Synthesis of significant nitroimidazole-triazole-pyridine hybrids and their molecular modeling as antimicrobial agents

2.1. 1-(2-Chloroacetyl)-2-methyl-5-nitro-1H-imidazole (2)

A 250 mL conical flask was charged with a suspension of 2-methyl-5-nitro-1H-imidazole (1) (1.90 g, 15 mmol) and potassium carbonate (2.07 g, 15 mmol) in dry acetone (40 mL). To this suspension, chloroacetyl chloride was added dropwise at 5-10°C with stirring for 4 h. The obtained solid after dilution with 50 mL of cold water was filtered, washed with cold ethanol, and used directly in the next step after drying.

Yield = 2.17 g (71.3%), m.p. = 189-190°C. IR (ν/cm^-1): 1636 (C=N), 1694 (C=O). ¹H NMR (δ/ppm): 2.58 (s, 3H, imidazole-CH₃), 4.40 (s, 2H, CO-CH₂-Cl), 8.04 (s, 1H, imidazole-H). Calculated analysis for C₆H₆ClN₃O₃ (203.01): C, 35.40; H, 2.97; N, 20.64%. Found: C, 35.52; H, 2.92; N, 20.58%.

2.2. 1-(2-Azidoacetyl)-2-methyl-5-nitro-1H-imidazole (3)

A mixture of 1-(2-chloroacetyl)-2-methyl-5-nitro-1H-imidazole (2) (1.62 g, 8 mmol) and sodium azide (0.65 g, 10 mmol) was stirred in dry DMSO (25 mL) at 50°C for 12 h. The mixture was diluted with 50 mL of cold water, and then the organic layer was extracted with ethyl acetate, dried with anhydrous sodium sulphate, and evaporated. The freshly obtained azide compound 3 was directly used in the next step.

Yield = 0.92 g (54.7%), m.p. = 223-224°C. IR (ν/cm^-1): 1698 (C=O), 2108 (-N=N=N). ¹H NMR (δ/ppm): 2.58 (s, 3H, imidazole-CH₃), 4.38 (s, 2H, CO-CH₂-N₃), 8.08 (s, 1H, imidazole-H). Calculated analysis for C₆H₆N₆O₃ (210.05): C, 34.29; H, 2.88; N, 39.99%. Found: C, 34.18; H, 2.94; N, 39.85%.

2.3. 2-(4-((1-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl) methoxy)benzylidene)malononitrile (5)

A mixture of 1-(2-azidoacetyl)-2-methyl-5-nitro-1H-imidazole (3) (0.94 g, 4.5 mmol) and 2-((4-propargyloxy)benzylidene)malononitrile (4) [14] (0.83 g, 4 mmol) was dissolved in 30 mL of dimethylformamide (DMF). To the obtained solution, sodium ascorbate (40 mol %) and copper sulphate pentahydrate (20 mol %) had been introduced in sequence. The mixture was stirred at 35-40°C for 12 h and then diluted with 100 mL ice-cold water. The precipitate that formed was collected and washed with 5 mL of ethyl acetate to furnish the precursor, 2-(4-(triazolyl-methoxy)benzylidene)-malononitrile compound 5.

Yield = 1.41 g (84.3%), m.p. = 160-161°C. IR (ν/cm^-1): 1688 (C=O), 2217 (C≡N). ¹H NMR (δ/ppm): 2.57 (s, 3H, imidazole-CH₃), 4.94 (s, 2H, N-CH₂-CO), 5.17 (s, 2H, O-CH₂-triazole), 6.93 (d, J = 8.5 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 7.90 (s, 1H, CH=C-CN), 8.11 (s, 1H, imidazole-H), 8.27 (s, 1H, triazole-H). MS for C₁₉H₁₄N₈O₄ [M]⁺: m/z = 418.2 (16.73%). Calculated analysis for C₁₉H₁₄N₈O₄ (418.11): C, 54.55; H, 3.37; N, 26.78%. Found: C, 54.68; H, 3.43; N, 26.70%.

2.4. 1-Aryl 6-amino-3,5-dicyano-4-(4-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-oxo-1,2-dihydropyridine hybrids 7a-d.

An RB-flask (100 mL) was charged with a solution of 2-(4-(triazolyl-methoxy)benzylidene)-malononitrile compound 5 (0.83 g, 2 mmol) in 30 mL ethanol and piperidine (0.1 mL). The corresponding N-aryl cyanoacetamide compound 6a, 6b, 6c, or 6d [15] (2 mmol) was added, and the mixture was refluxed for 2 h. The obtained solid was filtered and crystallized from an EtOH/DMF mixture (4:1) to afford the corresponding imidazole-triazole-pyridine hybrids 7a, 7b, 7c, and 7d, respectively.

