CN114685646A - 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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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, the fatty acid-amino acid conjugate or fatty diacid-amino acid conjugate in the obtained crude reaction liquid is reactedActivation of the exposed carboxyl groups of the compound; directly adding raw materials into the reactivated crude reaction solution
Or
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 increasing 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 (T2DM) with associated insulin resistance and insulin hyposecretion. Glucagon-like peptide-1 (GLP-1) is an important incretin. GLP-1 receptor agonists (GLP-1RAs) 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 a glucagon-like peptide-1 (GLP-1) analogue developed by Novonide, has 94% homology with human GLP-1, and has a molecular formula of C187H291N45O59Molecular weight 4113.58, half-life of about 7 days, is a long acting dosage form developed based on the basic structure of liraglutide. Side chain extension of somaglutide compared to liraglutideThe affinity to albumin is enhanced by 5-6 times, the molecular weight of the drug is increased after the albumin is combined, the drug is prevented from being rapidly cleared by the kidney and prevented from metabolic degradation, and 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 soma-Roudin side chain, and CN110423251A discloses a preparation method of a soma-Roudin side chain, although the prior art reports a liquid phase synthesis method of a soma-Roudin side chain, the synthesis route is multi-step, 2- (2- (2-aminoethoxy) ethoxy) acetic acid, Glu and octadecanedioic acid are coupled in sequence to obtain the soma-Roudin side chain, 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 is1Is 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 is
And 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 protected
The 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 activated
In the crude reaction solution of (1), wherein R3Is 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 group of the conjugate to obtain a fatty acid-amino acid conjugate or a fatty diacid-amino acid conjugate containing activated carboxyl at one end
Crude reaction solution of (2)Wherein R is4Is a carboxyl activating group; then directly adding raw materials into the crude reaction liquid
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 the feeding reaction is completed each time 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 the 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 invention omits the step of deprotection, so that the preparation process is simpler and more efficient.
(5) The method utilizes the one-pot 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 each feeding reaction is completed, and the crude reaction solution is not subjected to transfer, separation and purification treatment before being fed continuously; the crude reaction liquid is only subjected to temperature reduction treatment, and 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 protected
Wherein R is1Is 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 an amino acid 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 L-type or 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-substituted carboxylic acid in which one terminal carboxyl group is protectedGlu or D-Glu, preferably
In order 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. The carboxyl-protecting group is preferably an alkyl group (e.g., methyl, ethyl, t-butyl) or an aralkyl group (e.g., benzyl), more preferably a t-butyl group (tBu), methyl group (Me) or ethyl group (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 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 the carboxyl protecting group is selected from TFA and H2O, LiOH, NaOH, KOH, MeOH, EtOH, and combinations thereof, preferably TFA and H2A 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 C1-6Alkyl radical, C1-6Alkoxy radical C1-6Alkyl radical, C1-6Alkoxycarbonyl, aryloxycarbonyl, C1-6Alkylsulfonyl, arylsulfonyl, silyl, or the like. Amino protecting group is excellentSelected from Boc-t-butyloxycarbonyl, Moz p-methoxybenzyloxycarbonyl and Fmoc 9-fluorenylmethyloxycarbonyl. The reagent for removing the amino protecting group is selected from TFA and H2O, LiOH, MeOH, EtOH, and combinations thereof, preferably TFA and H2A 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 20% piperidine in N, N-Dimethylformamide (DMF).
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 "carboxy activating group" is the residue of a carboxy 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 NHS/EDCI, NHS/DCC, HONb/DCC, most preferably 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 4 h.
