CN114621243B

CN114621243B - Sulfonamide derivative and application thereof

Info

Publication number: CN114621243B
Application number: CN202111529297.7A
Authority: CN
Inventors: 李云飞; 莫明广; 杨煜文; 张芳
Original assignee: Shanghai Tuojie Biomedical Technology Co ltd
Current assignee: Shanghai Tuojie Biomedical Technology Co ltd
Priority date: 2020-12-14
Filing date: 2021-12-14
Publication date: 2024-03-01
Anticipated expiration: 2041-12-14
Also published as: CN114621243A

Abstract

The present disclosure relates to sulfonamide derivatives and uses thereof. Specifically, the present disclosure provides a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein ring A and B, X ¹ ～X ⁴ 、R ¹ ～R ⁸ As defined herein.

Description

Sulfonamide derivative and application thereof

Technical Field

The present disclosure belongs to the field of medicine, and relates to sulfonamide derivatives and uses thereof.

Background

Semicarbazide-sensitive amine oxidase (SSAO) is a class of amine oxidases containing a dopamine quinone group, and belongs to a family of semicarbazide-sensitive amine oxidases, also known as vascular adhesion protein-1, vap-1 (vascular adhesion protein 1). Numerous studies have demonstrated that SSAO and its metabolites are closely related to inflammatory-related diseases such as atherosclerosis, diabetes and its complications, obesity, stroke, chronic kidney disease, retinopathy, chronic Obstructive Pulmonary Disease (COPD), autoimmune diseases, multiple sclerosis, rheumatoid arthritis, alzheimer's disease, and the like.

Several known MAO inhibitors such as Morfejine have been synthesized, studies have shown that Morfejine inhibits experimental autoimmune encephalomyelitis (US 20060025438),

WO2009066152 describes 3-substituted 3-haloallylamine SSAO/VAP-1 inhibitors and claims them as treatment of inflammatory diseases,

WO2013163675 on the basis of this develops a new class of 3-haloallylamine SSAO/VAP-1 inhibitors, exemplified by the following compounds:

in addition, other 3-haloallylamine SSAO/VAP-1 inhibitors have also been reported successively, such as CN109251166, CN109810041, CN110938059, CN108778278, CN109988093, CN109988106, CN109988109, WO2018027892, WO2018149226, WO2020233583, WO2007120528, WO2018196677, WO2020063854, WO2020089025, WO2020089026, WO2020125776, etc., however SSAO/VAP-1 inhibitors have not been marketed yet, while the compounds of the present disclosure are not disclosed in any literature, and such compounds exhibit specific VAP-1 inhibitory effects.

Disclosure of Invention

The disclosure provides a compound of formula I or a pharmaceutically acceptable salt thereof

Wherein R is ¹ And R is ² Independently selected from hydrogen, deuterium, chlorine, and fluorine;

R ³ and R is ⁴ Independently selected from hydrogen, deuterium, C _1-6 Alkyl, said alkyl optionally being substituted with one or more R ^A1 Each independently substituted, R ^A1 Selected from halogen (e.g., fluorine, chlorine), deuterium, hydroxy, nitro, cyano or amino;

R ⁵ And R is ⁶ Independently selected from hydrogen, deuterium, C _1-6 Alkyl, said alkyl optionally being substituted with one or more R ^A2 Each independently substituted, R ^A2 Selected from halogen (e.g., fluorine, chlorine), deuterium, hydroxy, nitro, cyano or amino;

R ⁷ and R is ⁸ Independently selected from hydrogen, deuterium, C _1-6 Alkyl, said alkyl optionally being substituted with one or more R ^A3 Each independently substituted, R ^A3 Selected from halogen (e.g., fluorine, chlorine), deuterium, hydroxy, nitro, cyano or amino;

ring A is selected from 4To 6 membered heterocycles, said ring A optionally being substituted with one or more R ^A4 Substituted; r is R ^A4 Each independently selected from halogen (e.g., fluorine, chlorine), deuterium, oxo (=o), hydroxy, nitro, cyano, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -SR ^1a 、-S(O)R ^1a 、-S(O) ₂ R ^1a 、-NR ^1a (R ^1b )、-(CH ₂ ) _o COR ^1a 、-(CH ₂ ) _o NHCOR ^1a 、-(CH ₂ ) _o CONR ^1a (R ^1b )、-N(CH ₂ ) _o CONR ^1a (R ^1b )、-N(CH ₂ ) _o COR ^1a ；

R ^1a Or R is ^1b Each independently selected from hydrogen, deuterium, hydroxy, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl or heterocycloalkyl optionally being substituted by one or more groups selected from halogen (e.g. fluoro, chloro), deuterium, hydroxy, oxo, nitro, cyano or amino;

ring B is selected from a 3 to 6 membered carbocyclic ring, a 3 to 6 membered heterocyclic ring or a 5 to 6 membered heteroaromatic ring, said ring B optionally being substituted with one or more R ^A5 Substituted; r is R ^A5 Selected from halogen (e.g. fluorine, chlorine), deuterium, oxo (=o), hydroxy, nitro, cyano, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, aryl, heteroaryl, -SR ^2a 、-S(O)R ^2a 、-S(O) ₂ R ^2a 、-NR ^2a (R ^2b )、-(CH ₂ ) _p COR ^2a 、-(CH ₂ ) _p NHCOR ^2a 、-(CH ₂ ) _p CONR ^2a (R ^2b )、-(CH ₂ ) _p OCONR ^2a (R ^2b )、-N(CH ₂ ) _p CONR ^2a (R ^2b )、-N(CH ₂ ) _p COR ^2a ；

R ^2a Or R is ^2b Each independently selected from hydrogen, deuterium, hydroxy, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl or heterocycloalkyl optionally being substituted by one or more groups selected from halogen (e.g. fluoro, chloro), deuterium, hydroxy, oxo, nitro, cyano or amino;

and ring A and ring B are connected in a parallel or spiro form;

X ¹ 、X ² 、X ³ and X ⁴ Each independently selected from-CH-or-N-, and not simultaneously-N-;

o, p are each selected from integers between 0 and 3.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ¹ Selected from hydrogen, R ² Selected from fluorine.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ¹ Selected from fluorine, R ² Selected from hydrogen.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ¹ Selected from hydrogen, R ² Selected from chlorine.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ¹ Selected from chlorine, R ² Selected from hydrogen.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ⁷ And R is ⁸ Independently selected from C _1-6 Alkyl, said alkyl optionally being substituted with one or more R ^A3 Substituted, R ^A3 Each independently selected from halogen, deuterium, hydroxy, nitro, cyano or amino.

In other embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ⁷ And R is ⁸ Independently selected from methyl, ethyl or propyl, further optionally substituted with one or more R ^A3 Substituted, R ^A3 Each independently selected from halogen, deuterium, hydroxy, nitro, cyano or amino.

In some embodiments, a compound of formula I orR in pharmaceutically acceptable salts thereof ⁵ And R is ⁶ Independently selected from hydrogen or deuterium.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ⁵ 、R ⁶ 、R ⁷ And R is ⁸ Selected from hydrogen.

In another aspect, some embodiments provide compounds of formula I or a pharmaceutically acceptable salt thereof wherein ring A is selected fromFurther, ring A is optionally substituted with 1 to 3R ^A4 Substituted, R ^A4 The foregoing definition.

Other embodiments provide compounds of formula I or a pharmaceutically acceptable salt thereofSelected from the group consisting ofWherein n, m are each independently selected from integers from 0 to 4 and the sum of n and m is not more than 4, Y is selected from-NH-, O-, and-CH ₂ -or-S-, further->Is 1 to 3R ^A4 Or R is ^A5 Substituted, R ^A4 、R ^A5 As defined above. In some embodiments, wherein Y is selected from the group consisting of-NH-, O, -CH ₂ -; the sum of n and m is 1, 2, 3 or 4.

In some embodiments, ring B of the compound of formula I or a pharmaceutically acceptable salt thereof is selected from 3 to 6 carbocycles, preferably Further, ring B is optionally substituted with 1 to 3R ^A5 Substituted, R ^A5 As defined above.

In other embodiments, the compound of formula I or a pharmaceutically acceptable salt thereofSelected from the group consisting of Further->Is 1 to 3R ^A4 Or R is ^A5 Substituted, R ^A4 、R ^A5 As defined above.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A4 Each independently selected from halogen, deuterium, oxo (=o), hydroxy, cyano or amino.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A4 Each independently selected from C _1-6 Alkyl or C _1-6 An alkoxy group.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A4 Each independently of the otherSelected from C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, aryl or heteroaryl.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A4 Each independently selected from-NR ^1a (R ^1b )、-(CH ₂ ) _o COR ^1a 、-(CH ₂ ) _o NHCOR ^1a 、-(CH ₂ ) _o CONR ^1a (R ^1b )、-N(CH ₂ ) _o CONR ^1a (R ^1b )、-N(CH ₂ ) _o COR ^1a ，o、R ^1a Or R is ^1b As defined above. In some embodiments, o is 0, 1 or 2 in a compound of formula I or a pharmaceutically acceptable salt thereof.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^1a Or R is ^1b Selected from hydrogen, deuterium, hydroxy, C _1-6 Alkyl or C _1-6 Alkoxy, said alkyl or alkoxy being optionally substituted with 1 to 3 substituents selected from halogen. In other embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^1a Or R is ^1b Selected from hydrogen, deuterium, hydroxy, methyl, ethyl, methoxy or ethoxy.

On the other hand, in some embodiments R in the compound of formula I or a pharmaceutically acceptable salt thereof ^A5 Each independently selected from halogen, deuterium, oxo (=o), hydroxy, cyano or amino.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A5 Each independently selected from oxo (=O), hydroxy, halogen, C _1-6 Alkyl or C _1-6 An alkoxy group.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A5 Each independently selected from C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, aryl or heteroaryl.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A5 Each independently selected from oxo (=O), hydroxy, C _1-6 Alkyl, -NR ^2a (R ^2b )、-(CH ₂ ) _p COR ^2a 、-(CH ₂ ) _p NHCOR ^2a 、-(CH ₂ ) _p CONR ^2a (R ^2b )、-(CH ₂ ) _p CONR ^2a (R ^2b )、-N(CH ₂ ) _p CONR ^2a (R ^2b )、-N(CH ₂ ) _p COR ^2a ，p、R ^2a Or R is ^2b As defined above.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A5 Each independently selected from- (CH) ₂ ) _p COR ^2a Or- (CH) ₂ ) _p CONR ^2a (R ^2b ). In some embodiments, p is 0, 1 or 2 in a compound of formula I or a pharmaceutically acceptable salt thereof.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A5 Each independently selected from oxo (=O), hydroxy, C _1-6 Alkyl, -NR ^2a (R ^2b )、-COR ^2a 、-CH ₂ NHCOR ^2a 、-CONR ^2a (R ^2b )、-CH ₂ CONR ^2a (R ^2b )、-N(CH ₂ )CONR ^2a (R ^2b )、-NCH ₂ COR ^2a 、-NHCOR ^2a ，R ^2a Or R is ^2b As defined above.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A5 Each independently selected from oxo (=O), hydroxy, C _1-6 Alkyl, -COR ^2a 、-CONR ^2a (R ^2b )、-NHCOR ^2a ，R ^2a Or R is ^2b As defined above.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^A5 Selected from fluorine, oxo, C _1-3 Alkyl, -COOH, -COC _1-3 Alkoxy, -CONHC _1-3 An alkoxy group.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^2a Or R is ^2b Selected from hydrogen, deuterium, hydroxy, C _1-6 Alkyl or C _1-6 Alkoxy, said alkyl, alkoxy optionally substituted with 1 to 3 substituents selected from halogen. In other embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^2a Or R is ^2b Selected from hydrogen, deuterium, hydroxy, methyl, ethyl, methoxy or ethoxy.

