Polypeptide compounds and uses thereof

Abstract

The present application relates to the technical field of therapeutic polypeptide, in particular to polypeptide compounds and application thereof. The polypeptide compound is selected from the compounds shown in the following structural formula or chiral isomers, tautomers, or pharmaceutically acceptable salts, esters or amides thereof: wherein R1 represents a hydrogen atom, R a , R b , R c , R d , R e and R f are independently selected from unsubstituted C1-C5 alkyl; AA1 represents QGTFTSDYSI; x represents any integer from 1-5; R2 represents AA2 represents AFIEYLLEGGPSSGAPPPS; the polypeptide compound can be used as a new GLP drug, and has good biological activity and oral bioavailability.

Description

[0001] The present application claims priority to Chinese patent application No. 202411033707.2, entitled "Polypeptide compound and application thereof", the specification and claims of which are incorporated herein in their entirety by reference. TECHNICAL FIELD

[0002] The present application relates to the technical field of therapeutic polypeptides, in particular, to a polypeptide compound and application thereof. BACKGROUND

[0003] In the 1980s, scientists discovered glucagon-like peptide-1 (GLP-1). In 2005, the GLP-1 analogue exenatide was approved for marketing by the US FDA, but it needs to be injected twice a day, which makes the patient's compliance poor. In 2010, the FDA approved the second GLP-1 drug liraglutide, which is injected once a day. In 2017, semaglutide was approved by the US FDA, which is injected once a week. In 2023, the first dual-target agonist tirzepatide was approved by the FDA, which is injected once a week. In 2019, the FDA approved the oral dosage form Rybelsus (for diabetes) of semaglutide, which is the only oral GLP-1 drug approved in the world so far.

[0004] GLP-1 drugs have shown great clinical demand and value, and are actively expanding dozens of indications, which are expected to solve more clinical needs.

[0005] After decades of development, there are more than ten GLP-1 drugs on the market, and hundreds of drugs in clinical research, but most of them are injections, and only semaglutide oral dosage form Rybelsus is on the market, which needs to be administered daily.

[0006] GLP-1 drug development trends: 1. Long-acting: Long-acting GLP-1 drugs have achieved great success in sales due to better patient compliance; 2. Oral: Oral administration is more convenient and has better compliance, and can also avoid the so-called "needle-phobia" of some patients, that is, they are very afraid of injecting themselves; 3. Multi-target: Glucose-dependent insulinotropic polypeptide receptor (GIPR), glucagon receptor (GCGR), and fibroblast growth factor 21 receptor (FG21R) can play a synergistic role with GLP-1 in blood glucose balance regulation, fat metabolism, food intake, etc., and improve drug efficacy.

[0007] Currently, there is only one oral GLP-1 drug, a single-target drug that requires daily administration and has low compliance. Therefore, there is a need to develop longer-acting oral GLP-1 multi-target drugs, such as those that can be administered every two or three days or once a week, to better meet clinical needs.

[0008] In view of this, the present invention is proposed. Summary of the Invention

[0009] The present invention aims to provide polypeptide compounds and their applications. The polypeptide compounds provided in the embodiments of the present invention can be used as new GLP drugs, are multi-target drugs, can produce synergistic effects, and have good biological activity and oral bioavailability.

[0010] The present invention is achieved in that:

[0011] In a first aspect, the present invention provides a polypeptide compound selected from the compounds represented by the following structural formulas or chiral isomers, tautomers, or pharmaceutically acceptable salts, esters, or amides thereof:

[0012]

[0013] Wherein, R1 represents a hydrogen atom, R3 and R4 are independently selected from any one of a carboxyl group, a carboxylate group, a tetrazolyl group, a phosphate group and a phosphate group; n is any integer between 10 and 20;

[0014] R a 、R b 、R c 、R d 、R e and R f Each independently selected from unsubstituted C1-C5 alkyl;

[0015] AA1 means QGTFTSDYSI;

[0016] The sequence listing of AA1 is shown as SEQ ID NO. 1;

[0017] x represents any integer from 1 to 5;

[0018] R2 represents R7 represents any one of a carboxyl group, a carboxylate group, a tetrazolyl group, a phosphate group, and a phosphate group; p represents any integer between 10 and 20; m represents any value between 0 and 2;

[0019] AA2 stands for AFIEYLLEGGPSSGAPPPS;

[0020] The sequence listing of AA2 is shown in SEQ ID NO.2.

[0021] In a second aspect, the present invention provides a use of a polypeptide compound according to any one of the preceding embodiments in the preparation of a medicament for treating any one of the following diseases, the diseases including: obesity, diabetes, cardiovascular disease, chronic kidney disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, myocardial infarction, stroke, peripheral arterial disease, Alzheimer's disease, fatty liver disease, arrhythmia, diabetic nephropathy, osteoarthritis, chronic kidney disease, hypertension, cognitive impairment, coronary heart disease, stroke, neurodegenerative disorders, heart failure, behavioral bowel syndrome, dyspepsia and gastric ulcer.

[0022] The present invention has the following beneficial effects: the polypeptide compound provided in the embodiment of the present invention can be used as a GLP-1 drug, is a multi-target drug, can produce a synergistic effect, has good biological activity and oral bioavailability, and can be adapted to a variety of diseases. DETAILED DESCRIPTION

[0023] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0024] GLP 1 receptor agonists have been used to treat obesity, diabetes, cardiovascular risks, and dozens of chronic diseases are under clinical research. The only oral GLP 1 receptor agonist drug currently on the market is Rybelsus, a single-target drug, and is administered daily. Treatment regimens that reduce the frequency of dosing may improve patient convenience and compliance. Multiple targets can increase synergistic effects and thereby enhance drug efficacy. Therefore, the development of oral GLP 1 multi-target drugs with a dosing frequency lower than once a day will be a great improvement to the available treatment options. The polypeptide compound provided in the embodiments of the present invention is also a GLP-1 compound, which not only has the above-mentioned efficacy and can treat the corresponding diseases, but also can be used as a long-acting oral GLP-1 drug that can extend the dosing period, for example, it can be taken orally once a week.

[0025] An embodiment of the present invention provides a polypeptide compound selected from the compounds represented by the following structural formula or chiral isomers, tautomers, or pharmaceutically acceptable salts, esters, or amides thereof:

[0026]

[0027] Among them, R1 hydrogen atom, represents R3 and R4 are independently selected from any one of carboxyl, carboxylate, tetrazolyl, phosphate and phosphate groups; for example, R3 is selected from -COOR5, -PO(OR6)2 and wherein R5 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, for example, a methyl group, an ethyl group, a n-propyl group, and an isopropyl group. Preferably, R5 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; more preferably, it is a hydrogen atom, a methyl group, and an ethyl group, and most preferably, it is a hydrogen atom.

