WO2023005841A1 - Polypeptide compounds containing lactam bridges - Google Patents
Polypeptide compounds containing lactam bridges Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
Definitions
- the invention relates to a series of polypeptide compounds containing lactam bridges and their application in the preparation of medicines for treating related diseases.
- CVD cardiovascular disease
- Glucagon-like peptide-1 (GLP-1) protects islet ⁇ cells in islets, stimulates islet ⁇ cells to release insulin in a glucose-dependent manner, and effectively controls postprandial blood sugar. Because of its unique mechanism of action, the risk of hypoglycemia is greatly reduced. Although GLP-1R agonists have demonstrated excellent hypoglycemic effects in clinical practice, there are still many type 2 diabetics who have not achieved their hypoglycemic and weight loss goals.
- GLP-1R glucose-dependent insulinotropic polypeptide receptor
- GCGR glucagon receptor
- Glucose-dependent insulinotropic polypeptide receptor is a polypeptide secreted by neuroendocrine K cells of the small intestine. Its physiological effects are mediated by GIPR, mainly non-glucose-dependent stimulation of insulin secretion, enhancement of glucagon secretion, and enhancement of lipid metabolism. Although the beneficial effects of GIPR agonists appear to be attenuated in hyperglycemic symptoms in patients with type 2 diabetes, studies have shown that the diminished insulinotropic effect of GIP can be fully restored after a period of normalization of plasma glucose levels. Therefore, introducing GIPR agonist activity into GLP-1R agonists may obtain better hypoglycemic effect.
- Glucagon is secreted by the pancreas and combines with GCGR to produce a hormone with physiological functions. Glucagon promotes the rise of blood sugar by increasing gluconeogenesis and glycogenolysis. In addition, GCG can also reduce fatty acid synthesis in liver adipose tissue and promote fat decomposition. The introduction of GCGR agonistic activity into GLP-1R agonists can be more beneficial to patients' weight control. In summary, the development of GLP-1R/GIPR/GCGR triple agonists has great medical prospects for the treatment of diabetes, obesity and related diseases.
- the invention provides a polypeptide (SEQ ID NO: 1):
- a lactam bridge is formed between the amino acid side chains at positions i and i+4 or between the amino acid side chains at positions j and j+3, wherein i is 16, 20 or 24, and j is 17;
- K 0 represents lysine, and the amino group on the lysine side chain is linked to -X 0 ;
- n are each independently 1, 2 or 3;
- p 8, 9 or 10.
- the invention provides a polypeptide (SEQ ID NO: 1):
- a lactam bridge is formed between the amino acid side chains at positions i and i+4 or between the amino acid side chains at positions j and j+3, wherein i is 16, 20 or 24, and j is 17;
- K 0 represents lysine, and the amino group on the lysine side chain is linked to -X 0 ;
- n are each independently 1, 2 or 3;
- p 8, 9 or 10.
- the C-terminus of the amino acid at position 39 is amidated, and other variables are as defined in the present invention.
- m and n are each independently 2, and other variables are as defined in the present invention.
- the above-X is selected from
- polypeptide is selected from:
- a lactam bridge is formed between the amino acid side chains at positions i and i+4 or between the amino acid side chains at positions j and j+3, wherein i is 16, 20 or 24, and j is 17;
- Aib and K0 are as defined herein.
- the present invention also provides a polypeptide represented by the following formula,
- the present invention also provides the above-mentioned pharmaceutical composition, which comprises, as an active ingredient, a therapeutically effective amount of the above-mentioned polypeptide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
- the application of the above-mentioned polypeptide compound or its pharmaceutically acceptable salt or the above-mentioned composition in the preparation of a medicament for treating type 2 diabetes.
- the polypeptide of the present invention has strong agonistic activity to GIP/GLP-1/GCG; among them, there is a large difference in the agonistic activity of different compounds for GCG, which will be of guiding significance for the study of the balance of the three target activities; the compound of the present invention has Excellent pharmacokinetic properties and in vivo efficacy.
- pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
- pharmaceutically acceptable salt refers to a salt of a compound of the present invention, which is prepared from a compound having a specific substituent found in the present invention and a relatively non-toxic acid or base.
- base addition salts can be obtained by contacting such compounds with a sufficient amount of base, either neat solution or in a suitable inert solvent.
- acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent.
- Certain specific compounds of the present invention contain basic and acidic functional groups and can thus be converted into either base or acid addition salts.
- the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods.
- such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
- amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids.
- Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
- Amino acid analogs are compounds that have the same basic chemical structure (such as the alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, formazine Thionine sulfoxide, methionine methylsulfonium.
- Such analogs can have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
- Amino acid mimetics are chemical compounds whose structure differs from the general chemical structure of amino acids, but which function similarly to naturally occurring amino acids.
- a or Ala described herein represents alanine, and the structure is R or Arg means arginine, the structure is N or Asn means asparagine, the structure is D or Asp means aspartic acid, the structure is C or Cys means cysteine, the structure is Q or Gln means glutamine, the structure is E or Glu means glutamic acid, the structure is G or Gly means glycine, the structure is H or His means histidine, the structure is I or Ile means isoleucine, the structure is L or Leu means leucine, the structure is ⁇ -MeL or ⁇ -MeLeu means ⁇ -methylleucine, the structure is K or Lys means lysine, the structure is M or Met means methionine, the structure is F or Phe means phenylalanine, the structure is P or Pro means proline, the structure is S or Ser represents serine, the structure is T or Thr means threonine, the structure is W or Trp means tryptophan, the
- treating includes inhibiting, slowing, stopping or reversing the progression or severity of an existing symptom or condition.
- the compounds of the invention may exist in particular geometric or stereoisomeric forms.
- the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention.
- Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
- enantiomer or “optical isomer” refer to stereoisomers that are mirror images of each other.
- cis-trans isomers or “geometric isomers” arise from the inability to rotate freely due to the double bond or the single bond of the carbon atoms forming the ring.
- diastereoisomer refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of the molecules.
- keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key or straight dotted key
- the terms “enriched in an isomer”, “enriched in an isomer”, “enriched in an enantiomer” or “enantiomerically enriched” refer to one of the isomers or enantiomers
- the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
- the terms “isomer excess” or “enantiomeric excess” refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomeric excess (ee value) is 80% .
- Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
- a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
- the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds.
- compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
- radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
- heavy hydrogen can be used to replace hydrogen to form deuterated drugs.
- the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
- deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
- linking group listed does not indicate its linking direction
- its linking direction is arbitrary, for example,
- the connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right
- any one or more sites of the group can be linked to other groups through chemical bonds.
- connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group.
- the chemical bonds that the site is connected with other groups can use straight solid line bonds Straight dotted key or tilde express.
- the straight solid-line bond in -OCH 3 indicates that it is connected to other groups through the oxygen atom in the group;
- the straight dotted line bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups;
- the wavy lines in indicate that the 1 and 2 carbon atoms in the phenyl group are connected to other groups.
- the structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuK ⁇ radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
- SXRD single crystal X-ray diffraction
- the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
- the solvent used in the present invention is commercially available.
- the polypeptide WX002 was obtained.
- the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4902.6, and the detected value was 4902.3.
- the polypeptide WX003 was obtained.
- the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4915.7, and the detected value was 4915.8.
- the polypeptide WX004 was obtained.
- the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4910.6, and the detected value was 4910.4.
- the polypeptide WX005 was obtained.
- the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4968.7, and the detected value was 4968.6.
- the polypeptide WX006 was obtained.
- the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4901.5, and the detected value was 4901.4.
- the polypeptide WX007 was obtained.
- the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4857.5, and the detected value was 4858.2.
- the polypeptide WX008 was obtained.
- the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4901.5, and the detected value was 4902.0.
- Example 1 In vitro GLP-1R/GIPR/GCGR agonistic activity test
- the cell line was constructed by Shanghai WuXi PharmaTech. See Table 1 below for details.
- HSA Human Serum Albumin
- IBMX 3-Isobutyl-1-methylxanthine
- the compound to be tested is diluted 4 times at 10 points, the initial concentration is 30 ⁇ M, and Bravo completes the dilution
- the compound of the present invention has strong agonistic activity on GLP-1R/GIPR/GCGR. Among them, there are large differences in the agonistic activity of different compounds for GCG, which will be of guiding significance for the study of the balance of the activities of the three targets.
- the compounds of the present invention have excellent effects on improving glucose tolerance.
- the temperature in the breeding room is maintained at 20-24° C., and the humidity is maintained at 30-70%.
- the temperature and humidity in the feeding room were monitored in real time by a thermo-hygrometer, and the temperature and humidity were recorded twice a day (once in the morning and once in the afternoon).
- the daylighting in the animal breeding room is controlled by an electronic timing lighting system, and the lights are turned on for 12 hours and turned off for 12 hours every day (turn on at 7:00 in the morning and turn off at 19:00 in the afternoon).
- the animals were kept in single cages, and toys were provided for each cage.
- the animals had free access to food (feed for growth/breeding of rats and mice) and drinking water.
- Each group of animals was subcutaneously injected with the vehicle and the compound to be tested (10 nmol/kg), the administration time: 9:30 in the morning, the administration frequency was once every three days, and the administration cycle was 22 days.
