nandrolone ( 1 ) was obtained from sigma - aldrich . sabouraud dextrose agar used for culture growth was procured from merck . precoated tlc plates were used for thin layer chromatography ( merck , pf 254 , 20 × 20 , 0 . 25 mm , germany ). column chromatography was done with flash silica . compounds were purified on recycling preparative hplc - lc - 908 ( japan ), equipped with jaigel - ods - l - 80 ( l = 250 mm , i . d .= 20 mm . optical rotations were recorded on jasco p - 2000 polarimeter ( japan ). evolution 300 uv - visible spectrophotometer ( uk ) was used for recording uv spectrum . infrared ( ir ) spectrum was measured on vector 22 ir spectrophotometer ( bruker , france ). 1 h - and 13 c - nmr experiments were recorded in cd 3 od , cdcl 3 and dmso on bruker avance - nmr ( 300 mhz 500 mhz and 600 mhz ). electron impact mass spectra ( ei - ms ) were recorded on jeol jms - 600h mass spectrometer . solvents and reagents were of analytical grades . fungus culture were purchased from american type culture collection ( atcc ). cunninghamella echinulata ( atcc 9244 ) and cunninghamella blakesleeana ( atcc 8688a ) were cultured on sabouraud dextrose agar ( sda ) slant and maintained at 4 ° c . culture media for cunninghamella echinulata ( atcc 9244 ) and cunninghamella blakesleeana ( atcc 8688a ) were prepared by mixing the giving ingredients in one liter of distilled water ; glucose ( 10 g ), kh 2 po 4 ( 5 g ) peptone ( 5 g ), yeast extract ( 5 g ), nacl ( 5 g ) and glycerol ( 10 ml ). five liter of the medium was prepared by mixing of aforementioned chemicals and distributed equally in fifty 250 ml erlenmeyer flasks ( 100 ml each ). the media containing flasks were autoclaved at 121 ° c . the fungal spores were distributed to 3 - 4 seed flasks and incubated on shaker ( 121 rpm ) at 26 ± 2 ° c . till appropriate growth . the spores were then transferred to the remaining flasks and placed on shaker ( 121 rpm at 26 ± 2 ° c .). nandrolone ( 1 ) ( 1 g ) was dissolved in 50 ml of methanol , transferred to the flasks containing four day old culture of cunninghamella echinulata and cunninghamella blakesleeana . the fermentation experiment was continued for 12 days on shaker . on completion of fermentation , the reaction was inhibited by adding dichloromethane and fungal mass was separated by filtration . the extraction of filtrate was done with dcm ( 4 l × 3 ), extract was dried with anhydrous sodium sulfate and evaporated on rotary evaporator to obtain a brown thick material . one gram of nandrolone ( 1 ) was subjected to fermentation for 12 days with cunninghamella echinulata on a rotatory shaker ( 121 rpm ) at 26 + 2 ° c . the filtration , extraction and evaporation resulted brown gum of 1 . 5 g which was fractionated with silica gel column chromatography . the mobile phase was comprised of 10 % gradient hexanes enriched with ethyl acetate . four main fractions ( nano 1 - 4 ) were obtained after compilation of different fractions . compound 5 ( 2 . 5 mg , r t = 22 min ) and 6 ( 5 mg , r t = 31 min .) were isolated from fraction nano - 1 with reverse phase recycling hplc ( methanol : water 60 : 40 ). similarly , fraction nano - 2 was subjected to recycling hplc ( chloroform : isopropanol 94 : 06 ), which yielded metabolite 4 ( 6 mg , r t = 184 min ). fraction nano - 3 yielded metabolite 2 ( 2 mg ) on elution of silica gel column ( 10 % gradient hexanes and ethyl acetate = 15 : 85 ), and compound 3 ( 15 mg , r t = 204 min ) was isolated from fraction nano - 4 through purification with reverse phase recycling hplc ( methanol : water 70 : 30 ). fermentation of nandrolone ( 1 ) with c . blakesleeana yielded four metabolites ( 4 , 5 , 7 and 8 ). incubation , filtration , and extraction procedure was similar as mentioned before . silica gel column chromatography yielded four main fractions ( nacb 1 - 4 ). compound 4 ( 5 mg ) was purified from fraction nabc - 1 with recycling hplc ( chloroform : isopropanol , 94 : 06 r t = 184 min ), whereas compound 5 was isolated from nabc - 2 on elution with reverse phase recycling hplc ( meoh : h 2 o , 60 : 40 , r t = 42 min .). the metabolites 7 and 8 were purified with silica gel column chromatography by using hexanes and ethyl acetate as solvents ( 70 : 30 ). 10β , 12β , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one ( 2 ): white solid ; log ε = 1 . 97 ; [ α ] d 25 =− 126 . 2 ( c 0 . 018 , meoh ); ir ( kbr ); υ max 3419 ( o — h stretching ), 1663 ( c ═ o stretching ); hrei - ms m / z 306 . 1842 ( m + , [ c 18 h 26 o 4 ] + , calc . 306 . 1831 ); ei - ms : m / z 306 . 2 [ m + ]( 25 ), 288 . 2 ( 16 ), 278 . 2 ( 49 ), 190 . 2 ( 39 ); 1 h - nmr ( cd 3 od , 300 mhz ): table 2 ; 13 c - nmr ( cd 3 od , 125 mhz ): table 3 . 10β , 16α , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one ( 3 ): white solid ; log ε = 3 . 4 ; 2 . 8 [ α ] d 25 = 62 . 5 ( c 0 . 01 , meoh ); ir ( chcl 3 ); υ max 3388 ( o — h stretching ), 1664 ( c ═ o stretching ); hrei - ms m / z 306 . 1840 ( m + , [ c 18 h 26 o 4 ] + , calc . 306 . 1831 ); ei - ms : m / z 306 . 2 [ m + ]( 40 ), 288 . 3 ( 30 ), 278 . 3 ( 89 ), 264 . 2 ( 21 ), 213 . 2 ( 66 ); 1 h - nmr ( cd 3 od , 300 mhz ): table 2 ; 13 c - nmr ( cd 3 od , 150 mhz ): table 3 . 6β , 10β , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one ( 4 ): white solid ; log ε = 3 . 4 ; [ α ] d 25 =− 257 . 5 ( c 0 . 02 , meoh ); ir ( chcl 3 ); υ max 3392 ( o — h stretching ), 1669 ( c ═ o stretching ); hrei - ms m / z 306 . 1819 ( m + , [ c 18 h 26 o 4 ] + , calc . 306 . 1831 ); ei - ms : m / z 306 . 2 [ m + ] ( 100 ), 259 . 2 ( 10 ), 138 . 1 ( 18 . 8 ), 133 . 1 ( 16 ), 91 . 1 ( 14 . 4 ); 1 h - nmr ( cd 3 od , 300 mhz ): table 2 ; 13 c - nmr ( cd 3 od , 125 mhz ): table 3 . 10β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 5 ): white solid ; log e = 3 . 34 [ α ] d 25 = 274 . 2 ( c 0 . 07 , meoh ); ir ( chcl 3 ); υ max 3406 ( o — h stretching ), 1656 ( c ═ o stretching ); hrei - ms m / z 290 . 1900 ( m + , [ c 18 h 26 o 4 ] + , calc . 290 . 1882 ); ei - ms : m / z 290 . 0 [ m + ]( 60 . 9 ), 262 . 1 ( 100 . 0 ), 248 ( 21 . 2 ), 148 . 0 ( 69 . 0 ), 133 . 0 ( 71 . 0 ), 99 . 0 ( 21 . 7 ); 1 h - nmr ( cdcl 3 , 300 mhz ); 13 c - nmr ( cdcl 3 , 125 mhz ). 6β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 6 ): white solid ; log ε = 3 . 34 ; [ α ] d 25 = 126 . 2 ( c 0 . 012 , meoh ); ir ( chcl 3 ); υ max 3389 . 2 ( o — h stretching ), 1666 ( c ═ o stretching ); hrei - ms m / z 290 . 1897 ( m + , [ c 18 h 26 o 3 ] + , calc . 290 . 1882 ); ei - ms : m / z 290 . 1 [ m + ]( 23 . 5 ), 261 . 1 ( 15 . 0 ), 246 . 1 ( 9 . 3 ), 213 . 1 ( 27 . 7 ), 138 . 1 ( 52 . 5 ); 1 h - nmr ( cdcl 3 , 300 mhz ); 13 c - nmr ( cd 3 od , 150 mhz ). 10β - hydroxy - 19 - nor - 4 - androsten - 3 , 17 - dione ( 7 ): white solid ; log ε = 2 . 8 ; [ α ] d 25 =− 68 . 8 ( c 0 . 006 , meoh ); ir ( chcl 3 ); υ max 3413 . 9 ( o — h stretching ), 1723 ( c ═ o stretching ); hrei - ms m / z 288 . 1725 ( m + , [ c 18 h 24 o 3 ] + , calc . 288 . 1725 ); ei - ms : m / z 288 . 1 [ m + ]( 7 . 0 ), 272 ( 10 . 0 ), 261 . 1 ( 18 . 8 ), 138 . 1 ( 53 . 0 ); 1 h - nmr ( cdcl 3 , 300 mhz ); 13 c - nmr ( cd 3 od , 100 mhz ). 16β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 8 ): white solid ; log ε = 0 . 9 ; [ α ] d 25 = 34 . 4 ( c 0 . 012 , meoh ); ir ( chcl 3 ); υ max 3395 ( o — h stretching ), 1660 ( c ═ o stretching ); hrei - ms m / z 290 . 