WO2024120512A1 - Ep2 and ep4 receptor antagonist - Google Patents

Abstract

The present invention provides an EP2 and EP4 receptor antagonist. The antagonist is a compound represented by formula I, and may be used for treating EP2 and EP4 receptor-mediated diseases.

Description

An EP2, EP4 receptor antagonist

The present invention claims priority to a prior application with patent number 2022115749794, entitled “An EP2, EP4 receptor antagonist”, filed with the State Intellectual Property Office of China on December 8, 2022. The entire text of the prior application is incorporated herein by reference.

Technical Field

The present invention belongs to the field of pharmaceutical chemistry, and in particular, the present invention relates to an EP2, EP4 receptor antagonist and its use.

Background technique

Prostaglandin E2 (PGE2) is an endogenous bioactive lipid. PGE2 causes a wide range of upstream and downstream dependent biological responses by activating prostaglandin receptors, and participates in the regulation of many physiological and pathological processes including inflammation, pain, renal function, cardiovascular system, lung function, and cancer. It is reported that PGE2 is highly expressed in cancerous tissues of various cancers, and it has been confirmed that PGE2 is associated with the occurrence, growth and development of cancer and disease conditions in patients. It is generally believed that PGE2 is associated with the activation of cell proliferation and cell death (apoptosis), and plays an important role in the process of cancer cell proliferation, disease progression and cancer metastasis.

There are four subtypes of PGE2 receptors, EP1, EP2, EP3 and EP4, which are widely expressed in various tissues. EP1 receptor activates phospholipase C and inositol triphosphate pathway, EP2 and EP4 receptors activate adenylate cyclase and cAMP-protein kinase A, and activation of EP3 receptor can both inhibit adenylate cyclase and activate phospholipase C.

Among them, EP2 and EP4 are expressed in a variety of immune cells (such as macrophages, dendritic cells, NK cells and cytotoxic T lymphocytes (CTL)). Inhibition of EP2 and EP4 can enhance immune activity and inhibit tumor growth.

PGE2 continuously activates EP receptors in the tumor microenvironment (produced in large quantities by tumor cells), which promotes the accumulation and enhances the activity of various immunosuppressive cells, including type 2 tumor-associated macrophages (TAMS), Treg cells, and myeloid-derived suppressor cells (MDSCs). One of the main characteristics of the immunosuppressive tumor microenvironment is the presence of a large number of MDSCs and TAMs, which in turn are closely associated with the poor overall survival of patients with gastric cancer, ovarian cancer, breast cancer, bladder cancer, hepatocellular carcinoma (HCC), head and neck cancer, and other types of cancer. In addition, PGE2 has been reported to induce immune tolerance by inhibiting the accumulation of antigen-presenting dendritic cells (DC) in tumors and inhibiting the activation of tumor-infiltrating DCs, thereby helping tumor cells escape immune surveillance. PGE2 plays a very important role in promoting the occurrence and development of tumors. In various malignant tumors including colon cancer, lung cancer, breast cancer, and head and neck cancer, the expression levels of PGE2 and its related receptors EP2 and EP4 have been found to be elevated, and are often closely associated with poor prognosis. Therefore, selectively blocking the EP2 and EP4 signaling pathways can inhibit the occurrence and development of tumors by changing the tumor microenvironment and regulating tumor immune cells.

Selective and dual EP2 and/or EP4 antagonists may be used to treat other diseases and conditions. EP4 antagonists have been shown to be effective in relieving joint inflammation and pain in rodent models of rheumatoid arthritis and osteoarthritis, and have also been shown to be effective in rodent models of autoimmune disease.

PGE2 is the main prostaglandin that mediates proinflammatory function through the EP2 receptor, so EP2 antagonists may show utility as therapeutic agents for certain chronic inflammatory diseases, especially inflammatory neurodegenerative diseases such as epilepsy, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and traumatic brain injury (TBI). In an Alzheimer's disease mouse model, the EP4 antagonist ONO-AE3-208 reduced amyloid-β and improved behavioral performance.

Studies have confirmed that the expression of the pain-causing substance PGE2 in the peritoneal fluid of patients with endometriosis (endometriosis) is significantly increased, and the increase in PGE2 can further stimulate the expression of the rate-limiting enzyme (aromatase) for estrogen synthesis, increase the synthesis of estrogen, and thus promote the occurrence and development of endometriosis. Inhibition of EP2 and EP4 can inhibit the expression of progesterone (P4) signal transduction mechanism proteins in the epithelial and stromal cell-specific patterns in endometriosis lesions, inhibit the retention, invasion, biosynthesis and signal transduction of PGE2 and estrogen (E2), thereby inhibiting the production of proinflammatory cytokines, reducing the growth, survival and spread of peritoneal endometriosis lesions, alleviating pelvic pain, and restoring the receptivity of the endometrium.

Therefore, it is of great significance to develop novel compounds that can be used to treat diseases mediated by EP2 and/or EP4 receptors. Such compounds have the potential to be used to treat inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular diseases and cancer.

Summary of the invention

The object of the present invention is to provide an EP2, EP4 receptor antagonist, wherein the antagonist is a compound represented by formula I of the present invention.

In a first aspect, the present invention provides a compound of formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug:

Wherein, R ₁ is halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl;

L ₁ and L ₂ are each independently absent or C ₁ -C ₃ alkylene; the C ₁ -C ₃ alkylene is optionally substituted by C ₁ -C ₃ alkyl;

Ring A is thiophene, and ring B is cyclopropyl;

Alternatively, ring A is pyrazine, and ring B is a benzene ring;

The ring A and the ring B are optionally substituted by 1, 2 or 3 identical or different Ra, wherein Ra is selected from the group consisting of halogen, cyano, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, and -OC ₁ -C ₃ alkyl.

In a preferred embodiment, ring A is thiophene, ring B is cyclopropyl; R ₁ is halogen; L ₁ is absent or is methylene; and L ₂ is -CH(CH ₃ )-.

In a preferred embodiment, the halogen is F or Cl.

In a preferred embodiment, ring A is thiophene, ring B is cyclopropyl; R ₁ is halogen; L ₁ is absent; L ₂ is -CH(CH ₃ )-;

The halogen is Cl.

In a preferred embodiment, ring A is pyrazine, and ring B is a benzene ring, wherein the benzene ring is optionally substituted by 1, 2 or 3 substituents selected from the group consisting of halogen, cyano, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, -OC ₁ -C ₃ alkyl;

R ₁ is C ₁ -C ₃ alkyl or halogen; L ₁ is -CH ₂ -; L ₂ is -CH ₂ -.

In a preferred embodiment, the ring B is optionally substituted by 1 or 2 identical or different Ra, wherein Ra is selected from: halogen, cyano, methyl, methoxy;

In a preferred embodiment, the halogen is F.

In a preferred embodiment, R ₁ is Cl or methyl.

In a preferred embodiment, L ₁ and L ₂ are each independently absent or -CH ₂ - or -CH(CH ₃ )-.

