Synthesis and antimicrobial activity of aryldiazenyl/arylhydrazono pyrazoles

Abstract

We report the design, synthesis, and in vitro antimicrobial evaluation of functionalized pyrazoles containing a hydrazono/diazenyl moiety. Among these newly synthesized derivatives, 4-[2-(4-chlorophenyl)hydrazono]-5-methyl-2-[2-(naphthalen-2-yloxy)acetyl]-2,4-dihydro-3H-pyrazol-3-one is a promising antimicrobial agent against Staphylococcus aureus (minimum inhibitory concentration 0.19 μg mL⁻¹). Structure–activity relationship studies reveal that the electronic environment on the distal phenyl ring has a considerable effect on the antimicrobial potential of the hybrid analogues. Molecular docking studies into the active site of S. aureus dihydrofolate reductase also prove the usefulness of hybridizing a pyrazole moiety with azo and hydrazo groups in the design of new antimicrobial agents.

AHP as a promising antibacterial agent effective against dihydrofolate reductase (DHFR) enzyme of an S. aureus.

Introduction

The advent of antibiotics completely changed the course of modern medicine by significantly reducing human morbidity and mortality. ¹ However, the recent global spread of deadly infectious diseases and the emergence of drug-resistant microorganisms have claimed millions of lives. ² According to the World Health Organization (WHO), lower respiratory tract infections are the fourth leading cause of death globally. ³ The upsurge of multi-drug-resistant (MDR) Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and MDR Streptococcus pneumoniae, as well as MDR Gram-negative bacteria such as carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii are a major threat to healthcare systems. ⁴ Moreover, recent reports of MDR Candida auris and mucormycosis fungal infections in critically ill coronavirus disease patients are an increasing health concern worldwide. ⁵ Consequently, costly treatments due to drug resistance and their associated adverse reactions pose new challenges that prompt the development of novel antimicrobial lead compounds.

Azo (–N=N–) and related hydrazo (=N–NH–) groups are essential pharmacophores for the synthesis of antimicrobial agents.^6,7 Furthermore, pyrazole derivatives are an interesting class of compounds showing a broad range of biological activities such as anticancer, ⁸ antimalarial, ⁹ antiviral, ¹⁰ and antimicrobial. ¹¹

Recently, a molecular hybridization method offered a promising strategy to design new potent hybrid molecules with multi-targeting synergistic effects. ¹² Previous studies showed that molecular hybrids containing pyrazole and azo (–N=N–)/hydrazo (=N–NH–) pharmacophores in a single molecular framework resulted in improved antimicrobial potentials.^13,14 In continuation of our endeavors to develop potent and effective antimicrobial agents, ¹⁵ this study deals with the optimization of pyrazole-azo/hydrazo molecular hybrid leads as potential antimicrobial agents.

