WO2014101828A1

WO2014101828A1 - Method for preparing romidepsin

Info

Publication number: WO2014101828A1
Application number: PCT/CN2013/090669
Authority: WO
Inventors: 肖庆; 潘俊锋; 马亚平; 袁建成
Original assignee: 深圳翰宇药业股份有限公司
Priority date: 2012-12-27
Filing date: 2013-12-27
Publication date: 2014-07-03
Also published as: CN103897029A; CN103897029B

Abstract

Disclosed is a method for preparing Romidepsin. The present invention relates to the pharmaceutical synthesis. The present invention is based on a method of solid phase synthesis. First, coupling is performed between resin and carboxyl groups on 3-hydroxy-7-mercapto-4-heptenoic acid; then,coupling is performed in sequence between four pieces of amino acid on Romidepsin; then, hydroxyl groups are removed, and disulfide bonds and amido bonds are obtained by means of cyclization, so as to form Romidepsin. The purity the finally finished product is greater than 99%, the total yield is higher than 30%, and method features simple preparation, a short synthesis cycle and low cost, and helps to produce Romidepsin in a large scale.

Description

Preparation method of romidepsin

The present application claims priority to Chinese Patent Application No. 201210579007.4, entitled "Preparation of a Romidepsin" by the Chinese Patent Office on December 27, 2012, the entire contents of which is incorporated herein by reference. In the application. Technical field

The invention relates to the field of medical synthesis, in particular to a preparation method of romidepsin. Background technique

Romidisin, English name is Romidepsin, its chemical name is ( 1 S, 4S, 7Z, 10S, 16E,

21R) -7-ethylidene-4,21-diisopropyl-2-oxa-12,13-dithio-5, 8, 20, 23-tetraazabicyclo[8, 7, 6] Tridecyl-16-ene-3, 6, 9, 19, 22-pentanone, a molecular formula of C ₂₄ H ₃₆ N ₄ 0 ₆ S ₂ , is a bicyclic tetrapeptide with a stable hydrophobic structure and its structure A unique disulfide bond is an active group that acts. In 2009, Romidepsin was approved by the US Food and Drug Administration (FDA) for the treatment of skin T as follows:

Romidisin

Romidepsin, a inhibitor of histone deacetylase (HDACs), enters the cytoplasm through the tumor cell membrane, and the intracellular disulfide bonds are reduced to sulfhydryl groups by glutathione, which binds to zinc in zinc-dependent HDACs. It plays a role in inhibiting HDACs, thereby further inducing tumor cell differentiation and apoptosis.

At present, there are mainly two methods for preparing romidepsin, one is biological fermentation, and the other is by chemical synthesis. There are several articles on chemical synthesis, such as 1996 Knhn et al. Human is synthesized in the whole liquid phase with romidepsin (J.AM. Chem. Soc, 118:7237-7238). The method in this literature uses valine oxime ester as a raw material, coupling the corresponding amino acid, and then lithium hydroxide. The oxime ester is removed, and then esterified under DEAD and PPh ₃ conditions. Finally, the disulfide bond is synthesized by iodine oxidation, and the romidepsin acetate is synthesized through 14 steps (in clinical application, romidepsin itself does not Too stable, need to be made of romidepsin acetate). However, this method is cumbersome and not simple enough. More importantly, the total liquid phase yield is only about 18%. These problems have always been the main factors affecting the production efficiency of romidepsin. Summary of the invention

In view of the above, it is an object of the present invention to provide a process for the preparation of romidepsin which enables the process of the present invention to increase its overall yield while reducing the preparation steps.

