Discovery of a novel series of quinolone and naphthyridine derivatives as potential topoisomerase I inhibitors by scaffold modification

Article abstract of DOI:10.1021/jm900469e

A novel series of topoisomerase I (Top I) inhibitors were designed on the basis of camptothecin using scaffold modification strategy. Thirty-one new compounds were synthesized and evaluated for anticell proliferation activity. The most potent compound 26

Full text of DOI:10.1021/jm900469e

J. Med. Chem. 2009, 52, 5649–5661 5649
DOI: 10.1021/jm900469e
Discovery of a Novel Series of Quinolone and Naphthyridine Derivatives as Potential Topoisomerase I
Inhibitors by Scaffold Modification
Qi-Dong You,*,† Zhi-Yu Li,† Chiung-Hua Huang,§ Qian Yang,† Xiao-Jian Wang,† Qing-Long Guo,† Xiao-Guang Chen,‡
Xun-Gui He,† Tsai-Kun Li, and Ji-Wang Chern*,§,^
†School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China, ‡Institute of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College, Beijing 100050, China, §School of Pharmacy and Department of Microbiology, College of Medicine
and ^Department of Life Science, College of Life Science, National Taiwan University, No. 1, Section 1, Ren-Ai Road, Taipei 100, Taiwan
Received March 17, 2009
A novel series of topoisomerase I (Top I) inhibitors were designed on the basis of camptothecin using
scaffold modification strategy. Thirty-one new compounds were synthesized and evaluated for anticell
proliferation activity. The most potent compound 26 presented a significant inhibitory effect on Top I,
leading to Top I-mediated cleavage and influences on Top I expression at the cellular level. Moreover, 26
was proved to induce cell death via apoptosis and accelerated DNA strand breaks without significant
alteration in cell cycle populations. All of the experimental results herein indicated that 26 could interact
with DNA-Top I complex and induce cancer cell apoptosis to produce antitumor effects. The in vivo
evaluation of 26 on the growth of HT-29 tumor xenografts in nude mice suggested its therapeutic
potential for further development.
Introduction
DNA topoisomerase I (Top I)a is ubiquitous and essential in
mammals, and this enzyme is involved in tumor growth.
now in clinical evaluation. In view of the successful clinical
cases of these non-CPT derivatives, it was reasoned that the
better chemical stability at longer lifetimes of the trapped
cleavage complex of the non-CPT Top I inhibitors was due
to the absence of a lactone ring in their skeleton.12 The scaffold
modification approach has been generally accepted as a means
to transform one pharmacophoric template into another.13 The
rationale behind this type of transformation is that the new
template will retain some of the characteristics of the original
template but exhibit improved potency, selectivity, and phar-
macokinetics. Consequently, we attempted to synthesize com-
pounds lacking the lactone ring of 1 and change the rigid
framework of 1 into a combination of the quinolone skeleton
and benzimidazole group or its bioisoesters having a single
carbon bond (Figure 1A). The target scaffold depicted in
Figure 1B possesses an identical arrangement and similar
distance of every H-bond donors and acceptors when com-
pared with 1. We reasoned that the fluoro atom and piperazine
on the skeleton could be the scaffold to enhance the druglike
properties: To our best knowledge, the fluoro atom could
improve the metabolic characteristics of compounds by
prolonging the half-lives, and the piperazine could enhance
the water solubility of compounds. Herein, we present the
synthesis and biological studies of a series of 3-substituted
quinoline and naphthyridine compounds as potential anti-
cancer agents by inhibiting Top I.
Traditional Top
I inhibitors, like the natural penta-
cyclic alkaloid camptothecin (CPT, 1),1,2 can bind and stabilize
a binary DNA-Top I complex containing a single-strand
nick,3,4 thus inhibiting DNA replication and eventually causing
cell death during the S phase of the cell cycle. This unique
mechanism of action coupled with the initial success of 1 in
preclinical studies provoked a flurry of research on this im-
portant natural compound in the hope of developing a novel
anticancer drug for managing human malignancies. Unfortu-
nately, clinical applications of 1 have been thwarted by the
compound’s high toxicity and poor water solubility. Therefore,
tremendous efforts have been made to develop semisynthetic
and more water-soluble analogues of 1, such as topotecan
(TPT, 3) and irinotecan (4), which are derivatives of
10-hydroxycamptothecin (HCPT, 2) and have been approved
for clinical treatment of cancer patients.5,6 However, the
therapeutic potential of 1and its derivatives is severely hindered
due to their rapid inactivation through lactone ring hydrolysis
at physiological pH (Scheme 1).7 Considering that Top I is a
proven and effective target for cancer treatment, metabolically
stable non-CPT derivatives have been developed,8-11 like
indolocarbazoles and indenoisoquinolines, some of which are
*To whom correspondence should be addressed. (Q.-D.Y.) Phone:
þ86-25-83271351. Fax: þ86-25-83271351. E-mail: youqidong@
gmail.com. (J.-W.C.) Phone: þ886-2-2393-9462. Fax: þ886-2-2393-
4221. E-mail: jwchern@ntu.edu.tw.
a Abbreviations: CPT, camptothecin; EGFP, enhanced green fluor-
escent protein; HCPT, 10-hydroxycamptothecin; MTT; 1-N-methyl-5-
thiotetrazole; PI, propidium iodide; PPA, polyphosphoric acid; SRB,
sulforhodamine B; SSGE, single-cell gel electrophoresis; Top I, topo-
isomerase I; TPT, topotecan.
Results and Discussion
Chemistry. The targets 6-36 (Table 1) were synthesized as
outlined in Scheme 2. Quinolone-3-carboxylic acids or
naphthyridine-3-carboxylic acids were condensed with o-phenyl-
enediamine, o-aminophenol, or o-aminobenzenethiol in
polyphosphoric acid (PPA) at 170-250 ꢀC to obtain 6-20.
r
2009 American Chemical Society
Published on Web 08/28/2009
pubs.acs.org/jmc

5650 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
You et al.
heated for 1-2 h at 40-45 ꢀC to afford 21-33. Compounds 21,
22, and 24 were subjected to hydrogen reduction with Pd/C to
give 34-36.
Cytotoxicity of Quinolone and Naphthyridine Derivatives in
Various Human Cancer Cell Lines. Compound 1 derivatives,
which selectively target DNA-Top I, are among the most
effective anticancer drugs (especially against solid tumors)
recently approved by the U.S. Food and Drug Administra-
tion. We used the 1-N-methyl-5-thiotetrazole (MTT)-based
assay to evaluate the antiproliferative effect of quinolone and
naphthyridine derivatives 6-36 in three cancer cell lines.
Table 1 lists cytotoxic activities of two main skeleton quino-
line and naphthyridine derivatives containing three kinds
of heterocycles, such as benzimidazole, benzoxazole, and
benzothiazole at the 3-position. Certain compounds demon-
strated cytotoxicity comparable to 2. By comparing the basic
skeletons of these three heterocycles in 6-18, the most active
structural feature relevant to cytotoxicity against the three
tumor cell lines appeared to be the 3-benzothiazole-
naphthyridine skeleton 15 with an IC50 in the 2.4-2.7 μM
range. In general, the piperidinyl side chain at the 7-position
yielded greater cytotoxicity than compounds containing the
3-methyl- or 4-methyl-piperidinyl side chains. Interestingly,
20, containing a naphthyridine nucleus and a piperidinyl side
chain with a chlorine atom at the 6-position of benzimida-
zole, was more cytotoxic than 19, which contains a quinolone
nucleus. We presumed that an electron-withdrawing group
at the 6-position of the benzimidazole ring may contribute to
the cytotoxicity. Thus, a series of 21-33 with a nitro group
at the 6-position of the benzimidazole, benzoxazole, and
benzothiazole rings was synthesized and evaluated. Most of
these compounds, except for 22, 27, and 30, exhibited better
cytotoxicity (IC50 range, 0.2-7.7 μM) than 6-18. Among
21-33, 26, a quinolone nucleus with a 3-benzoxazole sub-
stituent, had an IC50 range of 0.2-0.6 μM, better than the
other compounds containing a 3-benzimidazole or 3-benzo-
thiazole ring. Interestingly, 34 and 35, obtained by further
reduction of the nitro group of 21 and 24, had dramatically
reduced cytotoxicity, lending support to the presumption
that an electron-withdrawing group at position 6 may be
important for cytotoxicity. Notably, a methyl group on the
piperidine substituent in 6-18 reduced cytotoxicity. How-
ever, this phenomenon was not observed in the nitro-com-
pound series 21-33. Table 1 shows that 6, 7, 20, 23, 26, 28,
and 31-36 showed potential cytotoxicity with IC50 values in
the submicromolar range, and thus, we further evaluated the
cytotoxicity of these compounds against 12 human cancer
cell lines using a colorimetric sulforhodamine B (SRB)
protein dye staining method or MTT assay. On the basis of
Table 2, 26 showed the greatest potential cytotoxicity with an
IC50 range of 0.7-5.5 μM, and long-term treatment (14 days)
of HT-29 human colorectal adenocarcinoma cells with 5 μM
26 almost completely inhibited anchorage-dependent colony
formation (Figure 2).
Figure 1. Compound generation by scaffold modification. (A) Scaf-
fold designed based on 1. (B) Superimposition of target scaffold with 1.
Scheme 1. Structures of 1 Derivativesa
Effect of Quinolone and Naphthyridine Derivatives on Top I
Activity. In principle, inhibitors of DNA topoisomerases
could act at any of the three major steps comprising the
enzyme reaction mechanism, namely, enzyme binding to
DNA, cleavage of DNA strands, and religation of the
DNA following the strand-passing step.10 To investigate
the mechanism(s) by which our synthetic compounds inhi-
bited Top I and thereby caused cytotoxicity, the effect of
quinolone and naphthyridine derivatives on Top I activity
was examined by measuring the relaxation of supercoiled
a (a) Lactone ring hydrolysis of 1. (b) Clinically approved 1 derivatives.
Subsequently, 6-18 were subjected to nitration in a mixture of
concentrated H2SO4 and HNO3 (1:1.2-1.3) at 5 ꢀC and then

