Title of Invention	AN IMPROVED PROCESS FOR THE PREPARATION OF 10-HYDROXY CAMPTOTHECIN USING ASPERGILLUS OCHRACEUS
Abstract	An improved process for the preparation of 10-hydroxy camptothecin using aspergillus ochraceus which comprises cultivating novel strain of aspergillus ochraceus having characteristics such as herein described in a conventional nutrient medium additionally containing glucose 2 to 4%, caseine hydrolysate 0.2 to 0.8% corn steep liquor, 0.02 to 0.05% in a known manner, addinq camptothecin in a solvent to the above said qrowinq culture followed by incubating for a period of atleast 24hr, extracting the reaction mixture with a water immiscible organic solvent preferably chloro solvent or esters, removing the solvent to get crude to hydroxy camptothecin and further purifying by known chromatographic methods.

Title of Invention

AN IMPROVED PROCESS FOR THE PREPARATION OF 10-HYDROXY CAMPTOTHECIN USING ASPERGILLUS OCHRACEUS

Abstract

An improved process for the preparation of 10-hydroxy camptothecin using aspergillus ochraceus which comprises cultivating novel strain of aspergillus ochraceus having characteristics such as herein described in a conventional nutrient medium additionally containing glucose 2 to 4%, caseine hydrolysate 0.2 to 0.8% corn steep liquor, 0.02 to 0.05% in a known manner, addinq camptothecin in a solvent to the above said qrowinq culture followed by incubating for a period of atleast 24hr, extracting the reaction mixture with a water immiscible organic solvent preferably chloro solvent or esters, removing the solvent to get crude to hydroxy camptothecin and further purifying by known chromatographic methods.

