Synthesis and preclinical evaluations of 2-(2-fluorophenyl)-6,7- methylenedioxyquinolln-4-one monosodium phosphate (CHM-I-P-Na) as a potent antitumor agent

Article abstract of DOI:10.1021/jm901292j

CHM-1 [2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one] (1) has a unique antitumor mechanism of action. However, because 1 has relatively low hydrophilicity, it was evaluated only via ip administration, which is not clinically acceptable. In this study, we synthesized the monosodium phosphate salt (CHM-1 -P-Na, 4) of 1 as a hydrophilic prodrug. Compound 4 was rapidly converted into 1 following iv and po administration and also possessed excellent antitumor activity in a SKOV-3 xenograft nude mice model. Compound 4 also had clear-cut pharmacological effects on enzymes related with tumor cells. Neither 4 nor 1 significantly affected, normal biological function in a safety pharmacology profiling study. Compound 1 caused apoptotic effects in breast carcinoma cells via accumulation of cyclin Bl, and importantly, the endogenous levels of the mitotic spindle checkpoint, proteins BubRl directly correlated with cellular response to microtubule disruption. With, excellent antitumor activity profiles, 4 is highly promising for development as an anticancer clinical trials candidate.

Full text of DOI:10.1021/jm901292j

1616 J. Med. Chem. 2010, 53, 1616–1626
DOI: 10.1021/jm901292j
Synthesis and Preclinical Evaluations of 2-(2-Fluorophenyl)-6,7-methylenedioxyquinolin-4-one
Monosodium Phosphate (CHM-1-P-Na) as a Potent Antitumor Agent
Li-Chen Chou,† Chien-Ting Chen,† Jang-Chang Lee,† Tzong-Der Way,§ Chi-Hung Huang, Shih-Ming Huang,†
Che-Ming Teng,^ Takao Yamori,# Tian-Shung Wu,3 Chung-Ming Sun,O Du-Shieng Chien,[ Keduo Qian,‡
Susan L. Morris-Natschke,‡ Kuo-Hsiung Lee,*,‡ Li-Jiau Huang,†,z and Sheng-Chu Kuo*,†,z
†Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan, ‡Natural Products Research Laboratories,
Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7568, §School of Biological Science and
Technology, China Medical University, Taichung, Taiwan, Taiwan Advance Biopharm, Inc., 12F, No. 25, Lane 169, Kangning Street, Xizhi City,
Taipei 221, Taiwan, ^Pharmacological Institute, College of Medicine, National Taiwan University, Taiwan, #Division of Molecular
Pharmacology, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 135-8550, Japan, 3Department of Chemistry,
National Cheng Kung University, No. 1, Dasyue Road, Tainan, Taiwan, ODepartment of Applied Chemistry, National Chiao-Tung University,
Hsinchu, Taiwan, and [SunTen Phytotech Co., Ltd. 4F-1, No. 268, Liancheng Road, Jhonghe City, Taipei County 235, Taiwan. z L.-J. Huang
and S.-C. Kuo contributed equally to this work.
Received August 28, 2009
CHM-1 [2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one] (1) has a unique antitumor mechanism
of action. However, because 1 has relatively low hydrophilicity, it was evaluated only via ip adminis-
tration, which is not clinically acceptable. In this study, we synthesized the monosodium phosphate salt
(CHM-1-P-Na, 4) of 1 as a hydrophilic prodrug. Compound 4 was rapidly converted into 1 following iv
and po administration and also possessed excellent antitumor activity in a SKOV-3 xenograft nude mice
model. Compound 4 also had clear-cut pharmacological effects on enzymes related with tumor cells.
Neither 4 nor 1 significantly affected normal biological function in a safety pharmacology profiling
study. Compound 1 caused apoptotic effects in breast carcinoma cells via accumulation of cyclin B1, and
importantly, the endogenous levels of the mitotic spindle checkpoint proteins BubR1 directly correlated
with cellular response to microtubule disruption. With excellent antitumor activity profiles, 4 is highly
promising for development as an anticancer clinical trials candidate.
Introduction
control group.15 Compound 1 was also evaluated against
OVCAR-3 ovarian tumor xenograft in nude mice by the
National Cancer Institute (NCI, USA) and was found to
exhibit excellent antitumor activity. At dosages of 200, 134,
and 79 mg/kg (Q7D ꢀ 3, ip), 1 extended the life span of tumor-
bearing mice by 124%, 133%, and 79%, respectively. Notably,
even at the highest test dosage (200 mg/kg), 1 did not reach the
maximum tolerance dose (MTD). The preliminary results
from the above in vivo studies clearly indicate that 1 has
significant antitumor activity while maintaining low toxicity.
Unfortunately, 1 has relatively low water solubility (13 ng/
mL); thus, its animal study could only be done via ip admini-
stration, which is not suitable for clinical use. Accordingly, to
enhance the developmental value of 1, we designed its conver-
sion to a prodrug, which could be administrated through both
oral and iv routes.
In our prior studies,1-14 substituted 2-phenylquinolin-4-
ones (2-PQs) (Figure 1A) were identified as novel antimitotic
agents, and their structure-activity relationships were esta-
blished from experimental results of numerous related syn-
thetic analogues. Many of the tested 2-PQ derivatives demon-
strated potent cytotoxicity against human cancer cell lines and
were selected for in vivo testing in our laboratory. To date,
among tested compounds, 2-(2-fluorophenyl)-6,7-methylene-
dioxyquinolin-4-one (CHM-1,a 1) (Figure 1B) has been identi-
fied as the most active compound in vivo. Namely, when 1 was
evaluated in SCID mice bearing HA-22T xenograft, the tumor
growth was remarkably suppressed and the survival life span
was significantly extended by 1at a dosage of 10 mg/kg injected
intraperitoneally (ip) Q4D ꢀ 3 when compared with the
In the present study, we selected the hydrophilic mono-
sodium phosphate of 1 (CHM-1-P-Na, 4) (Figure 1B) as the
target compound. This prodrug should be converted readily
to the parent molecule in the bloodstream or gastrointestinal
tract by reaction with nonspecific alkaline or acidic phos-
phatases.16-20 Similar prodrug strategies have successfully
improved the clinical usage of estramustine, etoposide, com-
bretastatin A-4, and 2-methoxyestradiol.21-25
*To whom correspondence should be addressed. For K.-H.L.: phone,
919-962-0066; fax, 1-919-966-3893; E-mail: khlee@unc.edu. For S.-C.
K.: phone, þ886-4-22053366-5608; fax, þ886-4-22030760; E-mail:
sckuo@mail.cmu.edu.tw.
a Abbreviations: CHM-1, 2-(2-fluorophenyl)-6,7-methylenedioxy-
quinolin-4-one; CHM-1-P, 2-(2-fluorophenyl)-6,7-methylenedioxyqui-
nolin-4-one dihydrogen phosphate; CHM-1-P-Na, 2-(2-fluorophenyl)-
6,7-methylenedioxyquinolin-4-one monosodium phosphate; ip, intra-
peritoneal; iv, intravenous; po, oral; SPP, safety pharmacology profil-
ing; MTD, maximum tolerance dose; EMCIT, enzyme-mediated cancer
imaging and therapy; JFCR, Japanese Foundation for Cancer Research;
AUC, area under curve; ADME, administration, distribution, metabo-
lism, excretion; PQ, phenylquinolone.
Recently, enzyme-mediated cancer imaging and therapy
(EMCIT)26-29 has been developed as a novel method
for enzyme-dependent, site-specific in vivo precipitation of
r
pubs.acs.org/jmc
Published on Web 01/26/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1617
Figure 1. (A) Structures of substituted 2-phenylquinolin-4-ones (2-PQs). (B) Synthesis of sodium 2-(2-fluorophenyl)-6,7-methylenedioxy-
quinolin-4-yl hydrogen phosphate (4).
water-insoluble radioactive molecules within solid tumors. A
radiolabeled prodrug containing a phosphate moiety is de-
phosphorylated both in vitro and in vivo to its water-insoluble
form by alkaline phosphatase overexpressed on the extra-
cellular space of some specific tumor cells, such as ovarian,
bladder, and hepatoma cells. On the basis of this methodo-
logy, we were encouraged to pursue the synthesis of 4 as a
reasonable target delivery strategy. Our phosphate-bearing
prodrug, 4, could reasonably be desphosphorylated to its
water-insoluble form and precipitated in situ on the extra-
cellular space of SKOV-3 ovarian tumors. In this paper, we
report the synthesis of 4 and the selective toxicity of 1 against
various human cancer cell lines. Also reported are the results
of a preliminary pharmacokinetic study and evaluation of the
antitumor activity of 4 following oral and iv administration.
