GLP-1 and PYY twin drug as well as preparation method and application thereof

Abstract

The invention discloses a GLP-1 and PYY twin drug and a preparation method and application thereof, the structure of the GLP-1 and PYY twin drug is shown in the following general formula (I), n is a natural number, and n is greater than or equal to 1 and less than or equal to 8. The GLP-1 and PYY twin drug can play the activities of GLP-1 and PYY at the same time to generate a synergistic effect. The GLP-1 and PYY twin drug disclosed by the invention is obtained by coupling a GLP-1 analogue and a PYY analogue through a pegylation scheme. The GLP-1 and PYY twin drugs can effectively reduce blood sugar and weight, and can be used for treating related diseases such as diabetes and obesity. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a GLP-1 and PYY twin drug and a preparation method and application thereof, belonging to the technical field of polypeptides. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar. According to statistics, the number of patients worldwide has reached 476 million, of which 463 million suffer from type 2 diabetes (T2DM). It is estimated that by 2045, there will be 700 million people suffering from the disease worldwide. Diabetes has the characteristics of high morbidity, high disability rate and high mortality rate, and has become a serious health problem facing the world. In addition, it is found in clinical practice that T2DM patients are often accompanied by obesity or overweight. In addition, the incidence and course of many metabolic syndromes such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and dyslipidemia are also closely related to obesity.

[0003] Incretins are hormones released by the gastrointestinal tract in response to food intake. Glucagon-like peptide-1 (GLP-1) is an incretin hormone produced by intestinal L cells in response to food intake, which achieves its various biological effects by stimulating GLP-1 receptors. GLP-1 has a blood sugar-dependent incretin secretion effect, which not only controls blood sugar but also avoids the risk of hypoglycemia in diabetes treatment. In addition to regulating blood sugar, GLP-1 can also organize the degeneration of pancreatic β cells, stimulate the proliferation and differentiation of β cells, and improve the course of diabetes from the source. In addition, GLP-1 can also increase satiety and suppress appetite by acting on the hypothalamic feeding center, delay gastrointestinal motility and gastric emptying, thereby achieving the effect of weight loss. However, the limited applicability of natural GLP-1 stems from its short half-life, which is mainly due to its inactivation by glomerular filtration and rapid removal of the N-terminal dipeptide by dipeptidyl peptide IV (DPP-IV). To solve this problem, various structural modification strategies have been developed around long-acting GLP-1 to extend the half-life of GLP-1. Currently, there are many drugs on the market, such as liraglutide, semaglutide, dulaglutide, loxenatide, etc. Although the current GLP-1 receptor agonists have shown commendable efficacy, inherent limitations still exist. Their weight loss efficacy will not continue to increase after the dose reaches a certain value, and the gastrointestinal side effects will become more obvious during the treatment of high-dose GLP-1 receptor agonists. Therefore, there is still a need to find safer and more tolerable GLP-1 receptor agonist therapeutic drugs that can effectively reduce weight and lower blood sugar.

[0004] PYY is a gastrointestinal hormone peptide composed of 36 amino acids. 1-36 and PYY 3-36There are two forms. After the N-terminal dipeptide is removed by the DPP-IV enzyme, the resulting PYY 3-36 Y 2 The receptor has good agonist selectivity. 3-36 It can bind to Y at the presynaptic terminal of NPY neurons in the hypothalamus 2 receptors induce anorexia. Therefore PYY 3-36 It can increase satiety, suppress appetite and thus reduce weight. 3-36 It can regulate energy homeostasis by increasing energy expenditure and reducing food intake, thus having a therapeutic effect on obesity.

[0005] At present, drugs for treating diabetes usually focus on a single target, and the therapeutic effect has certain limitations. Summary of the invention

[0006] Purpose of the invention: In order to solve the above technical problems, the present invention provides a GLP-1 and PYY twin drug and its preparation method and application, which can act on GLP-1 receptor, Y 2 Receptors that can simultaneously exert the activities of GLP-1 and PYY.

[0007] Technical solution: In order to achieve the above purpose, the present invention adopts the following technical solution:

[0008] A GLP-1 and PYY twin drug or a pharmaceutically acceptable salt thereof, wherein the structure of the GLP-1 and PYY twin drug is shown in the following general formula (I):

[0009]

[0010] Wherein, n is a natural number, and 1≤n≤8.

