Indian Patents. 220711:A NOVEL ANTI-FUNGAL MOLECULE 2-(3,4-DIMETHYL-2, 5-DIHYDRO-1H-PYRROL-2-YL)-1-METHYLETHYL PENTANOATE

Title of Invention	A NOVEL ANTI-FUNGAL MOLECULE 2-(3,4-DIMETHYL-2, 5-DIHYDRO-1H-PYRROL-2-YL)-1-METHYLETHYL PENTANOATE
Abstract	The present invention relates to a novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate. It particularly relates to a novel anti-fungal lead molecule isolated from a plant Datura metel.

Full Text	The present invention relates to a novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate. It particularly relates to a novel anti-fungal lead molecule isolated from a plant Datura metel. The main utility of the invention is to provide a process for identifying a new lead molecule for development of new drugs for treating diseases caused by pathogenic fungi. The incidence and mortality due to mycotic infections has been observed to be on the rise in the past decade (Saral, R., 1991, Rev. Inf. Dis. 13, 487-492., Koll, B.S. and Brown, A.E. 1993, Clin. Inf. Dis. 17, S-322-S-326). Infections induced by these species are increasingly recognized as emerging threat to the public health. In immuno-compromised host, the infections by Aspergillus, Candida, Histoplasma etc. become invasive and disseminate from primary site of infection to other parts of the body including gut, kidney, brain etc. Invasive aspergillosis is reported to be associated with a mortality rate of 55% (Denning, D.W. and Stevens, D.A., 1990, Rev. Inf. Dis., 12, 1147-1201). Mortality due to Aspergillus infection in bone marrow transplant recipients was observed to be as high as 80% despite appropriate chemotherapy (Meyer,'J.D., 1990, Semin. Oncol. 17 (suppl. 6), 10-13). Cerebral aspergillosis presents the symptoms of acute meningitis and is always fatal. Candida species are commensal organisms of human mucocutaneous surface. However, in immunocompromised host, Candida may progress to become opportunistic potential pathogen. It may be transmitted by contact to other immuno-compromised patients as a nosocomial pathogen. It is, therefore, important to diagnose and treat these fungal infections, at early stage to prevent irreversible damage. For the treatment of mycoses, several compounds belonging to azoles, polyenes and other proups of chemicals have been described in the literature. But all of these compounds have their own limitations. Polyenes are among the oldest and most frequently prescribed anti-fungal agents. Amphotericin B is an important polyene drug which has been found to have a broad range activity against fungi, but its therapeutic doses are frequently associated with severe chills, fever, vomiting, life threatening hypotension and respiratory distress. Amphotericin B produces renal dysfunction (Allende, M.C., Lee, J.W., Francis, P., Garrett, K., Dollenberg, H., Berenguer, J., Lyman, C. A., Pizzo, P.A. and Walsh, T.J. 1994; Antimicrob. Agents and Chemother. 38, 518-522) and necessity of its intravenous administration is its major limitation (Stevens, D.A. and Lee, J.Y., 1997, Arch. Int. Med., 57, 1857-1862). In addition to its adverse effects, Amphotericin B resistance in fungal species has been reported (Gokhale, P.C., Kshirsagar, N.A. and Pandya, S.K., 1993, Current Science 65(6), 448-454, Forthergill, A.W. and McGough, D.A., 1995, Clin. Microbiol. Procedure Handbook, In Vitro susceptibility Testing of Yeast, 5.15.1-5.15.15). Nystatin is another important drug, however, the development of resistance and its dose limited toxicity make this drug also less useful (Forthergill A.W. and McGough A.W., 1995, Clin. Microbiol. Procedure Handbook, In Vitro susceptibility Testing of Yeasts, 5.15.1-5.15.15). New azole anti-fungals such as ketoconazole, fluconazole and itraconazole provided the hope that these compounds might be useful to prevent or treat fungal infections. However, their liver toxicity, hypoglycemic nature and development of resistant strains of fungi did not keep this hope alive for longer period (Denning, D.W. Tucker, R.M., Hanson, L.H., Hamilton, J.R. and Stevens, D.A., 1989, Arch. Int. Med., 149, 2301-2308). The azoles are reported to decrease the secretion of stomach acid and thereby reducing the absorption of drug itself and other important biomolecules. These effects may cause severe drop in body glucose to a level which may be life threatening. Resistance against the azole derivatives also has been reported in fungi (Forthergill A.W. and McGough A.W., 1995, Clin. Microbiol. Procedure Handbook, In Vitro susceptibility Testing of Yeasts, 5.15.1-5.15.14). Currently available anti-fungal drugs are not sufficiently broad in their spectrum and not consistently effective against fungi including Aspergilli. These drugs are either toxic or immunosuppressive in nature. Further, the development of resistance in fungi to most of available drugs has also been reported. It is evident from the above that need exists for development of new anti-fungal drugs which obviate the drawbacks listed above. The novelty of the present invention is to provide a process for isolation of a novel anti-fungal lead molecule from Datura metel which is many fold less cytotoxic as compared to standard anti-fungal drug such as amphotericin B. The main object of the present invention is to provide a novel anti-fungal lead compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate for developing new drugs against the pathogenic fungi. Another object of the invention is to provide the process for isolation of the novel anti-fungal lead molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate from a plant Datura metel. Another object of the present invention is to provide a method for testing the novel compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate as an anti-fungal agent. Yet another object of the invention is to provide usage of the novel compound 2-(3.4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate as an anti-fungal agent. Yet another object is to provide a novel anti-fungal lead molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate which is 57.8 times less cytotoxic than the standard anti-fungal drug such as Amphotericin B. List of Figures accompanying the specification: Fig 1: Photograph of a plate showing TLC pattern of the active column sub-fractions and purified compound. Fig 2: HPLC profile of TLC purified compound Fig 3: Photograph of TLC of HPLC purified compound. Fig 4: HPLC profile of purified compound. Fig 5: Inhibition of growth of A.fumigatus by purified compound. Fig 6: Graph represents the percent spore germination inhibition by the compound against Aspcrgillus species. Fig 7: UV spectrum of the compound. Fig 8: IR spectrum of the compound. Fig 9: Mass spectrum of the compound Fig 10: NMR spectrum of the compound. Fig 11: COSY spectrum of the compound. Fig 12: Percent cytotoxicity of the compound Fig 13: The dose required to protect 50% of the animals (EDso) after treatment with PC-1. Summary of the invention: The present invention is directed to a novel anti-fungal compound 2-(3,4 dimethyl-2,5-dihydro-lh-pyrrole-2-yl)-l-methylethyl pentanoate of the formula 1 as given below. The compound has anti-fungal properties and is several times less cytotoxic as compared to the standard anti-fungal drugs. This compound may be useful for controlling systemic and superficial fungal infections in humans with fewer toxic effects. This compound has been isolated from an easily available plant Datura metel. Accordingly the present invention provides a novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate of formula 1 (Figure Removed) Formula 1 In an embodiment, the characterization of the active anti-fungal lead molecule is carried out by known analytical methods such as TLC, group specific chemical staining, UV, infra-red, mass and nuclear magnetic resonance spectroscopy and biological assays such as anti-mycotic assay and cyto-toxicity assay. In still another embodiment to the invention, the novel anti-fungal lead molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate is a heterocyclic alkaloid as shown by group specific chemical staining using Dragendorff reagent and has following characteristics: Rf value of 0.22 on TLC (Fig 3), Absorption maixma at 300 nm and 206 nm in UV spectrum (Fig . 7 ) IR V Max: 3434 (NH), 3024, 1711 (ester), 1646 (OC), 1524, 1426,1230, 938, 760 cm'1 (Fig 8). +ve FABMS m/z: 239 [M]+ (CM H25 O2 N) (16.3), 212 (13.2), 174 (16.2), 122 (34.2), 102 (96.4), 58 (100), and 57 (19.2) (Fig 9). 'H NMR (CDC13): 5 4.15 (lH,m, H-3), 3.75 (lH,m, H-l), 3.17 (2H,dd, J=7.44, 7.44Hz, H2-5), 2.31 (1H, t, J=6.48 Hz, H-2'a), 1.95 (1H, m, H-2TD), 1.62 (6H, brs, C-6 Me, C-7 Me), 1.42 (1H, d, J=6.96 Hz, H-2a), 1.35 (1H, d, J=7.28Hz, H-2b), 1.25 (7H,brs, Me-4, H2.3'-, H2.4'.), 0.87 (3H, t, J=5.96 Hz, Me-5') (Fig 10) COSY spectrum (Fig 11). The minimum inhibitory concentration (MIC) of the said novel anti-fungal lead molecule is 5.0 (ag/disc by disc diffusion assay (Fig. 5) and 87.5 \|ag/ml by percent spore germination inhibition (Fig. 6) respectively. The dose cytotoxic to 50% of the cells (CTso value) of the said anti-fungal molecule is 889.2 (ig/ml (Fig 12). The EDso for the said anti-fungal compound is 167.0 mg/kg body weight (Fig 13). The anti-fungal effective dose to the said subject ranges from 100 to 400 mg/kgbody weight (Table 3). The process for isolation of novel molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-1-methylethyl pentanoate is disclosed and claimed in our copending patent application no.NF343/2001. In another embodiment, the novel anti-fungal compound is 57.8 times less cyto-toxic than the standard anti-fungal drug. In further embodiment is provided a method for treatment as wherein, the dose of 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate, effective for survival of 50%(ED50) of the said subjects is 167.Omg/kg body weight (Fig. 13). In still another embodiment, the protective in vivo effective dose of the anti-fungal compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate to the said subject ranges from 100 to 400 mg/kg body weight (Table 3). Accordingly the present invention also provides a pharmaceutical composition comprising an acceptable amount of above compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate with pharmaceutically acceptable carrier, additives and adjuvants. hi still another embodiment, the acceptable additives are selected from the nutrients, which are pharmaceutically acceptable carrier. In another embodiment to the invention the novel anti-fungal molecule may be used in the form of tablet, capsule, syrup, powder, ointment. In yet another embodiment the pharmaceutically acceptable composition contains the effective amount of novel molecule at a concentration in the range of 100 to 400 mg/ml. A method for the treatment of fungal infections in a said subject comprising the steps of administration of an effective amount of 2-(3,4 dimethyl-2,5-dihydro-lh-pyrrole-2-yl)-l-methylethyl pentanoate through routes such as oral, nasal, intra venous, intra-peritoneal, intra muscular etc. In an embodiment of the invention the effective dose of 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate may be in the range of 100-400 mg/kg of body weight. Another aspect of the invention is to provide a process for isolation of a novel anti-fungal compound 2-(3,4 dimethyl-2,5-dihydro-lH-pyrrole-2-yl)-l-methylethyl pentanoate, from a plant, Datura metel, which comprises (i) extracting successively the powdered Datura metel plant material with an organic solvent at a temperature in the range of 15 to 45 °C (ii) removing the solvent to obtain residue (iii) extracting the above said residue obtained in step (ii) with an aliphatic hydrocarbon solvent followed by extraction with chloroform (iv) removing chloroform from chloroform fractions (v) screening the above obtained chloroform fractions for antimycotic activity (vi) separating the anti-fungal fractions and purifying the novel anti-fungal lead molecule from active antimycotic fractions by conventional chromatographic methods (viii) assaying the said anti-fungal lead molecule for its cytotoxicity. In an embodiment to the invention the solvent used for extraction may be selected from methanol, ethanol, acetone, chloroform. The aliphatic hydrocarbon solvent used may be selected from hexane, petroleum ether. In another embodiment to the invention, the purification of compound is carried out by column or thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). In yet another embodiment to the invention, the purification of novel compound maybe carried out by Thin Layer Chromatography using solvent systems selected from Chloroform: Acetone: Diethylamine (5.0:4.0:1.0), Chloroform: Methanol: Diethylamine (8.5:1.5:0.1) and Chloroform: Methanol: Formic acid (8.0:2.0:0.1) or different combinations of above said organic solvents. In yet another embodiment to the invention, the purification of novel compound may be effected by HPLC using solvent system 70:30 of acetonitrile and water using reverse phase RP-8 column. In still another embodiment, the invention provides a method of testing of novel compound (2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate) as an anti-fungal agent. In yet another embodiment to the present invention, the antimycotic activity may be tested after each purification step by known methods such as microbroth dilution (MD), disc diffusion (DD) and spore germination inhibition (SGI). Details of Invention 1. Purification of the compound . The invention provides a novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate a process for its isolation. It particularly relates to the isolation of a novel anti-fungal lead molecule from natural source. (i) Preparation of methanolic extract Plant material Datura metel (Solanaceae) was collected during the month of April/May from near about the railway station Kishanganj, Delhi, India. Hamdard College of Pharmacy, New Delhi, authenticated the identification of plant material where voucher sample has been preserved. The freshly collected plant material was dried in the shade in a well-ventilated enclosure. Dried material was powdered and extracted with methanol. All the extracts obtained after four cycles of extraction with methanol were pooled and evaporated to dryness under reduced pressure at 45 °C in Rotavapor R-l 14 (Buchi) attached to a Waterbath B-480 (Buchi). The residue was obtained and stored at 4°C till use. Methanolic extract was examined for its anti-fungal activity against species of Aspergillus. The extract having anti-fungal activity was subjected to further fractionation for isolation of the active anti-fungal component (ii) Fractionation of methanolic extract of Datura metel The constituents of crude methanolic preparation were fractionated by the method of Harbourne, with slight modifications (Harbourne J.B., 1997, Phytochemical Methods, Chapman and Hall, London, pp. 1-5). The Methanolic extract was successively extracted with hexane, chloroform and acetone. The methanolic extract was first extracted with hexane four times. All the four extracts of hexane were pooled and solvent was evaporated with the help of Rotavapour R-l 14 (Buchi). The dry fraction was collected and kept at 4°C till further use. The residue after the extraction with hexane was extracted stepwise with chloroform and acetone four times as in case of hexane to obtain chloroform and acetone fractions. The leftover residue was finally dissolved in methanol. Solvents were evaporated and dry fractions were recovered. All the fractions were labeled properly and examined for their anti-fungal activity using pathogenic species of Aspergillus. The chloroform fraction was found to be active, therefore, it was further sub-fractionated by column and thin layer chromatography. (iii) Column Chromatography The chloroform fraction was sub-fractionated by modified column chromatography using a silica gel column. Silica gel was suspended in hexane and packed in a glass column of 1.25x35 cm size. Slurry of chloroform extract was prepared and loaded on to the top of the pre-equilibrated silica gel (60-120 \|J) column. The components of chloroform fraction were eluted with 100 ml of chloroform at a flow rate of 1.0 ml/min followed by different ratio of chloroform: methanol ranging from 100:0 to 0:100. All the sub-fractions were analysed by TLC. The sub-fractions showing similar profile of Rf values were pooled and dried in vacua. The anti-fungal activity of each sub-fraction was tested using pathogenic strains of Aspergill/. The sub-fractions, which showed anti-fungal potential, were further subjected to the thin layer chromatography for identifying and separating out pure active component. (iv) Thin layer chromatography (TLC) The active anti-fiingal column fractions were spotted onto the Silica gel plates (E. Merck Cat No. 1.05554, ¥254) and subjected to TLC with three different solvent systems i.e. Chloroform: Acetone: Diethylamine (5.0:4.0:1.0), Chloroform: Methanol: Diethylamine (8.5:1.5:0.1) and Chloroform: Methanol: Formic acid (8.0:2.0:0.1). The bands on the plates were visualised with UV light at 254 nm and 366 nm and by spraying with Chloropalatinate, Dragendorf f reagent and Iodine. The components in different bands were scrapped and examined for anti-fungal activity. The preparative TLC was performed to obtain active component of our interest. (v) High pressure liquid chromatography (HPLC) The purity of the compound PC-1 obtained from preparative TLC was analyzed by HPLC using RP-8 column (Merck). The reverse phase HPLC was performed isocratically with the solvent system 70 parts acetonitrile: 30 parts water. The test samples were passed through a membrane of pore size before loading. 