CN114685646B - Preparation method and application of polypeptide side chain analogue - Google Patents
Preparation method and application of polypeptide side chain analogue Download PDFInfo
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- CN114685646B CN114685646B CN202011634857.0A CN202011634857A CN114685646B CN 114685646 B CN114685646 B CN 114685646B CN 202011634857 A CN202011634857 A CN 202011634857A CN 114685646 B CN114685646 B CN 114685646B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a method for preparing a polypeptide side chain analogue by four-step one-pot boiling and application thereof, belonging to the field of compound synthesis. The specific operation process is as follows: activating the naked carboxyl of fatty acid containing one naked carboxyl or fatty diacid with one end carboxyl protected; directly adding amino acid into the crude reaction liquid obtained after activation to carry out condensation reaction; after the reaction is completed, activating the exposed carboxyl of the fatty acid-amino acid conjugate or the fatty diacid-amino acid conjugate in the obtained crude reaction liquid; directly adding raw materials into the reactivated crude reaction solutionOr
Description
Technical Field
The invention relates to the field of compound synthesis, in particular to a preparation method and application of a polypeptide side chain analogue.
Background
In recent years, with rapid development of economy, lifestyle changes, and an increase in the prevalence of diabetes every year, it has become a worldwide problem threatening the health of all humans. According to epidemiological studies, most diabetic patients are primarily type 2 diabetes mellitus (T2 DM) with associated insulin resistance and insulin hyposecretion. Glucagon-like peptide-1 (GLP-1) is an important incretin. GLP-1 receptor agonists (GLP-1 RAs) are one of the most promising modern diabetes drugs, and can reduce the blood sugar by stimulating the insulin secretion of islet beta cells and inhibiting the glucagon secretion of islet alpha cells through glucose dependence, thereby reducing the occurrence risk of hypoglycemic events, delaying gastric emptying and central appetite inhibition, and controlling body weight while stably reducing blood sugar.
The polypeptide drug has unique advantages, such as remarkable activity, strong specificity, weak toxicity, difficult accumulation in vivo, less interaction with other drugs and the like. In recent years, peptides such as benralide, loxapide, exenatide, liraglutide, exenatide long-acting preparation, albiglutide, dulaglutide, lixisenatide and somaglutide have attracted much attention for the treatment of type 2 diabetes. Wherein the Somarlu peptide is glucagon-like peptide-1 (GLP-1) analogue developed by Novonide, has 94% homology with human GLP-1, and has molecular formula C 187 H 291 N 45 O 59 The molecular weight is 4113.58, the half-life is about 7 days, and the long-acting preparation is developed based on the basic structure of liraglutide. Compared with liraglutide, the affinity of the carbon chain of the side chain of the somaglutide for albumin is enhanced by 5-6 times, the molecular weight of the medicine is increased after the side chain of the somaglutide is combined with albumin, the medicine is prevented from being rapidly cleared by the kidney and prevented from metabolic degradation, and therefore the half-life period in vivo is prolonged. The oral preparation of the somaglutide, which is further developed by Noohandride company of 20 days in 9 months in 2019, is listed in the United states, is the first oral preparation of the glucagon-like peptide-1 receptor agonist which is listed on the market, and the convenience and the compliance of the patients with type 2 diabetes are greatly improved.
In the existing patent literature, the preparation of the side chain of the somaglutide mostly adopts a solid-phase synthesis method, the solid-phase synthesis method is similar and different, the cost of a multi-polymer support is expensive, the cost of the processing material is most, a large amount of polymer carrier waste is generated, the scale enlargement is difficult, and the industrial production is not facilitated. CN110041219A discloses a liquid phase synthesis method of a side chain of somatalmin, CN110423251A discloses a preparation method of a side chain of somatalmin, although the prior art reports a liquid phase synthesis method of a side chain of somatalmin, the synthesis route is multi-step, 2- (2- (2-aminoethoxy) ethoxy) acetic acid, glu and octadecanedioic acid are sequentially coupled to obtain the side chain of somatalmin, each intermediate is also subjected to separation and purification operations in the preparation process, and the defects of multiple reaction steps, long period, difficulty in controlling the generation of impurities and low final product yield (about 33% in CN 110041219A) exist. Therefore, there is a need to develop an improved method for preparing polypeptide side chain analogs, in particular, somaglutide side chain analogs.
Disclosure of Invention
Aiming at the technical bottlenecks of complicated preparation process, high cost and unsatisfactory yield in the conventional preparation method of the side chain of the somaglutide, the invention mainly aims to provide the preparation method of the side chain analogue of the polypeptide, which has the advantages of high yield, high purity and low cost and is suitable for large-scale production.
The invention also aims to provide application of the preparation method of the polypeptide side chain analogue.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a preparation method of a polypeptide side chain analogue is characterized in that the structural general formula of the polypeptide side chain analogue is shown as formula (1):
wherein R is 1 Is methyl or protected carboxy; n is an integer of 9 to 19; AA is an amino acid residue; p is 1 or 2; the structure of AEEA isAnd wherein the amino terminus is linked to an amino acid residue;
the polypeptide side chain analogue is prepared by the following method:
will contain a naked carboxyl groupFatty acids or fatty diacids protected at one end by a carboxyl groupThe exposed carboxyl group of the fatty acid derivative or the fatty diacid derivative is activated to obtain the fatty acid derivative or the fatty diacid derivative containing one end of the carboxyl group which is activatedIn the crude reaction solution of (1), wherein R 3 Is a carboxyl activating group, the carboxyl activation is activation treatment of carboxyl by a carboxyl activating agent, and the carboxyl activating group is a residue of the carboxyl activating agent; directly adding raw material amino acid into the crude reaction liquid to carry out condensation reaction; after the reaction is completed, the product containing fatty acid-amino acid conjugate or fatty diacid-amino acid conjugate is obtained>Directly activating the exposed carboxyl of the conjugate to obtain a fatty acid-amino acid conjugate or a fatty diacid-amino acid conjugate containing activated carboxyl at one endIn the crude reaction solution of (1), wherein R 4 Is a carboxyl activating group; then directly adding raw material into the crude reaction solution>Condensation reaction is carried out to obtain the compound shown in the formula (1).
The method of preparation of the polypeptide side chain analogue is particularly suitable for the preparation of insulin analogues; the insulin analogues are for use as a medicament, preferably for the treatment or prevention of hyperglycemia, type II diabetes, impaired glucose tolerance or type I diabetes.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention adopts a 'one-pot' method to prepare the polypeptide side chain analogue, strictly controls the feeding amount and the reaction temperature in the whole preparation process, directly puts new raw materials into crude reaction liquid obtained after each feeding reaction is complete without purification and treatment of an intermediate, and processes the reaction liquid containing a final product and separates and purifies the final product until the end, thereby simplifying the preparation process, reducing the using amount of organic reagents, lowering the cost, being more green and environment-friendly and being suitable for large-scale production.