Full Text	The. present invention relates to an improved process for the preparation of 10-hydroxy camp-tothecin using Aspergillus ochraceus. Particularly this - relates to a process of biotransformation of camptothecin to 10-HYDROXY CAMPTOTHECIN. The process of present invention uses a new strain of Aspergillus ochraceus designated as RRL 123. Camptothecin {4 (S)-4-ethyl-4-hydroxy-1-H-pyrano {(3,4,6,7)} indolizino (1,2,b)quinoline- 3,14(4H,12H)} dione of formula 1 as shown in the drawing accompanying this specification belongs to a group of anticancer agents with a unique mechanism; of action poisoning of eukaryotic DNA topoisomerase I. Camptothecin is an alkaloid isolated from the Chinese tree Camptotheca acuminata by Wall and coworkers (J.Am.Chem.Soc.88 3388 1966). Camptothecin and minor camptothecinoids have been obtained from the Indian tree Nothapodytes foetida Sleumer (formerly Mappia foetida Miers by Govindachari (Phytochemistry 11 3529 1972) and Japanese spieces of Nothapodytes obscura, N.obtusifolia, N.piltosporosides, N.tomento-sa and N.collina (Zhang Xiande Bao Juchen et al CN.1,045 266 (CIC07D491) 147.12 Sep.1990 C.A 114 164607v 1991). Other plant source of Camptotheci-noides are Ophiorrhizamungoes Linn. (Tafur et al, Lloydia 39 261 1976)Ervatamia heyneana Wall (Gunsekera S.P.et al J.Nat.Prod.42,4 75 1979) and Merriliodendron megacarpus Helms (Arisawa M.et.al.Planta Med.43404 1981). . Camptothecin has been evaluated clinically in China as sodium salt in the treatment of gastric cancer, head, neck, tumour and bladder carcinoma. 10-Hydroxycamptothecin was shownto possess therapeutic effect in Phase 2 clinical trials on liver carcinoma, leukemia,head and neck cancer .,10-Hydroxycamptothecin was reported to show improved antitumour activity and was found to be ten times more potent against P-388 and L1210 mouse leukemia than the parent camp¬tothecin and was less toxic.' The hydrophobicity of camptothecin precluded its development as clinical agent and necessitated the use of the hydrophilic synthetic congeners in various phases of clinical trials. Two water-soluble derivatives, topotecan(TPT) and irinotecan (CPT11) have shown promising chemotherapeu-tic efficacy in clinical trials: (Slichenmyer W.J. et a1.J.Natl.Cancer Inst 85 271-287 1983), The later releasing the prodrug, 10-hydroxy-7-ethylcamp-tothecin (SN-38) by metabolic conversion in liver (Potmesil.M.Cancer Res.54 1431-39 1994). Camptothecinoids belong to a group of anticancer agents with unique mode of action by inhibiting eu-karyotic DNA topoisomerase 1. The cytotoxicity and drug potency of these drugs towards human colon carci¬noma HT2 9 cells were mediated by topoisomerase I for DNA single strand breaks (SSB) during its cleavage (Tanizawa A.et al.J.Nat.Can.Inst. 86(11) 836 1994) . 10-Hydroxycamptothecin (0.005%) and 10-methoxy-camptothecin have been isolated as minor compounds from the extract of stem-wood of C.acuminata by Wall and Wani (J.Org.Ghent.34 1364 1969) . Chu.K.P. et al (K'o Hsuech Tung Pao 23 761 1970 C.A 10 19733q, 1979)obtained 10-hydroxycamp-tothecin by Camptothecin by Aspergillus T 36. Microbial conversion of 2g of camptothecin with this fungus yielded 140mg of 10-hydroxy camptothecin corresponding to 7% yield. Creasey and cowerkers(Cancer Treat. Rep.67 167-82 1983) have shown action of 10-Hydroxycamptothecin on P-388 leukemia. Wani M.C.(J.Med.Chem.23(5)554-560 1980) reported the total synthesis of dl 10-hydroxy-20(S)-camptothe¬cin with the ring E intact involving a number of steps. However,the method gives a low yield and there¬fore is only of academic value. Kingsbury W.D.(J.Med.Chem.37 98-107,1991) converted camptothecin to 10-Hydroxycamptothecin by reduction-oxidation sequence, using platinum catalyst to afford mixture of compounds including 10-acetoxy-camptothecin and unreacted camptothecin. This method of preparation of 10-hydroxy-20 (s)'-camptothecin was not economically viable. The main object of the present invention is to develop an improved process for the preparation of 10-hydroxy camptothecin of formula 2 by submerged fermen¬tation using a. strain of Aspergillus ochraceus RRL-123. Accordingly the present invention provides an improved process for the preparation of 10-hydroxy camptothecin using Aspergillus ochraceus which comprises cultivating novel strain of Aspergillus ochraceus having characteristics such as herein de¬scribed in a conventional nutrient medium additionally containing glucose 2 to 4%, caseine hydrolysate 0.2 to 0.8% corn steep liquor, 0.02 to 0.05% in a known man¬ner, adding camptothecin in a solvent to the above said growing culture followed by incubating for a period of atleast 24hr, extracting the reaction mixture with a water immiscible;organic solvent pref- erably chloro solvent or esters, removing the solvent to get crude to hydroxy camptothecin and further purifying by known chromatographic methods. In an embodiment of the invention the solvent used for dissolving camptothecin may be