Design and synthesis of 2-(3-benzo[b]thienyl)-6,7-methylenedioxyquinolin-4- one analogues as potent antitumor agents that inhibit tubulin assembly

Article abstract of DOI:10.1021/jm900456w

As part of our continuing investigation of azo-flavonoid derivatives as potential anticancer drug candidates, a series of 2-aryl-6,7- methylenedioxyquinolin-4-one analogues was designed and synthesized. The design combined structural features of 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4- one (CHM-1), a previously discovered compound with potent in vivo antitumor activity, and 2-arylquinolin-4-ones, identified by CoMFA models. The newly synthesized analogues were evaluated for cytotoxicity against seven human cancer cell lines, and structure -activity relationship (SAR) correlations were established. Analogues 1, 37, and 39 showed potent cytotoxicity against different cancer cell lines. Compound 1 demonstrated selective cytotoxicity against Hep 3B (hepatoma) cells. Compound 37 was cytotoxic against HL-60 (leukemia), HCT-116 (colon cancer), Hep 3B (hepatoma), and SK-MEL-5 (melanoma) cells. Compound 39 exhibited broad cytotoxicity against all seven cancer cell lines, with IC₅₀ values between 0.07 and 0.19 μM. Results from mechanism of action studies revealed that these new quinolone derivatives function as antitubulin agents.

Full text of DOI:10.1021/jm900456w

J. Med. Chem. 2009, 52, 4883–4891 4883
DOI: 10.1021/jm900456w
Design and Synthesis of 2-(3-Benzo[b]thienyl)-6,7-methylenedioxyquinolin-4-one Analogues as Potent
Antitumor Agents that Inhibit Tubulin Assembly
Yu-Hsun Chang,† Mei-Hua Hsu,† Sheng-Hung Wang,‡ Li-Jiau Huang,† Keduo Qian,§ Susan L. Morris-Natschke,§
Ernest Hamel, Sheng-Chu Kuo,*,† and Kuo-Hsiung Lee*,§
†Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan, ‡Institute of Cellular and Organismic Biology,
Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan, §Natural Products Research Laboratories, UNC Eshelman
School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7360, and Toxicology and Pharmacology Branch,
Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes
of Health, Frederick, Maryland 21702
Received April 8, 2009
As part of our continuing investigation of azo-flavonoid derivatives as potential anticancer drug
candidates, a series of 2-aryl-6,7-methylenedioxyquinolin-4-one analogues was designed and synthe-
sized. The design combined structural features of 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-
one (CHM-1), a previously discovered compound with potent in vivo antitumor activity, and
2-arylquinolin-4-ones, identified by CoMFA models. The newly synthesized analogues were evaluated
for cytotoxicity against seven human cancer cell lines, and structure-activity relationship (SAR)
correlations were established. Analogues 1, 37, and 39 showed potent cytotoxicity against different
cancer cell lines. Compound 1 demonstrated selective cytotoxicity against Hep 3B (hepatoma) cells.
Compound 37 was cytotoxic against HL-60 (leukemia), HCT-116 (colon cancer), Hep 3B (hepatoma),
and SK-MEL-5 (melanoma) cells. Compound 39 exhibited broad cytotoxicity against all seven cancer
cell lines, with IC50 values between 0.07 and 0.19 μM. Results from mechanism of action studies revealed
that these new quinolone derivatives function as antitubulin agents.
Introduction
2-arylquinolin-4-ones,8-15 2-arylnaphthyridin-4-ones,16-18
and 2-arylquinazolin-4-ones19,20 (Figure 1). Some of these
compounds, especially analogues belonging to the 2-aryl-
naphthyridin-4-one family, demonstrated significant antitu-
mor and antitubulin activities. In general, a good correlation
was found between the cytotoxicity of these analogues and
their inhibitory effects on tubulin polymerization. Additional
mechanism of action studies revealed that the most active of
these compounds are also potent inhibitors of colchicine
binding to tubulin. Further modification of the 2-phenylqui-
nolin-4-one series was directed by computer modeling studies
combined with SAR results from previously synthesized
compounds. In the current study, we report the design and
syntheses of novel 2-arylquinolin-4-one analogues, which
showed potent and selective inhibitory activities toward dif-
ferent human cancer cell lines.
Design. Both conventional comparative molecular field
analysis (CoMFA) and g2 GRS CoMFA were used to
identify structural requirements that may be essential for
increasing the binding affinity of 2-phenylquinolin-4-one
and 2-phenylnaphthyridin-4-one analogues for the colchi-
cine site of tubulin. The training set of the models contained
51 compounds, and the cross-validated R2 (q2) values for
conventional CoMFA and g2 GRS CoMFA were 0.637 and
0.692, respectively. These QSAR models predicted that a
larger heterocyclic aromatic ring at the C-2 position should
enhance activity. The predictive power of the models was
validated by the prediction for a test set of 53 compounds
with known antitubulin potencies, and the predictive R2
values were 0.546 and 0.426, respectively. On the basis of
this study, new analogues were designed in both compound
Microtubules of eukaryotic cells are known to play an
important role in mitosis.1 Therefore, compounds that target
microtubules have long been investigated as anticancer drugs.
Typically, such compounds arrest cells, at least transiently, in
the mitotic phase of the cell cycle. Currently, two groups of
antimitotic agents are used clinically for cancer treatment.
Vinca alkaloids and estramustine inhibit tubulin polymeriza-
tion into microtubules, while taxoids and epothilones stabilize
microtubules and consequently interfere with their normal
dynamic instability.2,3 Colchicine is another well-known anti-
mitotic agent used for treating gout and inflammatory dis-
eases. However, because of its high toxicity, colchicine is not
used in cancer therapy. Combretastatin A-4, with a modified
colchicine-like structure, exhibits a lower toxicity profile while
it still targets the same binding site as colchicine.4,5 The
phosphate prodrug of combretastatin A-4 (CA-4) is currently
in phase III clinical trials as an anticancer agent.6,7 Although
several antimitotic agents are available, due to the structural
complexity of vinca alkaloids and taxoids, there is still a need
to identifynovel anticancer drugs thattarget microtubules but
have simplified structures because such compounds can be
readily prepared in a more cost-effective way.
