WO2024098718A1 - Novel long-acting polypeptide compound, composition, and use thereof - Google Patents

Novel long-acting polypeptide compound, composition, and use thereof Download PDF

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WO2024098718A1
WO2024098718A1 PCT/CN2023/096029 CN2023096029W WO2024098718A1 WO 2024098718 A1 WO2024098718 A1 WO 2024098718A1 CN 2023096029 W CN2023096029 W CN 2023096029W WO 2024098718 A1 WO2024098718 A1 WO 2024098718A1
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compound
iva
aib
γglu
egtftsdysi
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PCT/CN2023/096029
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French (fr)
Chinese (zh)
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胡菲菲
李春艳
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内蒙古博睿精创科技有限公司
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  • the present invention belongs to the field of biochemical technology. Specifically, the present invention relates to a novel long-acting polypeptide compound, composition and application thereof that can be used to treat or prevent diabetes or obesity.
  • Diabetes is a group of clinical syndromes caused by the interaction of genetic and environmental factors, in which the body is in a state of high blood sugar levels for a long time. Diabetes is manifested in absolute or relative insufficiency of insulin secretion and decreased sensitivity of target tissue cells to insulin, accompanied by a series of metabolic disorders such as sugar, protein, fat, water and electrolytes.
  • ADA American Diabetes Association
  • type I diabetes accounts for 5-10% of all diabetic patients
  • type II diabetes accounts for about 90-95% of all diabetic patients.
  • Type I diabetes is caused by autoimmune ⁇ -cell destruction, which usually leads to absolute insulin deficiency, including latent autoimmune diabetes in adults (LADA).
  • Type II diabetes patients gradually lose insulin secretion from ⁇ cells on the basis of insulin resistance. It was once called “non-insulin-dependent diabetes” or "adult-onset diabetes”. Its significant pathophysiological characteristics are a decrease in insulin's ability to regulate glucose metabolism (insulin resistance) accompanied by a decrease in insulin secretion due to defective pancreatic ⁇ cell function.
  • insulin resistance is mainly caused by obesity, dyslipidemia, and an unhealthy lifestyle, resulting in a relative lack of insulin secretion (Report of a WHO Consultation. 1999).
  • GLP-1 is a pancreatic hormone secreted by intestinal L cells, which has pharmacological effects such as promoting insulin secretion, inhibiting the release of glucagon, stimulating pancreatic B cell regeneration, improving insulin sensitivity and increasing glucose utilization (Modern Medicine and Clinic, 2020.).
  • GLP-1 and its related receptors have been clinically studied as important targets for the treatment of type 2 diabetes, and have shown strong and broad application prospects in the field of diabetes treatment (GLP-1 receptor Expert guidance on the clinical application of agonists. Chinese Journal of Diabetes, 2018, 26(05): 353-361.).
  • GLP-1RA GLP-1 receptor agonists
  • multi-target agonists have been launched on the market. From the initial twice-daily administration to the recent once-a-week administration, the clinical practice has lasted for more than 10 years, and a large amount of clinical evidence has been accumulated for hypoglycemic, cardiovascular benefits and weight loss. With the launch of weekly preparations, it has greatly facilitated patients. At the same time, it has a good effect on the treatment of cardiovascular diseases, which has also led to the recognition of this type of drug in recent years.
  • Ultra-long-acting peptide drug molecule modification technology is the key to research and development in this field, and it is also a bottleneck to be broken through internationally. Therefore, how to further design and improve the hypoglycemic effect of compounds, reduce toxic side effects, and increase the duration of drug effect and/or half-life is still an important technical problem to be solved by technicians in this field, which has great social and clinical needs!
  • the object of the present invention is to provide a new type of long-acting polypeptide compound.
  • Another object of the present invention is to provide a composition containing the above-mentioned long-acting polypeptide compound.
  • Another object of the present invention is to provide the application of the long-acting polypeptide compound.
  • amino acid sequence of the long-acting polypeptide compound is as follows:
  • X3 is selected from E, Q or N; X27 is selected from L or I; X28 is selected from A or D; X35 is selected from A or Aib; X20 is K, K( Gx (SG) Z - ⁇ Glu-CO ( CH2 ) aCO2H ), K(( PEG2 ) b - ⁇ Glu-CO( CH2 ) cCO2H ) or K((AEEA) d - ⁇ Glu-CO( CH2 ) eCO2H ), wherein x is an integer of 0-5 , z is an integer of 1-5, a is an integer of 12-20, b is an integer of 1-8, c is an integer of 12-20, d is an integer of 1-8, and e is an integer of 12-20; R1 is selected from OH or NH2 ;
  • X2 is selected from Aib, Iva or Cba:
  • X1 is selected from H or Y;
  • X13 is selected from Aib, Iva or Cba;
  • X2 is D-Ser: X1 is H; X13 is selected from Aib, Iva or Cba.
  • X20 is selected from K( Gx (SG) Z - ⁇ Glu-CO( CH2 ) aCO2H ) , K(( PEG2 ) b - ⁇ Glu-CO( CH2 ) cCO2H ) or K((AEEA) d - ⁇ Glu-CO( CH2 ) eCO2H )
  • Gx (SG) Z - ⁇ Glu-CO( CH2 ) aCO2H, ( PEG2 ) b - ⁇ Glu-CO( CH2 ) cCO2H or (AEEA) d - ⁇ Glu-CO( CH2 ) eCO2H is the "sidearm" structure of the long-acting polypeptide compound described in the present application, and the "sidearm" structure is connected to the main peptide chain of the long-acting polypeptide compound by forming an amide bond with the side chain amino group of amino acid K on the main peptide chain of the long-acting polypeptide compound.
  • the 9th amino acid D and the 16th amino acid K on the long-acting polypeptide compound are connected via an amide bond.
  • X20 is K(G x (SG) Z - ⁇ Glu-CO(CH 2 ) a CO 2 H), wherein x is 2, z is 2 or 3, and a is 16 or 18.
  • X20 is K((PEG 2 ) b - ⁇ Glu-CO(CH 2 ) c CO 2 H), wherein b is 2, and c is 16 or 18.
  • X20 is K((AEEA) d - ⁇ Glu-CO(CH 2 ) e CO 2 H), wherein d is 2, and e is 16 or 18.
  • X35 is A.
  • X1 is Y
  • X2 is selected from Aib, Iva or Cba
  • X3 is E
  • X13 is selected from Aib, Iva or Cba.
  • X2 is Iva
  • X13 is Aib or Iva
  • X2 is Aib
  • X13 is Iva
  • X1 is H
  • X2 is selected from Aib, Iva or Cba
  • X3 is selected from E or Q
  • X13 is selected from Aib, Iva or Cba.
  • X2 is Aib or Iva
  • X13 is Iva
  • X2 is Aib
  • X1 is Y
  • X3 is E
  • X13 is selected from Iva or Cba
  • X27 is selected from L or I
  • X28 is selected from A or D
  • X35 is A.
  • X2 is selected from Iva or Cba, and X1 is Y; X3 is E; and X13 is selected from Aib, Iva or Cba; X27 is selected from L or I; X28 is selected from A or D; X35 is A.
  • X2 is selected from Aib, Iva or Cba, and X1 is H; X3 is selected from E or Q; X13 is selected from Aib, Iva or Cba; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
  • X2 is Aib, and X1 is Y; X3 is E; X13 is Iva; X20 is K( Gx (SG) Z - ⁇ Glu-CO( CH2 ) aCO2H ) , wherein x is 2, z is 2 or 3, and a is 16 or 18; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
  • X2 is Iva
  • X1 is Y
  • X3 is E
  • X13 is selected from Aib or Iva
  • X20 is K( Gx (SG) Z - ⁇ Glu-CO( CH2 ) aCO2H ), wherein x is 2, z is 2 or 3, and a is 16 or 18
  • X27 is selected from L or I
  • X28 is selected from A or D
  • X35 is A.
  • X2 is selected from Aib or Iva, and X1 is H; X3 is selected from E or Q; X13 is Iva; X20 is K( Gx (SG) Z - ⁇ Glu-CO ( CH2 ) aCO2H ), wherein x is 2, z is 2 or 3, and a is 16 or 18; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
  • the long-acting polypeptide compound is selected from any one of the following compounds:
  • Y-Iva-EGTFTS D
  • YSI-Iva-LD K
  • IAQKAFVQWLIAGGPSSGAPPPS-OH IAQKAFVQWLIAGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • Y-Aib-EGTFTS D
  • YSI-Iva-LD K
  • IAQKAFVQWLLDGGPSSGAPPPS-OH IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • Y-Iva-EGTFTS D
  • YSI-Iva-LD K
  • IAQKAFVQWLLDGGPSSGAPPPS-OH IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • H-Aib-QGTFTS D
  • YSI-Aib-LD K
  • IAQKAFVQWLLDGGPSSGAPPPS-OH IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • H-Iva-EGTFTS D
  • YSI-Aib-LD K
  • IAQKAFVQWLLDGGPSSGAPPPS-OH IAQKAFVQWLLDGGPSSGAPPPS-OH, in which the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
  • Y-Iva-EGTFTS D
  • YSI-Iva-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • Y-Aib-EGTFTS D
  • YSI-Iva-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • Y-Iva-EGTFTS D
  • YSI-Iva-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • H-Aib-QGTFTS D
  • YSI-Aib-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • H-Iva-EGTFTS D
  • YSI-Aib-LD K
  • IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH IAQK(GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
  • Compound 60 (SEQ ID NO. 60):
  • the -NH2 at the C-terminus of the compounds of the present invention means that the terminal amino acid is amidated to form a C-terminal primary amide.
  • the -OH at the end is the structure of the terminal amino acid itself.
  • the long-acting polypeptide compound is a pharmaceutically acceptable salt.
  • the preparation method of the above series of long-acting polypeptide compounds comprises the following steps:
  • Step 1 According to the Fmoc/t-Bu strategy, the main peptide resin corresponding to the main peptide chain of the polypeptide analog is synthesized.
  • Step 2 Based on the main peptide resin, according to the Fmoc/t-Bu strategy, coupling the corresponding "sidearm" structure to obtain the corresponding polypeptide resin; wherein the "sidearm" structure is PEG 2 -PEG 2 - ⁇ Glu-CO(CH 2 ) 18 CO 2 H, AEEA-AEEA- ⁇ Glu-CO(CH 2 ) 18 CO 2 H, GGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H or GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H.
  • the "sidearm” structure is PEG 2 -PEG 2 - ⁇ Glu-CO(CH 2 ) 18 CO 2 H, AEEA-AEEA- ⁇ Glu-CO(CH 2 ) 18 CO 2 H, GGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H or GGSGSGSG- ⁇ Glu-CO(CH 2 ) 18 CO 2 H.
  • Step 3 adding a cleavage solution to the polypeptide resin to perform a cleavage reaction, removing the polypeptide from the entire protection, and extracting a crude compound; and purifying the crude compound.
  • the coupling agents used are 1-hydroxybenzotriazole (HOBt) and N, N-diisopropylcarbodiimide (DIC), the solvent is N, N-dimethylformamide (DMF), and the Fmoc group is removed with a 20% piperidine/N, N-dimethylformamide solution;
  • the cleavage solution is composed of trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethylenethiol) (DODT), m-cresol and H2O in a volume ratio of 92.5:2.5:2.5:2.5;
  • the crude compound extraction method includes filtration, precipitation and/or methyl tert-butyl ether extraction.
  • the purity of the obtained compounds 31, 35 and 38 is greater than 96%.
  • Another object of the present invention is to provide a composition, which contains a long-acting polypeptide compound and a pharmaceutically acceptable carrier or excipient.
  • a carrier that can reduce drug degradation and loss and reduce side effects such as micelles, microemulsions, gels and other carriers
  • excipients refer to materials added to make the drug into a suitable dosage form, such as buffers, lyophilization excipients, etc., which can make the pharmaceutical composition containing the compound of the present invention into a solution or lyophilized powder for parenteral administration.
  • the lyophilized powder can be added with an appropriate solvent or other pharmaceutically acceptable carrier to reconfigure the powder.
  • the liquid formula is generally a buffer solution, an isotonic solution and an aqueous solution.
  • the buffer solution can be a phosphate buffer solution
  • the isotonic solution can be a 0.9% sodium chloride solution
  • the aqueous solution is a solution obtained by directly dissolving with purified water.
  • the pharmaceutical composition prepared by adding the long-acting polypeptide compound as an active ingredient to a pharmaceutically acceptable carrier and/or excipient is suitable for various modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc.
  • the pharmaceutical composition of the present invention can be prepared into various suitable dosage forms, which contain at least one effective dose of the compound of the present invention and at least one pharmaceutically acceptable Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions that can be used for injection.
  • the dosage of the pharmaceutical composition of the present invention can vary within a wide range, and those skilled in the art can determine it based on objective factors, such as the type of disease, severity of the disease, patient weight, dosage form, route of administration, and the like.
  • Another object of the present invention is to provide applications of long-acting polypeptide compounds and compositions.
  • the present invention obtains a series of long-acting polypeptide compounds, and studies the pharmacodynamic effects of this series of drugs. Studies have shown that the long-acting polypeptide compounds of the present invention have a longer half-life, have insulinotropic activity, have no adverse reactions, can be used to treat diabetes and obesity, and can potentially be used as a new generation of drugs for treating diabetes and obesity.
  • the applications of the long-acting polypeptide compound and composition of the present invention specifically include:
  • the polypeptide compounds involved in the present invention not only have a primary structure, but also have a secondary/tertiary structure that is crucial to the activity of the polypeptide compounds.
  • Various proteins/polypeptides have specific secondary/tertiary spatial conformations, and these specific spatial conformations are related to their specific biological functions, and the structure and function are highly unified; the secondary and tertiary spatial structures have a significant impact on whether the compound can bind to the target, how it binds to the target, and the strength of the binding, that is, the secondary/tertiary structure is crucial for the polypeptide compounds to exert their biological functions.
  • side chains in the present invention greatly affects the secondary and tertiary spatial structures of the polypeptide compounds, so in the present invention, different
  • the addition of side chains causes a great change in the spatial conformation of the main peptide chain of the polypeptide compound involved in the present application, and the change in the polypeptide conformation will affect the performance of its biological function.
  • the biological properties of the polypeptide are unpredictable. Whether it is effective for the long-term effect of a specific polypeptide or protein requires a large number of biopharmaceutical tests and experiments to know.
  • the design and synthesis of the novel long-acting polypeptide compound described in the present invention and the applied long-acting modification technology are only effective for the polypeptide compound described in the present invention or the undefined polypeptide compound, and are unpredictable.
  • the results of the test (Example 6) to further verify the creativity and novelty of the invention show that the long-acting polypeptide compound of the present invention greatly prolongs the half-life, and the drug half-life in rats can reach more than 22 hours, realizing the ultra-long-acting polypeptide drug, and achieving the frequency of drug administration once every 2 weeks or more for human use.
  • the drug half-life of the polypeptide drug in rats currently reported in this field is basically less than 10 hours, and the frequency of drug administration can only be achieved once a week.
  • the -NH 2 at the C-terminus of compound 61 indicates that the terminal amino acid is amidated, and the -OH at the C-terminus of compounds 62-64 is the terminal amino acid structure itself.
  • lixisenatide can bind to the extracellular domain and transmembrane pocket of GLP-1R, and form a suitable steric hindrance with the GLP-1R receptor.
  • the two modified peptides (compounds 63 and 64) have a change in conformation due to the entanglement of the long-acting side chain, thereby forming a large steric hindrance with GLP-1R, hindering ligand-receptor binding, and only showing entanglement with the extracellular domain of GLP-1R, but there is no binding site in the transmembrane pocket, which not only fails to prolong its hypoglycemic effect, but also causes the original parent peptide to lose its hypoglycemic effect.
  • novel long-acting polypeptide compound of the present invention utilizes lipophilic substituents to bind to albumin in the blood, protecting it from enzymatic degradation, thereby increasing the half-life.
  • the helical structure of the molecule is stabilized by an intramolecular bridge, thereby increasing the efficacy and/or selectivity against the target.
  • novel long-acting polypeptide compound of the present invention has high synthesis yield, good stability, is easy to scale up production and has low cost.
  • the novel long-acting polypeptide compound of the present invention has a better pharmacological effect of reducing body weight.
  • the long-acting polypeptide compound can be used to prevent weight gain or promote weight loss by causing a decrease in food intake and/or an increase in energy consumption. Therefore, the novel long-acting polypeptide compound of the present invention can also be used to directly or indirectly treat other diseases caused by or characterized by overweight, such as treating and/or preventing obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, and sleep apnea caused by obesity.
  • the effect of the present invention in these diseases may be due to the direct or indirect effect of the novel long-acting polypeptide compound on body weight, or the effect on other aspects of the body other than body weight.
  • DCM dichloromethane
  • DMF is N, N-dimethylformamide
  • MeOH is methanol
  • Piperidine is piperidine
  • HOBt is 1-hydroxybenzotriazole
  • DIC is N, N'-diisopropylcarbodiimide
  • Fmoc is fluorenylmethoxycarbonyl
  • resin is resin
  • FBS is fetal bovine serum
  • H and His are histidine
  • Y and Tyr are tyrosine
  • E and Glu are glutamic acid
  • Q and Gln are glutamine
  • N and Asn are asparagine
  • G and Gly are glycine
  • T and Th are r is threonine
  • F and Phe are phenylalanine
  • S and Ser are serine
  • D and Asp are aspartic acid
  • I and IIe are isoleucine
  • L and Leu are leucine
  • K and Lys are lysine
  • AEEA 8-amino-3,6-dioxaoctanoic acid
  • TFA trifluoroacetic acid
  • DODT 2,2'-(1,2-ethylenedioxy)bis(ethanediol)
  • ACN is acetonitrile.
