Synthesis and molecular docking of novel pyrazole, pyrimidine, and pyridine derivatives as potent antimicrobial, antibiofilm, and anticancer agents

Chemistry

General:

The Gallen Kamp melting point instrument was used to measure each melting point, and the results were uncorrected. A Pye-Unicam SP-3–300 infrared spectrophotometer was used to record the FTIR spectra, which were then represented in wavenumber (cm^–1). Using TMS as an internal standard in deuterated dimethyl sulfoxide (DMSO-d₆), ¹H-NMR spectra were recorded at 300 and 400 MHz and ¹³C-NMR spectra at 100 MHz on a Varian Gemini spectrometer. Chemical changes were linked to the solvents and are expressed in δ. Every coupling constant (J) value is expressed in hertz. The units of chemical shifts (δ) are ppm. A CHN analyzer was used for elemental analysis, and every compound was within ± 0.4 of the theoretical values. Thin-layer chromatography (TLC) sheets coated with UV fluorescent silica gel Merck 60 F₂₅₄ plates were used to monitor the reactions, and a UV lamp was used to observe the results.

Synthesis of 3-(furan-2-yl)-1-(4-nitrophenyl)prop-2-en-1-one (1)

With constant stirring, 10 ml of a 20% sodium hydroxide solution was added to a solution of p-nitroacetophenone (5 mmol, 0.82 g) in 15 ml of absolute ethanol. Furfural (5 mmol, 0.48 g) was then added to the reaction mixture. TLC was used to monitor the reaction, which was agitated overnight at 5–10 °C. The reaction liquid was put onto crushed ice and neutralized with HCl solution (1N) once the reaction was finished. The comparable pure product 1 was obtained as yellow crystals in a respectable yield of 88%, m.p. 140–142 °C, after the product was filtered and recrystallized from 1,4-dioxane. Anal. Calcd. for C₁₃H₉NO₄ (243.05): C, 64.20; H, 3.73; N, 5.76. Found: C, 64.07; H, 3.46; N, 5.72. FTIR (KBr, ν cm⁻¹): 1659 (C = O), 1587 (C = C). ¹H-NMR (300 MHz, DMSO‑d₆) δ (ppm): 8.34 (d, Ha, 2H J = 9.00 Hz), 8.25 (d, Hb, 2H, J = 9.00 Hz), 7.61 (d, Hd, 1H, J = 15.3 Hz), 7.49 (d, Hc, 1H, J = 15.6 Hz), 7.94–7.14 (m, 3H, Ar–H of furyl ring). MS (m/z, %): 243 (M^{∙ +}; 17.52%).

Synthesis of 5-(furan-2-yl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole (2)

Compound 1 (5 mmol, 1.22 g) and hydrazine hydrate (10 mmol, 0.49 g) were combined with ethanol (10 ml) and refluxed for six hours. After the reaction was completed, the reaction mixture was poured onto crushed ice and neutralized with 1N hydrochloric acid solution. A pure product 2 in the form of deep yellow crystals was obtained by collecting the solid product and recrystallizing it from ethanol, yield 82%, m.p. 122–124 °C. Anal. Calcd. for C₁₃H₁₁N₃O₃ (257.08): C, 60.70; H, 4.31; N, 16.33. Found: C, 60.58; H, 4.29; N, 16.07. FTIR (KBr, ν cm⁻¹): 3335 (NH), 1593 (C = N). ¹H-NMR (300 MHz, DMSO‑d₆) δ (ppm): 8.26- 8.11 (m, 4H, Ar–H of 4-nitrophenyl ring), 7.83–6.36 (m, 3H, Ar–H of furyl ring), 7.18 (s, 1H, NH, exchangeable with D₂O), 4.99 (t, 1H, CH-pyrazole), 3.40–3.07 (m, 2H, CH₂). MS (m/z, %): 257 (M^{∙ +}; 30.65%).