In a preferred embodiment, having the structure shown in Formula Ia

wherein m is 1 or 2; Ra is F, cyano, methyl or methoxy;

_L1 is absent or is _-CH2- .

In a preferred embodiment, having the structure shown in Formula Ib

Wherein, R ₁ is halogen; L ₁ is absent or -CH ₂ -; L ₂ is -CH(CH ₃ )-.

In a preferred embodiment, the structure shown in Formula Ic or Formula Id is

In a preferred embodiment, R ₁ is Cl.

In a preferred embodiment, L ₁ is absent or is -CH ₂ -.

In a preferred embodiment, _L2 is

In a preferred embodiment, the compound represented by formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug is selected from

Preferably, With structure

Preferably, With structure

Preferably, With structure

In a second aspect, the present invention provides a pharmaceutical composition, comprising: a compound of formula I as described in the first aspect of the present invention, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs; and a pharmaceutically acceptable carrier.

According to an embodiment of the present invention, the pharmaceutical composition further comprises a second drug.

In a preferred embodiment, the second drug comprises an antibody;

Preferably, the antibodies include anti-PD-L1 antibodies and anti-PD-1 antibodies.

In a third aspect, the present invention provides a use of the compound of formula I as described in the first aspect of the present invention, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, or the use of the pharmaceutical composition as described in the second aspect, the use comprising:

1) Antagonize EP2 and/or EP4;

2) Binding to EP2 and/or EP4 receptors;

3) Prevention and treatment of diseases mediated by EP2 and/or EP4 receptors;

4) preparing EP2 and/or EP4 antagonists;

5) Preparation of drugs, pharmaceutical compositions or preparations for preventing and treating diseases mediated by EP2 and/or EP4 receptors.

Preferably, the diseases mediated by the EP2 and/or EP4 receptors include inflammatory diseases (e.g., arthritis and endometriosis), autoimmune diseases (e.g., multiple sclerosis), neurodegenerative diseases (e.g., epilepsy, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and traumatic brain injury), cardiovascular diseases (e.g., atherosclerosis) and cancers (e.g., colon cancer, lung cancer, breast cancer and head and neck cancer).

In a fourth aspect, the present invention provides a method for preventing and/or treating diseases mediated by EP2 and/or EP4 receptors, the method comprising administering to an individual in need thereof an effective amount of the compound of formula I, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs.

In a preferred embodiment, the method further comprises use in combination with antibody therapy; preferably, the antibody therapy comprises PD-L1 antibody therapy and PD-1 antibody therapy.

In a preferred embodiment, the mass ratio of the compound of formula I as described in the first aspect, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug to the antibody is 1:100 to 100:1, preferably 10:1 to 100:1, more preferably 10:1 to 50:1, for example, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1.

In a preferred embodiment, the diseases mediated by the EP2 and/or EP4 receptors include inflammatory diseases (e.g., arthritis and endometriosis), autoimmune diseases (e.g., multiple sclerosis), neurodegenerative diseases (e.g., epilepsy, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and traumatic brain injury), cardiovascular diseases (e.g., atherosclerosis) and cancers (e.g., colon cancer, lung cancer, breast cancer and head and neck cancer).

Additional aspects and advantages of the present invention will be given in part in the following description and will become apparent in part from the following description. Or understand through the practice of the present invention.

Terms and Definitions

Unless otherwise specified, the definitions of groups and terms recorded in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in examples, etc., can be arbitrarily combined and combined with each other. The group definitions and compound structures after such combinations and combinations shall fall within the scope of the description of this application.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the subject matter of the claims pertains. Unless otherwise indicated, all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. If there are multiple definitions of a term herein, the definition in this chapter shall prevail.

It should be understood that the above brief description and the detailed description below are exemplary and are only used for explanation, and do not impose any restrictions on the subject matter of the present invention. In this application, unless otherwise specifically stated, the use of the singular also includes the plural. It must be noted that unless otherwise clearly stated in the text, the singular form used in this specification and claims includes the plural form of the referred thing. It should also be noted that unless otherwise stated, the "or" and "or" used mean "and/or". In addition, the term "including" and other forms, such as "including", "containing" and "containing" are not restrictive.

Unless otherwise indicated, conventional methods within the technical scope of the art are adopted, such as mass spectrometry, NMR, IR and UV/VIS spectroscopy and pharmacological methods. Unless specifically defined, the terms used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and drugs and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, preparation and delivery, and in the treatment of patients. For example, the instructions for use of the kit can be utilized by the manufacturer, or the reaction and purification can be implemented according to a manner well known in the art or the description of the present invention. The above-mentioned techniques and methods can usually be implemented according to the description in a plurality of summaries and more specific documents cited and discussed in this specification, according to conventional methods well known in the art. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural parts and compounds.

When substituents are described by conventional chemical formulas written from left to right, the substituents also include chemically equivalent substituents that would result if the formula were written from right to left. For example, CH ₂ O is equivalent to OCH ₂ . As used herein, Indicates the attachment site of a group. As used herein, "R ₁ ", "R1" and "R ¹ " have the same meaning and can be replaced with each other. For other symbols such as R ₂ , similar definitions have the same meaning.

The section headings used herein are only for the purpose of organizing the article and should not be interpreted as limitations on the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, operating manuals and papers, are incorporated herein by reference in their entirety.

In addition to the foregoing, when used in the specification and claims of the present application, the following terms have the meanings indicated below unless otherwise specifically stated.

In this application, the term "halogen" when used alone or as part of other substituents refers to fluorine, chlorine, bromine, iodine. As used herein, the term "alkyl" when used alone or as part of other substituents means a straight or branched hydrocarbon chain group consisting only of carbon atoms and hydrogen atoms, free of unsaturated bonds, having, for example, 1 to 6 carbon atoms and connected to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl As used herein, the term "alkenyl" refers to an unbranched or branched monovalent hydrocarbon chain containing one or more carbon-carbon double bonds. As used herein, the term "alkynyl" refers to an unbranched or branched monovalent hydrocarbon chain containing one or more carbon-carbon triple bonds. "C ₂ -C ₅ alkynyl" contains 2-5 carbon atoms.

The term "C ₁ -C ₃ alkyl" alone or as part of another substituent is understood to mean a straight-chain or branched saturated monovalent hydrocarbon radical having 1, 2 or 3 carbon atoms, such as methyl, ethyl, n-propyl or isopropyl.

The term "C ₁ -C ₃ alkoxy" should be understood to mean a linear or branched saturated monovalent hydrocarbon group having 1, 2, or 3 carbon atoms and an oxygen atom, or represented as C ₁ -C ₃ alkyl-O-. The definition of C ₁ -C ₃ alkyl is as described in the present specification, and the oxygen atom can be connected to any carbon atom of the linear or branched chain of the C ₁ -C ₃ alkyl group. Including but not limited to: methoxy (CH ₃ -O-), ethoxy (C ₂ H ₅ -O-), propoxy (C ₃ H ₇ -O-).