  • FIG1 is a graph showing the time-blood glucose results of an OGTT experiment in mice 0.5 h after administration in Example 2;
  • FIG2 is a graph showing the area under the blood glucose curve (AUC) in FIG1 ;
  • FIG3 is a graph showing the time-blood glucose results of an OGTT experiment in mice 1 hour after administration in Example 2;
  • FIG4 is a graph showing the area under the blood glucose curve (AUC) in FIG3 ;
  • FIG5 is a graph showing the time-blood glucose results of an OGTT experiment in mice 24 hours after administration in Example 3;
  • FIG6 is a graph showing the area under the blood glucose curve (AUC) in FIG5 ;
  • FIG. 7 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 for mice 48 hours after administration.
  • FIG8 is a graph showing the area under the blood glucose curve (AUC) in FIG7 ;
  • FIG. 9 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 72 hours after administration.
  • FIG10 is a graph showing the area under the blood glucose curve (AUC) in FIG9 ;
  • FIG. 11 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 96 hours after administration.
  • FIG12 is a graph showing the area under the blood glucose curve (AUC) in FIG11 ;
  • FIG. 13 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 120 hours after administration.
  • FIG14 is a graph showing the area under the blood glucose curve (AUC) in FIG13 ;
  • FIG. 15 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 144 hours after administration.
  • FIG16 is a graph showing the area under the blood glucose curve (AUC) in FIG15 ;
  • FIG. 17 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 168 hours after administration.
  • FIG18 is a graph showing the area under the blood glucose curve (AUC) in FIG17 ;
  • Figure 19 is a statistical diagram of the weight monitoring data of mice in Example 4.
  • Figure 20 is the fasting blood glucose monitoring data statistics of mice in Example 4.
  • FIG21 is a graph showing the time-blood glucose results of an OGTT experiment in mice 24 hours after administration in Example 5;
  • FIG. 22 is a graph showing the area under the blood glucose curve (AUC) in FIG. 21 .
  • Boc-His(Trt)-OH and Fmoc-Aib-OH were purchased from Shanghai Jier, and mono-tert-butyl eicosanedioate was self-made.
  • the remaining amino acids were purchased from Chengdu Zhengyuan Company, and the condensation agent was purchased from Suzhou Haofan Company. Unless otherwise specified, all other reagents were analytically pure, and the solvents were purchased from Shanghai Titan Company.
  • the centrifuge was purchased from Lu Xiangyi.
  • a 5.0 cm reverse phase C 18 preparative column (46 mm x 250 mm) was used to purify the peptide.
  • the high performance liquid chromatograph was a product of Thermo Fisher Scientific. Mass spectrometry was performed using a Waters mass spectrometer.
  • Step 1 Synthesize the main peptide resin corresponding to the main peptide chain
  • the synthesis scale was 0.5 mmol, and the following main peptide resin was synthesized:
  • the main peptide chain peptide resin was obtained: Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin.
  • Coupling Fmoc-Gly-OH Add Fmoc-Gly-OH, HOBt, and an appropriate amount of DMF to the main peptide resin product, stir evenly with nitrogen, add DIC, stir with nitrogen for 2 hours, and use hydrated ninhydrin to detect the coupling effect. If it is colorless and transparent, the reaction is over. Draw off the reaction solution, wash it with N, N-dimethylformamide (DMF) 3 times, remove the Fmoc group with 20% Piperidine/DMF solution (twice 5min + 7min), wash it with DMF 6 times after removing Fmoc, take a sample for ninhydrin detection, and if it is positive, proceed to the subsequent coupling step.
  • DMF N-dimethylformamide
  • Lysis solution TFA, DODT, m-cresol, and H 2 O were prepared in advance at a volume ratio of 92.5:2.5:2.5:2.5 and frozen in a refrigerator for 2 h.
  • the filtrate was collected by filtration, and the resin was washed 3 times with a small amount of lysis solution. After the filtrate was combined, it was concentrated under reduced pressure to about 1/4 of the original volume, and slowly poured into ice methyl tert-butyl ether under stirring, and the residue in the bottle was washed into methyl tert-butyl ether with a small amount of lysis solution. Let it stand for more than 2 hours until the precipitation is complete. The supernatant was removed, the precipitate was centrifuged, washed 3 times with methyl tert-butyl ether, centrifuged, and the solid was blown dry with nitrogen.
  • the HPLC purification was repeated once, starting with 29% ACN/H 2 O (containing 0.1% acetic acid), the column was eluted with a gradient (increasing the proportion of ACN at a rate of 0.33%/min) at a flow rate of 80 mL/min for 60 minutes, and the fractions containing the polypeptide were collected and freeze-dried to obtain 440 mg of refined peptide with a purity of greater than 98.96% and a total yield of 17%.
  • the isolated product peptide was identified by LC-MS, starting with 5% ACN/H 2 O (containing 0.1% formic acid), with a gradient (increasing the proportion of ACN at a rate of 6%/min), a flow rate of 0.4 mL/min, and elution analysis for 15 minutes.
  • the target compound 31 was determined to have a calculated [M+H] + value of 5083.73 and a measured [M+3H] 3+ value of 1695.20.
  • step 1 synthesizes the following main peptide resin different from compound 31:
  • the purification and product confirmation methods of compound 35 were the same as those of compound 31, and 332 mg of refined peptide was obtained with a purity greater than 98.46% and a total yield of 13%.
  • the separated product was identified by LC-MS and confirmed to be the target compound 35, with a calculated value of [M+H] + of 5097.76 and a measured value of [M+3H] 3+ of 1699.90.
  • the synthesis scale was 0.5 mmol and the following main peptide resin was synthesized manually: Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu-Asp(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Ser(tBu)-Rink Amide AM Resin.
  • the purification and product confirmation methods of compound 38 were the same as those of compound 31, and 385 mg of refined peptide was obtained with a purity greater than 98.35% and a total yield of 15%.
  • the separated product was identified by LC-MS and confirmed to be the target compound 38, with a calculated value of [M+H] + of 5128.69 and a measured value of [M+3H] 3+ of 1709.50.
  • mice 8-week-old C57BL/6J male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were used in this experiment, 7 mice/group, and the dosage of all compounds and Semaglutide (injection, purchased from Guangzhou Tonghui Pharmaceutical Co., Ltd.) was 50 ⁇ g/kg.
  • the weight and random blood glucose of mice were measured before the experiment, and the mice were regrouped according to the weight and random blood glucose to ensure that the average weight and random blood glucose of each group were similar.
  • the drugs were prepared on the day of administration, and the corresponding drugs were subcutaneously injected according to the groups.
  • OGTT tests were performed 0.5h and 1h after administration (parallel experiments, one group of mice underwent 0.5h OGTT test, and another group of mice underwent 1h OGTT test).
  • Glucose was administered by gavage at a dose of 2g/kg, and blood was collected from the tail vein to detect blood glucose values at 0, 15, 30, 60, 90 and 120min after gavage.
  • the data were processed using the software GraphPadPrism, and the time-blood glucose curve was plotted to calculate the area under the blood glucose curve AUC.
  • One-way ANOVA analysis was performed with the PBS control group without medication to calculate the significant difference. The test results are shown in Figures 1-4, where * indicates p ⁇ 0.05; ** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001; **** indicates p ⁇ 0.0001.
  • the weight and random blood glucose of mice were measured before the experiment, and the mice were regrouped according to the weight and random blood glucose to ensure that the average weight and random blood glucose of each group were similar.
  • the drugs were prepared on the day of administration and the corresponding drugs were subcutaneously injected according to the group.
  • Glucose OGTT test was performed 24h, 48h, 72h, 96h, 120h, 144h, and 168h after administration. Glucose was gavaged at a dose of 2g/kg, and blood was collected from the tail vein to detect the blood glucose values at 0, 15, 30, 60, 90 and 120min after gavage. The data were processed using the software GraphPadPrism, the time-blood glucose curve was drawn, and the area under the blood glucose curve AUC was calculated. One-way ANOVA analysis was performed with the PBS control group without medication to calculate the significant difference. The test results are shown in Figure 5-18, where * indicates p ⁇ 0.05; ** indicates p ⁇ 0.01; *** indicates p ⁇ 0.001; **** indicates p ⁇ 0.0001.
  • Example 4 Therapeutic effects of compounds 31, 33, 35, 38, 39, 43, 44, 49, 58, 60 and Semaglutide on BKS-db diabetic mice
  • mice 8-week-old BKS-db diabetic mice with the genotype of Lepr KO/KO (purchased from Guangdong Yaokang Biotechnology Co., Ltd.). Blood glucose and body weight were measured, and the mice were randomly divided into each compound group, positive control group (Semaglutide) and model control group (PBS) according to body weight and blood glucose.
  • Semaglutide positive control group
  • PBS model control group
  • Each group of mice was subcutaneously injected with compounds 31, 33, 35, 38, 39, 43, 44, 49, 58, 60, and Semaglutide (injection purchased from Guangzhou Tonghui Pharmaceutical Co., Ltd.) at a dose of 120 ⁇ g/kg, once every other day, and the control group was injected with an equal volume of saline.
  • the experimental period was 4 weeks.
  • Compound 62 synthesized by the present invention is the marketed GLP-1 receptor agonist polypeptide drug lixisenatide
  • compound 63 is a compound obtained by modifying the 12th Lys of lixisenatide with the side chain Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly- ⁇ -Glu-CO(CH 2 ) 18 CO 2 H of the present invention
  • compound 64 is a compound obtained by modifying the 26th Lys of lixisenatide with the side chain Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly- ⁇ -Glu-CO(CH 2 ) 18 CO 2 H of the present invention.
  • OGTT oral glucose tolerance
  • mice of about 8 weeks old Male C57BL/6J mice of about 8 weeks old (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were fed for one week to adapt to the environment and randomly divided into groups according to blood sugar, with 8 mice in each group.
  • Each peptide compound was administered at a dose of 80ug/kg.
  • the drug was administered by subcutaneous injection, and the control group was given an equal volume of PBS.
  • the subjects were fasted for 16 hours on the evening of the administration.
  • the data were processed using the software GraphPadPrism, and the time-blood glucose curve was plotted to calculate the area under the blood glucose curve AUC.
  • One-way ANOVA analysis was performed with the PBS control group that was not administered, and significant differences were calculated
  • Example 6 Pharmacokinetic study of compound 33, compound 35, compound 38, compound 39, compound 43, compound 44, compound 58 and compound 61 in SD rats
  • SD rats (SPF grade, source: Sbefor (Beijing) Biotechnology Co., Ltd., experimental animal production license number: SCXK (Beijing) 2019-0010, weight: 180-200g, age: 6-8 weeks) were raised for one week to adapt to the environment. The general condition of the animals was checked during the adaptation period. Unqualified animals were not included in this experiment.
  • the feeding environment control system used WINCC7.3 EMS series computer room environment monitoring system, and the feed used SPF mice and rats to maintain the feed. 20 qualified SD rats were randomly divided into groups according to their weight, with 4 rats in each group (half male and half female), for a total of 5 groups (animals were grouped and statistically analyzed using software Stata 15).
  • the compound was administered subcutaneously to the skin of the back of the neck at a dose of 0.15 mg/kg or 0.2 mg/kg, a dosing volume of 2 mL/kg, a dosing concentration of 0.075 mg/mL or 0.1 mg/mL, and a solvent of PBS.
  • Blood samples were collected from the jugular vein of rats in the single subcutaneous administration group before administration (0h) and 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, and 192h after administration.
  • About 0.2mL of whole blood was collected at each time point and placed in an EDTA- K2 anticoagulant tube.
  • the blood was centrifuged at 4°C and 1800g for 10min, and the supernatant was taken.
  • the separated plasma was transferred to a -80°C refrigerator for storage.
  • UPLC-MS/MS was used to establish the chemical and molecular weight distribution of plasma in SD rats.
  • the concentration analysis method of the compound was used to determine the drug concentration of the compound in plasma.
  • the data were processed using WinNonlin 8.1 software to calculate the pharmacokinetic parameters. The experimental results are shown in Table 2.

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Abstract

Provided are a novel long-acting polypeptide compound, a composition, and a use thereof. In the long-acting polypeptide compound, the substitution of an amino acid at a key particular position enhances the stability, activity and hydrolysis resistance of a polypeptide. By means of a large number of trials, after amino acid position 2 and/or 3 of the backbone of the polypeptide compound is substituted by an unnatural amino acid AiB, Iva or Cba, the stability, activity and hydrolysis resistance of polypeptide molecules are significantly enhanced, and the respective substitution of the amino acid position 27 and/or 28 by an L and/or D amino acid can also significantly enhance the activity of the polypeptide molecules. The long-acting polypeptide compound has a long half-life period, and has good pharmacodynamic effects of treating diabetes and reducing weight.

Description

一种新型长效多肽化合物、组合物及其应用A novel long-acting polypeptide compound, composition and application thereof 技术领域Technical Field
本发明属于生物化学技术领域，具体而言，本发明涉及一种新型的可用于治疗或预防糖尿病或肥胖的长效多肽化合物、组合物及其应用。The present invention belongs to the field of biochemical technology. Specifically, the present invention relates to a novel long-acting polypeptide compound, composition and application thereof that can be used to treat or prevent diabetes or obesity.
背景技术Background technique
糖尿病是一组由遗传和环境因素相互作用而引起的、机体长时间处于高血糖水平的临床综合征。糖尿病体现在胰岛素分泌绝对或相对不足以及靶组织细胞对胰岛素敏感性降低，并伴有糖、蛋白、脂肪、水和电解质等一系列代谢的紊乱。2021年美国糖尿病协会(ADA)将糖尿病分为四类：I-型糖尿病、II-型糖尿病、妊娠期糖尿病、其他特殊类型糖尿病。其中，I-型糖尿病占所有糖尿病患者的5-10％，II-型糖尿病，II-型糖尿病占所有糖尿病患者的90-95％左右。I-型糖尿病是由于自身免疫性β细胞破坏引起的，通常导致绝对胰岛素缺乏，包括成人隐匿性自身免疫性糖尿病(LADA)。II-型糖尿病患者在胰岛素抵抗的基础上，β细胞胰岛素分泌逐渐丧失，曾被称为“非胰岛素依赖型糖尿病”或“成人发病型糖尿病”，其显著的病理生理学特征为胰岛素调控葡萄糖代谢能力的下降(胰岛素抵抗)伴胰岛β细胞功能缺陷所导致的胰岛素分泌减少；尤其是起病初期，主要是因为肥胖、血脂异常、不良生活方式等原因引起胰岛素抵抗，胰岛素分泌相对不足(Report of a WHO Consultation.1999)。Diabetes is a group of clinical syndromes caused by the interaction of genetic and environmental factors, in which the body is in a state of high blood sugar levels for a long time. Diabetes is manifested in absolute or relative insufficiency of insulin secretion and decreased sensitivity of target tissue cells to insulin, accompanied by a series of metabolic disorders such as sugar, protein, fat, water and electrolytes. In 2021, the American Diabetes Association (ADA) divided diabetes into four categories: type I diabetes, type II diabetes, gestational diabetes, and other special types of diabetes. Among them, type I diabetes accounts for 5-10% of all diabetic patients, and type II diabetes accounts for about 90-95% of all diabetic patients. Type I diabetes is caused by autoimmune β-cell destruction, which usually leads to absolute insulin deficiency, including latent autoimmune diabetes in adults (LADA). Type II diabetes patients gradually lose insulin secretion from β cells on the basis of insulin resistance. It was once called "non-insulin-dependent diabetes" or "adult-onset diabetes". Its significant pathophysiological characteristics are a decrease in insulin's ability to regulate glucose metabolism (insulin resistance) accompanied by a decrease in insulin secretion due to defective pancreatic β cell function. Especially in the early stages of the disease, insulin resistance is mainly caused by obesity, dyslipidemia, and an unhealthy lifestyle, resulting in a relative lack of insulin secretion (Report of a WHO Consultation. 1999).
糖尿病不易治愈，而且其并发症给患者带来了极大的痛苦，通常需要终生用药来控制血糖。在糖尿病的治疗领域，这十几年来的发展非常快，在非胰岛素类药物方面，特别是GLP-1受体激动剂及其类似物被应用于临床，使临床治疗和治疗结局发生了非常大的变化(Frontiers in Endocrinology,2019,10:155)。已有报道：GLP-1是一种由肠道L细胞分泌的促胰素，具有促进胰岛素分泌、抑制胰高血糖素的释放、刺激胰岛B细胞再生、改善胰岛素敏感性和增加葡萄糖的利用等药理学作用(现代药物与临床，2020.)。临床研究表明，II型糖尿病患者，其表现为“肠促胰岛效应”受损，但其促进胰岛素分泌以及降血糖的作用并无明显受损。因此，在治疗糖尿病领域，GLP-1及其相关受体已经作为II型糖尿病治疗的重要靶点进行了临床研究，在糖尿病治疗领域已显示了强大和广阔的应用前景(GLP-1受体 激动剂临床应用专家指导意见.中国糖尿病杂志，2018，26(05)：353-361.)。Diabetes is not easy to cure, and its complications bring great pain to patients, usually requiring lifelong medication to control blood sugar. In the field of diabetes treatment, the development in the past decade has been very rapid. In terms of non-insulin drugs, especially GLP-1 receptor agonists and their analogs have been used in clinical practice, which has greatly changed clinical treatment and treatment outcomes (Frontiers in Endocrinology, 2019, 10: 155). It has been reported that GLP-1 is a pancreatic hormone secreted by intestinal L cells, which has pharmacological effects such as promoting insulin secretion, inhibiting the release of glucagon, stimulating pancreatic B cell regeneration, improving insulin sensitivity and increasing glucose utilization (Modern Medicine and Clinic, 2020.). Clinical studies have shown that patients with type 2 diabetes have impaired "incretin effect", but their effects on promoting insulin secretion and lowering blood sugar are not significantly impaired. Therefore, in the field of diabetes treatment, GLP-1 and its related receptors have been clinically studied as important targets for the treatment of type 2 diabetes, and have shown strong and broad application prospects in the field of diabetes treatment (GLP-1 receptor Expert guidance on the clinical application of agonists. Chinese Journal of Diabetes, 2018, 26(05): 353-361.).
