EA048725B1 - ANTIBODY-DRUG CONJUGATES - Google Patents

Description

Link to the sequence listing provided in electronic form

The contents of the electronic sequence listing (name: 3817_053PC03_Seqlisting_ST25; size: 7104 bytes and creation date: April 22, 2020) are incorporated herein by reference in their entirety.

Field of technology

The present invention provides antibody-drug conjugates comprising STING modulators. Also provided are compositions comprising the antibody-drug conjugates. The compounds and compositions are useful for stimulating an immune response in a subject in need thereof.

State of the art

Antibody-drug conjugates (ADCs), a rapidly growing class of targeted therapeutics, represent a promising new approach to enhancing the selectivity and cytotoxic activity of drugs. These therapeutic agents consist of an antibody (or antibody fragment) that can be linked to a useful drug to form an immunoconjugate. The antibody directs the ADC to bind to the target cell. The ADC can then be taken up and released from the payload, which delivers treatment to the cell. Because the ADC is directed to its target cell, the side effects of conjugated drugs may be lower than those encountered with systemic administration of the same agent.

The adaptor protein STING (Stimulator of Interferon Genes) has been shown to play an important role in the innate immune system. Activation of the STING pathway triggers an immune response that results in the production of specific killer T cells that shrink tumors and may provide long-lasting immunity to prevent tumors from recurring. Activated STING pathway also promotes antiviral response by producing antiviral and proinflammatory cytokines that fight the virus and mobilize both the innate and adaptive immune systems, ultimately leading to long-lasting immunity against the pathogenic virus. The potential therapeutic benefits of enhancing both the innate and adaptive immune responses make STING an attractive target for drug discovery. Cyclic dinucleotides can act as STING agonists and are in clinical trials. However, their anionic properties make them poorly membrane permeable, which may limit their ability to interact with STING inside the cell, often resulting in unwanted distribution of these compounds into the bloodstream.

There remains a need for new STING agonists and improved methods for their delivery to the target cell.

The essence of the invention

In a first aspect, the present invention provides a compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

b is an integer between 1 and 20;

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

n is 0 or 1;

D-NH- is part of an amino-substituted compound, where the amino-substituted compound has the formula D-NH2;

each R ¹ is independently selected from C ₁ -C ₄ -alkyl, O-C ₁ -C ₄ -alkyl and halogen;

R ² is selected from C ₁ -C ₄ -alkyl and -(CH ₂ CH ₂ O) _s -CH ₃ ; where s is an integer from 1 to 10;

R ³ and R ³ are each independently selected from hydrogen and C ₁ -C 3 alkyl;

L is a cleavable linker; and

Ab is an antibody, antibody fragment, or antigen-binding fragment.

In a first embodiment of the first aspect, a is an integer from 1 to 4; b is an integer from 1 to 10; and m is 0.

In a second embodiment of the first aspect, a is an integer from 1 to 4; m is 0; n is 0; and each of R ³ and R ³ is hydrogen.

In the third embodiment of the first aspect, L is

- 1 048725

where r represents the point of attachment to the nitrogen atom;

* represents the point of attachment to Ab;

t is an integer between 1 and 10;

W is absent or represents a self-cleaving group;

Z is absent or is a peptide of 2-5 amino acids;

U and U' are independently absent or a spacer; and

Q is a heterobifunctional group;

provided that W and Z are not absent simultaneously.

In a fourth embodiment of the first aspect, W is a self-splitting group selected from where

In a sixth embodiment of the first aspect, W is a point of attachment to a carbonyl group; and / is a point of attachment to Z.

In the fifth embodiment of the first aspect, W is selected from

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In a seventh embodiment of the first aspect, Z is a peptide capable of enzymatic cleavage. In an eighth embodiment, Z is cleaved by cathepsin. In a ninth embodiment, Z is a two-amino acid peptide selected from Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys, and Lys-Phe. In a tenth embodiment of the first aspect, Z is Val-Ala or Ala-Val.

In the eleventh embodiment of the first aspect, U and U' are independently absent or selected from

where ³¹ represents the attachment point to Z;

* represents the point of attachment to Q;

p is an integer from 1 to 6;

q is an integer between 1 and 20;

X is O or -CH2-; and each r is independently 0 or 1.

In the twelfth embodiment of the first aspect, U' is absent, and U

represents

In a thirteenth embodiment of the first aspect, Q is a heterobifunctional group that is capable of being attached to Ab via chemical or enzyme-mediated conjugation. In a fourteenth embodiment, Q is selected from

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where A represents the point of attachment to U or, when U is absent, the point of attachment to Z;

\ and * represents the attachment point to U' or, when U' is absent, the attachment point to Ab.

In a fifteenth embodiment of the first aspect, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein L is:

and where t is 1; W is absent or is a self-cleaving group; and Z is absent or is a 2-amino acid peptide.

In a sixteenth embodiment of the first aspect, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R ² is -CH3 or (CH ₂ CH ₂ O) _S -CH3, and s is an integer from 1 to 10.

In a seventeenth embodiment of the first aspect, the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D-NH2 is an amino-substituted compound that modulates the activity of STING. In an eighteenth embodiment of the first aspect, the amino-substituted compound that modulates the activity of STING comprises a guanine or adenine derivative.

In a nineteenth embodiment, the amino-substituted compound that modulates the activity of STING is a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

X ¹⁰ represents SH or OH;

X ²⁰ represents SH or OH;

Y ^a represents O, S or CH2;

Y ^b is O, S, NH or NR ^a , wherein R ^a is C1-C ₄ alkyl;

R ¹⁰ represents hydrogen, fluorine, OH, NH2, ORb or NHRb;

R ²⁰ represents hydrogen or fluorine;

R ³⁰ represents hydrogen; R ⁴⁰ represents hydrogen, fluorine, OH, NH2, ORb or NHRb; or R ³⁰ and R ⁴⁰ taken together to form CH ₂ O;

R ⁵⁰ represents hydrogen or fluorine;

Rb is C1-C6 alkyl, halo(C1-C6)alkyl or _C3 -C6 cycloalkyl;

ring A is an optionally substituted 5- or 6-membered monocyclic heteroaryl ring containing 1-4 heteroatoms selected from N,

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O or S, or an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 1-5 heteroatoms selected from N, O, or S; wherein ring A ¹⁰ contains at least one N atom in the ring, and wherein Y ^b is attached to a carbon atom of ring A ¹⁰ ; and ring B ¹⁰ is an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 2-5 heteroatoms selected from N, O, or S; wherein ring B contains at least two N atoms in the ring;

provided that ring A ¹⁰ or ring B ¹⁰ is attached to the carbonyl group of formula (I) via the -NH- group.

In a twentieth embodiment, the amino-substituted compound that modulates STING activity is

or a pharmaceutically acceptable salt thereof, wherein A represents the point of attachment to the carbonyl group of formula (I).

In a twenty-first embodiment of the first aspect, the amino-substituted compound that modulates the activity of STING is a compound of formula (III):

or a pharmaceutically acceptable salt thereof, where

X ¹⁰ represents SH or OH;

X ²⁰ represents SH or OH;

Y ^c represents O, S or CH ₂ ;

Y ^d represents O, S or CH ₂ ;

B ¹⁰⁰ is a group represented by the formula (B1-A) or the formula (B ¹ -B):

_R 1000

B-A B ¹ -B

R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ and R ¹⁷ each independently represent a hydrogen atom or a substituent;

R ¹⁰⁰⁰ represents hydrogen or a bond to the carbonyl group of formula (I);

Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ and Y ¹⁶ each independently represent N or CR ^1a , wherein R ^1a represents hydrogen or a substituent;

Z ¹¹ , Z ¹² , Z ¹³ , Z ¹⁴ , Z ¹⁵ and Z ¹⁶ each independently represent N or C;

R ¹⁰⁵ represents a hydrogen atom or a substituent;

B is a group represented by the formula (B ² -A) or the formula (B ² -B):

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R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ each independently represent a hydrogen atom or a substituent;

R ¹⁰⁰ represents hydrogen or a bond to the carbonyl group of formula (I);

Y ²¹ , Y ²² , Y ²³ , Y ²⁴ , Y ²⁵ and Y ²⁶ each independently represent N or CR ^2a , wherein R ^2a represents hydrogen or a substituent;

Z ²¹ , Z ²² , Z ²³ , Z ²⁴ , Z ²⁵ and Z ²⁶ each independently represent N or C; and

R ²⁰⁵ represents a hydrogen atom or a substituent; wherein R ¹⁰⁵ and R ²⁰⁵ are each independently attached to the 2- or 3-position of the 5-membered ring to which they are attached, respectively;

provided that:

one of B ¹⁰⁰ or B ²⁰⁰ is attached to the carbonyl group of formula (I) via an -NH- group.

In a twenty-second embodiment of the first aspect, the amino-substituted compound that modulates the activity of STING is a compound of formula (IIIa),

Oh, SI I

or a pharmaceutically acceptable salt thereof, where

B is a group represented by the formula (B1-A) or the formula (B1-B):

^1000

About HN^

B-A B ¹ -B

R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ and R ¹⁷ each independently represent a hydrogen atom or a substituent;

R ¹⁰⁰⁰ represents hydrogen or a bond to the carbonyl group of formula (I);

Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ and Y ¹⁶ each independently represent N or CR ^1a , wherein R ^1a represents hydrogen or a substituent;

Z ¹¹ , Z ¹² , Z ¹³ , Z ¹⁴ , Z ¹⁵ and Z ¹⁶ each independently represent N or C;

R ¹⁰⁵ represents a hydrogen atom or a substituent;

B is a group represented by the formula (B2-A) or the formula ( ^B2 -B):

R™'

About NH

R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ each independently represent a hydrogen atom or a substituent;

R ¹⁰⁰ represents hydrogen or a bond to the carbonyl group of formula (I);

Y ²¹ , Y ²² , Y ²³ , Y ²⁴ , Y ²⁵ and Y ²⁶ each independently represent N or CR ^2a , wherein R ^2a represents hydrogen or a substituent;

Z ²¹ , Z ²² , Z ²³ , Z ²⁴ , Z ²⁵ and Z ²⁶ each independently represent N or C; and

R ²⁰⁵ represents a hydrogen atom or a substituent; where R ¹⁰⁵ and R ²⁰⁵ are each independently attached

- 6 048725 diene to the 2- or 3-position of the 5-membered ring to which they are attached, respectively;

provided that:

one of B ¹⁰⁰ or B ²⁰⁰ represents:

where R ¹⁸ is hydrogen or C^alkyl; and

R ¹⁹ is a halogen atom;

and the other is attached to the carbonyl group of formula (I) through the -NH- group.

In a twenty-third embodiment of the first aspect, the amino-substituted compound that modulates the activity of STING is:

or a pharmaceutically acceptable salt thereof, a bonyl group of formula (I).

where + represents the point of attachment to carIn a second aspect, the present invention provides a compound of formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

b is an integer between 1 and 20;

k is 0, 1, 2, or 3;

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

D-NH- is part of an amino-substituted compound, where the amino-substituted compound has the formula D-NH2;

R ² is selected from H, C1-C ₄ -alkyl and -(CH ₂ CH ₂ O) _S -CH ₃ ; where s is an integer from 1 to 10;

R ² is selected from hydrogen and any side chain of a naturally occurring amino acid;

R ⁵ is selected from C ₁ -C ₄ -alkyl and O-C ₁ -C ₄ -alkyl;

L is a cleavable linker; and

Ab is an antibody, antibody fragment, or antigen-binding fragment.

In a first embodiment of the second aspect, L is where + is the point of attachment to the carbonyl group;

* represents the point of attachment to Ab;

W is a self-cleaving group;

Z is absent or is a peptide of 2-5 amino acids; and

U and U' are independently absent or a spacer; and

Q is a heterobifunctional group.

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In a third aspect of the present invention there is provided a compound of formula (V):

D-NH- is

or a pharmaceutically acceptable salt thereof, wherein:

X represents O or CH2;

f is an integer between 1 and 10;

g is an integer between 1 and 20;

U and U' are independently absent or represent a spacer;

Q is a heterobifunctional group; and

Ab is an antibody, antibody fragment, or antigen-binding fragment.

In the first embodiment of the third aspect, X represents O.

In a fourth aspect of the present invention there is provided a compound of formula (VI):

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

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

n is 0 or 1;

D-NH- is part of an amino-substituted compound, where the amino-substituted compound has the formula D-NH ₂ ;

each R ¹ is independently selected from C1-C ₄ -alkyl, O-C1-C ₄ -alkyl and halogen;

R ² is selected from C1-C ₄ -alkyl and -(CH ₂ CH ₂ O) _S -CH ₃ ; where s is an integer from 1 to 10;

R ³ and R ³ are each independently selected from hydrogen and C1-C ₃ -alkyl;

L is a cleavable linker; and

LP is a lipid.

In a first embodiment of the fourth aspect, the lipid is cholesterol or phospholipid. In a second embodiment of the fourth aspect, the lipid is a phospholipid. In a third embodiment of the fourth aspect, the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipid and egg yolk phospholipid. In a fifth embodiment of the fourth aspect, the phospholipid is phosphatidylethanolamine, wherein phosphatidylethanolamine is 1,2-distearoyl-n-glycero-3-phosphorylethanolamine.

In a sixth embodiment of the fourth aspect, the present invention relates to a lipid complex comprising a compound of formula (VI) or a pharmaceutically acceptable salt thereof. In the seventh

- 8 048725 In this embodiment, the lipid complex is in the form of a liposome.

In an eighth embodiment of the fourth aspect, the lipid complex further comprises one or more phospholipids. In a ninth embodiment, the one or more phospholipids are selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipid and egg yolk phospholipid. In a tenth embodiment, the phospholipid is phosphatidylcholine, wherein the phosphatidylcholine is 1,2-distearoyl-sn-glycero-3-phosphocholine.

In an eleventh embodiment of the fourth aspect, the lipid complex additionally contains fatty alcohol and/or cholesterol.

In a twelfth embodiment of the fourth aspect, the lipid complex further comprises at least one PEGylated lipid. In a thirteenth embodiment, the PEGylated lipid is selected from a PEGylated phospholipid and a PEGylated diacylglycerol. In a fourteenth embodiment, the PEGylated lipid is N(carbonylmethoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine.

In a fifth aspect, the present invention provides a pharmaceutical composition comprising a compound or complex described herein, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

In a sixth aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable amount of a compound or complex described herein.

In a seventh aspect, the present invention provides a method of stimulating an immune response in a subject in need thereof, the method comprising administering to the subject a pharmaceutically acceptable amount of a compound or complex described herein.

Brief description of graphic materials

Fig. 1 shows the preparation of Ab-STING agonist conjugates via stochastic cysteine conjugation.

Fig. 2 shows the preparation of Ab-STING agonist conjugates via transglutaminase conjugation.

Fig. 3 shows the preparation of Ab-STING agonist conjugates via transglutaminase conjugation.

Fig. 4 shows the preparation of Ab-STING agonist conjugates via sortase conjugation (SEQ ID3 and SEQ ID4)

Figure 5 shows the release of ADC-1 payload in a tritosome assay.

Figure 6 shows the release of ADC-3 payload in a tritosome assay.

Figure 7 shows the release of ADC-9 payload in a tritosome assay.

Fig. 8 shows the plasma PK of ADC-1 in GCC expressing CT26 bearing Balb/C mice (payload dose 0.1 mg/kg).

Fig. 9 shows the plasma PK of ADC-8 in GCC expressing CT26 bearing Balb/C mice (0.055 mg/kg payload dose, BLQ CDN released).

Fig. 10 shows the plasma PK of ADC-9 in GCC expressing CT26 bearing Balb/C mice (0.05 mg/kg payload dose, BLQ CDN released).

Fig. 11 shows the efficacy of ADC-1 in GCC expressing CT26 bearing Balb/C mice (useful doses are shown).

Fig. 12 shows the efficacy of ADC-5 in GCC expressing CT26 bearing Balb/C mice (useful doses are shown).

Fig. 13 shows the efficacy of ADC-8 in GCC expressing CT26 bearing Balb/C mice (useful doses are shown).

Fig. 14 shows the efficacy of ADC-9 in GCC expressing CT26 bearing Balb/C mice (useful doses are shown).

Detailed description

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 this invention pertains. All patents and publications mentioned in this document are incorporated by reference in their entirety.

Singular forms include plural references unless explicitly stated otherwise.

As used in this document, the term or is a logical distinction (i.e., and/or) and does not indicate an exclusive distinction unless expressly stated otherwise, such as by the terms or, except, alternatively, and words of similar effect.

As used in this document, the term approximately refers to ±10%.

Antibody-drug conjugates.

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In some embodiments of the present invention, a compound of formula (I) is provided,

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

b is an integer between 1 and 20;

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

n is 0 or 1;

D-NH- is part of an amino-substituted compound, where the amino-substituted compound has the formula D-NH2;

each R ¹ is independently selected from C ₁ -C ₄ -alkyl, O-C ₁ -C ₄ -alkyl and halogen;

R ² is selected from C1-C ₄ -alkyl and -(CH ₂ CH ₂ O) _s -CH ₃ ; where s is an integer from 1 to 10;

R and R ³ are each independently selected from hydrogen and C ₁ -C ₃ -alkyl;

L is a cleavable linker; and

Ab is an antibody, antibody fragment, or antigen-binding fragment.

In some embodiments of the present invention, a compound of formula (IV) is provided,

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

b is an integer between 1 and 20;

k is 0, 1,2 or 3;

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

D-NH- is part of an amino-substituted compound, where the amino-substituted compound has the formula D-NH ₂ ;

R ² is selected from H, C1-C ₄ -alkyl and -(CH ₂ CH ₂ O) _s -CH ₃ ; where s is an integer from 1 to 10;

R ⁴ is selected from hydrogen and any side chain of a naturally occurring amino acid;

R ⁵ is selected from C1-C ₄ -alkyl and O-C ₁ -C ₄ -alkyl;

L is a cleavable linker; and

Ab is an antibody, antibody fragment, or antigen-binding fragment.

In some embodiments of the present invention, a compound of formula (V) is provided:

or a pharmaceutically acceptable salt thereof, wherein:

D-NH is

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X is O or _CH2 ;

f is an integer between 1 and 10;

g is an integer between 1 and 20;

U and U' are independently absent or represent a spacer;

Q is a heterobifunctional group; and

Ab is an antibody, antibody fragment, or antigen-binding fragment.

In the compounds of formula (I) and (IV), the amino-substituted compound D-NH2 may be any amino-containing drug. In some embodiments, D- _NH2 is an immunomodulator. As used herein, the term immunomodulator refers to a compound that elicits an immune response in a subject in need thereof. Examples of immunomodulators include, but are not limited to, IDO inhibitors, MEK inhibitors, modulators

STING, CCR4 antagonists, PD-1/PD-L1 inhibitors, TLR7/8 agonists, etc. In some embodiments, D-NH2 is a STING modulator. In some embodiments, D-NH2 is a guanine or adenine derivative. In some embodiments, the STING modulator is a cyclic dinucleotide or a cyclic dinucleotide-like compound (each, CDN) of the formula:

wherein A' and A are each independently nucleosides or synthetic analogs thereof; and each of Q, Q', Q ² and Q ² is independently oxygen or sulfur. In some embodiments, Q' and Q ² are sulfur. Examples of CDNs, including cyclic dinucleotides and cyclic dinucleotide-like compounds, that are within the scope of this invention include, but are not limited to, those described in US 9724408, US 10106574, US 9549944, US 9695212, US 9718848, US 9994607, US 10047115, US 20180230178, US 20180064745, US 2018028132, US 20180002369, US 20180186828, US 20190016750, US 20180162899, US 20180369268, US 2018273578, US 20180092937, WO 2017/027646 and WO 2018/100558; as well as compounds such as c-di-GMP, 2'3'-cGAMP and SB 11285 (Spring Bank Pharmaceuticals).

In some embodiments, the STING modulator is a compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

X ¹⁰ represents -SH or -OH;

X ²⁰ represents -SH or -OH;

Ya represents -O-, -S-, or _-CH2- ;

Y ^b is -O-, -S-, -NH- or -NRa-, where Ra is C1- _C4 -alkyl;

R ¹⁰ represents hydrogen, fluorine, -OH, -NH ₂ , -OR ^b or -NHR ^b ;

R ²⁰ represents hydrogen or fluorine;

R ³⁰ represents hydrogen; R ⁴⁰ represents hydrogen, fluorine, -OH, -NH2, -OR ^b or -NHR ^b ; or R ³⁰ and R ⁴⁰ taken together form -CH2O-;

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R ⁵⁰ represents hydrogen or fluorine;

R ^b is C1-C ₆ alkyl, halo(C1-C ₆ )alkyl, or C3-C6 cycloalkyl;

ring A ¹⁰ is an optionally substituted 5- or 6-membered monocyclic heteroaryl ring containing 1-4 heteroatoms selected from N, O or S, or an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 1-5 heteroatoms selected from N, O or S; wherein ring A ¹⁰ contains at least one N atom in the ring, and wherein Y is attached to a carbon atom of ring A ¹⁰ ; and ring B ¹⁰ is an optionally substituted 9- or 10-membered bicyclic heteroaryl ring containing 2-5 heteroatoms selected from N, O or S; wherein ring B ¹⁰ contains at least two N atoms in the ring.

As described herein, the A ¹⁰ ring and the B ¹⁰ ring may contain one or more substituents and thus may be optionally substituted. Suitable substituents on the unsaturated carbon atom of the heteroaryl group include and are typically selected from halogen,

-NO ₂ , -CN, -R ⁺ , -C(R ⁺ )=C(R ⁺ ) ₂ , -OC-R ⁺ ,

- OR ⁺ , -SR°, -S(O)R°, -SO2R°, -SO3R ⁺ , -SO2N(R ⁺ )2, -N(R ⁺ )2, -NR ⁺ C(O)R ⁺ , -NR ⁺ C(S)R ⁺ , -NR ⁺ C(O)N(R ⁺ )2, -NR ⁺ C(S)N(R ⁺ )2, -N(R ⁺ )C(=NR ⁺ )-N(R ⁺ )2, -N(R ⁺ )C(=NR ⁺ )-R°, -nr ⁺ co2r ⁺ , -NR ⁺ SO2R ^o , -NR ⁺ SO2N(R ⁺ )2, -OC(O)R ⁺ , -O-CO2R ⁺ , -OC(O)N(R ⁺ )2, -C(O)R ⁺ , -C(S)R°, -CO2R ⁺ , -C(O)-C(O)R ⁺ , -C(O)N(R ⁺ )2, -C(S)N(R ⁺ )2. -C(O)N(R ⁺ )-OR ⁺ ,

- C(O)N(R ⁺ )C(=NR ⁺ )-N(R ⁺ )2, -N(R ⁺ )C(=NR ⁺ )-N(R ⁺ )-C(O)R ⁺ , -C(=NR ⁺ )-N(R ⁺ )2,

- C(=NR ⁺ )-OR ⁺ , -N(R ⁺ )-N(R ⁺ )2, -C(=NR ⁺ )-N(R ⁺ )-OR ⁺ , -C(R°)=N-OR ⁺ , -P(O)(R ⁺ )2, -P(O)(OR ⁺ )2, -OP(O)-OR ⁺ , and -P(O)(NR ⁺ )-N(R ⁺ )2, where R ⁺ is independently hydrogen or an optionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic or heterocyclyl group, or two independent occurrences of R ⁺ are taken together with their intervening atom(s) to form an optionally substituted 5- to 7-membered aryl, heteroaryl, cycloaliphatic or heterocyclyl group. In some embodiments, R is independently hydrogen, a C1.6 aliphatic group, or a C3-6 cycloaliphatic group. Each R° is independently an optionally substituted aliphatic, aryl, heteroaryl, cycloaliphatic, or heterocyclyl group.

As described in detail above, in some embodiments, two independent occurrences of R ⁺ (or any other variable similarly defined in the specification and claims) are taken together with their intervening atom(s) to form a monocyclic or bicyclic ring selected from a 3-13-membered cycloaliphatic ring, a 3-12-membered heterocyclyl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 6-10-membered aryl, or a 5-10-membered heteroaryl having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In some embodiments, the STING modulator is a compound of formula (IIA):

or a pharmaceutically acceptable salt thereof, wherein each of R ¹⁰ and R ⁴⁰ independently represents hydrogen, fluorine, -OH or -OCH2CF3 and rings A ¹⁰ and B ¹⁰ have the meanings specified for the compound of formula (II), provided that ring A ¹⁰ or ring B ¹⁰ is attached to the carbonyl group of the moiety of the starting molecule of compound (I), (IV) or (V) via the -NH- group.

In some embodiments, ring A ¹⁰ is an optionally substituted 6-membered monocyclic heteroaryl ring containing 1, 2, or 3 nitrogen atoms.

In some embodiments, ring B is:

where Z ¹⁰ , Z ²⁰ , Z ³⁰ and Z ⁴⁰ each independently represent N or CR ²⁰⁰ ; R ²¹⁰ represents hydrogen or C1-C ₆ -alkyl, halo(C1-C ₆ )alkyl or C ₃ -C ₆ cycloalkyl;

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R ²³⁰ represents hydrogen or -NH2; and

R ²⁰⁰ , R ²²⁰ , and R ²⁴⁰ each independently represent hydrogen, halogen, -OH, -NH ₂ , -CN, C1-C6 alkyl, halo(C1-C6)alkyl, or C _3- C6 cycloalkyl.

In some embodiments, the STING modulator is:

or a pharmaceutically acceptable salt thereof, where represents the point of attachment to the carbonyl group of the original molecular fragment.

In some embodiments, the STING modulator is a cyclic dinucleotide of formula (X):

or a pharmaceutically acceptable salt thereof, wherein: a partial structure represented by formula (A-1):

is a partial structure represented by the formula (Z-A) or the formula (Z-B):

R ¹⁰⁵ and R ²⁰⁵ each independently represent a hydroxy group or a halogen atom;

B ¹⁰⁰ is a group represented by the formula (B1-A) or the formula (B ¹ -B):

R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ and R ¹⁷ each independently represent a hydrogen atom or a substituent;

Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ and Y ¹⁶ each independently represent N or CR ^1a ;

Z ¹¹ , Z ¹² , Z ¹³ , Z ¹⁴ , Z ¹⁵ and Z ¹⁶ each independently represent N or C;

R ^1a represents a hydrogen atom or a substituent;

B ²⁰⁰ is a group represented by the formula (B ² -A) or the formula (B ² -B):

- 13 048725

R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ each independently represent a hydrogen atom or a substituent;

Y ²¹ , Y ²² , Y ²³ , Y ²⁴ , Y ²⁵ and Y ²⁶ each independently represent N or CR ^2a ;

Z ²¹ , Z ²² , Z ²³ , Z ²⁴ , Z ²⁵ and Z ²⁶ each independently represent N or C;

R ^2a represents a hydrogen atom or a substituent;

X ¹ and X ² each independently represent an oxygen atom or a sulfur atom; and

Q ^1a , Q ^2a , Q ^3a and Q ^4a each independently represents an oxygen atom or a sulfur atom; and one of B ¹⁰⁰ or B ²⁰⁰ is attached to the 5-membered ring of the parent structure through an -NH- group.

In another embodiment, one of B ¹⁰⁰ or B ²⁰⁰ is:

p1E O + G where R ¹⁸ is hydrogen or ^^alkyl; and the other is attached to the 5-membered ring of the parent structure through an -NH- group; and

R ¹⁹ is a halogen atom; and the other is attached to the carbonyl group of formula (I) through the -NH- group.

In another aspect, the present invention provides an antibody-drug conjugate as described herein.

As described herein, certain R groups are selected from hydrogen and a substituent. Examples of a substituent include a halogen atom, a cyano group, a nitro group, an optionally substituted hydrocarbon group, an optionally substituted heterocyclic group, an acyl group, an optionally substituted amino group, an optionally substituted carbamoyl group, an optionally substituted thiocarbamoyl group, an optionally substituted sulfamoyl group, an optionally substituted hydroxy group, an optionally substituted sulfanyl (SH) group, and an optionally substituted silyl group.

In the present specification, examples of the hydrocarbon group (including the hydrocarbon group of the optionally substituted hydrocarbon group) include a _C2-6 alkyl group, a C2-6 alkenyl group, a _C2-6 alkynyl group, _{a C3-10} cycloalkyl group, _{a C3-10 cycloalkenyl} group, a _C6-14 aryl group _, and _{a C7-16 aralkyl} group.

In this specification, examples of an optionally substituted hydrocarbon group include a hydrocarbon group optionally having a substituent(s) selected from the following substituent group A, which includes:

(1) a halogen atom, (2) a nitro group, (3) a cyano group, (4) an oxo group, (5) a hydroxy group, (6) an optionally halogenated N-alkyloxy group, (7) a C ₆ . ₁₄ aryloxy group (e.g. phenoxy, naphthoxy), (8) a C ₇ . ₁₆ aralkyloxy group (e.g. benzyloxy), (9) a 5- to 14-membered aromatic heterocyclyloxy group (e.g. pyridyloxy), (10) a 3- to 14-membered non-aromatic heterocyclyloxy group (e.g. morpholinyloxy, piperidinyloxy), (11) a N-alkylcarbonyloxy group (e.g. acetoxy, propanoyloxy), (12) C ₆ . ₁₄ arylcarbonyloxy group (e.g. benzoyloxy, 1-naphthoyloxy, 2-naphthoyloxy), (13) C 6 . 14 -arylcarbamoyloxy group (e.g. methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy), (14) mono- or di-C _{6 . 14 -alkylcarbamoyloxy group (e.g. methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy, diethylcarbamoyloxy), (15) C 6} . ₁₄ -arylcarbamoyloxy group (e.g. phenylcarbamoyloxy, naphthylcarbamoyloxy), (16) 5- to 14-membered aromatic heterocyclylcarbonyloxy group (e.g. nicotinoyloxy),

- 14 048725 (17) a 3- to 14-membered non-aromatic heterocyclylcarbonyloxy group (e.g. morpholinylcarbonyloxy, piperidinylcarbonyloxy), (18) an optionally halogenated C _1-6 alkylsulfonyloxy group (e.g. methylsulfonyloxy, trifluoromethylsulfonyloxy), (19) a C _6- 1 ₄ arylsulfonyloxy group optionally substituted by a _C1-6 alkyl group (e.g. phenylsulfonyloxy, toluenesulfonyloxy), (20) an optionally halogenated _C1-6 alkylthio group, (21) a 5- to 14-membered aromatic heterocyclic group, (22) a 3- to 14-membered non-aromatic heterocyclic group, (23) a formyl group, (24) a carboxy group, (25) optionally a halogenated _C1-6 alkyl carbonyl group, (26) a C _{6- 1-4} aryl carbonyl group, (27) a 5- to 14-membered aromatic heterocyclyl carbonyl group, (28) a 3- to 14-membered non-aromatic heterocyclyl carbonyl group, (29) a _C1-6 alkoxy carbonyl group, (30) a C _{6- 1-4} aryloxycarbonyl group (e.g. phenyloxycarbonyl, 1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl), (31) a C _{7- 1-6} aralkyloxycarbonyl group (e.g. benzyloxycarbonyl, phenethyloxycarbonyl), (32) a carbamoyl group, (33) a thiocarbamoyl group, (34) a mono- or di- _C1-6 alkylcarbamoyl group, (35) C _6- 1 ₄ arylcarbamoyl group (e.g. phenylcarbamoyl), (36) 5- to 14-membered aromatic heterocyclylcarbamoyl group (e.g. pyridylcarbamoyl, thienylcarbamoyl), (37) 3- to 14-membered non-aromatic heterocyclylcarbamoyl group (e.g. morpholinylcarbamoyl, piperidinylcarbamoyl), (38) optionally halogenated _C1-6 alkylsulfonyl group, (39) C _6- 1 ₄ arylsulfonyl group, (40) 5- to 14-membered aromatic heterocyclylsulfonyl group (e.g. pyridylsulfonyl, thienylsulfonyl), (41) optionally halogenated _C1-6 alkylsulfinyl group, (42) C _6- 1 ₄ arylsulfinyl group (e.g. phenylsulfinyl, 1-naphthylsulfinyl, 2-naphthylsulfinyl), (43) 5- to 14-membered aromatic heterocyclylsulfinyl group (e.g. pyridylsulfinyl, thienylsulfinyl), (44) amino group, (45) mono- or di-C _1-6 alkylamino group (e.g. methylamino, ethylamino, propylamino, isopropylamino, butylamino, dimethylamino, diethylamino, dipropylamino, dibutylamino, N-ethyl-K-methylamino), (46) mono- or di-C _6-14 arylamino group (e.g. phenylamino), (47) 5- to 14-membered aromatic heterocyclylamino group (e.g. pyridylamino), (48) C _7- 1 ₆ aralkylamino group (e.g. benzylamino), (49) formylamino, (50) _C1-6 alkylcarbonylamino (e.g. acetylamino, propanoylamino, butanoylamino), (51) (C _1-6 alkyl) (C _1-6 alkyl-arbonyl) amino (e.g. N-acetyl-N-methylamino), (52) C _6-14 aryl-carbonylamino (e.g. phenylcarbonylamino, naphthylcarbonylamino), (53) C _1-6 alkoxy-carbonylamino (e.g. methoxycarbonylamino, ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino, tert-butoxycarbonylamino), (54) C _7-16 aralkyloxy-carbonylamino (e.g. benzyloxycarbonylamino), (55) C _1-6 alkylsulfonylamino group (e.g. methylsulfonylamino, ethylsulfonylamino), (56) C _6-14 arylsulfonylamino group optionally substituted with a C _1-6 alkyl group (e.g. phenylsulfonylamino, toluenesulfonylamino), (57) optionally halogenated C _1-6 alkyl group, (58) C _2-6 alkenyl group, (59) C _2-6 alkynyl group, (60) C _3-10 cycloalkyl group, (61) C _3-10 cycloalkenyl group, (62) C _6-14 aryl group.

The number of the above-mentioned substituents in the optionally substituted hydrocarbon group is, for example, from 1 to 5, usually from 1 to 3. When the number of substituents is two or more,

- 15 048725 the corresponding substituents may be the same or different.

In the present specification, examples of the heterocyclic group (including the heterocyclic group of the optionally substituted heterocyclic group) include (i) an aromatic heterocyclic group, (ii) a non-aromatic heterocyclic group, and (iii) a 7- to 10-membered bridged heterocyclic group, each of which contains, as a ring-forming atom other than a carbon atom, 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom, and an oxygen atom.

In the present specification, examples of the aromatic heterocyclic group (including a 5- to 14-membered aromatic heterocyclic group) include a 5- to 14-membered (usually 5- to 10-membered) aromatic heterocyclic group containing, as a ring-forming atom other than a carbon atom, from 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.

Suitable examples of the aromatic heterocyclic group include 5- or 6-membered monocyclic aromatic heterocyclic groups such as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl and the like; and also 8- to 14-membered fused polycyclic (usually bi- or tricyclic) aromatic heterocyclic groups such as benzothiophenyl, benzofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl, imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl, pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazolopyrimidinyl, pyrazolotriazinyl, naphtho-EDthienyl, phenoxathiinyl, indolyl, isoindolyl, β-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl and the like.

In the present specification, examples of the non-aromatic heterocyclic group (including a 3- to 14-membered non-aromatic heterocyclic group) include a 3- to 14-membered (usually 4- to 10-membered) non-aromatic heterocyclic group containing, as a ring-forming atom, in addition to a carbon atom, from 1 to 4 heteroatoms selected from a nitrogen atom, a sulfur atom and an oxygen atom.

Suitable examples of the non-aromatic heterocyclic group include 3- to 8-membered monocyclic non-aromatic heterocyclic groups such as aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl, tetrahydroisooxazolyl piperidinyl, piperazinyl, tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, azepanyl, diazepanyl, azepinyl, oxepanil, azocanil, diazocanil and the like; and also 9- to 14-membered fused polycyclic (usually bi- or tricyclic) non-aromatic heterocyclic groups such as dihydrobenzofuranyl, dihydrobenzimidazolyl, dihydrobenzoxazolyl, dihydrobenzothiazolyl, dihydrobenzisothiazolyl, dihydronaphtho[2,3-b]thienyl, tetrahydroisoquinolyl, tetrahydroquinolyl, 4H-quinolizinyl, indolinyl, isoindolinyl, tetrahydrothieno[2,3-c]pyridinyl, tetrahydrobenzazepinyl, tetrahydroquinoxalinyl, tetrahydrophenanthridinyl, hexahydrophenothiazinyl, hexahydrophenoxazinyl, tetrahydrophthalazinyl, tetrahydronaphthyridinyl, tetrahydroquinazolinyl, tetrahydrocinnolinyl, tetrahydrocarbazolyl, tetrahydro-b-carbolinyl, tetrahydroacridinyl, tetrahydrophenazinyl, tetrahydrothioxanthenyl, octahydroisoquinolyl, and the like.

In this specification, suitable examples of the 7- to 10-membered bridged heterocyclic group include quinuclidinyl and 7-azabicyclo[2.2.1]heptanyl.

In the present specification, examples of the nitrogen-containing heterocyclic group include a heterocyclic group containing at least one nitrogen atom as a ring-forming atom.

In this specification, examples of the optionally substituted heterocyclic group include a heterocyclic group optionally having a substituent(s) selected from the above-mentioned Substituent Group A.

The number of substituents in an optionally substituted heterocyclic group is, for example, from 1 to 3. When the number of substituents is two or more, the respective substituents may be the same or different.

In the present specification, examples of the acyl group include a formyl group, a carboxy group, a carbamoyl group, a thiocarbamoyl group, a sulfino group, a sulfo group, a sulfamoyl group and a phosphono group, each of which optionally has 1 or 2 substituents selected from a _C16 alkyl group, a _C2-6 alkenyl group, a _C3-10 cycloalkyl group, a _C3-10 cycloalkenyl group, a _C6-14 aryl group, a _C7-16 aralkyl group, a 5-14-membered aromatic heterocyclic

- 16 048725 group and a 3-14 membered non-aromatic heterocyclic group, each of which optionally has from 1 to 3 substituents selected from a halogen atom, an optionally halogenated C.'i ₆ alkoxy group, a hydroxy group, a nitro group, a cyano group, an amino group and a carbamoyl group.

Examples of the acyl group also include a hydrocarbon sulfonyl group, a heterocyclyl sulfonyl group, a hydrocarbon sulfinyl group, and a heterocyclyl sulfinyl group.

In this document, a hydrocarbon-sulfonyl group means a sulfonyl group bonded to a hydrocarbon group, a heterocyclylsulfonyl group means a sulfonyl group bonded to a heterocyclic group, a hydrocarbon-sulfinyl group means a sulfinyl group bonded to a hydrocarbon group, and a heterocyclylsulfinyl group means a sulfinyl group bonded to a heterocyclic group.