To achieve the above object, the present invention provides the following technical solutions:

A method for preparing romidepsin comprises the following steps:

Step 1. Coupling the resin with a carboxyl group on 3-hydroxy-7-(R)mercapto-4-heptenoic acid by an activator to obtain a compound of formula 1;

Step 2. Fmoc-L-Val-OH is coupled with a hydroxyl group on the compound of formula 1, and after removing the Fmoc protecting group, Fmoc-L-Thr-OH, Fmoc-D-Cys(R)-OH, Fmoc- are sequentially successively. D-Val-OH undergoes polypeptide chain extension coupling to obtain a compound of formula 2;

Step 3, the compound of formula 2 is removed from the Fmoc protecting group and the hydroxyl group on the side chain of the L-Thr residue to obtain a compound of formula 3;

Step 4, the compound of formula 3 is cyclized by a deoxidation method to form a disulfide bond to obtain a compound of formula 4; Step 5, the compound of formula 4 is cleaved to remove the resin to obtain a compound of formula 5;

Step 6. The carboxyl group on the compound of formula 5 and the N-terminus of the D-Val residue are cyclized to form an amide bond to obtain romidepsin;

Compound of formula 1

Compound of formula 2

Compound of formula 4

The protecting groups described in the present invention are protecting groups which are commonly used in the field of amino acid synthesis to protect the amino acid backbone and the amino group, carboxyl group, sulfhydryl group and the like in the side chain, and prevent the amino group, the carboxyl group, the thiol group and the like from preparing the target product. The reaction takes place during the process to form impurities. For the amino acids in the present invention which need to protect the side chain, the side chain structure is known to those skilled in the art. And knowing to use a common protecting group to protect the amino group, carboxyl group, thiol group and the like on the side chain of the amino acid, wherein R is a thiol protecting group, the Fmoc is an N-terminal protecting group of the amino acid, and the Resin is a resin. Fmoc-L-Val-OH refers to the coupling of the Fmoc protecting group L-Val at the N-terminus, and Fmoc-L-Thr-OH refers to the L-Thr, Fmoc-D-Cys with the Fmoc protecting group coupled at the N-terminus. (R)-OH refers to D-Cys having an Fmoc protecting group coupled to the N-terminus and an R protecting group at the side chain thiol group. Fmoc-D-Val-OH means that an Fmoc protecting group is coupled at the N-terminus. D-Val, the above simplified form is a common form in the field.

Preferably, the mercapto protecting group is a triphenylsulfonyl group or an acetamidofluorenyl group, more preferably a triphenyl group; preferably, the resin is CTC Resin (CTC resin) or Wang Resin (Wang Resin), more preferably For CTC Resin, the most preferred is CTC Resin with a degree of substitution of 0.5 mmol/g.

The invention is directed to the problem that the preparation method of the existing romidepsin is cumbersome and the yield is low. Based on the solid phase synthesis method, the solid phase carrier (ie, the resin) and the 3-hydroxy-7-mercapto-4-heptene are firstly obtained. The carboxyl group on the acid is coupled, and then the four amino acids on the romidepsin are sequentially coupled, followed by removal of the hydroxyl group, cyclization into a disulfide bond, and an amide bond to form romidepsin, which significantly reduces the synthesis step. Moreover, the yield is increased to about 30%.

In the first step of the production method of the present invention, preferably, the molar ratio of the resin, activator and 3-hydroxy-7-(R)decyl-4-heptenoic acid is 1:6:3.

The most preferred solution, step 1 is:

3-Hydroxy-7-(R)mercapto-4-epenoic acid is dissolved and added to DIPEA for activation, and then coupled with a washed, swollen resin to give a compound of formula 1. Among them, the solvent for dissolving, washing and swelling the resin in this step is preferably DMF.

In step 2 of the preparation method of the present invention, the polypeptide chain extension coupling means that after Fmoc-L-Val-OH is coupled with the compound of formula 1, the remaining amino acids are sequentially linked according to their linkage sequence in the romidepsin structure. Coupling is carried out by a condensation reaction (condensation reaction of a main chain amino group and a carboxyl group) with the former coupled amino acid. In the polypeptide chain extension coupling, since each amino acid has a protecting group at the N-terminus, it is necessary to first remove the N-terminal protecting group and then re-coupling, which is common knowledge to those skilled in the art, and the present invention preferably uses DBLK to remove it. N-terminal protecting group.

As a preferred solution, step 2 is: After dissolving Fmoc-L-Val-OH, the coupling agent and DMAP are added, and then coupled with the compound of formula 1, the Fmoc protecting group is removed by DBLK, then the above coupling agent and DMAP are added, the amino acid is added, and the Fmoc protecting group is removed. The step of sequentially coupling the polypeptide chain extension of Fmoc-L-Thr-OH, Fmoc-D-Cys(R)-OH, Fmoc-D-Val-OH one by one to obtain a compound of formula 2.

