CN103897031A

CN103897031A - Chemically modified thymopentin and synthetic method thereof

Info

Publication number: CN103897031A
Application number: CN201210572767.2A
Authority: CN
Inventors: 粟武
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2012-12-25
Filing date: 2012-12-25
Publication date: 2014-07-02

Abstract

本发明公开了一种化学修饰的胸腺五肽及其合成方法，其具有以下结构：A-Arg-Lys-Asp-Val-Try-OH或H-Arg-Lys(A)-Asp-Val-Try-OH，所述A为小分子配体，所述小分子配体为与人血清白蛋白亲和性结合的脂肪酸，或与人血清白游离巯基偶联的马来酰亚胺衍生物。本发明的化学修饰的胸腺五肽修饰位点精确，化学结构明确，合成方法工艺简练，经化学修饰的胸腺五肽在静脉注射后立即特异性地与人体自身的血清白蛋白结合，将以人体自身的血清白蛋白作为缓释载体，从而大大延长其半衰期，显著增加有效药物浓度的持续时间，生物利用度高。The invention discloses a chemically modified thymopentin and its synthesis method, which has the following structure: A-Arg-Lys-Asp-Val-Try-OH or H-Arg-Lys(A)-Asp-Val-Try -OH, the A is a small molecule ligand, and the small molecule ligand is a fatty acid bound to human serum albumin with affinity, or a maleimide derivative coupled to a free sulfhydryl group of human serum albumin. The chemically modified thymopentin of the present invention has precise modification sites, clear chemical structure, and concise synthesis process. The chemically modified thymopentin specifically binds to the serum albumin of the human body immediately after intravenous injection and will Self-serum albumin acts as a slow-release carrier, thereby greatly extending its half-life, significantly increasing the duration of effective drug concentration, and high bioavailability.

Description

The thymopeptide-5 of chemically modified and synthetic method thereof

Technical field

The invention belongs to the synthetic field of biochemical pharmacology, more specifically, the present invention relates to a kind of thymopeptide-5 and synthetic method thereof of chemically modified.

Background technology

Thymopeptide-5 is the chemically synthesized polypeptide medicine of first independent research of China, one of chemosynthesis class polypeptide drugs that Ye Shi hospital sales volume is the highest, and the market value rapid growth year by year in China's medical market.Thymopeptide-5 can be used for the immunotherapy of Several Kinds of Malignancy clinically, and the assisting therapy of operation, radiation and chemotherapy, and effectively prevents and treats the complication such as secondary infection.In addition, thymopeptide-5 is clinical treatment or assisting therapy for diseases such as chronic viral hepatitis, great surgical operation and severe infections, autoimmune disorder (as rheumatoid arthritis and lupus erythematosus etc.), serious burn, type ii diabetes and old immunologic hypofunctions also, and clinical application field is very wide.

Pharmacokinetic shows: thymopeptide-5 is degraded by proteolytic enzyme and aminopeptidase very soon in human plasma, and the transformation period is about 30 seconds.Thymopentin lyophilization powder injection for injection at the preparation of clinical use at present, thereby the transformation period is very short, very easily degraded by enzymes in blood plasma and lose biological activity, bioavailability is very low, needs repeatedly repeatedly and the defect such as long-term injection is the subject matter of current thymopeptide-5 product when clinical use.

The at present main transformation period that adopts microball preparation or the large technical scheme prolongation of polyoxyethylene glycol (PEG) chemically modified two polypeptide drugs: micro-balloon injection is one of the most successful preparation technique of polypeptide slowly-releasing at present, this technology take biodegradable polymer if PLGA is as framework material parcel polypeptide drugs, make it reach in vivo slow release effect, many successful precedents over nearly 10 years, are obtained, as the Leuprolide microballoon of the triptorelin PLGA microballoon of French IPSEN company research and development, Japanese military field pharmacy exploitation etc.But microball preparation technology exists encapsulation rate low at present, burst effect is obvious and preparation process technical requirements is high, and therefore polypeptide drugs industry is also difficult to promote at home at present.

