Abstract
In the development of antimicrobial agents, we designed and synthesized novel tetrazole derivatives. The structures of compounds 6a–f and 7a–f were characterized by IR, 1H NMR, 13C NMR, MS and elemental analysis. These compounds were tested for their antimicrobial activity against a series of strains Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa and for antifungal activity against the strains Candida albicans, Candida glabrata, and Candida tropicalis. Compounds 6e, 6f, 7a, and 7f exhibit potent antimicrobial activities compared to the reference drugs streptomycin and miconazole. Tetrazole derivatives 7a–f also inhibit biofilm formation and compound 7f exhibits best anti-biofilm activity with a biofilm inhibitory concentration (BIC) as low as 0.9 μm.
Introduction
Microbial infections become an increasingly serious and challenging problem for human health across the world. Such infections most commonly affect patients with decreased immunity, neoplastic disorders and undergoing organ transplantation [1]. This situation stimulates an urgent need to develop novel antimicrobial agents from newer classes of compounds. Fluorinated compounds have proved invaluable as antimicrobial agents, and have been used for the treatment of obesity and various diseases associated with the cardiovascular and central nervous systems [2]. The incorporation of fluorine into a potential drug molecule can improve the therapeutic efficacy due to hydrogen bonding interactions at the active sites of enzyme [3].
The naturally occurring flavones display biological activity (Figure 1) [4], [5], [6], [7], [8], [9], [10]. One of the recognized functions of flavonoids in plants is their protective role against microbial incursion [4]. These compounds play an important role in drug discovery processes.
Naturally occurring flavones and pharmacologically important tetrazole drugs.
The synthetic versatility of tetrazole is due to its widespread applications in medicinal chemistry. Tetrazole scaffold is part of highly effective drugs such as candesartan, pamiroplast and pranlukast (Figure 1) [11]. Tetrazole can be considered a carboxylic acid analogue because of similar pKa values and planar delocalized systems. Tetrazole derivatives are resistant to various biological degradation processes, which contributes to bioavailability [12], [13]. These factors play a role in applications of tetrazole derivatives as anticancer [14], antifungal [15], antitubercular [16], anti-HIV [17], antioxidant [18] and hormonal agents [19].
It can be suggested that the presence of fluorine, flavone and tetrazole in a single molecular framework (Figure 2) would increase biological activity of the compound. In continuation of our earlier work on the synthesis of tetrazole derivatives [20], we now report the synthesis of such compounds and evaluation of their antimicrobial and anti-biofilm activities.
The designed scaffold containing fluorinated flavone and tetrazole as a main backbone.
Results and discussion
Chemistry
Substituted 3-hydroxychromones 5a–f were synthesized as described in our previous report [21] (Scheme 1). Claisen-Schmidt condensation of substituted 2-hydroxyacetophenones 3a–f with 4-fluorobenzaldehyde in ethanolic solution of potassium hydroxide at room temperature furnished substituted chalcones 4a–f which were converted into corresponding substituted 3-hydroxychromones 5a–f upon oxidative cyclization using hydrogen peroxide in ethanol.
![Scheme 1 Reagents and conditions: [i] acetic anhydride, pyridine, 100°C, 3–4 h, 80%–98%; [ii] AlCl3, 150°C, 3–4 h, 70%–75%; [iii] 4-fluorobenzaldehyde, KOH, EtOH, room temp, 2–3 h, 60%–80%; [iv] H2O2, NaOH, 0°C to room temp, 2–3 h, 70%–80%.](/document/doi/10.1515/hc-2017-0016/asset/graphic/j_hc-2017-0016_scheme_001.jpg)
Reagents and conditions: [i] acetic anhydride, pyridine, 100°C, 3–4 h, 80%–98%; [ii] AlCl3, 150°C, 3–4 h, 70%–75%; [iii] 4-fluorobenzaldehyde, KOH, EtOH, room temp, 2–3 h, 60%–80%; [iv] H2O2, NaOH, 0°C to room temp, 2–3 h, 70%–80%.