迄今为止，有关GLP-1受体激动剂(GLP-1RA)及其相关的多靶点激动剂已有多个药物上市，从最开始的每天两次给药到最近的一周一次，临床已经超过了10年，对降糖、心血管获益以及减重，都有大量的临床证据积累。随着周制剂的上市，极大的方便了患者。同时，对心血管疾病的治疗有良好的作用，也促使这类药物近几年被大家认可。然而，值得注意的是，目前上市的GLP-1受体及其相关靶点的药物也表现出了相当大的毒副作用及不足，如胃肠道反应剧烈，表现在恶心、呕吐，注射给药频率仍然有大的降低空间等等(The Lancet,2009,374(9683):39-47.)。So far, several drugs related to GLP-1 receptor agonists (GLP-1RA) and related multi-target agonists have been launched on the market. From the initial twice-daily administration to the recent once-a-week administration, the clinical practice has lasted for more than 10 years, and a large amount of clinical evidence has been accumulated for hypoglycemic, cardiovascular benefits and weight loss. With the launch of weekly preparations, it has greatly facilitated patients. At the same time, it has a good effect on the treatment of cardiovascular diseases, which has also led to the recognition of this type of drug in recent years. However, it is worth noting that the currently marketed GLP-1 receptor and related target drugs also show considerable toxic side effects and deficiencies, such as severe gastrointestinal reactions, manifested in nausea and vomiting, and there is still a lot of room for reducing the frequency of injection administration, etc. (The Lancet, 2009, 374 (9683): 39-47.).
超长效多肽药物分子修饰技术是本领域研究发展的关键，在国际上也是待突破的瓶颈。因此，如何进一步设计和提高化合物的降糖效果、降低毒副作用、提高药效持续时间和/或半衰期仍是本领域技术人员要解决的重要技术问题，具有重大的社会和临床需求！Ultra-long-acting peptide drug molecule modification technology is the key to research and development in this field, and it is also a bottleneck to be broken through internationally. Therefore, how to further design and improve the hypoglycemic effect of compounds, reduce toxic side effects, and increase the duration of drug effect and/or half-life is still an important technical problem to be solved by technicians in this field, which has great social and clinical needs!
发明内容Summary of the invention
针对以上问题，本发明的目的在于提供一种新型的长效多肽化合物。In view of the above problems, the object of the present invention is to provide a new type of long-acting polypeptide compound.
本发明的另一目的在于提供一种含有上述长效多肽化合物的组合物。Another object of the present invention is to provide a composition containing the above-mentioned long-acting polypeptide compound.
本发明的再一目的在于提供上述长效多肽化合物的应用。Another object of the present invention is to provide the application of the long-acting polypeptide compound.
根据本发明具体实施方式的长效多肽化合物，其氨基酸序列如下：According to a specific embodiment of the present invention, the amino acid sequence of the long-acting polypeptide compound is as follows:
X1-X2-X3-GTFTSDYSI-X13-LDKIAQ-X20-AFVQWL-X27-X28-GGPSSG-X35-PPPS-R1；X1-X2-X3-GTFTSDYSI-X13-LDKIAQ-X20-AFVQWL-X27-X28-GGPSSG-X35-PPPS-R 1 ;
其中，其中，X3选自E、Q或N；X27选自L或I；X28选自A或D；X35选自A或Aib；X20为K、K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)、K((PEG2)b-γGlu-CO(CH2)cCO2H)或K((AEEA)d-γGlu-CO(CH2)eCO2H)，其中，x为0-5的整数，z为1-5的整数，a为12-20的整数，b为1-8的整数，c为12-20的整数，d为1-8的整数，e为12-20的整数；R1选自OH或NH2；wherein X3 is selected from E, Q or N; X27 is selected from L or I; X28 is selected from A or D; X35 is selected from A or Aib; X20 is K, K( Gx (SG) Z -γGlu-CO ( CH2 ) aCO2H ), K(( PEG2 ) b -γGlu-CO( CH2 ) cCO2H ) or K((AEEA) d -γGlu-CO( CH2 ) eCO2H ), wherein x is an integer of 0-5 , z is an integer of 1-5, a is an integer of 12-20, b is an integer of 1-8, c is an integer of 12-20, d is an integer of 1-8, and e is an integer of 12-20; R1 is selected from OH or NH2 ;
当X2选自Aib、Iva或Cba时：X1选自H或Y；X13选自Aib、Iva或Cba；When X2 is selected from Aib, Iva or Cba: X1 is selected from H or Y; X13 is selected from Aib, Iva or Cba;
当X2为D-Ser时：X1为H；X13选自Aib、Iva或Cba。 When X2 is D-Ser: X1 is H; X13 is selected from Aib, Iva or Cba.
优选地，当X20选自K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)、K((PEG2)b-γGlu-CO(CH2)cCO2H)或K((AEEA)d-γGlu-CO(CH2)eCO2H)时，Gx(SG)Z-γGlu-CO(CH2)aCO2H、(PEG2)b-γGlu-CO(CH2)cCO2H或(AEEA)d-γGlu-CO(CH2)eCO2H为本申请所述长效多肽化合物的“侧臂”结构，“侧臂”结构通过与长效多肽化合物主肽链上的氨基酸K的侧链氨基形成酰胺键与所述长效多肽化合物的主肽链连接。Preferably, when X20 is selected from K( Gx (SG) Z -γGlu-CO( CH2 ) aCO2H ) , K(( PEG2 ) b -γGlu-CO( CH2 ) cCO2H ) or K((AEEA) d -γGlu-CO( CH2 ) eCO2H ) , Gx (SG) Z -γGlu-CO( CH2 ) aCO2H, ( PEG2 ) b -γGlu-CO( CH2 ) cCO2H or (AEEA) d -γGlu-CO( CH2 ) eCO2H is the "sidearm" structure of the long-acting polypeptide compound described in the present application, and the "sidearm" structure is connected to the main peptide chain of the long-acting polypeptide compound by forming an amide bond with the side chain amino group of amino acid K on the main peptide chain of the long-acting polypeptide compound.
优选地，所述的长效多肽化合物上的第9位氨基酸D与第16位氨基酸K通过酰胺键连接。Preferably, the 9th amino acid D and the 16th amino acid K on the long-acting polypeptide compound are connected via an amide bond.
优选地，所述长效多肽化合物，X20为K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)，其中x为2，z为2或3，a为16或18。Preferably, in the long-acting polypeptide compound, X20 is K(G x (SG) Z -γGlu-CO(CH 2 ) a CO 2 H), wherein x is 2, z is 2 or 3, and a is 16 or 18.
优选地，所述长效多肽化合物，X20为K((PEG2)b-γGlu-CO(CH2)cCO2H)，其中b为2，c为16或18。Preferably, in the long-acting polypeptide compound, X20 is K((PEG 2 ) b -γGlu-CO(CH 2 ) c CO 2 H), wherein b is 2, and c is 16 or 18.
优选地，所述长效多肽化合物，X20为K((AEEA)d-γGlu-CO(CH2)eCO2H)，其中d为2，e为16或18。Preferably, in the long-acting polypeptide compound, X20 is K((AEEA) d -γGlu-CO(CH 2 ) e CO 2 H), wherein d is 2, and e is 16 or 18.
优选地，所述长效多肽化合物，X35为A。Preferably, in the long-acting polypeptide compound, X35 is A.
优选地，所述长效多肽化合物，X1为Y，X2选自Aib、Iva或Cba，X3为E，X13选自Aib、Iva或Cba。Preferably, in the long-acting polypeptide compound, X1 is Y, X2 is selected from Aib, Iva or Cba, X3 is E, and X13 is selected from Aib, Iva or Cba.
优选地，所述长效多肽化合物，X2为Iva，X13为Aib或Iva；或者，X2为Aib，X13为Iva。Preferably, in the long-acting polypeptide compound, X2 is Iva, and X13 is Aib or Iva; or, X2 is Aib, and X13 is Iva.
优选地，所述长效多肽化合物，X1为H，X2选自Aib、Iva或Cba，X3选自E或Q；X13选自Aib、Iva或Cba。Preferably, in the long-acting polypeptide compound, X1 is H, X2 is selected from Aib, Iva or Cba, X3 is selected from E or Q; and X13 is selected from Aib, Iva or Cba.
优选地，所述长效多肽化合物，X2为Aib或Iva，X13为Iva。Preferably, in the long-acting polypeptide compound, X2 is Aib or Iva, and X13 is Iva.
优选地，所述长效多肽化合物，X2为Aib，且X1为Y；X3为E；X13选自Iva或Cba；X27选自L或I；X28选自A或D；X35为A。Preferably, in the long-acting polypeptide compound, X2 is Aib, and X1 is Y; X3 is E; X13 is selected from Iva or Cba; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
优选地，所述长效多肽化合物，X2选自Iva或Cba，且X1为Y；X3为E；X13选自 Aib、Iva或Cba；X27选自L或I；X28选自A或D；X35为A。Preferably, in the long-acting polypeptide compound, X2 is selected from Iva or Cba, and X1 is Y; X3 is E; and X13 is selected from Aib, Iva or Cba; X27 is selected from L or I; X28 is selected from A or D; X35 is A.
优选地，所述长效多肽化合物，X2选自Aib、Iva或Cba，且X1为H；X3选自E或Q；X13选自Aib、Iva或Cba；X27选自L或I；X28选自A或D；X35为A。Preferably, in the long-acting polypeptide compound, X2 is selected from Aib, Iva or Cba, and X1 is H; X3 is selected from E or Q; X13 is selected from Aib, Iva or Cba; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
优选地，所述长效多肽化合物，X2为Aib，且X1为Y；X3为E；X13为Iva；X20为K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)，其中x为2，z为2或3，a为16或18；X27选自L或I；X28选自A或D；X35为A。Preferably, in the long-acting polypeptide compound, X2 is Aib, and X1 is Y; X3 is E; X13 is Iva; X20 is K( Gx (SG) Z -γGlu-CO( CH2 ) aCO2H ) , wherein x is 2, z is 2 or 3, and a is 16 or 18; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
优选地，所述长效多肽化合物，X2为Iva，且X1为Y；X3为E；X13选自Aib或Iva；X20为K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)，其中x为2，z为2或3，a为16或18；X27选自L或I；X28选自A或D；X35为A。Preferably, in the long-acting polypeptide compound, X2 is Iva, and X1 is Y; X3 is E; X13 is selected from Aib or Iva; X20 is K( Gx (SG) Z -γGlu-CO( CH2 ) aCO2H ), wherein x is 2, z is 2 or 3, and a is 16 or 18; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
优选地，所述长效多肽化合物，X2选自Aib或Iva，且X1为H；X3选自E或Q；X13为Iva；X20为K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)，其中x为2，z为2或3，a为16或18；X27选自L或I；X28选自A或D；X35为A。Preferably, in the long-acting polypeptide compound, X2 is selected from Aib or Iva, and X1 is H; X3 is selected from E or Q; X13 is Iva; X20 is K( Gx (SG) Z -γGlu-CO ( CH2 ) aCO2H ), wherein x is 2, z is 2 or 3, and a is 16 or 18; X27 is selected from L or I; X28 is selected from A or D; and X35 is A.
优选地，所述长效多肽化合物，所述长效多肽化合物选自如下化合物中的任一种：Preferably, the long-acting polypeptide compound is selected from any one of the following compounds:
化合物1(SEQ ID NO.1)：Compound 1 (SEQ ID NO.1):
Y-Aib-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSG-Aib-PPPS-OH；Y-Aib-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSG-Aib-PPPS-OH;
化合物2(SEQ ID NO.2)：Compound 2 (SEQ ID NO. 2):
Y-Aib-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Aib-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物3(SEQ ID NO.3)：Compound 3 (SEQ ID NO. 3):
Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物4(SEQ ID NO.4)：Compound 4 (SEQ ID NO.4):
Y-Iva-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH;
化合物5(SEQ ID NO.5)：Compound 5 (SEQ ID NO.5):
Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH；Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH;
化合物6(SEQ ID NO.6)：Compound 6 (SEQ ID NO.6):
Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH； Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH;
化合物7(SEQ ID NO.7)：Compound 7 (SEQ ID NO.7):
Y-Iva-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物8(SEQ ID NO.8)：Compound 8 (SEQ ID NO.8):
Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物9(SEQ ID NO.9)：Compound 9 (SEQ ID NO.9):
Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物10(SEQ ID NO.10)：Compound 10 (SEQ ID NO. 10):
Y-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物11(SEQ ID NO.11)：Compound 11 (SEQ ID NO.11):
Y-Cba-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Cba-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物12(SEQ ID NO.12)：Compound 12 (SEQ ID NO.12):
Y-Cba-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Cba-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物13(SEQ ID NO.13)：Compound 13 (SEQ ID NO.13):
Y-Aib-EGTFTSDYSI-Cba-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Aib-EGTFTSDYSI-Cba-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物14(SEQ ID NO.14)：Compound 14 (SEQ ID NO. 14):
Y-Iva-EGTFTSDYSI-Cba-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Cba-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物15(SEQ ID NO.15)：Compound 15 (SEQ ID NO.15):
H-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH；H-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH;
化合物16(SEQ ID NO.16)：Compound 16 (SEQ ID NO. 16):
H-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH；H-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH;
化合物17(SEQ ID NO.17)：Compound 17 (SEQ ID NO. 17):
H-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH;
化合物18(SEQ ID NO.18)：Compound 18 (SEQ ID NO.18):
H-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH； H-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物19(SEQ ID NO.19)：Compound 19 (SEQ ID NO. 19):
H-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物20(SEQ ID NO.20)：Compound 20 (SEQ ID NO. 20):
H-Cba-QGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH；H-Cba-QGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH;
化合物21(SEQ ID NO.21)：Compound 21 (SEQ ID NO. 21):
H-Cba-QGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；H-Cba-QGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物22(SEQ ID NO.22)：Compound 22 (SEQ ID NO. 22):
H-(D-Ser)-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；H-(D-Ser)-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
化合物23(SEQ ID NO.23)：Compound 23 (SEQ ID NO. 23):
Y-Iva-EGTFTS(D)YSI-Iva-LD(K)IAQKAFVQWLIAGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Iva-EGTFTS (D) YSI-Iva-LD (K) IAQKAFVQWLIAGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
化合物24(SEQ ID NO.24)：Compound 24 (SEQ ID NO. 24):
Y-Aib-EGTFTS(D)YSI-Iva-LD(K)IAQKAFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Aib-EGTFTS (D) YSI-Iva-LD (K) IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
化合物25(SEQ ID NO.25)：Compound 25 (SEQ ID NO. 25):
Y-Iva-EGTFTS(D)YSI-Iva-LD(K)IAQKAFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Iva-EGTFTS (D) YSI-Iva-LD (K) IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
化合物26(SEQ ID NO.26)：Compound 26 (SEQ ID NO. 26):
H-Aib-QGTFTS(D)YSI-Aib-LD(K)IAQKAFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；H-Aib-QGTFTS (D) YSI-Aib-LD (K) IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
化合物27(SEQ ID NO.27)：Compound 27 (SEQ ID NO. 27):
H-Iva-EGTFTS(D)YSI-Aib-LD(K)IAQKAFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；H-Iva-EGTFTS (D) YSI-Aib-LD (K) IAQKAFVQWLLDGGPSSGAPPPS-OH, in which the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
化合物28(SEQ ID NO.28)： Compound 28 (SEQ ID NO.28):
Y-Aib-QGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSG-Aib-PPPS-NH2；Y-Aib-QGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSG-Aib-PPPS-NH 2 ;
化合物29(SEQ ID NO.29)：Compound 29 (SEQ ID NO. 29):
Y-Aib-QGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Aib-QGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物30(SEQ ID NO.30)：Compound 30 (SEQ ID NO. 30):
Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物31(SEQ ID NO.31)：Compound 31 (SEQ ID NO.31):
Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
化合物32(SEQ ID NO.32)：Compound 32 (SEQ ID NO.32):
Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(PEG2-PEG2-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
化合物33(SEQ ID NO.33)：Compound 33 (SEQ ID NO. 33):
Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
化合物34(SEQ ID NO.34)：Compound 34 (SEQ ID NO.34):
Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(PEG2-PEG2-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
化合物35(SEQ ID NO.35)：Compound 35 (SEQ ID NO.35):
Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
化合物36(SEQ ID NO.36)： Compound 36 (SEQ ID NO.36):
Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
化合物37(SEQ ID NO.37)：Compound 37 (SEQ ID NO. 37):
Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物38(SEQ ID NO.38)：Compound 38 (SEQ ID NO.38):
Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物39(SEQ ID NO.39)：Compound 39 (SEQ ID NO. 39):
Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物40(SEQ ID NO.40)：Compound 40 (SEQ ID NO.40):
Y-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物41(SEQ ID NO.41)：Compound 41 (SEQ ID NO.41):
Y-Cba-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Cba-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物42(SEQ ID NO.42)：Compound 42 (SEQ ID NO.42):
Y-Cba-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Cba-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物43(SEQ ID NO.43)：Compound 43 (SEQ ID NO.43):
Y-Aib-EGTFTSDYSI-Cba-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Cba-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物44(SEQ ID NO.44)： Compound 44 (SEQ ID NO.44):
Y-Iva-EGTFTSDYSI-Cba-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Cba-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
化合物45(SEQ ID NO.45)：Compound 45 (SEQ ID NO.45):
H-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLAGGPSSGAPPPS-OH；H-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLAGGPSSGAPPPS-OH;
化合物46(SEQ ID NO.46)：Compound 46 (SEQ ID NO.46):
H-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLAGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLAGGPSSGAPPPS-OH;
化合物47(SEQ ID NO.47)：Compound 47 (SEQ ID NO.47):
H-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLAGGPSSGAPPPS-OH；H-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLAGGPSSGAPPPS-OH;
化合物48(SEQ ID NO.48)：Compound 48 (SEQ ID NO.48):
H-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLAGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLAGGPSSGAPPPS-OH;
化合物49(SEQ ID NO.49)：Compound 49 (SEQ ID NO.49):
H-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH；H-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH;
化合物50(SEQ ID NO.50)：Compound 50 (SEQ ID NO.50):
H-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH;
化合物51(SEQ ID NO.51)：Compound 51 (SEQ ID NO.51):
H-Cba-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-OH；H-Cba-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-OH;
化合物52(SEQ ID NO.52)：Compound 52 (SEQ ID NO.52):
H-Cba-QGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH；H-Cba-QGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH;
化合物53(SEQ ID NO.53)： Compound 53 (SEQ ID NO.53):
H-Aib-QGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSG-Aib-PPPS-OH；H-Aib-QGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSG-Aib-PPPS-OH;
化合物54(SEQ ID NO.54)：Compound 54 (SEQ ID NO.54):
H-(D-Ser)-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH；H-(D-Ser)-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH;
化合物55(SEQ ID NO.55)：Compound 55 (SEQ ID NO.55):
Y-Iva-EGTFTS(D)YSI-Iva-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Iva-EGTFTS (D) YSI-Iva-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 , wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
化合物56(SEQ ID NO.56)：Compound 56 (SEQ ID NO.56):
Y-Aib-EGTFTS(D)YSI-Iva-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Aib-EGTFTS (D) YSI-Iva-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 , wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
化合物57(SEQ ID NO.57)：Compound 57 (SEQ ID NO.57):
Y-Iva-EGTFTS(D)YSI-Iva-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Iva-EGTFTS (D) YSI-Iva-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 , wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
化合物58(SEQ ID NO.58)：Compound 58 (SEQ ID NO.58):
H-Aib-QGTFTS(D)YSI-Aib-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；H-Aib-QGTFTS (D) YSI-Aib-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
化合物59(SEQ ID NO.59)：Compound 59 (SEQ ID NO.59):
H-Iva-EGTFTS(D)YSI-Aib-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；H-Iva-EGTFTS (D) YSI-Aib-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
化合物60(SEQ ID NO.60)：Compound 60 (SEQ ID NO. 60):
Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2。Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 .