[0028] R6 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, etc. Preferably, R6 is a hydrogen atom or an unsubstituted C1-C3 alkyl group, more preferably any one of a hydrogen atom, a methyl group, and an ethyl group, and most preferably a hydrogen atom.

[0029] R 10 is any one of a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, a metal ion, and an ammonium cation; for example, a substituted or unsubstituted C1-C3 alkyl group such as methyl, ethyl, n-propyl, and isopropyl; a monovalent cation such as potassium ion, sodium ion, etc. Preferably R 10 It is any one of a hydrogen atom, an unsubstituted C1-C3 alkyl group, a metal ion triethylamine, ethanolamine, arginine or lysine to form a cation; more preferably, it is any one of a hydrogen atom, a methyl group, an ethyl group and a monovalent metal ion; further preferably, it is any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion and a potassium ion; and most preferably, it is any one of a hydrogen atom, a methyl group and an ethyl group.

[0030] R4 is -COOR 12 、-PO(OR 13 )2 and Any one of the functional groups formed; wherein R 12 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; for example, methyl, ethyl, n-propyl and isopropyl. 12 It is a hydrogen atom or an unsubstituted C1-C3 alkyl group; more preferably, it is any one of a hydrogen atom, a methyl group and an ethyl group, and most preferably, it is a hydrogen atom.

[0031] R 13 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; for example, methyl, ethyl, n-propyl and isopropyl. 13 It is a hydrogen atom or an unsubstituted C1-C3 alkyl group; more preferably, it is any one of a hydrogen atom, a methyl group and an ethyl group, and most preferably, it is a hydrogen atom.

[0032] R 14 is any one of a hydrogen atom, a C1-C3 alkyl group, a metal ion, and an ammonium cation; for example, a C1-C3 alkyl group such as methyl, ethyl, n-propyl, and isopropyl; a monovalent cation such as a potassium ion, a sodium ion, etc. Preferably R 14It is any one of a hydrogen atom, an unsubstituted C1-C3 alkyl group, a metal ion, and a cation formed by triethylamine, ethanolamine, arginine or lysine; more preferably, it is any one of a hydrogen atom, a methyl group, an ethyl group and a monovalent metal ion; further preferably, it is any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion and a potassium ion; and most preferably, it is any one of a hydrogen atom, a methyl group and an ethyl group.

[0033] n is any integer between 12 and 20; 12, 13, 14, 15, 16, 17, 18, 19 and any number between 12 and 20, for example, preferably any integer between 13 and 19; more preferably 15, 16 or 17, most preferably 15 or 17.

[0034] R a 、R b 、R c 、R d 、R e and R f are independently selected from unsubstituted C1-C5 alkyl groups; for example, methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-butyl and other unsubstituted alkyl groups. Preferably, they are independently selected from unsubstituted C1-C3 alkyl groups. For example, R a 、R c , and R f When both are methyl, R b 、R d and R e are independently selected from unsubstituted C1-C5 alkyl groups, preferably, are independently selected from unsubstituted C1-C3 alkyl groups. a 、R b 、R c 、R d 、R e and R f The same or two different alkyl groups may be used.

[0035] AA1 means QGTFTSDYSI;

[0036] The sequence listing of AA1 is shown in SEQ ID NO. 1; the polypeptide sequence represented by AA1 is an existing sequence and will not be described in detail in the examples of the present invention.

[0037] x represents any integer between 1 and 5; for example, x represents any integer between 1 and 5, such as 1, 2, 3, 4, and 5. Preferably, x represents 2 to 4, more preferably 3.

[0038] R2 represents R7 represents any one of a carboxyl group, a carboxylate group, a tetrazolyl group, a phosphoric acid group and a phosphoric acid ester group; p represents any integer between 10 and 20; and m represents any value between 0 and 2.

[0039] Specifically, R7 is -COOR8, -PO(OR9)2 and wherein R8 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, for example, a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. Preferably, R8 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; more preferably, it is a hydrogen atom, a methyl group, and an ethyl group, and most preferably, it is a hydrogen atom.

[0040] R9 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, for example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, etc. Preferably, R9 is a hydrogen atom or an unsubstituted C1-C3 alkyl group, more preferably any one of a hydrogen atom, a methyl group, and an ethyl group, and most preferably a hydrogen atom.

[0041] R 11 is any one of a hydrogen atom, a C1-C3 alkyl group, a metal ion, and an ammonium cation; for example, a C1-C3 alkyl group such as methyl, ethyl, n-propyl, and isopropyl; a monovalent cation such as a potassium ion, a sodium ion, etc. Preferably R 11 It is any one of a hydrogen atom, an unsubstituted C1-C3 alkyl group, a metal ion, and a cation formed by triethylamine, ethanolamine, arginine or lysine; more preferably, it is any one of a hydrogen atom, a methyl group, an ethyl group and a monovalent metal ion; further preferably, it is any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion and a potassium ion; and most preferably, it is any one of a hydrogen atom, a methyl group and an ethyl group.

[0042] p is any integer between 10 and 20, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. Preferably, p is any integer between 12 and 20, preferably any integer between 16 and 18, and preferably 16 or 18.

[0043] m is 0, 1 or 2, preferably 1 or 2.

[0044] AA2 stands for AFIEYLLEGGPSSGAPPPS;

[0045] The sequence listing of AA2 is shown in SEQ ID NO. 2. The sequence listing of AA2 is shown in SEQ ID NO. 2. The polypeptide sequence represented by AA2 is an existing sequence and therefore will not be described in detail in the present embodiment.

[0046] More specifically, amino acids other than Aib2, Leu13, Lys17, and Aib20 are depicted using the standard single-letter amino acid code:

[0047] R1 is a hydrogen atom, Wherein, R3 is -COOR5, -PO(OR6)2 and Any one of the functional groups formed; wherein R5 is a hydrogen atom or a substituted or unsubstituted C1-C3 alkyl group; R6 is a hydrogen atom or a substituted or unsubstituted C1-C3 alkyl group; R 10 is any one of a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, a metal ion and an ammonium cation;

[0048] R4 is -COOR 12 、-PO(OR 13 )2 and Any one of the functional groups formed; wherein R 12 is a hydrogen atom or a substituted or unsubstituted C1-C3 alkyl group; R 13 is a hydrogen atom or a substituted or unsubstituted C1-C3 alkyl group; R 14 is any one of a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, a metal ion and an ammonium cation;

[0049] n is any integer between 10 and 20;

[0050] R a 、R b 、R c 、R d 、R e and R f Each independently selected from unsubstituted C1-C3 alkyl;

[0051] x represents any integer from 2 to 4;

[0052] R2 is in,

[0053] R7 is -COOR8, -PO(OR9)2 and Any one of the functional groups formed; wherein R8 is a hydrogen atom or a substituted or unsubstituted C1-C3 alkyl group; R9 is a hydrogen atom or a substituted or unsubstituted C1-C3 alkyl group; R 11 is any one of a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group, a metal ion and an ammonium cation;

[0054] p is any integer between 10 and 20; m is 0, 1 or 2.

