Salt of thiophene [2, 3-d] imidazole compound, crystal form of salt and application of salt in medicine

Abstract

The invention provides a compound with GLP-1R agonist activity, such as salt and / or crystal form as shown in formula (I), preparation and medical application, and the pharmaceutically acceptable salt is selected from tris (hydroxymethyl) aminomethane salt or triethanolamine salt: # imgabs0 #

Description

A salt of a thiophene[2,3-d]imidazole compound and its crystal form and its use in medicine Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a salt crystal form of a thiophene[2,3-d]imidazole compound and its use in medicine. Background Art

[0002] Diabetes is a group of metabolic diseases characterized by hyperglycemia. Hyperglycemia is caused by defects in insulin secretion, impaired insulin action, or both. Long-term high blood sugar levels in diabetes lead to chronic damage and dysfunction of various tissues, particularly the eyes, kidneys, heart, blood vessels, and nerves. Diabetes is primarily divided into two types. Type 1 diabetes: Destruction of pancreatic B cells leads to absolute insulin deficiency. Type 2 diabetes: Insulin resistance with relative insulin deficiency or impaired insulin secretion with insulin resistance.

[0003] Drugs for type 2 diabetes can be divided into six major categories (insulin, insulin secretagogues, biguanides, glucosidase inhibitors, thiazolidinediones, and SGLT2 inhibitors), each of which works through a different primary mechanism. However, with the exception of GLP-1 receptor agonists and SGLT2 inhibitors, these drugs have limited efficacy and do not address the most important issue, namely, decreased cellular function and associated obesity.

[0004] GLP-1 is a 30-amino acid long incretin hormone secreted by L cells in the intestine. GLP-1 stimulates insulin secretion in a physiological, glucose-dependent manner, reduces glucagon secretion, inhibits gastric emptying, reduces appetite, and stimulates β-cell proliferation. In nonclinical studies, GLP-1 has been shown to promote β-cell sustainability by stimulating the transcription of genes important for glucose-dependent insulin secretion and promoting β-cell neogenesis. In healthy individuals, GLP-1 plays a key role in regulating postprandial blood flow, increasing peripheral glucose absorption by stimulating glucose-dependent insulin secretion from the pancreas. GLP-1 also inhibits glucagon secretion, resulting in reduced hepatic glucose output. Furthermore, GLP-1 delays gastric emptying, slows small intestinal motility, and delays food absorption.

[0005] GLP-1 receptor agonists, such as GLP-1, liraglutide, and exendin-4, are peptide drugs, mostly administered by injection. Small molecule GLP-1 receptor agonists have become a hot topic in drug development in recent years due to their potential for high oral bioavailability.

[0006] Summary of the Invention

[0007] The present invention aims to provide a pharmaceutically acceptable salt (such as a tris(hydroxymethyl)aminomethane salt or a triethanolamine salt) of a compound represented by formula (I), a solvate thereof, a crystalline form thereof, and a preparation method thereof, as well as a pharmaceutical composition thereof and its use in preparing a medicament for treating diabetes or diabetes-related diseases.

[0008] The crystal of the present invention is easy to process, crystallize and handle; has good stability, is convenient for oral administration, and has good solubility and bioavailability.

[0009] The present invention provides a pharmaceutically acceptable salt of a compound represented by formula (I) and a solvate thereof.

[0010] In some embodiments, the pharmaceutically acceptable salt is selected from the tris or triethanolamine salts;

[0011] In some embodiments, the molar ratio of the compound represented by formula (I): the pharmaceutically acceptable salt is 1:0.9 to 1:1.1;

[0012] In some embodiments, the molar ratio of the compound represented by formula (I): the pharmaceutically acceptable salt is 1:1;

[0013] In some embodiments, the solvate is methanol;

[0014] The present invention provides a tris(hydroxymethyl)aminomethane salt crystalline form A of the compound represented by formula (I), which has an X-ray powder diffraction pattern using Cu-Kα radiation and has characteristic diffraction peaks at the following 2θ positions: 8.97°±0.2°, 9.32°±0.2°, 9.92°±0.2°, 10.68°±0.2°, 17.50°±0.2°, and 20.17°±0.2°;

[0015] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form A of the compound represented by the aforementioned formula (I) has an X-ray powder diffraction pattern using Cu-Kα radiation having characteristic diffraction peaks at the following 2θ positions: 8.97°±0.2°, 9.32°±0.2°, 9.92°±0.2°, 10.68°±0.2°, 15.07°±0.2°, 17.50°±0.2°, 17.71°±0.2°, 19.95°±0.2°, 20.17°±0.2°, 24.27°±0.2°, and 26.81°±0.2°;

[0016] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form A of the compound represented by the aforementioned formula (I) has characteristic diffraction peaks at the following 2θ positions using Cu-Kα radiation: 6.02°±0.2°, 8.97°±0.2°, 9.32°±0.2°, 9.92°±0.2°, 10.68°±0.2°, 12.03±0.2°, 13.77°±0.2°, 15.07°±0.2°, 16.18°±0.2°, 17.50°±0.2°, 17. 71°±0.2°, 18.27°±0.2°, 18.70°±0.2°, 19.15°±0.2°, 19.95°±0.2°, 20.17°±0.2°, 20.90°±0.2°, 21.89°±0.2°, 22.35°±0.2°, 24.05°±0.2°, 24.27°±0.2°, 24.65°±0.2°, 25.03°±0.2°, 25.54°±0.2°, 26.81°±0.2°, 28.03°±0.2°;

[0017] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form A of the compound represented by the aforementioned formula (I) has an X-ray powder diffraction pattern as shown in FIG1 using Cu-Kα radiation;

[0018] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form A of the compound represented by formula (I) has a differential scanning calorimetry (DSC) curve showing an endothermic curve, wherein T 初始 =159.5℃, T 峰值 =164.6°C, ΔH=84.9 J / g, and its thermogravimetric analysis curve (TGA) showed a weight loss of 1.6% before 120°C.

[0019] In some embodiments, the differential scanning calorimetry curve and thermogravimetric analysis curve of the tris(hydroxymethyl)aminomethane salt crystalline form A of the compound represented by formula (I) are shown in FIG2 .

[0020] The present invention provides a tris(hydroxymethyl)aminomethane salt crystalline form C of the compound represented by formula (I), which has an X-ray powder diffraction pattern using Cu-Kα radiation and has characteristic diffraction peaks at the following 2θ positions: 10.55°±0.2°, 13.69°±0.2°, 19.41°±0.2°, and 20.70±0.2°;

[0021] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form C of the compound represented by the aforementioned formula (I) has an X-ray powder diffraction pattern using Cu-Kα radiation having characteristic diffraction peaks at the following 2θ positions: 10.55°±0.2°, 10.92°±0.2°, 13.69°±0.2°, 17.98°±0.2°, 19.41°±0.2°, 20.70±0.2°, 21.97±0.2°, 22.31±0.2°, 26.44±0.2°;

[0022] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form C of the compound represented by the aforementioned formula (I) has a characteristic diffraction peak at the following 2θ positions using Cu-Kα radiation: 5.44°±0.2°, 8.12°±0.2°, 10.17°±0.2°, 10.55°±0.2°, 10.92°±0.2°, 11.56°±0.2°, 13.08°±0.2°, 13.69°±0.2°, 14.49°±0.2°, 15.26°±0.2°. .2°, 17.06°±0.2°, 17.78°±0.2°, 17.98°±0.2°, 18.41°±0.2°, 19.41°±0.2°, 20.70±0.2°, 21.25±0.2°, 21.97±0.2°, 22.31±0.2°, 23.27±0.2°, 24.23±0.2°, 25.24±0.2°, 25.70±0.2°, 26.44±0.2°, 27.49±0.2°, 28.71±0.2°;

[0023] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form C of the compound represented by the aforementioned formula (I) has an X-ray powder diffraction pattern as shown in FIG3 using Cu-Kα radiation;

[0024] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form C of the compound represented by formula (I) has a differential scanning calorimetry (DSC) curve showing three endothermic peaks, wherein T 峰值 =49.6℃、T 峰值 =112.8℃ and T 峰值 =161.4°C, and its thermogravimetric analysis curve (TGA) showed a weight loss of 4.6% before 130°C.