such as dimethyl formamide, propylene glycol, ethyl alcohol. In another embodiment of this invention, the culture medium may be sterilized at a pressure in the range 10-18psig for a period of 10-20 minutes. In still another embodiment of this invention, incubaton may be effected at temperature ranging from 20°C-30°C on rotary shaker at 220-250 rpm. In yet another embodiment the organic solvent used for extraction may be such as chloroform, dichloromethane, carbontetrachloride, ethylacetate. Purification of the product may be effected by conventional chromatography. Characteristics of Aspergillus ochraceus designated as (RKL 123) Colonies on Czapek's Dox agar at30°C spreading, reaching 50mm in diameter in 7 days,floccose,surface showing ochraceous shades, consisting of conidiophores and conidial heads with little aerial mycelium.Conidial heads compact,columnar,upto 60-70u in diameter, sterig-mata in two series, primary commonly 15-30u long, secondary 7-10x1.5-2u.Conidia globose to elliptical, yellow, 3.5-5u, deposited in RRL-Biotech repository. In the drawing accompanying this specification the scheme of hydroxylation of camptothecin to 10-hydroxy-camptothecin has been suggested which is accomplished by sub-merged fermentation using' a strain of Aspergil¬lus ochraceus RRL123. 10-Hydroxycamptothecin.can be used as sub strate for further modification and to prepare more potent, water-soluble drug which can prove its potency in all phases of clinical trials as hydroxy camptothecin congeners which can be more efficiently absorbed by the body system leading to the development of promising drug for industrial exploitation b>y entrepreneurs. Accordingly the present invention provides an improved process for the preparation of 10- hydroxycamptothecin by Aspergillus ochraceus, RRL 123 which comprises of following steps:- The organism, the developed strain of Aspergillus ochraceus RRL123 is inoculated on Sabouraud's agar slants and incubated for 5-10 days. After this the spore suspension from these slants are transferred in a liquid medium containing casein hydrolysate 0.2-0.8%, glucose 2-4%, cornsteep liquor 0.02-0.05%. The inocu¬lated medium is incubated and shaken continuously for a period of 24-72hrs when optimum growth of myceli¬um is obtained at 25-35°C. Camptothecin upto 99% pure was added to the grow¬ing culture in a suitable solvent and the enzymatic reaction was continued for a period of 72-2 00hrs. The reaction was monitored by chromatographic tech¬niques TLC,HPLC using different solvent systems and at different wavelengths. The formation of the product was also determined: by UV scanning. The product showed. bathochromic 'shift when treated with dilute base. The final purity of the product was determined by superimposable IR(KBR) 3480(OH)-1740-1755cm-1 (lactone >C=0) and HPLC scanning using diode array detector. In the embodiment of the present invention is described in detail a process for the selective hy-droxylation of camptothecin to 10-hydroxycamptothecin by submerged fermentation using a strain of Asper¬gillus ochraceus RRL-123. The following examples are given by way of illustration of the present invention and should not be construed-to limit the scope; of the present inven-tion: - EXAMPLE -I Two days old growth of" Aspergillus ochraceus RRLi 123 on sabouraud's agar slants was inoculated in sterilized medium containing casein hydrolysate 5g, glucose 25g, cornsteep liquor 3.15gl-1.Inoculated medium was incubated at30±l°C on rotary shaker at 220rpm. 500ml Erlenmeyer flask containing 100ml of medium was sterilized at 15psig for 15 minutes. 10% of precultured growth organism was added to production medium as described above and incubated at 3 0±1°C on rotary shaker at 220rpm. After 48hrs of incubation camptothecin 0.5gl dissolved in dimethy formamide (10%) was added. The reaiction mixture was incubated. Biotransformation was monitored after every 24hrs by ferric chloride test and the concentra¬ tion of the substrate and product EtOH was quantified by UV absorption spectra at max 288 (log 4.52) and max EtOH 267(log 4.48). After 168hrs of incubation on rotary max shaker the broth was extracted with chloroform. The solvent was evaporated in rotary evaporator under vacuum and the residue was analysed by TLC (Blue/violet: sports under UV light at 254 & 366nm wavelength),HPLC and UV absorption spectrum. The percentage conversion was 41.21% and total conversion of the substrate was 0.2 06gl The dry residue was chromatographed on silica gel 60 and eluted with 2% methanol in chlo¬roform and yellow soild was obtained, which was recrystallised from 2 0% methanol, chloroform and ethyl acetate and 15mg pale yellow solid was obtained m-p 266-270°c,UV222,2667,330 and382nm,IRvmav(KBr)3480 (OH), 1740(lactone)and 1655 (pyridone)cm-1. EXAMPLE-II Two days old growth of Aspergillus ochraceus RRL 123 on Sabouraud's agar slants was inoculated in sterilized medium containing casein hydrolysate 5g,glucose 25g,and cornsteep liquor 3.15gl . Inocu¬lated medium was incubated at 30+l°C on rotary shaker at 22 0rpm. 500ml Erlenmeyer flask containing 100ml of medium was sterilized at 15 psig for 15 minutes. 