Design, synthesis, biological evaluation, and molecular docking studies of quinolone derivatives as potential antitumor topoisomerase I inhibitors

Article abstract of DOI:10.1248/cpb.c13-00040

A novel series of quinolone derivatives (6a-n) were designed and synthesized, and their biological activities were evaluated as potential antitumor topoisomerase I (Top I) inhibitors. Among these compounds, 6j exhibited the most potent antitumor activities against multiple cancer cell lines. Docking simulation was performed to insert compound 6j into the crystal structure of DNA-Top I to determine the probable binding model.

Full text of DOI:10.1248/cpb.c13-00040

June 2013
Regular Article
Chem. Pharm. Bull. 61(6) 631–636 (2013)
631
Design, Synthesis, Biological Evaluation, and Molecular Docking Studies
of Quinolone Derivatives as Potential Antitumor Topoisomerase I
Inhibitors
,b,c
Kai-jun Shou,a Jie Li,b Yi Jin,b and Yan-wen Lv*
b
a People’s Hospital of Zhuji; Zhuji 312000, China: College of Chemistry and Materials Engineering, Quzhou
c
University; Quzhou 324000, China: and Key Laboratory of Pharmaceutical Engineering of Ministry of Education,
College of Pharmaceutical Sciences, Zhejiang University of Technology; Hangzhou 310014, China.
Received January 11, 2013; accepted April 1, 2013; advance publication released online April 5, 2013
A novel series of quinolone derivatives (6a–n) were designed and synthesized, and their biological ac-
tivities were evaluated as potential antitumor topoisomerase I (Top I) inhibitors. Among these compounds,
6j exhibited the most potent antitumor activities against multiple cancer cell lines. Docking simulation was
performed to insert compound 6j into the crystal structure of DNA-Top I to determine the probable binding
model.
Key words quinolone derivative; antitumor; topoisomerase I; molecular docking
DNA-topoisomerase I (Top I) is an important enzyme to derivatives as potential Top I inhibitors by scaffold modi-
relax supercoiled DNA for transcription, replication, and mito- fication. In addition, Al-Trawneh et al.24) also reported that
sis.1) Top I relaxes DNA by producing reversible single-strand tetracyclic fluoroquinolones exhibited high antiproliferative
DNA breaks. The generally accepted mechanism of Top I activity against breast MCF-7 and lung A549 tumor cell lines.
action involves a nucleophilic attack by the catalytic tyrosine
We attempted to design quinolone derivatives by scaffold
723 residue of Top I on the phosphate group at 3′-end of the modification (Fig. 2). Fragment A was designed based on
broken DNA strand, which leads to the break of the DNA Top I inhibitors CPUY01325) and A-62176.26) Fragment B was
phosphodiester bond and the formation of a binary Top I-DNA designed on the basis of Top I-DNA and CPT binding mode1)
covalent complex (Top I-DNAcc). The rotation of the 5′-end which showed that the amino acid residues Asn722 provides
around the intact strand allows for relaxation of the super- interactions with the A-ring of CPT. The target scaffold was a
coil.1,2) As it plays a pivotal role in cellular proliferation, Top combination of fragment A and B, with water soluble sulfonyl
I is often overexpressed in human tumors. Thus, DNA-Top I guanidine as a linker. Herein, we report the synthesis and in
has been identified as a promising cancer therapeutic target.3) vitro evaluation of the antitumor properties of novel N-substi-
Camptothecin (CPT) (1)4) and its clinically used analogues, tuted piperazinylquinolone derivatives (6a–n). The probable
topotecan (2) and irinotecan (3)5,6) (Fig. 1), were found to in- mechanism of the active compound (6j) was also examined by
hibit Top I activity by intercalating into the cleavage complex docking studies.
and preventing the relegation step of the catalytic cycle. As
a result, the covalent Top I-DNA adduct produces collisions
with advancing replication forks and transcription complexes,
which triggers irreversible DNA damage and apoptosis.7,8)
Results and Discussion
Chemistry N-Substituted piperazinylquinolone derivatives
(6a–n) were prepared via a two-step one-pot tandem process.
