Design, Synthesis and Molecular Docking of Novel Acetophenone-1,2,3-Triazoles Containing Compounds as Potent Enoyl-Acyl Carrier Protein Reductase (InhA) Inhibitors

New medications are desperately needed to combat rising drug resistance among tuberculosis (TB) patients. New agents should ideally work through unique targets to avoid being hampered by preexisting clinical resistance to existing treatments. The enoyl-acyl carrier protein reductase InhA of M. tuberculosis is one of the most crucial targets since it is a promising target that has undergone extensive research for anti-tuberculosis drug development. A well-known scaffold for a variety of biological activities, including antitubercular activity, is the molecular linkage of a1,2,3-triazole with an acetamide group. As a result, in the current study, which was aided by ligand-based molecular modeling investigations, 1,2,3-triazolesweredesigned and synthesized adopting the CuAAC aided cycloaddition of 1-(4-(prop-2-yn-1-yloxy)phenyl)ethanone with appropriate acetamide azides. Standard spectroscopic methods were used to characterize the newly synthesized compounds. In vitro testing of the proposed compounds against the InhA enzyme was performed. All the synthesized inhibitors completely inhibited the InhA enzyme at a concentration of 10 µM that exceeded Rifampicin in terms of activity. Compounds 9, 10, and 14 were the most promising InhA inhibitors, with IC50 values of 0.005, 0.008, and 0.002 µM, respectively. To promote antitubercular action and investigate the binding manner of the screened compounds with the target InhA enzyme’s binding site, a molecular docking study was conducted.


Introduction
Triazoles, a class of nitrogen-containing heterocycle, are well known as intriguing structural motifs with fascinating applications in many areas of disciplines such as materials research, synthetic organic chemistry, and drug discovery [1].
On the other hand, different aryl derivatives can be linked to the 1,2,3-triazole scaffold via an acetamide linker, as shown by lead structures ( Figure 3C,D), which have MICs of 6.25 and 0.08 g/mL, respectively, and have shown good antitubercular activity [34,35].
In light of these facts, and as a follow-up to our efforts on the synthesis of such bioactive scaffolds [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18], we have anticipated to design and synthesize a novel 1,4-disubstituted 1,2,3-triazolecontainingacetamide in order to link aryl derivatives to the triazole motif and an acetyl phenoxy methyl moiety substituting the 4-position of the triazole ring. The resulting 1,2,3-triazole adducts were investigated as inhibitors of the NADH-dependent enoyl-acyl carrier protein reductase enzyme. InhA contributes to the manufacture of key components of the mycobacterial cell walls through involvement in the type II fatty acid biosynthetic pathway. Among the studied compounds, one demonstrated good inhibitory activity against the InhA enzyme with an IC 50 of 0.002 µM and was considered the most active molecule. Molecular modeling was also used to evaluate the pattern of binding of the tested drugs to the target enzyme. Pharmaceuticals 2022, 15, x FOR PEER REVIEW 3 of 21

