Design, synthesis, and preclinical evaluation of new 5,6- (or 6,7-) disubstituted-2-(fluorophenyl)quinolin-4-one derivatives as potent antitumor agents

Article abstract of DOI:10.1021/jm100780c

Our previous exploration of 2-phenylquinolin-4-ones (2-PQs) has led to an anticancer drug candidate 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one monosodium phosphate (CHM-1-P-Na). In order to develop additional new drug candidates, novel 2-PQs were

Full text of DOI:10.1021/jm100780c

J. Med. Chem. 2010, 53, 8047–8058 8047
DOI: 10.1021/jm100780c
Design, Synthesis, and Preclinical Evaluation of New 5,6- (or 6,7-)
Disubstituted-2-(fluorophenyl)quinolin-4-one Derivatives as Potent Antitumor Agents
Li-Chen Chou,†,# Meng-Tung Tsai,†,# Mei-Hua Hsu,† Sheng-Hung Wang,‡ Tzong-Der Way,§ Chi-Hung Huang, Hui-Yi Lin,†
Keduo Qian,^ Yizhou Dong,^ Kuo-Hsiung Lee,^ Li-Jiau Huang,*,† and Sheng-Chu Kuo*,†
†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, §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, and ^Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina,
Chapel Hill, North Carolina 27599-7568, United States. # L.-C. Chou and M.-T. Tsai contributed equally to this work.
Received June 24, 2010
Our previous exploration of 2-phenylquinolin-4-ones (2-PQs) has led to an anticancer drug candidate
2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one monosodium phosphate (CHM-1-P-Na). In
order to develop additional new drug candidates, novel 2-PQs were designed, synthesized, and evaluated
for cytotoxic activity. Most analogues, including 1b, 2a,b, 3a,b, 4a,b, and 5a,b, exhibited significant
inhibitory activity (IC50 of 0.03-8.2 μM) against all tested tumor cell lines. As one of the most potent
analogue, 2-(3-fluorophenyl)-5-hydroxy-6-methoxyquinolin-4-one (3b) selectively inhibited 14 out of
60 cancer cell lines in a National Cancer Institute (NCI) evaluation. Preliminary mechanism of action
study suggested that 3b had a significant effect on the tyrosine autophosphorylation of insulin-like
growth factor-1 receptor (IGF-1R). Safety pharmacology profiling of 3b showed no significant effect on
normal biological functions of most enzymes tested. Furthermore, sodium 2-(3-fluorophenyl)-6-
methoxy-4-oxo-1,4-dihydroquinolin-5-yl phosphate (15), the monophosphate of 3b, exceeded the
activity of doxorubicin and was comparable to CHM-1-P-Na in a Hep3B xenograft nude mice model.
In summary, 15 is a promising clinical candidate and is currently under preclinical study.
Introduction
a class of promising anticancer agents, which prompted us to
design and develop additional new promising analogues as
potential clinical trials candidates.
During the past 2 decades, we have synthesized a series
of substituted 2-phenylquinolin-4-ones (2-PQsa)1-4 (Figure 1)
and identified them as a new class of anticancer agents. In a
recent in vivo evaluation of a series of 2-PQs with potent
cytotoxicity, excellent antitumor activity was identified with
2-(2-fluorphenyl)-6,7-methylenedioxyquinolin-4-one (CHM-1)4
(Figure 1) and the sodium salt of its phosphate derivative
2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one mono-
sodium phosphate (CHM-1-P-Na)4,5 (Figure 1). Further-
more, both acute toxicity and safety pharmacology profiling
(SPP) studies supported that CHM-1-P-Na is extremely safe
to use. On the basis of the above findings, 2-PQ derivatives are
As mentioned above, CHM-1-P-Na exhibited excellent
antitumor activity through both oral and iv administration.
Its unique structure possesses three key functional groups. The
first key moiety is a phosphate group located on the 4-position
of its quinoline ring. As stated in our previous report,4 a
pharmacokinetic study of CHM-1-P-Na confirmed its rapid
bioconversion into its active parent molecule CHM-1 follow-
ing iv or po administration. It was also found that CHM-1-
P-Na could be dephosphorylated by alkaline phosphatase into
CHM-1 on the extracellular space of SKOV-3 tumor. Because
alkaline phosphatase is known to be overexpressed on the
extracellular space of specific tumor cells, such as ovarian and
hepatic carcinoma cells, the introduction of a phosphate group
appears to be a reasonable strategy for target delivery. Second,
a methylenedioxy moiety bridges the 5- and 6-positions of the
quinoline ring. This moiety could form an orthoquinone upon
metabolism and subsequently be metabolized into more cyto-
toxic metabolites in hypoxic cells.4,6 Because severe hypoxia is
a common situation in locally advanced solid tumors, the
incorporation of a methylenedioxy moiety becomes a mean-
ingful antitumor approach. Third, a fluorine atom is located
on the 2-phenyl group. In certain medicines, the unordinary
nature of fluorine has been reported to impart a variety of
properties, including enhanced potency, improved duration of
action, and attenuation of biliary clearance.7
*To whom correspondence should be addressed. For L.-J.H.:
phone, þ886-4-22053366-5609; fax, þ886-4-22030760; e-mail, ljhuang@
mail.cmu.edu.tw. For S.-C.K.: phone, þ886-4-22030760; fax, þ886-4-
22030760; e-mail, sckuo@mail.cmu.edu.tw.
a Abbreviations: 2-PQ, 2-phenylquinolin-4-one; SARs, structure-
activity relationships; CHM-1, 2-(2-fluorophenyl)-6,7-methylenedioxy-
quinolin-4-one; CHM-1-P-Na, 2-(2-fluorophenyl)-6,7-methylenediox-
yquinolin-4-one monosodium phosphate; SPP, safety pharmacology
profiling; NCI, National Cancer Institute; GI50, 50% growth inhibition;
MG-MID, mean growth midpoint; TGI, total growth inhibition; 3-HP,
2-[(3-hydroxy-2-pyridinyl)methylene]hydrazinecarbothioamide; IGF-
1R, insulin-like growth factor-1 receptor; PDB, Protein Data Bank;
PQIP, cis-3-[3-(4-methylpiperazin-l-yl)cyclobutyl]-1-(2-phenylquinolin-
7-yl)imidazo[1,5-a]pyrazin-8-ylamine; CYP450, cytochrome P450 en-
zymes; PDE, phosphodiesterase; YES1, V-yes-1 Yamaguchi sarcoma
viral oncogene homologue 1; UGT1A1, UDP-glucuronosyltransferase
1A1; iv, intravenous; po, oral.
r
2010 American Chemical Society
Published on Web 10/25/2010
pubs.acs.org/jmc

8048 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Chou et al.
Meanwhile, our prior structure-activity relationships
(SARs)1-3 have established that the presence of a group with
a lone pair of electrons (for instance, OCH3, NRR, Cl, F) at
both the 6-position of the quinoline ring and 30-position of the
2-phenyl group enhanced the cytotoxicity of 2-PQs. With this
finding in mind, we have designed compounds 1-5 (Figure 2)
and their related phosphates as our target compounds based
on the following attributes: (1) the presence of an OR group at
the quinoline 6-position, (2) the presence of a fluorine atom on
the 2-phenyl group, and (3) ability to be metabolized into an
orthoquinone in vivo.
Among the target compounds 1-5 synthesized and eval-
uated, analogue 3b appeared to be the most potent cytotoxic
agent. Further studies, including safety pharmacology profil-
ing, mechanism of action, and molecular modeling of 3b, as
wellas invivo antitumor evaluationon its monophosphate15,
were performed.
Chemistry
The synthetic route to 5,6,7,20,30,40-substituted 2-phenyl-
quinolin-4-ones (1, 2, 6) is illustrated in Scheme 1A. First,
3,4,5-substituted 1-amino-2-acetylbenzenes 7a-c were reacted
separately with 2,3,4-substituted benzoyl chlorides 8a-c to
yield the corresponding amides 9a-i, which were subse-
quently cyclized in t-BuOH in the presence of t-BuOK to afford
the target compounds (1, 2, 6). Starting materials 6-amino-2,3-
dimethoxyacetophenone (7a),8 6-amino-2,3-methylenedioxya-
cetophenone (7b),9 and 4-benzyloxy-3-methoxy-6-nitroaceto-
phenone (10)10 were prepared according to previously
reported methods. As shown in Scheme 1B, nitro 10 was
reduced by reaction with SnCl2 to give 6-amino-3-methoxy-4-
benzyloxyacetophenone (7c).
Figure 1. Structures of 2-PQs, CHM-1, and CHM-1-P-Na.
As shown in Scheme 2, selective demethylation of compounds
1a-c with BCl3 provided the corresponding 2-(fluorophenyl)-5-
hydroxy-6-methoxyquinolin-4-ones 3a-c, whose structures
Figure 2. Structures of target compounds 1-5.
