Synthesis and biological evaluation of 6-fluoro-3-phenyl-7-piperazinyl quinolone derivatives as potential topoisomerase I inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.06.054

The design and synthesis of a new series of 6-fluoro-3-phenyl-7-piperazinyl quinolone derivatives, built on the structure of 1-ethyl-3-(6-nitrobenzoxazol-2-yl)-6,8-difluoro-7-(3-methylpiperazin-1-yl)-4(1H)-quinolone, are described. These compounds provide

Full text of DOI:10.1016/j.ejmech.2016.06.054

Accepted Manuscript
Synthesis and biological evaluation of 6-fluoro-3-phenyl-7-piperazinyl quinolone
derivatives as potential topoisomerase I inhibitors
Raoling Ge, Qian Zhao, Zhouling Xie, Lu Lu, Qinglong Guo, Zhiyu Li, Li Zhao
PII:
S0223-5234(16)30544-X
10.1016/j.ejmech.2016.06.054
EJMECH 8715
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 March 2016
Revised Date: 27 June 2016
Accepted Date: 28 June 2016
Please cite this article as: R. Ge, Q. Zhao, Z. Xie, L. Lu, Q. Guo, Z. Li, L. Zhao, Synthesis and biological
evaluation of 6-fluoro-3-phenyl-7-piperazinyl quinolone derivatives as potential topoisomerase I
inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.06.054.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of 6-fluoro-3-phenyl-7-piperazinyl Quinolone Derivatives as
Potential Topoisomerase I Inhibitors
Raoling Gea, Qian Zhaoa, Zhouling Xiea, Lu Lub, Qinglong Guob, Zhiyu Lia , Li Zhaob
a Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, The People's Republic of China
b Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24
Tongjiaxiang, Nanjing 210009, The People's Republic of China
Corresponding author. Zhiyu Li, tel: +86 13951678592; e-mail: zhiyuli@cpu.edu.cn
Corresponding author. Li Zhao, tel: +86 13851847586; e-mail: zhaoli@cpu.edu.cn
Abstract:
The design and synthesis of a new series of 6-fluoro-3-phenyl-7-piperazinyl quinolone derivatives,
built
on
the
structure
of
1-ethyl-3-(6-nitrobenzoxazol-2-yl)-6,8-difluoro-7-(3-methylpiperazin-1-yl)-4(1H)-quinolone,
are
described. These compounds provide new scaffold for the discovery of Topoisomerase I (Top I)
inhibitors and target based assay showed that they can obviously inhibited Top I at 100 ꢀM. The in
vitro anti-proliferative activity of these new compounds was evaluated against A549, Hela, BGC-823,
and HepG2 cell lines. Compounds 18a-g showed potent inhibitory activity against the growth of those
cancer cell lines. The most positive compounds 18f and 18g demonstrated as potent as camptothecin in
Top I inhibition assay and MTT assay. Compounds 18f and 18g led to an obvious increase in the
percentage of S phase of the cells in 24 h. The in vivo data showed that 18f and 18g inhibited tumor
growth with the inhibitory rate of 29.25% and 42.75% at 20 mg/kg, respectively. The data
suggested the therapeutic potential for further development.
Keywords: quinolone derivatives, synthesis, Top I inhibitors, anti-proliferative activity
Corresponding author. Zhiyu Li, tel: +86 13951678592; e-mail: zhiyuli@cpu.edu.cn
Corresponding author. Li Zhao, tel: +86 13851847586; e-mail: zhaoli@cpu.edu.cn
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1. Introduction
DNA topoisomerase I (Top I) is a ubiquitous DNA-cleaving enzyme which cuts one strand of a
DNA double helix to relax supercoiled DNA for transcription, replication, and mitosis [1]. Top I works
by uncoiling natural supercoiled DNA, transiently breaking one of the two strands of DNA, make
another single chain pass through the gap, followed by religation of the broken strand, thus Top I
changes supercoiling of DNA or helix insufficient [2]. Top I plays a critical role in the proliferation of
cancer cells, and it is recognized as an important target to prevent rapid proliferation of cancer cells.
Therefore, the discovery of new Top I inhibitors has attracted the attention of medicinal chemist
throughout the world.
Figure 1. Structure of Camptothecin, 10-Hydroxycamptothecin, Irinotecan and Toptecan.
Camptothecin (CPT, 1, Figure 1), a specific inhibitor of Top I, is widely used in clinics for its
curative effect on a wide range of cancer cell lines. Unfortunately, the clinic utility of CPT is hampered
by several disadvantages such as poor aqueous solubility and high toxicity [3]. As a result, these
problems promoted further development of more water-soluble analogues, such as
10-hydroxycamptothecin (HCPT, 2), irinotecan (3) and topotecan (TPT, 4, Figure 1). Irinotecan and
topotecan, which are the only current FDA-approved Top I inhibitors as anticancer drugs, are in clinical
treatment [4-7]. However, the usage of CPT and its analogues was hindered by its rapid inactivation,
which was derived from the rapid hydrolysis of the lactone ring under physiological condition [8]. As
Top I is proven to be an effective target for cancer treatment, a slice of metabolically stable non-CPT
Top I inhibitors with better pharmacokinetic features have been developed [9-14], like indolocarbazoles
[13] and indenoisoquinolines (Figure 2) [14], some of them are now in clinical assessment [8].
Therefore, in our present study, we made great efforts to improve their chemical stability and biological
activity.
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Figure 2. Structure of Rebeccamycin and NSC 314622.
2. Results and discussion
2.1 Design
With a view to the successful clinic utility of these non-CPT Top I inhibitors, we ascribe the better
chemical stability with longer lifetimes of the trapped cleavage complex to the disappearance of a
lactone ring in their skeleton [15]. It is well known that fluoro atom prolongs the half-life to improve
the metabolic characteristics and piperazine or methyl piperazine increases the water solubility of
compounds [3]. In 2009, our group discovered a novel series of Top I inhibitors with quinolone scaffold.
1-ethyl-3-(6-nitrobenzoxazol-2-yl)-6,8-difluoro-7-(3-methylpiperazin-1-yl)-4(1H)-quinolone
(5,
Figure 3) was the most potent compound we synthesized [3]. We also further improved biological
activity and water solubility. Using a scaffold modification strategy, our team changed the
6-nitrobenzoxazol group into phenyl with different substituent groups. As F atom at the 8-position had
little effect to enhance cytotoxicity and a methyl group on the piperazine substituent reduced
cytotoxicity [3], we removed them to simplify the structure (Figure 3). Besides, the NH in quinolone
was also substituted with ethyl and cyclopropyl. Herein we identified a suite of quinolone derivatives
as potential Top I inhibitors.
Figure 3. Compound generation by scaffold modification.
2.2 Chemistry
The synthesis of quinolone derivatives 17a-h, 18a-i is described in Scheme 1. Decarboxylation of
6, 7 by reacting with concentrated hydrochloric acid led to intermediate 8, 9. In the presence of
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bromine and acetic acid, 3-H was substituted by bromine to form 10, 11. Butyloxycarbonyl and methyl
were introduced to piperazine to get intermediates 12, 13 and 14. Afterwards, 12, 13 and 14 were
subsequently coupled with different substituted phenylboronic acids to give rise to compounds 15a-h,
16a-g and 18h-i. Then butyloxycarbonyl was removed by refluxing in the acetic acid to provide 17a-h,
18a-g.
