Pyrrolyl thiadiazoles as Mycobacterium tuberculosis inhibitors and their in silico analyses

Abstract

A novel series of pyrrolyl thiadiazoles was synthesized and tested for antimycobacterial activity against the Mycobacterium tuberculosis H₃₇Rv strain, using the microplate Alamar blue assay method. Molecular docking and in vitro minimum inhibitory concentration assays revealed that these molecules can be primarily screened for ENR inhibition, using the score values and H-bond interactions with amino acid residues Tyr158, Met98, and cofactor NAD⁺, which are the key interactions. For most of the molecules, hydrophobic interaction is the key factor affecting their antitubercular activity. The activity of -OCH₃, -NO₂, -F, pyridine, and sulfonamide substituted derivatives was better than that of -CH₃, -NH₂, -Cl, and -Br substituted derivatives, as per experimental and docking studies. Molecular modeling studies are in agreement with their biological evaluations.

Introduction

Among the many infectious diseases, tuberculosis (TB) is one of the major ones caused by Mycobacterium tuberculosis.¹ Since 1993, the World Health Organization has identified TB as a global health emergency, with more than nine million new cases arising every year and an annual death toll of around 1.8 million people worldwide. The treatment of TB is a major problem, in view of the emergence of monodrug- and multidrug-resistant strains of M. tuberculosis.² Thus, there is an increasing need to develop novel anti-TB agents for the effective treatment of TB with reduced toxicity and enhanced activity against multidrug-resistance (MDR) strains for a short duration of therapy.

There are two discrete enzymes in the biosynthesis of fatty acids in bacteria; namely, fatty acid synthase (FAS) I and II. Type II fatty acid elongation system (FAS-II) of bacteria, in which reactions are catalyzed by different enzymes and each is encoded by a discrete gene, constitutes an attractive target for inhibition, as these enzymes differ significantly from type I FAS (FAS-I) in mammalians, in which enzymatic activities are encoded in one or two multifunctional polypeptides. M. tuberculosis possesses both FAS-I and FAS-II systems, of which FAS-I is responsible for bimodal distribution of products,^3,4 centered on C16 and C24–C26, but the FAS-II system prefers C16 as the starting substrate, which can extend⁵ up to C56, indicating that mycobacterial FAS-II uses the products of FAS-I as the primers to extend fatty acyl chain lengths even further. The longer chain products of FAS-II are the precursors of mycolic acids, and both the systems provide precursors for biosynthesis of mycolic acids, which contain very long chain fatty acids that are the prominent and essential components of the mycobacterial cell wall.^6,7

The nicotinamide adenine dinucleotide-dependent enoyl-acyl carrier protein reductase encoded by Mycobacterium gene inhA has been validated as the primary molecular target of the frontline antitubercular drug, isoniazid.⁸ Recent studies have demonstrated that InhA is also the target for the second-line antitubercular drug ethionamide.⁹ As a prodrug, isoniazid must be activated by the mycobacterial catalase-peroxidase KatG into its acyl radical active form. However, some inhibitors can target InhA directly, without a requirement for activation similar to pyrazole derivatives, indole-5-amides,¹⁰ alkyl diphenyl ethers,¹¹ and pyrrolidine carboxamides.¹² During our study, it was found that the InhA inhibitor, that is, 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (pyrrolidine carboxamide or 641), contains three hydrophobic moieties, cyclohexyl, oxopyrrolidine, and 3,5-dichlorophenyl, which can be mapped by new designed molecules containing pyrrole, 1,3,4-thiadiazole, and substituted phenyl (see details in Figure S1). These findings prompted us to select InhA as the target for our newly designed molecules. Hence, by considering the nicotinamide adenine dinucleotide-dependent enoyl-acyl carrier protein reductase as the target receptor, we have performed molecular docking studies and screening for the supportive coordination between in silico studies and the in vitro results.

Pyrroles conform to an important class of heterocycles having a wide range of biological activities,¹³ such as antitubercular, antiinflammatory, antiviral, and antiproliferative activities. The versatile and eminent biological profiles of 1,3,4-thiadiazoles and their analogs are well known.¹⁴ Because of the presence of a toxophoric N=C-S moiety, 1,3,4-thiadiazoles exhibit a broad spectrum of biological activities. Recent literature suggests that 1,3,4-thiadiazole derivatives exhibit antibacterial and antitubercular activities.^15,16 On the basis these facts, and supported by the literature findings,^17–22 we propose synthesizing and testing the biological activities of a new type of pyrrolyl thiadiazole derivatives from 2-amino-5-substituted phenyl-1,3,4-thiadiazoles, with a hope that these new molecules would exhibit enhanced biological activity because of the presence of pharmacologically active heterocyclic and aromatic substituents. In our previous study, we have synthesized various heterocycles as antiinfective agents (Figure 1), where we determined that pyrrole and thiadiazole derivatives are good antitubercular and antimicrobial agents.^23–27 Keeping this in mind, we report here new prototype hybrid molecules (Figure 2) by combining pyrrole and thiadiazole moieties and investigating their in vitro antitubercular activity, as well as Surflex-Docking analyses. The present work, therefore reports on the structure and ligand-based drug design and discovery processes. The crystallographic 3D structural information of the biomolecular targets offers tremendous opportunities for establishing such novel drug design strategies to accelerate the drug discovery process. To accomplish this, docking simulation was performed to predict the binding orientation of small molecules to protein targets to predict their affinity and activity.²⁸

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Figure 1 Reported molecules; pyrrole connected to heterocycles (oxadiazole, triazole, pyrazolo[3,4-b]quinolin-1-yl, naphtha[2,1-b]furan-2-yl) through phenyl bridge. Abbreviations: CHEMBL, chemical database of bioactive molecules with drug-like properties; MIC, minimum inhibitory concentration.

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Figure 2 Designed molecules; pyrrole connected to thiadiazole directly.

Results and discussion

Synthesis and spectral studies

Compounds 4a–4i, 5a–5i, 8a, and 8b were synthesized as per Figures 3 and 4. In Figure 3, the 2-amino-5-(4-substituted phenyl)-1,3,4-thiadiazoles (3a–3i) were synthesized by condensation of aromatic acids (1a–i) with thiosemicarbazide (2) in the presence of a dehydration agent (POCl₃). The 2-amino-5-sulfonamido-1,3,4-thiadiazole (7) was obtained by the hydrolysis of acetazolamide (6) in concentrated HCl (Figure 4). The Paal-Knorr pyrrole synthesis involving the reaction of 1,4-dicarbonyl (2,5-hexanedione) or 2,5-dimethoxy tetrahydrofuran with amines is among the most classical methods of heterocyclic pyrrole ring synthesis. Pyrrole (4a–4i, 8a) and 2,5-dimethyl pyrrole (5a–5i, 8b) rings have been constructed by using the free amino group at the second position of 1,3,4-thiadiazoles (3a–3i, 7) in the presence of dry glacial acetic acid.

