G. Sumran, et al.
BioorganicChemistry88(2019)102932
N]CeH), 11.37 (s, 1H, NH, D2O exchangeable), 12.33 (s, 1H, amide
NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6) δ: 115.51,
116.81, 123.92, 124.25, 126.06, 128.91, 129.14, 131.75, 133.00,
142.02, 146.21, 147.51, 151.21. Anal. Calcd. for C13H9BrN4OS: C,
44.71; H, 2.60; N, 16.04. Found: C, 44.82; H, 2.81; N, 16.42%.
1H, Jm = 1.84 Hz, Ph-2′-H), 7.38–7.42 (m, 2H, quinox-5, 8-H), 12.08 (s,
1H, amide NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6) δ:
56.13, 56.24, 112.44, 113.55, 116.79, 117.57, 120.36, 121.42, 123.11,
123.22, 128.15, 129.87, 144.55, 149.40, 151.19, 152.39. HRMS: m/z
323.1132 [M + 1]+, 324.1159 [M + 2]+ (C17H14N4O3 [M + 1]+ re-
quires 323.1144). Anal. Calcd. for C17H14N4O3: C, 63.35; H, 4.38; N,
17.38. Found: C, 63.21; H, 4.66; N, 17.77%.
6.2. General procedure for cyclization of hydrazones (3a-k) to 1-aryl/
heteroaryl-[1,2,4]-triazolo[4,3-a]quinoxalin-4(5H)-ones (4a-k)
6.2.5. 1-(Thiophen-2′-yl)-[1,2,4]-triazolo[4,3-a]quinoxalin-4(5H)-one
(4j)
To a stirred suspension (or) solution of hydrazone (3) (1.0 mmol) in
dichloromethane (20 mL) was added IBD (0.354 g, 1.1 mmol) in small
portions over a period of five minutes at room temperature. The reac-
tion mixture was allowed to stir for about 2 h or till the completion of
reaction as monitored by TLC. Excess of dichloromethane was distilled
off in vacuo and the residual mass was triturated with petroleum ether
to give solid product which was recrystallized from ethanol.
IR: 3140 (NeH str), 1682 (C]O), 1612 (C]N); 1H NMR (300 MHz,
DMSO-d6) δ: 7.06–7.11 (t, 1H, Jo = 8.4 Hz, quinox-6-H), 7.22–7.25 (d,
1H, Jo = 8.4 Hz, quinox-7-H), 7.38–7.39 (dd, 1H, J5′, 4′=5.1 Hz, J3′,
4′=3.9 Hz, thiophene-4′-H), 7.39–7.43 (m, 2H, quinox-5, 8-H),
7.64–7.66 (d, 1H, J3′, 4′ = 3.3 Hz, thiophene-3′-H), 8.04–8.06 (d, 1H,
J4′, 5′ = 5.1 Hz, thiophene-5′-H), 12.12 (s, 1H, amide NH, D2O ex-
changeable); 13C NMR (100 MHz, DMSO-d6) δ: 116.35, 117.65, 121.27,
123.18, 127.25, 128.42, 128.69, 129.97, 131.68, 132.60, 145.20,
145.43, 152.21. HRMS: m/z 269.0501 [M + 1]+ (C13H8N4OS
[M + 1]+ requires 269.0497). Anal. Calcd. for C13H8N4OS: C, 58.20; H,
3.01; N, 20.88. Found: C, 57.92; H, 3.15; N, 20.92%.
6.2.1. 1-(4′-Bromophenyl)-[1,2,4]-triazolo[4,3-a]quinoxalin-4(5H)-one
(4d)
IR: 1682 (C]O), 1612 (C]N); 1H NMR (400 MHz, DMSO-d6) δ:
7.05–7.12 (m, 2H, quinox-6-H, 7-H), 7.37–7.42 (m, 2H, quinox-5-H, 8-
H), 7.69–7.72 (td, 2H, Jo = 8.56, Jp = 1.96, Jm = 2.36, Ph-3′, 5′-H),
7.88–7.91 (td, 2H, Jo = 8.56, Jm = 2.32, Jp = 1.96, Ph-2′, 6′-H), 12.12
(s, 1H, amide NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6)
δ: 116.70, 117.67, 121.25, 123.23, 125.19, 127.87, 128.26, 129.89,
132.44, 132.74, 144.90, 150.24, 152.33. HRMS: m/z 340.9970
[M + 1]+, 342.9951 [M + 1 + 2]+ in the ratio showing typical bro-
mine isotope profile (1:1), (C15H9BrN4O [M + 1]+ requires 341.0038).
Anal. Calcd. for C15H9BrN4O: C, 52.81; H, 2.66; N, 16.42. Found: C,
53.03; H, 2.88; N, 16.18%.
6.2.6. 1-(5′-Bromothiophen-2′-yl)-[1,2,4]-triazolo[4,3-a]quinoxalin-
4(5H)-one (4k)
IR: 1680 (C]O), 1612 (C]N); 1H NMR (300 MHz, CDCl3) δ:
6.87–6.89 (d, 1H, Jo = 8.1 Hz, quinox-6-H), 6.92–6.95 (m, 1H, quinox-
7-H), 7.10–7.15 (m, 2H, thiophene-4′, 3′-H), 7.17–7.20 (d, 1H,
Jo = 7.8 Hz, quinox-5-H), 7.33–7.36 (d, 1H, Jo = 8.7 Hz, quinox-8-H),
12.09 (s, 1H, amide NH, D2O exchangeable); 13C NMR (100 MHz,
DMSO-d6) δ: 115.63, 116.21, 118.00, 123.25, 123.40, 125.42, 128.43,
129.46, 130.82, 132.09, 144.97, 148.03, 152.71. Anal. Calcd. for
6.2.2. 1-(4′-Methylphenyl)-[1,2,4]-triazolo[4,3-a]quinoxalin-4(5H)-one
(4e)
C13H7BrN4OS: C, 44.97; H, 2.03; N, 16.14. Found: C, 45.29; H, 2.43; N,
16.45%.
IR: 1682 (C]O), 1612 (C]N); 1H NMR (400 MHz, DMSO-d6) δ:
2.48 (s, 3H, 4′-CH3), 7.00–7.05 (dt, 1H, Jo = 8.56 Hz, Jm = 2.08 Hz,
quinox-6-H), 7.11–7.13 (d, 1H, Jo = 8.08 Hz, quinox-7-H), 7.35–7.42
(m, 2H, quinox-5, 8-H), 7.47–7.49 (d, 2H, Jo = 7.88 Hz, Ph-3′, 5′-H),
7.60–7.62 (d, 2H, Jo = 8.04 Hz, Ph-2′, 6′-H), 12.10 (s, 1H, amide NH,
D2O exchangeable); 13C NMR (100 MHz, DMSO-d6) δ: 21.61, 116.56,
117.63, 121.37, 123.03, 125.67, 126.95, 128.16, 129.44, 129.90,
130.20, 141.25, 144.71, 151.20, 152.41. HRMS: m/z 277.1083
7. Biological activity
7.1. Material
The DMSO, ethylenediaminetetraacetic acid (EDTA) and ethidium
bromide (EtBr) were purchased from Sigma-Aldrich, USA. pMaxGFP
plasmid was obtained from Lonza. Stock solutions of compound 4
(15 mg/mL) and 3a, 3b, 3j (10 mg/mL) and 3i, 3k (5 mg/mL) were
prepared in DMSO and stored in brown containers in the refrigerator.
All gel electrophoresis experiments were performed in 1 × TAE buffer
(0.04 M Tris-borate, 0.114% acetic acid and 50 mM EDTA, pH 8.0).
