WO2025119372A2 - Mrgprx2 antagonist and use thereof - Google Patents

Abstract

Provided in the present invention are a compound as shown in formula IA, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof. The compound has a good MRGPRX2 antagonistic effect.

Description

MRGPRX2 antagonists and uses

This application requires the applicant to:

The priority benefit of the prior application for patent application number 202311693381.1, entitled “MRGPRX2 antagonists and uses”, filed with the State Intellectual Property Office of China on December 8, 2023;

The priority benefit of the prior application for patent application number 202410166370.6, entitled “MRGPRX2 antagonists and uses”, filed with the State Intellectual Property Office of China on February 5, 2024;

The priority benefit of the prior application for patent application number 202411392820.X, entitled “MRGPRX2 antagonists and uses”, filed with the State Intellectual Property Office of China on September 30, 2024;

The priority benefit of the prior application for patent application number 202411507978.7, entitled “MRGPRX2 antagonists and uses”, filed with the State Intellectual Property Office of China on October 25, 2024;

The priority benefit of the prior application for patent application number 202411754629.5, entitled “MRGPRX2 antagonists and uses”, filed with the State Intellectual Property Office of China on November 29, 2024;

The entirety of said prior application is incorporated into the present application by reference.

Technical Field

The present invention belongs to the field of medicine, and specifically, the present invention relates to a MRGPRX2 antagonist and its use.

Background Art

Mast cells (MCs) are tissue-resident immune cells that originate from the hematopoietic lineage. MCs reside in a variety of connective tissues and vascularized organs. Their numbers are largest and their density is highest at the interface between the internal and external environments. They respond to foreign organisms and antigens and play a sentinel role. These sites include the dermis, skeletal muscle, oral mucosa, gastrointestinal mucosa and submucosa, conjunctiva, alveoli and airways, and auricles. MCs in the dermis are often very close to blood vessels, nerves, and lymphatic vessels. After activation by human high-affinity immunoglobulin E (IgE) receptor (FcεRI) and Mas-related G protein-coupled receptor X2 (MRGPRX2), MCs degranulate, release a variety of bioactive substances (such as histamine and proteases) or synthesize new prostaglandins, leukotrienes, and some cytokines from scratch, and play a central role in host defense, inflammation, and allergic reactions.

Mas-related G protein-coupled receptors (MRGPRs) are divided into nine subfamilies according to the different receptor members: MRGPRA, B, C, D, E, F, G, H and MRGPRX, which is unique to primates. Each subfamily includes different subtypes, such as MRGPRX1, MRGPRX2, MRGPRX3, and MRGPRX4. Transcriptome analysis shows that most MRGPR family members are expressed in peripheral neurons, but MRGPRX2 is mainly expressed in skin MCs, and its expression rate is even higher than Fcε-RI. MRGPRX2 can sense a variety of endogenous or exogenous agonists including polycationic compounds and peptides to trigger mast cell degranulation reactions.

Studies have shown that inappropriate activation of MRGPRX2 may lead to mast cell-related diseases such as drug pseudoallergy, rosacea, atopic dermatitis, allergic contact dermatitis, urticaria, pruritus, mastocytosis, interstitial cystitis, pain, rheumatoid arthritis, asthma and ulcerative colitis. At present, the IND application of MRGPRX2 antagonist EP262 has been approved by FDA for the treatment of mast cell-related diseases (such as chronic urticaria). Preclinical studies have shown that EP262 can effectively inhibit mast cell activation and degranulation induced by various MRGPRX2 agonists, and block the release of trypsin and inflammatory factors in human mast cells. Oral administration of EP262 can effectively inhibit mast cell degranulation and increased vascular permeability in MRGPRX2-KI mice induced by agonists, showing significant potential for the treatment of mast cell-related diseases. All these indicate that the development of new MRGPRX2 antagonists for the treatment of various mast cell-related diseases is a potential direction.

Currently, there are no drugs on the market that act as MRGPRX2 antagonists. Therefore, the development of new compounds that can antagonize MRGPRX2 activity has positive significance for the treatment of diseases.

Summary of the invention

The object of the present invention is to provide a novel compound for use as a MRGPRX2 antagonist.

In the first aspect of the present invention, there is provided a compound of formula IA, a tautomer, a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof:

in,

X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ each independently represent a ring atom;

X ¹ , X ² , X ³ , X ⁵ and X ⁶ are each independently N, CH ₂ , CH or C;

X ⁴ is C;

The bond between ^X1 and ^X2 , and between ^X5 and ^X6 is a single bond or a double bond;

The group fragment formed by connecting ^X5 and ^X6 wherein A is absent, ^X5 and ^X6 are each independently substituted by ^R4 or ^R5 ; or when A is present, A, together with ^X5 and ^X6 ring atoms, forms a 6-10 membered aryl group, a 3-11 membered heterocycloalkyl group or a 5-10 membered heteroaryl group; and said A is further substituted by ^Rc ; said ^Rc is substituted by one or more substituents, and when there are multiple substituents ^Rc , said substituents are the same or different; the heteroatoms in said heterocycloalkyl group and heteroaryl group are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3;

When A, X ⁵ and X ⁶ together form a 6-membered aryl group, ^Ra is oxo;

Alternatively, when A, ^X5 and ^X6 ring atoms together form a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatom in the heteroaryl group is selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3;

Ring B is C _3-12 cycloalkyl;

Ring C is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3;

R ^a , R ^c , R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently H, halogen, cyano, amino, oxo, C _1-6 alkyl, C 1-6 alkyl substituted by hydroxy, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 cycloalkyl, halogenated C _3-8 cycloalkyl, -OR _f , -C(O) ^{OR f} , -OC(O)R ^f , -N(R ^f ) ₂ , -N(R ^f )C(O)R ^f , -N(R ^f )S(O) ₂ R ^f or -S(O) ₂ R ^f ; R ^f is selected from H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkylamino, -(CH ₂ ) r R ^g , 6-10 membered aryl, C _3-8 cycloalkyl, 5-10 membered heteroaryl or 5-10 membered heterocycloalkyl, or two R ^f groups together with the atoms to which they are connected form a 5-11 membered heterocycloalkyl; the R ^g is H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-8 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl or 5-10 membered heterocycloalkyl; the heteroatoms in the heterocycloalkyl and heteroaryl groups are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3;

R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C 3-8 halocycloalkyl, C _1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-10 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR ^j )-R ^k , -(CHR j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O ⁾ -N(R ^k ) ₂ , -(CHR ^j ) _0-3 -(N═)S(═O)(R ^k ) ₂ , -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; The C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl are optionally substituted by one or more R ^d ; the R ^d is selected from the following substituents: halogen, hydroxyl, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _{1-6 haloalkyl, C 1-6} alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl; when there are _multiple substituents R ^d , the R ^d are the same or different; the heteroatoms in the heterocycloalkyl are selected from one or more of N, O, S, and the number of heteroatoms is 1, 2 or 3;

R ^j and R ^k are each independently H, cyano, amino, C _1-6 alkyl, C _1-6 alkylamino, -(CH ₂ ) _0-3 C _3-8 cycloalkyl, -COC _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkyl substituted with hydroxyl, or a single 5-10 membered heterocycloalkyl; the heteroatom in the heterocycloalkyl is selected from one or more of N, O, and S, and the number of the heteroatoms is 1, 2, or 3;

m, n, and r are 0, 1, 2, or 3 respectively.

According to an embodiment of the present invention, R ^b is H, F, Cl, -CN, methyl, amino, methoxy, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH ₂ -SO ₂ -CH ₃ , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ ,

According to an embodiment of the present invention, ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, F, Cl, methyl, ethyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,}

According to an embodiment of the present invention, the group fragment formed by the connection of ^X5 and ^X6 In the formula (a), A is absent, ^X5 and ^X6 are each independently substituted by ^R4 or ^R5 , then the structural fragment is Selected from In some embodiments, R ⁴ and R ⁵ are each independently H, F, Cl, methyl, -CF ₃ , or -CHF ₂ .

In some embodiments, the group fragment formed by the connection of X ⁵ and X ⁶ In the formula (a), A is absent, ^X5 and ^X6 are each independently substituted by ^R4 or ^R5 , then the structural fragment is Selected from

According to an embodiment of the present invention, the group fragment formed by the connection of ^X5 and ^X6 When A exists, A, together with X ⁵ and X ⁶ ring atoms, forms And the A is further substituted by R ^c .

In some embodiments, when A is present, the structural fragment Selected from

In some embodiments, when A is present, the structural fragment Selected from

In some embodiments, the structural fragment Selected from the following structures:

According to an embodiment of the present invention, the ring C is a 6-8 membered aryl group or a 5-10 membered heteroaryl group; the heteroatoms in the heteroaryl group are selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2 or 3.

According to an embodiment of the present invention, the ring C is a 5- to 6-membered nitrogen-containing heteroaryl group, and the number of the nitrogen atoms is 1, 2 or 3.

According to an embodiment of the present invention, the ring C is phenyl, pyrazolyl or pyridyl.

According to an embodiment of the present invention, the ring B is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.

According to an embodiment of the present invention, the structural fragment Selected from

In some embodiments, the structural fragment Selected from

In some embodiments, the formula IA can be selected from the following IA-1:

wherein A, Ring B, Ring C, X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , R ¹ , R ² , R ³ , R ^b , m and n are as defined in Formula IA.

In a second aspect of the present invention, there is provided a compound of formula I, a tautomer, a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof:

According to an embodiment of the present invention, in the first aspect, the formula IA is selected from formula I, and the groups in the formula I have the definitions as described in formula IA;

According to an embodiment of the present invention,

In the formula I,

X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ each independently represent a ring atom;

X ¹ , X ² , X ³ , X ⁵ and X ⁶ are each independently N, CH ₂ , CH or C;

X ⁴ is C;

The bond between ^X1 and ^X2 , and between ^X5 and ^X6 is a single bond or a double bond;

The group fragment formed by connecting ^X5 and ^X6 wherein A is absent, ^X5 and ^X6 are each independently substituted by ^R4 or ^R5 ; or when A is present, A, together with ^X5 and ^X6 ring atoms, forms a 6-10 membered aryl group, a 3-11 membered heterocycloalkyl group or a 5-10 membered heteroaryl group; the heteroatoms are independently selected from one or more of N, O and S; and A is further substituted by ^Rc ; the ^Rc is substituted with one or more substitutions, and when there are multiple substituents ^Rc , the substituents are the same or different; the heteroatoms in the heterocycloalkyl group and the heteroaryl group are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3;

When A, X ⁵ and X ⁶ together form a 6-membered aryl group, ^Ra is oxo;

Alternatively, when A, ^X5 and ^X6 ring atoms together form a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatom in the heteroaryl group is selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3;

Ring B is C _3-12 cycloalkyl;

Ring C is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3;

R ^a , R ^c , R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently H, halogen, cyano, amino, oxo, C _1-6 alkyl, C 1-6 alkyl substituted by hydroxy, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 cycloalkyl, halogenated C _3-8 cycloalkyl, -OR _f , -C(O) ^{OR f} , -OC(O)R ^f , -N(R ^f ) ₂ , -N(R ^f )C(O)R ^f , -N(R ^f )S(O) ₂ R ^f or -S(O) ₂ R ^f ; R ^f is selected from H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkylamino, -(CH ₂ ) r R ^g , 6-10 membered aryl, C _3-8 cycloalkyl, 5-10 membered heteroaryl or 5-10 membered heterocycloalkyl, or two R ^f groups together with the atoms to which they are connected form a 5-11 membered heterocycloalkyl; the R ^g is H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-8 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl or 5-10 membered heterocycloalkyl; the heteroatoms in the heterocycloalkyl and heteroaryl groups are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3;

R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C 2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C 1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C 1-6 alkoxy, C _1-6 haloalkoxy or 3-10 membered heterocycloalkyl; the C 1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C 3-8 halocycloalkyl, C _1-6 alkylhydroxyl, C 1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or 3-10 membered heterocycloalkyl; the C _1-6 alkyl, C 3-8 cycloalkyl, C _2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C _3-8 halocycloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C 1-6 alkoxy, C _1-6 haloalkoxy is optionally substituted by one or more R ^d ; the R ^d is selected from the following substituents: halogen, hydroxyl, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _{C 1-6} alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy; when there are multiple substituents R ^d , the R ^d are the same or different; the heteroatom in the heterocycloalkyl is selected from one or more of N, O, and S, and the number of the heteroatoms is 1, 2 or 3;

m, n, and r are 0, 1, 2, or 3 respectively.

In the present invention, the definitions of certain substituents in the compounds of Formula IA or Formula I may be as described below, and the definitions of substituents not mentioned are as described in any of the above schemes.

In a preferred embodiment of the present invention, the compound shown in Formula IA or Formula I is selected from the following structures:

Among them, the group fragment formed by connecting ^X5 and ^X6 wherein A is as defined in the first aspect of the present invention; ring C is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3; and ^X1 , ^X2 , ^X3 , ^X4 , ^X5 , ^X6 , ^R1 , ^R2 , ^R3 , ^Ra , ^Rb , m, and n are as defined in the first aspect of the present invention.

In a preferred embodiment of the present invention, the compound as shown in formula IA is selected from the following structures: Among them, the group fragment formed by connecting ^X5 and ^X6 wherein A is as defined in the first aspect of the present invention; ring C is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3; and ^X1 , ^X2 , ^X3 , ^X4 , ^X5 , ^X6 , ^R1 , ^R2 , ^R3 , ^Ra , ^Rb , m, and n are as defined in the first aspect of the present invention.

In a preferred embodiment of the present invention, the compound as shown in formula I is selected from the following structures: Among them, the group fragment formed by connecting ^X5 and ^X6 wherein A is as defined in the first aspect of the present invention; ring C is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3; and ^X1 , ^X2 , ^X3 , ^X4 , ^X5 , ^X6 , ^R1 , ^R2 , ^R3 , ^Ra , ^Rb , m, and n are as defined in the first aspect of the present invention.

In a preferred embodiment of the present invention, the compound as shown in formula IA is selected from the following structures:

wherein A, together with ^X5 and ^X6 ring atoms, forms a 6- to 8-membered aryl group, a 3- to 8-membered heterocycloalkyl group, a 5-membered heteroaryl group or a 6-membered heteroaryl group; the heteroatoms are independently selected from one or more of N, O and S; A is further substituted by ^Rc ; the ^Rc is substituted with one or more substitutions, and when there are multiple substituents ^Rc , the substituents are the same or different; and when A, together with ^X5 and ^X6 ring atoms, forms a 6-membered aryl group, ^Ra is oxo; or, when A, together with ^X5 and ^X6 ring atoms, forms a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatoms in the heterocycloalkyl group and the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; Ring C is a 6- to 10-membered aryl group or a 5- to 8-membered heteroaryl group; the heteroatoms in the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C 1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-8 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR ^j )-R ^k , -(CHR j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O)-N(R ^k ) ₂ , -(CHR ^j ) _0-3 - ⁽ N═)S(═O)(R ^k ) ₂ , -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; the C The heterocycloalkyl radical is selected from one or more of N, O and S, and the number of heteroatoms is ₁ , _{2 or 3 ;} Rj ^{and Rk are} each independently H, cyano, amino, _C1-6 alkyl, _C2-6 alkenyl _{, C2-6} alkynyl, _{C1-6 haloalkyl} , _C3-8 halocycloalkyl, _C1-6 ^{alkylhydroxyl} , _C1-6 alkylcarbonyl, _C1-6 ^alkoxyl _{and C1-6 haloalkoxyl .} R ^a , R c , R ¹ , R ₂ , and R ³ are each independently H, halogen, _cyano , C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 cycloalkyl, or halogenated C _{3-8 cycloalkyl} ; _X ¹ , ^X ₂ , X ^{3 , X 4} _, ^{X 5 ,} X ⁶ , ^m and n are as defined in the first aspect of _the present invention. In a preferred embodiment of the present invention, R ^b is H, F, Cl, -CN, methyl, amino, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH ₂ -SO ₂ -CH ₃ , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ , In a preferred embodiment of the present invention, ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, F, Cl, methyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,}

In a preferred embodiment of the present invention, the compound as shown in formula IA is selected from the following structures:

wherein A, together with ^X5 and ^X6 ring atoms, forms a 6- to 8-membered aryl group, a 3- to 8-membered heterocycloalkyl group, a 5-membered heteroaryl group or a 6-membered heteroaryl group; the heteroatoms are independently selected from one or more of N, O and S; A is further substituted by ^Rc ; the ^Rc is substituted with one or more substitutions, and when there are multiple substituents ^Rc , the substituents are the same or different; and when A, together with ^X5 and ^X6 ring atoms, forms a 6-membered aryl group, ^Ra is oxo; or, when A, together with ^X5 and ^X6 ring atoms, forms a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatoms in the heterocycloalkyl group and the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; Ring C is a 6- to ₁₀ -membered aryl group or a 5- to 8-membered heteroaryl group; the heteroatoms in the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C 1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-8 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR ^j )-R ^k , -(CHR j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O)-N(R ^k ) ₂ , -(CHR ^j ) _0-3 - ⁽ N═)S(═O)(R ^k ) ₂ , -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; the C The heterocycloalkyl radical is selected from one or more of N, O and S, and the number of heteroatoms is ₁ , _{2 or 3 ;} Rj ^{and Rk are} each independently H, cyano, amino, _C1-6 alkyl, _C2-6 alkenyl _{, C2-6} alkynyl, _{C1-6 haloalkyl} , _C3-8 halocycloalkyl, _C1-6 ^{alkylhydroxyl} , _C1-6 alkylcarbonyl, _C1-6 ^alkoxyl _{and C1-6 haloalkoxyl .} R ^a , R c , R ¹ , R ₂ , and R ³ are each independently H, halogen, _cyano , C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 cycloalkyl, or halogenated C _{3-8 cycloalkyl} ; _X ¹ , ^X ₂ , X ^{3 , X 4} _, ^{X 5 ,} X ⁶ , ^m and n are as defined in the first aspect of _the present invention. In a preferred embodiment of the present invention, R ^b is H, F, Cl, -CN, methyl, methoxy, amino, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH 2 -SO _{2 -CH 3} , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ , In a preferred embodiment of the present invention, ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, F, Cl, methyl, ethyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,}

In a preferred embodiment of the present invention, the compound as shown in formula I is selected from the following structures:

wherein A, together with ^X5 and ^X6 ring atoms, forms a 6- to 8-membered aryl group, a 3- to 8-membered heterocycloalkyl group, a 5-membered heteroaryl group or a 6-membered heteroaryl group; the heteroatoms are independently selected from one or more of N, O and S; A is further substituted by ^Rc ; the ^Rc is substituted with one or more substitutions, and when there are multiple substituents ^Rc , the substituents are the same or different; and when A, together with ^X5 and ^X6 ring atoms, forms a 6-membered aryl group, ^Ra is oxo; or, when A, together with ^X5 and ^X6 ring atoms, forms a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatoms in the heterocycloalkyl group and the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; Ring C is a 6- to 10-membered aryl group or a 5- to 8-membered heteroaryl group; the heteroatoms in the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C 2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-8 membered heterocycloalkyl; the heteroatom in the heterocycloalkyl is selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3; ^Ra , Rc, ^R1 , ^R2 and R3 are each independently H, halogen, cyano, C 1-6 alkyl, C _{2-6 alkenyl, C 2-6} alkynyl, C _1-6 haloalkyl, C 3-8 cycloalkyl or halo C _3-8 cycloalkyl; ^X1 , ^X2 , ^X3 , ^X4 , ^X5 are each independently H, halogen, cyano, C _1-6 alkyl, C 2-6 alkenyl, C _2-6 alkynyl, C 1-6 haloalkyl, C _3-8 cycloalkyl or halo C ^3-8 cycloalkyl; ^X5 , ^X6 , m and n are as defined in the first aspect of the present invention. In a preferred embodiment of the present invention, ^Rb is H, F, Cl, methyl, amino, oxo, _-CH2CF3 , _{-CF3 .} In a preferred embodiment of the present invention, ^Ra , ^Rc , ^R1 , ^R2 , ^R3 are each independently H, F, Cl, methyl, amino, oxo, _-CH2CF3 , _{-CF3 ,}