In recent years, we have designed and synthesized three
series of azo-flavonoids as new classes of antimitotic agents:
*To whom correspondence should be addressed. For S.-C.K.: phone,
þ886-4-22053366-1006; fax, þ886-4-22030760; E-mail, sckuo@mail.
cmu.edu.tw. For K.-H.L.: phone, 1-919-962-0066; fax, 1-919-966-
3893; e-mail, khlee@email.unc.edu. Sheng-Chu Kuo and Kuo-Hsiung
Lee contributed equally to this work.
r
2009 American Chemical Society
Published on Web 07/16/2009
pubs.acs.org/jmc

4884 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Chang et al.
Figure 1. Three series of structural skeletons of azo-flavonoids: 2-arylquinolin-4-ones, 2-arylnaphthyridin-4-ones, and 2-arylquinazolin-
4-ones.
Figure 3. Structures of compound B and CHM-1.
Figure 2. Proposed models and newly designed compounds. Con-
ventional comparative molecular field analysis (CoMFA) and g2
GRS CoMFA are two computational programs performed to
identify the essential structure requirements for increasing affinity
at the colchicine site of tubulin. On the basis of the results of the
QSAR study, we proposed two concept models. As the proof of
concept, analogue A was synthesized and tested against cancer cell
lines and for binding to tubulin.
series (Figure 2).21 As a proof of concept, the newly designed
arylnaphthyridine analogue 2-(3-benzo[b]thienyl)naphthyr-
idin-4-one (A), which has a benzothienyl ring at the C-2
position of the naphthyridine, exhibited potent cytotoxicity
in the low micromolar to nanomolar concentration range.
Further, a mechanism of action study revealed that the
compound also inhibited tubulin polymerization with an
IC50 value of 0.37 μM. These results verified the success of
our modeling design.18 Thus, the same modifications were
carried out at the C-2 position of the 2-arylquinolin-4-one
Figure 4. Metabolic pathway of CHM-1-P-Na.
series in the present study.
In addition, when we reanalyzed results from our prior
in vivo antitumor study of selected 2-phenylquinolin-4-one
compounds, we found that analogues with a methylenedioxy
functional group at the C-6 and -7 positions showed superior
antitumor and safety profiles compared with C-6 monomo-
dified compounds. For instance, 2-(3-methoxyphenyl)-6-
pyrrolinylquinolin-4-one (B, Figure 3) exhibited only mod-
erate antitumor activity at the maximum tolerated dose
(MTD, 25 mg/kg, ip, once weekly for three courses) against
the OVCAR-3 xenograft model in nude mice. In con-
trast, 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one
(CHM-1, Figure 3) extended the life span of tumor-bearing
mice by 124%, 133%, and 79%, at dosages of 200, 134, and
79 mg/kg (ip, once weekly for three courses), respectively.
Additionally, the maximum tolerated dose of CHM-1 was
not reached at the highest tested dosage of 200 mg/kg.22
Currently, the phosphate prodrug of CHM-1 (CHM-1-P-
Na, Figure 4) is under preclinical study.22,23 Therefore, the
methylenedioxy ring of CHM-1 was also incorporated into
our newly designed 2-arylquinolin-4-one series, which led
to the syntheses of 2-aryl-6,7-methylenedioxyquinolin-4-one
analogues (1, 36-45). Compound 56, a ring isomer of
analogue 39, was also synthesized because ring isomerization
is a common approach in structure-activity relationship
studies of a compound series.
Chemistry. The synthetic procedure for target com-
pounds (1, 36-45) is illustrated in Scheme 1. The starting
arylcarboxylic acids (3-13) were first treated with oxalyl
chloride to form the corresponding acid chlorides (14-24),
which were then reacted with 2-amino-4,5- methylenediox-
yacetophenone (2) to give the corresponding amides (25-35).
The intermediates (25, 27-34) were subjected to a cycli-
zation reaction in t-BuOH, in the presence of t-BuOK, to
afford the corresponding 2-arylquinolin-4-one analogues
(1, 37-44). Cyclization was also carried out with 1,4-
dioxane as solvent, in the presence of NaOH, which yielded
the target products (1, 36, 39, and 45) in yields of 40-74%.
When the starting compounds (25-26, 28-32, and 34) were
treated with t-BuOK in toluene at 90 °C for 72 h, the

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4885
Scheme 1a
a Reagents and conditions: (a) toluene/DMF, 22-25 °C; (b) toluene/triethylamine, 22-25 °C; (c) t-BuOK/t-BuOH or NaOH/1,4-dioxane, reflux;
(d) t-BuOK/toluene, 90 °C, 72 h.
Scheme 2a
a Reagents and conditions: (a) toluene/triethylamine, 22-25 °C; (b) NaOH/1,4-dioxane, reflux.
corresponding deacetylated products 46-53 were obtained
unexpectedly.
yielded 4-methyl-6,7-methylenedioxy-3-(1-naphthyl)qui-
nolin-2-one (56) as the final product.
Scheme 2 depicts the synthesis of compound 56. The
starting material 2 was first treated with 1-naphthyl-
methyl chloride (54) to give the corresponding amide
(55). Compound 55 was then subjected to a cyclization
reaction in 1,4-dioxane, in the presence of NaOH, which
Results and Discussion
The newly synthesized 2-aryl-6,7-methylenedioxyquinolin-
4-ones (1, 36-45) and the ring isomer 56 were assayed for

4886 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Table 1. IC50 (μM) Values from In Vitro Cytotoxicity Testing of 1, 36-45, and 56
Chang et al.
* Not assayed
in vitro cytotoxicity against seven human cancer cell lines,
including HL-60 (leukemia), HCT-116 (colon cancer), A549
(nonsmall cell lung carcinoma), Hep 3B (hepatoma), KB
(epidermoid carcinoma of the nasopharynx), KB-VIN (p-gly-
coprotein-expressing epidermoid carcinoma of the nasophar-
ynx), and DU145 (prostate cancer). The cytotoxicity results are
summarized in Table 1. Compounds 46-53 were tested against
the HL-60, HCT-116, H226, A549, Hep G2, and A498 cancer
cell lines; however, none of the compounds showed significant
activity (IC50 values were >10 μM).
Compound 1 exhibited significant cytotoxicity against Hep
3B cells, with an IC50 value of 0.06 μM, and moderate activity
against HL-60 and HCT-116 cells, with IC50 values of 0.17
and 0.14 μM, respectively. This result, along with the prior
cytotoxicity data obtained with compound A, confirmed the
prediction generated by conventional and g2 GRS CoMFA
that a larger heterocyclic aromatic ring should be preferred
at the C-2 position. In comparison, replacement of the 3-ben-
zo[b]thienyl group (1) with a 2-benzo[b]thienyl group (36)
resulted in significantly reduced cytotoxicity, demonstrating
the importance of the exact linkage of the aromatic ring
moiety.
