Novel Hybrid 1,2,4- and 1,2,3-Triazoles Targeting Mycobacterium Tuberculosis Enoyl Acyl Carrier Protein Reductase (InhA): Design, Synthesis, and Molecular Docking

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis is still a serious public health concern around the world. More treatment strategies or more specific molecular targets have been sought by researchers. One of the most important targets is M. tuberculosis’ enoyl-acyl carrier protein reductase InhA which is considered a promising, well-studied target for anti-tuberculosis medication development. Our team has made it a goal to find new lead structures that could be useful in the creation of new antitubercular drugs. In this study, a new class of 1,2,3- and 1,2,4-triazole hybrid compounds was prepared. Click synthesis was used to afford 1,2,3-triazoles scaffold linked to 1,2,4-triazole by fixable mercaptomethylene linker. The new prepared compounds have been characterized by different spectroscopic tools. The designed compounds were tested in vitro against the InhA enzyme. At 10 nM, the inhibitors 5b, 5c, 7c, 7d, 7e, and 7f successfully and totally (100%) inhibited the InhA enzyme. The IC₅₀ values were calculated using different concentrations. With IC₅₀ values of 0.074 and 0.13 nM, 7c and 7e were the most promising InhA inhibitors. Furthermore, a molecular docking investigation was carried out to support antitubercular activity as well as to analyze the binding manner of the screened compounds with the target InhA enzyme’s binding site.

1. Introduction

Nitrogen heterocycles are among the most significant structural components of antimicrobial agents. Among them, 1,4-disubstituted 1,2,3-triazoles, afforded by copper (I) azide alkyne cyclo-addition, portrayed outstanding medicinal chemistry attributes that encouraged the Nobel Prize winner Prof. K. Barry Sharpless to describe them as aggressive pharmacophores [1]. 1,2,3-triazole cores were found in many FDA-approved drugs such as rufinamide (anticonvulsant), TSAO (anti-HIV), cefatrizine (antibiotic), tazobactam (antibacterial), CAI (anticancer), and ribavirin analogs (antiviral) [2,3,4,5,6]. In addition, a literature survey revealed that 1,2,3 and or 1,2,4-triazoles are privileged scaffolds with versatile biological activity, particularly in the area of antimycobacterium drug discovery that utilizes several mechanisms of action, most particularly InhA (Enoyl Acyl Carrier Protein Reductase) inhibitors (Figure 1 and Figure 2) [7,8,9,10,11,12,13,14,15,16,17].

Tuberculosis (TB) is one of the most immense diseases, ranked second after AIDS, which attracted researchers to try to find appropriate treatments. The World Health Organization (WHO) reported Mycobacterium tuberculosis (Mtb) infection as a leading cause of mortality and morbidity worldwide, with 1.6 million deaths annually [18]. Recently, the increasing incidence of multidrug-resistant tuberculosis (MDRTB) and extensively drug-resistant tuberculosis (XDRTB) has worsened the situation [19] and has made TB a more dreadful disease than before. One of the factors that made TB a more complicated infection compared to other microbial infections is their unique cell envelope. The cell envelope contains a protective layer of mycolic acid (which is a saturated chain of β-hydroxy fatty acids along with α-alkyl side chain) [20]. InhA is a NADH-dependent 2-trans enoyl-acyl carrier protein (ACP) reductase of type II fatty acid synthase, which is essential for mycolic acid biosynthesis. There is a portfolio of evidence demonstrating that it is the primary target of the potent and well-known antitubercular drug isoniazid (INH) [18].

A short time ago, there were several drug candidates at different stages of the development pipeline, including one morpholine-containing compound (I-A09) that exerted its antitubercular action through inhibition of protein tyrosine phosphatase B (mPTPB) (Figure 3) [5].

Morpholine is a versatile moiety, a privileged pharmacophore, and an extraordinary heterocyclic motif, especially in the field of antimicrobial agents. WHO, for example, recently reclassified a commercially available antimicrobial linezolid as a Group A drug for the treatment of MDRTB and XDRTB [19,20] (Figure 4).

Our lab has committed to discover novel lead structures that might be of value for the development of novel potent antitubercular agents. In the present study, a new set of hybrid derivatives containing 1,2,3-and 1,2,4 triazole moieties were designed based on the structural features of four pleiotropic lead compounds (Figure 3). The newly synthesized compounds were tested in vitro against the mycobacterium tuberculosis InhA enzyme in aspiration of lead structures A and B, which demonstrated remarkable inhibitory activity with IC₅₀ values of 0.906 and 0.057 μM, respectively, [4].

The first target structure-1 that exhibit good InhA inhibitory activity with IC₅₀ = 0.13 nM was designed by connecting both 1,2,4-triazole scaffold with the click modifiable 1,2,3-triazole via the flexible SCH₂-bonding and molecular hybridization with morpholine ring achieved by reaction of 4-morpholinobenzaldehyde with 1,2,4-triazole containing compound.

For further optimization and structural versatility, the second target structure was designed, where the 4-phenyl morpholine moiety was replaced by benzo[d][1,3]dioxole, affording a more potent candidate that displayed an IC₅₀ of 0.074 nM and was considered the most potent candidate compared to lead structures A, B, and the other tested compounds.

