Synthesis, Molecular Docking, and Antimalarial Activity of Hybrid 4-Aminoquinoline-pyrano[2,3-c]pyrazole Derivatives

Widespread resistance of Plasmodium falciparum to current artemisinin-based combination therapies necessitate the discovery of new medicines. Pharmacophoric hybridization has become an alternative for drug resistance that lowers the risk of drug–drug adverse interactions. In this study, we synthesized a new series of hybrids by covalently linking the scaffolds of pyrano[2,3-c]pyrazole with 4-aminoquinoline via an ethyl linker. All synthesized hybrid molecules were evaluated through in vitro screenings against chloroquine-resistant (K1) and -sensitive (3D7) P. falciparum strains, respectively. Data from in vitro assessments showed that hybrid 4b displayed significant antiplasmodial activities against the 3D7 strain (EC50 = 0.0130 ± 0.0002 μM) and the K1 strain (EC50 = 0.02 ± 0.01 μM), with low cytotoxic effect against Vero mammalian cells. The high selectivity index value on the 3D7 strain (SI > 1000) and the K1 strain (SI > 800) and the low resistance index value from compound 4b suggested that the pharmacological effects of this compound were due to selective inhibition on the 3D7 and K1 strains. Molecular docking analysis also showed that 4b recorded the highest binding energy on P. falciparum lactate dehydrogenase. Thus, P. falciparum lactate dehydrogenase is considered a potential molecular target for the synthesized compound.


Introduction
Malaysia has made significant progress in the control of malaria. However, the fatality rate fluctuated throughout the period 2001-2012. The National Strategic Plan for Malaria Elimination 2011-2020 was initiated to stop locally-acquired malaria in Peninsular Malaysia by 2015 and East Malaysia by 2020. To date, reports on the development of resistance towards artemisinin in Southeast Asian countries have initiated a 'Global Plan for Artemisinin Resistance Containment' by the World Health Organization (WHO) Global Malaria Program in 2011 to prevent the emergence and spread of artemisinin resistance. According to the public health perspective, the goal of treatment of malaria is to reduce transmission of the infection to others by reducing the infectious population and the spread of resistance to antimalarial medicines. The development of a multi-therapeutic length greater than three carbon atoms were less potent than chloroquine [8]. Besides conjugating 4-aminoquinolines with established antiplasmodial targets such as cyclic peroxides, newer antiplasmodial agents based on various scaffold, such as imipramine and pyrimidine and isatin derivatives, were introduced to help with the drug resistance.
Most of the published studies on the design and synthesis of antimalarial hybrids involved different pharmacophore components such as endoperoxide-quinoline-based hybrids, endoperoxide-chalcone-based hybrids, and trioxaquine-quinoline based hybrids [7,8].
whereas the isatin group inhibits P. falciparum cysteine proteases. The length and nature of the linker may exert a profound influence on the antimalarial effect of the conjugates. In this study, the 4-amino groups will be linked with the ethyl chain (two carbon atoms) that may increase activity against chloroquine-resistant strains. Hybrids with a linker chain length greater than three carbon atoms were less potent than chloroquine [8]. Besides conjugating 4-aminoquinolines with established antiplasmodial targets such as cyclic peroxides, newer antiplasmodial agents based on various scaffold, such as imipramine and pyrimidine and isatin derivatives, were introduced to help with the drug resistance. Most of the published studies on the design and synthesis of antimalarial hybrids involved different pharmacophore components such as endoperoxide-quinoline-based hybrids, endoperoxide-chalcone-based hybrids, and trioxaquine-quinoline based hybrids [7,8].
Nevertheless, to date, no attempt has been made in conjugating 4-aminoquinoline scaffold and pyrano [2,3-c]pyrazole as molecular hybrids for antimalarial agents. In this study, we would like to functionalize the -NH of ethyl linker with pyrano[2,3-c]pyrazole scaffold and study the effect on antimalarial activity. In such a case, functionalization at the -NH of the ethyl linker chain with a heterocyclic group may increase the lipophilicity of the molecule. Introducing this heterocyclic component at this alkyl side chain is vital for antimalarial activity as they help the hybrid drug accumulate in the parasite cell by becoming protonated. The prediction of good bioavailability was carried out using an in silico approach (ADMET/PK profile). Drugs with less than ten rotatable bonds and topological polar surface area less than 140 Å 2 are more likely to show good bioavailability. All hybrid compounds will be subjected to in vitro antimalarial assays against P. falciparum 3D7 and K1 cultures, whereas cytotoxicity assay will be carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. In addition, molecular docking analysis on P. falciparum lactate dehydrogenase will be conducted to study a potential molecular target of the hybrids ( Figure 2).