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5651
Table 1. Structure and in Vitro MTT Assay Results for Quinolone and Naphthyridine Derivatives
structure
cytotoxicity (IC50, μM)a
compound
no.
X
Y
Z
R1
R2
R3
KB
A2780
Bel7402
2
6
0.6 ( 0.1
10.5 ( 2.0
2.0 ( 0.3
0.7 ( 0.1
0.8 ( 0.3
4.8 ( 1.0
0.2 ( 0.1
27.4 ( 5.1
4.1 ( 0.3
NH
NH
NH
NH
O
C
N
C
C
C
C
C
N
C
N
C
C
H
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
piperazin-1-yl
piperazin-1-yl
7
H
H
H
H
H
H
H
H
H
H
H
8
F
3-methylpiperazin-1-yl
4-methylpiperazin-1-yl
chlorine
19.1 ( 4.0
36.7 ( 7.8
10.2 ( 1.5
18.9 ( 2.8
87.4 ( 11.3
11.7 ( 2.3
28.7 ( 3.2
2.4 ( 0.2
11.6 ( 0.9
>5018.2 ( 2.7
78.4 ( 8.8
83.5 ( 11.8
0.7 ( 0.2
15.3 ( 1.3
11.3 ( 0.5
2.7 ( 0.2
30.2 ( 3.5
9
H
H
F
10
11
12
13
14
15
16
17
28.9 ( 3.2
5.7 ( 1.4
2.0 ( 0.2
16.8 ( 2.0
24.3 ( 3.0
2.4 ( 0.7
24.0 ( 4.7
92.2 ( 21.1
O
3-methylpiperazin-1-yl
4-methylpiperazin-1-yl
piperazin-1-yl
O
H
O
S
H
piperazin-1-yl
piperazin-1-yl
S
S
H
4-methylpiperazin-1-yl
4-methylpiperazin-1-yl
19.0 ( 2.1
60.2 ( 11.1
114.0 ( 16.6
26.6 ( 2.2
S
(S)-CH
(CH3)CH2O-
F
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
S
C
N
C
C
N
C
C
C
C
C
N
C
N
C
C
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
Cl
3-methylpiperazin-1-yl
piperazin-1-yl
piperazin-1-yl
24.3 ( 2.7
10.3 ( 1.2
1.6 ( 0.2
1.1 ( 0.2
22.4 ( 2.7
2.3 ( 0.2
2.5 ( 1.0
0.5 ( 0.3
0.6 ( 0.2
40.3 ( 13.0
1.8 ( 0.2
0.7 ( 0.2
179.3 ( 22.2
5.8 ( 1.0
0.6 ( 0.2
9.8 ( 1.4
6.3 ( 0.7
4.9 ( 0.5
1.6 ( 0.2
12.4 ( 1.8
2.3 ( 0.4
4.7 ( 0.7
ND b
15.0 ( 2.0
21.5 ( 3.3
2.8 ( 0.5
2.1 ( 0.7
10.8 ( 1.8
1.1 ( 0.2
4.4 ( 0.9
1.8 ( 0.3
0.2 ( 0.2
50.2 ( 6.6
3.0 ( 0.5
1.8 ( 0.2
24.6 ( 6.6
7.7 ( 1.3
2.0 ( 0.4
NH
NH
NH
NH
NH
NH
O
H
H
Cl
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
NO2
piperazin-1-yl
piperazin-1-yl
F
3-methylpiperazin-1-yl
4-methylpiperazin-1-yl
chlorine
H
H
F
O
3-methylpiperazin-1-yl
4-methylpiperazin-1-yl
piperazin-1-yl
0.2 ( 0.2
39.6 ( 8.6
ND b
O
H
O
S
H
piperazin-1-yl
piperazin-1-yl
0.7 ( 0.2
200.2 ( 33.0
4.3 ( 1.3
1.2 ( 0.2
S
S
H
4-methylpiperazin-1-yl
4-methylpiperazin-1-yl
S
(S)-CH
(CH3)CH2O-
33
34
35
36
S
C
C
C
N
F
Et
Et
Et
Et
NO2
NH2
NH2
NH2
3-methylpiperazin-1-yl
piperazin-1-yl
4.7 ( 1.2
35.4 ( 5.2
>50
3.3 ( 1.2
51.5 ( 8.7
>50
4.1 ( 0.8
92.1 ( 11.6
>50
NH
NH
NH
H
H
4-methylpiperazin-1-yl
piperazin-1-yl
30.1 ( 4.1
42.3 ( 7.2
93.3 ( 13.9
a The cytotoxicity IC50 values are the concentrations corresponding to 50% cell death. b ND, not determined; KB, oral epidermal carcinoma cell line;
A2780, ovarian carcinoma cell line; and Bel-7402, hepatocellular carcinoma cells.
Scheme 2. Synthesis of Quinolone and Naphthyridine Derivativesa
a Reagents and conditions: (a) PPA, N2, 170-250 ꢀC. (b) HNO3,
H2SO4, 40-45 ꢀC. (c) Pd/C.
Figure 2. Long-term inhibitory effect of 26 showing inhibition of
anchorage-dependent colony formation. Cpd 1, CPT; Cpd 3, TPT;
and Cpd 26, compound 26.
DNA of plasmid pBR322. We also compared the inhi-
bitory activities of these series of derivatives with a known
DNA-Top I inhibitor, 1 (Figure 3). Although the inhibi-
tion at the higher 1 concentrations (100-1000 μM) was
abundant, the products were more heterogeneous as
compared with those observed at lower 1 concentrations
(Figure 3A,B). Top I inhibition was similar for 1 and

5652 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
You et al.
Table 2. In Vitro SRB Assay To Test the Effectiveness of 12 Quinolone and Naphthyridine Derivatives against 12 Cancer Cell Linesa
cell line
HeLa
nullb
HT-29
mtc
AGS
wtd
MCF-7
MDA-MB-261
A549
wt
HepG2
wt
Hep3B
null
PC-3
null
U937 THP-1 K562
mt mt null
p53 status
wt (ERþ)e
mt (ER-)f
compds (μM) GI50 IC50 GI50 IC50 GI50 IC50 GI50 IC50 GI50
IC50 GI50 IC50 GI50 IC50 GI50 IC50 GI50 IC50 IC50 IC50 IC50
1
0.04 0.2 0.03 0.4 0.01 0.02 0.01 1.1
0.08 1.6 0.08 0.4 0.1 0.3 0.8 2.0
0.08
0.9
1.7
2.0
0.01 0.04 0.1 0.2 0.01 0.06 0.7 1.4 0.3
0.9 1.9 1.2 1.6 0.4 1.4 1.3 2.0 1.1
0.6
1.5
1.4
1.7
3
6
>10 >10 >10 >10 9.5 >10 >10 >10 >10
>10
10
0.6 4.7 >10 >10 >10 >10 >10 >10 >10 >10 >10
4.9 7.8 6.8 9.1 3.7 7.8 >10 >10 >10 >10 9.1
7
6.4 10
>10 >10 2.1 6.6 8.3 9.5
3.3
0.9
1.8
0.7
20
23
26
28
31
32
33
34
35
36
1.2 4.2 1.8 4.7 4.5 1.0 4.2 4.5
1.4 2.4 4.2 7.5 2.6 8.2 2.6 4.9
1.1 2.4 1.0 5.5 0.7 3.5 3.0 6.8
2.3
5.5
1.0 0.5 1.8 3.7 0.9 2.3 3.3 4.8 4.2
0.6 2.4 1.5 4.2 1.6 5.5 7.9 9.6 >10 7.8
0.8 1.5 2.8 4.5 1.5 3.9 3.3 4.2 1.4 3.5
2.8
2.7
4.7
4.6
3.1
>10 >10 >10 >10 2.3 4.4 >10 >10 >10
>10 >10 >10 >10 2.6 4.5 >10 >10 >10
>10
>10
>10
7.4
2.1 7.3 >10 >10 >10 >10 5.8 9.8 >10 >10 >10
4.7 10 >10 >10 >10 >10 5.5 7.5 >10 >10 >10
3.9 >10 7.6 >10 >10 >10 1.8 9.9 >10 >10 >10
1.5 7.0 0.9 3.6 0.6 1.4 2.7 10 >10 >10 >10
>10 >10 >10 >10 >10 >10 >10 >10 >10 10 >10
1.0 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
0.5 >10 >10 >10 >10 >10 >10 >10 >10 8.2 >10
>10 >10 5.0 10
1.7 9.9 1.0 5.0 3.3 5.4 >10 >10 2.3
>10 >10 >10 >10 7.8 10 >10 >10 >10
7.0 8.8 >10 >10 >10
>10
>10
>10
>10 >10 >10 >10 10 >10 >10 >10 >10
>10 >10 >10 >10 6.7 9.2 >10 >10 >10
a HeLa, cervical adenocarcinoma; HT-29, colorectal adenocarcinoma; AGS, gastric adenocarcinoma; MCF-7 and MDA-MB-261, breast
adenocarcinoma; A549, lung carcinoma; HepG2 and Hep3B, hepatocarcinoma; PC-3, prostate adenocarcinoma; THP-1, acute monocytic leukemia
cells; K562, chronic myelogenous leukemia cells; and U937, acute myeloid leukemia cells. Titrated concentrations of test compounds: 10, 5, 2.5, 1.25,
0.63, 0.32, 0.15, 0.08, and 0 μM. Titrated concentrations of 1 and 3: 2, 1, 0.5, 0.25, 0.125, 0.063, 0.032, 0.015, and 0 μM. b Null, no expression. c mt, mutant
type. d wt, wild type. e ER(þ), estrogen receptor positive. f ER(-), estrogen receptor negative.
Figure 3. Effects of 1 and 26 on Top I-mediated DNA relaxation.
(A, C) The position of the origin (o) and the migration positions of
relaxed (r) and supercoiled (s) plasmids are shown on the left. (B, D)
Dose responses for the effects of 1 and 26, respectively, on Top
I-mediated DNA relaxation. The proportion of relaxed DNA to
total DNA was measured by scanning with an imaging system. Bars
Figure 4. Effects of quinolone and naphthyridine derivatives on
show the percentage of supercoiled vs relaxed DNA.
Top I-mediated DNA relaxation activity. (A) Lane 1, supercoiled
pBR322 DNA; and lane 2, with Top I but no test compounds
(Cpds). (B) Effects of test compounds on Top I-mediated DNA
relaxation. The proportion of relaxed DNA to total DNA was
measured by scanning with an imaging system. Bars show the
percentage of supercoiled vs relaxed DNA.
26 at lower concentrations up to 12.5 μM of 26 (Figure 3C,
D), but 1 was a much stronger inhibitor at higher concentra-
tions.
In parallel, we tested our other compounds for Top I
at higher concentrations of 50 μM, these compounds were
precipitated out under typical assay conditions.
Prompted by the above results, 26 was further investigated
for the influence of Top I protein expression by immuno-
inhibition (Figure 4A,B). Despite moderate inhibition of 26
and 32 on Top I at 25 μM, most of the test compounds did
not appreciably inhibit the enzyme at this concentration.
However, because of the poor solubility of these compounds,

