WO2013063877A1

WO2013063877A1 - Glp-1 derivate dlg3312 and solid-phase chemical synthesis method thereof

Info

Publication number: WO2013063877A1
Application number: PCT/CN2012/070451
Authority: WO
Inventors: 劳勋; 吴自荣; 黄静; 赵丽芬; 李娟�; 赵云; 耿旭; 汪正华
Original assignee: 华东师范大学
Priority date: 2011-11-03
Filing date: 2012-01-17
Publication date: 2013-05-10
Also published as: CN103087174A; CN103087174B

Abstract

Provided in the present invention is a GLP-1 derivate DLG3312 with the structure X-Y-X, wherein the sequence represented by X is H-X8-EGTFTSDVSSYLEGQAAKEFIAWLVKGRG, X8 is any one of A、G、dA or V, and Y represents diaminocarboxylic acid comprising Lys and Om. Also provided in the present invention are the solid-phase chemical synthesis method of the GLP-1 derivate DLG3312 and the use thereof in preparing a drug for treatment of diabetes.

Description

GLP-1 derivative DLG3312 and solid phase chemical synthesis method thereof

Technical field

The invention belongs to the technical field of biology and chemical engineering, and in particular relates to a GLP-1 derivative DLG3312 having human glucagon-like peptide-1 (GLP-1) activity and a solid phase chemical synthesis method thereof.

Background technique

In 1929, Zunz and Labbare named the body fluid factor, which is isolated from the intestine and promoted glucose-stimulated insulin secretion, as incretin, and GLP-1 is the main component. GLP-1 is a product of the glucagon preprogenitor gene secreted by intestinal L cells, which is secreted after food intake. There are two main biologically active forms in the body: GLP-1 (7-36) amide and GLP-1 (7-37), and about 80% of GLP-1 has circulating activity from the former. As a hormone, GLP-1 reduces postprandial blood glucose by stimulating insulin secretion while inhibiting glucagon secretion. Further, it has an effect of suppressing the emptying of gastrointestinal foods and the intake of food, and in addition, it can cause proliferation of islet β cells.

GLP-1 is indicated for the treatment of type 2 diabetes, and its effect of lowering blood glucose is blood glucose-dependent. When blood glucose concentration is higher than 6mmol/L, GLP-1 significantly promotes insulin secretion, and once blood sugar returns to normal, it is no longer Continue to play the role of hypoglycemic, so there will be no hypoglycemia during the medication. Through long-term treatment, it can effectively improve the amount of glycated hemoglobin in patients with type 2 diabetes, and has a good application prospect for the prevention and treatment of type 2 diabetes.

In practical applications, according to previous research results, the half-life of GLP-1 after entering the body is 2 minutes, which is due to the degradation of GLP-1 by various proteases in the body. Currently, DPP-4 is the most important enzyme. In order to resist the degradation of GLP-1 by this enzyme and prolong the time of action in vivo, various methods such as site-directed mutagenesis of amino acids and molecular modification have been made. For example, CJC-1131 extends the half-life of GLP-1 to 18 hours, which greatly extends the half-life by chemically synthesizing a linker molecule to link the polypeptide to plasma proteins. LY315902 is an 8C fat chain attached to GLP-1 in dogs. Half-life is 3-6 hours; Liraglutide, which binds a 16C fatty acid side chain at the 26th lysine of GLP-1, and mutates 34 to lysine, half-life in human body It is 8 hours; PEG-modified GLP-1 is 40 times longer than the natural GLP-1 plasma half-life.

Although the efficacy time can be prolonged by modifying the sequence of GLP-1, since the modified molecule has a low homology rate with native GLP-1, the difference is large, leading to different degrees of side effects such as allergies, vomiting, gastrointestinal discomfort. Wait.

In the prior art, a GLP-1 derivative has been prepared by DNA recombination technology, and the following invention is entitled "A human glucagon-like peptide-1 derivative and its preparation and application" (200610024355.X). Disadvantages: Using Escherichia coli as a host strain for expression of GLP-1 derivatives, in the subsequent purification process, it is necessary to detect and control the residual amount of E. coli endotoxin and E. coli DNA, increasing the production steps and costs.

GLP-1 has two forms in vivo, one is GLP-1 (7-36) amide, consisting of 30 amino acid residues, and the other It is GLP-1 (7-37) and consists of 31 amino acid residues, both of which are biologically active. The present invention relates to GLP-1 which refers to GLP-1 (7-37), the sequence of which is as follows: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG.