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Figure 3 Synthetic route for 2-substituted pyrrole-1H-5-(4-substituted phenyl)-1,3,4-thiadiazoles.

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Figure 4 Synthetic route for 2-substituted pyrrole-1H-5-sulfonamido-1,3,4-thiadiazoles. Abbreviation: Conc., concentration.

Structures of the compounds were assigned by the spectral and analytical data; namely, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and mass spectroscopy, as reported in the experimental section. In the ¹H NMR spectrum of 4c, four protons of pyrrole moiety resonate as two doublet of doublets at δ 7.43 and δ 6.40, whereas four protons of phenyl moiety resonate as two doublets at δ 7.93 and δ 7.56. The mass spectrum of 4c showed the molecular ion peak at a mass-to-charge ratio (m/z) of 261.78 that confirmed its molecular weight.

The disappearance of the NH₂ stretching band in the FTIR spectrum of 5c confirmed the formation of dimethyl pyrrole. The ¹H NMR spectrum of 5c showed a singlet at δ 2.25 that was accounted for two methyl groups. The C₃ and C₄ protons of pyrrole ring appeared as a singlet at δ 5.94. The four protons of phenyl moiety resonate as two doublets at δ 8.02 and δ 7.60. Electron impact mass spectra showed accurate molecular ion peaks at m/z 226.97, 257.49, 261.78 (263.78), 272.31 (273.31), 306.09 (308.09), 241.17, 242.28, 245.37, 228.11, 255.01, 285.11, 289.05 (291.05), 300.23 (301.23), 332.56 (334.56), 269.03, 270.17, 273.01, 256.13, 229.07, and 258.19 for compounds 4a–4i, 5a–5i, and 8a–8b, respectively.

Antitubercular activity

The results of antitubercular activities (expressed in minimum inhibitory concentration [MIC], which was converted to pMIC = −logMIC, calculated by Sybyl-X 2.0 software) of the compounds against selected M. tuberculosis H₃₇Rv are illustrated in Table 1. The compounds (4a–4i, 5a–5i, and 8a–8b) showed the activities against mycobacteria, with the MIC values ranging from 12.5 to 100 μg/mL (pMIC 4.903–4.000). Compounds 4b, 5b, and 5d inhibited mycobacterial growth effectively compared with others in the series, showing MIC values of 12.5 μg/mL (pMIC 4.903), followed by compounds 4d, 4h, 4i, 5h, 5i, 8a, and 8b at 25 μg/mL (pMIC 4.602). The compounds with -OCH₃, -NO₂, -F, pyridine, and sulphonamide substituents have shown better antitubercular activities than others. In these compounds, all the functional groups are H-bond acceptors, which might be responsible for achieving a better inhibitory action on M. tuberculosis.

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Table 1 Antimycobacterial activity (MIC values in μg/mL and pMIC values are −logMIC) for pyrrolyl thiadiazole derivatives (4a–4i, 5a–5i, and 8a–8b) Note: *Values calculated by Sybyl-X 2.0 software. Abbreviation: MIC, minimum inhibitory concentration.

Protein quality and active site identification

Ramachandran plots, which give an indication of the quality of the model, as well as hydrophobicity plots (Figures 5 and 6A and B), were obtained at the end of the minimization. In Figure 5, red color violation 2 means PRO in the generously allowed region and non-GLY in the disallowed region, and a magenta color violation 1 means PRO in the allowed region and non-GLY in the generously allowed region, but a blue color violation 0 means PRO in the favored region, non-GLY in the favored or allowed region, and GLY in any region. Almost 98% of the residues were found in the most favored region, but 2% were found in the additional allowed regions; 0% were found in the disallowed regions. As shown in Figure 6A, analyzing the shape of the plot gives information about partial structure of the protein. For instance, if a stretch of about 20 amino acids shows positive for hydrophobicity, these amino acids may be part of alpha-helix spanning across a lipid bilayer, which is composed of hydrophobic fatty acids. On the converse, amino acids with high hydrophilicity indicate these residues are in contact with the solvent or water and are, therefore, likely to reside on the outer surface of the protein (scores are given in Table S1).

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Figure 5 Ramachandran plot for analysis of residue shows the PHI (Φ) and PSI (ψ) torsion angles for all residues in structure; Φ values on X-axis and ψ values on Y-axis. Notes: Red color indicates Proline (PRO) in generously allowed region and non-Glycine (GLY) in disallowed region; magenta color indicates PRO in allowed region and non-GLY in generously allowed region; blue color indicates PRO in favored region, non-GLY in favored or allowed region and GLY in any region.

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Figure 6 (A) Hydropathy index averaged over a moving window of eleven residues. (B) Hydrophobicity at each residue.

The active site at InhA was identified using SiteID or Protomol generation suite. The SiteID method generated many possible spheres of radius of water inside the protein molecules that were in search of the largest space or cluster available, which could be identified as an active site (Figure 7). A flood-fill algorithm, similar to the one implemented in CAVITY, was used²⁹ for each solvent molecule in the pocket, and all atoms in the protein lying within the specified distance (default =3 Å) were considered. This generated four sites: site 1, yellow, Gly96, Met103, Gly104, Pro156, Ala157, Tyr158, Met161, Met199, Ile202, Ile215, Leu218, and NAD500; site 2, green, Ile15, Ile16, NAD500, Leu38, Thr39, Gly40, Phe41, Ile47, Leu60, and Leu63; site 3, cyan, Phe149, Asp150, Met155, Ala190, Ala191, Trp222, and Asp261; and site 4, white, Ser19, His24, and Ala235. In the case of the Protomol method, Protomols can be produced by one of three routes: automatic: Surflex-Dock finds the largest cavity in the receptor protein; ligand-based: by a ligand in the same coordinate space as the receptor; residue-based: by specified residues in the receptor. Thus, a Protomol can be generated automatically or be defined on the basis of a cognate ligand or known active site. The sites generated were compared with the active site of the template and we found that site 1 (yellow region) is highly conserved. In this article, a Protomol generated by the ligand-based approach was identical to SiteID site 1. Hence, we used the ligand-based generated Protomol for further study (Figure 8A). However, Figure 8B gives a clear picture of the obtained active site.

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Figure 7 The possible binding-sites (spacefill models) of ENR enzyme from Mycobacterium tuberculosis; site 1, yellow; site 2, green; site 3, cyan; site 4, white.

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Figure 8 (A) Protomol generated (yellow-colored spacefill model) at ENR enzyme, using a ligand-based approach and (B) active site at ENR enzyme (Tyr158 and NAD+ spacefill models in blue; magenta, Connolly surface; ribbon colored by secondary structure).

