Synthesis, In-vitro Antimalarial Activity and In silico Molecular Docking Study of Amino Chalcone Derivatives from 1-(2-aminophenyl)-3-(4- substituted-phenyl) prop-2-en-1-one and Dihydroquinolone Derivatives

Background: Malaria is one of the major global health problems in developing countries and faced to the increased resistance of Plasmodium falciparum against existing malarial agents, it is important to look for new antimalarial compounds that will be active in multiple stage of Plasmodium falciparum's life cycle. Objective: The goal of this work was to synthesize Amino Chalcone derivatives and Dihydroquinolone derivatives, then evaluate their antimalarial activity by standard computational and biological methods. Methods: These amino chalcones were synthesized by the ClaisenSchmidt condensation and by intramolecular cyclization of substituted amino chalcones for the Dihydroquinolones derivatives. Their structures have been determined by NMR (H and C). The in-vitro antimalarial assays were carried out by using the maturation test of trophozoites into schizonts. The molecular docking of these compounds was performed by AutoDock vina program using Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) (PDB ID 1J3I) as target protein. Results: All synthesized amino chalcones and dihydroquinolone derivatives were active against Fresh clinical isolates of Plasmodium falciparum with a range of EC50 ranging from 1.56 to 25μg/mL. However, the 2phenyl-2, 3-dihydroquinolin-4-(1H)-one (DHQ 2) and 2(4-methoxyphenyl)-2, 3-dihydroquinolin-4-(1H)-one (DHQ 4) showed excellent antimalarial activity with IC50 of 3.125 and 1.56 μg/mL, respectively. Whereas the IC50 of Chloroquine use as reference was 1.56μg/mL. Based on absorption, distribution, metabolism and excretion (ADME) properties, all synthetized compounds satisfied the Lipinski rule. Conclusion: The results suggest that these synthesized compounds (DHQ 2 and DHQ 4), could be used, after in vivo and clinical tests, like antimalarial supplement or even replace current drug therapies.


INTRODUCTION
For decades, Malarial caused by the Plasmodium parasite, which is transmitted by the Anopheles mosquitoes' bite, has been one of the major global health problems in the Tropical and subtropical regions. According to recent reports from the World Health Organization (WHO), the number of malaria cases worldwide had reached 229 million in 2019 in 87 malaria endemic countries 1,2 . In general, about fifty percent of the world's community lives under the continuing threat of malaria, which is currently treated by Artemisinin, its derivatives and chloroquine in endemic countries 1,2 . This disease caused approximately 619, 000 deaths (in 2021), 90% of which were in Sub -Saharan Africa, and 78% of these deaths for children under 5 years of age 3,4 . It is the most prevalent parasitic disease and the most common cause of hospital visitation in Democratic Republic of the Congo 3 . This is explained by the increased resistant of parasite to existing drugs such as Chloroquine and Artemisinin, which largely target the asexual blood stage of Plasmodium falciparum life cycle [3][4][5][6] . Faced to this alarming situation, much scientific research is directed towards the discovery of new antimalarial compounds with other mechanisms of action against Plasmodium falciparum and that will be active in multiple stage of life cycle of the latter.

Part 1-Chemistry
All the starting materials were commercially obtained (Merck). Thin-layer chromatography was carried out on silica gel plates (Merck Kieselgel 60 F254) and visualized by UV light (254 nm). The melting points are determined using a Büchi M-565 melting point apparatus (Büchi Labortechnick AG). NMR spectra were obtained using a Jeol ECA 400 (400 MHz) and Lambda 400 NMR spectrometers. All chemical shifts are expressed in ppm. FT-IR spectra were taken in KBr pellets (100 mg) using Shimadzu FT-IR spectrophotometer and the values were represented as wavenumber in cm −1 .

b)
Synthesis of four (4) Dihydroquinolones from amino chalcones. Preparation of catalyst PMA/SiO2 (1 mol %) 1 eq of PhosphoMolibdic acid (PMA: H3PMo12O40) and 0.9 eq of Silica gel were added methanol by stirring at the room temperature for 6 hours. After, the methanol solvent was removed using rotary evaporator and the solid catalyst PMA/SiO2 was collected.
The four (4) Dihydroquinolone derivatives were obtained by adding the mixture of 1 mol of previous obtained aminochalcone derivatives (AC1, AC2, AC3 and AC4, respectively) and the catalyst PMA/SiO2 (1 mol %) in 5 mL of methanol under N2 atmospheric. The mixture was stirred under reflux for 8 hours. After this,     Labortechnik). Samples with monoinfection due to Plasmodium falciparum and a parasite density between 1% and 2% were used for the in vitro antimalarial tests. The parasites were cultivated and maintained continuously in human erythrocytes according to previously described methods 35 .

2-(4-methoxyphenyl)-2, 3-dihydroquinolin-4-(1H)one (DHQ4
In-vitro Antimalarial Assay Procedure. The antiplasmodial activity of each synthesized compound was evaluated against the strain of Plasmodium falciparum using maturation test of trophozoites into schizonts described by Mulula and al 3 . A stock solution of 100µg/mL of compound was prepared in methanol (MeOH). These were further diluted in complete medium to attain the final concentrations of 50μg/mL. The stock solutions were prepared on the assay day. Chloroquine was used as the standard reference drug (positive control). Compounds were serially diluted two fold in complete medium (RPMI) up to 0.195µg/mL using a flat bottomed, 96well microtitre plate. Erythrocyte non parasitised was added to column 1 (blank) which had no drugs, while parasitized red blood cells were added to columns 2-12. The plate was incubated at 37°C for 48 hours. After incubation, 50µL of each well were placed on the glass slide and air -dried for 24 hours. GIEMSA was added and microscope lecture was realized. The concentration of any substance that inhibited 50% of the parasite growth (IC50) was determined in triplicata.