Although the camptothecins possess potent antitumor activ- The general synthetic pathway of target compounds is out-
ity, they also suffer from well-identified drawbacks, including lined in Chart 1. The description of substituents and yields are
solubility and bioactivity, dose-limiting toxicity.7–10) Addition- listed in Table 1.
ally, the E-ring lactone of camptothecin is readily opened to
its hydroxycarboxylate form in vivo.11) This form is less active
and binds strongly to human blood proteins.12) Thus, these
problems recommended further development of other non-
CPT Top I inhibitors with better pharmacokinetic features.
The non-CPT Top I inhibitors include indenoisoquino-
lines,13–15) indolocarbazones,16) saintopin,17) benzophena-
zines,18) terpyridines,19) and 3-arylisoquinolines.20) The suc-
cessful clinical cases of the non-CPT derivatives were attrib-
uted to the better chemical stability at longer lifetimes of the
trapped cleavage complex of the non-CPT Top I inhibitors at
the absence of a lactone ring in their skeleton.21)
The reaction of arylsulfonyl chloride (4) with cyanamide
Quinolones are among the most common frameworks pres-
ent in bioactive molecules and hence represent an attractive
starting point for the design of combinatorial libraries.22) Re-
cently, You and co-workers23) discovered series of quinolone
The authors declare no conflict of interest.
Fig. 1. Structures of 1 and Its Derivatives
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: lyw@qzu.zj.cn

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Vol. 61, No. 6
Fig. 2. Compound Generation by Scaffold Modification
Reagents and conditions: (a) NH2CN (50%), butanone, 40–60°C, 3h; (b) norfloxacin or ciprofloxacin, 80°C, 3h.
Chart 1. General Synthesis of Compounds (6a–n)
to form intermediates 5 is a typical nucleophilic substitution
Table 1. Structure and Synthetic Yield of the Target Compounds
reaction. The relatively weak nucleophilicity of the cyanamide
and the presence of dicyanamide may lead to byproducts.
The product arylsulfonyl cyanamide (5) can also react with
cyanamide to form byproducts. Therefore the reaction condi-
tions have to be carefully controlled. The target compound 6
was prepared by a subsequent nucleophilic addition of com-
mercially available norfloxacin hydrochloride or ciprofloxacin
hydrochloride with 5 in one-pot with yields ranging from 42%
to 85% (Table 1). The intermediates 5 are soluble in aqueous
medium, while the target compounds are not very soluble in
the reaction medium. Thus the pH of each reaction mixture
has to be well adjusted to precipitate the product.
Biological Activity and Discussion Evaluation of the
synthetic N-substituted piperazinylquinolone derivatives (6a–
n) for antiproliferative activity was performed by 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay using three different human cancer cell lines, human
Compound
R1
R2
Yield (%)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
H
H
Et
c-C3H5
Et
c-C3H5
Et
c-C3H5
Et
c-C3H5
Et
c-C3H5
Et
c-C3H5
Et
c-C3H5
55
70
76
85
75
84
76
83
74
58
71
76
58
42
4-Me
4-Me
4-Cl
4-Cl
4-CF3
4-CF3
2-Cl
2-Cl
2-CF3
2-CF3
3-Cl
3-Cl
lung carcinoma cell (A549), human promyelocytic leukemia skeleton exhibited remarkable antiproliferative effects. Among
cell (HL-60) and human cervical cancer cell (Hela). The as- them, compound 6j displayed the most potent inhibitory activ-
sessments of antiproliferative activities were expressed as the ity (IC50=0.072µm for A549, IC50=0.043µm for HL-60 and
concentration inhibiting 50% of cancer cell growth (IC50). The IC50=0.039µm for Hela), similar to the positive control Irino-
Top I inhibitory activity assays was carried out using a topo- tecan (IC50=0.032µm for A549, IC50=0.044µm for HL-60 and
isomerase I drug screening kit. The results were summarized IC50=0.038µm for Hela, respectively). As expected, compound
in Table 2.