Quantification of cleavage efficiency was performed by integration of
the optical density as a function of the band area using the Image J
Software (recommended by NIH, USA) [44].
[M + 1]+
,
278.1106 [M + 2]+ (C16H12N4O [M + 1]+ requires
277.1089). Anal. Calcd. for C16H12N4O: C, 69.55; H, 4.38; N, 20.28.
Found: C, 69.72; H, 4.61; N, 20.57%.
6.2.3. 1-(2′,5′-Dimethoxyphenyl)-[1,2,4]-triazolo[4,3-a]quinoxalin-
4(5H)-one (4g)
IR: 1682 (C]O), 1612 (C]N); 1H NMR (400 MHz, DMSO-d6) δ:
3.60 (s, 3H, 2′-OCH3), 3.79 (s, 3H, 5′-OCH3), 7.06–7.08 (m, 2H, quinox-
6, 7-H), 7.17–7.18 (d, 1H, Jm = 2.92 Hz, Ph-6′-H), 7.23–7.25 (d, 1H,
Jo = 9.04 Hz, Ph-3′-H), 7.28–7.31 (dd, 1H, Jo = 9.12 Hz, Jm = 3.0 Hz,
Ph-4′-H), 7.40–7.42 (m, 2H, quinox-5, 8-H), 12.14 (s, 1H, amide NH,
D2O exchangeable); 13C NMR (100 MHz, DMSO-d6) δ: 56.20, 56.48,
113.64, 116.14, 117.31, 117.49, 117.87, 118.49, 121.25, 123.37,
128.33, 129.55, 144.57, 148.35, 152.04, 152.23, 153.72. HRMS: m/z
323.1067 [M + 1]+, 324.1095 [M + 2]+ (C17H14N4O3 [M + 1]+ re-
quires 323.1144). Anal. Calcd. for C17H14N4O3: C, 63.35; H, 4.38; N,
17.38. Found: C, 63.69; H, 4.03; N, 17.81%.
7.2. DNA photocleavage assays
The DNA photoactivated cleavage experiments were performed by
treating 0.2 μg/μl of supercoiled pMaxGFP plasmid (Lonza) DNA in the
TAE buffer with the compound 4 or 3 (presence/absence) dissolved in
DMSO at room temperature and the solution was then irradiated at
ambient atmospheric pressure under aerobic condition for the indicated
time using a UV-lamp (312 nm, 15 W). Reactions were also performed
without irradiation as control. After illumination all reactions were
quenched by adding 2 μl of 6 × gel loading dye (containing 0.25%
bromophenol blue in 30% glycerol) in TAE buffer. Cleavage products
were electrophoresed for 45–60 min in TAE buffer at 120 V on a 0.8%
agarose gel by using BioRad electrophoresis system. The nicked and
supercoiled forms of pMaxGFP plasmid DNA were then visualized by
staining the gel with fluorescent and DNA intercalating dye i.e. ethi-
dium bromide (0.5 μg/mL). The gel was washed with water and DNA
bands were visualized and photographed on a ChemiDoc XRS + CCD
6.2.4. 1-(3′,4′-Dimethoxyphenyl)-[1,2,4]-triazolo[4,3-a]quinoxalin-
4(5H)-one (4h)
IR: 1682 (C]O), 1620 (C]N); 1H NMR (400 MHz, DMSO-d6) δ:
3.76 (s, 3H, 4′-OCH3), 3.90 (s, 3H, 3′-OCH3), 7.05–7.09 (dt, 1H,
Jo = 8.6 Hz, Jm = 2.32 Hz, quinox-6-H), 7.17–7.19 (d, 1H,
Jo = 8.12 Hz, quinox-7-H), 7.21–7.24 (d, 1H, Jo = 8.28 Hz, Ph-5′-H),
7.26–7.28 (dd, 1H, Jo = 8.2 Hz, Jm = 1.84 Hz, Ph-6′-H), 7.30–7.31 (d,
8