The term "alkylene" refers to a saturated divalent hydrocarbon group obtained by removing two hydrogen atoms from a saturated straight or branched hydrocarbon group. "C ₁ -C ₃ alkylene" refers to an alkylene group containing 1, 2 or 3 carbon atoms, examples of which include methylene (-CH ₂ -), ethylene (including -CH ₂ CH ₂ - or -CH(CH ₃ )-), isopropylene (including -CH(CH ₃ )CH ₂ - or -C(CH ₃ ) ₂ -), and the like.

The term "halo" can be used interchangeably with the term "halogen-substituted" when used alone or as part of other substituents. "Haloalkyl" or "halogen-substituted alkyl" refers to saturated aliphatic hydrocarbon groups including branched and straight chains having a specific number of carbon atoms, substituted with one or more halogens. The halogens include fluorine, chlorine, bromine, iodine, preferably fluorine.

Compounds provided herein include intermediates that can be used to prepare compounds provided herein, which contain reactive functional groups (such as but not limited to carboxyl, hydroxyl and amino moieties), and also include protected derivatives thereof. "Protected derivatives" are those compounds in which one or more reactive sites are blocked by one or more protecting groups (also referred to as protecting groups). Suitable carboxyl moiety protecting groups include benzyl, tert-butyl, etc., and isotopes, etc. Suitable amino and amido protecting groups include acetyl, trifluoroacetyl, tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable hydroxyl protecting groups include benzyl, etc. Other suitable protecting groups are well known to those of ordinary skill in the art.

In the present application, "optional" or "optionally" means that the event or situation described subsequently 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 the aryl is substituted or unsubstituted, and the description includes both substituted aryl and unsubstituted aryl. Further, the "optional" or "optionally" substitution situation covers the situation where the compound structure/group is unsubstituted, and the compound structure/group is substituted by one or more defined substituents. For example, "optionally substituted aryl" means unsubstituted aryl and aryl substituted by one or more defined substituents. "Multiple" means more than two, that is, including two or more than three.

In the present application, the term "salt" or "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

"Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. In addition to pharmaceutically acceptable salts, other salts are contemplated in the present invention. They can serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts or can be used in the present invention. Identification, characterization or purification of a compound.

The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers and conformational isomers.

Depending on the choice of starting materials and methods, the compounds of the 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, the prefixes D and L or R and S are used to indicate 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 the symbols used to specify the rotation of plane polarized light caused by the compound, where (–) or L indicates that the compound is levorotatory. Compounds prefixed with (+) or D are dextrorotatory.

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. The absolute configuration of a stereocenter is indicated by a wedge-shaped bond and a dashed bond.

The term "tautomer" refers to functional group isomers 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 physicochemical 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 predominates; while in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

In this application, "pharmaceutical composition" refers to a preparation of the compound of the present invention and a medium generally accepted in the art for delivering the biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote administration of the organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

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 "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of the term "excipient" include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. Excipients can enhance the handling characteristics of a pharmaceutical formulation, i.e., make the formulation more suitable for direct compression by increasing fluidity and/or adhesion.

The term "patient" refers to any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and most preferably humans.

The term "therapeutically effective amount" refers to the amount of an active compound or drug that elicits the biological or medical response that a researcher, veterinarian, physician or other clinician is seeking in a tissue, system, animal, individual or human, and includes one or more of the following: (1) Preventing disease: e.g., preventing a disease, disorder or condition in an individual who is susceptible to the disease, disorder or condition but does not yet experience or develop the pathology or symptoms of the disease. (2) Inhibiting disease: e.g., inhibiting a disease, disorder or condition (i.e., preventing further development of the pathology and/or symptoms) in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder or condition. (3) Alleviating disease: e.g., alleviating a disease, disorder or condition (i.e., reversing the pathology and/or symptoms) in an individual who is experiencing or developing the pathology or symptoms of the disease, disorder or condition.

As used herein, the term "treatment" and other similar synonyms include the following meanings:

(i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

(ii) inhibiting a disease or condition, i.e. arresting its development;

(iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

(iv) alleviating the symptoms caused by the disease or condition.

The term "antibody" includes all types of immunoglobulins. Antibodies can be monoclonal or polyclonal and can be of any species origin, including, for example, mouse, rat, rabbit, horse or human. Antibodies can be chimeric or humanized, especially when used for therapeutic purposes. Antibodies can be obtained or prepared by methods known in the art.

The term "PD-L1 antibody" or "anti-PD-L1" refers to an antibody directed against programmed death-ligand 1 (PD-L1).

The term "PD-1 antibody" or "anti-PD-1" refers to an antibody directed against programmed death protein 1 (PD-1).

The term "antibody therapy" refers to the medical use of antibodies that bind to target cells or target cell proteins to treat cancer and/or stimulate an immune response in a subject that results in recognition, attack and/or destruction of cancer cells in the subject, and in some embodiments of the invention, to activate or stimulate a memory immune response in a subject that results in subsequent recognition, attack and/or destruction of cancer cells in the subject.

The term "PD-L1 antibody therapy" refers to the use of antibodies against programmed death ligand 1 (anti-PD-L1) to modulate the immune response of a subject. In some embodiments, the PDL1 antibody inhibits or blocks the interaction of PD-L1 with programmed cell death protein 1 (PD-1), wherein the blockade of the interaction between PD-L1 and PD-1 inhibits the negative regulation of PD-1 on T cell activation, thereby attacking and destroying cancer cells.

The term "PD-1 antibody therapy" refers to the use of antibodies against programmed cell death protein 1 PD-1 (anti-PD-1) to regulate the immune response of the subject. In some embodiments, the PD-1 antibody inhibits or blocks the interaction between PD-1 and PD-L1, wherein the inhibition or blocking of the interaction between PD-L1 and PD-1 inhibits the negative regulation of PD-1 on T cell activation, thereby attacking and destroying cancer cells.

The reaction temperature of each step can be appropriately selected according to the solvent, starting materials, reagents, etc., and the reaction time can also be appropriately selected according to the reaction temperature, solvent, starting materials, reagents, etc. After the reaction of each step is completed, the target compound can be separated and purified from the reaction system by common methods, such as filtration, extraction, recrystallization, washing, silica gel column chromatography, etc. In the case of not affecting the next step reaction, the target compound can also be directly used in the next step reaction without separation and purification.

Beneficial Effects

After extensive and in-depth research, the inventors unexpectedly developed an EP2 and EP4 receptor antagonist, which is a compound shown in Formula I of the present invention, and can be used to treat inflammatory diseases (such as arthritis and endometriosis), autoimmune diseases (such as multiple sclerosis), neurodegenerative diseases (such as epilepsy, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and traumatic brain injury), cardiovascular diseases (such as atherosclerosis) and cancer (such as colon cancer, lung cancer, breast cancer and head and neck cancer). Experiments show that the compound of the present invention has a good antagonistic effect on EP2, shows a good inhibitory effect on EP4 calcium flow, has a good affinity with both EP2 and EP4 receptors, has a good thermodynamic solubility, has a low clearance rate for intravenous administration, a high exposure amount for oral administration, exhibits excellent pharmacokinetic properties, and has good drugability.

