Synthesis and in vitro SAR evaluation of natural vanillin-based chalcones tethered quinolines as antiplasmodial agents

A series of novel chalcone derivatives were synthesized and investigated against the chloroquine-sensitive P. falciparum 3D7 (Pf3D7) strain and chloroquine-resistant P. falciparum K1 strain to establish their structure-activity relationship. In this study, compound 7 was found most active as well as less cytotoxic (IC50 = 4.12 µM and 3.14 µM for Pf3D7 and PfK1 respectively; CC50 = 46.18 µM). Compound 7 was studied for effect on parasite growth and the microscopic examination showed excessive DNA damage in the trophozoite stage. The parasite recovery after drug removal was poor due to the dramatic genotoxic effect of compound 7. It suggested that 7-chloro quinoline and triazole linkage were crucial for antimalarial potential. Graphical abstract Graphical abstract


Introduction
Malaria is a mosquito-borne disease, which is associated with high morbidity and mortality [1]. Although diverse potent antimalarial agents including quinolones [2][3][4] (e.g. chloroquine) and endoperoxides [3a] (e.g., Artemisinin and its derivatives) [3b] are available, the emergence of resistance against front-line drugs has raised a major health concern. Thus, the search for newer efficacious drugs as well as new molecular frameworks possessing antimalarial activity remains a vital goal towards achieving control over malaria [5]. Among various antimalarial molecules, the design and development of novel hybrid molecules also represent a logical approach that has the potential to overcome the rapid development of drug resistance, decrease parasite burden, and reduce both the cost and the risk of drug-drug interactions compared to cocktails or multicomponent drugs [6,7]. In view of encouraging efficacies with good bioavailability and minimized toxicity; the next generation drugs may be hybrid molecules reducing the possibility of resistance. This was demonstrated by the compounds such as tetraoxaquine 1a, trioxaquine, trioxaferroquine and stilbene-chalcone hybrid 1b [8][9][10][11][12] (Fig. 1).
Chalcone (1,3-diaryl-2-propen-1-one), coumarin, triazole, and quinoline are major classes of naturally occurring compounds that have been reported to possess several effective therapeutic properties [11,[13][14][15]. These pharmacophores are derived from both natural and synthetic routes, each with a distinct mechanism of action, and could be beneficial in malaria treatment as well as minimize the chances of getting drug resistance. Ratifying this approach, various research groups have reported hybrid molecules by coupling chalcone, coumarin, curcumin, flavone, and quinoline with different other bioactive molecules, like resveratrol, maleimide, and alphalipoic acid [16][17][18][19][20] to address the problems with hybrid molecules, their solubility, and toxicity. Our group has reported chalcone, chalcone-stilbene hybrids, and distyrylbenzenes for their antimalarial activities [21,22]. In line with our earlier report on the stilbene-chalcone hybrid with the antimalarial property envisioned that a hybrid of the chalcone molecules with quinolines and coumarin may exhibit potential antimalarial activities [8,23]. Hence, we designed chalconequinoline and coumarin-based novel hybrid molecules and studied its structure-activity relationship (SAR) for the antimalarial activity, and also studied the effect of the active molecule on the growth of the human malaria parasite, Plasmodium falciparum.

Antimalarial activity
All the synthesized hybrid-compounds were tested for antimalarial activity against chloroquine-sensitive P. falciparum 3D7 (Pf3D7) strain and chloroquine-resistant P. falciparum K1 strain (Table 1) by SYBR-Green-I assay [24]. The fluorescence readout gives an indication of parasite growth in infected RBCs. SYBR green-based fluorescence plotted with respect to drug concentration gives a precise estimation of parasite inhibitory concentrations. Interestingly, the most active compound i.e. (E)-3-(4-(allyloxy)-3-methoxyphenyl)-1-(4-chlorophenyl)prop-2-en-1-one (IC 50 = 2.5 µM) was selected from our previous reports for further hybridization which is considered as the basis of the study and used as a precursor for further elaboration of new chemical entities in antimalarial activity [8]. Thereafter, the structure-activity relationship studies were carried out by changing the substitutions on ring B keeping the 4-chloro substituent constant on ring A. Amino methyl, allyl, and propargyl substituent on ring B exhibited no activity against both the strains but compound (7) was quite efficient in killing both chloroquine-sensitive and resistant strain. This clearly shows the benefit of the addition of triazole-linked 7-Chloro quinoline to the chalcone. We next ventured to evaluate the positional importance of the amino 7-chloroquinoline group on ring A (Table 1). It is known that activity was markedly affected by p-substitution of O-allyl group [8]. So, allylated vanillin substitution at the ring B was kept unchanged, and subsequently, the effect of changing the nature of the N-substituent (H, allyl, phenyl, C 4 H 9, and CH 2 C 6 H 4 Br) was evaluated, and reduced activity was observed in each instance (Table 1, 15,16) except, Nallylated chalcone (14) but lacked selectivity. It was observed that there was no further enhancement in antimalarial activity for any of the coumarin-chalcone hybrids (Table 1). Heteroaryl-substitution is an appealing strategy for desirable activity and several inspiring reports on the antimalarial activity of heterocyclic containing chalcone derivatives [25] boost us to synthesize such analogues. We designed chalcones by the condensation of benzaldehydes with different heterocyclic carbonyls like 7-chloroquinoline, and coumarin [26]. However, in each case, the antimalarial potential was not found, although, the 7-chloroquinoline is considered an excellent lead prototype for the development of antimalarial drugs [27,28].