2.5. Ethyl 2-cyano-3-(4-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)acrylate (9)

A mixture of 1-(2-azidoacetyl)-2-methyl-5-nitro-1H-imidazole (3) (0.94 g, 4.5 mmol) and ethyl 2-cyano-3-((4-propargyloxy)phenyl)acrylate (8) [16] (1.02 g, 4 mmol) was dissolved in 30 mL DMF. To the obtained solution, sodium ascorbate (40 mol %) and copper sulphate pentahydrate (20 mol %) had been introduced in sequence. The mixture was stirred at 35-40°C for 12 h and then diluted with 100 mL ice-cold water. The precipitate that formed was collected and washed with 5 mL of ethyl acetate to furnish the precursor, ethyl 2-(4-(triazolyl-methoxy)phenyl)-acrylate compound 9.

Yield = 1.54 g (82.8%), m.p. = 145-146°C. IR (ν/cm^-1): 1721 (C=O), 1690 (C=O), 2218 (C≡N). ¹H NMR (δ/ppm): 1.27 (t, 3H, OCH₂CH₃), 2.58 (s, 3H, imidazole-CH₃), 4.25 (t, 2H, OCH₂CH₃), 4.92 (s, 2H, N-CH₂-CO), 5.18 (s, 2H, O-CH₂-triazole), 6.94 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.96 (s, 1H, CH=C-CN), 8.10 (s, 1H, imidazole-H), 8.25 (s, 1H, triazole-H). Calculated analysis for C₂₁H₁₉N₇O₆ (465.14): C, 54.19; H, 4.11; N, 21.07%. Found: C, 54.30; H, 4.16; N, 21.12%.

2.6. 1-Aryl-3,5-dicyano-6-hydroxy-4-(4-((1-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-2-oxo-1,2-dihydropyridine hybrids 10a-d.

An RB-flask (100 mL) was charged with a solution of ethyl 2-(4-(triazolyl-methoxy)phenyl)-acrylate compound 9 (0.93 g, 2 mmol) in 30 mL of ethanol and piperidine (0.1 mL). The corresponding N-aryl cyanoacetamide compound 6a, 6b, 6c, or 6d [15] (2 mmol) was added, and the mixture was refluxed for 2 h. The obtained solid was filtered and crystallized from an EtOH/DMF mixture (4:1) to afford the corresponding imidazole-triazole-pyridine hybrids 10a, 10b, 10c, and 10d, respectively.

2.7. DFT modeling

All density functional theory (DFT) calculations have been carried out using the Gaussian 09W software [17]. Geometrical optimization of isolated hybrids has been achieved at the B3LYP level of theory with the 6-311⁺⁺G(d, p) basis set [18]. The optimized structures, frontier molecular orbitals (FMOs), and electronic parameters were resolved by GaussView [19].

2.8. Antimicrobial assay

The antimicrobic efficacy of the produced nitroimidazole derivatives was assessed by measuring both inhibition zones (IZ) and minimum inhibitory concentrations (MIC) against different pathogens. This encompassed Gram (+ve) bacteria, including S. aureus (ATCC 29213) and B. subtilis (ATCC 19659), Gram (-ve) bacteria like S. typhimurium (ATCC 13311) and E. coli (ATCC 25922), along with fungal strains such as C. albicans (ATCC 14053). The antibacterial agent was produced in a stock solution at a predetermined concentration. To establish a range of concentrations in the microtiter plate wells, the stock solution was serially diluted twice in the broth medium (Mueller-Hinton broth for bacteria and RPMI-1640 for fungi). The concentration range usually covers a number of orders of magnitude. A bacterial and fungal suspension’s turbidity was adjusted to a certain McFarland standard in order to provide a standardized inoculum of the test microorganism. To ensure uniform dispersion, a set volume of the standardized inoculum was applied to each well of the microtiter plate. For eighteen to twenty-four hours, the microtiter 96-well plates were incubated at the proper temperature (30°C for fungus and 37°C for bacteria). Following incubation, the microtiter plate was examined to determine the MIC of the antimicrobial agent that prevents the bacteria from growing visibly. The reference drugs were selected based on their established clinical use and relevance to the test microbial strains. Chloramphenicol, a broad-spectrum antibiotic, and Cephalothin, a first-generation cephalosporin, were employed as standards for antibacterial assays to allow for comparison with general as well as resistant bacteria. The use of two classes of standards allows for a more relevant comparison of the potency of the synthesized hybrids. Cycloheximide, a potent inhibitor of eukaryotic protein synthesis, was used as the standard reference for antifungal assay against C. albicans. The results for the drug references Chloramphenicol, Cephalothin, and Cycloheximide, obtained under identical experimental conditions (see the supporting information file) [20]. All the antimicrobial assays were performed in triplicate to reduce opportunities for error and maximize reproducibility and reliability of data.

