CN114085220B

CN114085220B - Substituted morpholine-4-carboxylic acid ester derivatives, compositions and pharmaceutical uses thereof

Info

Publication number: CN114085220B
Application number: CN202110208011.9A
Authority: CN
Inventors: 胡斌; 谢婧; 杨文�; 吴春艳; 张凯; 石晓永; 朱经峰; 关慧平
Original assignee: Yangtze River Pharmaceutical Group Co Ltd; Shanghai Haiyan Pharmaceutical Technology Co Ltd
Current assignee: Yangtze River Pharmaceutical Group Co Ltd; Shanghai Haiyan Pharmaceutical Technology Co Ltd
Priority date: 2020-06-22
Filing date: 2021-02-25
Publication date: 2023-06-16
Anticipated expiration: 2041-02-25
Also published as: CN114085220A

Abstract

The invention discloses a substituted morpholine-4-carboxylic ester derivative, the structure of which is shown as the formula (I-d). In addition, the invention also discloses pharmaceutically acceptable salts, solvates, stereoisomers, prodrugs and pharmaceutical compositions of the derivatives and application of the derivatives in medicines. The compound has remarkable P2X3 inhibition activity and selectivity and has practical value.

Description

Substituted morpholine-4-carboxylic acid ester derivatives, compositions and pharmaceutical uses thereof

The present application claims priority from chinese patent office, application No. 2020105735461, entitled "substituted morpholine-4-carboxylic acid ester derivatives, compositions thereof, and pharmaceutical uses," filed 22 months 2020, the entire contents of which are incorporated herein by reference.

Technical Field

The invention relates to the technical field of medicines, in particular to a substituted morpholine-4-carboxylic ester derivative, pharmaceutically acceptable salts, solvates, stereoisomers and prodrugs thereof, and a pharmaceutical composition and a medical application thereof.

Background

P2X purinergic receptors are a family of ion channels activated by extracellular Adenosine Triphosphate (ATP). Purine receptors are involved in a variety of biological functions, particularly in relation to pain sensitivity. The P2X3 receptor is one of the members of this family, originally cloned from the rat dorsal root ganglion. Chen et al Nature, volume 377, pages 428-431 (1995). The nucleotide and amino acid sequences of both rat P2X3 and human P2X3 are now known. Lewis et al, nature, vol 377, pp 432-435 (1995); and Garcia-Guzman et al, brainRes. Mol. BrainRes., vol.47, pp.59-66 (1997).

P2X3 is reported to be involved in controlling the afferent pathways of bladder volume reflex. Thus, inhibition of P2X3 can treat disorders associated with urine storage and urination, such as overactive bladder. Cockayne et al, nature, volume 407, pages 1011-1015 (2000).

P2X3 is also selectively expressed on nociceptive, small diameter sensory neurons (i.e., neurons stimulated by pain or injury), consistent with its role in pain sensitivity. In addition, blocking P2X3 receptors has also been reported to have analgesic effects in animal models of chronic inflammatory and neuropathic pain. Jarvis et al, PNAS,99,17179-17184 (2002). Thus, methods of reducing P2X3 levels or activity would be useful in modulating pain perception in a subject with pain.

P2X3 is also capable of forming a P2X2/3 heterodimer with P2X2, the P2X2 being another member of the P2X purinergic ligand-gated ion channel family. P2X2/3 is highly expressed on the peripheral (central and peripheral) of sensory neurons. Chen et al Nature, volume 377, pages 428-431 (1995). Recent studies also indicate that P2X2/3 is expressed predominantly in bladder sensory neurons (over P2X 3) and is likely to play a role in bladder filling and nociceptive sensation. Zhong et al, neuroscience, volume 120, pages 667-675 (2003).

In view of the foregoing, there is a need for new P2X3 and/or P2X2/3 receptor ligands, particularly antagonists, that may be useful and safe for treating various conditions associated with P2X3 and/or P2X 2/3.

Disclosure of Invention

The invention aims to provide a substituted morpholine-4-carboxylate derivative with higher activity and better selectivity. In a first aspect, the present invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof:

in the method, in the process of the invention,

ring a is a fused 5-to 6-membered heteroaryl ring or a fused benzene ring;

X ₁ 、X ₂ each represents a ring atom, independently C or N;

X ₃ 、X ₄ 、X ₅ each represents a ring atom, independently C, N, O or S;

X ₁ 、X ₂ 、X ₃ 、X ₄ 、X ₅ not both are N;

X ₃ 、X ₄ 、X ₅ not both O and S;

(R ₁ ) _n1 represents hydrogen on ring A by n1 Rs ₁ Substituted, n1 is 0, 1, 2, 3 or 4, each R ₁ The same or different, each independently is hydrogen, cyano, acetyl, hydroxy, carboxyl, halogen (preferably fluorine or chlorine), NR _a0 R _b0 、C _1-8 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-3 Alkyl group, C _1-8 Alkoxy (preferably C) _1-6 Alkoxy, more preferably C _1-3 Alkoxy group), C _3-8 Cycloalkyl (preferably C) _3-6 Cycloalkyl), 3-to 6-membered heterocycloalkyl, 5-to 6-membered heteroaryl or C _6-10 An aryl group; wherein said C _1-8 Alkyl, C _1-8 Alkoxy, C _3-8 Cycloalkyl, 3-to 6-membered heterocycloalkyl, 5-to 6-membered heteroaryl and C _6-10 Aryl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: deuterium, halogen, cyano, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, C _2-4 Alkenyl, C _2-4 Alkynyl, halo C _1-3 Alkyl, halogenated C _1-3 Alkoxy, -SO ₂ C _1-3 Alkyl, -S (O) C _1-3 Alkyl, -C (O) NR _a0 R _b0 、-C(O)OC _1-3 Alkyl, -OC (O) C _1-3 Alkyl, C _3-6 Cycloalkyl, C _3-6 Cycloalkyloxy, 3-to 6-membered heterocycloalkyl, phenyl, 5-to 6-membered heteroaryl;

(R ₂ ) _n2 represents hydrogen on five-membered ring by n 2R ₂ Substituted, n2 is 0, 1, 2 or 3, each R ₂ The same or different, each independently is hydrogen, cyano, acetyl, hydroxy, carboxyl, halogen (preferably fluorine or chlorine), NR _a0 R _b0 、C _1-8 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-3 Alkyl group, C _1-8 Alkoxy (preferably C) _1-6 Alkoxy, more preferably C _1-3 Alkoxy group), C _3-8 Cycloalkyl (preferably C) _3-6 Cycloalkyl), 3-to 6-membered heterocycloalkyl, 5-to 6-membered heteroaryl or C _6-10 An aryl group; wherein said C _1-8 Alkyl, C _1-8 Alkoxy, C _3-8 Cycloalkyl, 3-to 6-membered heterocycloalkyl, 5-to 6-membered heteroaryl and C _6-10 Aryl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: deuterium, halogen, cyano, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, C _2-4 Alkenyl, C _2-4 Alkynyl, halo C _1-3 Alkyl, halogenated C _1-3 Alkoxy, C _3-6 Cycloalkyl, C _3-6 Cycloalkyloxy, 3-to 6-membered heterocycloalkyl, phenyl, 5-to 6-membered heteroaryl;

ring B is a benzene ring, a 5-to 6-membered heteroaryl ring, or an 8-to 10-membered heteroaryl ring;

(R ₃ ) _m represents hydrogen on ring B by m R ₃ Substituted, m is 0, 1, 2, 3 or 4, each R ₃ The same or different, each independently is hydrogen, cyano, acetyl, hydroxy, carboxyl, halogen (preferably fluorine or chlorine), NR _a0 R _b0 、C _1-8 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-3 Alkyl group, C _1-8 Alkoxy (preferably C) _1-6 Alkoxy, more preferably C _1-3 Alkoxy group), C _3-8 Cycloalkyl (preferably C) _3-6 Cycloalkyl), 3-to 6-membered heterocycloalkyl, 5-to 6-membered heteroaryl or C _6-10 An aryl group; wherein said C _1-8 Alkyl, C _1-8 Alkoxy, C _3-8 Cycloalkyl, 3-to 6-membered heterocycloalkyl, 5-to 6-membered heteroaryl and C _6-10 Aryl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: deuterium, halogen, cyano, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, C _2-4 Alkenyl, C _2-4 Alkynyl, halo C _1-3 Alkyl, halogenated C _1-3 Alkoxy, C _3-6 Cycloalkyl, C _3-6 Cycloalkyloxy, 3-to 6-membered heterocycloalkyl, phenyl, 5-to 6-membered heteroaryl;

ring C is a 5 to 6 membered heteroaryl ring, an 8 to 10 membered heteroaryl ring, a 5 to 8 membered heterocycloalkenyl ring, or a 4 to 6 membered heterocycloalkyl ring;

(R ₄ ) _u For hydrogen on ring C by u R ₄ Substituted, u is 0, 1, 2, 3 or 4, each R ₄ The same or different, each independently is hydrogen, cyano, acetyl, hydroxy, carboxyl, halogen (preferably fluorine or chlorine), NR _a0 R _b0 、C _1-8 Alkyl (preferably C) _1-6 Alkyl, more preferably C _1-3 Alkyl group, C _1-8 Alkoxy (preferably C) _1-6 Alkoxy, more preferably C _1-3 Alkoxy group), C _3-8 Cycloalkyl (preferably C) _3-6 Cycloalkyl), 3 to 6 memberedHeterocycloalkyl, 5-to 6-membered heteroaryl or C _6-10 An aryl group; wherein said C _1-8 Alkyl, C _1-8 Alkoxy, C _3-8 Cycloalkyl, 3-to 6-membered heterocycloalkyl, 5-to 6-membered heteroaryl and C _6-10 Aryl is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: deuterium, halogen, cyano, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, C _2-4 Alkenyl, C _2-4 Alkynyl, halo C _1-3 Alkyl, halogenated C _1-3 Alkoxy, C _3-6 Cycloalkyl, C _3-6 Cycloalkyloxy, 3-to 6-membered heterocycloalkyl, phenyl, 5-to 6-membered heteroaryl;

R _a0 、R _b0 each independently is hydrogen or C _1-3 An alkyl group; or R is _a0 、R _b0 Together with the attached nitrogen atom, form a 4-to 6-membered saturated mono-heterocyclic ring; the 4-to 6-membered saturated mono-heterocycle is optionally substituted with 1, 2 or 3 substituents each independently selected from the group consisting of: deuterium, halogen, cyano, hydroxy, carboxyl, C _1-3 Alkyl, C _1-3 Alkoxy, C _2-4 Alkenyl, C _2-4 Alkynyl, halo C _1-3 Alkyl, halogenated C _1-3 Alkoxy, -SO ₂ C _1-3 Alkyl, -S (O) C _1-3 Alkyl, -C (O) NR _a0 R _b0 、-C(O)OC _1-3 Alkyl, -OC (O) C _1-3 Alkyl, C _3-6 Cycloalkyl, C _3-6 Cycloalkyloxy, 3-to 6-membered heterocycloalkyl;

R ₀ is hydrogen or C _1-6 An alkyl group.

In some embodiments, the compound of formula (I) is of the structure of formula (I-a):

wherein ring A, ring B, ring C, X ₁ 、X ₂ 、X ₃ 、X ₄ 、X ₅ 、R ₁ 、R ₂ 、R ₃ 、R ₄ 、n1、n2、m、u、R ₀ As defined above.

In some embodiments, the compound of formula (I) is of the structure of formula (I-b):

In some embodiments of the present invention, in some embodiments, ring B is a benzene ring, a thiophene ring, a furan ring, a thiazole ring, an isothiazole ring, an imidazole ring, an oxazole ring, a pyrrole ring, a pyrazole ring, a triazole ring, a 1,2, 3-triazole ring, a 1,2, 4-triazole ring, a 1,2, 5-triazole ring, a 1,3, 4-triazole ring, a tetrazole ring, an isoxazole ring, an oxadiazole ring, a 1,2, 3-oxadiazole ring, a 1,2, 4-oxadiazole ring, a 1,2, 5-oxadiazole ring, a 1,3, 4-oxadiazole ring, a thiadiazole ring, a pyridine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, a triazine ring, a tetrazine ring, an indole ring, an isoindole ring, an indazole ring, a benzotriazole ring, a benzothiophene ring an isobenzothiophene ring, a benzofuran ring, a benzisofuran ring, a benzimidazole ring, a benzoxazole ring, a benzisoxazole ring, a benzoxadiazole ring, a benzothiazole ring, a benzisothiazole ring, a benzothiadiazole ring, an indenazine ring, a purine ring, a pyrido [3,2-d ] pyrimidine ring, a pyrido [2,3-d ] pyrimidine ring, a pyrido [3,4-d ] pyrimidine ring, a pyrido [4,3-d ] pyrimidine ring, a 1, 8-naphthyridine ring, a 1, 7-naphthyridine ring, a 1, 6-naphthyridine ring, a 1, 5-naphthyridine ring, a pteridine ring, a quinoline ring, an isoquinoline ring, a cinnoline ring, a quinoxaline ring, a phthalazine ring, or a quinazoline ring.

In some embodiments, the compound of formula (I) is of the structure of formula (I-c):

wherein ring A, ring C, X ₁ 、X ₂ 、X ₃ 、X ₄ 、X ₅ 、R ₁ 、R ₂ 、R ₃ 、R ₄ 、n1、n2、m、u、R ₀ As defined above.

In some embodiments, ring a is a fused 5-to 6-membered heteroaryl ring or a fused benzene ring; wherein the fused 5-to 6-membered heteroaryl ring is selected from the group consisting of a thiophene ring, a furan ring, a thiazole ring, an isothiazole ring, an imidazole ring, an oxazole ring, a pyrrole ring, a pyrazole ring, a triazole ring, a 1,2, 3-triazole ring, a 1,2, 4-triazole ring, a 1,2, 5-triazole ring, a 1,3, 4-triazole ring, a tetrazole ring, an isoxazole ring, an oxadiazole ring, a 1,2, 3-oxadiazole ring, a 1,2, 4-oxadiazole ring, a 1,2, 5-oxadiazole ring, a 1,3, 4-oxadiazole ring, a thiadiazole ring, a pyridine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, a triazine ring, and a tetrazine ring.

