Discovery of 5-Chlorobenzimidazole-based as Promising Inhibitors of Chloroquine-Resistant Plasmodium Strains: Synthesis, Biological Evaluation, Molecular Docking and Computational Studies

Background: To overcome drug resistance to current antimalarial drugs, we propose the synthesis and in vitro evaluation of the antiplasmodial activity of a series of 5-chlorobenzimidazolyl-chalcones against chloroquino sensitive (CQ-S) and chloroquino resistant (CQ-R) strains of P. falciparum. Objective: This study aimed to establish through structure-activity relationship studies and docking, the structural elements essential for antiplasmodial activities. Original Research Article N’Guessan et al.; JPRI, 33(46B): 136-147, 2021; Article no.JPRI.74758 137 Methods: The antiplasmodial activity of these benzimidazolylchalcones was carried out according to the Rieckmann microtest technique, followed by the determination of the concentrations inhibiting 50% of the production of parasitic HRP2 antigens (IC50) by ELISA. Chloroquine was used as a reference molecule with a sensitivity threshold set at 100 μM. Molecular docking was performed using sensitive (PDB ID: 1J3I) and resistant (PDB ID: 4DP3) dihydrofolate reductasethymidylate synthase proteins (PfDHFR-TS). Results: All benzimidazolylchalcones tested expressed antiplasmodial activities especially against chloroquine resistant isolates (IC50 = 0.32-44.38 μM). The best profile against both isolates was the methoxylated derivative (3e) with an IC50 ranging from 0.32 to 1.96 μM. This compound had the best antimalarial activity against CQ-S isolates. On CQ-R isolates, the unsubstituted 5chlorobenzimidazole derivative (3b) had exalted activity (IC50 = 0.78 μM). We selected a weakly active non-chlorinated derivative 3a and chlorinated derivatives 3b, 3d, 3e and 3f) with IC50< 3μM against the chloroquine-resistant strain to perform docking studies. These revealed that the pyrrolic nitrogen of benzimidazole and the ketone of propenone are the main chemical entities involved in the interaction at the receptor. Moreover, ADMET studies showed favorable pharmacokinetic properties. Conclusion: Molecular docking studies confirmed the experimental findings and revealed the possible interactions pattern. Derivatives 3b and 3e, which showed promising binding affinities against PfDHFR-TS, can be proposed as lead compounds for the development of antimalarial drug candidates.