Detailed ways

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.

Example 1 Preparation of Compound I-1

The synthetic route is shown below

Step 1: Synthesis of (5-(3-fluorophenyl)pyrazine-2-yl)methanol (B1-3)

Under nitrogen protection, add 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (3.62g, 5mmol) and sodium carbonate (14.3g, 135mmol) to a mixed solution of (5-chloropyrazin-2-yl)methanol (7.2g, 50mmol) and (3-fluorophenyl)boronic acid (11.9g, 85mmol) in dioxane/water (85mL/15mL). Stir at 80°C for 3h. The reaction was monitored by TLC. After the reaction of (5-chloropyrazin-2-yl)methanol was completed, the reaction solution was filtered with diatomaceous earth, water (100 mL) was added to the filtrate, and the mixture was extracted with ethyl acetate (100 mL×3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 5/1 to 1/1) to obtain (5-(3-fluorophenyl)pyrazin-2-yl)methanol (B1-3) (9.6 g, yield 94%).

LC-MS, M/Z(ESI):205.1[M+H] ⁺

Step 2: Synthesis of 2-(chloromethyl)-5-(3-fluorophenyl)pyrazine (B1-4)

Under nitrogen protection, dichlorothionyl (8.53 mL) was slowly added dropwise to dichloromethane (120 mL) of (5-(3-fluorophenyl)pyrazine-2-yl)methanol (B1-3) (9.6 g, 47 mmol), and the mixture was reacted overnight at room temperature. The reaction solution was spin-dried and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 10/1 to 5/1) to obtain 2-(chloromethyl)-5-(3-fluorophenyl)pyrazine (B1-4) (8.24 g, yield 82.4%).

LC-MS, M/Z(ESI):223.0[M+H] ⁺

Step 3: Synthesis of 5-methyl-1H-indazole-7-carboxylic acid methyl ester (B1-6)

Under nitrogen protection, methylboric acid (6g, 100mmol), 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (725mg, 1mmol) and potassium carbonate (4.15g, 30mmol) were added to a solution of 5-bromo-1H-indazole-7-carboxylic acid methyl ester (B1-5) (2.54g, 10mmol) in dioxane (50mL), and the reaction solution was reacted at 90°C overnight. After the reaction was completed by TLC detection, the reaction solution was filtered with diatomaceous earth, water (50mL) was added to the filtrate, and it was extracted with ethyl acetate (50mL×3), the organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and mixed with silica gel column (volume ratio: petroleum ether/ethyl acetate = 5/1 to 1/1) to obtain 5-methyl-1H-indazole-7-carboxylic acid methyl ester (B1-6) (1.78g, yield 93%).

LC-MS, M/Z(ESI):191.1[M+H] ⁺

Step 4: Synthesis of methyl 1-((5-(3-fluorophenyl)pyrazine-2-yl)methyl)-5-methyl-1H-indazole-7-carboxylate (B1-7)

Under nitrogen protection, 2-(chloromethyl)-5-(3-fluorophenyl)pyrazine (B1-4) (4.1 g, 17.8 mmol) and cesium carbonate (7.6 g, 22.25 mmol) were added to 5-methyl-1H-indazole-7-carboxylic acid methyl ester (B1-6) (1.7 g, 8.9 mmol) in DMF (20 mL), and the mixture was reacted overnight at room temperature. After TLC detection, water (50 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and mixed with silica gel column (volume ratio: petroleum ether/ethyl acetate = 5/1 to 1/1) to obtain 1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxylic acid methyl ester (B1-7) (2.8 g, yield 83%).

LC-MS, M/Z(ESI):377.1[M+H] ⁺

Step 5: Synthesis of 1-((5-(3-fluorophenyl)pyrazine-2-yl)methyl)-5-methyl-1H-indazole-7-carboxylic acid (B1-8)

Under nitrogen protection, lithium hydroxide monohydrate (500 mg, 12 mmol), methanol (3 mL) and water (3 mL) were added to a solution of 1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxylic acid methyl ester (B1-7) (1.13 g, 3 mmol) in tetrahydrofuran (15 mL), and the mixture was reacted at room temperature overnight. 1N hydrochloric acid was added to the reaction solution to adjust the pH to 4, and the mixture was extracted with ethyl acetate (10 mL×3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo to obtain a crude product, which was slurried with petroleum ether/ethyl acetate (volume ratio: 10/1) to obtain 1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxylic acid (B1-8) (600 mg, 55%).

LC-MS, M/Z(ESI):363.1[M+H] ⁺

Step 6: Synthesis of ethyl 2-(6-(1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxamido)spiro[3.3]hept-2-yl)acetate (B1-10)

Under nitrogen protection, to a solution of 1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxylic acid (B1-8) (362 mg, 1 mmol) in DMF (15 mL) were added ethyl 2-(6-aminospiro[3.3]hept-2-yl)acetate (B1-9) (350 mg, 1.5 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (570 mg, 1.5 mmol), and N,N-diisopropylethylamine (0.87 mL), and the reaction was allowed to react at room temperature overnight. Water (20 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 5/1 to 1/1) to obtain ethyl 2-(6-(1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxamido)spiro[3.3]hept-2-yl)acetate (B1-10) (508 mg, yield 94%).

LC-MS, M/Z(ESI):542.3[M+H] ⁺

Step 7: Synthesis of 2-(6-(1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxamido)spiro[3.3]hept-2-yl)acetic acid (I-1)

Under nitrogen protection, lithium hydroxide monohydrate (158 mg, 3.75 mmol), methanol (1 mL) and water (1 mL) were added to a solution of ethyl 2-(6-(1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxamido)spiro[3.3]hept-2-yl)acetate (B1-10) (508 mg, 0.94 mmol) in tetrahydrofuran (5 mL) and the reaction was carried out at room temperature overnight. 1N hydrochloric acid was added to the reaction solution to adjust the pH to 4, and the mixture was extracted with ethyl acetate (10 mL×3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and slurried with petroleum ether/ethyl acetate (volume ratio: 10/1) to give 2-(6-(1-((5-(3-fluorophenyl)pyrazin-2-yl)methyl)-5-methyl-1H-indazole-7-carboxamido)spiro[3.3]hept-2-yl)acetic acid (I-1) (387 mg, 80%).

LC-MS, M/Z(ESI):514.2[M+H] ⁺

¹ H NMR (400 MHz, DMSO-d ₆ )δ11.91(brs,1H),9.10–9.06(m,1H),8.55(d,1H),8.18–8.16(m,1H),8.15–8.13(m ,1H),7.96–7.91(m,1H),7.90–7.84(m,1H),7.72–7.68(m,1H),7.60–7.53(m,1H),7.35–7.29( m,1H),7.27–7.23(m,1H),5.97(s,2H),4.12–4.0(m,1H),2.42(s,3H),2.39–2.29(m,1H),2.26–2.11( m, 4H), 2.08–2.0 (m, 1H), 1.90–1.82 (m, 1H), 1.76–1.62 (m, 3H), 1.49 (dd, 1H).

