0-20% EtOAc gradient in hexanes. The fractions containing the product were pooled and concentrated to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloroacetate (1.40 g, 2.84 mmol, 62.2%) as a white solid. UPLC/ELSD: RT: 3.49 min. MS (ES): m/z (MH ⁺ ) 492.2 for C ₃₁ H ₅₁ ClO ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.41 (m, 1H), 4.70 (br.m, 1H), 4.06 (s, 2H), 2.40 (d, 2H, J = 6Hz), 1.93 (br.m, 5H), 1.52 (br.m, 7H), 1.20 (br.m, 11H), 1.05 (s, 6H) ), 0.96 (d, 5H, J = 6Hz), 0.85 (q, 9H, J = 6Hz), 0.70 (s, 3H).

Step 2: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

(3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloroacetate (0.51 g, 1.04 mmol) and tert-butyl (3-((tert-butoxycarbonyl)amino)propyl)(4-((3-((tert-butoxycarbonyl)amino)propyl)amino)butyl)carbamate (0.71 g, 1.41 mmol) were combined in a vial and purged with three vacuum and nitrogen cycles. This was then dissolved in anhydrous THF (10.42 mL) and triethylamine (0.29 mL, 2.09 mmol) was added. The mixture is stirred under nitrogen, heated to 65 ℃, and stirred for 48h.Then, the mixture is cooled to room temperature and diluted with ethyl acetate (30mL) and saturated sodium bicarbonate aqueous solution (30mL).The aqueous layer is separated and extracted with EtOAc (3x30 mL).All organic layers are combined, washed with saline (1x20 mL), dried over sodium sulfate, filtered, and concentrated into a yellow oil.The oil is dissolved in DCM and purified with 0-70% EtOAc gradient in hexane on silica. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oate as a light yellow oil (0.47 g, 0.49 mmol, 46.8%). UPLC/ELSD: RT: 2.90 min. MS (ES): m/z (MH ⁺ ) 958.4 for C ₅₆ H ₁₀₀ N ₄ O ₈ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.39 (m, 1H), 4.64 (br.m, 1H), 4.14 (m, 1H), 3.26 (br.m, 9H), 2.60 (m, 4H), 2.35 (d, 2H, J = 6Hz), 2.05 (br.m, 6H), 1.65 (br.m, 8H), 1.47(br.s,30H),1.20(br.m,11H),1.03(s,5H),0.95(d,5H,J＝6Hz),0.86(q,8H,J＝6Hz),0.69(s,3H).

Step 3: N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)glycine (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester tetrahydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-olate (0.47 g, 0.49 mmol) in isopropanol (7 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 1.17 mL, 5.85 mmol) dropwise. The solution was heated to 40 ° C and overnight. The next morning, the solution was cooled to room temperature and anhydrous acetonitrile (15mL) was added to the mixture, the mixture was sonicated and stirred for another 1 hour. Then the white solid was filtered out from the solution, washed repeatedly with acetonitrile, and dried in a vacuum to obtain N- (3- aminopropyl) -N- (4- ((3- aminopropyl) amino) butyl) glycine (3S, 8S, 9S, 10R, 13R, 14S, 17R) -17- ((2R, 5R) -5- ethyl -6- methyl heptyl -2- bases) -10, 13- dimethyl -2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17- tetrahydro -1H- cyclopenta [a] phenanthrene -3- bases ester tetrahydrochloride (0.36g, 0.41mmol, 83.5%) as a white solid. UPLC/ELSD: RT=1.71min. MS (ES): m/z (MH ⁺ ) 658.1 for C ₄₁ H ₈₀ Cl ₄ N ₄ O ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm5.42(m,1H),4.73(br.m,2H),4.30(br.m,2H),3.93(m,1H),3.32(br.m,6H),3.10(br.m,8H),2.43(br.s,2H),2.17(br.m,4H),2.03 (br.m,10H),1.53(br.m,8H),1.31(br.m,9H),1.15(d,6H,J＝6Hz),1.05(s,6H),0.95(d,5H,J＝6Hz),0.86(q,9H,J＝6Hz),0.72(s,3H).

AT. Compound SA120: (4-aminobutyl)(3-aminopropyl)carbamic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)amino)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate

4-Nitrophenyl β-sitosterol carbonate (0.200 g, 0.345 mmol), tert-butyl N-[3-({4-[(tert-butoxycarbonyl)amino]butyl}amino)propyl]carbamate (0.155 g, 0.448 mmol), and triethylamine (0.15 mL, 1.1 mmol) were combined in toluene (3.5 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 21 h, the reaction mixture was cooled to room temperature, diluted with dichloromethane (20 mL), and washed with 5% aqueous NaHCO ₃ solution (3 x 10 mL). The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (10%-50% ethyl acetate in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)amino)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.210 g, 0.267 mmol, 77.4%). UPLC/ELSD: RT = 3.37 min. MS (ES): m/z of C ₄₇ H ₈₃ N ₃ O ₆ = 787.67 [M+H] ⁺ ; ¹ HNMR (300MHz, CDCl ₃ ): δ5.10-5.44 (m, 2H), 4.42-4.89 (m, 2H), 3.01-3.40 (br.m, 8H), 2.22-2.42 (m, 2H), 1 .76-2.09(m,5H),0.88-1.75(br.m,28H),1.44(s,18H),1.02(s,3H),0.92(d,3H,J＝6.4Hz),0.78-0.88(m,9H),0.68(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(4-aminobutyl)(3-aminopropyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) (4-((tert-butoxycarbonyl)amino)butyl)(3-((tert-butoxycarbonyl)amino)propyl)carbamate (0.203 g, 0.258 mmol) in isopropanol (3.0 mL) was added 5-6N HCl in isopropanol (0.37 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. Acetonitrile (9 mL) was added at 24 h and the reaction mixture was stirred for 5 min. After this time, the solid was collected by vacuum filtration and rinsed with 3:1 acetonitrile/isopropanol to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)(4-aminobutyl)(3-aminopropyl)carbamate dihydrochloride (0.151 g, 0.218 mmol, 84.4%) as a white solid. UPLC/ELSD: RT = 1.93 min. MS (ES): m/z of C ₃₇ H ₆₇ N ₃ O ₂ = 586.67 [M+H] ⁺ ; ¹ H NMR (300MHz, CD ₃ OD): δ 5.38-5.47 (m, 1H), 4.39-4.55 (m, 1H), 3.28-3.46 (m, 4H), 2.91-3.06 (m, 4H), 2. 31-2.47(m,2H),1.83-2.14(m,7H),0.93-1.79(br.m,26H),1.08(s,3H),0.98(d,3H,J＝6.4Hz),0.82-0.93(m,9H),0.75(s,3H).

AU. Compound SA118: N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)alanine (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester tetrahydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloropropanoate

1,8-diazabicyclo [5.4.0] 11-7-ene (1.28 mL, 8.56 mmol) was added to a solution of β-sitosterol (1.25 g, 2.85 mmol) in anhydrous DCM (15 mL) stirred under nitrogen. The reactant was cooled to 0 ° C, and a solution of 2-chloropropionyl chloride (0.55 mL, 5.71 mmol) in 5 mL of anhydrous DCM was added dropwise over 20 minutes, so that the solution changed from a clear colorless mixture to a turbid dark brown mixture. The reaction mixture was gradually warmed to room temperature and carried out overnight. The next morning, the reaction was not complete via thin layer chromatography (7: 3 hexane/ethyl acetate, PMA staining), so the reaction mixture was cooled to 0 ° C again, and 0.50 mL 1,8-diazabicyclo [5.4.0] 11-7-ene was added again. The reactant was warmed to room temperature. After 1 hour, the reaction was complete as shown by TLC. The reaction mixture was cooled to 0 ° C and quenched with 20 mL of water. After the mixture is warmed to room temperature, each layer is separated, and the aqueous layer is extracted with DCM (3x30 mL). All organic layers are combined, dried over sodium sulfate, filtered, and concentrated into a dark brown oil. The oil is dissolved and purified with a 0-20% ethyl acetate gradient on silica in hexane. The fractions containing the product are collected and concentrated to obtain 2-chloropropionic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R) -17- ((2R, 5R) -5- ethyl -6- methyl hept-2-yl) -10, 13- dimethyl -2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetrahydro -1H- cyclopenta [a] phenanthrene -3-yl ester (1.12 g, 2.22 mmol, 77.7%) as a white solid. UPLC / ELSD: RT = 3.39 min. MS (ES): m/z (MH ⁺ ) 506.2 for C ₃₂ H ₅₃ ClO ₂ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.44(m,1H),4.48(br.m,1H),3.95(m,1H),3.33(br.m,5H),3.11(br.m,8H),2.45(br.m,7H),1.99(br.m,7H),1.68(br.m,11H) ,1.37(br.m,9H),1.15(d,8H,J＝6Hz),1.08(s,6H),0.95(d,5H,J＝6Hz),0.86(q,9H),0.74(s,3H).

Step 2: 9-(tert-Butoxycarbonyl)-14-(3-((tert-Butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 2-chloropropanoate (1.12 g, 2.22 mmol) and tert-butyl N-{3-[(tert-butoxycarbonyl)amino]propyl}-N-[4-({3-[(tert-butoxycarbonyl)amino]propyl}amino)butyl]carbamate (1.34 g, 2.66 mmol) in anhydrous THF (22 mL) stirred under nitrogen was added triethylamine (0.62 mL, 4.43 mmol). The mixture was heated to 65°C for one week, during which time very little product was formed as monitored by LCMS. After one week, the mixture was cooled to room temperature and concentrated in vacuo to an oil. The oil was dissolved in DCM and purified on silica with a 0-50% ethyl acetate gradient in hexanes. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-oate as a light yellow oil (0.09 g, 0.10 mmol, 4.4%). UPLC/ELSD: RT = 2.79 min. MS (ES): m/z (MH ⁺ ) 972.5 for C ₅₇ H ₁₀₂ N ₄ O ₈ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.31 (m, 1H), 4.54 (br.m, 1H), 3.39 (m, 1H), 3.07 (br.m, 7H), 2.52 (br.m, 4H), 2.23 (d, 2H, J = 6Hz), 1.77 (br.m, 6H), 1.54 (br.m, 9 H), 1.36 (d, 30H, J = 9Hz), 1.17 (br.m, 14H), 0.95 (s, 5H), 0.86 (d, 5H, J = 6Hz), 0.75 (q, 8H), 0.61 (s, 3H).

Step 3: N-(3-aminopropyl)-N-(4-((3-aminopropyl)amino)butyl)alanine (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester tetrahydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 9-(tert-butoxycarbonyl)-14-(3-((tert-butoxycarbonyl)amino)propyl)-2,2,15-trimethyl-4-oxo-3-oxa-5,9,14-triazahexadecane-16-olate (0.09 g, 0.10 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.19 mL, 0.97 mmol) dropwise. The solution is heated to 40 DEG C and carried out overnight.The next morning, the mixture is cooled to room temperature and anhydrous acetonitrile (20mL) is added to the mixture, the mixture is sonicated and stirred for another 1 hour.Then white solid is filtered out from the solution, washed repeatedly with acetonitrile, and dried in a vacuum, N-(3-aminopropyl)-N-(4-((3-aminopropyl) amino) butyl) alanine (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methyl heptyl-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester tetrahydrochloride (0.07g, 0.07mmol, 76.9%) is obtained as white solid. UPLC/ELSD: RT=1.61min. MS (ES): m/z (MH ⁺ ) 672.2 for C ₄₂ H ₈₂ Cl ₄ N ₄ O ₂ . ¹ H NMR (300MHz, MeOD) δ: ppm5.44(m,1H),4.48(br.m,1H),3.95(m,1H),3.32(s,5H),3.11(br.m,8H),2.27(br.m,7H),1.99(br.m,7H),1.68(br.m,11H),1.37 (br.m,9H),1.15(d,8H,J＝6Hz),1.08(s,6H),0.95(d,5H,J＝6Hz),0.86(q,9H),0.74(s,3H).

AV. Compound SA121: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

Step 1: 14-(3-((tert-Butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 5-{[(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylhept-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl]oxy}-5-oxopentanoic acid (0.23 g, 0.45 mmol) was stirred in anhydrous DCM under nitrogen. To the solution in 4% paraformaldehyde (10 mL), tert-butyl N-(4-{[4-({3-[(tert-butoxycarbonyl)amino]-3-methylbutyl}amino)butyl]amino}-2-methylbutyl-2-yl)carbamate (0.23 g, 0.49 mmol), dimethylaminopyridine (0.01 g, 0.09 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.17 g, 0.89 mmol) were added. The resulting solution was cooled to 0 ° C and diisopropylethylamine (0.24 mL, 1.34 mmol) was added dropwise. The mixture was gradually warmed to room temperature and left overnight. The solution was then diluted with dichloromethane, washed with saturated sodium bicarbonate aqueous solution (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica with a 0-80% (70:25:5 DCM/MeOH/ _NH4OH ) gradient in DCM. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate as a light yellow oil (0.09 g, 0.09 mmol, 20.2%). UPLC/ELSD: RT: 2.74 min. MS (ES): m/z (MH ⁺ ) 942.4 for C ₅₆ H ₁₀₀ N ₄ O ₇ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm5.30(m,1H),4.52(br.m,2H),3.23(m,4H),2.93(s,1H),2.61(t,2H)2.54(t,2H),2.29(br.m,6H),1.87(br.m,10H),1.62(m,3H ),1.49(m,6H),1.36(d,24H,J＝3Hz),1.23(br.m,17H),1.06(br.m,7H),0.94(s,7H),0.85(d,4H,J＝9Hz),0.81(d,7H,J＝9Hz),0.61(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 14-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate (0.06 g, 0.06 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.12 mL, 0.61 mmol) dropwise. The solution was heated to 42° C. overnight. The next morning, anhydrous acetonitrile (15 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.03 g, 0.04 mmol, 58.0%) as a white solid. UPLC/ELSD: RT = 1.62 min. MS (ES): m/z (MH ⁺ ) 742.3 for C ₄₆ H ₈₇ Cl ₃ N ₄ O ₃ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.42(m,1H),4.57(br.m,1H),3.48(m,4H),3.33(br.m,3H),3.16(br.m,4H),2.48(br.m,5H),2.14(m,2H),1.91(br.m,10H),1.5 4(br.m,6H),1.42(br.m,14H),1.16(m,6H),1.06(s,5H),0.97(d,3H,J=6Hz),0.91(q,5H,J=6Hz),0.74(s,3H).

AW. Compound SA122: N-(8-aminooctyl)-N-(5-aminopentanoyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (8-((tert-Butyloxycarbonyl)amino)octyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

By N-(8-aminooctyl) tert-butyl carbamate (0.994g, 4.07mmol), cholesteryl chloroacetate (1.000g, 2.159mmol), potassium iodide (0.072g, 0.43mmol) and potassium carbonate (0.597g, 4.32mmol) are combined in dioxane (15mL) in a sealed tube. Monitor the reaction mixture by LCMS. While stirring, the reaction mixture is irradiated with microwaves at 140 ℃ for 3h. While stirring, the reaction mixture is irradiated with microwaves at 150 ℃ for 3h, cooled to room temperature, and filtered through a celite pad rinsed with EtOAc. The filtrate is concentrated and then dissolved in DCM (100mL). The organic matter is washed with water, passed through a hydrophobic glass frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (30%-70% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-(8-((tert-butoxycarbonyl)amino)octyl)glycine as a viscous amber oil (0.993 g, 1.48 mmol, 68.5%). UPLC/ELSD: RT = 2.56 min. MS (ES): m/z = 672.06 [M+H] ⁺ for C ₄₂ H ₇₄ N ₂ O ₄ . ¹ H NMR(300MHz,CDCl ₃ )：δ5.35-5.43(m,1H),4.60-4.76(m,1H),4.50(br.s,1H),3.46(s,2H),2.99-3.17(m,2H),2.69(t,2H,J＝7.3Hz),2.28-2.40(m,2H),1.72-2.08(m,5H),0.93-1.71(br.m,33H),1.44(s,9H),1.02(s,3H),0.91(d,3H,J＝6.5Hz),0.87(d,3H,J＝6.5Hz),0.86(d,3H,J＝6.6Hz),0.68(s,3H)。

Step 2: N-(8-((tert-Butoxycarbonyl)amino)octyl)-N-(5-((tert-Butoxycarbonyl)amino)pentanoyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

(8-((tert-butoxycarbonyl)amino)octyl)glycine (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.150 g, 0.224 mmol), 5-[(tert-butoxycarbonyl)amino]pentanoic acid (0.058 g, 0.268 mmol) and DMAP (catalyst) were combined in DCM (3.0 mL). The reaction mixture was cooled to 0 ° C in an ice bath and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.064 g, 0.34 mmol) was added. The reaction mixture was stirred at room temperature and monitored by LCMS. At 20 h, the reaction mixture was cooled to 0 °C in an ice bath, and then water (3 mL) was added. The biphasic mixture was diluted with DCM (5 mL). The layers were separated, and the aqueous solution was extracted with DCM ( ₅ mL). The combined organics were passed through a hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-60% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl N-(8-((tert-butoxycarbonyl)amino)octyl)-N-(5-((tert-butoxycarbonyl)amino)pentanoyl)glycine as a clear oil (0.185 g, 0.213 mmol, 95.1%). UPLC/ELSD: RT = 3.27 min. MS (ES): m/z of C ₅₂ H ₉₁ N ₃ O ₇ = 872.43 [M+H] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ5.32-5.47(m,1H),4.29-4.78(m,3H),3.92-4.06(m,2H),3.23-3.42(m,2H),3.01-3.21(m,4H),2.18-2.45(m,4H),1.05-2. 10(br.m,60H),1.01(s,3H),0.91(d,3H,J＝6.2Hz),0.87(d,6H,J＝6.5Hz),0.68(s,3H).

Step 3: N-(8-aminooctyl)-N-(5-aminopentanoyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

To a solution of (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-N-(8-((tert-butoxycarbonyl)amino)octyl)-N-(5-((tert-butoxycarbonyl)amino)pentanoyl)glycine (0.237 g, 0.272 mmol) in iPrOH (3.5 mL) was added 5-6N HCl (0.39 mL) in iPrOH. The reaction mixture was stirred at 40 °C and monitored by LCMS. At 16 h, the reaction mixture was cooled to room temperature and then ACN (10.5 mL) was added. The solid was collected by vacuum filtration and rinsed with 3:1 ACN/iPrOH to give N-(8-aminooctyl)-N-(5-aminopentanoyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride (0.150 g, 0.185 mmol, 68.1%) as a white solid. UPLC/ELSD: RT = 1.94 min. MS (ES): m/z = 336.11 [M+2H] ²⁺ for C ₄₂ H ₇₅ N ₃ O ₃ . ¹ H NMR (300MHz, DMSO): δ7.90(br.s,6H),5.31-5.40(m,1H),4.39-4.61(m,1H),3.89-4.24(m,2H),3.18-3.39(m,2H),2.65-2.88(m,4H),2.13-2.41(m, 4H),1.70-2.05(m,5H),0.91-1.67(br.m,37H),0.98(s,3H),0.89(d,3H,J＝6.3Hz),0.84(d,6H,J＝6.5Hz),0.65(s,3H).

AX. Compound SA123: N-(6-aminohexanoyl)-N-(8-aminooctyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: N-(6-((tert-butoxycarbonyl)amino)hexanoyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

(8-((tert-butoxycarbonyl)amino)octyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.200 g, 0.298 mmol), 6-[(tert-butoxycarbonyl)amino]hexanoic acid (0.090 g, 0.39 mmol) and DMAP (catalyst) were combined in DCM (4.0 mL). The reaction mixture was cooled to 0 °C in an ice bath and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.086 g, 0.45 mmol) was added. The reaction mixture was stirred at room temperature and monitored by LCMS. At 20 h, the reaction mixture was cooled to 0 °C in an ice bath, and then water (4 mL) was added. The biphasic mixture was diluted with DCM (5 mL). The layers were separated, and the aqueous solution was extracted with DCM ( ₅ mL). The combined organics were passed through a hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-60% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl N-(6-((tert-butoxycarbonyl)amino)hexanoyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine as a clear oil (0.260 g, 0.294 mmol, 98.6%). UPLC/ELSD: RT = 3.28 min. MS (ES): m/z of C ₅₃ H ₉₃ N ₃ O ₇ = 885.39 [M+H] ⁺ . ¹ HNMR (300MHz, CDCl ₃ ): δ5.27-5.51(m,1H),4.28-4.76(m,3H),3.94-4.05(m,2H),3.23-3.41(m,2H),3.02-3.20(m,4H),2.16-2.43(m,4H),0.93-2.1 1(br.m,65H),0.91(d,3H,J＝6.5Hz),0.87(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.63-0.75(m,3H).

Step 2: N-(6-aminohexanoyl)-N-(8-aminooctyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl N-(6-((tert-butoxycarbonyl)amino)hexanoyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine (0.251 g, 0.284 mmol) in iPrOH (4.0 mL) was added 5-6N HCl in iPrOH (0.40 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 16 h, the reaction mixture was cooled to room temperature and then ACN (12 mL) was added. The solid was collected by vacuum filtration and rinsed with 3:1 ACN/iPrOH to give N-(6-aminohexanoyl)-N-(8-aminooctyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride (0.163 g, 0.198 mmol, 69.8%) as a white solid. UPLC/ELSD: RT = 1.97 min. MS (ES): m/z = 343.02 [M+2H] ²⁺ for C ₄₃ H ₇₇ N ₃ O ₃ . ¹ H NMR (300MHz, CD ₃ OD): δ5.37-5.47(m,1H),4.51-4.70(m,1H),4.02-4.25(m,2H),3.35-3.49(m,2H),2.87-3.01(m,4H),2.28-2.56(m,4H),1.79-2. 14(m,5H),0.99-1.78(br.m,39H),1.07(s,3H),0.96(d,3H,J＝6.4Hz),0.90(d,6H,J＝6.6Hz),0.74(s,3H).

AY. Compound SA124: (2-(1-aminocyclopropyl)ethyl)(4-((2-(1-aminocyclopropyl)ethyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (1-(2-((2-nitrophenyl)sulfonamido)ethyl)cyclopropyl)carbamate

To a solution of tert-butyl N-[1-(2-aminoethyl)cyclopropyl]carbamate (2.00 g, 9.49 mmol) in anhydrous DCM (25 mL) stirred under nitrogen was added triethylamine (2.64 mL, 18.98 mmol). The solution was cooled to 0°C and then a solution of 2-nitrobenzenesulfonyl chloride (2.31 g, 10.44 mmol) in 25 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with another 10 mL of DCM, washed with 1 M aqueous sodium bicarbonate solution (2x15 mL), water (1x15 mL), 10% aqueous citric acid solution (2x15 mL), water (1x15 mL), and brine (2x15 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (1-(2-((2-nitrophenyl)sulfonamido)ethyl)cyclopropyl)carbamate (3.73 g, 9.70 mmol, quant.) as a white solid. UPLC/ _ELSD : RT = 0.59 min _. MS (ES): m/z (MH ⁺ ) 386.4 _{for Ci6H23N3O6S} . ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 8.13(m,1H),7.84(m,1H),7.73(m,2H),6.45(br.s,1H),4.89(br.s,1H),3.27(q,2H),1.73(t,2H),1.38(s,9H),0.82(br.m,2 H),0.69(br.s,2H).

Step 2: Di-tert-butyl (((butane-1,4-diylbis(azanediyl))bis(ethane-2,1-diyl))bis(cyclopropane-1,1-diyl))dicarbamate

To a solution of tert-butyl (1-(2-((2-nitrophenyl)sulfonamido)ethyl)cyclopropyl)carbamate (3.74 g, 9.70 mmol) in anhydrous DMF (50 mL) stirred under nitrogen was added potassium carbonate (3.89 g, 28.16 mmol) and 1,4-diiodobutane (0.61 mL, 4.62 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.46 mL, 3.83 mmol) was added and the reaction was allowed to proceed for 8 h at room temperature. Thiophenol (1.82 mL, 17.78 mmol), potassium carbonate (1.91 g, 13.85 mmol) and an additional 10 mL of anhydrous DMF were then added and the reaction was allowed to proceed overnight. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x5 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a gradient of 0-60% (70:20:10 DCM/MeOH/concentrated aqueous ammonium hydroxide solution). The fractions containing the product were combined and concentrated to give di-tert-butyl (((butane-1,4-diylbis(azanediyl))bis(ethane-2,1-diyl))bis(cyclopropane-1,1-diyl))dicarbamate (0.47 g, 1.04 mmol, 22.5%) as a colorless oil. UPLC/ELSD: RT=0.35 min. MS (ES): m/z (MH ⁺ ) 455.6 for C ₂₄ H ₄₆ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.03 (m, 2H), 2.76 (t, 4H), 2.64 (m, 4H), 1.72 (m, 4H), 1.59 (m, 4H), 1.44 (s, 17H), 0.80 (m, 4H), 0.66 (m, 4H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (2-(1-((tert-butoxycarbonyl)amino)cyclopropyl)ethyl)(4-((2-(1-((tert-butoxycarbonyl)amino)cyclopropyl)ethyl)amino)butyl)carbamate

To a solution of di-tert-butyl (((butane-1,4-diylbis(azanediyl))bis(ethane-2,1-diyl))bis(cyclopropane-1,1-diyl))dicarbamate (0.47 g, 1.03 mmol) in anhydrous toluene (20 mL) stirred under nitrogen was added triethylamine (0.43 mL, 3.08 mmol). Then add (3S, 8S, 9S, 10R, 13R, 14S, 17R) -10, 13- dimethyl -17- ((R) -6- methyl hept-2-yl) -2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17- tetrahydro -1H- cyclopenta [a] phenanthrene -3-yl ester (0.57g, 1.03mmol) of (4- nitrophenyl) carbonate, and the solution is heated to 90 ℃ and carried out overnight. The next morning, the reaction mixture is cooled to room temperature, diluted with toluene, washed with water (3x15 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil is dissolved in DCM and purified via silica gel chromatography in DCM with a gradient of 0-70% (70: 25: 5DCM / MeOH / concentrated aqueous ammonium hydroxide solution). Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (2-(1-((tert-butoxycarbonyl)amino)cyclopropyl)ethyl)(4-((2-(1-((tert-butoxycarbonyl)amino)cyclopropyl)ethyl)amino)butyl)carbamate as a colorless oil (0.35 g, 0.41 mmol, 39.4%). UPLC/ELSD: RT = 2.65 min. MS (ES): m/z (MH ⁺ ) 868.3 for C ₅₂ H ₉₀ N ₄ O ₆ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm5.80(m,1H),5.40(m,1H),4.96(br.m,2H),4.50(m,1H),3.42(m,2H),3.15(m,2H),2.74(t,2H),2.61(m,2H),2.34(m,2H),2. 00(m,5H),1.70(m,4H),1.56(br.m,8H),1.44(s,18H),1.15(br.m,10H),1.03(s,5H),0.94(d,4H,J＝6Hz),0.89(d,5H,J＝6Hz),0.69(br.m,11H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (2-(1-aminocyclopropyl)ethyl)(4-((2-(1-aminocyclopropyl)ethyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (2-(1-((tert-butoxycarbonyl)amino)cyclopropyl)ethyl)(4-((2-(1-((tert-butoxycarbonyl)amino)cyclopropyl)ethyl)amino)butyl)carbamate (0.35 g, 0.41 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.81 mL, 4.05 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature, and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (2-(1-aminocyclopropyl)ethyl)(4-((2-(1-aminocyclopropyl)ethyl)amino)butyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10, 13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.27 g, 0.32 mmol, 80.2%) as a white solid. UPLC/ELSD: RT=1.60 min. MS (ES): m/z (MH ⁺ ) 668.7 for C ₄₂ H ₇₇ Cl ₃ N ₄ O ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.41(m,1H),4.46(br.m,1H),3.93(br.m,1H),3.53(m,2H),3.33(m,6H),3.11(m,2H),2.40(m,2H),2.15(br.m,4H),1.93(br.m ,5H),1.55(br.m,15H),1.18(br.m,11H),1.08(m,8H),0.97(m,7H),0.89(m,7H),0.74(s,3H).

AZ. Compound SA125: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

Step 1: 14-(3-((tert-Butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 5-{[(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylhept-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl]oxy}-5-oxopentanoic acid (0.30 g, 0.59 mmol) was added to nitrogen. To a solution of anhydrous DCM (15 mL) stirred under vacuum was added di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-4,2-diyl))dicarbamate (0.77 g, 1.78 mmol), dimethylaminopyridine (0.15 g, 1.19 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.23 g, 1.19 mmol). The mixture was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-50% (50:45:5 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate as a light yellow oil (0.18 g, 0.20 mmol, 32.9%). UPLC/ELSD: RT: 2.50 min. MS (ES): m/z (MH ⁺ ) 914.4 for C ₅₄ H ₉₆ N ₄ O ₇ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.33(m,1H),4.82(br.m,3H),3.62(br.m,2H),3.24(m,4H),2.55(m,4H),2.31(m,6H),1.89(m,7H),1.54(m,12H),1.39(s, 20H), 1.28 (m, 6H), 1.11 (d, 12H, J = 6Hz), 0.97 (s, 6H), 0.87 (d, 4H, J = 6Hz), 0.82 (d, 6H, J = 6Hz), 0.63 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 14-(3-((tert-butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate (0.18 g, 0.20 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.39 mL, 1.95 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature and anhydrous acetonitrile (15 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.07 g, 0.08 mmol, 40.1%) as a white solid. UPLC/ELSD: RT=1.60 min. MS (ES): m/z (MH ⁺ ) 668.7 for C ₄₂ H ₇₇ Cl ₃ N ₄ O ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.41(m,1H),4.46(br.m,1H),3.93(br.m,1H),3.53(m,2H),3.33(m,6H),3.11(m,2H),2.40(m,2H),2.15(br.m,4H),1.93(br.m ,5H),1.55(br.m,15H),1.18(br.m,11H),1.08(m,8H),0.97(m,7H),0.89(m,7H),0.74(s,3H).

BA. Compound SA126: (4-aminopentan-2-yl)(4-((4-aminopentan-2-yl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (4-((2-nitrophenyl)sulfonamido)pentan-2-yl)carbamate

To a solution of tert-butyl (4-aminopentan-2-yl)carbamate (2.50 g, 11.74 mmol) in anhydrous DCM (50 mL) stirred under nitrogen was added triethylamine (3.27 mL, 23.48 mmol). The solution was cooled to 0°C and then a solution of 2-nitrobenzenesulfonyl chloride (2.86 g, 12.91 mmol) in 50 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with 10 mL of DCM, washed with saturated aqueous sodium bicarbonate (1 x 100 mL), water (1 x 100 mL), 10% aqueous citric acid (1 x 100 mL), water (1 x 100 mL), and brine (1 x 100 mL), dried over sodium sulfate, filtered, and concentrated to give tert-butyl (4-((2-nitrophenyl)sulfonamido)pentan-2-yl)carbamate (4.56 _g , 11.77 mmol, quantitative) as a white solid. UPLC/ELSD: RT = 0.78 min _. MS (ES): m/z (MH ⁺ ) _{for Ci6H25N3O6S} 388.4. ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 8.10(m,1H),7.79(m,1H),7.66(m,2H),5.31(br.s,1H),4.29(br.s,1H),3.59(m,2H),1.64(m,2H),1.38(s,9H),1.05(t,6H) .