(2) The invention adopts a one-pot method to prepare the polypeptide side chain analogue, introduces a carboxyl activated base, greatly improves the reaction activity, improves the yield and the purity of a final product, and reduces the difficulty of subsequent purification.
(3) In the glutamic acid raw material adopted by the invention, the amino group is deprotected, and compared with the glutamic acid raw material adopting amino Fmoc protection in the existing preparation method, the invention omits the deprotection step, so that the preparation process is simpler and more efficient.
(4) The raw material H-AEEA-OH or the amino group of H-AEEA-AEEA-OH adopted by the invention is also deprotected, and compared with the raw material adopting amino protection in the existing preparation method, the method omits the step of deprotection, so that the preparation process is simpler and more efficient.
(5) The method utilizes the one-pot boiling method of multi-step reaction for the first time, has the advantages of simple operation, labor and time saving, lower cost, greenness, environmental protection, high product quality and yield, suitability for large-scale production and the like, and is suitable for preparing high-purity insulin analogues.
Detailed description of the preferred embodiments
Description of the terms
In the present invention, unless otherwise specified, each term has the following meaning.
The preparation method of the invention of 'one-pot' refers to that the intermediate reaction liquid is not transferred, washed, extracted, recrystallized, and subjected to separation and purification treatment such as column chromatography after the feeding in the preparation process, a plurality of raw materials of the preparation method of 'one-pot' can be fed simultaneously or fed step by step, and in the invention, the feeding of a plurality of raw materials is preferably carried out step by step. The four-step one-pot boiling refers to that in the preparation process, the reaction raw materials are fed in four steps, the crude reaction liquid which is completely reacted after each step of feeding is not subjected to separation and purification treatment such as transfer, washing, extraction, recrystallization, column chromatography and the like, only the crude reaction liquid is subjected to temperature treatment, and then new raw materials are directly added for next reaction.
In the invention, the term "crude reaction solution" refers to a reaction solution obtained after the completion of each feeding reaction, and the crude reaction solution is not subjected to transfer, separation and purification treatment before the continuous feeding; the crude reaction liquid is only subjected to temperature reduction treatment, then new raw materials are directly added for the next reaction, and the temperature of the crude reaction liquid is reduced to-5-10 ℃, preferably 0-10 ℃.
In the present invention, the "fatty acid" contains one carboxyl group, and includes a straight or branched aliphatic carboxylic acid having at least two carbon atoms, preferably 12 to 22 carbon atoms, and may be a saturated fatty acid or an unsaturated fatty acid. Specific examples of fatty acids include, but are not limited to, myristic acid, palmitic acid, stearic acid, and the like.
In the present invention, "aliphatic diacids" include straight-chain or branched aliphatic diacids having at least two carbon atoms, preferably 12 to 22 carbon atoms, and may be saturated aliphatic diacids or unsaturated aliphatic diacids. Specific examples of aliphatic diacids include, but are not limited to, succinic acid, adipic acid, suberic acid, sebacic acid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid, octadecanedioic acid, nonadecanedioic acid, and didecanedioic acid, and the like.
In the present invention, a fatty acid having a naked carboxyl group or a fatty diacid in which a carboxyl group at one end is protectedWherein R is 1 Is methyl or protected carboxy; n is an integer from 9 to 19, preferably n is 12, 13, 14, 15, 16 or 17.
In the present invention, the "amino acid residue" includes amino acids in which a hydrogen atom is removed from an amino group and/or a hydroxyl group is removed from a carboxyl group and/or a hydrogen atom is removed from a mercapto group and/or an amino group is protected and/or a carboxyl group is protected and/or a mercapto group is protected. Not to be strict, an amino acid residue may be referred to as an amino acid. The source of the amino acid in the present invention is not particularly limited unless otherwise specified, and may be a natural source, a non-natural source, or a mixture of both. The amino acid structure type in the present invention is not particularly limited unless otherwise specified, and may be either an L-type or a D-type, or a mixture of both. In one embodiment of the present invention, the amino acid is a hydrophobic amino acid selected from any one of tryptophan (Trp), phenylalanine (Phe), valine (Val), isoleucine (Ile), leucine (Leu), and tyrosine (Tyr). In another embodiment of the present invention, the amino acid is a hydrophilic amino acid selected from any one of glutamic acid (Glu), aspartic acid (Asp), histidine (His), glutamine (gin), asparagine (Asn), serine (Ser), threonine (Thr), proline (Pro), glycine (Gly), lysine (Lys), and arginine (Arg), preferably glutamic acid or aspartic acid, more preferably glutamic acid. In the present invention, the amino acid is preferably any one of glutamic acid, aspartic acid, histidine, glutamine, asparagine, serine, threonine, proline, glycine, lysine and arginine; more preferably glutamic acid or aspartic acid; more preferably L-Glu or D-Glu in which one terminal carboxyl group is protected, preferably L-Glu or D-Glu
To prevent the functional group from affecting the reaction, the functional group is usually protected. When the number of functional groups is 2 or more, only the target functional group is selectively reacted, and thus the other functional groups are protected. The protecting group is required to be easily removed as needed in addition to stably protecting a functional group to be protected. It is therefore important in organic synthesis to deprotect only the protecting group bonded to the specified functional group under appropriate conditions.
In the present invention, the "carboxyl-protecting group" refers to a protecting group which can be converted into a carboxyl group by hydrolysis or a deprotection reaction of the carboxyl-protecting group. Carboxyl protecting group, preferably alkyl (e.g. methyl)Alkyl, ethyl, tert-butyl) or aralkyl (e.g. benzyl), more preferably tert-butyl (tBu), methyl (Me) or ethyl (Et). In the present invention, the "protected carboxyl group" refers to a group formed by protecting a carboxyl group with a suitable carboxyl protecting group, and is preferably a methoxycarbonyl group, an ethoxycarbonyl group, a tert-butoxycarbonyl group or a benzyloxycarbonyl group. The carboxyl protecting group can be removed by hydrolysis under the catalysis of acid or base, and occasionally can also be eliminated by pyrolysis, for example, tert-butyl can be removed under mild acidic conditions, and benzyl can be removed by hydrogenolysis. The reagent for removing carboxyl protecting group is selected from TFA and H 2 O, liOH, naOH, KOH, meOH, etOH, and combinations thereof, preferably TFA and H 2 A combination of O, liOH and MeOH, or LiOH and EtOH. Deprotection of a protected carboxyl group to produce the corresponding free acid, said deprotection being carried out in the presence of a base which forms a pharmaceutically acceptable salt with the free acid formed by said deprotection.