Scheme 1

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8049
Scheme 2
Scheme 3
were confirmed by NOSY spectra. Catalytic hydrogenation of
compounds 2a-cand 7-benzyloxy-2-(fluorophenyl)-6-methoxy-
quinolin-4-ones 6a-c yielded 2-(fluorophenyl)-5,6-dihydroxy-
quinolin-4-ones 4a-c and 2-(fluorophenyl)-7-hydroxy-
6-methoxyquinolin-4-ones 5a-c, respectively.
The phosphorylation of 2-(3-fluorophenyl)-5-hydroxy-6-
methoxyquinoline-4-one (3b) is shown in Scheme 3. Com-
pound 3b was first reacted with tetrabenzylpyrophosphate
(11) in the presence of NaH to yield 2-(3-fluorophenyl)-6-
methoxyquinoline-4,5-diyl bis(dibenzyl phosphate) (12) which
was treated withMeOH atroom temperatureprovided mono-
phosphate 13. Its structure was confirmed by the chemical
shift of the proton on the 3-position (δ 6.27) in the 1H NMR
spectra. Monophosphoric acid 14 was obtained by catalytic
hydrogenation of 13. Finally, 14 was converted into water-
soluble sodium salt 15.

8050 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Table 1. Cytotoxicities of CHM-1 and Target Compounds 1-5a
Chou et al.
IC50 b( μM)
0
0
0
R4
compd
R2
R3
HL-60
HCT116
Hep3B
H460
CHM-1
1a
1b
0.08
3.7
0.15
>20
1.2
0.13
>20
2.6
0.14
>20
3.5
F
H
F
H
H
F
H
H
F
1.3
2.0
1c
2a
H
H
F
>20
2.1
>20
1.9
>20
4.5
H
H
F
1.0
0.7
2b
2c
H
H
F
2.5
2.4
3.2
H
H
F
>10
0.07
0.03
1.8
>10
0.05
0.07
2.4
>10
0.05
0.07
11.0
3.9
>10
0.11
0.08
8.8
3a
3b
H
H
F
H
H
F
3c
4a
H
H
F
H
H
F
0.5
0.3
0.6
8.2
4.1
6.1
4b
4c
H
H
F
6.9
H
H
F
38.6
1.3
>100
5.8
1.1
100
5.3
100
4.4
5a
5b
H
H
F
H
H
0.9
38.2
5.3
>100
4.8
>100
5c
H
>100
a Human tumor cells were treated with different concentrations of compounds for 48 h. b Data are presented as IC50 (μM, the concentration of 50%
proliferation-inhibitory effect).
Results and Discussion
Compound 3b was submitted for evaluation of activity
against NCI’s 60 human tumor cell line panel (Figure 3). The
results demonstrated that 3b was a potent antitumor agent
against a broad range of human tumor cell lines [mean
log GI50 (MG-MID) of -7.35]. As to total growth inhibition
(TGI), 3b also displayed potent inhibitory activity (TGI <
0.1 μM) against the following 14 cancer cell lines: HL-60, SR,
NCI-H522, Colo 205, HCC-2998, HT-29, SF-539, SNB-75,
MDA-MB-435, IGROV1, OVCAR-3, NCI/ADR-RES,
RXF-393, and DU-145.
Unique COMPARE Fingerprint of 3b. The mean graph
pattern (fingerprint) of 3b was analyzed by a pattern-
recognition computer program (COMPARE), which contains a
database covering similar types of fingerprints from over 300
known anticancer agents with various mechanisms of action.
The results of COMPARE analysis in Table 2 suggested that
the mechanism of action of 3b matched best with 2-[(3-
hydroxy-2-pyridinyl)methylene]hydrazinecarbothioamide
(3-HP). However, the low correlation coefficient (r < 0.558)
implied that its mechanism of action was different from those
of the anticancer agents covered in the COMPARE data-
base.
Growth Inhibitory Activity of 1-5 against Human Cancer
Cell Lines. The 5,6- (6,7-) disubstituted 2-(fluorophenyl)-
quinolin-4-ones 1-5 and CHM-1 were screened against
HL-60, HCT-116, Hep3B, H-460 human tumor cell lines, and
the results are summarized in Table 1. Among the 5,6-dimethoxy
derivatives 1a-c, the 30-fluoro derivative 1b exhibited the
greatest cytotoxicity, although it was less potent than the
positive control CHM-1. Meanwhile, both 2a and 2b, with a
5,6-methylenedioxy entity on the quinoline ring, demon-
strated activity comparable to that of 1b, although again
they were less potent than CHM-1. While all three 5-hydroxy-
6-methoxy derivatives 3a-c showed significant cytotoxicity,
compounds 3a and 3b, with the fluorine in the 20 and 30
positions, respectively, were better than 3c, with the fluorine
in the 40 position. Changing the quinolone substitution
pattern again from 5-hydroxy-6-methoxy (3a-c) to 5,6-
dihydroxy (4a-c) or 7-hydroxy-6-methoxy (5a-c) led to
decreased potency. However, similar rank orders of po-
tency were found among all derivatives regarding the
position of the fluoro substituent. Generally, 40-fluorophe-
nyl derivatives (1c-5c) were much less potent than the 20-
(1a-5a) and 30-fluorophenyl (1b-5b) derivatives. In fact,
only 1c and 3c showed significant activity against some
tested cancer cell line. Overall, compounds 3a and 3b are
considered the most promising anticancer agents, as they
exhibited potent broad cytotoxic activity against all four
cancer cell lines tested.
Mechanism of Action of 3b. In our previous work,4 we
reported a multiple-targeting action mechanism for CHM-1.
More recently,11 we found that CHM-1 also affected the
insulin-like growth factor-1 receptor (IGF-1R), which is
gaining recognition as an attractive anticancer treatment
target.12 Because IGF-1R is frequently overexpressed in a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8051
Figure 3. Differential activity patterns for compound 3b against 60 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.
Table 2. Results of COMPARE cCorrelation at GI50 Level for Compound 3b
rank
compound (NCI number)
3HPb (NSC 95678)
r a
mechanism of action category
DNA antimetabolite
1
2
3
4
5
6
7
8
9
10
0.558
0.416
0.395
0.383
0.379
0.366
0.359
0.351
0.348
0.34
trimetrexate (NSC 352122)
methotrexate (NSC 740)
rhizoxin (NSC 332598)
maytansine (NSC 153858)
thalicarpine (NSC 68075)
pancratiastatin (NSC 349156)
paclitaxel (NSC 125973)
DUP 785c (NSC 368390)
5-fluorouracil (NSC 19893)
RNA/DNA antimetabolite
RNA/DNA antimetabolite
antimitotic agent
antimitotic agent
inhibits the synthesis of DNA, RNA, and protein
inhibition of the cell cycle from G0/G1 to S phase
antimitotic agent
RNA/DNA antimetabolite
RNA/DNA antimetabolite
c
a r: correlation coefficient. b 3HP: 2-[(3-hydroxy-2-pyridinyl)methylene]hydrazinecarbothioamide. DUP 785: brequinar sodium [6-fluoro-2-(20-
fluoro-1,10-biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid sodium salt].
number of human tumors and is considered an important
anticancer target,13,14 we investigated the effect of 3b on
IGF-1R expression in Hep3B cells, by examining receptor
phosphorylation by the IGF-1R tyrosine kinase, in the
presence or absence of 3b. As shown in Figure 4, the cells
were treated with different doses of 3b for 12 h with DMSO
as a control. Western blot analysis of IGF-1R phosphoryla-
tion with the antibody against PY1135/1136-IGF-1R showed
that the concentration of phosphor-IGF-1R decreased gradually
with increasing concentration of 3b. The expression of IGF-1R,
as evaluated by antibody to its β-subunit, served as loading
control. The preliminary result indicated that 3b had a significant
effect on the tyrosine autophosphorylation of IGF-1R.
To delineate the interaction between 3b and IGF-1R
kinase, structural models of Protein Data Bank (PDB)
entries 1K3A15 and 3D9416 were selected for molecular
modeling study. PDB 1K3A is a complex structure of human
IGF-1R kinase, which illustrates the ATP and Tyr-peptide
substrate binding sites. On the other hand, PDB entry 3D94
illustrates the binding mode of the ATP-competitive inhibi-
tor cis-3-[3-(4-methylpiperazin-l-yl)cyclobutyl]-1-(2-phenyl-
quinolin-7-yl)imidazo[1,5-a]pyrazin-8-ylamine (PQIP), which

8052 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Chou et al.
