CN110128526A

CN110128526A - Long-actingization Exenatide derivative and its salt and preparation method and purposes

Info

Publication number: CN110128526A
Application number: CN201910465730.1A
Authority: CN
Inventors: 谷海涛; 赵呈青; 王蔡典; 姜建军
Original assignee: Hangzhou Nortel O Sano Pharmaceutical Technology Development Co Ltd; Jiangsu Sinopep Macao Zaino Biological Pharmaceutical Ltd By Share Ltd
Current assignee: Hangzhou Nortel O Sano Pharmaceutical Technology Development Co Ltd; Jiangsu Sinopep Macao Zaino Biological Pharmaceutical Ltd By Share Ltd
Priority date: 2019-05-30
Filing date: 2019-05-30
Publication date: 2019-08-16
Anticipated expiration: 2039-05-30
Also published as: WO2020237709A1; CN110128526B

Abstract

The present invention is a kind of long-actingization Exenatide derivative, belongs to polypeptide compound technical field.The Exenatide derivative is to carry out structure optimization to Exenatide sequence, it is set to be provided simultaneously with hypoglycemic and weight loss effect, and the fatty acid chain of the Exenatide of doubling dose and single times of amount is conjugated, using fatty acid chain performance and sero-abluminous combination, the Exenatide derivative with longer pharmacological action time is obtained.The invention also discloses the preparation method of Exenatide derivative, its pharmaceutically acceptable Exenatide derivative salt, Exenatide derivative medicament, pharmaceutical composition and purposes, which is application of the Exenatide derivative in preparation treatment and/or the drug for preventing diabetes, obesity, hyperlipemia, nonalcoholic fatty liver.Long-actingization Exenatide derivative can have loss of weight effect, and biological half-life significantly extends compared with Exenatide prototype, has partially reached 36 hours or more, greatly extends hypoglycemic loss of weight action time on the basis of retaining hypoglycemic activity.

Description

Long-actingization Exenatide derivative and its salt and preparation method and purposes

Technical field

The present invention relates to polypeptide compound technical fields, and in particular to a kind of Exenatide derivative, preparation method, medicine Agent is to conjunction object and its purposes as drug.

Background technique

The cause of disease of metabolic syndrome is the metabolic disorder of many kinds of substance such as protein, fat and carbohydrate.Nutrition Surplus, physical exertion reduction etc. will lead to fat and obesity-related disease, such as diabetes.In recent years, diabetes B, blood lipid The disease incidence of metabolic disorder increasingly increases.

Glucagon-like-peptide-1 (GLP-1) is a kind of glucose dependency gut incretin hormones.It can excitement GLP-1 receptor plays blood sugar reducing function.Most significant function is to promote the regeneration and reparation of β cell, increases the number of beta Cell of islet Amount, while being also avoided that recurrent risk of hypoglycemia in treating diabetes, there is wide application in treating diabetes field Prospect.Although natural GLP-1 has many advantages, such as that it is in vivo easily by dipeptidyl peptidase IV (DPP-IV) in treatment diabetes Fast degradation, Half-life in vivo only 3 minutes or so.Exenatide (Exenatide) extracts from America gilamonster Yi salivary gland, Ratified to list by FDA within 2005, polypeptide sequence HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂。 Exenatide is not the substrate of DPP-4, therefore the DPP-4 degradation that will not be widely present in vivo, and Half-life in vivo is than endogenous Property GLP-1 extend, about 2.4h.But Exenatide still can quickly be filtered in kidney and be eliminated, and the degradation of anti-DPP-IV enzyme is only It can a degree of half-life period for extending GLP-1.

Summary of the invention

In view of the deficiencies of the prior art, it is an object of the present invention to provide a kind of long-actingization Exenatide derivative, the derivatives Object is on the basis of Exenatide, on the basis of keeping its height agonist activity to GLP-1 receptor, with glucagon pair It carries out site displacement, obtains that there are double target spot agonist activity Exenatides to spread out to the agonist activity of glucagon to increase it Raw sequence.

Two preferred Exenatide derived peptides sequences are innovatively utilized and are relied on the basis of above-mentioned purpose by the present invention Propylhomoserin connection, and be conjugated with the fatty acid chain of different length.When fatty acid chain can increase conjugate and sero-abluminous combination Between, while overall molecule volume ratio Exenatide increases, and can slow down kidney and quickly filter, two aspects work simultaneously, greatly prolong The action time in vivo of peptide chain is grown.Thus such economic benefits and social benefits Exenatide analog is having hypoglycemic and the active premise of loss of weight Under, significantly extend its internal action time.

