WO2022037601A1 - Pyrazolamide derivative serving as ep4 receptor antagonist and use thereof in preparation of drugs for treating cancer and inflammation - Google Patents

Abstract

A compound capable of effectively antagonizing an EP4 receptor, which compound is a compound as represented by formula (I), and a tautomer, a stereoisomer, a hydrate, a solvate, a pharmaceutically acceptable salt or a prodrug thereof, wherein R1 is selected from H or a halogen, and the halogen is selected from F, Cl, Br and I.

Description

[Title of invention formulated by ISA pursuant to Rule 37.2] Pyrazolamide derivatives as EP4 receptor antagonists and their use in the manufacture of medicaments for the treatment of cancer and inflammation

This application claims to enjoy the patent application number 202010835348.8 submitted by the applicant to the State Intellectual Property Office of China on August 18, 2020, and the name of the invention is "Pyrazolamide derivatives as EP4 receptor antagonists and their use in cancer and inflammation use" of the priority of the earlier application. The entire contents of this prior application are incorporated herein by reference.

technical field

The present invention relates to the fields of chemistry and medicine, in particular, the present invention relates to pyrazole amide derivatives and uses thereof.

Background technique

Prostaglandin E ₂ (Prostaglandin E ₂ , PGE ₂ ) is an endogenous bioactive lipid, and PGE ₂ induces a wide range of upstream and downstream-dependent biological responses by activating prostaglandin receptors (Legler, DF et al, hit. J Biochem .Cell Biol.2010,42,p.198-201), involved in the regulation of many physiological and pathological processes including inflammation, pain, renal function, cardiovascular system, lung function and cancer. PGE ₂ is reported to be highly expressed in cancerous tissues of various cancers, and it has been demonstrated that PGE ₂ is associated with the occurrence, growth and progression of cancer and disease conditions in patients. It is widely believed that PGE ₂ is associated with the activation of cell proliferation and cell death (apoptosis) and plays an important role in the processes of cancer cell proliferation, disease progression and cancer metastasis.

Among the receptors of PGE ₂ , there are four subtypes of EP1, EP2, EP3 and EP4, which are widely distributed in various tissues. Among these subtypes, PGE ₂ interferes with inflammatory responses (including immune inflammatory responses) via EP4 receptors, smooth muscle relaxation, pain, lymphocyte differentiation, hypertrophy or proliferation of mesangial cells, and secretion of gastrointestinal mucus. Therefore, it can be considered that EP4 receptor antagonists are promising as anti-inflammatory and/or analgesic drugs to treat diseases related to the PGE ₂ -EP4 pathway, such as inflammatory diseases, diseases accompanied by various pains, and the like.

EP4 is a major receptor involved in arthritic pain in rodent models of rheumatoid arthritis and osteoarthritis (see eg, J. Pharmacol. Exp. Ther., 325, 425 (2008)), which upon activation leads to intracellular The signaling molecule cAMP accumulates. EP4 receptor expression has been detected on peripheral nerve endings of pain receptors, macrophages, and neutrophils, and these cell types have been shown to be extremely important in endometriosis. Studies have reported that oral EP4 antagonists can alleviate proteinuria in type 2 diabetic mice and inhibit the progression of diabetic nephropathy. Another study reported that the activation of EP4 and the increased production of PGE ₂ in the bladder mucosa may be an important cause of overactive bladder caused by prostatitis. Intravesical injection of EP4 antagonists can effectively improve the overactive bladder after prostatitis. Thus, selective EP4 antagonists are useful in the treatment of arthritis, including arthritic pain as well as endometriosis, diabetic nephropathy, and overactive bladder. Existing treatments for arthritis are dominated by traditional NSAIDs (non-steroidal anti-inflammatory drugs) or selective COX-2 inhibitors, which can produce cardiovascular and/or gastrointestinal side effects. Selective EP4 antagonists are less likely to produce cardiovascular side effects.

PGE ₂ persistently activates EP receptors in the tumor microenvironment (produced abundantly 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), promotes the accumulation and enhances the activity of a variety of immunosuppressive cells, including type 2 tumor-associated macrophages (TAMS), Treg cells, and myeloid-derived suppressor cells (MDSCs) . One of the main features of the immunosuppressive tumor microenvironment is the presence of abundant MDSCs and TAMs, which in turn correlate with the development of cancers in patients with gastric, ovarian, breast, bladder, hepatocellular (HCC), head and neck, and other types of cancer. was closely related to poor overall survival. Furthermore, PGE ₂ has been reported to induce immune tolerance by inhibiting the accumulation of antigen-presenting dendritic cells (DCs) in tumors and inhibiting the activation of tumor-infiltrating DCs (Wang et al, Trends in Molecular Medicine 2016, 22, p. .1-3). All of these PGE2 _- mediated actions will collectively help tumor cells evade immune surveillance. PGE ₂ plays an important role in promoting tumorigenesis and development. The expression levels of PGE ₂ and its related receptors EP2 and EP4 have been found to be elevated in various malignant tumors, including colon cancer, lung cancer, breast cancer, and head and neck cancer, and are often closely related to poor prognosis (Bhooshan, N. et al. al. Lung Cancer 101, 88-91). Therefore, selectively blocking the EP2 and EP4 signaling pathways can inhibit the occurrence and development of tumors by changing the tumor microenvironment and regulating tumor immune cells.