Surflex-Dock was applied to studying the molecular docking, which uses an empirical scoring function and a patented search engine to dock the ligands into the protein's binding site.^30,31 In the docking procedure, ten binding poses per ligand were obtained, and the binding pose with the highest total score was considered for ligand–receptor interactions. Optimization of the results was carried out by allowing the protein movement. The strengths of the individual scoring functions were combined to produce a consensus that is more robust and accurate than any single function for evaluating the ligand–receptor interactions. Thus, CScore (consensus score) was used for ranking the affinity of ligands bound to the active site of a receptor. CScore integrates a number of popular scoring functions and provides several functions: D_Score, charge and van der Waals interactions between the protein and the ligand; PMF (potential of mean force) Score; G_Score, showing hydrogen bonding, complex (ligand–protein), and internal (ligand–ligand) energies; and Chem_Score, points for hydrogen bonding, lipophilic contact, and rotational entropy, along with an intercept term. CScore was automatically computed from the six scores (0, 1, 2, 3, 4, and 5); the best CScore is 5. Structures with scores of 3 or 4 merit further consideration. Structures with a CScore of 0 are consistently considered bad by all scoring functions and should be dropped. Additional scores were observed as we allowed the protein movement.

Surflex-Docking

Docking simulation plays a key role in structural molecular biology and computer-assisted drug designing. Binding models for receptors and ligands via the lowest energy pathway may be best represented by docking simulations. One of the most effective docking techniques is Surflex-Dock. The literature review showed that Surflex-Dock has several advantages in drug design studies.

In our previous communication, protein flexibility was not considered,¹⁷ and hence, in the present study, we have investigated the effect of protein flexibility on the docking process. To accomplish this process, the protein movement was allowed, which means whether to allow flexibility of protein atoms whose van der Waals surface distances from ligand atoms are less than 4 Å and to adapt the active site conformation to the docked ligand. Only hydrogens were allowed in protein flexibility to optimize hydroxyls and thiols, as well as all protons in the protein pocket. The binding models of 641, 4b, and 8a are depicted in Figure 9A–C. Model 641 showed two H-bonding interactions; the oxygen of the carbonyl group on pyrrolidine makes H-bonds with that of the OH group of the active site of Tyr158 (2.12 Å) and NAD⁺ ribose (1.95 Å). Compound 4b makes four hydrogen bonds: the oxygen atom of the methoxy group makes two H-bonds with Tyr158 (2.23 Å) and NAD⁺ ribose (1.82 Å), and that of nitrogens at the third and fourth positions of the oxadiazole ring makes two H-bonds with Met98 (1.97 and 2.29 Å). In the case of compound 8a, it makes five H-bonds; that is, at the third position nitrogen (Tyr158, 2.88 Å) and the fourth position nitrogen (NAD⁺ ribose, 2.21 Å) of the oxadiazole ring, compound 8a makes two H-bonds. Free NH₂ of sulphonamide group makes two H-bonds with NAD⁺ ribose (2.04 and 1.91 Å), and one more H-bond was observed between NAD⁺ ribose and the oxygen of SO₂ with a distance of 2.42 Å. However, in the case of nonprotein flexibility (compounds 641, 4b, and 8a), the H-bonding interactions at the active site with their respective distances are given in Table 2. The binding models of 641, 4b, and 8a by nonprotein flexibility docking are depicted in Figures 10–12.

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Figure 9 Docking conformation (capped sticks model in atom type color) of 641 (A), compounds 4b (B), and 8a (C) at the active site (yellow dotted lines indicate H-bond); flexible docking.

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Table 2 Key H-bonding interactions observed by simple and flexible docking processes with distance in Å.

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Figure 10 Docking conformation of 641 (capped sticks model in atom type color) at the active site (yellow dotted lines indicate H-bond).

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Figure 11 Docking conformation of compound 4b (capped sticks model in atom type color) at the active site (yellow dotted lines indicate H-bond).

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Figure 12 Docking conformation of compound 8a (capped sticks model in atom type color) at the active site (yellow dotted lines indicate H-bond).

Furthermore, interactions were also stabilized by the hydrophobic residues of the inner cavity, such as in Ile16, Ile21, Phe97, Met98, Pro99, Met103, Pro156, Ala157, Typ160, Met161, Pro193, and Ile194 and hydrophilic residues such as Gly14, Ser19, Ser20, Ser94, Gly96, Gln100, Gly102, Gly104, Gly119, Asp148, Asp150, Tyr158, Asn159, Thr162, Gly192, and Thr196. These amino acid residues were involved in the active cavity (shown in Figure 13A and B). Pmove score, the average movement of the protein atoms in the pocket for this pose, was observed in the range of 0.08–0.12, but not much change was observed when it was aligned with both the models. The docking scores, namely, C-score, Crash, Polar, D_Score, PMF_Score, G_Score, and Chem_Score, from Surflex-Dock are given in Tables 3 and 4. None of the molecules were observed with better scores than the re-docked 641 ligand, but they are making key interactions at the active site or substrate binding site. Comparing the predicted (CScore) and experimental (pMIC) results, it can be said that compounds with pMIC values of 4.903 and 4.602 showed the highest CScores (6.36–5.45) compared with other molecules in the series.

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Figure 13 Hydrophobic (brown) (A) and hydrophilic (blue) (B) amino acids surrounded to compound 4b (capped sticks model in atom type color).

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Table 3 Surflex-Dock scores of pyrrolyl thiadiazole derivatives Notes: CScore, consensus score, integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a receptor and reports the output of total score; Crash, the degree of inappropriate penetration by the ligand into the protein and of interpenetration (self-clash) between ligand atoms that are separated by rotatable bonds (crash scores close to 0 are favorable, negative numbers indicate penetration); Polar, contribution of the polar interactions to the total score; Pose, indication of which pose in the initial run has the best score after optimization with protein flexibility; Strain, nominal ligand strain relative to the nearby local minimum in units of pKd; Total, ligand's score corrected for strain energy; Ligmin, energy of the nearby ligand minimum (kcal/mol); Full, absolute energy of the optimized ligand, including protein interaction (kcal/mol); Complex, absolute energy of the complex including ligand, protein pocket, and intermolecular interactions (kcal/mol); Cscale, scaled complex score that normalizes the protein score components so that ligand poses that contact different numbers of protein atoms are more directly comparable; Pmove, average movement of the protein atoms in the pocket for this pose.