3-[(1H-tetrazol-5-yl)methoxy]-2-(4-fluorophenyl)-4H-chromen-4-ones 7a–f were synthesized from the corresponding 3-hydroxychromones 5a–f (Scheme 2). First, compounds 5a–f were alkylated with 2-chloroacetonitrile in the presence of K2CO3 in N,N′-dimethylformamide (DMF) at room temperature for 3–4 h to afford the substituted 2-(2-(4-fluorophenyl)-4-oxo-4H-chromen-3-yloxy)acetonitriles 6a–f in 71%–86% yield. Then, treatment of 6a–f with sodium azide and zinc bromide in water at 100°C for 4–5 h furnished the final products 7a–f in 73%–81% yield. All compounds were characterized by spectroscopic techniques of 1H NMR, 13C NMR and MS.
![Scheme 2 Reagents and conditions: [i] 2-chloroacetonitrile, K2CO3, DMF, room temp, 3–4 h, 71%–86%; [ii] sodium azide, zinc bromide, H2O, reflux, 4–5 h, 73%–81%.](/document/doi/10.1515/hc-2017-0016/asset/graphic/j_hc-2017-0016_scheme_002.jpg)
Reagents and conditions: [i] 2-chloroacetonitrile, K2CO3, DMF, room temp, 3–4 h, 71%–86%; [ii] sodium azide, zinc bromide, H2O, reflux, 4–5 h, 73%–81%.
Antimicrobial activity
The minimum inhibitory concentration (MIC) values were determined by micro-broth dilution method. The results were compared with activities of standard antibacterial drug streptomycin and antifungal drug miconazole (Table 1). All compounds 6a–f and 7a–f exhibit moderate to potent antibacterial and antifungal activity. It can be seen that compounds exhibit varying degrees of antifungal activity due to the nature of heterocyclic skeleton. Tetrazole derivatives 7a–f are more active compared to 6a–f analogues. Compounds 6f, 7a and 7f are equipotent with miconazole against Candida albicans. Compound 7f shows most potent activity against Candida glabrata. Compounds 6f, 7a, and 7c exhibit more potent activity than 6e, 7b, 7d and 7e against C. glabrata. In addition, compounds 6a, 6b, 6c and 6d show excellent activity against C. glabrata. Compound 7a shows highly potent activity against Candida tropicalis. Compounds 6f, 7b and 7f show more potent activity than 6a, 6e, 7c, 7d and 7e against C. tropicalis.
Antimicrobial screening data of the compounds 6a–f and 7a–f.
| Entry | Antimicrobial activity (MIC, μg/mL) | |||||||
|---|---|---|---|---|---|---|---|---|
| Antibacterial activity | Antifungal activity | |||||||
| Sa | Bs | Ec | Pa | Ca | Cg | Ct | ||
| 6a | 400 | 400 | 50 | 50 | 100 | 100 | 50 | |
| 6b | 400 | 400 | 100 | 100 | 100 | 200 | 200 | |
| 6c | 100 | 100 | 50 | 100 | 100 | 200 | 100 | |
| 6d | 400 | 400 | 50 | 50 | 50 | 100 | 100 | |
| 6e | 200 | 200 | 25 | 50 | 25 | 50 | 50 | |
| 6f | 100 | 100 | 100 | 100 | 12.5 | 25 | 25 | |
| 7a | 50 | 100 | 100 | 100 | 12.5 | 25 | 12.5 | |
| 7b | 100 | 200 | 100 | 50 | 25 | 50 | 25 | |
| 7c | 50 | 100 | 200 | 200 | 25 | 25 | 50 | |
| 7d | 200 | 200 | 100 | 50 | 50 | 50 | 50 | |
| 7e | 100 | 100 | 200 | 100 | 50 | 50 | 50 | |
| 7f | 50 | 50 | 400 | 200 | 12.5 | 12.5 | 25 | |
| Streptomycin | 12.5 | 400 | 400 | 200 | – | – | – | |
| Miconazole | – | – | – | – | 12.5 | 400 | 800 | |
Sa, Staphylococcus aureus; Bs, Bacillus subtilis; Ec, Escherichia coli; Pa, Pseudomonas aeruginosa; Ca, Candida albicans; Cg, Candida glabrata; Ct, Candida tropicalis.
