302
J. Yang et al. / European Journal of Medicinal Chemistry 109 (2016) 294e304
ꢀ
5
4
.85e5.93 (m, 1H, CH), 6.63 (s, 1H, NH), 7.42 (s, 2H, Ph-H),7.52 (s,
H, Ph-H); 13C NMR (100 MHz, CDCl
: 16.14, 44.82, 85.03, 86.28,
66%, mp: 179e181 C; 1H NMR (400 MHz, CDCl
3
)
d
: 2.19 (s, 6H,
3
)
d
2 ꢂ CH
3
), 2.72 (t, J ¼ 6.40 Hz, 2H, CH
2
), 3.66 (q, J ¼ 6.26 Hz, 2H, CH
2
),
103.54, 109.46, 116.42, 118.00, 118.51, 119.57, 132.84, 133.20, 133.27,
4.77 (t, J ¼ 6.16 Hz, 1H, NH), 5.37 (d, J ¼ 1.60 Hz, 1H, Py-H), 5.66 (d,
134.70, 144.86, 153.92,154.52, 159.27, 166.16; ESI-MS:396.4
J ¼ 1.60 Hz, 1H, Py-H), 6.66 (s, 1H, NH), 7.42 (s, 2H, Ph-H), 7.52 (q,
þ
13
[MþH] . C24
H
21
N
5
O (395.17).
J ¼ 8.88 Hz, 4H, Ph-H); C NMR (100 MHz, CDCl
3
) d: 16.15, 18.61,
3
8.12, 86.34, 86.86, 103.76, 109.53, 118.03, 118.44, 118.46, 119.43,
4
.1.5.11. 4-((6-(cyclopropylamino)-4-(mesityloxy)pyridin-2-yl)
132.90, 133.17, 133.32, 144.77, 153.82, 154.72, 158.23, 166.03; ESI-
ꢀ
þ
amino) benzonitrile (If). White solid, yield 38%, mp: 203e205 C;
20 6
MS: 409.5 [MþH] . C24H N O (408.17).
1
H NMR (400 MHz, CDCl
3
)
d
: 2.09 (s, 6H, 2 ꢂ CH
.13 (s, 1H, Py-H), 6.35 (d, J ¼ 0.88 Hz, 1H, Py-H), 6.75 (s, 1H, NH),
.92 (s, 2H, Ph-H), 7.49 (s, 4H, Ph-H); 13C NMR (100 MHz, CDCl
3 3
), 2.31 (s, 3H, CH ),
6
6
4.1.5.17. 4-((4-(mesityloxy)-6-((2-(piperazin-1-yl)ethyl)amino)pyri-
3
)
d:
dine-2 -yl)amino)benzonitrile (Ii). White solid, yield 68%, mp:
ꢀ
1
16.04, 20.80, 94.23, 104.24, 104.70, 118.27, 119.27, 129.90, 130.24,
114e116 C; H NMR (400 MHz, CDCl
(s, 3H, CH ), 2.46 (s,1H, NH), 2.64 (s, 6H, 3CH
3.32 (d, J ¼ 4 Hz, 2H, CH ), 4.83 (s,1H, NH), 5.40 (s,1H, Py-H), 5.63 (s,
H, Py-H), 6.55 (s, 1H, NH), 6.90 (s, 2H, Ph-H), 7.49 (q, J ¼ 8.52 Hz,
3
)
d
: 2.10 (s, 6H, 2 ꢂ CH
3
), 2.31
133.49, 135.81, 144.01, 147.46, 151.06, 154.95, 167.41; ESI-MS: 385.5
3
2
), 3.10 (s, 4H, 2 ꢂ CH
2
),
þ
[MþH] . C24
H N
24 4
O (384.20).
2
1
1
3
4
.1.5.12. 4-((2-((4-cyanophenyl)amino)-6-(cyclopropylamino)pyr-
3
4H, Ph-H); C NMR (100 MHz, CDCl ) d: 16.10, 20.81, 38.43, 44.32,
idin-4-y l) oxy)-3,5-dimethylbenzonitrile (IIf). White solid, yield
51.11, 56.82, 85.66, 86.69, 117.63, 129.57, 130.69, 133.27, 135.06,
ꢀ
1
þ
4
9%, mp: 216e218 C; H NMR (400 MHz, CDCl
J ¼ 4.28 Hz, 6.88 Hz, 2H, CH ), 0.73 (dt, J ¼ 1.60 Hz, 6.80 Hz, 2H,
CH ), 2.21 (s, 6H, 2CH ), 2.44e2.45 (m, 1H, CH), 5.00 (s, 1H, NH),
.49 (d, J ¼ 1.72 Hz, 1H, Py-H), 5.75 (d, J ¼ 1.68 Hz, 1H, Py-H), 6.55 (s,
3
)
d
: 0.54 (td,
145.25, 147.97, 154.33, 159.32, 167.37; ESI-MS: 457.6 [MþH] .
2
27 32 6
C H N O (456.26).
