WO2022111222A1 - 作为前列腺素ep4受体拮抗剂的酰胺衍生物及其用途 - Google Patents

作为前列腺素ep4受体拮抗剂的酰胺衍生物及其用途 Download PDF

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WO2022111222A1
WO2022111222A1 PCT/CN2021/127887 CN2021127887W WO2022111222A1 WO 2022111222 A1 WO2022111222 A1 WO 2022111222A1 CN 2021127887 W CN2021127887 W CN 2021127887W WO 2022111222 A1 WO2022111222 A1 WO 2022111222A1
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洪健
D•达斯
王景炳
乔丹丹
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安润医药科技（苏州）有限公司
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
    • C07D209/42—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present invention relates to the technical field of biomedicine, in particular to amide derivatives as prostaglandin EP4 receptor antagonists and uses thereof.
  • Prostaglandins are a group of biologically active lipid compounds that are mediators of pain, fever, and other symptoms associated with inflammation or cell membrane damage.
  • Prostaglandin E2 (PGE2), the major eicosane derivative in inflammation, is involved in a variety of biological processes, including inflammation, pain, fever, renal function, mucosal integrity, hyperalgesia, uterine contractions, bone metabolism, platelets Function, Angiogenesis and Tumor Growth and Cancer (Trends in Molecular Medicine 2012;18:233–43).
  • Cyclooxygenase (COX) is an important enzyme in the synthesis of prostaglandin PGD2, PGE2, PGF2a, prostaglandin PG12 and thromboxane TXA2 from arachidonic acid.
  • Prostaglandins act by activating seven G protein-coupled receptors (GPCRs), EP1, EP2, EP3 and EP4 being PGE2-mediated activating receptors.
  • GPCRs G protein-coupled receptors
  • the EP4 receptor is one of seven cell membrane receptors, and its activation is normally associated with an increase in intracellular cyclic adenosine monophosphate (cAMP) levels, triggering multiple downstream events.
  • cAMP cyclic adenosine monophosphate
  • PGE2 contributes to pro-inflammatory immune responses; however, PGE2 is considered an important component in the immunosuppressive milieu of many solid tumors (Whiteside, Expert Opinion in Biological Therapy, 2010.10, 1019-1035), a persistent tumor microenvironment Level-promoting tumor accumulation enhances the activity of multiple immunosuppressive cells, including tumor-associated macrophages (TAMs), Treg cells, and myeloid-derived suppressor cells (MDSCs), thereby promoting tumor immune escape. Accumulating evidence suggests that elevated cAMP levels through EP4 are the main signaling pathway leading to immune cell immunosuppression.
  • TAMs tumor-associated macrophages
  • MDSCs myeloid-derived suppressor cells
  • Prostaglandin E receptor subtypes EP2 and EP4 promote the differentiation and expansion of Thl and Thl 7 lymphocytes through distinct signaling modules (Nature Medicine, 2009, 15, 633-640; Eur. J. Immunol 2009, 39, 1301-1312). It has been reported in the literature that prostaglandin E2 and interleukin 23 synergize to facilitate the expansion of human Thl7 (Blood, 2008, 112, 3696-3703), prostaglandin E2 regulates Thl7 through cyclic adenosine monophosphate and EP2/EP4 receptor signaling Cell differentiation and function (J. Exp. Med.
  • PGE2 acts as a cytokine amplification system through activated EP4 receptors, such as interleukin-6 (IL-6), and Induces differentiation and expansion of pro-inflammatory T-helper lymphocytes (Th1) (Yokoyama et al., Pharmacol. Rev. 2013, 65:1 0 10-52).
  • IL-6 interleukin-6
  • Th1 pro-inflammatory T-helper lymphocytes
  • COX2 and PGE2 expression are associated with tumor transformation, cell growth, angiogenesis, invasiveness, metastasis and immune evasion.
  • COX2 promotes tumor growth mainly through PGE2 and is up-regulated in colorectal, gastric, esophageal, pancreatic, breast and ovarian cancers.
  • Functional PGE2 antagonists have potential therapeutic utility in a variety of diseases.
  • EP4 antagonism has brought beneficial results in human and mouse preclinical systems as a therapeutic strategy for abdominal aortic aneurysm (AAA) (Yokoyama et al., 2012, PLoS One 7, e36724).
  • EP4 knockout in mice showed delayed tumorigenesis, suggesting tumor-promoting activity of PGE2-EP4 signaling in host immune cells (Mutoh et al., 2002, Cancer Res 62, 28–32).
  • selective EP4 receptor antagonists slow tumor progression and metastasis (Yang et al., 2006, . Cancer Research 66, 9665–9672; Mao et al., 2014, Clin Cancer Res 20, 4096 –4106).
  • EP4 has been identified as a target for preventing the development of atherosclerosis through macrophage EP4 deficiency, and inhibiting atherosclerosis (Babaev et al., 2008, Cell Metabolism 8, 492-501).
  • EP4 receptors are involved in various anti-inflammatory, osteogenic and hemostatic effects; EP4 agonists increase bone deposition and promote bone resorption; EP4 antagonist treatment can inhibit the immunosuppressive and tumorigenic effects of PGE2 in tumors:
  • Colorectal cancer Increased EP4 receptor expression has been reported to promote cell growth and anchoring in colorectal cancer (Chell et al., 2006, Cancer Research 66, 3106–3113);
  • EP4 was identified as a potential target for the treatment of castration-resistant prostate cancer in animal models (Terada et al., 2010, Cancer Research 70, 1606–1615);
  • EP4 antagonists protect natural killer cells from PGE2-mediated immunosuppression and inhibit breast cancer metastasis (Ma et al., 2013, OncoImmunology 2, e22647). EP4 is a therapeutic target for breast cancer cells with stem-like properties (Kundu et al., 2014, Breast Cancer Research and Treatment 143, 19–31);
  • Ovarian cancer (eg, epithelial ovarian cancer: The role of PGE2 synthesis and signaling in malignant transformation and progression, Mol Cancer, 2006, 5, 62);
  • Pain and inflammation neuropathic pain, EP4 neuropathic pain in a rat model of chronic contractile injury (Murase et al, 2008, European Journal of Pharmacology 580, 116–121) and inflammatory pain (Lin et al, 2006, Journal of Pharmacology and Experimental Therapeutics 319, 1096–1103) was confirmed.
  • EP4 antagonism especially in combination with chemotherapy, endocrine therapy or immunotherapy, should be further investigated as a promising approach for cancer therapy.
  • the EP4 receptor is a promising new therapeutic target for the treatment of breast cancer.
  • Several EP4 antagonists are in early clinical trials for cancer treatments.
  • the multiple biological functions of EP4 receptors can explain the activation and opening of multiple signaling pathways by EP4.
  • the EP4 antagonist E7046 reduces myeloid immunosuppression, and synergistically reduces IL-2-diphtheria toxin fusion protein with Treg to restore antitumor immunity (Albu et al., 2017, OncoImmunology 6, e1338239).
  • Targeting COX-2 and EP4 is known to control tumor growth, angiogenesis, lymphangiogenesis, and lung and lymph node metastasis in breast cancer models (Xin et al., 2012, Laboratory Investigation 92, 1115–1128).
  • the EP4 antagonist CR6086 has been reported to be an antirheumatic drug (Caselli et al., 2018, Pharmacological characterisation of CR6086,).
  • EP4 receptor antagonists were reported for the treatment of NASH-related liver cancer.
  • EP4 receptor agonists show promising results in ulcerative colitis (UC).
  • Orally bioavailable KAG-308 inhibits the development of colitis and promotes mucosal healing in a mouse model (Watanabe et al., 2015, European Journal of Pharmacology 754, 179–189).
  • EP4 is associated with diabetic nephropathy and ASP7657, a selective EP4 receptor antagonist, dose-dependently reduces proteinuria in mice with type 2 diabetes (Mizukami et al., 2018, Naunyn Schmiedebergs Arch Pharmacol 391, 1319–1326).
  • the EP4 antagonist ONO-AE3-208 inhibits cell invasion and metastasis of prostate cancer (Xu et al., 2014, Cell Biochemistry and Biophysics 70, 521-527).
  • MF-766 has been reported as a selective EP4 antagonist for the treatment of inflammatory pain (Colucci et al., 2010, Bioorganic & Medicinal Chemistry Letters 20, 3760-3763).
  • the EP4 receptor antagonist CJ-042794 was shown to be effective in a rat model of pain and inflammation.
  • THU0085 Cr6086 has immunomodulatory properties and was reported to reduce bone loss in a rat collagen-induced arthritis (CIA) model (Lanza et al., 2018, BMJ Publishing Group Ltd and European League against Rheumatism, 265–265).
  • CIA collagen-induced arthritis
  • PGE2/EP4 signaling may have therapeutic value in inflammatory autoimmune diseases.
  • Antagonists of the EP4 subtype of the PGE receptor may be beneficial and useful in the treatment of diseases or conditions mediated by the EP4 receptor, such as cancer and inflammatory diseases or diseases such as acute and chronic pain, osteoarthritis, rheumatoid arthritis and multiple sexual sclerosis.
  • the purpose of the present invention is to provide an amide derivative as a prostaglandin EP4 receptor antagonist and its use.
  • This document relates to the synthesis of novel compounds or pharmaceutically acceptable salts thereof and their use as prostaglandin EP4 antagonists.
  • the compounds described herein are useful in the treatment or prevention of diseases involving the EP4 receptor.
  • the compounds of the present invention can be used as monotherapy or in combination with one or more other methods of therapy.
  • the first object of the present invention is to provide a compound whose structural formula is shown in formula I:
  • Ring A is selected from wherein, W 1 and 2 are independently selected from C or N satisfying the valence state;
  • R 1 is selected from substituted or unsubstituted C 1-10 straight or branched chain alkyl, substituted or unsubstituted C 3-10 cycloalkyl, substituted or unsubstituted C 1-3 deuterated alkyl, substituted or unsubstituted C 1-3 deuterated alkyl, substituted or unsubstituted C 1-3 deuterated alkyl unsubstituted C7-12 alkylaryl or substituted or unsubstituted C6-10 alkylheteroaryl ;
  • R2 is selected from hydrogen , substituted or unsubstituted C6-14 aryl, substituted or unsubstituted C5-10 heteroaryl, substituted or unsubstituted C 3-7 saturated carbocyclic ring or substituted or unsubstituted C 1-10 straight or branched chain alkyl;
  • R 2 is absent;
  • R 3 and R 4 are independently selected from hydrogen, halogen, CN, substituted or unsubstituted C 1-3 alkyl, or substituted or unsubstituted C 1-3 alkoxy;
  • R 5 and R 6 are independently selected from hydrogen, C 1-3 straight or branched chain alkyl; or R 5 and R 6 form C 3-4 cycloalkyl or C 3-4 heterocycloalkyl;
  • G is selected from hydrogen, hydrocarbyl alcohol, tetrazolyl, -CONHSO 2 R 7 , -CO 2 R 7 , -CONHR 7 , -SO 2 NHR 7 or NHSO 2 R 7 ; wherein R 7 is selected from hydrogen, substituted or Unsubstituted C 1-5 alkyl, aryl or heteroaryl.
  • Ring A is selected from Wherein, W 3 and W 4 are independently selected from C or N satisfying the valence state, and W 5 , W 6 , W 7 and W 8 are independently selected from CH 2 , NH, O or S;
  • R 1 is selected from substituted or unsubstituted C 1-10 straight or branched chain alkyl, substituted or unsubstituted C 3-10 cycloalkyl, substituted or unsubstituted C 1-3 deuterated alkyl, substituted or unsubstituted C 1-3 deuterated alkyl, substituted or unsubstituted C 1-3 deuterated alkyl unsubstituted C7-12 alkylaryl or substituted or unsubstituted C6-10 alkylheteroaryl ;
  • R2 is selected from hydrogen , substituted or unsubstituted C6-14 aryl, substituted or unsubstituted C5-10 heteroaryl, substituted or unsubstituted C 3-7 saturated carbocyclic ring or substituted or unsubstituted C 1-10 straight or branched chain alkyl;
  • R 2 is absent;
  • R 3 and R 4 are independently selected from hydrogen, halogen, CN, substituted or unsubstituted C 1-3 alkyl, or substituted or unsubstituted C 1-3 alkoxy;
  • R 5 and R 6 are independently selected from hydrogen, C 1-3 straight or branched chain alkyl; or R 5 and R 6 form C 3-4 cycloalkyl or C 3-4 heterocycloalkyl;
  • G is selected from hydrogen, hydrocarbyl alcohol, tetrazolyl, -CONHSO 2 R 7 , -CO 2 R 7 , -CONHR 7 , -SO 2 NHR 7 or NHSO 2 R 7 ; wherein R 7 is selected from hydrogen, substituted or Unsubstituted C 1-5 alkyl, aryl or heteroaryl.
  • A is selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphatic ring and 4-7 membered heterocarbocycle containing one or more N, O heteroatoms .
  • halogen is selected from fluorine, chlorine or bromine atom; substituted or unsubstituted C 1-10 alkyl, substituted or unsubstituted C 1-3 alkyl or substituted or unsubstituted C 1-3 alkyl
  • the C 1-5 alkyl groups include C 1-3 fluoroalkyl groups; fluoroalkyl groups are selected from trifluoromethyl, difluoromethyl, difluorodeuteromethyl or monofluoromethyl; substituted or unsubstituted
  • the C 1-3 deuterated alkyl group is selected from tri-deuteromethyl, difluoro-deuteromethyl or penta-deuteroethyl.
  • the hydrocarbyl alcohol group includes a methylene alcohol group (-CH 2 OH) or an ethylene alcohol group (-CH 2 CH 2 OH).
  • X is selected from NH, O, S or CH 2
  • R 2 is selected from hydrogen, substituted C 6-14 aryl, substituted C 5-10 heteroaryl, substituted C 5-6 saturated carbocycle; or
  • X is selected from Cl, Br, F, CF3 , CN or CONH2 , R2 is absent .
  • -XR 2 is selected from a group of the following structural formula:
  • G is selected from a group of the following structural formula:
  • R 1 is selected from methyl, ethyl, propyl, fluoromethyl, trifluoromethyl or deuterated methyl.
  • R 3 and R 4 are independently selected from hydrogen, methyl, trifluoromethyl, halogen (chlorine, bromine, fluorine), nitrile (-CN) or methoxy (- OMe).
  • W 5 , W 6 , W 7 and W 8 are independently selected from CH 2 , NH, O or S.
  • the present invention further provides the preparation method of the compound shown in the above-mentioned formula I, and the reaction scheme is as follows:
  • ester-containing functional group of the compound of formula 1 is subjected to halogenation to obtain the ester-containing halide of formula 2 substituted at the 3-position; the compound of formula 2 undergoes alkylation to obtain the compound of formula 3; the compound of formula 3 is in SNAr, Suzuki or Buchwald.
  • the second object of the present invention is to protect the use of the above-mentioned compounds, or pharmaceutically acceptable salts, stereoisomers, deuterium substituted derivatives, hydrates or solvates thereof in the preparation of medicaments for the treatment of EP A condition in which 4 receptor antagonism alleviates or inhibits the proliferation of cells or enzymes, ie the drug acts as an EP 4 receptor antagonist.
  • EP4 receptor is a receptor for prostaglandin E2 (PGE2).
  • the compound shown in the above formula I can prepare a preparation for inhibiting the proliferation of cells and enzymes.
  • the cells are contacted with an effective amount of the compound of the formula I or its deuterated derivatives, its stereoisomeric derivatives or its pharmaceutically acceptable compounds.
  • condition is selected from one or more of cancer, inflammatory disease and pain.
  • the disorder is selected from lymphoma, kidney cancer, skin cancer, colorectal cancer, prostate cancer, breast cancer, urothelial cancer, lung cancer, non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), ovarian cancer
  • NSCLC non-small cell lung cancer
  • TNBC triple negative breast cancer
  • ovarian cancer One or more of (eg, epithelial ovarian cancer), cervical cancer, liver cancer, COX-related pain, neuropathic pain, multiple sclerosis, endometriosis, inflammation, inflammatory pain, and migraine.