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Table 4 Different energy scores for pyrrolyl thiadiazole derivatives from the Surflex-Docking Notes: D_Score, charge and van der Waals interactions between the protein and the ligand; PMF_Score, indicating the Helmholtz free energies of interactions for protein–ligand atom pairs; PMF, potential of mean force; G_Score, showing hydrogen bonding, complex (ligand–protein), and internal (ligand–ligand) energies; Chem_Score, points for hydrogen bonding, lipophilic contact, and rotational entropy, along with an intercept term.

Experimental

All chemicals used were purchased either from Sigma-Aldrich, Fine-Chem Limited, or Spectrochem Pvt Ltd. Solvents were of reagent grade, and whenever necessary, they were purified and dried using the standard methods. The melting points of the compounds were determined using the Shital Scientific Industries melting point apparatus and are uncorrected. FTIR spectra were recorded on a Bruker spectrophotometer, using KBr pellets. The ¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE II 400 MHz instrument, using dimethyl sulfoxide (DMSO)-d₆ solvent and TMS as the internal standard. Chemical shifts are expressed in δ values (ppm).

Mass spectra (MS) were taken in JEOL GCMATE II GC-Mass and Waters Micromass Q-Tof Micro liquid chromatography-mass spectrometers. The compounds showed spectral data consistent with their proposed. Analytical thin-layer chromatography (TLC) was performed on precoated TLC sheets of silica gel 60 F₂₅₄ (Merck, Darmstadt, Germany), visualized by long- and short-wavelength ultraviolet lamps. Chromatographic purifications were performed on Merck aluminum oxide (70–230 mesh) and Merck silica gel (70–230 mesh).

General procedure for the preparation of 2-amino-5-(4-substituted phenyl)- 1,3,4-thiadiazoles (3a–3i)

A mixture of appropriate aromatic acid (50 mmol), N-aminothiourea (50 mmol), and POCl₃ (13 mL) was heated at 75°C for 30 minutes and cooled, to which 10 mL of water was added, and the mixture was refluxed for 4 hours. The pH was then adjusted to 8.0 by adding 50% sodium hydroxide solution. The separated solid was filtered and recrystallized from ethanol to give the desired compounds.³²

General procedure for the preparation of 5-(4-substituted phenyl)-2-(1H-pyrrol-1-yl) 1-1,3,4-thiadiazoles (4a–4i)

To a solution of 2-amino-5-(4-substituted phenyl)-1,3,4-thiadiazoles (10 mmol) in 20 mL glacial acetic acid, 2,5-dimethoxytetrahydrofuran (15 mmol) was added slowly at room temperature and refluxed for 1 hour (monitored by TLC). The reaction mixture was poured into ice-cold water and basified with sodium bicarbonate solution. The separated solid was collected, washed with water, and dried. All the compounds were recrystallized, using ethanol as the solvent.

4-Phenyl-5-(1H-pyrrol-1-yl)-1,3,4-thiadiazole (4a)

Yield, 78%: mp 136°C–138°C; FTIR (KBr): 2,923 and 2,848 (Ar-H), 1,509 (C=N) cm⁻¹; ¹H NMR (400 MHz, deuterated chloroform [CDCl₃]) δ ppm: 6.44 (s, 2H, pyrrole-C₃, and C₄-H), 7.55 (dd, 2H, pyrrole-C₂ and C₅-H), 7.57–7.59 (m, 3H, ph-C₃, C₄, C₅-H), 7.95 (d, 2H, ph-C₂, and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 113.54 (pyrrole-C₃ and C₄), 121.66 (pyrrole-C₂ and C₅), 127.68 (ph-C₂ and C₆), 129.82 (ph-C₄), 129.99 (ph-C₃ and C₅), 131.85 (ph-C₁), 162.22 (thiadiazole-C₂), 164.24 (thiadiazole-C₅); MS (EI): m/z = found 226.97 [M⁺]; calcd. 227.05. Anal. C₁₂H₉N₃S.

2-(4-Methoxyphenyl)-5-(1H-pyrrol-1-yl)-1,3,4-thiadiazole (4b)

Yield, 71%: mp 140°C–142°C; FTIR (KBr): 2,926 and 2,836 (Ar-H), 1,605 (C=N) cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ ppm: 3.87 (s, 3H, OCH₃), 6.39 (dd, 2H, pyrrole-C₃, and C₄-H), 7.05 (dd, 2H, ph-C₃, and C₅-H), 7.39 (dd, 2H, pyrrole-C₂, and C₅-H), 7.86 (dd, 2H, ph-C₂, and C₆-H); ¹³C NMR (300 MHz, DMSO) δ ppm: 55.27 (OCH₃), 112.65 (pyrrole-C₃ and C₄), 114.56 (ph-C₃ and C₅), 120.72 (pyrrole-C₂ and C₅), 121.83 (ph-C₁), 128.65 (ph-C₂ and C₆), 160.75 (ph-C₄), 161.52 (thiadiazole-C₂), 163.32 (thiadiazole-C₅); MS (EI): m/z = found 257.49 [M⁺]; calcd. 257.31. Anal. C₁₃H₁₁N₃OS.

2-(4-Chlorophenyl)-5-(1H-pyrrol-1-yl)-1,3,4-thiadiazole (4c)

Yield, 89%: mp 160°C–162°C; FTIR (KBr): 2,918 and 2,848 (Ar-H), 1,584 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 6.40 (dd, 2H, pyrrole-C₃, and C₄-H), 7.43 (dd, 2H, pyrrole-C₂, C₅-H), 7.56 (dd, 2H, ph-C_3, C₅-H), 7.93 (dd, 2H, ph-C_2, C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 113.62 (pyrrole-C₃ and C₄), 121.69 (pyrrole-C₂ and C₅), 128.68 (ph-C₂ and C₆), 129.36 (ph-C₃ and C₅), 130.05 (ph-C₁), 136.42 (ph-C₄), 162.50 (thiadiazole-C₂), 163.05 (thiadiazole-C₅); MS (electrospray ionisation [EI]): m/z = found 261.78 [M⁺], 263.78 [M⁺ +2]; calcd. 261.73. Anal. C₁₂H₈ClN₃S.

2-(4-Nitrophenyl)-5-(1H-pyrrol-1-yl)-1,3,4-thiadiazole (4d)

Yield, 63%: mp 216°C–219°C; FTIR (KBr): 2,916, 2,848 (Ar-H), 1,600 (C=N), 1,503, 1,339 (NO₂) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 6.44 (dd, 2H, pyrrole-C₃, and C₄-H), 7.54 (dd, 2H, pyrrole C₂, and C₅-H), 8.23 (dd, 2H, ph-C₂, and C₆-H), 8.41 (dd, 2H, ph-C₃, and C₅-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 113.27 (pyrrole-C₃ and C₄), 121.12 (pyrrole-C₂ and C₅), 124.48 (ph-C₃ and C₅), 128.28 (ph-C₂ and C₆), 134.99 (ph-C₁), 148.58 (ph-C₄), 161.39 (thiadiazole-C₂), 162.89 (thiadiazole-C₅); MS (EI): m/z = found 272.31 [M⁺], 273.31 [M⁺+1]; calcd. 272.28. Anal. C₁₂H₈N₄O₂S.