Moreover, the molecular docking study was accomplished both to support the antitubercular activity and to investigate the binding mode of the interactions of the screened compounds with the binding site of the target InhA enzyme.

2. Results and Discussion

2.1. Chemistry

A series of novel 1,2,3-triazoles tethered to a 1,2,4-triazol motif 7(a–f) were synthesized from 1-amino-1,2,4-triazole 3 as a starting material (Scheme 1). The synthetic strategies adopted in the present work are depicted in Scheme 1 and Scheme 2. The targeted 1,2,4-triazole-1,2,3-triazole molecular conjugates 7(a–f) were synthesized from three Schiff bases of 4-amino-1,2,4-triazole 4(a–c), which in turn were obtained through muli-steps synthesis from the appropriate phenylacetic acid hydrazide 2 according to the reported procedures [21,22] (Scheme 1). The synthesis of Schiff bases of 4-amino-5-phenylmethyl-1,2,4-triazole-3-thiol (3) has been achieved successfully by refluxing of the free amine 3 with the appropriate aromatic aldehyde.

The propargylaion reaction of the free thiol 4(a–c) with propargyl bromide successfully afforded the corresponding product in quantitative yield. 1,3-Dipolar cycloaddition reaction of propargylated thiol 5(a–c) with aryl azides 6(a,b) in the presence of CuSO₄·5H₂O and sodium L-ascorbate under stirring at room overnight resulted in the formation of the 4-ayl-1,2,3-triazole derivative 7(a–f) in yield = 50%.

The success of such 1,3-dipolar cycloaddition was supported by the spectroscopic results of the resulted 1,2,3-triazole 7, which were in agreement with its proposed structure. The ¹H-NMR spectrum of 7a as a representation example displayed two distinguishable singlets at 4.16 and 4.50 ppm of the two nonequivalent methylene groups. The singlet recorded at 8.9 ppm was attributed to the proton of the 1,2,3-triazole residue. Additionally, the ¹³C NMR spectrum revealed no signals on the sp-carbon regions, confirming their involvement in the cycloaddition reaction, and one additional signal appeared at 119.91 ppm characteristic for the CH of the triazole. The two methylene carbons (CH₂) resonated separately in the aliphatic region at 27.48 and 30.71 ppm.

2.2. Biological Evaluation

A brief screening of the synthesized compounds was tested as a direct InhA enzyme inhibitor, and the InhA inhibition (IC₅₀) was calculated. In the present study, 5b,c and 7c–f successfully and completely (100%) inhibited the InhA enzyme at 10 nM (Figure 5). Different concentrations were prepared and the IC₅₀ values were measured. 7c and 7e were the most promising agents as InhA inhibitor with IC₅₀ = 0.074 and 0.13 nM, respectively. Compared to the known IC₅₀ of Rifampicin and Isoniazid, which were reported as 0.8 nM and 54.6 nM, respectively [23], our compounds can pave the way as a new highly active TB drug (

Table 1. The inhibitory effect (IC₅₀) of the tested compounds against InhA.

Tested Compounds	IC50 (nM)
4a	53.2
4b	9.16
4c	8.16
5a	5.61
5b	0.66
5c	0.23
7a	6.18
7b	12.5
7c	0.074
7d	0.18
7e	0.13
7f	0.25

).

2.3. Molecular Modeling

2.3.1. Docking Simulations

Compounds 5c, 7d, 7c, and 7e were chosen for molecular docking studies into the binding site of inhA based on previous biological evaluation results in order to obtain insight into the hypothesized intermolecular interactions and investigate the possible binding pattern that underpins these drugs’ inhibitory effects. This was accomplished with the help of molecular operating environment software (MOE 2014.0802). The protein data bank provided the X-ray crystal structures of inhA (PDB ID: 4TRO) with its co-crystallized ligand (NAD) [24].

The top-scored conformation with the best binding interactions detected by the MOE search algorithm and scoring function was the basis for the selection of the docking poses. In addition, binding energy scores, formation of binding interaction with the neighboring amino acid residues, and the relative positioning of the docked poses in comparison to the co-crystallized ligands were the factors determining the binding affinities to the binding pockets of the enzymes.

2.3.2. Docking into inhA Active Site

With a binding energy score (S) of −9.99 kcal/mol and a root mean standard deviation (RMSD) of 1.04, compound 7c was shown to be optimally positioned in the active site of the inhA enzyme in its best-docked pose. It was lodged into the active site through a hydrogen bond of 3.61 Å between oxygen atom of the piperonal moiety and Met103. Furthermore, two hydrophobic interactions with 4.03 and 4.33 Å formed between the aromatic ring part of the 5-benzyl side chain and 1,2,3 triazol-1-yl benzoic acid with Lys165 and Ile95, respectively. In addition, the 1,2,4-triazole ring and Ile21 formed a hydrophobic interaction with 3.87 Å (Figure 6A,B).