[0055] Preferably, R1 is a hydrogen atom, Wherein, R3 is -COOR5, -PO(OR6)2 and Any one of the functional groups formed; wherein R5 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; R6 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; R 10 Any one of a hydrogen atom, an unsubstituted C1-C3 alkyl group, a metal ion, and a cation formed by triethylamine, ethanolamine, arginine, or lysine;

[0056] R4 is -COOR 12 、-PO(OR 13 )2 and Any one of the functional groups formed; wherein R 12 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; R 13 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; R 14 Any one of a hydrogen atom, an unsubstituted C1-C3 alkyl group, a metal ion, and a cation formed by triethylamine, ethanolamine, arginine, or lysine;

[0057] n is any integer between 12 and 20;

[0058] R a 、R c , and R f When both are methyl, R b 、R d and R e Each independently selected from unsubstituted C1-C5 alkyl;

[0059] x represents any integer from 2 to 4;

[0060] R2 is R7 is -COOR8, -PO(OR9)2 and Any one of the functional groups formed; wherein R8 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; R9 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; R 11 Any one of a hydrogen atom, an unsubstituted C1-C3 alkyl group, a metal ion, and a cation formed by triethylamine, ethanolamine, arginine, or lysine;

[0061] p is any integer between 12 and 20; m is 0, 1 or 2.

[0062] Preferably, R1 is a hydrogen atom, Wherein, R3 is -COOR5, -PO(OR6)2 and Any one of the functional group groups formed; wherein R5 is any one of a hydrogen atom, a methyl group or an ethyl group; R6 is any one of a hydrogen atom, a methyl group or an ethyl group; R10 is any one of a hydrogen atom, a methyl group, an ethyl group or a metal ion;

[0063] R4 is -COOR 12 、-PO(OR 13 )2 and Any one of the functional groups formed; R 12 is any one of a hydrogen atom, a methyl group or an ethyl group; R 13is any one of a hydrogen atom, a methyl group or an ethyl group; R 14 is any one of a hydrogen atom, a methyl group, an ethyl group or a metal ion;

[0064] n is any integer between 13 and 19;

[0065] R a 、R c , and R f When both are methyl, R b 、R d and R e Each independently selected from unsubstituted C1-C3 alkyl;

[0066] x represents any integer from 2 to 4;

[0067] R2 is Wherein, R7 is -COOR8, -PO(OR9)2 and Any one of the functional groups formed; wherein R8 is any one of a hydrogen atom, a methyl group or an ethyl group; R9 is any one of a hydrogen atom, a methyl group or an ethyl group; R 11 is any one of a hydrogen atom, a methyl group, an ethyl group or a metal ion;

[0068] p is any integer between 16 and 18; m is 0, 1 or 2.

[0069] Preferably, R1 is a hydrogen atom, Wherein, R3 is -COOH, -PO(OH)2 or Any one of R 10 is any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion or a potassium ion;

[0070] R4 is -COOH, -PO(OH)2, or Any one of R 14 is any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion or a potassium ion;

[0071] n is any one of 15, 16 or 17;

[0072] R a 、R c , and R f When both are methyl, R b 、R d and R e Each independently selected from unsubstituted C1-C3 alkyl;

[0073] x represents 3 or 4;

[0074] R2 is Wherein, R7 is -COOH, -PO(OH)2 or

[0075] Any one of the following; where R 11 is any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion or a potassium ion;

[0076] p is any one of 16, 17 or 18;

[0077] m is any one of 0, 1 or 2.

[0078] Preferably, R1 is a hydrogen atom, Wherein, R3 is -COOH, -PO(OH)2 or Any one of the functional groups formed; R 10 is a hydrogen atom or a methyl group;

[0079] R4 is -COOH, -PO(OH)2 or Any one of the functional groups formed; R 14 is a hydrogen atom or a methyl group;

[0080] n is 15 or 17;

[0081] R a 、R c , and R f When both are methyl, R b and R e Methyl or ethyl, R d isobutyl;

[0082] x represents 3;

[0083] R2 is Wherein, R7 is -COOH, -PO(OH)2 or

[0084] Any one of the functional groups formed; R 11 is a hydrogen atom or a methyl group;

[0085] p is 16 or 18; m is 1 or 2.

[0086] Specifically, the polypeptide compound is selected from any one of the compounds represented by the following structural formulas:

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] Preferably, it is selected from any one of the compounds represented by the following structural formulas:

[0098]

[0099]

[0100]

[0101]

[0102] The present invention also provides a method for preparing the above-mentioned polypeptide compound. The preparation method can refer to existing methods. The present invention provides examples. Specifically,

[0103] The present invention uses an Fmoc chemistry method based on SPPS to synthesize peptides on a polypeptide synthesis column. The Fmoc-protected amino acids used in the method are all purchased from Shanghai Jier Biochemical Co., Ltd., such as: Fmoc-Aib-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ile-OH, Fmoc-α-Me-Leu-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Pro-OH.

[0104] Wherein R1-Tyr(tBu)-Aib-OH and Fmoc-Lys(R2)-OH are prepared by the following method:

[0105] Synthesis of R1-Tyr(tBu)-Aib-OH: M2, M3, M4 were sequentially amidated and condensed, after activation of the naked carboxylic acid of M4, H-Tyr(tBu)-Aib-OH was condensed to give R1-Tyr(tBu)-Aib-OH. If R3 and R4 are carboxyl, the carboxyl group is protected with a common protecting agent (such as t-butyl, methyl, ethyl, benzyl).

[0106]

[0107] Synthesis of Fmoc-Lys(R2)-OH: M5, H-L-Glu-OtBu, AEEA were sequentially amidated and condensed, after activation of the naked carboxylic acid of AEEA, Fmoc-Lys-OH was condensed to give Fmoc-Lys(R2)-OH. If R7 is -COOH, -PO(OH)2, the carboxyl group is protected with a common protecting agent (such as t-butyl, methyl, ethyl, benzyl).