Synthesis of 2-chloro-1-(5-(furan-2-yl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (3)

A catalytic amount of triethylamine (TEA) was present when compound 2 (10 mmol, 2.57 g) was dissolved in 20 ml of ethanol. The mixture was then refluxed for eight hours while ethyl chloroacetate (10 mmol, 1.1 mL) was added dropwise. After cooling, the reaction mixture was added to 50 ml of ice-cold water. By filtering, washing, and recrystallizing the resulting precipitate from ethanol in 48% yield, m.p. 231–233 °C. Compound 3 was produced as a yellow powder. Anal. Calcd. for C₁₅H₁₂ClN₃O₄ (333.7): C, 53.99; H, 3.62; N, 12.59. Found: C, 53.77; H, 3.34; N, 12.38. FT-IR (KBr, ν /cm⁻¹): 3083 (CH_arom.), 2985, 2931 (CH_aliph), 1693 (C = O). ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 8.38–7.96 (m, 4H, 4-nitrophenyl ring), 7.59–6.57 (m, 3H, furyl ring), 5.63,5.60 (dd, 1H, (CH)pyrazole, J = 4.80 Hz, J = 12.00 Hz), 3.83, 3.79 (dd, 1H, (CH₂)pyrazole, J = 4.80 Hz, J = 18.00 Hz), 3.71 (s, 2H, CH₂Cl), 3.45, 3.41 (dd, 1H, (CH₂)pyrazole, J = 4.80 Hz, J = 18.00 Hz). ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 167.21 (C = O), 152.91, 148.36, 143.15, 137.43, 128.10, 126.41, 124.41, 111.11, 108.09, 55.53 (CH)pyrazole, 53.39 (CH₂Cl), 38.59 (CH₂)pyrazole.

Synthesis of 5-(furan-2-yl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazole-1-carbaldehyde (4)

Compound 1 (5 mmol, 1.22 g) and hydrazine hydrate (10 mmol, 0.49 g) were combined with formic acid (10 ml) and refluxed for nine hours under TLC monitoring. After cooling, the reaction mixture was transferred to crushed ice. Compound 4 was produced as weak yellow crystals after the precipitate was filtered, cleaned with water, dried, and recrystallized from 1,4-dioxane. Yield 75%, m.p. 164–166 °C. Anal. Calcd. for C₁₄H₁₁N₃O₄ (285.07): C, 58.95; H, 3.89; N, 14.73. Found: C, 58.78; H, 3.75; N, 14.57. FTIR (KBr, ν cm⁻¹): 3117, 3067 (CH_aromatic), 2921, 2851 (CH_aliphatic), 1672 (C = O). ¹H-NMR (400 MHz, DMSO‑d₆) δ (ppm): 8.89 (s, 1H, CHO), 8.31- 8.01 (m, 4H, Ar–H of 4-nitrophenyl), 7.95- 6.91 (m, 3H, Ar–H of furyl), 5.68, 5.72 (dd, 1H, CH-pyrazole), 3.90–3.41 (m, 2H, CH₂). ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 160.68 (C = O), 155.17, 151.73, 148.64, 143.24, 137.01, 128.28, 124.47, 111.10, 108.38, 53.07 (CH)pyrazole, 38.80 (CH₂)pyrazole. MS (m/z, %): 285 (M^{∙ +}; 22.00%).

Synthesis of 5-(furan-2-yl)-3-(4-nitrophenyl)-1H-pyrazole-1-carboxylic acid (5)

A vigorous stirring solution of pyrazole derivative 4 (10 mmol, 3.01 g) in ethanol (20 mL) was combined with 50% aqueous NaOH solution (5 mL), and the combination was allowed to stir at room temperature for six hours. The course of the reaction was monitored using precoated TLC plates. The mixture was neutralized with 1N hydrochloric acid to produce the pure product as yellow crystals. The resulting precipitate was filtered and recrystallized from ethanol (m.p.: 238–240 °C, yield 61%). Anal. Calcd. for C₁₄H₉N₃O₅ (299.24): C, 56.19; H, 3.03; N, 14.04. Found: C, 56.11; H, 2.98; N, 13.94. FT-IR (KBr, ν /cm⁻¹): br. 3441 (OH), 1680 (C = O). ¹H-NMR (400 MHz, DMSO-d₆) δ (ppm): 13.95 (s, 1H, OH, exchangeable with D₂O), 8.35–8.13 (m, 4H, Ar–H, 4-nitrophenyl ring), 7.79 (s, 1H, pyrazole ring), 7.19- 6.62 (m, 3H, ArH of furyl ring). ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 158.72 (C = O), 153.09, 146.97, 143.44, 141.11, 133.60, 126.52, 124.66, 112.34, 107.54, 100.79, 95.17.