In some embodiments of the present invention, in some embodiments,

the structure is selected from thiophene ring, furan ring, thiazole ring, isothiazole ring, imidazole ring, oxazole ring, pyrrole ring, pyrazole ring, triazole ring, 1,2, 3-triazole ring, 1,2, 4-triazole ring, 1,2, 5-triazole ring, 1,3, 4-triazole ring, tetrazole ring, isoxazole ring, oxadiazole ring, 1,2, 3-oxadiazole ring, 1,2, 4-oxadiazole ring, 1,2, 5-oxadiazole ring, 1,3, 4-oxadiazole ring, thiadiazole ring, pyridine ring, pyridazine ring, pyrimidine ring, pyrazine ring, triazine ring and tetrazine ring.

In some embodiments, X ₃ 、X ₅ Is selected from any one of the following:

(a)X ₃ is C; x is X ₅ Is C; (b) X is X ₃ Is C; x is X ₅ Is N; (c) X is X ₃ Is N; x is X ₅ Is C.

In some embodiments of the present invention, in some embodiments,

the structure is selected from:

in some embodiments, the compound of formula (I) is of the structure of formula (I-d):

wherein ring C, R ₁ 、R ₃ 、R ₄ 、n1、m、u、R ₀ As defined above.

In some embodiments, R ₀ Is methyl.

In some embodiments of the present invention, in some embodiments, ring C is selected from thiophene ring, furan ring, thiazole ring, isothiazole ring, imidazole ring, oxazole ring, pyrrole ring, pyrazole ring, triazole ring, 1,2, 3-triazole ring, 1,2, 4-triazole ring, 1,2, 5-triazole ring, 1,3, 4-triazole ring, tetrazole ring, isoxazole ring, oxadiazole ring, 1,2, 3-oxadiazole ring, 1,2, 4-oxadiazole ring, 1,2, 5-oxadiazole ring, 1,3, 4-oxadiazole ring, and thiadiazole ring, pyridine ring, pyridazine ring, pyrimidine ring, pyrazine ring, triazine ring, tetrazine ring, indole ring, isoindole ring, indazole ring, benzotriazole ring, benzothiophene ring, isobenzothiophene ring, benzofuran ring, benzisothiazole ring, benzimidazole ring, benzoxazole ring, benzisoxazole ring, benzoxadiazole ring, benzoxazole ring, and benzoxazole ring benzothiazole ring, benzisothiazole ring, benzothiadiazole ring, indenazine ring, purine ring, pyrido [3,2-d ] pyrimidine ring, pyrido [2,3-d ] pyrimidine ring, pyrido [3,4-d ] pyrimidine ring, pyrido [4,3-d ] pyrimidine ring, 1, 8-naphthyridine ring, 1, 7-naphthyridine ring, 1, 6-naphthyridine ring, 1, 5-naphthyridine ring, pteridine ring, quinoline ring, isoquinoline ring, cinnoline ring, quinoxaline ring, phthalazine ring, quinazoline ring, 4, 5-dihydro-1H-imidazole ring, 1,4,5, 6-tetrahydropyrimidine ring, 3,4,7, 8-tetrahydro-2H-1, 4, 6-oxadiazolazine ring, 1, 6-dihydropyrimidine ring, 4,5,6, 7-tetrahydro-1H-1, 3-diaza Zhuo Huan, 2,5,6, 7-tetrahydro-1, 3-dioxa-3, 37-dione (4, 3H) -2, 3-dioxa-37, 4-oxa-one (4H) -3H-dioxa-3-one, 3, 5-dihydro-4H-imidazol-4-one, 2, 4-dihydro-3H-1, 2, 4-triazol-3-one, 1,3, 4-thiadiazol-2 (3H) -one, thiazolidine-2, 4-dione, imidazolidine-2, 4-dione.

In some embodiments, ring C is of the structure shown in formula (a);

wherein Z is-N=, - (NR) _1a )-、-O-、-(CR _1b )＝、-S-、-(CR _1b R _1c )-；

R _1a Is hydrogen or C _1-3 An alkyl group; r is R _1b 、R _1c Each independently is hydrogen, halogen, cyano, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, halo C _1-3 Alkyl or halo C _1-3 An alkoxy group;

is a single bond or a double bond;

R _2a 、R _3a is linked to form, together with the attached Z and nitrogen atom, a 5-to 8-membered heterocycloalkenyl ring, a 5-to 6-membered heteroaryl ring, or an 8-to 10-membered heteroaryl ring; wherein the 5-to 8-membered heterocycloalkenyl ring has 2, 3 or 4 nitrogen atoms and 0, 1 or 2 oxygen atoms as ring atoms; the 5-to 6-membered heteroaryl ring has 2, 3 or 4 nitrogen atoms and 0 or 1 oxygen atom as ring atoms; the 5-to 8-membered heterocycloalkenyl ring, 5-to 6-membered heteroaryl ring, and 8-to 10-membered heteroaryl ring are unsubstituted or substituted with 1,2, or 3 substituents each independently selected from the group consisting of: halogen, cyano, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, halo C _1-3 Alkyl, halogenated C _1-3 An alkoxy group.

In some embodiments, R _2a 、R _3a The 5-to 8-membered heterocycloalkenyl ring formed together with the attached Z and nitrogen atom is selected from the group consisting of 4, 5-dihydro-1H-imidazole ring, 1,4,5, 6-tetrahydropyrimidine ring, 3,4,7, 8-tetrahydro-2H-1, 4, 6-oxadiazolazine ring, 1, 6-dihydropyrimidine ring, 4,5,6, 7-tetrahydro-1H-1, 3-diaza Zhuo Huan, 2,5,6, 7-tetrahydro-1, 3, 5-oxadiaza Zhuo Huan, 1,2, 4-oxadiazol-5 (4H) -one, pyrimidin-4 (3H) -one, 3, 5-dihydro-4H-imidazol-4-one, 2, 4-dihydro-3H-1, 2, 4-triazole-3-one, 1,3, 4-thiadiazol-2 (3H) -one.

In some embodiments, R _2a 、R _3a Is linked to form, together with the Z and nitrogen atom to which it is attached, a 5-to 8-membered heterocycloalkenyl ring selected from

In some embodiments, R _2a 、R _3a And a 5-to 6-membered heteroaryl ring formed together with the attached Z and nitrogen atom is selected from the group consisting of a thiophene ring, a furan ring, a thiazole ring, an isothiazole ring, an imidazole ring, an oxazole ring, a pyrrole ring, a pyrazole ring, a triazole ring, a 1,2, 3-triazole ring, a 1,2, 4-triazole ring, a 1,2, 5-triazole ring, a 1,3, 4-triazole ring, a tetrazole ring, an isoxazole ring, an oxadiazole ring, a 1,2, 3-oxadiazole ring, a 1,2, 4-oxadiazole ring, a 1,2, 5-oxadiazole ring, a 1,3, 4-oxadiazole ring, a thiadiazole ring, a pyridine ring, a pyridazine ring, a pyrimidine ring, a pyrazine ring, a triazine ring, and a tetrazine ring.

In some embodiments, R _2a 、R _3a Is linked to form a 5-to 6-membered heteroaryl ring selected from the group consisting of

In one embodiment, R _2a 、R _3a Is linked to form a 5-to 6-membered heteroaryl ring selected from the group consisting of

And->

Substituted with 1,2 or 3 substituents each independently selected from the group consisting of: halogen, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, halo C _1-3 Alkyl, halogenated C _1-3 An alkoxy group.

In one embodiment of the present invention, in one embodiment,

is->

In some embodiments, R _2a 、R _3a Is linked to form an 8-to 10-membered heteroaryl ring together with the linked Z and nitrogen atom selected from the group consisting of an indole ring, an isoindole ring, an indazole ring, a benzotriazole ring, a benzothiophene ring, an isobenzothiophene ring, a benzofuran ring, a benzisotofuran ring, a benzimidazole ring, a benzoxazole ring, a benzisoxazole ring, a benzoxadiazole ring, a benzothiazole ring, a benzisothiazole ring, an indenazine ring, a purine ring, a pyrido [3,2-d ]]Pyrimidine ring, pyrido [2,3-d ]]Pyrimidine ring, pyrido [3,4-d ]]Pyrimidine ring, pyrido [4,3-d ]]Pyrimidine ring, 1, 8-naphthyridine ring, 1, 7-naphthyridine ring, 1, 6-naphthyridine ring, 1, 5-naphthyridine ring, pteridine ring, quinoline ring, isoquinoline ring, cinnoline ring, quinoxaline ring, phthalazine ring, and quinazoline ring.

In some embodiments, R _2a 、R _3a Is linked to form, together with the Z and nitrogen atom to which it is attached, an 8-to 10-membered heteroaryl ring

In some embodiments, the 5-to 8-membered heterocycloalkenyl ring is of the structure shown in formula (b) or formula (c);

wherein W is ₁ 、W ₂ Independently is- (NR) _a ) -, -O-, -S-or- (CR) _b R _c )-；

L ₁ 、L ₂ independently-C (O) -or- (CR) _d R _e )-；

Y is- (NR) _f )-、-(CR _g R _h ) -, -S-or-O-;

is a single bond or a double bond;

R _a 、R _f each independently is hydrogen or C _1-3 An alkyl group;

R _b 、R _c 、R _d 、R _e 、R _g 、R _h each independently is hydrogen, halogen, cyano, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, halo C _1-3 Alkyl or halo C _1-3 An alkoxy group.

In some embodiments, the structure of formula (a) is selected from the group consisting of:

the above structure is unsubstituted or substituted with 1, 2 or 3 substituents each independently selected from the group consisting of: halogen, cyano, hydroxy, C _1-3 Alkyl, C _1-3 Alkoxy, halo C _1-3 Alkyl, halogenated C _1-3 An alkoxy group.

In some embodiments, ring C is selected from the following structures:

in some embodiments, the 4-to 6-membered heterocycloalkyl is selected from the group consisting of azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyrrolyl, oxazolidinyl, dioxolanyl, piperidinyl, piperazinyl, morpholinyl, dioxane, thiomorpholinyl, thiomorpholin-1, 1-dioxide, tetrahydropyranyl, pyrrolidin-2-one, dihydrofuran-2 (3H) -one, morpholin-3-one, piperazin-2-one, piperidin-2-one, thiazolidine-2, 4-dione, imidazolidine-2, 4-dione.

In some embodiments, the compound of formula (I) is any one of the following compounds:

in some embodiments, the compound of formula (I) is any one of the exemplified compounds.

The second aspect of the present invention provides a pharmaceutical composition comprising a compound according to the first aspect of the present invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof, and a pharmaceutically acceptable carrier.

In a third aspect, the invention provides the use of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof, in the manufacture of a medicament for the treatment of a disease associated with P2X3 activity (in particular overactivity) or with P2X2/3 activity (in particular overactivity).

In a fourth aspect, the invention provides the use of a pharmaceutical composition according to the second aspect of the invention for the manufacture of a medicament for the treatment of a disease associated with P2X3 activity (in particular overactivity) or with P2X2/3 activity (in particular overactivity).

In a fifth aspect, the present invention provides a method of treating a disease associated with P2X3 activity (in particular overactivity) or with P2X2/3 activity (in particular overactivity) comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof, or administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition according to the second aspect of the invention.

In some embodiments, the disease associated with P2X3 activity (particularly overactivity) or P2X2/3 activity (particularly overactivity) is pain, such as chronic pain, neuropathic pain, acute pain, back pain, cancer pain, pain caused by rheumatoid arthritis, migraine, visceral pain, and the like.

In some embodiments, the disease associated with P2X3 activity (particularly overactivity) or P2X2/3 activity (particularly overactivity) is a urinary tract disorder, such as overactive bladder (also known as urinary incontinence), pelvic hypersensitivity, urethritis, and the like.

In some embodiments, the disease associated with P2X3 activity (particularly overactivity) or with P2X2/3 activity (particularly overactivity) is a gastrointestinal disease, such as constipation and a functional gastrointestinal disorder, such as irritable bowel syndrome or functional dyspepsia, and the like.

In some embodiments, the disease associated with P2X3 activity (particularly overactive) or with P2X2/3 activity (particularly overactive) is a cancer, such as prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, endometrial cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, melanoma, basal cancer, mesothelial lining cancer, white blood cell cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, small cell lung cancer, non-small cell lung cancer, adrenal cancer, thyroid cancer, renal cancer, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma, etc.

In some embodiments, the disease associated with P2X3 activity (particularly overactive) or P2X2/3 activity (particularly overactive) is an immune-related disease such as rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergies, fibrosis, anaemia fibromyalgia, alzheimer's disease, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, parkinson's disease, infections, crohn's disease, ulcerative colitis, allergic contact dermatitis and other eczema, systemic sclerosis, and multiple sclerosis, and the like.

In some embodiments, the disease associated with P2X3 activity (particularly overactivity) or P2X2/3 activity (particularly overactivity) is cough, depression, anxiety, stress-related disorders, such as post-traumatic stress disorder, panic disorder, social phobia, or obsessive compulsive disorder, and the like.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Detailed Description

The present inventors have conducted extensive and intensive studies and have unexpectedly found that such substituted morpholine-4-carboxylic acid ester derivatives have a remarkable P2X3 inhibitory activity and a low P2X2/3 inhibitory activity. The series of compounds is therefore expected to be developed as medicaments for modulating P2X3 and/or P2X2/3 to treat various diseases mediated by (or otherwise associated with) P2X3 and/or P2X 2/3. On this basis, the inventors completed the present invention.

Definition of terms

In order that the technical content of the present invention can be more clearly understood, the following terms of the present invention will be further described.

"alkyl" refers to straight and branched chain saturated aliphatic hydrocarbon groups. "C _1-8 Alkyl "means an alkyl group having 1 to 8 carbon atoms, preferably C _1-6 Alkyl, more preferably C _1-3 An alkyl group; non-limiting examples of alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl A radical, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof.

"alkenyl" refers to a straight or branched unsaturated aliphatic hydrocarbon radical having one or more carbon-carbon double bonds (c=c), "C _2-8 Alkenyl "means alkenyl having 2 to 8 carbon atoms, preferably C _2-6 Alkenyl groups, more preferably C _2-4 Alkenyl groups, defined similarly; non-limiting examples include ethenyl, propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, hexenyl, and the like.

"alkynyl" refers to straight and branched chain unsaturated aliphatic hydrocarbon groups having one or more carbon-carbon triple bonds, "C _2-8 Alkynyl "means alkynyl having 2 to 8 carbon atoms, preferably C _2-6 Alkynyl, more preferably C _2-4 Alkynyl, defined similarly; non-limiting examples include ethynyl, propynyl, n-butynyl, isobutynyl, pentynyl, hexynyl, and the like.