The present invention improves the sequence and structure of GLP-1, and proposes a GLP-1 derivative DLG3312 and a solid phase chemical synthesis method thereof. The GLP-1 derivative DLG3312 proposed by the invention solves the problem that the natural GLP-1 has a short activity time in vivo, and has good biological activity, mainly because: the structure of the GLP-1 derivative DLG3312 of the invention is Χ-Υ-Χ In the connection between X and hydrazine, two X-terminal carboxyl groups are condensed with two amino groups of hydrazine to form a U-type homodimer of a specific structure consisting of two X and one hydrazine. In the present invention, the affinity of the dimer to the receptor is greatly increased relative to the monomer, thereby enabling activation of the receptor more efficiently; the dimer contains the mutated amino acid, and at the same time, the dimeric structure is itself , can hinder the binding of various enzymes to the body, thereby reducing the degradation rate thereof; the dimer has a relatively large molecular weight compared with the monomer, thereby relatively prolonging the clearance due to glomerular filtration Time, so that the retention time in the body increases, prolonging the efficacy time. Moreover, the side effects of various diabetes drugs currently used, such as hypoglycemia, weight gain, insulin resistance, islet β cell failure, and the like, are also avoided in terms of safety. In contrast, DLG3312 of the present invention has a function of improving insulin resistance and inhibiting apoptosis of islet β cells, thereby fundamentally improving the glucose metabolism ability of diabetic patients. In addition, the prior art method for preparing GLP-1 derivatives has many disadvantages such as high steps and high cost, and the preparation method proposed by the invention has the advantages of simple operation, low cost, and the like, and has greater application potential.

Summary of the invention

The present invention provides a GLP-1 derivative DLG3312, including tautomers, solvates and pharmaceutically acceptable salts thereof; the structure of the GLP-1 derivative DLG3312 is Χ-Υ-Χ; wherein, X The sequence represented is H-X8-EGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein: Χ8 is any one of A, G, dA or V, dA represents D-Ala; Y represents diaminocarboxylic acid, including Lys, Orn; The DLG3312 is condensed to form a homodimer by the carbon terminal carboxyl group of the X and the amino group of the Y.

among them,

When X8 is A, the sequence X is Seq ID No.1;

When X8 is G, the sequence X is Seq ID No. 2;

When X8 is dA, the sequence X is Seq ID No. 3;

When X8 is V, the sequence X is Seq ID No. 4.

among them,

When the sequence X is Seq ID No. 1, when Y is Lys, the GLP-1 derivative is DLG3312-1 as shown in the following structural formula (1); when Y is Orn, the GLP-1 derivative is DLG3312 -5 is as shown in the following structural formula (5): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys Orn HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(1) (5) When the sequence X is Seq ID No. 2, when Y is Lys, the GLP-1 derivative is DLG3312-2 and has the following structural formula.

(2), when Y is Orn, the GLP-1 derivative is DLG3312-6 as shown in the following structural formula (6):

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys Orn HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(2) (6) When the sequence X is Seq ID No. 3, when Y is Lys, the GLP-1 derivative is DLG3312-3 having the following structural formula

(3), when Y is Orn, the GLP-1 derivative is DLG3312-7 as shown in the following structural formula (7):

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(3)

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Orn

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(7)

When the sequence X is Seq ID No. 4, when Y is Lys, the GLP-1 derivative is DLG3312-4 having the following structural formula

(4), when Y is Orn, the GLP-1 derivative is DLG3312-8 as shown in the following structural formula (8):

HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys Orn HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(4) (8) The present invention also provides a solid phase chemical synthesis method of the GLP-1 derivative DLG3312. The resin is first placed in a polypeptide synthesizer, and the protective group-containing diaminocarboxylic acid Y is combined with the resin, and then The amino acid monomer having a protecting group is arranged in a polypeptide synthesizer from the C-terminus to the N-terminus according to the X sequence, and a polypeptide resin having a side chain protecting group is synthesized, and then subjected to deprotection, cleavage of the resin, purification by HPLC, and freeze-drying. The GLP-1 derivative DLG3312 is obtained; wherein the diaminocarboxylic acid Y having a protecting group comprises: Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Orn(Fmoc)-OH _; The amino acid monomers having a protecting group include: Fmoc-L-Ala-OH, Fmoc-D-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc- L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L -Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L- Trp-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OHo

Wherein, when the sequence X is Seq ID No.l,

The amino acid monomer having a protecting group is: Fmoc-L-Lys(Fmoc)-OH. Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L- Gln(Trt)-OH , Fmoc-L-Gly-OH , Fmoc-L-Glu(OtBu)-OH , Fmoc-L-Leu-OH , Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L -Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L- When Ala-OH or Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-1;

The amino acid monomer having a protecting group is: Fmoc-L-Om(Fmoc)-OH. Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L- Gln(Trt)-OH , Fmoc-L-Gly-OH , Fmoc-L-Glu(OtBu)-OH , Fmoc-L-Leu-OH , Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L -Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L- When Ala-OH or Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-5.

