CN103965293B - High-purity bivalirudin and industrial preparation method thereof - Google Patents

Abstract

The invention provides high-purity bivalirudin and an industrial preparation method thereof. The preparation method adopts a solid-phase polypeptide synthesis technology, and adopts a solvent ethyl acetate which is convenient to recycle and low in price as a washing agent, so that the reaction yield and the product quality are improved, the production cost is effectively reduced, and the waste discharge is reduced.

Description

High-purity bivalirudin and industrial preparation method thereof

Technical Field

The invention belongs to a high-purity medicinal polypeptide and an industrial preparation method thereof, and particularly relates to high-purity bivalirudin and an industrial preparation method thereof.

Background

Bivalirudin (bivalirudin), also known as bivalirudin, of formula C₉₈H₁₃₈N₂₄O₃₃CAS number 128270-60-0, an artificially synthesized hirudin derivative with thrombin inhibiting activity, is commonly used as anticoagulant in percutaneous transluminal coronary angioplasty (PCI) in clinic. Bivalirudin was first developed by Biogen and later marketed under the trade name Angiomax by the medicins company in the united states. At present, only Shenzhen Xin Li Tai pharmaceutical industry markets bivalirudin preparation products in China, and the product is named Taijianing^®。

Bivalirudin consists of 20 amino acids, and the amino acid sequence of bivalirudin is as follows: D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH, and the chemical name is as follows: D-phenylalanyl-L-prolyl-L-arginyl-L-prolyl-glycyl-L-asparaginyl-glycyl-aspartyl-L-phenylalanyl-L-glutamyl-L-isoleucyl-L-prolyl-L-glutamyl-L-tyrosyl-L-leucine.

The bivalirudin bulk drug is produced by chemical synthesis in industrial production, and specifically, a liquid phase synthesis method, a solid phase synthesis method or a combination of the two methods can be adopted. Among them, the solid-phase synthesis method is widely used in the chemical synthesis of polypeptide products due to its high yield and easy separation of products. At present, the bivalirudin is synthesized by a solid-phase synthesis method.

The solid-phase synthesis of bivalirudin involves a plurality of synthesis steps, and after each step of reaction is completed, a detergent is needed to wash the residual reactants in the peptide resin to carry out the next step of reaction. Otherwise, the residual reactant may cause a series of side reactions and accumulate in the subsequent reaction, thereby resulting in a decrease in the total yield of the product, an increase in the amount and number of impurities in the product, and difficulty in the subsequent purification of the crude peptide.

As for the selection of the detergent, DMF or DMF together with methanol and dichloromethane is commonly used as the detergent after each step of reaction in the prior art. Patent CN200610024611.5 reports the use of DMF as a detergent in bivalirudin solid phase synthesis; patent CN200810008131.9 reports the use of DMF, methanol and dichloromethane as detergents in bivalirudin solid phase synthesis. DMF has a boiling point as high as 152.8 ℃, has high recycling cost in industrial production, and causes environmental pollution if directly discharged; the methanol washing effect is poor, the methanol cannot be used as a washing agent independently, and the methanol has high toxicity, is easy to cause harm to human bodies and pollute the environment; methylene chloride has a boiling point which is too low (39.8 ℃), and is easily lost by volatilization when used alone or in combination with DMF, and is not suitable for industrial production, and therefore, methylene chloride is not an ideal washing solvent. Although in actual production, the washing effect can also be improved by increasing the number of washing times, washing the resin multiple times tends to consume a large amount of organic solvent. Therefore, the technical problem to be solved is to find an economical, low-toxicity and high-efficiency bivalirudin solid-phase synthesis washing solvent.

The inventor starts from solving the defects of the prior art, and discovers through a large number of experiments that when the solid-phase synthesis method is adopted to prepare the bivalirudin, the reaction yield can be obviously improved by using the ethyl acetate as a resin detergent, and the obtained bivalirudin has higher purity and pure peptide content.

Disclosure of Invention

One purpose of the invention is to provide a detergent for bivalirudin solid-phase synthesis and a using method thereof, which can effectively reduce the production cost of bivalirudin, and the yield and the purity of the obtained bivalirudin are higher than those of the methods in the prior art.