In the present invention, the "amino-protecting group" includes all groups that can be used as protecting groups for general amino groups, for example, aryl C 1-6 Alkyl radical, C 1-6 Alkoxy radical C 1-6 Alkyl radical, C 1-6 Alkoxycarbonyl, aryloxycarbonyl, C 1-6 Alkylsulfonyl, arylsulfonyl, silyl, or the like. The amino protecting group is preferably Boc-t-butoxycarbonyl, moz p-methoxybenzyloxycarbonyl, and Fmoc 9-fluorenylmethyloxycarbonyl. The reagent for removing the amino protecting group is selected from TFA and H 2 O, liOH, meOH, etOH, and combinations thereof, preferably TFA and H 2 A combination of O, liOH and MeOH, or LiOH and EtOH. The reagent for removing the Boc protecting group is TFA or HCl/EA; TFA is preferred. The deprotection agent used in the Fmoc protecting group removal reaction was a solution of N, N-Dimethylformamide (DMF) containing 20% piperidine.
In the present invention, "carboxyl group activation" refers to activation treatment of carboxyl group with a carboxyl group activating agent, which promotes better condensation reaction after carboxyl group activation, such as: inhibiting the generation of racemization impurities in condensation reaction, catalyzing and accelerating the reaction speed, and the like. A "carboxyl activating group" is the residue of a carboxyl activating agent. The carboxyl activating agent is one or more of N-hydroxysuccinimide (NHS), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI), N-hydroxy-5-norbornene-2, 3-dicarboximide (HONb) and N, N-Dicyclohexylcarbodiimide (DCC), preferably a combination of NHS/EDCI, NHS/DCC, HONb/DCC, most preferably a combination of NHS/EDCI. When the carboxyl activating agent is a combination of NHS/EDCI, the molar ratio of NHS to EDCI is 1:1-1.5, preferably 1:1-1.2, more preferably 1:1.14.
in the technical scheme of the invention, in the carboxyl activation process, the reaction time of the compound to be activated by carboxyl and the carboxyl activator is 3-8h, preferably 3-6h, and more preferably 4h.
In the present invention, AEEA means a structureWherein the amino terminus is linked to an amino acid of formula (1) and H-AEEA-OH is the amino acid NH 2 -(CH 2 CH 2 O) 2 -CH 2 -COOH, H-AEEA-AEEA-OH is the amino acid NH 2 -(CH 2 CH 2 O) 2 -CH 2 -CO-NH-(CH 2 CH 2 O) 2 -CH 2 -COOH. The starting materials H-AEEA-OH and H-AEEA-AEEA-OH can be purchased directly or obtained by a suitable coupling reaction. In a preferred embodiment of the present invention, the raw material is preferably selectedIs the case with p =2, i.e. preferably the starting material is ^ 4>
In the present invention, the "condensation reaction" may be carried out by the action of a condensing agent, each independently selected from 1-hydroxybenzotriazole (HOBt), N-Diisopropylcarbodiimide (DIC), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl or EDCI), N-Dicyclohexylcarbodiimide (DCC), benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), benzotriazole-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), 2- (7-aza-1H-benzotriazol-1-yl) -1, 3-tetramethyluronium Hexafluorophosphate (HATU), benzotriazole-N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HBTU), O-benzotriazol-N, N, N ', N-tetramethyluronium tetrafluoroborate (TBTU), diisopropylethylamine (DIEA), preferably DIC/HOBt, HBTU/HOBt/DIEA or PyBop/HOBt/DIEA combinations. In the present invention, the "condensation reaction" may be carried out without the action of a condensing agent, and the condensation reaction may be carried out directly by the action of a base after activating the reactive group of the reactant.
In the present invention, when the structure concerned has an isomer, any of the isomers may be used unless otherwise specified. For example, a cis-isomer or trans-isomer may be present in a structure; when the optical rotation property exists, the optical rotation property can be left-handed rotation or right-handed rotation.
In the present invention, the numerical range includes both the numerical range marked by the short horizontal line (e.g. 3-8) and the numerical range marked by the wavy line (e.g. 9-19). In the present invention, unless otherwise specified, the integer intervals marked as intervals may represent the group of all integers within the range of the interval, and the range includes both endpoints. For example, the integer range of 9 to 19 represents a group consisting of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and 19.
In the present invention, two or more objects "are independently preferred" and, when there are preferences in multiple stages, they are not required to be selected from the same preferred group of the same stage, but one may be selected from a wide range of preferences, one may be selected from a narrow range of preferences, one may be selected from a maximum range of preferences, and the other may be selected from any of the preferences, and the preferences in the same stage may be selected. For the purposes of the present invention, "two or more objects are each independently selected" does not limit two objects to being selected from the same thing, e.g., "R 2 And R 5 Each independently selected from any one of methyl, ethyl, tert-butyl and benzyl, and may be all methyl or all ethyl or all tert-butyl or all benzyl, or R 2 Is methyl and R 5 Any of ethyl, tert-butyl and benzyl, or R 5 Is methyl and R 2 Any one of ethyl, tert-butyl and benzyl, etc.
In the present invention, the divalent linking group such as alkylene, arylene, amide bond and the like is not particularly limited, and any of the two linking ends may be selected when other groups are linked, for example, in A-CH 2 CH 2 -and-CH 2 When an amide bond is used as a divalent linking group between-B, it may be A-CH 2 CH 2 -C(=O)NH-CH 2 -B or A-CH 2 CH 2 -NHC(=O)-CH 2 -B。
In the formula, when the terminal group of the linking group is easily confused with the substituent contained in the linking group, as in the formulaIn, adopted->To mark the position of the divalent linking group to which other groups are bonded, the two aforementioned structural formulas each represent-CH (CH) 2 CH 2 CH 3 ) 2 -、-CH 2 CH 2 CH(CH 3 ) 2 -CH 2 CH 2 -。
The numerical values referred to herein as "about" and "around" generally refer to a range of values of + -10%, and in some cases may be up to + -15%, but not more than + -20%. The preset value is used as a base number.
The numerical ranges in this disclosure, including but not limited to, whole, non-whole, percent, fractional, and inclusive of the two endpoints unless specifically stated otherwise.
The invention discloses a preparation method of a polypeptide side chain analogue, wherein the structural general formula of the polypeptide side chain analogue is shown as the formula (1):
wherein R is 1 Is methyl or protected carboxy; n is an integer of 9 to 19; AA is an amino acid residue; p is 1 or 2; the structure of AEEA isAnd wherein the amino terminus is linked to an amino acid residue;
the polypeptide side chain analogue is prepared by the following method:
reacting a fatty acid containing a naked carboxyl group or a fatty diacid in which one of the carboxyl groups is protectedThe exposed carboxyl group of the fatty acid derivative or the fatty diacid derivative is activated to obtain the fatty acid derivative or the fatty diacid derivative containing one end of the carboxyl group which is activatedIn the crude reaction solution of (1), wherein R 3 Is a carboxyl group activating group, the carboxyl group activation is activation treatment of a carboxyl group with a carboxyl group activating agent, the carboxyl group activating group is a residue of the carboxyl group activating agent; directly adding raw material amino acid into the crude reaction liquid to carry out condensation reaction; after the reaction is completed, the product containing fatty acid-amino acid conjugate or fatty diacid-amino acid conjugate is obtained>Directly activating the exposed carboxyl of the conjugate to obtain a fatty acid-amino acid conjugate or a fatty diacid-amino acid conjugate containing activated carboxyl at one endIn the crude reaction solution of (1), wherein R 4 Is a carboxyl activating group; then the raw material is directly added into the crude reaction solution>Condensation reaction is carried out to obtain the compound shown in the formula (1).