5.0 pi of the 0.22 (j filtered sample was loaded on to the pre-equilibrated HPLC column at room temperature. The flow rate was maintained at 1.0 ml/min. 2. Anti-fungal Activity Assays The anti-fungal activity of various fractions such as crude, methanolic extract, sub-fractions and purified compound was studied by microbroth dilution, disc diffusion and spore germination inhibition assays using pathogenic strains of fungi. (i) Pathogen Pathogenic strains of fungi were obtained from the Mycology Department of Vallabhbhai Patel Chest Institute, Delhi. All the strains were grown on Sabouraud dextrose agar at 37°C. The conidia form these cultures were harvested and suspended in Sabouraud maltose broth. The number of conidia was counted using haemocytometer and their number in the suspension was adjusted to as per need of the experiment. (ii) Microbroth Dilution Assay The anti-fungal susceptibility of the fungi to various fractions or the purified component was assayed by the microbroth dilution method (Forthergill A.W. and McGough A.W., 1995, Clin. Microbiol. Procedure Handbook. In Vitro susceptibility Testing of Yeast, 5.15.1-5.15.15). The fungal spores were harvested from 96 h cultures and their number adjusted to IxlO6 per ml. The Sabouraud Dextrose medium was dissolved in glass double distilled water and autoclaved at 10 psi for 15 min. 90 \|Jl medium was added into the wells of cell culture plates. The different concentrations of the extract, fractions or the purified compound were prepared in duplicate wells and then the wells were inoculated with 10 pi of spore suspension. The plates were incubated at 37 0 C and examined macroscopically after 48 h for the growth of fungal mycelia. The activity was represented as -ve if growth was there and +ve if medium appeared clear with out any growth of fungi. (iii) Disc Diffusion Assay The disc diffusion test was performed in 10 cm diameter (Tarsons) radiation sterilized petri plates as per the method described in Indian Pharmacopoeia (Indian Pharmacopoeia, 1996, Appendix 9, p A101-A110). Sabouraud Dextrose agar medium was dissolved in double glass-distilled water and autoclaved at 10 psi for 15 minutes. It was cooled to 45°C and 20.0 ml was poured into each petri dish. IxlO6 conidia in 1.0 ml of conidial suspension were prepared in Sabouraud maltose broth and overlaid on the surface of the agar plate. Different concentrations of the samples were impregnated on the sterilized discs measuring 5.0 mm in diameter from Whatman filter paper number. The discs were placed on the surface of the agar plates already inoculated with the fungal spores. The plates were incubated at 37°C and examined at 48 hrs for zone of inhibition, if any, around the disc. The diameter of zone of inhibition was measured with the help of a scale. The concentration, which developed the zone of inhibition of 6.0 mm diameter, was considered as minimum inhibitory concentration. Amphotericin B was used in assay as a standard control drug. An additional control disc without any sample but impregnated with equivalent amount of solvent was also used. The test was repeated three to five times with various test preparations of plant to ascertain activity. (iv) Preparation of Standard Curve Standard curve was prepared in accordance with the method given in the Indian Pharmacopoeia (Indian Pharmacopoeia, 1996, Appendix 9, p A101-A110). (v) Spore Germination Inhibition Assay The modified spore germination inhibition assay was performed as described earlier (Sureder P. and Janaiah C., 1987, Ind. J. Expt. Biol., 25, 233-234). The fungal species were grown on Sabouraud dextrose agar plates at 37°C for 96 h. Conidia were harvested from the plates and their homogenous suspension was prepared in the Sabouraud dextrose broth. The conidia were counted and their number in the suspension was adjusted to IxlO4 per ml. The standard drug Amphotericin B and test samples were initially dissolved in minimal quantity of dimethylsulfoxide (DMSO) and then diluted with Sabouraud dextrose medium. Various concentrations of the test samples in 90 \|Jl of culture medium were prepared in 96 well flat bottom micro-culture plates (Nunc) by double dilution method. The wells were prepared in triplicates for each concentration. Each well was then inoculated with 10 pi of spore suspension containing 100+5 spores. The plates were incubated at 37°C for 10 h and then examined for spore germination under inverted microscope (Nickon Diphot). The number of germinated and non-germinated spores was counted. The percent spore germination inhibition (PSGI) was calculated using following formula No. of spores germinated in drug treated well PSGI = 100 - x 100 No. of spores germinated in control well 3. Characterization studies: (i) Structure elucidation Characterisation of the compound was carried out using various techniques such as class identification by chemical methods, derivatisation of the compound, melting point of the purified compound, infra red spectroscopy (Fig 8), ultraviolet spectroscopy, mass spectroscopy (Fig 9), nuclear magnetic resonance spectroscopy (Fig 10) and. COSY (Fig 11). Details of the spectral data obtained with respect to novel compound is given above. (ii) In vitro toxicity evaluation: The in vitro toxicity of the active molecule was studied by MTT assay using RAW cells (Mossman, T., 1983, J. Immunol. Methods, 65, 55-63). Cell culture: The stock culture of RAW cells was obtained from National Facility for Cell and Tissue Culture, Pune. Cells were maintained in RPMI-1640 medium supplemented with glutamine (2.3 gm/L), fetal calf serum (10% v/v) and gentamycin (50.0 mg/L) at 37°C in Nuair IR Autoflow water Jacketed carbon dioxide incubator. Sample preparation: The stock solution of the compound was prepared by dissolving 2.5 mg of the purified compound in minimum amount of DMSO and then diluted with double distilled water to make the final volume 1.0 ml. The different concentrations ranging from 1250.0 }a,g/ml to 19.5 \|ag/ml were tested in the cytotoxicity assay. Cell cytotoxicity assay: The cells were harvested at the log phase of growth confluency from the flask. The homogeneous suspension of cells was prepared in 2.0 ml of culture medium. The number of cells in the suspension was counted using a hemocytometer and the cell suspension was diluted in such a way so as to obtain 5xl07 viable cells per ml. The viability of the cells was checked with trypan blue dye exclusion test. The cytotoxicity assay was performed in 96 wells flat bottom tissue culture plates (Nunc Nunclon). The cells (5xl06) in 100.0 yl volume were seeded into each well. The plate was incubated at 37 °C in atmosphere of 5% (v/v) COa for 8 h. The wells were examined microscopically for the formation of monolayer of the cells. Various concentrations ranging from 1250.0 ug/ml to 19.5 ug/ml of the compound PC-1 (metelatropinyl ester) were added to the monolayer of cells. In +ve control wells, a known cytotoxic protein obtained from A. fumigatus, was used. The ZnSC>4 (8.0 mg/L) was used as another +ve toxic material in another set of wells. In negative control wells equivalent amount of the solvent was used. The duplicate wells were used for each concentration of the test compound. The plates were incubated at 37 °C in 5% (v/v) COa incubator overnight (12 h). The medium along with floating dead cells from the wells was removed by inverting the plate. Thus only the live cells sticking to the surface of plate were left in the wells. The cells were stained with 40.0 ^1 of 2.5 % dye MTT. After adding the dye to the wells, the plate was kept at 37 °C in 5% CO2 for 1 h. The tissue culture plate was removed from the incubator and all the dye was aspirated. Only live cells took up the dye. The cells were lysed by adding 100.0 jj,l of isopropanol-HC1 to the wells. After lysis of the cells, the plates were read at 540 nm using plate reader (Spectra Max 190, Molecular Device). Interpretation of results: The toxicity of the compound was expressed as percentage with respect to that obtained in -ve control sample. The percent cytotoxicity was calculated using the formula given below. OD in -ve control - OD in the test % Cytotoxicity = — x 100 OD in -ve control (iii) In vivo Efficacy of PC-1 In vivo efficacy of the compound against A. fumigatus infection was studied in the Balb/C mice of 6-8 weak of age of either sex, weighing 15-20 g each. The mice model of aspergillosis described by Dixon et al (1989), was used to study the effects of compound on in vivo infection of A. fumigatus. The animals were housed in the micro-barrier cages on sterile bedding and fed ad libitum water and food. The mice were divided into 6 groups and each group contained 8-10 animals. The mice were infected experimentally with A. fumigatus. Three days prior to infection with conidia, mice were injected sub-cutaneously with 3 doses (250.0 mg/kg/day) of cortisone acetate in 400.0 ul of PBS. On the infection day, each mouse received 2xl07 conidia by nasal instillation of a single droplet of conidial suspension. The animals^ of this model developed invasive aspergillosis and all the infected animals died within 4-6 days. Inoculum prepration for A. fumizatus for experimental infection to mice: A. fumigatus (190/96) isolate was grown on Sabouraud dextrose agar plates at 37 °C for 4 days. The conidia were collected from the culture plates using PBS (pH 7.2) containing 0.05% Tween 80 (Sigma Chemicals) and the suspension was filtered through sterile glass wool. The conidia were pelleted by centrifugation at 2000 rpm and re-suspended in PBS, pH 7.2. The number of conidia was counted and adjusted to IxlO8 conidia/ml. The viability of the conidia was determined by plating the dilutions of suspensions on Sabouraud dextrose agar. The purified compound (metelatropinyl ester) isolated from the D. metel was administered orally in the concentration ranging from 0.0, 25.0 50.0 100.0 200.0 and 400.0 mg/kg body weight to the mice infected experimentally with A. fumigatus. Treatment: The dosing of the animals (cortisone treated and challenged) with compound in 6 different groups was initiated within 30 min after challenge with A. fumigatus conidia. Group I: The mice were treated orally with 7 doses of 25.0 mg/kg/day of the compound. Group II: The animals infected with A. fumigatus conidia were given 50.0 mg/kg/day of the compound for 7 days orally. Group ffl: The animals in this group were given 7 oral doses of 100.0 mg/kg/day of the compound. Group IV: In this group of animals, 7 oral doses of the compound (200.0 mg/kg/day) were given. Group V: The animals of this group were treated with 7 doses of 400.0 mg/kg/day orally over a period of 7 days. Group VI: The animals of this group acted as control and were treated orally with 7 doses of 400 ul PBS containing solvent. Survival Rate: The animals were housed in properly labeled cages and kept under close watch for weight loss and the mortality. The survival rate over a period of 10 days was calculated and the fungal burden in survivors determined (demons and Stevens, 1994). Of the animals treated with doses of 25.0, 50.0, 100.0, 200.0 and 400.0 mg/kg body weight, 1 out of 10 (10%), 1 out of 8 (12.5%), 3 out of 9 (33.3%), 4 out of 9 (44.4%) and 8 out of 10 (80%) respectively, survived up to 10th day. Quantification of Colony Forming Units: The mice were kept under constant watch and those getting moribund, dying or survived up to 10 days were sacrificed. The autopsy was performed on the mice who had died to remove their organs for quantification of colony forming units (CPU). The lungs, livers and kidneys of the mice were removed aseptically, placed in sterile PBS (pH 7.2) and homogenized with teflon pestle mortar. The CPU in the animals were determined by plating 10 fold dilutions of organ homogenates on Sabouraud dextrose agar containing 0.05% triton X-100. The triton X-100 limited the colony size and thus greatly facilitated colony counting (Frosco, 1992). After an incubation for 48 h at 37 °C, colonies were counted and results were expressed as CPU per organ. The survival time and CPU indicating the fungal burden in various organs were considered to evaluate the protective efficacy of the compound in vivo The following examples are given by way of illustration of the present invention and should not be construed to limit of the scope of the present invention. Example 1 Extraction and fractionation Aerial parts of the plant Datura metel (Solanaceae) were collected during the month of April from an area around Delhi, India. Hamdard College of Pharmacy, New Delhi, authenticated the identification of plant material where voucher sample has been preserved. The freshly collected plant material was dried in the shade in a well-ventilated enclosure. 