Synthesis, in silico studies , Antiprotozoal and Cytotoxic Activities of Quinoline-biphenyl Hybrids

The synthesis, in silico studies, antiprotozoal and cytotoxic activities of eleven quinoline-biphenyl hybrids are described herein. The structure of the synthesized products was elucidated by a combination of spectrometric analyses. The synthesized compounds were evaluated against Plasmodium falciparum, and amastigotes forms both Leishmania (V) panamensis and Trypanosoma cruzi . Cytotoxicity was evaluated against human U-937 macrophages. Hybrid 4a showed similar activity than meglumine antimoniate and compound 4b exhibited an activity similar to that of benznidazole. Hybrid 4k showed the best activity against P. falciparum . Although these compounds were toxic for mammalian U-937 cells, however they may still have potential to be considered as candidates for drug development because of their antiparasite activity. In addition, molecular docking was used to determine the in silico inhibition of some of the designed compounds against PfLDH and cruzipain, two important pharmacological targets involved in antiparasitic diseases. All hybrids were docked to the three-dimensional structures of PfLDH and T. cruzi cruzipain as enzymes using AutoDock Vina. Notably, the docking results showed that the most active compounds 4b (CE 50 : 11.33 μg/mL for P. falciparum ) and 4k (CE 50 : 8.84 μg/mL for T. cruzi ) showed the highest scoring pose (- 7.5 and -7.7 kcal/mol, respectively). This result show a good correlation between the predicted scores with the experimental data profile, suggesting that these ligands could act as competitive inhibitors of PfLDH or T. cruzi cruzipain enzymes, respectively. Finally, in silico ADMET studies of the quinoline hybrids showed that these novel compounds have suitable drug-like properties, making them potentially promising agents for antiprotozoal therapy.


Introduction
Protozoal diseases (PD) are a diverse group of diseases which are the cause of a significant mortality rate in various developing countries of tropical and subtropical regions. PDs include, among others, Chagas' disease (American trypanosomiasis), leishmaniasis and malaria which are caused by the parasitic protozoan of Trypanosoma cruzi (T. cruzi), Leishmania species and Plasmodium species, respectively (WHO 2018a). Human malaria is caused by at least five species of Plasmodium, the most important being P. falciparum and P. vivax (WHO 2018b). Leishmania (V) panamensis is one of the most prevalent Leishmania species involved in human cases of cutaneous leishmaniasis in Colombia and other countries in Central America (Alvar et al., 2012).
Current chemotherapies are still based on old drugs such as pentavalent antimonials (meglumine antimoniate and sodium stibogluconate), pentamidine isethionate and amphotericin B to treat cutaneous leishmaniasis (WHO 2019a); nitroaromatic compounds (benznidazole and nifurtimox) for treatment of Chagas disease (WHO 2019b) or chloroquine, amodiaquine, sulfadoxine/pyrimethamine to treat P. falciparum or P. vivax malaria, respectively. More recently new artemisinin-based combination therapy is recommended for the treatment of P. falciparum (WHO 2018b). Unfortunately, all of these drugs have several toxic effects on the patients that are associated with high doses and length of therapeutic schemes. Moreover, they are no longer as effective as before due to the emergence of drug resistance in the parasite, which complicates the control of these diseases (Chatelain and Ioset, 2011;Den Boer et al., 2011;Keenan and Chaplin, 2015;Fidock et al., 2004).
Another interesting compound is biphenyl derivative 1f, whose structure is based on that of methylglyoxal bis(guanylhydrazone). Compound 1f was active in vitro against several Trypanosoma species, including T. brucei rhodesiense and T. b. brucei (Brun et al. 1996). Finally, the furanchalconebiphenyl hybrid 1g, exhibited good activity against T. cruzi showing better activity than benznidazole (García et al., 2019) (fig. 1).

