Long-acting GLP-1 / GIP / Y2 receptor triple agonist as well as preparation method and application thereof

Abstract

The invention discloses a long-acting GLP-1 / GIP / Y2 receptor triple agonist as well as a preparation method and application thereof, the amino acid sequence of the receptor triple agonist is as shown in the following general formula (I), and the receptor triple agonist can act on a GLP-1 receptor, a GIP receptor and a Y2 receptor at the same time and can play the activities of GLP-1, GIP and PYY at the same time. The GLP-1 / GIP / Y2 receptor triple agonist disclosed by the invention has the effects of effectively reducing blood sugar and weight and regulating lipid. The compound has greater potential when being applied to medicines for treating metabolic syndromes, such as diabetes, obesity, non-alcoholic fatty liver diseases, non-alcoholic steatohepatitis, dyslipidemia and other diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a long-acting GLP-1 / GIP / Y2 receptor triple agonist and its preparation method and application, belonging to the technical field of polypeptides. Background Art

[0002] The sharp rise in the obesity epidemic has become a major public health crisis. Obesity is a major risk factor for several diseases, such as non-alcoholic steatohepatitis (NASH), hypertension, type 2 diabetes (T2DM), and cardiovascular diseases, all of which can shorten lifespan and quality of life. Currently, there are limited drugs for treating obesity, and many obesity drugs have relatively significant side effects. Currently, among the drugs for treating T2DM, only GLP-1 and SGLT2 inhibitors have good weight loss effects.

[0003] Glucagon-like peptide-1 (GLP-1) is an incretin polypeptide hormone secreted by intestinal L cells of the small intestine, and its hypoglycemic effect is glucose concentration-dependent. As an important enteric-islet axis regulator, GLP-1 promotes glucose-stimulated insulin secretion by activating GLP-1 receptors on the surface of pancreatic β cells. In addition, GLP-1 exerts its weight loss effect by acting on the central nervous system to produce satiety and delaying the gastric emptying rate. However, endogenous GLP-1 has significant pharmacokinetic limitations under physiological conditions. Its plasma half-life is only 1.5 - 2 minutes, mainly due to two factors: the renal clearance mechanism and dipeptidyl peptidase IV (DPP-IV)-mediated enzymatic inactivation, which renders GLP-1 inactive by cleaving the histidine-alanine dipeptide at the N-terminus of the GLP-1 molecule. To overcome this limitation, researchers have developed various molecular engineering strategies, including amino acid modification, fatty acid chain coupling, and albumin binding, etc., to enhance the enzymatic stability of GLP-1 analogs and extend their plasma half-life. Although existing GLP-1 receptor agonists have shown significant hypoglycemic and weight loss effects in clinical applications, there are still certain treatment limitations. To achieve an ideal weight control effect, it is often necessary to increase the dosage, which may trigger dose-dependent gastrointestinal adverse reactions (such as nausea, vomiting, etc.), resulting in reduced patient tolerance and a narrow therapeutic window. Therefore, developing novel GLP-1 receptor agonists with better safety and tolerance characteristics to achieve more optimized blood glucose control and weight management remains an important research direction in the current field of diabetes treatment.

[0004] Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone composed of 42 amino acids and is the earliest identified member of the incretin family. GIP binds to its specific G protein-coupled receptor (GPCR), activates the cAMP-PKA signaling pathway in pancreatic β cells, and thus promotes glucose-dependent insulin secretion. Notably, the regulation of blood glucose homeostasis by GIP is bidirectional: it inhibits glucagon secretion in hyperglycemic states and promotes its secretion in hypoglycemic states. In addition, GIP regulates lipid metabolism by activating GIP receptors in adipose tissue and acts on the central nervous system to produce an anorectic effect.

[0005] Peptide YY (PYY) is a gastrointestinal peptide hormone composed of 36 amino acids and belongs to the neuropeptide NPY family. In the body, it mainly exists in the full-length form of PYY 1-36 and PYY cleaved by DPP-IV enzyme 3-36 in two forms. After removing the N-terminal dipeptide by DPP-IV enzyme, the resulting PYY 3-36 exhibits highly selective agonist activity for the Y2 receptor. PYY 3-36 can induce anorexia by binding to the Y2 receptor at the presynaptic terminal of hypothalamic NPY neurons. Therefore, Y2 receptor agonists can increase satiety, suppress appetite, and thus reduce body weight. In addition, Y2 receptor agonists can regulate energy homeostasis by increasing energy expenditure and reducing food intake, and thus have a therapeutic effect on obesity.