Results and discussion

Chemistry

So far, the majority of previously reported syntheses of arylhydrazono pyrazoles (AHPs) and aryldiazenyl pyrazoles (ADPs) were carried out in two steps. The first step involved the reactions of aryl diazonium salts with acetylacetone/ethyl acetoacetate via the Japp–Klingemann reaction, followed by cyclo-condensation with aryl hydrazides in the second step. These procedures require extended reaction time at reflux and afford low yields.^13,14,16 We have reported a fast and expedient method for the preparation of AHPs and ADPs using one-pot, three-component condensations of ethyl acetoacetate/acetylacetone, aryl hydrazides, and aryldiazonium salts under solvent-free conditions. We demonstrated that the catalyst, triethylamine, being basic in character, facilitated proton removal from the active methylene compounds and increased the reaction rate. ¹⁷ Using this method, herein we report the one-pot synthesis of new AHPs 1a–f (75%–84% yield) and ADPs 2a–f (72%–85% yield). The synthetic protocols for the preparation of AHPs 1a–f and ADPs 2a–f are outlined in Scheme 1. All the prepared compounds were fully characterized by IR, NMR, and elemental analyses. The spectral data of new compounds are in full agreement with the previously reported spectra. ¹⁷ Literature revealed that the AHPs exist in different tautomeric forms whose relative stabilities depend on the influence of the substituents and on the medium. In the solid state, such compounds exist largely in the aryldiazenyl (OH) or the arylhydrazono (NH) forms with the latter predominating. Moreover, it is noteworthy that the 5-oxo group on the pyrazole nucleus and the carbonyl substituent at the N-1 position were reported to participate in hydrogen-bonding interactions. Compound 1a showed that a low-frequency C=O band at 1668 cm⁻¹ may be strongly attributed to its conjugation with the C=N group as well as intra-molecular hydrogen bonding with the NH group. A separate carbonyl frequency at 1692 cm⁻¹ due to amide carbonyl vibration and absence of N=N stretching vibration ruled out the aryldiazenyl (OH) form. The AHPs are therefore assigned in the hydrazono form which is consistent with our previous reports. The ¹H NMR spectra of prototype compound 1a showed two shielded singlets at δ 4.70 and δ 2.11 due to methylene and methyl protons, respectively. In addition, a one deshielded singlet at δ 11.55 was due to the –NH group. The 12 aromatic protons resonated between δ 7.35 and δ 7.83. The ¹³C NMR spectrum of compound 1a showed five deshielded signals at δ 141.55, 146.85, 155.57, 166.67, and 170.14 due to two C=N, one O–C (ether), and two carbonyl carbons, respectively. Two signals in the aliphatic range were observed at δ 11.62 and δ 64.55 due to methyl and methylene carbons, respectively. The remaining resonances in the aromatic region at δ 106.95, 107.28, 115.18, 115.65, 118.47, 123.80, 125.10, 126.48, 127.52, 128.10, 128.72, 129.60, and 134.10 along with the upfield and downfield signals confirm the presence of 22 carbons in compound 1a.

Scheme 1.

One-pot synthesis of AHPs 1a-f and ADPs 2a-f.

Biological activity

The newly synthesized compounds 1a–f and 2a–f were evaluated for their antibacterial activity against S. aureus (ATCC-25923) and Escherichia coli (ATCC-25922), along with their antifungal activity against Aspergillus niger (ATCC9029) and Candida albicans (ATCC-90028) in Dimethylformamide (DMF) using the serial plate dilution method. All the results are presented as minimum inhibitory concentration (MIC), which is the lowest concentration of antimicrobial agent that results in the complete inhibition of visible growth of microorganisms (Table 1). The standard antibiotic ofloxacin, belonging to the quinolone class, and antifungal drug fluconazole, belonging to the azole class, were used for comparison of the MIC values.

Table 1.

Antimicrobial activity of the AHPs and ADPs.

Compound	R	ClogP a	MIC (μg mL−1)
			S. aureus (Gram +ve)	E. coli (Gram −ve)	A. niger	C. albicans
1a	H	3.48	50	50	50	50
1b	CH3	5.66	>100	>100	>100	>100
1c	OCH3	5.08	50	100	100	100
1d	Cl	5.88	0.19	0.78	6.25	6.25
1e	Br	6.03	6.25	12.5	6.25	6.25
1f	NO2	4.91	12.5	12.5	25	25
2a	H	5.86	100	100	100	100
2b	CH3	6.36	>100	>100	>100	>100
2c	OCH3	6.04	>100	>100	>100	>100
2d	Cl	6.66	6.25	6.25	25	25
2e	Br	6.81	25	25	25	25
2f	NO2	5.78	12.5	25	25	25
Ofloxacin	‒	‒	0.19	0.19	‒	‒
Fluconazole	‒	‒	‒	‒	1.56	0.19

AHP: arylhydrazono pyrazole; ADP: aryldiazenyl pyrazole; MIC: minimum inhibitory concentration.

a

Calculated using ChemDraw version 14.0.0.117.