[0025] In some embodiments, the differential scanning calorimetry curve and thermogravimetric analysis curve of the tris(hydroxymethyl)aminomethane salt crystalline form C of the compound represented by formula (I) are shown in FIG4 .

[0026] The present invention provides a tris(hydroxymethyl)aminomethane salt crystalline form D of the compound represented by formula (I), which has an X-ray powder diffraction pattern using Cu-Kα radiation and has characteristic diffraction peaks at the following 2θ positions: 5.55°±0.2°, 10.46°±0.2°, 11.15°±0.2°, 13.12°±0.2°, 13.98°±0.2°, 19.66°±0.2°, and 22.47°±0.2°;

[0027] In some embodiments, the tris(hydroxymethyl)aminomethane salt form D of the compound represented by the aforementioned formula (I) is subjected to Cu-Kα radiation, 5.55°±0.2°, 8.34±0.2°, 10.46°±0.2°, 11.15°±0.2°, 11.50±0.2°, 13.12°±0.2°, 13.98°±0.2°, 19.66°±0.2°, 20.80±0.2°, 22.08±0.2°, 22.47°±0.2°, 23.26±0.2°, 26.73±0.2°;

[0028] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form D of the compound represented by the aforementioned formula (I) has an X-ray powder diffraction pattern as shown in FIG5 using Cu-Kα radiation;

[0029] In some embodiments, the tris(hydroxymethyl)aminomethane salt crystalline form D of the compound represented by formula (I) above has a differential scanning calorimetry (DSC) curve showing two endothermic curves, wherein T 峰值 =119.2℃, T 峰值 =133.9°C, and its thermogravimetric analysis curve (TGA) showed a weight loss of 3.7% before 140°C.

[0030] In some embodiments, the differential scanning calorimetry curve and thermogravimetric analysis curve of the tris(hydroxymethyl)aminomethane salt crystalline form D of the compound represented by formula (I) are shown in FIG6 .

[0031] The present invention provides a triethanolamine salt crystalline form A of the compound represented by formula (I), which has an X-ray powder diffraction pattern using Cu-Kα radiation and has characteristic diffraction peaks at the following 2θ positions: 10.42°±0.2°, 12.68°±0.2°, 14.80°±0.2°, 16.37°±0.2°, 16.94°±0.2°, and 23.39°±0.2°;

[0032] In some embodiments, the triethanolamine salt form A of the compound represented by the aforementioned formula (I) has an X-ray powder diffraction pattern using Cu-Kα radiation having characteristic diffraction peaks at the following 2θ positions: 8.42°±0.2°, 10.42°±0.2°, 12.68°±0.2°, 13.28°±0.2°, 14.80°±0.2°, 16.37°±0.2°, 16.94°±0.2°, 18.04°±0.2°, 22.26°±0.2°, 23.39°±0.2°, 24.98°±0.2°, and 25.55°±0.2°;

[0033] In some embodiments, the triethanolamine salt crystalline form A of the compound represented by the aforementioned formula (I) has a characteristic diffraction peak at the following 2θ positions using Cu-Kα radiation: 4.21±0.2°, 6.30±0.2°, 8.42±0.2°, 10.42±0.2°, 12.68±0.2°, 13.28±0.2°, 14.35±0.2°, 14.80±0.2°, 15.11±0.2°, 16.10±0.2°, 16.37±0.2°, 16.94±0.2°, 17.32±0.2°. °, 18.04°±0.2°, 18.72°±0.2°, 19.83°±0.2°, 20.96°±0.2°, 21.23°±0.2°, 21.39°±0.2°, 21.70°±0.2°, 21.85°±0.2°, 22.26°±0.2°, 22.61°±0.2°, 23.39°±0.2°, 24.22°±0.2°, 24.55°±0.2°, 24.98°±0.2°, 25.55°±0.2°, 26.48°±0.2°, 28.07°±0.2°, 29.90°±0.2°

[0034] In some embodiments, the triethanolamine salt crystalline form A of the compound represented by the aforementioned formula (I) has an X-ray powder diffraction pattern as shown in FIG7 using Cu-Kα radiation;

[0035] In some embodiments, the triethanolamine salt of the compound represented by the aforementioned formula (I) in Form A has a differential scanning calorimetry (DSC) curve showing an endothermic curve, wherein T 初始 =146.2℃, T 峰值 =149.9°C, ΔH=73.6 J / g, and its thermogravimetric analysis curve (TGA) showed a weight loss of 1% before 120°C.

[0036] In some embodiments, the differential scanning calorimetry curve and thermogravimetric analysis curve of the triethanolamine salt form A of the compound represented by formula (I) are shown in FIG8 .

[0037] The present invention also provides a method for preparing a pharmaceutically acceptable salt of the compound represented by formula (I), wherein the method comprises the step of forming a salt with the compound represented by formula (I) and an acid, wherein the acid is preferably a tris(hydroxymethyl)aminomethane salt or a triethanolamine salt.

[0038] In some embodiments, the solvent used in the aforementioned preparation method is selected from C 1-6 Halogenated alkane solvents, C 2-6 Ester solvents, C 2-6 Ether solvents, C 1-6 One or more of alcohol solvents or water;

[0039] In some embodiments, the solvent used in the aforementioned preparation method is selected from one or more of dichloromethane, 1,2-dichloroethane, ethyl acetate, methanol, ethanol, isopropanol, propanol, diethyl ether, tetrahydrofuran and water.

[0040] On the other hand, the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition contains a therapeutically effective amount of the pharmaceutically acceptable salt of the compound represented by formula (I) and its solvate or the tris(hydroxymethylaminomethane) salt crystalline form A of the compound represented by formula (I), the tris(hydroxymethylaminomethane) salt crystalline form C of the compound represented by formula (I), the tris(hydroxymethylaminomethane) salt crystalline form D of the compound represented by formula (I), the triethanolamine salt crystalline form A of the compound represented by formula (I) and a pharmaceutically acceptable carrier or excipient.

[0041] As used herein, "effective amount" or "therapeutically effective amount" refers to a sufficient amount of the compound disclosed herein to alleviate to some extent one or more symptoms of the disease or disorder (eg, diabetes) being treated.