[0108]

[0109] Resin: 2-Chlorotrityl chloride resin (1% DVB cross-linked) from Tianjin Nankai Hengcheng Co. Ltd, 100-200 mesh, degree of substitution 0.4-0.6 mmol / g. Fmoc-Lys(R2)-OH for lysine at position 17, R1-Tyr(tBu)-Aib for tyrosine 2-amino isobutyric acid at position 1, 2. First, 2-Chlorotrityl chloride resin was activated and coupled with Fmoc-Ser(tBu)-OH, where the molar ratio of 2-Chlorotrityl chloride resin, Fmoc-Ser(tBu)-OH, DIPEA was 1:1.1:4. Fmoc group was removed using 20% piperidine / DMF for 2x 10 min before each coupling step. All standard amino acid couplings were performed using 2-fold molar ratio of Fmoc-protected amino acid, 3-fold molar ratio of diisopropylcarbodiimide and 3-fold molar ratio of 1-hydroxybenzotriazole, coupling for 2-3 hours under nitrogen protection, where the coupling degree was detected by ninhydrin color reagent. In some cases, the coupling time was increased or the coupling step was repeated to achieve a satisfactory coupling level. After the synthesis was completed, the peptide resin was washed with DCM, and then fully air-dried to obtain the peptide resin.

[0110] Cleavage:

[0111] The dry peptide resin synthesized was cleaved with 10 volumes of cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane, 95:2.5:2.5 v / v) at room temperature for 2 hours. The resin was filtered off and washed twice with 2 volumes of pure TFA each time, and the combined filtrate was treated with 5 volumes of cold diethyl ether (-20°C) to precipitate the crude peptide. The crude peptide / diethyl ether suspension was then centrifuged at 3500 rpm for 10 minutes, the supernatant was removed, and the solid was washed twice with diethyl ether before being dried in vacuo to give the crude peptide.

[0112] Purification:

[0113] The crude peptide was dissolved in MQ water containing 40% acetic acid and purified by reverse phase preparative HPLC (Waters DeltaPrep 4000) on a column containing C18 silica gel. Elution was performed with a gradient of increasing MeCN in MQ water containing 0.1% TFA. Relevant fractions were analysed by UPLC. Fractions containing pure target peptide were combined. The resulting solution was analysed (UPLC, LCMS) and the product was aliquoted into glass vials. The vials were capped with Millipore glass fibre pre-filters. Lyophilisation gave the target compound as a white solid.

[0114] General LCMS method:

[0115] Mass spectrometry (MS) parameters:

[0116] Instrument: Agilent 6410 triple quadrupole LCMS; ionisation source: +ESI.

[0117] Liquid chromatography parameters:

[0118] Column: Agilent ZORBAX SB-C18, dimensions: 250 x 4.6 mm, 5 μm; detection wavelength: 195 nm; column temperature: 30 °C; injection volume: 10 mL; flow rate: 1 mL / min; mobile phase A: 0.05% glacial acetic acid; mobile phase B: acetonitrile.

[0119] The present application also provides the use of the polypeptide compound in the preparation of a medicament for treating any one of the following diseases: obesity, diabetes, cardiovascular disease, chronic kidney disease, nonalcoholic steatohepatitis, nonalcoholic fatty liver disease, myocardial infarction, stroke, peripheral arterial disease, Alzheimer's disease, fatty liver disease, arrhythmia, diabetic nephropathy, osteoarthritis, chronic kidney disease, hypertension, cognitive impairment, coronary heart disease, stroke, neurodegenerative disorders, heart failure, irritable bowel syndrome, indigestion and gastric ulcer.

[0120] The names of the compounds or raw materials used in the preparation method of the embodiments of the present application and their corresponding abbreviations are as follows:

[0121] Aib: α-aminoisobutyric acid; Boc: tert-butyloxycarbonyl; Fmoc: 9-fluorenylmethoxycarbonyl; tBu: tert-butyl; DIEA: N,N-diisopropylethylamine; EDCl: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOSu: N-hydroxysuccinimide; TFA: trifluoroacetic acid; AEEA-AEEA: 17-amino-10-oxo-3,6,12,15-tetraoxa-9-azaheptadecanoic acid; DCM: dichloromethane; DMF: N,N-dimethylformamide; PIP: piperidine; HOBt: 1-hydroxybenzotriazole; HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIPEA: N,N-diisopropylethylamine; TIPS: triisopropylsilane; PHSME: methylphenyl sulfide; Trt: trityl.

[0122] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0123] Example 1

[0124] The present invention provides a polypeptide compound (hereinafter referred to as Compound 1), the structural formula of which is as follows:

[0125]

[0126] The present invention also provides a method for preparing the above-mentioned polypeptide compound, comprising: synthesizing R1-Tyr(tBu)-Aib-OH(1-A) containing a protecting group according to the following method:

[0127]

[0128] Specifically, 5.00 g of compound 1-A1 (13.51 mmol) was added to a 100 ml reaction flask, followed by 25 ml of dichloromethane and 1.71 g of HOSu (14.86 mmol). 3.10 g of EDCl.HCl (16.22 mmol) was slowly added at 10°C. After addition, the mixture was kept warm for 1 hour. TLC observation showed that the reaction was complete. 2.27 g of N,N-diisopropylethylamine (17.56 mmol) and 2.88 g of compound 1-A2 (14.19 mmol) were added, and the mixture was naturally returned to room temperature for 3 hours. TLC monitoring showed that the reaction was complete. The mixture was washed twice with 1 mol / L hydrochloric acid and once with 5% brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure at 40-45°C until no fraction remained, yielding 6.84 g of compound 1-A3 with a yield of 91.2%.

[0129] Into a 100 ml reaction flask, was placed 5.00 g of compound 1-A3 (9.0 mmol), 25 ml of dichloromethane, 1.14 g of HOSu (9.9 mmol), and 2.07 g of EDCl.HCL (10.8 mmol) was added slowly at 10 °C. After the addition was completed, the reaction was allowed to proceed for 1 h. TLC was used to monitor the reaction. After the reaction was completed, 1.51 g of N,N-diisopropylethylamine (11.71 mmol), and 2.73 g of compound 1-A4 (9.9 mmol) were added. The reaction was allowed to proceed for 3 h at room temperature. The reaction was washed with 1 mol / L hydrochloric acid water twice, 5% brine once, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure at 40-45 °C to remove the solvent. This resulted in 6.75 g of compound 1-A5 with a yield of 92.3%.

