WO2016084100A2

WO2016084100A2 - Novel and efficient method for large scale synthesis of romidepsin

Info

Publication number: WO2016084100A2
Application number: PCT/IN2015/000435
Authority: WO
Inventors: Lakshmi Prasad ALAPARTHI; Ganga Ramu VASANTHAKUMAR; Talluri Bhushaiah CHOWDARY; Anand Vijaykumar MANTRI; Gaurav Kulkarni
Original assignee: Alaparthi Lakshmi Prasad
Priority date: 2014-11-26
Filing date: 2015-11-26
Publication date: 2016-06-02
Also published as: WO2016084100A3

Abstract

The present invention relates to intermediate compounds which are used as key intermediates in the synthesis of Romidepsin and process of preparation of these intermediates. The present invention also provides processes for synthesis of Romidepsin in high purity and good yields from said intermediate compounds.

Description

Novel and efficient method for large scale synthesis of Romidepsin Field of invention

Romidepsin or FK228 or FR901228, is a depsipeptide, approved by US FDA in November 2009 for the treatment of cutaneous T-cell lymphoma and other different types of cancers. Romidepsin is an exclusive natural product and was conventionally revealed from cultures of chromobacterium violaceum, a gram- negative bacterium isolated from soil sample. Because of its present synthetic challenges, and even fermentation process resulting in poor yields, development of commercial scale synthetic process is prerequisite. The present invention relates to novel intermediates and completely synthetic process to prepare Romidepsin in high purity and good yields.

Background of invention

Romidepsin (IUPAC name ( l S,4S,7Z, 10S,16E,21R)-7-Ethylidene -4,21 - diisopropyl-2-oxa-12,l 3-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene- 3,6,9,19,22-pentone and CAS Registry Number: 128517-07-7 ), represented by following formula, is a natural product obtained from the bacteria Chromobacterium violaceum, and works by blocking enzymes known as histone deacetylases an

Romidepsin was approved for cutaneous T-cell lymphoma under FDA's fast track development program. FDA Fast Track Designation is a designation of the United States Food and Drug Administration (FDA) that facilitates the development, and expedites the review, of drugs which treat a serious or life-threatening condition and fill an unmet medical need. Thus, Romidepsin forms one of the important therapeutic agents.

Different routes of preparation of Romidepsin are known in the art.

As described in US4977138 Romidepsin or FR901228 is prepared by fermentation of the strain WB968 (identified as Chromobacterium violaceum). The strain used in fermentation process is isolated from soil obtained from Yamagata-Ken, Japan.

US20090186382 (US8691534) disclose a method of preparing romidepsin, the method comprising the step of isolating Romidepsin from a fermentation broth, and the step of purifying Romidepsin to provide greater than 98% monomeric Romidepsin. The purifying step is performed at an apparent pH 4.0 to 6.0 to circumvent the reduction of the disulfide bridge and there by formation of dimerized, oligomerized, or polymerized adducts.

US7608280 discloses type B crystalline form of compound FR901228 and its compositions thereof.

US 761 1724 describes type A crystalline form of compound FR901228 and compositions thereof.

Analogues of Romidepsin include dimmers, reduced forms, racemates, enantiomers region-isomers, salts and their biological properties comprising immune mediated responses are described in WO2007061939 and CA2630216. CN 102241736 describes solution phase chemical synthesis of Romidepsin by employing both Boc as well as Fmoc-chemistry. Synthesized and characterized spectroscopically the intermediates of di, tri, tetra peptides and followed by coupling with heptenoic acid and finally cyclization of linear peptide and oxidation resulted the requisite API. Synthesis, isolation, and purification of many intermediates are tedious and which is the drawback of solution synthesis.

WO2014101828 discloses process for producing Romidepsin involving solid phase synthesis. However, when present inventors repeated this process, cyclization reaction could not take place probably due to steric hindrance.

Another methodology is a solution phase synthesis by employing fragment condensation approach, as described in J. Org. Chem. 2008, 73, 9353-9361. In this method, the requisite peptide has been synthesized by reacting a peptide fragment represented by the general formula Fmoc-D-Val-D -Cys(Trt)-Dhb-OH with another fragment represented by the following formula Fmoc-Val-OX (where X= 7-mercapto-[S-trityl]-4-heptenoic acid trimethylsilyl ester) by deprotection of Fmoc- and TMSE protecting groups followed by amide bond formation through lactamization between D-Val and heptenoic acid. It is widely observed that the removal of by product (dibenzofulvene . adduct) after deprotection of Fmoc-group is difficult.

The publication, J. Med. Chem. 2008, 51, 6639-6641 by Jung-Mo et al. describes solid phase approach for the synthesis of Romidepsin analogues. In this publication aminomethylpolystyrene resin with backbone amide linker has been employed for the synthesis and synthetically challenging heptenoic acid has been replaced with aspartylcysteamine there by result in Romidepsin analogues. Another variation in fragment coupling was disclosed in Chem. Eur. J. 2009, 42, 1 1 174-1 1 186. The publication describes the total synthesis of Spiruchostatins A and B, 5"-epi-Spiruchostatin B, and Romidepsin by employing solution phase chemistry by adopting Boc-protecting strategy. In this, Romidepsin has been synthesized by coupling fragments of the general formula H-D-Cys(Trf)-Dhb-Val- OMe and PMB-protected heptenoic acid D-valine conjugate through macrolactonization. Our efforts to cycilze the linear peptide through lactonization producing limited success.