400 gm dried material was powdered and extracted with 500 ml methanol in each cycle. All the extracts obtained after four cycles of extraction were pooled and evaporated to dryness under reduced pressure. The 25 gm residue was obtained and stored at 4°C till use. The methanolic extract was successively extracted with hexane, chloroform and acetone. The methanolic extract was first extracted with hexane four times. All the four extracts of hexane were pooled and solvent was evaporated. The dry hexane fraction was collected and kept at 4°C till further use. The residue after the extraction with hexane was extracted stepwise with chloroform and acetone four times as in case of hexane to obtain chloroform and acetone fractions. The leftover residue was finally dissolved in methanol. Dried fractions were examined for their anti-fungal activity. The fractions, which did not inhibit the growth of Aspergillus up to a concentration of 1500 pg/ml and 50 (Jg/disc in spore germination inhibition and disc diffusion assays respectively, were considered to be non-active. The chloroform fraction of Datura metel was found to be active as a concentration of 1250 pg/ml inhibited the germination of 100% of the spores in spore germination inhibition assay and a concentration of 25.0 pg/disc produced the zone of inhibition of activity significance. Therefore, this fraction was further used to identify and purify the active component using various chromatographic methods. Example 2 Purification of active compound The chloroform fraction was sub-fractionated by modified column chromatography as described above. Fractions were analyzed by TLC and those showing similar spots were pooled and dried in vacua to get sub-fractions. Total 15 sub-fractions were obtained. The anti-fungal activity of each sub-fraction thus obtained was tested using pathogenic strains of Aspergillus. The fraction numbers 10 and 11 showed anti- fungal potential and they were further subjected to the thin layer chromatography for separating out pure active component. Silica gel plates (Cat No. 1.05554, F254) were purchased from E. Merck and used for performing TLC. The column chromatographic active sub-fractions were spotted onto the plates and subjected to run with three different solvent systems i.e. chloroform: acetone: diethylamine (5.0:4.0:1.0), chloroform: methanol: diethylamine (8.5:1.5:0.1) and chloroform: methanol: formic acid (8.0:2.0:0.1). The bands on the plates were visualized with UV light at 254 nm and 366 nm and by spraying with chloropalatinate, Dragendorf f reagent and iodine. The components in three different major bands resolved with a solvent system chloroform: methanol: formic acid (8.0:2.0:0.1) (Fig 1) having Rf values 0.14, 0.22 and 0.37 were scrapped and examined for anti-fungal activity. The compound, which had efficacy against Aspergillus, was recovered from a band having Rf value of 0.22. The compound was purified by repeated preparative TLC and tested for its anti-fungal activity. The active compound reacted positively with Dragendorff s reagent showing thereby its nature to be alkaloidal. The purity of the compound obtained from preparative TLC was analyzed by HPLC using RP-8 column (Merck). The reverse phase HPLC was performed isocratically with the solvent system acetonitrile: water (70:30). Samples were passed through a membrane of 0.22 \|J pore size before loading. A total of 5.0 (Jl of the sample was loaded on to the pre-equilibrated column at room temperature. The flow rate was maintained at 1.0 ml/min. The HPLC profile of compound purified by TLC showed two peaks, peak-I and II, which had retention times 1.91 and 2.71 respectively (Fig 2). These two corresponding peaks at retention time 1.88 and 2.84 were present in the HPLC profile of active sub-fraction obtained from the column chromatography also. The active column sub-fraction had five major peaks corresponding to retention time 1.31, 1.88, 2.84, 3.82 and 4.35. Changing the solvent system in TLC carried out the further purification of partially purified compound. The solvent system comprised of chloroform: methanol: diethylamine (8.5:1.5:0.1) resolved the active molecule at Rf 0.42 (Fig 3). The retention time of compound in HPLC was found to be around 1.9 and it was showing purity from 97% to 100% in various runs (Fig 4). The retention time of the compound remained same in both chromatographic active sub-fraction and its purified form. Example 3 Anti-fungal activity of pure compound by microbroth dilution assay The anti-fungal susceptibility of the fungi to the purified compound was assayed by the microbroth dilution method The different concentrations of the purified compound ranging from 21.87 to 350 fJg/ml were prepared in duplicate wells by twofold dilution method. The test was done as described above. The anti-fungal activity of the compound is given in the Table 1. The MIC of the compound was found to be 87.5 (JQ/ml. At this concentration of compound none the three species of Aspergillus showed any visual growth in wells. Table 1: Activity of the compound against Aspergillus sp. after 48 hrs. (Table Removed) Example 4 Anti-fungal activity of pure compound by disc diffusion assay The disc diffusion test was performed as per described in the section of details of invention. Amphotericin B 1.25 pg/disc was used in assay as positive control standard drug. Additional discs impregnated with equivalent amount of solvent were also used in the assay as negative controls (Fig: 5). The MIC of the compound was observed to be 5 (jg/disc since this concentration developed zone of inhibition having mean diameter of 6.25, 6.15 and 6.4 mm against A.fumigatus, A. flavus and A. niger respectively (Table 2). Table 2: Anti-fungal activity of compound by disc diffusion method(Table Removed) Example 5 Comparison of anti-fungal activity of compound with that of amphotericin B The potency of the compound was determined with the help of standard curve constructed using amphotericin B as standard drug against A.fumigatus. Standard curve was prepared according to method given in the Indian Pharmacopoeia The corrected values of diameter of zone of inhibition along with lowest and highest values were determined. These values were plotted on semilog paper to prepare standard curve. The value of diameter of zone of inhibition obtained for compound was put into the standard curve and corresponding dose producing the effect equivalent to amphotericin B was determined to find out the potency of the compound (Fig: 6). A concentration of 10 (Jg/disc of the compound produced a diameter of zone^of inhibition equivalent to that produced by 2.67 \|Jg of amphotericin B. These observation showed that the compound was 3.74 time less potent as compared to amphotericin B. But more importantly it was a novel molecule. Example 6 Anti-fungal activity of pure compound by spore germination inhibition assay The spore germination inhibition assay was performed to evaluate the activity of the compound. Various concentrations of the compound ranging from 21.875 to 700 pg/ml in 90 \|jl of the culture medium were prepared by double dilution method. Wells were inoculated with 10 (Jl of spore suspension and incubated at 37° C for 10 hrs. The number of germinated and non-germinated spores was counted and the percent spore germination inhibition (PSGI) was calculated. The percent spore germination decrease with increased dose of the compound. The MIC of the compound was found to be 87.5 (Jg/ml (Fig 7). Example 7 Cell Cytotoxicity The in vitro cell cytotoxicity of the compound PC-1 was investigated by incubating RAW cells with varying concentrations of PC-1 and measuring the extent of cytotoxicity by MTT assay as described above. A concentration of compound up to 312.5 was completely non-toxic to the cells. The higher doses of the compound developed variable toxicity. A concentration of 1250.0 (J.g/ml of the compound was toxic to 75.95% of the cells. The percent cytotoxicity was plotted against log concentration of the compound to find cut concentration which was cytotoxic to 50% of the cells (CT 50). The CT 50 of the compound was found to be 889.2 (xg/ml (Fig 12). Example 8 Structure elucidation of the compound The characterisation of the compound was carried out by correlating all the spectral and chemical analysis information of the compound. (i) UV spectroscopic data The compound PC-1, named metelatropinyl ester, showed two maxima in the UV spectrum, which had Xmax 300 and 206 nm and absorbance 0.250 and 1.676 respectively. The prominent maxima at 206 nm in UV spectrum might be due to the presence of ester group. The Xmax for ketone has been shown to be 208 nm or little higher. Therefore, the peak at 206 nm suggested the presence of ester group in the PC-1. (ii) IR spectroscopic data The IR spectrum exhibited absorption bands for amino group (3434 cm"1), ester group (1711,1230 cm"1), and unsaturation at 1646 cm"1 (Fig 8). (iii) Mass spectroscopic data The molecular mass of the compound was found to be 239.2 on the basis of its FAB mass spectrum and the intensity of the peak was found to be 20%. The calculations made on the basis of peak intensity at m/z 239.2 (20) in the mass spectrum revealed the presence of 13.8 (= 14) carbon atoms in the molecule PC-1 (Furniss et al, 1989; Kemp, 1991). The chemical analysis and IR spectrum of PC-1 indicated the presence of at least one nitrogen and two oxygen atoms in the compound. The chemical formula thus derived for the compound PC-1 turned out to be Cnt^C^N. The nitrogen rule also supported the presence of single nitrogen atom in the molecule. The molecular weight derived from the chemical formula was found 239.188, which indeed was the same as shown by the mass spectrum. The mass spectrum of compound displayed the base peak at m/z 58 generated due to Ci-C2 and OC-O fission, suggesting secondary nature of the tropane ring. A prominent ion peak at m/z 102 arose due to formation of pentanoic acid moiety (CsHioO2+). Cleavage of Ci-Cr linkage resulted in the formation of an ion peak at m/z 57 (C4H9+)(Fig9). (iv) NMR spectroscopic data The *H NMR spectrum displayed two one-proton carbinol multiplets at 5 4.15 and 3.75 assigned to H-3 and H-l, respectively. A two-proton double doublet at 5 3.17 (1=7.44,7.44 Hz) was ascribed to Ha-S attached to amino group. Two one-proton signals at 8 2.31 (t, J=6.48 Hz) and 8 1.95 (m) were associated with C-2' methylene proton adjacent to ester carbonyl group. A six-proton broad singlet at 8 1.62 was attributed to C-6 and C-7 methyl protons attached to C-6 and C-7 olefmic carbons. Two one-proton doublets at 8 1.42 (J=6.96 Hz) and 8 1.35 (J=7.28 Hz) were accounted to C-2 methylene protons linked to ester bearing carbon. A three proton triplet at 8 0.87 (J=5.96 Hz) was due to C-5' primary methyl group. The remaining C-4 methyl and C-31 and C-4' methylene protons resonated as a seven-proton broad signal at 8 1.25 (Fig 10). The absence of any signal beyond 84.15 suggested tetrasubsituted olefinic linkage in the molecule. The existence of a methyl group linked to endo-nitrogen atom was resulted out due to absence of any signal between 8 2.0 and 8 3.0 in the 'H NMR spectrum, hi 'H-'H 2D COSY spectrum the correlation between H2-5 and H3-6, H-l and H2-2, H3-4 and H-3, H3-5 and Ha-4 was observed (Fig 11). Based on these evidences the structure of the molecule has been formulated as 2-(3,4-dimethyl-2,5-dihydro-lH-pyiTol-2-yl)-l-methylethylpentanoate. The molecule PC-1 identified in the present investigation as 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate is a new tropine derivative and the occurrence of this molecule is being reported for the first time from a plant or synthetic source. This is a novel anti-fungal compound useful as a lead molecule for the development of new antimycotic drugs. The CTso of amphotericin B and its liposomal derivatives (drug of choice) was reported to be in the range of 4.0-64.0 (ag/ml (Zager, R.A., 2000, Am. J. Kidney Dis., 2, 238). The CT50 value of novel compound is 889.2 jig/ml which indicated the compound to be many fold less cytotoxic than the amphotericin B. Example 9: In vivo Efficacy The Balb/C mice of 6-8 weak of age of either sex, weighing 15-20 g, were housed in micro-barrier cages on sterile bedding and fed ad libitum water and food. The animals were divided into 6 groups and each group contained 8-10 mice. The efficacy of PC-1 against aspergillosis was studied at five dose levels. Various doses of the compound were administered orally to the animals already challenged with 2xl07 spores of A. fumigatus shows the survival rate of the animals treated with compound. Of the animals treated with doses of 25.0, 50.0, 100.0, 200.0 and 400.0 mg/kg body weight, 1 out of 10 (10%), 1 out of 8 (12.5%), 3 out of 9 (33.3%), 4 out of 9 (44.4%) and 8 out of 10 (80%) respectively, survived up to 10th day. The percent survival rate in treated animals increased with the dose, indicating thereby the protective efficacy to be dose dependent. The probit values of percent survival were plotted against log dose of the compound PC-1 on the semilog paper to determine the effective dose required to confer protection in 50% of the animals (EDso). The EDso of the compound PC-1 was found to be 167.0 mg/kg body weight (Fig. 13 ). Example 10 Quantification of Colony Forming Units: The mice were kept under constant watch and those getting moribund, dying or survived up to 10 days were sacrificed. The autopsy was performed on the mice who had died to remove their organs for quantification of colony forming units (CPU). The lungs, livers and kidneys of the mice were removed aseptically, placed in sterile PBS (pH 7.2) and homogenized with teflon pestle mortar. The CPU in the animals were determined by plating 10 fold dilutions of organ homogenates on Sabouraud dextrose agar containing 0.05% triton X-100. The triton X-100 limited the colony size and thus greatly facilitated colony counting (Frosco, 1992). After an incubation for 48 h at 37 °C, colonies were counted and results were expressed as CPU per organ. The survival time and CPU indicating the fungal burden in various organs were considered to evaluate the protective in vivo efficacy of the compound 2-(3,4-dimethyl-2,5-dihydro-1 H-pyrrol-2-yl)-1 -methylethyl pentanoate. The fungal burden in the organs of the animals, which died or became moribund during study or those survived up to 10th day, was determined. The lungs, livers and kidneys of the animals were isolated, homogenized and the homogenate was used to culture the pathogen on Sabouraud dextrose agar plates. The colonies of the fungus were counted and expressed as colony forming units (CFU)/organ. Table 3 shows the number of CPU in different organs. The doses of 25.0 and 50.0 mg/kg body weight did not have any effect on the CPUs in the lung tissue. However, protective effect was observed in animals treated with higher doses. A dose of 400 mg/kg body weight of PC-1, reduced the colony counts significantly as compared to controls. The difference was found to be statistically significant (p were treated with 100.0 to 400.0 mg/kg body weight of the purified anti-fungal compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate (Table 3). Table 3: Colony forming units in mice treated with various doses of the purified anti-fungal compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate. (Table Removed) The main advantages of the present invention are (i) present invention provides a novel anti-fungal lead compound. (ii) this novel molecule can be used to develop new drugs for treating various fungal diseases of humans. (iii) invention also provides a method for isolation and purification of the novel anti-fungal compound from a plant Datura metel. We Claim: 1. A novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate of formula 1 (Figure Removed) 2. A novel anti-fungal molecule claimed in claim 1, wherein the said hetrocyclic molecule is characterized by known analytical methods such as TLC, group specific chemical staining, UV, infra-red, mass and nuclear magnetic resonance spectroscopy and biological assays such as anti-mycotic assay and cyto-toxicity assay and has following characteristics: Rf value of 0.22 on TLC (Fig 3), Absorption maixma at 300 nm and 206 nm in UV spectrum (Fig . 7 ) IR V Max: 3434 (NH), 3024, 1711 (ester), 1646 (C=C), 1524, 1426,1230, 938, 760 cm'1 (Fig 8). +ve FABMS m/z: 239 [M]+ (CM H25 O2 N) (16.3), 212 (13.2), 174 (16.2), 122 (34.2), 102 (96.4), 58 (100), and 57 (19.2) (Fig 9). 'H NMR (CDC13): 6 4.15 (lH,m, H-3), 3.75 (lH,m, H-l), 3.17 (2H,dd, 3=1.44, 7.44Hz, H2-5), 2.31 (1H, t, J=6.48 Hz, H-2'a), 1.95 (1H, m, H-2'b), 1.62 (6H, brs, C-6 Me, C-7 Me), 1.42 (1H, d, J=6.96 Hz, H-2a), 1.35 (1H, d, J=7.28Hz, H-2b), 1.25 (7H,brs, Me-4, H2.3'., H2.4'.), 0.87 (3H, t, J=5.96 Hz, Me-51) (Fig 10) COSY spectrum (Fig 11). The dose cytotoxic to 50% of the cells (CT5o value) of the said anti-fungal molecule is 889.2 ng/ml (Fig 12), EDso for the said anti-fungal compound is 167.0 mg/kg body weight (Fig 13). 3. Use of novel molecule of formula 1 given in claim 1 as anti-fungal agent. 4. The use of the novel anti-fungal molecule in the form of tablet, capsule, syrup, powder, ointment etc. 5. Use as claimed in claim 3 wherein the effective dose of novel molecule to the said subject ranges from 100 to 400 mg/kg body weight. 6. A novel anti-fungal compound claimed in claims 1 - 5 wherein the novel molecule is 57.8 times less cyto-toxic than the standard anti-fungal drug. 7. Use of novel anti-fungal compound may be by oral, intraperitoreal route. 8. A pharmaceutical composition comprising an acceptable amount of above compound 2- (3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate of formula 1 with pharmaceuticallyacceptable, additives and adjuvants. 9. A pharmacetically acceptable composition claimed in claims 8 wherein, the acceptable additives are selected from the nutrients, which are pharmaceutically acceptable carrier. 10. A pharmacetically acceptable composition claimed in claim 8 wherein, the effective dose of composition containing the novel molecule ranges from 100 to 400 mg/kg body weight. 11. A method for the treatment of fungal infections in a said subject comprising the steps of administration of an effective amount of 2-(3,4 dirnethyl-2,5-dihydro-lh-pyrrole-2-yl)-l- methylethyl pentanoate through routes such as oral, nasal, intra venous, intra-peritoneal, intra muscular etc. 12. A method as claimed in claim 11 wherein, the protective in vivo effective dose of the anti-fungal compound 2-(3,4-dimethyl-2,5-dihydro- lH-pyrrol-2-yl)-l-methylethyl pentanoate to the said subject ranges from 100 to 400 mg/kg body weight (Table 3). 13. A novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate substantially as herein described with reference to the examples and drawings accompanying this specification.

