CN107253985B - Design and application of long-acting hypoglycemic peptide - Google Patents

Abstract

The invention relates to a design of a novel long-acting hypoglycemic peptide and a synthetic method thereof. The Exendin-4 derivative with longer pharmacological action time is obtained by modifying Exendin-4, the synthesis of target polypeptide is quickly realized by an orthogonal protection strategy solid-phase synthesis method, and the long-acting glycopeptide is obtained by purifying and freeze-drying a crude product.

Description

Design and application of long-acting hypoglycemic peptide

Technical Field

The invention relates to a design and application of long-acting hypoglycemic peptide in the field of diabetes treatment.

Background

Diabetes mellitus is the third most serious chronic non-infectious disease threatening human health after tumor and cardiovascular disease. Currently, about 3 million diabetics worldwide are predicted to increase to 5 million by 2025. Clinically, intensive insulin therapy is used to delay the progression of diabetes, but insulin injections risk hypoglycemia. The treatment effect is influenced by factors such as dosage, injection position, injection route and the like, the individual difference is large, and serious hypoglycemia side effect can occur due to the fact that insulin is used carelessly.

Glucagon-like peptide-1 (GLP-1) is a glucose-dependent incretin, GLP-1 stimulates insulin secretion without hypoglycemia, and the glucose-dependent insulinotropic secretion characteristic avoids the hypoglycemia risk frequently existing in the treatment of diabetes. Therefore, GLP-1 has wide development prospect as a medicament for treating type 2 diabetes.

However, native GLP-1 has many advantages in treating diabetes, for example, it is rapidly degraded by dipeptidyl peptidase IV (DPP-IV) in vivo. DPP-IV specifically recognizes the alanine (Ala) residue at position 8 at the N-terminus of GLP-1, and cleaves the dipeptide from the alanine (Ala) at position 8 at the N-terminus of the peptide chain to convert it to an inactive form with an in vivo half-life of only about 5 min. The N-terminal of GLP-1 peptide chain is a binding site with GLP-1 receptor, and if the histidine residue of the GLP-1 peptide chain is lost, the GLP-1 completely loses bioactivity. The currently widely used modification strategy for prolonging the in vivo half-life of GLP-1 is mainly to modify the 8 th position, so that GLP-1 can resist the degradation of DPP-IV enzyme, and in addition, the purpose can be achieved by exchanging amino acids at the 8 th position and the 9 th position of the N end of a GLP-1 peptide chain. Exenatide is a typical short-acting GLP-1 receptor agonist that reduces the metabolism of the DPP-IV enzyme. However, since GLP-1 is eliminated by renal rapid filtration, the half-life of GLP-1 is only prolonged to some extent against degradation by the DPP-IV enzyme.

In the patent, a long-acting hypoglycemic peptide is designed and synthesized by adopting a cysteine-maleimide conjugation strategy on the basis of a GLP-1 receptor strong agonist exenatide (Exendin-4). According to the strategy, a small molecular group is conveniently and efficiently introduced through the Michael addition reaction between the sulfydryl of cysteine and maleimide, and the problems of poor selectivity, inconvenient reaction and the like caused by the fact that lysine is used as a small molecular group connecting arm in the early GLP-1 receptor long-acting agonist research and development process can be solved.

In the patent, the dicoumarin small molecule is conjugated with the exenatide for the first time, and the dicoumarin small molecule group has higher serum albumin binding rate, so that the binding of the conjugate and the serum albumin can be enhanced, the half-life period of the compound is prolonged to a great extent, and the rapid kidney filtration and metabolic inactivation of the compound can be reduced, so that the half-life period and the in-vivo blood glucose reduction action time of the compound are remarkably prolonged.

In a word, the compounds have better druggability, can reduce the pain of the patient caused by multiple injections and improve the compliance of the patient, and are medicaments with great development prospects in the field of type 2 diabetes treatment.