On the other hand, in some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^1a Or R is ^1b Selected from C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, said cycloalkyl or heterocycloalkyl being optionally substituted with 1 to 3 substituents selected from halogen, deuterium, hydroxy, oxo, nitro, cyano or amino.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ^2a Or R is ^2b Selected from C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, said cycloalkyl or heterocycloalkyl being optionally substituted with 1 to 3 substituents selected from halogen, deuterium, hydroxy, oxo, nitro, cyano or amino.

In another aspect, the compound of formula I, or a pharmaceutically acceptable salt thereof, is ring B and ring A, and ring B is selected from 5 membered heteroaromatic rings. In some embodiments, ring B of the compound of formula I or a pharmaceutically acceptable salt thereof is selected from Further, ring B is optionally substituted with 1 to 3R ^A5 Substituted, R ^A5 As defined above. Other embodiments provide compounds of formula I or a pharmaceutically acceptable salt thereofSelected from-> It is further substituted with 1 to 3R ^A4 Or R is ^A5 Substituted.

In some embodiments, ring B of the compound of formula I or a pharmaceutically acceptable salt thereof is selected fromFurther, ring B is optionally substituted with 1 to 3R ^A5 Substituted, R ^A5 As defined above. In other embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof +.>Selected from->It is further substituted with 1 to 3R ^A4 Or R is ^A5 Substituted, R ^A5 As defined above.

In other embodiments, ring B of the compound of formula I or a pharmaceutically acceptable salt thereof is selected fromFurther, ring B is optionally substituted with 1 to 3R ^A5 Substituted, R ^A5 As defined above.

In other embodiments, the compound of formula I or a pharmaceutically acceptable salt thereofSelected from the group consisting ofFurther->Is 1 to 3R ^A4 Or R is ^A5 Substituted, R ^A4 、R ^A5 As defined above. In other embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof +.>Is 1 to 3R ^A5 Substituted, R ^A5 Each independently oxo (=o) or C _1-6 Alkyl (e.g., methyl, ethyl, or propyl).

On the other hand, in some embodiments, X in a compound of formula I or a pharmaceutically acceptable salt thereof ² Selected from-N-, X ¹ 、X ³ 、X ⁴ Selected from-CH-; or X ³ Selected from-N-, X ¹ 、X ² 、X ⁴ Selected from-CH-,

in some embodiments, X in a compound of formula I or a pharmaceutically acceptable salt thereof ¹ 、X ² 、X ³ 、X ⁴ Selected from-CH-,

in some embodiments, X in a compound of formula I or a pharmaceutically acceptable salt thereof ¹ 、X ⁴ Selected from-N-, X ² 、X ³ Selected from-CH-,

in some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereofSelected from the group consisting of Further->Is 1 to 2R ^A4 Or R is ^A5 Substituted.

In some embodiments, a compound of formula I or a pharmaceutically acceptable salt thereofSelected from the group consisting ofFurther->Is 1 to 2R ^A4 Or R is ^A5 Substituted.

Other embodiments provide compounds of formula I or a pharmaceutically acceptable salt thereof as

In some embodiments, the compound of formula I or a pharmaceutically acceptable salt thereof is

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ³ Selected from hydrogen or C _1-6 Alkyl (including but not limited to methyl, ethyl or propyl).

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ³ Selected from C _1-6 Alkyl (including but not limited to methyl, ethyl or propyl) optionally substituted with one or more R ^A1 Substituted, R ^A1 Selected from halogen, deuterium, hydroxy, nitro, cyano or amino.

Further, in some embodiments, R ^A5 Selected from- (CH) ₂ ) _p COR ^2a Wherein R is ^2a Selected from C _1-6 Alkyl optionally substituted with one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, cyano or amino, p=0, 1 or 2.

In some embodiments, R ^A5 Selected from- (CH) ₂ ) _p CONR ^2a (R ^2b ) Wherein R is ^1a Selected from hydrogen, R ^2a Selected from C _1-6 Alkyl groups optionally substituted with one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, cyanoSubstituted by a group or amino group, p=0, 1 or 2. Further, in some embodiments, R ^A4 Selected from- (CH) ₂ ) _p COR ^2a Wherein R is ^2a Selected from C _1-6 Alkyl optionally substituted with one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, cyano or amino, p=0, 1 or 2.

In some embodiments, R ^A4 Selected from- (CH) ₂ ) _p CONR ^2a (R ^2b ) Wherein R is ^1a Selected from hydrogen, R ^2a Selected from C _1-6 Alkyl optionally substituted with one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, cyano or amino, p=0, 1 or 2.

In some embodiments, R in a compound of formula I or a pharmaceutically acceptable salt thereof ³ Selected from hydrogen, methyl or ethyl.

Typical compounds of formula I or pharmaceutically acceptable salts thereof include, but are not limited to:

wherein->Including the E or Z configuration.

In some embodiments, a compound of formula I, or a pharmaceutically acceptable salt thereof, comprises:

also provided in the present disclosure is a pharmaceutical composition comprising at least one therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, as described above, and a pharmaceutically acceptable excipient.

In some embodiments, the pharmaceutical composition is in a unit dose of 0.001mg to 1000mg.

In certain embodiments, the pharmaceutical composition comprises 0.01 to 99.99% of the foregoing compound, or a pharmaceutically acceptable salt thereof, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition comprises 0.1-99.9% of the foregoing compound or pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical composition comprises 0.5% to 99.5% of the foregoing compound or a pharmaceutically acceptable salt thereof. In certain embodiments, the pharmaceutical compositions comprise 1% to 99% of the foregoing compounds, or pharmaceutically acceptable salts thereof. In certain embodiments, the pharmaceutical composition comprises 2% to 98% of the foregoing compound or a pharmaceutically acceptable salt thereof.

In certain embodiments, the pharmaceutical composition contains 0.01% to 99.99% of a pharmaceutically acceptable excipient, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1% to 99.9% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 0.5% to 99.5% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 1% to 99% of a pharmaceutically acceptable excipient. In certain embodiments, the pharmaceutical composition contains 2% to 98% of a pharmaceutically acceptable excipient.

The present disclosure also provides a method of preventing and/or treating a patient suffering from a SSAO or SSAO/VAP-1 related disorder by administering to the patient a therapeutically effective amount of a compound of formula I as set forth above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as set forth above.

In some embodiments, the disorder associated with SSAO or SSAO/VAP-1 is selected from inflammation, diabetes, an ocular disease, fibrosis, a neuroinflammatory disease, or cancer.

The present disclosure also provides a method of preventing and/or treating a patient suffering from inflammation, diabetes, an ocular disease, fibrosis, a neuroinflammatory disease, or cancer comprising administering to the patient a therapeutically effective amount of a compound of formula I as set forth above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as set forth above.

The present disclosure also provides the use of a compound of formula I as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, in the manufacture of a medicament for the prevention and/or treatment of a condition associated with SSAO or SSAO/VAP-1. In some embodiments, the PDE-related disorder is preferably inflammation, diabetes, an ocular disease, fibrosis, a neuroinflammatory disease, or cancer.

The present disclosure also provides the use of a compound of formula I as described above, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described above, in the manufacture of a medicament for the prevention and/or treatment of inflammation, diabetes, an ocular disease, fibrosis, a neuroinflammatory disease, or cancer.

Pharmaceutically acceptable salts of the compounds described in this disclosure may be selected from inorganic or organic salts.

The compounds of the present disclosure may exist in particular geometric or stereoisomeric forms. The present disclosure contemplates all such compounds, including cis and trans isomers, (-) -and (+) -pairs of enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the disclosure. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present disclosure. The asymmetric carbon atom containing compounds of the present disclosure may be isolated in optically active pure or racemic forms. Optically pure forms can be resolved from the racemic mixture or synthesized by using chiral starting materials or chiral reagents.

Optically active (R) -and (S) -isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it may be prepared by asymmetric synthesis or derivatization with chiral auxiliary wherein the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereomeric resolution is carried out by conventional methods well known in the art, and then the pure enantiomer is recovered. Furthermore, separation of enantiomers and diastereomers is typically accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amine).

In the chemical structure of the compounds of the present disclosure, the bondIndicating the unspecified configuration, i.e.the bond +.>Can be +.>Or at the same time contain-> Two configurations. Key- >Indicating unspecified configurations including cis (E) or trans (Z) configurations. Or +.>Refers to a double bond, and the structure bonded by the bond can be a "cis isomer" or a "trans isomer" or a "cis isomer and a" trans isomerA mixture of isomers of formula (la) formed in any ratio ", for example formula E represents E-1, formula E-2 or a mixture of both formed in any ratio:

the compounds and intermediates of the present disclosure may also exist in different tautomeric forms, and all such forms are included within the scope of the disclosure. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that can interconvert via a low energy barrier. For example, proton tautomers (also known as proton transfer tautomers) include tautomers via proton transfer, such as keto-enol and imine-enamine, lactam-lactam isomerization. Examples of lactam-lactam balances are between a and B as shown below.

All compounds in the present disclosure may be drawn as form a or form B. All tautomeric forms are within the scope of the disclosure. The naming of the compounds does not exclude any tautomers.

The present disclosure also includes some isotopically-labeled compounds of the present disclosure which are identical to those recited herein, but for the replacement of one or more atoms by an atom having an atomic weight or mass number different from the atomic weight or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as, respectively ² H、 ³ H、 ¹¹ C、 ¹³ C、 ¹⁴ C、 ¹³ N、 ¹⁵ N、 ¹⁵ O、 ¹⁷ O、 ¹⁸ O、 ³¹ P、 ³² P、 ³⁵ S、 ¹⁸ F、 ¹²³ I、 ¹²⁵ I and ³⁶ cl, and the like.

