GIP / GLP-1 / GCG triple agonist and application thereof

Abstract

The invention provides a polypeptide compound with a sequence shown as a formula Z or a pharmaceutically acceptable salt thereof, the series of polypeptides act on three targets of GLP-1, GIP and GCG, and the polypeptide compound has a prospect of being developed into drugs for preventing or treating metabolic diseases. The formula Z is as follows: YX2QGT FTSDY SIX13X14D KX17AQX20X21 FIEX25X26X27X28X29G PSSX34A PPPS0, and the formula Z is shown in the description.

Description

Technical Field

[0001] The present invention relates to a series of polypeptides and applications thereof, and in particular, to a series of polypeptide compounds having relatively good agonistic activity on GLP-1R / GIPR / GCGR and applications thereof. Background Art

[0002] Obesity is a common chronic disease, often accompanied by various health problems or serious complications, which increases the risk of heart disease, stroke, diabetes, musculoskeletal diseases, cancer and other diseases. The World Obesity Federation predicts that by 2035, the world's overweight population will reach 4 billion, accounting for 51% of the total population. Solving the obesity problem is crucial to achieving global health goals, but there are still few available drugs.

[0003] Glucagon-like peptide-1 (GLP-1) and insulinotropic peptide (GIP) are incretins, and glucagon (GCG) is secreted by pancreatic α cells. The aforementioned substances can directly or indirectly affect the body's glucose metabolism and / or lipid metabolism. LY3437943 (Retatrutide) developed by Eli Lilly, as a GIP / GLP-1 / GCG triple receptor agonist, can regulate the secretion of insulin and gastrointestinal hormones by simulating the activity of GLP-1 and GCG, thereby achieving the effects of lowering blood sugar, increasing gastric satiety and reducing weight. This multi-target treatment method provides a new treatment option for metabolic diseases.

[0004] However, there are relatively few studies on new GLP-1 / GIP / GCG triple agonists, and no GLP-1R / GCGR / GIPR triple-target agonist drugs have been marketed so far.

[0005] International patent WO2023088140 discloses a variety of stapled peptides, and the different positions of the stapled modifications have different agonist activities on GLP-IR / GIPR / GCG, especially in terms of the agonist activity of GCG. China's patent application CN202410061582.8 studied a variety of stapled peptides with different sequences and different stapled structures, and recorded that a variety of polypeptides showed significant weight loss, blood sugar and blood lipid lowering effects.

[0006] Regarding the design of GLP-1R / GCGR / GIPR triple-target agonist molecules, although innovations have been introduced by introducing "staple"-like structural modifications, the molecular structure of the polypeptide has an important influence on its activity, efficacy and pharmacokinetic properties. Even a single change in the polypeptide sequence, such as the peptide amino acid sequence, stapling position, etc., may lead to significant changes in its activity at different targets.

[0007] It can be seen from this that there is still a need to continue to find more compounds that act on the GLP-1R / GIPR / GCGR targets and provide more polypeptide compounds with different structures that have three-target activity capabilities, in order to provide more therapeutic drug options in the field of metabolic diseases and fully demonstrate the advantages of multi-target drugs. Summary of the invention

[0008] The present invention aims to solve the deficiencies of the prior art and provides a series of polypeptides, which show good effects on GLP-1 receptor (GLP-1R), GIP receptor (GIPR) and GCG receptor (GCGR) in in vitro experiments and have good drug development prospects.

[0009] The object of the present invention is to provide a polypeptide compound having a sequence shown in Formula Z or a pharmaceutically acceptable salt thereof:

[0010] YX2QGT FTSDY SIX 13 X 14 D K 17 AQX 20 X 21 FIEX 25 X 26 X 27 X 28 X 29 GPSSX 34 APPPS0

[0011] Formula Z (SEQ ID NO: 1)

[0012] in:

[0013] X2 is Aib; X 13 is α-MeL (α-methyl substituted leucine); X 14 K or L; X 17 is K or dK (D-lysine); X 20 Aib;X 21 A or K; X 25 Y, F or dF (D-phenylalanine); X 26 is L or α-MeL; X 27 is L, α-MeL, or NLe (norleucine); X 28 is E or K; X 29 G or Aib; X 34 is G or Aib;

[0014] S0 is selected from (i.e., the C-terminal amino acid is optionally amidated to form a C-terminal primary amide);

[0015] And the compound has the following modifications:

[0016] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure similar to a "staple" structure, wherein i is independently selected from 14 or 17, and j is independently selected from 2, 3 and 4,

[0017] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connection; X a Selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0018] X3 is selected from

[0019] m is selected from 1, 2 and 3; p is selected from 1 and 2; n is selected from 8, 9 and 10.

[0020] In some embodiments of the present invention, when the modified amino acid position i in the sequence of formula Z is 14, then X 14 K, X 21 is A, and other variables are as defined in the present invention.

[0021] In some embodiments of the present invention, when the modified amino acid position i in the sequence of formula Z is 17, then X 14 L, X 21 is K, and other variables are as defined in the present invention.

[0022] In some embodiments of the present invention, when i is 14, j is 2, i+j is 16, and other variables are defined as in the present invention.

[0023] In some embodiments of the present invention, when i is 14, j is 3, i+j is 17, and other variables are defined as in the present invention.

[0024] In some embodiments of the present invention, when i is 17, j is 4, i+j is 21, and other variables are defined as in the present invention.

[0025] In some embodiments of the present invention, in the sequence shown in formula Z, X 27 is NLe, and X 34 is Aib, and other variables are as defined in the present invention.

[0026] The present invention provides a polypeptide compound having a sequence shown in formula ZI or a pharmaceutically acceptable salt thereof:

[0027] YX2QGT FTSDY SIX 13 KD KKAQX 20 AFIEX 25LX 27 EX 29 GPSSX 34 APPPS0

[0028] (ZI) (SEQ ID NO: 2)

[0029] Where: X2 is Aib; X 13 is α-MeL (α-methyl substituted leucine); X 20 Aib;X 25 is Y or F; X 27 is L, α-MeL, or NLe (norleucine); X 29 G or Aib; X 34 is G or Aib;

[0030] S0 is selected from (i.e., the C-terminal amino acid is optionally amidated to form a C-terminal primary amide);

[0031] And the compound has the following modifications:

[0032] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure similar to a "staple" structure, wherein i is 14, and j is independently selected from 2, 3,

[0033] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connection; X a Selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0034] X3 is selected from

[0035] m is selected from 1, 2 and 3; p is selected from 1 and 2; n is selected from 8, 9 and 10.

