WO2013029279A1 - Novel glp-i analogue, preparation method and use thereof - Google Patents

Abstract

Provided is a glucagon-like peptide-1 (GLP-1) analogue comprising a parent peptide having the amino acid sequence of GLP-1 (7-37) and two modifying groups coupled to amino groups of two lysine residues of the parent peptide via amide bonds, wherein one of the groups comprises a maleimide group (MPA). The GLP-1 analogue has the activity of insulin and has advantages of long half-life, good stability and less side effects, so as to be used for treating diseases such as diabetes. Also provided are a synthesis method, composition and pharmaceutical use of the GLP-1 analogue.

Description

New GLP-I analog and preparation method and use thereof

Technical field

The present invention relates to the field of medicinal chemistry and organic chemistry, and in particular to polypeptide analogs, to methods of preparing polypeptide analogs, compositions and their use in pharmacy.

Background technique

 Glucagon-like peptide-1 (GLP-I) is a small intestine-L A polypeptide hormone secreted by a cell. GLP-I is a 30 amino peptide fragment that is cleaved from a 160 amino acid proglucagon (PG) peptide chain. GLP- I can promote insulin secretion, inhibit glucagon release, promote the expression of proinsulin gene and delay gastric emptying and gastric acid secretion at high blood sugar levels, and find GLP- I can increase satiety (suppress appetite) and reduce energy intake. Long-term injection of GLP-I or exendin-4 (a long-acting analog of human GLP-I) can increase rat beta- The number of cell clusters. GLP- regulates blood glucose levels through a variety of independent mechanisms of action, causing widespread concern in the prevention and treatment of diabetes.

 L-cells in the gastrointestinal tract are regulated by blood glucose, secreting GLP-I peptide, with a half-life of 5 min and a metabolic clearance rate of 12-13 min. GLP-I is degraded by DDP IV (dipeptidyl peptidase IV), ie, the N-terminal two amino acid residues are removed and converted into inactive GLP- I peptide. Due to the extremely short half-life reduction of GLP-I, its clinical application has been limited, and some analogs with GLP-I-like biological activity have been studied. Such as exendin-4 isolated from the saliva of snakes, It is highly homologous to the GLP-I sequence, has similar physiological effects, and has a longer half-life. The N-terminal cleavage product exendin of E xendin-4 is capable of interacting with the GLP-I receptor on the surface of beta cells ( GLP-I-R) antagonizes and specifically inhibits GLP-I-mediated insulin secretion by duodenal glucose and oral nutrients.

 In order to meet the needs of clinical applications, GLP-I is molecularly engineered to resist enzyme degradation and enhance activity, including N-terminal The methylation, deamination, hydroxylation, etc. of the His free amino group, and the D2 type amino acid substitution of the second Ala have achieved the desired effect, possibly 2 The treatment of type 2 diabetes opens up new avenues. Essexide ( exenatide ) from Lilly Corporation , which is currently in clinical use , is the first novel GLP - I agonist injection . Glycemic control for type 2 diabetes patients with metformin and sulfonylureas that control uncontrolled blood glucose has been marketed in the United States. GLP-I developed by NovoNordisk of Denmark The analog liraglutide is resistant to the degradation of DDP IV with a half-life of up to 12 h and is linked to albumin with a slow release profile. One injection can maintain the efficacy of 24 hours. Conjuchem's CJC1131 is a stereoisomer of position 8 with a non-natural D-alanine GLP- I and a linker with a chemically active group, covalently bound to albumin after injection, with a half-life of about 10-12 h. Studies have shown that the product obtained by modifying the N-terminus of GLP-I, such as N-glutamic acid -GLP-I and N-acetyl-GLP-I, and GLP- Compared with I, it has a long half-life and a strong insulin-promoting effect. However, these drugs have strong side effects, which can cause side effects such as nausea and vomiting, and the steps of chemical synthesis are cumbersome and the price is very high.

 Therefore, people are eager to develop a high activity, good stability, easy to use chemical synthesis, and fewer side effects can be used to treat diabetes. GLP-I analogue.

Summary of the invention

 The object of the present invention is to develop a highly active GLP-I analogue for the treatment of diabetes and as a new generation of drugs for the treatment of diabetes.

 The inventors have carried out a large number of experimental studies to modify the GLP-I molecule, and the results show that the GLP- The I analog has a longer half-life, has insulinotropic activity, has no clinical adverse effects, and can be used for the treatment of diseases such as diabetes, thereby completing the present invention.

 A first aspect of the invention provides a GLP-I analog comprising a parent peptide of the following sequence:

H ₂ N-Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Xaa26-Glu-Phe-Ile- Ala-Trp-Leu-Val-Xaa34-Gly-Arg-Xaa37-COR ₁ ;

Wherein R ₁ = -OH or -NH ₂ ;

 Xaa7= histidine, D-histidine, deamination-histidine, 2-amino-histidine, β-hydroxyl Histidine, homohistidine, N α -acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridyl alanine, 2-pyridyl Alanine or 4- Pyridyl alanine;

 Xaa8=Ala, D-Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl) Carboxylic acid, (1-aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid or (1-amino group) Cyclooctyl)carboxylic acid;

 Xaa26=Lys;

 Xaa34=Lys, Glu, Asn or Arg,

 Xaa37=Gly, Ala, Glu, Pro or Lys, and Xaa34 or Xaa37 At least one of them is Lys;

 The GLP-I analog also contains Q1 and Q2 groups, and the Q1 and Q2 groups are simultaneously present in the parent peptide. Xaa26, Xaa34, Xaa37 When any two or all of them are Lys, any two Lys in Xaa26, Xaa34, Xaa37 are linked in the form of an amide bond. Residue

The Q1 group is a lipophilic substituent attached to a bridging group W, and the lipophilic substituent forms an amide bond with the amino group of a bridging group at its carboxyl group, a carboxyl group of the amino acid residue of the bridging group and a parent peptide An N-terminal residue of Lys forms an amide bond to be attached to the parent peptide; the bridging group W is 1-7 methylene-free branched paraffins α, ω-dicarboxy; the lipophilic substituent is An acyl group selected from the group consisting of CH ₃ (CH ₂ ) _n CO-, wherein n is an integer from _{4 to 38} ;

The Q2 group is ε(AEEA) _n -MPA, n=0-3, and its structural formula is as follows:

。

.

 The definition of each symbol is as follows, His : histidine, Ala: alanine, Glu: glutamic acid, Gln: Glutamine, Gly: glycine, Thr: threonine, Phe: phenylalanine, Ser: serine, Asp: aspartic acid, Val: proline , Tyr : Tyrosine , Leu : Leucine , Ile : Isoleucine , Lys : Lysine , Trp : Tryptophan , Arg : Arginine , Asn : Asparagine, Pro : Proline, Aib : 2-Aminoisobutyric acid, AEEA : 2-(2-(2-Aminoethoxy)ethoxy)acetic acid, MPA : 3- Maleimide propionic acid.

 In one embodiment of the invention, Xaa7 is preferably histidine.

 In one embodiment of the invention, Xaa8 is preferably D-Ala.

 In one embodiment of the invention, the bridging group W is preferably a non-branched alkane having 2 methylene groups, ω - Dicarboxyl; further preferred is glutamic acid.

In one embodiment of the invention, the lipophilic substituent is preferably CH ₃ (CH ₂ ) _n CO , wherein n is an integer from 4 to 24; further preferably CH ₃ (CH ₂ ) ₁₄ CO- .

 The GLP-I analog of the present invention has insulinotropic activity, good stability, long half-life, and no clinical adverse effects. Can be used for treatment Hyperglycemia, Type 2 Diabetes, Impaired Glucose Tolerance, Type 1 Diabetes, Obesity, Hypertension, X Syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, dyspepsia or gastric ulcer; or to reduce food intake, reduce beta Apoptosis, enhancement of β-cell function and β-cell mass and / or recovery of glucose sensitivity of β-cells; preferably treatment of hyperglycemia, type 2 diabetes, impaired glucose tolerance, 1 Type 2 diabetes; more preferably treatment of type 2 diabetes.

 Another aspect of the invention provides a method of preparing a GLP-I analog of the first aspect of the invention, comprising: synthesizing GLP- The parent peptide of the I analog, the free amino group and the free carboxyl group on the parent peptide are protected by a protecting group; the protecting group on the amino acid residue at the coupling position of the Q1 group at the parent peptide is removed, Q1 a group coupled to the parent peptide; a protecting group on the amino acid residue at the coupling position of the Q2 group on the parent peptide, Q2 The group is coupled to the parent peptide; the protecting group on the other amino acid residues on the parent peptide is removed, and the GLP-I analog is prepared.