Wherein, the coupling agent is preferably a HOBT/DIC dual system coupling agent, a PyBOP/HOBt dual system coupling agent or a TBTU/HOBt dual system coupling agent, and most preferably a PyBOP/HOBt dual system coupling agent. For these multi-system coupling agents, the ratio of the components thereof is specific and well known in the art and will not be described herein. The solvent for dissolving in the step 2 is preferably one or both of DMF, DCM, NMP, DMSO, and more preferably a mixed solvent having a volume ratio of DMF: NMP of 1:1. Preferably, the coupling agent is used in an amount of the resin in the step 1, and the molar ratio of the coupling agent is 3:1, and the amount of the DMAP is based on the amount of the resin in the step 1. The molar ratio of DMAP: resin is 0.2:1.

In step 3 of the preparation method of the present invention, as a preferred embodiment, step 3 is:

Adding triethylamine to the compound of formula 2 and dissolving DMAP after activation, then adding decylsulfonyl chloride dropwise for 1-5 hours, then adding the dissolved DABCO reaction for 1-5 hours to remove the entire hydroxyl group, and finally removing it with DBLK. The compound of formula 3 is obtained in addition to the Fmoc protecting group.

In the preferred embodiment of step 3, the reaction time is preferably 2 hours; the temperature of the reaction is preferably -5 ° C to 5 ° C, more preferably 0 ° C; the solvent for dissolution is preferably DMF , DCM or THF; the amount of the triethylamine is based on the amount of the resin in the step 1, the molar ratio of the two is triethylamine: the resin is preferably 2:1, and the amount of the mercapto-cross-acid chloride is the resin in the step 1. The amount is the reference, the molar ratio of the two is decyl cross-acid chloride: the resin is preferably 1.5:1, the amount of the DMAP is based on the amount of the resin in the step 1, the molar ratio of the two is DMAP: the resin is preferably 0.2:1. The amount of DABCO used is based on the amount of the resin in the step 1, and the molar ratio of the two is preferably 2:1.

In the step 4 of the preparation method of the present invention, as a preferred embodiment, the step 4 is:

After the iodine is dissolved, the compound of the formula 3 is added to react for 1-4 hours, and then washed, shrinked, and dried to obtain a compound of the formula 4.

In a preferred embodiment of step 4, the reaction time is preferably 2 hours; The solvent is preferably DMF or MeOH; the amount of the break is based on the amount of the resin in the step 1, and the molar ratio of the two is preferably 4 to 10:1, more preferably 6:1.

In the step 5 of the preparation method of the present invention, as a preferred embodiment, the step 5 is:

The cleavage reagent was added to the compound of the formula 4 for 2 hours, filtered, and the filtrate was precipitated with diethyl ether. The precipitate was collected as a compound of the formula 5, and the lysing reagent was a mixed lysate having a volume ratio of TFA:H ₂ 0 of 95:5, volume ratio. TFA: EDT: PHOH: H ₂ 0 is a mixed lysate of 95:5:3:2 or a mixed lysate having a volume ratio of TFA: EDT:TIS:PHOH:H ₂ 0 of 80:5:5:5:5.

In the step 6 of the preparation method of the present invention, as a preferred embodiment, the step 6 is:

After the compound of the formula 5 is dissolved, a coupling agent and DIPEA are added to react for 2 to 5 hours to obtain romidepsin. Wherein, the coupling agent is preferably a HOBT/DIC dual system coupling agent, a PyBOP/HOBt dual system coupling agent or a TBTU/HOBt dual system coupling agent, and most preferably a PyBOP/HOBt dual system coupling agent. For these multi-system coupling agents, the ratio of the components thereof is specific and well known in the art and will not be described herein. The solvent for dissolution described in the step 6 is preferably DMF; the reaction time is preferably 4 hours. Preferably, the coupling agent is used in an amount of the resin in the step 1, and the molar ratio of the coupling agent is 3:1, and the amount of the DIPEA is based on the amount of the resin in the step 1. The molar ratio of DIPEA: resin is 6:1.