According to a preferred scheme of the invention, the preparation process is 'one-pot' of multi-step reaction, the reaction raw materials are fed in four steps, the crude reaction liquid which is completely reacted after each step of feeding is not subjected to separation and purification treatment such as transfer, water washing, extraction, recrystallization, column chromatography and the like, only the crude reaction liquid is subjected to temperature reduction treatment, and then new raw materials are directly added for the next step of reaction.
In a preferred embodiment of the present invention, the feeding order is a fatty acid or a fatty diacid in which one end of carboxyl group is protected, a carboxyl group activator, an amino acid, a carboxyl group activator, a,(wherein p is 1 or 2, AEEA is) (ii) a The preferred charging sequence of the reaction is octadecanedioic acid, carboxyl activators, glutamic acid, carboxyl activators,. Sup.>
In a preferred embodiment of the present invention, the raw material fatty acid or fatty diacid, amino acid,(wherein p is 1 or 2) in a molar ratio of 1:1-1.5:1-1.2:1-1.5, preferably 1:1-1.2:1-1.05:1-1.2, more preferably 1:1.01:1.05.
in a preferred embodiment of the present invention, the amino acid is glutamic acid, and the polypeptide side chain analog has a structural general formula shown in formula (2):
wherein R is 2 And R 5 Are identical or different carboxyl protecting groups, preferably R 2 And R 5 Each independently selected from any one of methyl, ethyl, tert-butyl and benzyl, more preferably R 2 And R 5 And is simultaneously a tert-butyl group;
the polypeptide side chain analogue is prepared by the following method:
fatty diacid obtained by protecting carboxyl at one endIs activated to obtain a fatty diacid derivative which contains an activated terminal carboxyl group>In the crude reaction solution of (1), wherein R 3 Is a carboxyl activating group; directly adding glutamic acid with protected carboxyl at one end of amino acid raw material into the crude reaction solution>Condensation reaction is carried out to obtain the conjugate containing the fatty diacid-amino acid>Directly activating the naked carboxyl of the combination to obtain a fatty diacid-amino acid combination which has one end carboxyl activated>In the crude reaction solution of (1), wherein R 4 Is a carboxyl activating group; finally, the raw material is directly added into the coarse reaction solution>(wherein p is 1 or 2) to obtain a compound represented by the formula (2).
In a preferred embodiment of the present invention, the condensation reaction is carried out at a temperature of 20 to 35 deg.C, preferably 30 deg.C.
According to a preferable scheme of the invention, the crude reaction solution is not subjected to separation and purification treatment before continuous feeding; the crude reaction solution is cooled to-5-10 deg.C, preferably 0-10 deg.C.
In a preferred embodiment of the present invention, the condensation reaction is carried out under the action of an organic base selected from any one of N, N-Diisopropylethylamine (DIEA), monoethylamine, diethylamine, triethylamine, imidazole, 1, 8-diazabicycloundec-7-ene (DBU), pyridine and piperazine, preferably triethylamine.
Purification and characterization of the end product
The final product prepared in the present invention can be purified by purification methods including, but not limited to, extraction, recrystallization, adsorption treatment, precipitation, reverse precipitation, membrane dialysis, or supercritical extraction. The final product structure and molecular weight can be confirmed by characterization methods including but not limited to nuclear magnetism, electrophoresis, ultraviolet-visible spectrophotometer, FTIR, AFM, GPC, HPLC, MALDI-TOF, circular dichroism method, etc. For polypeptide side chain analogs, the molecular weight is preferably confirmed by MALDI-TOF.
Another objective of the invention is to provide application of the preparation method of the polypeptide side chain analogue.
The preparation method of the polypeptide side chain analogue is particularly suitable for preparing insulin analogues; the insulin analogues are for use as a medicament, preferably for the treatment or prevention of hyperglycemia, type II diabetes, impaired glucose tolerance or type I diabetes.
In a preferred embodiment of the present invention, the method for preparing the side chain analog of the polypeptide is used for preparing the insulin analog which is the somaglutide.
The terms treatment and care in the present invention refer to the treatment and care of a patient for the purpose of combating a disease, disorder or condition. The term is intended to include: delay the progression of the disease, disorder or condition, alleviate or alleviate symptoms and complications, and/or cure or eliminate the disease, disorder or condition. The patient to be treated is preferably a mammal, especially a human.
The present invention relates to the use of insulin analogues as medicaments for lowering blood glucose levels in mammals, in particular for the treatment of diabetes.
The insulin analogues contemplated in the present invention are administered in combination with one or more other active substances in any suitable ratio. Such other active agent may be selected from human insulin, fast-acting insulin analogues, anti-diabetic agents, anti-hyperlipidemic agents, anti-obesity agents, anti-hypertensive agents and agents for the treatment of complications originating from or associated with diabetes.
The preparation of the polypeptide side chain analogs is further described below with reference to some specific examples. The specific examples are intended to illustrate the present invention in further detail, and are not intended to limit the scope of the present invention. In the examples where the peptide side chain analogs were prepared, the final product was characterized by nuclear magnetism and the molecular weight was confirmed by MALDI-TOF.
Examples 1-7 were prepared according to the following routes, the specific methods of preparation being detailed in the examples.
Example 1: synthesis of compound 6d tBuO-Ste-Glu- (AEEA-AEEA-OH) -OtBu
20g of compound 1d octadecanedioic acid mono-tert-butyl ester tBuO-Ste-OH (n =15, 54.0 mmol) was dissolved in 400mL of DCM solution, placed in a nitrogen-protected flask, and after the above-mentioned mixed solution was cooled to 0 to 10 ℃, NHS (6.5g, 56.7 mmol) and EDCI (12.4g, 64.8mmol) were carefully added to the above-mentioned solution, and the reaction was continued after returning to room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 2d for the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, the compounds L-Glu (3a, 11.1g, 54.5mmol) and TEA (8.19g, 81.0mmol) were added to the reaction system under nitrogen protection, and the reaction was allowed to return to room temperature overnight. TLC showed complete consumption of the starting material and yielded a crude reaction containing compound 4d which was directly fed to the next step.