6j showed excellent Top I inhibitory activity (++++) com-
As shown in Table 2, these N-substituted piperazinylquino- parable to Irinotecan. Consistency between cytotoxicities and
lone derivatives bearing the fluoro atom and piperazine on the Top I inhibitory activities was observed in these series, sug-

June 2013
633
Table 2. IC50 Cytotoxicity and Top I Inhibitory of Target Compounds
IC50(μm)
Compd.
Top Ia)
A549
HL-60
Hela
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
0.161
0.153
>0.193
>0.189
0.130
0.123
0.101
0.096
0.079
0.071
0.101
0.098
0.089
0.085
0.032
>0.313
>0.302
0.104
0.099
0.146
0.139
0.084
0.079
0.065
0.062
0.052
0.043
0.066
0.063
0.059
0.056
0.044
>0.313
>0.302
0.093
0.087
0.122
0.118
0.063
0.068
0.057
0.055
0.045
0.032
0.058
0.056
0.052
0.048
0.038
>0.313
>0.302
+
+
−
−
++
++
++
++
++++
++++
+++
+++
+++
+++
++++
−
6n
Irinotecan
Norfloxacin
Ciprofloxacin
−
a) Activity is expressed semi-quantitatively as follows: − no activity, + very weak activity, ++ weak activity, +++ lower activity than Irinotecan, ++++ similar or
greater activity than Irinotecan.
gesting that the target of N-substituted piperazinylquinolones ing result, along with the biological assays, suggested that
is limited to Top I.
N-substituted piperazinylquinolone derivatives were potential
Subsequently, structure–activity relationships (SAR) stud- Top I inhibitors as anti-cancer agents.
ies were inferred from Table 2. In general, target compounds
with R2=cyclopropyl shown more potent activities than those
Conclusion
with R2=ethyl at N-1 position. And the substituent group on
In our present work, a novel series of quinolone derivatives
the benzene ring had decisive affect on the IC50 value. Com- (6a–n) have been synthesized and evaluated. These com-
pounds with electron-donating group on the benzene ring, pounds exhibited potent antiproliferative activities against
such as 6c and d, shown weaker cytotoxic activities, and elec- A549, HL-60, and Hela cells. Among them, compound 6j
tron-withdrawing group such as chlorine or trifluoromethyl demonstrated strong cytotoxicity as well as potent Top I in-
on the benzene ring would increase the activities. Moreover, hibitory activity. Consistency between cytotoxicities and Top I
the position of substituent (R1) on the benzene ring also influ- inhibitory activities suggesting that the target of N-substituted
enced the activities. Compounds with ortho substituent (6i–l) piperazinylquinolones is limited to Top I. Molecular docking
shown more potent activities than those with meta (6m,n) or studies shown that compound 6j bound to the DNA-Top I
para (1e–h) substituent.
binding site by three hydrogen bonds and two π-cation inter-
Docking Study Molecular docking is an application actions which might play crucial roles in its antiproliferative
wherein molecular modeling techniques are used to predict activities. Further discovery of more enhanced N-substituted
how a protein interacts with small molecules.27) In the pres- piperazinylquinolone derivatives with better anticancer activ-
ent study, to gain better understanding on the interactions ity and lower toxicity and side-effects is under the way.
between target compound and Top I (PDB code: 1T8I) and to
explore their binding mode, a docking study was performed
using the CDOCKER protocol in Discovery Studio 2.1 (Dis-
covery Studio 2.1, Accelrys, Inc., San Diego, CA, U.S.A.).