The pharmacophores AHP and ADP, which were reported to have promising antimicrobial activity in our previous studies, were the lead compounds in the design of these hybrid molecules (Figure 1).^13,14 We hypothesized that replacement of the quinoline and benzothiazole nucleus with a more lipophilic naphthyl ring would further enhance the antimicrobial activity of these AHP and ADP analogues. Finally, substituents were installed on the terminal phenyl ring to study the stereo-electronic effects of different functional groups on the antimicrobial activity of the pyrazole derivatives. The results of the antibacterial activity studies of the AHPs revealed that most of these compounds demonstrated excellent to moderate microbial growth inhibition, with the exception of compound 1b (R = 4-CH₃, MIC >100 μg mL⁻¹). Among the Gram-positive and Gram-negative bacterial strains, S. aureus was found to be most sensitive to the AHPs. Compound 1d (R = 4-Cl, MIC 0.19 μg mL⁻¹) was found to be the most potent compound of the series and was equipotent with the standard drug ofloxacin (MIC 0.19 μg mL⁻¹) against the S. aureus strain. Compounds 1e (R = 4-Br, MIC 6.25 μg mL⁻¹) and 1f (R = 4-NO₂, MIC 12.5 μg mL⁻¹) also showed moderate antibacterial effects, whereas compounds 1a (R = H, MIC 50 μg mL⁻¹) and 1c (R = 4-OCH₃, MIC 50 μg mL⁻¹) showed the lowest antibacterial effects against S. aureus. AHPs were less effective against the Gram-negative E. coli bacterial strain. The potency of compound 1d (R = 4-Cl, MIC 0.78 μg mL⁻¹) was only fourfold lower than that of the reference drug ofloxacin (MIC 0.19 μg mL⁻¹) against E. coli. The remaining AHPs (1a, 1c, 1e, and 1f) showed moderate to lower antibacterial activity (MIC 12.5-100 μg mL⁻¹) against E. coli. The antibacterial activity of the ADPs showed that only compound 2d (R = 4-Cl, MIC 6.25 μg mL⁻¹) had moderate antibacterial activity, whereas compounds 2e (R = 4-Br, MIC 25 μg mL⁻¹) and 2f (R = 4-NO₂, MIC 25 μg mL⁻¹) showed lower antibacterial effects, in fact being 131 times less potent than ofloxacin.

Figure 1.

Design strategy and lead optimization of the new AHP and ADP antimicrobial agents.

The AHPs and ADPs were also evaluated for their antifungal activity. Among these, AHP analogues 1d (R = 4-Cl, MIC 6.25 μg mL⁻¹) and 1e (R = 4-Br, MIC 6.25 μg mL⁻¹) showed moderate antifungal activities against both A. niger and C. albicans, respectively. The standard drug fluconazole showed antifungal growth inhibition with MIC values of 1.56 and 0.19 μg mL⁻¹ against A. niger and C. albicans, respectively. The remaining compounds were minimally effective against these fungal strains (MIC 25 to >100 μg mL⁻¹).

The structure–activity relationship of the AHP analogues showed antibacterial potency in the following order: 4-Cl > 4-Br,4-NO₂ > H,4-OCH₃ > 4-CH₃, whereas the ADP analogues had antibacterial potency in the order: 4-Cl > 4-Br,4-NO₂ > H > 4-CH₃,4-OCH₃. Thus, it was clearly observed that compounds with electron-withdrawing groups on the phenyl ring were more effective than those with electron-releasing substituents. Moreover, it is evident that the AHPs were more effective than the ADPs.

Furthermore, the partition coefficient (log P) is an imperative physicochemical property affecting penetration into microbial cells. ¹⁴ Therefore, the partition coefficients of all the compounds were calculated using ChemDraw to establish the correlation between Clog P and antimicrobial activity. The results showed that the lipophilicity of the compounds increased with lipophilic electron-withdrawing groups, for example, Cl (ClogP 5.88–6.66) and Br (ClogP 6.03–6.81), which consequently enhanced the antimicrobial potencies of the AHPs and ADPs.

