SSTR5-TGR5 double-effect compound as well as preparation method, pharmaceutical composition and application thereof

Abstract

The invention relates to an SSTR5-TGR5 double-effect compound as well as a preparation method, a pharmaceutical composition and application of the SSTR5-TGR5 double-effect compound. The compounds of the present invention are represented by the following general formula (I). When the compound is independently used, the gastrointestinal hormone level can be improved, and the compound is used for GLP-1, GLP-2, GCG, GIP and CCK related type 2 diabetes mellitus, obesity, non-alcoholic fatty liver disease, non-alcoholic fatty liver fibrosis, Alzheimer's disease, Parkinson's disease, inflammatory bowel disease, cholangitis, gall-stone and other diseases. Besides, the compound provided by the invention is combined with a DPP4 inhibitor for use, so that the gastrointestinal hormone level can be greatly improved, and the compound is used for preventing or treating diseases such as type 2 diabetes mellitus, obesity, non-alcoholic fatty liver disease, non-alcoholic fatty liver fibrosis, Alzheimer's disease, Parkinson's disease, inflammatory bowel disease and the like. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and in particular to a class of SSTR5-TGR5 ​​dual-effect compounds, and a pharmaceutical composition with the class of compounds as active components. The compounds are used for preventing and treating diseases related to SSTR5 and TGR5. Background Art

[0002] Gastrointestinal endocrine cells release a variety of gastrointestinal hormones in response to nutritional factors and external stimuli, such as glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), peptide tyrosine tyrosine (PYY), glucagon (GCG), gastric inhibitory peptide (GIP) and cholecystokinin (CCK), etc. These gastrointestinal hormones have important physiological functions in regulating physiological functions in the body. Among them, GLP-1 is secreted by intestinal endocrine L-cells (Physiol Rev 87:1409–1439, 2007). It can promote insulin release and inhibit glucagon production in a blood sugar-dependent manner on pancreatic islets, inhibit β-cell apoptosis, and promote β-cell proliferation; it can delay gastric emptying when acting on the stomach; it can increase the utilization of glucose and reduce the utilization of fatty acids when acting on the heart, and play a myocardial protective function; it can inhibit appetite and promote nerve cell growth when acting on the brain, and play a neuroprotective function; it can reduce liver sugar and liver fat when acting on the liver, and resist fibrosis; it can promote energy metabolism when acting on muscle and adipose tissue. Therefore, drug development based on GLP-1 has been successfully applied to type 2 diabetes and obesity, and has shown positive therapeutic effects in clinical trials of non-alcoholic steatohepatitis (NASH), Alzheimer's disease (AD) and Parkinson's disease (PD). GLP-2 is secreted by endocrine L cells (Annu. Rev. Physiol. 2014. 76: 561–83), which can promote intestinal absorption of nutrients, promote the growth of small intestinal villi and crypts, and its analogs have been successfully used in the treatment of short bowel syndrome and have shown positive therapeutic effects in inflammatory bowel disease. PYY is secreted by intestinal endocrine L cells and is mainly related to diet control. It is currently used to develop treatments for obesity and type 2 diabetes. GIP is secreted by endocrine K cells and mainly acts on the pancreatic islet glucose-dependent insulin release and promotes β-cell proliferation. GCG is secreted by endocrine L cells and mainly plays a role in regulating blood glucose homeostasis. In summary, these gastrointestinal hormones play an important role in energy metabolism and are potential therapeutic strategies for chronic metabolic diseases such as type 2 diabetes, obesity, non-alcoholic liver fibrosis, non-alcoholic fatty liver disease, Alzheimer's disease, Parkinson's disease, and gastrointestinal diseases.

[0003] Takeda G protein receptor 5 (TGR5) is widely expressed on endocrine cells in the gastrointestinal tract. When TGR5 receptor is activated, it can promote the release of gastrointestinal hormones GLP-1, GIP, GLP-2, PYY, and GCG through a cyclic adenosine monophosphate (cAMP)-dependent pathway; therefore, TGR5 agonists can be used in the development of drugs for chronic metabolic diseases and gastrointestinal diseases, such as type 2 diabetes, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, obesity, and inflammatory bowel disease (Digestive and Liver Disease 46 (2014) 302–312). SSTR5 is widely distributed in the gastrointestinal tract, and its endogenous ligand is somatostatin. When somatostatin binds to SSTR5 (somatostatin receptor 5), it activates SSTR5, thereby inhibiting the release of insulin and gastrointestinal hormones GLP-1, GLP-2, PYY, GIP, and CCK (Results Probl Cell Differ, DOI 10.1007 / 400_2009_29). SSTR5 antagonists can relieve this effect and promote the release of insulin and gastrointestinal hormones GLP-1, GLP-2, PYY, GIP and CCK; they are potential therapeutic strategies for chronic metabolic diseases and gallbladder-related diseases.

[0004] Further studies have shown that when GLP-1 levels increase, activating the GLP-1 receptor in the body will promote the secretion of somatostatin, which in turn activates SSTR5 and antagonizes the secretion of GLP-1. This negative feedback regulation prevents GLP-1 levels from increasing significantly (JCI Insight. 2021; 6(4): e143228.). In addition, GLP-1 is rapidly degraded by the DPP4 enzyme in the body, with a half-life of only 1-2 minutes. DPP4 inhibitors can significantly increase GLP-1 levels. In vivo studies in mice have found that TGR5 agonists, SSTR5 antagonists, and DPP4 inhibitors have significant synergistic effects on increasing GLP-1, and can achieve supratherapeutic levels of GLP-1 (Diabetes 2018; 67: 309–320, ACS Pharmacol. Transl. Sci. 2018, 1, 3-11); similarly, since GLP-1, GLP-2, and GCG are all processed from the same precursor, proglucagon (GASTROENTEROLOGY 2007; 132: 2131–2157), theoretically, GCG and GLP-2 will also be greatly increased; similarly, TGR5 agonists can promote the release of GIP, SSTR5 antagonists can relieve the inhibition of GIP release, and DPP4 inhibitors can prevent the hydrolysis and inactivation of GIP, so the combination of the three can theoretically also achieve a significant increase in GIP. Summary of the invention

[0005] The technical purpose of the present invention is to provide a class of compounds having SSTR5 antagonistic activity and TGR5 agonistic activity.

[0006] Another technical object of the present invention is to provide a pharmaceutical composition comprising the above compound.

[0007] Another technical objective of the present invention is to provide use of the above compound or the above pharmaceutical composition in the preparation of a drug for preventing or treating a disease mediated by TGR5 and / or SSTR5.

[0008] In one aspect, the present invention provides compounds of the following general formula (I), their enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts:

[0009]

[0010] in,

[0011] Ring A is selected from

[0012] R1 is hydrogen, C1-C6 alkyl or C3-C6 cycloalkyl;

[0013] X is O or CH2;

[0014] R2, R3, R4, R5 are each independently hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or C3-C6 cycloalkyl;

[0015] Ring B is derived from the core ring structure in the SSTR5 antagonist structural framework.

[0016] X1 is NH or absent;

[0017] R6 and R7 are each independently hydrogen, halogen, hydroxy, amino, C1-C6 alkyl, C1-C3 alkoxy, or amino mono- or di-substituted by C1-C6 alkyl;

[0018] R8 is a carboxyl group, a phosphoric acid group, a sulfonic acid group, a sulfonic acid ester group esterified with a C1-C6 alkyl group, a tetrazolyl group, a hydroxyl group or an amino group;

[0019] Y1 and Y2 are each independently CH or N;

[0020] Indicates the key connection location.