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5653
Figure 5. Effects of 26 on Top I expression in SKOV-3 and HT-29 cells. Cells were grown on microscope slides, fixed, permeabilized, and
double-stained with Top I antibodies. Compound 26 was included at the concentrations indicated. Control, no compound added.
cytochemistry.14 Cancer cell lines HT-29 and human ovarian
cancer cells SKOV-3 were selected because 26 strongly
inhibited the proliferation (for HT-29 cells, the IC50 value
is 0.37 μg/mL; for SKOV-3 cells, the IC50 value is 0.55 μg/
mL) in the MTT assay. As shown in Figure 5, Top I protein
expression in HT-29 and SKOV-3 cells treated with 26 was
significantly lower than in control cells, and the effect was
dose-dependent, suggesting that 26 is able to both directly
inhibit Top I and lower Top I protein expression at the
cellular level.
Effect of 26 on Top I-Mediated Cleavable Complexes.
Further studies of DNA cleavage in Top I were then per-
formed in the presence of purified recombinant human Top I
and a 3k base pair radiolabeled DNA. The extent of DNA
fragmentation that occurs in the presence of Top I and
various concentrations of 26 and 1 is shown in Figure 6A
and quantitated in Figure 6B. Compounds 1 and 26 have
Top I-targeting cleavage activity, as revealed by agarose gel
electrophoresis of alkali-denatured 32P-labeled linearized
pRYG plasmid DNA. The potency of Top I-mediated
cleavage activities of 26 was less than 1; however, the
cleavage pattern induced by 26 was not identical to that of
1, which indicated that the 26 consensus sequence
was different from that of 1. These results suggested that
26 at pharmacologically relevant doses was primarily Top I
poisons with DNA cleavage patterns exhibiting differences
from 1.
Effect of 26 on DNA Distribution in HT-29 Cells. Previous
studies in rapidly proliferating cells suggested that the cyto-
toxicity of the DNA-Top I inhibitor, 1, depends on active
DNA replication.15 Thus, the effects of 1 and 3, and 26 at 1, 5,
Figure 6. Stimulation of Top I-mediated DNA cleavage by com-
and 10 μM for 6, 12, 24, and 48 h on the percentage of HT-29
pound 26. (A) A representative agarose gel from Top I-mediated
cells in each phase of the cell cycle were evaluated via flow
cytometry. As shown in representative histograms presented
DNA cleavage carried out in the presence of positive compound 1
and compound 26. (B) Quantitative determination of the relative
intensity of the full length DNA substrate band using an imaging
system.
in Figure 7A-D, short-term (6, 12, and 24 h) and a low dose
(0.1 μM) of the Top I inhibitors 1 and 3 caused an increase in
the percentage of HT-29 cells in the S phase because of
diminished DNA synthesis. Prolonged exposure up to 48 h
resulted in an overall increase in the number of cells in the S
and sub-G1 phases, suggesting that 1 and 3 are highly
effective to HT-29 cells, shifting the cell cycle distribution
to the early S phase followed by induction of apoptosis.
Interestingly, 26-induced cytotoxicity did not coincide with a
significant alteration in cell cycle populations, as only 5% of
treated (up to 10 μM) cells were in the S phase, which was
similar to control (Figure 7E). To quantify the degree
of apoptosis, cells in the sub-G1 peak were counted and
expressed as a percentage of the total cell population
(Figure 7F). Remarkably, only about 4.4% of the control
cells were in the apoptotic peak at 48 h, whereas 0.1 μM 1,
0.1 μM 3, and 1 μM 26 increased the number of apoptotic
cells to 44.8, 40.5, and 35.5%, respectively. Compound 26
potently reduced the number of HT-29 cells with diploid
DNA content (G0/G1 region) as compared with control, and
it caused a corresponding increase in the number of cells with
hypodiploid DNA content (sub-G1 region), indicative of
apoptosis and necrosis. These cytotoxicity results indicated

5654 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
You et al.
Figure 7. Quantification of cell cycle distribution of HT-29 cells by flow cytometry. (A-D) DNA histograms of HT-29 cells at the indicated
time points after the addition of 26. (a) Control, (b) 1 (0.1 μM), (c) 3 (0.1 μM), (d) 26 (1 μM), (e) 26 (5 μM), and (f) 26 (10 μM). (E) The
population in the S phase. (F) The population in the sub-G1 phase.
that exposure of exponentially growing HT-29 cells to 26
induced time-dependent apoptosis and growth inhibition
and that the mechanism of 26’s toxicity in mammalian cells
was distinct from that of 1.
The effect of 26 on induction of apoptosis was also
evaluated by cell morphology (Figure 8A) and fluore-
scence-activated cell sorting (Figure 8B). Visual assessment
of the immunostaining suggested that the majority of SKOV-
3 and HT-29 cells were severely distorted or elongated and
displayed membrane blebbing and cell shrinkage, and some
cells turned round within 24 h after treatment with 26 (0.25
and 5 μg/mL), whereas the untreated control group dis-
played a normal shape and well-ordered cytoskeleton under
the same conditions. Additionally, flow cytometry with the
fluorescein isothiocyanate Annexin V/propidium iodide (PI)
double staining assay was carried out to evaluate whether 26-
induced cell death was due to apoptosis or necrosis. The
percentage of apoptotic cells (Annexin Vþ/PI- staining) in
the control group was 4.5% for SKOV-3 cells and 20.7% for
HT-29 cells (Figure 8B-D). After treatment with 5 and

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Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5655
Figure 8. Compound 26 induces apoptosis of SKOV-3 and HT-29 cells. (A) Morphological changes of SKOV-3 (a-c) and HT-29 (d-f) cells
after treatment with 2.5 or 5.0 μg/mL of 26. (B) Compound 26 mediates SKOV-3 (a-c) and HT-29 (d-f) cell death. SKOV-3 and HT-29 cells
were incubated in vitro without or with 5 or 10 μg/mL of 26 for 24 h and analyzed by Annexin V/PI cell viability assay. (C, D) Apoptotic (lower
right quadrant) and necrotic (upper right quadrant) cells were identified and expressed as a percentage of total dead cells.
10 μg/mL of 26 for 24 h, the percentage of apoptotic cells
increased to 7.4 and 13.6%, respectively, in the SKOV-3 cell line
and increased to 23.5 and 35.9%, respectively, in the HT-29 line
(Figure 8B-D). This assessment correlated well with the above
DNA distribution results showing that 26 reduced the diploid
DNA content and correspondingly increased the hypodiploid
DNA content in HT-29 cells. Taken together, these results
demonstrated that 26 induced cell death mainly via apoptosis.
We next evaluated the possibility that the pro-apoptotic
activity of 26 in HT-29 cells was attributable to a higher level
of induced DNA damage and/or altered DNA repair. The
potential of 26 to induce DNA strand breaks in HT-29 cells
was assessed via Comet assay using the Top I poison 1 as a
positive control. At a concentration of 10 μM for 1 h, 26
caused a significant increase in DNA strand breaks (48%) as
compared with untreated cells (9%); the percentage was 84%
for 1-treated cells (Figure 9A,B).
It is generally agreed that S phase arrest was a common
hallmark of the death induced by DNA-Top I inhibitors.
Taking all data above together, this novel lactone-free
compound 26 induced DNA damage and apoptosis despite
no alteration of cell cycle progression. According to the data
from Top I-mediated cleavage and DNA distribution on HT
29 cell lines, it appeared that 26 trapped Top I-DNA
covalent complexes with a potency and sequence selectivity
different to that of 1. Because it was previously reported that
certain quinolone compounds possessed inhibitory activity
against Top II,16 we subsequently evaluated the effects of 26
against purified calf thymus Top II by relaxation of pRYG
DNA according to the literature.17 However, no inhibitory
effect of 26 on Top II at 200 μM was observed (data not
shown). Therefore, we speculated that an antitumor effect of
26 might be only partially dependent on Top I, indicating
that 26 had additional targets besides Top I.