2-(4-Bromophenyl)-5-(1H-pyrrol-1-yl)-1,3,4-thiadiazole (4e)

Yield, 58%: mp 162°C–165°C; FTIR (KBr): 2,919 and 2,847 (Ar-H), 1,581 (C=N), 598 (C-Br) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 6.42 (dd, 2H, pyrrole-C₃, and C₄-H), 7.49 (dd, 2H, pyrrole C₂, and C₅-H), 7.75 (dd, 2H, ph-C₂, and C₆-H), 7.88 (dd, 2H, ph-C₃, and C₅-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 114.12 (pyrrole-C₃ and C₄), 122.02 (pyrrole-C₂ and C₅), 125.61 (ph-C₃ and C₅), 129.15 (ph-C₂ and C₆), 141.11 (ph-C₁), 150.02 (ph-C₄), 162.77 (thiadiazole-C₂), 164.03 (thiadiazole-C₅); MS (EI): m/z = found 306.09 [M⁺], 308.09 [M⁺ +2]; calcd. 306.18. Anal. C₁₂H₈BrN₃S.

2-(4-Methylphenyl)-5-(1H-pyrrol-1-yl)-1,3,4-thiadiazole (4f)

Yield, 83%: mp174°C–176°C; FTIR (KBr): 2,917, 2,863 (Ar-H), 1,593 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 2.41 (s, 3H, CH₃), 6.39 (s, 2H, pyrrole-C₃, and C₄-H), 7.34 (d, 2H, J = 4.90 ph-C₂ and C₆-H), 7.43 (s, 2H, J = 4.92 pyrrole C₂ and C₅-H), 7.81 (d, 2H, ph-C₃, and C₅-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 27.33 (CH₃), 112.36 (pyrrole-C₃ and C₄), 120.93 (pyrrole-C₂ and C₅), 126.03 (ph-C₃ and C₅), 128.23 (ph-C₂ and C₆), 143.12 (ph-C₁), 148.56 (ph-C₄), 161.87 (thiadiazole-C₂), 163.35 (thiadiazole-C₅); MS (EI): m/z = found 241.17 [M⁺]; calcd. 241.07. Anal. C₁₃H₁₁N₃S.

2-(4-Aminophenyl)-2-(1H-pyrrol-1-yl)-1,3,4-thiadiazole (4g)

Yield, 20%: mp 180°C–182°C; FTIR (KBr): 3,381 (NH₂), 2,923, 2,853 (Ar-H), 1,603 (C=N) cm⁻¹; ¹HNMR (400 MHz, CDCl₃) δ ppm: 6.33 (dd, 2H, pyrrole-C₃, and C₄-H), 6.42 (s, 2H, NH₂), 7.35 (dd, 2H, pyrrole C₂, and C₅-H), 7.44–7.7.77 (m, 4H, ph-C₂, C₃, C₅, and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 114.13 (pyrrole-C₃ and C₄), 123.00 (pyrrole-C₂ and C₅), 126.05 (ph-C₃ and C₅), 128.66 (ph-C₂ and C₆), 140.59 (ph-C₁), 149.55 (ph-C₄), 162.25 (thiadiazole-C₂), 164.12 (thiadiazole-C₅); MS (EI): m/z = found 242.28 [M⁺]; calcd. 242.30. Anal. C₁₂H₁₀N₄S.

2-(4-Fluorophenyl)-5-(1H-pyrrol-1-yl)-1,3,4-thiadiazole (4h)

Yield, 62%: mp 149°C–151°C; FTIR (KBr): 2,918 and 2,848 (Ar-H), 1,594 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 6.40 (dd, 2H, pyrrole-C₃, and C₄-H), 7.33 (dd, 2H, pyrrole-C₂, and C₅-H), 7.44 (dd, 2H, ph-C₃, and C₅-H), 7.98 (dd, 2H, ph-C₂, and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 112.79 (pyrrole-C₃ and C₄), 116.13 (ph-C₃ and C₅), 120.69 (pyrrole-C₂ and C₅), 129.24 (ph-C₂ and C₆), 129.33 (ph-C₁), 161.50 (thiadiazole-C₂), 162.26 (ph-C₄), 164.92 (thiadiazole-C₅); MS (EI): m/z = found 245.37[M⁺]; calcd. 245.28. Anal. C₁₂H₈FN₃S.

2-(Pyridin-3-yl)-5-(1H-pyrrol-1-yl)-1,3, 4-thiadiazole (4i)

Yield, 61%: mp 136°C–138°C; FTIR (KBr): 2,988 and 2,923 (Ar-H), 1,597 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 6.42 (dd, 2H, pyrrole-C₃, and C₄-H), 7.35 (dd, 2H, pyrrole-C₂, and C₅-H), 7.56–9.01 (m, 4H, pyridine-C₂, C₄-H, C₅-H, and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 109.05 (pyrrole-C₃ and C₄), 124.26 (pyridine-C₅), 130.02 (pyrrole-C₂ and C₅), 133.54 (pyridine-C₃), 134.06 (pyridine-C₄), 148.23 (pyridine-C₆), 149.08 (pyridine-C₂), 163.39 (thiadiazole-C₂), 174.57 (thiadiazole-C₅); MS (EI): m/z = found 228.11 [M⁺]; calcd. 228.05. Anal. C₁₁H₈N₄S.

General procedure for the preparation of 5-(4-substituted phenyl)-2-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3,4-thiadiazoles (5a–5i)

To a solution of 2-amino-4-(4-substituted phenyl) thiadiazoles (10 mmol) in 20 mL glacial acetic acid, acetonyl acetone (15 mmol) was added slowly at room temperature and refluxed for 1 hour (monitored by TLC). This mixture was poured into ice-cold water and basified with sodium bicarbonate solution. The separated solid was collected, washed with water, dried, and recrystallized, using n-hexane as the solvent.

2-(2,5-Dimethyl-1H-pyrrol-1-yl)-5-phenyl-1,3,4-thiadiazole (5a)

Yield, 58%: mp 234°C–238°C; FTIR (KBr): 2,951 and 2,853 (Ar-H), 1,624 (C=N) cm⁻¹; 1H NMR (400 MHz, CDCl₃) δ ppm: 2.28 (s, 6H, 2CH₃), 5.94 (s, 2H, pyrrole-C₃ and C₄-H), 7.37–7.44 (m, 3H, ph-C_3, C₄ and C₅-H), 7.74–7.77 (m, 2H, ph-C₂ and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 13.01 (2 CH₃), 111.24 (pyrrole-C₃ and C₄), 128.21 (pyrrole-C₂ and C₅), 129.57 (ph-C₂ and C₆), 132.08 (ph-C₃ and C₅), 132.90 (ph-C₁), 135.29 (ph-C₄), 162.06 (thiadiazole-C₂), 164.03 (thiadiazole-C₅); MS (EI): m/z = found 255.01 [M⁺]; calcd. 255.08. Anal. C₁₄H₁₃N₃S.

