CN107778350B

CN107778350B - Method for synthesizing romidepsin

Info

Publication number: CN107778350B
Application number: CN201610728784.9A
Authority: CN
Inventors: 马中刚; 郭德文; 曾德志; 文永均
Original assignee: Chengdu Shengnuo Biopharm Co ltd
Current assignee: Chengdu Shengnuo Biopharm Co ltd
Priority date: 2016-08-25
Filing date: 2016-08-25
Publication date: 2021-04-13
Anticipated expiration: 2036-08-25
Also published as: CN107778350A

Abstract

The invention relates to the field of medicine synthesis, and discloses a method for synthesizing romidepsin. The invention synthesizes the romidepsin linear peptide chain one by using proper protected amino acid, completes amido bond cyclization and disulfide bond cyclization in sequence by a solid phase means, optimizes the solid phase synthesis scheme in detail, and finally obtains the romidepsin with higher purity and total yield.

Description

Method for synthesizing romidepsin

Technical Field

The invention relates to the field of medicine synthesis, in particular to a method for synthesizing romidepsin.

Background

Romidepsin, the English name of which is Romidepsin, is an artificially synthesized 5-peptide cyclic compound, has a stable hydrophobic structure, and a specific disulfide bond in the structure is a key group for exerting activity. In 2009 romidepsin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma (CTCL), and its amino acid structural composition was as follows:

[Val¹-(R²-D-Val³-D-Cys⁴)-DH-Thr⁵]

wherein R is (3S,4E) -3-hydroxy-7-thio-4-heptenoic acid, which forms a disulfide bond with D-cysteine at position 4, and valine at position 1 forms an amide bond with threonine at position 5.

Romidepsin is a Histone Deacetylase (HDACs) inhibitor, enters cytoplasm through a tumor cell membrane, is reduced into sulfydryl by glutathione in intracellular disulfide bonds, and is combined with zinc in zinc-dependent HDACs to play a role in inhibiting the HDACs, so that the differentiation and apoptosis of tumor cells are further induced.

At present, two methods are mainly used for preparing romidepsin, one is a biological fermentation method, such as patent CN201310430220, but the whole process is relatively complex; secondly, the compound is prepared by chemical synthesis, for example, in patent CN201010133961, linear peptide is synthesized by liquid phase, liquid phase amide ring cyclization is carried out firstly, then liquid phase disulfide bond cyclization is carried out, the operation is complex, and the product yield is low; chinese patent CN201210579007 starts from (3S,4E) -3-hydroxy-7- [ (triphenylmethyl) thio ] -4-heptenoic acid, uses 2-Cl-Trt resin as starting resin, adopts solid phase method to synthesize linear peptide, uses iodine oxidation method to cyclize to form disulfide bond, removes resin to cyclize amide ring in liquid phase, but its crude product purity is only 67.53% at most, and its total yield after purification and transacetate is only 33.7% at most. The problems of yield and purity have always been the main factors affecting the production efficiency of romidepsin.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for synthesizing romidepsin, such that the method of the present invention has high crude product purity, finished product purity and total yield.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for synthesizing romidepsin, comprising the following steps:

step 1, coupling protected Thr-OAll with resin under the action of organic base to obtain peptide resin 1;

step 2, starting from the peptide resin 1, sequentially extending and coupling the protected D-Cys, the protected D-Val and the protected (3S,4E) -3-hydroxy-7-thio-4-heptenoic acid one by one under the action of a condensation reagent and an activation reagent according to the sequence of polypeptides from the C end to the N end of the romidepsin amino acid sequence to obtain a peptide resin 2;

step 3, starting from the peptide resin 2, under the action of a condensation reagent, an activation reagent and a catalyst, protected Val is accessed to obtain a peptide resin 3;

step 4, removing the All protecting group of Thr-OAll in the peptide resin 3 by using an All deprotecting agent, then performing intramolecular lactam cyclization reaction under the action of a condensation reagent and an activation reagent to couple valine and threonine, and then performing intramolecular disulfide bond cyclization reaction by an iodine oxidation method to obtain romidepsin peptide precursor resin;

step 5, carrying out acidolysis on the romidepsin peptide resin to obtain a romidepsin precursor;

step 6, dehydrating the romidepsin precursor by a dehydrating agent to obtain a crude romidepsin product (the precursor has one more hydroxyl group than the crude romidepsin product on a Thr structure, and the dehydrating agent is used for intramolecular dehydration);

and 7, purifying the crude product of the romidepsin and converting the purified product into acetate to obtain a purified product of the romidepsin.