Wherein, when the sequence X is Seq ID No. 2 ,

The amino acid monomers having a protecting group are: Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH Fmoc-L-Leu-OH, Fmoc-L-Trp -OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc- L-Ala-OH, Fmoc-L-Ala-OH Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH Fmoc-L-Asp(OtBu)-OH , Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Phe-OH Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc -L-Glu(OtBu)-OH, Fmoc-L-Gly-OH Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-2;

The amino acid monomers having a protecting group are: Fmoc-L-Om(Fmoc)-OH, Fmoc-L-Gly-OH Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc -L-Lys(Boc)-OH, Fmoc-L-Val-OH Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc -L-Phe-OH Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH Fmoc-L-Gln(Trt )-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser(tBu)- OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu) -OH, Fmoc-L-Phe-OH Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH Fmoc-L When -His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-6.

Wherein, when the sequence X is Seq ID No. 3,

The amino acid monomers having a protecting group are: Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Gly-OH Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc -L-Lys(Boc)-OH, Fmoc-L-Val-OH Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc -L-Phe-OH Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH Fmoc-L-Gln(Trt )-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser(tBu)- OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu) -OH, Fmoc-L-Phe-OH Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-D-Ala-OH Fmoc-L When -His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-3;

The amino acid monomers having a protecting group are: Fmoc-L-Om(Fmoc)-OH, Fmoc-L-Gly-OH Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc -L-Lys(Boc)-OH, Fmoc-L-Val-OH Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc -L-Phe-OH Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc -L-Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH , Fmoc-L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, When Fmoc-D-Ala-OH or Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-7.

Wherein, when the sequence X is Seq ID No. 4,

The amino acid monomer having a protecting group is: Fmoc-L-Lys(Fmoc)-OH. Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L- Gln(Trt)-OH , Fmoc-L-Gly-OH , Fmoc-L-Glu(OtBu)-OH , Fmoc-L-Leu-OH , Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser (tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L -Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L- When Val-OH or Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-4;

The amino acid monomers having a protecting group are: Fmoc-L-Om(Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH , Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L -Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L- Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L When -Val-OH or Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-8.

The present invention also proposes a pharmaceutical composition of the GLP-1 derivative DLG3312 comprising the GLP-1 derivative DLG3312 and a pharmaceutically acceptable excipient.

The invention also proposes the use of the GLP-1 derivative DLG3312 in the preparation of a medicament for treating diabetes and obesity. The GLP-1 derivative DLG3312 of the present invention has the structure XYX, wherein X represents GLP-1 (7-37) and its mutant amino acid sequence, that is, H-X8-EGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein: X8 is A, G, dA is D -Ala, V, 2-ME-A is any amino acid of alpha-Me-Ala, S and L; Y represents a diamino carboxylic acid, namely: Lys or Om or L-2, 4-di Aminobutyric acid or diaminopimelic acid. The present invention GLP-1 derivative DLG3312 In the structure XYX, the connection between X and Y is such that two X-terminal carboxyl groups are condensed with two amino groups of Y to form a U-type homodimer of a specific structure composed of two Xs and one Y. . The invention proposes an innovative structural configuration U-type homodimer, which has an enhanced effect on its biological activity and action time. The main reason is that the affinity of the dimer to the receptor is relative to the monomer. A larger increase, thereby enabling more efficient activation of the receptor; the dimeric structure, by itself, has the ability to block binding to various enzymes in the body, thereby reducing its degradation rate; dimer and monomer phase The ratio has a relatively large molecular weight, thereby relatively prolonging the time of clearance due to glomerular filtration, increasing the residence time in the body and prolonging the efficacy time. DLG3312 acts as an agonist of the GLP-1 receptor and retains most of the biological activity of native GLP-1. Moreover, the resistance to degradation of proteases in vivo (especially DPP-4) is superior to that of natural GLP-1, and it is expected to solve the problem that the current biological activity time is not satisfactory. The compounds of the invention can be administered by non-oral routes, such as subcutaneous or intramuscular, and oral routes, primarily for the treatment of type 2 diabetes. In the present invention, a synthetic compound is designed by solid phase synthesis of a polypeptide. The compound of the present invention has hypoglycemic activity in vivo, and greatly enhances its stability in vivo and prolongs the time of blood glucose lowering in vivo relative to natural GLP-1.

The present invention provides an idea for modifying peptide derivatives and a method for providing solid phase chemical synthesis of the above derivatives. The solid phase chemical synthesis method is very mature in the preparation of small peptides with less than 40 amino acids. It has the advantages of rapid and simple purification, and there is no endotoxin removal in the subsequent treatment process. The production steps are few and the purification process is simple. , low production cost, stable product quality, suitable for large-scale mass production.

detailed description

The invention will be further described in detail below with reference to examples.