Experimental
Melting points were determined on Büchi B-540 melting
point apparatus and are uncorrected. 1H-NMR (300MHz)
Docking model illustrates that the quinolone derivatives spectra were recorded on a Bruker AV 300MHz spectrom-
of 6j was well positioned in the binding sites of DNA-Top eter. Mass spectra were obtained on a Thermo Finnigan
I complex (Fig. 3). The structural model suggested that one LCQ-Advantage spectrometer (electrospray ionization (ESI),
of the two sulfonyl oxygens, fluoro atom on the quinolone atmospheric-pressure chemical ionization (APCI)). High reso-
skeleton and quinolone ring, interacted with enzyme (Fig. 3a). lution (HR)-MS was carried out on an APEX (Bruker) mass
One of the sulfonyl oxygen formed one hydrogen bond with III spectrometer. The compounds were dissolved in DMSO-d6.
the amino hydrogen of Lys425. And the fluoro atom on the Chemical shifts, δ, are given in ppm relative to tetramethyl-
quinolone ring of compound 6j associates with Arg364 by two silane (TMS) (δ=0) and are referenced by using the residual
hydrogen bonds. The quinolone skeleton of compound 6j is undeuterated solvent signal. Coupling constants, J, are re-
calculated to interact with Arg364 and is likely stabilized by ported in Hz, multiplicities being marked as: singlet (s), broad
π-stacking interactions.
singlet (brs), doublet (d), triplet (t), quartet (q), multiplet (m).
All the amino acid residues which had interactions with
General Experimental Procedure Benzenesulfonyl chlo-
compound 6j were displayed in Fig. 3b. The enzyme surface ride (0.01mol) in butanone (30mL) was heated with stirring
model was shown in Fig. 3c, which revealed that the molecule to 40°C, and cyanamide solution (50%) was added dropwise
was well embedded in the active pocket. This molecular dock- during 30min. The temperature was raised to 60°C and stir-

634
Vol. 61, No. 6
Fig. 3a. Compound 6j Bound into Top I
The dotted lines show the hydrogen bonds and the solid lines show the π-cation interactions.
Fig. 3b. 2D Ligand Interaction Diagram of Compound 6j with Top I Using Discovery Studio Program with the Essential Amino Acid Residues at the
Binding Site Are Tagged in Circles
ring continued for 3h. After cooling the reaction mixture to carboxylic Acid (6b): White crystals, mp 295–298°C; 70%
1
below 30°C, norfloxacin hydrochloride or ciprofloxacin hy- yield; H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.18 (m, 2H),
drochloride monohydrate (0.008mol) was added. The mixture 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.28
was heated to 80°C and maintained for 3h. After cooling to (d, 1H, J=3.0Hz), 7.33–7.42 (m, 2H), 7.48–7.65 (m, 3H), 7.90
below 40°C, the reaction mixture was poured into cold water (d, 1H, J=9.0Hz), 8.63 (s, 1H), 9.23 (brs, 2H), 15.05 (brs,
while stirring, white crystals or powders were precipitated, 1H); HR-MS Calcd for C24H25FN5O5S [M+H+]: 514.1560.
filtered, washed with water, and dried at 80°C. Analytically Found: 514.1553.
pure samples were obtained by recrystallization from aqueous
ethanol.
1-Ethyl-6-fluoro-4-oxo-7-(4-(N-tosylcarbamimidoyl)-
piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6c):
1
1-Ethyl-6-f luoro-4-oxo-7-(4-(N-(phenylsulfonyl)- White crystals, mp 257–260°C; 76% yield; H-NMR (DMSO-
carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3- d6, 300MHz) δ (ppm): 1.41 (t, 3H, J=4.5Hz), 2.45 (s, 3H), 3.19
carboxylic Acid (6a): White crystals, mp 221–225°C; 55% (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5Hz), 7.17 (d, 1H, J=
1
yield; H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.41 (t, 3H, 3.0Hz), 7.35 (m, 2H), 7.58 (m, 3H), 7.86 (d, 1H, J=9.0Hz),
J=4.5Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5Hz), 8.82 (s, 1H), 8.91 (brs, 2H), 15.05 (brs, 1H); HR-MS Calcd for
7.17 (d, 1H, J=3.0Hz), 7.35 (m, 2H), 7.58 (m, 3H), 7.86 (d, C24H27FN5O5S [M+H+]: 516.1717. Found: 516.1724.