Bioisosteric replacement of 3-benzo[b]thienyl (1) with 3-ben-
zo[b]furanyl (37) led to increased potency against two cancer
cell lines. Specifically, compared with 1, the cytotoxic activity
of 37 increased 5-fold against HL-60 (IC50 0.03 μM) and 3-fold
against HCT-116 (IC50 0.05 μM) cells. The two compounds
had similar potency against Hep 3B cells. Most importantly, 37
retained potency (IC50: 0.59 μM) against KB-VIN cells, a
vincristine-resistant epidermoid carcinoma of the nasophar-
ynx cell line, compared with KB cells (IC50 1.05 μM). As with
1 and 36, moving the attachment of the benzofuran moiety
from 30 in 37 to 20 in 38 was detrimental to cytotoxic activity.
Compound 39, with a 1-naphthyl group rather than a 3-ben-
zo[b]thienyl group (1) at the C-2 position, exhibited potent
cytotoxicity toward all seven cancer cell lines, with IC50 values
ranging from 0.07 to 0.19 μM. It should be noted that although
39 showed similar activity toward the HL-60, HCT-116, and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4887
Table 2. Anti-Tubulin Data for CA-4, 1, and 37-45
inhibition of tubulin assembly a IC50 (μM) ( SD
compd
inhibition of colchicine binding b % inhibition at tested concentration
1 μM
5 μM
CA-4
1
1.2 ( 0.2
0.76 ( 0.06
0.58 ( 0.02
>4
91
25
39
99
61
51
37
38
39
40
41
42
43
44
45
0.64 ( 0.07
>40
>40
33
67
>4
>40
27 ( 3
>40
a Assembly assay contained 10 μM tubulin. b Colchicine binding assay contained 1 μM tubulin and 5 μM [3H]colchicine.
Hep 3B cancer cell lines as compared with 1 and 37, it showed
significantly increased cytotoxicity toward A549, KB, KB-
VIN, and DU145 cells. Compound 40, the ring positional
isomer of 39, was much less cytotoxic, as was the case with
compounds 36 and 38.
Surprisingly, cytotoxicity was reduced significantly when
the C-2 aryl moiety was a quinoline group, no matter how it
was attached (41-44). In addition, expanding the naphtha-
lene ring of 39 to an anthracene (45) lowered the cytotoxicity
remarkably. Thus, we speculate that the size of the C-2
substitutedaromatic ringplaysanimportantrolein antitumor
activity and that the binding pocket for this portion of the
drug molecule is quite small.
Compound 56 was designed as a ring isomer of 39. The
in vitro bioassay data showed that moving the aromatic ring to
C-3 and the carbonyl to C-2 to give a cyclic amide completely
abolished cytotoxicity of the compound. Noncyclic amide
analogues 46-53 also exhibited poor in vitro activity.
Figure 5. Dose-response curves of 37 against different leukemia
cell lines.
In addition to the in vitro cytotoxicity study, we also
performed initial mechanism of action studies with the newly
synthesized 2-aryl-6,7-methylenedioxyquinolin-4-one analo-
gues (1, 37-45). As noted above, 2-arylquinolin-4-ones are
azo-flavonoids, which were shown to inhibit tubulin assembly
and the binding of colchicine to tubulin. Therefore, com-
pounds 1 and 37-45 were tested for their in vitro activities in
these assays in comparison with combretastatin A-4 (CA-4).
The results are summarized in Table 2. The data showed that
1, 37, and 39, the three compounds with the greatest in vitro
cytotoxicity, were potent inhibitors of tubulin assembly, with
IC50 values of 0.76, 0.58, and 0.64 μM, respectively. Although
all three compounds were better assembly inhibitors than
CA-4 (IC50 1.2 μM), they were less effective than CA-4 in
inhibiting colchicine binding to tubulin.
Among the active analogues, 37, which showed potent
cytotoxicity and reasonable solubility, was chosen for sub-
mission to the National Cancer Institute (NCI, USA) for
further screening against 60 human tumor cell lines. In pre-
liminary screening, 37 showed potent selective cytotoxicity
against many leukemia cell lines (Figure 5). In addition, it also
significantly inhibited the growth of several colon cancer
(HCC-2998 and KM-12), CNS cancer (SF-539 and SNB-
75), melanoma (SK-MEL-5), and ovarian cancer (IGROV1
and OVCAR-3) cell lines. On the basis of these results, 37
appears to be an attractive candidate for further development
as a potential anticancer agent in the clinic.
Furthermore, results from a pharmacokinetic study in a
mice model revealed that the 6,7-methylenedioxy moiety
of CHM-1-P-Na is metabolized to an ortho-quinone (D,
Figure 4, unpublisheddata). It is knownthatthepara-quinone
moiety in mitomycin C can be subjected to one-electron
reduction by NADPH-cytochrome C (P450) reductase to
form the corresponding semiquinone radical anion.24 We
postulate that similar reduction of the ortho-quinone moiety
of D may take place to form the radical anion product E,
which may be further metabolized or broken down into more
cytotoxic metabolites in hypoxic cells. Because severe hypoxia
is a common property of locally advanced solid tumors, this
postulate may explain our finding that 6,7-methylenedioxy-
quinoline analogues (e.g., CHM-1) showed enhanced in vivo
activityprofiles compared with 6-monosubstituted analogues.
In conclusion, a series of 2-aryl-6,7-methylenedioxyquino-
lin-4-one analogues (1, 36-45) were designed, synthesized,
and evaluated for in vitro cytotoxicity. This design combined
two structural features: larger heterocyclic aromatic rings at
the C-2 position as predicted to be favorable by CoMFA
models and a 6,7-methylenedioxy moiety rather than C-6
monosubstituted analogue based on the improved cytotoxi-
city of CHM-1 and the metabolic pathway of CHM-1-P-Na.
In our studies, 1 showed selective cytotoxicity against Hep 3B
cells, 37 was active against HL-60, HCT-116, Hep 3B, and
KB-VIN cells, and 39 had potent cytotoxicity against all seven
cancer celllines, with IC50 values between 0.07and 0.19μM. A
mechanism of action study demonstrated that 1, 37, and 39
also function as antitubulin agents. Compound 37 was further
selected for evaluation against 60 human cancer cell lines and

4888 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Chang et al.
was active against several types of cancer cells. The significant
in vitro cytotoxicity of 37 and 39 suggested that they can be
further developed as anticancer drugs.