O
O
O
F
F
Br
F
COOH
a
b
N
N
N
N
N
N
HN
R1
HN
R1
HN
R1
6: R1= ethyl
8: R1= ethyl
10: R1= ethyl
7: R1= cyclopropyl
9: R1= cyclopropyl
11: R1= cyclopropyl
c
f
O
O
F
Br
F
Br
N
N
N
N
N
R1
N
R1
Boc
12: R1= ethyl
14: R1= cyclopropyl
13: R1= cyclopropyl
HO
d
R2
HO
B
HO
R2
g
B
HO
O
O
R2
R2
F
F
N
N
N
N
N
R1
N
R1
Boc
18hꢁi: R1= cyclopropyl
R2= 3ꢁCl. 3ꢁOCH3
15aꢁh: R1= ethyl
R2= H, 4ꢁF. 4ꢁCl. 4ꢁOCH3. 4ꢁCN.
3ꢁF. 3ꢁCl, 3ꢁOCH3
16aꢁg: R1= cyclopropyl
R2= H, 4ꢁF. 4ꢁCl. 4OCH3.
3ꢁF. 3ꢁCl, 3ꢁOCH3
e
O
R2
F
N
N
HN
R1
17aꢁh: R1= ethyl
R2= H, 4ꢁF. 4ꢁCl. 4ꢁOCH3. 4ꢁCN.
3ꢁF. 3ꢁCl, 3ꢁOCH3
18aꢁg: R1= cyclopropyl
R2= H, 4ꢁF. 4ꢁCl. 4OCH3.
3ꢁF. 3ꢁCl, 3ꢁOCH3
Scheme 1. Synthesis of quinolone derivatives. Reagents and conditions: (a) con.HCl, reflux, 24h, 98%;
(b) CH3COOH, Br2, 25 , overnight, 95%; (c) (Boc)2O, Et3N, CH2Cl2, 25 , overnight, 95%; (d)
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Toluene, MeOH, H2O, K2CO3, Pd(PPh3)4, N2, reflux, 2h, 80-90%; (e) CH3COOH, reflux, 6h, 80-90%;
(f) CH3I, NaOMe, -10 , overnight, 60%; (g) Toluene, MeOH, H2O, K2CO3, Pd(PPh3)4, N2, reflux, 2h,
80-90%.
2.3 Results and discussions
2.3.1 MTT assays
To explore the in vitro antiproliferative activity of all quinolone derivatives,
1-N-methyl-5-thiotetrazole (MTT) assay was used in four human cancer cell lines such as Hela
(cervical adenocarcinoma), BCG-823 (gastric carcinoma), HepG2 (human hepatocellular liver
carcinoma) and A549 (non-small cell lung carcinoma) with CPT as a positive control compound.
Table 1. The structures and the in vitro cytotoxic activities of quinolone derivatives.
Compounds
R
R1
R2
Antitumor cell proliferation (IC50, µM)
Hela
BGC-823
1.27±0.11
2.74±0.27
HepG2
A549
CPT
5
1.41±0.22
2.98±0.24
1.52±0.22
2.65±0.21
0.46±0.05
1.47±0.14
17a
17b
17c
17d
17e
17f
17g
17h
18a
18b
18c
18d
H
H
H
H
H
H
H
H
H
H
H
H
ethyl
ethyl
H
35.12±2.11 75.41±9.11 30.11±2.76 10.84±1.71
60.93±3.98 69.59±8.31 56.20±5.09 11.39±1.13
15.39±2.01 14.60±0.88 24.00±3.06 1.25±0.08
4-F
4-Cl
ethyl
ethyl
4-OCH3 69.04±6.02 46.10±5.91 57.91±4.78 11.63±1.53
ethyl
4-CN
3-F
14.64±2.01 18.66±1.33 12.60±1.02 1.09±0.07
21.95±1.62 27.27±3.11 27.80±2.21 3.06±0.43
16.07±1.97 18.37±2.02 15.26±1.49 0.78±0.05
ethyl
ethyl
3-Cl
ethyl
3-OCH3 12.19±0.99 14.73±1.21 13.02±1.55 0.80±0.11
cyclopropyl
cyclopropyl
cyclopropyl
H
9.99±0.87
14.86±1.54 15.11±1.94 10.57±0.99 1.68±0.08
9.30±1.01 12.92±1.49 8.02±0.76 0.65±0.05
7.86±0.63
8.65±1.01
0.50±0.04
4-F
4-Cl
cyclopropyl 4-OCH3 16.47±1.95 18.90±2.09 20.76±1.99 1.87±0.26
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18e
18f
18g
18h
18i
H
H
H
cyclopropyl
cyclopropyl
3-F
9.16±1.03 12.04±1.31 7.38±0.44
1.10±0.08
0.11±0.01
0.12±0.01
3-Cl
0.89±0.04
0.61±0.08
0.69±0.03
0.86±0.10
0.91±0.11
cyclopropyl 3-OCH3 0.92±0.11
3-Cl
methyl cyclopropyl 3-OCH3 14.78±1.09 20.92±0.99 25.38±1.93 1.12±0.10
methyl cyclopropyl
15.83±1.09 20.17±1.39 26.49±2.05 1.34±0.11
The results are reported as mean value ± SEM, n = 3.
Table 1 lists cytotoxic activities of two main skeleton quinoline derivatives. In different
substituted groups to the phenyl ring, the data manifested that 4-substituted compounds follow this
trends: -CN, -Cl > -H, -F, -OCH3. However, this is not suitable for 3-substituted compounds. By
comparing the different substituent positions, the substitution scaffolds at the 3-position and 4-position
of phenyl have distinct effects on the biological activities of the molecules. Among these compounds, a
quinolone nucleus with a 3-substituted phenyl, had an IC50 range of 0.11-27.80 ꢀM, better than other
compounds containing a 4-substituted ones, showing that the 3-substituted phenyl quinolone
derivatives were more preferred, which may be due to steric-hindrance effect. We presumed that
piperazine was crucial to cytotoxicity, then compounds 18h and 18i with 4-methyl piperazine at the
7-position were synthesized and evaluated. The IC50 values of compounds 18h and 18i were immensely
decrease comparing with 18f and 18g as expected, supporting that piperazine substituted compounds
yield preferable cytotoxicity than 4-methyl piperazine ones. Notably, cyclopropyl substituted
compounds were more cytotoxic than ethyl substituted ones, which is in coincidence with the basic
skeletons. Compounds 18f and 18g, whose biological activities were greatly improved, were
discovered to be the best of all compounds we synthesized, with IC50 values of 0.89, 0.92 ꢀM (Hela),
0.61, 0.69 ꢀM (BCG-823), 0.86, 0.91 ꢀM (HepG2) and 0.11, 0.12 ꢀM (A549) respectively, which is as
potent as CPT in the biological assay. Further data showed that almost all the compounds induced
stronger growth inhibitory effect on A549 cells than on another cancer cells in MTT assay.
2.3.2 Top I activity
To explore the mechanism by which compounds inhibited Top I and thereby caused cytotoxicity,
seven compounds with the best anti-proliferation activity (18a-g) were examined at 100 ꢀM by
measuring the relaxation of supercoiled DNA of plasmid pBR322 with CPT as positive control. The
inhibitory activities on Top I of all the tested compounds were as potent as CPT, which coincided with
the antiproliferative activities in MTT assay. In the Top I assay, IC50 values were much higher than in
the cytotoxy assays. We speculated that an anti-proliferative activity of 18a-g might be only partially
dependent on Top I, indicating that 18a-g had additional targets besides Top I. At higher concentrations
of 50 ꢀM, compound 5 was precipitated out under typical assay conditions. However, the solubility of
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our compounds is higher than 400 ꢀM, indicating that the solubility of our compounds was improved.
Figure 4. Effects of quinolone derivatives on Top I-mediated DNA relaxation activity. (A) Lane 1:
marker; Lane 2: Top I + supercoiled pBR322 DNA (ScDNA); Lane 3: Top I + ScDNA + CPT; Lane 4:
Top I + ScDNA + compound 18a; Lane 5: Top I + ScDNA + compound 18b, Lane 6: Top I + ScDNA +
compound 18c; Lane 7: Top I + ScDNA + compound 18d; Lane 8: Top I + ScDNA + compound 18e;
Lane 9: Top I + ScDNA + compound 18f; Lane 10: Top I + ScDNA + compound 18g. (B) Effects of
test compounds on Top I-mediated DNA relaxation. The proportion of relaxed DNA to the total DNA
was measured by scanning with an imaging system. Bars show the percentage of supercoiled vs relaxed
DNA. Marker: product of Tanon, China. R for relax pBR322 DNA and s for supercoiled pBR322 DNA.
