Deuterated mimic peptide GIP / GLP-1 double-receptor agonist compound and application thereof

Abstract

The invention discloses a deuterated mimic peptide GIP / GLP-1 double-receptor agonist which is shown as a formula I. The invention relates to the deuterated mimic peptide GIP / GLP-1 double-receptor agonist, a pharmaceutical composition and application thereof. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals, and in particular relates to a deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and a pharmaceutically acceptable salt thereof and uses thereof. Background Art

[0002] Diabetes Mellitus (DM) is an endocrine metabolic disease characterized by high blood sugar, mainly caused by insulin secretion defects and / or insufficient insulin action. Diabetes can be divided into type 1 and type 2. Type 1 does not produce insulin and is an insulin-dependent disease, while type 2 is mainly due to the body's insensitivity to insulin and produces insulin resistance, accounting for more than 90% of diabetic patients. Diabetes can lead to a variety of fatal complications, such as obesity, cardiovascular disease, kidney disease, etc., which seriously endanger human health.

[0003] GLP-1R belongs to the pancreatic glucose receptor subfamily of the 7-transmembrane G protein-coupled receptor B family, which is expressed in the pancreas or extrapancreatic tissues, including the central nervous system, cardiovascular, gastrointestinal tract, etc. Human pancreatic tissue highly expresses GLP-1R. GLP-1 can bind to GLP-1R to promote the synthesis and secretion of insulin, stimulate the proliferation of pancreatic β cells, and inhibit their apoptosis. GLP-1 receptor agonists can stimulate human insulin secretion, and their degradation rate is slow. They also have the clinical effects of reducing weight, improving blood lipids, and lowering blood pressure. Tirzepatide is a dual receptor agonist of GIP / GLP-1. It activates the two incretin receptors GIP / GLP-1 involved in blood sugar control through a dual mechanism of action, which can achieve the effect of lowering blood sugar or reducing weight. Tirzepatide was first approved for marketing by the US FDA in May 2022, and was approved in the European Union, Japan and other countries and regions for the treatment of type 2 diabetes.

[0004] Deuterated drugs, that is, new drug molecules in which one or more carbon-hydrogen bonds of a drug molecule are replaced with carbon-deuterium bonds, can improve the pharmacokinetic properties of the original drugs, thereby overcoming the original defects of the drugs such as easy metabolism and large side effects.

[0005] CN116496361A discloses a deuterated mimetic peptide GIP and GLP-1 dual receptor agonist, which is a new class of drugs developed by the present invention. However, in subsequent efficacy studies, it was found that the compounds 1-3 still have problems such as poor affinity and activity with GLP-1 and GIP.

[0006] The present invention is a further improved deuterated peptide-mimicking GIP / GLP-1 dual receptor agonist drug, which improves the pharmacokinetic properties of the peptide-mimicking GIP / GLP-1 dual receptor agonist Tirzepatide through modification, thereby reducing the dosage and possible toxic side effects. Summary of the invention

[0007] The deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and a pharmaceutically acceptable salt thereof provided by the present invention can further improve the pharmacokinetic properties of the deuterated mimetic peptide GIP / GLP-1 dual receptor agonist Tirzepatide, and reduce the dosage and possible toxic side effects.

[0008] In order to achieve the above-mentioned purpose, the present invention provides a deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and a pharmaceutically acceptable salt thereof as shown in the following formula I:

[0009]

[0010] Wherein, R1 and R2 are independently selected from H or deuterium, and R1 and R2 are not H at the same time.

[0011] Further, the deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt described in the present invention have the structure:

[0012]

[0013]

[0014] The deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt described in the present invention are selected from the salts of hydrochloric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, benzoic acid, salicylic acid, nicotinic acid, 3-phenylpropionic acid, aminosulfonic acid, citric acid, tartaric acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, ascorbic acid, and aspartic acid.

[0015] The deuterated peptide-mimicking GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt described in the present invention include its use in treating type 2 diabetes, obesity or overweight and complications related to weight.

[0016] The deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt described in the present invention comprise the deuterated mimetic peptide GIP / GLP-1 dual receptor agonist and its pharmaceutically acceptable salt as active ingredients and a pharmaceutically acceptable carrier.