本发明化合物中C末端的-NH2指末端氨基酸被酰胺化为C末端伯酰胺，化合物中C末 端的-OH为末端氨基酸自身的结构。The -NH2 at the C-terminus of the compounds of the present invention means that the terminal amino acid is amidated to form a C-terminal primary amide. The -OH at the end is the structure of the terminal amino acid itself.
优选地，所述长效多肽化合物为药学上可接受的盐。Preferably, the long-acting polypeptide compound is a pharmaceutically acceptable salt.
上述系列长效多肽化合物的制备方法，包括以下步骤：The preparation method of the above series of long-acting polypeptide compounds comprises the following steps:
步骤1：按照Fmoc/t-Bu策略，合成所述多肽类似物主肽链对应的主肽树脂。Step 1: According to the Fmoc/t-Bu strategy, the main peptide resin corresponding to the main peptide chain of the polypeptide analog is synthesized.
步骤2：在所述主肽树脂的基础上，按照Fmoc/t-Bu策略，偶联对应的“侧臂”结构，得到对应的多肽树脂；其中，所述“侧臂”结构为PEG2-PEG2-γGlu-CO(CH2)18CO2H、AEEA-AEEA-γGlu-CO(CH2)18CO2H、GGSGSG-γGlu-CO(CH2)18CO2H或GGSGSGSG-γGlu-CO(CH2)18CO2H。Step 2: Based on the main peptide resin, according to the Fmoc/t-Bu strategy, coupling the corresponding "sidearm" structure to obtain the corresponding polypeptide resin; wherein the "sidearm" structure is PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H, AEEA-AEEA-γGlu-CO(CH 2 ) 18 CO 2 H, GGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H or GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H.
步骤3：向所述多肽树脂中加入裂解液，进行裂解反应，脱除多肽全保护，提取粗品化合物；纯化所述粗品化合物。Step 3: adding a cleavage solution to the polypeptide resin to perform a cleavage reaction, removing the polypeptide from the entire protection, and extracting a crude compound; and purifying the crude compound.
根据本发明具体实施方式的化合物31、35、38的制备方法，步骤2中，使用的偶联剂为1-羟基苯并三唑(HOBt)和N，N-二异丙基碳二亚胺(DIC)，溶剂为N，N-二甲基甲酰胺(DMF)，以20％的哌啶(Piperidine)/N，N-二甲基甲酰胺溶液脱除Fmoc基团；步骤3中，裂解液由三氟乙酸(TFA)、2，2'-(1，2-乙二基双氧代)双乙硫醇(DODT)、间甲酚、H2O按体积比为92.5：2.5：2.5：2.5的比例组成；所述粗品化合物提取方式包括过滤、沉淀和/或甲基叔丁基醚提取。获得的化合物31、35、38纯度大于96％。According to the preparation method of compounds 31, 35 and 38 of the specific embodiment of the present invention, in step 2, the coupling agents used are 1-hydroxybenzotriazole (HOBt) and N, N-diisopropylcarbodiimide (DIC), the solvent is N, N-dimethylformamide (DMF), and the Fmoc group is removed with a 20% piperidine/N, N-dimethylformamide solution; in step 3, the cleavage solution is composed of trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethylenethiol) (DODT), m-cresol and H2O in a volume ratio of 92.5:2.5:2.5:2.5; the crude compound extraction method includes filtration, precipitation and/or methyl tert-butyl ether extraction. The purity of the obtained compounds 31, 35 and 38 is greater than 96%.
本发明的再一目的在于提供一种组合物，其包含长效多肽化合物，还包含药学上可接受的载体或辅料。例如，能够减少药物降解及损失、降低副作用的载体，例如胶束、微乳液、凝胶等载体；辅料指为使药物制成适宜的剂型而加入的物料，例如缓冲剂、冻干用赋形剂等，能够将含有本发明化合物的药物组合物制成溶液或者冻干粉末以用于胃肠外给药，冻干粉末在使用前可加入适当溶剂或其他可药用的载体将粉末重新配置，液体配方一般是缓冲液、等渗溶液和水溶液。缓冲液可以是磷酸盐缓冲液，等渗溶液可以是0.9％的氯化钠溶液，水溶液即直接用纯净水溶解获得的溶液。Another object of the present invention is to provide a composition, which contains a long-acting polypeptide compound and a pharmaceutically acceptable carrier or excipient. For example, a carrier that can reduce drug degradation and loss and reduce side effects, such as micelles, microemulsions, gels and other carriers; excipients refer to materials added to make the drug into a suitable dosage form, such as buffers, lyophilization excipients, etc., which can make the pharmaceutical composition containing the compound of the present invention into a solution or lyophilized powder for parenteral administration. Before use, the lyophilized powder can be added with an appropriate solvent or other pharmaceutically acceptable carrier to reconfigure the powder. The liquid formula is generally a buffer solution, an isotonic solution and an aqueous solution. The buffer solution can be a phosphate buffer solution, the isotonic solution can be a 0.9% sodium chloride solution, and the aqueous solution is a solution obtained by directly dissolving with purified water.
本领域技术人员可以理解，以所述长效多肽化合物作为活性成分添加药学上可接受载体和/或辅料制成的药物组合物，适用于各种给药方式，例如口服给药、经皮给药、静脉给药、肌肉内给药、局部给药、经鼻给药等。根据所采用的给药方式，可将本发明的药物组合物制成各种合适的剂型，其中包含至少一种有效剂量的本发明的化合物和至少一种药学上可接受 的药用载体。适当剂型的实例为片剂、胶囊、糖衣片剂、粒剂、口服溶液和糖浆，用于皮肤表面的油膏和药贴，气雾剂、鼻喷剂，以及可用于注射的无菌溶液。Those skilled in the art will appreciate that the pharmaceutical composition prepared by adding the long-acting polypeptide compound as an active ingredient to a pharmaceutically acceptable carrier and/or excipient is suitable for various modes of administration, such as oral administration, transdermal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc. According to the mode of administration adopted, the pharmaceutical composition of the present invention can be prepared into various suitable dosage forms, which contain at least one effective dose of the compound of the present invention and at least one pharmaceutically acceptable Examples of suitable dosage forms are tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, ointments and patches for skin surfaces, aerosols, nasal sprays, and sterile solutions that can be used for injection.
本发明的药物组合物的用量可以在一个较大范围内变动，本领域技术人员可以根据一些客观的因素，如根据疾病的种类、病情严重程度、病人体重、剂型、给药途径等因素加以确定。The dosage of the pharmaceutical composition of the present invention can vary within a wide range, and those skilled in the art can determine it based on objective factors, such as the type of disease, severity of the disease, patient weight, dosage form, route of administration, and the like.
本发明还有一个目的在于提供长效多肽化合物以及组合物的应用。Another object of the present invention is to provide applications of long-acting polypeptide compounds and compositions.
本发明得到了一系列长效多肽化合物，并对这一系列药物的药效作用展开研究。研究表明，本发明的长效多肽化合物具有更长的半衰期，具有促胰岛素活性、没有不良反应发生，可用于糖尿病、肥胖的治疗，潜在地可作为新一代治疗糖尿病、肥胖的药物。The present invention obtains a series of long-acting polypeptide compounds, and studies the pharmacodynamic effects of this series of drugs. Studies have shown that the long-acting polypeptide compounds of the present invention have a longer half-life, have insulinotropic activity, have no adverse reactions, can be used to treat diabetes and obesity, and can potentially be used as a new generation of drugs for treating diabetes and obesity.
本发明的长效多肽化合物及组合物的应用具体包括：The applications of the long-acting polypeptide compound and composition of the present invention specifically include:
在制备用于预防或治疗糖尿病的药物方面，在制备用于预防或治疗肥胖的药物方面。In the preparation of drugs for preventing or treating diabetes, in the preparation of drugs for preventing or treating obesity.
本发明的创新性结果和有益效果：The innovative results and beneficial effects of the present invention are:
多肽半衰期和稳定性的解决是多肽药物设计是否能够成药的关键，也是本领域研究的重大科学和核心问题。其中，在关键特定位点氨基酸的替换，增强了多肽的稳定性、活性和抗水解性。通过大量的试验摸索，在本发明多肽化合物主肽链的第2位和/或第13位氨基酸位点用非天然氨基酸Aib、Iva或Cba替代后，多肽分子的稳定性、活性和抗水解性明显增强；第27位和/或第28位氨基酸位点分别用L和/或D氨基酸取代也会明显增强多肽分子的活性。这些发展创新多肽药物关键的发现都是建立在大量的试验摸索基础之上的(请见具体实施例)。除了主肽链的创新，超长效多肽药物分子修饰技术的发展也是关键，是本领域国际上待突破的瓶颈。本发明通过生物信息学、结构生物学、计算机辅助设计、构效关系研究等，开发出位点特异性侧链修饰技术，突破超长效多肽及蛋白药物分子修饰技术，大大延长了合成的化合物的半衰期，实现了多肽药物的超长效化。本发明涉及的多肽化合物不仅具有一级结构，还具有对多肽化合物活性发挥至关重要的二级/三级结构。各种蛋白质/多肽都有特定的二级/三级空间构象，而这些特定的空间构象又与它们特定的生物学功能相关，结构与功能具有高度统一性；二级和三级空间结构对化合物能否结合靶标，以何种方式结合靶标以及结合的强弱影响重大，也就是说二级/三级结构对多肽化合物发挥生物学功能至关重要。本发明侧链的加入极大地影响到了多肽化合物的二级和三级空间结构，因此本发明中，不同的 侧链的加入使得本申请涉及的多肽化合物主肽链空间构象发生极大变化，而多肽构象的改变则会影响到其生物学功能的发挥，其多肽的生物学性质是不可预测的，是否对特有多肽或蛋白长效化有效，需要开展大量的生物药效测试和试验才能知道。The solution to the half-life and stability of polypeptides is the key to whether the design of polypeptide drugs can be made into medicines, and it is also a major scientific and core issue studied in this field. Among them, the replacement of amino acids at key specific sites enhances the stability, activity and hydrolysis resistance of polypeptides. Through a large number of experimental explorations, after the 2nd and/or 13th amino acid sites of the main peptide chain of the polypeptide compound of the present invention are replaced with non-natural amino acids Aib, Iva or Cba, the stability, activity and hydrolysis resistance of the polypeptide molecule are significantly enhanced; the 27th and/or 28th amino acid sites are replaced with L and/or D amino acids respectively. The activity of the polypeptide molecule will also be significantly enhanced. These key discoveries in the development of innovative polypeptide drugs are based on a large number of experimental explorations (see specific examples). In addition to the innovation of the main peptide chain, the development of ultra-long-acting polypeptide drug molecule modification technology is also key and is a bottleneck to be broken through internationally in this field. The present invention has developed site-specific side chain modification technology through bioinformatics, structural biology, computer-aided design, structure-activity relationship research, etc., breaking through ultra-long-acting polypeptides and protein drug molecule modification technology, greatly extending the half-life of the synthesized compounds, and realizing the ultra-long-acting of polypeptide drugs. The polypeptide compounds involved in the present invention not only have a primary structure, but also have a secondary/tertiary structure that is crucial to the activity of the polypeptide compounds. Various proteins/polypeptides have specific secondary/tertiary spatial conformations, and these specific spatial conformations are related to their specific biological functions, and the structure and function are highly unified; the secondary and tertiary spatial structures have a significant impact on whether the compound can bind to the target, how it binds to the target, and the strength of the binding, that is, the secondary/tertiary structure is crucial for the polypeptide compounds to exert their biological functions. The addition of side chains in the present invention greatly affects the secondary and tertiary spatial structures of the polypeptide compounds, so in the present invention, different The addition of side chains causes a great change in the spatial conformation of the main peptide chain of the polypeptide compound involved in the present application, and the change in the polypeptide conformation will affect the performance of its biological function. The biological properties of the polypeptide are unpredictable. Whether it is effective for the long-term effect of a specific polypeptide or protein requires a large number of biopharmaceutical tests and experiments to know.
本发明所述的新型长效多肽化合物的设计合成及所应用的长效化修饰技术，仅对本发明所述的多肽化合物或不确定的多肽类化合物有效，具有不可预见性，作为进一步验证该发明创造性和新颖性的试验(实施例6)的结果显示，本发明的长效多肽化合物大大延长了半衰期，在大鼠的药物半衰期可达22小时以上，实现了多肽药物的超长效化，可实现人用2周及以上给一次药的频率。目前报道的该领域多肽药物大鼠的药物半衰期基本小于10小时，仅能做到1周给一次药的频率。The design and synthesis of the novel long-acting polypeptide compound described in the present invention and the applied long-acting modification technology are only effective for the polypeptide compound described in the present invention or the undefined polypeptide compound, and are unpredictable. The results of the test (Example 6) to further verify the creativity and novelty of the invention show that the long-acting polypeptide compound of the present invention greatly prolongs the half-life, and the drug half-life in rats can reach more than 22 hours, realizing the ultra-long-acting polypeptide drug, and achieving the frequency of drug administration once every 2 weeks or more for human use. The drug half-life of the polypeptide drug in rats currently reported in this field is basically less than 10 hours, and the frequency of drug administration can only be achieved once a week.
本申请的侧链修饰技术不具有普遍适用性，是否对特有多肽或蛋白在保持活性的情况下长效化有效，需要开展大量的创新性的生物药效测试和试验才能知道。为了说明这一点，我们也合成了如下对照化合物，化合物61为类似化合物，化合物62是上市多肽GLP-1药物利西拉肽(Lixisenatide)，化合物63和化合物64分别为侧链修饰利西拉肽不同位置得到的化合物：The side chain modification technology of this application is not universally applicable. Whether it is effective for long-term effects on specific peptides or proteins while maintaining activity requires a large number of innovative biopharmaceutical tests and experiments to determine. To illustrate this point, we also synthesized the following control compounds, compound 61 is a similar compound, compound 62 is the marketed peptide GLP-1 drug lixisenatide, and compounds 63 and 64 are compounds obtained by side chain modification of different positions of lixisenatide:
化合物61(SEQ ID NO.61)：Compound 61 (SEQ ID NO.61):
Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(AEEA-AEEA-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
化合物62(SEQ ID NO.62)：Compound 62 (SEQ ID NO. 62):
HGEGTFTSDLSKQMEEEVRLFIEWLKNGGPSSGAPPSKKKKKK-OH；HGEGTFTSDLSKQMEEEVRLFIEWLKNGGPSSGAPPSKKKKKK-OH;
化合物63(SEQ ID NO.63)：Compound 63 (SEQ ID NO. 63):
HGEGTFTSDLSK(GGSGSGSG-γGlu-CO(CH2)18CO2H)QMEEEVRLFIEWLKNGGPSSGAPPSKKKKKK-OH；HGEGTFTSDLSK(GGSGSGSG-γGlu-CO(CH 2 )1 8 CO 2 H)QMEEEVRLFIEWLKNGGPSSGAPPSKKKKKK-OH;
化合物64(SEQ ID NO.64)：Compound 64 (SEQ ID NO. 64):
HGEGTFTSDLSKQMEEEVRLFIEWLK(GGSGSGSG-γ-Glu-CO(CH2)18CO2H)NGGPSSGAPPSKKKKKK-OH；HGEGTFTSDLSKQMEEEVRLFIEWLK(GGSGSGSG-γ-Glu-CO(CH 2 ) 18 CO 2 H)NGGPSSGAPPSKKKKKK-OH;
化合物61的C末端的-NH2指末端氨基酸被酰胺化，化合物62-64的C末端的-OH为末端氨基酸自身结构。 The -NH 2 at the C-terminus of compound 61 indicates that the terminal amino acid is amidated, and the -OH at the C-terminus of compounds 62-64 is the terminal amino acid structure itself.
