D. Kaneko, et al.
BioorganicChemistry104(2020)104293
Scheme 3. Incorporation of 1,3,4-oxadiazole and 1,3,4-thiadiazole units into imiquimod.
spectrometers with tetramethylsilane as an internal standard. Silica gel
column chromatography (CC) was performed on silica gel 60 N
(Wakogel, 38–100 μm). Thin-layer chromatography (TLC) spots on
plates pre-coated with silica gel 70F254 were detected with a UV lamp
(254 nm). Fractionations for all CCs were based on TLC analyses.
Table 1
IC50 values of [1,2,4]triazolo[4,3-a]quinoxaline-1,3,4-oxadiazole derivatives
(7b, 7e, and 7f) against cancer cell lines.
IC50 (μM
SEM)
HL-60
U937
B16
HepG2
4.1.2. Synthetic methods
7b
3.23
3.37
2.72
2.07
46.7
0.17
0.10
0.27
0.06
1.3
1.56
3.73
2.65
0.82
40.2
0.25
0.32
0.13
0.15
1.3
11.3
0.2
0.1
0.1
5.35
4.86
3.84
60.9
> 80
0.22
0.25
0.13
1.2
Detailed synthetic conditions and spectroscopic data of compounds
are given in the Supplementary data. The physical data of 7a–7f are
shown here.
7e
10.6
7f
26.2
EAPB0203
Imiquimod
> 80
> 80
N-Phenyl-4-((5-phenyl-1,3,4-oxadiazol-2-yl)thio)-[1,2,4]triazolo[4,3-
a]quinoxalin-1-amine (7a): IR (ATR): νmax 2230, 1693, 1578, 1543,
1466, 1397, 1351, 1240, 1142, 946, 856, 839, 775, 636, 594, 496,
1
468 cm−1; H NMR (400 MHz, DMSO‑d6): δ 9.41 (1H, s, –NH-), 8.33
(1H, d, J = 8.2 Hz, H-6), 8.10–8.08 (2H, m, H-2″ and H-6″), 7.70–7.53
(6H, m, H-2′, H-4″, H-6′, H-7, H-8, and H-9), 7.25 (2H, t, J = 8.0 Hz, H-
3′ and H-5′), 7.10–7.08 (2H, m, H-3″ and H-5″), 6.93 (1H, t, J = 7.3 Hz,
H-4′); 13C NMR (100 MHz, DMSO‑d6): δ 168.4, 156.7, 149.4, 148.6,
143.2, 136.0, 133.3, 130.2 (3C), 129.7 (2C), 129.5, 129.2, 128.1, 127.4
(2C), 125.9, 123.4, 121.7, 117.2 (2C), 116.9; HRESITOFMS: m/z
438.1126 [M + H]+ (calcd. for C23H16N7OS, 438.1137).
N-Phenyl-4-((5-(p-tolyl)-1,3,4-oxadiazol-2-yl)thio)-[1,2,4]triazolo
[4,3-a]quinoxalin-1-amine (7b): IR (ATR): νmax 1598, 1544, 1493, 1456,
1410, 1354, 1248, 1145, 1102, 1080, 944, 824, 743, 733, 690, 657,
1
500, 463 cm−1; H NMR (400 MHz, DMSO‑d6): δ 9.42 (1H, s, –NH-),
Fig. 3. Proposed hydroxylable positions of EAPB0203 and [1,2,4]triazolo[4,3-
a]quinoxaline-1,3,4-oxadiazole derivatives (7b, 7e, and 7f) in hepatic meta-
bolism. Red arrows point hydroxylable positions.
8.31 (1H, d, J = 8.2 Hz, H-6), 7.97 (2H, d, J = 8.2 Hz, H-2″ and H-6″),
7.67 (1H, dd, J = 7.8 Hz and 1.4 Hz, H-9), 7.61–7.58 (1H, m, H-7 or H-
8), 7.53 (1H, t, J = 7.1 Hz, H-7 or H-8), 7.45 (2H, d, J = 8.2 Hz, H-2′
and H-6′), 7.25 (2H, t, J = 7.8 Hz, H-3′ and H-5′), 7.10 (2H, d,
J = 7.3 Hz, H-3″ and H-5″), 6.93 (1H, t, J = 7.6 Hz, H-4′), 2.42 (3H, s,
–CH3); 13C NMR (100 MHz, DMSO‑d6): δ 168.5, 156.3, 149.3, 148.7,
143.6, 143.2, 141.2, 136.0, 130.7 (2C), 129.7 (2C), 129.5, 129.1,
128.0, 127.3 (2C), 125.9, 121.7, 120.6, 117.2 (2C), 116.9, 21.7; HRE-
SITOFMS: m/z 452.1291 [M + H]+ (calcd. for C24H18N7OS, 452.1291).
4-((5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)-N-phenyl-[1,2,4]
triazolo[4,3-a]quinoxalin-1-amine (7c): IR (ATR): νmax 2997, 1602,
1577, 1556, 1502, 1414, 1305, 1253, 1176, 1147, 1032, 954, 829, 749,
692, 666, 627, 503, 479 cm−1; 1H NMR (500 MHz, DMSO‑d6): δ 9.41
(1H, s, –NH-), 8.32–8.30 (1H, m, H-6), 8.03 (2H, d, J = 8.6 Hz, H-2″
and H-6″), 7.67 (1H, d, J = 8.0 Hz, H-9), 7.60 (1H, t, J = 7.2 Hz, H-7 or
H-8), 7.53 (1H, t, J = 7.2 Hz, H-7 or H-8), 7.26 (2H, t, J = 8.0 Hz, H-3′
and H-5′), 7.18 (2H, d, J = 9.2 Hz, H-2′ and H-6′), 7.10 (2H, d,
J = 8.0 Hz, H-3″ and H-5″), 6.93 (1H, t, J = 7.4 Hz, H-4′), 3.87 (3H, s,
-OMe); 13C NMR (125 MHz, DMSO‑d6): δ 168.3, 163.2, 155.8, 149.3,
148.7, 146.8, 143.2, 141.2, 136.1, 129.7 (2C), 129.5, 129.3 (2C),
cancer cell lines. The 1,3,4-oxadiazole derivatives 7b, 7e, and 7f
bearing Me, OH, and NH2 groups on the phenyl ring respectively, were
found to be the most beneficial compounds when compared to imi-
quimod and EAPB0203. The new structural combination of [1,2,4]
triazolo[4,3-a]quinoxaline and 1,3,4-oxadiazole units is valuable for
accelerating anticancer drug development.
4. Experimental section
4.1. Chemistry
4.1.1. General
All solvents and reagents were purchased from the suppliers and
used without further purification. IR spectra were recorded on a
PerkinElmer FT-IR/FIR Spectrometer 400. MS spectra were obtained
using the Waters UPLC-MS system (Aquity UPLC XevoQTof). 1H and 13
C
NMR spectra were recorded with JEOL ECX-400P and ECA-500
4