Disclosure of Invention

The invention relates to a long-acting hypoglycemic peptide with ether bond, which is characterized in that the amino acid sequence of the polypeptide is as follows:

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-chemically modified Cys-Gln-Met-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂(ii) a Or His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-chemically modified Cys-NH₂(ii) a Or His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-chemically modified Cys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Glu-Gly-Pro-Ser-Gly-Arg-Pro-Pro-Pro-Ser-NH₂(ii) a Or His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Ala-Val-Arg-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Glu-Gly-Gly-Pro-Ser-Gly-Arg-Pro-Pro-Pro-Ser-chemically modified Cys-NH-₂；

Wherein the chemically modified Cys structure is:

n is selected from 0 to 20.

In a preferred embodiment of the present invention, the present invention is characterized in that,

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-chemically modified Cys-Gln-Met-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂(ii) a Or His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Gly-Ala-Pro-Pro-Pro-Ser-chemically modified Cys-NH₂(ii) a Or His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-chemically modified Cys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Glu-Gly-Pro-Ser-Gly-Arg-Pro-Pro-Pro-Ser-NH₂(ii) a Or His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Ala-Val-Arg-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Glu-Gly-Gly-Pro-Ser-Gly-Arg-Pro-Pro-Pro-Ser-chemically modified Cys-NH-₂；

Wherein the chemically modified Cys structure is:

n is taken from 11 to 16.

In one embodiment, the present invention relates to a long acting glycopeptide having the sequence:

the invention also provides a pharmaceutical composition comprising a therapeutically effective amount of at least one of the above compounds and pharmaceutically acceptable salts thereof, or a pharmaceutically acceptable carrier or diluent.

The invention further provides application of the compound and pharmaceutically acceptable salts thereof, or pharmaceutically acceptable carriers or diluents in preparing medicines for preventing and treating diabetes.

The compound provided by the invention has a remarkable blood sugar reducing effect, is stable in chemical property, has the blood sugar reducing effect maintaining time of more than 40 hours, and is remarkably improved compared with endogenous GLP-1 (half-life period of 2-3 min) or a marketed drug exenatide (half-life period of 2.4 hours). Meanwhile, adverse reactions such as local pruritus and the like caused by a long-acting method in pharmaceutics are avoided.

The invention also provides a preparation method of the compound, and the target compound is efficiently and quickly synthesized by adopting a solid-phase synthesis strategy.

The pharmacological test method and result of the in vivo and in vitro sugar reduction of the long-acting hypoglycemic peptide are as follows:

(1) receptor agonistic activity experiment of long-acting hypoglycemic peptide

HEK293 cells are co-transfected with cDNA encoding GLP-1R, the cell lines express and Western Blot is used for detecting the protein level of GLP-1R in the constructed HEK293 cells so as to investigate whether a GLP-R-HEK293 cell strain with stable and high expression is established. In the receptor agonistic activity assay, first, cells were seeded in a 96-well plate, and after 2h, the compounds were dissolved in DMSO, diluted to different fold using a medium containing 0.1% bovine serum albumin, and added to the co-transfected GLP-1R-HEK293 cells. After 20min incubation, the corresponding cAMP values were detected using an ELISA kit from Cisbo and the EC of the compound was calculated after non-linear regression₅₀Numerical values.

TABLE 1 Long acting hypoglycemic peptide receptor agonistic activity

As shown in Table 1, the agonistic activity of all long-acting hypoglycemic peptides on GLP-1R is reserved, and compared with the marketed drug liraglutide, the agonistic activity of the compound on GLP-1R is obviously improved. Wherein the compound of seq.id NO: the agonistic activity of 2 on GLP-1R is obviously improved, and compared with liraglutide, the agonistic activity is improved by 4.2 times.

(2) Abdominal glucose tolerance experiment of long-acting hypoglycemic peptide

Normal kunming mice, randomly grouped, 8 mice per group, were housed in standardized animal houses. Fasted for 12 hours prior to the experiment, only drinking water was given. Before administration, the initial blood sugar value of each group of mice is measured and determined to be-30 min, and then 25nmol/kg of exenatide or long-acting hypoglycemic peptide is injected into the abdominal cavity. After 30min, 18mmol/kg glucose solution was injected intraperitoneally for 0 min. Measuring blood glucose level with a blood glucose meter at 0, 15, 30, 45, 60, 120min, and detecting blood glucose lowering activity of long-acting hypoglycemic peptide.