[0130] Into a 100 ml reaction flask, was placed 5.00 g of compound 1-A5 (6.15 mmol), 25 ml of dichloromethane, 0.78 g of HOSu (6.78 mmol), and 1.41 g of EDCl.HCL (7.36 mmol) was added slowly at 10 °C. After the addition was completed, the reaction was allowed to proceed for 1 h. TLC was used to monitor the reaction. After the reaction was completed, the reaction was concentrated under reduced pressure at 40-45 °C to remove the solvent. This resulted in 5.51 g of compound 1-A6 with a yield of 98.4%.

[0131] Into a 100 ml reaction flask, was placed 5.00 g of compound 1-A6 (5.49 mmol), 25 ml of dichloromethane, 0.92 g of N,N-diisopropylethylamine (7.14 mmol), and 1.97 g of compound H-Tyr(tBu)-Aib-OH (6.1 mmol). The reaction was allowed to proceed for 3 h at room temperature. TLC was used to monitor the reaction. After the reaction was completed, the reaction was washed with 1 mol / L hydrochloric acid water twice, 5% brine once, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure at 40-45 °C to remove the solvent. This resulted in 5.65 g of compound 1-A with a yield of 92.1%.

[0132] Fmoc-Lys(R2)-OH (1-B) containing a protecting group was synthesized as follows:

[0133]

[0134] 7.54g of compound 1-B1 (13.51mmol) was added to a 100ml reaction flask, followed by 30ml of dichloromethane and 1.71g of HOSu (14.86mmol). 3.10g of EDCl.HCl (16.22mmol) was slowly added at 10°C. After addition, the mixture was allowed to react for 1h. TLC indicated that the reaction was complete. 2.27g of N,N-diisopropylethylamine (17.56mmol) and 2.88g of compound 1-A2 (14.19mmol) were added, and the mixture was allowed to react naturally at room temperature for 3h. TLC indicated that the reaction was complete. The mixture was washed twice with 1mol / L hydrochloric acid and once with 5% brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure at 40-45°C until no fraction remained. This afforded 9.22g of compound 1-B2 in a 91.7% yield.

[0135] 6.69 g of compound 1-B2 (9.0 mmol) was added to a 100 ml reaction flask, followed by 30 ml of dichloromethane and 1.14 g of HOSu (9.9 mmol). 2.07 g of EDCl.HCl (10.8 mmol) was slowly added at 10 °C. After addition, the mixture was allowed to react for 1 h. TLC indicated that the reaction was complete. 1.51 g of N,N-diisopropylethylamine (11.71 mmol) and 1.62 g of compound AEEA (9.9 mmol) were added, and the mixture was allowed to return to room temperature and react for 3 h. TLC indicated that the reaction was complete. The mixture was washed twice with 1 mol / L hydrochloric acid and once with 5% brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure at 40-45 °C until no fraction remained. This afforded 7.42 g of compound 1-B3, with a yield of 92.8%.

[0136] 5.49 g of compound 1-B3 (6.15 mmol) was added to a 100 ml reaction flask, followed by 30 ml of dichloromethane and 0.78 g of HOSu (6.78 mmol). 1.41 g of EDCl.HCl (7.36 mmol) was slowly added at 10 °C. After addition, the mixture was allowed to react for 1 h. TLC indicated that the reaction was complete. 1.32 g of N,N-diisopropylethylamine (8.00 mmol) and 2.49 g of Fmoc-Lys-OH (6.76 mmol) were added, and the mixture was allowed to react naturally at room temperature for 3 h. TLC indicated that the reaction was complete. The mixture was washed twice with 1 mol / L hydrochloric acid and once with 5% brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure at 40-45 °C until no fraction remained. This afforded 7.12 g of compound 1-B in a 93.5% yield.

[0137] Compound 1 was synthesized by SPPS on a peptide synthesis column using Fmoc chemistry. The synthetic route is as follows:

[0138]

[0139] At room temperature, 1-C1 (5 mmol) was reacted with Fmoc-Ser(tBu)-OH (5.5 mmol) in DIEA (20 mmol) in DMF (50 ml) for 2 hours to obtain 1-C2. 1-C2 was treated with a 20% piperidine / DMF solution to remove the Fmoc protecting group to obtain 1-C3. 1-C3 was sequentially subjected to an amide condensation reaction with the fully protected amino acids and side chain fragments in the sequence (15 mmol HOBt, 15 mmol HBTU, 15 mmol DIPEA, 50-100 ml DMF) to obtain 1-C4. 1-C4 was removed from the resin beads using 100 ml of a 1% TFA / DCM solution to obtain 1-C5. 1-C5 was hydrogenated to remove the benzyl group to obtain 1-C6. 1-C6 was subjected to removal of the side chain protecting groups to obtain compound 1.

[0140] Compound 1 was characterized by LCMS

[0141] Calculated masses: (M+3) / 3=1784.28, (M+4) / 4=1338.46, (M+5) / 5=1070.97.

[0142] Measured mass: (M+3) / 3=1784.22, (M+4) / 4=1338.43, (M+5) / 5=1070.94.

[0143] Example 2-12

[0144] Examples 2-12 provide a polypeptide compound, respectively, which is prepared according to the preparation method provided in Example 1. The obtained polypeptide compounds and characterization data are as follows:

[0145] Example 2

[0146] Compound 2 has the following structure:

[0147]

[0148] LCMS

[0149] Calculated masses: (M+3) / 3=1793.29, (M+4) / 4=1345.47, (M+5) / 5=1076.58.

[0150] Measured mass: (M+3) / 3=1793.22, (M+4) / 4=1345.43, (M+5) / 5=1076.54.

[0151] Example 3

[0152] Compound 3 has the following structure:

[0153]

[0154] LCMS

[0155] Calculated masses: (M+3) / 3=1795.94, (M+4) / 4=1347.46, (M+5) / 5=1077.97.

[0156] Measured masses: (M+3) / 3=1795.84, (M+4) / 4=1347.41, (M+5) / 5=1077.91.

[0157] Example 4:

[0158] Compound 4 has the following structure:

[0159]

[0160] LCMS

[0161] Calculated masses: (M+3) / 3=1801.63, (M+4) / 4=1351.22, (M+5) / 5=1081.38. Measured masses: (M+3) / 3=1801.68, (M+4) / 4=1351.29, (M+5) / 5=1081.44.