[0036] In some embodiments of the present invention, the formula ZI is further preferably a sequence shown in formula Z-II:

[0037] YX2QGT FTSDY SIX 13 KD KKAQX 20 AFIEX 25 LX 27 EX 29 GPSSX 34 A PPPS-NH2

[0038] (Z-II)

[0039] That is, S0 is (Amino acid C-terminally amidated at position 39), X 20 Aib;X 25 is Y or F; X 27 is L, α-MeL, or NLe (norleucine); X 29 G or Aib; X 34 is G or Aib; other variables are as defined in the present invention.

[0040] In some embodiments of the present invention, in the sequence shown in formula ZI, the above X 25 is Y, and other variables are as defined in the present invention.

[0041] In some embodiments of the present invention, in the sequence shown in formula ZI, the above X 27 is NLe, and other variables are as defined in the present invention.

[0042] In some embodiments of the present invention, in the sequence shown in formula ZI, the above X 27 is NLe, and X 29 is Aib, and other variables are as defined in the present invention.

[0043] In some embodiments of the present invention, in the sequence shown in formula ZI, the above X 27 is NLe, and X 34 is Aib, and other variables are as defined in the present invention.

[0044] In some embodiments of the present invention, the polypeptide of the sequence shown in formula ZI or a pharmaceutically acceptable salt thereof has the following sequence:

[0045] YAibQGT FTSDY SIα-MeLKD KKAQAib AFIEY LNLeEGGPSSGAPPPS-NH2

[0046] YAibQGT FTSDY SIα-MeLKD KKAQAib AFIEY LNLeEGGPSSAibAPPPS-NH2

[0047] YAibQGT FTSDY SIα-MeLKD KKAQAib AFIEY LNLeEAibGPSSGA PPPS-NH2

[0048] Other variables are as defined in the present invention.

[0049] In some embodiments of the present invention, the polypeptide of the sequence represented by formula ZI or a pharmaceutically acceptable salt thereof may be selected from SEQ ID NO: 4-6:

[0050] YAibQGT FTSDY SIα-MeL K DK K AQAib AFIEY LNLeEGGPSSGA PPPS-NH2 (SEQ IDNO: 4)

[0051] YAibQGT FTSDY SIα-MeL K DK K AQAib AFIEY LNLeEGGPSSAibA PPPS-NH2 (SEQ IDNO: 5)

[0052] YAibQGT FTSDY SIα-MeL K DK K AQAib AFIEY LNLeEAibGPSSGA PPPS-NH2 (SEQ IDNO: 6)

[0053] The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure similar to a "staple" structure, where i is 14 and j is 3, that is, the underlined K refers to the modified amino acid, and the other variables are as defined in the present invention.

[0054] The present invention provides a polypeptide compound having a sequence shown in formula Z-III or a pharmaceutically acceptable salt thereof:

[0055] YX2QGT FTSDY SIX 13 LD KX 17 AQX 20 KFIEX 25 LX 27 X 28 X 29 GPSSX 34 APPPS0

[0056] (Z-III) (SEQ ID NO: 3)

[0057] Where: X2 is Aib; X 13 is α-MeL (α-methyl substituted leucine); X 17 is K, dK (D-lysine); X 20 Aib;X 25 Y, F or dF (D-phenylalanine); X 27 is L, I or NLe (norleucine); X 28 is E or K; X 29 G or Aib; X 34 is G or Aib;

[0058] S0 is selected from (i.e., the C-terminal amino acid is optionally amidated to form a C-terminal primary amide);

[0059] And the compound has the following modifications:

[0060] 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure similar to a "staple" structure, where i is 17, j is 4,

[0061] 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connected;

[0062] X a Selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid;

[0063] X3 is selected from

[0064] m is selected from 1, 2 and 3; p is selected from 1 and 2; n is selected from 8, 9 and 10.

[0065] In some embodiments of the present invention, the above polypeptide compound or a pharmaceutically acceptable salt thereof, Formula Z-III is further preferably a sequence shown in Formula Z-IV:

[0066] YX2QGT FTSDY SIX 13 LD KX 17 AQX 20 KFIEX 25 LX 27 X 28 X 29 GPSSX 34 A PPPS-NH2

[0067] (Z-IV)

[0068] That is, S0 is (C-terminally amidated amino acid at position 39), and other variables are as defined in the present invention.

[0069] In some embodiments of the present invention, in the sequence shown in formula Z-III, the above X 27 is L or NLe, and other variables are as defined in the present invention.

[0070] In some embodiments of the present invention, in the sequence shown in formula Z-III, the above X 28 is E, and other variables are as defined in the present invention.

[0071] In some embodiments of the present invention, in the sequence shown in formula Z-III, the above X 27 is NLe, and X 34 is Aib, and other variables are as defined in the present invention.

[0072] In some embodiments of the present invention, the polypeptide of the sequence shown in formula Z-III or a pharmaceutically acceptable salt thereof can be selected from SEQ ID NO: 7-12:

[0073] YAibQGT FTSDY SIα-MeLLD K K AQ K FIEF LLEGGPSSAibAPPPS-NH2 (SEQ ID NO: 7)

[0074] YAibQGT FTSDY SIα-MeLLD K dK AQ K FIEF LLEGGPSSGA PPPS-NH2 (SEQ ID NO: 8)

[0075] YAibQGT FTSDY SIα-MeLLD K K AQ K FIEY LNLeEGGPSSAibA PPPS-NH2 (SEQ IDNO: 9)

[0076] YAibQGT FTSDY SIα-MeLLD K K AQ K FIEdF LNLeEGGPSSAibA PPPS-NH2 (SEQ IDNO: 10)

[0077] YAibQGT FTSDY SIα-MeLLD K K AQ K FIEF LNLeEAibGPSSGA PPPS-NH2 (SEQ IDNO: 11)

[0078] YAibQGT FTSDY SIα-MeLLD K K AQ K FIEY LNLeEGGPSSGA PPPS-NH2 (SEQ IDNO: 12)

[0079] Among them, the amino acid side chains at positions i and i+j in the sequence can be connected by modification to form a modified structure similar to a "staple" structure, where i is 17 and j is 4, i.e., the underline mark in the sequence K or dK refers to the modified amino acid position, and the other variables are as defined in the present invention.

