Title of Invention	FATTY ACID DERIVATIVES AND PROCESS FOR PREPARATION THEREOF
Abstract	The invention discloses a process for preperation of compound2 of formula I, by fermentation of microorganism capable of producing compound of formula I.

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I FATTY ACID DERIVATIVES AND PROCESS FOR PREPARATION THEREOF FIELD OF THE INVENTION The invention relates to a novel compound of formula I, process for the preparation of compound of formula I and process for the preparation of compound of formula II from compound of formula I. BACKGROUND OF THE INVENTION U.S. Pat. No. 4,598,089 discloses (2S,3S,5S,7Z,10Z)-5-[(S)-2-formamido-4-methyl-valeryloxy]-2-hexyl-3-hydroxy-7,10-hexadecadienoic 1,3 acid lactone, also known as lipstatin, a fermentative process for its production, a process for its isolation from the broth and a process for its hydrogenation to 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodecyl ester. 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodecyl ester, also known as orlistat is an anti-obesity agent. Conversion of lipstatin to tetrahydrolipstatin was the only available route for the manufacture of Orlistat. Since the prior art method for preparation of orlistat suffers from many drawbacks like low production levels of lipstatin, difficulties in the purification from impurities, low level of conversion of (2S,3S,5S,7Z,10Z)-5-[(S)-2-formamido-4-methyl-valeryloxy]-2-hexyl-3-hydroxy-7,10-hexadecadienoic 1,3 acid lactone to orlistat, high cost of linoleic acid used for the manufacture of (2S,3S,5S,7Z,10Z)-5-[(S)-2-formamido-4-methyl-valeryloxy]-2-hexyl-3-hydrox y-7,l(3-hexadecadienoic 1,3 acid lactone and the like, there is a need to find alternative method for production of orlistat which is cost effective, industrially applicable, requiring less steps of purification and ease of conversion to orlistat. We have now found that 2-Formylannino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodecyl ester can be prepared by employing novel compounds of formula I. We have also found that the novel compounds of formula I can be prepared by a novel fermentation method. SUMMARY OF THE INVENTION The present invention is related with compounds of the formula I wherein G signifies either structural formula la or structural formula lb. The novel compounds of the invention are I) Formula I wherein G=Ia (i.e. 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodec-3-enyl ester) and II) Formula I wherein G=Ib (i.e. 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodeca-3,6,9-trienyl ester). The present invention is also related to a fermentative process for production of compound of formula I. I The present invention is furtlner related to a novel process for preparation of compounds of formula II from compounds of formula I. DESCRIPTION OF THE DRAWINGS DRAWING 1: LC MS/MS OF Oleistatin DRAWING 2: H NMR of Oleistatin DRAWING 3: C13NMR of Oleistatin DRAWING 4: LC chromatogram of Linolenistatin DRAWING 5: LC chromatogram of tetrahydrolipstatin (Orlistat) DRAWING 6: LC chromatogram of Oleistatin before hydrogenation DRAWING 7: RT of orlistat after hydrogenation of oleistatin to orlistat DRAWING 8: HNMR of the olistat formed DRAWING 9: ^^C NMR of the orlistat formed DETAILED DESCRIPTION OF THE INVENTION The first aspect of present invention is compound of the formula I wherein G signifies either structural formula la or structural formula lb. Particularly, the compounds of the present invention are I) Formula I wherein G=Ia (i.e., 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodec-3-enyl ester) (Oleistatin) and II) Formula I wherein G=Ib (i.e. 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodeca-3,6,9-trienyl ester) (Linolenistatin). The second aspect of the invention is a process for preparation of compounds of formula I. The third aspect of the invention is preparation of compounds of formula II from compounds of formula I. The novel compounds of formula I can be manufactured by the following novel procedure disclosed below. Aerobic cultivation of a microorganism of the species Actinomycetales, preferably Streptomyces which produces these compounds in an aqueous Culture medium or solid substrate medium which contains suitable carbon, nitrogen sources and inorganic salts, feeding appropriate precursors for the production of compounds of interest and separating the compound of formula la produced from the culture broth. Streptomyces strains, which produce compounds of formula I can be isolated from, soil samples from various locations. An example is the microorganism isolated from a soil sample found in Bangalore, India, which was given the laboratory designation Streptomyces sp. BICC 7745. The media that can be used for seed as well as production fermentation comprises Soya bean meal 