Synthesis, molecular docking and biological evaluation of quinolone derivatives as novel anticancer agents

Article abstract of DOI:10.1248/cpb.c17-00035

A series of novel quinolone derivatives (8a-j) were synthesized, and their anticancer activities were tested in human cancer cell lines, human lung carcinoma cell (A549), human promyelocytic leukemia cell (HL-60), and human cervical cancer cell (Hela). Compound 8i was found to be 5-times more potent in cellkilling activity for cell lines A549, HL-60, and Hela than the positive control irinotecan or cisplatin, with IC₅₀ of 0.009, 0.008 and 0.010 μM, respectively. The docking study revealed that compound 8i might have strong interactions with the active site of DNA-topoisomerase I.

Full text of DOI:10.1248/cpb.c17-00035

Vol. 66, No. 1
Chem. Pharm. Bull. 66, 55–60 (2018)
55
Regular Article
Synthesis, Molecular Docking and Biological Evaluation of Quinolone
Derivatives as Novel Anticancer Agents
Jie Li, Tu-cai Zheng, Yi Jin, Jian-guo Xu, Jian-gang Yu, and Yan-wen Lv*
College of Chemistry and Materials Engineering, Quzhou University; Quzhou 324000, China.
Received January 6, 2017; accepted November 2, 2017; advance publication released online November 9, 2017
A series of novel quinolone derivatives (8a–j) were synthesized, and their anticancer activities were
tested in human cancer cell lines, human lung carcinoma cell (A549), human promyelocytic leukemia cell
(HL-60), and human cervical cancer cell (Hela). Compound 8i was found to be 5-times more potent in cell-
killing activity for cell lines A549, HL-60, and Hela than the positive control irinotecan or cisplatin, with
IC50 of 0.009, 0.008 and 0.010µM, respectively. The docking study revealed that compound 8i might have
strong interactions with the active site of DNA-topoisomerase I.
Key words quinolone derivative; anticancer; synthesis; molecular docking
Quinolone as a privileged scaffold represents one of the nificant potency against eukaryotic topoisomerases (Top) and
most important structural unit prevalent in various naturally showing cytotoxic activity against tumor cell lines.4–7)
occurring and bioactive compounds.1) Quinolones consist of
Li and colleagues designed and synthesized a series of
a bicyclic ring structure (Fig. 1) in which there is a substitu- quinolone derivatives as potential Top I inhibitors for cancer
tion at position N-1, with various moieties. Most of the current treatment.8–10) Rajulu et al. designed a series of fluoroqui-
agents have a carboxyl group at position 3, a keto group at nolones displaying good growth inhibition activities against
position 4, a fluorine atom at position 6 and a nitrogen het- human lung carcinoma cell (A549) and colon carcinoma
erocycle at position 7.2,3) In the late 1980s, reports emerged (HCT-116).11) Recently, our group discovered a novel series of
describing experimental antibacterial quinolones having sig- Top I inhibitors with quinolone scaffold. Quinolone derivative
1 was the most potent compound we synthesized.12) Several
important series of antimicrobial agents are associated with
particular N-1 substituents. Tosufloxacin (2) and difloxacin
(3) (Fig. 2) with the substituent of fluorine atom in the aro-
matic ring enhanced both potency against Gram-positives and
pharmacokinetics.13) The antibacterial tosufloxacin (2) can be
viewed as intermediate agents on the evolutionary path to-
ward both antibacterial and anticancer quinolone derivatives.7)
Using a scaffold modification strategy, our team changed the
N-1 cyclopropyl group into N-1 aryl substituents with fluorine
in the aromatic ring (Fig. 2).
Fig. 1. Structural Features of Quinolones
Fig. 2. Compound Generation by Scaffold Modification
*To whom correspondence should be addressed. e-mail: lyw@qzu.zj.cn
© 2018 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 66, No. 1 (2018)
Chart 1. Synthesis of Compounds (8a–j)
Results and Discussion
Table 1. IC50 Cytotoxicity of Target Compounds
Chemistry The method for the preparation of novel N-flu-
oroaromatic substituted piperazinylquinolone derivatives 8a–j
relies on the Grohe–Heitzer cycloacylation reaction.14,15) The
synthetic route is outlined in Chart 1. The commercially avail-
able compound 4 was subjected to an addition–elimination
reaction with a substituted primary amine, and the obtained
product 5 was cyclised in a tandem addition–elimination
reaction at the ortho position. Compound 7 was obtained by
displacing the chlorine atom in compound 6 with anhydrous
piperazine. Then the target compound 8 was prepared via a
two-step one-pot tandem process.12)
Biological Activity and Discussion All the target com-
pounds (8a–j) were evaluated for their in vitro cytotoxic ac-
tivity against three different human cancer cell lines, A549,
human promyelocytic leukemia cell (HL-60) and human
cervical cancer cell (Hela) by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay with irinotecan
and cisplatin as positive control compounds. The assessments
of anticancer activities were expressed as the concentration
IC50 (µM)
Compd.