In a preferred embodiment of the present invention, the compound as shown in formula IA is selected from the following structures:

wherein A, together with ^X5 and ^X6 ring atoms, forms a 6- to 8-membered aryl group, a 3- to 8-membered heterocycloalkyl group, a 5-membered heteroaryl group or a 6-membered heteroaryl group; the heteroatoms are independently selected from one or more of N, O and S; A is further substituted by ^Rc ; the ^Rc is substituted with one or more substitutions, and when there are multiple substituents ^Rc , the substituents are the same or different; and when A, together with ^X5 and ^X6 ring atoms, forms a 6-membered aryl group, ^Ra is oxo; or, when A, together with ^X5 and ^X6 ring atoms, forms a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatoms in the heterocycloalkyl group and the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; Ring C is a 6- to 10-membered aryl group or a 5- to 8-membered heteroaryl group; the heteroatoms in the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; R ^b is H, halogen, hydroxy, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C _1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-8 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR j )-R ^k , -(CHR ^j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O)-N(R ^k ) ₂ , -(CHR ^j ) _0-3 -(N═)S(═O)(R ^k ) ₂ ^, -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; the C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C 1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl are optionally substituted by one or more R ^d ; the R ^d is selected from the following substituents: halogen, hydroxyl, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C 1-6 alkoxyl, C _1-6 haloalkoxyl; the heteroatoms in the heterocycloalkyl are selected from one or more of N, O, S, and the number of heteroatoms is 1, 2 or 3; R ^j and R ^k are each independently H, cyano, amino, C _2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C _1-6 alkylhydroxyl, C 1-6 alkylcarbonyl, C 1-6 alkoxyl, C 1-6 haloalkoxyl R ^a , R c , R ₁ , R ₂ , and R ³ _are each independently _H , halogen, cyano, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 cycloalkyl, or halogenated C _3-8 cycloalkyl; _X ¹ , ^{X 2} , ^{X 3} , ^X ₄ , ^{X 5 , X 6} _, ^{m and} n are as defined in the _first aspect of the present invention. In a preferred embodiment of the present invention, R ^b is H, F, Cl, -CN, methyl, methoxy, amino, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH 2 -SO _{2 -CH 3} , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ , In a preferred embodiment of the present invention, ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, F, Cl, methyl, ethyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,}

In a preferred embodiment of the present invention, the compound as shown in formula IA is selected from the following structures:

wherein A, together with ^X5 and ^X6 ring atoms, forms a 6- to 8-membered aryl group, a 3- to 8-membered heterocycloalkyl group, a 5-membered heteroaryl group or a 6-membered heteroaryl group; the heteroatoms are independently selected from one or more of N, O and S; A is further substituted by ^Rc ; the ^Rc is substituted with one or more substitutions, and when there are multiple substituents ^Rc , the substituents are the same or different; and when A, together with ^X5 and ^X6 ring atoms, forms a 6-membered aryl group, ^Ra is oxo; or, when A, together with ^X5 and ^X6 ring atoms, forms a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatoms in the heterocycloalkyl group and the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; Ring C is a 6- to 10-membered aryl group or a 5- to 8-membered heteroaryl group; the heteroatoms in the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C 1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-8 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR ^j )-R ^k , -(CHR j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O)-N(R ^k ) ₂ , -(CHR ^j ) _0-3 - ⁽ N═)S(═O)(R ^k ) ₂ , -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; the C The heterocycloalkyl radical is selected from one or more of N, O and S, and the number of heteroatoms is ₁ , _{2 or 3 ;} Rj ^{and Rk are} each independently H, cyano, amino, _C1-6 alkyl, _C2-6 alkenyl _{, C2-6} alkynyl, _{C1-6 haloalkyl} , _C3-8 halocycloalkyl, _C1-6 ^{alkylhydroxyl} , _C1-6 alkylcarbonyl, _C1-6 ^alkoxyl _{and C1-6 haloalkoxyl .} R ^a , R c , R ¹ , R ₂ , and R ³ are each independently H, halogen, _cyano , C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 cycloalkyl, or halogenated C _{3-8 cycloalkyl} ; _X ¹ , ^X ₂ , X ^{3 , X 4} _, ^{X 5 ,} X ⁶ , ^m and n are as defined in the first aspect of _the present invention. In a preferred embodiment of the present invention, R ^b is H, F, Cl, -CN, methyl, methoxy, amino, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH 2 -SO _{2 -CH 3} , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ , In a preferred embodiment of the present invention, ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, F, Cl, methyl, ethyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,}

In a preferred embodiment of the present invention, the compound as shown in formula IA is selected from the following structures:

wherein A, together with ^X5 and ^X6 ring atoms, forms a 6- to 8-membered aryl group, a 3- to 8-membered heterocycloalkyl group, a 5-membered heteroaryl group or a 6-membered heteroaryl group; the heteroatoms are independently selected from one or more of N, O and S; A is further substituted by ^Rc ; the ^Rc is substituted with one or more substitutions, and when there are multiple substituents ^Rc , the substituents are the same or different; and when A, together with ^X5 and ^X6 ring atoms, forms a 6-membered aryl group, ^Ra is oxo; or, when A, together with ^X5 and ^X6 ring atoms, forms a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatoms in the heterocycloalkyl group and the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; Ring C is a 6- to 10-membered aryl group or a 5- to 8-membered heteroaryl group; the heteroatoms in the heteroaryl group are selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C 1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-8 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR ^j )-R ^k , -(CHR j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O)-N(R ^k ) ₂ , -(CHR ^j ) _0-3 - ⁽ N═)S(═O)(R ^k ) ₂ , -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; the C The heterocycloalkyl radical is selected from one or more of N, O and S, and the number _of heteroatoms is ₁ , _{2 or 3 ;} Rj ^{and Rk are} each independently H, cyano, amino, _C1-6 alkyl, _C2-6 alkenyl _{, C2-6} alkynyl, _C1-6 haloalkyl, _C3-8 halocycloalkyl, _C1-6 ^{alkylhydroxyl} , _C1-6 alkylcarbonyl, _C1-6 ^alkoxyl _{and C1-6 haloalkoxyl .} R ^a , R c , R ¹ , R ₂ , and R ³ are each independently H, halogen, _cyano , C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 cycloalkyl, or halogenated C _{3-8 cycloalkyl} ; _X ¹ , ^X ₂ , X ^{3 , X 4} _, ^{X 5 ,} X ⁶ , ^m and n are as defined in the first aspect of _the present invention. In a preferred embodiment of the present invention, R ^b is H, F, Cl, -CN, methyl, methoxy, amino, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH 2 -SO _{2 -CH 3} , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ , In a preferred embodiment of the present invention, ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, F, Cl, methyl, ethyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,}

In some embodiments of the present invention, the compound represented by formula I is selected from the following structures:

Wherein, Ring C, X ³ , R ¹ , R ⁴ , R ⁵ , R ^b and n are as defined above.

In a preferred embodiment of the present invention, the compound as shown in formula I is selected from the following structures:

wherein ring C is a 6-10 membered aryl or a 5-8 membered heteroaryl; the heteroatoms in the heteroaryl are selected from one or more of N, O, and S, and the number of the heteroatoms is 1, 2, or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C 1-6 alkyl, C _3-8 cycloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C _3-8 halocycloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C 1-6 haloalkoxy, or a _3-8 membered heterocycloalkyl; the heteroatoms in the heterocycloalkyl are selected from one _{or more} of N, O, and S, and the number of the heteroatoms is 1, ₂ , or 3; Ra, R 1, R ₂ , R ³ , R ⁴ , and R 5 are each independently H, halogen, cyano, amino, oxo, C _1-6 alkyl, C 3-8 cycloalkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 haloalkyl, C 3-8 halocycloalkyl, C ^1-6 alkylhydroxyl, C ^1-6 alkylcarbonyl, C _1-6 alkoxy, C ^1-6 haloalkoxy, or a 3-8 membered heterocycloalkyl; the heteroatoms in the heterocycloalkyl are selected from one ^{or more} of N, O, and S, and the number of the heteroatoms is 1, 2, or 3; wherein _X ¹ , X ² , _X 3 , X ⁴ , X ⁵ , X ⁶ , m and n are as defined in the _first aspect of the present invention. In a preferred embodiment of _the present invention, said ^{R b} _is H, F, Cl, methyl, amino, oxo, -CH ₂ CF ³ , -CF ₃ . In a preferred embodiment of the present invention, said ^Ra , R ¹ , R ² , R ₃ , R ⁴ , R ⁵ are each independently H ^, F, Cl, methyl, amino, oxo, -CH ₂ CF ₃ , -CF ₃ ,

In a preferred embodiment of the present invention, ring C is a 6-8 membered aryl group or a 5-10 membered heteroaryl group. The heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of the heteroatoms is 1, 2, or 3.

In a preferred embodiment of the present invention, ring C is a 5- to 6-membered nitrogen-containing heteroaryl group, and the number of the nitrogen atoms is 1, 2 or 3.

In a preferred embodiment of the present invention, ring C is phenyl, pyrazolyl or pyridinyl.

In a preferred embodiment of the present invention, the compound as shown in formula IA is selected from any one of the following compounds:

In a preferred embodiment of the present invention, the pharmaceutically acceptable salt of the compound of Formula IA is trifluoroacetate.

In a preferred embodiment of the present invention, the compound as shown in formula IA is selected from any one of the following compounds:

In a preferred embodiment of the present invention, the pharmaceutically acceptable salt of the compound of Formula IA or Formula I is trifluoroacetate.

The third aspect of the present invention provides a pharmaceutical composition, which comprises: a compound of formula IA or formula I as described in the first or second aspect of the present invention, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug; and a pharmaceutically acceptable carrier.

In the fourth aspect of the present invention, there is provided the use of the compound represented by formula IA or formula I as described in the first or second aspect of the present invention, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, or the use of the pharmaceutical composition described in the third aspect of the present invention, the uses including: antagonizing MRGPRX2; and/or, preventing and/or treating MRGPRX2-related diseases; and/or, preparing drugs, pharmaceutical compositions or preparations for antagonizing MRGPRX2, and/or preventing and/or treating MRGPRX2-related diseases.

Preferably, the MRGPRX2-related diseases include: mast cell-related diseases.

Preferably, the MRGPRX2-related diseases include: skin diseases, autoimmune diseases, and nervous system diseases.

Preferably, the skin disease is selected from atopic dermatitis, contact dermatitis, urticaria, and pruritus.

Preferably, the urticaria is selected from chronic spontaneous urticaria and induced urticaria.

Preferably, the autoimmune disease is selected from the group consisting of allergy, mastocytosis, rheumatoid arthritis, asthma, ulcerative colitis and interstitial cystitis.

Preferably, the neurological disease is selected from pain.

In the fifth aspect of the present invention, a method for antagonizing MRGPRX2, or preventing and/or treating MRGPRX2-related diseases is provided, comprising the steps of administering to a subject in need thereof a compound of formula IA or formula I, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs as described in the first or second aspect of the present invention.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

Terms and Definitions

Unless otherwise specified, the terms and definitions used in the present application, including the description and claims of this application, are as follows. It will be understood by those skilled in the art that, according to the conventions used in the art, in the structural formula of this application, Used to depict chemical bonds, which are the points at which a moiety or substituent is attached to a core or backbone structure.

Unless otherwise specified, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

Unless otherwise specified, the term "pharmaceutically acceptable salt" refers to salts of pharmaceutically acceptable non-toxic acids or bases including salts of inorganic acids and bases, and organic acids and bases.

Unless otherwise specified, the term "pharmaceutical composition" means a mixture of one or more compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs with other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.

Unless otherwise specified, the term "prodrug" refers to a compound of the present invention that can be converted into a biologically active compound under physiological conditions or by solvolysis. The prodrug of the present invention is prepared by modifying the functional groups in the compound, and the modification can be removed by conventional operations or in vivo to obtain the parent compound. The prodrug includes a compound formed by connecting a hydroxyl or amino group in the compound of the present invention to any group. When the prodrug of the compound of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free hydroxyl group and a free amino group, respectively.

Unless otherwise specified, the term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, and includes cis-trans isomers, enantiomers, diastereomers and conformational isomers.

Depending on the choice of raw materials and methods, the compounds of the present invention may exist in the form of one of the possible isomers or a mixture thereof, for example as a pure optical isomer, or as a mixture of isomers, such as a racemic and diastereomeric mixture, depending on the number of asymmetric carbon atoms. When describing optically active compounds, prefixes D and L or R and S are used to represent the absolute configuration of the molecule with respect to the chiral center (or multiple chiral centers) in the molecule. The prefixes D and L or (+) and (–) are symbols for the rotation of plane polarized light caused by the specified compound, where (–) or L indicates that the compound is left-handed. Compounds prefixed with (+) or D are right-handed. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be referred to as enantiomers, and mixtures of the isomers are generally referred to as mixtures of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process. Many geometric isomers of olefins, C=N double bonds, etc. may also exist in the compounds described herein, and all such stable isomers are contemplated in the present invention. When the compounds described herein contain olefinic double bonds, unless otherwise specified, such double bonds include both E and Z geometric isomers. If the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituents may be in the cis- or trans- configuration.

When the bonds to the chiral carbon in the formula of the present invention are depicted as straight lines, it should be understood that both the (R) and (S) configurations of the chiral carbon and the enantiomerically pure compounds and mixtures thereof produced therefrom are included within the scope of the general formula. The graphic representation of racemates or enantiomerically pure compounds herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, the absolute configuration of a stereocenter is indicated by a wedge-shaped bond and a dashed bond.

Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral preparations, or resolved using conventional techniques. Compounds of the invention containing asymmetrically substituted carbon atoms can be separated in optically active form or racemic form. Resolution of a racemic mixture of a compound can be carried out by any of a number of methods known in the art. Exemplary methods include fractional recrystallization using a chiral resolution acid that is an optically active salified organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids such as the D and L forms of β-camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include α-methyl-benzylamine (e.g., S and R forms or diastereomeric pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, etc. The resolution of the racemic mixture can also be carried out by eluting on a chromatographic column filled with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). High performance liquid chromatography (HPLC) can also be used to carry out supercritical fluid chromatography (SFC). The selection of specific methods and elution conditions, the selection of chromatographic columns can be selected by those skilled in the art according to the structure of the compound and the test results. Further, optically pure starting materials or reagents of known configurations can also be used to obtain any enantiomer or diastereomer of the compounds described in the present invention through stereo organic synthesis.

Unless otherwise specified, the term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom in a molecule between two positions. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Prototropic tautomers arise from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to separate a single tautomer usually produce a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical characteristics within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form is predominant; while in phenols, the enol form is predominant. The present invention includes all tautomeric forms of the compounds.

Unless otherwise specified, the key is a solid wedge. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicates the relative configuration of a stereocenter.

Unless otherwise indicated, the term "solvate" means that the compound of the present invention or a salt thereof includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent forces between molecules, and when the solvent is water, it is a hydrate.

With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or agent that is non-toxic but can achieve the desired effect. For oral dosage forms of the present invention, an "effective amount" of an active substance in a composition refers to the amount required to achieve the desired effect when used in combination with another active substance in the composition. The determination of the effective amount varies from person to person, depending on the age and general condition of the recipient, and also on the specific active substance. The appropriate effective amount in each case can be determined by a person skilled in the art based on routine experiments.

Unless otherwise specified, the terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

Unless otherwise specified, the term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, including deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is a keto group (i.e., =O), it means that two hydrogen atoms are replaced. Keto substitution does not occur on aromatic groups.

In the present application, "optional" or "optionally" means that the event or situation described later may or may not occur, and the description includes both the occurrence and non-occurrence of the event or situation. For example, "optionally substituted aryl" means that aryl is substituted or unsubstituted, and the description includes both substituted aryl and unsubstituted aryl.

Unless otherwise specified, the term "C _1-6 alkyl" is used to represent a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C _1-6 alkyl includes C _1-5 , C _1-4 , C _1-3 , C _{1-2, C 2-6 ,} C _2-4 , C ₆ and C ₅ alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C _1-6 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, etc.

Unless otherwise specified, the term "C _1-3 alkyl" is used to represent a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C _1-3 alkyl group includes C _1-2 and C _2-3 alkyl groups, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C _1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

The term "halo" by itself or as part of another substituent is used interchangeably with the term "halogen-substituted."

Unless otherwise specified, "haloalkyl" or "halogen-substituted alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted with one or more halogens.

Unless otherwise specified, "C _2-6 alkenyl" is used to represent a straight or branched hydrocarbon group consisting of 2 to 6 carbon atoms containing at least one carbon-carbon double bond, which may be located at any position of the group. The C _2-6 alkenyl includes C _2-4 , C _2-3 , C ₄ , C ₃ and C ₂ alkenyl, etc.; it may be monovalent, divalent or polyvalent. Examples of C _2-6 alkenyl include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, etc.

Unless otherwise specified, "C _2-6 alkynyl" is used to represent a straight or branched hydrocarbon group consisting of 2 to 6 carbon atoms containing at least one carbon-carbon triple bond, which may be located at any position of the group. The C _2-6 alkynyl includes C _2-4 , C _2-3 , C ₄ , C ₃ and C ₂ alkynyl, etc. It may be monovalent, divalent or polyvalent. Examples of C _2-6 alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, etc.

Unless otherwise specified, the term "C _1-6 alkoxy" refers to those alkyl groups containing 1 to 6 carbon atoms that are connected to the rest of the molecule through an oxygen atom. The C _1-6 alkoxy includes C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-4 , C ₆ , C ₅ , C ₄ and C ₃ alkoxy, etc. Examples of C _1-6 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, isobutoxy, s-butoxy and t-butoxy), pentoxy (including n-pentoxy, isopentyl and neopentyl), hexyl and the like.

Unless otherwise specified, the term "C _1-3 alkoxy" refers to those alkyl groups containing 1 to 3 carbon atoms connected to the rest of the molecule through an oxygen atom. The C _1-3 alkoxy includes C _1-2 , C _2-3 , C ₃ and C ₂ alkoxy, etc. Examples of C _1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), etc.

Unless otherwise specified, the term "C _1-6 alkylamino" refers to those alkyl groups containing 1 to 6 carbon atoms that are attached to the rest of the molecule through an amino group. The C _1-6 alkylamino group includes C _1-4 , C _1-3 , C _1-2 , C _2-6 , C _2-4 , C ₆ , C ₅ , C ₄ , C ₃ and C ₂ alkylamino groups, etc. Examples of _{C 1-6} alkylamino groups include, but are not limited to, -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCH ₂ CH ₃ , -N(CH ₃ )CH ₂ CH ₃ , -N(CH ₂ CH ₃ )(CH ₂ CH ₃ ), -NHCH ₂ CH ₂ CH ₃ , -NHCH ₂ (CH ₃ ) ₂ , -NHCH ₂ CH ₂ CH ₂ CH3, and the like.

Unless otherwise specified, the term "C _3-8 cycloalkyl" means a saturated cyclic hydrocarbon group consisting of 3 to 8 carbon atoms, including monocyclic and bicyclic systems, wherein the bicyclic system includes spirocyclic, fused and bridged rings. The C _3-8 cycloalkyl includes C _3-6 , C _3-5 , C _4-8 , C _4-6 , C _4-5 , C _5-8 or C _5-6 cycloalkyl, etc.; it can be monovalent, divalent or polyvalent. Examples of C _3-8 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, [2.2.2] bicyclooctane, etc.

Unless otherwise specified, the term "C _3-6 cycloalkyl" means a saturated cyclic hydrocarbon group consisting of 3 to 6 carbon atoms, which is a monocyclic and bicyclic system, and the C _3-6 cycloalkyl includes C _3-5 , C _4-5 and C _5-6 cycloalkyl, etc.; it can be monovalent, divalent or polyvalent. Examples of C _3-6 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

Unless otherwise specified, Cn _-n+m or _Cn - _Cn+m includes any specific case of n to n+m carbon atoms, for example, _C1-12 includes _C1 , _C2 , C3, _C4 , _{C5 , C6, C7, C8, C9, C10, C11 , and C12 , and also includes} any range from n to n+m, for example _{, C1-12} includes _C1-3 , _C1-6 , _C1-9 , _C3-6 , _C3-9 , _C3-12 , _C6-9 , _C6-12 , and C13. _9-12 , etc.; similarly, n-membered to n+m-membered means that the number of atoms in the ring is n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and also includes any range from n to n+m, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring, 5-7-membered ring, 6-7-membered ring, 6-8-membered ring, and 6-10-membered ring, etc.