The amino acid monomers with protecting groups and other chemical reagents used in the specification and the following examples can be purchased from related companies. The experimental methods without specific conditions can be carried out according to conventional conditions, or by commodity suppliers. The proposed conditions are carried out. All of the examples are operated using the apparatus and reagents specified by the synthetic methods in the Summary of the Invention and the steps specified in the synthetic methods of the Summary of the Invention, and are not repeated here. All of the examples only list the key steps associated with the respective products. .

There are 22 amino acid groups with protecting groups for use in the present invention, including: Fm _OC -L-Ala-OH, Fm _OC -D-Ala-OH, Fmoc-alpha-Me-Ala-OH, Fmoc-L- Arg(Pbf)-OH , Fmoc-L-Asp(OtBu)-OH , Fmoc-L-2,4-diaminobutyric acid, Fmoc-2,6-diaminopimelic acid, Fmoc-L-Gln(Trt)-OH, Fmoc -L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc)-OH, Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Orn(Fmoc)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L -Thr(tBu)-OH, Fmoc-L-Trp-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OHo

The abbreviations are: Fmoc: 9-fluorenylmethoxycarbonyl;

Boc: tert-butyloxycarbonyl; 艮卩tert-butyloxycarbonyl;

Trt: trityl, ie trityl;

OtBu: tert-butyl ester;

tBU: tert-butyl, ie tert-butyl;

Pbf: 2,2,5,7,8-pentamethylchroman-6-benzenesulfonyl;

The equipment and reagents used in the present invention are as follows:

Instrument: SYMPHONY 12-channel peptide synthesizer, model: SYMPHONY, American products.

Reagents: N-methylpyrrolidone, dichloromethane, hexahydropyridine, methanol, dimethylaminopyridine

Dimethylaminopyridinel/DMF, N,N-diisopropylethylamine, N,N-diisopropylethylamine/NMP, HBTU 100mmol/0.5M HOBT in DMF, N,N-dicyclohexylcarbodiimide, Ν-Dicyclohexylcarbodiimide/ NMP, wherein: DMF is hydrazine, hydrazine-dimethylformamide;

ΝΜΡ is Ν-methylpyrrolidone;

ΗΟΒΤ is 1-hydroxybenzotriazole;

HBTU is 2-(1H benzotriazolyl)-tetramethylurea hexafluorophosphate, ie

2-(lH-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphateo

The solid phase chemical synthesis method of the GLP-1 derivative of the present invention DLG3312 comprises the following steps:

Synthesis of Polypeptide Resin: Taking 0.25mmol as an example, weigh 0.25g of Wang's resin and put it into the reactor on the SYMPHONY type 12-channel peptide synthesizer. The diaminocarboxylic acid with protecting group is (I) Y , 艮卩: Fmoc-L-Lys(Fmoc)-OH or Fmoc-L-Orn(Fmoc)-OH or Fmoc-2,6-diaminopimelic acid or Fmoc-L-2,4-diaminobutyric acid is weighed in 1mmol Bottle, the carboxyl group of Y is combined with the resin, and then the protected amino acid monomer is weighed into 1 mmol bottling, according to Seq ID No. 1, Seq ID No. 2, Seq ID No. 3, or Seq ID No. The amino acid sequence of X of X is arranged in the synthesizer from the C-terminus to the N-terminus, and is automatically controlled by computer program to remove Fmoc protection, activation, and connection at 25 ° C, and then proceed to the next round. The synthesis was carried out in this manner to obtain a polypeptide resin having a side chain protecting group, which was dried and weighed on the synthesizer.

Deprotection group and cleavage resin: The polypeptide resin with side chain protecting group obtained in the first step was placed in a stoppered flask, and the following lysis reagent was added: 0.25 ml of water, 0.25 ml of EDT, namely 1,2-ethanedithiol, 1 ml of TIS is triethylpropylsilica, 9.45 ml of trifluoroacetic acid, and then electromagnetically stirred at 30 ° C for 2 hours, filtered, and the filtrate is collected. The resin is washed with trifluoroacetic acid, and the collected liquid and washing liquid are combined, and diethyl ether is added. Precipitating, filtering, washing with diethyl ether and drying to obtain a crude product;

Separation and purification by HPLC, freeze-drying: The crude product obtained in the second step was separated and purified by preparative HPLC, and then freeze-dried to obtain product DLG3312. The product DLG3312 prepared according to the preparation method of the present invention contains the amino acid sequence of the above-mentioned proposed GLP-1 and its derivatives.

Example 1

The DLG3312-1 of the present invention is synthesized by the solid phase chemical synthesis method of the present embodiment, and X is Seq ID Νο.1, and the same operation steps as those in the above method are not described again.