2-(4-Methoxyphenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3,4-thiadiazole (5b)

Yield, 38%: mp 110°C–112°C; FTIR (KBr): 2,922 and 2,845 (Ar-H), 1,606 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 2.24 (s, 6H, 2CH₃), 3.88 (s, 3H, OCH₃), 5.92 (s, 2H, pyrrole-C₃ and C₄-H), 7.10 (dd, 2H, ph-C₃ and C₅-H), 7.94 (dd, 2H, ph-C₂ and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 12.90 (2CH₃), 55.26 (OCH₃), 108.60 (pyrrole-C₃ and C₄), 114.54 (ph-C₃ and C₅), 121.93 (pyrrole-C₂ and C5), 129.28 (ph-C₂ and C₆), 128.92 (ph-C₁), 158.52 (ph-C₄), 161.79 (thiadiazole-C₂), 168.17 (thiadiazole-C₅); MS (EI): m/z = found 285.11 [M⁺]; calcd. 285.09. Anal. C₁₅H₁₅N₃OS.

2-(4-Chlorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3,4-thiadiazole (5c)

Yield, 42%: mp 128°C–130°C; FTIR (KBr): 2,917 and 2,852 (Ar-H), 1,590 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 2.25 (s, 6H, 2CH₃), 5.94 (s, 2H, pyrrole-C₃ and C₄-H), 7.60 (dd, 2H, ph-C₃ and C₅-H), 8.02 (dd, 2H, ph-C₂ and C_6-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 13.00 (2CH₃), 108.90 (pyrrole-C₃ and C₄), 128.16 (pyrrole-C₂ and C₅), 128.85 (ph-C₂ and C₆), 129.34 (ph-C₃ and C₅), 129.38 (ph-C₁), 136.50 (ph-C₄), 159.67 (thiadiazole-C₂), 166.99 (thiadiazole-C₅); MS (EI): m/z = found 289.05 [M⁺], 291.05 [M⁺ +2]; calcd. 289.04. Anal. C₁₄H₁₂ClN₃S.

2-(4-Nitrophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3,4-thiadiazole (5d)

Yield, 43%: mp 150°C–152°C; FTIR (KBr): 2,921 and 2,854 (Ar-H), 1,596 (C=N), 1,497, 1,331 (NO₂) cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ ppm: 2.30 (s, 6H, 2CH₃), 5.96 (s, 2H, pyrrole-C₃ and C₄-H), 8.28 (d, 2H, J = 8.5 Hz, ph-C₂ and C_6-H), 8.42 (d, 2H, J = 9 Hz, ph-C₃ and C₅-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 12.97 (2CH₃), 110.12 (pyrrole-C₃ and C₄), 127.59 (pyrrole-C₂ and C₅), 129.03 (ph-C₂ and C₆), 131.06 (ph-C₃ and C₅), 132.87 (ph-C₁), 137.57 (ph-C₄), 161.13 (thiadiazole-C₂), 164.25 (thiadiazole-C₅); MS (EI): m/z = found 300.23 [M⁺], 301.23 [M⁺+1]; calcd. 300.07. Anal. C₁₄H₁₂N₄O₂S.

2-(4-Bromophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3,4-thiadiazole (5e)

Yield, 44%: mp 108°C–110°C; FTIR (KBr): 2,917 and 2,854 (Ar-H), 1,585 (C=N) cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ ppm: 2.25 (s, 6H, 2CH₃), 5.94 (s, 2H, pyrrole-C₃ and C₄-H), 7.95 (d, 2H, J = 5.35 Hz, ph-C₃ and C₅-H), 8.07 (d, 2H, J = 5.33 Hz, ph-C₂ and C_6-H); ¹³C NMR (400 MHz, DMSO-d₆) δ ppm: 13.01 (2CH₃), 109.03 (pyrrole-C₃ and C₄), 129.22 (pyrrole-C₂ and C₅), 129.30 (ph-C₂ and C₆), 130.05 (ph-C₃ and C₅), 133.26 (ph-C₁), 138.54 (ph-C₄), 160.23 (thiadiazole-C₂), 165.15 (thiadiazole-C₅); MS (EI): m/z = found 332.56 [M⁺], 334.56 [M⁺ +2]; calcd. 332.99. Anal. C₁₄H₁₂BrN₃S.

2-(4-Methylphenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3,4-thiadiazole (5f)

Yield, 53%: mp 92°C–94°C; FTIR (KBr): 2,921 and 2,857 (Ar-H), 1,603 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 2.24 (s, 6H, 2CH₃), 2.42 (s, 3H, CH₃), 5.93 (s, 2H, pyrrole-C₃ and C₄-H), 7.37 (d, 2H, J = 5.05 ph-C₃ and C₅-H), 7.88 (d, 2H, J = 5.10 ph-C₂ and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 13.33 (2CH₃), 25.02 (CH₃), 110.06 (pyrrole-C₃ and C₄), 128.59 (pyrrole-C₂ and C₅), 129.17 (ph-C₂ and C₆), 131.00 (ph-C₃ and C₅), 133.22 (ph-C₁), 136.52 (ph-C₄), 159.69 (thiadiazole-C₂), 163.29 (thiadiazole-C₅); MS (EI): m/z =found 269.03 [M⁺]; calcd. 269.10. Anal. C₁₅H₁₅N₃S.

2-(4-Aminophenyl)-2-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3,4-thiadiazole (5g)

Yield, 21%: mp 196°C–198°C; FTIR (KBr): 3,244 (NH₂), 2,922 and 2,853 (Ar-H), 1,652 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 2.06 (s, 6H, 2CH₃), 5.85 (s, 2H, pyrrole-C₃ and C₄-H), 7.38 (s, 2H, NH₂), 7.98–8.15 (m, 4H, ph-C_2, C_3, C₅ and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 13.22 (2CH₃), 108.69 (pyrrole-C₃ and C₄), 127.53 (pyrrole-C₂ and C₅), 129.36 (ph-C₂ and C₆), 130.59 (ph-C₃ and C₅), 132.05 (ph-C₁), 138.42 (ph-C₄), 161.00 (thiadiazole-C₂), 164.67 (thiadiazole-C₅); MS (EI): m/z = found 270.17 [M⁺]; calcd. 270.09. Anal. C₁₄H₁₄N₄S.

