FORM2
THE PATENTS ACT, 1970
(39 of 1970)
&
The Patents Rules, 2003
PROVISIONAL SPECIFICATION
(See section 10; rule 13)

1. Title of the invention.

ISOLATION AND RECOVERY OF SIMVASTATIN IN LACTONE FORM OR IN THE FORM OF AN ACID SALT FROM THE HARVESTED FERMENTATION BROTH

2. Applicant(s)

(a) NAME :
(b) NATIONALITY
(c) ADDRESS :

THEMIS MEDICARE LIMITED
An Indian Company.
11/12, Udyognagar, S V Road, Goregaon (W), Mumbai 400 104, State of Maharashtra, India.

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

TITLE OF THE INVENTION
Isolation and recovery of simvastatin in lactone form or in the form of an acid salt from the harvested fermentation broth
TECHNICAL FIELD OF THE INVENTION
Present invention relates to a novel process for isolation of simvastatin in lactone form or in the form of an acid salt from the fermentation broth. In particular present invention relates to isolation of simvastatin in the form of simvastatin lactone or in the form of simvastatin acid salt from the harvested fermentation broth.
BACKGROUD AND PRIOR ART
Certain mevalonate derivatives are known to be active as anti-hyper-cholestrolemic agents, and these function by limiting cholesterol biosynthesis by inhibiting the enzyme HMG-CoA reductase, atorvastatin, lovastatin (also known as mevinolin or monacolin K), Mevastain (also known as compactin), and derivatives and analogs of these compounds such as pravastatin, simvastain are known HMG-CoA reductase inhibitors and are used as anti-hypercholesterolemic agents. Majority of them are produced by fermentation using different species belonging to Aspergillus, Monascus, Nocardia, Amycolaptosis, Mucor or Penicillium genus; others are obtained by treating the fermentation products using the method of chemical synthesis or they are produced by chemical synthesis.
Lovastatin is the first of the statin to be used widely and is manufactured by a fermentation-based process. It is produced as secondary metabolite of the fungus Aspergillus terreus (US 4231938).
mevastatin and lovastatin are natural fermentation products which possess a 2-methylbutyrate side chain in the 8-position of their hexahydronaphthalene ring system. It was later on proved that products having a 2,2-dimethylbutyrate side chain


in this position are more active inhibitors of HMG-CoA reductase than analogs with a 2-methylbutyrate side chain.
Lovastatin is a natural product obtained by fermentation using Aspergillus tereus possessing 2-methylbutyrate side chain in C-8 position, whereas simvastatin, its more potent analog possess 2,2-dimethylbutyrate side in C-8 position, simvastatin is produced semisynthetically from lovastatin.
Simvastatin is synthetically prepared from lovastatin in many different ways.
For example, US 4444784 discloses a process in which the methyl group is
introduced by using many chemical steps which ultimately result in low yield.
In US 4582915 (to Merck), in one component of the process the methyl group is introduced by direct alkylation of the methylbutyrate side chain using a metal alkylamide and methylhalide. The disadvantages of this process are many, like product contamination with by-products and starting material resulting in low purity of the required product.
CA 1287063 and US 4820850 (to Merck) describe a process wherein the said methylation is carried out using low boiling amines. This process also suffers from a drawback. Low boiling amines are unsafe to handle, the silyl intermediate involved in the process is oil and hence difficult to isolate after purification and characterisation, the deprotection is carried out by using hydrofluoric acid which is highly corrosive and the hydrolysis is carried out using a base thus leading to metal salt of mevilonic acid which needs an additional step of lactonisation.
Above mentioned chemical processes suffer from one or other drawbacks like use of silyl reagents or low yield of the final product.
Simvastatin exist in both the lactone as well as the hydroxyl-acid form as shown below. There is equilibrium between the two forms. Due to difference in the polarity


between the two forms, isolation and subsequent purification of simvastatin is difficult. Great care has to excercised during isolation and purification of simvastatin as the method to isolate one form may not be suitable for the isolation of the other form thus there will be reduction in the overall yield.

US 5202029 describes a process for purification of lactone form of statin compounds by dissolving the broth in an organic solvent and eluting through HPLC column. The statin compounds which were eluted were crystallized after lengthy procedure of partial evaporation of the solvent followed by addition of water. The disadvantage of this process is that this can not be used on industrial scale as the use of column for purification of the final product make the process un economical.
US 5616595 describes the technique of tangential filtration of water-insoluble compounds form the fermentation broth. This is suitable for the lactone form of the statin compounds. The drawback of this process is the need of solvents for solubilization as the compounds are water-insoluble and the multiple membranes required for filtration, and the repeated filtrations, which make the process very costly.
A process for isolation of lovastatin in the lactone form is described in US 5712130. According to this patent, lovastatin is extracted from the broth by extraction with butyl acetate. Crystals of lovastatin are obtained by removal of organic phase by vacuum distillation followed by cooling. Recrystallization yields the product of 90% purity. The


disadvatage of the process is the need of further purification of the product before it can be used as API, thus adding to the cost of production.
US 6387258 describes the process for isolation of compactin and lovastatin from fermentated microbial broth. However, the disadvantage of the process is that it is not only a lengthy and tedious non viable method and only hints for the purification of lovastatin, compactin and pravastatin by preferably in alkaline condition. Further it has no teaching of biochemically prepared simvastatin or its isolation.
WO03/010324 discloses a process of preparing simvastatin by providing a host having a customised gene encoding for a polyketide synthase and fermenting the said host to obtain simvastatin or analogs thereof or derivatives thereof. The crude product was isolated and purified by high-pressure liquid chromatography (HPLC) to get simvastain.
In the above mentioned prior art process a customised gene is used for biosynthesis of simvastatin which needs to be developed (cultivated), the product is isolated in crude form, and the yield of the crude product is very low, and is purified by HPLC for characterisation. The said process is also not suitable for producing Simvastaitn industrially as the product obtained in very low yield and purity.
Hence there is need to develop a process for production of simvastatin which is simple, economical, scalable and capable of producing simvastatin in high yield and purity. As the chemical processes of converting lovastatin to simvastatin are low yielding and involve the use of hazardous chemicals, it is necessary to develop an environmentally friendly process which uses the technique of fermentation for production of simvastatin. It is also desired to develop a simple, easily scalable, economical, and high yielding process for isolation of statins such as simvastatin from the said fermentation broth.


The inventors have found that simvastatin in lactone form or in the form of an acid salt can be isolated and recovered in high yield and purity, from the microbial broth without isolation of lovastatin.
Advantages of the process are, it does not require purification of lovastatin as a first step, followed by further semisynthetic procedures and use a single fermentation step to produce simvastatin. The process use the same fermentation facility as used for producing lovastatin with little modifications, materials used are relatively inexpensive and the purification steps are easily practicable.
OBJECT OF THE INVENTION
It is an object of the invention to provide a novel process for isolation and recovery of simvastatin in lactone form or in the form of an acid salt in high yield and purity, from harvested microbial broth.
It is yet another object of the invention to provide a simple process for isolation of statin compounds such as simvastatin in lactone form or in the form of an acid salt from harvested microbial broth.
It is further object of the invention to provide an industrially scalable process for isolation of statin compounds such as simvastatin in lactone form or in the form of an acid salt from harvested microbial broth.
It is yet another object of the invention to provide an environmentaly friendly process for isolation of statin compounds such as simvastatin in lactone form or in the form of an acid salt from harvested microbial broth.
It is further object of the invention to provide a cost effective process for isolation of statin compounds such as simvastatin in lactone form or in the form of an acid salt from harvested microbial broth.


SUMMARY OF THE INVENTION
The present invention relates to a novel process for isolation and recovery of compounds such as biosynthetically produced simvastatin in either lactone form or in the form of an acid salt in high yield and purity, from microbial fermentation broth and isolating the said statin from harvested microbial broth.
DESCRIPTION OF THE INVENTION
Simvastatin is a well-known cholesterol lowering drug. It is more potent derivative of lovastatin. It is marketed by Merck Co. as Zocor. Simvastain is produced semisynthetically.
The inventors of the present invention have found that statins such as simvastatin can be produced biosynthetically using microorganisms and can be isolated in lactone form or in the form of an acid salt in high yield and purity from harvested microbial broth.
The inventors have found that simvastatin can be isolated from the fermentation broth by recovering the said statins from the mixture by various novel techniques/methods as described.
There are various advantages of the process of the present invention such as:1) the process of the present invention does not require isolation of lovastatin in the first step; 2) simvastatin can be isolated from the fermentation broth in high yield and purity by using this process; 3) the process comprises of using simple microorganisms for producing simvastatin; 4) the methods used for isolation of simvastatin from the harvested broth are inexpensive, scalable and eco-friendly.
According an aspect of the invention there is provided a novel process for producing statin compounds such as simvastatin biosynthetically from fermentation broth and isolating the said statin in lactone form or in the form of an acid salt from harvested


microbial broth. In particular there is provided a novel process for isolation of simvastatin in lactone form or in the form of an acid salt from mirobial broth.
According to one aspect of the invention, there is provided a novel process for the isolation of smvastatin from microbial broth, the process comprises steps of adjusting the pH of the broth after diluting the broth or without dilution of the broth to convert the said simvastatin into the lactone form or in the acid salt form; separating the biomass containing the simvastatin compounds using standard methods of separation of solid from aqueous layer; extracting the simvastatin compound from the biomass into organic solvent using standard methods of extraction ; adjusting the pH of the extract to alkaline by treatment with a base to obtain the simvastatin compound in the form of its salt; and isolating the statin/simvastatin in the lactone form or in the form of its salt, from the mother liquor.
According to another aspect of the invention, there is provided a novel process for isolation of simvastatin in the lactone form or in the from of salt, from microbial broth; the process comprises the steps of adjusting the pH of the broth with an acid to pH 2-4; subjecting the broth to continuous Westfalia type extractor to extract the simvastatin into organic solvent; separating the organic phase; isolating the product from the organic phase by concentrating the organic phase to obtain simvastatin.
According to one more aspect of the invention, there is provided a novel process for isolation of simvastatin in the lactone form or in the from acid salt, from microbial broth comprising subjecting whole broth to multiple-stage continuous extractor where the organic solvent is introduced from one side and the broth is introduced from another side of the extractor and the two phases are discharged in the opposite directions at the end of the extraction; simvastatin is isolated from the organic phase by concentrating the organic phase to obtain simvastatin.
According to yet another aspect of the invention, the process for isolation statin/simvastatin from microbial broth comprises the steps of treating the microbial broth with a base to get pH to 9 - 12 separating the biomass by known techniques


used for the separation of solid from water; treating the aqueous layer with an acid to pH 2-4.5; extracting with organic solvent; treating the organic layer with a base and isolating the simvastatin as a salt; recovering the statin from the mother liquor by concentrating and crystallizing the lactone.
According to yet another aspect of the present invention there is provided a process for isolation of simvastatin from microbial broth in the form of lactone comprising the steps of: stirring the broth with an organic solvent and separating the organic layer from the biomass; concentrating the organic layer to get a concentrated mass; treating the concentrated mass with a hydrophilic solvent such as alcohol to precipitate simvastatin; filtering the precipitated simvastatin lactone. Alternatively the bomass is treated with a hydrophobic solvent such as toluene; the toluene extract is concentrated and is diluted with hexane to obtain simvastatin.
Simvastatin may be isolated in the form of lactone or in the form it's acid salt like inorganic salt or organic salt.
The organic solvent used for the extraction of the statin from the broth may be selected from hydrocarbons, arylhydrocarbons, esters, carbonyl compounds such as ketones, halohydrocarbons, alcohols, ethers, and their mixtures etc. either singly or in combination.
The acid used for acidification may be selected from inorganic acids like mineral acids, phosphoric acid, or organic acids.
The base used may be selected from inorganic bases like alkali metal hydroxides, alkali metal carbonates, alkaline earth metal hydroxides, alkali metal carbonates, metal hydroxides, aqueous ammonia, ammonia gas, or organic bases like alkyl amines, aryl amines and their amine derivatives.
Standard methods of separation of solids from water may be selected from the use of equipments like Filter press, Rotary vacuum drum filter, Solid/liquid separator, centrifuge, Decanters to separate out water.


Standard method of recovering the statin from the extraction solvent include concentration of under vacuum or without applying vacuum, addition of another solvent, salting out and by adding antisolvents and crystallization techniques known by an expert skilled in the art.
The invention is now illustrated by following non-limiting example.
Example :
Isolation of simvastatin in acidic condition.
Isolation of simvastatin ammonium salt or simvastatin in acidic condition.
1) A fermentation broth containing 12 -13 g per litre of simvastatin is prepared by the method known in the stated prior arts.
Chill the broth at 8-10° C and adjust the pH to 2.9 - 3.1 using ortho-phosphoric acid. The mixture is well stirred and filtered through rotary vaccum drum filter to separate the bio mass. The bio mass is again suspended three to four times in water and filtered to get the almost dried mass.
Ethyl acetate is added to the almost dried bio mass in a ratio 0.75 : 1. The mass is agitated for an hour and filtered. The operation is repeated twice using ethyl acetate. The total solvent is mixed and washed with 10% saline. Separated ethyl acetate layer is dried using anhydrous glauber salt. Chill the ethyl acetate layer . The ethyl acetate layer is purged with ammonia gas to get pH of 10-10.2 which results in to formation of simva ammonium salt crystals with the purity of more than 90% . This is further purified by treating crude Ammonium salt with water in a proportion of 10 times volume of water for original cake. The same is back extracted in ethyl acetate, followed by treatment with anhydrous glauber salt and precipitation by passing ammonia gas in ethyl acetate layer to get pH of 10 -10.2. The resultant simvastastin ammonium salt has a purity of more than 99% . Overall yield from fermentation broth to pharmaceutically acceptetable grade of simvastastin ammonium salt is 70 - 75 %.


Alternatively, toluene, water and ortho-phosphoric acid (pH to 2.9 - 3.1 ) is added to the dried biomass and is kept under agitation and refluxed at 58 - 60 deg. C for 8-10 hrs for the extraction and lactonization. of simvastatin The operation is repeated twice. The toluene layer is concentrated under vacuum to obtain a simvastatin in a concentration of 120 - 130 g/l The Hexane is added in a proportion for original simva to Hexane in a ratio of 1:8-9. The mass in Hexane is chilled at 5-10 deg.C which results in precipitation of simvastatin with a purity of more than 90%. The crude simvastatin is further purified and recrystalized using methanol as a solvent to get the pharmacopoeial grade of simvastastin achieving a purity more than 98%. The overall yield from fermentation broth to pharmaceutical acceptable grade Simvastastin is 67-72 %.