Fused ring compounds as ep4 antagonists and methods of making and using the same

Abstract

The present application provides a kind of compound shown in formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug;The compound has effective antagonism to EP4, has good affinity with EP4 receptor, and can be used as EP4 antagonist.

Description

[0001] Priority information

[0002] This application claims priority and benefits to patent application No. 202110098734.8, filed with the China National Intellectual Property Administration on January 25, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of medicine. Specifically, this invention relates to a fused-ring compound as an EP4 antagonist, its preparation method, and its uses. Background Technology

[0004] Prostaglandin E2 (PGE2) is an endogenous bioactive lipid. PGE2 activates prostaglandin receptors, inducing a wide range of upstream and downstream dependent biological responses (Legler, DF et al., hit. J Biochem. Cell Biol. 2010, 42, p. 198-201), participating in the regulation of numerous physiological and pathological processes, including inflammation, pain, renal function, cardiovascular system, lung function, and cancer. PGE2 has been reported to be highly expressed in cancerous tissues of various cancers, and its association with the occurrence, growth, and development of cancer and disease status in patients has been confirmed. It is generally believed that PGE2 is associated with the activation of cell proliferation and cell death (apoptosis) and plays an important role in cancer cell proliferation, disease progression, and cancer metastasis.

[0005] There are four subtypes of PGE2 receptors: EP1, EP2, EP3, and EP4, which are widely distributed in various tissues. Among these subtypes, PGE2, through the EP4 receptor, intervenes in inflammatory responses (including immune inflammatory responses), smooth muscle relaxation, pain, lymphocyte differentiation, hypertrophy or proliferation of mesangial cells, and gastrointestinal mucus secretion. Therefore, EP4 receptor antagonists can be considered promising anti-inflammatory and / or analgesic drugs for treating diseases related to the PGE2-EP4 pathway, such as inflammatory diseases and diseases accompanied by various types of pain.

[0006] EP4 is a major receptor involved in arthritic pain in rodent models of rheumatoid arthritis and osteoarthritis (see, for example, J. Pharmacol. Exp. Ther., 325, 425 (2008)). Its activation leads to the accumulation of the intracellular signaling molecule cAMP. Studies have detected EP4 receptor expression in peripheral nerve endings of pain receptors, macrophages, and neutrophils, confirming the crucial role of these cell types in endometriosis. Studies have reported that oral administration of EP4 antagonists can reduce proteinuria and inhibit the progression of diabetic nephropathy in type 2 diabetic mice. Other studies have reported that EP4 activation and increased PGE2 production in the bladder mucosa may be an important cause of overactive bladder due to prostatitis, and intravesical injection of EP4 antagonists can effectively improve overactive bladder after prostatitis. Therefore, selective EP4 antagonists can be used to treat arthritis, including arthritic pain, as well as endometriosis, diabetic nephropathy, and overactive bladder. Current treatments for arthritis primarily consist of traditional NSAIDs (nonsteroidal anti-inflammatory drugs) or selective COX-2 inhibitors, which can cause cardiovascular and / or gastrointestinal side effects. Selective EP4 antagonists, on the other hand, are less likely to cause cardiovascular side effects.

[0007] PGE2 persistently activates EP receptors (produced in large numbers by tumor cells) in the tumor microenvironment (Ochs et al, J Neurochem. 2016, 136, p. 1142-1154; Zelenay, S. et al, Cell 2015, 162, p. 1257-1270), promoting the accumulation and enhancing 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 low overall survival in patients with gastric cancer, ovarian cancer, breast cancer, bladder cancer, hepatocellular carcinoma (HCC), head and neck cancer, and other types of cancer. Furthermore, PGE2 has been reported to induce immune tolerance by inhibiting the accumulation of antigen-presenting dendritic cells (DCs) in tumors and suppressing the activation of tumor-invasive DCs (Wang et al., Trends in Molecular Medicine 2016, 22, pp. 1-3). All these PGE2-mediated effects collectively help tumor cells evade immune surveillance. PGE2 plays a crucial role in promoting tumorigenesis and development. Elevated expression levels of PGE2 and its related receptors EP2 and EP4 have been found in various malignant tumors, including colon cancer, lung cancer, breast cancer, and head and neck cancer, and are often closely associated with poor prognosis (Bhooshan, N. et al., Lung Cancer 101, 88-91). Therefore, selectively blocking the EP2 and EP4 signaling pathways can inhibit tumorigenesis and development by altering the tumor microenvironment and regulating tumor immune cells.

[0008] Existing preclinical research data show that EP2 and EP4 specific antagonists can prevent or inhibit tumor growth to varying degrees in animal models of colorectal cancer, esophageal cancer, lung cancer, and breast cancer. Among PGE2 receptor drugs that have entered clinical trials, Pfizer's EP4 antagonist Grapiprit has been approved by the FDA for the treatment of arthritis in dogs and entered a Phase II clinical trial in 2015 for the treatment of various types of solid tumors, including prostate cancer, non-small cell lung cancer, and breast cancer (De Vito, V. et al. J Pharm Biomed Anal 118, 251-258). Eisai's EP4 antagonist E7046 also began a Phase I clinical trial in 2015 and a Phase Ib clinical trial in 2017 for combination with radiotherapy or chemoradiotherapy for rectal cancer. Ono Pharmaceutical's ONO-4578 was initiated in a Phase I clinical trial for advanced or metastatic solid tumors in 2017, and in 2018 it was initiated in a Phase I / II clinical trial for the treatment of advanced solid tumors as a monotherapy or in combination with nivolumab.

[0009] Currently, EP4 antagonists have made some progress in the treatment of inflammatory diseases, pain, and cancer, but there is still a need to further develop new drugs to improve or replace current drugs. Summary of the Invention

[0010] The purpose of this invention is to provide a fused-ring compound for use as an EP4 antagonist, its preparation method, and its uses.

[0011] In a first aspect, the present invention provides compounds of Formula I, their tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs:

[0012]

[0013] Among them, ring A is absent or a 7-10 member bridged cycloalkyl or a 6 member oxygen-containing heterocyclic alkyl;

[0014] Furthermore, when ring A is a 6-membered oxygen-containing heterocyclic alkyl group, the functional group fragment... for

[0015] R1 is selected from C1-C6 alkyl groups, -CONH-R 11 -NHCO-R 12 ;R 11 R 12 Each is independently hydrogen or C1-C6 alkyl; R1, R 11 R 12 The C1-C6 alkyl group described herein is optionally surrounded by one or more R fSubstitution; when there are multiple substituents, the R f Same or different;

[0016] R2 is selected from halogen, cyano, C2-C6 alkenyl, C2-C6 alkynyl, -CO-R 21 ;R 21 The amino, hydroxyl, or C1-C6 alkyl group is used; wherein the amino or hydroxyl group is optionally independently substituted with a C1-C6 alkyl group; wherein the C2-C6 alkenyl group, the C2-C6 alkynyl group, and the C1-C6 alkyl group are optionally each independently substituted with one or more R groups. f Substitution; when there are multiple substituents, the R f Same or different;

[0017] R3 is a C1-C6 carboxyl group or a C1-C6 amide group;

[0018] R4 is selected from hydrogen, hydroxyl, halogen, cyano, C1-C4 alkyl or C1-C4 haloalkyl;

[0019] The R mentioned above f It is selected from hydroxyl, halogen or cyano groups.

[0020] In a preferred embodiment of the invention, the compound has the structure shown in Formula II, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs:

[0021]

[0022] In a preferred embodiment of the present invention, ring A is absent or a 7-membered bridged cycloalkyl, or a 6-membered oxygen-containing heterocyclic alkyl, and when ring A is a 6-membered oxygen-containing heterocyclic alkyl, the group fragment... for Preferably, the group for

[0023] When ring A is absent, the group fragment for Preferably, when ring A is absent, the group fragment for

[0024] Preferably, when ring A is a 7-membered bridged cycloalkyl group, the group fragment for More preferably, when ring A is a 7-membered bridged cycloalkyl group, the group fragment for

[0025] In a preferred embodiment of the present invention, R1 is -CONH-R 11 or -NHCO-R 12 Among them, R11 R 12 Each is independently a C1-C6 alkyl group; preferably, R1 is -CONH-R. 11 R 11 It is a C1-C6 alkyl group; preferably, R1 is -CONH-R 11 R 11 The derivatives are methyl, ethyl, propyl, or isopropyl; more preferably, R1 is -CONH-R. 11 R 11 R1 is propyl or isopropyl; preferably, R1 is -CONH-R 11 R 11 It is isopropyl.