A549
HL-60
Hela
1
0.071
>0.157
>0.086
>0.053
0.041
0.043
>0.157
0.081
0.049
0.039
0.048
0.016
0.050
0.044
0.033
0.008
0.018
0.044
0.057
0.032
>0.157
>0.086
0.040
0.043
0.049
0.037
0.069
0.045
0.029
0.010
0.010
0.038
0.047
4
8a
8b
8c
8d
0.032
8e
0.036
8f
>0.082
0.039
8g
8h
0.038
8i
0.009
8j
0.028
Irinotecan
Cisplatin
0.032
0.048
inhibiting 50% of cancer cell growth (IC50). The Top I inhibi- activity comparable to irinotecan and cisplatin.
tory activity assays was carried out using a topoisomerase I
Subsequently, structure–activity relationships (SAR) studies
drug screening kit. The results are summarized in Table 1 were inferred from Table 1. In general, target compounds with
and Fig. 3. As illustrated in Table 1, these compounds exhib- N-1 substituent of 2,4-difluorophenyl group (8f–j) showed
ited significant antitumor activity. Among them, compound 8i more potent activities than those with N-1 substituent of 4-flu-
displayed the most potent inhibitory activity (IC50=0.009µM orophenyl group (8a–e). In three different human cancer cell
for A549, IC50=0.008µM for HL-60, and IC50=0.010 µM for lines, using electron withdrawing group (Cl, CF3) in the phen-
Hela), which was better than the positive control irinotecan yl moiety as in compounds 8b–e and 8g–j increase the reac-
(IC50=0.032µM for A549, IC50=0.044µM for HL-60, and tivity rather than using electron donating group (CH3) in the
IC50=0.038µM for Hela) and cisplatin (IC50=0.048µM for phenyl moiety as in compounds 8a and 8f. And compounds
A549, IC50=0.057µM for HL-60, and IC50=0.047µM for Hela). 8i and 8j with trifluoromethyl in the phenyl moiety showed
As expected, compound 8i showed excellent Top I inhibitory higher cytotoxic activity than in compounds 8g and 8h with

Vol. 66, No. 1 (2018)
Chem. Pharm. Bull.
57
Fig. 3. Top I Inhibitory Activity
Inhibition of Top I at 1µM. Lane 1: supercoiled plasmid DNA (pBR322); Lane 2: DNA+Top I; Lane 3: DNA+Top I+Irinotecan; Lane 4: DNA+Top I+Cisplatin; Lane
5–15: DNA+Top I+compounds (1, 8a, 8b, 8c, 8d, 8e, 8f, 8g, 8h, 8i and 8j). Sc—supercoiled DNA, Nck—nicked open circular DNA.
Fig. 4A. Compound 8i Bound to Top I-DNA Complex
The dotted lines show the hydrogen bonds and the solid lines show the π–cation and π–π interactions.
Fig. 4B. 2D Ligand Interaction Diagram of Compound 8i with Top I-DNA Complex Using Discovery Studio Program with the Essential Amino Acid
Residues at the Binding Site Are Tagged in Circles
chlorine atom in the phenyl moiety. It is noted that the sub- of phenyl (8j), which may be due to steric-hindrance effect.
stitution R2 at the 2-position of phenyl (8i) exhibiting better
Docking Study Docking was performed against DNA-
biological activities than the substitution R2 at the 3-position Top I because it is reported as possible anticancer target of

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Chem. Pharm. Bull.
Vol. 66, No. 1 (2018)
Fig. 4C. 3D Model of the Interaction between Compound 8i and Top I-DNA Complex
The protein is represented by molecular surface. Compound 8i is depicted by balls.
quinolone derivatives.16–19) In order to understand the bind- 300MHz spectrometer. Mass spectra were obtained on a
ing conformation of the most potent cytotoxic activity of the Thermo Finnigan LCQ-Advantage spectrometer (electrospray
compound 8i, its flexible molecular docking was carried out ionization (ESI), atmospheric pressure chemical ionization
into the Top I (PDB code: 1K4T) active site using CDOCKER (APCI)), and high resolution (HR)-MS were carried out on an
protocol of Discovery Studio 2.1.