N-(6-Acetyl-1,3-benzodioxol-5-yl)naphthalene-1-carboxamide (29).
Obtained as a grayish-white solid from 2 and naphthalene-1-
carbonyl chloride (18); mp 143-144 °C; MS (ESI) 334 m/z [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6, δ): 2.59 (3H, s), 6.20 (2H, s),
7.60-7.68 (4H, m), 7.87 (1H, d, J= 7.2 Hz), 8.05-8.07 (1H, m), 8.15
(1H, d, J = 8.0 Hz), 8.33-8.38 (2H, m), 12.52 (1H, s). IR (KBr):
Experimental Section
1647, 1672 (CdO) cm-1
.
Chemistry. General Experimental Procedures. Reagents and
solvents were obtained commercially and used without further
purification. Reactions were monitored by thin-layer chroma-
tography, using Merck plates with fluorescent indicator (TLC
Silica Gel 60 F254). Flash column chromatography was per-
formed on silica gel (Merck Silica Gel 60, 40-63 μm) using a
mixture of CH2Cl2 and EtOH as eluant. Melting points were
determined on a Yanaco MP-500D melting point apparatus and
were uncorrected. IR spectra were recorded on a Shimadzu
IRPrestige-21 spectrophotometer as KBr pellets. 1H NMR
spectra were obtained on a Bruker NMR AV 400 spectrometer
in DMSO. The following abbreviations are used: s, singlet; d,
doublet; t, triplet; m, multiplet; dd, double doublet; td, triple
doublet; ddd, double double doublet. EI-MS spectra were
measured with an HP 5995 GC-MS instrument. ESI-MS
spectra were measured with a Bruker HCT ultra PTM Discov-
ery system (Proteineer fc, UltiMate 3000). Elemental analyses
(C, H, and N) were performed on a Perkin-Elmer 2400 Series II
CHNS/O analyzer, and the results were within (0.4% of the
calculated values.
Preparation of Arylcarbonyl Chlorides (14-24). Arylcar-
boxylic acids (3-13) were suspended in dry toluene (150 mL)
at 20 ( 2 °C. Oxalyl chloride (2.2 equiv) was added dropwise.
The reaction mixtures were stirred for 30 min at 20 ( 2 °C and
then DMF (2 drops) was added. The mixtures were stirred for
6 h and then evaporated to dryness. The residues were washed
with petroleum ether and used directly in the next step.
Preparation of Carboxamides (25-35, 55). Into solutions of
14-24, 54 (5.1 mmol) in 200 mL of dry toluene were added
triethylamine (4 mL) and 2-amino-4,5-methylenedioxy aceto-
phenone (2) (5 mmol). The mixtures were stirred at 20 ( 2 °C for
24 h and then evaporated. The residues were washed with
acetone and EtOH and then recrystallized from acetone or
EtOH to form the pure carboxamides.
N-(6-Acetyl-1,3-benzodioxol-5-yl)naphthalene-2-carboxamide (30).
Obtained as a pale-yellow solid from 2 and naphthalene-2-carbonyl
1
chloride (19); mp 172-173 °C; MS (ESI) 334 m/z [M þ H]þ. H
NMR (400 MHz, DMSO-d6, δ): 2.68 (3H, s), 6.19 (2H, s), 7.64-7.72
(3H, m), 7.99-8.06 (2H, m), 8.15 (2H, d, J = 8.8 Hz), 8.42 (1H, s),
8.58 (1H, s), 13.09 (1H, s). IR (KBr): 1636, 1670 (CdO) cm-1
.
N-(6-Acetyl-1,3-benzodioxol-5-yl)quinoline-4-carboxamide (31).
Obtained as a grayish-white solid from 2 and quinoline-4-carbo-
nyl chloride (20); mp 166-167 °C;MS(ESI)335m/z [M þ H]þ. 1H
NMR (400 MHz, DMSO-d6, δ): 2.59 (3H, s), 6.21 (2H, s), 7.69
(1H, s), 7.72 (1H, ddd, J = 1.2, 7.2 Hz), 7.81 (1H, d, J = 4.4 Hz),
7.87 (1H, ddd, J = 1.2, 7.4 Hz), 8.15 (1H, d, J = 8.4 Hz), 8.24 (1H,
s), 8.31 (1H, d, J = 8.4 Hz), 9.09 (1H, d, J = 4.2 Hz), 12.48 (1H, s).
IR (KBr): 1645, 1680 (CdO) cm-1
.
N-(6-Acetyl-1,3-benzodioxol-5-yl)quinoline-3-carboxamide (32).
Obtained as a pale-yellow solid from 2 and quinoline-3-carbonyl
chloride (21); mp 215-216 °C; MS (ESI) 335 m/z [M þ H]þ. 1H
NMR (400 MHz, DMSO-d6, δ): 2.66 (3H, s), 6.21 (2H, s), 7.73
(1H, s), 7.76 (1H, ddd, J = 1.2, 7.4 Hz), 7.94 (1H, ddd, J = 1.6, 6.8
Hz), 8.15 (1H, d, J = 8.4 Hz), 8.23 (1H, d, J = 7.2 Hz), 8.34 (1H,
s), 8.92 (1H, s), 9.38 (1H, s), 12.05 (1H, s). IR (KBr): 1639, 1670
(CdO) cm-1
.
N-(6-Acetyl-1,3-benzodioxol-5-yl)quinoline-2-carboxamide (33).
Obtained as a yellow solid from 2 and quinoline-2-carbonyl
chloride (22); mp 210-211 °C; MS (ESI) 335 m/z [M þ H]þ. 1H
NMR (400 MHz, DMSO-d6, δ): 2.69 (3H, s), 6.21 (2H, s), 7.73
(1H, s), 7.79 (1H, ddd, J = 1.2, 7.2 Hz), 7.95 (1H, ddd, J = 1.6, 7.6
Hz), 8.15 (1H, d, J= 7.2 Hz), 8.22 (1H, d, J= 8.0 Hz), 8.28 (1H, d,
J = 8.8 Hz), 8.55 (1H, s), 8.65-8.68 (1H, d, J = 8.8). IR (KBr):
1647, 1670 (CdO) cm-1
.
N-(6-Acetyl-1,3-benzodioxol-5-yl)quinoline-5-carboxamide (34).