Object of the invention

The first object of the present invention is to provide novel synthetic intermediates for preparation of Romidepsin.

Second object of the invention is to provide novel process for synthesis of the novel intermediates.

Third object is to provide novel process for the synthesis of Romidepsin. The process is completely synthetic and hence there is no dependence on any bacterial strain. The process can be used in any laboratory or manufacturing facility not competent to handle bacterial cultures.

Fourth object is to provide a completely novel process for the preparation of Romidepsin using sequentially following processes

i) solid phase synthesis

ii) release of intermediates from solid phase by hydrolysis

iii) lactamization

iv) intramolecular disulfide bond formation by oxidation. Yet another object is to provide a completely novel process for the preparation of Romidepsin using sequentially following processes

i) solid phase synthesis up to and involving intramolecular disulfide bridge / bond formation by oxidation

ii) release of intermediates from solid phase by hydrolysis

iii) lactamization

Summary of the Invention

Solid phase synthesis is a common technique for peptide synthesis. This technique allows an amino-protected amino acid to bind to a solid phase material forming a covalent bond between the carbonyl group of amino acid and the resin.

Lactamization process is process of preparing a cyclic amide by the elimination of a molecule of water from the amine and carboxylic acid functionality.

Disulfide bond or disulfide bridge formation is essentially an oxidation reaction between two thiols. Intramolecular Disulfide bond or disulfide bridge formation is the Disulfide bond formation between two thiols of the same peptide.

The present inventors have invented two different processes incorporating solid phase synthesis of peptides, lactamization and intramolecular disulfide bridge formation to synthesize Romidepsin. These two processes also involve synthesis of two intermediates namely a compound of formula I, in particular Intermediate I and Intermediate II. The present process to prepare Romidepsin using these intermediates is feasible at large scale.

In the first aspect, the invention provides a compound of formula I

Formula I

wherein R is selected from the group consisting of alkyl, aryl, substituted aryl, benzyl, substituted benzyl, acetamidomethyl, trityl and tert-butylthio.

In a second aspect the invention provides novel process to prepare Formula I compounds in a series of steps starting from Fmoc protected valine amino acid.

Fmoc protected valine Formula I

In yet another aspect, the invention provides Intermediate II, a disulfide bridged compound.

Intermediate II

In one more aspect, the invention provides novel process to prepare intermediate II in a series of steps starting from Fmoc protected valine amino acid, resulting through disulfide bridge formation while the peptide is loaded on resin and subsequent release of peptide.

Intermediate II

In another aspect the invention provides process of preparaion of Romidep from Formula I compound as follows:

Formula I Romidepsin In yet another aspect the invention provides process of preparaion of Romidep from intermediate II .

Intermediate II Romidepsin

Brief Description of the figures:

Sheet 1 of Sheet 8- HPLC profile of intermediate I

Sheet 2 of Sheet 8- NMR spectra of intermediate I

Sheet 3 of Sheet 8 - Mass spectra of intermediate I

Sheet 4 of Sheet 8- HPLC profile of Romidepsin API

Sheet 5 of Sheet 8 - NMR Spectra of Romidepsin API

Sheet 6 of Sheet 8 - MS spectra of Romidepsin API Sheet 7 of Sheet 8 - Mass spectra of Intermediate I A compound

Sheet 8 of Sheet 8 - HPLC profile of intermediate I which is stable up to 12 months. Detailed Description of the Invention

The details of the invention are as set forth.

All the technical and scientific terms used herein above have the meaning as commonly understood by a person skilled in the art to which the invention belongs, unless otherwise defined in this specification.

Following abbreviations are used and should be construed accordingly.

According to the first aspect, the present invention provides novel intermediate compound, in particular, Formula I compounds as follows:

wherein R is selected from the group consisting of alkyl, aryl, substituted aryl, benzyl, substituted benzyl, acetamidomethyl, trityl and tert-butylthio. These intermediate are synthetically produced and processed further to produce Romidepsin in a complete synthetic pathway.

In particular, preferably, R is acetamidomethyl or trityl.

Accordingly, the present invention provides novel key intermediate, in particular, Intermediate I compound as follows where R is Trityl. The process to prepare Romidepsin using intermediate I provides alternative synthesis to prepare Romidepsin by fermentation of Chromobacterium violaceum.

This intermediate I is easily produced in any laboratory from lab scale to manufacturing scale, and is a stable intermediate that can be stored for several months before further use. The intermediate I is produced in good yields and is having a purity of > 90%, preferably > 95% and most preferably > 99%.

The intermediate I is characterized as follows:

Physical characteristics

1. Appearance: White amorphous powder

2. Melting point: 95-98 °C

Spectroscopic characteristics:

Intermediate I is characterized by studying Ή-NMR spectra, mass spectra, and RP-HPLC profile; the details of which are provided in figures 1-3 from sheets 1 -3 of sheets 6. ¹ H-NMR spectra: The Ή NMR was recorded on Varian Mercury 300 MHz Spectrometer using CDC1₃ as solvent. The details of Ή NMR spectra are provided in a tabular form as follows:

Preparative (RP)-HPLC profile

Purity was confirmed by Shimadzu RP-HPLC

The chromatographic parameters used for the study is as follows:

Flow rate : 1 mL/minute

Detection : UV detector 210 nm Injection Volume : 10 μΐ,

Column Oven Temperature : Ambient

Run time : 20 min.