Synthesis of 1-(5-(furan-2-yl)-3-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-1-yl)ethan-1-one (6)

Method (A):

Compound 1 (5 mmol, 1.22 g) and hydrazine hydrate (10 mmol, 0.49 g) were mixed with glacial acetic acid (10 ml) and refluxed for nine hours under TLC monitoring. After cooling, the reaction mixture was transferred onto crushed ice. Compound 6 was produced as pale-yellow crystals after the precipitate was filtered, cleaned with water, dried, and recrystallized from 1,4-dioxane. Produce 57%.

Method (B):

Compound 2 (10 mmol, 2.57 g) was refluxed for three hours using 5 ml of recently distilled acetic anhydride. TLC was used to advance the reaction. Following the completion of the reaction, the precipitate was filtered, dried, and recrystallized to produce compound 6 in a 75% yield at m.p. 164–166 °C. Anal. Calcd. for C₁₅H₁₃N₃O₄ (299.29): C, 60.20; H, 4.38; N, 14.04. Found: C, 59.82; H, 4.10; N, 13.65. FTIR (KBr, ν cm⁻¹): 3117, 3067 (CH_aromatic), 2921, 2851 (CH_aliphatic), 1658 (C = O). ¹H-NMR (300 MHz, DMSO‑d₆) δ (ppm): 8.31- 8.01 (m, 4H, Ar–H of 4-nitrophenyl), 7.54- 6.35 (m, 3H, Ar–H of furyl), 5.72, 5.68 (dd, 1H, CH-pyrazole), 3.85–3.39 (m, 2H, CH₂), 2.29 (s, 3H, CH₃). MS (m/z, %): 299 (M^{∙ +}; 34.12%).

Synthesis of 4-(furan-2-yl)-6-(4-nitrophenyl)pyrimidin-2-amine (7)

Compound 1 (5 mmol, 1.22 g) and guanidine hydrochloride (5 mmol, 0.30 g) were combined and refluxed for 12 h in an alcoholic potassium hydroxide solution (20% w/v, 10 ml). TLC tracked the development of reaction. Once the reaction was finished, it was cooled, put onto crushed ice, and acidified with diluted hydrochloric acid (1N). Product 7 was prepared by filtering out the precipitate, washing it with water, drying it, and recrystallizing it from ethanol⁴⁷. Yield 77%; brown crystals; m.p. 220–222 °C. Anal. Calcd for: C₁₄H₁₀N₄O₃ (282.26): C, 59.57; H, 3.57; N, 19.85. Found: C, 59.29; H, 3.26; N, 19.52%. FTIR (KBr, ν cm⁻¹): 3347, 3217 (NH₂), 1654 (C = N), 1595 (C = C). MS (m/z, %): 282 (M^{∙ +}; 16.14%).

Synthesis of 4-(furan-2-yl)-6-(4-nitrophenyl)-1,3-diphenyl-3,4-dihydropyrimidin-2(1H)-imine (8)

