T.-S. Feng et al. / Biochemical Pharmacology 82 (2011) 236–247
239
2.1.8. Ugi adduct (6b)
1H, H14), 7.88 (d, J 1.8, 1H, H14), 7.88 (d, J 9.0, 1H, H12), 7.74 (d, J
9.0, 1H, H12), 7.55 (m, 4H, (H2 + H3) Â 2), 7.35 (dd, J 9.0 and 1.8,
1H, H13), 7.31 (dd, J 9.0 and 1.8, 1H, H13), 7.13 (d, J 7.5, 1H, H1),
7.13 (d, J 7.5, 1H, H1), 6.97 (br s, 1H, NH), 6.34 (d, J 5.4, 1H, H11),
6.33 (d, J 5.7, 1H, H11), 4.09 (s, 2H, H7),4.00 (s, 2H, H7), 3.88 (m, 2H,
H8), 3.70 (br s, 4H, H8 + H9), 3.50 (m, 2H, H9), 2.97 (t, J 7.8, 2H, H6),
2.72 (t, J 8.1, 2H, H5), 2.57 (t, J 7.8, 2H, H6), 2.28 (t, J 8.1, 2H, H5),
2.24 (s, 3H, H4), 1.92 (s, 3H, H4), 1.75 (m, 11H, cyclohexyl-H), 1.22
(m, 11H, cyclohexyl-H); dc (100 MHz; CDCl3) 174.9, 161.2, 151.6,
151.0, 137.3, 136.8, 136.2, 136.1, 132.0, 125.9, 125.8, 125.7, 123.9,
122.2, 122.0, 119.2, 119.1, 98.6, 98.0, 53.1, 43.0, 41.0, 32.9, 32.2,
31.5, 25.5, 25.2, 24.8, 24.7, 22.7, 22.4, 12.9; (Found: m/z 603.1182
[M+], C33H35ClN4O5 requires M, 603.1177) (Found: C, 66.67, H,
7.07, N, 7.29%; Requires C, 66.95, H, 7.28 N, 7.10%).
Purified via column chromatography, first with EtOAc and then
with EtOAc/MeOH 9.5:0.5 to yield the pure product as a white
powder (0.5009 g, 84%). (m.p. 121–123 8C. Rf (MeOH/EtOAc, 1:9)
0.48; nmax (CHCl3)/cmÀ1 3333 (N–H), 2926 (Ar–H), 2894 (alkyls),
1666 (C55O), 1580 (Ar–C), 1010 (C–N), 753 (C–Cl); dH (400 MHz;
CDCl3) 8.45 (d, J 5.2, 1H, H24), 8.23 (br s, 1H, H29), 7.99 (s, 2H, H31),
7.36 (br s, 5H, H30 + H18 + H19), 7.13 (br s, 1H, NH), 6.31 (br s, 1H,
H28), 6.10 (br s, 1H, NH), 5.44 (s, 1H, H12), 4.91 (d, J 12.4, 1H, H17),
4.90 (d, J 4.0, 1H, H10), 4.56 (d, J 12.4, 1H, H17), 4.11 (m, 2H, CH2,
H22), 3.81 (br s, 2H, CH2, H25), 3.52 (br s, 2H, CH2, H23), 3.48 (s, 2H,
H20), 3.26 (br s, 2H, CH2, H24), 2.52 (m, 1H, H9), 2.00–1.45 (m,
20H), 1.45 (s, 3H, H14), 0.96 (d, J 7.2, 3H, H15), 0.95 (d, J 5.6, 3H,
H16); dc (100 MHz; DMSO-d6) 170.9, 167.1, 151.8, 150.0, 149.0,
139.5, 135.7, 132.3, 127.4, 126.8, 126.6, 126.5, 124.4, 124.0, 123.9,
117.4, 103.3, 100.6, 98.6, 87.0, 80.4, 68.7, 52.0, 47.6, 45.8, 43.7,
42.3, 36.6, 36.0, 34., 32.4, 32.1, 30.5, 25.6, 24.7, 24.4, 20.0, 12.7;
(Found: m/z 789.4125 [M+], C44H57ClN4O7 requires M, 789.4122)
(Found: C, 66.80, H, 6.91, N, 6.92%; Requires C, 66.95, H, 7.28 N,
7.10%).