APEX (Bruker) mass III spectrometer. The compounds were
The binding modes of compound 8i and Top I-DNA com- dissolved in dimethyl sulfoxide (DMSO)-d6. Chemical shifts
plex were depicted in Fig. 4. The amino acid residue which were reported in ppm (δ) relative to tetramethylsilane (TMS)
had interaction with Top I-DNA complex were labeled in Figs. (δ=0). Coupling constants, J, are reported in Hz, multiplicities
4A and 4B. In the binding mode, compound 8i was nicely being marked as: singlet (s), broad singlet (brs), doublet (d),
bound to the Top I-DNA complex active site via six hydrogen triplet (t), quartet (q), multiplet (m).
bond, two cation–π interaction and ten π–π interaction. The
General Procedure for Synthesis of Compounds (5a,
nitrogen atom of guanidine group and one of the sulfonyl b) Firstly, a solution of commercially available compound 4
oxygen formed two hydrogen bond with amino of LYS 751. (8g, 25mmol) in 25mL ethanol, was added with the solution
The fluorine atom at the 2-position of phenyl and the carboxyl of fluoro-substituted aniline (26.25mmol) in 10mL ethanol
group of 8i formed two hydrogen bond with amino of ARG dropwise during 10min. The reaction mixture was then stirred
364 and two hydrogen bond with base pair of TGP 11, respec- at 45°C for 6h. The cooled reaction was filtered to give the
tively. And the benzene ring with two fluorine atom associated desired product 5 as light yellow solid.
with LYS 532 and ARG 364 by two cation–π interactions. The
General Procedure for Synthesis of Compounds (6a, b)
quinolone skeleton and the benzene ring of compound 8i sta- A solution of 5 (10mmol), K2CO3 (2.2g, 16mmol) in 20mL
bilized by base-stacking interactions with both the upstream N,N-dimethylformamide (DMF) was stirred at 140°C for 2h.
(−1) and downstream (+1) base pairs with ten π–π interac- The hot mixture was filtered rapidly, then the solution was
tions.
allowed to stir at room temperature for 10min until a yellow
The enzyme surface model was shown in Fig. 4C, which precipitate is formed. The solid was filtered, washed with
revealed that the molecule was well embedded in the active water twice, and dried in vacuo.
pocket. This molecular docking results and the biological
General Procedure for Synthesis of Compounds (7a,
assay data suggested that compound 8i was a potential Top I b) The compound 6 (3.5mmol), anhydrous piperazine (1.5g,
inhibitors as anti-cancer agents.
17.5mmol) and isopropanol (10mL) were mixed and stirred
at 130°C for 3h, then the solvent was removed under reduced
pressure. The residue was added with 10% NaOH (6mL), and
Conclusion
We have designed and synthesized a novel series of quino- stirred at 90°C for 1.5h, then added with activated carbon
lone derivatives (8a–j). These compounds exhibited in vitro and stirred at reflux for 1h, respectively. The hot mixture was
cytotoxic activity against A549, HL-60, and Hela cells. Dock- filtered and the aqueous phase was then acidified with hydro-
ing simulations were performed to position most active com- chloric acid to pH 7. The mixture was stirred at room tem-
pound 8i into the Top I-DNA complex active site to determine perature until a precipitated is formed. The solid was filtered
the probable binding conformation and the results confirmed on a Buchner funnel to give the desired product 7.
that the compound was a potential Top I inhibitor.
General Procedure for Synthesis of Compounds (8a,
j) Benzenesulfonyl chloride (1mmol) in butanone (5mL) was
heated with stirring to 40°C, and cyanamide solution (50%)
Experimental
Chemistry Melting points (m.p.) were determined on was added dropwise, then the temperature was raised to 60°C
Büchi B-540 melting point apparatus and are uncorrected. and stirring continued for 3h. The compound 7 (0. 8mmol)
1H-NMR (300MHz) spectra were recorded on a Bruker AV was added and heated to 80°C for 3h. After cooling to 40°C,

Vol. 66, No. 1 (2018)
Chem. Pharm. Bull.
59
the reaction mixture was poured into cold water while stir-
7-(4-(N-((2,6-Dichlorophenyl)sulfonyl)carbamimidoyl)-
ring, white crystals or powders were precipitated, filtered, piperazin-1-yl)-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-
washed with water, and dried. Analytically pure samples were dihydroquinoline-3-carboxylic Acid (8c)
obtained by recrystallization from aqueous ethanol.
