Pyrrolyl Pyrazoline Carbaldehydes as Enoyl-ACP Reductase Inhibitors: Design, Synthesis and Antitubercular Activity

2.1. General Information

Melting points of the synthesized compounds were determined using digital melting point apparatus (Shital, Mumbai) and are uncorrected. FTIR spectra were recorded on a Bruker FTIR spectrophotometer using KBr pellets. The ¹H and ¹³C NMR spectra were recorded on a Bruker AVANCE II at 400 and 100/75 MHz, respectively; chemical shifts are expressed in parts per million (δ ppm) relative to tetramethylsilane. Abbreviations used to describe the peak patterns are: (b) broad, (s) singlet, (d) doublet, (t) triplet, and (m) multiplet.

Mass spectra (MS) were recorded in a JEOL GCMATE II GC-Mass spectrometer and Schimadzu QP 20105 GC-Mass spectrometer. 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 UV lamps. Chromatographic purifications were performed on Merck silica gel (70-230 mesh).

2.2. General Procedure for the Synthesis of 1-(4-(1H-Pyrrol-1-yl) Phenyl)-3-Phenylprop-2-en-1-one (3a)

To a stirred alcoholic solution of pyrrolyl acetophenone 1 (1.57g, 0.01 mol), an alcoholic solution of substituted aldehydes 2(a-o) (0.01 mol) and 40% sodium hydroxide (8 mL) were added with continuous stirring for 24-30 h. The resultant mixture was poured onto crushed ice and neutralized with hydrochloric acid. The separated solid was filtered, washed with water, dried and purified by column chromatography on a silica gel with ethyl acetate/petroleum ether (6:4) mixture as the eluent. Similar procedure was used to synthesize compounds (3b-o) [16].

2.3. General Procedure for the Synthesis of 3-(4-(1H-Pyrrol-1-yl) Phenyl)-5-Phenyl-4,5-Dihydro-1H-Pyrazole-1-Carbaldehydes (4a)

A mixture of chalcones (3a) (0.01 mmol), hydrazine hydrate 99% (0.01 mmol) and formic acid (5 mL) was refluxed for 18 h to complete the reaction as confirmed by TLC. Then the reaction mixture was cooled to ambient temperature; the separated solid was filtered, washed with cold ethanol, dried and purified by column chromatography on a silica gel with ethyl acetate/petroleum ether (6:4) mixture as the eluent to afford the compounds in high purity with good yields. The same procedure was used to synthesize compounds (4b-o).

(Yield 85%). mp 178-180^oC; FTIR (KBr): υ, cm^-1 3138, 3029 (Ar-H), 1688 (C=O), 1607 (C=N); ¹H NMR (400 MHz, CDCl₃): δ, ppm: 3.28 (dd, J=4.88, 4.84 Hz, 1H, pyrazoline-C₄-H_a), 3.88 (dd, J=11.80, 11.76 Hz, 1H, pyrazoline-C₄-H_b), 5.60 (dd, J=4.80 Hz, 1H, pyrazoline-C₅-H_x), 6.41 (t, 2H, pyrrole-C₃, C₄-H), 7.17 (t, 2H, pyrrole-C₂, C₅-H), 7.28-7.49 (m, 7H, bridging phenyl-C₂, C₆ and ph-C₂, C₃, C₄, C₅, C₆-H), 7.83 (t, 2H, bridging phenyl-C₃, C₅-H), 9.00 (d, J = 0.72 Hz, 1H, -CHO); ¹³C NMR (100 MHz, CDCl₃): δ, ppm: 159.87, 154.62, 142.17, 133.60, 129.48, 128.66, 127.97, 120.14, 111.25, 55.71, 39.26; MS (EI): m/z = found 315 [M⁺]; calcd. 315.14.

3.1. Molecular Docking Using Surflex-Dock

Molecular docking was performed to elucidate the type of binding of the compounds to afford uncomplicated information for further structural optimization. Surflex-Dock that adopted an experimental scoring function and a patented searching engine [32, 33] was employed in molecular docking studies. The crystal structure of M. tuberculosis enoyl reductase (InhA) complexed with 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (PDB ID 4TZK, 1.62 Å X-ray resolution) was downloaded from the Brookhaven Protein Database (PDB http://www.rcsb.org/pdb). During the preparation of protein for docking study, ligands and water molecules present in the crystal structure were removed (except co-factor NAD⁺), then the enzyme 4TZK was assigned with polar hydrogens and united atom Amber7 FF99. Then, ligand-based mode was used to produce the “protomol”, keeping the threshold and bloat parameters at their default values of 0.50 and 0 Å all the inhibitors were docked within the prepared protein.

