Design, Synthesis and Biological Investigation of Some Novel Quinazolin-4(3H)-One Tethered 1, 3, 4- Thiadiazole-Thiol Motifs as Direct Enoyl Acyl Carrier Protein Reductase Inhibitors

Aims: In this study two noteworthy pharmacophores quinazolin-4(3H)-one and 1,3,4-thiadiazole through methylene bridge were utilized to design, synthesize and characterize some novel 2-methyl quinazolin-4(3H)-one and 6-chloro-2-methyl quinazolin-4(3H)-one tethered S-substituted-1,3,4Original Research Article Deshpande et al.; JPRI, 33(47A): 617-632, 2021; Article no.JPRI.76063 618 thiadiazole-thiol structural analogs respectively as direct Mycobacterium Tuberculosis (MTB) enoyl acyl carrier protein reductase (InhA) inhibitors. Study Design: Design of structural analogs of quinazolin-4(3H)-one tethered 1,3,4-thiadiazole-thiol through methylene bridge by functional group modifications in core scaffold followed by computational studies to select promising compounds. Synthesis of some novel compounds, structural characterization and screening of biological activity of the same. Methodology: The molecular docking of designed compunds was carried out using schrodinger Glide XP into the active site of MTB InhA with protein data bank code (PDB ID: 2H7M). The interactions were evaluated based on the glide G score compared with reference standard isoniazid. Ten new compounds 7(A1-A10) were synthesized, characterized and screened for their in-vitro antitubercular activity by Microplate Almar Blue Assay (MABA) method followed by cytotoxicity evaluation of compounds 7A4 and 7A10 using Vero cell line. Results: All the designed compounds of series 7(A1-A10) had drug-like characteristics and were non-toxic to normal cells. In the molecular docking studies, compounds 7A4, 7A5, and 7A10 demonstrated strong binding affinity in the active region of MTB InhA protein and retained necessary amino acid interaction, similar to co-crystal 2H7M. Synthesized compounds 7(A1-A10) were found to have good antitubercular activity. Out of the series the compounds 7A4 and 7A10 were found to possess excellent antitubercular activity equipotent to reference standard streptomycin with minimum inhibitory concentration (MIC) value of 6.25μg/ml. The cytotoxic potential of compounds 7A4 and 7A10 showed remarkable selectivity index against Vero cell line. Conclusion: The findings of this study highlights the importance of tethering two pharmacophoric motifs in one compound to develop novel antitubercular agents that can be exploited as promising leads as direct InhA inhibitors.


INTRODUCTION
Tuberculosis is one of the most dangerous infections, claiming the lives of around 1.21 million people worldwide in the year 2019. The long duration of therapy, combined with the microorganism's resistance, has resulted in tuberculosis recurrence, particularly in multidrugresistant (MDR) and extensively drug-resistant (XDR) tuberculosis. Currently, very few drugs are effective against MDR and XDR strains of Mycobacterium tuberculosis. Nonetheless, second-line drugs have been used for many decades are contributing to a rise in emergence of drug-resistant TB. As a result, new drugs are urgently needed to reduce the length of treatment and treat drug-resistant strains. The development of compounds that can serve as enzyme inhibitors is now the focus of the quest for new entities [1][2][3][4].  The biosynthesis of mycolic acid requires several enzymes. The InhA gene encodes a NADHdependent enoyl-acyl carrier protein (ACP) reductase that is involved in fatty acid prolongation in cell wall formation. Moreover, the first-line anti-TB drug isoniazid (INH) is reported to be a potent enoyl ACP reductase inhibitor but needs direct activation by KatG, a catalaseperoxidase enzyme. The fundamental issue, which is responsible for INH resistance, is mutations in KatG.As a result, the current investigation focuses on developing a new antitubercular medication that does not require KatG activation prior to acting as a direct InhA inhibitor [5,6].
Despite the wide range of biological activities demonstrated by quinazolinone and 1,3,4thiadiazole derivatives, there is scarcity of literature reported on the development of potential antitubercular agents based on the combination of these heterocycles in a single molecule which we have attempted in present study [24,25]. In the present study, with the help of computational drug discovery tools, we planned to explore quinazolin-4(3H)-one tethered 1,3,4-thiadiazole-thiol through methylene bridge having the general structure as represented in (Fig.1). The goal of this study was to design and synthesize a library of 2-methyl-quinazolin-4(3H)one and 6-chloro-2-methyl-quinazolin-4(3H)-one tethered S-substituted-1,3,4-thiadiazole thiol compounds respectively as Mycobacterium tuberculosis enoyl acyl carrier protein reductase (MTB InhA) inhibitors. The insertion of methyl tail at position-2, chloro group at position-6 and various phenacyl bromides at position-3 on right hand side through thiadiazole thiol methylene bridge were taken in the quinazolin-4(3H)-one core to establish the early structure-activity relationship (SAR) of the same. Moreover the SAR and computational studies gave an insight of synergistic impact within the two rings to obtain the novel antitubercular agents.