Obtained as a yellow solid from 2 and quinoline-5-carbonyl
1
chloride (23); mp 210-211 °C; MS (ESI) 335 m/z [MþH]þ. H
NMR (400 MHz, DMSO-d6, δ): 2.61 (3H, s), 6.21 (2H, s), 7.70
(1H, s), 7.82 (1H, dd, J=4.4, 8.8 Hz), 8.03 (1H, t, J=7.8 Hz), 8.11
(1H, d, J=7.2 Hz), 8.30-8.35 (2H, m), 9.03 (1H, d, J=8.4 Hz),
9.13 (1H, d, J = 4.4 Hz), 12.59 (1H, s). IR (KBr): 1636, 1672
N-(6-Acetyl-1,3-benzodioxol-5-yl)-1-benzothiophene-3-carbox-
amide (25). Obtained as a pale-yellow solid from 2-amino-4,5-
methylenedioxyacetophenone (2) and benzo[b]thiophene-3-car-
bonyl chloride (14); mp 213-214 °C; MS (ESI) 340 m/z [M þ H]þ.
1H NMR (400 MHz, DMSO-d6, δ): 2.63 (3H, s), 6.18 (2H, s),
7.43-7.53 (2H, m), 7.66 (1H, s), 8.10 (1H, dd, J = 1.2, 7.2 Hz),
8.30 (1H, s), 8.47 (1H, dd, J = 1.2, 7.2 Hz), 8.53 (1H, s), 12.75
(CdO) cm-1
.
N-(6-Acetyl-1,3-benzodioxol-5-yl)anthracene-1-carboxamide (35).
Obtained as a pale-yellow solid from 2 and anthracene-1-carbonyl
chloride (24); mp 206-207 °C; MS (ESI) 384 m/z [M þ H]þ. 1H
NMR (400 MHz, DMSO-d6, δ): 2.59 (3H, s), 6.21 (2H, s), 7.55-
7.61 (2H, m), 7.62-7.67 (1H, m), 7.70 (1H, s), 7.90 (1H, d, J =
6.8 Hz), 8.13-8.16 (2H, m), 8.32 (1H, d, J = 8.4 Hz), 8.46 (1H, s),
8.73 (1H, s), 9.03 (1H, s), 12.60 (1H, s). IR (KBr): 1643, 1674
(1H, s). IR (KBr): 1638, 1668 (CdO) cm-1
.
N-(6-Acetyl-1,3-benzodioxol-5-yl)-1-benzothiophene-2-carbox-
amide (26). Obtained as a grayish-white solid from 2 and benzo
[b]thiophene-2-carbonyl chloride (15); mp 233-235 °C; MS
1
(ESI) 340 m/z [M þ H]þ. H NMR (400 MHz, DMSO-d6, δ):
(CdO) cm-1
.
2.69 (3H, s), 6.19 (2H, s), 7.46-7.57 (2H, m), 7.71 (1H, s), 8.09-
8.13 (2H, m), 8.14 (1H, s), 8.25 (1H, s), 13.11 (1H, s). IR (KBr):
N-(6-Acetyl-1,3-benzodioxol-5-yl)-2-(naphthalen-1-yl)acetamide
(55). Obtained as a white solid from 2 and 1-naphthylmethyl
1640, 1655 (CdO) cm-1
.
1
chloride (54); mp 89-90 °C; MS (ESI) 384 m/z [M þ H]þ. H
N-(6-Acetyl-1,3-benzodioxol-5-yl)-1-benzofuran-3-carboxamide
(27). Obtained as a pale-yellow solid from 2 and benzo[b]furan-3-
carbonyl chloride (16); mp 144-145 °C; MS (ESI) 324 m/z [M þ
H]þ. 1H NMR (400 MHz, DMSO-d6, δ):2.63 (3H, s), 6.19 (2H, s),
7.41-7.50 (2H, m), 7.68 (1H, s), 7.75 (1H, dd, J = 1.6, 6.8 Hz),
8.15 (1H, dd, J = 2.0, 8.8 Hz), 8.27 (1H, s), 8.71 (1H, s), 12.63 (1H,
NMR (400 MHz, DMSO-d6, δ): 2.53 (3H, s), 4.21 (2H, s) 6.13 (2H,
s), 7.50-7.61 (5H, m), 7.90 (1H, d, J = 8.0 Hz), 7.95 (1H, dd, J =
2.0, 7.2 Hz), 8.03 (1H, dd, J = 1.2, 8.0 Hz), 8.15 (1H, s), 11.89 (1H,
s). IR (KBr): 1641, 1684 (CdO) cm-1
.
Preparation of 2-Aryl-6,7-methylenedioxyquinolin-4-ones (1,
37-44). Method A. Into a suspension of 25, 27-34 (2.95 mmol)
in t-butyl alcohol (100 mL) was added potassium t-butoxide
(1.66 g, 14.7 mmol). The mixture was refluxed under argon for
12 h, cooled, and poured into a 10% ammonium chloride
solution (100 mL). The solid precipitate was collected and
washed with EtOH. The crude product was purified by flash
chromatography (silica gel, CH2Cl2:EtOH 16:1-10:1).
s). IR (KBr): 1635, 1677 (CdO) cm-1
.
N-(6-Acetyl-1,3-benzodioxol-5-yl)-1-benzofuran-2-carboxa-
mide (28). Obtained as a grayish-white solid from 2 and ben-
zo[b]thiophene-2-carbonyl chloride (17); mp 184-185 °C;
1
MS (ESI) 324 m/z [M þ H]þ. H NMR (400 MHz, DMSO-
d6, δ): 2.69 (3H, s), 6.20 (2H, s), 7.40 (1H, t, J = 7.6 Hz), 7.55
(1H, td, J = 1.2, 7.8 Hz), 7.72-7.78 (3H, m), 7.84 (1H, d, J =
8.0 Hz), 8.36 (1H, s) 13.21 (1H, s). IR (KBr): 1667, 1682
2-(3-Benzo[b]thienyl)-6,7-methylenedioxyquinolin-4-one (1).
Yield 35% from 25 as a white solid; mp >330 °C; MS (ESI)
(CdO) cm-1
.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4889
322 m/z [M þ H]þ. 1H NMR (DMSO-d6, δ): 6.15 (2H, s), 6.19
(1H, s), 7.11 (1H, s), 7.43 (1H, s), 7.47-7.54 (2H, m), 7.93 (1H, d,
J = 7.6 Hz), 8.14 (1H, d, J = 7.6 Hz), 8.24 (1H, s), 11.80 (1H, s).
