In vitro Antiplasmodial Activity and Cytotoxicity of Vincadifformine and Its Semisynthetic Derivatives

An indole alkaloid with aspidospemane structure possessing a potential antiplasmodial activity, vincadifformine, has been isolated from Aspidosperma pyrifolium Mart. Moreover, 10 derivatives were prepared from the vincadifformine. The study was conducted to evaluate the in vitro antiplasmodial and cytotoxic activity of the vincadifformine and their semisynthetic derivatives. The in vitro antiplasmodial activity was evaluated on Plasmodium falciparum chloroquine-resistant (FcM ) and –sensitive (Nigerian) strains after 24-h and 72-h incubation, 29 while cytotoxic activity was estimated on Hela cells and Cytotoxicity Index (CI = IC on HeLa cells/IC on FcM strain) 50 50 29 was calculated to evaluate the safety of tested compounds. Experiment results showed that two compounds (4 and 8) exhibited good antiplasmodial activities in comparison with parent compound, vincadifformine and other tested compounds with IC ranging from 5.3 to 12.8 μM on FcM strain and 11.4 to 24.0 μM on Nigerian strain. In addition, 50 29 the CI of two compounds were also lower after 24-h incubation (CI, 2.0 and 4.8) than that of after 72-h incubation (CI, 9.5 and 11.5). Further study will be conducted to evaluate quantitative structure-activity relationship (QSAR) in order to design new antimalarial drugs.

plants like quinine from Cinchona ledgeriana or artemisinin from Artemisia annua (Li and Rieckmann, 1992).In South America, medicinal plants play an important role in the treatment of a variety of diseases such as malaria.Aspidosperma pyrifolium, Mart. is one of the medicinal plants traditionally used to treat malaria in Bolivia.As A. pyrifolium Mart. is used to treat malaria by traditional healers, it is retained as a potential antimalarial candidate.Previous studies reported the presence of numerous indole alkaloids in the A. pyrifolium, Mart., among other things indole alkaloid with aspidospermane structure having an potential antiplasmodial activity (Mitaine et al., 1996).Other aspidospremane structure viz.vincadifformine has been isolated from A. pyrifolium Mart.and a series of their semisynthetic derivatives have been

In vitro Antiplasmodial Activity and Cytotoxicity of Vincadifformine and Its Semisynthetic Derivatives Introduction
T h e d e v e l o p m e n t o f n e w antimalarial drugs is of paramount importance to combat the rapid spread of multi-drug resistant strains of Plasmodium falciparum.The growing reality that antimalarial drugs currently available may not be effective any more has led to the investigation of medicinal plant used in traditional medicine for hundreds of years.Medicinal plants for malaria treatment could be a promising source of new antimalarial agents.Significant success was achieved with the new agent extracted from Corresponding author : Mustofa, Department Pharmacology & Toxicology, Faculty of Medicine, Gadjah Mada University, Sekip Utara, Yogyakarta 55281, Indonesia.Telp.+62-274-511103, Fax. +62-274-511103;e-mail : mustofajogja@yahoo.comsynthesized.The aim of the present study was to investigate the in vitro antiplasmodial activity of these compounds on P. falciparum culture.These compounds were also tested for cytotoxic activity on HeLa cell lines.

Tested compounds
Vincadifformine and 10 semisynthetic derivatives were evaluated in this study (Figure 1).These compounds were supported by Professor Guy Lewin from Department of Pharmacognocy Center for Pharmaceutical Study, Châtenay Malabry, France.The vincadifformine and their s e m i s y n t h e t i c d e r i v a t i v e s w e r e differentiated from one another by the substitution difference on C (R ), C (R , R ),  Plasmodium falciparum strains and in vitro culture Two strains of P. falciparum were used to evaluate the in vitro antiplasmodial activities of the compounds.A FcM strain 2 9 (chloroquine-resistant strain with IC for 50 chloroquine about 230 ng/mL) and a Nigerian (chloroquine-sensitive strain with chloroquine IC , about 41 ng/mL) were 50 cultured continuously according to Trager and Jensen (1976).The IC values of 50 chloroquine were checked every two months and we observed no significant variations.
The parasites were maintained in vitro  (1978) followed by 5% of D-sorbitol lysis (Merck) as reported by Lambros and Venderberg (1979).

Assay for antiplasmodial activity
The antiplasmodial activity of the compounds was evaluated by a radioactive method described by Desjardins et al., (1979).Testing of each compound was performed tree times in triplicate in 96-well culture plates (TPP).Compounds testing were dissolved initially in dimethyl sulphoxide (DMSO) and then diluted in serum-free culture medium to provide stock solutions.Further dilutions were made before testing each compound at various concentrations.One hundred µL of vigorously growing culture with a predominance of young ring stages (synchronization interval, 16 h) at 0.5-1% parasitemia (hematocrit, 1%) were distributed in 96-well culture plates and 100 µL of the serum-free culture medium c o n t a i n i n g c o m p o u n d s a t v a r i o u s concentrations were added.Parasite culture was then incubated with each compound for

Assay for cytotoxicity
Cytotoxicity of the compounds was estimated on HeLa cells as conducted by Valentin et al. (1997).Cells were cultured in the same conditions as for P. falciparum, except for the 5% human serum, which was replaced by 5% fetal bovine serum (Boehringer).For the determination of compounds in vitro toxicity, 100 µL cell culture were distributed in 96-well plates at -4 2 x 10 cells per well and 100 µL of culture medium containing compounds at various concentration were added.Cell growth was 3 e s t i m a t e d b y [ H ] -h y p o x a n t h i n e incorporation after 24-h and 72-h incubation exactly as for the P. falciparum contact period. 3 The [ H]-hypoxanthine incorporation in the presence of compounds was compared with that of control cultures without compounds.IC values were also derived for each drug.

