Pyrazole amide derivatives as ep4 receptor antagonists and their use in cancer and inflammation

Abstract

This invention proposes a novel compound that effectively antagonizes the EP4 receptor, which is a compound of formula (I), or a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug of a compound of formula (I), wherein R 1 The compounds are selected from H or halogens; halogens are selected from F, Cl, Br, and I. The compounds of this invention can effectively antagonize the EP4 receptor, have better pharmacokinetic properties, higher in vivo exposure, lower dosage, and better compliance, and are of great significance and promising application for scientific research on the EP4 receptor and the prevention and treatment of related diseases.

Description

[0001] This application claims priority to an earlier application filed by the applicant with the China National Intellectual Property Administration on August 18, 2020, with patent application number 202010835348.8 and entitled "Pyrazolamide Derivatives as EP4 Receptor Antagonists and Their Use in Cancer and Inflammation". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0002] This invention relates to the fields of chemistry and medicine, and more specifically, to pyrazole amide derivatives and their uses. Background Technology

[0003] 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.

[0004] 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.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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

[0009] The present invention aims to provide a compound that can effectively antagonize EP4, which can serve as an improvement or replacement for current drugs or EP4 antagonists.

[0010] Therefore, the present invention proposes a compound, which is the compound of formula (I), or a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug of the compound of formula (I):

[0011]

[0012] Among them, R 1 Selected from H or halogens; halogens are selected from F, Cl, Br, and I.

[0013] The compounds according to embodiments of the present invention can effectively antagonize the EP4 receptor, have better pharmacokinetic properties, higher in vivo exposure, lower dosage, and better compliance. As EP4 receptor antagonists, they are of great significance and have promising applications for scientific research on the EP4 receptor and the prevention and treatment of related diseases.

[0014] According to an exemplary embodiment of the present invention, the compound shown in formula (I) may be further preferably any of the following compounds:

[0015]

[0016] According to embodiments of the present invention, the pharmaceutically acceptable salt is selected from at least one of the following: sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, hydrochloric acid, formic acid, acetic acid, propionic acid, benzenesulfonic acid, benzoic acid, phenylacetic acid, salicylic acid, alginic acid, aminoanilic acid, camphoric acid, citric acid, vinyl sulfonic acid, formic acid, fumaric acid, furoic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, mucoic acid, dihydroxynaphthyl acid, pantothenic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, p-toluenesulfonic acid, malonic acid, 2-hydroxypropionic acid, oxalic acid, glycolic acid, glucuronic acid, galacturonic acid, citric acid, lysine, arginine, aspartic acid, cinnamic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, or trifluoromethanesulfonic acid. Those skilled in the art will understand that, in addition to pharmaceutically acceptable salts, the present invention may employ other types of salts that can be used as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts, or for the identification, characterization, or purification of the compounds of the present invention.

[0017] In a second aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises: a pharmaceutically acceptable excipient and the aforementioned compound. The pharmaceutical composition according to embodiments of the invention can effectively antagonize the EP4 receptor, exhibits better pharmacokinetic properties, higher in vivo exposure, lower dosage, and better compliance. As an EP4 receptor antagonist, it has significant implications and promising applications for scientific research on the EP4 receptor and the prevention and treatment of related diseases.

[0018] In a third aspect, the invention provides for the use of the aforementioned compounds or pharmaceutical compositions in the preparation of a medicament. According to embodiments of the invention, the medicament is used to treat or prevent EP4-related diseases. As previously stated, the compounds or pharmaceutical compositions according to embodiments of the invention can effectively antagonize the EP4 receptor, thereby enabling their use in the prevention or treatment of EP4 receptor-related diseases.

[0019] According to embodiments of the present invention, the use may further include at least one of the following additional technical features:

[0020] According to embodiments of the present invention, the drug is used to treat or prevent diseases selected from at least one of the following: inflammatory diseases, pain, cancer, metabolic diseases, and urinary system diseases.

[0021] According to an embodiment of the present invention, the inflammatory disease includes at least one of the following: arthritis, rheumatoid arthritis.

[0022] According to embodiments of the present invention, the pain includes osteoarthritis pain and pain caused by endometriosis.