Unless otherwise indicated, when a position is specifically designated as deuterium (D), that position is understood to be deuterium (i.e., at least 10% deuterium incorporation) having an abundance that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%). The natural abundance of a compound in an example can be at least 1000 times greater than the abundance of deuterium, at least 2000 times greater than the abundance of deuterium, at least 3000 times greater than the abundance of deuterium, at least 4000 times greater than the abundance of deuterium, at least 5000 times greater than the abundance of deuterium, at least 6000 times greater than the abundance of deuterium, or higher than the abundance of deuterium. The present disclosure also includes various deuterated forms of the compounds of formula (I). Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. Those skilled in the art are able to refer to the relevant literature for the synthesis of deuterated forms of the compounds of formula (I). Commercially available deuterated starting materials may be used in preparing the deuterated form of the compound of formula (I) or they may be synthesized using conventional techniques with deuterated reagents including, but not limited to, deuterated boranes, trideuteroborane tetrahydrofuran solutions, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

"optionally" or "optionally" is intended to mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example "C optionally substituted by halogen or cyano _1-6 Alkyl "means that halogen or cyano may be, but need not be, present, and this description includes the case where alkyl is substituted with halogen or cyano and the case where alkyl is not substituted with halogen and cyano.

Term interpretation:

"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically acceptable salt or prodrug thereof, and other chemical components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

"pharmaceutically acceptable excipients" include, but are not limited to, any auxiliary agent, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that has been approved by the U.S. food and drug administration for use in humans or livestock animals.

The term "effective amount" or "therapeutically effective amount" as used in this disclosure includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: such as the condition to be treated, the general health of the patient, the route of administration and the dosage and severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"alkyl" refers to saturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms. Alkyl groups containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, and various branched isomers thereof, and the like. The alkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any useful point of attachment, preferably one or more groups independently selected from halogen, deuterium, hydroxy, nitro, cyano or amino.

The term "cycloalkyl" or "carbocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups. Cycloalkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably one or more groups independently selected from halogen, deuterium, oxo (=o), hydroxy, nitro, cyano, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, aryl, heteroaryl, -SR ', -S (O) R', -S (O) ₂ R’、-NR’(R”)、-(CH ₂ ) _p COR’、-(CH ₂ ) _p NHCOR’、-(CH ₂ ) _p CONR’(R”)、-(CH ₂ ) _p OCONR’(R”)、-N(CH ₂ ) _p CONR’(R”)、-N(CH ₂ ) _p COR'. The cycloalkyl ring may be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, and the like. Cycloalkyl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halogen, deuterium, oxo (=o), hydroxy, nitro, cyano, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, aryl, heteroaryl, -SR ', -S (O) R', -S (O) ₂ R’、-NR’(R”)、-(CH ₂ ) _p COR’、-(CH ₂ ) _p NHCOR’、-(CH ₂ ) _p CONR’(R”)、-(CH ₂ ) _p OCONR’(R”)、-N(CH ₂ ) _p CONR’(R”)、-N(CH ₂ ) _p COR’。

The term "heterocycloalkyl" or "heterocycle" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 6 ring atoms, non-limiting examples of "heterocycloalkyl" include: etc.

The heterocycloalkyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halogen, deuterium, oxo (=o), hydroxy, nitro, cyano, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, aryl, heteroaryl, -SR ', -S (O) R', -S (O) ₂ R’、-NR’(R”)、-(CH ₂ ) _p COR’、-(CH ₂ ) _p NHCOR’、-(CH ₂ ) _p CONR’(R”)、-(CH ₂ ) _p OCONR’(R”)、-N(CH ₂ ) _p CONR’(R”)、-N(CH ₂ ) _p COR’。

The term "heteroaryl" refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl groups are preferably 5-or 6-membered. For example. Non-limiting examples of which include:etc.

Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halogen, deuterium, oxo (=o), hydroxy, nitro, cyano, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, aryl, heteroaryl, -SR ', -S (O) R', -S (O) ₂ R’、-NR’(R”)、-(CH ₂ ) _p COR’、-(CH ₂ ) _p NHCOR’、-(CH ₂ ) _p CONR’(R”)、-(CH ₂ ) _p OCONR’(R”)、-N(CH ₂ ) _p CONR’(R”)、-N(CH ₂ ) _p COR’。

In the present disclosure R 'or R' is selected from hydrogen, deuterium, hydroxy, amino, C _1-6 Alkyl, C _1-6 Alkoxy, C _3-6 Cycloalkyl, 3-to 6-membered heterocycloalkyl, said alkyl, alkoxy, cycloalkyl, heterocycloalkyl, aryl or heteroaryl being optionally substituted by one or more groups selected from halogen, deuterium, hydroxy, oxo, nitro, cyano or amino.

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, cyclohexyloxy. The alkoxy groups may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from halogen, deuterium, hydroxy, oxo, nitro, cyano or amino.

The term "hydroxy" refers to an-OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "cyano" refers to-CN.

The term "cyano" refers to-NH ₂ 。

The term "nitro" refers to-NO ₂ 。

The term "oxo" refers to an =o substituent.

"substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort.

Detailed Description

The present disclosure is further described below in connection with examples, which are not intended to limit the scope of the disclosure.

Experimental methods for which specific conditions are not noted in the examples in this disclosure are generally in accordance with conventional conditions, or in accordance with conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or/and Mass Spectrometry (MS). NMR shift (. Delta.) of 10 ^-6 Units of (ppm) are given. NMR was performed using Bruker AVANCE-400 nuclear magnetic resonance apparatus with deuterated dimethyl sulfoxide (DMSO-d) ₆ ) Deuterated chloroform (CDCl) ₃ ) Deuterated Methanol (Methanol-d) ₄ ) The internal standard is Tetramethylsilane (TMS).

HPLC was determined using an Agilent1100 high pressure liquid chromatograph, GAS15B DAD ultraviolet detector, water Vbridge C18 150 x 4.6mm 5um column.

The mass of the sample is measured by an Agilent6120 triple quadrupole mass spectrometer, a G1315D DAD detector, a Waters Xbridge C18.6 x 50mm, a 5um chromatographic column, and the sample is scanned in a positive/negative ion mode, and the mass scanning range is 80-1200.

The thin layer chromatography silica gel plate is a smoke table yellow sea HSGF254 silica gel plate, the Thin Layer Chromatography (TLC) adopts a silica gel plate with the specification of 0.2mm plus or minus 0.03mm, and the thin layer chromatography separation and purification product adopts a specification of 0.4mm-0.5mm.

Flash column purification systems used Combiflash Rf150 (teldyne ISCO) or isolaraone (Biotage).

The forward column chromatography generally uses the yellow sea silica gel of the smoke table with 200-300 meshes or 300-400 meshes as a carrier, or uses the Santai prefill of Changzhou to prefill the ultra-pure phase silica gel column (40-63 mu m,60g,24g,40g,120g or other specifications).

Known starting materials in the present disclosure may be synthesized using or following methods known in the art, or may be purchased from Shanghai taitant technology, ABCR GmbH & Co.KG, acros Organics, aldrich Chemical Company, shaoshan chemical technology (Accela ChemBio Inc), pichia medicine, and the like.

The examples are not particularly described, and the reactions can all be carried out under nitrogen atmosphere.

The nitrogen atmosphere is defined as the reaction flask being connected to a nitrogen balloon of about 1L volume.

The hydrogen atmosphere is defined as the reaction flask being connected to a balloon of hydrogen gas of about 1L volume.

The hydrogen is prepared by a QPH-1L type hydrogen generator of Shanghai full-pump scientific instrument company.

The nitrogen atmosphere or the hydrogenation atmosphere is usually vacuumized, filled with nitrogen or hydrogen, and repeatedly operated for 3 times.

The examples are not specifically described, and the solution refers to an aqueous solution.

The reaction temperature is room temperature and is 20-30 deg.c without specific explanation in the examples.

The reaction progress in the examples was monitored by Thin Layer Chromatography (TLC), a developing agent used in the reaction, a system of column chromatography eluent used for purifying the compound and a developing agent system of thin layer chromatography, and the volume ratio of the solvent was adjusted according to the polarity of the compound, and may be adjusted by adding a small amount of an alkaline or acidic reagent such as triethylamine and acetic acid.

Example 1

Synthesis of 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-oxa-7-azaspiro [3.5] nonane (1)

Step 1: synthesis of 7- [ 6-chloropyridin-3-yl) sulfonyl ] -2-oxa-7-azaspiro [3.5] nonane (1 c)

Compound 1b (563 mg,2.59 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (716mg, 7.07 mmol) and compound 6-chloropyridine-3-sulfonyl chloride (1 a) (0.50 g,2.36 mmol) were added and the reaction was allowed to react at room temperature until LC-MS monitored to completion. Filtration using a flash chromatography apparatusIsolation was performed to give compound 1c (0.10 g, yield 14%).

MS(ESI)m/z:303.3[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ8.76(d,J＝2.3Hz,1H),7.99(dd,J＝2.6,8.3Hz,1H),7.53(d,J＝8.0Hz,1H),4.37(s,4H),3.07-2.98(m,4H),2.03-1.97(m,4H).

Step 2: synthesis of 7- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-oxa-7-azaspiro [3.5] nonane (1 d)

Compound 1c (59 mg,0.20 mmol) was dissolved in N-methylpyrrolidone (1 mL) and triethylamine (30 mg,0.29 mmol) was added and cooled to 0deg.C with an ice-water bath. A solution of BB-1 (40 mg,0.20 mmol) in tetrahydrofuran (2 mL) and a solution of sodium tert-butoxide (28 mg,0.29 mmol) in dimethyl sulfoxide (0.5 mL) were added. The reaction was completed at room temperature until LC-MS was monitored, water (10 mL) was added, extracted with ethyl acetate (10 mL. Times.2), washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 1d (50 mg, yield 54%).

MS(ESI)m/z:472.2[M+H] ⁺ 。

Wherein BB-1 is prepared by taking commercial BB-1a as a raw material:

compound BB-1a (6.62 g,21.80 mmol) was dissolved in tetrahydrofuran (300 mL), compound BB-1b (10.0 g,21.60 mmol) was added, cooled to-60℃and NaHMDS (32.7 mL,32.7mmol, 1.0M) was then slowly added, reacted at-50-60℃for 1 hour, saturated aqueous ammonium chloride solution (500 mL) was added, extracted with ethyl acetate (500 mL. Times.2), saturated aqueous salt was washed with water, dried over anhydrous magnesium sulfate, and the mixture was purified by a flash chromatography apparatus BB-1c (1.50 g, yield 21%) was isolated.

MS(ESI):m/z 220.2[M+H] ⁺ 。 ¹ H NMR(400MHz,CDCl ₃ )δ6.78-6.16(m,1H),4.34-3.96(m,2H),3.85-3.44(m,2H),1.34(d,J＝2.1Hz,9H),0.83-0.80(m,9H),0.01(d,J＝5.5Hz,6H).