PEG is modified at chemical modification method and extends protein medicaments and obtained success: as the PEGization IFN α-2a of the PEGization IFN α-2b of Schering Plough company, Roche Holding Ag etc.The shortcoming that PEG is modified at chemical modification method is: because PEG itself is polymkeric substance, be difficult to obtain decorating site accurately, the unique product of product chemical structure.In addition, PEG molecular weight is large, and after modifying, the biological activity of medicine declines greatly.

In recent years, obtained great success with albuminous affinity small molecule part as tetradecanoic acid modified protein and polypeptide drugs.The insulin product promise peace (Levemir) of Novo Nordisk (Novo Nordisk) company is exactly the polypeptide drugs of modifying with albuminous affinity small molecule part tetradecanoic acid, and its transformation period extends to 5-7 hour from original 4-6 minute.After this, Novo Nordisk Co.,Ltd utilized similar technology to release the hyperglycemic-glycogenolytic factor (GLP-1) (trade(brand)name: Victoza) that tetradecanoic acid is modified, and was extended to 11-15 hour its transformation period from 1.5-2 minute.But the technique of these two kinds of products of Novo Nordisk Co.,Ltd is all first to use gene engineering expression Regular Insulin or hyperglycemic-glycogenolytic factor, is then modified and is obtained final product by tetradecanoic acid.Owing to there are multiple amido modified sites in Regular Insulin or hyperglycemic-glycogenolytic factor, therefore apply this technique be difficult to obtain decorating site accurately, the unique product of product chemical structure.

Summary of the invention

Based on this, the invention provides a kind of thymopeptide-5 and synthetic method thereof of new chemically modified, the thymopeptide-5 decorating site of chemically modified is accurate, chemical structure is clear and definite, keeping on the basis of thymopeptide-5 curative effect, extend the transformation period of thymopeptide-5, improved the bioavailability of thymopeptide-5.

A thymopeptide-5 for chemically modified, has following structure:

A-Arg-Lys-Asp-Val-Try-OH or H-Arg-Lys (A)-Asp-Val-Try-OH, described A is small molecules part, described small molecules part is the lipid acid of being combined with human serum albumin affinity, or with the maleimide derivatives of human seralbumin free sulfhydryl groups coupling.

In three embodiment, described lipid acid is tetradecanoic acid therein, in albumin, have multiple binding sites can high-affinity, highly selective combines with tetradecanoic acid.The structure of the thymopeptide-5 of described chemically modified is: Myr-Arg-Lys-Asp-Val-Try-OH or H-Arg-Lys (Myr)-Asp-Val-Try-OH.

Therein in an embodiment, the carboxylic acid derivative that described maleimide derivatives is maleimide is as butyric acid, caproic acid or sad etc.Maleimide can be optionally and albumin free sulfhydryl groups generation nucleophilic addition.

The present invention also provides the synthetic method of the thymopeptide-5 of above-mentioned chemically modified, comprises the following steps:

(1) the tyrosine king resin of protecting take Fmoc is as starting raw material, according to the king's resin that is loaded with thymopeptide-5 of the synthetic Fmoc protection of standard Fmoc strategy;

(2) slough after terminal amino group Fmoc blocking group with the DMF solution (v/v) of 20% piperidines, small molecules part is dissolved in to dimethyl formamide, with HBTU and the rear king's mixed with resin with being loaded with thymopeptide-5 of DIEA activation, condensation was drained after 1 hour, DMF washs, and obtains being loaded with king's resin of the ligand modified thymopeptide-5 of small molecules; The mol ratio of the tyrosine king resin of described small molecules part and Fmoc protection is 2～6: 1;

(3) with trifluoroacetic acid cutting king resin, obtain the ligand modified thymopeptide-5 crude product of small molecules, purifying, freeze-drying obtain pure products.

In an embodiment, the mol ratio of the tyrosine king resin of described small molecules part and Fmoc protection is 4: 1 therein.