Molecular docking studies

To gain further support regarding the antibacterial effects of the most promising hybrid AHP 1d against S. aureus, a molecular docking study was carried out on the dihydrofolate reductase (DHFR) enzyme (PDB ID: 3SRQ) of an S. aureus bacterial strain ¹⁸ using the AutoDock program. AutoDock 4.2.2 with a Lamarkian genetic algorithm-implemented program suite was employed to identify appropriate binding modes and the conformations of the ligand molecules. DHFR catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate, which is essential for the synthesis of purines, some amino acids, and thymidine required for bacterial growth and proliferation. ¹⁹ Thus, DHFR represents an attractive antifolate drug target which produces antibacterial action by selectively disrupting the folate pathway. The docking protocol was validated by redocking the co-crystallized ligand 7-aryl-2,4-diaminoquinazoline at the active site (root-mean-square deviation (RMSD) 0.12). The results of docking studies clearly showed that compound 1d fits nicely into the active site and formed hydrogen bonds, van der Waals, π-σ, and π-alkyl interactions with the active site residues (Figures 2–4). The binding free energy of compound 1d was found to be −10.4 kcal mol⁻¹, indicating sufficient affinity between the hybrid analogue and the enzyme. The 3D and 2D docked conformations of the ligand 1d bound to the active site of DHFR are shown in Figures 3 and 4. The hydrazo −NH group of compound 1d formed a hydrogen bond with Thr47, whereas the carbonyl oxygen formed a hydrogen bond with the Asp28 active site residue. Lipophilicity also appears to play a crucial role in the antibacterial activity of 1d, as the naphthyl ring positioned itself into the lipophilic pocket of the binding site and formed π-σ and π-alkyl interactions with the Leu55, Leu29, and Val32 residues. This validates our hypothesis that the optimization of previous lead molecules by changing the quinoline and benzothiazole rings to a lipophilic naphthyl moiety greatly improved the antibacterial potential of these hybrid analogues.

Figure 2.

Overlay of the co-crystallized ligand (green) and compound 1d (magenta) at the same active site of DHFR.

Figure 3.

Binding mode of compounds 1d (stick) into the DHFR active site showing two hydrogen bonds (yellow dotted lines) with Asp28 (2.9 Å) and Thr47 (2.5 Å).

Figure 4.

2D binding interactions of compound 1d at the active site of DHFR.

Conclusion

In summary, we have synthesized 12 hybrid azo/hydrazo-pyrazole derivatives as potential antimicrobial agents. Most of the compounds demonstrated promising antimicrobial activities. The results revealed that compound 1d (ClogP 5.88) was the most potent antibacterial agent against S. aureus, with an MIC value of 0.19 μg mL⁻¹. This study provides a novel optimized azo/hydrazo-pyrazole hybrid as a potential lead molecule for the development of promising antimicrobial agents.

Experimental procedure

All the chemicals used in this study were purchased from Sigma-Aldrich and used without further purification. The progress of the reactions was monitored on pre-coated silica gel 60 Thin-layer chromatography (TLC) plates on a support of aluminum (CAMAG®). Melting points were determined in open capillary tubes in a Buchi automated melting point apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer 1720 FTIR spectrometer using KBr pellets. ¹H NMR spectra were obtained on a JEOL ECA 500-II 500 MHz NMR (125 MHz ¹³C) spectrometer using tetramethylsilane (TMS) as an internal reference (chemical shifts in δ ppm). The aryldiazonium salts were prepared by following previously reported methods. ²⁰ The amines (0.01 mol) were added to HBF₄ (5 mL, Sigma 48%), cooled to 0 °C, and diazotized by the dropwise addition of a solution of NaNO₂ (0.02 mol) in water (5 mL). Colorless shiny crystals of the diazonium salt were cooled on ice, collected, washed with ether, and used without purification.

General procedure for the synthesis of arylhydrazono/aryldiazenyl pyrazoles (1a-f and 2a-f)

An equimolar amount of aryldiazonium salts (1 mmol), ethyl acetoacetate/acetylacetone (1 mmol), and 2-(1-naphthyloxy)acetohydrazide (1 mmol) was mixed in the presence of 2–3 drops of triethylamine. The mixture was heated in an oil bath at 90 °C for 6–8 h. The completion of the reaction was ascertained by TLC using ethyl acetate:hexane (3:7) as the mobile phase. The resulting reaction mixture was poured onto crushed ice and the precipitate was filtered, washed with water, and recrystallized from ethanol.

5-Methyl-2-[2-(naphthalen-2-yloxy)acetyl]-4-(2-phenylhydrazono)-2,4-dihydro-3H-pyrazol-3-one (1a): Yield: 0.32 g (84%); pale yellow solid; m.p. 115 °C–117 °C. IR (KBr): 1040, 1668, 1692, 3412 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 11.55 (s, 1H, NH), 7.35–7.83 (m, 12H, ArH), 4.70 (s, 2H, OCH₂), 2.11 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d₆) δ = 170.14, 166.67, 155.57, 146.85, 141.55, 134.10, 129.60, 128.72, 128.10, 127.52, 126.48, 125.10, 123.80, 118.47, 115.65, 115.18, 107.28, 106.95, 64.55, 11.62. Anal. Calcd for C₂₂H₁₈N₄O₃: C, 68.38; H, 4.70; N, 14.50. Found: C, 68.46; H, 4.65; N, 14.43.