Purity of the compound of Intermediate I is > 99.67%. The gradient is provided under table 2.

Table 2: Gradient Program*

*A is mobile phase containing 0.01% trifluroacetic acid in Milli Q water and B is 0.01 % trifluroacetic acid in acetonitrile.

The novel Intermediate I elutes at the retention time of 1 1.553 min.

This intermediate I is stored at for 12 months and tested for the stability. The stability of Intermediate I at -20°C for 12 months is as recorded in sheet 8. It is observed that the intermediate I is stable up to 12 months and can be used for synthesis of Romidepsin. In second aspect, the invention relates to process of preparation of novel compounds of formula I particularly intermediate I.

The formula I compounds are prepared by emplyoing solid phase peptide synthesis approach by loading Fmoc-D-Val-OH (Fmoc protected valine) to resin and obtain the formula I compounds in a series of steps such as adding various Fmoc protected amino acids, repeated deprotection, coupling, esterification, and dehydration reactions as per the sequence provided in scheme I.

Further aspects of the invention cover preparation of Formula I compounds, particularly, intermediate I and then preparation of Romidepsin. Accordingly 4 schemes are developed as follows: Scheme Description

Scheme 1 Preparation of Formula I compounds and particularly

Intermediate I by Solid phase synthesis

Scheme 2 Preparation of Romidepsin from Formula I compounds, particularly preparation of Romidepsin from Intermediate I through lactamization and oxidation

Scheme 3 Preparation of Intermediate II by solid phase synthesis

Scheme 4 Preparation of Romidepsin from Intermediate II by lactamization

Accordingly, under second aspect, the present invention provides process to prepare formula I compounds and particularly intermediate I by solid phase synthesis as described in scheme 1. Accordingly the process to prepare formula I compounds comprising the steps of:

a) treating 2-chlorotrityl chloride resin (before use as solid phase in solid phase synthesis) with solvent.

b) loading of Fmoc-D-Val-OH to treated resin.

c) deprotecting Fmoc-group in the loaded resin.

d) coupling of (3S, 4E)-3-mercapto-7-hydroxy-4-heptenoic acid derivative. e) esterifying Fmoc-Val-OH and subsequently deprotecting of Fmoc-group. f) coupling of Fmoc-Thr-OH and subsequently deprotecting of Fmoc-group g) coupling of Fmoc-D-Cys-(R)-OH

h) dehydrating and subsequently deprotecting of Fmoc group

i) acidic cleavage to release Formula I compounds from the solid support.

The process to prepare the Formula I compounds is described in details as below. This process utilizes solid phase synthesis of peptides, using resin as solid support. The resin is selected from 2-chloro trityl resin, wang resin, TentaGel and HypoGel 4-alkyloxybenzyl alcohol resins, Rink acid resins. The preferred resin is 2-chlorotrityl chloride. 2-Chlorotrityl chloride resin is an acid labile resin for peptide synthesis using Fmoc-amino acids. The steric bulk and mild acid conditions for cleavage makes it a resin of choice. Step 1 : Swelling of 2-chlorotrityl chloride resin

The treatment given to 2-chlorotrityl chloride before use is as follows.

2-chlorotrityl chloride resin is washed repeatedly with dry dichlomethane and swelling is carried out by swirling the resin in dichlormethane for one hour and draining the solvent.

Step 2: Loading of Fmoc protected valine on resin and Fmoc deprotection i) The Fmoc protected valine also mentioned herein as Fmoc-D-Val-OH is loaded on resin with the help of organic base such as triethylamine or diisopropylethyl amine or other mild organic bases including collidene, N-methylmorpholine etc. The organic base is used in molar excess, preferably 1.5 - 4 times of Fmoc protected valine. The mixture of amino acid and organic base is loaded to the resin and swirled for three hours in presence of nitrogen atmosphere. The resin is washed with dichlormethane and dimethylformamide successively and drained. ii) Fmoc-deprotection of loaded Fmoc protected amino acid is carried out by using 2-40 % piperidine in DMF NMP (N-methylpyrrolidone) or 2 % DBU (1 ,8- Diazabicyclo[5.4.0]undec-7-ene) in a polar or nonpolar solvent or other amines like cyclohexylamine, diethylamine, triethylamine, dimethylamino pyridine, morpholine, ethanolamine, Tris(2-aminoethyl) amine, 1 ,3-cyclohexanebis- (methylamine) for about 2-60 min. The peptide loaded resin is repeatedly washed with DMF. The peptide is now deprotected. This deprotected peptide is termed as peptide A.

Step 3:

i) Coupling of deprotected peptide, peptide A with (3S, 4E)-3-hydroxy-7- mercapto-4-heptenoic acid derivative:

wherein preferably, R is acetamidomethyl or trityl.