  • the inflammation is rheumatoid arthritis.
  • the active compounds of the present invention are administered to a patient or subject for the treatment of various conditions, especially when "patient” or “subject” refers to an animal subject, preferably a rat, mouse, dog, cat, horse, Cow, sheep, goats, monkeys, etc., especially human subjects. Any or part of the subject's condition may be treated from the following list:
  • the drug is administered orally, parenterally, intravenously or transdermally.
  • the active compound can be administered to a subject by any suitable route, including oral, parenteral, inhalation spray, topical, rectal, nasal, oral, vaginal, or through an implanted reservoir.
  • parenteral injection includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, or perfusion techniques.
  • R and S are descriptors of the stereochemical configuration of asymmetrically substituted carbon atoms. Designation of asymmetrically substituted carbon atoms as “R” or “S” is accomplished by applying the Cahn-Ingold Prelog precedence rules well known to those skilled in the art and in the International Union of Pure and Applied Chemistry (lUPAC) nomenclature for organic chemistry described.
  • C ij as used herein means that the moiety has ij carbon atoms.
  • C1-10 alkyl means that the alkyl unit has any number between 1 and 10 carbon atoms.
  • Alkyl as used herein refers to a fully saturated straight, branched or cyclic hydrocarbon chain.
  • Ar or "aryl” refers to an aromatic carbocyclic moiety having one or more closed rings. Including, but not limited to, phenyl, naphthyl, anthracenyl, benzanthryl, biphenyl, and pyrenyl.
  • Heteroaryl refers to a cyclic moiety having one or more closed rings, wherein at least one ring has one or more heteroatoms (eg, oxygen, nitrogen, or sulfur), wherein at least one ring is aromatic, and wherein One or more rings can be independently fused and/or bridged.
  • pyridyl pyrrolyl, pyrazolinyl, quinolinyl, isoquinolinyl, indolyl, furyl, thienyl, quinoxolinyl, indazolinyl, thieno[2,3 -c] Pyrazolinyl, benzofuranyl, thienyl, benzothiazolyl.
  • “Pharmaceutically acceptable salts” refers to acid or base salts of the compounds of the present invention, the salts of which have the desired pharmacological activity and are not biologically or otherwise adversely affected. Including but not limited to acetate, adipate, benzoate, citrate, camphor ester, camphorsulfonate, digluconate, lauryl sulfate, ethyl ethanesulfonate, fumarate acid salt, glucosyl heptanoate, glycerophosphate, hemisulfate, heptanoate, caproate, hydrobromide, hydroiodate, 2-hydroxyethane sulfonate, lactate, maleate , oxalate.
  • the present invention has at least the following advantages:
  • the invention discloses a compound represented by formula I, which can be used to prepare an EP 4 receptor antagonist of PGE2 or a preparation for inhibiting the proliferation of cells and enzymes, and provides a new method for treating diseases related to EP 4 receptors. direction.
  • Figure 1 shows the results of the effect of different compounds on tumor growth in colorectal cancer CT-26 cell-bearing mice.
  • HATU 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DPCI:N,N'-diisopropylcarbodiimide ;DIEA:N,N-diisopropylethylamine;TEA:triethylamine;DMAP:4-dimethylaminopyridine;DMF:N,N-dimethylformamide;NMP:N-methylpyrrolidone;THF : tetrahydrofuran; DCM: dichloromethane; TFA: trifluoroacetic acid; TLC: thin layer chromatography; Structure: structural formula.
  • starting materials B-1 to B-42 were commercially available; B-24 was prepared according to the method of WO2013092979 or RSC Advances, 3(24), 9391-9401; 2013.
  • Compounds C-1 to C-10 are commercially available or prepared according to the literature. Specifically, compounds C-1, C-2, C-3 and C-5 are commercially available; C-4 preparation method reference: WO2017156179, WO2006057845; C-6 preparation method reference: ACS Catalysis, 10(1 ), 405-411; 2020, WO2011123678; C-7 preparation method reference: Bioorganic & Medicinal Chemistry Letters, 29(17), 2503-2510; 2019, WO2017156165; C-8 preparation method reference: WO2018214980, WO2009012375; -9 Preparation method reference: WO2018189340, US20080103182; C-10 preparation method reference: EP3101009, WO2016193461.
  • Step 2 Preparation of methyl 3-chloro-1-(3-(trifluoromethyl)benzyl)-1H-indole-2-carboxylate (8)
  • the third step preparation of 3-chloro-1-(3-(trifluoromethyl)benzyl)-1H-indole-2-carboxylic acid (9)
  • the first step the preparation step of methyl 3-bromo-1H-indole-2-carboxylate (10)
  • the second step the preparation step of methyl 3-bromo-1-methyl-1H-indole-2-carboxylate (11)
  • Compound 1-2 was prepared in an analogous manner to the preparation of intermediate 14 using intermediate 13a and methyl (S)-4-(1-aminoethyl)benzoate (C-1).
  • Compound 1-3 was prepared in an analogous manner to the preparation of intermediate 14 using intermediate 13b and methyl (S)-4-(1-aminoethyl)benzoate (C-1).
  • intermediates 14d-14u (Table 5) were prepared according to the preparation method of intermediate 14, and the starting materials were selected as intermediates 13d-13u, respectively.
  • Compound 1-7 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14d and sodium hydroxide.
  • Compound 1-8 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14e and sodium hydroxide.
  • Compound 1-9 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using compound 1-2 and sodium hydroxide.
  • Compound 1-10 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14f and sodium hydroxide.
  • Compound 1-11 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14g and sodium hydroxide.
  • Compound 1-12 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14h and sodium hydroxide.
  • Compound 1-13 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14i and sodium hydroxide.
  • Compound 1-14 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14j and sodium hydroxide.
  • Compound 1-15 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14k and sodium hydroxide.
  • Compound 1-16 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 141 and sodium hydroxide.
  • Compound 1-17 was prepared in a manner analogous to the preparation of compound 1-6 in Example 6 using intermediate 14m and sodium hydroxide.
  • Step 1 Using A-2, A-3, A-4, A-5, A-6 and NBS as starting materials, respectively, intermediates 19a-19e were prepared in a manner similar to that of intermediate 10.
  • Step 2 Starting from intermediates 19a-19d and methyl iodide (B-2), respectively, intermediates 20a-20e were prepared in a manner similar to the preparation of intermediate 11.
  • Step 1 Intermediates 21a-21e were prepared in a similar manner to the preparation of intermediate 12 using intermediates 20a-20e and 3-(trifluoromethyl)aniline (B-4) as starting materials, respectively.
  • Step 2 Intermediates 22a-22e were prepared in a similar manner to the preparation of intermediate 13 using intermediates 21a-21e and sodium hydroxide as starting materials, respectively.
  • intermediate 23 was prepared in a manner analogous to the preparation of intermediate 14.
  • Intermediates 23a-23e were prepared following the above routes.
  • Compound 1-20 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 23c and sodium hydroxide as starting materials.
  • intermediates 24a-24f were prepared in a similar manner to the preparation of intermediate 11 (Table 9 ).
  • Step 1 Intermediate 25 was prepared in a similar manner to the preparation of intermediate 12 using intermediate 24 and 3-(trifluoromethyl)aniline (B-4) as starting materials.
  • Step 2 Intermediate 26 was prepared in a manner analogous to the preparation of intermediate 13 using intermediate 25 and sodium hydroxide as starting materials.
  • Compound 1-24 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 27c and sodium hydroxide as starting materials.
  • Compound 1-25 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 27d and sodium hydroxide as starting materials.
  • Step 1 Starting from intermediate 13b and methyl(R)-4-(1-aminoethyl)benzoate (C-2), intermediate 28 was prepared in a manner analogous to the preparation of intermediate 14.
  • Step 2 Starting from intermediate 28 and sodium hydroxide, 1-27 was prepared in a manner analogous to the preparation of compound 1-6.
  • Compound 1-28 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 14n and sodium hydroxide as starting materials.
  • Intermediates 29a, 29b can be prepared from intermediates 11 and 3 - (trifluoromethyl)phenol (B-22) and 3-(trifluoromethyl)benzenethiol (B-23) following similar procedures in WO2019199979 prepared separately.
  • Intermediate 11 and 44,5,5-tetramethyl-2-(3-(trifluoromethyl)benzyl)-1,3,2-dioxolane (B-24) according to Intermediate 29c was prepared in a similar manner to WO2007068621.
  • Intermediates 30a-c were obtained from methyl esters of intermediates 29a-c in a manner similar to the synthesis of intermediates 12, which were combined with methyl (S)-4-(1-aminoethyl)benzoate (C- Coupling of 1) (step 2)
  • Intermediates 31a-c were prepared in a manner analogous to the synthesis of intermediate 14, respectively.
  • Compound 1-30 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 31a and sodium hydroxide as starting materials.
  • Compound 1-33 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 14p and sodium hydroxide as starting materials.
  • Compound 1-35 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 14r and sodium hydroxide as starting materials.
  • Compound 1-36 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 14s and sodium hydroxide as starting materials.
  • Step 1 was prepared using NBS in a similar manner to the preparation of intermediate 10.
  • step 2 was prepared in a manner analogous to the preparation of intermediate 11 using iodomethane (B-2).
  • the Buchwald reaction step was prepared in a manner analogous to the preparation of intermediate 12 using 3-(trifluoromethyl)aniline (B-4).
  • step 4 was prepared in a similar manner to the preparation of intermediate 13 using sodium hydroxide to provide intermediates 35a-c.
  • Intermediates 35a-c and methyl (S)-4-(1-aminoethyl)benzoate (c-1) were used as starting materials, and the intermediates were prepared by coupling reactions similar to those for the preparation of intermediates 14. 36a-c.
  • Compound 1-40 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 36b and sodium hydroxide as starting materials.
  • the intermediate 37 was prepared according to the method for the synthesis of I-1.
  • Compound 1-42 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 37 and sodium hydroxide as starting materials.
  • compound 1-43 was prepared in a similar manner to the preparation of compound 1-6.
  • compound 1-44 was prepared in a manner analogous to the preparation of compound 1-6.
  • compound 1-45 was prepared in a manner analogous to the preparation of compound 1-6.
  • intermediate 38 was prepared according to the method for preparing I-1; compound I-46 was prepared by a method similar to preparing compound I-5 using intermediate 38 and sodium hydroxide as starting materials.
  • Example 50 4-((1-Methyl-3-((3-(trifluoromethyl)phenyl)amino)-1H-indole 2-carboxamido)methyl)benzoic acid (1-50 )
  • intermediates 41a-41f were prepared in a manner analogous to the preparation of intermediate 14 ( Table 13).
  • Compound 1-50 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 41a and sodium hydroxide as starting materials.
  • Compound 1-51 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 41b and sodium hydroxide as starting materials.
  • Compound 1-52 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 41c and sodium hydroxide as starting materials.
  • Example 54 4-(3-(1-Methyl-3-((3-(trifluoromethyl)phenyl)amino)-1H-indole-2-carboxamido)oxetane- 3-yl)benzoic acid (I-54)
  • compound 1-54 was prepared in a manner analogous to the preparation of compound 1-6.
  • Intermediate 42 can be prepared from compound A-1 and N-iodosuccinimide (NIS) in a similar manner to intermediate 8
  • intermediates 43a and 43c can be prepared from intermediate 42 and vinylboronic acid pinacol, respectively Esters (B-32), cyclopropylboronic acid (B-34) were prepared according to the similar method of WO2020016453
  • intermediate 43b can be prepared from intermediate 42 and trimethylethynylsilicon (B-33) according to the similar method of WO2016154434
  • Intermediate 43d can be prepared from intermediate 42 and (difluoromethyl)trimethylsilane (B-35) in an analogous manner with reference to J. Am. Chem. Soc. 134:5524-5527.
  • intermediates 44a-d were obtained in a similar manner to the synthesis of intermediates 12, which were combined with (S)-methyl 4-(1-aminoethyl)benzoate (C Coupling of -1) (step 2)
  • Intermediates 45a-d were prepared in a manner analogous to the synthesis of intermediate 14, respectively.
  • Compound 1-60 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 45a and sodium hydroxide as starting materials.
  • Compound 1-61 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 45b and sodium hydroxide as starting materials.
  • Compound 1-62 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 45c and sodium hydroxide as starting materials.
  • Compound 1-63 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 45d and sodium hydroxide as starting materials.
  • Intermediates 46a-g can be sequentially prepared from compounds A-15, 16, 17, 18, 19, 20, and bromine in a manner similar to the preparation method of the document "Organic Chemistry" 2006:23(7)798-802, the intermediate Intermediates 47a-g can be prepared from intermediates 46a-g and CuCN under palladium catalysis, respectively, with reference to a similar method in WO2003044014, and then intermediates 47a-g are used as raw materials to obtain intermediates 48a- g, using intermediates 48a-g as raw materials, to obtain intermediates 49a-g in a method similar to the synthesis of intermediate 9, and finally the intermediates and (S)-4-(1-aminoethyl) methyl benzoate
  • the condensation reaction of (C-1) prepares intermediates 50a-g, respectively, in a manner similar to the synthesis of intermediate I-1. Product results are shown in Tables 15-19.
  • compound I-65 was prepared by a method similar to the preparation of compound I-6.
  • Compound 1-67 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 50b and sodium hydroxide as starting materials.
  • Compound 1-68 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 50c and sodium hydroxide as starting materials.
  • Intermediates 51a-b can be prepared sequentially from intermediates A-7, A-13, and trifluoroiodomethane (B-42), respectively, in a similar manner to Tetrahedr on Letters, 53(15), 2005-2008; 2012, and then Using intermediates 51a-b as raw materials, intermediates 52a-b were obtained in a manner similar to the synthesis of intermediate 8, and intermediates 52a-b were obtained by a method similar to the synthesis of intermediates 9.
  • Intermediates 53a-b and finally the condensation reaction of this intermediate with methyl (S)-4-(1-aminoethyl)benzoate (C-1) prepared intermediates 54a-b, respectively, in a manner similar to the synthesis of intermediate I-1.
  • the product results are shown in Table 20.
  • Intermediates 55a-d can be prepared sequentially from compounds A-13, 14, 8, 7 and N-bromosuccinimide (NBS) in a similar manner to the preparation of intermediate 8, and intermediates 56a-d can be prepared separately Prepared from intermediates 55a-d and CuCN under palladium catalysis with reference to the similar method of WO2003044014, and then using intermediates 56a-d as raw materials, intermediates 57a-d were obtained in a similar manner to the synthesis of intermediate 8, and then intermediates 57a-d were obtained.
  • NBS N-bromosuccinimide
  • compound 1-75 was prepared in a similar manner to compound 1-6.
  • Intermediates 60a-d can be prepared from intermediates 55a or 55d and B-33 or B-39, respectively, under palladium catalysis with reference to similar methods for 43b and 43e, respectively, and then intermediates 60a-d are used as raw materials.
  • Intermediates 61a-d are obtained by the method of intermediate 8, and intermediates 61a-d are used as raw materials to obtain intermediates 62a-d by a method similar to the synthesis of intermediate 9.
  • the intermediates are combined with (S)-4-( Condensation of methyl 1-aminoethyl)benzoate (C-1)
  • Intermediates 63a-d, respectively were prepared in a manner analogous to the synthesis of intermediate 1-1. The product results are shown in Table 22.
  • Compound 1-76 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 63a and sodium hydroxide as starting materials.
  • Compound 1-63 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 64 and sodium hydroxide as starting materials.
  • Example 78 (S)-4-(1-(1-(2-Chloro-3-fluorobenzyl)-3-cyano-1H-indole-2-carboxamido(ethyl)benzoic acid ( Trifluoromethyl)benzyl-1H-indole-2-carboxamido(ethyl)benzoic acid (I-78)
  • intermediate 1-(2-chloro-3-fluorobenzyl) was obtained in a manner similar to the synthesis of intermediate 8.