There are 4 and 1 hydroxy acids in the backbone amino acid of romidepsin, consisting of:

[Val¹-(R²-D-Val³-D-Cys⁴)-DH-Thr⁵]

wherein R is (3S,4E) -3-hydroxy-7-thio-4-heptenoic acid which forms a disulfide bond with D-cysteine at position 4 and a valine at position 1 forms an amide bond with threonine at position 5; DH-Thr means dehydroxythreonine.

The invention synthesizes the romidepsin linear peptide chain one by using proper protected amino acid, completes amido bond cyclization and disulfide bond cyclization in sequence by a solid phase means, optimizes the solid phase synthesis scheme in detail, and finally obtains the romidepsin with higher purity and total yield.

The protecting group is a protecting group which is required to protect groups which interfere with synthesis, such as amino, carboxyl, sulfydryl and the like on an amino acid main chain and a side chain in the field of amino acid synthesis, and prevents the amino, the carboxyl, the sulfydryl and the like from reacting to generate impurities in the process of preparing a target product, for example, a Trt or Acm protecting group is used as the protecting group of the sulfydryl in the invention. Amino acids protected by protecting groups are collectively referred to as protected amino acids (e.g., protected Thr-OAll, protected D-Cys, etc.). The alpha amino group at the N-terminal of the protected amino acid of the invention is preferably protected by Fmoc protecting group.

Preferably, the protected amino acids of the present invention are as follows:

Fmoc-Thr-OAll, Fomc-D-Cys (Trt) or Fomc-D-Cys (Acm), Fmoc-D-Val, (3S,4E) -3-hydroxy-7- [ (Trt) thio ] -4-heptenoic acid or (3S,4E) -3-hydroxy-7- [ (Acm) thio ] -4-heptenoic acid, Fmoc-D-Val.

Preferably, the resin is a trityl resin, more preferably a Trt resin or a 2-Cl-Trt resin.

The one-by-one coupling of the invention means that after the previous amino acid is coupled with resin or peptide resin, the rest amino acids are coupled with the previous coupled amino acid one by one according to the condensation reaction of the polypeptide sequence from the C end to the N end of romidepsin. In the coupling of the present invention, the molar ratio of the protected amino acid to the resin or the corresponding peptide resin at each coupling is preferably 1-6:1, more preferably 2.5-3.5: 1; the coupling reaction time is preferably 60 to 300 minutes, and more preferably 120 to 180 minutes.

The peptide resin of the present invention refers to a peptide resin formed by connecting any number of amino acids to a resin in the sequence of romidepsin polypeptide, and includes not only peptide resins 1 to 3 but also a plurality of peptide resins obtained during the synthesis of peptide resin 2. The corresponding peptide resin is illustrated, for example, the peptide resin 1 is the corresponding peptide resin coupled with the protected D-Cys, the peptide resin coupled with the protected D-Cys is the corresponding peptide resin when the protected D-Val is subjected to extension coupling, and the like, the protected (3S,4E) -3-hydroxy-7-sulfenyl-4-heptenoic acid and the protected Val respectively form corresponding relations when the peptide resin is coupled with the peptide resin after the last protected amino acid.

In the extension coupling, since each amino acid has a protecting group at the N-terminus, it is common knowledge to those skilled in the art that the protecting group at the N-terminus needs to be removed before coupling. The invention preferably uses PIP/DMF (piperidine/N, N-dimethylformamide) mixed solution to remove the Fomc protecting group at the N end, wherein the mixed solution contains 10-30% (V) of piperidine and the balance of DMF. The time for removing the N-terminal protecting group is preferably 10 to 60 minutes, and preferably 15 to 25 minutes. The amount of the N-terminal-protecting-group-removing agent to be used is preferably 10mL/g of the peptide resin.