2.9. Molecular docking

Molecular docking experiments were performed to determine the binding of interactions, focusing on the binding affinities of the new nitroimidazole-hybrids towards PDB: 1BDD protein, utilizing M.O.E. 2019. The E. coli UDP-N-acetylenolpyruvoyl glucosamine reductase (MurB) enzyme (PDB: 1BDD) was selected as the molecular target for the docking experiments. MurB is a crucial enzyme in the cytoplasmic peptidoglycan biosynthetic pathway in bacteria, an activity that is not present in humans, and hence making it an interesting and validated target for novel antibacterial research [21]. The use of one, clearly defined bacterial target provides the opportunity for regular and comparative examination of the mode of binding of all the synthesized hybrids against a therapeutically useful protein. While the initial mechanism of native nitroimidazoles is bio-reduction, the hybrids here are novel chemical entities. Nitroimidazole unit in these compounds is a pharmacophore that modulates redox potential or adds new sites of interaction, but the primary postulated mechanism for the hybrid structure is inhibition of critical enzymes like MurB with assistance from triazole and pyridine units. Docking against MurB attempts to confirm this hypothesis. However, preparation of the protein included water and co-crystallized ligand removal, addition of hydrogen atoms, and the assignment of partial charges using the AMBER10: EHT force field. The geometries of the hybrids synthesized were energy-minimized using the MMFF94x force field. The binding site of the native ligand was utilized to define the docking site. Docking simulations were executed using the Triangle Matcher placement method and London dG scoring function, followed by energy refinement using the Forcefield refinement method and GBVI/WSA dG scoring function. The conformations produced were compared based on binding affinity (S, kcal/mol) and interaction patterns with key amino acid residues.

3.1. Preparation of imidazole-triazole-pyridine hybrids 7 and 10

The synthetic approach of our targeting imidazole-triazole-pyridine hybrids 7a-d and 10a-d is demonstrated in Schemes 1-3. This strategy is initiated by the preparation of the precursor, 1-(2-azidoacetyl)-2-methyl-5-nitro-1H-imidazole (3), by chloroacetylation of 2-methyl-5-nitro-1H-imidazole (1) upon treatment with chloroacetyl chloride in stirred acetone and potassium carbonate. The produced 1-(2-chloroacetyl)-2-methyl-5-nitro-1H-imidazole (2) was dissolved in dimethyl sulfoxide and stirred with sodium azide to furnish the corresponding 1-azidoacetyl-imidazole compound 3 (Scheme 1). The relatively low yield (54.6%) of compound 3 was accounted for by partial hydrolysis on aqueous workup. Different solvent systems and bases were tested; however, potassium carbonate and dry DMSO resulted in the maximum yield without decomposition.

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Scheme 1.

Synthesis of 1-(2-azidoacetyl)-2-methyl-5-nitro-1H-imidazole (3).

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Scheme 2.

Synthesis of imidazole-triazole-pyridine hybrids 7a-d.

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Scheme 3.

Synthesis of imidazole-triazole-pyridine hybrids 10a-d.

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The azido-imidazole compound 3 has been employed as a precursor for the production of triazole ring system in the targeting 2-(4-(triazolyl-methoxy)benzylidene)-malononitrile compound 5 through its 1,3-dipolar cycloaddition reactions with the terminal alkyne, 2-((4-propargyloxy)benzylidene)malononitrile (4) °C in the presence of copper sulphate and sodium ascorbate in DMF as a solvent. The Huisgen cycloaddition reactions ran with pristine regioselectivity towards the 1,4-disubstituted triazole isomer, which was confirmed by the solo triazole proton resonance at δ 8.25-8.35 ppm and absence of any other triazole signals in the ¹H NMR spectra.

The 2-(4-(triazolyl-methoxy)benzylidene)-malononitrile compound 5 was refluxed with different N-aryl cyanoacetamide compounds 6a-d [15] in ethanol and piperidine to produce the conforming imidazole-triazole-pyridine hybrids 7a-d (Scheme 2). All the newly prepared hybrids 7a-d were fully characterized using IR, ¹H NMR, ¹³C NMR, and mass analyses. The mechanism was initiated by Michael in addition to the reaction of N-aryl cyanoacetamide compound 6 (donor) to unsaturated nitrile compound 5 (acceptor). The formed intermediate (A) underwent intramolecular addition of the N-H group of cyanacetamide part into the nitrile group and tautomerization, followed by air oxidation process to pick up the final targeted product, imidazole-triazole-pyridine hybrid 7. The infrared spectrum of hybrid 7a (as an example) revealed the characteristic absorption frequencies of carbonyl groups at 1651 and 1683 cm^-1. The nitrile group (-C≡N) was detected at its expected frequency at 2211 cm^-1. The absorptions of the amino function (-NH₂) were observed at 3341 and 3213 cm^-1. The ¹H NMR signals were detected as singlet signals at δ 2.61, 4.97, and 5.18 ppm for the protons of methyl (imidazole-CH₃) and two methylene groups, respectively. The aromatic protons were identified by the doublet and multiplet signals at δ 7.10-7.56 ppm. The protons of the amino group were recorded by the singlet signal at δ 7.83 ppm. The detected singlet signals at δ 8.08 and 8.31 ppm were attributed to the protons of imidazole-C and triazole-C, respectively. The mass analysis displayed the molecular ion peak for the formula C₂₈H₂₀N₁₀O₅ at m/z = 576.1 with a relative intensity of 24.38%.