Figure 4. Effect of 3b on basal receptor tyrosine phosphorylation
(pTyr) in Hep3B cells. Hep3B cells were treated with various
concentrations of 3b at 37 ꢀC for 12 h. Levels of phosphorylated
IGF-1R and IGF-1R were analyzed by immunoblotting. Immuno-
blotting with β-actin antibody demonstrated equivalent protein in
each lane. Western blot data presented are representative of those
obtained in at least three separate experiments.
Figure 6. Structural superimposition of IGF1R model 1K3A and
3D94. The structural features of IGF1R kinase were compared
between ATP-bound (1K3A, yellow color) and PQIP-bound (3D94,
magenta color) states. At the PQIP-bound state, the conformation
of the activation loop containing Tyr-1131, Tyr-1135, and Tyr-1136
changed dramatically, compared with ATP-bound state.
Figure 7. Predicted binding mode of compound 3b with IGF1R
kinase. Binding site of 3b is the same as that of 2-phenylquinolinyl
segment of PQIP, which is beside the ATP-binding site. The binding
sites of ATP and peptide substrate are indicated as shaded regions
according to model 1K3A.
The binding pocket of PQIP was then used for predicting
the interaction mode of compound 3b against IGF-1R
kinase. Molecular docking study showed that 3b interacted
with IGF-1R directly at the binding site of the 2-phenylqui-
nolinyl segment of PQIP (Figure 7). The results indicate that
the comparable binding is due to structural similarity.
On the basis of the molecular modeling study described
above, the structure of PQIP consists of two segments. One is
an ATP-superimposable scaffold, and the other is a 3b-
superimposable scaffold (Figure 8). Furthermore, the 3b-
binding site can be identified as a potential allosteric binding
site, which is unique and different from the ATP-binding site.
Thus, 3b might inhibit autophosphorylation of the activa-
tion loop by changing its conformation, without significantly
interfering with ATP-binding. Therefore, this model could
explain why the autophosphorylation of IGF-1R was in-
hibited by compound 3b. The exact action mechanism of 3b
will be explored further.
Figure 5. (A) Chemical structure of compound 3b and PQIP.
(B) Structural similarity between 3b and PQIP. PQIP possesses a
2-phenylquinolinyl segment, which is superimposable with 3b.
contains a 2-phenylquinolinyl segment that can be superimposed
on compound 3b because of structural similarity (Figure 5).
During molecular modeling, the structural features of
IGF-1R kinase with different ligand-bound states were first
compared through molecular 3D superimposition. As shown
in Figure 6, the conformation of the activation loop changed
dramatically between ATP-bound (model 1K3A, yellow
color) and PQIP-bound (model 3D94, magenta color) states.
The autophosphorylation of Tyr-1131, Tyr-1135, and Tyr-
1136 on the activation loop is also inhibited by PQIP.
Safety Pharmacology Profiling (SPP) of 3b. To explore its
general pharmacological activities, 3b was tested in 158
types of enzyme spectrum screening assays (Table 3). At
10 μM, 3b inhibited the activity of only the following five
enzymes: cytochrome P450 enzymes (CYP450), phospho-
diesterase 4 (PDE4), phosphodiesterase 5 (PED5), V-yes-1
Yamaguchi sarcoma viral oncogene homologue 1 (YES1),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8053
new analogues was established. As one of our most promis-
ing target compounds, 3b was submitted for evaluation of
activity against NCI’s 60 human tumor cell line panel.18,19
The NCI results confirmed that compound 3b had potent
inhibitory activity against 14 cancer cell lines. The prelim-
inary result of a mechanistic study indicated that 3b had a
significant effect on the tyrosine autophosphorylation of
IGF-1R, which is an attractive target for anticancer ther-
apy. The SPP results from enzyme assays suggested that 3b
has a clear-cut pharmacological effect on tumor-associated
UGT1A1. Furthermore, a second target compound, 15, the
monophosphate derivative of 3b, demonstrated excellent
antitumor activity in nude mice bearing Hep3B xenograft,
when administrated via po and iv routes. Excitingly, the
antitumor activity of 15 exceeded that of doxorubicin, the
most common drug used in current practice, and 15 was
comparable to CHM-1-P-Na. In summary, 15 is a promis-
ing antitumor agent, which merits further exploration as a
clinical candidate.
Figure 8. Structure of PQIP can be identified as two segments,
including ATP-superimposed scaffold and 3b-superimposed scaf-
fold. Thus, 3b is probably an allosteric inhibitor, which might
inhibit autophosphorylation of activation loop without interfering
with ATP-binding.
Experimental Section
Chemistry. General Experimental Procedures. All reagents
and solvents were obtained commercially and used without
further purification. Reactions were monitored by thin-layer
chromatography (TLC), using Merck plates with fluorescent
indicator (TLC silica gel 60 F254). The following adsorbent was
used for column chromatography: silica gel 60 (Merck, particle
size 0.063-0.200 mm). Melting points were determined on a
Yanaco MP-500D melting point apparatus and are uncorrected.
IR spectra were recorded on Shimadzu IR Prestige-21 spectro-
photometers as KBr pellets. NMR spectra were obtained on a
Bruker Avance DPX-200 FT NMR spectrometer in CDCl3 or
DMSO. The following abbreviations are used: s, singlet; d,
doublet; t, triplet; q, quartet; dd, double doublet; m, multiplet.
EI mass spectra were measured with an HP 5995 GC-MS
instrument. ESI mass spectra were measured with a Finnigan
LCQ ion-trap mass spectrometer (TSQ Quantum, Thermo
Finnigan Corporation, San Jose, CA). 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. The purity of tested compounds was g95% as
determined by HPLC conducted on a Shimadzu LC-10AT
apparatus equipped with a Shimadzu SPD-M10AVP diode
array detector and a Shimadzu SIL-10A autoinjector.
N-(2-Acetyl-3,4-dimethoxyphenyl)-2-fluorobenzamide (9a). To
a solution of 2-fluorobenzoyl chloride (8a, 0.48 g, 2.46 mmol) in
40 mL of dry toluene were added triethylamine (0.5 mL) and
compound 7a8 (0.70 g, 4.43 mmol). The mixture was stirred at
55-60 ꢀC for 30 min and then poured into crushed ice and
extracted with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and evaporated. The crude product was
purified by column chromatography (silica gel, EtOAc/n-hexane)
to give 9a (0.5 g, 1.58 mmol) as a yellow solid. Yield: 64.1%. Mp
106-108 ꢀC. MS (EI, 70 eV): m/z 317 (Mþ). 1H NMR (DMSO-
d6, 200MHz):δ2.45 (s, 3H), 3.76 (s, 3H), 3.81 (s, 3H), 7.14 (d, J =
2.6 Hz, 2H), 7.24-7.34 (m, 2H), 7.52-7.63(m, 2H), 10.07 (s, 1H).
13C NMR (DMSO-d6, 50 MHz): δ 31.91, 56.52, 61.42, 114.52,
116.70, 121.43, 124.16, 125.06, 126.81, 130.52, 131.50, 133.35
(d, J = 8.0 Hz), 145.98, 150.47, 159.61 (d, J = 247.5 Hz), 163.19,
201.38. Anal. (C17H16FNO4) C, H, N.
Table 3. Inhibitory Effects of 3b on Enzyme Spectrum Screen Assays
for SPPa
concn
species ( μM)
inhibtion
(%)
primary biochemical assay
CYP450, 1A2
hum
hum
hum
hum
hum
10
10
10
10
10
85
55
57
68
86
phosphodiesterase, PDE4
phosphodiesterase, PDE5
protein tyrosine kinase, YES1
UDP-glucuronosyltransferase,
UGT1A1
a hum = human. A standard error of the mean is presented where
results are based on multiple, independent determinations.
and UDP-glucuronosyltransferase 1A1 (UGT1A1). Its inhi-
bitory activity was most profound against UGT1A1. Inter-
estingly, a recent study by E. Selga identified UGT1A1 as an
important gene code in methotrexate resistance.17 The author
speculated that UGT1A1 may be associated with the drug
resistance of certain cancer cell lines. The above results
indicated that the SPP of 3b differed from that of CHM-1.
In Vivo Antitumor Activity of 15. The water-soluble mono-
phosphate of 3b (15) was evaluated in the Hep3B xenograft
nude mice model by oral route (po) at doses of 7.5, 15, and 30
(mg/kg)/day. As shown in Figure 9A-C, 15 induced dose- and
time-dependent inhibition of Hep3B tumor growth. Signifi-
cant tumor growth suppression, at an extent exceeding that
observed after 10 (mg/kg)/day oral dosing of doxorubicin, was
detected after 7.5 (mg/kg)/day oral dosing of 15. Nearly
complete tumor suppression was observed after 30 (mg/kg)/
day oral dosing. During the course of antitumor evaluation, no
significant body weight changes were detected in either the
tested or the control mice. Similarly, the dose- and time-
dependent antitumor test results by iv administration, sum-
marized in Figure 9D-F, resembled those obtained by oral
administration and generally indicated slightly better antitu-
mor activity. Overall, the antitumor activity of 15 admini-
strated po or iv is comparable with that of CHM-1-P-Na.