"cycloalkyl" and "cycloalkyl ring" are used interchangeably and refer to a saturated monocyclic, bicyclic, or polycyclic cyclic hydrocarbon group, which may be fused to an aryl or heteroaryl group. The cycloalkyl ring may be optionally substituted. In certain embodiments, the cycloalkyl ring contains one or more carbonyl groups, such as oxo groups. "C _3-8 Cycloalkyl "refers to a monocyclic cycloalkyl group having 3 to 8 carbon atoms, non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexylCycloheptyl, cyclooctyl, cyclobutanone, cyclopentanone, cyclopentane-1, 3-dione, and the like. Preferably C _3-6 Cycloalkyl including cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. "C _8-10 Cycloalkyl "means a fused bicyclic cyclic hydrocarbon group having 8 to 10 ring atoms, C _8-10 Non-limiting examples of cycloalkyl groups include

"cycloalkenyl" and "cycloalkenyl ring" are used interchangeably and refer to a monocyclic, bicyclic, or polycyclic, cyclic hydrocarbon group containing one or more carbon-carbon double bonds in the ring, which may be fused to an aryl or heteroaryl group. The cycloalkenyl ring may be optionally substituted. In certain embodiments, cycloalkenyl rings contain one or more carbonyl groups, such as oxo groups. "C _3-8 Cycloalkenyl "refers to a monocyclic cycloalkenyl group having 3 to 8 carbon atoms. Preferably C _3-6 A cycloalkenyl group. Non-limiting examples of cycloalkenyl groups include cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, cyclopentyl-2-en-1-one, cyclohexyl-2, 5-dien-1-one, cyclohexyl-2-en-1-one, cyclohex-2-en-1, 4-dione, and the like.

"heterocycloalkenyl" and "heterocycloalkenyl ring" are used interchangeably to refer to a heterocycloalkyl group containing one or more carbon-carbon double bonds or carbon-nitrogen double bonds within the ring, but are not intended to include heteroaryl moieties as defined herein. The group may be fused to an aryl or heteroaryl group. The heterocyclenyl ring may be optionally substituted. In certain embodiments, the heterocycloalkenyl ring contains one or more carbonyl or thiocarbonyl groups, e.g., groups comprising oxo and thio. "5-to 8-membered heterocycloalkenyl ring" means a heterocycloalkenyl ring having 5 to 8 ring atoms in which 1,2 or 3 ring atoms are heteroatoms selected from nitrogen, oxygen and sulfur. Preferably a 5 to 6 membered heterocycloalkenyl ring. Non-limiting examples of heterocycloalkenyl rings include 4, 5-dihydro-1H-imidazole ring, 1,4,5, 6-tetrahydropyrimidine ring, 3,4,7, 8-tetrahydro-2H-1, 4, 6-oxadiazolazine ring, 1, 6-dihydropyrimidine ring, 4,5,6, 7-tetrahydro-1H-1, 3-diaza Zhuo Huan, 2,5,6, 7-tetrahydro-1, 3, 5-oxadiazol-Zhuo Huan, 1,2, 4-oxadiazol-5 (4H) -one, pyrimidine-4 (3H) -one, 3, 5-dihydro-4H-imidazol-4-one, 2, 4-dihydro-3H-1, 2, 4-triazol-3-one, 1,3, 4-thiadiazol-2 (3H) -one.

"aryl" and "aromatic ring" are used interchangeably and refer to an all-carbon monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) group having a conjugated pi-electron system, which may be fused to a cycloalkyl ring, heterocycloalkyl ring, cycloalkenyl ring, heterocycloalkenyl ring, or heteroaryl group. "C _6-10 Aryl "refers to a monocyclic or bicyclic aryl group of 6 to 10 carbon atoms, non-limiting examples of aryl groups include phenyl, naphthyl, and the like.

"heteroaryl" and "heteroaryl ring" are used interchangeably and refer to a group of a monocyclic, bicyclic, or polycyclic 4n+2 aromatic ring system (e.g., having 6 or 10 pi electrons shared in a cyclic arrangement) having ring carbon atoms and ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. Heteroaryl groups in the present invention also include ring systems in which the heteroaryl ring described above is fused to one or more cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, or aromatic rings. Heteroaryl rings may be optionally substituted. "5-to 10-membered heteroaryl" refers to a monocyclic or bicyclic heteroaryl group having 5 to 10 ring atoms, wherein 1,2,3 or 4 ring atoms are heteroatoms. "5-to 6-membered heteroaryl" refers to a monocyclic heteroaryl group having 5 to 6 ring atoms, wherein 1,2,3 or 4 ring atoms are heteroatoms, non-limiting examples include thienyl, furyl, thiazolyl, isothiazolyl, imidazolyl, oxazolyl, pyrrolyl, pyrazolyl, triazolyl, 1,2, 3-triazolyl, 1,2, 4-triazolyl, 1,2, 5-triazolyl, 1,3, 4-triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, tetrazinyl. "8-to 10-membered heteroaryl" refers to a bicyclic heteroaryl having 8 to 10 ring atoms, wherein 1,2,3 or 4 ring atoms are heteroatoms, formed by the fusion of a 5-to 6-membered heteroaryl with a phenyl or by the fusion of a 5-to 6-membered heteroaryl with a 5-to 6-membered heteroaryl. Non-limiting examples include indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisotofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, thiophenofuranyl, thiophenopyrimidinyl, furanthiadiazolyl, furanopyrrolyl, imidazopyridine, imidazothiazole, oxazoloimidazolyl, oxazolopyridinyl, pyrrolopyridinyl, pyrrolopyrazoles, pyrazolopyridine, indenazinyl, purinyl, pyrido [3,2-d ] pyrimidinyl, pyrido [2,3-d ] pyrimidinyl, pyrido [3,4-d ] pyrimidinyl, pyrido [4,3-d ] pyrimidinyl, 1, 8-naphthyridinyl, 1, 7-naphthyridinyl, 1, 6-naphthyridinyl, 1, 5-naphthyridinyl, pteridinyl, isoquinolinyl, quinazolinyl, 1, 2-phthalazinyl, 1, 2-b, 1-quinoxalinyl, 1, 2-phthalazinyl, 1-b, 1-naphthyridinyl. "heteroatom" means nitrogen, oxygen or sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as the valency permits. The heteroaryl bicyclic ring system may include one or more heteroatoms in one or both rings.

"fused" refers to a structure in which two or more rings share one or more bonds.

"Phenylacetycloalkyl" refers to groups in which the benzene ring is fused to a heterocycloalkyl ring to form a bicyclic, tricyclic, or polycyclic ring system, wherein the heterocycloalkyl ring is as defined above. "7 to 11 membered phenylaminoheterocycloalkyl" means a bicyclic cyclic group having 7 to 11 ring atoms wherein 1,2,3 or 4 ring atoms are heteroatoms selected from nitrogen, oxygen and sulfur. Preferably an 8 to 10 membered phenylao-heterocycloalkyl group having 8 to 10 ring atoms, wherein 1,2 or 3 ring atoms are heteroatoms selected from nitrogen, oxygen and sulfur. Non-limiting examples include indoline, benzo [ d ] [1,3] dioxazole, 1,2,3, 4-tetrahydroisoquinoline, 3, 4-dihydro-2H-benzo [ b ] [1,4] oxazine, and the like.

"heteroaryl-heterocycloalkyl" refers to a group in which a heteroaryl ring is fused to a heterocycloalkyl ring to form a bicyclic, tricyclic, or polycyclic ring system, wherein the heterocycloalkyl ring is as defined above. "7 to 11 membered heteroarylheterocycloalkyl" refers to a bicyclic ring group having 7 to 11 ring atoms, where 1,2,3, or 4 ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur. Preferably an 8 to 10 membered heteroarylheterocycloalkylgroup having 8 to 10 ring atoms, wherein 1,2,3 or 4 ring atoms are heteroatoms selected from nitrogen, oxygen and sulfur. Non-limiting examples include 2, 3-dihydro-1H-pyrrolo [2,3-b ] pyridine, [1,3] dioxolo [4,5-b ] pyridine, 2, 3-dihydro-1H-pyrido [3,4-b ] [1,4] oxazine, 2,3,4, 6-tetrahydropyrrolo [3,4-b ] [1,4] oxazine, 2,4,5, 6-tetrahydropyrano [2,3-c ] pyrazole, 5,6,7, 8-tetrahydropyrido [3,4-d ] pyrimidine, and the like.

"alkoxy" refers to an-O-alkyl group, wherein alkyl is as defined above. Preferably C _1-8 Alkoxy, more preferably C _1-6 Alkoxy, most preferably C _1-3 An alkoxy group. Non-limiting examples include methoxy, ethoxy, n-propoxy, isopropoxy, butoxy, t-butoxy, isobutoxy, pentoxy, and the like.

"Cycloalkyloxy" refers to-O-cycloalkyl, wherein cycloalkyl is as defined above. Preferably C _3-8 Cycloalkyloxy, more preferably C _3-6 Cycloalkyl oxy. Non-limiting examples include cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.

"a bond" means that two groups joined by it are joined by a covalent bond.

"halogen" means fluorine, chlorine, bromine or iodine.

"halo" means that one or more (e.g., 1,2, 3, 4, or 5) hydrogens in the group are replaced with a halogen.

For example, "halo C _1-8 Alkyl "means that the alkyl is substituted with one or more (e.g., 1,2, 3, 4, or 5) halogens, wherein the alkyl is defined above. Selected as halogenated C _1-6 Alkyl, more preferably halogenated C _1-3 An alkyl group. Halogenated C _1-8 Examples of alkyl groups include, but are not limited to, monochloromethyl, dichloromethyl, trichloromethyl, monochloroethyl, 1, 2-dichloroethyl, trichloroethyl, monobromoethyl, monofluoromethyl, difluoromethyl, trifluoromethyl, monofluoroethyl, difluoroethyl, trifluoroethyl, and the like.

Also for example, "halo C _1-8 Alkoxy "means that the alkoxy group is substituted with one or more (e.g., 1,2. 3, 4 or 5) halogen substituents, wherein alkoxy is as defined above. Preferably halogenated C _1-6 Alkoxy, more preferably halo C _1-3 An alkoxy group. Including, but not limited to, trifluoromethoxy, trifluoroethoxy, monofluoromethoxy, monofluoroethoxy, difluoromethoxy, difluoroethoxy, and the like.

Also for example, "halo C _3-8 Cycloalkyl "refers to cycloalkyl substituted with one or more (e.g., 1, 2, 3, 4, or 5) halogens, wherein cycloalkyl is defined above. Preferably halogenated C _3-6 Cycloalkyl groups. Including, but not limited to, trifluorocyclopropyl, monofluorocyclopropyl, monofluorocyclohexyl, difluorocyclopropyl, difluorocyclohexyl, and the like.

"amino" means NH ₂ "cyano" means CN, "nitro" means NO ₂ "benzyl" means-CH ₂ -phenyl, "oxo" means =o, "carboxy" means-C (O) OH, "acetyl" means-C (O) CH ₃ "hydroxymethyl" means-CH ₂ OH, "hydroxyethyl" means-CH ₂ CH ₂ OH or-CHOHCH ₃ "hydroxy" refers to-OH, "thiol" refers to SH, "cyclopropylene" structure:

"substituted" means that one or more hydrogen atoms, preferably 1 to 5 hydrogen atoms, in the group are independently substituted with a corresponding number of substituents, more preferably 1 to 3 hydrogen atoms 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. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.

Unless otherwise defined, the "substituents each independently selected from … …" in the present invention means that when one or more hydrogens on the group are substituted with a substituent, the substituent species may be the same or different, and the substituents selected are each independent species.

Unless otherwise defined, "… …" as used herein, are the same or different and are each … … "and mean that when more than one identical substituent group is present in the formula, the groups may be the same or different and are each independent species. For example L is (CR) ₀₁ R ₀₂ ) _s When s is 2, i.e., L is (CR ₀₁ R ₀₂ )-(CR ₀₁ R ₀₂ ) Wherein two of R ₀₁ Or R is ₀₂ May be the same or different, and are each independently of the other, e.g., L may be C (CH) ₃ )(CN)-C(CH ₂ CH ₃ )(OH)，C(CH ₃ )(CN)-C(CH ₃ ) (OH) or C (CN) (CH ₂ CH ₃ )-C(OH)(CH ₂ CH ₃ )。

The present Wen Renyi groups may be substituted or unsubstituted, unless otherwise defined. When the above groups are substituted, the substituents are preferably 1 to 5 or less, independently selected from cyano, halogen (preferably fluorine or chlorine), C _1-8 Alkyl (preferably C _1-6 Alkyl, more preferably C _1-3 Alkyl group, C _1-8 Alkoxy (preferably C _1-6 Alkoxy, more preferably C _1-3 Alkoxy), halo C _1-8 Alkyl (preferably halogenated C) _1-6 Alkyl, more preferably halogenated C _1-3 Alkyl group, C _3-8 Cycloalkyl (preferably C) _3-6 Cycloalkyl, halo C _1-8 Alkoxy (preferably halo C) _1-6 Alkoxy, more preferably halo C _1-3 Alkoxy group), C _1-8 Alkyl-substituted amino, halo C _1-8 Alkyl-substituted amino, acetyl, hydroxy, hydroxymethyl, hydroxyethyl, carboxyl, nitro, C _6-10 Aryl (preferably phenyl), C _3-8 Cycloalkyloxy (preferably C _3-6 Cycloalkyl oxy group, C _2-8 Alkenyl (preferably C _2-6 Alkenyl groups, more preferably C _2-4 Alkenyl group, C _2-8 Alkynyl (preferably C) _2-6 Alkynyl, more preferably C _2-4 Alkynyl), -CONR _a0 R _b0 、-C(O)OC _1-10 Alkyl (preferably-C (O) OC) _1-6 Alkyl, more preferably-C (O) OC _1-3 Alkyl), -CHO, -OC (O) C _1-10 Alkyl (preferably-OC (O) C) _1-6 Alkyl, more preferably-OC (O) C _1-3 Alkyl), -SO ₂ C _1-10 Alkyl (preferably-SO) ₂ C _1-6 Alkyl, more preferably-SO ₂ C _1-3 Alkyl), -SO ₂ C _6-10 Aryl (preferably-SO) ₂ C ₆ Aryl radicals, e.g. -SO ₂ -phenyl), -COC _6-10 Aryl (preferably-COC) ₆ Aryl, such as-CO-phenyl), 4-to 6-membered saturated or unsaturated mono-heterocycle, 4-to 6-membered saturated or unsaturated monocyclic, 5-to 6-membered monocyclic heteroaryl ring, 8-to 10-membered bicyclic heteroaryl ring, spiro, bridged or bridged heterocycle, wherein R _a0 、R _b0 Each independently is hydrogen or C _1-3 An alkyl group.

