A PROCESS FOR THE PRODUCTION OF AN INHIBITOR OF HUMAN PLATELET AGGREGATION AND SOYBEAN LIPOXYGENASE

Abstract: The present invention relates to a process for the production of 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one useful as inhibitors of human platelet aggregation as well as soybean lipoxygenase, the said compound having formula 1 being obtained from Aspergillus niger. Formula 1

Description: A PROCESS FOR THE PRODUCTION OF AN INHIBITOR OF HUMAN PLATELET AGGREGATION AND SOYBEAN LIPOXYGENASE Technical Field The present invention relates to a process for the production of 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one useful as inhibitors of human platelet aggregation as well as soybean lipoxygenase. The said compound having formula 1 is obtained from Aspergillus niger. Formula 1 Background Art Japan Patent No.06256275 claimed a process for the production of a rabbit platelet aggregation inhibitor from streptomyces sps. However, a large number of medium constituents have been used for the production of the inhibitor. Japan Patent No.04159274 claimed a benzoquinone derivative through fermentation as a rabbit platelet aggregation inhibitor. The activity claimed in the prior art is very low i.e. activity with an IC 50 of 1 mm. United States Patent No.5208364 claimed a 5 -lipoxygenase inhibitor from Streptomyces sp. The prior art describes an unwieldy process for using HPLC for purifying the inhibitor from the broth. The preparation of lipoxygenase inhibitors is disclosed in Betina, V; Prog hid Microbiol 30 (1994) 121-135. The prior art describes the production of these inhibitors, such as 3- methoxytropolone and kf 8940, from streptoverticillium sps and pseudomonas methanica, respectively. However, these metabolites have been reported to be produced from complex medium components. Studies on sulphasalazine metabolites, such as N-acetylaminosalicylic acid and 5-amino salicylic acids showing effective soybean lipoxygenase inliibition is disclosed in Allgayer,H; Eisenburg ; Paumgartner,G. Eur J Clin Pharmacol. 26 (984) 449-451. However, this prior art has no disclosure or suggestion that soybean lipoxygenase inhibitors act as inhibitors against platelet aggregation as well. From the above discussion describing the state of the art it is clear that there were no known or apparently simple fermentation medium and conditions to obtain molecules with activity against human platelet aggregation and acting as soybean lipoxygenase inhibitors. Further, none of the above prior art disclosed preparation of the compound 8-methyl 13- phenyl trideca 4,6,8,10,12 pentene-3-one Formula 1 useful as a human platelet aggregation inhibitor and soybean lipoxygenase inhibitor. Summary of the invention The objective of the present invention is to develop a process for the production of 8- methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one and exploit its use as human platelet aggregation inhibitor and soybean lipoxygenase inhibitor. The said compound has formula 1 as shown herein below. Formula 1 Detailed description of the invention Accordingly, the invention provides a process for the production of 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one having formula 1 and obtained from Aspergillus niger. The said compound is useful as an inhibitor human platelet aggregation as well as soyabean lipoxygenase. The process for the production of 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one, comprises the steps of: i) developing a medium comprising potato dextrose broth and having pH of 5- 5.5, ii) inoculating the medium in step (i) with fungi and incubating the culture at 28° to 37°C for 5 to 8 days, iii) adding ethyl acetate to the culture and incubating it at room temperature for 1-3 hrs., iv) separating the solvent from the fermentation broth by filtration, v) removing all traces of water from the culture, and drying it to obtain a crude extract, vi) subjecting the crude