[0006] Currently, there are related studies on drugs that can act on both GLP-1 receptor and Y2 receptor simultaneously. A cysteine is connected to the C-terminal of the GLP-1 part, and maleimidopropionic acid is connected to the N-terminal of PYY. The two parts are combined into a GLP-1 / Y2 receptor dual agonist through the specific reaction of cysteine and maleimide. This method can rapidly synthesize polypeptides, and the synthesized dual receptor agonist has better effects on reducing blood sugar and body weight than GLP-1 receptor agonists. However, the introduction of cysteine and maleimide affects the activities of GLP-1 and Y2 receptors to a certain extent. In addition, the introduction of PYY enhances the weight loss effect but will exacerbate gastrointestinal adverse reactions such as nausea and vomiting. Summary of the Invention

[0007] Object of the Invention: To solve the above technical problems, the present invention provides a class of long-acting GLP-1 / GIP / Y2 receptor triple agonists and their preparation methods and applications. The triple agonist can act on GLP-1 receptor, GIP receptor, and Y2 receptor simultaneously, and can simultaneously exert the activities of GLP-1, GIP, and PYY.

[0008] Technical Solution: To achieve the above object, the present invention adopts the following technical solutions:

[0009] A class of long-acting GLP-1 / GIP / Y2 receptor triple agonists or pharmaceutically acceptable salts thereof, and the amino acid sequence of the long-acting GLP-1 / GIP / Y2 receptor triple agonist is shown in the following general formula (I):

[0010]

[0011] Wherein, X1 is M or L; X2 is V or A; X3 is I or P; X4 is E or K; X5 is L or W; X6 is S or D; X7 is L or W; X8 is V or L; n is a natural number, and 1 ≤ n ≤ 8.

[0012] Preferably, X1 is L; X2 is A; X3 is P; X4 is K; X5 is W; X6 is D; X7 is W; X8 is L; n is a natural number, and 1 ≤ n ≤ 8.

[0013] Preferably, the long-acting GLP-1 / GIP / Y2 receptor triple agonist is selected from one of the following sequences: Structure 1

[0014]

[0015] Structure 2

[0016]

[0017] Structure 3

[0018]

[0019] Structure 4

[0020]

[0021] Structure 5

[0022]

[0023] Structure 6

[0024]

[0025] Structure 7

[0026]

[0027] Structure 8

[0028]

[0029] Structure 9

[0030]

[0031] Structure 10

[0032]

[0033] Structure 11

[0034]

[0035] Structure 12

[0036]

[0037] Structure 13

[0038]

[0039] Structure 14

[0040]

[0041] Structure 15

[0042]

[0043] As a specific embodiment, the salt is a salt formed by a long-acting GLP-1 / GIP / Y2 receptor triple agonist and one of the following compounds: acetic acid, salicylic acid, lauric acid, cinnamic acid, lactic acid or succinic acid.

[0044] The present invention also provides a method for synthesizing the long-acting GLP-1 / GIP / Y2 receptor triple agonist, comprising the following steps:

[0045] First, swell the resin and remove the Fmoc protecting group, then synthesize Fmoc-Tyr-Rink amide-MBHA resin, and then extend the peptide chain according to the amino acid sequence, modify the Lys side chain, and finally cleave, purify the polypeptide on the resin to obtain the product.

[0046] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of at least one of the above-mentioned long-acting GLP-1 / GIP / Y2 receptor triple agonists or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0047] As a specific embodiment, the dosage form of the composition is a tablet, capsule, tincture, inhalant, spray, injection, film, patch, powder, granule, emulsion or suppository as described in pharmacy.

[0048] Finally, the present invention provides the application of the long-acting GLP-1 / GIP / Y2 receptor triple agonist or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition in the preparation of a drug for treating metabolic diseases.

[0049] Preferably, the metabolic disease is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and / or dyslipidemia. In certain aspects, the drug is used to treat more than one metabolic disease or disorder, for example, diabetes and obesity; obesity and dyslipidemia; diabetes and dyslipidemia; diabetes, dyslipidemia, and obesity.

[0050] More preferably, the diabetes is T1DM, T2DM, or gestational diabetes.