Unless otherwise specified, the term "aryl" refers to a monocyclic or polycyclic carbon ring having 6 to 20 carbon atoms, wherein at least one ring is an aromatic ring. When one of the rings is a non-aromatic ring, the group may be connected through an aromatic ring or a non-aromatic ring. "Aryl" may be a 6-8 membered, 6-10 membered aryl group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, 2,3-dihydroindanyl, biphenyl, phenanthrenyl, anthracenyl, and acenaphthenyl. The term "6-10 membered aryl" refers to a monocyclic or polycyclic carbon ring having 6 to 10 carbon atoms, wherein at least one ring is an aromatic ring.

Unless otherwise specified, the term "heterocycloalkyl" refers to a cycloalkyl group in which one or more (e.g., 1, 2, 3 or 4, in some embodiments, 1 to 3) carbon atoms are replaced by heteroatoms, such as, but not limited to, N, O, S and P. The term "m-n membered heterocycloalkyl" is understood to mean a saturated, unsaturated or partially saturated ring having m to n atoms, wherein the heteroatoms are selected from N, O, S, P, preferably N, O or S. The term "heterocycloalkyl" may be 3 to 8 members, 3 to 11 members, 6 to 8 members. The term "3-11 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 3 to 11 ring atoms, respectively. The term "6-8 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 6 to 8 ring atoms, 1, 2, 3 or 4 of which are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., NO and S(O)p, p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein the bicyclic ring system includes spirocyclic, cyclic and bridged rings. In addition, with respect to the "6-8 membered heterocycloalkyl", heteroatoms may occupy the position where the heterocycloalkyl is connected to the rest of the molecule. For example, 6-8 membered heterocycloalkyl includes, but is not limited to, 6-membered, 7-membered, and 8-membered. Examples of 6-8 membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, and hexahydropyridazinyl.

Unless otherwise specified, the term "heteroaromatic ring" or "heteroaryl" refers to a monocyclic or polycyclic carbon ring in which at least one ring atom (e.g., 1, 2, 3) is a heteroatom independently selected from oxygen, sulfur and nitrogen, and the remaining ring atoms are C, wherein at least one ring is an aromatic ring. The group may be a carbon group or a heteroatom group (i.e., it may be C-attached or N-attached, as long as it is possible). When one of the rings is a non-aromatic ring, the group may be attached through an aromatic ring or a non-aromatic ring. "Heteroaryl" may be a 5-10 membered (e.g., 5, 6, 7, 8, 9, 10 membered) heteroaryl, or a 5-8 membered heteroaryl. Examples of heteroaryl groups include, but are not limited to, imidazolyl, acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furanyl, thienyl, benzothienyl, benzofuranyl, quinolyl, isoquinolyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, N-methylpyrrolyl, and tetrahydroquinoline. The term "heteroaromatic ring" can be used interchangeably with the terms "heteroaromatic ring", "heteroaryl" or "heteroaromatic ring group".

Unless otherwise specified, the term "oxo" means that two hydrogen atoms on a methylene group are replaced by oxygen atoms, that is, a methylene group is replaced by a carbonyl group, representing =0.

Unless otherwise specified, the term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

In addition, it should be noted that, unless otherwise clearly indicated, the description method "...independently" used in the present invention should be understood in a broad sense, meaning that the individuals described are independent of each other and can be independently the same or different specific groups. In more detail, the description method "...independently" can mean that in different groups, the specific options expressed by the same symbols do not affect each other, and can also mean that in the same group, the specific options expressed by the same symbols do not affect each other.

In this application, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant governmental regulatory authorities as acceptable for human or livestock use.

The term "treat" refers to therapeutic treatment. When referring to a specific condition, treatment means: (1) ameliorating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

The term "prevent" refers to the reduction of the risk of acquiring or developing a disease or disorder.

The term "patient" refers to any animal, preferably a mammal, that is about to or has been administered the compound or composition according to embodiments of the present invention. The term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., preferably humans.

The term "therapeutically effective amount" refers to an amount of a compound that is effective in treating a disease or condition described herein when administered to a patient."Therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, and can be adjusted as needed by those skilled in the art.

Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

Beneficial Effects

After extensive and in-depth research, the inventors unexpectedly developed a compound or a pharmaceutically acceptable salt thereof, a preparation method and use. The present invention provides a compound shown in Formula IA, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, wherein the compound of Formula IA has a significant inhibitory effect on MRGPRX2, can be used as an antagonist of MRGPRX2, exhibits excellent pharmacokinetic properties, has good drugability, and has no significant inhibitory effect on BSEP bile efflux transporter, and has no risk of cholestatic toxicity, which reflects from multiple aspects that the compound of the present invention has high safety and drugability.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with specific examples. It should be understood that the following description is only the most preferred embodiment of the present invention and should not be considered as limiting the scope of protection of the present invention. On the basis of a full understanding of the present invention, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Those skilled in the art may make non-essential changes to the technical solution of the present invention, and such changes should be deemed to be included in the scope of protection of the present invention.

EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

HOBt: 1-Hydroxybenzotriazole

DIEA: N,N-diisopropylethylamine

Ruphos Pd G4: methanesulfonate (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II)

Intermediate A1: Preparation of N-((1R,3S)-3-aminocyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide

The synthetic route of target intermediate A1 is as follows:

Step 1: Synthesis of tert-butyl ((1S,3R))-3-(1-methyl-1H-pyrazole-4-carboxamide)cyclohexyl)carbamate

At room temperature, 1-methyl-1H-pyrazole-4-carboxylic acid (500 mg, 4 mmol) was dissolved in N,N-dimethylformamide (10 mL), and then tert-butyl ((1S, 3R)-3-aminocyclohexane (428 mg, 4 mmol), N,N-diisopropylethylamine (408 mg, 12 mmol) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.38 g, 6 mmol) were added in sequence, and then heated at room temperature. The reaction was carried out for 12 hours. After the reaction was completed, the reaction solution was poured into ice water and extracted with ethyl acetate (3×50 mL). The obtained organic phase was dried over anhydrous sodium sulfate, filtered and dried, and the obtained crude product was purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 10:1) to obtain compound ((1S, 3R))-3-(1-methyl-1H-pyrazole-4-carboxamide)cyclohexyl)carbamic acid tert-butyl ester (A1-3) (830 mg, 2.58 mmol, yield 64.5%).

LC-MS, M/Z(ESI):323.20[M+H] ⁺

Step 2: Synthesis of N-((1R,3S)-3-aminocyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide

At room temperature, tert-butyl ((1S,3R))-3-(1-methyl-1H-pyrazole-4-carboxamide)cyclohexyl)carbamate (830 mg, 2.58 mmol) was dissolved in anhydrous 1,4-dioxane (10 mL), stirred at room temperature, and then a solution of hydrogen chloride in 1,4-dioxane (4 mol/L, 1.76 mL, 4 mmol) was slowly added dropwise, and the reaction solution was stirred at room temperature for 30 min. After monitoring by thin layer chromatography showed that the raw materials had reacted completely, stirring was stopped, a saturated sodium bicarbonate solution was added to the reaction system to adjust the pH value to 7-8, and the mixture was extracted with ethyl acetate (30 mL×3). The organic phase was collected and dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column (dichloromethane: methanol (V/V) = 10:1) to obtain compound N-((1R, 3S)-3-aminocyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide (intermediate A1) (213 mg, yield 36.8%).

LC-MS, M/Z(ESI):223.13[M+H] ⁺

Intermediate A2: Preparation of compound 3-chloro-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxylic acid

The synthetic route of target intermediate A2 is as follows:

Step 1: Synthesis of compound 3-chloro-1-((methylthio)methyl)-1H-pyrazole-4-carboxylic acid ethyl ester

Under room temperature, the compound 3-chloro-1H-pyrazole-4-carboxylic acid ethyl ester (2.00 g, 11.46 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), stirred in an ice bath at 0°C for 10 min, then sodium hydride (504 mg, 12.60 mmol) was added in batches, and then chloromethyl methyl sulfide (1.33 g, 13.75 mmol) was slowly added. The reaction solution was slowly warmed to room temperature and stirred for 30 min. After the reaction was completed, stirring was stopped, the reaction solution was poured into water (30 mL), and then extracted with ethyl acetate (30 mL×3), the organic phase was collected and dried with anhydrous sodium sulfate, filtered and concentrated, and the residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 10:1) to obtain the compound 3-chloro-1-((methylthio)methyl)-1H-pyrazole-4-carboxylic acid ethyl ester (930 mg, yield 34.59%).

LC-MS, M/Z(ESI):235.3[M+H] ⁺

¹ H NMR (400MHz, DMSO-d6) δ8.50(s,1H),5.26(s,2H),4.23(q,2H),2.14(s,3H),1.27(t,3H).

Step 2: Synthesis of compound 3-chloro-1-((methylsulfonyl)methyl)-1H-pyrazole-4-carboxylic acid ethyl ester

Under room temperature, the compound 3-chloro-1-((methylthio)methyl)-1H-pyrazole-4-carboxylic acid ethyl ester (550mg, 2.34mmol) was dissolved in anhydrous dichloromethane (11mL), stirred at 0℃ for 10min, then m-chloroperbenzoic acid (1.21g, 7.03mmol) was added, the reaction solution was slowly heated to room temperature, and stirring was continued for 2h. After thin layer chromatography showed that the raw materials had reacted completely, stirring was stopped, the reaction solution was poured into a saturated sodium thiosulfate aqueous solution (30mL), and then extracted with ethyl acetate (30mL×3), the organic phase was collected and washed with a saturated sodium carbonate aqueous solution (40mL×3), and then dried with anhydrous sodium sulfate, filtered and concentrated to obtain the compound 3-chloro-1-((methylsulfonyl)methyl)-1H-pyrazole-4-carboxylic acid ethyl ester (615mg, yield 98.4%), and the obtained crude product can be directly used in the next step reaction.

LC-MS, M/Z(ESI):267.0[M+H] ⁺

Step 3: Synthesis of ethyl 3-chloro-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxylate

At room temperature, the compound 3-chloro-1-((methylsulfonyl)methyl)-1H-pyrazole-4-carboxylic acid ethyl ester (200 mg, 0.75 mmol) was dissolved in anhydrous DMF (2 mL), and then cesium carbonate (488.67 mg, 1.50 mmol) was added. After the reaction solution was stirred for 10 min, iodomethane (212.88 mg, 1.50 mmol) was slowly added dropwise, and then stirred at room temperature for 12 h. After TLC monitoring showed that the raw material had reacted, stirring was stopped, water was added to quench the reaction (5 mL), and then extracted with ethyl acetate (15 mL×3), the organic phase was collected and dried with anhydrous sodium sulfate, filtered and concentrated, and the residue was separated and purified by silica gel column chromatography (DCM: MeOH (V/V) = 100:2) to obtain the compound 3-chloro-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxylic acid ethyl ester (139 mg, yield 66.03%).

LC-MS, M/Z(ESI):281.0[M+H] ⁺

Step 4: Synthesis of compound 3-chloro-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxylic acid

At room temperature, the compound 3-chloro-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxylic acid ethyl ester (130 mg, 0.46 mmol) was dissolved in a mixed solution of methanol (2 mL) and water (0.8 mL), and then sodium hydroxide (148 mg, 3.70 mmol) was added, and the reaction solution was stirred at room temperature for 2 h. After TLC monitoring showed that the raw materials had reacted completely, stirring was stopped, and methanol was removed by distillation under reduced pressure. Water (5 mL) was added to the residue for dilution, and then the pH was adjusted to 2 with dilute hydrochloric acid (2 mol/L), and then extracted with ethyl acetate (15 mL×3), the organic phase was collected and dried with anhydrous sodium sulfate, and the organic phase was concentrated by distillation under reduced pressure to obtain the compound 3-chloro-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxylic acid (110 mg, yield 94.01%).

LC-MS, M/Z(ESI):252.8[M+H] ⁺

Intermediate A3: Preparation of 3-chloro-1-(2-hydroxy-2-methylpropyl)-1H-pyrazole-4-carboxylic acid

The synthetic route of target intermediate A3 is as follows:

Step 1: Synthesis of ethyl 3-chloro-1-(2-hydroxy-2-methylpropyl)-1H-pyrazole-4-carboxylate

1-Bromo-2-methyl-2-propanol (351 mg, 2.29 mmol), cesium carbonate (1.12 g, 3.44 mmol) and potassium iodide (190 mg, 1.15 mmol) were added to a solution of 3-chloro-1H-pyrazole-4-carboxylic acid ethyl ester (200 mg, 1.15 mmol) in N, N-dimethylformamide (3 mL), and the mixture was stirred at 70 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was diluted with water (100 mL), then extracted with ethyl acetate (200 mL × 3), and the organic phases were combined and concentrated to give a crude product, which was purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 100: 0-0: 100) to give compound 3-chloro-1- (2-hydroxy-2-methylpropyl) -1H-pyrazole-4-carboxylic acid ethyl ester (181 mg, yield 64.1%).

LC-MS, M/Z (ESI): 247.2 [M+H] ⁺ .

Step 2: Synthesis of 3-chloro-1-(2-hydroxy-2-methylpropyl)-1H-pyrazole-4-carboxylic acid

To a solution of ethyl 3-chloro-1-(2-hydroxy-2-methylpropyl)-1H-pyrazole-4-carboxylate (181 mg, 0.73 mmol) in methanol (3 mL) and water (3 mL) was added lithium hydroxide (308 mg, 7.34 mmol), and the resulting mixture was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated under reduced pressure to give a crude product, which was purified by column chromatography (water: acetonitrile (V/V) = 100: 0 to 80: 20) to give compound 3-chloro-1-(2-hydroxy-2-methylpropyl)-1H-pyrazole-4-carboxylic acid (150 mg, yield 93.5%).

LC-MS, M/Z(ESI):219.0[M+H] ⁺ .

Example 1: Preparation of N-((1S,4S)-4-((2,7-bis(trifluoromethyl))imidazo[1,2-a](pyridin-5-yl)amino)cyclohexyl)-3-chloro-1-methyl-1H-pyrazole-4-carboxamide (Compound 20A)

The synthetic route of the target compound 20A is as follows:

Step 1: Synthesis of 6-chloro-4-(trifluoromethyl)pyridin-2-amine

Under room temperature, the compound 2,6-dichloro-4-trifluoromethylpyridine (20A-1) (2.00 g, 9.26 mmol) was dissolved in 10 mL of ammonia water (25%) and stirred at 120°C for 12 h in a metal can reactor. After the reaction was complete, the heating was stopped, the mixture was cooled to room temperature, and the solvent was removed by rotary evaporation under reduced pressure. The residue was separated and purified by silica gel column (dichloromethane: methanol (V/V) = 100:5) to obtain the compound 6-chloro-4-(trifluoromethyl)pyridin-2-amine (20A-2) (1.30 g, yield 71.43%).

LC-MS,M/Z(ESI):196.80[M+H] ⁺ .

Step 2: Synthesis of 5-chloro-2,7-bis(trifluoromethyl)imidazo[1,2-a]pyridine

Under room temperature, compound 6-chloro-4-(trifluoromethyl)pyridin-2-amine (20A-2) (1.30 g, 6.61 mmol) and compound 3-bromo-1,1,1-trifluoroacetone (1.89 g, 9.92 mmol) were dissolved in ethylene glycol (15 mL), and the reaction solution was stirred at 120°C for 12 h. After TLC monitoring showed that the raw materials had reacted completely, stirring was stopped, the reaction solution was cooled to room temperature, and then water (80 mL) was added to dilute it, extracted with ethyl acetate (40 mL×5), the organic phase was collected and dried over anhydrous sodium sulfate, filtered and concentrated, and the obtained residue was separated and purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:5) to obtain compound 5-chloro-2,7-bis(trifluoromethyl)imidazo[1,2-a]pyridine (20A-3) (1.29 g, yield 67.59%).

LC-MS, M/Z(ESI):289.22[M+H] ⁺

Step 3: Synthesis of tert-butyl ((1S,4S)-4-((2,7-bis(trifluoromethyl))imidazo[1,2-a]pyridin-5-yl)amino)cyclohexyl)carbamate (20A-5)

At room temperature, the compound 5-chloro-2,7-bis(trifluoromethyl)imidazo[1,2-a]pyridine (20A-3) (1.00 g, 3.47 mmol) and the compound 1-N-Boc-cis-1,4-cyclohexanediamine (20A-4) (1.48 g, 6.93 mmol) were dissolved in anhydrous DMSO (10 mL), and then N,N-diisopropylethylamine (1.79 g, 13.96 mmol) was added dropwise, and the reaction solution was stirred at 100°C for 4 hours. After TLC monitoring showed that the raw material had reacted, 50 mL of water was added to dilute it, and it was extracted with ethyl acetate (30 mL×5). The organic phase was collected and dried over anhydrous sodium sulfate. The organic phase was concentrated under reduced pressure, and the residue was separated and purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:20) to obtain compound ((1S,4S)-4-((2,7-bis(trifluoromethyl))imidazo[1,2-a]pyridin-5-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (20A-5) (1.60 g, yield was 73.52%).

LC-MS, M/Z(ESI):467.01[M+H] ⁺

Step 4: Synthesis of (1S,4S)-N ¹ -(2,7-bis(trifluoromethyl))imidazo[1,2-a]pyridin-5-yl)cyclohexane-1,4-diamine hydrochloride

At room temperature, the compound ((1S,4S)-4-((2,7-bis(trifluoromethyl))imidazo[1,2-a]pyridin-5-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (20A-5) (1.60 g, 3.43 mmol) was dissolved in dichloromethane (20 mL), and then a solution of hydrogen chloride in 1,4-dioxane (4 mol/L, 4.0 mL, 16.0 mmol) was slowly added dropwise, and the reaction solution was stirred for 1 h at room temperature. After the reaction was completed as monitored by TLC, the solvent was distilled off under reduced pressure, ethyl acetate (40 mL) was added, and the reaction was stirred at room temperature for 2 h. The suspension was filtered and dried in vacuo to obtain the compound (1S,4S)-N ¹ -(2,7-bis(trifluoromethyl))imidazo[1,2-a]pyridin-5-yl)cyclohexane-1,4-diamine hydrochloride (20A-6) (1.25 g, yield 90.47%).

LC-MS, M/Z(ESI):367.30[M+H] ⁺

Step 5: Synthesis of N-((1S,4S)-4-((2,7-bis(trifluoromethyl))imidazo[1,2-a](pyridin-5-yl)amino)cyclohexyl)-3-chloro-1-methyl-1H-pyrazole-4-carboxamide

At room temperature, compound 3-chloro-1-methyl-1H-pyrazole-4-carboxylic acid (200 mg, 1.25 mmol) was dissolved in anhydrous DMF (5 mL) and stirred at 0°C for 10 min. Then, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) (947.28 mg, 2.49 mmol) and N,N-diisopropylethylamine (397.85 mg, 4.98 mmol) were added. The reaction solution was stirred at 0°C for 30 min. Then, compound (1S,4S)-N ¹ -(2,7-bis(trifluoromethyl)imidazo[1,2-a]pyridin-5-yl)cyclohexane-1,4-diamine hydrochloride (20A-6) (501.71 mg, 1.25 mmol) was added. The reaction solution was warmed to room temperature and stirred for 12 h. After TLC monitoring showed that the raw materials had reacted completely, stirring was stopped, water (50 mL) was added to the reaction solution to dilute it, and it was extracted with ethyl acetate (30 mL×5). The organic phase was collected and dried over anhydrous sodium sulfate, and the organic phase was concentrated by distillation under reduced pressure. The residue was separated and purified by column chromatography (dichloromethane:methanol (V/V)=100:5) to obtain compound N-((1S,4S)-4-((2,7-bis(trifluoromethyl))imidazo[1,2-a](pyridin-5-yl)amino)cyclohexyl)-3-chloro-1-methyl-1H-pyrazole-4-carboxamide (Compound 20A) (220 mg, 34.72%).

LC-MS, M/Z(ESI):509.20[M+H] ⁺

¹ H NMR (400MHz, CDCl ₃ )δ7.92(s,1H),7.86(t,1H),7.46(d,1H),6.68(d,1H),6.13(d,1H),4.36 (d,1H),4.21(dd,1H),3.88(s,3H),3.78–3.72(m,1H),2.13–1.79(m,8H).