[0023] According to embodiments 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.

[0024] According to an embodiment of the present invention, the cancer includes solid cancer.

[0025] According to embodiments of the present invention, the cancers include 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 / or urethral cancer.

[0026] According to embodiments of the present invention, the metabolic disease includes diabetes mellitus, and the urinary system disease includes overactive bladder.

[0027] According to embodiments of the present invention, the drug is adapted to inhibit calcium flow to the EP4 receptor; the drug is adapted to bind to the EP4 receptor.

[0028] According to embodiments of the present invention, the compounds or pharmaceutical compositions of the present invention can provide patients in need with better and more effective clinical treatments or regimens. According to embodiments of the present invention, the present invention proposes a series of EP4 antagonists with novel structures, superior pharmacokinetic properties, better efficacy, and better drug-likeness, which can effectively treat EP4-related diseases or conditions.

[0029] The present invention also relates to a method for treating diseases associated with EP4, the method comprising administering to a patient a therapeutically effective dose of a pharmaceutical preparation comprising the compound described herein or a pharmaceutically acceptable salt thereof.

[0030] Terminology Definitions and Explanations

[0031] 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.

[0032] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0033] The term "pharmaceutically acceptable salt" or "its pharmaceutically acceptable salt" refers to a pharmaceutically acceptable non-toxic salt of an acid or base, including salts of inorganic acids and bases, and salts of organic acids and bases. Salts derived from inorganic bases include, but are not limited to, metal salts formed from Al, Ca, Li, Mg, K, Na, and Zn; salts derived from organic bases include, but are not limited to, salts of primary, secondary, or tertiary amines, including naturally occurring substituted or unsubstituted amines, cyclic amines, and basic ion exchange resins, such as ammonium, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, caffeine, procaine, choline, betaine, penicillin, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, or organic salts formed from polyamine resins; salts derived from inorganic and organic acids include, but are not limited to, organic salts formed from sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, hydrochloric acid, formic acid, acetic acid, etc.

[0034] In addition to pharmaceutically acceptable salts, the present invention also contemplates other salts. These may serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts, or may be used for the identification, characterization, or purification of the compounds of the present invention.

[0035] The term "stereoisomer" refers to isomers resulting from differences in the spatial arrangement of atoms in a molecule. The stereochemical definitions and conventions used in this invention are generally in accordance with those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds," John Wiley & Sons, Inc., New York, 1994. The compounds of this invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is contemplated that all stereoisomeric forms of the compounds of this invention, including but not limited to diastereomers, enantiomers, atropisomers, and geometric (or conformational) isomers, and mixtures thereof, such as racemic mixtures, are within the scope of this invention.

[0036] Many organic compounds exist in an optically active form, meaning they possess the ability to rotate the plane of plane-polarized light. 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 light rotation caused by the compound, where (–) or L indicates that the compound is levorotatory. Compounds with the prefix (+) or D are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers may also be called enantiomers, and mixtures of such isomers are generally called mixtures of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in a chemical reaction or method.

[0037] Depending on the choice of raw materials and methods, the compounds of this invention can exist as one or a mixture of possible isomers, for example, as pure optical isomers, or as mixtures of isomers, such as racemic and diastereomeric mixtures, depending on the number of asymmetric carbon atoms. Optically active (R)- or (S)- isomers can be prepared using chiral synthons or chiral formulations, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the cycloalkyl substituent may be cis- or trans (cis- or trans-) configuration.

[0038] 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. Unless otherwise stated, wedge-shaped and dashed bonds represent the absolute configuration of a stereocenter.