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(pentane-4,2-diyl))dicarbamate

To a solution of tert-butyl (4-((2-nitrophenyl)sulfonamido)pentan-2-yl)carbamate (4.56 g, 11.77 mmol) in anhydrous DMF (50 mL) stirred under nitrogen was added potassium carbonate (4.72 g, 34.18 mmol) and 1,4-diiodobutane (0.74 mL, 5.60 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.55 mL, 4.65 mmol) was added and the reaction was allowed to proceed for 8 h at room temperature. Then, thiophenol (2.21 mL, 21.57 mmol), potassium carbonate (2.32 g, 16.81 mmol) and an additional 20 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL DCM, washed with water (2x10 mL) and brine (2x10 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-50% (50:45:5 DCM/MeOH/concentrated aqueous ammonium hydroxide solution) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(pentane-4,2-diyl))dicarbamate (2.07 g, 4.51 mmol, 80.5%) as a colorless oil. UPLC/ELSD: RT=0.27 min. MS (ES): m/z (MH ⁺ ) 459.6 for C ₂₄ H ₅₀ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.31 (m, 2H), 3.77 (m, 2H), 2.75 (m, 4H), 2.51 (m, 2H), 1.56 (m, 7H), 1.45 (s, 20H), 1.17 (m, 12H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(4-((4-((tert-butoxycarbonyl)amino)pentan-2-yl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(pentane-4,2-diyl))dicarbamate (0.83 g, 1.80 mmol) in anhydrous toluene (20 mL) stirred under nitrogen was added triethylamine (0.76 mL, 5.40 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.99 g, 1.80 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, diluted with toluene, washed with water (3x15 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (50:45:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)amino)pentan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)pentan-2-yl)amino)butyl)carbamate as a colorless oil (0.70 g, 0.80 mmol, 44.4%). UPLC/ELSD: RT = 2.74 _{min .} MS (ES): m/z (MH ⁺ ) 872.3 _{for C52H94N4O6} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.26(m,1H),4.61(m,1H),4.41(br.m,1H),3.69(br.m,4H),2.97(m,2H),2.58(m,2H),2.24(br.m,4H),1.89(m,6H),1.44(m, 11H), 1.33 (s, 20H), 1.24 (br.m, 5H), 1.04 (m, 19H), 0.92 (s, 5H), 0.83 (d, 4H, J = 6Hz), 0.77 (d, 6H, J = 6Hz), 0.58 (s, 3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(4-aminopentan-2-yl)(4-((4-aminopentan-2-yl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)amino)pentan-2-yl)(4-((4-((tert-butoxycarbonyl)amino)pentan-2-yl)amino)butyl)carbamate (0.70 g, 0.80 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 1.60 mL, 7.99 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.46 g, 0.57 mmol, 70.7%) as a white solid. UPLC/ELSD: RT=1.96 min. MS (ES): m/z (MH ⁺ ) 672.1 for C ₄₂ H ₈₁ Cl ₃ N ₄ O ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.42(m,1H),4.47(br.m,1H),4.28(m,1H),3.53(br.m,2H),3.53(m,2H),3.33(s,2H),3.15(m,4H),2.40(m,2H),1.93(br.m,1 8H), 1.42 (br.m, 12H), 1.28 (br.m, 4H), 1.16 (d, 8H, J = 6Hz), 1.08 (m, 6H), 0.98 (d, 4H, J = 9Hz), 0.89 (d, 6H, J = 6Hz), 0.74 (s, 3H).

BB. Compound SA127: (3-aminopentyl)(4-((3-aminopentyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (1-((2-nitrophenyl)sulfonamido)pentan-3-yl)carbamate

To a solution of tert-butyl (1-aminopentan-3-yl)carbamate (2.00 g, 9.39 mmol) in anhydrous DCM (50 mL) stirred under nitrogen was added triethylamine (2.62 mL, 18.78 mmol). The solution was cooled to 0°C and then a solution of 2-nitrobenzenesulfonyl chloride (2.29 g, 10.33 mmol) in 50 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with 10 mL of DCM, washed with saturated aqueous sodium bicarbonate (1 x 100 mL), water (1 x 100 mL), 10% aqueous citric acid (1 x 100 mL), water (1 x 100 mL), and brine (1 x 100 mL), dried over sodium sulfate, filtered, and concentrated to give tert-butyl (1-((2-nitrophenyl)sulfonamido)pentan-3-yl)carbamate (3.73 _g , 9.61 mmol, quantitative) as a white solid. UPLC/ELSD: RT = 0.81 min _. MS (ES): m/z (MH ⁺ ) _{for Ci6H25N3O6S} 388.4. ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 8.12(m,1H),7.83(m,1H),7.74(m,2H),6.26(br.s,1H),4.27(br.s,1H),3.54(m,1H),3.31(m,1H),3.03(m,1H),1.76(m,1 H),1.41(s,9H),0.87(t,3H).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(pentane-1,3-diyl))dicarbamate

To a solution of tert-butyl (1-((2-nitrophenyl)sulfonamido)pentan-3-yl)carbamate (3.73 g, 9.61 mmol) in anhydrous DMF (50 mL) stirred under nitrogen was added potassium carbonate (3.86 g, 27.93 mmol) and 1,4-diiodobutane (0.60 mL, 4.58 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.45 mL, 3.80 mmol) was added and the reaction was allowed to proceed for 8 h at room temperature. Thiophenol (1.80 mL, 17.63 mmol), potassium carbonate (1.90 g, 13.73 mmol) and an additional 20 mL of anhydrous DMF were then added and the reaction was allowed to proceed overnight. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL DCM, washed with water (2x10 mL) and brine (2x10 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-50% (50:45:5 DCM/MeOH/concentrated aqueous ammonium hydroxide solution) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(pentane-1,3-diyl))dicarbamate (1.40 g, 3.05 mmol, 66.7%) as a colorless oil. UPLC/ELSD: RT=0.34 min. MS (ES): m/z (MH ⁺ ) 459.6 for C ₂₄ H ₅₀ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 4.94 (m, 2H), 3.29 (m, 4H), 2.47 (m, 8H), 1.53 (m, 2H), 1.36 (m, 8H), 1.21 (s, 19H), 0.69 (t, 6H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)pentyl)(4-((3-((tert-butoxycarbonyl)amino)pentyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(pentane-1,3-diyl))dicarbamate (0.58 g, 1.27 mmol) in anhydrous toluene (20 mL) stirred under nitrogen was added triethylamine (0.54 mL, 3.82 mmol). Then (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.70 g, 1.27 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, diluted with toluene, washed with water (3x15 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (50:45:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)pentyl)(4-((3-((tert-butoxycarbonyl)amino)pentyl)amino)butyl)carbamate as a colorless oil (0.67 g, 0.77 mmol, 60.5%). UPLC/ELSD: RT = 3.06 _{min .} MS (ES): m/z (MH ⁺ ) 872.3 _{for C52H94N4O6} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.24(m,1H),4.61(br.m,3H),3.43(br.m,2H),3.11(br.m,4H),2.47(m,4H),2.22(m,2H),1.87(br.m,8H),1.42(m,13H),1.32 (s,24H),1.14(br.m,13H),1.04(m,19H),0.91(s,6H),0.79(d,9H,J＝6Hz),0.76(d,7H,J＝6Hz),0.56(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-aminopentyl)(4-((3-aminopentyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)pentyl)(4-((3-((tert-butoxycarbonyl)amino)pentyl)amino)butyl)carbamate (0.67 g, 0.77 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5N in isopropanol, 1.54 mL, 7.70 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3-aminopentyl)(4-((3-aminopentyl)amino)butyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.52 g, 0.65 mmol, 84.4%) as a white solid. UPLC/ELSD: RT=1.90 min. MS (ES): m/z (MH ⁺ ) 672.1 for C ₄₂ H ₈₁ Cl ₃ N ₄ O ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.31(m,1H),4.34(br.m,1H),3.81(m,2H),3.22(br.m,6H),3.01(m,5H),2.26(m,2H),1.98(m,2H),1.94(s,4H),1.81(br.m,5H) ,1.63(br.m,8H),1.44(br.m,7H),1.28(br.m,5H),1.06(d,15H,J＝9Hz),0.96(m,11H),0.84(d,4H,J＝6Hz),0.80(d,6H,J＝6Hz),0.63(s,3H).

BC. Compound SA128: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ((1-(aminomethyl)cyclopropyl)methyl)(4-(((1-(aminomethyl)cyclopropyl)methyl)amino)butyl)carbamate trihydrochloride

Step 1: tert-Butyl ((1-(((2-nitrophenyl)sulfonamido)methyl)cyclopropyl)methyl)carbamate

To a solution of tert-butyl ((1-(aminomethyl)cyclopropyl)methyl)carbamate (2.50 g, 11.86 mmol) in anhydrous DCM (25 mL) stirred under nitrogen was added triethylamine (3.31 mL, 23.72 mmol). The solution was cooled to 0°C and then a solution of 2-nitrobenzenesulfonyl chloride (2.89 g, 13.04 mmol) in 50 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for an additional 3 hours. The mixture was then diluted with another 10 mL of DCM, washed with saturated aqueous sodium bicarbonate solution (1 x 100 mL), water (1 x 100 mL), 10% aqueous citric acid solution (1 x 100 mL), water (1 x 100 mL), and brine (1 x 100 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl ((1-(((2-nitrophenyl)sulfonamido)methyl)cyclopropyl)methyl)carbamate (4.60 g, 11.92 mmol, quant.) as a white solid. UPLC/ELSD: RT = 0.83 min _{. MS} (ES): m/z (MH ⁺ ) 386.4 _{for Ci6H23N3O6S} . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 8.11 (m, 1H), 7.85 (m, 1H), 7.75 (m, 2H), 6.33 (br.s, 1H), 4.82 (br.s, 1H), 3.08 (d, 2H, J = 6Hz), 3.02 (d, 2H, J = 6Hz), 1.45 (s, 9H), 0 .48(m,4H).

Step 2: Di-tert-butyl ((((butane-1,4-diylbis(azanediyl))bis(methylene))bis(cyclopropane-1,1-diyl))bis(methylene))dicarbamate

To a solution of tert-butyl ((1-(((2-nitrophenyl)sulfonamido)methyl)cyclopropyl)methyl)carbamate (4.60 g, 11.92 mmol) in anhydrous DMF (50 mL) stirred under nitrogen was added potassium carbonate (4.79 g, 34.64 mmol) and 1,4-diiodobutane (0.75 mL, 5.68 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.56 mL, 4.71 mmol) was added and the reaction was allowed to proceed for 8 h at room temperature. Thiophenol (2.24 mL, 21.86 mmol), potassium carbonate (2.35 g, 17.03 mmol) and an additional 20 mL of anhydrous DMF were then added and the reaction was allowed to proceed overnight. The next morning, the salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x10 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-50% (50:45:5 DCM/MeOH/concentrated aqueous ammonium hydroxide solution) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((((butane-1,4-diylbis(azanediyl))bis(methylene))bis(cyclopropane-1,1-diyl))bis(methylene))dicarbamate (2.47 g, 5.43 mmol, 95.6%) as a colorless oil. UPLC/ELSD: RT=0.28 min. MS (ES): m/z (MH ⁺ ) 455.6 for C ₂₄ H ₄₆ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.67 (m, 2H), 2.87 (d, 4H, J = 6Hz), 2.41 (m, 4H), 2.34 (s, 4H), 1.36 (m, 5H), 1.28 (s, 19H), 0.25 (m, 4H), 0.17 (m, 4H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl((1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)methyl)(4-(((1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)methyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((((butane-1,4-diylbis(azanediyl))bis(methylene))bis(cyclopropane-1,1-diyl))bis(methylene))dicarbamate (0.92 g, 2.02 mmol) in anhydrous toluene (20 mL) stirred under nitrogen was added triethylamine (0.85 mL, 6.04 mmol). Then (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (1.11 g, 2.02 mmol) was added. The solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, diluted with toluene, washed with water (3x15 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (50:45:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ((1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)methyl)(4-(((1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)methyl)amino)butyl)carbamate as a colorless oil (0.83 g, 0.95 mmol, 47.3%). UPLC/ELSD: RT = 3.05 min. MS (ES): m/z (MH ⁺ ) 868.3 for C ₅₂ H ₉₀ N ₄ O ₆ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm5.77(br.m,1H),5.29(m,1H),4.44(br.m,1H),3.13(br.m,4H),2.97(m,2H),2.85(m,2H),2.50(t,2H),2.42(s,2H),2.26(br.m ,2H),1.85(br.m,5H),1.47(m,9H),1.34(s,19H),1.05(br.m,11H),0.94(s,6H),0.84(d,4H,J＝6Hz),0.78(d,6H,J＝6Hz),0.59(s,3H),0.50(m,2H),0. 35(m,2H),0.26(m,4H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl((1-(aminomethyl)cyclopropyl)methyl)(4-(((1-(aminomethyl)cyclopropyl)methyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ((1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)methyl)(4-(((1-(((tert-butoxycarbonyl)amino)methyl)cyclopropyl)methyl)amino)butyl)carbamate (0.83 g, 0.95 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 1.91 mL, 9.54 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature, and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in a vacuum to give ((1-(aminomethyl)cyclopropyl)methyl)(4-(((1-(aminomethyl)cyclopropyl)methyl)amino)butyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10, 13-dimethyl-17-((R)-6-methylhept-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetrahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.67 g, 0.83 mmol, 86.8%) as a white solid. UPLC/ELSD: RT=1.61 min. MS (ES): m/z (MH ⁺ ) 668.1 for C ₄₂ H ₇₇ Cl ₃ N ₄ O ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.44(m,1H),4.54(br.m,1H),3.86(m,2H),3.33(br.m,6H),3.15(m,6H),2.81(m,2H),2.44(m,2H),1.73(br.m,11H),1.55(br. m, 6H), 1.39 (m, 5H), 1.18 (d, 17H, J = 6Hz), 1.09 (s, 6H), 0.98 (d, 7H, J = 9Hz), 0.90 (d, 9H, J = 6Hz), 0.74 (br.m, 7H).

BD. Compound SA129: N-(8-aminooctyl)-N-((S)-2,5-diaminopentanoyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: N-((S)-2,5-bis((tert-butoxycarbonyl)amino)pentanoyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester

A solution of (8-((tert-butoxycarbonyl)amino)octyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.250 g, 0.373 mmol) and (2S)-2,5-bis[(tert-butoxycarbonyl)amino]pentanoic acid (0.161 g, 0.484 mmol) in DCM (3.75 mL) was cooled to 0° C. in an ice bath. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.107 g, 0.559 mmol) was then added. The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 h, the reaction mixture was cooled to 0 ° C in an ice bath, and then water (3.0 mL) was added. The layers were separated and the aqueous layer was extracted with DCM (10 mL). The combined organics were washed with 5% NaHCO ₃ aqueous solution, passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (10%-40% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl N-((S)-2,5-bis((tert-butoxycarbonyl)amino)pentanoyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine as a clear oil (0.380 g, quant.). UPLC/ELSD: RT = 4.03 min. MS (ES): m/z of C ₅₇ H ₁₀₀ N ₄ O ₉ = 987.54 [M+H] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ 5.20-5.43 (m, 2H), 4.24-4.80 (m, 4H), 4.29 (d, 1H, J = 17.0Hz), 3.72 (d, 1H, J = 17.2Hz), 3.02-3.53 (m, 6H), 2.20-2.42 (m, 2H), 0. 93-2.18(br.m,69H),1.01(s,3H),0.91(d,3H,J＝6.5Hz),0.86(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.63-0.76(m,3H).

Step 2: N-(8-aminooctyl)-N-((S)-2,5-diaminopentanoyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl N-((S)-2,5-bis((tert-butoxycarbonyl)amino)pentanoyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine (0.356 g, 0.347 mmol) in iPrOH (3.5 mL) was added 5-6N HCl in iPrOH (0.50 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 19 h, more 5-6N HCl in iPrOH (0.10 mL) was added. At 22 h, the reaction mixture was cooled to room temperature, and then ACN (7 mL) was added. The suspension was cooled in an ice bath, and then the solid was collected by vacuum filtration, rinsed with 2:1 ACN/iPrOH to give N-(8-aminooctyl)-N-((S)-2,5-diaminopentanoyl)glycine (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.182 g, 0.203 mmol, 58.6%) as a white solid. UPLC/ELSD: RT=1.94 min. MS (ES): m/z of C ₄₂ H ₇₆ N ₄ O ₃ = 343.40 [M+2H] ²⁺ . ¹ H NMR (300MHz, DMSO): δ7.85-8.59(m,9H),5.24-5.55(m,1H),4.25-4.68(m,2H),4.17(d,1H,J＝17.0Hz),3.95(d,1H,J＝17.2Hz),3.12-3.61(m,2H),2.65 -2.85(m,4H),2.20-2.42(m,2H),0.92-2.03(br.m,42H),0.98(s,3H),0.89(d,3H,J＝6.3Hz),0.84(d,3H,J＝6.6Hz),0.84(d,3H,J＝6.5Hz),0.65(s,3H).

BE. Compound SA130: N-(L-lysyl)-N-(8-aminooctyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: N-(N2,N6-bis(tert-butoxycarbonyl)-L-lysyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

A solution of (8-((tert-butoxycarbonyl)amino)octyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.235 g, 0.350 mmol) and (2S)-2,6-bis[(tert-butoxycarbonyl)amino]hexanoic acid (0.121 g, 0.350 mmol) in DCM (3.5 mL) was cooled to 0° C. in an ice bath. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.101 g, 0.525 mmol) was then added. The reaction mixture was stirred at room temperature and monitored by LCMS. At 19h, the reaction mixture was cooled to 0°C, and then (2S)-2,6-bis[(tert-butoxycarbonyl)amino]hexanoic acid (23mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (20mg) were added. The reaction mixture was stirred at room temperature. At 21h, the reaction mixture was cooled to 0°C in an ice bath, and then water (3.5mL) was added. The layers were separated, and the aqueous layer was extracted with DCM (10mL). The combined organic matter was washed with 5% NaHCO ₃ aqueous solution, passed through a hydrophobic glass frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (10%-40% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)-N-(N2,N6-bis(tert-butoxycarbonyl)-L-lysyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine as a clear oil (0.323 g, 0.323 mmol, 92.3%). UPLC/ELSD: RT = 4.09 min. MS (ES): m/z of C ₅₈ H ₁₀₂ N ₄ O ₉ = 1001.35 [M+H] ⁺ . ¹ HNMR (300MHz, CDCl ₃ ): δ5.21-5.46(m,2H),4.26-4.79(m,4H),4.30(d,1H,J＝17.1Hz),3.71(d,1H,J＝17.0Hz),3.01-3.52(m,6H),2.22-2.43(m,2H),0 .93-2.14(br.m,71H),1.01(s,3H),0.91(d,3H,J＝6.4Hz),0.86(d,6H,J＝6.6Hz),0.64-0.75(m,3H).

Step 2: N-(L-lysyl)-N-(8-aminooctyl)glycine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl N-(N2,N6-bis(tert-butoxycarbonyl)-L-lysyl)-N-(8-((tert-butoxycarbonyl)amino)octyl)glycine (0.303 g, 0.303 mmol) in iPrOH (3.0 mL) was added 5-6N HCl in iPrOH (0.45 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 19 h, additional 5-6N HCl in iPrOH (0.10 mL) was added. At 22h, the reaction mixture was cooled to room temperature, and then ACN (6mL) was added. The suspension was cooled to 0°C in an ice bath, and then the solid was collected by vacuum filtration, rinsed with 2:1 ACN/iPrOH to give N-(L-lysyl)-N-(8-aminooctyl)glycine (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.163g, 0.173mmol, 57.0%) as a white solid. UPLC/ELSD: RT=1.96min. MS (ES): m/z of C ₄₃ H ₇₈ N ₄ O ₃ = 350.43 [M+2H] ²⁺ . ¹ H NMR (300MHz, DMSO): δ7.89-8.51(m,9H),5.22-5.41(m,1H),4.23-4.61(m,2H),4.19(d,1H,J＝17.0Hz),3.93(d,1H,J＝17.1Hz),3.13-3.51(m,2H),2.6 4-2.85(m,4H),2.22-2.36(m,2H),0.92-2.04(br.m,44H),0.98(s,3H),0.90(d,3H,J＝6.3Hz),0.84(d,6H,J＝6.6Hz),0.65(s,3H).

BF. Compound SA131: (6-aminohexyl)-L-lysine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: N6-(tert-Butyloxycarbonyl)-N2-((2-nitrophenyl)sulfonyl)-L-lysine methyl ester

A mixture of (2S)-2-amino-6-[(tert-butoxycarbonyl)amino]hexanoic acid methyl ester hydrochloride (1.000 g, 3.369 mmol), DMAP (catalyst) and triethylamine (1.40 mL, 9.96 mmol) in DCM (15 mL) was cooled to 0 ° C, and then 2-nitrobenzenesulfonyl chloride (0.896 g, 4.04 mmol) in DCM (5 mL) was added dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 1 h, the reaction mixture was cooled to 0 ° C, and then water (20 mL) was added. The layers were separated, and the organic layer was washed with 5% NaHCO ₃ aqueous solution, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (30%-70% EtOAc in hexanes) to give N6-(tert-butoxycarbonyl)-N2-((2-nitrophenyl)sulfonyl)-L-lysine methyl ester (1.44 g, 3.23 mmol, 95.9%) as a viscous yellow _oil . UPLC/ELSD: RT = 0.78 min _{. MS (ES): m/z for Ci8H27N3O8S = 390.30} [(M+H)-(( _CH3 ) _2C = _CH2 )] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ 8.02-8.10 (m, 1H), 7.89-7.97 (m, 1H), 7.68-7.78 (m, 2H), 6.08 (d, 1H, J = 9.1Hz), 4.53 (br.s, 1H), 4.16 (td, 1H, J = 8.5, 5.0Hz), 3 .47(s,3H),3.09(td,2H,J=6.1,6.1Hz),1.33-1.94(m,6H),1.44(s,9H).

Step 2: N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-N2-((2-nitrophenyl)sulfonyl)-L-lysine methyl ester

(2S)-6-[(tert-butoxycarbonyl)amino]-2-(2-nitrobenzenesulfonamido)hexanoic acid methyl ester (0.603 g, 1.35 mmol), tert-butyl N-(6-bromohexyl)carbamate (0.504 g, 1.80 mmol), potassium carbonate (0.480 g, 3.48 mmol) and potassium iodide (0.046 g, 0.28 mmol) were combined in DMF (9.0 mL) and stirred at 80° C. The reaction was monitored by LCMS. At 18 h, the reaction mixture was cooled to room temperature, filtered, diluted with MTBE (100 mL), washed with water (3×) and brine, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (20%-70% EtOAc in hexanes) to give N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-N2-((2-nitrophenyl)sulfonyl)-L-lysine methyl ester (0.693 g, 1.08 mmol, 79.4%) _as a clear oil. UPLC/ELSD: RT = 1.53 min _. MS (ES): m/z for _C29H48N4O10S = 489.29 [(M ₊ H)-2(( _CH3 ) _2C = _CH2 ) _-CO2 ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ7.99-8.09(m,1H),7.64-7.75(m,2H),7.52-7.61(m,1H),4.42-4.73(m,3H),3.54(s,3H),3.31-3.45(m,1H),2.99-3.21(m, 5H),1.94-2.13(m,1H),1.20-1.89(br.m,13H),1.44(s,18H).

Step 3: N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-N2-((2-nitrophenyl)sulfonyl)-L-lysine

To a solution of (2S)-6-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)hexanoic acid methyl ester (0.690 g, 1.07 mmol) in THF (7.0 mL) and MeOH (1.4 mL) was added aqueous lithium hydroxide monohydrate (0.90 mL, 15 w/v%). The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 h, the reaction mixture was concentrated to remove volatile organics and then partitioned between water (50 mL) and EtOAc (50 mL). The biphasic mixture was washed with 5% _{aqueous K2CO3} and 0.1 _N aqueous HCl, dried over _Na2SO4 , and concentrated to give (2S)-6-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)hexanoic acid (0.578 g, 0.916 mmol, 85.6%) _as an amber _oil . UPLC/ELSD: RT = 1.27 _min . MS (ES): m/z for _C28H46N4O10S = 475.35 [(M+H)-2(( _CH3 ) _2C = _CH2 ) _-CO2 ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ8.03-8.12(m,1H),7.63-7.74(m,2H),7.53-7.62(m,1H),4.48-4.79(m,3H),2.94-3.42(m,6H),1.92-2.18(m,1H),1.20-1.8 3(br.m,13H),1.44(s,18H).

Step 4: N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-N2-((2-nitrophenyl)sulfonyl)-L-lysine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

A mixture of (2S)-6-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)hexanoic acid (0.560 g, 0.888 mmol), cholesterol (0.378 g, 0.977 mmol) and DMAP (catalyst) in DCM (8.5 mL) was cooled to 0 °C. Then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.238 g, 1.24 mmol) was added. While stirring, the reaction mixture was slowly warmed to room temperature and monitored by LCMS. At 19 h, the reaction mixture was cooled to 0 °C, and then 5% aqueous _NaHCO3 solution (8.5 mL) was added. Once the reaction mixture warmed to room temperature, the layers were separated. The aqueous layer was extracted with DCM (8 mL). The combined organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-40% EtOAc in hexanes) to afford N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-N2-((2-nitrophenyl)sulfonyl)-L-lysine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.562 g, 0.562 mmol, 63.3%) as a white foam. UPLC/ELSD: RT = 3.62 min. MS (ES): m/z of C ₅₅ H ₉₀ N ₄ O ₁₀ S = 900.19 [(M+H)-((CH ₃ ) ₂ C=CH ₂ )-CO ₂ ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ7.99-8.10(m,1H),7.64-7.74(m,2H),7.52-7.60(m,1H),5.24-5.36(m,1H),4.36-4.81(m,4H),3.33-3.51(m,1H),2.94-3. 24(m,5H),1.48(br.m,60H),0.93(s,3H),0.90(d,3H,J＝6.4Hz),0.86(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.66(s,3H).

Step 5: N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-L-lysine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a mixture of N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-N2-((2-nitrophenyl)sulfonyl)-L-lysine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.540 g, 0.540 mmol) and potassium carbonate (0.224 g, 1.621 mmol) in DMF (6.5 mL) was added thiophenol (0.10 mL, 0.980 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 h, LCMS data were consistent with the completion of the reaction. The reaction mixture was diluted with DCM (20 mL) and then purified by The filtrate was diluted to 80 mL with DCM and then washed with water (3x) and 5% aqueous NaHCO ₃ . The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (40%-80% EtOAc in hexanes) to afford N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-L-lysine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.400 g, 0.491 mmol, 90.9%) as a clear oil. UPLC/ELSD: RT = 2.92 min. MS (ES): m/z of C ₄₉ H ₈₇ N ₃ O ₆ = 815.18 [M+H] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ5.34-5.43(m,1H),4.30-4.75(m,3H),3.20-3.32(m,1H),3.00-3.20(m,4H),2.47-2.71(m,2H),2.22-2.43(m,2H),0.93-2. 13 (br.m, 58H), 1.02 (s, 3H), 0.91 (d, 3H, J = 6.4Hz), 0.86 (d, 3H, J = 6.6Hz), 0.86 (d, 3H, J = 6.6Hz), 0.68 (s, 3H).

Step 6: (6-aminohexyl)-L-lysine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

To a solution of N6-(tert-butoxycarbonyl)-N2-(6-((tert-butoxycarbonyl)amino)hexyl)-L-lysine (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.379 g, 0.465 mmol) in iPrOH (5.5 mL) was added 5-6N HCl (0.93 mL) in iPrOH. The reaction mixture was stirred at 40 °C and monitored by LCMS. At 16 h, the reaction mixture was cooled to room temperature, and then iPrOH (30 mL) was added. The suspension was centrifuged (10,000 x g for 30 min). The supernatant was decanted, and the solid was then suspended in MTBE (35 mL). The suspension was centrifuged (10,000 x g for 30 min). The supernatant was decanted, and the solid was suspended in heptane and then concentrated to give (6-aminohexyl)-L-lysine (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthrene-3-yl ester trihydrochloride (0.133 g, 0.166 mmol, 35.7%) as a white solid. UPLC/ELSD: RT = 1.85 min. MS (ES): m/z of C ₃₉ H ₇₁ N ₃ O ₂ = 328.58 [(M+2H)+CH ₃ CN] ²⁺ . ¹ H NMR (300MHz, DMSO): δ9.85(br.s,1H),9.33(br.s,1H),7.71-8.43(m,6H),5.26-5.52(m,1H),4.50-4.76(m,1H),3.87-4.07(m,1H),2.67-3.06(m,6H) ,2.25-2.44(m,2H),0.92-2.11(br.m,40H),0.99(s,3H),0.89(d,3H,J＝6.3Hz),0.84(d,6H,J＝6.5Hz),0.65(s,3H).

BG. Compound SA132: (S)-5-amino-2-((6-aminohexyl)amino)pentanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: Methyl (2S)-5-[(tert-butoxycarbonyl)amino]-2-(2-nitrobenzenesulfonamido)pentanoate

A solution of (S)-2-amino-5-((tert-butoxycarbonyl)amino)pentanoic acid methyl ester hydrochloride (1.000 g, 3.537 mmol) and triethylamine (1.50 mL, 10.7 mmol) in DCM (15 mL) was cooled to 0 ° C in an ice bath, and then 2-nitrobenzenesulfonyl chloride (0.940 g, 4.24 mmol) in DCM (5.0 mL) was added dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 h, the reaction mixture was cooled to 0 ° C in an ice bath, and then water (20 mL) was added. The layers were separated, and the organic matter was washed with 5% NaHCO ₃ aqueous solution, passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (30%-70% EtOAc in hexanes) to give (2S)-methyl 5-[(tert-butoxycarbonyl)amino]-2-(2-nitrobenzenesulfonamido)pentanoate (1.366 g, 3.166 mmol, 89.5%) as _a viscous yellow oil. UPLC/ELSD: RT = 0.68 min. MS _{(ES): m/z for Ci7H25N3O8S = 376.23} [(M+H)-( _CH3 ) _2C = _CH2 ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ 8.03-8.10 (m, 1H), 7.88-7.96 (m, 1H), 7.68-7.78 (m, 2H), 6.14 (d, 1H, J = 9.0Hz), 4.55 (br.s, 1H), 4.18 (td, 1H, J = 8.4, 5.2Hz), 3. 47(s,3H),3.14(dt,2H,J=6.0,5.7Hz),1.82-1.97(m,1H),1.55-1.80(m,3H),1.44(s,9H).

Step 2: Methyl (2S)-5-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)pentanoate

By (2S)-5-[(tert-butoxycarbonyl)amino]-2-(2-nitrobenzenesulfonamido)pentanoic acid methyl ester (0.600g, 1.39mmol), N-(6-bromohexyl)carbamic acid tert-butyl ester (0.506g, 1.81mmol), potassium carbonate (0.480g, 3.48mmol) and potassium iodide (0.046g, 0.28mmol) are combined in DMF (9.0mL). The reaction mixture is stirred at 80°C and monitored by LCMS. At 2.5h, the reaction mixture is cooled to room temperature and then filtered, rinsed with MTBE. The filtrate is diluted to 80mL with MTBE, washed with water (3x) and _brine , dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (30%-70% EtOAc in hexanes) to give (2S)-methyl 5-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)pentanoate (0.720 g, 1.141 mmol, 82.1%) _as a clear oil. UPLC/ELSD: _RT = 1.41 min _{. MS (ES): m/z for C28H46N4O10S =} 475.47 [(M+H)-2(( _CH3 ) _2C = _CH2 ) _-CO2 ] ⁺ . ¹ HNMR (300MHz, CDCl ₃ ): δ7.99-8.09(m,1H),7.64-7.73(m,2H),7.53-7.61(m,1H),4.42-4.76(m,3H),3.54(s,3H),3.32-3.45(m,1H),2.99-3.23(m, 5H),1.99-2.16(m,1H),1.20-1.91(br.m,11H),1.44(s,18H).

Step 3: (2S)-5-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)pentanoic acid

To a solution of (2S)-5-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)pentanoic acid methyl ester (0.716 g, 1.14 mmol) in THF (7.7 mL) and MeOH (1.5 mL) was added an aqueous solution of lithium hydroxide monohydrate (0.96 mL, 15 w/v%). The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 h, the reaction mixture was concentrated to remove volatile organics, dissolved in water (50 mL), and extracted with EtOAc (3 x 25 mL). The combined organics were washed with 5% _{aqueous K2CO3} and then 5% aqueous citric acid, dried over _Na2SO4 , and concentrated to give (2S)-5-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)pentanoic acid (0.619 _g , 1.00 mmol, 88.4%) as an amber oil. UPLC/ELSD: RT = 1.22 min _{. MS} (ES ₎ : m _/ z for _C27H44N4O10S = 461.4 [(M+H)-2(( _CH3 ) _2C = _CH2 ) _-CO2 ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ8.02-8.11(m,1H),7.62-7.72(m,2H),7.54-7.62(m,1H),4.47-4.84(m,3H),2.98-3.44(m,6H),1.94-2.13(m,1H),1.20-1.8 3(br.m,11H),1.44(s,18H).

