DESC:FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
A PROCESS FOR ISOLATION OF ROMIDEPSIN FROM FERMENTATION BROTH AND PREPARATION OF CRYSTALS OF ROMIDEPSIN

2. APPLICANT:
(a) NAME : CONCORD BIOTECH LIMITED
(b) NATIONALITY : Indian
(c) ADDRESS : 1482 – 1486, Trasad Road
Dholka - 387810
Dist: Ahmedabad
Gujarat, India

3. PREAMBLE TO THE DESCRIPTION:
The following specification describes the invention.

Field of the invention
The present invention provides a process for the isolation of romidepsin and a process for the preparation of romidepsin crystalline form.
Background of the invention
Romidepsin, also known as FK228 and FR901228, is a potent histone deacetylase inhibitor. The chemical structure of romidepsin is depicted below as Formula I.

Formula I
US4977138 describes preparation of romidepsin by culturing microorganism Chromobacterium violaceum WB968. Romidepsin is isolated from fermentation broth by extraction and column chromatography.
US7396665 provides an alternative process for the preparation of romidepsin which requires carrying out four column chromatographic purifications steps.
US2009/0186382 states that above published processes for the isolation of romidepsin does not produce pure romidepsin consistently. US’382 application then goes on to describe an alternative process for the preparation of pure romidepsin to avoid drawbacks of above published processes. US’382 process is carried out at specific pH range and involves use of four chromatographic purifications and two crystallization steps.
A process disclosed in US4977138 for the preparation of romidepsin crystal requires use of three solvents and provides crystals having higher residual solvent content. An improved process described in US7611724 involves use of two solvents which must be used in the specific concentration. Hence, there is a need to provide a simpler process which has a small number of purification steps and requires less time for the isolation of pure romidepsin.
Object of the invention
The main object of the present invention is to provide an improved process for the isolation of romidepsin.
Another object of the present invention is to provide a process for the preparation of crystals of romidepsin.
Summary of the invention
The present invention provides an improved process for the isolation of romidepsin from the fermentation broth. The fermentation broth is produced by culturing a microorganism,Chromobacterium violaceum, by the method known in the art to produce fermentation broth.
An embodiment of the invention provides a process for the whole broth extraction of romidepsin. The whole broth extraction does not require separation of any ingredient of the broth before the extraction of romidepsin. Hence, step of filtration or separation of any broth ingredient is not required which makes isolation process simpler.
The present invention provides single solvent process for the preparation of crystals of romidepsin by three steps:
a) dissolving romidepsin in a solvent to obtain a solution
b) initiating precipitation of romidepsin
c) recovering crystals of romidepsin
Brief description of the drawings
Figure 1 refers to x-ray diffractogram of crystals of romidepsin
Figure 2 refers to DSC thermogram of crystals of romidepsin
Figure 3 refers to MP data of crystals of romidepsin
Figure 4 refers to HPLC chromatogram of romidepsin
Detailed description of the invention
Before explaining the present invention in detail, it is to be understood that the invention is not limited in its steps and concentration, mentioned hereafter to obtain romidepsin from fermentation broth by said components. The nature of the invention along with various components is described in the following pages. It is to be understood that the phraseology and terminology employed herein is for the purpose of description and not of limitation.
The term “romidepsin”, “FK228” and “FR901228” are used interchangeably and refer to the compound of Formula I.
The term “isolation of romidepsin” refers to a process of separating romidepsin in a powder form from the fermentation broth. In general, isolation includes one or more steps selected from extraction, chromatography and crystallization.
The term “fermentation broth” refers to the broth obtained by culturing a microorganism capable of producing romidepsin, for example Chromobacterium violaceum. The fermentation broth can be prepared by the process described in prior art, for example US4977138.
The term “crystals of romidepsin” refers to the crystalline form of romidepsin which is similar to crystals obtained in US4977138 when compared by XRD, DSC and melting point.
