WO2020125045A1

WO2020125045A1 - Method for synthesizing romidepsin

Info

Publication number: WO2020125045A1
Application number: PCT/CN2019/101597
Authority: WO
Inventors: 陈学明; 林欣媛; 宓鹏程; 陶安进; 袁建成
Original assignee: 深圳翰宇药业股份有限公司
Priority date: 2018-12-19
Filing date: 2019-08-20
Publication date: 2020-06-25
Also published as: CN111333697B; CN111333697A

Abstract

Provided is a method for synthesizing romidepsin, comprising the following steps: 1) coupling a solid phase synthetic resin with Fmoc-L-Val-OH under the action of an activator to obtain an intermediate I; 2) according to an Fmoc solid phase synthesis strategy, sequentially using Fmoc-L-Thr-OH, Fmoc-D-Cys(R)-OH, Fmoc-D-Val-OH and (R)-3-tert-butoxy-7-mercapto-4-heptenoic acid in a one-by-one manner to perform polypeptide chain extension coupling to obtain an intermediate V; 3) removing the hydroxyl group on the L-Thr residue side chain of the intermediate V to form a double bond, so as to obtain an intermediate VI; 4) cracking the intermediate VI and removing the resin and side chain protecting groups, so as to obtain an intermediate VII; 5) oxidizing the intermediate VII to form an intramolecular disulfide bond, so as to obtain an intermediate VIII; and 6) subjecting the intermediate VIII to an intramolecular esterification reaction to obtain romidepsin.

Description

Romidepsin synthesis method

Technical field

The invention relates to the field of solid-phase synthesis of medicinal chemistry, in particular to a method for synthesizing romidepsin.

Background technique

Romidepsin, whose English name is Romidepsin, and its chemical name is (1S, 4S, 7Z, 10S, 16E, 21R)-7-ethylidene-4,21-diisopropyl-2-oxa-12, 13-disulfide-5,8,20,23-tetraazabicyclo[8,7,6]cosadec-16-ene-3,6,9,19,22-pentanone with molecular formula C ₂₄ H ₃₆ N ₄ O ₆ S ₂ is a bicyclic tetrapeptide with a stable hydrophobic structure. The unique disulfide bond in its structure is a key group for its activity. In 2009, Romidepsin was approved by the US Food and Drug Administration (FDA) for the treatment of cutaneous T-cell lymphoma (CTCL). Its chemical structure is as follows:

Romidepsin is a histone deacetylase (HDACs) inhibitor, enters the cytoplasm through the tumor cell membrane, and the intracellular disulfide bond is reduced by glutathione to a sulfhydryl group, which is combined with zinc in zinc-dependent HDACs. Play the role of inhibiting HDACs, thereby further inducing tumor cell differentiation and apoptosis.

Currently, there are two main methods for preparing romidepsin, one is biological fermentation and the other is preparation by chemical synthesis. There are several reports on chemical synthesis. For example, in 1996, Knhn et al. synthesized romidepsin (J. AM. Chem. Soc., 118: 7237-7238) in a full liquid phase. The method in this document uses valine Methyl ester is used as the raw material, coupled with the corresponding amino acid, then the methyl ester is removed by lithium hydroxide, and then the ester is formed under the conditions of DEAD and PPh ₃ , and finally the disulfide bond is synthesized by iodine oxidation, after a total of 14 steps to synthesize romidi Xin. However, this method is cumbersome and not simple enough. More importantly, the total liquid yield is only about 18%. These problems have always been the main factors affecting the production efficiency of romidepsin.

In the patent CN103897029A, a preparation method based on solid-phase synthesis of romidepsin is disclosed: first, the resin is coupled to the carboxyl group on 3-hydroxy-7-mercapto-4-heptenoic acid, and then romidepsin The upper 4 amino acids are coupled in sequence, and then the hydroxyl group is removed, cyclized into a disulfide bond, and an amide bond to form romidepsin.