- the compounds of the present invention exhibit excellent weight loss efficacy in DIO mice.
- mice After the db/db mice arrive at the facility, they are kept in an animal breeding room with strictly controlled environmental conditions.
- the temperature in the breeding room is maintained at 20-24° C., and the humidity is maintained at 30-70%.
- the temperature and humidity in the feeding room were monitored in real time by a thermo-hygrometer, and the temperature and humidity were recorded twice a day (once in the morning and once in the afternoon).
- the daylighting in the animal breeding room is controlled by an electronic timing lighting system, and the lights are turned on for 12 hours and turned off for 12 hours every day (turn on at 7:00 in the morning and turn off at 19:00 in the afternoon).
- the animals were kept in single cages, and toys were provided for each cage. During the experiment, the animals had free access to food (feed for growth/breeding of rats and mice) and drinking water.
- the vehicle and the compound to be tested (15 nmol/kg) were subcutaneously injected into each group of animals respectively, the administration time: 9:30-11:00 in the morning, the administration frequency was once a day, and the administration was continued for 4 weeks.
- the compound of the present invention exhibits excellent hypoglycemic efficacy in db/db mice.
- the pharmacokinetic characteristics of the compounds in rodents after subcutaneous injection were tested according to the standard protocol.
- the candidate compounds were made into clear solutions and given to mice for subcutaneous injection (SC, 10 nmol/kg).
- Whole blood was collected, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
- the compounds of the present invention have excellent pharmacokinetic properties in mice.
- the pharmacokinetic characteristics of the compounds in rodents after subcutaneous injection were tested according to the standard protocol.
- the candidate compounds were made into a clear solution and given to rats for a single subcutaneous injection (SC, 10nmol/kg).
- Whole blood was collected, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
- the compounds of the present invention have excellent pharmacokinetic properties in rats.
- the mammalian pharmacokinetic characteristics of the compound after subcutaneous injection were tested according to the standard protocol.
- the candidate compound was formulated into a clear solution and given to cynomolgus monkeys for single subcutaneous injection (SC, 4nmol/kg).
- Whole blood was collected, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
- the compound of the present invention has excellent monkey pharmacokinetic properties.
Abstract
A series of polypeptide compounds containing lactam bridges and the use in the preparation of a drug for treating related diseases.
Description
本申请要求申请日为2021/07/30的中国专利申请CN2021108730549、申请日为2021/09/16日的中国专利申请CN202111087337.7、申请日为2022/02/15的中国专利申请CN202210138834.3、申请日为2022/07/13日的中国专利申请CN202210828556.4的优先权。本申请引用上述中国专利申请的全文。This application requires the Chinese patent application CN2021108730549 with the filing date of 2021/07/30, the Chinese patent application CN202111087337.7 with the filing date of 2021/09/16, the Chinese patent application CN202210138834.3 with the filing date of 2022/02/15, The priority of Chinese patent application CN202210828556.4 with the filing date of 2022/07/13. This application cites the full text of the above-mentioned Chinese patent application.
本发明涉及一系列含内酰胺桥的多肽化合物，及其在制备治疗相关病症的药物中的应用。The invention relates to a series of polypeptide compounds containing lactam bridges and their application in the preparation of medicines for treating related diseases.
随着经济发展与生活方式的改变，糖尿病患病率居高不下，部分患者通常伴有肥胖或心血管疾病(CVD)。With economic development and changes in lifestyle, the prevalence of diabetes remains high, and some patients are usually accompanied by obesity or cardiovascular disease (CVD).
胰高血糖素样肽-1(GLP-1)在胰岛中发挥保护胰岛β细胞的作用，以葡萄糖依赖的方式刺激胰岛β细胞释放胰岛素，有效控制餐后血糖。因其独特的作用机制，低血糖风险大降低。虽然GLP-1R激动剂在临床中展现了优秀的降糖效果，但还是有很多二型糖尿病人未达到降糖及减重目标。因此，在胰高血糖素样肽-1受体(GLP-1R)激动剂基础上，引入葡萄糖依赖性促胰岛素多肽受体(GIPR)和胰高血糖素受体(GCGR)激动活性，提高药效，开发一款GLP-1R/GIPR/GCGR三重激动剂用于二型糖尿病治疗是一个迫切且有前景的需求。Glucagon-like peptide-1 (GLP-1) protects islet β cells in islets, stimulates islet β cells to release insulin in a glucose-dependent manner, and effectively controls postprandial blood sugar. Because of its unique mechanism of action, the risk of hypoglycemia is greatly reduced. Although GLP-1R agonists have demonstrated excellent hypoglycemic effects in clinical practice, there are still many type 2 diabetics who have not achieved their hypoglycemic and weight loss goals. Therefore, on the basis of glucagon-like peptide-1 receptor (GLP-1R) agonists, glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon receptor (GCGR) agonistic activities were introduced to improve drug Therefore, it is an urgent and promising need to develop a GLP-1R/GIPR/GCGR triple agonist for the treatment of type 2 diabetes.
葡萄糖依赖性促胰岛素多肽受体(GIP)是由小肠的神经内分泌K细胞分泌的多肽。由GIPR介导其生理作用，主要为非葡萄糖依赖的促胰岛素分泌、增强胰高血糖素分泌、增强脂质代谢等。虽然GIPR激动剂的有益作用似乎在2型糖尿病患者的高血糖症状中减弱，但研究表明，GIP减弱的促胰岛素分泌作用可以在血浆葡萄糖水平恢复正常一段时间后完全恢复。因此在GLP-1R激动剂中引入GIPR激动活性将有可能获得更优的降糖效果。Glucose-dependent insulinotropic polypeptide receptor (GIP) is a polypeptide secreted by neuroendocrine K cells of the small intestine. Its physiological effects are mediated by GIPR, mainly non-glucose-dependent stimulation of insulin secretion, enhancement of glucagon secretion, and enhancement of lipid metabolism. Although the beneficial effects of GIPR agonists appear to be attenuated in hyperglycemic symptoms in patients with type 2 diabetes, studies have shown that the diminished insulinotropic effect of GIP can be fully restored after a period of normalization of plasma glucose levels. Therefore, introducing GIPR agonist activity into GLP-1R agonists may obtain better hypoglycemic effect.
胰高血糖素(GCG)是由胰腺分泌的，与GCGR结合产生生理功能的激素。胰高血糖素通过增加糖异生及糖原分解的方式促进血糖的升高。另外，GCG还可以减少肝脏脂肪组织中的脂肪酸合成，促进脂肪的分解。GLP-1R激动剂中引入GCGR激动活性能够更有利于患者控制体重。综上所述，开发GLP-1R/GIPR/GCGR三重激动剂对于治疗糖尿病，肥胖及相关疾病具有很大的医疗前景。Glucagon (GCG) is secreted by the pancreas and combines with GCGR to produce a hormone with physiological functions. Glucagon promotes the rise of blood sugar by increasing gluconeogenesis and glycogenolysis. In addition, GCG can also reduce fatty acid synthesis in liver adipose tissue and promote fat decomposition. The introduction of GCGR agonistic activity into GLP-1R agonists can be more beneficial to patients' weight control. In summary, the development of GLP-1R/GIPR/GCGR triple agonists has great medical prospects for the treatment of diabetes, obesity and related diseases.
发明内容Contents of the invention
本发明提供了一种多肽(SEQ ID NO:1)：The invention provides a polypeptide (SEQ ID NO: 1):
SEQ ID NO:1 YAibQGT FTSDY SIα-MeLLD KKAQAib AFIEY LLEGG PSSGA PPPSSEQ ID NO:1 YAibQGT FTSDY SIα-MeLLD KKAQAib AFIEY LLEGG PSSGA PPPS
其具有以下修饰：It has the following modifications:
1)上述第17位或第24位的氨基酸有且只有一个被替换为K
0；和
1) one and only one of the amino acids at the 17th or 24th position above is replaced by K 0 ; and
2)SEQ ID NO:1的1-4个氨基酸被替换；和2) 1-4 amino acids of SEQ ID NO: 1 are replaced; and
3)i和i+4位置的氨基酸侧链之间或j和j+3位置的氨基酸侧链之间形成内酰胺桥，其中，i为16、20或 24，j为17；3) A lactam bridge is formed between the amino acid side chains at positions i and i+4 or between the amino acid side chains at positions j and j+3, wherein i is 16, 20 or 24, and j is 17;
其中，in,
Aib的结构为
The structure of Aib is
K
0表示赖氨酸，且该赖氨酸侧链上的氨基与-X
0相连；
K 0 represents lysine, and the amino group on the lysine side chain is linked to -X 0 ;
X
0选自
X 0 selected from
m和n分别独立地为1、2或3；m and n are each independently 1, 2 or 3;
p为8、9或10。p is 8, 9 or 10.