The various substituents described hereinabove may themselves be substituted with the groups described herein.

Compounds of the invention

The compound of the present invention includes a compound represented by the formula (I) of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug thereof.

The "pharmaceutically acceptable salts" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. By "pharmaceutically acceptable acid addition salt" is meant a salt with an inorganic or organic acid that retains the biological effectiveness of the free base without other side effects. Inorganic acid salts include, for example, carbonates and the like; organic acid salts include, for example, propionate, glycolate, gluconate, and the like. These salts can be prepared by methods known in the art. "pharmaceutically acceptable base addition salts" include, but are not limited to, salts with inorganic bases such as sodium, potassium, calcium, and magnesium salts, and the like, as well as salts with organic bases such as ammonium, triethylamine, lysine, arginine, and the like. These salts can be prepared by methods known in the art.

In the present invention, the compounds of formula (I) and pharmaceutically acceptable salts thereof include all solid state forms of the compounds of formula (I) and pharmaceutically acceptable salts thereof. The compounds of formula (I) and pharmaceutically acceptable salts thereof are also intended to encompass all solvated (e.g., hydrated) and unsolvated forms of the compounds of formula (I) and pharmaceutically acceptable salts thereof. Reference herein to a "solvate" is to a complex of a compound of formula (I) or a pharmaceutically acceptable salt thereof according to the invention with a solvent. They either react in the solvent or precipitate or crystallize out of the solvent.

The compounds of formula (I) of the present invention may have one or more chiral centers and thus the structure of formula (I) for which no chirality is indicated encompasses any single chiral stereoisomer (e.g. enantiomers and diastereomers) and any mixture of chiral stereoisomers (e.g. racemates) corresponding to any possible existence of the structure. The single chiral stereoisomer may be resolved by methods known in the art, such as crystallization and chiral chromatography. In other embodiments, a single chiral stereoisomer may be obtained via direct synthesis from, for example, a chiral starting material.

In the present invention, the compounds of formula (I) and pharmaceutically acceptable salts thereof also encompass any tautomer that may be formed. "tautomer" is any other structural isomer that exists in equilibrium form due to the migration of a hydrogen atom, such as amide-imide acid tautomerism.

The invention also includes prodrugs of the compounds of formula (I) above. Prodrugs include known amino protecting groups and carboxyl protecting groups, which are hydrolyzed under physiological conditions or released via enzymatic reactions to give the parent compound. Specific methods of prodrug preparation can be found in (saurnier, m.g.; frenneson, d.b.; deshpande, m.s.; hansel, S.B and Vysa, d.m. bioorg.med.chem lett.1994,4, 1985-1990; and Greenwald, r.b.; choke, y.h.; conover, c.d.; shu, k.; wu, d.; royzen, m.j.med.chem.2000, 43, 475).

As shown in the test examples below, the compounds of the invention may modulate P2X3, and in particular may act as antagonists against P2X 3. The compounds of the invention may therefore be used to modulate P2X3 and/or P2X2/3 to treat a variety of diseases mediated by (or otherwise associated with) P2X3 and/or P2X 2/3.

In some embodiments, the compounds of the invention may be used to treat, for example, pain. Such pain may be, for example, chronic pain, neuropathic pain, acute pain, back pain, cancer pain, pain caused by rheumatoid arthritis, migraine, visceral pain, and the like.

In some embodiments, the compounds of the invention may be used to treat urinary tract disorders. These conditions include, for example, overactive bladder (also known as urinary incontinence), pelvic hypersensitivity, urethritis, and the like.

In some embodiments, the compounds of the present invention may be used to treat gastrointestinal disorders. Such diseases include, for example, constipation and functional gastrointestinal disorders (e.g., irritable bowel syndrome or functional dyspepsia).

In some embodiments, the compounds of the invention may be used to treat cancer. Such cancers include, for example, prostate cancer, colon cancer, rectal cancer, pancreatic cancer, cervical cancer, endometrial cancer, brain cancer, liver cancer, bladder cancer, ovarian cancer, testicular cancer, head cancer, neck cancer, melanoma, basal carcinoma, mesothelial lining cancer, white blood cell cancer, esophageal cancer, breast cancer, muscle cancer, connective tissue cancer, small cell lung cancer, non-small cell lung cancer, adrenal cancer, thyroid cancer, kidney cancer, or bone cancer; or glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, testicular seminoma, etc.

In some embodiments, the compounds of the invention may be used as immunomodulators, in particular for the treatment of autoimmune related diseases (e.g. rheumatoid arthritis, renal failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergies, fibrosis, anemic fibromyalgia, alzheimer's disease, congestive heart failure, stroke, aortic stenosis, arteriosclerosis, osteoporosis, parkinson's disease, infections, crohn's disease, ulcerative colitis, allergic contact dermatitis and other eczema, systemic sclerosis and multiple sclerosis, etc.); can also be used for skin transplantation, organ transplantation or similar operation; for collagen diseases; for allergies; or as an antitumor or antiviral agent.

In some embodiments, the compounds of the invention may be used to treat cough, depression, anxiety, stress-related disorders (e.g., post-traumatic stress disorder, panic disorder, social phobia, or obsessive compulsive disorder), and also may be used to treat premature ejaculation, psychosis, traumatic brain injury, stroke, spinal cord injury, drug addiction (e.g., to treat alcohol, nicotine, opioid, or other drug abuse), or sympathological disorders (e.g., hypertension).

Pharmaceutical compositions of the invention

The compounds of the present invention are useful in the preparation of pharmaceutical compositions. In general, the pharmaceutical composition comprises a therapeutically effective amount of a compound of formula (I) of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug thereof. The pharmaceutical compositions comprising the compounds of the present invention may vary widely. In general, the compounds of the invention may be administered in a suitable dosage form with one or more pharmaceutically acceptable carriers. Wherein pharmaceutically acceptable carrier means a non-toxic, inert, solid, semi-solid substance or liquid filling machine, diluent, encapsulating material or co-formulation or any type of adjuvant compatible with the patient and which is suitable for delivering the active compound to the target site without stopping the activity of the compound. Patient means an animal, preferably a mammal, more preferably a human. The mammal is warm-blooded vertebrate mammal, including cat, dog, rabbit, bear, fox, wolf, monkey, deer, mouse, pig and human.

These dosage forms are suitable for oral, rectal, topical, intraoral, and other parenteral administration (e.g., subcutaneous, intramuscular, intravenous, etc.). For example, dosage forms suitable for oral administration include capsules, tablets, granules, syrups and the like. The compounds of the invention contained in these formulations may be solid powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or oil-in-water emulsions, and the like. The above-described dosage forms may be prepared from the compounds of the present invention and one or more carriers or excipients via conventional pharmaceutical methods. The above-mentioned carriers need to be compatible with the compounds of the present invention or other excipients. For solid formulations, common non-toxic carriers include, but are not limited to, mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, and the like. The carrier for liquid formulations includes water, physiological saline, aqueous dextrose, glycols, polyethylene glycols, and the like. The compounds of the present invention may form solutions or suspensions with the carriers described above.

The pharmaceutical compositions of the present invention are formulated, quantified and administered in a manner consistent with medical practice specifications. The therapeutically effective amount of the compound to be administered will be determined by the particular condition being treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration, among other factors. A therapeutically effective amount refers to an amount of a compound of the invention that will elicit a biological or medical response in an individual, e.g., reduce or inhibit enzyme or protein activity or ameliorate symptoms, alleviate a condition, slow or delay the progression of a disease or prevent a disease, etc. "treating" refers to alleviating, slowing progression, attenuating, preventing, or maintaining an existing disease or condition (e.g., cancer). Treatment also includes curing, preventing the development of, or alleviating to some extent, one or more symptoms of the disease or disorder.

In some embodiments, the therapeutically effective amount of a compound of formula (I) of the present invention, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a stereoisomer thereof, or a prodrug thereof, contained in a pharmaceutical composition of the present invention is from 0.1mg to 5g/kg (body weight).

Preparation method

The present invention provides methods for the preparation of compounds of formula (I), which may be synthesized using standard synthetic techniques known to those skilled in the art or using methods known in the art in combination with the methods described herein. The solvents, temperatures and other reaction conditions set forth herein may vary according to the art. The reactions may be used sequentially to provide the compounds of the invention, or they may be used to synthesize fragments that are subsequently added by the methods described herein and/or by methods known in the art.

The compounds described herein may be synthesized using methods analogous to those described below or exemplary methods described in the examples, or related publications used by those skilled in the art, by using appropriate alternative starting materials. The starting materials for the synthesis of the compounds described herein may be synthesized or may be obtained from commercial sources. The compounds described herein and other related compounds having different substituents can be synthesized using techniques and starting materials known to those skilled in the art. The general methods of preparing the compounds disclosed herein may come from reactions known in the art, and the reactions may be modified by reagents and conditions deemed appropriate by one skilled in the art to introduce various moieties into the molecules provided herein.

Compared with the prior art, the invention has the main advantages that:

provides a series of substituted morpholine-4-carboxylic ester derivatives with novel structure, which have high inhibition activity to P2X3 and IC ₅₀ A value of less than 1000nM, or less than 500nM, or less than 100nM, or less than 50nM, has low P2X2/3 inhibitory activity, and is IC ₅₀ The value is greater than 1. Mu.M, or greater than 5. Mu.M, or greater than 10. Mu.M. And thus are useful as antagonists against P2X3 for the treatment of various diseases mediated by (or otherwise associated with) P2X3 and/or P2X 2/3.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. Unless defined otherwise, terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention.

Reagents and apparatus

¹ HNMR: the Bruker AVANCE-400 nuclear magnetic instrument is internally marked by Tetramethylsilane (TMS).

LC-MS: agilent 1200HPLC System/6140MS liquid chromatography mass spectrometer (manufacturer: agilent), column Waters X-Bridge, 150X 4.6mm,3.5 μm.

An ISCO Combiflash-Rf75 or Rf200 automatic column passing instrument is adopted, and Agela 4g, 12g, 20g, 40g, 80g and 120g disposable silica gel columns are adopted.

Known starting materials can be synthesized using or according to methods known in the art, or can be purchased from ABCR GmbH & Co.KG, acros Organics, aldrich Chemical Company, shaoshao chemical technology (Accela ChemBio Inc), darui chemical, and other companies.

DCM: dichloromethane, DMF: n, N-dimethylformamide, DMSO: dimethyl sulfoxide, THF: tetrahydrofuran, TEA: triethylamine, DIEA: n, N-diisopropylethylamine, EA: ethyl acetate, PE: petroleum ether, BINAP: (2R, 3S) -2,2 '-bis-diphenylphosphino-1, 1' -binaphthyl, NBS: n-bromosuccinimide, NCS: n-chlorosuccinimide, pd ₂ (dba) ₃ : tris (dibenzacetylacetone) dipalladium, pd (dppf) Cl ₂ : [1,1' -bis (diphenylphosphorus) ferrocene]Palladium dichloride, DPPA: diphenyl azide phosphate, HATU:2- (7-azobenzotriazole) -N, N' -tetramethylurea hexafluorophosphate, DBU:1, 8-diazabicyclo undec-7-ene, CDI: n, N' -carbonyldiimidazole, TBAF: tetrabutylammonium fluoride, na Ascorbate: sodium ascorbate, t-BuXPhosPd-G3: methanesulfonic acid (2-di-tert-butylphosphino-2 ',4',6 '-triisopropyl-1, 1' -biphenyl) (2 '-amino-1, 1' -biphenyl-2-yl) palladium (II).

As used herein, room temperature refers to about 20-30 ℃.

Preparation of intermediate 3, 5-difluoro-4-formyl-N-methoxy-N-methylbenzamide

Step 1: 3, 5-Difluorobenzoic acid (10 g,63.3 mmol) was dissolved in dry THF (150 mL), tetramethyl ethylenediamine (20.9 mL,139.3 mmol) was added, cooled to-78deg.C, and n-butyllithium solution (63.3 mL,158.3mmol, 2.5M) was added dropwise and stirred at-78deg.C for 1 hour. Methyl formate (8.4 g,139.3 mmol) was then slowly added dropwise to the reaction, stirred for half an hour at-78 ℃ and gradually warmed to room temperature. The saturated ammonium chloride was quenched, acidified with aqueous hydrochloric acid, extracted with EA, and the organic phase concentrated under reduced pressure, and the crude product was separated by column chromatography (PE: ea=70:30) to give 3, 5-difluoro-4-formylbenzoic acid (8.7 g) in 74% yield. MS m/z (ESI) 187.0[ M+1].

Step 2: 3, 5-difluoro-4-formylbenzoic acid (3.0 g,16.1 mmol) and dimethylhydroxylamine hydrochloride (2.4 g,24.2 mmol) were dissolved in DMF (15 mL), HATU (6.1 g,16.1 mmol) and DIEA (5.3 mL,32.2 mmol) were added and stirred at room temperature overnight. After the completion of the reaction, EA was extracted, the saturated brine was washed, and the organic phase was concentrated under reduced pressure to give a crude product, which was subjected to column separation (PE: ea=60:40) to give 3, 5-difluoro-4-formyl-N-methoxy-N-methylbenzamide (1.5 g), yield 41%. MS m/z (ESI) 230.0[ M+1].

Preparation of intermediate methyl 3, 5-difluoro-4-formylbenzoate

3, 5-difluoro-4-formylbenzoic acid (5 g,26.87 mmol) and potassium carbonate (7.43 g,53.73 mmol) and methyl iodide (5.72 g,40.30 mmol) were dissolved in DMF (50 mL). The reaction was stirred for 8 hours at 20℃and monitored by TLC/LC-MS. After the completion of the reaction, it was cooled to/warmed to room temperature, quenched with brine solution, extracted with solvent, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was dried under reduced pressure and separated by column chromatography (80 g,0 to 50% EA/PE) to give methyl 3, 5-difluoro-4-formylbenzoate (3 g), in 55.79% yield. MS m/z (ESI) 201.1[ M+1].