In this process, in a separate flask, solution of (3S, 4E)-3-hydroxy-7-mercapto-4- heptenoic acid derivative, coupling agent, scavenger and a base is prepared in solvent and this solution is charged into peptide vessel containing deprotected peptide and reaction is allowed to proceed. After the reaction, the loaded resin beads are washed. This is a peptide having mercapto heptanoic acid derivative. This peptide is termed as peptide B.

ii) Esterification of peptide B with valine amino acid

This step does not require scavenger. The peptide B having hydroxyl group is esterified using Fmoc protected valine amino acid. In this step solution of Fmoc- Valine-OH, coupling agent, and a base is prepared separately in solvent and added to peptide vessel containing peptide B having mercapto heptanoic acid derivative. The reaction is allowed to proceed to give a peptide which has Fmoc protected valine added to peptide B through formation of an ester. After the deprotection, valine added peptide B is obtained which is termed as peptide C.

iii) Coupling of peptide C with threonine amino acid

Further, peptide C is coupled with Fmoc protected threonine. In this step solution of Fmoc-Threonine, coupling agent, scavenger and a base is prepared separately in solvent and added to peptide vessel containing peptide C. The Fmoc-Threonine is added to peptide C which after deprotection has threonine added to peptide C and is termed as peptide D.

iv) Further, peptide D is coupled with Fmoc protected Fmoc-D-Cys(R)-OH. In this process, solution of Fmoc protected D-Cys(R)-OH, coupling agent, scavenger and a base is prepared separately in solvent and added to peptide vessel containing peptide D. The Fmoc protected D-Cys(R)-OH is added to peptide D which after deprotection has D-Cys(R)-OH added to peptide D and is termed as peptide E. According to one embodiment the coupling of Fmoc-D-Cys(R)-OH with the growing peptide on the resin is carried out in presence of coupling agent, scavenger and a base. The molar ratios are as follows. 1 equivalent of peptide requires 3-4 equivalents of Fmoc-protected amino acid. 3-4 equivalents of coupling agent, 3-4 equivalents of scavenger. Base is used in further excess to that of Fmoc-protected amino acid, coupling agent and scavenger. In particular the ratio is 0.25 - 0.35 M peptide: 1 mole of Fmoc protected amino acid: 1 mole of coupling agent: 1 mole of scavenger: 1.5 - 2 moles of a base. The reaction time is around 0.5 hr - 4 hr, preferably for 1 -2 hr. Repeated washing of peptide loaded resin beads with DMF are given.

Fmoc-D-Cys(R)-OH Fmoc-D-Cys(trityl)-OH

The R group is selected from the group consisting of alkyl, aryl, substituted aryl, benzyl, substituted benzyl, acetamidomethyl, trityl, tert-butylthio. The preferred R group is trityl and therefor the preferred amino acid Fmoc-D-Cys(R)-OH is Fmoc- D-Cys(trityl)-OH as represented in above structure.

Fmoc-deprotection is carried out by using 2-40 % piperidine in DMF/NMP or 2 % DBU in a polar or nonpolar solvent. The reaction time is usually within an hour and preferably around 2-20 min. The synthesized peptide is repeatedly washed with multiple solvents including DMF, dichloromethane ether.

The above steps are carried out sequentially. After each step, Kaiser Test is performed to confirm whether amino acid is protected or deprotected as desired. When deprotection is desired, Kaiser Test should be positive and when protection is desired, it should be negative.

In all coupling reactions, where peptide bond is formed, one equivalent of loaded peptide (resin loaded compound) requires around 1.5 equivalents of sulfhydryl protected (3S, 4E)-3-hydroxy-7-mercapto-4-heptenoic acid, around 3-4 equivalents of Fmoc-protected amino acid, around 3-4 equivalents of coupling agent, around 3-4 equivalents of scavenger. The preferred sulfhydryl protected (3S,4E)-3-hydroxy-7-mercapto-4-heptenoic acid is (3S,4E)-3-Hydroxy-7- [(triphenylmethyl)thio]-4-heptenoic acid. Base is used in further excess to that of Fmoc-protected amino acid, coupling agent and scavenger. In particular the preferred ratio is for 0.25 - 0.35 M Fmoc-deprotected loaded amino acid: 1 mole of Fmoc protected amino acid: 1 mole of coupling agent: 1 mole of scavenger: 1.5 - 2 moles of a base.

The preferred base used is diisopropylethylamine. The preferred coupling agent is HBTU and the preferred scavenger is hydroxybenzotriazole or ethyl(hydroxyimino)cyanoacetate (Oxyma pure). The coupling reaction is carried out for about 1- 4 h. After the coupling, peptide loaded resin is repeatedly washed with solvents selected from DMF or dichloromethane. v) Dehydration and subsequent deprotection of Fmoc-group

Dehydration of threonine part of the synthesized peptide is carried in presence of dehydrating agent. The preferred dehydration agents are carbodiimides such as Ν,Ν'-dicyclohexylcarbodiimide and Ν,Ν'-diisopropylcarbodiimide and most preferred is l -Ethyl-3-(3-dimethylaminopropyl) carbodiimide. The dehydration reaction is carried out in presence of catalytic amount of CuCb. The reaction time is for 2-20 h (essentially 3-4 h). After the completion of reaction, repeated washings are given to the peptide loaded resin with several solvents including dichloromethane, DMF. The dehydration reaction introduces double bond as follows. Fmoc-deprotection was carried out using 2-40% piperidine in DMF NMP or 2 % DBU in a polar or nonpolar solvent for 2-20 min. Repeated washing of the peptide loaded resin beads with DMF.

vi) Mild acidic hydrolysis to release Formula I compounds particularly, Intermediate I. The peptide is released from resin by subjecting it to mild acid solution. The acid solution may consist of 0.1 % to 5.0 % TFA (trifluoro acetic acid) in dichloromethane or chloroform. Finally the peptide so synthesized is isolated by either precipitation or recrystallisation by slow addition of single or mixture of polar or nonpolar solvents. The solvents for precipitation preferably are diethylether, methyl-tert-butyl ether (MTBE), diisopropyl ether or the mixture thereof.