Microscopic examination of antimalarial activity
Compound (7) was studied for microscopic examination which revealed that the compound treatment caused drastic effects on parasite growth in comparison to control. After 24 h, the ring-stage parasite progressed into the late trophozoite and schizont stage in control, whereas, the compound-treated sample showed delayed parasite growth and they were arrested in the early trophozoite stage. After 48 h, healthy schizonts in the control sample  progressed into a new infection cycle and parasites were predominantly in the ring stage. While, the treated parasite showed mainly stressed trophozoite with reduced staining of parasite DNA, probably due to excessive DNA damage. We also performed experiments to check whether the parasite is able to recover after the removal compound (7). Due to dramatic genotoxic effect of compound (7), the parasite shows poor recovery from stress even after drug removal (Fig. 3).

Conclusion
The study revealed that among all synthesized hybrid molecules vanillin-quinoline hybrid molecules showed better antimalarial potency against Pf3D7 and PfK1, respectively) in comparison to vanillin-coumarin hybrid molecules ((IC 50 of greater than 5 µM) as well as natural licochalcone (IC 50 of 4.1 µM). Microscopic examination studies of compound (7) showed a drastic effect on parasite growth even after removal of the compound. The study suggests that hybrid molecules may exhibit promising activities, and their economical route of synthesis may provide useful leads towards future antimalarial drug discovery.

Chemical and reagents
All the reagents were obtained from commercial sources (Merck or Acros). The solvents used for isolation/purification of compounds were obtained from commercial sources (Merck) and used without further purification. 1 H and 13 C NMR spectra were recorded on a Bruker Avance-400 spectrometer. TMS was used as an internal reference for 1 H NMR. HRMS-ESI spectra were determined using micro mass Q-TOF ultima spectrometer.
Cytotoxicity (CC 50 ) was evaluated in VERO cells (C 1008; monkey kidney epithelial cells) using the MTT assay. VERO cells were maintained in RPMI media supplemented with HEPES, 0.2% sodium bicarbonate, 0.2% D-glucose, 10% FBS, fungi zone (0.25 mg/L) and gentamycin (50 mg/L) at 37°C in a humidified CO 2 incubator. VERO cells (104/well) were seeded in a 96 well plate and cells were treated with different dilutions of compounds (16-18 h post-seeding). Podophyllotoxin (P4405, Sigma) was used as the positive control. After 72 h, 25 µl of MTT (M2128, Sigma) (stock 5 mg/ml) was added to each well and incubated for 2 h in a CO 2 incubator. The supernatant was removed carefully without disturbing the cells and 150 μl DMSO was added to each well to dissolve the purple precipitate. Absorbance was recorded at 540 nm using an ELISA plate reader and data was analyzed to determine 50% cytotoxic concentration (CC 50 ). For microscopic examination, 3D7 was synchronized with 5% sorbitol. The ring-stage parasite was maintained at 6-8% parasitemia with 2% haematocrit and treated with compound 7 at 10 µM concentration. After 24 h and 48 h of treatment, thin blood smears of both control and treated culture were prepared. Smears were fixed and stained with methanol and Giemsa, respectively. In a parallel experiment, after 24 h of treatment, the culture was washed (twice) with RPMI media to remove the drug and was further incubated without the drug to check the revival of the parasite after drug removal.