Preparation of intermediate (S) -4-tert-butoxycarbonyl-2-acetylenic morpholine

Step 1: (R) -4-Boc-2-hydroxymethylmorpholine (10 g,46.1 mmol) was dissolved in acetonitrile (40 mL) and 2-iodoxybenzoic acid (19.4 g,69.1 mmol) was added and refluxed at 100℃for 2 hours. After the reaction was completed, cooled to room temperature, filtered to obtain a filtrate, washed with a saturated sodium bicarbonate solution, dried under reduced pressure to obtain a crude product, and separated by column chromatography (PE: ea=70:30) to obtain (R) -4-t-butoxycarbonyl-2-formylmorpholine (8.8 g) as a colorless oily liquid in 89% yield. MS m/z (ESI) 160.0[ M-55].

Step 2: dimethyl (1-diazo-2-oxopropyl) phosphonate (7.9 g,40.9 mmol) was dissolved in a mixed solution of acetonitrile (60 mL) and methanol (10 mL), potassium carbonate (11.3 g,81.8 mmol) was added, stirred at room temperature for half an hour, and then (R) -4-t-butoxycarbonyl-2-formylmorpholine (8.8 g,40.9 mmol) obtained in the above step was added thereto and stirred at room temperature overnight. After the completion of the reaction, the filtrate was filtered and dried under reduced pressure to give a crude product, which was separated by column chromatography (PE: ea=80:20) to give (S) -4-t-butoxycarbonyl-2-acetylenic morpholine (5.7 g) in 66% yield. MS m/z (ESI): 156.1[ M-55].

Example 1 preparation of (S) -2- ((2- (2, 6-difluoro-4- (1H-imidazol-2-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester

Step 1: 2-amino-4-methylpyridine (108 mg,1.0 mmol) and 3, 5-difluoro-4-formyl-N-methoxy-N-methylbenzamide (229 mg,1.0 mmol) were dissolved in absolute ethanol (5 mL) and refluxed overnight at 80 ℃. Ethanol was removed under reduced pressure, the imine intermediate was dissolved in toluene (5 mL), and (S) -4-t-butoxycarbonyl-2-acetylmorpholine (211 mg,1.0 mmol), copper (II) trifluoromethanesulfonate (72 mg,0.2 mmol), and cuprous chloride (20 mg,0.2 mmol) were added in this order under argon, followed by stirring at 85℃for 4 hours. Cooled to room temperature and the spin-dried solvent column chromatographed (DCM: methanol=90:10) to give tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (methoxy (methyl) carbamoyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (145 mg) in 27% yield. MS m/z (ESI): 531.2[ M+1].

Step 2: (S) -2- ((2- (2, 6-difluoro-4- (methoxy (methyl) carbamoyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid tert-butyl ester (140 mg,0.26 mmol) was dissolved in dry THF (5 mL), lithium aluminum hydride (30 mg,0.79 mmol) was added at 0deg.C, after half an hour of reaction, water quenching was added, EA extraction, drying over anhydrous sodium sulfate, and spin-drying to give the crude (S) -2- ((2- (2, 6-difluoro-4-formylphenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid tert-butyl ester (100 mg) which was directly cast into the next step. MS m/z (ESI) 472.1[ M+1].

Step 3: (S) -2- ((2- (2, 6-difluoro-4-formylphenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid tert-butyl ester (100 mg,0.21 mmol) was dissolved in DCM (4 mL), trifluoroacetic acid (1 mL) was added and stirred at room temperature for 4 hours. The organic phase was dried under reduced pressure to give the crude (S) -3, 5-difluoro-4- (7-methyl-3- (morpholin-2-ylmethyl) imidazo [1,2-a ] pyridin-2-yl) benzaldehyde (80 mg) which was directly taken to the next step. MS m/z (ESI) 371.1[ M+1].

Step 4: (S) -3, 5-difluoro-4- (7-methyl-3- (morpholin-2-ylmethyl) imidazo [1,2-a ] pyridin-2-yl) benzaldehyde (80 mg,0.21 mmol) was dissolved in dry DCM (5 mL), DIEA (0.1 mL,0.63 mmol) and methyl chloroformate (30 mg,0.32 mmol) were added and stirred at room temperature for 2 hours. The solvent was dried directly after completion of the reaction, and separated by column chromatography (DCM: methanol=90:10) to give methyl (S) -2- ((2- (2, 6-difluoro-4-formylphenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (45 mg) in 40% yield. MS m/z (ESI) 430.1[ M+1].

Step 5: (S) -2- ((2- (2, 6-difluoro-4-formylphenyl) -7-methylimidazole [1, 2-a)]Pyridin-3-yl) methyl morpholine-4-carboxylate (45 mg,0.10 mmol) and glyoxal (44 mg,0.30mmol,40% in water) were dissolved in ammonia in methanol (5 mL) and refluxed at 70 ℃ for 4 hours. Spin-drying the solvent after completion of the reaction and purifying the resulting residue by preparative liquid chromatography to give (S) -2- ((2- (2, 6-difluoro-4- (1H-imidazol-2-yl) phenyl) -7-methylimidazo [1, 2-a) ]Pyridin-3-yl) methyl morpholine-4-carboxylate (H-1, 7.18 mg) in 15% yield. MS m/z (ESI): 468.1[ M+1]]。 ¹ H NMR(400MHz,Methanol-d ₄ )δ8.40(d,J＝7.1Hz,1H),7.64(d,J＝8.6Hz,2H),7.33(s,1H),7.23(s,2H),6.86(dd,J＝7.2,1.4Hz,1H),3.88–3.71(m,3H),3.64(s,3H),3.62–3.54(m,1H),3.37(dd,J＝11.9,3.0Hz,1H),3.18–3.01(m,2H),2.89(m,1H),2.63(m,1H),2.45(s,3H).

Example 2 preparation of (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-1H-imidazol-2-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester

(S) -2- ((2- (2, 6-difluoro-4-formylphenyl) -7-methylimidazole [1, 2-a)]Pyridin-3-yl) methyl morpholine-4-carboxylate (30 mg,0.07 mmol) and methylglyoxal (38 mg,0.21mmol,40% in water) were dissolved in ammonia in methanol (4 mL) and refluxed at 70 ℃ for 4 hours. Spin-drying the solvent after completion of the reaction and purifying the resulting residue by preparative liquid chromatography to give (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-1H-imidazol-2-yl) phenyl) -7-methylimidazo [1, 2-a)]Pyridin-3-yl) methyl morpholine-4-carboxylate H-2 (1.72 mg) in 5% yield. MS m/z (ESI): 482.2[ M+1]]。 ¹ H NMR(400MHz,Methanol-d4)δ8.41(d,J＝7.1Hz,1H),7.60(d,J＝8.6Hz,2H),7.35(s,1H),6.93(s,1H),6.88(dd,J＝7.1,1.5Hz,1H),3.88–3.72(m,3H),3.64–3.54(m,4H),3.37(dd,J＝11.9,2.9Hz,1H),3.18–3.02(m,2H),2.88(s,1H),2.70–2.56(m,1H),2.46(s,3H),2.32(s,3H).

Example 3 preparation of (S) -2- ((2- (2, 6-difluoro-4- (1H-pyrazol-3-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester

Step 2 3, 5-difluoro-4-formylbenzoic acid (5 g,26.87 mmol) and potassium carbonate (7.43 g,53.73 mmol) and methyl iodide (5.72 g,40.30 mmol) were dissolved in DMF (50 mL). The reaction was stirred for 8 hours at 20℃and monitored by TLC/LC-MS. After the completion of the reaction, it was cooled to/warmed to room temperature, quenched with brine solution, extracted with solvent, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was dried under reduced pressure and separated by column chromatography (80 g,0 to 50% EA/PE) to give methyl 3, 5-difluoro-4-formylbenzoate (3 g), in 55.79% yield. MS m/z (ESI) 201.1[ M+1].

Step 3: methyl 3, 5-difluoro-4-formylbenzoate (3 g,14.99 mmol) and 4-methylpyridin-2-amine (1.62 g,14.99 mmol) were added to ethanol (30 mL), stirred at 80℃overnight, and concentrated under reduced pressure to give a yellow oil. The oil was added (S) -2-acetylenemorpholine-4-carboxylic acid tert-butyl ester (3.17 g,14.99 mmol), copper triflate (5.42 g,14.99 mmol) N, N-dimethylacetamide (1.31 g,14.99 mmol) cuprous chloride (1.48 g,14.99 mmol) and toluene (50 mL), stirred overnight under nitrogen at 85 degrees, concentrated under reduced pressure to give a black oil which was isolated by column chromatography (PE: ea=1:99) to give (S) -2- ((2- (2, 6-difluoro-4- (methoxycarbonyl) phenyl) -7-methylimidazol [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid tert-butyl salt (1.6 g) as a grey solid in 21.28% yield. MS m/z (ESI) 502.1[ M+1].

Step 4: (S) -2- ((2- (2, 6-difluoro-4- (methoxycarbonyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid tert-butyl salt (200 mg, 398.79. Mu. Mol) was dissolved in DCM (5 mL) and trifluoroacetic acid (136.41 mg,1.20 mmol) was added thereto. The reaction was stirred at 25℃for 4 hours and monitored by LC-MS. After completion of the reaction, the solvent was dried by spin-drying to give methyl (S) -3, 5-difluoro-4- (7-methyl-3- (morpholin-2-ylmethyl) imidazo [1,2-a ] pyridin-2-yl) benzoate (160 mg), which was directly taken into the next step without purification, with a yield of 99.95%. MS m/z (ESI) 402.1[ M+1].

Step 5: methyl (S) -3, 5-difluoro-4- (7-methyl-3- (morpholin-2-ylmethyl) imidazo [1,2-a ] pyridin-2-yl) benzoate (0.55 g,1.37 mmol) was dissolved in DCM (5 ml), TEA (0.42 g,4.11 mmol) and methyl chloroformate (0.258 g,2.74 mmol) were added at 0℃and stirred for 1 hour at 0 ℃. The reaction solution was added with DCM, washed with 3N hydrochloric acid solution, saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a yellow oil. The oil was separated by column chromatography (DCM: meoh=9:1) to give methyl (S) -2- ((2- (2, 6-difluoro-4- (methoxycarbonyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (0.4 g) in 63.5% yield. MS m/z (ESI) 460.1[ M+1].

Step 6: (S) -2- ((2- (2, 6-difluoro-4- (methoxycarbonyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (200 mg,0.4 mmol) was dissolved in methanol (6 ml), water (1 ml) was added, lithium hydroxide (28 mg,1.2 mmol) was added, and stirred at room temperature for 1 hour. The pH was adjusted to 1 with 3N hydrochloric acid and extracted with DCM. The organic layer was concentrated under reduced pressure to give (S) -3, 5-difluoro-4- (3- ((4- (methoxycarbonyl) morpholin-2-yl) methyl) -7-methylimidazole [1,2-a ] pyridin-2-yl) benzoic acid (160 mg) as a yellow oil, crude. MS m/z (ESI) 446.1[ M+1].

Step 7: (S) -3, 5-difluoro-4- (3- ((4- (methoxycarbonyl) morpholin-2-yl) methyl) -7-methylimidazole [1,2-a ] pyridin-2-yl) benzoic acid (140 mg,0.31 mmol) was dissolved in DMF (10 ml), and dimethylamine hydrochloride (61 mg,0.62 mmol) and TEA (94 mg,0.93 mmole 1) and HATU (178 mg,0.47 mmol) were added and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to give a yellow oil, which was separated by column chromatography (DCM: meoh=9:1) to give methyl (S) -2- ((2- (2, 6-difluoro-4- (methoxy (methyl) carbamoyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (140 mg) in 93% yield. MS m/z (ESI): 489.1[ M+1].

Step 8: (S) -2- ((2- (2, 6-difluoro-4- (methoxy (methyl) carbamoyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (120 mg,0.25 mmol) was dissolved in anhydrous THF (8 ml) solution, and a solution of magnesium methyl iodide (3.0M, 0.5ml,0.75 mmol) was added at 0℃and stirred at 0℃for 1 hour. The reaction solution was added with saturated ammonium chloride solution, and extracted with EA solution. The organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give (S) -2- ((2- (4-acetyl-2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (100 mg) as a yellow solid, crude product. MS m/z (ESI) 444.1[ M+1].

Step 9: (S) -2- ((2- (4-acetyl-2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (80 mg,0.18 mmol) was dissolved in a solution of N, N-dimethylformamide dimethyl acetal (4 ml) and stirred at 120℃for 2 hours. Concentration under reduced pressure afforded (S, E) -2- ((2- (4- (3- (dimethylamino) acryloyl) -2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (80 mg) as a yellow oil, crude. MS m/z (ESI): 499.1[ M+1].

Step 10: methyl (S, E) -2- ((2- (4- (3- (dimethylamino) acryloyl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (80 mg,0.16 mmol) was added to a solution of methanol (5 ml), hydrazine hydrate (1 ml) was added, stirred at room temperature for 4 hours, concentrated under reduced pressure, and the resulting residue was purified by preparative liquid chromatography to give methyl (S) -2- ((2- (2, 6-difluoro-4- (1H-pyrazol-3-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (H-3, 1.25 mg) in 1.67% yield. MS m/z (ESI): 468.1[ M+1].

Example 4 preparation of (S) -2- ((2- (2, 6-difluoro-4- (5-oxo-4, 5-dihydro-1H-1, 2, 4-triazol-3-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester

Step 1: methyl ((2- (2, 6-difluoro-4- (methoxycarbonyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (220 mg,0.49 mmol) was dissolved in DMF (10 ml), ammonium chloride (80 mg,1.48 mmol) and TEA (150 mg,1.48 mmole 1) and HATU (280 mg,0.74 mmol) were added and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to give a yellow oil, which was separated by column chromatography (DCM: meoh=4:1) to give methyl 2- ((2- (4-carbamoyl-2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (150 mg) in 69% yield. MS m/z (ESI) 445.1[ M+1].

Step 2: methyl 2- ((2- (4-carbamoyl-2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (150 mg,0.34 mmol) was dissolved in anhydrous DCM (5 ml), and the Bogis reagent (241 mg,1.01 mmol) was added and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure to give a yellow solid which was separated by column chromatography (DCM: meoh=19:1) to give methyl 2- ((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (90 mg) as a yellow solid in 62% yield. MS m/z (ESI): 427.1[ M+1].