Or comprise the steps:

(1) the tyrosine king resin of protecting take Fmoc and Fmoc-Lys (Dde)-OH are as starting raw material, according to the king's resin that is loaded with thymopeptide-5 of the synthetic Fmoc protection of standard Fmoc strategy;

(2) slough after the amino Dde blocking group of lysine side-chain, small molecules part is dissolved in to dimethyl formamide, with HBTU and the rear king's mixed with resin with being loaded with thymopeptide-5 of DIEA activation, condensation was drained after 1 hour, after DMF washing four times, obtain being loaded with king's resin of small numerator modified thymopeptide-5; The mol ratio of the tyrosine king resin of described small molecules part and Fmoc protection is 2～6: 1;

In an embodiment, in described DMF solution, the volumn concentration of hydrazine is 2% therein.

Or comprise the steps:

(1) small molecules ligand coupling is obtained to Fmoc-Lys (A)-OH to Fmoc-Lys-OH; Described small molecules part is 1～3: 1 with the mole dosage ratio of Fmoc-Lys-OH; The tyrosine king resin of protecting take Fmoc and Fmoc-Lys (A)-OH are as starting raw material, according to the king's resin that is loaded with thymopeptide-5 of the synthetic Fmoc protection of standard Fmoc strategy; Wherein A is small molecules part;

(2) slough after terminal amino group Fmoc blocking group, with trifluoroacetic acid cutting king resin, obtain the ligand modified thymopeptide-5 crude product of small molecules, purifying, freeze-drying obtain pure products.

In an embodiment, described small molecules part is 1: 1 with the mole dosage ratio of Fmoc-Lys-OH therein.

In an embodiment, in above-mentioned preparation method, the consumption of trifluoroacetic acid is the tyrosine king resin of 1ml/100mgFmoc protection therein.

The present invention according to the constitutional features of human serum albumin and with the combination of small molecules affinity ligands, the small molecules part pointed decoration thymopeptide-5 of choice and optimization high specific, high-affinity, small molecules part comprises that the lipid acid of human serum albumin affinity combination is as tetradecanoic acid etc., and can be specifically with maleimide (maleimido) derivative of human seralbumin free sulfhydryl groups coupling as dimaleoyl imino caproic acid etc.Because human serum albumin can specific binding small molecules part, therefore, the drug molecule of small molecules ligand coupling enters after blood fast and albumin bound, and the transformation period of human serum albumin reaches 19 days, therefore the thymopeptide-5 of ligand coupling is combined with the serum albumin of human body self specifically immediately after intravenous injection, by the serum albumin using human body self as slow-released carrier, thereby greatly extend its transformation period, significantly increase the time length of active drug concentration, bioavailability is high, while having overcome clinical use, need repeatedly repeatedly and the defect such as long-term injection, provide novel process for researching and developing long-acting thymopeptide-5 of new generation.

The present invention, by the complete synthesis thymopeptide-5 technique of improved chemistry, can utilize special blocking group optionally to protect the amino acid sites that will modify, thereby it is accurate to obtain decorating site, the product that chemical structure is unique.The synthetic of thymopeptide-5 of the present invention also can, directly by the side chain amino of the ligand modified Methionin of small molecules, then be incorporated into ligand modified Methionin in thymopeptide-5 by solid-phase polypeptide synthesis method, obtains the thymopeptide-5 product of chemically modified.

Adopt the synthetic method craft of thymopeptide-5 of chemically modified of the present invention terse, can on the basis of existing solid-phase polypeptide synthesis method, directly apply, also can be used for the synthetic thymopeptide-5 of Liquid phase peptides synthesis method, the decorating site of products therefrom is accurate, chemical structure is clear, meets State Food and Drug Administration completely

(SFDA) about the requirement of chemically synthesized polypeptide kind new medicine.

Embodiment

Describe the present invention in detail below in conjunction with specific embodiment.