The invention realizes the beneficial effects by using 3-6 ml/g of resin ethyl acetate as a detergent in the bivalirudin solid-phase synthesis method, and the washing times are 1-9 times. After the coupling reaction step and the deprotection step are completed, washing the resin by using a detergent to avoid bringing the residual reactants into the subsequent reaction, wherein if the amount of the used detergent is too small, the washing effect is possibly poor, and the residual reactants are brought into the subsequent reaction to further influence the reaction yield and the product quality; excessive washing results in waste and increases the production cost of the product. The metering unit ml/g resin refers to: 1 ml of solvent is used in each step of the solid phase reaction per 1 g of the resin to which the amino acid is attached; for example: using ethyl acetate of 3-6 ml/g resin in the deprotection step as follows: in the deprotection step, for each 1 g of resin to be deprotected, the amount of the detergent ethyl acetate used in the corresponding deprotection step is 3-6 ml, and preferably, the amount of the detergent ethyl acetate is 4ml/g of resin. Although impurities can be removed more effectively by multiple washes, multiple washes inevitably increase the amount of solvent used, extending the overall reaction time and thus increasing production costs. The inventor unexpectedly discovers through a large number of experiments that in the reaction of synthesizing bivalirudin by a solid-phase polypeptide method, under the same conditions, ethyl acetate is used as a detergent, impurities can be thoroughly washed off compared with other solvents, the used detergent amount is less, and the advantage of using ethyl acetate as the detergent is particularly obvious when the washing times are 1-9 times and the detergent amount is 3-6 ml/g. The amount of the detergent used in each step of the reaction and the washing times are independent of each other, the washing times of each step of the reaction are cumulative, specifically, for the coupling reaction step, the washing times of the corresponding ethyl acetate are preferably 1-3, and for the deprotection step, the washing times of the corresponding ethyl acetate are preferably 5-9.

The amino acid protecting group at the terminal position of the amino acid raw material used in the bivalirudin solid-phase synthesis reaction can be a protecting group commonly used in organic chemical reactions, such as Boc, Fmoc, Moz and the like, preferably, the amino protecting agent is Fmoc.

The Resin used for solid phase synthesis is Leu-Wang Resin, and the load (SD) is between 0.5 and 1 mmol/g, preferably 0.6 mmol/g. The resin with excessively low load amount has low reaction yield despite high washing efficiency, and further has high total production cost; the resin with excessively high load capacity means that the amount of loaded peptide is high, a large amount of washing solvent is needed, the yield is easily reduced due to the winding of peptide chains during reaction, the product quality is further reduced, the subsequent purification difficulty is increased, and the total production cost is increased.

Another objective of the present invention is to provide a bivalirudin production method, which is suitable for bivalirudin solid-phase synthesis using ethyl acetate as a detergent, has tightly combined steps, is economical and environment-friendly, and can be used in bivalirudin industrial production, and the method comprises the following steps:

1) condensing Leu-Wang Resin with SD of 0.5-1 mmol/g with coupling agent and activating agent and amino acid Tyr-OH with protected end amino group as raw material;

2) removing the reaction solution, and washing a solid phase by using ethyl acetate;

3) deprotection using piperidine DMF solution;

4) removing the reaction solution, and washing a solid phase by using ethyl acetate;

5) and (3) referring to the steps 1) to 4), sequentially condensing the rest eighteen amino acids with protected terminal amino groups according to a bivalirudin amino acid sequence;

6) cutting bivalirudin peptide resin by using a cracking reagent, collecting the cracking reagent, and precipitating crude peptide by using diethyl ether;

7) purifying the crude peptide by a reversed phase high performance liquid phase method, and drying to obtain a pure product.

In the production method of bivalirudin, the coupling agent used is one or a mixture of more of DCC, DIC, HBTU, TBTU and PyBOP; the used activating agent is one or a mixture of two of HOAt and HOBt; the coupling reaction solvent used is DMF; the lysis solution used was 95: 2.5: 2.5 TFA, TIS, H₂O mixed solution, the using amount is 6-12ml/g resin; the amount of the ether is 8-10 times of the volume of the cracking solution; for the technical scheme of the invention, in the reasonable raw material dosage and raw material proportioning range of the bivalirudin solid-phase synthesis method, the ethyl acetate with the dosage and the washing times can be used as the detergent to realize the beneficial effects, preferably,when the ratio of the coupling agent to the amino acid is 1-1.1: 1 and the ratio of the activating agent to the amino acid is 1-1.1: 1, the washing effect is particularly obvious.

