Polypeptide compound and application thereof

Abstract

The invention relates to the technical field of therapeutic polypeptides, in particular to a polypeptide compound and application thereof. The polypeptide compound is selected from a compound shown in the following structural formula or a chiral isomer, a tautomer, or pharmaceutically acceptable salt, ester or amide thereof: # imgabs0 #, R1 represents a hydrogen atom, # imgabs1 # Ra, Rb, Rc, Rd, Re and Rf are respectively and independently selected from unsubstituted C1-C5 alkyl; aA1 represents QGTFTSDYSI (Quantitative GTFTSDYSI) X represents any integer from 1 to 5; r < 2 > represents # imgabs < 2 > # AA2 represents AFIEYLLEGGPSSGAPPPS, R < 2 > represents # imgabs < 2 > # AA2; the polypeptide compound can be used as a novel GLP drug, and has good biological activity and oral bioavailability.

Description

[0001] The present invention claims the priority of Chinese patent application No.: 202411033707.2, entitled: Polypeptide compounds and their applications, and the contents recorded in the description and claims of the priority document are fully introduced into the description of the present invention and are regarded as part of the original record of the description of the present invention. Technical Field

[0002] The present invention relates to the technical field of therapeutic polypeptides, and in particular to polypeptide compounds and applications thereof. Background Art

[0003] In the 1980s, scientists discovered glucagon-like peptide-1 (GLP-1). In 2005, the GLP-1 analog exenatide was approved for marketing by the U.S. FDA, but it required twice-daily injections, resulting in poor patient compliance. In 2010, the FDA approved the world's second GLP-1 drug, liraglutide, for once-daily injection. In 2017, semaglutide was approved by the U.S. FDA for once-weekly injections. In 2023, the world's first dual-target agonist telpotide was approved by the FDA for once-weekly injections. In 2019, the FDA-approved oral semaglutide formulation Rybelsus (for diabetes) became the only approved oral GLP-1 drug in the world.

[0004] GLP-1 drugs have demonstrated great clinical demand and value, and are currently being actively expanded to include dozens of indications, which is expected to address more clinical needs.

[0005] After decades of development, GLP-1 drugs have more than a dozen on the market and hundreds of drugs are in clinical research, but most of them are injectables. Only the oral formulation of semaglutide, Rybelsus, is on the market and needs to be taken daily.

[0006] Development trends of GLP-1 drugs: 1. Long-acting. Long-acting GLP-1 drugs have achieved great sales success due to better patient compliance; 2. Oral administration. Oral administration is more convenient, 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. Multiple targets. Targets such as glucose-dependent insulinotropic polypeptide receptor (GIPR), glucagon receptor (GCGR), fibroblast growth factor 21 receptor (FG21R) can synergize with GLP-1 in the processes of blood sugar balance regulation, fat metabolism, food intake, etc., to improve the efficacy of the drug.

[0007] Currently, there is only one oral GLP-1 drug, which is a single-target drug that needs to be administered daily and has low compliance. Therefore, it is necessary to develop a longer-acting oral GLP-1 multi-target drug, such as one 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 purpose of the present invention is to provide a polypeptide compound and its application. The polypeptide compound provided in the embodiment of the present invention can be used as a new GLP-type drug, is a multi-target drug, can produce a synergistic effect, has 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 formula or their chiral isomers, tautomers, or pharmaceutically acceptable salts, esters or amides:

[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 in SEQ ID NO. 1;

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

[0018] R2 means 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; 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 described in any one of the aforementioned embodiments in the preparation of a medicament for treating any of the following diseases, the diseases including: obesity, diabetes, cardiovascular disease, chronic kidney disease, non-alcoholic fatty hepatitis, 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 diseases, heart failure, behavioral bowel syndrome, indigestion 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 adapt to a variety of diseases. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all 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 that is administered daily. Treatment regimens that reduce the frequency of dosing may improve patient convenience and compliance, and 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 less than once a day will be a great improvement in 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] The present invention provides a polypeptide compound, which is selected from the compounds represented by the following structural formula or their chiral isomers, tautomers, or pharmaceutically acceptable salts, esters or amides:

[0026]

[0027] Among them, R1 hydrogen atom, represents 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; for example, R3 is selected from -COOR5, -PO(OR6)2 and Any one of the functional groups formed; wherein R5 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; for example, methyl, ethyl, n-propyl and isopropyl, etc. Preferably, R5 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; more preferably, it is any one of a hydrogen atom, a methyl 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, such as a methyl group, an ethyl group, a 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, such as 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, such as 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 Each is independently selected from an unsubstituted C1-C5 alkyl group; for example, methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-butyl and other unsubstituted alkyl groups. Preferably, each is independently selected from an unsubstituted C1-C3 alkyl group. For example, R a , R c , and R f When both are methyl, R b , R d and R e R 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 alkyl group mentioned above may be used, or two different alkyl groups mentioned above 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 embodiments of the present invention.

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

[0038] R2 means 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 Any one of the functional groups formed; wherein R8 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; for example, methyl, ethyl, n-propyl and isopropyl, etc. Preferably, R8 is a hydrogen atom or an unsubstituted C1-C3 alkyl group; more preferably, it is any one of a hydrogen atom, a methyl 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, such as a methyl group, an ethyl group, a 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, it is any integer between 16 and 18; preferably, it is 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, it will not be described in detail in the embodiment of the present invention.

[0046] More specifically, amino acids except Aib2, Leu13, Lys17, Aib20 are depicted using standard single-letter amino acid codes:

[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 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 means 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 polypeptide compound. The preparation method can refer to the existing method. The present invention is illustrated by example. Specifically,

[0103] The present invention uses an Fmoc chemical method on a polypeptide synthesis column through SPPS to synthesize peptides. 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, and M4 are amidated and condensed in sequence, and after activation by the exposed carboxylic acid of M4, they are condensed with H-Tyr(tBu)-Aib-OH to obtain R1-Tyr(tBu)-Aib-OH. If R3 and R4 are carboxyl groups, the carboxyl groups are protected with a common protective agent (such as tert-butyl, methyl, ethyl, benzyl).

[0106]

[0107] Synthesis of Fmoc-Lys(R2)-OH: M5, HL-Glu-OtBu, and AEEA are sequentially amidated and condensed, and after activation with AEEA exposed carboxylic acid, condensed with Fmoc-Lys-OH to obtain Fmoc-Lys(R2)-OH. If R7 is -COOH or -PO(OH)2, the carboxyl group is protected with a common protecting agent (such as tert-butyl, methyl, ethyl, benzyl).

[0108]

[0109] The resin used was 1% DVB cross-linked 2-Chlorotrityl chloride resin from Tianjin Nankai Hecheng Co., Ltd., 100-200 mesh, and the degree of substitution was 0.4-0.6mmol / g. Fmoc-Lys(R2)-OH) was used for lysine at position 17, and R1-Tyr(tBu)-Aib was used for tyrosine 2-aminoisobutyric acid at positions 1 and 2. The activated 2-Chlorotrityl chloride resin was first coupled with Fmoc-Ser(tBu)-OH, where the molar ratio of 2-Chlorotrityl chloride resin to Fmoc-Ser(tBu)-OH and DIPEA was 1:1.1:4. Before each subsequent coupling step, 20% piperidine / DMF was used to remove the Fmoc group for 2x 10min. All standard amino acid couplings used 2 times the molar ratio of Fmoc protected amino acids, 3 times the molar ratio of diisopropylcarbodiimide and 3 times the molar ratio of 1-hydroxybenzotriazole, and were coupled under nitrogen protection for 2 to 3 hours, wherein the degree of coupling was detected by ninhydrin colorimetric reagent. In some cases, the coupling time was increased or the coupling steps were 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] Cutting:

[0111] The synthesized dry peptide resin was cleaved with 10 volumes of cleavage mixture (trifluoroacetic acid: water: triisopropylsilane, 95:2.5:2.5 v / v) at room temperature for 2 hours. The resin was filtered out and washed twice with 2 volumes of pure TFA each time, and the combined filtrate was treated with 5 times of cold ether (-20°C) to precipitate the crude peptide. The crude peptide / ether suspension was then centrifuged at 3500 rpm for 10 minutes, the supernatant was removed, and the solid was washed twice with ether and dried in a vacuum to obtain 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. The elution was performed with a gradually increasing gradient of MeCN in MQ water containing 0.1% TFA. The relevant fractions were analyzed by UPLC. The fractions containing the pure target peptide were combined. The resulting solution was analyzed (UPLC, LCMS) and the product was dispensed into glass vials. The vials were capped with Millipore glass fiber prefilters. Freeze drying gave the target compound as a white solid.

[0114] General LCMS method:

[0115] Mass spectrometry (MS) parameters:

[0116] Detection instrument: Agilent 6410 triple quadrupole liquid spectrometer; ionization source: +ESI.

[0117] Liquid phase parameters:

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

[0119] The embodiments of the present invention also provide the use of the above-mentioned polypeptide compound in the preparation of a drug for treating any of the following diseases, the diseases including: obesity, diabetes, cardiovascular disease, chronic kidney disease, non-alcoholic fatty hepatitis, 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 diseases, heart failure, speech and behavior bowel syndrome, indigestion and gastric ulcer.

[0120] The names of the compounds or raw materials used in the preparation methods of the embodiments of the present invention 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 in conjunction with 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, 5.00g of the raw material compound 1-A1 (13.51mmol) was added to a 100ml reaction bottle, 25ml of dichloromethane was added, and then 1.71g of HOSu (14.86mmol) was added, and 3.10g of EDCl.HCL (16.22mmol) was slowly added at 10°C. After the addition was completed, the reaction was kept warm for 1h. The reaction was observed by TLC spot plate. 2.27g of N, N-diisopropylethylamine (17.56mmol) and 2.88g of compound 1-A2 (14.19mmol) were added, and the reaction was naturally returned to room temperature for 3h. TLC monitoring showed that the reaction was complete, and the mixture was washed twice with 1mol / L hydrochloric acid water, washed once with 5% brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure at 40-45°C until there was no fraction, to obtain 36.84g of compound 1-A, with a yield of 91.2%.

[0129] 5.00g of compound 1-A3 (9.0mmol) was added to a 100ml reaction bottle, 25ml of dichloromethane was added, and then 1.14g of HOSu (9.9mmol) was added. 2.07g of EDCl.HCL (10.8mmol) was slowly added at 10℃. After the addition was completed, the reaction was kept warm for 1h. The reaction was observed by TLC spot plate. 1.51g of N,N-diisopropylethylamine (11.71mmol) and 2.73g of compound 1-A4 (9.9mmol) were added. The reaction was naturally returned to room temperature for 3h. The reaction was monitored by TLC. The reaction was complete, and the mixture was washed twice with 1mol / L hydrochloric acid water and once with 5% brine. The mixture was dried over anhydrous magnesium sulfate and concentrated under reduced pressure at 40-45℃ until there was no fraction. 6.75g of compound 1-A5 was obtained with a yield of 92.3%.

[0130] Add 5.00 g of compound 1-A5 (6.15 mmol) as the raw material into a 100 ml reaction bottle, add 25 ml of dichloromethane, then add 0.78 g of HOSu (6.78 mmol), control the temperature at 10°C and slowly add 1.41 g of EDCl.HCL (7.36 mmol). After the addition is complete, keep the temperature for 1 hour. The reaction is complete as observed by TLC spot plate. Concentrate under reduced pressure at 40-45°C until there is no fraction to obtain 5.51 g of compound 1-A6 with a yield of 98.4%.