In addition, according to the actual clinical application of romidepsin, it needs to be prepared into acetate for stabilization. Therefore, the present invention further comprises the step of purifying romidepsin and preparing the romidepsin acetate step after step 6, specifically :

The romidepsin water obtained in the step 6 was diluted, and then purified by RP-HPLC system to obtain the pure product of romidepsin, and then the RP-HPLC system was used to transfer the salt with the acetonitrile-acetic acid solution as the mobile phase, and the target peak fraction was collected and concentrated. Freeze-dried is romidepsin acetate.

Wherein, the mass percentage of the acetic acid solution is preferably 0.2%.

The romidepsin acetate prepared according to the preparation method of the present invention is compared with the prior art (that is, the preparation process mentioned in the background art, which is also prepared as romidepsin acetate), the yield is 18% is increased to about 30%, and the preparation process is greatly reduced.

It can be seen from the above technical solutions that a method for preparing romidepsin based on the principle of solid phase synthesis is provided, and the product prepared by the method of the invention has a purity of more than 99%, a total yield of more than 30%, and the method is simple to operate. , the synthesis cycle is short, the cost is low, and it is beneficial as romidepsin. Mass production. detailed description

The invention discloses a preparation method of romidepsin, and those skilled in the art can learn from the contents of the paper and appropriately improve the process parameters. It is to be noted that all such alternatives and modifications will be apparent to those skilled in the art and are considered to be included in the present invention. The method of the present invention has been described by the preferred embodiments, and it is obvious that the compounds and preparation methods described herein can be modified or appropriately modified and combined without departing from the spirit, scope and scope of the present invention. The technique of the present invention is applied.

In a specific embodiment of the present invention, all of the amino acids coupled to the protecting group are commercially available, and the protected amino acids in the present invention are purchased from Gil Biochemical Co., Ltd., using CTC Resin (CTC resin) and Wang Resin (Wang Resin). The grease was purchased from Tianjin Nankai Hecheng Co., Ltd. The meaning of the Chinese abbreviation used in the application documents is shown in Table 1.

Table 1 Explanation of English abbreviations

Fmoc 9-oxime carbonyl

Trt triphenyl fluorenyl

NMP N-decylpyrrolidone

DMSO dimercapto sulfoxide

DMF Ν, Ν-dimercaptoamide

DCM dichlorodecane

DIC Ν, Ν-diisopropylcarbodiimide

DIPEA Ν, Ν-diisopropylethylamine

DMAP 4-diammonium pyridine

PYBOP benzotriazole hexafluorophosphate-1-yl-oxytripyrrolidinyl

TBTU 0-benzotriazole-Ν,Ν,Ν',Ν'-tetradecylurea tetrafluoroboric acid HOBT 1-hydroxybenzotriazole

(3H-1 , 2,3-triazolo[4,5-b]pyridine-3-yloxy)tris-1-pyrrolidinium

PYAOP

Hexafluoride

2-(7-azobenzotriazole)-Ν,Ν,Ν',Ν'-tetradecylurea hexafluorophosphate

HATU

Ester

HOAt-hydroxy-7-azobenzotriazole

ACM acetamido fluorenyl

TFA trifluoroacetic acid

EDT 1,2-ethanediol

PHOH phenol

TIS triisopropylsilane

MeOH sterol

MsCl sulfhydryl chloride

DABCO 1,4-diazabicyclo [2.2.2] octane

DBLK 20% Hexahydropyridine / DMF Solution The invention is further illustrated by the following examples in connection with the Examples. Example 1: Preparation of a compound of formula 1

CTC Resin 2g (synthesis scale 1 mmol) with a degree of substitution of 0.5 mmol/g was weighed, added to a solid phase reaction column, washed twice with DMF, and swollen with DMF for 30 minutes, and then weighed 1.26 g of 3-hydroxy-7. - (Triphenyl decyl) decyl-4-heptenoic acid was dissolved in DMF, added to 0.6 mL of DIPEA in an ice water bath, and then added to the reaction column containing the resin. The reaction was completed for 2 hours and washed 6 times with DMF. Compound of formula 1. Example 2: Preparation of a compound of formula 2