extract to column chromatography and eluting the compound 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one as red crystals from the orange colored fraction, and vii) purifying the compound by conventional methods. To describe in detail, the process comprises the steps of developing an inoculum for fermentation by scraping the mycelia or spores of aspergillus niger from a 3-5 day old potato dextrose agar slant transferring to a 500-2000 ml potato dextrose broth solution and incubating to 28-37°C for 24-48 hours under agitation at 100-200 rpm with the fermentation being carried out in a fermentor of 10-15 litres capacity, wherein the medium used for both inoculum development and main fermentation is potato dextrose. The broth comprises boiled 15-20 gms of peeled and cut potatoes and 1-3 gms of glucose for 100 ml of the medium, with final pH being 5.1-5.5 and the medium being sterilized at 121°C for 10-20 minutes and allowed to cool to 30°C before inoculation. Thereafter, 0.5-2 litres of inoculum is transferred to the liquid medium and incubated at 28-30°C for 5-8 days at 100- 200 rpm. At the end of the fermentation process, 1-5 litres of solvent such as ethylacetate is added to the fermented medium and incubated at room temperature for 1-3 hours, after which the fermented broth is filtered through whatman no 1 filter paper and the solvent is separated using a separating funnel. The next step is to add 10-50 gms of anhydrous sodium sulphate to remove any traces of residual water. The solvent is distilled under vacuum to give a crude extract of 0.5-2gms, which is loaded on 10-50gms of silica gel (100-200 mesh) column in hexane, wherein the column has a bed volume of 20-60 ml. After which the column is first washed with 1-2 volumes of hexane after this the inhibitor is eluted using 1-2 volumes of hexanexhloroform (1:1) in the form of an orange coloured fraction, which is distilled under vacuum to remove the solvent and obtain 100-200 mgs of this semi purified inhibitor, to which 1-10 ml of hexane is added and stored at 1-10°C for at least 2-3 days to obtain 1-5 mgs of the purified said inhibitor in the form of red crystals which has a final ic 50 activity of 234 μm against human platelet aggregation and an ic 50 activity of 95.6 μm against soybean lipoxygenase. In an embodiment of the present invention, fungi such as aspergillus and penicillin species may be used for the production of the inliibitor. The aspergillus species may be selected from known species such as A.βavus, A. japonicus, A. niger, A. awamori. These strains are equivalent to strains such as ATCC 9029, ATCC 11414, ATCC 1015, ATCC 10522, ATCC 26550, NRRL 3, NRRL 556, NRRL 337, NRRL 1042, CBS 102,49, CMI 41876. In another embodiment of the present invention, the liquid medium used is potato dextrose broth consisting of 15-20gms of potato and 1-3 gms of glucose for 100 ml of the medium. In a further embodiment of the present invention, cultivation may be effected for 3-5 days and the age of the inoculum may be 24-48 hours grown under agitation at 100-200 rpm. In yet another embodiment of the present invention, the liquid medium employed for fermentation may be sterilized at 121°C for 10-20 minutes and inoculated with 5 to 15% inoculum. In still another embodiment of the present invention, the fermentation time for the production of the human platelet aggregation inhibitor may be between 5 to 8 days. In still another embodiment of the present invention the fermented liquid medium may be extracted with water immiscible solvents like chloroform, ethylacetate. hi still another embodiment of the present invention the purification of the human platelet aggregation inhibitor comprises recovering the said inhibitor by conventional column chromatography using combinations of polar and non polar solvents. The invention, thus, provides 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one useful as human platelet aggregation inhibitor