我们研究发现：利西拉肽能同GLP-1R的胞外结构域和跨膜口袋相结合，且与GLP-1R受体形成合适的空间位阻。而两条改造肽(化合物63和64)由于长效侧链的缠绕，构象发生变化，从而与GLP-1R形成较大的空间位阻，阻碍配体-受体结合，仅显示与GLP-1R的胞外结构域缠绕结合，但是在跨膜口袋中无结合位点，非但没有延长其降血糖药效，反而使原母肽失去了降糖药效。我们的活性试验(实施例5)结果显示：利西拉肽仅仅依靠侧链改造，并不能改善葡萄糖耐受，降血糖效果极大降低甚至失活。因此，本发明经过自主设计、改造、合成，并通过一系列细胞模型和动物模型的活性验证试验筛选而得到的具有全新活性的化合物具有独创性。Our research found that lixisenatide can bind to the extracellular domain and transmembrane pocket of GLP-1R, and form a suitable steric hindrance with the GLP-1R receptor. However, the two modified peptides (compounds 63 and 64) have a change in conformation due to the entanglement of the long-acting side chain, thereby forming a large steric hindrance with GLP-1R, hindering ligand-receptor binding, and only showing entanglement with the extracellular domain of GLP-1R, but there is no binding site in the transmembrane pocket, which not only fails to prolong its hypoglycemic effect, but also causes the original parent peptide to lose its hypoglycemic effect. The results of our activity test (Example 5) show that lixisenatide cannot improve glucose tolerance by relying solely on side chain modification, and the hypoglycemic effect is greatly reduced or even inactivated. Therefore, the compounds with new activity obtained by the present invention through independent design, modification, synthesis, and screening through a series of activity verification tests in cell models and animal models are original.
本发明的新型长效多肽化合物利用亲脂性取代基结合血液中的白蛋白，保护其免受酶降解，从而提高半衰期。通过分子内桥稳定分子的螺旋结构，提高了针对靶点的效力和/或选择性。The novel long-acting polypeptide compound of the present invention utilizes lipophilic substituents to bind to albumin in the blood, protecting it from enzymatic degradation, thereby increasing the half-life. The helical structure of the molecule is stabilized by an intramolecular bridge, thereby increasing the efficacy and/or selectivity against the target.
本发明的新型长效多肽化合物合成产率高，稳定性好，易于放大生产，成本低。The novel long-acting polypeptide compound of the present invention has high synthesis yield, good stability, is easy to scale up production and has low cost.
同时，本发明的新型长效多肽化合物具有更好的降低体重的药效作用，长效多肽化合物可以通过引起摄食量降低和/或能量消耗升高，用于防止体重增长或促进体重减轻，因此，本发明的新型长效多肽化合物还可用于直接或间接治疗由体重超重所引起的或者以其为特征的其他病症，例如治疗和/或预防肥胖症、病态肥胖症、肥胖症相关炎症、肥胖症相关的胆囊疾病、肥胖症引起的睡眠呼吸暂停，本发明在这些病症中的作用可以是由于新型长效多肽化合物直接或间接对体重的作用，或者对体重以外身体其他方面的作用。At the same time, the novel long-acting polypeptide compound of the present invention has a better pharmacological effect of reducing body weight. The long-acting polypeptide compound can be used to prevent weight gain or promote weight loss by causing a decrease in food intake and/or an increase in energy consumption. Therefore, the novel long-acting polypeptide compound of the present invention can also be used to directly or indirectly treat other diseases caused by or characterized by overweight, such as treating and/or preventing obesity, morbid obesity, obesity-related inflammation, obesity-related gallbladder disease, and sleep apnea caused by obesity. The effect of the present invention in these diseases may be due to the direct or indirect effect of the novel long-acting polypeptide compound on body weight, or the effect on other aspects of the body other than body weight.
本发明中所用缩写具体含义如下：The specific meanings of the abbreviations used in the present invention are as follows:
DCM为二氯甲烷；DMF为N，N-二甲基甲酰胺；MeOH为甲醇；Piperidine为哌啶；HOBt为1-羟基苯并三唑；DIC为N，N'-二异丙基碳二亚胺；Fmoc为芴甲氧羰基；resin为树脂；FBS为胎牛血清；H、His为组氨酸；Y、Tyr为酪氨酸；E、Glu为谷氨酸；Q、Gln为谷氨酰胺；N、Asn为天冬酰胺；G、Gly为甘氨酸；T、Thr为苏氨酸；F、Phe为苯丙氨酸；S、Ser为丝氨酸；D、Asp为天冬氨酸；I、IIe为异亮氨酸；L、Leu为亮氨酸；K、Lys为赖氨酸；A、Ala为丙氨酸；V、Val为缬氨酸；W、Trp为色氨酸；P、Pro为脯氨酸；Aib为2-氨基异丁酸；Iva(Isovaline)为异缬氨酸；Cba(1-Aminocyclobutanecarboxylic acid)为ɑ-氨基环丁酸；Alloc为烯丙氧羰基；PEG2为3-氧代-2，7，10-三氧杂-4-氮杂十 三碳-13-酸，AEEA为8-氨基-3，6-二氧杂辛酸；TFA为三氟乙酸；DODT为2，2'-(1，2-乙二基双氧代)双乙硫醇；ACN为乙腈。DCM is dichloromethane; DMF is N, N-dimethylformamide; MeOH is methanol; Piperidine is piperidine; HOBt is 1-hydroxybenzotriazole; DIC is N, N'-diisopropylcarbodiimide; Fmoc is fluorenylmethoxycarbonyl; resin is resin; FBS is fetal bovine serum; H and His are histidine; Y and Tyr are tyrosine; E and Glu are glutamic acid; Q and Gln are glutamine; N and Asn are asparagine; G and Gly are glycine; T and Th are r is threonine; F and Phe are phenylalanine; S and Ser are serine; D and Asp are aspartic acid; I and IIe are isoleucine; L and Leu are leucine; K and Lys are lysine; A and Ala are alanine; V and Val are valine; W and Trp are tryptophan; P and Pro are proline; Aib is 2-aminoisobutyric acid; Iva (Isovaline) is isovaline; Cba (1-Aminocyclobutanecarboxylic acid) is α-aminocyclobutyric acid; Alloc is allyloxycarbonyl; PEG 2 is 3-oxo-2,7,10-trioxa-4-azadecanoic acid. Tricarbon-13-acid, AEEA is 8-amino-3,6-dioxaoctanoic acid; TFA is trifluoroacetic acid; DODT is 2,2'-(1,2-ethylenedioxy)bis(ethanediol); ACN is acetonitrile.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为实施例2中小鼠给药0.5h后的OGTT实验的时间-血糖结果图；FIG1 is a graph showing the time-blood glucose results of an OGTT experiment in mice 0.5 h after administration in Example 2;
图2为图1中血糖曲线下面积图(AUC)；FIG2 is a graph showing the area under the blood glucose curve (AUC) in FIG1 ;
图3为实施例2中小鼠给药1h后的OGTT实验的时间-血糖结果图；FIG3 is a graph showing the time-blood glucose results of an OGTT experiment in mice 1 hour after administration in Example 2;
图4为图3中血糖曲线下面积图(AUC)；FIG4 is a graph showing the area under the blood glucose curve (AUC) in FIG3 ;
图5为实施例3中小鼠给药24h后的OGTT实验的时间-血糖结果图；FIG5 is a graph showing the time-blood glucose results of an OGTT experiment in mice 24 hours after administration in Example 3;
图6为图5中血糖曲线下面积图(AUC)；FIG6 is a graph showing the area under the blood glucose curve (AUC) in FIG5 ;
图7为实施例3中小鼠给药48h后的OGTT实验的时间-血糖结果图。FIG. 7 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 for mice 48 hours after administration.
图8为图7中血糖曲线下面积图(AUC)；FIG8 is a graph showing the area under the blood glucose curve (AUC) in FIG7 ;
图9为实施例3中小鼠给药72h后的OGTT实验的时间-血糖结果图。FIG. 9 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 72 hours after administration.
图10为图9中血糖曲线下面积图(AUC)；FIG10 is a graph showing the area under the blood glucose curve (AUC) in FIG9 ;
图11为实施例3中小鼠给药96h后的OGTT实验的时间-血糖结果图。FIG. 11 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 96 hours after administration.
图12为图11中血糖曲线下面积图(AUC)；FIG12 is a graph showing the area under the blood glucose curve (AUC) in FIG11 ;
图13为实施例3中小鼠给药120h后的OGTT实验的时间-血糖结果图。FIG. 13 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 120 hours after administration.
图14为图13中血糖曲线下面积图(AUC)；FIG14 is a graph showing the area under the blood glucose curve (AUC) in FIG13 ;
图15为实施例3中小鼠给药144h后的OGTT实验的时间-血糖结果图。FIG. 15 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 144 hours after administration.
图16为图15中血糖曲线下面积图(AUC)；FIG16 is a graph showing the area under the blood glucose curve (AUC) in FIG15 ;
图17为实施例3中小鼠给药168h后的OGTT实验的时间-血糖结果图。FIG. 17 is a graph showing the time-blood glucose results of the OGTT experiment in Example 3 in mice 168 hours after administration.
图18为图17中血糖曲线下面积图(AUC)；FIG18 is a graph showing the area under the blood glucose curve (AUC) in FIG17 ;
图19为实施例4中小鼠体重监测数据统计图；Figure 19 is a statistical diagram of the weight monitoring data of mice in Example 4;
图20为实施例4中小鼠的空腹血糖监测数据统计；Figure 20 is the fasting blood glucose monitoring data statistics of mice in Example 4;
图21为实施例5中小鼠给药24h后的OGTT实验的时间-血糖结果图；FIG21 is a graph showing the time-blood glucose results of an OGTT experiment in mice 24 hours after administration in Example 5;
图22为图21中血糖曲线下面积图(AUC)。 FIG. 22 is a graph showing the area under the blood glucose curve (AUC) in FIG. 21 .
具体实施方式Detailed ways
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.
材料与方法：Materials and Methods:
Boc-His(Trt)-OH，Fmoc-Aib-OH购自上海吉尔，二十烷二酸单叔丁酯自制。其余的氨基酸购自成都郑源公司，缩合剂购自苏州昊帆公司。如果没有特别说明，其他所有试剂均为分析纯，溶剂购自上海泰坦公司。离心机购自卢湘仪。5.0cm反相C18制备柱(46mm x 250mm)用来纯化多肽。高效液相色谱仪为赛默飞公司产品。质谱分析采用Waters质谱仪进行测定。Boc-His(Trt)-OH and Fmoc-Aib-OH were purchased from Shanghai Jier, and mono-tert-butyl eicosanedioate was self-made. The remaining amino acids were purchased from Chengdu Zhengyuan Company, and the condensation agent was purchased from Suzhou Haofan Company. Unless otherwise specified, all other reagents were analytically pure, and the solvents were purchased from Shanghai Titan Company. The centrifuge was purchased from Lu Xiangyi. A 5.0 cm reverse phase C 18 preparative column (46 mm x 250 mm) was used to purify the peptide. The high performance liquid chromatograph was a product of Thermo Fisher Scientific. Mass spectrometry was performed using a Waters mass spectrometer.
实施例1多肽化合物的合成Example 1 Synthesis of polypeptide compounds
一、化合物31的合成1. Synthesis of Compound 31
化合物31的氨基酸序列：Amino acid sequence of compound 31:
Tyr-Iva-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2)18CO2H)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 Tyr-Iva-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH 2 ) 18 CO 2 H)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH 2
简写为：Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2 Abbreviated as: Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2
方法：method:
步骤1.合成主肽链对应的主肽树脂Step 1. Synthesize the main peptide resin corresponding to the main peptide chain
按照Fmoc/t-Bu策略手工合成，合成规模：0.5mmol，合成如下主肽树脂：According to the Fmoc/t-Bu strategy, the synthesis scale was 0.5 mmol, and the following main peptide resin was synthesized:
Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin。Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin.
(1)：称取0.89克Rink Amide AM Resin Resin树脂(loading 0.56mmol/g，西安 蓝晓)，加入反应柱中，加入15ml的DMF中溶胀30min，称取Fmoc-Ser(tBu)-OH：1.587g(6eq)，HOBt：0.672g(7.2eq)，DMAP：0.06g(0.72eq)，待用。抽掉DMF，用DMF充分洗涤树脂2次，把上述称好的物料加入到反应柱中。加入适量的DMF，氮气搅拌均匀，加入DIC 0.83mL(7.8eq)。反应2h，反应结束。抽掉反应液，用DMF洗涤3次，加入乙酸酐/吡啶(7：6，v/v)封闭4h。抽掉封闭液，DMF洗涤6次，得Fmoc-Ser(tBu)-Rink Amide AM Resin。(1): Weigh 0.89 g of Rink Amide AM Resin (loading 0.56 mmol/g, Xi'an Lan Xiao), add to the reaction column, add 15ml of DMF to swell for 30min, weigh Fmoc-Ser(tBu)-OH: 1.587g (6eq), HOBt: 0.672g (7.2eq), DMAP: 0.06g (0.72eq), and set aside. Draw out DMF, wash the resin twice with DMF, and add the above weighed materials to the reaction column. Add an appropriate amount of DMF, stir evenly with nitrogen, and add DIC 0.83mL (7.8eq). React for 2h, and the reaction is over. Draw out the reaction solution, wash with DMF 3 times, and add acetic anhydride/pyridine (7:6, v/v) to block for 4h. Draw out the blocking solution, wash with DMF 6 times, and obtain Fmoc-Ser(tBu)-Rink Amide AM Resin.
(2)：以Fmoc-Ser(tBu)-Rink Amide AM Resin为载体，以HOBt、DIC为偶联剂，以DMF为溶剂，以20％的Piperidine/DMF溶液脱除Fmoc基团(两次5min+7min)，偶联过程用水合茚三酮监测偶联效果。进行手工投料，依次从C端到N端进行缩合反应连接Fmoc-Pro-OH，Fmoc-Pro-OH，Fmoc-Pro-OH，Fmoc-Ala-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Ser(tBu)-OH，Fmoc-Pro-OH，Fmoc-Gly-OH，Fmoc-Gly-OH，Fmoc-Ala-OH，Fmoc-Ile-OH，Fmoc-Leu-OH，Fmoc-Trp(Boc)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Val-OH，Fmoc-Phe-OH，Fmoc-Ala-OH，Fmoc-Lys(Alloc)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Ala-OH，Fmoc-Ile-OH，Fmoc-Lys(Boc)-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Leu-OH，Fmoc-Aib-OH，Fmoc-Ile-OH，Fmoc-Ser(tBu)-OH，Fmoc-Ser(tBu)-OH，Fmoc-Tyr(tBu)-OH，Fmoc-Asp(OtBu)-OH，Fmoc-Ser(tBu)-OH，Fmoc-Thr(tBu)-OH，Fmoc-Phe-OH，Fmoc-Thr(tBu)-OH，Fmoc-Gly-OH，Fmoc-Glu(tBu)-OH，Fmoc-Iva-OH，Boc-Tyr(tBu)-OH。以上氨基酸投料相当于合成规模5eq，得Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin。(2): Using Fmoc-Ser(tBu)-Rink Amide AM Resin as carrier, HOBt and DIC as coupling agents, and DMF as solvent, the Fmoc group was removed with 20% Piperidine/DMF solution (twice for 5 min + 7 min), and the coupling effect was monitored with ninhydrin during the coupling process. Manual feeding was performed, and condensation reactions were performed from C-terminus to N-terminus to connect Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Gln(Tr t)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Iva-OH, Boc-Tyr(tBu)-OH. The above amino acid feed is equivalent to a synthesis scale of 5eq, and Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin is obtained.
有几点需要说明：There are a few points to note:
1)Fmoc-Ser(tBu)-Rink Amide AM Resin的合成，因Rink Amide AM Resin本身替代度较低，Fmoc-Ser(tBu)-OH的投料量要大，不然替代度偏低，浪费物料。用乙酸酐/吡啶封闭，防止缺损肽的出现。1) Synthesis of Fmoc-Ser(tBu)-Rink Amide AM Resin. Because the substitution degree of Rink Amide AM Resin itself is low, the amount of Fmoc-Ser(tBu)-OH should be large, otherwise the substitution degree will be low and the material will be wasted. Use acetic anhydride/pyridine to block to prevent the appearance of defective peptides.
2)接下来的每次缩合反应中Fmoc保护氨基酸，HOBt，DIC投料量均为5倍，反应时间为2个小时。 2) In each subsequent condensation reaction, the amount of Fmoc-protected amino acid, HOBt, and DIC added was 5 times, and the reaction time was 2 hours.
3)偶联过程用水合茚三酮监测偶联效果，若检测为阴性，则反应完全，若为阳性，则需复投一次。Fmoc保护氨基酸，HOBt，DIC投料量均为2倍，反应时间为1个小时。若仍为阳性，则用加入乙酸酐/吡啶(7：6，v/v)封闭2h。3) During the coupling process, the coupling effect was monitored with ninhydrin. If the test result was negative, the reaction was complete. If it was positive, it needed to be repeated. The amount of Fmoc-protected amino acid, HOBt, and DIC was 2 times, and the reaction time was 1 hour. If it was still positive, acetic anhydride/pyridine (7:6, v/v) was added to block for 2 hours.