[0017] The pharmaceutical composition of the deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt described in the present invention is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories or patches.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0019] The present invention provides a class of deuterated mimetic peptide GIP / GLP-1 dual receptor agonist drugs, which further improve the pharmacokinetic properties of the GIP / GLP-1 dual receptor agonist and reduce the dosage and possible toxic side effects. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with the examples, but the present invention is not limited to the scope of the examples described. The present invention also includes new intermediates and methods that can be used to synthesize the compounds of the present invention or their pharmaceutically acceptable salts. The intermediates and compounds of the present invention can be prepared by a variety of methods known in the art. Further, the chemical synthesis methods used are illustrated in the following examples; the specific synthetic steps of each approach described can be combined in different ways to prepare the compounds of the present invention or their salts. Reagents and raw materials are readily available to those of ordinary skill in the art. These examples are only further illustrations of the present invention and are not intended to limit the scope of the present invention in any way.

[0021] Specific implementation methods

[0022] General method for solid phase synthesis

[0023] The synthesis of solid-phase amino acids was carried out on a solid-phase automatic peptide synthesizer. Rinamide-AM resin (Merrifield polymer loaded with (2,4-dimethoxyphenyl) (4-alkoxyphenyl) methane, where 4-alkoxy is the position and chemical bond type connected to the resin, and the loading amount is 0.7 mmol / g) was used. DMF and DCM were used to dissolve the reagents used in the reaction. The solvents and reagents used in the reaction were: DMF = N,N-dimethylformamide; DCM = dichloromethane; HATU = 1-[di(dimethylamino)methylene]-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridine hexafluorophosphate; DIPEA = diisopropylethylamine. The desired amino acid (0.6 mmol), HATU (0.6 mmol), DIPEA (1.2 mmol), and DMF (6 mL) were used for the reaction at 25°C.

[0024] After the desired linear peptide synthesis is completed, the resin is washed once with anhydrous DMF and then pumped to dryness. A cutting solution (trifluoroacetic acid: thioanisole: 1,2-ethanedithiol: anisole = 90:5:2.5:2.5, volume ratio) is added to the dried resin. The mixture is shaken at 10°C for 3 hours. After the reaction is completed, the cutting solution is filtered and concentrated to 1 / 5 of the original volume, and the concentrate is added dropwise to 10 times the volume of -20°C anhydrous ether. The obtained crude peptide is dissolved in a certain amount of pure water, the pH is adjusted to 7 using triethylamine or 2M HCl, acetonitrile is added to clarify, and the crude solid is lyophilized. A certain amount of acetonitrile is added to completely dissolve it and filtered through a 0.33μM filter. Aglient Eclipase XDB-C18 column is used for separation, mobile phase A: 0.1% TFA / H2O; mobile phase B: 0.1% TFA / MeCN. The chromatographic conditions were 10% B-100% B, 20 min.

[0025] Unnatural amino acid synthesis:

[0026] Synthesis of Intermediate 1

[0027]

[0028] Ammonium chloride (2.57 g, 48 mmol) and sodium chloride (2.35 g, 48 mmol) were dissolved in CH2Cl2 / H2O (1:2, 15 mL), and then deuterated acetone (2.94 mL, 40 mmol) was added, and the mixture was stirred under nitrogen protection. After 48 h, the mixture was stirred, and CH2Cl2 was added for extraction. The organic phase was collected and concentrated, and 12 M hydrochloric acid solution (20 mL) was added. The mixture was stirred at 100°C for two hours, cooled to room temperature, and the solvent was concentrated. After that, ethanol was added and stirred for half an hour, and the mixture was filtered. The filtrate was concentrated to obtain intermediate 1.