[0051] The triple receptor agonist provided by the present invention can act on GLP-1 receptor, GIP receptor, and Y2 receptor simultaneously, and can exert the activities of GLP-1, GIP, and PYY simultaneously. GLP-1 receptor agonist can reduce blood glucose and suppress appetite; GIP receptor agonist is an effective supplement to GLP-1 receptor agonist in reducing blood glucose, and GIP receptor agonist has certain antiemetic properties to reduce the gastrointestinal adverse reactions of GLP-1 receptor agonist; the appetite-suppressing effect of Y2 receptor agonist is an effective supplement to the weight loss effect produced by GLP-1 receptor agonist and GIP receptor agonist. The three activities of triple receptor activation of GLP-1 receptor, GIP receptor, and Y2 receptor cooperate with each other and form a feedback mechanism depending on blood glucose concentration, which can not only control blood glucose but also reduce body weight. For the treatment of diabetes and obesity, the triple receptor agonist of the present invention has significant advantages over single-target GLP-1 analogs and GLP-1-based dual receptor agonists.

[0052] Technical effects: Compared with the prior art, the present invention has the following advantages:

[0053] (1) The GLP-1 / GIP / Y2 receptor triple agonist of the present invention retains the therapeutic effect of GLP-1 analog on diabetes and simultaneously has the beneficial effects of GIP and PYY on metabolism, thus having a synergistic effect on sugar, lipid, and energy metabolism, having a weight loss effect and an excellent lipid-lowering effect while effectively reducing blood glucose.

[0054] (2) The N-terminal amino acid His of the GLP-1 / GIP / Y2 receptor triple agonist of the present invention is identical to that of natural GLP-1, instead of Tyr commonly used in the design of GLP-1 / GIP receptor dual agonists, which brings better GLP-1 receptor agonist activity and hypoglycemic activity. In addition, the sequence structure of the GLP-1 / GIP / Y2 receptor triple agonist of the present invention enables the GLP-1 / GIP / Y2 receptor triple agonist of the present invention to have potent GLP-1 and Y2 receptor agonist activities as well as certain GIP receptor agonist activity, and unexpectedly results in the GLP-1 / GIP / Y2 receptor triple agonist of the present invention having a more excellent hypoglycemic effect compared with the marketed GLP-1 / GIP receptor dual agonists, as well as a good effect of inhibiting food intake, bringing unexpected beneficial effects;

[0055] (3) The receptor triple agonist provided by the present invention has stable chemical properties and pharmacokinetic characteristics supporting once-weekly administration at least; the receptor triple agonist provided by the present invention has a better therapeutic effect on metabolic diseases such as T2DM, obesity, and dyslipidemia than existing marketed drugs. Therefore, the receptor triple agonist provided by the present invention is suitable as an active ingredient of drugs for treating metabolic diseases such as diabetes, obesity, non-alcoholic steatohepatitis, and dyslipidemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Shows the acute hypoglycemic effect of each test substance of the present invention after single administration in ICR mice.

[0057] Figure 2 Shows the effect of inhibiting food intake of each test substance of the present invention after single administration in ICR mice.

[0058] Figure 3 Shows the percentage change in body weight of the test substance of the present invention after 21 days of long-term administration in DIO mice. DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention will be further illustrated below in conjunction with specific examples.

[0060] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms and methods related to chemistry, biology, and pharmacology described herein are well-known and commonly used in the art.

[0061] In addition, the abbreviations in the structural formula of the present invention represent specific amino acids, and adjacent amino acids are connected by peptide bonds. The amino acids are abbreviated according to the naming rules of IUPAC-IUB as follows:

[0062] Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine (Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Tyr, Y); Valine (Val, V).

[0063] In addition, unless otherwise specified, all amino acid residues in the polypeptide compounds of the present invention are preferably in the L configuration.

[0064] In addition, in the sequences of the present invention, in addition to natural amino acids, α-aminoisobutyric acid (Aib) is also used.

[0065] In addition, the “-NH2” part at the C-terminus of the said sequence indicates an amide group (-CONH) at the C-terminus.

[0066] The present invention is illustrated by the following examples, which do not limit the interpretation of the rights of the present invention in any way.

[0067] Example 1

[0068] Synthesis of the polypeptide compound of Structure 1

[0069]

[0070] (1) Swelling of the resin

[0071] Weigh 0.303 g (0.1 mmol equivalent) of RinkAmide MBHA resin with a loading capacity of 0.330 mmol / g, put it into a 25 mL reactor, wash the resin alternately with 7 mL of dichloromethane and methanol once, wash the resin with 7 mL of dichloromethane twice, then swell the resin with 7 mL of dichloromethane for 1 h, and finally wash the resin with 7 mL of DMF three times.