[0011] Preferably, the GLP-1 and PYY twin drugs are selected from one of the following structures:

[0012] (1) Structure 1

[0013]

[0014] (2) Structure 2

[0015]

[0016] (3) Structure 3

[0017]

[0018] (4) Structure 4

[0019]

[0020] (5) Structure 5

[0021]

[0022] As a specific embodiment, the salt is a salt formed by the twin drugs of GLP-1 and PYY and one of the following compounds: acetic acid, salicylic acid, lauric acid, cinnamic acid, lactic acid or succinic acid.

[0023] The GLP-1 and PYY twin drugs of the present invention are synthesized by coupling a GLP-1 analog and a PYY analog through a PEGylation scheme.

[0024] Specifically, the present invention also provides a method for synthesizing the GLP-1 and PYY twin drugs, comprising the following steps:

[0025] reacting a GLP-1 analog (II) with a pegylated PYY analog (III) to synthesize the GLP-1 and PYY twin drug;

[0026]

[0027] Wherein, n is the same as described above.

[0028] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of at least one of the GLP-1 and PYY twin drugs or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0029] 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.

[0030] Finally, the present invention provides the use of the GLP-1 and PYY twin drugs or their pharmaceutically acceptable salts, and the pharmaceutical composition in the preparation of drugs for treating metabolic diseases.

[0031] Preferably, the metabolic disease is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and / or dyslipidemia. In particular aspects, the drug is used to treat more than one metabolic disease or condition, for example, diabetes and obesity; obesity and dyslipidemia; diabetes and dyslipidemia; diabetes, dyslipidemia and obesity.

[0032] Further preferably, the diabetes is T1DM, T2DM or gestational diabetes.

[0033] LP-1, GLP-1 receptor corresponding to PYY, and Y 2The receptors all belong to the GPCR receptor family, and have similar protein structures and binding mechanisms, making it possible to have twin drugs that can act on these two receptors at the same time. The GLP-1 and PYY twin drugs of the present invention can simultaneously exert the activities of GLP-1 and PYY, producing a synergistic effect. GLP-1 analogs can lower blood sugar and suppress appetite; the appetite-suppressing effect of PYY analogs serves as an effective supplement to the weight loss and hypoglycemic effects produced by GLP-1. The two activities in the GLP-1 and PYY twin drugs cooperate with each other to form a feedback mechanism based on blood sugar concentration, which can both control blood sugar and reduce body weight. For the treatment of diabetes and obesity, GLP-1 and PYY twin drugs have significant advantages over single-target GLP-1 analogs.

[0034] Technical effect: Compared with the prior art, the present invention has the following advantages:

[0035] (1) The GLP-1 and PYY twin drugs of the present invention retain the therapeutic effect of GLP-1 analogs on diabetes while having the beneficial effects of PYY on metabolism, thereby producing a synergistic effect on sugar, fat, and energy metabolism, and having an excellent weight loss effect while effectively lowering blood sugar.

[0036] (2) The N-terminal amino acid His of the GLP-1 and PYY twin drugs of the present invention is consistent with that of natural GLP-1, and Tyr, which is commonly used in the design of GLP-1 / GIP receptor dual agonists, is not used, which brings better GLP-1 receptor agonist activity and hypoglycemic activity. In addition, the sequence structure of the GLP-1 and PYY twin drugs of the present invention makes the GLP-1 and PYY twin drugs of the present invention have a better hypoglycemic effect than the GLP-1 receptor agonists that have been marketed, as well as an excellent effect of inhibiting eating, which brings unexpected beneficial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The acute hypoglycemic effect of each test substance of the present invention on ICR mice after a single administration;

[0038] Figure 2 The inhibitory effect of each test substance of the present invention on ICR mice after a single administration; DETAILED DESCRIPTION

[0039] The present invention is further illustrated below in conjunction with specific examples.

[0040] Unless otherwise defined herein, the scientific and technical terms used in this application should have the meanings commonly understood by those of ordinary skill in the art. Generally, the terms and methods used in association with chemistry, biology, and pharmacology described herein are well known and commonly used in the art.