In Scheme 3, we aimed at replacing one of the nitrile functionalities in the first series with an ester group. Ethyl 2-(4-(triazolyl-methoxy)phenyl)-acrylate compound 9 was synthesized in the same way that 2-(4-(triazolyl-methoxy)benzylidene)-malononitrile compound 5 was. Thereafter, the azido-imidazole compound 3 was further reacted with an ethyl 2-cyano-3-((4-propargyloxy)phenyl)acrylate (8) [16] through Azide-alkyne Huisgen cycloaddition to produce the target imidazole-triazole precursor 9. Furthermore, the addition of N-aryl cyanoacetamide 6a-d [15] to a refluxing solution of ethyl 2-(4-(triazolyl-methoxy)phenyl)-acrylate compound 9 in ethanol and piperidine resulted in the formation of imidazole-triazole-pyridine hybrids of the type 10a-d, in which the pyridine ring substituted with hydroxy group at position number six (Scheme 3). The structure of the prepared hybrids of imidazole-triazole-pyridine 10a-d was well characterized and confirmed through interpretation of the spectral analysis data. The IR spectrum of hybrid 10a (as an example) exhibited absorption frequencies of carbonyl groups at 1658 and 1685 cm^-1. The presence of nitrile and hydroxy groups was indicated by their expected frequencies at 2220 and 3304 cm^-1, respectively. The ¹H NMR spectrum displayed the protons of protons of methyl (imidazole-CH₃) and two methylene groups as singlet signals at δ 2.58, 4.96, and 5.21 ppm, respectively. The aromatic protons were identified by the doublet and multiplet signals at δ 7.12-7.58 ppm. The singlet signals at δ 8.06, 8.30, and 8.87 ppm were attributed to the protons of imidazole-C, triazole-C, and the hydroxy group, respectively.

3.2. Molecular modeling

The nitroimidazole malononitrile 5 and acrylate 9, along with their derivatives, amino-pyridyl 7a-d and hydroxy-pyridyl 10a-d, respectively, exhibited resemble non-planar structures (Figures 2-3). The dihedral angle data of these analogues revealed that:

• i)

  The nitro-group atoms have been shifted away from the imidazole’s plane, e.g., C²_(Imz)-N¹_(Imz)-C⁵_(Imz)-NO_2(Imz) ≈ -169.0°, whereas the methyl group displayed less shift (177.7°) (Table S1).

• ii)

  Likewise, the oxoethyl atoms were dislocated from the imidazole and triazole planes, CO_(OxEt)-N¹_(Imz)-C²_(Imz)-N³_(Imz) ≈ 171.0°, CH_2(OxEt)-CO_(OxEt)-N¹_(Imz)-C⁵_(Imz) ≈ 38.3°, C⁵_(Trz)-N¹_(Trz)-CH_2(OxEt)-CO_(OxEt) ≈ 96.3° and CH_2(OxEt)-N¹_(Trz)-N²_(Trz)-N³_(Trz) ≈ 176.5°, respectively.

• iii)

  As well, the ether linkage has been positioned away the triazole’s plane, N_3(Trz)-C_4(Trz)-CH_2(Ethr)-O_(Ethr) ≈ -173.2°, while it was coplanar with the phenyl ring, which was slanted on the pyridyl nucleus, C²_(Ph)-C¹_(Ph)-C⁴_(Py)-C³_(Py) ≈ -51.0° (Table S1).

• iv)

  Otherwise, the two cyano- and oxo-substituents were slightly moved from the pyridyl’s plane, C⁵_(Py)-C⁴_(Py)-C³_(Py)-CN¹_(Py) ≈ 176.9° and C³_(Py)-C⁴_(Py)-C⁵_(Py)-CN²_(Py) ≈ 176.2°, while the oxo-, amino- and hydroxy groups were coplanar.

• v)

  Else, the phenyl substituent was practically perpendicular to the pyridyl ring, C²_(Py)-N¹_(Py)-C¹_(PhPy)-C²_(PhPy) ≈ -91.8°.

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Figure 2.

Compounds 5 and 7a-d optimized structures.

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Figure 3.

Compounds 9 and 10a-d optimized structures.

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Moreover, the DFT estimated bond lengths and angles, in comparison to single crystal X-ray of comparable compounds [22], revealed a valuable agreement as the discrepancies were <0.12 Å and <10.0° (RMSD = 0.02-0.03 and 4.5-5.7), respectively. The disparity could be ascribed to that the DFT computations investigated a sole gaseous molecule where no columbic interactions (Tables S2-S3).