Another object of the present invention is to provide a kind of preparation methods of Exenatide derivative.

It is also another object of the present invention to provide the pharmaceutically acceptable Chinese mugworts for using Exenatide derivative to be prepared Fill in that peptide derivant salt.

There is provided the Exenatide derivative medicaments that Exenatide derivative is prepared for a further object of the present invention.

There is provided a kind of pharmaceutical compositions containing Exenatide derivative for a further object of the present invention.

There is provided the purposes of Exenatide derivative, the purposes of Exenatide derivative salt for a further object of the present invention With the purposes of Exenatide derivative medicament.

The purpose of the present invention is what is also realized by technical solution below.The present invention is that one kind long-actingization Exenatide spreads out Biology, amino acid sequence are as follows:

His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Asp-Ser-Arg- Arg-Al a-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro- Pro-Pro-Ser-Xaa2-S er-Pro-Pro-Pro-Ala-Gly-Ser-Ser-Pro-Gly-Gly-Asn-Lys-Leu- Trp-Glu-Ile-Phe-Asp-Gln-Ala-Arg-Arg-Ser-Asp-Met-Gln-Lys-Ser-Leu-Asp-Ser-Thr- Phe-Thr-Gly-Gln-Xaa1-Hi s

Wherein:

Xaa1 is derived from: Aib or Gly

Xaa2 is derived from:

N be natural number 5-17, preferably 11,13 or 15.

The preferred present invention long-actingization Exenatide derivative can indicate are as follows:

The invention also discloses the pharmaceutically acceptable Exenatide derivative salt that Exenatide derivative is prepared, It is Exenatide derivative and hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid, methanesulfonic acid, second with the salt Sulfonic acid, benzene sulfonic acid, p-methyl benzenesulfonic acid, formic acid, acetic acid, acetoacetate, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, heptan Acid, hendecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4- hydroxy benzoyl) benzoic acid, camphoric acid, cinnamic acid, ring penta Alkane propionic acid, didextrose acid, 3- hydroxy-2-naphthoic acid, niacin, flutter acid, pectinic acid, persulfuric acid, 3- phenylpropionic acid, picric acid, Pivalic acid, 2- ethylenehydrinsulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecyl sulphate, 2- naphthalene sulfonic acids, two sulphur of naphthalene Acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, horse Come sour, fumaric acid, D- gluconic acid, mandelic acid, ascorbic acid, glucoheptose, phosphoglycerol, aspartic acid, sulfosalicylic acid, half sulphur Salt formed by acid or thiocyanic acid.

The present invention also provides a kind of pharmaceutical compositions, and the above-mentioned Exenatide of at least one including therapeutically effective amount is derivative Compounds or its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or diluent.Meanwhile the present invention is further Provide above-mentioned Exenatide derivative compound and its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or dilution Agent is preparing the utilization in the drug for treating and preventing diabetes.

The invention also discloses the Exenatide derivative medicament being prepared with Exenatide derivative, pharmaceutical formulations Selected from tablet, capsule, elixir, syrup, pastille, inhalant, spray, injection, film, patch, powder, granule, block agent, Emulsion, suppository or compound preparation.

The invention also discloses the purposes of the Exenatide derivative, its main feature is that, which spreads out for Exenatide Application of the biology in preparation treatment and/or the drug for preventing diabetes, obesity, hyperlipemia, nonalcoholic fatty liver.

The invention also discloses the purposes of Exenatide derivative salt, its main feature is that, which is Exenatide derivative Application of the salt in preparation treatment and/or the drug for preventing diabetes, obesity, hyperlipemia, nonalcoholic fatty liver.

The invention also discloses the purposes of Exenatide derivative medicament, its main feature is that, the purposes is derivative for Exenatide Application of the object medicament in preparation treatment and/or the drug for preventing diabetes, obesity, hyperlipemia, nonalcoholic fatty liver.

The present invention also provides the Preparation Method And Their Intermediate of Exenatide derivative, Exenatide provided by the invention The preparation method of derivative polypeptides is gradually coupled each amino acid of such Polypeptide-k main chain using solid-phase synthesis, cracks To the main chain polypeptide of side chain protection, the lysine for being connected with aliphatic chain is reacted to obtain Exenatide derivative with main chain.The method Synthesis step is easy, and coupling efficiency is high, is easy to purify, is conducive to the industrialized production of such polypeptide.