[0041] In addition, the abbreviations in the structural formula of the present invention represent specific amino acids, and their connection mode is the same as that of conventional polypeptides. Each adjacent amino acid is connected by a peptide bond (amide bond) to form a polypeptide analog with a certain sequence. The amino acids are abbreviated as follows according to the naming rules of IUPAC-IUB:

[0042] 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).

[0043] In addition, unless otherwise specified, all amino acid residues in the polypeptide compounds of the present invention are preferably in the L configuration.

[0044] In addition, the "-NH 2 The " part indicates that the amide group (-CONH 2 ).

[0045] In addition, in addition to natural amino acids, α-aminoisobutyric acid (Aib) is also used in the sequence of the present invention.

[0046] The present invention is illustrated by the following examples, but these examples are not to be construed as limiting the rights of the present invention.

[0047] Example 1

[0048] Synthesis of GLP-1 analogs (II)

[0049]

[0050] (1) Swelling of resin

[0051] Weigh 0.303 g (0.1 mmol equivalent) of RinkAmide MBHA resin with a loading of 0.330 mmol / g and put it into a 25 mL reactor. Wash the resin once with 7 mL of dichloromethane and methanol alternately, wash the resin twice with 7 mL of dichloromethane, then swell the resin with 7 mL of dichloromethane for 1 hour, and finally wash the resin three times with 7 mL of DMF.

[0052] (2) Removal of Fmoc protecting group from resin

[0053] The swollen resin was transferred to a PSI-200 semi-automatic peptide synthesizer, and 7 mL of 20% piperidine / DMF (v / v) was added to react at room temperature for 30 min. The deprotection solution was filtered off and the resin was washed 4 times with 7 mL of DMF for 1.5 min each time to obtain the Rink resin with the Fmoc protecting group removed.

[0054] (3) Synthesis of Fmoc-Cys-Rink amide-MBHA Resin

[0055] Weigh Fmoc-Cys(Trt)-OH (0.4mmol), dissolve it in 3mL of DMF, add 2mL of DIC / HOBt (0.4mmol / 0.44mmol) condensation agent, pre-activate for 30min, add the activated amino acid into the reactor, shake and react at room temperature for 2h, filter out the reaction solution and wash the resin 4 times with 7mL of DMF, use Kaiser reagent to detect whether the reaction coupling is complete, if not, couple it twice.

[0056] (4) Peptide chain extension

[0057] According to the sequence of the peptide chain, the above deprotection and coupling steps are repeated to connect the corresponding amino acids in sequence until the peptide chain is synthesized. The Lys site with modified side chain at position 16 adopts the Fmoc-Lys(Dde)-OH protection strategy, and the His at the N-terminus uses Boc-His(Boc)-OH.

[0058] (5) Modification of Lys side chain

[0059] After the peptide chain is synthesized, 7mL2% hydrazine hydrate / DMF (v / v) is added to selectively remove the Dde protecting group of Lys at position 16. After the Dde protecting group is removed, 0.4mmol of Fmoc-AEEA-OH, 0.4mmol of DIC and 0.44mmol of HOBt are added, and the reaction is shaken for 2h. After the Fmoc protecting group is removed, 0.4mmol of Fmoc-AEEA-OH, 0.4mmol of DIC and 0.44mmol of HOBt are added again, and the reaction is shaken for 2h. After the Fmoc protecting group is removed, 0.4mmol of Fmoc-Glu-OtBu, 0.4mmol of DIC and 0.44mmol of HOBt are added, and the reaction is shaken for 2h. After the Fmoc protecting group is removed, 0.4mmol of tert-butyl octadecane dioate, 0.4mmol of DIC and 0.44mmol of HOBt are added, and the reaction is shaken for 2h. After the reaction was complete, the resin was washed four times with 7 mL DMF.

[0060] (6) Peptide cleavage

[0061] The resin with the polypeptide obtained above was transferred to a round-bottom bottle, and the resin was cut with 5 mL of cutting agent Reagent R (TFA / thioanisole / phenol / EDT, 90:5:3:2, V / V). The reaction was kept at 30°C in an oil bath for 2 h, and the cutting solution was poured into 20 mL of ice ether. After refrigerated centrifugation, the crude product was washed three times with 15 mL of ice ether and finally dried with nitrogen to obtain the crude peptide.

