NATURAL ISOTHIOCYANATE ANTI-MALARIA: MOLECULAR DOCKING, PHYSICOCHEMICAL, ADME, TOXICITY AND SYNTHETIC ACCESSIBILITY STUDY OF EUGENOL AND CINNAMALDEHYDE

Objective : This study aims to evaluate novel compounds of isothiocyanate (ITC) based on eugenol and cinnamaldehyde derivatives as the drug candidate of Plasmodium falciparum anti-malaria using in silico method, physicochemical, pharmacokinetics, toxicity, and synthetic accessibility prediction. This present study also describes molecular docking and pharmacoinformatics of natural ITC in Moringa oleifera leaves. Methods : A series of novel ITC compounds (3, 5, and 6) were designed and analyzed with a series of natural ITC compounds (7, 8, 9, 10) for P. falciparum anti-malaria. This research is descriptive qualitative and uses the reverse molecular docking method, proving the biological activity of compounds theoretically using software and database information. Results : Molecular docking study showed that compound 6 exhibits binding affinity (-5.3 Kcal/mol) on Van der Waals interaction with the residual active site (His159, Cys25) of cysteine protease. All designed ITC compounds are obeyed the Lipinski and Veber Rule, have a well-brain penetrant character and have a medium risk for mutagenic, tumorigenic, and reproductive prediction. They are also in the simple rate of synthetic accessibility (SA) estimation. In regards to natural ITCs, they all have better assay characteristics except the SA. Conclusion : Molecular docking, physicochemical, pharmacokinetic, and toxicity studies show that methyl eugenol isothiocyanate and cinnamaldehyde isothiocyanate are promising anti-malaria compounds. Substituents of hydroxy, acetate and tetrahydropyran groups in the building block ring are suggested for better in silico profiles enhancement.


INTRODUCTION
Plasmodium falciparum malaria is a mosquito infection disease in erythrocytes that demands public attention [1][2][3]. Malaria long-term treatment using multidrug such as chloroquine (C) and sulfadoxinepyrimethamine causes drug resistance [4][5][6][7][8], increased morbidity, mortality, and health care costs [9]. The discovery of new, better bioactivity and lower toxicities drugs is focused on the abundant and renewable natural building blocks.
Molecular drug discovery is also focused on the functional group (FG) that has better bioactivity prediction. In some areas, people have used Moringa oleifera leaves for malaria treatment. It is known as a pearl of local wisdom on malaria therapy. Rhamnosyloxy benzyl isothiocyanates, the natural isothiocyanate found in M. oleifera leaves, are predicted responsible FG of anti-plasmodial [27]. Natural ITCs are known to have several bioactivities [28][29][30][31][32][33][34][35][36][37][38]. The double bond FG has partially positive and negative properties, and it is possible to carry out additional reactions into other FG. Eugenol and cinnamaldehyde have a double bond and aldehyde FG that building blocks potentially for ITC compounds. The bioactivity of eugenol-ITC and cinnamaldehyde-ITC derivatives as an anti-malaria is not known.
In the past, drug discovery was made with these categories such as time-consuming, trial and error process, high cost, and petroleumbased. Molecular docking and other bioinformatics techniques speed up the process of drug finding with better efficacy [39][40][41]. This recent study attempted to design ITC compounds from eugenol and cinnamaldehyde, identify its falciparum anti-malaria activities using in silico, and compare its prediction with chloroquine and natural ITC from Moringa oleifera leaves. This study will also briefly discuss the synthetic accessibility of ITC compounds.

Preparation compound and receptor
The common structure of compounds (1, 2, 4, and C) was collected in fig. 1. Novel designed-ITC (3, 5, 6) compounds ( fig. 1) and ITC derived from Moringa oleifera leaves (7, 8, 9, and 10) were constructed and converted to 3D by MarvinSketch ( fig. 2). Receptor PDB ID 1YVB was obtained from the RCSB protein databank. All compounds were prepared into ligands by adding charge and hydrogen using the Chimera 1.13.1 program with the AMBERff12SB standard and Gesteiger method. The receptor was prepared and purified from water, native ligands, and its optimization was carried out by adding charge AM1-BCC and hydrogen. Molecular docking was done by PyRx and the grid dimension set as a center at x = 83.731069686; y =-36.3469508554, z =-92.6668557437 then size point of x, y and z as 25 Ǻ, 25 Ǻ, and 25 Ǻ respectively. Interaction of ligand with receptor was determined using affinity binding (Kcal/mol). The 3D complex structures were visualized through Discovery Studio Visualizer 2019 client.