The process of preparing Formula I compounds particularly intermediate I is represented in the scheme 1.

Scheme 1 In yet another aspect the invention provides process to prepare Romidepsin from compounds of formula I, in particular Intermediate I as described in scheme 2. Under this aspect, the process comprises the following steps:

1. Lactamization of formula I compounds, particularly intermediate I to get a cyclic intermediate compound

2. Deprotection of sulfhydril protecting groups and in situ intramolecular disulfide bridge formation to give Romidepsin.

3. When the sulfhydryl protecting group is trityl as in case of Intermediate I, no separate deprotection step is required.

4. Optionally, purification of Romidepsin.

Lactamization:

Lactamization is a cyclization process of preparing a cyclic amide by the elimination of a molecule of water from aminocarboxylic acid. In the present process of Lactamization takes place at D-Valine and D-Cysteine sites. The cyclization is effected in presence of a coupling agent selected from the group consisting of HBTU, HATU, BOP, PyBOP, COMU, DEPBT, carbodiimides, and any combinations thereof. Disulfide bridge formation by oxidation:

Intramolecular disulfide bridge formation by oxidation of the cyclized peptide / depsipeptide is done by using iodine (1 -10 equivalent), methanol and dichloromethane with 10 - 500 volumes; preferably 100-200 volumes and quenching the excess iodine with 0.1M sodium thiosulfate solution. The organic layer is separated and washed with brine solution. The organic layer is dried over anhydrous sodium sulfate and evaporated in vacuum.

Isolation and Purification of Romidepsin:

Crude peptide Romidepsin was isolated by any one of concentration, extraction, crystallization by using the solvents like dichloromethane, chloroform, ethyl acetate, toluene, hexane, and ethers. Purification was performed by preparative - RP-HPLC using the gradient of acetonitrile and water with 0.01 % to 5 % trifluoro acetic acid.

Process of preparing Romidepsin from Formula I compound wherein R is as defined above, in particular intermediate I is represented in scheme 2.

Formula I Formula II Romidepsin

Scheme 2

In one embodiment, the Formula II compound, wherein R is trityl group is represented as below

Intermediate IA

The compound Intermediate IA has been characterized by Mass spectra (Sheet no. 7)·

In yet another aspect, as described under scheme 3, the present invention provides novel intermediate II by solid phase synthesis.

Intermediate II

The process is almost similar to the process for preparing Intermediate I except in following aspects: 1. The intermediate I is directly released from the resin by mild acidic treatment, instead, intermediate II is prepared by subjecting the loaded unreleased peptide to intramolecular disulfide bridge formation by oxidation using iodine.

2. On resin oxidation has been carried out to form the intra-molecular disulfide bridge followed by treating the resin with mild acid results with intermediate II.

The process to prepare the intermediate II is represented in scheme 3.

FmooH

Fmoc-D-Vfline-OH 2-OTr ;i

A

H

Intermediate Π

Scheme 3 Further, the invention provides process of preparing Romidepsin from intermediate II, as described in scheme 4. This process is a one-step lactamization /cyclization reaction using a coupling agent. The coupling agent is selected from the group consisting of HBTU, HATU, BOP, PyBOP, COMU, DEPBT, carbodiimides, and any combinations thereof.

The process of preparing Romidepsin from intermediate II is represented in in scheme 4.

Intermediate II Romidepsin

Scheme 4

The non-limiting examples according to present invention are as follows:

Examples

Example 1 : Preparation of intermediate I

To a clean and dry sintered peptide synthesis vessel is charged 20g of 2- chlorotrityl chloride resin. The resin beads are washed with anhydrous MDC (2 x 200 mL) and kept for swelling with anhydrous MDC (200 mL) for 1 h. The solvent is drained and are added first amino acid Fmoc-D-Val-OH (32.5 g), anhydrous MDC (200 mL), DIPEA (29.2 mL) in to a peptide vessel containing swollen resin beads. It is maintained for 2 - 3 h at 20 °C to 30 °C. The reaction mixture is drained, the resin is washed with MDC (3 x 200 mL), DMF (3 x 200 mL). Further the deprotection is achieved by 20 % piperidine solution (2 x 200 mL) kept for 10 minutes, each time. The resin beads are washed with DMF (3 x 200 mL). The reaction is monitored by Kaiser Test. The remaining steps coupling, deprotection, esterification and dehydration reactions are performed as per the sequence. The linear peptide was released by charging 1 % TFA in MDC solution (4 x 300 ml) into the peptide vessel. Maintain for 3 - 4 minutes at 20°C to 25°C. The cleavage mixture is collected and concentrated by rotary evaporator. The desired intermediate I is precipitated by diethyl ether, filtered and dried and used further.