The fermentation broth is extracted using an organic solvent which is capable of separating romidepsin from the broth. The organic solvent for the extraction step includes, but not limited to, isobutyl acetate, isopropyl acetate, ethyl acetate and toluene. Preferably, the fermentation broth is extracted using toluene. Extraction is carried out using from about one to about two volume of an organic solvent at a room temperature. Extraction step may be repeated if required. After the completion of extraction, an organic solvent is evaporated under reduced pressure. Evaporation is carried out at a temperature from about 25o to 40o C.
An organic solvent is added to the obtained residue. Preferably, the organic solvent is a ketone or nitrile solvent for example acetone, methyl isobutyl ketone and acetonitrile. Most preferably, methyl isobutyl ketone is added to the residue. The solvent is added in an amount of about 50 to 100 volumes with respect to active romidepsin content in the residue. The content of active romidepsin in the residue is determined by HPLC method. Romidepsin gets dissolved in the organic solvent and non-dissolved impurities are removed by filtration. The solvent is evaporated from the filtrate under reduced pressure at a temperature from about 25o to 40o C to give a residue.
Preferably, an ester solvent is added to the obtained residue to get the clear solution. Most preferably, an ester solvent is ethyl acetate. The obtained solution is subjected to silica gel column chromatography. Chromatography is performed on silica gel of 60-120 mesh or 230-400 mesh using mobile phase prepared from acetone-heptane, ethyl acetate-cyclohexane or dichloromethane-hexane. Fractions containing romidepsin are pooled and evaporated under reduced pressure at a temperature from about 25o to 40o C to give a residue.
An organic solvent is added to the residue in an amount of about 15 to about 20 volumes. Preferably, the organic solvent is a ketone or a nitrile solvent for example methyl isobutyl ketone and acetonitrile. Most preferably, the organic solvent is methyl isobutyl ketone. Solid romidepsin, thus obtained is filtered and purified by crystallization from an organic solvent selected from ketone or alcohol. Preferably, crystallization solvent is acetone, 2-butanone, methanol, isopropyl alcohol, butyl alcohol or a mixture thereof.
An embodiment of the invention provides a process for the isolation of romidepsin which comprises
a) extracting the fermentation broth with an organic solvent and evaporating the solvent to obtain a residue;
b) treating the residue with an organic solvent and evaporating the solvent to obtain a residue;
c) subjecting the residue to silica gel column chromatography;
d) treating the product of step c) with an organic solvent;
e) crystallizing the product obtained in step d).
A preferred embodiment of the invention provides an improved process for the isolation of romidepsin from the fermentation broth which comprises
a) extracting the fermentation broth with an organic solvent;
b) evaporating the organic solvent to give oily residue;
c) adding an organic solvent to the residue;
d) evaporating the organic solvent to give oily residue;
e) adding an ester solvent to the residue;
f) subjecting the ester solution to the silica gel column;
g) evaporating the solvent from the romidepsin containing fractions;
h) adding an organic solvent to the residue;
i) filtering the solid thus obtained;
j) crystallizing the solid from an organic solvent to give pure romidepsin.
An embodiment of the invention provides an improved process for the isolation of romidepsin from the fermentation broth which provides romidepsin having purity of greater than 99.5% by area in HPLC.
The improved process of the invention involves simple purification steps and hence, does not require multiple chromatographic purification steps to achieve desired quality of romidepsin. The process is advantageous over reported processes in terms of time, cost and simplicity.
The present invention also provides a process for the preparation of crystals of romidepsin.
An embodiment of the invention provides a single solvent process for the preparation of crystals of romidepsin which comprises
a) dissolving romidepsin in a solvent to obtain a solution
b) initiating precipitation of romidepsin
c) recovering crystals of romidepsin
Step a) involves dissolution of romidepsin in a solvent by mixing romidepsin and a solvent. The resultant mixture may be heated and filtered to obtain a clear solution. Preferably, the solvent is a chlorinated hydrocarbon or an ester, for example dichloromethane, chloroform, ethyl acetate, isopropyl acetate, isobutyl acetate and like that.
Precipitation of romidepsin in step b) is initiated by either allowing the solution to stand at a temperature lower than 25o C or evaporating the solution to the reduced volume. After initiation of precipitation, the solution is stirred for a period of about 10 minutes to about 1 hour.
Crystals of romidepsin is recovered in step c) by conventional method like filtration.