2.3.3 Docking study
Based on molecular docking, we compared 18g with CPT to reveal the binding mode of them
with Top I/DNA binary complex (PDB ID: 1K4T). As shown in Figure 5, 18g and CPT overlapped
and had similar positions and orientations, lending support to the view that 18g is Top I inhibitor as
CPT. It could be found that the Pi interactions of 18g and CPT within the complex were similar.
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Nonetheless, they interacted with Top I/DNA binary complex in different patterns. Compound 18g is
capable of making two hydrogen bonding interactions to enhance the stabilization of the complex,
illustrated as green dashed lines in Figure 5. The 4-position carbonyl oxygen of 18g is shown to form a
hydrogen-bonding with the guanidine protons of Arg364 and this have been previously reported for
indenoisoquinoline analogues [16-18].The NH of the 7 position piperazine group of 18g participated in
hydrogen bonding with Glu356, further explained that piperazine is crucial for enhancing
antineoplastic activity because it may increase the binding affinity of the inhibitors with Top I/DNA
binary complex.
Figure 5. Docking results of CPT and 18g with Top I/DNA binary complex. Energy-minimized
hypothetical top-ranked binding pose of CPT and 18g (colored by atom type). The stereoview is
programmed for wall-eyed (relaxed) viewing. Top I amino acid residues not involved in bonding
interactions have been removed to improve clarity. Hydrogen bonds are indicated by green dashed lines.
Pi interactions are indicated by orange lines. Protein and compounds represented in stick model.
2.3.4 Effect of 18f and 18g on DNA distribution in human non-small lung cancer A549 cells
Previous works in rapidly proliferating cells revealed that the cytotoxicity of the famous Top I
inhibitor, CPT, depended on active DNA replication [19]. Based on compound 18f and 18g showed
positive activity in MTT assay and Top I inhibition assay, we further evaluated the effect of 18f and 18g
on the distribution of the cells in cell cycle with CPT as positive control. Synchronized A549 cells
treated with 0.5 ꢀM CPT, 18f and 18g were performed to the flowcytometric analysis (Figure 6). 18f
and 18g (0.5 ꢀM) led to an obvious increase in percentage of cells in S phase at 24 h. The data showed
that the percentage of cells in S phase was increased by around 20% at 0.5 ꢀM at 24 h, as compared
with the untreated group. However, the effects of the compounds were weaker than CPT.
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Figure 6. 18f and 18g induced S phase cell cycle arrest in lung cancer cells. (A) A549 cells were
treated with 0.5 ꢀM 18f and 18g for 24 h. Cells treated with 0.5 ꢀM CPT was used as positive control.
(B) Summary of the percentage of cells in S phase.
2.3.5 Compounds 18f and 18g inhibited the growth of A549 tumor xenografts in nude mice
Tumor xenografts transplanted by A549 cells were used to evaluate the antitumor effect of
compounds 18f and 18g in vivo. The tumor volume in drug-treated mice was less than that in negative
control mice at the same measurement day (Figure 7A). Values of T/C in the 20 mg/kg 18f group were
87.18% (day 3), 95.42% (day 6), 85.54% (day 9), 58.03% (day 12), 76.40% (day 15) and 62.96% (day
18), 73.07% (day 21), 74.32% (day 23). And the values of T/C in the 20 mg/kg 18g group were
140.57% (day 3), 114.41% (day 6), 118.67% (day 9), 85.88% (day 12), 73.12% (day 15) and 68.69%
(day 18), 71.52% (day 21), 64.72% (day 23). In Hydroxycamptothecin group, the values of T/C was
91.99% (day 3), 68.37% (day 6), 54.55% (day 9), 29.44% (day 12), 27.97% (day 15) and 23.92% (day
18), 20.00% (day 21), 21.68% (day 23). At the end of the experiment (Figure 7B and 7D), the weight
of tumors was reduced by 18f and 18g, the inhibitory rate was 29.25% and 42.75%, respectively.
Meanwhile, the body weight of the mice in the 18f and 18g groups remained essentially unchanged
compared with 20 mg/kg Hydroxycamptothecin (Figure 7C) and none of the mice died during the
treatment, which suggested compounds 18f and 18g possess lower toxicities than
Hydroxycamptothecin.
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Figure 7. 18f and 18g inhibited the growth of A549 transplantable tumors. (A) Tumor volume of
control, 18f, 18g and Hydroxycamptothecin-treated group. (B) The weight of tumor of control, 18f, 18g
and Hydroxycamptothecin-treated group. *P < 0.05, ** P < 0.01 compared with control. (C) The body
weight of the control group and drug-treated groups. (D) The photograph of tumors.
3. Conclusion
In summary, 17 novel quinolone derivatives were designed and synthesized. Their antitumor
activities and Top I inhibitory effects in vitro were detected. As a result, two compounds - 18f and 18g
exhibited potent activities as strong as CPT. Furthermore, Compounds 18f and 18g led to an obvious
increase in percentage of A549 cells in S phase at 24 h treatment. The results in vivo showed that 18f
and 18g had less morbidity and a change in body weight compared with Hydroxycamptothecin. Further
research about the biological activities and other molecular mechanism of these potent compounds are
currently going on in our group.
4. Experimental procedures
4.1 Chemistry
All reagents were from commercial sources. With tetramethylsilane (TMS) as internal standard,
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1
the H NMR and 13C NMR were recorded on Bruker AV-300 apparatus by using deuterated solvents.
HR-MS was collected on Agilent technologies 6520 Accurate-Mass Q-TOF LC/MS instruments.
Melting points were measured by XT-4 melting point apparatus.
4.1.1 Typical procedure for the preparation of 1-ethyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one
(8), 1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (9)
To the stirred solution of 200 ml concentrated hydrochloric acid, norfloxacin (6, 20 g, 62.70 mmol)
or ciprofloxacin (7, 20 g, 60.42 mmol) was added. After stirred and refluxed for 24h, the mixture was
cooled down, 8 and 9 were obtained by the vacuum distillation.
4.1.2 Typical procedure for the preparation of 3-bromo-1-ethyl-6-fluoro-7-(piperazin-1-yl)quinolin-4
(1H)-one (10), 3-bromo-1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (11)
To a solution of compounds 8 (20 g, 44.15 mmol) or 9 (20 g, 43.01 mmol) in 200 ml AcOH, 3.9
ml Br2 which was diluted by 20 ml AcOH was slowly added. After stirred in 25
overnight, the
mixture was filtrated and washed with EA (50 ml × 3) to yield compounds 10, 11.
4.1.3
Typical
procedure
for
the
preparation
of
tert-butyl
4-(3-bromo-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)piperazine-1-carboxylate (12), tert-butyl
4-(3-bromo-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)piperazine-1-carboxylate (13)
To a solution of compounds 10 (20 g, 72.73 mmol) or 11 (20 g, 69.67 mmol) in 200 ml CH2Cl2, 17
ml (Boc)2O (16 ml for 11) which was diluted by 20 ml CH2Cl2 was slowly added, then 1ml Et3N was
added. After stirred at 25 overnight, the mixture was filtrated and washed with CH2Cl2 (50 ml × 3) to
yield compounds 12, 13.