[0021] In a specific embodiment, in the general formula (I), ring B is selected from Ring B separates the general formula (I) into a portion containing ring A and a portion without ring A. For the two aforementioned substituent groups, the left side of the substituent group is connected to the portion containing ring A of the general formula (I), and the right side is connected to the portion without ring A of the general formula (I), wherein Z is selected from CH2, O and NH, and m and n are each independently an integer of 1-3, provided that when ring B is When X1 does not exist, when ring B is X1 is NH.

[0022] In a specific embodiment, in the general formula (I), ring A is And ring B is Ring A is And ring B is Ring A is And ring B is Ring A is And ring B is

[0023] In a specific embodiment, in the general formula (I), R1 is a C1-C3 alkyl group or a C3-C6 cycloalkyl group, preferably a methyl group or a cyclopropyl group.

[0024] In a specific embodiment, in the general formula (I), R2, R3, R4, and R5 are each independently hydrogen, methyl, methoxy, ethoxy, or cyclopropyl.

[0025] In a specific embodiment, in the general formula (I), R6 and R7 are both hydrogen.

[0026] In a specific embodiment, in the general formula (I), when ring B is When Z is selected from O or NH, m and n are both 2.

[0027] In a specific embodiment, in the general formula (I), R8 is a carboxyl group or a sulfonic acid group.

[0028] In a specific embodiment, in the general formula (I), Y1 and Y2 are both CH.

[0029] In a specific embodiment, the compound of formula (I) is selected from one of the following compounds:

[0030]

[0031]

[0032]

[0033] In another aspect, the present invention provides a method for preparing the compound of the above general formula (I):

[0034] A: For A Ring B is The compound of general formula (I) wherein X1 does not exist is synthesized by the following method:

[0035]

[0036]

[0037] In the above reaction formula, R1, R2, R3, R4, R5, R6, R7, R8, Y1, Y2, m, n, and Z are defined as above; R9 is a sulfonate group substituted with a C1-C3 alkoxycarbonyl group or a neopentyl group; W is a halogen;

[0038] (1) a and b undergo nucleophilic substitution reaction to obtain c;

[0039] (2) c undergoes hydrogenation reduction decarboxylation to give d;

[0040] (3)d is prepared by reacting the acyl chloride with N 1 Substituted benzotetrahydroquinoxaline is condensed to give e;

[0041] (4) f and g are reacted to prepare h through nucleophilic substitution reaction, Buchwald–Hartwig coupling reaction or Ullman condensation;

[0042] (5) h is deprotected under acidic conditions to obtain i;

[0043] (6) i and e are reacted by nucleophilic substitution to obtain j;

[0044] (7) j is deprotected under alkaline conditions or under sodium iodide-acetone conditions to obtain a compound of the general formula k;

[0045] B: For A Ring B is The compound of general formula (I) wherein X is O and X1 is NH is synthesized by the following route:

[0046]

[0047] wherein R1, R2, R3, R4, R5, R6, R7, R8, Y1 and Y2 are as defined above;

[0048] R9 is a sulfonate group substituted with a C1-C3 alkoxycarbonyl group or a neopentyl group;

[0049] W is a halogen;

[0050] (1) 2a and 2b were prepared to give 2c via nucleophilic substitution reaction, Buchwald–Hartwig coupling reaction or Ullman condensation;

[0051] (2) 2c was deprotected under acidic conditions to give 2d;

[0052] (3) 2d and e were reacted via nucleophilic substitution to give 2e;

[0053] (4) 2e is deprotected under alkaline conditions or sodium iodide-acetone conditions to obtain a compound of the general formula 2f,

[0054] The intermediate e in the above step (3) is shown in Scheme 1.

[0055] C: For A Ring B is The compound of general formula (I) wherein X is CH2 and X1 does not exist is synthesized by the following three routes:

[0056]

[0057] R1, R2, R3, R4, R5, R6, R7, R8, Y1 and Y2, m, n, Z are as defined above respectively;

[0058] R9 is a sulfonate group substituted with a C1-C3 alkoxycarbonyl group or a neopentyl group;

[0059] (1) 3a reacts with N by amide condensation reaction. 1 Substituted benzotetrahydroquinoxaline was prepared to give 3b;

[0060] (2) 3b is deprotected under acidic conditions to give 3c;

[0061] (3) 3c was reacted with the aldehyde fragment to give 3d;

[0062] (4) 3d and i were reacted to give 3e by reductive amination;

[0063] (5) 3e is deprotected under alkaline conditions or sodium iodide-acetone conditions to obtain compound 3k,

[0064] The intermediate i in the above step (4) is shown in Scheme 1,

[0065] D: For A Ring B is X is CH2, and X1 is NH, the compound of formula (I) is synthesized by the following route 4:

[0066]

[0067] Wherein, R1, R2, R3, R4, R5, R6, R7, R8, Y1 and Y2 are as defined above;

[0068] R9 is a sulfonate group substituted with a C1-C3 alkoxycarbonyl group or a neopentyl group;

[0069] (1) 4a was prepared by reductive amination of 3d and 2c;

[0070] (2) 4a is deprotected under alkaline conditions or sodium iodide-acetone conditions to obtain compound 4b.

[0071] In another aspect, the present invention provides a pharmaceutical composition comprising the compound of the above general formula (I), its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts, and pharmaceutically acceptable excipients.

[0072] In a specific embodiment, the pharmaceutical composition further comprises one or more other drugs for treating diseases mediated by TGR5 or SSTR5. Preferably, the other drugs are selected from one or more of hypoglycemic drugs, weight loss drugs, non-alcoholic fatty liver disease drugs (NAFLD), non-alcoholic steatohepatitis drugs (NASH), inflammatory bowel disease drugs, Alzheimer's disease drugs, Parkinson's disease drugs, cholestasis drugs, gallstone drugs, cholangitis drugs, and cholecystitis drugs.

[0073] In a specific embodiment, the pharmaceutical composition further comprises a DPP4 inhibitor, wherein the DPP4 inhibitor can be selected from sitagliptin, alogliptin, linagliptin, vildagliptin and saxagliptin, preferably, the DPP4 inhibitor is sitagliptin.

[0074] In another aspect, the present invention provides the use of the compound of the above general formula (I), its enantiomers, diastereoisomers, racemic mixtures, deuterated products and pharmaceutically acceptable salts, or the above pharmaceutical compositions in the preparation of drugs for preventing or treating diseases mediated by TGR5 and / or SSTR5.

[0075] In a specific embodiment, the diseases mediated by TGR5 and / or SSTR5 include but are not limited to type 2 diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, inflammatory bowel disease, Alzheimer's disease, Parkinson's disease and gallbladder system diseases.

[0076] In a specific embodiment, the gallbladder system disease includes but is not limited to cholestasis, cholelithiasis, cholangitis, and cholecystitis.

[0077] Beneficial Effects

[0078] Based on the successful marketing of existing DPP4 inhibitors, the present invention provides a class of SSTR5-TGR5 ​​dual-effect compounds. On the one hand, the compound can increase the levels of gastrointestinal hormones such as GLP-1, GLP-2, GIP, GCG and CCK when used alone, and is used for diseases such as type 2 diabetes, obesity, non-alcoholic fatty liver, non-alcoholic fatty liver fibrosis, Alzheimer's disease, Parkinson's disease, inflammatory bowel disease, cholangitis, gallstones, etc. related to GLP-1, GLP-2, GCG, GIP and CCK; on the other hand, when used in combination with DPP4 inhibitors, the gastrointestinal hormone levels can be greatly increased, and it is used to prevent or treat diseases such as type 2 diabetes, obesity, non-alcoholic fatty liver, non-alcoholic fatty liver fibrosis, Alzheimer's disease, Parkinson's disease, inflammatory bowel disease, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 The figure shows the effect of compound 5 of the present invention on oral glucose tolerance (OGTT) of normal C57BL / 6 mice.