Example 2: Preparation of Intermediate I

Step 1 : Swelling and coupling

In a clean and dry sintered peptide vessel 2-chlorotrityl chloride resin (7.5 kg) were charged and resin beads were washed with anhydrous MDC (2 x 75 L) and kept this solution for swelling resin with anhydrous MDC (75 L) for 30 to 45 minutes. Fmoc-D-Valine-OH (12.21 kg), anhydrous MDC (75 L), DIPEA (9.4 L) were charged into clean dry 100 L container and mixed for 5 minutes and this mixture was charged into peptide vessel containing swollen resin beads. The mixture was maintained for 2 to 3 hours at 20°C to 30°C and the resin was washed with anhydrous MDC (3 x 75 L). The coupling reaction was repeated using half of the quantities of above mentioned coupling reaction. The mixture was maintained for 2 to 3 hours and washed the resin with anhydrous MDC (3 x 75 L). Capping was performed by treating resin with beads with MDC (71.25 L), DIPEA (1.87 L), acetic anhydride (1.87 L) for 30 minutes and washing resin with anhydrous MDC (3 x 75 L) followed by washing with DMF (3 x 75 L).

Step 2: Deprotection

20% piperidine solution (2 x 75 L) was charged to resin beads (obtained from step 1) and kept for 10 minutes each to get deprotected resin compound. The deprotection was monitored by Kaiser Test.

Step 3: Coupling

In a clean and dry 100 L container (3S,4E)-3-Hydroxy-7-[(triphenylmethyl)thio]- 4-heptenoic acid (7.5 kg), HOBT.H20 (2.75 kg), DMF (75 L), DIPEA (4.7 L) were charged, the mixture was stirred for 5 minutes and cooled to 0°C to 10°C. The mixture was charged to peptide vessel and followed by addition of HBTU (6.82 kg) into peptide vessel and the mixture was maintained for 2 hours at 20°C to 25°C. Sample was taken from peptide vessel for Kaiser Test and found -ve. The resin beads were washed with DMF (3 x 75 L) followed by anhydrous MDC (3 x 75 L). Step 4: Esterification

In a 100 L container Fmoc-Valine-OH (12.21 kg), anhydrous MDC (75 L), DIPC (1 1.35 L), DMAP (2.2 kg) were charged and mixed for 5 minutes. The mixture was charged into peptide vessel and maintained for 1 to 2 hours at 20°C to 25°C. The resin beads were then washed with anhydrous MDC (3 x 75 L). The coupling reaction was repeated using half of the quantities of above mentioned coupling reaction. The reaction mixture was maintained for 2 hours and the resin beads were washed with anhydrous MDC (3 x 75 L) followed by washing with DMF (3 x 75 L).

Step 5 : Deprotection

Step 5: Coupling

In a dry and clean 100 L container Fmoc-Thr-OH (12.3 kg), DMF (75 L), HOBT.H20 (5.5 kg), DIPEA (9.4 L) were charged and mixed for 5 minutes. Slowly the above solution was charged into peptide vessel followed by addition of HBTU (13.65 kg) and the reaction mixture was maintained for 1 to 2 hours at 20°C to 25 °C. Sample was taken from peptide vessel for Kaiser Test and found - ve. The resin beads were washed with DMF (3 x 75 L). The coupling reaction was repeated using half of the quantities of above mentioned coupling reaction. The reaction mixture was maintained for^" 2 hours and the resin beads were washed with DMF (3 x 75 L).

Step 6: Deprotection

20% piperidine solution (2 x 75 L) was charged to resin beads (obtained from step 1) and kept for 10 minutes each to get F-moc deprotected resin beads which are washed with DMF (3 x 75 L). The deprotection was monitored by Kaiser Test (+ve).

Step 7: Coupling

In dry and clean 100 L container Fmoc-D-Cystiene(Trt)-OH (21.08 kg), DMF (75 L), HOBT.H20 (5.5 kg), DIPEA (9.4 L) were charged. The mixture was charged into peptide vessel followed by addition of HBTU (13.65 kg) in peptide vessel. The reaction mixture was maintained for 1 to 2 hours at 20°C to 25°C. Sample was taken from peptide vessel for Kaiser Test and found -ve. The resin beads were washed with DMF (2 x 75 L). The coupling reaction was repeated using half of the quantities of above mentioned coupling reaction. The reaction mixture was maintained for 2 hours and the resin beads were washed with DMF (5 x 75 L) followed by washing with anhydrous MDC (3 x 75 L).

Step 8: Dehydration

Further anhydrous MDC (75 L), EDC.HC1 (5.75 Kg), and CuC12.2H20 (0.266 Kg) were added into peptide vessel containing the resin beads (obtained from step

7) followed by addition of anhydrous MDC (37.5 L). The reaction mixture was maintained for 16 to 18 hours at 20°C to 30°C. After the completion of reaction the resin beads were washed with anhydrous MDC (3 x 75 L) followed by washing with DMF (3 x 75 L).

Step 9: Deprotection

20% piperidine solution (2 x 75 L) was charged to resin beads (obtained from step

8) and kept for 10 minutes each to get F-moc deprotected resin beads which are washed with DMF (3 x 75 L) followed by MDC (4 x 75 L).

