S. Lopez et al. / Bioorg. Med. Chem. 19 (2011) 1658–1665
1665
129.0, 129.6, 136.0, 136.5, 139.3, 147.2, 156.8, 161.0, 165.0. IR mmax
3398, 3352 (NH2); 3158 (NH2); 1555 (NO2) cmꢀ1. HRMS (ES)
[M+H]+ calcd for C24H23N6O3 444.1753; found 444.1757. Anal.
Calcd for C24H22N6O3ꢂ0.7EtOAc: C, 63.9; H, 5.5; N, 16.7. Found: C,
63.6; H, 5.9; N, 16.9.
continued at 37 °C until the reaction was complete (1 h). For each
assay, serial dilutions of PaTrin-2 were used as a positive control.
Following the incubations, DNA was hydrolysed to acid solubility
using 1 M perchloric acid and removed and washed once by centri-
fugation. The acid-insoluble [3H]-methylated MGMT protein was
quantitated by liquid scintillation counting. IC50 values were deter-
mined from the slope of the concentration-dependence plot and
essentially represent the mean of 5 determinations.
4.2.6.2.
2-Amino-4-benzyloxy-6-[N-(4-(4-aminophenylami-
The residue was puri-
no)anilino)]-5-nitropyrimidine (4b).
fied by column chromatography on silica gel (2:1 EtOAc/hexane)
to afford a brown solid (27 mg, 30%). mp 198–200 °C. 1H NMR
(DMSO-d6) d 4.30 (br s, 2H, C6H4NH2); 5.59 (s, 2H, C6H5CH2O);
6.50 (d, 2H, J 8.4, H200); 6.75 (d, 2H, J 8.4, H30); 6.80 (d, 2H, J 8.4,
H300); 7.48 (m, 5H, H4000, H3000, H20); 7.51 (m, 5H, H2000, C6H5NHC6H5,
NH2); 10.49 (s, 1H, NH). 13C NMR (DMSO-d6) d 68.5 (C6H5CH2O);
110.0 (C5–NO2); 114.0, 115.2, 122.7, 124.9, 128.2, 128.4, 128.9,
132.1, 136.6, 144.3, 144.5, 156.6, 160.9, 165.1. IR mmax 3451
(NH2); 3312, 3215 (NH2); 1555 (NO2) cmꢀ1. HRMS (ES) [M+H]+
calcd for C23H22N7O3 445.1706; found 445.1710. Anal. Calcd for
C23H21N7O3ꢂ1.1EtOAc: C, 60.9; H, 5.6; N, 18.1. Found: C, 61.4; H,
6.1; N, 18.5.
Acknowledgements
The authors are grateful to the Irish Cancer Society, Cancer
Research UK and CHEMORES for financial support. The authors
are indebted to Dr. John O’Brien for the NMR studies.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
4.2.6.3. 2-Amino-4-benzyloxy-6-[N-(4-(4-aminophenoxy)anili-
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no)]-5-nitropyrimidine (4c).
The residue was purified by col-
umn chromatography on silica gel (2:1 EtOAc/hexane) to afford a
bright yellow solid (76 mg, 85%). Mp 198–200 °C. 1H NMR (DMSO-
d6) d 4.91 (br s, 2H, C6H4NH2); 5.37 (s, 2H, C6H5CH2O); 6.51 (d, 2H,
J 8.5, H200); 6.79 (d, 2H, J 8.5, H300); 6.74 (d, 2H, J 8.5, H20); 6.74 (d,
2H, J 8.5, H30); 7.26 (t, 1H, J 7.5, H4000); 7.32 (t, 2H, J 7.5, H3000); 7.38–
7.44 (m, 2H, H2000); 7.49 (m, 4H, H20, NH2); 10.3 (s, 1H, NH). 13C
NMR (DMSO-d6) d 68.3 (C6H5CH2O); 109.9 (C5–NO2); 115.0, 116.6,
121.1, 125.0, 127.9, 128.1, 128.6, 132.2, 136.3, 145.7, 145.8, 156.0,
156.5, 160.7, 164.7. IR mmax 3450, 3409 (NH2); 3326, 3218 (NH2);
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1113.
1555 (NO2) cmꢀ1
.
HRMS (ES) [M+H]+ calcd for C23H21N6O4
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4.2.6.4.
none)]-5-nitropyrimidine (4d).
2-Amino-4-benzyloxy-6-[N-(4,40-diaminobenzophe-
The residue was treated with
water and extracted with EtOAc (4 ꢁ 25 mL). The organic layer was
washed with water, brine and dried over Na2SO4. Filtration and
evaporation of the solvent gave a yellow residue, which was purified
by column chromatography on silica gel (2:1 EtOAc/hexane) to af-
ford a bright yellow solid (78 mg, 82%). mp 222–224 °C. 1H NMR
(DMSO-d6) d 5.48 (s, 2H, C6H5CH2O); 6.15 (br s, 2H, C6H4NH2); 6.61
(d, 2H, J 8.5, H300); 7.36 (t, 1H, J 7.5, H4000); 7.42 (t, 2H, J 7.5, H3000);
7.49–7.58 (m, 4H, H200, H2000); 7.62 (d, 2H, J 8.5, H30); 7.76 (s(br),
1H, NH); 7.78 (br s, 1H, NH); 7.91 (d, 2H, J 8.5, H20); 10.7 (s, 1H,
C6H4NH). 13C NMR (DMSO-d6) d 68.4 (C6H5CH2O); 110.5 (C5–NO2),
112.7, 121.7, 124.1, 127.9, 128.2, 128.6, 130.0, 132.6, 134.4, 136.2,
141.1, 153.8, 156.2, 160.7, 164.7; 192.6 (CO). IR mmax 3484, 3414
(NH2); 3376, 3316 (NH2); 1632 (CO); 1538 (NO2) cmꢀ1. HRMS (ES)
[M+Na]+ calcd for C24H20N6O4Na 479.1444; found 479.1436. Anal.
Calcd for C24H20N6O4ꢂ0.9EtOAc: C, 61.9; H, 5.1; N, 15.7. Found: C,
61.9; H, 5.05; N, 15.9.
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Determination of the IC50 values for inactivation of MGMT by
the study compounds was by a method previously described.28 In
brief, the compounds were dissolved at 10 mM in dry DMSO and
serial dilutions prepared in DMSO. In each assay, fixed amounts
of MGMT (50–60 fmol) were incubated with aliquots of the dilu-
tion series of test compound in a total volume of 200
containing 10 g of calf thymus DNA at 37 °C for 1 h. Excess
[3H]-methylated DNA substrate (100
L containing 4 g of DNA
and 100 fmol of O6-[3H]methylguanine) was added and incubation
lL of buffer
l
l
l
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