[0062] (7) Purification and characterization of peptides

[0063] The crude target polypeptide was dissolved in water, filtered with a 0.25 μm microporous filter membrane, and then purified by Shimadzu preparative reverse phase HPLC system. The chromatographic conditions were C18 reverse phase preparative column (250 mmX20 mm, 12 μm); mobile phase A: 0.1% TFA / water (V / V); mobile phase B: methanol (V / V); flow rate was 8 mL / min; detection wavelength was 214 nm. A linear gradient (20% B ~ 70% B / 30 min) was used for elution, and the target peak was collected. After removing methanol, lyophilization was performed to obtain 0.13 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 mass is 5015.8. ESI-MS m / z: calculated value [M+3H] 3+ 1672.9, [M+4H] 4+ 1254.9, [M+5H] 5+ 1004.1; observed value [M+3H] 3+ 1672.9,

[0064] [M+4H] 4+ 1254.6, [M+5H] 5+ 1003.9.

[0065] Example 2

[0066] MAL-AEEA-PYY 3-36 Synthesis

[0067]

[0068] (1) Swelling of resin

[0069] Weigh 0.303 g (0.1 mmol equivalent) of RinkAmide MBHA resin with a loading of 0.330 mmol / g and put it into a 25 mL reactor. Wash the resin once with 7 mL of dichloromethane and methanol alternately, wash the resin twice with 7 mL of dichloromethane, then swell the resin with 7 mL of dichloromethane for 1 hour, and finally wash the resin three times with 7 mL of DMF.

[0070] (2) Removal of Fmoc protecting group from resin

[0071] The swollen resin was transferred to a PSI-200 semi-automatic peptide synthesizer, and 7 mL of 20% piperidine / DMF (v / v) was added to react at room temperature for 30 min. The deprotection solution was filtered off and the resin was washed 4 times with 7 mL of DMF for 1.5 min each time to obtain the Rink resin with the Fmoc protecting group removed.

[0072] (3) Synthesis of Fmoc-Tyr-Rink amide-MBHA Resin

[0073] Weigh Fmoc-Tyr(tBu)-OH (0.4mmol), dissolve it in 3mL of DMF, add 2mL of DIC / HOBt (0.4mmol / 0.44mmol) condensation agent, pre-activate for 30min, add the activated amino acid into the reactor, shake and react at room temperature for 2h, filter out the reaction solution and wash the resin 4 times with 7mL of DMF, use Kaiser reagent to detect whether the reaction coupling is complete, if not, couple it twice.

[0074] (4) Peptide chain extension

[0075] According to the sequence of the peptide chain, the above deprotection and coupling steps are repeated to connect the corresponding amino acids in sequence until the peptide chain is synthesized.

[0076] (5) Peptide cleavage

[0077] The resin with the polypeptide obtained above was transferred to a round-bottom bottle, and the resin was cut with 5 mL of cutting agent Reagent R (TFA / thioanisole / phenol / EDT, 90:5:3:2, V / V). The reaction was kept at 30°C in an oil bath for 2 h, and the cutting solution was poured into 20 mL of ice ether. After refrigerated centrifugation, the crude product was washed three times with 15 mL of ice ether and finally dried with nitrogen to obtain the crude peptide.

[0078] (6) Purification and characterization of peptides

[0079] The crude target polypeptide was dissolved in water, filtered with a 0.25 μm microporous filter membrane, and then purified by Shimadzu preparative reverse phase HPLC system. The chromatographic conditions were C18 reverse phase preparative column (250 mmX20 mm, 12 μm); mobile phase A: 0.1% TFA / water (V / V); mobile phase B: methanol (V / V); flow rate was 8 mL / min; detection wavelength was 214 nm. A linear gradient (20% B ~ 70% B / 30 min) was used for elution, and the target peak was collected. After removing methanol, lyophilization was performed 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 mass is 4345.81. ESI-MS m / z: calculated value [M+3H] 3+ 1449.7, [M+4H] 4+1087.4, [M+5H] 5+ 870.1; observed value [M+3H] 3+ 1449.4,

[0080] [M+4H] 4+ 1087.3, [M+5H] 5+ 870.0.