Preferably, the substitution value of the peptide resin 1 is 0.2 to 1.5mmol/g resin, more preferably 0.5 to 1.0mmol/g resin.

Preferably, the condensation reagent is N, N-Diisopropylcarbodiimide (DIC), N-Dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate/organic base (PyBOP/organic base), 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate/organic base (HATU/organic base), benzotriazole-N, N, N ', N' -tetramethyluronium hexafluorophosphate/organic base (HBTU/organic base), O-benzotriazole-N, N, N ', N' -tetramethyluronium tetrafluoroborate/organic base (TBTU/organic base). The molar amount of the condensation reagent is preferably 1 to 6 times, and more preferably 2.5 to 3.5 times of the total molar number of the resin or the synthesized peptide resin.

It should be noted that the PyBOP/organic base, HATU/organic base, HBTU/organic base, TBTU/organic base are four two-system condensation reagents in the present invention, i.e. PyBOP, HATU, HBTU need to be combined with organic base to be one condensation reagent when in use, wherein the molar ratio of the organic base to PyBOP, HATU, HBTU, TBTU is preferably 1.3-3.0:1, more preferably 1.3-2: 1.

Preferably, the organic base in the condensation reagent and the organic base in step 2 are both preferably N, N-Diisopropylethylamine (DIPEA), Triethylamine (TEA) or N-methylmorpholine (NMM), more preferably DIPEA.

Preferably, the activating reagent is 1-hydroxybenzotriazole (HOBt) or N-hydroxy-7-azabenzotriazole (HOAt). The amount of the activating agent is preferably 1 to 6 times, more preferably 2.5 to 3.5 times, of the total molar number of the resin or the synthesized peptide resin.

Preferably, the catalyst is 4-Dimethylaminopyridine (DMAP).

Preferably, the All deprotecting agent is a solution of palladium tetratriphenylphosphine and phenylsilane in Dichloromethane (DCM). The molar ratio of the tetrakistriphenylphosphine palladium to the phenylsilane is 1: 8-12, preferably 1: 10. the dosage of the palladium tetratriphenylphosphine is 0.2 to 0.3 time of the molar weight of the All, and preferably 0.25 time.

Preferably, the acidolysis is performed by using a mixed acidolysis solution consisting of 95-100% by volume of TFA and the balance of water. More preferably, the acidolysis is carried out with a mixed acidolysis solution consisting of 98% by volume of TFA and the balance of water. The dosage of the mixed acidolysis solution is preferably 4-15 mL per gram of romidepsin precursor peptide resin, and more preferably 9-11 mL. The acidolysis time is preferably 1-6 hours at room temperature, and more preferably 3-4 hours.

Preferably, the dehydrating agent is a pyridine solution of p-toluenesulfonyl chloride or a pyridine solution of methanesulfonyl chloride, more preferably a pyridine solution of p-toluenesulfonyl chloride. The dosage of the dehydrating agent is 1.2 to 4 times of the molar weight of OH in the Thr, and more preferably 2 times.

Preferably, the purified trans-acetate is specifically:

dissolving crude romidepsin product in 0.1% TFA/water solution, filtering the solution with 0.45 μm microporous membrane, and purifying;

purifying by high performance liquid chromatography, wherein the purification uses reversed phase C18 with chromatographic packing of 10 μm, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the flow rate of a chromatographic column of 77mm x 250mm is 90mL/min, eluting by a gradient system, purifying by circulating sample injection, sampling the crude product solution in the chromatographic column, starting the mobile phase for elution, collecting the main peak, and evaporating acetonitrile to obtain the intermediate concentrated solution of purified romidepsin;

filtering the purified intermediate concentrated solution of romidepsin with a 0.45-micron filter membrane for later use;

performing salt exchange by adopting a high performance liquid chromatography, wherein a mobile phase system is 10% acetic acid/water solution-acetonitrile, a reversed phase C18 with a chromatographic packing of 10 mu m for purification, a chromatographic column with a flow rate of 77mm x 250mm of 90mL/min, performing gradient elution and a circular sample loading method, loading the sample into the chromatographic column, starting mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, performing reduced pressure concentration to obtain a romidepsin acetic acid water solution, and performing freeze drying to obtain a finished romidepsin product.