Compound 1 (5 mmol, 1.22 g) and 1,3-diphenylguanidine (5 mmol, 1.06 g) were combined and refluxed for 21 h in an alcoholic potassium hydroxide solution (20% w/v, 10 ml). TLC tracked the development of reaction. Once the reaction was finished, it was cooled, put onto crushed ice, and acidified with diluted hydrochloric acid (1N). A pure product 8 in the form of deep brown crystals was obtained by filtering out the precipitate, washing it with water, drying it, and recrystallizing it from 1,4-dioxane. 49% yield; m.p. 160–162 °C.Anal. Calcd. for C₂₆H₂₀N₄O₃ (436.15): C, 71.55; H, 4.62; N, 12.84. Found: C, 71.42; H, 4.58; N, 12.72. FTIR (KBr, ν cm⁻¹): br. 3200 (NH), 1649 (C = N), 1587 (C = C). ¹H-NMR (300 MHz, DMSO‑d₆) δ (ppm): 8.67 (s, 1H, NH, exchangeable by D₂O), 8.18–6.97 (m, 17H, Ar–H), 6.56 (d, 1H, -CH of pyrimidine ring), 3.44 (d, 1H, = CH of pyrimidine). MS (m/z, %): 436 (M^{∙ +}; 17.21%).

Synthesis of 6-(furan-2-yl)-4-(4-nitrophenyl)-5,6-dihydropyrimidin-2(1H)-one (9)

Compound 1 (5 mmol, 1.22 g) and urea (5 mmol, 0.30 g) were combined and refluxed for eighteen hours in an alcoholic potassium hydroxide solution (20% w/v, 10 mL). TLC tracked how the reaction was going. The reaction mixture was then cooled, poured onto crushed ice, and acidified with diluted hydrochloric acid (1N). Compound 9 was produced as deep brown crystals after the precipitate was filtered out, cleaned with water, dried, and recrystallized from ethanol. 65% yield; m.p.: 300–302 °C. Anal. Calcd for: C₁₄H₁₁N₃O₄ (285.26): C, 58.95; H, 3.89; N, 14.73. Found: C, 58.68; H, 3.52; N, 14.41%. FT-IR (KBr, ν cm⁻¹): 3363 (NH), 3071 (CH_aromatic), 1687 (C = O), 1600 (C = N). ¹H-NMR (300 MHz, DMSO‑d₆) δ (ppm): 8.36- 7.41 (m, 7H, Ar–H of 4-nitrophenyl and furyl rings), 6.57 (t, 1H, CH-pyrimidinone), 2.66 (d, 2H, CH₂), 5.41 (s, 1H, NH, exchangeable with D₂O). MS (m/z, %): 285 (M^{∙ +}; 15.85%).

Synthesis of 1-allyl-6-(furan-2-yl)-4-(4-nitrophenyl)pyrimidine-2(1H)-thione (10)

Compound 1 (5 mmol, 1.22 g) and N-allylthiourea (5 mmol, 0.58 g) were combined in an alcoholic potassium hydroxide solution (20% w/v, 10 mL) and refluxed for 24 h. The development of reaction was tracked using TLC. When the reaction was finished, it was cooled, put onto crushed ice, and then acidified with diluted hydrochloric acid (1N). After filtering off the precipitate, it was cleaned with water, dried, and recrystallized from 1,4-dioxan to produce compound 10 as brown crystals. 57% yield; m.p.: 220–224 °C.Anal. Calcd for: C₁₇H₁₃N₃O₃S (339.07): C, 60.17; H, 3.86; N, 12.38. Found: C, 59.94; H, 3.78; N, 12.22. FT-IR (KBr, ν cm⁻¹): 1684 (-CH = CH₂), 1624 (C = N), 1215 (C = S). ¹H-NMR (300 MHz, DMSO‑d₆) δ (ppm): 8.38–8.10 (m, 4H, Ar–H of 4-nitrophenyl ring), 7.94–6.67 (m, 3H, Ar–H of furyl ring), 6.64 (s, 1H, CH-pyrimidine), 5.05–4.02 (br. m, 3H, -CH = CH₂), 3.43 (d, 2H, -CH₂-). MS (m/z, %): 339 (M^{∙ +}; 13.72%).

