Molecular similarity search for new antimalarial compounds Sergio Sifontes-Rodríguez 1 , Yanetsy Machado Tugores 1 , Alfredo Meneses Marcel 1 , Rori Nelson García 2 , Yovani Marrero Ponce 3 , Vicente Arán Redó 2 , José Antonio Escario García–Trevijano 2 1 Departamento Biológico, Centro de Biaoctivos Químicos, Santa Clara CP 54830, Villa Clara, Cuba, sifontes@uclv.edu.cu 2 Departamento de Parasitología, Facultad de Farmacia, UCM, 28040 Madrid, España 3 Facultad de Química-Farmacia, Universidad Central “Martha Abreu” de Las Villas, Santa Clara CP 54830, Villa Clara, Cuba INTRODUCTION Malaria is one of the most important infectious diseases; moreover, it is a serious public health problem mainly in the world’s poorest countries. About 20% of all childhood deaths in Africa are due to malaria. Every 30 seconds, a child dies from malaria in Africa. Antimalarial drug resistance is widespread. Currently, artemisinin-based combination therapy is the only choice for malaria treatment in those places. For this reason, searching and developing for new drugs that are cheaper and easier to synthesize is a fundamental task. In this context, our laboratory works in collaboration with diverse synthesis groups, who provide us new compounds to screening. Thus, the team of synthesis of the Industrial Santander University in Colombia, has prepared two new different series of quinoline derivatives to screening for their antimalarial activity, in continuation of their search small nitrogen-containing molecules based on quinoline skeleton. Tetrahydroquinoline and quinoline derivatives are an important class of natural and synthetic compounds, which have shown a wide range of biological activities, specially to combat diverse parasitic diseases. Chloroquine hybrids have shown, so far, promising antiplasmodial activity against chloroquine resistant and sensitive strains. 1-3 Chloroquine hybrids (FER and SV) were obtained in two step synthetic route from commercial 4,7-dichloroquiniline and diamines (Scheme 1). 2,4-diaryl-3-methyl-l-1,2,3,4-tetrahydroquinolines (AR and DM) were prepared through a three-component condensation between trans-anethole (or isoeugenol), anilines and benzaldehyde 4,5 (Scheme 2). CHEMICAL SYNTHESIS Scheme 2 Scheme 1 Heme (FP IX) β-hematin Hemozoin 50 μl hemine chloride in DMSO, 100 μl acetate buffer pH 4.5 and 50 μl solution of drug or control distributed in 96 well plate. Incubation at 37ºC for 18-24h Discard supernatant Calculation of percentage of inhibition: % Inh = (OD control – OD drugs) x 100 OD control Read optical density at 405 nm Addition of DMSO 200 μl Centrifugation 5 min, 1600 g Centrifugation 5 min, 1600 g FERRIPROTOPORPHYRIN IX BIOMINERALIZATION INHIBITION TEST (FBIT) Discard supernatant Addition NaOH 0,1 N 150 μl Parasite culture maintained at 37 °C in RPMI 1640 medium supplemented with 0,5% AlbuMAX II 50 μl of infected human erythrocytes suspended at 2% hematocrit and 1% initial parasitemia and 50 μl of compound solutions (from 10 to 0,02 μg/ml) distributed in 96-well plate in triplicate IC 50 values estimated by plotting concentration versus inhibition percentage Calculating of inhibition percentages as follow: % Inh = (FI Control –FI Compound) x100 FI Control Freezing at -80°C for 1 h and thawing 100 μl per well of SYBR® Green I (1X) in lysis buffer added and incubated for 1h at room temperature in the dark Reading of fluorescence intensity FI with excitation and emission wavelength at 485 and 530 nm Plate incubated for 48 h at 37 °C in an atmosphere of 5% CO 2 FLUORESCENCE-BASED MICROTEST AGAINST Plasmodium falciparum 3D7 STRAIN Macrophages culture maintained at 37°C in RPMI 1640 medium supplemented with 10% SBF in 5% CO 2 atmosphere 70.000 macrophages/well distributed in 96-well plate Discard medium, add 100 μl/well of MTT solution 0,4 mg/ml and incubate 1 h at 37°C in 5% CO 2 atmosphere Discard MTT solution, add 100μl of DMSO to dissolve