[0081] Example 3

[0082] MAL-2AEEA-PYY 3-36 Synthesis

[0083]

[0084] The synthesis method is the same as in Example 2. The target peak is collected, and after removing methanol, lyophilization is performed to obtain 0.10 g of pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by LC-MS. The theoretical relative molecular mass is 4491.0. ESI-MS m / z: calculated value [M+3H] 3+ 1497.9, [M+4H] 4+ 1123.7, [M+5H] 5+ 899.2; observed value [M+3H] 3+ 1497.8,

[0085] [M+4H] 4+ 1123.6, [M+5H] 5+ 899.1.

[0086] Example 4

[0087] MAL-4AEEA-PYY 3-36 Synthesis

[0088]

[0089] The synthesis method is the same as in Example 2. The target peak was collected, and after removing methanol, lyophilization was performed 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 mass was 4781.3. ESI-MS m / z: calculated value [M+3H] 3+ 1594.7, [M+4H] 4+ 1196.3, [M+5H] 5+ 957.2; observed value [M+3H] 3+ 1594.5,

[0090] [M+4H] 4+ 1196.1, [M+5H] 5+ 957.3.

[0091] Example 5

[0092] MAL-6AEEA-PYY 3-36 Synthesis

[0093]

[0094] The synthesis method is the same as in Example 2. The target peak was collected, and after removing methanol, lyophilization was performed 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 mass was 5071.6. ESI-MS m / z: calculated value [M+3H] 3+ 1691.5, [M+4H] 4+ 1268.9, [M+5H] 5+ 1015.3; observed value [M+3H] 3+ 1691.2,

[0095] [M+4H] 4+ 1268.6, [M+5H] 5+ 1015.3.

[0096] Example 6

[0097] MAL-8AEEA-PYY 3-36 Synthesis

[0098]

[0099] The synthesis method is the same as in Example 2. The target peak was collected, and after removing methanol, lyophilization was performed 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 mass was 5361.8. ESI-MS m / z: calculated value [M+3H] 3+ 1788.3, ​​[M+4H] 4+ 1341.4, [M+5H] 5+ 1073.3; observed value [M+3H] 3+ 1788.2,

[0100] [M+4H] 4+ 1341.4, [M+5H] 5+ 1073.3.

[0101] Example 7

[0102] Synthesis of structure 1

[0103]

[0104] 10.4 mg of GLP-1 analog (II) and 9.6 mg of MAL-AEEA-PYY3-36 Dissolve them separately in 50 ml NMP by ultrasonication, and then mix the two. Use DIPEA (30-60 ml) to catalyze the specific reaction between thiol and maleimide for 60 minutes. Then add 0.1% TFA / H20 to stop the reaction, and purify the reaction mixture directly by RP-HPLC using a preparative C18 reverse phase preparative column (250 mmX20 mm, 12 μm). Mobile phase A: 0.1% TFA / water (V / V); mobile phase B: methanol (V / V); flow rate is 7 mL / min; detection wavelength is 214 nm. Use a linear gradient (20% B~70% B / 30 min) to elute, collect the target peak, remove methanol and freeze-dry to obtain a pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9361.6. ESI-MS m / z: calculated value [M+5H] 5+ 1873.3, [M+6H] 6+ 1561.3, [M+7H] 7+ 1338.4, [M+8H] 8+ 1171.2; observed value [M+5H] 5+ 1873.4,

[0105] [M+6H] 6+ 1561.1, [M+7H] 7+ 1338.4, [M+8H] 8+ 1171.2.