5-Methyl-2-[2-(naphthalen-2-yloxy)acetyl]-4-[2-(p-tolyl)hydrazono]-2,4-dihydro-3H-pyrazol -3-one (1b): Yield: 0.32 g (80%); brown solid; m.p. 106 °C–108 °C. IR (KBr): 1080, 1656, 1690, 3418 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 11.52 (s, 1H, NH), 7.21–7.83 (m, 11H, ArH), 4.78 (s, 2H, OCH₂), 2.28 (s, 3H, CH₃), 2.13 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ = 170.09, 166.57, 155.61, 146.70, 139.13, 134.55, 134.05, 132.98, 130.03, 129.38, 128.66, 127.50, 126.72, 126.46, 115.63, 115.22, 107.32, 106.96, 64.52, 25.45, 11.61. Anal. Calcd for C₂₃H₂₀N₄O₃: C, 68.99; H, 5.03; N, 13.99. Found: C, 68.96; H, 5.10; N, 13.93.

4-[2-(4-Methoxyphenyl)hydrazono]-5-methyl-2-[2-(naphthalen-2-yloxy)acetyl]-2,4-dihydro-3H-pyrazol-3-one (1c): Yield: 0.32 g (76%); brown solid; m.p. 123 °C–125 °C. IR (KBr): 1076, 1665, 1686, 3422 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 11.47 (s, 1H, NH), 6.98–7.84 (m, 11H, ArH), 4.78 (s, 2H, OCH₂), 3.75 (s, 3H, OCH₃), 2.13 (s, 3H, CH₃). ¹³C NMR (100 MHz, DMSO-d₆): δ = 170.65, 167.13, 156.14, 147.10, 136.57, 134.57, 129.92, 129.19, 128.04, 127.27, 127.00, 124.32, 118.98, 117.75, 117.23, 115.38, 107.84, 107.48, 66.55, 55.93, 12.14. Anal. Calcd for C₂₃H₂₀N₄O₄: C, 66.34; H, 4.84; N, 13.45. Found: C, 66.39; H, 4.91; N, 13.38.

4-[2-(4-Chlorophenyl)hydrazono]-5-methyl-2-[2-(naphthalen-2-yloxy)acetyl]-2,4-dihydro-3H-pyrazol-3-one (1d): Yield: 0.34 g (82%); white solid; m.p. 152 °C–154 °C. IR (KBr): 1074, 1663, 1697, 3425 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 11.54 (s, 1H, NH), 7.42-7.84 (m, 11H, ArH), 4.78 (s, 2H, OCH₂), 2.37 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ = 170.13, 166.62, 155.62, 146.9, 141.53, 129.31, 128.68, 127.52, 126.75, 126.48, 123.19, 118.69, 118.47, 117.37, 116.75, 107.33, 107.29, 106.97, 66.04, 11.63. Anal. Calcd for C₂₂H₁₇ClN₄O₃: C, 62.79; H, 4.07; N, 13.31. Found: C, 62.84; H, 4.10; N, 13.43.

4-[2-(4-Bromophenyl)hydrazono]-5-methyl-2-[2-(naphthalen-2-yloxy)acetyl]-2,4-dihydro-3H-pyrazol-3-one (1e): Yield: 0.37 g (80%); white solid; m.p. 190 °C–192 °C; IR (KBr): 1067, 1661, 1698, 3405 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆) δ 11.57 (s, 1H, NH), 7.22–7.82 (m, 11H, ArH), 4.72 (s, 2H, OCH₂), 2.36 (s, 3H, CH₃). ¹³ C NMR (125 MHz, DMSO-d₆) δ 170.74, 167.13, 156.31, 147.42, 141.56, 134.62, 132.83, 132.69, 129.84, 129.12, 128.02, 127.24, 126.94, 124.22, 119.06, 118.24, 117.64, 107.44, 66.64, 12.17. Anal. Calcd for C₂₂H₁₇BrN₄O₃: C, 56.79; H, 3.68; N, 17.17. Found: C, 56.82; H, 3.74; N, 17.25.