[0131] Add 5.00g of compound 1-A6 (5.49mmol) into a 100ml reaction bottle, add 25ml of dichloromethane, add 0.92g of N,N-diisopropylethylamine (7.14mmol), 1.97g of compound H-Tyr(tBu)-Aib-OH (6.1mmol), and naturally return to room temperature to react for 3h. TLC monitoring, the reaction is complete, wash with 1mol / L hydrochloric acid water twice, wash with 5% brine once, dry with anhydrous magnesium sulfate, and concentrate under reduced pressure at 40-45℃ until there is no fraction, to obtain 5.65g 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] Add 7.54g of compound 1-B1 (13.51mmol) of the raw material to a 100ml reaction bottle, add 30ml of dichloromethane, then add 1.71g of HOSu (14.86mmol), control the temperature for 10 seconds, slowly add 3.10g of EDCl.HCL (16.22mmol), after the addition, keep warm for 1h, observe the TLC plate, the reaction is complete, add 2.27g of N,N-diisopropylethylamine (17.56mmol), 2.88g of compound 1-A2 (14.19mmol), return to room temperature and react for 3h. Monitor the reaction by TLC, the reaction is complete, wash twice with 1mol / L hydrochloric acid water, wash once with 5% brine, dry with anhydrous magnesium sulfate, and concentrate under reduced pressure at 40-45℃ until there is no fraction, to obtain 9.22g of compound 1-B2, with a yield of 91.7%.

[0135] 6.69g of compound 1-B2 (9.0mmol) was added to a 100ml reaction bottle, 30ml of dichloromethane was added, and then 1.14g of HOSu (9.9mmol) was added. The temperature was controlled at 10 °C and 2.07g of EDCl.HCL (10.8mmol) was slowly added. After the addition was completed, the reaction was kept warm for 1h. The reaction was observed by TLC. 1.51g of N,N-diisopropylethylamine (11.71mmol) and 1.62g of compound AEEA (9.9mmol) were added. The reaction was naturally returned to room temperature for 3h. The reaction was monitored by TLC. The reaction was complete. The mixture was washed twice with 1mol / L hydrochloric acid water and once with 5% brine. The mixture was dried over anhydrous magnesium sulfate and concentrated under reduced pressure at 40-45 °C until there was no fraction. 7.42g of compound 1-B3 was obtained with a yield of 92.8%.

[0136] Add 5.49g of compound 1-B3 (6.15mmol) of the raw material to a 100ml reaction bottle, add 30ml of dichloromethane, then add 0.78g of HOSu (6.78mmol), control the temperature for 10 seconds, slowly add 1.41g of EDCl.HCL (7.36mmol), after the addition, keep warm for 1h, observe the TLC plate, the reaction is complete, add 1.32g of N,N-diisopropylethylamine (8.00mmol), 2.49g of compound Fmoc-Lys-OH (6.76mmol), return to room temperature and react for 3h. Monitor the reaction by TLC, the reaction is complete, wash twice with 1mol / L hydrochloric acid water, wash once with 5% brine, dry with anhydrous magnesium sulfate, and concentrate under reduced pressure at 40-45℃ until there is no fraction, to obtain 7.12g of compound 1-B, with a yield of 93.5%.

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

[0138]

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

[0140] Compound 1 was characterized by: LCMS

[0141] Calculated mass: (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 by referring 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 mass: (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 mass: (M+3) / 3=1795.94, (M+4) / 4=1347.46, (M+5) / 5=1077.97.

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

[0157] Embodiment 4:

[0158] Compound 4 has the following structure:

[0159]

[0160] LCMS

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

[0162] Embodiment 5:

[0163] Compound 5 has the following structure:

[0164]

[0165] LCMS

[0166] Calculated mass: (M+3) / 3=1805.62, (M+4) / 4=1354.46, (M+5) / 5=1083.57. Measured mass: (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 mass: (M+3) / 3=1774.94, (M+4) / 4=1331.20, (M+5) / 5=1065.37. Measured mass: (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 mass: (M+3) / 3=1786.93, (M+4) / 4=1340.20, (M+5) / 5=1072.36. Measured mass: (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 mass: (M+3) / 3=1794.94, (M+4) / 4=1346.20, (M+5) / 5=1077.16. Measured mass: (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 mass: (M+3) / 3=1782.61, (M+4) / 4=1337.46, (M+5) / 5=1070.17. Measured mass: (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 mass: (M+3) / 3=1784.28, (M+4) / 4=1338.46, (M+5) / 5=1070.97. Measured mass: (M+3) / 3=1784.35, (M+4) / 4=1338.49, (M+5) / 5=1070.98.

