Journal of Medicinal Chemistry
Article
(s, 3H), 6.48 (s, 2H), 7.20 (d, J = 9.1 Hz, 1H), 7.47 (d, J = 9.3 Hz,
1H), 7.97 (d, J = 9.3 Hz, 1H), 8.05 (d, J = 9.1 Hz, 1H).
Hz, 1H), 7.69 (d, J = 9.5 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1H), 8.32 (d,
J = 9.0 Hz, 1H); MS (EI) m/z 404 (M+, 26%), 325 (100%); HRMS
(EI) for C19H20N2O6S (M+) calcd 404.1042, found 404.1045.
Biology. Materials. Regents for cell culture were obtained from
Gibco-BRL Life Technologies (Gaitherburg, MD). Microtubule-
associated protein (MAP)-rich tubulin was purchased from
Cytoskeleton, Inc. (Denver, CO). [3H]Colchicine (specific activity,
60−87 Ci/mmol) was purchased from PerkinElmer Life Sciences
(Boston, MA).
Cell Growth Inhibitory Assay. Human cancer cell lines (KB,
MKN45, and HT29) used in this study were procured from American
Type Culture Collection (Rockville, MD) and grown in Dulbecco’s
modified Eagle’s medium, minimal essential medium, or RPMI 1640
medium. Resistant cell lines KB-Vin10, KB-7D, and KB-S15 were
maintained in medium containing additional 10 nM vincristine, 7 μM
VP16, and 50 nM paclitaxel, respectively. All cell cultures were
supplemented with 10% fetal bovine serum, 2 μM glutamine, 100
units/mL penicillin, and 100 μg/mL streptomycin and incubated in a
humidified atmosphere (95% air and 5% CO2) at 37 °C. KB-Vin10
and KB-S15 cell lines were resistant to vincristine and paclitaxel,
respectively, and both overexpressed the MDR drug efflux protein. KB-
7D cells were resistant to VP16 and overexpressed MRP. All resistant
cell lines were incubated in drug-free medium for 3 days before being
harvested for the growth inhibition assay. In vitro growth inhibition
was assessed with the methylene blue assay.23 In brief, exponentially
growing cells were seeded into 24-well culture plates at a density of
8000−20000 cells per milliliter per well (depending on the doubling time
of the cell line) and allowed to adhere overnight. Cells were incubated
with various concentrations of drugs for 72 h. Then, we measured the
6-Methoxy-5-nitro-2-(3′,4′,5′-trimethoxyphenylamino)quinoline
(31). A mixture of 3,4,5-trimethoxyaniline (0.12 g, 0.63 mmol) and
compound 28 (0.1 g, 0.42 mmol) was heated to 200 °C for 10 min.
The residue was extracted with CH2Cl2 and aqueous NaHCO3. The
organic layers were combined and purified by flash chromatography to
1
give compound 31 (19% yield; 2:3 EtOAc/n-hexane): H NMR (500
MHz, CDCl3) δ 3.89 (s, 9H), 3.93 (s, 3H), 6.52 (s, 2H), 7.51 (d, J =
9.0 Hz, 1H), 7.52 (d, J = 9.8 Hz, 1H), 7.92 (d, J = 9.6 Hz, 1H), 9.02
(d, J = 9.1 Hz, 1H), 9.95 (s, 1H).
6-Methoxy-5-nitro-2-(3′,4′,5′-trimethoxyphenylthio)quinoline
(32). The title compound was obtained in 63% overall yield from
compound 28 in a manner similar to that described for the preparation
of 31: 1H NMR (500 MHz, CDCl3) δ 3.85 (s, 6H), 3.91 (s, 3H), 4.04
(s, 3H), 6.89 (s, 2H), 7.12 (d, J = 9.0 Hz, 1H), 7.51 (d, J = 9.5 Hz,
1H), 7.83 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 9.5 Hz, 1H).
6-Methoxy-5-nitro-2-(3′,4′,5′-trimethoxyphenylsulfonyl)-
quinoline (33). To a mixture of compound 32 (0.50 g, 1.25 mmol)
and CH2Cl2 (100 mL) was added m-chloroperbenzoic acid (0.65 g,
3.75 mmol) at 0 °C. The reaction mixture was stirred at ambient
temperature overnight and washed with 10% sodium sulfite, saturated
NaHCO3, and saturated NaCl. The combined organic layer was
evaporated to give a residue that was purified by column
chromatography to give 33 (78% yield; 1:1 EtOAc/n-hexane): 1H
NMR (500 MHz, CDCl3) δ 3.87 (s, 3H), 3.91 (s, 6H), 4.10 (s, 3H),
7.32 (s, 2H), 7.67 (d, J = 9.5 Hz, 1H), 8.27 (dd, J = 9.0, 2.5 Hz, 2H),
8.36 (d, J = 9.5 Hz, 1H).
5-Amino-6-methoxy-2-(3′,4′,5′-trimethoxyphenyl)quinoline
(11). The title compound was obtained in 80% overall yield from
compound 29 in a manner similar to that described for the preparation
of 14: mp 222−223 °C; 1H NMR (500 MHz, CDCl3) δ 3.91 (s, 3H),
4.00 (s, 6H), 4.00 (s, 3H), 4.25 (br, 2H), 7.37 (s, 2H), 7.45 (d, J = 9.1
Hz, 1H), 7.66 (d, J = 9.0 Hz, 1H), 7.74 (d, J = 8.9 Hz, 1H), 8.19 (d,
J = 8.8 Hz, 1H); MS (EI) m/z 340 (100%); HRMS (EI) for
C19H20N2O4 (M+) calcd 340.1423, found 340.1423.