In this example, Fmoc-L-Lys(Fmoc)-OH is weighed into 1mmol bottling, the carboxyl group of Fmoc-L-Lys(Fmoc)-OH is combined with the resin, and then the amino acid sequence of Seq ID No.1 is used. From the C-terminus to the N-terminus, in the SYMPHONY type 12-channel polypeptide synthesizer, the amino acid monomers with protecting groups are added in the order of synthesis:

Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH,

Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc- L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH,

Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc -L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH , Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-L-Ala-OH, Fmoc-L-His(Trt)-OH.

In the present example, DLG3312-1 was obtained, and its structure is shown in the following formula (1).

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (" In the DLG3312-1 represented by the formula (1), the carbon terminal carboxyl groups on the two Xs are respectively condensed with the two amino groups of Lys, and are linked to form a homodimer, which has a U-shaped structure.

Example 2

The DLG3312-2 of the present invention is synthesized by the solid phase chemical synthesis method of the present embodiment, and X is Seq ID No. 2. The same operation steps as those in the above method will not be described again.

In this embodiment, Fmoc-L-Lys(Fmoc)-OH is weighed into a lmmol bottle, and the carboxyl group of Fmoc-L-Lys(Fmoc)-OH is combined with the resin, and then the amino acid in Seq ID No. 2 is used. The sequence, from the C-terminus to the N-terminus, is arranged in a SYMPHONY type 12-channel polypeptide synthesizer. The amino acid monomers with a protecting group are added in the order of synthesis:

Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH,

Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH,

Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH.

In the present example, DLG3312-2 was obtained, and its structure is shown in the following formula (2).

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG ^ In DLG3312-2 represented by formula (2), the carbon terminal carboxyl groups of two X groups are condensed with two amino groups of Lys, and are linked to form a homodimer, which has a U-shaped structure.

Example 3

The DLG3312-3 of the present invention is synthesized by the solid phase chemical synthesis method of the present embodiment, and X is Seq ID No. 3. The same operation steps as those in the above method will not be repeated.

In this example, Fmoc-L-Lys(Fmoc)-OH is weighed into a lmmol bottle, and the carboxyl group of Fmoc-L-Lys(Fmoc)-OH is combined with the resin, and then the amino acid in Seq ID No. 3 is used. The sequence, from the C-terminus to the N-terminus, is arranged in a SYMPHONY type 12-channel polypeptide synthesizer. The amino acid monomers with a protecting group are added in the order of synthesis:

Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH,

Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-D-Ala-OH, Fmoc-L-His(Trt)-OH.

In the present example, DLG3312-3 was obtained, and its structure is shown in the following formula (3). HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG In DLG3312-3 represented by the formula (3), the carbon terminal carboxyl groups of the two X groups are condensed with the two amino groups of Lys, and are linked to form a homodimer having a U-shaped structure.

Example 4

The DLG3312-4 of the present invention is synthesized by the solid phase chemical synthesis method of the present embodiment, and X is Seq ID No. 4. The same operation steps as those in the above method will not be repeated.

In this example, Fmoc-L-Lys(Fmoc)-OH is weighed into 1mmol bottling, the carboxyl group of Fmoc-L-Lys(Fmoc)-OH is combined with the resin, and then the amino acid in Seq ID No. 4 is used. The sequence, from the C-terminus to the N-terminus, is arranged in a SYMPHONY type 12-channel polypeptide synthesizer. The amino acid monomers with a protecting group are added in the order of synthesis:

Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH,

Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-L-Val-OH, Fmoc-L-His(Trt)-OH.

In the present example, DLG3312-4 was obtained, and its structure is shown in the following formula (4).

HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys

HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (In DLG3312-4 represented by the formula (4), the carbon terminal carboxyl groups of the two X groups are condensed with the two amino groups of Lys, and are joined to form a homodimer, which has a U-shaped structure.

Example 5

The DLG3312-5 of the present invention is synthesized by the solid phase chemical synthesis method of the present embodiment, and X is Seq ID No. 1, and the same operation steps as those in the above method are not described again.

In this example, Fmoc-L-Orn(Fmoc)-OH is weighed into 1mmol bottling to make Fmoc-L-Orn(Fmoc)-OH The carboxyl group is bound to the resin, and then arranged in the SYMPHONY type 12-channel polypeptide synthesizer from the C-terminus to the N-terminus according to the amino acid sequence in Seq ID No. 1, and the amino acid monomer having a protecting group is added in the order of synthesis:

Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH,

Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-L-Ala-OH, Fmoc-L-His(Trt)-OH.

In the present example, DLG3312-5 was obtained, and its structure is shown in the following formula (5).

