WO2021164663A1 - Glp-1激动多肽化合物及其盐与合成方法及用途 - Google Patents

Glp-1激动多肽化合物及其盐与合成方法及用途 Download PDF

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WO2021164663A1
WO2021164663A1 PCT/CN2021/076308 CN2021076308W WO2021164663A1 WO 2021164663 A1 WO2021164663 A1 WO 2021164663A1 CN 2021076308 W CN2021076308 W CN 2021076308W WO 2021164663 A1 WO2021164663 A1 WO 2021164663A1
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acid
aeea
fmoc
glu
seq
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PCT/CN2021/076308
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English (en)
French (fr)
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赵呈青
施国强
李顺子
王蔡典
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江苏诺泰澳赛诺生物制药股份有限公司
杭州诺泰澳赛诺医药技术开发有限公司
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Priority to JP2022521757A priority Critical patent/JP2022551716A/ja
Priority to EP21756797.3A priority patent/EP4108682A4/en
Publication of WO2021164663A1 publication Critical patent/WO2021164663A1/zh
Priority to US17/820,251 priority patent/US20230220033A1/en

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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 present invention relates to the field of polypeptide compounds, in particular to a series of polypeptide compounds with dual incretin-like peptides, which stimulate the receptor of glucagon-like peptide-1 (GLP-1) and can be used to treat type 2 diabetes and Obesity.
  • the invention also relates to pharmaceutically acceptable salts of the polypeptide compound, polypeptide pharmaceutical compositions, medicaments, preparation methods and uses thereof.
  • the cause of metabolic syndrome is abnormal metabolism of various substances such as protein, fat and carbohydrates. Overnutrition and reduced physical activity can lead to obesity and obesity-related diseases such as diabetes.
  • type 2 diabetes is the most common form of diabetes, accounting for about 90% of all diabetes.
  • Type 2 diabetes is characterized by high blood sugar levels caused by insulin resistance.
  • the current standard of care for type 2 diabetes includes diet and exercise, as well as available oral and injectable hypoglycemic drugs. Despite this, many patients with type 2 diabetes are still not adequately controlled.
  • GLP-1 is a 37 amino acid peptide that stimulates insulin secretion, protects pancreatic ⁇ cells, inhibits glucagon secretion, gastric emptying and food intake, leading to weight loss.
  • GLP-1 is called incretin; incretin receptor signaling plays a key role in physiology-related effects on glucose homeostasis. In normal physiology, GLP-1 is secreted from the intestine after a meal, and these incretins enhance the physiological response to food, including satiety, insulin secretion and nutritional processing. The incretin response is impaired in patients with type 2 diabetes.
  • DPP-4 dipeptidyl peptidase IV
  • GLP-1 analogues are limited by side effects such as nausea and vomiting, and therefore the administration usually cannot achieve the full effect of blood sugar control and weight loss.
  • Natural GLP-1 is a ubiquitous proteolytic enzyme, especially Dipeptidyl peptidase-4 (DPP-4) is rapidly inactivated, so it can only be used for short-term metabolic control.
  • DPP-4 is an exopeptidase proteolytic enzyme; it is known that DPP-4 can quickly hydrolyze the peptide bond at positions 7 to 8 in the GLP-1 sequence, so it is not natural to introduce in positions 7-8 and other positions in the sequence.
  • Amino acids can increase the proteolytic stability of any given peptide; apparently the use of unnatural amino acids contributes to the stability of the peptide against DPP-4 proteolysis and other forms of degradation.
  • the purpose of the present invention is to provide a series of GLP-1 that utilizes dual mechanisms of action and has better glucose-lowering activity and weight-reducing effects in view of the existing technologies and compounds in vivo stability, blood sugar and weight-reducing effects and side effects.
  • Agonist polypeptide compounds which can be used to treat Type II diabetes, obesity and cardiovascular diseases.
  • Another object of the present invention is to provide a method for preparing the aforementioned GLP-1 agonistic polypeptide compound.
  • Another object of the present invention is to provide a pharmaceutically acceptable salt of the aforementioned GLP-1 agonistic polypeptide compound.
  • Another object of the present invention is to provide a pharmaceutical composition of GLP-1 agonistic polypeptide compound.
  • Another object of the present invention is to provide a GLP-1 agonizing polypeptide compound medicament.
  • Another object of the present invention is to provide the use of the aforementioned GLP-1 agonistic polypeptide compound, its pharmaceutically acceptable salt, its pharmaceutical composition and medicament.
  • the present invention discloses a type of GLP-1 agonistic polypeptide compound, which is characterized in that the amino acid sequence of this type of polypeptide compound is:
  • Xaa 1 is taken from:
  • Xaa 2 is taken from: G,
  • Xaa 1 and Xaa 2 are the same or different;
  • R 1 is: a straight or branched chain alkyl group containing 2-6 carbon atoms
  • R 2 is: H or CH 3 ,
  • X is: O, S or N-CH 3 ,
  • R 1 and R 2 alkyl groups can be optionally substituted with 1 to 6 halogen atoms
  • Xaa 3 is taken from:
  • n and n are natural numbers, preferably n is a natural number from 12 to 20; preferably m is a natural number from 0 to 3.
  • a further preferred technical solution of the GLP-1 agonistic polypeptide compound of the present invention is that the preferred amino acid sequence of this type of polypeptide compound is:
  • HX 2 EGTFTSDVSSYLEX 1 QAAK(HOOC-(CH 2 ) 16 -CO- ⁇ -Glu-AEEA-AEEA)EFIAWLVRGRG
  • the invention also discloses the method for synthesizing the GLP-1 agonizing polypeptide compound, which is characterized by:
  • amino acids 1-31 of the main sequence of the solid-phase synthesis compound polypeptide wherein the amino acids 1-4 are the fragment Boc-His(Trt)Xaa 1 Glu(OtBu)Gly-OH, where:
  • Xaa 1 is taken from:
  • R 1 is: a straight or branched chain alkyl group containing 2-6 carbon atoms
  • R 2 is: H or CH 3 ,
  • X is: O, S or N-CH 3 ,
  • R 1 and R 2 alkyl groups can be optionally substituted with 1 to 6 halogen atoms
  • the 20th Lys uses the fragment Fmoc-Lys (Xaa 3 ); the fragment structure is as follows:
  • the condensing agent used is one or more of DIC/HOBt, Oxymapure/DIC, HBTU/HOBT/DIEA, PyBop/HOBT/DIEA;
  • the reaction solvent used is one of DCM, DMF, NMP, DMSO One or more combinations;
  • the Fmoc removal reagent used is a v/v 25% piperidine/DMF solution;
  • Step one prepare Fmoc-Gly 31 -Wang resin
  • Step two prepare fully protected peptide resin
  • Step a adding Fmoc-Gly 31 -Wang resin to the solid phase reactor;
  • Step b solid-phase synthesis method one by one Fmoc-Arg 30 (Pbf)-OH, Fmoc-Gly 29 -OH, Fmoc-Arg 28 (Pbf)-OH, Fmoc-Val 27 -OH, Fmoc-Leu 26 -OH, Fmoc-Trp 25 (Boc)-OH, Fmoc-Ala 24 -OH, Fmoc-Ile 23 -OH, Fmoc-Phe 22 -OH, Fmoc-Glu 21 (OtBu)-OH, Fmoc-Lys 20 (Xaa 3 )- OH, Fmoc-Ala 19 -OH, Fmoc-Ala 18 -OH, Fmoc-Gln 17 (Trt)-OH, Fmoc-Gly 16 -OH, Fmoc-Glu 15 (OtBu)-OH, Fmoc-Leu 14 -OH, Fmoc-Tyr 13
  • the Xaa 1 is taken from:
  • R 1 is: a straight or branched chain alkyl group containing 2-6 carbon atoms
  • R 2 is: H or CH 3 ,
  • X is: O, S or N-CH 3 ,
  • R 1 and R 2 alkyl groups can be optionally substituted with 1 to 6 halogen atoms
  • Step three cleavage the peptide resin to obtain a crude peptide
  • Step 4 Use reversed-phase chromatography to prepare polypeptide compounds.