10% of precultured growth organism was added to production medium as dessribed above and incubated at. 30 + l°C on rotary shaker at 220rpm. After 48hrs of incubation camptothecin l.Ogl-1 dis¬solved in dimethylformamide(10%) was added. The reaction mixture was incubated. Biotransformation was monitoted after every 24hrs by ferric chloride test and the concentration of the substrate and EtOH product was determined by UV absorption spectra atmax 288 (log 4.52) and Et0H ;2 6 7 (log 4.48)nm re¬spectively. After 168 hrs max hrs of incubation on rotary shaker the broth was extracted with chloroform. The solvent was evaporated in rotary evaporator under vacuum and the residue was analysed by TLC.HPLC and UV absorption spectrum.. The percentage conversion was 13.54% and total conversion of the substrate was 0.13 5g 1" . The dry residue was chro-matographed on silica gel 60 and eluted with 2% metha¬nol in chloroform and yellow soild was ob¬tained, which was recrystallised from 20% metha¬nol, chloroform and ethyl acetate and 7.45mg pale yellow solid was obtained m.p 266-270°c, UV222, 2667,330 and 382nm, IRvmax(KBr)348 0 (OH) 1740(lac¬tone) and 1655 (pyidone)cm-1. EXAMPLE-III Two days old growth of Aspergillus ochraceus RRL 123 on sabouraud's agar slants was inoculated in sterilized medium containing casein hydrolysate 5g,glucose 25g,and cornsteep liquor 3.15gl~ .Inoculated medium was incubated at30+l°C on rotary shaker at 220rpm. 500ml Erlenmeyer flask containing 100ml of medium was sterilized at 15psig for 15 minutes. 10% of precultured growth organism was added to produc¬tion medium as described above and incubated at 3 0±1°C on rotary shaker at 220rpm. Aftier 48hrs of incubation camptothecin2.0gl dissolved in dimethylformamide(10%) was added. The reaction mixture was incubated. Bio- transformation was monitored after every 24hrs,and the concentration of the substrate and product was determined by UV absorption spectra at EtOH max 288 (log 4.52) and Et0H 267 (log:4.48) . After 168hrs of max incubation on rotary shaker the broth was extracted with chloroform. The'solvent was evaporated in rotary evaporator under vacuum and the residue was analysed by TLC,HPLC and UV absorption spectrum. The percentage conversion was 12.37% and total con¬version of the substrate was 0.247gl" . The dry residue was chromatographed oni: silica gel 60 and eluted with 2% methanol in chloroform and yellow soild was obtained, which was recrystallised from 20% methanol, chloroform and ethyl acetate and 18.6mg pale yellow solid was obtained m.p 266-270°c, UV222, 2667, 330 and 382nm, IRvmax(RBr) 3480 (OH), '1740 (lactone) and 1655 (pyidone) cm-1. The main advantages of the present process are- 1. Out of three substrate concentrations used for biotransformation of camptothecin in. lowest substrate concentration described herein1 i.e.0.5gl_1 is the best for making 10-hydroxycamptothecin as the actual product recovered is maximum in this case. 2. The yield of the product recovery is higher with the culture used in this invention. 3. The organism used in this invention has got capability for hydroxylation of camptothecin by electrophilic aromatic substitution and produces a product which is more potent and; hydrophilic in nature. We clalim: 1. An improved, process for . the preparation of 10-hydroxy camptothecin using Aspergillus ochraceus which comprises cultivating novel strain of Asper¬ gillus ochraceus having characteristics such as herein described in a conventional nutrient medium additional¬ ly containing glucose 2 to 4%, caseine hydrolysate 0.2 to 0.8% corn steep liquor, 0.02 to 0.05% in a known manner, adding camptothecin in a solvent to the above said growing culture followed by incubating for a period of atleast 24hr, extracting the reaction mixture with a. water immiscible organic solvent pref¬ erably chloro solvent or esters, removing the solvent to get crude to hydroxy camptothecin and further puri¬ fying by known chromatographic methods. 2. An improved process as clciimed in claim 1 wherein the culture medium is sterilized at pressure in the range 10-18psig for a period of 10-20 minutes. 3. An improved process as claimed in claims 1-2 wherein the solvent used for dissolving camptothecin is dimethyl formamide, propylene glycol and ethyl alcohol. 4. An improved process as claimed in claims 1-3 wherein incubaton is effected at temperature ranging from 20°C-30°C on rotary shaker at 220-250 rpm. 5. An improved process as claimed in claims 1-4 wherein the 10-hydroxycamptothecin is extracted by chloroform, ethylacetate, dichloromethane and carbon tetrachlorde. 6. An improved process for the prepara¬tion of 10-hydroxy camptothecin using Aspergillus ochraceus . substantially as herein described with reference to the examples and drawing accompanying these specifications.