48g/I, Glycerol 30g/I, Silicon Antifoam Ig/I. The pH of the medium was adjusted to 7.0. The inoculum can be 24-96 hr grown culture. For the production of 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)- dodec-3-enyl ester or 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodeca-3,6,9-trienyl ester, the fermentation media may comprise with Oleic acid or Linolenic acid, respectively, at a final concentration between 0.1 g/I and 50.0 g/I. Oleic acid or linolenic acid can be also be used as ingredients of the fermentation media. The fermentation media can also be fed with Leucine. All Streptomyces strains, which produce the lipase inhibitor, are suitable for the purpose of the present invention, especially Streptomyces sp. BICC 7745 and its subcultures, mutants and variants. The cultivation of these microorganisms for the manufacture of the novel compounds can be carried out according to various fermentation methods. It can be carried out, for example, in shake flasks or in 10 L or 200 L and 1000 L fermentors. A fixed amount of spore material or mycelium or a oleistatin-producing or linolenistatin producing strain can be introduced into a liquid or sold medium which contains suitable carbon, nitrogen sources and salts that are required for the growth and the mixture is aerobically incubated at a temperature of 20 °C to 40 °C for 1-10 days. The solid substrate for fermentation can comprise one or more among wheat bran, wheat rava, oat meal, broken wheat, boiled rice, rice bran, rice rava, beaten rice, maize bran, maize grits, oat bran, bagasse, tapioca residue, soy grits, soy flaks, rice flakes, ceramic beads, glass beads, sponge or a mixture of two or more of these. The fermentation can be a fed-batch fermentation. The feeding for fed-batch fermentation can be done at the beginning of the fermentation or at intervals throughout the fermentation. The carbon source for feeding can comprise one or more among glucose, sucrose, starch maize, wheat, tapioca, potato), modified starch, maltose, malto-dextrin, soybean oil, acetate or a mixture of two or more of these. The nitrogen source for feeding can comprise one or more among ammonium sulphate, dried yeast, ammonium nitrate, sodium nitrate, bacteriological peptone, yeast extract, casein hydrolyzate, soy peptone, soy flour, cotton seed flour, corn steep liquor, amino acids or a mixture of two or more of these. Additives can comprise one or more among fatty acids, preferably oleic acid and linolenic acid for the production of 2-Formylamino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylmethyl)-dodec-3-enyl ester and 2-Formylannino-4-methyl-pentanoic acid l-(3-hexyl-4-oxo-oxetan-2-ylnnethyl)-dodeca-3,6,9-trienyl ester, respectively, which can be added either to the media or can be fed in the course of fermentation. The fermentation can be carried out at pH 6-8. The isolation of the compounds can be carried out according to novel method as given below. The broth can be extracted with water immiscible solvent, preferably ethyl acetate (EA). The water immiscible solvent layer can be separated by centrifugation and optionally concentrated. A water miscible solvent, preferably an alcohol can be added to the extract (2:1) and mixed well. The alcohol extracts can be combined and diluted with water (1:1). The product from the mixture can be extracted with water immiscible organic solvent preferably petroleum ether (1:2). The product from the water immiscible solvent can be extracted with water miscible solvent, preferably acetonitrile (1:3). The water miscible solvent containing the product can be washed with water immiscible solvent, preferably petroleum ether (2:1). The water miscible organic solvent can be evaporated to syrup. Water immiscible organic solvent preferably petroleum ether can be added to the syrup (10:1). The mixture can be treated with an adsorbent, preferably charcoal (0.02% by weight based on syrup weight) and can be stirred for 10 min. the mixture can be filtered over celite bed. The filtrate can be evaporated to syrup stage (Purified compound of formula I) (at temperature less than 40 °C) a novel process for preparation of compound of formula II from compound of formula I as given below. The syrup can be dissolved in 10 V methanol. Palladium on carbon (10% by weight based on purified compound of formula I syrup weight) can be added. Pressure can be applied, preferably upto 3 kg for 5 hrs) and the reaction mixture can be checked by TLC for the completion of reaction i.e. conversion of compound of formula I to compound of formula II. Once the reaction is over, the mixture can be filtered over celite bed. The filtrate can be concentrated to syrup and water immiscible solvent, preferably petroleum ether (15v) can be added. The mixture can be cooled to - 15 to -20 for 20 hrs. The solidified cool reaction mixture can be filtered and dried at room temperature. The solid can be dissolved in water miscible solvent, preferably methanol (10 v, based on solid weight). Water can be added (1.2 v based on solid weight). The mixture can be cooled to 0 to 5 °C and stirred for 1 hr. The mass can be filtered and suck dried and then dried at room temperature. The prior art compound, lipstatin has the following properties: Mw = 491.4 RT in HPLC is 9 min. The compound, oleistatin, of the present invention has the following properties: Mw= 493.4 Da RRT In HPLC with respect to lipstatin: 1.22 The compound, linolenistatin, of the present invention has the following properties: Mw = 489.4 Da RRT in HPLC with respect to lipstatin: 0.55 The compound, orlistat has the following properties: Mw = 495.4 RRT in HPLC with respect to lipstatin: 2 The compounds of formula II as prepared by employing compound of formula I can be used as medicaments, for example in the form of pharmaceutical preparations. Illustratively, the pharmaceutical preparations can be administered orally, for example in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions or suspensions. If desired, the compounds also can be administered parenterally. For the manufacture of pharmaceutical preparations compound of formula II prepared by employing compound of formula I can be processed with pharmaceutically inert, inorganic or organic carriers. Examples of carriers which can be used for tablets, coated tablets, dragees and hard gelatine capsules are lactose, maize starch or derivatives thereof, talc, stearic acid or its salts and the like. Suitable carriers for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like; depending on the nature of the active ingredient no carriers are, however, generally required in the case of soft gelatin capsules. Suitable carriers for the manufacture of solutions and syrups are, for example, water polyols, saccharose, Invert sugar, glucose and the like. The pharmaceutical preparations can contain preservative agents, solubilizers, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, colouring agents, flavouring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They can also contain still other therapeutically valuable substances. The following Examples illustrate the present invention in more detail, but are not intended to limit its extent. EXAMPLE 1 PRODUCTION OF 2-FORMYLAMINO-4-METHYL-PENTANOIC ACID 1- (3-HEXYL-4-OXO-OXETAN-2-YLMETHYL)-DODEC-3-ENYL ESTER (a) Fermentation: A shake flask with seed medium inoculated with spores of Streptomyces sp. BICC 7745 was aerobically incubated as a shaking culture at 28°C for 72 hour. This culture is used to inoculate a pre-culture media of 10 I. Incubation is carried out at 28 °C for 4 days with aeration of 1 wm and stirring at 400 rpm. This 10 I pre-culture is used to inoculate a 200 I production fermentor containing production medium. Fermentation was carried out at 28 °C for 120 hours with aeration of 1.0 wm and stirring at 150 rpm. The fermentation media was fed with Oleic acid 50.0 g/I and Leucine 4 g/I. The seed medium has the following composition: soya bean meal (48 g/I), Glycerol (30 g/I), Silicon Antifoam (1 g/I). The pH was adjusted to 7.0. 1 The production medium lias the following composition: soya bean meal (48 g/I), Glycerol (30g/I) Silicon Antifoam (1 g/I). The pH was adjusted to 7.0. (b) Working-up and purification: The broth was extracted with ethyl acetate and concentrated to syrup at temperature less than 40 °C. To the above extract 2 volume methanol based on syrup weight) to the syrup and stirred for 5 min and the layer separated. The separated layer was further extracted with methanol (Ivolume). The methanol extract was extracted with petroleum ether (2 volume based on syrup weight) and later by using acetonitrile and the acetonitrile layer is concentrated to syrup. 10 volume of pet ether was added to the syrup, charcolized and filtered over celite bed and crystallized to get the final product as pure lipstatin. The final product was analyzed by HPLC for purity. DRAWING 1 shows the LC MS/MS of Oleistatin The retention time of the Oleistatin was around 11 min. The retention time of Lipstatin was around 9 min. The molecular mass of the product was 493 as compared to 491 of Lipstatin. DRAWING 2 shows HNMR of Oleistatin. DRAWING 3 shows ^^C NMR of Oleistatin. EXAMPLE 2 PRODUCTION OF 2-FORMYLAMINO-4-METHYL-PENTANOIC ACID 1- (3-HEXYL-4-OXO-OXETAN-2-YLMETHYL)-DODECA-3,6,9-TRIENYL ESTER (a) Fermentation: A shake flask with seed media inoculated with spores of Streptomyces sp. BICC 7745 was aerobically incubated as a shaking culture at 28 °C for 72 hour. About 2-5 % v/v of this culture is used to inoculate a preculture media of 10 I. Incubation is carried out at 28 °C for 4 days with aeration of 1 wm and stirring at 400 rpm. This 10 I pre-culture is used to inoculate a 200 I production fermentor containing production medium. Fermentation is carried out at 28 °C for 120 hours with aeration of 1.0 wm and stirring at 150 rpm. The fermentation media was fed with Linolenic acid 30 g/I and Leucine 2.5 g/I. The seed medium has the following composition: soya bean meal (48 g/I), Glycerol (30 g/I), Silicon Antlfoam (1 g/I). The pH was adjusted to 7.0. The production medium has the following composition: soya bean meal (48 g/I), Glycerol (30 g/I), Silicon Antlfoam (1 g/I). The pH was adjusted to 7.0. (b) Working-up and purification: The broth was extracted with ethyl acetate and concentrated to syrup at temperature less than 40°C. To the above extract 2 volume methanol (based on syrup weight) to the syrup and stirred for 5 min and the layer separated. The separated layer was further extracted with methanol (1 volume). This methanol extract was extracted with petroleum ether (2 volume based on syrup weight) and later by using acetonitrile and the acetonitrile layer is concentrated to syrup. 10 volume of pet ether was added to the syrup, charcolized and filtered over celite bed and crystallized to get the final product as pure lipstatin. The final product was analyzed by HPLC for purity. DRAVyiNG 4 shows LC-MS chromatogram of Linolenistatin. The retention time of the product was around 5- min. The retention time of Lipstatin was around 9- min. The molecular mass of the product was 489 as compared to 491 of Lipstatin. EXAMPLE 3 PREPARATION OF 2-FORMYLAMINO-4-METHYL-PENTANOIC ACID 1-(3-HEXYL-4-OXO-OXETAN-2-YLMETHYL)-DODECYL ESTER The final syrup from example 1 or example 2 is dissolved in 10 v methanol (based on purified oleistatin or linolenistatin syrup). To the methanolic extract palladium on carbon is added (10% by weight based on purified oleistatin or linolenistatin syrup weight) and 3 kg pressure for 5 hrs is maintained and filtered over celite bed. The filtrate is concentrated to syrup and 15 v pet ether added; cooled to - 15 to -20 for 20 hrs; the solid filtered and dried in VTD at room temperature for 20 hrs. The solid is dissolved in 10 v methanol (based on solid weight); 1.2 v water (based on solid weight) added; cooled to o to 5 °C and stir for I hr; filtered and suck dried and then dried in VTD at room temperature for 20 hrs. The final product was analyzed by HPLC for purity. DRAWING 5 shows LC chromatogram of Standard Orlistat. The retention time of the product was around 18 min. DRAWING 6 shows LC chromatogram of Oleistatin before hydrogenation. DRAWING 7 shows RT of orlistat formed after hydrogenation of Oleistatin. The molecular mass of the product was 495 as compared to 493 of compound of formula I wherein G is la. DRAWING 8 shows H NMR of the Orlistat formed. DRAWING 9 shows ^^C NMR of the Orlistat formed. CLAIMS: We claim: 1. A compound of the formula I wherein G signified either structural formula la or structural formula lb 2. A compound of claim 1, 2-Formylamino-4-methyl-pentanoic acid l-(3- hexyl-4-oxo-oxetan-2-ylmethyl)-dodec-3-enyl ester. 3. A compound of claim 1, 2-Formylamino-4-methyl-pentanoic acid 1(3- hexyl-4-oxo-oxetan-2-ylmethyl)-dodeca-3,6,9-trienyl ester. 4. A process for preparation of compounds of formula I of claim 1. 5. A process for claim 4, wherein the compound is prepared by fermentation of microorganism capable of producing compound of formula I. 6. A process of claim 5, wherein the fermentation is carried out under suitable condition wherein oleic acid or linolenic acid is added to the fermentation media or broth. 7. A process of claim 6, wherein tlie addition of oleic acid or linolenic acid is carried out at the beginning or at any stage of the fermentation. 8. A process of claim 6, wherein the process is batch fermentation. 9. A process of claim 6, wherein the process is fed-batch fermentation. 10. A process of claim 6, wherein the process is continuous fermentation. 11. A process wherein the compound of claim 1 is converted into compound of formula II. 12. A process of claim 11, wherein the compound of formula II is prepared by hydrogenation of compound of formula I. 13. A process of claim 12, wherein the hydrogenation is carried out in presence of palladium and carbon. 14. A compound of formula I substantially as herein described with reference to the accompanying drawings. 15. A process for preparation of compounds of formula I substantially as herein described with reference to the accompanying drawings. 16. A process wherein the compound of formula I is converted into compound of formula II substantially as herein described with reference to the accompanying drawings.

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508-che-2003-abstract.pdf

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