Step 10: Cleavage

1 % TFA in MDC solution (3 x 1 13 L) was charged into the peptide vessel containing F-moc deprotected resin beads and the mixture was maintained for 3 to 4 minutes at 20°C to 25°C. The separated layer was collected and washed the resin beads with anhydrous MDC (37.5 L). The collected organic layer was subjected to distillation under vacuum at below 40°C and solvent was completely removed to get the crude compound. Diethyl ether (75 L) was charged into crude compound and solvent was distilled out completely under vacuum at below 40°C. Again diethyl ether (75 L) was charged to the obtained compound and stirred the mixture for 5 minutes. The diethyl ether layer was decanted from the solid and the obtained solid was dried under vacuum at below 30°C to get the titled compound (13.0 kg). Example 3: Preparation of Romidepsin from Intermediate I

Step 1 : Cyclization through lactamization

To a clean four-neck RB flask (3.0 L) equipped with mechanical stirrer, thermometer pocket in a tub was added intermediate I (30.0 g), DMF (300 mL) and HOBt. H₂0 (1.97 g) and stirred the mixture at 20°C - 30°C. The reaction mixture was cooled to 0°C to 10°C and slowly DIPEA (1 1.25 ml), HBTU (10.3 gm) was added into the reaction mixture at 0°C to 10°C. The temperature of the reaction mixture was increased to 20°C to 30°C and maintained for 2- 3 h. The reaction mixture was diluted with MDC (300 mL) and stirred for 5 to 10 min, washed with water (7 x 300 mL) and brine solution (2 x 300 ml). The organic layer was dried over anhydrous sodium sulfate and evaporated on rotary evaporator. The cyclic peptide was precipitated by adding ether such diisopropyl ether or methyl tertiarybutyl ether, filtered, dried, and used further for oxidation. Step 2: Oxidation to form Disulfide Bridge

To a clean four-neck RB flask (450 L) equipped with mechanical stirrer, thermometer pocket in a tub charged Iodine (37.06 gm), MDC (9.0 L), Methanol (1.0 L) was charged and stirred the mixture at 20°C to 25°C. Then cyclized intermediate obtained in step 1 (20.0 gm), MDC (4.5 L), methanol (500 ml) are added to the flask and the mixture was stirred at 15°C to 20°C for 1 -2 h. Further sodium thiosulfate solution (0.1 M, 4.5 L) was added into the reaction mixture and allowed to settle the layers. The organic layer was separated and washed with brine solution (2 X 4.5 L) and dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the crude peptide was precipitated with cold ether (100 ml), filtered, washed and purified by RP-HPLC.

Example 4: Preparation of Intermediate II

To a clean and dry sintered peptide synthesis vessel 20 g 2- chlorotrityl chloride resin is charged. The resin beads are washed with anhydrous MDC (2 x 200 mL) and kept for swelling with anhydrous MDC (200 mL) for 1 h. The solvent is drained and Fmoc-D-Val-OH (32.5 g), anhydrous MDC (200 mL), DIPEA (29.2 mL) are added in to a peptide vessel containing swollen resin beads. The mixture is maintained for 2 - 3 h at 20°C to 30°C. The reaction mixture is drained and washed the resin with MDC (3 x 200 mL), DMF (3 x 200 mL). Further the F-moc deprotection is performed using 20 % piperidine solution (2 x 200 mL) each time kept for 10 minutes. The resin beads are washed with DMF (3 x 200 mL). The reaction is monitored by Kaiser Test. The remaining steps coupling, deprotection, esterification, and dehydration, reactions are performed in an analogous manner as per the sequence described in example 2. The linear peptide obtained is oxidized on resin by treating with iodine and thereby forming intramolecular disulfide bridge. The peptide is released by charging with 1% TFA in MDC solution (4 x 300 mL) into the peptide vessel and maintained for 3 - 4 minutes at 20°C to 25°C. The cleavage mixture is collected and concentrated by rotary evaporator. The desired intermediate II is precipitated by adding diethyl ether, filtered the solid, dried and used further.

Example 5 : Preparation of Romidepsin from Intermediate II

To a clean four-neck RB flask (3.0L) equipped with mechanical stirrer, thermometer pocket in a tub intermediate II (16.0 g), DMF (150 mL) and HOBt. H₂0 (1.97 g) are added and the mixture is stirred at 20°C - 30°C. The reaction mixture is cooled to 0°C to 10°C and slowly DIPEA (1 1.25 mL), HBTU (10.3 g) are added into the reaction mixture at 0°C to 10°C. The temperature of the reaction mixture is raised to 20°C to 30°C and maintained for 2-3 h. The reaction mixture is diluted with MDC (300 mL) and stirred for 5-10 min, washed with water (7 x 150 mL) and brine solution (2 x 300 mL). The organic layer is dried over anhydrous sodium sulfate and evaporated on rotary evaporator. The cyclic peptide is precipitated by adding ether, filtered, dried, and purified by prep. RP- HPLC.