[0080] In some embodiments of the present invention, the above m is selected from 1 and 2, and other variables are as defined in the present invention.

[0081] In some embodiments of the present invention, the above p is selected from 1, and other variables are as defined in the present invention.

[0082] In some embodiments of the present invention, the above n is selected from 9, and other variables are as defined in the present invention.

[0083] In some embodiments of the present invention, the above X3 is selected from

[0084] In some embodiments of the present invention, the above X3 is selected from

[0085] In some embodiments of the present invention, the above structural unit Selected from:

[0086] In some embodiments of the present invention, the above structural unit For S1:

[0087] In some embodiments of the present invention, the above structural unit For S2:

[0088] In some embodiments of the present invention, the above structural unit For S3:

[0089] Some other solutions of the present invention are obtained by any combination of the above variables.

[0090] In some embodiments of the present invention, the "staple" structure in the above polypeptide is shown in the following structure, and other variables are as defined in the present invention:

[0091]

[0092]

[0093] In some embodiments of the present invention, the above polypeptides are shown in Table 1 below:

[0094] Table 1 Structure of polypeptide compounds 1-6 of the present invention

[0095]

[0096] Underlined amino acids K Refers to the modified amino acid, the amino or carboxyl groups on the side chains of the two amino acid unit structures in the sequence are Condensation connects to form a modification group similar to a "staple" structure.

[0097] In some specific embodiments of the present invention, the unfolded structure of the above polypeptide compound is as follows:

[0098]

[0099]

[0100] In some embodiments of the present invention, the "staple" structure in the above polypeptide is shown in the following structure, and other variables are as defined in the present invention:

[0101]

[0102] In some embodiments of the present invention, the structure of the above polypeptide compound is shown in Table 2 below:

[0103] Table 2 Structure of polypeptide compounds 7-14 of the present invention

[0104]

[0105] Underlined amino acids K or dK Refers to the modified amino acid, the amino or carboxyl groups on the side chains of the two amino acid unit structures in the sequence are Condensation connects to form a modification group similar to a "staple" structure.

[0106] In some specific embodiments of the present invention, the unfolded structure of the above polypeptide compound is as follows:

[0107]

[0108]

[0109]

[0110] The present invention also provides a pharmaceutical composition, comprising as active ingredients a therapeutically effective amount of the above-mentioned polypeptide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0111] In some embodiments of the present invention, the above-mentioned compound or its pharmaceutically acceptable salt or the above-mentioned composition is used in the preparation of a drug for treating metabolic diseases.

[0112] In some embodiments of the present invention, the above polypeptide compound or its pharmaceutically acceptable salt or the above composition is used in the preparation of a drug for preventing or treating diabetes and its related diseases. The diabetes-related diseases include diabetic cardiovascular disease, diabetic cerebrovascular disease, retinopathy, nephropathy, cataract, coronary artery disease, diabetic neuropathy, diabetic foot and peripheral neuropathy.

[0113] In some embodiments of the present invention, the above polypeptide compound or its pharmaceutically acceptable salt or the above composition is used in the preparation of a drug for preventing or treating obesity and its related diseases, including liver disease, hyperlipidemia and hypertension.

[0114] In some embodiments of the present invention, the above polypeptide compound or its pharmaceutically acceptable salt or the above composition is used in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease and its related diseases. The non-alcoholic fatty liver disease includes simple fatty liver, non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis and liver cancer.

[0115] Technical Effects

[0116] The polypeptide compound of the present invention has relatively good agonistic activity on GLP-1R / GIPR / GCGR, and has good efficacy in lowering blood sugar and reducing weight.

[0117] Definition and Description

[0118] Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

[0119] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0120] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, prepared from a compound with a specific substituent discovered by the present invention and a relatively non-toxic acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting such a compound with a sufficient amount of a base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. Specific base addition salts include inorganic bases and organic bases, the inorganic bases include potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium hydride, calcium hydroxide and magnesium hydroxide, etc., and the organic bases include triethylamine, tri-n-butylamine, N,N-diisopropylethylamine and pyridine, etc. When the compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting such a compound with a sufficient amount of an acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, trifluoroacetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid and pamoic acid and the like; also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can be converted into either base or acid addition salts.

[0121] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

[0122] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (e.g., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group) as naturally occurring amino acids, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds whose structure is different from the general amino acid chemical structure, but function similarly to naturally occurring amino acids.

[0123] The natural amino acids and their various expressions described herein are well known to those skilled in the art, and their specific corresponding situations are shown in the following table:

[0124]

[0125]

[0126]

[0127] Some other unnatural amino acids, such as aminoisobutyric acid (Aib), correspond to the following table:

[0128]

[0129]

[0130] The term "treating" includes inhibiting, slowing, halting or reversing the progression or severity of existing symptoms or disorders.

[0131] Unless otherwise stated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereomers and tautomers.

[0132] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.

[0133] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0134] Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability of a ring to rotate freely about double bonds or single bonds of ring carbon atoms.

[0135] Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

[0136] Unless otherwise indicated, "(+)" indicates dextrorotatory, "(-)" indicates levorotatory, and "(±)" indicates racemic.

[0137] Unless otherwise specified, the key is a solid wedge. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond. and straight dashed key To indicate the relative configuration of a stereocenter, use a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Represents a straight solid bond or straight dashed key

[0138] Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

[0139] Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

[0140] Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (for example, a carbamate is generated from an amine).

[0141] The compounds of the invention may contain unnatural proportions of atomic isotopes on one or more of the atoms that make up the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C). For another example, deuterium can be used to replace hydrogen to form a deuterated drug. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing efficacy, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.

[0142] When the listed linking group does not indicate its linking direction, its linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of the reading order from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0143] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bonds are connected, the number of H atoms at the site will decrease with the number of connected chemical bonds to become a group with the corresponding valence. The chemical bond connecting the site to other groups can be a straight solid bond. Straight dotted key or wavy line For example, the straight solid bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group; The straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy lines in the phenyl group represent the connection to other groups through the carbon atoms at positions 1 and 2 in the phenyl group.