Biofilm inhibitory activity
Biofilm is surface-adhered bacterial area implanted in an extracellular matrix responsible for the growing resistance in bacteria to antibiotics [22]. Several pathogenic bacteria are found in environments that form biofilm which are structured microbial communities embedded with complex associations with each other. Many serious chronic infections are transmitted to humans via biofilms [23]. Developing new molecules having the ability to inhibit biofilm formation could be one of the solutions for the growing antibiotic resistance. In this regard, the synthesized flavones possessing tetrazole moiety 7a–f were evaluated for anti-biofilm activity against three bacterial strains, namely Staphylococcus aureus NCIM 2178, Bacillus subtilis NCIM 2250, Escherichia coli NCIM 2137 and Pseudomonas aeruginosa NCIM 2036. The results shown in Table 2 reveal that these compounds exhibit good to excellent anti-biofilm activity against all four strains tested. Particularly, compounds 7a, 7e and 7f demonstrate potent efficacy in inhibiting biofilm formation with IC50 values of 1.3, 1.4, and 0.9 μm, respectively, against P. aeruginosa NCIM 2036.
Biofilm inhibition assay of flavone-tetrazole conjugates 7a–f.
| Compound | IC50 values (μm) | |||
|---|---|---|---|---|
| Staphylococcus aureusNCIM 2178 | Bacillus subtilisNCIM 2250 | Escherichia coliNCIM 2137 | Pseudomonas aeruginosaNCIM 2036 | |
| 7a | 7.8±0.09 | 2.6±0.32 | 4.6±0.04 | 1.3±0.72 |
| 7b | 4.5±0.41 | 3.6±0.08 | 4.3±0.09 | 2.1±0.04 |
| 7c | 7.9±0.36 | 5.6±0.05 | 8.7±0.02 | 2.3±0.24 |
| 7d | 8.6±0.21 | 5.2±0.08 | 9.1±0.06 | 6.2±0.15 |
| 7e | 5.8±0.25 | 4.8±0.11 | 3.3±0.31 | 1.4±0.26 |
| 7f | 7.1±0.12 | 2.9±0.04 | 4.5±0.09 | 0.9±0.03 |
| Ciprofloxacin | 1.56±0.14 | 1.56±0.02 | 1.56±0.23 | 0.78±0.13 |
Conclusions
New tetrazole derivatives were synthesized and evaluated for antibacterial activity, antifungal activity, and bio-film inhibition. All compounds show moderate to potent activity compared to the standard drugs streptomycin and miconazole. Compounds 6e, 6f, 7a and 7f are potent antimicrobial agents. Compounds 7a–f which contains tetrazole ring are more potent antimicrobial agents compared to 6a–f series. Further, these compounds are also effective inhibitors of biofilm formation which may contribute to the development of antibiotic resistance in bacteria. Compound 7f is the most active against P. aeruginosa NCIM 2036. It can be concluded that incorporation of the tetrazole ring into fluorinated flavones enhances the biological effect.
Experimental
All reagents were purchased from commercial suppliers and used without purification. The progress of each reaction was monitored by ascending thin-layer chromatography (TLC) using Merck silica gel 60 F254 plates and visualized using UV light and iodine vapor. Melting points were determined in open capillaries and are uncorrected. Infrared spectra were recorded on a Carry 600 Series FT-IR spectrophotometer using KBr pellets. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker AVANCE II 400 spectrometer in CDCl3 or DMSO-d6. Mass spectra were recorded on a Waters Q-TOF micro mass instrument equipped with an electro-spray ionization (ESI) source. Elemental analysis was performed on a Perkin-Elmer EAL-240 elemental analyzer.
General procedure for the synthesis of substituted 2-(2-(4-fluorophenyl)-4-oxo-4H-chromen-3-yloxy)acetonitriles 6a–f
A mixture of 2-(4-fluorophenyl)-3-hydroxy-4H-chromen-4-one 5a–f (1 mmol), potassium carbonate (2 mmol) and 2-chloroacetonitrile (1 mmol) and DMF was stirred at room temperature for about 3–4 h and analyzed by TLC using petroleum ether/ethyl acetate as an eluent. Then the mixture was quenched with crushed ice and the precipitated solid was collected by filtration and crystallized from ethanol.
2-(2-(4-Fluorophenyl)-4-oxo-4H-chromen-3-yloxy)acetonitrile (6a) This compound was obtained from 5a as a white solid; yield 78%; mp 140–142°C; IR: υ 3012 (Ar-H), 2965 (C-H), 2346 (C≡N), 1634 (C=O), 1604 (C=C), 1238 (C-F) cm−1; 1H NMR (CDCl3): δ 5.11 (s, 2H, OCH2), 7.20–2.27 (m, 2H, ArH), 7.45 (ddt, 1H, J=8 Hz, 8 Hz and 1.2 Hz, ArH), 7.57 (dd, 1 H, J=9 Hz and 1.1 Hz, ArH), 7.74 (ddt, 1H, J=8 Hz, 8 Hz and 1.5 Hz, ArH), 8.08 (dd, 2H, J=9 Hz, ArH), 8.25 (dd, 1H, J=8 Hz and 1.5 Hz, ArH); 13C NMR (CDCl3): δ 56.1 (O-CH2), 115.1, 115.9, 116.1 (C≡N), 118.1, 123.8, 125.4, 125.8, 126.1, 131.3, 134.2, 137.5, 155.3, 156.4, 163.1, 165.6 (C-F), 174.1 (C=O). ESI-HRMS. Calcd. for C17H10FNO3 (M+H)+: m/z 296.0723. Found: 296.0372. Anal. Calcd for C17H10FNO3: C, 69.15; H, 3.41; N, 4.74. Found: C, 69.07; H, 3.35; N, 4.68.
2-(6-Chloro-2-(4-fluorophenyl)-4-oxo-4H-chromen-3-yloxy)acetonitrile (6b) This compound was obtained from 5b as a white solid; yield 86%; mp 168–170°C; IR: υ 3069 (Ar-H), 2952 (C-H), 2360 (C≡N), 1628 (C=O), 1600 (C=C), 1144 (C-F) 755 (C-Cl) cm−1; 1H NMR (DMSO-d6): δ 5.14 (s, 2H, OCH2), 7.34 (t, 2H, J=9 Hz, ArH), 7.73 (d, 1H, J=9 Hz, ArH), 7.79 (dd, 1H, J=9 Hz and 2.6 Hz, ArH), 8.07 (d, 1H, J=2.6 Hz, ArH), 8.13 (m, 2H, ArH); 13C NMR (DMSO-d6): δ 56.4 (O-CH2), 115.5, 115.7, 120.4 (C≡N), 124, 124.3, 125.7, 130.3, 131.1, 131.2, 134.1, 137.6, 153.1, 155.4, 162.4, 165 (C-F), 172.1 (C=O); ESI-MS: m/z 330 (M+H)+, m/z 352 (M+Na)+. Anal. Calcd for C17H9ClFNO3: C, 61.93; H, 2.75; N, 4.25. Found: C, 61.98; H, 2.82; N, 4.33.
2-(2-(4-Fluorophenyl)-8-methyl-4-oxo-4H-chromen-3-yloxy)acetonitrile (6c) This compound was obtained from 5c as a white solid; yield 71%; mp 176–178°C; IR: υ 3032 (Ar-H), 2915 (C-H), 2360 (C≡N), 1633 (C=O), 1604 (C=C), 1193 (C-F) cm−1; 1H NMR (CDCl3): δ 2.58 (s, 3H, CH3), 5.12 (s, 2H, OCH2), 7.23–7.27 (m, 2H, ArH), 7.34 (t, 1H, J=7 Hz, ArH), 7.57 (d, 1H, J=7 Hz, ArH), 8.07–8.13 (m, 3H, ArH); 13C NMR (CDCl3): δ 15.8 (CH3), 56, 1 (O-CH2), 115.1, 115.9, 116.2 (C≡N), 123.4, 123.8, 125.1, 126.5, 127.6, 131.3, 135, 137.4, 153.8, 155.8, 163.1, 165.6 (C-F), 174.4 (C=O); ESI-MS: m/z 310 (M+H)+, m/z 332 (M+Na)+. Anal. Calcd for C18H12FNO3: C, 69.90; H, 3.91; N, 4.53. Found: C, 69.96; H, 3.94; N, 4.56.
2-(2-(4-Fluorophenyl)-6-methyl-4-oxo-4H-chromen-3-yloxy)acetonitrile (6d) This compound was obtained from 5d as a white solid; yield 74%; mp 162–164°C; IR: υ 3087 (Ar-H), 2974 (C-H), 2369 (C≡N), 1632 (C=O), 1603 (C=C), 1187 (C-F) cm−1; 1H NMR (DMSO-d6): δ 2.53 (s, 3H, CH3), 5.15 (s, 2H, OCH2), 7.36 (s, 2H, ArH), 7.61 (s, 2H, ArH), 7.90 (s, 1H, ArH), 8.12 (s, 2H, ArH); 13C NMR (DMSO-d6): δ 20.5 (CH3), 56.4 (O-CH2), 115.5, 115.7, 118 (C≡N), 122.9, 124.1, 126.2, 131.1, 131.1, 134.8, 135.4, 136.3, 137.5, 153, 154.9, 162.3 (C-F), 173.1 (C=O); ESI-MS: m/z 310 (M+H)+, m/z 332 (M+Na)+. Anal. Calcd for C18H12FNO3: C, 69.90; H, 3.91; N, 4.53. Found: C, 69.82; H, 3.95; N, 4.66.
2-(6,8-Dichloro-2-(4-fluorophenyl)-4-oxo-4H-chromen-3-yloxy)acetonitrile (6e) This compound was obtained from 5e as a white solid; yield 77%; mp 177–179°C; IR: υ 3064 (Ar-H), 2916 (C-H), 2361 (C≡N), 1625 (C=O), 1601 (C=C), 1180 (C-F) 717 (C-Cl) cm−1; 1H NMR (CDCl3): δ 5.12 (s, 2H, OCH2), 7.26 (t, 2H, J=9 Hz, ArH), 7.77 (d, 1H, J=2.6Hz, ArH), 8.10 (d, 1H, J=2.6Hz, ArH), 8.18 (m, 2H, ArH); 13C NMR (CDCl3): δ 56.1 (O-CH2), 114.8, 116.2, 116.4 (C≡N), 123.8, 124.6, 125.5, 131.2, 131.5, 131.6, 134.2, 137.6, 149.4, 156.3, 163.5, 166 (C-F), 172.5 (C=O); ESI-MS: m/z 364 (M+H)+, m/z 386 (M+Na)+. Anal. Calcd for C17H8Cl2FNO3: C, 56.07; H, 2.21; N, 3.85. Found: C, 56.18; H, 2.24; N, 3.79.
2-(6-Chloro-2-(4-fluorophenyl)-7-methyl-4-oxo-4H-chromen-3-yloxy)acetonitrile (6f) This compound was obtained from 5f as a white solid; yield 82%; mp 170–172°C; IR: υ 3013 (Ar-H), 2915 (C-H), 2360 (C≡N), 1599 (C=O), 1552 (C=C), 1203 (C-F) 750 (C-Cl) cm−1; 1H NMR (DMSO-d6): δ 2.54 (s, 3H, CH3), 5.14 (s, 2H, OCH2), 7.35 (s, 2H, ArH), 7.70 (s, 1H, ArH), 8.04 (s, 1H, ArH), 8.12 (s, 2H, ArH); 13C NMR (DMSO-d6): δ 20.3 (CH3), 56.4 (O-CH2), 115.5, 115.6, 115.7 (C≡N), 120.3, 122.4, 124.2, 125.9, 125.9, 131.1, 131.1, 137.5, 142.9, 153.1, 155.1, 167.4 (C-F), 172.1 (C=O); ESI-MS: m/z 344 (M+H)+, m/z 366 (M+Na)+. Anal. Calcd for C18H11ClFNO3: C, 62.89; H, 3.23; N, 4.07. Found: C, 62.93; H, 3.28; N, 4.11.
General procedure for synthesis of 3-((1H-tetrazol-5-yl)methoxy)-2-(4-fluorophenyl)-4H-chromen-4-ones 7a–f
To a mixture of sodium azide (1.5 mmol) and zinc bromide (1.5 mmol) in water (20 mL) was added substituted 2-(2-(4-fluorophenyl)-4-oxo-4H-chromen-3-yloxy)acetonitrile 6a–f (1 mmol). The mixture was then heated under reflux for 4–5 h with vigorous stirring. After completion of the reaction, as evident by TLC analysis using chloroform/methanol as an eluent, the mixture was quenched with crushed ice and the precipitated solid was collected by filtration and crystallized from ethanol.
3-((1H-Tetrazol-5-yl)methoxy)-2-(4-fluorophenyl)-4H-chromen-4-one (7a) This compound was obtained from 6a as a white solid; yield 81%; mp 200–202°C; IR: υ 3444 (N-H), 3030 (Ar-H), 2917 (C-H), 1606 (C=O), 1554 (C=C), 1238 (C-F) cm−1; 1H NMR (DMSO-d6): δ 5.47 (s, 2H, OCH2), 7.26 (t, 2H, J=9 Hz, ArH), 7.48–7.52 (m, 1H, ArH), 7.70 (d, 1H, J=8 Hz, ArH), 7.82 (ddd, 1H, J=9 Hz, 7 Hz and 1.7 Hz, ArH), 8.01–8.05 (m, 2H, ArH), 8.16 (dd, 1H, J=8 Hz and 1.7 Hz, ArH); 13C NMR (DMSO-d6): δ 61.8 (O-CH2), 115.3, 115.6, 118.3, 123.4, 124.9, 125.1, 126.2, 130.9, 131, 134.1, 138.2, 154.7, 154.9, 162 (tetrazole C), 164.5 (C-F), 173.5 (C=O); ESI-MS: m/z 339 (M+H)+ and m/z 361 (M+Na)+. Anal. Calcd for C17H11FN4O3: C, 60.36; H, 3.28; N, 16.56. Found: C, 60.29; H, 3.25; N, 16.51.
3-((1H-Tetrazol-5-yl)methoxy)-6-chloro-2-(4-fluorophenyl)-4H-chromen-4-one (7b) This compound was obtained from 6b as a white solid; yield 85%; mp 203–205°C; IR: υ 3435 (N-H), 3035 (Ar-H), 2992 (C-H), 1622 (C=O), 1605 (C=C), 1197 (C-F), 718 (C-Cl) cm−1; 1H NMR (DMSO-d6): δ 5.42 (s, 2H, OCH2), 7.17 (t, 2H, J=9 Hz, ArH), 7.64 (d, 1H, J=9 Hz, ArH), 7.72 (dd, 1H, J=9 Hz and 2.6 Hz, ArH), 7.98 (dd, 2H, J=9 Hz and 5.5 Hz, ArH), 8.04 (d, 1H, J=2.6 Hz, ArH); 13C NMR (DMSO-d6): δ 61.7 (O-CH2), 115.4, 115.6, 120.7, 123.8, 124.5, 125.9, 125.9, 129.9, 131, 134, 138.2, 153.2, 155.2, 162.1 (tetrazole C), 164.6 (C-F), 172.5 (C=O); ESI-MS: m/z 395 (M+Na)+. Anal. Calcd for C17H10ClFN4O3: C, 54.78; H, 2.70; N, 15.03. Found: C, 54.84; H, 2.72; N, 15.23.
3-((1H-Tetrazol-5-yl)methoxy)-2-(4-fluorophenyl)-8-methyl-4H-chromen-4-one (7c) This compound was obtained from 6c as a white solid; yield 73%; mp 198–200°C; IR: υ 3435 (N-H), 3035 (Ar-H), 2992 (C-H), 1622 (C=O), 1605 (C=C), 1197 cm−1 (C-F); 1H NMR (DMSO-d6): δ 2.33 (s, 3H, CH3), 5.61 (s, 2H, OCH2), 6.97 (s, 1H, ArH), 7.10–7.22 (m, 2H, ArH), 7.53 (s, 1H, ArH), 7.69–7.80 (m, 3H, ArH), 8.41 (s, 1H, NH); 13C NMR (DMSO-d6): δ 15.1 (CH3), 63.5 (O-CH2), 114.9, 115.2, 122.3, 123, 124.3, 126.6, 127.1, 128.2, 130.4, 134.2, 139, 149.5, 152.6, 161.5 (tetrazole C), 164 (C-F), 173.8 (C=O); ESI-MS: m/z 353.3142 (M+H)+, m/z 375.2781 (M+Na)+. Anal. Calcd for C18H13FN4O3: C, 61.36; H, 3.72; N, 15.90. Found: C, 61.45; H, 3.69; N, 15.98.
3-((1H-Tetrazol-5-yl)methoxy)-2-(4-fluorophenyl)-6-methyl-4H-chromen-4-one (7d) This compound was obtained from 6d as a white solid; yield 75%; mp 202–204°C; IR: υ 3423 (N-H), 3021 (Ar-H), 2918 (C-H), 1602 (C=O), 1554 (C=C), 1174 cm−1 (C-F); 1H NMR (DMSO-d6): δ 2.46 (s, 3H, CH3), 5.47 (s, 2H, OCH2), 7.27 (t, 2H, J=8.8 Hz, ArH), 7.57–7.63 (m, 2H, ArH), 7.90 (s, 1H, ArH), 8.00 (dd, 2H, J=9.0 Hz and 5.3 Hz, ArH); 13C NMR (DMSO-d6): δ 20.4 (CH3), 61.8 (O-CH2), 115.3, 115.5, 118, 123.1, 124.1, 126.3, 126.3, 130.9, 134.7, 135.2, 138.2, 153, 154.7, 162 (tetrazole C), 164.5 (C-F), 173.4 (C=O); ESI-MS: m/z 353.0987 (M+H)+. Anal. Calcd for C18H13FN4O3: C, 61.36; H, 3.72; N, 15.90. Found: C, 61.34; H, 3.65; N, 15.81.
3-((1H-Tetrazol-5-yl)methoxy)-6,8-dichloro-2-(4-fluorophenyl)-4H-chromen-4-one (7e) This compound was obtained from 6e as a white solid; yield 80%; mp 194–196°C; IR: υ 3423 (N-H), 3040 (Ar-H), 2917 (C-H), 1660 (C=O), 1603 (C=C), 1158 (C-F), 760 cm−1 (C-Cl); 1H NMR (DMSO-d6): δ 5.50 (s, 2H, OCH2), 7.24 (t, 2H, J=8.4 Hz, ArH), 7.33–7.37 (m, 1H, ArH), 8.00 (d, 2H, J=5.5 Hz, ArH), 8.08 (dd, 1H, J=8.4 Hz and 5.5 Hz, ArH); 13C NMR (DMSO-d6): δ 62.0 (O-CH2), 115.6, 115.8, 123, 123.8, 125.2, 129.6, 131.0, 133.5, 138.6, 139.4, 148.9, 154.6, 162.2 (tetrazole C), 164.7 (C-F), 172.0 (C=O); ESI-MS: m/z 407.2 (M+H)+. Anal. Calcd for C17H9Cl2FN4O3: C, 50.15; H, 2.23; N, 13.76. Found: C, 50.33; H, 2.32; N, 13.87.