IR (KBr): 1616 (CdO) cm-1. Anal. Calcd for C18H11NO3S:
C, 67.00; H, 3.48; N, 4.25. Found: C, 67.28; H, 3.45; N, 4.36.
2-(3-Benzo[b]furyl)-6,7-methylenedioxyquinolin-4-one (37).
Yield 17% from 27 as a pale-yellow solid; mp >315 °C; MS
(ESI) m/z 306 [M þ H]þ. 1H NMR (DMSO-d6, δ): 6.12 (2H, s),
6.49 (1H, s), 7.13 (1H, s), 7.36-7.45 (3H, m), 7.69 (1H, d, J =
described in method A, except for the use of 1,4-dioxane as
solvent in place of t-butyl alcohol, and the use of NaOH in place
of potassium t-butoxide. Compounds 1 and 39 were confirmed
by comparison of mp and TLC with those of a sample obtained
from method A.
2-(3-Benzo[b]thienyl)-6,7-methylenedioxyquinolin-4-one (1). Yield
40% from 25 as a pale-yellow solid.
2-(2-Benzo[b]thienyl)-6,7-methylenedioxyquinolin-4-one (36).
Yield 74% from 26 as a white solid; mp >350 °C; MS (ESI)
m/z 322 [M þ H]þ. 1H NMR (DMSO-d6, δ): 6.20 (2H, s), 7.21
(1H, s), 7.39-7.45 (3H, m), 7.96-8.16 (3H, m), 11.55 (1H, s). IR
(KBr): 1616 (CdO) cm-1. Anal. Calcd for C18H11NO3S: C,
67.00; H, 3.48; N, 4.25. Found: C, 67.21; H, 3.47; N, 4.33.
2-(1-Naphthalenyl)-6,7-methylenedioxyquinolin-4-one (39). Yield
58% from 29 as a grayish-white solid.
8.0 Hz), 8.14 (1H, s), 8.52 (1H, s). IR (KBr): 1626 (CdO) cm-1
.
Anal. Calcd for C18H11NO4: C, 70.82; H, 3.63; N, 4.59. Found:
C, 70.52; H, 3.95; N, 4.21.
2-(2-Benzo[b]furyl)-6,7-methylenedioxyquinolin-4-one (38).
Yield 28% from 28 as a grayish-white solid; mp >320 °C;
1
MS (EI, 70 eV) m/z 305 [M]þ. H NMR (DMSO-d6, δ): 6.19
(2H, s), 6.75 (1H, s), 7.30 (1H, s), 7.35 (1H, t, J = 7.6 Hz), 7.40
(1H, s), 7.45 (1H, t, J = 7.6 Hz), 7.71-7.79 (3H, m). IR (KBr):
1630 (CdO) cm-1. Anal. Calcd for C18H11NO4: C, 70.82; H,
3.63; N, 4.59. Found: C, 70.62; H, 3.84; N, 4.24.
2-(1-Naphthalenyl)-6,7-methylenedioxyquinolin-4-one (39).
Yield 52% from 29 as a grayish-white solid; mp >350 °C;
MS (ESI) m/z 316 [M þ H]þ. 1H NMR (DMSO-d6, δ): 6.08 (1H,
s), 6.15 (2H, s), 7.03 (1H, s), 7.46 (1H, s), 7.56-7.63 (2H, m),
7.63-7.70 (2H, m), 7.83 (1H, d, J = 7.6 Hz), 8.06 (1H, d, J =
7.6 Hz), 8.11 (1H, d, J = 7.6 Hz), 11.90 (1H, s). IR (KBr): 1653
(CdO) cm-1. Anal. Calcd for C20H13NO3: C, 76.18; H, 4.16; N,
4.44. Found: C, 75.60; H, 3.94; N, 4.29.
2-(2-Naphthalenyl)-6,7-methylenedioxyquinolin-4-one (40).
Yield 48% from 30 as a white solid; mp >330 °C; MS (ESI) m/z
316 [M þ H]þ. 1H NMR (DMSO-d6, δ): 6.16 (2H, s), 6.49 (1H,
s), 7.25 (1H, s), 7.42 (1H, s), 7.59-7.64 (2H, m), 7.88-7.95 (1H,
m), 8.00-8.03 (1H, m), 8.06-8.12 (2H, m), 8.42 (1H, s). IR
(KBr): 1616 (CdO) cm-1. Anal. Calcd for C20H13NO3: C, 76.18;
H, 4.16; N, 4.44. Found: C, 76.04; H, 4.28; N, 4.28.
2-(1-Anthracenyl)-6,7-methylenedioxyquinolin-4-one (45). Yield
48% yield from 35 as a yellow solid; mp >320 °C; MS (ESI) m/z
366 [M þ H]þ. 1H NMR (DMSO-d6, δ): 6.15 (3H, s), 7.03 (1H, s),
7.43-7.70 (5H, m), 8.08 (2H, t, J = 7.6 Hz), 8.25 (1H, d, J = 8.4
Hz), 8.47 (1H, s), 8.70 (1H, s), 11.98 (1H, s). IR (KBr): 1632
(CdO) cm-1. Anal. Calcd for C24H15NO3: C, 78.89; H, 4.14;
N, 3.83. Found: C, 78.24; H, 4.40; N, 3.37.
4-Methyl-6,7-methylenedioxy-3-(1-naphthyl)quinolin-2-one (56).
Yield 61% from 55 as a white solid; mp >260 °C; MS (EI, 70 eV)
m/z 329 [M]þ. 1H NMR (DMSO-d6, δ): 2.02 (3H, s), 6.13 (2H, s),
6.91 (1H, s), 7.29-7.32 (2H, m), 7.39-7.47 (2H, m), 7.52 (1H, ddd,
J = 1.6, 6.4 Hz), 7.58 (1H, t, J =8.0 Hz), 7.93-8.01 (2H, m), 11.84
(1H, s). IR(KBr): 1632 (CdO) cm-1. Anal. Calcd for C21H15NO3:
C, 76.58; H, 4.59; N, 4.25. Found: C, 75.88; H, 5.07; N, 4.06.
Deacetylation of Compounds 25, 26, 28-32, and 34. Into a
suspension of compound (2.95 mol) in dry toluene (150 mL) was
added potassium t-butoxide (1.66 g, 14.75 mol). The mixture
was heated under argon at 90 °C for 72 h, and then concentrated
and neutralized with 20% HOAc The resulting solid precipitate
was collected and purified by flash chromatography (silica gel,
CH2Cl2-EtOH) to afford 46-53.