Compound BB-1c (1.50 g,0.46 mmol) was dissolved in tetrahydrofuran (30 mL), TBAF (7.0 mL,7.0mmol, 1.0M) was added, reacted at room temperature until the TLC detection was complete, saturated aqueous ammonium chloride solution (100 mL) was added, extracted with ethyl acetate (100 mL. Times.2), saturated brine, dried over anhydrous magnesium sulfate, and purified using a flash chromatography apparatusBB-1 (0.61 g, yield 63%) was isolated.

Step 3: synthesis of 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-oxa-7-azaspiro [3.5] nonane (1)

Compound 1d (50 mg,0.11 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature until the reaction was complete by LC-MS monitoring, and the reaction solution was concentrated to give a crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 1 (4 mg, yield 10%).

MS(ESI)m/z:372.2[M+H] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.57(d,J＝2.1Hz,1H),8.03(dd,J＝2.5,8.7Hz,1H),7.31-7.07(m,1H),7.02(d,J＝8.8Hz,1H),5.00(d,J＝3.7Hz,2H),4.36(s,4H),3.72(d,J＝1.8Hz,2H),3.11-2.91(m,4H),2.08-1.87(m,4H).

Example 2

Synthesis of 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-methyl-7-azaspiro [3.5] nonan-2-ol (2)

Step 1: synthesis of 7- [ 6-chloropyridin-3-yl) sulfonyl ] -2-methyl-7-azaspiro [3.5] nonan-2-ol (2 b)

Compound 1a (1.17 g,4.72 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (1.90 g,18.9 mmol) and compound 6-chloropyridine-3-sulfonyl chloride (1 a) (1.00 g,4.72 mmol) were added and the reaction was allowed to complete at room temperature until LC-MS monitored. Filtration using a flash chromatography apparatus 2b (0.80 g, 51% yield) was isolated. />

MS(ESI)m/z:331.1[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ8.76(dd,J＝0.6,2.4Hz,1H),8.00(dd,J＝2.6,8.3Hz,1H),7.65-7.48(m,1H),3.07-2.93(m,4H),1.92-1.85(m,4H),1.84-1.79(m,2H),1.72-1.64(m,2H),1.37(s,3H).

Step 2: synthesis of 7- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-methyl-7-azaspiro [3.5] nonan-2-ol (2 c)

Compound 2b (322 mg,0.98 mmol) was dissolved in N-methylpyrrolidone (4 mL) and triethylamine (150 mg,1.46 mmol) was added and cooled to 0deg.C with an ice-water bath. A solution of BB-1 (200 mg,0.98 mmol) in tetrahydrofuran (8 mL) and a solution of sodium tert-butoxide (141 mg,1.46 mmol) in dimethyl sulfoxide (2 mL) were added. The reaction was completed at room temperature until LC-MS was monitored, water (10 mL) was added, extracted with ethyl acetate (10 mL x 2), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 2c (200 mg, yield 41%).

MS(ESI)m/z:500.2[M+H] ⁺ 。

Step 3: synthesis of 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-methyl-7-azaspiro [3.5] nonan-2-ol (2)

Compound 2c (200 mg,0.40 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, stirred at room temperature until the reaction was complete by LC-MS monitoring, and the reaction solution was concentrated to give a crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 2 (31 mg, yield 14%).

MS(ESI)m/z:400.2[M+H] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.58(d,J＝2.3Hz,1H),8.05(dd,J＝2.6,8.8Hz,1H),7.47-7.13(m,1H),7.05(d,J＝8.7Hz,1H),5.02(d,J＝3.5Hz,2H),3.83(d,J＝2.0Hz,2H),2.96(br d,J＝4.3Hz,4H),1.91-1.81(m,4H),1.80-1.73(m,2H),1.71-1.63(m,2H),1.30(s,3H)

Example 3

Synthesis of methyl 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -7-azaspiro [3.5] nonane-2-carboxylate (3)

Step 1: synthesis of 7- [ 6-chloropyridin-3-yl) sulfonyl ] -2-methyl-7-azaspiro [3.5] nonan-2-ol (3 b)

Compound 3a (1.68 g,5.66 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (1.53 g,15.1 mmol), DMAP (50 mg,0.38 mmol) and compound 6-chloropyridine-3-sulfonyl chloride (1 a) (1.00 g,4.72 mmol) were added and the reaction was allowed to complete at room temperature until LC-MS monitored. Using flash chromatography apparatus3b (0.83 g, 61% yield) was isolated.

MS(ESI)m/z:359.1[M+H] ⁺¹ H NMR(400MHz,CDCl ₃ )δ8.75(d,J＝2.5Hz,1H),7.98(dd,J＝2.5,8.3Hz,1H),7.51(d,J＝8.3Hz,1H),3.67(s,3H),3.09-2.95(m,5H),2.03-1.93(m,4H),1.72(td,J＝5.6,13.7Hz,4H).

Wherein 3a can be prepared from commercial 3a-1 by the following procedure:

3a-1 (2.00 g,7.43 mmol) was dissolved in acetone (20 mL) and potassium carbonate (3.08 g,22.3 mmol) and methyl iodide (2.31 mL,37.1 mmol) were added and the reaction was allowed to react at room temperature until LC-MS monitored to completion. Filtration using a flash chromatography apparatus3a-2 (1.40 g, 67% yield) was isolated as a colorless oil.

MS(ESI)m/z:284.2[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ3.71-3.67(m,3H),3.38-3.33(m,2H),3.31-3.26(m,2H),3.09(quin,J＝8.8Hz,1H),2.06(d,J＝8.8Hz,4H),1.60-1.56(m,2H),1.55-1.50(m,2H),1.45(s,9H).

Compound 3a-2 (1.00 g,3.53 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (3.4 mL) was added, stirred at room temperature until the reaction was complete as monitored by TLC (ethyl acetate/petroleum ether=1:3), and the reaction solution was concentrated to give a crude product. The crude product was used directly in the next reaction.

MS(ESI)m/z:184.2[M+H] ⁺

Step 2: synthesis of methyl 7- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -7-azaspiro [3.5] nonane-2-carboxylate (3 c)

Compound 3b (350 mg,0.98 mmol) was dissolved in N-methylpyrrolidone (4 mL) and triethylamine (150 mg,1.46 mmol) was added and cooled to 0deg.C with an ice-water bath. A solution of BB-1 (200 mg,0.98 mmol) in tetrahydrofuran (8 mL) and a solution of sodium tert-butoxide (141 mg,1.46 mmol) in dimethyl sulfoxide (2 mL) were added. The reaction was completed at room temperature until LC-MS was monitored, water (10 mL) was added, extracted with ethyl acetate (10 mL. Times.2), the organic phases were combined, washed with saturated brine, and dried over anhydrous magnesium sulfateDrying, filtering, evaporating the filtrate to obtain crude 3c (500 mg, yield 97%). MS (ESI) m/z 428.0[ M-Boc ]] ⁺ 。

Step 3: synthesis of 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -7-azaspiro [3.5] nonane-2-carboxylic acid (3)

Compound 3c (500 mg,0.95 mmol) was dissolved in dichloromethane (4.5 mL), trifluoroacetic acid (1.5 mL) was added, stirred at room temperature until the reaction was complete by LC-MS monitoring, and the reaction solution was concentrated to give a crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 3 (46 mg, 9% yield).

MS(ESI)m/z:428.2[M+H] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.56(d,J＝2.3Hz,1H),8.03(dd,J＝2.5,8.8Hz,1H),7.39-7.14(m,1H),7.05(d,J＝8.8Hz,1H),5.01(d,J＝3.5Hz,2H),3.83(s,2H),3.63(s,3H),3.15-3.04(m,1H),3.01-2.96(m,2H),2.95-2.89(m,2H),2.03-1.90(m,4H),1.77-1.71(m,2H),1.68-1.62(m,2H)

Examples 4 and 5

2- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-azaspiro [3.3] heptane (4) and

synthesis of 2- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-azaspiro [3.3] heptane (5)

Step 1: synthesis of 2- [ 6-chloropyridin-3-yl) sulfonyl ] -2-azaspiro [3.3] heptane (4 b)

Compound 4a (38 mg,2.59 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (716mg, 7.07 mmol) and compound 6-chloropyridine were added3-Sulfonyl chloride (1 a) (0.50 g,2.36 mmol), reaction at room temperature until LC-MS monitored reaction was complete. The reactant is directly mixed with silica gel and a rapid chromatographic separation instrument is usedIsolation gave 4b (0.20 g, 31% yield) as a white solid. MS (ESI) m/z 273.1[ M+H ]] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ8.83(d,J＝2.3Hz,1H),8.07(dd,J＝2.4,8.3Hz,1H),7.55(d,J＝8.3Hz,1H),3.79(s,4H),2.09-2.03(m,4H),1.85-1.76(m,2H).

Step 2: synthesis of 2- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-azaspiro [3.3] heptane (4 c)

Compound 4b (53 mg,0.20 mmol) was dissolved in N-methylpyrrolidone (1 mL) and triethylamine (30 mg,0.29 mmol) was added and cooled to 0deg.C with an ice-water bath. A solution of BB-1 (40 mg,0.20 mmol) in tetrahydrofuran (2 mL) and a solution of sodium tert-butoxide (28 mg,0.29 mmol) in dimethyl sulfoxide (0.5 mL) were added. The reaction was completed at room temperature until LC-MS was monitored, water (10 mL) was added, extracted with ethyl acetate (10 mL x 2), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 4c (50 mg, yield 58%). MS (ESI) m/z 442.2[ M+H ]] ⁺ 。

Step 3: synthesis of 2- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-azaspiro [3.3] heptane (4) and 2- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -2-azaspiro [3.3] heptane (5)

Compound 4c (50 mg,0.11 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature until the reaction was complete by LC-MS monitoring, and the reaction solution was concentrated to give a crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 4 (11 mg, yield 28%) and compound 5 (3 mg, yield 6%). MS (ESI) m/z 342.1[ M+H ]] ⁺

Compound 4:

MS(ESI):m/z 342.1[M+H] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.63(d,J＝2.3Hz,1H),8.11(dd,J＝2.4,8.8Hz,1H),7.38-7.12(m,1H),7.08(d,J＝8.7Hz,1H),5.03(d,J＝3.3Hz,2H),3.79(d,J＝1.5Hz,2H),3.74(s,4H),2.10-1.97(m,4H),1.88-1.72(m,2H).

compound 5:

MS(ESI):m/z 342.1[M+H] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.64(d,J＝2.3Hz,1H),8.11(dd,J＝2.5,8.7Hz,1H),7.38-6.96(m,2H),5.19(d,J＝2.6Hz,2H),3.75(s,4H),3.69(d,J＝2.7Hz,2H),2.14-1.94(m,4H),1.87-1.69(m,2H).

example 6

Synthesis of 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -7-azaspiro [3.5] nonane-2-carboxamide (6)

Step 1: synthesis of 7- [ 6-chloropyridin-3-yl) sulfonyl ] -7-azaspiro [3.5] nonane-2-carboxamide (6 b)

Compound 6a (2.20 g,7.07 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (2.62 mL,18.9 mmol), DMAP (60 mg,0.47 mmol) and compound 6-chloropyridine-3-sulfonyl chloride (1 a) (1.00 g,4.71 mmol) were added and the reaction was allowed to complete at room temperature until LC-MS monitored. Using flash chromatography apparatusIsolation gave 6b (1.00 g, 57% yield). MS (ESI) m/z 272.1[ M+H ]] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ8.73(d,J＝2.5Hz,1H),7.97(dd,J＝2.5,8.3Hz,1H),7.50(d,J＝8.3Hz,1H),5.41(br s,1H),3.31-3.22(m,2H),3.06-2.93(m,4H),2.90-2.84(m,1H),2.05-1.86(m,4H),1.75-1.68(m,4H),1.10(t,J＝7.3Hz,3H).