Molecular docking studies of the target compound 7e displayed (S) −11.7 kcal/mol and (RMSD) of 1.29 revealed that the 4-morpholino ring oxygen forms a hydrogen bond of 3.34 Å with Val65. Furthermore, 1,2,3 triazol-1-yl benzoic acid forms a hydrogen bond of 3.10 Å with Ile194 along the same track, 3-thio atom part in the linker forms a hydrogen bond of 3.70 Å with Ser94. Besides, 2 Hydrophobic π-H bond interactions were encountered between 1,2,4 triazole and 4-benzylidene ring of 3.79 and 4.89 Å with Gly96 and Ser20, respectively (Figure 7A,B).

With regard to compound 7d, which showed (S) −9.63 kcal/mol and (RMSD) of 1.49, H-bonding was observed between the carbonyl oxygen component in 1H-1,2,3-triazol-1-yl)phenyl(Ethan-1-one of 3.63 Å and the Asp64. The complex formed was further stabilized by two hydrophobic interactions of 3.96 and 4.62 Å between the previous moiety and Gly96 and Ile95, respectively. This is in addition to the hydrogen bond of 3.24 Å that existed between the 3-thio atom in the methylene thio linker and Ser94 (Figure 8A,B).

Through a hydrogen bond of 3.70 Å between the thioether atom and Ser94, docking revealed that compound 5c appropriately occupied the enzyme active site. In addition, the 1,2,4-triazole ring and Ile21 have a hydrophobic interaction of 4.58 Å (Figure 9A,B).

The reported Inha-Inhibitors Lead structure B IC₅₀ = 0.057 μM was selected to docked into 4TRO active site (Figure 10).

Lead structure B was shown (S) −11.63 kcal/mol, and (RMSD) of 1.56 can occupy the active site of the enzyme through hydrogen bond of 3.19 Å between the carbonyl group part in amide side chain and Ile 194. Furthermore, two hydrophobic interaction 4.44, 4.38 Å was observed between 1,2,3-triazole ring and their benzyl moiety with Ile 16 and Ile 95, respectively (Figure 10).

The examination of the overlay complex between our most potent candidate compounds 7c and 7e and lead structure B revealed that Ile 95 in the active site can form the same hydrophobic interaction with compound 7c and lead structure B. On the other hand, Ile 194 can make the same hydrogen bond with lead structure B and compound 7e (Figure 11 and Figure 12).

The Isoniazid molecular docking study with (S) −7.81 kcal/mol and (RMSD) of 1.75 indicate that the pyridine nitrogen and NH₂ group in the hydrazide moiety form hydrogen bonds of 3.33 and 3.37 Å with Lys165 and Ile194, respectively, while hydrophobic interaction of 4.86 Å showed between pyridine ring and Phe149 (Figure 13). Besides, Rifampicin docking analysis displayed (S) −7.81 kcal/mol and (RMSD) of 1.51 showed two hydrophobic interactions of 4.23 Å and 4.31 Å formed between the naphthyl ring in rifampicin and Ser20 and Ile21, respectively. Furthermore, the carbonyl ester and hydroxyl group of rifampicin side chain form hydrogen bonds of 3.16 and 3.14 Å with Met103 (Figure 13 and Figure 14).

Molecular docking studies of the least active 7b displayed (S) −8.47 kcal/mol and (RMSD) of 1.73 revealed that 1,2,4-triazole nitrogen forms a hydrogen bond of 3.14 Å with Gly96. In addition, 1,2,3-triazole nitrogen forms a hydrogen bond of 2.96 with Thr196 Å while thioether atom forms a hydrogen bond of 2.60 Å with Gly14 (Figure 15).

The high in vitro activity of compounds 7c, d and e, as well as the explanation for the lowest activity of compound 7b, can be explained based on previous docking investigations. Rifampicin’s naphthyl ring forms a hydrophobic interaction with Ile21 and Ser20. Similarly, those two aminoacids form the same type of interaction with the 1,2,4-triazole moiety of compounds 7c, d and e. On the other hand, the docking study of the least active 7b demonstrates that there is no interaction found between previous aminoacids and the 7b 1,2,4-triazole ring, thus a decline in activity can be predicted. In addition, piperonal oxygen in compound 7c forms a hydrogen bond with Met103 Rifampicin side chain, sharing the same type of interaction with Met103. Besides, the morpholino phenyl ring in 7e forms a hydrophobic interaction with Gly96. In contrast, the thiophen ring in 7b cannot form any type of noncovalent bonding interaction with the surrounding aminoacid inside the active site.

When the activity of the intermediate 5c is compared to that of 7c, d and e, the relevance of the 1,2,3-triazole ring and/or its p-substituted phenyl ring becomes clear. The 1,2,3-triazole ring of Compound 7d creates a hydrophobic contact with Gly96 and Ile95. In addition, Ile95 forms a hydrophobic interaction with the 7c and d phenyl rings linked to the 1,2,3-triazole. Furthermore, Ile 194 forms a hydrogen bonding with 1,2,3 triazol-1-yl benzoic acid. Finally, Asp64 can form a hydrogen bond with the carbonyl oxygen component in 1H-1,2,3-triazol-1-yl) phenyl (Ethan-1-one) 7d.