2-(4-Quinolinyl)-6,7-methylenedioxyquinolin-4-one (41). Yield
54% from 31 as a pale-yellow solid; mp >320 °C; MS
1
(ESI) m/z 317 [M þ H]þ. H NMR (DMSO-d6, δ): 6.12-6.29
N-(1,3-Benzodioxol-5-yl)-1-benzothiophene-3-carboxamide (46).
Yield 28% from 25 as a white solid; mp 188-189 °C; MS (EI,
70 eV) m/z 297 [M]þ. 1H NMR (DMSO-d6, δ): 6.01 (2H, s), 6.90
(1H, d, J = 8.4 Hz), 7.17 (1H, dd, J = 2.0, 8.4Hz), 7.40-7.51 (3H,
m), 8.06 (1H, dd, J = 1.2, 7.2 Hz), 8.37 (1H, dd, J = 0.8, 8.0 Hz),
8.46 (1H, s), 10.29 (1H, s). IR (KBr): 1647 (CdO) cm-1. Anal.
Calcd for C16H11NO3S: C, 64.63; H, 3.73; N, 4.71. Found: C,
64.32; H, 3.94; N, 4.50.
(3H, m), 7.06 (1H, s), 7.47 (1H, s), 7.65-7.70 (2H, m), 7.86 (1H,
t, J = 7.6 Hz), 7.93 (1H, d, J = 8.4 Hz), 8.15 (1H, d, J = 8.0 Hz),
9.03 (1H, s). IR (KBr): 1618 (CdO) cm-1. Anal. Calcd for
C19H12N2O3: C, 72.15; H, 3.82; N, 8.86. Found: C, 72.35; H,
4.03; N, 8.60.
2-(3-Quinolinyl)-6,7-methylenedioxyquinolin-4-one (42). Yield
65% from 32 as a grayish-white solid; mp >320 °C; MS
(ESI) m/z 317 [M þ H]þ. 1H NMR (DMSO-d6, δ): 6.19 (2H, s),
6.62 (1H, s), 7.23 (1H, s), 7.44 (1H, s), 7.73 (1H, t, J = 7.6 Hz),
7.87 (1H, t, J = 7.6 Hz), 8.11-8.17 (2H, m), 8.88 (1H, s), 9.38
(1H, s). IR (KBr): 1618 (CdO) cm-1. Anal. Calcd for
C19H12N2O3: C, 72.15; H, 3.82; N, 8.86. Found: C, 71.86; H,
3.76; N, 8.63.
2-(2-Quinolinyl)-6,7-methylenedioxyquinolin-4-one (43). Yield
48% from 33 as a pale-yellow solid. mp 345-347 °C; MS (EI,
70 eV) m/z 316 [M]þ. 1H NMR (DMSO-d6, δ): 6.18 (2H, s), 6.98
(1H, s), 7.43 (1H, s), 7.63 (1H, s), 7.73 (1H, t, J = 7.6 Hz), 7.91
(1H, t, J = 7.6 Hz), 8.11 (1H, d, J = 8.0 Hz), 8.27 (1H, d,
J = 8.4 Hz), 8.32 (1H, d, J = 8.8 Hz), 8.60 (1H, d, J = 8.8 Hz),
11.85 (1H, s). IR (KBr): 1611 (CdO) cm-1. Anal. Calcd for
C19H12N2O3: C, 72.15; H, 3.82; N, 8.86. Found: C, 71.76; H, 3.88;
N, 8.46.
N-(1,3-Benzodioxol-5-yl)-1-benzothiophene-2-carboxamide (47).
Yield 36% from 26 as a grayish-white solid; mp 171-173 °C; MS
(EI, 70 eV) m/z297 [M]þ.1H NMR (DMSO-d6,δ): 6.01 (2H, s), 6.91
(1H, d, J = 8.4 Hz), 7.15 (1H, dd, J = 2.0, 8.4 Hz), 7.38 (1H, d, J =
2.4 Hz), 7.41-7.54 (2H, m), 7.96-8.05 (2H, m), 8.28 (1H, s), 10.41
(1H, s). IR (KBr): 1634 (CdO) cm-1. Anal. Calcd for C16H11NO3S:
C, 64.63; H, 3.73; N, 4.71. Found: C, 64.28; H, 3.98; N, 4.46.
N-(1,3-Benzodioxol-5-yl)-1-benzofuran-2-carboxamide (48).
Yield 28% from 28 as a yellow solid; mp 156-158 °C; MS
(EI, 70 eV) m/z 281 [M]þ. 1H NMR (DMSO-d6, δ): 6.03 (2H, s),
6.93 (1H, d, J = 8.4 Hz), 7.27 (1H, dd, J = 2.0, 8.4 Hz), 7.37 (1H,
t, J = 7.6 Hz), 7.47 (1H, d, J = 2.0 Hz), 7.51 (1H, ddd, J = 2.0,
8.4 Hz), 7.71-7.75 (2H, m), 7.82 (1H, d, J = 8.0 Hz), 10.49 (1H,
s). IR (KBr): 1668 (CdO) cm-1. Anal. Calcd for C16H11NO4: C,
68.32; H, 3.94; N, 4.98. Found: C, 68.22; H, 4.14; N, 4.22.
N-(1,3-Benzodioxol-5-yl)naphthalene-1-carboxamide (49).
Yield 32% from 29 as a white solid; mp 204-206 °C; MS (EI, 70
2-(5-Quinolinyl)-6,7-methylenedioxyquinolin-4-one (44). Yield
47% from 34 as a pale-yellow solid; mp >330 °C; MS (ESI) m/z
1
1
317 [M þ H]þ. H NMR (DMSO-d6, δ): 6.03-6.19 (3H, m),
eV) m/z 291 [M]þ. H NMR (DMSO-d6, δ): 6.02 (2H, s), 6.91
7.03 (1H, s), 7.47 (1H, s), 7.60 (1H, dd, J = 4.0, 8.4 Hz), 7.81 (1H,
d, J = 6.8 Hz), 7.91 (1H, t, J = 7.8 Hz), 8.20 (1H, d, J = 8.4 Hz),
8.30 (1H, d, J = 6.4 Hz), 8.99 (1H, s), 11.91 (1H, s). IR (KBr):
1614 (CdO) cm-1. Anal. Calcd for C19H12N2O3: C, 72.15; H,
3.82; N, 8.86. Found: C, 72.08; H, 3.94; N, 8.66.