Wherein 6a can be prepared using commercially available 3a-1, as follows:

synthesis of tert-butyl 7-carboxylate-7-azaspiro [3.5] nonane-2-formylethylamine (6 a-1)

3a-1 (2.50 g,9.28 mmol) was dissolved in DMF (20 mL), cooled to 0deg.C, HATU (4.24 g,11.1 mmol), HOBt (1.38 g,10.2 mmol) and DIEA (3.07 mL,18.60 mmol) were added and diethylamine hydrochloride (1.14 g,13.90 mmol) was slowly added with stirring. The reaction was completed at room temperature until LC-MS monitoring. Quenching the reaction with water (20 mL), extracting with ethyl acetate (20 mLx 3), mixing the organic phases, washing with saturated salt water, drying the organic phases with anhydrous sodium sulfate, filtering, directly mixing the filtrate with silica gel, and separating with a flash chromatography apparatus The reaction mixture was separated to give 6a-1 (2.51 g, yield 95%).

MS(ESI):m/z 297.2[M+H] ⁺

¹ H NMR(400MHz,DMSO-d ₆ )δ4.02(q,J＝7.1Hz,1H),3.28-3.12(m,4H),3.08-2.99(m,2H),2.94-2.84(m,1H),2.73-2.63(m,4H),1.85(d,J＝8.6Hz,4H),1.37(s,9H),0.98(t,J＝7.2Hz,3H).

Synthesis of 7-azaspiro [3.5] nonane-2-formylethylamine (6 a)

Compound 6a-1 (1.50 g,5.06 mmol) was dissolved in dichloromethane (15 mL), trifluoroacetic acid (5 mL) was added, stirred at room temperature until the reaction was complete as monitored by TLC (ethyl acetate/petroleum ether=1:2), and the reaction solution was concentrated to give crude product (2.00 g). The crude product was used directly in the next reaction. MS (ESI) m/z 197.2[ M+H ]] ⁺

Step 2: synthesis of 7- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -7-azaspiro [3.5] nonane-2-carboxamide (6 c)

Compound 6b (360 mg,0.97 mmol) was dissolved in N-methylpyrrolidone (4 mL) and triethylamine (0.20 mL,1.45 mmol) was added and cooled to 0deg.C with an ice-water bath. A solution of BB-1 (200 mg,0.98 mmol) in tetrahydrofuran (8 mL) andsodium t-butoxide (140 mg,1.45 mmol) in dimethyl sulfoxide (2 mL). The reaction was warmed to room temperature until completion of LC-MS monitoring, water (10 mL) was added, extracted with ethyl acetate (10 mL. X2), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 6c (507 mg, yield 100%). MS (ESI) m/z 541.2[ M+H ]] ⁺ 。

Step 3: synthesis of 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -7-azaspiro [3.5] nonane-2-carboxamide (6)

Compound 6c (523 mg,0.97 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, and the mixture was stirred at room temperature until the reaction was complete by LC-MS monitoring, and the reaction solution was concentrated to give a crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 6 (18 mg, yield 4%).

MS(ESI):m/z 441.1[M+H] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.55(d,J＝2.5Hz,1H),8.01(dd,J＝2.5,8.8Hz,1H),7.30-6.92(m,2H),4.98(d,J＝3.5Hz,3H),3.66(s,2H),3.15(q,J＝7.3Hz,2H),3.03-2.88(m,5H),1.89(d,J＝8.5Hz,4H),1.77-1.71(m,2H),1.68-1.62(m,2H),1.07(t,J＝7.2Hz,3H).

Example 7

8- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -1-oxo-2-methyl-2-8-diazaspiro [4.5] decane (7)

Step 1: synthesis of 8- [ 6-chloropyridin-3-yl) sulfonyl ] -7-azaspiro [3.5] nonane-2-carboxamide (7 b)

11a (2.00 g,7.07 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (2.62 mL,18.9 mmol), DMAP (60 mg,0.47 mmol) and 6-chloropyridine-3-sulfonyl chloride (1 a) (1.00 g,4.71 mmol) were added and the reaction was allowed to complete at room temperature until LC-MS monitored.The reactant is directly mixed with silica gel and a rapid chromatographic separation instrument is used7b (1.20 g, yield 56%) was isolated. MS (ESI) m/z 344.1[ M+H ]] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ8.77(d,J＝2.5Hz,1H),7.99(dd,J＝2.5,8.3Hz,1H),7.50(d,J＝8.3Hz,1H),3.53-3.48(m,2H),3.30(t,J＝6.9Hz,2H),3.50-3.03(m,2H),2.81(s,3H),2.01-1.92(m,2H),1.84(t,J＝6.9Hz,2H),1.65-1.55(m,2H).

Wherein 7a can be prepared using commercial 7a-1, as follows:

synthesis of tert-butyl 8-carboxylate-1-oxo-2-8-diazaspiro [4.5] decane (7 a-2)

Compound 7a-1 (2.50 g,9.83 mmol) was dissolved in DMF (25 mL), cooled to 0deg.C with an ice-water bath, and 60% sodium hydrogen (0.79 mg,19.70 mmol) was added and the reaction continued at 0deg.C for 10 min. Methyl iodide (1.22 mL,19.7 mmol) was slowly added, then the reaction was kept at 0deg.C until the LC-MS was monitored to be complete, water (200 mL) was added, extracted with ethyl acetate (150 mL x 3), the organic phases were combined, washed with saturated common salt, dried over anhydrous magnesium sulfate, filtered, the filtrate was evaporated to dryness to give a crude product, the reaction was directly stirred with silica gel, and the crude product was purified by flash chromatography using a flash chromatography apparatus 7a-2 (2.51 g, yield 95%) was isolated.

MS(ESI):m/z 169.2[M-Boc] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ4.00(br s,2H),3.32(t,J＝7.0Hz,2H),2.98(br t,J＝11.2Hz,2H),2.86(s,3H),1.97(br t,J＝6.5Hz,2H),1.89-1.89(m,1H),1.89-1.82(m,1H),1.46(s,9H),1.37(d,J＝13.1Hz,2H).

Synthesis of 1-oxo-2-8-diazaspiro [4.5] decane (7 a)

Compound 7a-2 (1.00 g,3.73 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (3 mL) was added, stirred at room temperature until the LC-MS monitoring reaction was complete, and the reaction mixture was concentrated to give crude product (2.00 g). The crude product is directly used for the next stepAnd (3) performing a step reaction. MS (ESI) m/z 169.1[ M+H ]] ⁺

Step 2: synthesis of 8- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -1-oxo-2-methyl-2-8-diazaspiro [4.5] decane (7 c)

Compound 7b (335 mg,0.97 mmol) was dissolved in N-methylpyrrolidone (4 mL) and triethylamine (0.20 mL,1.45 mmol) was added and cooled to 0deg.C with an ice-water bath. A solution of BB-1 (200 mg,0.98 mmol) in tetrahydrofuran (8 mL) and a solution of sodium tert-butoxide (140 mg,1.45 mmol) in dimethyl sulfoxide (2 mL) were added. The reaction was warmed to room temperature until completion of LC-MS monitoring, water (80 mL) was added, extracted with ethyl acetate (40 mL. Times.2), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 7c (499 mg). MS (ESI): m/z 413.0[ M-Boc ]] ⁺ 。

Step 3: synthesis of 8- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -1-oxo-2-methyl-2-8-diazaspiro [4.5] decane (7)

Compound 7c (499 mg,0.97 mmol) was dissolved in dichloromethane (4.5 mL), trifluoroacetic acid (1.5 mL) was added, stirred at room temperature until the LC-MS monitoring reaction was complete, and the reaction solution was concentrated to give the crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 7 (68 mg, 15% yield).

MS(ESI):m/z 413.1[M+H] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.61-8.50(m,2H),8.05(dd,J＝2.5,8.8Hz,1H),7.32-7.09(m,1H),7.03(d,J＝8.8Hz,1H),5.00(d,J＝3.5Hz,2H),3.76(s,2H),3.62-3.55(m,2H),3.36-3.33(m,1H),2.81-2.71(m,5H),1.91-1.82(m,4H),1.60-1.51(m,2H).

Example 8

Synthesis of 8- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -1-oxo-2-oxa-8-azaspiro [4.5] decane (8)

Step 1: synthesis of 8- [ 6-chloropyridin-3-yl) sulfonyl ] -1-oxo-2-oxa-8-azaspiro [4.5] decane (8 b)

Compound 8a (323 mg,3.77 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (1.43 g,14.1 mmol) and compound 6-chloropyridine-3-sulfonyl chloride (1 a) (1.00 g,4.71 mmol) were added and the reaction was allowed to react at room temperature until LC-MS monitored to completion. The reactant is directly mixed with silica gel and a rapid chromatographic separation instrument is used8b (0.50 g, 40% yield) was isolated.

MS(ESI):m/z 331.1[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ8.79(d,J＝2.1Hz,1H),8.22(dd,J＝2.4,8.4Hz,1H),7.83(d,J＝8.4Hz,1H),4.23(br t,J＝7.0Hz,2H),3.48(br d,J＝12.1Hz,2H),2.90-2.68(m,2H),2.07(br t,J＝6.9Hz,2H),1.71(br d,J＝4.5Hz,4H).