[0039] The compounds of the present invention containing asymmetrically substituted carbon atoms can be separated in either an optically active or racemic form. Resolution of racemic mixtures of the compounds can be performed by any of a number of methods known in the art. Exemplary methods include fractional recrystallization using a chiral resolving acid, which is an optically active salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods include, for example, optically active acids such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or various optically active camphorsulfonic acids such as the D and L forms of β-camphorsulfonic acid. Other resolving agents suitable for fractional recrystallization methods include stereoisomerically pure forms of α-methylbenzylamine (e.g., S and R forms or diastereoisomerically pure forms), 2-phenylglycine, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, etc. Resolution of racemic mixtures can also be achieved by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). This can be performed using high-performance liquid chromatography (HPLC) or supercritical fluid chromatography (SFC). The specific method, elution conditions, and column selection can be chosen by those skilled in the art based on the structure of the compound and experimental results. Furthermore, any enantiomer or diastereomeric form of the compound described in this invention can be obtained through stereoorganic synthesis using optically pure starting materials or reagents of known configuration.

[0040] Many geometric isomers of alkenes, C=N double bonds, etc., can also exist in the compounds described herein, and all such stable isomers are considered in this invention. When the compounds described herein contain an alkene double bond, unless otherwise stated, such double bond includes E and Z geometric isomers.

[0041] 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.

[0042] The term "pharmaceutical composition" refers to a mixture of one or more compounds described herein, or physiologically / pharmaceutical acceptable salts or prodrugs thereof, with other chemical components, such as physiologically / pharmaceutical acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to a living organism.

[0043] 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.

[0044] 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.

[0045] 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 tritium. 3 H), Iodine-125 125 I) or C-14 14 C). All isotopic variations of the compounds of the present invention, regardless of radioactivity, are included within the scope of the present invention.

[0046] 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. Typical examples of pharmaceutically acceptable carriers suitable for the above formulations include: sugars, such as lactose, sucrose, mannitol, and sorbitol; starches, such as corn starch, tapioca starch, and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; stearic acid; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil; nonionic, cationic, and anionic surfactants; ethylene glycol polymers; fatty alcohols; and hydrolyzed cereal solids, as well as other nontoxic and compatible excipients commonly used in pharmaceutical formulations, such as fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, and colorants.

[0047] Beneficial effects

[0048] According to embodiments of the present invention, the compounds or pharmaceutical compositions described herein can effectively antagonize EP4 receptor activity, exhibit better pharmacokinetic properties, higher in vivo exposure, lower dosage, and better compliance. They have broad application prospects in the scientific research of EP4 receptors and in the preparation of drugs for the prevention or treatment of EP4-related diseases.

[0049] 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. Detailed Implementation

[0050] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0051] Unless otherwise specified, the compounds of this invention are identified by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts are in units of 10⁻⁶. -6 (ppm). The solvents used for NMR determination were deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard was tetramethylsilane (TMS).

[0052] Liquid chromatography-mass spectrometry (LC-MS) was performed using a Waters Acquity H-class UPLC-SQD2 mass spectrometer, monitored using an Ultimate UHPLC XB C18 1.8µm 2.1mm*50mm column. Gradient elution conditions: Run at 0.6 mL / min for 3.0 min, initially holding 5% solvent B1 for 0.2 min, then increasing to 95% solvent B1 within 1.3 min, then holding at 95% solvent B1 for 1.0 min, decreasing to 5% solvent B1 within 0.1 min, and finally holding at 5% solvent B1 for 0.4 min. Percentages represent the volume percentage of a specific solvent in the total solvent volume. Solvent A1: 0.05% formic acid aqueous solution; Solvent B1: 0.05% formic acid in acetonitrile solution. Percentages represent the volume percentage of the solute in the solution. Injection volume: determined by the concentration of the reaction solution; for a typical 1.0 mg / mL sample, 1 μL is injected. Detection wavelength: 254 / 214 / 280 nm. Column temperature: 40℃. Sample tray temperature: 25℃. SQD2 mass spectrometry parameters: Ion source: ESI source. Molecular weight scan range: 150–1000. Capillary voltage: 3.5 kV. Desolvation temperature: 650℃. Ion source temperature: 150℃. Cone voltage: 30 V.

[0053] The abbreviations of this invention are defined as follows:

[0054] CuI: Cuprous iodide

[0055] DIPEA: Also written as DIEA, diisopropylethylamine, i.e., N,N-diisopropylethylamine.