Interaction mode of the ligand present in the crystal structure was used as a standard docked model against 4TZK PDB. The maximum of 20 numbers of maximum orientations per ligand was set with no constraints to perform molecular docking. Docking complex was believed to show the ligand-receptor interactions, which was selected based on three criteria: (i) docking score of the orientation possessed the highest docking score, (ii) orientation of aromatic rings of the ligand into the active site in a similar manner with the co-crystallized ligand orientations, and (iii) preservation of H-bonding interactions with Tyr158 and Co-factor NAD⁺. For comparative study of the designed molecules, the following parameters were estimated using the C-Score module of the Sybyl-X 2.0, D_score [34], PMF_score [35], G_score [36] and Chem_score [37].

4.1. Antitubercular Activities

Microplate almar blue assay method was used to study the inhibition of M. tuberculosis H₃₇Rv of all the synthesized compounds [38]. The 96 wells containing plate charged with 100 mL of Middlebrook 7H9 broth and serial dilution of compounds with the drug concentrations of 0.2, 0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 and 100 mg/mL were added directly on the plate, sealed with parafilm and incubated for 5 days at 37^o C. Then a mixture (1:1) of almar blue reagent and 10% Tween 80 (25 mL) were added to the plate and incubated for 24 h. Bacterial growth was interpreted by the development of pink color, while blue color suggests no bacterial growth. The MIC, defined as the lowest drug concentration, prevented the color change from blue to pink. Table 1 reveals anti-TB activity data expressed in MIC.

4.2. MTT-Based Cytotoxicity Activity

Cellular conversion of MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide] into a formazan product [39] was used to estimate cytotoxic activity (IC₅₀) of some of the compounds against cell-line A549 (lung adenocarcinoma) up to concentrations of 100 µg/mL using 5×10³cells/well in a 96-well flat-bottom micro plate and maintained at 37⁰C in 95% humidity and 5% CO₂ for overnight using paclitaxel as the positive control. The IC₅₀ values are the averages (± SEM) of three independent experiments, which are presented in Table 2.

5.1. Chemistry

Synthesis of all the compounds was carried out as depicted in Scheme (1). The Paal-Knorr synthesis involved the reaction of diketone like 2,5-dimethoxy tetrahydrofuran with amine, which is among the most conventional method for the synthesis of heterocyclic pyrrole ring. Hence, (4-pyrrol-1-yl)acetophenone (1) was obtained by forming pyrrole ring using the amino group present at the para- position of aromatic ring of 4-amino acetophenone in the presence of dried glacial acetic acid. The required key intermediates viz., chalcones (3a-o) were synthesized using Claisen-Schmidt condensation reaction between (4-pyrrol-1-yl)acetophenone (1) and substituted aldehydes (2a-o) in the presence of 40% alcoholic sodium hydroxide. Thus, synthesized chalcones (3a-o) were then cyclized to pyrazolyl carbaldehyde (4a-o) in a solvent free atmosphere by treating with hydrazine hydrate and formic acid.

All the synthesized compounds were characterized by FTIR, ¹H NMR, ¹³C NMR and mass spectroscopy. FTIR showed the presence of carbonyl and C=N groups as strong absorption bands in the regions 1664-1688 cm^-1 and 1521-1607 cm^-1, respectively. In ¹H NMR spectra resonating signals of formyl group (-CHO), protons are observed as doublets in the range δ 8.89-9.04 ppm. The pyrazoline protons at the 4-H position appeared as two doublet of doublet/multiplet from δ 3.11-3.97 and that of protons at the 5-H position appeared as a doublet of doublet/triplet from δ 5.52-6.26. The pyrrole protons at the 3-H and 4-H positions appeared as a triplet from δ 6.38-6.41 ppm and those protons at the 2-H and 5-H positions resonated as triplet/multiplet from δ 7.06-7.17 ppm. The ¹³C NMR spectra exhibited characteristic signals of carbonyl carbon atom in the range of δ 159.87-163.96 ppm. The mass spectra showed a precise molecular ion peak data for the respective compounds.