  • the reversed-phase chromatographic packing uses silica-based bonded C8 and C18 packings.
  • the mobile phase uses acetonitrile and aqueous solution, where the aqueous solution is TFA, phosphoric acid, sulfuric acid solution under certain pH conditions, or The sodium salt, potassium salt, ammonium salt, etc. formed by these acids.
  • the present invention also discloses a class of pharmaceutically acceptable salts of GLP-1 agonizing polypeptide compounds, which are characterized in that: the GLP-1 agonizing polypeptide compound is the polypeptide compound described in the above technical scheme, and this type of polypeptide compound C The terminal amino acid is amidated as a C-terminal primary amide or a pharmaceutically acceptable salt thereof.
  • the pharmaceutically acceptable salt of the GLP-1 agonizing polypeptide compound of the present invention is: the salt is a salt formed by the GLP-1 agonizing polypeptide compound and one of the following compounds: hydrochloric acid , Hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid, nitric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propylene Acid, butyric acid, caproic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentane propionic acid , Dig
  • the present invention also discloses a pharmaceutical composition of a GLP-1 agonistic polypeptide compound, which is characterized in that the pharmaceutical composition uses the GLP-1 agonistic polypeptide compound described in any one of the above technical solutions as an effective raw material, or is based on the above technical solution
  • the pharmaceutically acceptable salt of the GLP-1 agonistic polypeptide compound described in any one of them is an effective raw material, and is composed of a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutically acceptable salt of the GLP-1 agonizing polypeptide compound of the present invention uses the GLP-1 agonizing polypeptide compound and the above-mentioned compounds as raw materials, and is prepared by conventional methods disclosed in the prior art.
  • the present invention also discloses a medicament prepared from the GLP-1 agonistic polypeptide compound described in any one of the above technical solutions, which is characterized in that the medicament is any kind of tablets, capsules, and elixirs in pharmacy.
  • the medicament consists of GLP-1 agonist polypeptide compound and pharmaceutically acceptable It is composed of pharmaceutical excipients, carriers or diluents.
  • the medicament of the present invention can be prepared according to conventional methods in the prior art.
  • GLP-1 agonistic polypeptide compound described in the present invention is that it can be used as an effective raw material to prepare drugs for the treatment or prevention of diabetes, or for the preparation of anti-obesity drugs.
  • the pharmaceutically acceptable use of the GLP-1 agonist polypeptide compound described in the present invention is that it can be used as an effective raw material to prepare drugs for the treatment or prevention of diabetes, or for the preparation of anti-obesity drugs.
  • the pharmaceutical composition and medicament of the GLP-1 agonistic polypeptide compound described in the present invention can be used as drugs for the treatment and prevention of diabetes, or as anti-obesity drugs.
  • the substitution of unnatural amino acids not only has a greater effect on increasing the in vivo stability of GLP-1 agonists, but also has a greater impact on the activity.
  • a different amino acid Xaa 1 is used in the above sequence, its GLP-1 agonist activity has a great change, and when Xaa 1 has chirality, one of the optical isomers is higher than the other substituted GLP- 1 Agonist has stronger activity.
  • fatty acids through their albumin binding motifs can improve the pharmacokinetics of peptides by extending the half-life.
  • fatty acids can improve the half-life of peptides
  • the half-life of the peptide is extended.
  • the present invention has the following beneficial effects:
  • the present invention discloses a series of GLP-1 agonist polypeptide compounds with better hypoglycemic activity and weight-reducing effects that utilize dual mechanisms of action.
  • the polypeptide compounds of the present invention have both hypoglycemic and weight-reducing effects, and are beneficial in cardiovascular aspects.
  • the polypeptide compound of the present invention has the effect of producing weight loss in patients, has low immunogenicity characteristics and supports the pharmacokinetic (PK) characteristics of once-a-week dosing. It is suitable as an active ingredient of medicines for the treatment of diabetes and obesity. So it can be used to treat type 2 diabetes, obesity and cardiovascular diseases.
  • the GLP-1 agonistic polypeptide compound prepared by the preparation method of the present invention has good hypoglycemic and weight gain slowing activities, long drug effect time, high yield, short synthesis cycle, easy purification of crude product, low production cost, and easy industrial automation production.
  • Figure 1 is a graph showing the effect of each tested drug on random blood glucose of db/db mice in the experiment
  • Figure 2 is a graph of the change rate of each test drug on the random blood glucose of db/db mice in the experiment;
  • Figure 3 is a graph showing the influence of each tested drug on the body weight of ob/ob mice in the experiment.
  • Fig. 4 is a graph showing the weighting rate of each test drug on ob/ob mice in the experiment.
  • the steps of deprotection and coupling are repeated to connect the corresponding amino acids in sequence, and the corresponding amino acids are connected in sequence until the peptide chain is synthesized, and the peptide resin is obtained.
  • the specific coupling protection amino acids are as follows:
  • the solid-phase synthesis method combines Fmoc-Arg 30 (Pbf)-OH, Fmoc-Gly 29 -OH, Fmoc-Arg 28 (Pbf)-OH, Fmoc-Val 27 -OH, Fmoc-Leu 26 -OH, Fmoc-Trp one by one.
  • HX 2 EGTFTSDVSSYLEX 1 QAAK(HOOC-(CH 2 ) 16 -CO- ⁇ -Glu-AEEA-AEEA)EFIAWLVRGRG
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.93 g of pure product.
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 2.87 g of pure product.
  • the synthesis method is the same as in Example 1, and the purified sample solution is collected and lyophilized to obtain 2.55 g of pure product.
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 2.60 g of pure product.
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 2.61 g of pure product.
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.1 g of pure product.
  • Embodiment 8 SEQ ID NO: 8
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.35 g of pure product.
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 2.70 g of pure product.
  • Embodiment 10 SEQ ID NO: 10
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.90 g of pure product.
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.74 g of pure product.
  • Embodiment 12 SEQ ID NO: 12
  • the synthesis method was the same as that in Example 1.
  • the purified sample solution was collected and lyophilized to obtain 3.22 g of pure product.
  • Embodiment 13 SEQ ID NO: 13
  • the synthesis method was the same as that in Example 1.
  • the purified sample solution was collected and lyophilized to obtain 3.48 g of pure product.