Fig. 1 Biologically actives biphenyl and quinoline hybrids
Hybrid molecules are chemical entities with two (or more than two) structural domains having different biological functions and that can, therefore, to show a dual mode of action acting as two distinct pharmacophores (Cardona-G et al. 2018;Meunier 2008) without necessarily acting on the same biological target (Dunn et al. 2016). A promising strategy based on these class of compounds has recently emerged in medicinal chemistry for the discovery and development of new drugs. In the search for new therapeutic alternatives to treat cutaneous leishmaniasis, Chagas disease and malaria, we designed and synthesized a series of quinoline-biphenyl hybrids, whose general structures are indicated in fig. 2, and their in vitro cytotoxicity, antileishmanial, antitrypanosomal and antiplasmodial activities was in turn evaluated by us.

General remarks
Microwave reactions were carried out in a CEM Discover microwave reactor in sealed vessels (monowave, maximum power 300 W, temperature control by IR sensor, fixed temperature). 1 H and 13 C NMR spectra were recorded on a Varian instruments operating at 600 (300) MHz. The signals of the deuterated solvent (CDCl3) were used as reference (CDCl3:  = 7.27 ppm for 1 H NMR and  = 77.00 ppm for 13 C NMR. Carbon atom types (C, CH, CH2, CH3) were determined by using the DEPT (Distortion less Enhancement by Polarization Transfer) or APT (Attached Proton Test) pulse sequence.
High resolution mass spectra were recorded using electrospray ionization mass spectrometry (ESI-MS).
A QTOF Premier instrument with an orthogonal Z-spray-electrospray interface (Waters, Manchester, UK) was used operating in the W-mode. The drying and cone gas was nitrogen set to flow rates of 300 and 30 L/h, respectively. Methanol sample solutions (ca. 1 x 10 -5 M) were directly introduced into the ESI spectrometer at a flow rate of 10 L/min. A capillary voltage of 3.5 kV was used in the positive scan mode, and the cone voltage set to Uc = 10 V. For accurate mass measurements, a 2 mg/L standard solution of leucine enkephalin was introduced via the lock spray needle at a cone voltage set to 85 V and a flow rate of 30 L/min. IR spectra were recorded on a Spectrum RX I FT-IR system (Perkin-Elmer, Waltham, MA, USA) in KBr disks. Silica gel 60 (0.063-0.200 mesh, Merck, Whitehouse Station, NJ, USA) was used for column chromatography, and precoated silica gel plates (Merck 60 F254 0.2 mm) were used for thin layer chromatography (TLC).

In vitro Cytotoxicity
The cytotoxic activity of the compounds was assessed in the human promonocytic cell line U-937 (ATCC CRL-1593.2 TM ) based on the viability evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay as described elsewhere (Taylor et al. 2011). Briefly, U-937 cells grown in tissue flasks were harvested and washed with phosphate buffered saline (PBS) by centrifugation. Cells were counted and adjusted at 1×10 6 cells/mL of complete culture medium (RPMI-1640 supplemented with 10% Fetal Bovine Serum-FBS and 1% of antibiotics -100 U/mL penicillin and 0.1 mg/mL streptomycin. One hundred µL of cell suspension were dispensed into each well of a 96well cell-culture plate and then 100 L of two-fold serial dilutions of the compounds (starting at 200 µg/mL) in complete RPMI 1640 medium were added. Plates were incubated at 37 °C, 5% CO2 during 72 h in the presence of compounds. Then, 10 µL/well of MTT solution (0.5 mg/mL) was added into each well and plate was incubated at 37 °C for 3h. The formazan crystals were dissolved by adding 100 µL/well of dimethyl sulfoxide and 30 min incubation. Cell viability was determined according to the intensity of color (absorbance) registered as optical densities (O.D) obtained at 570 nm in a spectrophotometer (Varioskan™ Flash Multimode Reader -Thermo Scientific, USA). Cells cultured in absence of compounds were used as control of viability (negative control), while doxorrubicin was used as control for cytotoxic drugs. Non-specific absorbance was corrected by subtracting the O.D of the blank. Assays were conducted in two independent runs with three replicates per each concentration tested.

