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

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.


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     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 drugresistant 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,

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 (Schemes 1). The synthetic strategies adopted in the present work are depicted in Schemes 1 and 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. 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 IC 50 values of 0.906 and 0.057 µM, respectively, [4].
The first target structure-1 that exhibit good InhA inhibitory activity with IC 50 = 0.13 nM was designed by connecting both 1,2,4-triazole scaffold with the click modifiable 1,2,3-triazole via the flexible SCH 2 -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 IC 50 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.
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 1 H-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 13 C 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 (CH 2 ) resonated separately in the aliphatic region at 27 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 1 H-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 13 C 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.

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 7cf 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, respec-Scheme 2. Synthesis of novel 1,2,3-triazoles tethered Schiff bases of 4-amino-5-phenylmethyl-1,2,4triazole motif 7(a-f).

Biological Evaluation
A brief screening of the synthesized compounds was tested as a direct InhA enzyme inhibitor, and the InhA inhibition (IC 50 ) 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 IC 50 values were measured. 7c and 7e were the most promising agents as InhA inhibitor with IC 50 = 0.074 and 0.13 nM, respectively. Compared to the known IC 50 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). tively. 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).  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].
Rif 4a 4b 4c 5a 5b 5c 7a 7b 7c 7d 7e 7f Tested compounds  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.

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).
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.

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 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 hydro- 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 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 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). 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). 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 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). 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). 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 The reported Inha-Inhibitors Lead structure B IC50 = 0.057 μM was selected to docked into 4TRO active site ( Figure 10).  The reported Inha-Inhibitors Lead structure B IC50 = 0.057 μM was selected to docked into 4TRO active site ( 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 (Figures 11 and 12). 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 (Figures 11 and 12).     The Isoniazid molecular docking study with (S) −7.81 kcal/mol and (RMSD) of 1.75 indicate that the pyridine nitrogen and NH 2 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 (Figures 13 and 14). 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 (Figures 13 and 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). 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 Ri- 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.

(B)
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.

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 1 H NMR (400 MHz) and 13 C 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).

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). 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 NaNO 2 (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 abovementioned 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 MgSO 4 , 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.

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.

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 IC 50 values. The results showed that compounds 7c and 7e were the most promising InhA inhibitors, with IC 50 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.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.