[0072] (2) Removal of the Fmoc protecting group from the resin

[0073] Transfer the swollen resin to a PSI-200 semi-automatic polypeptide synthesizer, add 7 mL of 20% piperidine / DMF (v / v), react at room temperature for 30 min, filter off the deprotection solution, and wash the resin with 7 mL of DMF four times, 1.5 min each time, to obtain Rink resin with the Fmoc protecting group removed.

[0074] (3) Synthesis of Fmoc-Tyr-Rink amide-MBHAResin

[0075] Weigh Fmoc-Tyr(tBu)-OH (0.4 mmol), dissolve it in 3 mL of DMF, add 2 mL of HBTU / HOBt (0.4 mmol / 0.44 mmol) condensing agent, pre-activate for 30 min, then add the activated amino acid into the reactor, shake and react at room temperature for 2 h. After filtering the reaction solution, wash the resin 4 times with 7 mL of DMF, and use Kaiser reagent to detect whether the reaction coupling is complete. If it is incomplete, perform coupling twice.

[0076] (4) Elongation of the peptide chain

[0077] According to the sequence of the peptide chain, repeat the above steps of deprotection and coupling to sequentially connect the corresponding amino acids until the peptide chain synthesis is completed. Among them, the Lys site with a modified side chain at position 16 adopts the Fmoc-Lys(Dde)-OH protection strategy, and Boc-His(Boc)-OH is used for the His at the N-terminus.

[0078] (5) Modification of the Lys side chain

[0079] After the peptide chain synthesis is completed, add 7 mL of 2% hydrazine hydrate / DMF (v / v) to selectively remove the Dde protecting group of Lys at position 16. After the Dde protecting group is removed, add 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt, and shake and condense for 2 h. After removing the Fmoc protecting group, add 0.4 mmol of Fmoc-AEEA-OH, 0.4 mmol of DIC and 0.44 mmol of HOBt again, and shake and condense for 2 h. After removing the Fmoc protecting group, add 0.4 mmol of Fmoc-Glu-OtBu, 0.4 mmol of DIC and 0.44 mmol of HOBt, and shake and condense for 2 h. After removing the Fmoc protecting group, add 0.4 mmol of monoterbutyl octadecanedioate, 0.4 mmol of HBTU and 0.44 mmol of HOBt, and condense for 2 h. After the reaction is complete, wash the resin 4 times with 7 mL of DMF.

[0080] (6) Cleavage of the polypeptide

[0081] Transfer the resin with the polypeptide obtained above to a round-bottom flask, use 5 mL of the cutting agent ReagentR (TFA / thioanisole / phenol / EDT, 90:5:3:2, V / V) to cut the resin, react at a constant temperature of 30 °C in an oil bath for 2 h, pour the cutting solution into 40 mL of ice-cold diethyl ether, freeze and centrifuge, wash the crude product 3 times with 15 mL of ice-cold diethyl ether, and finally dry it with nitrogen to obtain the crude peptide.

[0082] (7) Purification and Characterization of Polypeptides

[0083] The crude target polypeptide was dissolved in water, filtered through a 0.25 μm microporous membrane, and then purified by a Shimadzu preparative reverse-phase HPLC system. The chromatographic conditions were as follows: C18 reverse-phase preparative column (250 mm X 20 mm, 12 μm); mobile phase A: 0.1% TFA / water (V / V); mobile phase B: methanol (V / V); flow rate: 8 mL / min; detection wavelength: 214 nm. Elution was carried out using a linear gradient (20% B - 70% B / 30 min). The target peak was collected, methanol was removed, and the product was freeze-dried to obtain 0.11 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9261.46. ESI-MS m / z: calculated value [M+7H] 7+ 1324.0, [M+8H] 8+ 1158.7, [M+9H] 9+ 1030.0; observed value [M+7H] 7+ 1324.0,

[0084] [M+8H] 8+ 1158.6, [M+9H] 9+ 1030.0.

[0085] Example 2

[0086] Synthesis of the Polypeptide Compound of Structure 2

[0087]

[0088] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9406.62. ESI-MS m / z: calculated value [M+7H] 7+ 1344.8, [M+8H] 8+ 1176.8, [M+9H] 9+ 1046.2; observed value [M+7H] 7+ 1344.7,

[0089] [M+8H] 8+ 1176.7, [M+9H] 9+ 1046.0.