Safety pharmacology profiling (SPP) is a reliable, fast, and
cost-effective means for determining potential drug liabilities
to aid lead selection and lead optimization. Analysis of
profiling data enables us to find off-target interactions for
predicting potential side-effects, efficacy, and safety of com-
pounds. SPP concentrates on early hazard identification to
guide drug discovery projects and to minimize or abolish
deleterious effects through structure-activity relationship
considerations. Early compound profiling by in vitro phar-
macology profiling can flag for receptor-, enzyme-, transpor-
ter-, and channel-related liabilities of compounds and
translates these data into conjunction with ADME and
toxicity (ADME-Tox) characteristics. Therefore, we can
use enzyme and radioligand binding assays to identify targets
associated with beneficial effects for treating cancer or with
possible side effects. Both 1 and 4 were evaluated, and their
safety profiling results were recorded. The mechanism of
action of 4’s antitumor activity was also explored and re-
ported in the current study.
Chemistry
Although the synthesis of monosodium phosphate salt of 1
was challenging due to the ready decomposition of CHM-1-
phosphate (3) back to 1 in MeOH, it was successfully
achieved. Although synthetic methods to produce quinolin-
4-phosphoric acid dialkyl esters (IA) (Figure 1A) have been
reported,3,30 a literature search revealed no information about
the preparation of the hydrolyzed quinolinol-4-phosphoric
acids (IB) (Figure 1A). To our knowledge, this is the first time
that phosphate has been linked to the 4-O position of a
quinoline. The synthesis of 2-(2-fluorophenyl)-6,7-methylene-
dioxyquinolin-4-yl phosphate monosodium salt (4) is illu-
strated in Figure 1B. Initially, 1 was dissolved in THF, and
reacted with tetrabenzyl pyrophosphate in the presence of
NaH, to yield dibenzyl 2-(2-fluorophenyl)-6,7-methylene-
dioxyquinolin-4-yl phosphate (2). Compound 2 was subjected
to Pd/C catalytic hydrogenolysis in MeOH to give 3 in high
yield(97%). Thecompoundprecipitatedfrom solution, which
prevented the further decomposition of the prodrug. Finally,

1618 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Chou et al.
Figure 2. Differential activity patterns for 1 against 39 human cancer cell lines. MG-MID: mean of log X values (X = GI50, TGI, and LC50).
Delta: logarithm of the difference between the MG-MID and the log X of the most sensitive cell line. Range: logarithm of the difference between
the log X of the most resistant cell line and the log X of the most sensitive cell line. H: High concentration (10-4 M) (highest concentration of 1
used during testing); L: Low concentration (10-8 M) (lowest concentration of 1 used during testing). H:1 indicates that the number of cell lines
tested at the highest concentration is 1. L:0 indicates that the number of cell lines tested at the lowest concentration is 0.
3 was treated with NaHCO3 to obtain the water-soluble
monosodium salt (4).
similar types of fingerprints from over 300 known anticancer
agents with various mechanisms of action. The low correla-
tion coefficient (r < 0.4) found in fingerprint-matching for 1
seemed to imply that its mechanism of action differed widely
from those of the anticancer agents covered in the COM-
PARE database.
Single Dose Pharmacokinetics of 4 in Male CD-1 (Crl.)
Mouse. One objective of this study was to find the dosage of
4 required to achieve significant antitumor activity via iv and
oral administration. As mentioned above, an ip dose of
10 mg/kg of 1 resulted in significant antitumor activity in
our animal study. After the dosing, pharmacokinetic para-
meters were determined. Similarly, 10 mg/kg of the prodrug
4 was administered via iv and oral routes, and the pharmaco-
kinetic parameters of 3 and its metabolite 1 were determined.
The dosage of 4 required to achieve significant antitumor
activity in an animal study could thus be predicted by
comparing the pharmacokinetic profiles of 1 and 4.
Selected pharmacokinetic parameters are summarized in
Table 1 for ip, iv, and po administration, respectively. The
mean plasma concentration-time curves of 1 and 3, deter-
mined after ip, iv, and po dosing, are shown in Figure 3A-D.
As shown in Figure 3A, a high concentration of active
metabolite 1 appeared almost instantly in the mouse blood-
stream after iv dosing of 10 mg/kg of 4, while the concentra-
tion of prodrug 3 declined rapidly to a very low level within
1 h. These results indicated that 4 was quickly converted into
its active parent compound 1 after iv dosing.
Biology
Growth Inhibitory Activity of 1 against Human Cancer Cell
Line Panel. Compound 1, the parent compound of 4, was
evaluated for its growth inhibitory activity against the
JFCR-39 human cancer cell panel by the Japanese Founda-
tion for Cancer Research (JFCR).31-33 The evaluation was
carried out to determine the selective responses of 1 toward
various human cancer cell lines so that the results could be
used as a guideline for selecting suitable cancer types in the
animal study of its prodrug, 4.
The growth inhibitory activity data of 1 were analyzed
computationally and presented as dose-response curves at
five different concentrations between 10-4 and 10-8 M. GI50,
TGI, and LC50 values were calculated, and the correspond-
ing mean graph or fingerprint (Figure 2) was obtained.
Our results showed that 1 was active against most tested
cancer cell lines with a mean log GI50 (MG-MID) value of
-6.65. In particular, 1 was profoundly cytotoxic (log GI50
<
-7.0) against the following 15 cell lines: U251, KM-12,
HCT-15, HCT-116, NCI-H522, DMS-273, DMS-114, OV-
CAR-4, SKOV-3, RXF-631 L, MKN-1, MKN-7, MKN-45,
DU-145, and PC-3.
Unique COMPARE Fingerprint of 1. The fingerprint of
the preferential responses of 1 against specific cells in the cell
panel was then analyzed by a pattern-recognition computer
program (COMPARE), which contains a database covering
The plasma levels of 1 following iv and ip dosing of 4 and 1,
respectively, are shown in Figure 3B. This comparison

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1619
Table 1. Pharmacokinetic Parameters of 1 and 4 in Male CD-1 (Crl.) Mice Following a Single Dose of either 1 or 4 at 10 mg/kg by Different Routes
Cmax
(ng/
mL)
t1/
b
compd
dosed
compds
examined
AUC(0-inf)
(ng h/mL)
Tmax
(h)
MRT
(h)
CL
(mL/(min kg))
2
(h)
3
3
1 (ip)
4 (iv)
1
3
1
3
1
155
29810
8702
91
417
3220
1956
28
0.3
nda
nda
0.3
0.5
7.3
0.1
0.7
0.3
1.4
nda
51.7
nda
nda
nda
7.3
nda
1.0
nda
1.0
4 (po)
1312
1230
a nd: not determined. b t1/2 was determined from 6 to 27 h time points.
Figure 3. (A) Mean plasma concentration-time profiles of 1 and 4 in male CD-1 (Crl.) mice following a single iv dose of 4 at 10 mg/kg. (B)
Mean plasma concentration-time profiles of 1 in male CD-1(Crl.) mice following a single iv dose of 4 and single ip dose of 1 at 10 mg/kg. (C)
Mean plasma concentration-time profile of 1 and 3 in male CD-1 (Crl.) mice following a single po dose of 4 at 10 mg/kg. (D) Mean plasma
concentration-time profiles of 1 in male CD-1 (Crl.) mice following a single po dose of 4 at 10 mg/kg and single ip dose of 1 at 10 mg/kg.
indicated that the plasma concentration of 1 (AUC=1956
ng h/mL, Table 1) following iv dosing of 4 was significantly
higher than that of 1 (AUC = 417 ng h/mL, Table 1)
following ip dosing of 1, particularly in the first hour
postdosing. Therefore, we expected that the antitumor acti-
vity of 4 at 10 mg/kg iv dosing would be superior to that
resulting from 1 at 10 mg/kg ip dosing.