In vitro antileishmanial activity
The activity of compounds was evaluated on intracellular amastigotes of L. (V) panamensis transfected with the green fluorescent protein gene (MHOM/CO/87/UA140-EGFP) (Pulido et al., 2012). The effect of each compound was determined according to the inhibition of the infection evidenced by both decrease of the infected cells and decrease of intracellular parasite amount. Briefly, U-937 human cells at a concentration of 3 × 10 5 cells/mL in RPMI 1640 containing 0.1 μg/mL of phorbol-12-myristate-13-acetate (PMA) were dispensed into each well of a 24-well cell culture plate and then infected with 5 days-old promastigotes in a 15:1 parasites per cell ratio. Plates were incubated at 34 °C, 5% CO2 during 3h and cells were washed two times with PBS to eliminate not internalized parasites. One mL of fresh complete RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics was added into each well, cells were incubated again to guarantee multiplication of intracellular parasites. After 24 h of infection, culture medium was replaced by fresh culture medium containing each compound at 50-6.12 and 1.56 μg/mL and plates were incubated at 37 °C, 5% CO2.
After 72 h, inhibition of the infection was determined. For this, cells were removed from the bottom plate with a trypsin/EDTA (250 mg) solution; recovered cells were centrifuged at 1100 rpm during 10 min at 4 °C, the supernatant was discarded and cells were washed with 1 mL of cold PBS and centrifuged at 1100 rpm during 10 min at 4 °C. The supernatant was discarded and cells were suspended in 500 μL of PBS and analyzed by flow cytometry (FC 500MPL, Cytomics, Brea, CA, US).
All determinations for each compound including and standard drugs were carried out in triplicate, in two independent experiments (Buckner et al., 1996;Pulido et al., 2012). Activity of tested compounds was carried out in parallel with infection progress in culture medium alone and in culture medium with amphotericin B and meglumine antimoniate as antileishmanial drugs (positive controls).

In vitro antitrypanosomal Activity
T. cruzi, Tulahuen strain transfected with β-galactosidase gene (donated by Dr. F. S. Buckner, University of Washington) were maintained in vitro as epimastigotes by culturing in modified Novy-MCNeal-Nicolle (NNN) medium. The U-937 cells were adjusted at 2.5×10 6 cells/mL of complete RPMI-1640 medium containing 0.1 μg/mL of phorbol myristate acetate to induce differentiation to macrophages. Then, 100 μL of this cell suspension were dispensed into each well of a 96-well cellculture plate. After 24 h of incubation at 37 °C, 5% CO2, macrophages were infected with early stationary growth phase (10 days in culture) epimastigotes, at the concentration of 12.5x10 5 parasites/mL of complete RPMI 1640 medium equivalent to 5:1 (parasites per cell) ratio. Plates were incubated at 34 °C, 5% CO2 during 24 hours to allow the conversion to intracellular amastigotes. Extracellular parasites were removed by washing twice with 100 μL of PBS. Then, 100 μL of each concentration (100 -25 -6.12 and 1.56 μg/mL) of compounds were added to infected cells, plates were incubated at 34 °C, 5% CO2. After 72 h of incubation plate wells were washed twice with PBS and the β-galactosidase activity was measured by spectrophotometry adding 100 μM of the chromogenic substrate CPRG (chlorophenol red-beta-D-galactopyranoside) and 0.1% nonidet P-40 to each well. After 3 h of incubation at 25 °C, absorbance was read at 570 nm in a spectrophotometer (Varioskan™ Flash Multimode Reader -Thermo Scientific, USA). Infected cells exposed to benznidazole were used as control for antitrypanosomal activity (positive control) while infected cells incubated in complete RPMI 1640 culture medium were used as control for infection (negative control). Non-specific absorbance was corrected by subtracting the O.D of the blank. Determinations were done by triplicate in at least two independent experiments (Insuasty et al. 2015).