2-(4-Fluorophenyl)-5-(2,5-dimethyl-1H-pyrrol-1-yl)-1,3,4-thiadiazole (5h)

Yield, 41%: mp 118°C–120°C; FTIR (KBr): 2,920 and 2,852 (Ar-H), 1,612 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 2.25 (s, 6H, 2CH₃), 5.94 (s, 2H, pyrrole-C₃ and C₄-H), 7.30–8.07 (m, 4H, ph-C_2, C_3, C₅ and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 12.97 (2CH₃), 105.83 (pyrrole-C₃ and C₄), 116.37 (ph-C₃ and C₅), 129.33 (pyrrole-C₂ and C₅), 129.53 (ph-C_1, C₂ and C₆), 132.23 (ph-C₄), 159.88 (thiadiazole-C₂), 163.05 (thiadiazole-C₅); MS (EI): m/z = found 273.01 [M⁺]; calcd. 273.07. Anal. C₁₄H₁₂FN₃S.

2-(2,5-Dimethyl-1H-pyrrol-1-yl)-5-(pyridin-3-yl)-1,3,4-thiadiazole (5i)

Yield, 39%: mp 103°C–105°C; FTIR (KBr): 2,918 and 2,855 (Ar-H), 1,602 (C=N) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 2.26 (s, 6H, 2CH₃), 5.83 (s, 2H, pyrrole-C₃ and C₄-H), 7.42–8.77 (m, 4H, pyridine-C_2, C_4, C₅ and C₆-H); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 12.89 (2CH₃), 105.73 (pyrrole-C₃ and C₄), 123.16 (pyridine-C₅), 129.31 (pyrrole-C₂ and C₅), 131.24 (pyridine-C₃), 133.57 (pyridine-C₄), 148.03 (pyridine-C₆), 148.99 (pyridine-C₂), 163.22 (thiadiazole-C₂), 174.03 (thiadiazole-C₅); MS (EI): m/z = found 256.13 [M⁺]; calcd. 256.08. Anal. C₁₃H₁₂N₄S.

Synthesis of 2-(1H-pyrrol-1-yl)-5-sulfonamido-1,3,4-thiadiazole (8a)

To a solution of 2-amino-5-sulfonamido-1,3,4-thiadiazole (10 mmol) in 20 mL glacial acetic acid, 2,5-dimethoxytetrahydrofuran (15 mmol) was added slowly at room temperature and was refluxed for 1 hour (monitored by TLC). The reaction mixture was poured into ice-cold water and basified with sodium bicarbonate solution. The separated solid was collected, washed with water, dried, and recrystallized from aqueous ethanol.

Yield, 40%: mp 188°C–190°C; FTIR (KBr): 3,358.53, 3,253.98 (SO₂NH₂), 3,108.56 (Ar-H), 1,514.81 (C=N), 1,347.45 (SO₂^as), 1,174.42 (SO₂^sym) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 6.46 (s, 2H, pyrrole-C₃ and C₄-H), 7.61 (s, 2H, pyrrole-C₂, C₅-H), 8.59 (s, 2H, NH₂); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 114.21 (pyrrole-C₃ and C₄), 122.13 (pyrrole-C₂ and C₅), 165.29 (thiadiazole-C₂), 166.32 (thiadiazole-C₅); MS (EI): m/z = found 229.07 [M⁺]; calcd. 229.99. Anal. C₆H₆N₄O₂S₂.

Synthesis of 2-(2,5-dimethyl-1H-pyrrol-1-yl)-5-sulfonamido-1,3,4-thiadiazole (8b)

To a solution of 2-amino-5-sulfonamido-1,3,4-thiadiazole (10 mmol) in 20 mL glacial acetic acid, acetonyl acetone (15 mmol) was added slowly at room temperature and was refluxed for 30 minutes. The reaction mixture was poured into ice-cold water and basified with sodium bicarbonate solution. The separated solid was collected, washed with water, dried, and recrystallized from ethanol.

Yield, 30%: mp 206°C–208°C; FTIR (KBr): 3,274.75, 3,085.68 (SO₂NH₂), 2,773.97 (Ar-H), 1,513.63 (C=N) 1,316.14 (SO₂^as), 1,129.71 (SO₂^sym) cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ ppm: 1.80 (s, 6H, pyrrole-2CH₃), 5.60 (s, 2H, pyrrole-C₃ and C₄-H), 8.52 (s, 2H, NH₂); ¹³C NMR (400 MHz, CDCl₃) δ ppm: 12.81 (2CH₃), 115.23 (pyrrole-C₃ and C₄), 121.98 (pyrrole-C₂ and C₅), 164.88 (thiadiazole-C₂), 166.03 (thiadiazole-C₅); MS (EI): m/z = found 258.19 [M⁺]; calcd. 258.02. Anal. C₈H₁₀N₄O₂S₂.

Biological evaluation

Antitubercular activity

The MIC values were determined for the newly synthesized compounds (4a–4i, 5a–5i, and 8a–8b) against M. tuberculosis strain H₃₇Rv, using a microplate Alamar blue assay.³³ Isoniazid was used as the standard drug. The 96-well plate received 100 μL of the Middlebrook 7H9 broth, and serial dilution of compounds was made directly on the plate. The final drug concentrations tested were 0.2, 0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50, and 100 μg mL⁻¹. Plates were covered and sealed with parafilm and incubated at 37°C for 5 days. After this, 25 μL freshly prepared 1:1 mixture of Alamar blue reagent and 10% Tween 80 was added to the plate and incubated for 24 hours. A blue color in the well was interpreted as no bacterial growth, and a pink color was scored as the growth. The MIC was defined as the lowest drug concentration that prevented the color change from blue to pink. Table 1 reveals the antitubercular activity (MIC) data of the newly synthesized compounds.