Meanwhile, as shown in Figure 3C, after po dosing of
10 mg/kg of 4, the plasma concentration-time profiles of 3
and its metabolite 1 appeared to follow the same pattern
(Figure 3A) found with iv dosing of 4. Namely, the active
metabolite 1 appeared instantly in the bloodstream, while the
concentration of 3 declined rapidly until it essentially dis-
appeared within an hour. This experiment demonstrated that
4 was also quickly converted into its active metabolite 1
following oral dosing.
expected that the antitumor activity for 4 at 10 mg/kg po dosing
of would be superior to that resulting from 1 at 10 mg/kg ip
dosing.
3
3
Acute Oral Toxicity of 4. The mortality of male mice orally
administered 4 from group 1 to group 5 was 0/5, 0/5, 0/5, 0/5,
and 5/5, and of female mice was 0/5, 0/5, 0/5, 2/5, and 5/5,
respectively. Dosages of test compound, administered by
gavage, were 0, 500, 1500, 2700, and 5000 mg/kg for groups
1-5, respectively. Hunched posture, tremors, and prostra-
tion were observed before animals died. Salivation, hypo-
activity, soft feces, and feces stain were observed in other
surviving animals. All observations were gone within three
days. None of the animals had obvious gross lesions. The
LD50 with 95% confidence interval was 2462 mg/kg in
females and 2720 mg/kg in males.
Safety Pharmacology Profiling (SPP) of 1 and 4. To
explore their general pharmacological activities, 1 and 4 were
further screened in 317 enzyme assays and 167 radioligand
binding assays. The results of inhibitory activity screening of
1 are summarized in Table 2. At 10 μM, 1 showed signi-
ficant responses (g50% inhibition) toward glutamate
Figure 3D compares the pharmacokinetic profiles of 4 and
1 following po and ip dosing, respectively. The plasma concen-
tration of 1 (AUC = 1230 ng h/mL, Table 1), following
3
oral dosing of 4, was higher than that of 1 (AUC=417 ng
h/mL, Table 1) following ip dosing of 1. Likewise, we
3

1620 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Chou et al.
Table 2. Inhibitory Effects of 1 on Enzyme and Radioligand Binding Assays for SPP
biological assaya
species
% of inhibition
glutamate decarboxylaseb
C. perfringens
human
human
human
human
pig
66
52
monoamine oxidase MAO-Ab
peptidase, CTSD (cathepsin D)b
peptidase, CTSE (cathepsin E)b
peptidase, CTSS (cathepsin S)b
peptidase, pepsinb
CYP450, 2D6b
protein serine/threonine kinase [MAPK10 (JNK3)]b
protein tyrosine phosphatase, PTP4A1(PRL-1)b
protein tyrosine phosphatase, PTP4A2(PRL-2)b
protein tyrosine phosphatase, PTP4A3(PRL-3)b
GABAA, benzodiazepine control c
GABAA, flunitrazepam, central c
27
12
1
85
human
human
human
human
human
rat
ndd
55
ndd
ndd
ndd
83
rat
ndd
a Concentration = 10 μM. b Enzyme assay. c Radioligand binding assay. d nd: no determination.
Table 3. Inhibitory Effects of 4 on Enzyme and Radioligand Binding Assays for SPPa
biological assayb
species
inhibition %
IC50 (μM)
glutamate decarboxylase c
C. perfringens
human
human
human
human
pig
nde
-24
50
nde
monoamine oxidase MAO-A c
nde
peptidase, CTSD (cathepsin D) c
peptidase, CTSE (cathepsin E) c
9.79
1.71
1.62
nde
78
peptidase, CTSS (cathepsin S) c
106
-19
73
peptidase, pepsin c
CYP450, 2D6 c
human
human
human
human
human
rat
3.22
nde
protein serine/threonine kinase [MAPK10 (JNK3)] c
protein tyrosine phosphatase, PTP4A1(PRL-1) c
protein tyrosine phosphatase, PTP4A2(PRL-2) c
protein tyrosine phosphatase, PTP4A3(PRL-3) c
GABAA, benzodiazepine control d
GABAA, flunitrazepam, central d
-20
98
0.676
0.314
4.93
nde
101
72
nde
62
rat
5.57
a A standard error of the means is presented where results are based on multiple, independent determination. b Concentration = 10 μM. c Enzyme
assay. d Radioligand binding assay. e ND: no determination.
decarboxylase, MAO-A, pepsin, MAPK10, and GABAA.
The same screening indicated that 4 exhibited significant
inhibitory activity against eight targets at 10 μM, and further
assessment was performed to determine IC50. The inhibitory
activity of 4 was greatest against protein tyrosine phospha-
tases PRL-1 and PRL-2 (IC50 < 10.0 μM) (Table 3). Among
those enzymes and ligand inhibited by 4, PRL-1, -2, and -3
are postulated to promote cell motility, invasion, and meta-
stasis, while cathepsin-D, -E, -S, and GABAA are also
associated with tumor activity.34-38
In Vivo Antitumor Activity of 4. On the basis of the above
cytotoxicity data of 1 and the pharmacokinetic parameters
of 4, we selected the SKOV-3 xenograft model using dosing
at 5, 10, and 20 mg/kg (iv and po) to evaluate the in vivo
antitumor activity of 4.
According to the results shown in parts A and B of
Figure 4, 4 induced dose- and time-dependent inhibition of
SKOV-3 tumor growth. Significant tumor growth suppres-
sion was detected at 5 mg/kg/day, and almost complete
tumor suppression was observed at 20 mg/kg/day. During
the course of antitumor evaluation, no significant body
weight changes were detected in either test or control mice
(Figure 4C,D).
The above findings indicated that 4 has excellent anti-
tumor activity when administered either iv or po and that the
same dosing led to similar antitumor efficacy, regardless of
administration route.
lines using the MTT assay. We chose three clinical trial
agents, taxol, colchicine, and doxorubicin, as positive con-
trols, and in comparison, 1 had a nearly equivalent effect
against cancer cells (Figure 5A). These data indicate that 1
induces potent cytotoxicity in human ovarian cancer cells.
To clarify the mechanism of 1-induced proliferation inhibi-
tion, we examined the effect of 1 on the cell cycle. Com-
pound 1 caused the accumulation of cells in the G2-M phase
with concomitant losses in G0-G1 phase, with a maximum
effect observed at 36 h (Figure 5B). The accumulation of
cells with G2-M DNA content was followed by an increase
in hypodiploid cells at the later time points (48 h), as
indicated as apoptotic cells. These results indicate that 1
induced G2-M arrest of the cell cycle followed by apoptosis.
To better understand the mechanism of 1-induced G2-M
arrest, the expression of cell cycle-related proteins
was analyzed. Cyclin B1 and securin serve as markers for
mitotic arrest induction. Accordingly, we next assessed the
effects of 1 on cyclin B1 and securin protein expre-
ssion. Treatment of SKOV-3 cells with 1 resulted in
increases in protein expression of cyclin B1 and securing
(Figures 5C,D).