Physicochemical, pharmacokinetics, and bioactivities analysis
Physicochemical, pharmacokinetics, and toxicities of ligands were examined respectively by Swiss ADME, Molinspiration, and Osiris.

Preparation compound and receptor for molecular docking
Docking system validation was done by redocking the native ligand of the 1YVB chain A. The precision of the docking process was determined by RMSD (Root Mean Standard Deviation) value less than 2.0. All ligands have good validation at 0.0 . The molecular docking principle is based on ligand binding interactions with active amino acids in the receptors via the presence of hydrogen bonds, Van der Waals, and electrostatic interactions. Ligand and amino acids bond distance will affect the affinity energy (ΔG) or complex stability between ligand and receptor [42]. The smaller the bond distance, the better the value of the ligand-receptor complex affinity. As starting material, eugenol has the same affinity with the cinnamaldehyde complex (table 1). However, OH-eugenol is predicted to inhibit the ITC group's entry, and methylation is required to form methyl eugenol (ME). It was showed that ME increased the stability complex and has a better affinity (AG =-5.2 Kcal/mol) than eugenol. Methyl eugenol isothiocyanate (ME-ITC) or 4-(2-isothiocyanatopropyl)-1,2-dimethoxybenzene has affinity -4.9 Kcal/mol. Inserting the ITC group into cinnamaldehyde resulted in 2 types of ITC prediction, namely 3-isothiocyanato-3-phenylpropanal (ligand 5) and 2-isothiocyanato-3-phenylpropanal (ligand 6). The affinity score of ligand 6 is better than its derivatives. However, both ME-ITC (ligand 3) and cinnamaldehyde-ITCs (ligand 5 and 6) could not achieve chloroquine affinity and natural ITC from Moringa oleifera leaves (ligands 7, 8. 9, and 10). In general, ligands 8 and 10 have better affinity than chloroquine. It is in accordance with the local tradition of using Moringa oleifera leaves as a traditional malaria medicine. Ligand 8 has the best energy affinity compared to chloroquine and other ligands (table 1).  Result docking analysis is should also notice the interaction between ligand with active site residue. His159 and Cys25 residues are the active sites in the surface layer of cysteine protease responsible for the proliferation of falciparum erythrocyte [43]. Ligand 6 and C make Van der Waals link at His159 and Cys25 with its affinities -5.3 Kcal/mol and -6.3 kcal/mol, respectively. Ligand 8, with the lowest affinity (-6.6 Kcal/mol), has hydrogen bond interaction between His159 and the hydroxy group of tetrahydropyran. The Cys25 forms. Van der Waals bonds around the sulfur double bond ( fig. 3). Hydrogen bonds in the complex's residue ligand are much stronger than the Van der Waals; they stabilize the complex bonds and reduce affinity energy.

