Title of Invention	"A PROCESS FOR THE PURIFICATION CILASTATIN"
Abstract	The present invention pertains to a process for the purification of cilastatin which comprises contacting a solution of crude cilastatin with a non-ionic adsorbent resin and recovering pure cilastatin from a solution thereof

Full Text

The present invention relates to an industrial process for the preparation of pure cilastatin. Cilastatin of Formula I, as shown in the accompanied drawings, possesses the ability to pre\enl nephrotoxicity associated with the use of P-lactam antibiotics such as imipenem. Cilaslaiin is chemically known as [R-{R*, S-(Z)]]-7-[(2-amino-2-carboxyethyl)thio]-2-[[2.2-dimelliylcyclopropyl)carbonyl]amino-2-heptenoic acid. It is a renal dehydropeptidase mhibiior and is co-administered as the sodium salt with imipenem in order to prevent its renal metabolism. Imipenem/cilastatin combination is used as a potent broad spectrum antibacterial agent. Cilaslaiin is claimed in the U.S. Pat. No. 5,147,868 which describes a multistep process for preparmg cilastatin involving condensing cysteine hydrochloride with heptenoic acid of Formula 11. as shown in the accompanied drawings, wherein X is chloro or bromo, in the presence of sodium hydroxide in aqueous medium. Cilastatin so obtained contains the corresponding undesired E-isomer in amounts from about 6 to 10% as detennined by HPLC. The U.S patent also recites a process for isomerising the E-isomer to cilastatin by heating the mixture at pH 3 (Example 19A). However, we have observed that the isomerization process results m the formation of impurities in the range of 5-8% due to the degradation of cilastatin w hich makes it unsuitable for human consumption. U.S. Pal. No. 5,147,868 further teaches a method for isolating cilastatin from the reaeiion mixlure invoking two purifications viz. chromatography using a cation exchange resin followed by solvent purification using ethanol and diethyl ether. The ion exchange chromatography removes inorganic salts such as sodium chloride v/hich is otherwise difficult to remove as cilastatin itself is also water soluble. How e\ er, our attempts to isolate pure cilastatin by following the process exemplified in said ixilent (Example 19) were unsuccessful. We could obtain cilastatin in the form of its ammonium salt on eluting the cation exchange resin with ammonia solution and not as the free acid. Obtaining the free acid by using an acid such as hydrochloric acid entailed formation of inorganic ammonium salts such as ammonium chloride thus defeating the very purpose of using a cation exchange resin. An alternative process for preparing cilastatin has been described in J. Med. Chem. 30, 1083 (1987) wherein cysteine is condensed with the halo-heptenoic acid of Formula II in sodium metal / liquid ammonia and the resultant mixture isomerized to obtain cilastatin using methyl iodide in methanol. The product i.e. cilastatin is isolated by using a cation exchange resin followed by treatment with an anion exchange resin to remove inorganic salts. The method taught in J. Med. Chem. is not suitable at an industrial scale as it involves the use of sodium metal / liquid ammonia which is very hazardous and also uses methyl iodide for isomerization, which is expensive and requires special storage conditions. Loading the ion exchange column requires repeated circulation of cilastatin solution. Also, ion exchange column operates on the principle of ionic bonding / acid base reaction. Such reaction being exothermic causes considerable degradation of cilastatin. The use of two stage ion exchange chromatography is cumbersome, tedious and not practicable at an industrial scale. Thus, there is a need for a simple, convenient and efficient process for the preparation of pure cilastatin. The present invention overcomes the disadvantages associated with the prior art. In one aspect, the present invention provides a process for purification of cilastatin using a non-ionic adsorbent resin. The process requires a single purification using chromatographic techniques to obtain the pure product. Loading of the non-ionic adsorbent resin with crude cilastatin is achieved by passing the solution only once through the resin. Since no acid base reaction takes place, no degradation of the product is observed. In particular, the present invention pertains to a process for the purification of cilastatin comprising: a. loading solution of crude cilastatin on conventional non-ionic adsorbent resin, b. washing the loaded column with water to remove halide ions c. eluting the pure cilastatin with polar organic or aqueous polar organic solvent, and d. isolating the pure cilastatin from the eluate by conventional method. "Crude cilastatin" in the meaning of the present invention comprises cilastatin containing impurities which may be inorganic salts such as sodium chloride, sodium bromide and the like, or organic impurities which may have formed due to degradation of cilastatin, or may be the side products, unreacted intermediates of the multi-step synthesis for the preparation of cilastatin. A solution of crude cilastatin may be obtained by dissolving crude cilastatin in a suitable solvent or may be obtained directly from a reaction for the preparation of cilastatin. Suitable solvents include water, polar organic solvents and mixtures thereof The polar organic solvents include methanol, ethanol, acetonitrile, acetone, and the like. Cilastain may be prepared by any of the methods reported in prior art. Any of the non-ionic adsorbent resins which are commercially available and on the surface of which cilastatin is adsorbed, may be used. In particular, non-ionic macroporous water insoluble polymers such as polyacrylates or copolymers of styrene and polyvinyl benzene may be used. Preferred adsorbent resin is a copolymer oi' styrene cross linked with di\iny I benzene. "Pure cilastatin" in the meaning of the present invention refers to cilastatin having a purity of 98"or more by HPLC. A txpical process for purification of cilastatin comprises loading a solution of crude cilastatin on a column of non-ionic adsorbent resin, washing it with deionized water till no halide ions can be detected. The resin is then eluted with polar organic or aqueous polar organic sohent and pure cilastatin isolated from the eluate by common methods known in the art such as concentration, precipitation and recrystallization as required. However, alternative method of purification such as slurrying with the adsorbent resin may also be used. .According to another aspect of the present invention, it provides a process for the isomerization of E-isomer to cilastatin. The process comprises heating a solution of cilastatin containing the corresponding undesired E-isomer at a pH of about 0.5 to 1.5. We have observed that cilastatin obtained using this process greatly reduces fonnation of degradation products. The solution of cilastatin containing the corresponding E-isomer is preferably obtained direcll) from a reaction for the preparation of cilastatin. Cilastatin may be prepared by any of the multi-step processes described in prior art. The isomerization is preferably performed at 85-95°C. The pH is adjusted to 0.5 to 1.5, more preferably to 0.5 to 1 and most preferably to about 0.5 to obtain best results. Any inorganic acid may be used for adjusting the pH of the solution. Preferably, hydrochloric acid is used. In a pieferred embodiment of the present invention, the two aspects of the invention are combined i.e. the isomerization process is followed by the purification process to obtain pure cilastatin. In the following section preferred embodiments are described by way of examples to illuslrale the process of the invention. However, these are not intended to in any way 'imit the scope of the present invention. EXAMPLE 1 Preparation of cilastatin Cysteine hydrochloride monohydrate (166.3g) was dissolved in water (1.2L). To this solution was added aqueous sodium hydroxide (113.7 g in 400ml water) and sodium salt of 7-chloro-2-[[( I S)-2.2-dimethylcyclopropane]carboxamido]-2-heptenoic acid (200g). Reaction mixture w as stirred at room temperature till complete conversion of the starting heptenoic acid to the product was achieved. The con-esponding E isomer (5% by HPLC) was isomenzed to cilastatin by heating the reaction mixture at SS-QO^C for 30 minutes after adjusting the pH to 0.5 with concentrated hydrochloric acid. Purification of cilastatin The reaction mixture obtained above was loaded on a column packed with diaion HP - 20 resin as the adsorbent. The column was washed with water to remove sodium chloride and then the product was eluted with aqueous methanol. Column fractions containing pure product were pooled and concentrated to obtain pure cilastatin (160g; Purity : 99 % by HPLC). EXAMPLE 2 Elhyl-7-ch!oro-2-oxo-heptanoate (25g), (S)-2,2-dimethylcyclopropane carboxamide (13.68g) and p-tokiene sulphonic acid (0.125g) were refluxed in toluene using dean - stark trap for the azeolropic removal of water from the reaction mixture. After the condensation was complete, the reaction mixture was washed with dilute hydrochloric acid and aqueous sodium bisulfite to remove unreacted (S)-2,2-dimethylcyclopropane carboxamide and ethyl-7-chloro-2-oxo- hcplanoale, respectively. The organic solution was then concentrated to recover toluene under reduced pressure. The resultant oily ethyl ester of 7-chloro-2-[[(lS)-2,2- dimethylcyclopropane]carboxamide]-2-heptenoic acid was hydrolyzed with aqueous sodium hydroxide in presence of denatured spirit at room temperature. After hydrolysis, the reaction mixture was concentrated to half of its volume under reduced pressure and washed \\ ith toluene. Cysteine hydrochloride monohydrate (29.7g) and aqueous sodium hydroxide solution wei'e added to the above aqueous layer. Reaction mixture was stirred at room temperature till the complete conversion of 7-chloro-2-[[(lS)-2,2- dimctliylcyclopropane]carboxamido]-2-heptenoic acid to the product was achieved. The corresponding E isomer was isomerized to cilastatin by heating the reaction mixture at 85-90o after adjusting the pH to 0.5 with concentrated hydrochloric acid. Fiiritlcation of cilastatin The reaction mixture obtained above was loaded on a column packed with diaion HP 20 resin as the adsorbent. The column was washed with water to remove sodium chloride and then the product was eluted with aqueous acetonitrile. Column fractions containing pure product were pooled and concentrated to obtain pure cilastatin (16.3g; Purity :99.2% by HPLC). WE CLAIM : 1. A process for the purification of cilastatin comprising : a. loading solution of crude cilastatin on conventional non-ionic adsorbent resin, b. washing the loaded column with water to remove halide ions c. eluting the pure cilastatin with polar organic or aqueous polar organic solvent, and d. isolating the pure cilastatin from the eluate by conventional method. 2. The process as claimed in claim 1 wherein the solution of crude cilastatin is obtained directly as a solution from a reaction mixture by methods known in the art. 3. The process as claimed in claim 1 wherein the solution of crude cilastatin is obtained by dissolving crude cilastatin in a solvent. 4. The process as claimed in claim 3 wherein the solvent is selected from water, a polar organic solvent, and mixtures thereof 5. The process as claimed in claim 1 or 4 wherein the polar organic solvent is selected from methanol, ethanol, acetonitrile and acetone. 6. The process as claimed in claim 1 wherein the non-ionic adsorbent resin comprises a non-ionic macroporous water insoluble polymer on the surface of which cilastatin is adsorbed. 7. The process as claimed in claim 6 wherein the polymer is selected from polyacrylates or copolymers of styrene and polyvinyl benzene. 8. The process as claimed in clam 7 wherein the adsorbent resin is a copolymer of styrene cross linked with divinylbenzene. 9. The process for the purification of cilastatin as herein described and illustrated by the examples herein.

Documents:

879-del-2001-abstract.pdf

879-del-2001-claims.pdf

879-del-2001-complete specification (granted).pdf

879-del-2001-correspondence-others.pdf

879-del-2001-correspondence-po.pdf

879-del-2001-description (complete).pdf

879-del-2001-drawings.pdf

879-del-2001-form-1.pdf

879-del-2001-form-2.pdf

879-del-2001-form-3.pdf

879-del-2001-form-9.pdf

879-del-2001-pct-210.pdf

879-DEL-2001-Post-Grant-Opposition-(24-12-2010).pdf