3. Experimental

3.1. Chemistry

All reactions were monitored by thin layer chromatography (TLC) on silica gel using 60 F254 aluminum sheets and were visualized under UV lamp at λ = 254 nm. The melting points were recorded and are uncorrected using a Stuart Scientific Melt-Temp apparatus. The IR spectra were recorded on BRUKER spectrometer using KBr disks. The ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded using BRUKER spectrometer and TMS as an internal standard to calibrate the chemical shifts (δ) reported in ppm (see Supplementary Materials).

3.2. Synthesis of 4-Amino-5-Benzyl-2,4-Dihydro-1,2,4-Triazole-3-Thiol, 3

The reaction of methyl phenylacetate (0.01 mole, 1.50 g) with hydrazine hydrate (0.1 mole, 4.86 mL) in ethanol under reflux for 4 h the benzyl hydrazide 1 was obtained. A mixture of 1 (0.1 mole, 15.01 g) and carbon disulfide (0.15 mole, 9.06 mL) in presence of potassium hydroxide (0.15 mole, 8.41 g) under reflux gave 5-benzyl-1,3,4-oxadiazole-3-thiol 2. Treatment of 2 (0.01 mole, 1.92 g) with hydrazine hydrate (0.1 mole, 5 g) under reflux gave 4-Amino-5-benzyl-2,4-dihydro-3H-1,2,4-triazole-3-thione. White crystals; Yield = 55%; mp = 172–173 °C, lit. mp = 170 °C [25,26].

4.-(. Arylideneamino)-5-benzyl-2,4-dihydro-3H-1,2,4-triazole-3-thione, 4(a–c).

3.2.1. General Procedure

The reaction of 3 (0.01 mole, 2.06 g) with aryl aldehyde (0.01 mole) in absolute ethanol with 4 drops conc. H₂SO₄ was refluxed for 4 h. A mixture of the reaction was left to cool then poured into cold water. The precipitate was collected via filtration, washed by water, and recrystallized by ethanol.

5.-benzyl-4-((thiophen-2-ylmethylene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione, 4a.

Yield = 64.58%, white crystals, mp = 184–186 °C. IR spectrum (KBr, ύ, cm⁻¹): 1571 (C=N), 3136 (N-H). ¹H NMR spectrum (DMSO-d₆, 400 MHz): δ, ppm = 4.09 (s, 2H, -CH₂-), 7.22 (t, 2H, J = 4 Hz, Ar-H), 7.30 (d, 4H, J = 4 Hz, Ar-H), 7.74 (d, 1H, J = 4 Hz, Ar-H), 7.91 (d, 1H, J = 4 Hz, Ar-H), 10.21 (s, 1H, -N=CH-), 13.85 (s, 1H, NH). ¹³C NMR spectrum (DMSO, 100 MHz): δ, ppm = 31.19 (Ph-CH₂), 127.37, 128.94, 129.42, 133.26, 135.48, 135.99, 136.99 (8C, Ar), 150.68 (N=CH) 157.16 (C-triazole), 162.06 (C=S). Anal. Calc. for C₁₄H₁₂N₄S₂ (%): C, 55.98; H, 4.03; N, 18.65; S, 21.35. Found: C, 55.68; H, 4.21, N, 18.90; S, 21.30.

4.-((benzo[d][dioxol-4-ylmethylene)amino)-5-benzyl-2,4-dihydro-3H-1,2,4-triazole-thione, 4b.

Yield = 67%, white crystals, mp = 164 °C. IR spectrum (KBr, ύ, cm⁻¹), 1200 (C-O), 1590 (C=N), 3083 (N-H. ¹H NMR spectrum (CDCl₃, 400 MHz): δ, ppm = 4.18 (s, 2H, -Ph-CH₂-), 6.08 (s, 2H, O-CH₂-O), 7.2–7.4 (m, 8H, Ar-H), 9.84 (s, 1H, -N=CH-), 10.10 (s, 1H, NH). ¹³C NMR spectrum (CDCl₃, 100 MHz): δ, ppm = 31.43 (Ph-CH₂), 101.82 (O-CH₂-O), 106.14, 108.46, 126.67, 126.88, 127.31, 128.72, 128.98, 134.42 (10C, Ar), 148.57 (C-triazole), 151.54 (N=CH), 161.04 (C=S). Anal. Calc. for C₁₇H₁₄N₄O₂S (%): C, 60.34; H, 4.17; N, 16.56. Found: C, 60.04; H, 4.40, N, 16.94.

(E)-5-benzyl-4-((4-morpholinobenzylidene)amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione, 4c.

Yield = 44%, pale brown crystals, mp = 220–222 °C. IR spectrum (KBr, ύ, cm⁻¹): 1118 (C-O), 1228 (C-N) 1595 (C=N), 3082 (N-H), ¹H NMR spectrum (CDCl₃, 400 MHz): δ, ppm = 3.33 (t, 4H, 2CH₂, J = 4 Hz, (morpholine)), 3.89 (t, 4H, J = 4 Hz, 2CH₂(morpholine)), 4.17 (s, 1H, Ph-CH₂), 6.94 (d, 2H, J = 8 Hz, Ar-H), 7.24–7.31 (m, 5H, Ar-H), 7.74 (d, 2H, J = 8 Hz, Ar-H), 9.91 (s, 1H, -N=CH-), 10.93 (s, 1H, NH). ¹³C NMR spectrum (CDCl₃, 100 MHz): δ, ppm = 31.46 (Ph-CH₂), 47.78 (C-N-(morpholine)), 66.56 (C-O-(morpholine)), 114.25, 122.85, 127.23, 128.67, 129.03, 130.47, 131.35, 134.60 (8C, Ar), 151.50 (N=CH), 153.99 (C-triazole), 162.09 (C=S). Anal. Calc. for C₁₇H₁₄N₄O₂S (%): C, 62.31; H, 7.06; N, 18.17. Found: C, 62.47; H, 6.98, N, 18.00.

