Synthesis and potential antitumor activity of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines based on ciprofloxacin and norfloxacin scaffolds: in silico studies

Article abstract of DOI:10.3109/14756366.2015.1069288

The potential antitumor activities of a series of 7-(4-substituted piperazin-1-yl)fluoroquinolone derivatives (1–14a,b) using ciprofloxacin and norfloxacin as scaffolds are described. These compounds exhibit potent and broad spectrum antitumor activities using 60 human cell lines in addition to the inherent antibacterial activity. Compounds 1a, 2a, 3b, 6b and 7a were found to be the most potent, while 2b, 5b, and 6a were found to have an average activity. The results of this study demonstrated that compounds 1a, 2a, 3b, 6b and 7a (mean GI₅₀; 2.63–3.09 μM) are nearly 7-fold more potent compared with the positive control 5-fluorouracil (mean GI₅₀; 22.60 μM). More interestingly, compounds 1a, 2a, 3b, 6b and 7a have an almost antitumor activity similar to gefitinib (mean GI₅₀; 3.24 μM) and are nearly 2-fold more potent compared to erlotinib (mean GI₅₀; 7.29 μM). In silico study and ADME-Tox prediction methodology were used to study the antitumor activity of the most active compounds and to identify the structural features required for antitumor activity.

Full text of DOI:10.3109/14756366.2015.1069288

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J Enzyme Inhib Med Chem, Early Online: 1–14
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1069288
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RESEARCH ARTICLE
Synthesis and potential antitumor activity of 7-(4-substituted
piperazin-1-yl)-4-oxoquinolines based on ciprofloxacin and
norfloxacin scaffolds: in silico studies
Alaa A.-M. Abdel-Aziz1,2, Adel S. El-Azab1,3, Amer M. Alanazi1, Yousif A. Asiri4, Ibrahim A. Al-Suwaidan1,
Azza R. Maarouf2,5, Rezk R. Ayyad6, and Taghreed Z. Shawer6
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 2Department of Medicinal Chemistry,
3
Faculty of Pharmacy, University of Mansoura, Mansoura, Egypt, Department of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University,
Cairo, Egypt, 4Department of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 5Department of Pharmaceutical
6
Chemistry, Faculty of Pharmacy, Delta University for Science & Technology, Gamasa City, Egypt, and Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
Abstract
Keywords
The potential antitumor activities of a series of 7-(4-substituted piperazin-1-yl)fluoroquinolone
derivatives (1–14a,b) using ciprofloxacin and norfloxacin as scaffolds are described. These
compounds exhibit potent and broad spectrum antitumor activities using 60 human cell lines
in addition to the inherent antibacterial activity. Compounds 1a, 2a, 3b, 6b and 7a were found
to be the most potent, while 2b, 5b, and 6a were found to have an average activity. The results
of this study demonstrated that compounds 1a, 2a, 3b, 6b and 7a (mean GI50; 2.63–3.09 mM)
ADME-T prediction, antitumor activity,
fluoroquinolones, in silico study, synthesis
History
Received 30 April 2015
Revised 29 May 2015
Accepted 30 May 2015
Published online 30 July 2015
are nearly 7-fold more potent compared with the positive control 5-fluorouracil (mean GI50
;
22.60 mM). More interestingly, compounds 1a, 2a, 3b, 6b and 7a have an almost antitumor
activity similar to gefitinib (mean GI50; 3.24 mM) and are nearly 2-fold more potent compared to
erlotinib (mean GI50; 7.29 mM). In silico study and ADME-Tox prediction methodology were used
to study the antitumor activity of the most active compounds and to identify the structural
features required for antitumor activity.
Introduction
and intercalates DNA and it is currently being evaluated in a
Phase 2 clinical trial for ovarian cancer22,23
.
Cancer is continuing to be a major health problem worldwide to
treat and is the leading cause of human death1–4. The development
of novel anticancer agents remains an important and a challenge
goal in medicinal chemistry and therefore, developing new
effective anticancer drugs is an important strategy in cancer
treatment3,4. The antibacterial fluoroquinolones have been found
Recently we reported the synthesis and antibacterial activity of
bulky arylsulfonylfluoroquinolones based on ciprofloxacin and
norfloxacin scaffolds6. It was found that compounds carrying
dimethoxy or dichloro substituents exhibited excellent antibacter-
ial activity compared with ciprofloxacin as a reference drug. From
the detailed analysis of the results of our studies, we conclude that
antibacterial activity of these compounds significantly depends on
the electronic effect of methoxy and chloro groups.
We thus initiated a screening program to search for novel
utility of 7-(4-substituted piperazin-1-yl)fluoroquinolones, such
as 7-(4-arylsulfonylpiperazin-1-yl)fluoroquinolones, 7-(4-((phe-
nylsulfonyl)carbamoyl)piperazin-1-yl)-fluoroquinolones and 7-(4-
alkylpiperazin-1-yl)fluoroquinolones, as potential antitumor
agents in addition to the reported antibacterial activity.6,24 To
the best of our knowledge, there are no reports concerning
antitumor activity of bulky arylsulfonylfluoroquinolones were
reported.
to be one of the fastest growing groups of drugs in recent years5–8
.
Quinolones are known for their antibacterial and antitumor
activities through alteration of the normal functions of bacterial
gyrase, and are found to be a topoisomerase II inhibitor in
humans9–18. Ciprofloxacin (Figure 1), a commonly used broad-
spectrum fluoroquinolone antibiotic, has shown anticancer activ-
ity in several cancer cell lines19,20. Other fluoroquinolone
derivatives such as levofloxacin and ofloxacin have also been
shown to inhibit the growth of cell bladder cancer cell lines
(Figure 1)21. Voreloxin is a quinolone derivative that shows potent
cytotoxicity towards eukaryotic cancer cell lines without antibac-
terial activity (Figure 1)22,23. Voreloxin inhibits topoisomerase II
In this context, the present work describes the investigation of
the antitumor properties of 7-(4-arylulfonylpiperazin-1-yl)fluoro-
quinolones (1–7ab), 7-(4-alkylpiperazin-1-yl)fluoroquinolones
(8–13ab) and 7-(4-((phenylsulfonyl)carbamoyl)piperazin-1-yl)-
fluoroquinolones (14ab) based on ciprofloxacin and norfloxacin
scaffolds and the achievement of a better antitumor profile at
lower concentrations using 60 human cancer cell lines and
Address for Correspondence: Alaa A.-M. Abdel-Aziz, Department of
Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia. Tel: +966-53-5991127. E-mail:
alaa_moenes@yahoo.com

2
A. A.-M. Abdel-Aziz et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
O
N
O
O
N
O
O
N
O
O
N
O
F
F
F
OH
OH
OH
OH
N
N
N
N
N
HN
HN
N
O
N
O
S
N
O
Ciprofloxacin (b)
Ofloxacin
Levofloxacin
F
Voreloxin (Vosaroxin)
O
O
O
O
O
O
F
F
OH
OH
OH
N
N
R
N
N
R
N
N
N
R
O
O
N
N
1
1
1
S
n
O
R
2
N
n= 1; 10a,b
n= 2; 11a,b
N
X= O; 8a,b
X
R = 4-chlorobenzene; 1a,b
R = 2,4-dichlorobenzene; 2a,b
2
X= CH ; 9a,b
2
O
2
R = 3,4-dichlorobenzene; 3b
2
R = 3,4-dimethoxybenzene; 4a,b
2
R = 2,4-dimethoxybenzene; 5b
2
R = 2,4,6-trimethylbenzene; 6a,b
2
R = 1-naphthyl; 7a
2
O
O
O
O
F
F
OH
OH
R
3
N
N
R
N
N
R
N
O
N
N
1
1
NH
R = H; 12a,b
3
3
S
14ab
R = Bn; 13a,b
O
O
Figure 1. Reported antitumor quinolones and designed N-piperazinyl fluoroquinolones 1–14ab.
keeping the inherent antibacterial activity. Moreover, in silico carried out for C, H and N, at Research Center, King Saud
study and ADME-T prediction were used to identify the structural University, Riyadh, Saudi Arabia. Solvent evaporation was
features required for the antitumor properties of the designed performed under reduced pressure using Buchan Rotatory
compounds. The rationale for testing of these 7-(4-substituted Evaporator at the Pharmaceutical Chemistry Department, King
piperazin-1-yl)fluoroquinolones as antitumor agents was the Saud University, Riyadh, Saudi Arabia. Thin layer chromatography
following: (i) delineate the structure-activity relationship (SAR) was performed on precoated (0.25 mm) silica gel GF254 plates (E.
for the antitumor activity of the arylsulfonylfluoroquinolones Merck, Darmstadt, Germany), compounds were detected
with compounds incorporating ciprofloxacin and norfloxacin with 254 nm UV lamp. Silica gel (60–230 mesh) was employed
scaffolds; (ii) investigate and compare the antitumor activity of 4- for routine column chromatography separations. Compounds
arylsulfonyl, 4-alkylpiperazinyl and 4-phenylsulfonylcarbamoyl 7-arylsulfonyl-piperazin-1-ylfluoroquinolones (1–7a,b) were pre-
derivatives; (iii) compare the efficacy of the mono and dichloro pared according to the reported procedure6,24
.
of arylsulfonylfluoroquinolones versus the methoxy derivatives
for the inhibitory power against various tumor cell lines, in General method for synthesis of ciprofloxacin and norfloxacin
compounds incorporating the same scaffold. Furthermore, deriva- containing 2-(morpholin-4-yl)ethyl and 2-(piperidin-1-yl)ethyl
tives incorporating the bulkier trimethylbenzenesulfonyl moieties fragments (8, 9ab)
were also included in the study, in order to explore as much
A mixture of norfloxacin (a) or ciprofloxacin (b) (1 mmol)
chemical space as possible.
and K2CO3 (1.1 mmol) was stirred in DMF (10 mL) at
room temperature for 20 min. To the resulted mixture, the 1-(2-
Materials and methods
chloroethyl)piperidine or 4-(2-chloroethyl)morpholine (1.1 mmol)
Chemistry
in DMF (5 mL) was added dropwise over a period of 10 min. The
reaction mixture was further stirred at room temperature for 24 h.
The separated solid was then filtered, washed with cold water,
dried and crystallized from the appropriate solvent.
Melting points (uncorrected) were recorded on Barnstead 9100
Electrothermal melting apparatus at the Pharmaceutical
Chemistry Department, King Saud University, Riyadh, Saudi
Arabia. IR spectra were recorded on a FT-IR Perkin-Elmer
spectrometer at Research Center, King Saud University, Riyadh,
Saudi Arabia. Nuclear magnetic resonance (1H and 13C NMR)
1-Ethyl-6-fluoro-4-oxo-7-(4-(2-(morpholin-1-yl)ethyl)piperazin-
1-yl)-1,4-dihydroquinoline-3-carboxylic acid (8a)
spectra were recorded on Bruker 700 MHz or 500 MHz spec- White powder, 52% yield; mp 217–219 ꢀC (MeOH/CH2Cl2); IR
trometer using CD3OD, CDCl3 and DMSO-d6 as solvents at (KBr) ꢀ max/cmꢁ1: 3440 (OH), 1719 (C¼O), 1641 (C¼O).
Research Center, King Saud University, Riyadh, Saudi Arabia. 1HNMR (CDCl3): d 1.54–1.58 (3H, t, J ¼ 7.5 Hz), 2.54-2.63
The chemical shifts are expressed in d ppm using TMS as internal (8H, m), 2.75–2.76 (4H, d, J¼ 2.5 Hz), 3.35–3.36 (4H, d, J¼ 3.5 Hz),
standard. Mass spectra were recorded on a Clarus 600 GC/MS 3.75–3.82 (4H, d, J ¼ 5.0 Hz), 4.31–4.36 (2H, q, J ¼ 7.5 Hz),
(Middletown, CT) and Varian, TQ 320 GC/MS/MS mass 6.88–6.89 (1H, d, J ¼ 6.5 Hz), 8.02–8.08 (1H, dd, J ¼ 12.5,
spectrometers (West Sussex, UK) at Research Center, King 7.5 Hz), 8.67–8.68 (1H, d, J ¼ 6.0 Hz), 15.29 (1H, s, br); 13CNMR
Saud University, Riyadh, Saudi Arabia. Elemental analysis was (CDCl3): d 14.44, 49.81, 49.85, 53.23, 54.15, 55.49, 56.34, 66.93,