Suitable examples of the acyl group include a formyl group, a carboxyl group, a Cl _-6 alkylcarbonyl group, a _C2-6 alkenylcarbonyl group (e.g. crotonoyl), a _C3-10 cycloalkylcarbonyl group (e.g. cyclobutanecarbonyl, cyclopentanecarbonyl, cyclohexanecarbonyl, cycloheptanecarbonyl), a _C3-10 cycloalkenylcarbonyl group (e.g. 2-cyclohexenecarbonyl), a _C6-14 arylcarbonyl group, a _C7-16 aralkylcarbonyl group, a 5-14-membered aromatic heterocyclylcarbonyl group, a 3-14-membered non-aromatic heterocyclylcarbonyl group, a C2-alkoxycarbonyl group, a _C6-14 aryloxycarbonyl group (e.g. phenyloxycarbonyl, naphthyloxycarbonyl), a C _7-16 aralkyloxycarbonyl group (e.g. benzyloxycarbonyl, phenethyloxycarbonyl), a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group, a mono- or di-C2-6 alkenylcarbamoyl group (e.g. diallylcarbamoyl), a mono- or di-C3-10 cycloalkylcarbamoyl group (e.g. cyclopropylcarbamoyl), a mono- or di-C6-14 arylcarbamoyl group (e.g. phenylcarbamoyl), a mono- or di-C7-16 aralkylcarbamoyl group, a 5- to 14-membered aromatic heterocyclylcarbamoyl group (e.g. pyridylcarbamoyl), a thiocarbamoyl group, a mono- or di-C ^ alkylthiocarbamoyl group (e.g. methylthiocarbamoyl, N-ethyl-N-methylthiocarbamoyl), a mono- or di-C _2-6 alkenylthiocarbamoyl group (e.g. diallylthiocarbamoyl), a mono- or di-C _3-10 cycloalkylthiocarbamoyl group (e.g. cyclopropylthiocarbamoyl, cyclohexylthiocarbamoyl), a mono- or di-C _6-14 arylthiocarbamoyl group (e.g. phenylthiocarbamoyl), a mono- or di-C _7-16 aralkylthiocarbamoyl group (e.g. benzylthiocarbamoyl, phenethylthiocarbamoyl), a 5- to 14-membered aromatic heterocyclylthiocarbamoyl group (e.g. pyridylthiocarbamoyl), a sulfino group, a C 2-6 alkylsulfinyl group (e.g. methylsulfinyl, ethylsulfinyl), sulfo group, C ₁₆ alkylsulfonyl group, C _6-14 arylsulfonyl group, phosphono group and mono- or di-C _{1 6} alkylphosphono group (e.g. dimethylphosphono, diethylphosphono, diisopropylphosphono, dibutylphosphono).

In the present specification, examples of the optionally substituted amino group include an amino group optionally having 1 or 2 substituents selected from a C alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, a C _7-16 aralkyl group, a C alkylcarbonyl group, a C _6-14 arylcarbonyl group, a C _7-16 aralkyl carbonyl group, a 5-14-membered aromatic heterocyclylcarbonyl group, a 3-14-membered non-aromatic heterocyclylcarbonyl group, a C 2-6 alkoxycarbonyl group, a 5-14-membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group, a mono- or di-C _7-16 aralkylcarbamoyl group, a C _1-6 alkylsulfonyl group and a C _6-14 arylsulfonyl group, each of which optionally has from 1 to 3 substituents selected from Substituent Group A.

Suitable examples of the optionally substituted amino group include an amino group, a mono- or di- (optionally halogenated C _1- ^km) amino group (e.g. methylamino, trifluoromethylamino, dimethylamino, ethylamino, diethylamino, propylamino, dibutylamino), a mono- or di- _C2-6 alkenylamino group (e.g. diallylamino), a mono- or di- _C3-10 cycloalkylamino group (e.g. cyclopropylamino, cyclohexylamino), a mono- or di- _C6-14 arylamino group (e.g. phenylamino), a mono- or di- _C7-16 aralkylamino group (e.g. benzylamino, dibenzylamino), a mono- or di- (optionally halogenated C2-6 alkylcarbonylamino group (e.g. acetylamino, propionylamino), mono- or di-C _6-14 arylcarbonylamino (e.g. benzoylamino), mono- or di-C _7-16 aralkylcarbonylamino (e.g. benzylcarbonylamino), mono- or di-5-14-membered aromatic heterocyclylcarbonylamino (e.g. nicotinoylamino, isonicotinoylamino), mono- or di-3-14-membered non-aromatic heterocyclylcarbonylamino (e.g. piperidinylcarbonylamino), mono- or di-C 6-14 alkoxycarbonylamino (e.g. tert-butoxycarbonylamino), 5-14-membered aromatic heterocyclylamino (e.g. pyridylamino), carbamoylamino, (mono- or di-C 6-14 alkoxycarbonylamino) amino (e.g. methylcarbamoylamino), (mono- or di-C _7-16 aralkyl-carbamoyl) amino group (e.g. benzylcarbamoylamino), C _1-6 alkylsulfonylamino group (e.g. methylsulfonylamino, ethylsulfonylamino), C _6-14 arylsulfonylamino group (e.g. phenylsulfonylamino), (C alkyl)(C 6-14 alkyl-carbonyl) amino group (e.g. N-acetyl-N-methylamino) and (C alkyl)(C _6-14 aryl-carbonyl) amino group (e.g.

- 17 048725 mer, N-benzoyl-N-methylamino).

In the present specification, examples of the optionally substituted carbamoyl group include a carbamoyl group optionally having 1 or 2 substituents selected from a _C1-6 alkyl group, a _C2 -balkenyl group, a _C3-10 cycloalkyl group, a _C6-14 aryl group, a _C7-16 aralkyl group, a _C1-6 alkylcarbonyl group, a _C6-14 arylcarbonyl group, a _C7-16 aralkylcarbonyl group, a 5-14-membered aromatic heterocyclylcarbonyl group, a 3-14-membered non-aromatic heterocyclylcarbonyl group, a _C1-6 alkoxycarbonyl group, a 5-14-membered aromatic heterocyclic group, a carbamoyl group, a mono- or di- _C1-6 alkylcarbamoyl group, and a mono- or di- _{C7-1 6} aralkyl-carbamoyl groups, each of which optionally has from 1 to 3 substituents selected from substituent group A.

Suitable examples of the optionally substituted carbamoyl group include a carbamoyl group, a mono- or di- _C1-6 alkylcarbamoyl group, a mono- or di- _C2-6 alkenylcarbamoyl group (e.g. diallylcarbamoyl), a mono- or di- _C3-10 cycloalkylcarbamoyl group (e.g. cyclopropylcarbamoyl, cyclohexylcarbamoyl), a mono- or di- _C6-14 arylcarbamoyl group (e.g. phenylcarbamoyl), a mono- or di- _C7-16 aralkylcarbamoyl group, a mono- or di-C, ₆ alkylcarbonylcarbamoyl group (e.g. _{acetylcarbamoyl} , propionylcarbamoyl), a mono- or di- _C6-14 arylcarbonylcarbamoyl group (e.g. benzoylcarbamoyl) and a 5- to 14-membered aromatic heterocyclylcarbamoyl group (e.g. pyridylcarbamoyl).

In the present specification, examples of the optionally substituted thiocarbamoyl group include a thiocarbamoyl group optionally having 1 or 2 substituents selected from a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, a C _7-16 aralkyl group, a C _1-6 alkylcarbonyl group, a C _6-14 arylcarbonyl group, a C _7-16 aralkylcarbonyl group, a 5-14-membered aromatic heterocyclylcarbonyl group, a 3-14-membered non-aromatic heterocyclylcarbonyl group, a C _1-6 alkoxycarbonyl group, a 5-14-membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group, and mono- or di-C _7-16 aralkyl-carbamoyl group, each of which optionally has from 1 to 3 substituents selected from substituent group A.

Suitable examples of the optionally substituted thiocarbamoyl group include a thiocarbamoyl group, a mono- or di-C _1-6 alkylthiocarbamoyl group (e.g. methylthiocarbamoyl, ethylthiocarbamoyl, dimethylthiocarbamoyl, diethylthiocarbamoyl, H-ethyl-H-methylthiocarbamoyl), a mono- or di-C _2-6 alkenylthiocarbamoyl group (e.g. diallylthiocarbamoyl), a mono- or di-C ₃₁₀ cycloalkylthiocarbamoyl group (e.g. cyclopropylthiocarbamoyl), a mono- or di-C _6-14 arylthiocarbamoyl group (e.g. phenylthiocarbamoyl), a mono- or di-C _7-16 aralkylthiocarbamoyl group (e.g. benzylthiocarbamoyl, phenethylthiocarbamoyl), a mono- or di-C _1-6 alkylcarbonylthiocarbamoyl group (e.g. acetylthiocarbamoyl, propionylthiocarbamoyl), a mono- or di-C _6-14 arylcarbonylthiocarbamoyl group (e.g. benzoylthiocarbamoyl) and a 5-14 membered aromatic heterocyclylthiocarbamoyl group (e.g. pyridylthiocarbamoyl).

In the present specification, examples _of the optionally substituted sulfamoyl group include a sulfamoyl group optionally having 1 or 2 substituents selected from a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, a C _7-16 aralkyl group, a C _1-6 alkylcarbonyl group, a C _6-14 arylcarbonyl group, a C 7-16 aralkylcarbonyl group, a 5-14-membered aromatic heterocyclylcarbonyl group, a 3-14-membered non-aromatic heterocyclylcarbonyl group, a C _1-6 alkoxycarbonyl group, a 5-14-membered aromatic heterocyclic group, a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group, and a mono- or di-C7-16 aralkyl-carbamoyl group, each of which optionally has from 1 to 3 substituents selected from substituent group A

Suitable examples of the optionally substituted sulfamoyl group include a sulfamoyl group, a mono- or di-C _1-6 alkyl sulfamoyl group (e.g. methylsulfamoyl, ethylsulfamoyl, dimethylsulfamoyl, diethylsulfamoyl, H-ethyl-H-methylsulfamoyl), a mono- or di-C ₂₆ alkenylsulfamoyl group (e.g. diallylsulfamoyl), a mono- or di-C _3-10 cycloalkylsulfamoyl group (e.g. cyclopropylsulfamoyl, cyclohexylsulfamoyl), a mono- or di-C _6-14 aryl sulfamoyl group (e.g. phenylsulfamoyl), a mono- or di-C _7-16 aralkylsulfamoyl group (e.g. benzylsulfamoyl, phenethylsulfamoyl), a mono- or di-C _1-6 alkylcarbonylsulfamoyl group (e.g. acetylsulfamoyl, propionylsulfamoyl), mono- or diC _6-14 arylcarbonylsulfamoyl group (e.g. benzoylsulfamoyl) and 5-14-membered aromatic heterocyclylsulfamoyl group (e.g. pyridylsulfamoyl).

In the present specification, examples of the optionally substituted hydroxy group include a hydroxy group optionally having substituents selected from a C _1-6 alkyl group, a C _2-6 alkenyl group, a C _3-10 cycloalkyl group, a C _6-14 aryl group, a C _7-16 aralkyl group, a C _1-6 alkylcarbonyl group, a C 6-14 arylcarbonyl group, a C _7-16 aralkylcarbonyl group, a 5-14-membered aromatic heterocyclylcarbonyl group, a 3-14-membered non-aromatic heterocyclylcarbonyl group, a C 1-6 alkoxycarbonyl group, a 5-14-membered aromatic heterocyclylcarbonyl group, a C 1-6 alkoxycarbonyl group, a 5-14-membered aromatic heterocyclylcarbonyl group, a C _1-6 alkoxycarbonyl group, a C _6-14 arylcarbonyl group, a C 7-16 aralkyl ...

- 18 048725 alkyl group, a carbamoyl group, a mono- or di-C _1-6 alkylcarbamoyl group and a mono- or di-C _7-16 aralkylcarbamoyl group, a C ^ alkylsulfonyl group and a C ₆ . ₁₄ arylsulfonyl group, each of which optionally has from 1 to 3 substituents selected from substituent group A.

Suitable examples of the optionally substituted hydroxy group include a hydroxy group, a _C1.6 alkoxy group, a _C2.6 alkenyloxy group (e.g. allyloxy, 2-butenyloxy, 2-pentenyloxy, 3-hexenyloxy), _{a C3.10 cycloalkyloxy} group (e.g. cyclohexyloxy) _{, a C6.14 aryloxy} group (e.g. phenoxy, naphthyloxy), _{a C7.16 aralkyloxy} group (e.g. benzyloxy, phenethyloxy), a C^alkylcarbonyloxy group (e.g. acetyloxy, propionyloxy, butyryloxy, isobutyryloxy, pivaloyloxy), _{a C6.14 arylcarbonyloxy} (e.g. benzoyloxy), a _C7 . ₁₆ aralkylcarbonyloxy (e.g. benzylcarbonyloxy), 5- to 14-membered aromatic heterocyclylcarbonyloxy group (e.g. nicotinoyloxy), 3- to 14-membered non-aromatic heterocyclylcarbonyloxy group (e.g. piperidinylcarbonyloxy), C1. ₆ alkoxycarbonyloxy group (e.g. tert-butoxycarbonyloxy), 5- to 14-membered aromatic heterocyclyloxy group (e.g. pyridyloxy), carbamoyloxy group, C1. ₆ alkylcarbamoyloxy group (e.g. methylcarbamoyloxy), C ₇ . ₁₆ aralkylcarbamoyloxy group (e.g. benzylcarbamoyloxy), C1. ₆ alkylsulfonyloxy group (e.g. methylsulfonyloxy, ethylsulfonyloxy) and C ₆ . ₁₄ arylsulfonyloxy (eg phenylsulfonyloxy).

In the present specification, examples of the optionally substituted sulfanyl group include a sulfanyl group optionally having a substituent selected from _{a C1.6 alkyl} group, a _C2.6 alkenyl group, _{a C3.10 cycloalkyl} group, _{a C6.14 aryl} group, a _C7.16 aralkyl group, _{a C1.6 alkylcarbonyl} group, a _C6.14 arylcarbonyl group, and a _5- to 14-membered aromatic heterocyclic group, each of which optionally has 1 to 3 substituents selected from Substituent Group A and a halogenated sulfanyl group.

Suitable examples of the optionally substituted sulfanyl group include a sulfanyl(SH) group, a _C1.6 alkylthio group, a _C2.6 alkenylthio group (e.g. allylthio, 2-butenylthio, ₂ -pentenylthio, 3-hexenylthio), _{a C3.10 cycloalkylthio} group (e.g. cyclohexylthio), _{a C6.14} arylthio group (e.g. phenylthio, naphthylthio), a _C7.16 aralkylthio group (e.g. benzylthio, phenethylthio), _{a C1.6 alkylcarbonylthio} group (e.g. acetylthio, propionylthio, butyrylthio, isobutyrylthio, pivaloylthio), _{a C6} . ₁₄ arylcarbonylthio group (e.g. benzoylthio), 5- to 14-membered aromatic heterocyclylthio group (e.g. pyridylthio) and halogenated thio group (e.g. pentafluorothio).

In the present specification, examples of the optionally substituted silyl group include a silyl group optionally having 1 to 3 substituents selected from _{a C1.6} alkyl group, _{a C2.6 alkenyl} group, _{a C3.10 cycloalkyl} group, a _C6.14 aryl group and _{a C7.16} aralkyl group, each of which optionally has 1 to 3 substituents selected from Substituent Group A. Examples of the optionally substituted silyl group include _a tri- _C1.6 alkylsilyl group (e.g., trimethylsilyl, tert-butyl(dimethyl)silyl).

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

or a pharmaceutically acceptable salt thereof, wherein:

R ¹⁰⁵ and R ²⁰⁵ each independently represent a hydroxyl group or a halogen atom;

B ¹⁰⁰ is a group represented by the formula (B1-A) or the formula (B1-B):

R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ and R ¹⁷ each independently represent a hydrogen atom or a substituent;

R ¹⁰⁰⁰ represents hydrogen or a bond to the carbonyl group of formula (I);

Y ¹¹ , Y ¹² , Y ¹³ , Y ¹⁴ , Y ¹⁵ and Y ¹⁶ each independently represent N or CR ^1a ;

Z ¹¹ , Z ¹² , Z ¹³ , Z ¹⁴ , Z ¹⁵ and Z ¹⁶ each independently represent N or C;

- 19 048725

R ¹⁰⁵ and R ²⁰⁵ each independently represent a hydroxyl group or a halogen atom;

R ^1a represents a hydrogen atom or a substituent;

B ²⁰⁰ is a group represented by the formula (B ² -A) or the formula (B ² -B):

R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ , each independently, represents a hydrogen atom or a substituent;

R ¹⁰⁰ represents hydrogen or a bond to the carbonyl group of formula (I);

γ ²¹ ,γ ²² ,γ ²³ ,γ ²⁴ ,γ ²⁵ and y ²⁶ each independently represent N or CR ^2a ;

Z ²¹ , Z ²² , Z ²³ , Z ²⁴ , Z ²⁵ and Z ²⁶ each independently represents N or C; R ^2a represents a hydrogen atom or a substituent;

X ¹ and X ² each independently represent an oxygen atom or a sulfur atom; and

Q ¹ , Q2, Q3 and Q ⁴ each independently represents an oxygen atom or a sulfur atom.

provided that:

one of B ¹⁰⁰ or B ²⁰⁰ represents:

R ¹³ θ πΎ

N'

Ά where R ¹⁸ is hydrogen or C1. ₆ alkyl; and

R ¹⁹ is a halogen atom and the other one of B ¹⁰⁰ or B ²⁰⁰ is attached to the 5-membered ring of the parent structure through an -NH- group.

In some embodiments, the STING modulator is a compound of formula (III) or a pharmaceutically acceptable salt thereof, wherein R ²⁰⁵ is F and B ¹⁰⁰ is

F θ

Cm

In some embodiments, the cyclic dinucleotide is:

or a pharmaceutically acceptable salt thereof, where represents the point of attachment to the carbonyl group of the original molecular fragment.

The L group is a cleavable linker. As used herein, the term linker refers to any chemical moiety capable of linking an antibody, antibody fragment, or antigen-binding fragment (Ab) to a drug-containing moiety in the compounds of formula (I) and (IV). The linker may be branched and may be substituted with 1 to 20 drug-containing moieties. In some embodiments, the linker may be substituted with 1 to 10 drug-containing moieties. In some embodiments, the linker may be substituted with 1 to 5 drug-containing moieties. In some embodiments, the linker may be substituted with one or two drug-containing moieties. In some embodiments, the linker may be substituted with one drug-containing moiety.

Linker L is cleavable. In some embodiments, the linker may be susceptible to acid-induced cleavage, photo-induced cleavage, enzymatic cleavage, etc. under conditions in which the drug and/or antibody may remain active.

- 20 048725 tive. In some embodiments, the cleavable linker can be cleaved enzymatically. In some embodiments, the cleavable linker can be cleaved by a protease, peptidase, esterase, glycosidase, phosphodiesterase, phosphatase, or lipase. In some embodiments, the cleavable linker can be cleaved by a protease. Examples of proteases include, but are not limited to, cathepsin B, VAGP tetrapeptide, and the like. In some embodiments, the linker can be any of the linkers disclosed in PCT publications WO 2018/200812, WO 2018/100558, which are incorporated by reference in their entireties.

In some embodiments, L has the formula:

where + represents the point of attachment to the nitrogen atom; and * represents the point of attachment to Ab.

In some embodiments, L has the formula:

Where:

Ch represents the point of attachment to the nitrogen atom;

* represents the point of attachment to the antibody;

The group W is absent or is a self-cleaving group. As used herein, the term self-cleaving refers to a group that undergoes an electron cascade resulting in the release of the group to which it is attached. In some embodiments, the self-cleaving group comprises one or more groups that can undergo 1,4-elimination, 1,6-elimination, 1,8-elimination, 1,6-cyclization-elimination, 1,5-cyclization-elimination, 1,3-cyclization-elimination, intramolecular 5-exo-trigger cyclization and/or 6-exo-trigger cyclization. In some embodiments, the self-cleaving group can be any of those disclosed in PCT publications WO 2018/200812, WO 2018/100558, which are incorporated by reference in their entireties.

The Z group is absent or is a peptide of 2-5 amino acids. In some embodiments, the peptide is a linker cleavage site, thereby facilitating drug release upon exposure to intracellular proteases such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Examples of peptides containing two amino acids include, but are not limited to, alanine-alanine (ala-ala), valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phehomolys); and N-methyl-valine-citrulline (Me-val-cit). Examples of peptides containing three amino acids include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid combinations listed above can also be present in the reverse order (i.e., cit-val).

The peptides of the present invention may comprise natural and/or unnatural amino acid residues. The term natural amino acid refers to Ala, Asp, Cys, Glu, Phe, Gly, His, He, Lys, Leu, Met, Asn, Pro, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. Unnatural amino acids (i.e., amino acids not found in nature) include, but are not limited to, homoserine, homoarginine, citrulline, phenylglycine, taurine, iodotyrosine, selenocysteine, norleucine (Me), norvaline (Nva), beta-alanine, L- or D-naphthalanine, ornithine (Orn), and the like. Peptides can be designed and optimized for enzymatic cleavage by a specific enzyme, such as tumor-associated protease, cathepsin B, C, and D, or plasmin protease.

Amino acids also include the D-forms of natural and unnatural amino acids. D- denotes an amino acid having the D (dextrorotatory) configuration, as opposed to the configuration in naturally occurring (L-) amino acids. Natural and unnatural amino acids can be purchased commercially (Sigma Chemical Co., Advanced Chemtech) or synthesized using methods known in the art.

The U and U' groups are independently absent or a spacer. As used herein, the term "spacer" refers to a chemical moiety that serves as a connector. In the present invention, a spacer may connect an antibody, antibody fragment, or antigen fragment to a heterobifunctional group and/or connect a heterobifunctional group to a peptide Z or, when Z is absent, to a W group. Non-limiting examples of spacers include -NH-, -S-, -O-, NHC(=O)CH2CH2-, -S(=O)2-CH2CH2-, -C(=O)NHNH-, -C(=O)O-, -C(=O)NH-, -CH2-, -CH2CH2-, CH2CH2CH2-, -CH ₂ =CH ₂ -, -C=C-, -CH=NO-, polyethyleneglycol (PEG),

- 21 048725

In the compounds of the present invention, when U is present, it can be a branched group substituted with 1 to 10 -C(O)-W-Z- groups. In some embodiments, U is substituted with 1 to 5 -C(O)-W-Z- groups. In some embodiments, U is substituted with 1 to 2 -C(O)-W-Z- groups. In some embodiments, U is substituted with 1 -C(O)-W-Z- group. In some embodiments, the spacer can be any of the spacers disclosed in PCT publications WO 2018/200812, WO 2018/100558, which are incorporated by reference in their entireties.

The Q group is a heterobifunctional group. As used herein, the term heterobifunctional group refers to a chemical moiety that connects a linker of which it is a part to an antibody, antibody fragment, or antigen-binding fragment. See, for example, WO 2017/191579. Heterobifunctional groups are characterized by the presence of different reactive groups at both ends of the chemical moiety. The heterobifunctional group can be attached directly to Ab or, alternatively, can be attached via a U' linker. Attachment to Ab can be accomplished by chemical or enzymatic methods. Chemical conjugation involves the controlled reaction of accessible amino acid residues on the surface of the antibody with a Q or U' reactive label. Examples of chemical conjugation include, but are not limited to, lysine amide coupling, cysteine coupling, and coupling via a non-natural amino acid introduced by genetic engineering, where non-natural amino acid residues with a desired reactive label are installed on the Ab. In enzymatic conjugation, an enzyme mediates the coupling of a linker to an available amino acid residue on the antibody, antibody fragment, or antigen-binding fragment. Examples of enzymatic conjugation include, but are not limited to, sortase-based transpeptidation, microbial transglutaminase-based transpeptidation, and N-glycan engineering. Chemical conjugation and enzymatic conjugation can also be used sequentially. For example, enzymatic conjugation can also be used to install unique reactive markers on the Ab that will be used in subsequent chemical conjugation. In some embodiments, the heterobifunctional group may be any of those disclosed in PCT publications WO 2018/200812, WO 2018/100558, which are incorporated by reference in their entireties.

In some embodiments, Q is selected from

- 22 048725

ABOUT

where ⁴ represents the point of attachment to U or, when U is absent, the point of attachment to Z; and represents the point of attachment to U' or, when U' is absent, the point of attachment to Ab.

In some embodiments, the present invention relates to a compound of formula (XX):

or a pharmaceutically acceptable salt thereof, wherein n, m, a, t, D-NH-, R ¹ , R ² , R ³ , R ³ , W, Z and U have the meanings specified herein, and wherein Q* is a reactive functional group capable of conjugation with an antibody, antibody fragment or antigen-binding fragment. Examples of suitable Q* groups include, but are not limited to, activated carboxylic acid groups such as -C(O)-Cl acid chloride and acid anhydrides, haloacetamide, maleimide, alkyne, cycloalkyne such as cyclooctyne, oxanoboradiene, norbornene, azide, diaryltetrazine, monoaryltetrazine, aldehyde, ketone, hydroxylamine, vinyl sulfone and aziridine. In some embodiments, the reactive functional group can be any of those disclosed in PCT publications WO 2018/200812, WO 2018/100558, which are incorporated by reference in their entireties.

An Ab group is an antibody, an antibody fragment, or an antigen-binding fragment. An antibody is a protein produced by the immune system that is capable of recognizing and binding to a specific antigen. The target antigen typically has multiple binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody, which specifically binds to different epitopes, has a different structure. Thus, one antigen may have more than one corresponding antibody. The term antibody is used herein in the broadest sense and specifically covers monoclonal antibodies, single-domain antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric, or of other species. (Janeway, S., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York).

The term "antibody" as used herein also refers to a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds a target antigen of interest or a portion thereof, such targets including, but not limited to, cancer cells or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of an immunoglobulin molecule. Immunoglobulins can be of any species. However, in one aspect, the immunoglobulin is of human, mouse, or rabbit origin.

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The term single-domain antibody, also known as nanobody, is an antibody fragment consisting of a single monomeric variable domain of an antibody with a molecular weight of about 12 kDa to about 15 kDa. Single-domain antibodies may be based on the variable domains of the heavy chain or the light chain. Examples of single-domain antibodies include, but are not limited to, VHH fragments and V _NAR fragments. See, e.g., Harmsen MM et al. Applied Microbiology and Biotechnology 77 (1): 13-22.

Antibody fragments comprise a portion of an intact antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region) and epitope-binding fragments of any of the foregoing that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

An intact antibody is an antibody that includes an antigen-binding variable region, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, _CH2 , and CH3. The constant domains may be native sequence constant domains (e.g., native human sequence constant domains) or amino acid sequence variants thereof.

As used herein, the term monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant of the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or can be produced by recombinant DNA techniques (see U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597; for example.

The monoclonal antibodies described herein specifically include chimeric antibodies in which a portion of the heavy and/or light chain is identical to or homologous with corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass and the remainder of the chain(s) is identical to or homologous with corresponding sequences in antibodies derived from another species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity (U.S. Patent No. 4,816,567 and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies of interest include primatized antibodies containing variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences.

Various methods have been used to produce monoclonal antibodies (MAbs). Hybridoma technology, which refers to a cloned cell line that produces one type of antibody, uses cells from different species, including mice, hamsters, rats, and humans. Another method for producing MAbs uses genetic engineering, including recombinant DNA techniques. Monoclonal antibodies produced by these methods include chimeric antibodies and humanized antibodies, among others. A chimeric antibody combines coding regions of DNA from more than one type of species. For example, a chimeric antibody may be made with a variable region from a mouse and a constant region from a human. A humanized antibody is predominantly human, even if it contains non-human parts. Like a chimeric antibody, a humanized antibody may contain a completely human constant region. But unlike a chimeric antibody, the variable region may be partially human. The non-human synthetic portions of a humanized antibody are often derived from the CDRs in mouse antibodies. In either case, these regions are critical for the antibody to recognize and bind to a specific antigen. Although mouse antibodies are useful for diagnostics and short-term therapy, they cannot be administered to humans over long periods of time without increasing the risk of a harmful immunogenic response. This response, called a human anti-mouse antibody (HAMA), occurs when the human immune system recognizes the mouse antibody as foreign and

- 24 048725 attacks him. The NAMA reaction can cause toxic shock or even death.

Chimeric and humanized antibodies reduce the likelihood of a HAMA response by minimizing the non-human portions of the administered antibodies. In addition, chimeric and humanized antibodies may have the added benefit of activating secondary human immune responses such as antibody-dependent cellular cytotoxicity.

An intact antibody may have one or more effector functions, which refer to biological activities inherent in the Fc region (either native sequence Fc regions or amino acid sequence variant Fc regions) of the antibody. Examples of antibody effector functions include Clq binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be classified into different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called a, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different immunoglobulin classes are well known.

Useful non-immunoreactive protein, polypeptide, or peptide antibodies include, but are not limited to, transferrin, epidermal growth factors (EGF), bombesin, gastrin, gastrin-releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors (TGFs) such as TGF-α and TGF-β, vaccinia growth factor (VGF), insulin and insulin-like growth factors I and II, lectins, and low-density lipoprotein apoprotein.

Suitable polyclonal antibodies are heterogeneous populations of antibody molecules obtained from sera of immunized animals. Various procedures well known in the art can be used to obtain polyclonal antibodies to an antigen of interest. For example, various domestic animals can be immunized by injection with an antigen of interest or a derivative thereof to produce polyclonal antibodies, including, but not limited to, rabbits, mice, rats, and guinea pigs. Various adjuvants can be used to enhance the immunological response depending on the host species, including but not limited to Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guérin) and corynebacterium parvum. Such adjuvants are also well known in the art.

Suitable monoclonal antibodies are homogeneous populations of antibodies to a specific antigenic determinant (e.g., a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, a nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen of interest can be obtained using any technique known in the art that enables the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma method originally described by Kohler and Milstein (1975, Nature 256, 495-497), the human B-cell hybridoma method (Kozbor et al., 1983, Immunology Today 4:72), and the EBV hybridoma method (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any class of immunoglobulin, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. The hybridoma producing the mAb used in this invention may be cultured in vitro or in vivo.

Suitable monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, antibody fragments, or chimeric human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies can be produced by any of numerous methods known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79; and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

The antibody may also be a bispecific antibody. Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies relies on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Milstein et al., 1983, Nature 305:537-539). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, typically accomplished using affinity chromatography steps, is cumbersome and the yield of product is low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO J. 10:3655-3659(1991).

- 25 048725

According to another approach, variable domains of antibodies with the desired binding specificity (antibody-antigen fusion sites) are fused to immunoglobulin constant domain sequences. The fusion may be with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. The first heavy chain constant region (CH1) may comprise a site necessary for light chain binding present in at least one of the fusions. Nucleic acids with sequences encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides greater flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construct provide optimal yields. However, it is possible to insert coding sequences for two or all three polypeptide chains into a single expression vector when expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are not particularly important.

Bispecific antibodies may have a hybrid immunoglobulin heavy chain with the first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing the second binding specificity) in the other arm. This asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy means of separation (WO 94/04690; Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigues et al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992, Bio/Technology 10:163-167; Carter et al., 1995, J. of Hematotherapy 4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681. Using such methods, bispecific antibodies can be prepared for conjugation as ADCs in the treatment or prophylaxis of diseases as defined in this document.

Hybrid or bifunctional antibodies may be produced either biologically, i.e. by cell fusion techniques, or chemically, especially by cross-linking agents or reagents forming a disulfide bridge, and may comprise whole antibodies or fragments thereof (EP 105360; WO 83/03679; EP 217577).

The antibody may be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to cancer cell antigens, viral antigens or microbial antigens or other antibodies associated with tumor cells or a matrix. In this regard, functionally active means that the fragment, derivative or analog is capable of eliciting anti-anti-idiotypic antibodies that recognize the same antigen as the antibody from which the fragment, derivative or analog is derived. In particular, in an exemplary embodiment, the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of the framework and CDR sequences that are C-terminal to the CDR sequence that specifically recognizes the antigen. To determine which CDR sequences bind antigen, synthetic peptides containing the CDR sequences can be used in antigen binding assays by any binding assay known in the art (e.g., core BIA assay) (see, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md.; Kabat E et al., 1980, J. of Immunology 125 (3):961-969).

Other useful antibodies include antibody fragments such as, but not limited to, F(ab')2 fragments, which contain the variable region, the constant region of the light chain, and the CH1 domain of the heavy chain, which can be obtained by pepsin digestion of the antibody molecule, and Fab fragments, which can be obtained by reducing the disulfide bridges of F(ab')2 fragments. Other useful antibodies are dimers of heavy and light chains of antibodies or any minimal fragments thereof, such as Fv or single chain antibodies (SCAs) (e.g., as described in U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., (1989) Nature 334:544-54), or any other molecule with the same specificity as an antibody.

In addition, recombinant antibodies, such as chimeric and humanized monoclonal antibodies containing both human and non-human portions, which can be produced using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as having a variable region derived from the constant regions of a mouse monoclonal and a human immunoglobulin. (See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; and Boss et al., U.S. Patent No. 4,816,397). Humanized antibodies are antibody molecules from a non-human species that have one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. (See, e.g., Queen, U.S. Patent No. 5,585,089). Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art,

- 26 048 725 for example, using the methods described in WO 87/02671; EP 184 187; EP 171 496; EP 173 494; WO 86/01533; U.S. Patent No. 4,816,567; EP 12023; Berter et al., 1988, Science 240: 1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4: 214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al., 1988, J. Immunol. 141:4053-4060.

Fully human antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chain genes, but which can express human heavy and light chain genes. The transgenic mice are immunized in the usual manner with a selected antigen, for example, all or part of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be produced using conventional hybridoma technology. The human immunoglobulin transgenes contained in the transgenic mice are rearranged during B cell differentiation and then undergo class switching and somatic mutations. Thus, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be produced using this method. For a review of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., U.S. Patent Nos. 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806. Other human antibodies are available commercially, e.g., from Abgenix, Inc. (Fremont, Calif.) and Genpharm (San Jose, Calif.).

Fully human antibodies that recognize a selected epitope can be generated using a method called guided selection. In this approach, a selected monoclonal antibody of non-human origin, such as a murine antibody, is used to select a fully human antibody that recognizes the same epitope. (Jespers et al. (1994) Biotechnology 12:899-903). Human antibodies can also be generated using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).

The antibody may be a fusion protein of an antibody or a functionally active fragment thereof, for example, in which the antibody is fused via a covalent bond (e.g., a peptide bond) either at the N-terminus or at the C-terminus to an amino acid sequence of another protein (or part thereof, for example, at least 10, 20 or 50 amino acid portion of the protein) that is not an antibody. The antibody or fragment thereof may be covalently linked to another protein at the N-terminus of a constant domain.

Antibodies include analogs and derivatives that are also modified, i.e., by covalent attachment of any type of molecule, so long as such covalent attachment allows the antibody to retain its antigen-binding immunospecificity. For example, but not by way of limitation, derivatives and analogs of antibodies include those that have been further modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, attachment to a cellular antibody unit or other protein, etc. Any of the numerous chemical modifications can be accomplished by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, an analog or derivative may contain one or more unnatural amino acids.

Antibodies in antibody-drug conjugates include antibodies having modifications (e.g., substitutions, deletions, or additions) at amino acid residues that interact with Fc receptors. In particular, antibodies include antibodies having modifications at amino acid residues identified as participating in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g., WO 97/34631). Immunospecific antibodies to a cancer cell antigen can be obtained commercially, e.g., from Genentech (San Francisco, Calif.), or prepared by any method known to those skilled in the art, such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific to a cancer cell antigen can be obtained, e.g., from the GenBank or similar database, from literature publications, or by routine cloning and sequencing.

The ADC antibody may be a monoclonal antibody, such as a murine monoclonal antibody, a chimeric antibody, or a humanized antibody. The antibody may be an antibody fragment, such as a Fab fragment.

Known antibodies for the treatment or prevention of cancer can be conjugated as ADCs. Immunospecific antibodies to a cancer cell antigen can be obtained commercially or produced by any method known to a person skilled in the art, such as, for example, recombinant methods.

- 27 048725 expression. The nucleotide sequence encoding antibodies immunospecific for a cancer cell antigen can be obtained, for example, from the GenBank or similar database, literature publications, or by routine cloning and sequencing. Examples of antibodies available for the treatment of cancer include, but are not limited to, a humanized monoclonal antibody against HER2 for the treatment of patients with metastatic breast cancer; RITUXAN.RTM. (rituximab; Genentech), which is a chimeric monoclonal antibody against CD20 for the treatment of patients with non-Hodgkin's lymphoma; OvaRex (AltaRex Corporation, MA), which is a murine antibody for the treatment of ovarian cancer; Panorex (Glaxo Wellcome, NC), which is a murine IgG 2a antibody for the treatment of colorectal cancer; Cetuximab Erbitux (Imclone Systems Inc., New York), which is a chimeric anti-EGFR IgG antibody for the treatment of epidermal growth factor positive malignancies such as head and neck cancer; Vitaxin (Medlmmune, Inc., MD), which is a humanized antibody for the treatment of sarcoma; Campath I/H (Leukosis, Massachusetts), which is a humanized IgG 1 antibody for the treatment of chronic lymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., California), which is a humanized IgG anti-CD33 antibody for the treatment of acute myeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ), which is a humanized IgG anti-CD22 antibody for the treatment of non-Hodgkin's lymphoma; Smart ID10 (Protein Design Labs, Inc., California), which is a humanized anti-HLA-DR antibody for the treatment of non-Hodgkin lymphoma; Oncolim (Techniclone, Inc., California), which is a radiolabeled murine anti-HLA-Dr10 antibody for the treatment of non-Hodgkin lymphoma; Allomune (BioTransplant, California), which is a humanized anti-CD2 mAb for the treatment of Hodgkin disease or non-Hodgkin lymphoma; Avastin (Genentech, Inc., California), which is a humanized anti-VEGF antibody for the treatment of lung and colorectal cancer; Epratuzamab (Immunomedics, Inc., New Jersey and Amgen, California), which is an anti-CD22 antibody for the treatment of non-Hodgkin lymphoma; and CEAcide (Immunomedics, NJ), which is a humanized anti-CEA antibody for the treatment of colorectal cancer.

Other antibodies useful in the treatment of cancer include, but are not limited to, antibodies against the following antigens: CA125 (ovary), CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y (carcinomas), Lewis X (carcinomas), alpha-fetoprotein (carcinomas), CA 242 (colorectal), placental alkaline phosphatase (carcinomas), prostate-specific antigen (prostate), prostatic acid phosphatase (prostate), epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2 (carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrin receptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA (colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2 receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma), CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionic gonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma), mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and oncogenic product Neu (carcinomas). Some specific useful antibodies include, but are not limited to, mAb BR96 (Trail, PA, et al., Science (1993) 261, 212-215), BR64 (Trail, PA, et al. Cancer Research (1997) 57, 100-105, mAbs against CD40 antigen such as mAb S2C6 (Francisco, JA, et al. Cancer Res. (2000) 60:3225-3231), mAbs against CD70 antigen such as mAb 1F6, and mAbs against CD30 antigen such as AC10 (Bowen, MA, et al. (1993) J. Immunol., 151:5896-5906; Wahl et al., 2002 Cancer Res. 62 (13):3736-42). Many other internalizing antibodies that bind to tumor antigens can be used and have been reviewed (Franke, A. E., et al Cancer Biother Radiopharm. (2000) 15:459-76; Murray, J. L., (2000) Semin Oncol., 27:64-70; Breitling, F., and Dubel, S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

Antibodies that bind to antigens associated with antigen-presenting cells such as CD40, OX40L, endoglin, DEC-205, 4-1BBL, CD36, CD36, CD204, MARCO, DC-SIGN, CLEC9A, CLEC5A, dectin 2, CLEC10A, CD206, CD64, CD32A, CD1A, HVEM, CD32B, PD-L1, BDCA-2, XCR-1, and CCR2 can also be conjugated as ADCs.