[0026] In a preferred embodiment of the present invention, R2 is a halogen or a cyano group; more preferably, R2 is a cyano group.

[0027] In a preferred embodiment of the present invention, R3 is a C1-C6 carboxyl group; more preferably, R3 is butyric acid.

[0028] In a preferred embodiment of the present invention, R4 is selected from hydrogen, hydroxyl, halogen, cyano, methyl or halomethyl; preferably, R4 is hydrogen.

[0029] In a preferred embodiment of the present invention, the compound has any of the following structures, tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs:

[0030]

[0031] In a second aspect, the present invention provides an intermediate B-1, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs:

[0032]

[0033] Among them, ring A is absent or a 7-10 member bridged cycloalkyl or a 6 member oxygen-containing heterocyclic alkyl;

[0034] R1 is selected from C1-C6 alkyl groups, -CONH-R 11 -NHCO-R 12 ;R 11 R 12 Each is independently hydrogen or C1-C6 alkyl; R1, R 11 R 12 The C1-C6 alkyl group described herein is optionally surrounded by one or more R f Substitution; when there are multiple substituents, the R f Same or different;

[0035] R4 is selected from hydrogen, hydroxyl, halogen, cyano, C1-C4 alkyl or C1-C4 haloalkyl; preferably R4 is hydrogen;

[0036] The R mentioned above f It is selected from hydroxyl, halogen or cyano groups.

[0037] In a preferred embodiment of the present invention, when ring A is a 6-membered oxygen-containing heterocyclic alkyl group... for

[0038] In a preferred embodiment of the present invention, ring A is absent or a 7-membered bridged cycloalkyl group or a 6-membered oxygen-containing heterocyclic alkyl group fragment. for R1 is -CONH-R 11 or -NHCO-R 12 , where R 11 R 12 Each is independently a C1-C6 alkyl group, and R4 is hydrogen;

[0039] Preferably, R1 is -CONH-R 11 R 11 It is a C1-C6 alkyl group, and R4 is hydrogen;

[0040] Preferably, R1 is -CONH-R 11 R 11 R4 represents hydrogen; methyl, ethyl, or propyl.

[0041] More preferably, R1 is -CONH-R 11 R 11 R4 is propyl, and R4 is hydrogen.

[0042] Better place, for Preferably, the group for Preferably, the propyl group is isopropyl; preferably, the group fragment... for

[0043] In a preferred embodiment of the present invention, the intermediate B-1 has any of the following structures, tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs:

[0044]

[0045] In a third aspect of the invention, an intermediate B-2, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs are provided:

[0046]

[0047] Among them, ring A is a 7-10 membered bridged cycloalkyl group or a 6 membered oxygen-containing heterocycloalkyl group;

[0048] R1 is selected from C1-C6 alkyl groups, -CONH-R 11 -NHCO-R 12 ;R 11 R 12 Each is independently hydrogen or C1-C6 alkyl; R1, R 11 R 12 The C1-C6 alkyl group described herein is optionally surrounded by one or more R f Substitution; when there are multiple substituents, the R f Same or different;

[0049] R4 is selected from hydrogen, hydroxyl, halogen, cyano, C1-C4 alkyl or C1-C4 haloalkyl; preferably, R4 is hydrogen;

[0050] The R mentioned above f Selected from hydroxyl, halogen, and cyano groups.

[0051] In a preferred embodiment of the present invention, ring A is a 6-membered oxygen-containing heterocyclic alkyl group. for

[0052] In a preferred embodiment of the present invention, R1 is -CONH-R 11 or -NHCO-R 12 Among them, R 11 R 12 Each is independently a C1-C6 alkyl group, and R4 is hydrogen;

[0053] Preferably, the group fragment for R1 is -CONH-R 11 R 11 It is a C1-C6 alkyl group, and R4 is hydrogen;

[0054] Preferably, R1 is -CONH-R 11 R 11 The compounds are methyl, ethyl, propyl, and isopropyl.

[0055] More preferably, R1 is -CONH-R 11 R 11 It is propyl or isopropyl;

[0056] Preferably, the group fragment for Preferably, the group fragment for Preferably, R1 is -CONH-R11 R 11 It is isopropyl.

[0057] In a preferred embodiment of the present invention, a substance having any of the following structures, tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs is included:

[0058]

[0059] In a fourth aspect, the present invention provides a method for preparing the compound as described in the first aspect, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts, or prodrugs, the method comprising the steps of:

[0060] i) Intermediate B-1 reacts with compound B-3 to give the compound shown in Formula I.

[0061] And / or,

[0062] ii) Intermediate B-2 reacts with compound B-4 to remove the protecting group PG1 of the carboxyl group in B-4, yielding intermediate B-1.

[0063]

[0064] The definitions of rings A, R1, R2, and R4 are as described in the first aspect of this invention;

[0065] R3 is a C1-C6 carboxyl group or a C1-C6 amide group.

[0066] In a preferred embodiment of the present invention, step i) is carried out under nitrogen protection; and / or, in the presence of a catalyst; preferably, the catalyst is a rhodium catalyst and a copper catalyst; more preferably, the catalyst is rhodium acetate.

[0067] In a preferred embodiment of the present invention, R3 is a C1-C6 carboxyl group; more preferably, R3 is butyric acid.

[0068] In a preferred embodiment of the present invention, step i) further includes the following steps: i-1) protecting the substituent R3 in compound B-3 with a carboxyl protecting group, and then reacting it with intermediate B-1 to obtain the compound of formula I protected by a carboxyl protecting group; i-2) removing the carboxyl protecting group from the compound of formula I protected by a carboxyl protecting group to obtain the compound of formula I.

[0069] In a preferred embodiment of the present invention, step i) further includes the step: i-3) chiral resolution of the compound represented by formula I.

[0070] In a preferred embodiment of the present invention, PG1 includes C1-C6 alkyl, phenyl-substituted C1-C6 alkyl; preferably C1-C6 alkyl, more preferably methyl, ethyl, propyl, or tert-butyl.

[0071] A fifth aspect of the present invention provides a pharmaceutical composition comprising: a compound, a tautomer, a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt, or a prodrug as described in the first aspect of the present invention; and a pharmaceutically acceptable carrier; or a compound, a tautomer, a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt, or a prodrug as described in the first aspect of the present invention; and at least one other pharmacological activity inhibitor; preferably, the other pharmacological activity inhibitor comprises a CTLA4 antibody, PDL1, and PD1.

[0072] The sixth aspect of the present invention provides the use of compounds of Formula I as described in the first aspect of the present invention, their tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs in the prevention and / or treatment of inflammatory diseases, pain, cancer, metabolic diseases, and urinary system diseases; and / or the use of pharmaceutical compositions as described in the fifth aspect of the present invention, said use including: the use in the preparation of medicaments, pharmaceutical compositions or formulations for the prevention and / or treatment of inflammatory diseases, pain, cancer, metabolic diseases, and urinary system diseases.

[0073] In a preferred embodiment of the present invention, the inflammatory disease is selected from arthritis and rheumatoid arthritis; the pain is selected from osteoarthritis pain and pain caused by endometriosis; the cancer is selected from solid cancer, preferably from breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and urethral cancer; the metabolic disease is diabetes; and the urinary system disease is selected from overactive bladder.

[0074] In a preferred embodiment of the present invention, the drug is administered in combination with radiotherapy and / or antibody therapy, wherein the antibody therapy is selected from one or a combination of CTLA4 antibody therapy, PDL1 antibody therapy and PD1 antibody therapy.

[0075] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

[0076] Terms and Definitions

[0077] Unless otherwise stated, the definitions of groups and terms recorded in this application specification and claims, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, and definitions of specific compounds in the examples, can be arbitrarily combined and combined with each other. Such combinations and combinations of group definitions and compound structures shall fall within the scope of this application specification.

[0078] Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent applications, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0079] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this application, unless specifically stated otherwise, the singular is used to include the plural. It must be noted that unless clearly stated otherwise, the singular form used in this specification and claims includes the plural form of the referred to. It should also be noted that unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0080] Definitions of standard chemical terms can be found in the references (including Carey and Sundberg, "Advanced Organic Chemistry 4th Edition," Vols. A (2000) and B (2001), Plenum Press, New York). Unless otherwise stated, conventional methods within the scope of the art, such as mass spectrometry, NMR, IR, UV / VIS spectroscopy, and pharmacological methods, are used. Unless specifically defined, the terminology used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry is known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. For example, reactions and purifications can be carried out using the manufacturer's instructions for use of kits, or in accordance with methods known in the art or the description of this invention. The techniques and methods described above can generally be carried out according to conventional methods well known in the art, based on the descriptions in the various summary and more specific references cited and discussed in this specification. In this specification, groups and their substituents can be selected by those skilled in the art to provide stable structural moieties and compounds.

