Synthesis of novel ketene dithioacetals via one pot reaction: Molecular modelling in-silico Admet studies and antimicrobial activity

Article history: Received October 1, 2020 Received in revised form November 30, 2020 Accepted January 4, 2021 Available online January 4, 2021 A simple and efficient method for the synthesis of fifteen novel ketene dithioacetals (2-(6amino5-cyano-4-aryl-4H-1,3-dithiin-2-ylidene) malononitrile) via a one-pot three-component reaction of activated methylene group malononitrile with carbon disulfide in the presence of arylidene malononitriles were reported. The effects of LiOH.H2O as a base at different concentrations have been investigated and can provide products in good yields at 40-50oC temperature (54-89%). All the synthesized ketene dithioacetals compounds (MCB1-MCB15) were checked for favorable pharmacokinetic parameters along with toxicities which are based on drug-likeness explained by Lipinski’s rule of five by Med chem designer software correlated with that of pkCSM online tool. Explorations of synthesized ketene dithioacetals compounds for the antimicrobial study were found to be effective towards Staphylococcus aureus (MCB5 and MCB13) with a zone of inhibition at 26mm and 22mm which is compared to that of standard ciprofloxacin (26mm). This made our study to explore the inhibition mechanism with the help of molecular docking studies with possible binding energies (-6.4 to -8.9 kJ/mol) by pyrx 0.8 software to represent a good prediction of interactions between the ligand and protein (2XCT). Further evaluation of druggability and ADMET predictions compounds MCB5 and MCB13 were found to be effective. Based on the in-vitro and in-silico studies a series of ketene dithioacetals compounds may be helpful for further studying SAR and designing more potent antimicrobials. © 2021 Growing Science Ltd. All rights reserved.

A simple and efficient method for the synthesis of fifteen novel ketene dithioacetals (2-(6-amino5-cyano-4-aryl-4H-1,3-dithiin-2-ylidene) malononitrile) via a one-pot three-component reaction of activated methylene group malononitrile with carbon disulfide in the presence of arylidene malononitriles were reported. The effects of LiOH.H2O as a base at different concentrations have been investigated and can provide products in good yields at 40-50ºC temperature (54-89%). All the synthesized ketene dithioacetals compounds (MCB1-MCB15) were checked for favorable pharmacokinetic parameters along with toxicities which are based on drug-likeness explained by Lipinski's rule of five by Med chem designer software correlated with that of pkCSM online tool. Explorations of synthesized ketene dithioacetals compounds for the antimicrobial study were found to be effective towards Staphylococcus aureus (MCB5 and MCB13) with a zone of inhibition at 26mm and 22mm which is compared to that of standard ciprofloxacin (26mm). This made our study to explore the inhibition mechanism with the help of molecular docking studies with possible binding energies (-6.4 to -8.9 kJ/mol) by pyrx 0.8 software to represent a good prediction of interactions between the ligand and protein (2XCT). Further evaluation of druggability and ADMET predictions compounds MCB5 and MCB13 were found to be effective. Based on the in-vitro and in-silico studies a series of ketene dithioacetals compounds may be helpful for further studying SAR and designing more potent antimicrobials.

Introduction
Development of organic molecules from one pot reaction has made considerable attention because no necessity for purification of organic intermediates and usage of solvents has been slashed. Environment friendly methodologies or processes are developing day to day for widely used organic compounds that are readily obtainable reagents which is one of the major challenges for chemists in organic synthesis. Attention has been made for organic synthesis which is green, mild and simple procedures. In this context, lithium hydroxide monohydrate (LiOH.H2O) is used as a green basic catalyst for the organic synthesis.
In, heterocyclic chemistry, organosulfur compounds which contain sulfur in their cyclic structure is well known for their pharmacological activities. 1 Sulfur containing drugs such as sulfonamides, thioethers, sulfones, penicillin's and cephalosporin's moieties which are well studied both on synthesis and application during the past decades but, little study was carried out so far for synthesis and pharmacological evaluation of dithianes.