Existing preclinical research data show that specific antagonists of EP2 and EP4 can prevent or inhibit tumor growth to varying degrees in animal models such as colon cancer, esophageal cancer, lung cancer and breast cancer. Among the PGE ₂ receptor drugs that have entered the clinic, Grapiprant, an EP4 antagonist developed by Pfizer, has been approved by the FDA for the treatment of arthritis in dogs. At the same time, it entered an anti-tumor clinical phase II study in 2015 for the treatment of prostate cancer. , non-small cell lung cancer and breast cancer and other types of solid tumors (De Vito, V. et al. J Pharm Biomed Anal 118, 251-258). E7046, an EP4 antagonist developed by Eisai, also launched a Phase I clinical study in 2015, and a Phase Ib clinical trial in combination with radiotherapy or chemoradiotherapy for rectal cancer in 2017. ONO-4578 developed by Ono Pharmaceutical was launched in 2017 for a phase I clinical study of advanced or metastatic solid tumors, and in 2018, a phase I/II clinical trial for the treatment of advanced solid tumors as a single agent or in combination with nivolumab was launched.

At present, EP4 antagonists have made certain progress in the treatment of inflammatory diseases, pain, cancer and other fields, but there is still a need to further develop new drugs as improvements or replacements of current drugs.

SUMMARY OF THE INVENTION

The present invention aims to provide a compound that can effectively antagonize EP4, which can be used as an improvement or replacement of current drugs or EP4 antagonists.

To this end, the present invention proposes a compound, which is a compound represented by formula (I), or a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable compound represented by formula (I) The salt or prodrug of:

Wherein, R ¹ is selected from H or halogen; halogen is selected from F, Cl, Br, I.

The compounds according to the embodiments of the present invention can effectively antagonize EP4 receptors, have better pharmacokinetic properties, higher in vivo exposure, lower administration doses, and better compliance, as EP4 receptor antagonists for EP4 The scientific research of receptors and the prevention and treatment of related diseases are of great significance and application prospects.

According to an exemplary embodiment of the present invention, the compound represented by formula (I) may be further preferably any of the following compounds:

According to an embodiment 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, benzene Acetic acid, salicylic acid, alginic acid, anthranilic acid, camphoric acid, citric acid, ethylene sulfonic acid, formic acid, fumaric acid, furoic acid, gluconic acid, glucuronic acid, glutamic acid, glycolic acid, isethionate acid, lactic acid, maleic acid, malic acid, mandelic acid, mucilic acid, pamoic 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 trifluoro Methanesulfonic acid. It will be appreciated by those skilled in the art that, in addition to pharmaceutically acceptable salts, the present invention can also employ other salt types that can be used as intermediates or useful in the purification of compounds or in the preparation of other pharmaceutically acceptable salts for the identification, characterization or purification of the compounds of the present invention.

In the second aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: a pharmaceutically acceptable excipient and the aforementioned compound. The pharmaceutical compositions according to the embodiments of the present invention can effectively antagonize EP4 receptors, have better pharmacokinetic properties, higher in vivo exposure, lower dosages, and better compliance, as EP4 receptor antagonists It has important significance and application prospects for the scientific research of EP4 receptor and the prevention and treatment of related diseases.

In the third aspect of the present invention, the present invention proposes the use of the aforementioned compound or the aforementioned pharmaceutical composition in the preparation of a medicament. According to an embodiment of the present invention, the medicament is used for treating or preventing EP4-related diseases. As mentioned above, the compounds or pharmaceutical compositions according to the embodiments of the present invention can effectively antagonize EP4 receptors, so that they can be used to prevent or treat EP4 receptor-related diseases.

According to an embodiment of the present invention, the use may further include at least one of the following additional technical features:

According to an embodiment of the present invention, the medicament is used to treat or prevent a disease selected from at least one of the following: inflammatory disease, pain, cancer, metabolic disease, and urinary system disease.