[0090] Example 3

[0091] Synthesis of the Polypeptide Compound of Structure 3

[0092]

[0093] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9696.94. ESI-MS m / z: calculated value [M+7H] 7+ 1386.3, [M+8H] 8+ 1213.1, [M+9H] 9+ 1078.4; observed value [M+7H] 7+ 1386.3,

[0094] [M+8H] 8+ 1213.0, [M+9H] 9+ 1078.4.

[0095] Example 4

[0096] Synthesis of the polypeptide compound of Structure 4

[0097]

[0098] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9987.25. ESI-MS m / z: calculated value [M+7H] 7+ 1427.7, [M+8H] 8+ 1249.4, [M+9H] 9+ 1110.7; observed value [M+7H] 7+ 1427.7,

[0099] [M+8H] 8+ 1249.3, [M+9H] 9+ 1110.7.

[0100] Example 5

[0101] Synthesis of the polypeptide compound of Structure 5

[0102]

[0103] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 10277.57. ESI-MS m / z: calculated value [M+7H] 7+ 1469.2, [M+8H] 8+ 1285.7, [M+9H] 9+ 1142.9; observed value [M+7H] 7+ 1469.2,

[0104] [M + 8H] 8+ 1285.7, [M + 9H] 9+ 1142.9。

[0105] Example 6

[0106] Synthesis of the polypeptide compound of Structure 6

[0107]

[0108] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9044.22. ESI-MS m / z: calculated value [M + 7H] 7+ 1293.0, [M + 8H] 8+ 1131.5, [M + 9H] 9+ 1005.9; observed value [M + 7H] 7+ 1292.8,

[0109] [M + 8H] 8+ 1131.3, [M + 9H] 9+ 1005.9。

[0110] Example 7

[0111] Synthesis of the polypeptide compound of Structure 7

[0112]

[0113] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9189.38. ESI-MS m / z: calculated value [M + 7H] 7+ 1313.8, [M + 8H] 8+ 1149.7, [M + 9H] 9+ 1022.0; observed value [M + 7H] 7+ 1313.7,

[0114] [M + 8H] 8+ 1149.6, [M + 9H] 9+ 1021.9。

[0115] Example 8

[0116] Synthesis of the polypeptide compound of Structure 8

[0117]

[0118] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9479.69. ESI-MS m / z: calculated value [M+7H] 7+ 1355.2, [M+8H] 8+ 1185.9, [M+9H] 9+ 1054.3; observed value [M+7H] 7+ 1355.1,

[0119] [M+8H] 8+ 1185.9, [M+9H] 9+ 1054.2.

[0120] Example 9

[0121] Synthesis of the polypeptide compound of Structure 9

[0122]

[0123] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9770.01. ESI-MS m / z: calculated value [M+7H] 7+ 1396.7, [M+8H] 8+ 1222.2, [M+9H] 9+ 1086.5; observed value [M+7H] 7+ 1396.6,

[0124] [M+8H] 8+ 1222.1, [M+9H] 9+ 1086.5.

[0125] Example 10

[0126] Synthesis of the polypeptide compound of Structure 10

[0127]

[0128] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 10060.33. ESI-MS m / z: calculated value [M+7H] 7+ 1438.2, [M+8H] 8+ 1258.5, [M+9H] 9+ 1118.8; observed value [M+7H] 7+1438.0,

[0129] [M + 8H] 8+ 1258.4, [M + 9H] 9+ 1118.7。

[0130] Example 11

[0131] Synthesis of the polypeptide compound of Structure 11

[0132]

[0133] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9215.38. ESI-MS m / z: calculated value [M + 7H] 7+ 1317.5, [M + 8H] 8+ 1152.9, [M + 9H] 9+ 1024.9; observed value [M + 7H] 7+ 1317.5,

[0134] [M + 8H] 8+ 1152.9, [M + 9H] 9+ 1024.9。

[0135] Example 12

[0136] Synthesis of the polypeptide compound of Structure 12

[0137]

[0138] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9360.52. ESI-MS m / z: calculated value [M + 7H] 7+ 1338.2, [M + 8H] 8+ 1171.0, [M + 9H] 9+ 1041.0; observed value [M + 7H] 7+ 1338.1,

[0139] [M + 8H] 8+ 1171.0, [M + 9H] 9+ 1041.0。

[0140] Example 13

[0141] Synthesis of the polypeptide compound of Structure 13

[0142]

[0143] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9650.86. ESI-MS m / z: calculated value [M+7H] 7+ 1379.7, [M+8H] 8+ 1207.3, [M+9H] 9+ 1073.3; observed value [M+7H] 7+ 1379.7,

[0144] [M+8H] 8+ 1207.3, [M+9H] 9+ 1073.3.