Example 2 Preparation of Compound I-2

The synthetic route is as follows:

Step 1: Synthesis of methyl 2-amino-5-chloro-3-methylbenzoate (B2-2)

Under nitrogen protection, iodomethane (1.4 mL, 20 mmol) was added dropwise to a solution of 2-amino-5-chloro-3-methylbenzoic acid (B2-1) (3.7 g, 20 mmol) and cesium carbonate (10.4 g, 20 mmol) in DMF (100 mL) and the mixture was reacted at room temperature for 12 hours. After the reaction was completed by TLC, water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL×3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 5/1 to 1/1) to obtain methyl 2-amino-5-chloro-3-methylbenzoate (B2-2) (3.9 g, yield 97%).

LC-MS, M/Z(ESI):200.0[M+H] ⁺

Step 2: Synthesis of 5-chloro-1H-indazole-7-carboxylic acid methyl ester (B2-3)

At 0°C, an aqueous solution (2 mL) of sodium nitrite (690 mg, 10 mmol) was added dropwise to a fluoroboric acid solution (50% aqueous solution, 20 mL) of methyl 2-amino-5-chloro-3-methylbenzoate (B2-2) (2 g, 10 mmol), and then reacted at 0°C for 1 hour. The reaction solution was filtered with diatomaceous earth, and the resulting solid was added to a dichloromethane suspension (20 mL) of potassium acetate (1.96 g, 20 mmol) and stirred at room temperature for 2 hours. Water (30 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (30 mL × 3). The organic phases were combined and washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 10/1 to 5/1) to obtain methyl 5-chloro-1H-indazole-7-carboxylate (B2-3) (450 mg, yield 21%).

LC-MS, M/Z(ESI):211.0[M+H] ⁺

Step 3: Synthesis of 1-(5-cyclopropylthiophen-2-yl)ethane-1-one (B2-5)

Under nitrogen protection, cyclopropylboric acid (10.3 g, 120 mmol), 1,1'-bis(diphenylphosphinoferrocene)palladium dichloride (1.45 g, 2 mmol) and potassium carbonate (8.3 g, 60 mmol) were added to a solution of 1-(5-bromothiophen-2-yl)ethane-1-one (B2-4) (4.1 g, 20 mmol) in dioxane (100 mL), and then reacted at 90°C overnight. After the reaction was completed by TLC, the reaction solution was filtered with diatomaceous earth, water (100 mL) was added to the filtrate, and extracted with ethyl acetate (100 mL×3), the combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and mixed with the sample and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 100/1 to 20/1) to obtain 1-(5-cyclopropylthiophen-2-yl)ethane-1-one (B2-5) (2.5 g, yield 75%).

LC-MS, M/Z(ESI):167.1[M+H] ⁺

Step 4: Synthesis of 1-(5-cyclopropylthiophen-2-yl)ethane-1-ol (B2-6)

At room temperature, sodium borohydride (1.2 g, 30.7 mmol) was added to a solution of 1-(5-cyclopropylthiophen-2-yl)ethane-1-one (B2-5) (2.5 g, 15.36 mmol) in ethanol (80 mL). After reacting at room temperature for 2 hours, the mixture was cooled to 0°C and water (10 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (20 mL × 3), and the combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 20/1 to 10/1) to obtain 1-(5-cyclopropylthiophen-2-yl)ethane-1-ol (B2-6) (2.5 g, yield 96%).

LC-MS, M/Z(ESI):169.1[M+H] ⁺

Step 5: Synthesis of 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid methyl ester (B2-7)

Under nitrogen protection and at 0°C, diethyl azodicarboxylate (0.63 mL, 4.0 mmol) was added dropwise to a solution of 1-(5-cyclopropylthiophen-2-yl)ethane-1-ol (B2-6) (403 mg, 2.4 mmol), 5-chloro-1H-indazole-7-carboxylic acid methyl ester (B2-3) (420 mg, 2.0 mmol) and triphenylphosphine (1.05 g, 4.0 mmol) in tetrahydrofuran (12 mL), and then reacted at room temperature for 2 hours. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL×3). The organic phases were combined, washed three times with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 100/1 to 20/1) to obtain 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid methyl ester (B2-7) (192 mg, yield 27%).

LC-MS, M/Z(ESI):361.1[M+H] ⁺

Step 6: Synthesis of 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid (B2-8)

Under nitrogen protection, lithium hydroxide monohydrate (90 mg, 2.13 mmol), methanol (0.8 mL) and water (0.8 mL) were added to a solution of 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid methyl ester (B2-7) (192 mg, 0.53 mmol) in tetrahydrofuran (4 mL) and reacted at room temperature overnight. 1N hydrochloric acid was added to the reaction solution to adjust pH = 4, extracted with ethyl acetate (5 mL × 3), the combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and mixed with the sample and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 10/1 to 5/1) to obtain 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid (B2-8) (175 mg, yield 95%).

LC-MS, M/Z(ESI):347.1[M+H] ⁺

Step 7: Synthesis of methyl 6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylate (B2-10)

Under nitrogen protection, to a solution of 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid (B2-8) (175 mg, 0.5 mmol), methyl 2-(6-aminospiro[3.3]hept-2-yl)carboxylate hydrochloride (142 mg, 0.75 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (285.2 mg, 0.75 mmol) in DMF (5 mL) was added N,N-diisopropylethylamine (0.44 mL) and the mixture was reacted at room temperature overnight. Water (10 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 5/1 to 1/1) to give 6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid methyl ester (B2-10) (243 mg, yield 98%).

LC-MS, M/Z(ESI):498.2[M+H] ⁺

Step 8: Synthesis of 6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid (I-2)

Under nitrogen protection, to a solution of methyl 6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylate (B2-10) (243 mg, 0.49 mmol) in tetrahydrofuran (4 mL) was added lithium hydroxide monohydrate (83 mg, 1.96 mmol), methanol (0.8 mL) and water (0.8 mL), and the reaction was carried out at room temperature overnight. 1N hydrochloric acid was added to the reaction solution to adjust the pH to 4, and the mixture was extracted with ethyl acetate (5 mL×3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 10/1 to 5/1) to obtain 6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid (I-2) (200 mg, yield 85%).

LC-MS, M/Z(ESI):484.1[M+H] ⁺

¹ H NMR (400 MHz, DMSO-d6) δ9.03 (d, 1H), 8.20 (s, 1H), 7.97 (d, 1H), 7.39 (d, 1H), 6.58–6.50 (m, 2H), 6.40 (q, 1H), 4.38–4.26 (m, 1H), 2.98–2.86 (m, 1H), 2.50–2.43 (m, 1H), 2.34–2.25 (m, 2H), 2.24–2.17 (m, 1H), 2.16–1.91 (m, 5H), 1.86 (d, 3H), 0.90–0.83 (m, 2H), 0.55–0.47 (m, 2H).

Step 9 Synthesis of Compounds I-2A and I-2B

Referring to the method of Example 3, compound I-2 was separated by SFC to obtain compounds I-2A and I-2B.