1H, J=9.0Hz), 8.82 (s, 1H), 8.91 (brs, 2H), 15.05 (brs, 1H);
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(N-tosylcarbamimidoyl)-
HR-MS Calcd for C23H25FN5O5S [M+H+]: 502.1560. Found: piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic Acid (6d):
1
502.1554.
White crystals, mp 262–265°C; 85% yield; H-NMR (DMSO-
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(N-(phenylsulfonyl)- d6, 300MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 2.45 (s,
carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3- 3H), 3.33 (m, 4H), 3.53 (m, 4H), 3.83 (m, 1H), 7.22 (d, 1H,

June 2013
635
7-(4-(N-(2-Chlorophenylsulfonyl)carbamimidoyl)-
piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic Acid (6i): Pale yellow crystals, mp 255–258°C; 74%
1
yield; H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.41 (t, 3H,
J=4.5Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, J=4.5Hz),
7.17 (d, 1H, J=3.0Hz), 7.46 (m, 3H), 7.90 (d, 1H, J=9.0Hz),
8.46 (m, 1H), 8.95 (s, 1H), 9.11 (brs, 2H), 15.10 (brs, 1H);
HR-MS Calcd for C23H24ClFN5O5S [M+H+]: 536.1171. Found:
536.1166.
7-(4-(N-(2-Chlorophenylsulfonyl)carbamimidoyl)-
piperazin-1-yl)-1-cyclopropyl- 6 -f luoro- 4-oxo-1,4-
dihydroquinoline-3-carboxylic Acid (6j): Pale yellow crystals,
mp 286–289°C; 58% yield; 1H-NMR (DMSO-d6, 300MHz)
δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m,
4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0Hz), 7.46 (m, 3H), 7.90
(d, 1H, J=9.0Hz), 8.46 (m, 1H), 8.63 (s, 1H), 9.23 (brs, 2H),
15.05 (brs, 1H); HR-MS Calcd for C24H24ClFN5O5S [M+H+]:
548.1171. Found: 548.1169.
Fig. 3c. 3D Model of the Interaction between Compound 6j and Top I
1-Ethyl-6-fluoro-4-oxo-7-(4-(N-(2-(trifluoromethyl)-
phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic Acid (6k): White-like crystals,
Bonding Site
The protein is represented by molecular surface. Compound 6j is depicted by
balls.
1
mp 218–221°C; 71% yield; H-NMR (DMSO-d6, 300MHz) δ
J=3.0Hz), 7.38 (m, 2H), 7.58 (m, 2H), 7.86 (d, 1H, J=9.0Hz), (ppm): 1.41 (t, 3H, J=4.5Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58
8.63 (s, 1H), 9.23 (brs, 2H), 15.05 (brs, 1H); HR-MS Calcd for (q, 2H, J=4.5Hz), 7.17 (d, 1H, J=3.0Hz), 7.64–7.85 (m, 3H),
C25H27FN5O5S [M+H+]: 528.1717. Found: 528.1723.
7.90 (d, 1H, J=9.0Hz), 8.50 (m, 1H), 8.95 (s, 1H), 9.11 (brs,
7-(4-(N-(4-Chlorophenylsulfonyl)carbamimidoyl)- 2H), 15.10 (brs, 1H); HR-MS Calcd for C24H24F4N5O5S [M+
piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3- H+]: 570.1434. Found: 570.1437.
carboxylic Acid (6e): Light yellow crystals, mp 297–299°C;
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(N-(2-(trifluoromethyl)-
75% yield; 1H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.41 phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4-
(t, 3H, J=4.5Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H, dihydroquinoline-3-carboxylic Acid (6l): White-like crystals,
1
J=4.5Hz), 7.17 (d, 1H, J=3.0Hz), 7.46 (m, 2H), 7.66 (m, 2H), mp 237–240°C; 76% yield; H-NMR (DMSO-d6, 300MHz) δ
7.90 (d, 1H, J=9.0Hz), 8.95 (s, 1H), 9.11 (brs, 2H), 15.10 (brs, (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H),
1H); HR-MS Calcd for C23H24ClFN5O5S [M+H+]: 536.1171. 3.83 (m, 1H), 7.28 (d, 1H, J=3.0Hz), 7.64–7.85 (m, 3H), 7.90
Found: 536.1164.