2.1.12. N-(2-(tert-Butylamino)-2-oxoethyl)-N-(2-(7-chloroquinolin-
4-ylamino)ethyl)-3-(3-methyl-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)propanamide (6e)
Purified via column chromatography, first with EtOAc and then
with EtOAc/MeOH 9.5:0.5 to yield the pure product as an orange
powder (0.123 g, 28%). m.p. 205–207 8C; Rf (EtOAc) 0.11; nmax
(CHCl3)/cmÀ1 3320 (N–H), 3026 (Ar–H), 1736 (C55O), 1430 (Ar–C),
1216 (C–N); 753 (C–Cl); dH (400 MHz; CDCl3) 8.35 (d, J 5.2, 1H,
H10), 8.34 (d, J 5.2, 1H, H10), 7.90 (d, J 9.2, 1H, H12), 7.85 (d, J 2.0,
1H, H14), 7.84 (d, J 9.2, 1H, H12), 7.79 (d, J 2.0, 1H, H14), 7.51 (m,
4H, (H2 + H3) Â 2), 7.29 (dd, J 9.2 and 2.0, 1H, H13), 7.28 (dd, J 9.2
and 2.0, 1H, H13), 7.15 (d, J 7.2, 1H, H1), 7.14 (d, J 7.2, 1H, H1), 6.36
(d, J 5.6, 1H, H11), 6.35 (d, J 5.6, 1H, H11), 3.99 (s, 2H, H7), 3.79 (s,
2H, H7), 3.77 (d, J 5.6, 2H, H8), 3.66 (d, J 5.6, 2H, H9), 3.63 (d, J 5.6,
2H, H8), 3.49 (d, J 5.6, 2H, H9), 2.91 (t, J 8.0, 2H, H6), 2.72 (t, J 8.0, 2H,
H6), 2.51 (t, J 8.0, 2H, H5), 2.30 (t, J 8.0, 2H, H5, 2.19 (s, 3H, H4), 1.93
2.1.9. Ugi adduct (6c)
Purified via column chromatography, first with EtOAc and then
with EtOAc/MeOH 9.5:0.5 to yield the pure product as a white
powder (0.453 g, 82%). (m.p. 127–129 8C. Rf (MeOH/EtOAc, 1:9)
0.18; nmax (CHCl3)/cmÀ1 3385 (N–H), 3017 (Ar–H), 1520 (C55O),
1215 (C–N), 756 (C–Cl); dH (400 MHz; CDCl3) 8.47 (br s, 1H, H24),
8.24 (br s, 1H, H26), 8.12 (d, J 8.6, 2H, H19), 8.00 (s, 1H, H28), 7.40
(d, J 8.6, 2H, H18), 7.39 (br s, 1H, H27), 7.04 (br s, 1H, NH), 6.35 (br s,
1H, H25), 5.42 (s, 1H, H12), 4.87 (br s, 2H, H17 + H10), 4.51 (br s,
1H, H17), 3.93 (br s, 2H, H22), 3.64 (br s, 2H, H23), 2.68 (m, 1H, H9),
2.38 (ddd, J 14.4, 13.2 and 4.2, 1H, not known), 2.10–1.57 (m, 4H),
1.44 (s, 3H, H14), 1.35 (s, 9H, t-butyl), 0.95 (d, J 6.0, 6H, H16 + H15);
(s, 3H, H4), 1.37 (s, 9H, t-butyl), 1.27 (s, 9H, t-butyl); dc (100 MHz;
CDCl3) 151.6, 150.3, 145.6, 136.1, 127.2, 126.4, 125.6, 123.7, 123.6,
122.5, 122.4, 119.0, 98.9, 98.2, 52.7, 52.0, 49.4, 14.2, 49.0, 48.7,
48.5, 47.7, 42.1, 40.8, 32.0, 31.4, 28.4, 22.5, 22.3, 12.6; (Found: m/z
577.0805 [M+], C31H33ClN4O5 requires M, 577.0798) (Found: C,
66.80, H, 7.39, N, 7.48%; Requires C, 66.95, H, 7.28 N, 7.10%).
d
c (100 MHz; CDCl3) 129.9, 127.2, 126.8, 125.9, 104.2, 104.2, 101.6,
101.6, 98.2, 88.1, 81.1, 70.7, 69.5, 69.1, 52.7, 52.6, 52.1, 44.5, 44.4,
37.5, 36.4, 34.6, 31.0, 30.9, 29.1, 28.7, 26.2, 24.7, 24.6, 20.3, 13.1;
(Found: m/z 735.3211 [M+], C40H51ClN4O7 requires M, 735.3205)
(Found: C, 65.68, H, 7.28, N, 7.56%; Requires C, 65.34, H, 6.99 N,
7.62%).