4.1.4
Typical
procedure
for
the
preparation
of
3-bromo-1-cyclopropyl-6-fluoro-7-(4-methylpiperazin-1-yl)quinolin-4(1H)-one (14)
Compound 11 (2 g, 6.97 mmol) was dissolved with 20 ml MeOH, ICH3 (7.67 mmol) and NaOMe
(7.67 mmol) were slowly added at -10 , After stirred overnight under N2, the solvent was
concentrated in vacuo, then 20 ml of water was added, the mixture was extracted with CHCl3 (20 ml ×
3). The combined organic layer was dried with anhydrous Na2SO4 and the product was obtained after
purification with PE:EA=30:1 by column chromatography.
4.1.5 Typical procedure for the preparation of 15a-g, 16a-g, 18h-i
To a solution of compounds 12-14 (1 g, 2.2 mmol for 12, 13, 2.6 mmol for 14) and phenylboronic
acid derivatives (2.4 mmol for 12, 13, 2.9 mmol for 14) in 20 ml toluene, 10 ml methanol and 3 ml
water, K2CO3 (5.5 mmol for 12, 13, 6.5 mmol for 14) and Pd(PPh3)4 (0.01 g) were added. After stirred
and refluxed 2 h under N2, the mixture was filtrated and washed with methanol, the solvent was
removed by reduced pressure distillation, and methanol was added to dissolve the solid. The mixture
was filtrated again washed with methanol to remove the K2CO3 and Pd(PPh3)4, then the solvent was
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removed by reduced pressure distillation. Recrystallized in acetonitrile to gave compounds 15a-h,
16a-g, 18h-i in yields of 80-90%.
4.1.6 Typical procedure for the preparation of 17a-h, 18a-g
Compounds 15a-h, 16a-g (1 g)were dissolved with 10 ml AcOH, after stirred and refluxed for 6 h,
the solution was cooled down and adjusted to pH=8 with 25% NaOH. The mixture was filtrated and
washed with ice water (10 ml × 3) to give compounds 17a-h, 18a-g in yield of 80-90%.
4.1.6.1 1-ethyl-6-fluoro-3-phenyl-7-(piperazin-1-yl)quinolin-4(1H)-one (17a)
It was obtained as a white solid in 87.1% yield. m.p. 228-229 °C, HRMS (ESI): m/z, calculated
1
for C21H22FN3O, 352.1747 (M+H)+, found 352.1808; H NMR (300 MHz, DMSO-d6): δ 8.27 (s, 1H,
5-H), 7.90 (s, 1H, 2-H), 7.74 (t, 2H, J = 7.2 Hz, Ar-H), 7.39 (d, 2H, J = 8.0 Hz, Ar-H), 7.30 (t, 1H, J =
8.9 Hz, Ar-H), 7.06 (s, 1H, 8-H), 4.40 (q, 2H, J = 7.7 Hz, N-CH2), 3.66 (t, 4H, J = 4.9 Hz, 2×CH2),
3.22 (t, 4H, J = 4.5 Hz, 2×CH2), 1.40 (t, 3H, J = 6.7 Hz, N-CH2CH3); 13C NMR (75 MHz, DMSO-d6):
δ 172.3, 159.5, 153.5, 150.0, 144.4, 142.7, 136.6, 132.9, 130.2, 118.8, 114.2, 111.3, 104.6, 51.0, 47.4,
45.5, 14.2 ppm.
4.1.6.2 1-ethyl-6-fluoro-3-(4-fluorophenyl)-7-(piperazin-1-yl)quinolin-4(1H)-one (17b)
It was obtained as a white solid in 85.6% yield. m.p. 229-230 °C, HRMS (ESI): m/z, calculated
1
for C21H21F2N3O, 370.1653 (M+H)+, found 370.1707; H NMR (300 MHz, DMSO-d6): δ 8.29 (s, 1H,
5-H), 7.87 (s, 1H, 2-H), 7.80 (d, 2H, J = 8.8 Hz, Ar-H), 7.22 (d, 2H, J = 8.9 Hz, Ar-H), 7.06 (s, 1H,
8-H), 4.40 (q, 2H, J = 6.9 Hz, N-CH2), 3.66 (t, 4H, J = 4.4 Hz, 2×CH2), 3.22 (t, 4H, J = 4.8 Hz, 2×CH2),
1.40 (t, 3H, J = 7.0 Hz, N-CH2CH3); 13C NMR (75 MHz, DMSO-d6): δ 172.8, 159.3, 153.7, 150.5,
144.6, 142.4, 136.3, 132.0, 130.2, 118.0, 114.7, 111.5, 104.6, 51.0, 47.3, 45.5, 14.2 ppm.
4.1.6.3 3-(4-chlorophenyl)-1-ethyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (17c)
It was obtained as a white solid in 83.2% yield. m.p. 229-230 °C, HRMS (ESI): m/z, calculated
for C21H21ClFN3O, 386.1357 (M+H)+, found 386.1419; 1H NMR (300 MHz, DMSO-d6): δ 8.33 (s, 1H,
5-H), 7.87 (s, 1H, 2-H), 7.82 (d, 2H, J = 8.5 Hz, Ar-H), 7.45 (d, 2H, J = 8.4 Hz, Ar-H), 7.06 (s, 1H,
8-H), 4.40 (q, 2H, J = 7.6 Hz, N-CH2), 3.66 (t, 4H, J = 4.9 Hz, 2×CH2), 3.22 (t, 4H, J = 4.7 Hz, 2×CH2),
1.40 (t, 3H, J = 6.8 Hz, N-CH2CH3); 13C NMR (75 MHz, DMSO-d6): δ 172.6, 160.7, 153.7, 150.4,
144.5, 142.6, 136.3, 134.6, 129.9, 127.7, 117.6, 111.5, 104.6, 51.0, 47.4, 45.4, 14.2 ppm.
4.1.6.4 1-ethyl-6-fluoro-3-(4-methoxyphenyl)-7-(piperazin-1-yl)quinolin-4(1H)-one (17d)
It was obtained as a white solid in 81.8% yield. m.p. 227-228 °C, HRMS (ESI): m/z, calculated
for C22H24ClFN3O2, 382.1853 (M+H)+, found 382.1911; 1H NMR (300 MHz, DMSO-d6): δ 8.20 (s, 1H,
5-H), 7.85 (s, 1H, 2-H), 7.68 (d, 2H, J = 8.8 Hz, Ar-H), 7.04 (s, 1H, 8-H), 6.95 (d, 2H, J = 8.8 Hz,
Ar-H), 4.39 (q, 2H, J = 7.4 Hz, N-CH2), 3.78 (s, 3H, -OCH3), 3.65 (t, 4H, J = 4.4 Hz, 2×CH2), 3.20 (t,
4H, J = 4.7 Hz, 2×CH2), 1.38 (t, 3H, J = 7.9 Hz, N-CH2CH3); 13C NMR (75 MHz, DMSO-d6): δ 172.8,
12

ACCEPTED MANUSCRIPT
158.0, 153.6, 150.4, 144.5, 141.8, 136.2, 129.4, 128.0, 118.9, 113.2, 111.5, 104.5, 55.0, 51.1, 47.2, 45.5,
14.2 ppm.
4.1.6.5 4-(1-ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinolin-3-yl)benzonitrile (17e)
It was obtained as a white solid in 86.7% yield. m.p. 228-229 °C, HRMS (ESI): m/z, calculated
1
for C22H21FN4O, 377.1699 (M+H)+, found 377.1756; H NMR (300 MHz, DMSO-d6): δ 8.45 (s, 1H,
5-H), 8.04 (d, 2H, J = 8.2 Hz, Ar-H), 7.88 (d, 2H, J = 8.1 Hz, Ar-H), 7.83 (s, 1H, 2-H), 7.07 (s, 1H,
8-H), 4.42 (q, 2H, J = 7.7 Hz, N-CH2), 3.65 (t, 4H, J = 4.8 Hz, 2×CH2), 3.25 (t, 4H, J = 4.4 Hz, 2×CH2),
1.40 (t, 3H, J = 6.8 Hz, N-CH2CH3); 13C NMR (75 MHz, DMSO-d6): δ 172.6, 153.8, 150.6, 144.7,
143.6, 140.8, 136.2, 128.5, 121.1, 119.2, 116.7, 111.6, 108.4, 104.7, 50.9, 47.6, 45.4, 14.2 ppm.