  • -3-cyano-1H-indole-2-carboxylic acid methyl ester the methyl ester hydrolysis in the subsequent step was prepared in a manner similar to the synthesis of intermediate 9, the product was similar to (S)-4-(1-aminoethyl) Condensation of methyl benzoate (C-1) to prepare compound 65 in a manner analogous to the synthesis of 1-1, namely (S)-4-(1-(1-(2-chloro-3-fluorobenzyl)-3 -cyano-1H--indole-2-carboxamide)ethyl)benzoic acid methyl ester;
  • Compound 1-78 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 65 and sodium hydroxide as starting materials.
  • Compound 1-80 was prepared in a manner analogous to the preparation of compound 1-6 using intermediate 54b and sodium hydroxide as starting materials.
  • Compound 1-82 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 59b and sodium hydroxide as starting materials.
  • Compound 1-83 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 59c and sodium hydroxide as starting materials.
  • Compound 1-86 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 63c and sodium hydroxide as starting materials.
  • Compound 1-87 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 63d and sodium hydroxide as starting materials.
  • compound I-90 was prepared in a similar manner to the preparation of compound I-6.
  • Compound 1-91 was prepared in an analogous manner to the preparation of compound 1-6 using intermediate 59d and sodium hydroxide as starting materials.
  • kit instructions prepare an activation buffer with a concentration of 1 times the working solution for later use;
  • EP1 positive compound It is AH6809
  • EP3 positive compound is L-798106
  • the 10 corresponding concentrations of the EP1 and EP3 receptor compounds to be tested are 200000, 6666.67, 2222.22, 740.74, 246.91, 82.30, 27.43, 9.14, 3.05, and 1.02 nM, respectively;
  • EP1-HEK and EP3-HEK cells were digested with trypsin, the medium was removed after centrifugation, the cells were resuspended in activation buffer with a concentration of 1 times the working solution, and the cells were counted and seeded in 384-well plates.
  • the seeding density was about 4500 cells/well/9.1 ⁇ L for EP1 and about 5000 cells/well/9.1 ⁇ L for EP3;
  • kit instructions prepare an activation buffer with a concentration of 1 times the working solution for later use;
  • EP2-HEK and EP4-HEK cells were digested with trypsin, the medium was removed after centrifugation, the cells were resuspended in activation buffer with a concentration of 1 times the working solution, and the cells were counted and seeded in a 384-well plate.
  • the seeding density was 1500 cells/well/5 ⁇ L;
  • step 4 Take 1 ⁇ L of the compound diluted in step 2 with the corresponding concentration of 10 times and add it to the corresponding experimental wells. Add 1 ⁇ L of the positive compound with the maximum concentration of 10 times the corresponding concentration to the Max hole, and add 1 ⁇ L of the corresponding concentration of the minimum concentration of 10 times to the Min hole. DMSO buffer (2v%), centrifuged and incubated at 37°C for 15min;
  • Tables 23-24 show the inhibition of human prostaglandin E2 receptors (EP1, EP2, EP3, EP4) by some examples in the cAMP assay.
  • Table 24 shows that the compound of Example 6 (I-6) has better selectivity for EP receptor subtype than E7046, stronger inhibition of EP4, and no inhibition of EP1/EP2/EP3.
  • the specific test plan is as follows:
  • Cells were digested with 0.25% Trypsin-EDTA, resuspended cells were counted in an automated cell counter. Dilute the cell suspension to the desired density according to the seeding density. Add 100 ⁇ L of cells to each well and incubate overnight at 37 °C, 5% CO 2 .
  • the IC50 was first calculated using GraphPad Prism 5 with the following formula:
  • %Inh (Max signal-Compound signal)/(Max signal-Min signal) ⁇ 100.
  • Max signal is the test result of adding only DMSO without adding compound in the orifice plate in the above-mentioned step (2)
  • Min signal is the result of adding the culture medium to the whole orifice plate in the above-mentioned step (2) and test. The results are shown in Table 25.
  • BALB/c mice were ordered from Shanghai Jihui Laboratory Animal Breeding Co., Ltd. and allocated to this study at 5-6 weeks of age. Animal breeding, rearing and health conditions follow animal welfare guidelines.
  • Mouse colorectal cancer CT-26 cells were cultured in RPMI-1640 medium + 10% FBS medium and passaged at least 3 times before inoculation. Each mouse was inoculated with about 2 ⁇ 10 5 mouse colorectal cancer CT-26 cells, and the cells were collected in sterile PBS solution, and the inoculation volume was about 100 ⁇ L.
  • BALB/c mice were previously anesthetized with 3-4% isoflurane.
  • mice with appropriate tumor size were randomly divided into 4 groups according to tumor size and body weight, with 6 mice in each group, namely Vehicle (G1 group), E7046 150 mg/kg ( G2 group), Example 6 (I-6) compound 75 mg/kg (G3 group), Example 6 (I-6) compound 150 mg/kg (G4 group), 4 groups were orally administered for 11 days.
  • Vehicle (G1 group) E7046 150 mg/kg ( G2 group)
  • Example 6 (I-6) compound 150 mg/kg (G4 group) 4 groups were orally administered for 11 days.
  • Table 26 The results of tumor volume inhibition rate are shown in Table 26.
  • Tumor inhibition rate [1-(TV t -TV initial )/(CV t -CV initial )]*100%;
  • TV t Tumor volume of Day 11 treatment group
  • TV initial Tumor volume of Day 0 treatment group
  • CV t Tumor volume of Day 11 control group
  • CV initial Tumor volume of Day 0 control group.
  • Example 6 (I-6) and E7046 have a significant inhibitory effect on the tumor growth of colorectal cancer CT-26 cell tumor-bearing mice. Under the same dose, Example 6 (I- 6) The compound is obviously better than E7046.

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Abstract

本发明公开了一系列式I酰胺衍生物或其医药上可接受的盐及其作为前列腺素E2受体4(EP4)拮抗剂的用途。本文描述式I酰胺衍生物的制备，其中G、X、R 1、R 2、R 3、R 4、R 5、R 6和A环如本文定义，并公开其用途。本发明还涉及式I或作为EP4拮抗剂的医药上可接受的盐及其在癌症和免疫相关疾病中的用途，包括但不限于减轻疼痛、炎症和其他炎症相关疾病如关节炎，以及治疗癌症如乳腺癌，结直肠癌、胰腺癌和前列腺癌。

Description

作为前列腺素EP4受体拮抗剂的酰胺衍生物及其用途 技术领域
本发明涉及生物医药技术领域，尤其涉及作为前列腺素EP4受体拮抗剂的酰胺衍生物及其用途。
背景技术
前列腺素是一组具有生物活性的脂类化合物，是疼痛、发热和其他炎症或细胞膜损伤相关症状的介质。
前列腺素E2(PGE2)是炎症中主要的二十烷类衍生物，它参与多种生物学过程，包括炎症、疼痛、发热、肾功能、粘膜完整性、痛觉过敏、子宫收缩、骨代谢、血小板功能，血管生成与肿瘤生长和癌症(Trends in Molecular Medicine 2012；18:233–43)。环氧化酶(COX)是花生四烯酸合成前列腺素PGD2、PGE2，PGF2a、前列腺素PGl2和血栓素TXA2的重要酶。前列腺素通过激活7个G蛋白偶联受体(GPCRs)发挥作用，EP1、EP2、EP3和EP4是PGE2介导的激活受体。EP4受体是七个细胞膜膜受体之一，其激活通常与细胞内环磷酸腺苷(cAMP)水平的增加有关，从而引发多个下游事件。PGE2有助于促炎性免疫反应；然而，PGE2被认为是许多实体瘤形成的免疫抑制环境中的重要成分(Whiteside,Expert Opinion in Biological Therapy,2010.10,1019-1035)，肿瘤微环境中持续的水平促进肿瘤的积聚增强多种免疫抑制细胞的活性，包括与肿瘤相关的巨噬细胞(TAM)、Treg细胞和骨髓源性抑制细胞(MDSCs)，从而促进肿瘤免疫逃逸。越来越多的证据表明，通过EP4升高cAMP水平是导致免疫细胞免疫抑制的主要信号通路。
前列腺素E受体亚型EP2和EP4通过不同的信号模块促进Thl和Thl 7淋巴细胞的分化和扩张(Nature Medicine,2009,15,633-640；Eur.J.Immunol 2009,39,1301-1312)。如有文献报道了前列腺素E2与白细胞介素23协同作用有利于人类Thl7的扩张(Blood，2008，112，3696-3703)、前列腺素E2通过环腺苷酸和EP2/EP4受体信号调节Thl7细胞的分化和功能(J.Exp.Med.2009,206,535-548)、前列腺素E2(PGE2)通过活化的EP4受体发挥细胞因子放大系统的作用，例如白介素-6(IL-6)，并诱导促炎性T-辅助淋巴细胞(Th1)的分化和扩张(Yokoyama et al.,Pharmacol.Rev.2013,65:1 0 10-52)。
COX2和PGE2的高水平表达与肿瘤转化、细胞生长、血管生成、侵袭性、转移和免疫逃避有关。很明显，COX2主要通过PGE2促进肿瘤生长、在结直肠癌、胃癌、食管癌、胰腺癌、乳腺癌和卵巢癌中上调。功能性PGE2拮抗剂在多种疾病中具有潜在的治疗用途。EP4拮抗作用在人类和小鼠的临床前系统中带来了有益的结果，可作为腹主动脉瘤(AAA)的治疗 策略(Yokoyama et al.，2012，PLoS One 7,e36724)。小鼠EP4基因敲除显示肿瘤发生延迟，表明宿主免疫细胞中PGE2-EP4信号的肿瘤促进活性(Mutoh等人，2002，Cancer Res 62,28–32)。在各种临床前肿瘤模型中，选择性EP4受体拮抗剂可减缓肿瘤进展和转移(Yang等人，2006，.Cancer Research 66,9665–9672；Mao等人，2014，Clin Cancer Res 20,4096–4106)。EP4已被确认为通过巨噬细胞EP4缺乏预防动脉粥样硬化发展的靶点，并抑制动脉粥样硬化(Babaev等人，2008，Cell Metabolism 8,492–501)。
EP4受体参与多种抗炎、成骨和止血作用；EP4激动剂增加骨沉积，促进骨吸收；EP4拮抗剂治疗可抑制PGE2在肿瘤中的免疫抑制和致瘤作用：
大肠癌：据报道，大肠癌中EP4受体表达增加促进了细胞生长和锚定(Chell等人，2006，Cancer Research 66,3106–3113)；
前列腺癌：EP4被确定为动物模型治疗去势抵抗前列腺癌的潜在靶点(Terada等人，2010年，Cancer Research 70,1606–1615)；
乳腺癌：EP4拮抗剂保护自然杀伤细胞免受PGE2介导的免疫抑制，并抑制乳腺癌转移(Ma等人，2013，OncoImmunology 2,e22647)。EP4是一个具有干细胞样特性的乳腺癌细胞的治疗靶点(Kundu等人，2014，Breast Cancer Research and Treatment 143,19–31)；
卵巢癌：(如卵巢上皮癌：PGE2合成和信号传导在恶性转化和进展中的作用，Mol癌症，2006,5,62)；
疼痛和炎症：神经病理性疼痛，EP4神经病理性疼痛在大鼠慢性收缩损伤模型(Murase等人，2008，European Journal of Pharmacology 580,116–121)和炎症性疼痛(Lin等人，2006，Journal of Pharmacology and Experimental Therapeutics 319,1096–1103)中的作用得到证实。
越来越多的证据表明，EP4拮抗作用，特别是与化疗、内分泌治疗或免疫治疗相结合，应作为癌症治疗的有前途的方法进行进一步研究。EP4受体是治疗乳腺癌的一个有希望的新的治疗靶点。几种EP4拮抗剂正处于癌症治疗的早期临床试验阶段。EP4受体的多种生物学功能可以解释EP4激活和开启多种信号通路。