1871 ( m + , [ c 18 h 26 o 3 ] + , calc . 290 . 1882 ); ei - ms : m / z 290 . 1 [ m + ] 290 . 1 ( 6 ), 272 . 2 ( 5 ), 149 . 0 ( 15 ), 110 ( 35 ), 82 . 9 ( 70 ). 1 h - nmr ( cdcl 3 , 300 mhz ); 13 c - nmr ( cdcl 3 , 100 mhz ). compound 2 was obtained as white solid . the molecular formula ( c 18 h 26 o 4 ) of compound 2 was assigned on the bases of its hrei - ms which showed [ m ] + at m / z 306 . 1842 ( calc . 306 . 1831 ). the molecular mass was 32 amu higher than 1 which indicated dihydroxylation . the ir spectrum showed characteristic absorption at 3419 cm - 1 ( hydroxyl ) and 1663 cm - 1 ( ketonic carbonyl ). 1h - nmr spectrum showed the presence of a downfield methine proton at δ 3 . 45 ( dd , j aa = 10 . 5 hz , j ae = 5 . 7 hz ). the 13 c - nmr spectrum showed two additional downfield carbon signals at δ 70 . 6 and 79 . 8 , which supported the dihyroxylation . the hmbc correlations of h - 4 ( δ 5 . 72 s ), h 2 - 6 ( δ 2 . 20 m , 1 . 52 m ) and h 2 - 2 ( δ 2 . 66 m , 2 . 32 m ) with c - 10 ( δ 70 . 6 ) suggested the position of one of the two hydroxyl groups at c - 10 . similarly , the position of second hydroxyl group was assigned at c - 12 by the hmbc correlations of h 2 - 11 ( δ 1 . 64 , 1 . 73 ), h - 17 ( δ 3 . 81 ) and h 3 - 18 ( δ 0 . 84 ) with c - 12 ( δ 79 . 8 ) the oh at c - 10 was assigned β ( axial ) stereochemistry on the basis of noe correlation of h - 11 ( δ 1 . 64 axial ) with oh ( δ 5 . 01 ) at c - 10 , and noesy correlation with h - 18 ( δ 0 . 84 ). the methine proton attached to c - 12 ( δ 3 . 45 ) was a oriented as it showed noesy correlations with h - 9 , h - 14 and h - 17 . the metabolite 2 was deduced as 10β , 12β , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one . the molecular composition of compound 3 ( c 18 h 26 o 4 ) was determined with hrei - ms where it showed the molecular ion peak [ m ] + at m / z 306 . 1840 ( calc . 306 . 1831 ). the molecular mass of the observed compound was 32 amu higher than substrate 1 . the ir spectrum showed characteristic absorption at 3352 ( o — h ) and 1664 cm − 1 ( c ═ o ). an additional methine proton at δ 4 . 02 ( q , j ee = 12 . 5 hz , j ea = 7 . 5 hz ) was observed in 1 h - nmr spectrum . the presences of two additional oh groups in compound 3 were supported by the 13 c - nmr spectrum where a new methine and a quaternary carbons were resonated at ( δ 70 . 8 ) and ( δ 70 . 9 ) respectively . the cosy - dfqf spectrum showed the coupling between the new methine proton ( δ 4 . 02 ) and h - 17 methine proton ( δ 3 . 30 d , j ae = 7 . 44 hz ). the hmbc spectrum showed correlations of h - 2 ( δ 2 . 58 , 2 . 27 ) and h - 4 ( δ 5 . 72 ) with c - 10 ( δ 70 . 9 ), indicating one hydroxylation at c - 10 . similarly , h - 15 ( δ 2 . 18 ) showed the hmbc correlations with new methine carbon ( δ 70 . 8 ). therefore second hydroxylation was occurred at c - 16 . the oh ( δ 4 . 59 ) at c - 10 was assigned β stereochemistry on the basis of its noe correlation with h - 8 ( δ 1 . 87 ) ( dmso - d 6 ). the β stereochemistry of h - 16 was assigned through noesy correlations of methine h - 16 ( δ 4 . 02 ) with h 3 - 18 ( δ 0 . 88 ). the compound 3 was identified as 10β , 16α , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one . the hrei - ms of compound 4 showed molecular ion peak [ m ] + at m / z 306 . 1819 ( c 18 h 26 o 4 , calc . 306 . 1831 ), indicating the presence of two additional oh . the ir absorbances were observed at 3392 ( oh ) and 1669 cm − 1 ( c ═ o ). the 1 h - nmr spectrum showed an additional broad singlet of methine proton at δ 4 . 39 ( w 1 / 2 = 1 . 2 hz ). two new downfield signals were resonated at δ 73 . 