Upregulation of the Mitotic Spindle Checkpoint Protein
BubR1 was Associated with a Sustained G2/M Cell Cycle
Arrest after Treatment with 1. The mitotic spindle checkpoint
protein BubR1 has been shown to monitor tension across
attached kinetochores and initiate mitotic arrest in response
to loss of microtubule tension. As shown in Figure 6A,
upregulation of BubR1 was predominantly found in cells
treated with 10 μM of 1 for the indicated durations. BubR1
Compound 1 Inhibits Cell Growth in Human Ovarian
Cancer Cells by Inducing Mitotic Arrest. We first deter-
mined the effect of 1 on the growth of ovarian cancer cell

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1621
Figure 4. (A) Mean tumor volume-time profiles in SKOV-3 xenograft nude mice (n = 11) following iv dosing of 4 at 5, 10, and 20 mg/kg
five days per week for four consecutive weeks. (B) Mean tumor volume-time profiles in SKOV-3 xenograft nude mice (n = 11) following po
dosing of 4 at 5, 10, and 20 mg/kg five days per week for four consecutive weeks. (C) Mean body weight-time profiles in SKOV-3 xeno-
graft nude mice (n = 11) following iv dosing of 4 at 5, 10, and 20 mg/kg five days per week for four consecutive weeks. (D) Mean
body weight-time profiles in SKOV-3 xenograft nude mice (n = 11) following po dosing of 4 at 5, 10, and 20 mg/kg five days per week for
four consecutive weeks.
Figure 5. Effect of 1 on cell growth and cell cycle progression in human ovarian cancer cells. (A) SKOV-3 cells were incubated in the absence or
presence of the 10 μM of 1, colchicine, doxorubicin, and taxol for 24 h. (B) The time effect of 1 on cell cycle distribution, cells were treated with
10 μM of 1 for 6, 12, 24, 36, and 48 h and analyzed for propidium iodide-stained DNA content by flow cytometry. (C) SKOV-3 cells were treated
with vehicle (DMSO), 1 (10 μM) for the indicated time. (D) SKOV-3 cells were treated with the indicated dose 1 for 24 h. Cells were then
harvested and lysed for the detection of cyclin B1, securin, and β-actin protein expression. Western blot data presented are representative of
those obtained in at least three separate experiments.
upregulation produced microtubule disruption, as indicated
by a significant increase in the percentage of cells arrested in
the G2/M phase of the cell cycle (Figure 6B). Moreover, 1
caused the accumulation of BuBR1 (Figure 6B). Our result

1622 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Chou et al.
Figure 6. BubR1 upregulation was associated with 1-induced G2/M cell cycle arrest in human ovarian cancer cells. (A) SKOV-3 cells were
treated with vehicle (DMSO), 1 (10 μM) for the indicated time. (B) SKOV-3 cells were treated with the indicated dose 1 for 24 h. Whole-cell
lysates were resolved by SDS-PAGE and probed with anti-BubR1 mouse mAb. Results are representative of three separate experiments. Blots
were probed with anti-β-actin mAb as a loading control.
suggested that BubR1 contributed to the mitotic checkpoint
induced by 1.
that antimicrotubule drug induced M-phase arrest, and in-
appropriate accumulation of B-type cyclins were associated
with the initiation of apoptotic pathways. In this study, 1
arrestedthe growth of cancer cellsatthe G2-M phaseand then
induced apoptotic cell death via the accumulation of cyclin
B1. More importantly, the endogenous levels of the mitotic
spindle checkpoint protein BubR1 directly correlated with the
cellular response to microtubule disruption. The mitotic
spindle checkpoint is a complex pathway conserved across
species and monitors the metaphase to anaphase transition.
The basic model for the spindle checkpoint defines that
tension defects or unattached chromosomes activate the
checkpoint, delaying the onset of anaphase until such aberra-
tions are corrected.
In this study, our results suggested that absolute levels of
BubR1 may determine the length of stay in G2/M. In support
of our hypothesis, a recent study demonstrated that knock-
down of BubR1 accelerates the normal progression of mito-
sis.40 Furthermore, in our study, the 1-induced G2/M block
was maintained until endogenous levels of BubR1 protein
became undetectable, and at this point, a marked increase in
apoptosis was observed. BubR1 cellular levels inversely cor-
related with the onset of apoptosis. Collectively, these results
suggest that endogenous levels of BubR1 may predict the
apoptotic efficacy and potential chemotherapeutic benefit of
microtubule-targeted drugs in human cancers. Because the
current antimitotic anticancer drugs, such as vinca alkaloids
and taxoids, are not cost-effective to synthesize due to struc-
tural complexity, 4 represents a promising novel class of
mitotic-arresting compounds, which may supplement current
cancer chemotherapy.
In conclusion, we have reported the synthesis of the mono-
sodium phosphate prodrug (4) of 1 in high yield. Prodrug 4
was readily converted to its parent molecule 1 during both iv
and po administration, and showed excellent antitumor acti-
vity when administered via iv and po routes to nude mice
bearing SKOV-3 xenograft. Results from SPP using enzyme
and radioligand binding assays suggested that 4 has clear-cut
pharmacological effects on several tumor-associated enzymes
and ligands. Further mechanism ofaction studyindicated that
1 arrested the growth of cancer cells at the G2-M phase and
then induced apoptotic cell death via the accumulation of
cyclin B1. The 1-induced G2/M block was maintained until
endogenous levels of BubR1 protein became undetectable. On
the basis of the above findings, we conclude that 4 is a
promising anticancer clinical trials candidate, which functions
as a novel antimitotic agent.
Results and Discussion
As we mentioned earlier, although 1 shows significant
antitumor activity with an interesting mechanism of action,
its low hydrophilicity limits its development. In this study, we
successfully synthesized the monosodium phosphate salt (4)
of 1, which could be administrated through oral and iv routes
instead of the original ip dosing. The single dose pharmaco-
kinetic study of 4 in male CD-1 mouse proved that 4 was
rapidly converted to its active form of 1 via iv or oral
administration. By comparing the pharmacokinetic para-
meters, we postulated that the same dosing of 4 by iv or po
routes would result in superior antitumor activity compared
withipdosing of 1. Thispostulate was confirmed inthe later in
vivo antitumor assay. All of these results demonstrated the
success of our prodrug strategy, which may highly improve
the clinical usage of our drug candidate 1.
Because 4 was rapidly converted to metabolites in mice with
both iv and po administration, we presumed that the anti-
tumor activity of 4 may depend on the overall plasma
exposure of 3 and its metabolites. However, comparison of
the data in Table 1 revealed a big difference between the total
AUC values (measured as the sum of plasma exposure of 1
and 3) determined after iv and po administration. Never-
theless, the in vivo antitumor activity test results revealed
similar antitumor activity between 4 administered via iv and
po routes. The dose-dependency of pharmacokinetics versus
pharmacodynamic response appears to be nonlinear. There-
fore, the contributions from factors such as protein binding,
drug distribution, and the possible presence of additional
active metabolites need to be further investigated.
The growth inhibition assay of 1 revealed that this com-
pound was particularly active against breast cancer (U251),
lung cancer (NCI-H522, DMS273, DMS114), colon cancer
(HCT-15, HCT-116), ovarian cancer (OVCAR-4, SKOV-3),
stomach cancer (MKN1, MKN7, MKN45), and prostate
cancer (DU-145, PC-3). Compound 1 displayed a signi-
ficant growth inhibition and apoptosis in breast carcinoma
cells and promising antitumor actions against human ovarian
carcinoma cells compared with those of taxol, colchicine, and
doxorubicin.
In our mechanism of action studies of 1, we observed
multiple functions, including antimitotic activity, apoptosis-
induction, antimetastasis, and antiangiogenesis.39 Of particu-
lar interest to us is that 1 causes mitotic arrest through the
expression of mitotic kinase BubR-1 and disrupts microtubule
organization by enhancing SIRT2-mediated tubulin deacetyl-
ation. It has been widely reported that cyclin B1/CDK1
complexes are involved in the regulation of the G2/M phase
and the M-phase transition. Many reports have demonstrated
Experimental Section
Materials. Compounds 1 and 4 were synthesized according to
Figure 1B. Melting points were determined with a Yanaco MP-
500D melting point apparatus and are uncorrected. IR spectra

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1623
were recorded on Shimadzu IRPrestige-21 spectrophotometers
as KBr pellets. NMR spectra were obtained on a Bruker Avance
DPX-300 FT-NMR spectrometer in CDCl3 or DMSO. The
following abbreviations are used: s, singlet; d, doublet; t, triplet;
dd, double doublet; and m, multiplet. MS spectra were mea-
sured with a Waters Quattro Micro LC/MS/MS instrument.