Chemistry
The propargylation of 4-hydroxyacetophenone (1) with 3-bromoprop-1-yne has been carried out in dimethylformamide as a solvent and in the presence of a catalytic amount of potassium carbonate as the basic catalyst, which resulted in the formation of precursor 1-(4-(prop-2-yn-1-yloxy) phenyl)4thenone (2) in 91% yield, Scheme 1. The formation of O-propargylated acetophenone 2 as the key intermediate has been inferred from its spectral information (IR, 1 H NMR and 13 C NMR).
The IR spectrum unveiled a significant characteristic absorption bands, confirming the proposed structure. Sharp bands at 2100 and 3210 cm −1 were detected, which were ascribed to the acetylenic hydrogen carbons (C≡C) and (≡C-H) groups, respectively. In addition, the vanishing of the hydroxyl absorption band proved that it had been propargylated.
Examination of 1 H and 13 C NMR spectra of alkyne showed the existence of an alkyne side chain; based on the presence of a diagnostic doublet at δH 4.77 ppm attributed to OCH2 protons. The ≡C-H proton resonated as a broad singlet at δH 2.57 ppm overlapping with the signal containing three protons of the methyl group. The aromatic protons resonated in the aromatic area as expected (δH 7.03-7.97 ppm).
In contrast, the existence of significant propargyl carbon signals (OCH2 and C≡C) at δC 51.08, 71.48 and 73.00 ppm was also compatible with such acetylenic side chains. The signals recorded at δC 109.68-156.51 ppm were assigned to the aromatic carbons of the phenyl ring. The carbonyl carbon was also observed in the downfield area at δC 192. 09 ppm.
The formation of O-propargylated acetophenone 2 as the key intermediate has been inferred from its spectral information (IR, 1 H NMR and 13 C NMR).
The IR spectrum unveiled a significant characteristic absorption bands, confirming the proposed structure. Sharp bands at 2100 and 3210 cm −1 were detected, which were ascribed to the acetylenic hydrogen carbons (C≡C) and (≡C-H) groups, respectively. In addition, the vanishing of the hydroxyl absorption band proved that it had been propargylated.
Examination of 1 H and 13 C NMR spectra of alkyne showed the existence of an alkyne side chain; based on the presence of a diagnostic doublet at δ H 4.77 ppm attributed to OCH 2 protons. The ≡C-H proton resonated as a broad singlet at δ H 2.57 ppm overlapping with the signal containing three protons of the methyl group. The aromatic protons resonated in the aromatic area as expected (δ H 7.03-7.97 ppm).
In contrast, the existence of significant propargyl carbon signals (OCH 2 and C≡C) at δ C 51.08, 71.48 and 73.00 ppm was also compatible with such acetylenic side chains. The signals recorded at δ C 109.68-156.51 ppm were assigned to the aromatic carbons of the phenyl ring. The carbonyl carbon was also observed in the downfield area at δ C 192.09 ppm.
The combination of IR, 1 H NMR, and 13 C NMR spectral analysis has verified the formation of triazoles 9-14. Compound 9 has been used as a model in order to assign, absorption bands in the IR spectra and resonance peaks in the NMR spectra.
Triazole 9 has an IR spectrum that matches its assigned structure. The disappearance of the peaks belonging to C≡C and ≡C-H absorption bands and the appearance of a sharp absorption at 3300 cm −1 attributed to the NH group of the acetamide linkage indicates in part that the cycloaddition reaction has taken place.
The 1 H NMR spectrum of triazole 9 reveals the disappearance of the signal attributed to the ≡C-H proton of its precursor O-alkyne 2 and the appearance of a diagnostic singlet at δ H 8.25 ppm, assigned to the CH-1,2,3-triazole proton. The spectrum also shows two pairs of singlets for the OCH 2 and NCH 2 protons at δ H 5.24 and 5.41 ppm, respectively. Moreover, the assigned CONH proton at δ H 10.29 ppm was also in agreement with the assigned 1,2,3-triazole 9. Further assignment of the aromatic protons revealed that they were resonated at their appropriate positions and listed in the experimental section.
In addition, the 13 C NMR spectrum of compound 9 also revealed the absence of the two sp-carbons and the presence of OCH 2 and NCH 2 -carbons at δ C 52.96 and δ C 61.74 ppm, respectively. A new signal also appeared at δ C 165.23 assigned to the carbonyl amide carbon (CONH). All sp 2 carbons were observed in the aromatic region (See experimental section).
On the other hand, the click synthesis has also been evidenced by the 19 F NMR spectrum of compound 9, which showed multiple signals recorded between δ F −124.75 to −124.69 ppm, which confirmed the presence of an aromatic fluorine atom in the structure.

Biological Evaluation
A brief screening of the synthesized compounds was tested as a direct InhA enzyme inhibitor. The InhA inhibition (IC 50 ) was calculated ( Table 1). All synthesized inhibitors completely inhibited the InhA enzyme at a concentration of 10 µM (Figure 4), excelling in terms of activity, with a rifampicin IC 50 of value 8.5 µM. Moreover, all synthesized inhibitors showed activity better than isoniazid IC 50 of value 0.054 µM except compound 13 IC 50 of value 0.084 µM [36]. Furthermore, 9,10, and 14 were the most promising InhA inhibitors, with IC 50 values of 0.005, 0.008, and 0.002 µM, respectively.

Docking Simulations
All synthesized compounds are subjected to molecular docking studies into the binding site of inhA in order to understand hypothesized intermolecular interactions and consider the potential binding pattern that supports these drugs' inhibitory effects. Molecular operating environment software was used to accomplish this (MOE 2016.0802). The pro-