The preparation method of currently preferred Exenatide derivative the following steps are included:

Step 1: taking resin, after activation, gradually coupling amino acid, obtains the first peptide resin；

Step 2: taking first peptide resin, cracked, purified, obtain the polypeptide backbone of side chain protection；

Step 3: the lysine for being connected with aliphatic chain being reacted with doubling dose main chain, after purification to obtain the final product；

Preferably, in preparation method provided by the invention, the resin in step 1 is 2-CTC Resin or Wang Resin.

Preferably, the mixing that lytic reagent used is TFA and DCM is cracked in preparation method provided by the invention, in step 2 Object.In some embodiments of the invention, in preparation method provided by the invention, TFA, DCM in agents useful for same are cracked in step 2 The ratio between volume be 0.1:99.9.

In other embodiment of the invention, in preparation method provided by the invention, purified in step 2 and step 3 Method therefor is chromatography.In other embodiment of the invention, in preparation method provided by the invention, step 2 It is C18 column with chromatographic column used is purified in step 3.

Inventor's research is thought, GLP-1 and glucagon, and the two can be obtained after preceding Proglucagon translation shearing With certain sequence homology.Therefore the high blood of introducing portion pancreas in the Exenatide that there is height agonist activity to GLP-1 The sequence of sugared element, available while excitement GLP-1R and GCGR dual agonist, to obtain hypoglycemic activity and weight loss effect All good polypeptide compound.

Compared with prior art, beneficial effects of the present invention:

1, a kind of long-actingization Exenatide derivative of amboceptor excitement proposed by the present invention can retain hypoglycemic activity On the basis of, there is loss of weight effect, and biological half-life significantly extends compared with Exenatide prototype (~2.4h), has partially reached 36 Hour or more, greatly extend hypoglycemic loss of weight action time.

2, the method for the present invention obtains the crude product of peptide chain using the synthesis in solid state Exenatide derivative of orthogonal Preservation tactics Purity is greater than 85%, greatly improves compared with conventional synthesis process, facilitates subsequent purification work.

3, the method for the present invention is low using the cost of solid phase method synthesis Exenatide derivative.Since coupling efficiency is higher, Required protected amino acid is average only to need 2 times of excess, and 4 to 5 times of excessive amino acid are needed in conventional synthesis process, greatly Saved cost.

4, the present invention is easy to using the method for the orthogonal protection synthesis in solid state strategy synthesis Exenatide derivative of Fmoc/tBu Realize automation, large-scale, this makes it be more suitable for industrialized production.

5, with it is provided by the invention by solid phase synthesis technique prepare Exenatide derivative, hypoglycemic and slow down weight increasing Add activity good, efficacy time is long, and high income, synthesis cycle are short, purifying crude is easy, and production cost is low, is easy to industrial automation Production.The Exenatide derivative being prepared is suitable as the active constituent for the treatment of diabetes, antiobesity agents.

Detailed description of the invention

Fig. 1 is the abdominal cavity sugar tolerance experimental result of NO:1~6 Exenatide derivative SEQ.ID；

Fig. 2 is the stabilizing blood sugar experimental result of NO:1~6 Exenatide derivative SEQ.ID.

Specific embodiment

Following abbreviation is used in this specification:

English abbreviation	Chinese
		DCM	Methylene chloride
NMP	N-Methyl pyrrolidone
		HBTU	Benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
HOBt	1- hydroxyl-benzotriazole
		DIEA/DIPEA	N, N'- diisopropylethylamine
Fmoc	N-9- fluorenylmethyloxycarbonyl
		ESI-MS	Electrospray ionization mass spectrum
EDT	Dithioglycol
		HPLC	High performance liquid chromatography
TFA	Trifluoroacetic acid
		tBu	Tert-butyl
DMSO	Dimethyl sulfoxide

The present invention is to be illustrated by the following example, but these embodiments do not do any restrictions solution of the invention It releases.

Embodiment 1:

Synthesis

1. the synthesis of peptide chain

The swelling of 1.1 resins

2-CTC Resin1g (degree of substitution 0.4mmol/g) is weighed, is swollen 30min through DCM10mL, suction filtration removes DCM, then uses NMP10mL is swollen 30min, uses NMP, DCM10mL to rinse well respectively.