Step 4: (S)-5-((tert-butoxycarbonyl)amino)-2-((N-(6-((tert-butoxycarbonyl)amino)hexyl)-2-nitrophenyl)sulfonamido)pentanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

A mixture of (2S)-5-[(tert-butoxycarbonyl)amino]-2-(N-{6-[(tert-butoxycarbonyl)amino]hexyl}-2-nitrobenzenesulfonamido)pentanoic acid (0.520 g, 0.843 mmol), cholesterol (0.359 g, 0.927 mmol) and DMAP (catalyst) in DCM (8.0 mL) was cooled to 0 ° C. Then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.226 g, 1.18 mmol) was added. While stirring, the reaction mixture was slowly warmed to room temperature and monitored by LCMS. At 22 h, the reaction mixture was cooled to 0 ° C., and then DMAP (catalyst) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (105 mg) were added. The reaction mixture was stirred at room temperature. At 26 h, the reaction mixture was cooled to 0 ° C. in an ice bath, and then water (8 mL) was added. The biphasic mixture was warmed to room temperature and then separated. The organics were washed with 5% aqueous NaHCO ₃ solution, passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-40% EtOAc in hexanes) to afford (S)-(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((tert-butoxycarbonyl)amino)-2-((N-(6-((tert-butoxycarbonyl)amino)hexyl)-2-nitrophenyl)sulfonamido)pentanoate as an amber foam (0.299 g, 0.303 mmol, 36.0%). UPLC/ELSD: RT = 3.59 min. MS (ES): m/z of C ₅₄ H ₈₈ N ₄ O ₁₀ S = 829.74 [(M+H)-2((CH ₃ ) ₂ C=CH ₂ )-CO ₂ ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ8.00-8.09(m,1H),7.63-7.74(m,2H),7.53-7.60(m,1H),5.23-5.35(m,1H),4.35-4.84(m,4H),3.33-3.53(m,1H),2.93-3.2 7(m,5H),0.94-2.17(br.m,58H),0.92(s,3H),0.90(d,3H,J＝6.5Hz),0.86(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.66(s,3H).

Step 5: (S)-5-((tert-butoxycarbonyl)amino)-2-((6-((tert-butoxycarbonyl)amino)hexyl)amino)pentanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a mixture of (S)-5-((tert-butoxycarbonyl)amino)-2-((N-(6-((tert-butoxycarbonyl)amino)hexyl)-2-nitrophenyl)sulfonamido)pentanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.284 g, 0.288 mmol) and potassium carbonate (0.119 g, 0.865 mmol) in DMF (5.0 mL) was added thiophenol (0.05 mL, 0.49 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 3 h, DCM (10 mL) was added, and The reaction mixture was filtered through a pad of Celite. The filtrate was diluted to 80 mL with DCM and then washed once with 5% aqueous NaHCO ₃ solution and three times with water. The organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (40%-80% EtOAc in hexanes) to afford (S)-(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((tert-butoxycarbonyl)amino)-2-((6-((tert-butoxycarbonyl)amino)hexyl)amino)pentanoate as a clear viscous oil (0.203 g, 0.254 mmol, 88.0%). UPLC/ELSD: RT = 2.92 min. MS (ES): m/z of C ₄₈ H ₈₅ N ₃ O ₆ = 801.37 [M+H] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ): δ5.34-5.42(m,1H),5.03-5.13(m,1H),4.45-4.81(m,2H),3.50-3.81(m,1H),2.82-3.32(m,6H),2.22-2.48(m,2H),0.94-2. 19(br.m,56H),1.02(s,3H),0.91(d,3H,J＝6.4Hz),0.87(d,3H,J＝6.6Hz),0.86(d,3H,J＝6.6Hz),0.68(s,3H).

Step 6: (S)-5-amino-2-((6-aminohexyl)amino)pentanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

To a solution of (S)-5-((tert-butoxycarbonyl)amino)-2-((6-((tert-butoxycarbonyl)amino)hexyl)amino)pentanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester (0.197 g, 0.246 mmol) in iPrOH (3.0 mL) was added 5-6N HCl in iPrOH (0.49 mL). The reaction mixture was stirred at 40 °C and monitored by LCMS. At 16 h, the reaction mixture was cooled to room temperature, and then ACN (9 mL) was added. The suspension was filtered, but the particles were passed through a glass frit. The suspension was concentrated, and the residue was suspended in MTBE (30 mL). The suspension was centrifuged (10,000 x g for 30 min), and the supernatant was then decanted. The solid was suspended in heptane, and then concentrated to give (S)-5-amino-2-((6-aminohexyl)amino)pentanoic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester trihydrochloride (0.147 g, 0.170 mmol, 69.0%) as a white solid. UPLC/ELSD: RT=1.86 min. MS (ES): m/z of C ₃₈ H ₆₉ N ₃ O ₂ = 321.42 [(M+2H)+CH ₃ CN] ²⁺ . ¹ H NMR (300MHz, DMSO): δ9.75(br.s,1H),9.40(br.s,1H),7.61-8.32(m,6H),5.31-5.48(m,1H),4.53-4.72(m,1H),3.92-4.16(m,1H),2.66-3.05(m,6H), 2.24-2.45(m,2H),0.92-2.13(br.m,38H),1.00(s,3H),0.90(d,3H,J＝6.3Hz),0.84(d,3H,J＝6.6Hz),0.84(d,3H,J＝6.6Hz),0.66(s,3H).

BH. Compound SA133: (3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: tert-Butyl (3-ethyl-1-((2-nitrophenyl)sulfonamido)pentan-3-yl)carbamate

To a solution of tert-butyl N-(1-amino-3-ethylpentan-3-yl)carbamate (2.50 g, 10.31 mmol) in anhydrous DCM (50 mL) stirred under nitrogen was added triethylamine (2.87 mL, 20.62 mmol). The solution was cooled to 0°C and then a solution of 2-nitrobenzenesulfonyl chloride (2.51 g, 11.34 mmol) in 50 mL anhydrous DCM was added dropwise over 30 minutes. The reaction was allowed to proceed at 0°C for 1 hour and then at room temperature for another 3 hours. The mixture was then diluted with 10 mL of DCM, washed with saturated aqueous sodium bicarbonate (1 x 100 mL), water (1 x 100 mL), 10% aqueous citric acid (1 x 100 mL), water (1 x 100 mL), and brine (1 x 100 mL), dried over sodium sulfate, filtered, and concentrated to afford tert-butyl (3-ethyl-1-((2-nitrophenyl)sulfonamido)pentan-3-yl)carbamate (4.48 _g , 10.31 mmol, quantitative) as a white solid. UPLC/ELSD: RT = 1.27 min _. MS (ES): m/z (MH ⁺ ) 415.5 _{for C18H29N3O6S} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 8.12(m,1H),7.85(m,1H),7.76(m,2H),5.41(br.s,1H),4.19(br.s,1H),3.14(m,2H),1.92(t,2H),1.63(m,2H),1.45(m,2 H),1.40(s,9H),0.78(t,6H).

Step 2: Di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(3-ethylpentane-1,3-diyl))dicarbamate

To a solution of tert-butyl (3-ethyl-1-((2-nitrophenyl)sulfonamido)pentan-3-yl)carbamate (4.48 g, 10.78 mmol) in anhydrous DMF (50 mL) stirred under nitrogen was added potassium carbonate (4.33 g, 31.32 mmol) and 1,4-diiodobutane (0.68 mL, 5.13 mmol). The solution was heated to 40 °C and allowed to proceed overnight. The next morning, benzyl bromide (0.51 mL, 4.26 mmol) was added and the reaction was allowed to proceed for 8 h at room temperature. Then, thiophenol (2.02 mL, 19.77 mmol), potassium carbonate (2.13 g, 15.40 mmol) and an additional 20 mL of anhydrous DMF were added and the reaction was allowed to proceed overnight. The next morning, salts were removed from the supernatant via multiple rounds of centrifugation and washing with DMF. The combined supernatant was concentrated in vacuo to an oil, which was dissolved in 40 mL of DCM, washed with water (2x10 mL) and brine (2x10 mL), dried over potassium carbonate, filtered, and concentrated to an oil. The oil was redissolved in DCM and purified via silica gel chromatography in DCM with a 0-50% (50:45:5 DCM/MeOH/concentrated aqueous ammonium hydroxide solution) gradient. The fractions containing the product were combined and concentrated to give di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(3-ethylpentane-1,3-diyl))dicarbamate (2.14 g, 4.17 mmol, 81.1%) as a colorless oil. UPLC/ELSD: RT=2.52 min. MS (ES): m/z (MH ⁺ ) 515.6 for C ₂₈ H ₅₈ N ₄ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 5.20 (m, 2H), 2.45 (br.m, 8H), 1.48 (m, 13H), 1.35 (s, 4H), 1.23 (s, 19H), 0.62 (t, 12H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)(4-((3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)amino)butyl)carbamate

To a solution of di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(3-ethylpentane-1,3-diyl))dicarbamate (0.50 g, 0.97 mmol) in anhydrous toluene (10 mL) stirred under nitrogen was added triethylamine (0.41 mL, 2.91 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.54 g, 0.97 mmol) was added and the solution was heated to 90° C. overnight. The next morning, the reaction mixture was cooled to room temperature, diluted with toluene, washed with water (3x15 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in DCM using a 0-30% (50:45:5 DCM/MeOH/concentrated aqueous ammonium hydroxide) gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)(4-((3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)amino)butyl)carbamate as a colorless oil (0.55 g, 0.59 mmol, 60.9%). UPLC/ELSD: RT = _3.06 min _. MS (ES): m/z (MH ⁺ ) 928.3 _{for C56H102N4O6} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm5.34(br.m,1H),5.02(m,1H),4.46(br.m,3H),3.18(br.m,4H),2.56(m,4H),2.28(m,2H),1.83(m,6H),1.58(br.m,16H),1.39 (s,18H),1.10(br.m,11H),0.97(s,5H),0.88(d,3H,J＝6Hz),0.79(m,18H),0.64(s,4H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)carbamate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)(4-((3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)amino)butyl)carbamate (0.55 g, 0.59 mmol) in isopropanol (10 mL) stirred under nitrogen was added hydrochloric acid (5N in isopropanol, 1.18 mL, 5.90 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.34 g, 0.40 mmol, 68.1%) as a white solid. UPLC/ELSD: RT=2.08 min. MS (ES): m/z (MH ⁺ ) 728.2 for C ₄₆ H ₈₉ Cl ₃ N ₄ O ₂ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.45(m,1H),4.48(br.m,1H),3.94(m,1H),3.37(br.m,3H),3.14(m,4H),2.40(m,2H),2.11(m,3H),1.93(br.m,6H),1.74(br.m, 13H), 1.55 (m, 12H), 1.18 (d, 14H, J = 6Hz), 1.04 (br.m, 17H), 0.98 (d, 4H, J = 6Hz), 0.91 (d, 6H, J = 6Hz), 0.74 (s, 3H).

BI. Compound SA134: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-aminooctyl)(3-aminopropyl)amino)-5-oxopentanoate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-5-oxopentanoate

To 5-{[(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylhept-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl]oxy}-5-oxopentanoic acid (0.24 g, 0.48 mmol) was added. To a solution in anhydrous DCM (10 mL) stirred under nitrogen was added tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.19 g, 0.48 mmol), dimethylaminopyridine (0.12 g, 0.95 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.18 g, 0.95 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-50% (50:45:5 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-5-oxopentanoate as a light yellow oil (0.32 g, 0.37 mmol, 77.0%). UPLC/ELSD: RT: 3.52 _{min .} MS (ES): m/z (MH ⁺ ) 885.4 _{for C53H93N3O7} . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm5.17(m,1H),5.02(m,1H),4.43(br.m,1H),4.29(br.m,1H),3.06(m,2H),2.95(m,1H),2.86(m,2H)2.73(m,4H),2.04(br.m,6H) ,1.62(br.m,4H),1.51(m,4H),1.20(br.m,12H),1.10(s,19H),0.97(br.m,13H),0.81(br.m,7H),0.68(s,6H),0.60(d,4H,J＝6Hz),0.54(d,6H,J＝6Hz),0.3 5(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-aminooctyl)(3-aminopropyl)amino)-5-oxopentanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-5-oxopentanoate (0.32 g, 0.37 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.73 mL, 3.66 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in a vacuum to give 5-((8-aminooctyl)(3-aminopropyl)amino)-5-oxopentanoic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride (0.16 g, 0.19 mmol, 53.1%) as a white solid. UPLC/ELSD: RT=1.99 min. MS (ES): m/z (MH ⁺ ) 684.9 for C ₄₃ H ₇₉ Cl ₂ N ₃ O ₃ . ¹ HNMR(300MHz,MeOD)δ:ppm 5.42(m,1H),4.55(br.m,1H),3.48(t,2H),3.36(m,2H),2.94(m,4H),2.49(t,2H),2.40(m,4H),1.92(br.m,9H),1.63(br.m,1 1H), 1.41 (br.m, 12H), 1.16 (m, 8H), 1.07 (s, 5H), 0.97 (d, 4H, J = 6Hz), 0.90 (d, 6H, J = 6Hz), 0.75 (s, 3H).

BJ. Compound SA135: 5-((8-aminooctyl)(3-aminopropyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-5-oxopentanoate

To 5-{[(1R,3aS,3bS,7S,9aR,9bS,11aR)-1-[(2R,5R)-5-ethyl-6-methylhept-2-yl]-9a,11a-dimethyl-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl]oxy}-5-oxopentanoic acid (0.24 g, 0.45 mL) was added. To a solution of tert-butyl N-[3-({8-[(tert-butoxycarbonyl)amino]octyl}amino)propyl]carbamate (0.18 g, 0.45 mmol), dimethylaminopyridine (0.11 g, 0.90 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.17 g, 0.90 mmol) in anhydrous DCM (10 mL) stirred under nitrogen was added. The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-50% (50:45:5 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-5-oxopentanoate as a light yellow oil (0.27 g, 0.30 mmol, 67.0%). UPLC/ELSD: RT: 3.60 min _. MS (ES): m/z (MH ⁺ ) _{913.4 for C55H97N3O7} . ¹ H NMR (300MHz, CDCl ₃ )δ:ppm 5.03(m,1H),4.44(m,1H),4.25(br.m,1H),3.06(br.m,2H),2.95(m,1H),2.86(m,2H)2.74(m,4H),2.04(br.m,6H),1.62(br.m, 4H),1.50(m,4H),1.32(br.m,11H),1.10(s,19H),0.97(br.m,12H),0.79(br.m,7H),0.68(s,5H),0.60(d,5H,J=6Hz),0.51(q,9H),0.35(s,4H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-aminooctyl)(3-aminopropyl)amino)-5-oxopentanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((8-((tert-butoxycarbonyl)amino)octyl)(3-((tert-butoxycarbonyl)amino)propyl)amino)-5-oxopentanoate (0.27 g, 0.30 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.59 mL, 2.96 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in a vacuum to give 5-((8-aminooctyl)(3-aminopropyl)amino)-5-oxopentanoic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride (0.12 g, 0.12 mmol, 44.6%) as a white solid. UPLC/ELSD: RT=2.23 min. MS (ES): m/z (MH ⁺ ) 712.8 for C ₄₅ H ₈₃ Cl ₂ N ₃ O ₃ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.40(m,1H),4.55(br.m,1H),3.48(t,2H),3.33(m,1H),2.91(m,3H),2.49(t,2H),2.40(m,4H),1.91(br.m,7H),1.66(br.m,1 1H), 1.41 (br.m, 15H), 1.18 (d, 6H, J = 6Hz), 1.07 (s, 6H), 0.99 (d, 5H, J = 6Hz), 0.89 (q, 9H), 0.75 (s, 3H).

BK. Compound SA136: 5-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 14-(3-((tert-Butyloxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 5-{[(1R,3aS,3bS,7S,9aR,9bS,11aR)-1-[(2R,5R)-5-ethyl-6-methylhept-2-yl]-9a,11a-dimethyl-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl]oxy}-5-oxopentanoic acid (0.33 g, 0.61 mmol) was added under nitrogen. To a solution in anhydrous DCM (10 mL) stirred at 37° C. was added tert-butyl N-(4-{[4-({3-[(tert-butoxycarbonyl)amino]butyl}amino)butyl]amino}butan-2-yl)carbamate (0.79 g, 1.83 mmol), dimethylaminopyridine (0.15 g, 1.22 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.24 g, 1.22 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM using a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate as a light yellow oil (0.12 g, 0.12 mmol, 20.2%). UPLC/ELSD: RT: 2.81 min. MS (ES): m/z (MH ⁺ ) 942.4 for C ₅₆ H ₁₀₀ N ₄ O ₇ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.33(m,1H),4.85(m,3H),3.70(br.m,1H),3.23(br.m,5H),2.56(br.m,4H),2.32(m,7H)1.91(m,8H),1.56(br.m,12H),1.40 (s,21H),1.21(m,6H),1.12(m,11H),0.98(s,5H),0.90(d,5H,J＝6Hz),0.79(q,9H),0.65(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate (0.12 g, 0.12 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.25 mL, 1.23 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.05 g, 0.05 mmol, 42.5%) as a white solid. UPLC/ELSD: RT=1.90min. MS (ES): m/z (MH ⁺ ) 742.0 for C ₄₆ H ₈₇ Cl ₃ N ₄ O ₃ . ¹ H NMR(300MHz,MeOD)δ:ppm 5.42(m,1H),4.57(br.m,1H),3.67(m,1H),3.48(m,5H),3.18(m,5H),2.42(m,6H),1.92(br.m,22H),1.39(m,10H),1.20(m,8 H), 1.07 (s, 5H), 0.98 (d, 5H, J = 6Hz), 0.87 (q, 9H), 0.75 (s, 3H).

BL. Compound SA137: 5-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 14-(3-((tert-Butyloxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a stirred anhydrous flask under nitrogen was added 5-{[(1R,3aS,3bS,7S,9aR,9bS,11aR)-1-[(2R,5R)-5-ethyl-6-methylhept-2-yl]-9a,11a-dimethyl-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl]oxy}-5-oxopentanoic acid (0.31 g, 0.58 mmol). To a solution in water DCM (10 mL) was added tert-butyl N-(4-{[4-({3-[(tert-butoxycarbonyl)amino]-3-methylbutyl}amino)butyl]amino}-2-methylbutan-2-yl)carbamate (0.80 g, 1.75 mmol), dimethylaminopyridine (0.14 g, 1.17 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.23 g, 1.17 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM using a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate as a light yellow oil (0.20 g, 0.20 mmol, 35.0%). UPLC/ELSD: RT: 2.86 min. MS (ES): m/z (MH ⁺ ) 970.4 for C ₅₈ H ₁₀₄ N ₄ O ₇ . ¹ H NMR (300MHz, CDCl ₃ )δ: ppm 5.54(m,1H),5.04(m,1H),4.23(m,2H),2.91(br.m,4H),2.35(br.m,4H),2.03(br.m,6H),1.61(m,8H)1.35(m,10H),1.10(s ,19H),0.94(m,15H),0.83(m,6H),0.68(s,6H),0.60(m,5H),0.49(q,9H),0.35(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-olate (0.20 g, 0.20 mmol) in isopropanol (5 mL) stirred under nitrogen was added hydrochloric acid (5 N in isopropanol, 0.41 mL, 2.04 mmol) dropwise. The solution was heated to 40°C overnight. The next morning, the mixture was cooled to room temperature and anhydrous acetonitrile (20 mL) was added to the mixture, which was sonicated and stirred for another hour. The white solid was then filtered from the solution, washed repeatedly with acetonitrile, and dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-5-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride (0.11 g, 0.11 mmol, 54.0%) as a white solid. UPLC/ELSD: RT = 1.94 min. MS (ES): m/z (MH ⁺ ) 770.0 for C ₄₈ H ₉₁ Cl ₃ N ₄ O ₃ . ¹ HNMR(300MHz,MeOD)δ:ppm 5.42(m,1H),4.55(br.m,1H),3.45(m,4H),3.16(m,4H),2.41(m,6H),1.89(br.m,22H),1.43(m,14H),1.27(m,7H),1.18(m,4H ), 1.07 (s, 6H), 0.98 (d, 5H, J = 6Hz), 0.89 (q, 9H), 0.75 (s, 3H).

BM. Compound SA138: N-(3-amino-3-methylbutyl)-N-(4-((3-amino-3-methylbutyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride

Step 1: tert-Butyl (9-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)-2,2,6,6,17-pentamethyl-4-oxo-3-oxa-5,9,14-triazaoctadec-17-yl)carbamate

3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.250 g, 0.493 mmol), N-(4-{[4-({ A solution of tert-butyl 3-[(tert-butoxycarbonyl)amino]-3-methylbutyl}amino)butyl]amino}-2-methylbutyl-2-yl)carbamate (0.452g, 0.986mmol) and triethylamine (0.20mL, 1.4mmol) in DCM (2.5mL) was cooled to 0°C in an ice bath, and then propanephosphonic anhydride (50wt% in DCM) (0.62g, 0.97mmol) was added dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 1.5h, the reaction mixture was cooled to 0°C in an ice bath, and 5% _NaHCO3 aqueous solution (10mL) was added. The reaction mixture was then stirred at room temperature for 10min. Thereafter, the mixture was extracted with DCM ( _3x 15mL). The combined organic matter was passed through a hydrophobic glass frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-12% (5% concentrated aqueous _NH4OH in MeOH) in DCM) to afford tert-butyl (9-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)propanoyl)-2,2,6,6,17-pentamethyl-4-oxo-3-oxa-5,9,14-triazaoctadec-17-yl)carbamate (0.269 g, 0.284 mmol, 57.6%) as a clear gel. UPLC/ELSD: RT = 2.90 min. MS (ES): m/z of C ₅₄ H ₉₈ N ₄ O ₅ S ₂ = 948.55 [M+H] ⁺ . ¹ HNMR (300MHz, CDCl ₃ ): δ5.32-5.39(m,1H),3.18-3.58(m,6H),2.43-3.03(m,9H),2.26-2.40(m,2H),0.91-2.18(br.m,64H),1.00(s,3H),0.91(d,3H) , J=6.5Hz), 0.86 (d, 3H, J=6.6Hz), 0.86 (d, 3H, J=6.6Hz), 0.67 (s, 3H).

Step 2: N-(3-amino-3-methylbutyl)-N-(4-((3-amino-3-methylbutyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride

To a solution of tert-butyl (9-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)propanoyl)-2,2,6,6,17-pentamethyl-4-oxo-3-oxa-5,9,14-triazaoctadec-17-yl)carbamate (0.266 g, 0.281 mmol) in DCM (2.6 mL) in a screw cap vial was added 4N HCl in dioxane (0.49 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 2h, the reaction mixture was diluted to 30mL with MTBE, and then centrifuged (10,000xg for 30min). The supernatant was decanted. The solid was suspended in MTBE and then concentrated to give N-(3-amino-3-methylbutyl)-N-(4-((3-amino-3-methylbutyl)amino)butyl)-3-(((3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylheptyl-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta [a] phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride (0.203g, 0.225mmol, 80.3%) as a white solid. UPLC/ELSD: RT=1.96min. MS (ES): m/z of C ₄₄ H ₈₂ N ₄ OS ₂ = 264.75 [(M+3H)+CH ₃ CN] ³⁺ . ¹ H NMR (300MHz, CD ₃ OD): δ5.35-5.42(m,1H),3.37-3.59(m,4H),3.05-3.25(m,4H),2.92-3.03(m,2H),2.76-2.89(m,2H),2.57-2.74(m,1H),2.25-2. 42(m,2H),0.96-2.18(br.m,46H),1.03(s,3H),0.95(d,3H,J＝6.5Hz),0.88(d,6H,J＝6.6Hz),0.72(s,3H).

BN. Compound SA139: N-(3-amino-3-methylbutyl)-N-(8-amino-8-methylnonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: 4-methoxybenzyl (9-(N-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)-2-methylnon-2-yl)carbamate

To the mixture was added 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.100 g, 0.197 mmol), N-(4-{[8 To a stirred solution of tert-butyl-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate (0.103 g, 0.197 mmol) and triethylamine (0.09 mL, 0.6 mmol) in DCM (1.0 mL) cooled to 0°C was added 50 wt% propanephosphonic anhydride (0.20 mL, 0.39 mmol) in DCM dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 hours, the reaction mixture was diluted with DCM (10 mL) and then washed with 5% NaHCO ₃ aqueous solution. The aqueous layer was extracted with DCM (10 mL). The combined organic layers were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-50% EtOAc in hexanes) to afford 4-methoxybenzyl (9-(N-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)-2-methylnonan-2-yl)carbamate (0.146 g, 0.144 mmol, 73.2%) as a clear oil. UPLC/ELSD: RT = 3.80 min. MS (ES): m/z of C ₅₉ H ₉₉ N ₃ O ₆ S ₂ = 1012.83 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ7.32-7.26(m,2H),6.93-6.84(m,2H),5.40-5.31(m,1H),4.97(s,2H),4.73-4.34(m,2H),3.80(s,3H),3.37-3.17(m,4H),3.03 -2.88(m,2H),2.76-2.56(m,3H),2.42-2.25(m,2H),2.12-0.94(m,61H),0.99(s,3H),0.91(d,J＝6.4Hz,3H),0.86(d,J＝6.6Hz,6H),0.67(s,3H).

Step 2: N-(3-amino-3-methylbutyl)-N-(8-amino-8-methylnonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a stirred solution of 4-methoxybenzyl (9-(N-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)-2-methylnonan-2-yl)carbamate (0.143 g, 0.142 mmol) in DCM (2.5 mL) cooled to 0° C. was added 4N HCl in dioxane (0.25 mL). The reaction mixture was slowly warmed to room temperature while stirring and monitored by LCMS. At 22 hours, 4N HCl in dioxane (0.10 mL) was added. At 27 hours, MTBE (20 mL) was added and the reaction mixture was kept at 4°C overnight. The suspension was centrifuged (10,000 xg at 4°C for 30 min). The supernatant was decanted and the solid was suspended in MTBE and then concentrated to give N-(3-amino-3-methylbutyl)-N-(8-amino-8-methylnonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.067 g, 0.078 mmol, 55.0%) as a white solid. UPLC/ELSD: RT = 2.33 min. MS (ES): m/z of C ₄₅ H ₈₃ N ₃ OS ₂ = 374.56 (M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.45-5.33(m,1H),3.53-3.34(m,4H),3.01-2.89(m,2H),2.85-2.75(m,2H),2.74-2.57(m,1H),2.40-2.27(m,2H),2.14-1.77(m ,7H),1.73-0.97(m,33H),1.37(s,6H),1.33(s,6H),1.03(s,3H),0.95(d,J＝6.5Hz,3H),0.89(d,J＝6.6Hz,3H),0.88(d,J＝6.7Hz,3H),0.73(s,3H).

BO. Compound SA141: 5-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-5-oxopentanoate

To 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.100 g, 0.200 mmol), N-(4-{[8-( To a stirred solution of tert-butyl {[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate (0.104 g, 0.200 mmol) and DMAP (0.049 g, 0.40 mmol) in DCM (2.0 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.077 g, 0.40 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 15 h, the reaction mixture was diluted with DCM (15 mL) and washed with 5% NaHCO ₃ aqueous solution. The aqueous solution was extracted with DCM (15 mL). The combined organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-5-oxopentanoate (0.158 g, 0.157 mmol, 78.8%) as a clear oil. UPLC/ELSD: RT = 3.68 min. MS (ES): m/z of C ₆₁ H ₁₀₁ N ₃ O ₈ = 1005.92 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ7.32-7.26(m,2H),6.92-6.84(m,2H),5.41-5.30(m,1H),4.97(s,2H),4.76-4.34(m,3H),3.80(s,3H),3.37-3.13(m,4H),2.41 -2.24(m,6H),2.08-0.94(m,63H),1.01(s,3H),0.91(d,J＝6.4Hz,3H),0.86(d,J＝6.6Hz,3H),0.86(d,J＝6.6Hz,3H),0.67(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-5-oxopentanoate dihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-5-oxopentanoate (0.143 g, 0.143 mmol) in DCM (2.2 mL) was added 4N HCl in dioxane (0.25 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 h, 4N HCl in dioxane (0.10 mL) was added. At 22 hours, MTBE (15mL) was added, and the reaction mixture was kept overnight at 4 ° C. The suspension was centrifuged (10,000xg was kept for 30min at 4 ° C). The supernatant was decanted. The solid was suspended in MTBE and concentrated to obtain 5-((3-amino-3-methylbutyl) (8-amino-8-methylnonyl) amino)-5-oxopentanoic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylheptyl-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester dihydrochloride (0.082g, 0.098mmol, 68.7%) as a white solid. UPLC/ELSD: RT = 2.24 min. MS (ES): m/z for C ₄₇ H ₈₅ N ₃ O ₃ = 371.23 (M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.43-5.35(m,1H),4.62-4.47(m,1H),3.51-3.33(m,4H),2.49-2.27(m,6H),2.11-1.78(m,9H),1.71-0.98(m,33H),1.37(s,6H) ,1.33(s,6H),1.05(s,3H),0.95(d,J＝6.4Hz,3H),0.89(d,J＝6.6Hz,3H),0.88(d,J＝6.6Hz,3H),0.73(s,3H).

BP. Compound SA142: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminobutyl)(8-aminononyl)amino)-5-oxopentanoate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-5-oxopentanoate

To 5-{[(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylhept-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl]oxy}-5-oxopentanoic acid (0.09 g, 0.18 mmol) was added. To a solution in anhydrous DCM (5 mL) stirred under nitrogen was added tert-butyl (9-((3-((tert-butoxycarbonyl)amino)butyl)amino)non-2-yl)carbamate (0.08 g, 0.18 mmol), dimethylaminopyridine (0.04 g, 0.35 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.07 g, 0.35 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/ _NH4OH ) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-5-oxopentanoate as a light yellow oil (0.13 g, 0.15 mmol, 82.9%). UPLC/ELSD: RT: 3.53 _{min .} MS (ES): m/z (MH ⁺ ) 913.4 _{for C55H97N3O7} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.35(br.m,1H),4.59(br.m,2H),4.38(br.s,1H),3.59(br.m,2H),3.22(br.m,4H),2.32(m,6H),1.93(m,4H),1.81(m,3H),1.50(br .m,10H),1.42(s,18H),1.27(s,14H),1.09(m,12H),0.99(s,6H),0.90(d,4H,J＝6Hz),0.83(d,6H,J＝6Hz),0.66(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminobutyl)(8-aminononyl)amino)-5-oxopentanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-5-oxopentanoate (0.13 g, 0.15 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4N in dioxane, 0.36 mL, 1.45 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (25 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-5-(( ₃ -aminobutyl)(8-aminononyl)amino)-5-oxopentanoate dihydrochloride (0.10 g, 0.12 mmol, 80.4%) as a _white solid. UPLC/ELSD: RT = 1.77 min _. MS (ES): m/z (MH ⁺ ) 713.3 _{for C45H83Cl2N3O3} . ¹ H NMR (300MHz, CDCl ₃ ) δ8.49(br.m,2H),8.29(br.m,3H),5.39(s,1H),4.63(br.m,1H),3.45(br.m,5H),2.61(br.m,2H),2.43(br.m,2H),2.31(d,2H,J＝9Hz) ,2.01(br.m,6H),1.86(br.m,5H),1.45(br.m,29H),1.14(br.m,8H),1.04(s,6H),0.94(d,4H,J＝6Hz),0.90(d,7H,J＝6Hz),0.70(s,3H).

BQ. Compound SA144: (3-aminobutyl)(8-aminononyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester dihydrochloride

Step 1: 8-Bromo-N-methoxy-N-methyloctanamide

Oxalyl chloride solution (16.81mL, 33.62mmol, 2M in DCM) is added dropwise to the solution of 8-bromooctanoic acid (5.00g, 22.41mmol) in anhydrous DCM (50mL) stirred under nitrogen. After adding the initial 3mL oxalyl chloride, catalytic dimethylformamide (0.17mL, 2.24mmol) is added, and gas is formed as seen by bubbling. Then the remaining oxalyl chloride solution is added dropwise. The reaction is carried out for 3h at room temperature, and then the solution is concentrated into a yellow oil in a vacuum. Residue is dissolved in 30mL DCM and added dropwise to a solution of N, O-dimethylhydroxylamine hydrochloride (2.69g, 27.57mmol) in 80mL DCM stirred under nitrogen. During addition, solution is discharged as HCl gas is formed. The turbid yellow reaction mixture is stirred at room temperature overnight. The mixture was then further diluted with DCM, washed with water (1 x 30 mL), 1 M HCl (1 x 30 mL), 1 M NaOH (1 x 30 mL), and brine (1 x 30 mL), dried over sodium sulfate, filtered, and concentrated to give 8-bromo-N-methoxy-N-methyloctanamide as a clear yellow liquid, which was used without further purification (5.87 g, 22.04 mmol, 98.4%). UPLC/ELSD: RT = 0.67 min. MS (ES): m/z (MH ⁺ ) 267.1 for C ₁₀ H ₂₀ BrNO ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 3.61 (s, 3H), 3.33 (t, 2H), 3.10 (s, 3H), 2.34 (t, 2H), 1.78 (qu, 2H), 1.56 (qu, 2H), 1.29 (br.m, 7H).