EP4拮抗剂E7046降低髓系免疫抑制，与Treg协同降低IL-2-白喉毒素融合蛋白恢复抗肿瘤免疫(Albu et al.，2017，OncoImmunology 6,e1338239)。已知靶向COX-2和EP4报道可在乳腺癌模型中控制肿瘤生长、血管生成、淋巴管生成和肺和淋巴结转移(Xin等人，2012，Laboratory Investigation 92,1115–1128)。据报道，EP4拮抗剂CR6086是一种抗风湿药物(Caselli等人，2018，Pharmacological characterisation of CR6086,)。在WO-2018084230中，EP4受体拮抗剂被报道用于治疗NASH相关肝癌。EP4受体激动剂对溃疡性结肠炎(UC)显示出有希望的结果。口服生物利用度高的KAG-308在小鼠模型中抑制结肠炎的发展并促进粘膜愈合(Watanabe等人，2015，European Journal of Pharmacology 754,179–189)。EP4与糖尿病肾病有关，ASP7657是一种选择性EP4受体拮抗剂，可剂量依赖性降低2型糖尿病小鼠的 蛋白尿(Mizukami等人，2018，Naunyn Schmiedebergs Arch Pharmacol 391,1319–1326)。EP4拮抗剂ONO-AE3-208抑制前列腺癌的细胞侵袭和转移(Xu等人，2014，Cell Biochemistry and Biophysics 70,521–527)。MF-766被报道为治疗炎症性疼痛的选择性EP4拮抗剂(Colucci等人，2010，Bioorganic & Medicinal Chemistry Letters 20,3760–3763)。EP4受体拮抗剂CJ-042794在大鼠疼痛和炎症模型中被证明是有效。EP4激动剂用于控制骨丢失和关节炎，THU0085 Cr6086具有免疫调节特性，并报道在大鼠胶原诱导关节炎(CIA)模型中骨丢失减少(Lanza et al.，2018，BMJ Publishing Group Ltd and European League Against Rheumatism，265–265)。
已发表的专利WO2020012305、WO20190255013、WO2019149286、WO2019152982、WO2019038156、WO2018216640、WO2018162562、WO2017085198、WO2013004290、WO201207063、WO2012039972、WO2010019796、WO2009139373、EP-3632898、EP 2649061公开了作为EP4拮抗剂并用于治疗前列腺素介导疾病的一些化合物。
据报道，抑制PGE2/EP4信号可能对炎症性自身免疫性疾病具有治疗价值。PGE受体的EP4亚型的拮抗剂可有益并应用于治疗由EP4受体介导的疾病或状况，例如癌症和炎症性疾病或诸如急慢性疼痛、骨关节炎、类风湿性关节炎和多发性硬化症。
发明内容
为解决上述技术问题，本发明的目的是提供一种作为前列腺素EP4受体拮抗剂的酰胺衍生物及其用途。本文涉及新化合物或其医药上可接受的盐的合成及其作为前列腺素EP4拮抗剂的用途。本文所述化合物可用于治疗或预防涉及EP4受体的疾病。本发明化合物可用作单一疗法或与一种或多种其他疗法方法组合使用。
本发明的第一个目的是提供一种化合物，其结构式如式I所示：
Figure PCTCN2021127887-appb-000001
其中：
环A选自
Figure PCTCN2021127887-appb-000002
其中，W 1和 2独立地选自满足价态的C或N；
R 1选自取代或未取代的C 1-10直链或支链烷基、取代或未取代的C 3-10环烷基、取代或未取代的C 1-3氘代烷基、取代或未取代的C 7-12烷基芳基或取代或未取代的C 6-10烷基杂芳基；
当X选自NH、O、S或CH 2时，则R 2选自氢、取代或未取代的C 6-14芳基、取代或未取代的C 5-10杂芳基、取代或未取代的C 3-7饱和碳环或取代或未取代的C 1-10直链或支链烷基；
当X选自卤素、CN、CONH 2、取代或未取代的C 1-3链状或环状烷基、乙烯基、乙炔基时，则R 2不存在；
R 3和R 4独立地选自氢、卤素、CN、取代或未取代的C 1-3烷基、或取代或未取代的C 1-3烷氧基；
R 5和R 6独立地选自氢、C 1-3直链或支链烷基；或R 5和R 6组成C 3-4环烷基或C 3-4杂环烷基；
G选自氢、烃基醇基、四氮唑基、-CONHSO 2R 7、-CO 2R 7、-CONHR 7、-SO 2NHR 7或NHSO 2R 7；其中R 7选自氢、取代或未取代的C 1-5烷基、芳基或杂芳基。
进一步地，上述化合物的结构式如式I所示：
Figure PCTCN2021127887-appb-000003
其中：
环A选自
Figure PCTCN2021127887-appb-000004
其中，W 3、W 4独立地选自满足价态的C或N，W 5、W 6、W 7和W 8独立地选自CH 2、NH、O或S；
R 1选自取代或未取代的C 1-10直链或支链烷基、取代或未取代的C 3-10环烷基、取代或未取代的C 1-3氘代烷基、取代或未取代的C 7-12烷基芳基或取代或未取代的C 6-10烷基杂芳基；
当X选自NH、O、S或CH 2时，则R 2选自氢、取代或未取代的C 6-14芳基、取代或未取代的C 5-10杂芳基、取代或未取代的C 3-7饱和碳环或取代或未取代的C 1-10直链或支链烷基；
当X选自卤素、CN、CONH 2、取代或未取代的C 1-3链状或环状烷基、乙烯基、乙炔基时，则R 2不存在；
R 3和R 4独立地选自氢、卤素、CN、取代或未取代的C 1-3烷基、或取代或未取代的C 1-3烷氧基；
R 5和R 6独立地选自氢、C 1-3直链或支链烷基；或R 5和R 6组成C 3-4环烷基或C 3-4杂环烷基；
G选自氢、烃基醇基、四氮唑基、-CONHSO 2R 7、-CO 2R 7、-CONHR 7、-SO 2NHR 7或NHSO 2R 7；其中R 7选自氢、取代或未取代的C 1-5烷基、芳基或杂芳基。
上述式I所示的化合物中，A选自取代或未取代的芳基、取代或未取代的杂芳基、脂肪环和含有一个或多个N、O杂原子的4-7元杂碳环。
进一步地，上述式I所示的化合物中，卤素选自氟、氯或溴原子；取代或未取代的C 1-10 烷基、取代或未取代的C 1-3烷基或取代或未取代的C 1-5烷基中包括C 1-3氟代烷基；氟代烷基选自三氟甲基、二氟甲基、二氟氘代甲基或一氟甲基；取代或未取代的C 1-3氘代烷基选自三氘代甲基、二氟氘代甲基或五氘代乙基。
进一步地，上述式I所示的化合物中，烃基醇基包括亚甲基醇基(-CH 2OH)或亚乙基醇基(-CH 2CH 2OH)。
进一步地，上述式I所示的化合物中，X选自NH、O、S或CH 2，R 2选自氢、取代的C 6-14芳基、取代的C 5-10杂芳基、取代的C 5-6饱和碳环；或者
X选自Cl、Br、F、CF 3、CN或CONH 2，R 2不存在。
进一步地，上述式I所示的化合物中，-XR 2选自一种如下结构式的基团：
Figure PCTCN2021127887-appb-000005
进一步地，上述式I所示的化合物中，G选自一种如下结构式的基团：
Figure PCTCN2021127887-appb-000006
优选地，式I所示的化合物中，R 1选自甲基、乙基、丙基、氟甲基、三氟甲基或氘代甲基。
优选地，式I所示的化合物中，R 3和R 4独立地选自氢、甲基、三氟甲基、卤素(氯、溴、氟)、腈(-CN)或甲氧基(-OMe)。
进一步地，上述式I所示的化合物中，其结构式如式Ia-Ic之一所示：
Figure PCTCN2021127887-appb-000007
进一步地，上述式I所示的化合物中，其结构式如式Id-If之一所示：
Figure PCTCN2021127887-appb-000008
进一步地，上述式I所示的化合物中，其结构式如式Ig-Ih所示：
Figure PCTCN2021127887-appb-000009
其中，W 5、W 6、W 7和W 8独立地选自的CH 2、NH、O或S。
式Ia-Ih中，G、X、R 1、R 2、R 3、R 4、R 5、R 6和A环如本文上文中所定义的。
进一步地，上述式I所示的化合物中，其结构式如下之一所示：
Figure PCTCN2021127887-appb-000010
Figure PCTCN2021127887-appb-000011
Figure PCTCN2021127887-appb-000012
Figure PCTCN2021127887-appb-000013
进一步地，上述式I所示的化合物中，其结构式如下之一所示：
Figure PCTCN2021127887-appb-000014
Figure PCTCN2021127887-appb-000015
Figure PCTCN2021127887-appb-000016
以G为羧基为例，本发明还进一步提供了上述式I所示的化合物的制备方法，反应路线如下：
Figure PCTCN2021127887-appb-000017
将式1化合物的含酯官能团发生卤化反应得到在3位取代的式2的含酯卤代物；式2的化合物发生烷基化反应得到式3的化合物；式3的化合物在SNAr、Suzuki或Buchwald反应条件下转化为式4的化合物；式4的甲酯衍生物在碱性条件下水解得到式5的酸衍生物；在合适的偶联反应条件和试剂存在下，将式5的酸衍生物与式6的化合物偶联得到式I的衍生物(其中G＝酯类、醇类、磺胺类、酰胺类，四唑)；在碱性反应条件下，将式I的酯基水解，得到式I的酸结构(其中G＝-CO 2H)。
本发明的第二个目的是保护上述化合物，或其药学上可接受的盐、立体异构体、氘取代衍生物、其水合物或溶剂化物在制备药物中的用途，药物用于治疗被EP 4受体拮抗作用减轻的病症或抑制细胞或酶的增殖，即该药物作为EP 4受体拮抗剂。
进一步地，EP4受体为前列腺素E2(PGE2)的受体。
上述式I所示的化合物可制备用于抑制细胞和酶增殖的制剂，使用上述制剂时，使细胞接触有效量的式I化合物或其氘代衍生物、其立体异构衍生物或其药学上可接受的盐或溶剂化物，或其定义的药物组合物。
进一步地，病症选自癌症、炎性疾病和疼痛中的一种或几种。
进一步地，病症选自淋巴瘤、肾癌、皮肤癌、结直肠癌、前列腺癌、乳腺癌、尿路上皮癌、肺癌、非小细胞肺癌(NSCLC)、三阴性乳腺癌(TNBC)、卵巢癌(例如，卵巢上皮性癌)、宫颈癌、肝癌、COX相关疼痛、神经病理性疼痛、多发性硬化症、子宫内膜异位症、炎症、炎症性疼痛和偏头痛中的一种或几种。
进一步地，炎症为类风湿关节炎。
将本发明活性化合物投与患者或受试者以治疗各种不同的状况，尤其是在“患者”或“受试者”指动物受试者，优选大鼠、小鼠、狗、猫、马、牛、绵羊、山羊、猴子等，尤其是人类受试者。受试者的任何或部分情况可从以下列表中进行治疗：
进一步地，药物经口、经肠外、静脉注射或经皮肤施用。
活性化合物可通过任何合适的途径给受试者，包括经口、非肠道、吸入喷雾、局部、直肠、鼻、口、阴道或通过植入的蓄水池。本文所用术语“非肠道注射”包括皮下注射、静脉注射、肌肉注射、关节内注射、滑膜内注射、胸骨内注射、鞘内注射、肝内注射、损伤内注射或灌注技术。
本发明中，如无特殊说明，术语解释如下：
“R”和“S”作为描述异构体的术语，是不对称取代碳原子立体化学构型的描述符。将不对称取代碳原子指定为“R”或“S”是通过应用本领域技术人员熟知的Cahn-Ingold Prelog优先规则完成的，并在国际纯粹与应用化学联合会(lUPAC)有机化学命名规则中进行了描述。
本文使用的术语C i-j意味着该部分具有i-j碳原子。例如，C 1-10烷基意味着烷基单元具有1到10之间的任意数量的碳原子。”如本文所用“烷基”系指完全饱和的直链、支链或环状烃链。
“Ar”或“芳基”指具有一个或多个闭环的芳香碳环部分。包括但不限于苯基、萘基、蒽基、苯蒽基、联苯和芘基。
“杂芳基”是指具有一个或多个闭环的环状部分，其中至少一个环具有一个或多个杂原子(例如，氧、氮或硫)，其中至少一个环是芳香族的，并且其中一个或多个环可以独立地熔融和/或桥接。包括但不限于吡啶基、吡咯基、吡唑啉基、喹啉基、异喹啉基、吲哚基、呋喃基、噻吩基、喹恶啉基、吲唑啉基、噻吩并[2,3-c]吡唑啉基、苯并呋喃基、噻吩基、苯并噻唑基。
“药学上可接受的盐”系指本发明化合物的酸或碱盐，其盐具有期望的药理活性，并且在生物上或其它方面并无不良影响。包括但不限于醋酸盐、己二酸酯、苯甲酸盐、柠檬酸盐、樟脑酯、樟脑磺酸盐、二葡萄糖酸盐、十二烷基硫酸酯、乙磺酸乙酯、富马酸盐、庚酸葡萄糖酯、甘油磷酸、半硫酸盐、庚酸盐、己酸盐、氢溴酸盐、氢碘酸盐、2-羟基乙烷磺酸盐、乳酸盐、马来酸盐、草酸盐。
借由上述方案，本发明至少具有以下优点：
本发明公开了一种式I所示的化合物，其可用于制备PGE2的EP 4受体拮抗剂或制备用于抑制细胞和酶增殖的制剂，为治疗与EP 4受体相关的疾病提供了新方向。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1是不同化合物对结直肠癌CT-26细胞荷瘤小鼠肿瘤生长影响结果。
具体实施方式
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
以下实施例中，如无特殊说明，柱层析法使用Biotage SP4；溶剂去除采用Buchii旋转蒸发器或Genevac离心蒸发器；在酸性流动相条件下，用Waters自动净化仪和19x100mmXTERRA 5微米MS CI8柱进行制备LC/MS；用瓦里安400MHz谱仪记录核磁共振谱。当“惰性”一词用于描述反应器(例如，反应容器、烧瓶、玻璃反应器等)时，意味着反应器中的空气已被基本上不含水或干燥的惰性气体(例如氮气、氩气等)所取代。
以下缩略语具有所示含义：
HATU:2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯；DPCI:N,N'-二异丙基碳二亚胺；DIEA:N,N-二异丙基乙胺；TEA:三乙胺；DMAP:4-二甲氨基吡啶；DMF:N,N-二甲基甲酰胺；NMP:N-甲基吡咯烷酮；THF:四氢呋喃；DCM：二氯甲烷；TFA:三氟乙酸；TLC:薄层色谱法；Structure:结构式。
表1中所示化合物可在市场上买到或按照已知文献方法制备：
化合物A-1，A-2，A-3，A-5，A-21可在商业上获得；化合物A-4制备方法参考文献:Journal of Medicinal Chemistry，57(7),3040-3052；2014，Tetrahedron Letters,55(13),2056-2060；2014；化合物A-6制备方法参考文献:European Journal of Medicinal Chemistry，49,379-396；2012，WO2014073627；化合物A-7制备方法参考文献:Journal of the American Chemical Society,140(27),8429-8433；2018，Synthesis,(16),2751-2757；2005；化合物A-8制备方法参考文献:WO 2012080450，WO2011078984；化合物A-9制备方法参考文献:WO2017100594；化合物A-10制备方法参考文献:ChemSusChem，12(13),3144-3151,2019；化合物A-11制备方法参考文献:CN 102675311，Tetrahedron Letters 53(15),2005-2008,2012；化合物A-12制备方法参考文献:WO 2003044014 A1，EP1314733A1；化合物A-13制备方法参考文献:EP1479680 A1，WO 2004031188；化合物A-14制备方法参考文献:WO2003022856；化合物A-15制备方法参考文献:WO9940913，JP 2004339176；化合物A-16和A-17制备方法参考文献:WO2011013752，WO 2018119395；化合物A-18、A-19和A-20制备方法参考文献:WO2009037001
表1 不同化合物结构式
Figure PCTCN2021127887-appb-000018
除化合物B-24外，起始物B-1至B-42(表2)可在商业上获得；B-24按照WO2013092979或RSC Advances,3(24),9391-9401；2013的方法制备。
表2 不同化合物结构式
Figure PCTCN2021127887-appb-000019
化合物C-1至C-10(表3)可通过商业获得或按照文献制备。具体，化合物C-1、C-2、C-3和C-5可从商业上获得；C-4制备方法参考文献:WO2017156179，WO2006057845；C-6制备方法参考文献:ACS Catalysis,10(1),405-411；2020，WO2011123678；C-7制备方法参考文献:Bioorganic & Medicinal Chemistry Letters,29(17),2503-2510；2019，WO2017156165；C-8制备方法参考文献:WO2018214980，WO2009012375；C-9制备方法参考文献:WO2018189340，US20080103182；C-10制备方法参考文献:EP3101009，WO2016193461。
表3 不同化合物结构式
Figure PCTCN2021127887-appb-000020
实施例1：甲基(S)-4-(1-(3-氯-1-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸甲酯(I-1)
Figure PCTCN2021127887-appb-000021
第一步：3-氯-1H-吲哚-2-羧酸甲酯(7)的制备
室温下，向1H-吲哚-2-羧酸甲酯(A-1，3.0g，17.1mmol)的无水DMF(30mL)溶液中加入NCS(3.4g，25.7mmol)。搅拌2小时后，将反应混合物倒入碎冰上，过滤掉沉淀物并干燥以获得粗产品。粗产物经硅胶柱层析，石油醚-乙酸乙酯(10:1)纯化，得到中间体7(2.6g，75％产率)。
1H NMR(400MHz,CDCl3)δ7.70(d,J＝8.1Hz,1H),7.42–7.31(m,2H),7.20(ddd,J＝7.9,6.5,1.2Hz,1H),3.97(s,3H),LCMS:m/z 210.1[M+H] +。
第二步：3-氯-1-(3-(三氟甲基)苄基)-1H-吲哚-2-羧酸甲酯(8)的制备
向3-氯-1H-吲哚-2-羧酸甲酯(7,3.3g，15.7mmol)的无水DMF(33mL)中的溶液中添加碳酸钾(2.6g，18.9mol，1.2eq)和1-(溴甲基)-3-(三氟甲基)苯(B-1，4.12g，17.2mmol，1.1eq)添加，在室温下搅拌2小时后，将反应混合物倒入碎冰中，将固体滤除，并将滤液干燥得到油状物。粗品经硅胶柱层析，石油醚-乙酸乙酯(10:1)纯化得到中间体8(4.5g，收率77.3％)。
1H NMR(400MHz,DMSO-d6)δ13.63(s,1H),7.69(dd,J＝8.2,5.2Hz,2H),7.60(d,J＝7.8Hz,1H),7.56(s,1H),7.51(t,J＝7.8Hz,1H),7.42(ddd,J＝8.4,7.1,0.9Hz,1H),7.31–7.20(m,2H),5.97(s,2H),3.99(s,3H)，LCMS:m/z368.1[M+H] +。
第三步：3-氯-1-(3-(三氟甲基)苄基)-1H-吲哚-2-羧酸(9)的制备
向3-氯-1-(3-(三氟甲基)苄基)-1H-吲哚-2-羧酸甲酯(8,5.6g，15.2mol，1eq)的THF(112mL)和水(28mL)的混合物中添加氢氧化钠(1.92g，45.7mol，3eq)。将反应混合物加热至50℃-60℃。用薄层色谱法监测，直到起始物质8完全消耗。在减压下浓缩反应混合物，并用稀盐酸调节pH至5-6，然后用乙酸乙酯(20ml×3)萃取。合并有机相并用无水硫酸钠干燥，减压浓缩得到粗品。产物经硅胶柱层析，二氯甲烷-甲醇(10:1)纯化，得到中间体9(4.0g，收率75％)。
1HNMR(400MHz,DMSO-d6)δ13.63(s,1H),7.69(dd,J＝8.2,5.2Hz,2H),7.60(d,J＝7.8Hz,1H),7.56 (s,1H),7.51(t,J＝7.8Hz,1H),7.42(ddd,J＝8.4,7.1,0.9Hz,1H),7.31–7.20(m,2H),5.97(s,2H)，LCMS:m/z354.1[M+H] +。