After the temperature of the crude reaction liquid in the previous step is reduced to 0-10 ℃, NHS (6.5g, 56.7mmol) and EDCI (12.4g, 64.8mmol) are added into the reaction system under the protection of nitrogen, and the reaction is continued after returning to room temperature. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 5d was directly fed to the next step.
The last stepAfter the temperature of the crude reaction solution is reduced to 0-10 ℃, H-AEEA-AEEA-OH (17.5g, 56.7mmol) and TEA (8.19g, 81.0mmol) are added into the reaction system under the protection of nitrogen, and the reaction is returned to room temperature for overnight reaction. TLC showed complete consumption of starting material. The reaction solution was washed twice with 200mL of 0.5N HCl/10% NaCl mixed solution and once with saturated brine, and the organic phase was anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by column separation on silica gel (DCM: meOH = 50. Ms [ M + H + ]846.7g/mol. The hydrogen spectrum data of the final product 6d are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.60(s,1H),8.05(d,J=7.5Hz,1H),7.90(t,J=5.6Hz,1H),7.67(t,J=5.7Hz,1H),4.09-3.99(m,3H),3.88(s,2H),3.55(tdd,J=9.1,6.0,3.3Hz,8H),3.43(dt,J=11.8,5.9Hz,4H),3.24(dq,J=28.1,5.8Hz,4H),2.20-2.06(m,6H),1.89(td,J=13.5,7.5Hz,1H),1.80-1.69(m,1H),1.54-1.43(m,4H),1.39(brs,18H),1.23(brs,24H)。
example 2: synthesis of compound 6a tBuO-Pen-Glu- (AEEA-AEEA-OH) -OtBu
10g of the compound 1a, i.e., the mono-tert-butyl pentadecanedioate, tBuO-Pen-OH (n =12, 30.4 mmol), was dissolved in 200mL of a DCM solution, the solution was placed in a nitrogen-protected flask, NHS (3.7g, 32.0mmol) and EDCI (7.0g, 36.5mmol) were carefully added to the solution after the temperature of the mixture was lowered to 0 to 10 ℃, and the reaction was continued at room temperature. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 2a was obtained and directly fed to the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, the compound L-Glu (3a, 6.3g, 30.7mmol) and TEA (4.6g, 45.6mmol) were added to the reaction system under nitrogen protection, and the reaction was returned to room temperature for overnight reaction. TLC showed complete consumption of the starting material, and the crude reaction mixture containing compound 4a was obtained and directly fed to the next step.
Cooling the crude reaction liquid in the last step to 0-10 ℃, adding NHS (3.7g, 32.0mmol) and EDCI (7.0g, 36.5mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for continuous reaction. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 5a for the next step.
After the temperature of the crude reaction solution in the previous step is reduced to 0-10 ℃, H-AEEA-AEEA-OH (9.9 g,31.9 mmol) and TEA (4.6 g,45.6 mmol) are added into the reaction system under the protection of nitrogen, and the reaction is returned to room temperature for overnight reaction. TLC showed complete consumption of starting material. The reaction mixture was washed twice with 100mL of a 0.5N HCl/10% NaCl mixture, once with a saturated brine, and the organic phase was over anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was isolated and purified by silica gel column (DCM: meOH = 50. Ms [ M + H + ]804.6g/mol. The hydrogen spectrum data for end product 6a are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.60(s,1H),8.05(d,J=7.5Hz,1H),7.90(t,J=5.5Hz,1H),7.67(t,J=5.7Hz,1H),4.09-4.00(m,3H),3.88(s,2H),3.55(tdd,J=9.0,6.0,3.3Hz,8H),3.43(dt,J=11.8,5.9Hz,4H),3.30-3.15(m,4H),2.13(ddd,J=25.3,13.6,7.3Hz,6H),1.89(td,J=13.5,7.5Hz,1H),1.73(td,J=16.5,7.7Hz,1H),1.47(s,4H),1.39(brs,18H),1.24(brs,18H)。
example 3: synthesis of compound 6b tBuO-Pal-Glu- (AEEA-AEEA-OH) -OtBu
12g of compound 1b mono-tert-butyl hexadecanedioate tBuO-Pal-OH (n =13, 35.0 mmol) was dissolved in 250mL DCM solution, placed in a nitrogen-protected flask, and after the mixture was cooled to 0-10 deg.C, NHS (4.3g, 36.8 mmol) and EDCI (8.1g, 42.0 mmol) were carefully added to the solution, and the reaction was continued at room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 2b for the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, the compounds L-Glu (3a, 7.2g,35.4 mmol) and TEA (5.3g, 52.5 mmol) were added to the reaction system under nitrogen protection, and the reaction was allowed to proceed overnight at room temperature. TLC showed complete consumption of starting material and yielded a crude reaction containing compound 4b which was directly fed to the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, NHS (4.3g, 36.8mmol) and EDCI (8.1g, 42.0mmol) were added to the reaction system under a nitrogen atmosphere, and the reaction was continued at room temperature. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 5b was obtained and directly fed to the next step.
After the crude reaction liquid in the previous step is cooled to 0-10 ℃, H-AEEA-AEEA-OH (11.3g, 36.8mmol) and TEA (5.3g, 52.5mmol) are added into the reaction system under the protection of nitrogen, and the reaction is returned to room temperature for overnight reaction. TLC showed complete consumption of starting material. The reaction mixture was washed twice with 150mL of a 0.5N HCl/10-vol NaCl mixture, once with saturated brine, and the organic phase was washed with anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was isolated and purified by silica gel column (DCM: meOH = 50. Ms [ M + H + ]818.8g/mol. The hydrogen spectrum data of the final product 6b are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.59(s,1H),8.05(d,J=7.5Hz,1H),7.90(t,J=5.5Hz,1H),7.67(t,J=5.7Hz,1H),4.10-3.99(m,3H),3.88(s,2H),3.55(dtd,J=8.5,5.6,2.9Hz,8H),3.43(dt,J=11.8,5.9Hz,4H),3.24(ddd,J=28.0,11.6,5.8Hz,4H),2.13(ddd,J=24.4,13.1,6.9Hz,6H),1.89(td,J=13.4,7.5Hz,1H),1.73(dt,J=16.4,7.8Hz,1H),1.46(d,J=6.4Hz,4H),1.39(brs,18H),1.24(brs,20H)。
example 4: synthesis of compound 6c tBuO-Hep-Glu- (AEEA-AEEA-OH) -OtBu
5g of compound 1c mono-tert-butyl heptadecanedioate, tBuO-Hep-OH (n =14, 14.0 mmol), was dissolved in 100mL of DCM solution, placed in a nitrogen-protected flask, and after the mixture had cooled to 0-10 deg.C, NHS (1.7g, 14.7mmol) and EDCI (3.2g, 16.8mmol) were carefully added to the solution, and the reaction was continued at room temperature. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 2c was obtained and directly fed to the next step.