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. HPLC purity analysis of
target compound 4 confirmed purity was 99.77%.
Dibenzyl 2-(2-Fluorophenyl)-6,7-methylenedioxyquinolin-4-yl
Phosphate (2). Sodium hydride (13.7 mg, 0.57 mmol) was added
at 0 ꢀC to a stirred solution of 1 (64.5 mg, 0.23 mmol) in dry THF
(10 mL). After 1 h, tetrabenzyl pyrophosphate (1) (100 mg, 0.19
mmol) was added and stirring was continued for 20 min. The
mixture was filtered and washed with CH2Cl2. Then the filtrate
was concentrated under vacuum at a temperature below 35 ꢀC.
The residue was purified by column chromatography (silica gel,
EtOAc/n-hexane) to give 2 (69.1 mg, 0.127 mmol). Yield: 67%;
mp 101-104 ꢀC. MS (ESI): m/z (M þ H)þ 544. 1H NMR
(CDCl3, 300 MHz): δ 5.20 (s, 2H), 5.26 (s, 2H), 6.12 (s, 2H),
7.05 (s, 1H), 7.16-7.43 (m, 14H), 7.77 (s, 1H), 8.01-8.02 (m,
1H). Anal. (C30H23FNO6P) C, H, N.
renal cancer RXF-631 L and ACHN; stomach cancer St-4,
MKN1, MKN7, MKN28, MKN45, and MKN74; prostate
cancer DU-145 and PC-3. The cell lines were cultured in
RPMI-1640 (GIBCO/BRL, NY) supplemented with 5% fetal
bovine serum (FBS; GIBCO/BRL), penicillin (100 units/mL)
(GIBCO/BRL), and streptomycin (100 mg/mL) (GIBCO/BRL)
at 37 ꢀC in humidified air containing 5% CO2. Dose-response
curves at five different concentrations between 10-4 and 10-8
M
were obtained from computer analysis. The 50% growth inhi-
bition (GI50), total growth inhibition (TGI), and 50% lethal
concentration (LC50) values for these cell lines were determined
using the sulforhodamine B (SRB) colorimetric method. Com-
puter processing of these values produced differential activity
patterns against the cell lines (mean graphs). The mean graph
was compared with those of standard compounds, including
various anticancer drugs by using COMPARE analysis.
Single Dose Pharmacokinetics of 4 in Male CD-1 (Crl.)
Mouse. a. Preparation of Dosing Formulations. Compound 1
was dissolved in cremophor/DMSO/saline (20/5/75, v/v) to
make a dosing solution of 2 mg/mL for ip administration. 4
was dissolved in 9% NaHCO3/water to make a dosing solution
of 1 mg/mL for iv and oral administration.
b. In Vivo Experiment. Male CD-1 (Crl.) mice (body weight:
22-24 g) were used in this study. Source of animals was
BioLasco Taiwan. Water was provided ad libitum, regardless
of administration route. The 1 solutions (2 mg/mL) were
injected ip with a bolus dosing volume of 0.11-0.12 mL
(5 mL/kg) per animal. For iv administration, 4 was administered
via tail vein with a bolus dosing volume of 0.22-0.24 mL per
animal (10 mL/kg). For oral dosing, the drug suspension was
given via oral gavages with a dosing volume of 0.22-0.24 mL
per animal (10 mL/kg). The animals were fasted for 4 h prior to
oral administration and allowed access to standard chow 4 h
postdosing.
2-(2-Fluorophenyl)-6,7-methylenedioxyquinolin-4-yl Dihydro-
gen Phosphate (3). A suspension of 2 (97.7 mg, 0.18 mmol) in
anhydrous MeOH (10 mL) was hydrogenated in the presence of
10% Pd/C (50 mg) at rt for 10 min. The catalyst and precipitate
were collected and dissolved in 10% NaHCO3 solution and then
filtered. The filtrate was acidified with diluted aq HCl, and the
solid was then collected by filtration and washed with acetone to
give 3 (63.5 mg, 0.175 mmol). Yield: 97.2%; mp >300 ꢀC. MS
1
(ESI): m/z 362 (M - H)-. H NMR (DMSO-d6, 300 MHz): δ
6.22 (s, 2H), 7.32-7.41 (m, 4H), 7.49-7.54 (m, 1H), 7.74 (s, 1H),
7.93-7.98 (m, 1H). Anal. (C16H11FNO6P) C, H, N.
c. Sample Collection. Each blood sample (0.5-0.7 mL) was
collected by decapitation and collected in a prechilling 1.5 mL
size Eppendorf safe-lock microcentrifuge tube containing
sodium fluoride, followed by centrifugation (12000 rpm, 4 ꢀC)
for 10 min. The plasma fraction was transferred to a clean
microcentrifuge tube and stored at -70 ꢀC for further analysis.
The sampling time points were 0, 5, 15, 30 min, 1, 1.5, 2, 4, 6, 9,
24, and 27 h after ip dosing. The sampling time points were 0, 2,
5, 15, 30 min, 1, 1.5, 2, 4, 6, 9, 24, and 27 h after iv dosing. For
oral administration, the blood samples were collected at pre-
dose, 15, 30 min, 1, 1.5, 2, 4, 6, 9, 24, and 27 h after dosing. Three
mice were used per time point.
Sodium 2-(2-Fluorophenyl)-6,7-methylenedioxyquinolin-4-yl
Hydrogen Phosphate (4). To a flask containing 3 (3.6 g, 10
mmol), a precooled (ice bath) solution of NaHCO3 (0.1 M
solution in H2O, 100 mL) was added dropwise. After the
addition was complete, the reaction mixture was removed from
the ice bath, stirred at rt for 5 min, and then filtered through
celite after no dissolution from the solid was observed. The
resulting yellow solution was poured into acetone (400 mL) and
kept in an ice bath for 1 h. The precipitate was filtered and
washed with ice-cooled acetone (20 mL ꢀ 4). The solid was dried
under vacuum to yield 4 (2.9 g, 7.5 mmol). Yield: 75.3%;
1
mp >300 ꢀC. MS(ESI): m/z 384 (M - H)-. H NMR (D2O,
d. Analytics and Pharmacokinetic Calculation. The plasma
concentrations of 1 and 3 were measured by LC-MS/MS (mass
spectrometer: Micromass Quattro Ultima; pump and autosam-
pler: Waters Alliance 2795 LC; data processor: MassLynx
version 3.5) method with a reversed-phase Biosil Pro-ODS
column. The plasma samples were mixed with acetonitrile,
centrifuged, and the supernatant was injected onto an LC
column. HPLC conditions were the following. Mobile phase:
CHM-1: 35% CH3CN þ 1.0% HCOOH; CHM-1-P: 30%
CH3CN þ 1.0% CH3COOH. Column: Biosil, ODS 4.6 mm ꢀ
150 mm, 5 μ. Flow rate: 1.0 mL/min with a post column split
1/10 to mass. Retention time: CHM-1: 2.96 min; CHM-1-P: 3.78
min. Tandem mass spectrometry was performed with electro-
spray/positive ionization mode, source temperature 80 ꢀC,
deesolvation temperature 400 ꢀC. The pharmacokinetic para-
meters were calculated from mean plasma concentrations by
WinNonlin Standard program (version 3.1, Pharsight Corp).
The standard curve calculation involved the following: method:
simple weighted linear regression with 1 over nominal concen-
tration (1/x) or concentration square (1/x2) as weighting factor,
response: peak area ratio, equation: y=bx þ a.
300 MHz): δ 6.22 (s, 2H), 7.23-7.34 (m, 3H), 7.45-7.49 (m,
1H), 7.61 (s, 1H), 7.64 (s, 1H), 7.70 (t, J=8.1 Hz, 1H). Anal.
(C16H10FNNaO6P) C, H, N. HPLC purity analysis. Column:
BEH Shield RP18 1.7 μm. Mobile phase: 0.01 M ammonium
formate/CAN=80/20. Detection wavelength: PDA Ch1 254 nm
at 1.2 mm. Retention time: 0.565 min. Flow rate: 0.4 mL/min.