[0081] When a substituent is described using a conventional chemical formula written from left to right, that substituent also includes chemically equivalent substituents obtained when the structural formula is written from right to left. For example, CH2O is equivalent to OCH2. As used herein, Indicates the linking site of a functional group.

[0082] The chapter headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter. All references or portions thereof cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are incorporated herein by reference in their entirety.

[0083] Except as otherwise specified, when used in the specification and claims of this application, the following terms shall have the following meanings.

[0084] The numerical ranges described in this application specification and claims, when interpreted as "integers," should be understood to include the two endpoints of the range and every integer within that range. For example, "integers from 1 to 6" should be understood to include every integer of 0, 1, 2, 3, 4, 5, and 6.

[0085] In this application, the term "halogen" refers to fluorine, chlorine, bromine, or iodine, either alone or as part of other substituents.

[0086] As used herein, the term "amino" means -NH2, either alone or as part of other substituents.

[0087] As used herein, the term "nitro" means -NO2, either alone or as part of other substituents.

[0088] As used herein, the term "cyano" means -CN, either alone or as part of other substituents.

[0089] As used herein, the term "alkyl" refers, alone or as part of other substituents, to a straight-chain or branched hydrocarbon chain consisting only of carbon and hydrogen atoms, without unsaturated bonds, having, for example, 1 to 6 carbon atoms, and connected to the rest of the molecule by single bonds. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl. Alkyl groups can be unsubstituted or substituted with one or more suitable substituents. Alkyl groups can also be isotopic isomers of naturally abundant alkyl groups rich in carbon and / or hydrogen isotopes (i.e., deuterium or tritium). 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.

[0090] When used alone or as part of other substituents, the term "C1-C6 alkyl" should be understood to mean a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers. In particular, the group has 1, 2 or 3 carbon atoms (“C1-C3 alkyl”), such as methyl, ethyl, n-propyl or isopropyl.

[0091] When used alone or as part of other substituents, the term "cycloalkyl" or "carbocycloalkyl" refers to a cyclic alkyl group. The terms "mn-membered cycloalkyl" or "C" refer to a cyclic alkyl group. m -C n "Cycloalkyl" should be understood as referring to a saturated, unsaturated, or partially saturated carbon ring having m to n atoms. For example, "3-15 membered cycloalkyl" or "C3-C 15 "Cycloalkyl" refers to a cyclic alkyl group containing 3 to 15, 3 to 9, 3 to 6, or 3 to 5 carbon atoms, and may contain 1 to 4 rings. "5-8 membered cycloalkyl" contains 5-8 carbon atoms. It includes monocyclic, bicyclic, tricyclic, spirocyclic, or bridged rings. Examples of unsubstituted cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl, or bicyclic hydrocarbon groups such as decahydronaphthalene. Cycloalkyl groups may be substituted with one or more substituents. In some embodiments, the cycloalkyl group may be a cycloalkyl group fused with an aryl or heteroaryl group. The term "C3-C6 cycloalkyl" should be understood to indicate a saturated monovalent monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms, including fused or bridged polycyclic systems. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0092] When used alone or as part of other substituents, the term "C2-C6 alkenyl" should be understood to refer to a straight or branched monovalent hydrocarbon group containing one or more double bonds and having, for example, 2, 3, 4, 5, or 6 carbon atoms (i.e., C2-C6 alkenyl), or having 2 or 3 carbon atoms (i.e., C2-C3 alkenyl). It should be understood that when the alkenyl group contains more than one double bond, the double bonds may be separable or conjugated. The alkenyl group is, for example, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, pent-4-enyl, (E)-pent-3-enyl, (Z)-pent-3-enyl, (E)-pent-2-enyl, (Z)-pent-2-enyl, (E)- Pentyl-1-enyl, (Z)-pentyl-1-enyl, hex-5-enyl, (E)-hex-4-enyl, (Z)-hex-4-enyl, (E)-hex-3-enyl, (Z)-hex-3-enyl, (E)-hex-2-enyl, (Z)-hex-2-enyl, (E)-hex-1-enyl, (Z)-hex-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl 2-Methylprop-1-enyl, (E)-1-methylprop-1-enyl, (Z)-1-methylprop-1-enyl, 3-methylbut-3-enyl, 2-methylbut-3-enyl, 1-methylbut-3-enyl, 3-methylbut-2-enyl, (E)-2-methylbut-2-enyl, (Z)-2-methylbut-2-enyl, (E)-1-methylbut-2-enyl, (Z)-1-methyl But-2-enyl, (E)-3-methylbut-1-enyl, (Z)-3-methylbut-1-enyl, (E)-2-methylbut-1-enyl, (Z)-2-methylbut-1-enyl, (E)-1-methylbut-1-enyl, (Z)-1-methylbut-1-enyl, 1,1-dimethylprop-2-enyl, 1-ethylprop-1-enyl, 1-propylvinyl, 1-isopropylvinyl.

[0093] When used alone or as part of other substituents, the term “C2-C6 ynyl” should be understood to mean a straight or branched monovalent hydrocarbon group that contains one or more triple bonds and has, for example, 2, 3, 4, 5 or 6 carbon atoms (i.e., “C2-C6 ynyl”), or has 2 or 3 carbon atoms (“C2-C3 ynyl”). The alkyne group is, for example, ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, but-3-ynyl, pent-1-ynyl, pent-2-ynyl, pent-3-ynyl, pent-4-ynyl, hex-1-ynyl, hex-2-ynyl, hex-3-ynyl, hex-4-ynyl, hex-5-ynyl, 1-methylprop-2-ynyl, 2-methylbut-3-ynyl, 1-methylbut-3-ynyl, 1-methylbut-2-ynyl, 3-methylbut-1-ynyl, 1-ethylprop-2-ynyl, 3-methylpent-4-ynyl, 2-methylpent-4-ynyl -Alynyl, 1-methylpentan-4-ynyl, 2-methylpentan-3-ynyl, 1-methylpentan-3-ynyl, 4-methylpentan-2-ynyl, 1-methylpentan-2-ynyl, 4-methylpentan-1-ynyl, 3-methylpentan-1-ynyl, 2-ethylbutan-3-ynyl, 1-ethylbutan-3-ynyl, 1-ethylbutan-2-ynyl, 1-propylpropan-2-ynyl, 1-isopropylpropan-2-ynyl, 2,2-dimethylbutan-3-ynyl, 1,1-dimethylbutan-3-ynyl, 1,1-dimethylbutan-2-ynyl, or 3,3-dimethylbutan-1-ynyl. In particular, the ynyl group is ethynyl, propan-1-ynyl, or propan-2-ynyl.

[0094] When used alone or as part of other substituents, "haloalkyl" refers to a saturated aliphatic hydrocarbon group (such as -CvFw, where v = 1 to 3 and w = 1 to (2v+1)) that comprises a specific number of carbon atoms and is branched and straight-chained and substituted with one or more halogens. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl.

[0095] The term "7-10 membered bridged ring" refers to a cyclic hydrocarbon containing 7-10 carbon atoms, where any two rings share two non-directly connected carbon atoms. Based on the number of rings, they are classified as bicyclic hydrocarbons, tricyclic hydrocarbons, tetracyclic hydrocarbons, etc. Non-limiting examples include:

[0096]

[0097] When used alone or as part of other substituents, the term "ester group" refers to an R-CO-O- or -CO-OR group, where R is an alkyl group and the alkyl group is as defined above herein. For example, "C2-C6 ester group" refers to a group with a C1-C5 alkyl-CO-O- structure or a group with a -CO-O-C1-C5 alkyl structure. Representative examples of ester groups include (but are not limited to): CH3COO-, C2H5COO-, C3H8COO-, (CH3)2CHCOO-, -COOCH3, -COOC2H5, -COOC3H8, or similar groups.

[0098] When used alone or as part of other substituents, the term "amide group" refers to a group having a -CO-NH- or -CO-NH-R group, where R is an alkyl group, and the alkyl group is as defined above herein, for example, "C1-C4 amide group" is a group with a C1-C3 alkyl-CO-NH- structure or a group with a -CO-NH-C1-C3 alkyl structure or -CO-NH2. Representative examples of ester groups include (but are not limited to): CH3CO-NH-, C2H5-CO-NH-, C3H8-CO-NH-, (CH3)2-CO-NH-, -CO-NH-CH3, -CO-NHC2H5, -CO-NH-C3H8, or similar groups.