The romidepsin synthesized by the method has the purity of a crude product of more than 70 percent, the purity of a product of more than 99.5 percent, the maximum single impurity of less than 0.15 percent and the total yield of more than 50 percent through HPLC detection.

According to the technical scheme, the romycin linear peptide chain is synthesized one by using proper protected amino acids, the amido bond cyclization and the disulfide bond cyclization are completed sequentially by a solid phase means, the solid phase synthesis scheme is optimized in detail, and finally the obtained romycin has high purity and total yield.

Detailed Description

The invention discloses a method for synthesizing romidepsin, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made and the techniques of the present invention employed without departing from the spirit and scope of the invention.

In the present invention, the protected amino acids were obtained from Yoghui Biotech Co., Ltd, the resins were obtained from Tianjin south China and Yoghui Biotech Co., Ltd, and the English abbreviations used in the application documents have the corresponding Chinese meanings as shown in Table 1.

TABLE 1 English abbreviation definitions

The invention is further illustrated by the following examples.

Example 1: synthesis of peptide resin 1 (coupling of Fmoc-Thr-OAll)

0.3mol of Trt resin (with a substitution value of about 0.6mmol/g) is taken and washed 3 times with DMF; dissolving 0.6mol of Fmoc-Thr-OAll in a proper amount of DMF, adding the solution into the resin, adding 1.2mol of DIPEA under stirring, reacting for 6 hours under stirring at 65 ℃, pumping out the reaction solution, washing 3 times with DMF, washing 3 times with 10% DIPEA/methanol, washing 3 times with DMF for 3 minutes each time, and obtaining the Fmoc-Thr-OAll-Trt resin, namely the peptide resin 1.

Example 2: synthesis of peptide resin 2

Dissolving 0.3mol of Fmoc-D-Cys (Trt) and 0.3mol of HOBt in a proper amount of DMF; and adding 0.3mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.

0.1mol of the peptide resin 1 prepared in example 1 is taken, deprotected with 20% PIP/DMF solution for 25 minutes, washed with DMF, filtered, added with activated Fmoc-D-Cys (Trt) solution, stirred at room temperature for reaction for 3 hours, reaction solution is pumped out, washed with DMF for 3 times, washed with DCM for 3 minutes each time, deprotected with 20% PIP/DMF solution for 25 minutes, washed and filtered, and then the grafting of Fmoc-D-Cys (Trt) is completed.

Fmoc-D-Val and (3S,4E) -3-hydroxy-7- [ (Trt) thio ] -4-heptenoic acid are inoculated in the same way, and the mixture is washed and filtered to obtain (3S,4E) -3-hydroxy-7- [ (Trt) thio ] -4-heptenoyl-D-Val-D-Cys (Trt) -Thr-OAll-Trt resin, namely the peptide resin 2.

Example 3: synthesis of peptide resin 2

Dissolving 0.3mol of Fmoc-D-Cys (Acm) and 0.3mol of HOBt in a proper amount of DMF; and adding 0.3mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.

0.1mol of the peptide resin 1 prepared in example 1 is taken, deprotected with 20% PIP/DMF solution for 25 minutes, washed with DMF, filtered, added with activated Fmoc-D-Cys (Acm) solution, stirred at room temperature for reaction for 3 hours, reaction solution is pumped out, washed with DMF for 3 times, washed with DCM for 3 minutes each time, deprotected with 20% PIP/DMF solution for 25 minutes, washed and filtered, and then the grafting of Fmoc-D-Cys (Acm) is completed.

The Fmoc-D-Val and (3S,4E) -3-hydroxy-7- [ (Acm) thio ] -4-heptenoic acid are grafted in the same way, and the (3S,4E) -3-hydroxy-7- [ (Acm) thio ] -4-heptenoyl-D-Val-D-Cys (Trt) -Thr-OAll-Trt resin, namely the peptide resin 2, is obtained by washing and filtering.