Purity: 99.77%.
Growth Inhibitory Activity of 1 against Human Cancer Cell
Line Panel. The system was developed according to the NCI
method,41,42 modified by the Japanese Foundation for Cancer
Research (JFCR).32,43 The cancer panel experiment for 1 was
carried out by JFCR, and the inhibition profile was compared
with those of more than 300 standard compounds, including
various anticancer drugs. The precise experimental method and
data analyses have been described elsewhere.43
Briefly, we describe the cell lines used and the method for
detecting growth inhibition. The following human cancer cell
lines were used in cancer panel experiments: breast cancer HBC-
4, BSY-1, HBC-5, MCF-7, and MDA-MB-231; brain cancer
U251, SF-268, SF-295, SF-539, SNB-75, and SNB-78; colon
cancer HCC2998, KM-12, HT-29, HCT-15, and HCT-116; lung
cancer NCI-H23, NCI-H226, NCI-H522, NCI-H460, A549,
DMS273, and DMS114; melanoma LOX-IMVI; ovarian cancer
OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, and SKOV-3;
Acute Oral Toxicity Assay. Acute toxicity of 4 was evaluated
with the lethal dose test, involving single oral administrations to
a group of male and female rats at increasing doses in order to

1624 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Chou et al.
determine the dose that would kill 50% of mice (LD50) within a
set time frame. The assay was conducted in compliance with the
Good Laboratory Practice (GLP) regulations. All procedures
were approved by the DCB Institutional Animal Care and Use
Committee (IACUC) and conducted in compliance with the
Guide for the Care and Use of Laboratory Animals.
Fifty SD rats (25 males and 25 females) were obtained from
BioLASCO Taiwan Co., Ltd. The rats were approximately 7-8
weeks old at time of dosing. The body weight range was
170-250 g for males and 150-210 g for females.
Rats were randomized into five groups, each consisting of
five males and five females. The animals were fasted over-
night before dosing. The treated rats were administered test
article solution by gavage at dose levels of 500, 1500, 2700, and
5000 mg/kg; the control rats were administered water by gavage
with dose volume 20 mL/kg once. Test article solutions were
freshly prepared with water for oral injection. Rats were ob-
served once daily for clinical signs of toxicity and twice daily for
moribundity/mortality. Body weights were recorded weekly and
at necropsy. All animals were euthanized and necropsied for
gross lesion examination.
In Vivo Antitumor Activity Assay. Female BALB/c nude mice
(18-20 g; 6-8 weeks of age) were purchased from the National
Animal Center and maintained in pressurized ventilated cages
according to institutional regulations.
SKOV-3, human ovarian cancer cells, were cultured in
DMEM/F12 in 10% heat-inactivated bovine serum (FBS)
and incubated at 37 ꢀC in humidified atmosphere containing
5% CO2.
Nude mice were subcutaneously inoculated with SKOV-3
cells at 3 ꢀ 106 cells per mouse in 0.5 mL PBS via a 24 gauge
needle. After appearance of a 100 mm3 tumor nodule, 88 tumor-
bearing mice were randomly divided into eight groups for
treatment with vehicle (PBS) or 4 with different administered
methods and doses.
Animals were weighed and tumors were measured using
calipers twice weekly before, during, and after drug treatments.
Tumor volume was calculated with the following formula:
1/2(L þ W2), where L is the length and W is the width.44,45 At
the end of the experiments, animals were euthanized with carbon
dioxide followed by cervical dislocation. The tumors were
excised, weighed, and sectioned, and the tumor sections were
embedded in OCT compound and frozen at -70 ꢀC.
a. . The mice were administered with single intravenous
dosing of 4 via tail vein five days per week for four consecutive
weeks at 5, 10, and 20 mg/kg.
Triton X-100, 1 mM PMSF, 10 μg/mL leupeptin, 1 mM sodium
orthovanadate, 1 mM EGTA, 10 mM NaF, 1 mM sodium
pyrophosphate, 100 mM β-glycerophosphate, 20 mM Tris-HCl,
137 mM NaCl, 5 mM EDTA, 0.1% sodium dodecyl sulfate, and
10 μg/mL aprotinin. Adjust pH to 7.9) was added to lyse the
cells. After the cells lysed, the suspension solution were centri-
fuged, and the Bio-Rad protein assay kit (Bio-Red
Laboratories) was used to determine the protein contents.
Taken were 50 μg proteins to be resolved with SDS-PAGE
and transferred to PVDF membrane (polyvinylidene fluoride
transfer membrane) (BioTrace, UK.). The membrane was
blocked by blocking buffer (nonfat milk 5%), NaN3 (0.2%),
and Tween 20 (0.2%, v/v) in TBS). Then the PVDF membrane
was incubated with primary antibodies, followed by incubation
with horseradish peroxidase-conjugated goat antimouse anti-
body (1:2500 dilution, Roche Applied Science, Indianapolis,
IN). Reactive bands were visualized with an enhanced chemi-
luminescence system (Amersham Biosciences, Arlington
Heights, IL). The intensity of the bands was scanned and
quantified with a Phosphor-Image system.
d. Cell Proliferation Assays. Cells (1 ꢀ 104) were seeded on the
24-well cell culture cluster overnight and then treated with indi-
cated drug and incubated for 24 h. Next, 40 μL of MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (stock
conc 2 mg/mL, Sigma Chemical Co.) was added to each well
(volume of each well was 500 μL) and then incubated for 2 h at
37 ꢀC. After MTT-formazan crystals were formed, 250 μL of
DMSO was added to dissolve the crystals. Finally, absorbance was
detected at O.D. 550 nm by an enzyme-linked immunosorbent
assay (ELISA) reader.
e. Flow Cytometry. The cell cycle analysis was determined by
flow cytometry. Cells were cultured in 60 mm Petri dishes and
incubated for various times. After being trypsinized and washed
twice with ice-cold PBS, the cells were fixed with 70% ice-
cold EtOH overnight at -20 ꢀC. After centrifugation, the cell
pellets were treated with RNAase A and exposed to PI and
then analyzed by flow cytometry (FACScan, BD Biosciences,
Mountain View, CA). The percentage was analyzed by Cell
Quest software (BD Biosciences).
Acknowledgment. The investigation was supported by re-
search grants from the National Science Council of the
Republic of China (NSC 96-2323-B-039-001; NSC 97-2323-
B-039-001) awarded to S.-C. Kuo. Thanks are also due to
partial support from CA17625 awarded to K. H. Lee.
b. . The mice were administered with single oral dosing of 4
five days per week for four consecutive weeks at 5, 10, and 20
mg/kg.
Enzyme Assay and Radioligand Binding Assay (Spectrum
Screen).46 MDS PharmaServices performed this testing under
standard protocols.
Supporting Information Available: Literature references for
spectrum screen (enzyme and radioligand binding assays);
analytical results. This material is available free of charge via
the Internet at http://pubs.acs.org.
Mechanism of Action Study. a. Antibodies and Reagents.
Antibodies and reagents were purchased from commercial
sources: antibodies against cyclin B1 and securing were pur-
chased from Cell Signaling Technology (Beverly, MA); anti-
body against BuBR1 was from BD Biosciences (Los Angeles,
CA); antimouse and antirabbit antibodies conjugated to horse-
radish peroxidase were obtained from Santa Cruz Biotechno-
logy (Santa Cruz, CA); β-actin antibody and PI were from
Sigma Chemical Co. (St. Louis, MO).
References
(1) Kuo, S. C.; Lee, H. Z.; Juang, J. P.; Lin, Y. T.; Wu, T. S.; Chang,
J. J.; Lednicer, D.; Paull, K. D.; Lin, C. M. Synthesis and cytotoxicity
of 1,6,7,8-substituted-2-(40-substituted phenyl)-4-quinolones and re-
lated compounds: identification as antimitotic agents interacting with
tubulin. J. Med. Chem. 1993, 36 (9), 1146–1156.