Chemistry: Reaction and Mechanism
A hybrid scaffold (4) was initially synthesized via deprotonation of the acidic proton

Chemistry: Reaction and Mechanism
A hybrid scaffold (4) was initially synthesized via deprotonation of the acidic proton of the pyranopyrazole (1) by the methoxide base. Subsequently, nucleophilic attack of the generated pyrazolium anion to the 4-(bromoethylamino)-7-chloroquinoline (3) via SN2 reaction furnished 4-aminoquinoline-pyrano [2,3-c]pyrazole hybrid (4), as depicted in Fig  According to the natural population analysis (NPA), the highest positively charged atom of the pyrano[2,3-c]pyrazole derivative is C3, sandwiched between two According to the natural population analysis (NPA), the highest positively charged atom of the pyrano[2,3-c]pyrazole derivative is C3, sandwiched between two electronegative oxygen atoms ( Figure 4). The next two highly positive atoms are C6 and C7, which are also located adjacent to heteroatoms of the pyranopyrazole moiety. Subsequently, the three most positively charged hydrogen atoms are associated with the N2 and N3 atoms, i.e., H6 (+0.449) > H8 (+0.441) > H7 (+0.429). This result suggests that H6 is most likely to attract a nucleophile; therefore, it corroborates the anticipated deprotonation of the secondary amine proton [24]. Consequently, the available lone pair of electrons of N2 undergoes a nucleophilic attack on the aminoquinoline to obtain the molecular hybrids 4a-e. Furthermore, the thermodynamic quantity, namely, the standard enthalpy (∆ r H θ ) and Gibbs free energy (∆ r G θ ) of the reaction, were obtained via DFT calculation. The measured enthalpy change involved synthesizing the hybrids from the deprotonated 1d, and aminoquinoline was (∆ r H θ = −555.0 kJ mol −1 . What is noteworthy is that this exothermic process is also exergonic with the ∆ r G θ = −518.6 kJ mol −1 , whereby ∆ r H θ > ∆ r G θ and implies that the entropy change, ∆ r S θ < 0. Therefore, the spontaneity of the reaction depends on the temperature and is spontaneous at lower temperat.

Antimalarial (P. falciparum) and Cytotoxic (Vero Cells) Activities
The antimalarial activities of the series of hybrid molecules were evaluated against chloroquine-sensitive P. falciparum 3D7 and chloroquine-resistant P. falciparum K1. The results in EC 50 values (µM) against the 3D7 and K1 strains are presented in Table 1. The thresholds of antimalarial activities were set as follows: EC 50 < 1 µM as potent, 2-20 µM as active, 21-100 µM as moderately active, 101-200 as weakly active, and EC 50 > 201 as inactive [25,26]. Data from antimalarial assessment showed that the hybrid molecules 4a, 4b, 4c, and 4e exhibited potent inhibitory effect against the 3D7 (chloroquine-sensitive) strain with EC 50 values of 0.19 ± 0.07 µM, 0.0130 ± 0.0002 µM, 0.113 ± 0.002 µM, and 0.026 ± 0.009 µM, respectively. Besides the profound effects against the chloroquine-sensitive strain, the hybrid molecules of 4a, 4b, and 4d also possessed potent inhibition on the chloroquineresistant strain (K1) with EC 50 values of 0.25 ± 0.03 µM, 0.02 ± 0.01 µM, and 0.30 ± 0.01 µM, respectively. The antimalarial effects of 4c and 4d against K1 and 3D7 were determined to be active, as observed by the EC 50 values in ranges of 2-20 µM. The EC 50 value of 4b against 3D7 was about ten times more than chloroquine, while the EC 50 value of 4b against K1 was 100 times more than artemisinin.
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 18 electronegative oxygen atoms ( Figure 4). The next two highly positive atoms are C6 and C7, which are also located adjacent to heteroatoms of the pyranopyrazole moiety. Subsequently, the three most positively charged hydrogen atoms are associated with the N2 and N3 atoms, i.e., H6 (+0.449) > H8 (+0.441) > H7 (+0.429). This result suggests that H6 is most likely to attract a nucleophile; therefore, it corroborates the anticipated deprotonation of the secondary amine proton [24]. Consequently, the available lone pair of electrons of N2 undergoes a nucleophilic attack on the aminoquinoline to obtain the molecular hybrids 4a-e. Furthermore, the thermodynamic quantity, namely, the standard enthalpy (∆rH θ ) and Gibbs free energy (∆rG θ ) of the reaction, were obtained via DFT calculation. The measured enthalpy change involved synthesizing the hybrids from the deprotonated 1d, and aminoquinoline was (∆rH θ = −555.0 kJ mol −1 . What is noteworthy is that this exothermic process is also exergonic with the ∆rG θ = −518.6 kJ mol −1 , whereby ∆rH θ > ∆rG θ and implies that the entropy change, ∆rS θ < 0. Therefore, the spontaneity of the reaction depends on the temperature and is spontaneous at lower temperat.