[0042] In some embodiments, the result is reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

[0043] For example, " effective dose " for therapeutic use is the amount of the composition comprising compound disclosed herein required for providing clinically significant disease symptoms to reduce. The example of therapeutic effective dose (calculated as free alkali form) includes but is not limited to 1-600mg, 2-600mg, 3-600mg, 4-600mg, 5-600mg, 6-600mg, 10-600mg, 20-600mg, 25-600mg, 30-600mg, 40-600mg, 50-600mg, 60-600mg, 70-600mg, 75-600mg, 80-600mg, 90-600mg, 100-600mg, 200-600mg, 1-500mg, 2-500mg, 3-500mg, 4-500mg, 5-500mg, 6-50 0mg, 10-500mg, 20-500mg, 25-500mg, 30-500mg, 40-500mg, 50-500mg, 60-500mg, 70-500mg, 75-500mg, 80-500mg, 90-500mg, 100-500mg, 125-5 00mg, 150-500mg, 200-500mg, 250-500mg, 300-500mg, 400-500mg, 5-400mg, 10-400mg, 20-400mg, 25-400mg, 30-400mg, 40-400mg, 50-400mg, 6 0-400mg, 70-400mg, 75-400mg, 80-400mg, 90-400mg, 100-400mg, 125-400mg, 150-400mg, 200-400mg, 250-400mg, 300-400mg, 1-300mg, 2-300m g, 5-300mg, 10-300mg, 20-300mg, 25-300mg, 30-300mg, 40-300mg, 50-300mg, 60-300mg, 70-300mg, 75-300mg, 80-300mg, 90-300mg, 100-300mg , 125-300mg, 150-300mg, 200-300mg, 250-300mg, 1-200mg, 2-200mg, 5-200mg, 10-200mg, 20-200mg, 25-200mg, 30-200mg, 40-200mg, 50-200mg , 60-200mg, 70-200mg, 75-200mg, 80-200mg, 90-200mg, 100-200mg, 125-200mg, 150-200mg, 1-100mg, 2-100mg, 5-100mg, 10-100mg, 15-100mg,20-100 mg, 25-100 mg, 30-100 mg, 40-100 mg, 50-100 mg, 60-100 mg, 70-100 mg, 75-100 mg, 80-100 mg, 90-100 mg; in some embodiments, examples of therapeutically effective amounts include but are not limited to 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 4 5mg, 50mg, 55mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 110mg, 120mg, 125mg, 130mg , 140mg, 150mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 300mg. ,

[0044] In another aspect, the present invention also provides the use of the pharmaceutically acceptable salts of the compounds of the aforementioned formula (I) and their solvates, or the tris(hydroxymethylaminomethane) salt crystalline form A of the compound of the formula (I), the tris(hydroxymethylaminomethane) salt crystalline form C of the compound of the formula (I), the tris(hydroxymethylaminomethane) salt crystalline form D of the compound of the formula (I), the triethanolamine salt crystalline form A of the compound of the formula (I), or the aforementioned pharmaceutical compositions in the preparation of drugs for treating diabetes or diabetes-related diseases.

[0045] It can be understood that the expressions such as “preferably, ..., its X-ray powder diffraction pattern further has a characteristic diffraction peak at the following 2θ position” or “more preferably, ..., its X-ray powder diffraction pattern further has a characteristic diffraction peak at the following 2θ position” described in the present invention mean that on the basis of having a characteristic diffraction peak at the aforementioned 2θ position, there is further a characteristic diffraction peak at the aforementioned “following 2θ position”.

[0046] The X-ray powder diffraction, DSC pattern, and TGA pattern disclosed in the present invention, and those substantially the same also fall within the scope of the present invention.

[0047] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0048] “IC 50 ” refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved.

[0049] The "ether solvent" described in the present invention refers to a chain compound or a cyclic compound containing an ether bond -O- and having 1 to 10 carbon atoms. Specific examples include but are not limited to: tetrahydrofuran, diethyl ether, propylene glycol methyl ether, methyl tert-butyl ether, isopropyl ether or 1,4-dioxane.

[0050] The "alcohol solvent" of the present invention refers to one or more "hydroxyl" substituted "C 1-6 The group is derived from one or more hydrogen atoms on the "alkyl" group, the "hydroxyl" and "C 1-6 "Alkyl" is as defined above, and specific examples include but are not limited to methanol, ethanol, isopropanol, n-propanol, isopentanol or trifluoroethanol.

[0051] The "ester solvent" mentioned in the present invention refers to a combination of a lower organic acid containing 1 to 4 carbon atoms and a lower alcohol containing 1 to 6 carbon atoms. Specific examples include but are not limited to ethyl acetate, isopropyl acetate or butyl acetate.

[0052] The "ketone solvent" described in the present invention refers to a compound in which a carbonyl group (-C(O)-) is connected to two hydrocarbon groups. Depending on the different hydrocarbon groups in the molecule, ketones can be divided into aliphatic ketones, alicyclic ketones, aromatic ketones, saturated ketones and unsaturated ketones. Specific examples include but are not limited to: acetone, acetophenone, and 4-methyl-2-pentanone.

[0053] The "nitrile solvent" of the present invention refers to one or more "cyano" substituted "C 1-6 The group derived from one or more hydrogen atoms on the "alkyl" group, the "cyano" and "C 1-6 "Alkyl" is as defined above, and specific examples include but are not limited to acetonitrile or propionitrile.

[0054] The "halogenated hydrocarbon solvent" of the present invention refers to one or more "halogen atoms" replacing "C 1-6 The group derived from one or more hydrogen atoms on the "alkyl", the "halogen atom" and "C 1-6 "Alkyl" is as defined above, and specific examples include but are not limited to: dichloromethane, 1,2-dichloroethane, chloroform or carbon tetrachloride.

[0055] As used herein, "the crystal of the present invention", "the crystalline form of the present invention", "the polymorph of the present invention" and the like can be used interchangeably.

[0056] The crystal structure of the present invention can be analyzed using various analytical techniques known to those skilled in the art, including but not limited to, X-ray powder diffraction (XRD), differential scanning calorimetry (DSC) and / or thermogravimetric analysis (TGA), also known as thermogravimetry (TG).

[0057] The "2θ or 2θ angle" mentioned in the present invention refers to the diffraction angle, θ is the Bragg angle, and the unit is ° or degree. The error range of the 2θ can be ±0.3, ±0.2 or ±0.1.

[0058] It is understood that the numerical values ​​described and protected by the present invention are approximate values. Variations in the numerical values ​​may be due to equipment calibration, equipment errors, crystal purity, crystal size, sample size and other factors.

[0059] It is understood that the crystal form of the present invention is not limited to the characteristic spectra that are exactly the same as the characteristic spectra described in the drawings disclosed in the present invention, such as XRD, DSC, and TGA. Any crystal form having characteristic spectra that are substantially the same or essentially the same as those described in the drawings falls within the scope of the present invention.

[0060] It is understood that, as is well known in the art of differential scanning calorimetry (DSC), the melting peak height of a DSC curve depends on many factors related to sample preparation and instrument geometry, while the peak position is relatively insensitive to experimental details. Therefore, in some embodiments, the crystalline compound of the present invention is characterized by a DSC pattern having characteristic peak positions, having substantially the same properties as the DSC pattern provided in the accompanying drawings of the present invention, with an error tolerance of ±3°C.

[0061] The crystal form disclosed in the present invention can be prepared by the following common methods for preparing crystal forms:

[0062] 1. The volatilization experiment is to evaporate the clear sample solution at different temperatures until the solvent is dry.

[0063] 2. The slurry experiment is to stir the supersaturated solution of the sample (with insoluble solids present) at a certain temperature in different solvent systems.

[0064] 3. The antisolvent test is to dissolve the sample in a good solvent, add an antisolvent, stir the precipitated solid for a short time and then filter it immediately.

[0065] 4. The cooling crystallization experiment is to dissolve a certain amount of sample into the corresponding solvent at high temperature, and then stir and crystallize directly at room temperature or low temperature.

[0066] 5. The polymer template experiment is to add different types of polymer materials to the sample clear solution and leave it open at room temperature to evaporate until the solvent is dry.

[0067] 6. The thermal method experiment is to treat the sample according to certain thermal method crystallization conditions and cool it to room temperature.

[0068] 7. The water vapor diffusion experiment is to place the sample in a certain humidity environment at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG1 is an X-ray powder diffraction pattern of the tris(hydroxymethyl)aminomethane salt crystalline form A of compound I.