[0145] Example 14

[0146] Synthesis of the polypeptide compound of Structure 14

[0147]

[0148] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 9941.17. ESI-MS m / z: calculated value,

[0149] [M+8H] 8+ 1243.7, [M+9H] 9+ 1105.6, [M+10H] 10+ 995.1; observed value, [M+8H] 8+ 1243.8,

[0150] [M+9H] 9+ 1105.6, [M+10H] 10+ 995.1.

[0151] Example 15

[0152] Synthesis of the polypeptide compound of Structure 15

[0153]

[0154] The synthesis method was the same as that in Example 1. The target peak was collected and freeze-dried to obtain 0.12 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide was confirmed by LC-MS. The theoretical relative molecular weight was 10231.49. ESI-MS m / z: calculated value [M+7H] 7+ 1462.6, [M+8H] 8+ 1280.0, [M+9H]9+ 1137.8; Observed value [M + 7H] 7+ 1462.6,

[0155] [M + 8H] 8+ 1280.0, [M + 9H] 9+ 1137.7.

[0156] Example 16

[0157] Acute hypoglycemic effect of polypeptide compounds in ICR mice

[0158] Male ICR mice were randomly divided into groups of 6. After 7 days of adaptive feeding, the mice were deprived of food, the bedding was changed, and the mice in each group were fasted for more than 12 hours but allowed free access to water. Then, the mice in each group were subcutaneously injected with normal saline (blank control group, 10 ml / kg), semaglutide (positive control group, 30 nmol / kg), and Structures 1 - 5 (30 nmol / kg). Immediately after injection, the mice were tail - bled to measure blood glucose, and the blood glucose value at this moment was taken as - 60 min. 60 minutes after administration, the mice in each group were orally administered glucose (1.5 g / kg) through a gavage needle. At the following time points, 0 min, 15 min, 30 min, 60 min, 120 min, the blood glucose levels of the mice in each group were measured using a blood glucose meter.

[0159] The acute hypoglycemic effects of Structures 6 - 15 were measured in the same way. The grouping was Structures 6 - 10, Structures 11 - 15, for a total of three experiments.

[0160] As Figure 1 shown, the acute hypoglycemic experiment in ICR mice showed that Structures 3, 8, and 13 significantly improved the glucose tolerance level of the mice, had excellent hypoglycemic effects, and their hypoglycemic effects were better than that of semaglutide. These three compounds were used as preferred compounds for the next step of the food intake inhibition experiment.

[0161] Example 22

[0162] Food intake inhibition effect of polypeptide compounds in ICR mice

[0163] Male ICR mice were randomly divided into groups of 6 each. After 7 days of adaptive feeding, the mice were deprived of food, placed individually in cages with new bedding, and fasted for more than 12 hours while allowed free access to water. Then, the mice in each group were subcutaneously injected with normal saline (blank control group, 10 ml / kg), tirzepatide (positive control group, 30 nmol / kg), and Structure 3, 8, 13 (30 nmol / kg). Immediately 30 minutes after administration, the pre-weighed maintenance feed for the experimental mice was given and the feed weight at this time was recorded as 0 h. At 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 16 h, 20 h, 24 h, 36 h, and 48 h after the feed was given, the remaining feed weights of the mice in each group were weighed respectively. The food intake of the mice was calculated based on the remaining feed amount data, and a time-food intake curve was plotted.

[0164] As Figure 2 shown, the feeding inhibition experiment in ICR mice showed that Structure 13 significantly inhibited the food intake of the mice, had excellent feeding inhibition effect, and was far superior to the normal saline group. Its inhibitory effect was comparable to that of tirzepatide.

[0165] Example 23

[0166] Determination of the agonistic activity of polypeptide compounds on GLP-1, GIP, and Y2 receptors

