704 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Pe´rez-Sacau et al.
(()-2,2-Dimethyl-5,6-dioxo-3,4,5,6-tetrahydro-2H-benzo[h]-
chromen-3-yl 4-Bromobenzoate (26). (()-52 (133 mg, 0.51 mmol)
was treated with 170 mg (1.5 equiv) of p-bromobenzoyl chloride
and 80 µL (2 equiv) of pyridine, in 15 mL of CH2Cl2 at 0 °C. The
crude product was purified by flash chromathography, eluted with
hexanes/EtOAc 9/1, to yield 12 mg (5%) of 26. 1H NMR (CDCl3,
300 MHz) δ 8.03 (m, 2H), 7.99 (d, J ) 7.8 Hz, 2H), 7.83 (d, J )
7.8 Hz, 1H), 7.68 (m, 2H), 7.50 (m, H), 5.40 (m, 1H), 2.95 (dd,
J ) 18.3, 4.7 Hz, 1H), 2.85 (dd, J ) 18.3, 4.0 Hz, 1H), 1.55 (s,
3H), 1.52 (s, 3H). 13C NMR (CDCl3, 75 MHz) δ 179.3 (s), 178.2
(s), 163.1 (s), 150.9 (s), 134.1 (d), 132.2 × 2 (d), 132.1 (s), 131.9
× 2 (d), 130.1 (s), 129.0 (d), 125.9 (d), 124.3 (d), 127.7 (s), 127.0
(s), 109.8 (s), 79.1 (s), 70.6 (d), 24.9 (q), 23.5 (q), 22.7 (t). EI-MS
m/z 442 (M+ + 2, 8), 440 (M+, 8), 257 [M+ - COp(Br)Ph, 61],
183 [COp(Br)Ph, 100]. HR-EI-MS m/z 440.0278 [(M+); calcd for
C22H17O5Br 440.0310].
(s), 69.8 (d), 57.4 (q), 24.7 (q), 22.6 (t), 22.3 (q). HR-EI-MS m/z
407.1501 [(M+ + 1); calcd for C24H23O6 407.1495].
(3S)-3-Hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]-
chromene-5,6-dione (16). Hydrolysis of 28 was carried out with
NaHCO3
(0.1 M) in methanol at rt for 2 h. The residue was
extracted several times with CH2Cl2. The combined organic extracts
were washed with water, dried over MgSO4 and then purified by
flash chromathography on silica gel (eluted with mixtures of
hexanes/AcOEt 30%), to yield 13 mg (36%) of 16 ([R]D25 (CHCl3)
) +47.2) which showed spectroscopic data identical to those
reported in the literature.
(3R)-3-Hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo[h]-
chromene-5,6-dione (17). Hydrolysis of 29 was carried out
following the procedure described before to obtain 21 mg (58%)
of 17, which showed spectroscopic data identical to those for 16
25
except the optical rotation ([R]D (CHCl3) ) -49.0).
Cell Culture. The human promyelocytic leukemia HL-60 cell
line established by Gallagher et al.29 was cultured in suspension in
RPMI-1640 medium (Invitrogen) supplemented with 10% heat-
inactivated fetal bovine serum, penicillin (10 000 units/mL), and
streptomycin in a humidified atmosphere of 95% air and 5% CO2
at 37 °C. Cells were maintained at a density <1 × 106 cells/mL.
Cells were resuspended in fresh medium 24 h before each treatment
to ensure exponential growth. Stock solutions (100 or 50 mM) of
lapachol derivatives were made in dimethyl sulfoxide (DMSO),
aliquoted, and stored at -80 °C. Further dilutions were made in
culture medium prior to use. In all experiments, the final concentra-
tion of DMSO did not exceed 0.5%, a concentration that is not
toxic to the cells.
Assay for Cytotoxicity. Cytotoxic assays were performed by
the MTT procedure.30 Cells (1 × 104/well) were exposed to different
concentrations of the compounds in 96-well plates for 72 h at
37 °C. Controls and samples were always treated with the same
concentrations of vehicle (DMSO). Surviving cells were detected
on the basis of their ability to metabolize 3-[4,5-dimethylthiazol-
2-yl]2,5-diphenyltetrazolium bromide (MTT) into formazan crystals.