TABLE 2 Abdominal glucose tolerance test results of long-acting hypoglycemic peptides

Results are expressed as mean±SD，*P＜0.05，**P＜0.01，***P＜0.001vs saline.

As shown in Table 2, the results of the hypoglycemic experiments show that the long-acting hypoglycemic peptide of the invention has the same hypoglycemic effect as exenatide.

(3) Alternate-day blood sugar reduction experiment of long-acting hypoglycemic peptide

After the abdominal glucose tolerance test is finished, the mice are subjected to the abdominal glucose tolerance test again after drinking water for 10h and fasting for 12 h. Each group of mice was intraperitoneally injected with 18mmol/kg of glucose solution for 0min, and blood glucose levels were measured with a glucometer at 0, 15, 30, 45, 60, and 120 min.

TABLE 3 Effect of long-acting hypoglycemic peptides on alternate days

Results are expressed as mean±SD，*P＜0.05，**P＜0.01，***P＜0.001vs saline.

As shown in Table 3, the results of hypoglycemic experiments show that the long-acting hypoglycemic peptide involved in the invention still has hypoglycemic effect after being metabolized for 24 hours in vivo, and the exenatide is inactive for a long time. The biological half-life period of the long-acting hypoglycemic peptide obtained after modification is obviously prolonged and reaches more than 30 h.

(4) Blood sugar stabilizing experiment of long-acting hypoglycemic peptide

The blood glucose of STZ-induced diabetes model mice was determined, and mice with blood glucose values higher than 20mmol/L were selected for random grouping, 6 mice per group, and mice were fed freely during the experiment. The positive control group is injected with exenatide or liraglutide in the abdominal cavity, the negative control group is injected with normal saline in the abdominal cavity, and the administration groups are respectively injected with long-acting hypoglycemic peptide. Compounds were administered at 0h and blood glucose levels were determined using a glucometer at 0, 1, 2, 3, 4, 6, 8, 12, 24, 36 and 48h, respectively. The evaluation index is the time when the blood sugar value of the mice is lower than 8.35mmol/L after the compound is injected into the abdominal cavity.

TABLE 4 Stable blood sugar test of long-acting hypoglycemic peptides

Results are expressed as mean±SD，*P＜0.05，**P＜0.01，***P＜0.001vs saline.

As can be seen from table 4, seq.id NO: 1 the time for stabilizing the blood sugar can reach 48.4h, which is far higher than 11.2h of the liraglutide. The long-acting hypoglycemic peptide can achieve better long-acting hypoglycemic effect and has the potential of being developed into a hypoglycemic medicament which is administrated once in 2 days under the same experimental conditions compared with the medicaments of liraglutide and exenatide on the market.

The invention has the advantages that:

1. the provided long-acting hypoglycemic peptide has stronger GLP-1 receptor agonistic activity, and can achieve better long-acting hypoglycemic effect compared with the marketed drugs exenatide and liraglutide.

2. The provided long-acting hypoglycemic peptide has an excellent long-acting hypoglycemic effect, the blood sugar stabilizing time is up to more than 48.4h, and the long-acting hypoglycemic peptide is obviously prolonged compared with the liraglutide which is administrated once every day, has better drug forming property, can reduce the pain of patients after being administrated for many times, and is a drug with great development prospect in the existing new chemical entities.

3. The provided long-acting hypoglycemic peptide has the advantages of high yield, short synthesis period, easy purification of crude products, low production cost and easy industrial automatic production.

In conclusion, the long-acting hypoglycemic peptide provided by the invention has a brand new structure, is more stable than an exenatide prototype or endogenous GLP-1, has a longer hypoglycemic action time than the marketed drug liraglutide, is suitable to be used as a novel active ingredient of a diabetes treatment drug, and brings a new breakthrough to the field of diabetes treatment.