4.1.6.6 1-ethyl-6-fluoro-3-(3-fluorophenyl)-7-(piperazin-1-yl)quinolin-4(1H)-one (17f)
It was obtained as a white solid in 84.3% yield. m.p. 229-230 °C, HRMS (ESI): m/z, calculated
1
for C21H21F2N3O, 370.1653 (M+H)+, found 370.1707; H NMR (300 MHz, DMSO-d6): δ 8.38 (s, 1H,
5-H), 7.89 (s, 1H, 2-H), 7.71 (t, 1H, J = 9.3 Hz, Ar-H), 7.64 (d, 1H, J = 10.4 Hz, Ar-H), 7.42 (d, 1H, J =
9.3 Hz, Ar-H), 7.12 (s, 1H, Ar-H), 7.07 (s, 1H, 8-H), 4.40 (q, 2H, J = 8.0 Hz, N-CH2), 3.66 (t, 4H, J =
4.4 Hz, 2×CH2), 3.22 (t, 4H, J = 4.5 Hz, 2×CH2), 1.40 (t, 3H, J = 6.7 Hz, N-CH2CH3); 13C NMR (75
MHz, DMSO-d6): δ 172.8, 163.5, 160.2, 153.7, 150.5, 144.9, 143.0, 138.5, 136.3, 129.6, 123.9, 114.8,
113.1, 111.6, 104.6, 51.1, 47.5, 45.5, 14.2 ppm.
4.1.6.7 3-(3-chlorophenyl)-1-ethyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (17g)
It was obtained as a white solid in 82.5% yield. m.p. 229-230 °C, HRMS (ESI): m/z, calculated
for C21H21ClFN3O, 386.1357 (M+H)+, found 386.1419; 1H NMR (300 MHz, DMSO-d6): δ 8.38 (s, 1H,
5-H), 7.91 (s, 1H, 2-H), 7.87 (s, 1H, Ar-H), 7.76 (t, 1H, J = 7.9 Hz, Ar-H), 7.44 (d, 1H, J = 7.9 Hz,
Ar-H), 7.35 (d, 1H, J = 8.8 Hz, Ar-H), 7.07 (s, 1H, 8-H), 4.40 (q, 2H, J = 7.3 Hz, N-CH2), 3.66 (t, 4H,
J = 4.4 Hz, 2×CH2), 3.22 (t, 4H, J = 4.9 Hz, 2×CH2), 1.40 (t, 3H, J = 7.0 Hz, N-CH2CH3); 13C NMR
(75 MHz, DMSO-d6): δ 172.7, 163.5, 160.5, 153.8, 150.7, 144.8, 143.1, 138.5, 136.4, 129.6, 123.8,
114.6, 113.2, 111.5, 104.6, 51.1, 47.5, 45.5, 14.2 ppm.
4.1.6.8 1-ethyl-6-fluoro-3-(4-methoxyphenyl)-7-(piperazin-1-yl)quinolin-4(1H)-one (17h)
It was obtained as a white solid in 85.2% yield. m.p. 227-228 °C, HRMS (ESI): m/z, calculated
1
for C22H24FN3O2, 382.1853 (M+H)+, found 382.1911; H NMR (300 MHz, DMSO-d6): δ 8.29 (s, 1H,
5-H), 7.87 (s, 1H, 2-H), 7.38 (t, 1H, J = 8.1 Hz, Ar-H), 7.31 (d, 1H, J = 6.0 Hz, Ar-H), 7.26 (s, 1H,
Ar-H), 7.05 (d, 1H, J = 7.0 Hz, Ar-H), 6.86 (s, 1H, 8-H), 4.40 (q, 2H, J = 7.8 Hz, N-CH2), 3.78 (s, 3H,
-OCH3), 3.66 (t, 4H, J = 4.9 Hz, 2×CH2), 3.21 (t, 4H, J = 4.5 Hz, 2×CH2), 1.39 (t, 3H, J = 6.9 Hz,
N-CH2CH3); 13C NMR (75 MHz, DMSO-d6): δ 172.9, 158.8, 153.6, 150.4, 144.5, 142.6, 137.1, 136.2,
128.7, 120.5, 118.8, 114.0, 111.6, 111.3, 104.5, 54.9, 51.0, 47.3, 45.5, 14.2 ppm.
4.1.6.9 1-cyclopropyl-6-fluoro-3-phenyl-7-(piperazin-1-yl)quinolin-4(1H)-one (18a)
13

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It was obtained as a white solid in 89.4% yield. m.p. 268-269 °C, HRMS (ESI): m/z, calculated
1
for C22H22FN3O, 364.1747 (M+H)+, found 364.1828; H NMR (300 MHz, DMSO-d6): δ 8.18 (s, 1H,
5-H), 7.84 (s, 1H, 2-H), 7.79 (t, 1H, J = 7.3 Hz, Ar-H), 7.70 (s, 1H, 8-H), 7.42 ( t, 2H, J = 7.6 Hz, Ar-H),
7.38 (d, 2H, J = 7.7 Hz, Ar-H), 3.64 (m, 1H, N-CH), 3.18 (t, 4H, J = 4.9 Hz, 2×CH2), 2.94 (t, 4H, J =
4.9 Hz, 2×CH2), 1.24 (m, 2H, N-CHCH2), 1.17 (m, 2H, N-CHCH2); 13C NMR (75 MHz, DMSO-d6): δ
169.0, 145.3, 144.0, 138.4, 132.7, 129.0, 128.5, 128.0, 119.6, 117.9, 110.6, 110.3, 105.9, 46.1, 42.7,
36.0, 7.7 ppm.
4.1.6.10 1-cyclopropyl-6-fluoro-3-(4-fluorophenyl)-7-(piperazin-1-yl)quinolin-4(1H)-one (18b)
It was obtained as a white solid in 89.9% yield. m.p. 266-267 °C, HRMS (ESI): m/z, calculated
1
for C22H21F2N3O, 382.1653 (M+H)+, found 382.1722; H NMR (300 MHz, DMSO-d6): δ 8.03 (s, 1H,
5-H), 7.81 (s, 1H, 2-H), 7.74 (d, 2H, J = 7.3 Hz, Ar-H), 7.39 (s, 1H, 8-H), 7.20 (d, 2H, J = 7.6 Hz,
Ar-H), 3.60 (m, 1H, N-CH), 3.15 ( t, 4H, J = 4.9 Hz, 2×CH2), 2.91 (t, 4H, J = 4.9 Hz, 2×CH2), 1.21 (m,
2H, N-CHCH2), 1.14 (m, 2H, N-CHCH2); 13C NMR (75 MHz, DMSO-d6): δ 162.6, 144.2, 141.6, 138.3,
131.9, 130.4, 126.5, 124.3, 120.1, 118.0, 114.6, 111.2, 105.2, 50.8, 45.3, 34.0, 7.7 ppm.