As they significantly manipulate the molecule’s attitude to either afford or gain electrons [23], the HOMO-LUMO’s energies, frontier molecular orbitals (FMOs), has gained noteworthy importance. The explored analogues displayed comparable configurations of their FMOs; for instance, the HOMO of nitroimidazole malononitrile 5 and acrylate 9 had been spanned on the phenyl malononitrile and acrylate, respectively, whereas their LUMO was confined on the oxoethyl nitroimidazole moiety (Figure 4 and S1). The corresponding amino-pyridyl 7a-d and hydroxy-pyridyl 10a-d displayed a coincided composition of the HOMO, which centered on the amino- and hydroxy-dicyanopyridyl methoxyphenyl fragment, and LUMO, which centered on the oxoethyl nitroimidazole moiety. The nitrophenyl derivatives 9c and 10c showed an alternative LUMO configuration, wherein centered on the nitrophenyl group (Figure 4 and S1).

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Figure 4.

The 3D-plot of FMOs for compounds 5, 7a, 7c, 9, 10a, and 10c.

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Hence, the nitroimidazole analogues presented close values for the HOMO-LUMO energies, E_H = -6.96 - -6.37 and E_L = -3.75 - -3.44 eV, respectively. As a result, the observed energy gap (ΔE_H-L) was spanned from 2.93 to 3.33 eV, where the conjugates 7b and 7a had equal and the lowest value, while 5 exhibited the maximum, following the arrangement 7b = 7a < 7d < 9 < 10c < 10b < 7c < 10a < 10d < 5. Accordingly, the data indicated that: i) the aminopyridyl hybrids 7 had lower energy gap than hydroxypyridyl’s 10; ii) In aminopyridyl series 7a-d, the nitrophenyl (7c) exhibited the lowest energy gap, whereas the corresponding hydoxypyridyl (10c) had the maximum value; iii) In both cases, the following order was obeyed methoxyphenyl (b) < phenyl (a) < chlorophenyl (d) (Figure 5).

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Figure 5.

FMO’s energy representation of explored hybrids.

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Moreover, the FMO’s energy values have been exploited in the evaluation of particular chemical reactivity signifiers [24]. Such as, the electronegativity (χ) data divulged that conjugates 10c and 7b owned the highest and lowest standards (5.31 and 4.90 eV), respectively. Also, hybrid 5 unveiled the maximum hardness (η = 1.66 eV) and minimum softness (δ = 0.60 eV). The electrophilicity index (ω), 8.01 (10b) - 9.07 (10c) eV, endorsed the satisfactory electrophilicity character of the hybrids, ω > 1.5 eV, with better tendency to deliver than receiving electrons, as they displayed lower electron donating (ω⁺) than accepting (ω^-) power [25] (Table 1).

The molecule’s charge-transfer and electronegativity may be comprehended more from studying the Mulliken’s atomic charges [26]. The studied derivatives results unveiled that the imidazolyl nitrogen atoms N¹_(Imz) and N³_(Imz) had alternative negative charges, -0.169 - -0.178 and -0.223 - -0.225, respectively. Likewise, the triazolyl nitrogen atoms N²_(Trz) and N³_(Trz) were negatively charged, while the nitrogen N¹_(Trz) exhibited a small positive charge (0.014-0.016), which may be attributed to the electron-withdrawing influence of the bonded oxoethyl group (OC_(OxEt) = -0.267 - -0.271) (Table S4). Moreover, the imidazolyl nitro-group acquired different charges, where the nitrogen NO_2(Imz) was positively charged (0.345), and the oxygen atoms had close negative charges (O¹N_(Imz) = -0.362 and O²N_(Imz) = -0.307). Also, the ether oxygen O_(Ethr) gained a negative charge (-0.352), which is clearly higher than the corresponding oxoethyl atom. In compounds 5 and 9, the cyano-groups nitrogen atoms have been negatively charged, NC¹_(Mln) = -0.147, NC²_(Mln) = -0.156, and NC_(Acr) = -0.172, respectively. Comparably, the cyano-groups nitrogen atoms in aminopyridyl conjugates 7a-d exhibited higher charge than the corresponding in parent 5, NC¹_(Py) = -0.173 - -0.181 and NC²_(Py) = -0.185 - -0.193, while those belonging to hydroxypyridyl’s displayed lower values, -0.166 - -0.175. Further, the pyridyl ring substituents data revealed that the oxo-atom had the lower negative charge (Ox_(Py) = -0.355) than the hydroxy-oxygen and amino-nitrogen (OH_(Py) = -0.494 and NH_2(Py) = -0.766), respectively (Table S4).

Elsewhere, the hybrids’ molecular polarizability (α_total), hyperpolarizabilities (β_total), and dipole moment (μ) were appraised [27] to afford an illustration of the electronic density distribution (Eq. 1) and softness, which essentially influence the intermolecular interactions [28] (Eqs. 1 and 2).