[0099] The term "inert solvent" includes, but is not limited to: toluene, benzene, water, methanol, ethanol, isopropanol, ethylene glycol, N-methylpyrrolidone, dimethyl sulfoxide, tetrahydrofuran dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dioxane, or combinations thereof.

[0100] The compounds described herein include intermediates that can be used to prepare the compounds described herein, containing reactive functional groups (e.g., but not limited to carboxyl, hydroxyl, and amino moieties), and also their protected derivatives. A “protected derivative” is a compound in which one or more reactive sites are blocked by one or more protecting groups (also called protecting groups). Suitable carboxyl moieties include benzyl, tert-butyl, etc., and isotopes, etc. Suitable amino and amide protecting groups include acetyl, trifluoroacetyl, tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable hydroxyl protecting groups include benzyl, etc. Other suitable protecting groups are well known to those skilled in the art.

[0101] In this application, "optional," "optionally," "preferably," or "more preferably" means that the event or condition described below may or may not occur, and the description includes both the occurrence and non-occurrence of the event or condition. For example, "optionally substituted aryl" means that the aryl group is substituted or not substituted, and the description includes both substituted and unsubstituted aryl groups.

[0102] In this 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 compounds, materials, compositions, and / or dosage forms that, to the extent of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0103] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the bioavailability 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 bioavailability of the free acid without other side effects. In addition to pharmaceutically acceptable salts, other salts are also considered in this invention. They can serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts, or can be used for the identification, characterization, or purification of the compounds of this invention.

[0104] The term "amine salt" refers to the product obtained by neutralizing an alkyl primary amine, secondary amine, or tertiary amine with an acid. The acid includes the inorganic or organic acids described in this application.

[0105] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, non-corresponding isomers, and conformational isomers.

[0106] Depending on the choice of raw materials and methods, the compounds of the present invention can exist as one or a mixture of possible isomers, for example as purely optical isomers, or as mixtures of isomers, such as racemic and diastereomeric mixtures, 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 centers (or multiple chiral centers) in the molecule. The prefixes D and L or (+) and (–) are symbols used to specify the plane-polarized rotation induced by the compound, where (–) or L indicates that the compound is levorotatory. Compounds with the prefix (+) or D are dextrorotatory.

[0107] When the bonds of the chiral carbon in the formulas of this invention are depicted as straight lines, it should be understood that both the (R) and (S) configurations of the chiral carbon and the resulting enantiomerically pure compounds and mixtures thereof are included within the scope of the general formula. The illustration of racemic or enantiomerically pure compounds in this document is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. The absolute configuration of a stereocenter is represented by wedge-shaped and dashed bonds.

[0108] The term "tautomer" refers to a functional group isomer resulting from the rapid movement of an atom between two positions within a molecule. The compounds of this invention can exhibit tautomerism. Tautomers can exist in two or more interconvertible forms. Proton-transfer tautomers arise from the migration of covalently bonded hydrogen atoms between two atoms. Tautomers generally exist in equilibrium form; attempts to isolate a single tautomer typically yield a mixture whose physicochemical properties are consistent with those of the mixture of compounds. The equilibrium position depends on the intramolecular chemical characteristics. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone form is dominant; while in phenols, the enol form is dominant. This invention encompasses all tautomeric forms of the compounds.

[0109] In this application, "pharmaceutical composition" refers to a formulation of the compounds of the present invention with a medium generally accepted in the art for delivering bioactive compounds to mammals (e.g., humans). This medium includes pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate administration to the organism, thereby promoting the absorption of the active ingredient and the exertion of its bioactivity.

[0110] In this application, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, flow aid, sweetener, diluent, preservative, dye / coloring agent, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that is permitted by the relevant government regulatory authority to be acceptable for human or animal use.

[0111] The term "solvent" refers to a compound of the present invention or a salt thereof comprising a stoichiometric or nonstoichiometric solvent bound by intermolecular noncovalent forces, and a hydrate when the solvent is water.

[0112] The term "prodrug" refers to a compound of the present invention that can be converted into a biologically active form under physiological conditions or by solvation. The prodrugs of the present invention are prepared by modifying functional groups in the compound; this modification can be performed conventionally or removed in vivo to obtain the parent compound. Prodrugs comprise compounds formed by attaching a hydroxyl or amino group to any group within the compound of the present invention. When a prodrug of the compound of the present invention is administered to a mammalian individual, the prodrug is cleaved to form a free hydroxyl group and a free amino group.

[0113] The compounds of this invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, the compounds may be labeled with radioactive isotopes, such as deuterium. 2 H), tritium ( 3 H), Iodine-125 125 I) or C-14 14C). All isotopic variations of the compounds of the present invention, regardless of radioactivity, are included within the scope of the present invention.

[0114] The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of the term "excipient" include, without limitation, binders, disintegrants, lubricants, flow aids, stabilizers, fillers, and diluents. Excipients enhance the handling properties of pharmaceutical formulations, i.e., by increasing flowability and / or adhesion, making the formulation more suitable for direct compression.

[0115] The term "treatment" and other similar synonyms used in this article include the following meanings:

[0116] (i) To prevent the occurrence of disease or condition in mammals, especially when such mammals are susceptible to the disease or condition but have not yet been diagnosed with it;

[0117] (ii) To suppress a disease or symptom, that is, to curb its development;

[0118] (iii) To alleviate a disease or symptom, that is, to cause the condition of the disease or symptom to subside; or

[0119] (iv) To alleviate the symptoms caused by the disease or condition.

[0120] The reaction temperature and time for each step can be selected appropriately based on the solvent, starting materials, and reagents. After each reaction step is completed, the target compound can be separated and purified from the reaction system using common methods such as filtration, extraction, recrystallization, washing, and silica gel column chromatography. Alternatively, the target compound can proceed directly to the next reaction step without separation or purification, provided it does not affect the subsequent reaction.

[0121] Beneficial effects

[0122] Through extensive and in-depth research, the inventors unexpectedly developed a fused-ring compound as an EP4 antagonist. This compound exhibits effective antagonism against EP4 and good affinity for the EP4 receptor. Furthermore, this invention provides a fused-ring compound as an EP4 antagonist, an intermediate for preparing this compound, and a method for its preparation. The method is simple to operate, has high yield and high purity, and can be used for industrial pharmaceutical production. Detailed Implementation

[0123] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that the following description is merely the most preferred embodiment of the present invention and should not be considered as a limitation on the scope of protection of the present invention. Based on a full understanding of the present invention, experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Those skilled in the art can make non-essential modifications to the technical solutions of the present invention, and such modifications should be considered to be included within the scope of protection of the present invention.

[0124] The compounds of Formula I described in this invention can be prepared by the synthetic methods described below, wherein the substituents of the general formula have the meanings given above. These methods are intended to illustrate the invention and not to limit its subject matter or the scope of the compounds claimed for these embodiments. The present invention describes a fused-ring compound as an EP4 antagonist, its preparation method, and its use, the method comprising any of the steps in the following synthetic routes:

[0125]

[0126] When ring A is absent, intermediate B-1 has a structure It is prepared via the following route:

[0127]

[0128] Intermediate B-6 is reacted with trimethylsulfonium iodide and sodium hydrogen to obtain intermediate B-5, which is then deprotected by the carboxyl protecting group PG2 to obtain intermediate B-1. The carboxyl protecting group PG2 includes C1-C6 alkyl groups and phenyl-substituted C1-C6 alkyl groups; preferably C1-C6 alkyl groups, and more preferably methyl, ethyl, propyl, or tert-butyl groups.

[0129] In the above formulas, R3 is a C1-C6 carboxyl group or a C1-C6 amide group; preferably, R3 is a C1-C6 carboxyl group; more preferably, R3 is butyric acid. The method further includes protecting R3 with a carboxyl protecting group, then reacting it with intermediate B-1 to obtain the compound of formula I protected with a carboxyl protecting group, and removing the carboxyl protecting group from the compound of formula I to obtain the compound of formula I. The carboxyl protecting group includes C1-C6 alkyl groups and phenyl-substituted C1-C6 alkyl groups; preferably C1-C6 alkyl groups, more preferably methyl, ethyl, propyl, or tert-butyl groups.

[0130] Step i) is optionally carried out under nitrogen protection; and / or in the presence of a catalyst; wherein the preferred catalysts are rhodium and copper catalysts, and the more preferred catalyst is rhodium acetate.