2.1.Reaction conditions and mechanism
The optimization of reaction conditions, including the concentration of base, the reaction temperature and the equivalents of the starting materials was investigated. Previously reactions were carried out by several bases like NaOH, K2CO3, and triethylamine [16][17] . So, an attempt has been made to use hydroxide like LiOH.H2O which has significant covalent character due to the small size of the Li + ion. LiOH. H2O is used in knoevenegal condensation reaction as in ethanol/water medium as solvent by elimination of water molecule by withdrawal of α-acidic proton from active methylene where adjacent two electron withdrawing groups are present to form the benzylidene derivatives. [18][19] Making the point of view in abstraction a model reaction was carried out by activated methylene compound like malononitrile 1 (1mmol) was reacted with carbon disulphide 2 (1mmol) for 0.5h and followed with addition of benzylidene malononitrile derivatives 3 (1mmol) in ethanol( Table 1, Entries 1-8) which was progressed under room temperature as a basic experiment, we found that the reaction was not accelerated ( Table 1, Entry 1). Then the reaction was progressed by increasing the temperature to 40-50°C trace amount of product was observed ( Table 1, Entry 2). This indicates that temperature affects the reaction so reflux was carried out but only trace amount was observed ( Table 1, Entry 3). This made us to increase the concentration of starting material in larger amounts by increasing the concentration of carbon disulphide to 2mmol ( Table 1, Entries 4-5) and further to 3mmol ( Table 1, Entries 6-7) and progressed the reactions under room temperature and in other by maintaining the temperature at 40-50°C. We found that the reaction was accelerated by an increase in carbon disulphide concentration along with maintaining the temperature at 40-50°C 30% yield at 2mmol and 85% yield was obtained at 3mmol. Increasing the concentration beyond 3mmol did not improve the yield more than 85% ( Table 1, Entry 8). So by taking into account of 1:3:1 ratio a series of experiments were performed when malononitrile 1 of 1mmol was reacted with carbon disulphide 2 of 3mmol and arylidene malononitrile 3 of 1mmol in ethanol at 40-50°C to obtain optimum results of optimized compound 2-(6-amino-5-cyano-4-aryl-4H-1,3-dithiin-2-ylidene) malononitrile.
To the best of our knowledge, all the synthesized compounds depicted in Table 2, were characterized by infrared spectroscopy, 1 H-NMR analysis. For instance, the 1 H NMR spectrum methine proton was observed as singlet at δ = 1.256 ppm, a D2O-exchangeable signal at δ =7.779 ppm which is attributed to the NH2 group, and a multiplet at δ = 7.527-7.658 ppm and 7.897-7.926 ppm for the aromatic protons of the phenyl ring were also observed.  A proposed mechanism for the formation of 2-(6-amino-5-cyano-4-phenyl-4H-1,3-dithiin-2ylidene)malononitrile 4 (MCB1) is illustrated in scheme 1. The chemical reaction involves with active methylene compound like malononitrile 1 with carbon disulfide 2 in presence of strong base like LiOH.H2O gave the intermediates lithium 2,2-dicyanoethanedithioate 5 and lithium 2,2-dicyano-1mercaptoethenethiolate 6. Nucleophilic attack of intermediates 5 and 6 by sulfur anion on the electron deficient double bond of benzylidene malononitrile 3 could lead to formation of intermediate 7 via michael addition reaction. Then, hydrogen transfer takes place intramolecularly which produces intermediate 8, which further undergoes nucleophilic attack by second sulfur anion with the cyanide carbon to form a cyclic structure affording the intermediate 9. The final steps involve a 1,3 hydrogenshift which leads to formation of final product 4 (MCB1). Scheme 1. The proposed mechanism for the synthesis of 2-(6-amino-5-cyano-4-phenyl-4H-1,3dithiin-2-ylidene) malononitrile