Detailed Description

The following abbreviations are used throughout the specification:

ala: alanine;arg: arginine; asn: asparagine; asp: aspartic acid; DCM: dichloromethane; DIC: n, N' -diisopropylcarbodiimide; DIEA: n, N' -diisopropylethylamine; DMAP: 4-dimethylaminopyridine; DMF: dimethylformamide; DMSO, DMSO: dimethyl sulfoxide; edc.hcl: 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride; ESI-MS: electrospray mass spectrometry; et (Et)₃N: triethylamine; fmoc: n-9-fluorenylmethyloxycarbonyl; gln: (ii) glutamine; glu: glutamic acid; gly: glycine; HBTU: benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate; his: (ii) histidine; HOBt: 1-hydroxy-benzotriazole; HPLC: high performance liquid chromatography; ile: isoleucine; leu: leucine; lys: lysine; met: methionine; NMP: n-methyl pyrrolidone; phe: phenylalanine; pro: (ii) proline; ser: serine; thr: threonine; trp: tryptophan; tyr: tyrosine; val: valine.

The present invention is illustrated by the following examples, which are not to be construed as limiting the invention in any way.

Example 1

Solid phase synthesis of (1).

1. Synthesis of cysteine-modified polypeptide peptide chain

1.1 swelling of the resin

Weighing 50mg of Fmoc-Rink amide-MBHA Resin (the substitution degree is 0.4mmol/g), swelling with 7mL of DCM for 30min, filtering off DCM by suction, swelling with 10mL of NMP for 30min, and finally washing with 7mL of NMP, DCM and NMP respectively.

1.2 removal of Fmoc protecting group

Putting the swelled resin into a reactor, adding 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt, reacting for 1min, and filtering the solution after the reaction is finished; then 7mL of a 25% piperidine/NMP (V/V) solution containing 0.1M HOBt was added, the reaction was carried out for 4min, and after completion, the solution was filtered off and washed with NMP. The resin was obtained with the Fmoc protecting group initially attached removed.

1.3 Synthesis of Fmoc-Ser (tBu) -Rink amide-MBHA Resin

Fmoc-Ser (tBu) -OH (15.3mg, 0.04mmol), HBTU (15.1mg, 0.04mmol), HOBt (5.4mg, 0.04mmol) and DIPEA (13.9. mu.L, 0.08mmol) were dissolved in NMP 10mL, and this solution was added to the resin obtained in step 1.1, reacted for 7min, after which the reaction was filtered off and the resin was washed 3 times with 7mL each of DCM and NMP.

1.4 detection of coupling efficiency

Washing a small amount of resin particles with DMF, putting into a transparent vial, adding 3 drops of 1% bromophenol blue solution, shaking at normal temperature for 3 minutes, and determining that the resin is positive if it is blue and transparent if it is negative. If negative, the next coupling cycle can be entered.

1.5 elongation of peptide chain

And repeating the steps of deprotection and coupling according to the sequence of the peptide chain, sequentially connecting corresponding amino acids, and sequentially connecting corresponding amino acids until the peptide chain is synthesized, thereby obtaining the resin connected with the polypeptide chain.

1.6 cleavage of the Polypeptides on the resin

The resin with polypeptide chain obtained above was placed in a reaction flask, 10mL of cleavage agent Reagent K (TFA/thioanisole/water/phenol/EDT, 82.5: 5: 2.5, V/V) was added, shaken at 0 ℃ for 30min, and reacted at room temperature for 3 h. After the reaction was completed, the reaction mixture was filtered with suction, washed three times with a small amount of TFA and DCM, and the filtrates were combined. Adding the filtrate into a large amount of glacial ethyl ether to separate out white flocculent precipitate, freezing and centrifuging to obtain a crude product of the target polypeptide. The final yield was 89.2% of crude 37.1mg of compound.

2. Synthesis of chemically modified groups

Synthesis of 12-maleicamidododecanoic acid

Dissolving 12-aminododecanoic acid (0.86g, 4mmol) and maleic anhydride (0.47g, 4.8mmol) in glacial acetic acid, heating at 120 ℃ for 6h, detecting the reaction on a thin-layer plate, cooling the reaction solution to room temperature, pouring into water, extracting with ethyl acetate three times (3X 20mL), combining the upper layer of extract, washing the extract with saturated saline for 3 times, and drying with anhydrous Na2SO4 overnight. Concentrating the extractive solution under reduced pressure, and purifying the obtained crude product by column chromatography to obtain yellowish pure product 0.88g, yield 75%, mp 91-92 deg.C.