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Orn

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG In DLG3312-5 represented by the formula (5), the carbon terminal carboxyl groups of the two X groups are condensed with two amino groups of Orn, and are linked to form a homodimer, which has a U-shaped structure.

Example 6

The solid phase chemical synthesis method of the present embodiment synthesizes DLG3312-6 of the present invention, and X thereof is Seq ID No. 2, and the same operation steps as those in the above method will not be described again.

In this embodiment, Fmoc-L-Orn(Fmoc)-OH is weighed into a lmmol bottle, and the carboxyl group of Fmoc-L-Orn(Fmoc)-OH is combined with the resin, and then the amino acid in Seq ID No. 2 is used. The sequence, from the C-terminus to the N-terminus, is arranged in a SYMPHONY type 12-channel polypeptide synthesizer. The amino acid monomers with a protecting group are added in the order of synthesis:

Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH,

Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH. In the present example, DLG3312-6 was obtained, and its structure is shown in the following formula (6).

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Om

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (In DLG3312-6 represented by the formula (6), the carbon terminal carboxyl groups of the two X groups are condensed with the two amino groups of Orn, and are joined to form a homodimer, which has a U-shaped structure.

Example 7

The DLG3312-7 of the present invention is synthesized by the solid phase chemical synthesis method of the present embodiment, and X is Seq ID No. 3. The same operation steps as those in the above method are not described again.

In this example, Fmoc-L-Orn(Fmoc)-OH is weighed into a lmmol bottle, and the carboxyl group of Fmoc-L-Orn(Fmoc)-OH is combined with the resin, and then the amino acid in Seq ID No. 3 is used. The sequence, from the C-terminus to the N-terminus, is arranged in a SYMPHONY type 12-channel polypeptide synthesizer. The amino acid monomers with a protecting group are added in the order of synthesis:

Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH,

Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-D-Ala-OH, Fmoc-L-His(Trt)-OH.

In the present example, DLG3312-7 was obtained, and its structure is shown in the following formula (7).

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Om

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (?) In DLG3312-7 represented by the formula (7), the carbon terminal carboxyl groups of the two X groups are condensed with two amino groups of Orn, and are linked to form a homodimer, which has a U-shaped structure.

Example 8

The solid phase chemical synthesis method of the present embodiment synthesizes the DLG3312-8 of the present invention, and X thereof is Seq ID No. 4. The same operation steps as those in the above method are not described again. In this example, Fmoc-L-Orn(Fmoc)-OH is weighed into 1mmol bottling, the carboxyl group of Fmoc-L-Orn(Fmoc)-OH is combined with the resin, and then the amino acid in Seq ID No. 4 is used. The sequence, from the C-terminus to the N-terminus, is arranged in a SYMPHONY type 12-channel polypeptide synthesizer. The amino acid monomers with a protecting group are added in the order of synthesis:

Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH,

Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH,

Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH,

Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH,

Fmoc-L-Val-OH, Fmoc-L-His(Trt)-OH.

In the present example, DLG3312-8 was obtained, and its structure is shown in the following formula (8).

HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Orn

HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG In DLG3312-8 represented by the formula (8), the carbon terminal carboxyl groups of the two X groups are condensed with two amino groups of Orn, and are linked to form a homodimer, which has a U-shaped structure.

Example 9

Experimental materials and methods:

Female healthy Kunming mice (clean grade, provided by Shanghai Slack);

35.67% glucose solution;

0.9% NaCl solution;

GLP-1;

DLG3312, which is prepared by the preparation of Examples 1-8: DLG3312-1, DLG3312-2, DLG3312-3, DLG3312-4, DLG3312-5, DLG3312-6, DLG3312-7, DLG3312-8;

Blood glucose tester (produced by Shanghai Xinli Medical Devices Co., Ltd.).

About 30 g of female healthy Kunming mice were fasted overnight and divided into 10 groups (n=8). 1, glucose control group; 2, GLP-1 administration control group; 3~10, DLG3312 administration group, DLG3312 used in the present example is the product prepared in Examples 1-8. GLP-1 administration group 2 was intraperitoneally injected with glucose solution and 0.337 mg/kg body weight of GLP-1, and DLG3312 group 3 to 10 by weight group of 18 mmol/kg body weight. Each group was intraperitoneally injected with glucose solution and 0.337 at 18 mmol/kg body weight. Mg/kg The difference in weight is DLG3312, this moment is recorded as zero moment. Blood samples were taken from the tail vein of the mice at 30 min, 2 h, 6 h, and 9 h, respectively, and the blood glucose concentration was measured by a blood glucose meter. In order to observe the hypoglycemic effect of GLP-1 and DLG3312 for a long time, glucose solution was intraperitoneally injected at 18 mmol/kg body weight at 1.5 h, 5.5 h and 8.5 to test the hypoglycemic effect of GLP-1 and DLG3312. In the glucose control group, only glucose solution was intraperitoneally injected at 18 mm ₀ l/kg body weight, and GLP-1 or DLG3312 was not administered, and blood glucose was measured at the same time interval. The hypoglycemic rate was calculated as follows: Hypoglycemic rate (%) = (glucose control blood glucose level - blood glucose level of the administration group) I glucose control value χΐοο%.