5656 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
You et al.
Conclusion
In summary, a novel series of Top I inhibitors were
constructed by using a scaffold modification strategy: The
rigid framework of 1 was changed into a combination of the
quinolone skeleton and benzimidazole group or its bioiso-
esters through a single carbon bond. Thirty-one new com-
pounds were synthesized and evaluated for anticell prolifera-
tion activity. The most potent compound 26 presented
significant inhibitory effects on Top I and influences on Top
I protein expression at the cellular level; moreover, 26 was
proved to induce cell death via apoptosis rather than necrosis.
Interestingly, further investigations of 26 to Top I-mediated
cleavage and DNA distribution on tumor cell lines indicated
that 26 trapped Top I cleavage complexes at different DNA
sequencesascomparedwith1. ItcouldaccelerateDNAstrand
breaks without significant alteration in cell cycle populations.
Therefore, it was assumed that 26’s cytotoxicity originated
from both interacting with DNA-Top I complex and other
mechanisms on the apoptotic pathway. Furthermore, the in
vivo evaluation of 26 on the growth of HT-29 tumor xeno-
grafts in nude mice suggested its therapeutic potential for
further development. Our scaffold modification approach
based on 1 has yielded new generations of Top I inhibitors
for cancer treatment. Further investigation on lead optimiza-
tion and other molecular mechanisms are still under active
investigation in our laboratories.
Figure 9. Compound 26 induces chromosomal DNA strand breaks
as revealed by Comet assay. HT-29 cells were treated with 1 or 26 for
1 h. After treatment, the Comet assay was performed to assess
chromosomal DNA integrity. (A) Comet images are representative
of duplicates in three independent experiments. (B) Statistical
significance was determined using Student’s t test as compared with
placebo control (*P < 0.05).
Experimental Section
General Experimental Procedures. Chemistry. All reagents
were purchased from commercial sources and were used without
further purification unless otherwise noted. Melting points (mp)
were determined with a Mel-Temp II apparatus and are re-
ported without any correction. Infrared spectra were obtained
with Nicolet Impact 410 spectrophotometer using KBr film. The
absorption band position is given in cm-1. The 1H NMR spectra
were collected on a Bruker ACF-300 spectrometer (300 MHz)
using Dimethyl sulfoxide (DMSO)-d6 as the solvent with tetra-
methylsilane as the internal standard. Mass spectra were ob-
tained on either a Mariner Mass Spectrum or a MAT-212 mass
spectrometer. Elemental analyses were performed with a Carlo
Erba 1106 elemental analysis apparatus. Each of the syntheses
employed commercially available quinolone-3-carboxylic acids
unless otherwise noted. Each of the target compounds was
Figure 10. Effect of 26 on the growth of HT-29 tumor xenografts
in nude mice. Nude mice bearing HT-29 tumor xenografts were
treated daily with placebo (control) (b), 3 (positive control) at
5 mg/day/kg (4), 26 at 75 mg/day/kg (O), or 150 mg/day/kg (1).
The mean tumor volume was calculated by dividing the volume at
each time point by the average volume of the tumors at the time
when the treatment began. Data are expressed as means ( SEM of
six samples from two experiments. Statistical significance was
determined using Student’s t test as compared with placebo control
(*P < 0.05).
˚
purified by silica gel (60 A, 70-230 mesh) column chromato-
graphy. Concentration and evaporation of the solvent after
reaction or extraction were carried out on a rotary evaporator
(Buchi Rotavapor) operated at reduced pressure.
General Procedures for Compounds 6-20. A mixture of
quinolone-3-carboxylic acids (0.1 mol) and o-phenylenediamine
(0.01 mol), o-aminophenol (0.01 mol), or o-aminobenzenethiol
(0.01 mol) in PPA (35 mL) was maintained at 130-140 ꢀC to
form a homogeneous phase. Each mixture was stirred at
170-250 ꢀC for 4 h. After it was cooled to 100 ꢀC, each mixture
was poured slowly into 350 g of smashed ice with vigorous
stirring. The pH of each solution was adjusted to neutral with
2.5 M NaOH. Each resulting solid was collected and purified by
silica gel column chromatography (eluent, methanol:EtOAc=
1:2-1:4). The desired fraction in each case was collected, and the
solvent was evaporated in vacuo. Each solid was recrystallized
from methanol.
1-Ethyl-3-(benzimidazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-4(1H)-
quinolone (6). Compound 6 was obtained in 23.4% yield, mp
263 ꢀC. IR (film): 3923 (NH), 2947, 1630 (CO), 1563, 1542, 1527,
1492, 1255, 879 cm-1. 1H NMR (DMSO-d6): δ 1.44 (t, J=7.0
Hz, 3 H, CH3), 2.91 (m, 4 H, 2 ꢀ CH2), 3.17 (m, 4 H, 2 ꢀ CH2),
4.57 (q, J=7.0 Hz, 2 H, CH2), 7.13 (m, 3 H, 8-H and 50-H and
Compound 26 Inhibits the Growth of HT-29 Tumor Xeno-
grafts in Nude Mice. We next investigated the in vivo effect of
26 on the growth of HT-29 tumor xenografts. All 24 nude
mice receiving a subcutaneous injection of HT-29 cells
showed growth of solid tumors. As shown in Figure 10, the
size of HT-29 tumor xenografts treated with the placebo
increased steadily over time. Treatment with 3 at 5 mg/day/
kg body weight, however, suppressed tumor growth by 76%
as compared with placebo-treated controls. For 26, tumor
growth was suppressed by 52% (75 mg/day/kg group) or
65% (150 mg/day/kg group). Because of heterogeneity in
tumor size, there was significant variation among the tumor
volumes in each group, but the suppressive effect of 26 on
tumor growth was clear when the representative tumors from
the placebo and treated groups were compared side by side.
None of the mice treated with 26 showed signs of morbidity
or a change in body weight.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5657
60-H), 7.61 (m, 2 H, 40-H and 70-H), 7.95 (d, J=13.5 Hz, 1 H,
5-H), 9.17 (s, 1 H, 2-H), 12.69 (s, 1 H, 10-H). ESIMS m/z (relative
intensity) 391 (Mþ), 349 (100), 306. Anal. (C22H22FN5O) C, H,
N. Calcd: 67.50, 5.66, 17.89. Found: 67.23, 5.77, 17.79.
28.9% yield, mp 261 ꢀC. IR (film): 3412 (NH), 3289, 2981, 1637
(CO), 1473, 1445, 1265, 792 cm-1. 1H NMR (DMSO-d6): δ 1.40
(t, J = 7.0 Hz, 3 H, CH3), 2.85 (m, 4 H, 2 ꢀ CH2), 3.67 (m, 4 H,
2 ꢀ CH2), 4.41 (q, J = 7.0 Hz, 2 H, CH2), 7.37 (m, 2 H, 50-H and
60-H), 7.71 (m, 2 H, 40-H and 70-H), 7.98 (d, J = 13.7 Hz, 1 H, 5-
H,), 8.93 (s, 1 H, 2-H). ESIMS m/z (relative intensity) 393 (Mþ),
351 (100). Anal. (C21H20F N5O2) C, H, N. Calcd: 64.11, 5.12,
17.80. Found: 63.76, 5.34, 17.97.
1-Ethyl-3-(benzimidazol-2-yl)-6-fluoro-7-(1-piperazin-1-yl)-1,
8-naphthyridine-4(1H)-one (7). Compound 7 was obtained in
18.9% yield, mp 225 ꢀC. IR (film): 3488 (NH), 3058, 2979, 1634
1
(CO), 1474, 1445, 1257, 1139, 793, 738, 567 cm-1. H NMR
(DMSO-d6): δ 1.42 (t, J=6.8 Hz, 3 H, CH3), 2.98 (m, 4 H, 2 ꢀ
CH2), 3.90 (m, 4 H, 2 ꢀ CH2), 4.51 (q, J = 6.8 Hz, 2 H, CH2),
7.14 (m, 2 H, 50-H and 60-H), 7.61 (m, 2 H, 40-H and 70-H), 8.13
(d, J = 13.4 Hz, 2 H, 40-H and 70-H), 9.17 (s, 1 H, 2-H). ESIMS
m/z (relative intensity). 392 (100, Mþ). Anal. (C21H21FN6O) C,
H, N. Calcd: 64.27, 5.39, 21.41. Found: 64.39, 5.55, 21.13.
1-Ethyl-3-(benzimidazol-2-yl)-6,8-difluoro-7-(3-methylpipera-
zin-1-yl)-4(1H)-quinolone (8). Compound 8 was obtained in
14.2% yield, mp 272-275 ꢀC. IR (film): 3313 (NH), 3057,
2980, 2936, 2736, 1626 (CO), 1574, 1544, 1527, 1477, 1385,
1282, 1250, 1143, 1051, 924, 790 cm-1. 1H NMR (DMSO-d6): δ
1.19 (d, J = 6.8 Hz, 3 H, CH3), 1.46 (t, J = 6.6 Hz, 3 H, CH3),
3.13 (m, 4 H, 2 ꢀ CH2), 3.39 (m, 3 H, CH2, CH), 4.57 (q, J = 6.6
Hz, 2 H, CH2), 7.14 (m, 2 H, 50 -H and 60-H), 7.61 (m, 2 H, 40-H
and 70-H), 7.88 (d, J = 11.9 Hz, 1 H, 5-H), 9.08 (s, 1 H, 2-H),
12.59 (br, 1 H, NH). ESIMS m/z (relative intensity) 424 (100,
MHþ). Anal. (C23H24F2N5O) C, H, N. Calcd: 65.24, 5.47, 8.97.
Found: 65.35, 5.59, 8.85.
1-Ethyl-3-(benzimidazol-2-yl)-6-fluoro-7-(4-methylpiperazin-
1-yl)-4(1H)- quinolone (9). Compound 9 was obtained in 26.4%
yield, mp 239-242 ꢀC (dec.). IR (film): 3329 (NH), 2925, 1620-
(CO), 1550, 1473, 1284, 789 cm-1. 1H NMR(DMSO-d6): δ 1.44
(t, 3H, -CH3), 2.25 (s, 3H, -CH3), 2.50 (m, 4H, 2 ꢀ CH2), 3.27
(m, 4H, 2 ꢀ CH2), 4.56 (q, 2H, CH2), 7.12 (m, 3H, 8-H, 50-H,
60-H), 7.61 (m, 2H, 40-H and 70-H), 7.95 (d, 1H, 5-H), 9þ.17 (s, 1H,
1-H), 12.67 (s, 1H, 10-H). EI-MS m/z 405 (M ). Anal.
1-Ethyl-3-(benzothiazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-4(1H)-
quinolone (14). Compound 14 was obtained in 22% yield, mp
290-295 ꢀC. IR (film): 3429 (NH), 2938, 1615 (CO), 1493, 1254,
1010, 759 cm-1. 1H NMR (DMSO-d6): δ 1.46 (t, J = 6.8 Hz, 3
H, -CH3), 3.08 (m, 4 H, 2 ꢀ CH2), 3.35 (m, 4H, 2 ꢀ CH2), 4.62
(q, J = 6.8 Hz, 2 H, CH2), 7.15 (d, J = 7.6 Hz, 1 H, 8-H), 7.38
(m, 1 H, 50-H), 7.49 (m, 1 H, 60-H), 7.95 (m, 2 H, 5-H and 70-H),
8.09 (d, J = 7.6 Hz, 1 H, 40-H), 9.23 (s, 1 H, 2-H). ESIMS m/z
(relative intensity) 408 (Mþ), 366 (100). Anal. (C22H21FN4OS)
C, H, N. Calcd: 64.69, 5.18, 13.72. Found: 64.55, 5.10, 13.70.
1-Ethyl-3-(benzothiazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-1,8-
naphthyridine-4(1H)-one (15). Compound 15 was obtained in
26.6% yield, mp 259-262 ꢀC. IR (film): 3419 (NH), 1627 (CO),
1474, 1446, 1372, 1276, 792 cm-1. 1H NMR (DMSO-d6): δ 1.44
(t, J = 6.8 Hz, 3 H, CH3), 2.87 (m, 4 H, 2 ꢀ CH2), 3.71 (m, 4 H, 2
ꢀ CH2), 4.53 (q, J = 6.8 Hz, 2 H, CH2), 7.37 (m, 1 H, 50-H), 7.50
(m, 1 H, 60-H), 7.96 (d, J = 8.0 Hz, 1 H, 70-H), 8.07 (m, 2 H, 5-H
and 40-H), 9.24 (s, 1 H, 2-H). ESIMS m/z (relative intensity) 409
(Mþ), 367 (100). Anal. (C21H20FN5OS H2O) C, H, N. Calcd:
3
59.00, 5.15, 16.38. Found: 58.88, 4.97, 16.26.
1-Ethyl-3-(benzothiazol-2-yl)-6-fluoro-7-(4-methylpiperazin-
1-yl)-4(1H)-quinolone (16). Compound 16 was obtained in
25.7% yield, mp 226 ꢀC. IR (film): 2931, 1617 (CO), 1570,
1497, 1452, 1259, 1009, 758 cm-1. 1H NMR (DMSO-d6): δ 1.47
(t, J = 7.0 Hz, 3 H, -CH3), 2.28 (s, 3 H, CH3), 2.54 (m, 4 H, 2 ꢀ
CH2), 3.33 (m, 4 H, 2 ꢀ CH2), 4.61 (q, J = 7.0 Hz, 2 H, CH2),
7.16 (d, J = 7.2 Hz, 1 H, 8-H), 7.37 (t, J = 7.8 Hz, 1 H, 50-H),
7.50 (t, J = 7.8 Hz, 1 H, 60-H), 7.92-7.97 (m, 2 H, 5-H and 70-H),
8.10 (d, J = 7.8 Hz, 1 H, 40-H), 9.24 (s, 1 H, 1-H). ESIMS m/z
(C23H24FN5O 2H2O) Calcd: 62.57, 6.34, 15.86. Found:
3
62.02, 6.13, 16.26. HR-MS: Found, 405.197192; Required,
405.196474; error, 0.718.
(relative
intensity)
422
(100,
Mþ),
323.
Anal.
(C23H23FN4OS 0.5H2O) C, H, N. Calcd: 64.02, 5.61, 12.98.
1-Ethyl-3-(benzoxazol-2-yl)-6-fluoro-7-chloro-4(1H)-quino-
lone (10). Compound 10 was obtained in 31.6% yield, mp
290 ꢀC. IR (film): 1651 (CO), 1612, 1487, 1262, 1018, 749
cm-1. 1H NMR (DMSO-d6): δ 1.41 (t, J = 7.1 Hz, 3 H, CH3),
4.54 (q, J = 7.1 Hz, 2 H, CH2), 7.38 (m, 2 H, 50-H and 60-H), 7.73
(m, 2 H, 40-H and 70-H), 8.13 (d, J = 9.1 Hz, 1H, 5-H), 8.27 (d,
J = 6.6 Hz, 1 H, 8-H), 9.05 (s, 1 H, 2-H). ESIMS m/z (relative
intensity) 342 (100, Mþ), 314. Anal. (C18H12ClFN2O2) C, H, N.
Calcd: 63.08, 3.53, 8.17. Found: 63.27, 3.61, 8.06.
3
Found: 63.83, 5.43, 13.25.
(S)-6-(Benzothiazol-2-yl)-9-fluoro-2,3-dihydro-3-methyl-10-
(4-methylpiperazin-1-yl)-7-oxo-7H-pyrido[1,2,3-de][1,4]-benzox-
azine (17). Levofloxacin (8 g, 0.02 mol), o-aminobenzenethiol
(2.9 g, 0.02 mol), and PPA (90 mL) were used following the
general procedure for the synthesis of 6-16. Compound 17 was
obtained in 50.9% yield, mp 259-262 ꢀC. IR (film): 3010, 2976,
2924, 2797, 1611 (CO), 1585, 1547, 1479, 1468, 1452, 1296, 1236,
1133, 1046, 1011, 786, 760 cm-1. 1H NMR (DMSO-d6): δ 1.49
(d, J = 7.0 Hz, 1 H, CH3), 2.25 (s, 3 H, CH3), 2.46 (m, 4 H, 2 ꢀ
CH2), 3.28 (m, 4 H, 2 ꢀ CH2), 4.40 (m, 1 H, NCH), 4.60 (m, 1 H,
OCH), 4.97 (m, 1 H, OCH), 7.38 (m, J = 7.6 Hz, 1 H, 50-H), 7.50
(m, J = 7.0 Hz, 1 H, 60-H), 7.58 (d, J = 12.5 Hz, 1 H, 80-H), 7.95
(d, J = 7.8 Hz, 1 H, 70-H), 8.10 (d, J = 7.8 Hz, 1 H, 40-H), 9.22 (s,
1 H, 2-H). ESIMS m/z (relative intensity) 450 (100, Mþ). Anal.
1-Ethyl-3-(benzoxazol-2-yl)-6,8-difluoro-7-(3-methylpipera-
zin-1-yl)-4(1H)-quinolone (11). Compound 11 was obtained in
32.0% yield, mp 257 ꢀC. IR (film): 3468 (NH), 2929, 1646 (CO),
1
1623, 1477, 1238, 1053, 1014, 833, 788 cm-1. H NMR (ppm,
DMSO-d6): δ 0.99 (d, J = 5.5 Hz, 3 H, CH3), 1.44 (t, J = 6.7 Hz,
3 H, CH3), 2.79-2.94 (m, 4 H, 2 ꢀ CH2), 3.13 (m, 1 H, CH), 3.30
(m, 2 H, CH2), 4.52 (q, 2 H, CH2), 7.38 (q, 2 H, 50-H and 60-H),
7.74 (m, 2 H, 40-H and 70-H), 7.80 (d, J = 12.5 Hz,
1 H, 5-H), 8.91 (s, 1 H, 2-H). ESIMS m/z (relative intensity)
424 (Mþ), 368 (100). Anal. (C23H22F2N4O2) C, H, N. Calcd:
65.09, 5.22, 13.20. Found: 65.20, 5.34, 12.35.
1-Ethyl-3-(benzoxazol-2-yl)-6-fluoro-7-(4-methylpiperazin-1-
yl)-4(1H)-quinolone (12). Compound 12 was obtained in 28%
yield, mp 263 ꢀC. IR (film): 2930, 1626 (CO), 1487, 1450, 1376,
1255, 1145, 1012, 742 cm-1. 1H NMR (DMSO-d6): δ 1.43 (t, J =
7.0 Hz, 3 H, CH3), 2.25 (s, 3 H, CH3), 2.50 (m, 4 H, 2 ꢀ CH2),
3.27 (m, 4 H, 2 ꢀ CH2), 4.51 (q, J = 7.0 Hz, 2 H, CH2), 7.10 (d,
J = 7.3 Hz, 1 H, 8-H), 7.37 (m, 2 H, 50-H and 60-H), 7.72 (m, 2 H,
40-H and 70-H), 7.87 (d, J = 13.6 Hz, 1 H, 5-H), 8.94 (s, 1H, 1-H).
ESIMS m/z (relative intensity) 406 (100, Mþ). Anal.
(C24H23FN4O2S H2O) C, H, N. Calcd: 61.52, 5.38, 11.96.
3
Found: 61.29, 5.15, 11.85.
1-Ethyl-3-(benzothiazol-2-yl)-6,8-difluoro-7-(3-methylpiper-
azin-1-yl)-4(1H)-quinolone (18). Compound 18 was obtained in
15% yield, mp 224-227 ꢀC. IR (film): 3428 (NH), 2956, 1611
(CO), 1491, 1323, 1054, 755 cm-1. 1H NMR (DMSO-d6): δ 0.99
(d, J = 5.5 Hz, 3 H, CH3), 1.44 (t, J = 7.0 Hz, 3 H, CH3),
2.80-2.91 (m, 4 H, 2 ꢀ CH2), 3.24-3.33 (m, 3 H, CH and CH2),
4.59 (q, J = 7.0 Hz, 2 H, CH2), 7.39 (t, J = 7.7 Hz, 1 H, 50-H),
7.50 (t, J = 7.7 Hz, 1 H, 60-H), 7.83 (d, J = 7.0 Hz, J = 10.9 Hz,
1 H, 5-H), 7.90 (d, J = 7.9 Hz, 1 H, 70-H), 8.11 (d, J = 7.6 Hz, 1
H, 40-H), 9.16 (s, 1 H, 2-H). ESIMS m/z (relative intensity) 440
(Mþ), 384 (100). Anal. (C23H22F2N4OS H2O) C, H, N. Calcd:
(C23H23FN4O2 0.5H2O) C, H, N. Calcd: 66.49, 5.82, 13.48.
3
3
Found: 66.31, 5.72, 13.60.
1-Ethyl-3-(benzoxazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-1,8-
naphthyridine-4(1H)-one (13). Compound 13 was obtained in
60.25, 5.28, 12.22. Found: 60.30, 5.06, 11.94.
1-Ethyl-3-(6-chlorobenzimidazol-2-yl)-6-fluoro-7-(piperazin-
1-yl)-1,8-naphthyridine-4(1H)-one (18). Enoxacin (8 g, 0.025