[0131] Depending on the different groups in the compound of Formula I, different synthetic routes and intermediates can be selected. When there are active groups (e.g., carboxyl, amino, hydroxyl, etc.) in the substituents, the active groups can be protected by protecting groups as needed before participating in the reaction. After the reaction is complete, the protecting groups are removed. Compounds in which one or more reactive sites are blocked by one or more protecting groups (also called protecting groups) are "protected derivatives" of the compound of Formula I described in this invention. For example, suitable carboxyl protecting groups include benzyl, tert-butyl, methyl, ethyl, etc. Suitable amino and amide protecting groups include acetyl, trifluoroacetyl, tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable hydroxyl protecting groups include benzyl, etc. Other suitable protecting groups are well known to those skilled in the art.

[0132] Preferably, the reaction requires protection by an inert gas, which includes, but is not limited to, nitrogen, helium, neon, and argon.

[0133] The reactions of this invention are preferably carried out in an inert solvent, which includes, but is not limited to: toluene, benzene, water, methanol, ethanol, isopropanol, ethylene glycol, N-methylpyrrolidone, dimethyl sulfoxide, tetrahydrofuran dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dioxane, or combinations thereof.

[0134] The abbreviations are defined as follows:

[0135] Symbols or units:

[0136] IC 50 The half-maximum inhibitory concentration (MCC) refers to the concentration at which half of the maximum inhibitory effect is achieved.

[0137] M: mol / L, for example, n-butyllithium (14.56 mL, 29.1 mmol, 2.5 M n-hexane solution) means a n-butyllithium n-hexane solution with a molar concentration of 2.5 mol / L.

[0138] N: Equivalent concentration, for example, 2N hydrochloric acid represents a 2 mol / L hydrochloric acid solution.

[0139] Reagents:

[0140] DCM: Dichloromethane

[0141] DIPEA: Also written as DIEA, diisopropylethylamine, i.e., N,N-diisopropylethylamine.

[0142] DMF: N,N-Dimethylformamide

[0143] DMSO: Dimethyl sulfoxide

[0144] EA: Ethyl acetate

[0145] Et3N: Triethylamine

[0146] MeOH: Methanol

[0147] PE: Petroleum ether

[0148] THF: Tetrahydrofuran

[0149] Test or detection methods:

[0150] HPLC: High Performance Liquid Chromatography

[0151] SFC: Supercritical Fluid Chromatography

[0152] Example 1: Synthesis of Compound I-1

[0153] The synthesis route is as follows:

[0154]

[0155] Step 1: Synthesis of 3-bromo-4-(hydroxymethyl)benzoic acid (B1-2)

[0156]

[0157] Methyl 3-bromo-4-(hydroxymethyl)benzoate (B1-1) (1.0 g, 4.08 mmol) was added to tetrahydrofuran (20 mL) and water (20 mL) at room temperature, followed by the addition of sodium hydroxide (0.5 g, 12.5 mmol). The mixture was stirred for 12 h. The pH was adjusted to 3 with 1 N hydrochloric acid, and the mixture was extracted with ethyl acetate (20 mL × 3). The liquid and liquid phases were separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give 3-bromo-4-(hydroxymethyl)benzoic acid (white crude solid, 1.0 g, 100% yield).

[0158] Step 2: Synthesis of 3-bromo-4-(hydroxymethyl)-N-isopropylbenzamide (B1-3)

[0159]

[0160] 3-Bromo-4-(hydroxymethyl)benzoic acid (1.0 g, 4.08 mmol) was added to dichloromethane (30 mL) at room temperature, followed by isopropylamine (300 mg, 5.08 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.6 g, 4.9 mmol), and N,N-diisopropylethylamine (1.05 g, 8.1 mmol). The mixture was stirred at room temperature for 16 h. The solution was diluted with water (50 mL), extracted with dichloromethane (30 mL × 3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 4:1) to give 3-bromo-4-(hydroxymethyl)-N-isopropylbenzamide (colorless liquid, crude product, 0.8 g, yield 72%). LC-MS, M / Z (ESI): 272.2 [M+H] + .

[0161] Step 3: Synthesis of 4-((allyloxy)methyl)-3-bromo-4-(hydroxymethyl)-N-isopropylbenzamide (B1-4)

[0162]

[0163] Compound 3-bromo-4-(hydroxymethyl)-N-isopropylbenzamide (800 mg, 2.94 mmol) was added to tetrahydrofuran (20 mL) at room temperature, followed by the addition of 60% sodium hydroxide (488 mg, 12.2 mmol). The mixture was stirred at room temperature for 0.5 h, then allyl bromide (532 mg, 4.4 mmol) was added, and the mixture was stirred at room temperature for 3 h. The mixture was diluted with water (30 mL), extracted with ethyl acetate (20 mL × 3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 4-((allyloxy)methyl)-3-bromo-4-(hydroxymethyl)-N-isopropylbenzamide (white crude solid, 1.0 g, 100% yield). LC-MS, M / Z (ESI): 312.2 [M+H] + .

[0164] Step 4: Synthesis of N-isopropyl-4-methyleneisosperane-6-carboxamide (B1-5)

[0165]

[0166] Compound 4-((allyloxy)methyl)-3-bromo-4-(hydroxymethyl)-N-isopropylbenzamide (1.0 g, 3.2 mmol) was added to N,N-dimethylacetamide (10 mL) at room temperature, followed by cesium carbonate (2.0 g, 6.4 mmol), tetrakis(triphenylphosphine)palladium (7.39 mg, 0.64 mmol), and tetrabutylammonium iodide (1.18 g, 3.2 mmol). The mixture was heated to 100 °C under nitrogen protection and stirred for 16 h. After cooling to room temperature, the mixture was diluted with water (50 mL), extracted with ethyl acetate (30 mL × 3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 10:1) to give compound N-isopropyl-4-methyleneisocyanane-6-carboxamide (white solid crude product, 350 mg, yield 47%). LC-MS, M / Z (ESI): 232.3 [M+H] + .

[0167] Step 5: Synthesis of ethyl 6'-(isopropylformamide)spirocyclic [cyclopropane-1,4'-isochromane]-2-carboxylate (B1-6)

[0168]

[0169] At room temperature, N-isopropyl-4-methyleneisocyanane-6-carboxamide (300 mg, 1.3 mmol) was added to dichloromethane (20 mL). Under nitrogen protection, rhodium acetate (265 mg, 0.6 mmol) was added, and the mixture was heated to 50 °C. Diazonyl ethyl acetate (0.5 mL, 6.0 mmol) was then added, and the mixture was stirred for 0.5 h. After cooling to room temperature, the mixture was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 5:1) to give compound 6'-(isopropylformamide)spirocyclic [cyclopropane-1,4'-isocyanane]-2-carboxylate (white solid, crude product, 80 mg, yield 20%). LC-MS, M / Z (ESI): 318.2 [M+H] + .

[0170] Step 6: Synthesis of 6'-(isopropylformamide)spirocyclic [cyclopropane-1,4'-isochromane]-2-carboxylic acid (B1-7)

[0171]

[0172] Ethyl 6'-(isopropylformamide)spirocyclic [cyclopropane-1,4'-isochromane]-2-carboxylic acid (38 mg, 0.12 mmol) was added to tetrahydrofuran (5 mL) and water (5 mL) at room temperature, followed by the addition of lithium hydroxide (15 mg, 0.36 mmol). The mixture was stirred at room temperature for 16 h. The pH was adjusted to 3 with 1 N hydrochloric acid, diluted with water (10 mL), and extracted with ethyl acetate (10 mL × 3). The liquid and liquid phases were separated, combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give 6'-(isopropylformamide)spirocyclic [cyclopropane-1,4'-isochromane]-2-carboxylic acid (white crude solid, 40 mg, 100% yield). LC-MS, M / Z (ESI): 290.3 [M+H] + .

[0173] Step 7: Synthesis of ethyl 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)spirocyclic[cyclopropane-1,4'-isocyanane]-2-carboxamide)phenyl)butyrate (B1-8)

[0174]

[0175] Compound 6'-(isopropylformamide)spirocyclic[cyclopropane-1,4'-isochroman]-2-carboxylic acid (28 mg, 0.096 mmol) was added to 5 mL of dichloromethane at room temperature, followed by pyridine (0.25 mL) and ethyl 4-(2-amino-4-cyanophenyl)butyrate (23 mg, 0.10 mmol). The mixture was cooled to 0 °C, and 1 drop of phosphorus oxychloride was added. The mixture was stirred at room temperature for 16 h. The solution was diluted with 30 mL of water, extracted with ethyl acetate (50 mL × 3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give ethyl 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)spirocyclic[cyclopropane-1,4'-isochroman]-2-carboxamide)phenyl)butyrate (white solid, 22 mg, yield 45%). LC-MS, M / Z (ESI): 504.3 [M+H] + .