The synthesis of 1.2 Fmoc-Ser (tBu) -2-CTC Resin

By Fmoc-Ser (tBu)-OH (0.8mmol), DIPEA (1.6mmol) is dissolved in NMP10mL, then this solution is added Enter in resin obtained in the previous step and react 2 hours, after filter off reaction solution, washed resin 3 times with each 10mL of DCM and NMP.

The removing of 1.3 Fmoc protecting groups

25% piperidines/NMP (V/V) the solution removal Fmoc for containing 0.1 M HOBt, reaction are added into the resin after washing After washed resin 3 times with each 10mL of DCM and NMP.

The extension of 1.4 peptide chains

According to the sequence of Exenatide derivative peptide chain, repeats above-mentioned deprotection and be sequentially connected the step of coupling corresponding Amino acid, be sequentially connected corresponding amino acid until peptide chain synthesis finish, obtain the peptide tree for being connected with Exenatide derivative Rouge.

The cracking of polypeptide on 1.5 resins

The resin obtained above for being connected with Exenatide derivative peptide chain is put into reaction flask, decomposition agent is respectively added (TFA/DCM, 0.1/99.9, V/V) 10mL first shakes 30min at 0 DEG C, then reacts 1h at normal temperature.It takes out after reaction Filter, merging filtrate, vacuum distillation.Concentrate is added in a large amount of ice ether, white flock precipitate is precipitated, refrigerated centrifuge obtains The polypeptide crude product of side chain protection.It is purified using preparative liquid chromatography, chromatographic condition are as follows: C18 column (320mm × 28mm, 5 μ m)；Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V)；Eluent gradient: Mobile phase B 40% ~90%, 20min；Flow velocity is that 6mL/min Detection wavelength is 214nm.

The synthesis of (2. S) -2,6- diamino-N-dodecyl caproamide

Boc-Lys (Boc)-NH-12 alkane (0.8mmol) is dissolved in 7mL DCM, 1mL TFA is slowly added dropwise, reacts 3h, It is concentrated under reduced pressure, 5mL1M NaOH aqueous solution is added, white solid is precipitated, precipitating is collected by centrifugation, obtains white powdery solid.

3. the synthesis of Exenatide derivative

By Exenatide derivative (0.02mmol), HBTU (0.02mmol), HOBt (0.08mmol) and DIPEA (0.16mmol) is dissolved in NMP10mL, then (S) -2,6- diamino-N-dodecyl caproamide is added in this solution.It uses HPLC monitoring reaction, chromatographic condition are as follows: C18 column (150mm × 4.6mm, 5 μm)；Mobile phase A: 0.1%TFA/ water (V/V), flowing Phase B:0.1%TFA/ acetonitrile (V/V)；Eluent gradient: Mobile phase B 35%~85%, 20min；Flow velocity 1mL/min；Column temperature 40 ℃；Detection wavelength 214nm.After reaction, it is purified using preparative liquid chromatography, chromatographic condition are as follows: C18 column (320mm × 28mm, 5 μm)；Mobile phase A: 0.1%TFA/ water (V/V), Mobile phase B: 0.1%TFA/ acetonitrile (V/V)；Eluent gradient: flowing Phase B40%~90%, 20min；Flow velocity is that 6mL/min Detection wavelength is 214nm.Sterling 23.8mg is lyophilized to obtain in the solution of collection. Theoretical relative molecular mass is 8710.7.ESI-MS m/z:Calcd.[M+5H]⁵⁺1743.1,[M+6H]⁶⁺1452.8；Found [M+5H]⁵⁺1743.5,[M+6H]⁶⁺1453.1。

Embodiment 2:

For synthetic method with embodiment 1, sterling 24.9mg is lyophilized to obtain in the solution of collection.Theoretical relative molecular mass is 8654.6。ESI-MS m/z:Calcd.[M+5H]⁵⁺1731.9,[M+6H]⁶⁺1443.4；Found[M+5H]⁵⁺1732.1,[M+ 6H]⁶⁺1443.7。

Embodiment 3:

For synthetic method with embodiment 1, sterling 26.5mg is lyophilized to obtain in the solution of collection.Theoretical relative molecular mass is 8738.8。ESI-MS m/z:Calcd.[M+5H]⁵⁺1748.8,[M+6H]⁶⁺1457.5；Found[M+5H]⁵⁺1748.9,[M+ 6H]⁶⁺1458.0。