[0192] Embodiment 11

[0193] Compound 11 has the following structure:

[0194]

[0195] LCMS

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

[0197] Measured mass: (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 mass: (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 test uses 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 by the reporter gene method.

[0208] 2. Experimental process:

[0209] 1). The compound provided in the examples of the present invention was prepared into a working concentration of 40 nM, and was diluted 5-fold in a series 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 a 96-well cell plate.

[0211] 3). According to the experimental plate layout, add the gradient diluted samples to the 96-well cell culture plate with 50μ1 wells, place them in a carbon dioxide incubator at 37°C and 5% CO, incubate for a period of time, add the fluorescent reagent, and read the fluorescence value on the microplate reader. Use GraphPadPrism to fit the curve 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 which 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 solution. (The final DMSO concentration in this solution is 20%)

[0227] 1.4 Preparation of the compound administration 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 Collecting 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 37°C shaker 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 0 and 5 hours) were vortexed for 10 minutes and then centrifuged at 5594 μg for 15 minutes.

[0239] 1.6.2 Preparation of test samples:

[0240] The compound administration test sample provided in the embodiment of the present invention: 25 μL of the administration side sample was taken, 25 μL of blank buffer solution was added and mixed, 200 μL of ACN containing internal standard was added, and the mixture was shaken at 600 rpm for 10 minutes, and then centrifuged at 5594g for 15 minutes in a centrifuge (Thermo, Multifuge×3R).

[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 administration 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 Embodiment 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 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 the 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, and are an ideal model of type 2 diabetes.

[0255] The 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 screened and formally included in the experiment.

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

[0257] After all mice were fed adaptively for one week, the db / db mice were randomly divided into a model blank group, a Retatrutide group, a compound group of the present invention, and normal mice as a control group. There were 8 mice in each group, and a total of five groups were administered subcutaneously. 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 pH7.2-7.4) and observed for four weeks; (3) Retatrutide group: Retatrutide was given, 10nmol / kg, once a week, for four consecutive weeks; (4) Compound group of the present invention: Compound of the present invention was given, 10nmol / kg, once a week, for four consecutive weeks. The details are as follows:

[0258] Group dose Dosing frequency Number of animals Control group Normal saline QUR 8 Model Blank Group Blank solvent (1M phosphate buffer) QUR 8 Retatrutide Group 10nmol / kg QUR 8 Example 1 10nmol / kg QUR 8 Example 2 10nmol / kg QUR 8 Example 3 10nmol / kg QUR 8 Example 6 10nmol / kg QUR 8 Example 12 10nmol / kg QUR 8

[0259] 4. Fasting blood glucose measurement:

[0260] The fasting blood glucose determination method is that the mice fasted for 12 hours before the test, but not water, and then blood was collected from the tail vein using a Roche blood glucose meter (active type) for blood glucose determination. The fasting blood glucose was measured on the 0th day before administration and on the 7th, 14th, 21st, and 28th days after administration.

[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 action of the drug, and no mice died. During the experiment, their fasting blood glucose and glycosylated hemoglobin values ​​were dynamically monitored, 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 showed good hypoglycemic effects on diabetic mice. Specifically, the compounds provided in the examples of the present invention were similar in reducing glycosylated hemoglobin in diabetic model mice and were significantly better than Retatrutide. The compounds provided in the examples of the present invention were similar in reducing fasting blood glucose in animals and were better than Retatrutide. It is generally believed that the compounds provided in the examples of the present invention have a hypoglycemic effect that is better than Retatrutide.