MATERIALS AND METHODS
All the reagents and chemicals used were of synthetic grade and were obtained from E. Merck and Sigma-Aldrich (Mumbai, India). The open tube capillary method was used to determine the melting points of synthesized compounds, and the results are uncorrected. Hexane:ethyl acetate (1:1) was used as the mobile phase for thin-layer chromatography (TLC) to examine the purity by observing a single spot on Merck silica gel 60 F254 coated alumina plates (0.25mm) and visualization was done by iodine chamber. All the solvents used for silica gel column chromatography were distilled before use. Fourier-transform infrared spectroscopy (FT-IR) absorption spectra were recorded in KBr pellets on Shimadzu FT-IR spectrometer 8400, νmax in cm -1 . 1 H and 13 C NMR spectra were recorded on a Bruker AscendTM FT-NMR spectrometer 500 using TMS as the internal standard in DMSO-d6 solvent. All the chemical shift values were reported in ppm (δ) downfield from TMS. Data was reported as s= singlet, d= doublet, t= triplet, dd= doublet of doublets and m= multiplet. The mass spectra were recorded on Bruker Daltonik GmbH, Mass spectrometer ESI. Satisfactory analysis for CHN on EuroVector E3000 elemental analyzer was obtained for the compounds within ± 0.4 % of the theoretical values.

Lipinski's rule and ADMET prediction
Before docking pre-validation of the ligand was done by computation of molecular properties, drug-likeness and prediction of pharmacokinetic parameters (ADME). All designed compounds were subjected to ADME prediction and Lipinski's rule using Qikprop module 3.2 available in Schrodinger [26,27]. Further these compounds were subjected to in silico toxicity prediction using OSIRIS molecular property explorer to indicate the mutagenic, irritating, tumorigenic, or reproductive effects of these compounds [28].

Molecular docking studies
The molecular docking tool Glide, version 6.7, Schrodinger, LLC, New York, NY, 2015 served the purpose of molecular docking studies. The ligand structures were created in MOL 2 format with ChemDraw and then transformed to 3D conformations with Schrodinger's LigPrep module. The geometry optimization for all the molecules was carried out using OPLS-2005 (optimized potential liquid simulations) force field until the root mean square deviation (RMSD) reached the value of 1.4A 0 or less. The EPIK module, which generates ionization states in the pH range of 7 ± 2, was used to create all probable tautomers and stereoisomers.
The X-ray crystal structure of (InhA) enoyl acyl carrier protein (ACP) reductase of MTB complexed with 1-cyclohexyl-N-(3,5dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (PDB ID: 2H7M) inhibitor having a resolution of 1.62 A o , retrieved from identifier protein data bank. Before calculations, the protein was optimized for docking using the protein preparation wizard available in Schrodinger's Maestro. In the first step, the water molecules beyond 5A 0 and cofactors from the proteins were removed, hydrogen bonds were optimized and ligand present in the crystal structure was deleted. The receptor grid was generated using the co-ordinates of the X-ray ligand with the standard settings using glide. Glide energy grids were generated for each prepared protein complex. A rectangular box surrounded the X-ray ligand marking the binding site. The ligand's of lowest energy conformation were chosen and docked into the grid created by the protein structure. The best docked compounds are selected based on Glide gscore. Extra precision (XP) visualizer of Glide module was utilized to analyze the results based on the active site amino acid interactions. Glide gscore is a modified and extended version of the empirically based chemscore function [29][30][31].