The results are shown in Table 1. The values in the table are the glucose reduction rates of DLG3312-1, DLG3312-2, DLG3312-3, DLG3312-4, DLG3312-5, DLG3312-6, DLG3312-7, DLG3312-8, and the values shown. Both are mean values of n=8. Compared with the glucose group mice, the DLG3312 administration group and the GLP-1 administration group reduced blood glucose in mice within 30 minutes after administration, while the DLG3312 administration group still reduced blood glucose in mice within 180 minutes. The duration of the drug is 4 to 5 times that of GLP-1 and can reach 11 hours. Thus, it was shown that DLG3312 prepared by the method of the present invention all exhibited hypoglycemic activity superior to that of natural GLP-1, and had more prolonged hypoglycemic activity than native GLP-1. In addition, when the blood glucose level of the mouse reached a certain low value, DLG3312 showed no hypoglycemic activity, indicating that DLG3312 does not cause hypoglycemia, and is safe for treating diabetes.

Table 1 Hypoglycemic effect of GLP-1 and GLP-1 derivatives DLG3312— (1-8)

Hypoglycemic rate%

30min 2h 6h 9h l lh

Control 0.00 0.00 0.00 0.00 0.00

GLP-1 34.98 42.36 0.00 0.00 0.00

DLG3312-1 37.88 62.10 48.24 31.90 15.32

DLG3312-2 37.94 64.70 50.12 32.88 13.42

DLG3312-3 39.65 64.35 49.52 33.08 17.11

DLG3312-4 38.20 58.37 48.47 30.64 16.63

DLG3312-5 36.60 50.86 51.03 11.16 5.47

DLG3312-6 36.98 63.75 49.93 34.50 8.71

DLG3312-7 37.12 61.41 51.80 24.10 12.22

DLG3312-8 38.11 57.62 54.54 22.31 11.53 Example 10

Composition containing DLG3312

Take 1mg of solid phase chemical synthesis method of DLG3312, dissolved in 0.5ml of deionized water; another 50mg of mannitol, dissolved in 0.5ml of deionized water; the two are mixed into a combination of DLG3312 and mannitol, including Lmg/ml DLG3312 and 50 mg/ml mannitol. The DLG3312-containing composition prepared in this example was used in the hypoglycemic test of Example 9, and the same results were obtained, indicating that the DLG3312-containing composition has good hypoglycemic activity and a long potency.

The above-described embodiments are intended to illustrate the preferred embodiments of the invention and not to limit the invention in any form. Various modifications made by those skilled in the art in light of the teachings of the present invention, which are incorporated in the scope of the present invention, fall within the scope of the appended claims.

Claims

O 2013/063877 Claims PCT/CN2012/070451 A GLP-1 derivative DLG3312, characterized in that the GLP-1 derivative DLG3312 comprises its tautomers, solvates and pharmaceutically acceptable salts; The structure of the GLP-1 derivative DLG3312 is XYX; wherein, the sequence represented by X is H-X8-EGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein: X8 is any one of A, G, dA or V, and dA is D-Ala;

Y represents a diaminocarboxylic acid, including Lys, Orn _;

The GLP-1 derivative DLG3312 is condensed to form a homodimer by the two X carbon terminal carboxyl groups and the Y amino group.

The GLP-1 derivative DLG3312 according to claim 1, wherein

When X8 is A, the sequence X is Seq ID No.1;

When X8 is G, the sequence X is Seq ID No. 2;

When X8 is dA, the sequence X is Seq ID No. 3;

When X8 is V, the sequence X is Seq ID No. 4.

The GLP-1 derivative DLG3312 according to claim 1, wherein

When the sequence X is Seq ID No. 1, when Y is Lys, the GLP-1 derivative is DLG3312-1 as shown in the following structural formula (1); when Y is Orn, the GLP-1 derivative is DLG3312 -5 is as shown in the following structural formula (5):

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys Orn

HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(1) (5) When the sequence X is Seq ID No. 2, when Y is Lys, the GLP-1 derivative is DLG3312-2 as shown in the following structural formula (2), and when Y is Orn, the GLP The -1 derivative is DLG3312-6 as shown in the following structural formula (6):