1.-Aryl-N-[3-benzyl -5-(prop-2-yn-1-yl thio)-4H-1,2,4-triazol-4-yl]methanimine, 5(a–c).

3.2.2. General Procedure

A mixture of Schiff base derivatives (0.01 mole) and propargyl bromide (0.015 mole, 1.13 mL) in the presence of a catalytic amount of triethylamine (0.015 mole, 0.002 mL) in acetone was refluxed for 8 h. The solvent was removed under reduced pressure and the product was poured into ice. It was crystallized from a mixture of ethanol and water (1:2).

(E)-N-(3-benzyl-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazol-4-yl)-1-(thiophen-2-yl)methanimine, 5a.

Yield = 60%, off white crystals, mp = 98 °C. IR spectrum (KBr, ύ, cm⁻¹): 1594 (C=N), 3247 (C≡C-H). ¹H NMR spectrum (DMSO-d₆, 400 MHz): δ, ppm = 2.49 (t, 1H, J = 2.4 Hz, C≡C-H), 3.94 (d, 2H, J = 2.4 Hz, S-CH₂), 4.19 (s, 2H, Ph-CH₂), 7.17–7.27 (m, 6H, Ar-H), 7.75 (d, 1H, J = 4 Hz, Ar-H), 7.98 (d, 1H, J = 4 Hz, Ar-H), 8.96 (s, 1H, -N=CH-). ¹³C NMR spectrum (DMSO, 100 MHz): δ, ppm = 21.39 (S-CH₂), 30.69 (Ph-CH₂), 74.87 (C≡C-H), 79.27 (C≡C-H), 126.79, 128.52, 128.74, 128.94, 133.92, 135.61, 135.64, 136.85 (8C, Ar), 144.78 (C-triazole), 151.87 (N=CH), 160.54 (C-triazole). Anal. Calc. for C₁₇H₁₄N₄S₂ (%): C, 59.24; H, 3.73; N, 17.27. Found: C, 59.07; H, 4.00, N, 16.98.

(E)-1-(benzo[d][1,3]dioxol-4-yl)-N-(3-benzyl-5-(prop-2-yn-1-ylthio)-4H-1,2,4-triazol-4-yl)methanimine, 5b.

Yield = 95%, buff crystals, mp = 140 °C. IR spectrum (KBr, ύ, cm⁻¹), 1260 (C-O), 1623 (C=N), 3219 (C≡C-H). ¹H NMR spectrum (CDCl₃, 400 MHz): δ, ppm =2.25 (s, 1H, C≡C-H), 3.98 (d, 2H, -S-CH₂-), 4.25 (s, 2H, -Ph-CH₂-), 6.09 (s, 2H, O-CH₂-O), 6.87 (d, 1H, J = 8 Hz, Ar-H), 7.10 (d, 1H, J = 8 Hz, Ar-H), 7.20–7.27 (m, 6H, Ar-H), 8.24 (s, 1H, -N=CH-). ¹³C NMR spectrum (CDCl₃, 100 MHz): δ, ppm =22.02 (S-CH₂), 31.51 (Ph-CH₂), 72.87 (C≡C-H), 77.95 (C≡C-H), 102.07 (O-CH₂-O), 106.36, 108.54, 125.91, 127.06, 127.13, 128.68, 128.87, 135.49, 145.18, 148.77 (10C, Ar), 152.23 (C-triazole), 152.75 (N=CH), 164.22 (C-triazole). Anal. Calc. for C₂₀H₁₆N₄O₂S (%): C, 63.81; H, 4.28; N, 14.88. Found: C, 64.07; H, 4.47, N, 15.25.

(E)-N-(3-benzyl-5-(prop-2-yn-1-ylthio)-1,5-dihydro-4H-1,2,4-triazol-4-yl)-1-(4-morpholinophenyl)methanimine, 5c.