N-(2-Acetyl-3,4-dimethoxyphenyl)-3-fluorobenzamide (9b). 9b
was obtained from 7a and 3-fluorobenzoyl chloride (8b). Yellow
solid. Yield: 65.0%. Mp 98-99 ꢀC. MS (EI, 70 eV): m/z 317
(Mþ). 1H NMR (DMSO-d6, 200 MHz): δ 2.43 (s, 3H), 3.76
(s, 3H), 3.81 (s, 3H), 7.03-7.15 (m, 2H), 7.39-7.71 (m, 4H), 10.18
(s, 1H). 13C NMR (DMSO-d6, 50 MHz): δ 31.74, 56.45, 61.38,
114.30, 114.73 (d, J = 23 Hz), 119.04 (d, J = 21 Hz), 121.98,
124.13, 126.91, 131.12 (d, J= 7.5 Hz), 132.27, 136.89 (d, J=6.5Hz),
Conclusion
Novel 2-PQ analogues (1-5) were designed, synthesized,
and evaluated for antitumor activity. Preliminary SAR of the

8054 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Chou et al.
Figure 9. (A) Mean tumor volume-time profiles, (B) mean tumor weight-time profiles, and (C) mean body weight-time profiles in Hep3B
xenograft nude mice (n = 11) following po dosing of doxorubicin at 10 mg/kg and 15 at 7.5, 15, and 30 mg/kg 5 days per week for 4 consecutive
weeks. (D) Mean tumor volume-time profiles, (E) mean tumor weight-time profiles (F), and mean body weight-time profiles in Hep3B
xenograft nude mice (n = 11) following iv dosing of doxorubicin at 5 mg/kg and 15 at 7.5, 15, and 30 mg/kg 5 days per week for 4 consecutive
weeks.
145.90, 150.65, 162.40 (d, J = 243 Hz), 164.67, 201.22. Anal.
(C17H16FNO4) C, H, N.
(m, 1H), 7.85 (d, J = 8.8 Hz, 1H), 11.50 (s, 1H). 13C NMR
(DMSO-d6, 50 MHz): δ 32.53, 102.57, 111.92, 112.52, 114.74,
116.94 (d, J=22.5 Hz), 123.55 (d, J=12.5 Hz), 125.41, 130.98,
131.91, 134.04 (d, J = 8.5 Hz), 144.46, 149.00, 157.18, 162.22,
199.61. Anal. (C16H12FNO4) C, H, N.
N-(2-Acetyl-3,4-methylenedioxyphenyl)-3-fluorobenzamide (9e).
9e was obtained from 7b and 8b. Yellow solid. Yield: 95.0%. Mp
170-171 ꢀC. MS (EI, 70 eV): m/z 301 (Mþ). 1H NMR (DMSO-
d6, 200 MHz): δ 2.56 (s, 3H), 6.14 (s, 2H), 7.13 (d, J = 8.4 Hz,
1H), 7.43 (t, J=8.6 Hz, 1H), 7.52-7.68 (m, 2H), 7.72 (d, J=8.6
Hz, 2H), 11.56 (s, 1H). 13C NMR (DMSO-d6, 50 MHz): δ 32.48,
102.61, 112.48, 112.62, 114.48 (d, J=23 Hz), 114.89, 119.30 (d,
J = 21.5 Hz), 123.56, 131.52 (d, J = 8.0 Hz), 131.96, 137.32,
144.61, 148.88, 162.61 (d, J = 243.5 Hz), 163.97, 199.88. Anal.
(C16H12FNO4) C, H, N.
N-(2-Acetyl-3,4-dimethoxyphenyl)-4-fluorobenzamide (9c). 9c
was obtained from 7a and 4-fluorobenzoyl chloride (8c). Yellow
solid. Yield: 64.7%. Mp 146-147 ꢀC. MS (EI, 70 eV): m/z 317
(Mþ). 1H NMR (DMSO-d6, 200 MHz): δ 2.43 (s, 3H), 3.76 (s,
3H), 3.81 (s, 3H), 7.03-7.14 (m, 2H), 7.26-7.35 (m, 2H),
7.88-7.95 (m, 2H), 10.14 (s, 1H). 13C NMR (DMSO-d6, 50
MHz): δ 31.76, 56.48, 61.38, 114.33, 115.83 (d, J = 22 Hz),
121.91, 127.17, 130.65 (d, J = 9.0 Hz), 131.06, 132.21, 145.90,
150.53, 164.57 (d, J = 247 Hz), 164.94, 201.25. Anal.
(C17H16FNO4) C, H, N.
N-(2-Acetyl-3,4-methylenedioxyphenyl)-2-fluorobenzamide (9d).
9d was obtained from 7b9 and 8a. Yellow solid. Yield: 90.0%.
Mp 165-166 ꢀC. MS (EI, 70 eV): m/z 301 (Mþ). 1H NMR
(DMSO-d6, 200 MHz): δ 2.53 (s, 3H), 6.13 (s, 2H), 7.12 (d, J=
8.6 Hz, 1H), 7.28-7.38 (m, 2H), 7.56-7.61 (m, 1H), 7.72-7.82
N-(2-Acetyl-3,4-methylenedioxyphenyl)-4-fluorobenzamide (9f).
9f was obtained from 7b and 8c. Yellow solid. Yield: 84.0%.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8055
Mp 185-186 ꢀC. MS (EI, 70 eV): m/z 301 (Mþ). 1H NMR
(DMSO-d6, 200 MHz): δ 2.51 (s, 3H), 6.13 (s, 2H), 7.12 (d, J=
8.6 Hz, 1H), 7.20-7.40 (m, 2H), 7.77 (d, J = 8.6 Hz, 1H),
7.89-7.97 (m, 2H), 11.58 (s, 1H). 13C NMR (DMSO-d6,
50 MHz): δ 32.55, 102.55, 111.20, 112.61, 114.54, 116.26 (d,
J=22 Hz), 130.22 (d, J=7.0 Hz), 131.47, 132.41, 144.36, 148.97,
162.22, 164.67 (d, J = 248 Hz), 200.04. Anal. (C16H12FNO4)
C, H, N.
1H NMR (DMSO-d6, 200 MHz): δ 3.73 (s, 3H), 3.81 (s, 3H),
6.35 (s, 1H), 7.28-7.40 (m, 1H), 7.46-7.60 (m, 3H), 7.64-
7.76 (m, 2H). Anal. (C17H14FNO3) C, H, N. HPLC purity anal-
ysis. Column: Thermo ODS Hypersil, 150 mm ꢀ 4.6 mm,
5 μm. Mobile phase: 0.01 M sodium hydrogen carbonate/
acetonitrile=30/70. Detection wavelength: PDA Ch1 254 nm
at 4 nm. Retention time: 2.067 min. Flow rate: 1.0 mL/min.
Purity: 98.54%.
2-Amino-4-benzyloxy-5-methoxyacetophenone (7c). To a solu-
tion of 1010 (1.0 g, 3.32 mmol) in anhydrous EtOH (100 mL) was
added SnCl2 2H2O (3.7 g, 16.4 mmol). The mixture was refluxed
2-(4-Fluorophenyl)-5,6-dimethoxyquinolin-4-one (1c). 1c was
obtained from 9c. White needles. Yield: 54.6%. Mp 227-
229 ꢀC. MS (EI, 70 eV): m/z 299 (Mþ). IR (KBr): 1607 (CdO)
cm-1. 1H NMR (DMSO-d6, 200 MHz): δ 3.72 (s, 3H), 3.80 (s,
3H), 6.26 (s, 1H), 7.31-7.40 (m, 2H), 7.44-7.54 (m, 2H),
7.83-7.90 (m, 2H). Anal. (C17H14FNO3) C, H, N. HPLC purity
analysis. Column: Thermo ODS Hypersil, 150 mm ꢀ 4.6 mm,
5 μm. Mobile phase: 0.01 M sodium hydrogen carbonate/
acetonitrile = 30/70. Detection wavelength: PDA Ch1 254 nm
at 4 nm. Retention time: 2.054 min. Flow rate: 1.0 mL/min.
Purity: 98.86%.