INTRODUCTION
Malaria is a parasitic infection transmitted to people through the bites of infected mosquitoes due to species of the genus Plasmodium whose main infectious agent, Plasmodium falciparum, is formidable [1]. In fact, Plasmodium falciparum and Plasmodium vivax are the two species responsible for the most deadly forms of malaria, which mainly affects children and pregnant women [2]. Around 3.4 billion people in the world -more than one third of the world's population today-are at risk of being infected with malaria and developing disease malaria [3,4]. Estimates point to 229 million malaria episodes in 2019, of which 94% (215 M) were in the WHO African Region. In this region, the number of deaths due to malaria has decreased from 620,000 to 409,000 over the period 2009-2019 representing a reduction in malaria cases and death rates of 18% and 34% since 2010 [5]. These figures are all the more alarming as we are witnessing an increase in morbidity caused by resurgence of P. falciparum chemoresistance to almost all available antimalarial drugs, including artemisinin derivatives, the most potent and safe antimalarial drugs [6]. In fact, available classes mainly used for treatment of malaria due to P. falciparum can be divided into five classes namely quinolone and arylamino alcohol, artemisinin and its derivatives, antifolates, hydroxynaphthoquinones and antibacterial agents [7].
However, the efficacy of the current first-line agents for curative treatment based on artemisinin-based combination therapy (ACT) in nearly all areas is seriously limited by frequent drug resistance [6,7]. In 2017, the WHO sounded the alarm, declaring that malaria control was at a crossroads of paths. Despite remarkable progress, advances in the fight against malaria worldwide have stabilized in recent years, particularly in high-burden countries [1,2]. Meanwhile, as a result of disruptions to planned routine activities and services due to the current coronavirus pandemic, a modeling study predicts an impact on the malaria burden with an additional loss of life of up to 36% over the next 5 years [8]. Global progress in malaria control is at risk of being undermined by gaps in access, COVID-19, and inadequate funding [3,4]. In such a context of development of multidrugresistant malaria and absence of vaccines, a revival of malaria control is needed.
To overcome drug resistant, one of the alternatives proposed is to search for suitable drug inhibitors that are effective against resistant strains of Plasmodium [9]. Recent developments in the understanding of parasite biology as well as current therapeutic approaches, promote action on innovative targets to generate promising new drugs against resistant malaria. Among the metabolic processes of cytoplasmic enzymes, inhibition of the dihydrofolate reductase (DHFR) pathway, whether or not linked to Thymidylate synthase (TS), has been established as a prime target for antimalarial chemotherapy [10]. In fact, the discovery of the chemoprotective activities against Plasmodium falciparum of P218, a potent inhibitor of Dihydrofolate reductase, has given new hope for preventive therapy of the pregnant women and infants may in malaria endemic areas [11]. Indeed, this new candidate for intermittent preventive treatment may become an alternative to the combination of sulfadoxine and pyrimethamine whose efficacy is threatened by resistance resulting from mutations in the P. falciparum dihydrofolate reductase [PfDHFR] genes targeted by pyrimethamine [12] (Figure 1). Similarly, antimalarial research has taken a leap forward with the identification of the WR99210, an analog of cycloguanil, which is a selective inhibitor of the bifunctional enzyme DHFR-TS of the parasite, especially as it has no action on human DHFR, which is not fused with TS ( Fig.  1).
Recently, in silico antimalarial studies performed for the protein dihydrofolate reductasesthymidylate synthase (PfDHFR-TS) revealed that the chalcone analogues interacted with both the susceptible (1J3I.pdb) and the resistance protein (4DP3.pdb), meaning that they were active against both the chloroquine-sensitive as well as the chloroquine-resistant Plasmodium strains [13]. It is in this perspective that we are interested in hybrids of chalcone and benzimidazole as potential new antimalarial agents. In a previous study, we demonstrated that the 5-chlorobenzimidazole group of a series of benzimidazolyl-chalcones would behave as an antiplasmodial pharmacophore similar to the 7chloroquinoline of amodiaquine and chloroquine [14] (Fig. 2). In the present work, we propose to extend the antiplasmodial evaluations to a wider variety of derivatives possessing this pharmacophore so as to establish the structural elements essential for good serial antiplasmodial activities of benzimidazole-supported chalcone hybrids. Also, in silico studies were used to explore the interactions of 4 compounds, selected for their excellent antimalarial activities on both chloroquine-sensitive and chloroquine-resistant strains, with respect to protein binding sites. These computational studies also aimed to address the pharmacokinetics properties of the designed compounds.
Therefore, the objective of this work is to evaluate the antiplasmodial activity of new 5chlorobenzimidazolyl-chalcones. Specifically, we aimed to determine the concentrations of chalcones capable of inhibiting 50% of the maturation of P.falciparum by ELISA assay of the HRP2 antigen. Then, the main observations were pooled with the previously obtained results to establish the structural elements favorable to the induction of antiplasmodial activities as well as understand the interactions between the designed compounds with their biological targets.