Optical density at 560 nm was used as a measure of cell viability.
The MTT dye reduction assay measures mitochondrial respiratory
function and can detect earlier than dye-exclusion methods. Cell
survival (%) ) (mean absorbance in treated wells/mean absorbance
in control wells) × 100. Concentrations inducing 50% inhibition
of cell growth (IC50) were determined graphically for each
experiment by use of the curve-fitting routine of Prism 2.0
(GraphPad) and the equation derived by De Lean et al.31
Pharmacophore Model. The pharmacophore model was gener-
ated with the HypoGen module of Catalyst 4.10. Compounds 1-51
were built de novo with standard options within the 2D/3D editor
sketcher of the program. In cases where the chirality of the active
form was not known, all possible stereoisomers were generated
and considered. The BEST conformational analysis procedure was
applied. The number of conformers was limited to a maximum of
250, with a 20 kcal/mol energy threshold above the calculated global
minimum. The experimentally determined IC50 values of com-
pounds 1-51 span about 2-3 orders of magnitude from IC50
(7) ) 68.2 µM to IC50 (28) ) 0.1 µM. Therefore, the inactivity
spread, uncertainty, and spacing parameters were changed from the
default value of 3.0 to 1.5, 1.6, and 1.8, respectively, as proposed
by Accelrys for training sets with narrower activity span than usual.
The hydrogen-bond-acceptor (HBA) and -donor (HBD), hydro-
phobic (HYD), and aromatic ring (AR) chemical features were
considered for hypothesis generation and up to 10 excluded
volumes. Hypothesis selection was done by a cost analysis
procedure (represented in bit units) based on three terms: weight
cost (increases in a Gaussian form as the feature weight deviates
from an idealized value of 2.0); error cost (penalizes the deviation
between the estimated activities of the training set and their
experimentally determined values); and configuration cost (penalizes
the complexity of the hypothesis, should not exceed a maximum
value of 18). The error cost contributes the most in determining
the overall cost of a hypothesis. In addition, the costs of the ideal
hypothesis, the simplest possible hypothesis that fits the data with
(()-2,2-Dimethyl-5,6-dioxo-3,4,5,6-tetrahydro-2H-benzo[h]-
chromen-3-yl 4-Cyanobenzoate (27). (()-52 (130 mg, 0.50 mmol)
in 10 mL of CH2Cl2 was treated with 170 mg (2 equiv) of
p-cyanobenzoyl chloride and 100 µL (2.5 equiv) of pyridine, at 0
°C for 24 h. The crude product was purified by flash chromathog-
raphy, with 5-40% hexanes/EtOAc, to yield 51 mg (35%) of 27.
1H NMR (CDCl3, 300 MHz) δ 8.11 (d, J ) 7.6 Hz, 1H), 8.06 (d,
J ) 7.7 Hz, 2H), 7.89 (d, J ) 7.6 Hz, 1H), 7.68 (m, 3H), 7.58 (m,
1H), 5.40 (m, 1H), 2.95 (dd, J ) 18.4, 4.8 Hz, 1H), 2.85 (dd, J )
18.4, 4.0 Hz, 1H), 1.57 (s, 3H), 1.51 (s, 3H). 13C NMR (CDCl3, 75
MHz) δ 179.1 (s), 178.5 (s), 163.9 (s), 161.1 (s), 135.0 (d), 133.2
(s), 132.3 × 2 (d), 131.8 (s), 131.2 (d), 130.6 (s), 130.2 × 2 (d),
129.0 (d), 124.3 (d), 117.7 (s), 116.9 (s), 109.8 (s), 79.1 (s), 70.6
(d), 24.9 (q), 23.5 (q), 22.7 (t). EI-MS m/z 387 (M+, 1), 258 [M+
- COp(CN)Ph, 1], 130 [COp(CN)Ph], 94 (100). HR-EI-MS m/z
387.3401 [(M+); calcd for C23H17O5N 387.3854].