In the present invention, AEEA means a structure
Wherein the amino terminus is linked to an amino acid of formula (1) and H-AEEA-OH is the amino acid NH2-(CH2CH2O)2-CH2-COOH, H-AEEA-AEEA-OH is the amino acid NH2-(CH2CH2O)2-CH2-CO-NH-(CH2CH2O)2-CH2-COOH. The starting materials H-AEEA-OH and H-AEEA-AEEA-OH can be purchased directly or obtained by a suitable coupling reaction. A preferred embodiment of the present invention is a compoundMaterial
In the case of p ═ 2, i.e., the preferred starting material is
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), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium Hexafluorophosphate (HATU), benzotriazol-N, any one or any two combination of N, N ', N ' -tetramethyluronium Hexafluorophosphate (HBTU), O-benzotriazole-N, N, N ', N-tetramethyluronium tetrafluoroborate (TBTU) and Diisopropylethylamine (DIEA), preferably DIC/HOBt, HBTU/HOBt/DIEA or PyBop/HOBt/DIEA. 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 value interval includes both the numerical value interval marked by the short horizontal line (e.g. 3-8) and the numerical value interval 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. The integer range of 9-19 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., "R2And R5Each independently selected from any one of methyl, ethyl, tert-butyl and benzyl, and can be methyl, ethyl, tert-butyl or benzyl, or R2Is methyl and R5Any of ethyl, tert-butyl and benzyl, or R5Is methyl and R2Any 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-CH2CH2-and-CH2When an amide bond is used as a divalent linking group between-B, it may be A-CH2CH2-C(＝O)NH-CH2-B or A-CH2CH2-NHC(＝O)-CH2-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 formula
In (1), adopt
To mark the position of the divalent linking group to which other groups are bonded, the two aforementioned structural formulas each represent-CH (CH)2CH2CH3)2-、-CH2CH2CH(CH3)2-CH2CH2-。
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 the present invention include, but are not limited to, numerical ranges expressed as integers, non-integers, percentages, and fractions, and include both endpoints unless otherwise specified.
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 is1Is 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 is
And 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 protected
The 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 activated
In the crude reaction solution of (1), wherein R3Is 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
Is coarseDirectly activating the exposed carboxyl of the conjugate to obtain a fatty acid-amino acid conjugate or a fatty diacid-amino acid conjugate with one activated carboxyl
In the crude reaction solution of (1), wherein R4Is a carboxyl activating group; then directly adding raw materials into the crude reaction liquid
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 and AEEA is
) (ii) a The preferable charging sequence of the reaction is octadecanedioic acid, carboxyl activating agent, glutamic acid, carboxyl activating agent,
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 is2And R5Are identical or different carboxyl protecting groups, preferably R2And R5Each independently selected from any one of methyl, ethyl, tert-butyl and benzyl, more preferably R2And R5And is also tert-butyl;
the polypeptide side chain analogue is prepared by the following method:
fatty diacid obtained by protecting one end of carboxyl
The exposed carboxyl is activated to obtain the fatty diacid derivative with activated carboxyl at one end
In the crude reaction solution of (1), wherein R3Is 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 fatty diacid-amino acid conjugate
Directly activating the exposed carboxyl of the conjugate to obtain the fatty diacid-amino acid conjugate with activated carboxyl at one end
In the crude reaction solution of (1), wherein R4Is a carboxyl activating group; finally, the raw materials are directly added into the crude reaction liquid
(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-diazabicycloundecen-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 a 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 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
After 20g of compound 1d octadecanedioic acid mono-tert-butyl ester tBuO-Ste-OH (n 15, 54.0mmol) was dissolved in 400mL of DCM solution and placed in a nitrogen-protected flask, NHS (6.5g, 56.7mmol) and EDCI (12.4g, 64.8mmol) were carefully added to the above solution after the above mixture was cooled to 0 to 10 ℃, 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 2d for the next step.
After the temperature of the crude reaction solution in the previous step was lowered to 0 to 10 ℃, the compound L-Glu (3a, 11.1g, 54.5mmol) and TEA (8.19g, 81.0mmol) were added to the reaction system under nitrogen protection, and the mixture was allowed to react 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 crude reaction liquid in the previous step to 0-10 ℃, adding NHS (6.5g, 56.7mmol) and EDCI (12.4g, 64.8mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for further reaction. 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.