Full Text

The. present invention relates to an improved process for the preparation of 10-hydroxy camp-tothecin using Aspergillus ochraceus. Particularly this - relates to a process of biotransformation of camptothecin to 10-HYDROXY CAMPTOTHECIN. The process of present invention uses a new strain of Aspergillus ochraceus designated as RRL 123.
Camptothecin {4 (S)-4-ethyl-4-hydroxy-1-H-pyrano {(3,4,6,7)} indolizino (1,2,b)quinoline- 3,14(4H,12H)} dione of formula 1 as shown in the drawing accompanying this specification belongs to a group of anticancer agents with a unique mechanism; of action poisoning of eukaryotic DNA topoisomerase I. Camptothecin is an alkaloid isolated from the Chinese tree Camptotheca acuminata by Wall and coworkers (J.Am.Chem.Soc.88 3388 1966). Camptothecin and minor camptothecinoids have been obtained from the Indian tree Nothapodytes foetida Sleumer (formerly Mappia foetida Miers by Govindachari (Phytochemistry 11 3529 1972) and Japanese spieces of Nothapodytes obscura, N.obtusifolia, N.piltosporosides, N.tomento-sa and N.collina (Zhang Xiande Bao Juchen et al CN.1,045 266 (CIC07D491) 147.12 Sep.1990 C.A 114 164607v 1991). Other plant source of Camptotheci-noides are Ophiorrhizamungoes Linn. (Tafur et al, Lloydia 39 261 1976)Ervatamia heyneana Wall (Gunsekera S.P.et al J.Nat.Prod.42,4 75 1979) and Merriliodendron
megacarpus Helms (Arisawa M.et.al.Planta Med.43404 1981).
. Camptothecin has been evaluated clinically in China as sodium salt in the treatment of gastric cancer, head, neck, tumour and bladder carcinoma. 10-Hydroxycamptothecin was shown*to possess therapeutic effect in Phase 2 clinical trials on liver carcinoma, leukemia,head and neck cancer .,10-Hydroxycamptothecin was reported to show improved antitumour activity and was found to be ten times more potent against P-388 and L1210 mouse leukemia than the parent camp¬tothecin and was less toxic.' The hydrophobicity of camptothecin precluded its development as clinical agent and necessitated the use of the hydrophilic synthetic congeners in various phases of clinical trials.
Two water-soluble derivatives, topotecan(TPT) and irinotecan (CPT11) have shown promising chemotherapeu-tic efficacy in clinical trials: (Slichenmyer W.J. et a1.J.Natl.Cancer Inst 85 271-287 1983), The later releasing the prodrug, 10-hydroxy-7-ethylcamp-tothecin (SN-38) by metabolic conversion in liver (Potmesil.M.Cancer Res.54 1431-39 1994).
Camptothecinoids belong to a group of anticancer agents with unique mode of action by inhibiting eu-karyotic DNA topoisomerase 1. The cytotoxicity and drug potency of these drugs towards human colon carci¬noma HT2 9 cells were mediated by topoisomerase I for DNA single strand breaks (SSB) during its cleavage (Tanizawa A.et al.J.Nat.Can.Inst. 86(11) 836 1994) .
10-Hydroxycamptothecin (0.005%) and 10-methoxy-camptothecin have been isolated as minor compounds from the extract of stem-wood of C.acuminata by Wall and Wani (J.Org.Ghent.34 1364 1969) .
Chu.K.P. et al (K'o Hsuech Tung Pao 23 761
1970 C.A 10 19733q, 1979)obtained 10-hydroxycamp-tothecin by Camptothecin by Aspergillus T 36. Microbial conversion of 2g of camptothecin with this fungus yielded 140mg of 10-hydroxy camptothecin corresponding to 7% yield.
Creasey and cowerkers(Cancer Treat. Rep.67 167-82 1983) have shown action of 10-Hydroxycamptothecin on P-388 leukemia.
Wani M.C.(J.Med.Chem.23(5)554-560 1980) reported the total synthesis of dl 10-hydroxy-20(S)-camptothe¬cin with the ring E intact involving a number of
steps. However,the method gives a low yield and there¬fore is only of academic value.
Kingsbury W.D.(J.Med.Chem.37 98-107,1991) converted camptothecin to 10-Hydroxycamptothecin by reduction-oxidation sequence, using platinum catalyst to afford mixture of compounds including 10-acetoxy-camptothecin and unreacted camptothecin. This method of preparation of 10-hydroxy-20 (s)'-camptothecin was not economically viable.
The main object of the present invention is to develop an improved process for the preparation of 10-hydroxy camptothecin of formula 2* by submerged fermen¬tation using a. strain of Aspergillus ochraceus RRL-123.
Accordingly the present invention provides an improved process for the preparation of 10-hydroxy camptothecin using Aspergillus ochraceus which comprises cultivating novel strain of Aspergillus ochraceus having characteristics such as herein de¬scribed in a conventional nutrient medium additionally containing glucose 2 to 4%, caseine hydrolysate 0.2 to 0.8% corn steep liquor, 0.02 to 0.05% in a known man¬ner, adding camptothecin in a solvent to the above said growing culture followed by incubating for a period of atleast 24hr, extracting the reaction mixture with a water immiscible;organic solvent pref-