Full Text

The present invention relates to a novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate. It particularly relates to a novel
anti-fungal lead molecule isolated from a plant Datura metel.
The main utility of the invention is to provide a process for identifying a new lead molecule for development of new drugs for treating diseases caused by pathogenic fungi.
The incidence and mortality due to mycotic infections has been observed to be on the rise in the past decade (Saral, R., 1991, Rev. Inf. Dis. 13, 487-492., Koll, B.S. and Brown, A.E. 1993, Clin. Inf. Dis. 17, S-322-S-326). Infections induced by these species are increasingly recognized as emerging threat to the public health. In immuno-compromised host, the infections by Aspergillus, Candida, Histoplasma etc. become invasive and disseminate from primary site of infection to other parts of the body including gut, kidney, brain etc. Invasive aspergillosis is reported to be associated with a mortality rate of 55% (Denning, D.W. and Stevens, D.A., 1990, Rev. Inf. Dis., 12, 1147-1201). Mortality due to Aspergillus infection in bone marrow transplant recipients was observed to be as high as 80% despite appropriate chemotherapy (Meyer,'J.D., 1990, Semin. Oncol. 17 (suppl. 6), 10-13). Cerebral aspergillosis presents the symptoms of acute meningitis and is always fatal. Candida species are commensal organisms of human mucocutaneous surface. However, in immunocompromised host, Candida may progress to become opportunistic potential pathogen. It may be transmitted by contact to other immuno-compromised patients as a nosocomial pathogen.