[0070] FIG2 is a differential scanning calorimetry curve and a thermogravimetric analysis spectrum of the tris(hydroxymethyl)aminomethane salt Form A of Compound I.

[0071] FIG3 is an X-ray powder diffraction pattern of the tris(hydroxymethyl)aminomethane salt Form C of Compound I.

[0072] FIG4 is a differential scanning calorimetry curve and a thermogravimetric analysis spectrum of the tris(hydroxymethyl)aminomethane salt Form C of Compound I.

[0073] FIG5 is an X-ray powder diffraction pattern of the tris(hydroxymethyl)aminomethane salt crystalline form D of compound I.

[0074] FIG6 is a differential scanning calorimetry curve and a thermogravimetric analysis spectrum of the tris(hydroxymethyl)aminomethane salt Form D of Compound I.

[0075] FIG7 is an X-ray powder diffraction pattern of the triethanolamine salt of Compound I, Form A

[0076] FIG8 is a differential scanning calorimetry curve and a thermogravimetric analysis spectrum of the triethanolamine salt Form A of Compound I. DETAILED DESCRIPTION

[0077] The technical solutions of the present invention are described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention includes but is not limited to them.

[0078] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). NMR shifts (δ) were expressed in 10 -6 The unit of ppm is given. NMR measurements were performed using a Bruker Avance III 400 and Bruker Avance 300 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0079] MS was measured using Agilent 6120B (ESI) and Agilent 6120B (APCI).

[0080] HPLC analysis was performed using a LC-20AT (Shimadzu) high pressure liquid chromatograph (Kromasil 100-5-C18, 4.6 mm×250 mm).

[0081] XRD analysis was performed using a Bruker D8 Advance Diffractometer. The 2θ scanning angle ranged from 3° to 45°, with a scan step size of 0.02° and an exposure time of 0.08 seconds. The tube voltage and current were 40 kV and 40 mA, respectively, and the sample pan was a zero-background pan.

[0082] TGA test conditions: Thermogravimetric analyzer (TA Discovery 550, US) was used. Samples weighing 1-10 mg were placed in a pre-equilibrated sample pan and automatically weighed in the TGA oven. The sample was heated to the final temperature at a rate of 10°C / min. A nitrogen purge rate of 60 mL / min was maintained at the sample and 40 mL / min at the balance.

[0083] DSC test conditions: A TA Discovery 250 differential scanning calorimeter (TA, US) was used. A 1-2 mg sample was accurately weighed and placed in a standard pan or a perforated DSC Tzero pan. The sample was heated at a rate of 10°C / min to the final temperature. A nitrogen purge rate of 50 mL / min was used.

[0084] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as Titan Technology, Anage Chemical, Shanghai Demer, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, and Bailingwei Technology.

[0085] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0086] Unless otherwise specified in the examples, the room temperature is 20°C to 30°C.

[0087] The following describes in detail the implementation process of the present invention and the beneficial effects produced by specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention and is not intended to limit the scope of implementation of this case.

[0088] Example 1: Preparation of Compound I

[0089] Step 1: 2-(4-bromo-2-fluorophenyl)acetylcarbamate hydrochloride (1b)

[0090] ethyl 2-(4-bromo-2-fluorophenyl)acetimidate hydrochloride

[0091] 1a (5.0 g, 26.04 mmol) was added to anhydrous ethanol (3.0 mL). The reaction was stirred at 0°C while dry hydrogen chloride gas was introduced until saturation. The reaction was stirred at this temperature for 10 hours. Ether (40 mL) was then added to the reaction mixture, filtered, and the resulting solid was washed with ether (100 mL x 2). The solid was dried to afford 1b (5.0 g, 72% yield).

[0092] 1 H NMR (400MHz, DMSO-d6) δ12.04(s,2H),7.61(d,1H),7.51–7.43(m,2H),4.46(q,2H),4.14(s,2H),1.26(t,3H).

[0093] 19 F NMR (400MHz, DMSO-d6) δ111.30.

[0094] Step 2: 4-Bromo-2-fluoro-1-(2,2,2-triethoxyethyl)benzene (1c)

[0095] 4-bromo-2-fluoro-1-(2,2,2-triethoxyethyl)benzene

[0096] 1b (5.0 g, 19.30 mmol) was added to anhydrous ethanol (40 mL) and stirred at room temperature (15°C) for 48 hours. The reaction mixture was directly filtered to obtain a filtrate, which was then spin-dried to dryness and then slurried with a mixed solvent of petroleum ether and ethyl acetate (100 mL) (petroleum ether / ethyl acetate (v / v) = 10:1). The mixture was filtered and the filtrate was spin-dried to obtain 1c (4.0 g, 66% yield).

[0097] 1H NMR (400MHz, CDCl3)7.45(t,1H),7.23–7.14(m,2H),3.50(q,6H),3.07(s,2H),1.16(t,9H).

[0098] Step 3: (S)-2-(4-bromo-2-fluorobenzyl)-1-(oxan-2-ylmethyl)-1H-thieno[2,3-d]imidazole-5-carboxylic acid methyl ester (1e)

[0099] methyl(S)-2-(4-bromo-2-fluorobenzyl)-1-(oxetan-2-ylmethyl)-1H-thieno[2,3-d]imidazole-5-carboxylate

[0100] 1d (500 mg, 2.06 mmol) and 1c (1.0 g, 3.19 mmol) were added to glacial acetic acid (20 mL) and stirred at 60°C for 10 hours. The reaction system was cooled to room temperature and slowly added to a saturated aqueous solution of sodium bicarbonate (200 mL) for quenching. The mixture was then extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was then spin-dried and purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 1:1) to obtain 1e (430 mg, 47% yield).

[0101] LCMS m / z=439.0[M+1] + .

[0102] Step 4: (S)-2-(2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxobenzofuran-2-yl)benzyl)-1-(oxoalkane-2-ylmethyl)-1H-thieno[2,3-d]imidazole-5-carboxylic acid methyl ester (1f)

[0103] methyl(S)-2-(2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-1-(oxetan-2-ylmethyl)-1H-thieno[2,3-d]imidazole-5-carboxylate

[0104] 1e (400 mg, 0.98 mmol), bis(pinacolato) borate (262 mg, 1.0 mmol), Pd(dppf)Cl2·DCM (81 mg, 0.1 mmol), and potassium acetate (196 mg, 2.0 mmol) were added to 1,4-dioxane (20 mL). The reaction mixture was replaced with nitrogen and stirred at 100°C for 5 hours. The reaction mixture was cooled to room temperature, added with water (100 mL), and extracted with ethyl acetate (50 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was spin-dried and purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 1:1) to obtain 1f (340 mg, 71% yield).

[0105] LCMS m / z=487.2[M+1] + .

[0106] Step 5: (S)-2-(4-(6-((4-cyano-2-fluorobenzyl)oxy)pyridin-2-yl)-2-fluorobenzyl)-1-(oxoalkyl-2-ylmethyl)-1H-thieno[2,3-d]imidazole-5-carboxylic acid methyl ester (1h)

[0107] methyl(S)-2-(4-(6-((4-cyano-2-fluorobenzyl)oxy)pyridin-2-yl)-2-fluorobenzyl)-1-(oxetan-2-ylmethyl)-1H-thieno[2,3-d]imidazole-5-carboxylate

[0108] 1f (340 mg, 0.70 mmol), 6g (220 mg, 0.84 mmol), Pd(dppf)Cl2·DCM (83 mg, 0.1 mmol), and potassium carbonate (290 mg, 2.1 mmol) were added to a mixture of 1,4-dioxane (10 mL) and water (2 mL). The reaction mixture was replaced with nitrogen and stirred at 100°C for 5 hours. The reaction mixture was cooled to room temperature, added with water (50 mL), and extracted with ethyl acetate (30 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was spin-dried and purified by silica gel column chromatography (petroleum ether / ethyl acetate (v / v) = 1:1) to obtain 1h (200 mg, 48% yield).