The nitroimidazole derivatives unveiled widespread dipole moment (μ) oscillated from 2.70 D (10c) to 8.81 D (7b), which represented 1.97-6.42 times of urea, reference material (Table 2). Whilst, the analog 5 displayed the minimum polarizability value, but the conjugate 7c possessed the utmost, α_total = 3.23 and 4.26×10^-23 esu, respectively. On the contrary, the first-order hyperpolarizability suggested that compound 10c has the minimum value (β_total = 4.50×10^-30 esu) and the analogue 10b has the top (β_total = 15.72×10^-30 esu). In contrast to the urea’s value (Ahmed et al., 2008), most of the considered conjugates presented larger hyperpolarizability, 12.02-41.93 times (Table 2).

3.3. Antimicrobial assay

The antimicrobial investigation for the nitroimidazole-hybrids was carried out and revealed significant variations in activity based on structural changes (Table 3). Meanwhile, nitroimidazole-hybrid 5 revealed proper antimicrobial effectiveness against S. aureus, B. subtilis, and E. coli, through an MIC = 12.5 µg.mL^-1 and IZ value = 39 mm, while showing lower effectiveness towards S. typhimurium (MIC = 50 µg.mL^-1) and C. albicans (MIC = 50 µg.mL^-1). This proposes moderate effectiveness, especially against Gram (+ve) bacteria. However, through the series 7a-d, nitroimidazole-hybrid 7a showed the strongest effectiveness among the examined hybrids, an MIC of 12.5 µg.mL^-1 MIC and IZ values of 46–52 mm, indicating strong efficacy. However, through the series 7a-d, nitroimidazole-hybrid 7a showed the strongest effectiveness among the examined hybrids, with MIC = 6.25 µg.mL^-1 versus S. aureus and B. subtilis, MIC = 12.5 µg.mL^-1 towards E. coli, MIC = 25 µg/mL versus S. typhimurium, and IZ values of 46–52 mm, suggesting enhanced potency due to structural modifications. Nitroimidazole-hybrid 7b demonstrated similar antibacterial activity to nitroimidazole-hybrid 7a (MIC = 6.25-12.5 µg.mL^-1 and IZ = 33–43 mm), suggesting its potential as a versatile antimicrobial agent. Nitroimidazole-hybrid 7c, despite showing high inhibition zones, exhibited weaker effectiveness overall, with MIC = 50 µg.mL^-1 against S. aureus and E. coli, and MIC =100 µg.mL^-1 vs. S. typhimurium and C. albicans, proposing reduced in activity due to structural differences. Nitroimidazole-hybrid 7d revealed a slight improvement in the activity compared to nitroimidazole-hybrid 7c, with MIC values ranged from 12.5 to 50 µg.mL^-1 against bacteria but was weak against C. albicans (MIC = 100 µg.mL^-1). Nitroimidazole-hybrid 9 developed as the strongest antifungal agent, unveiling a forceful MIC = 3.125 µg.mL^-1 against C. albicans comparable to the cycloheximide reference. Nitroimidazole-hybrid 9 also showed strong efficacy vs. B. subtilis (MIC = 6.25 µg.mL^-1), with modest effects against other established bacteria. Among the series 10a-d, nitroimidazole-hybrid 10a showed a moderate antibacterial effectiveness, with MIC = 12.5 µg.mL^-1 against Gram (+ve) bacteria and MIC values from 25 to 50 µg.mL^-1 towards Gram (-ve) bacteria, even though disclosing weak antifungal effectiveness (MIC = 100 µg.mL^-1). Nitroimidazole-hybrid 10b exhibited slightly weaker antibacterial effectiveness, mainly against S. aureus (MIC = 25 µg.mL^-1), while sustaining adequate Gram-negative bacterial inhibition. Nitroimidazole-hybrid 10c displayed enhanced activity against E. coli (MIC = 6.25 µg.mL^-1) and S. typhimurium (MIC = 6.25 µg.mL^-1), making it one of the most effective Gram (-ve) bacterial inhibitors, however, its antifungal effectiveness stay put sensible (MIC = 50 µg.mL^-1). Nitroimidazole-hybrid 10d recorded the wide-spectrum agent, with MIC = 3.125 µg/mL against B. subtilis and E. coli, MIC = 6.25 µg.mL^-1against S. aureus, and MIC = 12.5 µg.mL^-1against S. typhimurium and C. albicans. This recommended that nitroimidazole-hybrid 10d is recognized as a promising for further progress as a broad-spectrum antimicrobial agent.

Finally, a general trend relating the MIC and IZ values existed, whereby compounds with lower MICs (and consequently greater potency) tended to exhibit larger inhibition zones. A few exceptions to this trend for individual compound-strain combinations can be accounted for by differences that exist between the two assays. Disk diffusion (IZ) test measures the percentage of inhibition of diffusion and overall growth in agar medium, while the broth microdilution (MIC) test determines the exact lowest concentration that inhibits growth in liquid medium. Solubility of the compound, rate of diffusion through the agar, and specific growth kinetics in different media can cause discrepancies between the two tests.