It is, therefore, important to diagnose and treat these fungal infections, at early stage to prevent irreversible damage. For the treatment of mycoses, several compounds belonging to azoles, polyenes and other proups of chemicals have been described in the literature. But all of these compounds have their own limitations.
Polyenes are among the oldest and most frequently prescribed anti-fungal agents. Amphotericin B is an important polyene drug which has been found to have a broad range activity against fungi, but its therapeutic doses are frequently associated with severe chills, fever, vomiting, life threatening hypotension and respiratory distress. Amphotericin B produces renal dysfunction (Allende, M.C., Lee, J.W., Francis, P., Garrett, K., Dollenberg, H., Berenguer, J., Lyman, C. A., Pizzo, P.A. and Walsh, T.J. 1994; Antimicrob. Agents and Chemother. 38, 518-522) and necessity of its intravenous administration is its major limitation (Stevens, D.A. and Lee, J.Y., 1997, Arch. Int. Med., 57, 1857-1862). In addition to its adverse effects, Amphotericin B resistance in fungal species has been reported (Gokhale, P.C., Kshirsagar, N.A. and Pandya, S.K., 1993, Current Science 65(6), 448-454, Forthergill, A.W. and McGough, D.A., 1995, Clin. Microbiol. Procedure Handbook, In Vitro susceptibility Testing of Yeast, 5.15.1-5.15.15).
Nystatin is another important drug, however, the development of resistance and its dose limited toxicity make this drug also less useful (Forthergill A.W. and McGough A.W., 1995, Clin. Microbiol. Procedure Handbook, In Vitro susceptibility Testing of Yeasts, 5.15.1-5.15.15).
New azole anti-fungals such as ketoconazole, fluconazole and itraconazole provided the hope that these compounds might be useful to prevent or treat fungal

infections. However, their liver toxicity, hypoglycemic nature and development of resistant strains of fungi did not keep this hope alive for longer period (Denning, D.W. Tucker, R.M., Hanson, L.H., Hamilton, J.R. and Stevens, D.A., 1989, Arch. Int. Med., 149, 2301-2308). The azoles are reported to decrease the secretion of stomach acid and thereby reducing the absorption of drug itself and other important biomolecules. These effects may cause severe drop in body glucose to a level which may be life threatening. Resistance against the azole derivatives also has been reported in fungi (Forthergill A.W. and McGough A.W., 1995, Clin. Microbiol. Procedure Handbook, In Vitro susceptibility Testing of Yeasts, 5.15.1-5.15.14).
Currently available anti-fungal drugs are not sufficiently broad in their spectrum and not consistently effective against fungi including Aspergilli. These drugs are either toxic or immunosuppressive in nature. Further, the development of resistance in fungi to most of available drugs has also been reported.
It is evident from the above that need exists for development of new anti-fungal drugs which obviate the drawbacks listed above. The novelty of the present invention is to provide a process for isolation of a novel anti-fungal lead molecule from Datura metel which is many fold less cytotoxic as compared to standard anti-fungal drug such as amphotericin B.

The main object of the present invention is to provide a novel anti-fungal lead compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate for developing new drugs against the pathogenic fungi.
Another object of the invention is to provide the process for isolation of the novel anti-fungal lead molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate from a plant Datura metel.
Another object of the present invention is to provide a method for testing the novel compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate as an anti-fungal agent.
Yet another object of the invention is to provide usage of the novel compound 2-(3.4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate as an anti-fungal agent.
Yet another object is to provide a novel anti-fungal lead molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate which is 57.8 times less cytotoxic than the standard anti-fungal drug such as Amphotericin B.
List of Figures accompanying the specification:
Fig 1: Photograph of a plate showing TLC pattern of the active column sub-fractions and purified compound.
Fig 2: HPLC profile of TLC purified compound
Fig 3: Photograph of TLC of HPLC purified compound.
Fig 4: HPLC profile of purified compound.
Fig 5: Inhibition of growth of A.fumigatus by purified compound.
Fig 6: Graph represents the percent spore germination inhibition by the compound against Aspcrgillus species.
Fig 7: UV spectrum of the compound.
Fig 8: IR spectrum of the compound.
Fig 9: Mass spectrum of the compound
Fig 10: NMR spectrum of the compound.
Fig 11: COSY spectrum of the compound.
Fig 12: Percent cytotoxicity of the compound
Fig 13: The dose required to protect 50% of the animals (EDso) after treatment with PC-1.
Summary of the invention:
The present invention is directed to a novel anti-fungal compound 2-(3,4 dimethyl-2,5-dihydro-lh-pyrrole-2-yl)-l-methylethyl pentanoate of the formula 1 as given below. The compound has anti-fungal properties and is several times less cytotoxic as compared to the standard anti-fungal drugs. This compound may be useful for controlling systemic and superficial fungal infections in humans with fewer toxic effects. This compound has been isolated from an easily available plant Datura metel.
Accordingly the present invention provides a novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate of formula 1
(Figure Removed)
Formula 1
In an embodiment, the characterization of the active anti-fungal lead molecule is carried out by known analytical methods such as TLC, group specific chemical staining, UV, infra-red, mass and nuclear magnetic resonance spectroscopy and biological assays such as anti-mycotic assay and cyto-toxicity assay.
In still another embodiment to the invention, the novel anti-fungal lead molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate is a heterocyclic alkaloid as shown by group specific chemical staining using Dragendorff reagent and has following characteristics:
Rf value of 0.22 on TLC (Fig 3),
Absorption maixma at 300 nm and 206 nm in UV spectrum (Fig . 7 )
IR V Max: 3434 (NH), 3024, 1711 (ester), 1646 (OC), 1524, 1426,1230, 938, 760 cm'1 (Fig 8).
+ve FABMS m/z: 239 [M]+ (CM H25 O2 N) (16.3), 212 (13.2), 174 (16.2), 122 (34.2), 102 (96.4), 58 (100), and 57 (19.2) (Fig 9).
'H NMR (CDC13): 5 4.15 (lH,m, H-3), 3.75 (lH,m, H-l), 3.17 (2H,dd, J=7.44, 7.44Hz, H2-5), 2.31 (1H, t, J=6.48 Hz, H-2'a), 1.95 (1H, m, H-2TD), 1.62 (6H, brs, C-6 Me, C-7 Me), 1.42 (1H, d, J=6.96 Hz, H-2a), 1.35 (1H, d, J=7.28Hz, H-2b), 1.25 (7H,brs, Me-4, H2.3'-, H2.4'.), 0.87 (3H, t, J=5.96 Hz, Me-5') (Fig 10) COSY spectrum (Fig 11).
The minimum inhibitory concentration (MIC) of the said novel anti-fungal lead molecule is 5.0 (ag/disc by disc diffusion assay (Fig. 5) and 87.5 |ag/ml by percent spore germination inhibition (Fig. 6) respectively.
The dose cytotoxic to 50% of the cells (CTso value) of the said anti-fungal molecule is 889.2 (ig/ml (Fig 12).
The EDso for the said anti-fungal compound is 167.0 mg/kg body weight (Fig 13).
The anti-fungal effective dose to the said subject ranges from 100 to 400 mg/kgbody weight (Table 3).
The process for isolation of novel molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-1-methylethyl pentanoate is disclosed and claimed in our copending patent application no.NF343/2001.
In another embodiment, the novel anti-fungal compound is 57.8 times less cyto-toxic than the standard anti-fungal drug.
In further embodiment is provided a method for treatment as wherein, the dose of 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate, effective for survival of 50%(ED50) of the said subjects is 167.Omg/kg body weight (Fig. 13).
In still another embodiment, the protective in vivo effective dose of the anti-fungal compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate to the said subject ranges from 100 to 400 mg/kg body weight (Table 3).
Accordingly the present invention also provides a pharmaceutical composition comprising an acceptable amount of above compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate with pharmaceutically acceptable carrier, additives and adjuvants.
hi still another embodiment, the acceptable additives are selected from the nutrients, which are pharmaceutically acceptable carrier.
In another embodiment to the invention the novel anti-fungal molecule may be used in the form of tablet, capsule, syrup, powder, ointment.
In yet another embodiment the pharmaceutically acceptable composition contains the effective amount of novel molecule at a concentration in the range of 100 to 400 mg/ml.
A method for the treatment of fungal infections in a said subject comprising the steps of administration of an effective amount of 2-(3,4 dimethyl-2,5-dihydro-lh-pyrrole-2-yl)-l-methylethyl pentanoate through routes such as oral, nasal, intra venous, intra-peritoneal, intra muscular etc.
In an embodiment of the invention the effective dose of 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate may be in the range of 100-400 mg/kg of body weight.
Another aspect of the invention is to provide a process for isolation of a novel anti-fungal compound 2-(3,4 dimethyl-2,5-dihydro-lH-pyrrole-2-yl)-l-methylethyl pentanoate, from a plant, Datura metel, which comprises (i) extracting successively the powdered Datura metel plant material with an organic solvent at a temperature in the range of 15 to 45 °C (ii) removing the solvent to obtain residue (iii) extracting the above said residue obtained in step (ii) with an aliphatic hydrocarbon solvent followed by extraction with chloroform (iv) removing chloroform from chloroform fractions (v) screening the above obtained chloroform fractions for antimycotic activity (vi) separating the anti-fungal fractions and purifying the novel anti-fungal lead molecule from active antimycotic fractions by conventional chromatographic methods (viii) assaying the said anti-fungal lead molecule for its cytotoxicity.
In an embodiment to the invention the solvent used for extraction may be selected from methanol, ethanol, acetone, chloroform.
The aliphatic hydrocarbon solvent used may be selected from hexane, petroleum ether.
In another embodiment to the invention, the purification of compound is carried out by column or thin layer chromatography (TLC) and high performance liquid chromatography (HPLC).
In yet another embodiment to the invention, the purification of novel compound maybe carried out by Thin Layer Chromatography using solvent systems selected from Chloroform: Acetone: Diethylamine (5.0:4.0:1.0), Chloroform: Methanol: Diethylamine (8.5:1.5:0.1) and Chloroform: Methanol: Formic acid (8.0:2.0:0.1) or different combinations of above said organic solvents.
In yet another embodiment to the invention, the purification of novel compound may be effected by HPLC using solvent system 70:30 of acetonitrile and water using reverse phase RP-8 column.
In still another embodiment, the invention provides a method of testing of novel compound (2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate) as an anti-fungal agent.
In yet another embodiment to the present invention, the antimycotic activity may be tested after each purification step by known methods such as microbroth dilution (MD), disc diffusion (DD) and spore germination inhibition (SGI).
Details of Invention
1. Purification of the compound
. The invention provides a novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate a process for its isolation. It particularly relates to the isolation of a novel anti-fungal lead molecule from natural source.
(i) Preparation of methanolic extract
Plant material Datura metel (Solanaceae) was collected during the month of April/May from near about the railway station Kishanganj, Delhi, India. Hamdard College of Pharmacy, New Delhi, authenticated the identification of plant material where voucher sample has been preserved. The freshly collected plant material was dried in the shade in a well-ventilated enclosure. Dried material was powdered and extracted with methanol. All the extracts obtained after four cycles of extraction with methanol were pooled and evaporated to dryness under reduced pressure at 45 °C in Rotavapor R-l 14 (Buchi) attached to a Waterbath B-480 (Buchi). The residue was obtained and stored at 4°C till use. Methanolic extract was examined for its anti-fungal activity against species of Aspergillus. The extract having anti-fungal activity was subjected to further fractionation for isolation of the active anti-fungal component
(ii) Fractionation of methanolic extract of Datura metel
The constituents of crude methanolic preparation were fractionated by the method of Harbourne, with slight modifications (Harbourne J.B., 1997, Phytochemical