In vitro Antiplasmodial Activity
The antiplasmodial activity was evaluated in asynchronic cultures of P. falciparum (NF54 strain), maintained in standard culture conditions. The effect of each compound over the growth of the parasites was determined by Plasmodium lactate dehydrogenase assay (pLDH) (Nkhoma et al. 2007;Londoño et al., 2016). Parasites were plated in the trophozoite phase at 1% hematocrit and 0.5 %parasitemia in 100 L of each compound at an defined concentration (1000 -25 -6.25 -1.56 ug/mL). Plates were incubated in an atmosphere with a gas mixture of 4% O2, 3% CO2, and 97% N2, and incubated at 37°C for 72 hours. Meanwhile, two reagents for detecting and measuring the LDH enzyme were prepared. The first of these was the Malstat reagent (400 L of Triton X-100 in 80 mL of deionized water, 4.0 g L-lactate , 1.32 g Tris buffer and 0.022 g of 3-acetylpyridine adenine dinucleotide (APAD), adjusting the pH to 9 with hydrochloric acid, and a volume of 200 mL with deionized water. The second reagent is NBT/PES solution (1-6 g nitro blue tetrazolium salt and 0.008 g phenazine ethosulfate in 100 mL of deionized water. The solution was stored in a foil-covered container and kept at 4°C until required. When incubation was complete, plates were harvested and subjected to three 20-minute freeze-thaw cycles to resuspend the culture. Thereafter, 100 L of Malstat reagent and 25 L of NBT/PES solution were added to each well of a new, duplicate flat-bottomed 96-well microtiter plate. The culture in each of the wells of the original plate was resuspended by mixing with a multichannel pipette. Thereafter, 15 L of the culture was taken from each well and added to the corresponding well of the Malstat plate, thereby initiating the LDH reaction. Color development of the LDH plate was monitored colorimetrically at 650 nm in the Varioskan Flahs readet after an hour of incubation in the dark.

Data Analysis
Cytotoxicity was determined according to cell growth (viability) and mortality percentages obtained for each isolated experiment (compounds, doxorubicin and culture medium). Results were expressed as 50 lethal concentrations (LC50), corresponding to the concentration necessary to eliminate 50% of cells, calculated by Probit analysis (Finney, 1978).
The antiplasmodial activity of each compound was evidenced by the reduction of the O.D. The inhibition of parasitemia percentage was calculated by equation 3.

Protein Structure and Setup
To explore the potential mechanism of action of the hybrids 4a-k against two principal targets for antiparasitic drugs, the crystals structures of P. falciparum Lactate Dehydrogenase (pfLDH) enzyme in complex with their cofactor NADH and the structure of cruzipain, the mayor papaine-like cysteine protease in T.cruzi were obtained from the Protein Data Bank (PDB entry code 1LDG and 3I06, respectively) (Dunn et al., 1996). Discovery Studio (DS) Visualizer 2.5 was used to edit the protein structure to remove water molecules together with bound ligands. For docking studies on pfLDH in the absence of cofactor, the NADH cofactor was also removed. Both, the structures of the selected proteins were parameterized using AutoDock Tools (Morris et al., 2009). In general, hydrogens were added to polar side chains to facilitate the formation of hydrogen bonds, and the Gasteiger partial charges were calculated.

Ligand dataset preparation and optimization
Ligands used in this study are the new quinoline-hybrids 4a-k, quinoline-based drugs that have been used in the treatment of malaria (amodiaquine, mefloquine, quinine and chloroquine) and the cofactor NADH for comparison in the malaria-case. DS visualizer was used to rewrite the data files into pdb format. The structures of the ligands were parameterized using AutodockTools to add full hydrogens to the ligands, to assign rotatable bonds, to compute Gasteiger partial atomic charges and save the resulting structure in the required format for use with AutoDock. All possible flexible torsions of the ligand molecules were defined using AUTOTUTORS in AutoDockTools (Morris et al., 2009;Morris et al., 1998) to facilitate the simulated binding with the receptor structure.