Example 2: Preparation of 1-methyl-N-((1R,3S)-3-((6-methyl-2-(trifluoromethyl)-1,5-naphthyridin-4-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (Compound 21A)

The synthetic route of the target compound 21A is as follows:

Step 1: Synthesis of 6-methyl-2-(trifluoromethyl)-1,5-naphthyridin-4(1H)-one

5-amino-2-methylpyridine (2 g, 18.49 mmol), diphenyl ether-biphenyl eutectic (20 mL) and ethyl 4,4,4-trifluoroacetoacetate (4.43 g, 24.04 mmol) were added to a 100 mL single-mouth bottle in sequence, and the temperature was raised to 250° C. and stirred for 7 h. After the reaction was completed, the reaction solution was filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate (V/V) = 1:0 to 1:4) to obtain compound 6-methyl-2-(trifluoromethyl)-1,5-naphthyridin-4(1H)-one (21A-3) (0.3 g, yield 7.11%).

LC-MS, M/Z(ESI):229.10[M+H] ⁺

Step 2: Synthesis of 4-chloro-6-methyl-2-(trifluoromethyl)-1,5-naphthyridine

6-Methyl-2-(trifluoromethyl)-1,5-naphthyridin-4(1H)-one (0.2 g, 0.88 mmol) and dichloroethane (5 mL) were added to a 100 mL two-necked flask in sequence. Under nitrogen protection, the reaction solution was cooled to 0°C, and then phosphorus oxychloride (1.34 g, 8.77 mmol) was slowly added. After 10 min, the reaction solution was heated to 80°C and stirred for 5 h. After the reaction was completed, ice water was added to quench the reaction, the organic phase was separated, and concentrated to obtain a crude product, which was purified by silica gel column chromatography (petroleum ether/ethyl acetate (V/V) = 1:0 to 20:1) to obtain compound 4-chloro-6-methyl-2-(trifluoromethyl)-1,5-naphthyridin (21A-4) (120 mg, yield 55.51%).

LC-MS, M/Z(ESI):247.15[M+H] ⁺

Step 3: Synthesis of 1-methyl-N-((1R,3S)-3-((6-methyl-2-(trifluoromethyl)-1,5-naphthyridin-4-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide

To a 100 mL single-necked bottle, 4-chloro-6-methyl-2-(trifluoromethyl)-1,5-naphthyridine (80 mg, 0.32 mmol), N-((1R,3S)-3-aminocyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide (Intermediate A1) (108.16 mg, 0.49 mmol), BrettPhosPdG3 (14.70 mg, 0.16 mmol) and sodium tert-butoxide (77.94 mg, 0.81 mmol) were added in sequence, and 1,4-dioxane (8 mL) was added. The reaction solution was heated to 100°C and stirred for 16 h. After the reaction, the reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to obtain a crude product, which was purified by reverse phase preparative liquid chromatography (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 um); solvent: A = water + 0.1% ammonia water, B = acetonitrile; gradient: 20% -70%) to obtain 1-methyl-N-((1R, 3S)-3-((6-methyl-2-(trifluoromethyl)-1,5-naphthyridin-4-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (Compound 21A) (14 mg, yield 9.98%).

LC-MS, M/Z(ESI):433.40[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ )δ8.16(d,1H),8.09(s,1H),7.85(d,1H),7.81(s,1H),7.66(d,1H),7.24(d,1H),7.05(s,1H),3.99–3.91(m,1H),3.87-3 .83(m,1H),3.81(s,3H),2.69(s,3H),2.14(d,1H),1.94(d,1H),1.86-1.76(m,2H),1.53-1.38(m,3H),1.29-1.22(m,1H).

Example 3: Preparation of N-((1R,3S)-3-((2-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridin-4-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide (Compound 22A)

The synthetic route of compound 22A is as follows:

Step 1: Synthesis of Thiophene-2-amine

Thiophene-2-ylcarbamic acid tert-butyl ester (2.00 g, 10.0 mmol) was dissolved in a solution of hydrogen chloride in 1,4-dioxane (20 mL, 4 mol/L), and the reaction solution was stirred at 25°C for 1 hour under nitrogen protection. After the reaction was completed, the reaction solution was concentrated under reduced pressure, then dissolved in dichloromethane (10 mL), and concentrated under reduced pressure. The concentration was repeated three times to obtain a crude product of thiophene-2-amine (22A-2) (1.35 g, yield 98.4%), which was directly used in the next step.

LC-MS, M/Z (ESI): 100.2 [M+H] ⁺ .

Step 2: Synthesis of 6-(trifluoromethyl)thieno[3,2-b]pyridine-4-ol

Thiophene-2-amine (4.80 g, 35.0 mmol) was dissolved in polyphosphoric acid (50 mL), and ethyl 4,4,4-trifluoroacetoacetate (6.45 g, 35.0 mmol) was added under nitrogen protection. The reaction solution was stirred at 140 ° C for 18 hours under N ₂ protection. The reaction mixture was cooled to room temperature, diluted with saturated sodium bicarbonate solution (500 mL), and then extracted with ethyl acetate (500 mL×3). The organic layers were combined, washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 80:20, gradient elution) to obtain compound 6-(trifluoromethyl)thieno[3,2-b]pyridine-4-ol (22A-4) (750 mg, yield 9.3%).

¹ H NMR (400MHz, CDCl ₃ ): δ7.55 (d, J = 6.0 Hz, 1H), 7.46 (d, J = 6.0 Hz, 1H), 7.09 (s, 1H).

LC-MS, M/Z (ESI): 220.2 [M+H] ⁺ .

Step 3: Synthesis of 4-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridine

6-(Trifluoromethyl)thieno[3,2-b]pyridine-4-ol (683 mg, 3.12 mmol) was dissolved in toluene (7 mL), and N,N-dimethylformamide (0.1 mL) and thionyl chloride (1.85 g, 15.6 mmol) were added in sequence. The reaction solution was stirred at 25 ° C for 4 hours. LC-MS detected the formation of the target product, and water (20 mL) was added to the reaction system for dilution, and extracted with ethyl acetate (10 mL×3). The organic phase was washed with saturated brine (10 mL), then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 80:20, gradient elution) to obtain compound 4-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridine (22A-5) (490 mg, yield 62.91%).

LC-MS, M/Z (ESI): 238.0 [M+H] ⁺ .

Step 4: Synthesis of 2,4-dichloro-6-(trifluoromethyl)thieno[2,3-b]pyridine

2,2,6,6-Tetramethylpiperidine (0.46mL, 2.73mmol) was dissolved in tetrahydrofuran (10mL) and nitrogen was replaced. n-Butyl lithium (2.18mL, 5.45mmol, 2.5mol/L n-hexane solution) was added dropwise at -78°C. The reaction solution was stirred at -78°C for 1 hour, and a solution of 4-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridine (648mg, 2.73mmol) in tetrahydrofuran (2mL) was slowly added dropwise into the reaction system. The reaction solution was stirred at -78°C for 1 hour, and then a solution of hexachloroethane (775mg, 3.27mmol) in tetrahydrofuran (1mL) was slowly added dropwise into the reaction system. The obtained reaction solution was stirred at -78°C for 1 hour, and then gradually heated to 25°C and continued to stir for 14 hours. LC-MS detected that the starting material disappeared. Water (50 mL) was added to the reaction mixture to quench it, and it was extracted with ethyl acetate (50 mL×3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was separated and purified by silica gel column (petroleum ether:ethyl acetate (V/V) = 100:0 to 80:20, gradient elution) to obtain compound 2,4-dichloro-6-(trifluoromethyl)thieno[2,3-b]pyridine (22A-6) (377 mg, yield 48.4%).

¹ H NMR (400MHz, CDCl ₃ ): δ7.67(s,1H),7.35(s,1H).

LC-MS, M/Z (ESI): 272.0 [M+H] ⁺ .

Step 5: Synthesis of tert-butyl ((1R,3S))-3-((2-chloro-6-trifluoromethyl))thieno[2,3-b]pyridin-4-yl)amino)cyclohexyl)carbamate

2,4-Dichloro-6-(trifluoromethyl)thieno[2,3-b]pyridine (250 mg, 0.92 mmol) and tert-butyl (((1R,3S)-3-aminocyclohexyl)amino)formate (207 mg, 0.97 mmol) were dissolved in N-methylpyrrolidone (5 mL), N,N-diisopropylethylamine (166 mg, 1.29 mmol) was added, and the reaction solution was stirred at 130 ° C for 2 hours. The reaction solution was cooled to room temperature, water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was separated and purified by silica gel column (petroleum ether:ethyl acetate (V/V) = 100:0 to 80:20, gradient elution) to obtain compound ((1R,3S))-3-((2-chloro-6-trifluoromethyl))thieno[2,3-b]pyridin-4-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (22A-7) (270 mg, yield 64.00%).

LC-MS, M/Z (ESI): 450.2 [M+H] ⁺ .

Step 6: Synthesis of (1R,3S)-N ¹ -((2-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridin-4-yl)cyclohexane-1,3-diamine

Tert-butyl ((1R,3S))-3-((2-chloro-6-trifluoromethyl))thieno[2,3-b]pyridin-4-yl)amino)cyclohexyl)carbamate (150 mg, 0.33 mmol) was dissolved in anhydrous dichloromethane (5 mL), and trifluoroacetic acid (2 mL) was added. The reaction solution was stirred at 25°C for 1 hour. LC-MS detected the formation of the target product, and the reaction solution was concentrated under reduced pressure. The residue was dissolved in dichloromethane (10 mL) and further concentrated under reduced pressure to obtain a crude product compound (1R,3S)-N ¹ -((2-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridin-4-yl)cyclohexane-1,3-diamine (22A-8) (110 mg, yield 92.4%), which was directly used in the next step.

LC-MS, M/Z (ESI): 350.2 [M+H] ⁺ .

Step 7: Synthesis of N-((1R,3S)-3-((2-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridin-4-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide

(1R,3S)-N ¹ -((2-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridin-4-yl)cyclohexane-1,3-diamine (100 mg, 0.29 mmol) and 1-methylpyrazole-4-carboxylic acid (43.3 mg, 0.34 mmol) were dissolved in anhydrous dimethyl sulfoxide (2 mL), and N,N-diisopropylethylamine (185 mg, 1.43 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (164 mg, 0.86 mmol), and 1-hydroxybenzotriazole (116 mg, 0.86 mmol) were added in sequence. The reaction solution was heated at 25° C. under nitrogen protection. The mixture was stirred for 18 hours. LC-MS detected the formation of the target product, and the reaction solution was dissolved in saturated brine (20 mL), then extracted with ethyl acetate (10 mL×3), the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was separated and purified by reverse silica gel column C18 (water:acetonitrile (V/V)=100:0 to 70:30, gradient elution) to obtain N-((1R,3S)-3-((2-chloro-6-(trifluoromethyl)thieno[2,3-b]pyridin-4-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide (Compound 22A) (88.3 mg, yield 66.6%).

LC-MS, M/Z(ESI):458.2[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.10(s,1H),7.89(s,1H),7.87(d,1H),7.82(s,1H),7.23(d,1H),6.98(s,1H),4.02–3.90(m,1H),3.83(s,4H), 2.13–2.07(m,1H),1.96–1.90(m,1H),1.87–1.76(m,2H),1.55–1.46(m,1H),1.41–1.31(m,1H),1.25–1.21(m,2H).

Example 4: Preparation of N-((1R,3S)-3-((2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide (Compound 23A) trifluoroacetate

The synthetic route of compound 23A trifluoroacetate is as follows:

Step 1: Synthesis of 5-(trifluoromethyl)thieno[3,2-b]pyridine-7-ol

Ethyl trifluoroacetoacetate (0.54 g, 5.43 mmol) was added to a polyphosphoric acid (10 mL) solution of 3-aminothiophene (1.00 g, 5.43 mmol), and the reaction solution was stirred at 140 ° C for 16 hours under nitrogen protection. The reaction mixture was cooled to room temperature, then diluted with saturated sodium bicarbonate aqueous solution (100 mL), and then extracted with ethyl acetate (100 mL×3). The organic layers were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was separated and purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:1 to 10:1, gradient elution) to obtain compound 5-(trifluoromethyl)thieno[3,2-b]pyridine-7-ol (720 mg, yield 55.3%).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.17 (d, J = 5.2 Hz, 1H), 7.57 (d, J = 5.6 Hz, 1H), 7.02 (s, 1H), 3.87 (s, 1H).

LC-MS, M/Z (ESI): 220.2 [M+H] ⁺ .

Step 2: Synthesis of 7-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridine

5-(Trifluoromethyl)thieno[3,2-b]pyridine-7-ol (360 mg, 1.64 mmol) was dissolved in phosphorus oxychloride (3 mL), and the reaction solution was stirred at 60°C for 2 hours. The phosphorus oxychloride was removed by concentration under reduced pressure, water (10 mL) was added to the residue, and it was extracted with ethyl acetate (10 mL×3), the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was separated and purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:1 to 5:1, gradient elution) to obtain 7-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridine (292 mg, yield 71.1%).

LC-MS, M/Z (ESI): 238.2 [M+H] ⁺ .

Step 3: Synthesis of 2,7-dichloro-5-(trifluoromethyl)thieno[3,2-b]pyridine

2,2,6,6-Tetramethylpiperidine (256 mg, 1.81 mmol) was dissolved in tetrahydrofuran (5 mL), nitrogen was replaced, and n-butyl lithium (0.66 mL, 1.65 mmol, 2.5 M) was added dropwise at -78 °C. The reaction solution was reacted at -78 °C for 1 hour, and then a solution of 7-chloro-5-(trifluoromethyl)thieno(3,2-b)pyridine (200 mg, 0.82 mmol) in tetrahydrofuran (1 mL) was added dropwise. After stirring at -78 °C for 1 hour, a solution of hexachloroethane (234 mg, 0.99 mmol) in tetrahydrofuran (1 mL) was slowly added dropwise. The resulting reaction solution was stirred at -78 °C for 1 hour, and then gradually heated to 25 °C and stirred for 14 hours. Water (10 mL) was added to the reaction mixture to quench the reaction, and the mixture was extracted with ethyl acetate (10 mL × 3). The organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The residue was separated and purified by silica gel column (petroleum ether:ethyl acetate (V/V) = 100:1 to 10:1, gradient elution) to give 2,7-dichloro-5-(trifluoromethyl)thieno[3,2-b]pyridine (130 mg, yield 52.1%).

LC-MS, M/Z(ESI):272.0[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.21(s,1H),8.04(s,1H).

Step 4: Synthesis of tert-butyl (1R,3S)-3-((2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)amino)cyclohexyl)carbamate

2,7-Dichloro-5-(trifluoromethyl)thieno[3,2-b]pyridine (100 mg, 0.37 mmol) and tert-butyl (((1R,3S)-3-aminocyclohexyl)amino)formate (82.7 mg, 0.39 mmol) were dissolved in N-methylpyrrolidone (2 mL), N,N-diisopropylethylamine (0.06 mL, 0.37 mmol) was added, and the reaction solution was stirred at 130 ° C for 3 hours. Water (30 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated brine (10 mL×3), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product, which was separated and purified by silica gel column (petroleum ether:ethyl acetate (V/V) = 100:1 to 5:1, gradient elution) to obtain tert-butyl (1R,3S)-3-((2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)amino)cyclohexyl)carbamate (100 mg, yield 54.4%).

LC-MS, M/Z (ESI): 450.2 [M+H] ⁺ .

Step 5: Synthesis of (1S,3R)-N ¹ -(2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)cyclohexane-1,3-diamine

Tert-butyl (1R,3S)-3-((2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)amino)cyclohexyl)carbamate (200 mg, 0.45 mmol) was dissolved in anhydrous dichloromethane (4 mL), trifluoroacetic acid (2 mL) was added, and the reaction solution was stirred at 25°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain a crude product which was separated and purified by a silica gel column (dichloromethane:methanol (V/V) = 10:1 to 1:1, gradient elution) to obtain (1S,3R)-N ¹ -(2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)cyclohexane-1,3-diamine (20 mg, yield 11.6%).

LC-MS, M/Z (ESI): 350.2 [M+H] ⁺ .

Step 6: Synthesis of N-((1R,3S)-3-((2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide

(1S,3R)-N ¹ -(2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)cyclohexane-1,3-diamine (10.0 mg, 0.03 mmol) and 1-methylpyrazole-4-carboxylic acid (4.33 mg, 0.03 mmol) were dissolved in anhydrous dimethyl sulfoxide (1 mL), and N,N-diisopropylethylamine (2 drops), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (16.7 mg, 0.09 mmol), and 1-hydroxybenzotriazole (11.76 mg, 0.09 mmol) were added in sequence. The reaction solution was stirred at 25° C. for 18 hours under nitrogen protection. The reaction solution was added to saturated brine (10 mL), then extracted with ethyl acetate (10 mL×3), the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to give a crude product, which was purified by high performance preparative liquid chromatography (Xtimate C18, 21.2*250 mm, 5 μm; 0.1% TFA-ACN; 40-70; 20 mL/min) to give compound N-((1R, 3S)-3-((2-chloro-5-(trifluoromethyl)thieno[3,2-b]pyridin-7-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide trifluoroacetate (3.50 mg, yield 21.4%).

LC-MS, M/Z(ESI):458.0[M+H] ⁺ .

¹ H NMR (400MHz, MeOD): δ8.03(s,1H),7.88(s,1H),7.37(s,1H),7.04(s,1H),4.10–3.98(m,1H),3.90(s,3H),3.8 9–3.77(m,1H),2.40–2.29(m,1H),2.12–2.05(m,1H),2.01–1.91(m,2H),1.66–1.57(m,1H),1.51–1.35(m,3H).

Example 5: Preparation of 1-methyl-N-((1R,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (Compound 26A)

The synthetic route of compound 26A is as follows:

Step 1: Synthesis of 6-chloro-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one

4-Bromo-6-chloro-2-methylpyridazine-3(2H)-one (500mg, 2.24mmol) was dissolved in a solvent of N-methylpyrrolidone (5mL), methyl fluorosulfonyl difluoroacetate (12.9g, 6.71mmol) and cuprous iodide (85.3g, 0.448mmol) were added, and the reaction solution was stirred at 80°C for 18 hours. LC-MS detected that the reaction was complete. Water (30mL) was added to the reaction solution, extracted with ethyl acetate (50mL×3), the organic layers were combined, washed with saturated brine (100mL), then dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was separated and purified by silica gel column (dichloromethane: ethyl acetate (V/V) = 100:0 to 95:5) to obtain compound 6-chloro-2-methyl-4-(trifluoromethyl)pyridazine-3(2H)-one (300mg, yield 63.1%).

LC-MS, M/Z(ESI):213.0[M+H] ⁺ .

Step 2: Synthesis of tert-butyl ((1R,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate

6-Chloro-2-methyl-4-(trifluoromethyl)pyridazine-3(2H)-one (500 mg, 2.35 mmol) was dissolved in toluene (10 mL), and tert-butyl (1R, 3S)-3-aminocyclohexyl)carbamate (504 mg, 2.35 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2', 4', 6'-tri-isopropyl-1, 1'-biphenyl) (2'-amino-1, 1'-biphenyl-2-yl) palladium (II) (200 mg, 0.235 mmol) and cesium carbonate (1.53 g, 4.71 mmol) were added. The reaction solution was stirred at 80°C for 14 hours. LC-MS detected that the reaction was complete. Water (50 mL) was added to the reaction system, and the mixture was extracted with ethyl acetate (50 mL×3). The organic layers were combined, washed with saturated brine (30 mL×3), and then dried over anhydrous sodium sulfate, filtered and concentrated. The residue was separated and purified by silica gel column (petroleum ether:ethyl acetate (V/V)=100:0-90:10) to give tert-butyl ((1R,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate (600 mg, yield 65.3%).

LC-MS, M/Z (ESI): 391.4 [M+H] ⁺ .

Step 3: Synthesis of 6-(((1S,3R)-3-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate

Tert-butyl ((1R, 3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate (600 mg, 1.54 mmol) was dissolved in dichloromethane (5 mL) solvent, and trifluoroacetic acid (2 mL) was added. The reaction solution was reacted at 25 ° C for 2 hours. LC-MS detected that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain a crude compound 6-(((1S, 3R)-3-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate (600 mg, yield 80.7%).

LC-MS, M/Z (ESI): 291.4 [M+H] ⁺ .