[0106] Synthesis of Structure 2

[0107]

[0108] 10.5 mg of GLP-1 analog (II) and 10.1 mg of MAL-2AEEA-PYY 3-36 Dissolve them separately in 50 ml NMP by ultrasonication, and then mix the two. Use DIPEA (30-60 ml) to catalyze the specific reaction between thiol and maleimide for 60 minutes. Then add 0.1% TFA / H20 to stop the reaction, and purify the reaction mixture directly by RP-HPLC using a preparative C18 reverse phase preparative column (250 mmX20 mm, 12 μm). Mobile phase A: 0.1% TFA / water (V / V); mobile phase B: methanol (V / V); flow rate is 7 mL / min; detection wavelength is 214 nm. Use a linear gradient (20% B~70% B / 30 min) to elute, collect the target peak, remove methanol and freeze-dry to obtain a pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9506.7. ESI-MS m / z: calculated value [M+7H] 7+ 1359.1, [M+8H]8+ 1189.3, [M+9H] 9+ 1057.3; observed value [M+7H] 7+ 1359.0, [M+8H] 8+ 1189.4,

[0109] [M+9H] 9+ 1057.2.

[0110] Example 9

[0111] Synthesis of peptide compound of structure 3

[0112]

[0113] 10.4 mg of GLP-1 analog (II) and 10.2 mg of MAL-4AEEA-PYY 3-36 Dissolve them separately in 50 ml NMP by ultrasonication, and then mix the two. Use DIPEA (30-60 ml) to catalyze the specific reaction between thiol and maleimide for 60 minutes. Then add 0.1% TFA / H20 to stop the reaction, and purify the reaction mixture directly by RP-HPLC using a preparative C18 reverse phase preparative column (250 mmX20 mm, 12 μm). Mobile phase A: 0.1% TFA / water (V / V); mobile phase B: methanol (V / V); flow rate is 7 mL / min; detection wavelength is 214 nm. Use a linear gradient (20% B~70% B / 30 min) to elute, collect the target peak, remove methanol and freeze-dry to obtain a pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 9797.0. ESI-MS m / z: calculated value [M+7H] 7+ 1400.6, [M+8H] 8+ 1225.6, [M+9H] 9+ 1089.6; observed value [M+7H] 7+ 1400.6, [M+8H] 8+ 1225.6,

[0114] [M+9H] 9+ 1089.6.

[0115] Example 10

[0116] Synthesis of structure 4

[0117]

[0118] 11.0 mg of GLP-1 analog (II) and 11.2 mg of MAL-6AEEA-PYY 3-36Dissolve them separately in 50 ml NMP by ultrasonication, and then mix the two. Use DIPEA (30-60 ml) to catalyze the specific reaction between thiol and maleimide for 60 minutes. Then add 0.1% TFA / H20 to stop the reaction, and purify the reaction mixture directly by RP-HPLC using a preparative C18 reverse phase preparative column (250 mmX20 mm, 12 μm). Mobile phase A: 0.1% TFA / water (V / V); mobile phase B: methanol (V / V); flow rate is 7 mL / min; detection wavelength is 214 nm. Use a linear gradient (20% B~70% B / 30 min) to elute, collect the target peak, remove methanol and freeze-dry to obtain a pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 10087.3. ESI-MS m / z: calculated value [M+6H] 6+ 1682.2, [M+7H] 7+ 1442.0, [M+8H] 8+ 1261.9, [M+9H] 9+ 1121.8; observed value [M+6H] 6+ 1682.2,

[0119] [M+7H] 7+ 1441.9, [M+8H] 8+ 1261.9, [M+9H] 9+ 1121.8.

[0120] Embodiment 11

[0121] Synthesis of structure 5

[0122]

[0123] 10.8 mg of GLP-1 analog (II) and 11.6 mg of MAL-8AEEA-PYY 3-36 Dissolve them separately in 50 ml NMP by ultrasonication, and then mix the two. Use DIPEA (30-60 ml) to catalyze the specific reaction between thiol and maleimide for 60 minutes. Then add 0.1% TFA / H20 to stop the reaction, and purify the reaction mixture directly by RP-HPLC using a preparative C18 reverse phase preparative column (250 mmX20 mm, 12 μm). Mobile phase A: 0.1% TFA / water (V / V); mobile phase B: methanol (V / V); flow rate is 7 mL / min; detection wavelength is 214 nm. Use a linear gradient (20% B~70% B / 30 min) to elute, collect the target peak, remove methanol and freeze-dry to obtain a pure product with a purity greater than 98%. The molecular weight of the target polypeptide is confirmed by MS. The theoretical relative molecular mass is 10377.6. ESI-MS m / z: calculated value [M+7H]7+ 1483.5, [M+8H] 8+ 1298.2, [M+9H] 9+ 1154.1; observed value [M+7H] 7+ 1483.6, [M+8H] 8+ 1298.2,

[0124] [M+9H] 9+ 1154.1.