5.2. Molecular Docking Results

To examine the mechanism of anti-TB activity and to understand the comprehensive intermolecular interactions between the synthesized compounds and the enzyme, molecular docking studies were performed on the crystal structure of M. tuberculosis enoyl reductase (InhA) complexed with 1-cyclohexyl-N- (3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide (PDB ID 4TZK, 1.62 Å X-ray resolution) using surflex-dock programme of sybyl-X 2.0 software. Based on the greater level of resistance associated with INH isolates against InhA, docking studies were performed on InhA complex with 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-oxopyrrolidine-3-carboxamide, which indicates the presence of drug-receptor interactions. All the 15 inhibitors were docked into the active site of ENR as shown in Figs. (2A and B). The predicted binding energies of the compounds are listed in Table (3). Superimposition of compounds 4i and 4g with ligand of 4TZK is depicted in Fig. 3.

The interaction of 4TZK ligand with the enzyme depicted in Figs. (4A-C) shows that oxygen atom of carbonyl group present on pyrrolidine ring has two H-bonding interactions with the hydrogens of TYR158 and NAD⁺ (-C=O ------ H-TYR158, 1.84 Å; H-NAD⁺, 2.20 Å). As depicted in Figs. (5A-C), the oxygen atom of formyl group present at the 2^nd position of pyrazoline ring of compound 4i exhibited two H-bonding interactions with the hydrogens of TYR158 and NAD⁺ (-CHO ------ H-TYR158, 2.22 Å; H-NAD⁺, 1.99 Å), while the oxygen atom of -OCH₃ present at the 4^th position of aromatic ring makes H-bonding interactions with MET98 (-OCH₃ ----- H-MET98, 1.98 Å). In Figs. (6A-C), the oxygen atom of formyl group present at the 2^nd position of pyrazoline ring of compound 4g makes two H-bonding interaction with the hydrogens of TYR158 and NAD⁺ (-CHO ------ H-TYR158, 2.21 Å; H-NAD⁺, 2.01 Å), while the oxygen atom of -OCH₃ present at the 4^th position of aromatic ring exhibited H-bonding interaction with MET98 (-OCH₃ ----- H-MET98, 1.92 Å). On the other hand, hydrophilic (SER94, SER123, SER20, NAD500, SER19, THR196, ARG195, GLN100, GLN216, GLN214, GLU219, TYR158, ASN159, THR162, ASP150, LYS165, ASP148) and hydrophobic (GLY96, PHE97, MET98, PRO99, TRP160, MET161, ILE21, MET199, ALA157, PRO156, ILE215, ILE194, PRO193, PHE149,ALA198, LEU197) amino acid residues surrounded over the representative compound 4i are represented in (Figs. 7A and B).

All the compounds showed consensus scores in the range of 9.05-2.65, indicating the summary of all forces of interactions between ligands and InhA. Charge and van der Waals interactions between protein and ligands varied from -77.96 to -168.11. Helmholtz free energies of interactions for protein ligands atom pairs range between -33.56 and -66.28. However, its H-bonding, complex (ligand-protein), and internal (ligand-ligand) energies range from -151.65 to -285.29, while those values of -25.78 to -45.80 indicate the ligands due to H-bonding, lipophilic contact, and rotational entropy as well as intercept terms. These scores indicate that molecules preferentially bind to InhA in comparison to reference 4 TZK ligand (Table 3). In general, it is observed that -OCH₃ and CHO groups make the H-bond at substrate binding site and the presence of electron donating or withdrawing substitution on the aromatic ring attached to pyrazoline moiety may be helpful to show the activity, while those of pyrrole and pyrazoline moieties help to occupy or penetrate the molecule at the active sites.

The Lipinski’s ‘rule of 5’ [40] was calculated for compounds 4(a-o) to evaluate their drug likeness property, which in general states that an orally active drug did not have more than one violation viz., (i) no more than five H-bond donors, (ii) molecular weight < 500 daltons, (iii) cLogP not > 5, and (iv) no more than 10 H-bond acceptors [38]. We have calculated theoretical cLogP, molecular weight (MW) and number of H-bond donors and acceptors using Sybyl-X.2.0. Examination of the results summerized in (Table 4), suggests that compounds 4(a-o) satisfied the range of physicochemical parameters established by the Lipinski’s rule.