Step 4: 1-methyl-N-((1R,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide

6-(((1S,3R)-3-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazine-3(2H)-one trifluoroacetate (300 mg, 0.620 mmol) and 1-methylpyrazole-4-carboxylic acid (78.2 mg, 0.620 mmol) were dissolved in dimethyl sulfoxide (5 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (178 mg, 0.930 mmol), 1-hydroxybenzotriazole (126 mg, 0.930 mmol) and N,N-diisopropylethylamine (401 mg, 3.10 mmol) were added. The reaction solution was stirred at 25° C. for 2 hours under N ₂ protection. LC-MS detected that the reaction was complete. The reaction solution was concentrated under reduced pressure and the residue was purified by preparative HPLC (Xtimate C18 21.2*250mm 5um; 0.1% NH ₃ .H ₂ O-ACN; 40-52; 20 mL/min) to give 1-methyl-N-((1R,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (160 mg, yield 64.9%).

LC-MS, M/Z (ESI): 399.4 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.09(s,1H),7.84(d,1H),7.81(d,1H),7.41(s,1H),6.59(d,1H),3.83(s,3H),3.82–3.74(m,1H),3.53–3.46(m,4 H),2.20–2.14(m,1H),2.03–1.96(m,1H),1.83–1.73(m,2H),1.43–1.31(m,1H),1.25–1.11(m,2H),1.08–0.98(m,1H).

Example 6: Preparation of N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 27A)

The synthetic route of compound 27A is as follows:

Step 1: Synthesis of tert-butyl (1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate

To a solution of 6-chloro-2-methyl-4-(trifluoromethyl)pyridazine-3(2H)-one (1.00 g, 4.70 mmol) in 1,4-dioxane (15 mL) were added cis-4-aminocyclohexyl)carbamic acid tert-butyl ester (1.00 g, 21.2 mmol), cesium carbonate (3.06 g, 9.41 mmol) and methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II) (200 mg, 0.240 mmol). The reaction solution was stirred at 80 ° C for 18 hours under nitrogen protection. LCMS detected that the reaction was complete. The reaction solution was filtered, the filtrate was diluted with water (100 mL), extracted with ethyl acetate (100 mL×3), the organic layers were combined, washed with saturated brine (100 mL×3), dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was separated and purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 80:20) to obtain compound (1S, 4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (27A-2) (700 mg, yield 38.1%).

LC-MS, M/Z(ESI):391.2[M+H] ⁺

Step 2: Synthesis of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (27A-3)

Trifluoroacetic acid (3.00 mL) was added to a solution of cis-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (27A-2) (300 mg, 0.760 mmol) in dichloromethane (10 mL), and the reaction solution was stirred at 25 ° C for 1 hour. LCMS detected that the reaction was complete. The reaction solution was poured into a saturated sodium bicarbonate aqueous solution (50 mL), extracted with dichloromethane (20 mL*3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (27A-3) (300 mg, yield 80.5%), which was directly used in the next step reaction.

LC-MS, M/Z(ESI):291.2[M+H] ⁺

Step 3: Synthesis of N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 27A)

1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (193 mg, 0.990 mmol), EDCI (237 mg, 1.28 mmol), HOBt (174 mg, 224 mmol) and DIEA (0.64 mL) were added to a solution of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (27A-3) (250 mg, 0.860 mmol) in dimethyl sulfoxide (2.0 mL). The reaction solution was reacted at 25°C for 1 hour. LCMS detected that the reaction was complete. After the reaction solution was filtered, water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL×3), washed with saturated brine (50 mL×3), and the organic layers were combined, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was purified by preparation (Xtimate C ₁₈ 21.2*250mm 5um; 0.1% _NH3.H2O -ACN; 30-62; 40mL/min) to give N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 27A) (151 mg, yield 62.7%).

LC-MS, M/Z(ESI):467.4[M+H] ⁺

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.31(s,1H),8.00(s,1H),7.94(d,1H),7.56(s,1H),6.59(d,1H),5.18(q,2H),3.8 5–3.74(m,1H),3.67–3.60(m,1H),3.50(s,3H),1.91–1.80(m,2H),1.69–1.56(m,6H).

Example 7: Preparation of 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 28A)

The synthetic route of compound 28A is as follows:

Step 1: Synthesis of ethyl 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylate

To a solution of 3-chloro-1H-pyrazole-4-carboxylic acid ethyl ester (1.00 g, 5.72 mmol) in N,N-dimethylformamide (10 mL) was added trifluoromethanesulfonic acid 2,2,2-trifluoroethyl ester (3.90 g, 17.2 mmol) and cesium carbonate (5.60 g, 17.2 mmol), and the reaction solution was stirred at 25 ° C for 18 hours. LCMS detection reaction was complete. Water (100 mL) was added to the reaction solution, extracted with ethyl acetate (100 mL × 3), the organic layer was combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product separated and purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100: 0 to 75: 25), and compound 3-chloro-1- (2,2,2-trifluoroethyl) -1H-pyrazole-4-carboxylic acid ethyl ester (1.20 g, yield 81.6%) was obtained.

LC-MS, M/Z(ESI):257.4[M+H] ⁺

Step 2: Synthesis of 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (Compound 28A-4)

Methanol (3.0 mL), water (2.0 mL) and lithium hydroxide (0.590 g, 14.0 mmol) were added to a solution of ethyl 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylate (1.20 g, 4.67 mmol) in tetrahydrofuran (10 mL), and the reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete, water (100 mL) was added to the reaction solution, pH was adjusted to 4 with 1M dilute hydrochloric acid, and then extracted with dichloromethane (100 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (600 mg, yield 56.1%), which was directly used in the next step reaction.

LC-MS, M/Z(ESI):229.0[M+H] ⁺

Step 3: Synthesis of 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide

To a solution of 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (100 mg, 0.430 mmol) in dimethyl sulfoxide (2.0 mL) were added 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (152 mg, 0.520 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (126 mg, 0.650 mmol), 1-hydroxybenzotriazole (88.7 mg, 0.650 mmol) and N,N-diisopropylethylamine (0.36 mL), and the reaction solution was stirred at 25°C for 1 hour. LCMS detected that the reaction was complete, the reaction solution was filtered, the filtrate was diluted with water (50 mL), extracted with ethyl acetate (50 mL×3), the organic layers were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product, which was purified by preparative liquid chromatography (Xtimate C18 21.2*250mm 5um; 0.1% FA.H ₂ O-ACN; 35-60; 20 mL/min) to obtain compound 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (16.4 mg, yield 7.5%).

LC-MS, M/Z(ESI):501.0[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD): δ8.22(s,1H),7.43(s,1H),4.97(q,2H),4.03–3.95(m,1H),3.81–3.74(m,1H),3.63(s,3H),1.86–1.76(m,8H).

Example 8: Preparation of N-((1S,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclobutyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 29A)

The synthetic route of compound 29A is as follows:

Step 1: Synthesis of tert-butyl (1S,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclobutyl)carbamate

6-Chloro-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (100 mg, 0.470 mmol) was dissolved in toluene (2 mL), and tert-butyl ((1S,3S)-3-aminocyclobutyl)carbamate (105 mg, 0.565 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium (II) (40.0 mg, 0.0470 mmol) and cesium carbonate (306 mg, 0.941 mmol) were added, and the reaction solution was stirred at 80°C for 14 hours. LC-MS detected that the reaction was completed, and water (50 mL) was added to the reaction system. The mixture was extracted with ethyl acetate (50 mL×3). The organic layers were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product which was separated and purified by a silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 80:20) to obtain compound (1S, 3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclobutyl)carbamic acid tert-butyl ester (100 mg, yield 58.6%).

LC-MS, M/Z (ESI): 363.4 [M+H] ⁺ .

Step 2: Synthesis of 6-(((1S,3S)-3-aminocyclobutyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate

Tert-butyl (1S,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclobutyl)carbamate (100 mg, 0.276 mmol) was dissolved in dichloromethane (5 mL) solvent, trifluoroacetic acid (2 mL) was added, and the reaction solution was reacted at 25°C for 1.5 hours. LC-MS detected that the reaction was complete, and the reaction solution was concentrated under reduced pressure to obtain a crude compound 6-(((1S,3S)-3-aminocyclobutyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate (100 mg, yield 96.7%), which was directly used in the next step reaction.

LC-MS, M/Z (ESI): 263.4 [M+H] ⁺ .

Step 3: Synthesis of N-((1S,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclobutyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide

6-(((1S,3S)-3-Aminocyclobutyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate (70.0 mg, 0.240 mmol) and 1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (46.6 mg, 0.240 mmol) were dissolved in dimethyl sulfoxide (3 mL), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (92.1 mg, 0.480 mmol), 1-hydroxybenzotriazole (64.9 mg, 0.480 mmol) and N,N-diisopropylethylamine (155 mg, 1.20 mmol) were added, and the reaction solution was stirred at 25 ° C for 2 hours under _N2 protection. LC-MS detected that the reaction was completed, and the reaction solution was purified by preparative liquid chromatography (Xtimate C18 21.2*250mm 5um; 0.05% NH ₃ .H ₂ O-ACN; 20-45; 20mL/min) to obtain compound N-((1S,3S)-3-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclobutyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 29A) (74.0 mg, yield 70.3%).

LC-MS, M/Z(ESI):439.2[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.38(d,1H),8.28(s,1H),7.97(s,1H),7.41(s,1H),6.97(d,1H),5.19(q,2H),4.1 4–4.04(m,1H),3.76–3.67(m,1H),3.51(s,3H),2.72–2.65(m,2H),1.91–1.83(m,2H).

Example 9: Preparation of 3-chloro-N-((1S,4S)-4-((6-oxo-1-(2,2,2-trifluoroethyl)-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 30A)

The synthetic route of compound 30A is as follows:

Step 1: Synthesis of 4-bromo-6-chloro-2-(2,2,2-trifluoroethyl)pyridazin-3(2H)-one

To a solution of 4-bromo-6-chloropyridazine-3(2H)-one (500 mg, 2.39 mmol) in N,N-dimethylformamide (10 mL) were added trifluoromethanesulfonic acid 2,2,2-trifluoroethyl ester (831 mg, 3.58 mmol) and potassium carbonate (989 mg, 7.16 mmol), and the resulting mixture was stirred at 25 ° C for 18 hours. LCMS detected that the reaction was complete. The reaction solution was diluted with water (100 mL), extracted with ethyl acetate (50 mL×3), the organic phases were combined, washed with saturated brine (40 mL), and concentrated to obtain a crude product. The crude product was purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 80:20) to obtain compound 4-bromo-6-chloro-2-(2,2,2-trifluoroethyl)pyridazine-3(2H)-one (500 mg, yield 71.9%).

LC-MS, M/Z (ESI): 293.2 [M+H] ⁺ .

Step 2: Synthesis of 6-chloro-2-(2,2,2-trifluoroethyl)-4-(trifluoromethyl)pyridazin-3(2H)-one

To a solution of 4-bromo-6-chloro-2-(2,2,2-trifluoroethyl)pyridazine-3(2H)-one (2.00 g, 6.862 mmol) in N-methylpyrrolidone (40 mL) was added methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (3.95 g, 20.6 mmol) and cuprous iodide (261 mg, 1.37 mmol), and the resulting mixture was reacted at 25 °C for 14 hours. LCMS detected that the reaction was complete. The reaction solution was diluted with water (50 mL), then extracted with ethyl acetate (100 mL×3), the combined organic layers were washed with saturated brine (100 mL), and concentrated to obtain a crude product. The crude product was purified by silica gel column (petroleum ether:ethyl acetate (V/V) = 100:0 to 90:10) to give compound 6-chloro-2-(2,2,2-trifluoroethyl)-4-(trifluoromethyl)pyridazin-3(2H)-one (1.00 g, yield 51.9%).

LC-MS, M/Z(ESI):281.0[M+H] ⁺ .

Step 3: Synthesis of tert-butyl (1S,4S)-4-((6-oxo-1-(2,2,2-trifluoroethyl)-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate

To a solution of tert-butyl ((1S,4S)-4-aminocyclohexyl)carbamate (382 mg, 1.78 mmol) in 1,4-dioxane (10 mL) were added 6-chloro-2-(2,2,2-trifluoroethyl)-4-(trifluoromethyl)pyridazin-3(2H)-one (500 mg, 1.78 mmol), Ruphos Pd G4 (1.55 g, 1.82 mmol) and cesium carbonate (1.74 g, 5.35 mmol), and the resulting mixture was stirred at 80 ° C for 18 hours. LCMS detected that the reaction was complete. The reaction solution was diluted with water (100 mL), extracted with ethyl acetate (100 mL×3), and the organic phases were combined, washed with saturated brine (100 mL), and concentrated to obtain a crude product. The crude product was purified by silica gel column (petroleum ether:ethyl acetate (V/V) = 100:0 to 75:25) to obtain compound (1S,4S)-tert-butyl 4-((6-oxo-1-(2,2,2-trifluoroethyl)-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate (200 mg, yield 24.5%).

LC-MS, M/Z(ESI):459.2[M+H] ⁺ .

Step 4: Synthesis of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-(2,2,2-trifluoroethyl)-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate

Trifluoroacetic acid (2 mL, 0.218 mmol) was added to a solution of tert-butyl (1S, 4S)-4-((6-oxo-1-(2,2,2-trifluoroethyl)-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate (100 mg, 0.218 mmol) in dichloromethane (5 mL), and the resulting mixture was stirred at 25 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain compound 6-(((1S, 4S)-4-aminocyclohexyl)amino)-2-(2,2,2-trifluoroethyl)-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate (100 mg, crude product), which was directly used in the next step reaction.

LC-MS, M/Z (ESI): 359.0 [M+H] ⁺ .

Step 5: Synthesis of 3-chloro-N-((1S,4S)-4-((6-oxo-1-(2,2,2-trifluoroethyl)-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 30A)

To a solution of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-(2,2,2-trifluoroethyl)-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate (80.0 mg, 0.167 mmol) and 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (38.3 mg, 0.167 mmol) in dimethyl sulfoxide (2 mL) was added N,N-diisopropylethylamine (0.138 mL, 0.835 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (48.2 mg, 0.251 mmol) and 1-hydroxybenzotriazole (33.9 mg, 0.251 mmol), and the resulting mixture was stirred at 25° C. for 14 hours. The reaction was complete by LCMS. The reaction solution was purified by preparative liquid chromatography (Xtimate C18 21.2*250mm 5um; 0.05% _NH3.H2O -ACN; 20-45; 20mL/min) to give compound 3-chloro-N-((1S,4S)-4-((6-oxo-1-(2,2,2-trifluoroethyl)-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 30A) (37.0 mg, yield 38.8%).

LC-MS, M/Z(ESI):569.2[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.37(s,1H),7.87(d,1H),7.65(s,1H),6.82(d,1H),5.20(q,2H),4.75(q,2 H),3.85–3.74(m,1H),3.64–3.55(m,1H),1.85–1.75(m,2H),1.72–1.60(m,6H).

Example 10: Preparation of 3-chloro-1-(2,2-difluoroethyl)-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (Compound 34A)

The synthetic route of compound 34A is as follows:

Step 1: Synthesis of ethyl 3-chloro-1-(2,2-difluoroethyl)-1H-pyrazole-4-carboxylate

2,2-difluoroethyl trifluoromethanesulfonate (2.45 g, 11.5 mmol) and cesium carbonate (3.73 g, 11.5 mmol) were added to a solution of 3-chloro-1H-pyrazole-4-carboxylic acid ethyl ester (1.00 g, 5.30 mmol) in N,N-dimethylformamide (10 mL), and the resulting mixture was stirred at 25 ° C for 18 hours. LCMS detected that the reaction was complete, the reaction solution was diluted with water (100 mL), and then extracted with ethyl acetate (100 mL×3), the organic layers were combined, and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 80:20) to obtain compound 3-chloro-1-(2,2-difluoroethyl)-1H-pyrazole-4-carboxylic acid ethyl ester (500 mg, yield 36.6%).

LC-MS, M/Z(ESI):239.0[M+H] ⁺

Step 2: Synthesis of 3-chloro-1-(2,2-difluoroethyl)-1H-pyrazole-4-carboxylic acid

Lithium hydroxide (264 mg, 6.29 mmol) was added to a mixed solution of ethyl 3-chloro-1-(2,2-difluoroethyl)-1H-pyrazole-4-carboxylate (500 mg, 2.09 mmol) in tetrahydrofuran (5 mL), methanol (2 mL) and water (1 mL), and the resulting mixture was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete, the reaction solution was diluted with water (100 mL), then adjusted to pH = 4 with 1M hydrochloric acid, and extracted with dichloromethane (100 mL×3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 3-chloro-1-(2,2-difluoroethyl)-1H-pyrazole-4-carboxylic acid (420 mg, yield 95.2%), which was directly used in the next step reaction.

LC-MS, M/Z(ESI):211.0[M+H] ⁺

Step 3: Synthesis of 3-chloro-1-(2,2-difluoroethyl)-N-(cis-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide

To a solution of 3-chloro-1-(2,2-difluoroethyl)-1H-pyrazole-4-carboxylic acid (150 mg, 0.712 mmol) in dimethyl sulfoxide (2 mL) were added 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (207 mg, 0.712 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (205 mg, 1.069 mmol), 1-hydroxybenzotriazole (144 mg, 1.07 mmol) and N,N-diisopropylethylamine (0.588 mL, 3.56 mmol), and the reaction solution was reacted at 25° C. for 1 hour. LCMS detected that the reaction was complete. The reaction solution was diluted with saturated brine (50 mL), and then extracted with ethyl acetate (50 mL×3). The organic phases were combined and concentrated to give a crude product, which was purified by preparative liquid chromatography (Xtimate C18 21.2*550mm 5um; 0.1% FA.H ₂ O-ACN; 30-55; 20 mL/min) to give compound 3-chloro-1-(2,2-difluoroethyl)-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (59.8 mg, yield 17.2%).

LC-MS, M/Z(ESI):483.4[M+H] ⁺

¹ H NMR (400MHz, CD3OD): δ8.15(s,1H),7.42(s,1H),6.20(tt,1H),4.60–4.58(m ,2H),4.01–3.94(m,1H),3.80–3.74(m,1H),3.62(s,3H),1.87–1.74(m,8H).

Example 11: Preparation of 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 39A)

The synthetic route of compound 39A is as follows:

Step 1: Synthesis of 5-bromo-1-methyl-3-(trifluoromethyl)pyridin-2(1H)-one

Potassium carbonate (14.3 g, 103 mmol) and iodomethane (3.52 g, 24.8 mmol) were added to a solution of 5-bromo-3-(trifluoromethyl)pyridin-2(1H)-one (5.00 g, 20.7 mmol) in N,N-dimethylformamide (50 mL), and the resulting mixture was stirred at 25° C. for 2 hours. LCMS detected that the reaction was complete, the reaction solution was diluted with water (50 mL), extracted with ethyl acetate (50 mL×3), the organic phases were combined, washed with saturated brine (40 mL), and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 90:10) to obtain compound 5-bromo-1-methyl-3-(trifluoromethyl)pyridin-2(1H)-one (4.00 g, yield 75.6%).

LC-MS, M/Z(ESI):256.0[M+H] ⁺ .

Step 2: Synthesis of tert-butyl (1S,4S)4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)amino)cyclohexyl)carbamate

To a solution of 5-bromo-1-methyl-3-(trifluoromethyl)pyridin-2(1H)-one (200 mg, 0.781 mmol) and tert-butyl ((1S,4S)-4-aminocyclohexyl)carbamate (251 mg, 1.17 mmol) in N,N-dimethylformamide (10 mL) were added sodium tert-butoxide (225 mg, 2.34 mmol) and methanesulfonic acid (2-di-tert-butylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (62.1 mg, 0.078 mmol), and the resulting mixture was stirred at 120° C. for 18 hours. LCMS detected that the reaction was complete. Water (10 mL) was added to the reaction mixture to dilute it, and then it was extracted with ethyl acetate (10 mL×3). The organic layers were combined, washed with saturated brine (10 mL), and concentrated under reduced pressure to give a crude product, which was purified by silica gel column (dichloromethane: methanol (V/V) = 100:0 to 95:5) to give compound (1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (50.0 mg, yield 16.4%).

LC-MS, M/Z (ESI): 390.2 [M+H] ⁺ .

