M.M. Alam et al.
Bioorganic & Medicinal Chemistry 38 (2021) 116136
–
–
Hz, 1H, Ar H), 6.89 (br.s., 1H, Ar H), 7.16 (d, J = 8.50 Hz, 1H,
4.2. Biological activities
–
–
–
Ar H), 7.29–7.30 (m, 1H, Ar H), 7.44 (t, J = 8.50 Hz, 1H, Ar H),
–
–
7.57 (d, J = 8.50 Hz, 1H, Ar H), 7.65 (br.s, 1H, Ar H), 8.39 (br.s, 1H,
triazole proton). 13C NMR (214 MHz, CDCl3) δ ppm: 17.99, 21.48,
22.87, 26.60, 112.83, 117.99, 121.97, 126.09, 129.74, 134.31, 136.59,
140.21, 155.28; ESI MS :322[M+H]+ C20H23N3O(Calcd): C, 74.74; H,
7.21; N, 13.07; O, 4.98. Obsd:C, 74.72; H, 7.22; N, 13.07; O, 4.99
4.2.1. Cytotoxicity activity
The human breast tumour cell lines, MCF-7 and MDA-MB-231 and
normal epithelial embryonic kidney cells HEK-293 were cultured in
Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine
serum, 2 mM L-glutamine and 100 µg/mL penicillin/streptomycin. Cells
were maintained at 37 ◦C in 5% CO2 in a humidified incubator. All cells
were sub-cultured 3 times/week by trypsinization using TrypLE Express
(1×). The compounds 3–13 were evaluated for antiproliferative effect
using the MTT viability assay of MCF-7, MDA-MB-231, and normal
epithelial embryonic kidney cells HEK-293 to calculate the relative IC50
values for each compound. Cells were seeded in 96-well plates at a
density of 10 × 103 cells/mL in a total volume of 200 µL per well. 0.1%
of DMSO was used as a vehicle control. After 24 h, the cells were treated
with 2 µL test compound which had been pre-prepared as stock solutions
to furnish the concentration range of study, 0.01 µM to 100 µM, and re-
incubated for 72 h. The culture medium was then removed, and the cells
washed with phosphate buffered saline (PBS) and 100 µL MTT was
added (final concentration of 5 mg/mL MTT). Cells were incubated for 4
h in darkness at 37 ◦C. 200 µL DMSO was then added to each well and
the cells maintained at room temperature in darkness for 20 min.
Absorbance was detected with a microplate reader at 570 nm. Results
were expressed as percentage viability relative to vehicle control
(100%). Dose response curves were plotted and IC50 values were ob-
tained using Prism software (GraphPad Software, Inc., La Jolla, CA,
USA). All the experiments were repeated in three independent
experiments.
4.1.2.8. 4-((2-isopropyl-5-methylphenoxy)methyl)-1-o-tolyl-1H-1,2,3-tri-
azole (10). Yield 68%; white powder; M.p.: 75–76 ◦C; IR (ATR)
ν
cmꢀ 1:2956, 1502, 1456, 1411, 1383, 1287, 1247, 1166, 1093, 1030,
1
–
947, 796; H NMR (850 MHz, CDCl3) δ ppm:1.24 (br.s, 6H, Ar CH
–
–
(CH3)2, 2.26 (br.s, 3H, Ar CH3), 2.38 (br.s, 3H, Ar CH3), 3.35 (br.s,
–
– –
–
–
–
1H, Ar CH(CH3)2), 5.33 (br.s, 2H, O CH2 ), 6.83 (br.s, 1H, Ar H),
–
–
6.90 (br.s, 1H, Ar H), 7.15 (br.s, 1H, Ar H), 7.40 (br. s, 2H, Ar H),
7.43 (br.s, 2H, Ar H), 8.20 (br.s, triazole proton); 13C NMR (214 MHz,
CDCl3) δ ppm:17.93, 21.46, 22.84, 26.69, 62.32, 112.96, 122.00,
126.12, 126.94, 130.07, 131.57, 133.78, 134.32, 136.58, 155.29; ESI
MS: 322[M+H]+ C20H23N3O(Calcd): C, 74.74; H, 7.21; N, 13.07; O,
4.98. Obsd:C, 74.73; H, 7.22; N, 13.06; O, 4.99.
4.1.2.9. 4-((2-isopropyl-5-methylphenoxy)methyl)-1-(4-nitrophenyl)-1H-
1,2,3-triazole (11). Yield 85%; yellow powder; M.p.:144–145 ◦C; IR
(ATR)
ν
cmꢀ 1:3055, 2962, 1610, 1597, 1523, 1506, 1456, 1405, 1334,
1256, 1238, 1166, 1096, 1039, 1019, 848; 1H NMR (850 MHz, CDCl3) δ
–
–
ppm: 1.24 (d, J = 6.7 Hz, 6H, Ar CH(CH3)2), 2.37 (s, 3H, Ar CH3),
–
– –
3.34 (m, 1H, Ar CH(CH3)2), 5.34 (br.s, 2H, O CH2 ), 6.83–6.85 (m,
–
–
2H, Ar H), 7.16 (d, J = 7.78 Hz, 1H, Ar H), 8.03 (d, J = 8.30 Hz, 2H,
–
–
Ar H), 8.24 (br.s, 1H, triazole proton), 8.45 (d, J = 7.78 Hz, 2H,
Ar H); 13C NMR (214 MHz, CDCl3) δ ppm: 21.42, 22.87, 26.62, 62.18,
4.2.2. In vitro thymidylate synthase enzymatic activity
The activity was performed as reported earlier.44,45 It involves a
mixture containing 2-mercaptoethanol (0.1 M), (6R,S)-tetrahydrofolate
(0.0003 M), formaldehyde (0.012 M), MgCl2 (0.02 M), dUMP (0.001 M),
TrisHCl (0.04 M), and NaEDTA (0.00075 M). This assay was done
spectrophotometrically at 30◦ C and pH 7.4. The reaction was initiated
by the addition of an amount of enzyme giving a change in absorbance at
340 nm of 0.016/min in the absence of inhibitor. The percent inhibition
was determined at a minimum of four inhibitor concentrations within
20% of the 50% point. The standard deviations for determination of the
50% points were within ± 10% of the values given.
112.72, 120.67, 122.19, 125.65, 126.21, 134.25, 136.67, 147.26,
155.09; ESI MS: 353 [M+H]+ C19H20N4O3 (Calcd): C, 64.76; H, 5.72; N,
15.90; O, 13.62. Obsd:C, 64.74; H, 5.73; N, 15.90; O, 13.63.
4.1.2.10. 3-(4-((2-isopropyl-5-methylphenoxy)methyl)-1H-1,2,3-triazol-
1-yl)benzoic acid (12). Yield 85%; white powder; M.p.:145–146 ◦C; IR
(ATR)
ν
cmꢀ 1: br. peak 3000–2600, 1705, 1610, 1592, 1505, 1457,
1409, 1359, 1309, 1291, 1259, 1169, 1116, 1095, 1054, 810; 1H NMR
–
(850 MHz, CDCl3) δ ppm: 1.25 (d, J = 5.71 Hz, 6H, Ar CH(CH3)2), 2.38
–
–
(s, 3H, Ar CH3), 3.33–3.37 (br.s 1H, Ar CH(CH3)2), 5.32 (s, 2H,
– –
–
–
O
CH2 ), 6.84 (d, J = 7.78 Hz, 1H, Ar H), 7.16 (d, J = 7.27 Hz, 1H,
4.2.3. Cell cycle analysis
–
–
Ar H), 7.73 (t, J = 7.01 Hz, 1H, Ar H), 8.19 (br.s, 1H, Ar H), 8.25 (d,
MCF-7 cells were seeded at a density of 1 × 105 cells/well in 6–well
plates and treated with compound 10 (10 µM) for 48 h. After trypsini-
zation, the cells were collected by and centrifuged at 800g for 15 min.
Cells were fixed in ice-cold 70% ethanol overnight at ꢀ 20 ◦C. Fixed cells
J = 7.27 Hz, 1H, Ar H), 8.54 (br.s, 1H); 13C NMR (850 MHz, CDCl3) δ
–
ppm: 21.47, 22.86, 26.56, 62.69, 112.82, 114.10,116.69, 121.98,
122.46, 126.66, 134.21, 136.55, 155.32, 168.90; ESI MS: 352 [M+H]+
C20H21N3O3 (Calcd): C, 68.36; H, 6.02; N, 11.96; O, 13.66. Obsd:C,
68.33; H, 6.03; N, 11.96; O, 13.68.
were centrifuged at 800g for 15 min and stained with 50 μg/mL of PI,
containing 50 μ
g/mL of DNase-free RNase A, at 37 ◦C for 30 min. The
DNA content of cells (10,000 cells) was analyzed by flow cytometer at
488 nm using a FACSCalibur flow cytometer (BD Biosciences, San Jose,
CA, USA).
4.1.2.11. 4-((2-isopropyl-5-methylphenoxy)methyl)-1-(1-(4-((2-isopro-
pyl-5-methylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)naphthalen-5-yl)-1H-
1,2,3-triazole (13). Yield: 72%; M.p.: 188–189 ◦C; IR: 3073, 2977, 1515,
1509, 1451, 1420, 1253, 1160, 1011, 831, 807; 1H NMR (850 MHz,
4.2.4. Annexin V/PI apoptotic assay
–
CDCl3) δ ppm: 1.24 (d, J = 6.75 Hz, 6H, Ar CH(CH3)2) 2.40 (s, 3H,
Flow cytometry using Annexin V and propidium iodide (PI) was
performed to detect apoptotic cell death. MCF-7 cells were seeded in 6
well plates at density of 1 × 105 cells/well and treated with vehicle
(0.1% (v/v) EtOH), or compound 10 (10 µM) for 48 h. Cells were then
harvested and prepared for flow cytometric analysis. Cells were washed
in 1X binding buffer and incubated in the dark for 30 min on ice in
Annexin V-containing binding buffer [1:100]. Then, cells were re-
suspended in PI-containing binding buffer [1:1000]. Samples were
analyzed immediately using the BD Accuri flow cytometer and Graph-
Pad Prism software version 5.01 for analysis the data. Four populations
are produced during the assay Annexin V and PI negative (Q4, healthy
cells), Annexin V positive and PI negative (Q3, early apoptosis), Annexin
V and PI positive (Q2, late apoptosis) and Annexin V negative and PI
–
–
–
– –
Ar CH3), 3.35–3.38 (m, Ar CH(CH3)2), 5.43 (br.s, 2H, O CH2 ),
–
6.85 (d, J = 7.27 Hz, 1H, Ar H), 6.93 (br.s, 1H, Ar H), 7.17 (d, J =
–
–
7.27 Hz, 1H, Ar H), 7.70 (t, J = 7.53 Hz, 1H, Ar H), 7.76 (d, J = 6.75
–
–
Hz, 1H, Ar H), 7.85 (d, J = 7.27 Hz, 1H, Ar H), 8.21 (br.s, 1H, triazole
proton); 13C NMR (214 MHz, CDCl3) δ ppm: 21.45, 22.87, 26.66, 62.13,
112.99, 122.15, 124.92, 126.19, 127.15, 129.54, 134.36, 136.64,
155.21; ESI MS: 587 [M+H]+.C36H38N6O2 (Calcd): C, 73.69; H, 6.53; N,
14.32; O, 5.45. Obsd: C, 73.67; H, 6.54; N, 14.32; O, 5.46.
12