Protection and deprotection of free amino groups and free carboxyl groups of amino acid residues can be carried out using techniques well known in the art. The carboxyl group is generally protected in the form of a salt or an ester. Commonly used salts are potassium salt, sodium salt, triethylamine salt, and tributylamine salt; commonly used esters have methyl esters ( OMe), ethyl ester (OEt), benzyl ester (oBzl), tert-butyl ester (OtBu). Commonly used amino protecting groups are benzyloxycarbonyl (CBZ) and tert-butoxycarbonyl (Boc). ), p-toluenesulfonyl (Tosyl) and the like.

 In one embodiment of the invention, the method further comprises the step of: purifying the prepared GLP-I analog using reverse phase liquid chromatography.

 For the prepared GLP- I analog purification can be further purified using techniques well known in the art, such as molecular sieves, adsorption chromatography, affinity chromatography, hydrophobic chromatography, electrophoresis, concentrated crystallization, and the like.

 In one embodiment of the invention, the coupling of a Q1 group to the parent peptide comprises: The carboxyl group of the amino acid residue of the bridging group and the N- of the Lys of the parent peptide An amide bond is formed on the terminal residue to be attached to the parent peptide, and the lipophilic substituent is coupled to the parent peptide by an amide bond between the carboxyl group and the amino group of a bridging group.

In one embodiment of the invention, the coupling of a Q2 group to the parent peptide comprises: the carboxyl group of (AEEA) _n forms an amide bond with the N-terminal residue of a Lys of the parent peptide Attached to the parent peptide, MPA forms an amide bond with the amino group of (AEEA) _n at its carboxyl group; n = 1-3 in the (AEEA) _n .

 In one embodiment of the invention, the coupling of a Q2 group to the parent peptide comprises: MPA with its carboxyl group and one of the parent peptide An amide bond is formed on the N-terminal residue of Lys.

 The preparation method of the GLP-I analog of the invention is simple, the product yield is high, and the preparation cost is greatly reduced.

 Another aspect of the invention provides a pharmaceutical composition comprising the GLP- of the first aspect of the invention I analog or a pharmaceutically acceptable salt thereof.

 In one embodiment of the invention, the pharmaceutical composition comprises: 0.9 mg/ml of the GLP of the first aspect of the invention I analog or a pharmaceutically acceptable salt thereof, 5.0% (w/v) cresol, 5.2% (w/v) mannitol, 12.5 mg/ml propylene glycol, 8.0 mM Phosphate buffer. Wherein the pH is usually from about 5 to 8, preferably from 6 to 8, more preferably from 7 to 7.5.

 When the GLP-I analog is used in the preparation of a medicament, it is preferably a pharmaceutically acceptable salt thereof. GLP- of the present invention The I analog can be reacted with any of the inorganic bases, inorganic and organic acids to form a salt. The acid which is usually used to form an acid addition salt is an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., and an organic acid such as p- Toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid, hydrobromic acid, more preferably with hydrochloric acid. The base addition salts include salts derived from inorganic base derivatives such as ammonium or alkali metal or rare earth metal hydroxides, carbonates, hydrogencarbonates and the like. Such bases include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate and the like.

The pharmaceutical compositions of the invention may also include pharmaceutically acceptable carriers thereof. The term 'pharmaceutically acceptable carrier', as used herein, refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) A full discussion of pharmaceutically acceptable excipients can be found. Pharmaceutically acceptable carriers in the compositions can include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as disintegrants, wetting agents, emulsifiers, and the like may also be present in these carriers. pH buffer material, etc. Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually about 5-8, preferably, the pH is about 6-8. .

 In the compositions of the present invention, various dosage forms such as pills, tablets, capsules can be prepared using techniques well known in the art. An injection or the like is preferably an injection. The injection may be a solution type, a sterile powder, preferably a sterile powder. When preparing an injection, the GLP-I analog of the present invention or a pharmaceutically acceptable salt thereof is used according to a technique well known in the art. Formulated as a solution, the solvent used is selected from the group consisting of water for injection, soybean oil for injection, ethanol, glycerin, propylene glycol, polyethylene glycol, benzyl benzoate, dimethylethanolamine. Other substances such as Tween 80 can be added to the solution. , methyl cellulose, mannitol, glucose, sodium chloride, cresol, phenol, chlorobutanol and the like. The solution can be selected from acetic acid - sodium acetate, citric acid - A sodium citrate, lactic acid, phosphate buffer system, preferably a phosphate buffer system. The formulated solution is prepared as an injection after filtration to remove solid particles, remove heat, sterilize or sterilize, and if the injection is a sterile powder, it further includes freeze-drying, and these techniques are well known in the art.

The administration route of the injection of the present invention may be intravenous, spinal injection, intramuscular, subcutaneous or intradermal injection, preferably intravenous, intramuscular, subcutaneous, and more preferably intravenous, depending on medical needs.

 It should be understood that the GLP-I analog used The effective dose can vary depending on the severity of the subject to be administered or treated. The specific situation depends on the individual circumstances of the subject (such as the subject's weight, age, physical condition, and the desired effect) It is decided that this is within the scope of the skilled physician.

 The compositions of the present invention are stable, and vascular irritation, muscle irritation, allergic and hemolysis experiments demonstrate no adverse clinical response.

 Another aspect of the invention is the use of a GLP-I analogue of the invention in the manufacture of a medicament for the treatment or prevention of a disease.

 These diseases include hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, hypertension, X Syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, indigestion or gastric ulcer.

 In one embodiment of the invention, the GLP-I analog is for use in the manufacture of a medicament for delaying or preventing the development of type 2 diabetes.

 In a further aspect of the invention, the GLP-I analog of the invention reduces food intake, reduces beta-cell apoptosis, and enhances beta-preparation Use of cell function and beta-cell mass and/or recovery of beta-cell glucose sensitivity in drugs.

detailed description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Those who do not specify the specific conditions in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

 For the English abbreviations used in the embodiments of the present invention, the list gives a comparison of the English names therein, as shown in Table 1.0.

 Table 1.0 English and Chinese names in English abbreviations

 abbreviation  English name  Chinese name  Fmoc  9-fluorenylmethoxycarbonyl  9-fluorenylmethoxycarbonyl  tBu  Tert-butyl  Tert-butyl  Boc  Tert-butoxycarbonyl  Tert-butyl acyl  Otbu  Tert-butoxy  Tert-butoxy  Trt  Triphenylmethyl(trityl)  Tritylmethyl  Aloc  Allyloxycarbonyl  Allyloxycarbonyl  ivDDe 1-(4,4-dimethyl-2,6-dioxocyclohexyliden)-3-methylbutyl  1-( 4 , 4-dimethyl-2,6-dioxocyclohexyl)-3-methylbutyl Pd(PPh ₃ ) ₄  Tetrakis(triphenylphosphine)palladium  Tetrakis(triphenylphosphine) palladium  NMM  N-methylmorpholin  N-methylmorpholine  DIC  N,N-diisopropylcarbodiimide  N,N-diisopropylcarbodiimide  HOBt  1-hydroxy-1H-benzotriazole  N-hydroxybenzotriazole  NMP  N-methyl-2-pyrrolidinone  N-methylpyrrolidone  DMF  N,N-dimethylformamide  N,N-dimethylformamide  Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-yl-sulfonyl  2,2,4,6,7-pentamethyldihydrobenzofuran -5-yl-sulfonyl  Fmoc-AEEA-OH  N-Fmoc-2-[2-(2-aminoethoxy)ethoxy]acetic acid  N-Fmoc-2-[2-(2-aminoethoxy)ethoxy)acetic acid  MPA  3-maleimidopropionic acid  3-maleimidopropionic acid  TFA  Trifluoroacetic acid  Trifluoroacetate  EDT  Ethanedithiol  Ethylenedithiol  DCM  Dichloromethane  Dichloromethane  HBTU  2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium Hexafluorophosphate  Benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate  DIEA  2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium Tetrafluoroborate  N-diisopropylethylamine

 Example 1:

 Synthesis of Compound 1 ( Compound 1 )

 1a) Master peptide chain assembly:

 Synthesized on the CS336X peptide synthesizer (C S Bio, USA) according to the Fmoc/tbu strategy 0.25mmol scale Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)

-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala -Ala- Lys(Aloc)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys (Boc)-Gly-Arg(pbf)-Lys (ivDDe) -wang resin.

 1b) Removal of Aloc and introduction of Q1:

The peptide resin obtained in 1a) was added to chloroform, Pd(PPh ₃ ) ₄ , NMM was added under argon atmosphere, and the reaction was stirred for 2 hours. After washing with DMF and DCM, the resin was added to Fmoc-Glu-Otbu, DIC, HOBt. NMP mixed solution, coupled for 2 hours, piperidine / DMF to remove Fmoc group, add palmitic acid, DIC, HOBt NMP coupling solution, coupled for 3 hours, get: Boc-His (Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr

(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala- Lys(Palmitoyl-gama-Glu-Otbu)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys (Boc) -Gly-Arg(pbf)-Lys (ivDDe) -wang resin.