3
for 2 h and then cooled to 25 ꢀC and poured in 5% NaHCO3
solution. The precipitate was collected and washed with H2O and
then extracted with EtOAc. The extract was washed with H2O,
dried over MgSO4, and evaporated. The crude product was
purified by column chromatography (SiO2, n-hexane/EtOAc =
1:1) to give 7c. Yellow solid. Yield: 72.2%. Mp 135-137 ꢀC. 1H
NMR (DMSO-d6, 200 MHz): δ 2.39 (s, 3H), 3.66 (s, 3H), 5.03 (s,
2H), 6.38 (s,1H), 7.05 (s, 2H), 7.10 (s, 1H), 7.30-7.50 (m, 5H). 13
C
NMR (DMSO-d6, 50 MHz): δ 28.21, 56.94, 69.87, 100.05, 109.70,
115.40, 128.34, 128.49, 128.93, 136.83, 139.45, 148.74, 154.64,
198.06. Anal. (C16H17NO3) C, H, N.
2-(2-Fluorophenyl)-5,6-methylenedioxyquinolin-4-one (2a). 2a
was obtained from 9d. Yellow solid. Yield: 47.6%. Mp 282-283 ꢀC.
MS (EI, 70 eV): m/z 283 (Mþ). IR (KBr): 1605 (CdO) cm-1
.
1H NMR (DMSO-d6, 200 MHz): δ 5.92 (s, 1H), 6.11 (s, 2H),
7.09 (d, J=8.8 Hz, 1H), 7.27-7.38 (m, 3H), 7.55-7.70 (m, 2H),
11.71 (s, 1H). Anal. (C16H10FNO3) C, H, N. HPLC purity anal-
ysis. Column: Thermo ODS Hypersil, 150 mm ꢀ 4.6 mm, 5 μm.
Mobile phase: methanol/0.05% TFA in water = 75/25. Detec-
tion wavelength: PDA Ch1 254 nm at 4 nm. Retention time:
2.377 min. Flow rate: 1.0 mL/min. Purity: 99.16%.
N-(2-Acetyl-5-benzyloxy-4-methoxyphenyl)-2-fluorobenzamide
(9g). 9g was obtained from 7c and 8a. Yellow solid. Yield:
1
89.0%. Mp 142-143 ꢀC. MS (EI, 70 eV): m/z 393 (Mþ). H
NMR (DMSO-d6, 200 MHz): δ 2.60 (s, 3H), 3.82 (s, 3H), 5.16 (s,
2H), 7.10-7.50 (m, 8H), 7.56-7.67 (m, 1H), 7.80-7.89 (m, 1H),
8.57 (s, 1H), 12.45 (d, J = 4.0 Hz,1H). 13C NMR (DMSO-d6,
50 MHz): δ 29.08, 56.42, 70.41, 105.31, 115.28, 116.05, 117.08
(d, J = 22 Hz), 123.27 (d, J = 12.5 Hz), 125.60, 128.58, 128.95,
131.20, 134.45 (d, J = 8.5 Hz), 135.77, 136.50, 144.46, 152.98,
157.26, 162.35, 201.78. Anal. (C23H20FNO4) C, H, N.
2-(3-Fluorophenyl)-5,6-methylenedioxyquinolin-4-one (2b). 2b
was obtained from 9e. White solid. Yield: 44.9%. Mp 286-288 ꢀC.
MS (EI, 70 eV): m/z 283 (Mþ). IR (KBr): 1609 (CdO) cm-1
.
1H NMR (DMSO-d6, 200 MHz): δ 6.11 (s, 2H), 6.19 (s, 1H),
7.19-7.36 (m, 3H), 7.55-7.67 (m, 3H), 11.71 (s, 1H). Anal.
(C16H10FNO3) C, H, N. HPLC purity analysis. Column: Thermo
ODS Hypersil, 150 mmꢀ4.6 mm, 5 μm. Mobile phase: methanol/
0.05% TFA in water = 75/25. Detection wavelength: PDA
Ch1 254 nm at 4 nm. Retention time: 2.516 min. Flow rate:
1.0 mL/min. Purity: 98.63%.
N-(2-Acetyl-5-benzyloxy-4-methoxyphenyl)-3-fluorobenzamide
(9h). 9h was obtained from 7c and 8b. Yellow solid. Yield:
1
86.6%. Mp 162-163 ꢀC. MS (EI, 70 eV): m/z 393 (Mþ). H
NMR (DMSO-d6, 200 MHz): δ 2.62 (s, 3H), 3.81 (s, 3H), 5.15 (s,
2H), 7.26-7.52 (m, 7H), 7.54-7.78 (m, 3H), 8.51 (s, 1H), 12.70
(s, 1H). 13C NMR (DMSO-d6, 50 MHz): δ 29.10, 56.42, 70.40,
104.74, 114.45, 115.29, 115.83, 119.59 (d, J = 21.5 Hz), 123.36,
128.53, 128.95, 131.74 (d, J=7.5 Hz), 136.28, 136.45, 137.30 (d,
J = 6.5 Hz), 144.43, 153.26, 162.71 (d, J = 244 Hz), 163.91,
202.48. Anal. (C23H20FNO4) C, H, N.
2-(4-Fluorophenyl)-5,6-methylenedioxyquinolin-4-one (2c). 2c
was obtained from 9f. White solid. Yield: 45.9%. Mp 286-288 ꢀC.
MS (EI, 70 eV): m/z 283 (Mþ). IR (KBr): 1613 (CdO) cm-1
.
1H NMR (DMSO-d6, 200 MHz): δ 6.10 (s, 3H), 7.17-7.31 (m,
2H), 7.32-7.41 (m, 2H), 7.78-7.85 (m, 2H), 11.46 (s, 1H). Anal.
(C16H10FNO3) C, H, N. HPLC purity analysis. Column: Thermo
ODS Hypersil, 150 mmꢀ4.6 mm, 5 μm. Mobile phase: methanol/
0.05% TFA in water = 75/25. Detection wavelength: PDA
Ch1 254 nm at 4 nm. Retention time: 2.500 min. Flow rate:
1.0 mL/min. Purity: 97.93%.
7-Benzyloxy-2-(2-fluorophenyl)-6-methoxyquinolin-4-one (6a).
6a was obtained from 9g. White solid. Yield: 60.5%. Mp 132-
134 ꢀC. MS (EI, 70 eV): m/z 375 (Mþ). 1H NMR (DMSO-d6, 200
MHz): δ 3.82 (s, 3H), 5.16 (s, 2H), 6.21 (s, 1H), 7.20-7.80 (m,
11H). 13C NMR (DMSO-d6, 50 MHz): δ 56.02, 70.40, 101.86,
104.14, 108.80, 116.77 (d, J = 21.5 Hz), 118.86, 123.30 (d, J =
13 Hz), 125.43, 128.50, 128.97, 131.24, 132.56 (d, J = 8.0 Hz),
136.58, 137.08, 144.73, 147.73, 152.52, 159.64 (d, J = 247 Hz),
174.57. Anal. (C23H18FNO3) C, H, N.
7-Benzyloxy-2-(3-fluorophenyl)-6-methoxyquinolin-4-one (6b).
6b was obtained from 9h. White solid. Yield: 64.3%. Mp 154-
155 ꢀC. MS (EI, 70 eV): m/z 375 (Mþ). 1H NMR (DMSO-d6, 200
MHz): δ 3.83 (s, 3H), 5.17 (s, 2H), 6.56 (s, 1H), 7.30-7.50 (m,
8H), 7.55-7.60 (m, 1H), 7.60-7.80 (m, 2H). 13C NMR (DMSO-
d6, 50 MHz): δ 56.07, 70.45, 102.27, 103.72, 106.03, 114.71 (d,
J = 23.5 Hz), 117.56 (d, J = 20.5 Hz), 118.44, 123.95, 128.56,
128.99, 131.50, 136.49, 137.41, 148.02, 148.44, 152.72, 165.13,
173.61. Anal. (C23H18FNO3) C, H, N.
N-(2-Acetyl-5-benzyloxy-4-methoxyphenyl)-4-fluorobenzamide
(9i). 9i was obtained from 7c and 8c. Yellow solid. Yield: 67.1%.
Mp 168-169 ꢀC. MS (EI, 70 eV): m/z 393 (Mþ). 1H NMR
(DMSO-d6, 200 MHz): δ 2.63 (s, 3H), 3.81 (s, 3H), 5.15 (s, 2H),
7.2-7.5 (m, 7H), 7.9-8.1 (m, 3H), 8.54 (s, 1H), 12.69 (s, 1H). 13
C
NMR (DMSO-d6, 50 MHz): δ 29.11, 56.47, 70.41, 104.73,
115.39, 115.83, 116.27, 116.72, 128.53, 128.94, 130.17 (d, J =
9.0 Hz), 132.54 (d, J = 9.5 Hz), 136.48, 136.58, 144.33, 153.33,
164.24, 166.82, 202.48. Anal. (C23H20FNO4) C, H, N.
2-(2-Fluorophenyl)-5,6-dimethoxyquinolin-4-one (1a). To a
suspension of 9a (0.50 g, 1.58 mmol) in tert-butyl alcohol
(30 mL) was added potassium tert-butoxide (1.0 g, 8.93 mmol).
The mixture was refluxed under argon for 20 h and evaporated.