Resolution of 3-Hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo-
[h]chromene-5,6-dione (52): Preparation of Esters 28 and 29.
(()-52 (149 mg, 0.58 mmol) in 6 mL of dry CH2Cl2 was treated
with 192 mg (2 equiv) of (R)-R-methoxyphenylacetic acid, at rt
for 10 h in the presence of dicyclohexylcarbodiimide (2 equiv) and
dimethylaminopyridine (DMAP) in catalytic amounts. When the
starting material was consumed, the solvent was removed under
vacuum and the crude product was purified by flash chromathog-
raphy (silica gel, eluted with mixtures of hexanes/EtOAc increasing
the polarity from 5% to 40%) to yield 105 mg (45%) of the RS
diastereomer 28 and 117 mg (50%) of the RR diastereomer 29.
Both esters were hydrolyzed with NaHCO3 (0.1 M) in methanol at
rt, to yield enantiomeric alcohols 16 and 17 in 36% and 58% yield,
respectively.
(3S)-2,2-Dimethyl-5,6-dioxo-3,4,5,6-tetrahydro-2H-benzo[h]-
chromen-3-yl (2R)-Methoxy(phenyl)ethanoate (28). [R]D25 (CHCl3)
1
) -7. H NMR (CDCl3, 300 MHz) δ 8.08 (d, J ) 7.6 Hz, 1H),
7.76 (d, J ) 7.7 Hz, 1H), 7.67 (t, J ) 7.6 Hz, 1H), 7.55 (t, J ) 7.7
Hz, 1H), 7.37 (m, 2H), 7.28 (m, 3H), 5.10 (t, J ) 4.2 Hz, 1H),
4.74 (s, 1H), 3.40 (s, 3H), 2.82 (dd, J ) 18.1, 4.9 Hz, 1H), 2.67
(dd, J ) 18.1, 4.7 Hz, 1H), 1.22 (s, 3H), 1.06 (s, 3H). 13C NMR
(CDCl3, 75 MHz) δ 179.2 (s), 178.4 (s), 169.6 (s), 161.0 (s), 136.0
(s), 134.8 (d), 131.9 (s), 131.0 (d), 130.1 (s), 128.9 (d), 128.8 (d),
128.6 × 2 (d), 126.8 × 2 (d), 124.2 (d), 109.8 (s), 82.3 (d), 79.6
(s), 69.8 (d), 57.4 (q), 24.7 (q), 22.6 (t), 22.3 (q). EI-MS m/z 240
(M+ - MPA, 28), 225 (240 - Me, 9), 121 (MPA - CO2, 100). IR
(CHCl3) νmax (cm-1): 2924, 2853, 1752, 1654, 1608, 1573, 1455,
1395, 1114, 1027, 754.
(3R)-2,2-Dimethyl-5,6-dioxo-3,4,5,6-tetrahydro-2H-benzo[h]-
chromen-3-yl (2R)-Methoxy(phenyl)ethanoate (29). [R]D25 (CHCl3)
) -412. 1H NMR (CDCl3, 300 MHz) δ 8.05 (d, J ) 7.6 Hz, 1H),
7.78 (d, J ) 7.7 Hz, 1H), 7.66 (t, J ) 7.6 Hz, 1H), 7.55 (t, J ) 7.7
Hz, 1H), 7.24 (m, 2H), 7.09 (m, 3H), 5.04 (t, J ) 4.2 Hz, 1H),
4.73 (s, 1H), 3.38 (s, 3H), 2.57 (dd, J ) 18.3, 4.5 Hz, 1H), 2.41
(dd, J ) 18.3, 3.9 Hz, 1H), 1.46 (s, 3H), 1.34 (s, 3H). 13C NMR
(CDCl3, 75 MHz) δ 179.2 (s), 178.4 (s), 169.6 (s), 161.0 (s), 136.0
(s), 134.8 (d), 131.9 (s), 131.0 (d), 130.1 (s), 128.9 (d), 128.8 (d),
128.6 × 2 (d), 127.2 × 2 (d), 124.2 (d), 109.8 (s), 82.3 (d), 79.6