3.4. Structural activity relationship

The antimicrobial effectiveness of the synthesized nitroimidazole-hybrids was prejudiced according to their structural alterations, especially the presence of electron-donating or withdrawing groups, and the functionalization of the dihydropyridine scaffold. Nitroimidazole-hybrid 5, with a malononitrile moiety, demonstrated moderate antibacterial effectiveness over (MIC = 12.5 µg/mL) against Gram (+ve) and Gram (-ve) bacterial strains but was less effective towards fungi (C. albicans, MIC = 50 µg.mL^-1), representing limited fungal effectiveness. Meanwhile, nitroimidazole-hybrid 7a, including a dihydropyridine-3,5-dicarbonitrile core, exhibited enhanced antibacterial effectiveness, mainly towards S. aureus, B. subtilis, and S. typhimurium, indicating that the electron-rich aminopyridine group shows a responsibility in the bacterial inhibition. However, nitroimidazole-hybrid 7b has an additional methoxyphenyl branch, maintaining equivalent effectiveness towards Gram (+ve) bacteria while viewing an improvement on the antifungal effectiveness (C. albicans, MIC = 12.5 µg.mL^-1), indicating that the methoxy branch enhanced their fungal inhibition. On the other hand, nitroimidazole-hybrid 7c, joining to a nitrophenyl group, presented a significant reduction in the antibacterial inhibition against S. aureus and E. coli, probable due to steric hindrance affecting bacterial penetration, whereas its antifungal effect remained weak. Likewise, nitroimidazole-hybrid 7d, including a chlorophenyl moiety, retained moderate antibacterial effectiveness but demonstrated weaker antifungal inhibitions (C. albicans, MIC = 100 µg/mL), indicating that the electron-withdrawing chloro-group might limit fungal binding interactions. Systematic SAR evaluation of the N-aryl substituents of hybrids 7a-d and 10a-d is presented in Table 4. Data indicate that EDGs like methoxy in hybrids 7b and 10b tended to maintain or even increase broad-spectrum activity, while potent EWGs like nitro in hybrids 7c and 10c tended to reduce antibacterial activity perhaps through a shift in electronic properties affecting binding with targets or intracellular penetration. Notably, the most potent broad-spectrum agent was the p-chlorophenyl analogue 10d with a moderately EWG, suggesting that there needs to be an optimum level of electronic and steric effects to achieve maximum activity.

A clear relationship was observed between the DFT calculated parameters of the synthesized conjugates and their antimicrobial effectiveness. Compounds with smaller HOMO-LUMO energy gap separations (ΔE_H-L) and greater electrophilicity (ω) generally exhibited stronger inhibition effects, indicating that efficient charge transfer enhanced the interaction between the hybrid and microbial targets. For instance, the derivatives 7c (ΔE_H-L = 3.17 eV, ω = 8.31 eV) and 7a (ΔE_H-L = 2.93 eV, ω = 8.50 eV) displayed the most pronounced activity (44-54 mm), which can be attributed to their higher molecular softness and improved electron delocalization. On the other hand, the analogues 9 and 10b, characterized by slightly wider energy gaps and lower ω values, were less active. These observations suggest that molecular flexibility and high electrophilic character play key roles in improving antimicrobial efficiency. Therefore, fine-tuning the electronic distribution across the π-conjugated system represents an effective strategy for enhancing biological performance within this hybrids framework.

3.5. Molecular docking

However, the molecular docking results highlighted on the bindings affinities and the interactions of the produced nitroimidazole-hybrids with the target PDB: 1BDD protein (Table S5). For molecular docking validation, a reference drug Cephalosporin was employed. This reference possesses the central β-lactam structure common to Cephalothin and is a known antibiotic inhibitor of penicillin-binding proteins (PBPs). Although not the main target of cephalosporins, docking set a baseline binding property and pattern for a recognized antibiotic class within the same protein scaffold utilized by our hybrids to enable a relative assessment of docking scores. Though the key of interactions includes (H-acceptor, H-donor, π-π stacking, and pi-H) bonds with definite amino acids such as Lys5, Lys8, Asn4, Asn24, Asn44, Pro39, and Ser40. Nitroimidazole-hybrid 5 also exhibited strong binding (-7.1215 kcal/mol), interacting with Asn24 via a hydrogen bond (3.21 Å) (Figure 6). Nitroimidazole-hybrids 7a, 7c , and 7d demonstrated multiple hydrogen bonds involving nitrile and triazole groups, with binding scores (S = -6.7898, -6.9322, and -6.5246 kcal/mol, respectively (Figure S2). Meanwhile, the anisyl-ring in nitroimidazole-hybrid 7b contributed to π-H interaction with Lys5, with a binding score (S = -7.0394) (Figure 6).