[0167] The agonistic effect of a polypeptide compound on a receptor is determined by a functional assay that measures the cAMP response of a HEK-293 cell line stably expressing the human GLP-1 receptor, GIP receptor, and NPY-2 receptor. The HEK-293 cell line stably expressing the GLP-1 receptor, GIP receptor, or NPY-2 receptor is cultured in DMEM + 100 μg / ml Hygromycin B medium containing 10% fetal bovine serum at a culture temperature of 37 °C and a carbon dioxide concentration of 5%. Cell passage: Remove the old medium and wash once with PBS, then add 1 ml of cell dissociation buffer and incubate at 37 °C for about 2 min. When the cells detach from the bottom of the dish, add about 5 ml of pre-warmed complete medium at 37 °C. Gently pipette the cell suspension to separate the aggregated cells. Transfer the cell suspension to a sterile centrifuge tube, centrifuge at 1000 rpm for 5 min to collect the cells for experiments or subculture. To maintain the physiological activity of the cells, the cell confluence of the experimental cells is controlled at about 80%. Trypsin digestion to collect cells Next, prepare 1-fold stimulation buffer according to the kit instructions for use: Gradient dilute the polypeptide compound with DMSO, and then dilute the compound 10-fold with 1-fold stimulation buffer; Culture the stable transfection cell line to 80% confluence: Digest and collect the cells with cell dissociation buffer, and after counting, seed 9 μL / well in a 384-well plate (9 μL / human GLP-1R: 2000 cells; human GIPR: 2000 cells). Take 1 μL of the diluted positive compound and the test article and add them to the corresponding experimental wells, centrifuge and incubate at 37 °C for 30 min. Dilute Eu-cAMP to the working concentration with Detection buffer, and take 4.3 μL / well and add it to the corresponding experimental wells. Dilute ULight-anti-cAMP to the working concentration with Detection buffer, and then take 4.3 μL / well and add it to the corresponding experimental wells; Centrifuge and incubate at room temperature for 1 h. After incubation, use a PHERAstar FSX microplate reader to detect the readings at 665 nm and 620 nm. Plot Ratio (665 / 620) against the compound concentration, and use the nonlinear regression method of GraphPad Prism 10.0 software for curve fitting and EC 50 calculation.

[0168] Table 1: Agonistic activities of polypeptide compounds on human GLP-1R, GIPR, and Y2R

[0169]

[0170]

[0171] As shown in Table 1, for the GLP-1 / GIP / Y2 receptor triple agonist structures 11-15 with PYY-2 as the Y2R agonist moiety, the potencies for GLP-1R, GIPR, and Y2R were well retained. In particular, structure 13 with 4OEG as the linker showed significantly better GLP-1R and Y2R receptor activities than native GLP-1 and PYY 3-36 . Compared with native GIP, the GIPR activity of structure 13 was only reduced by about 100-fold, and the GLP-1R activity was well retained. Although the agonist activity of structure 8 for GLP-1R was comparable to that of native GLP-1, the agonist activities for both GLP-1R and GIPR were attenuated to varying degrees. Additionally, the Y2R activity of structure 8 was significantly weaker than that of structure 13, indicating that PYY 3-36 was less potent than PYY-2 in Y2R agonist activity and might have affected the interaction of the GLP-1R / GIPR dual agonist moiety linked to it with GIPR. Based on the high potencies of structure 13 for GLP-1R, GIPR, and Y2R, as well as the previous in vivo activity experimental results, structure 13 was selected for subsequent long-term studies in DIO mice

[0172] Example 24

[0173] Effects of polypeptide compounds on body weight and blood lipids in diet-induced obese mice (DIO)

[0174] Male C57BL / 6J mice, weighing about 22 g, were fed with a high-fat diet (D12492) from Research Diets for about 18 weeks to establish a DIO mouse model. If the body weight exceeded 42 g, the model was successfully established. The successfully modeled DIO mice were randomly divided into 4 groups of 6 mice each. They were subcutaneously injected once a day with normal saline (10 mL / kg), semaglutide (30 nmol / kg), cagrisema (30 nmol / kg), and structure 13 (30 nmol / kg) for an experiment with a treatment cycle of 21 days. During the experiment, the food intake and body weight of the mice were recorded daily. At the start and during the experiment, the fasting blood glucose level (measured after the mice had fasted overnight for 8 h) was measured using a blood glucose meter every 7 days, and the body weight of each DIO mouse was weighed using a balance. After the experiment ended, blood was collected from the fundus venous plexus of each group of mice. The blood was placed in the upper layer of the refrigerator and allowed to stand for 1.5 hours, and then centrifuged for 15 minutes to obtain serum. The prepared serum samples were sent to a testing company for determination of the contents of total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). After blood collection, the mice were sacrificed

[0175] Table 2: Changes in body weight of DIO mice after 21 days of treatment

[0176]

[0177]

[0178] ***: P < 0.001 compared with the blank control group; : P < 0.001 compared with semaglutide (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice per group.