[0144] Unless otherwise specified, the term “C 1-3 "Alkyl" is used to refer to a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl, etc.; it may be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0145] The structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0146] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention.

[0147] The materials and solvents used in the present invention are commercially available.

[0148] Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names.

[0149] In the present invention, compound groups or reagents known to those skilled in the art may be represented by abbreviations, including but not limited to compound groups: tert-butyloxycarbonyl (BOC), benzyl (Bn), p-toluenesulfonyl (Tos), benzyloxycarbonyl (Cbz), fluorenylmethyloxycarbonyl (Fmoc), p-toluenesulfonyl (Ts), allyloxycarbonyl (Alloc), etc.; including but not limited to conventional reagents: dimethyl sulfoxide (DMSO), dichloromethane (DCM), N,N-dimethylformamide (DMF), tetrahydrofuran (THF) , piperidine (PIP), trifluoroacetic acid (TFA), N,N'-diisopropylcarbodiimide (DIC), 1-hydroxybenzotriazole (HOBt), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxy-7-azabenzotriazole (HOAT), tetrakistriphenylphosphine palladium (Pd(PPh3)4); Fmoc-(2-(allyloxy)-2-oxoethyl)glycine or (Fmoc-Ida(OAll)-OH): Fmoc-AEEA-OH: N-Boc-N'-Fmoc-Lys-OH: etc. DETAILED DESCRIPTION

[0150] The present invention is described in detail below by way of examples, but it is not intended to impose any adverse limitations on the present invention. The present invention has been described in detail herein, and specific embodiments thereof are also disclosed therein. It will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0151] Example 1-14 Synthesis of polypeptide compound 1-14

[0152] Synthesis of the polypeptide compound of the present invention: Referring to the polypeptide solid phase synthesis method known in the art, starting from the resin, adopting the Fmoc-strategy, according to the peptide sequence from C-terminus to N-terminus, using protected amino acids to condense one by one (including condensation, deprotection, elution cycle, and removal of side chain protection to form a lactam ring), cleavage, filtration and concentration, precipitation and filtration and drying to obtain a branched cyclic peptide cleavage crude product, and then purifying, concentrating and freeze-drying to obtain the finished product. Specifically, the polypeptide compound of the present invention can refer to the polypeptide synthesis process of Example 1 of Chinese application CN202410061582.8 and Example 38 to prepare the compound of the present invention. Wherein when the compound structural unit for When the branched chain coupling is Fmoc-Ida(OAll)-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosandioate; when the compound structural unit for When , the side chain coupling sequence is Fmoc-Ida(OAll)-OH, N-Boc-N'-Fmoc-Lys-OH, N-Boc-N'-Fmoc-Lys-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosandioate.

[0153] The following uses polypeptide compound 3 (amino acid modification at positions 14 and 17) and polypeptide compound 10 (amino acid modification at positions 17-21) as examples to illustrate the synthesis and preparation method of the polypeptide compound of the present invention. The preparation of the remaining polypeptide compounds can be synthesized by replacing amino acids and / or branched compound raw materials according to the amino acid sequence order and branched chain structure.

[0154] 1) Synthesis of the polypeptide compound (Compound 3) shown in Example 3:

[0155]

[0156] The peptide resin was synthesized using a conventional solid phase synthesis process, with nitrogen bubbling reaction in a solid phase reaction column with a sintered sieve plate at the bottom, using Rink amide-AM-resin as the starting resin, conventional Fmoc protected amino acids as feed, DIC / HOBt system as the condensation agent, DMF as the main reaction solvent, 20% PIP-DMF solution is the solvent for removing Fmoc, and condensation is carried out one by one from the C-terminus to the N-terminus of the peptide main chain. After the main chain synthesis is completed, the -Mtt on the ε-amino group of Fmoc-Lys(Mtt)-OH at position A-26 (from the C-terminus to the N-terminus) is removed, and then the side 1 to side 4 positions ((2-2-oxoethyl)-glycine, AEEA, γGlu, mono-tert-butyl eicosandioic acid) are condensed in sequence, and then the ε-amino protecting group of Lys at position A-23 (from the C-terminus to the N-terminus) and the carboxyl protecting group of (2-2-oxoethyl)-glycine at position 1 are removed, and then a condensation agent is added to form a lactam ring to obtain the target peptide resin.

[0157] The specific steps are as follows:

[0158] Step 1: Resin activation and deprotection

[0159] 1. Take 15g (5.1mmol) of Rink amide-AM-resin (S=0.34mmol / g) and add it to the solid phase reaction column. Add 225mL of DCM and let it stand for 20min to fully swell the resin. Drain the DCM, add 150ml of DMF and wash the resin by nitrogen bubbling. Repeat the above steps once and drain the solvent. Add 20% piperidine / DMF (150mL) to the reaction column and react by nitrogen bubbling for 5min. Drain the waste until no liquid flows out. Add 20% piperidine / DMF (150mL) to the reaction column again and continue nitrogen bubbling for 15min.

[0160] 2. React at 25°C for more than 2 hours. If the sample is negative for ninhydrin test (the resin is gray-red), the reaction is terminated.

[0161] 3. Draw off the reaction liquid, wash with DMF (150 mL) 7 times, adjust the pH to 7, 1 min each time, and drain until no liquid flows out.

[0162] Step 2: Fmoc-Ser(tBu)-OH coupling

[0163] 1. Weigh 5.75g (3.0eq) of Fmoc-Ser(tBu)-OH, 2.52g (4.0eq) of DIC and 2.03g (3.0eq) of HOBt, dissolve them in 80mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0164] 2. React in an environment of 25°C for 2.0h, take samples for ninhydrin detection, the resin is colorless and transparent;

[0165] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0166] The steps for coupling the remaining amino acids of the main chain are:

[0167] Referring to the method and amount of Fmoc-Ser(tBu)-OH coupling in step 2, the amino acid sequence is sequentially connected, and the order and amino acids containing protective groups used are as follows:

[0168]

[0169]

[0170] Step 3: Branched chain coupling

[0171] 1. Add 300 mL of DCM to the reaction column in sequence to cross-wash the resin 4 times. After draining the solvent, add 150 mL of 20% hexafluoroisopropanol (HFIP)-DCM solution and bubble nitrogen for five times, each time for 30 min;

[0172] 2. Drain the waste until no liquid flows out, take a sample for ninhydrin testing, and the resin will be gray-green;

[0173] 3. Add 150 mL of DCM and 150 mL of DMF in sequence to cross-wash the resin 4 times and drain until no liquid flows out.