[0176] Step 8: Synthesis of 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)spirocyclic[cyclopropane-1,4'-isocyanane]-2-carboxamide)phenyl)butyric acid (I-1)

[0177]

[0178] Ethyl 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)spiro[cyclopropane-1,4'-isocyanane]-2-carboxamide)phenyl)butyrate (22 mg, 0.43 mmol) was added to tetrahydrofuran (5 mL) and water (5 mL) at room temperature, along with lithium hydroxide (8.9 mg, 0.21 mmol). The mixture was stirred at room temperature for 4 h. The pH was adjusted to 3 with 1N hydrochloric acid, and the mixture was concentrated. The residue was then subjected to acidic preparation method A (Acidic preparation method A: Welch, Ultimate C18 column, 10μm, 21.2mm×250mm. Mobile phase A was a 1‰ trifluoroacetic acid aqueous solution, and mobile phase B was an acetonitrile solution. Gradient conditions: 0–3 min, mobile phase A maintained at 90%, 3–18 min gradient elution, from 90% to 5%, 18–22 min maintained at 5%) to prepare compound 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)spirocyclic[cyclopropane-1,4'-isocyanane]-2-carboxamide)phenyl)butyric acid (white solid, 4.5 mg, yield 22%).

[0179] 1 H NMR (400mHz, DMSO-d6) δ10.33(s,1H),8.40(t,1H),8.96(s,1H),7.67(q,1H),7.56(q,1H),7.45(t,2H),7.15(d,1H),4.83( d,2H),4.12-4.04(m,2H),3.92(d,1H),2.70-2.64(m,3H),2.54(s,1H),2.16(t,2H),1.71(t,3H),1.56(t,1H),1.19(d,6H).

[0180] LC-MS, M / Z (ESI): 476.3 [M+H] + .

[0181] Example 2: Synthesis of Compound I-2

[0182] The synthesis route is as follows:

[0183]

[0184] Step 1: Synthesis of 6-bromo-4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthalene (B2-2)

[0185]

[0186] 6-Bromo-2,3-dihydro-1,3-mannaphthyl-4(1H)-one (1.48 g, 6.25 mmol) was added to tetrahydrofuran (30 mL) at room temperature, followed by μ-chloro-μ-methylene[di(cyclopentadienyl)titanium]-dimethylaluminum (3.56 g, 12.5 mmol), and stirred for 12 h. The mixture was diluted with water (50 mL), extracted with ethyl acetate (30 mL × 3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give 6-bromo-4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl (B2-2) (white crude solid, 850 mg, yield 58%).

[0187] Step 2: Synthesis of ethyl 4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl-6-carboxylate (B2-3)

[0188]

[0189] Compound 6-bromo-4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthalene (910 mg, 3.89 mmol) was added to ethanol (50 mL) at room temperature, followed by triethylamine (1.18 g, 11.7 mmol) and 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (477 mg, 0.58 mmol). Carbon monoxide was bubbled through the mixture, and the mixture was heated to 80 °C and stirred for 16 h. After cooling to room temperature, the mixture was diluted with water (100 mL), extracted with ethyl acetate (50 mL × 3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give compound ethyl 4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthalene-6-carboxylate (B2-3) (white crude solid, 870 mg, yield 98%). LC-MS, M / Z (ESI): 229.2 [M+H] + .

[0190] Step 3: Synthesis of 4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl-6-carboxylic acid (B2-4)

[0191]

[0192] Ethyl 4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl-6-carboxylic acid (705 mg, 3.1 mmol) was added to tetrahydrofuran (8 mL) and water (8 mL) at room temperature. Lithium hydroxide (650 mg, 15.5 mmol) was added, and the mixture was stirred at room temperature for 16 h. The pH was adjusted to 3 with 1 N hydrochloric acid, diluted with water (30 mL), and extracted with ethyl acetate (20 mL × 3). The liquid and liquid phases were separated, and the organic phases were dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give compound 4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl-6-carboxylic acid (B2-4) (white solid, crude product, 620 mg, 100% yield). LC-MS, M / Z (ESI): 201.2 [M+H] + .

[0193] Step 4: Synthesis of N-isopropyl-4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl-6-carboxamide (B2-5)

[0194]

[0195] Compound 4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl-6-carboxylic acid (620 mg, 3.1 mmol) was added to DMF (20 mL) at room temperature, along with isopropylamine (201 mg, 3.4 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.53 g, 4.0 mmol), and N,N-diisopropylethylamine (1.2 g, 9.3 mmol). The mixture was stirred at room temperature for 16 h. Dilute with water (50 mL), extract with EA (30 mL × 3), separate, combine organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give compound N-isopropyl-4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl-6-carboxamide (B2-5) (white crude solid, 750 mg, 100% yield). LC-MS, M / Z (ESI): 242.2 [M+H] + .

[0196] Step 5: Synthesis of ethyl 6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic [cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxylate (B2-6)

[0197]

[0198] At room temperature, N-isopropyl-4-methylene-1,2,3,4-tetrahydro-1,3-mannaphthyl-6-carboxamide (700 mg, 2.9 mmol) was added to dichloromethane (10 mL), followed by ethyl diazonium (1.65 g, 14.5 mmol) and rhodium acetate (257 mg, 0.58 mmol). The mixture was heated to 40 °C and stirred for 16 h. Cool to room temperature, dilute with water (50 mL), extract with DCM (30 mL × 3), separate, combine organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give compound 6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic [cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxylic acid ethyl ester (B2-6) (white solid crude product, 560 mg, yield 59%). LC-MS, M / Z (ESI): 328.2 [M+H] + .

[0199] Step 6: Synthesis of 6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic [cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxylic acid (B2-7)

[0200]

[0201] Compound 6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic [cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxylic acid ethyl ester (560 mg, 1.71 mmol) was added to tetrahydrofuran (6 mL) and water (6 mL) at room temperature, along with lithium hydroxide (360 mg, 8.56 mmol), and stirred at room temperature for 16 h. The pH was adjusted to 3 with 1N hydrochloric acid, diluted with water (30 mL), and extracted with ethyl acetate (20 mL × 3). The liquid and liquid phases were separated, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 1:1) to give compound 6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic [cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxylic acid (B2-7) (white crude solid, 405 mg, yield 79%). LC-MS, M / Z (ESI): 300.2 [M+H] + .

[0202] Step 7: Synthesis of ethyl 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic[cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxamide)phenyl)butyrate (B2-8)

[0203]

[0204] Compound 6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic [cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxylic acid (322 mg, 1.1 mmol) was added to pyridine (10 mL) at room temperature, followed by ethyl 4-(2-amino-4-cyanophenyl)butyrate (255 mg, 1.1 mmol). The mixture was cooled to 0 °C, and phosphorus oxychloride (843 mg, 5.5 mmol) was added. The mixture was stirred at room temperature for 16 h. Dilute with water (30 mL), extract with ethyl acetate (10 mL × 3), separate, combine organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 2:1) to give compound ethyl 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclopropane-1,4'-[1,3]mannaphthyl]-2-carboxamide)phenyl)butyrate (B2-8) (white solid, 300 mg, yield 54%). LC-MS, M / Z (ESI): 514.3 [M+H] + .

[0205] Step 8: Synthesis of 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic[cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxamide)phenyl)butyric acid (I-2)

[0206]

[0207] Ethyl 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclo[cyclopropane-1,4'-[1,3]naphthyl]-2-carboxamide)phenyl)butyrate (300 mg, 0.58 mmol) was added to tetrahydrofuran (6 mL) and water (6 mL) at room temperature, along with lithium hydroxide (50 mg, 1.2 mmol). The mixture was stirred at room temperature for 16 h. Dilute with water (30 mL), extract with ethyl acetate (20 mL × 3), separate, combine organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify the residue by silica gel column chromatography (petroleum ether: ethyl acetate (V / V) = 2:1) to give compound 4-(4-cyano-2-((1S,2R)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxamide)phenyl)butyric acid (I-2) (white solid, 120 mg, yield 42%).

[0208] 1H NMR(400mHz,DMSO-d6)δ12.09(s,1H),9.59(s,1H),7.73(t,3H),7.52-7.48(m,2H),7.32(d,1H),7.08(d,1H),4.08-3.9 9(m,1H),3.27(t,1H),2.46(t,4H),2.24(t,1H),2.07(t,1H),1.99-1.86(m,4H),1.48-1.39(m,4H),1.12-1.06(m,6H). LC-MS,M / Z(ESI):486.3[M+H] + .