Claims

We claim

1. A compound of formula I

Formula I

2. A compound of formula

Intermediate I

3. A compound of formula

Intermediate IA

4. A process to prepare compounds of formula I comprising the steps of a) treating 2-chlorotrityl chloride resin with solvent and loading of Fmoc-D-Val- OH to treated resin to get following compound

b) deprotecting Fmoc-group of protected amino acid.

c) coupling of deprotected amino acid with (3S, 4E)-3-mercapto-7-hydroxy-4- heptenoic acid derivative to get heptenoic acid protected amino acid.

d) esterifying heptenoic acid protected amino acid using Fmoc-Val-OH and subsequently deprotecting Fmoc group.

e) coupling with Fmoc-Thr-OH to get peptide D and subsequently deprotecting of F-moc group to get peptide E

-Fmoc f) coupling of F-moc deprotected peptide E with Fmoc-D-Cys(R)-OH peptide F

g) dehydrating peptide F using dehydrating agent and subsequently deprotecting of Fmoc group to get peptide G

h) releasing formula I compounds from solid support by acidic cleavage

Formula I

5. The process according to claim 4 wherein

a) coupling reactions under step c and e are conducted using coupling agent, scavenger and a base, and

b) esterification reaction under step d is conducted using coupling agent and a base, and deprotection reaction under step d), step e) and step g) is conducted using one or more from piperidine, N-methylpyrrolidone, 1 ,8- Diazabicyclo[5.4.0]undec-7-ene, cyclohexylamine, diethylamine, triethylamine, dimethylamino pyridine, morpholine, ethanolamine, Tris(2- aminoethyl) amine and l ,3-cyclohexanebis-(methylamine) and

c) coupling reaction under step f) is conducted using coupling agent, scavenger and a base

d) dehydration under step g) is conducted using carbodiimide and CuCl₂ and e) acidic cleavage under step h) is conducted using trifluoroacetic acid.

6. A process to prepare Intermediate I comprising the steps of

a) treating 2-chlorotrityl chloride resin with solvent and loading of Fmoc-D-Val- OH to treated resin to get following compound

b) deprotecting Fmoc-group of protected amino acid.

A c) coupling of deprotected amino acid with (3S, 4E)-3-mercapto-7-hydroxy-4- heptenoic acid derivative to get heptenoic acid protected amino acid.

OH O wherein R is acetamidomethyl or trityl.

e) coupling with Fmoc-Thr-OH to get peptide D and subsequently deprotecting of F-moc group to get peptide E.

f) coupling of F-moc deprotected peptide E with Fmoc-D-Cys(R)-OH, wherein R is as defined in claim 1 to get peptide F

G

h) releasing Intermediate I compounds from solid support by acidic cleavage

7. The process according to claim 6, wherein

a) coupling reactions under step c) and e) are conducted using coupling agent, scavenger and a base

b) esterification reaction under step d) is conducted using coupling agent and a base,

c) deprotection reaction under step d), step e) and step g) is conducted using one or more from piperidine, N-methylpyrrolidone, 1 ,8- Diazabicyclo[5.4.0]undec-7-ene, cyclohexylamine, diethylamine, triethylamine, dimethylamino pyridine, morpholine, ethanolamine, Tris(2- aminoethyl) amine and l ,3-cyclohexanebis-(methylamine),

d) coupling reaction under step f) is conducted using coupling agent, scavenger and a base, and

e) dehydration under step g) is conducted using carbodiimide and CuCl₂, and f) acidic cleavage under step h) is conducted using trifluoroacetic acid.

8. The process according to claim 4 or 6 wherein the coupling agent is selected from the group consisting of HBTU, HATU, BOP, PyBOP, COMU, DEPBT, carbodiimides and combinations thereof.

9. The process according to claim 4 or 6 wherein the scavenger is selected from the group consisting of hydroxybenzotriazole or ethyl(hydroxyimino)cyanoacetate.

10. The process according to claim 4 or 6 wherein the base is selected from triethylamine or diisopropylethyl amine or other mild organic bases including collidene, N-methylmorpholine.

1 1. A process to prepare Romidepsin comprising the steps of a) cyclization/lactamization of Formula I compounds to produce formula II compounds,

b) deprotection of sulfhydril protecting groups of Formula II compounds and in situ in ramolecular disulfide bridge formation to give Romidepsin.

Formula I compounds Formula II compounds Romidepsii c) optionally purifying Romidepsin.

12. The process as claimed in claim 1 1 , wherein the Formula I compound is Intermediate I and Formula II compound is Intermediate IA.

13. The process as claimed in claim 1 1 wherein cyclization/lactamization is conducted using i) a coupling agent selected from the group consisting of HBTU, HATU, BOP, PyBOP, COMU, DEPBT, carbodiimides and combinations thereof and ii) a base diethylisopropylamine.

14. The process as claimed in claim 1 1 wherein deprotection of sulfhydril protecting group is carried out using deprotecting agent and intramolecular disulfide bridge formation is carried out by oxidation reaction using iodine.

15. The process as claimed in claim 1 1 wherein the deprotecting agent is selected from the group consisting of piperidine, l,8-Diazabicyclo[5.4.0]undec-7-ene, amines like cyclohexylamine, diethylamine, triethylamine, dimethylamino pyridine, morpholine, ethanolamine, Tris(2-aminoethyl) amine amd 1,3- cyclohexanebis-(methylamine) and deprotection is carried out in presence of polar or non-polar solvent such as DMF or NMP (N-methylpyrrolidone).

Dated this 26^th day of November 2015