and inhibitor of soyabean lioxygenase activity. The invention is illustrated but not restricted by the description in the following example. EXAMPLE In a 15 litres capacity fermentor, 10 litres of fresh potato dextrose broth was prepared by boiling 200 gms of peeled and sliced potatoes for 20 minutes. After cooling to room temperature, 200 gms of dextrose was added, the pH was adjusted to 5.1. The flask was then autoclaved at 121°C for 15 minutes. To this 1 1 of a 24 hour old fungal inoculum was added and then incubated for 7 days at 30°C. At the end of the fermentation period, 4 litres of ethylacetate was added and the culture was incubated at room temperature for two hours. The solvent was filtered through whatman no.l filter paper. Subsequently, the solvent was separated from the broth using a separating funnel and 50 gms of anhydrous sodium sulphate was added to the solvent to remove any traces of residual water. The solvent was evaporated under vacuum to obtain 1.5gms crude extract with platelet aggregation inhibition activity of 22.5%. This was loaded on 50gms of silica gel (100-200 mesh) column in hexane to give bed volume of 40 ml. After washing the column with 100 ml of hexane, the inhibitor was eluted using 600 ml of hexanexhloroform (1:1). The orange colored fraction containing the semi purified said inhibitor was distilled under vacuum to remove the solvent. To 150 mgs of this semi purified inhibitor, 2 ml of hexane was added and stored in cold overnight to obtain 3.3 mgs of the purified inhibitor in the form of red crystals. 234 μm of the inhibitor gave 50% inhibition against human platelet aggregation and 95.6μm gave 50% inhibition against soybean lipoxygenase. Analytical data for the compound: 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one Molecular Formula: C 20 H O Molecular Weight: 278 Color and physical appearance of inhibitor: red crystals Solubility: highly soluble in chloroform, dimethylsulfoxide, acetonitrile Absorption maxima (nm) at 30 μg/ml dmso: 255, 304 and 406 nm 1H NMR spectrum (cdcl 3 , δtm S = 0 ppm): 71.4 2h d 9.03hz; 7.31 2h t 9.00hz; 7.25 3h m 9.03 hz; 6.92 lh dd lβ.lhz; 6.67 2h dd lδ.lhz; 6.96 lh dd 16.1hz; 6.39 2h dd 16.1hz; 6.21 lh d lδ.lhz; 1.14 3h 5 7.6hz; 1.97 3h s; 2.6 2h q 7.6hz. Mass spectrum: m + - ch 3 = 263; m + - ch 3 " ch 2 = 249; m + - ch 3 " ch 2 -co+ = 221; m + - 91 = 187; 187-26 = 161; t 91+ ch=105 Activity of purified inhibitor; IC 50 of the inliibitor against platelet aggregaxion:234 μm.

Claims: Claims 1. A method for the production of 8-methyl 13-phenyl, trideca 4,6,8,10,12 pentene-3- one, useful as platelet aggregation inhibitor and soybean lipoxygenase inhibitor, said process comprising the steps of: i) developing a medium comprising potato dextrose broth and having pH of 5- 5.5, ii) inoculating the medium in step (i) with fungi and incubating the culture at 28° to 37°C for 5 to 8 days, iii) adding ethyl acetate to the culture and incubating it at room temperature for 1-3 hrs., iv) separating the solvent from the fermentation broth by filtration, v) removing all traces of water from the culture, and drying it to obtain a crude extract, vi) subjecting the crude extract to column chromatography and eluting the compound 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one as red crystals from the orange colored fraction, and vii) purifying the compound by conventional methods. 