In-vitro Antitubercular Activity
The in-vitro antitubercular activity of the compounds 7(A1-A10) was assessed against Mycobacterium tuberculosis H37Rv, ATCC 27294 [39] using the microplate alamar blue assay method [40]. The methodology used was nontoxic, involving the use of a thermostable substance, and revealed an excellent correlation between radiometric and proportional methods Medium evaporation in the test wells was reduced by adding 200μl of sterile deionized water in sterile 96 well plates at outer-perimeter wells during incubation. This was followed by the addition of 100μl Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI, USA) to 96 plates. Initial serial dilutions for test compounds of series were prepared and added to the plate. The final drug concentrations in the range of 0.4-100μg/ml in DMSO were used for further testing. Plates were incubated at 37 o C beforehand and sealed with parafilm. On the seventh day of incubation, a freshly prepared 1:1 mixture of alamar blue dye solution (25μl) and 10% tween 80 (12.5ml) were added to the wells, and the plates were reincubated for 24 h. Bacterial growth was interpreted by observing the colour change from blue to pink. The reference drugs selected for comparison were pyrazinamide, ciprofloxacin, streptomycin and isoniazid. The lowest drug concentration that prevented a colour change from blue to pink was identified as the MIC [41,42].

In-vitro Cytotoxicity by MTT Assay
The selected compounds from series 7(A1-A10) were further assayed in-vitro for cytotoxic activity against Vero (kidney normal cell line of African monkey) colorimetrically [43]. This assay is based on the capacity of the mitochondrial succinate dehydrogenase enzyme to convert soluble 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into an insoluble, colored formazan product. The intensity was estimated by a spectrophotometer [44,45]. This type of MTT reduction can only be seen in metabolically active cells.

RESULTS AND DISCUSSION
The study was aimed to synthesize new compounds by tethering quinazolin-4(3H)-one and 1,3,4-thiadiazole-thiol as one entity with improved antitubercular potential against direct InhA inhibitors. The computational study was carried out first for determination of drug-like molecules, all the designed compounds presented values within an acceptable range for the properties analyzed indicating their potential as drug-like molecules (Table 1) based on Lipinski's rule of 5. This rule in general is considered to influence good membrane permeability and oral bioavailability associated with filters namely, molecular weight ≤ 500, QlogP ≤ 5, number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤ 5. As the cutoffs for each of the four parameters were all close to 5 this simple mnemonic was called the Lipinski's rule of 5. Drug candidate that comply with the Lipinski rule have less failure rate during clinical trial. Drug like physicochemical parameters of compounds affect drug pharmacokinetics (ADME) in human body.
An in silico evaluation of ADME profile of synthesized derivatives is necessary to understand pharmacological behavior of the lead. The ADME properties of the majority of designed compounds were within acceptable limits. However, it can be noted that PSA values predicted a poor bioavailability for the reference standards, and a good bioavailability for the compounds selected for synthesis. It was observed that the low hydrogen bonding ability of designed compounds could enhance the biological membrane permeating capacity. The toxicity profile evaluation of designed compounds was performed employing the Osiris Molecular Property explorer software, none of the compounds were found to be toxic. A low in silico toxicity risk profile, ADME predictions, and Lipinski's parameters made these compounds 7(A1-A10) a better leads for development of safe and efficient antitubercular agents.
Docking analysis of the crystal structure of 2H7M revealed that the reference inhibitor in the MTB InhA active site formed a hydrogen-bonding network with Lys165. The predicted bound conformation of the lead molecule 6a,b within the active site of MTB InhA protein showed that the oxygen atom of the carbonyl group of quinazolinone formed a hydrogen-bonding network with the side chain of Tyr158. It served as the key feature to explain the orientation of the compounds within the active site. The docking results of compounds 7(A1-A10) showed the highest Glide gscore in kcal/mol and were more tightly bound to the active site of MTB InhA than other designed compounds. All these compounds showed pi-stacking interactions but were weak as compared to observed hydrogen bonding interactions. The amino acid residues like Lys-165, Ile-21, Asn-247, Thr-28, Trp-32 and Phe-304 were involved in these interactions. These observations provided a good basis for the estimation of the inhibitor activity and indicated high binding affinity of these selected compounds to MTB InhA than the lead molecule ( Table 2). Based on the outcomes of computational studies, the new series of compounds 7(A1-A10) were synthesized in good yield by condensing 6a,b with various phenacyl bromides in absolute ethyl alcohol. The reaction time was greatly shortened, and the reflux temperature was lowered to get compounds 7(A1-A10). All the synthesized compounds 7(A1-A10) were accurately analyzed by spectroscopic techniques, and results were in full accordance with the proposed structures.