Docking and subsequent analysis
Docking simulations were performed with AutoDock 5.6 using the Lamarkian genetic algorithm and default procedures for docking a flexible ligand to a rigid protein were followed. First, the Metapocket 2.0 server (Huang 2009) was used to identify the best candidates to protein binding pockets by predictive calculation of the topology of tertiary structures of selected subunits. According to standard program parameters, five binding pockets were calculated in the protein model and reliability of the model was reviewed through the Z-value statistical test. Second, once potential binding sites were identified, docking of ligands to these sites was carried out to determine the most probable and most Value of Caco-2 permeability is classified into three classes: (1) If permeability < 4, low permeability; (2) if permeability < 70, moderate permeability; and (3) if permeability > 70, higher permeability.

Chemistry
The synthetic strategy for the preparation of quinoline-biphenyl hybrids is shown in Scheme 1.
Thus, reaction of 8-hydroxyquinoline (1)  were used, however the results did not improve those obtained with the method described above.

Scheme 1 Synthetic pathway to quinoline-biphenyl hybrids
The structure of each compound was elucidated by a combined study of IR, The antileishmanial, antitrypanosomal and antiplasmodial activities were measured by determining the EC50 that corresponds to the concentration of drug that gives the half-maximal reduction of the amount of parasites (Table 1). Dose-response relationship showed that compounds 4a,4b, 4e, 4f and 4k were active against intracellular amastigotes of L. (V) panamensis with EC50 < 20 μg/mL. The most active hybrid was 4a an EC50 of 8.95 ± 0.87 μg/mL which is comparable to referential drug meglumine antimoniate (EC50 = 9.4 ± 2.1 μg/mL). Compounds 4c, 4d, 4h and 4i showed a moderated activity.
Finally, hybrids 4g and 4j were not active. showed activity comparable to that of meglumine antimoniate, benznidazole and chloroquine, respectively, the SI of these compounds is affected by their high cytotoxicity. These results suggest that the biological activity of the quinoline derivatives reported here, except for 4g and 4j, is selective and more active against L. (V) panamensis than U-937 cells. Compounds 4b, 4d, 4e and 4g-4i were more actives against T. cruzi parasites than U-937 cells. On the other hand, hybrids 4a, 4c-4i and 4k, showed selectivity against P. falciparum. In this sense, compound 4h exhibited the best SI on both L. (V) panamensis and P. falciparum, with 4.78 and 6.08 values, respectively (Table 2). SAR analysis showed that 4a, bearing an unsubstituted phenyl group, was the most active in L. (V) panamensis. Compound 4b, with a hydroxyl group in the 4-position is most active than compound 4c with a methoxy instead of the hydroxy group. The activity decreases when the hydroxy group was changed from 4-position to 2-position (4b vs 4d). Electron withdrawing groups in 4-position increase the activity over the electron donating methoxy group (4e and 4f vs 4c).
As regards T. cruzi, we found that 4b was the most active compound. Changing the position of the hydroxyl group (4d) or the presence of electron withdrawing groups (4e and 4f) decrease the activity.
The methylation of the hydroxyl group (4c) leads to the loss of activity. These results agree with other reports for several chalcones, coumarins, cinnamic ester and triclosan-caffeic acid hybrids (Brenzan et al. 2008;Aponte et al. 2010;Otero et al. 2014;Otero et al. 2017;García et al. 2018). The effect of the hydroxyl groups may be due to a better molecular recognition ability towards target bioreceptors upon hydrogen bond formation (Patrick 2013). In both protozoal disease, all disubstituted hybrids, with exception of 4k, exhibited low activity. Overall, monosubstituted compounds (4a-4f) showed better activity than disubstituted hybrids (4g-4j) On the other hand, we found that the presence of methoxyl groups in 3-and 4-position (4k) is very important for the antiplasmodial activity. This activity decreased when the methoxyl groups were changed to 2,4-(4h), 2,5-(4i), 2,6-(4i) and 2,3-(4g) positions. The activity is also related to the presence in 4-position of electron withdrawing groups, such as nitro and fluorine groups, and the presence of the hydroxyl electron donating group in 2-position, as it appears in compound 4d. Electron donating groups, hydroxyl and methoxyl groups in the 4-position, such as in 4c and 4d, lead to a decrease in activity.