[0109] LCMS m / z=587.1[M+1] + .

[0110] Step 6: (S)-2-(4-(6-((4-cyano-2-fluorobenzyl)oxy)pyridin-2-yl)-2-fluorobenzyl)-1-(oxoalkyl-2-ylmethyl)-1H-thieno[2,3-d]imidazole-5-carboxylic acid (Compound I)

[0111] (S)-2-(4-(6-((4-cyano-2-fluorobenzyl)oxy)pyridin-2-yl)-2-fluorobenzyl)-1-(oxetan-2-ylmethyl)-1H-thieno[2,3-d]imidazole-5-carboxylic acid

[0112] 1h (100 mg, 0.17 mmol) and 1i (120 mg, 0.85 mmol) were added to a mixture of acetonitrile (5 mL) and water (1 mL) and stirred at 12°C for 10 hours. The reaction solution was then adjusted to pH 6 with 1M hydrochloric acid and stirred until a white solid precipitated. The solid was filtered and washed with water. The solid was dissolved in dichloromethane, dried over anhydrous sodium sulfate, filtered, and the organic phase was concentrated under reduced pressure and separated by HPLC to obtain the trifluoroacetate salt of Compound I. This was then purified on a silica gel column to afford Compound I (40 mg, 41% yield).

[0113] LCMS m / z=573.1[M+1] + .

[0114] 1 H NMR(400MHz,CD3OD)δ12.90(br,1H),7.91(d,1H),7.88–7.80(m,4H),7.79–7.68(m,2H)7.64(d,1H),7.39(t,1H),6.92(d,1H ),5.61(s,2H),5.06–4.98(m,1H),4.60(dd,1H),4.54–4.44(m,2H),4.43–4.28(m,3H),2.73–2.61(m,1H),2.40–2.27(m,1H).

[0115] Example 2: Preparation of Tris(hydroxymethyl)aminomethane Salt Form A of Compound I

[0116] Compound I (2.920 g) was weighed, 76.8 mL of n-propanol was added, and the mixture was suspended at room temperature (about 25°C) for about 10 minutes. Tris (hydroxymethyl)aminomethane (0.615 g) was weighed, 7.68 mL of water was added, the mixture was suspended at room temperature (about 25°C), and sonicated until the solution was clear. The clear aqueous solution of tris (hydroxymethyl)aminomethane was added dropwise to the free n-propanol suspension, the mixture was suspended at room temperature for about 16 hours, and after centrifugation (10,000 rpm, 4 minutes), the solid portion was vacuum dried at 50°C for about 17 hours.

[0117] 1 H NMR(400MHz,DMSO-d6)δ7.94-7.88(m,1H),7.88-7.80(m,3H),7.79-7.69(m,2H),7.66-7.60(m,1H),7.47(s,1H),7.40-7.32(m,1H ),6.95-6.87(m,1H),5.61(s,2H),5.02-4.93(m,1H),4.57-4.21(m,6H),3.50-3.36(m,6H),2.71-2.60(m,1H),2.38–2.27(m,1H).

[0118] LCMS m / z=573.1[M+1] + .

[0119] Example 3: Preparation of Tris(hydroxymethyl)aminomethane Salt Form A of Compound I

[0120] Weigh 220.0 mg of tris(hydroxymethyl)aminomethane salt Form C, add 0.5 mL of dimethyl sulfoxide, and stir at room temperature (about 25°C) until dissolved. Take 60 μL of the clear solution and add it dropwise to 0.6 mL of 4-methyl-2-pentanone at room temperature (about 25°C). Stir for about 1 hour. After centrifugation (10,000 rpm, 4 min), the solid portion is vacuum dried at room temperature for 17 hours.

[0121] Example 4: Preparation of Tris(hydroxymethyl)aminomethane Salt Form C of Compound I

[0122] Weigh 220.2 mg of tris(hydroxymethyl)aminomethane salt Form A, add 0.8 mL of N-methylpyrrolidone, and stir at room temperature (about 25°C) until dissolved; take 70 μL of the clear solution, add 3.0 mL of dichloromethane dropwise to the clear solution at room temperature (about 25°C), stir for about 1 hour, and after centrifugation (10000 rpm, 4 min), dry the solid portion in vacuum at room temperature for 17 hours.

[0123] Example 5: Preparation of Tris(hydroxymethyl)aminomethane Salt Form D of Compound I

[0124] 220.0 mg of tris(hydroxymethyl)aminomethane salt form A was weighed, 14.0 mL of methanol was added, and the mixture was suspended at room temperature (about 25° C.) for about 16 h. After centrifugation (10,000 rpm, 4 min), the solid portion was vacuum dried at room temperature for about 17 h.

[0125] Example 6: Preparation of Triethanolamine Salt Form A of Compound I

[0126] Weigh 20.5 mg of triethanolamine salt raw material, add 6.5 mL of chloroform, and stir at room temperature until it dissolves clearly; place the clear liquid at room temperature (about 25°C) and evaporate it open for 8 days until solid precipitates.

[0127] 1H NMR(400MHz,DMSO-d6)δppm 2.33(m,1H),2.64(m,7H),3.45(t,7H),4.40(m,5H),4.59(m,1H),5.00(br dd,1H),5.61(s,2H),6.92(d,1H),7.39(t,1H),7.65(d,1H),7.75(m,3H),7.85(m,3H),7.93(d,1H).

[0128] Crystal form test example

[0129] 1. XRD test of compounds

[0130] The compound of the present invention was subjected to X-ray powder diffraction test according to the following method.

[0131] Table 1-1 XRD test parameters

[0132] 2. DSC test of compounds

[0133] The DSC spectra were collected on TA Instruments discovery DSC 2500 and DSC 250 differential scanning calorimeters. The test parameters of the tris (hydroxymethyl)aminomethane salt of Compound I are shown in Table 2-1, and the test parameters of the triethanolamine salt of Compound I are shown in Table 2-2.

[0134] Table 2-1 DSC test parameters

[0135] Table 2-2 DSC test parameters

[0136] 3. TGA test of compounds

[0137] The TGA spectra were collected on TA Instruments discovery TGA 55 and TGA 550 thermogravimetric analyzers. The test parameters of the tris (hydroxymethyl)aminomethane salt of Compound I are shown in Table 3-1, and the test parameters of the triethanolamine salt of Compound I are shown in Table 3-2.

[0138] Table 3-1 TGA test parameters

[0139] Table 3-2 TGA test parameters

[0140] 4. Specific peak characterization results of XRD test of compounds

[0141] The X-ray powder diffraction pattern (XRD) of the tris(hydroxymethyl)aminomethane salt of Compound I, Form A, is shown in Figure 1. The specific peaks are shown in Table 4.

[0142] Table 4

[0143] The X-ray powder diffraction pattern (XRD) of the tris(hydroxymethyl)aminomethane salt of Compound I, Form C, is shown in Figure 3. The specific peaks are shown in Table 5.

[0144] Table 5

[0145] The X-ray powder diffraction pattern (XRD) of the tris(hydroxymethyl)aminomethane salt of Compound I, Form D, is shown in Figure 5. The specific peaks are shown in Table 6.