Synthesis of 2-amino-4-(furan-2-yl)-6-(4-nitrophenyl)nicotinonitrile (11)

For 12 h, a combination of 1 (5 mmol, 1.22 g) and malononitrile (5 mmol, 0.33 g) in 15 ml of absolute ethanol with excess ammonium acetate was refluxed. The resultant precipitate was filtered out, cleaned with water, dried, and recrystallized from 1,4-dioxane to get a pure product 11 as a brown powder after the reaction liquid was cooled and placed onto crushed ice. 82% yield, m.p. 180–182 °C.Anal. Calcd. for C₁₆H₁₀N₄O₃ (306.08): C, 62.74; H, 3.29; N, 18.29. Found: C, 62.66; H, 3.17; N, 18.11. FTIR (KBr, ν cm⁻¹): 3353–3241 (NH₂), 2214 (C≡N), and 1662 (C = N). ¹H-NMR (300 MHz, DMSO‑d₆) δ (ppm): 8.40- 7.95 (m, 4H, Ar–H of 4-nitrophenyl), 7.91- 6.76 (m, 3H, Ar–H of furyl rings), 7.18 (s, 1H, pyridine ring), 6.90 (s, 2H, NH₂, exchangeable with D₂O). MS (m/z, %): 306 (M^{∙ +}; 18.12%).

Synthesis of 5-(furan-2-yl)-7-(4-nitrophenyl)-4H-pyrido[2,3-d][1,3]oxazin-4-one (12)

Compound 11 (5 mmol, 1.53 g) and formic acid (10 mL) were refluxed for nine hours. The reaction mixture was added to water with crushed ice once it had cooled. Compound 12 was obtained as a white solid when the resultant solid was filtered off, cleaned with cold water, and recrystallized from ethanol. 75% yield, m.p. 202–205 °C. Anal. Calcd. for C₁₇H₉N₃O₅ (335.05): C, 60.90; H, 2.71; N, 12.53. Found: C, 60.76; H, 2.67; N, 12.31. FTIR (KBr, ν cm⁻¹): 1714 (C = O) and 1618 (C = N). ¹H-NMR (400 MHz, DMSO‑d₆) δ (ppm): 8.39- 8.24 (m, 4H, Ar–H of 4-nitrophenyl), 7.92- 6.77 (m, 3H, Ar–H of furyl ring), 7.98 (s, 1H, CH-oxazinone ring), 7.19 (s, 1H, CH-pyridine ring). ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 161.50 (C = O), 159.29, 154.82, 153.60, 149.21, 148.96, 148.35, 146.22, 144.13, 130.50, 124.19, 119.00, 116.06, 114.61, 105.86. MS (m/z, %): 335 (M^{∙ +}; 47.19%).

Synthesis of 5-(furan-2-yl)-7-(4-nitrophenyl)-2-phenyl-4H-pyrido[2,3-d][1,3]oxazin-4-one (13)

For 24 h, compound 11 (5 mmol, 1.53 g) and benzoyl chloride (10 mL) were refluxed together. A rotating evaporator was used to eliminate the surplus benzoyl chloride. Deep brown crystals of product 13 were obtained by recrystallizing the solid residue from 1,4-dioxane following evaporation in yield (76%), m.p. 155–157 °C. Anal. Calcd. for C₂₃H₁₃N₃O₅ (411.4): C, 67.15; H, 3.19; N, 10.21. Found: C, 67.09; H, 3.03; N, 10.17. FTIR (KBr, ν cm⁻¹): 1686 (C = O) and 1619 (C = N). ¹H-NMR (400 MHz, DMSO‑d₆) δ (ppm): 8.37- 7.94 (m, 4H, Ar–H of 4-nitrophenyl), 7.90 (s, 1H, pyridine ring), 7.61- 7.47 (m, 5H, Ar–H of phenyl ring), 7.17–6.77 (m, 3H, Ar–H of furyl ring). ¹³C-NMR (100 MHz, DMSO-d₆) δ (ppm): 167.89 (C = O), 154.85, 149.02, 146.23, 144.17, 137.01, 133.28, 131.28, 130.53, 129.71, 129.01, 124.20, 114.64, 113.91, 113.46, 93.62.