Results and Discussion
Among strategies in the antimalarial development is structure modification of existing agents by substitution of various functional groups to parent compound.In The results are summarized in Table 1.This screening showed that all the compounds were less active against P. falciparum chloroquine-sensitive strain (Nigerian) than chloroquine-resistant strain (FcM ).The IC -sensitive (Nigerian) strains after 24-h and 72-h incubation.
The vincadifformine is indole alkaloids with aspidospermane skeleton.The indole alkaloids have been isolated from some plants and their antiplasmodial activity and cytotoxicity have also been evaluated by some authors.Wright et al. (1992) evaluated in vitro antiplasmodial activity nine indole alkaloids isolated from Alstonia angustifolia roots.The more active alkaloids were macrocarpamine, macralstonine and villastonine with IC values ranged from 2.92 50 to 9.36 µM.The antipalsmodial activity of the macrocarpamine and the villastonine has been confirmed by Keawpradub et al. (1999) after he evaluated this indole alkaloids obtained from other Alstonia spp viz. A. scholaris, A. macrophylla and A. glaucescens.In addition, Wright et al. (1994) also evaluated other indole alkaloids from Strychnos species (Loganiaceae) and showed that among t w e n t y a l k a l o i d s e v a l u a t e d , 3 ' , 4 'dihydrousambarensine was the most active against P. falciparum (IC = 0.023 µM).The in 50 vitro antiplasmodial activity of indole alkaloids with aspidospermane skeleton was reported firstly by Mitaine et al. (1998) after investigate 12 alkaloids on P. falciparum.The results showed that the tetracyclic alkaloids possessing a free ethyl chain such aspidospermine were more active (IC ranged 3.2 -15.4 µM) than the 50 pentacyclic alkaloids with ethyl chain included in a tetrahydrofuran such haplocine (IC ranged 22.6 -52.6 µM).The 50 cytotoxicity of aspidospermane indole alkaloids including vincadifformine have also reported by Lewin (1995) and Lewin et al. (2002).The cytotoxicity of these compounds varied depending on their structures.
In order to obtain an estimate for the therapeutic index, the cytotoxicity of the derivatives showing similar and higher a n t i p l a s m o d i a l a c t i v i t i e s t h a n vincadifformine on HeLa cells were also investigated.The cytotoxicity to HeLa cells has been compared to antiplasmodial activity on FcM29 strain to obtain Cytotoxicity Index (CI = IC on HeLa/IC on 50 50 FcM29 strain).The CI obtained enable the selection of compounds which show some specificity of action in the in vitro situation, prior to more detailed in vivo investigations.addition, the CI of these compounds were also lower after 24-h incubation (CI, 0.9 -4.8) than that of after 72-h incubation (CI, 3.1 -11.5).Compounds 4 and 8 showing good antiplasmodial activities were less toxic (CI, 2.0-11.5 and 4.8-9.5 respectively) than the parent compound vincadifformine In contrast, compound 9 showing good antiplasmodial activities was more toxic than vincadifformine with CI ranged 1.4-3.7.
It could be seen that the activities of the compounds against HeLa cells were not parallel with the activities against P. falciparum suggesting that the mode of action of these compounds as antimalarial was not simple cytotoxic effect and lent support to further investigation of these compounds a n d t h e i r a n a l o g u e s a s p o t e n t i a l antimalarials drugs.This study also clearly demonstrated that the antiplasmodial activity and cytotoxicity of the vincadifformine derivatives was influenced by the substitution difference on atoms of the aspidospermane skeleton (Table 1 and 2

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in human red blood cells (O ), diluted to 1% hematocrit in RPMI 1640 medium (GIBCO BRL) supplemented with 25-mM Hepes and 30-mM NaHCO (GIBCO BRL) and 3 complemented with 5% human AB .Parasite cultures were synchronized by flotation on gelatin (Plasmagel) according to Jensen p r e v i o u s s t u d y , m o d i f i c a t i o n o f vincadifformine aspidospermane skeleton has been conducted by the substitution difference on C (R ), C (R , R ), C (R In this study, in vitro a n t i p l a s m o d i a l a c t i v i t i e s o f t h e vincadifformine derivatives and their cytotoxicity were evaluated.Two strains of P. falciparum (FcM and 29 Nigerian strains) were used to evaluate the in vitro antiplasmodial activities of the vincadifformine and its derivatives (1 -11).

Table 1 .
In vitro antiplasmodial activity (IC in µM) of