  • Embodiment 14 SEQ ID NO: 14
  • the synthesis method was the same as that in Example 1.
  • the purified sample solution was collected and lyophilized to obtain 5.30 g of pure product.
  • Embodiment 15 SEQ ID NO: 15
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 2.70 g of pure product.
  • Embodiment 16 SEQ ID NO: 16
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.67 g of pure product.
  • Embodiment 17 SEQ ID NO: 17
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 2.33 g of pure product.
  • Embodiment 18 SEQ ID NO: 18
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.440 g of pure product.
  • Embodiment 19 SEQ ID NO: 19
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 2.70 g of pure product.
  • Embodiment 20 SEQ ID NO: 20
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.92 g of pure product.
  • Embodiment 21 SEQ ID NO: 21
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.62 g of pure product.
  • Embodiment 22 SEQ ID NO: 22
  • the synthesis method is the same as in Example 1.
  • the purified sample solution is collected and lyophilized to obtain 3.80 g of pure product.
  • polypeptide compounds The following are the relevant pharmacological test methods and results of GLP-1 agonistic polypeptide compounds (hereinafter referred to as polypeptide compounds):
  • GLP-1R Glucagon-like peptide-1 receptor
  • the Chinese hamster ovary cell line (CHO) stably expressing human GLP-1R was used to determine the activity of polypeptide compound samples and control compounds to induce cAMP signals downstream of GLP-1R.
  • Wild-type human GLP-1R (NM_002062.5) was transiently transfected and screened with 600 ⁇ g/ml hygromycin-B for two weeks to obtain a cell line that was recombinantly integrated into the FlpInCHO (Invitrogen) cell stable expression system.
  • the culture condition of FlpInCHO cells is that DMEM medium is added with 10% heat-inactivated fetal bovine serum and cultured in a 5% carbon dioxide cell incubator.
  • the cells were grown overnight in a 6-well cell culture plate, then transferred to a 384-well plate at a concentration of 8000 cells per well, and continued to be cultured at 37°C and 5% CO 2 for 24 Hour.
  • the experiment uses LANCE cAMP detection kit to measure its cAMP signal intensity. After the cells were incubated for 30 minutes, the LANCE cAMP detection kit was used to measure the fluorescence readings using a microplate reader, a standard curve was established to convert the fluorescence readings into the corresponding cAMP values, and the non-linear regression of GraphpadPrism 7.0 software was used to calculate the EC 50 value of the compound.
  • mice After 5-7 days of adaptation to DB/DB hyperglycemic mice, the non-fasting random blood glucose value was measured, and after 12 hours of fasting, the fasting blood glucose value was measured by a rapid blood glucose meter.
  • the animals were randomly divided into groups (random block design) based on body weight, fasting blood glucose after 12h fasting, and random blood glucose results. According to random weight, random blood glucose and fasting blood glucose, they were divided into solvent control group and peptide compound group (select test drugs: SEQ.ID NO:3 and SEQ.ID NO:4), positive control Semaglutide group, known control group SPN013.
  • the mice in each test drug group and the control group were injected subcutaneously with each test drug or control solution respectively, and the mice in the model control group were injected subcutaneously with PBS buffer; the experimental grouping and dosage settings are shown in Table 1:
  • mice n Number of mice in each group; Dosing volume: 5 ⁇ l/g based on mouse body weight.
  • Weight After the animals are grouped, the weight is measured once a day at a fixed time period
  • Blood glucose level Take the tail tip blood of mice to measure the blood glucose level with a rapid blood glucose meter and blood glucose test paper.
  • test substances were administered subcutaneously once, and blood glucose values were measured at 0h, 2h, 4h, 6h, 10h, 24h, 34h, 48h, 58h, 72h, 82h, 96h after administration.
  • hypoglycemic efficacy of the test substance was evaluated by BG (mmol/L) or AUC (mmol/L ⁇ min).
  • AUC [(BG 0 +BG 2 )x2+(BG 2 +BG 4 )x2+(BG 4 +BG 6 )x2+(BG 6 +BG 10 )x4+(BG 10 +BG 24 )x14+(BG 24 +BG 34 ) x10+(BG 34 +BG 48 )x14+(BG 48 +BG 58 )x10+(BG 58 +BG 72 )x14+(BG 72 +BG 82 )x10+(BG 82 +BG 96 )x14]/2.
  • T test is used for comparison between two groups.
  • One-way ANOVA is used for comparison between three or more groups. If the F value is significantly different, multiple comparisons should be made after ANOVA analysis. Use SPSS 17.0 for all data analysis. p ⁇ 0.05 considered a significant difference.
  • the results of the hypoglycemic experimental data and graphs show that when the concentration of the polypeptide compound of the present invention is 30 nmol/kg, the hypoglycemic effect is almost the same as the hypoglycemic effect of Semaglutide; positive Both the control group and the peptide compound group are significantly different from the solvent control group.
  • Table 5-6 and Figure 3-4 a single subcutaneous injection of the solvent control group has no significant effect on the random body weight and change of db/db mice. Both the positive control group and the polypeptide compound group are significantly different from the solvent control group; the difference is especially significant after 24 hours of administration.
  • SEQ.ID NO:3 and SEQ.ID NO:4 are equivalent to the same dose of Semaglutide.
  • the SEQ. ID NO: 3 and SEQ. ID NO: 4 polypeptide compounds show better weight control effects.
  • Semaglutide, SPN009 (SEQ. ID NO: 3) and SPN010 (SEQ. ID NO: 4) pharmacokinetic comparison experiment Take 9 male rats, 3 rats in each group, and give semaglutide, SPN009 and SPN010. A single subcutaneous injection, the dose of 0.02mg/kg; cut the tail to collect blood, the blood collection time points are: 0h, 15min, 30min, 1h, 2h, 3h, 4h, 6h, 8h, 24h, 48h, 72h.
  • the LC-MS/MS method was used to determine the concentration of the compound in plasma. All data were collected and exported by Unifi 1.9.3 software, and Excel software was used for data calculation.
  • the non-compartmental model of DAS 3.0 software was used to calculate the pharmacokinetic parameters of rats after administration. The results are shown in Table 7.