The synthetic route is as follows:

Summary of the invention

The synthetic route of the present invention is shown below

The invention includes the following steps:

1) Under the action of an activator, the solid phase synthetic resin is coupled with Fmoc-L-Val-OH to obtain intermediate I;

2) In accordance with the Fmoc solid-phase synthesis strategy, sequentially Fmoc-L-Thr-OH, Fmoc-D-Cys(R)-OH, Fmoc-D-Val-OH, (R)-3-tert-butoxy- 7-mercapto-4-heptenoic acid undergoes polypeptide chain extension coupling to obtain intermediate V;

3) Intermediate V removes the hydroxyl group on the side chain of L-Thr residue to form a double bond to obtain intermediate VI;

4) Intermediate VI is cleaved to remove the resin and side chain protecting group to obtain intermediate VII;

5) Intermediate VII forms an intramolecular disulfide bond by oxidation to obtain intermediate VIII;

6) Intermediate VIII undergoes intramolecular esterification to obtain romidepsin.

The protective groups mentioned in the present invention are protective groups commonly used in the field of amino acid synthesis to protect the amino acid main chain and side chains such as amino groups, carboxyl groups, mercapto groups and other groups that interfere with synthesis. Reactions occur during the process to form impurities. For the amino acids that need to protect the side chains in the present invention, those skilled in the art are well aware of their side chain structure and know that common protecting groups are used to protect amino, carboxyl, mercapto and other groups on the amino acid side chains Group, wherein R is a thiol protecting group, and Fmoc is an N-terminal protecting group of amino acids. Fmoc-L-Val-OH refers to the Fmoc protective group L-Val coupled to the N-terminus, Fmoc-L-Thr-OH refers to the L-Thr coupled Fmoc protective group at the N-terminus, Fmoc-D-Cys (R)-OH refers to the D-Cys coupled with the Fmoc protecting group at the N-terminus and the R protecting group at the side chain mercapto group, and Fmoc-D-Val-OH refers to the Fmoc protecting group coupled to the N-terminus D-Val, the abbreviation above is a common form in the field.

Preferably, the mercapto protecting group is trityl, 4-methoxytrityl or benzyl, more preferably trityl.

Preferably, the solid phase synthetic resin is CTC Resin (CTC resin) or Wang Resin (king resin), more preferably CTC Resin, and most preferably CTC Resin with a substitution degree of 0.5 mmol/g.

In step 1) of the preparation method of the present invention, the activator is a combination of one or more of DIPEA, diethylamine, triethylamine, morpholine, and N-methylmorpholine, preferably DIPEA.

In step 1) of the preparation method of the present invention, preferably, the molar ratio of the resin, the activator and Fmoc-L-Val-OH is 1:2-10:10-60, preferably 1:6:30.

As a preferred solution, step 1) is:

After Fmoc-L-Val-OH is dissolved, DIPEA is added to activate, and then coupled with the washed and swollen resin to obtain intermediate I. Among them, the solvent used to dissolve, wash and swell 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 resin, the remaining amino acids and 3-tert-butoxy-7-(R)- Mercapto-4-heptenoic acid undergoes a condensation reaction (condensation reaction of main chain amino group and carboxyl group) with the previous coupled amino acid one by one according to the order of its connection in the romidepsin structure. In the polypeptide chain extension coupling, since each amino acid has a protecting group at the N-terminus, it is necessary to remove the N-terminus protecting group before coupling, which is common knowledge for those skilled in the art, and DBLK is preferred for the present invention N-terminal protecting group.

In the technical solution of the present invention, step 2) is:

Remove the Fmoc protecting group from intermediate I, then couple Fmoc-L-Thr-OH, then repeat the steps of removing the Fmoc protecting group and coupling, and then complete Fmoc-D-Cys(R)-OH, Fmoc- The coupling of D-Thr-OH, 3-tert-butoxy-7-(R)-mercapto-4-heptenoic acid yields intermediate V.

Preferably, step 2) removes the Fmoc protecting group with DBLK for intermediate I, and then adds Fmoc-L-Thr-OH, coupling agent and DMAP for coupling. Then repeat the above operation to complete the coupling of Fmoc-D-Cys(R)-OH, Fmoc-D-Thr-OH, 3-tert-butoxy-7-(R)-mercapto-4-heptenoic acid one by one Intermediate V is obtained.

Among them, 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 proportion of each component in the art is certain and well-known in the art, and will not be repeated here.

The solvent used for dissolution in step 2) is preferably one or two of DMF, DCM, NMP, and DMSO, and more preferably a mixed solvent with a volume ratio of DMF:NMP of 1:1.