DOI: 10.3109/14756366.2015.1069288
In silico studies of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines
3
103.72, 108.32, 112.81, 120.52, 137.11, 146.07, 147.08, 152.52, 1HNMR (CDCl3): d 1.24–1.30 (3H, t, J ¼ 13.5 Hz), 2.51 (1H, s),
154.52, 167.23, 176.97; C22H29FN4O4 m/z: 432.5 (34.6%). Anal. 2.65 (5H, s), 3.26 (4H, s), 3.77 (2H, s), 4.51–4.64 (2H, q,
Calcd: C, 61.10; H, 6.76; N, 12.95. Founded: C, 61.31; H, 6.74; N, J ¼ 6.5 Hz), 7.15–7.16 (1H, d, J ¼ 6.5 Hz), 7.85–7.92 (5H, m),
12.84.
8.94 (1H, s), 15.36 (1H, s, br); 13CNMR (CDCl3): d 14.44, 28.16,
34.93, 36.89, 39.28, 53.64, 55.62, 62.72, 103.89, 107.99, 112.54,
123.38, 128.99, 131.29, 132.04, 134.10, 134.30, 147.27, 152.52,
167.17, 168.14, 168.56, 176.96; C26H25FN4O5 m/z: 492.5 (7.7%).
Anal. Calcd: C, 63.41; H, 5.12; N, 11.38. Founded: C, 63.29; H,
5.22; N, 11.56.
1-Cyclopropyl-6-fluoro-7-(4-(2-morpholinoethyl)piperazin-1-yl)-
4-oxo-1,4-dihydroquinoline-3-carboxylic (8b)
White powder, 66% yield; mp 145–147 ꢀC (MeOH/CH2Cl2);
IR(KBr) ꢀ max/cmꢁ1: 3441 (OH), 1733 (C¼O), 1628 (C¼O).
1HNMR (CDCl3): d 1.16 (2H, s), 1.35–1.36 (2H, d, J ¼ 6.0 Hz),
2.61 (4H, s), 2.80–2.82 (2H, t, J ¼ 7.5 Hz), 3.24 (2H, s), 3.30 (2H,
s), 3.45 (1H, s), 3.63 (2H, s), 3.74 (4H, s), 3.78 (2H, s), 4.46–4.47
(2H, d, J ¼ 6.0 Hz), 7.28 (1H, s), 8.15 (1H, s), 8.56 (1H, s);
1-Cyclopropyl-7-(4-(2-(1,3-dioxoisoindolin-2-yl)ethyl)piperazin-
1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (10b)
13CNMR (CDCl3): d 8.29, 35.34, 39.69, 45.33, 49.28, 50.82, White powder, 61% yield; mp 189–192 ꢀC (CH2Cl2). 1HNMR
63.43, 105.50, 108.27, 112.59, 120.57, 138.96, 145.36, 147.64, (CDCl3): d 1.16–1.18 (2H, d, J ¼ 5.5 Hz), 1.34–1.36 (2H, d,
160.80, 166.78, 177.07; C23H29FN4O4 m/z: 444.2 (5.5%). Anal. J ¼ 6.0 Hz), 2.43 (2H, s), 3.36 (4H, s), 3.63 (6H, m), 3.98 (1H, s),
Calcd: C, 62.15; H, 6.58; N, 12.60. Founded: C, 62.24; H, 6.52; N, 6.66 (1H, s), 7.48–7.50 (4H, d, J ¼ 6.0 Hz), 8.15–8.16 (1H, d,
12.40.
J ¼ 12.5 Hz), 8.41 (1H, s), 15.12 (1H, s, br); 13CNMR (CDCl3): d
8.16, 35.03, 37.79, 45.08, 49.12, 55.51, 57.01, 63.32, 103.88,
109.07, 112.78, 123.01, 133.38, 134.40, 135.53, 149.65, 151.50,
165.19, 167.00, 168.82, 174.77; C27H25FN4O5 m/z: 504.5
(15.6%). Anal. Calcd: C, 64.28; H, 4.99; N, 11.11. Founded: C,
64.38; H, 5.10; N, 10.99.
1-Ethyl-6-fluoro-4-oxo-7-(4-(2-(piperidin-1-yl)ethyl)piperazin-1-
yl)-1,4-dihydroquinoline-3-carboxylic acid (9a)
Yellow powder, 69% yield; mp 259–260 ꢀC (MeOH/CH2Cl2);
IR(KBr) ꢀ max/cmꢁ1: 3440 (OH), 1730 (C¼O), 1636 (C¼O).
1HNMR (CDCl3/CD3OD): d 1.56–1.59 (3H, t, J ¼ 7.0 Hz), 1.71
(2H, s), 1.94 (4H, s), 2.79 (4H, s), 2.88 (2H, s), 3.30–3.33 (6H, d,
J ¼ 13.5 Hz), 3.43 (4H, s), 4.51–4.52 (2H, d, J ¼ 7.0 Hz), 7.08–
7.09 (1H, d, J ¼ 6.5 Hz), 7.90–7.93 (1H, d, J ¼ 13.5 Hz), 8.80 (1H,
s); 13CNMR (CDCl3/CD3OD): d 15.24, 22.89, 24.12, 50.85,
51.13, 53.51, 53.95, 54.43, 54.93, 106.24, 108.66, 113.00, 121.35,
138.88, 147.43, 149.22, 154.01, 169.72, 178.22; C23H31FN4O3
m/z: 430.6 (28.7%). Anal. Calcd: C, 64.17; H, 7.26; N, 13.01.
Founded: C, 64.28; H, 6.17; N, 13.09.
7-(4-(3-(1,3-Dioxoisoindolin-2-yl)propyl)piperazin-1-yl)-1-ethyl-
5-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (11a)
White powder, 63% yield; mp 205-206 oC (Hexane/CH2Cl2).
1HNMR (CDCl3): d 1.59–1.61 (3H, t, J ¼ 7.0 Hz), 1.92–1.94 (2H,
t, J ¼ 6.5 Hz), 2.54 (2H, s), 2.65 (4H, s), 3.23 (4H, s), 3.82–3.84
(2H, t, J ¼ 7.0 Hz), 4.33-4.34 (2H, d, J ¼ 7.0 Hz), 6.77–6.78 (1H,
d, J ¼ 6.5 Hz), 7.72–7.74 (2H, dd, J ¼ 5.0, 8.0 Hz), 7.86–7.88 (2H,
dd, J ¼ 5.5, 8.5 Hz), 8.01-8.04 (1H, d, J ¼ 13.0 Hz), 8.68 (1H, s),
15.14 (1H, s, br); 13CNMR (CDCl3): d14.48, 25.20, 36.42, 49.76,
52.65, 52.68, 103.63, 108.33, 112.62, 120.49, 123.19, 132.27,
133.96, 137.09, 146.06, 147.09, 152.50, 154.50, 167.27, 168.52,
176.99; C27H27FN4O5 m/z: 506.5 (6.4%). Anal. Calcd: C, 64.02;
H, 5.37; N, 11.06. Founded: C, 64.11; H, 5.35; N, 11.09.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-(piperidin-1-yl)ethyl)piper-
azin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (9b)
Yellow powder, 73% yield; mp 265–266 ꢀC (MeOH/CH2Cl2);
IR(KBr) ꢀ max/cmꢁ1: 3443 (OH), 1737 (C¼O), 1640 (C¼O).
1HNMR (CDCl3/CD3OD): d 1.23 (2H, s), 1.44–1.45 (2H, d,
J ¼ 6.0 Hz), 1.71 (2H, s), 1.92 (4H, s), 2.80 (4H, s), 2.87 (2H, s),
3.28–3.29 (6H, dd, J ¼ 5.5, 7.0 Hz), 3.45 (4H, s), 3.76 (1H, s),
7.54–7.55 (1H, d, J ¼ 7.0 Hz), 7.86–7.89 (1H, d, J ¼ 13.0 Hz),
8.77 (1H, s); 13CNMR (CDCl3/CD3OD): d 8.85, 22.96, 24.24,
37.01, 50.71, 53.62, 53.95, 54.53, 54.94, 107.00, 108.33, 112.63,
112.83, 140.79, 149.20, 169.61; C24H31FN4O3 m/z: 442.7 (5.6%).
Anal. Calcd: C, 65.14; H, 7.06; N, 12.66. Founded: C, 65.35; H,
7.15; N, 12.23.
1-Cyclopropyl-7-(4-(3-(1,3-dioxoisoindolin-2-yl)propyl)pipera-
zin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (11b)
White powder, 59% yield; mp 210–212 ꢀC (Hexane/CH2Cl2);
IR(KBr) ꢀ max/cmꢁ1: 3437 (OH), 1726 (C¼O), 1711 (C¼O),
1627 (C¼O).1HNMR (CDCl3): d 1.31–1.33 (4H, t, J ¼ 5.5 Hz),
1.84–1.93 (2H, m), 2.14–2.16 (2H, t, J ¼ 6.0 Hz), 3.60–3.62 (2H,
t, J ¼ 6.0 Hz), 3.78-4.01 (7H, m), 4.29–4.31 (2H, t, J ¼ 6.0 Hz),
7.16–7.17 (1H, d, J ¼ 6.5 Hz), 7.77–7.92 (5H, m), 8.68 (1H, s),
15.10 (1H, s, br); 13CNMR (CDCl3): d 8.13, 27.70, 31.33, 34.56,
36.49, 49.74, 52.79, 55.82, 59.06, 61.59, 104.71, 109.83, 112.95,
123.15, 132.09, 133.93, 137.94, 144.44, 148.32, 152.27, 164.98,
166.31, 168.48, 172.99; C28H27FN4O5 m/z: 518.8 (9.7%).
Anal. Calcd: C, 64.86; H, 5.25; N, 10.80. Founded: C, 64.76;
H, 5.20; N, 10.89.
General method for synthesis of norfloxacin and ciprofloxacin
containing phthalimide fragments (10, 11ab)
A mixture of norfloxacin (a) or ciprofloxacin (b) (1.0 mmol)
and K2CO3 (1.1 mmol) was stirred in DMF (10 mL) at room
temperature for 20 min. To the resulted mixture, the 2-(2-
bromoethyl)isoindoline-1,3-dione or 2-(3-bromopropyl)isoindo-
line-1,3-dione (1.1 mmol) in DMF (5 mL) was added drop-wise
over a period of 10 min. The reaction mixture was further stirred
at room temperature for 24 h. The separated solid was then
filtered, washed with cold water, dried and crystallized from the
appropriate solvent.
General method for synthesis of ciprofloxacin and norfloxacin
containing 4-(piperidin-1-ylmethyl) fragments (12, 13ab)
To a mixture of norfloxacin (a) or ciprofloxacin (b) (1.0 mmol)
and the piperidine or 4-benzylpiperidine (1.0 mmol) in methanol
(10 mL), 1 mL of formaline (37%) was added. The reaction
mixture was heated overnight at a reflux temperature. The
reaction mixture was cooled, the solvent was removed under
7-(4-(3-(1,3-Dioxoisoindolin-2-yl)ethyl)piperazin-1-yl)-1-ethyl-5-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (10a)
White powder, 64% yield; mp 250–252 ꢀC (CH2Cl2); IR(KBr) ꢀ vacuum, the solid obtained washed with water, dried and re-
max/cmꢁ1: 3441 (OH), 1731 (C¼O), 1719 (C¼O), 1637 (C¼O). crystallised from an appropriate solvent.

