betulinic acid was isolated from the bark of biscofia javanica through soxhlet apparatus in toluene . all the chemicals , solvents used were reagent grade and purified , where applicable , prior to their use . the nmr chemical shift was reported in ppm relative to 7.20 and 77.0 ppm of cdcl3 solvent as the standards . h spectra were recorded in 300 mhz frequencies and c nmr spectra were recorded in 75.4 mhz frequencies . coupling constant j n - bromosuccinimide ( nbs ) in ccl4-chcl3 ( 1:1 mixture ) was treated with the methyl ester of betulinic acid at room temperature for 48 h. the reaction mixture was then filtered and solvent was recovered in reduced pressure . it was then poured into ice cold water , a white precipitate appeared that on purification over a column of silica gel gave 29,30-dibromo-28-oxoallobetulin ( 3 ) , in 22 % yield . ir at 3452 ( -oh ) , 2942 , 2858 , 1764 ( lactone carbonyl ) , 1724 , 1453 , 1380 ( gem dimethyl ) , 1024 , 668 cm , molecular ion peak at m / z 614 corresponding to the molecular formula of c30h46br2o3 , h nmr dh 0.71 ( 24-me ) , 0.79 ( 25-me ) , 0.84 ( 23-me ) , 0.86 ( 27-me ) and 0.92 ( 26-me ) , dh 3.15 ( m , 1h , ) and dh 3.48 ( m , 2h ) and 3.65 ( m , 2h ) . the cytotoxicity assay was performed on three different strain of leishmania sp . in three independent experiments as per the guidelines of biosafety committee of west bengal state university . leishmania donovani ag83 ( mhom / in/83/ag83 ) was originally obtained from indian institute of chemical biology , kolkata , india . leishmania donovani ms10 , a field isolate , was obtained from ipgmer , kolkata , india as a gift . l. donovani ms10 strain was an isolate recently transformed from l. donovani - infected patients from the geographical location of west bengal , india . leishmania major lv39 ( mrho / sv/59/p strain ) was obtained university of lausanne , switzerland as a gift . the parasites were maintained in the animal facility as per the guidelines of institutional animal ethics committee of west bengal state university . promastigote morphs of leishmania sp . were transformed from intracellular amastigotes , acquired from splenic aspirates of infected balb / c mice in complete m 199 medium ( invitrogen ) supplemented with 1% penicillin - streptomycin ( invitrogen ) and 10% fcs ( gibco ) at requisite temperature , 22 for l. donovani ( ag83 and ms10 ) and at 26 for l. major lv39 . to estimate the percentage of inhibition the derivative , the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) micro method briefly , late - log - phase promastigotes were seeded in a 96-well flat - bottom plate ( 200 l per well ; bd falcon ) in complete m 199 medium in presence or absence of the derivative . to examine the effect of compound 3 , the cultures were additionally supplemented without ( dmso control ) or with increasing concentrations of the derivative ( 5 to 25 g / ml ) soluble in dmso ( 0.2% v / v ) . after 72 h of incubation in requisite temperature , mtt ( 10 mg / ml , 10 l per well ) was added to each well and the plates were incubated for an additional 4 h at 37. the enzyme reaction was then stopped by addition of acidic isopropanol ( 0.4 ml 10 nhcl in 100 ml isopropanol , 100 l per well ) , and the absorbance was measured at 570 nm . degree of cytotoxicity of conventional antileishmanial drug , sodium antimony gluconate ( sag ) , was also measured on leishmania donovani ag83 at the increasing concentration of 5 g / ml to 25 g / ml . percent inhibition was measured in respect to the proliferation of leishmania promastigotes of dmso control group . statistical analyses for all experimentswere performed by student 's test with the program sigma plotusing alpha adjustment . the nuclear dna content of a cell can be quantitatively measured at high speed by flow cytometry . initially , a fluorescent dye that binds stoichiometrically to the dna is added to a suspension of permeabilized single cells or nuclei . the principle is that the stained material has incorporated an amount of dye proportional to the amount of dna . the stained material is then measured in the flow cytometer and the emitted fluorescent signal yields an electronic pulse with a height ( amplitude ) proportional to the total fluorescence emission from the cell . thereafter , such fluorescence data are considered as measurement of the cellular dna content . for flow cytometry analysis of dna content , exponentially grown l. donovani promastigote cells ( 210 ) were incubated without or with 10 g / ml and 15 g / ml of test compound in complete m199 media for 48 h. cells were then harvested and washed three times with 1x pbs , fixed in 45% ethanol ( diluted in 1x pbs ) , treated with 500 g / ml rnase a and then suspended in 0.5 m sodium citrate containing 69 m pi . these samples were analyzed through flow cytometry ( becton and dickinson , usa ) after keeping them in the dark for 45 min . n - bromosuccinimide ( nbs ) in ccl4-chcl3 ( 1:1 mixture ) was treated with the methyl ester of betulinic acid at room temperature for 48 h. the reaction mixture was then filtered and solvent was recovered in reduced pressure . it was then poured into ice cold water , a white precipitate