In following examples; Fmoc-Tyr (tBu)-Wang resin is purchased from Tianjin Nankai Hecheng S&T Co., Ltd., and the amino acid starting material of various protections, peptide bond condensing agent (HBTU, DIEA) and trifluoroacetic acid are all purchased from the biochemical (Shanghai) Co., Ltd. of gill.

The synthetic method of the thymopeptide-5 of embodiment 1 chemically modified

Comprise the following steps:

(1) weigh tyrosine king resin (Fmoc-Tyr (tBu)-Wang Resin that 200 milligrams of Fmoc protect

0.25mmol/g) in manual solid-phase polypeptide synthesizer, add DCM (methylene dichloride) swelling 30 minutes.Successively with Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Boc)-OH and

Fmoc-Arg (Pbf)-OH is amino acid starting material; with benzotriazole-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HBTU) and diisopropylethylamine (DIEA) are polypeptide condensing agent; the equivalence ratio of resin and every kind of protected amino acid and polypeptide condensing agent is tyrosine king portions of resin amino acid: HBTU: DIEA=1: 3: 3: 6, and according to the king's resin that is loaded with thymopeptide-5 (compound 1) of the synthetic Fmoc protection of standard Fmoc strategy.

(2) slough after terminal amino group Fmoc blocking group with 2 milliliters of piperidines deprotection agents (piperidines: DMF=20: 80 (v/v)), 46 milligrams of tetradecanoic acids (0.2mmol) are dissolved in to 2 milliliters of dimethyl formamides (DMF), with HBTU and the rear king's mixed with resin with being loaded with thymopeptide-5 of DIEA activation, the equivalence ratio of resin and tetradecanoic acid and polypeptide condensing agent is tyrosine king portions of resin tetradecanoic acid: HBTU: DIEA=1: 4: 4: 8, room temperature condensation was drained after 1 hour, after DMF washing four times, obtain king's resin of the thymopeptide-5 that is loaded with tetradecanoic acid modification,

(3) with trifluoroacetic acid (TFA, 2 milliliters) room temperature cutting obtains the thymopeptide-5 crude product that tetradecanoic acid is modified for 2 hours, and after high-efficient liquid phase chromatogram purification, freeze-drying obtains thymopeptide-5---the compound 2 (yield 39%) that pure products tetradecanoic acid is modified.

The building-up reactions equation of the thymopeptide-5 of the chemically modified of this embodiment is as follows:

The synthetic method of the thymopeptide-5 of embodiment 2 chemically modifieds

Comprise the following steps:

0.25mmol/g) in manual solid-phase polypeptide synthesizer, add DCM (methylene dichloride) swelling 30 minutes.

Successively with Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Dde)-OH and

Boc-Arg (Pbf)-OH is amino acid starting material; with benzotriazole-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HBTU) and diisopropylethylamine (DIEA) are polypeptide condensing agent; the equivalence ratio of resin and every kind of protected amino acid and polypeptide condensing agent is tyrosine king portions of resin amino acid: HBTU: DIEA=1: 3: 3: 6, and according to the synthetic king's resin (compound 3) that is loaded with thymopeptide-5 of standard Fmoc strategy.

(2) under 2% dimethyl formamide (DMF) solution room temperature, react and within 2 minutes, slough after the amino Dde blocking group of lysine side-chain take the volumn concentration of 2ml hydrazine; 46 milligrams of tetradecanoic acids (0.2mmol) are dissolved in to 2 milliliters of dimethyl formamides (DMF); with HBTU and the rear king's mixed with resin with being loaded with thymopeptide-5 of DIEA activation, the equivalence ratio of resin and tetradecanoic acid and polypeptide condensing agent is tyrosine king portions of resin tetradecanoic acid:

HBTU: DIEA=1: 4: 4: 8, room temperature condensation was drained after 1 hour, DMF washs after four times, obtains king's resin of the thymopeptide-5 that is loaded with tetradecanoic acid modification;

(3) with trifluoroacetic acid (TFA, 2 milliliters) room temperature cutting obtains the thymopeptide-5 crude product that tetradecanoic acid is modified for 2 hours, and after high-efficient liquid phase chromatogram purification, freeze-drying obtains thymopeptide-5---the compound 4 (yield 37%) that pure products tetradecanoic acid is modified.