[0056] DMF: N,N-dimethylformamide

[0057] DMSO: Dimethyl sulfoxide

[0058] Et3N: Triethylamine

[0059] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate

[0060] SFC: Supercritical Fluid Chromatography

[0061] THF: Tetrahydrofuran

[0062] IC 50 The half-maximum inhibitory concentration (MCI) is the concentration at which half of the maximum inhibitory effect is achieved.

[0063] Unless otherwise indicated, the compounds exemplified herein are named and numbered using ChemBioDraw Ultra 13.0.

[0064] Example 1: Preparation of the control compound

[0065]

[0066] The control compound was synthesized with reference to patent application WO2012039972A1.

[0067] The control compounds in the test examples below refer to the compounds described in Control Example 1.

[0068] Example 1: Preparation of compound I-1

[0069] (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propane-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoic acid (compound I-1)

[0070] (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(prop-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamido)ethyl)benzoic acid (Compound I-1)

[0071]

[0072] The synthetic route for compound I-1 is shown below:

[0073]

[0074] Step 1: Preparation of (S)-4-(1-(5-(3-bromo-4-fluorophenoxy)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)methyl benzoate (I-1B)

[0075] methyl(S)-4-(1-(5-(3-bromo-4-fluorophenoxy)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamido)ethyl)benzoate(I-1B)

[0076]

[0077] Methyl (S)-4-(1-(5-chloro-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoate (3.0 g, 8.1 mmol) was added to DMF (40 mL) at room temperature, along with 3-bromo-4-fluorophenol (3.1 g, 16.2 mmol), cesium carbonate (7.9 g, 24.3 mmol), cuprous iodide (308 mg, 1.62 mmol), and 1,10-phenanthroline (583 mg, 3.24 mmol). The mixture was heated to 110 °C and stirred for 16 h. Cool to room temperature, dilute with water (200 mL), extract with ethyl acetate (60 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 a white solid (S)-4-(1-(5-(3-bromo-4-fluorophenoxy)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoate (I-1B) (1.4 g, yield 32.9%).

[0078] LC-MS, M / Z (ESI): 525.6 [M+H] +

[0079] Step 2: Preparation of (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propane-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)methyl benzoate (I-1C)

[0080] methyl(S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(prop-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamido)ethyl)benzoate(I-1C)

[0081]

[0082] At room temperature, methyl (S)-4-(1-(5-(3-bromo-4-fluorophenoxy)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoate (1.28 g, 2.4 mmol) was added to DMF (30 mL). Under nitrogen protection, a DMF solution of 1 mol / L propyne (7.2 mL, 7.2 mmol), triethylamine (7.3 g, 7.2 mmol), cuprous iodide (91 mg, 0.48 mmol), and palladium dichloride dichloride (168 mg, 0.24 mmol) were added. The mixture was heated to 100 °C and stirred for 12 h. Cool to room temperature, dilute with water (200 mL), extract with ethyl acetate (60 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 a white solid (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propane-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)methyl benzoate (I-1C) (1.0 g, yield 84.5%).

[0083] LC-MS, M / Z (ESI): 486.4 [M+H] +

[0084] Step 3: Preparation of (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propane-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoic acid (I-1)

[0085] (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(prop-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamido)ethyl)benzoic acid(I-1)

[0086]

[0087] Methyl (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propane-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoate (970 mg, 2.0 mmol) was added to THF (5 mL) at room temperature, along with water (5 mL) and lithium hydroxide (144 mg, 6.0 mmol). The mixture was stirred at room temperature for 16 h. The reaction solution was concentrated to prepare a white solid (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propane-1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoic acid (600 mg, yield 36.2%).

[0088] 1 H NMR(400m Hz,DMSO-d6)δ12.8(s,1H),8.15(d,1H),7.78(d,2H),7.27(t,1H),7.15(d,2H),7.09(d, 1H),7.05(d,1H),7.02(t,1H),4.93-4.86(m,1H),3.73(s,3H),2.08(s,3H),1.29(d,3H).