Molecular modeling

General procedure

The 3D structure building and all modeling protocols were performed using the Sybyl-X 2.0 programming package running on a dual-core Intel core i3-2130 CPU 3.40 GHz, RAM Memory 2 GB workstation running Windows 7. Each structure was geometrically optimized using a conjugate gradient method based on Tripos force field³⁴ and MMFF94 charge, with a distance-dependant dielectric and Powell conjugate gradient algorithm with a convergence criterion of 0.01 kcal/mol. Partial atomic charges were calculated using the semiempirical program MOPAC 6.0, as well as by applying the AM1 Hamiltonian (Austin Model 1).³⁵

Protein preparation

Crystal structure of M. tuberculosis enoyl reductase (InhA) complexed with 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide was retrieved from Research Collaboratory for Structural Bioinformatics Protein Data Bank (PDB ID code 4TZK).¹² Water molecules were removed, and essential hydrogens were added; united atoms Amber7FF9902 were assigned for the protein. Geometry optimization was carried out using the standard Tripos force field, with a distance-dependent dielectric function keeping the energy gradient of 0.001 kcal/mol. Surflex-Dock is one of the best docking suites employed for docking analysis.^36,37

Conclusion

Most of the synthesized compounds exhibited moderate activity against M. tuberculosis. Compounds 4b, 5b, and 5d inhibited growth of M. tuberculosis very effectively at a MIC value of 12.5 μg/mL, followed by compounds 4d, 4h, 4i, 5h, 5i, 8a, and 8b, with MIC values 25 μg/mL. Most of the molecules could effectively bind to the substrate binding site of ENR. The key H-bonding interactions with Tyr158, Met98, and cofactor NAD⁺, as well as hydrophobic amino acid residues, stabilized the ligand–receptor complex to conclude that molecules are efficiently bound at the active site of ENR and, hence, can be better ENR inhibitors. To accomplish the inhibitory action, H-bond acceptor atoms and hydrophobic fragments played a key role (Figure 14). The predicted in silico Surflex-Dock values obtained through docking studies have pointed toward 4b, 5d, and 8a as the most promising inhibitors. Conclusively, compounds substituted with -OCH₃, -NO₂, -F, pyridine, and sulfonamide groups or moieties were found to be better inhibitors than compounds substituted with -CH₃, -NH₂, -Cl, and -Br groups. All the synthesized compounds showed a reasonable correlation between the experimental and predicted results. The present study suggests that molecular docking and in vitro MIC assay analysis could serve to be an efficient prescreening technique for identifying new ENR inhibitors and may be useful in situations in which enzyme inhibition experimental data are insufficient or not available.

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Figure 14 Pharmacophoric and receptor binding features (cyan, hydrophobic; green, H-bond acceptor; magenta, H-bond donor) for compounds 4b (A) and 8a (B) (capped sticks model in atom type color).

Acknowledgments

We thank the Indian Council of Medical Research, New Delhi, India, for financial support (File 64/4/2011-BMS, IRIS Cell 2010-08710) and acknowledge partial financial support from the Vision Group on Science and Technology, Department of Information Technology, Biotechnology and Science and Technology, Dr S Ananth Raj, Bangalore (File VGST/P-3/SMYSR/GRD-277/2013-14/, dated January 28, 2014). We also thank Mr HV Dambal, president and Dr VH Kulkarni, principal, Soniya Education Trust's College of Pharmacy, Dharwad, India, for providing facilities. We thank Dr KG Bhat, Maratha Mandal's Dental College, Hospital and Research Centre, Belgaum, India, for providing facilities for antitubercular activity. The director, Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Chennai, Tamil Nadu, India, and the director, SAIF, Panjab University, Chandigarh, Panjab, India, have provided NMR and mass spectral data. We also thank Mr Shrikant A Tiwari for his technical assistance.

Disclosure

The authors report no conflicts of interest in this work.

Supplementary materials

Pharmacophore mapping

During the study, it was found that the InhA inhibitor [1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (pyrrolidine carboxamide or 641)], which contains three hydrophobic moieties, such as cyclohexyl, oxopyrrolidine, and 3,5-dichlorophenyl, can be replaced by new, designed molecules, which contain pyrrole, 1,3,4-thiadiazole, and substituted phenyl. Hydrogen bond acceptor atoms such as oxygen at the fifth position of pyrrolidine and nitrogens at 1,3,4-thiadiazole make an H-bond with key amino acid Tyr158 and cofactor NAD⁺, which helps in structure-based drug design and the selection of target.

Hydropathy plots

In hydropathy, produce scatter plots of the hydropathy indices as a function of the residue number. Such plots are frequently used to identify segments of a protein sequence that have hydrophobic properties consistent with a transmembrane helix.

Three columns are added to the spreadsheet: HYDRO is the hydrophobicity at each residue, with its value depending on the hydrophobicity scale chosen. HYD_5 is the hydropathy index averaged over a moving window of eleven residues (five on either side of a given residue). The window size determines the extent of smoothing for the calculation of hydropathy indices. SEQNUM is an integer corresponding to the serial position of the residue in the protein. It is a useful index when residue numbering does not start at one or is not sequential.

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Figure S1 Structure-based drug design concept.

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Table S1 Hydrophobicity scales for protein Notes: HYDRO, hydrophobicity at each residue, with value depending on the hydrophobicity scale chosen; HYD_5, the hydropathy index averaged over a moving window of eleven residues (five on either side of a given residue), and is the window size that determines the extent of smoothing for the calculation of hydropathy indices; SEQNUM, an integer corresponding to the serial position of the residue in the protein. It is a useful index when residue numbering does not start at one or is not sequential.

References

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37. Pham TA, Jain AN. Customizing scoring functions for docking. J Comput Aided Mol Des. 2008;22(5):269–286. [/RAW_REF_TEXT] Supplementary materials Pharmacophore mapping During the study, it was found that the InhA inhibitor [1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (pyrrolidine carboxamide or 641)], which contains three hydrophobic moieties, such as cyclohexyl, oxopyrrolidine, and 3,5-dichlorophenyl, can be replaced by new, designed molecules, which contain pyrrole, 1,3,4-thiadiazole, and substituted phenyl. Hydrogen bond acceptor atoms such as oxygen at the fifth position of pyrrolidine and nitrogens at 1,3,4-thiadiazole make an H-bond with key amino acid Tyr158 and cofactor NAD⁺, which helps in structure-based drug design and the selection of target. Hydropathy plots In hydropathy, produce scatter plots of the hydropathy indices as a function of the residue number. Such plots are frequently used to identify segments of a protein sequence that have hydrophobic properties consistent with a transmembrane helix. Three columns are added to the spreadsheet: HYDRO is the hydrophobicity at each residue, with its value depending on the hydrophobicity scale chosen. HYD_5 is the hydropathy index averaged over a moving window of eleven residues (five on either side of a given residue). The window size determines the extent of smoothing for the calculation of hydropathy indices. SEQNUM is an integer corresponding to the serial position of the residue in the protein. It is a useful index when residue numbering does not start at one or is not sequential. Figure S1 Structure-based drug design concept. [/RAW_REF_TEXT] Table S1 Hydrophobicity scales for protein Notes: HYDRO, hydrophobicity at each residue, with value depending on the hydrophobicity scale chosen; HYD_5, the hydropathy index averaged over a moving window of eleven residues (five on either side of a given residue), and is the window size that determines the extent of smoothing for the calculation of hydropathy indices; SEQNUM, an integer corresponding to the serial position of the residue in the protein. It is a useful index when residue numbering does not start at one or is not sequential.