[0080] Figure 2 The figure shows the effect of compound 5 of the present invention combined with the DPP4 inhibitor sitagliptin on the level of active GLP-1 in normal C57BL / 6 mice. DETAILED DESCRIPTION

[0081] definition

[0082] Unless otherwise specified, the terms used in this invention have the following definitions:

[0083] The term "substituted" as used herein means replacement by one or more groups. When multiple groups are selected from the same series of candidate substituents, they may be the same or different.

[0084] The term "optionally" used in the present invention means that the defined group may be selected from a series of candidate groups or may not be selected.

[0085] The "alkyl" mentioned in the present invention refers to a saturated straight-chain or branched alkyl group with a specific number of atoms, specifically including but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, etc. The "C1-6 alkyl" refers to a saturated straight-chain or branched alkyl group with 1 to 6 carbon atoms, specifically including but not limited to methyl, ethyl, propyl, isopropyl, etc.

[0086] The "cycloalkyl" mentioned in the present invention represents a non-aromatic, saturated, cyclic aliphatic hydrocarbon group having 3 to 6 ring atoms. Representative examples include but are not limited to: cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0087] The "alkoxy" described in the present invention refers to all straight-chain or branched isomers having a specific number of carbon atoms, and specifically may include but is not limited to methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and the like.

[0088] The "halogen" refers to fluorine, chlorine, bromine or iodine.

[0089] In the present invention, if the substitution on the alkyl or cycloalkyl group is not specified to occur on a specific carbon atom, it means that it can occur on any carbon atom that has not reached saturation with the number of substituents. When multiple substituents are selected from the same series, they can be the same or different.

[0090] In the present invention, substitution on a benzene ring, aromatic heterocycle or heterocycle, unless it is indicated that it occurs on a specific atom, means that it may occur at any position that is not substituted by an atom other than hydrogen. When multiple substituents are selected from the same series, they may be the same or different.

[0091] "Pharmaceutically acceptable salt" means that the compound of formula (I) retains the desired biological activity and has minimal toxic side effects. The pharmaceutically acceptable salt can be obtained directly during the preparation and purification of the compound, or indirectly by reacting the free acid or free base of the compound with another suitable base or acid.

[0092] The term "solvate" is used herein to describe a molecular complex comprising a compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules (eg, ethanol). The term "hydrate" is employed when the solvent is water.

[0093] How to use

[0094] When used for treatment, the compounds of the present invention are usually administered in the form of a standard pharmaceutical composition, which contains one or more compounds of the general formula (I) in an effective therapeutic dose, and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients are pharmaceutically acceptable carriers, excipients or sustained-release agents, etc.

[0095] The compound and pharmaceutical composition provided by the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and can be present in suitable solid or liquid carriers or diluents. The pharmaceutical composition of the present invention can also be stored in suitable sterilizing apparatus for injection or instillation. The pharmaceutical composition can also contain odorants, flavoring agents, etc.

[0096] In the present invention, the pharmaceutical composition contains a safe and effective amount (such as 0.1-99.9 parts by weight, preferably 1-90 parts by weight) of a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; and the remainder of pharmaceutically acceptable excipients, wherein the total weight of the composition is 100 parts by weight. Alternatively, the pharmaceutical composition of the present invention contains 0.1-99.9% by weight, preferably 1-90% by weight of a compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof; and the remainder of pharmaceutically acceptable excipients, wherein the total weight of the composition is 100% by weight.

[0097] The preferred ratio of the compound represented by general formula (I) to the pharmaceutically acceptable carrier, excipient or sustained-release agent is that the compound represented by general formula (I) as the active ingredient accounts for more than 60% of the total weight, and the remaining part accounts for 0-40% of the total weight, and the amount of the remaining part is preferably 1-20%, and most preferably 1-10%.

[0098] The compound represented by the general formula (I) or the pharmaceutical composition comprising the compound represented by the general formula (I) can be used clinically in mammals, including humans and animals, and the administration route can include oral administration, nasal inhalation, transdermal absorption, pulmonary administration or gastrointestinal tract, etc. The preferred administration route is oral administration. It is preferably a unit dosage form, and each dose contains 0.01 mg-200 mg of the active ingredient, preferably 0.5 mg-100 mg, which is taken once or in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined according to the specific treatment. Usually, it is started with a small dose and gradually increased until the most suitable dose is found.

[0099] The pharmaceutical composition of the present invention can be administered orally as well as intravenously, intramuscularly or subcutaneously. From the standpoint of ease of preparation and administration, the preferred pharmaceutical composition is a solid composition, especially tablets and solid-filled or liquid-filled capsules. Oral administration of the pharmaceutical composition is preferred.

[0100] Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose and kaolin, etc., while liquid carriers include sterile water, polyethylene glycol, nonionic surfactants and edible oils (such as corn oil, peanut oil and sesame oil), etc., as long as they are suitable for the characteristics of the active ingredient and the specific administration method required. Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavoring agents, pigments, preservatives and antioxidants such as vitamin E, vitamin C, BHT and BHA.

[0101] Injectable preparations include, but are not limited to, sterile, injectable, aqueous, oily solutions, suspensions, emulsions, etc. These preparations can also be formulated into suitable parenteral diluents, dispersants, wetting agents, suspending agents, etc. Such injectable preparations can be sterilized by filtration in a filter that retains bacteria. These preparations can also be formulated with bactericides that are dissolved or dispersed in an injectable medium or formulated with other methods known in the art.

[0102] Combination therapy:

[0103] The compounds of the present invention can be used in combination with other drugs for preventing or treating the above diseases.

[0104] The compounds of the present invention can be used in combination with one or more other drugs to treat, prevent or improve diseases for which the compounds of the present invention or other drugs may be effective, wherein these drugs are safer or more effective in combination than any one drug alone. The other drugs can be administered simultaneously with the compounds of the present invention or before or after the administration by commonly used routes of administration and dosages. When the compounds of the present invention are used simultaneously with one or more other drugs, a pharmaceutical composition containing the other drugs and the compounds of the present invention in unit dosage form is preferred. However, combination therapy may also include therapies in which the general formula compounds described herein and one or more other drugs are administered in different overlapping schemes. When used in combination with one or more other active ingredients, the compounds of the present invention and the other drugs may be used at a lower dose than when used alone. Wherein the other drugs include but are not limited to TGR5 agonists, GPR119 agonists, GPR40 agonists, PDE4 inhibitors, DPP4 inhibitors, SGLT2 inhibitors, metformin, insulin sensitizers, insulin and its analogs, α-glucosidase inhibitors, sulfonylureas or non-sulfonylurea insulin secretion promoters, incretin analogs, etc.

[0106] The present invention will be further described below with reference to examples. It should be particularly noted that these examples are only used to illustrate the present invention and are not intended to limit the present invention in any way. All parameters and other descriptions in the examples are based on mass unless otherwise specified. The fillers used for column chromatography separation are all silica gel unless otherwise specified. The experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0107] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0108] Example 1

[0109] 4-(8-(4-((3-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)pyridin-4-yl)oxy)-2,5-dimethylbenzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzoic acid (Compound 1)

[0110]

[0111] The preparation route of compound 1 is shown in the figure below:

[0112]

[0113] In the above reaction formula, (a) Pd2(dba)3, K3PO4, Xantphos, 1,4-dioxane, 100-110°C, 3-6h; (b) HCl / 1,4-dioxane, DCM, rt, 2h.