[0029] Synthesis of Intermediate 2

[0030]

[0031] The intermediate 1 (1 mmol) was dissolved in 13 mL of acetone, and 13 mL of 10% sodium carbonate aqueous solution was slowly added. Fmoc-OSu (1.5 mmol) was added at 0°C, and stirred for 5 min. The mixture was moved to room temperature for reaction for 24 hours. After the reaction was completed as monitored by TLC, 2 M hydrochloric acid was added to adjust the pH to 2-3. Ethyl acetate was added to extract 3 times, washed with water 2 times, washed with saturated brine 1 time, and the organic phase was concentrated. The intermediate 2 was obtained by column chromatography (dichloromethane: methanol = 100: 1).

[0032] Synthesis of side chain intermediate 3

[0033]

[0034] Referring to the synthesis method of INT4 in patent WO2023141044, intermediate 3 can be obtained.

[0035] Example 1. Preparation of Compound 1

[0036]

[0037] Rinamide-AM resin (Merrifield polymer loaded with (2,4-dimethoxyphenyl) (4-alkoxyphenyl) methane, where 4-alkoxy is the position and chemical bond type connected to the resin, loading amount 0.7mmol / g) was used. DMF and DCM were used to dissolve the reagents used in the reaction. The solvents and reagents used in the reaction were: DMF = N,N-dimethylformamide; DCM = dichloromethane; HATU = 1-[di(dimethylamino)methylene]-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridine hexafluorophosphate; DIPEA = diisopropylethylamine. The desired amino acid (0.6mmol), HATU (0.6mmol), DIPEA (1.2mmol), and DMF (6mL) were used to react at 25°C to complete the synthesis of the desired linear peptide as follows.

[0038]

[0039] Continue to splice intermediate 3 by solid phase synthesis, then wash the resin with anhydrous DMF once, and then pump it to dryness. Add cutting liquid (trifluoroacetic acid: thioanisole: 1,2-ethanedithiol: anisole = 90:5:2.5:2.5, volume ratio) to the dried resin. Shake the mixture at 10°C for 3 hours. After the reaction is completed, the cutting liquid is filtered and concentrated to 1 / 5 of the original volume, and the concentrate is added dropwise to 10 times the volume of -20°C anhydrous ether. The obtained crude peptide is dissolved in a certain amount of pure water, adjusted to pH 7 with triethylamine or 2M HCl, acetonitrile is added to clarify, and the crude solid is lyophilized. Add a certain amount of acetonitrile to completely dissolve it and filter it through a 0.33μM filter. Use Aglient Eclipase XDB-C18 column for separation, mobile phase A: 0.1% TFA / H2O; mobile phase B: 0.1% TFA / MeCN. The chromatographic conditions were 10% B-100% B, 20 min. The compound was confirmed to be correct by LC / MS ([(M+4H) / 4]+=1217.4).

[0040] Example 2. Preparation of Compound 2

[0041]

[0042] Referring to the synthesis of Example 1, LC / MS: [(M+4H) / 4]+=1214.8.

[0043] Example 3. Preparation of Compound 3

[0044]

[0045] Refer to the synthesis of Example 1, LC / MS: [(M+4H) / 4]+=1214.9.

[0046] Test Example 1: GLP-1, GIP receptor binding test

[0047] The binding data of the compound and GLP-1 or GIP protein were detected using the Biacore S200 system and CM5 chip. The experimental operation was completed in accordance with the Biacore S200 operation manual. The compound was prepared using a 5% DMSO PBS solution. The data was analyzed and processed using the Biacore S200 Evaluation Software. Among them, compound-1, compound-2 and compound-3 are from Examples 1-3 in patent CN116496361A.

[0048] Table 1. Test of compound binding to GLP-1 and GIP receptors

[0049]

[0050] The data are shown in Table 1, from which it can be seen that the affinity activity of the patented compound to GLP-1 and GIP is better than that of the positive control Tirzepatide and compound-1, compound-2 and compound-3.

[0051] Experimental Example 2: Compounds Test on cAMP Activity of HEK293 Cells

[0052] The hGLP-2R functional activity in the presence of the compounds of the invention was demonstrated by measuring intracellular cAMP in HEK293 cells. HEK293 cells were passaged in complete medium and, after approximately 48 hours of proliferation, the freezing medium was exchanged with Invitrogen 31053DMEM containing 0.5% FBS on the day of the cell assay. The compounds of the invention were dissolved in DMSO and treated for 30 minutes at 37°C. The final DMSO concentration did not exceed 1.1% and the final IBMX concentration was 250 μΜ. Cyclic AMP was measured using the Dynamic2 assay by homogeneous time-resolved fluorescence technology (Cisbio Bioassays, Bedford, MA). The corresponding cAMP concentrations were derived by ratio calculation and external standards. The sigmoidal dose response of the test compounds was examined using a four-parameter logistic equation and compared with the native C18 acylated ligand.