第四步：(S)-4-(1-(3-氯-1-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸甲酯(I-1)
在氮气氛围下，向中间体9(0.35g，1mmol，1eq)的混合物，DIPEA(0.4mL，2.0mmol，2eq)的DMF(5mL)中添加HATU(0.45g，1.2mmol，1.2eq)。将反应混合物在室温下搅拌30分钟后，再将(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1，0.22g，1.2mmol，1.2eq)加入上述溶液中，搅拌至中间体9在TLC上完全消失。将反应混合物倒在碎冰上，过滤掉沉淀物，得到粗混合物。粗产物经硅胶柱层析，石油醚-乙酸乙酯(10:1)纯化，得到化合物I-1(0.33g，收率65％)。
1H NMR(400MHz,DMSO)δ9.26(d,J＝7.8Hz,1H),7.85(d,J＝8.3Hz,2H),7.69–7.57(m,3H),7.55(s,1H),7.46(dd,J＝7.9,4.1Hz,3H),7.39–7.30(m,1H),7.25(t,J＝7.5Hz,2H),5.74-5.53(m,2H),5.18(p,J＝7.0Hz,1H),3.84(s,3H),1.44(d,J＝7.0Hz,3H)，LCMS:m/z515.1[M+H] +。
实施例2：(S)-4-(1-(3-((3-氯苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸酯(I-2)
1、合成中间体11
Figure PCTCN2021127887-appb-000022
第一步：3-溴-1H-吲哚-2-羧酸甲酯(10)的制备步骤
此反应步骤与实施例1的第一步相似，将NCS替换为NBS，得到中间体10(收率69.2％)。
1HNMR(400MHz,CDCl3)δ7.70(d,J＝8.1Hz,1H),7.42–7.31(m,2H),7.20(ddd,J＝7.9,6.5,1.2Hz,1H),3.97(s,3H),LCMS:m/z253.9[M+H] +。
第二步：3-溴-1-甲基-1H-吲哚-2-羧酸甲酯(11)的制备步骤
向中间体10(5.7g，22.4mmol，1eq)的无水DMF(57mL)溶液中加入碳酸钾(6.0g，44.89mmol，2eq)和碘甲烷(B-2，3.18g，22.4mmol，1eq),在室温下搅拌2小时后，将反应混合物倒入碎冰上，将固体过滤并干燥。中间体11经硅胶柱层析法纯化得到中间体11(4.6g，收率76.8％)。
1HNMR(400MHz,CDCl3)δ7.70(d,J＝8.1Hz,1H),7.42–7.31(m,2H),7.20(ddd,J＝7.9,6.5,1.2Hz,1H),3.99(s,3H),3.92(s,3H),LCMS:m/z267.9[M+H] +。
2、合成中间体13
Figure PCTCN2021127887-appb-000023
第一步：Buchwald反应条件的一般程序：
在氮气氛围下，向中间体11(1.0mmol)和原料RNH 2(1.2mmol)的甲苯(5mL)的混合物溶液中添加Pd(OAc) 2(0.1mmol)、BINAP(0.15mmol)和t-BuONa(2eq)。反应混合物在65-80搅拌约2h，用薄层色谱法监测，然后用水稀释反应混合物。分离有机层，盐水洗涤，无水Na 2SO 4干燥，减压浓缩得到粗混合物，用石油醚-乙酸乙酯(10:1)硅胶柱层析纯化得到中间产物12。
第二步：甲酯水解的一般步骤
向中间体12(1mmol)的THF(10mL)和水(2.5mL)的混合物中加入氢氧化钠(2mmol)，将反应混合物加热至50℃-60℃，直到中间体12在TLC上完全消失。减压浓缩反应混合物，用稀盐酸调节pH至5-6后，用乙酸乙酯(10ml×3)萃取，无水Na 2SO 4干燥，减压浓缩得到粗品，用石油醚-乙酸乙酯(10:1)硅胶柱层析法纯化得到中间体13。
中间体12a-12u和13a-13u(表4)的合成按照上述方案制备
表4 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000024
Figure PCTCN2021127887-appb-000025
3、合成中间体14
Figure PCTCN2021127887-appb-000026
在氮气氛围下，向中间体13(1mmol)、DIPEA(3mmol)的在DMF(8mL)混合溶液中加入HATU(1.5mmol)，在室温下将反应混合物搅拌30min后，将原料C-1(1.0mmol)添加到上述溶液中。将反应混合物搅拌约2小时，用薄层色谱法监测反应完全后，将反应混合物倒入碎 冰上，过滤沉淀物，得到粗品，用石油醚-乙酸乙酯(10:1)进行硅胶柱层析纯化，得到中间体14。
使用中间体13a及(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)以类似中间体14的制备方法制备化合物I-2。
1HNMR(400MHz,DMSO-d6)δ8.64(d,J＝7.6Hz,1H),7.97(s,1H),7.84(d,J＝8.2Hz,2H),7.64(d,J＝8.4Hz,1H),7.40(td,J＝16.3,7.8Hz,4H),7.16(dt,J＝11.8,7.7Hz,2H),6.75(d,J＝7.7Hz,1H),6.67–6.52(m,2H),5.19(p,J＝6.9Hz,1H),3.99(s,3H),3.88(s,3H),1.40(d,J＝7.0Hz,3H)，LCMS:m/z462.1[M+H] +。
实施例3：(S)-4-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸甲酯(I-3)
使用中间体13b及(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)以类似中间体14的制备方法制备化合物I-3。
1HNMR(400MHz,DMSO-6)δ8.62(d,J＝7.7Hz,1H),8.09(s,1H),7.77(d,J＝8.3Hz,2H),7.60(d,J＝8.4Hz,1H),7.41(d,J＝8.0Hz,1H),7.37-7.25(m,4H),7.10(t,J＝7.5Hz,1H),6.99(d,J＝7.7Hz,1H),6.90(s,1H),6.82(d,J＝8.2Hz,1H),5.15(t,J＝7.2Hz,1H),3.95(s,3H),3.83(s,3H),1.35(d,J＝7.0Hz,3H)，LCMS:m/z496.1[M+H] +。
实施例4:(S)-4-(1-(1-甲基-3-((3-硝基苯基)氨基)-1H-吲哚2-甲酰胺基)乙基)苯甲酸甲酯(I-4)
使用中间体13c及(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)以类似中间体14的制备方法制备化合物I-4。
1HNMR(400MHz,DMSO)δ8.71(d,J＝7.8Hz,1H),8.33(s,1H),7.84(d,J＝8.2Hz,2H),7.71(d,J＝8.4Hz,1H),7.58(dd,J＝8.0,1.5Hz,1H),7.45(ddd,J＝15.3,13.2,5.1Hz,6H),7.20(t,J＝7.5Hz,1H),7.08(dd,J＝8.0,1.2Hz,1H),5.25(p,J＝6.9Hz,1H),4.03(s,3H),3.93(s,3H),1.46(d,J＝7.0Hz,3H)，LCMS:m/z473.2[M+H] +。
类似地，按照中间体14的制备方法制备中间体14d-14u(表5)，原料分别选择中间体13d-13u。
表5 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000027
实施例5:(S)-4-(1-(3-氯-1-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-5)
Figure PCTCN2021127887-appb-000028
室温下，向化合物I-1(0.47gm，1mol)的THF(10mL)和水(2.5mL)中混合物中添加氢氧化钠(0.11g，3.1mmol)。将反应混合物加热至50℃-60℃，直到化合物I-1在TLC上完全消失。然后，减压浓缩反应混合物，用稀HCl调节pH至5-6，然后用乙酸乙酯萃取，无水Na 2SO 4干燥，减压浓缩得到粗产物。产物经硅胶柱层析(二氯甲烷-甲醇10:1)纯化，得到化合物I-5(0.32g，65％)。
1HNMR(400MHz,DMSO)δ9.25(d,J＝7.9Hz,1H),7.85(d,J＝8.3Hz,2H),7.69–7.57(m,3H),7.55(s,1H),7.47(dd,J＝11.1,8.1Hz,3H),7.39–7.30(m,1H),7.30–7.19(m,2H),5.72–5.56(m,2H),5.18(p,J＝7.0Hz,1H),1.44(d,J＝7.0Hz,3H)，LCMS:m/z501.1[M+H] +。
实施例6:(S)-4-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-6)
Figure PCTCN2021127887-appb-000029
室温下，向化合物I-3(0.49g，1mmol)的THF(10mL)和水(2.5mL)中混合物中添加氢氧化钠(0.11g，3.1mmol)。将反应混合物加热至50℃-60℃，直到化合物I-3在TLC上完全消失。然后，在减压下浓缩反应混合物，用稀HCl调节pH至5-6，然后用乙酸乙酯(10mL×3)萃取，在无水Na 2SO 4上干燥并减压浓缩得到粗产物。粗品经硅胶柱层析(二氯甲烷-甲醇10:1)纯化得到化合物I-6(0.65g，70％)。
1H NMR(400MHz,DMSO-d6)δ12.82(s,1H),8.58(d,J＝7.7Hz,1H),8.07(s,1H),7.73(d,J＝8.2Hz,2H),7.60(d,J＝8.4Hz,1H),7.38(d,J＝8.0Hz,1H),7.31(dd,J＝13.7,8.4Hz,4H),7.09(t,J＝7.5Hz,1H),6.98(d,J＝7.6Hz,1H),6.86(s,1H),6.81(d,J＝8.2Hz,1H),5.18–5.04(m,1H),3.94(s,3H),1.32(d,J＝6.9Hz,3H)，LCMS:m/z482.2[M+H] +，[α] 20＝+159.6(C＝1,CH 3OH)。
实施例7:(S)-4-(1-(3-((3,5-二甲基苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-7)
使用中间体14d和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-7。
LCMS:m/z442.2[M+H] +。
实施例8:(S)-4-(1-(3-((3-氟苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-8)
使用中间体14e和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-8。
1HNMR(400MHz,DMSO-d6)δ12.84(s,1H),8.58(d,J＝7.6Hz,1H),7.93(s,1H),7.75(d,J＝8.3Hz,2H),7.59(d,J＝8.4Hz,1H),7.38(d,J＝8.0Hz,1H),7.35–7.27(m,3H),7.11(dt,J＝23.7,7.8Hz,2H),6.46(ddd,J＝10.1,6.4,2.0Hz,2H),6.27(dt,J＝11.8,2.2Hz,1H),5.19–5.05(m,1H),3.93(s,3H),1.34(d,J＝7.0Hz,3H)，LCMS:m/z432.2[M+H] +，[α] 20＝+105.3(C＝1,CH 3OH)。
实施例9:(S)-4-(1-(3-((3-氯苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-9)
使用化合物I-2和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-9。
1HNMR(400MHz,DMSO-d6)δ12.85(s,1H),8.59(d,J＝7.6Hz,1H),7.93(s,1H),7.77(d,J＝8.2Hz,2H),7.59(d,J＝8.4Hz,1H),7.39(d,J＝8.0Hz,1H),7.32(d,J＝8.2Hz,3H),7.18–7.05(m,2H),6.71(dd,J＝7.8,1.1Hz,1H),6.62–6.55(m,1H),6.53(d,J＝1.8Hz,1H),5.14(p,J＝6.9Hz,1H),3.94(s,3H),1.35(d,J＝7.0Hz,3H),LCMS:m/z448.1[M+H] +，[α] 20＝+182.3(C＝1,CH 3OH)。
实施例10:(S)-4-(1-(3-((3-溴苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-10)
使用中间体14f和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-10。
LCMS:m/z492.1[M+H] +。
实施例11:(S)-4-(1-(1-甲基-3-(间甲苯胺基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-11)
使用中间体14g和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-11。
1HNMR(400MHz,DMSO-d6)δ12.84(s,1H),8.78(d,J＝7.6Hz,1H),7.81(d,J＝8.2Hz,2H),7.69(s,1H),7.63(d,J＝8.4Hz,1H),7.43(d,J＝7.9Hz,1H),7.35(dd,J＝13.5,7.9Hz,3H),7.09(dt,J＝21.6,7.6Hz,2H),6.60(d,J＝7.4Hz,1H),6.48(d,J＝7.7Hz,2H),5.17(p,J＝6.9Hz,1H),4.03(s,3H),2.21(s,3H),1.38(d,J＝6.9Hz,3H)，LCMS:m/z428.2[M+H] +，[α] 20＝+373.9(C＝1,CH 3OH)。
实施例12:(S)-4-(1-(3-((3-甲氧基苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-12)
使用中间体14h和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-12。
LCMS:m/z444.2[M+H] +。
实施例13:(S)-4-(1-(1-甲基-3-((4-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-13)
使用中间体14i和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-13。
1HNMR(400MHz,DMSO-d6)δ12.76(s,1H),8.52(d,J＝7.7Hz,1H),8.19(s,1H),7.73(d,J＝8.3Hz,2H),7.59(d,J＝8.4Hz,1H),7.42(d,J＝8.6Hz,2H),7.37(d,J＝8.0Hz,1H),7.30(dd,J＝16.2,7.8Hz,3H),7.09(t,J＝7.5Hz,1H),6.68(d,J＝8.5Hz,2H),5.12(p,J＝6.9Hz,1H),3.92(s,3H),1.34(d,J＝7.0Hz,3H)，LCMS:m/z482.2[M+H] +。
实施例14:(S)-4-(1-(3-((3,5-双(三氟甲基)苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-14)
使用中间体14j和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-14。
LCMS:m/z550.1[M+H] +。
实施例15:(S)-4-(1-(3-((4-氯-3-(三氟甲基)苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-15)
使用中间体14k和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-15。
LCMS:m/z516.1[M+H] +。
实施例16:(S)-4-(1-(1-甲基-3-((3-(吡咯烷-1-基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-16)
使用中间体14l和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-16。
LCMS:m/z483.2[M+H] +。
实施例17:(S)-4-(1-(1-甲基-3-((3-吗啉苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-17)
使用中间体14m和氢氧化钠以类似于实施例6中制备化合物I-6的方法制备化合物I-17。
LCMS:m/z499.2[M+H] +。
实施例18:(S)-4-(1-(5-氟-1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-18)
1、中间体19和中间体20的制备
Figure PCTCN2021127887-appb-000030
中间体19a-19e和20a-20e(表6)按照以上路线的方法制备：
步骤1：分别以A-2、A-3、A-4、A-5、A-6和NBS为起始原料，以类似于中间体10的制备方法制备中间体19a-19e。
步骤2：分别以中间体19a-19d和碘甲烷(B-2)为起始原料，以类似于制备中间体11的方法制备中间体20a-20e。
表6 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000031
2、中间体21和中间体22的制备
Figure PCTCN2021127887-appb-000032
中间体21a-21e和22a-22e以上路线制备：
步骤1：分别使用中间体20a-20e和3-(三氟甲基)苯胺(B-4)为原料，以类似于制备中间体12的方法制备中间体21a-21e。
步骤2：分别使用中间体21a-21e和氢氧化钠为原料，以类似于制备中间体13的方法制备中间体22a-22e。
表7 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000033
3、中间体23的制备
Figure PCTCN2021127887-appb-000034
使用中间体22及(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)为原料，以类似于制备中间体14的方法制备中间体23。中间体23a-23e(表8)按照上述路线制备。
表8 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000035
使用中间体23a和氢氧化钠为原料，以类似于制备化合物I-6的方法制备I-18。
LCMS:m/z500.1[M+H] +。
实施例19:(S)-4-(1-(5-氯-1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-19)