Yield = 69.4%, pale brown crystals, mp = 236 °C. IR spectrum (KBr, ύ, cm⁻¹): 1169 (C-O), 1363 (C-N) 1589 (C=N), 3227 (C≡C-H). ¹H NMR spectrum (CDCl₃, 400 MHz): δ, ppm = 2.24 (s, 1H, C≡C-H), 3.32 (t, 4H, J = 4 Hz, 2CH₂(morpholine)), 3.85 (t, 4H, J = 4 Hz, 2CH₂(morpholine)), 3.95 (d, 2H, -S-CH₂), 4.22 (s, 1H, Ph-CH₂), 7.1–7.6 (m, 9H, Ar-H), 8.15 (s, 1H, -N=CH-). ¹³C NMR spectrum (CDCl₃, 100 MHz): δ, ppm = 21.82 (-S-CH₂), 31.46 (Ph-CH₂), 46.08 (C-N-(morpholine)), 66.47 (C-O-(morpholine)), 72.77 (C≡C-H), 78.08 (C≡C-H), 114.00, 121.52, 126.96, 128.62, 128.92, 130.87, 135.66, 145.24 (8C, Ar), 152.65 (C-triazole), 154.40 (N=CH), 165.28 (C-triazole). Anal. Calc. for C₂₃H₂₃N₅OS (%): C, 64.52; H, 6.65; N, 17.10. Found: C, 64.85; H, 6.33, N, 16.88.

Aryl azide, 6(a–c).

A solution of the appropriate aromatic amine (0.015 mole) was dissolved in a mixture of water (10 mL) and sulfuric acid (3 mL). The solution was cooled to 0 °C to which a solution of NaNO₂ (0.015 mole, 1.06 g) in water (3 mL) was added under constant stirring. Solution of sodium azide (0.0185 mole, 1.20 g) in water (5 mL) was added to the above-mentioned mixture. After additional 15 min of stirring at 0 °C, the formed precipitate was filtered and washed several times with water. Then it was dissolved in ethyl acetate, dried over MgSO₄, and the solvent was removed under reduced pressure (3). The product was used without crystallization. Yields and melting points of products are listed in

Table 2. M.Ps and yield % of Substituted phenyl azide, 6(a–c).

ID.	R1	M.P. (°C)	M.P. (°C) [Lit]	Yield (%)
6a	COOH	190–193	(190) [27]	80
6b	COCH3	176–178	(178–180) [27]	77

.

1-(aryl)-N-(3-[[(1-(aryl)-1H-1,2,3-triazol-4-yl)methyl]thio]-5-benzyl-4H-1,2,4-triazol-4-yl)methanimine, 7(a–f).

3.2.3. General Procedure

A mixture of propargyl derivatives (0.001 mole) and substituted azide (0.003 mole) in 15 mL DMF were stirred for 5 min to form a homogenous solution, then the mixture of (0.2 g) sodium ascorbate and (0.05 g) copper sulfate in 5 mL water was added to the homogenous solution. The reaction mixture was stirred for 24 h. The result was poured into cold water and filtrated off. The product was crystallized from acetonitrile [27].

(E)-4-(4-(((5-benzyl-4-((thiophen-2-ylmethylene)amino)-4H-1,2,4-triazol-3-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid, 7a.

Yield = 45%, off white crystals, mp = 196 °C. IR spectrum (KBr, ύ, cm⁻¹): 1493 (C=N), 1700 (C=O), 2481 (COOH). ¹H NMR spectrum (DMSO-d₆, 400 MHz): δ, ppm = 4.16 (s, 2H, Ph-CH₂), 4.50 (s, 2H, S-CH₂), 7.14–7.24 (m, 6H, Ar-H), 7.71 (d, 1H, J = 4 Hz, Ar-H), 7.79 (d, 1H, J = 4 Hz, Ar-H), 7.98–8.09 (m, 4H, Ar-H), 8.74 (s, 1H, -N=CH-), 8.91 (s, 1H, H-1,2,3triazole), 13.29 (broad, 1H, COOH). ¹³C NMR spectrum (DMSO, 100 MHz): δ, ppm 27.48 (Ph-CH₂), 30.71 (S-CH₂), 119.91 (C-triazole), 122.02, 126.72, 128.46, 128.64, 128.70, 131.29, 131.29, 133.80 (12C, Ar), 135.60, 136.72, 139.36 (C-triazole), 160.29 (COOH). Anal. Calc. for C₂₄H₁₉N₇O₂S₂ (%): C, 57.47; H, 3.82; N, 19.55. Found: C, 57.16; H, 3.98; N, 19.85.

(E)-1-(4-(4-(((5-benzyl-4-((thiophen-2-ylmethylene)amino)-4H-1,2,4-triazol-3-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)phenyl(Ethan-1-one, 7b.

Yield = 49%, off white crystals, mp = 196 °C. IR spectrum (KBr, ύ, cm⁻¹): 157c (C=N), 1677 (C=O). ¹H NMR spectrum (CDCl₃, 400 MHz): δ, ppm = 2.65 (s, 3H, COCH₃), 4.18 (s, 2H, Ph-CH₂), 4.62 (s, 2H, S-CH₂), 7.13–7.28 (m, 6H, Ar-H), 7.41 (d, 1H, J = 4.4 Hz, Ar-H), 7.61 (d, 1H, J = 4.4 Hz, Ar-H), 7.81 (d, 2H, J = 6.4 Hz, Ar-H), 8.08 (d, 2H, J = 6.4 Hz, Ar-H), 8.27 (s, 1H, -N=CH-), 8.42 (s, 1H, H-1,2,3triazole). ¹³C NMR spectrum (CDCl₃, 100 MHz): δ, ppm= 26.59 (CH₃), 27.18 (Ph-CH₂), 31.47 (S-CH₂), 119.97 (C-triazole), 121.56, 126.93, 128.12, 128.55, 128.86, 129.90, 132.68 (12C, Ar), 135.09, 136.70, 139.80 (C-triazole), 156.85 (N=CH), 196.49 (C=O). Anal. Calc. for C₂₅H₂₁N₇OS₂ (%): C, 60.10; H, 4.24; N, 19.63. Found: C, 60.47; H, 4.40; N, 19.81.