Chemistry
For all the characterized compounds, the spectra of Nuclear Magnetic Resonance (NMR) spectra of the 1 H proton (300MHz) and of the 13 C (75 MHz) were recorded on a Brücker Avance 300 instrument. The tetramethylsilane (TMS) is used as a reference reference of the shifts expressed in ppm. The description of the NMR spectra uses symbols : singlet = s; doublet = d; split doublet = dd; triplet = t; quadruplet = q; quintuplet = quint; multiplet = m. The mass spectra were recorded on a JEOL JMS DX300 spectrometer in ESI mode (electrospray/quadrupole ionization). The melting points (FP) were determined using a Kofler bench and are not corrected. The thin layer chromatography (TLC) were performed on silica plates Macherey-Nagel Sil G/UV254 or on Macherey-Nagel ALOX N/UV254 alumina. The eluent system used for the TLC of the synthesized compounds was a DCM / MetOH mixture (95: 5). The products were then revealed with iodine. Solvents and reagents, including benzaldehydes, were obtained from Acros Organics (France) or from Aldrich (France). Chloroquine, an antimalarial drug, supplied as a pure powder comes from Sigma Chemical Co. (USA).
The first step of chemical synthesis is to prepare 2-acetyl benzimidazole (2a-b) by heterocyclization from suitably selected orthophenylene diamines (1a-b) according to the reported procedures [14,15]. The ketone intermediates were then reacted with various heterocyclic benzaldehydes or aldehydes to give benzimidazolyl-chalcones (3a-i) substituted or not on the benzimidazole ring (Scheme 1).

Antiplasmodial Assay
Clinical isolates of Plasmosium falciparum were obtained from patients brought to the laboratory of the general hospital of Abobo, Abidjan Ivory Coast, for suspected malaria. Patients were included if they had a monospecific P. falciparum infection and had not used any antimalarial treatment for at least 30 days. Once informed consent was obtained, samples with a positive thick drop and thin blood smear with a parasite density greater than 100 parasites/µl were stored in a cooler containing a cold accumulator and then transported to the cell culture room of the Immunology Unit of the Center for Diagnosis and Research on AIDS and Opportunistic Diseases (CeDReS). The protocol of the study was approved by the ethical research committee of CeDReS of the CHU of Treichville, Ivory Coast. The samples were analyzed at the Immunology Unit of CeDres, Treichville University Hospital.

Malaria parasites and cultivation
Briefly, the parasites were cultured in human erythrocytes (obtained from an o positive blood group donor) and RPMI-1640 culture medium (Gibco by Life Technologies, Grand Island, NY, USA) supplemented with HEPES. This medium was enriched with 10% albumax II (Gibco by life technologies, Grand Island, NY, USA) used as a substitute for human serum and bicarbonate maintained at the fixed temperature of 37° C in a water bath. Each parasitized blood sample (GRP) was washed three times with RPMI 1640 before use in culture. Before the incorporation of the parasitized red blood cells (GRP) in the culture inoculum, a step of dilution of the GRP pellet was carried out using group O red blood cells in order to reduce the parasitaemia to between 0.1 and 0.2% when the parasite density was greater than this interval. For incubation, the culture was kept in a humid atmosphere in an oven thermostatically controlled at 37° C and, containing a gas mixture of 5% CO2, 5% O2 and 90% N2, for 72 hours. At the end of this incubation time, the plates were transferred from the oven to the laboratory bench. A rapid visual assessment of the maintenance of the blood-red coloration of the wells, indicating an absence of bacterial contamination, was necessary for the validation of the culture. After validation of the culture, the culture plate was left in the still sealed state, then stored by freezing at -20° C before the ELISA test step. This final parasite culture was immediately used for antimalarial assay.