1.01 g of Fmoc-Val-OH, 0.38 g of HOBt, and 0.03 g of DMAP were dissolved in a mixed solution of DMF and NMP in a volume ratio of 1:1, and 0.3 mL of DIC was added in an ice water bath to be activated, and then added to the solid phase reaction in Example 1. The column is reacted with the compound of formula 1 and reacted at room temperature for 2 h (the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete, the resin develops color, indicating that the reaction is incomplete, and the coupling reaction is further required). The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF.

Then, the above steps of adding a coupling agent and DMAP, adding an amino acid, and removing the Fmoc protecting group are repeated, and Fmoc-L-Thr-OH, Fmoc-D-Cys(Trt)-OH, Fmoc-D-Val-OH are sequentially completed one by one. Coupling of the extended polypeptide chain provides a compound of formula 2. Example 3: Preparation of a compound of formula 3

After 0.03 g of DMAP was dissolved in anhydrous dichloromethane, 0.6 ml of triethylamine was added to lower the temperature of the mixture to zero. After C, it was added to the reaction column of Example 2, and the force was dropped. 0.3 ml of mercaptosulfonyl chloride, reacted for 2 hours, the reaction temperature was maintained at 0 ° C, and after washing, 6 times with DMF, and then 2.2 g of DABCO and 50 mm of DCM were added, and after 2 hours of reaction, the reaction was completed and washed 6 times with DMF. The compound of formula 3 is obtained. Example 4: Preparation of a compound of formula 3

After weighed 0.03 g of DMAP and dissolved in anhydrous dichloromethane, 0.6 ml of triethylamine was added, and the temperature of the mixture was lowered to 5 ° C, and then added to the reaction column of Example 2, and the dropping force was applied. 0.3 ml of mercaptosulfonyl chloride, reacted for 1 hour, the reaction temperature was kept at 5 ° C, and after washing, 6 times with DMF, and then 2.2 g of DABCO and 50 mm of DCM were added, and after 2 hours of reaction, the reaction was completed and washed 6 times with DMF. The compound of formula 3 is obtained. Example 5: Preparation of a compound of formula 3

After weighed 0.03 g of DMAP and dissolved in anhydrous dichloromethane, 0.6 ml of triethylamine was added, and the temperature of the mixture was lowered to -5 ° C, and then added to the reaction column of Example 2, and 0.3 ml of ruthenium was added dropwise. The acid chloride was reacted for 5 hours, the reaction temperature was maintained at 5 ° C, and the reaction was completed 6 times with DMF. Further, 2.2 g of DABCO and 50 mml of DCM were added, and after reacting for 2 hours, the reaction was completed and washed 6 times with DMF to obtain a compound of the formula 3. Example 6: Preparation of a compound of formula 4

After weigh 1.5 g of iodine dissolved in DMF, it was added to the reaction column of Example 3 and reacted with the compound of Formula 3 for 2 hours. After the reaction, it was washed 6 times with DMF, then condensed 3 times with decyl alcohol, and dried under vacuum to obtain 2.4 g of Formula 4 Compound. Example 7: Preparation of a compound of formula 4

After weigh 1.5 g of iodine dissolved in DMF, it was added to the reaction column of Example 4 and reacted with the compound of Formula 3 for 2 hours. After the reaction, it was washed 6 times with DMF, then with sterol for 3 times, and dried under vacuum to obtain 2.4 g of Formula 4. Compound. Example 8: Preparation of a compound of formula 4

After weigh 1.5 g of iodine dissolved in DMF, it was added to the reaction column of Example 5 and reacted with the compound of Formula 3 for 2 hours. After the reaction, it was washed 6 times with DMF, then condensed 3 times with decyl alcohol, and dried under vacuum to obtain 2.4 g of Formula 4 Compound. Example 9: Preparation of a compound of formula 5

2.4 g of the compound of formula 4 in Example 6 was added to a 50 ml flask, and a lysis reagent was disposed.

