770
Z. Zhang et al. / European Journal of Medicinal Chemistry 44 (2009) 764e771
the residue was purified by preparative thin layer chromatog-
raphy (silica gel). Elution with CHCl3eCH3OH (8:1) gave
pure 14 (9 mg, 62%) as a light yellow solid; mp 110e
113 ꢀC. 1H NMR (500 MHz; DMSO-d6) d 1.15e1.19 (m,
6H, 2 ꢁ CH3), 1.63e1.70 (m, 1H, CHaHb-7), 1.91e2.11 (m,
2H, CH2CH2CO), 2.16e2.23 (m, 1H, CHaHb-7), 2.40e2.44
(m, 3H, CH2CH2CO, CHaHb-8), 2.47e2.51 (1H, CHaHb-8),
2.89e2.99 (m, 1H, CHaHb-9), 3.16e3.22 (m, 2H, CHaHb-9,
CH-6), 3.33e3.39 (m, 2H, N5-CH2), 3.49e3.51 (m, 2H,
CH2Br), 3.97e4.00 (m, 1H, CHBr), 4.02e4.11 (m, 6H,
2 ꢁ OCH2), 4.36e4.40 (m, 1H, CHCH2CH2), 4.76e4.82 (m,
1H, CH2NH ), 5.55e5.71 (br s, NH2), 5.93 (br s, NH2), 6.68
(d, 2H, J ¼ 8.8 Hz, H-Ph), 7.73e7.75 (d, 1H, J ¼ 8.5 Hz, H-
Ph), 8.03e8.03 (m, 1H, H-Ph), 8.45e8.47 (d, 1H,
J ¼ 10 Hz, CHaHb]eN5), 5.17e5.20 (d, 1H, J ¼ 17.0 Hz,
CHaCHb]eN10), 5.70e5.77 (m, 1H, CH]eN5), 5.82e5.88
(m, 1H, CH]eN10), 6.59 (d, H, J ¼ 8.5 Hz, H-Ph), 7.67 (d,
4H, H-Ph), 8.27e8.28 (m, 1H, CONH); 13C NMR (125 MHz;
DMSO-d6) d 14.1 (2 ꢁ CH3), 17.7 (C-7), 24.0 (C-8), 25.8
(CH2CH2CO), 30.2 (CH2CH2CO), 51.3 (C-6), 51.6 (C-9),
51.8 (NHCHCO), 53.2 (CH2eN5), 55.5 (CH2eN10), 59.9
(OCH2), 60.4 (OCH2), 110.6 (2 ꢁ C-Ph), 115.3 (CH2]eN5),
115.8 (C-4a), 116.4 (CH2]eN10), 119.9 (C-Ph), 128. 9
(2 ꢁ C-Ph), 133.6 (CH]eN5), 136.6 (CH]eN10), 150.4 (C-
Ph), 152.5 (C-8a), 157.9 (C-2), 159.7 (C-4), 166.5 (CONH),
172.2 (CH2COO), 172.3 (CHCOO). Found [M þ H]þ:
580.3246, C30H41N7O5 requires: 580.3242.
J ¼ 7.5 Hz,
CONH).
C27H36BrN7O5 requires: 776.0401.
Found
[M þ H]þ:
776.0428,
6.1.10. N-[4-[[2-(2,4-Diamino-5,10-diallyl-5,6,7,
8-tetrahydropyrido[3,2-d]pyrimidin-6-
yl)methyl]amino]benzoyl]-L-glutamic acid or
6.1.8. N-[4-[[2-(2,4-Diamino-5-(2,3-
8-deaza-N5,N10-diallyl-5,6,7,8-tetrahydroaminopterin (17)
A procedure similar to the preparation of 9 was followed to
prepare 17 from 16. Compound 17 was obtained in 73% yield as
a white solid; mp 140 ꢀC (dec). 1H NMR (300 MHz; DMSO-d6)
d 1.65e1.75 (m, 1H, CH-7), 1.90e2.00 (m, 1H, CHCHaHb),
2.03e2.12 (m, 2H, CHCHaHbCH2, CH-7), 2.23e2.36 (m, 2H,
CH2CH2COO), 2.47e2.50 (m, 2H, CH2-8), 3.08e3.15 (m, 1H,
CHaHbeN10), 3.20e3.40 (m, 4H, CH-6, CH2-9, CHaHbeN10),
3.85e3.90 (m, 1H, CHaCHbeN5), 4.07e4.12 (m, 1H, CHa
CHbeN5), 4.28e4.38 (m, 1H, CONHCH ), 4.94e5.02 (m,
2H, CHaHb]eN10, CHaHb]eN5), 5.05e5.09 (d, 1H,
J ¼ 10.5 Hz, CHaHb]eN5), 5.16e5.21 (d, 1H, J ¼ 17.1 Hz,
CHaHb]eN10), 5.71e5.82 (m, 1H, CH]eN5), 5.84e5.90
(m, 1H, CH]eN10), 6.60 (d, 2H, J ¼ 8.4 Hz, H-Ph), 7.68 (d,
2H, H-Ph), 8.04e8.06 (m, 1H, CONH); 13C NMR (75 MHz;
DMSO-d6) d 17.3 (C-7), 22.5 (C-8), 26.7 (CH2CH2CO), 31.0
(CH2CH2CO), 51.3 (C-6), 51.5 (C-9), 52.3 (NHCHCO), 53.3
(CH2eN5), 55.5 (CH2eN10), 110.7 (2 ꢁ Ph-C), 115.3 (CH2]
eN5), 115.8 (C-4a), 116.8 (CH2]eN10), 120.5 (Ph-C), 128.7
(2 ꢁ C-Ph), 133.8 (CH]eN5), 136.4 (CH]eN10), 148.9
(Ph-C), 150.2 (C-8a), 156.4 (C-2), 166.0 (C-4), 166.1
(CONH), 174.5 (CH2COO), 174.7 (CHCOO). Found
[M þ H]þ: 524.2628, C26H33N7O5 requires: 524.2616.