According to an embodiment of the present invention, the inflammatory disease includes at least one selected from the group consisting of arthritis, rheumatoid arthritis.

According to an embodiment of the present invention, the pain includes osteoarthritis pain, pain caused by endometriosis.

According to an embodiment of the present invention, the medicament 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.

According to an embodiment of the present invention, the cancer comprises solid cancer.

According to an embodiment of the present invention, the cancer includes breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, ovarian cancer, Cancer of the pancreas, prostate, skin and/or urethra.

According to an embodiment of the present invention, the metabolic disease includes diabetes, and the urinary system disease includes overactive bladder.

According to an embodiment of the present invention, the drug is suitable for inhibiting EP4 receptor calcium flux; the drug is suitable for binding to EP4 receptor.

According to the embodiments of the present invention, using the compounds or pharmaceutical compositions of the present invention, patients in need can be provided with better and more effective clinical treatment drugs or regimens. According to the embodiments of the present invention, the present invention proposes a series of EP4 antagonists with novel structure, better pharmacokinetic properties, better efficacy and good druggability, which can effectively treat EP4-related diseases or conditions.

In the fourth aspect of the present invention, the present invention provides a method for treating or preventing EP4 receptor-related diseases. According to an embodiment of the present invention, the method comprises: administering to a subject a compound or pharmaceutical composition as previously described. Thus, the methods according to the embodiments of the present invention can have a better effect of preventing or treating EP4-related diseases.

According to an embodiment of the present invention, the EP4 receptor-related disease is selected from at least one of the following diseases: inflammatory disease, pain, cancer, metabolic disease, and urinary system disease.

According to an embodiment of the present invention, the inflammatory disease is selected from arthritis, rheumatoid arthritis; the pain is selected from osteoarthritis pain, pain caused by endometriosis; the cancer is selected from solid cancer; The metabolic disease is diabetes, and the urinary system disease is selected from overactive bladder.

According to an embodiment of the present invention, the cancer is selected from breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, medulloblastoma, ovarian cancer , pancreatic, prostate, skin and/or urethral cancer.

According to an embodiment of the invention, the subject is administered radiation therapy and/or antibody therapy in combination.

According to an embodiment of the present invention, the antibody therapy is selected from one or a combination of CTLA4 antibody therapy, PDL1 antibody therapy and PD1 antibody therapy.

Definition and Explanation of Terms

Unless otherwise stated, definitions of groups and terms set forth in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions set forth in tables, and definitions of specific compounds in the examples etc., can be arbitrarily combined and combined with each other. Such combinations, group definitions and compound structures after the combination should fall within the scope described in the specification of the present application.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without more toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" or "pharmaceutically acceptable salts thereof" refers to pharmaceutically acceptable salts of non-toxic acids or bases, including salts of inorganic acids and bases, 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, benzyl penicillin, ethylenediamine, glucosamine, Organic salts of methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine or polyamine resins; salts derived from inorganic and organic acids include But not limited to organic salts formed by sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, hydrochloric acid, formic acid, acetic acid, etc.

In addition to the pharmaceutically acceptable salts, other salts are also contemplated by the present invention. They may serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts or may be used in the identification, characterization or purification of the compounds of the present invention.

The term "stereoisomer" refers to isomers that result from different arrangements of atoms in a molecule in space. Stereochemistry definitions and conventions used herein are generally in accordance with SP 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”, defined by John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric centers or chiral centers and therefore exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present invention are contemplated, including but not limited to diastereomers, enantiomers, and atropisomers and geometric (or conformational) isomers and mixtures thereof, such as racemic mixtures, are within the scope of the present invention.

Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule with respect to the chiral center (or centers) in the molecule. The prefixes D and L or (+) and (–) are symbols used to designate the rotation of plane polarized light by the compound, where (–) or L indicates that the compound is levorotatory. Compounds prefixed with (+) or D are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as a mixture of enantiomers. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or method Spin body.

Depending on the choice of starting materials and methods, the compounds of the present invention may exist as one of the possible isomers or as a mixture thereof, for example, as pure optical isomers, or as mixtures of isomers, such as racemic and non-isomeric isomers. A mixture of enantiomers, depending on the number of asymmetric carbon atoms. Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral preparations, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be of E or Z configuration; if the compound contains a disubstituted cycloalkyl, the substituent of the cycloalkyl may be cis or trans (cis- or trans-) structure.

When the bond to the chiral carbon in the formula of the present invention is depicted in line, it should be understood that both the (R) and (S) configurations of the chiral carbon and the resulting enantiomerically pure compounds and Mixtures of both are included within the scope of this formula. A schematic representation of racemate or enantiomerically pure compounds herein is from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise stated, wedge and dashed bonds are used to indicate the absolute configuration of a stereocenter.

Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Resolution of racemic mixtures of compounds can be carried out by any of a number of methods known in the art. Exemplary methods include fractional recrystallization using chiral resolving acids, which are optically active salt-forming organic acids. Suitable resolving agents for the fractional recrystallization process are, 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 β- D and L forms of camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include α-methyl-benzylamine in stereoisomerically pure form (eg, S and R forms or diastereomerically pure form), 2-phenylglycinol, Norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, etc. Resolution of the racemic mixture can also be performed by elution on a column packed with an optically active resolving agent (eg, dinitrobenzoylphenylglycine). It can be carried out by high performance liquid chromatography (HPLC) or supercritical fluid chromatography (SFC). Selection of specific methods, elution conditions, and selection of chromatographic columns can be selected by those skilled in the art according to the structures of compounds and test results. Further, any enantiomer or diastereomer of the compounds described in the present invention can also be obtained by stereoorganic synthesis using optically pure starting materials or reagents of known configuration.

Many geometric isomers of olefins, C=N double bonds, etc. may also exist in the compounds described herein, and all such stable isomers are contemplated in the present invention. When the compounds described herein contain olefinic double bonds, unless otherwise specified, such double bonds include both E and Z geometric isomers.

The term "tautomer" refers to an isomer of a functional group resulting from the rapid movement of an atom in two positions in a molecule. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertible species. Proton tautomers arise from the migration of covalently bonded hydrogen atoms between two atoms. Tautomers generally exist in equilibrium, and attempts to separate individual tautomers usually result in a mixture whose physicochemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the ketone form predominates; in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

The term "pharmaceutical composition" means a mixture of one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as a physiologically/pharmaceutically acceptable carrier and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.

The term "solvate" means that a compound of the present invention or a salt thereof includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces, and when the solvent is water, it is a hydrate.

The term "prodrug" refers to a compound of the invention that can be converted under physiological conditions or by solvolysis to a biologically active compound. The prodrugs of the present invention are prepared by modifying functional groups in the compounds, which modifications can be removed by conventional procedures or in vivo to yield the parent compounds. Prodrugs include compounds formed by connecting a hydroxyl or amino group in the compounds of the present invention to any group. When the prodrugs of the compounds of the present invention are administered to mammalian individuals, the prodrugs are cleaved to form free hydroxyl, free the amino group.

The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of classes of the term "excipient" include, without limitation, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of a pharmaceutical formulation, ie make the formulation more suitable for direct compression by increasing flowability and/or stickiness. Examples of typical "pharmaceutically acceptable carriers" suitable for use in the above formulations are: carbohydrates such as lactose, sucrose, mannitol and sorbitol; starches such as corn starch, tapioca starch and potato starch; cellulose and derivatives thereof compounds such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; calcium phosphates such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid ; alkaline earth metal stearate salts 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 Glycol polymers; fatty alcohols; and cereal hydrolyzed solids and other non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants Excipients commonly used in pharmaceutical preparations.

beneficial effect

According to the embodiments of the present invention, the compounds or pharmaceutical compositions of the present invention can effectively antagonize EP4 receptor activity, have better pharmacokinetic properties, higher in vivo exposure, lower dosage and better compliance . It has broad application prospects in the scientific research of EP4 receptor and the preparation of drugs for preventing or treating EP4-related diseases.

Additional aspects and advantages of the present invention will be set forth, in part, from the following description, and in part will be apparent from the following description, or may be learned by practice of the invention.

detailed description

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

Unless otherwise specified, the compounds of the present invention are identified by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). The units of NMR shifts are ^10-6 (ppm). The solvents for NMR measurement are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS).

Liquid-mass spectrometry (LC-MS) was determined by Waters Acquity H-class UPLC-SQD2 mass spectrometer and monitored using Ultimate UHPLC XB C18 1.8um 2.1mm*50mm chromatographic column. Gradient elution condition 1: run at a flow rate of 0.6 mL/min for 3.0 min, start with 5% solvent B1 for 0.2 min, increase from 5% solvent B1 to 95% solvent B1 within 1.3 min, and then keep 95% solvent B1 for 1.0 min, 0.1 It is reduced to 5% solvent B1 within min, and then 5% solvent B1 is maintained for 0.4min, and the percentage is the volume percentage of a certain solvent in the total solvent volume. Wherein, solvent A1: an aqueous solution of 0.05% formic acid; solvent B1: an acetonitrile solution of 0.05% formic acid. The percentage is the volume percent of the solute in the solution. Injection volume: determined by the concentration of the reaction solution, generally 1.0mg/mL concentration sample, injection 1μL; detection wavelength: 254/214/280nm; chromatographic column temperature: 40°C; sample tray temperature 25°C; Source: ESI source; molecular weight scan range: 150-1000; capillary voltage: 3.5kV; desolvation temperature: 650°C; ion source temperature: 150°C; cone voltage: 30V.