4
A. A.-M. Abdel-Aziz et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
1-Ethyl-6-fluoro-4-oxo-7-(4-(piperidin-1-ylmethyl)piperazin-1-yl)-
1,4-dihydroquinoline-3-carboxylic acid (12a)
(b) (0.01 mol) and triethylamine (1.0 mL, 0.01 mol) in dry toluene
(50 mL). The reaction mixture was heated under reflux for 3 h,
then evaporated in vacuo and the obtained residue was triturated
with ice water, filtered, dried and crystallized from methanol to
afford N-(phenylsulfonyl)carbamoyl derivatives.
Yellow powder, 74% yield; mp4300 oC (MeOH); IR(KBr) ꢀ max/
1
cmꢁ1: 3438 (OH), 1728 (C¼O), 1629 (C¼O). HNMR (CDCl3):
d 1.14-1.15 (9H, d, J ¼ 7.0 Hz), 1.52-1.53 (3H, d, J ¼ 7.0 Hz),
2.72 (1H, s), 2.84 (3H, s), 3.28 (4H, s), 3.47–3.49 (1H, d,
J ¼ 7.0 Hz), 3.64–3.65 (1H, d, J ¼ 6.5 Hz), 4.07 (1H, s), 4.25–4.27
(2H, t, J ¼ 7.0 Hz), 6.76–6.77 (1H, d, J ¼ 6.5 Hz), 7.94–7.96 (1H,
1-Ethyl-6-fluoro-4-oxo-7-(4-((phenylsulfonyl)carbamoyl)pipera-
zin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (14a)
d, J ¼ 13.5 Hz), 8.59 (1H, s), 15.05 (1H, s, br); 13CNMR (CDCl3): Yellow powder, 81% yield; mp 255–257 oC (MeOH); IR(KBr) ꢀ
d 14.45, 18.45, 30.94, 49.20, 49.96, 51.07, 58.45, 63.43, 64.40, max/cmꢁ1: 3446 (OH), 3260 (NH), 1718 (C¼O), 1684 (C¼O),
88.07, 103.77, 108.31, 112.82, 120.39, 137.136, 146.28, 147.06, 1628 (C¼O). 1HNMR (CDCl3/TFA): d 1.36–1.42 (3H, dd,
152.53, 154.53, 167.26, 176.98; C22H29FN4O3 m/z: 416.9 (2.5%). J ¼ 6.5, 8.0 Hz), 3.66 (4H, s), 3.85 (4H, s), 4.73 (2H, s), 7.28
Anal. Calcd: C, 63.44; H, 7.02; N, 13.45. Founded: C, 63.79; H, (3H, s), 7.48–7.51 (1H, d, J ¼ 14.5 Hz), 7.93–7.94 (1H, d,
6.81; N, 13.47.
J ¼ 6.0 Hz), 8.08–8.22 (2H, t, J ¼ 14.5 Hz), 9.13 (1H, s);
13CNMR (CDCl3/TFA): d 13.49, 44.03, 46.20, 46.75, 52.29,
63.43, 104.81, 115.67, 116.12, 126.31, 127.45, 129.36, 133.32,
138.52, 141.03, 142.02, 146.56, 159.93, 160.27, 169.46, 170.99;
C23H23FN4O6S m/z: 502.4 (5.7%). Anal. Calcd: C, 54.97; H, 4.61;
N, 11.15. Founded: C, 55.17; H, 4.60; N, 11.02.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(piperidin-1-ylmethyl)pipera-
zin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (12b)
Yellow powder, 79% yield; mp 256–258 ꢀC (MeOH). 1HNMR
(CDCl3): d 1.14–1.18 (6H, m), 1.31–1.33 (4H, d, J ¼ 6.0 Hz), 2.09
(2H, s), 2.72 (2H, s), 2.84 (4H, s), 3.30 (4H, s), 3.46–3.50 (2H, q,
J ¼ 7.0 Hz), 4.07 (1H, s), 7.28–7.29 (1H, d, J ¼ 6.5 Hz), 7.83–7.87
(1H, dd, J ¼ 8.5, 12.5 Hz), 8.62–8.64 (1H, d, J ¼ 6.5 Hz), 14.96
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-((phenylsulfonyl)carbamoyl)-
piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (14b)
(1H, s, br); 13CNMR (CDCl3): d 8.21, 15.28, 18.44, 30.93, 35.31, Yellow powder, 78% yield; mp 185–187 ꢀC (MeOH); IR(KBr) ꢀ
49.21, 49.88, 51.09, 58.39, 64.39, 88.09, 104.82, 107.96, 112.34, max/cmꢁ1: 3446 (OH), 3264 (NH), 1718 (C¼O), 1682 (C¼O),
1
119.53, 139.08, 145.97, 147.31, 152.66, 154.66, 166.96, 177.01; 1628 (C¼O). HNMR (CDCl3/TFA): d 1.30 (2H, s), 1.51–1.53
C23H29FN4O3 m/z: 428.6 (8.3%). Anal. Calcd: C, 64.47; H, 6.82; (2H, d, J ¼ 6.0 Hz), 3.51 (4H, s), 3.57 (4H, s), 3.79 (1H, s), 7.28
N, 13.08. Founded: C, 64.57; H, 6.82; N, 13.14.
(2H, s), 7.44–7.46 (1H, dd, J ¼ 2.0, 8.5 Hz), 7.52–7.54 (1H, d,
J ¼ 6.0 Hz), 7.59–7.60 (1H, d, J ¼ 1.5 Hz), 8.00–8.03 (2H, t,
J ¼ 3.0 Hz), 8.97 (1H, s); 13CNMR (CDCl3/TFA): d 8.29, 36.84,
45.40, 49.57, 105.39, 106.18, 112.18, 115.94, 127.64, 132.98,
133.24, 134.05, 139.83, 140.27, 146.75, 148.38, 153.18, 155.21,
159.06, 169.89, 174.51; C24H23FN4O6S m/z: 515.0 (4.6%). Anal.
Calcd: C, 56.02; H, 4.51; N, 10.89. Founded: C, 55.84; H, 4.51; N,
11.00.
7-(4-((4-Benzylpiperidin-1-yl)methyl)piperazin-1-yl)-1-ethyl-6-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (13a)
White powder, 89% yield; mp 285–287 ꢀC (MeOH); IR(KBr) ꢀ
max/cmꢁ1: 3440 (OH), 1736 (C¼O), 1641 (C¼O). 1HNMR
(CDCl3): d 1.14-1.17 (8H, d, J ¼ 7.0 Hz), 1.51–1.52 (4H, d,
J ¼ 7.0 Hz), 2.71 (3H, s), 2.86 (3H, s), 3.26 (4H, s), 3.46–3.48
(1H, d, J ¼ 7.0 Hz), 4.09 (1H, s), 4.24–4.26 (2H, t, J ¼ 7.0 Hz),
6.77–6.79 (1H, d, J ¼ 6.5 Hz), 7.20–7.26 (5H, m), 7.90–7.94 (1H,
dd, J ¼ 8.5, 5.0 Hz), 8.56 (1H, s), 15.06 (1H, s, br); 13CNMR
(CDCl3): d 14.44, 29.69, 49.20, 49.75, 49.95, 51.05, 58.42, 64.39,
88.07, 103.77, 108.25, 112.57, 112.75, 120.32, 120.38, 128.14,
129.12, 137.13, 146.19, 146.27, 147.04, 152.52, 154.52, 167.25,
176.93; C29H35FN4O3 m/z: 506.4 (6.1%). Anal. Calcd: C, 68.75;
H, 6.96; N, 11.06. Founded: C, 68.91; H, 7.02; N, 11.21.
Antitumor screening
A primary anticancer assay was performed for an approximately
60 human tumor cell line panel derived from nine neoplastic
diseases, in accordance with the protocol of the Drug Evaluation
Branch, National Cancer Institute, Bethesda, MD25
.
Three dose response parameters were calculated for each
compound including GI50 (the drug concentration resulting in a
50% lower net protein increase in the treated cells measured by
SRB staining), TGI (the drug concentration resulting in total
growth inhibition), and LC50 (concentration of drug resulting
in a 50% reduction in the measured protein at the end of the
drug treatment compared to that at the beginning). Values were
calculated for each of these three parameters if the level of
activity was reached; however, if the effect was not reached
or was exceeded, the value for that parameter was expressed
as more or less for the maximum or minimum concentration
tested. The lowest values are obtained with the most sensitive cell
lines. The compounds having GI50 ꢂ100 mM were declared to be
active25.
7-(4-((4-Benzylpiperidin-1-yl)methyl)piperazin-1-yl)-1-cyclopro-
pyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (13b)
Yellow powder, 86% yield; mp 260–262 ꢀC (MeOH). 1HNMR
(CDCl3): d 1.03–1.22 (6H, m), 1.31 (2H, s), 1.55–1.57 (2H, d,
J ¼ 6.0 Hz), 2.23–2.39 (1H, m), 2.45–2.47 (2H, d, J ¼ 7.0 Hz),
2.65 (1H, s), 2.72 (1H, s), 2.76–2.91 (4H, m), 3.27–3.31 (4H, d,
J ¼ 15.0 Hz), 3.48–3.48 (2H, d, J ¼ 3.5 Hz), 4.11–4.22 (1H, m),
7.059 (2H, s), 7.09–7.10 (1H, d, J ¼ 6.0 Hz), 7.18–7.19 (2H, d,
J ¼ 6.5 Hz), 7.25–7.27 (1H, d, J ¼ 7.0 Hz), 7.80–7.85 (1H, m),
8.60–8.61 (1H, J ¼ 4.5 Hz); 13CNMR (CDCl3): d 7.18, 30.70,
31.24, 34.30, 36.67, 37.33, 42.24, 48.97, 50.07, 51.21, 80.09,
103.72, 106.86, 111.18, 118.45, 124.74, 127.12, 128.08, 138.05,
139.61, 145.03, 146.27, 151.63, 153.63, 165.98, 166.05, 175.94;
C30H35FN4O3 m/z: 518.6 (5.5%). Anal. Calcd: C, 69.48; H, 6.80;
N, 10.80. Founded: C, 69.61; H, 6.84; N, 10.57.
Results and discussion
Chemistry
Scheme 1 outlines the synthetic pathway used to obtain
compounds (1–14a,b). Compounds 1–7a,b were prepared accord-
ing to our previous report by the reaction of norfloxacin (a) or
ciprofloxacin (b) with the appropriate arylsulfonyl chloride in
acetone in the presence of K2CO3 at room temperature for 24 h6.
On the other hand, compounds 8–11a,b were prepared by the
General method for synthesis of ciprofloxacin and norfloxacin
containing 4-(phenylsulfonyl)carbamoyl fragments (14ab)
Benzenesulfonyl isocyanate (1.83 g, 1.34 ml, 0.01 mol) was added
dropwise to a stirred solution of norfloxacin (a) or ciprofloxacin

