ꢁꢀꢀꢀ
V.S. Dofe et al.: Bio-activity of novel tetrazole derivativesꢂ ꢂ5
then heated under reflux for 4–5 h with vigorous stirring. After com-
pletion 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.
ArH), 7.33–7.37 (m, 1H, ArH), 8.00 (d, 2H, Jꢀ=ꢀ5.5 Hz, ArH), 8.08 (dd,
1H, Jꢀ=ꢀ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 (Cꢀ=ꢀO); ESI-MS: m/z 407.2
+
(Mꢀ+ꢀ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)-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),
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 (Cꢀ=ꢀO), 1618 (Cꢀ=ꢀ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,
Jꢀ=ꢀ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),
1
1606 (Cꢀ=ꢀO), 1554 (Cꢀ=ꢀC), 1238 (C-F) cm−1; H NMR (DMSO-d6): δ 5.47
(s, 2H, OCH2), 7.26 (t, 2H, Jꢀ=ꢀ9 Hz, ArH), 7.48–7.52 (m, 1H, ArH), 7.70
(d, 1H, Jꢀ=ꢀ8 Hz, ArH), 7.82 (ddd, 1H, Jꢀ=ꢀ9 Hz, 7 Hz and 1.7 Hz, ArH),
8.01–8.05 (m, 2H, ArH), 8.16 (dd, 1H, Jꢀ=ꢀ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),
+
+
+
164.5 (C-F), 172.4 (Cꢀ=ꢀO);. ESI-MS: m/z 409.2642 (Mꢀ+ꢀNa) . Anal. Calcd
173.5 (Cꢀ=ꢀO); ESI-MS: m/z 339 (Mꢀ+ꢀH) and m/z 361 (Mꢀ+ꢀNa) . Anal.
Calcd for C17H11FN4O3: C, 60.36; H, 3.28; N, 16.56. Found: C, 60.29;
H, 3.25; N, 16.51.
for C18H12ClFN4O3: C, 55.90; H, 3.13; N, 14.49. Found: C, 55.97; H, 3.21;
N, 14.58.
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),
Antimicrobial activity
1
2992 (C-H), 1622 (Cꢀ=ꢀO), 1605 (Cꢀ=ꢀC), 1197 (C-F), 718 (C-Cl) cm−1; H
In vitro antibacterial activity of the synthesized compounds was
tested against Gram-positive bacteria S. aureus (NCIM 2178), B. sub-
tilis (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.
NMR (DMSO-d6): δ 5.42 (s, 2H, OCH2), 7.17 (t, 2H, Jꢀ=ꢀ9 Hz, ArH), 7.64
(d, 1H, Jꢀ=ꢀ9 Hz, ArH), 7.72 (dd, 1H, Jꢀ=ꢀ9 Hz and 2.6 Hz, ArH), 7.98 (dd,
2H, Jꢀ=ꢀ9 Hz and 5.5 Hz, ArH), 8.04 (d, 1H, Jꢀ=ꢀ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
+
(Cꢀ=ꢀO); ESI-MS: m/z 395 (Mꢀ+ꢀ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 (Cꢀ=ꢀO), 1605 (Cꢀ=ꢀ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 (tetra-
Biofilm inhibition assay
+
zole C), 164 (C-F), 173.8 (Cꢀ=ꢀO); ESI-MS: m/z 353.3142 (Mꢀ+ꢀH) , m/z
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 5ꢀ×ꢀ105 cfu mL−1. Aliquots of 100 μL were distributed
in each well and then incubated at 37°C for 24 h under static con-
ditions. 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 tem-
perature. 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 experi-
ments were carried out in triplicates and the values are indicated
as meanꢀ ꢀS.D.
+
375.2781 (Mꢀ+ꢀ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 (Cꢀ=ꢀO), 1554 (Cꢀ=ꢀ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, Jꢀ=ꢀ8.8 Hz, ArH),
7.57–7.63 (m, 2H, ArH), 7.90 (s, 1H, ArH), 8.00 (dd, 2H, Jꢀ=ꢀ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 (Cꢀ=ꢀO); ESI-MS: m/z 353.0987 (Mꢀ+ꢀ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 (Cꢀ=ꢀO), 1603 (Cꢀ=ꢀC), 1158 (C-F), 760 cm−1
(C-Cl); 1H NMR (DMSO-d6): δ 5.50 (s, 2H, OCH2), 7.24 (t, 2H, Jꢀ=ꢀ8.4 Hz,
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