Step 2: 9-Bromononan-2-one

The solution of 8- bromo-N- methoxyl-N- methyloctanamide (5.87g, 22.04mmol) in anhydrous THF (100mL) is stirred under nitrogen and cooled to 0 DEG C. Then, methylmagnesium bromide solution (11.02ml, 33.06mmol, 3M in diethyl ether) is added dropwise to the stirred mixture. The mixture is stirred at 0 DEG C for 2.5h, and then it is gradually warmed to room temperature and kept for another 2h. Then the mixture is cooled to 0 DEG C again, and the reaction is quenched by dropping hydrochloric acid (66.13mL, 66.13mmol, 1M). The mixture is stirred continuously and gradually warmed to room temperature over 30 minutes. THF is removed under vacuum, and the mixture is extracted with EtOAc (3x50 mL). The organic phase merged is washed with brine (1x50 mL), dried over sodium sulfate, filtered, and concentrated into an oil in a vacuum. The oil was dissolved in DCM and purified on silica with a 0-50% EtOAc gradient in hexanes. Fractions containing product were combined and concentrated to give 9-bromononan-2-one (4.44 g, 20.08 mmol, 91.1%) as a colorless oil. UPLC/ELSD: RT = 0.84 min. MS (ES): m/z (MH ⁺ ) 222.1 for C ₉ H ₁₇ BrO. ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 3.33 (t, 2H), 2.36 (t, 2H), 2.06 (s, 3H), 1.78 (qu, 2H), 1.50 (qu, 2H), 1.30 (br. m, 7H).

Step 3: 9-Bromononan-2-amine

To a solution of 9-bromononan-2-one (3.44g, 15.56mmol) in anhydrous MeOH (100mL), ammonium acetate (10.79g, 140.00mmol) and sodium cyanoborohydride (1.27g, 20.22mmol) are added. The solution is vigorously stirred at room temperature for 36h. Then, the reaction is quenched by slowly adding HCl (100mL, 2M). Then, 10M NaOH is added dropwise until the pH of the solution reaches 11-12, qualitatively measured with pH paper. Then, the mixture is extracted with DCM (3x150 mL), and the combined organic phase is washed with brine (1x100 mL), dried over sodium sulfate, filtered, and concentrated into a yellow oil. The oil is dissolved in DCM and purified with 0-50% (1:1DCM/MeOH) gradient in DCM on silica. Fractions containing product were pooled and concentrated in vacuo to give 9-bromononan-2-amine (1.23 g, 5.53 mmol, 35.6%) as a colorless oil. UPLC/ELSD: RT = 0.89 min. MS (ES): m/z (MH ⁺ ) 223.1 for C ₉ H ₂₀ BrN. ¹ H NMR (300 MHz, CDCl ₃ ) δ: ppm 3.29 (t, 2H), 2.82 (br. m, 2H), 2.66 (s, 3H), 1.74 (qu, 2H), 1.21 (br. m, 11H), 0.99 (d, 3H).

Step 4: tert-Butyl (9-bromononan-2-yl)carbamate

To the solution of 9-brominonanyl-2-amine (0.57g, 2.54mmol) in the anhydrous THF (10mL) stirred at 0 ℃ under nitrogen, ditert-butyl dicarbonate (0.64mL, 2.80mmol) is added. Then, triethylamine (0.39mL, 2.80mmol) is added dropwise, and the solution is gradually warmed to room temperature and stirred overnight. Then the solvent is removed under vacuum, and the gained residue is dissolved in DCM, washed with 5% HCl aqueous solution (1x15 mL) and water (1x15 mL), dried over sodium sulfate, filtered, and concentrated into oily matter in a vacuum. The oily matter is dissolved in DCM and purified with 0-20% EtOAc gradient in hexane on silica. The fractions containing product are collected and concentrated in a vacuum to obtain (9-brominonanyl-2-yl) tert-butyl carbamate (0.47g, 1.47mmol, 57.6%) in oily state. UPLC/ELSD: RT=1.54min. MS (ES): m/z (MH ⁺ ) 323.3 for C ₁₄ H ₂₈ BrNO ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ: ppm 4.35 (br.m, 1H), 3.60 (br.m, 1H), 3.37 (t, 2H), 1.84 (qu, 2H), 1.41 (br.s, 11H), 1.28 (br.m, 8H), 1.08 (d, 3H).

Step 5: tert-Butyl (9-((3-((tert-butoxycarbonyl)amino)butyl)amino)nonan-2-yl)carbamate

N-[4-(2-nitrobenzenesulfonamido)butyl-2-yl]t-butyl carbamate (0.88 g, 2.37 mmol) and (9-bromononyl-2-yl)t-butyl carbamate (0.76 g, 2.37 mmol) were dissolved in 20 mL of anhydrous DMF and stirred under nitrogen. Then, potassium carbonate (1.96 g, 14.21 mmol) was added, and the solution was heated to 40 ° C and stirred overnight. The next morning, the mixture was cooled to room temperature, and benzyl bromide (0.17 mL, 1.42 mmol) was added. The solution was stirred at room temperature for 5 hours. Then, thiophenol (0.727 mL, 7.10 mmol), potassium carbonate (0.98 g, 7.10 mmol) and additional 10 mL of anhydrous DMF were added, and the reactants were stirred for 2 days. Then, salts were removed from the solution by centrifugation, and the supernatant was evaporated to residue. The residue was dissolved in 40 mL of DCM and washed with water (2x10 mL) and brine (2x10 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was resuspended in DCM and purified on silica in DCM with a 0-50% (50:45:5 DCM/MeOH/NH ₄ OH aqueous solution) gradient. The fractions containing the product were pooled and concentrated to give tert-butyl (9-((3-((tert-butoxycarbonyl)amino)butyl)amino)non-2-yl)carbamate (0.77 g, 1.80 mmol, 76.1%) as a colorless oil. UPLC/ELSD: RT=0.61 min. MS (ES): m/z (MH ⁺ ) 430.6 for C ₂₃ H ₄₇ N ₃ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ 4.94 (br.s, 1H), 4.33 (br.s, 1H), 3.62 (br.m, 2H), 2.61 (m, 4H), 1.66 (br.s, 1H), 1.45 (s, 21H), 1.29 (s, 9H), 1.14 (m, 6H).

Step 6: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)carbamate

To a solution of tert-butyl (9-((3-((tert-butoxycarbonyl)amino)butyl)amino)nonan-2-yl)carbamate (0.11 g, 0.26 mmol) in anhydrous toluene (5 mL) stirred under nitrogen was added triethylamine (0.11 mL, 0.78 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.15 g, 0.26 mmol) was added and the solution was heated to 90° C. for 2 days. The reaction mixture was then cooled to room temperature, diluted with toluene, washed with water (3 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography with a 0-30% EtOAc gradient in hexanes. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)carbamate as _a light yellow oil (0.18 g, 0.21 mmol, 81.3%). UPLC/ELSD: RT = 3.79 min _. MS (ES): m/z (MH ⁺ ) 871.4 _{for C53H95N3O6} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.07(s,1H),4.14(br.m,3H),3.33(br.s,2H),2.91(br.m,4H),2.07(m,2H),1.66(m,6H),1.26(br.m,10H),1.14(s,21H),0.98(s, 15H), 0.83 (d, J = 21.1Hz, 12H), 0.73 (s, 6H), 0.65 (s, 5H), 0.56 (s, 9H), 0.39 (s, 3H).

Step 7: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-aminobutyl)(8-aminononyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)carbamate (0.18 g, 0.21 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4N in dioxane, 0.53 mL, 2.11 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (25 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)(3-aminobutyl)(8-aminononyl)carbamate dihydrochloride (0.14 g, 0.18 mmol, 83.4%) as a white solid. UPLC/ELSD: RT = 1.89 min. MS (ES): m/z (MH ⁺ ) 671.3 for C ₄₃ H ₈₁ Cl ₂ N ₃ O ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ8.52(br.m,3H),8.33(br.m,3H),5.40(br.s,1H),4.52(br.s,1H),3.33(br.m,6H),2.37(m,2H),2.01(br.m,7H),1.34(br.m,31H),1 .18(dd,J＝13.1,7.6Hz,4H),1.11(s,2H),1.04(br.m,4H),0.93(br.s,4H),0.85(q,8H),0.68(s,3H).

BR. Compound SA145: N-(3-amino-3-methylbutyl)-N-(4-amino-4-methylpentyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: tert-Butyl (4-(N-(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)-2-methylbutan-2-yl)carbamate

To a stirred anhydrous solution of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.11 g, 0.22 mmol) was added to a stirred anhydrous solution of 1% ethanol under nitrogen. To a solution in DCM (5 mL) was added tert-butyl (5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-2-methylpentan-2-yl)carbamate (0.09 g, 0.22 mmol), dimethylaminopyridine (0.06 g, 0.45 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.09 g, 0.45 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give tert-butyl (4-(N-(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)-2-methylbutan-2-yl)carbamate (0.17 g, 0.19 mmol, 86.7%) as a light yellow oil. UPLC/ELSD: RT: 3.70 min. MS (ES): m/z (MH ⁺ ) 891.4 for C ₅₁ H ₉₁ N ₃ O ₅ S ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.27(br.s,1H),4.68(s,1H),4.41(br.s,1H),4.03(q,1H),3.20(br.m,4H),2.87(br.m,2H),2.62(br.m,3H),2.26(br.m,2H),1.96(br .m,8H),1.50(br.m,9H),1.37(s,19H),1.28(br.m,3H),1.20(m,14H),1.04(br.m,6H),0.92(s,5H),0.85(d,4H,J＝6Hz),0.80(d,6H,J＝6Hz),0.60(s,3H) .

Step 2: N-(3-amino-3-methylbutyl)-N-(4-amino-4-methylpentyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a solution of tert-butyl (4-(N-(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)propionamido)-2-methylbutan-2-yl)carbamate (0.17 g, 0.19 mmol) in DCM (5 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.49 mL, 1.94 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (10 mL) was added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give N-(3-amino-3-methylbutyl)-N-(4-amino-4-methylpentyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.13 g, 0.17 mmol, 85.9%) as a white solid. UPLC/ELSD: RT=2.12 min. MS (ES): m/z (MH ⁺ ) 691.3 for C ₄₁ H ₇₇ Cl ₂ N ₃ OS ₂ . ¹ H NMR (300MHz, MeOD) δ5.39(br.s,1H),3.48(br.m,4H),3.33(br.s,2H),2.98(br.m,2H),2.86(br.m,2H),2.67(br.m,1H),2.37(d,2H,J＝6Hz),1.97(br.m,7H) ,1.66(br.m,12H),1.46(s,4H),1.41(s,13H),1.31(s,3H),1.17(br.m,8H),1.05(s,5H),0.96(d,4H,J＝6Hz),0.90(d,8H,J＝6Hz),0.75(s,3H).

BS. Compound SA149: (3-amino-3-methylbutyl)(4-amino-4-methylpentyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)carbamate

To a solution of tert-butyl (5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-2-methylpentan-2-yl)carbamate (0.09 g, 0.22 mmol) in anhydrous toluene (5 mL) stirred under nitrogen was added triethylamine (0.07 mL, 0.09 mmol). Then, (1R,3aS,3bS,7S,9aR,9bS,11aR)-1-[(2R,5R)-5-ethyl-6-methylhept-2-yl]-9a,11a-dimethyl-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl 4-nitrophenyl carbonate (0.13 g, 0.22 mmol) was added and the solution was heated to 90° C. for 2 days. The reaction mixture was then cooled to room temperature, diluted with toluene, and washed with water (3 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography with a 0-50% EtOAc gradient in hexanes. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)carbamate as a light yellow oil (0.14 g, 0.17 mmol, 74.7%). UPLC/ELSD: RT = 3.78 _{min .} MS (ES): m/z (MH ⁺ ) 843.4 _{for C51H91N3O6} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.30(br.s,1H),4.42(br.m,3H),3.12(br.s,4H),2.28(br.m,2H),1.80(br.m,7H),1.52(br.m,11H),1.35(s,18H),1.20(s,18H),1 .08(br.m,5H),0.94(s,6H),0.86(d,5H,J＝6Hz),0.75(q,9H),0.61(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-3-methylbutyl)(4-amino-4-methylpentyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)carbamate (0.14 g, 0.17 mmol) in DCM (5 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.42 mL, 1.67 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (10 mL) was added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)carbamate dihydrochloride (0.08 g, 0.10 mmol, 61.2%) as a white solid. UPLC/ELSD: RT=2.07 min. MS (ES): m/z (MH ⁺ ) 643.3 for C ₄₁ H ₇₇ Cl ₂ N ₃ O ₂ . ¹ H NMR (300MHz, MeOD) δ5.44(br.s,1H),4.47(br.m,1H),3.34(br.m,7H),2.40(br.m,2H),1.97(br.m,7H),1.66(br.m,11H),1.37(d,14H,J＝6Hz),1.20(br.m,8 H), 1.08 (s, 5H), 0.99 (d, 5H, J = 6Hz), 0.87 (q, 8H), 0.75 (s, 3H).

BT. Compound SA151: (3-aminobutyl)(8-aminononyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)carbamate

To a solution of tert-butyl (9-((3-((tert-butoxycarbonyl)amino)butyl)amino)nonan-2-yl)carbamate (0.12 g, 0.27 mmol) in anhydrous toluene (5 mL) stirred under nitrogen was added triethylamine (0.12 mL, 0.82 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.15 g, 0.27 mmol) was added and the solution was heated to 90° C. for 2 days. Then, the reaction mixture is cooled to room temperature, diluted with toluene, washed with water (3x20 mL), dried over sodium sulfate, filtered, and concentrated into an oil. The oil is dissolved in DCM and purified by silica gel chromatography in hexane with 0-50% EtOAc gradient. The fractions containing the product are merged and concentrated to obtain (3-((tert-butoxycarbonyl)amino)butyl) (8-((tert-butoxycarbonyl)amino)nonyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenta [a] phenanthrene-3-yl ester (0.17 g, 0.21 mmol, 75.6%). UPLC/ELSD: RT=3.70min. MS (ES): m/z (MH ⁺ ) 843.4 for C ₅₁ H ₉₁ N ₃ O ₆ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.35(br.s,1H),4.46(br.m,3H),3.59(br.m,2H),3.19(br.m,4H),2.29(m,2H),2.01(m,6H),1.59(br.m,10H),1.40(s,20H),1.25 (br.m,15H),1.10(q,12H),0.99(s,6H),0.90(d,4H,J＝6Hz),0.83(d,6H,J＝6Hz),0.65(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-aminobutyl)(8-aminononyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)carbamate (0.17 g, 0.21 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4N in dioxane, 0.52 mL, 2.05 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (25 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-aminobutyl)(8-aminononyl)carbamate dihydrochloride (0.15 g, 0.20 mmol, 95.7%) as a white solid. UPLC/ELSD: RT = 1.83 min. MS (ES): m/z (MH ⁺ ) 643.3 for C ₄₁ H ₇₇ Cl ₂ N ₃ O ₂ . ¹ H NMR (301MHz, CDCl ₃ ) δ8.51(br.s,3H),8.32(br.s,3H),5.39(br.m,1H),4.50(br.m,1H),3.34(br.m,6H),2.37(m,2H),2.01(br.m,7H),1.45(br.m,29H),1. 11(br.m,8H),1.04(s,4H),0.93(d,3H,J＝6Hz,3H),0.90(d,3H,J＝6Hz,6H),0.70(s,3H).

BU. Compound SA152: (3-amino-3-methylbutyl)(8-amino-8-methylnonyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: 9-Chloro-2,2-dimethylnonanoic acid methyl ester

Methyl isobutyrate (3.0 mL, 26 mmol) was added to a solution of THF (30 mL) and lithium diisopropylamide (19 mL, 2.0 M in THF) cooled to -78 ° C. The reaction mixture was stirred at 0 ° C for 50 min and then cooled to -78 ° C. 1-bromo-7-chloroheptane (4.2 mL, 27 mmol) was added dropwise. The reaction mixture was stirred while slowly warming to room temperature and monitored by TLC. At 20 hours, the reaction mixture was cooled to 0 ° C, and then 1N HCl aqueous solution (30 mL) was added dropwise. The biphasic mixture was separated, and the aqueous layer was extracted with EtOAc (2x 30 mL). The combined organic matter was washed with brine, dried over Na ₂ SO ₄ , and concentrated to provide 9-chloro-2,2-dimethylnonanoic acid methyl esters (6.205 g, quantitative) in an amber oil. The material was used as it was. ¹ HNMR (300MHz, CDCl ₃ ): δ3.65 (s, 3H), 3.52 (t, J = 6.7Hz, 2H), 1.83-1.63 (m, 2H), 1.63-1.17 (m, 10H), 1.15 (s, 6H).

Step 2: 9-Chloro-2,2-dimethylnonanoic acid

A mixture of 9-chloro-2,2-dimethylnonanoic acid methyl ester (6.2 g, 26 mmol), THF (60 mL), MeOH (45 mL) and 10% NaOH aqueous solution (31 mL, 78 mmol) was stirred at 50 ° C. The reaction was monitored by TLC. At 23 hours, the reaction mixture was concentrated to remove volatile organics. The residue was dissolved in water (70 mL), washed with MTBE (2 x 50 mL), and then acidified to pH about 1 with 2N HCl aqueous solution. The aqueous solution was extracted with EtOAc (3 x 50 mL), dried over Na ₂ SO ₄ , and then concentrated to give 9-chloro-2,2-dimethylnonanoic acid (4.997 g, 22.64 mmol, 85.7%) as an amber oil. UPLC/ELSD: RT=1.00 min. MS (ES): m/z of C ₁₁ H ₂₁ ClO ₂ = 174.98 (M-CO ₂ H) ⁺ . ¹ H NMR (300MHz, CDCl3): δ9.71 (br.s, 1H), 3.53 (t, J = 6.7Hz, 2H), 1.88-1.66 (m, 2H), 1.62-1.22 (m, 10H), 1.19 (s, 6H).

Step 3: (4-methoxyphenyl)methyl N-(9-chloro-2-methylnonan-2-yl)carbamate

To a stirred solution of 9-chloro-2,2-dimethylnonanoic acid (2.00 g, 9.06 mmol) and triethylamine (1.8 mL, 13 mmol) in PhMe (30 mL) was added diphenylphosphoryl azide (2.4 mL, 11 mmol). The reaction mixture was stirred at room temperature for 1.25 hours and then stirred at 80 °C. Gas evolution occurred. At 2 hours, the reaction mixture was cooled to room temperature and then washed with 5% NaHCO ₃ aqueous solution (2x), water and brine. The organics were dried over Na ₂ SO ₄ , and then 4-methoxybenzyl alcohol (2.2 mL, 18 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (2.8 mL, 19 mmol) were added in sequence. The reaction mixture was stirred at 80 °C and monitored by LCMS. At 18 h, the reaction mixture was cooled to room temperature, diluted with EtOAc (150 mL), washed with 5% aqueous citric acid (2x), water, and _brine , dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-30% EtOAc in hexanes) to afford (4-methoxyphenyl)methyl N-(9-chloro-2-methylnon-2-yl)carbamate (1.613 g, 4.532 mmol, 50.0%) as a clear oil. UPLC/ELSD: RT = 1.70 _{min . MS (ES): m/z for C19H30ClNO3 =} 378.33 (M+Na) ⁺ . ¹ H NMR (300MHz, CDCl3): δ7.29 (d, J = 8.3Hz, 2H), 6.88 (d, J = 8.1Hz, 2H), 4.98 (s, 2H), 4.58 (s, 1H), 3.81 (s, 3H), 3.53 (t, J = 6.7Hz, 2H), 1.84-1.69 (m, 2H), 1 .68-1.16(m,16H).

Step 4: tert-Butyl N-(4-{N-[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]-2-nitrobenzenesulfonamido}-2-methylbutan-2-yl)carbamate

By N-[2-methyl-4-(2-nitrobenzenesulfonamido)butyl-2-yl]carbamic acid tert-butyl ester (0.907g, 2.34mmol), N-(9-chloro-2-methylnon-2-yl)carbamic acid (4-methoxyphenyl) methyl ester (0.700g, 1.97mmol), potassium carbonate (0.544g, 3.93mmol), potassium iodide (0.164g, 0.983mmol) and propionitrile (10.5mL) are combined in a sealed tube. The reaction mixture is heated at 150°C via microwave irradiation while stirring and monitored by LCMS. At 12 hours, the reaction mixture is cooled to room temperature and filtered, rinsed with ACN, and the filtrate is concentrated. The residue is dissolved in EtOAc ( _100mL ), then washed with 5% _NaHCO3 aqueous solution and brine, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-50% EtOAc in hexanes) to give tert-butyl N-(4-{N-[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8 _- methylnonyl]-2-nitrobenzenesulfonamido}-2-methylbutan- ₂ -yl)carbamate (1.267 g, 1.792 mmol, 91.1%) as a yellow oil. UPLC/ELSD: RT = 2.00 min. _MS (ES): m/z for _C35H54N4O9S = 607.64 [(M+H)-( _CH3 ) _2C = _CH2 - _CO2 ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ8.06-7.93(m,1H),7.77-7.52(m,3H),7.37-7.20(m,2H),6.97-6.78(m,2H),4.97(s,2H),4.59(s,1H),4.39(s,1H),3.80(s, 3H),3.41-3.20(m,4H),2.00-1.84(m,2H),1.66-1.10(m,33H).

Step 5: tert-Butyl N-(4-{[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate

To a mixture of tert-butyl N-(4-{N-[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]-2-nitrobenzenesulfonamido}-2-methylbutan-2-yl)carbamate (1.258 g, 1.780 mmol) and potassium carbonate (0.738 g, 5.34 mmol) in DMF (19 mL) was added thiophenol (0.33 mL, 3.2 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 2 h, the reaction mixture was filtered and rinsed with EtOAc. The filtrate was diluted to 125 mL with EtOAc, washed with 5% aqueous NaHCO ₃ solution, water (3x), and brine, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-16% (5% concentrated _NH4OH in MeOH) in DCM) to afford tert-butyl N-(4-{[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2 _- methylbutan-2-yl)carbamate (0.699 g, 1.34 mmol, 75.3%) as a yellow oil. UPLC/ELSD: RT = _0.89 min. MS (ES): m/z = 522.74 (M+H) ^{+ for} _{C29H51N3O5 .} ¹ H NMR (300MHz, CDCl ₃ ) δ7.33-7.27(m,2H),6.91-6.84(m,2H),5.61(s,1H),4.97(s,2H),4.59(s,1H),3.80(s,3H),2.75(t,J＝7.2Hz,2H),2.64(t,J＝7. 3Hz, 2H), 1.81 (t, J = 7.2Hz, 2H), 1.70-1.13 (m, 33H).

Step 6: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)carbamate

Cholesteryl carbonate 4-nitrophenyl ester (0.150 g, 0.272 mmol), tert-butyl N-(4-{[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate (0.184 g, 0.353 mmol), and triethylamine (0.12 mL, 0.86 mmol) were combined in PhMe (2.05 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. At 20 h, the reaction mixture was cooled to room temperature, diluted with DCM (30 mL), and then washed with 5% _NaHCO3 aqueous solution (3x). The organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-30% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)carbamate (0.227 g, 0.243 mmol, 89.4%) as a clear oil. UPLC/ELSD: RT = 3.79 min. MS (ES): m/z of C ₅₇ H ₉₅ N ₃ O ₇ = 935.72 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ7.34-7.24(m,2H),6.95-6.83(m,2H),5.42-5.32(m,1H),4.97(s,2H),4.64-4.32(m,3H),3.80(s,3H),3.33-3.07(m,4H),2.4 8-2.21(m,2H),2.12-0.94(m,61H),1.02(s,3H),0.91(d,J＝6.4Hz,3H),0.87(d,J＝6.6Hz,6H),0.68(s,3H).

Step 7: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-amino-3-methylbutyl)(8-amino-8-methylnonyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)carbamate (0.222 g, 0.238 mmol) in DCM (2.6 mL) was added 4N HCl in dioxane (0.43 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 18 h, hexanes (30 mL) were added and the mixture was centrifuged (10,000 x g for 30 min). The supernatant was decanted, the solid was suspended in hexane (30 mL), and the suspension was centrifuged (10,000 x g for 30 min). The supernatant was decanted, and the solid was dried under reduced pressure to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride (0.125 g, 0.163 mmol, 68.8%) as a white solid. UPLC/ELSD: RT = 1.87 min. MS (ES): m/z of C ₄₃ H ₇₉ N ₃ O ₂ = 335.74(M+2H) ²⁺ . ¹ H NMR (300MHz, DMSO) δ8.29-7.87(m,6H),5.41-5.26(m,1H),4.39-4.22(m,1H),3.29-3.06(m,4H),2.36-2.11(m,2H),2.09-0.90(m,52H),0.98(s,3H ), 0.89 (d, J = 6.2Hz, 3H), 0.84 (d, J = 6.6Hz, 6H), 0.65 (s, 3H).

BV. Compound SA153: (3-amino-3-methylbutyl)(8-amino-8-methylnonyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)carbamate

Sitosterol carbonate 4-nitrophenyl ester (0.175g, 0.302mmol), tert-butyl N-(4-{[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate (0.205g, 0.392mmol) and triethylamine (0.13mL, 0.93mmol) were combined in PhMe (2.3mL). The reaction mixture was stirred at 90°C and monitored by LCMS. At 20 hours, the reaction mixture was cooled to room temperature, diluted with DCM (30mL), and then washed with 5% _NaHCO3 aqueous solution (3x). The organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-30% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)carbamate (0.252 g, 0.262 mmol, 86.8%). UPLC/ELSD: RT = 3.89 min. MS (ES): m/z of C ₅₉ H ₉₉ N ₃ O ₇ = 963.23 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ7.32-7.24(m,2H),6.94-6.85(m,2H),5.42-5.32(m,1H),4.97(s,2H),4.66-4.40(m,3H),3.81(s,3H),3.33-3.08(m,4H),2. 47-2.20(m,2H),2.13-0.77(m,71H),1.02(s,3H),0.92(d,J＝6.3Hz,3H),0.68(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-3-methylbutyl)(8-amino-8-methylnonyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)carbamate (0.248 g, 0.258 mmol) in DCM (2.6 mL) was added 4N HCl in dioxane (0.46 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 18 hours, hexanes (30 mL) were added, and the mixture was centrifuged (10,000 xg for 30 min). The supernatant was decanted, the solid was suspended in hexanes (30 mL), and the suspension was centrifuged (10,000 xg for 30 min). The supernatant was decanted and the solid was dried under reduced pressure to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)(3-amino-3-methylbutyl)(8-amino-8 _{-methylnonyl)carbamate dihydrochloride (0.107 g, 0.130 mmol, 50.6%) as a white solid. UPLC/ELSD: RT = 3.89 min. MS (ES): m/z for C45H83N3O2 = 370.68} [(M+ _2H )+ _CH3CN ] ²⁺ . ¹ H NMR (300MHz, DMSO) δ8.34-7.91(m,6H),5.39-5.29(m,1H),4.41-4.21(m,1H),3.30-3.07(m,4H),2.37-2.16(m,2H),2.05-0.74(m,62H),0.98(s,3H ), 0.90 (d, J = 6.3Hz, 3H), 0.65 (s, 3H).

BW. Compound SA154: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminobutyl)(8-aminononyl)amino)-5-oxopentanoate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-5-oxopentanoate

To 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.09 g, 0.18 mmol) 1) To a solution in anhydrous DCM (5 mL) stirred under nitrogen was added tert-butyl (9-((3-((tert-butoxycarbonyl)amino)butyl)amino)non-2-yl)carbamate (0.08 g, 0.18 mmol), dimethylaminopyridine (0.04 g, 0.35 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.07 g, 0.35 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-5-oxopentanoate as a light yellow oil (0.09 g, 0.10 mmol, 57.3%). UPLC/ELSD: RT: 3.63 _min . MS (ES): m/z (MH ⁺ ) _{941.4 for C57H101N3O7} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.35(br.s,1H),4.60(br.m,2H),4.34(br.m,1H),3.63(br.m,3H),3.20(br.m,3H),2.35(m,6H),1.94(m,4H),1.83(br.m,3H),1.55( br.m,10H),1.43(s,18H),1.28(br.m,16H),1.11(m,12H),1.01(s,5H),0.92(d,5H,J＝6Hz),0.82(q,10H),0.67(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-aminobutyl)(8-aminononyl)amino)-5-oxopentanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-5-oxopentanoate (0.09 g, 0.10 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4 N in dioxane, 0.25 mL, 1.00 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (25 mL) was added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-5-oxopentanoate dihydrochloride (0.06 g, 0.06 mmol, 63.4%) as a white solid. UPLC/ELSD: RT=1.96 min. MS (ES): m/z (MH ⁺ ) 741.3 for C ₄₇ H ₈₇ Cl ₂ N ₃ O ₃ . ² _.01 (br.m,6H),1.86(br.m,3H),1.62(br.m,9H),1.46(br.m,16H),1.19(br.m,11H),1.04(s,5H),0.96(d,5H,J=6Hz),0.85(q,10H),0.70(s,3H).

BX. Compound SA155: 5-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-5-oxopentanoate

To the mixture was added 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.100 g, 0.189 mmol), N-(4- To a stirred solution of tert-butyl {[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate (0.099 g, 0.19 mmol) and DMAP (0.046 g, 0.38 mmol) in DCM (2.0 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.073 g, 0.38 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 15 h, the reaction mixture was diluted with DCM (15 mL) and washed with 5% _aqueous NaHCO. The aqueous solution was extracted with DCM (15 mL). The combined organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-5-oxopentanoate (0.121 g, 0.117 mmol, 62.0%) as a clear oil. UPLC/ELSD: RT = 3.77 min. MS (ES): m/z of C ₆₃ H ₁₀₅ N ₃ O ₈ = 1034.04 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ7.33-7.27(m,2H),6.92-6.84(m,2H),5.42-5.31(m,1H),4.97(s,2H),4.77-4.30(m,3H),3.81(s,3H),3.36-3.14(m,4H),2. 43-2.24(m,6H),2.16-0.76(m,73H),1.01(s,3H),0.92(d,J＝6.5Hz,3H),0.67(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-5-oxopentanoate dihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-5-oxopentanoate (0.116 g, 0.112 mmol) in DCM (2.0 mL) was added 4N HCl in dioxane (0.20 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 hours, 4N HCl in dioxane (0.10 mL) was added. At 22 hours, MTBE (20 mL) was added and the reaction mixture was kept at 4°C overnight. The suspension was centrifuged (10,000 xg at 4°C for 30 min). The supernatant was decanted. The solid was suspended in MTBE and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-methylbutyl)(8-amino-8- _{methylnonyl)amino)-5-oxopentanoate dihydrochloride (0.073 g, 0.080 mmol, 71.4%) as a white solid. UPLC/ELSD: RT = 2.38 min. MS (ES): m/z for C49H89N3O3 = 385.65 (} M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.43-5.32(m,1H),4.62-4.47(m,1H),3.50-3.33(m,4H),2.51-2.23(m,6H),2.13-1.77(m,9H),1.76-0.77(m,43H),1.37(s,6H) ,1.33(s,6H),1.05(s,3H),0.96(d,J＝6.4Hz,3H),0.75-0.70(m,3H).

BY. Compound SA156: 5-(bis(3-aminobutyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-(bis(3-((tert-butoxycarbonyl)amino)butyl)amino)-5-oxopentanoate

To 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.100 g, 0.189 m To a stirred solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.073 g, 0.38 mmol), tert-butyl N-[4-({3-[(tert-butoxycarbonyl)amino]butyl}amino)butan-2-yl]carbamate (0.075 g, 0.21 mmol), and DMAP (0.051 g, 0.42 mmol) in DCM (2.0 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.073 g, 0.38 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 h, the reaction mixture was diluted with DCM (10 mL) and then washed with 5% aqueous NaHCO. The _aqueous solution was extracted with DCM (10 mL). The combined organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-65% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-(bis(3-((tert-butoxycarbonyl)amino)butyl)amino)-5-oxopentanoate as a white foam (0.097 g, 0.11 mmol, 58.9%). UPLC/ELSD: RT = 3.57 min. MS (ES): m/z of C ₅₂ H ₉₁ N ₃ O ₇ = 871.11 (M+H) ⁺ . ¹ HNMR (300MHz, CDCl3) δ5.48-5.30(m,1H),4.77-4.42(m,3H),3.79-3.07(m,6H),2.52-2.19(m,6H),2.14-0.76(m,66H),1.01(s,3H),0.92(d,J＝6.4Hz ,3H),0.67(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-(bis(3-aminobutyl)amino)-5-oxopentanoate dihydrochloride

To a stirred solution of (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-5-(bis(3-((tert-butoxycarbonyl)amino)butyl)amino)-5-oxopentanoate (0.086 g, 0.099 mmol) in DCM (1.8 mL) was added 4N HCl (0.25 mL) in dioxane. The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 hours, MTBE (20 mL) was added and the reaction mixture was centrifuged (10,000 x g for 15 min at 4 °C). The supernatant was removed by suction and the solid was rinsed sparingly with MTBE. The solid was suspended in MTBE and then concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-5-oxopentanoate dihydrochloride (0.066 g, 0.082 mmol, 82.8%) as a white solid. UPLC/ELSD: RT = 2.22 min. MS (ES): m/z = 670.59 (M+H) ⁺ for C ₄₂ H ₇₅ N ₃ O ₃ . ¹ H NMR (300MHz, MeOD) δ5.43-5.31(m,1H),4.62-4.48(m,1H),3.73-3.33(m,5H),3.24-3.11(m,1H),2.60-2.23(m,6H),2.11-0.76(m,42H),1.37(d,J＝ 6.6Hz, 3H), 1.33 (d, J = 6.6Hz, 3H), 1.05 (s, 3H), 0.96 (d, J = 6.4Hz, 3H), 0.73 (s, 3H).