[0089] LCMS(ESI) m / z: 472.4 [M+H] +

[0090] Example 2: Preparation of compound I-2

[0091] (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamide)ethyl)benzoic acid (compound I-2)

[0092] (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(prop-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carbo xamido)ethyl)benzoic acid (Compound I-2)

[0093]

[0094] The synthetic route for compound I-2 is shown below:

[0095]

[0096] Step 1: Preparation of 5-(3-bromophenol)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxaldehyde (I-2B)

[0097] 5-(3-bromophenoxy)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carbaldehyde(I-2B)

[0098]

[0099] 5-Chloro-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxaldehyde (194 mg, 1.0 mmol) (synthesized according to patent application WO2011151369A1) was added to DMSO (3 mL) at room temperature, along with 3-bromophenol (346 mg, 2.0 mmol) and potassium hydroxide (118 mg, 3.0 mmol). The mixture was heated to 130 °C and stirred for 1 h. After cooling to room temperature, the mixture was diluted with water (20 mL) and extracted with ethyl acetate (15 mL × 3). The liquid phases were separated, combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate (V / V) = 5:1) to give a colorless liquid 5-(3-bromophenol)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxaldehyde (I-2B) (140 mg, yield 42.3%).

[0100] LC-MS, M / Z (ESI): 331.2 [M+H] + .

[0101] Step 2: Preparation of 3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxaldehyde (I-2C)

[0102] 3-(difluoromethyl)-1-methyl-5-(3-(prop-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carbaldehyde(I-2C)

[0103]

[0104] At room temperature, 5-(3-bromophenol)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxaldehyde (330 mg, 1.0 mmol) was added to DMF (5 mL), along with CuI (38 mg, 0.20 mmol), palladium dichloride dichloride (70 mg, 0.10 mmol), triethylamine (305 mg, 3.0 mmol), and a DMF solution of propyne (1 mol / L, 3 mL). The mixture was then microwaved to 110 °C under nitrogen protection and stirred for 4 h. Cool to room temperature, dilute with water (20 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) = 5:1) to give a colorless liquid 3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxaldehyde (I-2C) (220 mg, yield 75.8%).

[0105] LC-MS, M / Z (ESI): 291.4 [M+H] + .

[0106] Step 3: Preparation of (3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxylic acid (I-2D)

[0107] 3-(difluoromethyl)-1-methyl-5-(3-(prop-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxylic acid(I-2D)

[0108]

[0109] Compound 3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxaldehyde (220 mg, 0.76 mmol) was added to tert-butanol (10 mL) and water (5 mL) at room temperature, along with 2-methyl-2-butene (160 mg, 2.28 mmol), sodium chlorite (206 mg, 2.28 mmol), and sodium dihydrogen phosphate (228 mg, 1.9 mmol). The mixture was stirred at room temperature for 16 h. Dilute with water (5 mL), extract with ethyl acetate (20 mL × 3), separate the liquid and combine the organic phases, dry the organic phase with anhydrous sodium sulfate, filter, concentrate, and give a white solid crude product (3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxylic acid (I-2D) (230 mg, yield 99.0%).

[0110] LC-MS, M / Z (ESI): 307.6 [M+H] + .

[0111] Step 4: Preparation of (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamide)ethyl)methyl benzoate (I-2E)

[0112] methyl(S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(prop-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamido)ethyl)benzoate(I-2E)

[0113]

[0114] Compound (3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxylic acid (100 mg, 0.30 mmol)) was added to DMF (5 mL) at room temperature, along with methyl (S)-4-(1-aminoethyl)benzoate (64 mg, 0.36 mmol), HATU (171 mg, 0.45 mmol), and DIEA (58 mg, 0.45 mmol). The mixture was stirred at room temperature for 16 h, and then water was added. Dilute with 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) = 3:1) to give a white solid (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamide)ethyl)benzoate (I-2E) (110 mg, yield 72.0%).

[0115] LC-MS, M / Z (ESI): 468.5 [M+H] + .

[0116] Step 5: Preparation of (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamide)ethyl)benzoic acid (I-2)

[0117] (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(prop-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carbo xamido)ethyl)benzoic acid(I-2)

[0118]

[0119] Methyl (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamide)ethyl)benzoate (110 mg, 0.24 mmol) was added to THF (5 mL) and water (5 mL) at room temperature, followed by the addition of lithium hydroxide (17 mg, 0.72 mmol). The mixture was stirred at room temperature for 16 h. The reaction solution was concentrated to prepare a white solid (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamide)ethyl)benzoic acid (18.5 mg, yield 17.3%).