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Abstract

提供了一类GLP-1激动多肽化合物，其氨基酸序列为：HXaa1EGTFTSDVSSYLE Xaa2QAA Xaa3EFIAWLVRGRG，且C末端为羧基或羧基被酰胺化。还提供了该类多肽化合物的制备方法、药学上可接受的盐、药物组合物和药剂。该类多肽化合物同时具备降糖及减重效果，可以作为有效原料用于制备治疗或者预防糖尿病的药物或者减肥药物。

Description

GLP-1激动多肽化合物及其盐与合成方法及用途 技术领域
本发明涉及多肽化合物领域，具体涉及一系列具有双重肠促胰岛素肽类似化合物多肽化合物，其激动人胰高血糖素样肽-1(GLP-1)的受体，并且可用于治疗2型糖尿病和肥胖症。本发明还涉及该多肽化合物药学上可接受的盐，多肽药物组合物、药剂、制备方法及其用途。
背景技术
代谢综合征的病因是蛋白质、脂肪及碳水化合物等多种物质的代谢异常。营养过剩、体力活动减少等会导致肥胖以及肥胖相关疾病，如糖尿病等。近年来，2型糖尿病、血脂代谢异常的发病率日益增高；2型糖尿病是最常见的糖尿病形式，占所有糖尿病的约90％。2型糖尿病的特征在于由胰岛素抗性引起的高血糖水平。目前的2型糖尿病护理标准包括饮食和运动以及可用的口服和注射降糖药物。尽管如此，许多患有2型糖尿病的患者仍然未得到充分控制。
GLP-1是一种37个氨基酸的肽，可刺激胰岛素分泌，保护胰腺β细胞，抑制胰高血糖素分泌，胃排空和食物摄入，从而导致体重减轻。GLP-1被称为肠促胰岛素；肠降血糖素受体信号传导对葡萄糖体内平衡起关键作用的生理学相关作用。在正常生理学中，GLP-1在餐后从肠道分泌，并且这些肠降血糖素增强对食物的生理反应，包括饱腹感，胰岛素分泌和营养处理。2型糖尿病患者的肠促胰岛素反应受损。目前市售的肠降血糖素类似物或二肽基肽酶IV(DPP-4)抑制剂仅利用单一的作用机制用于血糖控制；如果利用双重作用机制的2型糖尿病化合物，就可以得到降糖活性和减重效果俱佳的多肽化合物。
已经发现GLP-1类似物的剂量受到诸如恶心和呕吐的副作用的限制，并且因此给药通常不能达到血糖控制和体重减轻的完全功效，天然GLP-1均被普遍存在的蛋白水解酶，尤其是二肽基肽酶-4(DPP-4)快速灭活，因此，只能用于短期代谢控制。DPP-4属于外肽酶类蛋白水解酶；已经知道DPP-4能快速水解GLP-1序列中第7到8位的肽键，所以在序列中的7-8位和其它位点引入非天然氨基酸可以增加任何给定肽的蛋白水解稳定性；显然使用非天然氨基酸有助于肽对DPP-4蛋白水解和其他形式降解的稳定性。
发明内容
本发明的目的是针对现有技术和化合物体内稳定性，降糖减重效果及副作用等存在的不足，提供一系列利用双重作用机制、具有更优的降糖活性和减重效果的GLP-1激动剂多肽化合物，从而用于治疗二类糖尿病，肥胖症和心血管等方面疾病。
本发明的另一个目的是提供了前述GLP-1激动多肽化合物的制备方法。
本发明的再一个目的是提供了前述GLP-1激动多肽化合物药学上可接受的盐。
本发明的又一个目的是提供了GLP-1激动多肽化合物的药物组合物。
本发明的又一个目的是提供了GLP-1激动多肽化合物的药剂。
本发明的又一个目的是提供了前述GLP-1激动多肽化合物、其药学上可接受的盐、其药物组合物和药剂的用途。
本发明的目的是通过以下的技术方案来实现的；本发明公开了一类GLP-1激动多肽化合物，其特点是，该类多肽化合物的氨基酸序列为：
H Xaa 1EGTFTSDVSSYLE Xaa 2QAA Xaa 3EFIAWLVRGRG，且该类多肽化合物C末端氨基酸作为羧基或羧基被酰胺化；
其中：
Xaa 1取自：
Figure PCTCN2021076308-appb-000001
Xaa 2取自：G，
Figure PCTCN2021076308-appb-000002
Xaa 1、Xaa 2相同或不同；
R 1为：含有2-6个碳原子的直链或支链烷基，
R 2为：H或CH 3,
X为：O,S或N-CH 3,
式中R 1和R 2烷基可任选地被1-6个卤素原子取代；
Xaa 3取自：
Figure PCTCN2021076308-appb-000003
上式中，m、n为自然数，优选的n为自然数从12到20；优选的m为自然数0到3。
本发明所述的GLP-1激动多肽化合物，其进一步优选的技术方案是，该类多肽化合物的优选氨基酸序列为：
(1)SEQ ID NO：1
HX 2EGTFTSDVSSYLEX 2QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(2)SEQ ID NO：2
HX 2EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(3)SEQ ID NO：3
HX 3EGTFTSDVSSYLEX 3QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(4)SEQ ID NO：4
HX 3EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(5)SEQ ID NO：5
HX 4EGTFTSDVSSYLEX 4QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(6)SEQ ID NO：6
HX 4EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(7)SEQ ID NO：7
HX 5EGTFTSDVSSYLEX 5QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(8)SEQ ID NO：8
HX 5EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(9)SEQ ID NO：9
HX 6EGTFTSDVSSYLEX 6QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(10)SEQ ID NO：10
HX 6EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(11)SEQ ID NO：11
HX 7EGTFTSDVSSYLEX 7QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(12)SEQ ID NO：12
HX 7EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(13)SEQ ID NO：13
HX 8EGTFTSDVSSYLEX 8QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(14)SEQ ID NO：14
HX 8EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(15)SEQ ID NO：15
HX 9EGTFTSDVSSYLEX 9QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(16)SEQ ID NO：16
HX 9EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(17)SEQ ID NO：17
HX 10EGTFTSDVSSYLEX 10QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(18)SEQ ID NO：18
HX 10EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(19)SEQ ID NO：19
HX 11EGTFTSDVSSYLEX 11QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(20)SEQ ID NO：20
HX 11EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(21)SEQ ID NO：21
HX 12EGTFTSDVSSYLEX 12QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
(22)SEQ ID NO：22
HX 12EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
式中：
X 1＝G；
Figure PCTCN2021076308-appb-000004
Figure PCTCN2021076308-appb-000005