Known antibodies for the treatment or prevention of autoimmune disorders can be conjugated as ADCs. Autoimmune disorders include systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjogren's syndrome, immune thrombocytopenia, and multiple sclerosis. Immunospecific antibodies to an antigen of a cell responsible for the production of autoimmune antibodies can be produced by any method known to those skilled in the art, such as, for example, chemical synthesis or recombinant expression techniques. SLE is characterized by overexpression of interferon-alpha (IFN-alpha) cytokine genes (Bennett et al (2003) Jour. Exp. Med. 197:711-723). Type 1 interferons (IFN-.alpha./.beta.) play an important role in the pathogenesis of lupus (Santiago-Raber (2003) Jour. Exp. Med. 197:777-788). Knockout mice (-IFN-.alpha./.beta.) showed significantly reduced red blood cell autoantibodies, erythroblastosis, hemolytic anemia, DNA autoantibodies, renal disease, and mortality. These results suggest that type 1 IFNs mediate murine lupus and that their downregulation in humans may be beneficial. Bis-conjugated anti-IFN Ab

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1,8-naphthalimide components of the drug may be effective therapeutic agents against SLE and other autoimmune diseases.

In another embodiment, useful antibodies in the ADC are immunospecific for the treatment of autoimmune diseases, including, but not limited to, antinuclear antibodies; anti-dsDNA; anti-ssDNA, anti-cardiolipin IgM, IgG antibodies; anti-phospholipid IgM, IgG antibodies; anti-SM antibody; anti-mitochondria antibody; anti-thyroid antibodies; microsomal antibodies; anti-thyroglobulin antibody; anti-SCL-70; anti-Jo; anti-U1RNP; anti-La/SSB; anti-SSA; anti-SSB; anti-perital cell antibody; anti-histones; anti-RNP; C-ANCA; P-ANCA; anti-centromere; anti-fibrillarin and anti-GBM antibody.

ADC antibodies may bind to either a receptor or a receptor complex expressed on an activated lymphocyte. The receptor or receptor complex may comprise a member of the immunoglobulin gene superfamily, a member of the TNF receptor superfamily, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members include CD2, CD3, CD4, CD8, CD 19, CD22, CD28, CD79, CD90, CD 152/CTLA-4, PD-1, and ICOS. Non-limiting examples of suitable members of the TNF receptor superfamily include CD27, CD40, CD95/Fas, CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins include CD11a, CD11b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD 103, and CD 104. Non-limiting examples of suitable lectins include C-type, S-type, and I-type lectins.

As used herein, the term viral antigen includes, but is not limited to, any viral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV glycoprotein F, influenza virus neuraminidase, influenza virus hemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., Gb, Gc, Gd, and Ge), and hepatitis B surface antigen) that is capable of eliciting an immune response. As used herein, the term microbial antigen includes, but is not limited to, any microbial peptide, polypeptide, protein, saccharide, polysaccharide, or lipid molecule (e.g., bacterial, fungal, pathogenic protozoan, or yeast polypeptide, including, for example, LPS and capsular polysaccharide 5/8) that is capable of eliciting an immune response.

Immunospecific antibodies to a viral or microbial antigen can be obtained commercially, for example, from BD Biosciences (San Francisco, CA), Chemicon International, Inc. (Temecula, CA), or Vector Laboratories, Inc. (Burlingame, CA), or produced by any method known to those skilled in the art, such as, for example, chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies that are immunospecific for a viral or microbial antigen can be obtained, for example, from the GenBank or similar database, literature publications, or by routine cloning and sequencing.

In some embodiments, the useful antibodies in the ADCs are antibodies that treat or prevent a viral or microbial infection in accordance with the methods disclosed herein. Examples of antibodies available for treating a viral infection or microbial infection include, but are not limited to, SYNAGIS (Medlmmune, Inc., MD), which is an anti-humanized respiratory syncytial virus (RSV) monoclonal antibody useful for treating patients with RSV infection; PRO542 (Progenies), which is a CD4 chimeric antibody used to treat HIV infection; OSTAVIR (Protein Design Labs, Inc., CA), which is a human antibody useful for treating hepatitis B virus; PROTOVIR (Protein Design Labs, Inc., CA), which is a humanized IgG antibody useful for treating cytomegalovirus (CMV); and anti-LPS antibodies.

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Other antibodies useful in ADCs for the treatment of infectious diseases include, but are not limited to, antibodies against antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrheae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenas, Klebsiella rhinoscleromotis, Staphylococcus aureus, Vibrio colerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoeba histolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or helminths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms).

Other antibodies used in ADCs for the treatment of a viral disease include, but are not limited to, antibodies against antigens of pathogenic viruses, including, by way of example but not limitation:

Poxviridae, Herpesviridae,

Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, non-A/non-B hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae and human immunodeficiency virus.

The term amino acid sequence variant refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Typically, amino acid sequence variants will have at least about 70% sequence identity to at least one receptor binding domain of a native antibody or at least one ligand binding domain of a native receptor, and will typically be at least about 80%, more typically at least about 90%, homologous in sequence to such receptor or ligand binding domains. Amino acid sequence variants contain substitutions, deletions, and/or insertions at certain positions in the amino acid sequence of the native amino acid sequence. Amino acids are designated by conventional names, one-letter codes, and three-letter codes.

Sequence identity is defined as the percentage of residues in an amino acid sequence variant that are identical after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignment are well known in the art. One such computer program is Align 2, created by Genentech, Inc., which was filed with user documentation in the U.S. Copyright Office, Washington, D.C. 20559, December 10, 1991.

The terms Fc receptor or FcR are used to describe a receptor that binds to the Fc region of an antibody. An exemplary FcR is the native-sequence human FcR. Additionally, an FcR may be one that binds an IgG antibody (a gamma receptor) and includes the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII receptors, including allelic variants and alternatively spliced forms of these receptors. The Fc.gamma.RII receptors include Fc.gamma.RIIA (the activating receptor) and Fc.gamma.RIIB (the inhibitory receptor), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor

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Fc.raMMa.RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See the review by M. in Daeron, Annu. Rev. Immunol., 15:203–234 (1997).) FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457–92 (1991); Capel et al., Immunomethods, 4:25–34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330–41 (1995). Other FcRs, including those that will be identified in the future, are encompassed by the term FcR in this document. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

Complement-dependent cytotoxicity or CDC refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) in complex with a cognate antigen. To assess complement activation, a CDC assay, such as that described in GazzanoSantoro et al., J. Immunol. Methods, 202:163 (1996), can be performed.

Native antibodies are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has symmetrically arranged intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a series of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Certain amino acid residues are thought to form the interface between the light chain and heavy chain variable domains.

The term variable refers to the fact that certain portions of the variable domains vary widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, variability is not evenly distributed across the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light chain and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FRs, mostly in a β-sheet configuration, connected by three hypervariable regions that form loops that connect and in some cases form part of the β-sheet structure.

The hypervariable regions in each chain are held together in close proximity by FR and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.). The constant domains are not directly involved in binding of the antibody to antigen, but have various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term hypervariable region, as used herein, refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region typically contains amino acid residues from the complementarity determining region or CDR (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al supra) and/or those residues from the hypervariable loop (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol., 196:901-917). Framework region or FR residues are variable domain residues other than hypervariable region residues as defined herein.

Papain digestion of antibodies produces two identical antigen-binding fragments called Fab'' fragments, each with one antigen-binding site, and a residual Fc'' fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

Fv is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or

- 31 048725 half of Fv, containing only three hypervariable regions specific for the antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear at least one free thiol group.

F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

The light chains of antibodies from any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains.

Single-chain Fv or scFv antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). Anti-ErbB2 scFv antibody fragments are described in WO 93/16185; U.S. Patent Nos. 5,571,894; and 5,587,458.

The term diabodies refers to small antibody fragments with two antigen-binding sites, the fragments comprising a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to pair the two domains on one chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al (1993) Proc. Natl. Acad. Sci. USA 90:6444–6448.

Humanized forms of non-human (e.g. rodent) antibodies are chimeric antibodies that contain minimal sequence derived from a non-human immunoglobulin. Humanization is a method of transferring the murine antigen-binding information to a non-immunogenic human antibody acceptor and has resulted in many therapeutically useful drugs. The humanization method typically begins with the transfer of all six murine complementarity determining regions (CDRs) to the human antibody framework (Jones et al, (1986) Nature 321:522-525). These CDR-grafted antibodies typically do not retain their original antigen-binding affinity, and in fact the affinity is often severely impaired. In addition to the CDRs, selected framework residues of the non-human antibody must also be incorporated to maintain the proper conformation of the CDRs (Chothia et al (1989) Nature 342:877). Transfer of key murine framework residues to a human acceptor to maintain the structural conformation of the grafted CDRs has been shown to restore antigen binding and affinity (Riechmann et al., (1992) J. Mol. Biol. 224, 487-499; Foote and Winter, (1992) J. Mol. Biol. 224:487-499; Presta et al., (1993) J. Immunol. 151, 2623-2632; Werther et al., (1996) J. Immunol. Methods 157:4986-4995; and Presta et al (2001) Thromb. Haemost. 85:379-389). For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which hypervariable region residues of the recipient have been replaced by hypervariable region residues of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and potency. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding residues of non-human origin. In addition, humanized antibodies may contain residues that are not present in either the recipient antibody or the donor antibody. These modifications are made to further improve the performance of the antibody. In general, a humanized antibody will include substantially all of at least one, and usually two, variable domains in which all or substantially all of the hypervariable loops correspond to loops of a non-human immunoglobulin and all or substantially all of the FRs are human immunoglobulin sequences. The humanized antibody will optionally also include at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. For more information, see U.S. Patent No. 6,407,213; Jones et al (1986) Nature, 321:522-525; Riechmann et al (1988) Nature 332:323-329; and Presta, (1992) Curr. Op. Struct. Biol., 2:593-596.

A parent antibody is an antibody comprising an amino acid sequence in which one or more amino acid residues have been replaced by one or more cysteine residues. A parent antibody may comprise a natural or wild type sequence. A parent antibody may have pre-existing amino acid sequence modifications (such as additions, deletions, and/or substitutions) compared to other native, wild type, or modified forms of the antibody. A parent antibody is directed against an intere

- 32 048725 of the target antigen. Antibodies directed against non-polypeptide antigens (such as tumor-associated glycolipid antigens; see U.S. Patent No. 5,091,178) are also provided.

An isolated antibody is an antibody that has been identified and separated and/or recovered from a component of its natural environment. Contaminating components of its natural environment are materials that may interfere with diagnostic or therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In specific embodiments, the antibody will be purified to (1) greater than 95% by weight of the antibody as determined by the Lowry method, or greater than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a gas-phase protein sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or silver staining. An isolated antibody includes an antibody in situ in recombinant cells, since at least one component of the antibody's natural environment will not be present. However, typically the isolated antibody is obtained by at least one purification step.

An antibody that binds a molecular target or antigen of interest is capable of binding that antigen with sufficient affinity such that the antibody can be used to target a cell expressing the antigen.

The terms treat or cure refer to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (reduce) an undesirable physiological change or disorder, such as the development or spread of cancer. For the purposes of this invention, useful or desired clinical results include, but are not limited to, relief of symptoms, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease state, delay or slowing of the progression of the disease, improvement or temporary alleviation of the disease state, and remission (partial or complete), detectable or undetectable. Treatment may also mean an increase in life expectancy compared to the expected survival in the absence of treatment. Those in need of treatment include those who already have the condition or disorder, as well as those who are prone to the condition or disorder, or those in whom the condition or disorder is to be prevented.

Phage display is a technique by which variant polypeptides are displayed as fusion proteins with a coat protein on the surface of phage particles, such as filamentous phage. One advantage of phage display is that large libraries of randomized protein variants can be quickly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide and protein libraries on phage has been used to screen millions of polypeptides for those that have specific binding properties. Multivalent phage display techniques have been used to display small random peptides and small proteins, usually through fusion to PIII or PVIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct. Biol., 3:355–362 (1992), and references cited therein. In monovalent phage display, a library of proteins or peptides is fused to the phage coat protein or part of it and expressed at low levels in the presence of the wild-type protein. Avidity effects are reduced compared with polyvalent phage so that sorting is based on intrinsic ligand affinity and phagemid vectors are used to facilitate DNA manipulation. Lowman and Wells, Methods: A companion to Methods in Enzymology, 3:2050216 (1991). Phage display includes methods for producing antibody-like molecules (Janeway, C, Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., Garland Publishing, New York, p 627628).

A phagemid is a plasmid vector that has a bacterial replication pattern, such as Co1E1, and a copy of the intergenic region of a bacteriophage. A phagemid can be used with any known bacteriophage, including filamentous bacteriophage and lambdoid bacteriophage. The plasmid will also typically contain a selectable antibiotic resistance marker. DNA segments cloned into these vectors can be propagated as plasmids. When cells carrying these vectors are equipped with all the genes necessary to produce phage particles, the plasmid replication mode changes to rolling circle replication to create copies of a single strand of plasmid DNA and package the phage particles. A phagemid can produce infectious or non-infectious phage particles. This term includes phagemids that contain a phage coat protein gene or fragment thereof linked to a heterologous polypeptide gene in a gene fusion such that the heterologous polypeptide is displayed on the surface of the phage particle. The compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some embodiments, such salts are prepared from inorganic or organic acids or bases. For a review of suitable salts, see, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19 and Remington: The Science and Practice of Pharmacy, 20th Ed., A. Gennaro (ed.), Lippincott Williams & Wilkins (2000).

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In the present invention, an Ab moiety (i.e., antibodies, antibody fragments, and/or antigen fragments) may be conjugated to more than one drug-containing moiety. In some embodiments, an Ab may be conjugated to 1-20 drug-containing moieties. In some embodiments, an Ab may be conjugated to 1-10 drug-containing moieties. In some embodiments, an Ab may be conjugated to 1-5 drug-containing moieties. In some embodiments, an Ab may be conjugated to 1 or 2 drug-containing moieties. In some embodiments, an Ab may be conjugated to one drug-containing moiety.

The compounds described herein may be in the form of pharmaceutically or pharmaceutically acceptable salts. In some embodiments, such salts are prepared from inorganic or organic acids or bases. For a review of suitable salts, see, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19 and Remington: The Science and Practice of Pharmacy, 20th Ed., A. Gennaro (ed.), Lippincott Williams & Wilkins (2000).

Examples of suitable acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

Examples of suitable base addition salts include ammonium salts; alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine; and salts with amino acids such as arginine, lysine, and the like.

For example, Berge lists the following FDA-approved commercially sold salts: anions acetate, besylate (benzenesulfonate), benzoate, bicarbonate, bitartrate, bromide, calcium edetate (ethylenediaminetetraacetate), camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate (ethylenediaminetetraacetate), edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate), esylate (ethanesulfonate), fumarate, gluceptate (glucoheptonate), gluconate, glutamate, glycollylarsanilate (glycollamidophenylarsonate), hexylresorcinate, hydrabamine (N,I-di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate (2-hydroxyethanesulfonate), lactate, lactobionate, malate, maleate, mandelate, mesylate (methanesulfonate), methyl bromide, methyl nitrate, methyl sulfate, mucate, napsylate (2-naphthalenesulfonate), nitrate, pamoate (embonate), pantothenate, phosphate/saltacturonate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate (8-chlorotheophyllinate), and triethiodide; organic cations benzathine (N,N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), and procaine; and metal cations of aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.

Berge additionally lists the following non-FDA approved commercially sold (outside the US) salts: adipate, alginate, aminosalicylate, anhydromethylene citrate, arecoline, aspartate, bisulfate, butyl bromide, camphorate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, hydrofluoride, hydroiodide, methylenebis(salicylate), napadisylate (1,5-naphthalenedisulfonate), oxalate, pectinate, persulfate, phenylethylbarbiturate, picrate, propionate, thiocyanate, tosylate, and undecanoate; organic cations benetamine (N-benzylphenethylamine), clemizole (1-chlorobenzyl-2-pyrrolyldin-1'-ylmethylbenzimidazole), diethylamine, piperazine, and tromethamine (tris(hydroxymethyl)aminomethane); and the metal cations barium and bismuth.

The compounds described herein may also include suitable carriers, excipients and excipients, which may vary depending on the route of administration.

In some embodiments, the pharmaceutical compositions can be formulated into a suitable dosage form for parenteral administration. These formulations can be prepared by various methods known in the art. The pharmaceutical compositions can be administered directly into the bloodstream, into a muscle, or directly into an organ. Suitable routes of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous. Suitable devices for parenteral administration include needle injectors, needle-free injectors, and infusion methods.

Compositions for parenteral administration are usually aqueous solutions which may contain auxiliary substances such as salts, carbohydrates and buffering agents. However, the composition can also be prepared as a sterile non-aqueous solution or as a dried form for use in combination with a suitable vehicle such as sterile pyrogen-free water.

The preparation of parenteral compositions under sterile conditions, for example by lyophilization, can readily be accomplished using standard techniques well known to those skilled in the art.

- 34 048725

Compositions for parenteral administration may be formulated with immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release. Thus, the compositions may be formulated as a solid, semi-solid or thixotropic liquid for administration as an implanted depot providing modified release of the active agent.

Parenteral formulations may be mixed with other suitable pharmaceutically acceptable excipients used in parenteral dosage forms, such as, but not limited to, preservatives.

In another embodiment, the pharmaceutical compositions can be formulated into suitable oral dosage forms such as tablets, capsules, powders, granules, suspensions, solutions, emulsions, and the like. Other suitable carriers such as disintegrants, diluents, chelating agents, binders, glidants, lubricants, fillers, excipients, anti-adhesive agents, and the like may be present.

Preparations for oral administration may also contain other suitable pharmaceutical excipients such as sweeteners, carriers/wetting agents, colorants, flavors, preservatives, viscosity enhancing/thickening agents, etc.

The dosage of the pharmaceutical compositions of the present invention can be adapted to the individual patient.

Lipid conjugates.

In some embodiments, the present invention relates to a compound of formula (VI):

or a pharmaceutically acceptable salt thereof, wherein:

a is an integer from 1 to 20;

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

n is 0 or 1;

D-NH- is part of an amino-substituted compound, where the amino-substituted compound has the formula D-NH2;

each R ¹ is independently selected from C ₁ -C ₄ -alkyl, O-C ₁ -C ₄ -alkyl and halogen;

R ² is selected from C ₁ -C ₄ -alkyl and -(CH ₂ CH ₂ O) _s -CH ₃ ; where s is an integer from 1 to 10;

R ³ and R ³ are each independently selected from hydrogen and C ₁ -C ₃ -alkyl;

L is a cleavable linker; and

LP is a lipid.

In some embodiments, D-NH2 is a STING modulator. In some embodiments, D-NH2 is a cyclic dinucleotide (CDN) or a CDN-like compound. In some embodiments, D- _NH2 is one of the formulas described herein.

In some embodiments of the present invention, a compound of formula (VI) is provided, wherein L has the formula

where W, Z, t, U, Q and U' have the meanings described herein.

In some embodiments, the compound of formula (VI) is associated with a lipid complex. The term lipid complex is a well-recognized term in the preparation of pharmaceutical compounds. Lipid complexes are characterized by a non-covalent association between the lipid and the compound of formula (VI). Without being limited by any particular theory, the compound of formula (VI) may be associated with the lipid complex through an electrostatic interaction, a hydrophilic interaction, a hydrophobic interaction, or any combination thereof.

In some embodiments, the lipid complexes described herein are in the form of lipid particles. In one embodiment, the complexes are in the form of lipid nanoparticles. In one embodiment, the complexes are in the form of liposomes. In some embodiments, the liposomes of the invention comprise a lipid monolayer or lipid multilayer. In some embodiments, the liposome comprises a lipid bilayer and an aqueous core. In some embodiments, the liposome comprises a lipid bilayer and an aqueous core.

- 35 048725 embodiments, the average particle size of the liposome is from about 5 nm to about 1 μm. In some embodiments, the average particle size of the lipid nanoparticle or liposome is from about 5 nm to about 500 nm, from about 10 nm to about 150 nm, and from about 30 nm to about 100 nm. In some embodiments, the compound of formula (V) is in an aqueous core, a lipid bilayer, or a combination thereof.

In some embodiments, the LP may be cholesterol or a phospholipid. In some embodiments, the LP is a phospholipid. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, soybean phospholipid, and egg yolk phospholipid. In some embodiments, the phospholipid is phosphatidylethanolamine. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine.

In some embodiments, the present invention provides a lipid complex comprising a compound of formula (VI). In some embodiments, the lipid complex may be a liposome.

In some embodiments, the lipid complex comprises from about 0.5 to about 40 mol % of a compound of formula (VI) or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complex comprises from about 1 to about 35 mol % of a compound of formula (VI) or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complex comprises from about 1 to about 30 mol % of a compound of formula (VI) or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complex comprises from about 1 to about 20 mol % of a compound of formula (VI) or a pharmaceutically acceptable salt thereof. In some embodiments, the lipid complex comprises from about 1 to about 15 mol % of a compound of formula (VI) or a pharmaceutically acceptable salt thereof.

In some embodiments, the lipid complex further comprises one or more additional phospholipids. In some embodiments, the phospholipid is selected from phosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, soybean phospholipid, and egg yolk phospholipid.

In some embodiments, the lipid complex comprises from about 30 to about 90 mol% of one or more phospholipids. In some embodiments, the lipid complex comprises from about 40 to about 85 mol% of one or more phospholipids. In some embodiments, the lipid complex comprises from about 50 to about 80 mol% of one or more phospholipids.

In some embodiments, the lipid complex further comprises a fatty alcohol or cholesterol. In some embodiments, the lipid complex comprises from about 5 to about 35 mole percent cholesterol. In some embodiments, the lipid complex comprises from about 5 to about 30 mole percent cholesterol. In some embodiments, the lipid complex comprises from about 5 to about 25 mole percent cholesterol.

In some embodiments, the lipid complex further comprises at least one PEGylated phospholipid. In some embodiments, the at least one PEGylated lipid is a PEGylated phospholipid. In some embodiments, the PEGylated phospholipid is selected from distearoyl-sn-glycero-3-phosphoethanolamine, distearoyl-sn-glycero-3-phosphoethanolamine, dipalmitoyl-sn-glycero-3-phosphoethanolamine, dipalmitoyl-sn-glycero-3-phosphoethanolamine, dimyristoyl-sn-glycero-3-phosphoethanolamine, dimyristoyl-sn-glycero-3-phosphoethanolamine,

5000)-1,22000)-1,22000)-1,25000)-1,22000)-1,25000)-1,22000)-1,22000)of N(carbonyl methoxypolyethylene glycol), N(carbonyl methoxypolyethylene glycol), N(carbonyl methoxypolyethylene glycol), N(carbonyl methoxypolyethylene glycol), N(carbonyl methoxypolyethylene glycol), N(carbonyl methoxypolyethylene glycol), N(carbonyl methoxypolyethylene glycol, distearoyl-rac-glycerol, and N(carbonyl methoxypolyethylene glycol 5000)-distearoyl-rac-glycerol, a pharmaceutically acceptable salt, or a mixture thereof. In some embodiments The PEGylated phospholipid is N-(carbonylmethoxypolyethyleneglycol 2000)-1,2-distearoyl-snglycero-3-phosphoethanolamine.

In some embodiments, the lipid complex comprises from about 0 to about 6 mol % of one or more PEGylated phospholipids. In some embodiments, the lipid complex comprises from about 0.5 and 5 mol % of one or more PEGylated phospholipids. In some embodiments, the lipid complex comprises from about 1 and 5 mol % of one or more PEGylated phospholipids. In some embodiments, the lipid complex comprises about 3 mol % of one or more PEGylated phospholipids. In some embodiments, the lipid complex comprises about 4 mol % of one or more PEGylated phospholipids. In some embodiments, the lipid complex comprises about 5 mol % of one or more PEGylated phospholipids.

Method of using compounds and compositions.

Some of the compounds described in this document are STING agonists and thus

- 36 048725 zom, are useful for stimulating the immune response in their subjects. The compositions can be used in the treatment of viruses.

The compounds of the present invention exhibit STING modulating/agonistic activity. Some compounds of the present invention may be superior in terms of potency, pharmacokinetics (e.g. absorption, distribution, metabolism, excretion), solubility (e.g. water solubility), drug interactions (e.g. drug metabolizing enzyme inhibitory effect), safety (e.g. acute toxicity, chronic toxicity, genetic toxicity, reproductive toxicity, cardiotoxicity, carcinogenicity, central toxicity) and/or stability (e.g. chemical stability, enzyme resistance) and may be useful as a drug.

The compound of the present invention can be used to increase STING activity in a mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, human).

The compound of the present invention can be used as a medicine, such as an agent for the prevention or treatment of diseases that can be affected by STING (herein, sometimes abbreviated as STING-related diseases), such as cancer diseases, such as colorectal cancer (e.g., colorectal cancer, rectal cancer, anal cancer, familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, malignant mesothelioma), mesothelioma, pancreatic cancer (e.g., pancreatic ductal carcinoma, pancreatic endocrine tumor), pharyngeal cancer, laryngeal cancer, esophageal cancer, gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma), cancer duodenal cancer, small bowel cancer, breast cancer (e.g., invasive ductal carcinoma, non-invasive ductal carcinoma, inflammatory breast cancer), ovarian cancer (e.g., epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian tumor of low malignant potential), testicular tumor, prostate cancer (e.g., hormone-dependent prostate cancer, non-hormone-dependent prostate cancer, castration-resistant prostate cancer), liver cancer (e.g., hepatocellular carcinoma, primary liver cancer, extrahepatic bile duct cancer), thyroid cancer (e.g., medullary thyroid carcinoma), kidney cancer (e.g., renal cell carcinoma (e.g., clear cell carcinoma of the renal cell), transitional cell carcinoma of the renal pelvis and ureter), uterine cancer (e.g., cervical cancer, endometrial cancer, sarcoma uterus), gestational choriocarcinoma, brain tumors (eg, medulloblastoma, glioma, pineal astrocytic tumors, pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma), retinoblastoma, skin cancer (eg, basal cell carcinoma, malignant melanoma), sarcomas (eg, rhabdomyosarcoma, leiomyosarcoma, soft tissue sarcoma, spindle cell sarcoma), malignant bone tumor, bladder cancer, blood cancer (eg, multiple myeloma, leukemia (eg, acute myelogenous leukemia), malignant lymphoma, Hodgkin's disease, chronic myeloproliferative disorder), primary cancer of unknown form; cancer growth inhibitor; inhibitor of cancer metastasis; apoptosis promoter; agent for the treatment of precancerous lesions (eg, myelodysplastic syndromes); and the like.

In some embodiments, a compound of this invention can be used as a medicine for colorectal cancer, breast cancer, skin cancer, malignant lymphoma, or lung cancer.

In addition, the compound of the present invention or the combination agent of the present invention can be used simultaneously with non-drug therapy. More specifically, the compound of the present invention or the combination agent of the present invention can be combined with non-drug therapy such as (1) surgery, (2) hypertensive chemotherapy using angiotensin II, etc., (3) gene therapy, (4) thermotherapy, (5) cryotherapy, (6) laser cauterization, and (7) radiation therapy.

For example, by using the compound of the present invention before or after the above-mentioned surgical operation and the like, or before or after a combination treatment of two or three kinds thereof, effects such as preventing the emergence of resistance, prolonging pain-free survival, suppressing metastasis or recurrence of cancer, prolonging life, etc. can be obtained.

In addition, it is possible to combine the treatment with the compound of the present invention or the combination agent of the present invention with the supportive therapy of (i) administration of an antibiotic (e.g., a β-lactam type such as pansporin, etc., a macrolide type such as clarithromycin, etc.) for complications with various infectious diseases, (ii) administration of high-calorie blood transfusion, an amino acid preparation, or a general vitamin preparation to improve malnutrition, (iii) administration of morphine to reduce pain, (iv) administration of a pharmaceutical agent to alleviate side effects such as nausea, vomiting, anorexia, diarrhea, leukopenia, thrombocytopenia,

- 37 048725 disease, decreased hemoglobin concentration, hair loss, hepatopathy, renal disease, DIC syndrome, fever and the like, and (v) administration of a pharmaceutical preparation for suppressing multidrug resistance of cancer, etc.

Examples

General synthetic methods and intermediates.

The compounds of the present invention can be prepared by one of ordinary skill in the art in light of the present invention and knowledge in the art and/or by reference to the schemes shown below and the synthetic examples. Typical synthetic routes are provided in the schemes below and in the examples. It should be understood that variables (e.g., R groups) shown in the following schemes and examples are to be read independently of variables occurring elsewhere in the invention. One skilled in the art will readily understand how the schemes and examples shown below illustrate the preparation of the compounds described herein.

Scheme 1. General method for converting a carboxylic acid to the corresponding acid chloride or (isobutyl carbonate) propionic anhydride.

About R ⁶

BUT Yc Vos

I

or (COCI) ₂ . THF, DCM or Ghose reagent, DCM

About R ⁶

W A

E Yc Vos a

Scheme 1 shows a general route for the activation of a carboxylic acid i to the corresponding acid chloride or (isobutylcarboxylic)propionic acid such as ii, where Yc is an alkyl or aromatic chain, ^R6 is methyl or an alkyl chain containing a polyether. The preparation of ii, where E is _OCO2CH2CH ( _CH3 ) ₂ , can be achieved by treating i with isobutyl chloroformate in the presence of base. When E is Cl, formation of ii is achieved by treating i with oxalyl chloride or Ghosez's reagent (see α-Chloro enamines, reactive intermediates for synthesis: 1-Chloro-N,N,2-trimethylpropenylamine. Haveaux B., et al. Org. Synth. 1980, 59, 26.)

Scheme 2. General method for attaching a spacer to an amino group in a drug moiety using a stepwise approach.

1. TMSCI, pyridine

2.

D-NH2____ε

Vos

D

3. N&3-3HF. МеОН vl

Scheme 2 shows the general method for attaching a spacer such as ii to an amino group in a drug moiety such as iii. After compound iii is globally silyl protected by treatment with trimethylsilyl chloride and pyridine, it is reacted with the activated spacer ii. Removal of the silyl groups with NEt ₃ -3HF yields iv. Deprotection of the amine iv is accomplished by treatment with an acid such as TFA or HCl, and the resulting amine salt is treated with activated dipeptide carbonate v to yield vi.

Scheme 3. General method for attaching a spacer to an amino group in a drug moiety using a stepwise approach.

- 38 048725

Scheme 3 shows a general method for attaching a spacer such as ii to an amino group in a drug moiety such as iii-a, where X ¹ and X ³ are oxygen, sulfur, or CH2, X ³ is nitrogen or CH, R ³ is hydrogen or fluorine, R ⁴ is hydroxyl, hydrogen, fluorine, or taken together with R ³ to form OCH2, R ^c is OH or SH, R ^d is methyl or (CH2) ₃ NHC(O)NH2. After compound iii-a is globally silyl protected by treatment with trimethylsilyl chloride and pyridine, it is reacted with the activated spacer ii. Removal of the silyl groups with NEt ₃ -3HF gives iv-a. Deprotection of the amine iv-a is accomplished by treatment with an acid such as TFA or HCl, and the resulting amine salt is treated with an activated dipeptide carbonate v to yield vi-a.

Scheme 4. General method for attaching a spacer to an amino group in a drug moiety using a stepwise approach.

Scheme 4 shows a general method for attaching a spacer group such as ii to an amino group in a drug moiety such as iii-b, according to the general method described in Scheme 3.

- 39 048725

Scheme 5. General pathway for the creation of a spacer peptide acid chloride.

But

Vos (COCIh

THF.DCM

Ϊ f=^ ^N Y ^A 'N ^Jl V ^N Boc o ⁿ Zh o

AI

Scheme 5 shows a general method for preparing the acid chloride viii. Deprotection of the Boc amine i with an acid such as TFA or HCl is followed by treatment with an activated carbonate v to give the carboxylic acid vii. Conversion of vii to the acid chloride viii can be accomplished by treatment with oxalyl chloride or sulfonyl chloride.

Scheme 6. Alternative general method for attaching a spacer to an amino group in a drug moiety.

Scheme 6 shows an alternative general method for the direct conversion of the payload iii to compound ix. After general silyl protection with trimethylsilyl chloride, iii is treated with the acid chloride viii. Removal of the silyl group yields ix.

Scheme 7. Alternative general method for attaching a spacer to an amino group in a drug moiety.

Scheme 7 shows an alternative general method for directly converting a iii-a payload to an ix-a compound in accordance with the general method described in Scheme 6.

- 40 048725

Scheme 8. Alternative general method for attaching a spacer to an amino group in a drug moiety.

gx-b

Scheme 8 shows an alternative general method for attaching a spacer to an amino group in a drug moiety such as iii-b, in accordance with the general method described in Scheme 6.

Figure 9. General path for preparing linker payloads.

Scheme 9 shows a general method for preparing a linker payload xi from vi, where Yd is a linear or branched alkyl chain or a polyether chain, and Z is a conjugation marker such as maleimide, BCN, or DBCO. Deprotection of the amine vi with an acid such as TFA or HCl is followed by reaction with an activated ester x to yield the linker payload xi.

- 41 048725

Figure 10. General path for preparing linker payloads

ABOUT

Scheme 10 shows a general method for preparing the linker payload xi-a from vi-a according to the general method described in Scheme 9.

Figure 11. General path for preparing linker payloads

Scheme 11 shows a general method for attaching a conjugation marker to compounds such as vi-b, following the general method described in Scheme 9.

- 42 048725

Figure 12. General pathway for preparing linker payloads

XI

Scheme 12 shows an alternative method for preparing the linker payload xi from iv. Deprotection of the amine iv with an acid such as TFA or HCl is followed by reaction xii to give xi.

Figure 13. General pathway for preparing linker payloads

Scheme 13 shows an alternative method for preparing the linker payload xi-a from iv-a according to the general method described in Scheme 12.

Figure 14. General path for preparing linker payloads

Scheme 14 shows a general method for preparing a glucuronidate-containing linker payload xvi. Deprotection of amine iv with an acid such as TFA or HCl is followed by reaction with an activated carbonate xiii to yield xiv. Generalized hydrolysis of the acetate and removal of the FMOC group in xiv with a base such as lithium hydroxide yields xv. Treatment of xv with an activated conjugation marker x yields a glucuronidate-containing linker payload xvi.

Scheme 15. General route for preparing linker payloads • ι;·Λί

Scheme 15 shows a general method for preparing a glucuronidate-containing linker payload xvi-a, according to the general method described in Scheme 14.

Figure 16. General path for preparing vJ linker payloads

TFA D-CM

MIV".

3. TFA, DCM w® o Ai

Scheme 16 shows a general method for preparing a terminal amine-containing linker payload xviii, where Ye is a linear alkyl chain or an alkyl chain containing a polyether. Deprotection of the amine vi with an acid such as TFA or HCl is followed by reaction with an activated conjugation marker xvii. Removal of the terminal protecting group with an acid such as TFA yields a terminal amine-containing linker payload xviii.

- 44 048725

Figure 17. General path for preparing linker payloads

Scheme 17 shows a general method for preparing the terminal amine-containing linker payload xviii-a following the general method described in Scheme 16.

Figure 18. General pathway for preparing linker payloads

1.TMSCI, pyridine d-nh ₂

Ui

NEt _:i DMF

He is χχϋ

Scheme 18 shows a general method for preparing a payload linker such as xxii, which includes a dipeptide spacer where R ⁷ is hydrogen, an alkyl, or an aromatic group. Compound iii is globally protected by treatment with trimethylsilyl chloride and pyridine and then treated with Boc-proline succinimide ester. Removal of the silyl group with a fluoride source such as NEt3-3HF gives xix. Deprotection of amine xix with an acid such as TFA or HCl is followed by reaction with a succinimide ester-activated amino acid xx to give xxi. Deprotection of amine xxi with an acid such as TFA or HCl is followed by installation of a maleimide marker to yield payload linker xxii.

- 45 048725

Figure 19. General pathway for preparing linker payloads.

Scheme 19 shows a general method for preparing a linker payload such as xxii-a according to the general method described in Scheme 18.

Preparation of exemplary ADCs and intermediates AA determinations LCMS method using ammonium acetate

Ac acetate

ACN acetonitrile atm atmosphere water, aqueous

BCN bicyclo[6.1.0]nonine

LQO below limit of quantification

- 46 048725

Bn benzyl

Vos m/tem-butoxycarbonyl tBu shreti-butyl

Bz benzoyl

From Celsius calculated

Cbz benzoyloxycarbonyl

DAR drug-to-antibody ratio

DBCO dibenzocyclooctyne

DCC N,N-dicyclohexylcarbodiimide

DCM dichloromethane

DIPEA N, N-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMA N,N-dimethylacetamide

DMB 2,4-dimethoxybenzyl

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

DTT dithiothreitol ε extinction coefficient

E 0.1% 0.1% solution extinction coefficient

EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide

EDTA ethylenediaminetetraacetic acid

Et ethyl

EtOH ethanol

EtOAc ethyl acetate

MA LCMS method using formic acid

Fmoc fluorenylmethyloxycarbonyl h hours

HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3 oxide hexafluorophosphate

HIC hydrophobic interaction chromatography

HOAt 1-hydroxy-7-azabenzotriazole

HPLC high pressure liquid chromatography

HRMS high resolution mass spectrometry

IC50 inhibitory concentration 50%

LC liquid chromatography

LCMS liquid chromatography mass spectrometry.

M/z mass to charge

MHz mega hertz

Me methyl

- 47 048725

MeOH methanol μΜ microns min minutes ml milliliters

MS mass spectrum

MWCO molecular weight cutoff

NMR nuclear magnetic resonance

PBS Phosphate buffered saline

PE petroleum ether

Ph phenyl psi pounds per square inch psig gauge pressure in pounds per square inch

Rug pyridine

QTOF quadrupole time of flight ct room temperature

SEC Size Exclusion Chromatography

SFC supercritical fluid chromatography

TBS otreot-butylmethylsilyl

TCER (tris(2-carboxyethyl)phosphine)

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TIPS triisopropylsilyl

TMS trimethylsilyl

Tris tris(hydroxymethyl)aminomethane

UPLC Ultra-Performance Liquid Chromatography

Analytical methods.

NMR conditions.

1H NMR spectra were recorded on a Bruker 400 MHz spectrometer unless otherwise stated. ³¹ P NMR spectra were recorded at 162 MHz on a Bruker 400 MHz spectrometer and obtained with 1H decoupling unless otherwise stated. ¹⁹ F NMR spectra were recorded at 376 MHz on a Bruker 400 MHz spectrometer unless otherwise stated.

MSVR conditions.