The residue was treated with a 10% NH4Cl solution (30 mL).
The solid precipitate was collected and washed with n-hexane
and Me2CO. The crude product was recrystallized from MeOH
to afford 1a as yellow needles (0.27 g, 0.9 mmol). Yield: 57.1%.
Mp 215-217 ꢀC. MS (EI, 70 eV): m/z 299 (Mþ). IR (KBr): 1628
(CdO) cm-1. 1H NMR (DMSO-d6, 200 MHz): δ 3.72 (s, 3H),
3.81 (s, 3H), 6.06 (s, 1H), 7.3-7.6 (m, 5H), 7.60-7.71 (m, 1H).
Anal. (C17H14FNO3) C, H, N. HPLC purity analysis. Column:
Thermo ODS Hypersil, 150 mm ꢀ 4.6 mm, 5 μm. Mobile phase:
0.01 M sodium hydrogen carbonate/acetonitrile = 30/70. De-
tection wavelength: PDA Ch1 254 nm at 4 nm. Retention time:
1.999 min. Flow rate: 1.0 mL/min. Purity: 98.35%.
2-(3-Fluorophenyl)-5,6-dimethoxyquinolin-4-one (1b). 1b was
obtained from 9b. Yellow needles. Yield: 53.1%. Mp 190-192 ꢀC.
7-Benzyloxy-2-(4-fluorophenyl)-6-methoxyquinolin-4-one (6c).
6c was obtained from 9i. White solid. Yield: 64.4%. Mp 248-
MS (EI, 70 eV): m/z 299 (Mþ). IR (KBr): 1599 (CdO) cm-1
.
1
249 ꢀC. MS (EI, 70 eV): m/z 375 (Mþ). H NMR (DMSO-d6,

8056 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Chou et al.
200 MHz): δ 3.80 (s, 3H), 5.13 (s, 2H), 6.26 (s, 1H), 7.20-7.60
(m, 9H), 7.80-8.00 (m, 2H). 13C NMR (DMSO-d6, 50 MHz): δ
55.96, 70.36, 101.41, 104.51, 106.61, 116.30 (d, J = 21.5 Hz),
119.27, 128.56, 128.99, 130.05 (d, J = 8.0 Hz), 136.60, 147.39,
148.06, 152.19, 163.63 (d, J = 246.5 Hz), 176.10. Anal.
(C23H18FNO3) C, H, N.
5 μm. Mobile phase: methanol/0.05% TFA in water = 75/25.
Detection wavelength: PDA Ch1 254 nm at 4 nm. Retention
time: 2.384 min. Flow rate: 1.0 mL/min. Purity: 96.50%.
2-(3-Fluorophenyl)-5,6-dihydroxyquinolin-4-one (4b). 4b was
obtained from 2b. White solid. Yield: 15.0%. Mp 307-308 ꢀC.
MS (EI, 70 eV): m/z 271 (Mþ). IR (KBr): 1608.63 (CdO) cm-1
.
1H NMR (DMSO-d6, 200 MHz): δ 6.25 (s, 1H), 7.15 (d, J =
8.8 Hz, 1H), 7.30-7.50 (m, 2H), 7.50-7.80 (m, 4H), 9.72 (s, 1H),
11.60 (s, 1H). 13C NMR (DMSO-d6, 50 MHz): δ 106.38, 107.57,
114.65 (d, J = 23 Hz), 117.38 (d, J = 21.5 Hz), 120.91, 122.62,
123.91, 126.81, 131.52 (d, J = 8.5 Hz), 134.45, 137.17, 147.66,
154.36, 162.70 (d, J=242 Hz), 176.82. Anal. (C15H10FNO3) C,
H, N. HPLC purity analysis. Column: Thermo ODS Hypersil,
150 mmꢀ4.6 mm, 5 μm. Mobile phase: methanol/0.05% TFA in
water=75/25. Detection wavelength: PDA Ch1 254 nm at 4 nm.
Retention time: 2.485 min. Flow rate: 1.0 mL/min. Purity:
99.51%.
2-(2-Fluorophenyl)-5-hydroxy-6-methoxyquinolin-4-one (3a).
To a solution of 1a (0.2 g, 0.67 mmol) in CH2Cl2 (3 mL) was
added 5 mL of BCl3 solution (1 M in CH2Cl2) dropwise at 0 ( 1 ꢀC.
The mixture was stirred at 25 ( 1 ꢀC for 2 h and then poured
into crushed ice and extracted with EtOAc. The organic layer
was washed with H2O, dried over MgSO4, and evaporated. The
crude was purified by column chromatography (SiO2, CHCl3/
MeOH = 15:1) and recrystallized from MeOH to give 3a.
Yellow solid. Yield: 24.1%. Mp 268-270 ꢀC. MS (EI, 70 eV):
m/z 285 (Mþ). IR (KBr): 1604.77 (CdO) cm-1 1H NMR
.
(DMSO-d6, 200 MHz): δ 3.78 (s, 3H), 6.11 (s, 1H), 7.01 (d,
J=7.4 Hz,1H), 7.36-7.48 (m, 3H), 7.54-7.72 (m, 2H), 12.25 (s,
1H), 14.54 (s, 1H). 13C NMR (DMSO-d6, 50 MHz): δ 55.80,
106.22, 106.43, 112.88, 116.36 (d, J=23 Hz), 120.81, 121.96 (d,
J = 13.5 Hz), 125.04, 130.85, 132.67 (d, J = 8.6 Hz), 135.09,
141.02, 146.27, 149.29, 158.92 (d, J = 247.7 Hz), 181.97. Anal.
(C16H12FNO3) C, H, N. HPLC purity analysis. Column: Thermo
ODS Hypersil, 150 mm ꢀ 4.6 mm, 5 μm. Mobile phase: 0.01 M
sodium hydrogen carbonate/acetone = 30/70. Detection wave-
length: PDA Ch1 254 nm at 4 nm. Retention time: 1.954 min.
Flow rate: 1.0 mL/min. Purity: 96.04%.
2-(3-Fluorophenyl)-5-hydroxy-6-methoxyquinolin-4-one (3b).
3b was obtained from 1b and BCl3. Yellow solid. Yield:
26.7%. Mp 274-276 ꢀC. MS (EI, 70 eV): m/z 285 (Mþ). IR
(KBr): 1606.70 (CdO) cm-1. 1H NMR (DMSO-d6, 200 MHz): δ
3.77 (s, 3H), 6.33 (s, 1H), 7.11 (d, J=8.8 Hz,1H), 7.33-7.48 (m,
2H), 7.51-7.76 (m, 3H), 12.09 (s, 1H), 14.56 (s, 1H). 13C NMR
(DMSO-d6, 50 MHz): δ 57.19, 104.82, 106.97, 113.39, 115.09 (d,
J=23 Hz), 118.06 (d, J=21 Hz), 121.07, 124.32, 131.64 (d, J=
9.0 Hz), 135.61, 136.16 (d, J = 8.0 Hz), 141.49, 149.64, 150.12,
162.64 (d, J=242.5 Hz), 182.69. Anal. (C16H12FNO3) C, H, N.
HPLC purity analysis. Column: Thermo ODS Hypersil, 150 mmꢀ
4.6 mm, 5 μm. Mobile phase: 0.01 M sodium hydrogen carbonate/
acetonitrile=30/70. Detection wavelength: PDA Ch1 254 nm at
4 nm. Retention time: 1.820 min. Flow rate: 1.0 mL/min. Purity:
99.13%.
2-(4-Fluorophenyl)-5-hydroxy-6-methoxyquinolin-4-one (3c).
3c was obtained from 1c and BCl3. Yellow solid. Yield: 23.0%.
Mp 307-309 ꢀC. MS(EI, 70 eV):m/z 285 (Mþ). IR (KBr): 1610.56
(CdO) cm-1. 1H NMR (DMSO-d6, 200 MHz): δ 3.76 (s, 3H), 6.25
(s, 1H), 7.08 (d, J=9.0 Hz, 1H), 7.34-7.43 (m, 3H), 7.82-7.89 (m,
2H), 12.01 (s, 1H), 14.60 (s, 1H). 13C NMR (DMSO-d6, 50 MHz):
δ 57.19, 104.53, 106.84, 113.23, 116.47 (d, J = 22 Hz), 120.99,
130.55 (d, J=9.0 Hz), 135.62, 141.45, 149.69, 150.64, 164.02 (d,
J=247 Hz), 182.59. Anal. (C16H12FNO3) C, H, N. HPLC purity
analysis. Column: Thermo ODS Hypersil, 150 mm ꢀ 4.6 mm,
5 μm. Mobile phase: 0.01 M sodium hydrogen carbonate/
acetonitrile=30/70. Detection wavelength: PDA Ch1 254 nm at
4 nm. Retention time: 1.822 min. Flow rate: 1.0 mL/min. Purity:
98.00%.