BZ. Compound SA157: (3-amino-3-methylbutyl)(4-amino-4-methylpentyl)carbamic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: 4-Methylbenzenesulfonic acid 4-((tert-butoxycarbonyl)amino)-4-methylpentyl ester

To a solution of tert-butyl N-(5-hydroxy-2-methylpentan-2-yl)carbamate (2.50 g, 11.50 mmol) in anhydrous DCM (30 mL) stirred under nitrogen, triethylamine (8.02 mL, 57.52 mmol), dimethylaminopyridine (0.14 g, 1.15 mmol) and p-toluenesulfonyl chloride (4.39 g, 23.01 mmol) were added. The solution was stirred at room temperature for 6 hours, after which it turned dark red. The mixture was then further diluted with DCM, washed with water (1x30 mL), saturated aqueous sodium bicarbonate solution (1x30 mL) and brine (1x30 mL), dried over sodium sulfate, filtered, and concentrated into a dark brown oil. The oil was dissolved in DCM and purified on silica with a 0-20% (80:19:1 DCM/MeOH/NH ₄ OH) gradient in DCM. Fractions containing product were pooled and concentrated to give 4-((tert-butoxycarbonyl)amino)-4-methylpentyl 4-methylbenzenesulfonate (3.55 g, 9.64 mmol, 83.0%) as a light brown oil. UPLC/ELSD: RT: 1.20 min. MS (ES): m/z (MH ⁺ ) 372.4 for C ₁₈ H ₂₉ NO ₅ S. ¹ H NMR (300 MHz, CDCl ₃ ) δ 7.78 (d, 2H, J=9 Hz), 7.36 (d, 2H, J=9 Hz), 4.37 (br. s, 1H), 4.00 (br. m, 2H), 2.45 (s, 3H), 1.64 (br. s, 4H), 1.40 (s, 10H), 1.20 (s, 6H).

Step 2: tert-Butyl (5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-2-methylpentan-2-yl)carbamate

To a solution of tert-butyl N-[2-methyl-4-(2-nitrobenzenesulfonamido)butan-2-yl]carbamate (0.91 g, 2.34 mmol) in anhydrous DMF (20 mL) stirred under nitrogen at room temperature was added tert-butyl N-{2-methyl-5-[(4-methylbenzenesulfonyl)oxy]pentan-2-yl}carbamate (0.87 g, 2.34 mmol) and potassium carbonate (1.97 g, 14.25 mmol). The solution was warmed to 40 ° C and stirred overnight. The next morning, the reaction was incomplete according to LC-MS, so it was heated to 100 ° C and stirred for another 3 hours. The mixture was then cooled to room temperature and benzyl bromide (0.23 mL, 1.94 mmol) was added. The solution was stirred at room temperature for 4 hours and then thiophenol (0.92 mL, 8.99 mmol) was added, followed by potassium carbonate (0.97 g, 7.01 mmol) and DMF (20 mL). The solution is stirred at room temperature overnight. The next morning, salt is removed from the mixture by centrifugation, and the supernatant is concentrated into residue. The residue is dissolved in 40mL DCM, washed with water (2x10mL) and saline (2x10 mL), dried over potassium carbonate, filtered, and concentrated into an oily substance. The oily substance is dissolved in DCM and purified by 0-50% (50:45:5DCM/MeOH/NH ₄ OH) gradient in DCM on silica. The fractions containing the product are collected and concentrated to obtain (5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-2-methylpentan-2-yl) tert-butyl carbamate (0.49g, 1.22mmol, 52.24%) in a colorless oil. UPLC/ELSD:RT:0.30min. MS (ES): m/z (MH ⁺ ) 402.4 for C ₂₁ H ₄₃ N ₃ O ₄ . ¹ H NMR (300MHz, CDCl ₃ ) δ 5.88 (br.s, 1H), 4.69 (br.s, 1H), 2.61 (t, 2H), 2.52 (t, 2H), 1.61 (br.m, 4H), 1.35 (s, 21H), 1.18 (s, 12H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)carbamate

To a solution of tert-butyl (5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-2-methylpentan-2-yl)carbamate (0.09 g, 0.22 mmol) in anhydrous toluene (5 mL) stirred under nitrogen was added triethylamine (0.07 mL, 0.09 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.12 g, 0.22 mmol) was added and the solution was heated to 90° C. for 2 days. The reaction mixture was then cooled to room temperature, diluted with toluene, washed with water (3 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography with a 0-50% EtOAc gradient in hexanes. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)carbamate as _a light yellow oil (0.16 g, 0.20 mmol, 89.9%). UPLC/ELSD: RT = 3.67 min _. MS (ES): m/z (MH ⁺ ) 815.4 _{for C49H87N3O6} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.29(br.s,1H),4.41(br.m,3H),3.13(br.m,4H),2.26(br.m,2H),1.78(br.m,7H),1.45(br.m,10H),1.35(s,19H),1.26(br.m,3H),1 .20(s,14H),1.06(br.m,6H),0.94(s,6H),0.85(d,3H,J＝6Hz),0.81(d,6H,J＝6Hz),0.61(s,3H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-amino-3-methylbutyl)(4-amino-4-methylpentyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)carbamate (0.16 g, 0.20 mmol) in DCM (5 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.50 mL, 2.01 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (10 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-3-methylbutyl)(4-amino-4-methylpentyl)carbamate dihydrochloride (0.09 g, 0.12 mmol, 60.2%) as a white solid. UPLC/ELSD: RT = 1.90 min. MS (ES): m/z (MH ⁺ ) 614.3 for C ₃₉ H ₇₃ Cl ₂ N ₃ O ₂ . ¹ H NMR(300MHz,MeOD)δ5.41(br.s,1H),4.45(br.m,1H),3.33(br.m,6H),2.37(br.m,2H),1.93(br.m,7H),1.60(br.m,11H),1.37(s,15H),1.18(br.m,6H),1. 08 (s, 5H), 0.98 (d, 3H, J = 6Hz), 0.89 (d, 7H, J = 6Hz), 0.75 (s, 3H).

CA. Compound SA158: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-amino-3-methylbutyl)carbamate dihydrochloride

Step 1: (Azanediylbis(2-methylbutane-4,2-diyl))dicarbamic acid di-tert-butyl ester

By (4-amino-2-methylbutyl-2-yl) tert-butyl carbamate (0.50g, 2.36mmol) and (2-methyl-4-oxobutyl-2-yl) tert-butyl carbamate (0.50g, 2.36mmol) be dissolved in 10mL anhydrous methanol and stir under nitrogen at room temperature.After 2 hours, sodium triacetoxy borohydride (1.25g, 5.90mmol) is added, and reactant is continued to be stirred at room temperature overnight.The next morning, amber reaction mixture is quenched with a few drops of water, condensed into orange oil, and dissolved back in DCM.Then it is washed with saturated sodium bicarbonate (1x15 mL) and saline (1x15 mL), dried over sodium sulfate, filtered, and condensed into yellow oil.The oil is resuspended in DCM and on silica in DCM with 0-40% (50:45:5DCM/MeOH/NH ₄ OH aqueous solution) gradient purification. Fractions containing product were pooled and concentrated to give di-tert-butyl (azanediylbis(2-methylbutane-4,2-diyl))dicarbamate (0.53 g, 1.37 mmol, 58.2%) as a yellow oil. UPLC/ELSD: RT = 0.38 min. MS (ES): m/z (MH ⁺ ) 388.6 for C ₂₀ H ₄₁ N ₃ O _4. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.56 (br. s, 2H), 2.67 (t, 4H), 1.77 (br. m, 5H), 1.62 (br. s, 4H), 1.44 (s, 18H), 1.30 (s, 12H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)carbamate

To a solution of di-tert-butyl (azanediylbis(2-methylbutane-4,2-diyl))dicarbamate (0.10 g, 0.26 mmol) in anhydrous toluene (5 mL) stirred under nitrogen was added triethylamine (0.11 mL, 0.77 mmol). Then, (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-nitrophenyl)carbonate (0.14 g, 0.26 mmol) was added and the solution was heated to 90° C. overnight. The next day, the reaction was incomplete, so 3 more equivalents of triethylamine were added and the reaction was allowed to proceed for another 24 hours at 90°C. The reaction mixture was then cooled to room temperature, diluted with toluene, washed with water (3x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in hexanes using a 0-50% EtOAc gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)carbamate as a light yellow oil (0.15 g, 0.19 mmol, _72.2 %). UPLC/ELSD: RT = 3.41 _min . MS (ES): m/z (MH ⁺ ) _801.4 for _C48H85N3O6 . ¹ H NMR (300MHz, CDCl ₃ ) δ5.29(br.s,1H),4.57(br.s,1H),4.44(br.m,2H),3.15(br.m,4H),2.26(m,2H),1.80(m,9H),1.42(br.m,7H),1.35(s,19H),1.27( m,3H),1.21(s,13H),1.05(br.m,8H),0.95(s,6H),0.85(d,4H,J＝6Hz),0.80(d,6H,J＝6Hz),0.60(s,3H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-amino-3-methylbutyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)carbamate (0.15 g, 0.19 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.47 mL, 1.86 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (15 mL) was added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-amino-3-methylbutyl)carbamate dihydrochloride (0.10 g, 0.14 mmol, 74.1%) as a white solid. UPLC/ELSD: RT = 1.78 min. MS (ES): m/z (MH ⁺ ) 601.3 for C ₃₈ H ₇₁ Cl ₂ N ₃ O ₂ . ¹ H NMR (300MHz, MeOD) δ5.42 (br.s, 1H), 4.45 (br.m, 1H), 3.40 (m, 6H), 2.41 (d, 2H, J = 6Hz), 1.94 (br.m, 10H), 1.56 (br.m, 7H), 1.41 (s, 16H), 1.17 (br.m, 7H), 1. 06 (s, 6H), 0.98 (d, 4H, J = 6Hz), 0.90 (d, 6H, J = 6Hz), 0.75 (s, 4H).

CB. Compound SA159: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-amino-3-methylbutyl)carbamate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)carbamate

To a solution of di-tert-butyl (azanediylbis(2-methylbutane-4,2-diyl))dicarbamate (0.10 g, 0.26 mmol) in anhydrous toluene (5 mL) stirred under nitrogen was added triethylamine (0.11 mL, 0.77 mmol). Then, (1R,3aS,3bS,7S,9aR,9bS,11aR)-1-[(2R,5R)-5-ethyl-6-methylhept-2-yl]-9a,11a-dimethyl-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9bH,10H,11H-cyclopenta[a]phenanthrene-7-yl 4-nitrophenylcarbonate (0.15 g, 0.26 mmol) was added and the solution was heated to 90° C. overnight. The next day, the reaction was not complete, so 3 more equivalents of triethylamine were added and the reaction was allowed to proceed for another 24 hours at 90°C. The reaction mixture was then cooled to room temperature, diluted with toluene, washed with water (3 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified via silica gel chromatography in hexanes with a 0-50% EtOAc gradient. Fractions containing product were combined and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)carbamate as a light yellow oil (0.17 g, 0.20 mmol, 78.6%). UPLC/ELSD: RT ₌ 3.55 _min . MS (ES): m/z (MH ⁺ ) 823.4 _{for C50H89N3O6} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.41(br.s,1H),4.53(br.m,3H),3.25(br.m,4H),2.40(m,2H),1.90(br.m,9H),1.58(m,7H),1.45(s,18H),1.30(s,13H),1.18(br. m,7H),1.04(s,5H),0.95(d,5H,J=6Hz),0.83(q,9H),0.70(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-amino-3-methylbutyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)carbamate (0.17 g, 0.20 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.51 mL, 2.03 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (15 mL) was added to the mixture, which was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)bis(3-amino-3-methylbutyl)carbamate dihydrochloride (0.12 g, 0.16 mmol, 77.4%) as a white solid. UPLC/ELSD: RT = 1.94 min. MS (ES): m/z (MH ⁺ ) 629.3 for C ₄₀ H ₇₅ Cl ₂ N ₃ O ₂ . ¹ H NMR(300MHz,MeOD)δ5.42(br.s,1H),4.45(br.m,1H),3.38(br.m,6H),2.41(d,2H,J＝6Hz),1.94(br.m,9H),1.56(br.m,8H),1.41(s,15H),1.22(br.m,6H), 1.09(s,6H),0.99(d,4H,J＝6Hz),0.87(q,9H),0.75(s,4H).

CC. Compound SA160: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride

Step 1: 14-(3-((tert-Butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.15 g, 0.31 mmol) was added under nitrogen. To a stirred solution in anhydrous DCM (10 mL) was added di-tert-butyl ((butane-1,4-diylbis(azanediyl))bis(butane-4,2-diyl))dicarbamate (0.33 g, 0.76 mmol), dimethylaminopyridine (0.08 g, 0.61 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.12 g, 0.61 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oate as a light yellow oil (0.08 g, 0.09 mmol, 30.2%). UPLC/ELSD: RT: 2.49 min. MS (ES): m/z (MH ⁺ ) 900.4 for C ₅₃ H ₉₄ N ₄ O ₇ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.37(br.s,1H),4.79(br.m,1H),4.62(br.m,2H),3.71(br.m,3H),3.29(m,5H),2.64(br.m,8H),2.35(t,3H),2.24(s,2H),2.00(br.m ,6H),1.62(br.m,14H),1.45(s,20H),1.27(br.m,7H),1.15(m,12H),1.03(s,5H),0.94(d,4H,J＝6Hz),0.90(d,6H,J＝6Hz),0.69(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 14-(3-((tert-butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oate (0.08 g, 0.09 mmol) in isopropanol (3 mL) stirred under nitrogen was added hydrochloric acid (5.5 M in isopropanol, 0.19 mL, 0.92 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, acetonitrile (25mL) was added to the mixture, the mixture was cooled to 0 ° C and stirred for 30 minutes. The solution was then filtered and washed with 3: 1 acetonitrile / isopropanol. The resulting solid was dried in a vacuum to give 4- ((3-aminobutyl) (4- ((3-aminobutyl) amino) butyl) amino) -4-oxobutanoic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R) -10,13- dimethyl -17- ((R) -6- methyl hept-2-yl) -2,3,4,7,8,9,10,11,12,13,14,15,16,17- tetrahydro -1H- cyclopenta [a] phenanthrene -3-yl ester trihydrochloride (0.05g, 0.06mmol, 60.0%) as a white solid. UPLC / ELSD: RT = 1.36min. MS (ES): m/z (MH ⁺ ) 700.3 for C ₄₃ H ₈₁ Cl ₃ N ₄ O ₃ . ¹ HNMR(300MHz,MeOD)δ5.28(br.s,1H),4.41(br.m,1H),3.85(m,1H),3.39(br.m,5H),3.20(s,2H),3.02(br.m,5H),2.60(br.m,4H),2.23(br.m,4H),1.92(s ,5H),1.71(br.m,9H),1.43(br.m,8H),1.28(m,12H),1.06(d,11H,J＝6Hz),0.95(s,6H),0.86(d,4H,J＝6Hz),0.80(d,6H,J＝6Hz),0.63(s,3H).

CD. Compound SA161: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride

Step 1: 14-(3-((tert-Butyloxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a stirred anhydrous solution of 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.10 g, 0.19 mmol) was added to a stirred anhydrous solution of 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.10 g, 0.19 mmol) under nitrogen was added to a stirred anhydrous solution of 4-(((3S,8S,9S,10R,13R,14S,17R)-17- To a solution in DCM (5 mL) was added tert-butyl N-(4-{[4-({3-[(tert-butoxycarbonyl)amino]-3-methylbutyl}amino)butyl]amino}-2-methylbutan-2-yl)carbamate (0.22 g, 0.48 mmol), dimethylaminopyridine (0.05 g, 0.39 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.07 g, 0.39 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oate as a light yellow oil (0.03 g, 0.03 mmol, 16.3%). UPLC/ELSD: RT: 2.77 min. MS (ES): m/z (MH ⁺ ) 956.4 for C ₅₇ H ₁₀₂ N ₄ O ₇ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.35(br.s,1H),4.60(br.m,1H),3.25(br.m,5H),3.00(br.m,2H),2.60(br.m,6H),2.34(d,3H,J＝6Hz),2.23(m,4H),1.99(br.m,3H),1 .87(br.m,4H),1.63(br.m,12H),1.42(s,18H),1.28(d,15H,J＝6Hz),1.12(br.m,7H),1.02(s,5H),0.93(d,5H,J＝6Hz),0.83(q,8H),0.68(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oate (0.03 g, 0.03 mmol) in DCM (1 mL) under nitrogen was added hydrochloric acid (4 M in dioxane, 0.08 mL, 0.31 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (10 mL) were added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride (0.03 g, 0.03 mmol, 89.0%) as a white solid. UPLC/ELSD: RT = 1.69 _{min . MS} (ES): m/z (MH ⁺ ) _{for C47H89Cl3N4O3} 756.3. ² .15(br.m,5H),1.84(br.m,8H),1.63(br.m,9H),1.43(t,15H),1.25(br.m,10H),1.07(s,5H),0.97(d,5H,J＝6Hz),0.89(q,9H),0.75(s,3H).

CE. Compound SA162: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-aminobutyl)(8-aminononyl)amino)-4-oxobutanoate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-4-oxobutanoate

To 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.09 g, 0.18 mmol) was added to nitrogen. To a solution in anhydrous DCM (5 mL) stirred under vacuum was added tert-butyl (9-((3-((tert-butoxycarbonyl)amino)butyl)amino)non-2-yl)carbamate (0.08 g, 0.18 mmol), dimethylaminopyridine (0.04 g, 0.35 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.07 g, 0.35 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-4-oxobutanoate as a light yellow oil (0.11 g, 0.12 mmol, 67.6%). UPLC/ELSD: RT: 3.29 _{min .} MS (ES): m/z (MH ⁺ ) 899.4 _{for C54H95N3O7} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.30(br.s,1H),4.53(br.m,2H),4.34(br.s,1H),3.54(br.m,2H),3.14(br.m,4H),2.56(m,4H),2.26(d,2H,J＝9Hz),1.85(br.m,5H), 1.49(br.m,9H),1.36(s,19H),1.24(br.m,13H),1.02(m,13H),0.94(s,5H),0.85(d,4H,J＝6Hz),0.81(d,6H,J＝6Hz),0.60(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-aminobutyl)(8-aminononyl)amino)-4-oxobutanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-4-oxobutanoate (0.11 g, 0.12 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4N in dioxane, 0.30 mL, 1.18 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (25 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-aminobutyl)(8-aminononyl)amino)-4-oxobutanoate dihydrochloride (0.08 g, 0.10 mmol, 86.1%) as a white solid. UPLC/ELSD: RT = 1.75 min. MS (ES): m/z (MH ⁺ ) 699.3 for C ₄₄ H ₈₁ Cl ₂ N ₃ O ₃ . ¹ H NMR (300MHz, CDCl ₃ ) δ 8.40 (m, 6H), 5.39 (br.s, 1H), 4.64 (br.m, 1H), 3.39 (br.m, 5H), 2.66 (s, 4H), 2.35 (d, 2H, J = 6Hz), 2.04 (br.m, 4H), 1.86 (m, 3H), 1.5 8(br.m,9H),1.46(br.m,21H),1.12(br.m,7H),1.03(s,6H),0.92(d,4H,J＝6Hz),0.88(d,7H,J＝6Hz),0.69(s,3H).

CF. Compound SA163: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-aminobutyl)(8-aminononyl)amino)-4-oxobutanoate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-4-oxobutanoate

To 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.10 g, 0.19 mmol) was added. ) in anhydrous DCM (5 mL) stirred under nitrogen was added tert-butyl N-[4-({8-[(tert-butoxycarbonyl)amino]nonyl}amino)butan-2-yl]carbamate (0.08 g, 0.19 mmol), dimethylaminopyridine (0.05 g, 0.39 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.07 g, 0.39 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-4-oxobutanoate as a light yellow oil (0.16 g, 0.17 mmol, 86.7%). UPLC/ELSD: RT: 3.65 _min . MS (ES): m/z (MH ⁺ ) _{927.4 for C56H99N3O7} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.28(br.s,1H),4.56(br.m,3H),3.54(br.m,3H),3.16(br.m,3H),2.57(m,4H),2.26(d,2H,J＝3Hz),1.76(br.m,5H),1.52(br.m,9H) ,1.36(s,20H),1.23(br.m,15H),1.08(br.m,12H),0.94(s,6H),0.86(d,5H,J＝6Hz),0.75(q,9H),0.60(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-aminobutyl)(8-aminononyl)amino)-4-oxobutanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)butyl)(8-((tert-butoxycarbonyl)amino)nonyl)amino)-4-oxobutanoate (0.16 g, 0.17 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.42 mL, 1.68 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (10 mL) was added to the mixture, which was cooled to 0°C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-aminobutyl)(8-aminononyl)amino)-4-oxobutanoate dihydrochloride (0.13 g, 0.13 mmol, 79.3%) as a white solid. UPLC/ELSD: RT = 2.09 min. MS (ES): m/z (MH ⁺ ) 728.3 for C ₄₆ H ₈₅ Cl ₂ N ₃ O ₃ . ¹ H NMR(300MHz,MeOD)δ5.40(br.s,1H),4.54(br.m,1H),3.69(br.m,1H),3.33(s,9H),2.65(br.m,4H),2.32(d,2H,J=6Hz),1.92(br.m,7H),1.63(br.m,11H), 1.43(br.m,11H),1.32(t,8H),1.20(br.m,7H),1.07(s,5H),0.99(d,5H,J＝6Hz),0.87(q,9H),0.75(s,3H).

CG. Compound SA164: 4-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-4-oxobutanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-4-oxobutanoate

To a stirred solution of cholesteryl hemisuccinate (0.100 g, 0.205 mmol), tert-butyl N-(4-{[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate (0.107 g, 0.205 mmol) and DMAP (catalyst) in DCM (2 mL) cooled to 0° C., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.079 g, 0.411 mmol) was added. The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 h, DMAP (0.050 g, 0.41 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (40 mg) were added. At 43 h, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (65 mg) was added. At 64 h, the reaction mixture was diluted with DCM (15 mL) and then washed with 5% aqueous NaHCO _3. The aqueous solution was extracted with DCM (15 mL). The combined organics were washed with 5% aqueous NaHCO ₃ , passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-4-oxobutanoate as a clear oil (0.097 g, 0.098 mmol, 47.7%). UPLC/ELSD: RT = 3.68 min. MS (ES): m/z of C ₆₀ H ₉₉ N ₃ O ₈ = 990.87 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ7.33-7.27(m,2H),6.92-6.84(m,2H),5.41-5.31(m,1H),4.97(s,2H),4.77-4.38(m,3H),3.81(s,3H),3.37-3.18(m,4H),2.71 -2.51(m,4H),2.38-2.26(m,2H),2.17-1.04(m,61H),1.01(s,3H),0.91(d,J＝5.9Hz,3H),0.86(d,J＝6.6Hz,3H),0.86(d,J＝6.6Hz,3H),0.67(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-4-oxobutanoate dihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-4-oxobutanoate (0.093 g, 0.094 mmol) in DCM (1.5 mL) was added 4N HCl in dioxane (0.17 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 h, 4N HCl in dioxane (0.07 mL) was added. At 22 hours, MTBE (10 mL) was added, and the reaction mixture was kept at 4°C overnight. The reaction mixture was blown to a gel under a stream _{of N2} . Then, ice-cold MTBE (10 mL) was added, and the suspension was centrifuged (10,000 x g at 4°C for 1 h). The supernatant was decanted. The solid was rinsed with cold MTBE, suspended in MTBE, and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-4-oxobutanoate dihydrochloride (0.058 g, 0.068 mmol, 72.8%) as a white solid. UPLC/ELSD: RT = 2.23 min. MS (ES): m/z = 364.70 (M+2H) ²⁺ for C ₄₆ H ₈₃ N ₃ O ₃ . ¹ H NMR (300MHz, MeOD) δ5.43-5.34(m,1H),4.62-4.45(m,1H),3.56-3.34(m,4H),2.73-2.57(m,4H),2.42-2.26(m,2H),2.14-1.77(m,7H),1.75-0.97(m ,33H),1.05(s,3H),1.36(s,6H),1.33(s,6H),0.95(d,J＝6.5Hz,3H),0.88(d,J＝6.5Hz,6H),0.73(s,3H).

CH. Compound SA165: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-4-oxobutanoate dihydrochloride

Step 1: 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid

Sitosterol (3.00g, 7.23mmol) and succinic anhydride (0.941g, 9.40mmol) are combined in pyridine (6.0mL). The reaction mixture is stirred at 80°C and monitored by TLC. At 19 hours, DMAP (catalyst) is added. At 89 hours, the reaction mixture is cooled to room temperature, diluted with DCM (100mL), and washed with water. The organic matter is extracted with 1N NaOH aqueous solution (3x50mL). A precipitate is formed. The mixture is filtered. The solid is dissolved in 1N HCl aqueous solution, and then extracted with DCM (3x50mL). The organic extract is washed with 1NHCl aqueous solution (2x) and water, passed through a hydrophobic glass frit, dried over Na ₂ SO ₄ , and concentrated. The residue is dissolved in DCM (5mL) and hexane (30mL) is added while heating. Solvent is removed using heating (37°C hot water bath) until solid is formed. The solution is cooled to room temperature, and further cooled to 0°C. After 1.5 hours, a white solid is formed. The mixture was warmed to room temperature and the solids were collected by vacuum filtration, rinsing with cold 9:1 hexanes/DCM to afford 4-4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.436 g, 0.847 mmol, 11.7%) as an off-white solid. ¹ H NMR (300MHz, CDCl ₃ ) δ5.42-5.30(m,1H),4.71-4.56(m,1H),2.72-2.55(m,4H),2.36-2.23(m,2H),2.09-1.75(m,5H),1.73-0.75(m,31H),1.02(s,3 H), 0.92 (d, J = 6.4Hz, 3H), 0.68 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-4-oxobutanoate

To 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.100 g, 0.194 mmol), N To a stirred solution of tert-butyl-(4-{[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate (0.111 g, 0.214 mmol) and DMAP (catalyst) in DCM (2.0 mL) cooled to 0° C. was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.074 g, 0.39 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 h, DMAP (0.047 g, 0.39 mmol) was added followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (45 mg). At 43 h, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (65 mg) was added. At 64 h, the reaction mixture was diluted with DCM (15 mL) and washed with 5% aqueous NaHCO _3. The aqueous solution was extracted with DCM (15 mL). The combined organics were washed with 5% aqueous NaHCO ₃ , passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-4-oxobutanoate (0.097 g, 0.095 mmol, 49.0%) as a clear oil. UPLC/ELSD: RT = 3.74 min. MS (ES): m/z of C ₆₂ H ₁₀₃ N ₃ O ₈ = 1018.87 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ7.34-7.26(m,2H),6.92-6.83(m,2H),5.39-5.32(m,1H),4.97(s,2H),4.80-4.36(m,3H),3.80(s,3H),3.39-3.18(m,4H),2.6 9-2.52(m,4H),2.35-2.23(m,2H),2.12-0.77(m,71H),1.01(s,3H),0.92(d,J=6.4Hz,3H),0.67(s,3H).

Step 3: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-4-oxobutanoate dihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)amino)-4-oxobutanoate (0.093 g, 0.091 mmol) in DCM (1.5 mL) was added 4N HCl in dioxane (0.17 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 hours, 4N HCl (0.07 mL) in dioxane was added. At 22 hours, MTBE (10 mL) was added, and the reaction mixture was kept overnight at 4°C. The reaction mixture was blown under a stream of _N2 until it was gel-like. Cold MTBE (10 mL) was added, and the suspension was centrifuged (10,000 x g at 4°C for 1 h). The supernatant was decanted and the solids were rinsed with cold MTBE and then concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-amino-3-methylbutyl)(8-amino-8-methylnonyl)amino)-4-oxobutanoate dihydrochloride (0.034 g, 0.039 mmol, 42.2%) as a white solid. UPLC/ELSD: RT = 2.34 min. MS (ES): m/z of C ₄₈ H ₈₇ N ₃ O ₃ = 377.76 (M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.46-5.32(m,1H),4.65-4.43(m,1H),3.54-3.34(m,4H),2.74-2.50(m,4H),2.45-2.21(m,2H),2.13-1.79(m,7H),1.77-0.78( m, 43H), 1.36 (s, 6H), 1.32 (s, 6H), 1.05 (s, 3H), 0.96 (d, J = 6.5Hz, 3H), 0.73 (s, 3H).

CI. Compound SA166: 4-((3-amino-3-methylbutyl)(4-amino-4-methylpentyl)amino)-4-oxobutanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)amino)-4-oxobutanoate

To a stirred solution of 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.11 g, 0.22 mmol) under nitrogen was added. To a solution in anhydrous DCM (5 mL) was added tert-butyl (5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-2-methylpentan-2-yl)carbamate (0.09 g, 0.22 mmol), dimethylaminopyridine (0.06 g, 0.45 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.09 g, 0.45 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)amino)-4-oxobutanoate as a light yellow oil (0.17 g, 0.19 mmol, 85.6%). UPLC/ELSD: RT: 3.55 _{min .} MS (ES): m/z (MH ⁺ ) 871.4 _{for C52H91N3O7} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.37(br.s,1H),4.62(br.m,3H),3.27(br.m,4H),2.64(br.m,4H),2.35(d,2H,J=6Hz),2.01(br.m,3H),1.85(br.m,4H),1.56(br.m,1 1H),1.44(s,17H),1.35(br.m,3H),1.30(m,13H),1.14(br.m,6H),1.03(s,5H),0.95(d,3H,J＝6Hz),0.90(d,6H,J＝6Hz),0.70(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-amino-3-methylbutyl)(4-amino-4-methylpentyl)amino)-4-oxobutanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)amino)-4-oxobutanoate (0.17 g, 0.19 mmol) in DCM (5 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.48 mL, 1.92 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (10 mL) was added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) 4-((3-amino-3-methylbutyl)(4-amino-4-methylpentyl)amino)-4-oxobutanoate dihydrochloride (0.12 g, 0.14 mmol, 74.0%) as a white solid. UPLC/ELSD: RT=1.99 min. MS (ES): m/z (MH ⁺ ) 671.3 for C ₄₂ H ₇₇ Cl ₂ N ₃ O ₃ . ¹ H NMR(300MHz,MeOD)δ5.52(s,1H),5.39(br.s,1H),4.56(br.m,1H),3.68(s,1H),3.47(br.m,3H),3.32(br.s,2H),2.64(br.m,4H),2.35(br.m,2H),1.90(br .m,6H),1.55(br.m,10H),1.46(s,3H),1.40(br.s,14H),1.16(br.m,5H),1.07(s,5H),0.98(d,5H,J＝6Hz),0.89(d,8H,J＝6Hz),0.75(s,3H).