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Figure 6.

Binding images between nitroimidazole-hybrid 5, 7b and 10d with PDB: 1BDD.

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Similarly, the imidazole-rings in nitroimidazole-hybrid 9 donated to π-H binding, over binding scores (S = -6.0546 kcal.mol^-1) with Leu52 (Figure S2). Moreover, nitroimidazole-hybrids 10a-c showed moderate binding affinities (S= -6.5245 to -6.3506 kcal.mol^-1), with interactions involving carbonyl and nitrile groups (Figure S2). However, nitroimidazole-hybrid 10d developed as the most potent binder, with a strong docking score (S = -7.2123 kcal.mol^-1, forming a H-bond with Tyr15 (2.98 Å) and a π-H stacking with Gln11 (4.06 Å) (Figure 6). Furthermore, the reference drugs, Chloramphenicol and Cephalosporin, displayed lower binding affinities (S = -5.7485 and -6.7186 kcal.mol^-1, respectively), primarily interacting with Ser40, Lys5, and Lys8 residues (Figure S2). These results suggest that the thiazolidinone-triazole hybrids, particularly those with nitrile and carbonyl groups, exhibit enhanced binding affinities and diverse interaction profiles compared to the reference compounds.

Finally, from the total SAR, docking data, and structural features, one can hypothesize an anticipated mechanism of action. The conserved nitroimidazole-triazole-oxoethyl chain appears to act as an anchor, potentially binding to the active site of the MurB enzyme, as predicted from docking. The dihydropyridine-N-aryl spacer domain variable is likely to control spectrum and potency; electron-donating systems, as in hybrid 7b possibly prefer more extensive contacts, while specific EWGs (such as p-chloro moiety in hybrid 10d can optimize fit as well as electronic complementarity in a sub-pocket to yield the resultant high potency. The imidazole nitro group, while not necessarily committed to its standard bio-reductive mechanism here, is participating in the electron affinity of the molecule and might be participating in significant hydrogen bonding or dipole interactions, as seen in the docking orientations with residues Asn24 and Tyr15.

3.6. Pharmacokinetic analysis

The SwissADME analysis of the novel nitroimidazole-hybrids revealed detailed pharmacokinetic properties, highlighting their potential as drug candidates (Table S6 and Figure S3). Nitroimidazole-hybrid 5 (M. Wt.: 418.37) exhibited high solubility, moderate lipophilicity (iLogP: 2.19), and a TPSA of 168.23 Ų, indicating good membrane permeability. With low GI absorption, no BBB permeability, and no Pgp substrate activity, it is suitable for peripheral targets, adhering to Lipinski’s rule with one violation and achieving a bioavailability score of 0.55. Similarly, nitroimidazole-hybrid 9 (M. Wt.: 465.42) demonstrated high solubility, a favorable iLogP (2.5), and a TPSA of 170.74 Ų, with low GI absorption and no BBB permeability, making it a promising candidate for non-CNS applications. It also adhered to Lipinski’s rule with one violation and scored 0.55 in bioavailability. In contrast, nitroimidazole-hybrids 7a-d and 10a-d displayed more complex profiles, with higher molecular weights (576.52–622.50), moderate solubility, and iLogP values ranging from 2.09 to 2.71. These compounds showed low GI absorption, no BBB permeability, and, except for nitroimidazole-hybrids 7b and 10b, were identified as Pgp substrates, which could limit their intracellular concentrations. Their TPSA values (210.46–262.07 Ų) and increased rotatable bonds (RT: 9–10) contributed to higher polarity and two Lipinski violations, resulting in uniformly low bioavailability scores (0.17). Among these hybrids, nitroimidazole-hybrid 7a (M. Wt.: 576.52) and nitroimidazole-hybrid 10a (M. Wt.: 577.51) revealed moderate-solubility and affirmative Pgp substrate, while nitroimidazole-hybrid 7b (M. Wt.: 606.55) and hybrid 10b (M. Wt.: 607.53) weren’t Pgp substrates, hypothetically presenting recompenses for certain therapies. Nitroimidazole-hybrids 7c (M. Wt.: 621.52) and 10c (M. Wt.: 622.50) showed the highest molecular-weights and TPSA values, further dropping their bioavailability, however nitroimidazole-hybrids 7d (M. Wt.: 610.97) and 10d (M. Wt.: 611.95) reflected the profiles of nitroimidazole-hybrids 7a and 10a. While nitroimidazole-hybrids 5 and 9 stand displayed the most drug-like candidates, the nitroimidazole-hybrids 7a-d and 10a-d current chances for structural variation to improve their pharmacokinetic possessions, predominantly for targeted therapies where Pgp effluence may be alleviated. This comprehensive analysis provides a foundation for further development of these hybrids as potential therapeutic agents.