[0120] 1 H NMR(400m Hz,DMSO-d6)δ12.8(s,1H),8.08(d,1H),7.75(d,2H),7.39(t,1H),7.24(d,1H),7.13(d, 2H),7.11(t,1H),7.11-6.96(m,2H),4.92(t,1H),3.73(s,3H),2.03(s,3H),1.24(d,3H).

[0121] LC-MS, M / Z (ESI): 454.5 [M+H] +

[0122] Preparation conditions: Welch, Ultimate C18 column, 10 nm, 21.2 nm × 250 mm. Mobile phase A was a 1‰ aqueous solution of trifluoroacetic acid, and mobile phase B was a 1‰ solution of trifluoroacetic acid acetonitrile. Gradient conditions: 0–3 min, mobile phase A maintained at 90%; 3–18 min, gradient elution, changing from 90% to 5%; 18–22 min, maintaining 5%.

[0123] Biological activity and related property test examples

[0124] Test Example 1: Determination of the inhibitory effect on EP4 receptor calcium flow

[0125] The inhibitory effect of the compounds on EP4 calcium flux was determined in 293 cells overexpressing the human EP4 receptor. Cells were rapidly thawed in a 37°C water bath, centrifuged, resuspended, and counted. Cell suspension was seeded at 20 μL / well in two 384-well plates (20,000 cells / well) and incubated overnight at 37°C in a 5% CO2 incubator. 2X Fluo-4 Direct was prepared. TM (Invitrogen, Cat#F10471) Loading buffer: Add 77 mg of probenecid (250 mM) to 1 mL of FLIPR buffer. Dispense one Fluo-4 Direct sample per tube. TM Add 10 mL of FLIPR buffer and 0.2 mL of 250 mM probenecid to crystals (F10471).

[0126] Remove a cell plate from the incubator and discard the culture medium. Add 20 μL of analysis buffer and 2X Fluo-4 Direct. TM Transfer no-wash loading buffer to a 384-well cell culture plate to a final volume of 40 μL. Incubate at 37°C, 5% CO2 for 50 min, then at room temperature for 10 min, and place in a FLIPR. Transfer 10 μL of buffer to the cell culture plate and read the fluorescence signal. Prepare a 10 mM stock solution of the agonist PGE2 in DMSO and serially dilute it with buffer to 10 concentration points of the 6X working solution. Transfer 10 μL of the agonist PGE2 to the cell culture plate, read the fluorescence signal, and calculate EC50. 80 value.

[0127] Preparing 6X EC 80 The concentration of the agonist PGE2 was determined, and the test compound was prepared into a 10 mM stock solution in DMSO solvent. The working solutions of the 6X compound at 10 concentration points were then serially diluted with buffer solution.

[0128] Take another cell plate, remove the culture medium, and add 20 μL of analysis buffer and 2X Fluo-4 Direct. TM No-wash loading buffer. Incubate at 37°C for 50 minutes in a 5% CO2 incubator, then incubate at room temperature for 10 minutes before placing in a FLIPR. Transfer 10 μL of the compound working solution, DMSO, and EP4 complete antagonist to the cell plate and read the fluorescence signal. Add 10 μL of 6X EC... 80 A concentration of the agonist PGE2 was transferred to a cell plate, fluorescence signals were read, and inhibition rate was calculated.

[0129] Inhibition rate (%) = 100 - (Test group - EP4 complete antagonist group) / (DMSO group - EP4 complete antagonist group) * 100

[0130] Based on the inhibition rates of different compound concentrations, the IC50 of the compound on EP4 calcium flux inhibition was calculated. 50 Value (i.e., intracellular Ca2+ value after overexpression of human EP4 receptor) 2+ (Drug concentration when flow is inhibited by half).

[0131] Table 1. Inhibitory effect of tested compounds on EP4 calcium flow.