Embodiment 4:

For synthetic method with embodiment 1, sterling 22.8mg is lyophilized to obtain in the solution of collection.Theoretical relative molecular mass is 8682.7。ESI-MS m/z:Calcd.[M+5H]⁵⁺1737.5,[M+6H]⁶⁺1448.1；Found[M+5H]⁵⁺1737.8,[M+ 6H]⁶⁺1448.7。

Embodiment 5:

For synthetic method with embodiment 1, sterling 24.5mg is lyophilized to obtain in the solution of collection.Theoretical relative molecular mass is 8766.9。ESI-MS m/z:Calcd.[M+5H]⁵⁺1754.4,[M+6H]⁶⁺1462.2；Found[M+5H]⁵⁺1754.9,[M+ 6H]⁶⁺1462.3。

Embodiment 6:

For synthetic method with embodiment 1, sterling 23.9mg is lyophilized to obtain in the solution of collection.Theoretical relative molecular mass is 8710.7。ESI-MS m/z:Calcd.[M+5H]⁵⁺1743.1,[M+6H]⁶⁺1452.8；Found[M+5H]⁵⁺1743.8,[M+ 6H]⁶⁺1453.0。

Embodiment 7: being the related pharmacological experimental method and result of Exenatide derivative involved in the present invention below:

1, the GLP-1 receptor agonist activity screening of Exenatide derivative

The cDNA of HEK293 cell cotransfection coding GLP-1R or GCGR.In the test for measuring compound, 2h will be thin in advance Born of the same parents plant in 96 orifice plates, and compound is dissolved with DMSO, are diluted to different times using the culture medium containing 0.1% bovine serum albumin Number, is added in the cell of cotransfection.After cell hatches 20min, using the ELISA kit of Cisbo company, surveyed using microplate reader Determine fluorescence reading, establishes standard curve for fluorescence reading and be converted into corresponding cAMP numerical value, it is soft using Graphpad Prism5.0 The nonlinear regression of part calculates the EC of compound₅₀Numerical value.

1 Exenatide derivative of table is to GLP-1R and GCGR agonist activity

Results are expressed as mean±SD,**P<0.01vs Glucagon,##P<0.01vs Exenatide.

As shown in table 1, compared with Exenatide prototype, all compounds still maintain the agonist activity of GLP-1R The agonist activity of suitable high level.There is large increase compared with Exenatide prototype to the agonist activity of GCGR simultaneously, is conjugated There is no influence too much agonist activity to fatty acid chain.

2, the abdominal cavity glucose tolerance test of Exenatide derivative

Normal kunming mice, random to be grouped, every group 8, mouse raising is in standardization animal house.Prohibit within experiment first 12 hours Food, is only given drinking-water.Every group of mouse surveys initial blood glucose value before Exenatide derivative is administered, it is determined as -30min, then abdomen The Exenatide derivative of chamber injection 50nmol/kg.After 30min, the glucose solution of 18mmol/kg is injected intraperitoneally, it is determined as 0min, control group inject the physiological saline of same volume or the Exenatide of 50nmol/kg.It is used 0,15,30,45,60,120min Blood glucose meter measures blood glucose level, detects the hypoglycemic activity of Exenatide derivative.

As shown in Figure 1, it is hypoglycemic the experimental results showed that, the present invention involved in Exenatide derivative administration concentration be When 50nmol/kg, blood sugar decreasing effect is suitable with the hypoglycemic effect of Exenatide.

3, the stabilizing blood sugar experiment of Exenatide derivative

The blood glucose for measuring the diabetic mice of STZ induction selects mouse of the numerical value higher than 20mmol/L to carry out random Grouping, every group six, mouse is freely eaten during experiment.Exenatide or Liraglutide, dosage is injected intraperitoneally in positive controls For 50nmol/kg, negative control group intraperitoneal injection of saline, the Exenatide that administration group injects 50nmol/kg respectively is derivative Object.0h gives compound, is measured respectively in 0,0.5,1,2,3,4,6,8,10,12,16,24,36,48 and 60h using blood glucose meter Blood glucose level.Evaluation index is time of the mouse blood sugar numerical value lower than 8.35mmol/L after intraperitoneal injection compound.