Step 3: Synthesis of 5-(((1S,4S)-4-aminocyclohexyl)amino)-1-methyl-3-(trifluoromethyl)pyridin-2(1H)-one trifluoroacetate

Trifluoroacetic acid (0.5 mL, 0.051 mmol) was added to a solution of tert-butyl (1S, 4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)amino)cyclohexyl)carbamate (20.0 mg, 0.051 mmol) in dichloromethane (2 mL), and the resulting mixture was stirred at 25° C. for 1 hour. LCMS detected that the reaction was complete, and the reaction solution was concentrated under reduced pressure to give compound 5-(((1S, 4S)-4-aminocyclohexyl)amino)-1-methyl-3-(trifluoromethyl)pyridin-2(1H)-one trifluoroacetate (20.0 mg, crude product), which was used directly in the next step.

LC-MS, M/Z (ESI): 290.2 [M+H] ⁺ .

Step 4: Synthesis of 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 39A)

To a solution of 5-(((1S,4S)-4-aminocyclohexyl)amino)-1-methyl-3-(trifluoromethyl)pyridin-2(1H)-one trifluoroacetate (20.0 mg, 0.069 mmol) and 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (15.8 mg, 0.069 mmol) in dimethyl sulfoxide (3 mL) were added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (26.5 mg, 0.138 mmol), 1-hydroxybenzotriazole (18.7 mg, 0.138 mmol) and N,N-diisopropylethylamine (0.057 mL, 0.346 mmol), and the resulting mixture was reacted at 25° C. for 18 hours. LCMS detected that the reaction was complete, and the reaction solution was concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (Xtimate C18 21.2*250mm 5um; 0.05% NH ₃ .H ₂ O-ACN; 15-35; 25mL/min) to give compound 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 39A) (7.60 mg, yield 21.4%).

LC-MS, M/Z (ESI): 500.2 [M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.39(s,1H),7.81(d,1H),7.68(d,1H),7.15(d,1H),5.20(q,2H),4.93(d ,1H),3.93–3.70(m,1H),3.44(s,3H),3.20–3.13(m,1H),1.75–1.56(m,8H).

Example 12: Preparation of 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxamide (Compound 42A)

The synthetic route of compound 42A is as follows:

Step 1: Synthesis of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one hydrochloride

At room temperature, tert-butyl (1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate (100 mg, 0.26 mmol) was dissolved in dichloromethane (2 mL), stirred for 10 minutes in an ice bath at 0°C, and then a solution of hydrogen chloride in 1,4-dioxane (0.13 mL, 0.13 mmol) was slowly added dropwise. After the addition was completed, the reaction solution was naturally warmed to room temperature and continued to stir at room temperature for 1 hour. After TLC monitoring showed that the raw material had reacted completely, stirring was stopped and the reaction solution was concentrated under reduced pressure to obtain compound 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one hydrochloride (100 mg, crude product), which was directly used in the next step.

LC-MS, M/Z(ESI):291.1[M+H] ⁺

Step 2: Synthesis of 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxamide

At room temperature, compound 3-chloro-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxylic acid (32.00 mg, 0.13 mmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL), and then N,N-diisopropylethylamine (65.47 mg, 0.50 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (96.98 mg, 0.25 mmol) were added, and the mixture was stirred at room temperature for 30 minutes. Then, 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one hydrochloride (50.00 mg, 0.15 mmol) was added, and stirring was continued at room temperature for 4 hours. After TLC monitoring showed that the reaction of the raw materials was completed, stirring was stopped, the reaction solution was cooled to room temperature, then 10 mL of water was added to dilute it, extracted with ethyl acetate (5 mL×3), the organic phase was collected and dried over anhydrous sodium sulfate, and the organic phase was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 100:20) to obtain compound 3-chloro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(1-(methylsulfonyl)ethyl)-1H-pyrazole-4-carboxamide.

LC-MS, M/Z(ESI):525.0[M+H] ⁺

¹ H NMR (400MHz, DMSO-d6): δ8.52(s,1H),7.87(d,1H),7.54(s,1H),6.62(d,1H),5.92(q,1H),3.85–3. 76(m,1H),3.62(s,1H),3.50(s,3H),2.99(s,3H),1.83(d,J＝7.1Hz,3H),1.75(s,2H),1.65(d,6H).

Example 13: Preparation of 3-chloro-1-(2-hydroxy-2-methylpropyl)-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (Compound 48A)

The synthetic route of compound 48A is as follows:

To a solution of 3-chloro-1-(2-hydroxy-2-methylpropyl)-1H-pyrazole-4-carboxylic acid (200 mg, 0.915 mmol) in dimethyl sulfoxide (2 mL) were added 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (266 mg, 0.915 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (263 mg, 1.37 mmol), 1-hydroxybenzotriazole (185 mg, 1.37 mmol) and N,N-diisopropylethylamine (0.756 mL, 4.58 mmol), and the reaction solution was reacted at 25° C. for 1 hour. LCMS detected that the reaction was complete. The reaction solution was diluted with saturated brine (50 mL), and then extracted with ethyl acetate (50 mL×3). The organic phases were combined and concentrated to give a crude product, which was purified by preparative liquid chromatography (Xtimate C1821.2*250mm 5μm; 0.1% _FA.H2O -ACN; 25-55; 20 mL/min) to give compound 3-chloro-1-(2-hydroxy-2-methylpropyl)-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (115 mg, yield 25.6%).

LC-MS, M/Z(ESI):491.2[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD): δ8.08(s,1H),7.43(s,1H),4.06(s,2H),4.02–3.97(m,1H),3.80–3.75(m,1H),3.63(s,3H),1.86–1.76(m,8H),1.19–1.17(m,6H).

Example 14: Preparation of 3-chloro-1-methyl-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (Compound 49A)

The synthetic route of compound 49A is as follows:

3-Chloro-1-methyl-1H-pyrazole-4-carboxylic acid (111 mg, 0.689 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (198 mg, 1.03 mmol), 1-hydroxybenzotriazole (140 mg, 1.03 mmol) and N,N-diisopropylethylamine (0.569 mL, 3.45 mmol) were added to a solution of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (200 mg, 0.689 mmol) in dimethyl sulfoxide (2 mL), and the resulting mixture was stirred at 25° C. for 1 hour. The reaction was complete by LCMS. The reaction solution was diluted with saturated brine (50 mL), and then extracted with ethyl acetate (50 mL×3). The organic phases were combined and concentrated to give a crude product, which was purified by preparative liquid chromatography (Xtimate C18 30*150mm 5um; 0.1% _FA.H2O -ACN; 28-48; 25 mL/min) to give compound 3-chloro-1-methyl-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (16.7 mg, yield 2.6%).

LC-MS, M/Z(ESI):433.2[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD): δ8.04(s,1H),7.43(s,1H),4.02–3.95(m,1H),3.87(s,3H),3.81–3.74(m,1H),3.63(s,3H),1.87–1.73(m,8H).

Example 15: Preparation of 4-fluoro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)benzamide (Compound 50A)

The synthetic route of compound 50A is as follows:

At room temperature, the compound p-fluorobenzoic acid (18.00 mg, 0.13 mmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL), and then N,N-diisopropylethylamine (65.47 mg, 0.50 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (96.98 mg, 0.25 mmol) were added. The reaction solution was stirred at room temperature for 30 minutes, and then 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazine-3(2H)-one hydrochloride (50.00 mg, 0.15 mmol) was added, and the reaction solution continued to stir at room temperature for 4 hours. After TLC monitoring showed that the reaction of the raw materials was completed, stirring was stopped, the reaction solution was cooled to room temperature, and then 10 mL of water was added to dilute it, extracted with ethyl acetate (5 mL×3), the organic phase was collected and dried over anhydrous sodium sulfate, and the organic phase was concentrated by distillation under reduced pressure. The residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 100:15) to obtain compound 4-fluoro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)benzamide.

LC-MS, M/Z(ESI):413.0[M+H] ⁺

¹ H NMR (400MHz, DMSO-d6): δ8.26(d,1H),7.98–7.87(m,2H),7.57(s,1H),7.33-.23(m,2H),6 .62(d,1H),3.86-.80(m,1H),3.64(s,1H),3.51(s,3H),1.87(dd,2H),1.73-1.61(m,6H).

Example 16: Preparation of 3-chloro-N-((1S,4S)-4-((1-cyclopropyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 31A)

The synthetic route of compound 31A is as follows:

Step 1: Synthesis of 4-bromo-6-chloro-2-cyclopropylpyridazin-3(2H)-one

Pyridine (23.1 mL, 286 mmol), triethylamine (24.9 mL, 179 mmol), cyclopropylboronic acid (9.23 g, 107 mmol) and cupric acetate (6.50 g, 35.8 mmol) were added to a solution of 4-bromo-6-chloropyridazine-3 (2H) -one (7.50 g, 35.8 mmol) in dioxane (60 mL), and the resulting reaction solution was stirred at 80 ° C for 30 minutes under nitrogen protection. LCMS detected that the reaction was complete. The reaction solution was cooled to room temperature, diluted with water (50 mL), and then extracted with ethyl acetate (50 mL × 3), the organic phases were combined, and concentrated to give a crude product, which was purified by silica gel column (petroleum ether: ethyl acetate (V / V) = 100: 0 to 80: 20) to give compound 4-bromo-6-chloro-2-cyclopropylpyridazine-3 (2H) -one (1.40 g, yield 15.7%).

LC-MS, M/Z(ESI):249.0[M+H] ⁺

Step 2: Synthesis of 6-chloro-2-cyclopropyl-4-(trifluoromethyl)pyridazin-3(2H)-one

Cuprous iodide (534 mg, 2.81 mmol) and methyl fluorosulfonyl difluoroacetate (5.39 g, 28.1 mmol) were added to a solution of 4-bromo-6-chloro-2-cyclopropylpyridazine-3 (2H) -one (1.40 g, 5.61 mmol) in N-methylpyrrolidone (15 mL), and the resulting reaction solution was stirred at 100 ° C for 14 hours in a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was cooled to room temperature, diluted with water (50 mL), and then extracted with ethyl acetate (50 mL × 3), the organic phases were combined, and the crude product was concentrated under reduced pressure to obtain a crude product, which was purified by a silica gel column (petroleum ether: ethyl acetate (V / V) = 100: 0 to 90: 10) to obtain compound 6-chloro-2-cyclopropyl-4- (trifluoromethyl) pyridazine-3 (2H) -one (1.20 g, yield 89.6%).

LC-MS, M/Z(ESI):239.0[M+H] ⁺

Step 3: Synthesis of tert-butyl (1S,4S)-4-((1-cyclopropyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate

To a solution of 6-chloro-2-cyclopropyl-4-(trifluoromethyl)pyridazin-3(2H)-one (1.20 g, 5.03 mmol) in 1,4-dioxane (15 mL) were added tert-butyl ((1S,4S)-4-aminocyclohexyl)carbamate (2.16 g, 10.1 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II) (428 mg, 0.503 mmol) and cesium carbonate (4.92 g, 15.1 mmol), and the resulting reaction solution was stirred at 80° C. for 18 hours under nitrogen protection. The reaction was completed by LCMS. The reaction solution was cooled to room temperature, diluted with water (100 mL), and then extracted with ethyl acetate (50 mL×3). The organic layers were combined and concentrated under reduced pressure to give a crude product, which was purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 80:20) to give compound (1S, 4S)-4-((1-cyclopropyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (1.50 g, yield 71.6%).

LC-MS, M/Z(ESI):417.4[M+H] ⁺

Step 4: Synthesis of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-cyclopropyl-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate

Trifluoroacetic acid (2 mL) was added to a solution of tert-butyl (1S, 4S)-4-((1-cyclopropyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate (500 mg, 1.20 mmol) in dichloromethane (5 mL), and the resulting mixture was stirred at 25 ° C for 14 hours. LCMS detected that the reaction was complete. The reaction solution was directly concentrated under reduced pressure to obtain compound 6-(((1S, 4S)-4-aminocyclohexyl)amino)-2-cyclopropyl-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate (500 mg, crude product), which was directly used in the next step.

LC-MS, M/Z(ESI):317.2[M+H] ⁺

Step 5: Synthesis of 3-chloro-N-((1S,4S)-4-((1-cyclopropyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide

To a solution of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-cyclopropyl-4-(trifluoromethyl)pyridazin-3(2H)-one trifluoroacetate (500 mg, 1.11 mmol) and 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (253 mg, 1.11 mmol) in dimethyl sulfoxide (10 mL) was added N,N-diisopropylethylamine (0.914 mL, 5.53 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (318 mg, 1.66 mmol) and 1-hydroxybenzotriazole (224 mg, 1.66 mmol), and the resulting mixture was stirred at 25° C. for 14 hours. The reaction was complete by LCMS. The reaction solution was purified by preparative liquid chromatography (Xtimate C18 21.2*550mm 5um; 0.1% _FA.H2O -ACN; 30-60; 20mL/min) to obtain compound 3-chloro-N-((1S,4S)-4-((1-cyclopropyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (350 mg, yield 60.0%).

LC-MS, M/Z(ESI):527.2[M+H] ⁺

¹ H NMR (400MHz, CD3OD): δ8.21(s,1H),7.38(s,1H),4.96(q,2H),4.13–4.04(m,1H),4.01–3 .93(m,1H),3.74–3.67(m,1H),1.84–1.71(m,8H),1.14–1.08(m,2H),0.97–0.90(m,2H).

Example 17: Preparation of 3-fluoro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 45A)

The synthetic route of compound 45A is as follows:

Step 1: Synthesis of 4-bromo-3-fluoro-1-(2,2,2-trifluoroethyl)-1H-pyrazole

2,2,2-trifluoroethyl trifluoromethanesulfonate (2.25 g, 9.70 mmol) and cesium carbonate (3.16 g, 9.70 mmol) were added to a solution of 4-bromo-3-fluoro-1H-pyrazole (0.80 g, 4.85 mmol) in N,N-dimethylformamide (5 mL), and the resulting mixture was stirred at 25 ° C for 14 hours. LCMS detected that the reaction was complete. The reaction solution was diluted with water (50 mL), then extracted with ethyl acetate (30 mL × 3), the organic layers were combined, and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (PE: EA (V / V) = 100: 0 to 90: 10) to obtain compound 4-bromo-3-fluoro-1- (2,2,2-trifluoroethyl) -1H-pyrazole (0.70 g, yield 58.4%).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.11 (s, 1H), 5.06 (q, 2H).

Step 2: Synthesis of methyl 3-fluoro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylate

To a solution of 4-bromo-3-fluoro-1-(2,2,2-trifluoroethyl)-1H-pyrazole (300 mg, 1.22 mmol) in methanol (10 mL) were added (1,1'-bis(diphenylphosphino)ferrocene)palladium dichloride (88.9 mg, 0.121 mmol) and triethylamine (0.506 mL, 3.64 mmol), and the resulting mixture was stirred at 80 ° C for 18 hours in a CO atmosphere. LCMS detected that the reaction was complete. The reaction solution was cooled to room temperature, diluted with water (20 mL), and then extracted with dichloromethane (10 mL×3). The organic phases were combined and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column (PE:EA (V/V) = 100:0 to 80:20) to obtain compound 3-fluoro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid methyl ester (170 mg, yield 61.9%).

LC-MS, M/Z(ESI):227.0[M+H] ⁺

Step 3: Synthesis of 3-fluoro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid

Lithium hydroxide (37.1 mg, 0.884 mmol) was added to a mixed solvent of tetrahydrofuran (2 mL) and water (2 mL) of 3-fluoro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid methyl ester (40.0 mg, 0.177 mmol), and the resulting mixture was stirred at 25 ° C for 14 hours. LCMS detected that the reaction was complete. The reaction solution was adjusted to pH = 3 with 1M hydrochloric acid, then extracted with dichloromethane (10 mL×3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 3-fluoro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (30.0 mg, yield 79.9%), and the crude product was used directly in the next step without purification.

LC-MS, M/Z(ESI):213.0[M+H] ⁺

Step 4: Synthesis of 3-fluoro-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (54.2 mg, 0.283 mmol), 1-hydroxybenzotriazole (38.2 mg, 0.283 mmol) and N,N-diisopropylethylamine (91.4 mg, 0.707 mmol) were added to a solution of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one (41.1 mg, 0.141 mmol) and 3-fluoro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (30.0 mg, 0.141 mmol) in dimethyl sulfoxide (0.117 mL, 0.707 mmol), and the reaction solution was stirred at 25° C. for 14 hours. The reaction was complete by LCMS. The reaction solution was directly purified by preparative liquid chromatography (Xtimate C18 21.2*550mm 5um; 0.1% _FA.H2O -ACN; 30-60; 20mL/min) to obtain compound 3-chloro-1-(2,2-difluoroethyl)-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1H-pyrazole-4-carboxamide (30.1 mg, yield 32.1%).

LC-MS, M/Z(ESI):485.4[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD): δ8.10–8.07(m,1H),7.43(s,1H),4.91–4.87(m,2H),3.97–3.90(m,1 H),3.80–3.75(m,1H),3.62(s,3H),1.91–1.84(m,2H),1.82–1.68(m,6H).

Example 18: Preparation of 3-chloro-N-((1S,4S)-4-((5-(difluoromethyl)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 51A)

The synthetic route of compound 51A is as follows:

Step 1: Synthesis of (E)-6-chloro-2-methyl-4-phenylvinylpyridazin-3(2H)-one

To a solution of 4-bromo-6-chloro-2-methylpyridazin-3(2H)-one (1.21 g, 5.41 mmol) in 1,4-dioxane (15 mL) were added (E)-phenylvinylboronic acid (1.00 g, 6.76 mmol), potassium phosphate (1.72 g, 8.11 mmol) and bis(triphenylphosphine)palladium dichloride (0.38 g, 0.541 mmol), and the resulting mixture was stirred at 85° C. for 18 hours under nitrogen protection. The reaction was complete by LCMS. The reaction solution was cooled to room temperature, diluted with water (100 mL), and then extracted with ethyl acetate (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by silica gel column (PE:EA (V/V) = 100:0-85:15) to obtain compound (E)-6-chloro-2-methyl-4-phenylvinylpyridazine-3(2H)-one (1.20 g, yield 90.2%).

LC-MS, M/Z(ESI):247.0[M+H] ⁺

Step 2: Synthesis of 6-chloro-2-methyl-3-oxo-2,3-dihydropyridazine-4-carbaldehyde

At 0°C, ruthenium trichloride (84.8 mg, 0.324 mmol) and sodium periodate (2.08 mg, 9.73 mmol) were added to a mixed solvent of (E)-6-chloro-2-methyl-4-phenylvinylpyridazine-3(2H)-one (800 mg, 3.24 mmol) in acetonitrile (3 mL), ethyl acetate (2 mL) and water (3 mL), and the resulting mixture was stirred at 25°C for 18 hours. LCMS detected that the reaction was complete. The resulting mixture was diluted with water (50 mL), then extracted with ethyl acetate (20 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 6-chloro-2-methyl-3-oxo-2,3-dihydropyridazine-4-carboxaldehyde (500 mg, crude product), which was directly used in the next step reaction.

LC-MS, M/Z(ESI):173.4[M+H] ⁺

Step 3: Synthesis of 6-chloro-4-(difluoromethyl)-2-methylpyridazin-3(2H)-one

At 0°C, diethylaminosulfur trifluoride (841 mg, 5.22 mmol) was added to 6-chloro-2-methyl-3-oxo-2,3-dihydropyridazine-4-carboxaldehyde (500 mg, 1.74 mmol) in dichloromethane (5 mL), and the resulting mixture was stirred at 20°C for 18 hours. LCMS detected that the reaction was complete. The reaction solution was quenched with saturated aqueous sodium carbonate solution (30 mL), then extracted with dichloromethane (20 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to give a crude product, which was purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100: 0-90: 10) to give compound 6-chloro-4-(difluoromethyl)-2-methylpyridazine-3(2H)-one (160 mg, yield 33.1%).

LC-MS, M/Z(ESI):195.0[M+H] ⁺

Step 4: Synthesis of tert-butyl (1S,4S)-4-((5-(difluoromethyl)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate

To a solution of 6-chloro-4-(difluoromethyl)-2-methylpyridazin-3(2H)-one (150 mg, 0.771 mmol) in 1,4-dioxane (2 mL) were added tert-butyl ((1S,4S)-4-aminocyclohexyl)carbamate (330 mg, 1.54 mmol), methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II) (65.6 mg, 0.077 mmol) and cesium carbonate (754 mg, 2.31 mmol), and the resulting mixture was stirred at 25° C. under nitrogen protection for 14 hours. The reaction was complete by LCMS. The reaction solution was diluted with water (30 mL), and then extracted with ethyl acetate (50 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product which was purified by silica gel column (petroleum ether:ethyl acetate (V/V) = 100:0-75:35) to obtain compound (1S,4S)-4-((5-(difluoromethyl)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (100 mg, yield 34.8%).