 1c) Removal of ivDDe and introduction of Q2:

 In NMP: DCM is 1:1 (volume ratio) solution will be 1b The resulting protected peptidyl resin was washed twice, freshly prepared 2% hydrazine NMP solution was added, and the reaction mixture was shaken at room temperature for 12 minutes and then filtered. Repeat the 肼 processing step twice. After that NMP, DCM and NMP wash the resin thoroughly. Add Fmoc-AEEA-OH, HBTU, DIEA NMP mixed coupling solution, shake 3 After the hour, the mixture was filtered, washed, and the Fmoc group was removed by piperidine/DMF. The reaction was carried out for 3 hours with 3-M maleimidopropionic acid, DIC, HOBt DMF coupling solution to obtain: Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala- Lys(Palmitoyl-gama-Glu-Otbu)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys (Boc)-Gly-Arg(pbf)-Lys(AEEA-MPA)-wang resin.

 1d) Removal of full protection

Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala-Lys(Palmitoyl-gama-Glu-Otbu)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc )-Leu-Val-Lys(Boc)-Gly-Arg(pbf)-Lys(AEEA-MPA)-wang Resin was added to the round bottom flask and the cutting solution was added to the ice bath TFA/EDT/Phenol/

 Water (88/2/5/5, volume ratio), temperature rise, control lysate temperature 25 °C, reaction 90 min . After filtration, the filter cake was washed 3 times with a small amount of TFA and the filtrate was combined. The filtrate was slowly poured into ice diethyl ether with stirring. Allow to stand for more than 1 h until the precipitation is complete. The supernatant was decanted, the pellet was centrifuged and washed with ice diethyl ether 6 The crude product was obtained: H-His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp

-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Palmitoyl-gama-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg- Lys(AEEA-MPA)-OH .

 1e) Purification and purification

The crude product obtained in 1d) was dissolved in 5% acetic acid / H ₂ O and purified by two semi-preparative HPLC on a 10 μm reversed C18 packed 50 mm x 250 mm column. The column was eluted with a gradient of 32-50% CH ₃ CN-0.1% TFA/H ₂ O at 50 ml/min for 45 minutes, and the peptide-containing fraction was collected, concentrated to remove CH ₃ CN, and then lyophilized. Pure product with HPLC purity greater than 98% is obtained: H-His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala- Ala-Lys (Palmitoyl-gama-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys (AEEA-MPA)-OH.

 The isolated product was analyzed by PDMS and the m/z value of the protonated molecular ion peak was found to be 4106.31 ± 3 . Thus, the molecular weight of Compound 1 prepared in Example 1 was found to be 4105.31 ± 3 Da (theoretical value 4105.31). Staphylococcus aureus V8 The target compound is subjected to enzymatic cleavage, followed by mass spectrometry of the peptide fragment by PDMS to determine the acylation position (Lys26, Lys37).

 The compounds prepared in Example 1 were examined from three experiments: pharmacokinetics, in vitro activity assay, and pharmacodynamic analysis. Animal experiments, the results are as follows:

 Pharmacokinetic analysis of compound 1 and GLP- I

 For male SD rats, intravenous injection (IV) or subcutaneous injection (SC) at a dose of 0.1 mg/kg The compound 1 and GLP-I prepared in Example 1 were separately administered. The animals were bled at different times within 0-360 hours after dosing, and the plasma of each sample was collected and used with N- End-specific radioimmunoassay analysis. Calculate pharmacokinetic parameters using model-dependent (data for IV) and model-independent (for SC-derived data) data as shown in Table 1-1 and Table 1-2 is shown. The elimination half-life of Compound 1 administered by IV was approximately 19 hours, and the elimination half-life of GLP-I was approximately 12 hours. And administered by SC Compound 1 has an elimination half-life of approximately 15 hours and a GLP-I elimination half-life of approximately 8 hours. Compound 1 and GLP-I were administered by IV or SC route , no clinical adverse hair should occur. From Table 1-1 and Table 1-2, it can be observed that Compound 1 prolongs the elimination half-life, reduces the clearance rate, and the like.

 Table 1-1 Mean value of compound 1 (± SD) pharmacokinetic data

 Route of administration Cmax (ng/mL) Tmax (h) AUC0-last (ng*h/mL) T1/2 (h) CL/F (mL/h/kg) Vss/F (mL/kg)  1 IV
(SD) 2251
(186) 0.10
(0.00) 53425
(1803) 18.4
(3.3) 2.1
(0.4) 56
(1.2) SC
(SD) 205
(33) 27.0
(0.0) 15847
(2935) 14.2
(1.1) 6.8
(1.5) 265
(65)

 Table 1-2 Mean of GLP-I (± SD) pharmacokinetic data

 Route of administration C _max (ng/mL) T _max (h) AUC _0-last (ng*h/mL) T _1/2 (h)  CL/F (mL/h/kg)  Vss/F (mL/kg) IV
(SD) 1752
(223) 0.008
(0.00) 33124
(2644) 11
(4.3) 1.8
(0.8) 101
(1.7) SC
(SD) 164
(38) 8.0
(0.0) 7942
(1985) 7
(2.1) 2.8
(1.7) 158
(45)

Wherein C _max represents the maximum observed plasma concentration; T _max represents the time at which the observed maximum plasma concentration is reached; AUC _0-last represents the area under the plasma concentration-time curve measured from 0 to infinity; _1/2 represents the elimination half-life in hours; CL/F represents the total body clearance as a function of bioavailability; Vss/F represents the volume of distribution at steady state as a function of bioavailability.

 In vitro activity assay of compound 1 and GLP- I

 HEK-293 cells stably expressing the human GLP-I receptor for the CRE-luciferase system, per well 120 μ l Low serum DMEM FBS medium, 30,000 cells were seeded into 96-well plates. On the second day after inoculation, a 20 μl aliquot of the sample to be tested was dissolved in 0.5% BSA. Medium, mixed with the cells and incubated for 5 hours. Before the cells are added to obtain a dose response curve (determining the EC50 value from the curve), generally the preparation of the test compound 1 is from 0.001 nM to 15 dilutions of 10nM for 15 samples of GLP-I from 0.001nM to 10nM and Val8-GLP-I (7-37) OH Standards Prepare 10 standard solutions of 0.3nM and 3nM. After incubation, add 100 μl of luciferase reagent directly to each plate and mix gently 2 Minutes. The plate was placed in a Tri-lux luminometer and the light output due to luciferase expression was calculated. The average EC50 values for Compound 1 and GLP-I are as follows: Average for Compound 1 The EC50 values were 0.42 ± 0.05 nM and the average EC50 of GLP-I was 0.28 ± 0.04 nM.

 Pharmacodynamic analysis of compound 1 and GLP- I

 Compound 1 and GLP- I were administered by subcutaneous injection (SC) according to 0.01 mg/kg, respectively. The dose is administered to male cynomolgus monkeys. The control solution phosphate buffer was also injected by a subcutaneous injection (SC) route at a dose of 0.01 mg/kg. Subcutaneous injection of 0.01mg/kg dose of compound After 1 and GLP-I, glucose solution was infused step by step on 1, 2, 3, 5, 7, and 10 days. Subcutaneous injection (SC) 0.01mg/kg Immediately after the injection of the control solution, the glucose solution was infused stepwise. The stepwise infusion of glucose solution was performed on monkeys given sedatives after fasting for 15 hours. At the beginning of the infusion of glucose solution 10 A minute before the blood sample is taken to define the baseline. Then, re-infusion for 30 minutes at a rate of 15 mg/kg/min. During the infusion, to 15 Blood samples were taken at intervals and insulin levels were determined by immunoassay. The data are shown in Tables 1-3 and 1-4.

 Table 1-3 Average value of compound 1 (± SD) Pharmacodynamic parameter value Area under the insulin curve

 Injection AUC _0-last
(pM*min) Day 1 Day 2 Day 3 Day 5 Day 7 Day 10 Average SD Average SD Average SD Average SD Average SD Average SD  Control solution  15432  3529  14251  2234  14012  2014  13984  1819  12759  1587  11845  1096  Compound 1  35453  3102  33123  1852  31721  1542  30456  1432  29652  1254  28121  1022

 Table 1-4 Average value of GLP-I (± SD) Pharmacodynamic parameter value Area under the insulin curve

 Injection AUC _0-last
(pM*min) Day 1 Day 2 Day 3 Day 5 Day 7 Day 10  Average SD  Average SD  Average SD  Average SD  Average SD  Average SD  Control solution  15432  3529  14251  2234  14012  2014  13984  1819  12759  1587  11845  1096  GLP- I  31254  3542  30045  2432  21123  1563  15542  1348  11764  1141  10548  1248

 As can be seen from Tables 1-3 and 1-4, after a single subcutaneous injection of 0.01 mg/kg of Compound 1, at least 10 It was shown to have insulinotropic activity, and only 3 days after injection of GLP-I had insulinotropic activity.

 Example 2:

 Compound 2 ( Compound 2 ) synthesis

 1a) Master peptide chain assembly:

 Synthesis of 0.25mmol scale on CS336X peptide synthesizer according to Fmoc/tbu strategy Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala-Lys( Boc )-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys (ivDDe) -Gly-Arg(pbf)-Lys (Aloc) -wang resin.