CJ. Compound SA167: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-amino-3-methylbutyl)(4-amino-4-methylpentyl)amino)-4-oxobutanoate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)amino)-4-oxobutanoate

To 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.12 g, 0.22 mmol) was added under nitrogen. To a solution in anhydrous DCM (5 mL) stirred at 37° C. was added tert-butyl (5-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-2-methylpentan-2-yl)carbamate (0.09 g, 0.22 mmol), dimethylaminopyridine (0.06 g, 0.45 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.09 g, 0.45 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1×20 mL) and brine (1×20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)amino)-4-oxobutanoate (0.14 g, 0.15 mmol, 67.6%) as a light yellow oil. UPLC/ELSD: RT: 3.64 min. MS (ES): m/z (MH ⁺ ) 899.4 for C ₅₄ H ₉₅ N ₃ O ₇ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.31(br.s,1H),4.57(br.m,3H),3.24(br.m,4H),2.57(br.m,4H),2.30(d,2H,J＝6Hz),2.00(br.m,4H),1.82(br.m,4H),1.53(br.m,1 1H), 1.38 (s, 18H), 1.31 (br.m, 2H), 1.24 (m, 16H), 1.11 (br.m, 6H), 0.98 (s, 6H), 0.88 (d, 5H, J = 6Hz), 0.79 (q, 10H), 0.64 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-amino-3-methylbutyl)(4-amino-4-methylpentyl)amino)-4-oxobutanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-((tert-butoxycarbonyl)amino)-3-methylbutyl)(4-((tert-butoxycarbonyl)amino)-4-methylpentyl)amino)-4-oxobutanoate (0.14 g, 0.15 mmol) in DCM (5 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.38 mL, 1.51 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (10 mL) was added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)4-((3-amino-3-methylbutyl)(4-amino-4-methylpentyl)amino)-4-oxobutanoate dihydrochloride (0.10 g, 0.12 mmol, 80.3%) as a white solid. UPLC/ELSD: RT=2.18 min. MS (ES): m/z (MH ⁺ ) 699.3 for C ₄₄ H ₈₁ Cl ₂ N ₃ O ₃ . ¹ H NMR(300MHz,MeOD)δ5.41(br.s,1H),4.53(br.m,1H),3.46(br.m,3H),3.32(m,5H),2.65(br.m,4H),2.33(br.m,2H),1.91(br.m,7H),1.64(br.m,11H),1.4 5(s,3H),1.40(s,13H),1.20(br.m,7H),1.07(s,5H),0.99(d,4H,J=6Hz),0.87(q,9H),0.75(s,4H).

CK. Compound SA168: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-aminobutyl)amino)-4-oxobutanoate dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-((tert-butoxycarbonyl)amino)butyl)amino)-4-oxobutanoate

To 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.100 g, 0.194 To a stirred solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.074 g, 0.39 mmol), tert-butyl N-[4-({3-[(tert-butoxycarbonyl)amino]butyl}amino)butan-2-yl]carbamate (0.077 g, 0.21 mmol), and DMAP (0.052 g, 0.43 mmol) in DCM (2.0 mL) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.074 g, 0.39 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 h, the reaction mixture was diluted with DCM (15 mL) and then washed with 5% _aqueous NaHCO. The aqueous solution was extracted with DCM (15 mL). The combined organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-65% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-((tert-butoxycarbonyl)amino)butyl)amino)-4-oxobutanoate (0.129 g, 0.151 mmol, 77.6%) as a clear oil. UPLC/ELSD: RT = 3.53 min. MS (ES): m/z of C ₅₁ H ₈₉ N ₃ O ₇ = 856.81 (M+H) ⁺ . ¹ HNMR (300MHz, CDCl ₃ ) δ5.41-5.32(m,1H),4.70-4.35(m,3H),3.78-3.08(m,6H),2.74-2.46(m,4H),2.38-2.23(m,2H),2.10-0.75(m,64H),1.01(s,3 H), 0.92 (d, J = 6.3Hz, 3H), 0.67 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-aminobutyl)amino)-4-oxobutanoate dihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-(bis(3-((tert-butoxycarbonyl)amino)butyl)amino)-4-oxobutanoate (0.125 g, 0.146 mmol) in DCM (2.5 mL) was added 4N HCl (0.37 mL) in dioxane. The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 hours, MTBE (20 mL) was added and the reaction mixture was centrifuged (10,000 x g for 15 min at 4 °C). The supernatant was removed by suction and the solid was rinsed with a small amount of MTBE. The solid was suspended in MTBE and then concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-(bis(3-aminobutyl)amino)-4-oxobutanoate dihydrochloride (0.084 g, 0.11 mmol, 74.8%) as a white solid. UPLC/ELSD: RT = 2.11 min. MS (ES): m/z of C ₄₁ H ₇₃ N ₃ O ₃ = 349.41 [(M+2H)+CH ₃ CN] ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.43-5.34(m,1H),4.62-4.46(m,1H),3.75-3.34(m,5H),3.26-3.15(m,1H),2.83-2.59(m,4H),2.42-2.25(m,2H),2.16-0.78( m, 40H), 1.38 (d, J = 6.6Hz, 3H), 1.32 (d, J = 6.5Hz, 3H), 1.05 (s, 3H), 0.96 (d, J = 6.4Hz, 3H), 0.73 (s, 3H).

CL. Compound SA169: 4-(bis(3-amino-3-methylbutyl)amino)-4-oxobutanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-4-oxobutanoate

To 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.13 g, 0.26 mmol) was added. ) was added di-tert-butyl (azanediylbis(2-methylbutane-4,2-diyl))dicarbamate (0.10 g, 0.26 mmol), dimethylaminopyridine (0.06 g, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.10 g, 0.52 mmol) to a solution of anhydrous DCM (5 mL) stirred under nitrogen. The resulting solution was stirred overnight at room temperature. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-4-oxobutanoate as a light yellow _oil (0.22 g, 0.26 mmol, 100.0%). UPLC/ELSD: RT: 3.58 _{min .} MS (ES): m/z (MH ⁺ ) 857.4 for _C51H89N3O7 . ¹ H NMR (300MHz, CDCl ₃ ) δ5.07(br.s,1H),4.55(br.s,1H),4.32(br.m,1H),4.20(br.s,1H),3.04(br.m,4H),2.33(s,4H),2.05(br.s,2H),1.65(br.m,9H),1.2 8(br.m,7H),1.14(s,19H),1.00(s,15H),0.83(br.m,7H),0.74(s,6H),0.65(d,4H,J＝6Hz),0.60(d,6H,J＝6Hz),0.39(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-amino-3-methylbutyl)amino)-4-oxobutanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-4-oxobutanoate (0.22 g, 0.26 mmol) in DCM (5 mL) stirred under nitrogen was added hydrochloric acid (4M in dioxane, 0.65 mL, 2.59 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (15 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-(bis(3-amino-3-methylbutyl)amino)-4-oxobutanoate dihydrochloride (0.12 g, 0.16 mmol, 62.4%) as a white solid. UPLC/ELSD: RT = 2.03 min. MS (ES): m/z (MH ⁺ ) 657.3 for C ₄₁ H ₇₅ Cl ₂ N ₃ O ₃ . ¹ H NMR(300MHz,MeOD)δ5.39(br.s,1H),4.53(br.m,1H),3.48(br.m,4H),3.33(br.s,3H),2.67(br.m,4H),2.33(br.m,2H),2.04(br.m,3H),1.91(br.m,6H),1. 55(br.m,7H),1.46(s,6H),1.40(s,8H),1.16(br.m,11H),1.07(s,6H),0.96(d,4H,J=6Hz),0.89(d,8H,J=6Hz),0.75(s,4H).

CM. Compound SA170: 4-(bis(3-amino-3-methylbutyl)amino)-4-oxobutanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester dihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-4-oxobutanoate

To 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.13 g, 0.2 To a solution of (2-methylbutane-4,2-diyl)-di-tert-butyl (0.10 g, 0.26 mmol) in anhydrous DCM (5 mL) stirred under nitrogen, dimethylaminopyridine (0.06 g, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.10 g, 0.52 mmol) were added. The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-4-oxobutanoate as a light yellow oil (0.18 g, 0.21 mmol, 80.2%). UPLC/ELSD: RT: 3.65 min _{. MS} (ES): m/z (MH ⁺ ) 885.4 _{for C53H93N3O7} . ¹ HNMR (300MHz, CDCl ₃ ) δ5.06(br.s,1H),4.54(br.s,1H),4.33(br.m,1H),4.21(s,1H),3.03(m,4H),2.32(s,4H),2.02(d,2H,J＝6Hz),1.63(br.m,9H),1.29 (br.m,7H),1.14(s,19H),0.99(s,15H),0.84(br.m,6H),0.73(s,5H),0.65(d,5H,J=6Hz),0.54(q,9H),0.39(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-amino-3-methylbutyl)amino)-4-oxobutanoate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(bis(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)amino)-4-oxobutanoate (0.18 g, 0.21 mmol) in DCM (5 mL) stirred under nitrogen was added hydrochloric acid (4M in dioxane, 0.52 mL, 2.07 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (15 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-(bis(3-amino-3-methylbutyl)amino) _-4 -oxobutanoate dihydrochloride (0.16 g, 0.19 mmol, 91.5%) _as a white solid. UPLC/ELSD: RT = 2.13 min _. MS (ES): m/z (MH ⁺ ) 685.3 _{for C43H79Cl2N3O3 .} ¹ H NMR (300MHz, MeOD) δ5.39(br.s,1H),4.53(br.m,1H),3.53(m,4H),3.33(br.s,3H),2.67(d,4H,J＝3Hz),2.33(d,2H,J＝6Hz),2.04(br.m,3H),1.93(br.m,6H ),1.58(br.m,8H),1.46(s,7H),1.40(s,8H),1.25(br.m,11H),1.07(s,5H),0.99(m,5H),0.87(q,10H),0.75(s,3H).

CN. Compound SA171: N-(3-aminobutyl)-N-(4-((3-aminobutyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride

Step 1: tert-Butyl (9-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)-2,2,6-trimethyl-4-oxo-3-oxa-5,9,14-triazaoctadec-17-yl)carbamate

To a stirred anhydrous D-2-ol of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.15 g, 0.30 mmol) was added 0.1% 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.15 g, 0.30 mmol) under nitrogen was added 0.1% 2-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.15 g, 0.30 mmol) To a solution in CM (10 mL) was added tert-butyl N-(4-{[4-({3-[(tert-butoxycarbonyl)amino]butyl}amino)butyl]amino}butan-2-yl)carbamate (0.32 g, 0.74 mmol), dimethylaminopyridine (0.07 g, 0.59 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.11 g, 0.59 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give tert-butyl (9-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)-2,2,6-trimethyl-4-oxo-3-oxa-5,9,14-triazaoctadec-17-yl)carbamate (0.08 g, 0.09 mmol, 28.7%) as a light yellow oil. UPLC/ELSD: RT: 2.79 min. MS (ES): m/z (MH ⁺ ) 920.4 for C ₅₂ H ₉₄ N ₄ O ₅ S ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.37(br.s,1H),4.79(br.m,2H),3.64(br.m,3H),3.31(br.m,4H),2.95(t,2H),2.69(br.m,7H),2.34(d,2H,J=6Hz),2.24(m,1H),1.9 4(br.m,4H),1.60(br.m,11H),1.44(s,22H),1.31(br.m,5H),1.16(br.m,13H),1.00(s,6H),0.93(d,4H,J=6Hz),0.88(d,4H,J=6Hz),0.68(s,3H).

Step 2: N-(3-aminobutyl)-N-(4-((3-aminobutyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride

To a solution of tert-butyl (9-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)propanoyl)-2,2,6-trimethyl-4-oxo-3-oxa-5,9,14-triazaoctadec-17-yl)carbamate (0.08 g, 0.09 mmol) in DCM (2 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.21 mL, 0.85 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (10 mL) was added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give N-(3-aminobutyl)-N-(4-((3-aminobutyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride (0.05 g, 0.05 mmol, 63.1%) as a white solid. UPLC/ELSD: RT=1.77 min. MS (ES): m/z (MH ⁺ ) 720.3 for C ₄₂ H ₈₁ Cl ₃ N ₄ OS ₂ . ¹ HNMR(300MHz,MeOD)δ5.39(br.s,1H),3.68(br.m,1H),3.46(br.m,6H),3.33(s,4H),3.14(br.m,6H),3.00(br.m,5H),2.63(br.m,1H),2.37(d,2H,J＝6Hz),1 .99(br.m,16H),1.53(br.m,8H),1.40(m,13H),1.18(br.m,8H),1.05(s,4H),0.98(d,5H,J=6Hz),0.90(d,8H,J=6Hz),0.74(s,3H).

CO. Compound SA172: N-(3-aminobutyl)-N-(8-aminononyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: tert-Butyl (4-(N-(8-((tert-butoxycarbonyl)amino)nonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)butan-2-yl)carbamate

To 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.09 g, 0.18 mmol) was added under nitrogen. To a stirred solution in anhydrous DCM (5 mL) was added tert-butyl (9-((3-((tert-butoxycarbonyl)amino)butyl)amino)non-2-yl)carbamate (0.08 g, 0.18 mmol), dimethylaminopyridine (0.04 g, 0.35 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.07 g, 0.35 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1 x 20 mL) and brine (1 x 20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give tert-butyl (4-(N-(8-((tert-butoxycarbonyl)amino)nonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)butan- ₂ -yl)carbamate (0.11 g, 0.12 mmol, 66.1%) as a light yellow oil. UPLC/ELSD: RT: 3.50 _{min .} MS (ES): m/z (MH ⁺ ) 919.4 _{for C53H95N3O5S2} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.29(br.s,1H),4.35(br.s,1H),3.55(br.m,3H),3.17(br.m,3H),2.89(t,2H),2.63(br.m,3H),2.27(br.m,2H),1.86 (m,5H),1.51(br.m,9H),1.36(s,19H),1.24(br.m,14H),1.05(m,13H),0.93(s,6H),0.86(d,4H,J＝6Hz),0.80(d,6H,J＝6Hz),0.61(s,3H).

Step 2: N-(3-aminobutyl)-N-(8-aminononyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a solution of tert-butyl (4-(N-(8-((tert-butoxycarbonyl)amino)nonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)butan-2-yl)carbamate (0.11 g, 0.12 mmol) in DCM (3 mL) stirred under nitrogen was added hydrochloric acid (4N in dioxane, 0.29 mL, 1.15 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (25 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give N-(3-aminobutyl)-N-(8-aminononyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.09 g, 0.11 mmol, 94.3%) as _a white solid. UPLC/ELSD: RT = 1.86 min _. MS (ES): m/z (MH ⁺ ₎ 719.3 _{for C43H81Cl2N3OS2} . ¹ H NMR (300MHz, CDCl ₃ ) δ8.39(br.m,6H),5.38(br.s,1H),3.41(br.m,5H),3.00(br.s,2H),2.85(br.s,2H),2.70(br.m,1H),2.35(br.m,2H),2.01(m,8H),1.4 4(br.m,28H),1.12(br.m,7H),1.02(s,6H),0.94(d,3H,J＝6Hz),0.87(d,7H,J＝6Hz),0.69(s,3H).

CP. Compound SA173: N,N-bis(3-aminobutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: Di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(butane-4,2-diyl))dicarbamate

To a stirred solution of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)propanoic acid (0.100 g, 0.197 mmol), tert-butyl N-[4-({3-[(tert-butoxycarbonyl)amino]butyl}amino)butan-2-yl]carbamate (0.078 g, 0.22 mmol) and triethylamine (0.08 mL, 0.6 mmol) in DCM (1.6 mL) cooled to 0 °C was added 50 wt% propanephosphonic anhydride in DCM (0.20 mL, 0.39 mmol) dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 17 hours, the reaction mixture was diluted with DCM (10 mL) and then washed with 5% aqueous NaHCO _3. The aqueous solution was extracted with DCM (10 mL). The combined organics were passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. The crude material was purified via silica gel chromatography (0-50% EtOAc in hexanes) to afford di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(butane-4,2-diyl))dicarbamate (0.124 g, 0.146 mmol, 74.1%) as a clear oil. UPLC/ELSD: RT = 3.60 min. MS (ES): m/z of C ₄₈ H ₈₅ N ₃ O ₅ S ₂ = 849.65 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl3) δ5.39-5.32(m,1H),4.68-4.38(m,2H),3.77-3.12(m,6H),3.06-2.85(m,2H),2.80-2.54(m,3H),2.41-2.23(m,2H),2.03-0.94 (m,54H),1.00(s,3H),0.91(d,J＝6.4Hz,3H),0.87(d,J＝6.6Hz,3H),0.86(d,J＝6.6Hz,3H),0.67(s,3H).

Step 2: N,N-bis(3-aminobutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a stirred solution of di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(butane-4,2-diyl))dicarbamate (0.121 g, 0.143 mmol) in DCM (2.5 mL) cooled to 0° C. was added 4N HCl in dioxane (0.36 mL) dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 h, MTBE (20 mL) was added and the reaction mixture was centrifuged (10,000×g for 15 min at 4° C.). The supernatant was removed and the solid was rinsed sparingly with MTBE. The solid was suspended in MTBE and then concentrated to give N,N-bis(3-aminobutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.089 g, 0.12 mmol, 83.6%) as a white solid. UPLC/ELSD: RT = 2.12 min. MS (ES): m/z = 648.64 (M+H) ⁺ for C ₃₈ H ₆₉ N ₃ OS ₂ . ¹ H NMR (300MHz, MeOD) δ5.45-5.31(m,1H),3.77-3.33(m,5H),3.27-3.17(m,1H),3.05-2.74(m,4H),2.73-2.59(m,1H),2.42-2.27(m,2H),2.15-1.75(m ,9H),1.72-0.96(m,21H),1.39(d,J＝6.8Hz,3H),1.33(d,J＝6.5Hz,3H),1.03(s,3H),0.95(d,J＝6.4Hz,3H),0.88(d,J＝6.6Hz,6H),0.73(s,3H).

CQ. Compound SA174: N,N-bis(3-amino-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: Di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(2-methylbutane-4,2-diyl))dicarbamate

To a solution of 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.13 g, 0.26 mmol) in anhydrous DCM (5 mL) stirred under nitrogen was added di-tert-butyl (azanediylbis(2-methylbutane-4,2-diyl))dicarbamate (0.10 g, 0.26 mmol), dimethylaminopyridine (0.06 g, 0.52 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.10 g, 0.52 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate solution (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM using a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(2-methylbutane-4,2-diyl))dicarbamate (0.21 _g , 0.24 mmol, 92.9%) as a light yellow oil. UPLC/ELSD: RT: 3.72 _{min .} MS (ES): m/z (MH ⁺ ) 877.4 _{for C50H89N3O5S2} . ¹ H NMR (300MHz, CDCl ₃ ) δ5.07(br.s,1H),5.02(s,1H),4.50(s,1H),4.18(s,1H),3.01(br.m,4H),2.64(t,2H),2.43(br.m,3H),2.03(br.m,2H),1.61(br.m, 9H), 1.27 (br.m, 5H), 1.14 (s, 19H), 0.99 (s, 15H), 0.83 (br.m, 8H), 0.71 (s, 5H), 0.64 (d, 4H, J = 6Hz), 0.58 (d, 6H, J = 6Hz), 0.39 (s, 3H).

Step 2: N,N-bis(3-amino-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a solution of di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(2-methylbutane-4,2-diyl))dicarbamate (0.21 g, 0.24 mmol) in DCM (5 mL) stirred under nitrogen was added hydrochloric acid (4M in dioxane, 0.60 mL, 2.40 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexanes (15 mL) were added to the mixture and the mixture was cooled to 0 °C and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give N,N-bis(3-amino-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.14 _{g ,} 0.17 mmol, 71.9%) as a white solid. UPLC/ELSD: RT = 2.17 min. MS (ES): m/z (MH ⁺ ) _{677.3 for C40H75Cl2N3OS2} . ¹ H NMR(300MHz,MeOD)δ5.40(br.s,1H),3.51(br.m,4H),3.32(br.s,2H),2.99(t,2H),2.86(t,2H),2.64(br.m,1H),2.37(d,2H,J＝6Hz),2.07(br.m,9H),1.55 (br.m,8H),1.47(s,6H),1.41(s,8H),1.18(br.m,11H),1.05(s,5H),0.96(d,4H,J＝6Hz),0.92(d,7H,J＝6Hz),0.74(s,3H).

CR. Compound SA175: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride

Step 1: 14-(3-((tert-Butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 4-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.10 g, 0.19 mmol) was added under nitrogen. To a stirred solution in anhydrous DCM (5 mL) was added tert-butyl N-(4-{[4-({3-[(tert-butoxycarbonyl)amino]butyl}amino)butyl]amino}butan-2-yl)carbamate (0.21 g, 0.48 mmol), dimethylaminopyridine (0.05 g, 0.39 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.07 g, 0.39 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oate as a light yellow oil (0.03 g, 0.03 mmol, 14.6%). UPLC/ELSD: RT: 2.76 min. MS (ES): m/z (MH ⁺ ) 928.4 for C ₅₅ H ₉₈ N ₄ O ₇ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.30(br.s,1H),4.70(br.m,1H),4.53(br.m,2H),3.57(br.m,2H),3.25(br.m,4H),2.57(br.m,8H),2.26(d,3H,J=6Hz),1.77(br.m,6H ),1.54(br.m,13H),1.37(s,20H),1.17(br.m,5H),1.08(br.m,12H),0.94(s,5H),0.86(d,5H,J=6Hz),0.78(q,9H),0.61(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 14-(3-((tert-butoxycarbonyl)amino)butyl)-2,2,6-trimethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oate (0.03 g, 0.03 mmol) in DCM (1 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.07 mL, 0.28 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (5 mL) was added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-4-((3-aminobutyl)(4-((3-aminobutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride (0.02 g, 0.02 mmol, 63.9%) as a white solid. UPLC/ELSD: RT=1.75 min. MS (ES): m/z (MH ⁺ ) 728.3 for C ₄₅ H ₈₅ Cl ₃ N ₄ O ₃ . ¹ H NMR(300MHz,MeOD)δ5.40(br.s,1H),4.55(br.m,1H),3.68(br.s,1H),3.50(br.m,4H),3.33(br.m,3H),3.14(br.m,5H),2.66(br.m,4H),2.33(br.m,3H),1. 81(br.m,19H),1.37(m,11H),1.20(br.m,6H),1.07(s,5H),0.99(d,5H,J=6Hz),0.89(q,9H),0.75(s,3H).

CS. Compound SA176: 4-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-4-oxobutanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 14-(3-((tert-Butyloxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To a stirred solution of 4-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-4-oxobutanoic acid (0.15 g, 0.31 mmol) in anhydrous DCM ( To a solution in 4% paraformaldehyde (0.10 mL) was added tert-butyl N-(4-{[4-({3-[(tert-butoxycarbonyl)amino]-3-methylbutyl}amino)butyl]amino}-2-methylbutan-2-yl)carbamate (0.35 g, 0.76 mmol), dimethylaminopyridine (0.8 g, 0.61 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.12 g, 0.61 mmol). The resulting solution was stirred at room temperature overnight. The solution was then diluted with dichloromethane, washed with saturated aqueous sodium bicarbonate (1x20 mL) and brine (1x20 mL), dried over sodium sulfate, filtered, and concentrated to an oil. The oil was dissolved in DCM and purified on silica in DCM with a 0-100% (80:19:1 DCM/MeOH/NH ₄ OH) gradient. Fractions containing product were pooled and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oate as a light yellow oil (0.10 g, 0.10 mmol, 33.6%). UPLC/ELSD: RT: 2.70 min. MS (ES): m/z (MH ⁺ ) 928.4 for C ₅₅ H ₉₈ N ₄ O ₇ . ¹ H NMR (301MHz, CDCl ₃ ) δ5.70(br.s,1H),5.29(br.s,1H),4.50(br.m,2H),3.21(br.m,4H),2.54(br.m8H),2.24(d,2H,J=6Hz),1.87(br.m,7H),1.53(br.m,10 H), 1.36 (s, 22H), 1.23 (s, 15H), 1.02 (br.m, 7H), 0.94 (s, 6H), 0.85 (d, 4H, J = 6Hz), 0.80 (d, 6H, J = 6Hz), 0.60 (s, 3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-((3-amino-3-methylbutyl)(4-((3-amino-3-methylbutyl)amino)butyl)amino)-4-oxobutanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 14-(3-((tert-butoxycarbonyl)amino)-3-methylbutyl)-2,2,6,6-tetramethyl-4,15-dioxo-3-oxa-5,9,14-triazaoctadecane-18-oate (0.10 g, 0.10 mmol) in DCM (2 mL) stirred under nitrogen was added hydrochloric acid (4 M in dioxane, 0.26 mL, 1.02 mmol) dropwise. The solution was stirred at room temperature overnight. The next morning, hexane (5 mL) was added to the mixture, which was cooled to 0° C. and stirred for 30 minutes. The solution was then centrifuged for 20 minutes, the supernatant discarded, and the white pellet dried in vacuo to give (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.09 g, 0.08 mmol, 74.4%) as a white solid. UPLC/ELSD: RT=1.72 min. MS (ES): m/z (MH ⁺ ) 728.3 for C ₄₅ H ₈₅ Cl ₃ N ₄ O ₃ . ^1. 05(br.m,10H),1.66(br.m,16H),1.43(br.m,16H),1.32(br.s,16H),1.16(br.m,8H),1.07(s,5H),0.92(br.m,25H),0.75(s,3H).

CT. Compound SA177: N,N-bis(3-amino-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: Sitosterol Chloride

Sitosterol (3.000 g, 7.234 mmol) and thionyl chloride (3.00 mL, 41.4 mmol) were combined in PhMe (30 mL). The reaction mixture was stirred at 80 ° C and monitored by TLC. At 20 hours, the reaction mixture was concentrated and then reconcentrated from PhMe (2x). The solid was dissolved in hot 3: 1 EtOH / EtOAc (45 mL) and then cooled to room temperature. The solid precipitated from the solution. The mother liquor was decanted and the solid was rinsed in small amounts with cold 3: 1 EtOH / EtOAc to obtain sitosterol chloride (2.534 g, 5.850 mmol, 80.9%) as a transparent solid. UPLC / ELSD: RT = 3.74 min. ¹ H NMR (300MHz, CDCl ₃ ) δ5.41-5.33(m,1H),3.87-3.67(m,1H),2.69-2.39(m,2H),2.17-1.75(m,6H),1.73-0.76(m,30H),1.03(s,3H),0.92(d,J＝6.5Hz, 3H),0.68(s,3H). ¹³ C NMR (75MHz, CDCl ₃ ) δ140.96,122.64,60.50,56.85,56.19,50.22,45.98,43.56,42.46,39.85,39.27,36.53,36.29,34.09,33.53,31.98,31.93, 29.30,28.38,26.22,24.43,23.22,21.11,19.98,19.41,19.19,18.93,12.13,12.00.

Step 2: Sitosterol Thiocyanate

Sitosterol chloride (2.748 g, 6.344 mmol) and sodium thiocyanate (19.235 g, 237.27 mmol) were refluxed in EtOH (105 mL). The reaction was monitored by TLC. At 64 hours, the reaction mixture was filtered hot and rinsed with a large amount of DCM. The filtrate was concentrated, dissolved in DCM (150 mL), and washed with water. The organic layer was passed through a hydrophobic glass frit, dried over Na ₂ SO ₄ , and concentrated. The resulting solid was dissolved in 1:1 EtOAc/hexane (19 mL) near boiling and then slowly cooled. Once room temperature was reached, the mixture was immediately further cooled to 4 ° C. The solid was collected by vacuum filtration and rinsed with cold 1:1 EtOAc/hexane in small amounts to obtain thiocyanate (2.183 g, 4.789 mmol, 75.5%) as an off-white solid. UPLC/ELSD: RT=3.47 min. ¹ H NMR (300MHz, CDCl ₃ ) δ 5.48-5.35 (m, 1H), 3.23-2.97 (m, 1H), 2.65-2.35 (m, 2H), 2.10-0.76 (m, 36H), 1.03 (s, 3H), 0.92 (d, J = 6.4Hz, 3H), 0.68 (s, 3H). ¹³ C NMR (75MHz, CDCl3) δ140.10,123.29,111.40,56.80,56.18,50.20,48.23,45.98,42.45,39.89,39.79,39.51,36.61,36.28,34.08,31.96,31.86, 30.10,29.29,28.37,26.21,24.41,23.21,21.06,19.97,19.35,19.18,18.93,12.13,12.00.

Step 3: Thiositosterol

To a stirred solution of THF (30 mL) and 2.3 M lithium aluminum hydride in 2-methyltetrahydrofuran (4.7 mL) was added a solution of thiocyanate sitosterol (2.100 g, 4.607 mmol) in PhMe (20 mL) over 15 min. The reaction mixture was stirred at room temperature and monitored by TLC. At 2.5 hours, the reaction mixture was cooled to 0 ° C, and then 3N HCl aqueous solution (50 mL) was slowly added dropwise over 10 min. After the addition was completed, the layers were separated. The aqueous layer was extracted with MTBE (3 x 30 mL). The combined organic layers were washed with water and brine, dried over Na ₂ SO ₄ , and concentrated to give thiositosterol (1.944 g, 4.513 mmol, 97.9%) as a white solid. UPLC/ELSD: RT = 3.70 min. ¹ H NMR (300MHz, CDCl ₃ ) δ5.43-5.22(m,1H),2.80-2.60(m,1H),2.45-2.23(m,2H),2.10-1.75(m,5H),1.74-0.78(m,32H),1.00(s,3H),0.92(d,J＝6.4Hz, 3H),0.67(s,3H). ¹³ C NMR (75MHz, CDCl ₃ ) δ142.07,121.19,56.90,56.20,50.35,45.98,44.35,42.45,40.08,39.89,39.60,36.49,36.30,34.22,34.09,31.95,29.29, 28.39,26.21,24.43,23.21,21.04,19.98,19.48,19.18,18.93,12.13,12.00.

Step 4: 2-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pyridine

Thiositosterol (1.92 g, 4.46 mmol) and Aldrithiol (1.08 g, 4.90 mmol) were combined in chloroform (12 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 24 hours, Aldrithiol (0.25 g) was added. At 6 days, the reaction mixture was concentrated, dissolved in MeOH (30 mL), and sonicated. The solid was collected by vacuum filtration and rinsed sparingly with MeOH to afford 2-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pyridine (2.098 g, 3.886 mmol, 87.2%) as a light yellow solid. UPLC/ELSD: RT = 3.56 min. MS (ES): m/z = 540.62 (M+H) ⁺ for C ₃₄ H ₅₃ NS ₂ . ¹ H NMR (300MHz, CDCl ₃ ) δ8.51-8.39(m,1H),7.83-7.71(m,1H),7.69-7.57(m,1H),7.13-7.00(m,1H),5.40-5.28(m,1H),2.89-2.69(m,1H),2.41-2.2 8(m,2H),2.08-1.75(m,5H),1.74-0.74(m,31H),0.98(s,3H),0.91(d,J=6.3Hz,3H),0.67(s,3H).

Step 5: 2-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)-1-methylpyridin-1-ium trifluoromethanesulfonate

To a solution of 2-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)pyridine (2.000 g, 3.704 mmol) in heptane (30 mL) and DCM (3.0 mL) was added methyl trifluoromethanesulfonate (0.51 mL, 4.5 mmol) dropwise over 10 min. The reaction mixture was stirred at room temperature and monitored by TLC. At 19 h, the solids were collected by vacuum filtration, rinsing with heptane to afford 2-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)-1-methylpyridin-1-ium trifluoromethanesulfonate (2.443 g, 3.470 mmol, 93.7%) as a white solid. UPLC/ELSD: RT = 2.67 min. MS (ES): m/z = 554.80 (M) ⁺ for C ₃₅ H ₅₆ NS ₂ . ¹ HNMR (300MHz, CD ₃ CN) δ8.60-8.49(m,2H),8.39-8.30(m,1H),7.75-7.66(m,1H),5.40-5.35(m,1H),4.19(s,3H),3.04-2.89(m,1H),2.49-2.32(m, 2H), 2.08-0.76 (m, 36H), 1.01 (s, 3H), 0.93 (d, J = 6.5Hz, 3H), 0.69 (s, 3H).

Step 6: 3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid

To a solution of 2-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)-1-methylpyridin-1-ium trifluoromethanesulfonate (2.400 g, 3.409 mmol) in DMF (15 mL) was added 3-mercaptopropionic acid (0.34 mL, 3.9 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 23 h, the reaction mixture was poured into water (30 mL) and sonicated. The solid was collected by vacuum filtration and rinsed with water. The solid was dissolved in DCM, passed through a hydrophobic frit, dried over Na ₂ SO ₄ , and concentrated. ACN (15 mL) was added to the residue. The suspension was sonicated and cooled in an ice bath. The solid was then collected by vacuum filtration and rinsed in small amounts with cold ACN. The solid was dissolved in ACN (15 mL) and sonicated. The solid was collected by vacuum filtration and rinsed with ACN to give 3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (1.333 g, 2.492 mmol, 73.1%) as a white solid. UPLC/ELSD: RT = 3.28 min. ¹ H NMR (300MHz, CDCl ₃ ) δ11.10(br.s,1H),5.46-5.25(m,1H),2.96-2.85(m,2H),2.84-2.74(m,2H),2.74-2.56(m,1H),2.42-2.22(m,2H),2.09-1.75(m ,5H),1.75-0.75(m,31H),1.00(s,3H),0.92(d,J＝6.5Hz,3H),0.68(s,3H).

