WO2024120512A1 - Antagoniste des récepteurs ep2 et ep4 - Google Patents

Antagoniste des récepteurs ep2 et ep4 Download PDF

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WO2024120512A1
WO2024120512A1 PCT/CN2023/137359 CN2023137359W WO2024120512A1 WO 2024120512 A1 WO2024120512 A1 WO 2024120512A1 CN 2023137359 W CN2023137359 W CN 2023137359W WO 2024120512 A1 WO2024120512 A1 WO 2024120512A1
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compound
formula
ring
pharmaceutically acceptable
halogen
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PCT/CN2023/137359
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Chinese (zh)
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张学军
臧杨
卓君明
汤亚敏
孙红娜
李禹琼
李莉娥
杨俊�
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武汉人福创新药物研发中心有限公司
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  • 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.
  • Prostaglandin E2 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.
  • EP1 receptor activates phospholipase C and inositol triphosphate pathway
  • EP2 and EP4 receptors activate adenylate cyclase and cAMP-protein kinase A
  • activation of EP3 receptor can both inhibit adenylate cyclase and activate phospholipase C.
  • 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.
  • immune cells such as macrophages, dendritic cells, NK cells and cytotoxic T lymphocytes (CTL)
  • 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).
  • TAMS type 2 tumor-associated macrophages
  • Treg cells Treg cells
  • MDSCs myeloid-derived suppressor cells
  • 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.
  • HCC hepatocellular carcinoma
  • 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.
  • DC antigen-presenting dendritic cells
  • PGE2 plays a very important role in promoting the occurrence and development of tumors.
  • 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.
  • 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).
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • ALS amyotrophic lateral sclerosis
  • TBI traumatic brain injury
  • the EP4 antagonist ONO-AE3-208 reduced amyloid- ⁇ and improved behavioral performance.
  • 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.
  • P4 progesterone
  • 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.
  • the present invention provides a compound of formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug:
  • R 1 is halogen, C 1 -C 3 alkyl, C 1 -C 3 haloalkyl
  • Ring A is thiophene, and ring B is cyclopropyl
  • ring A is pyrazine
  • 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.
  • ring A is thiophene
  • ring B is cyclopropyl
  • R 1 is halogen
  • L 1 is absent or is methylene
  • L 2 is -CH(CH 3 )-.
  • the halogen is F or Cl.
  • the halogen is Cl.
  • ring A is pyrazine
  • 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 -.
  • the ring B is optionally substituted by 1 or 2 identical or different Ra, wherein Ra is selected from: halogen, cyano, methyl, methoxy;
  • the halogen is F.
  • R 1 is Cl or methyl.
  • L 1 and L 2 are each independently absent or -CH 2 - or -CH(CH 3 )-.
  • L1 is absent or is -CH2- .
  • R 1 is Cl
  • L 1 is absent or is -CH 2 -.
  • L2 is
  • the compound represented by formula I its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug is selected from
  • 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.
  • the second drug comprises an antibody
  • the antibodies include anti-PD-L1 antibodies and anti-PD-1 antibodies.
  • 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:
  • 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).
  • 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
  • cancers e.g., colon cancer, lung cancer, breast cancer and head and neck cancer.
  • 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.
  • the method further comprises use in combination with antibody therapy; preferably, the antibody therapy comprises PD-L1 antibody therapy and PD-1 antibody therapy.
  • 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.
  • 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).
  • 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
  • cancers e.g., colon cancer, lung cancer, breast cancer and head and neck cancer.
  • substituents 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 .
  • halogen when used alone or as part of other substituents refers to fluorine, chlorine, bromine, iodine.
  • 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.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl
  • alkenyl refers to an unbranched or branched monovalent hydrocarbon chain containing one or more carbon-carbon double bonds.
  • 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.
  • 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.
  • 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-).
  • 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.
  • 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.
  • “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.
  • “optionally substituted aryl” means that the aryl is substituted or unsubstituted, and the description includes both substituted aryl and unsubstituted aryl.
  • 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.
  • “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.
  • salt or “pharmaceutically acceptable salt” includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.
  • 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.
  • 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.
  • stereoisomer refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers and conformational isomers.
  • 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.
  • 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.
  • 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.
  • the keto form predominates; while in phenols, the enol form predominates.
  • the present invention encompasses all tautomeric forms of the compounds.
  • 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.
  • 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.
  • excipient refers to a pharmaceutically acceptable inert ingredient.
  • 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.
  • 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.
  • terapéuticaally 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.
  • Preventing disease e.g., preventing a disease,
  • treatment and other similar synonyms include the following meanings:
  • 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.
  • P-L1 antibody or "anti-PD-L1” refers to an antibody directed against programmed death-ligand 1 (PD-L1).
  • PD-1 antibody or "anti-PD-1” refers to an antibody directed against programmed death protein 1 (PD-1).
  • 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.
  • PDL1 antibody therapy refers to the use of antibodies against programmed death ligand 1 (anti-PD-L1) to modulate the immune response of a subject.
  • 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.
  • PD-1 programmed cell death protein 1
  • 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.
  • 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.
  • 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.
  • 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).
  • 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
  • cancer such as colon cancer, lung cancer, breast cancer and head and neck cancer.
  • 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.
  • 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.
  • 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.
  • Step 4 Synthesis of methyl 1-((5-(3-fluorophenyl)pyrazine-2-yl)methyl)-5-methyl-1H-indazole-7-carboxylate (B1-7)
  • Step 5 Synthesis of 1-((5-(3-fluorophenyl)pyrazine-2-yl)methyl)-5-methyl-1H-indazole-7-carboxylic acid (B1-8)
  • lithium hydroxide monohydrate 500 mg, 12 mmol
  • methanol 3 mL
  • water 3 mL
  • 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)
  • 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).
  • 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)
  • 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)
  • lithium hydroxide monohydrate 158 mg, 3.75 mmol
  • methanol 1 mL
  • water 1 mL
  • 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
  • tetrahydrofuran 5 mL
  • 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 synthetic route is as follows:
  • 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).
  • 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.
  • 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.
  • B2-4 1-(5-bromothiophen-2-yl)ethane-1-one
  • Step 5 Synthesis of 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid methyl ester (B2-7)
  • 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.
  • Step 6 Synthesis of 5-chloro-1-(1-(5-cyclopropylthiophen-2-yl)ethyl)-1H-indazole-7-carboxylic acid (B2-8)
  • 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.
  • 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)
  • 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)
  • compound I-2 was separated by SFC to obtain compounds I-2A and I-2B.
  • 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)
  • 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)
  • 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)
  • 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)
  • 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)
  • 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 500 ⁇ M IBMX), and 8000 cells were inoculated in each well of a 384-well plate with an inoculation volume of 15 ⁇ L.
  • 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 2.5 ⁇ L 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 ⁇ L 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.
  • 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 ⁇ L/well in 2 polylysine-coated 384-well plates (20,000 cells/well) and placed in a 37°C, 5% CO 2 incubator overnight.
  • 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.
  • 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 ⁇ M 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).
  • Inhibition rate (%) 100 - (test group - PGE2 group) / (DMSO group - PGE2 group) * 100
  • the IC50 and Ki values of the compound determined by radioligand EP2 binding were calculated.
  • 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 ⁇ M stock solution using DMSO reagent, and 200 ⁇ M was used as the starting concentration, and 4-fold gradient dilution was made to 8 concentration points of working solution.
  • 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).
  • 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 ⁇ L 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.
  • Inhibition rate (%) 100 - (test group - PGE2 group) / (DMSO group - PGE2 group) * 100
  • the IC50 and Ki values of the compound were calculated by radioligand EP4 binding assay.
  • 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°C for 6 minutes, and the plasma was collected and stored at -80°C.
  • mice 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.
  • 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.