After the crude reaction solution in the previous step is cooled to 0-10 ℃, the compounds L-Glu (3a, 2.9g and 14.2mmol) and TEA (2.2g and 21mmol) are added into the reaction system under the protection of nitrogen, and the reaction system is returned to room temperature for overnight reaction. TLC showed complete consumption of starting material and yielded a crude reaction containing compound 4c which was directly fed to the next step.
Cooling the crude reaction liquid in the last step to 0-10 ℃, adding NHS (1.7g, 14.7mmol) and EDCI (3.2g, 16.8mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for continuous reaction. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 5c was obtained and directly fed to the next step.
After the temperature of the crude reaction liquid in the previous step is reduced to 0-10 ℃, H-AEEA-AEEA-OH (4.5g, 14.7 mmol) and TEA (2.2g, 21mmol) are added into the reaction system under the protection of nitrogen, and the mixture is returned to room temperature for reaction overnight. TLC showed complete consumption of starting material. The reaction mixture was washed twice with 50mL of 0.5N HCl/10% NaCl mixture, once with saturated brine, and the organic phase was washed with anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by separation on a silica gel column (DCM: meOH = 50. Ms [ M + H + ]830.8g/mol. The hydrogen spectrum data for final product 6c are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.55(s,1H),8.02(d,J=7.5Hz,1H),7.86(t,J=5.6Hz,1H),7.63(t,J=5.7Hz,1H),4.09-4.01(m,3H),3.88(s,2H),3.61-3.52(m,8H),3.43(dt,J=12.0,5.9Hz,4H),3.24(ddd,J=28.3,11.8,5.9Hz,4H),2.19-2.07(m,6H),1.89(td,J=13.5,7.5Hz,1H),1.74(td,J=16.4,7.8Hz,1H),1.53-1.44(m,4H),1.39(brs,18H),1.24(brs,22H)。
example 5: synthesis of compound 6e tBuO-Non-Glu- (AEEA-AEEA-OH) -OtBu
10g of compound 1e nonadecanoic diacid mono-tert-butyl ester tBuO-Non-OH (n =16, 26.0 mmol) was dissolved in 200mL of DCM solution, placed in a nitrogen-protected flask, and after the mixture was cooled to 0-10 deg.C, NHS (3.2g, 27.3 mmol) and EDCI (6.0 g,31.2 mmol) were carefully added to the solution, and the reaction was continued at room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 1e for the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, the compounds L-Glu (3 a,5.4g,26.3 mmol) and TEA (3.9g, 39.0 mmol) were added to the reaction system under nitrogen protection, and the reaction was allowed to proceed overnight at room temperature. TLC showed complete consumption of the starting material and yielded a crude reaction containing compound 4e which was directly fed to the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, NHS (3.2g, 27.3mmol) and EDCI (6.0 g, 31.2mmol) were added to the reaction system under the protection of nitrogen, and the reaction was continued at room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 5e for the next step.
After the temperature of the crude reaction solution in the previous step is reduced to 0-10 ℃, H-AEEA-AEEA-OH (8.4g, 27.3mmol) and TEA (3.9g, 39.0mmol) are added into the reaction system under the protection of nitrogen, and the reaction is returned to room temperature for overnight reaction. TLC showed complete consumption of starting material. The reaction mixture was washed twice with 100mL of 0.5N HCl/10% NaCl mixture solution, once with saturated brine, and the organic phase was washed with anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was isolated and purified by silica gel column (DCM: meOH = 50. Ms [ M + H + ]858.9g/mol. The hydrogen spectrum data of the final product 6e are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.58(s,1H),8.04(d,J=7.5Hz,1H),7.89(t,J=5.6Hz,1H),7.65(t,J=5.7Hz,1H),4.09-4.01(m,3H),3.88(s,2H),3.61-3.51(m,8H),3.43(dt,J=11.9,5.9Hz,4H),3.24(dq,J=28.2,5.9Hz,4H),2.19-2.07(m,6H),1.89(td,J=13.5,7.6Hz,1H),1.73(td,J=16.5,7.8Hz,1H),1.52-1.43(m,4H),1.39(brs,18H),1.24(brs,26H)。
example 6: synthesis of compound 6f tBuO-Ara-Glu- (AEEA-AEEA-OH) -OtBu
15g of the compound 1f di-tert-butyl ditodecanedioate tBuO-Ara-OH (n =17, 37.6 mmol) was dissolved in 300mL DCM solution, placed in a nitrogen-protected flask, and after the above mixture was cooled to 0-10 deg.C, NHS (4.6 g,39.5 mmol) and EDCI (8.6 g,45.1 mmol) were carefully added to the above solution, and the reaction was continued after returning to room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 2f which was directly fed to the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, compounds L-Glu (3a, 7.7g, 38.0mmol) and TEA (5.7g, 56.4mmol) were added to the reaction system under nitrogen protection, and the reaction was allowed to return to room temperature overnight. TLC showed complete consumption of the starting material and yielded a crude reaction containing compound 4f which was directly fed to the next step.
After the temperature of the crude reaction liquid in the previous step is reduced to 0-10 ℃, NHS (4.6 g,39.5 mmol) and EDCI (8.6 g,45.1 mmol) are added into the reaction system under the protection of nitrogen, and the reaction is continued after returning to room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 5f which was directly fed to the next step.
After the temperature of the crude reaction liquid in the previous step is reduced to 0-10 ℃, H-AEEA-AEEA-OH (12.2g, 27.3mmol) and TEA (5.7g, 56.4mmol) are added into the reaction system under the protection of nitrogen, and the reaction is returned to room temperature for overnight reaction. TLC showed complete consumption of starting material. The reaction mixture was washed twice with 150mL of a 0.5N HCl/10% NaCl mixture, once with saturated brine, and the organic phase was anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by column separation on silica gel (DCM: meOH = 50. Ms [ M + H + ]858.9g/mol. The hydrogen spectrum data of the final product 6f are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.58(s,1H),8.04(d,J=7.5Hz,1H),7.89(t,J=5.6Hz,1H),7.65(t,J=5.7Hz,1H),4.09-4.01(m,3H),3.88(s,2H),3.61-3.51(m,8H),3.43(dt,J=11.9,5.9Hz,4H),3.24(dq,J=28.2,5.9Hz,4H),2.19-2.07(m,6H),1.89(td,J=13.5,7.6Hz,1H),1.73(td,J=16.5,7.8Hz,1H),1.52-1.43(m,4H),1.39(brs,18H),1.24(brs,28H)。
example 7: synthesis of Compound 6g tBuO-Ste-Glu- (AEEA-OH) -OtBu
10g of compound 1d octadecanedioic acid mono-tert-butyl ester tBuO-Ste-OH (n =15, 27 mmol) was dissolved in 200mL of DCM solution, placed in a nitrogen-protected flask, and after the above-mentioned mixture was cooled to 0-10 ℃, NHS (3.3g, 28.4 mmol) and EDCI (6.2g, 32.4 mmol) were carefully added to the above-mentioned solution, and the reaction was continued back to room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 2d for the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, the compounds L-Glu (3a, 5.6g,27.3 mmol) and TEA (4.1g, 40.5 mmol) were added to the reaction system under nitrogen protection, and the reaction was allowed to proceed overnight at room temperature. TLC showed complete consumption of starting material and yielded a crude reaction containing compound 4d which was directly fed to the next step.