[0146] Table 6

[0147] The X-ray powder diffraction pattern (XRD) of the triethanolamine salt of Compound I, Form A, is shown in Figure 7. The specific peaks are shown in Table 7.

[0148] Table 7

[0149] 5. Stability data of the pharmaceutical salt of Compound I

[0150] The samples were tested at 40°C + 75% RH and 30°C + 60% RH respectively, and the purity (expressed as a percentage) was detected by HPLC. The experimental results are shown in Table 10.

[0151] The preparation method of the test solution and the HPLC purity test conditions are shown in Tables 8 and 9;

[0152] Table 8 Preparation method of test solution

[0153] Table 9 HPLC purity test conditions

[0154] Table 10 Chemical stability of compound 1 and different salts and crystal forms under different conditions (purity determined by HPLC)

[0155] Conclusion: The tris(hydroxymethyl)aminomethane salt form A of compound 1 has good chemical stability.

[0156] 6. Solubility test

[0157] The solubility of the test compounds was tested according to the method of the Chinese Pharmacopoeia 2020 edition, and the results are shown in Table 11.

[0158] Table 11 Solubility data of free form and pharmaceutical salt of Compound I

[0159] Conclusion: At pH 6.8, the solubility of the tris(hydroxymethyl)aminomethane salt form A of compound I is significantly improved compared to the free form.

[0160] Biological test cases

[0161] 1. HEK293 / CRE-luc / GLP-1R cell activity

[0162] Cells: HEK293 / CRE-luc / GLP-1R

[0163] Cell culture medium: DMEM + 10% FBS + 400 μg / ml G418 + 100 μg / ml Hygromycin B

[0164] Freezing medium: 90% FBS, 10% (V / V) DMSO

[0165] Assay buffer: DMEM + 1% FBS

[0166] Experimental procedures

[0167] The cells were cultured in DMEM medium + 10% FBS + 400 μg / ml G418 + 100 μg / ml Hygromycin B in a 37°C CO2 incubator and passaged every 3-4 days.

[0168] Cell plating: Trypsin digestion was used to adjust the cell density to 1.67 × 10 5 cells / mL; 60 μL of cells were seeded into each well of a 384-well plate (10,000 cells / well); NC wells (negative control) and background wells (no cells) were set up. Incubate in an incubator for approximately 18 ± 2 h.

[0169] The compound was diluted with detection buffer in a gradient manner, with the detection concentration ranging from 0.01 nM to 1000 nM.

[0170] Remove the cell culture plate, aspirate all supernatant from the cells, and gently wash twice with 1X PBS.

[0171] Add the diluted compound to a 384-well plate (10 μL / well), with three replicates for each concentration. Add 10 μL of detection buffer to the NC wells, seal, and incubate at 37°C for 6 h.

[0172] The plate was removed and the cells were allowed to equilibrate to room temperature (at least 15 min), after which all the supernatant was aspirated from the cells.

[0173] Add 10 μL / well to the sample wells Reagent and incubate at room temperature for 5 min to lyse the cells.

[0174] The test results were read using a BMG microplate reader.

[0175] Data processing

[0176] Calculate the average background value. Calculate the fold of induction (FI) = (value of the induced wells – background value) / (value of the negative control wells – background value).

[0177] Graphpad Prim 8.0 software was used to calculate the EC of the samples using a four-parameter fitting analysis.50 Numeric value.

[0178] Statistical analysis

[0179] All results are presented as mean ± SEM, and statistical analysis was performed using Graphpad Prism software. Specific data are presented in graphical form. P < 0.05 was considered statistically significant.

[0180] Conclusion: Compound I has a good agonist effect on GLP-1 receptor, and its EC 50 The value was less than 10 nM.

[0181] 2. Effects of compounds on hERG potassium channels

[0182] Experimental platform: electrophysiology manual patch clamp system

[0183] Cell line: Chinese hamster ovary (CHO) cells stably expressing hERG potassium channel

[0184] Experimental method: CHO (Chinese Hamster Ovary) cells stably expressing hERG potassium channel were used to record hERG potassium channel current using whole-cell patch clamp technique at room temperature. Glass microelectrodes were pulled from glass electrode blanks (BF150-86-10, Sutter) using a puller. The tip resistance after perfusing the electrode liquid was about 2-5MΩ. The glass microelectrode was inserted into the amplifier probe to connect to the patch clamp amplifier. The clamping voltage and data recording were controlled and recorded by a computer using pClamp 10 software, with a sampling frequency of 10kHz and a filter frequency of 2kHz. After obtaining the whole-cell recording, the cell was clamped at -80mV to induce the hERG potassium current (I hERG ) was administered with a 2-second depolarization step from -80 mV to +20 mV, followed by repolarization to -50 mV for 1 second before returning to -80 mV. This voltage stimulus was administered every 10 seconds, and administration began after confirming that the hERG potassium current was stable (for at least 1 minute). Compound administration was continued for at least 1 minute at each test concentration, and at least two cells (n ≥ 2) were tested for each concentration.

[0185] Data processing:

[0186] Data were analyzed and processed using pClamp 10, GraphPad Prism 5, and Excel software. The degree of inhibition of hERG potassium current (peak hERG tail current evoked at -50 mV) by different compound concentrations was calculated using the following formula: Inhibition% = [1–(I / Io)] × 100%.

[0187] Wherein, Inhibition% represents the inhibition percentage of the compound on hERG potassium current, and I and Io represent the amplitude of hERG potassium current before and after drug addition, respectively.

[0188] Compound IC 50 The results were calculated using GraphPad Prism 5 software by fitting the following equation: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC 50 -X)×HillSlope))

[0189] Where X is the Log value of the test sample concentration, Y is the inhibition percentage at the corresponding concentration, and Bottom and Top are the minimum and maximum inhibition percentages, respectively.

[0190] Conclusion: Compound I has a small hERG inhibitory effect, IC 50 >40μM.

[0191] 3. Mouse Liver Microsome Stability Test

[0192] At 37°C, 1 μM of the test compound was incubated with mouse liver microsomes (0.5 mg / mL) supplemented with an NADPH regeneration system for 5, 10, 20, 30, and 60 minutes. The concentration of the test compound in the resulting samples was determined by LC-MS / MS. The half-life (T) of the compound in the mouse liver microsome solution was obtained by calculating the remaining percentage of the compound at each time point. 1 / 2 ) and intrinsic clearance (CL int(mic) ).

[0193] Biological test results:

[0194] Conclusion: The compounds of the present invention have good stability in mouse liver microsomes. For example, the clearance rate of compound I in mouse liver microsomes is significantly slowed down and the half-life is significantly prolonged compared with the control compound. 1 / 2 The ratio is greater than 2.

[0195] 4. Human Liver Microsome Stability Test

[0196] At 37°C, 1 μM of the test compound was incubated with human liver microsomes (0.5 mg / mL) supplemented with NADPH regeneration system for 5, 10, 20, 30, and 60 minutes. The concentration of the test compound in the resulting samples was determined by LC-MS / MS. The half-life (T) of the compound in the human liver microsome solution was obtained by calculating the remaining percentage of the compound at each time point. 1 / 2 ) and intrinsic clearance (CL int(mic) ).

[0197] Biological test results:

[0198] Conclusion: The compounds of the present invention have good stability in human liver microsomes. For example, the clearance rate of compound 1 in human liver microsomes is significantly slowed down and the half-life is significantly prolonged compared with the control compound. 1 / 2 The ratio is greater than 3.