The amount of the coupling agent used in step 2) is, on a molar basis, the coupling agent: the amount of solid phase synthetic resin used in step 1) is 2-4:1, preferably 3:1,

The amount of DMAP used in step 2) is on a molar basis. The amount of DMAP: the amount of solid phase synthetic resin used in step 1) is 0.1-0.5:1, preferably 0.2:1.

Step 3) of the preparation method of the present invention is:

Piperidine and dissolved DMAP were added to intermediate V for activation, then p-toluenesulfonyl chloride was added to react to completion, and then dissolved DABCO was added to react to remove hydroxyl groups to form double bonds to obtain intermediate VI.

In step 3), the reaction time is 1-5 hours, preferably 2 hours; the reaction temperature is preferably -5°C to 100°C, more preferably 30°C.

In step 3), the solvent used for dissolution is preferably DMF, DCM or THF.

In step 3), the amount of piperidine used is on a molar basis. The amount of piperidine: solid phase synthetic resin used in step 1) is 1-3:1, preferably 2:1.

In step 3), the dosage of the p-toluenesulfonyl chloride is on a molar basis, and the dosage ratio of the solid-phase synthetic resin in the step 1) is 1-2:1, preferably 1.5:1.

In step 3), the amount of DMAP used is on a molar basis, and the ratio of the amount of solid phase synthetic resin used in DMAP: step 1) is 0.1-1:1, preferably 0.2:1.

In step 3), the amount of DABCO used is on a molar basis. The solid phase synthetic resin in DABCO: step 1) is 1-5:10, preferably 2:10.

In step 4) of the preparation method of the present invention,

A cleavage reagent was added to Intermediate VI for reaction for 2 hours, filtered, and the filtrate was precipitated with diethyl ether. The precipitated peptide was collected and purified by reverse phase high-phase liquid phase to obtain Intermediate VII.

In step 4), the aqueous solution of the lysis reagent TFA is preferably a mixed lysis solution with a volume ratio of TFA:H ₂ O of 95:5 and a volume ratio of TFA:EDT:PhOH:H ₂ O of 95:5:3: 2. Mixed lysate or volume ratio TFA:EDT:TIS:PhOH:H ₂ O is 80:5:5:5:5.

In step 5) of the preparation method of the present invention, after the intermediate VII is dissolved, an oxidizing agent is added to react to completion to obtain the intermediate VIII.

In step 5), the reaction time is 1-10 hours, preferably 5 hours.

In step 5), the reaction temperature is preferably -5°C to 100°C, more preferably 35°C.

In step 5), the oxidant is hydrogen peroxide, horseradish peroxidase, DMSO, ferric chloride hexahydrate, air, ammonium persulfate, preferably ferric chloride hexahydrate.

In step 5), the solvents used are methanol, ethanol, acetone, tetrahydrofuran, acetonitrile, and mixed solutions of the above solvents and water in different ratios; preferably, a mixed solution of acetonitrile and water.

In step 6), after the intermediate VIII is dissolved, a coupling agent is added, the reaction is performed for 1-10 hours, and romidepsin is prepared by purification.

In step 6), the reaction time is preferably 3 hours; the reaction temperature is preferably -5°C to 100°C, more preferably 35°C.

In step 6), the coupling agent is preferably DIAD, PPh ₃ and TsOH.

In step 6), the solvent is methanol, ethanol, acetone, tetrahydrofuran, acetonitrile; preferably tetrahydrofuran.

In step 6), the purification is purification by reverse-phase high-pressure liquid chromatography. Preferably, the reverse-phase high-pressure liquid chromatography includes: taking reverse-phase octadecylsilane as a stationary phase, and 0.2% aqueous acetic acid solution /Acetonitrile is the mobile phase, and the ratio of the mobile phase is preferably 98:2 to 50:50, more preferably 80:20 to 60:40, and most preferably 70:30. The desired peak fractions were collected, concentrated and lyophilized.

The invention discloses a method for preparing romidepsin. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve. In particular, it should be noted that all similar substitutions and modifications will be obvious to those skilled in the art, and they are all considered to be included in the present invention. The method of the present invention has been described through preferred embodiments, and it is obvious that relevant persons can modify or appropriately modify and combine the compounds and preparation methods described herein without departing from the content, spirit, and scope of the present invention. Apply the technology of the present invention.