Step 2: synthesis of 8- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -1-oxo-2-oxa-8-azaspiro [4.5] decane (8 c)

Compound 8b (65 mg,0.20 mmol) was dissolved in N-methylpyrrolidone (1 mL) and triethylamine (30 mg,0.29 mmol) was added and cooled to 0deg.C with an ice-water bath. A solution of BB-1 (40 mg,0.20 mmol) in tetrahydrofuran (2 mL) and a solution of sodium tert-butoxide (28 mg,0.29 mmol) in dimethyl sulfoxide (0.5 mL) were added. The reaction was warmed to room temperature until completion of LC-MS monitoring, water (10 mL) was added, extracted with ethyl acetate (10 mL. X2), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 8c (50 mg, yield 58%). MS (ESI): m/z 400.1[ M-Boc ] ] ⁺ 。

Step 3: synthesis of 8- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -1-oxo-2-oxa-8-azaspiro [4.5] decane (8)

Compound 8c (50 mg,0.10 mmol) was dissolved in dichloromethane (4.5 mL), trifluoroacetic acid (1.5 mL) was added, stirring at room temperature was continued until the reaction was complete by LC-MS monitoring, and the reaction mixture was concentrated to give a crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 8 (10 mg, yield 23%).

MS(ESI):m/z 400.0[M+H] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ8.61(d,J＝2.4Hz,1H),8.08(dd,J＝2.5,8.7Hz,1H),7.43-7.15(m,1H),7.07(d,J＝8.8Hz,1H),5.03(d,J＝3.5Hz,2H),4.31(t,J＝7.0Hz,2H),3.84(d,J＝2.0Hz,2H),3.44-3.37(m,2H),3.06-3.01(m,2H),2.15(t,J＝7.0Hz,2H),2.03-1.87(m,2H),1.79-1.76(m,2H).

Example 9

6- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylic acid methyl ester (9) and

synthesis of methyl 6- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylate (10)

Step 1: synthesis of methyl 6- [ 6-chloropyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylate (9 b)

Compound 9a (1.00 g,3.53 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (1.90 g,18.90 mmol) and compound 6-chloropyridine-3-sulfonyl chloride (1 a) (1.00 g,4.71 mmol) were added and the reaction was allowed to react at room temperature until LC-MS monitored to completion. The reactant is directly mixed with silica gel and a rapid chromatographic separation instrument is used9b (0.80 g, yield 49%) was isolated. MS (ESI) m/z 331.1[ M+H ]] ⁺

¹ H NMR(400MHz,CDCl3)δ8.78(d,J＝2.0Hz,1H),8.01(dd,J＝2.5,8.4Hz,1H),7.53(d,J＝8.3Hz,1H),3.67(s,3H),3.35-2.94(m,4H),1.95-1.79(m,2H),1.67-1.61(m,1H),1.58-1.51(m,2H),1.13(t,J＝5.1Hz,1H),0.92(dd,J＝4.8,8.2Hz,1H).

Wherein 9a can be prepared from commercial 9a-1 by the following procedure:

Synthesis of methyl 6-tert-Ding Tansuan ester-6-azaspiro [2.5] octane-1-carboxylate (9 a-2)

9a-1 (1.50 g,5.88 mmol) was dissolved in acetone (15 mL) and potassium carbonate (1.62 g,11.80 mmol) and methyl iodide (1.97 mL,11.80 mmol) were added and reacted at room temperature until LC-MS monitored reaction was complete. Filtering to remove insoluble substances, mixing the filtrate with silica gel, and separating with a rapid chromatographic separation instrument9a-2 (1.40 g, yield 89%) was isolated. MS (ESI) m/z 270.1[ M+H ]] ⁺

¹ H NMR(400MHz,CDCl3)δ3.68(s,3H),3.56-3.38(m,3H),3.28(d,J＝7.5Hz,1H),1.70(dt,J＝4.3,7.2Hz,2H),1.58-1.54(m,1H),1.49-1.36(m,11H),1.18(t,J＝5.0Hz,1H),0.95(dd,J＝4.6,8.0Hz,1H).

Synthesis of methyl 6-azaspiro [2.5] octane-1-carboxylate trifluoroacetate salt (9 a)

Compound 9a-2 (1.00 g,5.17 mmol) was dissolved in dichloromethane (10 mL), trifluoroacetic acid (3 mL) was added, stirred at room temperature until the reaction was complete as monitored by TLC (ethyl acetate/petroleum ether=1:3), and the reaction solution was concentrated to give the crude product. The crude product was used directly in the next reaction. MS (ESI) m/z 170.2[ M+H ]] ⁺

Step 2: synthesis of methyl 6- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylate (9 c)

Compound 9b (336 mg,0.98 mmol) was dissolved in N-methylpyrrolidone (4 mL) and triethylamine (148 mg,1.46 mmol) was added and cooled to 0deg.C with an ice-water bath. A solution of BB-1 (200 mg,0.98 mmol) in tetrahydrofuran (8 mL) and sodium tert-butoxide (140 mg,1.46 mmol) in dimethyl sulfoxide (2 mL) were added And (3) liquid. The reaction was warmed to room temperature until completion of LC-MS monitoring, water (80 mL) was added, extracted with ethyl acetate (40 mL. Times.2), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 9c (200 mg, yield 46%). MS (ESI): m/z 414.0[ M-Boc ]] ⁺ 。

Step 3: methyl 6- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylate (9) and methyl 6- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylate (10)

Compound 9c (200 mg,0.39 mmol) was dissolved in dichloromethane (4.5 mL), trifluoroacetic acid (1.5 mL) was added, stirred at room temperature until the LC-MS monitoring reaction was complete, and the reaction solution was concentrated to give a crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 9 (130 mg, yield 58%) as compound 10 (5 mg, yield 2%). MS (ESI) m/z 413.9[ M+H ]] ⁺

Compound 9:

MS(ESI):m/z 413.9[M+H] ⁺

¹ H NMR(400MHz,CDCl3)δ8.60(d,J＝2.2Hz,1H),8.07(dd,J＝2.5,8.7Hz,1H),7.42-7.13(m,1H),7.06(d,J＝8.8Hz,1H),5.03(d,J＝3.5Hz,2H),3.84(d,J＝2.0Hz,2H),3.62(s,3H),3.16-3.05(m,3H),2.98(br dd,J＝3.7,7.3Hz,1H),2.03-1.73(m,2H),1.66-1.51(m,3H),1.05(s,2H).

compound 10:

MS(ESI):m/z 413.9[M+H] ⁺

¹ H NMR(400MHz,CDCl3)δ8.61(d,J＝2.3Hz,1H),8.07(dd,J＝2.5,8.7Hz,1H),7.31-6.98(m,2H),5.18(d,J＝2.4Hz,2H),3.72(d,J＝2.4Hz,2H),3.62(s,3H),3.17-3.07(m,3H),3.02-2.85(m,1H),1.92-1.81(m,2H),1.63-1.55(m,3H),1.08-0.93(m,2H).

example 10

6- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-formylmethylamine trifluoroacetate (11) and

synthesis of 6- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-formylmethylamine trifluoroacetate salt (12)

Step 1: synthesis of 6- [ 6-chloropyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylic acid (11 a)

Compound 9b (1.10 g,3.19 mmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL), lithium hydroxide hydrate (268 mg,6.38 mmol) was added and the reaction was allowed to proceed to completion at room temperature until LC-MS monitored. The pH was adjusted to pH=5 to 7 with 1N hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 11a (1.00 g). MS (ESI) m/z 331.1[ M+H ]] ⁺ 。

Step 2: synthesis of 6- [ 6-chloropyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxamide (11 b)

Compound 11a (1.00 g,3.02 mmol) was dissolved in DMF (10 mL) and HOBt (449 mg,3.33 mmol), DIPEA (781 mg,6.05 mmol), HATU (1.38 g,3.33 mmol) and methylamine hydrochloride (370 mg,4.54 mmol) were added. The reaction was complete at room temperature until LC-MS monitoring. Water was added, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to give a crude product, which was separated by column chromatography to give compound 11b (0.60 g, 58%). MS (ESI) m/z 344.1[ M+H ]] ⁺ 。

Step 3: synthesis of 6- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxamide (11 c)

Compound 11b (0.67 g,1.95 mmol) was dissolved in NMP (5 mL) and triethylamine (292 mg,2.92 mmol) was added, cooled to 0℃with an ice-water bath, followed by BB-room temperature1 (400 mg,1.95 mmol) in tetrahydrofuran (10 mL) and sodium tert-butoxide (281mg, 2.92 mmol) in DMSO (2.5 mL). The reaction was warmed to room temperature until completion of LC-MS monitoring, water (30 mL), ethyl acetate (30 mL. Times.2) were added, the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 11c (500 mg). MS (ESI): m/z 413.2[ M-Boc+H] ⁺ 。

Step 3: synthesis of 6- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-formylmethylamine trifluoroacetate (11) and 6- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-formylmethylamine trifluoroacetate (12)

Compound 11c (300 mg,0.58 mmol) was dissolved in dichloromethane (3.0 mL), trifluoroacetic acid (1.0 mL) was added, stirred at room temperature until the reaction was complete by LC-MS monitoring, and the reaction solution was concentrated to give a crude product. The crude product is directly separated by preparative high performance liquid chromatography to give compound 11 (127 mg, yield 53%) MS (ESI) m/z 413.2[ M+H ]] ⁺

¹ H NMR(400MHz,METHANOL-d ₄ )δ8.59(d,J＝2.5Hz,1H),8.06(dd,J＝2.5,8.8Hz,1H),7.47-7.12(m,1H),7.06(d,J＝8.8Hz,1H),5.02(d,J＝3.5Hz,2H),3.85(s,2H),3.19(ddd,J＝3.8,7.1,11.2Hz,1H),3.13-2.95(m,3H),2.69(s,3H),1.79(t,J＝5.5Hz,2H),1.66-1.41(m,3H),1.03(t,J＝4.9Hz,1H),0.77(dd,J＝4.6,8.1Hz,1H)。

And Compound 12 (9 mg, yield 3%). MS (ESI): m/z 413.2[ M+H ] ] ⁺

¹ H NMR(400MHz,METHANOL-d ₄ )δ8.60(d,J＝2.1Hz,1H),8.53(br s,1H),8.07(dd,J＝2.4,8.7Hz,1H),7.24-6.93(m,1H),5.18(d,J＝2.3Hz,2H),3.68(d,J＝2.4Hz,2H),3.25-2.96(m,4H),2.68(s,3H),1.80(br t,J＝5.4Hz,2H),1.68-1.31(m,3H),1.04(t,J＝4.9Hz,1H),0.77(dd,J＝4.5,8.0Hz,1H)。

Example 11

6- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylic acid trifluoroacetate salt (13) and

synthesis of trifluoroacetate salt (14) of 6- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylic acid

Step 1: synthesis of 6- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylic acid (11 a)

Compound 9c (0.90 g,1.75 mmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL) to which lithium hydroxide hydrate (0.15 g,3.51 mmol) was added and reacted at room temperature until the reaction was complete as monitored by LC-MS. The pH was adjusted to pH=5 to 7 with 1N hydrochloric acid, extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 13a (800 mg, yield 91%). MS (ESI): m/z 444.1[ M-tBut ]] ⁺ 。

Step 2: synthesis of 6- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylic acid trifluoroacetate (13) and 6- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -6-azaspiro [2.5] octane-1-carboxylic acid trifluoroacetate (14)

Compound 13a (500 mg,1.00 mmol) was dissolved in dichloromethane (3.0 mL), trifluoroacetic acid (1.0 mL) was added, stirring at room temperature until LC-MS monitoring reaction was complete, and the reaction solution was concentrated to give a crude product. The crude product is directly separated by preparative high performance liquid chromatography to obtain compound 13 (127 mg, yield 30%). MS (ESI) m/z 400.1[ M+H ] ] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.60(d,J＝2.0Hz,1H),8.07(dd,J＝2.5,8.8Hz,1H),7.39-7.15(m,1H),7.06(d,J＝8.8Hz,1H),5.03(d,J＝3.3Hz,2H),3.84(d,J＝1.8Hz,2H),3.22-3.04(m,3H),3.02-2.83(m,1H),1.98-1.85(m,2H),1.66-1.47(m,3H),1.11-0.84(m,2H).