[0199] 5. Pharmacokinetic testing

[0200] Pharmacokinetic evaluation in mice

[0201] Male C57 mice (purchased from Hunan Slake Jingda Experimental Animal Co., Ltd., license number: SCXK (Xiang) 2019-0004), 20-25g, fasted overnight. On the day of the experiment, 18 male C57 mice were divided into two groups, 9 in each group, and the test compound was administered intravenously and orally at doses of 5 mg / kg and 20 mg / kg, respectively. 0.1 ml of blood was collected from the orbital venous plexus before administration and 5, 15, 30 minutes, 1, 2, 4, 6, 8 and 24 hours after administration. The blood was placed in an EDTAK2 centrifuge tube and centrifuged at 6000 rpm and 4 ° C for 5 minutes to collect plasma. The plasma sample was added to an acetonitrile solution containing an internal standard (dexamethasone, 500 ng / mL), vortexed and centrifuged at 10000 rpm for 10 minutes, and the supernatant was taken for LC-MS / MS analysis. Pharmacokinetic parameters were calculated using the non-compartmental model in WinNonlin Version 8.0 (Pharsight, Mountain View, CA) pharmacokinetic software.

[0202] Conclusion: The compounds of the present invention have good oral properties. For example, the bioavailability of compound 1 in C57 mice is >25%.

[0203] Pharmacokinetic evaluation in cynomolgus monkeys

[0204] Male cynomolgus macaques (housed at Suzhou Leo Biotechnology Co., Ltd.) weighing 3-5 kg ​​were fasted overnight (14-18 hours). On the day of the experiment, six male cynomolgus macaques were divided into two groups of three and administered the test compound at doses of 2 mg / kg and 10 mg / kg intravenously and orally. Blood (1 ml) was collected from the four limb veins before administration and at 5, 15, 30 minutes, 1, 2, 4, 6, 8, 10, 12, and 24 hours after administration. The blood was placed in EDTAK2 centrifuge tubes and centrifuged at 6000 rpm at 4°C for 5 minutes to collect plasma. Plasma samples were spiked with acetonitrile containing an internal standard (dexamethasone, 500 ng / mL), vortexed, and centrifuged at 10,000 rpm for 10 minutes. The supernatant was then analyzed by LC-MS / MS. Pharmacokinetic parameters were calculated using a non-compartmental model in WinNonlin Version 8.0 (Pharsight, Mountain View, CA) pharmacokinetic software.

[0205] Conclusion: The compounds of the present invention have good metabolic properties. For example, Compound I has a low clearance rate and a long half-life in monkeys, which has the advantages of longer drug action and less frequent dosing.

[0206] 6. Cytochrome P450 isoenzyme inhibition test

[0207] Experimental purpose: To determine the inhibitory effect of the test compounds on the activities of human liver microsomal cytochrome P450 isoenzymes (CYP1A2, CYP2C19, CYP2D6 and CYP3A4).

[0208] Experimental procedure: The test compounds at incubation concentrations of 0, 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 μM were pre-incubated with human liver microsome solutions containing 5 enzyme mixed substrates (liver microsome concentration: 0.1 mg / mL) in a 37°C water bath for 10 minutes, and then the coenzyme factor (NADPH) was added to initiate the reaction; after further incubation in a 37°C water bath for 10 minutes, the reaction was terminated by adding glacial acetonitrile solution containing the internal standard, shaken for 1 minute, and centrifuged at 4°C and 10,000 rpm for 10 minutes. The supernatant was then subjected to LC / MS / MS analysis to detect the amount of metabolites produced by each CYP enzyme substrate.

[0209] The production rate of each metabolite reflects the activity of each CYP isoenzyme. The activity of each isoenzyme in the solvent control incubation system without the test substance was set to 100%, and the ratio of the production rate of the metabolite in the presence of different concentrations of the test substance to the production rate of the metabolite in the solvent control sample was used as the residual activity percentage of each isoenzyme. The data were plotted using Graphpad Prism 5.0 with the residual activity percentage as the ordinate and the inhibitor concentration as the abscissa, and the IC value of the test substance for each CYP isoenzyme was calculated. 50 value.

[0210] Conclusion: The compounds of the present invention have weak inhibitory activity against cytochrome P450 isoenzymes. For example, compound I has inhibitory activity IC against CYP2C19, CYP1A2, CYP2D6 and CYP3A4-M. 50 >50μM.

[0211] 7. Intravenous Glucose Tolerance Test in Cynomolgus Monkeys

[0212] Experimental animals: 6 male cynomolgus macaques (raised at Crown Biotech (Taicang) Co., Ltd.), weighing 6.5-7.5 kg.

[0213] Experimental procedure: During the entire experimental period, before each blood collection (ivGTT operation), the experimental animals were required to fast overnight for approximately 16 hours. The animals were grouped and dosed according to the following Table 10-1.

[0214] Intravenous Glucose Tolerance Test (ivGTT): After anesthesia, baseline blood was collected, followed by an IV bolus and infusion. Pre-glucose baseline blood glucose was collected 15 minutes after the infusion, and 50% glucose solution (0.25 g / kg, 0.5 mL / kg) was administered 30 minutes after the infusion. The total infusion duration was 1.0 hour.

[0215] Blood was collected before administration, at -15 minutes (15 minutes before glucose administration), at 0 minutes (before glucose administration), and at 2, 5, 7, 10, 20, and 30 minutes after glucose administration. The collected blood was allowed to stand at room temperature for at least 30 minutes and then centrifuged at 3500 g / min for 10 minutes at room temperature. The supernatant was aliquoted and stored at 2-8°C until glucose and insulin analysis. All experimental data were analyzed using GraphPad Prism 9.3.1.

[0216] Table 10-1 Grouping and Dosage

[0217] Note: *Intravenous infusion was performed at a constant rate using a Harvard syringe pump. The solvent consisted of 5% polyethylene glycol 400 (PEG400) + 95% [20% sulfobutyl-β-cyclodextrin (SEBCD)]. After brief sonication, the solvent was completely dissolved and filtered through a disposable syringe filter before preparation. Prepare the solution immediately and adjust the pH to 7.0-7.4.

[0218] The test results are shown in Table 10-2:

[0219] Table 10-2 Blood glucose and insulin analysis results

[0220] Conclusion: Compound I can promote insulin secretion, improve glucose tolerance and increase glucose clearance rate.

[0221] 8.Caco2 permeability test

[0222] The assay used Caco-2 cell monolayers in 96-well Transwell plates, incubated in triplicate. Transport buffer (HBSS, 10 mM HEPES, pH 7.4 ± 0.05) containing a compound of the invention (2 μM) or control compounds: Compound DZ-6, digoxin (10 μM), nadolol (2 μM), and metoprolol (2 μM) was added to the apical or basolateral wells of the cell monolayer. Transport buffer containing DMSO was added to the corresponding receiving wells. After incubation at 37 ± 1°C for 2 hours, the cell plate was removed and appropriate samples were taken from the apical and basolateral wells to a new 96-well plate. Proteins were then precipitated by adding acetonitrile containing an internal standard. Samples were analyzed by LC-MS / MS, and the concentrations of the compound of the invention and control compound were determined. Concentration data were used to calculate the apparent permeability coefficients for transport from the apical to basolateral and basolateral to apical sides of the cell monolayer, thereby calculating the efflux rate. Leakage of Lucifer Yellow was used to assess the integrity of the cell monolayer after 2 hours of incubation.

[0223] Table 11-1. Caco2 test results of the compounds of the present invention

[0224] Conclusion: Compound I is a low permeability compound, and its efflux rate is lower than that of the control compound DZ-6.

Claims

1. A pharmaceutically acceptable salt of a compound represented by formula (I) and a solvate thereof, The pharmaceutically acceptable salt is selected from tris(hydroxymethylaminomethane) salt or triethanolamine salt.