5-Methyl-2-[2-(naphthalen-2-yloxy)acetyl]-4-[2-(4-nitrophenyl)hydrazono]-2,4-dihydro-3H-pyrazol-3-one (1f): Yield: 0.32 g (75%); yellow solid; m.p. 103 °C–105 °C; IR (KBr): 1060, 1658, 1697, 3406 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 11.68 (s, 1H, NH), 7.58–8.27 (m, 11H, ArH), 4.78 (s, 2H, OCH₂), 2.42 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ = 170.64, 167.36, 156.13, 147.75, 143.75, 134.58, 129.92, 129.19, 128.04, 127.27, 127.01, 126.10, 124.33, 119.21, 118.98, 116.31, 115.38, 107.48, 66.63, 12.19. Anal. Calcd for C₂₂H₁₇N₅O₅: C, 61.25; H, 3.97; N, 16.23. Found: C, 61.32; H, 4.01; N, 16.17.

1-[3,5-Dimethyl-4-(phenyldiazenyl)-1H-pyrazol-1-yl]-2-(naphthalen-2-yloxy)ethan-1-one (2a): Yield: 0.31 g (80%); pale yellow solid; m.p. 112 °C–114 °C; IR (KBr): 1050, 1625, 1695 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 7.77 (d, J = 8.5 Hz, 1H, ArH), 7.70 (d, J = 6.5 Hz, 1H, ArH), 7.56 (d, J = 8.5 Hz, 1H, ArH), 7.49 (t, J = 8, 7.5 Hz, 1H, ArH), 7.40–7.45 (m, 2H, ArH), 7.33 (t, J = 5, 10 Hz, 1H, ArH), 7.24 (d, J = 5 Hz, 1H, ArH), 7.17–7.20 (m, 2H, ArH), 4.77 (s, 2H, OCH₂), 2.46 (s, 3H, CH₃), 2.43, (s, 3H, CH₃), 7.81–7.83 (d, J = 7 Hz, 2H, ArH). ¹³C NMR (125 MHz, DMSO-d₆): δ = 168.03, 155.66, 141.77, 134.16, 133.31, 129.62, 129.39, 129.18, 128.67, 127.52, 126.75, 126.48, 125.42, 123.80, 121.39, 118.49, 116.31, 106.96, 64.60, 20.48, 11.92. Anal. Calcd for C₂₃H₂₀N₄O₂: C, 71.86; H, 5.24; N, 14.57. Found: C, 71.03; H, 5.18; N, 14.71.

1-[3,5-Dimethyl-4-(p-tolyldiazenyl)-1H-pyrazol-1-yl]-2-(naphthalen-2-yloxy)ethan-1-one (2b): Yield: 0.29 g (75%); white solid; m.p. 176 °C–178 °C; IR (KBr): 1043, 1620, 1691 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 7.79 (d, J = 7.5 Hz, 2H, ArH), 7.75 (d, J = 8.0 Hz, 1H, ArH), 7.58 (d, J = 8.0 Hz, 1H, ArH), 7.40–7.45 (m, 2H, ArH), 7.31 (t, J = 7.5 Hz, 1H, ArH), 7.27 (d, J = 8.0 Hz, 1H, ArH), 7.19–7.22 (m, 2H, ArH), 7.15 (dd, J = 2.0, 7.0 Hz, 1H, ArH), 4.75 (s, 2H, OCH₂), 2.43 (s, 3H, CH₃), 2.40 (s, 3H, CH₃), 2.36 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ = 170.06, 155.61, 151.03, 139.40, 139.17, 134.03, 130.00, 129.62, 129.34, 128.64, 127.47, 126.69, 126.42, 123.75, 121.30, 118.41, 116.29, 106.97, 64.55, 26.34, 20.50, 11.82. Anal. Calcd for C₂₄H₂₂N₄O₂: C, 72.34; H, 5.57; N, 14.06. Found: C, 72.38; H, 5.64; N, 14.12.