Synthesis of ethyl-N-(3-cyano-4-(furan-2-yl)-6-(4-nitrophenyl)pyridin-2-yl)formimidate (14)

Compound 11 (5 mmol, 1.53 g) and 10 mL of triethyl orthoformate (TEOF) were refluxed for 24 h. TLC was used to track the progress of reaction, and the excess TEOF was vacuum-removed once it was finished. The residual solid was repeatedly cleaned with n-hexane and recrystallized from benzene to produce pale-yellow crystals of ethyl formimidate derivative 14. 75% yield, m.p. 144–146 °C.Anal. Calcd. for C₁₉H₁₄N₄O₄ (362.35): C, 62.98; H, 3.89; N, 15.46. Found: C, 62.81; H, 3.91; N, 15.34. FTIR (KBr, ν cm⁻¹): 2217 (C≡N), 1648 (C = N). ¹H-NMR (300 MHz, DMSO‑d₆) δ (ppm): 8.41–6.80 (m, 7H, Ar–H of 4-nitrophenyl and furyl rings), 8.03 (s, 1H, CH-pyridine ring), 7.77 (s, 1H, N＝CH), 4.42 (q, 2H, CH₂, J = 6.9 Hz), 1.39 (t, 3H, CH₃, J = 7.2 Hz). MS (m/z, %): 362 (M^{∙ +}; 33.10%).

Microbial strains and general growth conditions

In the present study, we used five microbial strains to screen the antimicrobial activity of synthesized compounds distributed as follows, two Gram Bacillus subtilis (B. subtilis) ATCC6051, Staphylococcus aureus (S. aureus) ATCC 9144, two Gram negative Escherichia coli (E. coli) O157:H7 ATCC 51,659 and Pseudomonas aeruginosa (P. aeruginosa) ATCC 27,853 and one fungal strains Candida albicans (C. albicans) ATCC 90,028.

Generally, Muller Hinton (MH) or Sabouraud dextrose agar and broth were used for bacterial and fungal growth respectively, at 37 °C for 24–30 h. Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% fetal bovine serum (FBS) and 0.1% antibiotic–antimycotic solution was used for culture of the HepG2 cell lines. Culture media and all reagents used were obtained from Sigma-Aldrich (USA), Oxoid (UK), or Fluka (Switzerland). All experiments were conducted in triplicate.

Determination of minimum inhibitory concentration (MIC)

The antimicrobial potential of the synthesized compounds were evaluated by determination of minimum inhibitory concentrations (MICs) following the standard broth dilution method described by CLSI^50,51. In summary, overnight cultures of reference microbial strains were diluted 1:1000 to obtain a final concentration of approximately 1.5 × 10⁵ CFU.mL⁻¹. MIC values were assessed using a two-fold serial dilution across concentrations from 7.8 to 1000 µg. mL⁻¹. The inoculated cultures were incubated at 37 °C with shaking at 150 rpm, and growth was monitored by measuring optical density at 600 nm. DMSO was used as control. All experiments were conducted in triplicate. Additional intermediate concentrations were tested (interdilution approach) to accurately determine the endpoint. The synergistic effect of the selected promising compounds in combination with N-acetylcysteine (NAC) was assessed using the checkerboard method to determine the fractional inhibitory concentration (FIC) index.

Cytotoxicity testing

The Holding Company for Biological Products and Vaccines (VACSERA, Giza, Egypt) provided the HepG2 cell line used in this investigation. To investigate the cytotoxic effect of tested compounds, MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) on HepG2 cell viability have been employed. In summary, 0.5 × 10⁵ cells/well in serum-free medium were plated in a flat bottom 96-well microplate and exposed to 20 µl of various doses 5–100 µg.mL⁻¹ of the tested compounds over 48 h at 37º C in 5% CO₂. Following 4 h of incubation, the media were withdrawn, 40 µl of MTT solution/well was applied, and the absorbance at 570 nm was measured photometrically using microplate reader (Biotek Elx-808)⁵¹.