In the production method of bivalirudin, the used deprotection reagent is piperidine DMF solution with the dosage of 8-12 ml/g resin volume percentage of 20%, because in actual production, in order to remove the protection group more completely, the operation of deprotection can be repeated for multiple times, and the deprotection reagent is distributed to each operation, the dosage of the deprotection reagent refers to cumulative dosage. For example, in the reaction for removing the protecting group from the same peptide resin, the protecting group removing reagent used in an amount of 10 ml/g resin may be divided into two portions, and the deprotection operation may be repeated twice, and each time the deprotection operation is followed by washing with ethyl acetate.

One preferred embodiment of the production method of bivalirudin comprises the following steps:

1) coupling Resin and amino acid Fmoc-Tyr-OH by taking Leu-Wang Resin with SD of 0.6mmol/g as a raw material, DIC as a coupling agent, HOBt as an activating agent and DMF as a solvent; the molar ratio of the coupling agent to the amino acid is 1.1:1, and the molar ratio of the activating agent to the amino acid is 1.1: 1;

2) removing the reaction solution, and washing the solid phase for 1 time by ethyl acetate with the proportion of 4ml/g resin;

3) adding 20 percent by volume of piperidine DMF solution of 5ml/g resin for deprotection;

4) removing the reaction solution, and washing the solid phase by ethyl acetate with the proportion of 4ml/g resin for 2 times;

5) adding 20 volume percent piperidine DMF of 5ml/g resin for repeated deprotection;

6) removing the reaction solution, and washing the solid phase by 6 times with ethyl acetate with the proportion of 4ml/g resin;

7) referring to steps 1) to 6), sequentially coupling the rest eighteen Fmoc-protected amino acids according to a bivalirudin amino acid sequence by using DIC as a coupling agent, HOBt as an activating agent and DMF as a solvent, wherein the molar ratio of the coupling agent to the amino acids is 1.1:1, and the molar ratio of the activating agent to the amino acids is 1.1: 1;

8) cutting bivalirudin peptide resin by using 6-12ml/g of lysate of the resin, collecting the lysate, and precipitating crude peptide by using ether with 8-10 times of volume of lysate;

9) purifying the crude product by gradient elution with a reversed-phase high-performance liquid phase method, and eluting with an eluent w_(TFA)：V_{(acetonitrile)}：V_(Water)=1:250: 750-1: 400: and 600, collecting eluent, and drying to obtain a pure product.

The third purpose of the invention is to provide a bivalirudin product, which is prepared by the preparation method and has the characteristics of low production cost, high purity, high pure peptide content, small impurity amount and low impurity content.

The invention provides high-purity bivalirudin and a preparation method thereof, and the high-purity bivalirudin has the following beneficial effects:

1. the ethyl acetate which is low in cost and easy to recover is used as a bivalirudin solid-phase synthesis detergent, so that the production cost is reduced, and the waste liquid discharge is reduced;

2. the optimal dosage range of the detergent is provided, and a bivalirudin solid-phase synthesis method suitable for ethyl acetate as the detergent is designed according to the optimal dosage range, has the characteristics of economy and environmental protection, and is suitable for bivalirudin industrial production;

3. the bivalirudin product has the characteristics of low production cost, high purity, high pure peptide content, small impurity amount and low impurity content.

Drawings

FIG. I is a chromatogram of the crude bivalirudin obtained in example 1

FIG. two is a chromatogram of the purified bivalirudin obtained in example 1

Detailed Description

The present invention is further described in detail with reference to the following specific examples, but the embodiments of the present invention are not limited by the following examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.

Example 1

The technical scheme of the invention is adopted to prepare bivalirudin

1.1 kg of Leu-Wang Resin (SD =0.60 mmol/g) was weighed into a silanized glass reactor, 6L of DMF was added to completely immerse the Resin therein, stirred for 30 min, and DMF was suction filtered. At room temperature, Fmoc-Tyr-OH (0.55 kg, 1.2 mol) and HOBt (0.173 kg, 1.3 mol) are dissolved in 3.3L DMF, added into the resin and stirred uniformly, DIC (0.2L, 1.3 mol) is added immediately, and the mixture is stirred and reacted for 3-4 h. After the resin is colorless, the reaction liquid is pumped out by Kaiser reagent detection.