erably chloro solvent or esters, removing the solvent to get crude to hydroxy camptothecin and further purifying by known chromatographic methods.
In an embodiment of the invention the solvent used for dissolving camptothecin may be such as dimethyl formamide, propylene glycol, ethyl alcohol.
In another embodiment of this invention, the culture medium may be sterilized at a pressure in the range 10-18psig for a period of 10-20 minutes.
In still another embodiment of this invention, incubaton may be effected at temperature ranging from 20°C-30°C on rotary shaker at 220-250 rpm.
In yet another embodiment the organic solvent used for extraction may be such as chloroform, dichloromethane, carbontetrachloride, ethylacetate.
Purification of the product may be effected by conventional chromatography.
Characteristics of Aspergillus ochraceus designated as (RKL 123)
Colonies on Czapek's Dox agar at30°C spreading, reaching 50mm in diameter in 7 days,floccose,surface showing ochraceous shades, consisting of conidiophores
and conidial heads with little aerial mycelium.Conidial heads compact,columnar,upto 60-70u in diameter, sterig-mata in two series, primary commonly 15-30u long, secondary 7-10x1.5-2u.Conidia globose to elliptical, yellow, 3.5-5u, deposited in RRL-Biotech repository.
In the drawing accompanying this specification the scheme of hydroxylation of camptothecin to 10-hydroxy-camptothecin has been suggested which is accomplished by sub-merged fermentation using' a strain of Aspergil¬lus ochraceus RRL123.
10-Hydroxycamptothecin.can be used as sub strate for further modification and to prepare more potent, water-soluble drug which can prove its potency in all phases of clinical trials as hydroxy camptothecin congeners which can be more efficiently absorbed by the body system leading to the development of promising drug for industrial exploitation b>y entrepreneurs.
Accordingly the present invention provides an
improved process for the preparation of 10-
hydroxycamptothecin by Aspergillus ochraceus, RRL 123
which comprises of following steps:-
The organism, the developed strain of Aspergillus ochraceus RRL123 is inoculated on Sabouraud's agar slants and incubated for 5-10 days. After this the spore suspension from these slants are transferred in a
liquid medium containing casein hydrolysate 0.2-0.8%, glucose 2-4%, cornsteep liquor 0.02-0.05%. The inocu¬lated medium is incubated and shaken continuously for a period of 24-72hrs when optimum growth of myceli¬um is obtained at 25-35°C.
Camptothecin upto 99% pure was added to the grow¬ing culture in a suitable solvent and the enzymatic reaction was continued for a period of 72-2 00hrs. The reaction was monitored by chromatographic tech¬niques TLC,HPLC using different solvent systems and at different wavelengths. The formation of the product was also determined: by UV scanning. The product showed. bathochromic 'shift when treated with dilute base. The final purity of the product was determined by superimposable IR(KBR) 3480(OH)-1740-1755cm-1 (lactone >C=0) and HPLC scanning using diode array detector.
In the embodiment of the present invention is described in detail a process for the selective hy-droxylation of camptothecin to 10-hydroxycamptothecin by submerged fermentation using a strain of Asper¬gillus ochraceus RRL-123.
The following examples are given by way of illustration of the present invention and should not be construed-to limit the scope; of the present inven-tion: -
EXAMPLE -I
Two days old growth of" Aspergillus ochraceus
RRLi 123 on sabouraud's agar slants was inoculated
in sterilized medium containing casein hydrolysate
5g, glucose 25g, cornsteep liquor 3.15gl-1.Inoculated
medium was incubated at30±l°C on rotary shaker at
220rpm. 500ml Erlenmeyer flask containing 100ml of
medium was sterilized at 15psig for 15 minutes. 10% of
precultured growth organism was added to production
medium as described above and incubated at 3 0±1°C on
rotary shaker at 220rpm. After 48hrs of incubation
camptothecin 0.5gl dissolved in dimethy
formamide (10%) was added. The reaiction mixture was
incubated. Biotransformation was monitored after
every 24hrs by ferric chloride test and the concentra¬