本发明还公开了所述的GLP-1激动多肽化合物的合成方法，其特点是：
(1)固相合成化合物多肽的主序列第1-31位氨基酸，其中第1-4位氨基酸采用片段Boc-His(Trt)Xaa 1Glu(OtBu)Gly-OH，其中：
其中：
Xaa 1取自：
Figure PCTCN2021076308-appb-000006
R 1为：含有2-6个碳原子的直链或支链烷基，
R 2为：H或CH 3,
X为：O,S或N-CH 3,
式中R 1和R 2烷基可任选地被1-6个卤素原子取代；
(2)第20位Lys采用片段Fmoc-Lys(Xaa 3)；片段结构式如下：
Figure PCTCN2021076308-appb-000007
所述的n为自然数从12到20；m为自然数0到3；
(3)所用的缩合剂为DIC/HOBt，Oxymapure/DIC，HBTU/HOBT/DIEA，PyBop/HOBT/DIEA其中的一种或多种；所用的反应溶剂为DCM、DMF、NMP、DMSO中的一种或多种组合；所使用的Fmoc脱除试剂为v/v25％哌啶/DMF溶液；
(4)包括以下步骤：
步骤一，制备Fmoc-Gly 31-Wang树脂；
步骤二，制备全保护肽树脂；
步骤a，将Fmoc-Gly 31-Wang树脂加入到固相反应器中；
步骤b，固相合成方法逐一将Fmoc-Arg 30(Pbf)-OH、Fmoc-Gly 29-OH、Fmoc-Arg 28(Pbf)-OH、Fmoc-Val 27-OH、Fmoc-Leu 26-OH、Fmoc-Trp 25(Boc)-OH、Fmoc-Ala 24-OH、Fmoc-Ile 23-OH、Fmoc-Phe 22-OH、Fmoc-Glu 21(OtBu)-OH、Fmoc-Lys 20(Xaa 3)-OH、Fmoc-Ala 19-OH、Fmoc-Ala 18-OH、Fmoc-Gln 17(Trt)-OH、Fmoc-Gly 16-OH、Fmoc-Glu 15(OtBu)-OH、Fmoc-Leu 14-OH、Fmoc-Tyr 13(tBu)-OH、Fmoc-Ser 12(tBu)-OH、Boc-Ser 11-OH、Fmoc-Val 10-OH、Fmoc-Asp 9(OtBu)-OH、Fmoc-Ser 8(tBu)-OH、Fmoc-Thr 7(tBu)-OH、Fmoc-Phe 6-OH、Fmoc-Thr 5(tBu)-OH、Boc-His 1(Trt)Xaa 1Glu 3(OtBu)Gly 4-OH偶联到Fmoc-Gly 31-Wang上；
所述Xaa 1取自：
Figure PCTCN2021076308-appb-000008
R 1为：含有2-6个碳原子的直链或支链烷基，
R 2为：H或CH 3,
X为：O,S或N-CH 3,
式中R 1和R 2烷基可任选地被1-6个卤素原子取代；
所述片段Fmoc-Lys 20(Xaa 3)-OH结构式如下：
Figure PCTCN2021076308-appb-000009
所述的n为自然数从12到20；m为自然数0到3。
步骤三，将所述肽树脂进行裂解，得到粗肽，
裂解试剂采用复合试剂，配比为：TFA:(苯甲硫醚/TIS/EDT/苯酚/水)＝90:5，其中捕获剂为苯甲硫醚/TIS/EDT/水中的任意组合。
步骤四，采用反相色谱方法制备多肽化合物，反相色谱填料采用硅胶基键合的C8、C18填料，流动相采用乙腈和水溶液，其中水溶液为一定pH条件下的TFA、磷酸、硫酸溶液，或这些酸形成的钠盐、钾盐、铵盐等。
本发明还公开了一类GLP-1激动多肽化合物药学上可接受的盐，其特点是：所述的GLP-1激动多肽化合物为以上技术方案中所述的多肽化合物，且该类多肽化合物C末端氨基酸作为C末端伯酰胺或其药学上可接受的盐被酰胺化。
本发明所述的GLP-1激动多肽化合物药学上可接受的盐，其优选的技术方案是：所述的盐为GLP-1激动多肽化合物与下述化合物中的一种所形成的盐：盐酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸、硝酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、肥酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或者硫氰酸。
本发明还公开了GLP-1激动多肽化合物的药物组合物，其特点是，该药物组合物以上述技术方案中任意一项所述的GLP-1激动多肽化合物为有效原料，或者以上述技术方案中任意一项所述的GLP-1激动多肽化合物药学上可接受的盐为有效原料，再加上药学上可接受的载体或稀释剂组成。
本发明所述的GLP-1激动多肽化合物药学上可接受的盐，以GLP-1激动多肽化合物和上述化合物为原料，再采用现有技术中公开的常规方法进行制备。
本发明还公开了上述技术方案中任意一项所述的GLP-1激动多肽化合物所制备的药剂，其特点是，所述的药剂是任何一种药剂学上所说的片剂、胶囊、酏剂、糖浆、锭剂、吸入剂、喷雾剂、注射剂、膜剂、贴剂、散剂、颗粒剂、块剂、乳剂、栓剂或者复方制剂，药剂由GLP-1激动多肽化合物和药学上可接受的药用辅料、载体或稀释剂组成。本发明所述的药剂，可以按照现有技术常规方法进行制备。
本发明记载的GLP-1激动多肽化合物的用途是，它可以作为有效原料用来制备用于治疗或者预防糖尿病的药物，或者用于制备减肥药物。
本发明记载的GLP-1激动多肽化合物药学上可接受的用途是，它可以作为有效原料在用来制备治疗或者预防糖尿病的药物，或者用于制备减肥药物。
本发明记载的GLP-1激动多肽化合物的药物组合物、药剂，可以用作治疗和预防糖尿病的药物，或者用作减肥药物。
在本说明书全文中，采用以下缩写的含义见下表：
英文缩写 中文
DCM 二氯甲烷
DMF N-甲基吡咯烷酮
HBTU 苯并三氮唑-N，N，N'，N'-四甲基脲六氟磷酸酯
HOBt 1-羟基-苯并三氮唑
DIEA/DIPEA N，N'-二异丙基乙胺
Fmoc N-9-芴甲氧羰基
EDT 乙二硫醇
HPLC 高效液相色谱
TFA 三氟乙酸
tBu 叔丁基
作为本发明的一部分，非天然氨基酸的取代不仅对增加GLP-1激动剂的体内稳定性较大的作用，而且对活性也产生了影响较大的影响。例如，当上述序列中的用不同氨基酸Xaa 1时，其GLP-1激动剂活性有很大的变化，并且当Xaa 1具有手性时，其中的一个光学异构体比另一个取代的GLP-1激动剂具有更强的活性。另外，脂肪酸通过其白蛋白结合基序可以通过延长半衰期来改善肽的药代动力学。虽然使用脂肪酸可以改善肽的半衰期，但申请人发现，作为本发明的一部分，脂肪酸链的长度，组成和位置以及肽和脂肪酸链之间的连接体可能具有意外对GLP-1激动剂活性影响，同时延长了肽的半衰期。
与现有多肽化合物和制备技术相比，本发明具有以下有益效果：
本发明公开了一系列利用双重作用机制、具有更优的降糖活性和减重效果的GLP-1激动剂多肽化合物，本发明多肽化合物同时具备降糖、减重效果，此外心血管方面具有益处，可以作为有效原料用于制备治疗或者预防糖尿病的药物或者减肥药物。基于动物能量消耗数据证明，本发明多肽化合物具有在患者中产生体重减轻的作用，具有低免疫原性特性和支持每周一次给药的药代动力学(PK)特征。适合作为治疗糖尿病、肥胖药物的活性成分。从而用于治疗二类糖尿病，肥胖症和心血管等方面疾病。
本发明制备方法所制得的GLP-1激动多肽化合物降糖和减缓体重增加活性好，药效时间长，收率高、合成周期短、粗品纯化容易，生产成本低、易于工业自动化生产。
附图说明
图1为实验中各受试药对db/db小鼠随机血糖的影响图；
图2为实验中各受试药对db/db小鼠随机血糖的变化率图；
图3为实验中各受试药对ob/ob小鼠的体重影响图；
图4为实验中各受试药对ob/ob小鼠的体重化率图。
具体实施方式
本发明通过下列实施例来进行进一步的说明，但这些实施例不做任何限制本发明权利的解释。
式中：
X 1＝G；
Figure PCTCN2021076308-appb-000010
Figure PCTCN2021076308-appb-000011
实施例1，SEQ ID NO：1多肽化合物的合成：
HX 2EGTFTSDVSSYLEX 2QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
1.SEQ ID NO：1多肽化合物肽链的合成
1.1 树脂的溶胀
称取Wang-Resin 10g(取代度0.53mmol/g)，经DCM 100mL溶胀30min，抽滤去DCM，再用DMF 100mL溶胀30min，分别用DMF，DCM 100mL冲洗干净。
1.2 Fmoc-Gly-Wang-Resin的合成
将Fmoc-Gly(tBu)-OH(15mmol)，HOBT(18mmol)和DIC(18mmol)溶于DMF 100mL中，再将此溶液加入上一步得到的树脂中反应2小时，结束后滤去反应液，用DCM和DMF各100mL洗涤树脂3次。