[0179] As Figure 3 As shown in and Table 2, the structure 13 of the present invention can significantly reduce the body weight of DIO mice after continuous administration for 3 weeks in vivo, and the weight loss effect is significantly stronger than that of the positive control drugs semaglutide and cagrisema.

[0180] Table 3: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of DIO mice after 21 days of treatment

[0181] Sample (dose) Alanine aminotransferase (U / L) Aspartate aminotransferase (U / L) Blank control (saline group) 190±66 199±40 Semaglutide (30 nmol / kg) <![CDATA[101±29 *** > <![CDATA[116±25 *** > Structure 13 (30 nmol / kg) <![CDATA[79±11 *** > <![CDATA[108±11 *** >

[0182] ***: P < 0.001 compared with the blank control group (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice per group.

[0183] Table 4: Serum triglyceride (TG) and total cholesterol (TC) of DIO mice after 21 days of treatment

[0184] Sample (dose) Triglyceride (mmol / L) Total cholesterol (mmol / L) Blank control (saline group) 2.15±0.3 7.35±1.0 Semaglutide (30 nmol / kg) <![CDATA[1.23±0.1 *** > <![CDATA[5.15±0.8 *** > Structure 13 (30 nmol / kg) <![CDATA[0.99±0.1 *** > <![CDATA[4.24±0.5 *** >

[0185] ***: P < 0.001 compared with the blank control group (One-Way ANOVA, Tukey post hoc test). The results are expressed as the mean ± SD of 6 mice per group.

[0186] As shown in Table 3 and Table 4, the structure 13 of the present invention can significantly reduce the serum alanine aminotransferase and aspartate aminotransferase of DIO mice after continuous administration for 3 weeks in vivo, and significantly reduce the serum triglyceride and total cholesterol. And the effect is better than that of the control group semaglutide. It shows that the structure 13 of the present invention has a good effect on regulating lipids and improving liver function, and thus has the prospect of treating NAFLD and NASH.

[0187] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A long-acting GLP-1 / GIP / Y2 receptor triple agonist or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the long-acting GLP-1 / GIP / Y2 receptor triple agonist is shown in the following general formula (I): Among them, X1 is M or L; X2 is V or A; X3 is I or P; X4 is E or K; X5 is L or W; X6 is S or D; X7 is L or W; X8 is V or L; n is a natural number, and 1≤n≤8.

2. The long-acting GLP-1 / GIP / Y2 receptor triple agonist or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: X1 is L; X2 is A; X3 is P; X4 is K; X5 is W; X6 is D; X7 is W; X8 is L; n is a natural number, and 1≤n≤8.

3. The long-acting GLP-1 / GIP / Y2 receptor triple agonist or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The long-acting GLP-1 / GIP / Y2 receptor triple agonist is selected from one of the following sequences: Structure 1 Structure 2 Structure 3 Structure 4 Structure 5 Structure 6 Structure 7 Structure 8 Structure 9 Structure 10 Structure 11 Structure 12 Structure 13 Structure 14 Structure 15 4. The long-acting GLP-1 / GIP / Y2 receptor triple agonist or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The salt is a salt formed by a long-acting GLP-1 / GIP / Y2 receptor triple agonist and one of the following compounds: acetic acid, salicylic acid, lauric acid, cinnamic acid, lactic acid or succinic acid.

5. The method for synthesizing the long-acting GLP-1 / GIP / Y2 receptor triple agonist according to any one of claims 1 to 4, characterized in that: The following steps are involved: First, the resin is swollen, the Fmoc protecting group is removed, and then the Fmoc-Tyr-Rink amide-MBHA resin is synthesized. Then, the peptide chain is extended according to the amino acid sequence, the Lys side chain is modified, and finally the polypeptide on the resin is cleaved and purified to obtain the product.

6. A pharmaceutical composition comprising a therapeutically effective amount of at least one long-acting GLP-1 / GIP / Y2 receptor triple agonist or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable carrier and / or excipient.

7. The composition according to claim 6, characterized in that The dosage form of the composition is tablets, capsules, tinctures, inhalants, sprays, injections, films, patches, powders, granules, emulsions or suppositories as described in pharmacy.

8. Use of the long-acting GLP-1 / GIP / Y2 receptor triple agonist or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, or the pharmaceutical composition according to claim 6 or 7 in the preparation of drugs for treating metabolic diseases.

9. The use according to claim 8, characterized in that: The metabolic disease is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and / or dyslipidemia.

10. The use according to claim 9, characterized in that: The diabetes mellitus is T1DM, T2DM or gestational diabetes.

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