2. The pharmaceutically acceptable salt and solvate thereof according to claim 1, wherein the molar ratio of the compound represented by formula (I): the pharmaceutically acceptable salt is 1:0.9 to 1:1.

1.

3. The pharmaceutically acceptable salt and solvate thereof according to claim 1, wherein the molar ratio of the compound represented by formula (I): the pharmaceutically acceptable salt is 1:

1. The pharmaceutically acceptable salt and solvate thereof according to claim 1 , wherein the solvate is methanol.

5. A crystalline form A of a tris(hydroxymethyl)aminomethane salt of a compound represented by formula (I), using Cu-Kα radiation, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ positions: 8.97°±0.2°, 9.32°±0.2°, 9.92°±0.2°, 10.68°±0.2°, 17.50°±0.2°, and 20.17°±0.2°.

6. The crystalline form A according to claim 5, using Cu-Kα radiation, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ positions: 8.97°±0.2°, 9.32°±0.2°, 9.92°±0.2°, 10.68°±0.2°, 15.07°±0.2°, 17.50°±0.2°, 17.71°±0.2°, 19.95°±0.2°, 20.17°±0.2°, 24.27°±0.2°, and 26.81°±0.2°.

7. The crystalline form A according to claim 5, using Cu-Kα radiation, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ positions: 6.02°±0.2°, 8.97°±0.2°, 9.32°±0.2°, 9.92°±0.2°, 10.68°±0.2°, 12.03±0.2°, 13.77°±0.2°, 15.07°±0.2°, 16.18°±0.2°, 17.50°±0.2°, 17.71°±0.2°, 18.27°±0.2°, 18.70°±0.2°, 19.15°±0.2°, 19.95°±0.2°, 20.17°±0.2°, 20.90°±0.2°, 21.89°±0.2°, 22.35°±0.2°, 24.05°±0.2°, 24.27°±0.2°, 24.65°±0.2°, 25.03°±0.2°, 25.54°±0.2°, 26.81°±0.2°, 28.03°±0.2°.

8. The crystal form A according to claim 5, using Cu-Kα radiation, has an X-ray powder diffraction pattern as shown in Figure 1.

9. A tris(hydroxymethyl)aminomethane salt crystalline form C of the compound represented by formula (I), having an X-ray powder diffraction pattern using Cu-Kα radiation having characteristic diffraction peaks at the following 2θ positions: 10.55°±0.2°, 13.69°±0.2°, 19.41°±0.2°, and 20.70±0.2°.

10. The crystalline Form C according to claim 9, using Cu-Kα radiation, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ positions: 10.55°±0.2°, 10.92°±0.2°, 13.69°±0.2°, 17.98°±0.2°, 19.41°±0.2°, 20.70±0.2°, 21.97±0.2°, 22.31±0.2°, and 26.44±0.2°.

11. The crystalline form C according to claim 9, using Cu-Kα radiation, has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ positions: 5.44°±0.2°, 8.12°±0.2°, 10.17°±0.2°, 10.55°±0.2°, 10.92°±0.2°, 11.56°±0.2°, 13.08°±0.2°, 13.69°±0.2°, 14.49°±0.2°, 15.26°±0.2°, 17.06 °±0.2°, 17.78°±0.2°, 17.98°±0.2°, 18.41°±0.2°, 19.41°±0.2°, 20.70±0.2°, 21.25±0.2°, 21.97±0.2°, 22.31±0.2°, 23.27±0.2°, 24.23±0.2°, 25.24±0.2°, 25.70±0.2°, 26.44±0.2°, 27.49±0.2°, 28.71±0.2°.

12. The crystal form C according to claim 9, using Cu-Kα radiation, has an X-ray powder diffraction pattern as shown in FIG3.

13. A crystalline form D of the tris(hydroxymethyl)aminomethane salt of the compound represented by formula (I), having an X-ray powder diffraction pattern using Cu-Kα radiation having characteristic diffraction peaks at the following 2θ positions: 5.55°±0.2°, 10.46°±0.2°, 11.15°±0.2°, 13.12°±0.2°, 13.98°±0.2°, 19.66°±0.2°, and 22.47°±0.2°.

14. The crystalline form D according to claim 13, using Cu-Kα radiation, has an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2θ positions: 5.55°±0.2°, 8.34±0.2°, 10.46°±0.2°, 11.15°±0.2°, 11.50±0.2°, 13.12°±0.2°, 13.98°±0.2°, 19.66°±0.2°, 20.80±0.2°, 22.08±0.2°, 22.47°±0.2°, 23.26±0.2°, and 26.73±0.2°. 15 . The crystal form D according to claim 13 , using Cu-Kα radiation, has an X-ray powder diffraction pattern as shown in FIG5 .

16. A triethanolamine salt crystalline form A of the compound represented by formula (I), having an X-ray powder diffraction pattern using Cu-Kα radiation having characteristic diffraction peaks at the following 2θ positions: 10.42°±0.2°, 12.68°±0.2°, 14.80°±0.2°, 16.37°±0.2°, 16.94°±0.2°, and 23.39°±0.2°.

17. The crystalline form A according to claim 16, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ positions using Cu-Kα radiation: 8.42°±0.2°, 10.42°±0.2°, 12.68°±0.2°, 13.28°±0.2°, 14.80°±0.2°, 16.37°±0.2°, 16.94°±0.2°, 18.04°±0.2°, 22.26°±0.2°, 23.39°±0.2°, 24.98°±0.2°, and 25.55°±0.2°.

18. The crystalline form A according to claim 16, wherein the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2θ positions using Cu-Kα radiation: 4.21±0.2°, 6.30±0.2°, 8.42±0.2°, 10.42±0.2°, 12.68±0.2°, 13.28±0.2°, 14.35±0.2°, 14.80±0.2°, 15.11±0.2°, 16.10±0.2°, 16.37±0.2°, 16.94±0.2°, 17.32±0.2°, 18.04 .2°, 24.55°±0.2°, 24.98°±0.2°, 25.55°±0.2°, 26.48°±0.2°, 28.07°±0.2°, and 29.90°±0.2°.

19. The crystal form A according to claim 16, using Cu-Kα radiation, has an X-ray powder diffraction pattern as shown in FIG7.

20. A method for preparing a pharmaceutically acceptable salt of a compound represented by formula (I), wherein: The method comprises the step of forming a salt with the compound represented by formula (I) and an acid.

21. The preparation method according to claim 20, wherein The solvent used is selected from C 1-6 Halogenated alkane solvents, C 2-6 Ester solvents, C 2-6 Ether solvents, C 1-6 One or more of alcohol solvents or water.

22. The preparation method according to claim 21, wherein The solvent used is selected from one or more of dichloromethane, 1,2-dichloroethane, ethyl acetate, methanol, ethanol, isopropanol, propanol, ether, tetrahydrofuran and water.

23. A pharmaceutical composition, wherein The pharmaceutical composition contains a therapeutically effective amount of a pharmaceutically acceptable salt of the compound according to any one of claims 1 to 4 and a solvate thereof, or a crystalline form according to any one of claims 5 to 22, and a pharmaceutically acceptable carrier or excipient.

24. The pharmaceutical composition according to claim 23, wherein the therapeutically effective amount is 1-600 mg calculated as the free base.

25. Use of a pharmaceutically acceptable salt of the compound according to any one of claims 1 to 4 and a solvate thereof, or a crystalline form according to any one of claims 5 to 19, or a pharmaceutical composition according to any one of claims 23 to 24 in the preparation of a medicament for treating diabetes or diabetes-related diseases.

Images