(volume ratio, TFA: H ₂ 0 = 95:5), the cleavage reagent was poured into a flask, and reacted at room temperature for 2 hours. At the end of the reaction, the filtrate was collected by filtration. It was added dropwise to 240 ml of diethyl ether reagent, centrifuged, washed with anhydrous diethyl ether, and the precipitate was dried in vacuo to give 0.52 g of the compound of formula 5 with a purity of 72.52%. Example 10: Preparation of a compound of formula 5

2.4 g of the compound of formula 4 in Example 7 was added to a 50 ml flask, and a lysis reagent (volume ratio, TFA: EDT: PHOH: H ₂ 0 = 95: 5: 3: 2) was placed, and the lysis reagent was poured into the flask. The reaction was carried out at room temperature for 2 hours. At the end of the reaction, the filtrate was collected by filtration. Add dropwise to 240 ml of ether reagent, The precipitate was washed by centrifugation, anhydrous diethyl ether, and dried under vacuum to give 0.50 g of compound of formula 5 with a purity of 65.42%. Example 11: Preparation of a compound of formula 5

2.4 g of the compound of formula 4 in Example 8 was added to a 50 ml flask, and a lysis reagent was disposed.

(volume ratio, TFA: EDT: TIS: PHOH: H ₂ O = 80: 5: 5: 5: 5), the cleavage reagent was poured into the flask, and reacted at room temperature for 2 hours. At the end of the reaction, the filtrate was collected by filtration. It was added dropwise to 240 ml of diethyl ether reagent, centrifuged, washed with anhydrous diethyl ether, and the precipitate was dried in vacuo to give 0.48 g of compound of formula 5 with a purity of 61.48%. Example 12: Preparation of crude romidepsin

0.52 g of the compound of the formula 5 in Example 9 was added with DMF solvent, 0.38 g of HOBt and 1.56 g of PyBOP, and 0.3 ml of DIPEA was added dropwise to the reaction flask for 4 hours to obtain crude romidepsin having a purity of 68.13%. Example 13: Purification of crude romidepsin to prepare romidepsin acetate

The crude romidepsin water of Example 12 was diluted 10 times, and the target peak fraction was collected by using an RP-HPLC system, a wavelength of 230 nm, a 50×250 mm reverse phase C18 column, and a 0.2% TFA/acetonitrile mobile phase purification. A fine romidepsin having a purity greater than 98.5% is obtained. The fine romidepsin solution was prepared by RP-HPLC system, the column was 50×250 mm reverse phase C18 column, 0.2% acetic acid solution/acetonitrile mobile phase was transferred to salt, the peak fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain romidepsin. The acetate was 0.17 g, the HPLC purity was 98.5%, and the total yield was 31.5%. Example 14: Preparation of a compound of formula 1

CTC Resin 2g (synthesis scale 1 mmol) with a substitution of 0.5 mmol/g was weighed, added to a solid phase reaction column, washed twice with DMF, and swollen with DMF for 30 minutes, and then weighed 1.15 g of 3-hydroxy-7. - (Acetylamino) decyl-4-heptenoic acid was dissolved in DMF, added to 0.6 mL of DIPEA in an ice water bath, and then added to the reaction column containing the resin. The reaction was completed for 2 hours and washed 6 times with DMF. The compound of formula 1 is obtained. Example 15: Preparation of a compound of formula 2

1.llg Fmoc-Val-OH, 0.38 g HOBt, 0.03 g DMAP, dissolved in a volume ratio of 1:1 DMF and NMP mixed solution, added to 0.3 mL DIC after ice water bath activation, and then added to the solid phase reaction of Example 14. The column is reacted with the compound of formula 1 and reacted at room temperature for 2 h (the end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete, the resin develops color, indicating that the reaction is incomplete, and the coupling reaction is further required). The Fmoc protecting group was then removed with DBLK and washed 6 times with DMF.

