SYNTHESIS OF 4-BENZYLSULFANYL AND 4-BENZYLSULFONYL CHALCONES. BIOLOGICAL EVALUATION AS ANTIMALARIAL AGENTS

Malaria management has been complicated in recent times, perhaps, by increasing the Plasmodium spp. resistance to the drugs of clinical use for its treatment. The study describes the synthesis of a new series of chalcones 7 40 and their antimalarial efficacy against a chloroquine-susceptible strain of Plasmodium berghei. Furthermore, we carried out hemindependent studies to unfold the mechanism of action of these synthesized hybrid molecules. Twenty compounds showed inhibitory activity on the formation of β-hematin higher than 75%, compared to chloroquine. The compounds 9, 21, 33 and 37 showed enhanced antimalarial activity in vivo and may reduce malaria progression in this model through a mechanism related to the inhibition of hemozoin formation. Critical aspects of the structure-activity relationship (SAR) are also discussed to better understand the effect shown by these compounds.


Introduction
Human malaria, a tropical, re-emerging infectious disease caused by several types of protozoan parasites of the genus Plasmodium, has been a primary concern to humanity for centuries and is now extended to more than 40% of the world's population. It is a disease primarily of the tropics but is also found in many temperate regions. The disease is responsible for 229 million cases resulting in 409000 deaths in 2019 [1]. Humans are affected by six species of the genus Plasmodium: falciparum, vivax, ovale curtisi, ovale wallikeri, malariae, and occasionally knowlesi. Of these, P. falciparum and P. vivax are the most aggressive species worldwide, often fatal to humans [2]. Several factors contribute to the increasing spread of malaria. Besides climatic and environmental factors, the Anopheles mosquito has become increasingly resistant to insecticides and has adapted to avoid insecticide-treated surfaces. The other cause for the re-emergence of malaria is the development of parasite resistance to antimalarial drugs, in particular, some of the commonly used synthetic quinolines such as chloroquine (CQ), antifolates. Resistance even to the artemisinin derivatives has been recently reported [3,4]. Vaccine development has been an imperative for decades. Still, today it seems unlikely because of the extreme degree of antigenic variation exhibited by these parasites and the lack of resources for translational work and large vaccine trials. In October 2019, WHO announced the phase 3 trial results for malaria vaccine RTS,S/AS01 in Africa [5]. Therefore, chemotherapy remains the only treatment option for controlling infection once acquired, but none of the different chemotherapeutic strategies used in the past has proven consistently successful.
To overcome the drug resistance issue, extensive modification of existing antimalarial chemotypes and the search for novel targets (and new chemotypes interacting with them) have been undertaken and are currently being pursued. Thus, many interesting compounds have been selected for use in monotherapy or, even better, in combination chemotherapies able to improve efficacy and delay the onset of resistance [6][7][8][9][10][11]. Since the antimalarial activity of licochalcone A, a natural product isolated from Chinese liquorice roots, has been reported, several research groups around the world have isolated from natural sources and synthesized a wide variety of chalcone derivatives to study their chemotherapeutic activities and modify their pharmacokinetic properties [12][13][14][15][16][17][18][19][20][21][22][23][24][25]. Chalcone compounds have a common chemical scaffold of 1,3-diaryl-2-propen-1-one, that exists as trans and cis isomers, with the trans isomer being thermodynamically more stable (Figure 1) [26].