2. 4L of ethyl acetate was added thereto, the mixture was stirred for 5min, and the washing solution was removed by suction.

3. Adding 5L of 20 volume percent piperidine DMF solution into the resin, stirring for reaction for 3-10 min, and filtering off the reaction solution.

4. Add 4L ethyl acetate, stir for 5min, pump off the wash and repeat the wash 1 time.

5. Adding 5L of 20% piperidine DMF solution in volume percent into the resin, stirring for reaction for 20 min, and filtering the reaction solution.

6. Add 4L ethyl acetate, stir for 5min, pump off the wash and repeat the wash 5 times.

7. Sequentially coupling 4 th to 20 th amino acids at the C terminal according to the steps 1 to 6;

wherein the dosage of the 3 rd to 20 th amino acids is as follows:

Fmoc-L-Glu(OtBu)-OH： 0.51 kg，1.2mol

Fmoc-L-Glu(OtBu)-OH： 0.51 kg，1.2mol

Fmoc-L-Pro-OH： 0.404 kg，1.2mol

Fmoc-L-Ile-OH： 0.424 kg，1.2mol

Fmoc-L-Glu(OtBu)-OH： 0.51 kg，1.2mol

Fmoc-L-Glu(OtBu)-OH： 0.51 kg，1.2mol

Fmoc-L-Phe-OH： 0.464 kg，1.2mol

Fmoc-L-Asp(OtBu)-OH： 0.494 kg，1.2mol

Fmoc-Gly-OH： 0.356 kg，1.2mol

Fmoc-L-Asn(Trt)-OH： 0.716 kg，1.2mol

Fmoc-Gly-OH： 0.356 kg，1.2mol

Fmoc-Gly-OH： 0.356 kg，1.2mol

Fmoc-Gly-OH： 0.356 kg，1.2mol

Fmoc-Gly-OH： 0.356 kg，1.2mol

Fmoc-L-Pro-OH： 0.404 kg，1.2mol

Fmoc-L-Arg(Pbf)-OH： 0.776 kg，1.2mol

Fmoc-L-Pro-OH： 0.404 kg，1.2mol

Fmoc-D-Phe-OH： 0.464 kg，1.2mol

after the deprotection is finished and the washing is finished, carrying out vacuum drying treatment to obtain 2 kg of peptide resin;

8. cracking

The resulting peptide resin was added portionwise to a solution containing 10L of lysis solution (V) at room temperature_TFA：V_TIS：V_H2O= 95: 2.5: 2.5) in the reactor, stirring was maintained for 2.5 h. The lysate is filtered with suction, the resin is washed twice with 3L of lysate, the washing liquid is incorporated into the lysate, the lysate is dropped into about 120L of cold ether and vigorously stirred, centrifuged and washed. Drying to obtain 1.2 kg crude product, and detecting by high performance liquid chromatography to obtain product with purity of 93% (shown in figure I).

9. Purification of

Purifying and separating the obtained crude product by reversed-phase high performance liquid chromatography gradient elution, using octadecylsilane chemically bonded silica as stationary phase, and eluting with w_(TFA)：V_{(acetonitrile)}：V_(Water)And the flow rate is 500 ml/min, the detection wavelength is 214nm, and the ratio is 1:250: 750-1: 400: 600. The collected eluent is rotary evaporated, and after freeze drying, bivalirudin 0.54kg, the purity of 99.8 percent and the total yield of 37 percent are obtained (the spectrogram is shown in figure two).

Comparative example 1

The detergent ethyl acetate was changed to DMF according to the synthesis process and the charge ratio of example 1. The purity of the crude reaction product is 84 percent, the purity after purification is 99.8 percent, and the total yield is 26 percent.

