Anti-proliferative activity, molecular modeling studies and interaction with calf thymus DNA of novel ciprofloxacin analogues

Article abstract of DOI:10.1007/s12039-018-1528-y

Abstract: In our pursuit to expand new potential anticancer leads, a series of eighteen novel 1-cyclopropyl-6-fluoro-4-oxo-7-(4-substituted piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid analogues have been synthesized, characterized and evaluated anti-proliferative activity against five human cancer cell lines such as A549 (lung cancer), Mia Paca (pancreatic cancer), HeLa (cervical cancer), MDA MB-231 (breast cancer), MCF-7 (breast cancer) and normal embryonic kidney?cell line (HEK) were carried out using MTT assay. Few of the synthesized analogues exhibited potent anticancer activity against the cancer cell lines at a lower concentration. The synthesized compounds showed the less toxic effect on normal human embryonic kidney?cell line (HEK) compared with doxorubicin. Noticeably, compound 3o exhibited potent activity against all five cancer cell lines compared with ciprofloxacin. Further study exposed that compound 3o could competently intercalate into calf thymus DNA to form 3o-DNA complex which might block DNA replication to apply anti-proliferative activity. Docking simulation studies supported by molecular interactions with DNA type II topoisomerase. These derivates can become lead structures for the development of potential anticancer drugs. Graphical Abstract: Eighteen CP analogues were synthesized and evaluated for anti-proliferative activity. The interactions with DNA topoisomerase II were supported by molecular docking studies. 3o showed promising anticancer activity than CP against MCF7 cell line and interaction with calf thymus DNA was studied by fluorescence spectroscopy.[Figure not available: see fulltext.].