本发明提供了一种多肽(SEQ ID NO:1)：The invention provides a polypeptide (SEQ ID NO: 1):
SEQ ID NO:1 YAibQGT FTSDY SIα-MeLLD KKAQAib AFIEY LLEGG PSSGA PPPSSEQ ID NO:1 YAibQGT FTSDY SIα-MeLLD KKAQAib AFIEY LLEGG PSSGA PPPS
其具有以下修饰：It has the following modifications:
1)上述第17或24位的氨基酸有且只有一个替换为K
0；
1) There is one and only one replacement of the amino acid at the 17th or 24th position above with K 0 ;
2)SEQ ID NO:1的1-4个氨基酸被替换；2) 1-4 amino acids of SEQ ID NO:1 are replaced;
3)i和i+4位置的氨基酸侧链之间或j和j+3位置的氨基酸侧链之间形成内酰胺桥，其中，i为16、20或24，j为17；3) A lactam bridge is formed between the amino acid side chains at positions i and i+4 or between the amino acid side chains at positions j and j+3, wherein i is 16, 20 or 24, and j is 17;
其中，in,
Aib的结构为
The structure of Aib is
K
0表示赖氨酸，且该赖氨酸侧链上的氨基与-X
0相连；
K 0 represents lysine, and the amino group on the lysine side chain is linked to -X 0 ;
X
0选自
X 0 selected from
m和n分别独立地为1、2或3；m and n are each independently 1, 2 or 3;
p为8、9或10。p is 8, 9 or 10.
在本发明的一些方案中，上述第39位的氨基酸C-末端酰胺化，其他变量如本发明所定义。In some embodiments of the present invention, the C-terminus of the amino acid at position 39 is amidated, and other variables are as defined in the present invention.
在本发明的一些方案中，上述m和n分别独立地为2，其他变量如本发明所定义。In some solutions of the present invention, the aforementioned m and n are each independently 2, and other variables are as defined in the present invention.
在本发明的一些方案中，上述p为9，其他变量如本发明所定义。In some solutions of the present invention, the above p is 9, and other variables are as defined in the present invention.
在本发明的一些方案中，上述-X
0选自
In some solutions of the present invention, the above-X is selected from
本发明还有一些方案由上述变量任意组合而来。Some solutions of the present invention are formed by any combination of the above variables.
在本发明的一些方案中，上述多肽，其选自：In some aspects of the present invention, the above-mentioned polypeptide is selected from:
(I-1)YAibQGT FTSDY SIα-MeLLD KK
0AQK AFIEY LLEGG PSSGA PPPS-NH
2(SEQ ID NO:2)
(I-1) YAibQGT FTSDY SIα-MeLLD KK 0 AQK AFIEY LLEGG PSSGA PPPS-NH 2 (SEQ ID NO: 2)
(I-2)YAibEGT FTSDY SIα-MeLLD KK
0AQK AFIEY LLEGG PSSGA PPPS-NH
2(SEQ ID NO:3)
(I-2) YAibEGT FTSDY SIα-MeLLD KK 0 AQK AFIEY LLEGG PSSGA PPPS-NH 2 (SEQ ID NO: 3)
(I-3)YAibQGT FTSDY SIα-MeLLE KK
0AQK AFIEY LLEGG PSSGA PPPS-NH
2(SEQ ID NO:4)
(I-3) YAibQGT FTSDY SIα-MeLLE KK 0 AQK AFIEY LLEGG PSSGA PPPS-NH 2 (SEQ ID NO: 4)
(I-4)YAibQGT FTSDY SIα-MeLLD KK
0AQK AFVEW LLEGG PSSGA PPPS-NH
2(SEQ ID NO:5)
(I-4) YAibQGT FTSDY SIα-MeLLD KK 0 AQK AFVEW LLEGG PSSGA PPPS-NH 2 (SEQ ID NO: 5)
(I-5)YAibQGT FTSDY SIα-MeLLD KK
0AQK EFVEW LLEGG PSSGA PPPS-NH
2(SEQ ID NO:6)
(I-5) YAibQGT FTSDY SIα-MeLLD KK 0 AQK EFVEW LLEGG PSSGA PPPS-NH 2 (SEQ ID NO: 6)
(I-6)YAibQGT FTSDY SIα-MeLLD KEAQK AFIK
0Y LLEGG PSSGA PPPS-NH
2(SEQ ID NO:7)
(I-6) YAibQGT FTSDY SIα-MeLLD KEAQK AFIK 0 Y LLEGG PSSGA PPPS-NH 2 (SEQ ID NO: 7)
(I-7)YAibQGT FTSDY SIα-MeLLD KK
0AQAib AFIEY LLKGG PSSGA PPPS-NH
2(SEQ ID NO:8)
(I-7) YAibQGT FTSDY SIα-MeLLD KK 0 AQAib AFIEY LLKGG PSSGA PPPS-NH 2 (SEQ ID NO: 8)
(I-8)YAibQGT FTSDY SIα-MeLLD EKAQK AFIK
0Y LLEGG PSSGA PPPS-NH
2(SEQ ID NO:9)
(I-8) YAibQGT FTSDY SIα-MeLLD EKAQK AFIK 0 Y LLEGG PSSGA PPPS-NH 2 (SEQ ID NO:9)
其中，in,
i和i+4位置的氨基酸侧链之间或j和j+3位置的氨基酸侧链之间形成内酰胺桥，其中，i为16、20或24，j为17；A lactam bridge is formed between the amino acid side chains at positions i and i+4 or between the amino acid side chains at positions j and j+3, wherein i is 16, 20 or 24, and j is 17;
Aib和K
0如本发明所定义。
Aib and K0 are as defined herein.
本发明还提供了下式所示多肽，The present invention also provides a polypeptide represented by the following formula,
本发明还提供了上述药物组合物，包括作为活性成分的治疗有效量根据上述的多肽化合物或其药学上可接受的盐以及药学上可接受的载体。The present invention also provides the above-mentioned pharmaceutical composition, which comprises, as an active ingredient, a therapeutically effective amount of the above-mentioned polypeptide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
在本发明的一些方案中，上述多肽化合物或其药学上可接受的盐或者上述的组合物在制备治疗二型糖尿病的药物上的应用。In some aspects of the present invention, the application of the above-mentioned polypeptide compound or its pharmaceutically acceptable salt or the above-mentioned composition in the preparation of a medicament for treating type 2 diabetes.
技术效果technical effect
本发明的多肽对GIP/GLP-1/GCG具有很强的激动活性；其中，不同化合物对于GCG的激动活性存在较大差异，对于研究三靶点活性的平衡将具有指导意义；本发明化合物具有优异的药代动力学性质和体内药效。The polypeptide of the present invention has strong agonistic activity to GIP/GLP-1/GCG; among them, there is a large difference in the agonistic activity of different compounds for GCG, which will be of guiding significance for the study of the balance of the three target activities; the compound of the present invention has Excellent pharmacokinetic properties and in vivo efficacy.
定义和说明Definition and Description
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A specific term or phrase should not be considered indeterminate or unclear if it is not specifically defined, but should be understood according to its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding trade name or its active ingredient.
这里所采用的术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is prepared from a compound having a specific substituent found in the present invention and a relatively non-toxic acid or base. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base, either neat solution or in a suitable inert solvent. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat solution or in a suitable inert solvent. Certain specific compounds of the present invention contain basic and acidic functional groups and can thus be converted into either base or acid addition salts.
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods. In general, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
“氨基酸”是指天然存在的和合成的氨基酸，以及起到与天然存在的氨基酸类似的作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸，以及后来修饰的那些氨基酸，例如，羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指具有与天然存在的氨基酸相同的基本化学结构(例如与氢、羧基基团、氨基基团和R基团结合的α碳)的化合物，例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。这样的类似物可以具有修饰的R基团(例如，正亮氨酸)或修饰的肽骨架，但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指其结构不同于一般的氨基酸化学结构，但起到与天然存在的氨基酸相似的作用的化学化合物。"Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure (such as the alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, formazine Thionine sulfoxide, methionine methylsulfonium. Such analogs can have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics are chemical compounds whose structure differs from the general chemical structure of amino acids, but which function similarly to naturally occurring amino acids.
本文所述的A或Ala表示丙氨酸，结构为
R或Arg表示精氨酸，结构为
N或Asn表示天冬酰胺，结构为
D或Asp表示天冬氨酸，结构为
C或Cys表示半胱氨酸，结构为
Q或Gln表示谷氨酰胺，结构为
E或Glu表示谷氨酸，结构为
G或Gly表示甘氨酸， 结构为
H或His表示组氨酸，结构为
I或Ile表示异亮氨酸，结构为
L或Leu表示亮氨酸，结构为
α-MeL或α-MeLeu表示α-甲基亮氨酸，结构为
K或Lys表示赖氨酸，结构为
M或Met表示甲硫氨酸，结构为
F或Phe表示苯丙氨酸，结构为
P或Pro表示脯氨酸，结构为
S或Ser表示丝氨酸，结构为
T或Thr表示苏氨酸，结构为
W或Trp表示色氨酸，结构为
Y或Tyr表示酪氨酸，结构为
V或Val表示缬氨酸，结构为
Fmoc-AEEA-OH表示
A or Ala described herein represents alanine, and the structure is R or Arg means arginine, the structure is N or Asn means asparagine, the structure is D or Asp means aspartic acid, the structure is C or Cys means cysteine, the structure is Q or Gln means glutamine, the structure is E or Glu means glutamic acid, the structure is G or Gly means glycine, the structure is H or His means histidine, the structure is I or Ile means isoleucine, the structure is L or Leu means leucine, the structure is α-MeL or α-MeLeu means α-methylleucine, the structure is K or Lys means lysine, the structure is M or Met means methionine, the structure is F or Phe means phenylalanine, the structure is P or Pro means proline, the structure is S or Ser represents serine, the structure is T or Thr means threonine, the structure is W or Trp means tryptophan, the structure is Y or Tyr means tyrosine, the structure is V or Val represents valine, the structure is Fmoc-AEEA-OH said
术语“治疗”包括抑制、减缓、停止或逆转现有症状或病患的进展或严重程度。The term "treating" includes inhibiting, slowing, stopping or reversing the progression or severity of an existing symptom or condition.