(3)：烯丙氧羰基(Alloc)的脱除(3): Removal of allyloxycarbonyl (Alloc)
树脂中加入DCM，加入吗啉0.5mL(12eq)，称取0.173g Pd(PPh3)4(0.3eq)加入反应柱中，反应1h。反应结束，抽掉反应液，用DMF洗涤3次，DCM洗涤6次。得到主肽链肽树脂：Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin。DCM was added to the resin, 0.5 mL (12 eq) of morpholine was added, 0.173 g of Pd(PPh 3 ) 4 (0.3 eq) was weighed and added to the reaction column, and the reaction was continued for 1 hour. After the reaction was completed, the reaction solution was removed and washed with DMF 3 times and DCM 6 times. The main peptide chain peptide resin was obtained: Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin.
步骤2.偶联“侧臂”结构：Step 2. Coupling of the "sidearm" structure:
偶联Fmoc-Gly-OH：向主肽树脂产物中加入Fmoc-Gly-OH、HOBt、适量DMF，氮气搅拌均匀，加入DIC，氮气搅拌反应2小时，用水合茚三酮检测偶联效果，无色透明，反应结束。抽掉反应液，用N，N-二甲基甲酰胺(DMF)洗涤3次，以20％的Piperidine/DMF溶液脱除Fmoc基团(两次5min+7min)，脱除Fmoc后用DMF洗涤6次，取样茚三酮检测，呈阳性则进入后续偶联步骤。Coupling Fmoc-Gly-OH: Add Fmoc-Gly-OH, HOBt, and an appropriate amount of DMF to the main peptide resin product, stir evenly with nitrogen, add DIC, stir with nitrogen for 2 hours, and use hydrated ninhydrin to detect the coupling effect. If it is colorless and transparent, the reaction is over. Draw off the reaction solution, wash it with N, N-dimethylformamide (DMF) 3 times, remove the Fmoc group with 20% Piperidine/DMF solution (twice 5min + 7min), wash it with DMF 6 times after removing Fmoc, take a sample for ninhydrin detection, and if it is positive, proceed to the subsequent coupling step.
重复上述操作依次偶联Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gly-OH，Fmoc-Ser(tBu)-OH，Fmoc-Gly-OH，Fmoc-Glu-OtBu，二十烷二酸单叔丁酯。得到Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu-OtBu-CO(CH2)18CO2-tBu)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin。用DMF洗3次，DCM洗3次，MeOH收缩2次，真空抽干，得干燥多肽树脂3.2g。Repeat the above operation to sequentially couple Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu-OtBu, and mono-tert-butyl eicosandioate. Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu-OtBu-CO(CH 2 ) 18 CO 2 -tBu)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin. Wash with DMF 3 times, DCM 3 times, shrink with MeOH 2 times, and vacuum dry to obtain 3.2 g of dry polypeptide resin.
步骤3.脱除多肽全保护Step 3. Deprotection of peptides
裂解液：TFA、DODT、间甲酚、H2O按体积比为92.5：2.5：2.5：2.5的比例事先配好，冰箱中冷冻2h。 Lysis solution: TFA, DODT, m-cresol, and H 2 O were prepared in advance at a volume ratio of 92.5:2.5:2.5:2.5 and frozen in a refrigerator for 2 h.
按10mL裂解液/g多肽树脂，往干燥的多肽树脂Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu-OtBu-CO(CH2)18CO2-tBu)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin中加入裂解液，升温到室温，裂解反应3个小时。过滤收集滤液，树脂用少量裂解液洗涤3次，合并滤液后减压浓缩至原体积的1/4左右，在搅拌下缓慢倒入冰的甲基叔丁基醚中，用少量裂解液将瓶中残余物一并洗至甲基叔丁基醚中。静置2个小时以上，待沉淀完全。去除上清液，沉淀离心，用甲基叔丁基醚洗涤3次，离心，固体用氮气吹干。得到粗品化合物Tyr-Iva-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2)18CO2H)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2。粗品重1.48g。At a rate of 10 mL of lysate/g of peptide resin, add the dried peptide resin Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Gly-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Ser(tBu)-Gly-Glu-OtBu-CO(CH 2 ) 18 CO 2 -tBu)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin was added with lysis solution, and the temperature was raised to room temperature, and the lysis reaction was carried out for 3 hours. The filtrate was collected by filtration, and the resin was washed 3 times with a small amount of lysis solution. After the filtrate was combined, it was concentrated under reduced pressure to about 1/4 of the original volume, and slowly poured into ice methyl tert-butyl ether under stirring, and the residue in the bottle was washed into methyl tert-butyl ether with a small amount of lysis solution. Let it stand for more than 2 hours until the precipitation is complete. The supernatant was removed, the precipitate was centrifuged, washed 3 times with methyl tert-butyl ether, centrifuged, and the solid was blown dry with nitrogen. The crude compound Tyr-Iva-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln -Lys(Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γGlu-CO(CH2)18CO2H ) -Ala -Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser- NH2 was obtained. The crude product weighed 1.48 g.
步骤4.精制纯化粗品化合物Step 4. Purification of crude compound
将步骤3中所得粗品化合物溶于ACN：H2O＝1：3(v/v)的溶液中，通过5.0cm反相C18的填充的46mm x 250mm柱上进行制备型HPLC纯化。用39％ACN/H2O(含0.1％三氟乙酸)为起始，以梯度(0.33％/min的速度增加ACN的比例)，流速为80mL/min将该柱洗脱60分钟，收集含有多肽的组分，得到HPLC纯度大于90％的样品。重复一次HPLC纯化，用29％ACN/H2O(含0.1％醋酸)为起始，以梯度(0.33％/min的增加ACN的比例)，流速为80mL/min，将该柱洗脱60分钟，收集含有多肽的组分，冷冻抽干，得到精肽440mg，纯度大于98.96％，总收率为17％。The crude compound obtained in step 3 was dissolved in a solution of ACN:H 2 O=1:3 (v/v) and purified by preparative HPLC on a 46 mm x 250 mm column packed with 5.0 cm reverse phase C 18. Starting with 39% ACN/H 2 O (containing 0.1% trifluoroacetic acid), the column was eluted with a gradient (increasing the proportion of ACN at a rate of 0.33%/min) at a flow rate of 80 mL/min for 60 minutes, and the fractions containing the polypeptide were collected to obtain a sample with an HPLC purity of greater than 90%. The HPLC purification was repeated once, starting with 29% ACN/H 2 O (containing 0.1% acetic acid), the column was eluted with a gradient (increasing the proportion of ACN at a rate of 0.33%/min) at a flow rate of 80 mL/min for 60 minutes, and the fractions containing the polypeptide were collected and freeze-dried to obtain 440 mg of refined peptide with a purity of greater than 98.96% and a total yield of 17%.
步骤5.产物确认Step 5. Product confirmation
用液质联用鉴定分离出的产物多肽，用5％ACN/H2O(含0.1％甲酸)为起始，以梯度(6％/min的速度增加ACN的比例)，流速为0.4mL/min，洗脱分析15分钟，确定为目标化合物31，[M+H]+计算值为5083.73，[M+3H]3+实测值为1695.20。The isolated product peptide was identified by LC-MS, starting with 5% ACN/H 2 O (containing 0.1% formic acid), with a gradient (increasing the proportion of ACN at a rate of 6%/min), a flow rate of 0.4 mL/min, and elution analysis for 15 minutes. The target compound 31 was determined to have a calculated [M+H] + value of 5083.73 and a measured [M+3H] 3+ value of 1695.20.
二、化合物35的合成2. Synthesis of Compound 35
由于化合物35与化合物31的区别只在于主肽链的序列中的X13不同，35中的X13是Iva，因此二者的合成步骤的区别在于步骤1合成不同于化合物31的以下主肽树脂： Since the difference between compound 35 and compound 31 is only that X13 in the sequence of the main peptide chain is different, and X13 in 35 is Iva, the difference between the synthesis steps of the two is that step 1 synthesizes the following main peptide resin different from compound 31:
Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin，合成规模为：0.5mmol。Boc-Tyr(tBu)-Iva-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin, synthesis scale: 0.5 mmol.
化合物35后续的侧链偶联及裂解过程同化合物31。The subsequent side chain coupling and cleavage process of compound 35 was the same as that of compound 31.
化合物35的精制纯化与产物确认方法同化合物31，得到精肽332mg，纯度大于98.46％，总收率为13％。用液质联用鉴定分离出的产物，确定为目标化合物35，[M+H]+计算值为5097.76，[M+3H]3+实测值为1699.90。The purification and product confirmation methods of compound 35 were the same as those of compound 31, and 332 mg of refined peptide was obtained with a purity greater than 98.46% and a total yield of 13%. The separated product was identified by LC-MS and confirmed to be the target compound 35, with a calculated value of [M+H] + of 5097.76 and a measured value of [M+3H] 3+ of 1699.90.
三、化合物38的合成3. Synthesis of Compound 38
由于化合物38与化合物31的区别在于主肽链的序列中的X2、X13、X27、X28不同，化合物38中的X2是Aib，X13是Iva，X27是Leu、X28是Asp，因此二者合成步骤的区别在于步骤1合成主肽树脂不同：Since the difference between compound 38 and compound 31 lies in the difference in X2, X13, X27, and X28 in the sequence of the main peptide chain, X2 in compound 38 is Aib, X13 is Iva, X27 is Leu, and X28 is Asp, the difference in the synthesis steps of the two is that the resins for synthesizing the main peptide in step 1 are different:
按照Fmoc/t-Bu策略手工合成，合成规模：0.5mmol，合成如下主肽树脂：Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu-Asp(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin。According to the Fmoc/t-Bu strategy, the synthesis scale was 0.5 mmol and the following main peptide resin was synthesized manually: Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Iva-Leu-Asp(OtBu)-Lys-Ile-Ala-Gln(Trt)-Lys(Alloc)-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Leu-Asp(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Rink Amide AM Resin.
化合物38后续的侧链偶联及裂解过程同化合物31。The subsequent side chain coupling and cleavage process of compound 38 was the same as that of compound 31.
化合物38的精制纯化与产物确认方法同化合物31，得到精肽385mg，纯度大于98.35％，总收率为15％。用液质联用鉴定分离出的产物，确定为目标化合物38，[M+H]+计算值为5128.69，[M+3H]3+实测值为1709.50。The purification and product confirmation methods of compound 38 were the same as those of compound 31, and 385 mg of refined peptide was obtained with a purity greater than 98.35% and a total yield of 15%. The separated product was identified by LC-MS and confirmed to be the target compound 38, with a calculated value of [M+H] + of 5128.69 and a measured value of [M+3H] 3+ of 1709.50.
基于以上合成步骤，精制纯化与产物确认方法，根据化合物1-60肽链中的区别位点，调整化合物合成步骤1或步骤2中树脂的偶联顺序，最终合成对应的目标产物，再通过液质联用鉴定分离出的产物，确认质子化分子离子峰的m/z值(表1中的实测值)，并将实测值与分子量理论值比较，确认合成纯化的产物为目标产物。表1分别给出了化合物1-60的分子量理论值、液质鉴定的实测值、序列和分子式。 Based on the above synthesis steps, purification and product confirmation methods, according to the distinguishing sites in the peptide chain of compound 1-60, the coupling order of the resin in step 1 or step 2 of the compound synthesis is adjusted, and the corresponding target product is finally synthesized, and then the separated product is identified by liquid chromatography-mass spectrometry, and the m/z value of the protonated molecular ion peak (measured value in Table 1) is confirmed, and the measured value is compared with the theoretical molecular weight value to confirm that the synthesized and purified product is the target product. Table 1 shows the theoretical molecular weight value, measured value of liquid chromatography-mass spectrometry, sequence and molecular formula of compound 1-60, respectively.
表1 长效多肽化合物氨基酸序列及液质联用鉴定结果






Table 1 Amino acid sequence of long-acting peptide compounds and LC-MS identification results






实施例2 化合物1-27和索马鲁肽(Semaglutide)对C57BL/6J小鼠葡萄糖耐受量的影响Example 2 Effects of Compound 1-27 and Semaglutide on Glucose Tolerance in C57BL/6J Mice
我们对化合物1-27进行体内药效探究，通过口服葡萄糖耐量实验(OGTT)研究了化合物1-27与同等剂量下Semaglutide对正常小鼠葡萄糖耐受量的影响和维持时间。We investigated the in vivo efficacy of compound 1-27 and studied the effects and maintenance time of compound 1-27 and semaglutide at the same dose on glucose tolerance in normal mice through an oral glucose tolerance test (OGTT).
实验方法：本次试验采用8周龄的C57BL/6J雄性小鼠(购自广东斯嘉景达生物科技有限公司)，7只/组，所有化合物及Semaglutide(注射液，购自广州市桐晖药业有限公司)给药剂量均为50μg/kg。首先，在试验前测量小鼠体重和随机血糖，根据体重和随机血糖重新分组，保证每组的平均体重和随机血糖平均值相近。给药当天配制好药物，按分组皮下注射对应药物，给药后0.5h和1h后分别进行OGTT试验(平行实验，一批小鼠进行0.5h OGTT实验，另一批小鼠进行1h OGTT试验)。按照2g/kg的剂量灌胃给予葡萄糖，尾静脉采血检测灌胃后0、15、30、60、90和120min共6个时间点的血糖值。使用软件GraphPadPrism处理数据，绘制时间-血糖曲线图，计算血糖曲线下面积AUC。与未给药的PBS对照组进行One-way ANOVA分析，计算出显著性差异。试验结果如图1-4所示，其中，*表示p<0.05；**表示p<0.01；***表示p<0.001；****表示p<0.0001。Experimental methods: 8-week-old C57BL/6J male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were used in this experiment, 7 mice/group, and the dosage of all compounds and Semaglutide (injection, purchased from Guangzhou Tonghui Pharmaceutical Co., Ltd.) was 50μg/kg. First, the weight and random blood glucose of mice were measured before the experiment, and the mice were regrouped according to the weight and random blood glucose to ensure that the average weight and random blood glucose of each group were similar. The drugs were prepared on the day of administration, and the corresponding drugs were subcutaneously injected according to the groups. OGTT tests were performed 0.5h and 1h after administration (parallel experiments, one group of mice underwent 0.5h OGTT test, and another group of mice underwent 1h OGTT test). Glucose was administered by gavage at a dose of 2g/kg, and blood was collected from the tail vein to detect blood glucose values at 0, 15, 30, 60, 90 and 120min after gavage. The data were processed using the software GraphPadPrism, and the time-blood glucose curve was plotted to calculate the area under the blood glucose curve AUC. One-way ANOVA analysis was performed with the PBS control group without medication to calculate the significant difference. The test results are shown in Figures 1-4, where * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.
结果分析：由图1-2可知，在给药后0.5h，各采血时间点与PBS相比，所有给药组均 明显改善葡萄糖耐量，降糖效果明显(****，即P≤0.0001)，各组之间没有明显差异。Results analysis: As shown in Figures 1-2, at 0.5 h after administration, all the blood sampling time points were compared with PBS. Glucose tolerance was significantly improved, and the blood sugar lowering effect was significant (****, i.e., P≤0.0001), with no significant difference among the groups.
由图3-4可知，在给药1h后，与PBS相比，化合物4-14、化合物16-21和化合物23-27仍能改善葡萄糖耐量，具有明显降糖效果(****，即P≤0.0001)，但AUC值有所升高其降糖效果弱于给药后0.5h。在给药1h后，与PBS相比，化合物1-3、化合物15和化合物22不具有统计学明显差异，失去降糖效果。As shown in Figures 3-4, 1 hour after administration, compared with PBS, compounds 4-14, compounds 16-21 and compounds 23-27 can still improve glucose tolerance and have a significant hypoglycemic effect (****, i.e., P≤0.0001), but the AUC value is increased and the hypoglycemic effect is weaker than 0.5 hours after administration. 1 hour after administration, compared with PBS, compounds 1-3, compounds 15 and compounds 22 have no statistically significant difference and lose their hypoglycemic effect.
实施例3：化合物28-61和Semaglutide对C57BL/6J小鼠葡萄糖耐受量的影响Example 3: Effects of Compounds 28-61 and Semaglutide on Glucose Tolerance in C57BL/6J Mice
我们对化合物28-60进行体内药效探究，并以化合物61作为对照组，通过口服葡萄糖耐量实验(OGTT)实验研究了化合物28-61与同等剂量下Semaglutide对正常小鼠葡萄糖耐受量的影响和维持时间。We investigated the in vivo efficacy of compounds 28-60 and used compound 61 as the control group. We conducted an oral glucose tolerance test (OGTT) to study the effects and maintenance time of compounds 28-61 and Semaglutide at the same dose on glucose tolerance in normal mice.
实验方法：本次试验采用8周龄的C57BL/6J雄性小鼠(购自广东斯嘉景达生物科技有限公司)，7只/组，所有化合物及Semaglutide(注射液，购自广州市桐晖药业有限公司)给药剂量均为80μg/kg。首先，在试验前测量小鼠体重和随机血糖，根据体重和随机血糖重新分组，保证每组的平均体重和随机血糖平均值相近。给药当天配制好药物，按分组皮下注射对应药物。给药后24h、48h、72h、96h、120h、144h、168h后进行灌胃葡萄糖OGTT试验，按照2g/kg的剂量灌胃给予葡萄糖，尾静脉采血检测灌胃后0、15、30、60、90和120min共6个时间点的血糖值。使用软件GraphPadPrism处理数据，绘制时间-血糖曲线图，计算血糖曲线下面积AUC。与未给药的PBS对照组进行One-way ANOVA分析，计算出显著性差异。试验结果如图5-18所示，其中，*表示p<0.05；**表示p<0.01；***表示p<0.001；****表示p<0.0001。Experimental methods: This experiment used 8-week-old C57BL/6J male mice (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.), 7 mice/group, and the dosage of all compounds and Semaglutide (injection, purchased from Guangzhou Tonghui Pharmaceutical Co., Ltd.) was 80μg/kg. First, the weight and random blood glucose of mice were measured before the experiment, and the mice were regrouped according to the weight and random blood glucose to ensure that the average weight and random blood glucose of each group were similar. The drugs were prepared on the day of administration and the corresponding drugs were subcutaneously injected according to the group. Glucose OGTT test was performed 24h, 48h, 72h, 96h, 120h, 144h, and 168h after administration. Glucose was gavaged at a dose of 2g/kg, and blood was collected from the tail vein to detect the blood glucose values at 0, 15, 30, 60, 90 and 120min after gavage. The data were processed using the software GraphPadPrism, the time-blood glucose curve was drawn, and the area under the blood glucose curve AUC was calculated. One-way ANOVA analysis was performed with the PBS control group without medication to calculate the significant difference. The test results are shown in Figure 5-18, where * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.
结果分析：Result analysis:
(1)24h结果。由图5-6可知，所有化合物相较于PBS对照组均有明显改善葡萄糖耐量，降低血糖效果。其中，化合物31-36，化合物38-39，41-52化合物55-60以及Semaglutide降糖效果最为显著。(1) 24h results. As shown in Figures 5-6, all compounds significantly improved glucose tolerance and lowered blood sugar compared to the PBS control group. Among them, compounds 31-36, compounds 38-39, 41-52, compounds 55-60 and Semaglutide had the most significant blood sugar lowering effects.
(2)48h结果。由图7-8可知，所有化合物相较于PBS对照组均有明显改善葡萄糖耐量，降低血糖效果。其中，化合物31、33、35，化合物38-39，化合物41-52和化合物55-60效果最优，与24h接近；化合物53、54药效次之；再然后是化合物37、40、61；最后是 化合物29、30；化合28、32、34、36和Semaglutide效果相当，药效相较于24h有所降低。(2) 48h results. As shown in Figures 7-8, all compounds significantly improved glucose tolerance and lowered blood sugar compared to the PBS control group. Among them, compounds 31, 33, 35, compounds 38-39, compounds 41-52 and compounds 55-60 had the best effects, close to those at 24h; compounds 53 and 54 were second; followed by compounds 37, 40, and 61; and finally Compounds 29, 30; Compounds 28, 32, 34, 36 and Semaglutide have comparable effects, but their efficacy is somewhat lower than that at 24h.
(3)72h结果。由图9-10可知，化合物31、33、35、38，化合物39-60仍然具有较好的药效，稍弱于48h药效；化合物29、30、61药效次之，相较于PBS对照组化合物28、32、34、36和Semaglutide已无显著性差异，失去药效。(3) 72h results. As shown in Figures 9-10, compounds 31, 33, 35, 38, and compounds 39-60 still have good efficacy, slightly weaker than the 48h efficacy; compounds 29, 30, and 61 have the second best efficacy, and compared with the PBS control group, compounds 28, 32, 34, 36 and Semaglutide have no significant difference and have lost their efficacy.
(4)96h结果。由图11-12可知，化合物38、39、43、44、55、58、59、60仍然具有药效，但弱于72h药效；化合物31、33、35、37、40、49、50药效次之；随后是化合物51、52、53、54；最后后是化合物29、30；相较于PBS对照组化合物28、32、34、36、41、42、45-48、53、56-57、61和Semaglutide已无显著性差异，失去药效。(4) 96h results. As shown in Figures 11-12, compounds 38, 39, 43, 44, 55, 58, 59, and 60 still have efficacy, but are weaker than the 72h efficacy; compounds 31, 33, 35, 37, 40, 49, and 50 are second in efficacy; followed by compounds 51, 52, 53, and 54; and finally compounds 29 and 30; compared with the PBS control group, compounds 28, 32, 34, 36, 41, 42, 45-48, 53, 56-57, 61 and Semaglutide have no significant difference and have lost their efficacy.
(5)120h结果。由图13-14可知，其中，化合物38、39、43、44、55、58、59、60仍然具有药效，药效弱于96h；化合物31、33、35、37、40、49-54药效次之；然后是化合物29、30，具有较弱药效；相较于PBS对照组化合物28、32、34、36、41、42、45-48、56-57、61和Semaglutide已无显著性差异，无药效，与72h、96h的无药效结果一致。(5) 120h results. As shown in Figures 13-14, compounds 38, 39, 43, 44, 55, 58, 59, and 60 still have efficacy, but the efficacy is weaker than that at 96h; compounds 31, 33, 35, 37, 40, and 49-54 have the second best efficacy; followed by compounds 29 and 30, which have weaker efficacy; compared with the PBS control group, compounds 28, 32, 34, 36, 41, 42, 45-48, 56-57, 61 and Semaglutide have no significant difference and no efficacy, which is consistent with the no efficacy results at 72h and 96h.
(6)144h结果。由图15-16可知，化合物38、39、43、44、55、58仍然具有药效，药效弱于120h；化合物49、50、51、52、53、54、59药效次之，再然后是化合物31、33、35、37、40，具有较弱药效；相较于PBS对照组化合物28、29、30、32、34、36、41、42、45-48、56-57、60、61和Semaglutide均已无显著性差异，失去药效。(6) 144h results. As shown in Figures 15-16, compounds 38, 39, 43, 44, 55, and 58 still have efficacy, but the efficacy is weaker than that at 120h; compounds 49, 50, 51, 52, 53, 54, and 59 have the second best efficacy, followed by compounds 31, 33, 35, 37, and 40, which have weaker efficacy; compared with the PBS control group, compounds 28, 29, 30, 32, 34, 36, 41, 42, 45-48, 56-57, 60, 61 and Semaglutide have no significant difference and have lost their efficacy.
(7)168h结果。由图17-18可知，相较于PBS对照组化合物38、39、43、44、55、58仍然具有弱药效；49、50、51、52、53、54、59效果次之；其余各化合物则失去药效。(7) 168h results. As shown in Figures 17-18, compared with the PBS control group, compounds 38, 39, 43, 44, 55, and 58 still have weak efficacy; 49, 50, 51, 52, 53, 54, and 59 have the second best effect; the remaining compounds have lost their efficacy.
结论：分析以上结果发现，尽管在改善葡萄糖耐量方面，Semaglutide与各化合物都具有良好的效果。但是，在药效维持时间方面即在长效性降糖方面，各化合物药效持续时间不一。其中，化合物38、39、43、44、55、58、49、50、51、52、53、54、59在药效和药效维持时间(长效性)这两方面均具有明显的优势，且远优于Semaglutide。Conclusion: Analysis of the above results shows that although Semaglutide and each compound have good effects in improving glucose tolerance, the duration of drug effect varies among the compounds, that is, in terms of long-term glucose lowering. Among them, compounds 38, 39, 43, 44, 55, 58, 49, 50, 51, 52, 53, 54, and 59 have obvious advantages in both drug effect and drug effect duration (long-term effect), and are far superior to Semaglutide.
实施例4：化合物31、33、35、38、39、43、44、49、58、60和Semaglutide对BKS-db糖尿病小鼠的治疗作用Example 4: Therapeutic effects of compounds 31, 33, 35, 38, 39, 43, 44, 49, 58, 60 and Semaglutide on BKS-db diabetic mice
根据OGTT实验结果，我们进一步研究化合物31、33、35、38、39、43、44、49、 58、60在BKS-db糖尿病疾病小鼠模型上的药效，考察化合物对体重和血糖的影响。Based on the results of OGTT experiments, we further studied compounds 31, 33, 35, 38, 39, 43, 44, 49, The efficacy of 58 and 60 in the BKS-db diabetic mouse model was investigated to examine the effects of the compounds on body weight and blood glucose.
实验方法：本次试验采用8周龄基因型为Lepr KO/KO的BKS-db糖尿病小鼠(购自广东药康生物科技有限公司)。测定血糖和体重，按照体重和血糖随机分组，每组6只，分为各化合物组，阳性对照组(Semaglutide)和模型对照组(PBS)。每组小鼠分别皮下注射化合物31、33、35、38、39、43、44、49、58、60、Semaglutide(注射液购自广州市桐晖药业有限公司)，剂量为120μg/kg，隔天给药一次，对照组注射等体积生理盐水。试验周期为4周。每次给药之后，隔天禁食6h，检测小鼠的血糖和体重并记录数据，使用软件GraphPadPrism处理数据，实验结果如图19-20，其中，*表示p<0.05；**表示p<0.01；***表示p<0.001；****表示p<0.0001。Experimental methods: This experiment used 8-week-old BKS-db diabetic mice with the genotype of Lepr KO/KO (purchased from Guangdong Yaokang Biotechnology Co., Ltd.). Blood glucose and body weight were measured, and the mice were randomly divided into each compound group, positive control group (Semaglutide) and model control group (PBS) according to body weight and blood glucose. Each group of mice was subcutaneously injected with compounds 31, 33, 35, 38, 39, 43, 44, 49, 58, 60, and Semaglutide (injection purchased from Guangzhou Tonghui Pharmaceutical Co., Ltd.) at a dose of 120μg/kg, once every other day, and the control group was injected with an equal volume of saline. The experimental period was 4 weeks. After each administration, fasting for 6 hours every other day, blood glucose and body weight of the mice were measured and the data were recorded. The data were processed using GraphPadPrism software. The experimental results are shown in Figures 19-20, where * indicates p<0.05; ** indicates p<0.01; *** indicates p<0.001; **** indicates p<0.0001.
结果分析：Result analysis:
(1)小鼠的体重监测数据统计分析见图19。结果显示，与模型对照组(PBS)相比，化合物31、33、35、38、39、43、44、49、58、60均可有效降低小鼠体重，且降体重效果优于Semaglutide。(1) The statistical analysis of the weight monitoring data of mice is shown in Figure 19. The results showed that compared with the model control group (PBS), compounds 31, 33, 35, 38, 39, 43, 44, 49, 58, and 60 could effectively reduce the weight of mice, and the weight reduction effect was better than Semaglutide.
(2)小鼠的空腹血糖监测数据统计分析见图20。结果显示，与模型对照组(PBS)相比，化合物31、33、35、38、39、43、44、49、58、60和Semaglutide均能显著降低BKS-db糖尿病小鼠的空腹血糖水平，说明这些化合物均有显著的降糖效果。在第1周的时候血糖已经降到正常水平，且此后化合物31、33、35、38、39、43、44、49、58、60组的血糖相对稳定并且效果优于Semaglutide。(2) Statistical analysis of fasting blood glucose monitoring data of mice is shown in Figure 20. The results show that compared with the model control group (PBS), compounds 31, 33, 35, 38, 39, 43, 44, 49, 58, 60 and Semaglutide can significantly reduce the fasting blood glucose level of BKS-db diabetic mice, indicating that these compounds have significant hypoglycemic effects. In the first week, blood glucose has dropped to normal levels, and thereafter, the blood glucose of the compound 31, 33, 35, 38, 39, 43, 44, 49, 58, 60 group is relatively stable and the effect is better than Semaglutide.
实施例5：利西拉肽及其改造肽的药效学验证试验Example 5: Pharmacodynamics validation test of lixisenatide and its modified peptides
本发明合成的化合物62为已上市GLP-1受体激动剂多肽药物利西拉肽(Lixisenatide)，化合物63为本发明涉及的侧链Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2)18CO2H修饰在利西拉肽第12位Lys得到的化合物，化合物64为本发明涉及的侧链Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH2)18CO2H修饰在利西拉肽第26位Lys得到的化合物。研究化合物62、化合物63、化合物64对小鼠口服葡萄糖耐量(OGTT)的药效作用。Compound 62 synthesized by the present invention is the marketed GLP-1 receptor agonist polypeptide drug lixisenatide, compound 63 is a compound obtained by modifying the 12th Lys of lixisenatide with the side chain Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2 ) 18 CO 2 H of the present invention, and compound 64 is a compound obtained by modifying the 26th Lys of lixisenatide with the side chain Gly-Gly-Ser-Gly-Ser-Gly-Ser-Gly-γ-Glu-CO(CH 2 ) 18 CO 2 H of the present invention. The pharmacodynamic effects of compound 62, compound 63 and compound 64 on oral glucose tolerance (OGTT) in mice were studied.
实验方法：将8周龄左右的雄性C57BL/6J小鼠(购自广东斯嘉景达生物科技有限公司)，饲养一周适应环境，按照血糖随机分组，每组8只。各多肽化合物均按照80ug/kg的剂量， 皮下注射给药，对照组给予同等体积的PBS。给药当天晚上禁食16h，在给药24h后，按照2g/kg的剂量灌胃给予葡萄糖，并在t＝0min、t＝15min、t＝30min、t＝60min、t＝90min和t＝120min时检测血糖值。使用软件GraphPadPrism处理数据，绘制时间-血糖曲线图，计算血糖曲线下面积AUC。与未给药的PBS对照组进行One-way ANOVA分析，计算出显著性差异。Experimental method: Male C57BL/6J mice of about 8 weeks old (purchased from Guangdong Sijia Jingda Biotechnology Co., Ltd.) were fed for one week to adapt to the environment and randomly divided into groups according to blood sugar, with 8 mice in each group. Each peptide compound was administered at a dose of 80ug/kg. The drug was administered by subcutaneous injection, and the control group was given an equal volume of PBS. The subjects were fasted for 16 hours on the evening of the administration. 24 hours after administration, glucose was administered by gavage at a dose of 2g/kg, and blood glucose levels were tested at t=0min, t=15min, t=30min, t=60min, t=90min and t=120min. The data were processed using the software GraphPadPrism, and the time-blood glucose curve was plotted to calculate the area under the blood glucose curve AUC. One-way ANOVA analysis was performed with the PBS control group that was not administered, and significant differences were calculated.
结果分析：根据OGTT结果数据统计图(图21-22)得知：在给药24h后，与载剂(PBS)相比，化合物62(利西拉肽)能显著降低AUC，说明利西拉肽具有显著的葡萄糖耐受性，可有效降低血糖；而对利西拉肽进行侧链修饰后的化合物63、化合物64则对AUC没有显著的影响，说明仅仅依靠侧链修饰改造肽并不能改善葡萄糖耐受，不能有效降低血糖。Result analysis: According to the statistical diagram of OGTT result data (Figures 21-22), it was learned that: 24 hours after administration, compared with the vehicle (PBS), compound 62 (lixisenatide) could significantly reduce the AUC, indicating that lixisenatide has significant glucose tolerance and can effectively lower blood sugar; while compounds 63 and 64 after side chain modification of lixisenatide had no significant effect on AUC, indicating that peptide modification relying solely on side chain modification cannot improve glucose tolerance and cannot effectively lower blood sugar.
结论：将本申请涉及的侧链添加到利西拉肽，非但没有延长其降血糖药效，反而使原肽失去了降糖药效。因此可说明：本申请发明涉及的多肽化合物的侧链连接并不是普遍适用于其他肽，而是具有独自的创造性。Conclusion: Adding the side chain involved in this application to lixisenatide not only fails to prolong its hypoglycemic effect, but makes the original peptide lose its hypoglycemic effect. Therefore, it can be explained that the side chain connection of the polypeptide compound involved in the invention of this application is not generally applicable to other peptides, but has its own creativity.
实施例6：化合物33、化合物35、化合物38、化合物39、化合物43、化合物44、化合物58、化合物61在SD大鼠体内的药代动力学研究Example 6: Pharmacokinetic study of compound 33, compound 35, compound 38, compound 39, compound 43, compound 44, compound 58 and compound 61 in SD rats
将SD大鼠(SPF级，来源斯贝福(北京)生物技术有限公司，实验动物生产许可证号：SCXK(京)2019-0010，体重：180～200g，年龄：6～8周)，饲养一周适应环境，适应期检查动物的一般状态，不合格的动物不纳入本试验，饲养环境控制系统采用WINCC7.3 EMS系列机房环境监控系统，饲料使用SPF大小鼠维持饲料。将20只饲养合格的SD大鼠按照体重随机分组，每组4只(雌雄各半)，共5组(使用软件Stata 15对动物进行分组、统计分析)。SD rats (SPF grade, source: Sbefor (Beijing) Biotechnology Co., Ltd., experimental animal production license number: SCXK (Beijing) 2019-0010, weight: 180-200g, age: 6-8 weeks) were raised for one week to adapt to the environment. The general condition of the animals was checked during the adaptation period. Unqualified animals were not included in this experiment. The feeding environment control system used WINCC7.3 EMS series computer room environment monitoring system, and the feed used SPF mice and rats to maintain the feed. 20 qualified SD rats were randomly divided into groups according to their weight, with 4 rats in each group (half male and half female), for a total of 5 groups (animals were grouped and statistically analyzed using software Stata 15).
本实施例中化合物按照0.15mg/kg或0.2mg/kg的剂量，给药体积2mL/kg，给药浓度0.075mg/mL或0.1mg/mL，溶媒为PBS，进行单次的颈背部皮肤皮下给药。In this example, the compound was administered subcutaneously to the skin of the back of the neck at a dose of 0.15 mg/kg or 0.2 mg/kg, a dosing volume of 2 mL/kg, a dosing concentration of 0.075 mg/mL or 0.1 mg/mL, and a solvent of PBS.