Molecular Modeling Docking Simulations
All synthesized compounds are subjected to molecular docking studies into the binding site of inhA in order to understand hypothesized intermolecular interactions and consider the potential binding pattern that supports these drugs' inhibitory effects. Molecular operating environment software was used to accomplish this (MOE 2016.0802). The protein data bank provided the X-ray crystal structures of InhA (PDB ID: 4UVD) with its co-crystallized ligand (HRW) (PDB DOI: 10.2210/pdb4UVG/pdb).
The docking poses were chosen based on the top-scored conformation with the best binding interactions found by the MOE search algorithm and scoring function. Furthermore, binding energy scores, creation of binding interactions with neighboring amino acid residues, and relative placement of docked poses in respect to co-crystallized ligands all played a role in defining binding affinities to the enzyme binding pockets and docking to the InhA active site.
The most active compounds, 9, 10, and 14, were found to be at the best-docked position in the active site of the inhA enzyme (4UVD), with binding energy scores (S) of −8.63, −8.97, and −9. kcal/mol and RMSDs of 1.78, 1.85, and 1.91, respectively. With key amino acids inside the pocket, these compounds can create a non-covalent bonding interaction. For instance, the compound 9 triazole ring forms a hydrophobic bond of 3.86 Å with Met98. Similarly, the compound 10 triazole ring forms a hydrogen bond of 3.25 Å with Met98. Along the same track, the compound 14 4-methoxy benzyl ring can interact via a hydrophobic bond of 4.59 Å with met98. Moreover, Met103 forms hydrogen bonds of 4.15 Å and 3.83 Å with compound 9 triazole ring and acetamide nitrogen of compound 14, respectively. Furthermore, Met161 shares hydrogen bonds of 4.35 and 3.88 Å with acetamide linker of compounds 10 and 14, respectively. Figures 5A,B, 6A,B and 7A,B. Accordingly, the importance of triazole scaffold and acetamide linker in future design of InhA inhibitors cannot be neglected. Compounds 11,12, and 13, on the other hand, which exhibit less invitro activity, showed main interactions with Met98. For example, the acetamide oxygen of compounds 11 and 12 forms hydrogen bonds of 3.12 Å, whereas compound 13, triazole ring, forms a hydrophobic interaction of 4.09 Å with Met98. There were no interactions between compounds 11, 12, and 13 and Met 103 and Met 161, which may explain why these compounds had lower in vitro activity ( Figures 7A,B, 8A

General Information
All reagents and solvents used were of the highest quality of analytical reagent grade and were used without further purification. Melting points were measured on a Stuart Scientific SMP1 and are uncorrected. TLC was performed on UV fluorescent Silica gel Merck 60 F254 plates, and the spots were visualized using a UV lamp (254 nm). SHI-MADZU FTIR-Affinity-1S spectrometer was used for identification of functional groups in the range of 400-4000 cm −1 . The NMR spectra were run with Bruker spectrometers (500 and 400 MHz) in the presence of TMS as internal reference. All azides used in this study were prepared according to reported procedures [37][38][39][40] 4.1.2. Synthesis Andof 1-(4-(Prop-2-Yn-1-Yloxy) Phenyl) Ethan-1-One (2) A mixture of 4-hydroxyacetophenone (1) (10 mmol) in dimethylformamide (10 mL) and potassium carbonate (12 mmol) was stirred for 1 h. Then, propargyl bromide (1.2 mmol) was added, and the stirring was continued for 4 h at 80 °C until the consumption of the starting material as indicated by TLC (hexane-ethyl acetate). After cooling, the mixture was poured onto crushed ice water and the precipitate formed was filtered, washed with water, dried and crystallized from ethanol to give the targeted O-propargylated acetophenone 2 as pale yellow powder in 91% yield, mp: 150-151 °C IR (υ, cm

General Information
All reagents and solvents used were of the highest quality of analytical reagent grade and were used without further purification. Melting points were measured on a Stuart Scientific SMP1 and are uncorrected. TLC was performed on UV fluorescent Silica gel Merck 60 F254 plates, and the spots were visualized using a UV lamp (254 nm). SHIMADZU FTIR-Affinity-1S spectrometer was used for identification of functional groups in the range of 400-4000 cm −1 . The NMR spectra were run with Bruker spectrometers (500 and 400 MHz) in the presence of TMS as internal reference. All azides used in this study were prepared according to reported procedures [37][38][39][40] 4.1.2. Synthesis Andof 1-(4-(Prop-2-Yn-1-yloxy) phenyl) ethan-1-one (2) A mixture of 4-hydroxyacetophenone (1) (10 mmol) in dimethylformamide (10 mL) and potassium carbonate (12 mmol) was stirred for 1 h. Then, propargyl bromide (1.2 mmol) was added, and the stirring was continued for 4 h at 80 • C until the consumption of the starting material as indicated by TLC (hexane-ethyl acetate). After cooling, the mixture was poured onto crushed ice water and the precipitate formed was filtered, washed with water, dried and crystallized from ethanol to give the targeted O-propargylated acetophenone 2 as pale yellow powder in 91% yield, mp: 150-151 • C IR (υ, cm   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 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, PG Instruments lmited woodway lane, Leicester, 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 µM and the compounds' inhibitory activity was expressed as the IC 50 inhibition of InhA activity with respect to the control experiments [41].

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: 4UVD) with its co-crystallized ligand (HRW). 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 ligand (HRW) was 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
To target M. tuberculosis' enoyl-acyl carrier protein reductase, the CuAAC of 1-(4-(prop-2-yn-1-yloxy) phenyl) ethanone with suitable acetamide azides was employed to develop and synthesize the 1,2,3-triazole molecular series. At a dose of 10 µM, all of the synthetic inhibitors completely inhibited the InhA enzyme and outperformed rifampicin in terms of activity. The most promising InhA inhibitors were 9,10, and 14, which had IC 50 values of 0.005, 0.008, and 0.002 µM, respectively. As a result, our compounds may open the path for new, extremely effective anti-TB drugs.