dibromopropane)-5,6,7,8-tetrahydropyrido
[3,2-d]pyrimidin-6-yl)methyl]amino]-3-bromo-
benzoyl]-L-glutamate (15)
A procedure similar to the preparation of 9 was followed to
prepare 15 from 14. Compound 15 was obtained in 82% yield
as a white solid; mp 180 ꢀC (dec). 1H NMR (300 MHz;
DMSO-d6) d 1.63e1.67 (m, 1H, CHaHb-7), 1.82e2.10 (m,
2H, CH2CH2CO), 2.12e2.21 (m, 1H, CHaHb-7), 2.26e2.35
(m, 3H, CH2CH2CO, CHaHb-8), 2.41e2.61 (m, 2H, CH2-9),
2.95e3.06 (m, 1H, CH-6), 3.12e3.34 (m, 2H, N5-CH2) 4.00e
4.12 (m, 1H, CHBr), 4.25e4.38 (t, 1H, CONHCHCH2), 5.73
(br s, NH2), 6.19 (br s, NH2), 6.57 (br s, NH-10), 6.64e6.67
(d, 1H, J ¼ 6.9 Hz, H-Ph), 7.71e7.74 (d, 1H, H-Ph), 8.03 (s,
1H, H-Ph), 8.31e8.34 (d, 1H, J ¼ 7.8 Hz, CONHCH). Found
[M þ H]þ: 719.9765, C23H28Br3N7O5 requires: 719.9775.
6.1.9. Diethyl N-[4-[[2-(2,4-diamino-5,10-diallyl-5,6,7,
8-tetrahydropyrido[3,2-d]pyrimidin-6-
yl)methyl]amino]benzoyl]-L-glutamate (16)
To a solution of diethyl 4-amino-8-deaza-5,6,7,8-tetrahy-
drofolate 8 (560 mg, 0.9 mmol) in anhydrous DMF (22 ml)
were added allyl bromide (2.8 ml, 3.2 mmol) and powdered
K2CO3 (220 mg), and the reaction mixture was stirred at
room temperature for about 24 h. Solvent was removed under
reduced pressure. To the resulting residue water (20 ml) was
added and the mixture was extracted with CHCl3
(3 ꢁ 20 ml). The extracts were pooled and dried over anhy-
drous Na2SO4. After evaporation of solvent to a small volume
the residue was purified by column chromatography (silica
gel). Elution with CHCl3eCH3OH (22:1) gave pure 16
6.2. Biological evaluation
Human dihydrofolate reductase and MTX were purchased
from Sigma Chemical Co. RPMI-1640 medium was produced
from GIBCO. Sulforhodamine B (SRB), 3-[4,5-dimethylthia-
zol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), tetrazo-
lium salt and propidium iodide were purchased from Sigma
Chemical Co. Microplate reader from FLUOsta OPTIMA,
Germany and BECScan flow cytometer from Becton Sickin-
son FACScan, American.
1
(448 mg, 69%) as a yellow solid; mp 67e69 ꢀC. H NMR
(500 MHz; DMSO-d6) d 1.14e1.18 (m, 6H, 2 ꢁ CH3), 1.65e
1.68 (m, 1H, CH-7), 1.95e2.01 (m, 1H, CHCHaHb), 2.05e
2.09 (m, 2H, CHCHaHbCH2, CH-7), 2.36e2.44 (m, 4H, CH2-
8, CH2CH2COO), 3.09e3.13 (m, 1H, CHaHbeN10), 3.16e
3.21 (m, 1H, CH-6), 3.25e3.38 (m, 3H, CH2-9, CHaHbeN10),
3.85e3.88 (m, 1H, CHaHbeN5), 4.01e4.09 (m, 5H, OCH2 ꢁ 2,
CHaCHbeN5), 4.35e4.39 (m, 1H, CONHCH ), 4.93e4.97 (m,
2H, CHaHb]eN10, CHaCHb]eN5), 5.04e5.06 (d, 1H,
6.2.1. Cell lines and culture conditions
Human promyelocytic leukemic cell line (HL-60), human
hepatocellular carcinoma cell line (Bel-7402), stomach adeno-
carcinoma (BCG-823) and cervical cancer (Hela) were grown
and maintained in RPMI-1640 medium supplemented with