HRMS spectra were acquired using an Agilent 1260 Infinity II Bioinert Multisampler, bioinert column compartment, bioinert quaternary pump, DAD multiwavelength detector, and Agilent 6545 LC-QTOF mass spectrometers with an Osaka Soda Capcell PAK. C1 UG120 (5 μm, 2.0 mm i.d. x 35 mm long) reversed-phase column. LC solvent systems consisted of 0.1% formic acid in water for mobile phase A and 0.1% formic acid in acetonitrile for mobile phase B or equivalent solvent system. Typically, LC elution was initiated at a mobile phase A/B ratio of 95%/5%. After an isocratic flow interval of typically 0.5 min, the mobile phase was replaced with 100% B over a 1.5 min linear gradient. The flow rate was 0.7 ml/min over a 5 min run.

Typically, 2 μL of 1 mM DMSO solution was injected into the mass spectrometer. The QTOF settings were: gas temperature 35°C, drying gas flow rate = 10 L/min, nebulizer pressure = 55 psi, sheath gas temperature and flow = 35°C and 12 L/min, respectively. The following voltage settings were used: capillary voltage = 4500 V, nozzle voltage = 2000 V, fragmenter voltage = 75 V.

Data were analyzed using Agilent Mass Hunter Qualitative Analysis software, version 8.07.00 SP2. Mass error [ppm] was expressed as the difference between the exact mass and the observed mass divided by the exact mass multiplied by 1 x 10 ⁶ .

LCMS conditions.

LCMS spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100 Series LC system coupled to a Micromass mass spectrometer using reversed-phase C18 columns. Different gradients and run times were chosen to best characterize the compounds.

- 48 048725 The mobile phases were based on ACN/water or MeOH/water gradients and contained either 0.1% formic acid (methods designated as MC) or 10 mM ammonium acetate (methods designated as AA). One example of a solvent gradient that was used was from 100% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid) at a flow rate of 1 mL/min over 16.5 min.

In some cases, LCMS spectra were recorded on an Agilent 1290 Infinity UPLC system coupled to an Agilent 6130 mass spectrometer, a Waters Acquity UPLC system coupled to a Waters Acquity SQ mass spectrometer, or an Agilent 1100 Series LC system coupled to a Waters Micromass ZQ mass spectrometer using reversed-phase C18 columns. Different gradients and run times were chosen to best characterize the compounds. Mobile phases were based on ACN/water or MeOH/water gradients and contained either 0.1% formic acid (methods designated as MC) or 10 mM ammonium acetate (methods designated as AA). One example of a solvent gradient that was used was from 95% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid) at a flow rate of 0.5 mL/min for 5 min.

Preparative HPLC.

Preparative HPLC separations were performed using Sunfire C-18 18x150 mm columns eluting with water-ACN gradients using a Gilson instrument driven by 322 pumps with a 155 UV/visible detector set to 200 to 400 nm. Mass-locked fractions were collected on an Agilent 1100 LC/MSD.

One of ordinary skill in the art will appreciate that modifications to the gradient, column length, and flow rate are possible and that some conditions may be more suitable for compound characterization than others, depending on the chemicals being analyzed.

Preparative SFC.

Preparative SFC was performed using ChiralPak 10, 20, or 30 mm x 250 mm columns (typically IA, IB, IC, ID, IE, and IF), 10 or 20 mm x 250 mm Phenomenex Lux Cellulose-4, or 2-ethylpyridine columns, eluting with the appropriate percentage of supercritical carbon dioxide and an alcohol containing 0.3% diethylamine, 0.3% TEA, 0.3% formic acid, or no acidic or basic additives. Isocratic conditions with flow rates in the range of 10-100 mL/min and a column temperature of 40°C are typical. Preparative SFC was performed on a Jasco SFC precleaner with a UV/Vis fraction set from 200 to 400 nm and a backpressure adjustment of 10 MPa.

One of ordinary skill in the art will appreciate that modifications to the gradient, column length, and flow rate are possible and that some conditions may be more suitable for compound characterization than others, depending on the chemicals being analyzed.

SEC Analytical Terms.

SEC spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100 Series LC systems with a diode array detector using an SEC column (typically Tosoh Biosep TSK Gel, G3000SWxl; P/N 8541; 250A; 5 μm; 7.8 mm x 300 mm) at 280 nm. The mobile phase was 100 mM sodium phosphate, 300 mM sodium chloride, pH 6.8, 10% acetonitrile (v/v), or 1xPBS. A typical run was isocratic at a flow rate of 1 ml/min for 20 min.

HIC analytical conditions.

HIC spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100 Series LC systems with a diode array detector using a HIC column (typically Tosoh Butyl-NPR, 4.6 x 35 mm, 2.5 μm, P/N: 14947) at 280 nm. Mobile phase A was 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7, and mobile phase B was 75% 25 mM sodium phosphate, pH 7, 25% isopropanol. For a typical 20 min run between the initial and final isocratic flow intervals, a 12 min linear gradient from 95%/5% A/B to 100% B would be used.

LC-QTOF conditions:

LCMS spectra were recorded on an Agilent 1260 Bioinert Series LC system coupled to an Agilent 6545 QTOF mass spectrometer using a reversed-phase column heated to 80 °C (typically Agilent, PLRP-S, 5 μm, 1000 A, 2.1 mm x 50 mm). Different gradients and run times were chosen to best characterize the compounds. Mobile phases were based on ACN/water gradients and contained 0.1% formic acid. One example of a solvent gradient that was used was from 95% mobile phase A (mobile phase A=99% water+1% ACN+0.1% formic acid) to 100% mobile phase B (mobile phase B=95% ACN+5% water+0.1% formic acid) with the conditions shown in Table 1.

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Table 1

Time (min) Flow (ml/min) %A %IN 0 0.35 82 18 1 0.35 82 18 2 0.35 70 30 19 0,5 50 50 19,5 0,5 10 90 21 0,5 10 90 21,1 0,5 82 18 22 0,5 82 18

Samples were either native or reduced (20 µL of 1~5 mg/mL ADC solution was treated with 4 µL of 0.5 M DTT at 37°C for 30 min). Raw data were deconvoluted to the appropriate mass range using Agilent BioConfirm software to obtain protein molecular weight, and Agilent DAR calculator was used to calculate DAR.

LC/MS/MS conditions.

LC/MS/MS analysis was performed using a Shimadzu UFLC LC-20AD XR binary pump and a SIL-30AC MP autosampler and AB SCIEX Triple Quad 4500 ESI mass spectrometry system.

Typically, 5 µL aliquots were injected into the LC/MS/MS after passing through a Waters Xselect C18 CSH 3.5u 2.1 mm ID x 30 mm column. Mobile phase A contained 0.1% formic acid in water and mobile phase B contained 0.1% formic acid in 5% water with 95% acetonitrile. The total run time was 3 min at 1.5 mL/min with a linear gradient from 100% A to 100% B in 1.5 min flow rate. The instrument was initially run with 100% aqueous mobile phase solvent for 0.5 min and then ramped up to 100% organic solvent over the next 1.5 min.

Preparative SEC.

Preparative SEC cleanup was performed on a Gilson Preparative HPLC system with UV detection using an SEC column (typically GE Superdex 200 Increase 10/300 GL). The mobile phase was 1xPBS (pH 7.4). A typical run was isocratic at a flow rate of 1 ml/min for 30 min. Fraction collection was triggered based on UV threshold (at 214 and 280 nm).

ADC Concentration:

The ADC concentration was calculated from the UV absorbance at 280 nm measured using the NanoDrop coefficient (2000c; Fisher Scientific) after subtracting the UV absorbance from the corresponding linker-payload constructs.

Example 1.

tert-Butyl N[(2-chlorocarbonylphenyl)methyl]4-methylcarbamate (intermediate 1)

PS-1

Step 1: 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid

To a solution of 2-[(methylamino)methyl]benzoic acid (7.50 g, 44 mmol) in 1,4-dioxane (136 mL) and 1 N sodium hydroxide in water (182 mL) was added di-tert-butyl dicarbonate (19.2 g, 88 mmol). The reaction mixture was allowed to stir at room temperature for 2 h. The reaction mixture was then evaporated to remove the organic solvent and the pH was adjusted to 3 with 1 N HCl. The crude product was extracted with EtOAc (3x). The combined organic phases were washed with brine, dried (Na2SO4), filtered and evaporated. The crude compound was purified by silica gel chromatography (0-50% EtOAc/DCM) to give 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid (8.88 g, 75%). LCMS (AA): m/z=264,l (M-H).

Step 2: tert-butyl N-[(2-chlorocarbonylphenyl)methyl]4-methylcarbamate, intermediate 1.

1-Chloro4,^2-trimethylpropenylamine (0.52 mL, 4.0 mmol) was slowly added to a solution of 2(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid (525 mg, 2.0 mmol) in DCM (16 mL) at

- 50 048725 °C. The reaction mixture was then warmed to room temperature and stirred for 1 hour. The reaction mixture was then evaporated to dryness to give crude tert-butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate (intermediate 1, 562 mg, 100%).

Example 2.

tert-Butyl N[[2-(2-chloro-2-oxoethyl)phenyl]methyl]-N-methylcarbamate (intermediate 2)

PS-2

Step 1: 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid.

To a solution of 2-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)acetic acid (1.50 g, 5.7 mmol) in THF (20 mL) was added iodomethane (2.8 mL, 45.2 mmol) at room temperature. The reaction mixture was cooled to 0 °C and sodium hydride (60% dispersion in mineral oil, 905 mg, 22.6 mmol) was added in 3 portions. The reaction mixture was then allowed to stir at room temperature for 16 h. The reaction mixture was quenched with water and the organic solvent was removed by evaporation. The aqueous phase was acidified to pH 3 using 1 N HCl and the crude product was extracted with Et ₂ O (2x). The combined organic phases were washed with brine, dried (Na2SO4), filtered and evaporated to give 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid (1.36 g, 86%). LCMS (AA): m/z 280.2 (M+H).

Step 2: tert-butyl N-[[2-(2-chloro-2-oxoethyl)phenyl]methyl]-N-methylcarbamate, intermediate 2.

The title compound was prepared according to the procedure described in Example 1, step 2, starting from 2-[2-[[tert-butoxycarbonyl(methyl)amino]methyl]phenyl]acetic acid (650 mg, 2.33 mmol) to give crude intermediate 2 (693 mg, 100%).

Example 3.

Isobutoxycarbonyl 4-[tert-butoxycarbonyl(methyl)amino]butanoate (intermediate 3) o

PS-3

Step 1: 4-(((tert-butoxycarbonyl)(methyl)amino)methyl)butanoic acid

The title compound was prepared according to the procedure described in Example 2, Step 1 substituting 4-(tert-butoxycarbonylamino)butanoic acid (2.92 g, 14.4 mmol) for 2-(2-(((tert-butoxycarbonyl)amino)methyl)phenyl)acetic acid to give 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (2.89 g, 93%). LCMS (AA): m/z=216.1 (M-H).

Step 2: isobutoxycarbonyl 4-[tert-butoxycarbonyl(methyl)amino]butanoate, intermediate 3.

To a solution of 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (360 mg, 1.57 mmol) and triethylamine (0.24 mL, 1.73 mmol) dissolved in DCM (7 mL) and cooled to 0 °C was added dropwise isobutyl chloroformate (0.22 mL, 1.73 mmol). After addition, the reaction mixture was stirred at room temperature for 2 h. Then, the mixture was evaporated to dryness to give crude isobutoxycarbonyl 4-[tert-butoxycarbonyl(methyl)amino]butanoate (intermediate 3, 499 mg, 100%).

Example 4.

The compounds listed below were prepared as described in Example 3, replacing 2,4-(tert-butoxycarbonylamino)butanoic acid with the starting material shown in the Table.

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Table 2

Source material Product Protocol noAHA. _to; O O ·, . । . d d X 'Vos Prom. soed.-4 ^Η0 γΟ O Vos .Λ^γγΟ о О Вос Prom. conn.-5 Stage 1 is skipped

Example 5.

N-{ 9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1Hpurin-2-yl}-2-[(methylamino)methyl]benzamide (Intermediate 6)

Step 1: tert-Butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-Spurin-2-yl}carbamoyl)benzyl]methylcarbamate

2-Amuho-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclocyclotetradecin-7-yl]-1,9-dihydro-6Hpurin-6-one as the N,N-diethylethanamine salt (Compound I-5c, see PCT/IB2O18/O58846, 160 mg, 0.19 mmol) was dissolved in dry pyridine and evaporated to dryness (3x2 ml), then placed under vacuum for 15 min. The residue was dissolved in pyridine (3 mL) under argon and chlorotrimethylsilane (0.15 mL, 1.13 mmol) was added. The reaction mixture was allowed to stir at room temperature for 30 min. Intermediate 1 (800 mg, 2.82 mmol) dissolved in pyridine (3 mL) was then added via syringe. The reaction mixture was stirred at room temperature under argon for 16 h. The reaction mixture was then evaporated to dryness, MeOH (10 mL) and ammonium hydroxide (28-30% solution in water, 10 mL) were added and the mixture was allowed to stir for 30 min. The reaction mixture was evaporated to dryness and the residue was dissolved in MeOH (15 mL). Triethylamine trihydrofluoride (0.12 mL, 0.75 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was evaporated to dryness, the crude residue was adsorbed onto celite and purified by reverse phase flash column chromatography (0-50% ACN/aqueous triethylammonium acetate (10 mM)) to give tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8

- 52 048725 methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1Hpurin-2-yl}carbamoyl)benzyl]methylcarbamate in the form of N,N-diethylethanamine salt (120 mg, 58%). LCMS (AA): m/z=897.3 (M+H). 1H NMR (400 MHz, CD3OD) δ 8.70 (s, 1H), 8.49 (s, 1H), 8.41 (d, J=6.0 Hz, 1H), 7.70 (d, J=7.5 Hz, 1H), 7.58-7.52 (m, 1H), 7.44-7.39 (m, 1H), 7.33 (d, J=7.8 Hz, 1H), 6.85 (d, J=6.0 Hz, 1H), 6.17 (d, J=8.3 Hz, 1H), 5.64-5.58 (m, 1H), 5.49-5.44 (m, 1H), 5.08-5.02 (m, 1H), 4.88-4.78 (m, 1H), 4.76-4.67 (m, 2H), 4.37-4.21 (m, 3H), 4.06-4.00 (m, 1H), 3.83-3.74 (m, 1H), 2.82 (s, 3H), 2.60-2.32 (m, 4H), 1.54-1.46 (m, 1H), 1.42 (s, 9H). ³¹ P NMR (162 MHz, CD3OD) δ 55.19 (s, 1P), 53.20 (s, 1P).

Step 2: N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}-2-[(methylamino)methyl]benzamide, intermediate 6.

tert-Butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}carbamoyl)benzyl]methylcarbamate as N,N-diethylethanamine salt (50 mg, 0.045 mmol) was added to a round-bottomed flask and cooled to 0 °C. Then a solution of trifluoroacetic acid (0.24 mL, 3.2 mmol) and DCM (0.58 mL) was added via syringe and the reaction mixture was stirred at 0 °C for 30 min. The reaction mixture was then evaporated to dryness and placed under vacuum for 2 h to afford intermediate 6 as the 2,2,2-trifluoroacetate salt (41 mg, 100%). LCMS (AA): m/z 797.1 (M+H).

Example 6.

4-{ [(2S)-2-{ [(2S)-2-{ [6-(2,5-dioxo-2,5-dihydro-III-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}carbamoyl)benzyl]methyl carbamate, Compound 1 (C-1).

- 53 048725

Step 1: 4-['[(^)-2-(['(^)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-rHgpoKCH-2,10-gHOKCHgo-14-(nHpHMHdin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, intermediate product 7

To a solution of tert-butyloxycarbonyl valyl-alanyl-(4-aminobenzyl)-(4-nitrophenyl)carbonate (58 mg, 0.10 mmol) and 4-dimethylaminopyridine (12 mg, 0.10 mmol) in DMF (0.38 ml) and triethylamine (0.055 ml, 0.40 mmol) was added a solution of Intermediate 6 (45 mg, 0.05 mmol) in DMF (1.5 ml) at room temperature. The reaction mixture was allowed to stir at room temperature for 15 min. Celite was added and the mixture was evaporated to dryness. The crude residue absorbed on Celite was purified by reverse phase flash column chromatography (0-40% ACN/aqueous triethylammonium acetate (10 mM)) to give 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-Spurin-2-yl}carbamoyl)benzyl]methylcarbamate as N,N-diethylethanamine salt (Intermediate 7, 10 mg, 16%). LCMS (AA): m/z=1216.3 (M+H). ^1H NMR (400 MHz, CD3OD) δ 8.70 (s, 1H), 8.43 (br s, 1H), 8.40 (d, J=5.9 Hz, 1H), 7.70 (d, J=7.5 Hz, 1H), 7.56-7.46 (m, 3H), 7.42-7.38 (m, 1H), 7.32-7.19 (m,

3H), 6.84 (d, J=5.5 Hz, 1H), 6.15 (d, J=7.3 Hz, 1H), 5.62-5.56 (m, 1H), 5.54-5.48 (m, 1H), 5.08-5.02 (m, 1H),

5.04 (s, 2H), 4.88-4.83 (m, 1H), 4.81-4.76 (m, 2H), 4.53-4.47 (m, 1H), 4.37-4.22 (m, 3H), 4.08-4.03 (m, 1H),

3.92-3.89 (m, 1H), 3.82-3.74 (m, 1H), 2.88 (s, 3H), 2.58-2.30 (m, 4n), 2.10-2.03 (m, 1H), 1.54-1.47 (m, 1H),

- 54 048725

1.46-1.44 (m, 3H), 1.44 (s, 9H), 0.98 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8 Hz, 3H). ³¹ P NMR (162 MHz, CD3OD) δ 55.01 (s, 1P), 53.03 (s, 1P).

Step 2: 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl [2-({9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, intermediate 8

The title compound was prepared according to the procedure described in Example 5, Step 2 substituting Intermediate 7 (10 mg, 0.008 mmol) for tert-butyl [2-({9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate and stirring at room temperature for 10 min with by obtaining 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}carbamoyl)benzyl]methylcarbamate as 2,2,2-trifluoroacetate salt (intermediate 8.9 mg, 100%). LCMS (AA): m/z=1116.3 (M+H).

Stage 3: 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl] amino}propanoyl]amino}benzyl[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxide o-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}carbamoyl)benzyl]methylcarbamate, compound S-1.

To a solution of intermediate 8 (8 mg, 0.007 mmol) and succinimidyl-6-maleimidohexanoate (3.1 mg, 0.010 mmol) in THF (0.20 mL) and DMF (0.10 mL) was added dropwise N,N-diisopropylethylamine (2.5 μL, 0.014 mmol). The reaction mixture was allowed to stir at room temperature for 1 h. The reaction mixture was then evaporated to dryness and the crude residue was purified by reverse phase flash column chromatography (0-100% ACN/aqueous ammonium acetate (10 mM)) to give 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl[2-({9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-3-purin-2-yl}carbamoyl)benzyl]methylcarbamate as the ammonium salt (C-1, 4.2 mg, 45%). LCMS (AA): m/z=1309.4 (M+H). MCBP (m/z): [M+H] ⁺ calcd for C ₅₅ H6 ₆ N ₁₂ O ₁₈ P ₂ S ₂ 1309.3607; found, 1309.3639.

Example 7.

2-[tert-Butoxycarbonyl(methyl)amino]ethyl carbonochloridate (Intermediate 9) triphosgene

DIPEA, THF

CI^O'^ ^x ^' ^N 'Boc ps-9

Step 1: 2-[tert-butoxycarbonyl(methyl)amino]ethyl carbonyl chloride, Intermediate 9

To a solution of tert-butyl N-(2-hydroxyethyl)-N-methylcarbamate (296 mg, 1.69 mmol) in THF (4.2 mL) and N,N-diisopropylethylamine (1 mL, 5.9 mmol) was slowly added triphosgene (752 mg, 2.53 mmol) cooled to 0°C. The reaction mixture was warmed to room temperature and stirred for 1 h. The reaction mixture was filtered, the solids were washed with THF and the filtrate was evaporated to dryness to give crude 2-[tert-butoxycarbonyl(methyl)amino]ethyl carbonochloridate (intermediate 9, 402 mg, 100%).

Example 8.

[4-[[(2S)-2-[[(2S)-2-(tert-Butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[(2-chlorocarbonylphenyl)methyl]-N-methyl carbamate (intermediate 10)

- 55 048725

Step 1: 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid.

The title compound was prepared according to the procedure described in Example 6, Step 1, starting from 2-[(methylamino)methyl]benzoic acid HCl (150 mg, 0.72 mmol) in place of Intermediate 6. Purification by silica gel chromatography (0-25% MeOH/DCM) gave 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid (306 mg, 67%). LCMS (AA): m/z=583.4 (MH).

Step 2: [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N-[(2-chlorocarbonylphenyl)methyl]-N-methyl-carbamate, Intermediate 10.

To a solution of 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid (295 mg, 0.50 mmol) in THF (1.5 mL) cooled to 0 °C was added oxalyl chloride (2.0 M solution in DCM, 0.25 mL, 0.50 mmol) followed by 3 drops of DMF. The reaction mixture was allowed to stir at 0 °C for 45 min. Then the mixture was evaporated to dryness to give crude Intermediate 10 (304 mg, 100%).

Example 8A.

4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]3-methylbutanoyl}amino)propanoyl]amino}benzyl(4-chloro-4-oxobutyl)methylcarbamate.

(Intermediate 34)

Step 1: methyl 4-[{[(4-{[(^)-2-({(^)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl] amino } benzyl)oxy] carbonyl } (methyl)amino]butanoate.

The title compound was prepared according to the procedure described in Example 6, step 1, starting from methyl 4-(methylamino)butanoate HCl (1.44 g, 8.16 mmol) in place of intermediate 6. Purification by silica gel chromatography (0-70% EtOAc/DCM) gave methyl 4-[{[(4{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoate (3.45 g, 92%). LCMS (AA): m/z=549.3 (M-H).

Step 2: 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid.

To a solution of methyl 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoate (3.45 g, 6.27 mmol) in THF (40.7 mL) and water (20.3 mL) was added lithium hydroxide monohydrate (657 mg, 15.7 mmol) at 0 °C. The homogeneous mixture was then warmed to room temperature and stirred for 2 h.

- 56 048725 sov. This reaction mixture was cooled to 0 °C and diluted with hydrochloric acid (1.0 mol/L in water, 1.83 mL). This mixture was adjusted to pH < 4 by adding additional 1 N HCl. This solution was warmed to room temperature and diluted with EtOAc and water. The aqueous phase was further extracted with EtOAc. The combined organic phases were washed with brine and dried over magnesium sulfate. After filtration and removal of solvents, the residue was purified by silica gel chromatography (0-10% MeOH/EtOAc). Removal of solvents gave 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid (3.17 g, 94%) as a white solid. LCMS (AA): m/z=535.3 (M-H).

Step 3: 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]3-methylbutanoyl}amino)propanoyl]amino}benzyl(4-chloro-4-oxobutyl)methylcarbamate.

The title compound was prepared according to the procedure described in Example 8, step 2, starting with 4-[{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}(methyl)amino]butanoic acid (387 mg, 0.721 mmol) instead of 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylmethylamino]methyl]benzoic acid to give crude intermediate 34 (400 mg, 100%) upon evaporation of the reaction mixture.

Example 9.

4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2D0-dioxido-14-(pyrimidin-4-yloxy)2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methyl carbamate (Intermediate 7)

ABOUT

Connection I - 5c

3. NEt _r 3HF. MeOH

4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methyl carbamate, intermediate 7

The title compound was prepared according to the procedure described in Example 5, Step 1, replacing tert-butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate (Intermediate 1) with Intermediate 10. The crude product was purified by reverse phase flash column chromatography (0-50% ACN/aqueous ammonium bicarbonate (5 mM)) to afford Intermediate 7 as the ammonium salt. The material was then dissolved in MeOH (2 mL) and triethylamine (5 mL) was added. The mixture was shaken for 1 min, the solvents were removed and lyophilized overnight to give 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-7-yl]6-oxo-6,9-dihydro-N-purin-2-yl}carbamoyl)benzyl]methylcarbamate as N,N-diethylethanamine salt (147 mg, 48%). LCMS (AA): m/z=1216.3 (M+H). ¹ H NMR identical to example 6, step 1.

- 57 048725

Example 10.

4-{[(^)-2-({(^)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino} benzyl [2-(chlorocarbonyl)benzyl]2,5,8,11-tetraoxatridecan-13-ylcarbamate (intermediate 11)

Step 1: 2-(2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid.

To a solution of 2-carboxybenzaldehyde (1.10 g, 7.3 mmol) dissolved in MeOH (15 mL) was added 2,5,8,11-tetraoxatridecane-13-amine (1.82 g, 8.8 mmol) and the reaction mixture was stirred at rt for 1 h. Sodium borohydride (139 mg, 3.7 mmol) was then added slowly in portions. The reaction mixture was stirred at rt for 1 h. The reaction mixture was quenched by addition of acetone (0.65 mL) and then evaporated to dryness. The crude residue was purified by silica gel chromatography (0-25% MeOH/DCM) to give 2-(2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (2.25 g, 87%). LCMS (AA): m/z=342.3 (M+H).

Step 2: 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid

The title compound was prepared according to the procedure described in Example 6, Step 1, starting from 2-(2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (764 mg, 2.24 mmol) instead of Intermediate 6 using DCM as solvent and stirring for 16 h. Purification by silica gel chromatography (0-25% MeOH/DCM) afforded 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (855 mg, 47%). LCMS (AA): m/z=759.4 (M-H).

Step 3: 4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-(chlorocarbonyl)benzyl]2,5,8,11-tetraoxatridecan-13-ylcarbamate, intermediate 1.1

The title compound was prepared according to the procedure described in Example 8, step 2, starting from 2-(14-{[(4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl)oxy]carbonyl}-2,5,8,11-tetraoxa-14-azapentadecan-15-yl)benzoic acid (591 mg, 0.777 mmol) instead of 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methyl-amino]methyl]benzoic acid to give crude Intermediate 11 (605 mg, 100%) upon evaporation of the reaction mixture.

Example 11.

The compounds listed below in Table 3 were prepared as described in Example 5, replacing tert-butyl N-[(2-chlorocarbonylphenyl)methyl]-N-methylcarbamate (Intermediate 1) with the starting material shown in the table.

- 58 048725

Table 3

Source material Product Analytical data ^C1 GP o _g I - ^L '- Vos Prom. comp.-2 10 equiv. used in Stage 1 ⁰ < tlT 1 X) θ'· 1 Ο 'ϊ Ν -'Χ-'-'ΧΧ » 4 n 1 / \ j / ΝΗ Ν οΊ ^Η< ^ Λ ⁽ f Τ Ό^ρ/ HS' ^υ Industrial connection-12 LCMS (AA): m/z=811.2 (M+H) O 0 | 'Vos Prom. conn.-Z 6 eq. used at Stage 1 O o ^p -o-dd-1 _n + _y L.^mn nJ HO b O ^ν % HS ^υ Prom. connection-13 LCMS (AA): m/z=749.2 (M+H) O o , , I ^xaXch 0 O Vos Prom. com.-4 6 equiv. used in Stage 1 0 o -5Н // ^N J~NH 0,, I Ух VLxxKA ⁰ VO.) NN GA \ / N. ,0 ⁴ ^ ^Н ° ^e Р Ao HS ^and Prom. connection-14 LCMS (AA): m/z=777.2 (M+H) ^°т°тА О О Вос Prom. komp.-5* 8 equiv. used at Stage 1 0 o. - ^SH DuAn o 'Р-п \,Л Ά A, - о ' _Ν Ν ^Α γ^ _> /•-\ \_х ngхУ К И 4 i 1 x ---1 £ ί Уг у ^no /° nJ 'J HS' ^u Prom. connection-15 LCMS (AA): m/z=747O (M+H) DMB : A X Voc G ¥% d 1 A ^{n α} IL L Prom. comp.-24 6 equiv. used in Stage 1 Ϊ ^н ' „ SH ^N У, NH О r' ^N 'H^'NH- Р-0 1 J. б ' 3 YJ ^N г VJ _N . 1 ^Η θ ί О HS' ⁰ Prom. connection-16 LCMS (AA): m/z=854.l (M+H)

* In Step 2, 4 M HCl in 1,4-dioxane with methanol as solvent was used for the deprotection step.

- 59 048725

Example 12.

N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7Hpyrrolo[2,3A]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4(methylamino)butanamide (Intermediate 17)

Step 1: tert-Butyl [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo3,4-dihydro-7H-pyrrolo[2,3A]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6yl}amino)-4-oxobutyl]methylcarbamate.

The title compound was prepared according to the procedure outlined in Example 5, Step 1 using 7-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-14-(6-amino-9H-purin-9-yl)-15fluoro-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-5-fluoro-3,7-dihydro-4H-pyrrolo[2,3A]pyrimidin-4-one as the sodium salt (Compound No. 14, see WO2018100558A2, 200 mg, 0.265 mmol) and Intermediate 3 (503 mg, 1.59 mmol) instead of 2-amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one and intermediate 1, respectively. Purification by reverse phase flash column chromatography (0-50% ACN/aqueous ammonium bicarbonate (5 mM)) afforded tert-butyl [4-({9[(2R,5R,7R,8R, 10R, 12aR, 14R, 15R, 15aR, 16R)-15-fluoro-7-(5-Fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3A]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfaniloctahydro- 12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate as ammonium salt (148 mg, 59%). LCMS (AA): m/z=910.5 (M+H). ^1H NMR (400 MHz, D2O) δ 8.70 (s, 1H), 8.34 (s, 1H), 8.01 (s, 1H), 7.28 (s, 1H), 6.56 (d, J=16.1 Hz, 1H), 6.43 (d, J=8.4 Hz, 1H), 5.67 (dd, J=51.0, 3.9 Hz, 1H), 5.18-4.99 (m, 2H), 4.82-4.77 (m, 1H), 4.63-4.58 (m, 1H), 4.49-4.38 (m, 3H), 4.314.25 (m, 1H), 4.12-4.06 (m, 1H), 3.43-3.36 (m, 2H), 2.88 (s, 3H), 2.73-2.68 (m, 2H), 2.04-1.97 (m, 2H), 1.34 (s, 9H). ³¹ P NMR (162 MHz, D2O) δ 55.47 (s, 1P), 52.07 (s, 1P). ^19F NMR (376 MHz, CD3OD) δ -165.50 (m, 1F), -203.45 (m, 1F).

Step 2: N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro7H-pyrrolo[2,3A]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide, intermediate 17.

The title compound was prepared according to the procedure outlined in Example 5, step 2 using tert-butyl [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3A]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9Hpurin-6-yl}amino)-4-oxobutyl] methylcarbamate as the ammonium salt (42 mg, 0.045 mmol) instead of tert-butyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate and stirring at room temperature for 30 minutes. N-{9[(2R,5R,7R,8R, 10R, 12aR, 14R, 15R, 15aR,16R)-15-Fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3

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(d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,21][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}-4-(methylamino)butanamide was obtained as the 2,2,2-trifluoroacetate salt (intermediate 17, 41 mg, 100%). LCMS (AA): m/z=810.2 (M+H).

Example 13.

The compounds listed below in Table 5 were prepared as described in Example 12, replacing isobutoxycarbonyl 4-[tert-butoxycarbonyl(methyl)amino]butanoate (Intermediate 3) with the starting material shown in Table 4.

Table 4

Source material Product Analytical data O | Prom. conn.-9* 15 equiv. used in Stage 1 Prom. connection-18 LCMS (AA): m/z=812.l (M+H) С1'' ^Л лА ^г *''1 - о ТО н ; н Н zrx χΝν A·. A Г ,·ί^ уд дк Ν зГ Boe Η η 1 Ο ,χΎ Prom. compound-10 6 equiv. used in Stage 1 Prom. connection-31 LCMS (AA): m/z^l 177.1 (M+H) n IS n i । GPG ]G YY _c| A/^yYJ θ ' O Prom. com.-34 6 equiv. used in Stage 1 Prom. connection-35 LCMS (AA): w/2^1129.2 (M+N)

* In step 2, 4 M HCl in 1,4-dioxane was used for the deprotection step.

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Table 5

Product Structure Prom, connection, - 18 X \ — / , \ Enr O / '-'СЛ X / Ох сл,. / X .....Ζ о^С.Т θ θ''' 4/+ ⁷¹ ~Z ϊ=θ Prom, connection, - 31 F 0 O SH ^F . u z ^p \ Si''nh F o 0 \ J ] ΛΛ V ³ / ^N / ^N Y> \ Η ό ά their „ ΜΑ ^HS ' ⁰ O J r Y й J jQyA ⁰ NA ¥ α - Prom, connection, - 35 ο ^{SH F} 1 P NII C V? N iQ N _N < a = i II ¹ θ^ ^{0Η0 0} T ^N % JL n JL. ,JL JI o ΗζΝ'γ' γΉΆ^ 0 *

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Table 6

Source material Product Analytical data o _n ЗН Д'-if ''NH O (АХ 'P-η b J । LL ϊ \·^ ^Ν '^; Ή ' T o Уч Чн N 0 1 ^Но р ¹ О Άζ V HS' О Пром. сод.-12 C-2 LCMS (AA): m/z=1323.4(M+H); HRMS (m/z): [M+H] ⁺ calcd. for C56H68N12O18P2S2 1323.3764; found, 1323.3790. o _n SH PxfSffl 0 | A. Jk J g ,Ν^,Ο 1 ^but p O HS' ^and Prom. conn.LZ* C-3 LCMS (AA): m/z=1261.4(M+H); HRMS (m/z): [M+H] ⁺ calcd for C ₅ 1H ₆₆ N12O18P2S2 1261.3607; found, 1261.3630. °'^;?н Vjl 7γ \ V Ο NH HN— γ • о Prom. komp.-18 C-4 LCMS (AA): m/^1324.1 (M+H); MSVR (m/z): [M+N] ⁺ calc. for C51H61F2N13O19P2S2 1324.3164; found, 1324.3186.

* In step 2, p-cresol was added to the reaction mixture in a ratio of 2:1 (p-cresol:TFA).

Table 7

Product Structure C-2 0 o -SH 0 AX A _o (JI 1 LJ 0' \ 0 -χΑΖγ ₀ ZA \ 7 ^H k Д Λ Λ A A nM να-να 0 _H v 9 Ya z -54 \ / N y if N (I О °X ^H * I and Ь

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The compound shown in Table 8 (Intermediate 19) was prepared as described in Example 6, step 1, replacing tert-butyloxycarbonyl valyl-alanyl-(4-aminobenzyl)-(4-nitrophenyl)carbonate with the starting material shown in the table.

Table 8

Source material Product Analytical e data ОуМН ₂ НН^ Н = 9 Н Α|Γ ^ν υ ^χ 'η J ^Ν ' ^{Βκ on} A ⁰ o °γ ^ΝΗ2 (Y A O' 107 ^NN lA u - θ u ? V ^u 4 η hr η h R Y ) ί IY if π N Boc i¥A ₀ ^H0 / -YO o ^H A nA Ao ⁰ Prom. connection-19 LCMS (AA): m/z= 1302.4 (M+H)

Example 16.

The compound shown in Table 10 was prepared as described in Example 6, steps 2 and 3, replacing intermediate 7 with the starting material shown in Table 9.

Table 9

Source material Product Analytical data ₀ °γ ^ΝΗ 2 _o a e 1I ^N i “Ί O ί P w NN tj _u % 0 ί, I Η H » 81 H O „Н i '' । 'fl 'th G L..O I HO 0 ζΝγΑν ^{1 0} A. ί AA „/ Y nA °Ά ⁰ HS' ^u Prom. connection-19 C-5 LCMS (AA): m/z=1395.2(M+H); HRMS (m/z): [M+H] ⁺ calcd. FOR C58H72N14O19P2S2 1395.4088; found, 1395.4125.

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Table 10

Example 17.

The compounds listed in Table 12 were prepared as described in Example 6, Step 3, substituting N-succinimidyl 6-maleimidohexanoate for the starting material shown in Table 11.

Table 11

Source material Product Analytical data g ⁰ 0 o C-6 LCMS (AA): m/z=1431.2(M+H); HRMS (m/z): [M+H] ⁺ calcd. FOR C66H72N12O17P2S2 1431.4128; found, 1431.4153.

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o. o o V-\ / \/ Η k. κζ ⁿ C-7 LCMS (AA): m/z=726.3 (M/2+H); HRMS (m/z): [M+H] ⁺ calcd. for C63H3O12O20P2S2 1451.4601; found, 1451.4608. Ό θκ 4~f ° Va у- ^Ν 4 0 0 C-8 LCMS (AA): m/z=1487.2(M+H); HRMS (m/z): [M+H] ⁺ calcd. FOR С6Н3ОХ 12O23P2S2 1487.4449; found, 1487.4474. ΖΡ ο oV\ C-9 LCMS (AA): m/z=913.9 (M/2+H) 0 Ο^ΝΗ 0 K.,+х„л4-о. _и J KI о K/ 0 ο Prom. connection-20 S-10 LCMS (AA): m/z=1586.0(M+H); HRMS (m/z): [M+H] ⁺ calcd. FOR C67H89N13O24P2S2 1586.5133; found, 1586.5150.

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^{n n} ° C-11* LCMS (AA): w/2^1715.8 (M+N) „о о, /Ύ о Ад \Jl ЛА А /f '0 0 0 C-12 LCMS (AA): nVz= 1253.3 (M+H) s Z, l ζθ /A 2 n 9 1 \\ +—Χ,Α,Λ^Η, JI J ΐ-Ζ N γ /k ι L D no 'yes s°l5 Prom. connection-27 C-13 LCMS (AA): m/z^920.9 (M/2+H); HRMS (m/z): [M+H] ⁺ calcd. for C83H104N14O26P2S2 1839.6236; found, 1839.6307. ₀ ζ·«^^0 _{4 0 n} n/D kA 1 ⁿ / N D Prom. connection-Zb S-35 LCMS (AA): nVz= 1726.3 (M-H); MSVR (m/z): [M+N] ⁺ calc. FOR C75H103N13O26P2S2 1728.6127; found, 1728.6148 ΟΎ ₀ -D ₀ D oD J o^D ^ο χχΊ θΑ A 0 hnAd o °y^ Χ _Ν ^γΑ^χ^ _Ν Aa y ^Akon o 0 Prom. conn.-40 S-39 LCMS (AA): m/z= 1775.8 (M+H); HRMS (m/z): [M+H] ⁺ calcd. for C75H104N14O28P2S2 1775.6134; found, 1775.6138.

- 67 048725 * Triethylamine was used as a base instead of N,N-diisopropylethylamine.