(Z)-Methyl 2-(2,4-Dichloro-5-fluorobenzoyl)-3-((4-fluoro- δ (ppm): 3.40–3.54 (m, 4H, –CH2, piperazine), 3.76–3.84
phenyl)amino)acrylate (5a)
M.p. 211–213°C, Yield 71%, 1H-NMR (DMSO-d6, 300MHz)
(m, 4H, –CH2, piperazine), 6.83 (d, 1H, J=4.0Hz, 8-H),
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 3.80 (s, 3H, 7.10–7.13 (m, 4H, –C6H4), 7.49–7.73 (m, 3H, –C6H3), 8.01
–CH3), 6.74–6.94 (m, 4H, –C6H4), 7.55 (d, 1H, J=4.0Hz, 6-H), (d, 1H, J=4.0Hz, 5-H), 8.26 (s, 1H, 2-H); HR-MS Calcd for
7.81 (d, 1H, J=4.0Hz, 3-H), 9.79 (s, 1H, –NH). MS (ESI): C27H21Cl2F2N5O5S [M−]: 635.0609. Found: 635.0601.
385.0.
6-Fluoro-1-(4-f luorophenyl)-4-oxo-7-(4-(N-((2-(tri-
(Z)-Methyl 2-(2,4-Dichloro-5-fluorobenzoyl)-3-((2,4-diflu- fluoromethyl)phenyl)sulfonyl)carbamimidoyl)piperazin-1-yl)-
orophenyl)amino)acrylate (5b)
1,4-dihydroquinoline-3-carboxylic Acid (8d)
M.p. 207–209°C, Yield 60%, H-NMR (DMSO-d6, 300MHz)
1
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 3.83 (s, 3H, –CH3),
6.60–6.78 (m, 3H, –C6H3), 7.61 (d, 1H, J=4.0Hz, 6-H), 7.83 δ (ppm): 3.35–3.57 (m, 4H, –CH2, piperazine), 3.75–3.84
(d, 1H, J=4.0Hz, 3-H), 9.56 (s, 1H, –NH). MS (ESI): 403.0. (m, 4H, –CH2, piperazine), 7.01 (d, 1H, J=4.0Hz, 8-H),
Methyl 7-Chloro-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4-di- 7.08–7.09 (m, 4H, –C6H4), 7.54–7.75 (m, 4H, –C6H4), 8.01
hydroquinoline-3-carboxylate (6a) (d, 1H, J=4.0Hz, 5-H), 8.21 (s, 1H, 2-H); HR-MS Calcd for
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 3.82 (s, 3H, –CH3), C28H22F5N5O5S [M−]: 635.1262. Found: 635.1260.
7.09–7.18 (m, 4H, –C6H4), 7.25 (d, 1H, J=4.0Hz, 8-H), 7.66 (d,
1H, J=4.0Hz, 5-H), 8.29 (s, 1H, 2-H). MS (ESI): 349.0.
6-Fluoro-1-(4-f luorophenyl)-4-oxo-7-(4-(N-((3-(tri-
fluoromethyl)phenyl)sulfonyl)carbamimidoyl)piperazin-1-yl)-
Methyl 7-Chloro-1-(2,4-difluorophenyl)-6-fluoro-4-oxo-1,4- 1,4-dihydroquinoline-3-carboxylic Acid (8e)
1
dihydroquinoline-3-carboxylate (6b)
M.p. 205–206°C, Yield 65%, H-NMR (DMSO-d6, 300MHz)
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 3.82 (s, 3H, –CH3), δ (ppm): 3.44–3.54 (m, 4H, –CH2, piperazine), 3.76–3.84 (m,
6.82–7.12 (m, 3H, –C6H3), 7.18 (d, 1H, J=4.0Hz, 8-H), 7.66 (d, 4H, –CH2, piperazine), 6.99 (d, 1H, J=4.0Hz, 8-H), 7.10–7.14
1H, J=4.0Hz, 5-H), 8.25 (s, 1H, 2-H). MS (ESI): 367.0.
(m, 4H, –C6H4), 7.53–7.92 (m, 3H, –C6H4), 7.93 (d, 1H,
6-Fluoro-1-(4-fluorophenyl)-4-oxo-7-(piperazin-1-yl)-1,4- J=4.0Hz, 5-H), 8.19 (s, 1H, –C6H4), 8.37 (s, 1H, 2-H); HR-MS
dihydroquinoline-3-carboxylic Acid (7a)
Calcd for C28H22F5N5O5S [M−]: 635.1262. Found: 635.1259.