1.3 Fmoc保护基的脱除
向洗涤后的树脂中加入含0.1M HOBt的25％哌啶/DMF(V/V)溶液脱除Fmoc，反应结束后用DCM和DMF各100mL洗涤树脂3次。
1.4 肽链的延长
按照序列，重复上述脱保护和偶合的步骤依次连接上相应的氨基酸，依次连接上相应的氨基酸直至肽链合成完毕，得到肽树脂。具体偶联保护氨基酸如下：
固相合成方法逐一将Fmoc-Arg 30(Pbf)-OH、Fmoc-Gly 29-OH、Fmoc-Arg 28(Pbf)-OH、Fmoc-Val 27-OH、Fmoc-Leu 26-OH、Fmoc-Trp 25(Boc)-OH、Fmoc-Ala 24-OH、Fmoc-Ile 23-OH、Fmoc-Phe 22-OH、Fmoc-Glu 21(OtBu)-OH、Fmoc-Lys 20(Oct(OtBu)-γ-Glu(OtBu)-AEEA-AEEA)、Fmoc-Ala 19-OH、Fmoc-Ala 18-OH、Fmoc-Gln 17(Trt)-OH、Fmoc-Gly 16-OH、Fmoc-Glu 15(OtBu)-OH、Fmoc-Leu 14-OH、Fmoc-Tyr 13(tBu)-OH、Fmoc-Ser 12(tBu)-OH、Boc-Ser 11-OH、Fmoc-Val 10-OH、Fmoc-Asp 9(OtBu)-OH、Fmoc-Ser 8(tBu)-OH、Fmoc-Thr 7(tBu)-OH、Fmoc-Phe 6-OH、Fmoc-Thr 5(tBu)-OH、Boc-His 1(Trt)X 2Glu 3(OtBu)Gly 4-OH偶联到Fmoc-Gly 31-Wang上；
1.5 肽树脂的裂解
量取三氟乙酸至反应器中，冷却至-10～0℃，加入三异丙基硅烷、1,2-乙二硫醇和纯化水(TFA:TIS:EDT＝95:2.5:2.5)，启动搅拌，混合均匀。缓慢加入肽树脂，升温至20～30℃，裂解反应115～125min。反应结束后滤除树脂，滤除的树脂用M×8×20％ml的TFA洗涤，滤除液和洗涤液全部转移到M×8×1.2×4ml的乙醚中，搅拌5～10min，静置沉降15min以上。将沉降悬浊液放入离心机中，离心收集固体；用乙醚洗涤固体6次，每次用不少于5L。将固体于20～35℃、真空干燥6～10h，得粗肽10.90g。
2 SEQ ID NO：1多肽化合物的纯化
采用制备液相色谱进行纯化，色谱条件为：C18柱(100mm×250mm，10μm)；流动相A：0.1％H 3PO 4/水(V/V)，流动相B：0.1％H 3PO 4/乙腈(V/V)；流动相梯度：流动相B 20％～60％，60min；流速为200mL/min检测波长为214nm，收集纯度大于98.0％的馏分，旋蒸浓缩后冷冻干燥得3.10g样品。
多肽化合物SEQ ID NO：2-30的合成方法同实例1：
实施例2，SEQ ID NO：2多肽化合物的合成：
HX 2EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.93g。
实施例3，SEQ ID NO：3多肽化合物的合成：
HX 3EGTFTSDVSSYLEX 3QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品2.87g。
实施例4，SEQ ID NO：4多肽化合物的合成：
HX 3EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品2.55g。
实施例5，SEQ ID NO：5
HX 4EGTFTSDVSSYLEX 4QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品2.60g。
实施例6，SEQ ID NO：6
HX 4EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品2.61g。
实施例7，SEQ ID NO：7
HX 5EGTFTSDVSSYLEX 5QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.1g。
实施例8，SEQ ID NO：8
HX 5EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.35g。
实施例9，SEQ ID NO：9
HX 6EGTFTSDVSSYLEX 6QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品2.70g。
实施例10，SEQ ID NO：10
HX 6EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.90g。
实施例11，SEQ ID NO：11
HX 7EGTFTSDVSSYLEX 7QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.74g。
实施例12，SEQ ID NO：12
HX 7EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.22g。
实施例13，SEQ ID NO：13
HX 8EGTFTSDVSSYLEX 8QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.48g。
实施例14，SEQ ID NO：14
HX 8EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品5.30g。
实施例15，SEQ ID NO：15
HX 9EGTFTSDVSSYLEX 9QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品2.70g。
实施例16，SEQ ID NO：16
HX 9EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.67g。
实施例17，SEQ ID NO：17
HX 10EGTFTSDVSSYLEX 10QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品2.33g。
实施例18，SEQ ID NO：18
HX 10EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.440g。
实施例19，SEQ ID NO：19
HX 11EGTFTSDVSSYLEX 11QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品2.70g。
实施例20，SEQ ID NO：20
HX 11EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.92g。
实施例21，SEQ ID NO：21
HX 12EGTFTSDVSSYLEX 12QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.62g。
实施例22，SEQ ID NO：22
HX 12EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
合成方法同实施例1，收集纯化后的样品溶液冻干得纯品3.80g。
以下是GLP-1激动多肽化合物(下称多肽化合物)的相关药理实验方法以及结果：
1、多肽化合物的GLP-1受体激动活性
胰高血糖素样肽-1受体(GLP-1R)属于B类G蛋白偶联受体，且在血糖额体重条件中发挥重要作用，是公认的重要抗糖尿病药物作用靶点。利用稳定表达人GLP-1R的中国仓鼠卵巢细胞系(CHO)，测定多肽化合物样品及对照化合物对GLP-1R下游cAMP信号的诱导活力。
野生型人源GLP-1R(NM_002062.5)通过瞬时转染，并以600μg/ml的hygromycin-B筛选两周，获得获得重组整合入FlpInCHO(Invitrogen)细胞稳定表达系统的细胞系。FlpInCHO细胞的培养条件为DMEM培养液加入10％热灭活的胎牛血清，于5％二氧化碳细胞培养箱中培养。
下游cAMP信号检测的试验中，将细胞种于6孔细胞培养板中过夜培养后，以每孔8000个细胞的浓度转入384孔板中，在37℃，5％CO 2条件下继续培养24小时。实验采用LANCE cAMP检测试剂盒测定其cAMP信号强度。细胞孵化30min后，使用LANCE cAMP检测试剂盒，使用酶标仪测定荧光读数，建立标准曲线将荧光读数转化为相应的cAMP数值，使用GraphpadPrism 7.0软件的非线性回归计算化合物的EC 50数值。
