A deuterated indazole compound, pharmaceutical composition and application thereof

Abstract

The present invention discloses a compound represented by Formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate, or hydrate thereof, a pharmaceutical composition, and uses thereof. The compound represented by Formula I provided by the present invention has a good therapeutic effect on non-insulin-dependent diabetes mellitus (type 2 diabetes) and obesity, among other conditions. #imgabs0#

Description

[0001] This application is a divisional application of the application with application date of November 20, 2022, application number 202211451463.0, and invention name “A deuterated indazole compound, pharmaceutical composition and its application”. Technical Field

[0002] The present invention belongs to the field of innovative medicinal chemistry and relates to a deuterated indazole compound, a pharmaceutical composition and an application thereof. Background Art

[0003] Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by elevated blood glucose concentrations and carries significant morbidity and mortality. The global prevalence of T2DM is increasing at an alarming rate. In 2019, over 463 million people were living with diabetes, primarily T2DM. Obesity is considered a significant risk factor for T2DM, with approximately 85% of patients with T2DM being overweight or obese. Furthermore, moderate weight loss can prevent the development of T2DM in overweight / obese individuals and reduce the need for antidiabetic medications in established T2DM patients. Glucagon-like peptide-1 (GLP-1) is an incretin secreted by intestinal L cells in response to nutrient transport through the digestive tract. GLP-1 exerts multiple physiological effects by activating the GLP-1 receptor, including glucose-dependent insulin secretion and biosynthesis, inhibition of glucagon release, pancreatic β-cell survival, improved insulin sensitivity, and delayed gastric emptying. Although GLP-1 analogs have been used clinically for the treatment of diabetes, they must be administered subcutaneously, leading to poor patient compliance. The development of non-peptide GLP-1 receptor small molecule agonists to improve patient compliance is of great significance and has become a research hotspot in the field of diabetes. Currently, GLP-1 receptor agonists are already used in the clinical treatment of T2DM. In addition, GLP-1 receptor agonists can also induce satiety and reduce weight. However, in most obese patients, currently approved GLP-1 receptor agonists are limited by dose-dependent gastrointestinal side effects and can only achieve moderate weight loss effects. LY3502970 is a GLP-1 receptor agonist currently in Phase II clinical research.

[0004] Deuterated drugs are drugs in which some of the hydrogen atoms in the drug molecule are replaced with deuterium. Because deuterium's shape and volume in the drug molecule are similar to hydrogen, deuterated drugs generally retain the original drug's biological activity and selectivity. Because CD bonds are more stable than C-H bonds, deuterated drugs are less likely to break during chemical reactions, resulting in a longer half-life. Deuterated drugs have been widely used in drug research since 2000.

[0005] Summary of the Invention

[0006] The present invention provides a compound as shown in Formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, the structure of which is as follows:

[0007]

[0008] Wherein, R1 is hydrogen or deuterium,

[0009] R2, R3, R4, R5, R6 or R7 are independently selected from CD3 or CH3,

[0010] When R1 is hydrogen, at least one of R2, R3, R4, R5, R6 or R7 is CD3.

[0011] In some embodiments, R1 is deuterium.

[0012] In some embodiments, the R2 is CD3.

[0013] In some embodiments, the compound is represented by any of the following structural formulas:

[0014]

[0015] The present invention provides a use of a compound as shown in Formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in the preparation of a GLP-1 receptor agonist.

[0016] The present invention provides a use of a compound shown in I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in the preparation of a medicament for non-dependent diabetes mellitus, hyperglycemia, impaired glucose tolerance and insulin-dependent diabetes mellitus, obesity, diabetic complications, hypertension, hyperlipidemia, atherosclerosis, coronary heart disease, cerebral infarction, non-alcoholic steatohepatitis, Parkinson's disease or dementia.

[0017] In some embodiments, the non-dependent diabetes mellitus, hyperglycemia, impaired glucose tolerance and insulin-dependent diabetes mellitus, obesity, diabetic complications, hypertension, hyperlipidemia, atherosclerosis, coronary heart disease, cerebral infarction, non-alcoholic steatohepatitis, Parkinson's disease or dementia is non-dependent diabetes mellitus, hyperglycemia, impaired glucose tolerance and insulin-dependent diabetes mellitus, obesity, diabetic complications, hypertension, hyperlipidemia, atherosclerosis, coronary heart disease, cerebral infarction, non-alcoholic steatohepatitis, Parkinson's disease or dementia associated with abnormal GLP-1 receptor activity.

[0018] The present invention provides a pharmaceutical composition comprising a compound as shown in Formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, and a pharmaceutically acceptable carrier or excipient.