[0162] Example 5:

[0163] Compound 5 has the following structure:

[0164]

[0165] LCMS

[0166] Calculated masses: (M+3) / 3=1805.62, (M+4) / 4=1354.46, (M+5) / 5=1083.57. Measured masses: (M+3) / 3=1805.69, (M+4) / 4=1354.49, (M+5) / 5=1083.59.

[0167] Example 6

[0168] Compound 6 has the following structure:

[0169]

[0170] LCMS

[0171] Calculated masses: (M+3) / 3=1774.94, (M+4) / 4=1331.20, (M+5) / 5=1065.37. Measured masses: (M+3) / 3=1774.98, (M+4) / 4=1331.25, (M+5) / 5=1065.42.

[0172] Example 7

[0173] Compound 7 has the following structure:

[0174]

[0175] LCMS

[0176] Calculated masses: (M+3) / 3=1786.93, (M+4) / 4=1340.20, (M+5) / 5=1072.36. Measured masses: (M+3) / 3=1786.97, (M+4) / 4=1340.29, (M+5) / 5=1072.39.

[0177] Example 8

[0178] Compound 8 has the following structure:

[0179]

[0180] LCMS

[0181] Calculated masses: (M+3) / 3=1794.94, (M+4) / 4=1346.20, (M+5) / 5=1077.16. Measured masses: (M+3) / 3=1794.91, (M+4) / 4=1346.13, (M+5) / 5=1077.11.

[0182] Example 9

[0183] Compound 9 has the following structure:

[0184]

[0185] LCMS

[0186] Calculated masses: (M+3) / 3=1782.61, (M+4) / 4=1337.46, (M+5) / 5=1070.17. Measured masses: (M+3) / 3=1782.66, (M+4) / 4=1337.56, (M+5) / 5=1070.21.

[0187] Example 10

[0188] Compound 10 has the following structure:

[0189]

[0190] LCMS

[0191] Calculated masses: (M+3) / 3=1784.28, (M+4) / 4=1338.46, (M+5) / 5=1070.97. Measured masses: (M+3) / 3=1784.35, (M+4) / 4=1338.49, (M+5) / 5=1070.98.

[0192] Example 11

[0193] Compound 11 has the following structure:

[0194]

[0195] LCMS

[0196] Calculated masses: (M+3) / 3=1795.94, (M+4) / 4=1347.46, (M+5) / 5=1077.97.

[0197] Measured masses: (M+3) / 3=1795.87, (M+4) / 4=1347.41, (M+5) / 5=1077.93.

[0198] Example 12

[0199] Compound 12 has the following structure:

[0200]

[0201] Compound 12 can be obtained by replacing 1-A in the above synthetic route with Fmoc-Tyr(tBu)-Aib-OH.

[0202] LCMS

[0203] Calculated masses: (M+3) / 3=1589.16, (M+4) / 4=1192.12, (M+5) / 5=953.90.

[0204] Measured mass: (M+3) / 3=1589.12, (M+4) / 4=1192.08, (M+5) / 5=953.85.

[0205] Pharmacological test evaluation

[0206] Experimental Example 1 GLP-1R / GIPR / GCGR receptor agonist activity detection test

[0207] 1. Experimental Method: This study used transfected HEK293 cells overexpressing human GIP receptor (GIPR), GLP 1 receptor (GLP 1R), or human glucagon receptor (GcgR) to detect the GLP-1 receptor activity of the compounds of the present invention using a reporter gene assay.

[0208] 2. Experimental process:

[0209] 1) The compound provided in the examples of the present invention was prepared to a working concentration of 40 nM and subjected to 5-fold serial gradient dilution, with a total of 10 dilutions, with duplicate wells at each dilution point.

[0210] 2) Digest the cultured cells with trypsin, precipitate, resuspend and count, and inoculate 50 mL into 96-well cell plates.

[0211] 3) According to the experimental plate layout, add the serially diluted samples to 50 μl wells of a 96-well cell culture plate. Place the plate in a carbon dioxide incubator at 37°C and 5% CO2 for a period of time. Then add the fluorescent reagent and read the fluorescence value on a microplate reader. Use GraphPadPrism to perform curve fitting and calculate the EC 50 value.

[0212] 3. See Table 1 for experimental results.

[0213] Table 1 Agonist activity test results of the compounds in the examples

[0214]

[0215]

[0216] 4. Conclusion

[0217] The compounds provided in the examples of the present invention have strong agonist effects on GLP-1, GIP, and GCG receptors. Among them, the agonist activity of the compound in Example 12 on GLP-1, GIP, and GCG receptors is comparable to the agonist activity of Retatrutide on GLP-1R, GIPR, and GCGR receptors.

[0218] Experimental Example 2 Plasma protein binding rate test experiment

[0219] 1. Experimental steps

[0220] 1.1 Prepare 50 mM sodium phosphate buffer solution, pH 7.4.

[0221] Prepare 50 mM sodium phosphate buffer solution with sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate (Na2HPO4), and sodium chloride (NaCl), and adjust the pH to 7.4 + / - 0.1 with NaOH or H3PO4.

[0222] 1.2 Example compounds were dissolved in DMSO to prepare 10 mM stock solutions.

[0223] 1.3 Preparation of dosing solution:

[0224] 1.3.1: Configuration of the compound solution provided in the embodiment of the present invention:

[0225] Prepare a 10 mM DMSO stock solution;

[0226] 2mM Solution B: Add 20μL of 10mM DMSO stock solution to 80μL of 50mM sodium phosphate buffer. (The final DMSO concentration in this solution is 20%)

[0227] 1.4 Preparation of the compound delivery matrix provided in the embodiments of the present invention:

[0228] 380 μL of plasma was added to each 96-deep-well plate;

[0229] Add 20 μL of solution B (2 mM of the example compound) to the above 96-well plate. (Test compound: final concentration 100 μM containing 1% DMSO)

[0230] 1.5 Collect 0-hour samples: Take 25 μL of the matrix containing the compound provided in the examples of the present invention and add it to a blank 96-well collection plate and store it at -20°C.

[0231] 1.5 Preparing the Equilibrated Dialysis Apparatus

[0232] Add 100 µL of buffer to the receiving side of the equilibrated dialysis plate.

[0233] Then, 100 μL of the dosing medium containing the example compound was added to the dosing side of the equilibrium dialysis plate.

[0234] The prepared balanced dialysis plate was placed in a shaker at 37°C and shaken at 60 rpm for 5 hours.

[0235] 1.6 At the end of incubation (5 hours), sample preparation:

[0236] 1.6.1 Preparation of receiving side samples:

[0237] 25 μL of the sample from the receiving side was taken out and placed in a 96-well sample collection plate, and the same volume of matrix (blank plasma) was added and mixed.