 1b) Removal of Aloc and introduction of Q1:

The peptide resin obtained in 1a) was added to chloroform, Pd(PPh ₃ ) ₄ , NMM was added under argon atmosphere, and the reaction was stirred for 2 hours. The resin was washed with DMF, DCM, and then added with Fmoc-Glu-OtBu, DIC, HOBt. NMP coupling solution, coupled for 3 hours, piperidine / DMF to remove Fmoc group, add palmitic acid, DIC, HOBt, NMP coupling solution, coupled for 3 hours, get: Boc -His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)

-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala-Lys (Boc)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys(ivDDe)-Gly-Ar g(pbf)-Lys( Palmitoyl-gama-Glu-Otbu )- wang resin .

 1c) Removal of ivDDe and introduction of Q2:

 There will be 1b in a solution where NMP:DCM is 1:1 (volume ratio) The resulting protected peptidyl resin was washed twice, freshly prepared 2% hydrazine NMP solution was added, and the reaction mixture was shaken at room temperature for 12 minutes and then filtered. Repeat the 肼 processing step twice. After that NMP, DCM and NMP wash the resin thoroughly. Add 3-maleimidopropionic acid, DIC, HOBt The DMF coupling solution was coupled for 3 hours to give: Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe

-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala -Lys( Boc )-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys (MPA) -Gly-Arg(pbf)-Lys( Palmitoyl-gama-Glu-Otbu) - wang resin.

 1d) Removal of full protection

Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala-Lys( Boc) -Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys(MPA)-Gly-Arg(pbf)-Lys( Palmitoyl-gama-Glu-Otbu )- wang resin resin was added to the round bottom flask, and the cutting solution was added under ice bath. TFA/EDT/Phenol/Water

 (88/2/5/5, volume ratio), temperature rise, control lysate temperature 25 °C, reaction 90 min . After filtration, the filter cake was washed 3 times with a small amount of TFA and the filtrate was combined. The filtrate was slowly poured into ice diethyl ether with stirring.

 Allow to stand for more than 1 h until the precipitation is complete. The supernatant was decanted, the precipitate was centrifuged, and washed with ice diethyl ether 6 times to give a crude product: H-His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser

-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (MPA )-Gly-Arg-Lys( Palmitoyl-gama-Glu) -OH.

 1e) Purification and purification

The crude product obtained in 1d) was dissolved in 5% acetic acid / H ₂ O and purified by two semi-preparative HPLC on a 10 μm reverse phase C18 packed 50 mm x 250 mm column. The column was eluted with a gradient of 34-46% CH3CN-0.1% TFA/H ₂ O at 50 ml/min for 45 minutes, and the peptide-containing fraction was collected, concentrated to remove CH ₃ CN, and then lyophilized. Pure product with HPLC purity greater than 98% is obtained: H-His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala- Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (MPA)-Gly-Arg-Lys (Palmitoyl-gama-Glu)-OH.

 The separated product was analyzed by PDMS and the m/z value of the protonated molecular ion peak was found to be 3949.31 ± 3 . Thus, the molecular weight of the compound 2 prepared in Example 2 was found to be 3948.31 ± 3 Da (theoretical value: 3948.31).

 The compounds prepared in Example 2 were examined from three experiments of pharmacokinetics, in vitro activity assay, and pharmacodynamic analysis. Animal experiments, the results are as follows:

 Pharmacokinetic analysis of compound 2 and GLP- I

 For male SD rats, intravenous injection (IV) or subcutaneous injection (SC) at a dose of 0.1 mg/kg The compound 2 and GLP-I prepared in Example 2 were separately administered. The animals were bled at different times within 0-360 hours after dosing, and the plasma of each sample was collected and used with N- End-specific radioimmunoassay analysis. Calculate pharmacokinetic parameters using model-dependent (data for IV) and model-independent (for SC-derived data) data as shown in Table 2-1 and Table 2-2 is shown. The elimination half-life of Compound 2 administered by IV is approximately 23 hours, and the elimination half-life of GLP-I is approximately 12 hours. And administered by SC The elimination half-life of Compound 2 is approximately 18 and the elimination half-life of GLP-I is approximately 8 hours. Administration of Compound 2 and GLP-I by IV or SC route , no clinical adverse hair should occur. Compound 2 extended elimination half-life, reduced clearance, etc. can be observed by Table 2-1 and Table 2-2.

 Table 2-1 Mean (±) pharmacokinetic data of Compound 2

 Route of administration  Cmax (ng/mL)  Tmax (h)  AUC0-last (ng*h/mL)  T1/2 (h)  CL/F (mL/h/kg)  Vss/F (mL/kg)  2 IV
(SD) 2239
(201) 0.11
(0.00) 53439
(1631) 21.9
(3.0) 2.3
(0.5) 48
(1.4) SC
(SD) 211
(32) 23.8
(0.0) 15812
(1914) 16.9
(1.2) 5.7
(1.6) 259
(twenty two)

 Table 2-2 Mean (±) pharmacokinetic data of GLP-I

 Route of administration  Cmax (ng/mL)  Tmax (h)  AUC0-last (ng*h/mL)  T1/2 (h)  CL/F (mL/h/kg)  Vss/F (mL/kg) IV
(SD) 1752
(223) 0.008
(0.00) 33124
(2644) 11
(4.3) 1.8
(0.8) 101
(1.7) SC
(SD) 164
(38) 8.0
(0.0) 7942
(1985) 7
(2.1) 2.8
(1.7) 158
(45)

Wherein C _max represents the maximum observed plasma concentration; T _max represents the time at which the observed maximum plasma concentration is reached; AUC _0-last represents the area under the plasma concentration-time curve measured from 0 to infinity; _1/2 represents the elimination half-life in hours; CL/F represents the total body clearance as a function of bioavailability; Vss/F represents the volume of distribution at steady state as a function of bioavailability.

 In vitro activity assay of compound 2 and GLP-I

 HEK-293 cells stably expressing the human GLP-I receptor for the CRE-luciferase system, per well 120 μl of low serum DMEM FBS medium and 30,000 cells were seeded into 96-well plates. On the second day after inoculation, dissolve the 20 μl aliquot of the sample to be tested. In 0.5% BSA, mix with the cells and incubate for 5 hours. Before the cells are added to obtain a dose response curve (determining the EC50 value from the curve), generally the preparation of the test compound 1 contains 15 dilutions from 0.001nM to 10nM, 15 dilutions from 0.001nM to 10nM for GLP-I to be tested, and Val8- The GLP-I (7-37) OH standard was prepared in 10 standard solutions of 0.3 nM and 3 nM. After incubation, 100 μl Luciferase reagent was added directly to each plate and gently mixed for 2 minutes. The plate was placed in a Tri-lux luminometer and the light output due to luciferase expression was calculated. Average of compound 2 and GLP-I The EC50 values were as follows: Compound 2 had an average EC50 of 0.44 ± 0.06 nM; GLP-I had an average EC50 of 0.28 ± 0.04 nM. .

 Pharmacodynamic analysis of compound 2 and GLP-I

 Compound 2 and GLP- I were administered by subcutaneous injection (SC) according to 0.01 mg/kg The dose is administered to male cynomolgus monkeys. The control solution phosphate buffer was also injected by a subcutaneous injection (SC) route at a dose of 0.01 mg/kg. Subcutaneous injection of 0.01mg/kg dose of compound 2 and GLP- I, 1 , 2, 3, 5, 7 and 10 days, infusion of glucose solution. Subcutaneous injection (SC) 0.01mg/kg Immediately after the injection of the control solution, the glucose solution was infused stepwise. The stepwise infusion of glucose solution was performed on monkeys given sedatives after fasting for 15 hours. At the beginning of the infusion of glucose solution 10 A minute before the blood sample is taken to define the baseline. Then, re-infusion for 30 minutes at a rate of 15 mg/kg/min. During the infusion, to 15 Blood samples were taken at intervals and insulin levels were determined by immunoassay. The data are shown in Tables 2-3 and 2-4.

 Table 2-3 Average value of compound 2 (± SD) Pharmacodynamic parameter value Area under the insulin curve

 Injection AUC _0-last
(pM*min) Day 1 Day 2 Day 3 Day 5 Day 7 Day 10 Average SD Average SD Average SD Average SD Average SD Average SD  Control solution  15432  3529  14251  2234  14012  2014  13984  1819  12759  1587  11845  1096  Compound 2  33251  2545  31545  1531  30123  1266  29142  1148  27964  1041  26418  1048

 Table 2-4 Average value of GLP-I (± SD) Pharmacodynamic parameter value Area under the insulin curve

 Injection AUC _0-last
(pM*min) Day 1 Day 2 Day 3 Day 5 Day 7 Day 10  Average SD  Average SD  Average SD Average  SD  Average SD Average  SD  Control solution  15432  3529  14251  2234  14012  2014  13984  1819  12759  1587  11845  1096  GLP- I  31254  3542  30045  2432  21123  1563  15542  1348  11764  1141  10548  1248

 As can be seen from Tables 2-3 and 2-4, after a single subcutaneous injection of 0.01 mg/kg of Compound 2, at least 10 It was shown to have insulinotropic activity, and only 3 days after injection of GLP-I had insulinotropic activity.