[0019] In the pharmaceutical composition, the compound of formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof is used in a therapeutically effective amount.

[0020] The present invention provides an application of a pharmaceutical composition in the preparation of a GLP-1 receptor agonist.

[0021] The present invention provides a use of a pharmaceutical composition in the preparation of a medicament for treating non-adherent diabetes, hyperglycemia, impaired glucose tolerance and insulin-dependent diabetes, obesity, diabetic complications, hypertension, hyperlipidemia, atherosclerosis, coronary heart disease, cerebral infarction, non-alcoholic steatohepatitis, Parkinson's disease or dementia.

[0022] In some embodiments, the non-dependent diabetes mellitus, hyperglycemia, impaired glucose tolerance and insulin-dependent diabetes mellitus, obesity, diabetic complications, hypertension, hyperlipidemia, atherosclerosis, coronary heart disease, cerebral infarction, non-alcoholic steatohepatitis, Parkinson's disease or dementia is non-dependent diabetes mellitus, hyperglycemia, impaired glucose tolerance and insulin-dependent diabetes mellitus, obesity, diabetic complications, hypertension, hyperlipidemia, atherosclerosis, coronary heart disease, cerebral infarction, non-alcoholic steatohepatitis, Parkinson's disease or dementia associated with abnormal GLP-1 receptor activity.

[0023] The pharmaceutical excipients may be those widely used in the field of pharmaceutical production. Excipients are primarily used to provide a safe, stable, and functional pharmaceutical composition. They may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration of the composition to a subject. The pharmaceutical excipients may be inert fillers, or may provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient in the composition. The pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0024] The pharmaceutical compositions of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0025] The pharmaceutical compositions of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ophthalmic, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention can also be controlled-release or delayed-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0026] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in neat solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in neat solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid (forming sulfate or bisulfate), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid. The organic acid salts also include salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups and can be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner and the parent compound is separated to regenerate the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

[0027] The "pharmaceutically acceptable salts" of the present invention can be synthesized from parent compounds containing acid or basic groups by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

[0028] The term "isomers" refers to compounds that have the same chemical formula but different arrangements of the atoms.

[0029] The term "metabolite" refers to a pharmaceutically active product produced by the in vivo metabolism of a compound of Formula I or a salt thereof. Such a product may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronidation, enzymatic cleavage, or the like of the administered compound. Thus, the present invention includes metabolites of the compounds of the present invention, including compounds produced by methods in which a compound of the present invention is contacted with a mammal for a period of time sufficient to yield a metabolite thereof.

[0030] Metabolites are typically identified by preparing a radiolabeled isotope of a compound of the invention, administering it parenterally to an animal, such as a rat, mouse, guinea pig, monkey, or human, at a detectable dose (e.g., greater than about 0.5 mg / kg), allowing sufficient time for metabolism to occur (typically about 30 seconds to 30 hours), and isolating the conversion products from urine, blood, or other biological samples. These products are easily isolated because they are labeled (others are isolated by utilizing antibodies that can bind to antigenic epitopes present in the metabolites). Metabolite structures are determined in a conventional manner, for example, by MS, LC / MS, or NMR analysis. Typically, analysis of metabolites is performed using the same methods as conventional drug metabolism studies known to those skilled in the art. As long as the metabolite products are not otherwise undetectable in vivo, they can be used in assays for therapeutic dosing of the compounds of the invention. The compounds of the invention may contain unnatural ratios of atomic isotopes on one or more of the atoms that constitute the compound. For example, compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0031] In addition to the form of salts, the compounds provided by the present invention also exist in prodrug form. The prodrugs of the compounds described herein are easily chemically changed under physiological conditions to be converted into the compounds of the present invention. Any compound that can be converted in vivo to provide a bioactive substance (i.e., a compound shown in Formula I) is a prodrug within the scope and spirit of the present invention. For example, a compound containing a carboxyl group can form a physiologically hydrolyzable ester, which acts as a prodrug by being hydrolyzed in vivo to obtain the compound shown in Formula I itself. The prodrug is preferably administered orally, because hydrolysis occurs primarily under the influence of digestive enzymes in many cases. When the ester itself is active or hydrolysis occurs in the blood, parenteral administration can be used.

[0032] The positive progress effect of the present invention is:

[0033] (1) The compounds of the present invention have significant agonist activity on GLP-1 receptors.

[0034] (2) The pharmacokinetic properties of the compounds of the present invention are significantly improved, the half-life is prolonged, and they have good oral bioavailability.