(E)-4-(4-(((4-((benzo[d][1,3]dioxol-4-ylmethylene)amino)-5-benzyl-4H-1,2,4-triazol-3-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid, 7c.

Yield = 59%, off white powder crystals, mp = 188 °C. IR spectrum (KBr, ύ, cm⁻¹), 1266 (C-O), 1448 (C=N), 1695 (C=O), 2465 (COOH). ¹H NMR spectrum (DMSO-d₆, 400 MHz): δ, ppm =4.19 (s, 2H, -Ph-CH₂-), 4.49 (s, 2H, -S-CH₂-), 6.13 (s, 2H, O-CH₂-O), 7.02 (d, 1H, J = 8 Hz, Ar-H), 7.18–8.11 (m, 11H, Ar-H), 8.58 (s, 1H, H-1,2,3-triazole) 8.71 (s, 1H, -N=CH-), 13.11 (broad, 1H, COOH). ¹³C NMR spectrum (DMSO, 100 MHz): δ, ppm =28.22 (Ph-CH₂), 31.15 (S-CH₂), 102.59 (O-CH₂-O), 106.29, 109.10, 122.38, 126.29, 127.12, 127.41, 128.89, 129.07, 136.25 (14C, Ar), 120.59, 139.78, 144.70, 148.56 (C-triazole), 152.08 (N=CH), 166.04 (COOH). Anal. Calc. for C₂₇H₂₁N₇O₄S (%): C, 60.10; H, 3.92; N, 18.17. Found: C, 60.7c; H, 4.15, N, 18.41.

(E)-1-(4-(4-(((4-((benzo[d][1,3]dioxol-4-ylmethylene)amino)-5-benzyl-4H-1,2,4-triazol-3-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)phenyl(Ethan-1-one, 7d.

Yield = 55%, off white crystals, mp = 196 °C. IR spectrum (KBr, ύ, cm⁻¹): 1210 (C-O), 1450 (C=N), 1672 (C=O). ¹H NMR spectrum (DMSO-d₆, 400 MHz): δ, ppm =2.49 (s, 3H, COCH₃), 4.21 (s, 2H, -Ph-CH₂-), 4.49 (s, 2H, -S-CH₂-), 6.11 (s, 2H, O-CH₂-O), 7.02 (d, 1H, J = 6.4 Hz, Ar-H), 7.14–8.10 (m, 11H, Ar-H), 8.12 (s, 1H, H-1,2,3-triazole) 8.57 (s, 1H, -N=CH-). ¹³C NMR spectrum (DMSO, 100 MHz): δ, ppm = 26.86 (CH₃), 27.73 (Ph-CH₂), 30.62 (S-CH₂), 102.19 (O-CH₂-O), 105.68, 108.68, 125.70, 126.74, 127.19, 128.47, 128.60, 130.04 (14C, Ar), 119.62, 139.78, 144.70, 148.65 (C-triazole). 165.95 (N=CH), 196.96 (C=O). Anal. Calc. for C₂₈H₂₃N₇O₃S (%): C, 62.56; H, 4.31; N, 18.24. Found: C, 62.49; H, 4.46, N, 18.16.

(E)-4-(4-(((5-benzyl-4-((4-morpholinobenzylidene)amino)-4H-1,2,4-triazol-3-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid, 7e.

Yield = 35%, pale yellow powder, mp = 192 °C. IR spectrum (KBr, ύ, cm⁻¹): 1232 (C-O), 1588 (C=N), 1706 (C=O), 2615 (COOH). ¹H NMR spectrum (DMSO-d₆, 400 MHz): δ, ppm = 3.29 (s, 4H, 2CH₂(morpholine)), 3.74 (s, 4H, 2CH₂(morpholine), 4.19 (s, 1H, Ph-CH₂), 4.22 (s, 2H, -S-CH₂), 6.98–8.33 (m, 13H, Ar-H), 8.45 (s, 1H, H-1,2,3-triazole), 8.71 (s, 1H, -N=CH-), 13.00 (broad, 1H, COOH). ¹³C NMR spectrum (DMSO, 100 MHz): δ, ppm = 28.08 (Ph-CH₂), 31.12 (-S-CH₂), 47.22 (C-N-(morpholine)), 66.27 (C-O-(morpholine)), 114.13, 122.51, 127.14, 128.89, 129.02, 131.05 (12C, Ar), 121.15, 136.12, 139.48, 145.09 (C-triazole), 154.57 (N=CH), 167.40 (COOH). Anal. Calc. for C₃₀H₂₈N₈O₃S (%): C, 62.05; H, 4.86; N, 19.30. Found: C, 61.75; H, 4.60, N, 19.24.

(E)-1-(4-(4-(((5-benzyl-4-((4-morpholinobenzylidene)amino)-4H-1,2,4-triazol-3-yl)thio)methyl)-1H-1,2,3-triazol-1-yl)phenyl(Ethan-1-one, 7f.