Antimicrobial Evaluation
The fifteen newly synthesized ketene dithioacetals derivatives MCB1-MCB15 were tested for their antimicrobial activity against two Gram-positive bacterial strains Bacillus Subtilis and Staphylococcus aureus and Gram-negative bacterial strain Escherichia Coli along with their fungal strain Candida Albicans with their resulting inhibition zones were measured in mm diameter, in Table 3. Among the tested compounds, MCB5 and MCB13 were found to be most active against Gram-positive bacterial strains Bacillus Subtilis (24 and 22 mm respectively) and Staphylococcus aureus (26 and 22 mm respectively) and MCB13 were found to be active for fungal strain Candida Albicans (22mm respectively). Among the different substituent's attached to the ketene dithioacetals which exerted potential antimicrobial activity against gram-positive and gram-negative bacteria are the electron withdrawing groups to an aromatic ring like fluoro, chloro and nitro groups attached in ortho positions compared to that of an electron donating groups.

Molecular docking studies
Considering the outcome of antimicrobial activity, it was thought worthy to execute computational methods like molecular docking studies by substantiating the in-vivo results with in-silico studies. The enzyme DNA Gyrase is selected for the present study which is one of the topoisomerases II classes involved in winding and unwinding of DNA during the process of replication and transcription. Gyrase enzyme is present in both prokaryotic and eukaryotic cell but the enzymes are not entirely similar in structure or sequence, which affects the topological state of DNA, and have different affinities for different molecules hence it is considered as an important intracellular target for antibacterial agents as a representative model for other DNA topoisomerases. 20 Further investigation was made based on fact to study for binding mode, docking score energy and the predictable type of interactions between the new chemical entities and binding site of the target protien of DNA Gyrase enzyme. The fifteen synthesized compounds showed good binding energy towards the target protien 2XCT ranging from -6.4 to -8.9 kcal/mol, among them MCB5 (-7.5 kcal/mol), MCB11 (-8.9 kcal/mol), MCB12 (-7.6 kcal/mol), and MCB13 (-7.4 kcal/mol), exhibited good DNA gyrase binding affinity along with standard ciprofloxacin. Moreover, the changes in binding energies coincided well with the experimental data of antimicrobial studies found that MCB5 and MCB13 found to be effective. With the observations from the biological data and the molecular docking results, might suggest that the antibacterial activities of these compounds are seemingly derived from the interaction between the compounds and the enzyme DNA Gyrase. However, there was not always a correlation among the in vitro and in-silico study outcome. Although compounds MCB11 and MCB12 showed good binding affinity for 2XCT with value -8.9 kcal/mol and -7.6 kcal/mol they showed low antimicrobial activity against all the microorganisms. The compounds exhibit good poses that are represented in the form of hydrogen bonds, binding affinities and amino acids depicted in Table 4. All the docked molecules were subjected to 2D and 3D protein-ligand interaction analysis. Fig. 1 represents the further extrapolation of binding conformation of the docked molecules.

Druggability Validations through Lipinski's Filter
To upsurge efficacy in relation to the biological activity and fastidiousness while settling the conformity of the drug-like physicochemical properties termed by Lipinski's rule which is a characteristic approach in drug discovery process. 21 Lipinski's rule of five (Ro5) support as a widespread and considerable rule of thumb in the consideration of drug-likeness and also in the determination of the molecule's potential as an orally active drug or as a candidate drug with respect to its pharmacological or biological activity. Lipinski's rule covers some of the important molecular properties of a drug's pharmacokinetics like ADME, but still it fails to anticipate if a compound is pharmacologically active. A conformity of the drug candidate's is purely based on Rule of 5 that offers more promise to the candidate drug for ultimate success during clinical trials and therefore the higher chance of reaching the market. 22 As depicted in Table 5, the predicted Lipinski's filters almost positive for all proposed 2-(6-amino-5-cyano-4-aryl-4H-1,3-dithiin-2-ylidene) malononitrile compounds. None of the synthesized compounds (MCB1-MCB15) showed negative values. These most potent compounds were found to have substituted with electron withdrawing groups like chlorine, fluorine and nitro present in the ortho position of the aromatic ring.