[0035] (3) The compounds of the present invention have good therapeutic effects on type 2 diabetes and obesity. DETAILED DESCRIPTION

[0036] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0037] Example 1: Synthesis of Compound I-1

[0038]

[0039] Step 1: Synthesis of compound b

[0040] At 0°C, NaH (12 mmol, 480 mg, 60%) was added to a solution of compound a (2.13 g, 10 mmol) in DMF (15 mL), stirred at room temperature for 10 min, and then deuterated iodomethane (1.7 g, 12 mmol) was added dropwise to the reaction solution. The reaction was stirred at room temperature for 2 h. After the reaction was completed, saturated ammonium chloride was added to quench the reaction. The solvent was removed under reduced pressure, extracted with ethyl acetate (10 mL × 3), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound b (1.5 g, 63%). MS (ESI, m / z): 231 (M + +1).

[0041] Step 2: Synthesis of compound d

[0042] Compound c (542 mg, 1 mmol) was dissolved in deuterated DMSO (2 mL) under nitrogen protection. Potassium tert-butoxide (40 mol%, 22 mg) was added to the above solution and the temperature was raised to 35°C and stirred for 6 h. After the reaction was completed, water was added and the mixture was extracted with ethyl acetate (5 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound d (247 mg, 45%). MS (ESI, m / z): 548 (M + +1).

[0043] Step 3: Synthesis of compound 1

[0044] Compound d (548 mg, 1 mmol) was dissolved in ethyl acetate (5 mL). EA / HCl (4 M, 4 mmol, 1 mL) was added to the above solution and stirred at room temperature for 4 h. After the reaction was completed, the filter cake was collected by suction filtration and vacuum dried to obtain compound 1 (295 mg, 85%). MS (ESI, m / z): 348 (M + +1).

[0045] Step 4: Synthesis of compound 3

[0046] HATU (575 mg, 1.5 mmol) and DIPEA (0.53 mL, 3 mmol) were added to a DMF (3 mL) solution of compound 1 (347 mg, 1 mmol) and compound 2 (411 mg, 1 mmol), and the mixture was stirred at room temperature for 6 h. After the reaction was completed, water was added to the solution to quench the reaction, and the mixture was extracted with ethyl acetate (5 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain compound 3 (481 mg, 65%). MS (ESI, m / z): 741 (M + +1).

[0047] Step 5: Synthesis of Compound I-1

[0048] To a solution of compound b (13 mg, 0.056 mmol), compound 3 (21 mg, 0.028 mmol), (1S,2S)-1-N,2-N-dimethylcyclohexane-1,2-diamine (1.6 mg, 0.011 mmol), and potassium carbonate (12 mg, 0.085 mmol) in NMP (0.2 mL) was added cuprous iodide (1.1 mg, 0.0056 mmol). Under nitrogen, the reaction mixture was heated to 130°C and stirred for 3 h. After cooling to room temperature, the reaction mixture was purified by HPLC to yield compound I-5 (19 mg, 77%). 1H NMR(500MHz,DMSO-d6)δ9.09(s,1H),7.78(s,1H),7.76(dd,J=8.4,4.9Hz,1H),7.55–7.46(m,2H),7.29(d,J=5.0Hz,2H),7.21–7.12(m,2 H),6.80(d,J=7.0Hz,1H),6.62(d,J=7.1Hz,1H),5.35(q,J=7.0Hz,1H),3.68(ddd,J=12.1,5.9,4.7Hz,1H),3.65–3.54(m,1H),3.56–3.47 (m,2H),3.15(dddd,J=7.5,6.4,5.1,4.2Hz,1H),2.93–2.82(m,2H),2.60(dd,J=6.9,2.8Hz,1H),2.44(dp,J=6.8,5.2Hz,2H),2.36(dd,J= 5.3,2.8Hz,2H),2.07–1.96(m,2H),1.93–1.83(m,2H),1.55(s,3H),1.25(s,3H),1.20(s,2H),1.01(d,J=5.2Hz,3H).MS(ESI,m / z):892(M + +1).

[0049] Example 2: Synthesis of Compound I-2

[0050]

[0051] Step 1: Synthesis of compound e

[0052] Potassium hydroxide (105.5 mg, 1.88 mmol) and elemental iodine (239 mg, 0.94 mmol) were added to a solution of compound b (107 mg, 0.47 mmol) in N,N-dimethylformamide (15 mL) and reacted at room temperature for 3 hours. The reaction was complete as monitored by TLC. A saturated sodium sulfite solution was added to quench the reaction. The aqueous phase was extracted with ethyl acetate (10 mL × 2), washed with water (20 mL × 2), washed with saturated sodium chloride (20 mL), dried over anhydrous sodium sulfate, and concentrated by column chromatography to obtain compound e (67 mg, 40%). MS (ESI, m / z): 357 (M + +1).