Step 3: 2- ((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazole [1, 2-a) ]Methyl pyridin-3-yl) morpholine-4-carboxylate (60 mg,0.14 mmol) was dissolved in ethyl hydrazinoformate (1 ml), DMAP (20 mg) was added and stirred at 170℃for 1.5 h under microwave conditions. The reaction solution was purified by preparative liquid chromatography to give a residue, which was concentrated under reduced pressure to give (S) -2- ((2- (2, 6-difluoro-4- (5-oxo-4, 5-dihydro-1H-1, 2, 4-triazol-3-yl) phenyl) -7-methylimidazo [1, 2-a) as a white solid]Pyridin-3-yl) methyl morpholine-4-carboxylate (H-4, 4 mg) in 5.9% yield. MS m/z (ESI): 485.1[ M+1]]。 ¹ H NMR(400MHz,DMSO-d6)δ11.88(s,1H),8.40-8.38(d,J＝8Hz,1H),7.56-7.54(d,J＝8Hz,2H),7.32(s,1H),6.81-6.79(m,1H),3.72-3.58(m,3H),3.51(s,3H),3.47-3.42(m,1H),3.24-3.13(m,2H),3.02-3.00(m,2H),2.77-2.75(m,1H),2.29(s,3H).

Example 5 preparation of (S) -2- ((2- (2, 6-difluoro-4- (5-oxo-4, 5-dihydro-1, 3, 4-thiadiazol-2-yl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester

Step 1: (S) -methyl 2- (((2- (2, 6-difluoro-4- (methoxycarbonyl) phenyl)) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (150 mg,0.326 mmol) and hydrazine hydrate (163 mg,3.26 mmol) were added to 15mL of ethanol and reacted overnight at 90 ℃. Concentration under reduced pressure afforded (S) -2- (((2- (2, 6-difluoro-4- (hydrazinocarbonyl) phenyl) -7) -methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (150 mg, yellow oily liquid) and the crude product was used directly in the next step. MS m/z (ESI) 460.2[ M+1].

Step 2: (S) -2- (((2- (2, 6-difluoro-4- (hydrazinocarbonyl) phenyl) -7) -methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (150 mg,0.326 mmol), di-tert-butyl dicarbonate (143 mg, 0.015 mmol) and TEA (99 mg,0.978 mmol) were added to 20mL DCM and reacted overnight at room temperature. The residue was purified by silica gel column chromatography with an eluent system (DCM/methanol: 1/0 to 10/1) to give methyl (S) -2- ((2- (4- (2- (tert-butoxycarbonyl) hydrazine-1-carbonyl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (140 mg, yellow foamy solid), yield: 77%. MS m/z (ESI) 560.2[ M+1].

Step 3: methyl (S) -2- ((2- (4- (2- (tert-butoxycarbonyl) hydrazine-1-carbonyl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (100 mg, 0.178 mmol) and lawson reagent were added to THF (3 mL) and reacted in microwaves at 110 ℃ for 6 hours. The residue was purified by preparative thin layer chromatography (DCM/methanol: 100/7) to give methyl (S) -2- ((2- (4- (2- (tert-butoxycarbonyl) hydrazino-1-carbosulfanyl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (45 mg, white solid) in 44% yield. MS m/z (ESI): 476.2[ M+1].

Step 4: (S) -2- ((methyl 2- (4- (2- (tert-butoxycarbonyl) hydrazine-1-carbosulfanyl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (45 mg,0.078 mmol) was dissolved in DCM (3 mL), trifluoroacetic acid (2 mL) was added and reacted at room temperature for 1 hour. Concentrated under reduced pressure, saturated sodium bicarbonate (10 mL) was added and extracted with DCM (30 mL. Times.3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to give methyl (S) -2- ((2- (2, 6-difluoro-4- (hydrazinothiocarbonyl) phenyl) -7) -methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (30 mg, yellow solid) in 81% yield. MS m/z (ESI): 476.1[ M+1].

Step 5: (S) -2- ((2- (2, 6-difluoro-4- (hydrazinothiocarbonyl) phenyl) -7) -methylimidazo [1, 2-a)]Methyl pyridin-3-yl) morpholine-4-carboxylate (30 mg,0.063 mmol) and N' N-carbonyldiimidazole (11 mg,0.068 mmol) were added to 5mL THF and reacted at room temperature for 1 hour. Concentrating under reduced pressure, and subjecting to preparative liquid chromatography (preparation column: 21.2X250mM C18 column; system: 10mM NH) ₄ HCO ₃ H ₂ O; wavelength: 254/214nm; gradient: 30% -60% acetonitrile change) purification stationThe residue is obtained to give (S) -2- ((2- (2, 6-difluoro-4- (5-oxo-4, 5-dihydro-1, 3, 4-thiadiazol-2-yl) phenyl) -7-methylimidazole [1, 2-a) ]Pyridin-3-yl) methyl morpholine-4-carboxylate (H-5, 7.61mg, white solid). The yield was 11%. MS m/z (ESI) 502.2[ M+1]]。 ¹ H NMR(400MHz,DMSO-d ₆ )δ8.40(d,J＝7.1Hz,1H),7.54(d,J＝7.2Hz,2H),7.33(s,1H),6.81(d,J＝6.9Hz,1H),3.72(d,J＝13.5Hz,1H),3.61(t,J＝10.3Hz,2H),3.52(s,3H),3.46-3.44(m,1H),3.23-3.17(m,1H),3.08–2.95(m,2H),2.77(s,1H),2.51(s,1H),2.35(s,3H).

Example 6 preparation of (S) -2- ((2- (4- (5-, 5-dimethyl-4-oxo-4, 5-dihydro-1H-imidazol-2-yl) -2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methylmorpholine-4-carboxylic acid methyl ester

Step 1: (S) -4- (3- ((4- (t-Butoxycarbonyl) morpholin-2-yl) methyl) -7-methylimidazo [1,2-a ] pyridin-2-yl) -3, 5-difluorobenzoic acid (preparation method of reference example 3, steps 5 and 6, except that methyl chloroformate was replaced with t-butyl chloroformate) (60 mg,0.123 mmol), methyl 2-amino-2-methylpropionate hydrochloride (21 mg,0.137 mmol), HATU (94 mg,0.247 mmol) and DIEA (80 mg,0.62 mmol) were added to DMF (3 mL) and reacted overnight at room temperature. Concentrated under reduced pressure and the resulting residue was purified by preparative thin layer chromatography (DCM/methanol: 20/1) to give tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (1-methoxy-2-methyl-1-oxopropan-2-ylcarbamoyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (60 mg, colorless oily liquid) in 83% yield. MS m/z (ESI): 587.3[ M+1].

Step 2: (S) -2- ((tert-butyl 2- (2, 6-difluoro-4- (1-methoxy-2-methyl-1-oxopropan-2-ylcarbamoyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (60 mg,0.102 mmol), 7N methanolic ammonia (3 mL) was sealed in a tube and reacted at 100℃for 2 days. Concentration under reduced pressure afforded (S) -2- ((2- (4- (1-amino-2-methyl-1-oxopropan-2-ylcarbamoyl) -2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid tert-butyl ester (58 mg, colorless oily liquid) and the crude product was used directly in the next step. MS m/z (ESI) 572.3[ M+1].

Step 3: (S) -2- ((2- (4- (1-amino-2-methyl-1-oxopropan-2-ylcarbamoyl) -2, 6-difluorophenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid tert-butyl ester (58 mg,0.101 mmol) was dissolved in methanol (10 mL), 6N sodium hydroxide solution (0.08 mL,0.48 mmol) was added and heated to reflux for 2 hours. The resulting residue was purified by preparative thin layer chromatography (DCM/methanol: 100/8) to give tert-butyl (S) -2- ((2- (4- (5, 5-dimethyl-4-oxo-4, 5-dihydro-1H-imidazol-2-yl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (20 mg, colorless oily liquid) in 36% yield. MS m/z (ESI): 554.2[ M+1].

Step 4: (S) -2- ((2- (4- (5, 5-dimethyl-4-oxo-4, 5-dihydro-1H-imidazol-2-yl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid tert-butyl ester (20 mg,0.036 mmol) was dissolved in DCM (2 mL) and trifluoroacetic acid (1 mL) was added and reacted at room temperature for 1 hour. Concentrated under reduced pressure to give (S) -2- (3, 5-difluoro-4- (7-methyl-3- (morpholin-2-ylmethyl) imidazo [1,2-a ] pyridin-2-yl) phenyl) -5, 5-dimethyl-1H-imidazol-4 (5H) -one (16 mg, yellow oily liquid), the crude product was used directly in the next step. MS m/z (ESI) 454.2[ M+1].

Step 5: (S) -2- (3, 5-difluoro-4- (7-methyl-3- (morpholin-2-ylmethyl) imidazo [1, 2-a)]Pyridin-2-yl) phenyl) -5, 5-dimethyl-1H-imidazol-4 (5H) -one (16 mg,0.035 mmol) was dissolved in DCM (6 mL) and N, N-diisopropylethylamine (14 mg,0.108 mmol) and methyl chloroformate (5 mg,0.053 mmol) were added and reacted at room temperature for 1 hour. Concentrating under reduced pressure, and subjecting to preparative liquid chromatography (preparation column: 21.2X250mM C18 column; system: 10mM NH) ₄ HCO ₃ H ₂ O; wavelength: 254/214nm; gradient: the resulting residue was purified by varying from 30% to 60% acetonitrile to give (S) -2- ((2- (4- (5-, 5-dimethyl-4-oxo-4, 5-dihydro-1H-imidazol-2-yl) -2, 6-difluorophenyl) -7-methylimidazole [1, 2-a)]Pyridin-3-yl) methylmorpholine-4-carboxylic acid methyl ester (H-6, 10.78mg, white solid). The yield was 60%. MS m/z (ESI) 512.2[ M+1 ]]。 ¹ H NMR(400MHz,CD3OD)δ8.39(d,J＝7.1Hz,1H),7.70(s,2H),7.33(s,1H),6.86(dd,J＝7.1,1.5Hz,1H),3.86–3.70(m,3H),3.63(s,3H),3.56-3.54(m,1H),3.35-3.32(m,1H),3.08(d,J＝6.5Hz,2H),2.85(s,1H),2.60(s,1H),2.44(s,3H),1.41(s,6H).

EXAMPLE 7 preparation of Compound H-7

Step 1: (S) -methyl 2- (((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (60 mg,0.14 mmol), hydroxylamine hydrochloride (15 mg,0.22 mmol) and DIEA (55 mg,0.43 mmol) were added to ethanol (15 mL) and reacted at 80℃for 2 hours.

Step 2: (S) -methyl 2- (((2- (2, 6-difluoro-4- (N-hydroxycarbamoyl)) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate 3 (64 mg,0.14 mmol), CDI (34 mg,0.21 mmol) and DBU (23 mg,0.15 mmol) were added to 1, 4-dioxane (15 mL) and reacted at 110℃for 1 hour. The mixture was concentrated under reduced pressure, and the resulting residue was purified by preparative liquid chromatography (preparative column: 21.2X250mm C18 column; system: 10mM NH4HCO 3H 2O; wavelength: 254/214nm; gradient: 30% -60% acetonitrile change) to give compound H-7 (35.59 mg, white solid). The yield thereof was found to be 53%. MS m/z (ESI): 486.2[ M+1],1H NMR (400 MHz, CD3 OD) delta 8.45 (d, J=7.1 Hz, 1H), 7.59 (d, J=7.8 Hz, 2H), 7.37 (s, 1H), 6.93 (dd, J=7.1, 1.5Hz, 1H), 3.86-3.69 (m, 3H), 3.63 (s, 3H), 3.58-3.56 (m, 1H), 3.35-3.30 (m, 1H), 3.10 (d, J=6.0 Hz, 2H), 2.83 (s, 1H), 2.59 (s, 1H), 2.46 (s, 3H).

EXAMPLE 8 preparation of Compound H-8

Step 1: (S) -3, 5-difluoro-4- (3- ((4- (methoxycarbonyl) morpholin-2-yl) methyl) -7-methylimidazo [1,2-a ] pyridin-2-yl) benzoic acid (110 mg,0.25 mmol), 1, 2-o-diphenylamine (30 mg,0.25 mmol), HATU (100 mg,0.25 mmol) and diethylamine (50 mg,0.375 mmol) were dissolved in DMF (10 ml), stirred at room temperature overnight, concentrated and purified by silica gel column chromatography (methanol/DCM=1/25) to give (S) -methyl 2- (((2- (4- (((2-aminophenyl) carbamoyl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (100 mg, Y: 74.6%) MS m/z (ESI): 536.2[ M+1]

Step 2: (S) -methyl 2- (((2- (4- (((2-aminophenyl) carbamoyl) -2, 6-difluorophenyl) -7-methylimidazo [1, 2-a)]Pyridin-3-yl) methyl morpholine-4-carboxylate (100 mg,0.187 mmol) was dissolved in acetic acid (15 ml), warmed to 100 ℃, stirred for 3 hours, concentrated, and purified by preparative HPLC (preparative column: 21.2X250mm C18 column; the system comprises: 10mM NH ₄ HCO ₃ H ₂ O; wavelength: 254/214nm; gradient: 30% -60% acetonitrile change) to give compound H-8 (40 mg, y: 41.3%) MS m/z (ESI) 518.2[ M+1 ]]。 ¹ H NMR(400MHz,DMSO-d ₆ )δ13.14(s,1H),8.41(d,J＝7.1Hz,1H),7.96-7.94(m,2H),7.70(d,J＝7.9Hz,1H),7.57(d,J＝7.7Hz,1H),7.35-7.32(m,1H),7.23-7.20(m,2H),6.83-6.81(m,1H),3.75-3.77(m,1H),3.63-3.61(m,2H),3.52-3.50(s,3H),3.46-3.41(m,1H),3.27-3.16(m,1H),3.06-3.07(m,2H),2.77(s,1H),2.52(s,1H),2.36(s,3H).