1-{4-[(4-Methoxyphenyl)diazenyl]-3,5-dimethyl-1H-pyrazol-1-yl}-2-(naphthalen-2-yloxy) ethan-1-one (2c): Yield: 0.33 g (80%); brown solid; m.p. 165 °C–167 °C. IR (KBr): 1040, 1651, 1685 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 7.82 (d, J = 8.5 Hz, 2H, ArH), 7.77 (d, J = 8.0 Hz, 1H, ArH), 7.69 (dd, J = 2.0, 8.5 Hz, 1H, ArH), 7.53 (dt, J = 2.5, 9.0 Hz, 1H, ArH), 7.43 (t, J = 7.0 Hz, 1H, ArH), 7.33 (t, J = 7.0 Hz, 1H, ArH), 7.24 (d, J = 2.5 Hz, 1H, ArH), 7.18 (dd, J = 2.5, 11.5 Hz, 1H, ArH), 7.04 (dd, J = 2.0, 7.0 Hz, 1H, ArH), 7.00 (dd, J = 2.0, 9.0 Hz, 1H, ArH), 4.78 (s, 2H, OCH₂), 3.81 (s, 3H, OCH₃), 2.42 (s, 3H, CH₃), 2.38 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ = 170.65, 156.14, 147.67, 135.66, 134.57, 132.93, 129.89, 129.17, 128.02, 127.25, 126.97, 124.29, 123.47, 118.98, 118.39, 115.35, 114.79, 107.45, 65.07, 55.95, 20.97, 12.39. Anal. Calcd for C₂₄H₂₂N₄O₃: C, 69.55; H, 5.35; N, 13.52. Found: C, 69.60; H, 5.39; N, 13.46.

1-{4-[(4-Chlorophenyl)diazenyl]-3,5-dimethyl-1H-pyrazol-1-yl}-2-(naphthalen-2-yloxy) ethan-1-one (2d): Yield: 0.35 g (85%); white solid; m.p. 202 °C–204 °C; IR (KBr): 1045 (C-O), 1628 (C=N), 1666 (C=O) cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 7.82 (dd, J = 2.0, 7.0 Hz, 2H, ArH), 7.77 (d, J = 8.0 Hz, 1H, ArH), 7.71 (dt, J = 2.5, 9.0 Hz, 1H, ArH), 7.59–7.61 (d, J = 7.0 Hz, 1H, ArH), 7.55 (dt, J = 2.0, 1H, 7.0 Hz, ArH), 7.43–7.47 (m, 2H, ArH), 7.34 (td, J = 1.5, 5.5 Hz, 1H, ArH), 7.24 (d, J = 3.0 Hz, 1H, ArH), 7.18 (dd, J = 3.0, 8.5 Hz, 1H, ArH), 4.77 (s, 2H, OCH₂), 2.42 (s, 3H, CH₃), 2.38 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ = 170.21, 155.69, 141.50, 134.13, 133.75, 129.54, 129.47, 129.36, 129.05, 128.73, 127.59, 126.81, 126.55, 123.88, 123.13, 118.54, 118.01, 107.01, 64.63, 21.15,12.03. Anal. Calcd for C₂₃H₁₉ClN₄O₂: C, 69.95; H, 4.57; N, 13.38. Found: C, 70.01; H, 4.64; N, 13.30.

1-{4-[(4-Bromophenyl)diazenyl]-3,5-dimethyl-1H-pyrazol-1-yl}-2-(naphthalen-2-yloxy) ethan-1-one (2e): Yield: 0.37 g (80%); pale yellow solid; m.p. 106 °C–108 °C; IR (KBr): 1052, 1641, 1696 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 7.78–7.81 (dd, 2H, J = 2.5, 9.0 Hz, ArH), 7.74–7.76 (d, 1H, J 8.0 Hz, ArH), 7.51–7.67 (m, 4H, ArH), 7.41 (t, J = 7.5 Hz, 1H, ArH), 7.31 (t, J = 7.5 Hz, 1H, ArH), 7.21 (d, J = 3.0 Hz, 1H, ArH), 7.16 (dd, J = 2.5, 8.5 Hz, 1H, ArH), 4.76 (s, 2H, OCH₂), 2.40 (s, 3H, CH₃), 2.37 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ = 170.65, 156.14, 141.86, 134.59, 132.85, 132.73, 129.92, 129.19, 128.04, 127.27, 127.00, 124.33, 123.84, 122.93, 118.99, 118.81, 117.62, 107.48, 65.03, 26.89, 12.43. Anal. Calcd for C₂₃H₁₉BrN₄O₂: C, 59.62; H, 4.13; N, 12.09. Found: C, 59.58; H, 4.15; N, 12.03.