(d, 1H, J=9.0Hz), 8.50 (m, 1H), 8.73 (s, 1H), 9.18 (brs, 2H),
7-(4-(N-(4-Chlorophenylsulfonyl)carbamimidoyl)- 15.05 (brs, 1H); HR-MS Calcd for C25H24F4N5O5S [M+H+]:
piperazin-1-yl)-1-cyclopropyl- 6 -f luoro- 4 -oxo-1,4- 582.1434. Found: 582.1427.
dihydroquinoline-3-carboxylic Acid (6f): Light yellow
7-(4-(N-(3-Chlorophenylsulfonyl)carbamimidoyl)-
crystals, mp 288–291°C; 84% yield; 1H-NMR (DMSO-d6, piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
300MHz) δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), carboxylic Acid (6m): White-like crystals, mp 222–225°C;
3.53 (m, 4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0Hz), 7.36 (m, 58% yield; 1H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.41
2H), 7.60 (m, 2H), 7.90 (d, 1H, J=9.0Hz), 8.63 (s, 1H), 9.23 (t, 3H, J=4.5Hz), 3.19 (m, 4H), 3.43 (m, 4H), 4.58 (q, 2H,
(brs, 2H), 15.05 (brs, 1H); HR-MS Calcd for C24H24ClFN5O5S J=4.5Hz), 7.17 (d, 1H, J=3.0Hz), 7.35–7.60 (m, 3H), 7.86
[M+H+]: 548.1171. Found: 548.1179.
(s, 1H), 7.95 (d, 1H, J=9.0Hz), 8.86 (s, 1H), 9.16 (brs, 2H),
1-Ethyl-6-fluoro-4-oxo-7-(4-(N-(4-(trifluoromethyl)- 15.00 (brs, 1H); HR-MS Calcd for C23H24ClFN5O5S [M+H+]:
phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4- 536.1171. Found: 536.1178.
dihydroquinoline-3-carboxylic Acid (6g): White-like crystals,
mp 217–220°C; 76% yield; H-NMR (DMSO-d6, 300MHz) δ piperazin-1-yl)-1-cyclopropyl- 6 -f luoro- 4-oxo-1,4-
(ppm): 1.41 (t, 3H, J=4.5Hz), 3.19 (m, 4H, –CH2), 3.43 (m, dihydroquinoline-3-carboxylic Acid (6n): White-like crystals,
7-(4-(N-(3-Chlorophenylsulfonyl)carbamimidoyl)-
1
1
4H), 4.58 (q, 2H, J=4.5Hz), 7.17 (d, 1H, J=3.0Hz), 7.56 (m, mp 228–232°C; 42% yield; H-NMR (DMSO-d6, 300MHz) δ
2H), 7.90 (d, 1H, J=9.0Hz), 8.22 (m, 2H), 8.86 (s, 1H), 9.15 (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, 4H),
(brs, 2H), 15.08 (brs, 1H); HR-MS Calcd for C24H24F4N5O5S 3.83 (m, 1H), 7.28 (d, 1H, J=3.0Hz), 7.35–7.60 (m, 3H), 7.86
[M+H+]: 570.1434. Found: 570.1427.
(s, 1H), 7.90 (d, 1H, J=9.0Hz), 8.63 (s, 1H), 9.23 (brs, 2H),
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(N-(4-(trifluoromethyl)- 15.05 (brs, 1H); HR-MS Calcd for C24H24ClFN5O5S [M+H+]:
phenylsulfonyl)carbamimidoyl)piperazin-1-yl)-1,4- 548.1171. Found: 548.1177.