 Example 3:

 Compound 3 ( Compound 3 ) synthesis

 1a) Master peptide chain assembly:

 Synthesis of 0.25mmol scale on CS336X peptide synthesizer according to Fmoc /tbu strategy Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala-Lys( Aloc )-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys( ivDDe )-Gly-Arg(pbf)- Pro -wang resin.

 1b) Removal of Aloc and introduction of Q1:

 Add the peptide resin obtained in 1a to chloroform, add Pd(PPh3)4, NMM under argon atmosphere, and stir the reaction 2 After the resin was washed with DMF and DCM, a mixed solution of Fmoc-Asp-Otbu, DIC, HOBt in NMP was added and coupled for 2 hours, piperidine ( Piperidine ) /DMF Remove Fmoc group, stearic acid, DIC, HOBt, coupled for 3 hours, to obtain: Boc-His (Boc)

-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu) -Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala-Lys(Stearyl -beta-Asp-Otbu )-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys( ivDDe )-Gly-Arg(pbf)-Pro-wang resin.

 1c) Removal of ivDDe and introduction of Q2:

 In NMP: DCM is 1:1 (volume ratio) solution will be 1b The resulting protected peptidyl resin was washed twice, freshly prepared 2% hydrazine NMP solution was added, and the reaction mixture was shaken at room temperature for 12 minutes and then filtered. Repeat the 肼 processing step twice. After that NMP, DCM and NMP wash the resin thoroughly. Add Fmoc-AEEA-AEEA-OH, HBTU, DIEA NMP mixed coupling solution, shake 3 After the hour, the mixture was filtered, washed, and the Fmoc group was removed by piperidine/DMF. The reaction was carried out for 3 hours with 3-M maleimidopropionic acid, DIC, HOBt DMF coupling solution to obtain: Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala-Lys(Stearyl -beta-Asp-Otbu )-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys( AEEA-AEEA-MPA )-Gly-Arg(pbf)- Pro-wang resin.

 1d) Removal of full protection

Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-Ala-Lys(Stearyl -beta-Asp-Otbu )-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys( AEEA-AEEA-MPA )-Gly-Arg(pbf)- Pro -wang resin was added to the round bottom flask and the cutting solution was added to the ice bath TFA/EDT/Phenol/

 Water (88/2/5/5, volume ratio), temperature rise, control lysate temperature 25 °C, reaction 90 min . After filtration, the filter cake was washed 3 times with a small amount of TFA and the filtrate was combined. The filtrate was slowly poured into ice diethyl ether with stirring. Allow to stand for more than 1 h until the precipitation is complete. The supernatant was decanted, the pellet was centrifuged and washed with ice diethyl ether 6 Second, the crude product was obtained: H -His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp

-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Stearyl-beta-Asp -Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (AEEA-AEEA-MPA) -Gly-Arg-Pro-OH.

 1e) Purification and purification

The crude product obtained in 1d) was dissolved in 5% acetic acid / H ₂ O and purified by two semi-preparative HPLC on a 10 μm reverse phase C8 packed 50 mm x 250 mm column. The column was eluted with 35-54% CH3CN-0.1% TFA / H 2 O gradient at 50ml / min 45 minutes to collect the fraction containing the peptide, was concentrated to remove CH3CN after lyophilization. Pure product with HPLC purity greater than 98% is obtained: H -His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala- Ala-Lys(Stearyl-beta-Asp)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys (AEEA-AEEA-MPA)-Gly-Arg-Pro-OH.

 The isolated product was analyzed by PDMS and the m/z value of the protonated molecular ion peak was found to be 4205.78 ± 3 . Thus, the molecular weight of Compound 3 prepared in Example 3 was found to be 4204.78 ± 3 Da (theoretical value 4204.78). Staphylococcus aureus V8 The target compound is subjected to enzymatic cleavage, followed by mass spectrometry of the peptide fragment by PDMS to determine the acylation position (Lys26, Lys34).

 The compounds prepared in Example 3 were examined from three experiments of pharmacokinetics, in vitro activity assay, and pharmacodynamic analysis. Animal experiments, the results are as follows:

 Pharmacokinetic analysis of compound 3 and GLP-I

 For male SD rats, intravenous injection (IV) or subcutaneous injection (SC) at a dose of 0.1 mg/kg The compound 3 and GLP-I prepared in Example 3 were separately administered. The animals were bled at different times within 0-360 hours after dosing, and the plasma of each sample was collected and used with N- End-specific radioimmunoassay analysis. Calculate pharmacokinetic parameters using model-dependent (data for IV) and model-independent (for SC-derived data) data as shown in Table 3-1 and Table 3-2 is shown. The elimination half-life of Compound 3 administered by IV is approximately 16 hours, and the elimination half-life of GLP-I is approximately 12 hours. Compound administered by SC The elimination half-life of 3 is about 13 and the elimination half-life of GLP-I is about 8 hours. Compound 3 and GLP- are administered separately via IV or SC routes I, no clinical adverse reactions should occur. Compound 3 extended elimination half-life, reduced clearance, etc. can be observed by Table 3-1 and Table 3-2.

 Table 3-1 Average value of compound 3 (± SD) pharmacokinetic data

 Route of administration  Cmax (ng/mL)  Tmax (h)  AUC0-last (ng*h/mL)  T1/2 (h)  CL/F (mL/h/kg)  Vss/F (mL/kg)  3 IV
(SD) 2191
(209) 0.09
(0.00) 53335
(1565) 14.5
(3.5) 2.2
(0.5) 51
(1.3) SC
(SD) 217
(30) 22.7
(0.0) 15742
(1454) 11.3
(1.3) 4.9
(1.4) 245
(28)

 Table 3-2 Mean of GLP-I (± SD) pharmacokinetic data

 Route of administration C _max (ng/mL) T _max (h) AUC _0-last (ng*h/mL) T _1/2 (h)  CL/F (mL/h/kg)  Vss/F (mL/kg) IV
(SD) 1752
(223) 0.008
(0.00) 33124
(2644) 11
(4.3) 1.8
(0.8) 101
(1.7) SC
(SD) 164
(38) 8.0
(0.0) 7942
(1985) 7
(2.1) 2.8
(1.7) 158
(45)

 Wherein Cmax represents the maximum observed plasma concentration; Tmax represents the time at which the observed maximum plasma concentration is observed; AUC0-last represents the measured plasma concentration from 0 to infinity - the area under the time curve; T 1/2 represents the elimination half-life in hours; CL/F Indicates the total body clearance as a function of bioavailability; Vss/F represents the volume of distribution at steady state as a function of bioavailability.

 In vitro activity assay of compound 3 and GLP-I

 HEK-293 cells stably expressing human GLP-I receptor for CRE-luciferase system, per well 120 μl of low serum DMEM FBS medium and 30,000 cells were seeded into 96-well plates. On the second day after inoculation, dissolve the 20 μl aliquot of the sample to be tested. In 0.5% BSA, mix with the cells and incubate for 5 hours. Before the cells are added to obtain a dose response curve (determining the EC50 value from the curve), generally the test compound 3 is prepared to contain 15 dilutions from 0.001nM to 10nM for 15 dilutions from 0.001nM to 10nM for the GLP-I to be tested, and Val8- The GLP-I (7-37) OH standard was prepared in 10 standard solutions of 0.3 nM and 3 nM. After incubation, 100 μl Luciferase reagent was added directly to each plate and gently mixed for 2 minutes. The plate was placed in a Tri-lux luminometer and the light output due to luciferase expression was calculated. Average of compound 3 and GLP-I The EC50 values were as follows: Compound 3 had an average EC50 of 0.33 ± 0.06 nM; GLP-I had an average EC50 of 0.28 ± 0.04 nM. .

 Pharmacodynamic analysis of compound 3 and GLP-I

 Compound 3 and GLP-I were administered by subcutaneous injection (SC) according to 0.01 mg/kg, respectively. The dose is administered to male cynomolgus monkeys. The control solution phosphate buffer was also injected by a subcutaneous injection (SC) route at a dose of 0.01 mg/kg. Subcutaneous injection of 0.01 mg/kg of compound 3 After GLP-I, the glucose solution was infused in steps of 1, 2, 3, 5, 7, and 10 days. Subcutaneous injection (SC) 0.01mg/kg Immediately after the injection of the control solution, the glucose solution was infused stepwise. The stepwise infusion of glucose solution was performed on monkeys given sedatives after fasting for 15 hours. At the beginning of the infusion of glucose solution 10 A minute before the blood sample is taken to define the baseline. Then, re-infusion for 30 minutes at a rate of 15 mg/kg/min. During the infusion, to 15 Blood samples were taken at intervals and insulin levels were determined by immunoassay. The data are shown in Tables 3-3 and 3-4.