除非另有说明，术语“异构体”意在包括几何异构体、顺反异构体、立体异构体、对映异构体、旋光异构体、非对映异构体和互变异构体。Unless otherwise stated, the term "isomer" is intended to include geometric isomers, cis-trans isomers, stereoisomers, enantiomers, optical isomers, diastereoisomers and interconversions isomer.
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。The compounds of the invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise from the inability to rotate freely due to the double bond or the single bond of the carbon atoms forming the ring.
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。Unless otherwise indicated, the term "diastereoisomer" refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of the molecules.
除非另有说明，“(+)”表示右旋，“(-)”表示左旋，“(±)”表示外消旋。Unless otherwise specified, "(+)" means dextrorotation, "(-)" means levorotation, and "(±)" means racemization.
除非另有说明，用楔形实线键
和楔形虚线键
表示一个立体中心的绝对构型，用直形实线键
和直形虚线键
表示立体中心的相对构型，用波浪线
表示楔形实线键
或楔形虚线键
或用波浪线
表示直形实线键
或直形虚线键
Unless otherwise noted, keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key or straight dotted key
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。Unless otherwise stated, the terms "enriched in an isomer", "enriched in an isomer", "enriched in an enantiomer" or "enantiomerically enriched" refer to one of the isomers or enantiomers The content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。Unless otherwise stated, the terms "isomer excess" or "enantiomeric excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomeric excess (ee value) is 80% .
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(
3H)，碘-125(
125I)或C-14(
14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds. For example, compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C). For another example, heavy hydrogen can be used to replace hydrogen to form deuterated drugs. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，
中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
When the linking group listed does not indicate its linking direction, its linking direction is arbitrary, for example, The connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right Combinations of the described linking groups, substituents and/or variations thereof are permissible only if such combinations result in stable compounds.
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键
直形虚线键
或波浪线
表示。例如-OCH
3中的直形实线键表示通过该基团中的氧原子与其他基团相连；
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连。
Unless otherwise specified, when a group has one or more linkable sites, any one or more sites of the group can be linked to other groups through chemical bonds. When the connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group. The chemical bonds that the site is connected with other groups can use straight solid line bonds Straight dotted key or tilde express. For example, the straight solid-line bond in -OCH 3 indicates that it is connected to other groups through the oxygen atom in the group; The straight dotted line bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy lines in indicate that the 1 and 2 carbon atoms in the phenyl group are connected to other groups.
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：
扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
The structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuKα radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
本发明所使用的溶剂可经市售获得。The solvent used in the present invention is commercially available.
本发明采用下述缩略词：aq代表水；eq代表当量、等量；DCM代表二氯甲烷；MeOH代表甲醇；BOC代表叔丁氧羰基是一种胺保护基团；Boc
2O代表二叔丁基二碳酸酯；TFA代表三氟乙酸；DIEA代表二异丙基乙基胺；DMF代表N,N-二甲基甲酰胺；HBTU代表苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯；PhSiH
3代表苯硅烷；Pd(PPh
3)
4代表四三苯基膦钯。
The present invention uses the following abbreviations: aq stands for water; eq stands for equivalent, equivalent; DCM stands for dichloromethane; MeOH stands for methanol; BOC stands for tert - butoxycarbonyl, which is an amine protecting group; Butyl dicarbonate; TFA stands for trifluoroacetic acid; DIEA stands for diisopropylethylamine; DMF stands for N,N-dimethylformamide; HBTU stands for benzotriazole-N,N,N',N '-Tetramethylurea hexafluorophosphate; PhSiH 3 represents phenylsilane; Pd(PPh 3 ) 4 represents tetrakistriphenylphosphine palladium.
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。The present invention will be described in detail through examples below, but it does not imply any unfavorable limitation to the present invention. The present invention has been described in detail herein, and its specific embodiments are also disclosed. For those skilled in the art, various changes and improvements can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention. will be obvious.
实施例1Example 1
1.挂树脂1. Hanging resin
1.1称取0.74g 4-(2’,4’-二甲氧基苯基-芴甲氧羰基-氨甲基)-苯氧基乙酰氨基-甲基二苯甲胺树脂Rink Amide MBHA Resin(替代度Sub＝0.28mmol/g)，加入到反应柱中，再加入DMF(20mL)至反应柱氮气鼓气体2h，排废，直至没有液体流出，加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。1.1 Weigh 0.74g of 4-(2',4'-dimethoxyphenyl-fluorenylmethoxycarbonyl-aminomethyl)-phenoxyacetamido-methylbenzhydrylamine resin Rink Amide MBHA Resin (substitute Sub=0.28mmol/g), added to the reaction column, then added DMF (20mL) to the reaction column nitrogen blowing gas for 2h, drained until no liquid flowed out, added DMF (20mL) to wash 5 times, each time for 1min, Drain until no liquid comes out.
1.2加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1.2 Add 20% piperidine/DMF (20 mL) to the reaction column, blow nitrogen gas for 20 min, and drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.氨基酸的偶联2. Coupling of Amino Acids
2.1 Fmoc-Ser(tBu)-OH的偶联2.1 Coupling of Fmoc-Ser(tBu)-OH
1.称取Fmoc-Ser(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。1. Weigh Fmoc-Ser(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
2.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。2. React in an environment of 25°C for 0.5h, detect with ninhydrin, the resin is colorless and transparent.
3.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。3. Remove the reaction liquid, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.2 Fmoc-Pro-OH的偶联2.2 Coupling of Fmoc-Pro-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Pro-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Pro-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.3 Fmoc-Pro-OH的偶联2.3 Coupling of Fmoc-Pro-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。四氯苯醌检测，树脂绿色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Chlorobenzoquinone detection, resin green.
2.称取Fmoc-Pro-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Pro-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，四氯苯醌检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, and the resin is colorless and transparent when detected by chloranil.
4.抽掉反应液，用DMF洗涤5次，每次1min，排废，直至没有液体流出。4. Take out the reaction liquid, wash with DMF 5 times, each time for 1 min, drain until no liquid flows out.
2.4 Fmoc-Pro-OH的偶联2.4 Coupling of Fmoc-Pro-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。四氯苯醌检测，树脂绿色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Chlorobenzoquinone detection, resin green.
2.称取Fmoc-Pro-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Pro-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，四氯苯醌检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, and the resin is colorless and transparent when detected by chloranil.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.5 Fmoc-Ala-OH的偶联2.5 Coupling of Fmoc-Ala-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。四氯苯醌检测，树脂绿色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Chlorobenzoquinone detection, resin green.
2.称取Fmoc-Ala-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ala-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，四氯苯醌检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, and the resin is colorless and transparent when detected by chloranil.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.6 Fmoc-Gly-OH的偶联2.6 Coupling of Fmoc-Gly-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Gly-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Gly-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.7 Fmoc-Ser(tBu)-OH的偶联2.7 Coupling of Fmoc-Ser(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Ser(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ser(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.8 Fmoc-Ser(tBu)-OH的偶联2.8 Coupling of Fmoc-Ser(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Ser(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ser(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.9 Fmoc-Pro-OH的偶联2.9 Coupling of Fmoc-Pro-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Pro-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Pro-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.10 Fmoc-Gly-OH的偶联2.10 Coupling of Fmoc-Gly-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。四氯苯醌检测，树脂绿色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Chlorobenzoquinone detection, resin green.