6-chloro-2-methyl-3-[(5-{[2-(4methylphenyl)-2-oxoethyl] sulfanyl}-1, 3, 4-thiadiazol -2-yl) methyl]-3,4dihydroquinazolin-4-one (7A3)
Yield: 51%, m.p.          Out of the ten only two compounds 7A4 and 7A10 were further examined for cytotoxicity in the African green monkey kidney normal cell line (Vero) at various concentrations. The % inhibitory cytotoxicity data along with IC 50 µg/ml of respective compounds is given in (Table 3) and graphically represented in (Fig. 3) whose columns reflect the viable cells in each treatment, DMSO denotes an experimental control. The most active MTB InhA inhibitors 7A4 and 7A10 showed no toxicity with % cell inhibition of 42.62 and 47.10 at IC 50 of 50μg/ml and 30 μg/ml respectively (Fig. 4). By comparing in-vitro antitubercular and cytotoxicity data it was found that most of the active compounds 7A4 and 7A10 were non-toxic (≥50% inhibition). Especially the compounds 7A4 and 7A10 showed an outstanding selectivity index of 8 and 4.8 respectively. The results confirmed that variation in selectivity over the Vero cell line was an outcome of variations in the substitution pattern.   Fig.1) with methyl tail at position-2, side-chain at position-3 with electron-withdrawing and electron-donating substituents on phenyl ring have confirmed that the presence and position of the substituents have a great impact on their antitubercular activity. The compounds 7A4 (R=H, R 1 = 4-F) and 7A5 (R=Cl, R 1 = 4-F) due to the presence of halogens, and 7A10 with methoxy moiety (R=H, R 1 = 4-OCH 3 ) exhibited the highest antitubercular activity and were found to be important fragments for hydrogen bonding that could be formed at the receptor site. Thus, low lipophilicity, less steric hindrance, electronwithdrawing groups, and electron-donating groups are summarized to be preferential for design and synthesis of novel quinazolin-4(3H)one tethered S-substituted-1,3,4-thiadiazole-thiol derivatives as antitubercular leads. According to the Glide gscore and experimentally determined MIC values against MTB InhA, compound 7A5 (MIC 12.5 µg/ml) exhibited good binding affinities with a Glide gscore (kcal/mol) of -8.533. The most active compounds 7A4 (Fig. 5) and 7A10 (Fig. 6) showed Glide gscore of -8.302 and -8.676 respectively (Table 2) both with MIC, 6.25 µg/ml have displayed hydrogen bonding interactions, one with the side chain of Lys165 and with similar orientation to that of crystal ligand respectively. This was considered to be critical for bioactivity, making these compounds more potent.

CONCLUSION
The present investigation concludes that the newly synthesized compounds 7 (A1-A10) showed superior inhibitions as direct MTB InhA inhibitors. Compounds 7A4 and 7A10 emerged as highly active agents against Mycobacterium tuberculosis H37Rv and inhibited drug-sensitive MTB with MIC value of 6.25µg/ml, which was equipotent to the reference standard streptomycin. Compounds 7A4 and 7A10 were found to be non-cytotoxic with superior potency against the Vero cell line. The ADME profiling, virtual toxicity and Lipinski's parameters of compounds were found to be as effective as the reference standards. The molecular docking results were in good agreement with the outcomes of biological activity. Therefore, taking into account the results presented herein, it can be inferred that these novel MTB InhA inhibitors are promising candidates for the effective treatment of TB.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.