Table 3-3 Average value of compound 3 (± SD) Pharmacodynamic parameter value Area under the insulin curve Injection AUC _0-last
(pM*min) Day 1 Day 2 Day 3 Day 5 Day 7 Day 10 average value SD average value SD average value SD average value SD average value SD average value SD Control solution 15432 3529 14251 2234 14012 2014 13984 1819 12759 1587 11845 1096 Compound 3 32996 2981 30121 1854 26731 1843 20454 1312 15342 1534 11131 832

Table 3-4 Average value of GLP-I (± SD) Pharmacodynamic parameter value Area under the insulin curve Injection AUC _0-last
(pM*min) Day 1 Day 2 Day 3 Day 5 Day 7 Day 10 average value SD average value SD average value SD average value SD average value SD average value SD Control solution 15432 3529 14251 2234 14012 2014 13984 1819 12759 1587 11845 1096 GLP- I 31254 3542 30045 2432 21123 1563 15542 1348 11764 1141 10548 1248

 As can be seen from Tables 3-3 and 3-4, after a single subcutaneous injection of 0.01 mg/kg of Compound 3, at least 5 It was shown to have insulinotropic activity, and only 3 days after injection of GLP-I had insulinotropic activity.

 Example 4:

 Compound 4 ( Compound 4 ) Synthesis

 1a) Master peptide chain assembly:

 Synthesis of 0.25mmol scale on CS336X peptide synthesizer according to Fmoc/tbu strategy Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-A la-Lys(ivDDe)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys(ivDDe)-Gly-Arg(pbf)-Lys(Aloc) -wang resin.

 1b) Removal of Aloc and introduction of Q1:

The peptide resin obtained in 1a) was added to chloroform, Pd(PPh ₃ ) ₄ , NMM was added under argon atmosphere, and the reaction was stirred for 2 hours. The resin was washed with DMF, DCM, and then added with Fmoc-Glu-OtBu, DIC, HOBt. NMP coupling solution, coupled for 3 hours, piperidine / DMF to remove Fmoc group, add palmitic acid, DIC, HOBt, NMP coupling solution, coupled for 3 hours, get: Boc -His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-

Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala -Ala-Lys(ivDDe)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys(ivDDe)-Gly-A Rg(pbf)-Lys (Palmitoyl-gama-Glu-Otbu)- wang resin.

 1c) Removal of ivDDe and introduction of Q2:

 In NMP: DCM is 1:1 (volume ratio) solution will be 1b The resulting protected peptidyl resin was washed twice, freshly prepared 2% hydrazine NMP solution was added, and the reaction mixture was shaken at room temperature for 12 minutes and then filtered. Repeat the 肼 processing step twice. After that NMP, DCM and NMP wash the resin thoroughly. A DMF coupling solution of 3-maleimidopropionic acid, DIC, HOBt was added thereto, and the coupling reaction was carried out for 3 hours to obtain: Boc-His(Boc)-D-Ala

-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu)-Tyr(tbu)-Leu-Glu(Otbu)-Gly -Gln(Trt)-Ala-Ala-Lys( MPA)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys(MPA)-Gly-Ar g(pbf)-Lys( Palmitoyl-gama-Glu-Otbu) -wang resin.

 1d) Removal of full protection

Boc-His(Boc)-D-Ala-Glu(Otbu)-Gly-Thr(tbu)-Phe-Thr(tbu)-Ser(tbu)-Asp(Otbu)-Val-Ser(tbu)-Ser(tbu )-Tyr(tbu)-Leu-Glu(Otbu)-Gly-Gln(Trt)-Ala-A la-Lys(MPA)-Glu(Otbu)-Phe-Ile-Ala-Trp(Boc)-Leu-Val-Lys(MPA)-Gly-Arg(pbf)-Lys(Palmitoyl-gama-Glu-Otbu)- Wang resin resin was added to the round bottom flask, and the cutting solution was added under ice bath TFA/EDT/Phenol/Water

 (88/2/5/5, volume ratio), temperature rise, control lysate temperature 25 °C, reaction 90 min . After filtration, the filter cake was washed 3 times with a small amount of TFA and the filtrate was combined. The filtrate was slowly poured into ice diethyl ether with stirring.

 Allow to stand for more than 1 h until the precipitation is complete. The supernatant was decanted, the precipitate was centrifuged, and washed with ice diethyl ether 6 times to give a crude product: H-His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser

 -Ser-Tyr-Leu-Glu -Gly-Gln-Ala-Ala-Lys(MPA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys(MPA)-Gly-Arg-Lys(Palmitoyl-gama-Glu) -OH.

 1e) Purification and purification

The crude product obtained in 1d was dissolved in 5% acetic acid / H ₂ O and purified by two semi-preparative HPLC on a 10 μm reversed C ₁₈ packed 50 mm x 250 mm column. The column was eluted with a gradient of 34-46% CH3CN-0.1% TFA/H ₂ O at 50 ml/min for 45 minutes, and the peptide-containing fraction was collected, concentrated to remove CH3CN, and lyophilized. Obtain pure product with HPLC purity greater than 98%: H-His-

D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Al a-Lys(MPA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys(MPA)-Gly-Arg-Lys(Palmitoyl-gama-Glu)-OH .

 The separated product was analyzed by PDMS and the m/z value of the protonated molecular ion peak was found to be 4,097.65 ± 3 . Therefore, the molecular weight of the compound 4 prepared in Example 4 was found to be 4096.65 ± 3 Da (theoretical value 4096.65). Staphylococcus aureus V8 The target compound is subjected to enzymatic cleavage, and then the mass of the peptide fragment is determined by PDMS to determine the acylation position (Lys26, Lys34, Lys37).

 The compounds prepared in Example 4 were examined from three experiments: pharmacokinetics, in vitro activity assay, and pharmacodynamic analysis. Animal experiments, the results are as follows:

 Pharmacokinetic analysis of compound 4 and GLP- I

 For male SD rats, intravenous injection (IV) or subcutaneous injection (SC) at a dose of 0.1 mg/kg The compounds prepared in Example 4 were separately administered with GLP-I. The animals were bled at different times within 0-360 hours after dosing, and the plasma of each sample was collected and used with N- End-specific radioimmunoassay analysis. Calculate pharmacokinetic parameters using model-dependent (data from IV) and model-independent (data from SC), as shown in Table 4-1 and Table 4-2 is shown. The elimination half-life of Compound 4 administered by IV is approximately 18 hours, and the elimination half-life of GLP-I is approximately 12 hours. And administered by SC The elimination half-life of Compound 4 is approximately 14 and the elimination half-life of GLP-I is approximately 8 hours. Compound 4 and GLP-I were administered by IV or SC route , no clinical adverse hair should occur. Compound 4 extended elimination half-life, reduced clearance, etc. can be observed by Table 4-1 and Table 4-2.

 Table 4-1 Mean (± SD) pharmacokinetic data of compound 4

 Route of administration  Cmax (ng/mL)  Tmax (h)  AUC0-last (ng*h/mL)  T1/2 (h)  CL/F (mL/h/kg)  Vss/F (mL/kg)  4 IV
(SD) 2211
(211) 0.15
(0.00) 53245
(1613) 16.8
(2.8) 2.7
(0.3) 46
(1.1) SC
(SD) 228
(34) 23.8
(0.0) 15458
(1918) 13.2
(0.9) 4.2
(1.2) 252
(twenty one)

Table 4-2 Mean of GLP-I (± SD) pharmacokinetic data Route of administration C _max (ng/mL) T _max (h) AUC _0-last (ng*h/mL) T _1/2 (h) CL/F (mL/h/kg) Vss/F (mL/kg) IV
(SD) 1752
(223) 0.008
(0.00) 33124
(2644) 11
(4.3) 1.8
(0.8) 101
(1.7) SC
(SD) 164
(38) 8.0
(0.0) 7942
(1985) 7
(2.1) 2.8
(1.7) 158
(45)

Wherein C _max represents the maximum observed plasma concentration; T _max represents the time at which the observed maximum plasma concentration is reached; AUC _0-last represents the area under the plasma concentration-time curve measured from 0 to infinity; _1/2 represents the elimination half-life in hours; CL/F represents the total body clearance as a function of bioavailability; Vss/F represents the volume of distribution at steady state as a function of bioavailability.