Antimalarial (P. falciparum) and Cytotoxic (Vero Cells) Activities
The antimalarial activities of the series of hybrid molecules were evaluated against chloroquine-sensitive P. falciparum 3D7 and chloroquine-resistant P. falciparum K1. The results in EC50 values (μM) against the 3D7 and K1 strains are presented in Table 1. The thresholds of antimalarial activities were set as follows: EC50 < 1 μM as potent, 2-20 μM as active, 21-100 μM as moderately active, 101-200 as weakly active, and EC50 >201 as inactive [25,26]. Data from antimalarial assessment showed that the hybrid molecules 4a, 4b, 4c, and 4e exhibited potent inhibitory effect against the 3D7 (chloroquine-sensitive) strain with EC50 values of 0.19 ± 0.07 μM, 0.0130 ± 0.0002 μM, 0.113 ± 0.002 μM, and 0.026 ± 0.009 μM, respectively. Besides the profound effects against the chloroquine-sensitive strain, the hybrid molecules of 4a, 4b, and 4d also possessed potent inhibition on the chloroquine-resistant strain (K1) with EC50 values of 0.25 ± 0.03 μM, 0.02 ± 0.01 μM, and 0.30 ± 0.01 μM, respectively. The antimalarial effects of 4c and 4d against K1 and 3D7 were determined to be active, as observed by the EC50 values in ranges of 2-20 μM. The EC50 value of 4b against 3D7 was about ten times more than chloroquine, while the EC50 value of 4b against K1 was 100 times more than artemisinin.  The results of the MTT assay against the Vero mammalian cell line revealed that the hybrid molecules 4a, 4b, 4c, and 4d exhibited low to moderate cytotoxic activities, with CC 50 ranging from 17 to 103 µM. Based on the cytotoxic threshold from Burger and Fiebig, pure compounds are classified as highly toxic to normal mammalian cells when the IC 50 value obtained is less than 10 µM or 4 µg/mL [27,28]. To understand the efficacy and potential of the hybrid molecules as safer and selective drugs, we calculated the selectivity indexes from EC 50 values of antimalarial and CC 50 values of cytotoxic tests. The calculation of SI values for the inhibition on 3D7 strain revealed that the hybrid molecules (4a, 4b, 4c, and 4e) that exhibited potent antimalarial effects have higher SI values that range from 355-1354. The high SI value showed that the antimalarial efficacy of these hybrid molecules is higher than the cytotoxic effect as an indication of its selective inhibitory properties toward the parasite. The calculation of SI values for the inhibition on the K1 strain showed that the hybrid molecules 4a, 4b, and 4e exhibited high SI values (ranges of SI = 287-880). The high SI values demonstrated by these hybrids are parallel to the potent inhibitory effects on the chloroquine-resistant strain. All hybrid molecules have high selective inhibition depending on the strain of parasites. Furthermore, the low resistance index values shown by the hybrid molecules 4a, 4b, and 4d indicate that these molecules' antimalarial effects against the K1 and 3D7 strains were comparable for both parasite strains.
4b has the most potent parasite inhibitory effects from all tested hybrid molecules, showing nanomolar range potency against both sensitive and resistant P. falciparum strains with high SI and low RI values. The combination of both 4-aminoquinoline and pyrano [2,3c]pyrazole has increased the potency of 4b against the K1 and 3D7 strains. We suggest that both quinolines and pyrazole pharmacophores are responsible for the antimalarial effects shown by 4b. As of now, this is the first report on the antimalarial properties of the hybrid 4-aminoquinoline-pyrano[2,3-c]pyrazole derivatives against P. falciparum 3D7 and KI. Previous studies revealed that hybrid quinoline-pyrimidine showed significant antimalarial activities in the nanomolar range [29]. Smit and N'Da [15] reported novel 4-aminoquinolinyl-chalcone amides with EC 50 values of 0.04-0.5 µM against chloroquinesensitive 3D7 strain comparable to the EC 50 reported in this study. Pyrazole derivatives have also been tested against P. falciparum, exerting potent antimalarial activity [20]. Furthermore, Insuasty et al. [21] have evaluated pyrazolines and pyrazolo [3,4-b] [1,4]diazepines derivatives against Plasmodium, and the derivatives showed strong antimalarial activities.