Step 7: Di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(2-methylbutane-4,2-diyl))dicarbamate

To 3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.110 g, 0.206 mmol) was added. To a stirred solution of DCM (1.1 mL) cooled to 0°C was added 50 wt% propanephosphonic anhydride (0.21 mL, 0.41 mmol), tert-butyl N-[4-({3-[(tert-butoxycarbonyl)amino]-3-methylbutyl}amino)-2-methylbutan-2-yl]carbamate (0.088 g, 0.226 mmol) and triethylamine (0.09 mL, 0.6 mmol) in DCM (1.1 mL) was added dropwise 50 wt% propanephosphonic anhydride (0.21 mL, 0.41 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 h, the reaction mixture was diluted to 10 mL with DCM and then washed with 5% _NaHCO3 aqueous solution. The aqueous solution was extracted with DCM (10 mL). The combined organics were washed with water, passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-50% EtOAc in hexanes) to afford di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(2-methylbutane-4,2-diyl))dicarbamate (0.139 g, 0.154 mmol, 74.7%) as a clear oil. UPLC/ELSD: RT = 3.65 min. MS (ES): m/z of C ₅₂ H ₉₃ N ₃ O ₅ S ₂ = 905.77 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.39-5.30(m,1H),4.73(s,1H),4.39(s,1H),3.40-3.18(m,4H),3.04-2.90(m,2H),2.78-2.56(m,3H),2.42-2.22(m,2H),2.1 1-0.78(m,70H),1.00(s,3H),0.92(d,J=6.3Hz,3H),0.68(s,3H).

Step 8: N,N-bis(3-amino-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a solution of di-tert-butyl (((3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)azanediyl)bis(2-methylbutane-4,2-diyl))dicarbamate (0.131 g, 0.145 mmol) in DCM (2.0 mL) was added 4N HCl in dioxane (0.26 mL) dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 hours, the reaction mixture was diluted to 30 mL with MBTE and then centrifuged (10,000 x g at 4°C for 15 min). The supernatant was removed by aspiration. The solid was suspended in MTBE and then concentrated to give N,N-bis(3-amino-3-methylbutyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.089 g, 0.109 mmol, 75.4%) as a white solid. UPLC/ELSD: RT = 2.25 min. MS (ES): m/z of C ₄₂ H ₇₇ N ₃ OS ₂ = 373.20 [(M+2H)+CH ₃ CN] ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.46-5.31(m,1H),3.61-3.39(m,4H),3.01-2.91(m,2H),2.88-2.77(m,2H),2.74-2.58(m,1H),2.40-2.24(m,2H),2.16-1.79( m,9H),1.77-0.78(m,31H),1.44(s,6H),1.39(s,6H),1.03(s,3H),0.96(d,J＝6.4Hz,3H),0.73(s,3H).

CU. Compound SA178: N-(3-aminobutyl)-N-(8-aminononyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: tert-Butyl (4-(N-(8-((tert-butoxycarbonyl)amino)nonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)butan-2-yl)carbamate

To 3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.100 g, 0.187 To a stirred solution of 1,4-dihydro-2-nitropropane (0.1447 g, 0.206 mmol), tert-butyl N-[4-({8-[(tert-butoxycarbonyl)amino]nonyl}amino)butan-2-yl]carbamate (0.088 g, 0.206 mmol), and triethylamine (0.08 mL, 0.569 mmol) in DCM (2.5 mL) cooled to 0°C was added dropwise 50 wt% propanephosphonic anhydride in DCM (0.19 mL, 0.37 mmol). The reaction mixture was stirred at room temperature. At 19 h, the reaction mixture was diluted to 10 mL with DCM and then washed with 5% aqueous _NaHCO3 . The aqueous solution was extracted with DCM (10 mL). The combined organic layers were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20%-50% EtOAc in hexanes) to afford tert-butyl (4-(N-(8-((tert-butoxycarbonyl)amino)nonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamido)butan-2-yl)carbamate (0.130 g, 0.137 mmol, 73.5%) as a white foam. UPLC/ELSD: RT = 3.65 min. MS (ES): m/z of C ₅₅ H ₉₉ N ₃ O ₅ S ₂ = 946.96 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.45-5.29(m,1H),4.70-4.18(m,2H),3.80-3.10(m,6H),3.05-2.87(m,2H),2.83-2.56(m,3H),2.43-2.25(m,2H),2.14-0.7 5(m,74H),1.00(s,3H),0.92(d,J=6.4Hz,3H),0.67(s,3H).

Step 2: N-(3-aminobutyl)-N-(8-aminononyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a stirred solution of tert-butyl (4-(N-(8-((tert-butoxycarbonyl)amino)nonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propionamido)butan-2-yl)carbamate (0.126 g, 0.133 mmol) in DCM (1.9 mL) was added 4N HCl in dioxane (0.24 mL) dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 hours, the reaction mixture was diluted with MTBE (30 mL) and the reaction mixture was centrifuged (10,000 x g for 15 min at 4°C). The supernatant was aspirated and the solid was suspended in MTBE and concentrated to give N-(3-aminobutyl)-N-(8-aminononyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.101 g, 0.115 mmol, 86.7%). UPLC/ELSD: RT = 2.28 min. MS (ES): m/z of C ₄₅ H ₈₃ N ₃ OS ₂ =373.82(M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.42-5.33(m,1H),3.76-3.59(m,1H),3.56-3.10(m,5H),3.03-2.57(m,5H),2.42-2.25(m,2H),2.12-0.78(m,56H),1.04(s,3H ), 0.96 (d, J = 6.4Hz, 3H), 0.73 (s, 3H).

CV. Compound SA179: N-(3-amino-3-methylbutyl)-N-(8-amino-8-methylnonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

Step 1: tert-Butyl (4-(3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)-N-(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)propionamido)-2-methylbutan-2-yl)carbamate

To the reaction mixture was added 3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.102 g, 0.191 mmol), N- To a stirred solution of tert-butyl (4-{[8-({[(4-methoxyphenyl)methoxy]carbonyl}amino)-8-methylnonyl]amino}-2-methylbutan-2-yl)carbamate (0.113 g, 0.217 mmol) and triethylamine (0.08 mL, 0.6 mmol) in DCM (2.5 mL) cooled to 0°C was added 50% propanephosphonic anhydride (0.19 mL, 0.37 mmol) in DCM dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 hours, the reaction mixture was diluted to 10 mL with DCM and then washed with 5% _NaHCO3 aqueous solution. The aqueous solution was extracted with DCM (10 mL). The combined organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (20-50% EtOAc in hexanes) to afford tert-butyl (4-(3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)-N-(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)propionamido)-2-methylbutan-2-yl)carbamate (0.160 g, 0.154 mmol, 80.8%) as a clear oil. UPLC/ELSD: RT = 3.68 min. MS (ES): m/z of C ₆₁ H ₁₀₃ N ₃ O ₆ S ₂ = 1039.59 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ7.33-7.27(m,2H),6.92-6.85(m,2H),5.39-5.30(m,1H),4.97(s,2H),4.71-4.35(m,2H),3.81(s,3H),3.38-3.16(m,4H),3. 01-2.89(m,2H),2.79-2.55(m,3H),2.39-2.25(m,2H),2.09-0.77(m,71H),1.00(s,3H),0.92(d,J＝6.4Hz,3H),0.68(s,3H).

Step 2: N-(3-amino-3-methylbutyl)-N-(8-amino-8-methylnonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride

To a solution of tert-butyl (4-(3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)-N-(8-((((4-methoxybenzyl)oxy)carbonyl)amino)-8-methylnonyl)propionamido)-2-methylbutan-2-yl)carbamate (0.154 g, 0.148 mmol) in DCM (2.4 mL) was added 4N HCl in dioxane (0.26 mL) dropwise. The reaction mixture was stirred at room temperature and monitored by LCMS. At 19 hours, the reaction mixture was diluted to 30 mL with MTBE and then centrifuged (10,000 x g for 15 min at 4°C). The supernatant was removed by aspiration. The solid was suspended in MTBE (30 mL) and then centrifuged (10,000 x g for 15 min at 4°C). The supernatant was removed by aspiration. The solid was suspended in MBTE and then concentrated to give N-(3-amino-3-methylbutyl)-N-(8-amino-8-methylnonyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide dihydrochloride (0.042 g, 0.047 mmol, 31.6%) as a white solid. UPLC/ELSD: RT = 2.33 min. MS (ES): m/z of C ₄₇ H ₈₇ N ₃ OS ₂ = 388.12(M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.45-5.33(m,1H),3.52-3.34(m,4H),3.02-2.89(m,2H),2.87-2.74(m,2H),2.73-2.57(m,1H),2.40-2.26(m,2H),2.13-1.79( m,7H),1.37(s,6H),1.33(s,6H),1.77-0.78(m,43H),1.04(s,3H),0.96(d,J＝6.4Hz,3H),0.73(s,3H).

CW. Compound SA180: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)(4-(methylamino)butyl)(3-(methylamino)propyl)carbamate dihydrochloride

Step 1: tert-Butyl N-methyl-N-[4-(2-nitrobenzenesulfonamido)butyl]carbamate

To a stirred solution of tert-butyl N-(4-aminobutyl)-N-methylcarbamate (1.000 g, 4.943 mmol) and triethylamine (0.95 mL, 6.8 mmol) in DCM (15 mL) cooled to 0 ° C was added a solution of 2-nitrobenzenesulfonyl chloride (1.315 g, 5.932 mmol) in DCM (5 mL). After addition, the reaction mixture was stirred at room temperature and monitored by TLC. At 17 hours, the reaction mixture was cooled to 0 ° C, and then 5% NaHCO ₃ aqueous solution (10 mL) was added. After warming to room temperature, the layers were separated. The aqueous layer was extracted with DCM (15 mL). The combined organics were washed with 5% aqueous citric acid and water (2x), passed through a hydrophobic frit, dried _{over Na2SO4} , and concentrated to afford tert-butyl N-methyl-N-[4-(2-nitrobenzenesulfonamido)butyl]carbamate (1.932 g, quant.) as an amber oil. UPLC/ELSD: RT = 0.76 min _. MS ( _ES ): m/z _{for Ci6H25N3O6S} = 288.09 [(M+H)-( _CH3 ) _2C =CH2 _{- CO2} ] ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ8.18-8.08(m,1H),7.90-7.80(m,1H),7.80-7.68(m,2H),5.34(br.s,1H),3.23-3.06(m,4H),2.79(s,3H),1.60-1.46(m,4H),1. 43(s,9H).

Step 2: tert-Butyl N-[3-(N-{4-[(tert-butoxycarbonyl)(methyl)amino]butyl}-2-nitrobenzenesulfonamido)propyl]-N-methylcarbamate

By N-methyl-N-[4-(2-nitrobenzenesulfonamido)butyl]carbamic acid tert-butyl ester (0.750g, 1.94mmol), N-(3-bromopropyl)-N-methylcarbamic acid tert-butyl ester (0.586g, 2.32mmol) and potassium carbonate (0.535g, 3.87mmol) in DMF (11.25mL). The reaction mixture was stirred at 80 ° C and monitored by LCMS. At 19 hours, the reaction mixture was filtered and rinsed with EtOAc. The filtrate was diluted to 150mL with EtOAc, then washed with 5% NaHCO ₃ aqueous solution, water (3x) and brine. The organics were dried over Na ₂ SO ₄ and concentrated. The crude material was purified via silica gel chromatography (20%-80% EtOAc in hexanes) to give tert-butyl N-[3-(N-{4-[(tert-butoxycarbonyl)(methyl)amino]butyl}-2-nitrobenzenesulfonamido)propyl]-N-methylcarbamate (0.836 g, 1.50 mmol, _82.8 %) as a white solid. UPLC/ELSD: RT = _1.46 min _{. MS (ES): m/z for C25H42N4O8S =} 559.37 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ8.06-7.93(m,1H),7.76-7.55(m,3H),3.42-3.10(m,8H),2.80(s,6H),1.89-1.70(m,2H),1.70-1.46(m,4H),1.44(s,18H).

Step 3: tert-Butyl N-[3-({4-[(tert-butoxycarbonyl)(methyl)amino]butyl}amino)propyl]-N-methylcarbamate

To a mixture of tert-butyl N-[3-(N-{4-[(tert-butoxycarbonyl)(methyl)amino]butyl}-2-nitrobenzenesulfonamido)propyl]-N-methylcarbamate (0.830 g, 1.49 mmol) and potassium carbonate (0.616 g, 4.46 mmol) in DMF (12.5 mL) was added thiophenol (0.28 mL, 2.7 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 23 h, the reaction mixture was filtered and rinsed with EtOAc. The filtrate was diluted to 150 mL with EtOAc and then washed with 5 _{% K2CO3} in water ( _2x ), water (3x) and brine. The organics were dried over _Na2SO4 and then concentrated. The crude material was purified via silica gel chromatography (0-20% (5% concentrated aqueous _NH4OH in MeOH) in DCM) to afford tert-butyl N-[3-({4-[(tert-butoxycarbonyl)(methyl)amino]butyl}amino)propyl]-N-methylcarbamate (0.477 g, 1.28 mmol, 86.0%) as _a yellow oil. UPLC/ELSD: RT = 0.45 min. MS (ES): m/z = 374.56 (M+H) ^{+ for} Ci9H39N3O4 ^{. 1H} _NMR (300 MHz _{, CDCl3} ) _δ 3.33-3.11 (m, 4H), 2.83 (s, 6H), 2.70-2.50 (m, 4H), 1.76-1.62 (m, 2H), 1.62-1.37 (m, 22H).

Step 4: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)(methyl)amino)butyl)(3-((tert-butoxycarbonyl)(methyl)amino)propyl)carbamate

Cholesteryl 4-nitrophenyl carbonate (0.120 g, 0.207 mmol), tert-butyl N-[3-({4-[(tert-butoxycarbonyl)(methyl)amino]butyl}amino)propyl]-N-methylcarbamate (0.077 g, 0.207 mmol), and triethylamine (0.09 mL, 0.6 mmol) were combined in PhMe (1.8 mL). The reaction mixture was stirred at 90 °C and monitored by LCMS. Over 24 hours, the reaction mixture was cooled to room temperature, diluted with DCM to 10 mL, and washed with 5% _K2CO3 in water (2x). The aqueous solution was extracted with DCM (10 mL). The combined organics were _passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-50% EtOAc in hexanes) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)(methyl)amino)butyl)(3-((tert-butoxycarbonyl)(methyl)amino)propyl)carbamate as a clear oil (0.152 g, 0.187 mmol, 90.2%). UPLC/ELSD: RT = 3.59 min. MS (ES): m/z of C ₄₉ H ₈₇ N ₃ O ₆ = 814.88 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.41-5.33(m,1H),4.58-4.42(m,1H),3.32-3.10(m,8H),2.88-2.79(m,6H),2.41-2.20(m,2H),2.09-0.75(m,60H),1.02(s,3 H), 0.92 (d, J = 6.4Hz, 3H), 0.68 (s, 3H).

Step 5: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(4-(methylamino)butyl)(3-(methylamino)propyl)carbamate dihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (4-((tert-butoxycarbonyl)(methyl)amino)butyl)(3-((tert-butoxycarbonyl)(methyl)amino)propyl)carbamate (0.143 g, 0.176 mmol) in DCM (2.2 mL) was added 4N HCl in dioxane (0.31 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 18 h, the reaction mixture was diluted to 20 mL with MTBE and then centrifuged (10,000 x g for 30 min at 4 °C). The supernatant was removed by suction. The solid was rinsed with MTBE, suspended in MTBE, and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)(4-(methylamino)butyl)(3-(methylamino)propyl)carbamate dihydrochloride (0.102 g, 0.131 mmol, 74.8%) as a white solid. UPLC/ELSD: RT = 2.03 min. MS (ES): m/z of C ₃₉ H ₇₁ N ₃ O ₂ = 328.45 [(M+2H)+CH ₃ CN] ²⁺ . ¹ HNMR(300MHz,MeOD)δ5.44-5.33(m,1H),4.52-4.37(m,1H),3.47-3.33(m,4H),3.10-2.90(m,4H),2.78-2.63(m,6H),2.44-2.27(m,2H),2.19-0.78(m ,42H),1.06(s,3H),0.96(d,J＝6.4Hz,3H),0.73(s,3H).

CX. Compound SA181: N-(3-amino-3-ethylpentyl)-N-(4-((3-amino-3-ethylpentyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride

Step 1: tert-Butyl (6,6,17-triethyl-9-(3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazanonadecan-17-yl)carbamate

To the reaction mixture was added 3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.150 g, 0.280 mmol), N tert-Butyl-(1-{[4-({3-[(tert-butoxycarbonyl)amino]-3-ethylpentyl}amino)butyl]amino}-3-ethylpentyl-3-yl)carbamate (0.217g, 0.421mmol) and triethylamine (0.14mL, 1.0mmol) in DCM (3.75mL) were added dropwise 50wt% propanephosphonic anhydride (0.29mL, 0.56mmol) in DCM. The reaction mixture was stirred at room temperature and monitored by LCMS. At 17h, the reaction mixture was diluted to _15mL with DCM and then washed with 5% _NaHCO3 aqueous solution. The aqueous solution was extracted with DCM (2x 15mL). The combined organics were dried over _Na2SO4 and concentrated. Via silica gel chromatography (3:2EtOAc/hexanes, then 0-20% (5% concentrated _NH4 in MeOH) in DCM The crude material was purified by HPLC-MS/MS (50% by volume, 1% by volume, 0.1% by volume, 0.2% by volume, 0.4% by volume, 0.6% by volume, 0.7% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.1% by volume, 0.2% by volume, 0.3% by volume, 0.4% by volume, 0.6% by volume, 0.7% by volume, 0.8% by volume, 0.9 ... MS (ES): m/z of C ₆₀ H ₁₁₀ N ₄ O ₅ S ₂ = 1032.19 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.44-5.32(m,1H),4.89(br.s,1H),4.53-4.16(m,1H),3.43-3.14(m,4H),3.11-2.86(m,2H),2.84-2.43(m,5H),2.43-2.22(m ,2H),2.10-0.73(m,84H),0.99(s,3H),0.92(d,J=6.6Hz,3H),0.67(s,3H).

Step 2: N-(3-amino-3-ethylpentyl)-N-(4-((3-amino-3-ethylpentyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride

To a stirred solution of tert-butyl (6,6,17-triethyl-9-(3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)propanoyl)-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazanonadecan-17-yl)carbamate (0.103 g, 0.100 mmol) in DCM (2.1 mL) was added 4N HCl in dioxane (0.17 mL, 0.68 mmol). The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 h, the reaction mixture was diluted to 20 mL with MTBE and then centrifuged (10,000 xg for 30 min at 4° C.) The supernatant was aspirated and the solids were rinsed with MTBE. The solid was suspended in MTBE and concentrated to give N-(3-amino-3-ethylpentyl)-N-(4-((3-amino-3-ethylpentyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride (0.092 g, 0.088 mmol, 87.8%) as a white solid. UPLC/ELSD: RT = 2.17 min. MS (ES): m/z of C ₅₀ H ₉₄ N ₄ OS ₂ = 416.72 (M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.41-5.33(m,1H),3.58-3.37(m,4H),3.19-3.06(m,4H),3.03-2.92(m,2H),2.88-2.78(m,2H),2.73-2.57(m,1H),2.39-2.27(m ,2H),2.20-0.77(m,70H),0.72(s,3H).

CY. Compound SA182: 5-((3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 14-(3-((tert-Butoxycarbonyl)amino)-3-ethylpentyl)-6,6-diethyl-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oic acid (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 5-(((3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.150 g, 0.284 mmol) was added. , N-(1-{[4-({3-[(tert-butoxycarbonyl)amino]-3-ethylpentyl}amino)butyl]amino}-3-ethylpentyl-3-yl)t-butyl carbamate (0.219g, 0.425mmol) and triethylamine (0.14mL, 1.0mmol) in DCM (3.75mL) in a stirred solution was added dropwise 50wt% propanephosphonic anhydride (0.29mL, 0.56mmol) in DCM. The reaction mixture was stirred at room temperature and monitored by LCMS. At 17h, the reaction mixture was diluted to 15mL with DCM, then washed with 5% _NaHCO3 aqueous solution. The aqueous solution was extracted with DCM ( _2x 15mL). The combined organics were passed through a hydrophobic glass frit, dried over _Na2SO4 , and concentrated. Via silica gel chromatography (3:2EtOAc/hexanes, then 0-20% (5% concentrated _NH4 in MeOH) in DCM The crude material was purified by HPLC-MS/MS (50% by volume, 4% by volume, 0.1% by volume, 0.2% by volume, 0.4% by volume, 0.6% by volume, 0.7% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.1% by volume, 0.2% by volume, 0.3% by volume, 0.4% by volume, 0.6% by volume, 0.7% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.6% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, 0.8% by volume, 0.9% by volume, 1.2% by volume, MS (ES): m/z of C ₆₂ H ₁₁₂ N ₄ O ₇ = 1026.39 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.41-5.31(m,1H),5.00-4.70(m,1H),4.69-4.51(m,1H),4.51-4.16(m,1H),3.37-3.11(m,4H),3.11-2.53(m,4H),2.43-2.21 (m,6H),2.12-0.72(m,84H),1.01(s,3H),0.92(d,J=6.4Hz,3H),0.67(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

To a solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)-6,6-diethyl-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate (0.119 g, 0.116 mmol) in DCM (2.4 mL) was added 4N HCl in dioxane (0.20 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 h, the reaction mixture was diluted to 20 mL with MTBE and then centrifuged (10,000 xg for 30 min at 4° C.) The supernatant was aspirated and the solids were rinsed with MTBE. The solid was suspended in MTBE and concentrated to give (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride (0.105 g, 0.092 mmol, 79.5%) as an off-white solid. UPLC/ELSD: RT = 2.09 min. MS (ES): m/z of C ₅₂ H ₉₆ N ₄ O ₃ = 413.39 (M+2H) ²⁺ . ¹ H NMR(300MHz,MeOD)δ5.42-5.35(m,1H),4.62-4.46(m,1H),3.57-3.36(m,4H),3.18-3.01(m,4H),2.56-2.25(m,6H),2.20-0.77(m,72H),0.72(s,3H)。

CZ. Compound SA183: (3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)carbamate (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl) ester trihydrochloride

Step 1: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)(4-((3-ethyl-3-(((neopentyloxy)carbonyl)amino)pentyl)amino)butyl)carbamate

4-Nitrophenyl carbonate sitosteryl ester (0.140 g, 0.241 mmol), tert-butyl N-(1-{[4-({3-[(tert-butoxycarbonyl)amino]-3-ethylpentyl}amino)butyl]amino}-3-ethylpentan-3-yl)carbamate (0.187 g, 0.362 mmol), and triethylamine (0.14 mL, 1.0 mmol) were combined in PhMe ( _3.5 mL). The reaction mixture was stirred at 100 °C and monitored by LCMS. At 16 h, the reaction mixture was cooled to room temperature, diluted with DCM to 15 mL, and then washed with 5% _K2CO3 in water (2x). The combined washings were extracted with DCM (2x 15 mL). The combined organics were passed through a _hydrophobic frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-20% (5% concentrated aqueous _NH4OH in MeOH) in DCM). The material was further purified via silica gel chromatography (1:1 EtOAc/hexanes then 0-20% (5% concentrated aqueous _NH4OH in MeOH) in DCM) to afford (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)(4-((3-ethyl-3-(((neopentyloxy)carbonyl)amino)pentyl)amino)butyl)carbamate as a white foam (0.097 g, 0.10 mmol, 41.5%). UPLC/ELSD: RT=2.95min. MS (ES): m/z of C ₅₈ H ₁₀₆ N ₄ O ₆ = 956.34 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.44-5.29(m,1H),5.17-4.91(m,1H),4.59-4.41(m,1H),4.35-4.06(m,1H),3.35-3.02(m,4H),2.70-2.53(m,4H),2.46-2.17 (m,2H),2.08-0.73(m,82H),1.01(s,3H),0.92(d,J＝6.5Hz,3H),0.68(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl(3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)carbamate trihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylhept-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl (3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)(4-((3-ethyl-3-(((neopentyloxy)carbonyl)amino)pentyl)amino)butyl)carbamate (0.094 g, 0.098 mmol) in DCM (1.9 mL) was added 4N HCl in dioxane (0.17 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 16h, the reaction mixture was diluted to 20mL with MTBE and then centrifuged (10,000xg at 4°C for 30min). The supernatant was removed and the solid was rinsed with MTBE. The solid was suspended in MTBE and then concentrated to give (3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)carbamic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-17-((2R, 5R)-5-ethyl-6-methylheptyl-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.083g, 0.089mmol, 90.1%) as a white solid. UPLC/ELSD: RT=1.99min. MS (ES): m/z of C ₄₈ H ₉₀ N ₄ O ₂ = 378.75 (M+2H) ²⁺ . ¹ H NMR(300MHz,MeOD)δ5.45-5.36(m,1H),4.52-4.35(m,1H),3.44-3.34(m,4H),3.17-3.05(m,4H),2.42-2.31(m,2H),2.16-0.78(m,70H),0.73(s,3H)。

DA. Compound SA184: N-(3-amino-3-ethylpentyl)-N-(4-((3-amino-3-ethylpentyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride

Step 1: tert-Butyl (14-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)-6,6,17-triethyl-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazanonadecan-17-yl)carbamate

To the mixture was added 3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoic acid (0.140 g, 0.276 mmol), N-(1- To a stirred solution of {[4-({3-[(tert-butoxycarbonyl)amino]-3-ethylpentyl}amino)butyl]amino}-3-ethylpentyl-3-yl)carbamate (0.213g, 0.414mmol) and triethylamine (0.14mL, 1.0mmol) in DCM (3.5mL) was added dropwise 50wt% propanephosphonic anhydride (0.24mL, 0.468mmol) in DCM. The reaction mixture was stirred at room temperature and monitored by LCMS. At 3h, the reaction mixture was diluted to 15mL with DCM and then washed with 5% _NaHCO3 aqueous solution. The aqueous solution was extracted with DCM ( _2x 15mL). The combined organics were passed through a hydrophobic glass frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-20% (5% concentrated aqueous _NH4OH in MeOH) in DCM) to afford tert-butyl (14-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)disulfanyl)propanoyl)-6,6,17-triethyl-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazanonadecan-17-yl)carbamate (0.097 g, 0.097 mmol, 35.1%) as a clear yellow oil. UPLC/ELSD: RT=2.95min. MS (ES): m/z of C ₅₈ H ₁₀₆ N ₄ O ₅ S ₂ = 1004.81 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.39-5.31(m,1H),5.03-4.74(m,1H),4.52-4.25(m,1H),3.39-3.10(m,4H),3.10-2.85(m,3H),2.85-2.42(m,6H),2.42-2.22 (m,2H),2.09-0.72(m,78H),0.99(s,3H),0.91(d,J＝6.5Hz,3H),0.67(s,3H).

Step 2: N-(3-amino-3-ethylpentyl)-N-(4-((3-amino-3-ethylpentyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride

To a stirred solution of tert-butyl (14-(3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanoyl)-6,6,17-triethyl-2,2-dimethyl-4-oxo-3-oxa-5,9,14-triazanonadecan-17-yl)carbamate (0.091 g, 0.091 mmol) in DCM (2.3 mL) was added 4N HCl in dioxane (0.16 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 16 h, the reaction mixture was diluted to 20 mL with MTBE and then centrifuged (10,000 x g at 4 ° C for 30 min). The supernatant was removed. The solid was rinsed with MTBE, then suspended in MTBE and concentrated to give N-(3-amino-3-ethylpentyl)-N-(4-((3-amino-3-ethylpentyl)amino)butyl)-3-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)disulfanyl)propanamide trihydrochloride (0.080 g, 0.080 mmol, 88.4%) as a white solid. UPLC/ELSD: RT=2.04min. MS (ES): m/z of C ₄₈ H ₉₀ N ₄ OS ₂ = 402.54(M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.45-5.31(m,1H),3.58-3.36(m,4H),3.19-3.04(m,4H),3.03-2.89(m,2H),2.89-2.76(m,2H),2.73-2.55(m,1H),2.42-2.23( m, 2H), 2.18-0.96 (m, 57H), 0.94 (d, J = 6.5Hz, 3H), 0.88 (d, J = 6.6Hz, 6H), 0.72 (s, 3H).

DB. Compound SA185: 5-((3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)amino)-5-oxopentanoic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride

Step 1: 14-(3-((tert-Butyloxycarbonyl)amino)-3-ethylpentyl)-6,6-diethyl-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oic acid (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester

To 5-(((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)oxy)-5-oxopentanoic acid (0.140 g, 0.28 mmol), N-(1 -{[4-({3-[(tert-butoxycarbonyl)amino]-3-ethylpentyl}amino)butyl]amino}-3-ethylpentyl-3-yl)carbamate (0.216g, 0.419mmol) and triethylamine (0.14mL, 1.0mmol) in DCM (3.5mL) were added dropwise 50wt% propanephosphonic anhydride (0.28mL, 0.546mmol) in DCM. The reaction mixture was stirred at room temperature and monitored by LCMS. At 3h, the reaction mixture was diluted to 15mL with DCM, then washed with 5% _NaHCO3 aqueous solution. The aqueous solution was extracted with DCM ( _2x 15mL). The combined organics were passed through a hydrophobic glass frit, dried over _Na2SO4 , and concentrated. The crude material was purified via silica gel chromatography (0-14% (5% concentrated aqueous _NH4OH in MeOH) in DCM) to afford (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 14-(3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)-6,6-diethyl-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate (0.071 g, 0.071 mmol, 25.4%) as a clear yellow oil. UPLC/ELSD: RT=2.89min. MS (ES): m/z of C ₆₀ H ₁₀₈ N ₄ O ₇ = 998.15 (M+H) ⁺ . ¹ H NMR (300MHz, CDCl ₃ ) δ5.43-5.31(m,1H),5.11-4.87(m,1H),4.68-4.51(m,1H),4.40-4.12(m,1H),3.37-3.12(m,4H),2.72-2.51(m,4H),2.43-2.21 (m,6H),2.07-0.74(m,80H),1.01(s,3H),0.91(d,J=6.5Hz,3H),0.67(s,3H).

Step 2: (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 5-((3-amino-3-ethylpentyl)(4-((3-amino-3-ethylpentyl)amino)butyl)amino)-5-oxopentanoate trihydrochloride

To a stirred solution of (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl)-14-(3-((tert-butoxycarbonyl)amino)-3-ethylpentyl)-6,6-diethyl-2,2-dimethyl-4,15-dioxo-3-oxa-5,9,14-triazanonadecan-19-oate (0.067 g, 0.067 mmol) in DCM (1.8 mL) was added 4N HCl in dioxane (0.12 mL). The reaction mixture was stirred at room temperature and monitored by LCMS. At 16h, the reaction mixture was diluted to 20mL with MTBE and then centrifuged (10,000xg at 4°C for 30min). The supernatant was removed. The solid was rinsed with MTBE, then suspended in MTBE and concentrated to give 5-((3-amino-3-ethylpentyl))(4-((3-amino-3-ethylpentyl)amino)butyl)amino)-5-oxopentanoic acid (3S, 8S, 9S, 10R, 13R, 14S, 17R)-10,13-dimethyl-17-((R)-6-methylheptyl-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl ester trihydrochloride (0.061 g, 0.066 mmol, 97.5%) as a white solid. UPLC/ELSD: RT=1.96min. MS (ES): m/z of C ₅₀ H ₉₂ N ₄ O ₃ = 399.58(M+2H) ²⁺ . ¹ H NMR (300MHz, MeOD) δ5.45-5.33(m,1H),4.62-4.46(m,1H),3.56-3.36(m,4H),3.18-3.03(m,4H),2.55-2.25(m,6H),2.18-0.98(m,59H),0.95(d,J＝ 6.5Hz, 3H), 0.88 (d, J = 6.6Hz, 6H), 0.73 (s, 3H).