LC-MS, M/Z(ESI):373.2[M+H] ⁺

Step 5: Synthesis of 6-(((1S,4S)-4-aminocyclohexyl)amino)-4-(difluoromethyl)-2-methylpyridazin-3(2H)-one

To tert-butyl (1S, 4S)-4-((5-(difluoromethyl)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate (30.0 mg, 0.073 mmol) was added hydrochloric acid (2 mL, 4 M dioxane solution), and the resulting mixture was stirred at 25 ° C for 1 hour. LCMS detected that the reaction was complete. The reaction solution was directly concentrated to give 6-(((1S, 4S)-4-aminocyclohexyl)amino)-4-(difluoromethyl)-2-methylpyridazin-3(2H)-one hydrochloride (30.0 mg, crude product), which was directly used in the next step reaction.

LC-MS, M/Z(ESI):273.4[M+H] ⁺

Step 6: Synthesis of 3-chloro-N-((1S,4S)-4-((5-(difluoromethyl)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide

To a solution of 6-(((1S,4S)-4-aminocyclohexyl)amino)-4-(difluoromethyl)-2-methylpyridazin-3(2H)-one (60.0 mg, 0.220 mmol) and 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (50.4 mg, 0.220 mmol) in dimethyl sulfoxide (3 mL) was added N,N-diisopropylethylamine (142 mg, 1.10 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (84.6 mg, 0.440 mmol) and 1-hydroxybenzotriazole (59.5 mg, 0.440 mmol), and the resulting mixture was stirred at 25° C. for 18 hours. The reaction was complete by LCMS. The reaction solution was diluted with water (5 mL), then extracted with ethyl acetate (5 mL×3), the organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. The crude product was purified by preparative liquid phase (Prime C18, 30*150 mm, 5 μm; 0.05% NH ₃ .H ₂ O-ACN; 35-56; 25 mL/min) to obtain compound 3-chloro-N-((1S,4S)-4-((5-(difluoromethyl)-1-methyl-6-oxo-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (12.0 mg, yield 11.3%).

LC-MS, M/Z(ESI):483.2[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD): δ8.22(s,1H),7.27(s,1H),6.76(t,1H),4.97(q,2H),4.02–3.92(m,1H),3.82–3.73(m,1H),3.62(s,3H),1.86–1.73(m,8H).

Example 19: Preparation of 3-chloro-N-((1S,4S)-4-((1-ethyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (Compound 52A)

The synthetic route of compound 52A is as follows:

Step 1: Synthesis of 4-bromo-6-chloro-2-ethylpyridazin-3(2H)-one

At 0°C, sodium hydride (955 mg, 23.9 mmol, 60% Wt) was added to a solution of 4-bromo-6-chloropyridazin-3(2H)-one (2.00 g, 9.55 mmol) in N,N-dimethylformamide (10 mL). After stirring at 0°C for 30 minutes, iodoethane (2.98 g, 19.1 mmol) was added, and the resulting mixture was stirred at 25°C for 1 hour. LCMS detected that the reaction was complete. The reaction solution was diluted with water (50 mL), then extracted with ethyl acetate (20 mL×3), the organic phases were combined and concentrated to obtain a crude product, which was purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100:0 to 90:0) to obtain compound 4-bromo-6-chloro-2-ethylpyridazin-3(2H)-one (1.00 g, yield 44.1%).

LC-MS, M/Z(ESI):237.0[M+H] ⁺

Step 2: Synthesis of 6-chloro-2-ethyl-4-(trifluoromethyl)pyridazin-3(2H)-one

Cuprous iodide (541 mg, 1.26 mmol) and methyl fluorosulfonyl difluoroacetate (2.63 g, 12.6 mmol) were added to a solution of 4-bromo-6-chloro-2-ethylpyridazine-3 (2H) -one (1.00 g, 4.21 mmol) in N-methylpyrrolidone (10 mL), and the resulting mixture was stirred at 90 ° C for 18 hours under nitrogen protection. LCMS detected that the reaction was complete. The reaction solution was cooled to room temperature, diluted with water (50 mL), and then extracted with ethyl acetate (20 mL × 3), and the organic phases were combined and concentrated to give a crude product. The crude product was purified by silica gel column (petroleum ether: ethyl acetate (V/V) = 100: 0 to 90: 0) to give compound 6-chloro-2-ethyl-4-(trifluoromethyl)pyridazine-3 (2H) -one (500 mg, yield 52.4%).

LC-MS, M/Z(ESI):227.0[M+H] ⁺

Step 3: Synthesis of tert-butyl (1S,4S)-4-((1-ethyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate

To a solution of 6-chloro-2-ethyl-4-(trifluoromethyl)pyridazin-3(2H)-one (200 mg, 0.883 mmol) and tert-butyl ((1S,4S)-4-aminocyclohexyl)carbamate (378 mg, 1.77 mmol) in 1,4-dioxane (4 mL) was added methanesulfonic acid (2-dicyclohexylphosphino-2',4',6'-tri-isopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (375 mg, 0.441 mmol) and cesium carbonate (719 mg, 2.21 mmol), and the resulting mixture was stirred at 80° C. for 18 hours. The reaction was complete by LCMS. The reaction solution was cooled to room temperature, diluted with water (20 mL), extracted with ethyl acetate (10 mL×3), the organic phases were combined and concentrated to obtain a crude product, which was purified by silica gel column (petroleum ether:ethyl acetate (V/V) = 100:0 to 80:20) to obtain compound (1S,4S)-4-((1-ethyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (180 mg, yield 50.4%).

LC-MS, M/Z(ESI):405.4[M+H] ⁺

Step 4: Synthesis of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-ethyl-4-(trifluoromethyl)pyridazin-3(2H)-one hydrochloride

To a solution of tert-butyl (1S, 4S)-4-((1-ethyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)carbamate (180 mg, 0.445 mmol) in dichloromethane (2 mL) was added a hydrogen chloride solution (2 mL, 4 M 1,4-dioxane solution), and the resulting mixture was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was directly spin-dried to obtain compound 6-(((1S, 4S)-4-aminocyclohexyl)amino)-2-ethyl-4-(trifluoromethyl)pyridazin-3(2H)-one (120 mg, yield 88.6%), which was directly used in the next step reaction.

LC-MS, M/Z(ESI):305.4[M+H] ⁺

Step 5: Synthesis of 3-chloro-N-((1S,4S)-4-((1-ethyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide

To a solution of 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-ethyl-4-(trifluoromethyl)pyridazin-3(2H)-one (60.0 mg, 0.197 mmol) and 3-chloro-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxylic acid (54.1 mg, 0.237 mmol) in dimethyl sulfoxide (4 mL) were added N,N-diisopropylethylamine (76.5 mg, 0.591 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (56.7 mg, 0.296 mmol) and 1-hydroxybenzotriazole (39.9 mg, 0.296 mmol), and the resulting mixture was stirred at 25° C. for 18 hours. The reaction was complete by LCMS. The reaction solution was purified by preparative liquid chromatography (Xtimate C18 21.2*550mm 5um; 0.1% _FA.H2O -ACN; 20-60; 20mL/min) to obtain compound 3-chloro-N-((1S,4S)-4-((1-ethyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)-1-(2,2,2-trifluoroethyl)-1H-pyrazole-4-carboxamide (45.9 mg, yield 45.2%).

LC-MS, M/Z(ESI):515.4[M+H] ⁺

¹ H NMR (400MHz, CD ₃ OD): δ8.22(s,1H),7.41(s,1H),4.96(q,2H),4.06(q,2H),4.01–3.94(m,1H),3.81–3.73(m,1H),1.90–1.69(m,8H),1.31(t,3H).

Example 20: Preparation of 4-methoxy-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)benzamide (Compound 53A)

The synthetic route of compound 53A is as follows:

At room temperature, the compound p-methoxybenzoic acid (45.00 mg, 0.30 mmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL), and then N,N-diisopropylethylamine (152.90 mg, 1.18 mmol) and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (224.92 mg, 0.59 mmol) were added, and stirred at room temperature for 30 minutes. Then, 6-(((1S,4S)-4-aminocyclohexyl)amino)-2-methyl-4-(trifluoromethyl)pyridazin-3(2H)-one hydrochloride (103.03 mg, 0.35 mmol) was added, and stirring was continued at room temperature for 4 hours. After TLC monitoring showed that the reaction of the raw materials was completed, stirring was stopped, water (10 mL) was added to the reaction solution to dilute it, and it was extracted with ethyl acetate (4 mL×3). The organic phase was collected, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 50:50) to obtain compound 4-methoxy-N-((1S,4S)-4-((1-methyl-6-oxo-5-(trifluoromethyl)-1,6-dihydropyridazin-3-yl)amino)cyclohexyl)benzamide (46.6 mg, yield was 37.10%).

LC-MS, M/Z(ESI):425.1[M+H] ⁺

¹ H NMR (400MHz, CDCl ₃ )δ7.76–7.68(m,2H),7.08(s,1H),6.97–6.89(m,2H),5.99(d,1H),4.17 –4.08(m,2H),3.85(s,3H),3.79(s,1H),3.67(s,3H),1.97–1.62(m,8H).

Example 21: Preparation of N-((1R,3S)-3-((7-chloro-4-oxo-3-(2,2,2-trifluoroethyl)-3,4-dihydrophthalazin-1-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide (Compound 24A)

The synthetic route of compound 24A is as follows:

Step 1: Synthesis of compound 4,6-dichlorophthalazin-1-ol

Under room temperature, the compound 1,4,6-trichlorophthalazine (5.00 g, 21.42 mmol) was dissolved in acetic acid (50 mL), and the reaction solution was stirred at 120°C for 5 hours. After TLC monitoring showed that the raw material was completely reacted, stirring was stopped, cooled to room temperature, and then water (300 mL) was added to dilute the reaction solution, extracted with ethyl acetate (50 mL×3), the organic phase was collected and dried with anhydrous sodium sulfate, and the organic phase was concentrated by vacuum distillation. The residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 100:20) to obtain compound 4,6-dichlorophthalazine-1-ol (4.32 g, yield 93.81%).

LC-MS, M/Z(ESI):216.2[M+H] ⁺

Step 2: Synthesis of compound 4,6-dichloro-2-(2,2,2-trifluoroethyl)phthalazin-1(2H)-one

At room temperature, compound 4,6-dichlorophthalazin-1-ol (3.00 g, 13.95 mmol) and potassium carbonate (1.93 g, 27.90 mmol) were dissolved in anhydrous N,N-dimethylformamide (30 mL), and then 2-iodo-1,1,1-trifluoroethane (4.39 g, 20.93 mmol) was added. After the addition was completed, the reaction solution was stirred in an oil bath at 80°C for 12 hours. After TLC monitoring showed that the raw materials had reacted completely, stirring was stopped, the reaction solution was cooled to room temperature, and then water (200 mL) was added to dilute it, extracted with ethyl acetate (40 mL×3), the organic phase was collected and dried over anhydrous sodium sulfate, and the organic phase was concentrated by distillation under reduced pressure. The residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 100:10), and compound 4,6-dichloro-2-(2,2,2-trifluoroethyl)phthalazin-1(2H)-one (580 mg, yield 14%) was obtained.

LC-MS, M/Z(ESI):298.3[M+H] ⁺

Step 3: Synthesis of tert-butyl ((1R, 3S)-3-((7-chloro-4-oxo-3-(2,2,2-trifluoroethyl)-3,4-dihydrophthalazin-1-yl)amino)cyclohexyl)carbamate

At room temperature, the compound, 6-dichloro-2-(2,2,2-trifluoroethyl)phthalazin-1(2H)-one (400.00 mg, 1.35 mmol) and tert-butyl ((1R,3S)-3-aminocyclohexyl)carbamate (346.27 mg, 1.62 mmol) were dissolved in toluene (5 mL), and sodium tert-butoxide (258.80 mg, 2.70 mmol), 2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl (125.07 mg, 0.27 mmol) and tris(dibenzylidene-BASE acetone)dipalladium (123.30 mg, 0.13 mmol) were added, and the reaction solution was stirred at 80°C for 4 hours under nitrogen protection. After TLC monitoring showed that the raw material reaction was complete, stirring was stopped, the mixture was cooled to room temperature, and then water (20 mL) was added to dilute the reaction solution, extracted with ethyl acetate (8 mL×5), the organic phase was collected and dried over anhydrous sodium sulfate, and the organic phase was concentrated by distillation under reduced pressure. The residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 100:40) to obtain the compound ((1R, 3S)-3-((7-chloro-4-oxo-3-(2,2,2-trifluoroethyl)-3,4-dihydrophthalazin-1-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (100.00 mg, yield 15.64%).

LC-MS, M/Z(ESI):475.9[M+H] ⁺

Step 4: Synthesis of compound 4-(((1S, 3R)-3-aminocyclohexyl)amino)-6-chloro-2-(2,2,2-trifluoroethyl)phthalazin-1(2H)-one hydrochloride

Under room temperature, the compound ((1R, 3S)-3-((7-chloro-4-oxo-3-(2,2,2-trifluoroethyl)-3,4-dihydrophthalazin-1-yl)amino)cyclohexyl)carbamic acid tert-butyl ester (100 mg, 0.21 mmol) was dissolved in dichloromethane (2 mL), stirred at 0°C for 10 minutes, and then a solution of hydrogen chloride in 1,4-dioxane (0.11 mL, 0.11 mmol) was slowly added dropwise, and the reaction solution was naturally warmed to room temperature and stirred for 2 hours. After TLC monitoring showed that the reaction of the raw materials was completed, stirring was stopped, and the reaction solution was evaporated under reduced pressure to remove the solvent to obtain the compound 4-(((1S, 3R)-3-aminocyclohexyl)amino)-6-chloro-2-(2,2,2-trifluoroethyl)phthalazin-1(2H)-one hydrochloride (80 mg, crude product), which can be directly used in the next step reaction.

LC-MS, M/Z(ESI):375.3[M+H] ⁺

Step 5: Synthesis of compound N-((1R,3S)-3-((7-chloro-4-oxo-3-(2,2,2-trifluoroethyl)-3,4-dihydrophthalazin-1-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide

At room temperature, compound 1-methyl-1H-pyrazole-4-carboxylic acid (20 mg, 0.16 mmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and then N,N-diisopropylethylamine (81.98 mg, 0.64 mmol) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (120.60 mg, 0.32 mmol) were added. The mixture was stirred at room temperature for 30 minutes, and then 4-(((1S, 3R)-3-aminocyclohexyl)amino)-6-chloro-2-(2,2,2-trifluoroethyl)phthalazin-1(2H)-one hydrochloride (80 mg, 0.19 mmol) was added, and stirring was continued at room temperature for 4 hours. After TLC monitoring showed that the reaction of the raw materials was completed, stirring was stopped, the reaction solution was cooled to room temperature, and then water (10 mL) was added to dilute it, extracted with ethyl acetate (5 mL×3), the organic phase was collected and dried over anhydrous sodium sulfate, and the organic phase was concentrated by distillation under reduced pressure. The residue was separated and purified by silica gel column chromatography (petroleum ether: ethyl acetate (V/V) = 100:30) to obtain compound N-((1R, 3S)-3-((7-chloro-4-oxo-3-(2,2,2-trifluoroethyl)-3,4-dihydrophthalazin-1-yl)amino)cyclohexyl)-1-methyl-1H-pyrazole-4-carboxamide (23.4 mg, yield 24.81%).

LC-MS, M/Z(ESI):483.1[M+H] ⁺

¹ H NMR(400MHz,DMSO-d6)δ8.41(d,1H),8.26(d,1H),8.10(s,1H),7.90(dd,1H),7.86(d,1H),7.82(s,1H),6.73(d,1H) ,4.87–4.70(m,2H),3.83(s,3H),3.77–3.67(m,2H),2.23(d,1H),2.08(d,1H),1.87–1.76(m,2H),1.42–1.25(m,4H).

The preparation methods of the following compounds are described in the above examples.

Test Example 1: MRGPRX2 in vitro calcium flow assay

The determination of the antagonistic effect of the compounds on MRGPRX2 was carried out in a CHO stable cell line that highly expresses human MRGPRX2. 18 hours before the test, the cells were seeded at a certain density in a black-walled transparent bottom plate containing DMEM/F12 (1:1) medium and kept at 37°C, 5% CO ₂ for 18 hours, and then the corresponding amount of dye solution was added to each well of the cells, and the cells were returned to the 37°C incubator for further incubation in the dark for 30 minutes, and then incubated at room temperature in the dark for 10 minutes, and then different final concentrations of compounds were added to each well, and balanced for 20 minutes. Finally, a certain amount of C48/80 solution was added to the cells, and the fluorescence signal value was detected by FLIPR. The antagonistic effect (IC ₅₀ value) of the compound was calculated by the software GraphPad Prism 8.0, with the compound concentration as the X-axis and the fluorescence signal value as the Y-axis. Among them, A means IC ₅₀ value ≤ 100nM, B means 100nM < IC ₅₀ value ≤ 500nM, and C means IC ₅₀ value greater than 500nM.

The results of the MRGPRX2 calcium flux test showed that the compounds of the present invention have a good antagonistic effect on MRGPRX2.

Table 1 Results of MRGPRX2 calcium flux assay

Test Example 2: MRGPRX2 in vitro IP-1 assay

The determination of the antagonistic effect of the compound on MRGPRX2 was carried out in a CHO stable cell line that highly expressed human MRGPRX2. The drug diluted in DMSO gradient was added to the microplate, the stable cells were digested with trypsin, the cells were collected, inoculated into the microplate after counting, and placed at 37°C for incubation for 10 minutes. A certain concentration of Cortistatin-14 solution was added, centrifuged and placed at 37°C for incubation for 60 minutes. After the incubation was completed, the diluted d2-IP1 and Anti-IP1-Cryptate were added to each test well, centrifuged and left to stand at room temperature for 1 hour. After the incubation was completed, the readings at 665nm and 620nm were detected. The antagonistic effect (IC ₅₀ value) of the compound was calculated by the software Graphpad with the compound concentration as the X-axis and the fluorescence signal ratio as the Y-axis.

Table 2: MRGPRX2 IP-1 test results

The IP-1 test results of MRGPRX2 showed that the compounds of the present invention have a good antagonistic effect on MRGPRX2.

Test Example 3: Test of the toxicity of compounds to liver cells

The toxicity test of the compound on hepatocytes was conducted on HepG2 (ATCC, HB-8065) cells, and the cell viability was measured using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, G7573) to characterize the toxicity of the compound by inhibiting the viability of HepG2 cells. HepG2 cells in the logarithmic phase were collected, the concentration of the cell suspension was adjusted, and 5000 cells/well were plated in a 96-well cell culture plate. The cells were placed in a cell culture incubator at 5% CO ₂ and 37°C and incubated overnight. The next day, the medium was changed and different concentrations of compound solutions were added. At the same time, a negative control group (cells + DMSO) and a blank control group (medium + DMSO) were set up, and the cells were incubated in a cell culture incubator at 5% CO ₂ and 37°C for 72 hours. After the treatment, the kit instructions were followed and the luminescence signal values in different wells were detected on the EnVision plate reader (2104). The inhibition of HepG2 cell viability by compounds at different concentrations was calculated according to the following formula, with the compound concentration as the X-axis and the inhibition rate as the Y-axis. The toxic effect (IC ₅₀ value) of the compound on HepG2 was calculated using the software GraphPad Prism 8.0.

Table 3: HepG2 toxicity results

The results of the hepatocellular toxicity test showed that the compound of the present invention exhibited good safety and low hepatocellular toxicity.

Test Example 4: Pharmacokinetics test in mice

Mouse pharmacokinetic test, using male ICR mice, 20-25g, fasted overnight. Three mice were taken and 10mg/kg was orally administered by gavage. Blood was collected before administration and at 15, 30 minutes, and 1, 2, 4, 8, and 24 hours after administration. The blood sample was centrifuged at 6800g, 2-8℃ for 6 minutes, and the plasma was collected and stored at -80℃. Take the plasma at each time point, add 3-5 times the amount of acetonitrile solution containing the internal standard, mix, vortex mix for 1 minute, centrifuge at 13000 rpm and 4℃ for 10 minutes, take the supernatant, add 3 times the amount of water and mix, and take an appropriate amount of the mixed solution for LC-MS/MS analysis. The main pharmacokinetic parameters were analyzed by non-compartmental model using WinNonlin 7.0 software.