Methods, Chapman and Hall, London, pp. 1-5). The Methanolic extract was successively extracted with hexane, chloroform and acetone. The methanolic extract was first extracted with hexane four times. All the four extracts of hexane were pooled and solvent was evaporated with the help of Rotavapour R-l 14 (Buchi). The dry fraction was collected and kept at 4°C till further use. The residue after the extraction with hexane was extracted stepwise with chloroform and acetone four times as in case of hexane to obtain chloroform and acetone fractions. The leftover residue was finally dissolved in methanol. Solvents were evaporated and dry fractions were recovered. All the fractions were labeled properly and examined for their anti-fungal activity using pathogenic species of Aspergillus. The chloroform fraction was found to be active, therefore, it was further sub-fractionated by column and thin layer chromatography.
(iii) Column Chromatography
The chloroform fraction was sub-fractionated by modified column chromatography using a silica gel column. Silica gel was suspended in hexane and packed in a glass column of 1.25x35 cm size. Slurry of chloroform extract was prepared and loaded on to the top of the pre-equilibrated silica gel (60-120 |J) column. The components of chloroform fraction were eluted with 100 ml of chloroform at a flow rate of 1.0 ml/min followed by different ratio of chloroform: methanol ranging from 100:0 to 0:100. All the sub-fractions were analysed by TLC. The sub-fractions showing similar profile of Rf values were pooled and dried in vacua. The anti-fungal activity of each sub-fraction was tested using pathogenic strains of Aspergill/. The sub-fractions, which showed anti-fungal potential, were further subjected to the thin layer chromatography for identifying and separating out pure active component.

(iv) Thin layer chromatography (TLC)
The active anti-fiingal column fractions were spotted onto the Silica gel plates (E. Merck Cat No. 1.05554, ¥254) and subjected to TLC with three different solvent systems i.e. Chloroform: Acetone: Diethylamine (5.0:4.0:1.0), Chloroform: Methanol: Diethylamine (8.5:1.5:0.1) and Chloroform: Methanol: Formic acid (8.0:2.0:0.1). The bands on the plates were visualised with UV light at 254 nm and 366 nm and by spraying with Chloropalatinate, Dragendorf f reagent and Iodine. The components in different bands were scrapped and examined for anti-fungal activity. The preparative TLC was performed to obtain active component of our interest.
(v) High pressure liquid chromatography (HPLC)
The purity of the compound PC-1 obtained from preparative TLC was analyzed by HPLC using RP-8 column (Merck). The reverse phase HPLC was performed isocratically with the solvent system 70 parts acetonitrile: 30 parts water. The test samples were passed through a membrane of pore size before loading. 5.0 pi of the 0.22 (j filtered sample was loaded on to the pre-equilibrated HPLC column at room temperature. The flow rate was maintained at 1.0 ml/min.
2. Anti-fungal Activity Assays
The anti-fungal activity of various fractions such as crude, methanolic extract, sub-fractions and purified compound was studied by microbroth dilution, disc diffusion and spore germination inhibition assays using pathogenic strains of fungi.

(i) Pathogen
Pathogenic strains of fungi were obtained from the Mycology Department of Vallabhbhai Patel Chest Institute, Delhi. All the strains were grown on Sabouraud dextrose agar at 37°C. The conidia form these cultures were harvested and suspended in Sabouraud maltose broth. The number of conidia was counted using haemocytometer and their number in the suspension was adjusted to as per need of the experiment.
(ii) Microbroth Dilution Assay
The anti-fungal susceptibility of the fungi to various fractions or the purified component was assayed by the microbroth dilution method (Forthergill A.W. and McGough A.W., 1995, Clin. Microbiol. Procedure Handbook. In Vitro susceptibility Testing of Yeast, 5.15.1-5.15.15). The fungal spores were harvested from 96 h cultures and their number adjusted to IxlO6 per ml. The Sabouraud Dextrose medium was dissolved in glass double distilled water and autoclaved at 10 psi for 15 min. 90 |Jl medium was added into the wells of cell culture plates. The different concentrations of the extract, fractions or the purified compound were prepared in duplicate wells and then the wells were inoculated with 10 pi of spore suspension. The plates were incubated at 37 0 C and examined macroscopically after 48 h for the growth of fungal mycelia. The activity was represented as -ve if growth was there and +ve if medium appeared clear with out any growth of fungi.
(iii) Disc Diffusion Assay
The disc diffusion test was performed in 10 cm diameter (Tarsons) radiation sterilized petri plates as per the method described in Indian Pharmacopoeia (Indian Pharmacopoeia, 1996, Appendix 9, p A101-A110). Sabouraud Dextrose agar medium was dissolved in double glass-distilled water and autoclaved at 10 psi for 15 minutes. It was cooled to 45°C and 20.0 ml was poured into each petri dish. IxlO6 conidia in 1.0 ml of conidial suspension were prepared in Sabouraud maltose broth and overlaid on the surface of the agar plate.
Different concentrations of the samples were impregnated on the sterilized discs measuring 5.0 mm in diameter from Whatman filter paper number. The discs were placed on the surface of the agar plates already inoculated with the fungal spores. The plates were incubated at 37°C and examined at 48 hrs for zone of inhibition, if any, around the disc. The diameter of zone of inhibition was measured with the help of a scale. The concentration, which developed the zone of inhibition of 6.0 mm diameter, was considered as minimum inhibitory concentration. Amphotericin B was used in assay as a standard control drug. An additional control disc without any sample but impregnated with equivalent amount of solvent was also used. The test was repeated three to five times with various test preparations of plant to ascertain activity.
(iv) Preparation of Standard Curve
Standard curve was prepared in accordance with the method given in the Indian Pharmacopoeia (Indian Pharmacopoeia, 1996, Appendix 9, p A101-A110).
(v) Spore Germination Inhibition Assay
The modified spore germination inhibition assay was performed as described earlier (Sureder P. and Janaiah C., 1987, Ind. J. Expt. Biol., 25, 233-234). The fungal species were grown on Sabouraud dextrose agar plates at 37°C for 96 h. Conidia were harvested from the plates and their homogenous suspension was prepared in the Sabouraud dextrose broth. The conidia were counted and their number in the suspension was adjusted to IxlO4 per ml. The standard drug Amphotericin B and test samples were initially dissolved in minimal quantity of dimethylsulfoxide (DMSO) and then diluted with Sabouraud dextrose medium. Various concentrations of the test samples in 90 |Jl of culture medium were prepared in 96 well flat bottom micro-culture plates (Nunc) by double dilution method. The wells were prepared in triplicates for each concentration. Each well was then inoculated with 10 pi of spore suspension containing 100+5 spores. The plates were incubated at 37°C for 10 h and then examined for spore germination under inverted microscope (Nickon Diphot). The number of germinated and non-germinated spores was counted. The percent spore germination inhibition (PSGI) was calculated using following formula
No. of spores germinated in drug treated well
PSGI = 100 - x 100
No. of spores germinated in control well
3. Characterization studies:
(i) Structure elucidation
Characterisation of the compound was carried out using various techniques such as class identification by chemical methods, derivatisation of the compound, melting point of the purified compound, infra red spectroscopy (Fig 8), ultraviolet spectroscopy, mass spectroscopy (Fig 9), nuclear magnetic resonance spectroscopy (Fig 10) and. COSY (Fig 11). Details of the spectral data obtained with respect to novel compound is given above.
(ii) In vitro toxicity evaluation:
The in vitro toxicity of the active molecule was studied by MTT assay using RAW cells (Mossman, T., 1983, J. Immunol. Methods, 65, 55-63).
Cell culture:
The stock culture of RAW cells was obtained from National Facility for Cell and Tissue Culture, Pune. Cells were maintained in RPMI-1640 medium supplemented with glutamine (2.3 gm/L), fetal calf serum (10% v/v) and gentamycin (50.0 mg/L) at 37°C in Nuair IR Autoflow water Jacketed carbon dioxide incubator.
Sample preparation:
The stock solution of the compound was prepared by dissolving 2.5 mg of the purified compound in minimum amount of DMSO and then diluted with double distilled
water to make the final volume 1.0 ml. The different concentrations ranging from 1250.0 }a,g/ml to 19.5 |ag/ml were tested in the cytotoxicity assay.
Cell cytotoxicity assay:
The cells were harvested at the log phase of growth confluency from the flask. The homogeneous suspension of cells was prepared in 2.0 ml of culture medium. The number of cells in the suspension was counted using a hemocytometer and the cell suspension was diluted in such a way so as to obtain 5xl07 viable cells per ml. The viability of the cells was checked with trypan blue dye exclusion test. The cytotoxicity assay was performed in 96 wells flat bottom tissue culture plates (Nunc Nunclon). The cells (5xl06) in 100.0 yl volume were seeded into each well. The plate was incubated at 37 °C in atmosphere of 5% (v/v) COa for 8 h. The wells were examined microscopically for the formation of monolayer of the cells. Various concentrations ranging from 1250.0 ug/ml to 19.5 ug/ml of the compound PC-1 (metelatropinyl ester) were added to the monolayer of cells. In +ve control wells, a known cytotoxic protein obtained from A. fumigatus, was used. The ZnSC>4 (8.0 mg/L) was used as another +ve toxic material in another set of wells. In negative control wells equivalent amount of the solvent was used. The duplicate wells were used for each concentration of the test compound. The plates were incubated at 37 °C in 5% (v/v) COa incubator overnight (12 h). The medium along with floating dead cells from the wells was removed by inverting the plate. Thus only the live cells sticking to the surface of plate were left in the wells. The cells were stained with 40.0 ^1 of 2.5 % dye MTT. After adding the dye to the wells, the plate was kept at 37 °C in 5% CO2 for 1 h. The tissue culture plate was removed from the incubator and all the dye was aspirated.
Only live cells took up the dye. The cells were lysed by adding 100.0 jj,l of isopropanol-HC1 to the wells. After lysis of the cells, the plates were read at 540 nm using plate reader (Spectra Max 190, Molecular Device).
Interpretation of results:
The toxicity of the compound was expressed as percentage with respect to that obtained in -ve control sample. The percent cytotoxicity was calculated using the formula given below.
OD in -ve control - OD in the test
% Cytotoxicity = — x 100
OD in -ve control
(iii) In vivo Efficacy of PC-1
In vivo efficacy of the compound against A. fumigatus infection was studied in the Balb/C mice of 6-8 weak of age of either sex, weighing 15-20 g each. The mice model of aspergillosis described by Dixon et al (1989), was used to study the effects of compound on in vivo infection of A. fumigatus.
The animals were housed in the micro-barrier cages on sterile bedding and fed ad libitum water and food. The mice were divided into 6 groups and each group contained 8-10 animals. The mice were infected experimentally with A. fumigatus. Three days prior to infection with conidia, mice were injected sub-cutaneously with 3 doses (250.0 mg/kg/day) of cortisone acetate in 400.0 ul of PBS. On the infection day, each mouse received 2xl07
conidia by nasal instillation of a single droplet of conidial suspension. The animals^ of this model developed invasive aspergillosis and all the infected animals died within 4-6 days.
Inoculum prepration for A. fumizatus for experimental infection to mice:
A. fumigatus (190/96) isolate was grown on Sabouraud dextrose agar plates at 37 °C for 4 days. The conidia were collected from the culture plates using PBS (pH 7.2) containing 0.05% Tween 80 (Sigma Chemicals) and the suspension was filtered through sterile glass wool. The conidia were pelleted by centrifugation at 2000 rpm and re-suspended in PBS, pH 7.2. The number of conidia was counted and adjusted to IxlO8 conidia/ml. The viability of the conidia was determined by plating the dilutions of suspensions on Sabouraud dextrose agar.
The purified compound (metelatropinyl ester) isolated from the D. metel was administered orally in the concentration ranging from 0.0, 25.0 50.0 100.0 200.0 and 400.0 mg/kg body weight to the mice infected experimentally with A. fumigatus.
Treatment:
The dosing of the animals (cortisone treated and challenged) with compound in 6 different groups was initiated within 30 min after challenge with A. fumigatus conidia.
Group I: The mice were treated orally with 7 doses of 25.0 mg/kg/day of the compound.
Group II: The animals infected with A. fumigatus conidia were given 50.0 mg/kg/day of the compound for 7 days orally.