1-{3,5-Dimethyl-4-[(4-nitrophenyl)diazenyl]-1H-pyrazol-1-yl}-2-(naphthalen-2-yloxy)ethan -1-one (2f): Yield: 0.30 g (72%); yellow solid; m.p. 95 °C–97 °C; IR (KBr): 1046, 1661, 1694 cm⁻¹. ¹H NMR (500 MHz, DMSO-d₆): δ = 8.35 (d, J = 8.5 Hz, 1H, ArH), 8.26 (d, J = 8.5 Hz, 1H, ArH), 7.89 (d, J = 9.0 Hz, 1H, ArH), 7.83 (d, J = 9.0 Hz, 2H, ArH), 7.75 (d, J = 8.5 Hz, 1H, ArH), 7.70 (d, J = 9.0 Hz, 1H, ArH), 7.41 (t, J = 7.5 Hz, 1H, ArH), 7.35 (t, J = 7.5 Hz, 1H, ArH), 7.24 (d, J = 3.0 Hz, 1H, ArH), 7.16 (dd, J = 2.5, 9.0 Hz, 1H, ArH), 4.78 (s, 2H, OCH₂), 2.46 (s, 3H, CH₃), 2.43 (s, 3H, CH₃). ¹³C NMR (125 MHz, DMSO-d₆): δ = 170.12, 155.62, 143.10, 137.10, 135.20, 134.10, 129.40, 128.67, 127.52, 126.75, 126.48, 125.58, 125.01, 123.80, 122.26, 118.46, 116.06, 106.96, 64.51 (OCH₂), 26.23 (CH₃), 21.15 (CH₃). Anal. Calcd for C₂₃H₁₉N₅O₄: C, 64.33; H, 4.46; N, 16.31. Found: C, 64.37; H, 4.50; N, 16.24.

Antimicrobial activity

The newly synthesized compounds 1a–f and 2a–f were evaluated for their antibacterial activity against S. aureus (ATCC-25923) and E. coli (ATCC-25922),^21,22 and their antifungal activity against A. niger (ATCC-9029) and C. albicans (ATCC-90028) in DMF using the serial plate dilution method.^23,24 A microbial broth was freshly prepared in normal saline using Mueller–Hinton agar and Sabouraud dextrose agar media for the antibacterial and antifungal activity, respectively. Compounds were serially diluted as 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, and 0.19 μg mL⁻¹ concentrations. The microtiter plates were inoculated with 0.1 mL inoculums of 1 × 10⁷ cfu mL⁻¹ and incubated at 37 °C for 24 h. The lowest concentration of the compounds that completely inhibited visible microbial growth (no turbidity) was assigned as the MIC. Ofloxacin and fluconazole were used as the standard drugs for antibacterial and antifungal activity, respectively.

Molecular docking studies

Molecular docking studies were performed using AutoDock v. 4.2.2 to identify appropriate binding modes and the conformation of the ligand molecule. The crystal structure of S. aureus dihydrofolate reductase complexed with novel 7-aryl-2,4-diaminoquinazolines (PDB code: 3SRQ) was retrieved from the RCSB protein data bank in PDB format. ¹⁸ The structures of all the ligands were drawn using ChemDraw Ultra 13.0 and converted into 3D structures using Hyperchem Pro 8.0 software (www.hyper.com). Autodock Tools (ADT) version 1.5.6 (www.autodock.scrips.edu) was used for the molecular docking. The active site was considered as a rigid molecule, while the ligands were treated as being flexible. Using default parameters, grid-based docking studies were carried out and docking was performed on all compounds using standard ligand 7-aryl-2,4-diaminoquinazolines. The best binding conformation was selected from the docking log (.dlg) file for each ligand and further interaction analysis was performed using PyMol and Discovery Studio Visualizer 4.0.