Then, the above steps of adding a coupling agent and DMAP, adding an amino acid, and removing the Fmoc protecting group are repeated, and Fmoc-L-Thr-OH, Fmoc-D-Cys(Acm)-OH, Fmoc-D-Val-OH are sequentially completed one by one. Coupling of the extended polypeptide chain provides a compound of formula 2. Example 16: Preparation of a compound of formula 3

After weighed 0.03 g of DMAP and dissolved in anhydrous dichloromethane, 0.6 ml of triethylamine was added, the temperature of the mixture was lowered to 0 ° C, and then added to the reaction column of Example 15, and 0.3 ml of decyl cross-acid chloride was added dropwise. The reaction was carried out for 2 hours, the reaction temperature was maintained at 0 ° C, and the reaction was completed 6 times with DMF. Then, 2.2 g of DABCO and 50 mm of DCM were added. After 2 hours of reaction, the reaction was completed and washed 6 times with DMF to obtain a compound of the formula 3. Example 17: Preparation of a compound of formula 4

1.5 g of iodine was weighed and dissolved in DMF, and then added to the reaction column of Example 16 and reacted with the compound of the formula 3 for 2 hours. After the completion of the reaction, the mixture was washed 6 times with DMF, and then condensed 3 times with decyl alcohol, and dried under vacuum to obtain 2.3 g of the formula 4. Compound. Example 18: Preparation of a compound of formula 5

2.3 g of the compound of the formula 4 in Example 17 was placed in a 50 ml flask, and a lysis reagent (volume ratio, TFA: H20 = 95:5) was placed, and the cleavage reagent was poured into the flask, and reacted at room temperature for 2 hours. At the end of the reaction, the resin was filtered and the filtrate was collected. Add dropwise to 240 ml of ether reagent, centrifuge, The precipitate was washed with anhydrous diethyl ether, and the precipitate was dried in vacuo to give 0.50 g of compound of formula 5 with a purity of 70.45. Example 19: Preparation of crude romidepsin

0.50 g of the compound of the formula 5 in Example 18 was added with DMF solvent, 0.38 g of HOBt and 1.56 g of PyBOP, and 0.3 ml of DIPEA was added dropwise to the reaction flask for 4 hours to obtain crude romidepsin having a purity of 65.83%. Example 20: Purification of crude romidepsin to prepare romidepsin acetate

The crude romidepsin water of Example 19 was diluted 10 times, and the target peak fraction was collected by using a RP-HPLC system, a wavelength of 230 nm, a 50×250 mm reverse phase C18 column, and a 0.2% TFA/acetonitrile mobile phase. A fine romidepsin having a purity greater than 98.5% is obtained. The fine romidepsin solution was prepared by RP-HPLC system, the column was 50×250 mm reverse phase C18 column, 0.2% acetic acid solution/acetonitrile mobile phase was transferred to salt, the peak fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain romidepsin. The acetate sperm peptide was 0.16 g, the HPLC purity was 98.5%, and the total yield was 30.0%. Example 21: Preparation of crude romidepsin

0.50 g of the compound of the formula 5 in Example 18 was added to 0.38 g of HOBt and 1.56 g of PyBOP, and 0.3 ml of DIPEA was added dropwise to the reaction flask for 4 hours to obtain crude romidepsin having a purity of 67.53%. Example 22: Purification of crude romidepsin to prepare romidepsin acetate

The crude romidepsin water of Example 21 was diluted 10 times, and the target peak fraction was collected by using an RP-HPLC system, a wavelength of 230 nm, a 50×250 mm reverse phase C18 column, and a 0.2% TFA/acetonitrile mobile phase purification. A fine romidepsin having a purity greater than 98.5% is obtained. The ruthenium rhodamine solution was prepared by RP-HPLC system, the color column was 50×250 mm reverse phase C18 column, 0.2% acetic acid dissolved acetonitrile mobile phase was transferred to salt, the peak fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain romided ground. The octyl acetate sperm peptide was 0.18 g, the HPLC purity was 98.5%, and the total yield was 33.7%. The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

Claims

Rights request

1. A method for preparing romidepsin, which is characterized in that it includes the following steps: Step 1. Under the action of an activator, the resin and 3-hydroxy-7-(R)mercapto-4-heptenic acid are Carboxyl coupling gives compound of formula 1;