After the temperature of the crude reaction solution in the previous step 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 mixture is returned to room temperature for reaction overnight. TLC showed complete consumption of starting material. The reaction solution was washed twice with 200mL of a 0.5N HCl/10% NaCl mixture, then once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation (DCM: MeOH ═ 50:1-30:1-20:1), the target eluent was collected, concentrated, and dried to give the final product 6d tBuO-Ste-Glu- (AEEA-OH) -OtBu in 51% yield and 98.2% HPLC purity. Ms [ M + H+]846.7 g/mol. The hydrogen spectrum data of the final product 6d are as follows:1H 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 compound 1a, i.e., the mono-tert-butyl pentadecanedioate, i.e., tBuO-Pen-OH (n: 12, 30.4mmol), was dissolved in 200mL of a DCM solution, the solution was placed in a nitrogen-protected flask, and after the temperature of the mixture was lowered to 0 to 10 ℃, NHS (3.7g, 32.0mmol) and EDCI (7.0g, 36.5mmol) 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 2a was obtained and directly fed to the next step.
After the temperature of the crude reaction solution in the previous step is reduced to 0-10 ℃, the compound L-Glu (3a, 6.3g, 30.7mmol) and TEA (4.6g, 45.6mmol) 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 and yielded a crude reaction containing compound 4a which was directly fed to the next step.
And (3) cooling the crude reaction liquid in the previous 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 further reaction. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 5a 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 (9.9g, 31.9mmol) and TEA (4.6g, 45.6mmol) 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 100mL of a 0.5N HCl/10% NaCl mixture, once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation (DCM: MeOH ═ 50:1-30:1-20:1), and the desired eluate was collected, concentrated, and dried to give the final product 6a tBuO-Pen-Glu- (AEEA-OH) -OtBu in 48% yield and 97.6% HPLC purity. Ms [ M + H+]804.6 g/mol. The hydrogen spectrum data of the end product 6a are as follows:1H 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, 35.0mmol) 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, 36.8mmol) and EDCI (8.1g, 42.0mmol) 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 2b was obtained and directly fed to the next step.
After the crude reaction solution in the previous step is cooled to 0-10 ℃, the compound L-Glu (3a, 7.2g, 35.4mmol) and TEA (5.3g, 52.5mmol) 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 4b which was directly fed to the next step.
And (3) cooling the crude reaction liquid in the previous step to 0-10 ℃, adding NHS (4.3g, 36.8mmol) and EDCI (8.1g, 42.0mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for further reaction. 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 temperature of the crude reaction liquid in the previous step is reduced 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 mixture is returned to room temperature for reaction overnight. TLC showed complete consumption of starting material. The reaction solution was washed twice with 150mL of a 0.5N HCl/10% NaCl mixture, then once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation (DCM: MeOH ═ 50:1-30:1-20:1), and the desired eluate was collected, concentrated, and dried to give the final product 6b tBuO-Pal-Glu- (AEEA-OH) -OtBu in 50% yield and 98.0% HPLC purity. Ms [ M + H+]818.8 g/mol. The hydrogen spectrum data of the end product 6b are as follows:1H 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 1 c-heptadecanedioic acid mono-tert-butyl ester tBuO-Hep-OH (n ═ 14, 14.0mmol) was dissolved in 100mL of DCM solution, placed in a nitrogen-protected flask, and after the mixture was 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 temperature of the crude reaction liquid in the previous step is reduced to 0-10 ℃, the compound L-Glu (3a, 2.9g, 14.2mmol) and TEA (2.2g, 21mmol) 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 and yielded a crude reaction containing compound 4c which was directly fed to the next step.
And (3) cooling the crude reaction liquid in the previous 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 further reaction. After 4h of reaction, TLC showed complete consumption of starting material to give crude reaction solution containing compound 5c 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 (4.5g, 14.7mmol) 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 solution was washed twice with 50mL of a 0.5N HCl/10% NaCl mixture, then once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. Separating and purifying the crude product with silica gel column (DCM: MeOH: 50:1-30:1-20:1), collecting the target eluate, concentrating, and drying to obtain the final productThe product 6c tBuO-Hep-Glu- (AEEA-AEEA-OH) -OtBu, yield 52%, HPLC purity 97.3%. Ms [ M + H+]830.8 g/mol. The hydrogen spectrum data of the final product 6c are as follows:1H 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, 26.0mmol) was dissolved in 200mL of DCM solution, placed in a nitrogen-protected flask, and after the mixture cooled to 0-10 deg.C, NHS (3.2g, 27.3mmol) and EDCI (6.0g, 31.2mmol) 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 1e was obtained and directly fed to the next step.
After the temperature of the crude reaction solution in the previous step was lowered to 0-10 ℃, the compound L-Glu (3a, 5.4g, 26.3mmol) and TEA (3.9g, 39.0mmol) were added to the reaction system under nitrogen protection, and the mixture was allowed to react overnight at room temperature. TLC showed complete consumption of starting material and yielded a crude reaction containing compound 4e which was directly fed to the next step.
And (3) cooling the crude reaction liquid in the previous step to 0-10 ℃, adding NHS (3.2g, 27.3mmol) and EDCI (6.0g, 31.2mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for further reaction. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 5e 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 (8.4g, 27.3mmol) and TEA (3.9g, 39.0mmol) 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 solution used 100mL of 0.5N HWashing twice with Cl/10% NaCl mixture, washing once with saturated saline solution, and washing the organic phase with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation (DCM: MeOH ═ 50:1-30:1-20:1), and the desired eluate was collected, concentrated, and dried to give the final product 6e tBuO-Non-Glu- (AEEA-OH) -OtBu in 44% yield and 97.5% HPLC purity. Ms [ M + H+]858.9 g/mol. The hydrogen spectrum data of the final product 6e are as follows:1H 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 compound 1f di-tert-butyl ditecanedioate, tBuO-Ara-OH (n. RTM.17, 37.6mmol), was dissolved in 300mL of DCM solution, placed in a nitrogen-protected flask, and after the mixture was cooled to 0-10 deg.C, NHS (4.6g, 39.5mmol) and EDCI (8.6g, 45.1mmol) 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 2f which was directly fed to the next step.
After the crude reaction solution in the previous step was cooled to 0-10 ℃, the compound L-Glu (3a, 7.7g, 38.0mmol) and TEA (5.7g, 56.4mmol) were added to the reaction system under nitrogen protection, and the mixture was allowed to react overnight at room temperature. TLC showed complete consumption of starting material and yielded a crude reaction containing compound 4f which was directly fed to the next step.
And (3) cooling the crude reaction liquid in the previous step to 0-10 ℃, adding NHS (4.6g, 39.5mmol) and EDCI (8.6g, 45.1mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for further reaction. After 4h of reaction, TLC showed complete consumption of starting material and the crude reaction solution containing compound 5f 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 (12.2g, 27.3mmol) and TEA (5.7g, 56.4mmol) 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 solution was washed twice with 150mL of a 0.5N HCl/10% NaCl mixture, then once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation (DCM: MeOH ═ 50:1-30:1-20:1), and the desired eluate was collected, concentrated, and dried to give the final product 6f tBuO-Ara-Glu- (AEEA-OH) -OtBu in 43% yield and 96.8% HPLC purity. Ms [ M + H+]858.9 g/mol. The hydrogen spectrum data of the final product 6f are as follows:1H 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, 27mmol) was dissolved in 200mL of DCM solution, the solution was placed in a nitrogen-protected flask, and after the mixture was cooled to 0 to 10 ℃, NHS (3.3g, 28.4mmol) and EDCI (6.2g, 32.4mmol) 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 2d was directly fed to the next step.
After the crude reaction solution in the previous step was cooled to 0-10 ℃, the compound L-Glu (3a, 5.6g, 27.3mmol) and TEA (4.1g, 40.5mmol) were added to the reaction system under nitrogen protection, and the mixture was allowed to react overnight at room temperature. TLC showed complete consumption of the starting material and yielded a crude reaction containing compound 4d which was directly fed to the next step.
And (3) cooling the reaction liquid in the previous step to 0-10 ℃, adding NHS (3.3g, 28.4mmol) and EDCI (6.2g, 32.4mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for further reaction. 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.
After the temperature of the crude reaction liquid in the previous step is reduced to 0-10 ℃, H-AEEA-OH (4.6g, 28.4mmol) and TEA (4.1g, 40.5mmol) 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 solution was washed twice with 200mL of a 0.5N HCl/10% NaCl mixture, then once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation (DCM: MeOH ═ 50:1-30:1-20:1), and the desired eluate was collected, concentrated, and dried to give the final product 6g tBuO-Ste-Glu- (AEEA-OH) -OtBu in 60% yield and 97.0% HPLC purity. Ms [ M + H+]701.7 g/mol. The hydrogen spectrum data of the final product 6g are as follows:1H 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, 27mmol) was dissolved in 200mL of DCM solution, the solution was placed in a nitrogen-protected flask, and after the mixture was cooled to 0 to 10 ℃, NHS (3.3g, 28.4mmol) and EDCI (6.2g, 32.4mmol) 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 2d for the next step.
After the crude reaction solution in the previous step was cooled to 0-10 ℃, the compound D-Glu (3b, 5.6g, 27.3mmol) and TEA (4.1g, 40.5mmol) were added to the reaction system under nitrogen protection, and the mixture was allowed to react 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.
And (3) cooling the crude reaction liquid in the previous step to 0-10 ℃, adding NHS (3.3g, 28.4mmol) and EDCI (6.2g, 32.4mmol) 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 5h of compound 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 (8.8g, 28.4mmol) and TEA (4.1g, 40.5mmol) 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 solution was washed twice with 200mL of a 0.5N HCl/10% NaCl mixture, then once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation (DCM: MeOH ═ 50:1-30:1-20:1), the desired eluent was collected, concentrated and dried to give the final product 6h tBuO-Ste- (D) -Glu- (AEEA-OH) -OtBu in 46% yield and 96.9% HPLC purity. Ms [ M + H+]846.7 g/mol. The hydrogen spectrum data of the final product 6h are as follows:1H 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, 35.2mmol) was dissolved in 200mL DCM solution, placed in a nitrogen-protected flask, and after the mixture cooled to 0-10 deg.C, NHS (4.3g, 37.0mmol) and EDCI (8.1g, 42.2mmol) 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 2i was obtained and directly fed to the next step.
After the crude reaction solution in the previous step is cooled to 0-10 ℃, the compound L-Glu (3a, 7.2g, 35.6mmol) and TEA (5.3g, 52.8mmol) 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 4i which was directly fed to the next step.
And (3) cooling the crude reaction liquid in the previous step to 0-10 ℃, adding NHS (4.3g, 37.0mmol) and EDCI (8.1g, 42.2mmol) into the reaction system under the protection of nitrogen, and returning to room temperature for further reaction. 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 temperature of the crude reaction liquid in the previous step is reduced to 0-10 ℃, H-AEEA-AEEA-OH (11.4g, 37.0mmol) and TEA (5.3g, 52.8mmol) 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 solution was washed twice with 200mL of a 0.5N HCl/10% NaCl mixture, then once with saturated brine, and the organic phase was washed with anhydrous MgSO4Drying, filtering and spin-drying to obtain a crude product. The crude product was purified by silica gel column separation (DCM: MeOH ═ 50:1-30:1-20:1), and the desired eluate was collected, concentrated, and dried to give the final product 6i Ste-Glu- (AEEA-OH) -OtBu in 53% yield and 97.0% purity by HPLC. Ms [ M + H+]759.8 g/mol. The hydrogen spectrum data of the final product 6i are as follows:1H 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 (17)
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 is1Is 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 is
And 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 protected
The 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 activated
In the crude reaction solution of (1), wherein R3Is 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 group of the conjugate to obtain a fatty acid-amino acid conjugate or a fatty diacid-amino acid conjugate containing activated carboxyl at one end
In the crude reaction solution of (1), wherein R4Is a carboxyl activating group; then directly adding raw materials into the crude reaction liquid
Condensation reaction is carried out to obtain the compound shown in the formula (1).
2. The method for preparing a polypeptide side chain analog according to claim 1, wherein the "one-pot" reaction of the multi-step reaction is performed, the reaction raw materials are fed in four steps, the crude reaction solution which has completely reacted after each step of feeding is not subjected to separation and purification treatments such as transfer, water washing, extraction, recrystallization, column chromatography and the like, and only the crude reaction solution is subjected to temperature reduction treatment and then new raw materials are directly added for the next step of reaction.
3. The method for preparing a polypeptide side chain analog according to claim 1, wherein the order of feeding is fatty acid or carboxyl group at one endA group-protected aliphatic diacid, a carboxyl activator, an amino acid, a carboxyl activator,
The preferable charging sequence of the reaction is octadecanedioic acid, carboxyl activating agent, glutamic acid, carboxyl activating agent,
4. The method for preparing a polypeptide side chain analog according to claim 1, wherein the amino acid is any one selected from the group consisting of glutamic acid, aspartic acid, histidine, glutamine, asparagine, serine, threonine, proline, glycine, lysine and arginine; preferably glutamic acid or aspartic acid.
5. The method for producing a polypeptide side chain analog according to claim 4, wherein the amino acid is L-Glu or D-Glu whose carboxyl group at one terminal is protected, preferably
Wherein R is2The carboxyl-protecting group is preferably any of methyl, ethyl, tert-butyl and benzyl, and more preferably tert-butyl.
6. The method for preparing a polypeptide side chain analog according to claim 1, wherein n is 12, 13, 14, 15, 16 or 17.
7. The method for preparing a polypeptide side chain analog according to claim 1, wherein the method comprises
Is composed of
8. The method of claim 1, wherein 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 NHS/EDCI, NHS/DCC, HONb/DCC, and most preferably NHS/EDCI.
9. The method for preparing a polypeptide side chain analog according to claim 1, wherein the amino acid is glutamic acid, and the structural general formula of the polypeptide side chain analog is represented by formula (2):
wherein, R is2And R5Are identical or different carboxyl protecting groups, preferably R2And R5Each independently selected from any one of methyl, ethyl, tert-butyl and benzyl, more preferably R2And R5And is also tert-butyl;
the polypeptide side chain analogue is prepared by the following method:
fatty diacid obtained by protecting one end of carboxyl
The exposed carboxyl is reacted with a carboxyl activating agent for activation to obtain the fatty diacid derivative with activated carboxyl at one end
The crude reaction solution of (3); 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 fatty diacid-amino acid conjugate
Directly reacting the exposed carboxyl of the conjugate with a carboxyl activating agent to activate the crude reaction liquid to obtain the aliphatic diacid-amino acid conjugate with one end of carboxyl being activated
The crude reaction solution of (3); finally, the raw materials are directly added into the crude reaction liquid
Condensation reaction is carried out to obtain the compound shown in the formula (2).
10. The method of claim 9, wherein the carboxyl activating agent is a combination of NHS/EDCI, and the molar ratio of NHS to EDCI is 1: 1-1.5, preferably 1: 1-1.2, more preferably 1: 1.14.
11. the method for preparing a polypeptide side chain analog according to claim 9, 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, preferably 3-6h, and more preferably 4 h.
12. The method for preparing a polypeptide side chain analog according to claim 1, wherein the starting fatty acid or fatty diacid, amino acid,
The feeding 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.
13. the method for preparing a polypeptide side chain analog according to claim 1, wherein the reaction temperature of the condensation reaction is 20 to 35 ℃, preferably 30 ℃.
14. 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; the crude reaction liquid is cooled, and the temperature of the crude reaction liquid is reduced to-5-10 ℃, preferably 0-10 ℃.
15. The method for preparing a polypeptide side chain analog according to claim 1, wherein the condensation reaction is performed under the action of an organic base selected from any one of N, N-Diisopropylethylamine (DIEA), monoethylamine, diethylamine, triethylamine, imidazole, 1, 8-diazabicycloundecen-7-ene (DBU), pyridine and piperazine, preferably triethylamine.
16. Use of the process for the preparation of a side chain analogue of a polypeptide according to any of claims 1-15 for the preparation of an insulin analogue for use as a medicament, preferably for the treatment or prevention of hyperglycemia, type II diabetes, impaired glucose tolerance or type I diabetes.
17. Use of the process for the preparation of a side chain analog of a polypeptide of claim 16 in the preparation of an insulin analog, wherein the insulin analog is somaglutide.
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