[0174] 4. Weigh 3.30 g (5.0 eq) of Fmoc-(2-(allyloxy)-2-oxoethyl)glycine (Fmoc-Ida(OAll)-OH), 1.26 g (6.0 eq) of DIC and 1.13 g (5.0 eq) of HOBt, dissolve them in 50 mL of DMF and add them to the reaction column. Add nitrogen and adjust the nitrogen to make the resin swell evenly.

[0175] 5. React in an environment of 25°C for 2.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0176] 6. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0177] The remaining side chain coupling steps: refer to the coupling method and feed equivalents of Fmoc-Ida(OAll)-OH in step 3 and sequentially add the following compounds:

[0178] Amino acids containing protecting groups used Fmoc-AEEA-OH Fmoc-Glu-OtBu Eicosanedioic acid monotert-butyl ester

[0179] Step 4: Ring closure

[0180] 1. Add 300 mL of DMF and 300 mL of DCM to the reaction column in sequence, cross-wash the resin 4 times, and drain until no liquid flows out;

[0181] 2. Add 400 ml of the mixed solvent at a volume ratio of DCM: CH3CN: NMP: DEA = 3:3:2:2, and then add 0.521 g (0.27 eq) of Pd(PPh3)4, and react for 10 min with nitrogen bubbling, and drain until no liquid flows out;

[0182] 3. Repeat the above steps 1 to 2 times;

[0183] 4. Take samples for ninhydrin testing, the resin is red;

[0184] 5. Add 300 mL of DMF and 300 mL of DCM in sequence and cross-wash the resin 8 times;

[0185] 6. Dissolve 1.26g (6.0eq) of DIC and 1.13g (5.0eq) of HOBt in 100mL of DMF, add to the reaction column, react for 26h with nitrogen, take a sample for ninhydrin detection, and the resin is yellow;

[0186] 7. Add 300 mL of DMF and 300 mL of DCM in sequence and cross-wash the resin 8 times;

[0187] 8. Add 200 mL of MeOH and 300 mL of DCM in sequence to cross-wash the resin 4 times;

[0188] 9. Take out the resin and vacuum dry it until the weight stops decreasing. Weigh it to obtain 12.26 g of peptide resin;

[0189] Step 5: Peptide resin cleavage

[0190] Prepare 229mL of cleavage reagent, the reagent formula is trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), 1,2-ethanedithiol (EDT), phenol (ArOH), according to the volume ratio of TFA: H2O: EDT: TIS: ArOH = 86.5: 5: 2.5: 1: 5, and precool to 10 ± 3 ° C after preparation. Slowly add the resin to the cleavage reagent under stirring, and continue to stir until the temperature of the reaction system is stable; then control the temperature at 18 ± 3 ° C and continue to stir and react for 4 hours. Filter out the lysate, concentrate it to a viscous state, and use methyl tert-butyl ether 12-15 times the volume of the concentrate to precipitate it. After filtering out the precipitate, wash it, and dry it under reduced pressure at room temperature to obtain 4.0g of crude cyclopeptide.

[0191] Step 6: Chromatographic purification

[0192] The above 1.0 g crude cyclopeptide was dissolved in 5% acetonitrile and 2% aqueous ammonia solution, and then purified by high performance liquid chromatography using C8 silica gel matrix filler. The fractions with purity > 90% were collected and combined. The purification chromatographic system is listed as follows:

[0193]

[0194]

[0195] Step 7: Concentrated freeze-dried

[0196] The fraction obtained in the previous step was concentrated to remove acetonitrile, filtered through a 0.22 μm microporous filter membrane, loaded into a hanging bottle freeze dryer, and freeze-dried to obtain 288 mg of the target polypeptide product (purity: 95.17%); the molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value [M+4H] / 4 was 1218.9, and the detected value was 1218.9.

[0197] 2) Synthesis of the polypeptide compound (Compound 10) shown in Example 10:

[0198]

[0199] The peptide resin was synthesized by conventional solid phase synthesis process, with nitrogen bubbling reaction in a solid phase reaction column with a sintered sieve plate at the bottom, using Rink The amide-AM-resin was used as the starting resin, conventional Fmoc-protected amino acids were added, the condensation agent used was a DIC / HOBt system, DMF was used as the main reaction solvent, and a 20% piperidine-DMF solution was used as the Fmoc removal solvent. The peptide main chain was condensed one by one from the C-terminus to the N-terminus. After the main chain synthesis was completed, the -Mtt on the ε-amino group of Fmoc-Lys(Mtt)-OH at position A-23 (from the C-terminus to the N-terminus) was removed, and then the side 1 to side 4 positions ((2-2-oxoethyl)-glycine, AEEA, γGlu, mono-tert-butyl eicosanedioic acid) were condensed in sequence, and then the ε-amino protecting group of Lys at position A-19 (from the C-terminus to the N-terminus) and the carboxyl protecting group of (2-2-oxoethyl)-glycine at position 1 were removed, and then a condensation agent was added to form a lactam ring to obtain the target peptide resin.

[0200] The specific steps are as follows:

[0201] Step 1: Resin activation and deprotection

[0202] 1. Take 15g (5.1mmol) of Rink amide-AM-resin (S=0.34mmol / g) and add it to the solid phase reaction column. Add 225mL of DCM and let it stand for 20min to fully swell the resin. Drain the DCM, add 150mL of DMF and wash the resin by nitrogen bubbling. Repeat the above steps once and drain the solvent. Add 20% piperidine / DMF (150mL) to the reaction column and react by nitrogen bubbling for 5min. Drain the waste until no liquid flows out. Add 20% piperidine / DMF (150mL) to the reaction column again and continue nitrogen bubbling for 15min.

[0203] 2. React at 25°C for more than 2 hours. If the sample is negative for ninhydrin test (the resin is gray-red), the reaction is terminated.