Abbreviations of the present invention are defined as follows:

CuI: cuprous iodide

DIPEA: can also be written as DIEA, diisopropylethylamine, that is, N,N-diisopropylethylamine

DMF: N,N-Dimethylformamide

DMSO: Dimethyl Sulfoxide

Et ₃ N: triethylamine

HATU: 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate

SFC: Supercritical Fluid Chromatography

THF: Tetrahydrofuran

IC ₅₀ : the half inhibitory concentration, which refers to the concentration at which half of the maximum inhibitory effect is achieved.

Unless indicated to the contrary, compounds exemplified herein are named and numbered using ChemBioDraw Ultra 13.0.

Comparative Example 1: Preparation of Control Compounds

The reference compound was synthesized with reference to patent application WO2012039972A1.

The control compounds in the following test examples all refer to the compounds described in Control Example 1.

Example 1: Preparation of Compound I-1

(S)-4-(1-(3-(Difluoromethyl)-5-(4-fluoro-3-(propan-1-yn-1-yl)phenoxy)-1-methyl-1H - Pyrazole-4-carboxamide)ethyl)benzoic acid (Compound I-1)

(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)

The synthetic route of compound I-1 is as follows:

The first step: (S)-4-(1-(5-(3-bromo-4-fluorophenoxy)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4- Preparation of methyl formamide)ethyl)benzoate (I-1B)

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

Compound (S)-methyl 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), 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) were added ), 1,10-phenanthroline (583 mg, 3.24 mmol), heated to 110 °C and stirred for 16 h. It was cooled to room temperature, diluted with water (200 mL), extracted with ethyl acetate (60 mL×3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column ( Petroleum ether:ethyl acetate (V/V)=2:1) to give white solid (S)-4-(1-(5-(3-bromo-4-fluorophenoxy)-3-(difluoromethyl) yl)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoic acid methyl ester (1-1B) (1.4 g, 32.9% yield).

LC-MS, M/Z(ESI): 525.6[M+H] ⁺

The second step: (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propan-1-yn-1-yl)phenoxy)-1- Preparation of methyl-1H-pyrazole-4-carboxamide)ethyl)benzoic acid methyl ester (I-1C)

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)

Compound (S)-4-(1-(5-(3-bromo-4-fluorophenoxy)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4- Formamide)ethyl)methyl benzoate (1.28g, 2.4mmol) was added to DMF (30mL), 1mol/L propyne in DMF solution (7.2ml, 7.2mmol), triethylamine (7.3ml) were added under nitrogen protection g, 7.2 mmol), cuprous iodide (91 mg, 0.48 mmol), bistriphenylphosphine palladium dichloride (168 mg, 0.24 mmol), heated to 100° C. and stirred for 12 h. It was cooled to room temperature, diluted with water (200 mL), extracted with ethyl acetate (60 mL×3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column ( Petroleum ether:ethyl acetate (V/V)=2:1) to give white solid (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propane-) Methyl 1-yn-1-yl)phenoxy)-1-methyl-1H-pyrazole-4-carboxamide)ethyl)benzoate (1-1C) (1.0 g, 84.5% yield).

LC-MS, M/Z(ESI): 486.4[M+H] ⁺

The third step: (S)-4-(1-(3-(difluoromethyl)-5-(4-fluoro-3-(propan-1-yn-1-yl)phenoxy)-1- Preparation of methyl-1H-pyrazole-4-carboxamide)ethyl)benzoic acid (I-1)

(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)

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

¹ H NMR (400m Hz, DMSO-d ₆ )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).

LCMS(ESI)m/z:472.4[M+H] ⁺

Example 2: Preparation of Compound 1-2

(S)-4-(1-(3-(Difluoromethyl)-1-methyl-5-(3-(propan-1-yn-1-yl)phenoxy)-1H-pyrazole- 4-Carboxamide)ethyl)benzoic acid (Compound 1-2)

(S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(prop-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamido)ethyl)benzoic acid (Compound I-2)

The synthetic route of compound I-2 is as follows:

The first step: the preparation of 5-(3-bromophenol)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carbaldehyde (I-2B)

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

Compound 5-chloro-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carbaldehyde (194 mg, 1.0 mmol) (synthesized with reference to patent application WO2011151369A1) was added to DMSO (3 mL) at room temperature , added 3-bromophenol (346 mg, 2.0 mmol), potassium hydroxide (118 mg, 3.0 mmol), heated to 130 °C, and stirred for 1 h. It was cooled to room temperature, diluted with water (20 mL), extracted with ethyl acetate (15 mL×3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column ( Petroleum ether:ethyl acetate (V/V)=5:1) to get colorless liquid 5-(3-bromophenol)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4- Formaldehyde (I-2B) (140 mg, 42.3% yield).