(2) Li, L.; Wang, H. K.; Kuo, S. C.; Wu, T. S.; Lednicer, D.; Lin, C. M.;
Hamel, E.; Lee, K. H. Antitumor agents. 150. 20,30,40,50,5,6,7-
Substituted 2-phenyl-4-quinolones and related compounds: their
synthesis, cytotoxicity, and inhibition of tubulin polymerization.
J. Med. Chem. 1994, 37 (8), 1126–1135.
(3) Li, L.; Wang, H. K.; Kuo, S. C.; Wu, T. S.; Mauger, A.; Lin, C. M.;
Hamel, E.; Lee, K. H. Antitumor agents. 155. Synthesis and
biological evaluation of 30,6,7-substituted 2-phenyl-4-quinolones
as antimicrotubule agents. J. Med. Chem. 1994, 37 (20), 3400–3407.
(4) Chen, K.; Kuo, S. C.; Hsieh, M. C.; Mauger, A.; Lin, C. M.; Hamel,
E.; Lee, K. H. Antitumor agents. 174. 20,30,40,5,6,7-Substituted 2-
phenyl-1,8-naphthyridin-4-ones: their synthesis, cytotoxicity, and
inhibition of tubulin polymerization. J. Med. Chem. 1997, 40 (14),
2266–2275.
b. Cell Lines and Cell Cultures. The human ovarian cancer cell
line SKOV-3 was obtained from The American Type Culture
Collection (Rockville, MD) and propagated in 100 mm culture
dishes at the desired density in DMEM/F-12 supplemented
with 10% fetal calf serum and 1% penicillin-streptomycin.
These cells were grown at 37 ꢀC in a humidified atmosphere of
5% CO2.
c. Western Blot Analyses. Cells (1 ꢀ 106) were washed twice
with PBS, and then the gold lysis buffer (10% glycerol, 1%

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1625
(5) Chen, K.; Kuo, S. C.; Hsieh, M. C.; Mauger, A.; Lin, C. M.; Hamel,
E.; Lee, K. H. Antitumor agents. 178. Synthesis and biological
evaluation of substituted 2-aryl-1,8-naphthyridin-4(1H)-ones as
antitumor agents that inhibit tubulin polymerization. J. Med.
Chem. 1997, 40 (19), 3049–3056.
(6) Xia, Y.; Yang, Z. Y.; Xia, P.; Bastow, K. F.; Tachibana, Y.; Kuo,
S. C.; Hamel, E.; Hackl, T.; Lee, K. H. Antitumor agents. 181.
Synthesis and biological evaluation of 6,7,20,30,40-substituted-
(23) Vincent, L.; Kermani, P.; Young, L. M.; Cheng, J.; Fan, Z.; Shido,
K.; Lam, G.; Bompais-Vincent, H.; Zhu, Z.; Hicklin, D. J.; Bohlen,
P.; Chaplin, D. J.; May, C.; Rafii, S. Combretastatin A4 phosphate
induces rapid regression of tumor neovessels and growth through
interference with vascular endothelial-cadherin signaling. J. Clin.
Invest. 2005, 115 (11), 2992–3006.
(24) Pettit, G. R.; Temple, C.; Narayanan, V. L.; Varma, R.; Simpson,
M. J.; Boyd, M. R.; Rener, G. A.; Bansal, N. Antineoplastic agents
322. Synthesis of combretastatin A-4 prodrugs. Anti-Cancer Drug
Des. 1995, 10 (4), 299–309.
(25) Edsall, A.; Agoston, G.; Treston, A.; Plum, S.; McClanahan, R.;
Lu, T.; Song, W.; Cushman, M. Synthesis and in vivo antitumor
evaluation of 2-methoxyestradiol 3-phosphate, 17-phosphate, and
3,17-diphosphate. J. Med. Chem. 2007, 50 (26), 6700–6705.
(26) Ho, N. H.; Harapanhalli, R. S.; Dahman, B. A.; Chen, K.; Wang,
K.; Adelstein, S. J.; Kassis, A. I. Synthesis and biologic evaluation
of a radioiodinated quinazolinone derivative for enzyme-mediated
insolubilization therapy. Bioconjugate Chem. 2002, 13 (2), 357–364.
(27) Chen, K; Wang, K; Kirichian, A. M.; Al Aowad, A. F.; Iyer, L. K.;
Adelstein, S. J.; Kassis, A. I. In silico design, synthesis, and
biological evaluation of radioiodinated quinazolinone derivatives
for alkaline phosphatase-mediated cancer diagnosis and therapy.
Mol. Cancer Ther. 2006, 5 (12), 3001–3013.
(28) Posoisil, P.; Wang, K.; Al Aowad, A. F.; Iyer, L. K.; Adelstein, S.
J.; Kassis, A. I. Computational modeling and experimental evalua-
tion of a novel prodrug for targeting the extracellular space of
prostate tumors. Cancer Res. 2007, 67, 2197–2205.
(29) Wang, K.; Kirichian, A. M.; Al Aowad, A. F.; Adelstein, S. J.;
Kassis, A. I. Evaluation of chemical, physical, and biologic proper-
ties of tumor-targeting radioiodinated quinazolinone derivative.
Bioconjugate Chem. 2007, 18, 754–764.
(30) Andrews, K. J. M.; Atherton, F. R.; Bergel, F.; Morrison, A. L.
Hydroxypyridine and hydroxyquinoline phosphates as anti-
cholinesterases. J. Chem. Soc. 1954, 1638–1640.
(31) Horst, G.; Harald, K.; Hilmar, M.; Gerhard, S. New 4-quinolinol-
and 5,6,7,8-tetrahydro-4-quinolinol derivatives with biocidal
effect. Liebigs Ann. Chem. 1982, 1656–1676.
1,2,3,4-tetrahydro-2-phenyl-4-quinolones as
a new class of
antimitotic antitumor agents. J. Med. Chem. 1998, 41 (7), 1155–
1162.
(7) Zhang, S. X.; Bastow, K. F.; Tachibana, Y.; Kuo, S. C.; Hamel, E.;
Mauger, A.; Narayanan, V. L.; Lee, K. H. Antitumor agents. 196.
Substituted 2-thienyl-1,8-naphthyridin-4-ones: their synthesis,
cytotoxicity, and inhibition of tubulin polymerization. J. Med.
Chem. 1999, 42 (20), 4081–4087.
(8) Zhang, S. X.; Feng, J.; Kuo, S. C.; Brossi, A.; Hamel, E.; Tropsha,
A.; Lee, K. H. Antitumor agents. 199. Three-dimensional quanti-
tative structure-activity relationship study of the colchicines
binding site ligands using comparative molecular field analysis.
J. Med. Chem. 2000, 43 (2), 167–176.
(9) Hour, M. J.; Huang, L. J.; Kuo, S. C.; Xia, Y.; Bastow, K.;
Nakanishi, Y.; Hamel, E.; Lee, K. H. 6-Alkylamino- and 2,3-
dihydro-30-methoxy-2-phenyl-4-quinazolinones and related com-
pounds: their synthesis, cytotoxicty, and inhibition of tubulin
polymerization. J. Med. Chem. 2000, 43 (23), 4479–4487.
(10) Xia, Y.; Yang, Z. Y.; Xia, P.; Hackl, T.; Hamel, E.; Mauger, A.;
Wu, J. H.; Lee, K. H. Antitumor agents. 211. Fluorinated 2-phenyl-
4-quinolone derivatives as antimitotic antitumor agents. J. Med.
Chem. 2001, 44 (23), 3932–3936.
(11) Xia, T.; Yang, Z. Y.; Hour, M. J.; Kuo, S. C.; Xia, P.; Bastow,
K. F.; Nakanishi, Y.; Nampoothiri, P.; Hackl, T.; Hamel, E.; Lee,
K. H. Antitumor agents. Part 204: Synthesis and biological evalua-
tion of substituted 2-aryl quinazolinones. Bioorg. Med. Chem. Lett.
2001, 11 (9), 1193–1196.