[0204] 3. Draw off the reaction liquid, wash with DMF (150 mL) 7 times, adjust the pH to 7, 1 min each time, and drain until no liquid flows out.

[0205] Step 2: Fmoc-Ser(tBu)-OH coupling

[0206] 1. Weigh 5.75g (3.0eq) of Fmoc-Ser(tBu)-OH, 2.52g (4.0eq) of DIC and 2.03g (3.0eq) of HOBt, dissolve them in 80mL of DMF and add them to the reaction column. Blow nitrogen and adjust the nitrogen to make the resin swell evenly.

[0207] 2. React in an environment of 25°C for 2.0h, take samples for ninhydrin detection, the resin is colorless and transparent;

[0208] 3. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0209] The steps for coupling the remaining amino acids of the main chain are:

[0210] Referring to the method and amount of Fmoc-Ser(tBu)-OH coupling in step 2, the amino acid sequence is sequentially connected, and the order and amino acids containing protective groups used are as follows:

[0211]

[0212]

[0213] Step 3: Branched chain coupling

[0214] 1. Add 300 mL of DCM to the reaction column in sequence to cross-wash the resin 4 times. After draining the solvent, add 150 mL of 20% hexafluoroisopropanol (HFIP)-DCM solution and bubble nitrogen for five times, each time for 30 min;

[0215] 2. Drain the waste until no liquid flows out, take a sample for ninhydrin testing, and the resin will be gray-green;

[0216] 3. Add 150 mL of DCM and 150 mL of DMF in sequence to cross-wash the resin 4 times and drain until no liquid flows out.

[0217] 4. Weigh 2.64 g (4.0 eq) of Fmoc-(2-(allyloxy)-2-oxoethyl)glycine (Fmoc-Ida(OAll)-OH), 1.05 g (5.0 eq) of DIC and 0.903 g (4.0 eq) of HOBt, dissolve them in 50 mL of DMF and add them to the reaction column. Add nitrogen and adjust the nitrogen to make the resin swell evenly.

[0218] 5. React in an environment of 25°C for 2.0 hours, take samples for ninhydrin detection, and the resin is colorless and transparent;

[0219] 6. Draw off the reaction liquid, wash with DMF (150 mL) 5 times, 1 min each time, and drain until no liquid flows out.

[0220] The remaining branch coupling steps

[0221] Referring to the coupling method and feed equivalents of Fmoc-Ida(OAll)-OH in step 3, the following compounds were added in sequence:

[0222] Amino acids containing protecting groups used Fmoc-AEEA-OH Fmoc-Glu-OtBu Eicosanedioic acid monotert-butyl ester

[0223] Step 4: Ring closure

[0224] 1. Add 300 mL of DMF and 300 mL of DCM to the reaction column in sequence, cross-wash the resin 4 times, and drain until no liquid flows out;

[0225] 2. Add 400 ml of the mixed solvent at a volume ratio of DCM: CH3CN: NMP: DEA = 3:3:2:2, and then add 0.521 g (0.27 eq) of Pd(PPh3)4, and react for 10 min with nitrogen bubbling, and drain until no liquid flows out;

[0226] 3. Repeat the above steps 1 to 2 times;

[0227] 4. Take samples for ninhydrin testing, the resin is red;

[0228] 5. Add 300 mL of DMF and 300 mL of DCM in sequence and cross-wash the resin 8 times;

[0229] 6. Dissolve 2.05 g (5.0 eq) of DIC and 1.80 g (4.0 eq) of HOBt in 100 mL of DMF, add to the reaction column, react for 26 h with nitrogen, take a sample of ninhydrin for detection, and the resin is light yellow;

[0230] 7. Add 300 mL of DMF and 300 mL of DCM in sequence and cross-wash the resin 8 times;

[0231] 8. Add 200 mL of MeOH and 300 mL of DCM in sequence to cross-wash the resin 4 times;

[0232] 9. Take out the resin and vacuum dry it until the weight stops decreasing, then weigh it to obtain 14 g of peptide resin;

[0233] Step 5: Peptide resin cleavage

[0234] Prepare 210ml of cleavage reagent, the reagent formula is trifluoroacetic acid (TFA), purified water (H2O), triisopropylsilane (TIS), 1,2-ethanedithiol (EDT), phenol (ArOH), according to the volume ratio of TFA: H2O: EDT: TIS: ArOH = 86.5: 5: 2.5: 1: 5, and pre-cool to 10 ± 3 ° C after preparation. Slowly add the resin to the cleavage reagent under stirring, and continue to stir until the temperature of the reaction system is stable; then control the temperature at 18 ± 3 ° C and continue to stir and react for 4 hours. Filter out the lysate, concentrate it to a viscous state, and use methyl tert-butyl ether 12-15 times the volume of the concentrate to precipitate it. After filtering out the precipitate, wash it, and dry it under reduced pressure at room temperature to obtain 4.0g of crude cyclopeptide.

[0235] Step 6: Chromatographic purification

[0236] The above 1.0 g crude cyclopeptide was dissolved in 5% acetonitrile and 2% aqueous ammonia solution, and then purified by high performance liquid chromatography using C8 silica gel matrix filler. The fractions with purity > 90% were collected and combined. The purification chromatographic system is listed as follows:

[0237] Process Water Phase Organic phase gradient One-time purification 0.2% TFA Acetonitrile B% (0-51min): 42%-46% Secondary purification <![CDATA[50mmol / L(NH3)2HPO4(pH 7.5)]]> Acetonitrile B% (0-71min): 31%-37% Salt conversion 0.2mmol / L NaOH Acetonitrile B％：55％

[0238] Step 7: Concentrated freeze-dried

[0239] The fraction obtained in the previous step was concentrated to remove acetonitrile, filtered through a 0.22 μm microporous membrane, loaded into a hanging bottle freeze dryer, and freeze-dried to obtain 80 mg of the target polypeptide product (purity of 98.16%); the molecular weight of the polypeptide was confirmed by ESI-MS, and the calculated value was [M+4H] / 4 + The value is 1229.4 and the detection value is 1229.2.