Table 12

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C-8 0 o - ^{SH ΝΗ 0} °''ρ\ _η < JJI °' °Ί 0 1 'tL'uA Η - ⁰ and 0 υΛ 9 5хгГ "УГ"°^ ⁰ ^ ⁰ ^ ⁰ ~ ⁰ ^ p V/ ΐ US' ° C-9 ο. *? A ° d Q Μ “ ⁰ Η Η ⁰ Г ³ “ . L .·- . ·, f JW ϊ..... HS' ⁰ S-10 < τ^τ ι S °' Ί O . ο Ο^ΝΗ 0 η and ^th AJ ^VnV>A^nA ,Ν„Ο °/ ^γΟ'.-Μ Λ ' +2 Ο °-4 ϊ C-11* '•α «Ά a ? Ά°·ΐ ^Ν ίΐ ΑΊι η ? " η η ^and a ) Ν„ο Ί " 5 4 ..γΟ-^ΧΑ ϊ " D, 4 <Γ 1J po ϊ

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C-12 0 o SH /-γ^'ΝΗ O Ap .1 ϊί l ν YES A- yes,YxU N .Al A O ^N D oA U ϊ ⁰ Hi ⁰ C-13 n S 9 o. . _n D > g 5 ΆΑ-Λ-'Α V' « J .11. ,Β Ϊ B ΐ ' A » 11 η.; O ." o j: ί J ί з - i 1 ^ϋ f ., ί _H <. 'J S-35 Q -4^·--Q χ 0 %% e j[A ^H £ Ί θ n G I ^Ν «Vl ai iW^r Ο Η Γ^ [Ar γ ^Λ ''Ν ^Λ τ ^η Α ° A l HO 0 J _o rj 0 HIJ [IT o—Pt Y NA HS' ⁰ ° S-39 0 o ~ ^SH /ii nh ° °A °A °A °A Λ- ^Ρ Ό, SAkA..A^\ J oA O A oA D D A ¹ JO 'JU. X »A ГД У-< ДЧ-^ A y о _H hnA о A \ ^nb / zAoJU LL^ IJ °A s ⁿ ΛI ^no HS' ^u

Example 18.

The compounds listed in Table 14 were prepared as described in Example 6, steps 2 and 3, replacing Intermediate 7 and Asuccinimidyl 6-maleimidohexanoate, respectively, with the starting materials shown in Table 13.

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Table 13

Stage 2 source material Stage 3 source material Product Analytical data Prom. connection-19 °.h G U- ² ' O D—\ dy ^N 1 C-14 LCMS (AA): m/z^759.5 (M/2+H); MCBP (m/z): [M+H] ⁺ calcd for C69H78N14O18P2S2 1517.4608; found, 1517.4635. Prom. connection-19 n 0 ο °VA Cz ^{h H} C-15 LCMS (AA): m/z^769.8 (M/2+H); MCBP (m/z): [M+H] ⁺ calcd. for CeeHseN 14O21P2S2 1537.5081; found, 1537.5120. Prom. connection-21 0 0 b o' C-16 LCMS (AA): m/z=1485.3(M+H); MCBP (m/z): [M+H] ⁺ calcd. FOR C63H82N12O22P2S2 1485.4656; found, 1485.4680.

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Prom. connection-21 o o ,^ _O -D _M γ 1 \s_ n l C-17 LCMS (AA): m/z=l 627.2 (M+H) Prom. connection-28* O D C-18 LCMS (AA): m/z=1718.3(M+H); MSVR (m/z): [M+N] ⁺ calc. FOR C73H101N13O27P2S2 1718.5919; found, 1718.5936. Prom. connection-29 O O U i >-/ o (U C-19 LCMS (AA): m/z=1789.0(M+H); HRMS (m/z): [M+H] ⁺ calcd. FOR C76H106N14O28P2S2 1789.6290; found, 1789.6286. Prom. connection-31** /° O. γγ o γ-Ά γΝ^. _ο ^Ο^ _ο ^Ο^ _οΛ ΥΙ _ο . _Ν γ 0 O S-32 LCMS (AA): m/z= 1547.9 (M+H); HRMS (m/z): [M+H] ⁺ calcd. FOR C63H77F2N13O23P2S 2 1548.4213; found, 1548.4236.

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Prom. connection-ЗЗ ,0 0, 0 0 S-33 LCMS (AA): nVz= 1498.3 (M+H); MSVR (m/z): [M+N] ⁺ calc. FOR C ₆ 2H ₇₇ FNi ₅ O ₂₂ PS ₂ 1498.4603; found, 1498.4604. Prom. connection-38 0 0 Χ _Ν Α^ζχ/\Ζ'··· y -'y VA o λ-ί Ό s S-36 LCMS (AA): m/z=1659,l (M-H); HRMS (m/z): [M+H] ⁺ calcd. FOR C ₇ iH9sNi ₂ O ₂ 6P ₂ S ₂ 1661.5705; found 1661.5759. Prom. connection-31** .0 Ok 0 X A. A. F. N.Z ⁰ 0 Prom. connection-39 S-38 LCMS (AA): nVz= 1777.6 (M-H); HRMS (m/z): [M+H] ⁺ calcd. FOR C ₇₄ H ₉₈ F ₂ N mO ₂₇ P ₂ S 2 1779.5683; found, 1779.5716. Prom. connection-29 o υ Ζ“~-η· ' O g'^' ^{4 l} 1 i ·. ' ^N. Ж N ' Г ' ⁰ '1 0 0 S-40 LCMS (AA): m/z=1731.4 (M-H); HRMS (m/z): [M+H] ⁺ calcd. for;? found,?.

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Prom. connection-38 .0 A' ? V'/ \ ,Ν Ж Ν. Z y 0 D 0 ό C-41 LCMS (AA): m/z=1605.4 (M+H); HRMS (m/z): [M+H] ⁺ calcd. FOR C67H90N12O26P2S2 1605.5079; found, 1605.5148 Prom. connection-41 A W 0 yA O 0 C-42 LCMS (AA): m/z=1542.l (M-H); MSVR (m/z): [M+N] ⁺ calc. for C64H83N13O24P2S2 1544.4663; found, 1544.4722. Prom. connection-31** o °D'\ 0 Ο^ΝΗ O °χ/ ^χ _ο ^ζ Κ'°χ/4 ^ζ Prom. connection-44 S-45 LCMS (AA): m/z= 1777.5 (M-H).

* Triethylamine was used as a base instead of N,N-diisopropylethylamine.

**Step 2 in Example 6 was skipped.

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Table 14

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O

HS ³¹ P NMR (162 MHz, Methanol-f) δ ppm 52.93 (s, 1 P) 54.97 (s, 1 P).

o m 11

ЬН NH O

P-tJ mJ -J U o D ,- N 'N N

Ή NMR (400 MHz, Methanol-sc) δ ppm 0.97 (t, J=7.15 Hz, 6 N) 1.24-1.40 (m, 2 N) 1.45 (d, J=7.09 Hz, 3 N) 1.55-1.88 (m, 5 N) 2.13 (m, 1 N) 2.25-2.40 (m, 1 N) 2.44-2.62 (m, 5 N) 2.89 (s, 3 N) 3.35 (s, 3 N) 3.46 (t, J=7.03 Hz, 2 N) 3.50-3.57 (m, 2 N) 3.62 (m, 28 N) 3.67-3.83 (m, 3 N) 3.98-4.08 (m, 1 N) 4.18 (d, J=6.97 Hz, 1 N) 4.22-4.40 (m, 4 N) 4.48 (d, J=7.09 Hz, 1 N) 4.78 (m, 2 N) 5.04 (s, 2 N) 5.455.64 (m, 2 N) 6.08-6.21 (m, 1 N) 6.78 (s, 2 N) 6.83 (d, J=6.11 Hz, 1 N) 7.12-7.35 (m, 3 N) 7.35-7.44 (m, 1 N) 7.45-7.61 (m, 3 N) 7.70 (br. d, J=7.58 Hz, 1 N) 8.40 (d, J=5.99 Hz, 2 N) 8.70 (s, 1 N).

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³¹ P NMR (162 MHz, Methanol-SC) δ ppm 52.26 (s, 1 P) 56.88 (s, 1 P)

Ή NMR (400 MHz, Methanol-SC) δ ppm 0.97 (m, 6 Η) 1.34 (m, 2 Η) 1.47 (d, J=7.09 Hz, 3 Η) 1.52-1.61 (m, 2 Η) 1.63-1.75 (m, 1 Η) 1.77-1.88 (m, 1 Η) 2.1-1-2.24 (m, 1 Η) 2.44-2.59 (m, 2 Η) 2.94 (br s, 3 Η) 3.35 (s, 3 Η) 3.45 (m, 2 Η) 3.49-3.56 (m, 2 Η) 3.58-3.66 (μ, 28 Η) 3.73 (τ, J=6.05 Hz, 2 Η) 4.01 (dd, J=11.25, 3.42 Hz, 1 Η) 4.20 (d, J=6.85 Hz, 1 Η) 4.29 (s, 1 Η) 4.33-4.59 (m, 5 Η) 4.86 (m, 2 Η) 4.96-5.08 (m, 3 Η) 5.15-5.30 (m, 1 Η) 5.72 (d, J=52.0 Hz, 1 Η) 6.35-6.53 (m, 2 Η) 6.77 (s, 2 Η) 7.09-7.24 (m, 2 Η) 7.26-7.35 (m, 1 Η) 7.38 (s, 1 Η) 7.43-7.59 (m, 4 Η) 7.76 (s, 1 Η) 7.80 (br. d, J=7.46 Hz, 1 N) 8.42 (s, 1 N) 8.62-8.69 (s, 1 N)

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P NMR (162 MHz, Methanol-SC) δ ppm 52.35 (s, 1 P) 56.91 (s, 1 P)

H NMR (400 MHz, Methanol-SC) δ ppm 0.98 (dd, J=12.90, 6.91 Hz, 6 H) 1.23-1.40 (m, 2 H) 1.47 (d, J=7.21 Hz, 3 H) 1.58 (m, 2 H) 1.73 (m, 2 H) 2.17-2.30 (m, 2

N) 2.35 (m, 1 N) 2.91 (s, 3 N) 3.31 (s, 3 N) 3.40 (m, 2 N) 3.46-3.60 (m, 30 N) 3.97 (dd, J=11.55, 3.36 Hz, 1 N) 4.12 (d, J=5.62 Hz, 1 N) 4.19-4.28 (m, 2 N) 4.30-4.54 (m, 6 N) 4.83 (m, 2 N) 4.93-5.07 (m, 3 N) 5.10-5.25 (m, 1 N) 5.72 (d, J=52.0 Hz, 1

N) 6.36-6.48 (m, 2 N) 6.76 (s, 2 N) 7.05-7.32 (m, 3 N) 7.35 (s, 1 N) 7.38-7.68 (m, 4

N) 7.75 (s, 1 N) 7.76-7.83 (m, 1 N) 8.41 (s, 1 N) 8.61-8.68 (s, 1 N)

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Example 19.

4-{[(^)-2-{[(^)-2-{[6-(2,5-Dioxo-2,5-dihydro-III-pyrrol-1-yl)hexanoyl]amino}-3 - methylbutanoyl]amino}propanoyl]amino}benzyl [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-rugpoKCu-2,10dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphatecyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}amino)2,2-dimethyl-4-oxobutyl]methylcarbamate, Compound 20 (C-20)

Step 1: 4-{ [(2S)-2-{ [(2S)-2-{[6-(2,5-Dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoyl]amino }-3methylbutanoyl]amino}propanoyl]amino}benzyl [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphate cyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1 H-purin-2-yl } amino)-2,2-dimethyl-4-oxobutyl]methylcarbamate, compound 20.

To a solution of [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (47.9 mg, 0.037 mmol) and 1-hydroxy-7-azabenzotriazole (4.08 mg, 0.03 mmol) in DMF (0.60 mL) and N,N-diisopropylethylamine (26 μL, 0.15 mmol) was added a solution of N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-3,3-dimethyl-4-(methylamino)butanamide as 2,2,2-trifluoroacetate salt (Intermediate 14, 20 mg, 0.02 mmol) in DMF (0.54 mL) at room temperature. The reaction mixture was allowed to stir at room temperature for 16 h. Celite was added and the mixture was evaporated to dryness. The crude residue absorbed on Celite was purified by reverse phase flash column chromatography (0-100% ACN/aqueous ammonium acetate (10 mM)) to give 4-{[(2S)-2{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl[4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}amino)-2,2-dimethyl-4-oxobutyl]methylcarbamate as an ammonium salt (C-20, 4 mg, 17%). LCMS (AA): m/z=1287.4 (M-H).

Example 20.

The compounds listed in Table 16 were prepared as described in Example 19, replacing Intermediate 14 and [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate, respectively, with starting materials A and B shown in Table 15.

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Table 15

Source material A Source material B Product Analytical data Prom. connection-17* oa/Ch ....... /=° ο S-21 LCMS (AA): m/z= 1322.3 (M+H); HRMS (m/z): [M+H] ⁺ calcd. for C52H63F2N13O18P2S2 1322.3371; found, 1322.3394. Prom. connection-17** ' s - s Ο ...from K Prom. connection-25 S-22 LCMS (AA): m/z= 1444.8 (M+H) Prom. connection-17** Η τ 0 _Η Ο / Λ θ ^Η Α ° 1, ) 1 J ¥ /A 0^-^++ ⁰ Prom. connection-26 S-23 LCMS (AA): t/^1416.8 (M+H) Prom. connection-18** _γ θ JU o ^Η Α ° α+ _ΟΝ Οΐ Ο ₂ Ν ' C-44 LCMS (AA): nVz= 1304.4 (M+H); HRMS (m/z): [M+H] ⁺ calcd. for C50H63N15O19P2S2 1304.3414; found 1304.3465.

* 1-hydroxybenzotriazole and triethylamine were used in the reaction instead of 1-hydroxy-7-azabenzotriazole and N,N-diisopropylethylamine.

** 4-dimethylaminopyridine and triethylamine were used in the reaction instead of 1-hydroxy-7-azabenzotriazole and N,N-diisopropylethylamine.

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Table 16

Example 21.

^-{(^)-6-(2,5-Dioxo-2,5-dihydro-III-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1oxohexan-2-yl}-2,5,8,11,14-pentaoxaheptadecane-17-amide (Intermediate 20)

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Step 1: (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14pentaoxa-18-azaicosan-20-oic acid.

To a solution of 1-[(17-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl)oxy]pyrrolidine-2,5-dione (4.0 g, 10.5 mmol) in anhydrous DCM (10 mL) was added (N)-2-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)-hexanoic acid hydrochloride (3.4 g, 12.9 mmol) dissolved in DMF (40 mL) followed by N,N-diisopropylethylamine (6.9 mL, 42 mmol). The reaction mixture was allowed to stir at room temperature for 18 h. The reaction mixture was filtered and evaporated to dryness. The crude residue was purified by preparative HPLC to give (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-oic acid (1.26 g, 24%). LCMS (AA): m/z=489.3 (M+H).

Step 2: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14-pentaoxaheptadecan-17-amide, intermediate 20.

To a solution of (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14pentaoxa-18-azaicosan-20-oic acid (65 mg, 0.133 mmol) and N,N'-dicyclohexylcarbodiimide (30 mg, 0.145 mmol) in 1,4-dioxane (0.68 mL) was added N-hydroxysuccinimide (15.3 mg, 0.133 mmol) at room temperature. The reaction mixture was allowed to stir at room temperature for 16 h. Celite was added and the mixture was filtered, washed with 1,4-dioxane and evaporated to dryness. The crude residue absorbed on celite was purified by silica gel chromatography (0-10% MeOH/DCM) to give intermediate 20 (14.6 mg, 19%). LCMS (AA): m/z=586.3 (M+H).

Example 22.

The compounds listed in Table 17 were prepared as described in Example 9, replacing Intermediate 10 with the starting material shown in the table.

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Table 17

Source material Product Analytics strong data ,о. -х z. ΑΑ,αΑ - — о ^ν 'ЛА..... о о А. Prom. conn.-П 6 equiv. used n f jj n ⁰ °A (ХХЗГХ 1( ^N '® ^K _o ^н Φ0-ΝΗ 0 ?у°А> 0 X / ° γХА'Х ° К ' 4 Αν, HS ^υ Prom. soed.-21 LCMS (AA): m/z=1392.2 (M+H) _o / _o + -JU-XLa 0 1 · >1 i ?, - Prom. conn.-37 6 equiv. used । o o +A O-X 0-4 y 9 n 1 θ n o S fY ^N YAV' ^B « °0 I χ _γ °·ΆΥ ⁰ o- yu N 01 ^НС ^ I /U K/ Ν Ηί? θ Prom. soed.-38 LCMS (AA): m/zA785, 3 (M/2+H) u’ Prom. conn.-42 6 equiv. used .—0 [ί /° I An / 0 iV “ / ^?Jl °K ci /^'li NH o X ZPX 0 0 Y\ ο' ^₽ · ⁰ >ο ν A α _ν Ay. o-Jy Αο-α+Ο _Ν th L ^Μ ο ί χ Ν-Υ HS' ^υ Prom. connection-43 LCMS (AA): m/z=1923.2 (M+H)

Example 23.

(2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, Compound 24 (C-24)

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Step 1: Methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluorene-9unMeTOKCu)kap6oHun]aMUHo}nponaHOun)aMUHo]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulf anildecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate.

The title compound was prepared according to the procedure described in Example 6, Step 1, starting from Intermediate 6 (104 mg, 0.094 mmol) and methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-[2-[3-(9H-fluoren-9-ylmethoxycarbonylamino)propanoylamino]-4-[(4-nitrophenoxy)carbonyloxymethyl]phenoxy]tetrahydropyran-2-carboxylate (150 mg, 0.16 mmol) (for synthesis, see Jeffrey, S.C. et al. Bioconjugate Chem. 2006, 17, 831-840) using N,N-diisopropylethylamine as base to provide methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulf anildecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-(3-purin-2-yl)carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate as an ammonium salt (71 mg, 48%). LCMS (AA): m z 1570.9(\|·| 1).

Step 2: (2S,3S,4S,5R,6S)-6-{2-[(3-aminopropanoyl)amino]-4-[({[2-({9[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, intermediate 22.

To the solution of methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfa To nildecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphate acyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-21-pyran-2-carboxylate as ammonium salt (66 mg, 0.042 mmol) dissolved in THF (13 mL) was added LiOH (0.5 M aqueous solution, 1.25 mL, 0.625 mmol) at room temperature, and the reaction mixture was stirred for 2.5 h. The reaction mixture was neutralized to pH 7 with 1 M HCl and the mixture was evaporated to dryness. The crude residue was purified by reverse phase flash column chromatography (0-30% ACN/aqueous ammonium bicarbonate (5 mM)) to afford Intermediate 22 as an ammonium salt (26 mg, 50%). This material was then dissolved in MeOH (5 mL) and triethylamine (1 mL) was added. The mixture was shaken for 1 min. The solvents were removed and the resulting residue was lyophilized overnight to afford (2S,3S,4S,5R,6S)-6-{2-[(3-aminopropanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,

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14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1Hpurin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid salt as N,N-diethylethanamine (Intermediate 22, 30 mg, 100%). LCMS (AA): m/z=1209.0 (M+H).

Step 3: (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9- [(2R,5R,7R, 8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxide-14(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphatetetradecin-7-yl]-6-oxo-6,9-dihydro-3H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid, Compound 24

The title compound was prepared according to the procedure described in Example 6, step 3, using Intermediate 22 (24 mg, 0.02 mmol) instead of Intermediate 8 to give (2S,3S,4S,5R,6S)-6-{2-[(3-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}propanoyl)amino]-4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1 H-purin-2-yl carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid as the ammonium salt (C-24, 17 mg, 71%). LCMS (AA): m/z=1402.4 (M+H). MCBP (m/z): [M+H] ⁺ calcd. for C56H65N11O24P2S2 1402.3193; found, 1402.3232.

Example 24.

The compound shown in Table 18 (Compound 25) was prepared as described in Example 23, step 3, replacing N-succinimidyl 6-maleimidohexanoate with the starting material shown in the table, as appropriate.

Table 18

Analytical data

Source material

Product found, 1544.4232.

C-25 Structure:

LCMS (AA): m/z=l 544.8(M+H); MCBP (m/z): [M+H] ⁺ calc. for C ₆ 4H ₇₉ N11O26P ₂ S2 1544.4187;

Example 25.

4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3 - methylbutanoyl]amino}propanoyl]amino}benzyl{2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-(III-purin-2-yl)carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate, compound 26 (C-26).

- 85 048725

PS-15

C-26

Step 1: 2-[[4-[[(^)-2-[[(^)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]acetic acid.

To a solution of glycine (24 mg, 0.32 mmol) in triethylamine (0.10 mL, 0.72 mmol), DMF (0.50 mL) and DMSO (0.50 mL) was added [4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3methylbutanoyl]amino]propanoyl]amino]phenyl]methyl (4-nitrophenyl) carbonate (220 mg, 0.34 mmol) at room temperature. The reaction mixture was stirred at room temperature for 4 h. Then, the reaction mixture was cooled to 0 °C. Water (1 mL) was added and the solution was acidified to pH 4 by adding formic acid. The mixture was evaporated to dryness and the crude residue was purified by reverse phase flash column chromatography (0-100% ACN/aqueous formic acid (0.1%)) to give 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]acetic acid (87 mg, 44%). LCMS (AA): m/z=586.3 (M-H).

Step 2: 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3methylbutanoyl]amino}propanoyl]amino}benzyl{2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)16-hydroxy-2,10-dioxido-1 4-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphatetetradecin-7-yl]-6-oxo-6,9-dihydro-3-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate, compound 26.

To a solution of 2-[[4-[[(2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylamino]acetic acid (40 mg, 0.068 mmol) and triethylamine (35 μL, 0.25 mmol) in DMF (1 mL) was added 1-[bis(dimethylamino)methylene]-1H-1,2,3-triczolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (46 mg, 0.12 mmol) at room temperature, and the reaction mixture was stirred for 10 min. Then, to this solution was added dropwise at room temperature a solution of (2R)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}pyrrolidine-2-carboxamide as 2,2,2-trifluoroacetate salt (Intermediate 15, 50 mg, 0.047 mmol) in DMF (0.60 ml). The reaction mixture was stirred at room temperature for 30 minutes. The mixture was then cooled to 0 °C, water (1 ml) was added, and the solution was acidified to pH 4 by adding formic acid. The mixture was evaporated to dryness and the crude residue was purified by reverse phase flash column chromatography (0-100% ACN/aqueous formic acid (0.1%)) to give 4-{[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3methylbutanoyl]amino}propanoyl]amino}benzyl{2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1 H-purine-2

- 86 048725 ({{{\text{yl}}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate (C-26, 32 mg, 50%). LCMS (AA): m/z=1316.4 (M+H). MCBP (m/z): [M+H] ⁺ calcd for C ₅₃ H ₆₇ N ₁₃ O ₁₉ P ₂ S ₂ 1316.3666; found, 1316.3705.

Example 26.

(2R)-1-({ [(2S)-2- {[(2S)-2-{ [6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoyl]amino }-3methylbutanoyl]amino}propanoyl]amino}acetyl)-X- {9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}pyrrolidine-2-carboxamide, Compound 27 (C-27)

Step 1: tert-Butyl {2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-3-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl} carbamate.

To a mixture of Intermediate 15 (60 mg, 0.05 mmol) and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate (19 mg, 0.07 mmol) in DMF (1.6 mL) was added triethylamine (50 μL, 0.355 μmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched with water, celite was added and the reaction mixture was evaporated to dryness. The crude residue absorbed on celite was purified by reverse phase column chromatography (0-100% ACN/aqueous ammonium bicarbonate (5 mM)). To the aqueous solution of the product was added triethylamine (14 μL, 0.1 mmol). Lyophilization overnight gave tert-butyl {2-[(2R)-2-({9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldeclotetradecanehydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphatidyl-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl} carbamate as the N,N-diethylethanamine salt (49 mg, 87%). LCMS (AA): m/z=904.4 (M+H).

Step 2: (2R)-1-(Aminoacetyl)^- {9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1Hpurin-2-yl}pyrrolidine-2-carboxamide

To a solution of tert-butyl (2-[(2R)-2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}carbamoyl)pyrrolidin-1-yl]-2-oxoethyl}carbamate as N,N-diethylethanamine salt (37 mg, 0.033 mmol) in MeOH (1 ml) was added hydrochloric acid (4 M solution in dioxane, 1 ml, 4 mmol), and the reaction mixture was stirred at room temperature for 1 h. Then the reaction mixture was evaporated to dryness and placed under vacuum for 2 h to give (2R)-1-(aminoacetyl)-N-{9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7 yl]-6-oxo-6,9-dihydro-N-purin-2-yl}pyrrolidine-2-carboxamide as a hydrochloride salt (37 mg, 100%). LCMS (AA): m/z=804.2 (M+H).

Step 3: (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}3-methylbutanoyl]amino}propanoyl]amino}acetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8

- 87 048725 ethanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1Hpurin-2-yl}pyrrolidine-2-carboxamide, compound 27

The title compound was prepared according to the procedure outlined in Example 26, step 1, starting from (2R)-1-(aminoacetyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}pyrrolidine-2-carboxamide as the hydrochloride salt (30 mg, 0.027 mmol) and (2,5-dioxopyrrolidin-1-yl) (2S)-2-[[(2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]propanoate (24 mg, 0.04 mmol) (for synthesis see US 20180015176 A1). Purification by reverse phase flash column chromatography (0-100% ACN/aqueous formic acid (0.1%)) gave (2R)-1-({[(2S)-2-{[(2S)-2-{[6-(2,5dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}acetyl)-N-{9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}pyrrolidine-2-carboxamide (C-27, 20 mg, 64%). LCMS (AA): m/z=1167.1 (M+H). MSVR (m/z): [M+H] ⁺ calc. for C ₄₅ H ₆₀ N ₁₂ O ₁₇ P ₂ S ₂ 1167.3189; found, 1167.3206.

Example 27.

4-{[(2S)-2-({(2S)-2-[(Aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7yl]-6-oxo-6,9-dihydro-III-purin-2-yl}carbamoyl)benzyl]methylcarbamate, Compound 28 (C-28)

JSH

DIPEA.THF,DMF

Step 1: tert-Butyl (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopene Ta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}carbamoyl)benzyl](meth yl)carbamoyl}oxy)methyl]phenyl}amino)-1-oxopropan-2-yl]amino}-3-methyl-1oxobutan-2-yl]amino}-2-oxoethyl)carbamate.

The title compound was prepared according to the procedure described in Example 6, Step 3, starting with Intermediate 8 (23 mg, 0.016 mmol) and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate (11 mg, 0.039 mmol) instead of N-succinimidyl 6-maleimidohexanoate. The reaction mixture was stirred at room temperature for 30 min. Purification by reverse phase flash column chromatography (0-100% ACN/aqueous triethylammonium acetate (10 mM)) afforded tert-butyl (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1 ] [ 1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1 N-purin-2yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}amino)-1-oxopropan-2-yl]amino}-3-methyl

- 88 048725

1-oxobutan-2-yl]amino}-2-oxoethyl)carbamate as N,N-diethylethanamine salt (13 mg, 57%). LCMS (AA): m/z=1273.l (M+H).

Step 2: 4-{[(2S)-2-({(2S)-2-[(Aminoacetyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, Compound 28

The title compound was prepared according to the procedure outlined in Example 5, Step 2, starting from tert-butyl (2-{[(2S)-1-{[(2S)-1-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1 N-purin-2yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}amino)-1-oxopropan-2-yl]amino}-3-methyl1-oxobutan-2-yl]amino}-2-oxoethyl)carbamate as N,N-diethylethanamine salt (6.6 mg, 0.005 mmol) instead of tert-butyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}carbamoyl)benzyl]methylcarbamate. The reaction mixture was stirred at room temperature for 5 min to give 4-{[(2S)2-({ (2S)-2-[(aminoacetyl)amino]-3-methylbutanoyl } amino)propanoyl]amino } benzyl [2-( { 9[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, (16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7yl]-6-oxo-6,9-dihydro-16R-purin-2-yl}carbamoyl)benzyl]methylcarbamate as 2,2,2-trifluoroacetate salt (C-28, 5.6 mg, 91%). LCMS (AA): m/z=1173.3 (M+H).

Example 28.

The compounds listed in Table 20 were prepared as described in Example 27, replacing 2,5-dioxopyrrolidin-1-yl(tert-butoxycarbonyl)glycinate with the starting material shown in Table 19.

Table 19

Source material Product Analytical data Vos J. JL, l J i d i d I / O 0 0- Prepared as described in U.S. Patent No. 6,022,966 S-29 LCMS (AA): m/z=l 287.2 (M+H) Vos l ,0. J. A n 1/ Prom. connection-23 S-30 LCMS (AA): m/z= 1349.3 (M+H)

- 89 048725

Table 20

Product Structure S-29 o 0 /ХЧн О P-о \ 1 JJ Г νΓΧγΊΐ Н; 9 N 9 N ,.....L ..n ( ! G; ': *;'· '.t L ¹ S-30 0 o _g i e X'F ⁰

- 90 048725

Step 1: 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid The title compound was prepared according to the procedure outlined in Example 10, Step 1, starting from 2-carboxybenzaldehyde (6.0 g, 40 mmol) and 2,4-dimethoxybenzylamine (12 mL, 80 mmol) to give 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid (7.6 g, 61%). LCMS (AA): m/z=302.2 (m+h).

Step 2: 2-[[[(2S)-2-(ret-butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid.

To a solution of 2-[[(2,4-dimethoxyphenyl)methylamino]methyl]benzoic acid (380 mg, 1.26 mmol) and triethylamine (0.5 mL, 3.55 mmol) in DMF (6 mL) was added 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)-E-alaninate (640 mg, 2.2 mmol). Water (2 mL) was added to help solubilize the reaction mixture, which was allowed to stir at room temperature for 1 day. The reaction mixture was then evaporated to dryness and the crude residue was purified by reverse phase flash column chromatography (0-100% ACN/aqueous formic acid (0.1%)) to give 2-[[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid (322 mg, 51%). LCMS (AA): m/z=473.3 (M+H).

Step 3: tert-butyl N[(^)-2-[(2-chlorocarbonylphenyl)methyl-[(2,4-dimethoxyphenyl)methyl]amino]1-methyl-2-oxoethyl]carbamate, Intermediate 24

The title compound was prepared according to the procedure described in Example 1, step 2, starting with 2-[[[(2S)-2-(tert-butoxycarbonylamino)propanoyl]-[(2,4-dimethoxyphenyl)methyl]amino]methyl]benzoic acid (270 mg, 0.57 mmol) instead of 2-(((tert-butoxycarbonyl)(methyl)amino)methyl)benzoic acid to give crude intermediate 24 (280 mg, 100%).

Example 31.

2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1 ] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide, Compound 31 (C-31)

Step 1: tert-Butyl [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]amino }-1-oxopropan-2-yl] amino}-3-methyl-1-oxobutan-2-yl] carbamate.

The title compound was prepared according to the procedure described in Example 26, step 1, starting from Intermediate 16 (45 mg, 0.53 mmol) and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl) Glycinate (100 mg, 0.32 mmol) instead of Intermediate 15 and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate, respectively. Purification by reversed-phase flash column chromatography (0-100% ACN/aqueous ammonium acetate (10 mM)) afforded tert-butyl [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]amino}-1-oxopropan-2yl]amino}-3-methyl-1-oxobutan-2-yl]carbamate as ammonium salt (24 mg, 44%). LCMS (AA):

- 91 048725 m/z=1053.0 (M+H).

Step 2: 2-({[(2S)-2-{[(2S)-2-aMUHO-3-MeTun6ymaHOun]aMUHo}nponaHOun]aMUHo}MeTun)-N-{9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide.

The title compound was prepared according to the procedure described in Example 5, Step 2, starting from tert-butyl [(2S)-1-{[(2S)-1-{[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}carbamoyl)benzyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]carbamate as an ammonium salt (24 mg, 0.023 mmol) instead of tert-butyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}carbamoyl)benzyl]methyl carbamate to give 2-({[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R, 10R, 12aR, (14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-(III-purin-2-yl}benzamide as 2,2,2-trifluoroacetate salt (24 mg, 97%). LCMS (AA): m/z=953,l (M+H).

Step 3: 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1Hpurin-2-yl}benzamide, compound 31.

The title compound was prepared according to the procedure outlined in Example 6, Step 3, starting from 2-({[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-N-purin-2-yl}benzamide as 2,2,2-trifluoroacetate salt (24 mg, 0.022 mmol) instead of Intermediate 8 to obtain 2-({[(2S)-2-{[(2S)-2-{[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}methyl)-N-{9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}benzamide as the ammonium salt (C-31, 6.2 mg, 23%). LCMS (AA): m/z=1146.1 (M+H). MCBP (m/z): [M+H] ⁺ calcd for C ₄₆ H ₅₇ NnO ₁₆ P ₂ S ₂ 1146.2974; found, 1146.2998.

Example 32.

4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3methylbutanoyl]amino}propanoyl]amino}benzyl 4-nitrophenyl carbonate (Intermediate 25)

Step 1: 6-(11,12-didehydrodibenzo[b, f]azocin-5(6H)-yl)-N-[(2S)-1-{[(2S)-1-{[4(hydroxymethyl)phenyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-oxohexanamide

To a solution of (2S)-2-amino-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxoethyl]-3methylbutanamide (240 mg, 0.82 mmol) dissolved in DMF (2 mL) and THF (5.5 mL) was added N,N-diisopropylethylamine (0.39 mL, 2.24 mmol). The reaction mixture was cooled to 0 °C and 1-{[6(11,12-didehydrodibenzo[N-azocine-5(6H)-yl)-6-oxohexanoyl]oxy}pyrrolidine-2,5-dione (320 mg, 0.75 mmol) was added. The reaction mixture was warmed to room temperature and stirred for 30 min. The reaction mixture was diluted with water and extracted with EtOAc (3x). The combined organic phases were washed with saturated aqueous sodium bicarbonate and _brine , dried ( _Na2SO4 ), filtered and evaporated to dryness. The crude residue was purified by silica gel chromatography (50-100% EtOAc/DCM) to give 6-(11,12-didehydrodibenzo[b, £]azocine-5(6H)-yl)-6-(11,12-dimethyl-6-(11,12-dimethyl-6-methyl-6-yl) ...

- 92 048725

[(2S)-1-{ [(2S)-1-{ [4-(hydroxymethyl)phenyl]amino }-1-oxopropan-2-yl]amino }-3-methyl-1-oxobutan-2yl]-6-oxohexanamide (347 mg, 76%). LCMS (AA): m/z=609.4 (M+H).

Step 2: 4-{[(2S)-2-{[(2S)-2-{[6-(11,12-didehydrodibenzo[b, £]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3-methylbutanoyl]amino}propanoyl]amino}benzyl 4-nitrophenyl carbonate, Intermediate 25.

To a solution of 6-(11,12-didehydrodibenzo[b, f]azocine-5(6H)-yl)-N-[(2S)-1-{[(2S)-1-{[4-(hydroxymethyl)phenyl]amino}-1-oxopropan-2-yl]amino}-3-methyl-1-oxobutan-2-yl]-6-oxohexanamide (257 mg, 0.42 mmol) in THF (4.2 mL) and N,N-diisopropylethylamine (0.37 mL, 2.1 mmol) cooled to 0 °C was added 4-nitrophenyl chloroformate (362 mg, 1.69 mmol) in one portion. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction mixture was diluted with water and extracted with DCM (3x). The combined organic phases were washed with saturated aqueous sodium bicarbonate and brine, dried ( _Na2SO4 ), filtered and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-100% EtOAc/DCM) to give ₄ -{[(2S)-2{[(2S)-2-{[6-(11,12-didehydrodibenzo[b, £]azocin-5(6H)-yl)-6-oxohexanoyl]amino}-3methylbutanoyl]amino}propanoyl]amino}benzyl 4-nitrophenyl carbonate (Intermediate 25, 137 mg, 42%). LCMS (AA): m/z = 774.4 (M+H).

Example 33.

The compound shown in Table 21 (intermediate 26) was prepared as described in Example 32, replacing the starting material shown in the table with 1-{[6-(11,12-didehydrodibenzo[b,f]azocine-5(6H)-yl)-6-oxohexanoyl]oxy}pyrrolidine-2,5-dione.

Table 21

Example 34.

(2S)-2-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosan-22-yl)-4-oxobutanamide (Intermediate 27) and (25S)-25-{[4-(11,12-didehydrodibenzo[b,f]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-oic acid (Intermediate 27A)

- 93 048725

Step 1: tert-Butyl (25S)-25-{[(6eH3unoKCu)Kap6oHun]aMUHo}-24-OKCO-2,5,8,11,14,17,20-renTaoKca23-azaheptacosane-27-oate.

The title compound was prepared according to the procedure described in Example 26, step 1, starting from tert-butyl (3S)-3-{[(benzyloxy)carbonyl]amino}-4-[(2,5-dioxopyrrolidin-1yl)oxy]-4-oxobutanoate (2.2 g, 5.3 mmol) and 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (2.0 g, 5.9 mmol) instead of intermediate 15 and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate, respectively. Purification by silica gel chromatography (0-10% MeOH/DCM) gave tert-butyl (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20heptaoxa-23-azaheptacosane-27-oate (3.8 g, 100%).

Step 2: tert-Butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate tert-Butyl (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (3.8 g, 5.9 mmol) was dissolved in ethanol (100 mL). Palladium (10% on carbon, 1.38 g, 1.17 mmol) was added and the mixture was stirred under hydrogen atmosphere (15 psi) for 3 h. The reaction mixture was filtered, washed with methanol and the filtrate was evaporated to dryness to give tert-butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (2.56 g, 85%).

Step 3: tert-Butyl (25S)-25-{[4-(11,12-didehydrodibenzo[b, £]azocin-5(6H)-yl)-4oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate

To a solution of tert-butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-oate (3.32 g, 6.5 mmol), 4-(11,12-didehydrodibenzo[b, dazocin-5(6H)-yl)-4-oxobutanoic acid (1.5 g, 4.91 mmol) and 1-hydroxy-7-azabenzotriazole (884 mg, 6.57 mmol) in DCM (50 mL) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1.25 g, 6.56 mmol) and triethylamine (1.26 mL, 9.1 mmol) at 0°C. The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was diluted with EtOAc and potassium carbonate (20 mL, 4.0 M in water) was added. The mixture was stirred for 15 min and the organic phase was separated, washed with saturated aqueous sodium bicarbonate, brine, dried (Na ₂ SO ₄ ), filtered and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-10% MeOH/DCM) to give tert-butyl

- 94 048725 (25S)-25{[4-(11,12-didehydrodibenzo [b, f]azocine-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-oate (2.27 g, 44%).

Step 4: (25S)-25-{[4-(11,12-gugerugpogu6eH3o[b, ^azocine-5(6H)-yl)-4-oxobutanoyl]amino}-24oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-oic acid.

The title compound was prepared according to the procedure outlined in Example 5, Step 2, starting from tert-butyl (25S)-25-{[4-(11,12-didehydrodibenzo[b, £]azocin-5(6H)-yl)-4oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (278 mg, 0.35 mmol) instead of tert-butyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, (16R)-16-hydroxy-2,10-dioxido-14(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}carbamoyl)benzyl]methylcarbamate. Purification by silica gel chromatography (0-20% MeOH/DCM) afforded (25S)-25-{[4-(11,12-didehydrodibenzo[C £]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23azaheptacosan-27-oic acid (Intermediate 27A, 180 mg, 70%). LCMS (AA): m/z=742.4 (M+H).