Molecular docking studies
The molecular docking of the synthesized compounds (4 Amino Chalcone and 4 dihydroquinolone derivatives) on the target proteins of microorganisms was carried out by using Autodock vina program with standard protocol according the literature for understanding the possible interactions between the compounds and parasites 18 . Plasmodium falciparum target was Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) (PDB ID 1J3I), which was obtained from the protein data bank (http://www.rcsb.org ).

In silico drug-likeness predictions
In silico Drug-likeness helps to know whether a particular pharmacological agent has properties consistent with being an orally active drug. The properties of these 4 Amino Chalcone and 4 dihydroquinolone derivatives were evaluated for their in silico parameters using SwissADME web [http://www.swissadme.ch/ (accessed on 8th February 2023).

Part-1 Biology Antimalarial Activity
Antimalarial activity of four synthesized amino chalcones and four dihydroquinolone derivatives against Fresh clinical isolates of Plasmodium falciparum were determined using the method described by Mulula and al 3 . Chloroquine was used as reference compounds for antimalarial activity. The IC50 (µg/mL) values of these synthesized compounds and Chloroquine are reported in Table 1. IC50 is the concentration of each test compound needed to hinder multiplication of parasites by 50%. Among all the tested compounds, DHQ2 and DHQ4 were found to be the most active with IC50 values of 3.125µg/mL and 1.56µg/mL, respectively. In addition, the compound DHQ4 showed also best antimalarial activity than Chloroquine (IC50= 1.56 µg/mL) which was use as reference. This could be explained by the structure of this synthesized compound that has electron donating groups -OCH3 in para position of Aryl ring B. According the literature, the presence of methoxy group (-OCH3) in para position of Aryl ring B of Chalcones and quinolones compounds, enhances the antimalarial activity because this substituent has the potential to increase the lipophilicity of a compound which is an important property in antimalarial activity 2,36 .
In this work, the presence of the substituent (methoxy, Benzyl, nitro, Chloro) in para position of Aryl ring B of Chalcones and quinolones compounds plays a very important role in the antimalarial activity. This increases the antimalarial activity following the order -OCH3> -Phenyl> -Cl> -NO2. In addition, amino chalcones AC1, AC2, AC3 and AC4 have shown less antimalarial activity against fresh clinical isolates of Plasmodium falciparum compared to the dihydroquinolones compounds (DHQ1, DHQ2, DHQ3 and DHQ4) derived from these amino chalcones. This could be explained by the presence of heterocyclic nitrogen associated with the carbonyl group already present in this cycle.

Molecular docking studies
The in-silico antimalarial activity results of synthesized amino chalcones and dihydroquinolone derivatives (Ligands) against Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) (PDB ID 1J3I) was reported in terms of binding energy and ligand interactions in order to predict the binding energy of ligands within the binding site of target proteins. These results are reported in Table 2, and Figures 1, 2 and 3.
Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) (PDB ID 1J3I) is one of the important enzymes responsible for the production of folates and thymidylates, which are required in DNA synthesis of Plasmodium falciparum 2 . Thus, the molecules that would block this enzyme will play a key role on the antimalarial activity. The in silico antimalarial results indicated that all synthesized amino chalcones and dihydroquinolone derivatives showed strong binding affinity (ranges from −7.5 to −8.2 Kcal/mol, given in Table 2) towards the amino acid residues in active pocket Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) (PDB ID 1J3I) protein through H-bond, Van der Wals and residual interactions, compared to standard drug Chloroquine (-5.9 Kcal/mol). Dihydroquinolone 4 (DHQ4), which exhibited best antimalarial activity like the drug standard, showed good affinity by interacting with 12 target protein amino acids including PHE-D520; GLY-D517; ARG-D345; THR-D346; ASN-D555; ASP-D605, HIS-D556; LEU-D516; VAL-D348; UMP-D711; TPR-D553 and ASP-D513 (Figure 2). Whereas the antimalarial drug reference (Choloroquine) showed less affinity against Plasmodium falciparum dihydrofolate reductasethymidylate synthase (-5.9 Kcal/mol) and interacted with

In-silico drug-likeness predictions
In silico Drug-likeness helps to know whether a particular pharmacological agent has properties consistent with being an orally active drug. To be effective, a compound must have optimal hydrophilic and hydrophobic properties to carry in the blood before penetrating the cell membrane. A simple method for evaluating drug properties is to verify compliance with the Lipinski rule (rule of 5), which specifies the number of hydrophilic; molecular groups weight and  18 . Lipinski rule proposed that drug target must have; the molecular weight (MW) ≤500, hydrogen bond acceptor (HBA)≤10, hydrogen bond donor (HBD)≤5, lipophilicity (logP)≤5. The absorption, distribution, metabolism and excretion (ADME) properties of synthetized amino chalcones and dihydroquinolone derivatives are represented in Table 3. With regard to Table 3, all synthetized amino chalcones and dihydroquinolone derivatives satisfied the Lipinski rule, Ghose, Veber, Egan and muegge rule of five and also showed very good solubility because the logP is between 2 and 6 (2< log P<6). Thus, these molecules could be used as orally active drug.

Funding Acknowledgment
No external funding was received.