In the specific embodiment of the present invention, all amino acids coupled with a protecting group are commercially available, and the protected amino acids in the present invention are purchased from Shanghai Jier Biochemical Co., Ltd. The CTC Resin (CTC resin) and Wang Resin (Wang Resin) was purchased from Tianjin Nankai Hecheng Co., Ltd.

Abbreviations and English meaning

Beneficial effect

The method of the invention has simple steps and high synthesis efficiency, and can increase the total yield of romidepsin to about 40%.

detailed description

Example 1 Preparation of Intermediate I

Weigh CTC Resin 2g (synthesis scale 1mmol) with a substitution degree of 0.5mmol/g, add it to the solid phase reaction column, wash it twice with DMF, and after swelling the resin with DMF for 30 minutes, weigh 1.01g Fmoc-Val-OH , 0.38g HOBt, 0.03g DMAP was dissolved in DMF, added 0.6mL DIPEA under ice water bath to activate, added to the above reaction column equipped with resin, the reaction was completed after 2 hours of reaction, washed 6 times with DMF to obtain intermediate I.

Example 2 Preparation of Intermediate V

In the solid-phase reaction column, the intermediate I was removed with 20% DBLK to remove the Fmoc protecting group, and then washed with DMF 6 times. A small amount of resin was taken for the ninhydrin test, and the resin developed color. Weigh 1.02g Fmoc-Thr-OH, 0.38g HOBt, 0.03g DMAP in a mixed solution of DMF and NMP with a volume ratio of 1:1, add 0.3mL DIC under ice water bath to activate, add it to a solid phase reaction column, and react at room temperature 2h (The end point of the reaction is determined by the ninhydrin method. If the resin is colorless and transparent, the reaction is complete and the resin is colored, indicating that the reaction is incomplete, and the coupling reaction needs to be re-coupled for 1h). Repeat the above steps to remove Fmoc protection and add corresponding amino acid coupling to complete Fmoc-D-Cys(Trt)-OH, Fmoc-D-Val-OH and (R)-3-tert-butoxy-7-mercapto -4-heptenoic acid gives intermediate V.

Example 3 Preparation of Intermediate VI

After weighing 0.03 g of DMAP and 0.20 g of p-toluenesulfonyl chloride in 10 mL of dichloromethane, 0.60 ml of piperidine was added. Then, the mixed solution was added to a solid-phase reaction column containing intermediate V, and the reaction was carried out for 2 hours. After the reaction was completed, it was washed 6 times with DMF, shrinked 3 times with methanol, and dried in vacuo to obtain 3.0 g of intermediate VI.

Example 4 Preparation of intermediate VII

3.0g of the intermediate VI resin obtained in Example 3 was added to a 50ml flask, a lysis reagent (volume ratio, TFA:EDT:PhOH:H ₂ O=95:5:3:2) was placed, and 30mL of the lysis reagent was poured Put into a flask and react at room temperature for 2 hours. At the end of the reaction, the resin was filtered and the filtrate was collected. It was added dropwise to 300 ml of ether reagent, centrifuged, washed with anhydrous ether and dried in vacuum to obtain 0.70 g of intermediate VII with a purity of 80.52%.

Example 5 Preparation of intermediate VIII

0.70 g of intermediate VII obtained in Example 4 was dissolved in 5 mL of a 70% acetonitrile aqueous solution, 0.20 g of ferric chloride hexahydrate was added, and the mixture was reacted at 35°C for 5 hours. After the reaction was completed, 10 mL of saturated ammonium chloride solution was added, and then extracted three times with dichloromethane, washed successively with saturated brine and water, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 0.60 g of intermediate VIII, with a purity of 75.5%.

Example 6 Preparation of romidepsin

In a 1000 mL reaction bottle, 3.8 g of triphenylphosphine, 0.6 g of p-toluenesulfonic acid, 2.2 mL of diisopropyl azodicarboxylate and 500 mL of tetrahydrofuran were added in sequence. The mixed solution was stirred at room temperature for 30 minutes, then cooled to 0°C, and a solution of 0.60 g of the intermediate VIII prepared in Example 5 in tetrahydrofuran solution 100 mL was added dropwise, and the drop was completed in two hours. After reaction at 0°C for three hours, it was concentrated to give an oil. This oil was diluted 10 times with water using RP-HPLC system, wavelength 230nm, chromatographic column was 50×250mm reverse phase C18 column, 0.2% acetic acid aqueous solution/acetonitrile solution mobile phase was purified, the target peak fraction was collected, concentrated by rotary evaporation, and lyophilized to obtain Romidepsin acetate refined peptide 0.25g, HPLC purity 98.5%, total yield 40.5%.