tion of the substrate and product EtOH was quantified
by UV absorption spectra at max 288 (log 4.52) and
max EtOH 267(log 4.48). After 168hrs of incubation
on rotary max shaker the broth was extracted with
chloroform. The solvent was evaporated in rotary
evaporator under vacuum and the residue was
analysed by TLC (Blue/violet: sports under UV
light at 254 & 366nm wavelength),HPLC and UV
absorption spectrum. The percentage conversion was 41.21% and total conversion of the substrate was 0.2 06gl The dry residue was chromatographed on
silica gel 60 and eluted with 2% methanol in chlo¬roform and yellow soild was obtained, which was recrystallised from 2 0% methanol, chloroform and ethyl acetate and 15mg pale yellow solid was obtained m-p 266-270°c,UV222,2667,330 and382nm,IRvmav(KBr)3480 (OH), 1740(lactone)and 1655 (pyridone)cm-1.
EXAMPLE-II
Two days old growth of Aspergillus ochraceus RRL 123 on Sabouraud's agar slants was inoculated in sterilized medium containing casein hydrolysate 5g,glucose 25g,and cornsteep liquor 3.15gl . Inocu¬lated medium was incubated at 30+l°C on rotary shaker at 22 0rpm. 500ml Erlenmeyer flask containing 100ml of medium was sterilized at 15 psig for 15 minutes. 10% of precultured growth organism was added to production medium as dessribed above and incubated at. 30 + l°C on rotary shaker at 220rpm. After 48hrs of incubation camptothecin l.Ogl-1 dis¬solved in dimethylformamide(10%) was added. The reaction mixture was incubated. Biotransformation was monitoted after every 24hrs by ferric chloride test and the concentration of the substrate and EtOH product was determined by UV absorption spectra atmax 288 (log 4.52) and Et0H ;2 6 7 (log 4.48)nm re¬spectively. After 168 hrs max hrs of incubation on rotary shaker the broth was extracted with chloroform.
The solvent was evaporated in rotary evaporator under vacuum and the residue was analysed by TLC.HPLC and UV absorption spectrum.. The percentage conversion was 13.54% and total conversion of the substrate was 0.13 5g 1" . The dry residue was chro-matographed on silica gel 60 and eluted with 2% metha¬nol in chloroform and yellow soild was ob¬tained, which was recrystallised from 20% metha¬nol, chloroform and ethyl acetate and 7.45mg pale yellow solid was obtained m.p 266-270°c, UV222, 2667,330 and 382nm, IRvmax(KBr)348 0 (OH) 1740(lac¬tone) and 1655 (pyidone)cm-1.
EXAMPLE-III
Two days old growth of Aspergillus ochraceus RRL 123 on sabouraud's agar slants was inoculated in sterilized medium containing casein hydrolysate 5g,glucose 25g,and cornsteep liquor 3.15gl~ .Inoculated medium was incubated at30+l°C on rotary shaker at 220rpm. 500ml Erlenmeyer flask containing 100ml of medium was sterilized at 15psig for 15 minutes. 10% of precultured growth organism was added to produc¬tion medium as described above and incubated at 3 0±1°C on rotary shaker at 220rpm. Aftier 48hrs of incubation camptothecin2.0gl dissolved in dimethylformamide(10%) was added. The reaction mixture was incubated. Bio-
transformation was monitored after every 24hrs,and the concentration of the substrate and product was determined by UV absorption spectra at EtOH max 288 (log 4.52) and Et0H 267 (log:4.48) . After 168hrs of max incubation on rotary shaker the broth was extracted with chloroform. The'solvent was evaporated in rotary evaporator under vacuum and the residue was analysed by TLC,HPLC and UV absorption spectrum. The percentage conversion was 12.37% and total con¬version of the substrate was 0.247gl" . The dry residue was chromatographed oni: silica gel 60 and eluted with 2% methanol in chloroform and yellow soild was obtained, which was recrystallised from 20% methanol, chloroform and ethyl acetate and 18.6mg pale yellow solid was obtained m.p 266-270°c, UV222, 2667, 330 and 382nm, IRvmax(RBr) 3480 (OH), '1740 (lactone) and 1655 (pyidone) cm-1.
The main advantages of the present process are-
1. Out of three substrate concentrations used for biotransformation of camptothecin in. lowest substrate concentration described herein1 i.e.0.5gl_1 is the best for making 10-hydroxycamptothecin as the actual product recovered is maximum in this case.
2. The yield of the product recovery is higher with the culture used in this invention.
3. The organism used in this invention has got capability for hydroxylation of camptothecin by electrophilic aromatic substitution and produces a product which is more potent and; hydrophilic in nature.