2.称取Fmoc-Gly-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Gly-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，四氯苯醌检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, and the resin is colorless and transparent when detected by chloranil.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.11 Fmoc-Gly-OH的偶联2.11 Coupling of Fmoc-Gly-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Gly-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Gly-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect with ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.12 Fmoc-Glu(OtBu)-OH的偶联2.12 Coupling of Fmoc-Glu(OtBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Glu(OtBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Glu(OtBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect with ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.13 Fmoc-Leu-OH的偶联2.13 Coupling of Fmoc-Leu-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Leu-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Leu-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect with ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.14 Fmoc-Leu-OH的偶联2.14 Coupling of Fmoc-Leu-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Leu-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Leu-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.15 Fmoc-Tyr(tBu)-OH的偶联2.15 Coupling of Fmoc-Tyr(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Tyr(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Tyr(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.16 Fmoc-Glu(OAll)-OH的偶联2.16 Coupling of Fmoc-Glu(OAll)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Glu(OAll)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Glu(OAll)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.17 Fmoc-Ile-OH的偶联2.17 Coupling of Fmoc-Ile-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Ile-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ile-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.18 Fmoc-Phe-OH的偶联2.18 Coupling of Fmoc-Phe-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Phe-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Phe-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.19 Fmoc-Ala-OH的偶联2.19 Coupling of Fmoc-Ala-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Ala-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ala-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.20 Fmoc-Lys(Alloc)-OH的偶联2.20 Coupling of Fmoc-Lys(Alloc)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Lys(Alloc)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Lys(Alloc)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应过夜，茚三酮检测，树脂无色透明。3. React overnight in an environment of 25°C, detect ninhydrin, and the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.21 Fmoc-Gln(Trt)-OH的偶联2.21 Coupling of Fmoc-Gln(Trt)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Gln(Trt)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Gln(Trt)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.22 Fmoc-Ala-OH的偶联2.22 Coupling of Fmoc-Ala-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Ala-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ala-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HATU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.23 Fmoc-Lys(Dde)-OH的偶联2.23 Coupling of Fmoc-Lys(Dde)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Lys(Dde)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Lys(Dde)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.24 Fmoc-Lys(Boc)-OH的偶联2.24 Coupling of Fmoc-Lys(Boc)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Lys(Boc)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Lys(Boc)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.25 Fmoc-Asp(OtBu)-OH的偶联2.25 Coupling of Fmoc-Asp(OtBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Asp(OtBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Asp(OtBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, add HBTU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.26 Fmoc-Leu-OH的偶联2.26 Coupling of Fmoc-Leu-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Leu-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Leu-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.27 Fmoc-α-MeLeu-OH的偶联2.27 Coupling of Fmoc-α-MeLeu-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times for 1 min each time, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-α-MeLeu-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-α-MeLeu-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HATU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应2h，茚三酮检测，树脂蓝色。3. React in an environment of 25°C for 2 hours, detect ninhydrin, and the resin is blue.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.28 Fmoc-Ile-OH的偶联2.28 Coupling of Fmoc-Ile-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。四氯苯醌检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Chlorobenzoquinone detection, resin blue.
2.称取Fmoc-Ile-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ile-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应过夜，四氯苯醌检测，树脂无色透明。3. Reacted overnight in an environment of 25°C, and the resin was colorless and transparent when detected by chlorobenzoquinone.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.29 Fmoc-Ser(tBu)-OH的偶联2.29 Coupling of Fmoc-Ser(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Ser(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ser(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.30 Fmoc-Tyr(tBu)-OH的偶联2.30 Coupling of Fmoc-Tyr(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Tyr(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Tyr(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, add HATU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.31 Fmoc-Asp(OtBu)-OH的偶联2.31 Coupling of Fmoc-Asp(OtBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Asp(OtBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Asp(OtBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.32 Fmoc-Ser(tBu)-OH的偶联2.32 Coupling of Fmoc-Ser(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Ser(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Ser(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.33 Fmoc-Thr(tBu)-OH的偶联2.33 Coupling of Fmoc-Thr(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Thr(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Thr(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.34 Fmoc-Phe-OH的偶联2.34 Coupling of Fmoc-Phe-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Phe-OH(3.0q)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Phe-OH (3.0q) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, add HATU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.35 Fmoc-Thr(tBu)-OH的偶联2.35 Coupling of Fmoc-Thr(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Thr(tBu)-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Thr(tBu)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (2.85eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.36 Fmoc-Gly-OH的偶联2.36 Coupling of Fmoc-Gly-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Gly-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Gly-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HATU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.37 Fmoc-Gln(Trt)-OH的偶联2.37 Coupling of Fmoc-Gln(Trt)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Gln(Trt)-OH(3.0eq)加入到上述树脂中加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Gln(Trt)-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HATU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应过夜，茚三酮检测，树脂无色透明。3. React overnight in an environment of 25°C, detect ninhydrin, and the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.38 Fmoc-Aib-OH的偶联2.38 Coupling of Fmoc-Aib-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Aib-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Aib-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, and add HATU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.39 Boc-Tyr(tBu)-OH的偶联2.39 Coupling of Boc-Tyr(tBu)-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。四氯苯醌检测，树脂绿色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Chlorobenzoquinone detection, resin green.
2.称取Boc-Tyr(tBu)-OH(6.0eq)加入到上述树脂中，加入DIEA(12.0eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HATU(5.70eq)。调节好氮气使树脂均匀鼓起。2. Weigh Boc-Tyr(tBu)-OH (6.0eq) and add it to the above resin, add DIEA (12.0eq) and add 5mL DMF to the reaction column, blow nitrogen gas, add HATU (5.70eq) after the amino acid is dissolved . Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，四氯苯醌检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, and the resin is colorless and transparent when detected by chloranil.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.40脱Alloc和OAll2.40 De-Alloc and OAll
1.加入PhSiH
3(20.0eq)和DCM(8mL)至反应柱中，氮气鼓起后加入Pd(PPh
3)
4(0.2eq)，氮气鼓起20min，反应二次，排废，直至没有液体流出。
1. Add PhSiH 3 (20.0eq) and DCM (8mL) to the reaction column, add Pd(PPh 3 ) 4 (0.2eq) after nitrogen blowing, nitrogen blowing for 20min, react twice, drain until there is no liquid flow out.
2.用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。2. Wash 5 times with DMF (20mL each time), 1min each time, drain until no liquid flows out.
2.41酰胺关环2.41 Amide ring closure
1.加入DIEA(3.00eq)补加10mL DMF至反应柱中，鼓氮气，待溶解后加入HATU(1.5eq)。调节好氮气使树脂均匀鼓起。1. Add DIEA (3.00eq) and add 10mL DMF to the reaction column, blow nitrogen gas, and add HATU (1.5eq) after dissolving. Adjust the nitrogen to make the resin bulge evenly.
2.在25℃的环境中反应0.5h,用DMF洗涤5次(30mL每次)，每次1min，排废，直至没有液体流出。2. React in an environment of 25°C for 0.5h, wash with DMF 5 times (30mL each time), 1min each time, drain until no liquid flows out.
3.重复上述1.2操作，再关环一次。茚三酮检测，树脂无色透明。3. Repeat the above 1.2 operation, and then close the ring again. Ninhydrin detection, the resin is colorless and transparent.
4.用DMF洗涤5次(30mL每次)，每次1min，排废，直至没有液体流出。4. Wash 5 times with DMF (30 mL each time), 1 min each time, drain until no liquid flows out.
2.42脱Dde2.42 off Dde
1.加入3％的水合肼/DMF(20mL)至反应柱中，氮气鼓起15mim，排废，加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 3% hydrazine hydrate/DMF (20mL) to the reaction column, blow up nitrogen for 15mim, drain, add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.43 Fmoc-AEEA-OH的偶联2.43 Coupling of Fmoc-AEEA-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-AEEA-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-AEEA-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.44 Fmoc-AEEA-OH的偶联2.44 Coupling of Fmoc-AEEA-OH
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-AEEA-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-AEEA-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.45 Fmoc-Glu-OtBu的偶联2.45 Coupling of Fmoc-Glu-OtBu
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次,每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20mL) to wash 5 times, each time for 1min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取Fmoc-Glu-OtBu-OH(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh Fmoc-Glu-OtBu-OH (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen gas, and add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
2.46 C20DA的偶联2.46 Coupling of C20DA
1.加入20％的哌啶/DMF(20mL)至反应柱中，氮气鼓起20min，排废，直至没有液体流出。加入DMF(20mL)洗涤5次，每次1min，排废，直至没有液体流出。茚三酮检测，树脂蓝色。1. Add 20% piperidine/DMF (20 mL) into the reaction column, blow nitrogen gas for 20 min, drain until no liquid flows out. Add DMF (20 mL) to wash 5 times, each time for 1 min, drain until no liquid flows out. Ninhydrin detection, resin blue.
2.称取C20DA(3.0eq)加入到上述树脂中，加入DIEA(6.00eq)补加5mL DMF至反应柱中，鼓氮气，待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。2. Weigh C20DA (3.0eq) and add it to the above resin, add DIEA (6.00eq) and add 5mL DMF to the reaction column, blow nitrogen, add HBTU (2.85eq) after the amino acid is dissolved. Adjust the nitrogen to make the resin bulge evenly.
3.在25℃的环境中反应0.5h，茚三酮检测，树脂无色透明。3. React in an environment of 25°C for 0.5h, detect ninhydrin, the resin is colorless and transparent.
4.抽掉反应液，用DMF洗涤5次(20mL每次)，每次1min，排废，直至没有液体流出。4. Take out the reaction solution, wash with DMF 5 times (20 mL each time), 1 min each time, drain until no liquid flows out.
5.用MeOH(20mL)收缩树脂三次，每次3min，排废，直至没有液体流出，将树脂倒出干燥，备用。5. Use MeOH (20mL) to shrink the resin three times, 3 minutes each time, drain until no liquid flows out, then pour out the resin and dry it for later use.
3切割及粗肽干燥3 cutting and drying of crude peptide
3.1按如下体积配置切割液3.1 Configure the cutting fluid according to the following volume
三氟乙酸：三异丙基硅烷：H
2O：3-巯基丙酸＝92.5：2.5：2.5：2.5
Trifluoroacetic acid: triisopropylsilane: H 2 O: 3-mercaptopropionic acid = 92.5: 2.5: 2.5: 2.5
3.2将干燥后的肽树脂加入到配好的切割液中，在摇床震荡2.5h，过滤，滤液加入到10倍体积冰异丙醚中，离心，再用异丙醚洗涤3次。在真空干燥2h得到粗肽，纯化。多肽分子量经ESI-MS确认，计算值m/z为4901.6，实测值为4902.0。3.2 Add the dried peptide resin to the prepared cutting solution, shake on a shaker for 2.5 hours, filter, add the filtrate to 10 times the volume of ice isopropyl ether, centrifuge, and wash 3 times with isopropyl ether. The crude peptide was obtained by drying in vacuo for 2 h and purified. The molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value m/z was 4901.6, and the measured value was 4902.0.
实施例2Example 2
参考WX001的合成，得到多肽WX002。多肽分子量经ESI-MS进行确认，计算值为4902.6，检测值为4902.3。Referring to the synthesis of WX001, the polypeptide WX002 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4902.6, and the detected value was 4902.3.
实施例3Example 3
参考WX001的合成，得到多肽WX003。多肽分子量经ESI-MS进行确认，计算值为4915.7，检测值为4915.8。Referring to the synthesis of WX001, the polypeptide WX003 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4915.7, and the detected value was 4915.8.
实施例4Example 4
参考WX001的合成，得到多肽WX004。多肽分子量经ESI-MS进行确认，计算值为4910.6，检测值为4910.4。Referring to the synthesis of WX001, the polypeptide WX004 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4910.6, and the detected value was 4910.4.
实施例5Example 5
参考WX001的合成，得到多肽WX005。多肽分子量经ESI-MS进行确认，计算值为4968.7，检测值为4968.6。Referring to the synthesis of WX001, the polypeptide WX005 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4968.7, and the detected value was 4968.6.
实施例6Example 6
参考WX001的合成，得到多肽WX006。多肽分子量经ESI-MS进行确认，计算值为4901.5，检测值为4901.4。Referring to the synthesis of WX001, the polypeptide WX006 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4901.5, and the detected value was 4901.4.
实施例7Example 7
参考WX001的合成，得到多肽WX007。多肽分子量经ESI-MS进行确认，计算值为4857.5，检测值为4858.2。Referring to the synthesis of WX001, the polypeptide WX007 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4857.5, and the detected value was 4858.2.
实施例8Example 8
参考WX001的合成，得到多肽WX008。多肽分子量经ESI-MS进行确认，计算值为4901.5，检测值为4902.0。Referring to the synthesis of WX001, the polypeptide WX008 was obtained. The molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4901.5, and the detected value was 4902.0.
生物测试数据biological test data
实施例1：体外GLP-1R/GIPR/GCGR激动活性测试Example 1: In vitro GLP-1R/GIPR/GCGR agonistic activity test
A：主要材料：A: Main material:
1)细胞株1) cell line
该细胞株由上海药明康德构建。详情见下表1。The cell line was constructed by Shanghai WuXi PharmaTech. See Table 1 below for details.
表1细胞株信息Table 1 Cell line information
| 靶点target | 宿主细胞host cell | 克隆clone |
| GLP-1RGLP-1R | HEK293HEK293 | N/AN/A |
| GCGRGCGR | HEK293HEK293 | N/AN/A |
| GIPRGIPR | CHOCHO | N/AN/A |
2)试剂与耗材2) Reagents and consumables
详情见下表2。See Table 2 below for details.
表2试剂与耗材信息Table 2 Reagents and consumables information
| 名称name | 批次.batch. | 货号Item No. | 厂家factory |
| cAMP检测盒cAMP detection kit | 29F29F | 62AM4PEJ62AM4PEJ | CisbioCisbio |
| 1M HEPES1M HEPES | 21209192120919 | 15630-10615630-106 | InvitrogenInvitrogen |
| Hanks平衡盐溶液(HBSS)Hanks Balanced Salt Solution (HBSS) | 21857752185775 | 1402514025 | InvitrogenInvitrogen |
| 人血清白蛋白(HSA)Human Serum Albumin (HSA) | SLCF7301SLCF7301 | A1653-10GA1653-10G | 西格玛Sigma |
| 酪蛋白casein | SLCC9458SLCC9458 | C4765-10mLC4765-10mL | 西格玛Sigma |
| 3-异丁基-1-甲基黄嘌呤(IBMX)3-Isobutyl-1-methylxanthine (IBMX) | STBF6061VSTBF6061V | I5879-5GI5879-5G | 西格玛Sigma |
| ECHO qualified 384孔板ECHO qualified 384-well plate | 00064336720006433672 | PP-0200PP-0200 | LabcyteLabcyte |
| OptiPlate-384OptiPlate-384 | 8210-194818210-19481 | 60072996007299 | 珀金埃尔默PerkinElmer |
3)仪器3) Instrument
详情见下表3。See Table 3 below for details.
表3仪器信息Table 3 Instrument information
| 名称name | 型号model | 厂家factory |
| EnVisionEnVision | envision2014envision2014 | 珀金埃尔默PerkinElmer |
| Vi-cell counterVi-cell counter | Vi-CELL TM XR Cell Viability Analyzer Vi-CELL ™ XR Cell Viability Analyzer | 贝克曼Beckman |
| BravoBravo | Bravo V11Bravo V11 | 安捷伦Agilent |
| ECHOECHO | ECHO 555ECHO 555 | LabcyteLabcyte |
| CentrifugeCentrifuge | Allegra TM 25R Centrifuge AllegraTM 25R Centrifuge | 贝克曼Beckman |
B.方法B. method
Ⅰ)实验材料：详情见下表4和表5。Ⅰ) Experimental materials: see Table 4 and Table 5 below for details.
表4实验缓冲液信息Table 4 Experiment buffer information
表5检测试剂制备信息Table 5 Detection reagent preparation information
| 试剂Reagent | 储存浓度storage concentration | 体积volume | 终浓度Final concentration |
| 细胞裂解液Cell Lysates | 1×1× | 9.5mL9.5mL | ≈1×≈1× |
| D2-cAMP溶液D2-cAMP solution | 40×40× | 250μL250μL | 1×1× |
| cAMP-抗体溶液cAMP-antibody solution | 40×40× | 250μL250μL | 1×1× |
Ⅱ)实验方法Ⅱ) Experimental method
a)制备化合物板：a) Preparation of compound plates:
待测化合物做10个点4倍稀释，起始浓度为30μM，Bravo完成稀释The compound to be tested is diluted 4 times at 10 points, the initial concentration is 30 μM, and Bravo completes the dilution
b)转移化合物：b) transfer compound:
1)使用Echo转移100nL化合物至OptiPlate-384 plate。1) Use Echo to transfer 100nL of compound to OptiPlate-384 plate.
2)将OptiPlate-384 plate在1000rpm离心5秒。2) Centrifuge the OptiPlate-384 plate at 1000rpm for 5 seconds.
c)细胞悬液的制备c) Preparation of cell suspension
1)将一支GLP-1R/GIPR/GCGR细胞冻存管迅速置于37℃温水中解冻。1) Quickly thaw a GLP-1R/GIPR/GCGR cell cryopreservation tube in warm water at 37°C.
2)将细胞悬液转移至Transfer15mL离心管中，用10mL HBSS轻柔冲洗。2) Transfer the cell suspension to a Transfer15mL centrifuge tube and gently rinse with 10mL HBSS.
3)将离心管在1000rpm室温离心1分钟。3) Centrifuge the centrifuge tube at 1000 rpm for 1 minute at room temperature.
4)弃去上清。4) Discard the supernatant.
5)轻柔打散底部细胞并再用10mL HBSS轻柔冲洗，离心沉降细胞，最后用实验缓冲液重悬细胞。5) Gently break up the bottom cells and gently rinse with 10mL HBSS, centrifuge to settle the cells, and finally resuspend the cells with the experimental buffer.
6)利用Vi-cell测量细胞密度与活度。6) Use Vi-cell to measure cell density and activity.
7)用实验缓冲液将GLP-1R/GIPR/GCGR细胞浓度稀释至2.0*10
5/mL。
7) Dilute the GLP-1R/GIPR/GCGR cell concentration to 2.0*10 5 /mL with the assay buffer.
8)在OptiPlate-384 plate中转入100nL稀释好的细胞悬液。8) Transfer 100nL of the diluted cell suspension into the OptiPlate-384 plate.
9)室温孵育30分钟。9) Incubate at room temperature for 30 minutes.
d)加入检测试剂：d) Add detection reagent:
1)在OptiPlate-384 plate空孔中加入10μL 800nM梯度稀释好的cAMP标准品。1) Add 10 μL 800nM gradient diluted cAMP standard to the empty wells of the OptiPlate-384 plate.
2)加入10μL cAMP检测试剂。2) Add 10 μL cAMP detection reagent.
3)用TopSeal-A film覆盖OptiPlate-384 plate，室温孵育60分钟。3) Cover the OptiPlate-384 plate with TopSeal-A film and incubate at room temperature for 60 minutes.
揭去TopSeal-A,在EnVision读数。Peel off TopSeal-A and read in EnVision.
C实验结果C Experimental results
实验结果如表6所示。The experimental results are shown in Table 6.