Examples
The following examples are presented to illustrate the invention and do not limit the scope of the invention. It should be understood that although specific embodiments are outlined in the examples, modifications can be made which are included within the scope of the invention.
Isolation of romidepsin
Harvested broth (1000 lit) received from fermentation was charged to Reactor. Toluene (1000 lit) was added to the broth and agitated for two hour. The mixture was filtered through hyflo bed and layers were separated. Toluene was distilled under reduced pressure at 35oC up to 500 L. The precipitated solid was filtered out through hyflo bed. Hyflo bed was washed with toluene. Toluene was distilled under reduced pressure at 35oC to give oily residue. MIBK (Methyl isobutyl keton) was added to the oily residue and stirred for half hour. The solution was filtered through hyflo bed. MIBK (Methyl isobutyl keton) was distilled under reduced pressure at 30oC to give oily residue. Ethyl acetate was added to the oily residue to get a solution.
Ethyl acetate solution was loaded to packed column [Silica gel (230-400 mesh): 30-35 lit]. The column was eluted using a mobile phase prepared from dichloromethane and hexane. Fractions containing compound were pooled and distilled under reduced pressure at 35oC to give oily residue. MIBK (Methyl isobutyl keton) was added to the residue and stirred for half hour. The solid obtained was filtered and suck dried for one hour to obtain a crude romidepsin. Crude romidepsin was dissolved in acetone at 50o C and stirred overnight at room temperature. The solid obtained was filtered and suck dried for 1 hour to obtain pure romidepsin. Figure 4 shows HPLC purity of isolated romidepsin which was found to be 99.89% by area.
Preparation of romidepsin crystals
Romidepsin was dissolved in dichloromethane at 50¬¬oC. The solution was filtered through hyflo bed. Hyflo bed was washed with dichloromethane. The solution was reduced to 75% under vacuum. Solid obtained was filtered and dried under reduced pressure at 50oC to obtain crystals of romidepsin. The result of this process is shown in Figure 1-3 as they respectively shows X-ray diffraction, DCS thermogram and Melting point study of romidepsin drug crystals.
,CLAIMS:We claim:
1. A process for the isolation of romidepsin from fermentation broth which comprises,
a) extracting the fermentation broth with an organic solvent and evaporating the solvent to obtain a residue;
b) treating the residue obtained in step a) with an organic solvent and evaporating the solvent to obtain a residue;
c) subjecting the residue obtained in step b) to silica gel column chromatography;
d) treating the product of step c) with an organic solvent;
e) crystallizing the product obtained in step d).
2. The process according to claim 1 wherein step c) is performed on silica gel of 60-120 mesh or 230-400 mesh.
3. The process according to claim 1 wherein an organic solvent in step b) is selected from a ketone or a nitrile solvent.
4. The process according to claim 1, wherein step c) is carried out using a mobile phase selected from acetone-heptane, ethyl acetate-cyclohexane and dichloromethane-hexane.
5. The process according to claim 1, wherein romidepsin is obtained with a purity of 99.5% or more by area.
6. A single solvent process for the preparation of crystals of romidepsin which comprises,
a) dissolving romidepsin in a solvent to obtain a solution;
b) initiating precipitation of romidepsin;
c) recovering crystals of romidepsin.
7. The process according to claim 6, wherein romidepsin is dissolved in a chlorinated hydrocarbon or an ester solvent in step a).
8. The process according to claim 6, wherein precipitation of romidepsin crystals in step b) is initiated by allowing the solution to stand at a temperature lower than 25° C or evaporating the solution to the reduced volume.
Dated this on February 24, 2015