Step 5: (2S)-2-{[4-(11,12-didehydrodibenzo[b,^azocine-5(6H)-yl)-4-oxobutanoyl]amino}-4[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2,5,8,11,14,17,20-heptaoxadocosan-22-yl)-4-oxobutanamide, Intermediate 27.

The title compound was prepared according to the procedure described in Example 29 starting from (25S)-25-{[4-(11,12-didehydrodibenzo[b,^azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-oic acid (60 mg, 0.08 mmol) instead of 2,2-dimethyl-4oxo-3,8,11,14,17-pentaoxa-5-azanonedecane-19-oic acid. The reaction mixture was stirred for 30 min at room temperature to give (2S)-2-{[4-(11,12-didehydrodibenzo[b, £]azocine-5(6H)-yl)-4-oxobutanoyl]amino}-4-[(2,5-dioxopyrrolidin-1-yl)oxy]-N-(2,5,8,11,14,17,20heptaoxadocosan-22-yl)-4-oxobutanamide (Intermediate 27, 68 mg, 100%). LCMS (AA): m/z=839.4 (M+H).

Example 35.

The compounds listed in Table 23 were prepared as described in Example 6, Step 3, substituting N-succinimidyl 6-maleimidohexanoate with the starting material shown in Table 22.

Table 22

Source material Product Analytical data , , _1G ,Vos O HN O N ' ° rt' D O O ' Prom, connection, - 28* LCMS (AA): m/z= 1424.0 (M+H) Vos^ O ΝΗ _n O A- Ν 'θ Prom. connection-ZO Prom. connection-29 LCMS (AA): m/z= 1696.0 (M+H) O A· Ν '° ' ^Vos VL o ⁿ Ό Prom. connection-41 LCMS (AA): m/z= 1504.7 (M-H)

* Triethylamine was used as a base instead of N,N-diisopropylethylamine.

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Table 23

Example 36.

tert-Butyl {(29S)-30-[(2,5-dioxopyrrolidin-1-yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23octaoxa-27-azatriacontan-29-yl}carbamate (Intermediate 30)

O

Step 1: (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27azatriacontane-30-oic acid.

The title compound was prepared according to the procedure outlined in Example 21, Step 1, starting from (S)-3-amino-2-((tert-butoxycarbonyl)amino)propanoic acid (238 mg, 1.17 mmol) and 1-[(26-oxo-2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)oxy]pyrrolidine-2,5-dione (300 mg, 0.58 mmol) instead of (S)-2-amino-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid hydrochloride and 1-[(17-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl)oxy]pyrrolidine-2,5-dione using DMF as solvent and stirring at room temperature for 2 h. Purification by reverse phase flash column chromatography (0-100% ACN/aqueous triethylammonium acetate (10 mM)) gave (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa27-azatriacontane-30-oic acid (344 mg, 99%). LCMS (AA): m/z=599.4 (M+H).

Step 2: tert-butyl {(29S)-30-[(2,5-dioxopyrrolidin-1-yl)oxy]-26,30-dioxo

- 96 048725

2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-29-yl} carbamate, Intermediate 30.

The title compound was prepared according to the procedure described in Example 21, Step 2, starting from (29S)-29-[(tert-butoxycarbonyl)amino]-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27azatriacontane-30-oic acid (100 mg, 0.166 mmol) instead of (19S)-19-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-17-oxo-2,5,8,11,14-pentaoxa-18-azaicosan-20-oic acid using DCM as solvent and stirring at room temperature for 1 h. The reaction mixture was filtered and evaporated to give crude tert-butyl {(29S)-30-[(2,5-dioxopyrrolidin-1yl)oxy]-26,30-dioxo-2,5,8,11,14,17,20,23-octaoxa-27-azatriacontan-29-yl}carbamate (Intermediate 30, 116 mg, 100%).

Example 37.

[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N[[2-[[9-[(2R,3R^)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-hydroxy-tetrahydrofuran-2-yl]-6-oxo-N-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl carbamate (Intermediate 32) o

O

PS-32

Step 1: [4-[[^)-2-[[^)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5 S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate.

2-Amino-9-[(2N,3N^)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-1H-purin-6-one (1.62 g, 3.27 mmol) (for synthesis see Prakash, T.E. J. Med. Chem. 2005, 48, 1199-1210) was dissolved in dry pyridine and evaporated to dryness (3x3 ml) and then placed under vacuum for 15 min. The residue was dissolved in pyridine (43 ml) under argon and a solution of Intermediate 10 (5.91 g, 9.80 mmol) in pyridine (32 ml) was added. The reaction mixture was allowed to stir at room temperature for 16 h. MeOH (20 ml) was added and the reaction mixture was evaporated to dryness. MeOH (100 ml) and ammonium hydroxide (28-30% solution in water, 50 ml) were added and the resulting mixture was allowed to stir for 60 min. The reaction mixture was evaporated to dryness, the crude residue was adsorbed on Celite and purified by silica gel chromatography (0-100% EtOAc/hexanes) to give [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2N,3N-)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate (2.17 g, 63%). LCMS (AA): m4 = 1062.0 (M+H).

Step 2: [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6oxo-N-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl-carbamate.

[4-[ ^[( 2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-3-[tert-butyl(dimethyl)silyl]oxy-5-[[tert-butyl(dimethyl)silyl]oxymethyl]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methyl carbamate (2.17 g, 2.04 mmol) dissolved in THF (27 mL) was added to a polypropylene tube. Triethylamine (2.26 mL, 16.1 mmol) was added, followed by triethylamine trihydrofluoride (1.19 mL, 7.33 mmol), and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was slowly quenched by adding aqueous saturated sodium bicarbonate solution and then diluted with EtOAc. The organic phase was separated and washed with brine, dried ( _{Na2SO4 )} , filtered and evaporated to dryness. The crude residue was purified by silica gel chromatography (0-20% MeOH/DCM) to give [4-[[^)-2-[[^)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate (1.18 g, 69%). LCMS (AA): m/z=834.4 (M+H).

Step 3: [4-[[^)-2-[[^)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)phenylmethoxy]methyl]-3-hydroxytetrahydrofuran-2-yl]-6-oxo-N-purin-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate, Intermediate 32

[4-[[^)-2-[[^)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-3-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]-6-oxo-1H-purin2-yl]carbamoyl]phenyl]methyl]-N-methyl carbamate (1.13 g, 1.36 mmol) was dissolved in dry pyridine and evaporated to dryness (3 x 15 ml). The residue was dissolved in pyridine (13.3 ml) and 4,4'-dimethoxytrityl chloride (650 mg, 1.9 mmol) was added, followed by 4-dimethylaminopyridine (8 mg, 0.068 mmol). The reaction mixture was allowed to stir at room temperature for 2 h. The reaction mixture was evaporated to dryness and adsorbed onto celite and purified by silica gel chromatography (0-5% MeOH/DCM, with 0.5% _NEt3 ) to give [4-[[^)-2-[[^)-2-(tert-butoxycarbonylamino)-3methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)phenylmethoxy]methyl]-3-hydroxytetrahydrofuran-2-yl]-6-oxo-N-purin-2yl]carbamoyl]phenyl]methyl]-N-methylcarbamate (Intermediate 32, 1.12 g, 73%). LCMS (AA): m/z=1136.5 (M+H).

Example 38.

4-{[^)-2-({^)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxido-14-(9H-purin-9-yl)-2sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]6-oxo-6,9-dihydro-III-purin-2-yl}carbamoyl)benzyl]methylcarbamate or 4-{ [^)-2-({^)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9[(2R,5S,7R,8R,12aR,14R,15R,15aR)-fluoro-2,10,10-trioxido-14-(9H-purin-9-yl)-2-sulfanyldecahydro5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methyl carbamate (Intermediate 33)

- 98 048725

Step 1: [4-[[(2S)-2-[[(2S)-2-(TpeT-0yTOi<cui<ap00Hu.iaMUHo)-3-MeTu.i0yTaHOu.i]aMUHo]iipoiiaHOu.i] amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-pyric-9-yltetrahydrofurac-2yl]methylsulfamoyloxy]tetrahydrofurac-2-yl]-6-oxo-1H-pyric-2-yl]carbamoyl]phenyl]methyl]-N-methyl carbamate

A mixture of [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarboxylamico)-3-methylbutacoyl]amico]propacoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]-3-hydroxytetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate (Intermediate 32, 1.1 g, 0.97 mmol), (4-nitrophenyl) N-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-purin-9-yl-tetrahydrofuran-2-yl]methyl]sulfamate (667 mg, 1.17 mmol) (for the synthesis, see PCT Application No. PCT/IB2019/052364), 4-dimethylaminopyridine (122 mg, 0.97 mmol) and molecular sieves 4A (1.74 g) in dry DCM (13.6 mL) were stirred under argon at room temperature for 10 min. Triethylamine (0.60 mL, 4.3 mmol) was then added and the reaction mixture was allowed to stir at 40 °C for 16 h. The reaction mixture was filtered and evaporated, and the resulting residue was purified by reverse phase column chromatography (10–100% ACN/aqueous ammonium bicarboxylate (5 mM)) to give [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarboxylamico)-3methylbutacoyl]amico]propacoyl]amico]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)phenylmethoxy]methyl]-3-[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-pyric-9-yltetrahydrofurac-2-yl]methylsulfamoyloxy]tetrahydrofurac-2-yl]-6-oxo-1H-pyric-2yl]carbamoyl]phenyl]methyl]-N-methylcarbamate (0.94 g, 62%). LCMS (AA): m/z=1565.0 (M+H).

Step 2: [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbokylamico)-3-methylbutacoyl]amico]propacoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyfekyl)fekylmethoxy]methyl]-3

- 99 048725

[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purin-9-yltetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate.

To a solution of [4-[[^)-2-[[^)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9-[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)phenylmethoxy]methyl]-3[[(2R,3R,4R,5R)-3-[tert-butyl(dimethyl)silyl]oxy-4-fluoro-5-purin-9-yl-tetrahydrofuran-2yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-III-purin-2-yl]carbamoyl]phenyl]methyl]-N-methylcarbamate (939 mg, 0.60 mmol) in THF (12 mL) in a polypropylene tube was added triethylamine trihydrofluoride (0.52 mL, 3.17 mmol) and triethylamine (0.98 mL, 6.95 mmol). The reaction solution was stirred at room temperature overnight, then diluted with dichloromethane and washed successively with saturated aqueous NaHCO ₃ and brine. The organic phase was dried over anhydrous MgSO ₄ , filtered and evaporated. The resulting residue was purified by reverse phase column chromatography (10-100% ACN/aqueous ammonium bicarbonate (5 mM)) to give [4-[[(2S)-2[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2-[[9[(2R,3R,5S)-5-[[bis(4-methoxyphenyl)phenylmethoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2yl]carbamoyl]phenyl]methyl]-N-methylcarbamate (795 mg, 89%). LCMS (AA): m/z=1452.2 (M+H).

Step 3: [^^^^)-2-[[[(2K3^)-2-[2-[[2-[[[4-[[^)-2-[[^)-2-(tert-Butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methoxycarbonyl-methylamino]methyl]benzoyl]amino]-6-oxo-1-(3-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid

Diphenyl phosphite (0.225 ml, 1.17 mmol) was added at 0°C to a solution of [4-[[(2S)-2[[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butanoyl]amino]propanoyl]amino]phenyl]methyl N-[[2[[9-[(2R3R5S)-5-[[bis(4-methoxyphenyl)-phenyl-methoxy]methyl]-3-[[(2R,3R,4R,5R)-4-fluoro-3-hydroxy-5-purin-9-yl-tetrahydrofuran-2-yl]methylsulfamoyloxy]tetrahydrofuran-2-yl]-6-oxo-1H-purin-2yl]carbamoyl]phenyl]methyl]-N-methyl carbamate (792 mg, 0.55 mmol) in pyridine (2 ml). After complete addition, the reaction mixture was stirred at room temperature for 1 h. Triethylamine (0.62 ml, 4.37 mmol) and water (0.51 ml, 28.3 mmol) were then added and the reaction mixture was stirred for another 60 min at room temperature and then evaporated to dryness. The residue was then dissolved in acetic acid (1.25 ml, 21.8 mmol) and water (0.33 ml, 18.6 mmol) and allowed to stir at room temperature for 1 h. The reaction mixture was quenched by adding triethylsilane (4.3 ml, 26.2 mmol) and stirred at room temperature for 1 h. The reaction mixture was then evaporated to dryness and the residue was purified by reverse phase column chromatography (10-100% ACN/aqueous ammonium bicarbonate (5 mM)) to give [(2R,3R,4R,5R)-2-[[[(2R,3R,5S)-2-[2-[[2-[[[4[[^)-2-[[^)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylmethylamino]methyl]benzoyl]amino]-6-oxo-1H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid as an ammonium salt (396 mg, 59%). LCMS (AA): m/z=1213.4 (M+H).

Step 4: 4-{[^)-2-({^)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl[2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxido-14-(9H-purin-9yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin7-yl]-6-oxo-6,9-dihydro-3H-purin-2-yl}carbamoyl)benzyl]methylcarbamate or 4-{ [(2S)-2-({(2S)-2[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl[2-({9[(2R,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxido-14-(9H-purin-9-yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-3H-purin-2-yl}carbamoyl)benzyl]methylcarbamate, intermediate 33.

Diphenylphosphorochloridate (0.055 mL, 0.26 mmol) was added to pyridine (1.6 mL) and the solution was cooled to -35°C. A solution of [^^^^)-2-[[[(2R3^)-2-[2-[[2-[[[4-[[^)-2-[[^)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylmethylamino]methyl]benzoyl]amino]-6-oxo-1H-purin-9-yl]-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxysulfonylamino]methyl]-4-fluoro-5-purin-9-yl-tetrahydrofuran-3-yl]oxyphosphinic acid as an ammonium salt (80 mg, 0.065 mmol) in DCM (2.5 mL) and pyridine (3.2 mL) was added dropwise to the cooled reaction mixture over 15 min. The resulting solution was stirred at -35 °C for 30 min. Solid 3H-1,2-benzodithiol-3-one (25 mg, 0.15 mmol) and water (0.056 mL) were added respectively to the cooled reaction mixture, which was further stirred at room temperature for 1 h. Then, the reaction solution was cooled in an ice bath to 0 °C and quenched by adding aqueous sodium thiosulfate solution (0.2 M in water, 0.14 mL, 0.70 mmol). The mixture was stirred at room temperature for 5 min and concentrated under reduced pressure. The resulting residue was purified by reverse phase column chromatography (0-50% ACN/aqueous triethylammonium acetate (10 mM)) to give 4-{[^)-2-({^)-2-[(tert-butoxycarbonyl)amino]-3methylbutanoyl}amino)propanoyl]amino}benzyl[2-({9-[(2S,5S,7R,8R,12aR,14R,15R,15aR)-15-fluoro-2,10,10-trioxido-14-(9H-purin-9-yl)-2-sulfanyldecahydro-5,8-methanofuro[2,3-m][1,3,6,9,10,11,2]tet- 100 048725 raoxathiazaphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate or 4-['[(A)-2-(['(A)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-( {9-[(2R, 5 S,7R, 8RJ2aR, 14R, 15R45aR)-15-Top-2,10,10-trioxido-14-(9H-purin-9-yl)2-sulfanyldecahydro-5,8-methanofuro[2,3-t][1,3,6,9,10,11,2]tetraoxathiazaphosphacyclotetradecin-7yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate as N,N-diethylethanamine salt (intermediate 33, 27 mg, 31%). LCMS (AA): m/z=1227.9 (M+H). ^1H NMR (400 MHz, CD3OD) δ 9.14 (s, 1H), 8.75 (s, 1H), 8.63 (s, 1H), 8.30 (s, 1H), 7.53-7.36 (m, 5H), 7.34-7.17 (m, 3H), 6.47 (d, J=19.3 Hz, 1H), 6.16 (d, J=5.1 Hz, 1H), 5.87-5.82 (m, 1H), 5.76-5.55 (m, 1H), 5.50-5.40 (m, 1H), 5.00 (s, 2H), 4.79-4.75 (m, 2H), 4.68-4.61 (m, 1H), 4.58-4.47 (m, 2H), 4.44-4.39 (m, 1H), 4.09-4.02 (m, 1H), 3.92-3.89 (m, 1H), 3.67-3.62 (m, 1H), 3.57-3.53 (m, 1H), 3.01-2.94 (m, 1H), 2.87 (s, Zn), 2.74-2.68 (m, 1H), 2.11-2.03 (m, 1H), 1.45-1.43 (m, 3H), 1.44 (s, 9H), 0.98 (d, J=6.7 Hz, 3H), 0.93 (d, J=6.7 Hz, 3H). ³¹ P NMR (162 MHz, CD3OD) δ 57.35 (s, 1P).

Example 3 8A

4-{[(25S,29S,32S)-25-{[4-(11,12-didehydrodibenzo[b, G]azocin-5(6H)-yl)-4-oxobutanoyl]amino}29-isopropyl-32-methyl-24,27,30,33-tetraoxo-2,5,8,11,14,17,20-heptaoxa-23,28,31-triazatritriacontan33-yl]amino}benzyl [4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3^]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6yl}amino)-4-oxobutyl]methyl carbamate (C-34)

PS-35

C-34

To the suspension of 4-{[(2S)-2-{ [(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl[4-({9[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,21][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl} amino)-4-oxobutyl]methylcarbamate (Prom. comp.-35, 17.4 mg, 0.0154 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.49 mg, 0.0182 mmol) and (25S)-25-{[4-(l 1,12-didehydrodibenzo[b, £]azocin-5(6H)-yl)-4-oxobutanoyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid (Prom. comp.-27A) (10.4 mg, 0.0140 mmol) were added triethylamine (0.004 ml, 0.0266 mmol) in DCM (0.37 ml). The mixture was stirred at room temperature for 20 min, then DMF (0.15 ml, 2.23 mmol) was added. The mixture was then stirred at room temperature for 2 h, and the solvent was evaporated. The crude residue was purified by reverse phase flash column chromatography (0-50% ACN/aqueous triethylammonium acetate (10 mM)) to give 4-{[(25S,29S,32S)-25-{[4-(11,12-didehydrodibenzo[b, G]azocine-5(6H)-yl)-4-oxobutanoyl]amino}-29isopropyl-32-methyl-24,27,30,33-tetraoxo-2,5,8,11,14,17,20-heptaoxa-23,28,31-triazatritriacontan-33yl]amino}benzyl [4-({9-[(2R5R7R8R10R12aR14R15R15aR16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3^]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofur[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}amino)-4-oxobutyl]methylcarbamate (C-34) (2.0 mg, 6.9%) as N,N-diethylethanamine salt LCMS (AA): m/z=926.9 (M/2+I) HRMS (m/z): [M+H] ⁺ calc. for C ₈₀ H ₁₀₁ F ₂ N ₁₅ O ₂₆ P ₂ S ₂ 1852.6000; found, 1852.6006.

Example 38B.

(^88.98)-bicyclo[6.1.0]non-4-yn-9-ylmethyl{(258)-27-[(2,5-dioxopyrrolidin-1-yl)oxy]-24.27

- 101 048725 dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-25-yl}carbamate (intermediate 36)

Step 1: tert-butyl (25S)-25-{[(0eH3u.ioi<cu)i<ap0OHu.i]aMUHo}-24-oi<co-2.5.8.11.14.17.20)-he11oxa23-azaheptacosane-27-oate.

The title compound was prepared according to the procedure described in Example 26, step 1, starting from tert-butyl (3S)-3-{[(benzyloxy)carbonyl]amino}-4-[(2,5-dioxopyrrolidin-1yl)oxy]-4-oxobutanoate (2.2 g, 5.3 mmol) and 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (2.0 g, 5.9 mmol) instead of intermediate 15 and 2,5-dioxopyrrolidin-1-yl (tert-butoxycarbonyl)glycinate, respectively. Purification by silica gel chromatography (0-10% MeOH/DCM) gave tert-butyl (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20heptaoxa-23-azaheptacosane-27-oate (3.8 g, 100%).

Step 2: tert-Butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate.

tert-Butyl (25S)-25-{[(benzyloxy)carbonyl]amino}-24-oxo-2,5,8,11,14,17,20-heptaoxa-23azaheptacosane-27-oate (3.8 g, 5.9 mmol) was dissolved in ethanol (100 mL). Palladium (10% on carbon, 1.38 g, 1.17 mmol) was added and the mixture was stirred under hydrogen atmosphere (15 psi) for 3 h. The reaction mixture was filtered, washed with methanol and the filtrate was evaporated to dryness to give tert-butyl (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oate (2.56 g, 85%).

Step 3: (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-oic acid.

To a solution of (25S)-25-amino-24-oxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-27-oate (587 mg, 1.15 mmol) in DCM (11.3 mL) was added TFA (11.3 mL). The mixture was stirred at room temperature for 1 h. The reaction mixture was evaporated and azeotroped with toluene and then dried under high vacuum to give the crude intermediate as an oil. To the crude oil intermediate was added 1-({[(^^^)-bicyclo[6.1.0]non-4-yn-9ylmethoxy]carbonyl}oxy)pyrrolidine-2,5-dione (279 mg, 0.959 mmol) and DCM (9.8 mL), triethylamine (1.34 mL, 9.59 mmol) at room temperature. The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc (50 mL) and potassium carbonate solution (10 mL, 4.0 M in water). The separated organic layer was washed with brine, dried over Na2SO4, filtered and evaporated. The residue was purified by reverse phase column chromatography (0-100% ACN/aqueous ammonium bicarbonate (5 mM)). The desired fractions were evaporated in vacuo to give (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo-2,5,8,11,14,17,20heptaoxa-23-azaheptacosan-27-oic acid (19 mg, 3.1% yield). LCMS (AA): m/z=629.2 (M-H); 1H NMR (400 MHz, Methanol^4) δ=4.46 (dd, J=5.7, 7.4 Hz, 1 N), 4.25-4.12 (m, 2 N), 3.68-3.51 (m, 28 N), 3.413.34 (m, 5 N), 2.80-2.60 (m, 2 N), 2.33-2.12 (m, 4 N), 1.69-1.54 (m, 2 N), 1.40 (quin, J=8.7 Hz, 1 N), 1.02-0.89 (m, 2 N).

Stage 4: (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethyl{(25S)-27-[(2,5-dioxopyrrolidin-1yl)oxy]-24,27-dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-25-yl}carbamate (intermediate product 36).

The title compound was prepared according to the procedure described in Example 29 starting from (25S)-25-({[(1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-24-oxo2,5,8,11,14,17,20-heptaoxa-23-azaheptacosane-27-oic acid instead of 2,2-dimethyl-4-oxo-3,8,11,14,17pentaoxa-5-azanonedecane-19-oic acid. The reaction mixture was stirred for 30 min at room temperature to give (^^^)-bicyclo[6.1.0]non-4-yn-9-ylmethyl {(25S)-27-[(2,5dioxopyrrolidin-1-yl)oxy]-24,27-dioxo-2,5,8,11,14,17,20-heptaoxa-23-azaheptacosan-25-yl}carbamate (intermediate 36, 22 mg, 100%). LCMS (AA): m/z=728.4 (M+H).

- 102 048725

Example 38C.

The compounds listed in Table 22A (Intermediate 37 and Intermediate 42) were prepared as described in Example 10, substituting 2,5,8,11,14,17,20-heptaoxadocosan-22-amine (Intermediate 37 and Intermediate 42) and methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2-[(3-{[(9H-fluoren-9ylmethoxy)carbonyl]amino}propanoyl)amino]-4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenoxy}tetrahydro-2H-pyran-2-carboxylate (Intermediate 42) for the starting materials shown in Step 1 and Step 2.

Table 22A

Original

Raw materials (stage 1) materials (stage 2)

Analytical data of the intermediate acid of stage 2 of the benzoic acid product

LCMS (AA): m/z=935.6 (M-H);

LCMS (AA): m/z= 1290.2 (M-H);

* DMAP was not used in stage 2.

Example 38D.

4-{[(2S,5S)-57-amino-5-isopropyl-2-methyl-4,7-dioxo10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecaoxa-3,6-diazaheptapentacontane-1-oil]amino}benzyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopentau[1] [ 1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (C-37)

- 103 048725

Step 1: 4-{[(53S,56S)-1-a3ugo-53-u3onponun-56-MeTun-51,54,57-TpuoKCO3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-hexadecaoxa-52,55-diazaheptapentacontan-57-yl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate.

To a solution of intermediate 8 (18.68 mg, 0.01674 mmol), 1-[(1-azido-51-oxo3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48-hexadecaoxahenpentacontan-51-yl)oxy]pyrrolidine-2,5dione (32.3 mg, 0.03348 mmol), THF (0.6 mL), and DMF (0.2 mL) was added DIEA (0.044 mL, 0.2511 mmol). The mixture was stirred at room temperature for 3 h, then quenched with MeOH (5 mL). After evaporation of most of the solvent, the residue was purified by reverse phase column chromatography (0-50% ACN/aqueous ammonium bicarbonate (5 mM)). The desired fractions were evaporated and lyophilized to give the title compound (17.8 mg, 55%) as a white powder. LCMS (AA): m/z=1915 (M-H).

Step 2: 4-{[(2S,5S)-57-amino-5-isopropyl-2-methyl-4,7-dioxo10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecaoxa-3,6-diazaheptapentacontan-1-oyl]amino}benzyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecyn-7-yl]-6-oxo-6,9-dihydro-III-purin-2-yl}carbamoyl)benzyl]methylcarbamate (C-37).

In a 50 mL round bottom flask was added 4-{[(53S,56S)-1-azido-53-isopropyl-56-methyl-51,54,57trioxo-3,6,9,12,15,8,21,24,27,30,33,36,39,42,45,48-hexadecaoxa-52,55-diazaheptapentacontan-57-yl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methylcarbamate (17.7 mg, 0.00923 mmol), THF (1.0 mL) and triphenylphosphine (24.2 mg, 0.0923 mmol). This mixture was stirred at room temperature overnight. Sodium hydroxide (0.0554 mL, 1.0 mol/L in water) was added to the reaction mixture. After stirring the mixture at room temperature for 1 h, it was evaporated. The residue was purified by reverse phase column chromatography (0-50% ACN/aqueous ammonium bicarbonate (5 mM)). The desired fractions were collected and lyophilized to give 4-{[(2S,5S)-57amino-5-isopropyl-2-methyl-4,7-dioxo-10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55-hexadecaoxa-3,6diazaheptapentacontan-1-oyl]amino}benzyl [2-({ 9-[(2R, 5R,7R,8R,10R, 12aR, 14R, (15aS,16R)-16-hydroxy2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-Spurin-2-yl}carbamoyl)benzyl]methylcarbamate (C-37) (5.7 mg, 32%) as a white fluffy powder. LCMS (AA): m/z=1889 (MH). MCBP (m/z): [M+H]+ calcd for C ₈₀ Hi ₂₄ N ₁₂ O ₃₂ P ₂ S ₂ 1891.7434; found, 1891.7433.

Example 38E.

N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (intermediate 39)

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Step 1: (28S)-28-[4-(2,5-guoKCO-2,5-gurugpo-1 H-pyrrol-1 -yl)butyl]-26-oxo-2,5,8,11,14,17,20,23octaoxa-27-azanonacosan-29-oic acid.

To a mixture of (2S)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride (1.05 g, 4.00 mmol) and 1-[(26-oxo-2,5,8,11,14,17,20,23-octaoxahexacosan-26-yl)oxy]pyrrolidine-2,5-dione (2.08 g, 4.04 mmol) in pyridine (21.0 mL) was added DIEA (1.43 mL, 8.20 mmol), and the mixture was stirred at room temperature for 30 min, then cooled to 0 °C and stirred overnight. The volatile solvent was removed and the residue was azeotroped with toluene (3x20 mL) and acetonitrile (10 mL) to give (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacozan-29-oic acid (4.00 g, 100% yield) as a colorless oil. LCMS (AA): m/z=619.3 (M-H).

Step 2: N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (intermediate product 39).

A mixture of N-hydroxysuccinimide (469 mg, 4.08 mmol), N,N'-dicyclohexylcarbodiimide (824 mg, 4.00 mmol), and crude (28S)-28-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butyl]-26-oxo2,5,8,11,14,17,20,23-octaoxa-27-azanonacozan-29-oic acid from Step 1 (4.00 g, 4.00 mmol) in DCM (76.7 mL, 1200 mmol) was stirred at room temperature for 16 h. The solid that precipitated from this reaction was filtered and the filtrate was evaporated to give a crude oil. Purification by silica gel chromatography (0-20% IPA/DCM) gave N-{(2S)-6-(2,5-dioxo-2,5-dihydro-1Hpyrrol-1-yl)-1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23octaoxahexacosan-26-amide (Intermediate 39) (1.70 g, 52%) as a colorless oil. ¹ H NMR (400 MHz, methanol®) δ ppm. 1.42-1.55 (m, 2 N) 1.64 (m, 2 N) 1.81-1.93 (m, 1 N) 1.94-2.01 (m, 1 N) 2.52 (m, 2 N) 2.81-2.86 (m, 4 N) 3.36 (s, 3 N) 3.51-3.57 (m, 4 N) 3.60-3.66 (m, 26 N) 3.68-3.76 (m, 2 N) 4.03-4.17 (m, 2 N) 4.74-4.80 (m, 1 N) 6.74-6.86 (s, 2 N).

Example 38F.

N-{(2S)-6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetyl]amino}-1-[(2,5-dioxopyrrolidin-1yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17 ,20,23-octaoxahexacosane-26-amide (Intermediate 40)

Step 1: (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa27-azanonacosan-29-oic acid.

The title compound was prepared according to the procedure outlined in Example 38E, step 1, starting with N-(tert-butoxycarbonyl)N-lysine (0.49 g, 1.99 mmol) instead of (2S)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride. Purification by reverse phase column chromatography (0-60% ACN/aqueous formic acid (0.1%)) gave (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacozan-29-oic acid (1.14 g, 90%) as a colorless oil. LCMS (AA): m/z=639.4 (M-H).

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Step 2: (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)-26-oxo2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-oic acid.

To a solution of (28S)-28-{4-[(tert-butoxycarbonyl)amino]butyl}-26-oxo-2,5,8,11,14,17,20,23octaoxa-27-azanonacozan-29-oic acid (148 mg, 0.2310 mmol) in DCM (2.0 mL) was added TFA (2.0 mL) at room temperature. The mixture was stirred at room temperature for 15 min, evaporated, and then co-evaporated with toluene (x3) to give the crude TFA salt. After further drying of this crude product under high vacuum for 2 h, NHS maleimidoacetic acid ester (69.89 mg, 0.2772 mmol) in DCM (2650 mg, 2.00 mL, 31.2 mmol) and DIEA (0.121 mL, 0.6929 mmol) were added and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was evaporated. Purification by reverse phase column chromatography (1030% ACN/aqueous ammonium acetate (10 mM)) gave (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1Hpyrrol-1-yl)acetyl]amino}butyl)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacozan-29-oic acid (120 mg, 38%) as an oil. LCMS (AA): m/z=676.3 (M-H).

Step 3: N-{(2S)-6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}-1-[(2,5dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23 -Octaoxahexacosane-26-amide (Intermediate 40).

To a flask containing (28S)-28-(4-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}butyl)26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacozan-29-oic acid (311 mg, 0.459 mmol) and DCM (2.94 mL) were added N,N'-dicyclohexylcarbodiimide (94.7 mg, 0.459 mmol) and N-hydroxysuccinimide (53.9 mg, 0.459 mmol). The suspension was stirred under argon at room temperature for 1 h. Then the reaction mixture was cooled to 0 °C, filtered through celite and washed with MeCN. The filtrate was evaporated. The residue was redissolved in DCM, washed with water (x2), dried over MgSO4 and evaporated to give N-{(2S)-6-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}1-[(2,5-dioxopyrrolidin-1-yl)oxy]-1-oxohexan-2-yl}-2,5,8,11,14,17,20,23-octaoxahexacosan-26amide (Intermediate 40) (300 mg, 84.4%) as a sticky oil. LCMS (AA): m/z=773.2 (M-H).

Example 38G.

The compound shown in Table 22C (C-43) was prepared as described in Example 23, Step 2 and Step 3, replacing the starting material shown in the table with methyl (2S,3S,4S,5R,6S)-3,4,5-triacetoxy-6-{2[(3-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}propanoyl)amino]-4-[({[2-({9[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)-2,10disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-3-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenoxy}tetrahydro-2H-pyran-2-carboxylate.

Table 22B

Source material Product Analytical data 0 g ° ° > / o I 1 L Ai o Al /tA ii GA° PL 9 ?' L A) ° aM H<5 b O A nA HS' ⁰ Prom. comp.-43 C-43 LCMS (AA): m/z=1755 (M+H); MSVR (m/z): [M+N] ⁺ calc. FOR C72H97N11032P2S2 1754.5291; found, 1754.5321.

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Table 22C

Example 38H.

Intermediate 44, shown in Table 22D, was prepared as described in Example 38E, replacing (^)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride in step 1 with the starting material (2R)-2-amino-6-(2,5-dioxopyrrol-1-yl)hexanoic acid hydrochloride shown in Table 22D.

Table 22D

Example 39.

The procedure for preparing Ab-STING agonist conjugates via stochastic cysteine conjugation (shown in Fig. 1).

To a solution of anti-GCC antibody (clone 5F9, see PCT Publication No. WO 2011/050242, 60 mg/ml) in 50 mM histidine, 100 mM arginine, pH 6.0 buffer was added 0.5 M Tris/25 mM EDTA pH 8.0 buffer until the pH of the solution was 7.5-8.0. To the above solution was added TCEP (10 mM solution in H2O, 3-5 equiv). The reaction mixture was purged with argon and incubated at room temperature or 37°C for 2 hours with gentle shaking. Then, the desired linker-payload construct (10 mM solution in DMA, 5-9 equiv) was slowly added to the above mixture. The reaction mixture was purged with argon and incubated at 37°C for an additional 1.5-2 h with gentle shaking. The reaction mixture was purified following the preparative SEC method described herein to yield ADC. ADC concentration, percent aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

The above procedure can be used to obtain conjugates of other antibodies. Example 40.

The antibody-drug conjugates listed in Table 24 were prepared as described in Example 39 using the parent linker-payload constructs shown as starting material.

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Table 24

Source material ADC product Payload DAR Aggregation % Exit % S-21 ADC-1 Connection #14 4,5 3.09 100 S-27 ADC-2 Connection 1-5s 3.7 2.08 56 C-3 ADC-3 Connection 1-5s 2.9 npko 57 C-16 ADC-4 Connection 1-5s 3.5 2,13 69 C-8 ADC-5 Connection 1-5s 3.6 1.54 50 S-24 ADC-6 Connection 1-5s 2,5 2.58 56 S-26 ADC-7 Connection 1-5s 4.1 NPKO 42 C-18 ADC-8 Connection 1-5s 3.9 0.59 83 C-1 ADC-9 Connection 1-5s 4.3 NPKO 29 C-4 ADC-15 Connection #14 3.1 npko 63 S-38 ADC-16 Connection #14 3.7 npko 60

Example 41.

The procedure for preparing Ab-STING agonist conjugates using transglutaminase conjugation (shown in Fig. 2).

Deglycosylation: A solution of anti-GCC antibody (clone 5F9, see PCT Publication No. WO 2011/050242, 60 mg/ml) in 50 mM histidine, 100 mM arginine, pH 6.0 buffer was diluted with an equal volume of PBS, pH 7.2. N-glycosidase F (New England Biolabs, P0704S, 500,000 units/ml, 300 units per mg antibody) was added to the solution and the reaction mixture was heated to 37°C with gentle stirring overnight. The resulting deglycosylated 5F9 was replaced with PBS, pH 7.2 buffer.

Transglutaminase conjugation: To the deglycosylated 5F9 (10-20 mg/mL) solution in PBS prepared above, 0.1 M DMSO solution of 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-1amine (azido-PEG9-amine, BroadPharm, BP-23556, 40 equivalents) was added, followed by transglutaminase (ACTIVA™, Ajinomoto, 5~10 mg per 1 mg antibody). The reaction mixture was heated to 37°C with gentle stirring overnight. The product was purified by the preparative SEC method described herein to obtain 5F9-NH-PEG-azide.

Strain-promoted azide-alkyne cycloaddition: To the 5F9-NH-PEGazide conjugate solution prepared above (2-15 mg/mL in PBS), 4~10 mM DMSO solution of strained alkyne containing linker-payload constructs (3-5 equivalents, in which DMSO <10% of the total solvent volume) was added. The reaction mixture was gently stirred at room temperature overnight. The product was purified by the preparative SEC method described herein to afford ADC. ADC concentration, percentage of aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

The above procedure can be used to obtain conjugates of other antibodies.

Example 42.

The antibody-drug conjugates listed in Table 25 were prepared as described in Example 41 using the linker-payload parent constructs shown as starting material in the table.

Table 25

Source material ADC product Payload DAR Aggregation % Exit % C-15 ADC-10 Connection 1-5s 1,8 NPKO 40 C-13 ADC-11 Connection 1-5s 2 npko 81

Example 43.

Preparation of Ab-STING agonist conjugates by transglutaminase conjugation (shown in Fig. 3).

To the deglycosylated 5F9 solution (10~20 mg/mL, prepared according to the deglycosylation procedure described in Example 41) in PBS, 1 M Tris, 5 M NaCl, pH 8.0 buffer (10~20% of the total volume) was added to adjust the pH to 8.0. A 10 mM DMSO solution of the primary amine-containing linker-payload constructs (20 equiv.) was added to the solution, followed by transglutaminase (ACTIVA™, Ajinomoto, 100-150 mg per 1 mg of antibody). The reaction mixture was heated to 37 °C at room temperature.

- 108 048725 with stirring overnight. The product was purified using a HiTrap Protein A HP column (GE Healthcare, 17-0402-01), first washing with 20 mM phosphate, pH 7.0, and then eluting the ADC with 0.1 M citric acid, pH 4.0.

The above procedure can be used to obtain conjugates of other antibodies.

Example 44.

The antibody-drug conjugates listed in Table 26 were prepared as described in Example 43 using the parent linker-payload constructs shown as starting material in the table.

Table 26

Source material ADC product Payload DAR Aggregation % Exit % S-30 ADC-17 Connection 1-5s 1,6 npko 72 S-37 ADC-18 Connection 1-5s 1,2 npko 72

Example 45.