单次皮下给药组大鼠在给药前(0h)和给药后0.5h、1h、2h、4h、6h、8h、24h、48h、72h、96h、120h、144h、168h、192h通过颈静脉进行血样采集，每个时间点采集量约0.2mL全血于EDTA-K2抗凝管中，1h内，在4℃和1800g离心力下离心10min，取上清，分离的血浆转移至-80℃冰箱保存。利用UPLC-MS/MS分别建立SD大鼠血浆中化 合物的浓度分析方法，测定血浆中化合物的药物浓度。数据处理采用WinNonlin 8.1软件计算药动学参数，实验结果如表2所示。Blood samples were collected from the jugular vein of rats in the single subcutaneous administration group before administration (0h) and 0.5h, 1h, 2h, 4h, 6h, 8h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, and 192h after administration. About 0.2mL of whole blood was collected at each time point and placed in an EDTA- K2 anticoagulant tube. Within 1h, the blood was centrifuged at 4℃ and 1800g for 10min, and the supernatant was taken. The separated plasma was transferred to a -80℃ refrigerator for storage. UPLC-MS/MS was used to establish the chemical and molecular weight distribution of plasma in SD rats. The concentration analysis method of the compound was used to determine the drug concentration of the compound in plasma. The data were processed using WinNonlin 8.1 software to calculate the pharmacokinetic parameters. The experimental results are shown in Table 2.
由实验结果可知，相比化合物61，单次皮下给药化合物33、化合物35、化合物38、化合物39、化合物43、化合物44、化合物58后，化合物在大鼠体内吸收较慢，达峰时间Tmax均为24h左右，明显高于化合物61的8h，半衰期t1/2平均分别为12.2h、17.8h、18.8h、22.3h、25.5h、21.7h、20.4h，明显高于化合物61的10.8h，且化合物39的半衰期能够达到22h以上。由此可以证实，本发明的长效多肽化合物具有更长的半衰期。From the experimental results, it can be seen that compared with compound 61, after a single subcutaneous administration of compound 33, compound 35, compound 38, compound 39, compound 43, compound 44, and compound 58, the compounds were absorbed more slowly in rats, and the peak time Tmax was about 24h, which was significantly higher than 8h of compound 61, and the half-life t1 /2 was 12.2h, 17.8h, 18.8h, 22.3h, 25.5h, 21.7h, and 20.4h on average, which was significantly higher than 10.8h of compound 61, and the half-life of compound 39 could reach more than 22h. It can be confirmed that the long-acting polypeptide compound of the present invention has a longer half-life.
表2 SD大鼠体内药代动力实验结果以及化合对应的给药剂量
Table 2 Results of pharmacokinetic experiments in SD rats and corresponding dosages of the compounds
以上仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以权利要求的保护范围为准。 The above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims (13)

  1. 一种新型长效多肽化合物，其特征在于，所述长效多肽化合物的氨基酸序列如下：A novel long-acting polypeptide compound, characterized in that the amino acid sequence of the long-acting polypeptide compound is as follows:
    X1-X2-X3-GTFTSDYSI-X13-LDKIAQ-X20-AFVQWL-X27-X28-GGPSSG-X35-PPPS-R1；X1-X2-X3-GTFTSDYSI-X13-LDKIAQ-X20-AFVQWL-X27-X28-GGPSSG-X35-PPPS-R 1 ;
    其中，X3选自E、Q或N；X27选自L或I；X28选自A或D；X35选自A或Aib；X20为K、K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)、K((PEG2)b-γGlu-CO(CH2)cCO2H)或K((AEEA)d-γGlu-CO(CH2)eCO2H)，其中，x为0-5的整数，z为1-5的整数，a为12-20的整数；b为1-8的整数，c为12-20的整数；d为1-8的整数，e为12-20的整数；R1选自OH或NH2；wherein X3 is selected from E, Q or N; X27 is selected from L or I; X28 is selected from A or D; X35 is selected from A or Aib; X20 is K, K( Gx (SG) Z -γGlu-CO ( CH2 ) aCO2H ), K(( PEG2 ) b -γGlu-CO( CH2 ) cCO2H ) or K((AEEA) d -γGlu-CO( CH2 ) eCO2H ), wherein x is an integer of 0-5 , z is an integer of 1-5, a is an integer of 12-20; b is an integer of 1-8, c is an integer of 12-20; d is an integer of 1-8, and e is an integer of 12-20; R1 is selected from OH or NH2 ;
    当X2选自Aib、Iva或Cba时：X1选自H或Y；X13选自Aib、Iva或Cba；When X2 is selected from Aib, Iva or Cba: X1 is selected from H or Y; X13 is selected from Aib, Iva or Cba;
    当X2为D-Ser时：X1为H；X13选自Aib、Iva或Cba。When X2 is D-Ser: X1 is H; X13 is selected from Aib, Iva or Cba.
  2. 根据权利要求1所述的长效多肽化合物，其特征在于，当X20选自K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)、K((PEG2)b-γGlu-CO(CH2)cCO2H)或K((AEEA)d-γGlu-CO(CH2)eCO2H)时，Gx(SG)Z-γGlu-CO(CH2)aCO2H、(PEG2)b-γGlu-CO(CH2)cCO2H或(AEEA)d-γGlu-CO(CH2)eCO2H通过与K的侧链氨基形成酰胺键与所述长效多肽化合物的主肽链连接。The long-acting polypeptide compound according to claim 1, characterized in that, when X20 is selected from K( Gx (SG) Z -γGlu-CO ( CH2 ) aCO2H ), K(( PEG2 ) b - γGlu -CO( CH2 ) cCO2H ) or K((AEEA) d -γGlu-CO( CH2 ) eCO2H ) , Gx (SG) Z -γGlu-CO( CH2 ) aCO2H , ( PEG2 ) b - γGlu -CO( CH2 ) cCO2H or (AEEA) d -γGlu-CO( CH2 ) eCO2H is connected to the main peptide chain of the long-acting polypeptide compound by forming an amide bond with the side chain amino group of K.
  3. 根据权利要求1所述的长效多肽化合物，其特征在于，X20为K(Gx(SG)Z-γGlu-CO(CH2)aCO2H)，其中x为2，z为2或3，a为16或18。The long-acting polypeptide compound according to claim 1, characterized in that X20 is K(G x (SG) Z -γGlu-CO(CH 2 ) a CO 2 H), wherein x is 2, z is 2 or 3, and a is 16 or 18.
  4. 根据权利要求1所述的长效多肽化合物，其特征在于，X35为A。The long-acting polypeptide compound according to claim 1, characterized in that X35 is A.
  5. 根据权利要求1所述的长效多肽化合物，其特征在于，X1为Y，X2选自Aib、Iva或Cba，X3为E，X13选自Aib、Iva或Cba。The long-acting polypeptide compound according to claim 1, characterized in that X1 is Y, X2 is selected from Aib, Iva or Cba, X3 is E, and X13 is selected from Aib, Iva or Cba.
  6. 根据权利要求5所述的长效多肽化合物，其特征在于，The long-acting polypeptide compound according to claim 5, characterized in that
    X2为Iva，X13为Aib或Iva；X2 is Iva, X13 is Aib or Iva;
    或者，X2为Aib，X13为Iva。 Alternatively, X2 is Aib and X13 is Iva.
  7. 根据权利要求1所述的长效多肽化合物，其特征在于，X1为H，X2选自Aib、Iva或Cba，X3选自E或Q；X13选自Aib、Iva或Cba。The long-acting polypeptide compound according to claim 1, characterized in that X1 is H, X2 is selected from Aib, Iva or Cba, X3 is selected from E or Q; X13 is selected from Aib, Iva or Cba.
  8. 根据权利要求7所述的长效多肽化合物，其特征在于，X2为Aib或Iva，X13为Iva。The long-acting polypeptide compound according to claim 7, characterized in that X2 is Aib or Iva, and X13 is Iva.
  9. 根据权利要求1所述的长效多肽化合物，其特征在于，第9位氨基酸D与第16位氨基酸K通过酰胺键连接。The long-acting polypeptide compound according to claim 1, characterized in that the 9th amino acid D and the 16th amino acid K are connected by an amide bond.
  10. 根据权利要求1所述的长效多肽化合物，其特征在于，所述长效多肽化合物选自如下化合物中的任一种：The long-acting polypeptide compound according to claim 1, characterized in that the long-acting polypeptide compound is selected from any one of the following compounds:
    化合物1：Compound 1:
    Y-Aib-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSG-Aib-PPPS-OH；Y-Aib-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSG-Aib-PPPS-OH;
    化合物2：Compound 2:
    Y-Aib-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Aib-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物3：Compound 3:
    Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物4：Compound 4:
    Y-Iva-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH;
    化合物5：Compound 5:
    Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH；Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH;
    化合物6：Compound 6:
    Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH;
    化合物7：Compound 7:
    Y-Iva-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物8：Compound 8:
    Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Aib-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物9： Compound 9:
    Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物10：Compound 10:
    Y-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物11：Compound 11:
    Y-Cba-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Cba-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物12：Compound 12:
    Y-Cba-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Cba-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物13：Compound 13:
    Y-Aib-EGTFTSDYSI-Cba-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Aib-EGTFTSDYSI-Cba-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物14：Compound 14:
    Y-Iva-EGTFTSDYSI-Cba-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；Y-Iva-EGTFTSDYSI-Cba-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物15：Compound 15:
    H-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH；H-Aib-EGTFTSDYSI-Aib-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH;
    化合物16：Compound 16:
    H-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH；H-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH;
    化合物17：Compound 17:
    H-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLAGGPSSGAPPPS-OH;
    化合物18：Compound 18:
    H-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；H-Iva-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物19：Compound 19:
    H-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物20：Compound 20:
    H-Cba-QGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH；H-Cba-QGTFTSDYSI-Aib-LDKIAQKAFVQWLIAGGPSSGAPPPS-OH;
    化合物21：Compound 21:
    H-Cba-QGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH； H-Cba-QGTFTSDYSI-Iva-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物22：Compound 22:
    H-(D-Ser)-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH；H-(D-Ser)-QGTFTSDYSI-Aib-LDKIAQKAFVQWLLDGGPSSGAPPPS-OH;
    化合物23：Compound 23:
    Y-Iva-EGTFTS(D)YSI-Iva-LD(K)IAQKAFVQWLIAGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Iva-EGTFTS (D) YSI-Iva-LD (K) IAQKAFVQWLIAGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
    化合物24：Compound 24:
    Y-Aib-EGTFTS(D)YSI-Iva-LD(K)IAQKAFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Aib-EGTFTS (D) YSI-Iva-LD (K) IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
    化合物25：Compound 25:
    Y-Iva-EGTFTS(D)YSI-Iva-LD(K)IAQKAFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Iva-EGTFTS (D) YSI-Iva-LD (K) IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
    化合物26：Compound 26:
    H-Aib-QGTFTS(D)YSI-Aib-LD(K)IAQKAFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；H-Aib-QGTFTS (D) YSI-Aib-LD (K) IAQKAFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
    化合物27：Compound 27:
    H-Iva-EGTFTS(D)YSI-Aib-LD(K)IAQKAFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；H-Iva-EGTFTS (D) YSI-Aib-LD (K) IAQKAFVQWLLDGGPSSGAPPPS-OH, in which the 9th amino acid D is linked to the 16th amino acid K through an amide bond;
    化合物28：Compound 28:
    Y-Aib-QGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSG-Aib-PPPS-NH2；Y-Aib-QGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSG-Aib-PPPS-NH 2 ;
    化合物29：Compound 29:
    Y-Aib-QGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Aib-QGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物30： Compound 30:
    Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物31：Compound 31:
    Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
    化合物32：Compound 32:
    Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(PEG2-PEG2-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
    化合物33：Compound 33:
    Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
    化合物34：Compound 34:
    Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(PEG2-PEG2-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(PEG 2 -PEG 2 -γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
    化合物35：Compound 35:
    Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
    化合物36：Compound 36:
    Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(AEEA-AEEA-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 ;
    化合物37：Compound 37:
    Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物38： Compound 38:
    Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物39：Compound 39:
    Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物40：Compound 40:
    Y-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物41：Compound 41:
    Y-Cba-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Cba-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物42：Compound 42:
    Y-Cba-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Cba-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物43：Compound 43:
    Y-Aib-EGTFTSDYSI-Cba-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Aib-EGTFTSDYSI-Cba-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物44：Compound 44:
    Y-Iva-EGTFTSDYSI-Cba-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2；Y-Iva-EGTFTSDYSI-Cba-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 ;
    化合物45：Compound 45:
    H-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLAGGPSSGAPPPS-OH；H-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLAGGPSSGAPPPS-OH;
    化合物46： Compound 46:
    H-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLAGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLAGGPSSGAPPPS-OH;
    化合物47：Compound 47:
    H-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLAGGPSSGAPPPS-OH；H-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLAGGPSSGAPPPS-OH;
    化合物48：Compound 48:
    H-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLAGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLAGGPSSGAPPPS-OH;
    化合物49：Compound 49:
    H-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH；H-Iva-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH;
    化合物50：Compound 50:
    H-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH；H-Iva-EGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH;
    化合物51：Compound 51:
    H-Cba-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-OH；H-Cba-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-OH;
    化合物52：Compound 52:
    H-Cba-QGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH；H-Cba-QGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH;
    化合物53：Compound 53:
    H-Aib-QGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSG-Aib-PPPS-OH；H-Aib-QGTFTSDYSI-Iva-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSG-Aib-PPPS-OH;
    化合物54：Compound 54:
    H-(D-Ser)-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH； H-(D-Ser)-QGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH;
    化合物55：Compound 55:
    Y-Iva-EGTFTS(D)YSI-Iva-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Iva-EGTFTS (D) YSI-Iva-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 , wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
    化合物56：Compound 56:
    Y-Aib-EGTFTS(D)YSI-Iva-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Aib-EGTFTS (D) YSI-Iva-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 , wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
    化合物57：Compound 57:
    Y-Iva-EGTFTS(D)YSI-Iva-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-NH2，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；Y-Iva-EGTFTS (D) YSI-Iva-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-NH 2 , wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
    化合物58：Compound 58:
    H-Aib-QGTFTS(D)YSI-Aib-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；H-Aib-QGTFTS (D) YSI-Aib-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
    化合物59：Compound 59:
    H-Iva-EGTFTS(D)YSI-Aib-LD(K)IAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLLDGGPSSGAPPPS-OH，其中，第9位氨基酸D与第16位氨基酸K通过酰胺键连接；H-Iva-EGTFTS (D) YSI-Aib-LD (K) IAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLLDGGPSSGAPPPS-OH, wherein the 9th amino acid D is linked to the 16th amino acid K via an amide bond;
    化合物60：Compound 60:
    Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH2)18CO2H)AFVQWLIAGGPSSGAPPPS-NH2。Y-Aib-EGTFTSDYSI-Aib-LDKIAQK(GGSGSGSG-γGlu-CO(CH 2 ) 18 CO 2 H)AFVQWLIAGGPSSGAPPPS-NH 2 .
  11. 根据权利要求1所述的长效多肽化合物，其特征在于，所述长效多肽化合物为药学上可接受的盐。The long-acting polypeptide compound according to claim 1, characterized in that the long-acting polypeptide compound is a pharmaceutically acceptable salt.
  12. 一种组合物，其特征在于，所述组合物包含权利要求1-11任一所述的长效多肽化合物和药学上可接受的载体或辅料。A composition, characterized in that the composition comprises the long-acting polypeptide compound according to any one of claims 1 to 11 and a pharmaceutically acceptable carrier or excipient.
  13. 权利要求1-11任一所述的长效多肽化合物或权利要求12所述的组合物在制备用于预防或治疗糖尿病的药物中的应用； Use of the long-acting polypeptide compound according to any one of claims 1 to 11 or the composition according to claim 12 in the preparation of a medicament for preventing or treating diabetes;
    或者，所述长效多肽化合物或所述组合物在制备用于预防或治疗肥胖的药物中的应用。 Alternatively, the long-acting polypeptide compound or the composition is used in the preparation of a drug for preventing or treating obesity.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016065090A1 (en) * 2014-10-24 2016-04-28 Merck Sharp & Dohme Corp. Co-agonists of the glucagon and glp-1 receptors
CN107207576A (en) * 2015-01-09 2017-09-26 伊莱利利公司 GIP and the co-agonists compounds of GLP 1
WO2021169512A1 (en) * 2020-02-24 2021-09-02 中山大学 Polypeptide compound and application thereof in prevention or treatment of diabetes or diabetes complication
CN113429471A (en) * 2021-07-19 2021-09-24 青岛博睿精创科技有限公司 Long-acting GLP-1 polypeptide analogue and preparation method and application thereof
WO2021260530A1 (en) * 2020-06-22 2021-12-30 Sun Pharmaceutical Industries Limited Long acting glp-1/gip dual agonists
WO2022079639A1 (en) * 2020-10-17 2022-04-21 Sun Pharmaceutical Industries Limited Glp-1/gip dual agonists

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016065090A1 (en) * 2014-10-24 2016-04-28 Merck Sharp & Dohme Corp. Co-agonists of the glucagon and glp-1 receptors
CN107108715A (en) * 2014-10-24 2017-08-29 默沙东公司 The co-agonists of hyperglycemic factor and the acceptors of GLP 1
CN107207576A (en) * 2015-01-09 2017-09-26 伊莱利利公司 GIP and the co-agonists compounds of GLP 1
WO2021169512A1 (en) * 2020-02-24 2021-09-02 中山大学 Polypeptide compound and application thereof in prevention or treatment of diabetes or diabetes complication
WO2021260530A1 (en) * 2020-06-22 2021-12-30 Sun Pharmaceutical Industries Limited Long acting glp-1/gip dual agonists
WO2022079639A1 (en) * 2020-10-17 2022-04-21 Sun Pharmaceutical Industries Limited Glp-1/gip dual agonists
CN113429471A (en) * 2021-07-19 2021-09-24 青岛博睿精创科技有限公司 Long-acting GLP-1 polypeptide analogue and preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, J. ET AL.: "Efficient synthesis of Aib 8 -Arg 34 -GLP-1 (7–37) by liquid-phase fragment condensation", J PEPT SCI., vol. 28, 22 January 2022 (2022-01-22) *

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