[0238] 200 μL of ACN containing internal standard was added, and all samples (including those at 0 and 5 h) were vortexed for 10 min and then centrifuged at 5594 μg for 15 min.

[0239] 1.6.2 Preparation of test samples:

[0240] For the compound administration test samples provided in the examples of the present invention, 25 μL of the administration side sample was taken and mixed with 25 μL of blank buffer solution. 200 μL of ACN containing the internal standard was added, and the mixture was shaken at 600 rpm for 10 minutes, followed by centrifugation at 5594 g for 15 minutes in a Thermo, Multifuge × 3R centrifuge.

[0241] 1.6.3 Sample preparation:

[0242] Preparation of Example Compound Samples: 150 μL of supernatant was added to a new 96-well plate;

[0243] Preparation of 0-hour samples: The 0-hour samples were re-thawed at 37°C and the sample preparation was the same as that of the drug-administered side samples.

[0244] 1.6.4 All samples were centrifuged and sent for LC-MS / MS analysis.

[0245] 2. The experimental results are shown in Table 2 below.

[0246] Table 2 Test results of plasma protein binding rate of the compounds in the examples

[0247] Example compounds Human plasma protein binding rate (%) Canine plasma protein binding rate (%) Example 1 99.97 99.96 Example 2 99.95 99.96 Example 3 99.91 99.93 Example 4 99.89 99.92 Example 5 99.94 99.94 Example 6 99.98 99.89 Example 7 99.94 99.97 Example 8 99.92 99.89 Example 9 99.94 99.93 Example 10 99.96 99.95 Example 11 99.94 99.95 Example 12 99.96 99.97

[0248] Experimental results:

[0249] The human plasma protein binding rate and canine plasma protein binding rate of the experimental example compound of the present invention are both very high.

[0250] Experimental Example 3 In vivo drug efficacy experiment in diabetic mouse model

[0251] 1. Experimental purpose:

[0252] The hypoglycemic effect of the compound provided in the examples of the present invention on an animal diabetic model was studied and compared with the positive control, Retatrutide.

[0253] 2. Animal model selection:

[0254] The experimental animals were specific pathogen-free (SPF) male db / db mice. These mice exhibit symptoms similar to those of human type 2 diabetes, such as polyphagia, obesity, polydipsia, polyuria, hyperglycemia, and insulin resistance, making them an ideal model of type 2 diabetes.

[0255] Screened db / db test mice with a body weight of more than 30 g and a fasting blood glucose value greater than 25 mmol / L were formally included in the experiment.

[0256] 3. Grouping of experimental animals and drug intervention measures:

[0257] After one week of adaptive feeding for all mice, the db / db mice were randomly divided into a model blank group, a Retatrutide group, a compound of the present invention group, and normal mice as the control group. There were 8 mice in each group, and a total of five groups. All mice were administered with subcutaneous injections. The intervention measures for each group were as follows: (1) Control group: db / db mice were given normal saline and observed for four weeks; (2) Model blank group: db / db mice were given blank solvent (1M phosphate buffer pH 7.2-7.4) and observed for four weeks; (3) Retatrutide group: Retatrutide was administered at 10 nmol / kg once a week for four consecutive weeks; (4) Compound of the present invention group: Compound of the present invention was administered at 10 nmol / kg once a week for four consecutive weeks. The details are as follows:

[0258] Group Dose Dosing frequency Number of animals Control group Saline Qw 8 Model blank group Blank vehicle (1M phosphate buffer) Qw 8 Retatrutide group 10 nmol / kg Qw 8 Example 1 10 nmol / kg Qw 8 Example 2 10 nmol / kg Qw 8 Example 3 10 nmol / kg Qw 8 Example 6 10 nmol / kg Qw 8 Example 12 10 nmol / kg Qw 8

[0259] 4. Fasting blood glucose measurement:

[0260] Fasting blood glucose was measured by fasting mice for 12 hours prior to testing. Blood was then collected from the tail vein using a Roche glucometer (Active). Fasting blood glucose was measured on day 0 before dosing and on days 7, 14, 21, and 28 after dosing.

[0261] 5. Results and Analysis

[0262] During the experiment, the mice were in good health and mental state. The mice in the drug group had varying degrees of suppression in their food intake under the effects of the drug, and no mice died. Their fasting blood glucose and glycosylated hemoglobin levels were dynamically monitored during the experiment, and their fasting blood glucose values ​​were as follows:

[0263]

[0264]

[0265] The results of the change rate of glycosylated hemoglobin values ​​are as follows:

[0266]

[0267] Note: “-” represents a decrease

[0268] Conclusion: The compounds provided in the examples of the present invention all demonstrated significant hypoglycemic effects in diabetic mice. Specifically, the compounds provided in the examples of the present invention were comparable in reducing glycated hemoglobin in diabetic mice and significantly superior to retatrutide. The compounds provided in the examples of the present invention were also comparable in reducing fasting blood glucose in animals and significantly superior to retatrutide. Overall, it is concluded that the compounds provided in the examples of the present invention have superior hypoglycemic effects to retatrutide.

[0269] Experimental Example 4: In vivo pharmacokinetic study of compounds

[0270] Experimental Method 1: This experiment was designed to determine the oral pharmacokinetic parameters of the compound in beagle dogs. Each beagle dog was orally administered 10 mg of the test compound (each tablet containing 10 mg of the test compound, 300 mg of sodium N-(8-(2-hydroxybenzoyl)amino)octanoate (SNAC), and 7.7 mg of magnesium stearate). Beagles were provided by the Sichuan Musk Deer Breeding Research Institute.

[0271] Tablet preparation: After the test substance, SNAC and excipients are fully mixed, the mixture is added to a granulator for tableting to obtain tablets of the test substance.

[0272] Experimental process: Oral administration, collection of plasma for drug concentration analysis, and acquisition of relevant pharmacokinetic parameters.

[0273] The experimental results are as follows:

[0274] 1. PK parameters of the compounds in beagle dogs. We tested the blood concentrations of the test compounds. The results are shown in Table 3.

[0275] Table 3 Pharmacokinetic parameters of the test compounds in beagle dogs

[0276] Compound number Cmax (ng / mL) AUC (h*ng / mL) T1 / 2 (h) Example 1 153.4 6464 74.8 Example 2 146.3 6348 73.9 Example 3 148.3 6208 72.5 Example 6 150.5 6254 73.6 Example 12 275 15200 65.5

[0277] Note: Cmax is the maximum drug concentration in the body; AUC is the drug exposure in the body; T1 / 2 (h) is the half-life;

[0278] Experimental conclusion: The compound provided in the embodiment of the present invention has good absorption and exposure after oral administration, has a relatively long half-life, is suitable for oral administration, and can effectively reduce the frequency of administration.