2-amino-N(2-((2-(2-azidoacetamido)ethyl)amino)-2-oxoethyl)acetamide

EDC

HOAT

NEEE

Cbz

Vos

DMF

H ₂ , Pd/C ethanol

Vos

EDC, HOAt. iPrNEt ₂ , DMF

Step 1: Benzyl (2,2-dimethyl-4,7,10-trioxo-3-oxa-5,8,11-triazatridecan-13-yl)carbamate

To a solution of Boc-Gly-Gly-OH (550 mg, 2.32 mmol), EEDC-HCl (437 mg, 2.28 mmol), and HOAt (317 mg, 2.33 mmol) in DMF (8.0 mL) was added triethylamine (0.340 mL, 2.44 mmol) at 0 °C. The resulting yellow heterogeneous mixture was allowed to stir for 15 min. A solution of benzyl (2-aminoethyl)carbamate (500 mg, 2.44 mmol) in DMF (6.0 mL) was slowly added. After 10 min, the mixture was allowed to warm to room temperature and stirred for 3 days. The reaction mixture was partitioned into water (100 mL) and EtOAc (50 mL). The separated aqueous layer was extracted with EtOAc (50 mL). The combined organic layer was washed with 50 mL of water, then with brine, dried over Na2SO4 and evaporated. The crude product was purified by silica gel chromatography (0-6% MeOH/ _{CH2Cl2 )} to give tert-butyl N-[2-[[2-[2-(benzyloxycarbonylamino)ethylamino]-2-oxoethyl]amino]-2-oxoethyl]carbamate (617 mg, 1.51 mmol, 65% yield) as a white solid. LCMS (MC): m/z=431.2 (M+Na). ^1H NMR (400 MHz, DMSO-06) δ 7.97-7.97 (m, 1H), 7.86-7.84 (m, 1H), 7.39-7.29 (m, 5H), 7.27-7.24 (m, 1H), 7.01-6.98 (m, 1H), 5.02 (s, 2H), 3.67 (d, J=5.62 Hz, 2H), 3.58 (d, J=5.87 Hz, 2H), 3.03-3.18 (m, 4H), 1.39 (s, 9H).

Step 2: tert-butyl (2-((2-((2-aminoethyl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate.

To a solution of tert-butyl N-[2-[[2-[2-(benzyloxycarbonylamino)ethylamino]-2-oxoethyl]amino]-2-oxoethyl]carbamate (615 mg, 1.51 mmol) in ethanol (20.0 mL) was added 10% Pd/C (62.0 mg, 0.0583 mmol). The mixture was purged with _H2 (1 atm) and stirred at room temperature for 3 h. Celite was added to the reaction mixture and the suspension was filtered through a short pad of Celite. The filtrate was evaporated and the residue was dried by coevaporation with toluene followed by high vacuum to give tert-butyl N-[2-[[2-(2-aminoethylamino)-2-oxoethyl]amino]-2-oxo-ethyl]carbamate (407 mg, 1.48 mmol, 98.5% yield) as an off-white solid. LCMS (MC): m/z=275.2 (M+H). 1H NMR (400 MHz, DMSO-06) δ 8.05-7.89 (m, 1H), 7.80-7.63 (m, 1H), 7.08-6.96 (m, 1H), 3.67 (d, J=5.75 Hz, 2H), 3.56 (d, J=5.87 Hz, 2H), 3.06 (k, J=6.24 Hz, 2H), 2.59-2.53 (m, 2H), 1.87-1.61 (m, 2H), 1.39 (s, 9H).

Step 3: tert-butyl No[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxoethyl]amino]-2oxoethyl]carbamate.

A mixture of EDC-HCl (57.6 mg, 0.300 mmol), HOAt (43.6 mg, 0.320 mmol), and 2-azidoacetic acid (27.0 μL, 0.315 mmol) in dichloromethane (2.70 mL) was stirred for 10 min. The mixture was cooled in an ice bath, and then tert-butyl N-[2-[[2-(2-aminoethylamino)-2-oxoethyl]amino]-2

- 109 048725 oxoethyl]carbamate (75.0 mg, 0.273 mmol) was added followed by the addition of N,N-diisopropylethylamine (55.8 μL, 0.320 mmol) after a few minutes. The mixture was stirred for 5 min at 0 °C and then at room temperature overnight. Water (2 mL) and EtOAc (5 mL) were added and the mixture was stirred vigorously for 20 min. The mixture was diluted with EtOAc (25 mL) and then washed with 50% brine/water (20 mL) and brine. The separated organic layer was dried over Na2SO4 and evaporated. Purification by silica gel chromatography (0-8% MeOH/CH ₂ Cl ₂ ) gave tert-butyl N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxoethyl]amino]-2-oxoethyl]carbamate (88.0 mg, 0.246 mmol, 90.1% yield) as a glassy solid. LCMS (MC): m/z=356.2 (M-H). 1H NMR (400 MHz, DSO-de) δ 8.16-8.08 (m, 1H), 8.04-7.92 (m, 1H), 7.92-7.81 (m, 1H), 7.05-6.94 (m, 1H), 3.82 (s, 2H), 3.67 (d, J=5.62 Hz, 2H), 3.58 (d, J=5.87 Hz, 2H), 3.10-3.19 (m, 4H), 1.39 (s, 9H).

Step 4: 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxoethyl]acetamide (Intermediate 34).

To a solution of tert-butyl N-[2-[[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxoethyl]amino]-2-oxoethyl]carbamate (86.0 mg, 0.241 mmol) in dichloromethane (4.0 mL) was added trifluoroacetic acid (1.00 mL, 8.77 mmol). The mixture was stirred at room temperature for 30 min. The mixture was diluted with 5 mL of toluene and evaporated. The residue was dissolved in 5 ml of water and lyophilized to give 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxoethyl]acetamide 2,2,2-trifluoroacetic acid salt (102 mg, 0.275 mmol, quantitative) colorless syrup. LCMS (MC): m/z=258.1 (M+H). ^1H NMR (400 MHz, DMSOL ₆ ) δ 8.55-8.62 (m, 1H), 8.12-8.20 (m, 1H), 8.02-8.09 (m, 1H), 7.90-8.02 (m, ZN), 3.743.82 (s, 2H), 3.80 (m, 2H), 3.55-3.68 (m, 2H), 3.11-3.19 (m, 4H).

Example 46.

Production and purification of 5F9-LPETGG-His6.

The 5F9-LPETGG-His6 protein was produced using the Expi293F Mammalian Transient Protein Expression System Kit from Thermo Fisher, Cat. #A14635, which includes Expi293FTM cells, Expi293TM Expression Growth Medium, and Expifectamine 293TM Transfection Kit. 1.8 liters of Expi293FTM cells at a density of 2.8 x 10 cells/mL in a 5 L vented shake flask were transfected with 0.666 mg light chain DNA plus 0.334 mg heavy chain DNA mixed with 5.4 mL Expifectamine 293TM Transfection Reagent and incubated overnight in a 37°C, 8% _CO2 incubator shaking at 100 rpm. The next day, 10 ml of enhancer 1 and 100 ml of enhancer 2 from the kit were added to the flask. The flask was incubated under the above conditions and harvested on day 5 post-transfection. The cells were centrifuged at 9000 rpm for 10 min at 4°C, then filter sterilized through a 0.22 μm filter element and stored at 4°C until column purification with 30 ml of MabSelect SuRe LX Protein A (GE Healthcare, catalog 17-5474-03), which was freshly packed. After the supernatant was loaded onto the column using a peristaltic pump, the resin was washed with 60 ml [2 column volumes (CV)] of low salt buffer (25 mM trisodium citrate pH 7.6, 125 mM NaCl), then washed with 60 ml (2CV) of high salt buffer (25 mM trisodium citrate pH 7.6, 2 M NaCl), followed by another 60 ml (2CV) of low salt buffer. Protein was eluted with a pH gradient elution of 25 mM citric acid, 125 mM NaCl, pH 8 to pH 2.2, and peak fractions were collected. Each fraction was neutralized with 1 M trisodium citrate pH 8.2 to 70 mM to approximate pH 6. Fractions in the major peak (2A5-2C5) were pooled. The pooled fractions were concentrated and the buffer was exchanged with 25 mM sodium citrate, pH 5.5, 125 mM NaCl buffer using a Sartorius VIVAFLOW200 cartridge with a molecular weight cutoff of 50 kDa. The sample was then filtered through a 0.22 μM syringe filter unit, aliquoted and stored at -80°C. The final yield was 623 mg of purified 5F9-LPETGG-His6 antibody with a purity of > 95% and an endotoxin of 0.64 EU/mL.

Heavy chain sequence:

MGWSCIILFLVATATGVHSOVOLOOOWGAGLLKPSETLSLTCAVFGGSFSGYYWSWIRO PPGKGLEWIGEINHRGNTNDNPSLKSRVTISVDTSKNQFALKLSSVTAADTAVYYCARE RGYTYGNFDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVT VSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKA GGGSLPETGGHHHHHH (signal sequence underlined) (SEQ ID NO. 1) Light chain sequence:

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MGWSCIILFLVATATGVHSEIVMTQSPATLSVSPGERATLSCRASOSVSRNLAWYOOKP GQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTIGSLQSEDFAVYYCQQYKTWPRTFGQ GTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (signal sequence underlined) (SEQ ID NO. 2)

Example 47.

Procedure for the Preparation of Ab-STING Agonist Conjugates by Sortase Conjugation (Depicted in Fig. 4) Sortase Conjugation: The 5F9 antibody containing LPETGG-His6 at its C-terminus of the heavy chains was prepared according to the procedures described in Example 46. A solution of 5F9 containing LPETG-His6 tags at the C-terminus (5F9-LPETGG-His6) of the heavy chains in 25 mM citrate, 125 mM NaCl, pH 5.5 buffer was diluted with 50 mM HEPES, 150 mM NaCl, pH 7.45 buffer so that the protein concentration was 48 μM. _CaCl2 (4 mM aqueous solution, 80~100 equivalents) and 2-amino-N-[2-[2-[(2-azidoacetyl)amino]ethylamino]-2-oxoethyl]acetamide (Intermediate 34, 800 μM solution in 50 mM HEPES, 150 mM NaCl, buffer pH 7.45 with 0.1% DMSO, 20 equivalents) were added to the solution, followed by Sortase A Q60-K206-P94S-D160N-D165A-K196 His6 (expressed as described in Antos, J. M., Ingram, J., Fang, T., Pishesha, N., Truttmann, M. C., Ploegh, H. L. (2017). Site-specific labeling of proteins via RNA-mediated transpeptidation sorting. Current Protocols in Protein Science, 89, 15.3.115.3.19, 2.0 μM solution in 50 mM HEPES, 150 mM NaCl, buffer pH 7.45, 0.03 equivalents). The reaction mixture was stirred at room temperature for 4 h. The product was purified on a Capturem nickel column (Takara, washed with PBS) followed by dialysis or the preparative SEC method described herein to obtain 5F9-LPETG azide.

Strain-promoted azide-alkyne cycloaddition: To the 5F9-LPETGGNH( _CH2 ) _{2NHCOCH2 -azide} conjugate solution prepared above (2~15 mg/mL in PBS), a 4~10 mM DMSO solution of the strained alkyne containing the linker-payload construct (3-5 equivalents, in which DMSO < 10% of the total solvent volume) was added. The reaction mixture was gently stirred at room temperature overnight. The product was purified by the preparative SEC method described herein to afford ADC. The ADC concentration, percentage of aggregation, and DAR were determined by UV absorbance, analytical SEC, and LC-QTOF, respectively, as described in the analytical methods.

The above procedure can be used to obtain conjugates of other antibodies.

Example 48.

The antibody-drug conjugates listed in Table 27 were prepared as described in Example 47 using the linker-payload parent constructs shown as starting material in the table.

Table 27

Source material ADC product Payload DAR Aggregation % Exit % C-15 ADC-12 Connection 1-5s 1,4 3.67 60 C-9 ADC-13 Connection 1-5s 1,8 npko 66 C-13 ADC-14 Connection 1-5s 1,4 3.7 84

Example 49.

Biological protocols and cell-based sting analysis data.

Tritosome assay conditions.

The test compound (121 μM linker-payload construct solution in DMSO or ADC solution in PBS, 4.65 μL) was added to a mixture of 125 μL rat liver tritosomes (purchased from XenoTech) and 433 μL aqueous buffer solution (54.7 mg/mL phosphate, 1.7 mg/mL EDTA, pH 6.0), and the solution was incubated at 37°C. 40 μL of sample were removed after 10 and 30 min, 1, 3, 5, and 24 h and treated with 160 μL 0.1% formic acid in methanol solution in a 96-well plate and then stored at 80°C. After collection of the last time point, each sample was thawed and treated with 200 μl of 0.1% formic acid in methanol containing 150 nM carbutamide (internal standard). The samples were centrifuged at 4000 g for 10 min and analyzed according to the LC/MS/MS method described in this paper. Excel-Fit was used to calculate t _1/2 . The calculated t _1/2 value is presented in Table 28. As shown in Table 28, free payload release from ADC-3 in this tritosomal assay was not observed within 24 hours, while ADC9, which has the same payload, successfully released the payload with a t _1/2 of 7 hours, indicating that the ADC-9 linker resulted in a markedly improved payload release rate under surrogate cell release conditions compared to ADC-3.

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Table 28

ADC Payload ti/2 (hour) ADC-1 Connection #14 2,4 ADC-3 Connection 1-5s .* ADC-9 Connection 1-5s 7.0 ADC-15 Connection #14 2.9 ADC-16 Connection #14 3.7

* No free payload release was observed within 24 hours.

A graphical representation of the payload release over time for ADC-1, ADC-3, and ADC-9 in Table 28 is shown in Figs. 5, 6, and 7.

Conditions for plasma stability analysis.

Test compounds were spiked into 1 ml plasma at a concentration of 10 μg/ml and 5 equal volume aliquots were then distributed into 2 ml Eppendorf microcentrifuge tubes (labeled 0, 24, 48, 72 and 96 hours). Time 0 sample tubes were immediately stored at -80°C and the remaining tubes were incubated at 37°C with moderate shaking. Aliquots were removed from the incubator at the appropriate time point and stored at -80°C. After all samples were collected, they were thawed at room temperature and placed on wet ice. 50 μl of each sample was dispensed in triplicate into a 96-well microtiter plate. Samples were quenched with 200 μl of ice-cold methanol containing 50 nM internal standard. The samples were vortexed for 2 min and then centrifuged at 3000 rpm for 10 min. 185 μL of the supernatant was transferred to a clean injection plate and then dried under N2 gas at 40°C. The dried sample extracts were reconstituted with 100 μL of LCMS grade water and then vortexed for 1 min to prepare for LC-MS/MS analysis.

Each sample was separated by reversed-phase HPLC using a Synergi 2.5 µm Polar-RP 100A C18 column (2.0 mm x 30 mm) (Phenomenex®) at 40°C using a gradient of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The analytes were detected by positive ion spray in multiple reaction monitoring (MRM) mode using a SCIEX API 4500 QTRAP instrument. The percentage payload loss in human, primate, and mouse plasma at various time points is presented in Table 29.

Table 29

AD C Loss of payload (%) in human plasma Payload loss (%) in monkey plasma Loss of payload (%) in mouse plasma 1d 2d Hello 4d 1d 2d Hello 4d 1d 2d Hello 4d AD C-9 0,1 0,7 1,1 1,7 0,3 0,3 1,0 1,2 1,4 2,1 3.0 3,4 AD C-2 5.7 ID 13.2 20.8 7.3 14.2 20.9 25.8 5.8 9.4 14.1 18.8 AD 0,1 0.8 1,6 1,8 0 0,7 1,3 1,6 0,4 0.8 1,0 5,6

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C-4 AD C-3 1,3 3.7 5.4 7.3 1,4 3.2 5.5 7.6 0,7 1,5 1,0 1,3 AD C-1 11,5 21.6 32.1 40.9 No tribute No tribute No data No data 9.2 16,0 23.3 30.4 AD C- 10 0 0 1,2 1,7 0 0 1,1 1,5 2 1,8 1,9 2.6 AD C-5 0 0.9 1,6 2,1 0,5 0,7 1,3 1,8 2,2 1,5 2,2 6.3 AD C-6 0 0 1,7 2,4 0,6 1,1 1,5 2,3 0,3 0,7 1,0 0.9 AD C-7 6.3 12,0 15,5 23.4 6.0 11.6 16.3 20,5 4.0 6.0 3.6 19.9 AD C-8 0,2 0,6 0.9 1,2 0,3 0,5 0,7 1,0 0,5 0,7 1,3 1,3 AD C- 12 0 0 0,3 1,7 0 0,4 1,1 1,3 21.2 25.6 27.2 28.8 AD C- 14 0 0,7 2,2 2.6 0,5 1,0 1,6 2,4 16,5 15,0 17.6 17.8 AD C- 15 0.9 2.0 3.0 4.1 0 1,6 2,2 3.1 3.6 3.7 6,6 8.0 AD C- 16 1,0 2.0 2,5 3.6 0.9 1,1 1,7 2.6 2,5 8.6 8.4 11.7

Conditions for the THP1 Dual Lucia reporter gene assay.

THP1-Dual™ KI-hSTING-R232 cells (InvivoGen#thpd-r232) were generated from the human monocyte cell line THP-1 by stable biallelic knockout of the endogenous human STING gene HAQ and knockout of the R232 variant of human STING. These cells also stably express the inducible secreted Lucia luciferase reporter gene under the control of the minimal ISG54 (interferon-stimulated gene) promoter in combination with five IFN-stimulated response elements (ISREs). Expression of the reporter gene allows study of the IFN regulatory factor (IRF) pathway by assessing Lucia luciferase activity. In addition to human STING and luciferase, these cells were engineered to stably express human guanylyl cyclase C (GCC), allowing study of target-mediated activation of the IRF pathway. Non-GCC vector-expressing cells were used as a control.

On the day of the experiment, cells were seeded on a white 384-well plate (Corning 356661) at a density of 15,000 cells/25 μl per well in growth medium (RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% heat-inactivated fetal bovine serum, 100 μg/ml Normocin™, 100 U/μλ-ΙΟΟ μg/ml Pen-Strep, 10 μg/ml blasticidin, 100 μg/ml zeocin, and 1 μg/ml puromycin). 5 μl of GCC-targeting ADC samples were added to the cell plates and then incubated at 37°C for 20 hours. At the end of incubation, 10 µl/well of QUANTI-Luc™ (InvivoGen # rep-qlc1) was added and luminescence was immediately measured using LeadSeeker.

For the above-described analysis method, the percentage of luminescence signal induction was calculated for

- 113 048 725 of each tested ADC at various concentrations relative to untreated and treated controls. Compound concentration versus percent signal induction curves were fitted to obtain EC50 values. One skilled in the art will appreciate that values obtained as EC50 values may vary experimentally. The observed EC50 and Emax are presented in Table 30. As shown in Table 30 below, ADC-16 was approximately 21-fold more potent than ADC-15 in THP1 vector cells, likely due to a more rapid release of intracellular payload from ADC-16 compared to ADC-15.

__________________________________________________________________Table 30

ADC hGCC expression of TIR 1 THP Vector 1 EC ₅₀ hM Emax EC ₅₀ hM Emax ADC-1 0,068 123 3.9 82.3 ADC-9 0,10 91.6 >280 39.6 ADC-2 0.28 80.7 >370 17,0 ADC-10 0,10 81.3 >180 3.8 ADC-3 >290 0.9 >290 0,4 ADC-4 0,047 96.6 3.92 67.6 ADC-5 0,089 82.5 11,0 49.8 ADC-11 0,12 86.9 >200 12.6 ADC-6 0.11 90.8 >250 28.3 ADC-7 0.57 90.1 >380 15.1 ADC-8 0.17 102.2 >430 40.2 ADC-12 0,10 94.8 >140 22.4 ADC-13 0,060 95 No data No data ADC-14 0,074 86.9 >140 15.4 ADC-15 0,067 108 242 54.9 ADC-16 0,040 97.7 11,2 83,0 ADC-17 0,032 105 >500 3.46 ADC-18 <0.025 100 >500 1,18

Example 50.

Pharmacokinetic evaluation in mice.

For in vivo evaluation of ADC in Balb/C mice bearing GCC-expressing CT26 murine colon carcinoma tumors, 6-8 week-old female Balb/C mice (purchased from Jackson Laboratory) were used. Parental CT26.WT cells were obtained from ATCC, Catalog #CRL-2638. GCC-expressing CT26 cells were constructed at Takeda by transducing CT26.WT with the lentiviral vector pLenti6.3 containing the human GCC gene, yielding CT26 pLenti6.3 GCC clone A5. CT26 pLenti6.3 GCC clone A5 cells were grown under sterile conditions in an incubator at 37°C with 5% CO2 for 12-14 days. Cells were grown in RPMI-1640 medium with 10% fetal bovine serum. Cells were passaged every 3-4 days using 0.05% trypsin/EDTA to detach cells. On the day of implantation, cells were lifted and resuspended in serum-free RPM-1640 medium at a concentration of 0.5 x 106 cells/100 μl. Clone A5 CT26 pLenti6.3 GCC cells were implanted by subcutaneous injection into the lower flank of mice using a 27-gauge needle (0.5 x 106 cells/100 μl injection volume per mouse). After cell implantation, tumors were measured with calipers, and mice were weighed twice weekly after tumor palpation. Tumors were measured in two dimensions. Caliper measurements were calculated as (L x W2)/2. Mice were fed a normal diet and housed in an SPF animal facility in accordance with the Guide for the Care and Use of Laboratory Animals and the Institutional Animal Care and Use Committee regulations. Animals were maintained at 18–26°C, 50 ± 20% relative humidity, and 12-hour light/dark cycles with food and water available ad libitum.

The pharmacokinetics of ADC were studied following injection of ADC into Balb/C mice bearing CT26-GCC tumors (^=3/treatment group) when the mean tumor volume (MTV) reached 500-800 ^mm3 . Serum samples were collected at various time points and stored frozen for analysis.

Total antibody and conjugated payload levels in mouse plasma were measured using

- 114 048725 by 2-in-1 immunocapture LC/MS analysis on a Shimadzu UHPLC system coupled to a Sciex 6500 QTRAP mass spectrometer. Briefly, mouse plasma samples were incubated with anti-human IgG-coated magnetic beads for 45 min at room temperature, then non-specifically bound proteins were removed by washing the magnetic beads with PBST (PBS buffer at pH 7.4 containing 0.05% Tween 20) and PBS buffer sequentially. Thereafter, both naked antibodies (DAR=0) and ADC (DAR>1) were eluted from the magnetic beads with 0.1% trifluoroacetic acid. After neutralization of the eluents and addition of stable isotope-labeled internal standards, one aliquot of the sample was pipetted and digested with papain for 1 h at 37°C and then used for LC/MS analysis of conjugated payloads. The remaining samples were digested with trypsin/Lys-C for 1 h at 70°C and then used for LC/MS analysis of total antibodies.

The free payload in the bloodstream was also measured by LC/MS after performing plasma protein precipitation. Briefly, mouse plasma was mixed with 8 volumes of methanol containing a stable isotope-labeled internal standard, then the supernatants were evaporated to dryness at 40°C under a gentle stream of nitrogen. Finally, the residues were reconstituted in LC/MS-grade water prior to LC/MS analysis. The PK profile of ADC-1, ADC-8, and ADC-9 is presented in Table 31. The graphical representation of the plasma PK is shown in Figs. As shown in Table 24, the DAR values for ADC-1, ADC-8, and ADC-9 are 4.5, 3.9, and 4.3, respectively, resulting in a higher molar concentration of conjugated CDN obtained at time 0 than the molar concentration of total Ab as shown in each of Figs. 8–10. However, as shown in Fig. 8, the molar concentration of conjugated CDN from ADC-1 dropped faster than the molar concentration of total Ab, and the two concentrations converged on day 7 (resulting in a DAR of about 1 on that day), indicating that the DAR of ADC-1 decreased over time due to the lower stability of its linker. In contrast, as shown in Figs. 9 and 10, the molar concentration of conjugated CDN from both ADC-8 and ADC-9 continued to be higher than that of their respective total Ab throughout the experiment. Furthermore, the longer half-lives of conjugated payload/CDN from ADC-8 and ADC-9 compared to ADC-1, as shown in Table 31, are likely a result of the increased linker stability in ADC-8 and ADC9. Thus, this experiment demonstrates that ADC-8 and ADC-9 linkers have improved stability in mouse plasma compared to ADC-1.

Table 31

ADC (payload dose) Half-life (hour) AUC (last) (hr*nM) ADC-1 (0.1 mg/kg) Conjugated payload 33 51432 Antibody 45 23280 ADC-9 (0.05 mg/kg) Conjugated payload 50 46262 Antibody 62 13313 ADC-8 (0.055 mg/kg) Conjugated payload 60 37315 Antibody 65 12540

Example 51.

Evaluation of antitumor activity in mice.

Balb/C mice bearing GCC-expressing CT26 murine colon carcinoma tumors were generated as described in Example 50.

When the mean tumor volume (MTV) reached approximately 75 ^mm3 , animals were randomized into several treatment groups (n=8/group) and given a single intravenous injection of various doses of ADC. Tumors continued to be measured twice weekly. The effects of ADC treatment on tumor growth were assessed by measuring tumor volumes and comparing them with vehicle-treated tumors.

The endpoint for the efficacy studies was reached when tumor size reached 10% of the animal's body weight or 2 cm in any direction. Mice were also closely observed for signs of clinical deterioration after injection. If for any reason mice showed any signs of disease, including respiratory distress, hunched posture, decreased activity, hind limb paralysis, tachypnea as evidence of pleural effusion, weight loss approaching 20% or 15% plus other signs, or if their ability to perform normal activities (feeding, mobility) was impaired, mice were sacrificed.

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Graphical representation of the observed antitumor activity for ADC-1, ADC-5, ADC-8 and ADC-9 is shown in Figs. 7, 8, 9 and 10, respectively.

Lipid conjugates.

Abbreviations.

AA LCMS method using ammonium acetate (LCMS AA) Ace acetate ACN acetonitrile atm atmosphere aq water BBN borabicyclo(3.3.1)nonane Bn benzyl Sunday /77/9C/77-butOXYCARBONYL (Bpin) ₂ bis(pinacolato)diborone

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tBu tert-butyl Bz benzoyl C Celsius CAD Charged Aerosol Detector cGAMP Cyclic guanosine monophosphate-adenosine monophosphate DBAD Ditert-butyl azodicarboxylate DBU 1,8-Diazabicyclo[5.4.0]undec-7-enedichloroacetic acid DCA dichloroacetic acid DCE dichloroethane DCM dichloromethane DEAD diethyl azodicarboxylate DIAD diisopropyl azodicarboxylate DIPEA N, A-diisopropylethylamine DMAP 4-dimethylaminopyridine DMFA N,N-dimethylformamide DMSO dimethyl sulfoxide DMTr 4,4'-dimethoxytrityl DOTMA 1,2-di-O-octadecenyl-3-trimethylammonium propane DPPC 1,2-dipalmitoyl-8n-glycero-3-phosphocholine DSPC 1,2-distearoyl-8p-glycero-3-phosphocholine Et ethyl EtOH ethanol EtOAc ethyl acetate MK LCMS using formic acid 4 watch Ind. connection intermediate product vzhh high performance liquid chromatography MCBP high resolution mass spectrometry IC _5O inhibitory concentration 50% IPA isopropyl alcohol IPC diisopinocampheil LCMS liquid chromatography, mass spectrometry. zhh/ms/ms liquid chromatography/tandem mass spectrometry. LDA lithium diisopropylamide LHMDS lithium bis(trimethylsilyl)amide

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m/z mass to charge MHz mega hertz Me methyl MeOH methanol min minutes ml milliliters MMR methylmorpholine MS mass spectrum nBu n-butane NMP 1-methyl-2-pyrrolidinone NMR nuclear magnetic resonance RE petroleum ether Ph phenyl psi pounds per square inch RUG pyridine kt room temperature SFC supercritical fluid chromatography TZR 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6- TDA-1 trioxide from/?is[2-(2-methoxyethoxy)ethyl]amine TBAF tetrabutylammonium fluoride TBS otreothbutyldimethylsilyl TBTU O-(benzotriazol-1-yl)-EG, N, N\N-tetramethyl! tetrafluoroborate TEA triethylamine TFA Trifluoroacetic acid TIDPSi 1,1,3,3-tetraisopropyldisiloxane TIPS triisopropylsilyl THF tetrahydrofuran SPLCH ultra performance liquid chromatography Xantphos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

Analytical methods NMR conditions.

NMR spectra were recorded on a Bruker Avance III 400 spectrometer operating at 400 MHz 1H unless otherwise stated. ³¹ P NMR and ¹⁹ F NMR spectra were recorded on the same spectrometer at 162 MHz and 376 MHz, respectively. All heteronuclear experiments were performed with 1H separation unless otherwise stated.

LCMS conditions.

LCMS spectra were recorded on a Hewlett-Packard HP1100 or Agilent 1100 Series LC system coupled to a Micromass mass spectrometer using reversed-phase C18 columns. Different gradients and run times were chosen to best characterize the compounds. Mobile phases were based on ACN/water or MeOH/water gradients and contained either 0.1% formic acid (methods designated as FA) or 10 mM ammonium acetate (methods designated as AA). One example of a solvent gradient that was used was from 100% mobile phase A (mobile phase A=99% water+1% ACN+0.1% formic acid) to 100% mobile phase B (mobile phase B=95% ACN+5% water+0.1% formic acid) at a flow rate of 1 mL/min over 16.5 min.

In some cases, LCMS spectra were acquired on an Agilent 1290 Infinity UPLC system coupled to an Agilent 6130 mass spectrometer, a Waters Acquity UPLC system coupled to a Waters Acquity SQ mass spectrometer, or an Agilent 1100 Series HPLC system coupled to a Waters Micro

- 118 048725 mass ZQ. mass spectrometer using reversed phase C18 columns. Different gradients and analysis times were chosen to best characterize the compounds. Mobile phases were based on ACN/water or MeOH/water gradients and contained either 0.1% formic acid (methods designated as MC) or 10 mM ammonium acetate (methods designated as AA). One example of a solvent gradient that was used was from 95% mobile phase A (mobile phase A = 99% water + 1% ACN + 0.1% formic acid) to 100% mobile phase B (mobile phase B = 95% ACN + 5% water + 0.1% formic acid) at a flow rate of 0.5 mL/min for 5 min.

Preparative HPLC.

Preparative HPLC was performed using a Phenomenex 5 micron C5 21.2 x 250 mm, with a gradient elution mode of solvent A (99% 10 mM ammonium acetate/1% acetonitrile)/solvent B (5% 10 mM ammonium acetate/95% acetonitrile) using a Gil son 322 pump, with a 155 UV/Vis detector, triggered by a fraction detector set to 200 to 400 nm. Mass-locked fractions were collected on an Agilent 1100 LC/MSD.

One of ordinary skill in the art will appreciate that modifications to the gradient, column length, and flow rate are possible and that some conditions may be more suitable for compound characterization than others, depending on the chemicals being analyzed.

Preparative SFC:

Preparative SFC is performed using ChiralPak 10, 20 or 30 mm x 250 mm columns (typically IA, IB, IC, ID, IE and IF), 10 or 20 mm x 250 mm Phenomenex Lux Cellulose-4 columns, or 2-ethylpyridine columns eluted with the appropriate percentage of supercritical carbon dioxide and alcohol containing 0.3% diethylamine or 0.3% TEA or 0.3% formic acid or without any acidic or basic additives. Isocratic conditions with flow rates in the range 10-100 mL/min and column temperatures of 40°C are typical. Preparative SFC is performed in a Jasco SFC pre-cleaner system with a UV/VIS trigger fraction set in the 200 to 400 nm range and a backpressure control set at 10 MPa.

One of ordinary skill in the art will appreciate that modifications to the gradient, column length, and flow rate are possible and that some conditions may be more suitable for compound characterization than others, depending on the chemicals being analyzed.

HPLC conditions for example B.

HPLC analysis was performed using a SHIMADZU LC-2010C with SHISELDO 3 micron C18 4.x75 mm, gradient elution mode of solvent A (80% water/10% 50 mM ammonium acetate/10% acetonitrile)/solvent B (10% 50 mM ammonium acetate/90% acetonitrile), flow rate of 1 ml/min, column temperature of 40°C and UV detection at 254 nm.

LC/MS/MS conditions for example B.

LC/MS/MS analysis was performed using a Shimadzu UltraFast liquid chromatograph and autosampler coupled to an AB SCIEX 4500 or AB SCLEX 5500 Triple Quad mass spectrometer or equivalent system.

For the analysis of Compound No. 14 and Compound 1-5c, 10 mM ammonium acetate in 99% water and 1% acetonitrile or 0.1% formic acid in water was prepared as the mobile phase A solvent, and 10 mM ammonium acetate in 5% water and 95% acetonitrile or 0.1% formic acid in 5% water and 95% acetonitrile was used as the mobile phase B solvent. Liquid chromatography was performed at 1.5 ml/min for 3 minutes in a gradient. The HPLC column was a 3.5-micron Waters Xselect C18 CSH column with an internal diameter of 2.1 mm and a length of 30 mm, and the column temperature was room temperature.

For the analysis of Compound CP-1, 10 mM ammonium acetate in 99% water and 1% acetonitrile was prepared as mobile phase A solvent, and 10 mM ammonium acetate in 5% water and 95% acetonitrile was prepared as mobile phase B solvent. Liquid chromatography was performed at 1.5 mL/min for 3.5 minutes in a gradient. The HPCL column was Osaka Soda Capcell PAK C1 UG120 5 micron, 2.0 mm i.d. x 35 mm length, and the column temperature was room temperature.

Analyst Software 1.7 was used for MS/MS peak area analysis and sample concentration calculation.

UPLC-CAD Terms.

UPLC-CAD analysis was performed using a Thermo Scientific™ Dionex™ Corona™ Veo™ RS (fast separation) Charged Aerosol Detector and a Waters ACQUITY UPLC system with an ACE Excel C4 2 micron 2.1 x 100 mm or equivalent column eluting with a gradient method using solvent A (10 mM ammonium acetate in water, 2.6 mL ammonium hydroxide in 4 L)/solvent B (10 mM ammonium acetate in methanol, 2.6 mL ammonium hydroxide 95% in 4 L) using a flow rate of 0.8 mL/min and a column temperature of 35°C. UPLC-CAD analysis may result in a variation of up to ±20%.

Example A1.

- 119 048725 tert-Butyl[4-({9-[(^^^ Ж10^12а^14^15^15а^^)-15-fluoro-7-(5-fluoro-4-oxo-3,4dihydro-7H-pyrrolo [2,3 -d]nupuMuguH-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfaniloctahydro12H-5,8-methanofuro[3,2-1] [1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl} amino)-4-oxobutyl]methylcarbamate salt TEA, intermediate-1

Compound #14 (300 mg, 0.40 mmol) was dissolved in dry pyridine and evaporated to dryness (5 ml x 3 times). The residue was dissolved in pyridine (6.41 ml) under argon and treated with chlorotrimethylsilane (0.30 ml, 2.38 mmol) cooled in an ice-water bath. The reaction mixture was allowed to warm to room temperature and stirred for 2 h to give TMS-protected compound #14. Separately (after ~30 min of the above reaction with stirring at room temperature), 4-methylmorpholine (0.28 mL, 2.50 mmol) was added to an ice-water bath-cooled solution of 4-((tert-butoxycarbonyl)(methyl)amino)butanoic acid (520.0 mg, 2.27 mmol) in tetrahydrofuran (10.7 mL), followed by the slow addition of isobutyl chloroformate (0.29 mL, 2.27 mmol) in THF. The cloudy suspension was allowed to warm to room temperature and stirred for 1 h, then slowly added to the above TMS-protected intermediate via syringe. The reaction mixture was stirred at room temperature overnight. MeOH (10 mL) was added to the above reaction mixture, and the reaction mixture was evaporated. The residue was redissolved in MeOH (3.8 mL). Triethylamine trihydrofluoride (0.26 mL, 1.59 mmol) was added to the reaction mixture cooled with an ice-water bath and allowed to warm to room temperature and stir for 30 min. The reaction mixture was absorbed onto diatomaceous earth (Celite®) (1 g) and purified by reverse phase flash column chromatography (0-50% ACN in aqueous triethylammonium acetate (10 mM)) to afford intermediate-1 as the TEA salt (214.0 mg, 48.5%). LCMS (AA): m/z=910.2 (M+H). ^1H NMR (D2O) δ 8.55 (s, 1H), 8.20 (s, 1H), 7.87 (s, 1H), 7.14 (d, J=4.0 Hz, 1H), 6.42 (d, J=16.0 Hz, 1H), 6.29 (d, J=8.0 Hz, 1H), 5.5d (dd, J=4.0, 52.0 Hz, 1H), 5.04-4.93 (m, 1H), 4.91-4.86 (m, 1H), 4.66 (d, J=4.0 Hz, 1H), 4.46 (d, J=8.0 Hz, 1H), 4.334.25 (m, 3H), 4.14 (dd, J=4.0, 12.0 Hz, 1H), 3.94 (dd, J=4.0, 8.0 Hz, 1H), 3.25 (br., m, 2H), 3.05 (k, J=8.0 Hz, 15H), 2.73 (s, 3H), 2.59-2.54 (m, 1H), 1.90-1.82 (m, 1H), 1.20 (s, 9H), 1.13 (t, J=8.0 Hz, 22H). ³¹ P NMR (D2O) δ 55.46 (s, 1P), 52.06 (s, 1P). ¹⁹ F NMR (D2O) δ -164.19 (s, br., 1F), -200.75 to -200.99 (m, 1F).

Example A2.

[(2R)-3-[Hydroxy-[2-[[5-[[(1S)-1-[[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxo-ethyl]carbamoyl]-2-methyl-propyl]amino]-5-oxo-pentanoyl]amino]ethoxy]phosphoryl]oxy-2-octadecanoyloxypropyl]octadecanoate, intermediate-2

(2S)-2-Amino-N-[(1S)-2-[4-(hydroxymethyl)anilino]-1-methyl-2-oxoethyl]-3-methylbutanamide (112.1 mg, 0.38 mmol) was suspended in toluene in a vial and evaporated to dryness (1 mL x 3). The residue was dissolved in DMF (1.5 mL) and treated with DIPEA (88.9 μL, 0.51 mmol). The DMF solution was then added to sodium [(2R)-2,3-di(octadecanoyloxy)propyl]-2-[[5-(2,5-dioxopyrrolidin-1-yl)oxy-5-oxopentanoyl]amino]ethyl phosphate (250.0 mg, 0.25 mmol) in DCM (15 mL). The reaction mixture was stirred at room temperature for 4 h. Most of the solvent (DCM) was evaporated and the residual solution (DMF) was poured into ice-cold aqueous HCl (0.1 N, 5 mL). The solid was collected

- 120 048725 by filtration and washed with water (3 mL x 3) and DCM (3 mL x 3) and dried in vacuo to give intermediate-2 (247.0 mg, 85.2%). LCMS (AA): m/z=1137.8 (M+H). 1H NMR (MeOD/CDCl ₃ ) δ 7.55 (hidden inside the peak of CDCl ₃ , 2H), 7.29 (d, J = 8.0 Hz, 1H), 5.25-5.22 (m, 1H), 4.53-4.49 (hidden inside the peak of water residue MeOD, 3H), 4.40 (dd, J = 4.0, 12.0 Hz, 1H), 4.19-4.00 (m, 6 H), 3.49-3.39 (m, 2H), 2.35-2.30 (m, 6H), 2.22 (t, J = 8.0 Hz, 2H), 2.17-2.09 (m, 1H), 1.97-1.89 (m, 2H), 1.65-1.58 (m, 4H), 1.45 (d, J= 8.0 Hz, 3H), 1.35-1.28 (broad, m, 56H), 0.99-0.97 (m, 6H), 0.89-0.86 (6H). ^31P NMR (MeOD/ _CDCl3 ) δ 0.77 (s, 1P).

Example A3.