Table 4 Results of pharmacokinetic tests in mice

The results of the mouse pharmacokinetic test showed that the compound of the present invention exhibited excellent pharmacokinetic properties and good drugability.

Test Example 5: Pharmacokinetics test in rats

For the pharmacokinetic test in rats, male SD rats, 180-240g, fasted overnight were used. Three rats were taken and 10 mg/kg was orally administered by gavage. Blood was collected before administration and at 15, 30 minutes, and 1, 2, 4, 8, and 24 hours after administration. The blood sample was centrifuged at 6800g for 6 minutes at 2-8℃, and the plasma was collected and stored at -80℃. The plasma was taken at each time point, mixed with 3-5 times the amount of acetonitrile solution containing the internal standard, vortexed for 1 minute, centrifuged at 13000 rpm and 4℃ for 10 minutes, the supernatant was taken and mixed with 3 times the amount of water, and an appropriate amount of the mixed solution was taken for LC-MS/MS analysis. The main pharmacokinetic parameters were analyzed by non-compartmental model using WinNonlin 7.0 software.

Table 5 Pharmacokinetic test results in rats

The results of the rat pharmacokinetic test show that the compound of the present invention exhibits excellent rat pharmacokinetic properties and good drugability.

Test Example 6: Determination of the inhibitory effect of compounds on BSEP bile efflux transporter

The inhibitory effect of compounds on BSEP (Bile salt export pump) bile efflux transporter was tested using vesicles expressing human BSEP bile efflux transporter (GenoMembrane). Different concentrations of compounds were pre-incubated with vesicles for 5 minutes, and negative control (NC) and positive control (PC) groups were set up: NC group was pre-incubated with blank buffer at 37℃ for 5 minutes, and PC group was pre-incubated with positive inhibitor and vesicles at 37℃ for 5 minutes. Subsequently, ATP or AMP was added, and the probe substrate was incubated at 37℃ for 5 minutes. The test was terminated with pre-cooled Buffer B1 (10×Buffer B1 (Stopping and Washing Buffer): 100mM Hepes-Tris, 1000mM KNO3, 500mM Sucrose). The test sample was transferred to a 96-well filter plate, filtered with a vacuum pump, and then washed repeatedly with 0.2 mL of pre-cooled Buffer B1 for 5 times. The vesicles on the filter plate were dissolved with 50 μL of 80% methanol, and the filtrate was collected by centrifugation at 2000 rpm for 2 minutes. Repeat once, combine the two filtrates together, mix well, and obtain about 100 μL of filtrate. Pre-cooled methanol containing internal standard was added, and centrifuged at 12,000 rpm for 5 minutes. The supernatant was taken for LC-MS/MS quantitative detection of the content of the transported substrate. With the compound concentration as the X-axis and the relative activity (% of NC) as the Y-axis, the IC ₅₀ value and inhibition rate of the compound's inhibition of bile efflux transporter activity were calculated by Prism software.

The transport rate (activity) and relative activity under different conditions were calculated according to the following formula:

Table 6 Inhibitory effects of test compounds on BSEP bile efflux transporter

The results of the BSEP bile efflux transporter inhibition test showed that the compounds of the present invention had no obvious inhibitory effect on the BSEP bile efflux transporter and had no risk of cholestatic toxicity.

The structure of reference compound 1 is: Test Example 7: Thermodynamic Solubility Test

Prepare phosphate buffered saline (PBS) at pH 7.4. Accurately weigh the compound and add the prepared phosphate buffer at pH 7.4 to a solution with a concentration of 4 mg/mL. Shake at 1000 rpm for 1 hour and then incubate at room temperature overnight. Centrifuge the incubated solution at 12000 rpm for 10 minutes to remove undissolved particles and transfer the supernatant to a new centrifuge tube. After appropriately diluting the supernatant, add acetonitrile solution containing the internal standard and quantify using a standard curve prepared with the same matrix.

The results of the thermodynamic solubility test show that the compound of the present invention has good thermodynamic solubility and good drugability.

Test Example 8: Human liver microsome stability test

The stability test of human liver microsomes was performed by incubating the compound with human liver microsomes in vitro. First, the compound to be tested was prepared into a 10mM stock solution in DMSO solvent, and then the compound was diluted to 0.5mM with acetonitrile. Human liver microsomes (Corning) were diluted with PBS to form a microsome/buffer solution, and the solution was used to dilute 0.5mM of the compound to form a working solution. The compound concentration in the working solution was 1.5μM and the human liver microsome concentration was 0.75mg/mL. Take a deep well plate, add 30μL of working solution to each well, and then add 15μL of preheated 6mM NADPH solution to start the reaction and incubate at 37°C. At 0, 5, 15, 30, and 45 minutes of incubation, add 135μL of acetonitrile to the corresponding wells to terminate the reaction. After terminating the reaction with acetonitrile at the last 45 minutes, the deep well plate was vortexed for 10 minutes (600rpm/min) and then centrifuged for 15 minutes. After centrifugation, the supernatant was collected and purified water was added in a 1:1 ratio for LC-MS/MS detection. The ratio of the peak area of the compound to the peak area of the internal standard at each time point was obtained. The peak area ratios of the compound at 5, 15, 30, and 45 minutes were compared with the peak area ratio at 0 minute, and the remaining percentage of the compound at each time point was calculated. T1/2 was calculated using Graphpad 5 software.

Table 8 Human liver microsome stability data of compounds

The results of the human liver microsome stability test show that the compounds of the present invention exhibit excellent human liver microsome stability and good drugability.

Test Example 9: Inhibition test of compounds on cytochrome P450

The inhibitory potential of the compounds on the cytochrome P450 (CYP450) subtype CYP3A4 (two substrates midazolam and testosterone) was tested. The test compound was first prepared into a 10mM stock solution in DMSO solvent, and the CYP3A4 inhibitor ketoconazole was prepared into 10mM, 2.5mM, and 2.5mM stock solutions in DMSO solvent. The test compound and ketoconazole were diluted to 400 times the final concentration (compound: 10μM, ketoconazole: 2.5μM) with acetonitrile.

Potassium phosphate buffer (0.1 M, pH 7.4) was used to prepare 4 times the final concentration of NADPH cofactor (66.7 mg NADPH was added to 10 mL potassium phosphate buffer) and substrate. The final concentration of CYP3A4 substrate midazolam was 320 μM, and the final concentration of CYP3A4 substrate testosterone was 20 μM.

Prepare human liver microsome solution with potassium phosphate buffer on ice at a concentration of 0.2 mg/mL. Prepare test compound and control inhibitor solution at 2 times the final concentration with human liver microsome solution on ice. Add 30 μL of test compound and control inhibitor solution to the test wells, respectively, and add 15 μL of substrate for duplicate well operation. Incubate the 96-well assay plate and NADPH solution at 37°C for 5 minutes, and add 15 μL of preheated 8 mM NADPH solution to the assay plate to start the reaction. The CYP3A4 assay plate was preincubated at 37°C for 5 minutes. Add 120 μL of acetonitrile to terminate the reaction. After quenching, shake the plate on a shaker (IKA, MTS2/4) for 10 minutes (600 rpm/min), and then centrifuge for 15 minutes. After centrifugation, the supernatant was taken, purified water was added in a 1:1 ratio, and then LC-MS/MS detection was performed to obtain the ratio of the peak area of the compound to the peak area of the internal standard. The peak area ratio of the compound was compared with the peak area ratio of the control inhibitor to calculate the inhibition rate.

The results of the inhibition test of the compounds on cytochrome P450 show that the compounds of the present invention have no obvious inhibitory effect on CYP3A4 (two substrates, midazolam and testosterone) and have good drugability.

Test Example 10: Evans blue vascular permeability test

The Evans blue vascular permeability test used C57 mice, with the left hind toe of the mouse as the model group and the right hind toe as the negative control group. Different doses of drugs were given by gavage, and 0.4% Evans blue (Sigma-Aldrich, Lot#SHBP1253) solution was injected into the tail vein 2h after administration. 5-10min later, C48/80 (Sigma-Aldrich, Lot#C2313) solution was injected subcutaneously into the left hind toe, and saline was injected into the right hind toe. The mice were killed 15min later, and the thickness of the left and right hind toes of the mice was measured with a vernier caliper. The mouse feet were cut off, dried, and weighed. The toes were crushed, and the Evans blue in the toes was dissolved with acetone: saline (7:3), and the absorbance was measured at 620nm, and the absorbance per unit volume was calculated. The swelling rate of the mouse ears and the amount of Evans blue permeation per unit mass were calculated.

The results of the Evans blue vascular permeability test showed that the compound of the present invention exhibited good efficacy, could significantly inhibit C48/80-induced vascular leakage, and had good drugability.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (10)

The compound represented by formula IA, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug:
in, X ¹ , X ² , X ³ , X ⁴ , X ⁵ and X ⁶ each independently represent a ring atom; X ¹ , X ² , X ³ , X ⁵ and X ⁶ are each independently N, CH ₂ , CH or C; X ⁴ is C; The bond between ^X1 and ^X2 , and between ^X5 and ^X6 is a single bond or a double bond; The group fragment formed by connecting ^X5 and ^X6 wherein A is absent, ^X5 and ^X6 are each independently substituted by ^R4 or ^R5 ; or when A is present, A, together with ^X5 and ^X6 ring atoms, forms a 6-10 membered aryl group, a 3-11 membered heterocycloalkyl group or a 5-10 membered heteroaryl group; and said A is further substituted by ^Rc ; said ^Rc is substituted by one or more substituents, and when there are multiple substituents ^Rc , said substituents are the same or different; the heteroatoms in said heterocycloalkyl group and heteroaryl group are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3; When A, X ⁵ and X ⁶ together form a 6-membered aryl group, ^Ra is oxo; Alternatively, when A, ^X5 and ^X6 ring atoms together form a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatom in the heteroaryl group is selected from one or more of N, O and S, and the number of the heteroatoms is 1, 2 or 3; Ring B is C _3-12 cycloalkyl; Ring C is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3; R ^a , R ^c , R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently H, halogen, cyano, amino, oxo, C _1-6 alkyl, C 1-6 alkyl substituted by hydroxy, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 cycloalkyl, halogenated C _3-8 cycloalkyl, -OR _f , -C(O) ^{OR f} , -OC(O)R ^f , -N(R ^f ) ₂ , -N(R ^f )C(O)R ^f , -N(R ^f )S(O) ₂ R ^f or -S(O) ₂ R ^f ; R ^f is selected from H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkylamino, -(CH ₂ ) r R ^g , 6-10 membered aryl, C _3-8 cycloalkyl, 5-10 membered heteroaryl or 5-10 membered heterocycloalkyl, or two R ^f groups together with the atoms to which they are connected form a 5-11 membered heterocycloalkyl; the R ^g is H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _3-8 cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl or 5-10 membered heterocycloalkyl; the heteroatoms in the heterocycloalkyl and heteroaryl groups are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C 3-8 halocycloalkyl, C _1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-10 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR ^j )-R ^k , -(CHR j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O ⁾ -N(R ^k ) ₂ , -(CHR ^j ) _0-3 -(N═)S(═O)(R ^k ) ₂ , -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; The C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl are optionally substituted by one or more R ^d ; the R ^d is selected from the following substituents: halogen, hydroxyl, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _{1-6 haloalkyl, C 1-6} alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl; when there are _multiple substituents R ^d , the R ^d are the same or different; the heteroatoms in the heterocycloalkyl are selected from one or more of N, O, S, and the number of heteroatoms is 1, 2 or 3; R ^j and R ^k are each independently H, cyano, amino, C _1-6 alkyl, C _1-6 alkylamino, -(CH ₂ ) _0-3 C _3-8 cycloalkyl, -COC _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkyl substituted with hydroxy, or a single 5- to 10-membered heterocycloalkyl; m, n, and r are 0, 1, 2, or 3 respectively. The compound of formula IA as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that: The R ^b is H, F, Cl, -CN, methyl, amino, methoxy, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH ₂ -SO ₂ -CH ₃ , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ , and/or, said ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, F, Cl, methyl, ethyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,} And/or, the group fragment formed by the connection of ^X5 and ^X6 In the formula (a), A is absent, ^X5 and ^X6 are each independently substituted by ^R4 or ^R5 , then the structural fragment is Selected from and/or, said R ⁴ and R ⁵ are each independently H, F, Cl, methyl, -CF ₃ , -CHF ₂ ; And/or, the group fragment formed by the connection of ^X5 and ^X6 In the formula (a), A is absent, ^X5 and ^X6 are each independently substituted by ^R4 or ^R5 , then the structural fragment is Selected from And/or, the group fragment formed by the connection of ^X5 and ^X6 When A exists, A, together with X ⁵ and X ⁶ ring atoms, forms and said A is further substituted by R ^c ; and/or, when A is present, the structural fragment Selected from and/or, when A is present, the structural fragment Selected from and/or, the ring B is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; and/or, the structural fragment Selected from and/or, the structural fragment Selected from The compound of formula IA as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that: The formula IA can be selected from the following IA-1:
wherein A, ring B, ring C, X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , R ¹ , R ² , R ³ , R ^b , m and n are as defined in claim 1; Or the formula IA is selected from the structures shown in the following formulas I-1, I-2 or I-3,
in, The group fragment formed by connecting ^X5 and ^X6 A is defined as in claim 1; Ring C is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3; X ¹ , X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , R ¹ , R ² , R ³ , ^Ra , R ^b , m and n are as defined in claim 1 ; Or the formula IA is selected from the structure shown in the following formula II-1 or II-3,
wherein A, together with ^X5 and ^X6 ring atoms, forms a 6- to 8-membered aryl group, a 3- to 8-membered heterocycloalkyl group, a 5-membered heteroaryl group or a 6-membered heteroaryl group; the heteroatoms are independently selected from one or more of N, O and S; A is further substituted by ^Rc ; the ^Rc is substituted with one or more substituents, and when there are multiple substituents ^Rc , the substituents are the same or different; and when A, together with ^X5 and ^X6 ring atoms, forms a 6-membered aryl group, ^Ra is oxo; or, when A, together with ^X5 and ^X6 ring atoms, forms a 5-membered heteroaryl group and ^Rc is not H, ^R1 is a _C1-6 haloalkyl group; the heteroatoms in the heterocycloalkyl group and the heteroaryl group are selected from one or more of N, O and S, and the number of heteroatoms is 1, 2 or 3; Ring C is a 6- to 10-membered aryl group or a 5- to 8-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C 3-8 halocycloalkyl, C _1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-8 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR ^j )-R ^k , -(CHR j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O ⁾ -N(R ^k ) ₂ , -(CHR ^j ) _0-3 -(N═)S(═O)(R ^k ) ₂ , -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; The C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl are optionally substituted by one or more R ^d ; the R ^d is selected from the following substituents: halogen, hydroxyl, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl; the heteroatoms in the heterocycloalkyl are selected from one or more of N, O, S, and the number of heteroatoms is 1, 2 or 3; R ^j and R ^k are each independently H, cyano, amino, C _1-6 alkyl, C _1-6 alkylamino, -(CH ₂ ) _0-3 C _3-8 cycloalkyl, -COC _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkyl substituted with hydroxy, or a single 5- to 10-membered heterocycloalkyl; ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, halogen, cyano, amino, oxo, _C1-6 alkyl, C1-6 alkyl substituted with hydroxy, _C2-6 alkenyl, _C2-6 alkynyl, _C1-6 haloalkyl, _{C3-8 cycloalkyl} , or halogenated _C3-8 cycloalkyl; X ¹ , X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , m and n are as defined in claim 1 ; and/or, R ^b is H, F, Cl, -CN, methyl, amino, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH ₂ -SO ₂ -CH ₃ , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ , and/or, said ^Ra , ^Rc , ^R1 , ^R2 , and ^R3 are each independently H, F, Cl, methyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,} The compound of formula IA as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that it has a structure shown in formula II-2, II-4 or II-5,
in, Ring C is a 6- to 10-membered aryl group or a 5- to 8-membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2, or 3; R ^b is H, halogen, hydroxyl, amino, cyano, carbonyl, oxo, C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C 3-8 halocycloalkyl, C _1-6 alkylhydroxy, C _1-6 alkylcarbonyl, C _1-6 alkoxy, C _1-6 haloalkoxy or _3-8 membered heterocycloalkyl, -(CHR ^j ) _0-3 -S(═O)(═NR ^j )-R ^k , -(CHR j ) _1-3 -S(O) ₂ -R ^k , -(CHR ^j ) _1-3 -OC(O ⁾ -N(R ^k ) ₂ , -(CHR ^j ) _0-3 -(N═)S(═O)(R ^k ) ₂ , -(CHR ^j ) _0-3 -P(═O)(R ^k ) ₂ ; The C _1-6 alkyl, C _3-8 cycloalkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _3-8 halocycloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl are optionally substituted by one or more R ^d ; the R ^d is selected from the following substituents: halogen, hydroxyl, amino, nitro, cyano, carbonyl, oxo, carboxyl, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 haloalkyl, C _1-6 alkylhydroxyl, C _1-6 alkylcarbonyl, C _1-6 alkoxyl, C _1-6 haloalkoxyl; the heteroatoms in the heterocycloalkyl are selected from one or more of N, O, S, and the number of heteroatoms is 1, 2 or 3; R ^j and R ^k are each independently H, cyano, amino, C _1-6 alkyl, C _1-6 alkylamino, -(CH ₂ ) _0-3 C _3-8 cycloalkyl, -COC _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkyl substituted with hydroxy, or a single 5- to 10-membered heterocycloalkyl; ^Ra , ^R1 , ^R2 , ^R3 , ^R4 , and ^R5 are each independently H, halogen, cyano, amino, oxo, _C1-6 alkyl, _C2-6 alkenyl, _C2-6 alkynyl, _C1-6 haloalkyl, _C3-8 cycloalkyl, or halogenated _C3-8 cycloalkyl; X ¹ , X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , m and n are as defined in claim 1 ; and/or, R ^b is H, F, Cl, -CN, methyl, methoxy, amino, oxo, -CH ₂ CF ₃ , -CF ₃ , -CHF ₂ , -CH ₂ CHF ₂ , -CH ₂ -SO ₂ -CH ₃ , -CH(CH ₃ )SO ₂ CH ₃ , -CH ₂ S(=O)(=NCH ₃ )-CH ₃ , and/or, said ^Ra , ^R1 , ^R2 , ^R3 , ^R4 , ^R5 are each independently H, F, Cl, methyl, ethyl, amino, oxo, _-CHF2 , _-CH2CF3 , _{-CF3 ,} The compound of formula IA, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug as described in any one of claims 1 to 4, characterized in that ring C is a 6-8 membered aryl group or a 5-10 membered heteroaryl group; the heteroatom in the heteroaryl group is selected from one or more of N, O, and S, and the number of heteroatoms is 1, 2 or 3; and/or, Ring C is a 5- to 6-membered nitrogen-containing heteroaryl group, and the number of the nitrogen atoms is 1, 2 or 3; and/or, Ring C is phenyl, pyrazolyl or pyridyl. The compound of formula IA as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that the compound comprises:



Optionally, the pharmaceutically acceptable salt of the compound is trifluoroacetate. The compound of formula IA as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that the compound comprises:



Optionally, the pharmaceutically acceptable salt of the compound is trifluoroacetate. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: a compound represented by formula IA as described in any one of claims 1 to 7, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug; and a pharmaceutically acceptable carrier. A use of a compound of formula IA as claimed in any one of claims 1 to 7, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, or a use of a pharmaceutical composition as claimed in claim 8, comprising: Antagonizes MRGPRX2; and/or, preventing and/or treating MRGPRX2-related diseases; And/or, preparing drugs, pharmaceutical compositions or preparations for antagonizing MRGPRX2, and/or preventing and/or treating MRGPRX2-related diseases. The use according to claim 9, characterized in that the MRGPRX2-related diseases include: mast cell-related diseases; Or the MRGPRX2-related diseases include: skin diseases (such as atopic dermatitis, contact dermatitis, urticaria, chronic spontaneous urticaria, induced urticaria, pruritus), autoimmune diseases (such as allergies, mastocytosis, rheumatoid arthritis, asthma, ulcerative colitis and interstitial cystitis), and nervous system diseases (such as pain).