[0114]

[0115] In the above reaction formula, (a) K2CO3, DMF, 90-110℃, 3h; (b) H2, Pd / C, THF / MeOH, rt, 3h; (c) SOCl2, DCM, 50℃, 1h; (d) TEA, 1-cyclopropyl-1,2,3,4-benzotetrahydroquinoxaline, DCM, rt, 2h; (e) K2CO3, DMF, 60-70℃, 8h; (f) LiOH, 1,4-dioxane / H2O; 40-50℃, 12h.

[0116] Preparation of intermediate 1a-1

[0117]

[0118] 2-Oxo-1-oxo-3,8-diazaspiro[4.5]decane-8-carboxylic acid tert-butyl ester (CAS: 169206-55-7, 1 eq), methyl p-bromobenzoate (1.5 eq), anhydrous potassium phosphate (3 eq), and Xantphos ligand (0.1 eq) were dissolved in 1,4-dioxane, and Pd2(dba)3 (0.05 eq) was added after nitrogen replacement, and the mixture was reacted in a pressure tube at 100-110°C in an oil bath for 3 hours. After cooling to room temperature, the reaction solution was filtered to remove the solid, and the filtrate was mixed with silica gel for column chromatography to obtain intermediate 1a-1 with a yield of about 65%.

[0119] Preparation of intermediate 1a-2

[0120]

[0121] The intermediate 1a-1 was dissolved in dichloromethane, and an excess of hydrogen chloride dioxane solution was added, and the mixture was reacted at room temperature for 2 hours. The reaction solution was then evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step without purification. The yield was about 90%.

[0122] Preparation of intermediate 1b-1

[0123]

[0124] 2,5-Dimethyl-4-hydroxybenzaldehyde (1 eq), benzyl p-chloronicotinate (1 eq), and potassium carbonate (1.5 eq) were dissolved in DMF, and reacted in an oil bath at 100° C. for 3 hours under nitrogen protection. After thin layer chromatography confirmed that the reaction was complete, the reaction solution was cooled to room temperature, extracted three times with ethyl acetate, and the organic phases were combined, washed three times with saturated sodium chloride solution, and then dried and concentrated; column chromatography was performed to obtain intermediate 1b-1 with a yield of about 78%.

[0125] Preparation of intermediate 1b-2

[0126]

[0127] The intermediate 1b-1 (850 mg, 2.35 mmol) was dissolved in methanol-tetrahydrofuran (v / v=1:1), and after nitrogen replacement, 100 mg of palladium carbon hydrogenation catalyst (10% palladium) was added, and the reaction was carried out overnight under a hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered to remove the solid, and the filtrate was evaporated to dryness to obtain 520 mg of intermediate 1b-2, which can be directly used in the next step without purification, and the yield is about 81%. MS (ESI): m / z 274.1 [M+H] + .

[0128] Preparation of intermediate 1b-3

[0129]

[0130] The intermediate 1b-2 (500 mg, 1.83 mmol) was dissolved in dichloromethane, 0.5 mL of thionyl chloride (excess) was added, and the mixture was heated to 50°C in an oil bath for 3 h. The reaction solution was concentrated under reduced pressure until no fraction was distilled out, and then the reaction solution was redissolved in dichloromethane and added dropwise to a dichloromethane solution of cyclopropyl-benzotetrahydroquinoxaline (318 mg, 1.83 mmol) and triethylamine (0.8 mL, 5.5 mmol) in an ice bath. The reaction was continued for 2 h. After the reaction was confirmed to be complete by thin layer chromatography, the reaction solution was directly mixed with silica gel for column chromatography to obtain 670 mg of intermediate 1b-3, with a yield of about 82%. MS (ESI): m / z 448.2 [M+H] + .

[0131] Preparation of intermediate 1b-4

[0132]

[0133] Intermediate 1b-3 (100 mg, 0.22 mmol), intermediate 1a-2 (72 mg, 0.22 mmol), potassium carbonate (61 mg, 0.44 mmol) were dissolved in DMF, heated to 60-70°C in an oil bath under nitrogen protection and reacted overnight. After TLC confirmed that the reaction was complete, the reaction solution was cooled to room temperature, added with saturated sodium chloride aqueous solution, and then extracted with ethyl acetate three times. The organic phases were combined and washed with saturated sodium chloride aqueous solution three times, dried, concentrated under reduced pressure, mixed with silica gel, and separated by column chromatography to obtain 100 mg of intermediate 1b-4, with a yield of about 65%. MS (ESI): m / z 702.3 [M+H] + .

[0134] Preparation of final product compound 1

[0135] The intermediate 1b-4 (90 mg) was dissolved in a mixed solvent of 1,4-dioxane and water (v / v=4:1), and 25 mg of lithium hydroxide monohydrate was added. The mixture was reacted at 40°C overnight. After the reaction was confirmed to be complete by thin layer chromatography, the reaction solution was concentrated and purified by semi-preparative liquid phase to obtain 50 mg of the final product compound 1, with a yield of about 57%. The semi-preparative liquid phase purification system used acetonitrile-water (containing 0.1% trifluoroacetic acid), and the gradient was 0 min: 30% acetonitrile-70% water; 45 min: 75% acetonitrile-25% water; 60 min: 90% acetonitrile-10% water. MS (ESI): m / z 688.3 [M+H] + . 1 H NMR (500MHz, DMSO-d6) δ8.68(s,1H),8.34(s,1H),7.83(d,J=8.7Hz,2H),7.44(d,J=8.7Hz,2H),7.22(s ,1H),7.13(s,1H),7.02(t,J=7.7Hz,1H),6.91(s,1H),6.65(s,1H),6.53(s,1H),6.37(s,1H),6.29(s, 1H),5.43(s,1H),4.71(s,1H),3.87(s,2H),3.15(s,2H),2.32–2.20(m,2H),2.19–2.10(m,2H),2.06–1 .96(m,5H),1.87(dd,J=18.4,11.2Hz,6H),1.53–1.42(m,2H),0.72–0.31(m,3H),-0.24–-0.48(m,1H).

[0136] Example 2

[0137] 4-(8-(4-((3-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)pyridin-4-yl)oxy)-3-ethoxybenzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzoic acid (Compound 2)

[0138]

[0139] Except that 3-ethoxy-4-hydroxybenzaldehyde was used instead of 2,5-dimethyl-4-hydroxybenzaldehyde, the rest of the preparation method was the same as Example 1. MS (ESI): m / z 704.3 [M+H] + .1H NMR(500MHz,Methanol-d4)δ9.09(s,1H),8.65(d,J=6.5Hz,1H),8.02(d,J=8.7Hz,2H),7.69(d,J=8.6Hz,2H ),7.60(s,1H),7.14(ddd,J=25.2,10.4,4.9Hz,4H),6.69(d,J=7.7Hz,1H),6.44(t,J=7.5Hz,1H),6.35(s,1 H),4.48(d,J=42.7Hz,2H),4.12(dp,J=14.2,7.2Hz,2H),4.03(s,2H),3.69–3.49(m,5H),3.38(t,J=12.5Hz ,2H),2.54–2.25(m,6H),1.20(t,J=6.9Hz,3H),0.80–0.69(m,2H),0.60–0.43(m,1H),-0.02–-0.12(m,1H).