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

[0270] Experimental method 1: The purpose of this experiment is to determine the oral pharmacokinetic parameters of the compound in beagle dogs. Each beagle dog is orally administered with 10 mg of the test compound (each tablet contains 10 mg of the test drug, 300 mg of sodium N-(8-(2-hydroxybenzoyl)amino)caprylate (SNAC) and 7.7 mg of magnesium stearate). Beagle dogs were provided by 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 No. 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 oral absorption and exposure, has a relatively long half-life, is suitable for oral administration, and can effectively reduce the frequency of administration.

[0279] 2. Compounds are metabolized to the original drug in beagle dogs For compound 12 in Example 12, we tested the blood concentration of the original drug after each test substance was metabolized. The results are shown in Table 4.

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

[0281] Compound No. 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 effectively metabolize the original drug ingredient compound 12 in the beagle dog.

[0284] Through pharmacokinetic experiments, the compounds of the embodiments of the present invention have good oral absorption in beagle dogs, and the prodrug can be effectively metabolized to the original drug component compound 12. The compounds provided in the embodiments of the present invention metabolize the original drug component compound 12 in beagle dogs with low blood drug concentration, flat drug-time curve, long half-life, and can reduce the toxic and side effects of the drug at effective concentrations.

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

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

[0287] Experimental process: Drugs were 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 tested compounds. The results are shown in Table 5:

[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 in the embodiment of the present invention has a high oral bioavailability (3.99%) and a half-life of >65h, and can be used for the development of long-acting oral preparations. The prodrug of the present invention has a good oral bioavailability (1.39%) and a half-life of >70h, and can be used for the development of long-acting oral preparations.

[0294] 2. Compounds metabolized to original drug in beagle dogs For compound 12 in Example 12, we tested the blood concentration of the original drug after each test substance 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 in 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 the same agonist activity as Retatrutide on GLP-1 receptor, GCGR receptor, and GIPR receptor, and the agonist activity is equivalent, has a very high plasma protein binding rate, is superior to Retatrutide in reducing blood sugar in vivo, and has good oral absorption; the prodrug compound of the present invention has a lower agonist activity on GLP-1 receptor, GCGR receptor, and GIPR receptor, has a very high plasma protein binding rate, is superior to Retatrutide in reducing blood sugar in vivo, has good oral bioavailability, and can effectively release the original drug. The prodrug compound of the present invention controls the concentration of the original drug, and can reduce the blood concentration of the original drug while ensuring the efficacy, and can effectively reduce the toxic and side effects of the original drug.

[0300] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide compound, characterized in that: It is selected from the compounds represented by the following structural formulas or their chiral isomers, tautomers, or pharmaceutically acceptable salts, esters or amides: 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; 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 listing of AA1 is shown in SEQ ID NO. 1; x represents any integer from 1 to 5; R2 means 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; m represents any value between 0 and 2; AA2 stands for AFIEYLLEGGPSSGAPPPS; The sequence listing of AA2 is shown in SEQ ID NO.

2.