DOI: 10.3109/14756366.2015.1069288
In silico studies of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines
5
O
O
O
O
O
O
R = 4-chlorobenzene; 1a,b
F
F
2
F
OH
Acetone, K CO
OH
OH
R = 2,4-dichlorobenzene; 2a,b
2
N
N
R
DMF, K CO
2
N
N
R
2
3
N
N
R
3
R = 3,4-dichlorobenzene; 3b
O
S
R
2
N
O
N
1
HN
1
rt, 24h
1
rt, 24h
R = 3,4-dimethoxybenzene; 4a,b
2
X= O; 8a,b
2
N
R = 2,4-dimethoxybenzene; 5b
2
R = ethyl (a), cyclopropyl (b)
X= CH ; 9a,b
1
2
X
R = 2,4,6-trimethylbenzene; 6a,b
2
R = 1-naphthyl; 7a
Toluene, TEA
2
R
3
PhSO NCO
MeOH, HCHO
Reflux, 24h
2
DMF, K CO
2
rt, 24h
Reflux, 3h
3
N
H
O
O
O
O
F
F
O
O
OH
OH
F
OH
N
N
R
N
N
R
R
3
N
O
N
1
N
N
R
1
n
O
N
N
N
NH
O
1
S
O
14a,b
O
n= 1; 10a,b
n= 2; 11a,b
R = H; 12a,b
3
R = Bn; 13a,b
3
Scheme 1. Synthesis of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines (1–14a,b).
reaction of alkyl halides with norfloxacin (a) or ciprofloxacin (b)
The screening results of the selected molecules at 10 mM
in DMF containing anhydrous K2CO3. Mannich bases 12, 13a,b concentration showed that 7-(4-arylsulfonylpiperazin-1-yl)fluoro-
were prepared (74–89% yield) by heating at reflux temperature of quinolones (1–7a,b) are more active compared with 7-(4-
norfloxacin (a) or ciprofloxacin (b) and piperidine derivatives as alkylpiperazin-1-yl)fluoroquinolones (8b, 10b, 11b and 12b)
secondary amine with excess formaldehyde in methanol. and
7-(4-((phenylsulfonyl)carbamoyl)piperazin-1-yl)-fluoro-
Moreover, compounds 14a,b were also prepared in a better quinolone (14b) in addition to the parent ciprofloxacin (b)
yield (79–85%) through the reaction of norfloxacin (a) or scaffold as indicated by the number of sensitive cell lines and
ciprofloxacin (b) with benzenesulfonyl isocyanate in toluene cytotoxic effect (Table 1, Supporting Information Figure S1).
(Scheme 1). The structural formulae and the purity of the
The synthesized 7-(4-arylsulfonylpiperazin-1-yl)fluoroquino-
synthesized compounds were checked by thin layer chromatog- lones (1–7a,b) displayed significant activity in the in vitro
raphy and spectral analysis. The NMR spectra were identified by screening on the tested cell lines in 10 mM concentration with
their chemical shifts, multiplicities and coupling constants. In positive cytotoxic effect (PCE) of 6/58 ꢃ 58/58. (Tables 1 and 2,
general, 1H NMR spectra showed the characteristic chemical Figure S1, Supporting Information). These molecules showed a
shifts for the fluoroquinolone nucleus.
cytotoxic effect on the most of the cancer cell lines especially
compounds 1a, 2a, 3b, 6b and 7a with mean growth inhibition
percentages (MGI%) of 75.5%, 94.1%, 89.2%, 43.8% and 93.7%
respectively. Arylsulfonylpiperazinyl fluoroquinolones (2b, 5b
Antitumor activity
The designed compounds 1–14a,b in addition to the main scaffold and 6a) showed moderate activity with MGI% of 17.3%, 7.3% and
ciprofloxacin (b) were tested for their in vitro antitumor activity. 18.3% respectively. Derivatives based on norfloxacin (a) scaffold
Sixteen compounds and ciprofloxacin (b) were selected by were more active compared with the corresponding ciprofloxacin
National Cancer Institute, Bethesda, MD (Table 1) on the basis (b) derivatives as indicated by mean growth inhibition percent-
of degree of the structure variation and computer modelling ages (MGI% ¼ 75.5, 1a; 3.5, 1b; 94.1, 2a and 17.3, 2b). Moreover
techniques for evaluation of their antitumor activity25. The the dimethoxy derivatives of arylsulfonylpiperazinyl fluoroquino-
selected compounds were subjected to in vitro antitumor assay lones (MGI% ¼ 2.3, 4a; 1.8, 4b and 7.3, 5b) were less active than
against tumor cells in a full panel of 60-cell lines taken from nine the corresponding dichloro derivatives of 7-arenesulfonyl-piper-
different organs (lung, colon, breast, ovary, blood, kidney, skin, azinyl fluoroquinolones (MGI% ¼ 94.1, 2a; 17.3, 2b and 89.2, 3b)
prostate and brain). The compounds were grouped into three emphasizing the importance of chloro fragment which may
series, including 7-(4-arenesulfonylpiperazin-1-yl)fluoroquino- attributed to lipophilic character and electronic effect of the
lone (1–7a,b), 7-(4-alkylpiperazin-1-yl)fluoroquinolones (8b, chloro derivatives. Replacement of the methoxy group with
10b, 11b and 12b) and 7-(4-((phenylsulfonyl)carbamoyl)piper- trimethyl substituent lead to improvement of the antitumor
azin-1-yl)-fluoroquinolone (14b) (Table 1, Figure S1, activity such as compounds 6a and 6b with mean growth
Supplementary file), which were first evaluated at a single dose inhibition percentages (MGI%) of 18.3% and 43.8% respectively.
concentration of 10 mM, the percentages of growth inhibitions On the other hand, introducing 1-naphthalenesulfonyl moiety in
over the 60 tested cell lines were determined and the results were compound 7a led to sharp increase of the antitumor activity
compared with 5-fluorouracil (5-Flu)26, gefitinib (IressaTM 27–29
and erlotinib (TarcevaTM 30
as reference drugs. In the screening molecules containing trimethylbenzenesulfonyl moiety such as
)
(MGI% ¼ 93.7) compared with parent ciprofloxacin (b) and
)
methodology, each cell line was inoculated and incubated for compounds 6a and 6b with mean growth inhibition percentages
24–47 h. Molecules were then added at a single concentration and (MGI%) of ꢁ1.4, 18.3 and 43.8% respectively (Table 1, Figure
the culture was incubated for further 48 h. End point determin- S1, Supporting Information).
ation were made with a protein binding dye, sulforhodamine
By investigating the variation in selectivity and broad spec-
(SRB)25. Results for each tested molecule were reported as the trum of the tested compounds over the full panel of cell lines,
percentage growth of the treated cells comparing to the untreated it was revealed that nearly all of arylsulfonylpiperazinyl fluoro-
control cells. The screening results and percentages of growth quinolones (1–7a,b) (Tables 1 and 2) showed significant
inhibitions over sensitive cell lines are shown in Tables 1 and 2. inhibition for the most cell lines used in this assay (leukemia,

6
A. A.-M. Abdel-Aziz et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Table 1. Antitumor activity of the designed 7-(4-substituted piperazin-1-yl)fluoroquinolones at 10 mM concentration.
60 cell lines assay in one dose 10.0 mM concentration
Compd
No
MGI%
75.59
PCE
RGI%
Most sensitive cell lines
1a
56/56 4100.0 to 31.3
Leukemia (CCRF-CEM, HL-60 (TB), K-562, MOLT-4, PRMI-8226), NSC Lung
Cancer (A549/ATCC, NCI-H226, HOP-92, NCI-H23, NCI-H322M, NCI-H460,
NCI-H522), Colon Cancer (COLO 205, HCC-2998, HCT-116, HCT-15, HT29,
KM12, SW-620), CNS Cancer (SF-268, SF-295, SF-539, SNB-19,SNB-75, U251),
Melanoma (LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-
28, SK-MEL-5, UACC-257, UACC-62), Ovarian Cancer (IGROV1, OVCAR-3,
OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES, SK-OV-3), Renal Cancer (786-0,
A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31), Prostate Cancer (PC-3,
DU-145), Breast Cancer (MCF7, MDA-MB-231/ATCC, HS 578T, BT-549, T-47D)
Melanoma (LOX IMVI, M14), Renal Cancer (A498, CAKI-1, UO-31), Prostate Cancer
(PC-3), Breast Cancer (T-47D)
Leukemia (CCRF-CEM, HL-60(TB), K-562, MOLT-4, PRMI-8226, SR), NSC Lung
Cancer (A549/ATCC, HOP-62, NCI-H226, HOP-92, NCI-H23, NCI-H322M, NCI-
H460, NCI-H522), Colon Cancer (COLO 205, HCC-2998, HCT-116, HCT-15,
HT29, KM12, SW-620), CNS Cancer (SF-268, SF-295, SF-539, SNB-19,SNB-75,
U251), Melanoma (LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2,
SK-MEL-5, UACC-257, UACC-62), Ovarian Cancer (IGROV1, OVCAR-3,
OVCAR-5, OVCAR-8, NCI/ADR-RES, SK-OV-3), Renal Cancer (786-0, A498,
ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31), Prostate Cancer (PC-3, DU-
145), Breast Cancer (MCF7, MDA-MB-231/ATCC, BT-549, T-47D, MDA-
MB-468)
1b
2a
3.5
7/53
59.75 to 10.12
94.17
56/58 4100.0 to 25.22
2b
3b
17.39
89.25
30/58
79.75 to 11.78
Leukemia (CCRF-CEM, HL-60(TB), MOLT-4, PRMI-8226, SR), NSC Lung Cancer
(HOP-62, NCI-H226, NCI-H23, NCI-H322M), Colon Cancer (COLO 205, HCT-
116, HCT-15), CNS Cancer (SF-268, SNB-75), Melanoma (LOX IMVI, SK-MEL-
28, SK-MEL-5, UACC-62), Ovarian Cancer (OVCAR-4), Renal Cancer (A498,
ACHN, CAKI-1, RXF 393, SN12C, UO-31), Prostate Cancer (PC-3), Breast Cancer
(MCF7, BT-549, T-47D, MDA-MB-468)
Leukemia (CCRF-CEM, HL-60(TB), K-562, MOLT-4, PRMI-8226, SR), NSC Lung
Cancer (A549/ATCC, HOP-62, NCI-H226, HOP-92, NCI-H23, NCI-H322M, NCI-
H460, NCI-H522), Colon Cancer (COLO 205, HCC-2998, HCT-116, HCT-15,
HT29, KM12, SW-620), CNS Cancer (SF-268, SF-295, SF-539, SNB-19,SNB-75,
U251), Melanoma (LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2,
SK-MEL-28, SK-MEL-5, UACC-257, UACC-62), Ovarian Cancer (IGROV1,
OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES, SK-OV-3), Renal
Cancer (786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31), Prostate
Cancer (PC-3, DU-145), Breast Cancer (MCF7, MDA-MB-231/ATCC, BT-549,
T-47D, MDA-MB-468)
58/58 4100.0 to 46.49
4a
4b
5b
2.33
1.84
7.33
9/58
6/58
55.93 to 10.04
35.09 to 10.43
46.83 to 10.48
Leukemia (MOLT-4, SR), CNS Cancer (SF-268, SNB-75), Melanoma (LOX IMVI,
M14), Renal Cancer (UO-31), Prostate Cancer (PC-3), Breast Cancer (T-47D)
NSC Lung Cancer (HOP-62, HOP-92), CNS Cancer (SNB-75), Melanoma (LOX
IMVI), Renal Cancer (UO-31), Breast Cancer (T-47D)
Leukemia (CCRF-CEM, HL-60(TB), K-562, MOLT-4, PRMI-8226, SR), NSC Lung
Cancer (HOP-62, NCI-H226), CNS Cancer (SF-268, SF-539, SNB-19, SNB-75),
Melanoma (LOX IMVI, SK-MEL-5, UACC-62), Renal Cancer (A498, UO-31),
Breast Cancer (T-47D, MDA-MB-468)
19/58
6a
18.31
46/58
66.90 to 10.12
Leukemia (CCRF-CEM, HL-60(TB), K-562, MOLT-4, PRMI-8226, SR), NSC Lung
Cancer (A549/ATCC, HOP-62, NCI-H226, HOP-92, NCI-H23, NCI-H322M, NCI-
H460, NCI-H522), Colon Cancer (HCC-2998, HCT-116, HCT-15, KM12), CNS
Cancer (SF-268, SF-295, SF-539, SNB-19,SNB-75, U251), Melanoma (LOX IMVI,
M14, MDA-MB-435, SK-MEL-2, SK-MEL-5, UACC-62), Ovarian Cancer
(IGROV1, OVCAR-3, OVCAR-5, OVCAR-8, NCI/ADR-RES), Renal Cancer
(A498, ACHN, CAKI-1, RXF 393, SN12C, UO-31), Prostate Cancer (PC-3), Breast
Cancer (MDA-MB-231/ATCC, BT-549, T-47D, MDA-MB-468)
6b
43.83
56/58
91.28 to 13.08
Leukemia (CCRF-CEM, HL-60(TB), K-562, MOLT-4, PRMI-8226, SR), NSC Lung
Cancer (A549/ATCC, HOP-62, NCI-H226, HOP-92, NCI-H23, NCI-H322M, NCI-
H460, NCI-H522), Colon Cancer (COLO 205, HCC-2998, HCT-116, HCT-15,
HT29, KM12, SW-620), CNS Cancer (SF-268, SF-295, SF-539, SNB-19,SNB-75,
U251), Melanoma (LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-28,
SK-MEL-5, UACC-257, UACC-62), Ovarian Cancer (IGROV1, OVCAR-4,
OVCAR-5, OVCAR-8, NCI/ADR-RES, SK-OV-3), Renal Cancer (786-0, A498,
ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31), Prostate Cancer (PC-3, DU-
145), Breast Cancer (MCF7, MDA-MB-231/ATCC, BT-549, T-47D, MDA-MB-
468)
7a
93.76
58/58 4100.0 to 40.83
Leukemia (CCRF-CEM, HL-60(TB), K-562, MOLT-4, PRMI-8226, SR), NSC Lung
Cancer (A549/ATCC, HOP-62, NCI-H226, HOP-92, NCI-H23, NCI-H322M, NCI-
H460, NCI-H522), Colon Cancer (COLO 205, HCC-2998, HCT-116, HCT-15,
HT29, KM12, SW-620), CNS Cancer (SF-268, SF-295, SF-539, SNB-19,SNB-75,
(continued )

DOI: 10.3109/14756366.2015.1069288
In silico studies of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines
7
60 cell lines assay in one dose 10.0 mM concentration
Most sensitive cell lines
Compd
No
MGI%
PCE
RGI%
U251), Melanoma (LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2,
SK-MEL-28, SK-MEL-5, UACC-257, UACC-62), Ovarian Cancer (IGROV1,
OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI/ADR-RES, SK-OV-3), Renal
Cancer (786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31), Prostate
Cancer (PC-3, DU-145), Breast Cancer (MCF7, MDA-MB-231/ATCC, BT-549,
T-47D, MDA-MB-468)
Ciprofloxacin (b)
8b
ꢁ1.41
ꢁ2.20
2/57
5/57
28.85 to 13.76
24.51 to 10.05
Renal Cancer (UO-31, A498)
NSC Lung Cancer (HOP-92, NCI-H522), Melanoma (MALME-3M), Renal Cancer
(UO-31), Prostate Cancer (PC-3)
10b
11b
12b
14b
ꢁ2.69
ꢁ4.16
ꢁ4.72
ꢁ2.46
3/58
3/57
2/57
4/57
36.83 to 13.18
38.76 to 13.64
32.52 to 13.02
26.16 to 10.00
CNS Cancer (SNB-75), Renal Cancer (UO-31), Breast Cancer (MCF7)
Leukemia (CCRF-CEM, PRMI-8226), Renal Cancer (UO-31)
Leukemia (SR), Renal Cancer (UO-31)
Leukemia (MOLT-4, SR), Renal Cancer (UO-31, A498)
MGI%: Mean growth inhibition percentage. RGI%: Range of growth inhibition percentage.
PCE: Positive cytotoxic effect which is ratio between number of cell lines with percentage growth inhibition from10 to4100 and total number of cell
lines.
non-small cell lung (NSCLC), colon, CNS, melanoma, ovarian, cells. Respecting melanoma; compounds 1a, 2a, 3b, 6b and 7a are
renal and breast cancer cell lines) with growth inhibition reached active against LOX IMVI cell lines with GI% values of 80.1,
to 4100% (Tables 1 and 2), while alkylpiperazinyl fluoroquino- 4100, 98.7, 70.3 and 4100, respectively. Pertaining to renal
lones (8b, 10b, 11b and 12b), phenylsulfonylcarbamoylpiperazi- cancer; compounds 1a, 2a, 3b and 7a were active against A498
nyl fluoroquinolone (14b) and ciprofloxacin (b) (Table 1, cell line with GI% values of 4100, 4100, 86.2 and 4100,
Figure S1, Supporting Information) are mainly active against respectively. Renal ACHN and UO-31 cell lines are sensitive to
renal cell lines (A498 and UO-31) with growth inhibition reached compounds 2a and 7a with GI% value of4100. Relating to breast
to 36%. It is clear that the agreement of the compounds under cancer; MCF7, MDA-MB-231/ATCC, T-47D and MDA-MB-468
investigation in the inhibition of renal cell lines could be cell lines possess convinced response to compounds 1a, 2a, 3b,
correlated to a similar inhibitory mechanism related to the 6b and 7a with GI% range of 46.0 – 4100. On the other hand,
common structural feature in ciprofloxacin core, while the ovarian IGROV1, OVCAR-3, OVCAR-5 and NCI/ADR-RES cell
selectivity of arylsulfonylpiperazinyl fluoroquinolones (1–7a,b) lines are receptive to compound 1a, 2a, 3b, 6b and 7a with GI%
(Tables 1 and 2, Figure S1, Supporting Information) over other range of 40.5 – 4100.
cell lines is probably caused by the differences in the hydrocarbon
skeleton around the core structure (ciprofloxacin scaffold).
Finally, compounds 1a, 2a, 3b, 6b and 7a were selected in
advanced assay against a panel of approximately 60 tumor cell
Moreover, among the 7-arenesulfonyl-piperazin-1-ylfluoroqui- lines at 10-fold dilution of five concentration (100, 10, 1, 0.1, and
nolones (1–7a,b), compounds 1a, 2a, 2b, 3b, 6a, 6b and 7a showed 0.01 mM) and the results were compared with 5-fluorouracil (5-
broad spectrum and significant inhibition for cancer cells (58 cell Flu), gefitinib (IressaTM) and erlotinib (TarcevaTM) as reference
lines; Tables 1 and 2, Figure S1, Supporting Information) and drugs (Tables 3 and 4; Figure S2, Supporting Information; http://
possessed a considerable cytotoxic activity against cell lines of dtp.nci.nih.gov/dtpstandard/dwindex/index.jsp). Based on the
leukemia (GI% ¼ 60.9 –4100), non-small cell lung (GI% ¼ 53.0 – cytotoxicity assays, three antitumor activity dose-response par-
4100), colon (GI% ¼ 52.3 – 4100), CNS (GI% ¼ 53.5 – 4100), ameters were calculated for tested molecules against each cell
melanoma (GI% ¼ 51.5 – 4100), ovarian (GI% ¼ 57.7 – 4100), lines: GI50, molar concentration of the compound that inhibits
renal (GI% ¼ 56.3 – 4100), prostate (GI% ¼ 68.3 – 98.4) and 50% net cell growth; TGI, molar concentration of the compound
breast (GI% ¼ 58.8 –4100) cancer cells. This potential inhibition leading to total inhibition; and LC50, molar concentration of the
at the mentioned concentration indicates a high potency for compounds leading to 50% net cell death. Furthermore mean
the compounds 1a, 2a, 2b, 3b, 6a, 6b and 7a with a strong lethal graph midpoints (MG_MID) were calculated for each of the
effect over cancer cells. parameters, giving an average activity parameter over all cell lines
Regarding the activity toward individual cell lines (Table 2); for the tested molecules.
compounds 1a, 2a, 3b, 6b and 7a showed selective activity
The screening data analysis indicated that compounds 1a, 2a,
against leukemia cell lines such as CCRF-CEM (GI % values of 3b, 6b and 7a possessed potent in vitro antitumor activity, with
4100, 97.2, 83.0, 62.6 and 87.9, respectively), HL-60 (GI % GI50 values across the 60 cell lines ranging from 1.22 to 5.37 mM
values of 91.0, 4100, 4100, 91.2 and 4100, respectively), and (Tables 3 and 4; Figure S2, Supporting Information). Mean GI50
PRMI-8226 (GI % values of 86.5, 90.9, 95.1, 77.6 and 93.5, of compounds 1a, 2a, 3b, 6b and 7a in comparison with
respectively). Non-small cell lung; NCI-H23 cell line proved to be 5-fluorouracil (5-Flu), gefitinib (IressaTM
)
and erlotinib
selectively sensitive to 1a, 2a, 3b, 6b and 7a with GI% values of (TarcevaTM) as standard antitumor drugs are given in Table 3
4100, 4100, 98.2, 42.6 and 99.9, respectively. In addition, and Figure S2, Supporting Information.
compounds 1a, 2a and 7a proved to have equal susceptibility to
Compounds 1a, 2a, 3b, 6b and 7a (Tables 3 and 4) exhibited
the HOP-92 cell line with GI% value of 4100. Concerning colon remarkable growth inhibitory activity pattern against leukemia
cancer; compounds 2a, 3b and 7a showed GI% values of 4100 (GI50 ¼ 2.89, 3.07, 3.02, 2.57 and 3.48 mM), non-small cell lung
with colon COLO 205 cancer cells. On the other hand, cancer (GI50 ¼ 2.87, 2.49, 3.07, 2.88 and 3.04 mM), colon cancer
compounds 1a, 2a, 3b, 6b and 7a verified sensitivity with GI% (GI50 ¼ 3.38, 3.15, 3.79, 3.46 and 3.39 mM), CNS (GI50 ¼ 3.57,
values of 92.1, 98.1, 99.7, 71.7 and 96.0 to colon HT-29 cancer 3.03, 3.68, 2.95 and 3.09 mM), breast cancer (GI50 ¼ 2.95, 2.51,

8
A. A.-M. Abdel-Aziz et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Table 2. Percentage of growth inhibition of the most active fluoroquinolones against individual cell lines.
% Growth Inhibition (GI %)
Subpanel tumor cell lines
1a
2a
2b
3b
6a
6b
7a
5-Flu
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
PRMI-8226
SR
4100
91.04
78.59
76.39
86.57
–
97.22
4100
89.06
98.2
90.93
4100
18.97
13.13
–
24.07
21.26
17.66
83.11
4100
83.06
96.56
95.14
4100
18.2
62.67
91.28
36.45
38.96
77.65
60.94
87.9
57.1
47.9
42.3
43.1
41.4
24.8
26.95
20.11
24.12
35.93
35.52
4100
84.58
91.59
93.53
97.85
Non-small cell lung cancer
A549/ATCC
HOP-62
NCI-H226
HOP-92
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
84.43
–
97.95
80.5
–
11.78
39.72
–
16.79
12.84
–
93.98
84.8
58.6
21.96
28.48
23.12
12.64
22.28
16.46
17.22
13.46
79.7
96.22
84.87
78.11
4100
99.94
4100
91.96
4100
34.2
47.8
69.5
50.6
39.0
59.5
13.0
58.0
42.73
28.19
53.06
42.63
25.29
60.54
32.68
39.86
4100
4100
54.08
94.08
91.61
86.87
4100
4100
4100
89.1
87.8
98.24
78.73
94.23
4100
4100
–
87.87
75.54
95.2
84.34
92.14
95.7
4100
69.37
93.63
92.42
98.13
90.6
11.94
4100
75.16
96.05
93.76
99.74
85.43
79.29
–
42.44
52.34
67.81
44.94
71.74
39.33
20.17
4100
68.42
94.55
88.9
96.04
88.28
77.19
40.2
4100
17.8
26.5
27.1
40.7
50.1
–
13.45
16.18
–
–
–
17.51
20.85
28.47
–
12.79
–
HT29
KM12
SW-620
76.04
83.65
CNS cancer
SF-268
SF-295
SF-539
SNB-19
77.28
96.06
78.3
79.39
58.16
87.97
75.44
4100
91.42
73.77
25.22
87.91
16.35
–
–
–
70.20
–
80.36
4100
88.63
75.33
86.8
16.29
51.60
20.08
10.12
29.22
16.65
43.33
66.75
53.5
33.73
63.38
80.32
83.61
92.32
82.56
71.16
85.92
85.77
59.0
69.1
4100
65.9
65.9
50.3
SNB-75
U251
87.56
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
80.12
76.25
81.1
4100
4100
84.12
87.23
4100
–
4100
4100
83.85
38.57
–
–
–
–
49.08
79.75
–
98.79
4100
81.47
87.01
4100
51.5
97.94
91.13
70.38
38.72
–
24.44
10.88
11.31
–
20.46
–
24.14
70.39
40.29
36.45
23.91
–
30.87
45.06
13.08
23.40
4100
4100
82.14
88.61
91.18
40.83
4100
85.98
68.78
30.4
58.2
–
36.6
95.5
–
33.7
19.5
39.7
74.9
56.19
55.42
73.4
70.99
78.52
14.58
90.63
71.75
62.33
48.09
87.42
31.3
92.5
92.11
–
92.08
94.16
90.66
98.3
–
70.7
20.62
10.76
–
11.41
18.51
15.18
–
64.31
–
86.31
4100
76.15
84.54
91.57
78.65
84.38
51.2
47.4
59.4
44.3
–
–
35.94
–
–
–
–
90.13
79.24
87.52
94.52
46.49
73.19
24.39
57.79
40.52
30.14
18.39
47.6
77.5
59.23
76.35
4100
71.35
56.36
86.6
88.39
46.4
48.92
81.27
4100
4100
95.25
72.06
89.73
87.63
4100
–
72.2
–
24.42
37.03
48.7
32.91
48.66
86.08
13.54
83.86
75.73
4100
4100
99.06
70.98
88.09
74.01
4100
48.7
4100
39.3
39.4
34.3
54.0
66.9
41.3
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
23.58
14.87
12.12
67.74
13.79
–
86.25
95.05
96.94
70.95
89.63
66.81
89.98
21.15
24.24
10.66
11.11
11.46
–
UO-31
40.04
66.90
Prostate cancer
PC-3
DU-145
74.74
68.32
98.4
88.44
18.91
–
83.79
82.81
38.94
–
32.08
28.56
92.18
81.12
58.2
35.5
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
85.71
58.88
81.1
87.41
58.92
–
10.75
74.46
–
4100
98.3
4100
82.38
–
–
13.68
26.38
65.11
86.55
63.36
–
4100
83.7
4100
–
46.0
15.37
–
22.67
37.05
34.85
85.4
76.08
–
4100
94.71
95.21
11.5
78.1
4100
37.8
56.7
–
16.33
–
26.26
25.94
19.73
T-47D
MDA-MB-468

DOI: 10.3109/14756366.2015.1069288
In silico studies of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines
9
Table 3. Average antitumor activity of compounds 1a, 2a, 3b, 6b and 7a against tumor cell lines from nine different organs at 10-fold dilution of five
concentrations; median growth inhibitory (GI50, mM), total growth inhibitory (TGI, mM) and median lethal (LC50, mM).
Subpanel tumor cell lines
NSC lung
cancer
Colon
cancer
CNS
cancer Melanoma
Ovarian
cancer
Renal
cancer
Prostate Breast
cancer
Compound
Activity Leukemia
cancer MG-MIDa
1a
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50
TGI
2.89
80.96
c
3.07
62.54
c
3.02
62.55
c
2.57
76.70
c
3.48
63.22
c
15.1
c
2.87
53.38
c
2.49
19.61
73.15
3.07
84.28
c
2.88
c
c
3.04
46.59
c
3.38
73.15
95.24
3.15
72.89
77.21
3.79
87.41
c
3.46
c
c
3.39
67.85
86.79
8.4
3.57
70.26
c
3.03
34.84
77.16
3.68
c
2.56
29.36
89.65
2.30
6.36
39.48
2.54
52.41
87.08
22.31
c
3.16
54.29
c
2.96
39.54
76.67
3.39
88.90
c
2.89
c
c
3.07
62.35
c
61.4
c
2.93
54.00
c
2.73
23.97
82.47
3.19
87.07
c
2.78
c
c
3.50
63.09
c
45.6
c
3.21
54.11
c
2.62
8.72
c
3.29
c
c
3.40
c
c
3.71
c
c
2.95
54.63
c
2.51
8.44
88.06
3.04
81.37
c
2.85
76.89
c
2.81
38.49
c
2.95
29.51
95.49
2.63
14.12
57.54
3.09
60.25
95.49
3.01
89.12
c
3.09
29.51
95.49
22.60
c
2a
3b
c
2.95
c
6b
c
c
7a
3.09
63.40
c
72.1
c
3.20
28.33
99.66
70.6
c
5-Flub,c
c
c
22.7
c
76.4
c
c
LC50
GI50
TGI
LC50
GI50
TGI
c
c
c
c
c
c
c
c
c
c
Gefitinibc (Iressa)
Erlotinibc (Tarceva)
3.54
12.64
39.64
23.66
96.57
100
7.79
28.67
65.05
12.09
73.76
97.71
7.01
31.16
66.38
51.68
100
8.13
21.98
49.2
16.98
82.10
100
5.27
14.24
36.11
25.34
80.09
93.98
6.62
28.31
70.79
5.50
76.66
97.06
2.66
16.15
42.22
2.46
42.59
89.15
1.65
22.53
75.05
20.90
100
100
7.80
24.34
65.47
24.72
70.53
96.57
3.24
19.3
49.3
7.29
64.3
95.1
LC50
100
aFull panel mean-graph midpoint (mM). c: Compounds showed values 4100 mM.
b5-Fluorouracil as reference drug.
chttp://dtp.nci.nih.gov/dtpstandard/dwindex/index.jsp.
3.04, 2.85 and 2.81 mM), ovarian cancer (GI50 ¼ 3.16, 2.96, 3.39, compounds contain norfloxacin scaffold is more active in
2,89 and 3.07 mM), melanoma cancer (GI50 ¼ 2.56, 2.30, 2.54, comparison with that contain ciprofloxacin scaffold such as
22.31 and 3.20 mM), prostate cancer (GI50 ¼ 3.21, 2.62, 3.29, 3.40 compounds 1a and 1b (MGI% ¼ 75.5 and 3.5, respectively).
and 3.71 mM) and renal cancer (GI50 ¼ 2.93, 2.73, 3.19, 2.78 and
3.50 mM), respectively. Compounds 1a, 2a, 3b, 6b and 7a (mean
In silico studies
GI50; 2.63–3.09 mM) are nearly seven-fold more potent compared
with the positive control 5-Flu (mean GI50; 22.60 mM). More Lipinski’s rule of five (the effect of lipophilic and steric
interestingly, compounds 1a, 2a, 3b, 6b and 7a have an almost parameters)31–33
.
As a part of our study; the compliance of
antitumor activity similar to gefitinib (mean GI50; 3.24 mM) and compounds to the Lipinski’s rule of five was evaluated31. In
are nearly 2-fold more potent compared to erlotinib (mean GI50; addition, the polar surface area (PSA) of the compounds was also
7.29 mM).
calculated (Table 5), since it is another key property that has been
The structure correlation study revealed that (1) potent linked to drug bioavailability, where passively absorbed com-
2
˚
antitumor activity of tested compounds depended on the presence pounds with a PSA4140 A are thought to have low oral
of a combination of quinolone scaffold and arylsulfonyl fragment bioavailability32. The results disclosed in Table 5 show that all of
in one molecule; such as arylsulfonylquinolones (1–7ab) (7.3– the synthesized compounds comply with these rules. Hence;
94.1 MGI%) were more active in comparison with alkylated theoretically, all of these compounds should present good passive
quinolones 8b, 10b and 11b (ꢁ4.1 MGI%); (2) attachment of a oral absorption and differences in their bioactivity cannot be
halogen atom to arylsulfonyl derivative such as compounds 1, 2ab attributed to this property.
allowed sharp increase of activity (17.3–94.1 MGI%) in com-
The introduction of electron withdrawal/donating groups
parison with ciprofloxacin scaffold (ꢁ1.4 MGI%); (3) introduc- incorporating the arylsulfonyl fragment and the variation of the
tion of a methoxy group at the arylsulfonyl fragment such as substituents on the N-piperazine moiety of the ciprofloxacin and
compounds 4a, 4b and 5b led to the decrease of activity; (4) the norfloxacin scaffolds have allowed us to evaluate the influence of
substituent in the arylsulfonyl fragment proved crucial and lipophilicity and steric parameters at the pharmacophoric part of
manipulated the antitumor activity; accordingly the introduction the molecules. Table 5 gathers physicochemical properties of
of an electron-withdrawing moiety, such as chloro fragment some selected compounds such as ClogP (lipophilic factors),
(compounds 1a, 2a, 2b and 3b), improved the antitumor activity molar refractometry and polar surface area (steric factors) for
in comparison with electron-donor moiety, such as methoxy group each compound, determined by using iLab2 online program
(compounds 4a, 4b and 5b), and methyl group (compounds 6a (https://ilab.acdlabs.com/iLab2/index.php).
Although
molar
and 6b); (5) replacement of the steric bulky trimethylbenzene- refractometry (MR) does not exert a significant effect on activity
sulfonyl substituent (compounds 6a and 6b; MGI% ¼ 18–43) with in tested compounds, an increase in potency was observed in
1-naphthylsulfonyl moiety (compound 7a; MGI% ¼ 93) led to compounds 1a, 2a, 3b, 6b and 7a with MR values of 120–
improvement of the antitumor activity; (6) it is clear also that the 132 cm3/mol. Regarding lipophilicity (logP); from the data

10 A. A.-M. Abdel-Aziz et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Table 4. Influence of compounds 1a, 2a, 3b, 6b and 7a on the growth of
the individual tumor cell lines; median growth inhibitory (GI50, mM).
gathered in Table 5, there is a clear influence of lipophilicity on
antitumor activity compared to polar surface area for all
compounds. The optimal lipophilicity for the most active
compounds was found to lie in the range of 2.14ꢃ2.87 (Table 5).
GI50 (mM)
Subpanel tumor cell lines
1a
2a
3b
6b
7a
ADME-Tox evaluation34,35
.
To estimate the prospect of designed
Leukemia
CCRF-CEM
HL-60(TB)
K-562
compounds as antitumor agents compared with the reported
antitumor agents ciprofloxacin (b) and 5-Flu; their drug-likeness
were calculated according to absorption, distribution, metabolism,
elimination, toxicity (ADME-T) program, and defined human
intestinal absorption (HIA) model34,35. It was predicted that the
examined compounds could be transported across the intestinal
epithelium, and they can cross the blood–brain barrier and are of
medium aqueous soluble. The values of HIA, BBB crossing and
solubility prediction for all compounds are presented in Table 5
using iLab2 online program (https://ilab.acdlabs.com/iLab2/
index.php). In general, all compounds presented some advantages
when compared to ciprofloxacin and 5-Flu. No marked differ-
ences, in human oral bioavailability (470%), human intestinal
absorption (100%), human jejunum permeability and health
effects in rodent toxicity profiles, were observed among the
compounds. However, the absorption related parameters call for
attention, since the promising compounds were calculated to be at
least as soluble as the reported compounds, and are predicted to
have oral bioavailability and absorption significantly higher than
that of the reported antitumor agents ciprofloxacin (b) and 5-Flu.
Accordingly; it can be deduced from these results that the
pharmacokinetic profile of the designed compounds is affected
and modified by the presence of arylsulfonyl moiety connected to
7-piperazinyl moiety of fluoroquinolone scaffolds.
2.93
2.18
2.91
3.83
3.08
3.03
2.22
4.00
3.55
2.55
2.61
2.04
3.92
4.00
2.53
2.80
NT
NT
NT
3.67
2.58
3.93
4.46
2.79
MOLT-4
PRMI-8226
Non-small cell lung cancer
A549/ATCC
HOP-62
NCI-H226
HOP-92
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
2.34
2.81
4.47
3.02
NT
2.67
NT
2.89
3.08
2.60
1.22
2.33
2.86
3.13
1.81
3.08
4.77
3.58
1.54
2.90
3.24
3.66
1.99
2.23
3.65
1.23
1.52
3.41
5.19
3.04
2.80
2.87
4.6
3.71
1.25
3.13
2.6
3.16
1.73
3.43
2.78
2.62
4.10
3.18
3.62
2.64
4.07
3.43
2.44
2.29
3.56
2.82
3.63
3.42
3.91
3.50
4.47
3.86
3.30
3.77
3.62
4.05
3.40
5.37
2.98
2.55
3.15
3.38
3.40
2.02
4.26
3.88
3.15
3.95
2.96
3.57
HT29
KM12
SW-620
CNS cancer
SF-268
SF-539
SNB-19
SNB-75
U251
3.85
4.12
3.97
2.60
3.10
3.86
1.92
3.07
NT
4.77
3.39
3.25
NT
3.96
3.14
2.56
2.23
2.89
4.39
2.43
3.47
1.89
3.29
3.30
3.33
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
Toxicities, drug score and drug-likeness profiles. Currently
there are many approaches that assess a compound drug-likeness
based on topological descriptors, fingerprints of molecular drug-
2.40
1.99
2.85
2.47
2.57
4.88
1.80
2.16
2.02
1.48
1.95
3.24
1.97
2.75
3.21
1.87
2.65
1.61
1.44
2.30
4.06
2.97
3.16
2.67
2.04
2.48
1.75
1.42
NT
4.69
NT
NT
NT
3.03
4100
2.43
1.63
2.83
4.37
3.13
3.55
6.09
2.24
2.72
2.25
likeness structure keys or other properties36,37 In the Osiris
.
program (http://www.organic-chemistry.org/prog/peo) the occur-
rence frequency of each fragment is determined within the
collection created by shredding 3300 traded drugs as well as
15 000 commercially available chemicals (Fluka) yielding a
complete list of all available fragments36,37. In this work, we
used the Osiris program for calculating the fragment based drug-
likeness of the active compounds also comparing them with
voreloxin, ciprofloxacin, levofloxacin, 5-Flu, erlotinib and
gefitinib (Figure 2). Interestingly, the derivatives 1a, 2a, 2b, 3b,
5b and 7a presented better drug-likeness values (from 2.53 to
4.72) than ciprofloxacin, 5-Flu, erlotinib and gefitinib (2.07,
ꢁ4.5, ꢁ6.73 and ꢁ2.62 respectively) and possessed similar
results to levofloxacin and voreloxin (5.77 and 4.71 respectively).
In this study we also verified the drug-score37 as the theoretical
data showed that compounds 1a, 2a, 2b, 3b, 5b, 6a, 6b and 7a
presented values once again higher than 5-Flu, erlotinib and
gefitinib (Figure 2). Moreover, we used the Osiris program to
predict the overall toxicity of the most active derivatives as it may
point to the presence of some fragments generally responsible for
the irritant, mutagenic, tumorigenic, or reproductive effects in
these molecules36,37. Interestingly, all of the active derivatives
presented a low in silico toxicity risk profile, better than 5-Flu and
similar to voreloxin, ciprofloxacin, levofloxacin, erlotinib and
gefitinib (Figure 2). These theoretical data reinforced the
cytotoxicity experimental data described in this work pointing
these compounds as lead compounds for further study.
3.14
3.51
2.20
4.25
2.80
2.98
3.28
2.33
2.54
2.58
3.43
3.30
3.28
3.00
2.73
3.90
2.28
4.14
3.33
4.01
NT
2.66
3.17
2.37
NT
2.87
3.29
3.02
2.47
3.69
2.60
3.38
3.09
3.20
3.11
3.77
1.81
3.09
2.75
3.32
2.92
3.73
2.07
3.19
2.99
2.62
2.01
2.08
2.77
4.02
2.20
4.02
2.91
3.10
2.71
2.98
2.97
4.31
2.58
4.08
2.37
2.46
1.80
2.41
2.61
4.57
1.96
4.50
2.74
2.88
2.33
3.19
2.59
7.60
2.21
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
2.31
4.11
2.43
2.81
2.46
4.31
NT
3.40
2.66
3.97
2.97
2.51
4.52
2.44
3.20
2.10
2.95
1.88
2.53
2.64
3.11
1.98
2.84
2.14
4.26
2.98
3.77
2.25
2.59
2.03
2.40
3.34
4.81
1.97
2.72
1.91
2.68
3.7
3.35
2.52
Conclusion
NT: Not tested.
Sixteen different quinolones based on ciprofloxacin and nor-
floxacin scaffolds were evaluated for their antitumor activity,

DOI: 10.3109/14756366.2015.1069288
In silico studies of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines 11

12 A. A.-M. Abdel-Aziz et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
Toxicity effects
Compd No.
M
T
I
R
1a
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
2a
2b
3b
5b
6a
6b
7a
Ciprofloxacin
Levofloxacin
Voreloxin
5-Flu
Low
High
Low
Low
Low
High
Low
Low
Low
High
Low
Low
Low
High
Low
Low
Erlotinib
Gefitinib
Drug Score
Drug-Likeness
1
8
0.87
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0.82
5.77
6
0.73
4.72
4.71
4.36
4.22
0.62
3.72
3.56
0.58
4
2
0.52
0.51
2.53
0.46 0.46
2.07
0.35
0.29
0.26
0.26
0
0.06
−2
−4
−6
−8
−2.62
−4.5
−6.49
−6.66
−6.73
Figure 2. In silico toxicity risks (upper panel), drug-Likeness (lower left panel) and drug-Score (lower right panel) of the reported and the most active
antitumor fluoroquinolone derivatives compared with reference drugs 5-Flu, Erlotinib and Gefitinib (M, mutagenic; T, tumorigenic; I, irritant; R,
reproductive).
O
N
O
O
N
O
O
N
O
O
N
O
O
N
O
F
F
F
F
F
OH
Cl
OH
Cl
OH
OH
OH
N
N
N
N
N
O
S
O
S
O
S
O
S
O
S
O
N
O
N
O
N
O
N
O
N
Cl
Cl
Cl
1a
2a
3b
6b
7a
(mean GI ; 2.95 µM)
(mean GI ; 2.63 µM)
(mean GI ; 3.09 µM)
(mean GI ; 3.01 µM)
(mean GI ; 3.09 µM)
50
50
50
50
50
(MGI% = 94.1)
(MGI% = 89.2)
(MGI% = 43.8)
(MGI% = 93.7)
(MGI% = 75.5)
F
O
N
F
O
HN
N
HN
N
Cl
HN
O
O
O
O
N
O
N
O
H
N
O
TM
TM
5-Fluorouracil
(mean GI ; 22.60 µM)
Erlotinib (Tarceva
)
Gefitinib (Iressa
)
(mean GI ; 7.29 µM)
(mean GI ; 3.24 µM)
50
50
50
Figure 3. Outcomes of GI50 (mM) of compounds 1a, 2a, 3b, 6b, 7a and reference drugs 5-Flu, Erlotinib and Gefitinib using tumor cell lines from nine
different.

DOI: 10.3109/14756366.2015.1069288
In silico studies of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines 13
10. Blondeau JM. Fluoroquinolones: mechanism of action, classifica-
tion, and development of resistance. Surv Ophtalmol 2004;49:
S73–8.
11. Anderson VE, Osheroff N. Type II topoisomerases as targets for
quinolone antibacterials: turning Dr. Jekyll into Mr. Hyde. Curr
Pharm Des 2001;7:337–53.
12. Alovero F, Pan XS, Morris J, et al. Engineering the specificity of
antibacterial fluoroquinolones: benzenesulfonamide modifications
at C-7 of ciprofloxacin change its primary target in Streptococcus
pneumoniae from topoisomerase IV to gyrase. Antimicrob Agents
Chemother 2000;44:320–5.
13. Pan XS, Hamlyn P, Talens-Visconti R, et al. Small-colony mutants
of Staphylococcus aureus allow selection of gyrase-mediated
resistance to dual-target fluoroquinolones. Antimicrob Agents
Chemother 2002;46:2498–506.
where most of the tested compounds exhibited significant
antitumor activity. Compounds 1a, 2a, 3b, 6b and 7a were
possessed the most potent broad-spectrum antitumor activities
(mean GI50; 2.63-3.09 mM) and were nearly seven-fold (Figure 3)
more potent compared with the positive control 5-Flu (mean GI50;
22.60 mM). More interestingly, compounds 1a, 2a, 3b, 6b and 7a
(Figure 3) have an almost antitumor activity similar to gefitinib
(mean GI50; 3.24 mM) and are nearly 2-fold more potent compared
to erlotinib (mean GI50; 7.29 mM). The results of this study
demonstrated that the electronic effect in the arylsulfonyl
fragment proved crucial and manipulated the antitumor activity.
The designed 7-substituted 1-piperazinyl fluoroquinolones
are compatible with Lipinski’s rule of five (molecular weight,
ClogP, hydrogen bond-donating and accepting capabilities).
In vitro antitumor activity, together with in silico studies,
revealed that compounds 1a, 2a, 3b, 6b and 7a could be
considered as promising leads for further development of more
potent antitumor agents.
14. Heddle J, Maxwell A. Quinolone-binding pocket of DNA
gyrase: role of GyrB. Antimicrob Agents Chemother 2002;46:
1805–15.
15. Fang KC, Chen YL, Sheu JY, et al. Synthesis, antibacterial, and
cytotoxic evaluation of certain 7-substituted norfloxacin derivatives.
J Med Chem 2000;43:3809–12.
´
16. Azema J, Guidetti B, Dewelle J, et al. 7-((4-Substituted)piperazin-1-
yl) derivatives of ciprofloxacin: synthesis and in vitro biological
evaluation as potential antitumor agents. Bioorg Med Chem Lett
2009;17:5396–407.
Acknowledgements
The authors would like to express their gratitude and thanks to the
National Cancer Institute (NCI), Bethesda Maryland, USA, http://
dtp.cancer.gov/ for doing the antitumor testing of the new compounds.
17. Yamashita Y, Ashizawa T, Morimoto M, et al. Antitumor quinolones
with mammalian topoisomerase II mediated DNA cleavage activity.
Cancer Res 1992;52:2818–22.
18. Paul M, Gafter-Gvili A, Fraser A, Leibovici L. The anti-cancer
effects of quinolone antibiotics. Eur J Clin Microbiol Infect Dis
2007;26:825–31.
19. Herold C, Ocker M, Ganslmayer M, et al. Ciprofloxacin induces
apoptosis and inhibits proliferation of human colorectal carcinoma
cells. Br J Cancer 2002;86:443–8.
20. El-Rayes BF, Grignon R, Aslam N, et al. Ciprofloxacin inhibits cell
growth and synergises the effect of etoposide in hormone resistant
prostate cancer cells. Int J Oncol 2002;21:207–11.
Declaration of interest
The authors extend their appreciation to the Deanship of Scientific
Research at King Saud University for funding the work through the
research group project No. RG-1435-046.
21. Yamakuchi M, Nakata M, Kawahara K, et al. New quinolones,
ofloxacin and levofloxacin, inhibit telomerase activity in transitional
cell carcinoma cell lines. Cancer Lett 1997;119:213–19.
22. Advani RH, Hurwitz HI, Gordon MS, et al. Voreloxin, a first-in-
class anticancer quinolone derivative, in relapsed/refractory solid
tumors: a report on two dosing schedules. Clin Cancer Res 2010;16:
2167–75.
23. Hawtin RE, Stockett DE, Byl JA, et al. Voreloxin is an anticancer
quinolone derivative that intercalates DNA and poisons topoisom-
erase II. PLoS One 2010;5:e10186.
24. Abdel-Aziz AA-M, El-Azab AS, Alanazi AM, et al. 7-substituted
piperazin-1-yl fluoroquinolone compounds, their use in the treat-
ment of cancer, pharmaceutical compositions and a method for
preparation. EPO patent application pending No.: EP 14181731.2,
filing date: August 21, 2014.
25. (a) Grever MR, Schepartz SA, Chabner BA. The National Cancer
Institute: cancer drug discovery and development program. Semin
Oncol 1992;19:622–38.
References
´
1. Avendan˜o C, Menendez JC. Medicinal chemistry of anticancer
drugs. Amsterdam, Netherlands: Elsevier; 2008.
2. Harris CC, Hollstein M. Clinical implications of the p53 tumor-
suppressor gene. N Engl J Med 1993;329:1318–27.
3. Varmus H. The new era in cancer research. Science 2006;312:
1162–5.
4. Eckhardt S. Recent progress in the development of anticancer
agents. Curr Med Chem 2002;2:419–39.
5. Hooper DC, Rubinstein E. Quinolone antimicrobial agents.
Washington, USA: ASM Press American Society for
Microbiology; 2003.
6. (a) Abdel-Aziz AA-M, Asiri YA, Al-Agamy MHM. Design,
synthesis and antibacterial activity of fluoroquinolones containing
bulky arenesulfonyl fragment: 2D-QSAR and docking study. Eur J
Med Chem 2011;46:5487–97.
(b) Monks A, Scudiero D, Skehan PJ. Feasibility of a high-flux
anticancer drug screen using a diverse panel of cultured human
tumor cell lines. Natl Cancer Inst 1991;83:757–66.
(b) Nieto MJ, Alovero FL, Manzo RH, Mazzieri MR. A new class of
fluoroquinolones: benzenesulfonamidefluoroquinolones (BSFQs),
antibacterial activity and SAR studies. Eur
1999;34:209–14.
J Med Chem
(c) Boyd MR, Paull KD. Some practical considerations and
applications of the national cancer institute in vitro anticancer
drug discovery screen. Drug Dev Res 1995;34:91–109.
26. Le VM, Wang JJ, Yuan M, et al. An investigation of antitumor
efficiency of novel sustained and targeted 5-fluorouracil
Nanoparticles. Eur J Med Chem 2015;92:882–9.
27. Barlesi F, Tchouhadjian C, Doddoli C, et al. Gefitinib (ZD1839,
Iressa) in non-small-cell lung cancer: a review of clinical trials from
a daily practice perspective. Fundam Clin Pharmacol 2005;19:
385–93.
(c) Nieto MJ, Alovero FL, Manzo RH, Mazzieri MR.
Benzenesulfonamide analogs of fluoroquinolones. Antibacterial
activity and QSAR studies. Eur J Med Chem 2005;40:361–9.
(d) Alovero F, Barnes A, Nieto M, et al. Comparative study
of new benzenesulphonamide fluoroquinolones structurally related
to ciprofloxacin against selected ciprofloxacin-susceptible and
-resistant Gram-positive cocci.
2001;48:709–12.
J
Antimicrob Chemother
7. Chen YL, Fang KC, Sheu JY, Hsu SL, Tzeng CC. Synthesis and
antibacterial evaluation of certain quinolone derivatives. J Med
Chem 2001;44:2374–7.
28. Arteaga CL, Johnson DH. Tyrosine kinase inhibitors-ZD1839
(Iressa). Curr Opin Oncol 2001;13:491–8.
29. Baker AJ, Gibson KH, Grundy W, et al. Studies leading to
the identification of ZD1839 (IRESSA): an orally active, select-
ive epidermal growth factor receptor tyrosine kinase inhibitor
targeted to the treatment of cancer. Bioorg Med Chem Lett 2001;11:
1911–14.
8. Mirzaei M, Foroumadi A. Synthesis and in-vitro antibacterial
activity of N-piperazinyl quinolone derivatives with a 2-thienyl
group. Pharm Pharm Commun 2000;6:351–4.
9. Hooper DC. Mechanisms of action of antimicrobials: focus on
fluoroquinolones. Clin Infect Dis 2001;32:S9–15.

14 A. A.-M. Abdel-Aziz et al.
J Enzyme Inhib Med Chem, Early Online: 1–14
30. Ganjoo KN, Wakelee H. Review of erlotinib in the treatment of 34. Moda TL, Torres LG, Carrara AE, Andricopulo AD. PK/DB:
advanced non-small cell lung cancer. BiolTargets Therapy 2007;1: database for pharmacokinetic properties and predictive in silico
335–46. ADME models. Bioinformatics 2008;24:2270–1.
31. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental 35. El-Deeb IM, Bayoumi SM, El-Sherbeny MA, Abdel-Aziz AA-M.
and computational approaches to estimate solubility and permeabil-
ity in drug discovery and development settings. Adv Drug Deliv Rev
2001;46:3–26.
Synthesis and antitumor evaluation of novel cyclic arylsulfonylureas:
ADME-T and pharmacophore prediction. Eur J Med Chem 2010;45:
2516–30.
32. Clark DE, Pickett SD. Computational methods for the prediction of 36. Tetko IV. Computing chemistry on the web. Drug Discov Today
‘drug-likeness’. Drug Discov Today 2000;5:49–58. 2005;10:1497–500.
33. El-Azab AS, Al-Omar MA, Abdel-Aziz AA-M, et al. Design, 37. Ghose AK, Viswanadhan VN, Wendoloski JJ. A knowledge-based
synthesis and biological evaluation of novel quinazoline derivatives
as potential antitumor agents: molecular docking study. Eur J Med
Chem 2010;45:4188–98.
approach in designing combinatorial or medicinal chemistry libraries
for drug discovery. 1. A qualitative and quantitative characterization
of known drug databases. J Comb Chem 1999;1:55–68.
Supplementary material available online
Supplementary Figures S1 and S2