[0140] Example 3

[0141] 4-(8-(4-((3-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)pyridin-4-yl)oxy)-2,5-diethoxybenzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzoic acid (Compound 3)

[0142]

[0143] Except that 2,5-diethoxy-4-hydroxybenzaldehyde was used instead of 2,5-dimethyl-4-hydroxybenzaldehyde, the rest of the preparation method was the same as Example 1. MS (ESI): m / z 748.3 [M+H] + . 1H NMR(500MHz,Chloroform-d)δ8.65(d,J=12.8Hz,1H),8.33(s,1H),8.18(d,J=8.8Hz,1H),8.02(s,1H),7.75 (d,J=8.8Hz,1H),7.50(s,1H),7.09(d,J=8.5Hz,2H),7.00(s,1H),6.56(s,1H),6.37(s,1H),6.29(s,1H),5. 65(s,1H),4.69(s,4H),4.05–3.60(m,6H),3.62–3.20(m,2H),3.20–2.58(m,4H),2.53–2.32(m,1H),2.06(d ,J=12.5Hz,4H),1.41(dd,J=12.7,6.2Hz,3H),1.16(d,J=19.0Hz,3H),0.87–0.44(m,3H),0.03–0.01(m,1H).

[0144] Example 4

[0145] 4-(8-(2-cyclopropyl-4-((3-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)pyridin-4-yl)oxy)-5-methoxybenzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzoic acid (Compound 4)

[0146]

[0147] The preparation method is the same as Example 1 except that 2-cyclopropyl-4-hydroxy-5-methoxybenzaldehyde is used instead of 2,5-dimethyl-4-hydroxybenzaldehyde. MS (ESI): m / z 729.3 [M+H] + . 1 H NMR(500MHz,Chloroform-d)δ8.55(s,1H),8.21(s,1H),7.74(s,2H),7.20–6.89 (m,5H),6.47(s,1H),6.30(s,1H),6.13(s,1H),5.72(s,1H),4.60(s,2H),3.99– 3.53(m,3H),3.51–3.31(m,4H),3.29–2.81(m,6H),2.80–2.40(m,2H),2.31(s,1 H),1.99–1.57(m,4H),0.92–0.75(m,2H),0.75–0.27(m,6H),0.14–0.01(m,2H).

[0148] Example 5

[0149] (S)-4-(8-(4-((4-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)thiazolidin-3-yl)methyl)-2,5-dimethoxybenzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzoic acid (Compound 5)

[0150]

[0151] Preparation of intermediate 5a-1;

[0152]

[0153] N-Boc-(R)-thiazole-4-carboxylic acid (500 mg, 2.15 mmol), DIPEA (750 μL, 4.3 mmol) and HATU (980 mg, 2.6 mmol) were dissolved in dichloromethane and reacted at room temperature for half an hour before adding 374 mg (2.15 mmol) of cyclopropyl-benzotetrahydroquinoxaline and continuing to react at room temperature for 3 hours. After TLC confirmed that the reaction was complete, the reaction solution was mixed with silica gel for column chromatography to obtain 590 mg of intermediate 5a-1, with a yield of about 71%. MS (ESI): m / z 390.2 [M+H] + ,290.2[M-Boc+H] + .

[0154] Preparation of intermediate 5a-2

[0155]

[0156] Dissolve the intermediate 5a-1 (580 mg, 1.49 mmol) in dichloromethane, add 3 mL of hydrogen chloride-1,4-dioxane solution (molar concentration: 4N), react at room temperature for 2 hours to precipitate an off-white solid, filter the reaction solution to obtain the intermediate 5a-2, which can be directly used in the next step without purification. MS (ESI): m / z 290.2 [M+H] + .

[0157] Preparation of intermediate 5a-3

[0158]

[0159] Intermediate 5a-2 (350 mg, 1.2 mmol), 1,4-bis(chloromethyl)-2,5-dimethoxybenzene (CAS: 3752-97-4, 280 mg, 1.2 mmol), potassium carbonate (165 mg, 1.2 mmol) were dissolved in acetonitrile and reacted at 60°C for 3 hours under nitrogen protection. After TLC confirmed that the reaction was complete, the reaction solution was mixed with silica gel for column chromatography to obtain 210 mg of intermediate 5a-3, with a yield of 36%. MS (ESI): m / z 488.2 [M+H] + ,452.2[M–Cl - ] + .

[0160] Intermediate 5a-4

[0161]

[0162] Except that intermediate 5a-3 was used instead of intermediate 1b-3, the rest of the preparation method was the same as 1b-4. MS (ESI): m / z 742.3 [M+H] + .

[0163] Preparation of final product compound 5

[0164]

[0165] Except that intermediate 5a-4 was used instead of intermediate 1b-4, the rest of the preparation method was the same as Example 1. MS (ESI): m / z 728.3 [M+H] + .1H NMR(500MHz,Chloroform-d)δ8.10(s,1H),7.73(d,J=8.5Hz,1H),7.62(s,1H),7.50(s,1H), 7.24–7.05(m,3H),6.98(s,1H),6.70(s,1H),4.42(s,2H),4.09(s,2H),3.96–3.73(m,8H),3. 67(s,2H),3.47–3.30(m,2H),3.20–3.12(m,1H),3.05–2.95(m,1H),2.51–2.38(m,1H),2.25( t,J=7.6Hz,2H),2.22–2.01(m,4H),1.44–1.31(m,4H),0.88–0.81(m,2H),0.70–0.52(m,2H).

[0166] Example 6

[0167] (S)-4-(8-(4-((4-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)thiazolidin-3-yl)methyl)-2,5-diethoxybenzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzoic acid (Compound 6)

[0168]

[0169] The preparation method is the same as that of Example 5 except that 1,4-bis(chloromethyl)-2,5-diethoxybenzene is used instead of 1,4-bis(chloromethyl)-2,5-dimethoxybenzene. 1 H NMR (500MHz, DMSO-d6) δ7.88(d,J=8.3Hz,2H),7.54(d,J=8.4Hz,2H),7.23(d,J=12.8Hz,1H),7.09(d,J=8 .2Hz,1H),6.98(s,2H),6.66(s,1H),6.58(t,J=7.7Hz,1H),4.52(s,1H),4.12(s,1H),4.05–3.82(m,6H), 3.81–3.48(m,8H),3.24(s,2H),3.03(s,2H),2.37(d,J=8.4Hz,2H),1.99(dt,J=17.2,6.8Hz,1H),1.92(s ,4H),1.52–1.41(m,1H),1.30(t,J=7.1Hz,6H),0.85(t,J=6.7Hz,1H),0.79(d,J=6.4Hz,2H),0.48(s,2H).

[0170] Example 7

[0171] 4-((1-(2-cyclopropyl-4-((3-(4-cyclopropyl-1,2,4-tetrahydroquinoxaline-1-carbonyl)pyridin-4-yl)oxy)-5-methoxybenzyl)piperidin-4-yl)amino)benzoic acid (Compound 7)

[0172]

[0173] Preparation of intermediate 7a-1

[0174]

[0175] The preparation method is the same as 1a-2 except that 1-Boc-4-aminopiperidine is used instead of tert-butyl 2-oxo-1-oxa-3,8-diazaspiro[4.5]decane-8-carboxylate. MS (ESI): m / z 235.2 [M+H] + .

[0176] Preparation of final product compound 7

[0177] Except for using intermediate 7a-1 instead of 1a-2, the rest of the preparation method is the same as Example 4. 1 H NMR (500MHz, DMSO-d6) δ8.60(s,1H),8.30(s,1H),7.61(d,J=8.1Hz,2H),7.08(s,1H),7.04(d,J=4.4Hz,2H),6.50(d,J=11.5Hz,1H),6. 47(d,J=8.4Hz,2H),6.36(s,1H),6.17(d,J=5.7Hz,1H),5.66(s,1H),5.48(s,1H),4.60(s,1H),3.67(s,3H),3.60(s,2H),3.27(d,J=6.0 Hz,2H),3.19(d,J=6.7Hz,1H),2.85(d,J=10.9Hz,2H),2.31(s,1H),2.17(t,J=11.5Hz,2H),2.07–1.97(m,1H),1.96–1.88(m,2H),1.42 (q,J=10.7Hz,2H),1.28–1.22(m,2H),0.94–0.81(m,2H),0.74–0.56(m,2H),0.56–0.46(m,1H),0.45–0.32(m,2H),-0.03–-0.16(m,1H).