¹H-NMR(DMSO-d₆，300MHz)：δppm：12.42(s，1H，-COOH)，7.50(s，2H，-COCH＝CHCO-)， 3.88(t，2H，J＝7.0Hz，-NCH2-)，2.68(t，J＝7.3Hz，2H，-CH₂COOH)，2.00-1.96(m，4H， -NCH₂CH₂(CH₂)₇CH₂)，1.73(s，14H，-NCH₂CH₂(CH₂)₇CH₂)。MS(ESI，m/z)：294.1[M+H]⁺.

Synthesis of 3, 3' - (4-nitrobenzylidene) -di-4-hydroxycoumarin

Weighing p-nitrobenzaldehyde (3.02g, 0.02mol), and dissolving with 35ml of absolute ethyl alcohol; 4-hydroxycoumarin (6.6g, 0.041mol) was added and 15ml of absolute ethanol was added to dissolve completely. Reacting for 4h at 80 ℃, filtering while hot, washing a filter cake for 3 times by using 30ml of hot ethanol to obtain 8.2g of a product, wherein the yield is 90.0 percent and the mp is 227 ℃.

¹H-NMR(CDCl₃，300MHz)δppm：6.13(s，H，-CH-)，7.43(m，8H，Ar-H)，7.68(m，2H，Ar-H)， 8.18(m，2H，Ar-H).MS(ESI，m/z)：456.0[M+H]⁺.

Synthesis of 3, 3' - (4-aminobenzylidene) -di-4-hydroxycoumarin

Weighing 3, 3' - (4-nitrobenzylidene) -di-4-hydroxycoumarin (1.14g, 0.0025mol), suspending with 30ml of acetic acid, adding 0.3g of 5% Pd/C, stirring, extracting for 3 times by a hydrogen tee joint, coating Vaseline on a bottle mouth, hydrogenating at normal temperature, reacting overnight, filtering, evaporating part of a solvent from a filtrate, recrystallizing with acetone to obtain 0.8g of a product, wherein the yield is 75.1%, and the mp is 220 ℃.

¹H-NMR(DMSO-d₆，300MHz)δppm：6.27(s，H，-CH-)，7.23(m，8H，Ar-H)，7.49(m，2H，Ar-H)，7.8l(m，2H，Ar-H).MS(ESI，m/z)：426.0[M+H]⁺.

Synthesis of 3, 3' - (4- (12-maleimidododecanamido) benzylidene) -di-4-hydroxycoumarin

12-Maleamidododecanoic acid (294.1mg, 1mmol) was dissolved in tetrahydrofuran, and DIC (17. mu.L, 1.1mmol) and HOBt (148.5 mmol) were addedmg, 1.1mmol), stirred at room temperature for 30min to activate the carboxyl group, and then a solution of 3, 3' - (4-aminobenzylidene) -di-4-hydroxycoumarin and DIPEA (17.4. mu.L, 0.1mmol) in tetrahydrofuran was slowly added dropwise thereto and stirred at room temperature overnight. Pouring the reaction solution into water and extracting with ethyl acetate for three times after the thin-layer plate detection reaction is finished, combining the extracts, and respectively using K₂CO₃HCl 1M, saturated brine washed three times. Adding anhydrous Na into the extract₂SO₄Drying overnight, concentrating under reduced pressure to obtain crude product, purifying by column chromatography to obtain pure product with yield of 69%, mp 204-.

¹H-NMR(DMSO-d₆，300MHz)：δppm：10.17(s，1H，-CONH-)，8.31(d，J＝7.8Hz，2H，Ar-H)， 8.00(t，J＝7.2Hz，2H，Ar-H)，7.84(d，J＝8.0Hz，2H，Ar-H)，7.76-7.72(m，6H，Ar-H)，7.49(s，2H，-COCH＝CHCO-)，6.70(s，1H，-CH-)，3.87(t，J＝7.0Hz，2H，-NCH₂-)，2.74(t，J＝7.2Hz，2H， -COCH₂-)，2.05-1.97(m，4H，-NCH₂CH₂(CH₂)₇CH₂-)，1.70(s，14H，-NCH₂CH₂(CH₂)₇CH₂-). MS(ESI，m/z)：703.1[M+H]⁺.