(12) Xia, T.; Yang, Z. Y.; Xia, P.; Bastow, K. F.; Nakanishi, Y.;
Nampoothiri, P.; Hamel, E.; Brossi, A.; Lee, K. H. Antitumor
agents. Part 226: Synthesis and cytotoxicity of 2-phenyl-4-quinolone
acetic acids and their esters. Bioorg. Med. Chem. Lett. 2003, 13 (17),
2891–2893.
(13) Lai, Y. Y.; Huang, L. J.; Lee, K. H.; Xiao, Z. Y.; Bastow, K. F.;
Yamori, T.; Kuo, S. C. Synthesis and biological relationships of
30,6-substituted 2-phenyl-4-quinolone-3-carboxylic acid deriva-
tives as antimitotic agents. Bioorg. Med. Chem. 2005, 13 (1), 265–
275.
(14) Nakamura, S; Kozuka, M.; Bastow, K. F.; Tokuda, H.; Nishino,
H.; Suzuki, M.; Tatsuzaki, J.; Morris-Natschke, S. L.; Kuo, S. C.;
Lee, K. H. Cancer preventive agents, Part 2: Synthesis and evalua-
tion of 2-phenyl-4-quinolone and 9-oxo-9,10-dihydroacridine de-
rivatives as novel antitumor promoters. Bioorg. Med. Chem. 2005,
13 (14), 4396–4401.
(32) Yamori, T.; Matsunaga, A.; Sato, S.; Yamazaki, K.; Komi, A.;
Ishizu, K.; Mita, I.; Edatsugi, H.; Matsuba, Y.; Takezawa, K.;
Nakanishi, O.; Kohno, H.; Nakajima, Y.; Komatsu, H.; Andoh,
T.; Tsuruo, T. Potent antitumor activity of MS-247, a novel DNA
minor groove binder, evaluated by an in vitro and in vivo human
cancer cell line panel. Cancer Res. 1999, 59, 4042–4049.
(33) Yaguchi, S.; Fukui, Y.; Koshimizu, I.; Yoshimi, H.; Matsuno, T.;
Gouda, H.; Hirono, S.; Yamazaki, K.; Yamori, T. Antitumor
activity of ZSTK474, a new phosphatidylinositol 3-kinase inhibi-
tor. J. Natl. Cancer Inst. 2006, 98 (8), 545–556.
(34) Radke, I.; Gotte, M.; Kersting, C.; Mattsson, B.; Kiesel, L.;
Wulfing, P. Expression and prognostic impact of the protein
tyrosine phosphatases PRL-1, PRL-2, and PRL-3 in breast cancer.
Br. J. Cancer 2006, 95 (3), 347–354.
(15) Wang, S. W.; Pan, S. L.; Huang, Y. C.; Guh, J. H.; Chiang, P. C.;
Huang, D. Y; Kuo, S. C.; Lee, K. H.; Teng, C. M. CHM-1, a novel
synthetic quinolone with potent and selective antimitotic anti-
tumor activity against human hepatocellular carcinoma in vitro
and in vivo. Mol. Cancer Ther. 2008, 7 (2), 350–360.
(35) Rochefort, H.; Garcia, M.; Glondu, M.; Laurent, V.; Liaudet, E.;
Rey, J.-M.; Roger, P. Cathepsin D in breast cancer: mechanisms
and clinical application, a 1999 overview. Clin. Chim. Acta 2000,
291 (2), 157–170.
(36) Liaudet-Coopman, E.; Beaujouin, M.; Derocq, D.; Garcia, M.;
ꢀ
(16) Heimbach, T; Oh, D. M.; Li, L. Y.; Forsberg, M.; Savolainen, J.;
Glondu-Lassis, M.; Laurent-Matha, V.; Prebois, C.; Rochefort,
€
Leppanen, J.; Matsunaga, Y.; Flynn, G.; Fleisher, D. Absorption
rate limit considerations for oral phosphate prodrug. Pharm. Res.
2003, 20 (6), 848–856.
H.; Vignon, F. Cathepsin D: newly discovered functions of a long-
standing aspartic protease in cancer and apoptosis. Cancer Lett.
2006, 237 (2), 167–179.
(17) Jordan, M. A. Mechanisms of action of antitumor drugs that
interact with microtubules and tubulin. Curr. Med. Chem. Anti-
cancer Agents 2002, 2, 1–17.
(37) Chang, W. S.; Wu, H. R.; Yeh, C. T.; Wu, C. W.; Chang, J. Y.
Lysosomal cysteine proteinase cathepsin S as a potential target for
anti-cancer therapy. J. Cancer Mol. 2007, 3, 5–14.
(18) Casagrande, C.; Merlo, L.; Ferrini, R.; Miragoli, G.; Semeraro, C.
Cardiovascular and renal action of dopaminergic prodrugs.
J. Cardiovasc. Pharmacol. 1989, 14, S40–S59.
(38) Takehara, A.; Hosokawa, M.; Eguchi, H.; Ohigashi, H.; Ishikawa,
O.; Nakamura, Y.; Nakagawa, H. γ-Aminobutyric acid (GABA)
stimulates pancreatic cancer growth through overexpressing
GABAA receptor π subunit. Cancer Res. 2007, 67, 9704–9712.
(39) Wang, S. W.; Pan, S. L.; Peng, C. Y.; Huang, D. Y.; Tsai, A. C.;
Chang, Y. L.; Guh, J. H.; Kuo, S. C.; Lee, K. H.; Teng, C. M.
CHM-1 inhibits hepatocyte growth factor-induced invasion of SK-
Hep-1 human hepatocellular carcinoma cells by suppressing matrix
metalloproteinase-9 expression. Cancer Lett. 2007, 257, 87–96.
(40) Meraldi, P.; Draviam, V. M.; Sorger, P. K. Timing and checkpoints
in the regulation of mitotic progression. Dev. Cell 2004, 7, 45–60.
(41) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero,
D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R. Display and
analysis of patterns of differential activity of drugs against human
tumor cell lines: development of mean graph and COMPARE
algorithm. J. Natl. Cancer Inst. 1989, 81 (14), 1088–1092.
(19) Juntunen, J.; Huuskonen, J.; Laine, K.; Niemi, R.; Taipale,
€
H.; Nevalainen, T.; Pate, D. W.; Jarvinen, T. Anandamide pro-
drugs 1. Water-soluble phosphate esters of arachidonylethanol-
amide and R-methanandamide. Eur. J. Pharm. Sci. 2003, 19 (1),
37–43.
(20) Pettit, G. R.; Grealish, M. P.; Jung, M. K.; Hamel, E.; Pettit, R. K.;
Chapuis, J. C.; Schmidt, J. M. Antineoplastic agents. 465. Struc-
tural modification of resveratrol: sodium resverastatin phosphate.
J. Med. Chem. 2002, 45 (12), 2534–2542.
(21) Perry, C. M.; McTavish, D. Estramustine phosphate sodium. A
review of its pharmacodynamic and pharmacokinetic properties,
and therapeutic efficacy in prostate cancer. Drugs Aging 1995, 7 (1),
49–74.
(22) Witterland, A. H. I.; Koks, C. H. W.; Beijnen, J. H. Etoposide
phosphate, the water soluble prodrug of etoposide. Pharm. World
Sci. 1996, 18, 163–170.
(42) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.;
Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A.;
Gray-Goodrich, M.; Campbell, H.; Mayo, J.; Boyd, M. Feasibility

1626 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Chou et al.
of a high-flux anticancer drug screen using a diverse panel of
cultured human tumor cell lines. J. Natl. Cancer Inst. 1991, 83,
757–766.
(44) Euhus, D. M.; Hudd, C.; LaRegina, M. C.; Johnson, F. E. Tumor
measurement in the nude mouse. J. Surg. Oncol. 1986, 31, 229–234.
(45) Tomayko, M. M.; Reynolds, C. P. Determination of subcutaneous
tumor size in athymic (nude) mice. Cancer Chemother. Pharmacol.
1989, 24, 148–154.
(43) Yamori, T. Panel of human cancer cell lines provides valuable
database for drug discovery and bioinformatics. Cancer Che-
mother. Pharmacol. 2003, 52, S74–S79.
(46) References provided in Supporting Information.