 Compound 4 and GLP- I in vitro activity assay

HEK-293 cells stably expressing human GLP-I receptor for CRE-luciferase system were inoculated into 96-well plates according to 120 μl of low serum DMEM FBS medium per well and 30,000 cells per well. On the second day after inoculation, a 20 μl aliquot of the test sample was dissolved in 0.5% BSA, mixed with the cells and incubated for 5 hours. Before the cells were added to obtain a dose response curve (the EC ₅₀ value was determined from the curve), 15 dilutions ranging from 0.001 nM to 10 nM were generally prepared for the test compound 4, and the preparation for the GLP-I to be tested included from 0.001 nM to 15 dilutions of 10 nM and 10 standard solutions of 0.3 nM and 3 nM were prepared for the Val ₈ - GLP-I (7-37) OH standard. After the incubation, 100 μl of luciferase reagent was added directly to each plate and gently mixed for 2 minutes. The plate was placed in a Tri-lux luminometer and the light output due to luciferase expression was calculated. The mean EC ₅₀ values for Compound 4 and GLP-I were as follows: Compound 4 had an average EC ₅₀ value of 0.44 ± 0.06 nM; and GLP-I had an average EC ₅₀ value of 0.28 ± 0.04 nM.

 Pharmacodynamic analysis of compound 4 and GLP-I

 Compound 4 and GLP- I were administered by subcutaneous injection (SC) according to 0.01 mg/kg The dose is administered to male cynomolgus monkeys. The control solution phosphate buffer was also injected by a subcutaneous injection (SC) route at a dose of 0.01 mg/kg. Subcutaneous injection of 0.01mg/kg dose of compound After 4 and GLP-I, glucose solution was infused in steps of 1, 2, 3, 5, 7, and 10 days. Subcutaneous injection (SC) 0.01mg/kg Immediately after the injection of the control solution, the glucose solution was infused stepwise. The stepwise infusion of glucose solution was performed on monkeys given sedatives after fasting for 15 hours. At the beginning of the infusion of glucose solution 10 A minute before the blood sample is taken to define the baseline. Then, re-infusion for 30 minutes at a rate of 15 mg/kg/min. During the infusion, to 15 Blood samples were taken at intervals and insulin levels were determined by immunoassay. The data are shown in Tables 4-3 and 4-4.

Table 4-3 Average value of compound 4 (± SD) Pharmacodynamic parameter value Area under the insulin curve Injection AUC _0-last
(pM*min) Day 1 Day 2 Day 3 Day 5 Day 7 Day 10 average value SD average value SD average value SD average value SD average value SD average value SD Control solution 15432 3529 14251 2234 14012 2014 13984 1819 12759 1587 11845 1096 Compound 4 31953 2982 30123 1862 25728 1382 23449 1162 20632 1034 15121 990

Table 4-4 Average value of GLP-I (± SD) Pharmacodynamic parameter value Area under the insulin curve Injection AUC _0-last
(pM*min) Day 1 Day 2 Day 3 Day 5 Day 7 Day 10 average value SD average value SD average value SD average value SD average value SD average value SD Control solution 15432 3529 14251 2234 14012 2014 13984 1819 12759 1587 11845 1096 GLP- I 31254 3542 30045 2432 21123 1563 15542 1348 11764 1141 10548 1248

 As can be seen from Tables 4-3 and 4-4, after a single subcutaneous injection of 0.01 mg/kg of Compound 4, at least 7 It was shown to have insulinotropic activity, and only 3 days after injection of GLP-I had insulinotropic activity.

 Example 5

 Composition Formulation: Compound 1 prepared in Example 1 at a concentration of 0.9 mg/ml at a concentration of 8.0 mM Phosphate buffer, 5.0% (w/v) cresol, 5.2% (w/v) mannitol, 12.5 mg/ml propylene glycol, pH about 7.5 .

 The preparation process was as follows: 0.9 g of the compound prepared in Example 1 was added to a 100 00 beaker. g mannitol, 50g cresol, 12.5g propylene glycol, 750ml water, add phosphate to a concentration of 8 mM, and adjust the pH to 7.5 with 1N NaOH , add water for injection to volume. Before filtration, 12.5 g of activated carbon was added to the injection, and the pyrogen was adsorbed for 30 minutes while stirring, and decarburized and filtered. The filtrate was filtered through a 0.22 μm titanium rod filter and passed through 0.22 μ m microporous membrane was sterilized and filtered. Each vial was filled into a 10 ml glass vial in an amount of 1.25 ml, lyophilized, plugged, and capped to obtain the compound prepared in Example 1 Formulation.

 Preparation of Example 5 from Example 5 The stability of the branch and the accelerated test were investigated. Local irritation was examined by animal vascular irritation, muscle irritation, hemolysis, and allergic experiments.

 Accelerated test:

 A batch of samples prepared in Example 5 was placed at a temperature of 40 ± 2 ° C and a relative humidity of 75% ± 5%. In the constant temperature and humidity chamber, the samples were taken at 0, 1, 2, 3 and 6 months, and the results are shown in Table 5-1.

 Table 5-1 Example 5 Sample Accelerated Test Results

 lot number  Time (month)  Appearance color  pH  Solution clarity  relative substance(%)  content(%) Single impurity Total impurity  100401  0  Colorless solution  7.5  clarify  0.14 0.85 100.2 1 Colorless solution 7.3 Clarification 0.18  0.79  101.1 2 Colorless solution 7.4  Clarification 0.26 0.76  100.8 3 Colorless solution 7.7 Clarification 0.30 0.97  99.5 6 Colorless solution 7.3 Clarification 0.33 1.13  98.9

 It can be seen from Table 5-1 that the preparation prepared in Example 5 was examined by accelerated test for 6 months, and the appearance color and pH were observed. There was no significant change in the clarity index of the solution, no significant increase in impurities, and no significant decrease in the content, indicating that the preparation prepared by the present invention can be stored at room temperature with high stability.

 Vascular irritation, muscle irritation, allergic and hemolysis experiments:

 Vascular irritation:

 Six healthy rabbits with no ears were selected, and the left ear vein was injected with 1 ml of the injection solution of Example 5, and the right ear was injected with a capacity of 5%. Glucose injection, once a day, for 7 consecutive days.

 During the injection, the irritative response of the ear vein was observed regularly every day. number 8 The rabbits were sacrificed at night, and the bilateral ear veins and surrounding tissues were taken and fixed with formaldehyde. Conventional tissue sections were taken at the proximal end of the injection site, and pathological changes were observed under light microscope. The observation indicators and judgment criteria are shown in Table 5-2.

眼 周围组织
0 无水肿
1 轻微水肿
2 明显水肿
3 重度水肿 < 0.5 无刺激性
< 2.5 轻度刺激性
< 4.5 中度刺激性
> 4.5 重度刺激性 血管
0正常
1 血管轻度充血发红，纹路清晰
2 血管充血发红，纹路不清
3 血管重度充血,呈紫红色 光
镜 周围组织
0正常
1 水肿
2 出血
3 炎症细胞浸润 < 0.5 无刺激性
< 2.5 轻度刺激性
< 4.5 中度刺激性
> 4.5 重度刺激性 血管
0内皮及血管壁完整
1 内皮损伤
2 血管栓塞
3 血管破裂 Table 5-2 Vascular irritation score and judgment criteria Observation method Vascular irritation scoring criteria Vascular irritation criteria Observation index score standard Cumulative score judgment standard meat

eye Surrounding organization
0 no edema
1 mild edema
2 obvious edema
3 severe edema < 0.5 non-irritating
< 2.5 mild irritation
< 4.5 Moderate irritancy
> 4.5 Severe irritancy Blood vessel
0 normal
1 The blood vessels are slightly congested and red, and the lines are clear.
2 vascular congestion is red, the lines are unclear
3 The blood vessels are heavily congested and are purple Light microscopy Surrounding organization
0 normal
1 edema
2 bleeding
3 inflammatory cell infiltration < 0.5 non-irritating
< 2.5 mild irritation
< 4.5 Moderate irritancy
> 4.5 Severe irritancy Blood vessel
0 endothelium and vascular wall integrity
1 Endothelial injury
2 vascular embolization
3 vascular rupture

 The results showed that the stimulation of the injection of Example 5 in the rabbit ear vein was 5%, and 5% There was no significant difference in glucose injection. Visual observation showed no inflammatory reaction such as vascular congestion and surrounding tissue edema. Tissue biopsy showed no abnormalities in vascular structure, endothelial damage, thrombosis, and other pathological changes. The cumulative scores of blood vessels and surrounding tissues observed by the naked eye and light microscope are less than 0.5, indicating no irritation.

 Muscle irritation:

 Take 6 healthy rabbits, and each rabbit was injected into the quadriceps of the left quadrant. Example 5 Injection 1ml On the right side, the same volume of normal saline was injected. After the injection, observe the muscles at the injection site for congestion, edema, etc., half of the animals after 48 hours (day 3) The blood was sacrificed, the skin was cut longitudinally, and the injection sites on both sides of the injection were observed with or without hyperemia and edema, and the tissues were taken for pathological examination. Then evaluate the stimulatory response of the drug according to the criteria in Table 1-3. The remaining animals continue to observe 14d The above operation was repeated after the bloodletting was performed on the 18th day, and the evaluation criteria are shown in Table 5-3.