Example 3 Preparation of Compounds I-3A and I-3B

The synthetic route is as follows:

Step 1: Preparation of (S)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid methyl ester (B3-1) and (R)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid methyl ester (B3-2) by supercritical fluid chromatography (SFC)

6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamidocarboxyl)spiro[3.3]heptane-2-carboxylic acid methyl ester (B2-10) (1.14 g, 2.29 mmol) was subjected to supercritical fluid chromatography (SFC) (column model: Chiralpak IF-3 50*4.6 mm I.D., 3 um; mobile phase: phase A is carbon dioxide, phase B is isopropanol (containing 0.05% diethanolamine); gradient eluent: isopropanol (containing 0.05% diethanolamine)/carbon dioxide: from 5% to 40%, flow rate: 3 mL/min; detector: P DA; column temperature: 35 ° C; back pressure: 100 Bar) and freeze-dried to obtain (S)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid methyl ester (B3-1) (61 mg, 0.12 mmol, retention time: 2.566 min) and (R)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid methyl ester (B3-2) (60 mg, 0.12 mmol, retention time: 2.206 min).

Step 2: Synthesis of (S)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid (I-3A)

Under nitrogen protection, (S)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro To a solution of [3.3]heptane-2-carboxylic acid methyl ester (B3-1) (61 mg, 0.12 mmol) in tetrahydrofuran (1.5 mL), lithium hydroxide monohydrate (21 mg, 0.49 mmol), methanol (0.3 mL) and water (0.3 mL) were added and reacted at room temperature overnight. 1N hydrochloric acid was added to the reaction solution to adjust the pH to 4, and the mixture was extracted with ethyl acetate (5 mL×3). The organic phases were combined and washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and mixed and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 10/1 to 1/1) to obtain (S)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid (I-3A) (40 mg, yield 69%).

LC-MS, M/Z(ESI):484.1[M+H] ⁺

¹ H NMR (400 MHz, DMSO-d6) δ8.99 (d, 1H), 8.28 (s, 1H), 7.39 (d, 1H), 7.25 (d, 1H), 6.58 (d, 1H), 6.54 (d, 1H), 6.45 (q, 1H), 4.40–4.27 (m, 1H), 2.90–2.80 (m, 1H), 2.50–2.40 (m, 1H), 2.33–2.07 (m, 4H), 2.06–1.91 (m, 4H), 1.88 (d, 3H), 0.92–0.83 (m, 2H), 0.57–0.47 (m, 2H).

Step 3: Synthesis of (R)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid (I-3B)

Under nitrogen protection, to a solution of (R)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamide)spiro[3.3]heptane-2-carboxylic acid methyl ester (B3-2) (60 mg, 0.12 mmol) in tetrahydrofuran (1.5 mL) were added lithium hydroxide monohydrate (21 mg, 0.49 mmol), methanol (0.3 mL) and water (0.3 mL), and the reaction was carried out at room temperature overnight. 1N hydrochloric acid was added to the reaction solution to adjust the pH to 4, and the mixture was extracted with ethyl acetate (5 mL×3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 10/1 to 1/1) to obtain (S)-6-(4-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-carboxylic acid (I-3B) (39 mg, yield 68%).

LC-MS, M/Z(ESI):484.1[M+H] ⁺

¹ H NMR (400 MHz, DMSO-d6) δ8.99 (d, 1H), 8.28 (s, 1H), 7.39 (d, 1H), 7.26 (d, 1H), 6.57 (d, 1H), 6.54 (s, 1H), 6.45 (q, 1H), 4.41–4.28 (m, 1H), 2.94–2.82 (m, 1H), 2.50–2.40 (m, 1H), 2.33–2.17 (m, 3H), 2.16–1.90 (m, 3H), 2.00–1.90 (m, 2H), 1.88 (d, 3H), 0.92–0.80 (m, 2H), 0.56–0.47 (m, 2H).

Example 4 Preparation of Compound I-4

The synthetic route is as follows:

Step 1: Synthesis of ethyl 2-(6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]hept-2-yl)acetate (B4-1)

Under nitrogen protection, to a solution of 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid (B2-8) (210 mg, 0.6 mmol), ethyl 2-(6-aminospiro[3.3]hept-2-yl)acetate hydrochloride (210 mg, 0.9 mmol), and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (350 mg, 0.9 mmol) in DMF (5 mL) was added N,N-diisopropylethylamine (0.53 mL) and the mixture was reacted at room temperature overnight. Water (10 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 10/1 to 5/1) to obtain ethyl 2-(6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]hept-2-yl)acetate (B4-1) (276 mg, yield 88%).

LC-MS, M/Z(ESI):526.19[M+H] ⁺

Step 2: Synthesis of 2-(6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-yl)acetic acid (I-4)

Under nitrogen protection, to a solution of ethyl 2-(6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]hept-2-yl)acetate (B4-1) (276 mg, 0.525 mmol) in tetrahydrofuran (4 mL) was added lithium hydroxide monohydrate (91 mg, 2.16 mmol), methanol (0.8 mL) and water (0.8 mL), and the reaction was carried out at room temperature overnight. 1N hydrochloric acid was added to the reaction solution to adjust the pH to 4, and the mixture was extracted with ethyl acetate (5 mL×3). The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated in vacuo, and passed through a silica gel column (volume ratio: petroleum ether/ethyl acetate = 10/1 to 1/1) to obtain 2-(6-(5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxamido)spiro[3.3]heptane-2-yl)acetic acid (I-4) (200 mg, yield 77%).

LC-MS, M/Z(ESI):498.15[M+H] ⁺

¹ H NMR (400 MHz, DMSO-d6) δ 11.94 (s, 1H), 9.02 (d, 1H), 8.20 (s, 1H), 7.97 (d, 1H), 7.42–7.36 (m, 1H), 6.58–6.50 (m, 2H), 6.40 (q, 1H), 4.40–4.25 (m, 1H), 2.48–2.37 (m, 2H), 2.33–2.19 (m, 4H), 2.10–1.91 (m, 4H), 1.86 (d, 3H), 1.79–1.71 (m, 1H), 1.70–1.62 (m, 1H), 0.93–0.81 (m, 2H), 0.57–0.46 (m, 2H).

Step 3: Synthesis of compounds I-4A and I-4B

Referring to the method of Example 3, compound I-4 was separated by SFC to obtain compounds I-4A and I-4B

The following compounds were prepared by the methods of Examples 1 to 4.