[0132] control compound 21 Compound I-1 10.9 Compound I-2 12.36

[0133] Experimental results show that the compound of the present invention exhibits good inhibitory effect on EP4 calcium flow, which is superior to that of the control compound. The compound of the present invention shows a more excellent inhibitory effect on EP4 calcium flow.

[0134] Test Example 2: Assay of Radioligand EP4 Receptor Binding

[0135] 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 […]. 3 H]-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.

[0136] Analyze the data using GraphPad Prism 5 and calculate the inhibition rate using the following formula:

[0137] Inhibition rate (%) = 100 - (Test group - PGE2 group) / (DMSO group - PGE2 group) * 100

[0138] Based on the inhibition rate of the compound at different concentrations, the IC50 of the compound, determined by binding to the radioligand EP4, was calculated. 50 And Ki value.

[0139] Table 2 shows the IC50 values ​​of the tested compounds determined by binding to the radioligand EP4. 50 and Ki value

[0140] control compound 30 16 Compound I-1 9.1 5.0

[0141] Experimental results show that, compared with the control compound, the compound of the present invention has a better affinity for the EP4 receptor and is superior to the control compound, demonstrating a superior affinity for the EP4 receptor.

[0142] Test Example 3: Pharmacokinetic Study

[0143] Pharmacokinetic studies were conducted in mice using male ICR mice (20-25g, fasted overnight). Three mice were administered 5 mg / kg orally via gavage. Blood samples were collected before administration and at 15, 30 minutes, and 1, 2, 4, 8, and 24 hours post-administration. Three other mice were administered 1 mg / kg intravenously, with blood samples collected before administration and at 15, 30 minutes, and 1, 2, 4, 8, and 24 hours post-administration. 6800g of blood samples were centrifuged at 2-8℃ for 6 minutes, and plasma was collected and stored at -80℃. Plasma samples from each time point were mixed with 3-5 times the volume of acetonitrile solution containing an internal standard, vortexed for 1 minute, centrifuged at 13000 rpm at 4℃ for 10 minutes, and the supernatant was mixed with 3 times the volume of water. An appropriate amount of the mixture was analyzed by LC-MS / MS. Key pharmacokinetic parameters were analyzed using a non-compartmental model with WinNonlin 7.0 software.

[0144] For the canine pharmacokinetic study, male Beagle dogs weighing 8-10 kg were used and fasted overnight. Three Beagle dogs were administered 3 mg / kg orally via gavage. Another three Beagle dogs were administered 1 mg / kg intravenously. The remaining procedures were the same as for the mouse pharmacokinetic study.

[0145] Table 3 Results of mouse pharmacokinetics test

[0146]

[0147] Table 4 Results of canine pharmacokinetics studies

[0148]

[0149] Experimental results show that, compared with the control compound, the compound of the present invention has a lower or comparable clearance rate after intravenous administration and a higher or comparable exposure after oral administration. The exposure after oral administration in mice is about 4 times that of the control compound, and the exposure after oral administration in dogs is about 2 times that of the control compound. The compound of the present invention exhibits superior pharmacokinetic properties and good drug-likeness.

Claims

1. A compound, characterized in that, It is a compound of formula (I), or a pharmaceutically acceptable salt of a compound of formula (I): ， Among them, R 1 Selected from H or halogen; Halogens are selected from F, Cl, Br, and I.

2. The compound according to claim 1, characterized in that, The compound represented by formula (I) is any of the following compounds: 。 3. A pharmaceutical composition, characterized in that, It comprises: pharmaceutically acceptable excipients and the compound of any one of claims 1 to 2.

4. The use of the compound according to any one of claims 1 to 2 or the pharmaceutical composition according to claim 3 in the preparation of a medicament, characterized in that, The drug is used to treat or prevent EP4 receptor-related diseases.

5. The use as described in claim 4, characterized in that, The EP4 receptor-related diseases are selected from at least one of the following diseases: inflammatory diseases, pain, cancer, metabolic diseases, and urinary system diseases.

6. The use as described in claim 5, characterized in that, 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 tumors; the metabolic disease is diabetes; and the urinary system disease is selected from overactive bladder.

7. The use as described in claim 5, characterized in that, The cancers mentioned are 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 / or urethral cancer.

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