And 14 (30 mg, yield 7%). MS (ESI) m/z 400.1[ M+H ]] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.61(d,J＝2.3Hz,1H),8.08(dd,J＝2.5,8.8Hz,1H),7.32-6.77(m,2H),5.18(d,J＝2.5Hz,2H),3.72(d,J＝2.5Hz,2H),3.20-2.93(m,4H),1.95-1.78(m,2H),1.75-1.45(m,3H),1.04(t,J＝4.9Hz,1H),0.93(dd,J＝4.6,7.9Hz,1H)

Example 12

7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [1,5-a ] pyrazine trifluoroacetate (15) and

synthesis of 7- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [1,5-a ] pyrazine trifluoroacetate (16)

Step 1: synthesis of 7- [ 6-chloropyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [1,5-a ] pyrazine (15 b)

Compound 15a (227 g,4.24 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (0.86 g,8.49 mmol) and compound 6-chloropyridine-3-sulfonyl chloride (1 a) (0.90 g,4.24 mmol) were added and the reaction was allowed to proceed to completion at room temperature until LC-MS monitored. The reactant is directly mixed with silica gel and a rapid chromatographic separation instrument is used15b (0.80 g, yield 62%) was isolated as a white solid. MS (ESI) m/z 300.1[ M+H ]] ⁺

¹ H NMR(400MHz,CDCl ₃ )δ8.86(d,J＝2.5Hz,1H),8.07(dd,J＝2.5,8.4Hz,1H),7.91(s,1H),7.55(d,J＝8.4Hz,1H),4.54(s,2H),4.32(t,J＝5.4Hz,2H),3.79-3.73(m,2H).

Step 2: synthesis of 7- [6- [ -2- (((tert-butoxycarbonyl) amino) methyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [1,5-a ] pyrazine (15 c)

Compound BB-1 (684 mg,3.34 mmol) was dissolved in tetrahydrofuran (10 mL), cooled to 0℃with an ice-water bath, and 60% sodium hydride (227 mg,6.68 mmol) was added to react at 0℃for 10 minutes. 15b (1.00 g,3.34 mmol) was added. The reaction was warmed to room temperature until completion of LC-MS monitoring, water (30 mL) was added, extracted with dichloromethane (30 mL. Times.2), the organic phases were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and the filtrate was evaporated to dryness to give crude 15c (500 mg). MS (ESI) m/z 469.2[ M+H ] ] ⁺ 。

Step 3: synthesis of 7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [1,5-a ] pyrazine trifluoroacetate (15) and 7- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [1,5-a ] pyrazine trifluoroacetate (16)

Compound 15c (500 mg,1.71 mmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (1 mL) was added, stirred at room temperature until the reaction was complete by LC-MS monitoring, and the reaction solution was concentrated to give a crude product. The crude product was directly separated by preparative high performance liquid chromatography to give compound 15 (94 mg, 23% yield).

MS(ESI):m/z 369.1[M+H] ⁺ 。

¹ H NMR(400MHz,METHANOL-d4)δ8.72(d,J＝2.1Hz,1H),8.16(dd,J＝2.6,8.8Hz,1H),7.96(s,1H),7.40-7.10(m,1H),7.05(d,J＝8.8Hz,1H),5.19-4.97(m,2H),4.54(s,2H),4.27(t,J＝5.6Hz,2H),3.89-3.73(m,4H).

And compound 16 (42 mg, yield 10%). MS (ESI) m/z 369.1[ M+H ]] ⁺

¹ H NMR(400MHz,METHANOL-d4)δ8.73(d,J＝2.3Hz,1H),8.17(dd,J＝2.5,8.8Hz,1H),7.96(s,1H),7.33-6.91(m,2H),5.18(d,J＝2.5Hz,2H),4.54(s,2H),4.27(t,J＝5.5Hz,2H),3.80(t,J＝5.5Hz,2H),3.71(d,J＝2.8Hz,2H).

Example 13

7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2] diazo-o [1,2-a ] pyrazine trifluoroacetate (17) and

the procedure for the synthesis of 7- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [1,2-a ] pyrazine trifluoroacetate (18) was analogous to example 11, substituting 5,6,7, 8-tetrahydroimidazo [1,2-a ] pyrazine hydrochloride for compound 15a to afford compound 17 (335 mg, 53% yield).

MS(ESI):m/z 368.1[M+H] ⁺ 。

¹ H NMR(400MHz,METHANOL-d4)δ8.73(d,J＝2.2Hz,1H),8.18(dd,J＝2.6,8.8Hz,1H),7.61-7.49(m,2H),7.42-7.14(m,1H),7.09(d,J＝8.8Hz,1H),5.04(d,J＝3.2Hz,2H),4.69(s,2H),4.35(t,J＝5.4Hz,2H),3.84(d,J＝2.1Hz,2H),3.80-3.67(m,2H).

And Compound 18 (22 mg, yield 3%), MS (ESI): m/z 368.1[ M+H ] ] ⁺ 。 ¹ H NMR(400MHz,METHANOL-d4)δ8.75(d,J＝2.6Hz,1H),8.19(dd,J＝2.6,8.8Hz,1H),7.51(s,2H),7.32-6.95(m,2H),5.19(d,J＝2.3Hz,2H),4.68(s,2H),4.45-4.28(m,1H),4.34(t,J＝5.4Hz,1H),3.88-3.74(m,2H),3.72(d,J＝2.3Hz,2H)。

Example 14

2-methyl-7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [1,5-a ] pyrazine trifluoroacetate (19) and

2-methyl-7- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy]Pyridin-3-yl) sulfonyl]-5,6,7, 8-tetrahydro- [1,2,4]]Triazolo [1,5-a ]]The synthesis of pyrazine trifluoroacetate (20) was similar to that of example 11 using 2-methyl-5, 6,7, 8-tetrahydro- [1,2,4]Triazolo [1,5-a ]]The corresponding procedure was followed using pyrazine instead of compound 15a to give compound 19 (72 mg, yield 18%), MS (ESI): m/z 383.1[ M+H ]] ⁺ ， ¹ H NMR(400MHz,METHANOL-d4)δ8.71(d,J＝2.0Hz,1H),8.16(dd,J＝2.6,8.8Hz,1H),7.45-7.12(m,1H),7.05(d,J＝8.8Hz,1H),5.02(dd,J＝0.7,3.7Hz,2H),4.48(s,2H),4.18(t,J＝5.6Hz,2H),3.83(s,2H),3.78(t,J＝5.6Hz,2H),2.31(s,3H)。

And compound 20 (22 mg, yield 54%). MS (ESI) m/z 383.1[ M+H ]] ⁺ ， ¹ H NMR(400MHz,METHANOL-d4)δ8.72(d,J＝2.1Hz,1H),8.17(dd,J＝2.6,8.8Hz,1H),7.39-6.93(m,2H),5.18(d,J＝2.7Hz,2H),4.48(s,2H),4.18(t,J＝5.5Hz,2H),3.78(t,J＝5.6Hz,2H),3.71(d,J＝2.3Hz,2H),2.31(s,3H)。

Example 15

7- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -5,6,7, 8-tetrahydro- [1,2,4] triazolo [4,3-a ] pyrazine trifluoroacetate (21) and

7- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy]Pyridin-3-yl) sulfonyl]-5,6,7, 8-tetrahydro- [1,2,4]]Triazolo [4,3-a ]]The synthesis of pyrazine trifluoroacetate (22) was similar to that of example 11 using 5,6,7, 8-tetrahydro- [1,2,4]]Triazolo [4,3-a ]]Pyrazine is used to replace compound 15a to perform corresponding process operationCompound 21 (35 mg, yield 7%), MS (ESI): m/z 369.1[ M+H ]] ⁺ ， ¹ H NMR(400MHz,METHANOL-d4)δ8.72(d,J＝2.1Hz,1H),8.48(s,1H),8.18(dd,J＝2.6,8.9Hz,1H),7.46-7.14(m,1H),7.09-7.00(m,1H),5.08-4.99(m,2H),4.57(s,2H),4.24(t,J＝5.6Hz,2H),3.83(d,J＝1.2Hz,2H),3.72(t,J＝5.6Hz,2H)。

And Compound 22 (18 mg, yield 4%), MS (ESI): m/z 369.1[ M+H ] ] ⁺ ， ¹ H NMR(400MHz,METHANOL-d4)δ8.74(d,J＝2.4Hz,1H),8.48(s,1H),8.19(dd,J＝2.5,8.9Hz,1H),7.40-6.87(m,2H),5.18(d,J＝2.7Hz,2H),4.58(s,2H),4.24(t,J＝5.6Hz,2H),3.89-3.67(m,4H)。

Example 16

3-methyl-8- [6- [ (E) -2- (aminomethyl) -3-fluoroallyloxy ] pyridin-3-yl) sulfonyl ] -1,3, 8-triazaspiro [4.5] decane-2, 4-dione trifluoroacetate (23) and

3-methyl-8- [6- [ (Z) -2- (aminomethyl) -3-fluoroallyloxy]Pyridin-3-yl) sulfonyl]-1,3, 8-triazaspiro [4.5]]The synthesis of decane-2, 4-dione trifluoroacetate (24) was carried out in analogy to example 14, using 23a instead of compound 15a and the corresponding procedure gave compound 23 (388 mg, yield 68%), MS (ESI): m/z 428.0[ M+H ]] ⁺ ， ¹ H NMR(400MHz,METHANOL-d4)δ8.60(d,J＝2.4Hz,1H),8.07(dd,J＝2.6,8.8Hz,1H),7.39-7.13(m,1H),7.06(d,J＝8.7Hz,1H),5.02(d,J＝3.7Hz,2H),3.82(d,J＝2.1Hz,2H),3.60-3.44(m,2H),3.31(td,J＝1.6,3.3Hz,3H),3.10-3.07(m,2H),2.05-2.00(m,2H),1.81-1.76(m,2H)。