Chemistry
General: The synthesis of intermediate (4 -6) and target compounds (7 -40) was performed as outlined in Figure 2. All moisture and oxygen-sensitive reactions were carried out in flame-dried glassware under N2. All solvents were dried and distilled under a nitrogen  [40,41,43].
Four-day suppressive test: One million P. berghei, injected in the caudal vein i.v, were used to infect the mice (18 -22 g, n = 6). After two hours of inoculation, the compounds that were active in vitro were dissolved in DMSO (0.1 M) and subsequently diluted with Saline-Tween 20 solution (2%) and administered i.p. for 4 days (20 mg/kg). The parasite load was evaluated on day four by examining Giemsastained smears. The positive control was chloroquine (20 mg/kg), and the negative control was a saline solution. Non-treated mice were used as a baseline control for survival times [40,41,44]. The survival days and percentage of parasitaemia were used to express the results. Statistical analysis: One-way ANOVA and T-tests for specific group comparisons were used for data analysis. Significance was only considered when p < 0.05 for all analyses. Data were expressed as mean SD. The software used was Graph Pad Prism 4.02 [45].

Chemistry
We prepared 34 chalcone derivatives through a facile synthesis of two and three steps. The most important change in the design of these derivatives is the addition of benzyl mercaptan moiety to 4-chlorobenzaldehyde or 4-chloroacetophenone ( Figure 2) [38]. We designed this inclusion because we wanted to evaluate the antimalarial activity of these compounds with a sulphur atom and possible changes when sulphur is oxidized to sulfone (Figure 2) [39]. The final compounds 7 -40 were synthesized through aldol condensation of Claisen-Schmidt between 4 and several substituted acetophenones and compounds 5 and 6 with several substituted benzaldehydes, using sodium hydroxide as a catalyst in ethanol at room temperature. These conditions were found to be satisfactory for the synthesis in good yields. Only (E) isomers were obtained, which were confirmed by the doublets in the 1 H-NMR spectra between 7.35 -7.83 and 7.55 -8.24 ppm, with coupling constants around (15)(16), assigned to the two protons in the Hβ and Hα position, respectively, and confirmed by COSY (Correlated Spectroscopy) experiment. We can see that the type of substituent on the phenyl rings (A and B) had a significant effect on the potential antimalarial activity of the target compounds. When the phenyl group (A) had a withdrawing electron substituent group at position 4, the corresponding compound exhibited excellent activity as an inhibitor of β-hematin formation and an antimalarial in vivo.
In the compounds 15 -40, when group phenyl (B) was mono-substituted in position 2 with group Me or F, but with an S in position 4 of the ring (A), the activity was marginal, however with these same substituents in positions 2, 3 or 4, but with a functional group SO2 in position 4 of the ring (A) the activity was increased. Compounds di-substituted with F in (B) are showing marginal activity as inhibitors of βhematin formation. It was found that when the phenyl group (B) was substituted with OMe in position 2, 3 and 4 or 2, 4 and 6 the antimalarial activity was marginal; however, di-substituted with the OMe group in position 2 and 3, 2 and 4 or 2 and 5 the corresponding compounds were able to inhibit very well the βhematin formation in vitro, but the compounds 2,4-OMe (B) with an S or SO2 group in position 4 of (A) exhibited an excellent activity in vivo. Our studies have demonstrated that compounds 9, 21, 33 and 37 may inhibit malaria progression in this model through a mechanism of action related to the inhibition of the hemozoin formation. Three types of interactions for these compounds are possible: hydrogen bonding, π-π stacking and coordination. These interactions have been proposed for quinidine, quinine, chloroquine and mefloquine [14,[46][47][48].

Conclusions
We synthesized 34 compounds derived from chalcones using synthetic strategies that were very useful and feasible since they allowed us to obtain moderate to very good yields. Nineteen compounds showed inhibitory activity on the formation of β-hematin greater than 75%. Concerning the in vivo antimalarial evaluation, we observed that the derivatives 9, 21, 33 and 37 decreased the parasitaemia on the fourth day after infection and increased the survival days in a more significant way of the mice. The results reported allowed us to infer and propose some characteristics that must possess the different functional groups present in both rings A and B to achieve that activity, however, noting that none of the compounds eliminates the parasites, we propose that this could be due to low bioavailability. Our results are still in the introductory phase and need further experiments with chloroquine-susceptible and resistant Plasmodium falciparum strains and studies to improve bioavailability.