1-(2,4 -Dif luorophenyl)- 6 -f luoro- 4 -oxo-7-(4 -(N-
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.20 (s, 1H, –NH),
2.82–2.85 (m, 4H, –CH2, piperazine), 3.53–3.63 (m, 4H, –CH2, tosylcarbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-
piperazine), 6.48 (d, 1H, J=4.0Hz, 8-H), 7.07–7.19 (m, 4H, carboxylic Acid (8f)
1
–C6H4), 7.87 (d, 1H, J=4.0Hz, 5-H), 8.32 (s, 1H, 2-H). MS
M.p. 203–204°C, Yield 62%, H-NMR (DMSO-d6, 300MHz)
δ (ppm): 2.28 (s, 3H, –CH3), 3.37–3.58 (m, 4H, –CH2, pi-
(ESI): 385.1.
1-(2,4-Difluorophenyl)-6-fluoro-4-oxo-7-(piperazin-1- perazine), 3.78–3.87 (m, 4H, –CH2, piperazine), 7.01 (d, 1H,
yl)-1,4-dihydroquinoline-3-carboxylic Acid (7b)
J=4.0Hz, 8-H), 7.09–7.42 (m, 3H, –C6H3), 7.44–7.92 (m,
1H-NMR (DMSO-d6, 300MHz) δ (ppm): 1.22 (s, 1H, –NH), 4H, –C6H4), 8.27 (d, 1H, J=4.0Hz, 5-H), 8.93 (s, 1H, 2-H);
2.81–2.85 (m, 4H, –CH2, piperazine), 3.34–3.70 (m, 4H, –CH2, HR-MS Calcd for C28H24F3N5O5S [M−]: 599.1450. Found:
piperazine), 6.75 (d, 1H, J=4.0Hz, 8-H), 6.85–7.13 (m, 4H, 599.1449.
–C6H4), 8.02 (d, 1H, J=4.0Hz, 5-H), 8.20 (s, 1H, 2-H). MS
(ESI): 403.1.
7-(4-(N-((2-Chlorophenyl)sulfonyl)carbamimidoyl)-
piperazin-1-yl)-1-(2,4-difluorophenyl)-6-fluoro-4-oxo-1,4-
6-Fluoro-1-(4-f luorophenyl)-4-oxo-7-(4-(N-tosyl- dihydroquinoline-3-carboxylic Acid (8g)
1
carbamimidoyl)piperazin-1-yl)-1,4-dihydroquinoline-3-car-
M.p. 215–218°C, Yield 72%, H-NMR (DMSO-d6, 300MHz)
δ (ppm): 3.36–3.58 (m, 4H, –CH2, piperazine), 3.79–3.86
boxylic Acid (8a)
1
M.p. 200–202°C, Yield 61%, H-NMR (DMSO-d6, 300MHz) (m, 4H, –CH2, piperazine), 7.06 (d, 1H, J=4.0Hz, 8-H),
δ (ppm): 2.45 (s, 3H, –CH3), 3.38–3.59 (m, 4H, –CH2, pi- 7.18–7.44 (m, 3H, –C6H3), 7.52–8.03 (m, 4H, –C6H4), 8.26
perazine), 3.78–3.88 (m, 4H, –CH2, piperazine), 6.98 (d, 1H, (d, 1H, J=4.0Hz, 5-H), 8.95 (s, 1H, 2-H); HR-MS Calcd for
J=4.0Hz, 8-H), 7.08–7.10 (m, 4H, –C6H4), 7.30–7.65 (m, C27H21ClF3N5O5S [M−]: 619.0904. Found: 619.0902.
4H, –C6H4), 8.01 (d, 1H, J=4.0Hz, 5-H), 8.20 (s, 1H, 2-H);
7-(4-(N-((2,6-Dichlorophenyl)sulfonyl)carbamimidoyl)-
HR-MS Calcd for C28H25F2N5O5S [M−]: 581.1544. Found: piperazin-1-yl)-1-(2,4-difluorophenyl)-6-fluoro-4-oxo-1,4-
581.1541.