[0209] Example 3: Synthesis of Compound I-3

[0210] The synthesis route is as follows:

[0211]

[0212] Step 1: Synthesis of (E)-3-(3-(tert-butoxy)-3-oxopropane-1-en-1-yl)methyl benzoate (B3-2)

[0213]

[0214] 2.5 g (23.4 mmol) of methyl 3-bromobenzoate was added to toluene (30 mL) at room temperature, followed by tert-butyl acrylate (4.5 g, 35.1 mmol), palladium dichloride dichloride (0.4 g, 0.58 mmol), and triethylamine (4.8 mL, 35 mmol). The mixture was heated to 100 °C under nitrogen protection and stirred for 16 h. After cooling to room temperature, 30 mL of water was added, and the mixture was extracted with ethyl acetate (30 mL × 3). The liquid and liquid phases were separated, and the organic phases were dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and the residue was purified by silica gel column chromatography (pure petroleum ether) to give compound (E)-3-(3-(tert-butoxy)-3-oxopropane-1-en-1-yl)benzoate (B3-2) (white crude solid, 1.3 g, yield 43%).

[0215] Step 2: Synthesis of methyl 3-(2-(tert-Butoxycarbonyl)cyclopropyl)benzoate (B3-3)

[0216]

[0217] Methyl (E)-3-(3-(tert-butoxy)-3-oxopropane-1-en-1-yl)benzoate (2.3 g, 8.8 mmol) was added to dimethyl sulfoxide (50 mL) and tetrahydrofuran (50 mL) at room temperature, followed by trimethylsulfonium iodide (3.9 g, 17.6 mmol) and 60% sodium hydroxide (352 mg, 8.8 mmol). The mixture was stirred at room temperature for 16 h. The solution was diluted with water (100 mL), extracted with ethyl acetate (200 mL × 3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 20:1) to give methyl 3-(-2-(tert-butoxycarbonyl)cyclopropyl)benzoate (B3-3) (colorless liquid crude product, 643 mg, yield 27%). LC-MS, M / Z (ESI): 277.2 [M+H] + .

[0218] Step 3: Synthesis of 3-(2-(tert-Butoxycarbonyl)cyclopropyl)benzoic acid (B3-4)

[0219]

[0220] Methyl 3-(2-(tert-Butoxycarbonyl)cyclopropyl)benzoate (640 mg, 2.3 mmol) was added to tetrahydrofuran (10 mL) and water (3 mL) at room temperature, followed by lithium hydroxide (290 mg, 6.9 mmol). The mixture was stirred at room temperature for 3 h. The pH was adjusted to 7 with 1 N hydrochloric acid, diluted with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The liquid and liquid phases were separated, combined, dried over anhydrous sodium sulfate, filtered, and concentrated to give a white crude solid of 3-(2-(tert-Butoxycarbonyl)cyclopropyl)benzoic acid (B3-4) (white crude solid, 565 mg, yield 93%). LC-MS, M / Z (ESI): 263.2 [M+H] + .

[0221] Step 4: Synthesis of tert-butyl 2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxylate (B3-5)

[0222]

[0223] Compound 3-(2-(tert-butoxycarbonyl)cyclopropyl)benzoic acid (565 mg, 2.16 mmol) was added to dichloromethane (10 mL) at room temperature, along with N,N-diisopropylethylamine (413 mg, 3.2 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (1.2 g, 3.2 mmol), and isopropylamine (153 mg, 2.6 mmol). The mixture was stirred at room temperature for 2 h. Dilute with water (10 mL), extract with dichloromethane (10 mL × 3), separate, combine organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 3:1) to give compound 2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxylic acid tert-butyl ester (B3-5) (white solid, 535 mg, yield 82%). LC-MS, M / Z (ESI): 304.3 [M+H] + .

[0224] Step 5: Synthesis of 2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxylic acid (B3-6)

[0225]

[0226] At room temperature, tert-butyl 2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxylate (535 mg, 1.7 mmol) was added to dichloromethane (20 mL), followed by trifluoroacetic acid (4 mL), and the mixture was stirred for 2 h. The mixture was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate (V / V) = 10:1) to give compound 2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxylic acid (B3-6) (white crude solid, 400 mg, yield 92%). LC-MS, M / Z (ESI): 248.2 [M+H] + .

[0227] Step 6: Synthesis of ethyl 4-(4-cyano-2-((1R,2R)-2-(3-(isopropylformamide)phenyl)cyclopropane-1-formamide)phenyl)butyrate (B3-7)

[0228]

[0229] Compound 2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxylic acid (100 mg, 0.4 mmol) was added to pyridine (3 mL) at room temperature, followed by ethyl 4-(2-amino-4-cyanophenyl)butyrate (93 mg, 0.4 mmol). The mixture was cooled to 0 °C, and phosphorus oxychloride (367 mg, 2.40 mmol) was added. The mixture was stirred at low temperature for 2 h. The mixture was diluted with water (30 mL), extracted with ethyl acetate (50 mL × 3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 3:2) to give compound ethyl 4-(4-cyano-2-((1R,2R)-2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxamide)phenyl)butyrate (B3-7) (white solid, 130 mg, yield 70%). LC-MS, M / Z (ESI): 462.6 [M+H] + .

[0230] Step 7: Synthesis of 4-(4-cyano-2-((1R,2R)-2-(3-(isopropylformamide)phenyl)cyclopropane-1-formamide)phenyl)butyric acid (I-3)

[0231]

[0232] Ethyl 4-(4-cyano-2-((1R,2R)-2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxamide)phenyl)butyrate (130 mg, 0.28 mmol) was added to tetrahydrofuran (5 mL) and water (3 mL) at room temperature, followed by the addition of lithium hydroxide (35 mg, 0.84 mmol). The mixture was stirred at room temperature for 16 h. The pH was adjusted to 7 with 1 N hydrochloric acid, and the mixture was extracted with ethyl acetate (10 mL × 3). The liquid and liquid phases were separated, and the organic phases were dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and the residue was purified by silica gel column chromatography (dichloromethane:methanol (V / V) = 20:1) to give compound 4-(4-cyano-2-((1R,2R)-2-(3-(isopropylformamide)phenyl)cyclopropane-1-carboxamide)phenyl)butyrate (I-3) (white solid, 6.7 mg, yield 5.4%).

[0233] 1H NMR(400mHz,DMSO-d6)δ12.14(s,1H),9.83(s,1H),8.21(d,1H),8.04(s,1H),7.68(d,1H),7.67(s,1H),7.57(t,1H),7.43(dd,2H),4 .14-4.06(m,2H),2.78-2.62(m,2H),2.49-2.38(m,1H),2.34(s,1H),2.24-2.20(m,2H),1.76(t,2H),1.52-1.46(m,2H),1.18(d,6H).

[0234] LC-MS, M / Z (ESI): 434.6 [M+H] + .

[0235] Example 4: Synthesis of Compound I-4

[0236] The synthesis route is as follows:

[0237]

[0238] Step 1: Synthesis of ethyl 4-(4-cyano-2-((1S,2S)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic[cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxamide)phenyl)butyrate (B4-1)

[0239]

[0240] Compound 6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic [cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxylic acid (B2-7) (322 mg, 1.1 mmol) was added to pyridine (10 mL) at room temperature, followed by ethyl 4-(2-amino-4-cyanophenyl)butyrate (255 mg, 1.1 mmol). The mixture was cooled to 0 °C, and phosphorus oxychloride (843 mg, 5.5 mmol) was added. The mixture was stirred at room temperature for 16 h. Dilute with water (30 mL), extract with ethyl acetate (10 mL × 3), separate, combine organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify the residue by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 2:1) to give compound ethyl 4-(4-cyano-2-((1S,2S)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxamide)phenyl)butyrate (4-1) (white solid, 240 mg, yield 43%). LC-MS, M / Z (ESI): 514.3 [M+H] + .

[0241] Step 2: Synthesis of 4-(4-cyano-2-((1S,2S)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclic[cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxamide)phenyl)butyric acid (I-4)

[0242]

[0243] Ethyl 4-(4-cyano-2-((1S,2S)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spirocyclo[cyclopropane-1,4'-[1,3]naphthyl]-2-carboxamide)phenyl)butyrate (200 mg, 0.39 mmol) was added to tetrahydrofuran (4 mL) and water (4 mL) at room temperature, along with lithium hydroxide (33 mg, 0.78 mmol). The mixture was stirred at room temperature for 16 h. Dilute with water (20 mL), extract with ethyl acetate (10 mL × 3), separate, combine organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and purify the residue by silica gel column chromatography (petroleum ether: ethyl acetate (V / V) = 2:1) to give compound 4-(4-cyano-2-((1S,2S)-6'-(isopropylformamide)-2',3'-dihydro-1'H-spiro[cyclopropane-1,4'-[1,3]mannaphthalene]-2-carboxamide)phenyl)butyric acid (I-4) (white solid, 150 mg, yield 79%).