Docking studies against PfLDH structure and binding pose prediction
The P. falciparum lactate dehydrogenase enzyme (PfLDH) has been is considered as a potential molecular target for antimalarials, since it is a key enzyme that catalyses the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD + and provides the energy for the survival of the parasite. One of the products of hemoglobin degradation by malarial parasites is the ferriprotoporphyrin IX ( When the cofactor was absent a comparable binding within the NADH-binding site and the allosteric site was achieved for all ligands, showing that the NADH-binding pocket (Site 1) was the preferred binding site for all the ligands studied. The most active compound 4k (CE50: 11.33 μg/mL, Malaria parasite) fitted well in the NADH pocket (Docking score of -7.7 kcal/mol) and showed the best docking energy values among all hybrids, that is, close to NADH (which has a docking energy of -9.4 kcal/mol). For comparison, docking score affinities against PfLDH structure for four quinoline-based market drugs (inhibitors used for malaria treatment) were also calculated in this study. It has been shown that the chloroquine interacts specifically with PfLDH in the NADH binding site, occupying a position analogous to that of the adenyl ring cofactor and therefore acts as a competitive inhibitor, suggesting that the mechanism of parasite growth inhibition by the different compounds results from drug competition with NADH for the PfLDH (Read et al. 1999;Vennerstrom et al. 1999;Menting et al. 1997). For all hybrids studied, there was comparable docked solutions within the cofactor-binding site similar to that of drugs currently used. In the absence of the cofactor, the most stable bound conformation of the hybrid 4k (the docking score was -7.7 kcal/mol) showed higher affinity than that observed for the marketed drugs chloroquine, amodiaquine and quinine (docking energy of -7.4, -7.4 and -7.1 kcal/mol, respectively). The binding energies for representative ligand structures, as calculated by Autodock, are given in Table 3. The superposition of NADH (in blue) and the best conformation obtained theoretically for all hybrids (in red), shown in Figure 1, reveals that ligands endowed with conformational mobility (calculated in this docking study) can rearrange themselves into favorable conformations in order to fill the cofactor-binding site.
All ligands studied showed good binding affinity compared to NADH in the NADH-binding pocket pointing to a possible competitive inhibition. In general, the compounds docked similarly across the delimited binding site, with a set of hydrophobic interactions that potentially confers stability during the binding event. The molecules also form diverse types of interactions, especially π-anion between aryl ring of hybrids and carboxylic group of Asp53 residue in the protein, π-sigma interaction between aromatic quinoline ring and the catalytic residues Ala98 and Ile54, located adjacent to the nicotinamide end of the cofactor-binding domain. The best docked conformation of highly active molecule (compound 4k) and the active site residues that interact inside PfLDH are shown in Figure 4A and Figure 4B. According to these docking results, PfLDH could be a potential therapeutic target for the evaluated hybrids, despite the fact that the docking scores when compared to natural ligand NADH showed highest binding values than the typical antimalarial-drugs. In addition, docking studies showed that the affinity of the designed hybrids is correlated with the results obtained from in-vitro studies.