Example 4: synthesis of peptide resin 3

Dissolving 0.3mol of Fmoc-Val and 0.3mol of HOBt by using a proper amount of DMF; and adding 0.3mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain the activated protected amino acid solution.

The above activated protected amino acid solution was added to the peptide resin 2 obtained in example 2, and 0.1mol of dmap (catalyst)/DMF solution was added, and stirred at room temperature for reaction for 8 hours, and washed and filtered to obtain a peptide resin 3.

Example 5: synthesis of peptide resin 3

Dissolving 0.3mol of Fmoc-Val and 0.3mol of HOBt by using a proper amount of DMF; and adding 0.3mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution.

The above activated protected amino acid solution was added to the peptide resin 2 obtained in example 3, and 0.1mol of dmap (catalyst)/DMF solution was added, and stirred at room temperature for reaction for 8 hours, and washed and filtered to obtain the peptide resin 3.

Example 6: synthesis of romidepsin precursor peptide resin

1. Deprotection of the All protecting group

0.025mol of tetratriphenylphosphine palladium and 0.25mol of phenylsilane were dissolved with an appropriate amount of DCM, added to the peptide resin 3 of example 4, stirred at room temperature for more than 10 hours, and after the reaction was completed, washed 3 times with DMF.

2. Fmoc-deprotecting group

Deprotection was then carried out for 25 min with 20% PIP/DMF and after completion of the reaction, it was washed 3 times with DMF.

3. Cyclization of amide ring

Dissolving 0.3mol DIC and 0.3mol HOAt in proper amount of DMF, slowly adding into the above peptide resin with the All and Fmoc protecting groups removed, performing cyclization reaction for 240-300 minutes, and washing with DMF for 3 times after the reaction is completed.

4. Cyclization reaction of disulfide

Taking 5% of I₂Adding DMF solution (10 mL/g resin), stirring at 40 deg.C for 4 hr, removing reaction solution, and washing with DMF for 3 times to obtain romidepsin precursor peptide resin.

Example 7: synthesis of romidepsin precursor peptide resin

1. Deprotection of the All protecting group

0.025mol of tetratriphenylphosphine palladium and 0.25mol of phenylsilane were dissolved with an appropriate amount of DCM and added to the peptide resin 3 of example 5, and the mixture was stirred at room temperature for a reaction time of 10 hours or more, and after the reaction was completed, it was washed 3 times with DMF.

2. Fmoc-deprotecting group

3. Cyclization of amide ring

Dissolving 0.3mol DIC and 0.3mol HOAt in a proper amount of DMF, slowly adding the solution into the peptide resin with the All and Fmoc protecting groups removed under stirring, carrying out cyclization reaction for 240-300 minutes, and washing 3 times with DMF after the reaction is finished.

4. Cyclization reaction of disulfide

Example 8: preparation of romidepsin precursor

The romidepsin peptide precursor resin prepared in example 6 was added with an acid hydrolysis solution (TFA: H)₂O98: 2, acidolysis solution 10 mL/clomiphene precursor peptide resin), stirring for reaction for 3 hours, filtering and collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, and concentrating under reduced pressure until the solvent is evaporated to dryness to obtain the romidepsin precursor.

Example 9: preparation of romidepsin precursor

An acidolysis solution (TFA: H) was added to the romidepsin precursor resin obtained in example 7₂O98: 2, acidolysis solution 10 mL/clomiphene precursor peptide resin), stirring for reaction for 3 hours, filtering and collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, and concentrating under reduced pressure until the solvent is evaporated to dryness to obtain the romidepsin precursor.

Example 10: preparation of crude romidepsin

Dissolving the romidepsin peptide precursor prepared in example 8 with a proper amount of pyridine, cooling to-5 ℃, adding 0.2mol of p-toluenesulfonyl chloride under stirring, keeping the temperature for reaction for 12 hours, pouring the reaction mixture into ice water, stirring to adjust the pH to 6, filtering, collecting solids, and washing to obtain a crude romidepsin product with the purity of 75.2%.