2-(2-Fluorophenyl)-5,6-dihydroxyquinolin-4-one (4a). A solu-
tion of 2a (0.1 g, 0.35 mmol) in anhydrous MeOH (30 mL) was
hydrogenated in the presence of 10% Pd/C (0.2 g) at 25 ( 2 ꢀC
for 40 h. The catalyst was filtered off, and the filtrate was
evaporated. The crude was purified by column chromatography
(SiO2, EtOAc/MeOH = 30:1) to give 4a. White solid. Yield:
13.7%. Mp 152-154 ꢀC. MS (EI, 70 eV): m/z 271 (Mþ). IR
(KBr): 1622.13 (CdO) cm-1. 1H NMR (DMSO-d6, 200 MHz): δ
6.03 (s, 1H), 7.15 (d, J=8.8 Hz,1H), 7.30-7.70 (m, 6H), 9.72 (s,
1H), 11.76 (s, 1H). 13C NMR (DMSO-d6, 50 MHz): δ 107.67,
108.57, 116.75 (d, J = 21.5 Hz), 120.54, 122.67, 123.36, 125.42,
126.70, 131.22, 132.49, 134.35, 144.30, 154.29, 159.43 (d, J =
248.5 Hz), 176.82. Anal. (C15H10FNO3) C, H, N. HPLC purity
analysis. Column: Thermo ODS Hypersil, 150 mm ꢀ 4.6 mm,
2-(4-Fluorophenyl)-5,6-dihydroxyquinolin-4-one (4c). 4c was
obtained from 2c. White solid. Yield: 13.9%. Mp 332-334 ꢀC.
MS (EI, 70 eV): m/z 271 (Mþ). IR (KBr): 1614.42 (CdO) cm-1
.
1H NMR (DMSO-d6, 200 MHz): δ 6.18 (s, 1H), 7.14 (dd, J =
9.0, 2.8 Hz, 1H), 7.33-7.42 (m, 3H), 7.59 (d, J = 8.8 Hz, 1H),
7.79-7.86 (m, 2H), 9.70 (s, 1H), 11.59 (s, 1H). 13C NMR
(DMSO-d6, 50 MHz): δ 106.24, 107.68, 116.39 (d, J = 21.5 Hz),
120.71, 122.48, 126.73, 130.19 (d, J = 8.5 Hz), 131.38, 134.42,
148.24, 154.20, 163.70 (d, J=247.5 Hz), 176.81. Anal. (C15H10-
FNO3) C, H, N. HPLC purity analysis. Column: Thermo ODS
Hypersil, 150 mm ꢀ 4.6 mm, 5 μm. Mobile phase: methanol/
0.05% TFA in water = 75/25. Detection wavelength: PDA
Ch1 254 nm at 4 nm. Retention time: 2.502 min. Flow rate:
1.0 mL/min. Purity: 99.40%.
2-(2-Fluorophenyl)-7-hydroxy-6-methoxyquinolin-4-one (5a).
Compound 6a (0.3 g, 0.80 mmol) was allowed to react in the
same manner as described in the preparation of compound 4a to
give 5a. White solid. Yield: 61.3%. Mp 277-279 ꢀC. MS (EI,
70 eV): m/z 285 (Mþ). IR (KBr): 1622.13 (CdO) cm-1. 1H NMR
(DMSO-d6, 200 MHz): δ 3.82 (s, 3H), 6.04 (s, 1H), 7.01 (s, 1H),
7.32-7.50 (m, 3H), 7.50-7.67 (m, 2H), 10.22 (s, 1H), 11.68 (s,
1H). 13C NMR (DMSO-d6, 50 MHz): δ 55.52, 102.72, 105.37,
108.20, 116.28 (d, J =22.5 Hz), 118.07, 122.94, 124.92, 130.75,
131.99 (d, J = 7.95 Hz), 136.45, 143.61, 146.58, 151.59, 158.98
(d, J=246.9 Hz), 175.30. Anal. (C16H12FNO3) C, H, N. HPLC
purity analysis. Column: Thermo ODS Hypersil, 150 mm ꢀ
4.6 mm, 5 μm. Mobile phase: methanol/0.05% TFA in water=75/
25. Detection wavelength: PDA Ch1 254 nm at 4 nm. Retention
time: 2.667 min. Flow rate: 1.0 mL/min. Purity: 99.39%.
2-(3-Fluorophenyl)-7-hydroxy-6-methoxyquinolin-4-one (5b).
5b was obtained from 6b. White solid. Yield: 44.8%. Mp
326-328 ꢀC. MS (EI, 70 eV): m/z 285 (Mþ). IR (KBr):
1606.70 (CdO) cm-1. 1H NMR (DMSO-d6, 200 MHz): δ 3.81
(s, 3H), 6.24 (s, 1H), 7.12 (s, 1H), 7.27-7.42 (m, 2H), 7.47-7.70
(m, 3H), 10.20 (s, 1H), 11.44 (s, 1H). 13C NMR (DMSO-d6, 50
MHz): δ 55.92, 103.35, 104.70, 106.67, 114.59 (d, J = 23 Hz),
117.26 (d, J = 21 Hz), 118.85, 123.85, 131.47 (d, J = 8.0 Hz),
136.85, 137.16, 146.94, 147.33, 151.93, 162.69 (d, J=242.5 Hz),
176.37. Anal. (C16H12FNO3) C, H, N. HPLC purity analysis.
Column: Thermo ODS Hypersil, 150 mm ꢀ 4.6 mm, 5 μm.
Mobile phase: methanol/0.05% TFA in water = 75/25. Detec-
tion wavelength: PDA Ch1 254 nm at 4 nm. Retention time:
2.754 min. Flow rate: 1.0 mL/min. Purity: 99.38%.
2-(4-Fluorophenyl)-7-hydroxy-6-methoxyquinolin-4-one (5c).
5c was obtained from 6c. White solid. Yield: 42.5%. Mp
352-354 ꢀC. MS (EI, 70 eV): m/z 285 (Mþ). IR (KBr):
1610.56 (CdO) cm-1. 1H NMR (DMSO-d6, 200 MHz): δ 3.80
(s, 3H), 6.19 (s, 1H), 7.11 (s, 1H), 7.20-7.50 (m, 3H), 7.70-7.90
(m, 2H). 13C NMR (DMSO-d6, 50 MHz): δ 55.89, 103.50,
104.55, 106.18, 116.30 (d, J = 21.5 Hz), 118.41, 130.03 (d, J =
8.5 Hz), 131.57, 137.22, 147.00, 148.01, 152.21, 163.58 (d, J =
246 Hz), 175.93. Anal. (C16H12FNO3) C, H, N. HPLC purity
analysis. Column: Thermo ODS Hypersil, 150 mm ꢀ 4.6 mm,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8057
5 μm. Mobile phase: methanol/0.05% TFA in water = 75/25.
Detection wavelength: PDA Ch1 254 nm at 4 nm. Retention
time: 2.736 min. Flow rate: 1.0 mL/min. Purity: 99.40%.
2-(3-Fluorophenyl)-6-methoxyquinoline-4,5-diyl Bis(dibenzyl
phosphate) (12). To a stirred solution of 3b (0.12 g, 0.42 mmol)
in dry THF (20 mL) was added NaH (96 mg, 4 mmol) at 0 (
1 ꢀC. After the mixture was stirred for 1 h, tetrabenzyl pyrophos-
phate (11) (430 mg, 0.8 mmol) was added and stirring was
continued for 25 min. The reaction mixture was filtered and
washed with CH2Cl2. The filtrate was concentrated under
vacuum at a temperature below 30 ꢀC. The residue was purified
by column chromatography (SiO2, n-hexane/EtOAc) to give 12.
Liquid. Yield: 95.0%. MS (EI, 70 eV): m/z 805 (Mþ). 1H NMR
(DMSO-d6, 200 MHz): δ 3.87 (s, 3H), 5.10 (s, 2H), 5.14 (s, 2H),
5.18 (s, 2H), 5.22 (s, 2H), 7.20-7.36 (m, 21H), 7.47-7.60 (m,
1H), 7.72-7.84 (m, 4H), 8.01 (d, J = 9.4 Hz, 1H). 13C NMR
(DMSO-d6, 50 MHz): δ 57.27, 69.63, 69.74, 70.12, 70.23, 110.20,
113.57, 114.03, 116.23, 116.92, 117.35, 119.48, 123.28, 128.10,
128.38, 128.70, 128.79, 128.85, 128.95, 131.35, 131.51, 135.79,
135.94, 136.32, 136.47, 140.41, 140.56, 145.39, 149.74, 149.82,
153.44, 153.57, 153.92, 160.71, 165.56. Anal. (C44H38FNO9P2)
C, H, N.