Step 2. Fmoc-L-Val-OH is coupled with the hydroxyl group on the compound of formula 1. After removing the Fmoc protecting group, Fmoc-L-Thr-OH, Fmoc-D-Cys(R)-OH, Fmoc- D-Val-OH performs polypeptide chain extension coupling to obtain compound of formula 2;

Step 3. Remove the Fmoc protecting group and the hydroxyl group on the side chain of the L-Thr residue from the compound of formula 2 to obtain the compound of formula 3;

Step 4. The compound of Formula 3 is cyclized to form a disulfide bond through the oxidation breaking method to obtain the compound of Formula 4; Step 5. The compound of Formula 4 is cracked and released from the resin to obtain the compound of Formula 5;

Compound of formula 1

Compound of formula 2

Formula 3 compound

Compound of formula 4 Compound of formula 5

Wherein, the R is a thiol protecting group, the Fmoc is an amino acid N-terminal protecting group, and the Resin is a resin.

2. The preparation method according to claim 1, characterized in that the thiol protecting group is triphenylmethyl or acetamidomethyl.

3. The preparation method according to claim 1, characterized in that the resin is CTC Resin or Wang Resin.

4. The preparation method according to claim 1, characterized in that step 1 is specifically: 3-hydroxy-7-(R)mercapto-4-heptenoic acid is dissolved and activated by adding DIPEA, and then washed and swollen. The resin is coupled to obtain the compound of formula 1.

5. The preparation method according to claim 1, characterized in that step 2 is specifically:

After Fmoc-L-Val-OH is dissolved, add the coupling agent and DMAP, and then couple it with the compound of formula 1. Use DBLK to remove the Fmoc protecting group. Then repeat the above steps to add the coupling agent and DMAP, add amino acids, and remove the Fmoc protecting group. Steps to complete Fmoc-L-Thr-OH, Fmoc-L-Thr-OH, The compound of formula 2 is obtained by coupling the polypeptide chain extension of Fmoc-D-Cys(R)-OH and Fmoc-D-Val-OH.

6. The preparation method according to claim 1, characterized in that step 3 is specifically: adding triethylamine and dissolved DMAP to the compound of formula 2 for activation, then adding methylsulfonyl chloride dropwise to react for 1-5 hours, and then Then add dissolved DABCO and react for 1-5 hours to remove the entire hydroxyl group. Finally, use DBLK to remove the Fmoc protecting group to obtain the compound of formula 3.

7. The preparation method according to claim 1, wherein step 4 is specifically: after iodine is dissolved, the compound of formula 3 is added to react for 1-4 hours, and then the compound of formula 4 is obtained after washing, shrinking the resin, and drying.

8. The preparation method according to claim 1, wherein step 5 is specifically: adding a cleavage reagent to the compound of formula 4 and reacting for 2 hours, filtering, and precipitating the filtrate with diethyl ether, and collecting the precipitate to obtain the compound of formula 5. The cleavage The reagent is a mixed lysate with a volume ratio of TFA:H ₂ 0 of 95:5, a mixed lysate with a volume ratio of TFA:EDT:PHOH:H ₂ 0 of 95:5:3:2, or a volume ratio of TFA:EDT:TIS: PHOH:H ₂ 0 is a mixed lysis solution of 80:5:5:5:5.

9. The preparation method according to claim 1, characterized in that step 6 is specifically: after the compound of formula 5 is dissolved, a coupling agent is added and DIPEA is reacted for 2-5 hours to obtain romidepsin.

10. The preparation method according to claim 5 or 9, characterized in that the coupling agent is a HOBT/DIC dual-system coupling agent, a PyBOP/HOBt dual-system coupling agent or a TBTU/HOBt dual-system coupling agent.

11. The preparation method according to claim 1, characterized in that, after step 6, it also includes the step of purifying romidepsin and preparing romidepsin acetate, specifically:

Dilute the romidepsin obtained in step 6 with water, and then purify it through the RP-HPLC system to obtain pure romidepsin. Then use acetonitrile-acetic acid solution as the mobile phase and continue to use the RP-HPLC system to transfer salt, and collect the target peak fraction for concentration. Freeze-dried is romidepsin acetate.