 Table 5-3 Evaluation criteria for muscle stimulation

 level  Stimulating response  Level 0  No obvious reaction at the site of administration  Level 1  Mild hyperemia at the site of administration, less than 0.15 cm in diameter  level 2  Moderately hyperemia at the site of administration, diameter 0.15 to 1.0 cm  Level 3  Severe congestion, redness, and muscle degeneration at the site of administration  level 4  Muscle brown degeneration, necrosis  Level 5  Severe muscle degeneration, large area necrosis

 The results showed that rabbits were injected into the quadriceps of the left quadrant. After the injection, the muscles at the injection site were visually observed to be free from congestion and edema, and no obvious stimulatory response such as tissue degeneration or necrosis was observed in the pathological examination, and there was no significant difference compared with the saline side.

 Sensitization to guinea pigs:

 Six healthy guinea pigs were selected, and 0.5 ml of the injection of Example 5 was injected intraperitoneally, and once every other day, a total of 3 injections were given. Times. Then, they were randomly divided into 2 groups, and 1 ml of the injection of Example 5 was intravenously administered 14 or 21 days after the first administration. Observe the guinea pig with allergies such as excitement and difficulty in breathing.

 Results The guinea pigs in both groups were normal and no respiratory abnormalities were observed.

 In vitro hemolytic test:

 A 2% rabbit red blood cell suspension was prepared. Take 7 tubes and add various liquids according to Table 5-4. Gently shake each tube and set it 37 Incubate in a constant temperature water bath at °C and observe the results for 0.5, 1, 2, 3, and 6 hours. The criteria for judging agglutination and hemolysis of erythrocytes are shown in Table 5-5.

 Table 5-4 Sample in vitro hemolysis test

 Pipe number  1  2  3  4  5  6  7  Example 5 (ml)  0.1  0.2  0.3  0.4  0.5  0  0  0.9% sodium chloride injection (ml)  2.4  2.3  2.2  2.1  2.0  2.5  0  Distilled water (ml)  0  0  0  0  0  0  2.5  2% red blood cell suspension (ml)  2.5  2.5  2.5  2.5  2.5  2.5  2.5  Example 5 final concentration (mg/ml)  0.18  0.36  0.54  0.72  0.90  0  0

 Table 5-5 Judging criteria for hemolysis and agglutination test of erythrocytes in vitro

 Observation index  Grading  Judgment criteria  Hemolysis  Complete hemolysis  The solution is clear red and there is no cell residue at the bottom of the tube. Partial hemolysis The solution is clear red or brownish red with a small amount of cell residue at the bottom of the tube. Not hemolyzed The supernatant was colorless and clear, and the red blood cells all sank.  Red blood cell agglutination  False agglutination  The red blood cells are aggregated and can be dispersed after shaking, and the preparation can be used for clinical use. True agglutination After shaking, the aggregates cannot be dispersed, and the preparation is not suitable for clinical use.

 As a result, the distilled water control tube was completely hemolyzed at 0.5 hour. Saline and Example 5 Sample Concentrations at 6 There is no hemolysis in the hour. After shaking gently, the physiological saline and the red blood cells deposited at the bottom of each sample of the sample of Example 5 were completely dispersed, indicating that the injection prepared in this example had no red blood cell agglutination reaction.

Vascular irritation, muscle irritation, in vitro hemolytic and allergic experiments showed that the Example 5 injection had no obvious irritation, allergies, and did not cause hemolysis.

Claims (1)

A GLP-I analog comprising a parent peptide of the following sequence: H ₂ N-Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys26-Glu-Phe-Ile- Ala-Trp-Leu-Val-Xaa34-Gly-Arg-Xaa37-COR1; Wherein R1=-OH or -NH ₂ ; Xaa7= histidine, D-histidine, deamination-histidine, 2-amino-histidine, β-hydroxy-histidine, homohistidine, N α -acetyl - histidine, α - fluoromethyl - histidine, α - methyl - histidine, 3-pyridyl alanine, 2-pyridyl alanine or 4- Pyridyl alanine; Xaa8=Ala, D-Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl)carboxylic acid, ( 1- Aminocyclobutyl)carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl)carboxylic acid, (1-aminocycloheptyl)carboxylic acid or (1-aminocyclooctyl)carboxylic acid ; Xaa26=Lys; Xaa34=Lys, Glu, Asn or Arg; Xaa37=Gly, Ala, Glu, Pro or Lys; and at least one of Xaa34 or Xaa37 is Lys ; The GLP-I analog also contains Q1 and Q2 groups, and the Q1 and Q2 groups are simultaneously present in the parent peptide. Xaa26, Xaa34, Xaa37 Any two or all of Lys are attached to any two of Lya Xaa26, Xaa34, Xaa37 in the form of an amide bond. Residue The Q1 group is a lipophilic substituent attached to a bridging group W, and the lipophilic substituent forms an amide bond with the amino group of a bridging group at the carboxyl group thereof, and the carboxyl group and the parent peptide of the amino acid residue of the bridging group An N-terminal residue of one Lys forms an amide bond to be attached to the parent peptide; the bridging group W is 1-7 methylene-free branched paraffins α, ω-dicarboxy; the lipophilic substitution The base is an acyl group selected from the group consisting of CH ₃ (CH ₂ ) _n CO-, wherein n is an integer from _{4 to 38} ; The Q2 group is ε(AEEA) _n -MPA, n=0-3, and its structural formula is as follows:

. The GLP-I analogue according to claim 1, wherein the Xaa7 is histidine. 3. The GLP-I analog of claim 2, wherein said Xaa8 is D-Ala . The GLP-I analogue according to claim 2, wherein the bridging group W is an unbranched paraffin α having 2 methylene groups. Omega-dicarboxyl. The GLP-I analogue according to claim 4, wherein the bridging group W is glutamic acid. 6. The GLP-I analogue according to claim 2, wherein the CH ₃ (CH ₂ ) _n CO , wherein n is an integer from 4 to 24. The GLP-I analogue according to claim 6, wherein the lipophilic substituent is CH ₃ (CH ₂ ) ₁₄ CO- . A method for producing the GLP-I analogue according to any one of claims 1 to 7, which comprises: synthesizing GLP- The parent peptide of the I analog, the free amino group and the free carboxyl group on the parent peptide are protected by a protecting group; the protecting group on the amino acid residue at the coupling position of the Q1 group at the parent peptide is removed, Q1 a group coupled to the parent peptide; a protecting group on the amino acid residue at the coupling position of the Q2 group on the parent peptide, Q2 The group is coupled to the parent peptide; the protecting group on the other amino acid residues on the parent peptide is removed, and the GLP-I analog is prepared. 9. The method of claim 8 further comprising: preparing the prepared GLP- The I analog was purified using reverse phase liquid chromatography. 10. The method of claim 8, wherein the coupling of a Q1 group to the parent peptide is characterized by comprising: The carboxyl group of the amino acid residue of the bridging group and the N- of the Lys of the parent peptide An amide bond is formed on the terminal residue to be attached to the parent peptide, and the lipophilic substituent is coupled to the parent peptide by an amide bond between the carboxyl group and the amino group of a bridging group. 11. The method of claim 8, wherein the Q2 group is coupled to the parent peptide, comprising: a carboxyl group of (AEEA) _n and an N-terminal residue of a Lys of the parent peptide An amide bond is formed to attach to the parent peptide, and MPA forms an amide bond with the amino group of (AEEA) _n at its carboxyl group; n = 1-3 in the (AEEA) _n . 12. The method of claim 8 wherein said coupling a Q2 group to said parent peptide comprises: MPA An amide bond is formed on the N-terminal residue of a Lys with its carboxyl group and the parent peptide. A pharmaceutical composition comprising the GLP- according to any one of claims 1-7. I analog or a pharmaceutically acceptable salt thereof. The pharmaceutical composition according to claim 13, comprising: 0.9 mg/ml of the GLP- according to any one of claims 1 to 7. I analog or a pharmaceutically acceptable salt thereof, 5.0% (w/v) cresol, 5.2% (w/v) mannitol, 12.5 mg/ml propylene glycol, 8.0 mM Phosphate buffer. Use of the GLP-I analogue according to any one of claims 1 to 7 for the preparation of a medicament for the treatment or prevention of a disease. The use according to claim 15, wherein the disease is hyperglycemia, type 2 diabetes, impaired glucose tolerance, 1 Type 2 diabetes, obesity, high blood pressure, X Syndrome, dyslipidemia, cognitive impairment, atherosclerosis, myocardial infarction, coronary heart disease and other cardiovascular diseases, stroke, inflammatory bowel syndrome, indigestion or gastric ulcer. 17. Use according to claim 15 wherein said GLP-I analogue is used for the preparation of delay or prevention 2 Drugs for the development of type 2 diabetes. 18. The GLP-I analog according to any one of claims 1 to 7 which reduces food intake and reduces β- in preparation Use in apoptosis, a drug that enhances beta-cell function and beta-cell mass and/or restores glucose sensitivity of beta-cells.