使用中间体23b和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-19。
LCMS:m/z516.1[M+H] +。
实施例20:(S)-4-(1-(6-氯-1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-20)
使用中间体23c和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-20。
LCMS:m/z516.1[M+H] +。
实施例21:(S)-4-(1-(1,5-二甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-21)
使用中间体23d和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-21。
LCMS:m/z496.2[M+H] +。
实施例22:(S)-4-(1-(1-(甲基-d3)-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-22)
1、合成中间体24
Figure PCTCN2021127887-appb-000036
分别使用中间体10和B-1、B-15、B-16、B-17、B-18和B-19为原料，以类似于制备中 间体11的方法制备中间体24a-24f(表9)。
表9 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000037
2、合成中间体26
Figure PCTCN2021127887-appb-000038
中间体25a-25f和26a-26f(表10)按照以上路线制备：
步骤1：使用中间体24和3-(三氟甲基)苯胺(B-4)为原料，以类似于制备中间体12的方法制备中间体25。
步骤2：使用中间体25和氢氧化钠为原料，以类似于制备中间体13的方法制备中间体26。
表10 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000039
3、合成中间体27
Figure PCTCN2021127887-appb-000040
中间体27a-27f(表11)是按照以上路线制备的：使用中间体26和(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)为原料，以类似于制备中间体14的方法制备中间体27。
表11 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000041
使用中间体27a和氢氧化钠为原料，以类似于方案7中制备化合物I-6的方法制备化合物I-22。LCMS:m/z485.2[M+H] +。
实施例23:(S)-4-(1-(1-乙基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-23)
使用中间体27b和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-23。LCMS:m/z496.2[M+H] +。
实施例24:(S)-4-(1-(1-(2-羟乙基)-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-24)
使用中间体27c和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-24。
LCMS:m/z512.2[M+H] +。
实施例25:(S)-4-(1-(1-异丁基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-25)
使用中间体27d和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-25。
LCMS:m/z524.2[M+H] +。
实施例26:(S)-4-(1-(1-苄基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-26)
使用中间体27e和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-26。
LCMS:m/z558.2[M+H] +。
实施例27:(R)-4-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-27)
Figure PCTCN2021127887-appb-000042
步骤1：利用中间体13b和甲基(R)-4-(1-氨基乙基)苯甲酸盐(C-2)为原料，以类似于制备中间体14的方法制备中间体28。
步骤2：使用中间体28和氢氧化钠为原料，以类似于制备化合物I-6的方法制备I-27。
1HNMR(400MHz,DMSO-d6)δ12.82(s,1H),8.62(d,J＝7.7Hz,1H),8.11(s,1H),7.78(d,J＝8.3Hz,2H),7.64(d,J＝8.4Hz,1H),7.43(d,J＝8.0Hz,1H),7.40–7.29(m,4H),7.13(t,J＝7.4Hz,1H),7.03(d,J＝7.6Hz,1H),6.92(s,1H),6.86(d,J＝8.2Hz,1H),5.17(p,J＝7.0Hz,1H),3.98(s,3H),1.37(d,J＝7.0Hz,3H)，LCMS:m/z496.2[M+H] +，[α] 20＝-177.2(C＝1,CH 3OH)。
实施例28:(S)-4-(1-(1-甲基-3-(吡啶-2-基氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-28)
使用中间体14n和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-28。
LCMS:m/z415.2[M+H] +。
实施例29:(S)-4-(1-(1-甲基-3-((6-甲基吡啶-2-基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-29)
使用中间体14o和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-29。
LCMS:m/z429.2[M+H] +。
实施例30:(S)-4-(1-(1-甲基-3-(3-(三氟甲基)苯氧基)-1H-吲哚2-甲酰胺基)乙基)苯甲酸(I-30)
1、合成中间体31
Figure PCTCN2021127887-appb-000043
中间体29a、29b可由中间体11与中间体3制备-(三氟甲基)苯酚(B-22)和3-(三氟甲基)苯硫醇(B-23)遵循WO2019199979中的类似方法分别制备。使用中间体11和4,4,5,5-四甲基-2-(3-(三氟甲基)苄基)-1,3,2-二氧杂环戊烷(B-24)按照WO2007068621的类似方法制备中间体29c。以中间体29a-c甲酯为原料以类似于合成中间体12的方法得到中间体30a-c，该中间体与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的偶联(步骤2)以类似于合成中间体14的方法分别制备中间体31a-c。
31a：LCMS:m/z497.2[M+H] +。
31b： 1H NMR(400MHz,DMSO-d 6)δ9.29(d,J＝7.7Hz,1H),7.83(d,J＝8.3Hz,2H),7.67(d,J＝8.3Hz,1H),7.52(dd,J＝13.2,8.1Hz,3H),7.45–7.30(m,4H),7.26–7.08(m,2H),5.20(p,J＝7.1Hz,1H),3.85(s,3H),3.83(s,3H),1.46(d,J＝7.0Hz,3H)，LCMS:m/z513.2[M+H] +。
31c： 1H NMR(400MHz,DMSO-d 6)δ9.18(d,J＝7.9Hz,1H),7.91(d,J＝8.1Hz,2H),7.59(d,J＝7.8Hz,1H),7.53(d,J＝7.8Hz,3H),7.48(t,J＝7.3Hz,2H),7.45–7.36(m,2H),7.24(t,J＝7.6Hz,1H),7.05(t,J＝7.5Hz,1H),5.30–5.15(m,1H),4.35–4.16(m,2H),3.84(s,3H),3.74(s,3H),1.48(d,J＝7.0Hz,3H),LCMS:m/z495.2[M+H] +。
2、合成I-30
使用中间体31a和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-30。
LCMS:m/z483.2[M+H] +。
实施例31:(S)-4-(1-(1-甲基-3-((3-(三氟甲基)苯基)硫基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-31)
使用中间体31b和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-31。
1H NMR(400MHz,DMSO-d 6)δ12.85(s,1H),9.27(d,J＝7.6Hz,1H),7.81(d,J＝8.2Hz,2H),7.75–7.62(m,2H),7.50(dd,J＝8.1,3.7Hz,2H),7.46–7.31(m,4H),7.20(t,J＝7.7Hz,2H),5.28–5.09(m,1H),3.85(s,3H),1.45(d,J＝7.0Hz,3H).
LCMS:m/z499.1[M+H] +。
实施例32:(S)-4-(1-(1-甲基-3-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-32)
使用中间体31c和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-32。
1H NMR(400MHz,DMSO)δ12.87(s,1H),9.16(d,J＝7.9Hz,1H),7.89(d,J＝8.2Hz,2H),7.59(d,J＝8.0Hz,1H),7.56–7.35(m,7H),7.24(t,J＝7.6Hz,1H),7.05(t,J＝7.5Hz,1H),5.24(p,J＝7.0Hz,1H),4.34–4.16(m,2H),3.74(s,3H),1.47(d,J＝7.0Hz,3H).
LCMS:m/z481.2[M+H] +。
实施例33:(S)-4-(1-(3-(环己基氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-33)
使用中间体14p和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-33。
LCMS:m/z420.2[M+H] +。
实施例34:(S)-4-(1-(3-(叔丁胺基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-34)
使用中间体14q和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-34。
LCMS:m/z394.2[M+H] +。
实施例35:(S)-4-(1-(1-甲基-3-(萘-1-基氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-35)
使用中间体14r和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-35。
LCMS:m/z464.2[M+H] +。
实施例36:(S)-4-(1-(1-甲基-3-(吡啶-4-氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-36)
使用中间体14s和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-36。
LCMS:m/z415.2[M+H] +。
实施例37:(S)-4-(1-(1-(3-(三氟甲基)苄基)-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-37)
使用中间体27f和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-37。
LCMS:m/z626.2[M+H] +。
实施例38:(S)-4-(1-(1-甲基-5-(三氟甲基)-3-((3-(三氟-甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-38)
使用中间体23e和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-38。
LCMS:m/z550.2[M+H] +。
实施例39:(S)-4-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吡咯[2,3-b]吡啶-2-甲酰胺基)乙基)苯甲酸(I-39)
1、合成中间体36a-c
Figure PCTCN2021127887-appb-000044
采用A-7，A-8和A-9为起始材料，按照以上路线制备中间体36a-c(表12)。具体地，溴化步骤(步骤1)与以类似于制备中间体10的方法使用NBS制备。烷基化步骤(步骤2)采用碘甲烷(B-2)以类似于制备中间体11的方法制备。Buchwald反应步骤(步骤3)采用3-(三氟甲基)苯胺(B-4)以类似于制备中间体12的方法制备。水解步骤(步骤4)采用氢氧化钠以类似于制备中间体13的方法制备，从而获得中间体35a-c。以中间体35a-c与甲基(S)-4-(1-氨基乙基)苯甲酸甲酯(c-1)为原料，以类似于制备中间体14的方法发生偶联反应制备中间体36a-c。
表12 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000045
2、合成化合物I-39
使用中间体36a和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-39。
1H NMR(400MHz,DMSO)δ12.78(s,1H),8.67(d,J＝7.7Hz,1H),8.44(dd,J＝4.5,1.2Hz,1H),8.13(s,1H),7.83(dd,J＝7.9,1.1Hz,1H),7.74(d,J＝8.2Hz,2H),7.38–7.26(m,3H),7.16(dd,J＝7.9,4.6Hz,1H),7.00(d,J＝7.7Hz,1H),6.91(s,1H),6.83(d,J＝8.2Hz,1H),5.13(p,J＝7.1Hz,1H),3.97(s,3H),1.33(d,J＝7.0Hz,3H).
LCMS:m/z483.2[M+H] +。
实施例40:(S)-4-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吡咯[3,2-c]吡啶-2-甲酰胺基)乙基)苯甲酸(I-40)
使用中间体36b和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-40。
LCMS:m/z483.2[M+H] +。
实施例41:(S)-4-(1-(7-甲基-5-((3-(三氟甲基)苯基)氨基)-7H-吡咯[2,3-d]嘧啶-6-甲酰胺基)乙基)苯甲酸(I-41)
使用中间体36c和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-41。
LCMS:m/z484.2[M+H] +。
实施例42:(S)-4-(1-(5-氯-7-((3-((二氟-l3-甲基)-l2-氟酰基)苯基)甲基)-7H吡咯[2,3-d]嘧啶-6-羧肟)乙基)苯甲酸(I-42)
Figure PCTCN2021127887-appb-000046
以化合物A9为起始原料，按照合成I-1的方法制备中间体37。使用中间体37和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-42。LCMS:m/z503.1[M+H] +.
实施例43:(S)-4-(1-(1-甲基-3-((3-硝基苯基)氨基)-1H吲哚-2-甲酰胺基)乙基)苯甲酸(I-43)
使用产物I-4和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-43。
1HNMR(400MHz,DMSO-d6)δ12.78(s,1H),8.59(d,J＝7.8Hz,1H),8.24(s,1H),7.73(d,J＝8.3Hz,2H),7.61(d,J＝8.4Hz,1H),7.49(dd,J＝8.0,1.5Hz,1H),7.39(d,J＝8.2Hz,2H),7.33(dd,J＝12.0,4.6Hz,4H),7.10(t,J＝7.4Hz,1H),7.01(dd,J＝8.1,1.7Hz,1H),5.23-5.05(m,1H),3.94(s,3H),1.35(d,J＝7.0Hz,4H)，LCMS:m/z459.2[M+H] +，[α] 20＝+104.4(C＝1,CH 3OH)。
实施例44:(S)-4-(1-(3-((3-氰基苯基)氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-44)
使用中间体14t和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-44。
LCMS:m/z439.2[M+H] +。
实施例45:(S)-4-(1-(3-([1,1'-联苯基]-3-氨基)-1-甲基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-45)
使用中间体14u和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-45。
LCMS:m/z490.2[M+H] +。
实施例46:(S)-4-(1-(3-(三氟甲基)-1-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-46)
Figure PCTCN2021127887-appb-000047
以化合物A-11为起始原料，按照制备I-1的方法制备中间体38；以中间体38和氢氧化钠为原料，以类似于制备化合物I-5的方法制备化合物I-46。LCMS:m/z535.1[M+H] +。
实施例47:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-47)
Figure PCTCN2021127887-appb-000048
以化合物A-12和B-1为起始原料，以类似于合成中间体8的方法得到中间体3-氰基-1-(3-三氟甲基)苄基)-1H-吲哚-2-甲酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备化合物64，即(S)-4-(1-(3-氰基-1-(3-三氟甲基)苄基)-1H-吲哚-2-甲酰胺)乙基)苯甲酸甲酯39；以中间体39和氢氧化钠为原料，以类似于制备化合物I-5的方法制备化合物I-47。
1H NMR(400MHz,DMSO-d 6)δ12.78(s,1H),9.78(d,J＝7.8Hz,1H),7.84(d,J＝8.3Hz,2H),7.80–7.73(m,2H),7.63(d,J＝7.9Hz,1H),7.59(s,1H),7.53–7.34(m,5H),7.30(d,J＝7.8Hz,1H),5.80–5.66(m,2H),5.18(p,J＝7.0Hz,1H),1.44(d,J＝7.0Hz,3H).