Full text of DOI:10.1007/s12039-018-1528-y

J. Chem. Sci. (2018) 130:121
© Indian Academy of Sciences
https://doi.org/10.1007/s12039-018-1528-y
REGULAR ARTICLE
Anti-proliferative activity, molecular modeling studies and
interaction with calf thymus DNA of novel ciprofloxacin analogues
NARVA SURESHa, AMAROJU SURESHa, SURESH YERRAMSETTYb,
MANIKA PAL BHADRAb, MALLIKA ALVALAc and
KONDAPALLI VENKATA GOWRI CHANDRA SEKHARa,∗
aDepartment of Chemistry, Birla Institute of Technology and Science-Pilani, Hyderabad Campus, Jawahar
Nagar, Hyderabad, Telangana 500 078, India
bCentre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, Telangana
500 076, India
cNational Institute of Pharmaceutical Education and Research-Hyderabad, Hyderabad, Telangana 500 037, India
E-mail: kvgc@hyderabad.bits-pilani.ac.in; kvgcs@yahoo.com
MS received 17 April 2018; revised 18 July 2018; accepted 18 July 2018
Abstract. In our pursuit to expand new potential anticancer leads, a series of eighteen novel 1-cyclopropyl-
6-fluoro-4-oxo-7-(4-substituted piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid analogues have been
synthesized, characterized and evaluated anti-proliferative activity against five human cancer cell lines such as
A549 (lung cancer), Mia Paca (pancreatic cancer), HeLa (cervical cancer), MDA MB-231 (breast cancer), MCF-
7 (breast cancer) and normal embryonic kidney cell line (HEK) were carried out using MTT assay. Few of the
synthesized analogues exhibited potent anticancer activity against the cancer cell lines at a lower concentration.
The synthesized compounds showed the less toxic effect on normal human embryonic kidney cell line (HEK)
compared with doxorubicin. Noticeably, compound 3o exhibited potent activity against all five cancer cell
lines compared with ciprofloxacin. Further study exposed that compound 3o could competently intercalate into
calf thymus DNA to form 3o-DNA complex which might block DNA replication to apply anti-proliferative
activity. Docking simulation studies supported by molecular interactions with DNA type II topoisomerase.
These derivates can become lead structures for the development of potential anticancer drugs.
Keywords. Ciprofloxacin; anti-proliferative activity; docking studies; DNA binding.
1. Introduction
cancer cell lines such as prostate cancer cell lines
(HRPC and PC-3),4 transitional cell carcinoma cell lines
(MBT-2 and T24),5 colon carcinoma cell lines (CC-
531, SW-403 and HT-29) and lung cancer cell lines
(A549).6,7
Also, these are known to inhibit proliferation of jurkat
cells without any symptoms of cell death through inhibi-
tion of mitosis.8 CP-induced significant morphological
alterations in non-small-cell lung cancer cell line (NCI-
H460),9 and exhibited a cytotoxic effect against A549
cells (lung cancer),10 ovarian cancer cell line (CHO
AA8) and murine glioma cell line (GL26).11,12 CP after
oral administration might reach higher concentration
in urine than in serum and exhibit potential anticancer
Cancer is a group of diseases; it can cause different signs
in the human body where it is located. In recent times
it has been declared as the second reason for death in
the United States.1 Fluoroquinolones (FQs) represent
a family of broad-spectrum antibacterial agents, which
inhibit the bacterial DNA gyrase and type II DNA topoi-
somerase in mammalian cells.
Transitional cell carcinoma growth was inhibited by
FQ derivatives like ofloxacin and levofloxacin.2 In par-
ticular, ciprofloxacin (CP) had low side effects, one of
the broad-spectrum FQ antibiotics and known to exhibit
antiproliferative,3 and apoptotic activities in numerous
*
For correspondence
Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12039-018-1528-y) contains
supplementary material, which is available to authorized users.
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121 Page 2 of 11
J. Chem. Sci. (2018) 130:121
activity against bladder cancer cell lines (HTB9) and spectral studies were performed on a spectrophotometer
(JASCO model V-650). The fluorescence spectra performed
also known to exhibit cell cycle arrest at the S/G2-M
checkpoints.13 CP prevents topoisomerase II, which
leads to cell death by apoptosis in malignant cells but
not in normal cells.14
1
on a spectrofluorometer (JASCO model FP-6300). H and
13C NMR spectra were recorded on Bruker 400 (400 MHz
1
for H, 100 MHz for 13C), in CDCl3. Chemical shifts have
been expressed in parts per million (δ) relative to tetramethyl-
silane (δ = 0.0) as aninternal standard and couplingconstants
(J) in Hertz. Low-resolution mass spectra (ESI-MS) were
recorded on Shimadzu.
FQ derivatives possess anticancer activity; further,
several reports cited that introduction of a substituent on
N-4 piperazinyl moiety of FQ, alters the physicochemi-
cal properties and enhances the activity.15–22 Lipophilic-
ity of camptothecin was enhanced due to C-7 substitu-
tion and it is lead to the discovery of phase II clinical
trial agent gimatecan.23 Lipophilicity of the substituent
in bis-quinolinium compounds improved their activity
against colon cancer cell line (HT-29).24 Based on these
studies, we recently reported 1-cyclopropyl-6-fluoro-
2.2 Synthesis
2.2a Synthesis of 2-chloro-N-(substituted phenyl) ace-
tamide (2a–r): 2-Chloroacetyl chloride (24 mmol) was
slowly added dropwise to a mixture of various anilines
(20 mmol) and Et3N (24 mmol) in anhydrous CH2Cl2
4-oxo-7-(4-substituted piperazine-1-yl)-1,4-dihydroqui (20 mL) at 0 ◦C. The reaction mixture was warmed to room
temperature and stirred for an additional 20 h. After the sol-
vent was removed under reduced pressure, the residue was
washed with ice water, and the precipitate was separated
by filtration. The crude product was purified by crystalliza-
tion using a mixture of ether/hexane (2a–r). Spectral data
of 2-chloro-N-(4-chlorophenyl) acetamide (2a): 1H NMR
noline-3-carboxylic acid derivatives as antiproliferative
agents.25
These fruitful anticancer results have attracted us
to explore various CP derivatives. Pigeon et al., syn-
thesized aniline or acetanilide hooked 2-ferrocenyl-
1,1-diphenyl-but-l-ene derivatives and demonstrated
that aniline or acetanilide group enhanced the anti-
cancer activity of the molecule when evaluated against
breast cancer cells.26 Hence, we envisaged synthesiz-
ing C7-piperazinyl CP acetanilide hybrids anticipating
enhanced physicochemical properties of CP and/or syn-
ergistic effect through combining CP and acetanilide in
one compact structure. In this study, we concerted on the
synthesis, anti-proliferative evaluation and DNA bind-
(400 MHz, CDCl3) δ 4.26 (s, 2H), 7.47 (d, 2H, J
=
8.7 Hz), 7.72 (d, 2H J = 8.7 Hz), 9.05 (s, 1H), 13C NMR
(100.61 MHz, CDCl3) 165.46, 136.43, 133. 129.12, 120.42,
49.16. ESI-MS (m/z): calcd. for C8H7Cl2NO 202.99, found
203.23[M + H]+.
2.2b Synthesis of title compounds (3a–r): To a solu-
tion of CP (0.6036 mmol) in dry DMF (2 mL), triethylamine
(1.8108 mmol) and potassium iodide (0.0603 mmol) were
ing. The synthesized compounds (3a–r) were evaluated added at RT under N2 atmosphere. To the resultant mixture,
2a (0.6036 mmol) was added and heated at 125 ◦C. After
for their in vitro anticancer activity on five human can-
the reaction was complete, as indicated by TLC, DMF was
cer cell lines. There is a curiosity in discovering the
evaporated in vacuo. The obtained residue was diluted with
binding of molecules with DNA for the rational design
20 mL of water. The compound was extracted with CH2Cl2
and construction of efficient drugs. In the current study,
we evaluated the DNA-binding interactions of newly
synthesized CP derivative by using fluorescence spec-
troscopic technique.
(3 × 5 mL). The organic layers were collected, washed with
saturated brine solution, dried over anhydrous Na2SO4 and
concentrated in vacuo. The resultant crude was purified by
column chromatography [CH2Cl2/MeOH (1–10%)] to get
the title compounds (3a–r).
2.2b1 7-(4-(2-(4-Chlorophenylamino)-2-oxoethyl)piperazin-
1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylicacid (3a) IR (KBr): ∼3465 cm−1(O-H stretch),
∼3285 cm−1 (N-H stretch of amide), ∼2970 cm−1 (alkene
2. Experimental
2.1 Materials and methods
Ciprofloxacin, CtDNA and dried solvents were purchased and aromatic C-H stretch), ∼2890 cm−1 (cyclopropyl C-H
from Sigma Aldrich, USA and used as received. All reactions stretch), ∼1725 cm−1 (C=O stretch of acid carbonyl group),
were monitored by analytical Thin Layer Chromatography ∼1670 cm−1 (C=O stretch of amide), ∼1590 cm−1 (N-H
(TLC) performed on E-Merck 0.25 mm pre-coated silica gel bending of amide), ∼1420 cm−1 (C-O stretch of carbonyl
glass plates (60 F254). Visualization of the spots on TLC group), ∼1285 cm−1 (O-H bending), ∼1035 cm−1 (C-
plates was achieved by exposure to UV light (254 nm). Col- F stretch), ∼820 cm−1 (C-H out-of-plane bending of para
umn chromatography was performed using silica gel (Acme, disubstituted benzene), ∼770 cm−1 (C- Cl stretch). 1H NMR
100–200 mesh). Melting points were determined using Stuart (400 MHz, CDCl3) δ 1.22 (t, 2H, J = 7.2 Hz), 1.28 (t,
SMP30 system and are uncorrected. IR spectra were recorded 2H, J = 6.7 Hz), 2.89 (m, 4H), 3.27 (s, 2H), 3.44 (m,
as KBr pellets on Jasco FTIR-4200 spectrometer. The UV 4H), 3.56 (tt, 1H, J = 7.0 Hz, J = 4.0 Hz), 7.29–7.31