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Abstract

La présente invention concerne un antagoniste des récepteurs EP2 et EP4. L'antagoniste est un composé représenté par la formule I, et peut être utilisé pour traiter des maladies médiées par les récepteurs EP2 et EP4.
PCT/CN2023/137359 2022-12-08 2023-12-08 Antagoniste des récepteurs ep2 et ep4 WO2024120512A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110891935A (zh) * 2017-04-18 2020-03-17 泰普斯特医疗公司 双环化合物及其在癌症治疗中的用途
CN112313208A (zh) * 2018-04-17 2021-02-02 泰普斯特医疗公司 双环羧酰胺及其使用方法
CN115448882A (zh) * 2021-06-08 2022-12-09 武汉人福创新药物研发中心有限公司 用于治疗ep2、ep4受体介导的疾病的苯并杂环化合物
CN115448913A (zh) * 2021-06-08 2022-12-09 武汉人福创新药物研发中心有限公司 用于治疗ep2、ep4受体介导的疾病的双环化合物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110891935A (zh) * 2017-04-18 2020-03-17 泰普斯特医疗公司 双环化合物及其在癌症治疗中的用途
CN112313208A (zh) * 2018-04-17 2021-02-02 泰普斯特医疗公司 双环羧酰胺及其使用方法
CN115448882A (zh) * 2021-06-08 2022-12-09 武汉人福创新药物研发中心有限公司 用于治疗ep2、ep4受体介导的疾病的苯并杂环化合物
CN115448913A (zh) * 2021-06-08 2022-12-09 武汉人福创新药物研发中心有限公司 用于治疗ep2、ep4受体介导的疾病的双环化合物

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