appeared that on purification over a column of silica gel gave 29,30-dibromo-28-oxoallobetulin ( 3 ) , in 22 % yield . ir at 3452 ( -oh ) , 2942 , 2858 , 1764 ( lactone carbonyl ) , 1724 , 1453 , 1380 ( gem dimethyl ) , 1024 , 668 cm , molecular ion peak at m / z 614 corresponding to the molecular formula of c30h46br2o3 , h nmr dh 0.71 ( 24-me ) , 0.79 ( 25-me ) , 0.84 ( 23-me ) , 0.86 ( 27-me ) and 0.92 ( 26-me ) , dh 3.15 ( m , 1h , ) and dh 3.48 ( m , 2h ) and 3.65 ( m , 2h ) . the cytotoxicity assay was performed on three different strain of leishmania sp . in three independent experiments as per the guidelines of biosafety committee of west bengal state university . leishmania donovani ag83 ( mhom / in/83/ag83 ) was originally obtained from indian institute of chemical biology , kolkata , india . leishmania donovani ms10 , a field isolate , was obtained from ipgmer , kolkata , india as a gift . l. donovani ms10 strain was an isolate recently transformed from l. donovani - infected patients from the geographical location of west bengal , india . leishmania major lv39 ( mrho / sv/59/p strain ) was obtained university of lausanne , switzerland as a gift . the parasites were maintained in the animal facility as per the guidelines of institutional animal ethics committee of west bengal state university . promastigote morphs of leishmania sp . were transformed from intracellular amastigotes , acquired from splenic aspirates of infected balb / c mice in complete m 199 medium ( invitrogen ) supplemented with 1% penicillin - streptomycin ( invitrogen ) and 10% fcs ( gibco ) at requisite temperature , 22 for l. donovani ( ag83 and ms10 ) and at 26 for l. major lv39 . to estimate the percentage of inhibition the derivative , the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) micro method briefly , late - log - phase promastigotes were seeded in a 96-well flat - bottom plate ( 200 l per well ; bd falcon ) in complete m 199 medium in presence or absence of the derivative . to examine the effect of compound 3 , the cultures were additionally supplemented without ( dmso control ) or with increasing concentrations of the derivative ( 5 to 25 g / ml ) soluble in dmso ( 0.2% v / v ) . equal volume of dmso only was added in control experiments . after 72 h of incubation in requisite temperature , mtt ( 10 mg / ml , 10 l per well ) was added to each well and the plates were incubated for an additional 4 h at 37. the enzyme reaction was then stopped by addition of acidic isopropanol ( 0.4 ml 10 nhcl in 100 ml isopropanol , 100 l per well ) , and the absorbance was measured at 570 nm . degree of cytotoxicity of conventional antileishmanial drug , sodium antimony gluconate ( sag ) , was also measured on leishmania donovani ag83 at the increasing concentration of 5 g / ml to 25 g / ml . percent inhibition was measured in respect to the proliferation of leishmania promastigotes of dmso control group . statistical analyses for all experimentswere performed by student 's test with the program sigma plotusing alpha adjustment . the nuclear dna content of a cell can be quantitatively measured at high speed by flow cytometry . initially , a fluorescent dye that binds stoichiometrically to the dna is added to a suspension of permeabilized single cells or nuclei . the principle is that the stained material has incorporated an amount of dye proportional to the amount of dna . the stained material is then measured in the flow cytometer and the emitted fluorescent signal yields an electronic pulse with a height ( amplitude ) proportional to the total fluorescence emission from the cell . thereafter , such fluorescence data are considered as measurement of the cellular dna content . for flow cytometry analysis of dna content , exponentially grown l. donovani promastigote cells ( 210 ) were incubated without or with 10 g / ml and 15 g / ml of test compound in complete m199 media for 48 h. cells were then harvested and washed three times with 1x pbs , fixed in 45% ethanol ( diluted in 1x pbs ) , treated with 500 g / ml rnase a and then suspended in 0.5 m sodium citrate containing 69 m pi . these samples were analyzed through flow cytometry ( becton and dickinson , usa ) after keeping them in the dark for 45 min . the modifications in the present work focused on the introduction of a halogen atom namely bromine into the pentacyclic triterpene skeleton . the compound 29,30-dibromo-28-oxoallobetulin was previously synthesized from methylbetulinate in a reaction that took at least ten days without any report of its biological activity . the present synthetic method involves the treatment of n - bromo succinimide ( nbs ) in ccl4-chcl3 ( 1:1 mixture ) on the methyl ester of betulinic acid efficiently in 22 % yield . methyl ester of betulinic acid was treated with recrystallized nbs in a 1:1 mixture of ccl4-chcl3 for 48 h. purification of the reaction mixture over a column of silica gel ( 60 - 120 mesh ) gave a white powdered compound of melting point 285 . in the ir spectrum it gave peaks at 3452 ( -oh ) , 2942 , 2858 , 1764 ( lactone carbonyl ) , 1724 , 1453 , 1380 ( gem dimethyl ) , 1024 , 668 cm . in the mass spectrum it showed a molecular ion peak