X-100, 137 mM NaCl, 10 mM NaF, 1 mM EGTA, 5 mM
EDTA, 1 mM sodium pyrophosphate, 20 mM Tris-HCl, pH 7.9,
100 mM β-glycerophosphate, 1 mM sodium orthovanadate,
0.1% SDS, 10 μg/mL aprotinin, 1 mM phenylmethylsulfonyl
fluoride, and 10 μg/mL leupeptin). The suspensions were centri-
fuged at 9000g for 1 min in a model 3200 Eppendorf/Brinkman
centrifuge, and the supernatant fraction was subsequently centri-
fuged at 10000g for 60 min. Protein content was determined
against a standardized control, using the Bio-Rad protein assay
kit (Bio-Rad Laboratories).
Molecular Modeling Study. Molecular flexible docking was
performed by Dock 5.1.22 The Kollman partial charges were
applied to protein models for force field calculation. Energy-
optimized 3D coordinates and partial charges of small mole-
cules were calculated by Marvin 5.2.2,23 Balloon 0.6,24 and
OpenBabel 2.2.3.25 There were 1000 orientations searched and
200 conformers generated per cycle identified in the Dock
program. The docked conformers were then rescored by
HotLig26 to predicted protein-ligand binding modes. Protein
structural superimposition was calculated and represented by
Chimera 1.4.1.27
Enzyme Spectrum Screen Assay.28 MDS PharmaServices
performed this testing under standard protocols.
Dibenzyl 2-(3-Fluorophenyl)-6-methoxy-4-oxo-1,4-dihydro-
quinolin-5-yl phosphate (13). A suspension of 12 (2.42 g,
3.0 mmol) in anhydrous MeOH (10 mL) was stirred at 25 ꢀC for
24 h. The precipitates were collected and purified by column
chromatography (SiO2, n-hexane/EtOAc) to give 13. Yellow
solid. Yield: 80.0%. Mp 136-138 ꢀC. MS (ESI): m/z 544.5
In Vivo Antitumor Activity Assay. The Hep-3B tumor cell line
was purchased from American Type Culture Collection (ATCC
HB-8064, human ovarian carcinoma cells). A culture medium of
DMEM, 90%, and fetal bovine serum, 10%, supplemented with
1% penicillin-streptomycin was used. The tumor cells were
incubated in an atmosphere containing 5% CO2 at 37 ꢀC.
Balb/c Nude mice used in this study were male, 4-6 weeks
age, weighing 18-20 g and provided by National Animal
Center. All animals were housed in individually ventilated cages
racks (IVC Racks, 36 Mini Isolator system) under specific
pathogen-free (SPF) conditions throughout the experiment.
Each cage (26.7 cm length ꢀ 20.7 cm width ꢀ 14.0 cm height)
was sterilized with autoclave and contained eight mice. The
animals were maintained in a hygienic environment under
controlled temperature (20-24 ꢀC) and humidity (40%-70%)
with a 12 h light/dark cycle. The animals were given free access
to sterilized lab chow and sterilized distilled water ad libitum. All
aspects of this work, i.e., housing, experimentation, and disposal
of animals, were performed in general accordance with the
Guide for the Care and Use of Laboratory Animals (National
Academy Press, Washington, DC, 1996).
In the xenograft tumor model of human ovarian carcinoma
cell lines (Hep-3B, ATCC HB-8064) in male Balb/c Nude mice,
compound 15 at doses 7.5, 15, and 30 mg/kg (iv or po, q.d.) was
administered 5 days per week for 4 consecutive weeks and ceased
at day 28. The tumor size and body weight were monitored and
recorded for 28 days. Human ovarian carcinoma cells (HEP-3B,
ATCC HB-8064) with 2 ꢀ 106 cells in 0.1 mL were injected
subcutaneously into the right flank of the mice. When the tumor
growth reached >100 mm3 in volume (assumed as day 0), the
tumor-bearing animals were assigned into several groups (eight
animals in each group) for study.
1
(M - H)-. H NMR (DMSO-d6, 200 MHz): δ 3.75 (s, 3H),
5.28 (s, 2H), 5.31 (s, 2H), 6.27 (s, 1H), 7.26-7.50 (m, 11H),
7.50-7.78 (m, 6H). 13C NMR (DMSO-d6, 50 MHz): δ 57.19,
69.32, 69.44, 108.51, 114.46, 114.93, 116.74, 117.38, 119.24,
123.92, 128.04, 128.51, 128.82, 131.49, 131.65, 136.74, 137.07,
137.23, 147.00, 160.29, 176.88. Anal. (C30H25FNO6P) C, H, N.
2-(3-Fluorophenyl)-6-methoxy-4-oxo-1,4-dihydroquinolin-5-yl
Dihydrogen Phosphate (14). A suspension of 13 (250 mg, 0.46 mmol)
in anhydrous MeOH (10 mL) was hydrogenated in the presence of
10% Pd/C (125 mg) at 25 ꢀC for 15 min. The catalyst and precipitate
were collected and dissolved in 10% NaHCO3 solution and then
filtered. The filtrate was acidified with dilute HCl and the precipitate
was then collected and washed with acetone to give 14. Yellow solid.
Yield: 63.7%. Mp 179-181 ꢀC. MS(ESI): m/z 366 (M þ H)þ.
1H NMR (D2O þ NaOD, 200 MHz): δ 3.76 (s, 3H), 6.53 (s, 1H),
7.05 (t, J=8.4 Hz, 1H), 7.24-7.60 (m, 5H). Anal. (C16H13FNO6P)
C, H, N.
Sodium 2-(3-Fluorophenyl)-6-methoxy-4-oxo-1,4-dihydroqui-
nolin-5-yl Phosphate (15). To a stirred solution of NaHCO3
(0.34 g, 4.0 mmol) in H2O (20 mL) was added 14 (0.73 g, 2.0
mmol) at 0 ( 1 ꢀC. After the addition was complete, the reaction
mixture was removed from the ice bath, stirred at 25 ꢀC for 10
min, and then filtered though Celite, after no dissolution from
the solid was observed. The resulting filtrate (15 mL) was poured
into acetone (60 mL) and kept in an ice bath for 1 h. The
precipitate was collected and washed with ice-cooled acetone (10
mLꢀ5). The solid was dried under vacuum to give 15. Yellow
solid. Yield: 48.0%. Mp >300 ꢀC. MS(ESI): m/z 410 (M þ H)þ.
1H NMR (D2O, 200 MHz): δ 3.72 (s, 3H), 6.54 (s, 3H), 6.99 (t,
J=7.8 Hz, 1H), 7.15-7.55 (m, 5H). Anal. (C16H11FNNa2O6P)
C, H, N.
The body weight and tumor size were measured and recorded
every 7 days during the experiment period of 28 days. Tumor
volume (mm3) was estimated according to the formula of
length ꢀ (width)2 ꢀ 0.5 in mm3. Tumor growth inhibition was
calculated as T/C (treatment/control) by the following formula:
T/C=(Tn - T0)/(Cn - C0)ꢀ100% (T0, tumor volume of treated
group on day 0; Tn, tumor volume of treated group on day n; C0,
tumor volume of control group on day 0; Cn, tumor volume of
control group on day n).
Biological Assays. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium Bromide) Assays.20,21 HL-60, HCT-116, Hep
3B, and H460 cells were treated with tested compounds for
the indicated periods. After treatment, cells were washed once
with PBS and incubated 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.
Acknowledgment. The investigation was supported by re-
search grants from the National Science Council of the
Republic of China awarded to S.-C.K. (NSC Grant 96-2323-
B-039-001, NSC Grant 97-2323-B-039-001) and to L.-J.H.
(NSC Grant 95-2320-B039-011-MY3). The work was also
Mechanism of Action Study. Hep3B cells (5.3 ꢀ 107) were
treated with 3b at different concentration or duration and then
placed in 1 mL of the lysis buffer (10% glycerol, 1% Triton

8058 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Chou et al.
supported by Grant CA17625-32 from the National Cancer
Institute, NIH (K.-H.L.).
(13) Mayer, S. C.; Banker, A. L.; Boschelli, F.; Di, L.; Johnson, M.;
Kenny, C. H.; Krishnamurthy, G.; Kutterer, K.; Moy, F.; Petusky,
S.; Ravi, M.; Tkach, D.; Tsou, H. R.; Xu, W. Lead identification to
generate isoquinolinedione inhibitors of insulin-like growth factor
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(14) Wood, E. R.; Shewchuk, L.; Hassel, A.; Nichols, J.; Truesdale,
A. T.; Smith, D.; Carter, H. L.; Weaver, K.; Barrett, G.; Leesnitzer,
T.; Alvarez, E.; Bardera, A. I.; Alamillo, A.; Cantizani, J.; Martin,
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Supporting Information Available: Literature references for
enzyme spectrum screen assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
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