Example 2

Generation of Nanoparticle Compositions

The lipid was dissolved in ethanol at a concentration of 24 mg/mL and a molar ratio of 49.0:11.2:39.3:0.5 (IL1:DSPC:cholesterol:PEG-DMG-2K) and mixed with an acidified buffer (45 mM acetate buffer at pH 4). The lipid solution and the acidified buffer were mixed using a multi-inlet vortex mixer with a 3:7 volume ratio of lipid:buffer (for mixers 1 and 2) and a 1:3 volume ratio of lipid:buffer (25% ethanol) (for mixer 3). After a residence time of 5 seconds, the resulting nanoparticles were mixed with 55 mM sodium acetate (pH 5.6) at a 5:7 volume ratio of nanoparticle:buffer. See Table 2a for mixing parameters. The resulting dilute nanoparticle buffer was then exchanged and concentrated into a final buffer containing 5 mM sodium acetate (pH 5.0) using tangential flow filtration (TFF). See Table 2b for TFF parameters. Then, a 70% sucrose solution in 5 mM acetate buffer (pH 5) was subsequently added.

Table 2a: Mixing parameters

Table 2b: TFF parameters

TFF parameters Mixer Value Feed flux 240L/ ^m2 /hr load 100-300g/ ^m3 Initial concentration: Factor 10x Diafiltration Volume (DV) 8DV Final concentration: Factor 4x TMP 4-6psi

The mRNA (luciferase or CFTR) of the 0.625mg/mL concentration in 42.5mM sodium acetate (pH 5.0) of the gained nanoparticle of 7.33mg/mL lipid concentration in 5mM acetate (pH 5) and 75g/L sucrose is mixed, N:P is 4.93.Multi-inlet vortex mixer is used by nanoparticle solution and nanoparticle with nanoparticle: the 3:2 volume ratio of mRNA is mixed.Once loaded with mRNA, these intermediate nanoparticles just stand 300 seconds residence time immediately, then with nanoparticle: the 5:1 volume ratio of buffer adds the neutralization buffer containing 120mM TRIS (pH 8.12).

For HeLa studies evaluating luciferase protein expression, PEG-DMG-2K dissolved in 20 mM TRIS buffer (pH 7.5) was added to the neutralized intermediate nanoparticle solution at a ratio of 1:6 to bring the solution to a final molar ratio of IL1:DSPC:cholesterol:PEG-DMG-2K of 48.5:11.1:38.9:1.5%. This nanoparticle formulation was then modified with a lipid amine. In a typical example, a nanoparticle formulation at a concentration of 0.18 mg/mL mRNA and a volume of 0.56 mL was modified with lipid amine SA50 (467.2 nmol), which was prepared in a buffer containing 20 mM TRIS, 14.3 mM sodium acetate, 32 g/L sucrose, and 140 mM NaCl (pH 7.5) at a volume ratio of 1:1 nanoparticle:buffer.

For HeLa studies evaluating CFTR protein expression, an intermediate nanoparticle preparation (1 mL, 0.415 mg mRNA) was mixed with a buffer containing 20 mM TRIS (pH 7.5), 0.9 mg/mL PEG-DMG-2K and lipid amine SA50 (647.1 nmol) at a volume ratio of 6:1 nanoparticle:buffer. The resulting nanoparticle suspension was concentrated using a centrifugal filter device (100 kDa molecular weight cutoff) and diluted to a final buffer matrix containing 70 mM NaCl in a running buffer (20 mM TRIS, 14.3 mM sodium acetate and 32 g/L sucrose, pH 7.5) containing 300 mM NaCl solution.

The two resulting nanoparticle suspensions were filtered through a 0.8/0.2 μm capsule filter and filled into glass vials at an mRNA strength of approximately 0.1-1 mg/mL. The biophysical data (diameter and PDI from DLS measurements and % encapsulation determined using Ribogreen) of the luciferase and CFTR mRNA nanoparticles containing lipidamine are shown in Table 2c and Table 2d, respectively.

Table 2c: Biophysical data of luciferase mRNA nanoparticles

Table 2d: CFTR mRNA Nanoparticle Biophysical Data

Example 3

Protein expression in human cervical cancer epithelial cells (HeLa)

Lipid nanoparticle compositions were prepared in a similar manner as in Example 2. To evaluate in vitro LNP cellular uptake and protein expression, HeLa cells (ATCC CCL-2) from ATCC.org were used. Prior to running the experiment, cells were cultured in complete minimal essential medium (MEM) and plated in 96-well Cell Carrier Ultra plates (PerkinElmer) with a PDL-coated surface.

Luciferase protein expression assay in HeLa cells

Cells were transfected with buffer control (PBS) or LNPs (25ng/well; N=4 replicate wells) of luciferase mRNA encapsulated in serum-free MEM medium. The cells transfected with LNPs were incubated for 5h, followed by removal of the culture medium and supplementation with complete MEM culture medium. The cells were further incubated overnight (24h) in complete MEM culture medium. After incubation for 24hr, luciferase protein expression was measured using ONE-Glo ^™ luciferase assay (Promega). Cells were dissolved for 10min using 1x passive lysis buffer (Catalog No. E194A) in a microplate mixer at room temperature. Luciferase in the supernatant was measured by adding a luciferase assay reagent (Catalog No. E151A) containing luciferin. Bioluminescence was then measured immediately on a Synergy H1 plate reader (BioTek). The results shown in Table 3a show the average relative light units (RLU) of each test sample.

Table 3a: Luciferase expression results

CFTR protein expression assay in HeLa cells

In the absence of serum, buffer control (PBS) and LNPs encapsulating cystic fibrosis transmembrane conductance regulator (CFTR) mRNA were administered with MEM medium (N=4 replicate wells). LNP-transfected cells were incubated for 5 h, followed by removal of the medium and supplementation with complete MEM medium. Cells were further incubated overnight (24 h) in complete MEM medium.

After incubation for 24 hr, cells were fixed with PFA and treated with anti-CFTR rabbit monoclonal antibody for immunofluorescence (IF). In short, cells were permeabilized with 0.5% TX-100 for 10 min, blocked with 3% bovine serum albumin (BSA) + PBST at room temperature for 1 hr, and incubated overnight at 4 ° C with primary anti-CFTR monoclonal antibody. After primary antibody incubation, cells were incubated with Alexa 488-conjugated secondary antibodies for 30 min and stained with DAPI and HCS CellMask blue stain. Between different incubation steps, cells were washed with PBS or PBST. Cells were imaged using Opera Phoenix spinning disk confocal microscope (PerkinElmer), and CFTR protein expression was detected using 488nm channel. Image analysis was implemented in Harmony 4.9, where the main analysis output was the CFTR intensity of each cell. The results shown in Table 3b show the multiple changes (NA=not obtained) in the CFTR signal intensity of each cell compared with the buffer (PBS) control.

Table 3b: CFTR protein expression results

A number of embodiments have been described. In any case, it should be understood that various modifications may be made without departing from the spirit and scope described. Therefore, other embodiments are within the scope of the appended claims. Each reference cited in this application (including all patents, patent applications, and publications) is incorporated herein by reference in its entirety.

Claims (102)

1. A lipid amine compound of formula A1, or a salt thereof, wherein: Z is N or CH; ^R1 is _C1-14 alkyl, _C1-14 alkenyl or _C1-14 hydroxyalkyl; ^R2 and ^R3 are each _C2-20 alkyl, wherein: (i) the C _2-20 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; (ii) 1, 2, 3 or 4 non-terminal carbons of the C _2-20 alkyl group are optionally replaced by O; (iii) 1, 2, 3 or 4 non-terminal carbons of the C _2-20 alkyl group are optionally replaced by NR ¹⁰ ; (iv) 1, 2, 3 or 4 non-terminal carbons of the _C2-20 alkyl group are optionally replaced by C(=O); and (v) 1, 2, 3 or 4 non-terminal carbons of the _C2-20 alkyl group are optionally replaced ^{by CRaRb} , wherein ^Ra and ^Rb together with the C atom to which they are attached form a _C3-6 cycloalkyl group; wherein ^R2 and ^R3 are the same or different; or R ² and R ³ together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group comprising 1, 2 or 3 ring-forming NR ¹⁰ groups, wherein the 7-18 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo; or R ² , R ³ and R ⁶ together with the atoms to which they are attached and any intervening atoms form a 7-18 membered bridged heterocycloalkyl group optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo; R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently selected from H, halo and C _1-4 alkyl; or ^R4 and ^R5 together with the carbon atom to which they are attached form a _C3-7 cycloalkyl group; or R ⁶ and R ⁷ together with the carbon atom to which they are attached form a C _3-7 cycloalkyl group; R ⁸ , R ⁹ and R ¹⁰ are each independently selected from H and C _1-4 alkyl; j is 0 or 1; k is 0, 1, 2, 3, 4, 5, or 6; l is 0 or 1; m is 0, 1, 2, 3, 4, 5 or 6; and n is 0 or 1; When j is 0, l is 1. Where j and l are not both 0, Provided that the compound is not: 2. A lipid amine compound of formula A1: or a salt thereof, wherein: Z is N or CH; ^R1 is _C1-14 alkyl, _C1-14 alkenyl or _C1-14 hydroxyalkyl; ^R2 and ^R3 are each _C2-20 alkyl, wherein: (i) the C _2-20 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; (ii) 1, 2, 3 or 4 non-terminal carbons of the _C2-20 alkyl group are optionally replaced by O; and (iii) 1, 2, 3 or 4 non-terminal carbons of the C _2-20 alkyl group are optionally replaced by NR ¹⁰ ; wherein ^R2 and ^R3 are the same or different; or R ² and R ³ together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group comprising 1, 2 or 3 ring-forming NR ¹⁰ groups, wherein the 7-18 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo; or R ² , R ³ and R ⁶ together with the atoms to which they are attached and any intervening atoms form a 7-18 membered bridged heterocycloalkyl group optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting of C _1-4 alkyl, -NR ⁸ R ⁹ , OH and halo; R ⁴ , R ⁵ , R ⁶ and R ⁷ are each independently selected from H, halo and C _1-4 alkyl; or ^R4 and ^R5 together with the carbon atom to which they are attached form a _C3-7 cycloalkyl group; or R ⁶ and R ⁷ together with the carbon atom to which they are attached form a C _3-7 cycloalkyl group; R ⁸ , R ⁹ and R ¹⁰ are each independently selected from H and C _1-4 alkyl; j is 0 or 1; k is 0, 1, 2, 3, 4, 5, or 6; l is 0 or 1; m is 0, 1, 2, 3, 4, 5 or 6; and n is 0 or 1; When j is 0, l is 1. Where j and l are not both 0, Provided that the compound is not: 3. The compound or salt thereof according to claim 1 or 2, wherein Z is N. 4. The compound or salt thereof according to claim 1 or 2, wherein Z is CH. 5. The compound or salt thereof according to any one of claims 1 to 4, wherein R ¹ is a C _1-14 alkyl group. 6. The compound or salt thereof according to any one of claims 1 to 4, wherein R ¹ is a C _1-14 hydroxyalkyl group. 7. The compound or salt thereof according to any one of claims 1 to 4, wherein R ¹ is C _1-14 alkenyl. 8. The compound according to any one of claims 1 to 4, wherein R ¹ is 9. The compound or salt thereof according to any one of claims 1 to 8, wherein j is 0. 10. The compound or salt thereof according to any one of claims 1 to 8, wherein j is 1. 11. The compound or salt thereof according to any one of claims 1 to 10, wherein k is 0, 2, 3 or 4. 12. The compound or salt thereof according to any one of claims 1 to 10, wherein k is 0. 13. The compound or salt thereof according to any one of claims 1 to 10, wherein k is 2. 14. The compound or salt thereof according to any one of claims 1 to 10, wherein k is 3. 15. The compound or salt thereof according to any one of claims 1 to 10, wherein k is 4. 16. The compound or salt thereof according to any one of claims 1 to 15, wherein l is 0. 17. The compound or salt thereof according to any one of claims 1 to 15, wherein l is 1. 18. The compound or salt thereof according to any one of claims 1 to 17, wherein m is 0, 1, 2 or 4. 19. The compound or salt thereof according to any one of claims 1 to 17, wherein m is 0. 20. The compound or salt thereof according to any one of claims 1 to 17, wherein m is 1. 21. The compound or salt thereof according to any one of claims 1 to 17, wherein m is 2. 22. The compound or salt thereof according to any one of claims 1 to 17, wherein m is 4. 23. The compound or salt thereof according to any one of claims 1 to 22, wherein n is 0. 24. The compound or salt thereof according to any one of claims 1 to 22, wherein n is 1. 25. The compound or salt thereof as claimed in any one of claims 1 to 8, wherein j is 0, k is 0, l is 1, m is 1, and n is 1. 26. The compound or salt thereof as claimed in any one of claims 1 to 8, wherein j is 0, k is 0, l is 1, m is 2, and n is 1. 27. The compound or salt thereof as claimed in any one of claims 1 to 8, wherein j is 0, k is 0, l is 1, m is 4, and n is 1. 28. The compound or salt thereof as claimed in any one of claims 1 to 8, wherein j is 1, k is 0, l is 0, m is 0, and n is 0. 29. The compound or salt thereof as claimed in any one of claims 1 to 8, wherein j is 1, k is 1, l is 0, m is 0, and n is 0. 30. The compound or salt thereof as claimed in any one of claims 1 to 8, wherein j is 1, k is 1, l is 0, m is 0, and n is 1. 31. The compound or salt thereof as described in any one of claims 1 to 8, wherein j is 1, k is 1, l is 0, m is 2, and n is 0. 32. The compound or salt thereof of any one of claims 1-8, wherein j is 1, k is 1, l is 1, m is 1, and n is 1. 33. The compound or salt thereof as described in any one of claims 1 to 8, wherein j is 1, k is 2, l is 0, m is 0, and n is 0. 34. The compound or salt thereof as described in any one of claims 1 to 8, wherein j is 1, k is 2, l is 0, m is 0, and n is 1. 35. The compound or salt thereof as described in any one of claims 1 to 8, wherein j is 1, k is 3, l is 0, m is 0, and n is 1. 36. The compound or salt thereof of any one of claims 1 to 8, wherein j is 1, k is 4, l is 0, m is 0, and n is 1. 37. A compound or salt thereof as described in any one of claims 1 and 3-36, wherein: ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein: (i) the C _2-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; (ii) one or two non-terminal carbons of the C _2-10 alkyl group are optionally replaced by O; (iii) one or two non-terminal carbons of the C _2-10 alkyl group are optionally replaced by NR ¹⁰ ; (iv) one or two non-terminal carbons of the _C2-10 alkyl group are optionally replaced by C(=O); and (v) One or two non-terminal carbons of the _C2-10 alkyl group are optionally replaced ^{by CRaRb} , wherein ^Ra and ^Rb together with the C atom to which they are attached form a _C3-6 cycloalkyl group. 38. A compound or salt thereof as described in any one of claims 1 to 36, wherein: ^R2 and ^R3 are each independently selected from _C2-10 alkyl, wherein: (i) the C _2-10 alkyl group is substituted by 1, 2, 3 or 4 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; (ii) 1 or 2 non-terminal carbons of the C _2-10 alkyl group are optionally replaced by O; and (iii) One or two non-terminal carbons of the C _2-10 alkyl group are optionally replaced by NR ¹⁰ . 39. The compound or salt thereof as described in any one of claims 1 and 3-36, wherein one of R ² and R ³ is C _2-5 alkyl, wherein: the C _2-5 alkyl is substituted with 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; And wherein the other of R ² and R ³ is a C _7-10 alkyl group, wherein: (i) the C _7-10 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; (ii) 1, 2, 3 or 4 non-terminal carbons of the C _7-10 alkyl group are optionally replaced by O; (iii) 1, 2, 3 or 4 non-terminal carbons of the C _7-10 alkyl group are optionally replaced by NR ¹⁰ ; (iv) 1, 2, 3 or 4 non-terminal carbons of the _C7-10 alkyl group are optionally replaced by C(=O); and (v) 1, 2, 3 or 4 non-terminal carbons of the C _7-10 alkyl group are optionally replaced by CR ^a R ^b , wherein R ^a and R ^b together with the C atom to which they are attached form a C _3-6 cycloalkyl group. 40. The compound or salt thereof according to any one of claims 1 to 36, wherein one of R ² and R ³ is a C _2-5 alkyl group, wherein: (i) the C _2-5 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; And wherein the other of R ² and R ³ is a C _7-10 alkyl group, wherein: (i) the C _7-10 alkyl group is substituted by 1, 2, 3, 4 or 5 substituents independently selected from -NR ⁸ R ⁹ , OH and halogen, wherein at least one substituent is -NR ⁸ R ⁹ ; (ii) 1, 2, 3 or 4 non-terminal carbons of the _C7-10 alkyl group are optionally replaced by O; and (iii) 1, 2, 3 or 4 non-terminal carbons of the C _7-10 alkyl group are optionally replaced by NR ¹⁰ . 41. The compound or salt ^thereof as claimed in any one of claims 1 to 36, wherein one of ^R2 and ^R3 is _C3 alkyl substituted with at least one ^-NR8R9 group and further optionally substituted with one or two groups selected from OH and halo. 42. A compound or salt thereof as described in any one of claims 1 to 36, wherein: ^R2 and ^R3 together with the N atom to which they are attached form a 7-18 membered heterocycloalkyl group containing 1, 2 or 3 ring-forming ^NR10 groups, wherein the 7-18 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group ^consisting of _C1-4 alkyl, ^-NR8R9 , OH and halo. 43. A compound or salt thereof as described in any one of claims 1 to 36, wherein: ^R2 and ^R3 together with the N atom to which they are attached form an 8-10 membered heterocycloalkyl group containing 1, 2 or 3 ring-forming ^NR10 groups, wherein the 8-10 membered heterocycloalkyl group is optionally substituted with 1, 2 or 3 substituents independently selected from the group consisting ^of _C1-4 alkyl, ^-NR8R9 , OH and halo. 44. A compound or salt thereof as described in any one of claims 1 to 36, wherein: ^R2 and ^R3 together with the N atom to which they are attached form a heterocycloalkyl group of the formula: 45. A compound or salt thereof as described in any one of claims 1-11, 13-24 and 29-36, wherein: ^R2 , ^R3 and ^R6 together with the atoms to which they are attached and any intervening atoms form a 7-18 membered bridged heterocycloalkyl optionally substituted with 1, 2 or 3 substituents independently selected from _C1-4 ^alkyl , ^-NR8R9 , OH and halo. 46. A compound or salt thereof as described in any one of claims 1-11, 13-24 and 29-36, wherein: R ² , R ³ and R ⁶ together with the atoms to which they are attached and any intervening atoms form a 7-18 membered bridged heterocycloalkyl selected from: 47. The compound or salt thereof as described in any one of claims 1-11, 13-24 and 29-46, wherein ^R4 and ^R5 are each independently H or _C1-4 alkyl. 48. A compound or salt thereof as described in any one of claims 1-11, 13-24 and 29-46, wherein ^R4 and ^R5 are each independently H or methyl. 49. The compound or salt thereof as described in any one of claims 1-11, 13-24 and 29-46, wherein ^R4 and ^R5 are both H. 50. The compound or salt thereof as described in any one of claims 1-11, 13-24 and 29-46, wherein ^R4 and ^R5 are both _C1-4 alkyl. 51. The compound or salt thereof as described in any one of claims 1-11, 13-24 and 29-46, wherein one of ^R4 and ^R5 is H and the other is _C1-4 alkyl. 52. The compound or salt thereof as described in any one of claims 1-18, 20-27, 31, 32 and 37-51, wherein ^R6 and ^R7 are each independently H or _C1-4 alkyl. 53. A compound or salt thereof as described in any one of claims 1-18, 20-27, 31, 32 and 37-51, wherein ^R6 and ^R7 are each independently H or methyl. 54. A compound or salt thereof as described in any one of claims 1-18, 20-27, 31, 32 and 37-51, wherein ^R6 and ^R7 are both H. 55. The compound or salt thereof as described in any one of claims 1-18, 20-27, 31, 32 and 37-51, wherein ^R6 and ^R7 are both _C1-4 alkyl. 56. The compound or salt thereof as described in any one of claims 1-18, 20-27, 31, 32 and 37-51, wherein one of ^R6 and ^R7 is H and the other is _C1-4 alkyl. 57. The compound or salt thereof as claimed in any one of claims 1 to 43, 45 and 47 to 56, wherein ^R8 , ^R9 and ^R10 are each independently selected from H and methyl. 58. A compound or salt thereof as described in any one of claims 1-43, 45 and 47-56, wherein R ⁸ and R ⁹ are both H. 59. The compound or salt thereof as described in any one of claims 1-43, 45 and 47-56, wherein ^R8 and ^R9 are both _C1-4 alkyl. 60. The compound or salt thereof as described in any one of claims 1-43, 45 and 47-56, wherein R ⁸ and R ⁹ are both methyl. 61. The compound or salt thereof as described in any one of claims 1-43, 45 and 47-56, wherein one of ^R8 and ^R9 is H and the other is _C1-4 alkyl. 62. The compound or salt thereof as described in any one of claims 1-43 and 47-56, wherein R ¹⁰ is H. 63. A compound as described in any one of claims 1 to 62, wherein the compound has formula A2: or a salt thereof. 64. The compound of claim 1, selected from the group consisting of: or a salt of any one of the above mentioned. 65. The compound of claim 1, selected from the group consisting of: or a salt of any one of the above mentioned. 66. The compound of claim 1, selected from the group consisting of: or a salt of any one of the above mentioned. 67. A lipid nanoparticle composition comprising a lipid amine compound or a salt thereof according to any one of claims 1-66. 68. The lipid nanoparticle composition of claim 67, wherein the lipid nanoparticle composition further comprises: (i) ionizable lipids, (ii) phospholipids, (iii) structural lipids, and (iv) optionally a PEG-lipid, and (v) Optionally, a payload for delivery into the cell. 69. The lipid nanoparticle composition of claim 68, wherein the weight ratio of the lipid amine to the payload is about 1:1 to about 4:1. 70. The lipid nanoparticle composition of claim 68, wherein the weight ratio of the lipid amine to the payload is about 1.25:1, about 2.5:1, or about 3.75:1. 71. The lipid nanoparticle composition of any one of claims 68-70, wherein the ionizable lipid is a compound of formula (I): or an N-oxide or salt thereof, wherein: ^R1 is in Indicates a connection point; ^Raα , ^Raβ , ^Raγ and ^Raδ are each independently selected from H, _C2-12 alkyl and _C2-12 alkenyl; ^R2 and ^R3 are each independently selected from _C1-14 alkyl and _C2-14 alkenyl; R ⁴ is selected from -(CH ₂ ) _n OH and wherein n is selected from 1, 2, 3, 4 and 5; in Represents a connection point, wherein R ¹⁰ is N(R) ₂ ; wherein each R is independently selected from C _1-6 alkyl, C _2-3 alkenyl and H; wherein n2 is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Each R ⁵ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H; Each R ⁶ is independently selected from C _1-3 alkyl, C _2-3 alkenyl and H; M and M' are each independently selected from -C(O)O- and -OC(O)-; R' is a C _1-12 alkyl group or a C _2-12 alkenyl group; l is selected from 1, 2, 3, 4 and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12 and 13. 72. The lipid nanoparticle composition of any one of claims 68-70, wherein the ionizable lipid is or an N-oxide or salt thereof. 73. The lipid nanoparticle composition of any one of claims 68-70, wherein the ionizable lipid is IL1: or a salt thereof. 74. The lipid nanoparticle composition of any one of claims 68-73, wherein the lipid nanoparticle composition comprises about 30 mol% to about 60 mol% ionizable lipids. 75. The lipid nanoparticle composition of any one of claims 68-74, wherein the phospholipid is a compound of formula (IV): or a salt thereof, wherein: Each R ¹ is independently H or optionally substituted alkyl; or optionally two R ¹ are linked together with an intermediate atom to form an optionally substituted monocyclic cycloalkyl or an optionally substituted monocyclic heterocyclyl; or optionally three R ¹ are linked together with an intermediate atom to form an optionally substituted bicyclic cycloalkyl or an optionally substituted bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; A has the following formula: ^each instance of L is independently a bond or optionally substituted Ci _-6 alkylene, wherein one methylene unit of the optionally substituted Ci _-6 alkylene is optionally replaced by -O-, -N( ^RN )-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C( ^O )O-, -OC(O)-, -OC(O)O-, -OC(O)N( ^RN )-, -NRNC ⁽ O)O-, or ^{-NRNC (} O)N( ^RN )-; each instance ^of R is independently optionally substituted _C1-30 alkyl, optionally substituted _C1-30 alkenyl, or optionally substituted _C1-30 alkynyl; optionally wherein one or more methylene units of ^R are independently replaced by optionally substituted cycloalkylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N( ^RN )-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C( ^O )O-, -OC(O)-, -OC(O) ^O- , -OC( ^O )N( ^RN )-, -NRNC ⁽ O)O-, ^-C (O)S-, -SC(O)-, -C(═NRN ⁾ -, -C(═NRN ⁾ -, or )N(R ^N )-, -NR ^N C(＝NR ^N )-, -NR ^N C(＝NR ^N )N(R ^N )-, -C(S)-, -C(S)N(R ^N )-, -NR ^N C(S)-, -NR ^N C(S)N(R ^N )-, -S(O)-, -OS(O)-, -OS(O)O-, -OS(O) ₂ -, _{-S(O) 2 O-, -OS(O) 2 O-} , -N(R ^N )S(O)-, -S(O)N(R ^N )-, -N(R ^N )S(O)N(R ^N )-, -OS(O)N(R ^N )-, -N(R ^N )S(O)O-, -S(O) ₂ -, -N(R ^N )S(O) ₂ -, -S(O) ₂ N(R ^N )-, -N(R ^N )S(O) ₂ N(R ^N )-, -OS(O) ₂ N(R ^N )- or -N(R ^N )S(O) ₂ O-; Each instance of ^RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl; and p is 1 or 2; Provided that the compound does not have the formula: wherein each instance of R ² is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl. 76. The lipid nanoparticle composition of any one of claims 68-74, wherein the phospholipid is 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-Dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecene-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-Hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0PE), 1,2-diphytanoyl-sn-glycero-3-phosphocholine (4ME 16:0PC), 1,2-Diphytanoyl-sn-glycero-3-phospho-(1'-racemic-glycerol) (sodium salt) (4ME 16:0PG), 1,2-Diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt) (4ME 16:0PS), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-Dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-Diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-di(docosahexaenoyl)-sn-glycero-3-phosphoethanolamine, 1,2-Dioleoyl-sn-glycero-3-phospho-racemic-(1-glycerol) sodium salt (DOPG) or Sphingomyelin. 77. The lipid nanoparticle composition of any one of claims 68-74, wherein the phospholipid is DSPC. 78. The lipid nanoparticle composition of any one of claims 68-77, wherein the lipid nanoparticle composition comprises about 5 mol% to about 15 mol% phospholipids. 79. The lipid nanoparticle composition of any one of claims 68-78, wherein the structural lipid is cholesterol, coproposterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine glycoside, ursolic acid, α-tocopherol, hopanes, phytosterols, steroids, or a mixture thereof. 80. The lipid nanoparticle composition of any one of claims 68-78, wherein the structural lipid is α-tocopherol, cholesterol, β-sitosterol, or cholesteryl hemisuccinate. 81. The lipid nanoparticle composition of any one of claims 68-80, wherein the lipid nanoparticle composition comprises about 20 mol% to about 60 mol% structural lipids. 82. The lipid nanoparticle composition of any one of claims 68-81, wherein the PEG-lipid is a compound of formula (V): or a salt thereof, wherein: ^R3 is ^-ORO ; R ^O is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive; ^L is optionally substituted C _1-10 alkylene, wherein at least one methylene group of the optionally substituted C _1-10 alkylene is independently replaced by optionally substituted cycloalkylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -O-, -N( ^RN )-, -S-, -C(O) ^- , -C(O)N(RN)-, -NRNC(O)-, -C(O)O-, -OC( ^O )-, -OC(O)O-, -OC(O)N( ^RN )-, -NRNC ⁽ O)O-, or ^-NRNC (O)N( ^RN )-; D is a moiety obtained by click chemistry or a moiety that can be cleaved under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; A has the following formula: ^each instance of L is independently a bond or optionally substituted Ci _-6 alkylene, wherein one methylene unit of the optionally substituted Ci _-6 alkylene is optionally replaced by -O-, -N( ^RN )-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -C( ^O )O-, -OC(O)-, -OC(O)O-, -OC(O)N( ^RN )-, -NRNC ⁽ O)O-, or ^{-NRNC (} O)N( ^RN )-; each instance ^of R is independently optionally substituted _C1-30 alkyl, optionally substituted _C1-30 alkenyl, or optionally substituted _C1-30 alkynyl; optionally wherein one or more methylene units of ^R are independently replaced by optionally substituted cycloalkylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, -N( ^RN )-, -O-, -S-, -C(O)-, -C(O)N(RN)-, -NRNC(O)-, -NRNC(O)N(RN)-, -C( ^O )O-, -OC(O)-, -OC(O) ^O- , -OC( ^O )N( ^RN )-, -NRNC ⁽ O)O-, ^-C (O)S-, -SC(O)-, -C(═NRN ⁾ -, -C(═NRN ⁾ -, or )N(R ^N )-, -NR ^N C(＝NR ^N )-, -NR ^N C(＝NR ^N )N(R ^N )-, -C(S)-, -C(S)N(R ^N )-, -NR ^N C(S)-, -NR ^N C(S)N(R ^N )-, -S(O)-, -OS(O)-, -OS(O)O-, -OS(O) ₂ -, _{-S(O) 2 O-, -OS(O) 2 O-} , -N(R ^N )S(O)-, -S(O)N(R ^N )-, -N(R ^N )S(O)N(R ^N )-, -OS(O)N(R ^N )-, -N(R ^N )S(O)O-, -S(O) ₂ -, -N(R ^N )S(O) ₂ -, -S(O) ₂ N(R ^N )-, -N(R ^N )S(O) ₂ N(R ^N )-, -OS(O) ₂ N(R ^N )- or -N(R ^N )S(O) ₂ O-; Each instance of ^RN is independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group; Ring B is optionally substituted cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl; and p is 1 or 2. 83. The lipid nanoparticle composition of any one of claims 68-81, wherein the PEG-lipid is PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG DMPE, PEG-DPPC, PEG-DSPE or PL1: 84. The lipid nanoparticle composition of any one of claims 63-78, wherein the lipid nanoparticle composition comprises about 0.1 mol% to about 5.0 mol% PEG-lipid. 85. A pharmaceutical composition comprising the lipid nanoparticle composition or a salt thereof according to any one of claims 62-79 and a pharmaceutically acceptable carrier. 86. The pharmaceutical composition of claim 85, wherein the composition is suitable for inhalation. 87. A method of delivering a payload into a cell, the method comprising contacting the cell with a lipid nanoparticle composition of any one of claims 67-84. 88. The method of claim 87, wherein the cells are airway epithelial cells. 89. A method of delivering a therapeutic or prophylactic payload to a patient, the method comprising administering to the patient a lipid nanoparticle composition of any one of claims 67-84. 90. The method of claim 89, wherein the therapeutic or prophylactic payload is mRNA. 91. The method of claim 89 or 90, wherein the therapeutic or prophylactic payload is delivered to airway epithelial cells of the patient. 92. The method of any one of claims 89-91, wherein the lipid nanoparticle composition is administered by intranasal, intrabronchial, or pulmonary administration. 93. The method of any one of claims 89-91, wherein the lipid nanoparticle composition is administered via a nebulizer or an inhaler. 94. A method for preparing a lipid nanoparticle composition, the method comprising contacting a lipid nanoparticle core with a lipid amine compound or a salt thereof as described in any one of claims 1-66. 95. The method of claim 94, wherein the lipid nanoparticle core comprises: (i) ionizable lipids, (ii) phospholipids, (iii) structural lipids, and (iv) optionally PEG-lipid. 96. The method of claim 95, wherein the lipid nanoparticle core comprises: about 40 mol % to about 50 mol % ionizable lipid; about 10 mol % to about 12 mol % of phospholipids; about 37 mol % to about 42 mol % structural lipids; and About 0.25 mol % to about 0.75 mol % PEG-lipid; each relative to total lipids. 97. The method of claim 95, wherein the lipid nanoparticle core comprises: about 49 mol% ionizable lipids; about 11 mol % to about 12 mol % phospholipids; about 39 mol % structural lipids; and About 0.5 mol % PEG-lipid; each relative to total lipids. 98. The method of any one of claims 95-97, wherein the lipid nanoparticle core further comprises a payload encapsulated for delivery into a cell. 99. The method of any one of claims 94-98, wherein the lipid amine compound is present in a liquid composition. 100. The method of claim 99, wherein the liquid composition comprises a non-ionic excipient. 101. The method of claim 100, wherein the nonionic excipient is macrogol 15 hydroxystearate (HS 15). 102. A lipid nanoparticle composition, which is prepared by the method described in any one of claims 94-101.