Test Example 1: EP2 antagonism assay

The antagonistic effect of the compound on EP2 was carried out on a CHO stable cell line that highly expresses the human EP2 receptor. After trypsin digestion, the cells were resuspended in a buffer (1×HBSS, 0.1% BSA, 20mM HEPES and 500μM IBMX), and 8000 cells were inoculated in each well of a 384-well plate with an inoculation volume of 15μL. The test compound, EP2 complete antagonist TG4-155 and PGE ₂ were prepared into 10mM stock solutions using DMSO reagent, and the 8X concentration of the test compound working solution, EP2 complete antagonist TG4-155 (purchased from MedChemExpress) working solution and PGE2 working solution were prepared with experimental buffer, and then 2.5μL of 8X compound working solution, 8X EP2 complete antagonist TG4-155 and DMSO (final concentration 0.2%) were added to the above 384-well plate, and incubated at 37°C for 10min. Add 2.5 μL of 8X concentration of agonist PGE ₂ working solution to each well in the above 384-well plate (PGE ₂ final concentration is 0.3 nM) and incubate at 37 ° C for 30 min. After the reaction is completed, dilute Eu-cAMP 1:50 with cAMP detection buffer in the kit (Perkin Elmer, Cat# TRF0263), add 10 μL of Eu-cAMP to each well, dilute ULight-anti-cAMP 1:150 with cAMP detection buffer, add 10 μL of ULight-anti-cAMP to each well, and incubate at room temperature for 1 h. Read the data at 665nm and 615nm wavelengths on the Envision 2105 plate reader. Calculate the antagonistic effect (IC ₅₀ value) of the test compound.

Table 1 Antagonistic effect of test compounds on EP2

The results show that the compounds of the present invention have a good antagonistic effect on EP2.

Test Example 2: Determination of the inhibitory effect on EP4 receptor calcium flow

The inhibitory effect of the compound on EP4 calcium flux was tested on 293 cells overexpressing the human EP4 receptor. The well-grown cells were resuspended in cell culture medium and the cell density was adjusted to 1×10 ⁶ cells per ml. The cell suspension was inoculated at 20 μL/well in 2 polylysine-coated 384-well plates (20,000 cells/well) and placed in a 37°C, 5% CO ₂ incubator overnight. Prepare 2X Fluo-4 Direct ^TM (Invitrogen, Cat#F10471) loading buffer: Add 77 mg of probenecid to 1 mL of FLIPR buffer at a concentration of 250 mM. Add 10 mL of FLIPR buffer and 0.2 mL of 250 mM probenecid to each tube of Fluo-4 Direct ^TM crystals (F10471), vortex and keep it still for 5 minutes in the dark.

Remove a cell plate from the incubator and remove the culture medium. Add 20 μL of assay buffer and 2X Fluo-4 Direct ^TM No-Wash Loading Buffer to a 384-well cell culture plate to a final volume of 40 μL. Incubate in a 37°C, 5% CO ₂ incubator for 50 minutes, incubate at room temperature for 10 minutes, and place in the FLIPR. Transfer 10 μL of buffer to the cell plate and read the fluorescence signal. Prepare the agonist PGE ₂ in DMSO solvent to a 10mM stock solution and use the buffer to grade dilute the 6X working solution at 10 concentration points. Transfer 10 μL of agonist PGE ₂ to the cell plate, read the fluorescence signal, and calculate the EC ₈₀ value.

Agonist PGE ₂ at 6X EC ₈₀ concentration was prepared, and the compound to be tested was prepared into a 10 mM stock solution in DMSO solvent, and the 6X compound working solution was diluted to 10 concentration points using a buffer gradient.

Take another cell plate and remove the culture medium. Add 20 μL of analysis buffer and 2X Fluo-4Direct ^TM wash-free loading buffer. Incubate in a 37°C, 5% CO ₂ incubator for 50 minutes, incubate at room temperature for 10 minutes, and place in FLIPR. Transfer 10 μL of compound working solution, DMSO, and EP4 complete antagonist to the cell plate and read the fluorescence signal. Transfer 10 μL of 6X EC ₈₀ concentration of agonist PGE ₂ to the cell plate, read the fluorescence signal, and calculate the IC ₅₀ value of the compound's inhibition of EP4 calcium flow.

Table 2 Inhibitory effects of test compounds on EP4 calcium flux

The experimental results show that the compounds of the present invention have a good inhibitory effect on EP4 calcium flow.

Test Example 3: Radioligand EP2 receptor binding assay

Radioligand EP2 binding assay was performed using recombinant human EP2 receptor membrane protein (Perkin Elmer #ES-562-M400UA, prepared from 293 cells overexpressing human EP2 receptor). The test compound was prepared into a 10 μM stock solution using DMSO solvent, and the test compound and radioligand [ ³ H]-PGE ₂ (Perkin Elmer #NET428025UC) were prepared into 10× working solutions using binding assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl 2 , 0.5 mM EDTA). 10 μL of compound working solution, 1% DMSO, PGE ₂ working solution (final concentration 10 μM) were added to the assay plate, 80 μL of EP2 receptor membrane protein (10 μg/well) and 10 μL of radioligand [ ³ H]-PGE ₂ (Perkin Elmer #NET428025UC) (final concentration 7.5 nM) were added, and incubated at 25°C for 1.5 hours. The reaction mixture was filtered through a 0.3% PEI-coated GF/C plate using Cell Harvester, and the filter plate was washed three times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4), and the filter plate was dried at 37°C for 2 hours. After drying, 50 μL of scintillation cocktail was added and the top of the filter plate was sealed. The ³ H counts captured on the filter were read using Topcount.

The data were analyzed using GraphPad Prism 5, and the inhibition rate was calculated according to the following formula:
Inhibition rate (%) = 100 - (test group - _PGE2 group) / (DMSO group - _PGE2 group) * 100

According to the inhibition rate of different concentrations of the compound, the _IC50 and Ki values of the compound determined by radioligand EP2 binding were calculated.

Experimental data show that the compound of the present invention has a very good affinity with the EP2 receptor.

Test Example 4: Radioligand EP4 receptor binding assay

Radioligand EP4 binding assay was performed using recombinant human EP4 receptor membrane protein (prepared from 293 cells overexpressing human EP4 receptor). The test compound and PGE ₂ were prepared into 10 μM stock solution using DMSO reagent, and 200 μM was used as the starting concentration, and 4-fold gradient dilution was made to 8 concentration points of working solution. Then, EP4 receptor membrane protein and radioligand [ ³ H]-PGE ₂ (PerkinElmer, Cat: NET428250UC, Lot: 2469552) were prepared into working solution concentration using buffer (50 mM HBSS, 0.1% BSA, 500 mM NaCl). 1 μL of compound working solution, DMSO, and PGE ₂ working solution were added to the assay plate, 100 μL of EP4 receptor membrane protein (20 μg/well) and 100 μL of radioligand [ ³ H]-PGE ₂ (PerkinElmer, Cat: NET428250UC, Lot: 2469552) (final concentration 1.5 nM) were added, and the plate was sealed and incubated at room temperature for 1 hour. At room temperature, 0.5% BSA, 50 μL/well, was soaked in a Unifilter-96 GF/C filter plate (Perkin Elmer) for at least 30 minutes. After binding, the reaction mixture was filtered through the GF/C plate using a Perkin Elmer Filtermate Harvester, and then the filter plate was washed and dried at 50°C for 1 hour. After drying, the bottom of the filter plate well was sealed with Perkin Elmer Unifilter-96 sealing tape, and 50 μL of MicroScint ^TM -20 cocktail (Perkin Elmer) was added to seal the top of the filter plate. The ³ H counts captured on the filter were read using a Perkin Elmer MicroBeta2 Reader.