Molecular Docking Analysis
Because P. falciparum highly depends on glycolysis for energy production, we hypothesize that hybrid molecules 4a-e exerted the antimalarial effects by inhibiting the activity of Plasmodium falciparum lactate dehydrogenase (PfLDH). It is known that there are two crucial ligand binding sites in PfLDH. The first is the enzyme active site, while the second is the cofactor binding site (Figure 5a). To check the docking efficiency, the co-crystallized ligands, 3,5-dihydroxy-2-naphthoic acid and chloroquine were extracted from their respective protein crystal structures (PDB IDs: 1CET and 1U5A) and re-docked back to their putative binding sites. The docking results showed that the RMSD. between the top-ranked pose and the crystal structure was within the acceptable range (<2 Å) (Figure 5b).
The docking results of compounds 4a-e revealed that they prefer to bind to the active site over the cofactor binding site (data not shown here) of PfLDH based on their predicted binding energies and visual inspection. Compound 4b recorded the highest binding energy out of the five compounds. This finding suggested that PfLDH is a plausible molecular target of the compound. The compound also exerted the highest inhibitory effect on both P. falciparum strains (Table 1). In general, two distinct binding conformations of compound 4b were observed based on the results clustering. The first binding conformation showed that the pyrano[2,3-c]pyrazole ring of compound 4b positioned itself in the hydrophobic groove in the active site by forming multiple van der Waals contacts with the adjacent residues, as listed in Table 2. One of the interesting observations is the ability of the carboxylate ester group to form hydrogen bonding interactions with the guanidinium side chain of Arg171 and His195. These interactions are pretty similar to the co-crystallized ligand, 3,5-dihydroxy-2-naphthoic acid. It would be interesting to see how the activity would be affected if a free carboxylic group replaces the ester. Furthermore, it is known that Arg171 is highly conserved in the active site. Apart from that, the quinoline ring lies in the hydrophobic groove made up of Gly29-Ile31 and Thr97-Gly99. The nitrogen atom in the quinoline ring forms a hydrogen bond with the hydroxyl side chain of Thr101. Lastly, the cyano group interacts with the carboxamide side-chain of Asn102 via a hydrogen bond (Figure 5c,d).   ofile of Compounds 4a-e mmarizes the ADMET properties for compounds 4a-e. In general, all comed a poor level of human intestinal absorption and low aqueous solubility. olved by hydrolyzing the ester group to a carboxylic acid. In terms of proding properties capability, all hybrid compounds are predicted to be nonmportantly, the in silico prediction revealed that compounds 4a-e are genxic. In future studies, we will be focusing on removing the toxicophores or with safer substituents or groups.