Docking of hybrids on Cruzipain active site and binding pose prediction
Cysteine proteases are essential for T. cruzi survival. Among them, cruzipain is a relevant protein target to design novel inhibitors for Chagas disease treatment (Otto and Shirmeister 1997;Sajid and McKerrow 2002). This enzyme hydrolyzes chromogenic peptides at arginine or lysine carboxyl terminals and plays a key role in the development and differentiation of the parasite during various life cycle stages (Beaulieu et al. 2010). In order to investigate the specific interactions from each ligand with Cruzipain (PDB: 3I06), we first identify the best pockets calculated from Metapocket server. The data-set suggested that the highest docking score resulted when the pocket used is characterized by the presence of amino acids residues: Lys17, Phe28, Glu50, Asp18, Asn47, Leu48, Ser49, Glu86, Asp87, Pro90, Tyr91, Ala15, Gln19, Val16, Thr185, Asp18, Ile31, Met52, Ser88, Thr85, Gln51 and Tyt89 ( Figure 1B). These amino acids residues were near to the enzyme surface. This site was defined as binding pocket for the docking runs.
Molecular docking studies were performed on all hybrids 4a-k, to investigate the importance of the motif that contribute to T. cruzi cruzipain binding when docked into the selected pocket. All quinolineligands showed similar binding affinities for the selected pocket (Table 3). Notably, the most active compound 4b showed the highest scoring pose (binding energy of -7.5 kcal/mol). The results from the docking analyses suggest a slowly reversible mechanism of inhibition that is aided by strong noncovalent interactions. In general, the compounds docked showed the formation of significant interactions with residues within the binding pocket. Then, a closer look at the best possible binding pose of hybrid 4b (highly active molecule, CE50: 8.84 μg/mL) reveals that strong interactions into the active site, which are shown in Figure 4C, involved one hydrogen bond interaction between hydroxyl group of 4b and the Tyr89 and Glu86 residues; π-cation between Lys17 residue of the protein and the quinoline-ring motif of the compound. Hydrophobic interactions surrounded by side chains of predominantly nonpolar residues confers stability during the binding event in the pocket ( Figure 4D). Table 3 summarizes the best binding energy per evaluated compound on T. cruzi cruzipain. Docking results suggest that hybrid 4b represents a novel hit cruzipain inhibitor that can be exploited for further analog design as potential antichagasic agents. Moreover, these findings are also supported by previous reports of active quinolines against this parasite protein target (Kaur et al 2010;Foley and Tilley 1998). -7.6 -6.7 -7.2 4f -7.6 -6.5 -7.1 4g -7.1 -6.4 -6.8 4h -7.5 -6.3 -6.9 4i -7.3 -6.2 -6.9 4j -6.9 -6.5 -6.9 4k -7.7 -6.5 -6.9 NADH -9.7 N/A ---Chloroquine -7.4 -6.3 ---Amodiaquine -7.4 -6.8 ---Quinine -7.1 -6.5 ---Mefloquine -8.3 -7.5 --a NADH binding site b Allosteric binding site