Antiplasmodial screening method
An in vitro histidine-rich protein 2 (HRP2) assay was performed to assess anti-P. falciparum activity. HRP2 assays were performed according to standard procedures. Briefly, P. falciparum isolates were grown in the presence of increasing dilutions of synthetic chalcone derivative and chloroquine, the reference antimalarial drug. In practice, one culture plate was used to culture 2 isolates. Each drug concentration was tested in duplicate in the culture wells. Thus, 4 concentration ranges (50 µg/ml, 10 µg/ml, 2 µg/ml and 0.04 µg/ml) of the tested chalcones were present in duplicate in the culture wells. For chloroquine, a range of 7 daughter solutions from 800 nM to 12.5 nM was obtained by a series of dichotomous dilutions in RPMI of a 1600 nM stock solution. In each well of a microplate, 100 µl of each dilution tested were added to 100 µl of final parasite culture. After incubation at 37°C for 72 h, the plates were then freeze-thawed twice to achieve complete hemolysis and 100 µl of each hemolysis culture sample was transferred to the ELISA plates. The microwells were previously coated with P. falciparum monoclonal anti-HRP II antibody and incubated at laboratory room temperature for 1 hour in a humidified chamber. Plates were washed five times with the PBS/Tween mixture called wash buffer and 100 µl of the diluted antibody conjugate was added to each well. After incubation for an additional 1 hr in a humidified chamber, the plates were washed with the wash buffer mixture (200 µl/well) and 100 µl of the substrate enzyme was added to each well. The plates were then incubated for an additional 15 minutes at room temperature and protected from light; and 50 µl of the stop solution was added. Absorbance values were read using an ELISA plate reader (iMarkTM, Bio-Rad, USA) at a maximum absorbance of 450 nm. The measurement of antimalarial activity of the drugs was expressed as 50% inhibitory concentration (IC50) which is the concentration of antimalarial drug inhibiting 50% of the parasite HRP2 released compared to the control without antimalarial drug. IC50 values were calculated using a nonlinear dose-response curve fitting analysis via ICEstimator 1.2 software. The threshold value for the chloroquine IC50 was 0.1 µM. This is a conventional value used by several authors. Thus, if the IC50 was lower than 0.1 µM, the isolate was said to be chloroquinosensitive. It was qualified as chlororesistant for an IC50 greater than or equal to 0.1 µM.

Molecular Docking and MM-GBSA studies
Molecular docking studies were conducted to explore protein ligands interactions. Crystal structures of sensitive (PDB ID: 1J3I) and resistant (PDB ID: 4DP3) proteins of dihydrofolate reductases-thymidylate synthase (PfDHFR) were obtained from protein data bank (RCSB). The proteins were prepared using protein preparation wizard of maestro v12.7 of Schrodinger suite. The compounds were prepared using ligprep. The binding cavities of the two proteins were identified using the receptor grid generation tool using the cocrystalized ligands. Docking was carried out using XP mode of Glide. MM-GBSA binding free energies were calculated using Prime module of Schrodinger.

ADME prediction
Pharmacokinetics properties of the compound were predicted using qikprop tool of Schrodinger.