[0240] The molecular weight information of the polypeptide compounds 1-14 synthesized in this application is summarized in the following table:

[0241]

[0242]

[0243] Biological test data Test 1: In vitro GLP-1R / GIPR / GCGR agonist activity test A: Main materials:

[0244] 1) Cell line: Cell source: Zhongsheng Ruichuang, see the table below for details.

[0245]

[0246] 2) Reagents and consumables

[0247]

[0248]

[0249] 3) Instruments

[0250] Instrument Name Manufacturer model Autoclave ZEALWAY GI54W Automatic cell analyzer Count Star Countstar S2 <![CDATA[CO2 Incubator]]> Thermo 3111 Biological Safety Cabinets Haier Medical HR40-IIA2 Inverted microscope LEICA LEICA DMi1 Multifunctional ELISA reader PE EnVision 2014

[0251] B. Cell Function Activity Test Method

[0252] 1) In a CO2 incubator at 37°C, HEK-293T-CRE-Luc2-GLP1R, HEK-293T-CRE-Luc2-GIPR, and HEK-293T-CRE-Luc2-GCGR cells were cultured in complete medium (DMEM+10% FBS+150 μg / mL Hygromycin B+1 μg / mL Puromycin) until the logarithmic growth phase, and the cell density and confluence reached 70-80%, and the cell suspension was collected.

[0253] 2) The cells were seeded into a 96-well white opaque-bottom microplate at a density of about 20,000 cells / well in 90 μL serum-free DMEM medium and incubated in a CO2 incubator at 37°C for 16 to 24 hours.

[0254] 3) Compound preparation: GLP-1(7-37) / GIP(1-42) / GCG(Human) or the test compound was first prepared into 1000X working solution concentration using DMSO, GLP-1(7-37) / GIP(1-42) / GCG(Human) was diluted 3 times, and the test compound was diluted 5 times, 10 points, duplicate wells or single wells. Then, the compound was diluted 100 times using serum-free DMEM and prepared into 10X working solution concentration (the working concentration should be 10 times the final concentration). The final DMSO concentration was 0.1%.

[0255] 4) Take out the 96-well plate incubated overnight in step 2, and add 10 μL of 10× prepared drug concentration to each well.

[0256] 5) Cover the plate and incubate at 37°C in a CO2 incubator for 6 h.

[0257] 6) Use Bright-lite prepared according to the recommended instructions TM Luciferase Assay Substrate Buffer was used for luciferase assay. Lyse the cells for 15 min and mix well, then detect luciferase using EnVision 2014 microplate reader.

[0258] 7) Data analysis: GraphPad Prism 8.0 was used to process the experimental data and calculate the EC 50 .

[0259] %Activity=(detection data-VC) / (PC-VC)*100%;

[0260] PC: average value of data of corresponding wells of 1nM GLP-1(7-37) / GIP(1-42) / GCG(Human);

[0261] VC: 0.1% DMSO corresponding to the average data of the wells.

[0262] C Experimental Results

[0263] serial number <![CDATA[GLP-1EC 50 (nM)]]> <![CDATA[GIP EC 50 (nM)]]> <![CDATA[GCG EC 50 (nM)]]> Human GLP-1 (7-37) 0.00135 NA NA Human GIP (1-42) NA 0.0040 NA Glucagon NA NA 0.0021 LY3437943 0.00020 0.00043 0.00027 Example 1 (Compound 1) 0.00763 0.00616 0.0750 Example 3 (Compound 3) 0.00022 0.00079 0.00041 Example 5 (Compound 5) 0.00444 0.00194 0.0259 Example 8 (Compound 8) 0.0124 0.00272 0.1938 Example 10 (Compound 10) 0.00385 0.00021 0.0351

[0264] Conclusion: The polypeptide compound of the present invention and LY3437943 have strong agonist activity on GLP-1R / GIPR / GCGR. The changes in the "staple" structure and the amino acid of the peptide sequence have a certain influence on the activity of the polypeptide.

[0265] Experiment 2: Pharmacodynamic study of the compound in the DIO obese mouse model

[0266] Experimental purpose: To evaluate the effects of repeated administration of the test substance (compound) on body weight and glucose and lipid metabolism.

[0267] In this experiment, mice induced from 4-5 weeks of age on a high-fat diet (Research Diets: D12492) for 8 weeks were used as test animals. The animals were grouped according to their body weight and fasting blood glucose levels. The average body weight of the model animals was 47.2±0.3g, and the average body weight of the control animals was 27.9±0.3g. The average fasting blood glucose of the model animals was 8.8±0.2mmol / L, and the average fasting blood glucose of the control animals was 6.0±0.2mmol / L. There were 6 animals in each group. They were given drugs immediately after grouping. All animals were given drugs by subcutaneous injection, once every 3 days, for 22 consecutive days. Both the normal control group and the model group were given solvent (20mM citric acid buffer), and the body weight and food intake were tested twice a week. Fasting blood glucose was tested on D10 and D20 after drug administration, OGTT test was performed on D20, blood was collected on D23 to test liver function indicators (ALT, AST, ALP), serum lipid metabolism indicators (TC, TG, HDL, LDL), liver tissue was taken to calculate the organ index, liver lipid metabolism indicators (liver TC, TG), and liver tissue was subjected to pathological examination (HE, oil red) to evaluate the effects of the test substances on body weight and glucose and lipid metabolism in the obese mouse model induced by a high-fat diet.

[0268] The experimental data of each group of animals were described by mean ± standard error (X ± SEM). P < 0.05 was considered statistically significant. All statistical analyses were performed using Graphpad 8.0 software.

[0269] Table 3 Effects of compounds on body weight changes in DIO mice

[0270]

[0271]

[0272] Conclusion: From the results in Table 3, the compound 3 polypeptide of the present invention has an obvious weight loss effect, and is comparable to the weight loss effect of LY3437943.

[0273] Table 4 Effects of compounds on fasting blood glucose changes in DIO mice

[0274]

[0275] Conclusion: It can be seen from Table 4 that the polypeptide of compound 3 of the present invention has an obvious hypoglycemic effect.

[0276] Table 5 Effects of compounds on changes in liver-to-body ratio in DIO mice

[0277] / Control (n = 6) Model(n=6) Compound 3 (n=6) LY3437943 (n=6) Liver to body ratio (g / g) 0.040±0.001 0.037±0.004 0.030±0.002 0.032±0.003

[0278] Conclusion: From the results in Table 5, it can be seen that the polypeptide of compound 3 of the present invention has the effect of improving the liver-to-body ratio.