5658 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
You et al.
mol), 4-chloro-1, 2-phenyldiamine (2.7 g, 0.025 mol), and PPA
(90 mL) were used following the general procedure for the
synthesis of 3-12. Compound 18 was obtained in 18.1% yield,
mp 261-264 ꢀC. IR (film): 3408 (NH), 3067, 2982, 2867, 2462,
1632 (CO), 1565, 1525, 1477, 1446, 1275, 1256, 1141, 1055, 921,
(C23H23F2N6O3) C, H, N. Calcd: 58.97, 4.73, 17.94. Found:
58.93, 4.86, 17.82.
1-Ethyl-3-(6-nitrobenzimidazol-2-yl)-6-fluoro-7-(4-methyl-1-
piperazinyl)-4(1H)-quinolone (24). Compound 24 was obtained
in 68% yield following the synthetic procedure described for 21,
mp 230-232 ꢀC. IR (film): 3249 (NH), 2937, 1629 (CO), 1492,
1310, 1256, 1139, 1006, 790 cm-1. 1H NMR(DMSO-d6): δ 1.45
(t, 3H, -CH3), 3.06 (s, 3H, -CH3), 2.58 (s, 4H, 2 ꢀ CH2), 3.29 (s,
4H, 2 ꢀ CH2), 4.59 (q, 2H, -CH2), 7.10(d, 1H, 60-H), 7.8
(dd, 1H, 70-H), 7.93 (m, 1H, 5-H), 8.05 (m, 1H, 8-H), 8.78
(d, 1H, 40-H), 9.18 (d, 1H, 2-H), 13.23 (s, 1H, 10-H). EI-MS
m/z 450 (Mþ). HR-MS: (C23H23FN6O3) Found, 450.181602;
Required, 450.181551; error, 0.101.
1
794 cm-1. H NMR (DMSO-d6): δ 1.41 (t, J = 7.0 Hz, 3 H,
CH3), 2.97 (m, 4 H, 2 ꢀ CH2), 3.78 (m, 4 H, 2 ꢀ CH2), 4.47 (q,
J = 7.0 Hz, 2 H, CH2), 7.16 (d, J = 8.1 Hz, 1 H, 40-H), 7.61 (m, 2
H, 50-H, 70-H), 8.03 (d, J = 13.7 Hz, 1 H, 5-H), 9.11 (s, 1 H, 2-H),
12.79 (br, 1 H, NH). ESIMS m/z (relative intensity) 427 (100,
MHþ). Anal. (C21H20N6OFCl) C, H, N. Anal.
(C21H20ClFN6O) C, H, N. Calcd: 59.09, 4.72, 19.69. Found:
59.10, 4.80, 19.74.
1-Ethyl-3-(6-chlorobenzimidazol-2-yl)-6-fluoro-7-(piperazin-
1-yl)-4(1H)-quinolone (20). Compound 20 was obtained in 21%
yield, mp 261-262 ꢀC. IR (film): 3422 (NH), 1629 (CO), 1492,
1255, 791 cm-1. 1H NMR (DMSO-d6): δ 1.44 (t, J = 7.0 Hz, 3
H, -CH3), 2.92 (m, 4 H, 2 ꢀ CH2), 3.20(m, 4 H, 2 ꢀ CH2), 4.57
(q, J = 7.0 Hz, J = 13.6 Hz, 2 H, CH2), 7.13 (m, 2 H, 8-H and 40-
H), 7.56 (m, 1 H, 40-H), 7.69 (m, 1 H, 50-H), 7.94 (d, 1 H, 5-H),
9.14 (s, 1 H, 2-H), 12.84 (b, 1 H, 10-H). ESIMS m/z (relative
1-Ethyl-3-(6-nitrobenzoxazol-2-yl)-6-fluoro-7-chloro-4(1H)-
quinolone (25). Following the synthetic procedure of 21, 25 was
obtained from 10 in 65.6% yield, mp 291-294 ꢀC. IR (film):
3010, 2930, 1648 (CO), 1609, 1537, 1482, 1340, 1328, 1278, 1265,
1055, 1013, 827, 790, 760 cm-1. 1H NMR (DMSO-d6): δ 1.43 (t,
J = 7.0 Hz, 3 H, CH3), 4.51 (q, J = 7.0 Hz, 2 H, CH2), 7.80 (d,
J = 7.0 Hz, 1 H, 40-H), 8.00 (m, 1 H, 50-H), 8.18 (m, 2 H, 5-H, 70-
H), 8.48 (s, 1 H, 8-H), 9.00 (s, 1 H, 2-H). ESIMS m/z (relative
intensity) 388 (100, MHþ). Anal. (C18H11ClFN3O4) C, H, N.
Calcd: 55.76, 2.86, 10.84. Found: 55.83, 2.96, 10.73.
intensity) 425 (Mþ), 383 (100). Anal. (C22H21ClFN5O 2H2O)
3
C, H, N. Calcd: 57.20, 5.45, 15.16. Found: 56.97, 5.27, 15.27.
General Procedures for Compounds 21-33. 1-Ethyl-3-(6-
nitrobenzimidazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-4(1H)-quino-
lone (21). To a solution of 6 (0.8 g, 2.5 mmol) in concentrated
sulfuric acid (3.20 mL) at 0 ꢀC, concentrated nitric acid (0.26
mL, 3.7 mmol) was added dropwise with vigorous stirring. The
internal temperature of the reaction flask was kept below 5 ꢀC
during the addition of nitric acid. After the addition of nitric
acid, the solution was stirred at room temperature for 1 h and
then at 35-40 ꢀC for 2 h in a warm water bath. The solution was
poured slowly into 350 g of smashed ice with vigorous stirring.
The pH of the resulting solution was adjusted to 8 with 2.5 M
NaOH. The precipitated solid was collected by suction filtration
and purified by silica gel column chromatography (eluent,
methanol:EtOAc=1:6-1:8). The desired fraction was collected,
and the solvent was evaporated in vacuo. The solid was recrys-
tallized from methanol. Compound 21 was obtained in 33%
yield, mp 236-240 ꢀC. IR (film): 3414 (NH), 2978, 2841, 1628
(CO), 1568, 1524, 1493, 1451, 1385, 1311, 1269, 1191, 1140,
1057, 828 cm-1. 1H NMR (DMSO-d6): δ 1.46 (t, J = 7.0 Hz, 3
H, CH3), 3.07 (m, 4 H, 2 ꢀ CH2), 3.17 (m, 4 H, 2 ꢀ CH2), 4.59 (q,
J = 7.0 Hz, 2 H, CH2), 7.16 (s, 1 H, 8-H), 7.68, 7.84 (m, 1 H, 40-
H), 7.96 (d, J = 13.2 Hz, 1H, 5-H), 8.07 (m, 1 H, 50-H), 8.38, 8.59
(m, 1 H, 70-H), 9.22 (s, 1 H, 2-H), 13.23 (NH). ESIMS m/z
(relative intensity) 436 (100, Mþ). Anal. (C22H21FN6O3) C, H,
N. Calcd: 60.54, 4.85, 19.26. Found: 60.37, 4.92, 19.10.
1-Ethyl-3-(6-nitrobenzimidazol-2-yl)-6-fluoro-7-(piperazin-
1-yl)-1,8-naphthyridine-4(1H)-one (22). Following the synthetic
procedure of 21, 22 was obtained from 7 in 24% yield, mp
230-233 ꢀC. IR (film): 3401 (NH), 3307 (NH), 2979, 1633 (CO),
1529, 1477, 1446, 1310, 1139, 795 cm-1. 1H NMR (DMSO-d6): δ
1.42 (t, J = 7.0 Hz, 3 H, CH3), 3.07 (m, 4 H, 2 ꢀ CH2), 3.82 (m, 4
H, 2 ꢀ CH2), 4.52 (q, J = 7.0 Hz, 2 H, CH2), 7.69,7.82 (m, 1 H,
40-H), 8.10 (m, 2 H, 50-H and 5-H), 8.38, 8.59 (m, 1 H, 40-H), 9.24
(s, 1 H, 2-H). ESIMS m/z (relative intensity) 437 (100, Mþ), 395.
Anal. (C21H20N7O3F) C, H, N. Calcd: 57.66, 4.61, 22.41.
Found: 57.41, 4.78, 22.32.
1-Ethyl-3-(6-nitrobenzoxazol-2-yl)-6,8-difluoro-7-(3-methyl-
piperazin-1-yl)-4(1H)-quinolone (23). Following the synthetic
procedure of 21, 26 was obtained from 11 in 25.8% yield, mp
218 ꢀC (dec.). IR (film): 3423 (NH), 3100, 3062, 2975, 2931,
2849, 2727, 1644, 1623 (CO), 1539, 1521, 1476, 1340, 1269, 1057,
827, 788, 734 cm-1. 1H NMR(DMSO-d6): δ 1.06 (d, J = 7.0 Hz,
3 H, CH3), 1.45 (t, J = 7.0 Hz, 3 H, CH3), 2.90 (m, 4 H, 2 ꢀ
CH2), 3.46 (m, 3 H, CH2 and CH), 4.53 (q, J = 7.0 Hz, 2 H,
CH2), 7.80 (d, J = 12.0 Hz, 1 H, 5-H), 7.91 (d, J = 8.7 Hz, 1 H,
40-H), 8.27 (d, J = 8.7 Hz, 1 H, 50-H), 8.62 (m, 1 H, 70-H), 8.99 (s,
1 H, 2-H). ESIMS m/z (relative intensity) 470 (100, MHþ). Anal.
(C23H21F2N5O4) C, H, N. Calcd: 58.85, 4.51, 14.92. Found:
58.96, 4.42, 14.94.
1-Ethyl-3-(6-nitrobenzoxazol-2-yl)-6-fluoro-7-(4-methylpiper-
azin-1-yl)-4(1H)-quinolone (27). Following the synthetic proce-
dure of 21, 27 was obtained from 12 (2.0 g, 4.93 mmol) in 5.4%
yield, mp 245-248 ꢀC. IR (film): 3100, 3050, 2926, 1628 (CO),
1606, 1551, 1535, 1489, 1453, 1341, 1268, 1168, 1042, 998, 829,
790, 762 cm-1. 1H NMR (DMSO-d6): δ 1.44 (t, J = 7.0 Hz, 3 H,
CH3), 2.26 (s, 3 H, NCH3), 2.50 (m, 4 H, 2 ꢀ CH2), 3.56 (m, 4 H,
2 ꢀ CH2), 4.53 (q, J = 7.0 Hz, 2 H, CH2), 7.08 (d, J = 7.4 Hz, 1
H, 8-H), 7.84 (m, 2-H, 5-H, 40-H), 8.27 (d, J = 7.8 Hz, 1 H, 50-H),
8.60 (d, J = 1.6 Hz, 1 H, 70-H), 9.02 (s, 1 H, 2-H). ESIMS m/z
(relative intensity) 452 (100, MHþ). Anal. (C23H22FN5O4) C, H,
N. Calcd: 61.19, 4.91, 15.51. Found: 61.20, 4.71, 15.62.
1-Ethyl-3-(6-nitrobenzoxazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-
1,8-naphthyridine-4(1H)-one (28). Following the synthetic pro-
cedure of 21, 28 was obtained from 13 in 17% yield, mp 258 ꢀC
(dec.). IR (film): 3411 (NH), 3099, 3059, 2983, 2934, 1632 (CO),
1606, 1584, 1540, 1477, 1446, 1383, 1341, 1271, 1233, 1163, 1128,
1091, 1054, 1003, 828, 794, 735 cm-1. 1H NMR (DMSO-d6): δ
1.39 (t, J = 7.3 Hz, 3 H, CH3), 3.00 (m, 4 H, 2 ꢀ CH2), 3.75 (m, 4
H, 2 ꢀ CH2), 4.42 (q, J = 7.3 Hz, 2 H, CH2), 7.83 (d, J = 13.8
Hz, 1 H, 5-H), 8.21 (m, 2 H, 40-H and 50-H), 8.49 (s, 1 H, 70-H),
8.98 (s, 1 H, 2-H). ESIMS m/z (relative intensity) 439 (100,
MHþ). Anal. (C21H19FN6O4) C, H, N. Calcd: 57.53, 4.37, 19.17.
Found: 57.62, 4.45, 19.08.
1-Ethyl-3-(6-nitrobenzimidazol-2-yl)-6,8-difluoro-7-(3-methyl-
piperazin-1-yl)- 4(1H)-quinolone (23). Following the synthetic
procedure of 21, 23 was obtained from 8 in 27.1% yield, mp
269 ꢀC (dec.). IR (film): 3420 (NH), 3069, 2982, 2937, 2853,
2733, 1622 (CO), 1580, 1526, 1477, 1389, 1333, 1249, 1143, 1089,
1053, 829, 736 cm-1. 1H NMR (DMSO-d6): δ 1.06 (d, J = 6.8
Hz, 3 H, CH3), 1.48 (t, J = 7.0 Hz, 3 H, CH3), 3.16 3.33 (m, 7 H,
3 ꢀ CH2, CH), 4.57 (q, J = 7.0 Hz, 2 H, CH2), 7.65, 7.80 (m, 1 H,
40-H), 7.84 (d, J = 11.7 Hz, 1H, 5-H), 8.03 (d, J = 8.0 Hz, 1H, 5-
H), 8.42 (m, 1 H, 70-H), 9.10 (s, 1 H, 2-H), 13.11 (br, 1 H, NH).
ESIMS m/z (relative intensity) 469 (100, MHþ). Anal.
1-Ethyl-3-(6-nitrobenzothiazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-
4(1H)-quinolone (29). Following the synthetic procedure of 21,
29 was obtained from 14 in 31.4% yield, mp 244 ꢀC (dec.). IR
(film): 3416 (NH), 3026, 2944, 2839, 1616 (CO), 1573, 1496,
1444, 1328, 1264, 1126, 894, 752 cm-1. 1H NMR (DMSO-d6): δ
1.48 (t, J = 7.0 Hz, 3 H, CH3), 3.08 (m, 4 H, 2 ꢀ CH2), 3.32 (m, 4
H, 2 ꢀ CH2), 4.61 (q, J = 7.0 Hz, 2 H, CH2), 7.12 (d, J = 7.2 Hz,
1 H, 8-H), 7.88 (d, J = 13.3 Hz, 1 H, 5-H), 7.98 (d, J = 9.0 Hz, 1
H, 40-H), 8.24 dd, J1=9.0 Hz, J2=2.4 Hz, 1 H, 50-H), 9.08 (d, J =
2.4 Hz, 1 H, 70-H), 9.24 (s, 1 H, 2-H). ESIMS m/z (relative