Biological Evaluation
The threshold value of the chloroquine IC50 was 0.1 µM. This is a conventional value used by several authors. Thus, if the IC50 was less than 100nM, the isolate was said to be chloroquino sensitive. It was qualified as chlororesistant for an IC50 greater than or equal to 0.1 µM. The results of the antiplasmodial screening of the height 5-chlorobenzimidazolylchalcone derivatives and IC50 of chloroquine and Chalcone (1,3-diphenylpropenone) used as reference for comparison is presented in table 1.
The results showed a higher efficacy of our synthetic chalcones on chloroquine-resistant isolates with IC50s ranging from 0.78 to 31.28µM compared to chloroquine-sensitive isolates with IC50s ranging from 0.32 to 44.38µM. Indeed, in a previous study it had been shown that 1,3diphenylpropenone or chalcone had a moderate antiplasmodial activity on both chloroquinesensitive and chloroquine-resistant isolates, with IC50 of 38.56 and 1.44 µM respectively. Benzimidazolyl-chalcones (3a-3i) were designed as a result of the juxtaposition of the benzimidazole ring and the arylpropenone linkage of chalcones. These two chemical entities have proven their strong antiplasmodial potentialities [13,18]. Moreover 3a, first compound directly derived from this concept of molecular hybridization by replacing the phenyl in position 1 of the 1,3-diphenylpropenone by benzimidazole, has also shown good antiplasmodial properties. Indeed, this compound not substituted on benzimidazole, showed significant activity against both chloroquineresistant P. falciparum isolate (IC50 = 6.81 µM) and chloroquine-sensitive P. falciparum isolate (IC50 = 44.38 µM). However, this activity remainsunsatisfactory compared to the chalcone without the benzimidazole ring. Tests to improve the antiplasmodial activities of compound 3a consisted in introducing a chlorine atom on the benzimidazole ring in positon 5 (compound 3b). This modulation contributes to the exaltation of antiplasmodial activities on both chloroquinesensitive (IC50 = 10.65 μM) and chloroquineresistant (IC50 = 0.78 μM) P. falciparum isolates by 4 to 9 times compared to its non-chlorinated analogue 3a.
Therefore, in order to optimize the antiplasmodial activities of compound 3b, two types of chemical modulations were undertaken on the benzene homocycle. The first modulation consisted in the introduction on the benzene ring of various substituents known as anti-infectious activity performers. In general, the presence of an electron-donor group (OH, OCH3, N(CH3)2 on the benzene homocycle of 5chlorobenzimidazolylchalcone 3b led to an improvement in the antiplasmodial activity. Indeed, compounds 3d, 3e and 3f had very good antimalarial activity on chloroquinosensitive isolates, with IC50s ranging from 9.40 µM to 0.32 respectively. These derivatives were at least 4fold more potent than the reference chalcone with an IC50 of 38.56 µM. Remarkably, among the screened hybrid of chalcones, the paramethylated derivative 3e was the most active against the chloroquine-sensitive isolate. Although these activities are below that of chloroquine (IC50 = 0.076 µM), these compounds remain remarkably effective against the chloroquino-sensitive isolate of P. falciparum. On the other hand, the presence of an electronwithdrawing group of the chloro or nitro type reduced the antiplasmodial activity of the corresponding derivatives towards the susceptible strain.
Even if, on the chloroquinoresistant isolate, the tendency of electron-withdrawing groups to induce less good antimalarial activities remains. Among the electron-donor derivatives, the best activity on the chloroquinoresistant isolate was obtained by the dimethyl amine derivative 3f. Similarly, with an IC50 of 1.8 µM, the presence of a dialkylamine group like the one present in Chloroquine, but this time of dimethylamine type, didn't allow 3f to improve antiplasmodial activity on chloroquine-resistant isolates. In the end, no substitution of the benzene homocycle allowed to obtain a derivative with a better activity than the unsubstituted compound 3b.
The second modulation consisted in replacing the benzene homocycle by a pyridine or furanic heterocycle. On the one hand, the pyridine derivative 3h with an activity around 25 μM, did not improve the activities obtained with compound 3b. On the other hand, the presence of the furanic heterocycle induced a loss of activities against the susceptible P. falciparum isolate while maintaining a minimal activity on the resistant strain (IC50 = 9.34 μM).
Overall, as summarize in figure 3, general trends of SAR in benzimidazole chalcone series established that the replacement of the benzene homocycle by a heterocycle is not appropriate to have excellent antiplasmodial activities.
the electro withdrawing groups seems to induce a reduction of activity on chloroquinosensitive isolates as well as on chloroquine-resistant isolates the presence of methoxygroup, a modulator of the antiplasmodial activity of quinine, increased the antiplasmodial efficacy only on chloroquinosensitive isolates.

Fig. 3. SAR of benzimidazolyl-chalcones with antiplasmodial properties
This activity would be related to the basic character of the benzimidazole heterocycle, like the quinoline nucleus of antimalarial drugs such as chloroquine and amodiaquine. This better performance on chloroquinoresistant isolate can be explained by either a different mechanism of action or a conformational modification of the target receptors of resistant strains in favor of chalcones with Benzimidazole vector.