[0279] Table 6 Effects of compounds on changes in liver indicators in DIO mice

[0280]

[0281] From the results in Table 6, it can be seen that the polypeptide of Compound 3 of the present invention has a significant effect of reducing liver triglycerides.

[0282] In summary, the present application newly designs and synthesizes a series of polypeptide compounds. The polypeptide (especially the compound 3 polypeptide) has strong in vitro agonist activity on GLP-1R / GIPR / GCGR, excellent in vitro and in vivo pharmacokinetic properties, better stability properties, and has obvious weight loss, hypoglycemic and hypolipidemic effects. The polypeptide compound of the present invention has a drug development prospect as a long-acting GLP-1 / GIP / GCG multi-agonist.

Claims

1. A polypeptide compound having a sequence as shown in Formula Z and a pharmaceutically acceptable salt thereof: YX2QGT FTSDY SIX 13 X 14 D KX 17 AQX 20 X 21 IN WHAT 25 LX 27 X 28 X 29 GPSX 34 A PPPS0 Formula Z in: X2 is Aib; X 13 is α-MeL (α-methyl substituted leucine); X 14 is K or L; X 17 is K or dK (D-lysine); X 20 for Aib; X 21 A or K; X 25 Y, F or dF (D-phenylalanine); X 27 is L, α-MeL or NLe (norleucine); X 28 is E or K; X 29 is G or Aib; X 34 is G or Aib; S0 is selected from And the compound has the following modifications: 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure similar to a "staple" structure, wherein i is independently selected from 14 or 17, and j is independently selected from 2, 3 and 4, 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connection; X a Selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid; X3 is selected from m is selected from 1, 2 and 3; p is selected from 1 and 2; n is selected from 8, 9 and 10.

2. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: When the modified amino acid position i in the sequence of formula Z is 14, X 14 K, X 21 is A.

3. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: When the modified amino acid position i in the sequence of formula Z is 17, X 14 L, X 21 For K.

4. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Its polypeptide sequence is shown in formula Z-1, YX2QGT FTSDY SIX 13 KD KKAQX 20 AFIEX 25 LX 27 EX 29 GPSSX 34 A PPPS0 (ZI) in: X2 is Aib; X 13 is α-MeL; X 20 for Aib; X 25 is Y or F; X 27 is a-MeL, L or NLe; X 29 is G or Aib; X 34 is G or Aib; S0 is selected from And the compound has the following modifications: 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure similar to a "staple" structure, wherein i is independently selected from 14, j is independently selected from 2, 3, 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connection; X a Selected from Wherein, "*" indicates the position connected to X3, and the two ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid; X3 is selected from m is selected from 1, 2 and 3; p is selected from 1 and 2; n is selected from 8, 9 and 10.

5. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Its polypeptide sequence is shown in formula Z-III, YX2QGT FTSDY SIX 13 LD KX 17 AQX 20 HOW 25 LX 27 X 28 X 29 GPSX 34 A PPPS0 (Z-III) in: X2 is Aib; X 13 is α-MeL; X 17 is K or dK; X 20 for Aib; X 25 is Y, F or dF; X 27 is L, I or NLe; X 28 is E or K; X 29 is G or Aib; X 34 is G or Aib; S0 is selected from And the compound has the following modifications: 1) The amino acid side chains at positions i and i+j in the sequence can be modified to form a modified structure similar to a "staple" structure, where i is 17, j is 4, 2) The modification is that the amino or carboxyl groups on the side chains of the two amino acid unit structures are Condensation connection; X a Selected from Wherein, "*" indicates the position connected to X3, and both ends are the positions connected to the corresponding amino or carboxyl groups in the modified amino acid; X3 is selected from m is selected from 1, 2 and 3; p is selected from 1 and 2; n is selected from 8, 9 and 10.

6. The polypeptide compound and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The polypeptide sequence is selected from SEQ ID NO: 4-12: YAibQGT FTSDY SIα-MeL K D K K AQAib AFIEY LNLeEGGPSSGA PPPS-NH2(SEQ ID NO:4)YAibQGT FTSDY SIα-MeL K D K K AQAib AFIEY LNLeEGGPSSAibA PPPS-NH2(SEQ ID NO:5) YAibQGT FTSDY SIα-MeL K D K K AQAib AFIEY LNLeEAibGPSSGA PPPS-NH2(SEQ ID NO:6) YAibQGT FTSDY SIα-MeLLD K K AQAib K FIEF LLEGGPSSAibA PPPS-NH2(SEQ ID NO:7) YAibQGT FTSDY SIα-MeLLD K dK AQAib K FIEF LLEGGPSSGAPPPS-NH2 (SEQ ID NO:8) YAibQGT FTSDY SIα-MeLLD K K AQAib K FIEY LNLeEGGPSSAibAPPPS-NH2(SEQ ID NO:9) YAibQGT FTSDY SIα-MeLLD K K AQAib K FIEdF LNLeEGGPSSAibAPPPS-NH2(SEQ ID NO:10) YAibQGT FTSDY SIα-MeLLD K K AQAib K FIEF LNLeEAibGPSSGA PPPS-NH2(SEQ ID NO:11) YAibQGT FTSDY SIα-MeLLD K K AQAib K FIEY LNLeEGGPSSGAPPPS-NH2(SEQ ID NO:12) The underscore in the sequence K or dK Refers to modified amino acids, the amino or carboxyl groups on the side chains of the two amino acids are Condensed together, Xa and X3 are as defined in claim 1.

7. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X3 is selected from Preferably, X3 is selected from 8. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The structural unit Selected from: Preferably, the structural unit Selected from 9. The polypeptide and pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The staple structure in the polypeptide structure is as follows:

10. The following polypeptide compounds and pharmaceutically acceptable salts thereof:

11. The polypeptide compound and pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, characterized in that: The pharmaceutically acceptable salt is a salt formed by the polypeptide compound and an organic acid and / or an inorganic acid and / or an organic base and / or an inorganic base.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the polypeptide compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof.

13. Use of the polypeptide compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition according to claim 12 in the preparation of a medicament for treating metabolic diseases.

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