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys Orn

HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(2) (6) When the sequence X is Seq ID No. 3, when Y is Lys, the GLP-1 derivative is DLG3312-3 as shown in the following structural formula (3), and when Y is Orn, the GLP The -1 derivative is DLG3312-7 as shown in the following structural formula (7): WO 2013/063877 Claim PCT/CN2012/070451

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(3)

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Orn

HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(7)

When the sequence X is Seq ID No. 4, when Y is Lys, the GLP-1 derivative is DLG3312-4 as shown in the following structural formula (4), and when Y is Orn, the GLP-1 derivative is DLG3312. -8 is shown in the following structural formula (8):

HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

Lys Orn

HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

(4) (8). A solid phase chemical synthesis method of the GLP-1 derivative DLG3312 according to any one of claims 1 to 3, characterized in that the resin is first placed in a polypeptide synthesizer, and the diaminocarboxylic acid Y having a protecting group is The resin is bound, and the protecting group-containing amino acid monomer is arranged in the polypeptide synthesizer from the C-terminus to the N-terminus according to the X sequence according to claim 1 or 2, and the polypeptide resin having a side chain protecting group is synthesized. Protecting group, cleavage resin, HPLC purification, freeze-drying, to obtain the GLP-1 derivative DLG3312; wherein the protecting group-containing diaminocarboxylic acid Y comprises: Fmoc-L-Lys(Fmoc)-OH, Fmoc -L-Orn(Fmoc)-OH, the protected amino acid monomer comprises: Fmoc-L-Ala-OH, Fmoc-D-Ala-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc- L-Asp(OtBu)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Phe-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L- Thr(tBu)-OH, Fmoc-L-Trp-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Val-OH.

The solid phase chemical synthesis method of the GLP-1 derivative DLG3312 according to claim 4, wherein when the sequence X is Seq ID No.l,

The amino acid monomers having a protecting group are: Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Gly-OH, WO 2013/063877 Claim PCT/CN2012/070451

Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L- Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L- Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp (OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L -Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Ala-OH, Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-1;

The amino acid monomers having a protecting group are: Fmoc-L-Orn(Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH , Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L -Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L- Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L In the case of -Ala-OH, Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-5.

The solid phase chemical synthesis method of the GLP-1 derivative DLG3312 according to claim 4, wherein when the sequence X is Seq ID No. 2,

The amino acid monomers having a protecting group are: Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH , Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L -Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L- Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L -Gly-OH, Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-2;

The amino acid monomers having a protecting group are: Fmoc-L-Orn(Fmoc)-OH, Fmoc-L-Gly-OH,

Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH,

Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH, Fmoc-L-Phe-OH, WO 2013/063877 Claim PCT/CN2012/070451

Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc -L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Gly-OH, Fmoc-L- In the case of His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-6.

The solid phase chemical synthesis method of the GLP-1 derivative DLG3312 according to claim 4, wherein when the sequence X is Seq ID No. 3,

The amino acid monomers having a protecting group are: Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH , Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L -Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L- Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-D -Ala-OH, Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-3;

The amino acid monomers having a protecting group are: Fmoc-L-Orn(Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH , Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L -Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L- Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-D In the case of -Ala-OH, Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-7.

The solid phase chemical synthesis method of the GLP-1 derivative DLG3312 according to claim 4, wherein when the sequence X is Seq ID No. 4,

The amino acid monomers having a protecting group are: Fmoc-L-Lys(Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH , Fmoc-L-Phe-OH, WO 2013/063877 Claim PCT/CN2012/070451

Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L-Gln(Trt)-OH, Fmoc -L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Val-OH, Fmoc-L- When His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-4;

The amino acid monomers having a protecting group are: Fmoc-L-Orn(Fmoc)-OH, Fmoc-L-Gly-OH, Fmoc-L-Arg(Pbf)-OH, Fmoc-L-Gly-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, Fmoc-L-Leu-OH, Fmoc-L-Trp-OH, Fmoc-L-Ala-OH, Fmoc-L-Ile-OH , Fmoc-L-Phe-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Ala-OH, Fmoc-L-Ala-OH, Fmoc-L -Gln(Trt)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L- Ser(tBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc-L-Val-OH, Fmoc-L-Asp(OtBu)-OH, Fmoc-L-Ser(tBu)-OH, Fmoc- L-Thr(tBu)-OH, Fmoc-L-Phe-OH, Fmoc-L-Thr(tBu)-OH, Fmoc-L-Gly-OH, Fmoc-L-Glu(OtBu)-OH, Fmoc-L When -Val-OH, Fmoc-L-His(Trt)-OH, the obtained GLP-1 derivative is DLG3312-8.

A pharmaceutical composition comprising the GLP-1 derivative DLG3312 according to claims 1-3 and a pharmaceutically acceptable excipient.

The use of the GLP-1 derivative DLG3312 according to any of claims 1-3 for the preparation of a medicament for the treatment of diabetes and obesity.