3. Chemically modified Cys₁₂-Synthesis and purification of Exenatide conjugates

Dissolving the 3, 3' - (4- (12-maleimidododecanamido) benzylidene) -bis-4-hydroxycoumarin obtained in the above step in DMSO to prepare a solution of about 10mg/mL, dissolving the modified exenatide polypeptide analog with Cys replaced in DMSO, mixing the two solutions, stirring the mixture at room temperature to react, adding 20. mu.l DIEPA to accelerate the reaction, and monitoring the reaction by LC-MS. The chromatographic conditions are as follows: c18 reverse phase column (1.7 μm 2.1X 50mm, Waters); mobile phase A: 0.1% formic acid/water (V/V), mobile phase B: 0.1% formic acid/acetonitrile (V/V), mobile phase gradient: 10-90% of mobile phase B, 2min, 90-90% of mobile phase B, 3 min; the flow rate is 0.3 ml/min; the ultraviolet detection wavelength is 214 nm. After the reaction, the reaction solution was diluted with acetonitrile containing 1% trifluoroacetic acid, centrifuged at high speed, filtered through a 0.45 μm microporous membrane, and then used to prepare a liquid phaseThe spectrum is purified, and the chromatographic conditions are as follows: c18 reversed phase column (320 mm. times.28 mm, 5 μm); mobile phase A: 0.1% trifluoroacetic acid/water (V/V), mobile phase B: 0.1% trifluoroacetic acid/acetonitrile (V/V); gradient of mobile phase: 40-80% of mobile phase B for 30 min; 80-85% for 10 min; 85-95% for 10 min; 95-40% for 10 min; the flow rate was 5ml/min and the detection wavelength was 214 nm. Collecting the solution, concentrating under reduced pressure to remove acetonitrile, and lyophilizing to obtain pure product. The theoretical relative molecular mass is 4900.0. ESI-MS m/z: calcd. [ M +3H ]]³⁺1634.3，[M+4H]⁴⁺1226.0；Found[M+3H]³⁺1634.0，[M+4H]⁴⁺1226.1。

Example 2

The synthesis was performed as in example 1, with a theoretical relative molecular mass of 5028.2. ESI-MS m/z: calcd [ M +3H ]]³⁺1677.1， [M+4H]⁴⁺1258.1；Found[M+3H]³⁺1677.8，[M+4H]⁴⁺1258.5。

Example 3

The synthesis was performed as in example 1, with a theoretical relative molecular mass of 5015.1. ESI-MS m/z: calcd [ M +3H ]]³⁺1672.7， [M+4H]⁴⁺1254.8；Found[M+3H]³⁺1672.2，[M+4H]⁴⁺1254.3。

Example 4

The synthesis was performed as in example 1, with a theoretical relative molecular mass of 5143.3. ESI-MS m/z: calcd [ M +3H ]]³⁺1715.4， [M+4H]⁴⁺1286.8；Found[M+3H]³⁺1715.4，[M+4H]⁴⁺1286.2。

Claims (7)

1. A long-acting hypoglycemic peptide, which is characterized in that

2. A pharmaceutically acceptable salt prepared from a compound according to claim 1, wherein said pharmaceutically acceptable salt is a salt with hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, succinic acid, maleic acid, acetic acid, fumaric acid, citric acid, tartaric acid, benzoic acid, benzenesulfonic acid, methanesulfonic acid, or naphthalenesulfonic acid.

3. A pharmaceutical preparation prepared from the compound of claim 1, wherein said pharmaceutical preparation is any one of the pharmaceutically acceptable tablets, capsules, elixirs, syrups, lozenges, inhalants, sprays, injections, films, patches, powders, granules, blocks, emulsions, suppositories, and combinations thereof.

4. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of diabetes.

5. Use of a pharmaceutically acceptable salt prepared from a compound according to claim 1 for the preparation of a medicament for the treatment of diabetes.

6. Use of a medicament prepared from a compound according to claim 1 for the manufacture of a medicament for the treatment of diabetes.

7. The method of claim 1, wherein the method comprises biological expression, liquid phase synthesis, and solid phase synthesis.