[0178] Example 8

[0179] (S)-4-((1-(4-((4-(4-cyclopropyl-1,2,4-tetrahydroquinoxaline-1-carbonyl)thiazolidin-3-yl)methyl)-2,5-dimethoxybenzyl)piperidin-4-yl)amino)benzoic acid (Compound 8)

[0180]

[0181] Except for using intermediate 7a-1 instead of intermediate 1a-2, the rest of the preparation method is the same as Example 5. 1H NMR (500MHz, DMSO-d6) δ7.60(d,J=8.3Hz,2H),7.46(s,1H),7.10(d,J=8.1Hz,1H),6.98(s,2H),6.59(t ,J=7.7Hz,1H),6.48(d,J=8.3Hz,2H),5.85(s,1H),4.52(s,1H),3.71(d,J=6.6Hz,6H),3.47(s,2H),3.2 9–3.23(m,2H),3.07–2.97(m,1H),2.82(d,J=11.1Hz,2H),2.40–2.33(m,1H),2.12(t,J=11.2Hz,2H),1 .89(d,J=11.4Hz,2H),1.41(q,J=10.2Hz,2H),1.28–1.20(m,2H),0.83–0.73(m,2H),0.57–0.43(m,3H).

[0182] Example 9

[0183] 4-(8-(2-cyclopropyl-4-((3-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)pyridin-4-yl)oxy)-5-methoxybenzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzenesulfonic acid (Compound 9)

[0184]

[0185] Preparation of intermediate 9a-1

[0186]

[0187] The preparation method is the same as that of intermediate 1a-2 except that 4-bromobenzenesulfonic acid neopentyl ester is used instead of methyl p-bromobenzoate; MS (ESI): m / z 383.3 [M+H] + .

[0188] Preparation of intermediate 9b-1

[0189]

[0190] The preparation method is the same as 1b-3 except that 2-cyclopropyl-4-hydroxy-5-methoxybenzaldehyde is used instead of 2,5-dimethyl-4-hydroxybenzaldehyde.

[0191] Preparation of intermediate 9a-2

[0192]

[0193] The preparation method is the same as that of intermediate 1b-4, except that intermediate 9a-1 is used instead of intermediate 1a-2, and 9b-1 is used instead of 1b-3;

[0194] Final product

[0195]

[0196] The intermediate 9a-2 (1 eq) and sodium iodide (5 eq) were dissolved in acetone, reacted at 120°C in a pressure tube for 18 h, cooled to room temperature, and the reaction solution was filtered to remove the solid. The filtrate was concentrated and then purified by semi-preparative liquid phase to obtain 50 mg of compound 9 with a yield of about 57%. The semi-preparative liquid phase purification system used acetonitrile-water (containing 0.1% trifluoroacetic acid) with a gradient of 0 min: 20% acetonitrile-70% water; 45 min: 75% acetonitrile-25% water; 60 min: 90% acetonitrile-10% water. MS (ESI): m / z 766.3 [M+H] + . 1 HNMR(500MHz,DMSO-d6)δ8.58(s,1H),8.29(s,1H),7.59(d,J=8.7Hz,2H),7.51(d,J=8.6Hz,2H) ,7.08(s,1H),7.03(d,J=4.9Hz,2H),6.49(d,J=7.8Hz,1H),6.34(s,1H),6.16(s,1H),5.48(s,1 H),3.89(s,2H),3.66(s,3H),3.63(s,2H),2.54(s,2H),2.33–2.26(m,1H),2.07–1.97(m,1H),1 .95–1.84(m,4H),0.94–0.79(m,2H),0.74–0.56(m,2H),0.53–0.33(m,3H),-0.07–-0.22(m,1H).

[0197] Example 10

[0198] 4-(8-(2-cyclopropyl-5-methoxy-4-((3-(4-methyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)pyridin-4-yl)oxy)benzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzoic acid (Compound 10)

[0199]

[0200] The preparation method is the same as that of Example 4 except that methyl-benzotetrahydroquinoxaline is used instead of cyclopropyl-benzotetrahydroquinoxaline. 1H NMR (500MHz, DMSO-d6) δ8.54(s,1H),8.28(d,J=5.8Hz,1H),7.65(d,J=8.3Hz,2H),7.07(s,1H),6.99(t,J=7.7Hz,1H),6.70(d ,J=8.4Hz,1H),6.58(d,J=8.4Hz,2H),6.47(d,J=8.0Hz,1H),6.26(t,J=7.7Hz,1H),6.14(d,J=5.8Hz,1H),5.78(s,1H),5.64(s ,1H),4.48(d,J=62.3Hz,2H),3.66(s,3H),3.58(s,3H),3.03(d,J=5.6Hz,2H),2.88(d,J=9.9Hz,1H),2.78(s,2H),2.60–2.53 (m,1H),2.47–2.34(m,3H),2.07–1.96(m,1H),1.64–1.52(m,3H),1.24(d,J=3.5Hz,2H),0.92–0.80(m,2H),0.63–0.34(m,2H).

[0201] Embodiment 11

[0202] (S)-4-(8-(4-((4-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)thiazolidin-3-yl)methyl)-2,5-diethoxybenzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzenesulfonic acid (Compound 11)

[0203]

[0204] Preparation of intermediate 11a-1

[0205]

[0206] The preparation method is the same as that of intermediate 5a-3, except that intermediate 2,5-diethoxybenzene-1,4-dicarboxaldehyde is used instead of intermediate 2,5-dimethoxybenzene-1,4-dicarboxaldehyde.

[0207] Preparation of final product compound 11

[0208]

[0209] The preparation method is the same as Example 9 except that intermediate 11a-1 is used instead of intermediate 9b-1. 1H NMR (500MHz, DMSO-d6) δ7.59(d,J=8.7Hz,2H),7.52(d,J=8.8Hz,2H),7.11(d,J=8.2Hz,1H),7.0 0(s,4H),6.60(t,J=7.9Hz,1H),4.54(s,1H),4.14(s,1H),3.97(s,4H),3.97–3.90(m,1H),3.88( s,2H),3.77(s,1H),3.64–3.58(m,3H),3.52(s,2H),3.33(s,4H),3.25(s,2H),3.06–3.01(m,1H) ,2.40(s,1H),1.90(h,J=7.7,6.9Hz,4H),1.32(t,J=6.9Hz,6H),0.83–0.78(m,2H),0.50(s,2H).

[0210] Example 12

[0211] (S)-4-(8-(2,5-diethoxy-4-((4-(4-methyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)thiazolidin-3-yl)methyl)benzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzoic acid (Compound 12)

[0212]

[0213] The preparation method is the same as that of Example 6 except that methyl-benzotetrahydroquinoxaline is used instead of cyclopropyl-benzotetrahydroquinoxaline. 1 H NMR(500MHz,DMSO-d6)δ7.85(d,J=8.4Hz,2H),7.46(dd,J=8.1,3.6Hz,2H),6.98(s,4H),6.69( d,J=8.4Hz,1H),6.53(t,J=7.6Hz,1H),4.55(s,1H),4.15(s,1H),3.96(s,6H),3.93(d,J=6.6Hz ,1H),3.88(s,2H),3.80(s,2H),3.64(s,1H),3.54(d,J=13.1Hz,1H),3.52(s,2H),3.26(t,J=5. 8Hz,2H),3.04(s,2H),2.86(s,4H),2.43(s,1H),1.89(q,J=7.1,6.2Hz,4H),1.35–1.23(m,6H).