Drug-likeness prediction studies
We calculated and analyzed various drug-likeness properties for the 11 quinoline-biphenyl hybrids.
The prediction results are summarized in Table 4. All compounds showed typical values for the parameters analyzed, exhibiting suitable drug like characteristics. The predicted values are within the range of properties of 95% of currently known drugs. According to Lipinski's rule of five (Lipinski et al. 1997, an orally active drug that has no more than one violation is acceptable) the tested hybrids 4a-k could be orally active drugs in human. It was observed that all the title compounds exhibited high human intestinal absorption (% HIA) and high percent of human oral absorption (% F) ranging from 80 to 100%. Greater HIA and F values denote that the hybrids 4a-k could be better absorbed from the intestinal tract upon oral administration. Among the predicted physico-chemical properties, the molecular PSA is a descriptor that was shown to correlate well with passive molecular transport through membranes and allows the prediction of drug-membrane interactions. Calculated PSA (Ertl et al. 2000) values for compounds 4a-k showed high PSA values, suggesting that perhaps these polar compounds tend to have a greater affinity and good ability to penetrate through infected cells. In addition, lipophilicity influences a number of physiological properties, including transport through lipid bilayers, and therefore it is an important property that a drug should exhibit. LogP gives a measure of the lipophilicity of a compound and is a good indicator of permeability across the cell wall (Veber et al. 2002). In this study, all the tested compounds exhibited LogP values below 5, ranging from 3.346 to 4.555, suggesting good permeability and permeation across the cell membrane of infected cells.
Moreover, in silico artificial membrane permeation rate across Caco-2 cell monolayers or MDCK cells was calculated for all quinoline-hybrids derivatives. It was found that the passive transmembrane permeation of the novel compounds displayed good permeability values (from 1356 to 9180 nm/s), except for nitro-substituted 4f which displayed poor cell permeability values (less than 867 nm/s).
Finally, the early prediction of plasma protein binding (calculated as log KHSA) has vital importance in the characterization of drug distribution in the systemic circulation. Unfavorable log KHSA values can represent a negative effect on clinical development of promising drug candidates for human parasitic diseases chemotherapy. For all compounds, were obtained high binding affinity values (more than 0.25) compared to reference values taken from 95% of currently known drugs (Log KHSA from -1.5 to 1.2).
From the therapeutic point of view, the interpretation of predicted ADMET properties showed recommended values ranges for an ideal drug, demonstrating the potential of the hybrids 4a-k as therapeutic candidates to discover novel drugs for specific treatment for T. cruzi or P. falciparum infections. These in-silico ADMET predictions suggest that quinoline-biphenyl hybrids here reported follow the criteria for orally active drugs and thus represent a potential pharmacologically active framework that should be considered in progressing further potential hits.

Conclusions
The synthesis, in silico studies, antileishmanial, antitrypanosomal and antiplasmodial screening of eleven quinoline-biphenyl hybrids are reported. Five of them were active against L. (V) panamensis, two of them against T. cruzi and four of them against P. falciparum with EC50 values lower than 20 μg/mL. Hybrid 4a showed similar activity than meglumine antimoniate and compound 4b exhibited an activity similar to that of benznidazole. Hybrid 4k showed the best activity against P. falciparum.
Studies on an animal model are needed to confirm the results observed in vitro. These compounds were toxic for mammalian U-937 cells, however they may still have potential to be considered as candidates for antileishmanial, antitrypanosomal and antiplasmodial drug development. More studies on toxicity using other cell lines are needed to discriminate whether the toxicity shown by these compounds is specific against tumor or non-tumor cells. SAR study revealed the importance of hydroxyl group in 4-position of the phenyl group for antitrypanosomal activity. On the other hand, for antileishmanial activity the presence of substituents in the phenyl group decrease de activity. As regards antiplasmodial activity, our studies have shown that the presence of methoxyl groups in 3-and 4-position and electron withdrawing groups in the 4-position are important in order to achieve biological action.
. Molecular docking was used to investigate the in silico inhibition effects of the eleven quinolinebiphenyl hybrids on two important antiparasitic drug targets (PfLDH and T. cruzi cruzipain enzymes).
Docking studies against PfLDH structure suggest that the hybrids could act as competitive inhibitors as they had higher binding energy than reference drugs, and close to the cofactor, NADH. In addition, the present findings further support that these molecules may be potentially inhibitors of T. cruzi cruzipain enzyme and could be a potential molecular target for the evaluated compounds.
Physicochemical and ADMET profile of these molecules, such as polar Surface area (PSA), LogP and the number of rotatable bonds (Nrot), membrane permeation rate, Plasma Protein Binding (KHSA) and human oral absorption (%F) showed that these hybrids have potential for an eventual development as oral agents and can be significant active drug candidates in search of better and safe antiprotozoal agents.