dihydroquinoline-3-carboxylic Acid (6h): White-like crystals,
Anti-proliferation Assay The antiproliferative activities
mp 248–251°C; 83% yield; 1H-NMR (DMSO-d6, 300MHz) of the prepared compounds against A549, HL-60 and Hala
δ (ppm): 1.18 (m, 2H), 1.33 (m, 2H), 3.33 (m, 4H), 3.53 (m, cells were evaluated using a standard MTT-based colorimet-
4H), 3.83 (m, 1H), 7.28 (d, 1H, J=3.0Hz), 7.47 (m, 2H), 7.90 ric assay. Target tumor cell lines were grown to log phase
(d, 1H, J=9.0Hz), 8.06 (m, 2H), 8.63 (s, 1H), 9.23 (brs, 2H), in RPMI 1640 medium supplemented with 10% fetal bovine
15.05 (brs, 1H); HR-MS Calcd for C25H24F4N5O5S [M+H+]: serum. After diluting to 1×106 cellsmL−1 with the complete
582.1434. Found: 582.1440.
medium, 100µL of the obtained cell suspension was added to

636
Vol. 61, No. 6
(2002).
each well of 96-well culture plates. The subsequent incubation
was permitted at 37°C, 5% CO2 atmosphere for 24h before the
cytotoxicity assessments. Each concentration was in triplicate,
and Irinotecan was used as the positive control. After 72h in-
cubation at 37°C, 5% CO2 atmosphere, 10µL of MTT solution
in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen,
U.S.A.) was added to each well. After three hours incubation
at 37°C, 150µL DMSO was added to each well. The plates
were then vibrated for 10min for complete dissolution. The
optical absorbance was measured at 570nm on an automated
microplate spectrophotometer (Bio-Rad, U.S.A.). In all experi-
ments three replicate wells were used for each drug concentra-
tion. The IC50 value was defined as the concentration at which
50% of the cells could survive. The results were summarized
in Table 2.
Docking Simulations The pdb file about the crystal struc-
ture of DNA-Top I bound to SA315F (PDB code: 1T8I)28,29)
was obtained from the RCSB Protein Data Bank (http://www.
pdb.org). The molecular docking procedure was performed by
using CDOCKER protocol for receptor-ligand interactions of
Discovery Studio 2.5. For ligand preparation, the 3D struc-
tures of 6j were generated and minimized using Discovery
Studio 2.5. For protein preparation, the hydrogen atoms were
added. The whole DNA-Top I domain defined as a receptor
and the site sphere was selected based on the ligand binding
location of SA315F, then the SA315F removed and the ligands
prepared by us was placed during the molecular docking
procedure. CHARMm was selected as the force field. The
molecular docking was performed with a simulated annealing
method.
9) Schaeppi U., Fleischman R. W., Cooney D. A., Cancer Chemother.
Rep. 3, 5, 25–36 (1974).
10) Chrencik J. E., Staker B. L., Burgin A. B. Jr., Pourquier P., Pom-
mier Y., Stewart L., Redinbo M. R., J. Mol. Biol., 339, 773–784
(2004).
11) Luzzio M. J., Besterman J. M., Emerson D. L., Evans M. G., Lackey
K., Leitner P. L., McIntyre G., Morton B., Myers P. L., Peel M.,
Sisco J. M., Sternbach D. D., Tong W.-Q., Truesdale A., Uehling D.
E., Vuong A., Yates J., J. Med. Chem., 38, 395–401 (1995).
12) Mi Z., Burke T. G., Biochemistry, 33, 10325–10336 (1994).
13) Meng L. H., Liao Z. Y., Pommier Y., Curr. Top. Med. Chem., 3,
305–320 (2003).
14) Morrell A., Antony S., Kohlhagen G., Pommier Y., Cushman M., J.
Med. Chem., 49, 7740–7753 (2006).
15) Nagarajan M., Morrell A., Ioanoviciu A., Antony S., Kohlhagen G.,
Agama K., Hollingshead M., Pommier Y., Cushman M., J. Med.
Chem., 49, 6283–6289 (2006).
16) Pereira E. R., Belin L., Sancelme M., Prudhomme M., Ollier M.,
Rapp M., Sevère D., Riou J. F., Fabbro D., Meyer T., J. Med. Chem.,
39, 4471–4477 (1996).