表6体外GLP-1R/GIPR/GCGR激动活性测试结果Table 6 GLP-1R/GIPR/GCGR agonistic activity test results in vitro
结论：本发明化合物对GLP-1R/GIPR/GCGR具有很强的激动活性。其中，不同化合物对于GCG的激动活性存在较大差异，对于研究三靶点活性的平衡将具有指导意义。Conclusion: the compound of the present invention has strong agonistic activity on GLP-1R/GIPR/GCGR. Among them, there are large differences in the agonistic activity of different compounds for GCG, which will be of guiding significance for the study of the balance of the activities of the three targets.
实验例2：化合物小鼠腹腔糖耐量(ipGTT)实验-体内药效评价Experimental Example 2: Compound Mouse Intraperitoneal Glucose Tolerance (ipGTT) Experiment - In vivo Drug Efficacy Evaluation
A.实验目的A. The purpose of the experiment
研究受试化合物对于正常小鼠糖耐量的改善作用。To study the improvement effect of the test compound on the glucose tolerance of normal mice.
B.实验操作B. Experimental operation
1.根据小鼠体重和血糖分组后，对每组动物分别注射待测化合物(0.3nmol/kg)和溶媒(20mM)柠檬酸盐缓冲液)，过夜禁食，18小时后腹腔注射葡萄糖溶液(2g/kg,10mL/kg)；1. After grouping according to mouse body weight and blood sugar, inject compound to be tested (0.3nmol/kg) and vehicle (20mM) citrate buffer) to each group of animals respectively, fast overnight, intraperitoneally inject glucose solution after 18 hours ( 2g/kg, 10mL/kg);
2.使用血糖仪测定给葡萄糖后-60，0，15，30，60和120分钟的血糖浓度。2. Use a blood glucose meter to measure blood glucose concentrations at -60, 0, 15, 30, 60 and 120 minutes after glucose administration.
C.实验结果C. Experimental results
实验结果如表7所示。The experimental results are shown in Table 7.
表7 ipGTT测试结果Table 7 ipGTT test results
| 化合物编号Compound number | WX002WX002 | WX005WX005 | WX007WX007 |
| Δ血糖AUC 0-120min(与溶媒组相比) ΔBlood glucose AUC 0-120min (compared with vehicle group) | -52％-52% | -42％-42% | -39％-39% |
结论：本发明化合物具有优异的糖耐量改善作用。Conclusion: The compounds of the present invention have excellent effects on improving glucose tolerance.
实验例3：在DIO小鼠中的药效研究-体内药效评价Experimental Example 3: Drug Efficacy Study in DIO Mice - In Vivo Evaluation of Drug Efficacy
A.实验目的A. The purpose of the experiment
研究受试化合物在DIO小鼠中的减重作用。The weight loss effect of test compounds in DIO mice was studied.
B.实验操作B. Experimental operation
1.DIO小鼠动物到达设施后，将其饲养于严格控制环境条件的动物饲养间中，饲养间的温度维持在20～24℃，湿度维持在30～70％。通过温湿度计对饲养间的温度和湿度进行实时监控，并且每天对温度和湿度记录两次(上午和下午各1次)。动物饲养间的采光由一个电子定时开灯系统来控制，每天开灯12小时关灯12小时(上午7:00开，下午19:00关)。实验过程中，动物单笼饲养，并给每个笼中提供玩具。实验过程中动物自由采食(大小鼠生长/繁殖饲料)和饮水。1. After the DIO mice arrive at the facility, they are kept in an animal breeding room with strictly controlled environmental conditions. The temperature in the breeding room is maintained at 20-24° C., and the humidity is maintained at 30-70%. The temperature and humidity in the feeding room were monitored in real time by a thermo-hygrometer, and the temperature and humidity were recorded twice a day (once in the morning and once in the afternoon). The daylighting in the animal breeding room is controlled by an electronic timing lighting system, and the lights are turned on for 12 hours and turned off for 12 hours every day (turn on at 7:00 in the morning and turn off at 19:00 in the afternoon). During the experiment, the animals were kept in single cages, and toys were provided for each cage. During the experiment, the animals had free access to food (feed for growth/breeding of rats and mice) and drinking water.
2.对每组动物分别皮下注射溶媒和待测化合物(10nmol/kg)，给药时间：早上9:30，给药频率三天一次，给药周期为22天。2. Each group of animals was subcutaneously injected with the vehicle and the compound to be tested (10 nmol/kg), the administration time: 9:30 in the morning, the administration frequency was once every three days, and the administration cycle was 22 days.
C.实验结果C. Experimental results
实验结果如表8所示。The experimental results are shown in Table 8.
表8测试化合物在DIO小鼠中的药效The drug effect of table 8 test compound in DIO mice
| 化合物编号Compound number | 溶媒solvent | WX002WX002 | WX005WX005 | WX007WX007 |
| Δ体重％(22天后与第1天相比)ΔBody weight % (after 22 days compared with day 1) | -5％-5% | -28％-28% | -23％-twenty three% | -33％-33% |
结论：本发明化合物在DIO小鼠中展现了优异的减重药效。Conclusion: The compounds of the present invention exhibit excellent weight loss efficacy in DIO mice.
实验例4：在db/db小鼠中的药效研究-体内药效评价Experimental Example 4: Drug efficacy study in db/db mice - in vivo drug efficacy evaluation
A.实验目的A. The purpose of the experiment
研究受试化合物对于II型糖尿病db/db小鼠的血糖控制作用。To study the blood sugar control effect of the test compound on type II diabetic db/db mice.
B.实验操作B. Experimental operation
1.db/db小鼠动物到达设施后，将其饲养于严格控制环境条件的动物饲养间中，饲养间的温度维持在20～24℃，湿度维持在30～70％。通过温湿度计对饲养间的温度和湿度进行实时监控，并且每天对温度和湿度记录两次(上午和下午各1次)。动物饲养间的采光由一个电子定时开灯系统来控制，每天开灯12小时关灯12小时(上午7:00开，下午19:00关)。实验过程中，动物单笼饲养，并给每个笼中提供玩具。实验过程中动物自由采食(大小鼠生长/繁殖饲料)和饮水。1. After the db/db mice arrive at the facility, they are kept in an animal breeding room with strictly controlled environmental conditions. The temperature in the breeding room is maintained at 20-24° C., and the humidity is maintained at 30-70%. The temperature and humidity in the feeding room were monitored in real time by a thermo-hygrometer, and the temperature and humidity were recorded twice a day (once in the morning and once in the afternoon). The daylighting in the animal breeding room is controlled by an electronic timing lighting system, and the lights are turned on for 12 hours and turned off for 12 hours every day (turn on at 7:00 in the morning and turn off at 19:00 in the afternoon). During the experiment, the animals were kept in single cages, and toys were provided for each cage. During the experiment, the animals had free access to food (feed for growth/breeding of rats and mice) and drinking water.
2.对每组动物分别皮下注射溶媒和待测化合物(15nmol/kg)，给药时间：早上9:30-11:00，给药频率一天一次，连续给药4周。2. The vehicle and the compound to be tested (15 nmol/kg) were subcutaneously injected into each group of animals respectively, the administration time: 9:30-11:00 in the morning, the administration frequency was once a day, and the administration was continued for 4 weeks.
C.实验结果C. Experimental results
实验结果如表9所示。The experimental results are shown in Table 9.
表9测试化合物在db/db小鼠中的降糖效果The hypoglycemic effect of table 9 test compound in db/db mice
| 化合物编号Compound number | WX001WX001 | WX002WX002 | WX007WX007 |
| ΔHbA1c％(连续给药四周后与溶媒组相比)ΔHbA1c% (compared with vehicle group after four weeks of continuous administration) | -26.8％-26.8% | -33.9％-33.9% | -33.4％-33.4% |
结论：本发明化合物在db/db小鼠中展现了优异的降糖药效。Conclusion: the compound of the present invention exhibits excellent hypoglycemic efficacy in db/db mice.
实验例5：化合物小鼠药代动力学评价Experimental Example 5: Pharmacokinetic evaluation of compounds in mice
A.实验目的A. The purpose of the experiment
测试化合物在C57BL/6小鼠体内药代动力学。Pharmacokinetics of test compounds in C57BL/6 mice.
B.实验操作B. Experimental operation
以标准方案测试化合物皮下注射给药后的啮齿类动物药代特征，实验中候选化合物配成澄清溶液，给予小鼠皮下注射给药(SC,10nmol/kg)。皮下注射溶媒为柠檬酸盐缓冲液(20mM,pH＝7)。收集全血，制备得到血浆，以LC-MS/MS方法分析药物浓度，并用Phoenix WinNonlin软件计算药代参数。The pharmacokinetic characteristics of the compounds in rodents after subcutaneous injection were tested according to the standard protocol. In the experiment, the candidate compounds were made into clear solutions and given to mice for subcutaneous injection (SC, 10 nmol/kg). The vehicle for subcutaneous injection is citrate buffer (20mM, pH=7). Whole blood was collected, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
C.实验结果C. Experimental results
实验结果如表10所示。The experimental results are shown in Table 10.
表10药代动力学测试结果Table 10 Pharmacokinetic test results
结论：本发明化合物具有优异的小鼠药代动力学性质。Conclusion: The compounds of the present invention have excellent pharmacokinetic properties in mice.