Example 11: preparation of crude romidepsin

Dissolving the romidepsin peptide precursor prepared in example 9 with a proper amount of pyridine, cooling to-5 ℃, adding 0.2mol of p-toluenesulfonyl chloride under stirring, keeping the temperature for reaction for 12 hours, pouring the reaction mixture into ice water, stirring to adjust the pH to 6, filtering, collecting solids, and washing to obtain a crude romidepsin product with the purity of 71.3%.

Example 12: purification of crude romidepsin

Dissolving the crude product of the romidepsin obtained in the embodiment 10 by using a 30% acetic acid solution, filtering the solution by using a 0.45 mu m microporous filter membrane, and purifying for later use;

performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the purification is performed by reversed phase C18 with chromatographic packing of 10 μm, the flow rate of a chromatographic column of 77mm × 250mm is 90mL/min, gradient elution and cyclic sample loading method are adopted, the sample is loaded in the chromatographic column, the mobile phase elution is started, the chromatogram is collected, the change of the absorbance is observed, the main peak of salt exchange is collected and the purity is detected by analyzing the liquid phase, the main peak solution of salt exchange is combined, the reduced pressure concentration is performed to obtain the water solution of the romidepsin acetic acid, and the freeze drying is performed to obtain 30.6g pure romidepsin product

Total yield 56.7%, molecular weight: 541.6, purity: 99.6 percent and 0.11 percent of maximum single impurity.

Example 13: purification of crude romidepsin

Dissolving the crude product of romidepsin obtained in the example 11 by using 30 percent acetic acid solution, filtering the solution by using a 0.45 mu m microporous membrane, and purifying for later use;

performing salt exchange by adopting a high performance liquid chromatography, wherein a mobile phase system is 1% acetic acid/water solution-acetonitrile, a reversed phase C18 with a chromatographic packing of 10 mu m for purification, a chromatographic column with a flow rate of 77mm x 250mm of 90mL/min, performing gradient elution and a circular sample loading method, loading the sample into the chromatographic column, starting mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, performing reduced pressure concentration to obtain a romidepsin acetic acid water solution, and performing freeze drying to obtain 28.5g of a purified romidepsin product.

Total yield 52.8%, molecular weight: 541.8, purity: 99.7% and maximum single impurity 0.09%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for synthesizing romidepsin is characterized by comprising the following steps:

step 6, dehydrating the romidepsin precursor by using a dehydrating agent to obtain a crude romidepsin product;

step 7, purifying and converting the crude product of romidepsin into acetate to obtain a purified product of romidepsin;

the protected Thr-OAll, the protected D-Cys, the protected D-Val, the protected (3S,4E) -3-hydroxy-7-thio-4-heptenoic acid and the protected Val are as follows:

2. The method of claim 1, wherein the resin is a trityl resin.

3. The method of claim 1, wherein the condensation reagent is one of N, N-diisopropylcarbodiimide, N, N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate/organic base, 2- (7-aza-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate/organic base, benzotriazol-N, N, N ', N' -tetramethyluronium hexafluorophosphate/organic base, O-benzotriazol-N, N, N ', N' -tetramethyluronium tetrafluoroborate/organic base.

4. The process according to claim 1 or 3, characterized in that the organic base is N, N-diisopropylethylamine, triethylamine or N-methylmorpholine.

5. The method of claim 1, wherein the activating reagent is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole.

6. The process of claim 1, wherein the catalyst is 4-dimethylaminopyridine.

7. The method of claim 1, wherein the All deprotecting agent is palladium tetratriphenylphosphine in dichloromethane with phenylsilane.

8. The method according to claim 1, wherein the dehydrating agent is pyridine solution of p-toluenesulfonyl chloride or pyridine solution of methanesulfonyl chloride.

9. The method as claimed in claim 1, wherein the acidolysis is performed by using a mixed acidolysis solution comprising 95-100% by volume of TFA and the balance of water.