[0053] Table 2. GLP-2R functional activity assay in HEK293 cells

[0054] Compound No. <![CDATA[EC 50 (nM)]]> Example 1 104 Example 2 90 Example 3 98 Tirzepatide 264 Compound-1 104

[0055] The data are shown in Table 2, from which it can be seen that the EC50 activity of the patented compound in the HEK293 cell cAMP activity test is better than that of the positive control Tirzepatide and compound-1.

[0056] Test Example 3: Pharmacokinetics of Compounds

[0057] 1. Experimental instruments and materials

[0058] High-speed refrigerated centrifuge, vortex oscillator (Vortex Genius3), high-speed centrifuge (Eppendorf5415D), disposable syringes, pipettes (Eppendorf), SD male rats used in the experiment were purchased from Yangzhou University, EDTA-K2 vacuum blood collection tubes, and normal saline. All rats in the oral group fasted for 12 hours before administration, had free access to water, and had free access to water and food during administration.

[0059] (II) Experimental steps

[0060] Example 1, intermediate 1 and compound-1 were dissolved in DMSO / solutol / water (10 / 10 / 80) to prepare a clear solution. The intranasal administration dose of the compound was 25 mg / kg, and the tail vein administration dose of the compound was 5 mg / kg. At 2min, 10min, 30min, 1h, 2h, 3h, 5h, 8h, 12h, 16h, and 24h after tail vein administration, 0.5mL of blood was continuously collected from the fundus venous plexus into a heparin tube. At 5min, 15min, 30min, 1h, 2h, 3h, 5h, 8h, 12h, 16h, and 24h after intranasal administration, 0.5mL of blood was continuously collected from the fundus venous plexus into a heparin tube. After the sample was centrifuged at 8000r and 4°C for 10min, 0.15mL of the upper plasma was collected and stored at -20°C, and then analyzed by LC-MS / MS. The data was analyzed by WinNolin non-compartmental model to obtain key pharmacokinetic parameters.

[0061] (III) Experimental results

[0062] Table 3 Pharmacokinetic parameters

[0063]

[0064] It can be seen from Table 3 above that, compared with Tirzepatide and Compound-1 in CN116496361A, the half-life of Compound 1 after oral administration is significantly improved, which can effectively improve the dosage and thus reduce the toxic side effects of high-dose administration.

[0065] Finally, it is necessary to point out that the above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A deuterated peptide mimetic GIP / GLP-1 dual receptor agonist compound as shown in formula I and a pharmaceutically acceptable salt thereof, in, R1 and R2 are independently selected from H or deuterium, and are not H at the same time.

2. The deuterated peptide mimetic GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt according to claim 1, characterized in that The structure is selected from the following compounds:

3. The deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt according to claim 1 or 2, characterized in that The pharmaceutically acceptable salt is selected from the group consisting of hydrochloric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, benzoic acid, salicylic acid, nicotinic acid, 3-phenylpropionic acid, aminosulfonic acid, citric acid, tartaric acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, ascorbic acid, and aspartic acid.

4. Use of the deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt according to claim 1 or 2 in drugs for treating or preventing type 2 diabetes.

5. Use of the deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of a drug for treating obesity or overweight and complications related to weight.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition is composed of the deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and a pharmaceutically acceptable salt thereof according to claim 1 or 2 as an active ingredient and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of the deuterated mimetic peptide GIP / GLP-1 dual receptor agonist compound and its pharmaceutically acceptable salt according to claim 6, characterized in that: The pharmaceutical composition is selected from capsules, powders, tablets, granules, pills, injections, syrups, oral solutions, inhalants, ointments, suppositories or patches.

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