The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles of the present invention, several improvements and retouches can be made. These improvements and retouches also It should be regarded as the protection scope of the present invention.

Claims

A method for synthesizing romidepsin includes the following steps:

1) Under the action of an activator, the solid phase synthetic resin is coupled with Fmoc-L-Val-OH to obtain intermediate I;

2) In accordance with the Fmoc solid-phase synthesis strategy, sequentially Fmoc-L-Thr-OH, Fmoc-D-Cys(R)-OH, Fmoc-D-Val-OH, (R)-3-tert-butoxy- 7--Mercapto-4-heptenoic acid undergoes polypeptide chain extension coupling to obtain intermediate V;

3) Intermediate V removes the hydroxyl group on the side chain of L-Thr residue to form a double bond to obtain intermediate VI;

4) Intermediate VI is cleaved to remove the resin and side chain protecting group to obtain intermediate VII;

5) Intermediate VII forms an intramolecular disulfide bond by oxidation to obtain intermediate VIII;

6) Intermediate VIII undergoes intramolecular esterification to obtain romidepsin;

Wherein, said R is a mercapto protecting group; preferably trityl, 4-methoxytrityl or benzhydryl, more preferably trityl.
The synthesis method according to claim 1, in step 1), the activator is a combination of one or more of DIPEA, diethylamine, triethylamine, morpholine and N-methylmorpholine, preferably DIPEA.
The synthesis method according to claim 1, step 2) is:

Remove the Fmoc protecting group from intermediate I, then couple Fmoc-L-Thr-OH, then repeat the steps of removing the Fmoc protecting group and coupling, and then complete Fmoc-D-Cys(R)-OH, Fmoc- The coupling of D-Thr-OH, (R)-3-tert-butoxy-7-mercapto-4-heptenoic acid gives intermediate V.
The synthesis method according to claim 1, 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, most preferably PyBOP/HOBt Double system coupling agent.
According to the synthesis method of claim 1, step 3) is: adding piperidine and dissolved DMAP to intermediate V to activate, then adding p-toluenesulfonyl chloride to complete reaction, and then adding dissolved DABCO to remove The hydroxyl group forms a double bond to give intermediate VI.
The synthesis method according to claim 1, step 4) is to add a cleavage reagent to the intermediate VI and react for 2 hours, filtering, the filtrate is precipitated with ether, and the crude peptide is collected by precipitation and purified by reverse phase high-phase liquid phase to obtain the intermediate VII;

Preferably, the lysis reagent is an aqueous solution of TFA, more preferably a mixed lysis solution with a volume ratio of TFA:H 2 O of 95:5, and a volume ratio of TFA:EDT:PhOH:H 2 O of 95:5:3:2 The mixed lysate or volume ratio TFA:EDT:TIS:PhOH:H 2 O is 80:5:5:5:5.
The synthesis method according to claim 1, step 5) after dissolving intermediate VII, adding an oxidizing agent to complete reaction to obtain intermediate VIII;

Preferably, the oxidizing agent is hydrogen peroxide, horseradish peroxidase, DMSO, ferric chloride hexahydrate, air, ammonium persulfate, more preferably ferric chloride hexahydrate.
According to the synthesis method of claim 1, in step 6), after the intermediate VIII is dissolved, a coupling agent is added, the reaction is performed for 1-10 hours, and romidepsin is purified and prepared,

Preferably, the coupling agent is DIAD, PPh 3 and TsOH.
The synthesis method according to claim 1, in step 6), the purification is purification by reverse-phase high-pressure liquid chromatography,

Preferably, the reversed-phase high-pressure liquid chromatography includes: reversed-phase octadecylsilane as the stationary phase, and 0.2% aqueous acetic acid/acetonitrile as the mobile phase, and the ratio of the mobile phase is preferably 98:2 to 50:50 ,

More preferably 80:20 to 60:40, most preferably 70:30.