Physicochemical, pharmacokinetics, and bioactivities of compound
The physicochemical of drug candidates was measured by its properties covered by the Lipinski Rule of Five (RO5) and Veber Rule [44][45][46]. The n-octanol/water partition coefficient (Log P) is a parameter that determines the hydrophobicity of a compound. Drug compounds' hydrophilic/lipophilic properties affect drug absorption, drug-receptor interactions, molecular metabolism, and toxicity [47]. Topological Polar Surface Area (TPSA) is a predictor of drug transport properties such as intestinal absorption and penetration of the blood-brain barrier. TPSA deals with hydrogen bonds in compounds. The number of rotatable bonds (RB) measures the flexibility of the compound related to drug absorption and bioavailability. All the ligands obeyed Lipinski and Veber Rule (table 2).
Drug candidates should have a pharmacokinetics character such as ADMET (Absorption, Distribution, Metabolism, Elimination, and Toxicological) as an integral part of screening to get the promising drug candidates [48]. ADMET is covered in drug-likeness (properties and bioactivities). The bioactivity of a drug candidate can be determined by calculating the G-Protein-Coupled Receptor (GPCR) ligand score, ion channel modulator, nuclear receptor ligand, a kinase inhibitor, protease inhibitor, enzyme inhibitor [47]. Ligands' biological activity scores of more than 0.00 are recognized as active, and less than-0.50 are inactive [49]. The potential bioactivity of building block compounds (ligands 1, 2, and 4) and ITC designed ligands 3, 5, and 6 are moderately active. Furthermore, the native ITC in M. oleifera has a variation score around active and moderate (table 3). All ligands are equipped with gastrointestinal absorption and brain access assays via the Brain or IntestinaL EstimateD (Boiled-Egg) permeation method for predicting lipophilicity and polarity of the drug candidate. The white egg illustrates the physicochemical space of compounds with the highest absorbed probability by the gastrointestinal tract (GI absorption), and the yellow region (yolk) is the highest permeate space to the brain (BBB permeant). Well-absorbed compound (blue point), wellbrain penetrant compound symbolized as pink point [50]. Analysis of all ligands shows that the building blocks (molecule 1, 2, and 4) have a well-brain penetrant character and are distributed in egg yolk (table 4). It also occurred to the ITCdesigned ligands and chloroquine (molecule 11). However, natural ITC (molecules 7, 8, 9, and 10) are stacked at the egg white border and assumed to be absorbed by the gastrointestinal tract ( fig. 4). Ligands 1 and 2 were observed in high-risk mutagenic, tumorigenic, and irritant categories, but ligand 4 was only tumorigenic toxic. Ligands 3 and 6 dominantly have medium-risk toxicity properties, and it is a promising drug for anti-malaria. Chloroquine has a high risk of mutagenicity and irritant. Natural ITC from M. oleifera leaves showed mutagenic, tumorigenic, and reproductive effects at medium risk (table 5).

Fig. 4: Boiled-Egg model of all ligands: white area = GI absorption, yolk area = BBB
Synthetic Accessibility (SA) score is an estimation parameter of a drug designed to be synthesized on a laboratory scale. It was measured based on complexity, starting materials, or retrosynthetic-based [51]. SA score of ITC-designed ligands is between 2.14-2.51 for easily synthesized and natural-ITCs in 4.22-4.38 for moderately categories (table 4). The 8 compound has an OR group in the para position where R is a tetrahydropyran molecule containing an acetate group. The therapeutic activity of ITC is also influenced by aromatic and aliphatic side chains [28]. In this regard, in the design of eugenol-ITC and cinnamaldehyde-ITC for malaria drug purposes, the substitution of tetrahydropyran, hydroxy, or acetate groups in the building block rings should be recommended to determine better anti-malaria potential.   [52].
Synthesis of ITC in several natural compounds using amine groups has also been successfully formed with raw materials noscapine, bile acids, amino acids, and several aromatic compounds performed by transforming the-NH2 group into an ITC group [53]. The natural product that has a triple bond group, -8,15-diisocyano-11(20)-amphilectene, has been isolated from the Caribbean sponge Svenzea flava and used as a building block to form isothiocyanate derivatives [54]. Various degrees of commercial amine (primary, secondary or tertiary), cyclic, aromatic, and all amine positions (ortho, meta, or para) have been used to synthesize the ITC group by one-pot method and water-based at room temperature [55]. Separation and purification of ITC compounds are generally done by column chromatography or preparative Thin Layer Chromatography. Its identification is implemented mainly by Infrared, Liquid Chromatography-Mass Spectrometry (LCMS), Gas Chromatography-Mass Spectroscopy (GCMS), and High-Performance Liquid Chromatography-Mass Spectrometry [55][56][57] because these compounds are unstable.

CONCLUSION
Eugenol and cinnamaldehyde availability allow them to be the raw materials and building block for isothiocyanate compounds. In silico studies show that methyl eugenol isothiocyanate and cinnamaldehyde isothiocyanate are promising antimalarial compounds in terms of substituents variation such as natural isothiocyanates. This research is an invaluable essential reference for the synthesis of isothiocyanates as anti-malaria.

ACKNOWLEDGEMENT
This article is part of the doctoral thesis. Sincerely grateful to the Indonesian Ministry of Research and Technology/National Agency for Research and Innovation and the Indonesian Ministry of Education and Culture. Authors thank Universitas Brawijaya was supporting through Hibah Guru Besar 2020-2021.

AUTHORS CONTRIBUTIONS
All authors have contributed equally.