GLP-1R的cAMP信号分析
Figure PCTCN2021076308-appb-000012
Figure PCTCN2021076308-appb-000013
针对多肽化合物SEQ.ID NO:3和SEQ.ID NO:4进一步开展了动物体内实验，在降糖作用和减重方面开展了详细比对实验，实验方案及结果如下。
2、多肽化合物的降糖及对体重的影响实验
对DB/DB高血糖的小鼠动物适应5-7天后，测定非禁食随机血糖值，禁食12小时后，快速血糖仪测定空腹血糖值。综合考虑体重、12h禁食后空腹血糖、随机血糖结果对动物进行随机分组(随机区组设计)，根据随机体重、随机血糖和空腹血糖分为溶剂对照组、多肽化合物组(选取受试药：SEQ.ID NO:3和SEQ.ID NO:4)、阳性对照Semaglutide组、已知对照组SPN013。各受试药组以及对照组小鼠分别单次皮下注射各受试药或对照组溶液，模型对照组小鼠单次皮下注射PBS缓冲液；实验分组和剂量设置情况详见表1:
表1.实验动物给药前分组情况
Figure PCTCN2021076308-appb-000014
注：n每组小鼠数目；给药容积：根据小鼠体重5μl/g。
实验仪器:快速血糖仪(强生,One Touch UltraEasy,仪器序列号：MGC23B4ER，MGC23B5ER)
实验观察:
临床症状：本实验方案的拟定及任何修改将在上海药物研究所苏研院实验动物伦理委员会(IACUC)进行评估核准后方可实行。每天监测动物的健康状况及死亡情况。
体重：自动物分组后，体重每天固定时间段测量一次
实验指标：
血糖值：取小鼠尾尖血以快速血糖仪及血糖试纸测定血糖值。
分组后，皮下给予不同受试物1次，分别于给药后0h、2h、4h、6h、10h、24h、34h、48h、58h、72h、82h、96h测定血糖值。
受试物的降糖疗效以BG(mmol/L)或AUC(mmol/L·min)评价。
AUC的计算：
AUC＝[(BG 0+BG 2)x2+(BG 2+BG 4)x2+(BG 4+BG 6)x2+(BG 6+BG 10)x4+(BG 10+BG 24)x14+(BG 24+BG 34)x10+(BG 34+BG 48)x14+(BG 48+BG 58)x10+(BG 58+BG 72)x14+(BG 72+BG 82)x10+(BG 82+BG 96)x14]/2。
数据分析：
T检验用于两组间比较。三组或多组间比较用one-way ANOVA。如果F值有显著性差异，应在ANOVA分析之后再进行多重比较。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。
如表2－4及图1-2所示，降血糖实验数据及图表的结果表明，本发明的多肽化合物给药浓度为30nmol/kg时，降血糖效果与Semaglutide的降糖效果几乎一致；阳性对照组和多肽化合物组均与溶剂对照组均有显著差异。如表5－6及图3-4结果显示，溶剂对照组单次皮下注射给药后对db/db小鼠的随机体重及其变化量没有明显影响。阳性对照组和多肽化合物组均与溶剂对照组均有显著差异；尤其给药24小时后差异最显著。同时实验结果显示多肽化合物SEQ.ID NO:3、SEQ.ID NO:4、SPN013在单次皮下注射给药后24h、48h、72h可显著降低db/db小鼠的随机体重变化量，优于Semaglutide组。
因此，GLP-1激动化合物单次皮下注射给药可显著降低2型糖尿病db/db小鼠的随机血糖，其中 SEQ.ID NO:3、SEQ.ID NO:4与等剂量Semaglutide的作用相当。其中SEQ.ID NO:3、SEQ.ID NO:4多肽化合物表现出了更好的体重控制效果。
3、多肽化合物的药代动力学实验
索马鲁肽、SPN009(SEQ.ID NO:3)和SPN010(SEQ.ID NO:4)药代动力学比较实验：取9只雄性大鼠，每组3只，分别给予索马鲁肽、SPN009和SPN010。单次皮下注射，给药剂量0.02mg/kg；剪尾采血，采血时间点为：0h、15min、30min、1h、2h、3h、4h、6h、8h、24h、48h、72h。采用LC-MS/MS法测定血浆中化合物浓度，所有数据由Unifi 1.9.3软件采集和导出，采用Excel软件进行数据计算。采用DAS 3.0软件的非室模型计算大鼠给药后的药动学参数。结果见表7。
如表7所示，大鼠单次皮下给药，给药浓度为0.02mg/kg时，SEQ ID NO:3和SEQ ID NO:4的主要药代动力学参数t 1/2和Tmax，综合优于索玛鲁肽。
Figure PCTCN2021076308-appb-000015
Figure PCTCN2021076308-appb-000016
Figure PCTCN2021076308-appb-000017

Claims (10)

  1. GLP-1激动多肽化合物，其特征在于，该类多肽化合物的氨基酸序列为：
    H Xaa 1EGTFTSDVSSYLE Xaa 2QAA Xaa 3EFIAWLVRGRG，且该类多肽化合物C末端氨基酸作为羧基或羧基被酰胺化；
    其中：
    Xaa 1取自：
    Figure PCTCN2021076308-appb-100001
    Xaa 2取自：G，
    Figure PCTCN2021076308-appb-100002
    Xaa 1、Xaa 2相同或不同；
    R 1为：含有2-6个碳原子的直链或支链烷基，
    R 2为：H或CH 3,
    X为：O,S或N-CH 3,
    式中R 1和R 2烷基可任选地被1-6个卤素原子取代；
    Xaa 3取自：
    Figure PCTCN2021076308-appb-100003
    m、n为自然数。
  2. 根据权利要求1所述的GLP-1激动多肽化合物，其特征在于：所述的n为自然数从12到20；m为自然数0到3。
  3. 根据权利要求1或2所述的GLP-1激动多肽化合物，其特征在于：该类多肽化合物的氨基酸序列选自：
    (1)SEQ ID NO：1
    HX 2EGTFTSDVSSYLEX 2QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (2)SEQ ID NO：2
    HX 2EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (3)SEQ ID NO：3
    HX 3EGTFTSDVSSYLEX 3QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (4)SEQ ID NO：4
    HX 3EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (5)SEQ ID NO：5
    HX 4EGTFTSDVSSYLEX 4QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (6)SEQ ID NO：6
    HX 4EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (7)SEQ ID NO：7
    HX 5EGTFTSDVSSYLEX 5QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (8)SEQ ID NO：8
    HX 5EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (9)SEQ ID NO：9
    HX 6EGTFTSDVSSYLEX 6QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (10)SEQ ID NO：10
    HX 6EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (11)SEQ ID NO：11
    HX 7EGTFTSDVSSYLEX 7QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (12)SEQ ID NO：12
    HX 7EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (13)SEQ ID NO：13
    HX 8EGTFTSDVSSYLEX 8QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (14)SEQ ID NO：14