LC-MS, M/Z (ESI): 331.2 [M+H] ⁺ .

The second step: 3-(difluoromethyl)-1-methyl-5-(3-(propan-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carbaldehyde (I- 2C) Preparation

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

5-(3-Bromophenol)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carbaldehyde (330 mg, 1.0 mmol) was added to DMF (5 mL) at room temperature and CuI was added (38mg, 0.20mmol), ditriphenylphosphonium palladium dichloride (70mg, 0.10mmol), triethylamine (305mg, 3.0mmol), propyne in DMF (1mol/L, 3mL), microwave under nitrogen protection Heat to 110°C and stir for 4h. It was cooled to room temperature, diluted with water (20 mL), extracted with ethyl acetate (20 mL×3), separated, and the organic phases were combined. The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was separated and purified by silica gel column ( Petroleum ether:ethyl acetate (V/V)=5:1) to give a colorless liquid 3-(difluoromethyl)-1-methyl-5-(3-(propan-1-yn-1-yl) Phenoxy)-1H-pyrazole-4-carbaldehyde (I-2C) (220 mg, 75.8% yield).

LC-MS, M/Z (ESI): 291.4 [M+H] ⁺ .

The third step: (3-(difluoromethyl)-1-methyl-5-(3-(propane-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxylic acid (I -2D) preparation

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

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

LC-MS, M/Z (ESI): 307.6 [M+H] ⁺ .

The fourth step: (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propan-1-yn-1-yl)phenoxy)-1H - Preparation of methyl pyrazole-4-carboxamide)ethyl)benzoate (I-2E)

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

Compound (3-(difluoromethyl)-1-methyl-5-(3-(propan-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxylic acid (100 mg) was prepared at room temperature , 0.30 mmol) was added to DMF (5 mL), (S)-methyl 4-(1-aminoethyl)benzoate (64 mg, 0.36 mmol), HATU (171 mg, 0.45 mmol), DIEA (58 mg, 0.45 mmol) were added mmol), stirred at room temperature for 16 h, added water (20 mL) to dilute, extracted with ethyl acetate (10 mL×3), separated, combined the organic phases, dried the organic phase with anhydrous sodium sulfate, filtered, concentrated, and the residue was filtered through a silica gel column Separation and purification (petroleum ether:ethyl acetate (V/V)=3:1) gave white solid (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3) -(Propan-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamide)ethyl)benzoic acid methyl ester (I-2E) (110 mg, 72.0% yield).

LC-MS, M/Z (ESI): 468.5 [M+H] ⁺ .

The fifth step: (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propan-1-yn-1-yl)phenoxy)-1H - Preparation of pyrazole-4-carboxamide)ethyl)benzoic acid (I-2)

(S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(prop-1-yn-1-yl)phenoxy)-1H-pyrazole-4-carboxamido)ethyl)benzoic acid(I-2)

The starting material (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propan-1-yn-1-yl)phenoxy)-1H -Pyrazole-4-carboxamide)ethyl)benzoic acid methyl ester (110mg, 0.24mmol) was added to THF (5mL) and water (5mL), lithium hydroxide (17mg, 0.72mmol) was added, and stirred at room temperature for 16h . The reaction solution was concentrated to prepare a white solid (S)-4-(1-(3-(difluoromethyl)-1-methyl-5-(3-(propan-1-yn-1-yl)phenoxy yl)-1H-pyrazole-4-carboxamide)ethyl)benzoic acid (18.5 mg, 17.3% yield).

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).

LC-MS, M/Z(ESI): 454.5[M+H] ⁺

Preparation conditions: Welch, Ultimate C18 column, 10 nm, 21.2 nm x 250 mm. Mobile phase A is 1‰ trifluoroacetic acid pure aqueous solution, and mobile phase B is 1‰ trifluoroacetic acid acetonitrile solution. Gradient conditions: 0 to 3 minutes, mobile phase A maintained at 90%, 3 to 18 minutes for gradient elution, from 90% to 5%, and 18 to 22 minutes to maintain 5%.