at m / z 614 . in the mass spectrum three distinct peaks atm / z 612 , 614 and 616 appeared in the ratio of 1:2:1 , thus signifying the presence of two bromine atoms in the synthesized molecule . from the elemental analysis and mass spectral data the molecular formula was determined to be c30h46br2o3 . in the h nmr spectrum it showed the presence of five methyl groups at h 0.71 ( 24-me ) , 0.79 ( 25-me ) , 0.84 ( 23-me ) , 0.86 ( 27-me ) and 0.92 ( 26-me ) . the absence of any methyl peak beyond dh 1.00 ( c-30 methyl group of the isopropelene side chain of lupane skeleton ) signified that during the reaction rearrangement of the isopropelene side chain could have been take place at the e ring of the pentacyclic triterpenoid . the h at c-3 appeared as a multiplet centered at dh 3.15 ( m , 1h ) . upto this it was clear that the two newly incorporated bromine atoms might be at c-29 and c-30 . this assumption was supported by the presence of two multiplets each with an integration of two protons centered at dh 3.48 ( m , 2h ) and 3.65 ( m , 2h ) . the presence of bromine atoms might be the chief cause for the observed deshielding of the two methylene groups . another sharp singlet at dh 4.29 ( s , 1h ) with an integration of one proton was due to the lactonic proton at c-19 . the lactone carbonyl appeared at dc 178.6 and the two bromine bearing carbon atoms appeared at dc 78.9 and 65.5 . all these data can only be explained by considering structure 3 to the synthesized compound . antiproliferative effect of 3 was evaluated on leishmania donovani , causative agent of visceral leishmaniasis and leishmania major , causative agent of cutaneous leishmaniasis ( fig . it has been estimated that 25 g / ml derivative inhibit the l. donovani ag83 promastigote proliferation by 63.27 % ( p<0.001 ) in respect to dmso control ( table 1 ) . at the same dose significantly , it has been found that 3 could inhibit the l. donovani ms10 strain , a field isolate obtained from an active visceral leishmaniasis patient very recently , by 85.92% ( p<0.001 ) in respect to dmso control . however , the effect of 3 was found costly against l. major lv39 , causing 39.22% ( p<0.001 ) inhibition in respect to dmso control . our data clearly indicated that 3 have a noteworthy antiproliferative effect on l. donovani ag83 and ms10 strain . we assumed that the finding is significant as 3 is highly effective against the recently isolated leishmania donovani from active visceral leishmaniasis patient and should be further investigated for its immunomodulatory consequences . antiproliferative effect of compound 3 i.e. 29,30-dibromo-28-oxoallobetulin on three different strains of leishmania donovani ag83 ( a ) , leishmania major lv39 ( b ) , leishmania donovani ms10 ( c ) by mtt assay . effect of 3 was compared with conventional antileishmanial drug sodium antimony gluconate ( sag ) on leishmania donovani ag83 ( d ) . percentage of proliferation inhibition of leishmania promastigotes in respect to dmso control cell - cycle analysis complemented the previous data obtained by mtt assay . it demonstrated that at 48 h of culture , both 10 and 15 g / ml of test compound caused l. donovani promastigotes to remain as resting g0/g1 ( m2 ) cells and inhibited their entry into the s phase ( m3 ) . the percent dead cells ( m1 ) increased during this incubation period and growth arrest was also visible . test compound at a concentration of 10 g / ml started to block the entry of l. donovani promastigotes into s phase from g0/g1 , however , at 15 g / ml completely blocked the entry dose dependently ( table 2 and fig . our results suggested that 29,30-dibromo-28-oxoallobetulin , 3 preferentially active against l. donovani promastigotes at inducing cell - cycle arrest followed by death in vitro . l. donovani ag83 promastigotes ( 210/ml ) were incubated with dmso ( 0.2% ) , 10 and 15 g / ml of test compound ( dissolved in dmso ) in complete m199 media at 22 , and analyzed for dna content by flow cytometry as described in experimental section . the percentage of cells in different phases of life cycle after treatment with test compound we have developed a simple method for the synthesis of 29,30-dibromo-28-oxoallobetulin . the compound can inhibit the growth of l. donovani ag83 promastigote proliferation by 63.27% ( p<0.001 ) in respect to dmso control . at the same dose the conventional drug sag inhibited only by 10.02% in respect to dmso control . our results suggested that compound 3 preferentially active against l. donovani promastigotes at inducing cell - cycle arrest followed by death in vitro . we assumed that the finding is significant as 3 is highly effective against the recently isolated leishmania donovani from active visceral leishmaniasis patient and should be further investigated for its immunomodulatory consequences .
a simple synthesis and in vitro antileishmanial activity of 29,30-dibromo-28-oxoallobetulin against the parasitic protozoans , leishmania donovani and leishmania major is described . 
 the structure of the compound is established on the basis of spectral data ( ir , nmr , ms ) . 
 both the antiproliferative effect and the cell cycle progression were studied .