ADMET and Pharmacokinetics Predictions
For accelerating opportunities in the discovery of new targets and to find out lead compounds with predicted biological activity depends on in-silico drug likeness prediction along with further ADME/Tox tools. For effective metabolism and action, a good drug candidate is absorbed in specified time and well distributed throughout the organism/system. Table 5 depicts the drug likeness properties of the synthesized new ketene dithioacetals as test compounds predicted using Med Chem designer. From Table 5, it can be seen that the partition coefficient (S+log P) value ranges from -0.817 to 5.017 and TPSA (Topological Polar Surface Area), ranges from 97.39 to 143.21, indicates the surface belonging to polar atoms in the compound where increased TPSA is associated with diminished membrane permeability and compounds with higher TPSA were better substrates for p-glycoprotein which is responsible for drug efflux from cell. In order to ensure the druggability potential of the synthesized ketene dithioacetals compounds (MCB1-MCB15), ADMET properties along with the other pharmacokinetic properties were predicted using pkCSM online tool. The established outcome was substituted and found to have correlated well with ADMET values that were predicted by using Med chem designer software. Studies predicted that all the synthesized ketene dithioacetals compounds will be well absorbed and intestinal absorption was found to be 84.073 to 100.
Skin permeability is a vital property for enhancing drug effectiveness in the development of the transdermal drug delivery systems. A drug barely penetrates the skin if log Kp is more than -2.5 cm/h. 23 From Table 6 it can be seen that the skin permeability (Kp) of synthesized ketene dithioacetals derivatives ranges from -2.559 to -3.233 cm/h (< -2.5). Therefore, it can be predicted that all the synthesized derivatives have good skin penetration.
The Caco-2 (human epithelial colorectal adenocarcinoma cells) monolayer of cells is widely used as an in vitro model of the human intestinal mucosa to predict the intestinal permeability for the absorption of orally administered drugs and investigate drug efflux by measuring the log of the evident permeability coefficient (log Papp; log cm/s). A compound is considered to have a high Caco-2 permeability if it has Papp > 8 x 10 -6 cm/s. High Caco-2 permeability is translated into predicted log Papp values > 0.90cm/s. 23 From Table 6 it can be seen that the Caco-2 permeability value (log Papp) of the synthesized ketene dithioacetals compounds ranges from -0.365 to 0.923 cm/s, in which MCB1, MCB5, and MCB13 have high Caco-2 permeability and other predicted that the compounds have a moderate to low Caco-2 permeability.
The volume of distribution (VD) is a parameter representing the calculated value of an individual drug that will be circulated at an equal level of blood plasma. This model is established from the estimation of the steady-state volume of distribution (VDss) which is represented as log L/kg. According to Pires et al 23 VDss >2.81 L/kg (log VDss > 0.45) is categorized as high whereas VDss <0.71 L/kg (log VDss < -0.15) is categorized as low. From Table 6 it can be seen that VDss values of the synthesized ketene dithioacetals ranges from 0.612 to 0.078, and one compound has a VDss value of < -0.15 i.e. MCB10, so it can be predicted that all the derivatives can be distributed evenly providing an equal level of blood plasma.
The ability of a drug to cross the Blood-Brain Barrier (BBB) is an important parameter to consider for reducing side effects and toxicities or to improve the efficacy of drugs whose pharmacological activity is within the brain. The BBB permeability is calculated in vivo as log BB, the logarithmic ratio of the brain-to-plasma drug concentration. Compounds are able to pass through the BBB promptly when log BB is higher than 0.3, but compounds with log BB smaller than -1 barely reach the brain. 23 From Table 6 it can be seen that the ranges of log BB value of synthesized ketene dithioacetals compounds range from -0.86 to -1.016, which means greater than -1, so it can be predicted that all the derivatives are able to penetrate the blood-brain barrier moderately (except MCB10). The central nervous system permeability of blood-surface area product (log PS) is a direct measurement that can be obtained from in situ brain perfusions with the drug directly injected into the carotid artery. This lacks the systemic distribution effects which may demolish brain penetration. Compounds with a log PS > -2 are considered to penetrate, while those with log PS < -3 are considered as unable to penetrate the central nervous system. 23 From Table 6 it can be seen that the log PS value of the synthesized ketene dithioacetals derivatives ranges from -1.293 to -2.999 which penetrate the central nervous system easily.
Cytochrome P450 is an important detoxification enzyme that oxidizes xenobiotics to facilitate their excretion from the body. The cytochrome P450's is responsible for the metabolism of many drugs. Inhibitors of the P450's can dramatically alter the pharmacokinetics of these drugs, so it is significant to evaluate whether a given compound is likely to be a cytochrome P450 substrate. The two main isoforms responsible for drug metabolism are cytochrome P2D6 (CYP2D6) and Cytochrome P3A4 (CYP3A4). 23 From Table 6 it can be seen that almost all synthesized ketene dithioacetals compounds do not affect or inhibit the CYP2D6 and CYP3A4 enzymes (except MCB11).
Drug clearance occurs primarily as a combination of hepatic clearance and renal clearance which is measured by the proportionality constant. To achieve steady-state concentrations depends on bioavailability and administered at the appropriate dosing rate. The higher the CLtot value of the drug, the faster the clearance process. 23 From Table 6 it can be seen that the CLtot value of synthesized ketene dithioacetals ranges from 0.177 to 0.62 ml/min/kg which predicts the rate of excretion of drugs.
Ames toxicity test is to determine the toxicity of the compound. The AMES test is used to predict the mutagenic potential of a compound using bacteria. A positive test indicates that the compound is mutagenic and therefore may act as a carcinogen. 23 From Table 6 it can be seen that except MCB2 and MCB4 all other synthesized ketene dithioacetals compounds are predicted to have mutagenic effects.
Hepatotoxicity is the most concerning safety aspect in the drug development process. The toxicity of the compounds shows at least one pathological or physiological hepatic event was considered hepatotoxic and highly related to liver disruption. 23 From Table 6 it can be seen that all the ketene dithioacetals derivatives are not hepatotoxic.