Group ffl: The animals in this group were given 7 oral doses of 100.0 mg/kg/day of the compound.
Group IV: In this group of animals, 7 oral doses of the compound (200.0 mg/kg/day) were given.
Group V: The animals of this group were treated with 7 doses of 400.0 mg/kg/day orally over a period of 7 days.
Group VI: The animals of this group acted as control and were treated orally with 7 doses of 400 ul PBS containing solvent.
Survival Rate:
The animals were housed in properly labeled cages and kept under close watch for weight loss and the mortality. The survival rate over a period of 10 days was calculated and the fungal burden in survivors determined (demons and Stevens, 1994). Of the animals treated with doses of 25.0, 50.0, 100.0, 200.0 and 400.0 mg/kg body weight, 1 out of 10 (10%), 1 out of 8 (12.5%), 3 out of 9 (33.3%), 4 out of 9 (44.4%) and 8 out of 10 (80%) respectively, survived up to 10th day.
Quantification of Colony Forming Units:
The mice were kept under constant watch and those getting moribund, dying or survived up to 10 days were sacrificed. The autopsy was performed on the mice who had died to remove their organs for quantification of colony forming units (CPU). The lungs, livers and kidneys of the mice were removed aseptically, placed in

sterile PBS (pH 7.2) and homogenized with teflon pestle mortar. The CPU in the animals were determined by plating 10 fold dilutions of organ homogenates on Sabouraud dextrose agar containing 0.05% triton X-100. The triton X-100 limited the colony size and thus greatly facilitated colony counting (Frosco, 1992). After an incubation for 48 h at 37 °C, colonies were counted and results were expressed as CPU per organ.
The survival time and CPU indicating the fungal burden in various organs were considered to evaluate the protective efficacy of the compound in vivo
The following examples are given by way of illustration of the present invention and should not be construed to limit of the scope of the present invention.
Example 1
Extraction and fractionation
Aerial parts of the plant Datura metel (Solanaceae) were collected during the month of April from an area around Delhi, India. Hamdard College of Pharmacy, New Delhi, authenticated the identification of plant material where voucher sample has been preserved. The freshly collected plant material was dried in the shade in a well-ventilated enclosure. 400 gm dried material was powdered and extracted with 500 ml methanol in each cycle. All the extracts obtained after four cycles of extraction were pooled and evaporated to dryness under reduced pressure. The 25 gm residue was obtained and stored at 4°C till use.

The methanolic extract was successively extracted with hexane, chloroform and acetone. The methanolic extract was first extracted with hexane four times. All the four extracts of hexane were pooled and solvent was evaporated. The dry hexane fraction was collected and kept at 4°C till further use. The residue after the extraction with hexane was extracted stepwise with chloroform and acetone four times as in case of hexane to obtain chloroform and acetone fractions. The leftover residue was finally dissolved in methanol. Dried fractions were examined for their anti-fungal activity. The fractions, which did not inhibit the growth of Aspergillus up to a concentration of 1500 pg/ml and 50 (Jg/disc in spore germination inhibition and disc diffusion assays respectively, were considered to be non-active. The chloroform fraction of Datura metel was found to be active as a concentration of 1250 pg/ml inhibited the germination of 100% of the spores in spore germination inhibition assay and a concentration of 25.0 pg/disc produced the zone of inhibition of activity significance. Therefore, this fraction was further used to identify and purify the active component using various chromatographic methods.
Example 2
Purification of active compound
The chloroform fraction was sub-fractionated by modified column chromatography as described above. Fractions were analyzed by TLC and those showing similar spots were pooled and dried in vacua to get sub-fractions. Total 15 sub-fractions were obtained. The anti-fungal activity of each sub-fraction thus obtained was tested using pathogenic strains of Aspergillus. The fraction numbers 10 and 11 showed anti-

fungal potential and they were further subjected to the thin layer chromatography for separating out pure active component.
Silica gel plates (Cat No. 1.05554, F254) were purchased from E. Merck and used for performing TLC. The column chromatographic active sub-fractions were spotted onto the plates and subjected to run with three different solvent systems i.e. chloroform: acetone: diethylamine (5.0:4.0:1.0), chloroform: methanol: diethylamine (8.5:1.5:0.1) and chloroform: methanol: formic acid (8.0:2.0:0.1). The bands on the plates were visualized with UV light at 254 nm and 366 nm and by spraying with chloropalatinate, Dragendorf f reagent and iodine. The components in three different major bands resolved with a solvent system chloroform: methanol: formic acid (8.0:2.0:0.1) (Fig 1) having Rf values 0.14, 0.22 and 0.37 were scrapped and examined for anti-fungal activity. The compound, which had efficacy against Aspergillus, was recovered from a band having Rf value of 0.22. The compound was purified by repeated preparative TLC and tested for its anti-fungal activity. The active compound reacted positively with Dragendorff s reagent showing thereby its nature to be alkaloidal.
The purity of the compound obtained from preparative TLC was analyzed by HPLC using RP-8 column (Merck). The reverse phase HPLC was performed isocratically with the solvent system acetonitrile: water (70:30). Samples were passed through a membrane of 0.22 |J pore size before loading. A total of 5.0 (Jl of the sample was loaded on to the pre-equilibrated column at room temperature. The flow rate was maintained at 1.0 ml/min. The HPLC profile of compound purified by TLC showed two peaks, peak-I and II, which had retention times 1.91 and 2.71 respectively (Fig 2). These two corresponding peaks at retention time 1.88 and 2.84 were present in the HPLC profile of