EXAMPLE 9 preparation of Compound H-9

Step 1: 2- ((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazo [1, 2-a)]Methyl pyridin-3-yl) morpholine-4-carboxylate (50 mg, 117. Mu. Mol) and sodium azide (15 mg, 234. Mu. Mol), ammonium chloride (12.4 mg, 234. Mu. Mol) were dissolved in DMF (2 mL). The reaction was stirred at 120℃for 4 hours. LCMS showed complete reaction. Cooled to room temperature, 40mLEA was added, washed with saturated sodium chloride solution (10 mL. Times.3), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by preparative chromatography(preparation of column: 21.2X250mm C18 column, system: 10mM NH4HCO 3H 2O wavelength: 254/214nm, gradient: 30% -60% acetonitrile change) to give Compound H-9 (15.13 mg, 32.2. Mu. Mol,27.5% yield) as a white solid. MS m/z (ESI) 470.2[ M+1 ] ]。 ¹ H NMR(400MHz,CD ₃ OD)δ8.63(d,J＝7.4Hz,1H),7.85(d,J＝8.8Hz,2H),7.53(s,1H),7.14(d,J＝7.0Hz,1H),3.92(d,J＝12.9Hz,1H),3.83–3.69(m,2H),3.69–3.57(m,4H),3.35(d,J＝10.7Hz,1H),3.18(d,J＝5.4Hz,2H),2.88(s,1H),2.67(d,J＝8.8Hz,1H),2.54(s,3H)

EXAMPLE 10 preparation of Compound H-10

Step 1: a solution of compound 2- ((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (100 mg,0.23 mmol) in ammonia/methanol was stirred in a closed tube at 95℃for 16h. LC-MS was followed until the reaction was complete. The reaction solution was concentrated under reduced pressure to give methyl 2- ((2- (4-carbamoyl-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (110 mg, yellow solid). MS m/z (ESI) 444[ M+1].

Step 2:2- ((2- (4-carbamoyl-2, 6-difluorophenyl) -7-methylimidazo [1, 2-a)]Methyl pyridin-3-yl) morpholine-4-carboxylate (50 mg,0.11 mmol), ethyl propiolate (54 mg,0.55 mmol) and potassium carbonate (23 mg,0.17 mmol) were reacted at 85℃for 16 hours in ethanol. LC-MC was followed until the reaction was complete. The reaction solution was diluted with methanol (10 mL) and filtered. The filtrate was concentrated under reduced pressure to remove the solvent, and the residue was purified by prep-HPLC and freeze-dried to give Compound H-10 (4.78 mg, white solid) in 6.11% yield. MS m/z (ESI) 496[ M+1]]。 ¹ H NMR(400MHz,DMSO-d ₆ )：δ12.75(br,1H),8.44(d,J＝7.2Hz,1H),8.24(s,1H),7.98(d,J＝8.0Hz,2H),7.36(s,1H),7.61(s,1H),6.85(d,J＝7.2Hz,1H),6.52(s,1H),3.71-3.77(m,1H),3.61-3.64(m,2H),3.55(s,3H),3.45-3.54(m,1H),3.19-3.25(m,2H),3.02-3.07(m,2H),2.70-2.85(m,1H),2.38(s 3H),

EXAMPLE 11 preparation of Compound H-11

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Step 1: (S) -2- (((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (213 mg,0.5 mmol) was dissolved in ethanol (5 mL) and hydroxylamine hydrochloride (94 mg,1 mmol) and TEA (101 mg,1 mmol) were then added the reaction was stirred at 50℃for 2 hours and monitored by LC-MS.

Step 2: methyl (S, E) -2- (((2- (2, 6-difluoro-4- (N' -hydroxycarbamoyl) phenyl)) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (92 mg,0.2 mmol) was dissolved in ethanol (2 mL), followed by ethyl propiolate (39 mg,0.4 mmol) and TEA (41 mg,0.4 mmol). The reaction was stirred at 90℃for 4 hours and monitored by LC-MS. After the reaction was completed, the solvent was dried by spin-drying. After spin-drying, the mixture was separated by column chromatography (4 g, 0-40% EA/PE) to give (S) -2- ((2- (4- ((E) -N' - (((E) -3-ethoxy-3-oxoprop-1-en-1-yl) oxy) carbamoyl) -2-methyl, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methylmorpholine-4-carboxylate 3 (89 mg, yellow oil): yield 79.89%. MS M/z (ESI): 558.2 (M+H)

Step 3: (S) -2- ((2- (4- ((E) -N' - (((E) -3-ethoxy-3-oxoprop-1-en-1-yl) oxy) carbamoyl) -2 methyl, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methylmorpholine-4-carboxylate (89 mg,0.16 mmol) was dissolved in diphenyl ether (1 mL). The reaction was stirred at 170℃for 3 hours and monitored by LC-MS after completion of the reaction, the solvent was spun dry after spinning dry was separated by Combiflash column chromatography (4 g,0 to 10% MeOH/DCM) to give methyl (S) -2- ((2- (4- (4- (ethoxycarbonyl) -1H-imidazol-2-yl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (72 mg, yellow oil) in 83.49%. M/z (ESI) 540.2

Step 4: (S) -2- ((2- (4- (4- (ethoxycarbonyl) -1H-imidazol-2-yl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylic acid methyl ester (72 mg,0.13 mmol) was dissolved in methanol (3 mL) and water (3 mL). The reaction was stirred at room temperature for 3 hours and monitored by LC-MS. After the reaction was completed, pH2 was adjusted with 1M hydrochloric acid, followed by three times of extraction with EA, and EA was combined and dried over anhydrous sodium sulfate. Spin-drying the solvent afforded (S) -2- (3, 5-difluoro-4- (3- ((4- (methoxycarbonyl) morpholin-2-yl) methyl) -7-methylimidazo [1,2-a ] pyridin-2-yl) phenyl) -1H-imidazole-4-carboxylic acid (51 mg, yellow solid) as the next step directly. The yield was 76.77%. MS m/z (ESI): 512.2

Step 5: (S) -2- (3, 5-difluoro-4- (3- ((4- (methoxycarbonyl) morpholin-2-yl) methyl) -7-methylimidazo [1,2-a ] pyridin-2-yl) phenyl) -1H-imidazole-4-carboxylic acid (51 mg,0.1 mmol) and NH4CL (16 mg,0.3 mmol) were dissolved in DMF (5 mL) and then HATU (46 mg,0.12 mmol) and DIEA (19 mg,0.15 mmol) were added. The reaction was stirred at room temperature for 2 hours and monitored by LC-MS. After the reaction was completed, the solvent was dried by spin-drying. The resulting residue was purified by preparative liquid chromatography (preparative column: 21.2X250mm C18 column; system: 10mM NH4HCO 3H 2O; wavelength: 254/214nm; gradient: 30% -60% acetonitrile change) to give compound H-11 (10 mg, white solid). The yield was 19.61%. MS m/z (ESI): 511.2[ M+1]. NMR (400 MHz, DMSO-d 6) delta 13.17 (s, 1H), 8.39 (d, J=8 Hz, 1H), 7.82-7.67 (m, 3H), 7.48 (s, 1H), 7.32 (s, 1H), 7.16 (s, 1H), 3.72-3.51 (m, 3H), 3.44 (s, 3H), 3.33-3.29 (m, 1H), 3.24-3.18 (m, 2H), 3.02 (d, J=8 Hz, 3H), 2.77 (s, 1H), 2.46 (s, 3H).

EXAMPLE 12 preparation of Compound H-12

Step 1: tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (methoxycarbonyl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (650 mg,1.3 mmol) was dissolved in ethanol (10 mL), hydrazine hydrate (2 mL) was added and stirred at 70℃for 2 hours. Concentrating under reduced pressure gave a yellow oil. The oil was isolated by column chromatography over CombiFlash (12 g, 0-10% dcm/MeOH) to give tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (hydrazinocarbonyl) phenyl) -7-methylimidazol [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (450 mg,69% yieldd) as a yellow oil. MS m/z (ESI): 502.1[ M+1]

Step 2 tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (hydrazinocarbonyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (220 mg,0.44 mmol) was dissolved in ethanol (10 mL), acetamidine hydrochloride (84 mg,0.88 mmol) and sodium methoxide (72 mg,1.32 mmol) were added and stirred overnight at room temperature. Concentrating under reduced pressure gave a yellow oil. The oil was isolated by column chromatography over Combiflash (12 g, 0-10% DCM/MeOH) to give tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (2- (1-iminoethyl) hydrazine-1-carbonyl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (120 mg,50% yield) MS m/z (ESI): 543.1[ M+1]

Step 3: tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (2- (1-iminoethyl) hydrazine-1-carbonyl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (100 mg,0.092 mmol) was dissolved in DMSO (2 ml) and stirred at 150 degrees for 30 minutes. The solution was separated by Combiflsh column chromatography (4 g, 0-10% DCM/MeOH) to give tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-4H-1, 2, 4-triazol-3-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (30 mg,31% yield) MSm/z (ESI): 525.1[ M+1]

Step 4: tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-4H-1, 2, 4-triazol-3-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (18 mg,0.057 mmol) was dissolved in dichloromethane (4 ml), trifluoroacetic acid (0.5 ml) was added and stirred at room temperature for 4 hours. The reaction solution was concentrated under reduced pressure to give (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-4H-1, 2, 4-triazol-3-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine (18 mg,75% yield) as a yellow solid. MSm/z (ESI): 425.1[ M+1]

Step 5: (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-4H-1, 2, 4-triazol-3-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine (18 mg,0.042 mmol) was dissolved in dichloromethane (5 ml), triethylamine (9 mg,0.084 mmol) and methyl chloroformate (6 mg,0.064 mmol) were added at 0℃and stirred at 0℃for 1 hour. Dichloromethane was added, the mixture was washed with saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a yellow solid. The solid was purified by preparative liquid chromatography to give the desired product as a white solid H-12 (1.40 mg,6.7% yield) MSm/z (ESI): 483.1[ M+1].

EXAMPLE 13 preparation of Compound H-13

Step 1: tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (hydrazinocarbonyl) phenyl) -7-methylimidazole [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (200 mg,0.4 mmol) was dissolved in xylene (10 mL), triethylorthopropionate (2 mL) was added, stirred for 1 hour at 150℃and concentrated under reduced pressure to give a yellow oil. The oil was isolated by column chromatography over Combiflash (12 g, 0-10% DCM/MeOH) to give tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-1, 3, 4-oxadiazol-2-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (120 mg,57% yield) MS m/z (ESI): 526.1[ M+1]

Step 2: tert-butyl (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-1, 3, 4-oxadiazol-2-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (100 mg,0.19 mmol) was dissolved in methanol (3 ml), trifluoroacetic acid (1 ml) at room temperature and stirred at room temperature for 4 hours. Concentration under reduced pressure afforded (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-1, 3, 4-oxadiazol-2-yl) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine (80 mg,95% yield) MSm/z (ESI): 426.1[ M+1]

Step 3: (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-1, 3, 4-oxadiazol-2-yl) phenyl) -7-methylimidazo [1, 2-a) ]Pyridin-3-yl) methyl morpholine (80 mg,0.19 mmol) was dissolved in dichloromethane (6 ml) and triethylamine (58 mg,0.57 mmol) and methyl chloroformate (27 mg,0.28 mmol) were added at 0℃and stirred at 0℃for 1 hour. Adding dichloromethane, washing with saturated sodium chloride solution, and removing organic phaseDried over sodium sulfate and concentrated under reduced pressure to give a yellow solid. The resulting residue was purified by preparative liquid chromatography to give the desired product methyl (S) -2- ((2- (2, 6-difluoro-4- (5-methyl-1, 3, 4-oxadiazol-2-yl) phenyl) -7-methylimidazo [1, 2-a)]Pyridin-3-yl) methyl morpholine-4-carboxylate (26 mg,20% yield) MSm/z (ESI) 484.1[ M+1]]； ¹ H NMR(400MHz,DMSO-d ₆ )δ8.42-8.40(d,J＝8Hz,1H),7.77-7.75(d,J＝8Hz,2H),7.33(s,1H),6.83-6.80(m,1H),3.75-3.71(m,1H),3.51-3.43(m,2H),3.35(s,3H),3.44-3.41(m,1H),3.20-3.18(m,1H),3.05-2.97(m,2H),2.80-2.73(m,2H),2.58(s,3H),2.35(s,3H)。

EXAMPLE 14 preparation of Compound H-14

(S) -3, 5-difluoro-4- (3- ((4- (methoxycarbonyl) morpholin-2-yl) methyl) -7-methylimidazole [1,2-a ] pyridin-2-yl) benzoic acid (50 mg,0.11 mmol) was dissolved in dichloromethane (6 mL), and (isocyanatoimino) triphenylphosphine (51 mg,0.17 mmol) was added and stirred at room temperature for 1 hour. Dichloromethane was added, the mixture was washed with saturated sodium chloride solution, and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a yellow solid. The solid was purified by preparative liquid chromatography to give compound H-14 (20 mg,38% yield) MSm/z (ESI): 470.1[ M+1];1H NMR (400 MHz, DMSO-d 6) delta 9.45 (s, 1H), 8.43-8.41 (d, J=8 Hz, 1H), 7.86-7.81 (m, 2H), 7.34 (s, 1H), 6.84-6.81 (m, 1H), 3.75-3.73 (m, 1H), 3.61-3.58 (m, 2H), 3.52 (s, 3H), 3.48-3.45 (m, 1H), 3.20-3.16 (m, 1H), 3.10-2.99 (m, 2H), 2.76-2.66 (m, 2H), 2.36 (s, 3H).

EXAMPLE 15 preparation of Compound H-15

Step 1: (S) -methyl 2- (((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (50 mg, 117.26. Mu. Mol) and hydroxylamine hydrochloride (12.22 mg, 175.88. Mu. Mol) were added to ethanol (10 mL), followed by the addition of DIEA (30.31 mg, 234.51. Mu. Mol.) the reaction solution was stirred at 80℃for 2 hours, the reaction solution was concentrated under reduced pressure directly to give (S) -methyl 2- (((2- (2, 6-difluoro-4- (N-hydroxycarbamoylamino)) phenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (52 mg, pale yellow oil),. MS m/z (ESI): 460.2)

Step 2: (S) -methyl 2- (((2- (2, 6-difluoro-4- (N-hydroxycarbamoylamino)) phenyl) -7-methylimidazo [1, 2-a)]Pyridin-3-yl) methyl morpholine-4-carboxylate (52 mg, 113.18. Mu. Mol) and N, N' -thiocarbonyldiimidazole (24.20 mg, 135.82. Mu. Mol) were added to THF (10 mL) and the reaction stirred at 30℃for 1 hour. Water (10 mL) was added, DCM (10 mLx 3) was used for extraction, and the organic phase was dried and concentrated under reduced pressure. The residue was dissolved in THF (10 mL), cooled to 0deg.C, and boron trifluoride etherate (48.19 mg, 339.54. Mu. Mol) was added. The reaction solution was naturally warmed to room temperature and stirred for 1 hour. Water (10 mL) was added, extracted with DCM (20 mLx 3), and the organic phase was dried and concentrated under reduced pressure. The residue obtained was purified by preparative liquid chromatography (preparative column: water-surfire; system: a: water+0.045% formic acid, B: acetonitrile; wavelength: 254/214nm; gradient: 5% -30% acetonitrile change) to give compound H-15 (2.84 mg, white solid), after lyophilization, yield: 5.00%. MS m/z (ESI): 502.2. ¹ H NMR(400MHz,CD ₃ OD)δ8.76(d,J＝7.2Hz,1H),7.81(t,J＝5.6Hz,2H),7.64(s,1H),7.31(d,J＝7.1Hz,1H),3.95(d,J＝12.9Hz,1H),3.78(d,J＝13.5Hz,1H),3.70(d,J＝11.7Hz,1H),3.65(s,4H),3.36–3.30(m,1H),3.21(d,J＝7.3Hz,2H),2.88(s,1H),2.66(s,1H),2.59(s,3H).