4.1.6.11 3-(4-chlorophenyl)-1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (18c)
It was obtained as a white solid in 89.9% yield. m.p. 268-269 °C, HRMS (ESI): m/z, calculated
for C22H21ClFN3O, 398.1357 (M+H)+, found 398.1444; 1H NMR (300 MHz, DMSO-d6): δ 8.09 (s, 1H,
5-H), 7.78 (s, 1H, 2-H), 7.76 (s, 1H, 8-H), 7.44 (d, 2H, J = 3.9 Hz, Ar-H), 7.41 (d, 2H, J = 2.6 Hz,
Ar-H), 3.62 (m, 1H, N-CH), 3.17 (t, 4H, J = 4.9 Hz, 2×CH2), 2.92 (t, 4H, J = 4.8 Hz, 2×CH2), 1.23 (m,
2H, N-CHCH2), 1.17 (m, 2H, N-CHCH2); 13C NMR (75 MHz, DMSO-d6): δ 152.6, 144.2, 141.8, 138.3,
134.5, 131.0, 130.1, 127.7, 123.2, 117.6, 111.3, 111.0, 105.3, 50.8, 45.3, 34.1, 7.7 ppm.
4.1.6.12 1-cyclopropyl-6-fluoro-3-(4-methoxyphenyl)-7-(piperazin-1-yl)quinolin-4(1H)-one (18d)
It was obtained as a white solid in 87.2% yield. m.p. 265-266 °C, HRMS (ESI): m/z, calculated
1
for C23H24FN3O2, 394.1853 (M+H)+, found 394.1923; H NMR (300 MHz, DMSO--d6): δ 7.97 (s, 1H,
5-H), 7.80 (s, 1H, 2-H), 7.64 (d, 2H, J = 9.9 Hz, Ar-H), 7.40 (s, 1H, 8-H), 6.95 (d, 2H, J = 9.3 Hz,
Ar-H), 3.78 (s, 3H, -OCH3), 3.60 (m, 1H, N-CH), 3.14 (t, 4H, J = 4.6 Hz, 2×CH2), 2.90 (t, 4H, J = 4.6
Hz, 2×CH2), 1.22 (m, 2H, N-CHCH2), 1.13 (m, 2H, N-CHCH2); 13C NMR (75 MHz, DMSO-d6): δ
158.0, 155.2, 150.6, 144.1, 140.9, 138.3, 129.6, 127.9, 120.3, 113.3, 111.2, 110.9, 105.2, 55.0, 51.0,
45.5, 33.9, 7.7 ppm.
4.1.6.13 1-cyclopropyl-6-fluoro-3-(3-fluorophenyl)-7-(piperazin-1-yl)quinolin-4(1H)-one (18e)
It was obtained as a white solid in 84.7% yield. m.p. 266-267 °C, HRMS (ESI): m/z, calculated
1
for C22H21F2N3O, 382.1653 (M+H)+, found 382.1722; H NMR (300 MHz, DMSO-d6): δ 8.09 (s, 1H,
5-H), 7.78 (s, 1H, 2-H), 7.61 (t, 1H, J = 10.1 Hz, Ar-H), 7.50 (s, 1H, 8-H), 7.40 (d, 1H, J = 7.4 Hz,
Ar-H), 7.38 (s, 1H, Ar-H), 7.17 (d, 1H, J = 8.2 Hz, Ar-H), 3.60 (m, 1H, N-CH), 3.19 (t, 4H, J = 4.6 Hz,
14

ACCEPTED MANUSCRIPT
2×CH2), 2.94 (t, 4H, J = 4.4 Hz, 2×CH2), 1.23 (m, 2H, N-CHCH2), 1.15 (m, 2H, N-CHCH2); 13C NMR
(75 MHz, DMSO-d6): δ 162.4, 144.3, 142.1, 138.5, 130.0, 129.6, 129.5, 124.2, 124.1, 120.0, 119.8,
116.9, 115.1, 114.8, 105.4, 50.8, 45.4, 34.2, 7.7 ppm.
4.1.6.14 3-(3-chlorophenyl)-1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (18f)
It was obtained as a white solid in 86.8% yield. m.p. 268-269 °C, HRMS (ESI): m/z, calculated
for C22H21ClFN3O, 398.1357 (M+H)+, found 398.1444; 1H NMR (300 MHz, DMSO-d6): δ 8.13 (s, 1H,
5-H), 7.86 (s, 1H, 2-H), 7.79 (d, 1H, J = 8.5 Hz, Ar-H), 7.68 (t, 1H, J = 7.9 Hz, Ar-H), 7.43 (s, 1H, 8-H),
7.41 (s, 1H, Ar-H), 7.33 (d, 1H, J = 7.1 Hz, Ar-H), 3.63 (m, 1H, N-CH), 3.17 (t, 4H, J = 4.4 Hz,
2×CH2), 2.91 (t, 4H, J = 4.0 Hz, 2×CH2), 1.23 (m, 2H, N-CHCH2), 1.17 (m, 2H, N-CHCH2); 13C NMR
(75 MHz, DMSO-d6): δ 162.2, 144.1, 142.2, 138.3, 137.8, 133.5, 132.5, 129.6, 129.5, 128.0, 126.8,
126.2, 118.4, 111.3, 105.3, 50.8, 45.4, 34.2, 7.7 ppm.
4.1.6.15 1-cyclopropyl-6-fluoro-3-(3-methoxyphenyl)-7-(piperazin-1-yl)quinolin-4(1H)-one (18g)
It was obtained as a white solid in 88.8% yield. m.p. 265-266 °C, HRMS (ESI): m/z, calculated
1
for C23H24FN3O2, 394.1853 (M+H)+, found 394.1923; H NMR (300 MHz, DMSO-d6): δ 8.06 (s, 1H,
5-H), 7.77 (s, 1H, 2-H), 7.40 (t, 1H, J = 7.5 Hz, Ar-H), 7.31 (s, 1H, 8-H), 7.26 (s, 1H, Ar-H), 7.23 (d,
1H, J = 6.3 Hz, Ar-H), 6.85 (d, 1H, J = 6.0 Hz, Ar-H), 3.78 (s, 3H, -OCH3), 3.60 (m, 1H, N-CH), 3.16
(t, 4H, J = 4.5 Hz, 2×CH2), 2.92 (t, 4H, J = 4.2 Hz, 2×CH2), 1.23 (m, 2H, N-CHCH2), 1.15 (m, 2H,
N-CHCH2); 13C NMR (75 MHz, DMSO-d6): δ 173.0, 158.9, 153.6, 150.8, 144.2, 141.7, 138.2, 128.7,
120.7, 118.8, 114.2, 112.0, 111.3, 111.0, 105.2, 55.0, 50.8, 45.3, 34.0, 7.7 ppm.
4.1.6.16
3-(3-chlorophenyl)-1-cyclopropyl-6-fluoro-7-(4-methylpiperazin-1-yl)quinolin-4(1H)-one
(18h)
It was obtained as a white solid in 88.4% yield. m.p. 268-269 °C, HRMS (ESI): m/z, calculated
for C23H23ClFN3O, 412.1514 (M+H)+, found 412.1604; 1H NMR (300 MHz, DMSO-d6): δ 8.15 (s, 1H,
5-H), 7.90 (s, 1H, 2-H), 7.76 (d, 1H, J = 8.5 Hz, Ar-H), 7.65 (t, 1H, J = 7.9 Hz, Ar-H), 7.48 (s, 1H, 8-H),
7.42 (s, 1H, Ar-H), 7.30 (d, 1H, J = 7.1 Hz, Ar-H), 3.63 (m, 1H, N-CH), 3.17 (t, 4H, J = 4.4 Hz,
2×CH2), 2.91 (t, 4H, J = 4.0 Hz, 2×CH2), 2.23 (s, 3H, N-CH3), 1.23 (m, 2H, N-CHCH2), 1.17 (m, 2H,
N-CHCH2); 13C NMR (75 MHz, DMSO-d6): δ 162.2, 144.1, 142.2, 138.3, 137.8, 133.5, 132.5, 129.6,
129.5, 128.0, 126.8, 126.2, 118.4, 111.3, 105.3, 50.8, 45.4, 40.6, 34.2, 7.7 ppm.
4.1.6.17
1-cyclopropyl-6-fluoro-3-(3-methoxyphenyl)-7-(4-methylpiperazin-1-yl)quinolin-4(1H)-one
(18i)
It was obtained as a white solid in 87.1% yield. m.p. 265-266 °C, HRMS (ESI): m/z, calculated
1
for C24H26FN3O2, 407.2009 (M+H)+, found 407.2073; H NMR (300 MHz, DMSO-d6): δ 8.16 (s, 1H,
5-H), 7.75 (s, 1H, 2-H), 7.43 (t, 1H, J = 7.5 Hz, Ar-H), 7.30 (s, 1H, 8-H), 7.25 (s, 1H, Ar-H), 7.20 (d,
1H, J = 6.3 Hz, Ar-H), 6.85 (d, 1H, J = 6.0 Hz, Ar-H), 3.78 (s, 3H, -OCH3), 3.60 (m, 1H, N-CH), 3.16
15

ACCEPTED MANUSCRIPT
(t, 4H, J = 4.5 Hz, 2×CH2), 2.92 (t, 4H, J = 4.2 Hz, 2×CH2), 2.23 (s, 3H, N-CH3), 1.23 (m, 2H,
N-CHCH2), 1.15 (m, 2H, N-CHCH2); 13C NMR (75 MHz, DMSO-d6): δ 173.0, 158.9, 153.6, 150.8,
144.2, 141.7, 138.2, 128.7, 120.7, 118.8, 114.2, 112.0, 111.3, 111.0, 105.2, 55.0, 50.8, 45.3, 40.6, 34.0,
7.7 ppm.
4.2 Docking study
Glide was selected as the molecular docking tool. The crystallized complex structure of Top I
(PDB ID: 1K4T) was prepared using the Protein PreparationWizard workflow. A receptor grid was
generated on the center of the co-crystallized ligand, which was defined as the ligand-binding site
search region. The compound to be docked was confirmed by an enclosing box that was similar in size
to the co-crystallized ligand. Furthermore, the compound set was minimized using the LigPrep module.
The best conformation of each compound was output on the basis of the Glide score and interactions
formed between the compounds and the active site. Finally, the potential compounds were flexibly
docked into the binding site using the extra precision (XP) docking mode. All the remaining parameters
were kept as default.
4.3 Biology
4.3.1 Cytotoxicity of quinolone derivatives in various human cancer cell lines.
Cells were seeded into a 96-well plate at 6×104 to 8×104 cells per well and incubated for 24h.
Over a range of concentrations from 0.01 to 100 ꢀM, all compounds were added and treated for 48 h.
20 ꢀl of MTT solution were added to each well, and plates were incubated for 4 h. DMSO (150 ꢀl) was
added to each well and shaken ten minutes in shaking table to dissolve the Formazan crystals. The
absorbance (at wavelength of 570 nm) was measured on an enzyme-linked immunosorbent detector.
Then the cytotoxicity IC50 values, which are the concentrations leading to 50% cell death in vitro, were
obtained.
4.3.2 Top I inhibition assay.
Reaction mixtures 20 ꢀl final volume contained 1 ꢀl (0.25 ꢀg/ul) of supercoile pBR322 DNA in
the 10×Tris/Glycine/SDS (TGS) buffer (10 mM Tris-HCl, pH 7.9, 0.15M NaCl, 10% BSA, 1 mM
EDTA, 0.1 mM Spermidine, 5% glycerol.), 3-5 units Top 1, 5 ꢀl compounds and appropriate distilled
water to make the final reaction volumes 20 ꢀl. Reactions were incubated at 37 °C for 30 min and
terminated by the addition of 2 ꢀl 10% SDS (0.1 volume), and then proteinase K was added to 50
ꢀg/ml. The reactions were digested at 37 °C for 30 min. 2 ꢀl of loading buffer (0.25% Bromophenol
Blue, 50% glycerol, 0.1 volume) was then added. Aliquots of 20 ꢀl were subjected to electrophoresis in
1% agarose gel at 60 V for 2.5 h in 1×Tris/acetate (TAE) buffer (20 mM Tris-acetate, 0.5 mM EDTA,
pH 8.3). After electrophoresis, DNA bands were stained in 0.5 mg/mL of ethidium bromide and
visualized by transillumination with UV light (510 nm).
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4.3.3 Flow cytometry for DNA content analysis.
A549 cells were seeded into a 6-well plate at 2.5×105 cells per well. The cells were incubated for
24 h in medium with 0.5% FBS and synchronized with serum-free medium for 12 h, then treated with
0.5 ꢀM CPT or 18f or 18g for another 24 h. Then the cells were harvested and fixed in cold 70%
ethanol over night at 4 . After washing with PBS, the cells were incubated with 100 ml RNaseA
(KeyGEN, Nanjing, China) at 37 for 30 min. Subsequently, the cells were incubated with 400 ml PI
(KeyGEN, Nanjing, China) for 30 min at 4 in the dark and analyzed by flow cytometry (Becton
Dickinson, California).
4.3.4 In vivo activity of 18f and 18g against human tumor xenografts in nude mice
This study was approved by the SPF Animal Laboratory of China Pharmaceutical University.
Five-to six-week-old male BALB/c nude mice were purchased from SLAC Laboratory Animals
(Shanghai, China) and were raised in air-conditioned rooms under controlled lighting(12h light/day)
and were fed with standard laboratory food and water ad libitum. A549 tumors were induced in the
mice by subcutaneously injecting of A549 cells (5.0×106) into the flanks of the mice. After 12-14 days,
tumor sizes were determined using micrometer calipers, and then the mice with similar tumor volumes
(eliminating mice with tumors that were too large or too small) were randomly divided into five groups
(with six nude mice per group). The mice were treated with 18f (20 mg/kg), 18g (20 mg/kg) and
Hydroxycamptothecin (20 mg/kg). The negative group received 0.9% normal saline. Treatments were
done by intravenous injection at a frequency of once every 3 days. At the end of 23 days, the mice were
sacrificed, and the tumor xenografts were removed and measured .Tumor volume (TV) was calculated
every 3 days using the following formula: TV(mm3)=D/2×d2, where d and D are the shortest and the
longest diameters, respectively. At the same time the animals were weighed twice per week and
monitored for mortality throughout the experimental period to assess toxicity of the treatments.
Relative tumor volume (RTV) was calculated according to the equation RTV=Vt/V0, where V0 is the
tumor volume at day 0 and Vt is the tumor volume at day t. And the evaluation index for inhibition was
the relative tumor growth ratio T/C=TRTV/CRTV×100%, where TRTV and CRTV represent the RTV of the
treated and control groups, respectively.
Acknowledgements
This work was supported by Research Grants 21372260 to Z.-Y.L. from the National Natural
Science Foundation (NSFC) of China.
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References
1. Redinbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.; Hol, W. G. Crystal Structures of Human
Topoisomerase I in Covalent and Noncovalent Complexes with DNA. Science. 1998, 279, 1504-1513.
2. Nguyen, T. X.; Morrell, A.; Conda-Sheridan, M.; Marchand, C.; Agama, K.; Bermingam, A.; Stephen,
A. G.; Chergui, A.; Naumova, A.; Fisher, R.; O’Keefe, B. R.; Pommier, Y.; Cushman, M. Synthesis and
Biological Evaluation of the First Dual Tyrosyl-DNA Phosphodiesterase I (Tdp1)−Topoisomerase I
(Top1) Inhibitors. J. Med. Chem. 2012, 55, 4457−4478.
3. You, Q. D.; Li, Z. Y.; Huang, C. H.; Yang, Q.; Wang, X. J.; Guo, Q. L.; Chen, X. G.; He, X. G.; Li, K.
T.; Chern, J. W. Discovery of a Novel Series of Quinolone and Naphthyridine Derivatives as Potential
Topoisomerase I Inhibitors by Scaffold Modif ication. J. Med. Chem. 2009, 52, 5649-5661.
4. Takimoto, C.H.; Wright, J.; Arbuck, S.G. Clinical applications of the camptothecins. Biochim.
Biophys. Acta. 1998, 1400, 107–119.
5. Garcia-Carbonero, R.; Supko, J. G. Clin. Current perspectives on the clinical experience,
pharmacology, and continued development of the camptothecins. Cancer Res. 2002, 8, 641-661.
6. Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Synthesis of nitrated
indenoisoquinolines as topoisomerase I inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 3659-3663.
7. Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen, G.; Agama, K.; Hollingshead, M.;
Pommier, Y.; Cushman, M. Synthesis and Evaluation of Indenoisoquinoline Topoisomerase I Inhibitors
Substituted with Nitrogen Heterocycles. J. Med. Chem. 2006, 49, 6283-6289.
8. Zhang, J. P.; Huang, J.; Liu, C.; Lu, X. F.; Wu, B. X.; Zhao, L.; Lu, N.; Guo, Q. L.; Li, Z. Y.; Jiang, C.
Discovery of a series of pyridopyrimidine derivatives as potential topoisomerase I inhibitors. Chin.
Chem. Lett. 2014, 25, 1025-1028.
9. Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Staker, B. L.; Stewart, L.; Cushman,
M. Design, synthesis, and biological evaluation of cytotoxic 11-alkenylindenoisoquinoline
topoisomerase I inhibitors and indenoisoquinoline-camptothecin hybrids. J. Med. Chem. 2003, 46,
3275-3282.
10. Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. A systematic study of nitrated
indenoisoquinolines reveals a potent topoisomerase I inhibitor. J. Med. Chem. 2006, 49, 7740-7753.
11. Pommier, Y.; Topoisomerase I inhibitors: camptothecins and beyond. Nat. Rev. Cancer. 2006, 6,
789-802.
12. Xiao, X. S.; Antony, S.; Pommier, Y.; Cushman, M.; Total synthesis and biological evaluation of
22-hydroxyacuminatine. J. Med. Chem. 2006, 49, 1408-1412.
13. Li, Z. Y.; Zhai, F. M.; Zhao, L; Guo, Q. L.; You, Q. D. Design and synthesis of N-methylmaleimide
indolocarbazole bearing modified 2-acetamino acid moieties as Topoisomerase I inhibitors. Bioorg.
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Med. Chem. Lett. 2009, 19, 406-409.
14. Zhang, X. Y.; Wang, R.; Zhao, L.; Lu, N.; Wang, J. B.; You, Q. D.; Li, Z. Y.; Guo, Q. L. Synthesis
and biological evaluations of novel indenoisoquinolines as topoisomerase I inhibitors. Bioorg. Med.
Chem. Lett. 2012, 22, 1276-1281.
15. Pommier, Y. DNA topoisomerase I inhibitors: chemistry, biology, and interfacial inhibition. Chem.
Rev. 2009, 109, 2894-2902.
16. Ioanoviciu, A.; Antony, S.; Pommier, Y.; Staker, B. L.; Stewart, L.; Cushman, M. Synthesis and
Mechanism of Action Studies of a Series of Norindenoisoquinoline Topoisomerase I Poisons Reveal an
Inhibitor with a Flipped Orientation in the Ternary DNA-Enzyme-Inhibitor Complex As Determined by
X-ray Crystallographic Analysis. J. Med. Chem. 2005, 48, 4803-4814.
17. Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembower, D.; Stewart, L.; Burgin, A. B.
Structures of Three Classes of Anticancer Agents Bound to the Human Topoisomerase I-DNA Covalent
Complex. J. Med. Chem. 2005, 48, 2336-2345.
18. Marchand, C.; Antony, S.; Kohn, K. W.; Cushman, M.; Ioanoviciu, A.; Staker, B. L.; Burgin, A. B.;
Stewart, L.; Pommier, Y. A Novel Norindenoisoquinoline Structure Reveals a Common Interfacial
Inhibitor Paradigm for Ternary Trapping of the Topoisomerase I-DNA Covalent Complex. Mol. Cancer
Ther. 2006, 5, 287-295.
19. Holm, C.; Covey, J. M.; Kerrigan, D.; Pommier, Y. Differential requirement of DNA replication for
the cytotoxicity of DNA topoisomerase I and II inhibitors in Chinese hamster DC3F cells. Cancer Res.
1989, 49, 6365–6368.
Figure caption:
Figure 1. Structure of Camptothecin, 10-Hydroxycamptothecin, Irinotecan and Toptecan.
Figure 2. Structure of Rebeccamycin and NSC 314622.
Figure 3. Compound generation by scaffold modification.
Figure 4. Effects of quinolone derivatives on Top I-mediated DNA relaxation activity. (A) Lane 1:
marker; Lane 2: Top I + supercoiled pBR322 DNA (ScDNA); Lane 3: Top I + ScDNA + CPT; Lane 4:
Top I + ScDNA + compound 18a; Lane 5: Top I + ScDNA + compound 18b, Lane 6: Top I + ScDNA +
compound 18c; Lane 7: Top I + ScDNA + compound 18d; Lane 8: Top I + ScDNA + compound 18e;
Lane 9: Top I + ScDNA + compound 18f; Lane 10: Top I + ScDNA + compound 18g. (B) Effects of
test compounds on Top I-mediated DNA relaxation. The proportion of relaxed DNA to the total DNA
was measured by scanning with an imaging system. Bars show the percentage of supercoiled vs relaxed
DNA. Marker: product of Tanon, China. r for relax pBR322 DNA and s for supercoiled pBR322 DNA.
Figure 5. Docking results of CPT and 18g with Top I/DNA binary complex. Energy-minimized
hypothetical top-ranked binding pose of CPT and 18g (colored by atom type). The stereoview is
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programmed for wall-eyed (relaxed) viewing. Top I amino acid residues not involved in bonding
interactions have been removed to improve clarity. Hydrogen bonds are indicated by green dashed lines.
Pi interactions are indicated by orange lines. Protein and compounds represented in stick model.
Figure 6. 18f and 18g induced S phase cell cycle arrest in lung cancer cells. (A) A549 cells were
treated with 0.5 ꢀM 18f and 18g for 24 h. Cells treated with 0.5 ꢀM CPT was used as positive control.
(B) Summary of the percentage of cells in S phase.
Figure 7. 18f and 18g inhibited the growth of A549 transplantable tumors. (A) Tumor volume of
control, 18f, 18g and Hydroxycamptothecin-treated group. (B) The weight of tumor of control, 18f, 18g
and Hydroxycamptothecin-treated group. *P < 0.05, ** P < 0.01 compared with control. (C) The body
weight of the control group and drug-treated groups. (D) The photograph of tumors.
Scheme caption:
Scheme 1. Synthesis of quinolone derivatives. Reagents and conditions: (a) con.HCl, reflux, 24h, 98%;
(b) CH3COOH, Br2, 25 , overnight, 95%; (c) (Boc)2O, Et3N, CH2Cl2, 25 , overnight, 95%; (d)
Toluene, MeOH, H2O, K2CO3, Pd(PPh3)4, N2, reflux, 2h, 80-90%; (e) CH3COOH, reflux, 6h, 80-90%;
(f) CH3I, NaOMe, -10 , overnight, 60%; (g) Toluene, MeOH, H2O, K2CO3, Pd(PPh3)4, N2, reflux, 2h,
80-90%.
Table caption:
Table 1. The structures and the in vitro cytotoxic activities of quinolone derivatives.
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Highlights:
ꢀ
ꢀ
Some of our compounds demonstrate potent Top I inhibitory activity as Camptothecin.
18f and 18g show comparable ability as Camptothecin in Top I inhibitory activity and celluar
inhibitory activity.
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18f and 18g impair the cell cycle progression in the s phase.
18f and 18g represent more safety compared with hydroxycamptothecin.