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second conformation, the pyrano[2,3-c]pyrazole ring occupies the center of the hile the quinoline ring extends out of the binding region into the solvent area. negative cyano group and the nitrogen atom of the pyrazole ring form hydrowith the Arg171 and Asn102 residues via hydrogen bonding interactions. Not y, the quinoline ring also participates in ᴨ-alkyl interaction with Ala236. On the , the ethylbenzene ring forms ᴨ-ᴨ stacking with Trp102 (Figure 5e,f). Based on interactions and energies displayed by the docking results, it is likely that 4b prefers the former.
Profile of Compounds 4a-e summarizes the ADMET properties for compounds 4a-e. In general, all comibited a poor level of human intestinal absorption and low aqueous solubility. be solved by hydrolyzing the ester group to a carboxylic acid. In terms of probinding properties capability, all hybrid compounds are predicted to be nonre importantly, the in silico prediction revealed that compounds 4a-e are gentotoxic. In future studies, we will be focusing on removing the toxicophores or em with safer substituents or groups.

ure-Activity Relationship
studies on the 4-aminoquinoline pharmacophore show that 7-chloro and 4ups are required for π-π interactions and associations with heme to increase the inhibitory activity [10]. Similarly, the quinoline moiety is essential and needed interaction; brown, salt bridge, attractive charge interaction.
In the second conformation, the pyrano[2,3-c]pyrazole ring occupies the center of the active site, while the quinoline ring extends out of the binding region into the solvent area. The electronegative cyano group and the nitrogen atom of the pyrazole ring form hydrogen bonds with the Arg171 and Asn102 residues via hydrogen bonding interactions. Not surprisingly, the quinoline ring also participates in In the second conformation, the pyrano[2,3-c]pyrazole ring occupies the center of the active site, while the quinoline ring extends out of the binding region into the solvent area.
The electronegative cyano group and the nitrogen atom of the pyrazole ring form hydrogen bonds with the Arg171 and Asn102 residues via hydrogen bonding interactions. Not surprisingly, the quinoline ring also participates in ᴨ-alkyl interaction with Ala236. On the other hand, the ethylbenzene ring forms ᴨ-ᴨ stacking with Trp102 (Figure 5e,f). Based on the binding interactions and energies displayed by the docking results, it is likely that compound 4b prefers the former. Table 3 summarizes the ADMET properties for compounds 4a-e. In general, all compounds exhibited a poor level of human intestinal absorption and low aqueous solubility. This could be solved by hydrolyzing the ester group to a carboxylic acid. In terms of protein plasma binding properties capability, all hybrid compounds are predicted to be nonbinders. More importantly, the in silico prediction revealed that compounds 4a-e are generally hepatotoxic. In future studies, we will be focusing on removing the toxicophores or replacing them with safer substituents or groups.  The electronegative cyano group and the nitrogen atom of the pyrazole ring form hydrogen bonds with the Arg171 and Asn102 residues via hydrogen bonding interactions. Not surprisingly, the quinoline ring also participates in ᴨ-alkyl interaction with Ala236. On the other hand, the ethylbenzene ring forms ᴨ-ᴨ stacking with Trp102 (Figure 5e,f). Based on the binding interactions and energies displayed by the docking results, it is likely that compound 4b prefers the former. Table 3 summarizes the ADMET properties for compounds 4a-e. In general, all compounds exhibited a poor level of human intestinal absorption and low aqueous solubility. This could be solved by hydrolyzing the ester group to a carboxylic acid. In terms of protein plasma binding properties capability, all hybrid compounds are predicted to be nonbinders. More importantly, the in silico prediction revealed that compounds 4a-e are generally hepatotoxic. In future studies, we will be focusing on removing the toxicophores or replacing them with safer substituents or groups.  In the second conformation, the pyrano[2,3-c]pyrazole ring occupies the center of the active site, while the quinoline ring extends out of the binding region into the solvent area.

ADMET Profile of Compounds 4a-e
The electronegative cyano group and the nitrogen atom of the pyrazole ring form hydrogen bonds with the Arg171 and Asn102 residues via hydrogen bonding interactions. Not surprisingly, the quinoline ring also participates in ᴨ-alkyl interaction with Ala236. On the other hand, the ethylbenzene ring forms ᴨ-ᴨ stacking with Trp102 (Figure 5e,f). Based on the binding interactions and energies displayed by the docking results, it is likely that compound 4b prefers the former. Table 3 summarizes the ADMET properties for compounds 4a-e. In general, all compounds exhibited a poor level of human intestinal absorption and low aqueous solubility.