A
595 of the resulting solution from 1% N-lauroylsarcosine extraction. Fifty
percent growth inhibition (IC50) was calculated on the basis of the A595
of untreated cells (taken as 100%). The values shown are the means and
standard errors of at least three independent experiments performed in
duplicate.
Tubulin Polymerization in Vitro Assay.21,24 Turbidimetric assays
of microtubules were performed as described by Bollag et al.25 In brief,
microtubule-associated protein (MAP)-rich tubulin (from bovine
brain, Cytoskeleton, Inc.) was dissolved in reaction buffer [100 mM
PIPES (pH 6.9), 2 mM MgCl2, and 1 mM GTP] in preparing the 4
mg/mL tubulin solution. The tubulin solution (240 μg of MAP-rich
tubulin per well) was placed in a 96-well microtiter plate in the
presence of test compounds or 2% (v/v) DMSO as a vehicle control.
The increase in absorbance was measured at 350 nm in a PowerWave
X Microplate Reader (BIO-TEK Instruments, Winooski, VT) at 37 °C
and recorded every 30 s for 30 min. The area under the curve (AUC) was
used to determine the concentration that inhibited tubulin polymeriza-
tion by 50% (IC50). The AUCs of the untreated control and 10 μM
colchicine were set to 100 and 0% polymerization, respectively, and the IC50
was calculated by nonlinear regression in at least three experiments.
Tubulin Competition Binding Scintillation Proximity Assay.26−28 This
assay was performed in a 96-well plate. In brief, 0.08 μM [3H]colchicine
was mixed with the test compound and 0.5 μg of special long-chain
biotin-labeled tubulin (0.5 μg) and then incubated in 100 μL of
reaction buffer [80 mM PIPES (pH 6.8), 1 mM EGTA, 10% glycerol,
1 mM MgCl2, and 1 mM GTP] for 2 h at 37 °C. Then 80 μg of
streptavidin-labeled SPA beads was added to each reaction mixture.
The radioactive counts were then directly measured with a scintillation
counter.
5-Amino-6-methoxy-2-(3′,4′,5′-trimethoxyphenoxy)quinoline
(12). The title compound was obtained in 80% overall yield from
compound 30 in a manner similar to that described for the preparation
of 14: mp 209−210 °C; 1H NMR (500 MHz, DMSO) δ 3.66 (s, 3H),
3.72 (s, 6H), 3.83 (s, 3H), 5.45 (s, 2H), 6.53 (s, 2H), 6.91 (d, J = 9.0
Hz, 1H), 6.97 (d, J = 9.1 Hz, 1H), 7.33 (d, J = 9.0 Hz, 1H), 8.53 (d,
J = 9.1 Hz, 1H); MS (EI) m/z 356 (100%); HRMS (EI) for
C19H20N2O5 (M+) calcd 356.1372, found 356.1375.
5-Amino-6-methoxy-2-(3′,4′,5′-trimethoxyphenylamino)-
quinoline (13). The title compound was obtained in 68% overall yield
from compound 31 in a manner similar to that described for the
1
preparation of 14: mp 222−223 °C; H NMR (500 MHz, DMSO) δ
3.60 (s, 3H), 3.79 (s, 6H), 3.80 (s, 3H), 5.23 (s, 2H), 6.81 (d, J = 9.2
Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 7.40 (s,
2H), 8.20 (d, J = 9.2 Hz, 1H), 9.12 (s, 1H); MS (EI) m/z 355 (M+,
85%), 340 (100%); HRMS (EI) for C19H21N3O4 (M+) calcd 355.1532,
found 355.1530.
5-Amino-6-methoxy-2-(3′,4′,5′-trimethoxyphenylthio)quinoline
(14). A mixture of 32 (0.1 g, 0.25 mmol), iron powder (0.04 g,
0.75 mmol), and ammonium chloride (0.03 g, 0.5 mmol) in IPA (2.5 mL)
and water (0.6 mL) was heated to reflux for 3 h. The reaction solvent
was removed under reduced pressure, the reaction quenched with
water, and the mixture extracted with CH2Cl2. The combined organic
layer was dried over anhydrous MgSO4, concentrated under reduced
pressure, and purified by column chromatography over silica gel to
afford compound 14 (47% yield): mp 180−182 °C; 1H NMR (500 MHz,
CDCl3) δ 3.84 (s, 6H), 3.90 (s, 3H), 3.97 (s, 3H), 6.90 (s, 2H), 6.93
(d, J = 9.0 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.53 (br, 2H), 7.95 (d,
J = 9.0 Hz, 1H); MS (EI) m/z 372 (100%); HRMS (EI) for
C19H20N2O4S (M+) calcd 372.1144, found 372.1145.
Capillary Disruption Assays.22 Capillary disruption assays were
conducted in a 96-well plate format using human umbilical vein
endothelial cells (HUVECs) plated at a density of 2 × 104 cells/well in
20% FBS M199 medium containing 20 ng/mL VEGF on a Matrigel
layer (BD Biosciences). Capillaries were allowed to form over a 4 h
period before the addition of test compound or vehicle control. Images
were acquired immediately following addition of the compound and
4 h after exposure to the test compound. Tube formation was quantified
by measuring the network number of capillary structures manually by
counting under a microscope (original magnification of 100×).
5-Amino-6-methoxy-2-(3′,4′,5′-trimethoxyphenylsulfonyl)-
quinoline (15). The title compound was obtained in 76% overall yield
from compound 33 in a manner similar to that described for the
1
preparation of 14: mp 168−169 °C; H NMR (500 MHz, CDCl3) δ
3.86 (s, 3H), 3.90 (s, 6H), 4.00 (s, 3H), 7.35 (s, 2H), 7.51 (d, J = 9.0
8523
dx.doi.org/10.1021/jm201031f | J. Med. Chem. 2011, 54, 8517−8525