And (3) cooling the reaction solution in the previous step to 0-10 ℃, adding NHS (3.3 g,28.4 mmol) and EDCI (6.2 g,32.4 mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for continuous reaction. After 4h of reaction, TLC showed complete consumption of starting material and yielded crude reaction solution containing compound 5d for the next step.
After the temperature of the crude reaction solution in the previous step was lowered to 0 to 10 ℃, H-AEEA-OH (4.6g, 28.4 mmol) and TEA (4.1g, 40.5 mmol) were added to the reaction system under the protection of nitrogen, and the mixture was allowed to react overnight at room temperature. TLC showed complete consumption of starting material. The reaction solution was washed twice with 200mL of 0.5N HCl/10% NaCl mixed solution and once with saturated brine, and the organic phase was anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was isolated and purified by silica gel column (DCM: meOH =50- (AEEA-OH) -OtBu, yield 60%, HPLC purity 97.0%. Ms [ M + H + ]701.7g/mol. The hydrogen spectrum data of the final product 6g are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.60(s,1H),8.05(d,J=7.5Hz,1H),7.90(t,J=5.5Hz,1H),4.08-4.00(m,3H),3.58(dd,J=5.7,3.2Hz,2H),3.52(dd,J=5.7,3.2Hz,2H),3.40(t,J=5.9Hz,2H),3.19(q,J=5.8Hz,2H),2.19-2.05(m,6H),1.88(td,J=13.5,7.5Hz,1H),1.73(td,J=16.5,7.8Hz,1H),1.46(d,J=6.3Hz,4H),1.39(brs,18H),1.23(brs,24H)。
example 8: synthesis of compound 6h tBuO-Ste- (D) -Glu- (AEEA-AEEA-OH) -OtBu
10g of compound 1d octadecanedioic acid mono-tert-butyl ester tBuO-Ste-OH (n =15, 27 mmol) was dissolved in 200mL of DCM solution, placed in a nitrogen-protected flask, and after the above-mentioned mixture was cooled to 0-10 ℃, NHS (3.3g, 28.4 mmol) and EDCI (6.2g, 32.4 mmol) were carefully added to the above-mentioned solution, and the reaction was continued back to room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 2d for the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, the compound D-Glu (3b, 5.6g,27.3 mmol) and TEA (4.1g, 40.5 mmol) were added to the reaction system under nitrogen protection, and the reaction was allowed to proceed overnight at room temperature. TLC showed complete consumption of the starting material, and the crude reaction solution containing 4h of compound was obtained and directly fed to the next step.
After the temperature of the crude reaction liquid in the previous step is reduced to 0-10 ℃, NHS (3.3 g,28.4 mmol) and EDCI (6.2 g,32.4 mmol) are added into the reaction system under the protection of nitrogen, and the reaction is continued after returning to the room temperature. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing 5h of compound was directly fed to the next step.
Cooling the crude reaction liquid in the last step to 0-10 ℃, and then reacting downwards under the protection of nitrogenH-AEEA-AEEA-OH (8.8g, 28.4mmol) and TEA (4.1g, 40.5mmol) were added to the reaction system, and the reaction was returned to room temperature overnight. TLC showed complete consumption of starting material. The reaction solution was washed twice with 200mL of 0.5N HCl/10% NaCl mixed solution and once with saturated brine, and the organic phase was anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was isolated and purified by silica gel column (DCM: meOH = 50. Ms [ M + H + ]846.7g/mol. The hydrogen spectrum data of the final product 6h are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.58(s,1H),8.04(d,J=7.5Hz,1H),7.89(t,J=5.6Hz,1H),7.66(t,J=5.7Hz,1H),4.09-4.00(m,3H),3.88(s,2H),3.56(tdd,J=8.9,6.0,3.2Hz,8H),3.43(dt,J=11.8,5.9Hz,4H),3.24(dq,J=28.1,5.8Hz,4H),2.20-2.05(m,6H),1.89(td,J=13.5,7.5Hz,1H),1.73(td,J=16.5,7.8Hz,1H),1.52-1.43(m,4H),1.39(brs,18H),1.24(brs,24H)。
example 9: synthesis of Compound 6i Ste-Glu- (AEEA-AEEA-OH) -OtBu
10g of compound 1i octadecanoic acid Ste-OH (n =15, 35.2 mmol) was dissolved in 200mL DCM solution, placed in a nitrogen-protected flask, and after the mixture was cooled to 0-10 deg.C, NHS (4.3 g,37.0 mmol) and EDCI (8.1 g,42.2 mmol) were carefully added to the solution, and the reaction was continued at room temperature. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 2i for the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, the compounds L-Glu (3a, 7.2g, 35.6mmol) and TEA (5.3g, 52.8mmol) were added to the reaction system under nitrogen protection, and the reaction was allowed to proceed overnight at room temperature. TLC showed complete consumption of starting material and yielded a crude reaction containing compound 4i which was directly fed to the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, NHS (4.3 g,37.0 mmol) and EDCI (8.1 g,42.2 mmol) were added to the reaction system under nitrogen protection, and the reaction was continued at room temperature. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 5i was obtained and directly fed to the next step.