active sub-fraction obtained from the column chromatography also. The active column sub-fraction had five major peaks corresponding to retention time 1.31, 1.88, 2.84, 3.82 and 4.35. Changing the solvent system in TLC carried out the further purification of partially purified compound. The solvent system comprised of chloroform: methanol: diethylamine (8.5:1.5:0.1) resolved the active molecule at Rf 0.42 (Fig 3). The retention time of compound in HPLC was found to be around 1.9 and it was showing purity from 97% to 100% in various runs (Fig 4). The retention time of the compound remained same in both chromatographic active sub-fraction and its purified form.
Example 3
Anti-fungal activity of pure compound by microbroth dilution assay
The anti-fungal susceptibility of the fungi to the purified compound was assayed by the microbroth dilution method The different concentrations of the purified compound ranging from 21.87 to 350 fJg/ml were prepared in duplicate wells by twofold dilution method. The test was done as described above. The anti-fungal activity of the compound is given in the Table 1. The MIC of the compound was found to be 87.5 (JQ/ml. At this concentration of compound none the three species of Aspergillus showed any visual growth in wells.
Table 1: Activity of the compound against Aspergillus sp. after 48 hrs. (Table Removed)
Example 4
Anti-fungal activity of pure compound by disc diffusion assay
The disc diffusion test was performed as per described in the section of details of invention. Amphotericin B 1.25 pg/disc was used in assay as positive control standard drug. Additional discs impregnated with equivalent amount of solvent were also used in the assay as negative controls (Fig: 5). The MIC of the compound was observed to be 5 (jg/disc since this concentration developed zone of inhibition having mean diameter of 6.25, 6.15 and 6.4 mm against A.fumigatus, A. flavus and A. niger respectively (Table 2).
Table 2: Anti-fungal activity of compound by disc diffusion method(Table Removed)
Example 5
Comparison of anti-fungal activity of compound with that of amphotericin B
The potency of the compound was determined with the help of standard curve constructed using amphotericin B as standard drug against A.fumigatus. Standard curve was prepared according to method given in the Indian Pharmacopoeia The corrected values of diameter of zone of inhibition along with lowest and highest values were determined. These values were plotted on semilog paper to prepare standard curve. The value of diameter of zone of inhibition obtained for compound was put into the standard curve and corresponding dose producing the effect equivalent to amphotericin B was determined to find out the potency of the compound (Fig: 6).
A concentration of 10 (Jg/disc of the compound produced a diameter of zone^of inhibition equivalent to that produced by 2.67 |Jg of amphotericin B. These observation showed that the compound was 3.74 time less potent as compared to amphotericin B. But more importantly it was a novel molecule.
Example 6
Anti-fungal activity of pure compound by spore germination inhibition assay
The spore germination inhibition assay was performed to evaluate the activity of the compound. Various concentrations of the compound ranging from 21.875 to 700 pg/ml in 90 |jl of the culture medium were prepared by double dilution method. Wells were inoculated with 10 (Jl of spore suspension and incubated at 37° C for 10 hrs. The number of germinated and non-germinated spores was counted and the percent spore germination inhibition (PSGI) was calculated. The percent spore germination decrease with increased dose of the compound. The MIC of the compound was found to be 87.5 (Jg/ml (Fig 7).
Example 7 Cell Cytotoxicity
The in vitro cell cytotoxicity of the compound PC-1 was investigated by incubating RAW cells with varying concentrations of PC-1 and measuring the extent of cytotoxicity by MTT assay as described above. A concentration of compound up to 312.5 was completely non-toxic to the cells. The higher doses of the compound developed
variable toxicity. A concentration of 1250.0 (J.g/ml of the compound was toxic to 75.95% of the cells. The percent cytotoxicity was plotted against log concentration of the compound to find cut concentration which was cytotoxic to 50% of the cells (CT 50). The CT 50 of the compound was found to be 889.2 (xg/ml (Fig 12).
Example 8
Structure elucidation of the compound
The characterisation of the compound was carried out by correlating all the spectral and chemical analysis information of the compound.
(i) UV spectroscopic data
The compound PC-1, named metelatropinyl ester, showed two maxima in the UV spectrum, which had Xmax 300 and 206 nm and absorbance 0.250 and 1.676 respectively. The prominent maxima at 206 nm in UV spectrum might be due to the presence of ester group. The Xmax for ketone has been shown to be 208 nm or little higher. Therefore, the peak at 206 nm suggested the presence of ester group in the PC-1.
(ii) IR spectroscopic data
The IR spectrum exhibited absorption bands for amino group (3434 cm"1), ester group (1711,1230 cm"1), and unsaturation at 1646 cm"1 (Fig 8).
(iii) Mass spectroscopic data
The molecular mass of the compound was found to be 239.2 on the basis of its FAB mass spectrum and the intensity of the peak was found to be 20%. The calculations made on the basis of peak intensity at m/z 239.2 (20) in the mass spectrum revealed the presence of 13.8 (= 14) carbon atoms in the molecule PC-1 (Furniss et al, 1989; Kemp, 1991). The chemical analysis and IR spectrum of PC-1 indicated the presence of at least one nitrogen and two oxygen atoms in the compound. The chemical formula thus derived for the compound PC-1 turned out to be Cnt^C^N. The nitrogen rule also supported the presence of single nitrogen atom in the molecule. The molecular weight derived from the chemical formula was found 239.188, which indeed was the same as shown by the mass spectrum. The mass spectrum of compound displayed the base peak at m/z 58 generated due to Ci-C2 and OC-O fission, suggesting secondary nature of the tropane ring. A prominent ion peak at m/z 102 arose due to formation of pentanoic acid moiety (CsHioO2+). Cleavage of Ci-Cr linkage resulted in the formation of an ion peak at m/z 57 (C4H9+)(Fig9).
(iv) NMR spectroscopic data
The *H NMR spectrum displayed two one-proton carbinol multiplets at 5 4.15 and 3.75 assigned to H-3 and H-l, respectively. A two-proton double doublet at 5 3.17 (1=7.44,7.44 Hz) was ascribed to Ha-S attached to amino group. Two one-proton signals at 8 2.31 (t, J=6.48 Hz) and 8 1.95 (m) were associated with C-2' methylene proton adjacent to ester carbonyl group. A six-proton broad singlet at 8 1.62 was attributed to C-6 and C-7 methyl protons attached to C-6 and C-7 olefmic carbons. Two one-proton
doublets at 8 1.42 (J=6.96 Hz) and 8 1.35 (J=7.28 Hz) were accounted to C-2 methylene protons linked to ester bearing carbon. A three proton triplet at 8 0.87 (J=5.96 Hz) was due to C-5' primary methyl group. The remaining C-4 methyl and C-31 and C-4' methylene protons resonated as a seven-proton broad signal at 8 1.25 (Fig 10). The absence of any signal beyond 84.15 suggested tetrasubsituted olefinic linkage in the molecule. The existence of a methyl group linked to endo-nitrogen atom was resulted out due to absence of any signal between 8 2.0 and 8 3.0 in the 'H NMR spectrum, hi 'H-'H 2D COSY spectrum the correlation between H2-5 and H3-6, H-l and H2-2, H3-4 and H-3, H3-5 and Ha-4 was observed (Fig 11).
Based on these evidences the structure of the molecule has been formulated as 2-(3,4-dimethyl-2,5-dihydro-lH-pyiTol-2-yl)-l-methylethylpentanoate.
The molecule PC-1 identified in the present investigation as 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate is a new tropine derivative and the occurrence of this molecule is being reported for the first time from a plant or synthetic source. This is a novel anti-fungal compound useful as a lead molecule for the development of new antimycotic drugs. The CTso of amphotericin B and its liposomal derivatives (drug of choice) was reported to be in the range of 4.0-64.0 (ag/ml (Zager, R.A., 2000, Am. J. Kidney Dis., 2, 238). The CT50 value of novel compound is 889.2 jig/ml which indicated the compound to be many fold less cytotoxic than the amphotericin B.
Example 9:
In vivo Efficacy
The Balb/C mice of 6-8 weak of age of either sex, weighing 15-20 g, were housed in micro-barrier cages on sterile bedding and fed ad libitum water and food. The animals were divided into 6 groups and each group contained 8-10 mice. The efficacy of PC-1 against aspergillosis was studied at five dose levels. Various doses of the compound were administered orally to the animals already challenged with 2xl07 spores of A. fumigatus shows the survival rate of the animals treated with compound. Of the animals treated with doses of 25.0, 50.0, 100.0, 200.0 and 400.0 mg/kg body weight, 1 out of 10 (10%), 1 out of 8 (12.5%), 3 out of 9 (33.3%), 4 out of 9 (44.4%) and 8 out of 10 (80%) respectively, survived up to 10th day.
The percent survival rate in treated animals increased with the dose, indicating thereby the protective efficacy to be dose dependent. The probit values of percent survival were plotted against log dose of the compound PC-1 on the semilog paper to determine the effective dose required to confer protection in 50% of the animals (EDso). The EDso of the compound PC-1 was found to be 167.0 mg/kg body weight (Fig. 13 ).
Example 10
Quantification of Colony Forming Units: The mice were kept under constant watch and those getting moribund, dying or survived up to 10 days were sacrificed. The autopsy was performed on the mice who had died to remove their organs for quantification of colony forming units (CPU). The lungs, livers and kidneys of the mice were removed aseptically, placed in sterile PBS (pH 7.2) and homogenized with teflon pestle mortar. The
CPU in the animals were determined by plating 10 fold dilutions of organ homogenates on Sabouraud dextrose agar containing 0.05% triton X-100. The triton X-100 limited the colony size and thus greatly facilitated colony counting (Frosco, 1992). After an incubation for 48 h at 37 °C, colonies were counted and results were expressed as CPU per organ.
The survival time and CPU indicating the fungal burden in various organs were considered to evaluate the protective in vivo efficacy of the compound 2-(3,4-dimethyl-2,5-dihydro-1 H-pyrrol-2-yl)-1 -methylethyl pentanoate.
The fungal burden in the organs of the animals, which died or became moribund during study or those survived up to 10th day, was determined. The lungs, livers and kidneys of the animals were isolated, homogenized and the homogenate was used to culture the pathogen on Sabouraud dextrose agar plates. The colonies of the fungus were counted and expressed as colony forming units (CFU)/organ. Table 3 shows the number of CPU in different organs.
The doses of 25.0 and 50.0 mg/kg body weight did not have any effect on the CPUs in the lung tissue. However, protective effect was observed in animals treated with higher doses. A dose of 400 mg/kg body weight of PC-1, reduced the colony counts significantly as compared to controls. The difference was found to be statistically significant (p
were treated with 100.0 to 400.0 mg/kg body weight of the purified anti-fungal compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate (Table 3).
Table 3: Colony forming units in mice treated with various doses of the purified anti-fungal compound 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate. (Table Removed)
The main advantages of the present invention are
(i) present invention provides a novel anti-fungal lead compound.
(ii) this novel molecule can be used to develop new drugs for treating various fungal diseases of humans.
(iii) invention also provides a method for isolation and purification of the novel anti-fungal compound from a plant Datura metel.

We Claim:
1. A novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate of formula 1
(Figure Removed)
2. A novel anti-fungal molecule claimed in claim 1, wherein the said hetrocyclic molecule is characterized by known analytical methods such as TLC, group specific chemical staining, UV, infra-red, mass and nuclear magnetic resonance spectroscopy and biological assays such as anti-mycotic assay and cyto-toxicity assay and has following characteristics:
Rf value of 0.22 on TLC (Fig 3),
Absorption maixma at 300 nm and 206 nm in UV spectrum (Fig . 7 )
IR V Max: 3434 (NH), 3024, 1711 (ester), 1646 (C=C), 1524, 1426,1230, 938, 760 cm'1 (Fig 8).
+ve FABMS m/z: 239 [M]+ (CM H25 O2 N) (16.3), 212 (13.2), 174 (16.2), 122 (34.2), 102 (96.4), 58 (100), and 57 (19.2) (Fig 9).
'H NMR (CDC13): 6 4.15 (lH,m, H-3), 3.75 (lH,m, H-l), 3.17 (2H,dd, 3=1.44, 7.44Hz, H2-5), 2.31 (1H, t, J=6.48 Hz, H-2'a), 1.95 (1H, m, H-2'b), 1.62 (6H, brs, C-6 Me, C-7 Me), 1.42 (1H, d, J=6.96 Hz, H-2a), 1.35 (1H, d, J=7.28Hz, H-2b), 1.25 (7H,brs, Me-4, H2.3'., H2.4'.), 0.87 (3H, t, J=5.96 Hz, Me-51) (Fig 10) COSY spectrum (Fig 11).
The dose cytotoxic to 50% of the cells (CT5o value) of the said anti-fungal molecule is 889.2 ng/ml (Fig 12), EDso for the said anti-fungal compound is 167.0 mg/kg body weight (Fig 13).
3. Use of novel molecule of formula 1 given in claim 1 as anti-fungal agent.
4. The use of the novel anti-fungal molecule in the form of tablet, capsule, syrup, powder,
ointment etc.
5. Use as claimed in claim 3 wherein the effective dose of novel molecule to the said
subject ranges from 100 to 400 mg/kg body weight.
6. A novel anti-fungal compound claimed in claims 1 - 5 wherein the novel molecule is
57.8 times less cyto-toxic than the standard anti-fungal drug.
7. Use of novel anti-fungal compound may be by oral, intraperitoreal route.
8. A pharmaceutical composition comprising an acceptable amount of above compound 2-
(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl pentanoate of formula 1 with
pharmaceuticallyacceptable, additives and adjuvants.
9. A pharmacetically acceptable composition claimed in claims 8 wherein, the acceptable
additives are selected from the nutrients, which are pharmaceutically acceptable carrier.
10. A pharmacetically acceptable composition claimed in claim 8 wherein, the effective dose
of composition containing the novel molecule ranges from 100 to 400 mg/kg body
weight.
11. A method for the treatment of fungal infections in a said subject comprising the steps of
administration of an effective amount of 2-(3,4 dirnethyl-2,5-dihydro-lh-pyrrole-2-yl)-l-
methylethyl pentanoate through routes such as oral, nasal, intra venous, intra-peritoneal,
intra muscular etc.
12. A method as claimed in claim 11 wherein, the protective in vivo effective dose of the
anti-fungal compound 2-(3,4-dimethyl-2,5-dihydro- lH-pyrrol-2-yl)-l-methylethyl
pentanoate to the said subject ranges from 100 to 400 mg/kg body weight (Table 3).
13. A novel anti-fungal molecule 2-(3,4-dimethyl-2,5-dihydro-lH-pyrrol-2-yl)-l-methylethyl
pentanoate substantially as herein described with reference to the examples and
drawings accompanying this specification.

Documents:

1008-del-2001-abstract.pdf

1008-del-2001-claims.pdf

1008-del-2001-correspondence-others.pdf

1008-del-2001-correspondence-po.pdf

1008-del-2001-description (complete).pdf

1008-del-2001-drawings.pdf

1008-del-2001-form-1.pdf

1008-del-2001-form-18.pdf

1008-del-2001-form-2.pdf

1008-del-2001-form-3.pdf

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Patent Number

220711

Indian Patent Application Number

1008/DEL/2001

PG Journal Number

30/2008

Publication Date

25-Jul-2008

Grant Date

03-Jun-2008

Date of Filing

28-Sep-2001

Name of Patentee

COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH

Applicant Address

RAFI MARG, NEW DELHI-110001, INDIA

Inventors:

#	Inventor's Name	Inventor's Address
1	GAINDA LAL SHARMA
2	RAJESH

PCT International Classification Number

C07D 207/20

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA