Synthesis, Biological Evaluation and Structure-Activity Relationships of New Quinoxaline Derivatives as Anti-Plasmodium falciparum Agents

We report the synthesis and antimalarial activities of eighteen quinoxaline and quinoxaline 1,4-di-N-oxide derivatives, eight of which are completely novel. Compounds 1a and 2a were the most active against Plasmodium falciparum strains. Structure-activity relationships demonstrated the importance of an enone moiety linked to the quinoxaline ring.


Introduction
Malaria is an infectious disease with an estimated 219 million cases and 660,000 deaths in 2010. Of these cases 86% correspond to children under 5 years old. In 2012, there were a total of 104 countries where malaria is considered to be endemic [1].
Plasmodium falciparum is the most dangerous form of the malaria parasite and it is responsible for a very high percentage of clinical attacks [2]. Artemisinin-based combination therapies (ACTs) are the standard treatment against uncomplicated P. falciparum malaria. Nevertheless, resistance to ACTs containing dihydroartemisinin has been reported in Pailin (Cambodia) [3] and resistance to pyrethroids (used as insecticides) has been detected in 64 countries around the World [1]. Other antimalarial drugs, such as chloroquine or mefloquine, are not effective enough [4]. FCR-3 P. falciparum is a chloroquine-resistant strain, but sensitive to pyrimethamine and sulfadoxine [5].
Some of the compounds belonging to this study were previously tested against L. amazonensis [40] (series 1), T. cruzi and L. peruviana [41] (series 3) and as cytotoxic agents [42] (compound 1a) and anti-inflammatory/antioxidant [28,43] agents (series 1, 3 and 7). According to the structure-activity relationships, these reports affirm that compounds in series 3 have better antioxidant activity than their analogs in series 1 due to the fact that the former group of molecules lacks the N-oxide groups [28]. On the other hand, compounds 1d and 3d have an interesting activity against different Leishmania strains, so the activity is associated with R 6 /R 7 = Me/Me substitution [40,41]. Compound 1a stands out as both a good anti-inflammatory and cytotoxic agent [42,43].
With the aim of expanding the SAR study of these chalcone analogs and obtaining new compounds with improved antimalarial activity, we describe herein the synthesis and the relationships between structure and antiplasmodial activity against the FCR-3 P. falciparum strain of eighteen quinoxaline and quinoxaline 1,4-di-N-oxide derivatives.
Chalcone analogs 1a-d were synthesized by a previously reported base-catalyzed Claisen-Schmidt condensation [49], using 3% NaOH in methanol as the catalyst and establishing an optimum reaction temperature of −10 °C [43]. Their reduced analogs 3a, 3c were synthesized using the same catalyst, but in this case, the condensation was performed at room temperature, as previously reported [28]. The compounds 1a-d and 3a, 3c obtained have been previously described [28,47]. Compound 2a was first synthesized according to a previously reported method [50]. It was a spontaneous reaction that gives the desired compound in good yield.
New cyclopropyl derivatives 4a-d, 5a and 6a, 6c were obtained according to a previously reported method [51]. These reactions were carried out between compounds 1a-d, 2a and 3a, 3c and trimethylsulfoxonium iodide (TMSOI) in the presence of an aqueous solution of NaOH, with tetrabutylammonium bromide (TBAB) as a phase transfer catalyst and dichloromethane as the solvent. With regard to the reaction mechanism, Corey and Chaykovsky [52] demonstrated that trimethylsulfoxonium halides in the presence of a strong base allow the formation of a reactive compound named dimethylsulfoxonium methylide (DMSY), commonly known as Corey's reagent [53]. The reaction of DMSY with an ,-unsaturated ketone involves the cyclopropanation of C=C double bond [54].
As their coupling constant values in the 1 H-NMR spectra showed J values ≈ 16 Hz [47], the double bond geometries of compounds 1a-d and 3a, 3c were trans, just like the newly synthesized 2a.

Biological Results
In this study, eighteen quinoxaline and quinoxaline 1,4-di-N-oxide derivatives were tested against a chloroquine-resistant FCR-3 strain of P. falciparum. All the synthesized compounds and their biological activities are shown in Tables 1, 2 and 3.   Almost all the synthesized compounds exhibited some activity against P. falciparum, but none of them had better IC 50 values than chloroquine itself. Compounds 1a and 2a have been chosen as lead compounds with interesting activities against the parasite. Although both leads were chalcone analogs with R 6 /R 7 = H/H, compounds 7b and 4b had the best activities among inverted chalcone and cyclopropyl derivatives. In these cases, the presence of a halogen atom in position 7 led to an increase in the activity. In fact, 4b was the third most active compound of the study. These results coincide with previous reports [18], where the most active compounds were those without any substitution on the quinoxaline ring and with monosubstitution by an electron withdrawing group.
Apart from compound 4b, changing the double bond in chalcones (compounds 1a-d, 2a and 3a, 3c) for a cyclopropyl structure led to a dramatic drop in the antiplasmodial activity in each case (compounds 4a-d, 5a and 6a, 6c). The same trend was noted when comparing cyclopropyl derivatives 4a-d with their analogs in series 7.
With regard to series 1 and 7, the inversion of the chalcone did not lead to a change in the antimalarial activity, except for compound 1a, whose IC 50 was significantly lower than that of the others. Therefore, the orientation of the enone moiety does not appear to be a determining factor for the antimalarial activity. Finally, only reduced chalcones 3a and 3c showed some interesting activity, while reduced cyclopropyl derivatives 6a and 6c were completely inactive. According to previous reports [58], a minimum requirement for the antimalarial activity was the oxygenation of N-1 and N-4 of the quinoxaline ring, because mono and reduced compounds were not active. Nevertheless, in this study the presence of N-oxides was not decisive for the synthesized compounds to be active.

General Information
All of the synthesized compounds were chemically characterized by thin layer chromatography (TLC), melting point, proton nuclear magnetic resonance ( 1 H-NMR), infrared spectroscopy (IR) and

In Vitro Antiplasmodial Drug Assay
Chloroquine-resistant FCR-3 strain of P. falciparum was cultivated at 37 °C in 5% CO 2 , 5% O 2 in a balanced N 2 atmosphere environment on RPMI 1640 medium supplemented with gentamicin 0.1 mg/mL and 10% heat-inactivated A + human serum, as previously described [59]. The drugs, dissolved in dimethyl sulfoxide, were added at final concentrations ranging from 250 to 0.1 M. The final DMSO concentration was never greater than 0.1%. In vitro antimalarial activity was measured using the [ 3 H]-hypoxanthine (MP Biomedicals, Santa Ana, CA, USA) incorporation assay [60]. Briefly, 250 L of total culture medium with the diluted drug and the suspension of human red blood cells in medium (A + group, 5% hematocrit) with 1% parasitemia were placed into the wells of 96-well microtiter plates. On the third day of the test, radioactivity was assessed. All experiments were performed in triplicate. Results were expressed as the concentration resulting in 50% inhibition (IC 50 ), which was calculated by a nonlinear regression logistic dose response model. The mean IC 50 values and standard deviation for each compound was calculated.

Conclusions
Eighteen quinoxaline and quinoxaline 1,4-di-N-oxide derivatives have been synthesized with the aim of studying their antimalarial activity. The SAR study suggested that the chalcone and inverted chalcone moieties can act as useful linkers in the search for antimalarial ligands. Moreover, the similar activities of series 1, 3 and 7 allow us to affirm that the enone moiety plays an important role in the antimalarial activity of these compounds, but not its orientation. Compounds 1a and 2a showed the best antiplasmodial activity. In general, the addition of cyclopropyl moiety dramatically reduces the biological activity in each case. We are not able to unequivocally confirm that N-oxides are essential for the antiplasmodial activity. These results show that further structural modifications may provide better antimalarial compounds.