Conclusion
Based on a Michael addition reaction novel fifteen new ketene dithioacetals derivatives were designed and synthesized by the reaction of active methylene compounds, carbon disulfide, and arylidene malononitrile in the presence of lithium hydroxide monohydrate as a base by the one-pot reaction. All the synthesized compounds were screened against three bacterial strains and one fungal species. Among the synthesized compounds, MCB5 and MCB13 compounds were found to be effective against S. aureus with a zone of inhibition at 26mm and 22mm which is compared to that of standard ciprofloxacin (26mm). Further, molecular docking simulations were carried out with protein 2XCT for receptor-ligand interactions which revealed that the compound MCB5 and MCB13 have binding mode and an affinity to DNA gyrase binding site comparable to that of ciprofloxacin. Further, in-silico studies were carried out for druggability by Lipinski's rule in which none of the molecules fails. ADMET studies reveal that all compounds come under category 5 which states low acute toxic compounds.
Herein, further studies can be made to optimize and develop new antimicrobial compounds, and modifications in the structures can be made to identify more biologically potent compounds.

4.1.General
All the chemicals were purchased from Sigma Aldrich Ltd., (Germany) and Merck with the quality of analytical grade and were used without any further purification. The melting points of synthesized compounds were determined by the open capillary tube method and the results are uncorrected. The progress of the reaction was monitored by thin layer chromatography (TLC) plates, which were precoated with aluminum silica gel 60F 254 procured from Merck (Germany) using solvent system were n-hexane: Ethyl acetate (1.5:0.5) by UV absorption for visualization. Spectral data of the compounds were routinely checked by IR, 1 H-NMR spectroscopies. FTIR spectra were recorded in KBr on Bruker Alpha. 1 H-NMR spectra were recorded on Bruker Avance-III 400 MHz instrument with CDCl3 as solvent and chemical shift values are reported relative to tetramethylsilane (TMS) as an internal standard.