The synthetic method of the thymopeptide-5 of embodiment 3 chemically modifieds

Comprise the following steps:

(1) first tetradecanoic acid (460 milligrams, 2mmol) is dissolved in to 100 milliliters of methylene dichloride (DCM), with the HBTU (759 milligrams, 2mmol) of equivalent and DIEA (260 milligrams, 2mmol) activation after 2 minutes,

Add Fmoc-Lys-OH (736 milligrams, 2mmol), room temperature reaction is after 1 hour, reduction vaporization DCM, and the tetradecanoic acid that crude product is obtained after HPLC purifying to Fmoc protection is modified the Methionin of side chain

(Fmoc-Lys (Myr)-OH) 872 milligrams, yield is 76%.

Reaction equation is as follows:

(2) weigh tyrosine king resin (Fmoc-Tyr (tBu)-Wang Resin that 200 milligrams of Fmoc protect

Successively with Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Myr)-OH and

Boc-Arg (Pbf)-OH is amino acid starting material; with benzotriazole-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HBTU) and diisopropylethylamine (DIEA)) be polypeptide condensing agent; the equivalence ratio of resin and every kind of protected amino acid and polypeptide condensing agent is tyrosine king portions of resin amino acid: HBTU: DIEA=1: 3: 3: 6, and according to the king's resin (compound 5) that is loaded with tetradecanoic acid and modifies thymopeptide-5 of the synthetic Fmoc protection of standard Fmoc strategy.

(3) slough terminal amino group Fmoc blocking group with 20% piperidines DMF solution, DMF washs after four times, obtains king's resin of the thymopeptide-5 that is loaded with tetradecanoic acid modification;

(4) with trifluoroacetic acid (TFA, 2 milliliters) room temperature cutting 2 hours, obtain the thymopeptide-5 crude product that tetradecanoic acid is modified, after high-efficient liquid phase chromatogram purification, freeze-drying obtains thymopeptide-5---the compound 6 (yield 42%) that pure products tetradecanoic acid is modified.

The reaction equation of this embodiment is as follows:

The synthetic method of the thymopeptide-5 of embodiment 4 chemically modifieds

Comprise the following steps:

(1) first by (422 milligrams of maleimide caproic acids; 2mmol) be dissolved in 100 milliliters of methylene dichloride (DCM); with (759 milligrams of the HBTU of equivalent; 2mmol) and DIEA (260 milligrams, 2mmol) activation after 2 minutes, add (736 milligrams of Fmoc-Lys-OH; 2mmol); after room temperature reaction 1 hour, reduction vaporization DCM obtains crude product the maleimide Methionin of acid modification side chain that Fmoc protects after HPLC purifying

(Fmoc-Lys (Mal)-OH) 960 milligrams, yield is 83%.

Reaction equation is as follows:

Successively with Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Lys (Mal)-OH and

Boc-Arg (Pbf)-OH is amino acid starting material; with benzotriazole-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HBTU) and diisopropylethylamine (DIEA) are polypeptide condensing agent; the equivalence ratio of resin and every kind of protected amino acid and polypeptide condensing agent is tyrosine king portions of resin amino acid: HBTU: DIEA=1: 3: 3: 6, according to the synthetic Fmoc protection of standard Fmoc strategy be loaded with maleimide acid modify king's resin (compound 7) of thymopeptide-5.

(3) slough terminal amino group Fmoc blocking group with 20% piperidines DMF solution, DMF washs after four times, obtains king's resin of the thymopeptide-5 that is loaded with the modification of maleimide caproic acid;

(4) with trifluoroacetic acid (TFA, 2 milliliters) room temperature cutting 2 hours, obtain the thymopeptide-5 crude product that maleimide caproic acid is modified, after high-efficient liquid phase chromatogram purification, freeze-drying obtains thymopeptide-5--the compound 8 (yield 38%) that pure products maleimide caproic acid is modified.