the formazan crystals and read optical density at 595 nm Calculating of citotoxicity percentages as follow: % Cx = (OD control – OD drugs) x100 OD control NONSPECIFIC CYTOTOXICITY AGAINST MURINE J774 MACROPHAGES Discard medium, addition of 200 μl of drug solutions (600, 60 and 6 μM) per triplicate and incubation 24 h at 37 ºC in 5% CO 2 Incubation at 37 ºC 24 h in 5% CO 2 atmosphere RESULTS AND DISCUSSION Five compounds, all of them chloroquine hybrids, showed very good in vitro activity against P. falciparum, with IC 50 values lower than 1μM, from 0.69 to 0.02 μM (See Table 1). The compound FER102 was twice more active than chloroquine, which showed an IC 50 value of 0.04 μM. The IC 50 values of the majority of tetrahydroquinolines were higher than 12 μM (data not showed). Regarding to nonspecific cytotoxicity test, these 5 compounds were cytotoxic at 600 μM, however, none of them were cytotoxic at concentrations lower than 60 μM, between 80 and 160 times higher than active concentrations against Plasmodium. Since the active compounds have the same functional group that chloroquine, we consider important measure the ability of these compounds to inhibit the crystallization of FPIX. This is a Plasmodium specific process in which the toxic FPIX derived from the digestion of ingested haemoglobin is converted to insoluble nontoxic crystal called haemozoin. Predictably, all compounds showed good activity. FER93, FER95 and FER102 were as active as chloroquine, while FER87 and FER90 were twice and five times lower respectively. Hence, these results suggest that active compounds could have the same mechanism of action, although, it does not exist a direct relation between the activity shown on FBIT and in vitro against the parasite. Additionally, we carried out an experiment to measure synergy or antagonism of three of the active compounds, FER93, FER95 and FER102 with chloroquine on FBIT. An decrease of 50% of IC 50 values of chloroquine was observed with low concentrations of all compounds (IC 50 /2). In vitro experiments will be conducted shortly to verify the activity of these compounds against chloroquine resistant strain of P. falciparum, as well as, in vivo test against Plasmodium berghei. MATERIALS AND METHODS We evaluated 33 new quinoline derivatives: 10 chloroquine hybrids and 23 2,4-diaryl-3-methyl-l-1,2,3,4-tetrahydroquinolines (see scheme 1 and 2) for in vitro antimalarial activity against Plasmodium falciparum 3D7 strain by fluorimetric microtest using SYBR ® Green I. Nonspecific cytotoxicity of the most active compounds was measured in murine J774 macrophages in vitro test. Moreover, we tested the ability of these compounds to inhibit the ferriporotoporphyrin IX (FPIX) biocrystallization process on FBIT assay. COMPOUNDS In vitro P. falciparum 3D7 % Cytotoxicity μM FBIT Structure IC 50 μM 600 60 6 IC 50 μM FER 87 0.24 60.0 2.1 0.0 244.2 FER 90 0.69 64.7 3.1 0.0 101.8 FER 93 0.09 92.1 1.8 0.0 27.9 FER 95 0.35 99.0 0.0 0.0 43.0 FER 102 0.02 97.8 0.0 0.0 48.5 Cloroquina 0.04 92.0 0.0 0.0 44.2 REFERENCES 1. Chiyanzu, I.; Clarkson C.; Smith, P.J.; Lehman J.; Gut, J.; Rosental, P.J.; Chibale, K., Bioorg. Med. Chem. 2005, 13, 3249-3261. 2. Burgess, S.J.; Selzer, A.; Kelly, J.X.; Smilkstein, M.J.; Riscoe, M.K.; Peyton, D.H., J. Med. Chem. 2006, 49, 5623-5625. 3. Musonda, C.C.; Gut, J.; Rosenthal, P.J.; Yardley, V.; Carvalho de Souza R.C.; Chibale, K., Bioorg. Med. Chem. 2006, 14, 5605-5615. 4. Kouznetsov V.V., Bohórquez Romero A.R., Stashenko E.E., Tetrahedron Lett., 2007, 48, 8855-8860. 5. Kouznetsov V.V., Merchan Arenas D.R., Bohórquez Romero A.R., Tetrahedron Lett., 2008, 49, 3097-3100. N Cl HN N S O N Cl HN N S O OCH 3 OH N Cl HN N S O OH OCH 3 N Cl HN N Cl HN N S O N Cl HN N Table 1. In vitro antimalarial activity against P. falciparum 3D7 strain, percentages of cytotoxicity on murine J774 macrophages, activity on FBIT and structures of chloroquine hybrids.