Biological Activity and Related Properties Test Examples

Test Example 1: Determination of Inhibitory Effect on EP4 Receptor Calcium Flux

Determination of the inhibitory effect of compounds on EP4 calcium flux was performed in 293 cells overexpressing the human EP4 receptor. Cells were rapidly thawed in a 37°C water bath, centrifuged, resuspended, and counted. The cell suspension was seeded in 2 384-well plates (20,000 cells/well) at 20 μL/well and placed in a 37° C., 5% CO ₂ incubator overnight. Prepare 2X Fluo-4 Direct ^™ (Invitrogen, Cat# F10471) loading buffer: To 1 mL of FLIPR buffer add 77 mg probenecid at 250 mM. Add 10 mL of FLIPR buffer, and 0.2 mL of 250 mM probenecid to each tube of Fluo-4 Direct ^™ crystals (F10471).

Remove a cell plate from the incubator and remove the medium, add 20 μL of Assay Buffer and 2X Fluo-4 Direct ^™ No-Wash Loading Buffer to a 384-well cell culture plate in a final volume of 40 μL. Incubate in a 37 °C, 5% _CO2 incubator for 50 min and room temperature for 10 min before placing in the FLIPR. Transfer 10 μL of buffer to the cell plate and read the fluorescent signal. The agonist PGE ₂ was prepared as a 10 mM stock solution in DMSO solvent, and 10 concentration point 6X working solutions were serially diluted in buffer. Transfer 10 μL of the agonist PGE ₂ to the cell plate, read the fluorescent signal, and calculate the _EC80 value.

Prepare 6X _EC80 concentration of agonist PGE ₂ and make up 10 mM stock solutions of test compounds in DMSO solvent, and use buffer to dilute 10 concentration point 6X compound working solutions.

Take another cell plate to remove the medium and add 20 μL of Assay Buffer and 2X Fluo-4 Direct ^™ No-Wash Loading Buffer. Incubate in a 37 °C, 5% _CO2 incubator for 50 min and room temperature for 10 min before placing in the FLIPR. Transfer 10 μL of compound working solution, DMSO, and EP4 complete antagonist to the cell plate, and read the fluorescent signal. Transfer 10 μL of 6X EC ₈₀ concentration of agonist PGE ₂ to the cell plate, read the fluorescent signal, and calculate the inhibition rate:

Inhibition rate (%)=100-(test group-EP4 complete antagonist group)/(DMSO group-EP4 complete antagonist group)*100

According to the inhibition rates of different concentrations of the compounds, the _IC50 values of the compounds for the inhibition of EP4 calcium flux (ie, the drug concentration when the intracellular Ca ²⁺ flux is inhibited by half after the overexpression of human EP4 receptors) were calculated.

Table 1 The inhibitory effect of test compounds on EP4 calcium flux

test compound _IC50 (nM) control compound twenty one Compound I-1 10.9 Compound I-2 12.36

The experimental results show that the compounds of the present invention have a better inhibitory effect on EP4 calcium flow, which is better than that of the control compounds, and the compounds of the present invention have a better inhibitory effect on EP4 calcium flow.

Test Example 2: Radioligand EP4 Receptor Binding Assay

Radioligand EP4 binding assays were performed using recombinant human EP4 receptor membrane protein (prepared from 293 cells overexpressing human EP4 receptor). Compounds to be tested and PGE ₂ were prepared as 10 mM stock solutions in DMSO solvent, and then 8 concentration point 4x working solutions were serially diluted in buffer (50 mM HBSS, 0.1% BSA, 500 mM NaCl). Add 1 μL of compound working solution, DMSO and PGE ₂ working solution to the assay plate respectively, add 100 μL of EP4 receptor membrane protein (20 μg/well) and 100 μL of radioligand [ ³ H]-PGE ₂ (PerkinElmer, Cat: NET428250UC) , Lot: 2469552) (final concentration 1.5nM), sealed and incubated for 1 hour at room temperature. Unifilter-96 GF/C filter plates (Perkin Elmer) were soaked in 0.5% BSA, 50 μL/well for at least 30 min at room temperature. After binding was complete, the reaction mixture was filtered through a 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 were sealed using Perkin Elmer Unifilter-96 sealing tape, and 50 μL of MicroScint ^™ -20 cocktail (Perkin Elmer) was added to seal the top of the filter plate. ^3H counts captured on the filters were read using a Perkin Elmer MicroBeta2 Reader.

Data were analyzed using GraphPad Prism 5, and inhibition rates were calculated as follows:

Inhibition rate (%)=100-(test group-PGE ₂ group)/(DMSO group-PGE ₂ group)*100

Based on the inhibition rates of different concentrations of the compounds, the _IC50 and Ki values of the compounds determined by the binding of the radioligand EP4 were calculated.