The reaction equation of this embodiment is as follows:

The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.

Claims

1. a chemically modified thymopentin, characterized in that it has the following structure:

A-Arg-Lys-Asp-Val-Try-OH or H-Arg-Lys(A)-Asp-Val-Try-OH, the A is a small molecule ligand, and the small molecule ligand is human serum Albumin affinity-bound fatty acid, or maleimide derivative conjugated to free sulfhydryl groups of human serum albumin.

2. the thymopentin of chemical modification according to claim 1, is characterized in that, described fatty acid is myristic acid.

3. the thymopentin of chemical modification according to claim 1, is characterized in that, described maleimide derivative is maleimide butyric acid, maleimide hexanoic acid or maleimide Amino caprylic acid.

4. a synthetic method of the thymopentin of chemical modification according to claim 1, is characterized in that, comprises the following steps:

(1) Using Fmoc-protected tyrosine king resin as the starting material, synthesize Fmoc-protected king resin loaded with thymopentin according to the standard Fmoc strategy;

(2) After the terminal amino Fmoc protecting group is removed, the small molecule ligand is dissolved in dimethylformamide, activated with HBTU and DIEA, mixed with Wang resin loaded with thymopentin, condensed at room temperature and then drained , washed with DMF to obtain Wang resin loaded with small molecule ligand-modified thymopentin; the molar ratio of the small molecule ligand to Fmoc-protected tyrosine king resin is 2 to 6:1;

(3) Cleavage of Wang resin with trifluoroacetic acid to obtain a crude product of thymopentin modified by a small molecule ligand, which was purified and freeze-dried to obtain a pure product.

5. the synthetic method of the Thymopentin of chemical modification according to claim 4, is characterized in that, the mol ratio of small molecular ligand and Fmoc protected king tyrosine resin described in step (2) is 4: 1 .

6. a synthetic method of the chemically modified Thymopentin of claim 1, is characterized in that, comprises the following steps:

(1) Using Fmoc-protected tyrosine king resin and Fmoc-Lys(Dde)-OH as starting materials, synthesize Fmoc-protected thymopentin-loaded king resin according to the standard Fmoc strategy;

(2) After removing the lysine side chain amino Dde protecting group with a DMF solution of 2-10% by volume of hydrazine, the small molecule ligand is dissolved in dimethylformamide and activated with HBTU and DIEA Mix with Wang resin loaded with thymopentin, condense at room temperature, drain, and wash with DMF to obtain Wang resin loaded with small-molecule modified thymopentin; the small molecule ligand and Fmoc-protected tyrosine king resin The molar ratio is 2～6:1;

7. the synthetic method of the Thymopentin of chemical modification according to claim 6 is characterized in that, the mol ratio of small molecular ligand described in step (2) and the tyrosine king resin of Fmoc protection is 4: 1 .

8. the synthetic method of the thymopentin of chemical modification according to claim 6 is characterized in that, the volume percentage composition of hydrazine in the DMF solution described in the step (2) is 2%.

9. a synthetic method of the chemically modified Thymopentin of claim 1, is characterized in that, comprises the following steps:

(1) coupling the small molecule ligand to Fmoc-Lys-OH to obtain Fmoc-Lys(A)-OH, the molar ratio of the small molecule ligand to Fmoc-Lys-OH is 1 to 3:1; Using Fmoc-protected tyrosine Wang resin and Fmoc-Lys(A)-OH as starting materials, Fmoc-protected Wang resin loaded with small molecule ligand-modified thymopentin was synthesized according to the standard Fmoc strategy; wherein A is small Molecular ligand;

(2) After the terminal amino Fmoc protecting group was removed, Wang resin was cleaved with trifluoroacetic acid to obtain a crude product of thymopentin modified with a small molecule ligand, which was purified and lyophilized to obtain a pure product.

10. The synthetic method of the chemically modified Thymopentin according to claim 9, characterized in that, the molar ratio of small molecule ligand and Fmoc-Lys-OH described in step (1) is 1:1.