ADMET Profile of Compounds 4a-e
This could be solved by hydrolyzing the ester group to a carboxylic acid. In terms of protein plasma binding properties capability, all hybrid compounds are predicted to be nonbinders. More importantly, the in silico prediction revealed that compounds 4a-e are generally hepatotoxic. In future studies, we will be focusing on removing the toxicophores or replacing them with safer substituents or groups.  Level of aqueous solubility prediction; 0 (extremely low), 1 (very low), 2 (low), 3 (good), 4 (optimal), 5 (too soluble), 6 (warning: molecules with one or more unknown Alog P calculation); f Very high penetrants (log BBP ≥7); g Level blood brain barrier penetration prediction; 0 (very high penetrate), 1 (high), 2 (medium), 3 (low), 4 (undefined); h Prediction plasma-protein binding (0: i j stacking with Trp102 (Figure 5e,f). Based on the binding interactions and energies displayed by the docking results, it is likely that compound 4b prefers the former. Table 3 summarizes the ADMET properties for compounds 4a-e. In general, all compounds exhibited a poor level of human intestinal absorption and low aqueous solubility. This could be solved by hydrolyzing the ester group to a carboxylic acid. In terms of protein plasma binding properties capability, all hybrid compounds are predicted to be non-binders. More importantly, the in silico prediction revealed that compounds 4a-e are generally hepatotoxic. In future studies, we will be focusing on removing the toxicophores or replacing them with safer substituents or groups.

Structure-Activity Relationship
SAR studies on the 4-aminoquinoline pharmacophore show that 7-chloro and 4-amino groups are required for π-π interactions and associations with heme to increase the βhematin inhibitory activity [10]. Similarly, the quinoline moiety is essential and needed to accumulate the compound in the digestive vacuole. This relationship is the reason why the 4-aminoquinoline scaffold is still relevant in malaria drug discovery. Shorter linkers consisting of two carbon chains are optimal to enhance activity against CQ-resistant and CQ-sensitive strains of the parasite. Pyrano [2,3-c]pyrazole core significantly increases the potency of the synthesized compounds.

Synthesis of 4-(Ethanolamino)-7-chloroquine (2)
A mixture of 4,7-dichloroquinoline (0.99 g, 5 mmol) and ethanolamine (1.5 mL, 25 mmol) was mixed in a three-neck round-bottom flask. The reaction was refluxed for 180 min, and the temperature was monitored using a thermometer. The reaction flask was cooled with an ice-water bath and stirring was continued until the precipitation of a colorless solid. Yield To a solution of hydrogen bromide, 33% (5 mL, 30 mmol), 4-(ethanolamine)-7-chloroquinoline (1.1135 g, 5 mmol) was added dropwise, together with sulfuric acid (0.5 mL, 10 mmol). The resulting mixture was refluxed for 60 min, and the progress of the reaction was monitored using TLC. The reaction mixture was left to cool at room temperature and poured into ice water. The solution was added with ammonia hydroxide until pH 9 to yield a solid colorless precipitate. The solid was filtered and heated in a solution of toluene and activated charcoal. The mixture was filtered and refrigerated at 4 • C to yield a colorless solid. Yield

Cytotoxicity Assay
The cytotoxicity of the hybrid 4-aminoquinoline-pyrano[2,3-c]pyrazole derivatives was measured using the MTT assay described by Mossmann [34]. Before performing the assay, 100 µL of Vero cells (ATCC CCL-81) in culture medium supplemented with 10% fetal bovine serum (FBS) were seeded in a 96-well flat-bottom microtiter plate at 2 × 10 4 cells/well and allowed to adhere for 48 h at 37 • C in a CO 2 incubator. After 48 h of incubation, the confluent cells were treated with the test compounds (10 to 0.01 µg/mL) to each well accordingly. The cell suspension without test compounds served as the positive control for cell growth. After 48 h incubation, MTT reagent in phosphate buffer saline (5 mg/mL) was added to each well, followed by 3 h incubation at 37 • C. The medium was then aspirated, and formazan crystals were solubilized by adding 100 µL of DMSO per well for 10 min at 37 • C in a CO 2 incubator. The solution was measured at 540 nm with a microplate reader. Three independent tests were conducted to determine the cytotoxicity of each test compound. The percentage of growth inhibition and the 50% cytotoxic concentration (CC 50 ) was estimated from a non-linear regression using Graphpad Prism. Cytotoxicity was expressed as the concentration of the test compounds inhibiting cell growth by 50% (CC 50 ).