实验例6：化合物大鼠药代动力学评价Experimental Example 6: Pharmacokinetic Evaluation of Compound Rats
A.实验目的A. The purpose of the experiment
测试化合物在SD大鼠体内药代动力学。Pharmacokinetics of test compounds in SD rats.
B.实验操作B. Experimental operation
以标准方案测试化合物皮下注射后的啮齿类动物药代特征，实验中候选化合物配成澄清溶液，给予大鼠单次皮下注射(SC,10nmol/kg)。注射溶媒为柠檬酸盐缓冲液(20mM,pH＝7)。收集全血，制备得到血浆，以LC-MS/MS方法分析药物浓度，并用Phoenix WinNonlin软件计算药代参数。The pharmacokinetic characteristics of the compounds in rodents after subcutaneous injection were tested according to the standard protocol. In the experiment, the candidate compounds were made into a clear solution and given to rats for a single subcutaneous injection (SC, 10nmol/kg). The injection vehicle is citrate buffer (20 mM, pH=7). Whole blood was collected, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
C.实验结果C. Experimental results
实验结果如表11所示。The experimental results are shown in Table 11.
表11药代动力学测试结果Table 11 Pharmacokinetic test results
结论：本发明化合物具有优异的大鼠药代动力学性质。Conclusion: The compounds of the present invention have excellent pharmacokinetic properties in rats.
实验例7：化合物食蟹猴药代动力学评价Experimental Example 7: Pharmacokinetic evaluation of compounds in cynomolgus monkeys
A.实验目的A. The purpose of the experiment
测试化合物在食蟹猴体内药代动力学。Pharmacokinetics of test compounds in cynomolgus monkeys.
B.实验操作B. Experimental operation
以标准方案测试化合物皮下注射给药后的哺乳类动物药代特征，实验中候选化合物配成澄清溶液，给予食蟹猴单次皮下注射给药(SC,4nmol/kg)。皮下注射溶媒为柠檬酸盐缓冲液(20mM,pH＝7)。收集全血，制备得到血浆，以LC-MS/MS方法分析药物浓度，并用Phoenix WinNonlin软件计算药代参数。The mammalian pharmacokinetic characteristics of the compound after subcutaneous injection were tested according to the standard protocol. In the experiment, the candidate compound was formulated into a clear solution and given to cynomolgus monkeys for single subcutaneous injection (SC, 4nmol/kg). The vehicle for subcutaneous injection is citrate buffer (20mM, pH=7). Whole blood was collected, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software.
C.实验结果C. Experimental results
实验结果如表12所示。The experimental results are shown in Table 12.
表12药代动力学测试结果Table 12 Pharmacokinetic test results
| 化合物编号Compound number | 最大血浆浓度maximum plasma concentration | 达峰时间peak time | 半衰期half life | 药时曲线下面积area under drug time curve |
| the | C max(nM) C max (nM) | T max(h) T max (h) | T 1/2(h) T 1/2 (h) | AUC 0-240h(nM.hr) AUC 0-240h (nM.hr) |
| WX001WX001 | 4141 | 21twenty one | 4141 | 35323532 |
| WX002WX002 | 2626 | 1919 | 4747 | 20682068 |
| WX005WX005 | 3535 | 4848 | 7777 | 45794579 |
| WX006WX006 | 4747 | 24twenty four | 7373 | 45114511 |
| WX007WX007 | 2828 | 24twenty four | 5454 | 26982698 |
结论：本发明化合物具有优异的猴药代动力学性质。Conclusion: The compound of the present invention has excellent monkey pharmacokinetic properties.
Claims (9)
- 一种多肽(SEQ ID NO:1)，a polypeptide (SEQ ID NO: 1),SEQ ID NO:1 YAibQGT FTSDY SIα-MeLLD KKAQAib AFIEY LLEGG PSSGA PPPSSEQ ID NO:1 YAibQGT FTSDY SIα-MeLLD KKAQAib AFIEY LLEGG PSSGA PPPS其具有以下修饰：It has the following modifications:1)上述第17位或第24位的氨基酸有且只有一个被替换为K 0；和 1) one and only one of the amino acids at the 17th or 24th position above is replaced by K 0 ; and2)SEQ ID NO:1的1-4个氨基酸被替换；和2) 1-4 amino acids of SEQ ID NO: 1 are replaced; and3)i和i+4位置的氨基酸侧链之间或j和j+3位置的氨基酸侧链之间形成内酰胺桥，其中，i为16、20或24，j为17；3) A lactam bridge is formed between the amino acid side chains at positions i and i+4 or between the amino acid side chains at positions j and j+3, wherein i is 16, 20 or 24, and j is 17;其中，in,Aib的结构为The structure of Aib is
K 0表示赖氨酸，且该赖氨酸侧链上的氨基与-X 0相连； K 0 represents lysine, and the amino group on the lysine side chain is linked to -X 0 ;X 0选自X 0 selected from
m和n分别独立地为1、2或3；m and n are each independently 1, 2 or 3;p为8、9或10。p is 8, 9 or 10. - 根据权利要求1所述的多肽，其中，第39位的氨基酸C-末端酰胺化。The polypeptide according to claim 1, wherein the C-terminus of the amino acid at position 39 is amidated.
- 根据权利要求1或2所述的多肽，其中，m和n分别独立地为2。The polypeptide according to claim 1 or 2, wherein m and n are 2 independently.
- 根据权利要求1或2所述的多肽，其中，p为9。The polypeptide according to claim 1 or 2, wherein p is 9.
- 根据权利要求1或2所述的多肽，其中，-X 0选自 The polypeptide according to claim 1 or 2 , wherein -X is selected from
- 根据权利要求1～5任意一项所述的多肽，其选自：The polypeptide according to any one of claims 1-5, which is selected from:(I-1)YAibQGT FTSDY SIα-MeLLD KK 0AQK AFIEY LLEGG PSSGA PPPS-NH 2 (I-1) YAibQGT FTSDY SIα-MeLLD KK 0 AQK AFIEY LLEGG PSSGA PPPS-NH 2(I-2)YAibEGT FTSDY SIα-MeLLD KK 0AQK AFIEY LLEGG PSSGA PPPS-NH 2 (I-2) YAibEGT FTSDY SIα-MeLLD KK 0 AQK AFIEY LLEGG PSSGA PPPS-NH 2(I-3)YAibQGT FTSDY SIα-MeLLE KK 0AQK AFIEY LLEGG PSSGA PPPS-NH 2 (I-3) YAibQGT FTSDY SIα-MeLLE KK 0 AQK AFIEY LLEGG PSSGA PPPS-NH 2(I-4)YAibQGT FTSDY SIα-MeLLD KK 0AQK AFVEW LLEGG PSSGA PPPS-NH 2 (I-4) YAibQGT FTSDY SIα-MeLLD KK 0 AQK AFVEW LLEGG PSSGA PPPS-NH 2(I-5)YAibQGT FTSDY SIα-MeLLD KK 0AQK EFVEW LLEGG PSSGA PPPS-NH 2 (I-5) YAibQGT FTSDY SIα-MeLLD KK 0 AQK EFVEW LLEGG PSSGA PPPS-NH 2(I-6)YAibQGT FTSDY SIα-MeLLD KEAQK AFIK 0Y LLEGG PSSGA PPPS-NH 2 (I-6) YAibQGT FTSDY SIα-MeLLD KEAQK AFIK 0 Y LLEGG PSSGA PPPS-NH 2(I-7)YAibQGT FTSDY SIα-MeLLD KK 0AQAib AFIEY LLKGG PSSGA PPPS-NH 2 (I-7) YAibQGT FTSDY SIα-MeLLD KK 0 AQAib AFIEY LLKGG PSSGA PPPS-NH 2(I-8)YAibQGT FTSDY SIα-MeLLD EKAQK AFIK 0Y LLEGG PSSGA PPPS-NH 2 (I-8) YAibQGT FTSDY SIα-MeLLD EKAQK AFIK 0 Y LLEGG PSSGA PPPS-NH 2其中，in,i和i+4位置的氨基酸侧链之间或j和j+3位置的氨基酸侧链之间形成内酰胺桥，其中，i为16、20或24，j为17；A lactam bridge is formed between the amino acid side chains at positions i and i+4 or between the amino acid side chains at positions j and j+3, wherein i is 16, 20 or 24, and j is 17;Aib和K 0如权利要求1～5任意一项所定义。 Aib and K 0 are as defined in any one of claims 1-5.
- 下式所示多肽，A polypeptide represented by the following formula,
- 一种药物组合物，包括作为活性成分的治疗有效量的根据权利要求1～7任意一项所述的多肽化合物或其药学上可接受的盐以及药学上可接受的载体。A pharmaceutical composition comprising, as an active ingredient, a therapeutically effective amount of the polypeptide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1-7 and a pharmaceutically acceptable carrier.
- 根据权利要求1～7任意一项所述的多肽化合物或其药学上可接受的盐或者根据权利要求8所述的组合物在制备治疗二型糖尿病药物上的应用。Use of the polypeptide compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof or the composition according to claim 8 in the preparation of a drug for treating type 2 diabetes.
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