    HX 8EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (15)SEQ ID NO：15
    HX 9EGTFTSDVSSYLEX 9QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (16)SEQ ID NO：16
    HX 9EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (17)SEQ ID NO：17
    HX 10EGTFTSDVSSYLEX 10QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (18)SEQ ID NO：18
    HX 10EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (19)SEQ ID NO：19
    HX 11EGTFTSDVSSYLEX 11QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (20)SEQ ID NO：20
    HX 11EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (21)SEQ ID NO：21
    HX 12EGTFTSDVSSYLEX 12QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    (22)SEQ ID NO：22
    HX 12EGTFTSDVSSYLEX 1QAAK(HOOC-(CH 2) 16-CO-γ-Glu-AEEA-AEEA)EFIAWLVRGRG
    式中：
    X 1＝G；X 2＝
    Figure PCTCN2021076308-appb-100004
    X3＝
    Figure PCTCN2021076308-appb-100005
    X 4＝
    Figure PCTCN2021076308-appb-100006
    X 5＝
    Figure PCTCN2021076308-appb-100007
    X 6＝
    Figure PCTCN2021076308-appb-100008
    X 7＝
    Figure PCTCN2021076308-appb-100009
    X 8＝
    Figure PCTCN2021076308-appb-100010
    X 9＝
    Figure PCTCN2021076308-appb-100011
    X 10＝
    Figure PCTCN2021076308-appb-100012
    X 11＝
    Figure PCTCN2021076308-appb-100013
    X 12＝
    Figure PCTCN2021076308-appb-100014
  4. 权利要求1或2或3所述的一类GLP-1激动多肽化合物的合成方法，其特征在于：
    (1)固相合成化合物多肽的主序列第1-31位氨基酸，其中第1-4位氨基酸采用片段Boc-His(Trt)Xaa 1Glu(OtBu)Gly-OH，其中：
    其中：
    Xaa 1取自：
    Figure PCTCN2021076308-appb-100015
    R 1为：含有2-6个碳原子的直链或支链烷基，
    R 2为：H或CH 3,
    X为：O,S或N-CH 3,
    式中R 1和R 2烷基可任选地被1-6个卤素原子取代；
    (2)第20位Lys采用片段Fmoc-Lys(Xaa 3)；片段结构式如下：
    Figure PCTCN2021076308-appb-100016
    所述的n为自然数从12到20；m为自然数0到3；
    (3)所用的缩合剂为DIC/HOBt，Oxymapure/DIC，HBTU/HOBT/DIEA，PyBop/HOBT/DIEA其中的一种或多种；所用的反应溶剂为DCM、DMF、NMP、DMSO中的一种或多种组合；所使用的Fmoc脱除试剂为v/v25％哌啶/DMF溶液；
    (4)包括以下步骤：
    步骤一，制备Fmoc-Gly 31-Wang树脂；
    步骤二，制备全保护肽树脂；
    步骤a，将Fmoc-Gly 31-Wang树脂加入到固相反应器中；
    步骤b，固相合成方法逐一将Fmoc-Arg 30(Pbf)-OH、Fmoc-Gly 29-OH、Fmoc-Arg 28(Pbf)-OH、Fmoc-Val 27-OH、Fmoc-Leu 26-OH、Fmoc-Trp 25(Boc)-OH、Fmoc-Ala 24-OH、Fmoc-Ile 23-OH、Fmoc-Phe 22-OH、Fmoc-Glu 21(OtBu)-OH、Fmoc-Lys 20(Xaa 3)-OH、Fmoc-Ala 19-OH、Fmoc-Ala 18-OH、Fmoc-Gln 17(Trt)-OH、Fmoc-Gly 16-OH、Fmoc-Glu 15(OtBu)-OH、Fmoc-Leu 14-OH、Fmoc-Tyr 13(tBu)-OH、Fmoc-Ser 12(tBu)-OH、Boc-Ser 11-OH、Fmoc-Val 10-OH、Fmoc-Asp 9(OtBu)-OH、Fmoc-Ser 8(tBu)-OH、Fmoc-Thr 7(tBu)-OH、Fmoc-Phe 6-OH、Fmoc-Thr 5(tBu)-OH、Boc-His 1(Trt)Xaa 1 Glu 3(OtBu)Gly 4-OH偶联到Fmoc-Gly 31-Wang上；
    所述Xaa 1取自：
    Figure PCTCN2021076308-appb-100017
    R 1为：含有2-6个碳原子的直链或支链烷基，
    R 2为：H或CH 3,
    X为：O,S或N-CH 3,
    式中R 1和R 2烷基可任选地被1-6个卤素原子取代；
    所述片段Fmoc-Lys 20(Xaa 3)-OH结构式如下：
    Figure PCTCN2021076308-appb-100018
    所述的n为自然数从12到20；m为自然数0到3；
    步骤三，将所述肽树脂进行裂解，得到粗肽，
    裂解试剂采用复合试剂，配比为：TFA:(苯甲硫醚/TIS/EDT/苯酚/水)＝90:5，其中捕获剂为苯甲硫醚/TIS/EDT/水中的任意组合。
  5. GLP-1激动多肽化合物药学上可接受的盐，其特征在于：所述的GLP-1激动多肽化合物为权利要求1或2或3所述的多肽化合物，且该多肽化合物C末端氨基酸作为C末端伯酰胺或其药学上可接受的盐被酰胺化。
  6. 根据权利要求5所述的GLP-1激动多肽化合物药学上可接受的盐，其特征在于：所述的盐为GLP-1激动多肽化合物与下述化合物中的一种所形成的盐：盐酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸、硝酸、甲磺酸、乙磺酸、苯磺酸、对甲苯磺酸、甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、肥酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或者硫氰酸。
  7. 权利要求1－3项中任意一项所述的GLP-1激动多肽化合物所制备的药剂，其特征在于，该药剂以GLP-1激动多肽化合物为有效成份，药剂剂型是任何一种药剂学上所说的片剂、胶囊、酏剂、糖浆、锭剂、吸入剂、喷雾剂、注射剂、膜剂、贴剂、散剂、颗粒剂、块剂、乳剂、栓剂或者复方制剂。
  8. GLP-1激动多肽化合物的药物组合物，其特征在于，该药物组合物以权利要求1－3中任何一项所述的GLP-1激动多肽化合物为有效原料，或者以权利要求4或5所述的GLP-1激动多肽化合物药学上可接受的盐为有效原料，再加上药学上可接受的载体或稀释剂组成。
  9. 权利要求1－3中任意一项所述的GLP-1激动多肽化合物或者权利要求4所述制备方法所制得的GLP-1激动多肽化合物的用途，其特征在于，所述的用途为GLP-1激动多肽化合物作为有效原料在制备用于治疗或者预防糖尿病的药物中的用途，或者用于制备减肥药物中的用途。
  10. 权利要求5或6所述的GLP-1激动多肽化合物药学上可接受的盐或者权利要求7所述的药剂或者权利要求8所述的药物组合物的用途，其特征在于，所述的用途多肽化合物药学上可接受的盐或者药剂或者药物组合物作为有效原料在制备用于治疗或者预防糖尿病的药物中的用途，或者用于制备减肥药物中的用途。
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