LCMS:m/z492.2[M+H] +。
实施例48:(S)-4-(1-(3-氨甲酰基)-1-(3-(三氟甲基)苄基)-1H吲哚-2-甲酰胺基)乙基)苯甲酸(I-48)
Figure PCTCN2021127887-appb-000049
向稀硫酸(100mg，84％)中加入50mg产物I-47，在20℃-30℃搅拌反应混合物，通过TLC监测反应，反应完全后，经过一系列后处理并在真空中干燥得到粗产物，粗产物经硅胶柱层析用乙酸乙酯纯化，得到化合物I-48(15mg，32％)。LCMS:m/z510.2[M+H] +。
实施例49:(S)-4-(1-(3-氯-1-新戊基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-49)
Figure PCTCN2021127887-appb-000050
使用中间体7和化合物1-溴-2,2-二甲基丙烷(B-40)为原料，以类似制备化合物8的方法制备化合物3-氯-1-新戊基-1H-吲哚-2-甲酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备(S)-4-(1-(3-氯-1-新戊基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸甲酯(40)。；
使用中间体40和氢氧化钠为原料，以类似于制备化合物I-5的方法制备化合物I-49。LCMS:m/z413.2[M+H] +。
实施例50:4-((1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚2-甲酰胺基)甲基)苯甲酸(I-50)
1、合成中间体41a-41f
Figure PCTCN2021127887-appb-000051
使用中间体13b和胺(C-3、C-4、C-5、C-6、C-7和C-8)为原料，以类似于中间体14的制备方法制备中间体41a-41f(表13)。
表13 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000052
2、合成I-50
使用中间体41a和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-50。
LCMS:m/z468.2[M+H] +。
实施例51:4-(2-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)丙-2-基)苯甲酸(I-51)
使用中间体41b和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-51。
LCMS:m/z468.2[M+H] +。
实施例52:4-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)环丙基)苯甲酸(I-52)
使用中间体41c和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-52。
LCMS:m/z494.2[M+H] +。
实施例53:4-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)环丁基)苯甲酸(I-53)
使用中间体41d和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-53。
LCMS:m/z508.2[M+H] +。
实施例54:4-(3-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)氧杂环丁烷-3-基)苯甲酸(I-54)
使用中间体41e和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-54。
LCMS:m/z510.2[M+H] +。
实施例55:3-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-55)
使用中间体41f和氢氧化钠为原料，以类似于制备化合物I-6的方法制备化合物I-55。
LCMS:m/z482.2[M+H] +。
实施例56:N-(4-(1H-四唑-5-基)苄基)-1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺(I-56)
Figure PCTCN2021127887-appb-000053
使用中间体13b和(4-(1H-四唑-5-基)苯基)甲烷胺(C-9)为原料，以类似于制备中间体14的方法制备化合物I-56，反应路线如上。产物LCMS：m/z492.2[M+H] +。
实施例57:N-(4-(N-环丙基氨磺酰基)苄基)-1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺(I-57)
使用中间体13b和4-(氨甲基)-N-环丙基苯磺酰胺(C-10)为原料，以类似于制备化合物I- 56的方法制备化合物I-57。产物LCMS：m/z543.2[M+H] +。
实施例58:(S)-N-(1-(4-(羟甲基)苯基)乙基)-1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺(I-58)
Figure PCTCN2021127887-appb-000054
向产品I-3(50mg，0.1mmol)的THF(5ml)溶液中，分批加入硼氢化钠(11.4mg，0.3mmol)。将上述混合物冷却至0℃，并在约10分钟内逐滴加入三氟化硼乙醚溶液(0.5mL，133mmol)。将反应加热至20℃-30℃，通过TLC监测反应完成后，将反应冷却至0℃并用氢氧化钠水小心地淬灭。将所述内容物搅拌3h，在真空下去除THF，将所得水悬浮液冷却至0℃，并过滤产物，在硅胶上使用正己烷-乙酸乙酯(1:1)对粗产物进行柱色谱纯化，得到化合物I-58(20mg，43％)。产物LCMS:m/z468.2[M+H] +。
实施例59:(S)-N-(1-(4-氨甲酰苯基)乙基)-1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吲哚-2-甲酰胺(I-59)
Figure PCTCN2021127887-appb-000055
氮气氛围下，向烧瓶中加入产品I-6(300mg，0.62mmol)、二氧化硅负载氯化铵(50mg，0.93mmol，1.5equiv)和TsCl(178mg，0.93mmol，1.5eq)、TEA(93mg，0.93mmol，1.5eq)并充分混合。TLC检测反应完成后，加入乙酸乙酯(50mL)，然后过滤，用0.02NHCl溶液洗涤滤液。有机层在无水硫酸镁上干燥并蒸发。粗品经硅胶柱层析，正己烷-乙酸乙酯(1:1)纯化，得到化合物I-59(149mg，50％)。
1HNMR(400MHz,DMSO)δ8.54(d,J＝7.7Hz,1H),8.07(s,1H),7.86(s,1H),7.69(d,J＝8.3Hz,2H),7.59(d,J＝8.5Hz,1H),7.37(d,J＝7.9Hz,1H),7.32(ddd,J＝8.3,3.1,1.7Hz,2H),7.26(d,J＝8.2Hz,3H),7.09(t,J＝7.3Hz,1H),6.99(d,J＝7.8Hz,1H),6.90–6.80(m,2H),5.18–4.99(m,1H),3.94(s,3H),1.29(d,J＝7.0Hz,3H)，LCMS:m/z481.2[M+H] +。
实施例60:(S)-4-(1-(-1(-3(三氟甲基)苄基)-3-乙烯基-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-60)
1、中间体45a-d的合成
Figure PCTCN2021127887-appb-000056
中间体42可由化合物A-1及N-碘代丁二酰亚胺(NIS)以类似于中间体8的制备方法制备，中间体43a、43c可以分别由中间体42和乙烯基硼酸频哪醇酯(B-32)、环丙基硼酸(B-34)参照WO2020016453的类似方法制备，中间体43b可以由中间体42和三甲基乙炔基硅(B-33)参照WO2016154434的类似方法制备，中间体43d可以由中间体42和(二氟甲基)三甲基硅烷(B-35)参照J.Am.Chem.Soc.134:5524–5527的类似方法制备。以中间体43a-d甲酯为原料，以类似于合成中间体12的方法得到中间体44a-d，该中间体与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的偶联(步骤2)以类似于合成中间体14的方法分别制备中间体45a-d。
表14 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000057
2、化合物I-60的合成
使用中间体45a和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-60。
产物LCMS：m/z493.2[M+H] +。
实施例61:(S)-4-(1-(3-乙炔基-1-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I- 61)
使用中间体45b和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-61。
产物LCMS：m/z491.1[M+H] +。
实施例62:(S)-4-(1-(3-环丙基-1-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-62)
使用中间体45c和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-62。
产物LCMS：m/z507.2[M+H] +。
实施例63:(S)-4-(1-(3-(二氟甲基)-1-(3(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-63)
使用中间体45d和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-63。
产物LCMS：m/z517.2[M+H] +。
实施例64:(S)-4-(1-(3-氯-1-(环丙基甲基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸(I-64)
使用中间体7和化合物B-36为原料，以类似制备化合物8的方法制备化合物3-氯-1-(环丙基亚甲基)-1H-吲哚-2-甲酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备(S)-4-(1-(3-氯-1-(环丙基甲基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸甲酯。
使用中间体(S)-4-(1-(3-氯-1-(环丙基甲基)-1H-吲哚-2-甲酰胺基)乙基)苯甲酸甲酯和氢氧化钠为原料，以类似制备化合物I-5的方法制备化合物I-64。
产物LCMS：m/z397.1[M+H] +。
实施例65:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吲哚-2-甲酰胺基(乙基)苯甲酸(I-65)
1、中间体50a-g的合成
Figure PCTCN2021127887-appb-000058
中间体46a-g可分别依次由化合物A-15,16,17,18,19,20,及溴素以类似于文献《有机化学》 2006:23(7)798-802的制备方法制备，中间体47a-g可以分别由中间体46a-g和CuCN在钯催化作用下参照WO2003044014的类似方法制备，然后以中间体47a-g为原料，以类似于合成中间体8的方法得到中间体48a-g，再以中间体48a-g为原料，以类似于合成中间体9的方法得到中间体49a-g，最后该中间体与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合反应以类似于合成中间体I-1的方法分别制备中间体50a-g。产物结果如表15-19所示。
表15 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000059
表16 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000060
表17 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000061
表18 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000062
表19 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000063
2、使用中间体50a和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-65。
产物LCMS：m/z496.2[M+H] +。
实施例66:(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吲哚-2-甲酰胺基(乙基)苯甲酸(I-66)
使用中间体46a和1-(溴甲基)-3-(三氟甲基)苯(B-1)为原料，以类似制备化合物8的方法制备化合物3-溴-1-(3-(三氟甲基)苄基)-4,5,6,7-4-四氢-1H-吲哚-2-甲酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吲哚-2-甲酰胺基(乙基)苯甲酸。
使用中间体(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吲哚-2-甲酰胺基(乙基) 苯甲酸和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-66。
产物LCMS：m/z549.1[M+H] +。
实施例67:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-1,5,6,7-四氢吡喃[3,2-b]吡咯-2-甲酰胺基)乙基)苯甲酸(I-67)
使用中间体50b和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-67。
产物LCMS：m/z498.2[M+H] +。
实施例68:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-1,4,6,7-四氢吡喃[4,3-b]吡咯-2-甲酰胺基)乙基)苯甲酸(I-68)
使用中间体50c和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-68。
产物LCMS：m/z498.2[M+H] +。
实施例69:(S)-4-1-(3-氰基-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[2,3-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-69)
使用中间体50d在三氟乙酸和二氯甲烷体系中脱除Boc反应得到中间体(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[2,3-b]吡啶-2-甲酰胺基)乙基)苯甲酸甲酯，以该中间体和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-69。
产物LCMS：m/z497.2[M+H] +。
实施例70:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[2,3-c]吡啶-2-甲酰胺基(乙基)苯甲酸(I-70)
使用中间体50e在三氟乙酸和二氯甲烷体系中脱除Boc反应得到中间体(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[2,3-c]吡啶-2-甲酰胺基)乙基)苯甲酸甲酯，以该中间体和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-70。
产物LCMS：m/z497.2[M+H] +。
实施例71:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[3,2-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-71)
使用中间体50f在三氟乙酸和二氯甲烷体系中脱除Boc反应得到中间体(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[3,2-b]吡啶-2-甲酰胺基)乙基)苯甲酸甲酯，以该中间体和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-71。
产物LCMS：m/z497.2[M+H] +。
实施例72:(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[3,2-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-72)
使用中间体46f和1-(溴甲基)-3-(三氟甲基)苯(B-1)为原料，以类似制备化合物8的方法制备化合物4-(叔丁基)2-甲基3-溴-1-(3-(三氟甲基)苄基)-1,5,6,7-四氢-4H-吡咯并[3,2-b]吡啶-2,4-二羧酸酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1- 氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[3,2-b]吡啶-2-甲酰胺基(乙基)苯甲酸甲酯。
使用中间体(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-4,5,6,7-四氢-1H-吡咯并[3,2-b]吡啶-2-甲酰胺基(乙基)苯甲酸甲酯和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-72。
产物I-72LCMS：m/z550.1[M+H] +。
实施例73:(S)-4-(1-(3-(三氟甲基)-1-(3-(三氟甲基)苄基)-1H-吡咯并[3,2-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-73)
1、中间体54a-b的合成
Figure PCTCN2021127887-appb-000064
中间体51a-b可以分别依次由中间体A-7、A-13和三氟碘甲烷(B-42)参照文献Tetrahedr on Letters,53(15),2005-2008；2012的类似方法制备，然后以中间体51a-b为原料，以类似于合成中间体8的方法得到中间体52a-b，再以中间体52a-b为原料，以类似于合成中间体9的方法得到中间体53a-b，最后该中间体与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合反应以类似于合成中间体I-1的方法分别制备中间体54a-b。产物结果如表20所示。
表20 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000065
2、使用中间体54a和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-73。
产物I-73LCMS：m/z536.2[M+H] +。
实施例74:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-1H-吡咯[3,2-b]吡啶-2-甲酰胺)乙基)苯甲酸(I-74)
1、中间体59a-d的合成
Figure PCTCN2021127887-appb-000066
中间体55a-d可分别依次由化合物A-13,14,8,7及N-溴代丁二酰亚胺(NBS)以类似于中间 体8的制备方法制备，中间体56a-d可以分别由中间体55a-d和CuCN在钯催化作用下参照WO2003044014的类似方法制备，然后以中间体56a-d为原料，以类似于合成中间体8的方法得到中间体57a-d，再以中间体57a-d为原料，以类似于合成中间体9的方法得到中间体58a-d，最后该中间体与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合反应以类似于合成中间体I-1的方法分别制备中间体59a-d。产物结果如表21所示。
表21 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000067
使用中间体59a和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-74。I-74表征结果如下：
1H NMR(400MHz,DMSO)δ12.54(s,1H),8.92(d,J＝8.5Hz,1H),8.71(d,J＝5.7Hz,1H),8.58(dd,J＝8.0,0.7Hz,1H),7.86(d,J＝8.2Hz,2H),7.73(d,J＝8.8Hz,2H),7.66–7.52(m,2H),7.49–7.36(m,3H),6.18(s,2H),5.18(p,J＝7.0Hz,1H),1.52(d,J＝7.0Hz,3H).
产物LCMS：m/z493.2[M+H] +。
实施例75:(S)-4-(1-(1-甲基-3-((3-(三氟甲基)苯基)氨基)-1H-吡咯并[2,3-c]吡啶-2-甲酰胺基(乙基)苯甲酸(I-75)
以化合物A-14为起始原料(以A-14替换原料A-1)，以类似制备化合物I-6的方法制备化合物I-75。
1H NMR(400MHz,DMSO-d 6)δ13.04(s,1H),9.03(s,1H),8.83(d,J＝7.6Hz,1H),8.18(d,J＝5.5Hz,1H),8.08(s,1H),7.74(d,J＝8.2Hz,2H),7.39–7.24(m,4H),6.98(d,J＝7.5Hz,1H),6.88(s,1H),6.80(d,J＝8.0Hz,1H),5.22–5.04(m,1H),4.00(s,3H),1.34(d,J＝7.0Hz,3H).
产物LCMS：m/z483.2[M+H] +。
实施例76:(S)-4-(1-(3-乙炔基-1-(3-(三氟甲基)苄基)-1H-吡咯[3,2-b]吡啶-2-甲酰胺)乙基)苯甲酸(I-76)
1、中间体63a-d的合成
Figure PCTCN2021127887-appb-000068
中间体60a-d可以分别由中间体55a或55d和B-33或B-39在钯催化作用下分别参照43b，43e的类似方法制备，然后以中间体60a-d为原料，以类似于合成中间体8的方法得到中间体61a-d，再以中间体61a-d为原料，以类似于合成中间体9的方法得到中间体62a-d，最后该中间体与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合反应以类似于合成中间体I-1的方法分别制备中间体63a-d。产物结果如表22所示。
表22 不同化合物的结构及分子量
Figure PCTCN2021127887-appb-000069
使用中间体63a和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-76。
1H NMR(400MHz,DMSO)δ12.84(s,1H),9.32(d,J＝7.8Hz,1H),8.51(dd,J＝4.5,1.2Hz,1H),8.09(dd,J＝8.5,1.2Hz,1H),7.86(d,J＝8.3Hz,2H),7.62(d,J＝7.8Hz,1H),7.57(s,1H),7.49(t,J＝8.4Hz,3H),7.37–7.25(m,2H),5.75(s,2H),5.25–5.08(m,1H),4.51(s,1H),1.42(d,J＝7.0Hz,3H).
产物LCMS：m/z492.2[M+H] +。
实施例77:(S)-4-(1-(3-氰基-1-(3-(三氟甲氧基)苄基)-1H-吲哚-2-羧酰胺基(乙基)苯甲酸(I-77)
Figure PCTCN2021127887-appb-000070
以化合物A-12和3-三氟甲氧基苄溴(B-37)为起始原料，以类似于合成中间体8的方法得 到中间体3-氰基-1-(3-三氟甲氧基)苄基)-1H-吲哚-2-甲酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备化合物64，即(S)-4-(1-(3-氰基-1-(3-三氟甲氧基)苄基)-1H-吲哚-2-甲酰胺)乙基)苯甲酸甲酯；
使用中间体64和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-63。
产物LCMS：m/z517.2[M+H] +。
实施例78:(S)-4-(1-(1-(2-氯-3-氟苄基)-3-氰基-1H-吲哚-2-羧酰胺基(乙基)苯甲酸(三氟甲基)苄基-1H-吲哚-2-甲酰胺基(乙基)苯甲酸(I-78)
Figure PCTCN2021127887-appb-000071
以化合物A-12和2-氯-3-氟苄溴(B-38)为起始原料，以类似于合成中间体8的方法得到中间体1-(2-氯-3-氟苄基)-3-氰基-1H-吲哚-2-甲酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备化合物65，即(S)-4-(1-(1-(2-氯-3-氟苄基)-3-氰基-1H--吲哚-2-甲酰胺)乙基)苯甲酸甲酯；
使用中间体65和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-78。
产物LCMS：m/z517.2[M+H] +。
实施例79:(S,E)-4-(1-(3-(丙基-1-烯-1-基)-1-(3-(三氟甲基)苄基)-1H-吲哚-2-甲酰胺基(乙基)苯甲酸(I-79)
使用中间体45e和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-79。
产物LCMS：m/z507.2[M+H] +。
实施例80:(S)-4-(1-(3-(三氟甲基)-1-(3-(三氟甲基)苄基)-1H-吡咯并[2,3-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-80)
使用中间体54b和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-80。
产物LCMS：m/z536.2[M+H] +。
实施例81:(S)-4-(1-(1-新戊基-3-(三氟甲基)-1H-吡咯[2,3-b]吡啶-2-甲酰胺)乙基)苯甲酸(I-81)
使用中间体51b和1-溴-2,2-二甲基丙烷(B-40)为原料，以类似于合成中间体8的方法得到中间体1-新戊基-3-(三氟甲基)-1H-吡咯并[2,3-b]吡啶-2-甲酸甲酯，后续步骤中的甲酯水解 以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备(S)-4-(1-(1-新戊基-3-(三氟甲基)-1H-吡咯[2,3-b]吡啶-2-甲酰胺)乙基)苯甲酸甲酯。
使用中间体(S)-4-(1-(1-新戊基-3-(三氟甲基)-1H-吡咯[2,3-b]吡啶-2-甲酰胺)乙基)苯甲酸甲酯和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-81。
产物LCMS：m/z448.2[M+H] +。
实施例82:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-1H-吡咯[3,2-c]吡啶-2-甲酰胺)乙基)苯甲酸(I-82)
使用中间体59b和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-82。
产物LCMS：m/z493.2[M+H] +。
实施例83:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-1H-吡咯[3,2-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-83)
使用中间体59c和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-83。
产物LCMS：m/z493.2[M+H] +。
实施例84:(S)-4-(1-)(3-溴-1-(3-(三氟甲基)苄基)-1H-吡咯[3,2-b]吡啶-2-甲酰胺基乙基)苯甲酸(I-84)
以中间体55a和和1-(溴甲基)-3-(三氟甲基)苯(B-1)为原料，以类似制备化合物8的方法制备化合物3-溴-1-(3-(三氟甲基)苄基)-1H-吡咯并[3,2-b]吡啶-2-甲酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备(S)-4-(1-)(3-溴-1-(3-(三氟甲基)苄基)-1H-吡咯[3,2-b]吡啶-2-甲酰胺基乙基)苯甲酸甲酯。
使用中间体(S)-4-(1-)(3-溴-1-(3-(三氟甲基)苄基)-1H-吡咯[3,2-b]吡啶-2-甲酰胺基乙基)苯甲酸甲酯和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-84。
产物LCMS：m/z546.1[M+H] +。
实施例85:(S)-4-(1-(3-乙炔基-1-(3-(三氟甲基)苄基)-1H-吡咯[2,3-b]吡啶-2-甲酰胺基乙基)苯甲(I-85)
使用中间体63b和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-85。
产物LCMS：m/z491.2[M+H] +。
实施例86:(S,E)-4-(1-(3-(丙基-1-烯-1-基)-1-(3-(三氟甲基)苄基)-1H-吡咯并[3,2-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-86)
使用中间体63c和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-86。
产物LCMS：m/z508.2[M+H] +。
实施例87:(S,E)-4-(1-(3-(丙基-1-烯-1-基)-1-(3-(三氟甲基)苄基-1H-吡咯并[2,3-b]吡啶-2- 甲酰胺基(乙基)苯甲酸(I-87)
使用中间体63d和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-87。
产物LCMS：m/z508.2[M+H] +。
实施例88:(S)-4-(1-(3-氰基-1-(3-氟苄基)-1H-吡咯[2,3-c]吡啶-2-甲酰胺)乙基)苯甲酸(I-88)
使用中间体55b和三氟溴苄(B-41)为原料，以类似制备化合物8的方法制备化合物3-氰基-1-(3-氟苄基)-1H-吡咯并[2,3-c]吡啶-2-羧酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备(S)-4-(1-(3-氰基-1-(3-氟苄基)-1H-吡咯[2,3-c]吡啶-2-甲酰胺)乙基)苯甲酸甲酯。
使用中间体(S)-4-(1-(3-氰基-1-(3-氟苄基)-1H-吡咯[2,3-c]吡啶-2-甲酰胺)乙基)苯甲酸甲酯和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-88。
产物LCMS：m/z443.2[M+H] +。
实施例89:(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-1H-吡咯并[2,3-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-89)
以中间体55d和1-(溴甲基)-3-(三氟甲基)苯(B-1)为原料，以类似制备化合物8的方法制备化合物3-溴-1-(3-(三氟甲基)苄基)-1H-吡咯并[2,3-b]吡啶-2-甲酸甲酯，后续步骤中的甲酯水解以类似于合成中间体9的方法制备，产物与(S)-4-(1-氨基乙基)苯甲酸甲酯(C-1)的缩合以类似于合成I-1的方法制备(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-1H-吡咯并[2,3-b]吡啶-2-甲酰胺基(乙基)苯甲酸甲酯。
使用中间体(S)-4-(1-(3-溴-1-(3-(三氟甲基)苄基)-1H-吡咯并[2,3-b]吡啶-2-甲酰胺基(乙基)苯甲酸甲酯和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-89。
产物LCMS：m/z546.1[M+H] +。
实施例90:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-1,4,6,7-四氢硫吡喃并[4,3-b]吡咯-2-甲酰胺基)乙基)苯甲酸(I-90)
使用中间体50g和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-90。
产物LCMS：m/z514.2[M+H] +。
实施例91:(S)-4-(1-(3-氰基-1-(3-(三氟甲基)苄基)-1H-吡咯并[2,3-b]吡啶-2-甲酰胺基(乙基)苯甲酸(I-91)
使用中间体59d和氢氧化钠为原料，以类似制备化合物I-6的方法制备化合物I-91。
1H NMR(400MHz,DMSO-d 6)δ12.74(s,1H),8.81(d,J＝8.5Hz,1H),8.65(d,J＝5.7Hz,1H),8.49(dd,J＝8.0,0.7Hz,1H),7.78(d,J＝8.2Hz,2H),7.67(d,J＝8.8Hz,2H),7.65–7.51(m,2H),7.51–7.35(m,3H),6.20(s,2H),5.21(p,J＝7.0Hz,1H),1.55(d,J＝7.0Hz,3H).