After the crude reaction solution in the previous step was cooled to 0 to 10 ℃, H-AEEA-AEEA-OH (11.4 g, 37.0mmol) and TEA (5.3g, 52.8mmol) were added to the reaction system under the protection of nitrogen, and the mixture was allowed to react overnight at room temperature. TLC showed complete consumption of starting material. The reaction solution was washed twice with 200mL of 0.5N HCl/10% NaCl mixed solution, once with saturated brine, and the organic phase was washed with anhydrous MgSO 4 Drying, filtering and spin-drying to obtain a crude product. The crude product was isolated and purified by silica gel column (DCM: meOH = 50. Ms [ M + H + ]759.8g/mol. The hydrogen spectrum data of the final product 6i are as follows: 1 H NMR(400MHz,DMSO)δ(ppm):12.58(s,1H),8.04(d,J=7.5Hz,1H),7.89(t,J=5.6Hz,1H),7.66(t,J=5.7Hz,1H),4.09-4.00(m,3H),3.88(s,2H),3.56(tdd,J=8.9,6.0,3.2Hz,8H),3.43(dt,J=11.8,5.9Hz,4H),3.24(dq,J=28.1,5.8Hz,4H),2.20-2.05(m,4H),1.89(td,J=13.5,7.5Hz,1H),1.73(td,J=16.5,7.8Hz,1H),1.52-1.43(m,2H),1.39(brs,9H),1.24(brs,31H)。
the above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be understood that it is capable of further modifications. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (30)
1. A preparation method of a polypeptide side chain analogue is characterized in that the structural general formula of the polypeptide side chain analogue is shown as formula (1):
wherein R is 1 Is methyl or carboxyl protected by tert-butyl ester; n is an integer of 9 to 19; AA is a glutamic acid residue, and the glutamic acid is L-Glu or D-Glu with one end protected by carboxyl; p is 1 or 2; the structure of AEEA isAnd wherein the amino terminus is linked to an amino acid residue;
the preparation method of the polypeptide side chain analogue is 'one-pot' of multi-step reaction, wherein after each step of feeding, the completely reacted crude reaction liquid is only subjected to cooling treatment, and then new raw materials are directly added for the next step of reaction;
the polypeptide side chain analogue is prepared by the following method:
fatty acid containing one naked carboxyl or fatty diacid with one end carboxyl protected by tert-butyl esterThe exposed carboxyl group is activated to obtain a fatty acid derivative or a fatty diacid derivative containing activated carboxyl at one endIn the crude reaction solution of (1), wherein R 3 Is a carboxyl group activating group, the carboxyl group activation is activation treatment of a carboxyl group with a carboxyl group activating agent, the carboxyl group activating group is a residue of the carboxyl group activating agent; immediately thereafter, directly to the foregoingAdding raw material L-Glu or D-Glu with protected carboxyl at one end into the crude reaction solution for condensation reaction; after the reaction is completed, the product containing fatty acid-amino acid conjugate or fatty diacid-amino acid conjugate is obtained>Directly activating the naked carboxyl of the conjugate to obtain a fatty acid-amino acid conjugate or a fatty diacid-amino acid conjugate which has one activated carboxyl>The crude reaction solution of (1), wherein AA is a glutamic acid residue; r is 4 Is a carboxyl activating group; then the raw material is directly added into the crude reaction solution>Condensation reaction is carried out to obtain the compound shown in the formula (1).
2. The method for preparing a polypeptide side chain analog as claimed in claim 1, wherein the reaction raw material is fed in four steps, and the crude reaction solution after each feeding reaction is completely reacted is not subjected to transfer, water washing, extraction, recrystallization, column chromatography separation and purification treatment.
6. The method for preparing a polypeptide side chain analog of claim 5, wherein R is 2 Is a tert-butyl group.
7. The method for preparing a polypeptide side chain analog according to claim 1, wherein n is 12, 13, 14, 15, 16 or 17.
9. The method of claim 1, wherein the carboxyl activating agent is one or more of N-hydroxysuccinimide (NHS), 1-ethyl- (3-dimethylaminopropyl) carbodiimides hydrochloride (EDCI), N-hydroxy-5-norbornene-2, 3-dicarboximide (HONb), and N, N-Dicyclohexylcarbodiimide (DCC).
10. The method for preparing a polypeptide side chain analog according to claim 9, wherein the carboxyl activating agent is any one combination of NHS/EDCI, NHS/DCC, HONb/DCC.
11. The method of claim 10, wherein the carboxyl activating agent is a combination of NHS/EDCI.
12. The method for preparing a polypeptide side chain analog according to claim 1, wherein the structural general formula of the polypeptide side chain analog is shown in formula (2):
wherein R is 2 And R 5 Are identical or different carboxyl protecting groups, R 5 Is tert-butyl, R 2 Any one selected from methyl, ethyl, tert-butyl and benzyl;
the polypeptide side chain analogue is prepared by the following method:
fatty diacid obtained by protecting carboxyl at one endThe naked carboxyl is reacted with a carboxyl activating agent to be activated to obtain a fatty diacid derivative which has one end carboxyl activated>The crude reaction solution of (3); directly adding the amino acid raw material glutamic acid with protected carboxyl at one end into the crude reaction solution>Condensation reaction is carried out to obtain a substance which contains fatty diacid-amino acid combination>Directly reacting the naked carboxyl of the conjugate with a carboxyl activator to activate the crude reaction solution to obtain the fat diacid-amino acid conjugate which has one activated carboxyl>The crude reaction solution of (3); finally, the raw material is directly added into the coarse reaction solution>Condensation reaction is carried out to obtain the compound shown in the formula (2).
13. The method for preparing a polypeptide side chain analog as claimed in claim 12, wherein R is 2 And R 5 And is also a tert-butyl group.
14. The method of claim 12, wherein when the carboxyl activating agent is a combination of NHS/EDCI, the molar ratio of NHS to EDCI is 1:1-1.5.
15. The method of claim 14, wherein the molar ratio of NHS to EDCI is 1:1-1.2.
16. The method of claim 15, wherein the molar ratio of NHS to EDCI is 1:1.14.
17. the method for preparing a polypeptide side chain analog according to claim 12, wherein in the carboxyl group activation process, the reaction time of the compound to be activated by the carboxyl group and the carboxyl group activating agent is 3-8h.
18. The method of claim 17, wherein the reaction time of the compound to be activated by carboxyl groups with the carboxyl activating agent is 3-6 hours.
19. The method of claim 18, wherein the reaction time of the compound to be activated by carboxyl group and the carboxyl group activating agent is 4 hours.
23. the method for producing a polypeptide side chain analog according to claim 1, wherein the reaction temperature of the condensation reaction is 20 to 35 ℃.
24. The method of claim 23, wherein the condensation reaction is carried out at a temperature of 30 ℃.
25. The method for preparing a polypeptide side chain analog according to claim 1, wherein the crude reaction solution is not subjected to separation and purification treatment before further feeding; and cooling the crude reaction liquid to-5-10 ℃.
26. The method for preparing a polypeptide side chain analog according to claim 25, wherein the temperature of the crude reaction solution is lowered to 0-10 ℃.
27. The method for preparing a polypeptide side chain analog according to claim 1, wherein the condensation reaction is carried out under the action of an organic base selected from any one of N, N-diisopropylethylamine, monoethylamine, diethylamine, triethylamine, imidazole, 1, 8-diazabicycloundecen-7-ene, pyridine and piperazine.
28. The method of claim 27, wherein the organic base is triethylamine.
29. Use of the process for the preparation of a side chain analogue of a polypeptide according to any of claims 1-28 for the preparation of an insulin analogue for use as a medicament for the treatment or prevention of hyperglycemia, type II diabetes, impaired glucose tolerance or type I diabetes.
30. Use of the process for the preparation of a side chain analog of a polypeptide of claim 29 in the preparation of an insulin analog, wherein the insulin analog is somaglutide.
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