[0214] Example 13

[0215] (S)-4-(8-(2,5-diethoxy-4-((4-(4-methyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)thiazolidin-3-yl)methyl)benzyl)-2-oxo-1-oxo-3,8-diazaspiro[4.5]dec-3-yl)benzenesulfonic acid (Compound 13)

[0216]

[0217] The preparation method is the same as that of Example 11 except that methyl-benzotetrahydroquinoxaline is used instead of cyclopropyl-benzotetrahydroquinoxaline. 1 H NMR(500MHz,DMSO-d6)δ7.59(d,J=8.7Hz,2H),7.52(d,J=8.8Hz,2H),7.00–6.96(m,4H),6.68(dd, J=8.4,1.2Hz,1H),6.53(t,J=7.6Hz,1H),4.55(s,1H),4.15(s,2H),3.92(q,J=7.6,7.0Hz,4H),3. 88(s,2H),3.80(s,2H),3.64(s,2H),3.54(d,J=13.1Hz,1H),3.52(s,2H),3.39(s,1H),3.33(s,3H ),3.25(s,2H),3.05(s,1H),2.85(s,3H),1.90(dp,J=13.8,7.7,7.1Hz,4H),1.31(t,J=6.8Hz,6H).

[0218] Embodiment 14

[0219] (S)-4-(8-(4-((4-(4-cyclopropyl-1,2,3,4-tetrahydroquinoxaline-1-carbonyl)thiazolidin-3-yl)methyl)-2,5-diethoxybenzyl)-2-oxo-1,3,8-triazaspiro[4.5]dec-3-yl)benzoic acid (Compound 14)

[0220]

[0221] The preparation method is the same as that of Example 6, except that tert-butyl 2-oxo-1-oxa-3,8-diazaspiro[4,5]decane-8-carboxylate (CAS: 169206-55-7) is used instead of tert-butyl 2-oxo-1-oxa-3,8-diazaspiro[4.5]decane-8-carboxylate (CAS: 183673-70-3).

[0222] Pharmacological Examples:

[0223] Pharmacological Example 1: In vitro SSTR5 antagonistic activity test of compounds

[0224] Test method: Calcium flow detection method

[0225] 1) Experimental principle: After the receptor binds to the ligand and is activated, it can cause the activation of Gα16 protein, which in turn activates phospholipase C (PLC) to produce IP3 and DAG. IP3 can bind to IP3 receptors on the endoplasmic reticulum and mitochondria in the cell, thereby causing the release of intracellular calcium. Therefore, measuring the changes in intracellular calcium can be used as a method to detect the activation state of hSSTR5. Fluo-4 / AM is a calcium fluorescent probe indicator used to measure calcium ions. As a non-polar, lipid-soluble compound, after entering the cell, under the action of cellular lipolytic enzymes, the AM group dissociates and releases Fluo-4; because Fluo-4 is a polar molecule and is not easy to pass through the lipid bilayer membrane, it can keep Fluo-4 in the cell for a long time. Finally, the level of Gα protein activation can be reflected by measuring the excited fluorescence intensity. Somatostatin, an agonist of SSTR5, can excite SSTR5 receptors and greatly increase the calcium flow response. The SSTR5 antagonist to be tested can inhibit the agonist activity of the SSTR5 receptor, reducing the calcium flow response of the receptor stimulated by the agonist.

[0226] 2) Experimental steps:

[0227] (1) hSSTR5-CHO-Gɑ16 and mSSTR5-CHO-Gɑ16 cell lines (purchased from ATCC (Manassas, VA, USA)) stably expressing hSSTR5 or mSSTR5 receptors were seeded at a density of 30,000 cells / well in a 96-well plate and cultured overnight in a 37°C incubator.

[0228] (2) Aspirate the culture medium, add 40 μl / well of freshly prepared dye, and incubate in a 37°C incubator for 45 min.

[0229] (3) Aspirate and discard the dye, wash once with freshly prepared calcium buffer, and then replace with 50 μl calcium buffer.

[0230] (4) Dilute the agonist Somatostatin-14 with calcium buffer and mix well.

[0231] (5) Detection with Flextation instrument. Starting from the 15th second, the instrument automatically adds 25 μl of pre-prepared agonist and finally reads the fluorescence value at 525 nm. Agonist EC 50 The agonist concentration was 9.7 nM, and 100 nM agonist concentration was selected to detect antagonist activity.

[0232] (6) Dilute the positive control antagonist and the test compound with calcium buffer and mix them evenly; wherein the positive control antagonist is 4-(8-((2,6-diethoxy-4'-fluoro-[1,1'-biphenyl]-4-yl)methyl)-3-oxo-2,8-diazaspiro[4.5]dec-2-yl)benzoic acid, which is from the article ACS Med.Chem.Lett.2018,9,1082-1087.

[0233] (7) Aspirate and discard the dye, wash once with freshly prepared calcium buffer, and then replace with 50 μl of 10-fold concentration gradient diluted antagonist.

[0234] (8) Detection using a Flextation instrument. Starting from the 15th second, the instrument automatically adds 25 μl of a pre-prepared 100 nM agonist and finally reads the fluorescence value at 525 nm.

[0235] The results are shown in Table 1 below.

[0236] Pharmacological Example 2: TGR5 agonist activity test of compounds

[0237] The cells were seeded in a 100 mm cell culture dish and cultured for 24 h. After digestion with trypsin, they were resuspended in serum-free DMEM and IBMX (final concentration 0.5 mmol / L) was added. 3 Cells / well / 5μL cell density were inoculated into 384-well plates, and then 5μL of compounds with different concentrations (0-10μmol / L) were added and mixed evenly, and incubated at room temperature in the dark for 30 minutes. Finally, 5μL of diluted cAMP-d2 and 5μL of anti-cAMP-Cryptate were added, and centrifuged and mixed evenly. Reacted at room temperature in the dark for 1h. Finally, the Envision2101 multi-function microplate reader was used to read the data. The data was plotted using GraphPad software to obtain the EC50 value. The results are shown in Table 1 below.

[0238] Table 1: In vitro activity test results of some compounds

[0239]

[0240]

[0241] Activity range expression method:

[0242] A:0-50nM:

[0243] B:50-200nM

[0244] C:200-10000nM

[0245] D:>10000nM

[0246] -:Untested

[0247] The above data show that this type of compound has good SSTR5 antagonistic activity and TGR5 agonistic activity.

[0248] Pharmacological Example 3: Oral glucose tolerance (OGTT) experiment of C57BL / 6 normal mice with the compound of the present invention. C57BL / 6J mice were fasted for 12 hours before the experiment, and a treatment group and a blank control group were set up, with 8 mice in each group. The mice in the blank control group orally administered 200 μL of pure water, and the mice in the treatment group orally administered 200 μL of an aqueous solution containing compound 5. Glucose (4 g / kg) was orally administered 60 minutes after administration. Blood was collected from the tail before administration, and 0, 15, 30, 60, 90, and 120 minutes after administration, and blood glucose was measured using Accu-Chek Advantage II Glucose Monitor (Roche, Indianapolis, IN, USA). The experimental results are shown in the following table. Figure 1 shown.

[0249] from Figure 1 It can be seen that compound 5 has a significant anti-hyperglycemic effect in mice.

[0250] Pharmacological Example 4: Experimental study on the active GLP-1 level of C57BL / 6 normal mice by combining compound 5 of the present invention with a DPP4 inhibitor

[0251] C57BL / 6J mice were fasted for 12 hours before the experiment and divided into a blank control group, a sitagliptin group, and a compound 5 combined with sitagliptin group; 8 mice in each group. Mice in the blank control group orally administered 200 μL of pure water, and mice in the drug administration group orally administered 200 μL of an aqueous solution containing the compound. Blood was collected 60 minutes after administration and centrifuged to determine the active GLP-1. The experimental results are shown in Figure 2 shown.