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5659
intensity) 454 (100, MHþ). Anal. (C22H21FN5O3S) C, H, N.
Calcd: 53.27, 4.45, 15.44. Found: 53.38, 4.46, 15.29.
for 34, mp 350 ꢀC. IR (film): 3410 (NH), 3334, 1629 (CO), 1495,
1258, 1139, 790 cm-1 1H NMR (DMSO-d6):δ1.43 (t, 3H,
.
1-Ethyl-3-(6-nitrobenzothiazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-
1,8-naphthyridine-4(1H)-one (30). Following the synthetic pro-
cedure of 21, 30 was obtained from 15 in 10.2% yield, mp 298 ꢀC
(dec.). IR (film): 3413 (NH), 3262, 3057, 2931, 2712, 1626 (CO),
-CH3), 2.29 (s, 3H, -CH3), 2.50(s, 2H, -NH2), 2.57 (m, 4H,
2 ꢀ CH2), 3.28 (m, 4H, 2 ꢀ CH2), 4.55 (q, 2H, -CH2-), 6.50
(s, 1H, 60-H) 6.77 (s, 1H, 40-H), 7.12 (d, 1H, 70-H), 7.26
(d, 1H, 8-H), 7.94 (d, 1H, 5-H), 9.05 (s, 1H, 2-H). EI-MS
m/z 420 (Mþ). HR-MS: (C23H25FN6O) Found, 420.207218;
Required, 420.207372; error, 0.154.
1574, 1508, 1477, 1442, 1371, 1331, 1276, 1263, 1123, 792 cm-1
.
1H NMR (DMSO-d6): δ 1.46 (t, J = 7.0 Hz, 3 H, CH3), 3.30 (m,
1 H, 2 ꢀ CH2), 3.96 (m, 4 H, ꢀ CH2), 4.57 (q, J = 7.0 Hz, 2 H,
CH2), 8.04 (d, J = 9.0 Hz, 1 H, 40-H), 8.16 (d, J = 13.1 Hz, 1 H,
5-H), 8.30 (dd, J1=9.0 Hz, J2=2.6 Hz, 1 H, 50-H), 9.14 (d, J =
2.6 Hz, 1 H, 70-H), 9.35 (s, 1 H, 2-H). ESIMS m/z (relative
intensity) 455 (100, MHþ). Anal. (C21H20FN6O3S) C, H, N.
Calcd: 55.50, 4.21, 18.49. Found: 55.49, 4.19, 18.38.
1-Ethyl-3-(6-aminobenzoimidazol-2-yl)-6-fluoro-7-(piperazin-
1-yl)-1,8-naph- thyridin-4(1H)-one (36). Compound 36 was ob-
tained from 22 in 40.3% yield by an approach similar to that for
34, mp 291 ꢀC. IR (film): 3414 (NH), 3330, 3222, 3039, 2965,
2934, 2722, 1631 (CO), 1570, 1555, 1473, 1444, 1273, 792 cm-1
.
1H NMR (DMSO-d6): δ 1.44 (t, 3H, -CH3), 3.33 (m, 4H, 2 ꢀ
CH2), 3.98 (m, 4H, 2 ꢀ CH2), 4.52 (q, 2H, -CH2-), 6.65 (dd,
1H, 50-H), 6.87 (s, 1H, 70-H), 7.36 (d, 1H, 40-H), 8.17 (d, 1H, 5-
H), 9.46 (s, 1H, 2-H). HR-ESI-MS (M þ 1): (C21H23FN7O)
Found, 408.1938; Required, 408.1943; error, 0.5.
1-Ethyl-3-(6-nitrobenzothiazol-2-yl)-6-fluoro-7-(4-methylpiper-
azin-1-yl)-4(1H)-quinolone (31). Following the synthetic proce-
dure of 21, 31 was obtained from 16 in 20.8% yield, mp 248 ꢀC
(dec.). IR (film): 3050, 2972, 2934, 2838, 1617 (CO), 1568, 1511,
1494, 1334, 1306, 1294, 1260, 1195, 1121, 1009, 896, 756 cm-1
.
Materials and Methods for Biological Assay. Reagents.
DMSO, 1, SRB, and MTT were from Sigma Chemical Co.
(St. Louis, MO). Top I and the plasmid pBR322 DNA were
purchased from Takara-Bio (Shiga, Japan). All other reagents
were of analytical grade or the highest grade available. Stock
solutions of 1, 3, and all test compounds were dissolved in
DMSO and stored at -20 ꢀC.
Cell Lines and Cell Culture. Human cancer cell lines used in
this study were obtained from the Culture Collection and
Research Center (CCRC, Hsinchu, Taiwan) and cultured in
the recommended media.
SRB Assay and Tetrazolium-Based MTT Assay for Cell
Viability. The cytotoxicity of compounds was assessed by the
MTT (for suspension cells)18 and SRB (for adherent cells)19,20
methods. Briefly, various cell lines were cultured at 5 ꢀ 103 cells
per well containing 100 μL of culture medium (the seeding
density depended on the doubling time of the different culture
cell lines). After overnight incubation, the medium was dis-
carded and then replaced with serum-free medium containing
the desired concentrations (0, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, 5,
and 10 μM) of test compounds dispensed from serial dilutions,
and the plates were incubated for another 48 h at 37 ꢀC. For SRB
assay, the incubation was terminated by the addition of tri-
chloroacetic acid (10% w/v final concentration in PBS), and
then, SRB dye (0.4% w/v final concentration in 1% v/v acetic
acid/H2O) was added to stain the cells. Bound dye was subse-
quently dissolved with 10 mM Tris base (Sigma), and absor-
bance of the solution at 515 nm was determined using an Emax
Microplate readers and SoftMax Pro software (Molecular
Devices, United States). For MTT assay, cells were
washed once before adding 50 μL of fetal bovine serum-free
medium containing MTT (0.5 mg/mL final concentration in
PBS). After 4 h of incubation at 37 ꢀC, the medium was
discarded, and the formazan blue that formed in the cells was
dissolved in DMSO. The optical density was measured at 570
nm. The GI50 and IC50 values were estimated by plotting the
colorimetric data on SigmaPlot 10.0 version (Systat Software
Inc., United States), and the data were fitted to an exponential
1H NMR (DMSO-d6): δ 9.28 (s, 1 H, 2-H), 9.11 (s, J = 2.6 Hz, 1
H, 70-H), 8.28 (dd, J1=8.8 Hz, J2=2.6 Hz, 1 H, 50-H), 8.04 (d,
J = 8.8 Hz, 1 H, 40-H), 7.92 (d, J = 13.2 Hz, 1 H, 5-H), 7.14 (d,
J = 7.7 Hz, 1 H, 8-H), 4.63 (q, J = 7.0 Hz, 2 H, CH2), 3.71 (m, 4
H, 2 ꢀ CH2), 2.63 (m, 4 H, 2 ꢀ CH2), 2.34 (s, 3 H, CH3), 1.40 (t,
J = 7.0 Hz, 3 H, CH3). ESIMS m/z (relative intensity) 468 (100,
MHþ). Anal. (C23H23FN5O3S) C, H, N. Calcd: 59.09, 4.74,
14.98. Found: 59.16, 4.89, 14.83.
(S)-6-(6-Nitrobenzothiazol-2-yl)-9-fluoro-2,3-dihydro-3-methyl-
10-(4-methylpiperazin-1-yl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benz-
oxazine (32). Following the synthetic procedure of 21, 32 was
obtained from 17 in 17% yield, mp 266 ꢀC. IR (film): 3100, 3010,
2973, 2931, 2840, 1614 (CO), 1573, 1513, 1476, 1440, 1332, 1294,
1235, 1126, 1047, 1006, 784, 753 cm-1. 1H NMR (DMSO-d6): δ
1.51 (d, J = 6.5 Hz, 3 H, CH3), 2.26 (s, 3 H, CH3), 2.48 (m, 4 H, 2
ꢀ CH2), 3.24 (m, 4 H, 2 ꢀ CH2), 4.42 (m, 1 H, NCH), 4.60 (d,
J = 10.8 Hz, 1 H, OCH), 4.99 (d, J = 10.8 Hz, 1 H, OCH), 7.54
(d, J = 12.6 Hz, 1 H, 5-H), 8.02 (d, J = 9.0 Hz, 1 H, 40-H), 8.29
(dd, J1=9.0 Hz, J2=1.9 Hz, 1 H, 50-H), 9.12 (d, J = 1.9 Hz, 1 H,
70-H), 9.26 (s, 1 H, 2-H). ESIMS m/z (relative intensity) 496 (100,
MHþ). Anal. (C22H23FN5O4S) C, H, N. Calcd: 58.17, 4.48,
14.13. Found: 58.27, 4.62, 14.22.
1-Ethyl-3-(6-nitrobenzothiazol-2-yl)-6,8-difluoro-7-(3-methyl-
piperazin-1-yl)-4(1H)-quinolone (33). Following the synthetic
procedure of 21, 33 was obtained from 18 in 14.6% yield, mp
217 ꢀC (dec.). IR (film): 3418 (NH), 3081, 3055, 2981, 2841, 1609
(CO), 1546, 1509, 1482, 1381, 1333, 1250, 1124, 1056, 787, 753
cm-1. 1H NMR (DMSO-d6): δ 1.04 (d, J = 7.1 Hz, 3 H, CH3),
1.48 (t, J = 7.0 Hz, 3 H, CH3), 2.78 (m, 4 H, 2 ꢀ CH2), 3.03 (m, 3
H, CH2, CH), 4.65 (q, J = 7.0 Hz, 2 H, CH2), 7.88 (d, J = 13.2
Hz, 1 H, 5-H), 8.08 (d, J = 9.1 Hz, 1 H, 40-H), 8.18 (dd, J1=9.1
Hz, J2=2.4 Hz, 1 H, 50-H), 8.31 (d, J = 2.4 Hz, 1 H, 70-H), 9.25
(s, 1 H, 2-H). ESIMS m/z (relative intensity) 486 (100, MHþ).
Anal. calcd for (C23H22F2N5O3S): C, 56.90; H, 4.36; N, 14.42.
Found: C, 56.95; H, 4.49; N, 14.33.
General Procedure for Compounds 34-36. 1-Ethyl-3-(6-ami-
nobenzoimidazol-2-yl)-6-fluoro-7-(piperazin-1-yl)-4(1H)-quino-
lone (34). To a solution of 21 (0.001 mol) in 1 N hydrochloric
acid, 10% Pd/C was added with H2 flow for 4 h. The mixture was
filtered to 2.5 M NaOH. The precipitated solid was collected and
dried to obtain 34 in 65.1% yield, mp 244-246 ꢀC. IR (film):
3408 (NH), 3310, 3211, 2926, 2815, 2769, 2719, 2655, 2488, 1632
decay function to generate
a common log concentra-
tion-response curve.
Anchorage-Dependent Growth. The anchorage-dependent
colony-forming assay was used to evaluate the long-term growth
inhibitory effect of 26.21 Briefly, HT-29 cells were seeded at a
density of 500 cells/well in six-well plates, and after a 24 h
attachment period, the cultures were continuously treated with
the indicated concentrations of test compounds or DMSO for 14
days. Medium containing 1, 3, 26, or DMSO was replaced every
72 h during the 14 day period. The cell colonies were then fixed in
1% (v/v) formaldehyde in phosphate-buffered saline (PBS) and
stained with 0.1% (w/v) crystal violet in PBS (Sigma). Images
were collected by single rapid scans (MicroTek ScanMaker
5900, China).
(CO), 1571, 1520, 1496, 1272, 1186, 1124, 1035, 828 cm-1
.
1H NMR (DMSO-d6): δ 1.50 (t, 3H, -CH3), 2.51 (m, 4H, 2 ꢀ
CH2), 3.55 (m, 4H, 2 ꢀ CH2), 4.55 (q, 2H, -CH2-), 6.82 (dd,
1H, 50-H), 6.99 (s, 1H, 8-H), 7.26 (d, 1H, 70-H), 7.50 (d, 1H, 40-
H), 8.00 (d, 1H, 5-H), 9.36 (br), 9.46 (s, 1H, 2-H). HR-ESI-MS:
(C22H24FN6O) Found, 407.1991; Required, 407.1990; error, 0.1.
1-Ethyl-3-(5-aminobenzimidazol-2-yl)-6-fluoro-7-(4-methyl-
1-piperazinyl)-4(1H)-quinolone (35). Compound 35 was ob-
tained from 24 in 73.3% yield by an approach similar to that