[0053] Step 2: Synthesis of compound f

[0054] Sodium acetate (97.9 mg, 0.72 mmol) was added to a solution of compound e (127 mg, 0.36 mmol) in deuterated acetic acid (8 mL) over 2 hours. The mixture was allowed to react at room temperature for 24 hours. The reaction was complete after TLC analysis. The mixture was concentrated under reduced pressure and purified by column chromatography to obtain compound 3 (70 mg, 85%). MS (ESI, m / z): 232 (M + +1).

[0055] Step 3: Synthesis of compound I-2

[0056] The synthesis of compound 4 was carried out according to the synthesis method of compound 3 in Example 1.

[0057] To a solution of compound f (13 mg, 0.056 mmol), compound 4 (21 mg, 0.028 mmol), (1S,2S)-1-N,2-N-dimethylcyclohexane-1,2-diamine (1.6 mg, 0.011 mmol), and potassium carbonate (12 mg, 0.085 mmol) in NMP (0.2 mL) was added cuprous iodide (1.1 mg, 0.0056 mmol). Under nitrogen, the reaction mixture was heated to 130°C and stirred for 3 h. After cooling to room temperature, the reaction mixture was purified by HPLC to yield compound I-2 (17 mg, 67%). 1 H NMR (500MHz, DMSO-d6) δ7.78(s,1H),7.76(dd,J=8.4,4.9Hz,1H),7.55–7.46(m,2H),7.29(d,J=5.0Hz,2H),7.21–7.12(m,2H),6.80(d,J=7. 0Hz,1H),6.62(d,J=7.1Hz,1H),5.35(q,J=7.0Hz,1H),3.68(ddd,J=12.1,5.9,4.7Hz,1H),3.65–3.54(m,1H),3.56–3.47(m,2H),3.15(dddd, J=7.5,6.4,5.1,4.2Hz,1H),2.93–2.82(m,2H),2.60(dd,J=6.9,2.8Hz,1H),2.44(dp,J=6.8,5.2Hz,2H),2.36(dd,J=5.3,2.8Hz,2H),2.07– 1.96(m,2H),2.01(s,3H),1.99(s,3H),1.93–1.83(m,2H),1.55(s,3H),1.25(s,3H),1.20(s,2H),1.01(d,J=5.2Hz,3H).MS(ESI,m / z):887(M + +1).

[0058] Example 3: Synthesis of Compound I-3

[0059]

[0060] Synthesis of compound i: The synthesis method is the same as Example 2, except that the raw material compound b is replaced by compound g.

[0061]

[0062] Synthesis of compound I-3: The synthesis method is the same as in Example 1. Replace with That's it. 1 H NMR(500MHz, DMSO-d6)δ7.78(s,1H),7.76(dd,J=8.4,4.9Hz,1H),7.55–7.46(m,2H),7.29(d,J=5.0Hz,2H),7.21–7.12(m,2H),6.80(d,J= 7.0Hz,1H),6.62(d,J=7.1Hz,1H),5.35(q,J=7.0Hz,1H),3.68(ddd,J=12.1,5.9,4.7Hz,1H),3.65–3.54(m,1H),3.56–3.47(m,2H),3.15( dddd,J=7.5,6.4,5.1,4.2Hz,1H),2.93–2.82(m,2H),2.77(s,3H),2.60(dd,J=6.9,2.8Hz,1H),2.44(dp,J=6.8,5.2Hz,2H),2.36(dd,J=5 .3,2.8Hz,2H),2.07–1.96(m,2H),1.93–1.83(m,2H),1.55(s,3H),1.25(s,3H),1.20(s,2H),1.01(d,J=5.2Hz,3H)..MS(ESI,m / z):890(M + +1).

[0063] Example 4: Synthesis of Compound I-4

[0064]

[0065] The synthesis method is as in Example 2, and only Replace with That's it. 1H NMR(500MHz,DMSO-d6)δ9.09(s,1H),7.78(s,1H),7.76(dd,J=8.4,4.9Hz,1H),7.55–7.46(m,2H),7.29(d,J=5.0Hz,2H),7.21–7.12(m,2H),6.8 0(d,J=7.0Hz,1H),6.62(d,J=7.1Hz,1H),5.35(q,J=7.0Hz,1H),3.68(ddd,J=12.1,5.9,4.7Hz,1H),3.65–3.54(m,1H),3.56–3.47(m,2H),3.15 (dddd,J=7.5,6.4,5.1,4.2Hz,1H),2.93–2.82(m,2H),2.60(dd,J=6.9,2.8Hz,1H),2.44(dp,J=6.8,5.2Hz,2H),2.36(dd,J=5.3,2.8Hz,2H),2. 07–1.96(m,2H),2.01(s,3H),1.99(s,3H),1.93–1.83(m,2H),1.55(s,3 H),1.25(s,3H),1.20(s,2H),1.01(d,J=5.2Hz,3H).MS(ESI,m / z):886(M + +1).