Comparative example 2

Referring to the synthesis process and the charge ratio of example 1, the detergents in the 2 nd and 4 th steps were changed to DMF, the detergent in the 6 th step was changed to DMF (5L × 4 times), methanol (5L × 1 times), and dichloromethane (5L × 1 times), and the detergents for condensation and deprotection of other amino acids in the 7 th step were also changed accordingly. The purity of the crude reaction product is 86 percent, the purity after purification is 99.8 percent, and the total yield is 28 percent.

In this specification, the reagent names corresponding to the respective acronyms are as follows:

Claims (6)

1. a solid-phase synthesis method of bivalirudin is characterized by comprising the following steps:

1) Leu-Wang Resin with SD of 0.6mmol/g is used as a raw material, and a coupling agent and an activating agent are used for condensing the Resin and amino acid Tyr-OH with protected amino at the end position;

2) removing the reaction solution, and washing the solid phase for 1 time by ethyl acetate with the proportion of 4ml/g resin;

3) adding 20 percent by volume of piperidine DMF solution of 5ml/g resin for deprotection;

4) removing the reaction solution, and washing the solid phase by ethyl acetate with the proportion of 4ml/g resin for 2 times;

5) adding 20 volume percent piperidine DMF of 5ml/g resin for repeated deprotection;

6) removing the reaction solution, and washing the solid phase by 6 times with ethyl acetate with the proportion of 4ml/g resin;

7) and (2) referring to steps 1) to 6), sequentially condensing the rest eighteen amino acids with protected terminal amino groups according to a bivalirudin amino acid sequence;

8) cutting bivalirudin peptide resin by using a cracking reagent, collecting the cracking reagent, and precipitating crude peptide by using diethyl ether;

9) purifying the crude peptide by a reversed phase high performance liquid phase method, and drying to obtain a pure product.

2. The synthesis method according to claim 1, wherein the coupling agent used in the synthesis method is one or more of DCC, DIC, HBTU, TBTU and PyBOP, and the molar ratio of the coupling agent to the amino acid is 1-1.1: 1.

3. The synthesis method according to claim 1, wherein the activating agent used in the synthesis method is one or a mixture of two of HOAt and HOBt, and the molar ratio of the activating agent to the amino acid is 1-1.1: 1.

4. The method of claim 1, wherein the cleavage reagent of step 8) is TFA, TIS, H₂O, and the volume ratio is 95: 2.5: 2.5, the dosage of the cracking reagent is 6-12ml/g resin.

5. The method of claim 1, wherein the amount of diethyl ether used in step 8) is 8-10 times the volume of the lysis reagent.

6. A method of synthesis according to any one of claims 2 to 5, characterised in that it comprises the following steps:

1) coupling Resin and amino acid Fmoc-Tyr-OH by taking Leu-Wang Resin with SD of 0.6mmol/g as a raw material, DIC as a coupling agent, HOBt as an activating agent and DMF as a solvent; the molar ratio of the coupling agent to the amino acid is 1.1:1, and the molar ratio of the activating agent to the amino acid is 1.1: 1;

2) removing the reaction solution, and washing the solid phase for 1 time by ethyl acetate with the proportion of 4ml/g resin;

3) adding 20 percent by volume of piperidine DMF solution of 5ml/g resin for deprotection;

4) removing the reaction solution, and washing the solid phase by ethyl acetate with the proportion of 4ml/g resin for 2 times;

5) adding 20 volume percent piperidine DMF of 5ml/g resin for repeated deprotection;

6) removing the reaction solution, and washing the solid phase by 6 times with ethyl acetate with the proportion of 4ml/g resin;

7) referring to steps 1) to 6), sequentially coupling the rest eighteen Fmoc-protected amino acids according to a bivalirudin amino acid sequence by using DIC as a coupling agent, HOBt as an activating agent and DMF as a solvent, wherein the molar ratio of the coupling agent to the amino acids is 1.1:1, and the molar ratio of the activating agent to the amino acids is 1.1: 1;

8) cutting bivalirudin peptide resin by using 6-12ml/g of lysate of the resin, collecting the lysate, and precipitating crude peptide by using ether with 8-10 times of volume of lysate;

9) purifying the crude product by gradient elution with a reversed-phase high-performance liquid phase method, and eluting with an eluent w_(TFA)：V_{(acetonitrile)}：V_(Water)1:250: 750-1: 400: and 600, collecting eluent, and drying to obtain a pure product.