We clalim:
1. An improved, process for . the preparation of
10-hydroxy camptothecin using Aspergillus ochraceus
which comprises cultivating novel strain of Asper¬
gillus ochraceus having characteristics such as herein
described in a conventional nutrient medium additional¬
ly containing glucose 2 to 4%, caseine hydrolysate 0.2
to 0.8% corn steep liquor, 0.02 to 0.05% in a known
manner, adding camptothecin in a solvent to the above
said growing culture followed by incubating for a
period of atleast 24hr, extracting the reaction
mixture with a. water immiscible organic solvent pref¬
erably chloro solvent or esters, removing the solvent
to get crude to hydroxy camptothecin and further puri¬
fying by known chromatographic methods.
2. An improved process as clciimed in claim 1 wherein the culture medium is sterilized at pressure in the range 10-18psig for a period of 10-20 minutes.
3. An improved process as claimed in claims 1-2 wherein the solvent used for dissolving camptothecin is dimethyl formamide, propylene glycol and ethyl alcohol.
4. An improved process as claimed in claims 1-3 wherein incubaton is effected at temperature ranging from 20°C-30°C on rotary shaker at 220-250 rpm.
5. An improved process as claimed in claims 1-4 wherein the 10-hydroxycamptothecin is extracted by chloroform, ethylacetate, dichloromethane and carbon tetrachlorde.
6. An improved process for the prepara¬tion of 10-hydroxy camptothecin using Aspergillus ochraceus . substantially as herein described with reference to the examples and drawing accompanying these specifications.

Documents:

3356-del-1997-abstract.pdf

3356-del-1997-claims.pdf

3356-del-1997-complete specification granted.pdf

3356-del-1997-correspondence-others.pdf

3356-del-1997-correspondence-po.pdf

3356-del-1997-description (complete).pdf

3356-del-1997-drawings.pdf

3356-del-1997-form-1.pdf

3356-del-1997-form-2.pdf

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Patent Number

232443

Indian Patent Application Number

3356/DEL/1997

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

17-Mar-2009

Date of Filing

21-Nov-1997

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	VINAY KUMAR GUPTA	REGIONAL RESEARCH, LABORATORY (CSIR) JAMMU ,INDIA
2	SATISH CHANDER PURI	REGIONAL RESEARCH, LABORATORY (CSIR) JAMMU ,INDIA
3	PRITI SOMAL	REGIONAL RESEARCH, LABORATORY (CSIR) JAMMU ,INDIA

PCT International Classification Number

A61K 31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA