Y.-H. Liang et al. / Bioorg. Med. Chem. 18 (2010) 4601–4605
4605
J = 4.0 Hz, 1H), 7.27–7.52 (m, 4H, Ph), 8.02–8.54 (m, 5H, naph), 8.82
4.2. Biological study
(s, 1H, CH), 10.12 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
100.18, 103.15, 109.78, 118.81, 119.09, 119.72, 123.09, 125.27,
125.55, 129.19, 130.08, 131.49, 132.81, 132.93, 135.93, 144.72,
159.33, 161.18, 168.88. MS (EI) m/z: 393.1 (M+); Anal.
(C22H12ClN5O) C, H, N.
4.2.1. Anti-HIV assay in MT-4 cells
The anti-HIV activity and cytotoxicity were evaluated against
wild-type HIV-1 strain IIIB in MT-4 cells using the 3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) meth-
od.11,12 Briefly, virus stocks were titrated in MT-4 cells and
expressed as the 50% cell culture infective dose (CCID50). MT-4 cells
were suspended in culture medium at 1 Â 105 cells/mL and in-
fected with HIV at a multiplicity of infection of 0.02. Immediately
4.1.7.5. 5-Chloro-6-(2-(4-cyanophenylamino)pyrimidin-4-yloxy)-
7-methoxy-2-naphthonitrile (3e). Yield 33.4%; flash chromatog-
raphy separation and recrystallized from 1,4-dioxane, mp
268.4–269.2 °C; 1H NMR (400 MHz, DMSO-d6) d 12 13C NMR
(400 MHz, DMSO-d6) d 3.89 (s, 3H, CH3), 6.82 (d, J = 4.4 Hz, 1H),
7.25–7..49 (m, 4H, Ph), 7.82–8.63 (m, 4H, naph), 8.52 (d,
J = 4.4 Hz, 1H), 10.11 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d
57.21, 99.71, 103.15, 108.27, 110.23, 118.66, 119.25, 119.72,
124.69, 125.54, 126.74, 127.57, 131.95, 132.94, 133.76, 141.45,
144.75, 152.66, 159.35, 161.13, 168.47. MS (EI) m/z: 427.1 (M+);
Anal. (C23H14ClN5O2) C, H, N.
after virus infection, 100 lL of the cell suspension was placed in
each well of a flat-bottomed microtiter tray containing various
concentrations of the test compounds. Stock solutions of the test
compounds were dissolved in DMSO at 50 mM or higher. After
4 days of incubation of virus-infected cells with the compounds
at 37 °C, the number of viable cells was determined using the
MTT method. Compounds were tested in parallel for cytotoxic ef-
fects in uninfected MT-4 cells.
4.2.2. HIV RT assay
4.1.7.6. 6-(2-(4-Cyanophenylamino)-5-methylpyrimidin-4-yloxy)-
2-naphthonitrile (3f). Yield 85.6%; recrystallized from 1,4-diox-
ane, mp 258.4–259.2 °C; 1H NMR (400 MHz, DMSO-d6) d 2.28 (s,
3H, CH3), 7.27–7.57 (m, 4H, Ph), 7.65–8.40 (m, 4H, naph), 8.72(s,
1H, CH), 9.93 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d 12.25,
102.46, 108.57, 118.40, 119.48, 119.64, 119.89, 124.61, 127.55,
129.40, 130.30, 130.95, 132.92, 134.79, 135.85, 145.16, 152.43,
157.75, 159.89, 167.90; MS (EI) m/z: 377.1 (M+); Anal.
(C23H15N6BrO2) C, H, N.
Reactions were performed under the conditions described for
the HIV-1 RT RNA-dependent DNA polymerase activity assay.
Incorporation of Biotin-dUTP into poly(rA)/oligo(dT) at different
concentrations of DNA or Biotin-dUTP was monitored in the pres-
ence of increasing fixed amounts of inhibitors. The activity of RT
was obtained through testing the amount of Biotin-dUTP by HRP-
labeled Streptavidin. For IC50 determinations, a range of inhibitor
concentrations between 0.2 IC50 and 5 IC50 was used.
Acknowledgments
4.1.7.7. 5-Bromo-6-(2-(4-cyanophenylamino)-5-methylpyrimi-
din-4-yloxy)-2-naphthonitrile (3g). Yield 83.3%; recrystallized
from 1,4-dioxane, mp 268.4–269.2 °C; 1H NMR (400 MHz, DMSO-
d6) d 2.43 (s, 3H, CH3), 6.70 (s, 1H, CH), 7.23–7.52 (m, 4H, Ph),
7.76–8.82 (m, 5H, naph), 10.09 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6) d 24.16, 98.81, 102.94, 109.70. 115.32, 118.69, 119.10,
119.77, 125.40, 128.19, 129.36, 130.78, 131.58, 132.89, 134.24,
135.47, 144.91, 151.15, 158.93, 169.35, 171.15; MS (EI) m/z:
457.0 (M+); Anal. (C23H15N5BrO2) C, H, N.
This work was financially supported by the National Natural
Science Foundation of China (No. 20872018 and 30672536) and
the Science and Technology Commission of Shanghai Municipality
(No. 09431902800).
References and notes
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Clercq, E. ChemMedChem 2009, 4, 219.
4.1.7.8. 6-(2-(4-Cyanophenylamino)-6-methylpyrimidin-4-yloxy)-
2-naphthonitrile (3h). Yield 82.4%; recrystallized from 1,4-diox-
ane, mp 223.4–224.2 °C; 1H NMR (400 MHz, DMSO-d6) d 2.24 (s,
3H, CH3), 7.24–7.54 (m, 4H, P), 7.62–8.68 (m, 6H, naph), 8.36 (s,
1H, CH), 9.90 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6) d 12.25,
102.45, 108.57, 118.39 (2C), 119.48, 119.65, 119.89, 124.61,
127.54, 129.40, 130.29, 132.94 (2C), 134.78, 135.84, 145.15,
153.41, 157.73, 159.87, 167.89; MS (EI) m/z: 377.1 (M+); Anal.
(C23H15N6O2) C, H, N.
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4.1.7.9. 5-Bromo-6-(2-(4-cyanophenylamino)-6-methylpyrimi-
din-4-yloxy)-2-naphthonitrile (3i). Yield 81.8%; recrystallized
from 1,4-dioxane, mp 254.4–225.2 °C; 1H NMR (400 MHz, DMSO-
d6) d 2.30 (s, 3H, CH3), 7.17–7.43 (m, 4H, Ph), 7.78–8.80 (m, 5H,
naph), 8.40 (s, 1H, CH), 9.91 (s, 1H, NH); 13C NMR (100 MHz,
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DMSO-d6)
d 12.17, 102.57, 109.22, 109.69, 115.26, 118.33,
119.10, 119.81, 125.64, 128.17, 129.35, 130.70, 131.57, 132.86,
134.22, 135.46, 145.01, 151.42, 157.67, 160.17, 167.10; MS (EI)
m/z: 457.0 (M+); Anal. (C23H15N6BrO2) C, H, N.
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