[0125] Example 12

[0126] Acute hypoglycemic effect of peptide compounds in ICR mice

[0127] Male ICR mice were randomly divided into groups of 6 mice per group. After 7 days of adaptive feeding, the mice were fed with feed and replaced with new bedding, and each group of mice was fasted for more than 12 hours but allowed to drink water freely. Then each group of mice was subcutaneously injected with normal saline (blank control group, 10ml / kg), semaglutide (positive control group, 30nmol / kg), and structure 3 (30nmol / kg). The mice were immediately cut off and blood was collected to measure blood sugar, and the blood sugar value at this moment was taken as -60min. After 60 minutes of administration, each group of mice was orally given glucose (1.5g / kg) by gavage needle, and the blood sugar level of each group of mice was measured with a blood glucose meter at the following time points: 0min, 15min, 30min, 60min, and 120min.

[0128] like Figure 1 As shown, the acute hypoglycemic experiment in ICR mice showed that structure 3 significantly improved the glucose tolerance level of mice and had an excellent hypoglycemic effect, which was better than semaglutide.

[0129] Example 13

[0130] Inhibitory effect of peptide compounds on feeding in ICR mice

[0131] Male ICR mice were randomly divided into groups of 6 mice per group. After 7 days of adaptive feeding, the mice were deprived of feed, and the mice were placed in single cages for feeding, and new bedding was replaced. The mice in each group were fasted for more than 12 hours but allowed to drink water freely. Then each group of mice was subcutaneously injected with normal saline (blank control group, 10ml / kg), tirzepatide (positive control group, 30nmol / kg), and structure 3 (30nmol / kg). Immediately after 30min of administration, the pre-weighed experimental mice were given maintenance feed and the feed weight at this time was recorded as 0h. At 1h, 2h, 3h, 4h, 5h, 6h, 8h, 10h, 12h, 16h, 20h, 24h, 36h and 48h after the feed was given, the weight of the remaining feed of each group of mice was weighed respectively. The food intake of the mice was calculated based on the remaining feed amount data, and the time-food intake curve was drawn.

[0132] like Figure 2 As shown, the feeding inhibition experiment in ICR mice showed that structure 3 significantly inhibited the food intake of mice and had an excellent feeding inhibition effect compared with normal saline, but its inhibitory effect was slightly lower than that of tirzepatide, but there was no significant difference.

[0133] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A GLP-1 and PYY twin drug or a pharmaceutically acceptable salt thereof, wherein the structure of the GLP-1 and PYY twin drug is shown in the following general formula (I): in, n is a natural number, and 1≤n≤8.

2. The GLP-1 and PYY twin drug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The GLP-1 and PYY twin drugs are selected from one of the following structures: (1) Structure 1 (2) Structure 2 (3) Structure 3 (4) Structure 4 (5) Structure 5 3. The GLP-1 and PYY twin drug or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The salt is a salt formed by the twin drugs of GLP-1 and PYY and one of the following compounds: acetic acid, salicylic acid, lauric acid, cinnamic acid, lactic acid or succinic acid.

4. The method for synthesizing the GLP-1 and PYY twin drugs according to any one of claims 1 to 3, characterized in that: The following steps are involved: reacting a GLP-1 analog (II) with a pegylated PYY analog (III) to synthesize the GLP-1 and PYY twin drug; Wherein, n is the same as described in any one of claims 1-3.

5. A pharmaceutical composition comprising a therapeutically effective amount of at least one GLP-1 and PYY twin drug according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

6. The composition according to claim 5, 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.

7. Use of the GLP-1 and PYY twin drug or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, or the pharmaceutical composition according to claim 5 or 6 in the preparation of drugs for treating metabolic diseases.

8. The use according to claim 7, characterized in that: The metabolic disease is diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and / or dyslipidemia.

9. The use according to claim 8, characterized in that: The diabetes mellitus is T1DM, T2DM or gestational diabetes.

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