[(2R)-3-[hydroxy-[2-[[5-[[(1S)-2-methyl-1-[[(1S)-1-methyl-2-[4-[(4-nitrophenoxy)carbonyloxymethyl]anilino]-2-oxo-ethyl]carbamoyl]propyl]amino]-5-oxopentanoyl]amino]ethoxy]phosphoryl]oxy-2-octadecanoyloxy-propyl]octadecanoate, intermediate-3

Intermediate 2 (247.0 mg, 0.22 mmol), THF (4.0 mL), and DIPEA (83.3 μL, 0.48 mmol) were added to the vial. The solution was cooled in an ice water bath under N2, and then bis(4-nitrophenyl)carbonate (97%, 74.9 mg, 0.24 mmol) was added in one portion. The pale yellow solution was stirred at 50 °C for 1 h. Additional bis(4-nitrophenyl)carbonate (97%, 145.3 mg, 0.48 mmol) was added and stirred at 50 °C for 1 h and then at room temperature overnight. The reaction mixture was poured into ice-cold aqueous HCl (0.05 N, 30 mL). The solid was collected by filtration and washed with water (10 mL x 3) and EtOAc (8 mL x 6) to give solid intermediate-3 (282.8 mg, 79.5%). LCMS (FA): m/z=1302.8 (M+H). 1H NMR (MeOD/CDCl ₃ ) δ 8.28 (d, J=8.0 Hz, 1H), 7.65 (d, J=8.0 Hz, 1H), 7.42-7.38 (m, 4H), 5.25-5.22 (m, 3H), 4.53-4.49 (hidden within the water peak of the MeOD residue, 1H), 4.38 (dd, J=4.0, 12.0 Hz, 1H), 4.19-4.02 (m, 6 H), 3.61-3.387 (m, 2H), 2.35-2.29 (m, 6H), 2.22-2.20 (m, 2H), 2.17-2.08 (m, 1H), 1.98-1.90 (m, 2H), 1.64-1.56 (m, 4H), 1.46 (d, J=8.0 Hz, 3H), 1.35-1.28 (br, m, 56H), 0.99-0.97 (m, 6H), 0.89-0.85 (6H). ^31P NMR (MeOD/ _CDCl3 ) δ 0.74 (s, 1P).

Example A4.

(2S,5S,19R)-2-({4-[({[4-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo3,4-dihydro-7H-pyrrolo[2,3^]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6yl}amino)-4-oxobutyl](methyl)carbamoyl}oxy)methyl]phenyl} carbamoyl)-16-hydroxy-5-isopropyl-16oxido-4,7,11-trioxo-19-(stearoyloxy)-15,17-dioxa-3,6,12-triaza-16 lambda~5-phosphaicosan-20-yl octadecanoate, CP-1

Intermediate-1 (15.0 mg, 0.013 mmol) was dissolved in 1,4-dioxane (0.29 mL) and treated with 4 M HCl in 1,4-dioxane (0.15 mL, 0.6 mmol) at room temperature for 1 h. The volatiles were removed and dried under vacuum for 1 h to afford 4-A as a crude product, which was used directly in the next step. A separate vial was charged with intermediate-3 (17.6 mg, 0.013 mmol), DMAP (3.3 mg, 0.027 mmol), TEA (0.015 mL, 0.11 mmol), and DCM

- 121 048725 (0.35 mL). To this mixture was slowly added a solution of 4-A in DMF (0.42 mL). The reaction mixture was stirred at room temperature for 15 min and evaporated to a small volume (~0.5 mL). The crude compound was purified by preparative HPLC (Phenomenex 5 micron C5 21.2 x 250 mm, eluting with solvent A [99% 10 mM ammonium acetate/1% acetonitrile] solvent B [5% 10 mM ammonium acetate/95% acetonitrile] 60% B to 100% B over 16 min at a flow rate of 20 mL/min) to afford CP-1 as an ammonium salt (11.0 mg, 40.3%). LCMS (AA): m/z=1972.8 (M+H). ¹ H NMR (MeOD) δ 8.55 (broad, s, 1H), 8.28 (broad, s, 1H), 7.75 (broad, s, 1H) 7.52-7.42 (m, 2H), 7.24-7.17 (m, 3H), 6.43-6.36 (m, 2H), 5.60 (dd, J = 4.0 and 52.0 Hz, 1H), 5.17-5.12 (m, 1H), 5.10-5.00 (m, 1H), 4.99 (s, 2H), 4.95- 4.89 (m, 1H), 4.78-4.76 (hidden inside the aqueous peak of MeOD residue, 1H), 4.48-4.28 (m, 6H), 4.23 (m, 1H), 4.13-4.09 (m, 2H), 3.96-3.82 (m, 5H), 3.42-3.33 (m, 4H), 2.91 (s, 3H), 2.73-2.64 (m, 2H), 2.31-2.16 (m, 8H), 2.11-2.03 (m, 1H), 1.99-1.92 (m, 2H), 1.89-1.82 (m, 2H), 1.57-1.47 (m, 4H), 1.49 (d, J=8.0 Hz, 3H), 1.27-1.28 (wide, 56H), 0.93-0.91 (m, 6H), 0.85-0.81 (m, 6H). ^31P NMR (MeOD) δ 57.00 (s, 1P), 52.26 (s, 1P), -0.20 (s, 1P). ^19F NMR (MeOD) δ -165.24 to -165.46 (s, broad, 1F), -202.98 to -203.24 (m, 1F). HRMS (C88H138F2N13O25P3S2) Expected: 1972.8622; Observed: 1972.8674

Example A5.

2-[(tert-butoxycarbonyl)(methyl)amino]ethyl {9-[( 1 R,3R,6R,8R,9R, 10R, 12R, 15R,17R, 1 8R)-9^mop17-(5-Fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3^]pyrimidin-7-yl)-18-hydroxy-3,12-dioxido-3,12disulfanyl-2,4,7,11,13,16-hexaoxa-3,12-diphosphate tricyclo[13.2.1.0~6,10~]octadec-8-yl]-9H-purin-6yl}carbamate, intermediate-4

O\f Phosgene in toluene 9\/

ΗΟ^ΛθΧ -55— ογο^Α ₀ Χ

Stage -1 _ .

Step 1: 2-[tert-butoxycarbonyl(methyl)amino]ethyl carbonyl chloride (5-A).

tert-Butyl (2-hydroxyethyl)(methyl)carbamate (1.40 g, 7.99 mmol) in DCM (100 mL) with pyridine (1.26 mL) was slowly added to a 15% solution of phosgene in toluene (16 mL, 22.4 mmol) at -78 °C. The reaction mixture was then warmed to room temperature and stirred for 1.5 h. The solvent was then evaporated to dryness to give crude 5-A (1.90 g, 100%).

Stage 2: 2-[(tert-butoxycarbonyl)(methyl)amino]ethyl{9-[(1R,3R,6R,8R,9R,10R,12R,15R,17R,18R)9-fluoro-17-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3^]pyrimidin- 7-yl)-18-hydroxy-3,12-dioxido3,12-disulfanyl-2,4,7,11,13,16-hexaoxa-3,12-diphosphate tricyclo[13.2.1.0~6,10~]octadec-8-yl]-9Hpurin-6-yl}carbamate, intermediate product-4.

Compound #14 (313 mg, 0.40 mmol) was dissolved in dry pyridine and evaporated to dryness (3 x 5 mL). The residue was dissolved in pyridine (6.70 mL) under argon and treated with chlorotrimethylsilane (0.378 mL, 2.97 mmol) cooled in an ice water bath. The reaction mixture was allowed to warm to room temperature and stirred for 20 min to give TMS-protected compound #14 in solution. 5-A (1.90 g, 7.52 mmol) was dissolved in DCM (12.5 mL) and then added to the above solution via syringe. The reaction mixture was stirred at room temperature under argon for 16 h. Then the reaction mixture was concentrated to dryness. To the residue were added MeOH (10 mL) and ammonium hydroxide (28-30% solution in water, 10 mL) and allowed to stir for 30 min. The reaction mixture was evaporated to dryness and the residue was dissolved in MeOH (15 mL). Triethylamine trihydrofluoride (0.12 mL, 0.75 mmol) was added and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was evaporated to dryness and the crude residue was adsorbed onto celite and purified by reverse phase flash column chromatography (0-50% ACN in aqueous triethylammonium acetate (10 mM)) to afford intermediate-4 as a TEA salt (181 mg, 39.3%). LCMS (AA): m/z=912.3 (M+H). 1H NMR (400 MHz, DMSO-do) δ 12.06(br., s, 1H), 10.56 (br., s, 1H), 9.16 (br., 2H), 8.62 (s, 1H), 8.52 (s, 1H), 7.89(d, J=4.0 Hz, 1H), 7.63 (s, 1H), 6.35 (dd, J=4.0 and 12.0 Hz, 1H), 6.26 (d, J=8.0 Hz, 1H), 5.80 (d, J=52.0 Hz, 1H), 5.19-5.14 (m, 1H), 4.95-4.93 (m, 1H), 4.79-4.78 (m, 1H), 4.56 (br., s, 1H), 4.33 (br., s, 1H), 4.21-4.12 (m, 5H), 4.00-3.90 (m, 1H), 3.72-3.69 (m, 1H), 3.45-3.43 (m, 2H), 3.05 (br., 12H), 2.86-2.83 (m, 3H), 1.34 (br., 9H), 1.15-1.11 (m, 18H). ³¹ P NMR (DMSO-06) δ 54.04 (s, 1P), 47.46 (s, 1P). ¹⁹ F NMR (DMSO-06) δ -165.06 (s, br, 1F), -207.23 to -207.54 (m, 1F)

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Example A6.

(2S,5S,19R)-2-{[4-({[{2-[({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-154TOp-7-(54TOp-4-OKCO3,4- dihydro-7H-pyrrolo[2,3^]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfaniloctahydro-12H-5 ,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6yl}carbamoyl)oxy]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}-16-hydroxy-5-isopropyl16-oxido-4,7,11-trioxo-19-(stearoyloxy)-15,17-dioxa-3,6,12-triaza-16-lambda~5-phosphaicosan-20yl octadecanoate, CP-2

The title compound was prepared using a procedure similar to that shown in Example A4, substituting intermediate-4 (77.0 mg, 0.069 mmol) for intermediate-1 to give CP-2 as an ammonium salt (42.0 mg, 30.0%). LCMS (AA): m/z=1974.6 (M+H). 1H NMR (MeOD) δ 8.52 (broad, d, J=12.0 Hz, 1H), 8.33 (broad, d, J=8.0 Hz, 1H), 7.74 (s, 1H), 7.50 (broad, d, J=8.0 Hz 1H), 7.40 (broad, d, J=8.0 Hz, 1H), 7.28 (broad, 1H), 7.24 (broad, d, J= 8.0 Hz, 1H), 7.14 (broad, d, J= 8.0 Hz, 1H), 6.43-6.34 (m, 2H), 5.56 (d, J=52.0 Hz, 1H), 5.17-5.06 (m, 2H), 5.01-4.93 (m, 3H), 4.78-4.76 (water peak hidden inside MeOD residue, 1H), 4.47-4.29 (m, 8H), 4.21 (m, 1H), 4.13-4.09 (dd, J=8.0 and 12.0 Hz, 2H), 3.95-3.82 (m, 5H), 3.63-3.58 (m, 2H), 3.33 (b, d, J=8.0 Hz, 2H), 2.99 (s, 3H), 2.30-2.22 (m, 6H), 2.17 (t, d, J=8.0 Hz, 2H), 2.10-2.04 (m, 1H), 1.88-1.81 (m, 2H), 1.56-1.48 (m, 4H), 1.49 (w, d, J=8.0 Hz, 3H), 1.27-1.21 (w, m, 56H), 0.93-0.90 (m, 6H), 0.84-0.81 (m, 6H). ³¹ P NMR (DMSO^b) δ 53.64 (s, 1P), 46.87 (s, 1P), -0.45 (s, 1P). ¹⁹ NMR (MeOD) δ -165.49 to -165.66 (m, 1F), -203.23 to -203.70 (m, 1F). MSVR (C87H136F2N13O26P3S2) Expected: 1974.8415; Observed: 1974.8455

Example A7.

[4-(Hydroxymethyl)phenyl]4-(aminomethyl)benzoate, intermediate-5

Step 1: [4-(hydroxymethyl)phenyl]4-[(tert-butoxycarbonylamino)methyl]benzoate.

A 100 mL round-bottomed flask was charged with 4-(Boc-aminomethyl)benzoic acid (503.0 mg, 1.98 mmol), 4-hydroxybenzyl alcohol (97%, 304.3 mg, 2.38 mmol), and suspended in DCM (15 mL) and treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (97%, 634.3 mg, 3.96 mmol). The reaction mixture was kept at room temperature for 2 h and washed with ice-cold HCl (0.1 N, 10 mL), then water (10 mL), and sat. NaHCO ₃ (10 mL), dried and purified by silica gel chromatography (0-100% EtOAc/hexanes) to give 7-A (385.0 mg, 54.4%). LCMS (AA): m/z=356.00 (MH). ¹ H NMR (DMSO-6) δ 8.09 (d, J=8.0 Hz, 1H), 7.53 (t, J=4.0 Hz, 1H), 7.47-7.39 (m, 4H), 7.22 (d, J=8.0 Hz, 1H), 5.25 (t, J=8.0 Hz, 1H), 4.53 (d, J=8.0 Hz, 2H), 4.24 (br, d, J=4.0 Hz, 2H), 1.41 (s, 9H).

Step 2: [4-(hydroxymethyl)phenyl]4-(aminomethyl)benzoate, intermediate 5.

[4-(hydroxymethyl)phenyl]4-[(tert-butoxycarbonylamino)methyl]benzoate 7-A (100.0 mg, 0.28 mmol) was dissolved in DCM (2 mL) and treated with TFA (0.8 mL, 10 mmol) at room temperature for 30 min. The volatiles were removed by rotary evaporation and the residue was redissolved in MeOH (2 mL) and heated at 37 °C for 90 min. After complete removal of volatiles, the residue was co-evaporated with MeOH (2 mL x 2) and DCM (4 mL x 2) to afford intermediate 5 as the TFA salt (110.0 mg, quantitative yield). LCMS (AA): m/z = 258.20 (M+H). 1H NMR (DMSO-6) δ 8.37 (br, 3H), 8.19 (d, J=8.0 Hz, 2H), 7.68 (d, J=8.0 Hz, 2H), 7.42 (d, J=8.0 Hz, 2H), 7.24 (d, J=8.0 Hz, 2H), 4.54 (s, 2H), 4.20 (br, s, 2H).

Example A8.

(2R)-3-((Hydroxy(2-(5-((4-((4-(hydroxymethyl)phenoxy)carbonyl)benzyl)amino)-5oxopentanamido)ethoxy)phosphoryl)oxy)propane-1,2-diyl distearate, intermediate-6

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DIPEA/DCM/DMF

OH

connection-6

Trifluoroacetic acid salt [4-(hydroxymethyl)phenyl]4-(aminomethyl)benzoate (64.1 mg, 0.17 mmol) was dissolved in DMF (0.8 mL). The solution was added to another solution of sodium [(2R)-2,3di(octadecanoyloxy)propyl]2-[[5-(2,5-dioxopyrrolidin-1-yl)oxy-5-oxopentanoyl]amino]ethyl phosphate (113.0 mg, 0.115 mmol) in DCM (3.2 mL). Then DIPEA (0.04 mL, 0.23 mmol) was added to the above mixture and stirred for 3.5 h at room temperature. Most of the solvent (DCM) was evaporated and the residual solution (DMF) was poured into ice-cold HCl (0.1 N, 5 mL). The solid was collected by filtration and washed with ice-cold HCl (0.1 N, 4 mL x 6) and water (3 mL x 6) and dried under vacuum to give the intermediate® (106.0 mg, 83.6%). LCMS (AA): weak signal. ^1H NMR (MeOD/CDCl3) δ 8.03 (d, J=8.0 Hz, 2H), 7.31 (d, J=8.0 Hz, 4H), 7.06 (d, J=8.0 Hz, 2H), 5.13-5.10 (m, 1H), 4.55 (s, 2H), 4.37 (s, 2H), 4.07-4.05 (m, 1H), 4.07-3.93 (m, 5H), 3.37-3.34 (br., 2H), 2.24-2.13 (m, 8H), 1.86-1.82 (m, 2H), 1.49 (br., 4H), 1.14 (br., 56H), 0.78 (m, 6H). ³¹ P NMR (MeOD/CDCl3) δ 3.06 (s, 1P).

Example A9.

(2R)-3-((Hydroxy(2-(5-((4-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenoxy)carbonyl)benzyl)amino)-5-oxopentanamido)ethoxy)phosphoryl)oxy)propane-1,2-diyl distearate, intermediate-7

Intermediate 6 (106 mg, 0.096 mmol) was dissolved in THF (2 mL) and treated with DIPEA (0.037 mL, 0.21 mmol). To this solution was added bis(4-nitrophenyl)carbonate (64.4 mg, 0.21 mmol). The reaction mixture was maintained at room temperature for 1 h, and then additional bis(4-nitrophenyl)carbonate (64.4 mg, 0.21 mmol) and DIPEA (0.037 mL, 0.21 mmol) were added. The reaction mixture was heated at 50 °C for another 1 h. The reaction mixture was added to ice-cold HCl (0.05 N, 100 mL). The precipitate was collected by filtration and washed with water (2 mL x 4). The solid was suspended in EtOAc (3 mL) and sonicated for 5 sec and filtered. The solid was washed with EtOAc (2 mL x 3) to give solid intermediate-7 (121.9 mg, 57.4%). LCMS (AA): weak signal. ¹ H NMR (MeOD/CDCl ₃ ) δ 8.26 (d, J=8.0 Hz, 2H), 8.12 (d, J=8.0 Hz, 2H), 7.50 (d, J=8.0 Hz, 4H), 7.40 (d, J=8.0 Hz, 2H), 7.37 (d, J=8.0 Hz, 2H), 7.23 (d, J=8.0 Hz, 2H), 5.29 (s, 2H), 5.21-5.18 (m, 1H), 4.48 (s, 2H), 4.31 (dd, J=4.0 and 12.0 Hz, SH), 4.14-4.01 (m, 5H), 3.45 (br, t, J=4.0 Hz, 2H), 2.32-2.23 (m, 8H), 1.96-1.92 (m, 2H), 1.57 (br., 4H), 1.22 (br., 56H), 0.84 (m, 6H).

Example A10.

4-({[{2-[({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-3-oxo-3,4-dihydro7H-pyrrolo[2,3®]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}carbamoyl)oxy]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl 4-[(15R)-12-hydroxy-12-oxido-3,7,18-trioxo-15(stearoyloxy)-11,13,17-trioxa-2,8-diaza-12lambda~5-phosphapentatriacont-1-yl] benzoate, CP-3

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The title compound was prepared using a procedure similar to that shown in Example A4, substituting intermediate 7 (8.0 mg, 0.0072 mmol) for intermediate-3 and intermediate-4 (9.1 mg, 0.0072 mmol) for intermediate-1 to give CP-3 as the ammonium salt (4.2 mg, 29.0%). LCMS (AA): m/z=1939.1 (M+H). ^1H NMR (MeOD) δ 8.65-8.59 (m, 1H), 8.42 (broad, d, J= 16.0 Hz, 1H), 8.14 (d, J= 8.0 Hz, 2H), 7.85 (s, 1H), 7.74 (s, 1H), 7.51 (d, J=8.0 Hz, 2H), 7.48-7.39 (m, 3H), 7.20 (w, d, J= 8.0 Hz, 1H), 7.11 w, d, J=8.0 Hz, 1H), 6.50 (dd, J=l,2 and 8.0 Hz, 1H), 6.42 (w, d, J=8.0 Hz, 1H), 5.80-5.61 (m, 1H), 5.27-5.10 (m, 4H), 5.08 -5.0 (m, 1H), 4.82 (m, 1H), 4.55-4.32 (m, 9H), 4.30 (wide, 1H), 4.04-3.98 (m, 3H), 3.96-3.91 (m, 2H), 3.83-3.65 (m, 2H), 3.44 (t, J=4.0 Hz, 2H), 3.10 (broad, d, J=12.0 Hz, 3H), 2.38-2.28 (m, 8H), 2.02-1.94 (m, 2H), 1.65-1.57 (m, 4H), 1.30 (wide, 56H), 0.93-0.90 (m, 6H). ³¹ P NMR (MeOD) δ 57.08 (s, 1P), 52.51 (s, 1P), 0.36 (s, 1P). ¹⁹ F NMR (MeOD) δ -165.58 to -165.67 (m, 1F), -203.66 to -203.99 (m, 1F) MCBP (C87H128F2N11O26P3S2) Expected: 1938.7727; Observed: 1938.7713

Example A11.

4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3 A]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10disulfaniloctahydro-12H-5,8-methanofuro[3,2-CH1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14yl]-9H-purin-6-yl}carbamoyl)benzyl]methylcarbamate, intermediate-8

Step 1: 2-[[[4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methoxycarbonylmethyl-amino]methyl]benzoic acid, 11-A.

The title compound was prepared according to the procedure described in Example A4, step 2, starting from 2-[(methylamino)methyl]benzoic acid HCl (150 mg, 0.72 mmol) instead of deBoc intermediate-1. Purification by silica gel chromatography (0-25% MeOH/DCM) gave 11-A (306 mg, 67%). LCMS.

(AA): m/z=583.4 (M-H).

Step 2: [4-[[(2S)-2-[[(2S)-2-(tert-butoxycarbonylamino)-3-methylbutanoyl]amino]propanoyl]amino]phenyl]methyl U-[(2-chlorocarbonylphenyl)methyl]-U-methylcarbamate, intermediate-8

To a solution of 11-A (295 mg, 0.50 mmol) in THF (1.5 mL) cooled to 0 °C was added oxalyl chloride (2.0 M solution in DCM, 0.25 mL, 0.50 mmol) followed by 3 drops of DMF. The reaction mixture was allowed to stir at 0 °C for 45 min. The mixture was then evaporated to dryness to give crude intermediate-8, which was used directly in the next step (304 mg, 100%).

Example A12

4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3 A]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin14-yl]-9H-purin-6-yl}carbamoyl)benzyl]methylcarbamate, intermediate-9

- 125 048725

Compound #14 (107 mg, 0.117 mmol) was added to a round bottom flask (50 mL) and co-evaporated with dry pyridine (2 mL x 3 times). Then the residue was redissolved in dry pyridine (4.26 mL, 52.7 mmol), treated with chlorotrimethylsilane (0.121 mL, 0.934 mmol) and stirred at room temperature for 30 min. To the above solution was added intermediate-8 (423 mg, 0.70 mmol) in pyridine (1.89 mL, 23.4 mmol) and stirred overnight at room temperature. The reaction mixture was quenched with 10 mL MeOH and stirred for 30 min, all solvent was removed and ammonium hydroxide (28-30% solution in water, 10 mL) was added and allowed to stir for 30 min. The reaction mixture was evaporated to give a crude dark solid. The solid was redissolved in MeOH (1.42 mL) and treated with triethylamine trihydrofluoride (0.12 mL, 0.75 mmol) at 0 °C and allowed to warm to room temperature over 30 min. The mixture was evaporated and purified by reverse phase flash column chromatography (0-50% ACN in aqueous ammonium bicarbonate (5 mM)) and further purified by preparative HPLC (Phenomenex 5 micron C5 21.2 x 250 mm, eluting with solvent A (99% 10 mM ammonium acetate/1% acetonitrile)/solvent B (5% 10 mM ammonium acetate/95% acetonitrile) from 60% B to 100% B over 16 min at 20 mL/min) to afford intermediate-9 as the ammonium salt. LCMS (AA): m/z=1277.8 (M+H). 1H NMR (MeOD) δ 8.68 (s, 1H), 8.44 (s, 1H), 7.82 (br., d, J=8.0 Hz, 1H), 7.77 (s, 1H), 7.52-7.39 (m, 4H), 7.39 (s, 1H), 7.23-7.26 (m, 3H), 6.50-6.43 (m, 2H), 5.74 (dd, J=4.0 and 52.0 Hz, 1H), 5.255.15 (m, 1H), 5.07-5.04 (m, 3H), 4.93-4.89 (m, 2H), 4.56-4.49 (m, 2H), 4.46-4.39(m, 3H), 4.30 (br., 1H), 4.03 (dd, J=4.0 and 12.0 Hz, 1H), 3.95 (d, J=4.0 Hz, 1H), 2.95 (br., 3H), 2.13-2.11 (m, 1H), 1.48 (d, J=8.0 Hz, 1H), 1.45 (s, 9H), 1.00 (d, J=4.0 Hz, 3H), 0.94 (J=4.0 Hz, 3H), ³¹ P NMR (MeOD) δ 56.91 (s, 1P), 52.36 (s, 1P). ^19F NMR (MeOD) δ -165.65 (s, 1F), -203.16 to -203.35 (m, 1F).

Example A13.

(2S,5S,19S)-2-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5-fluoro-4-oxo3,4-dihydro-7H-pyrrolo[2,3D]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6yl}carbamoyl) benzyl](methyl)carbamoyl}oxy)methyl]phenyl}carbamoyl)-16-hydroxy-5-isopropyl-16oxido-4,7,11-trioxo-19-(stearoyloxy)-15,17-dioxa-3,6,12-triaza-16lambda~5-phosphaicosan-20-yl octadecanoate, (CP-4)

Step 1: 4-{[(2S)-2-{[(2S)-2-amino-3-methylbutanoyl]amino}propanoyl]amino}benzyl [2-({9[(2R,5R,7R,8R, 10R,12aR, 14R, 15R, 15aR, 16R)-15-fluoro-7-(5-fluoro-4-oxo-3,4-dihydro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-l][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9H-purin-6-yl}carbamoyl)benzyl]methyl

- 126 048725 carbamate (13-A).

Intermediate-9 (56 mg, 0.045 mmol) was added to a round-bottomed flask and cooled to 0 °C. Then, a solution of TFA (0.24 mL, 3.2 mmol) and DCM (0.58 mL) was added via syringe and the reaction mixture was stirred at 0 °C for 30 min. Then, the reaction mixture was evaporated to dryness and placed under vacuum for 2 h to give 13-A as a TFA salt (56 mg, 100%). LCMS (AA): m/z=1177.0 (M+H).

Step 2: (2S,5S,19S)-2-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15R,15aR,16R)-15-fluoro-7-(5fluoro-4-oxo-3,4-dihydro-7H-pyrrolo [2,3 (^]pyrimidin-7-yl)-16-hydroxy-2,10-dioxido-2,10-disulfanyloctahydro-12H-5,8-methanofuro[3,2-1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-14-yl]-9Hpurin-6-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}carbamoyl)-16-hydroxy-5-isopropyl-16-oxido-4,7,11-trioxo-19-(stearoyloxy)-15,17-dioxa-3,6,12-triaza-16lambda~5—phosphaicosan-20-yl octadecanoate, CP-4.

To a solution of 13-A (56 mg, 0.037 mmol) and 13-B (40.0 mg, 0.041 mmol) in THF (1.4 mL) and DMF (0.7 mL) was added dropwise N,N-diisopropylethylamine (65 μL, 0.37 mmol). The reaction mixture was allowed to stir at room temperature for 16 h. The reaction mixture was then evaporated to dryness and the crude residue was purified by preparative HPLC (Phenomenex 5 micron C5 21.2 x 250 mm, eluting with solvent A (99% 10 mM ammonium acetate/1% acetonitrile)/solvent B (5% 10 mM ammonium acetate/95% acetonitrile) from 60% B to 100% B over 16 min at a flow rate of 20 ml/min) to afford CP-4 as the ammonium salt (32.8 mg, 42%). LCMS (AA): ¹ / ₂ m/z = 1011.1 (M+H). ¹ H NMR (MeOD) δ 8.78 (s, 1H), 8.74 (s, 1H), 7.90-7.88 (m, 1H) 7.78 (s, 1H), 7.64-7.46 (m, 4H), 7.36 (s, 1H), 7.33-7.13 (m, 3H), 6.54-6.48 (m, 2H), 5.79 (dd, J= 4.0 and 52.0 Hz, 1H), 5.26-5.13 (m, 2H), 5.05-4.99 (m, 3H), 4.84 (m, 2H hidden in water peak), 4.78-4.77 (m, 1H), 4.59-4.36 (m, 6H), 4.30-4.29 (m, 1H), 4.22-4.17 (m, 2H), 4.03-3.92 (m, 5H), 3.43 (t, J=4.0 Hz, 2H), 2.96 (s, 3H), 2.89-2.24 (m, 8H), 2.20-2.12 (m, 1H), 1.98-1.90 (m, 2H), 1.67-1.56 (m, br, 4H), 1.48 (d, J=8.0 Hz, 3H), 1.30 (br, 56H), 1.02-0.99 (m, 6H), 0.93-0.90 (m, 6H), ³¹ P NMR (MeOD) δ 57.47 (s, 1P), 52.87 (s, 1P), 0.12 (s, 1P) ¹⁹ F NMR (MeOD) δ -165.91 to -166.08 (m, 1F), -203.70 to -203.94 (m, 1F). MCBP (C92H138F2N13O25P3S2) Expected: 2020.8622; Observed: 2020.8601.

Example A14.

4-{[(2S)-2-({(2S)-2-[(tert-butoxycarbonyl)amino]-3-methylbutanoyl}amino)propanoyl]amino}benzyl [2-({9-[(2R,5R,7R,8R, 10R, 12aR, 14R, 15aS, 16R)-16-hydroxy-2,10-dioxido-14-(pyrimidin-4-yloxy)2,10-disulfanyldecahydro-5,8-methanocyclopenta[ 1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl]methyl carbamate, intermediate-10

1.TMSCI, pyridine

IT NH ₂

3. NEt ₃ -3HF. MeOH

ΝΗ O

Intermediate connection-10

The title compound was prepared according to the procedure described in Example A12 substituting 2-amino-9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10-dioxido-14(pyrimidin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphacyclotetradecin-7-yl]-1,9-dihydro-6H-purin-6-one as the TEA salt (Compound 1-5c) (for synthesis, see PCT/IBO18/O58846) for Compound I-5c. The crude product was purified by reverse phase flash column chromatography (0-50% ACN in aqueous ammonium bicarbonate (5 mM)) to afford intermediate-10 as an ammonium salt. For salt conversion, this material was then dissolved in MeOH (2 mL) and triethylamine (5 mL) was added. The mixture was shaken for 1 min. The solvents were removed.

- 127 048725 was added and lyophilized overnight to give intermediate-10 as the TEA salt (147 mg, 48%). LCMS (AA): m/z=1216.3 (M+H). ^1H NMR (400 MHz, MeOD) δ 8.70 (s, 1H), 8.43 (br s, 1H), 8.40 (d, J=5.9 Hz, 1H), 7.70 (d, J=7.5 Hz, 1H), 7.56-7.46 (m, 3H), 7.42-7.38 (m, 1H), 7.32-7.19 (m, 3H), 6.84 (d, J=5.5 Hz, 1H), 6.15 (d, J=7.3 Hz, 1H), 5.62-5.56 (m, 1H), 5.54-5.48 (m, 1H), 5.08-5.02 (m, 1H), 5.04 (s, 2H), 4.88-4.83 (m, 1H), 4.81-4.76 (m, 2H), 4.53-4.47 (m, 1H), 4.37-4.22 (m, 3H), 4.08-4.03 (m, 1H), 3.92-3.89 (m, 1H), 3.82-3.74 (m, 1H), 2.88 (s, 3H), 2.58-2.30 (m, 4H), 2.10-2.03 (m, 1H), 1.54-1.47 (m, 1H), 1.46-1.44 (m, 3H), 1.44 (s, 9H), 0.98 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8 Hz, 3H). ³¹ P NMR (162 MHz, MeOD) δ 55.01 (s, 1P), 53.03 (s, 1P).

Example A15.

(2S,5S,19R)-16-hydroxy-2-({4-[({[2-({9-[(2R,5R,7R,8R,10R,12aR,14R,15aS,16R)-16-hydroxy-2,10dioxido-14-(pyri midin-4-yloxy)-2,10-disulfanyldecahydro-5,8-methanocyclopenta[1][1,3,6,9,11,2,10]pentaoxadiphosphatetetradecine-7- yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}carbamoyl)benzyl](methyl)carbamoyl}oxy)methyl]phenyl}carbamoyl)-5-isopropyl- 16-oxido-4,7,11-trioxo-19(stearoyloxy)-15,17-dioxa-3,6,12-triaza-16-lambda~5—phosphaicosan-20-yloctadecanoate, SR-5

The title compound was prepared using a procedure similar to that shown for Example A13, starting from intermediate-10 instead of intermediate-9 to give CP-5 (96.0 mg, 48.8% for 2 steps). LCMS (AA): m/z=1959.8 (M+H). 1H NMR (MeOD) δ 8.58 (s, 1H), 8.33 (s, 1H), 8.28 (d, J=8.0 Hz, 1H), 7.58 (d, J=8.0 Hz, 1H), 7.45-7.36 (m, 3H), 7.29 (t, J=8.0 Hz, 1H), 7.14 (br, 3H), 6.72 (d, J=8.0 Hz, 1H), 6.04 (d, J=8.0 Hz, 1H), 5.48 (br, 1H), 5.38-5.33 (m, 1H), 5.13-5.08 (m, 1H), 4.94-4.89 (m, 3H), 4.71 (deepened inside the aqueous peak of the residue MeOD, 1H), 4.67 (m, 2H), 4.42 -4.38 (m, 1H), 4.34-4.30 (m, 1H), 4.25-4.04 (m, 5H), 3.95-3.91 (m, 1H), 3.87 (t, J=8.0 Hz, 2H), 3.82-3.78 (m, 2H), 3.693.62 (m, 1H), 3.29 (t, J=4.0 Hz, 2H), 2.77 (s, 3H), 2.47-2.17 (m, 10H), 2.13 (t, J=8.0 Hz, 2H), 2.05-1.97 (m, 1H), 1.82-1.77 (m, 2H), 1.53-1.43 (b, m, 4H), 1.41-1.38 (m, 1H), 1.34 (d, J=8.0 Hz, 3H), 1.17 (b, 56H), 0.89-0.86 (m, 6H), 0.80-0.76 (m, 6H). ^31P NMR (DMSO-06) δ 55.29 (s, 1P), 53.05 (s, 1P), 0.29 (s, 1P). MCBP (C _{91H 141N 12} O2 _{5P 3} S2) Expected: 1959.8858, Observed: 1959.8886.

It should be understood that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more, but not all, exemplary embodiments of the present invention as contemplated by the inventor(s), and thus are not intended to limit the present invention or the appended claims in any way.

The present invention has been described above using functional building blocks illustrating the implementation of certain functions and their interrelations. The boundaries of these functional building blocks have been defined arbitrarily in this document for the convenience of description. Alternative boundaries may be defined, provided that the specified functions and their interrelations are properly performed.

The foregoing description of specific embodiments will so fully disclose the general nature of the invention that others, using knowledge within the skill of the art, can easily modify and/or adapt such specific embodiments to various applications without undue experimentation, without departing from the general concept of the invention. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments based on the idea and guidance provided herein. It should be understood that the phraseology or terminology in this document is intended to be descriptive and not limiting, so that the terminology or phraseology of this description should be interpreted by a skilled person in light of the ideas and guidance.

The scope and extent of this invention should not be limited by any of the above

- 128 -

Claims (12)

exemplary embodiments, but should be determined only in accordance with the following claims and their equivalents. CLAUSE OF THE INVENTION 1. Compound of formula (I): or a pharmaceutically acceptable salt thereof, where a is an integer from 1 to 20; b is an integer between 1 and 20; m is 0, 1, 2, 3, or 4; n is 0 or 1; D-NH- selected from a) a fragment of formula (II): or a pharmaceutically acceptable salt thereof, wherein each of R ¹⁰ and R ⁴⁰ independently represents hydrogen, fluorine, -OH or -OCH2CF ₃ ; ring A ¹⁰ is a 6-membered monocyclic heteroaryl ring containing 1, 2 or 3 nitrogen atoms; and ring B ¹⁰ is each of Z ¹⁰ , Z ²⁰ , Z ³⁰ and Z ⁴⁰ independently represents N or CR ²⁰⁰ ; R ²¹⁰ represents hydrogen, C1-C ₆ alkyl, halo(C1-C ₆ )alkyl or C ₃ -C ₆ cycloalkyl; R ²³⁰ is hydrogen or -NH ₂ ; and each of R ²⁰⁰ and R ²²⁰ independently is hydrogen, halogen, -OH, -NH2, -CN, C1-C ₆ alkyl, halo(C1-C ₆ )alkyl or C ₃ -C ₆ cycloalkyl; provided that either ring A ¹⁰ or ring B ¹⁰ is attached to the carbonyl group of formula (I) through a group -NH-; or b) a fragment of formula (III): or a pharmaceutically acceptable salt thereof, wherein each of R ¹⁰⁵ and R ²⁰⁵ independently represents a hydroxy group or a halogen atom; - 129 048725 one of B ¹⁰⁰ and B ²⁰⁰ is where R ¹⁸ represents hydrogen or C1-C6 alkyl; and R ¹⁹ represents a halogen atom; and the other one is: each R ¹ is independently selected from C1-C ₄ -alkyl, O-C ₁ -C ₄ -alkyl and halogen; R ² is selected from C1-C ₄ -alkyl and -(CH ₂ CH2O) s -CH _3; where s is an integer from 1 to 10; R ³ and R ³ are each independently selected from hydrogen and C1-C ₃ -alkyl; L represents where Y represents the point of attachment to the nitrogen atom; represents the point of attachment to Ab; t is an integer between 1 and 10; W is missing or selected from where r represents the point of attachment to the carbonyl group; and / represents the point of attachment to Z; Z is absent or is a peptide of 2-5 amino acids; U is selected from * represents the point of attachment to Q; where represents the point of attachment to Z; - 130 048725 p is an integer from 1 to 6; q is an integer between 1 and 20; X is O or -CH2-; and each r is independently 0 or 1; U' is absent; and Q is selected from Ab is an antibody, antibody fragment, or antigen-binding fragment. 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein a is an integer from 1 to 4; b is an integer between 1 and 10; and m is 0. 3. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein m is 0; n is 0; and R ³ and R ³ each represent hydrogen. 4. A compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein Z is a peptide capable of enzymatic cleavage. 5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein Z is cleaved by cathepsin. 6. A compound according to any one of claims 4 or 5 or a pharmaceutically acceptable salt thereof, wherein Z is a two amino acid peptide selected from Val-Cit, Cit-Val, Val-Ala, Ala-Val, Phe-Lys and LysPhe. 7. The compound according to claim 1, where t is 1; and Z is absent or is a peptide of 2 amino acids. 8. A compound according to any one of claims 1 to 7, wherein R ² is -CH ₃ or -(CH ₂ CH ₂ O) _s -CH ₃ and s is an integer from 1 to 10. 9. The compound according to claim 1, wherein D-NH is: or a pharmaceutically acceptable salt thereof, where a represents the point of attachment to the carbonyl group of formula (I). 10. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers. 11. A method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable amount of a compound according to any one of claims 1 to 9. 12. A method for stimulating an immune response in a subject in need thereof, comprising administering to the subject a pharmaceutically acceptable amount of a compound according to any one of claims 1 to 9 or a composition according to claim 10. - 131 -