产物LCMS：m/z493.2[M+H] +。
实施例92:生物活性测试
选择上述实施例制备的一些化合物与对照化合物进行以下测试：
一、不同化合物对EP1及EP3受体抑制活性的测定-IP1实验：
1.按照试剂盒说明书配制1倍工作液浓度的激活缓冲液待用；
2.利用DMSO将阳性化合物及待测化合物进行梯度稀释10个浓度，然后用1倍工作液浓度激活缓冲液将10个浓度的化合物分别稀释至10倍对应浓度，振荡混匀备用；EP1阳性化合物为AH6809，EP3阳性化合物为L-798106；其中，EP1及EP3受体待测化合物的10个对应浓度均分别为200000、6666.67、2222.22、740.74、246.91、82.30、27.43、9.14、3.05、1.02nM；
3.EP1-HEK和EP3-HEK细胞经胰酶消化处理，离心后去除培养基，将细胞重悬于1倍工作液浓度的激活缓冲液中，经细胞计数后接种于384孔板中。接种密度分别为EP1约4500细胞/孔/9.1μL，EP3约5000细胞/孔/9.1μL；
4.取1.4μL步骤2中稀释好的10倍对应浓度分别加入到相应实验孔中(2个重复)，其中Max孔加入1.4μL浓度最大的10倍对应浓度的阳性化合物溶液，Min孔加入1.4μL浓度最小的10倍对应浓度的DMSO缓冲液(2v％)，离心后放置于37℃孵育10min；
5.用1倍工作液浓度的激活缓冲液配制120nM PGE2缓冲液，取3.5μL加入到EP1受体各实验孔中；配制60nM PGE2缓冲液，取3.5μL加入到EP3受体各实验孔中；离心后放置于37℃孵育1h；
6.用测试缓冲液将IP1和Anti-IP1稀释至工作浓度；
7.孵育完成后，加入3μL IP1至所有实验孔中；
8.然后加入3μL Anti-IP1至所有实验孔中，离心后于室温静置1h；
9.孵育完成后，检测665nm和620nm读值。
二、化合物对EP2及EP4受体抑制活性的测定-cAMP实验：
1.按照试剂盒说明书配制1倍工作液浓度的激活缓冲液待用；
2.利用DMSO将阳性化合物及待测化合物进行梯度稀释10个或11个浓度，然后用1倍工作液浓度的激活缓冲液将阳性化合物和DMSO分别稀释至10倍对应浓度；EP2及EP4受体待测化合物的初始浓度均为20μM，EP2受体的10个对应浓度分别为20000、6666.67、2222.22、740.74、246.91、82.30、27.43、9.14、3.05、1.02nM；EP4受体的11个对应浓度分别为20000、6666.67、2222.22、740.74、246.91、82.30、27.43、9.14、3.05、1.02、0.34nM；
3.EP2-HEK和EP4-HEK细胞胰酶消化处理，离心后去除培养基，将细胞重悬于1倍工作液浓度的激活缓冲液中，经细胞计数后接种于384孔板中。接种密度均为1500细胞/孔/5μL；
4.取1μL步骤2中稀释好的10倍对应浓度化合物分别加入至相应实验孔中,其中Max 孔加入1μL浓度最大的10倍对应浓度的阳性化合物，Min孔加入1μL浓度最小的10倍对应浓度DMSO缓冲液(2v％)，离心后放置于37℃孵育15min；
5.用1倍工作液浓度的激活缓冲液配制0.125nM PGE2缓冲液，取4μL加入到EP2受体各实验孔中；配制100nM PGE2缓冲液，取4μL加入到EP4受体各实验孔中；离心后放置于37℃孵育30min；
6.用测试缓冲液将Eu-cAMP稀释至工作浓度，取5μL/孔加入相应实验孔中；
7.将ULight TM-anti-cAMP用测试缓冲液稀释至工作浓度，然后取5μL/孔加入相应实验孔中；离心后放置于室温孵育1h；
8.孵育完成后，检测665nm和620nm读值。
cAMP试验中一些实施例对人前列腺素E2受体(EP1、EP2、EP3、EP4)活性抑制情况如表23-24所示。
表23 化合物对EP4酶抑制浓度IC 50
Figure PCTCN2021127887-appb-000072
表24 化合物对人前列腺素E2受体抑制浓度IC 50
Figure PCTCN2021127887-appb-000073
Figure PCTCN2021127887-appb-000074
表24显示实施例6(I-6)化合物比E7046对EP受体亚型有更好的选择性，对EP4的抑制较强，对EP1/EP2/EP3无任何抑制。
三、细胞活性测试
具体试验方案如下：
(1)第0天:铺板
将细胞用0.25％Trypsin-EDTA消化，重悬细胞用自动细胞计数器计数。根据播种密度,将细胞悬浮液稀释至所需密度。每个孔加入100μL细胞37℃，5％CO 2培养过夜。
(2)第1天:化合物配制
用DMSO将表20中的化合物配成200倍终溶液(化合物终溶液浓度为10uM)。在197μL的培养基中加入3μL的200倍终溶液，配成3倍终溶液。将50μL的3倍终溶液加入孔板中，37℃，5％CO 2，培养72小时。
(3)第4天:检测
将测试孔板平衡到室温。每孔加入40μL的CellTiter-
Figure PCTCN2021127887-appb-000075
试剂，振荡2分钟，室温静置孵育60分钟。用Envision检测。
(4)检测完毕后，进行数据分析
首先使用GraphPad Prism 5计算IC 50，计算公式如下：
％Inh＝(Max signal-Compound signal)/(Max signal-Min signal)×100.
其中，Max signal是上述第(2)步骤中的孔板中不加入化合物只加入DMSO所测试的结果，Min signal是上述第(2)步骤中整个孔板中只加入培养基所测试的结果。结果如表25所示。
表25 化合物对肿瘤细胞增殖抑制IC 50
Figure PCTCN2021127887-appb-000076
四、体内测试
BALB/c小鼠从上海吉辉实验动物饲养有限公司订购，并在5-6周龄分配到该研究中。动物养殖、饲养和健康条件遵照动物福利指南。小鼠结直肠癌CT-26细胞由RPMI-1640培养基+10％FBS培养基培养，在接种前至少传代3次。每只小鼠接种约2×10 5小鼠结直肠癌 CT-26细胞，细胞收集在无菌的PBS溶液中，接种体积为100μL左右。BALB/c小鼠事先用3-4％异氟烷麻醉。当肿瘤生长到平均约100-200mm 3左右时，24只肿瘤大小合适的小鼠根据肿瘤大小和体重被随机分成4组，每组6只，分别是Vehicle(G1组)、E7046 150mg/kg(G2组)、实施例6(I-6)化合物75mg/kg(G3组)、实施例6(I-6)化合物150mg/kg(G4组)，4组均口服给药11天。肿瘤体积抑瘤率的结果如表26所示。
肿瘤体积(V)的计算方法如下：V＝(长×宽 2)/2。
抑瘤率(TGI)＝[1-(TV t-TV initial)/(CV t-CV initial)]*100％；其中，
TV t:Day 11治疗组的肿瘤体积，TV initial：Day 0治疗组的肿瘤体积CV t：Day 11对照组肿瘤体积，CV initial：Day 0对照组的肿瘤体积。
表26 不同化合物对结直肠癌CT-26细胞荷瘤小鼠肿瘤增值率的影响
Figure PCTCN2021127887-appb-000077
注释：p.o.q.d.*11D表示口服/天，给药11天；Day11表示给药后第11天。
结合图1和表26可以看出，实施例6(I-6)化合物和E7046对结直肠癌CT-26细胞荷瘤小鼠肿瘤生长有明显的抑制作用，同等剂量下实施例6(I-6)化合物明显优于E7046。
以上仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。

Claims (13)

  1. 一种化合物，其特征在于，其结构式如式I所示：
    Figure PCTCN2021127887-appb-100001
    其中：
    环A选自
    Figure PCTCN2021127887-appb-100002
    其中，W 1和W 2独立地选自满足价态的C或N；或者
    环A选自
    Figure PCTCN2021127887-appb-100003
    其中，W 3、W 4独立地选自满足价态的C或N，W 5、W 6、W 7和W 8独立地选自CH 2、NH、O或S；
    R 1选自取代或未取代的C 1-10直链或支链烷基、取代或未取代的C 3-10环烷基、取代或未取代的C 1-3氘代烷基、取代或未取代的C 7-12烷基芳基或取代或未取代的C 6-10烷基杂芳基；
    当X选自NH、O、S或CH 2时，则R 2选自氢、取代或未取代的C 6-14芳基、取代或未取代的C 5-10杂芳基、取代或未取代的C 3-7饱和碳环或取代或未取代的C 1-10直链或支链烷基；
    当X选自卤素、CN、CONH 2、取代或未取代的C 1-3链状或环状烷基、乙烯基、乙炔基时，则R 2不存在；
    R 3和R 4独立地选自氢、卤素、CN、取代或未取代的C 1-3烷基、或取代或未取代的C 1-3烷氧基；
    R 5和R 6独立地选自氢、C 1-3直链或支链烷基；或R 5和R 6组成C 3-4环烷基或C 3-4杂环烷基；
    G选自氢、烃基醇基、四氮唑基、-CONHSO 2R 7、-CO 2R 7、-CONHR 7、-SO 2NHR 7或NHSO 2R 7；
    其中R 7选自氢、取代或未取代的C 1-5烷基、芳基或杂芳基。
  2. 根据权利要求1所述的化合物，其特征在于，式I中的-XR 2选自一种如下结构式的 基团：
    Figure PCTCN2021127887-appb-100004
  3. 根据权利要求1所述的化合物，其特征在于，式I中的G选自一种如下结构式的基团：
    Figure PCTCN2021127887-appb-100005
  4. 根据权利要求1所述的化合物，其特征在于，所述卤素选自氟、氯或溴原子；所述取代或未取代的C 1-10烷基、取代或未取代的C 1-3烷基或取代或未取代的C 1-5烷基中包括C 1-3氟代烷基；所述氟代烷基选自三氟甲基、二氟甲基、二氟氘代甲基或一氟甲基；取代或未取代的C 1-3氘代烷基选自三氘代甲基、二氟氘代甲基或五氘代乙基。
  5. 根据权利要求1所述的化合物，其特征在于：
    X选自NH、O、S或CH 2，R 2选自氢、取代的C 6-14芳基、取代的C 5-10杂芳基或取代的C 5-6饱和碳环；或者
    X选自Cl、Br、F、CF 3、CN或CONH 2，R 2不存在。
  6. 根据权利要求1所述的化合物，其特征在于，其结构式如式Ia-Ic之一所示：
    Figure PCTCN2021127887-appb-100006
  7. 根据权利要求1所述的化合物，其特征在于，其结构式如式Id-If之一所示：
    Figure PCTCN2021127887-appb-100007
  8. 根据权利要求1所述的化合物，其特征在于，其结构式如式Ig-Ih所示：
    Figure PCTCN2021127887-appb-100008
    其中，W 5、W 6、W 7和W 8独立地选自CH 2、NH、O或S。
  9. 根据权利要求1所述的化合物，其特征在于，其结构式如下之一所示：
    Figure PCTCN2021127887-appb-100009
    Figure PCTCN2021127887-appb-100010
    Figure PCTCN2021127887-appb-100011
    Figure PCTCN2021127887-appb-100012
  10. 根据权利要求1所述的化合物，其特征在于，其结构式如下之一所示：
    Figure PCTCN2021127887-appb-100013
    Figure PCTCN2021127887-appb-100014
    Figure PCTCN2021127887-appb-100015
  11. 权利要求1-10中任一项所述的化合物，或其药学上可接受的盐、立体异构体、氘取代衍生物、其水合物或溶剂化物在制备药物中的用途，所述药物用于治疗被EP 4受体拮抗作用减轻的病症或抑制细胞或酶的增殖。
  12. 根据权利要求11所述的应用，其特征在于，所述病症选自癌症、炎性疾病和疼痛中的一种或几种。
  13. 根据权利要求11所述的应用，其特征在于，所述病症选自淋巴瘤、结直肠癌、前列腺癌、乳腺癌、尿路上皮癌、肺癌、非小细胞肺癌、三阴性乳腺癌、宫颈癌、肝癌、COX相关疼痛、炎症、炎症性疼痛和偏头痛中的一种或几种。
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