2.2. Biological evaluation
2.1.10. Citrate salt of Ugi adduct 6c (6c-citrate)
2.2.1. Parasite cultivation
Citric acid (0.131 g, 0.680 mmol, 2.5 eq) in acetone (5 ml) was
added to a solution of 6c (0.200 g, 0.272 mmol, 1.0 eq) in acetone
(5 ml). The product precipitated out over 2 days at 4 8C to yield
D10 and K1 strains of P. falciparum were maintained in
continuous in vitro culture by standard methods [28]. The cultured
parasites were, whenever necessary, synchronized by treatment
white solid (0.247 g, 98%).
3317 (N–H), 3017 (Ar–H), 2962 (OH (acid)), 1707 (C55O), 1612 (Ar–
H), 1215 (C–N), 1007 (C–O), 752 (C–Cl); H (300 MHz; CD3OD) 8.51
n
max (CHCl3)/cmÀ1 3419 (OH (alcohol)),
with 5% D-sorbitol (Sigma) at the ring stage [29].
d
2.2.2. Evaluation of in vitro antiplasmodial activity
The in vitro antiplasmodial assays were carried out against D10
and K1 strains of P. falciparum using the parasite lactate
dehydrogenase assay, as previously reported by Guantai et al.
2010 [30].
(br s, 1H, H24), 8.43 (br s, 1H, H26), 7.91 (br s, 1H, H28), 7.61 (br s,
1H, H27), 7.39 (br s, 2H, H19), 6.99 (br s, 2H, H18), 6.42 (br s, 1H,
H25), 5.44 (s, 1H, H12), 4.04 (br s, 2H, H22), 3.95 (br s, 2H, H23),
2.58 (m, 1H, H9), 2.33 (td, J 14.4 and 13.0, 4.2 Hz, 1H, not known),
2.20–1.50 (m, 4H, not known), 1.44 (br s, 4H, H29 + H30), 1.37 (s,
3H, H14), 1.32 (s, 9H, t-butyl), 0.94 (d, J 6.0, 6H, H16 + H15);
(Found:, m/z 927.4471 [M+], C40H51N4ClO7 requires M, 927.4459)
(Found: C, 59.76, H, 6.63, N, 5.74%; Requires C, 59.57, H, 6.41, N,
6.04%).
2.2.3. Evaluation of
b-hematin inhibition
b-hematin inhibition assays were conducted as previously
described by Ncokazi et al. 2005 [31].
2.2.4. Cytotoxicity assays
2.1.11. N-(2-(7-Chloroquinolin-4-ylamino)ethyl)-N-(2-
Cytotoxicity assays were carried out on a human cervical
carcinoma cell line (HeLa) using the MTT [3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, cell cul-
tures were routinely maintained in Eagle’s Minimum Essential
Medium (EMEM) supplemented with 10% bovine foetal calf serum
(cyclohexylamino)-2-oxoethyl)-3-(3-methyl-1,4-dioxo-1,4-
dihydronaphthalen-2-yl)propanamide (6d)
Purified via column chromatography, first with EtOAc and then
with EtOAc/MeOH 9.5:0.5 to yield the pure product as an orange
powder (0.1138 g, 25%). (m.p. 135–138 8C. Rf (MeOH/EtOAc, 1:9)
0.11; nmax (CHCl3)/cmÀ1 3273 (N–H), 3020 (Ar–H), 2926 (alkyls),
1606 (C55O), 1430 (Ar–C), 1216 (C–N), 770 (C–Cl); dH (300 MHz;
CDCl3) 8.48 (d, J 5.7, 1H, H10), 8.44 (d, J 5.4, 1H, H10), 7.96 (d, J 1.8,
(FCS). For cytotoxicity evaluations, 5 Â 102 cells in 180
ml medium
were seeded in each well of a 96-well plate and incubated at 37 8C
under a 5% CO2 atmosphere for 1 h. Aliquots of 20
ml of serial
dilutions of the test compounds (stocks in DMSO) were added to