Molecular docking and MM-GBSA binding free energy studies
To understand the mechanism of the antiplasmodial activity of the designed compounds, molecular docking studies were conducted between the non-chlorinated derivative 3a less active and chlorinated derivatives 3b, 3d, 3e, and 3f, which exhibited the best antimalarial activities towards the chloroquine-resistant strain with IC50< 3µM. These studies were performed against the dihydrofolate reductases-thymidylate synthase (PfDHFR-TS) enzyme using both sensitive (PDB ID: 1J3I) and resistant proteins (PDB ID: 4DP3). As depicted in table 2, the docking scores of these hybrids of chalcone 3a, 3b, 3d, 3e, and 3f were-6.62, -7.68, -8.31, -5.55 and -7.73 kcal, respectively, in the case of the sensitive protein, and -8.22,-7.98, -6.43, -8.19 and -8.74 kcal/mol in the case of the resistant protein for the respective five compounds, in succession. All the compounds exhibited strong binding towards the two proteins in agreement with experimental results. MM-GBSA studies showed compounds 3a, 3b, 3d, 3e, and 3f possess some higher negative binding free energy values, viz., -44.4, -57.89 kcal/mol, -63.2 kcal/mol, -53.45 kcal/mol and -52.01 kcal/mol, respectively in case of sensitive protein; while, in case of resistant protein, binding free energies were better, -46.54, -63.09 kcal/mol, -53.59 kcal/mol, -60.13 kcal/mol and -68.41kcal/mol respectively for the four compounds.
In silico antimalarial studies performed for the PfDHFR-TS resistance protein (PDB ID: 4DP3) confirm that the ketone of propenone and the pyrrolic nitrogen of benzimidazole establish key hydrogen bonds with the target amino acids (Leu 164, Leu 40 and ALA 16) (Figure 4a-e). These key interactions as well as the chlorine on the benzimidazole, which allows for better hydrophobic pole occupancy, would account for the better antiplasmodial activities of the 5-chlorobenzimidazolylchalcones (3b-3i) compared to benzimidazolylchalcone 3a.    3 Predicted caco cell permeability in nm/s (acceptable range: <25 is poor and >500 is great), 4 Predicted blood brain barrier permeability (acceptable range -3-1.2). 5 Predicted apparent MDCK cell permeability in nm/s (acceptable range in nm/s (acceptable range: <25 is poor and >500 is great), 6 Percentage of human oral absorption (acceptable range: <25 is poor and >80% is high. 7 Lipinski rule of five.

In silico ADME analysis
The interesting in vitro and in silico docking results encouraged us to conduct ADME prediction studies for the synthesized molecules to study the pharmacokinetic properties. ADME prediction was performed using qikprop of Schrodinger suite. The predicted properties are presented in table 3. All five synthesized molecules showed acceptable lipophilicity (QPlogPo/w), high aqueous solubility (QPlogS), excellent cell permeability (QPPMDCK and QPPCaco), good CNS penetration (QPlogBB) and excellent oral absorption. None of the compounds violated Lipinski rule of five.

CONCLUSION
The present study was carried out with the aim of extending the evaluation of the antiplasmodial activities of benzimidazolyl-chalcones initiated in a previous study and which highlighted the excellent potentiality of the 5-chlorinated derivative. Ultimately, the 5chlorobenzimidazolyl-chalcones were found to be chalcone hybrids with strong antiplasmodium activity. In summary, based on the MICs, it should be noted that all 5-chlorobenzimidazolylchalcones have better antiplasmodium activity on chloroquine-resistant isolates (CQ-R) than on chloroquinosensitive isolates (CQ-S). In addition to the important role played by the 5chlorobenzimidazole core in the appearance of the antiplasmodial activities of the new chalcones, it appears from the pharmacomodulations that only the introduction of electrodonor substituents such as hydroxyl, méthoxy or a dimethylamine group on the phenyl ring, allowed to improve the activities on chloroquinosensitive isolates but not on chloroquinoresistant isolates. On chloroquinoresistant isolates, the unsubstituted derivative remains the one with the most remarkable antimalarial performance in this series of 5-chlorobenzimidazoles. However, for development as a drug candidate in the treatment of drug-resistant malaria, we can propose the methoxylated derivative. We selected 5 compounds namely 3a, 3b, 3d, 3e and 3f, to perform docking studies, as these compounds presented the best antimalarial activities. Molecular docking studies showed that all potent compounds presented significantly high binding affinity against resistant and sensitive dihydrofolate reductase plasmid-thymidylate synthase proteins. In addition, the compounds exhibited favorable in silico ADME properties.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The protocol of the study was approved by the ethical research committee of CeDReS of the CHU of Treichville, Ivory Coast.