3-((1H-Tetrazol-5-yl)methoxy)-6-chloro-2-(4-fluorophenyl)-7-methyl-4H-chromen-4-one (7f) This compound was obtained from 6f as a white solid; yield 78%; mp 178–180°C; IR: υ 3495 (N-H), 3077 (Ar-H), 2924 (C-H), 1638 (C=O), 1618 (C=C), 1166 (C-F) 771 cm−1 (C-Cl); 1H NMR (DMSO-d6): δ 2.44 (s, 3H, CH3), 5.45 (s, 2H, OCH2), 7.30 (t, 2H, J=9.0 Hz, ArH), 7.73–7.76 (m, 1H, ArH), 7.92–7.96 (m, 3H, ArH); 13C NMR (DMSO-d6): δ 20.1 (CH3), 61.8 (O-CH2), 115.4, 115.7, 120.6, 122.5, 123.9, 126.1, 130.6, 130.9, 131, 138.1, 142.7, 153.0, 154.9, 162.0 (tetrazole C), 164.5 (C-F), 172.4 (C=O);. ESI-MS: m/z 409.2642 (M+Na)+. Anal. Calcd for C18H12ClFN4O3: C, 55.90; H, 3.13; N, 14.49. Found: C, 55.97; H, 3.21; N, 14.58.
Antimicrobial activity
In vitro antibacterial activity of the synthesized compounds was tested against Gram-positive bacteria S. aureus (NCIM 2178), B. subtilis (NCIM 2250) and Gram-negative bacteria E. coli (NCIM 2137), P. aeruginosa (NCIM 2036). The compounds were also screened for antifungal activity against C. albicans (MTCC 277), C. glabrata (NCIM 3236), C. tropicalis (NCIM 3110). Compounds were diluted in DMSO with 1 μg/mL concentrations for bioassays. Micro-broth dilution method was used to determine MIC values of compounds in 96-well micro-titre plates [24]. Test compounds were serially diluted in growth medium. Plates were incubated at 30°C for fungi and 37°C for bacteria for 24 h. All experiments were carried out in triplicates and mean values are reported.
Biofilm inhibition assay
The flavone–tetrazole conjugates 7a–f were screened in sterile 96 well polystyrene micro-titre plates using the modified bio-film inhibition assay [25] against a panel of pathogenic bacterial strains S. aureus NCIM 2178, B. subtilis NCIM 2250, E. coli NCIM 2137 and P. aeruginosa NCIM 2036, which were cultured overnight in tryptone soy broth (supplemented with 0.5% glucose). The test compounds of predetermined concentrations ranging from 0 to 200 μg/mL were mixed with the bacterial suspensions having an initial inoculums concentration of 5×105 cfu mL−1. Aliquots of 100 μL were distributed in each well and then incubated at 37°C for 24 h under static conditions. Then medium was discarded and washed with phosphate buffered saline to remove the non-adherent bacteria. Micro-titre plate well was stained with 100 μL of 0.1% crystal violet solution followed by 30-min incubation at room temperature. Afterwards the crystal violet solution from the plates was discarded, thoroughly washed with distilled water 3–4 times and air dried at room temperature. The crystal violet stained biofilm was solubilized in 95% ethanol (100 μL) and the absorbance was recorded at 540 nm using a TRIAD multimode reader (Dynex Technologies, USA). All experiments were carried out in triplicates and the values are indicated as mean±S.D.
Acknowledgments
V.S.D. is grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing Senior Research Fellowship and Sophisticated Analytical Instrumentation Facility (SAIF). The authors thank Head, Department of Chemistry, for providing laboratory facility and the Rajarshi Shahu College, Latur, for providing biological activity results.
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