[0066] Example 5: Synthesis of Compound I-5

[0067]

[0068] The synthesis method is the same as in Example 1, and only the raw materials Replace with That's it. 1HNMR(500MHz,DMSO-d6)δ7.78(s,1H),7.76(dd,J=8.4,4.9Hz,1H),7.55–7.46(m,2H),7.29(d,J=5.0Hz,2H),7.21–7.12(m,2H),6.80( d,J=7.0Hz,1H),6.62(d,J=7.1Hz,1H),5.35(q,J=7.0Hz,1H),3.68(ddd,J=12.1,5.9,4.7Hz,1H),3.65–3.54(m,1H),3.56–3.47(m,2H) ,3.15(dddd,J=7.5,6.4,5.1,4.2Hz,1H),2.93–2.82(m,2H),2.60(dd,J=6.9,2.8Hz,1H),2.44(dp,J=6.8,5.2Hz,2H),2.36(dd,J=5.3 ,2.8Hz,2H),2.07–1.96(m,2H),1.93–1.83(m,2H),1.55(s,3H),1.25(s,3H),1.20(s,2H),1.01(d,J=5.2Hz,3H)..MS(ESI,m / z):893(M + +1).

[0069] Example 7: Detection of the degree of cAMP signal activation by compounds in cells

[0070] This experiment was performed in hGLP1R-HEK293, a cell line stably expressing the human GLP1R. hGLP1R-HEK293 cells were cultured in DMEM (DMEM) supplemented with 10% fetal bovine serum, 100 units / mL penicillin G, 100 μg / mL streptomycin sulfate, and 500 μg / mL geneticin in a humidified atmosphere containing 5% CO₂ at 37°C. hGLP1R-HEK293 cells were seeded at 2.0 × 10⁴ cells / well in a 96-well plate and cultured overnight. The next day, the culture medium was switched to 50 μL of Medium A (DMEM, 20 mM HEPES, 0.05% BSA, 0.5 mM 3-isobutyl-1-methylxanthine) and incubated at 37°C for 30 minutes. Then, 50 μL of culture medium B (DMEM, 20 mM HEPES, 0.05% BSA, 0.5 mM 3-isobutyl-1-methylxanthine) containing GLP-1 or compound was added and incubated at 37°C for another 30 minutes. Then, 100 μL of analytical lysis buffer was added and incubated at 37°C for 30 minutes. The cAMP concentration was quantified using the cAMP HiRange kit. By setting the cAMP concentration, the cAMP concentration of each well was converted into a reaction rate (%) when human GLP-1 (7-37) was placed under action at concentrations ranging from 1 nM to 100%. By applying a 4-parameter logistic regression analysis using XL fit, a dose-response curve of the test compound was created, and the half-maximal (50%) effective concentration (EC 50 ).

[0071] Table 1 The activation degree of cAMP signal of hGLP1R-HEK293 cells by the tested compounds (EC 50 nM)

[0072] name cAMP signaling activation I-1 1.85 I-2 1.80 I-3 1.90 I-4 1.83 I-5 1.84 LY3502970 4.55

[0073] As shown in Table 1, compounds I-1 to I-6 significantly increased the accumulation of cAMP in hGLP1R-HEK293 cells, and the effect was better than that of the positive control LY3502970.

[0074] Example 8: Pharmacokinetic properties of test compounds

[0075] Male SD rats were selected and administered orally (10 mg / kg) or intravenously (2 mg / kg). 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h, and 24 h after administration, blood was continuously collected from the retinal venous plexus and placed in EP tubes containing heparin. The blood was centrifuged and the upper plasma was collected for LC-MS / MS analysis. Based on the blood drug concentration-time data obtained from the test, WinNonlin software was used to calculate the pharmacokinetic parameters and the oral bioavailability.

[0076] The results of the study showed that the oral bioavailability of LY3502970 in rats was 15% and the half-life was 1.2h; the oral bioavailability of compound I-1 was increased to 42% and the half-life was extended to 3.7h, which indicates that the single dose of compound I-1 can be reduced.

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, which is any of the following: 。

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