[0252] from Figure 2 It can be seen that compound 5 combined with a DPP4 inhibitor can significantly increase the level of active GLP-1 in mice. Therefore, it will have great potential to develop a GLP-1-based drug by combining the compound of the present application with a DPP4 inhibitor.

Claims

1. A compound of the general formula (I) as shown below, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts: in, Ring A is selected from R1 is hydrogen, C1-C6 alkyl or C3-C6 cycloalkyl; X is O or CH2; R2, R3, R4, R5 are each independently hydrogen, halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy or C3-C6 cycloalkyl; Ring B is derived from the core ring structure in the SSTR5 antagonist structural framework; X1 is NH or absent; R6 and R7 are each independently hydrogen, halogen, hydroxy, amino, C1-C6 alkyl, C1-C3 alkoxy, or amino mono- or di-substituted by C1-C6 alkyl; R8 is a carboxyl group, a phosphoric acid group, a sulfonic acid group, a sulfonic acid ester group esterified with a C1-C6 alkyl group, a tetrazolyl group, a hydroxyl group or an amino group; Y1 and Y2 are each independently CH or N; Indicates the key connection location.

2. The compound of the general formula (I) according to claim 1, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts, wherein: In the general formula (I), ring B is selected from Ring B separates the general formula (I) into a portion containing ring A and a portion without ring A. For the two aforementioned substituent groups, the left side of the substituent group is connected to the portion containing ring A of the general formula (I), and the right side is connected to the portion without ring A of the general formula (I), wherein Z is selected from CH2, O and NH, and m and n are each independently an integer of 1-3, provided that when ring B is When X1 does not exist, when ring B is X1 is NH.

3. The compound of the general formula (I) according to claim 2, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts, wherein: In the general formula (I), X is O or CH2; R1 is a C1-C3 alkyl group or a C3-C6 cycloalkyl group, preferably a methyl group or a cyclopropyl group; R2, R3, R4, and R5 are each independently hydrogen, methyl, methoxy, ethoxy, or cyclopropyl; R6 and R7 are both hydrogen; R8 is a carboxyl group or a sulfonic acid group; Y1 and Y2 are both CH, Ring A is And ring B is or Ring A is And ring B is or Ring A is And ring B is or Ring A is And ring B is 4. The compound of the general formula (I) according to claim 3, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts, wherein: Ring B is wherein Z is selected from O or NH, and m and n are both 2.

5. The compound of the general formula (I) according to claim 1, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts, wherein: The compound of general formula (I) is selected from one of the following compounds:

6. The method for preparing the compound of general formula (I) as claimed in claim 1, comprising the following 4 situations: A: For A Ring B is The compound of general formula (I) wherein X is O and X1 does not exist is synthesized by the following route: In the above reaction formula, R1, R2, R3, R4, R5, R6, R7, R8, Y1, Y2, m, n, and Z are defined as any one of claims 1 to 5; R9 is a C1-C3 alkoxycarbonyl or neopentyl substituted sulfonate group; W is a halogen; (1) a and b undergo nucleophilic substitution reaction to obtain c; (2) c undergoes hydrogenation reduction decarboxylation to give d; (3)d is prepared by reacting acyl chloride with N 1 Substituted benzotetrahydroquinoxaline is condensed to give e; (4) f and g are reacted to prepare h through nucleophilic substitution reaction, Buchwald–Hartwig coupling reaction or Ullman condensation; (5) h is deprotected under acidic conditions to obtain i; (6) i and e are reacted by nucleophilic substitution to obtain j; (7) j is deprotected under alkaline conditions or under sodium iodide-acetone conditions to obtain a compound of the general formula k, B: For A Ring B is The compound of formula (I) wherein X is O and X1 is NH is synthesized by the following route: wherein R1, R2, R3, R4, R5, R6, R7, R8, Y1 and Y2 are as defined in any one of claims 1 to 5; R9 is a sulfonate group substituted with a C1-C3 alkoxycarbonyl group or a neopentyl group; W is a halogen; (1) 2a and 2b were prepared to give 2c via nucleophilic substitution reaction, Buchwald–Hartwig coupling reaction or Ullman condensation; (2) 2c was deprotected under acidic conditions to give 2d; (3) 2d and e were reacted via nucleophilic substitution to give 2e; (4) 2e is deprotected under alkaline conditions or sodium iodide-acetone conditions to obtain a compound of the general formula 2f, The intermediate e in the above step (3) is shown in Scheme 1. C: For A Ring B is The compound of general formula (I) wherein X is CH2 and X1 does not exist is synthesized by the following three routes: The definitions of R1, R2, R3, R4, R5, R6, R7, R8, Y1 and Y2, m, n, and Z are the same as those in any one of claims 1 to 5; R9 is a sulfonate group substituted with a C1-C3 alkoxycarbonyl group or a neopentyl group; (1) 3a reacts with N by amide condensation reaction. 1 Substituted benzotetrahydroquinoxaline was prepared to give 3b; (2) 3b is deprotected under acidic conditions to give 3c; (3) 3c and N are R 1 Substituted benzotetrahydroquinoxaline was prepared by reductive amination to give 3d; (4) 3d and i were reacted to give 3e by reductive amination; (5) 3e is deprotected under alkaline conditions or sodium iodide-acetone conditions to obtain compound 3k, The intermediate i in the above step (4) is shown in Scheme 1, D: For A Ring B is X is CH2, and X1 is NH, the compound of formula (I) is synthesized by the following route 4: in, R1, R2, R3, R4, R5, R6, R7, R8, Y1 and Y2 are as defined in any one of claims 1 to 5; R9 is a sulfonate group substituted with a C1-C3 alkoxycarbonyl group or a neopentyl group; (1) 4a was prepared by reductive amination of 3d and 2c; (2) 4a is deprotected under alkaline conditions or sodium iodide-acetone conditions to obtain compound 4b.

7. A pharmaceutical composition comprising a compound of the general formula (I) as claimed in any one of claims 1 to 5, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts, and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, further comprising one or more other drugs for treating diseases mediated by TGR5 or SSTR5, preferably, the other drugs are selected from one or more of hypoglycemic drugs, weight loss drugs, non-alcoholic fatty liver disease drugs (NAFLD), non-alcoholic steatohepatitis drugs (NASH), inflammatory bowel disease drugs, Alzheimer's disease drugs, Parkinson's disease drugs, cholestasis drugs, gallstone drugs, cholangitis drugs, cholecystitis drugs; and / or The pharmaceutical composition comprises a DPP4 inhibitor, wherein The DPP4 inhibitor is selected from sitagliptin, alogliptin, linagliptin, vildagliptin and saxagliptin. Preferably, the DPP4 inhibitor is sitagliptin.

9. Use of a compound of formula (I) as claimed in any one of claims 1 to 5, its enantiomers, diastereomers, racemic mixtures, deuterated compounds and pharmaceutically acceptable salts, or a pharmaceutical composition as claimed in claim 7 or 8 in the preparation of a medicament for preventing or treating a disease mediated by TGR5 and / or SSTR5.

10. The use according to claim 9, wherein The diseases mediated by TGR5 and / or SSTR5 include type 2 diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic fatty hepatitis, inflammatory bowel disease, Alzheimer's disease, Parkinson's disease and gallbladder system diseases. Wherein, the gallbladder system diseases include cholestasis, cholelithiasis, cholangitis or cholecystitis.

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