Antibiofilm activity

Determination of colony count

To determine the colony count of promising compounds 4 and 13, biofilms were developed in 96-well plates by adding 100 μL of Pseudomonas aeruginosa suspension (10⁶ CFU/mL) to each well and allowing bacterial adhesion for 1 h. The suspension was then removed, and the wells were gently rinsed once with 100 μL of PBS. Afterwards, 200 μL of fresh growth medium was added, and the plates were incubated at 37 °C for 48 h⁵². Following incubation, the medium was discarded, and the wells were washed again with 100 μL of PBS. Treatments consisting of 100 μL PBS (control) or varying sub-MIC concentrations of the tested formulations were applied for 2 or 4 h. The biofilms were then disrupted by pipetting, serially diluted, and plated on LB agar⁵³. Colony-forming units (CFU) were counted after incubation at 37 °C for 18 h. All assays were performed in triplicate.

Potential antimicrobial activity

Assessment of respiratory chain dehydrogenases activity (TTC assay)

The respiratory chain dehydrogenase activity of P. aeruginosa biofilms was evaluated using a tetrazolium-based colorimetric assay (TTC or XTT), which reflects the metabolic reducing capacity of viable cells. After treatment with the tested compounds at different sub-MIC concentrations for the indicated times, cultures were exposed to the tetrazolium reagent at 37 °C for 3–4 h, allowing enzymatic reduction to the colored formazan product. The developed color intensity, corresponding to dehydrogenase activity, was measured spectrophotometrically using microplate reader (Biotek Elx-808) at 490 nm, after the incubation period. Control wells containing untreated cells and reagent blanks were included to correct for background absorbance. The metabolic activity for each treatment was expressed as a percentage relative to untreated control. All experiments were performed in triplicate, and data were presented as mean ± SD.

In-Silico study

Molecular docking studies were carried out to evaluate the binding affinity and interaction modes of 14 synthetic compounds against the bacterial DNA gyrase enzyme, a key enzyme responsible for introducing negative supercoiling during DNA replication and transcription. AutoDock Vina, integrated within the PyRx 0.8 virtual screening platform, was employed for all docking experiments^54,55. Two crystal structures were retrieved from the Protein Data Bank: Escherichia coli DNA gyrase subunit B (PDB ID: 6KZX) complexed with a quinoline derivative and Staphylococcus aureus DNA gyrase subunit B (PDB ID: 3G75) complexed with a thiazole inhibitor^56,57,58. All water molecules were removed, and the co-crystallized ligands were retained to define the active site. Ligands were energy-minimized and converted into PDBQT format before docking. The grid box coordinates were centered at (x = 50.57, y = –3.38, z = 17.16) for 6KZX and (x = 27.02, y = 5.84, z = –8.37) for 3G75, with an exhaustiveness value of 20. The resulting docked complexes were analyzed and visualized using Schrödinger Maestro 14.6 (academic version) to study hydrogen bonding, salt bridges, and hydrophobic interactions within the active site. Docking protocol was validated by redocking the co-crystallized ligand, yielding an RMSD of 0.9 and 1.12 Å for E. coli and S. aureus respectively, between the predicted and experimental poses, confirming the reliability of the docking parameters. To validate the docking protocol, the co-crystallized ligand was redocked into the corresponding active site using the same docking parameters. The RMSD values between the crystallographic and redocked ligand poses were 0.90 Å for E. coli DNA gyrase (6KZX) and 1.12 Å for S. aureus DNA gyrase (3G75). Both values are below the generally accepted threshold of 2.0 Å, confirming that the docking protocol reliably reproduced the experimental binding mode and validating the docking methodology.

Molecular targeting

Total RNA was extracted from E. coli cultures exposed to the formula at 0.5 × MIC and from untreated control groups using the RNeasy Mini Kit (Qiagen, Germany) following the manufacturer’s instructions. The purity and concentration of the isolated RNA were measured spectrophotometrically. Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using a reverse transcription kit.

Quantitative real-time PCR (qRT-PCR) was then performed with gene-specific primers targeting the gyrB gene to evaluate the effect of the formula on its expression. The rrsE gene was used as a housekeeping reference for normalization. Amplification reactions were conducted in a real-time thermal cycler using SYBR Green Master Mix under optimized cycling conditions. Relative gene expression was calculated using the ΔΔCt method (Table 7).