Selectivity and Resistance Indexes Calculation
Selectivity indexes of the hybrid 4-aminoquinoline-pyrano[2,3-c]pyrazole derivatives were determined by a ratio of values from CC 50 of cytotoxicity and EC 50 of antimalarial activities (MTT/pLDH). Test pure compounds with SI values greater than 100 (SI > 100) are considered strong and selective antimalarial agents [35]. Resistance index is the ratio of the EC 50 for the resistant versus the sensitive strain (K1/3D7). The RI is a quantitative measurement of the antimalarial activity against the chloroquine-resistance strain relative to that against the chloroquine-sensitive strain. A lower RI index value indicates higher potency of a compound against chloroquine-resistant malarial parasites [36].

DFT Calculations
All compounds were modeled (GaussView 5.0) and optimized using density functional theory (DFT) with the B3LYP/6-31++G theoretical model and the Gaussian09 program [37]. The vibrational frequency calculations were established and validated to ensure that only positive eigenvalues were obtained.

Preparation and Optimization of Ligands
A series of hybrid compounds were constructed using Marvin Sketch version 1.5.6. These ligand structures were converted into 3D formats and geometrically optimized with OPLS3 using Ligand Preparation Wizard in Schrodinger Small Molecule Drug Discovery Suite 2017-1.

Preparation and Optimization of Protein Receptors and Molecular Docking
The X-ray crystal structures of the protein-ligand complex for PfLDH (PDB IDs: 1CET and 1U5A) were retrieved from the Protein Database Bank (PDB) (http://www.rcsb.org, accessed on 12 May 2020). All water molecules of the crystal structures were removed, while hydrogen atoms were added in their standard geometry, adjusting the bond orders and formal charges. The ligand-binding site was identified based on the position of the inhibitorbinding site, as reported in the literature. The in-house CDOCKER protocol was used for the docking analysis of active compounds to the binding site. Different conformations were performed for each ligand through high-temperature molecular dynamics. The ligands were heated (700 K in 2000 steps) followed by cooling (300 K), and they were then subjected to refinement by grid-based (GRID 1) simulated annealing and full force minimization after random rotation. The ligand was allowed to flex while the receptor was held rigid during the refinement process. The generated ligand conformations were clustered according to their binding interactions. For validation of the docking protocol, the co-crystallized ligands were re-docked to the binding site.

Pharmacological Properties of the Hybrid Compounds
The pharmacological properties of the synthesized hybrid compounds were measured using the standard ADMET descriptors protocol implemented in Discovery Studio 3.1. The parameters involved in the analysis were plasma-protein binding (PPB), human intestinal absorption (HIA), atom-based log P (Alog P98), aqueous solubility, polar surface area (PSA), blood-brain barrier (BBB) penetration, cytochrome P450 2D6 (CYP2D6) enzyme inhibition, and hepatotoxicity.

Conclusions
In summary, we developed a simple reaction protocol for a new series of antimalarial hybrid compounds containing pyrano [2,3-c]pyrazole-aminoquinoline pharmacophoric units. From the antimalarial assessment, compound 4b is considered the most potent, with high a selectivity index against both the 3D7 and K1 strains and a low resistance index value. The compound also exerted high-affinity binding on PfLDH, which suggests a potential molecular target in malarial infection. The findings from pharmacophoric hybridization in this study are crucial as an alternative therapy to combat drug resistance in malarial infection. Further, in vivo, antimalarial activity needs to be carried out, and we envisage the expansion of a hybrid antimalarial medicine series based on these derivatives.