2. The polypeptide compound according to claim 1, characterized in that R3 is selected from -COOR5, -PO(OR6)2 and Any one of the functional groups formed; wherein R5 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; R6 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; R 10 is any one of a hydrogen atom, an unsubstituted C1-C3 alkyl group, a metal ion and an ammonium cation; Preferably, R5 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, a hydrogen atom; Preferably, R6 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; Preferably, R 10 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 any one of a hydrogen atom, a methyl group, an ethyl group, and a monovalent metal ion; further preferably any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion, and a potassium ion; most preferably any one of a hydrogen atom, a methyl group, and an ethyl group; Preferably, 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; R 13 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; R 14 is any one of a hydrogen atom, a C1-C3 alkyl group, a metal ion, and an ammonium cation; Preferably, R 12 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; Preferably, R 13 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; Preferably, R 14 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 any one of a hydrogen atom, a methyl group, an ethyl group, and a monovalent metal ion; further preferably any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion, and a potassium ion; most preferably any one of a hydrogen atom, a methyl group, and an ethyl group; Preferably, n is any integer between 12 and 20; preferably any integer between 13 and 19; more preferably 15, 16 or 17, most preferably 15 or 17; Preferably, R7 is -COOR8, -PO(OR9)2 and Any one of the functional groups formed; wherein R8 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; R9 is a hydrogen atom, a substituted or unsubstituted C1-C3 alkyl group; R 11 is any one of a hydrogen atom, a C1-C3 alkyl group, a metal ion, and an ammonium cation; Preferably, R8 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; Preferably, R9 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, a hydrogen atom; 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 any one of a hydrogen atom, a methyl group, an ethyl group, and a monovalent metal ion; further preferably any one of a hydrogen atom, a methyl group, an ethyl group, a sodium ion, and a potassium ion; most preferably any one of a hydrogen atom, a methyl group, and an ethyl group; Preferably, p is any integer between 12 and 20; preferably any integer between 16 and 18; preferably 16 or 18; Preferably, m is 1 or 2; Preferably, R a , R b , R c , R d , R e and R f are independently selected from unsubstituted C1-C3 alkyl groups, Preferably, R a , R c , and R f When both are methyl, R b , R d and R e Each is independently selected from an unsubstituted C1-C5 alkyl group, preferably, each is independently selected from an unsubstituted C1-C3 alkyl group; Preferably, x represents 2-4, preferably 3.

3. The polypeptide compound according to claim 1 or 2, characterized in that: 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; 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; 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-C3 alkyl; x represents any integer from 2 to 4; R2 is in, 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; p is any integer between 10 and 20; m is 0, 1 or 2.

4. The polypeptide compound according to claim 1 or 2, characterized in that: 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; 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; n is any integer between 12 and 20; 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; x represents any integer from 2 to 4; R2 is R7 is -COOR8, -PO(OR9)2 and Base form; R 11 The functional group is any substituted hydrogen atom in the C1-C3 alkyl group; R8 is any substituted hydrogen atom in the C1-C3 alkyl group; R is any substituted ethyl amine, C1-ethanol, C3 amine, alkyl group; 9 It is either an amino acid or a C1-C3 alkane that can form a cation; p is any integer between 12 and 20; m is 0, 1 or 2.

5. The polypeptide compound according to claim 1 or 2, characterized in that: R1 is a hydrogen atom, Wherein, R3 is -COOR5, -PO(OR6)2 and Any one of the functional 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; 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 13 is 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; n is any integer between 13 and 19; 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; x represents any integer from 2 to 4; 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; p is any integer between 16 and 18; m is 0, 1 or 2.

6. The polypeptide compound according to claim 1 or 2, characterized in that: 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; 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; n is any one of 15, 16 or 17; 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; x means 3 or 4; R2 is Wherein R7 is -COOH, -PO(OH)2 or 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; p is any one of 16, 17 or 18; m is any one of 0, 1 or 2.

7. The polypeptide compound according to claim 1 or 2, characterized in that: 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; R4 is -COOH, -PO(OH)2 or Any one of the functional groups formed; R 14 is a hydrogen atom or a methyl group; n is 15 or 17; R a , R c , and R f When both are methyl, R b and R e Methyl or ethyl, R d isobutyl; x represents 3; R2 is Wherein R7 is -COOH, -PO(OH)2 or Any one of the functional groups formed; R 11 is a hydrogen atom or a methyl group; p is 16 or 18; m is 1 or 2.

8. The polypeptide compound according to claim 1 or 2, characterized in that: It is selected from any one of the compounds represented by the following structural formulas:

9. The polypeptide compound according to claim 1 or 2, characterized in that: It is selected from any one of the compounds represented by the following structural formulas:

10. Use of the polypeptide compound according to any one of claims 1 to 9 in the preparation of a drug for treating any of the following diseases, characterized in that: Diseases include: 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, cardiac arrhythmias, diabetic nephropathy, osteoarthritis, chronic kidney disease, hypertension, cognitive impairment, coronary heart disease, stroke, neurodegenerative disorders, heart failure, behavioral bowel syndrome, dyspepsia, and gastric ulcer.

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