[0279] 2. Compound Metabolism to Original Drug in Beagle Dogs For compound 12 in Example 12, we tested the plasma concentration of the original drug after each test compound was metabolized. The results are shown in Table 4.

[0280] Table 4 Pharmacokinetic parameters of compound 12 in beagle dogs

[0281] Compound number Cmax (ng / mL) AUC (h*ng / mL) T1 / 2 (h) Example 1 24.2 6450 74.7 Example 2 25.6 6341 73.8 Example 6 25.2 6795 73.2 Example 10 26.5 6857 74.3 Example 12 275 15200 65.5

[0282] Note: Cmax is the maximum drug concentration in the body; AUC is the drug exposure in the body; T1 / 2 (h) is the half-life;

[0283] Experimental conclusion: The compound provided in the embodiment of the present invention can be effectively metabolized into the original drug component Compound 12 in beagle dogs.

[0284] Pharmacokinetic experiments showed that the compounds embodied in the embodiments of the present invention were well orally absorbed in beagle dogs, and the prodrug was effectively metabolized to the original drug component, Compound 12. The compounds provided in the embodiments of the present invention metabolized the original drug component, Compound 12, to low plasma concentrations in beagle dogs, with a flat drug-dose curve and a long half-life, which can reduce drug toxicity and side effects at effective concentrations.

[0285] Experimental Method 2: The purpose of this experiment was to further determine the oral bioavailability of the compound in beagle dogs. Each beagle dog was intravenously administered 1.0 mg of the test compound. The beagle dogs were provided by the Sichuan Musk Deer Breeding Research Institute.

[0286] Preparation of injection: Dissolve the test substance in physiological saline and mix well.

[0287] Experimental process: Drug was administered by intravenous injection, plasma was collected to analyze drug concentration, and relevant pharmacokinetic parameters were obtained.

[0288] The experimental results are as follows:

[0289] 1. PK parameters of the compounds in beagle dogs. We tested the blood concentrations of the test compounds. The results are shown in Table 5 below:

[0290] Table 5 Pharmacokinetic parameters of the test compounds in beagle dogs

[0291]

[0292] Note: AUC is the drug exposure in vivo; T1 / 2 (h) is the half-life; F% is the bioavailability;

[0293] Experimental Conclusion: Compound 12 provided by the present invention has a high oral bioavailability (3.99%) and a half-life >65h upon oral administration, and can be used for the development of long-acting oral formulations. The prodrug of the present invention has a good oral bioavailability (1.39%) and a half-life >70h upon oral administration, and can be used for the development of long-acting oral formulations.

[0294] 2. Compound Metabolism to Original Drug Compound 12 of Example 12 in Beagle Dogs. We tested the plasma concentration of the original drug after each test compound was metabolized. The results are shown in Table 6 below:

[0295] Table 6 Pharmacokinetic parameters of compound 12 in beagle dogs

[0296]

[0297] Note: AUC is the drug exposure in vivo; T1 / 2 (h) is the half-life; F% is the bioavailability;

[0298] Experimental conclusion: After oral administration, the prodrug provided by the embodiment of the present invention is metabolized to the original drug with an oral bioavailability of 4.39% and a half-life of >70h. The compound of the embodiment of the present invention has excellent kinetic properties for oral administration and is suitable for the development of long-acting oral drugs.

[0299] In summary, the original drug compound 12 of the present invention has comparable agonist activity to Retatrutide at the GLP-1 receptor, GCGR receptor, and GIPR receptor, has a high plasma protein binding rate, is superior to Retatrutide in reducing blood glucose in vivo, and has excellent oral absorption. The prodrug compound of the present invention has lower agonist activity at the GLP-1 receptor, GCGR receptor, and GIPR receptor, has a high plasma protein binding rate, is superior to Retatrutide in reducing blood glucose in vivo, has excellent oral bioavailability, and can effectively release the original drug. The prodrug compound of the present invention controls the concentration of the original drug, reducing the blood concentration of the original drug while maintaining its efficacy, effectively reducing the toxic and side effects of the original drug.

[0300] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A polypeptide compound, characterized in that It is selected from the compounds represented by the following structural formula or pharmaceutically acceptable salts thereof: ， Among them, R1 represents , R3 and R4 are independently selected from any one of a carboxyl group, a tetrazolyl group and a phosphate group; n is any integer between 10 and 20; R a 、R b 、R c 、R d 、R e and R f Each independently selected from unsubstituted C1-C5 alkyl; AA1 means QGTFTSDYSI; The sequence of AA1 is shown in SEQ ID NO. 1; x represents any integer from 1 to 5; R2 represents , R7 represents any one of a carboxyl group, a tetrazolyl group and a phosphate group; p represents any integer between 10 and 20; m is 1 or 2; AA2 stands for AFIEYLLEGGPSSGAPPPS; The sequence of AA2 is shown in SEQ ID NO.

2.

2. The polypeptide compound according to claim 1, characterized in that n is any integer between 13 and 19.

3. The polypeptide compound according to claim 1, characterized in that n is 15, 16 or 17.

4. The polypeptide compound according to claim 1, characterized in that n is 15 or 17.

5. The polypeptide compound according to claim 1, characterized in that p is any integer between 16 and 18.

6. The polypeptide compound according to claim 1, characterized in that p is 16 or 18.

7. The polypeptide compound according to claim 1, characterized in that R a 、R b 、R c 、R d 、R e and R f are each independently selected from unsubstituted C1-C3 alkyl.

8. The polypeptide compound according to claim 1, characterized in that R a 、R c , and R f When both are methyl, R b 、R d and R e are each independently selected from unsubstituted C1-C5 alkyl.

9. The polypeptide compound according to claim 1, characterized in that R a 、R c , and R f When both are methyl, R b 、R d and R e are each independently selected from unsubstituted C1-C3 alkyl.

10. The polypeptide compound according to claim 1, characterized in that x represents 2-4.

11. The polypeptide compound according to claim 1, characterized in that x is 3.

12. The polypeptide compound according to any one of claims 1 to 11, characterized in that It is selected from any one of the compounds represented by the following structural formulas: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 or .

13. The polypeptide compound according to any one of claims 1 to 11, characterized in that It is selected from any one of the compounds represented by the following structural formulas: 、 、 、 、 、 、 、 、 、 、 or .

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