4.2.General procedure for the preparation of compounds (MCB1-MCB15)
A mixture of active methylene compound like malononitrile 1 (1.0 mmol), carbon disulfide 2 (3.0 mmol), and lithium hydroxide monohydrate (0.1 mmol) was stirred in 10 ml of ethanol at room temperature for 0.5 h. Arylidene malononitrile 3 (1 mmol) was added and the reaction was stirred by maintaining 40-50°C for 3-11.25 h. After completion of the reaction, determined by TLC, the reaction mixture was poured in cold water and precipitate was collected. The crude product was recrystallized from ethanol to give pure compounds 4 (MCB1-MCB15) with 54 to 89% yields.

2-(6-amino-5-cyano-4-(3-nitrophenyl)-4H-1,3-dithiin-2-ylidene)malononitrile (MCB3)
Dark   Four bacterial strains of Gram-positive (Bacillus Subtilis ATCC 6633, and Staphylococcus aureus ATCC 29213), Gram-negative (Escherichia coli ATCC 25922) and representative fungi (Candida albicans ATCC 10231) were used in our in-vitro antimicrobial activity. The antimicrobial potential of the newly synthesized ketene dithioacetals was investigated towards the tested microorganisms and expressed as the diameter of the inhibition zones according to the agar plate diffusion method. 24 100 μL of the test bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately 10 8 cells/mL for bacteria or 10 5 cells/mL for fungi. One mL of each sample (at 1 mg/mL) was added to each well (10 mm diameter holes cut in the agar gel). The plates were incubated for 24 h at 37 °C (for bacteria and yeast) and for 72 h at 27 °C (for filamentous fungi), each test was determined in triplicate. After incubation, the microorganism's growth was observed. Ciprofloxacin was used as standard antibacterial drugs while Fluconazole was used as standard antifungal drug. The resulting zone of inhibition were measured in millimeters and used as criterion for the antimicrobial activity. Solvent controls (DMSO) were included in each experiment as negative control. DMSO was used for dissolving the test compounds and showed no inhibition zones, confirming that it has no influence on growth of the tested microorganisms.

. Accession of ligand and protein
The crystal structure of DNA gyrase in complex with ciprofloxacin (PDB ID: 2XCT) 27 with resolution of 3.35A º was accessed from protein data bank (http://www.rcsb.org). For preparation of protein, water molecules were deleted, polar hydrogen's were added and kollman united atom charges were assigned. Protien energy was minimized with spdbv and saved as .pdb format. For the preparation of ligands, software such as Chemdraw 12.0 in which 2D structure was converted into 3D structure and energy minimization was carried by MM2 for each ligand. Molecular Docking was carried out by virtual screening tool by pyrx 0.8. The grid centre was set at X=14.53, Y= 36.95, Z= 93.94. The grid size was set to 64.51, 84.97, 109.22 xyz points with grid spacing of 0.5Aº by keeping all parameters in defaults for docking. The visualization was done using discovery studio visualizer 2020 for receptorligand interactions.

4.5.1.2.Pharmacokinetic Properties and toxicity prediction studies.
Drug-likeness prediction was carried out based on the Lipinski rule of five with consistent properties and satisfies the rule containing good absorption when taken orally as active pharmacological agent. It is applied to evaluate in vivo capabilities of the synthesized molecules. Any newly synthesized molecule with more than 5H-bond donors(-OH, NH), more than 10H-bond acceptors (N, O), molecular weight greater than 500 and the log P greater than 5 unsatisfies the rule of five. Med chem. Designer TM and pkCSM prediction tool was used for drug-likeness and toxicity prediction of the synthesized ligands. Med chem. Designer TM programme estimates the physicochemical properties like rule of five, octonal/water partition coefficient (S+LogP), water/octanol partition coefficient at specicific pH 7.4 (S+logD), Topological polar surface area (TPSA), molecular weight, count of nitrogen and oxygens (M_NO), etc. Meanwhile, pkCSM predictor provides information regarding absorption parameters like Human Intestinal Absorption (HIA), Oral bioavailability, Caco-2 permeability, distribution parameters like Plasma Protein Binding (PPB), Blood Brain Barrier (BBB), metabolism parameters like Cytochrome P450 (CYP450) substrate, inhibitor, excretion parameters like half time (t1/2), renal clearance and toxicity parameters like organ toxicity, genomic toxicities for drug candidates.