7-(4-(N-((2-Chlorophenyl)sulfonyl)carbamimidoyl)-
dihydroquinoline-3-carboxylic Acid (8h)
M.p. 210–212°C, Yield 79%, H-NMR (DMSO-d6, 300MHz)
1
piperazin-1-yl)-6-fluoro-1-(4-fluorophenyl)-4-oxo-1,4- δ (ppm): 3.47–3.52 (m, 4H, –CH2, piperazine), 3.79–3.84
dihydroquinoline-3-carboxylic Acid (8b) (m, 4H, –CH2, piperazine), 7.06 (d, 1H, J=4.0Hz, 8-H),
1
M.p. 216–218°C, Yield 73%, H-NMR (DMSO-d6, 300MHz) 7.18–7.26 (m, 3H, –C6H3), 7.48–7.72 (m, 3H, –C6H3), 8.28
δ (ppm): 3.35–3.52 (m, 4H, –CH2, piperazine), 3.75–3.81 (d, 1H, J=4.0Hz, 5-H), 8.95 (s, 1H, 2-H); HR-MS Calcd for
(m, 4H, –CH2, piperazine), 6.91 (d, 1H, J=4.0Hz, 8-H), C27H20Cl2F3N5O5S [M−]: 653.0514. Found: 653.0519.
7.06–7.7.08 (m, 4H, –C6H4), 7.40–7.78 (m, 4H, –C6H4), 7.92
1-(2,4-Dif luorophenyl)-6-f luoro-4-oxo-7-(4-(N-((2-
(d, 1H, J=4.0Hz, 5-H), 8.43 (s, 1H, 2-H); HR-MS Calcd for (trifluoromethyl)phenyl)sulfonyl)carbamimidoyl)piperazin-1-
C27H22ClF2N5O5S [M−]: 601.0998. Found: 601.0998.
yl)-1,4-dihydroquinoline-3-carboxylic Acid (8i)
M.p. 205–207°C, Yield 68%, 1H-NMR (DMSO-d6, 300MHz)
δ (ppm): 3.36–3.58 (m, 4H, –CH2, piperazine), 3.74–3.83
(m, 4H, –CH2, piperazine), 6.84 (d, 1H, J=4.0Hz, 8-H),

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Chem. Pharm. Bull.
Vol. 66, No. 1 (2018)
7.08–7.22 (m, 3H, –C6H3), 7.53–7.75 (m, 4H, –C6H4), 8.25 Discovery Studio 2.1. For ligand preparation, the 3D struc-
(d, 1H, J=4.0Hz, 5-H), 8.67 (s, 1H, 2-H); HR-MS Calcd for tures of 8i were generated and minimized using Discovery
C28H21F6N5O5S [M−]: 653.1168. Found: 653.1163.
Studio 2.1. For protein preparation, the hydrogen atoms were
1-(2,4-Dif luorophenyl)-6-f luoro-4-oxo-7-(4-(N-((3- added. The whole DNA-Top I domain defined as a receptor
(trifluoromethyl)phenyl)sulfonyl)carbamimidoyl)piperazin-1- and the site sphere was selected based on the ligand binding
yl)-1,4-dihydroquinoline-3-carboxylic Acid (8j)
location of Topotecan, then the Topotecan removed and the
M.p. 202–204°C, Yield 69%, 1H-NMR (DMSO-d6, prepared ligand was placed during the molecular docking
300MHz) δ (ppm): 3.40–3.55 (m, 4H, –CH2, piperazine), procedure. CHARMm was selected as the force field. The
3.75–3.83 (m, 4H, –CH2, piperazine), 6.82 (d, 1H, J=4.0Hz, molecular docking was performed with a simulated annealing
8-H), 7.08–7.23 (m, 3H, –C6H3), 7.53–7.92 (m, 3H, –C6H4), method.
8.24 (d, 1H, J=4.0Hz, 5-H), 8.28 (s, 1H, –C6H4), 8.67 (s, 1H,
2-H); HR-MS Calcd for C28H21F6N5O5S [M−]: 653.1168. Found:
653.1161.
Acknowledgments We are grateful to the National Natu-
ral Science Foundation of China (No. 21306104, 21476128) and
Anti-proliferation Assay The antiproliferative activities the Foundation of Educational Committee of Zhejiang Prov-
of the prepared compounds against A549, HL-60 and Hela ince of China (No. Y201329284) for financial support.
cells were evaluated using a standard MTT-based colorimet-
ric assay. Target tumor cell lines were grown to log phase
Conflict of Interest The authors declare no conflict of
in RPMI 1640 medium supplemented with 10% fetal bovine interest.
serum. After diluting to 1×106 cells mL−1 with the complete
medium, 100µL of the obtained cell suspension was added to
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 1.
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