8 ( c - 6 ) and 72 . 3 ( c - 10 ) in 13 c - nmr spectrum which further supported the dihydroxylation in compound 4 . the methine proton at δ 4 . 39 showed hmbc correlations with c - 4 ( δ 126 . 2 ), c - 5 ( δ 162 . 4 ) and c - 7 ( δ 39 . 4 ), indicated second oh at c - 6 . the h 2 - 1 ( δ 2 . 64 , 1 . 26 ), h 2 - 2 ( δ 2 . 18 , 1 . 87 ) and h - 4 ( δ 5 . 81 ) showed hmbc correlations with c - 10 ( δ 72 . 3 ), hence second hydroxylation was inferred at c - 10 . the methine proton at δ 4 . 39 showed noesy correlations with h - 4 ( δ 5 . 81 ) and h 2 - 7 ( δ 2 . 01 , 1 . 26 ). therefore , β - oh was placed at c - 6 . the noesy correlation of oh ( δ 5 . 01 ) ( dmso - d 6 ) with c - 6 oh ( δ 4 . 49 ) indicated a β - oh at c - 10 . thus the compound 4 was identified as 6β , 10β , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one . the structures of four known compounds 5 - 8 were characterized by comparison of their literature reported data with the observed data . the molecular mass of compounds 5 , 6 and 8 were 16 amu greater than 1 which showed mono - hydroxylation . compounds 5 , 6 and 8 showed ei - ms [ m ] + m / z at 290 whereas compound 7 at m / z 288 . metabolites 5 - 8 were identified as 10β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 5 ), 6β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 6 ), 10β - hydroxy - 19 - nor - 4 - androsten - 3 , 17 - dione ( 7 ) and 16β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 8 ). compounds 5 and 6 were previously synthesized by the biotransformation of 1 with aspergillus wentii mrc 200316 , whereas compound 7 was synthesized from androstene - 3 , 17 , 19 - trione by aromatase cytochrome p - 450 mediated transformation . metabolite 8 was synthesized from 1 through fermentation with molds . leishmania major was obtained from desto laboratories , pakistan . blood agar basic medium from bd company ( france ) was used in assay , rpmi - 1640 medium was purchase from sigma aldrich ( usa ) and fetal bovine serum was obtained from paa laboratories ( uk ). 96 well plates used in assay were purchase from coster ( italy ) and the neubauer chamber was purchase from marine field germany leishmania major were grown in modified nnn biphasic medium ( rpmi - 1640 + fetal bovine serum + blood agar in blood ) by using normal physiological saline . rpmi 1640 medium was used for culturing of leishmania promastigotes using 10 % heat inactivated fetal bovine serum ( fbs ) as supplemented . parasites were centrifuged ( at log phase ) at 2000 rpm for 10 minutes , and washed three times with saline at same speed and time . fresh culture medium was used for dilution of parasites to acquire a final density of 10 6 cells / ml . a 180 μl of medium was added in wells of first row and 100 μl of medium was added in remaining wells of a 96 - well microtiter plate . a 20 μl aliquot of the experimental compound was added in medium and serially diluted . a 100 μl aliquot of parasite culture was added in all wells , and two rows were left for negative and positive controls . negative controls contained only medium , while the positive control received the varying concentrations of standard leishmanicidal compound amphotericin or pentamidine . this plate was than incubated at 21 - 22 ° c . for 72 h . the culture was examined microscopically on an improved neubauer counting chamber . the ic 50 values of test compounds were calculated by software ezfit 5 . 