From Figure 2 it can be seen that the time of the stabilizing blood sugar of Exenatide is only 4h, the stabilizing blood sugar time of Liraglutide is 10h, The stabilizing blood sugar time of long-actingization Polypeptide-k involved in the present invention partially can be more than 35h more than for 24 hours.Stabilizing blood sugar Experiment shows that Exenatide derivative has good long-actingization hypoglycemic effect, can achieve more preferably long-actingization hypoglycemic effect, With the potentiality for being developed into the hypoglycemic medicine being administered once every day.

4, the weight gain that slows down of OXM hybrid peptide is tested

Male C57bl/6 mouse, high lipid food are fed 4 weeks, and mouse of the weight greater than 30g is selected to be tested.Mouse is random Grouping, 8 are one group, totally 8 groups, are administered daily Exenatide derivative (50nmol/kg, 10mL/kg) within continuous 56 days, negative right It is administered daily physiological saline according to group, Liraglutide is administered in positive controls.

The empty body weight of the 1st day and the 56th day each group mouse is tested, the average weight variation of each group mouse is investigated.

The loss of weight effect of 2 Exenatide derivative of table

Results are expressed as mean±SD.

As can be drawn from Table 2, after long term administration, all compounds all show preferable body weight control effect.

Sequence table

<110>Jiangsu Nuo Tai Australia Sino bio-pharmaceuticals limited liability company

Sino medical science development corporation, Ltd., Hangzhou Nuo Tai Australia

<120>long-actingization Exenatide derivative and its salt and preparation method and purposes

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 79

<212> PRT

<213>artificial sequence (artificial chromosome)

<400> 1

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser

1 5 10 15

Arg Arg Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Xaa Ser Pro Pro Pro Ala Gly Ser Ser

35 40 45

Pro Gly Gly Asn Lys Leu Trp Glu Ile Phe Leu Arg Val Arg Arg Ser

50 55 60

Asp Met Gln Lys Ser Leu Asp Ser Thr Phe Thr Gly Glu Gly His

65 70 75

<210> 2

<211> 77

<212> PRT

<213>artificial sequence (artificial chromosome)

<400> 2

His Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser Arg

1 5 10 15

Arg Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser

20 25 30

Gly Ala Pro Pro Pro Ser Xaa Ser Pro Pro Pro Ala Gly Ser Ser Pro

35 40 45

Gly Gly Asn Lys Leu Trp Glu Ile Phe Leu Arg Val Arg Arg Ser Asp

50 55 60

Met Gln Lys Ser Leu Asp Ser Thr Phe Thr Gly Glu His

65 70 75

<210> 3

<211> 79

<212> PRT

<213>artificial sequence (artificial chromosome)

<400> 3

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser

1 5 10 15

Arg Arg Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Xaa Ser Pro Pro Pro Ala Gly Ser Ser

35 40 45

Pro Gly Gly Asn Lys Leu Trp Glu Ile Phe Leu Arg Val Arg Arg Ser

50 55 60

Asp Met Gln Lys Ser Leu Asp Ser Thr Phe Thr Gly Glu Gly His

65 70 75

<210> 4

<211> 77

<212> PRT

<213>artificial sequence (artificial chromosome)

<400> 4

His Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser Arg

1 5 10 15

Arg Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser

20 25 30

Gly Ala Pro Pro Pro Ser Xaa Ser Pro Pro Pro Ala Gly Ser Ser Pro

35 40 45

Gly Gly Asn Lys Leu Trp Glu Ile Phe Leu Arg Val Arg Arg Ser Asp

50 55 60

Met Gln Lys Ser Leu Asp Ser Thr Phe Thr Gly Glu His

65 70 75

<210> 5

<211> 79

<212> PRT

<213>artificial sequence (artificial chromosome)

<400> 5

His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser

1 5 10 15

Arg Arg Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser

20 25 30

Ser Gly Ala Pro Pro Pro Ser Xaa Ser Pro Pro Pro Ala Gly Ser Ser

35 40 45

Pro Gly Gly Asn Lys Leu Trp Glu Ile Phe Leu Arg Val Arg Arg Ser

50 55 60

Asp Met Gln Lys Ser Leu Asp Ser Thr Phe Thr Gly Glu Gly His

65 70 75

<210> 6

<211> 77

<212> PRT

<213>artificial sequence (artificial chromosome)

<400> 6

His Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Asp Ser Arg

1 5 10 15

Arg Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser Ser

20 25 30

Gly Ala Pro Pro Pro Ser Xaa Ser Pro Pro Pro Ala Gly Ser Ser Pro

35 40 45

Gly Gly Asn Lys Leu Trp Glu Ile Phe Leu Arg Val Arg Arg Ser Asp

50 55 60

Met Gln Lys Ser Leu Asp Ser Thr Phe Thr Gly Glu His

65 70 75

Claims

1. a kind of long-actingization Exenatide derivative, which is characterized in that its amino acid sequence are as follows:

His-Xaa1-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Asp-Ser-Arg-Arg- Ala-Gln-Asp-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro- Pro-Ser-Xaa2-Ser-Pro-Pro-Pro-Ala-Gly-Ser-Ser-Pro-Gly-Gly-Asn-Lys-Leu-Trp-Glu- Ile-Phe-Asp-Gln-Ala-Arg-Arg-Ser-Asp-Met-Gln-Lys-Ser-Leu-Asp-Ser-Thr-Phe-Thr- Gly-Gln-Xaa1-His；

Wherein:

Xaa1 is derived from Aib or Gly；

Xaa2 is derived from

N is natural number 5-17.

2. Exenatide derivative according to claim 1, it is characterised in that: n is natural number 11,13 or 15 in Xaa2.

3. Exenatide derivative according to claim 1, it is characterised in that: the Exenatide derivative is following chemical combination One of object:

4. a kind of preparation method of Exenatide derivative as described in claim 1, which comprises the following steps:

(1) resin is taken, after activation, gradually coupling amino acid, obtains the first peptide resin；

(2) first peptide resin is taken, cracked, purified, the polypeptide backbone of side chain protection is obtained；

(3) lysine for being connected with aliphatic chain is reacted with doubling dose main chain, after purification to obtain the final product.

5. a kind of Exenatide derivative as described in any one of claim 1-3 is prepared pharmaceutically acceptable Exenatide derivative salt, which is characterized in that the pharmaceutically acceptable Exenatide derivative salt is Ai Saina Peptide derivant salt and acid formed salt, the acid be selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid, Methanesulfonic acid, ethanesulfonic acid, benzene sulfonic acid, p-methyl benzenesulfonic acid, formic acid, acetic acid, acetoacetate, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, Caproic acid, enanthic acid, hendecanoic acid, lauric acid, benzoic acid, salicylic acid, 2- (4- hydroxy benzoyl) benzoic acid, camphoric acid, cortex cinnamomi Acid, pentamethylene propionic acid, didextrose acid, 3- hydroxy-2-naphthoic acid, niacin, flutter acid, pectinic acid, persulfuric acid, 3- phenylpropionic acid, Picric acid, pivalic acid, 2- ethylenehydrinsulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecyl sulphate, 2- naphthalene sulfonic acids, Naphthalenedisulfonic acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, algae Acid, maleic acid, fumaric acid, D- gluconic acid, mandelic acid, ascorbic acid, glucoheptose, phosphoglycerol, aspartic acid, sulfosalisylic Acid, hemisulfic acid or thiocyanic acid.

6. a kind of Exenatide that the Exenatide derivative as described in any one of claim 1-3 is prepared is derivative Object medicament, which is characterized in that the pharmaceutical formulation is selected from tablet, capsule, elixir, syrup, pastille, inhalant, spray, note Penetrate agent, film, patch, powder, granule, block agent, emulsion, suppository or compound preparation.

7. a kind of pharmaceutical composition containing the Exenatide derivative as described in any one of claim 1-3, feature It is, the composition includes Exenatide derivative described in any one and its pharmacy in the claim 1-3 of therapeutically effective amount Upper acceptable carrier or diluent.

8. a kind of purposes of the Exenatide derivative as described in any one of claim 1-3, which is characterized in that the use Way is Exenatide derivative in preparation treatment and/or prevention diabetes, obesity, hyperlipemia, nonalcoholic fatty liver Application in drug.

9. a kind of purposes of pharmaceutically acceptable Exenatide derivative salt as claimed in claim 5, which is characterized in that should Purposes is Exenatide derivative salt in preparation treatment and/or prevention diabetes, obesity, hyperlipemia, non-alcoholic fatty Application in the drug of liver.

10. a kind of purposes of Exenatide derivative medicament as claimed in claim 6, which is characterized in that the purposes is Ai Sai That peptide derivant medicament is in preparation treatment and/or the drug of prevention diabetes, obesity, hyperlipemia, nonalcoholic fatty liver In application.