2
3
5
1
4.1.5.18. 4-((2-((4-cyanophenyl)amino)-6-((2-(piperazin-1-yl)ethyl)
ami no) pyridin-4-yl)oxy)-3,5-dimethylbenzonitrile (IIi). White
13
H, NH), 7.43 (s, 2H, Ph-H), 7.51 (q, J ¼ 7.32 Hz, 4H, Ph-H); C NMR
: 7.47, 16.16, 23.97, 85.18, 86.50, 103.39, 109.42,
17.86, 118.05, 118.53, 119.61, 132.82, 133.06, 133.23, 144.94, 154.01,
ꢀ
1
(
100 MHz, CDCl
3
)
d
solid, yield 64%, mp: 190e191 C; H NMR (400 MHz, CDCl
(s, 6H, 2 ꢂ CH ), 2.47 (s, 1H, NH), 2.64 (s, 6H, 3CH ), 3.07 (s, 4H,
2CH ), 4.91 (s, 1H, NH), 5.33 (d, J ¼ 1.4 Hz,
), 3.33 (d, J ¼ 4 Hz, 2H, CH
H, Py-H), 5.58 (d, J ¼ 1.28 Hz, 1H, Py-H), 6.56 (s, 1H, NH), 7.42 (s,
3
) d: 2.19
1
3
2
þ
154.62, 160.60, 166.21; ESI-MS: 396.4 [MþH] . C24
H
21
N
5
O (395.17).
2
2
1
13
4
.1.5.13. 4-((4-(mesityloxy)-6-(((tetrahydrofuran-2-yl)methyl)
2H, Ph-H), 7.52 (dd, J ¼ 2.96 Hz, 9.12 Hz, 4H, Ph-H); C NMR
amino)pyri dine-2-yl)amino)benzonitrile (Ig). White solid, yield
(100 MHz, CDCl : 16.16, 38.40, 44.55, 51.55, 56.83, 86.23, 109.43,
3
) d
ꢀ
1
3
6%, mp: 87e89 C; H NMR (400 MHz, CDCl
3
)
d: 1.88e1.95 (m, 2H,
117.84, 132.87, 133.20, 133.29, 145.03, 153.96, 154.72, 159.47, 165.99;
þ
CH ), 1.97e2.05 (m, 2H, CH
2
2
), 2.09 (s, 6H, 2 ꢂ CH
3
), 2.30 (s, 3H, CH ),
3
ESI-MS: 468.5 [MþH] . C27
29 7
H N O (467.24).
3
1
.17e3.23 (m, 1H, CH), 3.45e3.50 (m, 1H, CH), 3.76 (q, J ¼ 6.84 Hz,
H, CH), 3.88 (q, J ¼ 6.80 Hz, 1H, CH), 4.06e4.10 (m, 1H, CH), 4.73 (t,
4.1.5.19. 4-((6-((2,2-dimethoxyethyl)amino)-4-(mesityloxy)pyridine-
2-yl) amino)benzonitrile (Im). White solid, yield 77%, mp:
J ¼ 5.48 Hz, 1H, NH), 5.41 (d, J ¼ 1.44 Hz, 1H, Py-H), 5.59 (d,
J ¼ 1.32 Hz, 1H, Py-H), 6.56 (s, 1H, NH), 6.89 (s, 2H, Ph-H), 7.49 (q,
ꢀ
1
143e145 C; H NMR (400 MHz, CDCl
(s, 3H, CH
3
)
d
: 2.09 (s, 6H, 2 ꢂ CH
3
), 2.30
), 4.52
13
J ¼ 4.84 Hz, 4H, Ph-H); C NMR (100 MHz, CDCl
3
)
d: 16.08, 20.80,
3
), 3.40 (s, 6H, 2 ꢂ CH ), 3.42 (d, J ¼ 5.60 Hz, 2H, CH
3
2
2
5.82, 28.94, 46.21, 68.08, 85.91, 86.54,103.01,117.67,119.72,129.53,
(t, J ¼ 5.40 Hz, 1H, CH), 4.58 (t, J ¼ 5.68 Hz, 1H, NH), 5.40 (d,
J ¼ 1.36 Hz,1H, Py-H), 5.63 (d, J ¼ 1.20Hz, 1H, Py-H), 6.57 (s,1H, NH),
1
30.70, 133.23, 135.03, 145.23, 147.99, 154.15, 159.43, 167.34; ESI-
þ
13
MS: 429.5 [MþH] . C26
28
H N
4
O
2
(428.22).
6.89 (s, 2H, Ph-H), 7.51 (q, J ¼ 5.36 Hz, 4H, Ph-H); C NMR
(
3
100 MHz, CDCl ) d: 15.10, 20.81, 43.56, 54.03, 85.95, 86.80, 102.69,
4
.1.5.14. 4-((2-((4-cyanophenyl)amino)-6-(((tetrahydrofuran-2-yl)
102.98, 117.64, 119.75, 129.55, 130.68, 133.26, 135.06, 146.24, 147.97,
þ
methyl) amino)pyridin-4-yl)oxy)-3,5-dimethylbenzonitrile (IIg).
White solid, yield 66%, mp: 109e111 C; H NMR (400 MHz, CDCl )
3
28 4 3
154.31, 160.16,167.29; ESI-MS: 433.6 [MþH] . C25H N O (432.22).
ꢀ
1
d
1
: 1.89e2.05 (m, 4H, 2 ꢂ CH
2
), 2.19 (s, 6H, 2 ꢂ CH
3
), 3.16e3.22 (m,
4.1.5.20. 4-((2-((4-cyanophenyl)amino)-6-((2,2-dimethoxyethyl)
H, CH), 3.46e3.51 (m, 1H, CH), 3.77 (q, J ¼ 6.96 Hz, 1H, CH), 3.88 (q,
amino) pyridin-4-yl)oxy)-3,5-dimethylbenzonitrile (IIm). White
ꢀ
1
J ¼ 6.84, 1H, CH), 4.07e4.10 (m, 1H, CH), 4.80 (t, J ¼ 5.36 Hz, 1H, NH),
solid, yield 64%, mp: 167e169 C; H NMR (400 MHz, CDCl
(s, 6H, 2 ꢂ CH ), 3.41 (s, 6H, 2 ꢂ CH ), 3.43 (s, 2H, CH ), 4.53 (t,
J ¼ 4 Hz, 1H, CH), 4.73 (s, 1H, NH), 5.34 (s, Py-H), 5.61 (s, 1H, Py-H),
3
) d: 2.19
5
1
CDCl
1
.33 (d, J ¼ 1.36 Hz, 1H, Py-H), 5.57 (d, J ¼ 1.28 Hz, 1H, Py-H), 6.60 (s,
3
3
2
13
H, NH), 7.42 (s, 2H, Ph-H), 7.52 (s, 4H, Ph-H); C NMR (100 MHz,
: 16.14, 25.82, 28.96, 46.21, 68.10, 85.43, 86.14, 103.40,
09.43, 117.90, 118.53, 119.59, 132.83, 133.21, 133.25, 144.98, 153.96,
13
3
)
d
6.84 (s, 1H, NH), 7.42 (s, 2H, Ph-H), 7.53 (s, 4H, Ph-H); C NMR
(100 MHz, CDCl : 16.16, 43.63, 54.14, 85.40, 86.20, 102.60, 109.48,
3
) d
þ
1
54.50, 159.54, 165.99; ESI-MS: 440.6 [MþH] .
C
H
26 25
N
5
O
2
118.12, 118.49, 119.56, 132.87, 133.17, 133.30, 144.78, 153.87, 154.37,
þ
(
439.20).
158.92, 166.15; ESI-MS: 444.6 [MþH] . C25
25 5 3
H N O (443.20).
4
.1.5.15. 4-((6-((2-cyanoethyl)amino)-4-(mesityloxy)pyridin-2-yl)
4.2. In vitro anti-HIV assay
ꢀ
amino) benzonitrile (Ih). White solid, yield 67%, mp: 173e175 C;
1
H NMR (400 MHz, CDCl
3
)
d
: 2.09 (s, 6H, 2 ꢂ CH
3
), 2.30 (s, 3H, CH
3
),
By using MTT method described previously [40,41], the syn-
thesized compounds were evaluated for their activity against WT
HIV-1 [42] (strain HIV-IIIB), double RT mutant strain HIV-1 IIIB
(RES056 and F227L/V106A), five single RT mutant strain HIV-1 IIIB
(L100I, K103N, E138K, Y181C, Y188L), and HIV-2 [43] (strain ROD)
in MT-4 cells. At the beginning of each experiment, stock solutions
(10 ꢂ final concentration) of test compounds were added in 25 ìL
volumes to two series of triplicate wells to allow simultaneous
evaluation of their effects on mock-and HIV-infected cells. Using a
Biomek 3000 robot (Beckman instruments, Fullerton, CA), serial
five-fold dilutions of the test compounds were made directly in
2
.71 (t, J ¼ 6.4 Hz, 2H, CH
2
), 3.65 (q, J ¼ 6.44 Hz, 2H, CH
2
), 4.64 (t,
J ¼ 6.28 Hz, 1H, NH), 5.42 (d, J ¼ 1.36 Hz, 1H, Py-H), 5.70 (d,
J ¼ 1.28 Hz, 1H, Py-H), 6.50 (s, 1H, NH), 6.90 (s, 2H, Ph-H), 7.50 (q,
13
J ¼ 1.80 Hz, 4H, Ph-H); C NMR (100 MHz, CDCl
3
) d: 16.08, 18.62,
2
0.80, 38.16, 86.75, 87.36, 103.38, 117.73, 118.52, 119.56, 129.61,
130.62,133.29,13520,145.04,147.88,154.42,158.14,167.36; ESI-MS:
þ
3
98.4 [MþH] . C24
23 5
H N O (397.19).
4
.1.5.16. 4-((2-((2-cyanoethyl)amino)-6-((4-cyanophenyl)amino)
pyridine -4-yl)oxy)-3,5-dimethylbenzonitrile (IIh). White solid, yield