Yield = 56%, pale brown crystals, mp = 198–200 °C. IR spectrum (KBr, ύ, cm⁻¹): 1554 (C=N), 1676 (C=O). ¹H NMR spectrum (DMSO-d₆, 400 MHz): δ, ppm = 2.64 (s, 3H, CH₃), 3.29 (s, 4H, 2CH₂(morpholine)), 3.73 (s, 4H, 2CH₂(morpholine), 4.17 (s, 1H, Ph-CH₂), 4.50 (s, 2H, -S-CH₂), 6.98 (d, 2H, J = 8 Hz, Ar-H), 7.18–7.24 (m, 5H, Ar-H), 7.61 (d, 2H, J = 8 Hz, Ar-H), 7.98 (d, 2H, J = 8 Hz, Ar-H), 8.13 (d, 2H, J = 8 Hz, Ar-H), 8.46 (s, 1H, H-1,2,3-triazole), 8.75 (s, 1H, -N=CH-). ¹³C NMR spectrum (DMSO, 100 MHz): δ, ppm = 27.26 (CH₃), 27.96 (Ph-CH₂), 31.15 (-S-CH₂), 47.25 (C-N-(morpholine)), 66.27 (C-O-(morpholine)), 114.16, 121.31, 122.43, 127.11, 128.88, 129.07, 130.49, 130.99, 136.36, 136.88, 139.9 (12C, Ar), 120.16, 139.91, 144.82, 154.54 (C-triazole), 166.92 (N=CH), 197.34 (C=O). Anal. Calc. for C₃₁H₃₀N₈O₂S (%): C, 64.34; H, H,5.23; N, 19.36. Found: C, 64.34; H, 5.44, N, 19.7c.

3.2.4. Enzymatic Inhibition Experiments

M. tuberculosis InhA was overexpressed in E. coli while isoniazid and NADH were obtained from Sigma–Aldrich. The concentration of the pool INH–NAD was determined on the basis of ε₃₃₀ equaling 6900 M⁻¹ cm⁻¹. The substrate 2-trans-decenoyl-CoA concentration was determined on the basis of ε₂₆₀ equaling 22 600 M⁻¹ cm⁻¹.

For the inhibition assays with InhA, the pre-incubation reactions were performed in 80 μL (total volume) of 30 mM PIPES buffer solution, 150 mM NaCl, pH 6.8 at 25 °C containing 70 nM InhA and the tested compounds (at different concentrations). DMSO was used as a co-solvent and its final concentration was 0.5%. After 2 h of pre-incubation, the addition of 35 μM substrate (trans-2-decenoyl-CoA) and 200 μM cofactor (NADH) initiated the reaction which was measured at 25 °C and at 340 nm (oxidation of NADH) using a spectrophotometer (PG-T80, UK). Control reactions were done under the same conditions, but without the ligands. The pool of INH–NAD adducts was used as a positive control. The initial rates of the reactions were calculated. Rifampicin was used as a reference commercially known drug. The inhibition percentage of each compound was measured at 10 nM and the compounds’ inhibitory activity was expressed as the IC₅₀ inhibition of InhA activity with respect to the control experiments [28].

Docking Study

Computer-aided docking experiments were performed using Molecular Operating Environment (MOE 2014.0802) software (Chemical Computing Group, Montreal, QC, Canada).

Preparation of the Protein Crystal Structures

The protein data bank provided the X-ray crystal structures of inhA (PDB ID: 4TRO) with its co-crystallized ligand (NAD).

Redundant chains, water molecules, and any surfactants were discarded, explicit hydrogen atoms were added to the receptor complex structure, and partial charges were calculated. The preparation was completed with structure preparation module employing protonated 3D function. The co-crystal ligands were extracted from their corresponding proteins and used as reference molecules for the validation study.

Preparation of the Selected Compounds for Docking

The target compounds were constructed using the builder module of MOE. The compounds were then collected in a database and prepared by adding hydrogens, calculating partial charges and energy minimizing using Force field MMFF94x.

The top-scored conformation with the best binding interactions detected by the MOE search algorithm and scoring function was the basis for the selection of the docking poses. In addition, binding energy scores, formation of binding interaction with the neighboring amino acid residues, and the relative positioning of the docked poses in comparison to the co-crystallized ligands were the factors determining the binding affinities to the binding pockets of the enzyme.

4. Conclusions

A new set of hybrid derivatives containing 1,2,4-and click modifiable 1,2,3 triazole moieties were designed and synthesized in order to target M. tuberculosis’ enoyl-acyl carrier protein reductase InhA. In vitro study results revealed a successful and complete (100%) inhibition for some compounds at certain concentration. Of the investigated compounds, 5b, 5c, 7c, 7d, 7e, and 7f completely inhibited the InhA enzyme at 10 nM. Different concentrations were used to calculate the IC₅₀ values. The results showed that compounds 7c and 7e were the most promising InhA inhibitors, with IC₅₀ values of 0.074 and 0.13 nM, respectively, so our compounds have the potential to pave the way for new highly active anti-TB medications.