5660 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18
You et al.
Top I Inhibition Assay. Top I activity was measured by the
relaxation of superhelical DNA in plasmid pBR322.22 Each
20 μL assay contained 50 mM Tris-HCl (pH 8.0), 120 mM KCl,
10 mM MgCl2, 0.5 mM EDTA, 0.01% (w/v) bovine serum
albumin, pBR322 DNA (0.5 μg), and test compounds at various
concentrations (see the Results and Discussion) or vehicle alone
(for controls). The reaction was started by the addition of 1 U of
Top I and allowed to proceed at 37 ꢀC for 30 min. Aliquots of
20 μL were subjected to electrophoresis in 1% agarose gel (Bio-
Rad, United Kingdom) using Mupid-2 mini-Gel units (Cosmo
Bio, Japan) at 100 V for 1 h in 0.5ꢀ Tris/acetate (TAE) buffer
(20 mM Tris-acetate, 0.5 mM EDTA, pH 8.3). The gels were
stained with 0.5 mg/mL ethidium bromide for 30 min and
destained in distilled water for 10 min. The DNA bands were
visualized by illumination from below with short-wave ultra-
violet light and photographed. For quantitative determinations,
the integrated intensity of the ethidium bromide fluorescence of
the bands (relaxed form) was acquired and measured using
BioDoc-IT Imaging System (UVP, Inc., CA). Plasmid
pBR322 DNA alone (lane 1 in all gels) and pBR322 DNA
relaxed by Top I (lane 2 in all gels) were taken as the negative and
positive (100% activity) controls, respectively. DNA bands were
quantified and calculated from gel photographs using Image-
Quant image analysis software (GE Healthcare, United
Kingdom) and SigmaPlot 10.0.
Top I-Mediated DNA Cleavage Reactions. Human recombi-
nant Top I and pRYG plasmid were purchased from TopoGEN
(Columbus, OH). The full length fragment of pRYG plasmid
was cleaved with the restriction endonuclease Hind III (New
England Biolabs, MA) in supplied NE buffer 2 (50 μL reactions)
for 1 h at 37 ꢀC and checked by electrophoresis in a 1% agarose
gel made in 0.5ꢀ TAE buffer. Approximately 3 μg of the
fragment was terminal end-labeled at the Hind III site by fill-
in reaction with [R-32P]dCTP, and 10 μM dATP and dGTP, in
Klenow buffer (50 mM Tris, 5 mM MgCl2, and 0.01% Triton
X-100) with 5 units of Exofree Klenow fragment (Genemark
Technology Co., Ltd., Taiwan). Unincorporated 32P-dCTP was
removed using Gel/PCR DNA Fragments Extraction Kit
(Geneaid Biotech Ltd., Taiwan), and the eluate containing the
terminal end-labeled fragment was collected and measured the
specific activity of the labeled DNA by measuring 1 μL in a
scintillation counter (Beckman LS 6000IC Liquid Scintillation
Systems). Aliquots (approximately 100000 cpm/ reaction) were
incubated with Top I in reaction buffer [40 mM Tris-HCl (pH
8.0), 100 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol,
0.5 mM EDTA, and 30 μg/mL bovine serum albumin] at
25 ꢀC for 30 min in the presence of the tested compounds.
Reactions were terminated by adding SDS (5% final con-
centration) and proteinase K (4 mg/mL final concentration)
for further incubation at 45 ꢀC for 30 min. Samples were then
alkali-denatured by the addition of NaOH (0.15 N final con-
centration), mixed with loading buffer (80% formamide, 10 mM
sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol,
and 0.1% bromophenol blue, pH 8.0), and analyzed by electro-
phoresis in 1% agarose gel in 0.5ꢀ TAE electrophoresis buffer.
After electrophoresis, gels were dried onto Whatman 3MM
chromatographic paper using a gel dryer (Bio-Rad Lab.,
Hercules, CA) at 80 ꢀC for 1 h and autoradiographed at
-80 ꢀC using Kodak XAR-5 film.
immunoperoxidase activity by freshly prepared substrate
(0.05%, 3,30-diaminobenzidine tetrahydrochloride, DAB),
counter staining, and mounting by dimethyl benzene. Cells were
distinguished based on their immediate location along scalloped
trabecular surfaces, multiple nuclei, enrichment of vacuoles in
the basophilic cytoplasm, and a ruffled border appearance.
Flow Cytometry for DNA Content Analysis. HT-29 cells were
treated with vehicle or the indicated concentrations of test
compounds for the indicated times and then released by incu-
bated with trypsin-EDTA. Both attached and floating cells were
collected and fixed with 70% ethanol. Cells were then incubated
for 30 min with 0.1% (v/v) Triton X-100, washed again, and
subsequently stained with 50 μg/mL PI and 50 μg/mL RNaseA
in PBS for 30 min in the dark at room temperature. For cell cycle
analysis, 10000 cells were determined using flow cytometry on a
BD FACSCalibur cytometer, and the data were analyzed by
CellQuest software (Becton Dickinson, CA). The red fluore-
scence (FL2) of single events was recorded using an argon ion
laser (488 nm excitation, 610 nm emission) to measure the DNA
index. The percentage of apoptotic cells was calculated from
the number of cells in sub-G1 phase, representing fragmented
cell vesicles.
Immunostaining for Nuclei. All floating and attached cells
were harvested with 0.02% (w/v) EDTA and 0.25% (w/v)
trypsin. The cell suspension in RMPI 1640 medium containing
10% heated-inactivated calf serum was then added to bring the
total volume of each well to 500 μL. Different concentrations of
26 were added after 24 h and incubated for an additional 48 h.
The cell suspension was washed with PBS followed by the
addition of 4% (w/v) formaldehyde in PBS and fixation for
20 min, and then, the mixture was washed with PBS. Hoechst
33342 (10 μg/mL) was added and retained for 5-10 min. The cell
morphology was monitored under an inverted light microscope.
The cells were then observed under a fluorescence microscope
(Olympus IX51, Japan) with a peak excitation wavelength
of 490 nm.
Annexin V/PI Double-Staining Assay. SKOV-3 and HT-29
cells (1-5 ꢀ 106 cells/mL) were treated with 26 for 24 h,
harvested, and then washed and resuspended with PBS. Apop-
totic cells were identified by double supravital staining with
recombinant enhanced green fluorescent protein (EGFP)-con-
jugated Annexin Vþ/PI-, using the Annexin V-EGFP Apopto-
sis Detection kit (KeyGen, China) according to the
manufacturer’s instructions. Flow cytometric analysis was per-
formed immediately after supravital staining. Data acquisition
and analysis were performed with FACSCalibur and CellQuest
software. Cells not binding EGFP-Annexin V and excluding PI
were classified as Annexin V negative. Cells that bound EGFP-
Annexin V [excitation wavelength (λex) = 488 nm, emission
wavelength (λem) = 520 nm] but excluded PI (λex = 540 nm,
λ
em=630 nm) were termed Annexin V positive. Cells permeant
to PI (regardless of whether they bound EGFP-Annexin V) were
deemed necrotic.
Comet Assay for Top I-Cleavable Complexes. The Comet
assay, or single-cell gel electrophoresis (SSGE), has been used
extensively to monitor the integrity of chromosomal DNA and
the amount of Top I-cleavable complexes; the assay was per-
formed essentially as described.24,25 Briefly, exponentially grow-
ing HT-29 cells were subcultured in 24-well plates at 1 ꢀ 104
cells/well and allowed to attach overnight. Negative control cells
were not treated with 26, whereas positive control cells for DNA
strand breaks were treated with 1. Cells were then treated with
10 μM 26 for 1 h. The first layer of agarose on microscope slides
was prepared by dipping the slides into 1% normal-melting
agarose followed by drying; separately, 50 μL of HT-29 cell
suspension (1 ꢀ 104 cells) was mixed with 100 μL of 1.5% low-
melting point agarose and then poured onto a fully frosted slide
that had been precoated with 1% normal-melting agarose A
coverslip was then applied to each slide. The slides were sub-
merged in prechilled lysis solution (1% N-lauryl sarcosine, 1%
Immunocytochemical Studies. Immunocytochemistry was
used to detect Top I expression in cancer cell lines. Staining
was performed according to the standard protocol for
immunoperoxidase enzyme detection.23 To inhibit peroxidase
activity, slides were placed in freshly prepared 0.3% H2O2 in
methanol for 10 min before blocking in PBS containing 10%
normal goat serum. A primary antibody to Top I was applied for
10 h at a dilution of 1:100 in PBS. Secondary biotinylated
antigoat IgG was then applied for 2 h at room temperature.
The remaining steps of incubation involved avidin-biotinylated
horseradish peroxidase solution for 2 h, then detection of