[0244] 1 H NMR (400mHz, DMSO-d6) δ12.07(s,1H),9.87(s,1H),8.13(d,1H),7.77(d,1H),7.60-7.55(m,2H),7.42(d,2H),7.13(d,1H),4.14-4.09( m,1H),3.29-3.24(m,2H),2.63-2.57(m,4H),2.42(t,1H),2.18(t,2H),1.72(t,2H),1.68-1.58(m,2H),1.52(t,2H),1.18-1.10(m,6H).

[0245] LC-MS, M / Z (ESI): 486.3 [M+H] + .

[0246] Test Example 1: EP4 Antagonism Determination Experiment

[0247] The antagonistic effect of the compounds on EP4 was determined in a stable CHO cell line highly expressing the human EP4 receptor. Cells were trypsinized and resuspended in buffer (1×HBSS, 0.1% BSA, 20 mM HEPES, and 500 μM IBMX), and 8000 cells were seeded per well in a 384-well plate at a seed volume of 15 μL. An 8X working solution of the compound was prepared using experimental buffer, and 2.5 μL of this solution was added to each well of the 384-well plate, incubated at 37°C for 30 min. An 8X working solution (4 nM) of the agonist PGE2 was prepared using experimental buffer, and 2.5 μL of this solution was added to each well of the 384-well plate (final PGE2 concentration 0.5 nM), incubated at 37°C for 30 min. After the reaction, the cAMP concentration in the cells was quantified according to the instructions of the cAMP assay kit (Perkin Elmer, Cat#TRF0263). Calculate the antagonistic effect (IC50) of the test compound. 50 value).

[0248] Table 1 shows the antagonistic effects of the tested compounds on EP4.

[0249] Test compounds <![CDATA[IC 50 (nM)]]> I-1 42 I-2 38 I-3 27 I-4 >100

[0250] Experimental results show that the compound of this invention has a good antagonistic effect on EP4.

[0251] Test Example 2: Assay of Radioligand EP4 Receptor Binding

[0252] Radioligand EP4 binding was measured using recombinant human EP4 receptor membrane protein (prepared from 293 cells overexpressing human EP4 receptor). The test compound and PGE2 were prepared into 10 mM stock solutions in DMSO, and then serially diluted with buffer (50 mM HBSS, 0.1% BSA, 500 mM NaCl) to 8 concentration points of 4× working solution. 1 μL of the compound working solution, DMSO, and PGE2 working solution were added to the assay plate, along with 100 μL of EP4 receptor membrane protein (20 μg / well) and 100 μL of radioligand […]. 3H]-PGE2 (PerkinElmer, Cat: NET428250UC, Lot: 2469552) (final concentration 1.5 nM) was incubated at room temperature for 1 hour in a sealed container. At room temperature, the Unifilter-96 GF / C filter plate (PerkinElmer) was soaked in 0.5% BSA, 50 μL / well, for at least 30 min. After conjugation, the reaction mixture was filtered through the GF / C plate using a Perkin Elmer Filtermate Harvester, then the filter plate was washed and dried at 50°C for 1 hour. After drying, the bottom of the filter plate wells was sealed with Perkin Elmer Unifilter-96 sealing tape, and 50 μL of MicroScint was added. TM -20 cocktail (PerkinElmer), sealed filter top. Read the captured image on the filter using a Perkin Elmer MicroBeta2 Reader. 3 H count.

[0253] Analyze the data using GraphPad Prism 5 and calculate the inhibition rate using the following formula:

[0254] Inhibition rate (%) = 100 - (Test group - PGE2 group) / (DMSO group - PGE2 group) * 100

[0255] Based on the inhibition rate of the compound at different concentrations, the Ki value of the compound and the EP4 binding were calculated.

[0256] Table 2. Ki values ​​of compounds determined by binding to radioligand EP4.

[0257] Test compounds Ki(nM) I-1 22 I-2 94 I-3 77 I-4 ＞100

[0258] Experimental results show that the compound of this invention has a good affinity for the EP4 receptor.

[0259] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The compound represented by Formula I, its tautomers, stereoisomers, and pharmaceutically acceptable salts: I， in, Ring A is absent, group fragment for ; R1 is selected from -CONH-R 11 ;R 11 It is hydrogen or C1-C6 alkyl; R 11 The C1-C6 alkyl group described herein is optionally surrounded by one or more R f Substitution; when there are multiple substituents, the R f Same or different; R2 is selected from halogens and cyano groups; R3 is a C1-C6 carboxyl group or a C1-C6 amide group; The R mentioned above f It is selected from hydroxyl, halogen or cyano groups.

2. The compound according to claim 1, characterized in that, When ring A is absent, the group fragment for .

3. The compound according to claim 1, characterized in that, R1 is -CONH-R 11 R 11 It is a C1-C6 alkyl group.

4. The compound according to claim 1, characterized in that, R1 is -CONH-R 11 R 11 The derivatives are methyl, ethyl, propyl, and isopropyl.

5. The compound according to claim 1, characterized in that, R1 is -CONH-R 11 R 11 It is propyl or isopropyl.

6. The compound according to claim 1, characterized in that, R1 is -CONH-R 11 R 11 It is isopropyl.

7. The compound according to claim 1, characterized in that, R2 is a halogen.

8. The compound according to claim 1, characterized in that, R2 is a cyano group.

9. The compound according to claim 1, characterized in that, R3 is a C1-C6 carboxyl group.

10. The compound according to claim 9, characterized in that, R3 is butyric acid.

11. The compound according to claim 1, characterized in that, Having the following structures, their tautomers, stereoisomers, and pharmaceutically acceptable salts:

12. A compound, characterized in that, Having the following structures, their tautomers, stereoisomers, and pharmaceutically acceptable salts:

13. A method for preparing the compound of claim 1, its tautomers, stereoisomers, or pharmaceutically acceptable salts, characterized in that, The method includes the following steps: i) Intermediate B-1 reacts with compound B-3 to give the compound shown in Formula I. ; and / or, ii) Intermediate B-2 reacts with compound B-4, removing the protecting group PG1 from the carboxyl group in B-4 to obtain intermediate B-1. ； The definitions of rings A, R1, R2, and R4 are as described in claim 1; R3 is a C1-C6 carboxyl group or a C1-C6 amide group.

14. The method according to claim 13, characterized in that, R3 is a C1-C6 carboxyl group.

15. The method according to claim 14, characterized in that, R3 is butyric acid.

16. The method of claim 13, characterized in that, Step i) is carried out under nitrogen protection; and / or, in the presence of a catalyst.

17. The method of claim 16, characterized in that, The catalyst is a rhodium catalyst and a copper catalyst.

18. The method of claim 17, characterized in that, The catalyst is rhodium acetate.

19. The method as described in claim 13, characterized in that, Step i) further includes the step: i-1) After protecting the substituent R3 in compound B-3 with a carboxyl protecting group, it reacts with intermediate B-1 to obtain the compound shown in Formula I protected by a carboxyl protecting group. i-2) Remove the carboxyl protecting group from the compound of formula I that is protected by the carboxyl protecting group to obtain the compound of formula I.

20. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: a compound as described in any one of claims 1-11; and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, characterized in that, A further step includes at least one other pharmacologically active inhibitor selected from CTLA4 antibodies, PDL1, or PD1.

22. Use of the compound of any one of claims 1-11 or the pharmaceutical composition of any one of claims 20-21 in the preparation of a medicament or formulation for the prevention and / or treatment of EP4-mediated inflammatory diseases, pain, cancer, metabolic diseases, and urinary system diseases.

23. The use as described in claim 22, wherein the inflammatory disease is selected from arthritis; the pain is selected from osteoarthritis pain, pain caused by endometriosis; the cancer is selected from breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, and urethral cancer; the metabolic disease is diabetes mellitus; and the urinary system disease is selected from overactive bladder.

24. The use as claimed in claim 22, wherein the drug is administered in combination with radiotherapy and / or antibody therapy, wherein the antibody therapy is selected from one or a combination of CTLA4 antibody therapy, PDL1 antibody therapy and PD1 antibody therapy.

25. The use as claimed in claim 23, wherein the arthritis is selected from rheumatoid arthritis.

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