(1H, d, J = 8.4 Hz), 7.22 (1H, dd, J = 2.0, 8.4 Hz), 7.51 (1H, d,
J = 2.0 Hz), 7.56-7.63 (3H, m), 7.72 (1H, d, J = 6.4 Hz), 7.99-
8.03 (1H, m), 8.06 (1H, d, J = 8.4 Hz), 8.16-8.20 (1H, m), 10.46
(1H, s). IR (KBr): 1643 (CdO) cm-1. Anal. Calcd for
C18H13NO3: C, 74.22; H, 4.50; N, 4.81. Found: C, 73.83; H,
4.82; N, 4.26.
Preparation of 2-Aryl-6,7-methylenedioxyquinolin-4-ones (1,
36, 39, 45) and 4-Methyl-6,7-methylenedioxy-3-(1-naphthyl)qui-
nolin-2-one (56). Method B. Following the same procedure
N-(1,3-Benzodioxol-5-yl)naphthalene-2-carboxamide (50).
Yield 21% yield from 30 as a pale-yellow solid; mp 184-185 °C;

4890 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Chang et al.
MS (EI, 70 eV) m/z 291 [M]þ. 1H NMR (DMSO-d6, δ): 6.02 (2H,
s), 6.94 (1H, d, J = 8.4 Hz), 7.24 (1H, dd, J = 2.0, 8.4 Hz), 7.50
(1H, d, J = 2.0 Hz), 7.60-7.68 (2H, m), 7.98-8.15 (4H, m), 8.56
(1H, s), 10.38 (1H, s). IR (KBr): 1636 (CdO) cm-1. Anal. Calcd
for C18H13NO3: C, 74.22; H, 4.50; N, 4.81. Found: C, 74.19; H,
4.62; N, 4.56.
N-(1,3-Benzodioxol-5-yl)quinoline-4-carboxamide (51). Yield
31% from 31 as a white solid; mp 167-168 °C; MS (EI, 70 eV)
m/z 292 [M]þ. 1H NMR (DMSO-d6, δ): 6.02 (2H, s), 6.93 (1H,
d, J = 8.4 Hz), 7.16 (1H, dd, J = 1.6, 8.4 Hz), 7.43 (1H, d, J =
2.0 Hz), 7.66-7.72 (2H, m), 7.84 (1H, t, J = 8.0 Hz), 8.11 (2H,
d, J = 8.8 Hz), 9.01 (1H, s), 10.65 (1H, s). IR (KBr): 1636
(CdO) cm-1. Anal. Calcd for C17H12N2O3: C, 69.86; H, 4.14;
N, 9.58. Found: C, 69.65; H, 4.32; N, 9.34.
N-(1,3-Benzodioxol-5-yl)quinoline-3-carboxamide (52). Yield
23% from 32 as a yellow solid; mp 254-256 °C; MS (EI, 70 eV)
m/z 292 [M]þ. 1H NMR (DMSO-d6, δ): 6.05 (2H, s), 6.95 (1H,
d, J = 8.4 Hz), 7.23 (1H, dd, J = 2.0, 8.4 Hz), 7.49 (1H, d, J =
2.0 Hz), 7.74 (1H, ddd, J = 1.2, 8.0 Hz), 7.90 (1H, ddd, J = 1.6,
8.4 Hz), 8.10-8.18 (2H, m), 8.92 (1H, s), 9.33 (1H, s), 10.56
(1H, s). IR (KBr): 1659 (CdO) cm-1. Anal. Calcd for
C17H12N2O3: C, 69.86; H, 4.14; N, 9.58. Found: C, 69.73; H,
4.35; N, 9.42.
N-(1,3-Benzodioxol-5-yl)quinoline-5-carboxamide (53). Yield
20% from 34 as a white solid; mp 202-203 °C; MS (EI, 70 eV)
m/z 292 [M]þ. 1H NMR (DMSO-d6, δ): 6.02 (2H, s), 6.95 (1H,
d, J = 8.4 Hz), 7.21 (1H, dd, J = 2.0, 8.0 Hz), 7.50 (1H, d, J =
2.0 Hz), 7.63 (1H, dd, J = 4.0, 8.4 Hz), 7.83-7.90 (2H, m),
8.16-8.20 (1H, m), 8.63 (1H, d, J = 8.0 Hz), 8.98 (1H, dd, J =
1.6, 4.0 Hz), 10.58 (1H, s). IR (KBr): 1643 (CdO) cm-1. Anal.
Calcd for C17H12N2O3: C, 69.86; H, 4.14; N, 9.58. Found: C,
69.68; H, 4.33; N, 9.52.
due to support (in part) by grant CA 17625 from the National
Cancer Institute, NIH (K. H. Lee).
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Biological Evaluations. MTT (3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) Assays.25,26 HL-60, HCT-116,
and Hep 3B cells were treated with tested compounds for the
indicated periods. After treatment, cells were washed once with
PBS andincubated with MTT (Sigma, St. Louis, MO) for 2 h. The
formazan precipitate was dissolved in 150 μL of DMSO, and the
absorbance was measured with an ELISA reader at 570 nm.
SRB Assays.27 A549, KB, KB-VIN, and DU145 cells were
treated with tested compounds for the indicated periods. After
additional incubation with DMSO or compounds, cells were
fixed with 10% TCA, and SRB at 0.4% (w/v) in 1% acetic acid
was added to stain cells. Unbound SRB was washed out with 1%
acetic acid, and SRB bound to the cells was solubilized with
10 mM Trizma base. Absorbance was read at 515 nm.
Tubulin Assays.25,26 Tubulin assembly was measured by
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contained 1.0 mg/mL (10 μM) tubulin and varying drug con-
centrations and were preincubated 15 min at 30 °C in the
absence of GTP. The samples were placed on ice, and 0.4 mM
GTP was added. Reaction mixtures were transferred to cuvettes
held at 0 °C, and turbidity development was followed for 20 min
at 30 °C after a rapid temperature jump. Drug concentrations
that inhibited increase in turbidity by 50% relative to a control
sample were determined.
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Acknowledgment. We acknowledge special appreciation
and esteem for support received from Wen-Chih Chang,
who passed away because of nasopharyngeal carcinoma be-
fore this paper was completed. The investigation was sup-
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of the Republic of China (NSC 96-2323-B-039-001 and NSC
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