The data were analyzed using GraphPad Prism 5, and the inhibition rate was calculated according to the following formula:
Inhibition rate (%) = 100 - (test group - _PGE2 group) / (DMSO group - _PGE2 group) * 100

According to the inhibition rate of different concentrations of the compound, the _IC50 and Ki values of the compound were calculated by radioligand EP4 binding assay.

The experimental results show that the compound of the present invention has a very good affinity with the EP4 receptor.

Test Example 5: Thermodynamic Solubility Experiment

Prepare the fasting state simulated gastric fluid FaSSGF (1L solution contains 80μM sodium taurocholate, 20μM oocytes) at pH 1.6. Phospholipids, 0.1 g of pepsin, 34.2 mM sodium chloride), fasting simulated intestinal fluid FaSSIF at pH 6.5 (1 L solution contained 3 mM sodium taurocholate, 0.2 mM lecithin, 38.4 mM sodium hydroxide, 68.62 mM sodium chloride, 19.12 mM maleic acid), and phosphate buffered saline PBS at pH 7.4 (1 L solution contained 100 mM phosphate buffer, 11 g of disodium bicarbonate, 3.5 g of sodium dihydrogen phosphate dihydrate) were used as assay buffers.

Accurately weigh the compound, use different pH assay buffers to prepare the compound into a 4 mg/mL working solution, shake at 1000 rpm for 1 hour, and equilibrate at room temperature overnight. Centrifuge the sample at 12000 rpm for 10 minutes to remove undissolved particles. Transfer the supernatant to a new centrifuge tube and detect the compound concentration in the supernatant by LCMS-MS.

The experimental results show that the compound has good thermodynamic solubility.

Test Example 6: Pharmacokinetic Test

Mouse pharmacokinetic test, using male ICR mice, 20-25g, fasted overnight. Take 3 mice and orally gavage 5mg/kg. Blood was collected before and 15, 30 minutes and 1, 2, 4, 8, 24 hours after administration. Take another 3 mice and intravenously inject 1mg/kg, and blood was collected before and 15, 30 minutes and 1, 2, 4, 8, 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 and add 3 times the amount of water to 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 WinNonlin7.0 software.

For the rat pharmacokinetic test, male SD rats, 180-240g, were used and fasted overnight. Three rats were given 5 mg/kg by oral gavage. Another three rats were given 1 mg/kg by intravenous injection. The rest of the operation was the same as the mouse pharmacokinetic test.

For the canine pharmacokinetic test, male Beagle dogs, 8-10 kg, were fasted overnight. Three Beagle dogs were given 3 mg/kg by oral gavage. Another three Beagle dogs were given 1 mg/kg by intravenous injection. The rest of the procedures were the same as those for the mouse pharmacokinetic test.

For monkey pharmacokinetic experiments, male cynomolgus monkeys, 5-7 kg, fasted overnight were used. Three cynomolgus monkeys were given 5 mg/kg by oral gavage. Another three cynomolgus monkeys were given 3 mg/kg by intravenous injection. The rest of the operations were the same as the mouse pharmacokinetic experiments.

Table 3 Pharmacokinetic results of oral administration in monkeys

The experimental results show that the compound of the present invention has a low clearance rate after intravenous administration and a high exposure after oral administration, exhibits excellent pharmacokinetic properties, and has 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)

1. The compound represented by formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug:
   

   Wherein, R ₁ is halogen, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl;

   L ₁ and L ₂ are each independently absent or C ₁ -C ₃ alkylene; the C ₁ -C ₃ alkylene is optionally substituted by C ₁ -C ₃ alkyl;

   Ring A is thiophene, and ring B is cyclopropyl;

   Alternatively, ring A is pyrazine, and ring B is a benzene ring;

   The ring A and the ring B are optionally substituted by 1, 2 or 3 identical or different Ra, wherein Ra is selected from the group consisting of halogen, cyano, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, and -OC ₁ -C ₃ alkyl.
2. The compound of formula I as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that ring A is thiophene and ring B is cyclopropyl;

   R ₁ is halogen; L ₁ is absent or is methylene; L ₂ is -CH(CH ₃ )-;

   Preferably, the halogen is Cl.
3. The compound of formula I as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that ring A is pyrazine, ring B is a benzene ring, and the benzene ring is optionally substituted by 1, 2 or 3 substituents selected from the following: halogen, cyano, C ₁ -C ₃ alkyl, C ₁ -C ₃ haloalkyl, -OC ₁ -C ₃ alkyl;

   R ₁ is C ₁ -C ₃ alkyl, halogen; L ₁ is -CH ₂ -; L ₂ is -CH ₂ -;

   Preferably, the benzene ring is optionally substituted by 1 or 2 identical or different Ra, wherein Ra is selected from: halogen, cyano, methyl, methoxy;

   Preferably, the halogen is F;

   Preferably, R ₁ is Cl or methyl;

   Preferably, L ₁ and L ₂ are each independently absent or -CH ₂ - or -CH(CH ₃ )-.
4. The compound of formula I as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that it has the structure shown in formula Ia
   

   wherein m is 1 or 2; Ra is F, cyano, methyl or methoxy;

   _L1 is absent or is _-CH2- .
5. The compound of formula I as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that it has the structure shown in formula Ib
   

   wherein R ₁ is halogen; L ₁ is absent or -CH ₂ -; L ₂ is -CH(CH ₃ )-;

   Preferably, it has the structure shown in Formula Ic or Formula Id
   

   Preferably, R ₁ is Cl;

   Preferably, L ₁ is absent or is -CH ₂ -;

   Preferably, _L2 is
6. The compound of formula I as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that it is selected from
   

   Preferably, With structure

   Preferably, With structure

   Preferably, With structure
7. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: a compound of formula I as described in any one of claims 1 to 6, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug; and a pharmaceutically acceptable carrier.
8. A pharmaceutical composition, characterized in that the pharmaceutical composition comprises: a compound of formula I as described in any one of claims 1 to 6, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof; and a second drug;

   Preferably, the second drug comprises an antibody;

   Preferably, the antibodies include anti-PD-L1 antibodies and anti-PD-1 antibodies.
9. A use of a compound of formula I as claimed in any one of claims 1 to 6, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, or a use of a pharmaceutical composition as claimed in claim 7 or 8, comprising:

   1) Antagonize EP2 and/or EP4;

   2) Binding to EP2 and/or EP4 receptors;

   3) Prevention and treatment of diseases mediated by EP2 and/or EP4 receptors;

   4) preparing EP2 and/or EP4 antagonists;

   5) Preparation of drugs, pharmaceutical compositions or preparations for preventing and treating diseases mediated by EP2 and/or EP4 receptors.
10. The use according to claim 9, wherein the diseases mediated by the EP2 and/or EP4 receptors include inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cardiovascular diseases and cancer.