Table 2 _IC50 and Ki values of test compounds determined by radioligand EP4 binding

test compound _IC50 (nM) Ki(nM) control compound 30 16 Compound I-1 9.1 5.0

The experimental results show that, compared with the control compound, the compound of the present invention has better affinity with the EP4 receptor, and is better than the control compound, and the compound of the present invention shows a better affinity for the EP4 receptor.

Test Example 3: Pharmacokinetic Test

Pharmacokinetic test in mice, using male ICR mice, 20-25 g, fasted overnight. Three mice were taken and administered orally orally at 5 mg/kg. Blood was collected before administration and at 15, 30 minutes and 1, 2, 4, 8, and 24 hours after administration; another 3 mice were taken and administered 1 mg/kg intravenously, before and after administration Blood was collected at 15, 30 minutes and 1, 2, 4, 8, and 24 hours. Blood samples were centrifuged at 6800g at 2-8°C for 6 minutes, and plasma was collected and stored at -80°C. Take the plasma at each time point, add 3-5 times the volume of acetonitrile solution containing the internal standard to mix, vortex for 1 minute, centrifuge at 13,000 rpm for 10 minutes at 4°C, take the supernatant and add 3 times the volume of water to mix, take an appropriate amount of the mixture LC-MS/MS analysis was performed. The main pharmacokinetic parameters were analyzed by non-compartmental model using WinNonlin 7.0 software.

Canine pharmacokinetic test using male Beagle dogs, 8-10 kg, fasted overnight. Three Beagle dogs were taken and administered by oral gavage at 3 mg/kg. Another 3 Beagle dogs were taken and administered intravenously at 1 mg/kg. The rest of the operation is the same as the mouse pharmacokinetic test.

Table 3 Pharmacokinetic test results in mice

Table 4 Canine pharmacokinetic test results

The experimental results show that, compared with the control compound, the compound of the present invention has a lower or equivalent clearance rate by intravenous administration, and a higher or equivalent exposure by oral administration, and the exposure in mice after oral administration is about 4 times that of the control compound, After oral administration to dogs, the exposure is about twice that of the control compound, and the compound of the present invention exhibits better pharmacokinetic properties and good druggability.

Claims (16)

A compound is characterized in that, it is the compound shown in formula (I), or the tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt of the compound shown in formula (I) or Prodrugs:

wherein, R ¹ is selected from H or halogen; Halogen is selected from F, Cl, Br, I. The compound of claim 1, wherein the compound represented by the formula (I) is any of the following compounds:

A pharmaceutical composition, characterized by comprising: a pharmaceutically acceptable excipient and the compound of any one of claims 1-2. Use of the compound according to any one of claims 1 to 2, or the use of the pharmaceutical composition according to claim 3 in the preparation of a medicament, wherein the medicament is used to treat or prevent EP4 receptor-related diseases. The use according to claim 4, wherein the EP4 receptor-related disease is selected from at least one of the following diseases: inflammatory disease, pain, cancer, metabolic disease, and urinary system disease. The use of claim 5, wherein the inflammatory disease is selected from arthritis and rheumatoid arthritis; the pain is selected from osteoarthritis pain, pain caused by endometriosis; the cancer is selected from solid cancer; the metabolic disease is diabetes, and the urinary system disease is selected from overactive bladder. The use of claim 5, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, bone marrow Blastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer and/or urethral cancer. The use of claim 5, wherein the medicament is administered in combination with radiotherapy and/or antibody therapy. The use of claim 8, wherein the antibody therapy is selected from one or a combination of CTLA4 antibody therapy, PDL1 antibody therapy, and PD1 antibody therapy. The use of claim 5, wherein the medicament is suitable for inhibiting EP4 receptor calcium flux; The drug is suitable for binding to the EP4 receptor. A method for treating or preventing EP4 receptor-related diseases, comprising: The compound of claim 1 or 2 or the pharmaceutical composition of claim 3 is administered to a subject. The method of claim 11, wherein the EP4 receptor-related disease is selected from at least one of the following diseases: inflammatory disease, pain, cancer, metabolic disease, and urinary system disease. The method of claim 12, wherein the inflammatory disease is selected from arthritis, rheumatoid arthritis; the pain is selected from osteoarthritis pain, pain caused by endometriosis; the cancer is selected from solid cancer; the metabolic disease is diabetes, and the urinary system disease is selected from overactive bladder. The method of claim 12, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, colorectal cancer, endometrial cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, bone marrow Blastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer and/or urethral cancer. The method of claim 11, wherein radiation therapy and/or antibody therapy are administered to the subject in combination. The method of claim 15, wherein the antibody therapy is selected from one or a combination of CTLA4 antibody therapy, PDL1 antibody therapy, and PD1 antibody therapy.