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 18 5661
Triton X-100, 2.5 M NaCl, and 10 mM EDTA, pH 10.5) for
30 min at 4 ꢀC. After lysing, the slides were drained and placed
on a horizontal electrophoresis tray with freshly made alkaline
buffer (300 mM NaOH and 1 mM EDTA; pH > 13) for 30 min
at 4 ꢀC to allow the DNA to unwind. After they were soaked
with alkaline buffer, the slides were subjected to electrophoresis
for 10 min at 16 V (25 mA) at room temperature. After
electrophoresis, slides were stained with SyBr Gold
(Molecular Probes, United States), and images of nuclei were
captured with a Zeiss Axiophot 2 microscope (Carl Zeiss Inc.,
United States) connected to a Spot CCD camera and SPOT
software (Diagnostic Instruments Inc., United States). DNA
damage, reported as the mean percentage of DNA in the tail,
was quantified in at least 50 randomly selected nuclei for each
experimental point.
In Vivo Activity of 26 against Established Tumor Xenografts in
Nude Mice. Female athymic BALB/c nude mice (35-40 days
old) with body weights ranging from 18 to 22 g were supplied by
the Beijing Institute of Materia Medica, Chinese Academy of
Medical Sciences. Animal care and surgery protocols were
approved by the Animal Care Committees of the China
Pharmaceutical University. All animals were appropriately
treated in a scientifically valid and ethical manner. For inocula-
tion of nude mice, a suspension of 1 ꢀ 107 HT-29 cells in 200 μL
of culture medium was subcutaneously transplanted into the
axillary fossa of each BALB/c nude mouse. When the diameter
of a resultant tumor reached ∼1 mm, as measured with a vernier
caliper, the tumor was excised and then sterilely and uniformly
transplanted into the axillary fossa of a nude mouse (24 mice
were used, total). Treatment was begun when implanted tumors
had reached a volume of about 100-300 mm3 (after 1 week, on
average). Mice were randomized into four groups (n=6 each)
and treated daily by intraperitoneal injection with 400 μL of
physiological saline for 10 days, 3 at 5 mg/kg body weight in
400 μL for 2 days, or 26 at 75 or 150 mg/kg body weight in
400 μL for 10 consecutive days. Tumor volumes were monitored
by caliper measurement of the length and width and calculated
using the formula of V=1/2 ꢀ a ꢀ b2, where a is the tumor length
and b is the width. Tumor volumes and body weights were
monitored every 4 days over the course of treatment. Mice were
sacrificed on day 30 following initiation of treatment, and
tumors were removed and recorded for analysis.
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Acknowledgment. This work was supported by Research
Grants 30271543 and 30472084 to Q.-D.Y. from the National
Natural Science Foundation (NSFC) of China and by a
research grant to J.-W.C. from the National Science Council
of Taiwan (NSC97-2323-B-002-010).
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