And 24 (11 mg, yield 2%), MS (ESI): m/z 428.0[ M+H ]] ⁺ ， ¹ H NMR(400MHz,METHANOL-d4)δ8.61(d,J＝2.1Hz,1H),8.07(dd,J＝2.6,8.8Hz,1H),7.18-6.87(m,2H),5.17(d,J＝2.4Hz,2H),3.62-3.50(m,4H),3.10-2.99(m,2H),2.90(s,3H),2.06-2.02(m,2H),1.80-1.75(m,2H)。

Wherein 23a can be prepared using commercially available 23a-1, as follows:

synthesis of 3-methyl-8-t-butoxycarbonyl-1, 3, 8-triazaspiro [4.5] decane-2, 4-dione (23 a-2)

Compound 23a-1 (5.90 g,99.80 mmol) was dissolved in DMSO (40 mL) and cooled to 0deg.C, methyl iodide (1.16 g,8.17 mmol) and potassium carbonate (3.08 g,22.30 mmol) were added. The reaction was warmed to room temperature until LC-MS monitored the reaction was complete. Dilute with 200mL water and extract with ethyl acetate (100 mL x 2). The organic phases were combined, washed with saturated brine and dried over anhydrous sodium sulfate. The compound 23a-2 (1.74 g, yield 83%) was isolated by column chromatography as MS (ESI) m/z 184.4[ M+H] ⁺ 。 ¹ H NMR(400MHz,CDCl ₃ )δ6.95(s,1H),4.12-3.87(m,2H),3.20(ddd,J＝2.9,10.5,13.5Hz,2H),3.02(s,3H),2.08-1.93(m,2H),1.63(s,2H),1.47(s,9H)。

Synthesis of 3-methyl-1, 3, 8-triazaspiro [4.5] decane-2, 4-dione (23 a)

Compound 23a-2 (3.00 g,13.90 mmol) was dissolved in dichloromethane (30 mL), trifluoroacetic acid (10 mL) was added, stirred at room temperature until the LC-MS monitoring reaction was complete, and the reaction solution was concentrated to give a crude product. The crude product was directly isolated by preparative high performance liquid chromatography to give compound 23a (1.13 g) which was directly used in the next reaction.

Biological evaluation

The following further description explains the present disclosure in connection with test examples, which are not meant to limit the scope of the present disclosure.

Test example 1: assessment of inhibitory Activity of rhVAP-1 enzyme

(1) Instrument consumable and reagent

Multifunctional enzyme labelling instrument (MD, flexStation 3), black impermeable bottom 96-1L plate (Corning), rhVAP-1 (PeproTech)

(2) Self-preparation of compound concentration gradient solution

And taking a proper amount of the compound to be tested, dissolving the compound to 10mM in DMSO, and storing. Before the experiment, a proper amount of 10mM compound mother solution to be tested is diluted to 1mM solution by DMSO, then 3-time gradient dilution is carried out by DMSO, 10 concentration gradients are added, and 100-time dilution is carried out by PBS to prepare 10X series concentration compound solutions.

(3) Enzyme solution preparation

An appropriate amount of protein diluent was added to the rhVAP-1 powder to give 1mg/mL of mother liquor for storage. The enzyme solution was diluted with PBS to give a 4X concentration prior to the experiment.

(4) 2x concentration substrate mixed solution preparation

An appropriate amount of benzylamine was weighed, dissolved in PBS to obtain 200mM benzylamine solution, added with 2mM of Amplex Red mother liquor and 500U/mL of HRP mother liquor, and diluted with PBS to obtain a substrate mixture with 2X concentration.

(5) Test method

First, 10. Mu.L of a compound solution of different concentrations, 25. Mu.L of 4 XrhVAP-1 enzyme solution and 15. Mu.L of PBS were added to a 96-well plate, and after shaking and mixing, incubated at 37℃for 30min. Then 50 mu L of 2x substrate mixed solution is added into each hole, the detection is immediately carried out by using an enzyme-labeling instrument, excitation light is 565nm, emission light is 590nm, the fluorescence intensity of each hole is detected for 5 min/time, the total detection is 25min, and the inhibition rate is calculated according to the following formula:

V(RFU/min)＝(F _t (RFU)-F ₀ (RFU))/(time (min))

Inhibition (%) =100% -V _cmpd (RFU/min)/V _max (RFU/min)x 100％

V: rate of fluorescence change F _t Fluorescent reading F at time t ₀ : initial fluorescence readings; time: duration t; v (V) _cmpd Rate of change of fluorescence of test compound V _max Max Kong Yingguang rate of change.

(6) Fitting dose-response curve

The log value of the concentration is taken as an X axis, the percent inhibition rate is taken as a Y axis, and a log (inhibitor) vs. response-Variable slope fit quantitative effect curve of analysis software GraphPad Prism 5 is adopted, so that the IC of each compound on the enzyme activity is obtained ₅₀ Values.

The examples of the present disclosure inhibit VAP-1 enzyme activity in vitro by the above test Testing and measuring IC ₅₀ The values are shown in Table 1.

TABLE 1

Remarks:(PXS-4728A)；(BI-38-Z)。

test example 2: selectivity of MAO-A/B enzyme

(1) Instrument consumable and reagent

Microplate reader (Perkin Elmer, enVision), 384 well plate (Perkin Elmer), centrifuge (Eppendorf), MAO-GloTM (Promega), MAO-A (Active Motif) and MAO-B (Active Motif).

(2) Self-preparation of compound concentration gradient solution

An appropriate amount of the test compound was taken, dissolved in DMSO to 10mM, and stored, and then subjected to 4-fold gradient dilution with DMSO for a total of 6 concentration gradients.

(3) Enzyme solution preparation

MAO-A/B stock was diluted with MAO-A/B assay buffer to 2X concentration enzyme solution.

(4) 2x concentration substrate mixed solution preparation

The MAO-A/B substrate mixture stock solution was diluted with MAO-A/B assay buffer to A2X concentration substrate mixture.

(5) Test method

200nL of compound solution or solvent with different concentrations and 10 mu L of 2xMAO-A/B enzyme solution are added into A384-well plate, the mixture is centrifuged at 1000rpm for 60s, and the mixture is stirred and mixed uniformly and then incubated for 15min at room temperature. Then 10. Mu.L of 2 Xsubstrate mixture was added to each well to initiate the reaction. The 384-well plate is centrifuged at 1000rpm for 60s, and the mixture is stirred and mixed uniformly and then incubated for 60min at room temperature. And adding 20 mu L of stop detection solution, stopping reaction, centrifuging at 1000rpm for 60s, and shaking and mixing uniformly. After standing for 30min, reading is carried out by an enzyme-labeled instrument.

The inhibition rate was calculated according to the following formula:

inhibition ratio (%) = (signal_max-signal_sample)/(signal_max-signal_min) x 100

(6) Fitting dose-response curve

Examples of the present disclosure in vitro inhibition of MAO-A and MAO-B enzyme activity was determined by the above assay, IC measured ₅₀ The values are shown in Table 2.

TABLE 2

From tables 1 and 2 above, it can be seen that the compounds of the present disclosure have good inhibitory activity against rhVAP-1 enzyme, and that the compounds of the present disclosure exhibit excellent selective inhibitory effect against rhVAP-1 enzyme compared to monoamine oxidase (MAO). Taken together with tables 1 and 2 above, it can be seen that the compounds of the present disclosure do not have side effects due to inhibition of rhAOC1 and MAO enzymes while treating and/or preventing diseases associated with SSAO/VAP-1 enzymes.

Test example 3: in vivo pharmacokinetic studies in mice

1. Summary

The concentration of the drug in plasma was measured at various times after the mice were used as test animals and the LC/MS method was used to determine the concentration of the drug in plasma after the mice had been given compound 16 by intragastric administration. Pharmacokinetic behavior of the compounds of the present disclosure in mice was studied and their pharmacokinetic profile was assessed.

2. Test protocol

2.1 test drug

Compound 16

2.2 test animals

The C57 mice, 9 females, were equally divided into 3 groups, purchased from Shanghai Jieshijie laboratory animal Co., ltd., animal production license number: SCXK 2013-0006 (Shanghai)

2.3 pharmaceutical formulation

A certain amount of the compound was weighed, and 1% of hydroxyethylcellulose (w/v) and 0.25% of Tween 80 were added to dissolve the compound, so that a colorless clear solution of 1mg/ml was prepared.

2.4 administration of drugs

The C57 mice were fed by gastric lavage after overnight fast, the doses were 10mg/kg, and the volumes were 0.1ml/10g.

3. Operation of

Mice were given by gavage, 0.1ml of blood was collected before and after administration for 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, and 24.0 hours, and placed in heparinized tubes, centrifuged at 3500 rpm for 10 minutes, and plasma was isolated and stored at-20 ℃.

Determination of the content of test compounds in the plasma of mice following administration of different concentrations of drug by injection: 25. Mu.L of plasma from mice at each time after administration was added with 50. Mu.L (100 ng/mL) of camptothecin (China biological product assay), 200. Mu.L of acetonitrile, vortexed for 5 minutes, centrifuged for 10 minutes (4000 rpm), and 4. Mu.L of supernatant was obtained from the plasma sample for LC/MS/MS analysis.

4. Pharmacokinetic parameter results

TABLE 3 Table 3

In addition, ER=24 in the caco-2 permeability experiment shows that the brain permeability of the compound is very low, and the compound has very good safety.

Claims

1. A compound represented by the formula II-a or a pharmaceutically acceptable salt thereof,

wherein R is ¹ Selected from hydrogen, R ² Selected from fluorine; or R is ¹ Selected from fluorine, R ² Selected from hydrogen;

R ³ selected from hydrogen;

selected from-> Wherein ring A is optionally substituted with 1 to 3R ^A4 Ring B is optionally substituted with 1 to 3R ^A5 Substituted, R ^A4 、R ^A5 Each independently selected from halogen, deuterium, hydroxy, C _1-6 Alkyl or C _1-6 An alkoxy group.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ¹ Selected from F, R ² Selected from hydrogen.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula II-a is selected from

Wherein->Including the E or Z configuration.

4. A compound according to claim 3, or a pharmaceutically acceptable salt thereof, selected from

5. A pharmaceutical composition comprising at least one therapeutically effective amount of a compound of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

6. Use of a compound according to any one of claims 1-4 or a pharmaceutical composition according to claim 5 for the manufacture of a medicament for the prevention and/or treatment of a disorder associated with SSAO or SSAO/VAP-1.