WO2013063877A1 - Dérivé dlg3312 de glp-1 et son procédé de synthèse chimique en phase solide - Google Patents

Dérivé dlg3312 de glp-1 et son procédé de synthèse chimique en phase solide Download PDF

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WO2013063877A1
WO2013063877A1 PCT/CN2012/070451 CN2012070451W WO2013063877A1 WO 2013063877 A1 WO2013063877 A1 WO 2013063877A1 CN 2012070451 W CN2012070451 W CN 2012070451W WO 2013063877 A1 WO2013063877 A1 WO 2013063877A1
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fmoc
tbu
dlg3312
gly
otbu
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PCT/CN2012/070451
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Chinese (zh)
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劳勋
吴自荣
黄静
赵丽芬
李娟�
赵云
耿旭
汪正华
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华东师范大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • GLP-1 derivative DLG3312 having human glucagon-like peptide-1 (GLP-1) activity and a solid phase chemical synthesis method thereof.
  • GLP-1 is a product of the glucagon preprogenitor gene secreted by intestinal L cells, which is secreted after food intake.
  • GLP-1 (7-36) amide and GLP-1 (7-37) 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.
  • 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 ⁇ cells.
  • GLP-1 is indicated for the treatment of type 2 diabetes, and its effect of lowering blood glucose is blood glucose-dependent.
  • 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.
  • GLP-1 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.
  • 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.
  • DPP-4 is the most important enzyme.
  • various methods such as site-directed mutagenesis of amino acids and molecular modification have been made.
  • 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.
  • 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.
  • 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 ⁇ - ⁇ - ⁇ 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.
  • 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.
  • various diabetes drugs currently used such as hypoglycemia, weight gain, insulin resistance, islet ⁇ cell failure, and the like, are also avoided in terms of safety.
  • DLG3312 of the present invention has a function of improving insulin resistance and inhibiting apoptosis of islet ⁇ cells, thereby fundamentally improving the glucose metabolism ability of diabetic patients.
  • 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.
  • 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 ⁇ - ⁇ - ⁇ ; wherein, X The sequence represented is H-X8-EGTFTSDVSSYLEGQAAKEFIAWLVKGRG, wherein: ⁇ 8 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.
  • the GLP-1 derivative 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
  • HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
  • the GLP-1 derivative is DLG3312-4 having the following structural formula
  • 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-
  • the amino acid monomer having a protecting group is: Fmoc-L-Lys(Fmoc)-OH.
  • the amino acid monomer having a protecting group is: Fmoc-L-Om(Fmoc)-OH.
  • 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-
  • 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-
  • 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
  • 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
  • the amino acid monomer having a protecting group is: Fmoc-L-Lys(Fmoc)-OH.
  • 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
  • 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
  • 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.
  • 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.
  • the resistance to degradation of proteases in vivo 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.
  • 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.
  • 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. .
  • 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-L
  • Boc tert-butyloxycarbonyl; ⁇ tert-butyloxycarbonyl;
  • Trt trityl, ie trityl
  • tBU tert-butyl, ie tert-butyl
  • the equipment and reagents used in the present invention are as follows:
  • ⁇ is ⁇ -methylpyrrolidone
  • ⁇ is 1-hydroxybenzotriazole
  • HBTU 2-(1H benzotriazolyl)-tetramethylurea hexafluorophosphate, ie
  • the solid phase chemical synthesis method of the GLP-1 derivative of the present invention DLG3312 comprises the following steps:
  • 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;
  • the 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.
  • 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 ⁇ .1, and the same operation steps as those in the above method are not described again.
  • 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:
  • DLG3312-1 was obtained, and its structure is shown in the following formula (1).
  • 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.
  • 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.
  • 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:
  • DLG3312-2 was obtained, and its structure is shown in the following formula (2).
  • 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.
  • 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.
  • 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:
  • DLG3312-3 was obtained, and its structure is shown in the following formula (3).
  • 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.
  • 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.
  • 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:
  • DLG3312-4 was obtained, and its structure is shown in the following formula (4).
  • 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.
  • 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.
  • 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:
  • DLG3312-5 was obtained, and its structure is shown in the following formula (5).
  • 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.
  • 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.
  • 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:
  • 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.
  • 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.
  • 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:
  • DLG3312-7 was obtained, and its structure is shown in the following formula (7).
  • HdAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (?)
  • 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.
  • 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.
  • 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:
  • DLG3312-8 was obtained, and its structure is shown in the following formula (8).
  • 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.
  • 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;
  • the results are shown in Table 1.
  • 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.
  • 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.
  • DLG3312 showed no hypoglycemic activity, indicating that DLG3312 does not cause hypoglycemia, and is safe for treating diabetes.

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  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Obesity (AREA)
  • Emergency Medicine (AREA)
  • Child & Adolescent Psychology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un dérivé DLG3312 de GLP-1 ayant la structure X-Y-X, la séquence représentée par X est H-X8-EGTFTSDVSSYLEGQAAKEFIAWLVKGRG, X8 est l'un quelconque de A, G, dA ou V, et Y représente un acide diaminocarboxylique comprenant Lys et Om. La présente invention concerne également un procédé de synthèse chimique en phase solide du dérivé DLG3312 de GLP-1 et l'utilisation de celui-ci pour la préparation d'un médicament pour le traitement du diabète.
PCT/CN2012/070451 2011-11-03 2012-01-17 Dérivé dlg3312 de glp-1 et son procédé de synthèse chimique en phase solide WO2013063877A1 (fr)

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CN201110343357.6 2011-11-03

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CN104098689A (zh) * 2013-04-12 2014-10-15 华东师范大学 一种glp-1衍生物dlg3312及其固相化学合成方法
CN117186206B (zh) * 2023-10-20 2024-07-23 广东药科大学 新型不同glp1类似肽共价聚合体及其制备方法和应用

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CN101220088A (zh) * 2007-09-24 2008-07-16 中国人民解放军第四军医大学 人胰高血糖素样肽-1类似物的融合蛋白及其应用
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