17) Yamashita Y., Kawada S., Fujii N., Nakano H., Biochemistry, 30,
5838–5845 (1991).
18) Vicker N. Burgess L., Chuckowree I. S., Dodd R., Folkes A. J.,
Hardick D. J., Hancox T. C., Miller W., Milton J., Sohal S., Wang
S., Wren S. P., Charlton P. A., Dangerfield W., Liddle C., Mistry P.,
Stewart A. J., Denny W. A., J. Med. Chem., 45, 721–739 (2002).
19) Thapa P., Karki R., Thapa U., Jahng Y., Jung M. J., Nam J. M., Na
Y., Kwon Y., Lee E. S., Bioorg. Med. Chem., 18, 377–386 (2010).
20) Cho W. J., Min S. Y., Le T. N., Kim T. S., Bioorg. Med. Chem.
Lett., 13, 4451–4454 (2003).
21) Pommier Y., Chem. Rev., 109, 2894–2902 (2009).
22) Mugnaini C., Pasquini S., Corelli F., Curr. Med. Chem., 16, 1746–
1767 (2009).
23) You Q. D., Li Z. Y., Huang C. H., Yang Q., Wang X. J., Guo Q. L.,
Chen X. G., He X. G., Li T. K., Chern J. W., J. Med. Chem., 52,
5649–5661 (2009).
24) Al-Trawneh S. A., Zahra J. A., Kamal M. R., El-Abadelah M. M.,
Zani F., Incerti M., Cavazzoni A., Alfieri R. R., Petronini P. G.,
Vicini P., Bioorg. Med. Chem., 18, 5873–5884 (2010).
25) Ji Y. B., Zhou J. H., Zou M. X., You Q. D., Acta Pharmacol. Sin.,
43, 811–818 (2008).
26) Kang D. H., Kim J. S., Jung M. J., Lee E. S., Jahng Y., Kwon Y., Na
Y., Bioorg. Med. Chem. Lett., 18, 1520–1524 (2008).
27) Friesner R. A., Banks J. L., Murphy R. B., Halgren T. A., Klicic J.
J., Mainz D. T., Repasky M. P., Knoll E. H., Shelley M., Perry J. K.,
Shaw D. E., Francis P., Shenkin P. S., J. Med. Chem., 47, 1739–1749
(2004).
28) Staker B. L., Feese M. D., Cushman M., Pommier Y., Zembower D.,
Stewart L., Burgin A. B., J. Med. Chem., 48, 2336–2345 (2005).
29) Fox B. M., Xiao X., Antony S., Kohlhagen G., Pommier Y., Staker
B. L., Stewart L., Cushman M., J. Med. Chem., 46, 3275–3282
(2003).
Acknowledgement We are grateful to the Natural Science
Foundation of Zhejiang Province (No. Y2100908) for financial
support.
References
1) Redinbo M. R., Stewart L., Kuhn P., Champoux J. J., Hol W. G. J.,
Science, 279, 1504–1513 (1998).
2) Koster D. A., Croquette V., Dekker C., Shuman S., Dekker N. H.,
Nature (London), 434, 671–674 (2005).
3) Rasheed Z. A., Rubin E. H., Oncogene, 22, 7296–7304 (2003).
4) Wall M. E., Wani M. C., Cook C. E., Palmer K. H., McPhail A. T.,
Sim G. A., J. Am. Chem. Soc., 88, 3888–3890 (1966).
5) Thomas C. J., Rahier N. J., Hecht S. M., Bioorg. Med. Chem., 12,
1585–1604 (2004).
6) Teicher B. A., Biochem. Pharmacol., 75, 1262–1271 (2008).
7) Hsiang Y. H., Hertzberg R., Hecht S., Liu L. F., J. Biol. Chem., 260,
14873–14878 (1985).
8) Staker B. L., Hjerrild K., Feese M. D., Behnke C. A., Burgin A.
B. Jr., Stewart L., Proc. Natl. Acad. Sci. U.S.A., 99, 15387–15392