03 perella scientific ( usa ). all assays were repeated thrice . nandrolone ( 1 ) and its derivatives , 10β , 12β , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one ( 2 ), 10β , 16α , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one ( 3 ), 6β , 10β , 17β - trihydroxy - 19 - nor - 4 - androsten - 3 - one ( 4 ), along with four known metabolites , 10β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 5 ), 6β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 6 ) 10β - hydroxy - 19 - nor - 4 - androsten - 3 , 17 - dione ( 7 ) and 16β , 17β - dihydroxy - 19 - nor - 4 - androsten - 3 - one ( 8 ) were subjected to leishmanicidal assay . the compounds 1 - 8 showed varying ranges of ic 50 values , i . e . 29 . 55 to 80 . 23 μm against leishmaniasis . compound 8 showed significant leishmanicidal activity with an ic 50 value of 29 . 55 μm as compared to the standard pentamidine ic 50 5 . 09 ± 0 . 09 μm and it was the most active compound of this series . compound 3 , 4 and 6 showed very low activity , having ic 50 values of 77 . 39 , 70 . 90 and 80 . 23 μm , respectively . compound 5 and 7 showed moderate leishmanicidal activity with ic 50 values of 54 . 94 and 61 . 12 μm , respectively . the metabolite 8 was found to be more active against leishmania as compared to nandrolone ( 1 ) and all other transformed metabolites . since the only difference between compounds 1 to 8 is the position and number of oxygen atoms , it is suggested that the carbon - oxygen bonding plays an important role in leishmanicidal activity . nandrolone ( 1 ) has one ketonic carbonyl group at position c - 3 and a hydroxyl group at position c - 17 . it showed significant activity with ic 50 32 . 0 μm . compound 2 was found to be inactive against the leishmaniasis with an ic 50 greater than 100 μm . compound 2 has two hydroxyl groups , one at c - 10 and second at c - 12 , along with ketonic carbonyl at c - 3 , both have 13 stereochemistry . compound 3 also has two more hydroxyl groups as compared to nandrolone ( 1 ) and it was very low activity with an ic 50 of 77 . 39 μm . compound 4 also showed very low activity with an ic 50 of 70 . 90 μm . it was also dihydroxylated having hydroxyl groups at c - 6 and c - 10 , both having β orientated hydroxyl groups . the position of the hydroxyl group has a significant effect on activity . monohydroxylated compound 5 showed low activity having activity ic 50 of 54 . 94 μm . in this compound , hydroxylation occurs at c - 10 . on the other hand when hydroxylation occurs at c - 6 , activity again decreases . this was observed in case of compound 6 . compound 7 has a ketonic carbonyl group instead of a hydroxyl group at c - 17 , and also showed a very low activity against leishmaniasis , with an ic 50 of 80 . 23 μm . compound 8 showed significant activity against leishmaniasis . compound 8 is mono hydroxylated having β hydroxylation at c - 16 . the ic 50 was 29 . 55 m , which is comparable to compound 1 . compounds 1 and 8 were considered as having significant activity when compared to the standards , i . e . pentamidine ( ic 50 = 5 . 09 + 0 . 09 m ) and amphotericin b ( ic 50 = 0 . 29 ± 0 . 05 μm ).
this invention provides new therapeutic potential of nandrolone or a derivative thereof , against protozoal diseases . more specifically , nandrolone or a derivative thereof exhibits anti - protozoal activity against leishmania major . anti - protozoal potential of nandrolone derivatives , such as compounds 1 - 8 , can contribute in the development of effective therapies against protozoal diseases , such as leishmaniasis , trypanosomiasis , malaria , toxoplasmosis , babeosis , amoebic dysentery and lambliasis . another aspect of the invention is a method of testing derivatives of nandrolone for anti - protozoal activity comprising growing leishmania in the presence of the test derivative and determining the ic 50 value .