2. A process for the production of a human platelet aggregation inhibitor with soybean lipoxygenase inhibition, which comprises developing inoculum for the fermentation by scraping the mycelia or spores of Aspergillus from a 3-5 day old potato dextrose agar slant and transferred to a 500-2000 ml potato dextrose agar slant and transferred to a 500-2000 ml potato dextrose broth solution and incubated at 28-37°C for 24-48 hours under agitation at 100-200 rpm and thereby fermenting the broth, wherein the medium used for both inoculum development and main fermentation is potato dextrose broth comprising boiled 15-20 gms of peeled and cut potatoes and 1-3 gms of glucose for 100 ml of the medium, with the final pH being 5.1-5.5 and the medium being sterilized at 121°C for 10-20 minutes and allowed to cool to 30°C before inoculation, into which 0.5-2 litres of inoculum is transferred to the liquid medium and incubated at 28-30°C for 5-8 days at 100-200 rpm, and at the end of fermentation 1-5 litres of solvent such as ethylacetate is added to the fermented medium and incubated at room temperature for 1-3 hours, after which the fermented broth is filtered through whatman no.l filter paper and the solvent is separated using a separating funnel, adding 10-50 gms of anhydrous sodium sulphate to remove any traces of residual water, and the solvent being distilled under vacuum to give a crude extract of 0.5-2 gms, which is loaded on 10-50 gms of silica gel (100-200 mesh) column in hexane, wherein the column has a bed volume of 20-60 ml, after which the column is first washed with 1-2 volumes of hexane after which the inhibitor is eluted using 1-2 volumes of hexane hloroform (1:1) in the form of an orange coloured fraction, which is distilled under vacuum to remove the solvent and obtain 100-200 mgs of this semi purified inhibitor, to which 1-10 ml of hexane is added and stored at 1-10°C for at least 2-3 days to obtain 1-5 mgs of the purified said inhibitor in the form of red crystals which has a final ic 50 activity of 234 μm and 95.6 μm against human platelet aggregation and lipoxygenase inhibition, respectively. 3. A process as claimed in claim 2 wherein the fUngi are selected from Aspergillus and Pencillium species. 4. A process as claimed in claim 2 wherein the fϊingi are selected from Aspergillus flavus, Aspergillus japonicus, Aspergillus niger and Aspergillus awamori. 5. A process as claimed in claim 2 wherein the medium in step (i) comprises potato and glucose. 6. A process as claimed in claim 2 wherein the fungi are cultivated for 3-5 days in a medium containing potato, dextrose and agar under agitation at 100-200 rpm. 7. A process as claimed in claim 2 wherein the fermentation time for the production of the human platelet aggregation inhibitor with soybean lipoxygenase inhibition is between 5 to 8 days. 8. A process as claimed in claim 2 wherein the medium is inoculated with 5 to 15% of the inoculum. 9. A process as claimed in claim 2 wherein the fermented medium is extracted with water immiscible solvents such as chloroform and ethylacetate 10. A process as claimed in claim 2 wherein 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one is purified by column chromatography employing a combination of polar and non polar solvents. 11. A process as claimed in claim 2 wherein the solvents for column chromatography are hexane and chloroform. 12. A method of using 8-methyl 13-phenyl trideca 4,6,8,10,12 pentene-3-one as a human platelet aggregation inhibitor and as inhibitor of soyabean lipoxygenase.

Inventors: DIVAKAR SOUNDAR, KARANTH NAIKANAKATTE GANESH, KRISHNAKANTHA THIRUMALAI PARTHASARATHY, RAO KADIVALA CHANDRA S, SATTUR AVINASH PRAHLAD, SUNEETHA WESLEY JESSIE, VISHWANATHA SURYANARAYANA, DIVAKAR, SOUNDAR, KARANTH, NAIKANAKATTE, GANESH, KRISHNAKANTHA, THIRUMALAI, PARTHASA, RAO, KADIVALA, CHANDRA, S., SATTUR, AVINASH, PRAHLAD, SUNEETHA, WESLEY, JESSIE, VISHWANATHA, SURYANARAYANA
Applicants: COUNCIL SCIENT IND RES, DIVAKAR SOUNDAR, KARANTH NAIKANAKATTE GANESH, KRISHNAKANTHA THIRUMALAI PARTHASARATHY, RAO KADIVALA CHANDRA S, SATTUR AVINASH PRAHLAD, SUNEETHA WESLEY JESSIE, VISHWANATHA SURYANARAYANA, COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH, DIVAKAR, SOUNDAR, KARANTH, NAIKANAKATTE, GANESH, KRISHNAKANTHA, THIRUMALAI, PARTHASA, RAO, KADIVALA, CHANDRA, S., SATTUR, AVINASH, PRAHLAD, SUNEETHA, WESLEY, JESSIE, VISHWANATHA, SURYANARAYANA
Application year: 2001
Main classification: C12P7
IPC codes: A61K31, A61P7, A61P43, C12N9, C12P7, C12R1