EXAMPLE 16 preparation of Compound H-16

Step 1: (S) -2- (((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (350 mg, 820.80. Mu. Mol) was dissolved in pyridine (10 mL), then (NH 4) 2S (306.98 mg, 902.88. Mu. Mol,20% purity) and TEA (91.36 mg, 902.88. Mu. Mol, 125.93. Mu. L) were added, the reaction was stirred at 60℃for 5 hours, and the reaction was monitored by LC-MS. After completion of the reaction, the solvent was dried by spin-drying, EA was washed twice with water, dried over anhydrous sodium sulfate, and dried by spin-drying under reduced pressure to give (S) -2- (((2- (4-aminomethyl-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (360 mg, yellow) as a solid in a yield of 95.461.24%. Z/MS (1.461)

Step 2: (S) -2- (((2- (4-Aminomethylsulfonyl-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (100 mg, 217.16. Mu. Mol) was dissolved in acetone (5 mL.) then the reaction solution was cooled to-20℃and oxalyl chloride (27.56 mg, 217.16. Mu. Mol) was added and the reaction stirred at-20℃for 3 hours and monitored by LC-MS for reaction conditions, spin-dry solvent to give (S) -2- ((2- (4, 5-dioxo-4, 5-dihydrothiazol-2-yl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (65 mg, yellow solid) as a direct step at 58.18% MS m/z (ESI 533.1)

Step 3: methyl (S) -2- ((2- (4, 5-dioxo-4, 5-dihydrothiazol-2-yl) -2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (65.00 mg, 126.34. Mu. Mol) was dissolved in xylene (2 mL) and trimethylsilyl azide (29.11 mg, 252.67. Mu. Mol) was then added. The reaction was stirred at 120℃for 4 hours and monitored by LC-MS. After the reaction was completed, the solvent was spin-dried, water and EA were added, EA was washed twice with water, and dried over anhydrous sodium sulfate. The resulting residue was purified by preparative liquid chromatography (preparative column: 21.2X250mm C18 column; system: 10mM NH4HCO 3H 2O; wavelength: 254/214nm; gradient: 30% -60% acetonitrile change) to give compound H-16 (6.46 mg, white solid). The yield was 9.89%. MS m/z (ESI) 502.0[ M+1]. NMR (400 mhz, dmso-d 6) δ:12.90 (s, 1H), 8.40 (d, j=8 hz, 1H), 7.77 (d, j=8 hz, 2H), 7.33 (s, 1H), 6.81 (d, j=8 hz, 1H), 3.75-3.72 (m, 1H), 3.63-3.58 (m, 2H), 3.51 (s, 3H), 3.45 (s, 1H), 3.22-3.16 (m, 2H), 3.08-2.97 (m, 2H), 2.77 (s, 1H), 2.35 (s, 3H).

EXAMPLE 17 preparation of Compound H-17

Step 1: (S) -methyl 2- (((2- (2, 6-difluoro-4- (N-hydroxycarbamoylamino)) phenyl) -7-methylimidazo [1, 2-a) ]Pyridin-3-yl) methyl morpholine-4-carboxylate (95 mg, 112.52. Mu. Mol) and N, N' -thiocarbonyldiimidazole (30.08 mg, 168.79. Mu. Mol) were added to 1, 4-dioxane (10 mL) followed by DBU (68.52 mg, 450.10. Mu. Mol). The reaction solution was stirred at 110℃for 3 hours. Concentrating under reduced pressure to obtain yellow crude product. The resulting residue was purified by preparative liquid chromatography (preparative column: 21.2x250mm c18 column; system: 10mm nh4hco 3H 2o; wavelength: 254/214nm; gradient: 30% -60% acetonitrile change) to give compound H-17 (5.58 mg, white solid), yield: 9.86%. MS m/z (ESI): 502.2. ¹ H NMR(400MHz,CD ₃ OD)δ8.40(d,J＝7.1Hz,1H),7.68(d,J＝8.2Hz,2H),7.33(s,1H),6.87(d,J＝7.2Hz,1H),3.85–3.69(m,3H),3.61(d,J＝14.0Hz,3H),3.56(dd,J＝16.3,8.3Hz,1H),3.36–3.30(m,1H),3.14–3.03(m,2H),2.85(s,1H),2.62(d,J＝18.1Hz,1H),2.44(s,3H).

EXAMPLE 18 preparation of Compound H-18

Methyl (S) -2- ((2- (4-carbamoyl-2, 6-difluorophenyl) -7-methylimidazo [1, 2-a)]Pyridin-3-yl) methyl morpholine-4-carboxylate (80 mg,0.18 mmol) was dissolved in methanol (5 ml) and ethyl 2-chloro-3-oxopropionate (50.93 mg, 338.26. Mu. Mol) and potassium tert-butoxide (60.5 mg,0.54 mmol) were stirred at 50℃for 3 hours. Concentrated to 5ml of solvent under reduced pressure, extracted with DCM, the organic phase dried and concentrated under reduced pressure to give a yellow oil. The oil was subjected to pre-HPLC to give Compound H-18 (11.68 mg, 21.80. Mu. Mol,9.67% yield,98.92% purity) MSm/z (ESI): 530.1[ M+1 ] ]。 ¹ H NMR(400MHz,DMSO-d ₆ )δ8.41-8.37(m,2H),7.92-7.90(d,J＝8Hz,2H),7.33(s,1H),6.82-6.80(m,1H),4.16-4.10(m,2H)3.74-3.72(m,1H),3.65-3.62(m,2H),3.52(s,3H),3.45-3.43(m,1H),3.19-3.17(m,1H),3.04-3.01(m,2H),2.46(s,3H)

EXAMPLE 19 preparation of Compound H-19

Step 1: (S) -methyl 2- (((2- (4-cyano-2, 6-difluorophenyl) -7-methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (50 mg,0.12 mmol) was dissolved in ethanol (10 mL), hydrazine hydrate (61 mg,1.05 mmol) was added, stirring overnight at room temperature, and the reaction mixture was directly dried by spinning to give (S) -methyl 2- (((2- (2, 6-difluoro-4- (hydrazino (imino) methyl) phenyl) -7) -methylimidazo [1,2-a ] pyridin-3-yl) methyl) morpholine-4-carboxylate (crude, 55 mg). MS m/z (ESI) 460.1[ M+1].

Step 2: (S) -methyl 2- (((2- (2, 6-difluoro-4- (hydrazino (imino) methyl) phenyl) -7) -methylimidazo [1, 2-a)]Pyridine-3-yl) methyl morpholine-4-carboxylate (crude 55 mg) was dissolved in absolute ethanol (10 ml), ethyl 2-oxoacetate (24 mg,0.24 mmol) and 0.2ml trifluoroacetic acid were added, and the temperature was raised to 130℃under microwave conditions to react for 20 minutes. Cooled to room temperature, concentrated and purified by preparative HPLC (preparation column: 21.2X250mM C18 column; system: 10mM NH) ₄ HCO ₃ H ₂ O; wavelength: 254/214nm; gradient: 30% -60% acetonitrile change) to give H-19 (7 mg, yield 11.8%) as a white solid MS m/z (ESI): 497.1[ M+1]]。 ¹ H NMR(400MHz,DMSO-d ₆ )δ8.43(d,J＝7.1Hz,1H),7.88-7.85(m,3H),7.35(s,1H),6.84(d,J＝7.1Hz,1H),3.75-3.73(m,1H),3.61-3.59(m,2H),3.52(s,3H),3.44(s,1H),3.19-3.16(m,2H),3.10–2.96(m,2H),2.76(s,1H),2.53–2.48(m,1H),2.36(s,3H).

Test example 1: FLIPR assay screening of Compounds against hP2X ₃ /hP2X _2/3 Antagonistic activity of receptors

Materials:

cell preparation: cells 1321N1/hP2X3 and 1321N1/hP2X2/3 (supplier Chempartner) were stably transfected with Versene digest, centrifuged, resuspended in plating medium (DMEM+10% DFBS) and used to obtain a final productCount, adjust cells to 3 x 10 ⁵ mu.L of cells per well in 384-well test plates were plated at 5% CO per mL ₂ Culturing in an incubator at 37 ℃ for 16-24h.

Cell culture medium formula:

hP2X3	working fluid concentration	Concentration of stock solution	Dilution factor	Required volume (ml)
					DMEM	1*	1*	1	447.006
FBS	10％	100％	10	50
					G418 disulfate salt	300μg/ml	50mg/ml	167	2.994012
			Sample preparation amount	500

hP2X2/3	Working fluid concentration	Concentration of stock solution	Dilution factor	Required volume (ml)
					DMEM	1*	1*	1	447.7489
FBS	10％	100％	10	50
					G418 disulfate salt	150μg/ml	50mg/ml	333	1.501502
Hygromycin B	75μg/ml	50mg/ml	667	0.749625
								Sample preparation amount	500

Experiment Dye formula:

hP2X3	working fluid concentration	Concentration of stock solution	Dilution factor	Required volume (ml)
					10*dye stock	0.5*	10*	20	0.7
Probenecid	1.25	0.25	200	0.07
					Test buffer	1*	1*	1	13.23
ATP hydrolytic enzyme	0.5U/ml	10ul＝1U	200	0.07
								Sample preparation amount	14

					hP2X2/3	Working fluid concentration	Concentration of stock solution	Dilution factor	Required volume (ml)
10*dye stock	0.5*	10*	20	0.7
					Probenecid	1.25	0.25	200	0.07
Test buffer	1*	1*	1	13.23
								Sample preparation amount	14

Compound preparation: 1. test article: in 384 wells meeting the Echo standardIn a polypropylene microplate, test compounds (54 mM DMSO stock) were prepared at 180-fold required concentrations with DMSO, 500nL per well was added to 384-well compound plates, and 30. Mu.L of test buffer (containing 1.26mM Ca) was supplemented ²⁺ Hbss+2mm CaCl ₂ +20mM HEPES), and shaken for 20-40min to mix well.

2. Agonists: preparation of agonist (. Alpha.,. Beta. -meATP) (hP 2X) at 3-fold required concentration with test buffer ₃ And hP2X _2/3 Cells were all required at a final concentration of 3000 nM), 45. Mu.L of agonist was added to 384 well compound plates per well.

Dye incubation: removing cell plate, sucking cell supernatant, and adding 30 μL Dye per well

Caltium 4Assay Kit, test buffer dilution) and incubation for 1h.

FLIPR detection: 15uL of compound (FLIPR instrument) was added to each well of the cell plate, and after 15 minutes, 22.5 uL of agonist was added to each well, and fluorescence signal was detected (excitation wavelength 470nm-495nm, emission wavelength 515nm-575 nm).

And (3) data processing: taking the difference between the peak value and the valley value of the signal as basic data, taking the highest concentration data of the positive medicine as 100% inhibition rate and taking the DMSO data as 0% inhibition rate, fitting an inhibition effect curve of the compound on the software Graphpad prism 6 by (log (inhibitor) vs. response-Variable slope) and calculating the IC50 value.

Experimental uniformity criteria: and each plate is subjected to more than or equal to 12 max values (DMSO action results) and more than or equal to 12 min values (highest concentration positive drug action results), Z values are calculated, and if Z is more than or equal to 0.5, parallel holes are considered to be uniform, and data are credible. The calculation formula of Z value: z=1-3 (sdmax+sdmin)/(MEANmax-MEANmin).

Inhibitory Activity of the Compounds of Table 1 on P2X3

As shown in Table 1, the compound of the embodiment of the invention has higher inhibition activity to P2X3, lower inhibition activity to P2X2/3 and obvious inhibition selectivity.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims

1. A compound of formula (I-d) or a pharmaceutically acceptable salt thereof:

in the method, in the process of the invention,

n1 is 1, R ₁ Is methyl;

m is 2, R ₃ Is fluorine;

ring C is thiazolidine-2, 4-dione, imidazolidine-2, 4-dione or a structure represented by formula (a);

wherein Z is- (NR) _1a )-、-O-、-S-；

R _1a Is hydrogen or C _1-3 An alkyl group;

is a single bond or a double bond;

R _2a 、R _3a is linked to form, together with the attached Z and nitrogen atom, a 5-to 8-membered heterocycloalkenyl ring, a 5-to 6-membered heteroaryl ring, or an 8-to 10-membered heteroaryl ring;

wherein the 5-to 8-membered heterocycloalkenyl ring has 2, 3 or 4 nitrogen atoms and 0, 1 or 2 oxygen atoms as ring atoms, and the 5-to 8-membered heterocycloalkenyl ring is selected from

The 5-to 6-membered heteroaryl ring is selected from oxadiazole rings;

the 8-to 10-membered heteroaryl ring is selected from benzimidazole rings;

u is 0, 1, 2, 3 or 4, R ₄ Is cyano, acetyl, hydroxy, carboxyl, halogen, C _1-8 Alkyl, C _1-8 An alkoxy group; wherein said C _1-8 Alkyl, C _1-8 Alkoxy is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from the group consisting of: halogen;

R ₀ is methyl.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the structure of formula (a) is selected from the group consisting of:

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the structure of formula (a) is selected from the group consisting of:

4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (I-d) is any one of the following:

5. A compound as shown below or a pharmaceutically acceptable salt thereof:

6. a pharmaceutical composition comprising: a compound of any one of claims 1-5, or a pharmaceutically acceptable salt thereof; a pharmaceutically acceptable carrier.

7. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease associated with P2X3 activity or with P2X2/3 activity.

8. Use of the pharmaceutical composition of claim 6 for the manufacture of a medicament for the treatment of a disease associated with P2X3 activity or with P2X2/3 activity.

9. The use according to claim 7 or 8, wherein the disease associated with P2X3 activity or P2X2/3 activity is pain, a urinary tract disorder, a gastrointestinal disorder, cancer, an immune-related disorder, cough, depression, anxiety or a stress-related disorder.