J. Chem. Sci. (2018) 130:121
Page 3 of 11 121
(d, 2H, J = 8.7 Hz), 7.37–7.39 (d, 1H, JH−F = 7.0 Hz), 121.91, 120.42, 119.91 (d, JC−F = 8.1 Hz), 112.97 (d,
7.54–7.56 (d, 2H, J = 8.7 Hz), 7.99–8.02 (d, 1H, JH−F
=
JC−F = 24.14 Hz), 107.12, 104.89 (d, JC−F = 3.7 Hz),
12.7 Hz), 8.74 (s, 1H), 9.05 (s, 1H), 14.97 (s, 1H). 13C NMR 60.67, 51.12, 49.16, 35.58, 8.12. ESI-MS (m/z): calcd. for
(100.61 MHz, CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56, C25H24ClFN4O4 498.14, found 499.32 [M + H]+.
166.43, 153.21 (d, JC−F = 249.3 Hz), 147.23, 145.12 (d,
JC−F = 10.3 Hz), 138.95, 136.61, 133.31, 129.62, 120.42,
119.91 (d, JC−F = 8.1 Hz), 111.97 (d, JC−F = 24.14 Hz),
2.2b41-Cyclopropyl-6-fluoro-7-(4-(2-(3-methoxypheny
107.12, 104.89 (d, JC−F = 3.7 Hz), 60.67, 51.12, 49.16,
lamino)-2-oxoethyl)piperazin-1-yl)-4-oxo-1,4-dihydroq
uinoline-3-carboxylicacid (3d) IR (KBr): ∼3470 cm−1
35.58, 8.12. ESI-MS (m/z): calcd. for C25H24ClFN4O4
498.14, found 499.23 [M + H]+.
(O-H stretch), ∼3285 cm−1 (N-H stretch of amide),
∼2975cm−1 (alkeneandaromaticC-Hstretch), ∼2880 cm−1
2.2b2 7-(4-(2-(3-Chlorophenylamino)-2-oxoethyl) pip- (cyclopropyl C-H stretch), ∼1715 cm−1 (C=O stretch of
erazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1, 4-dihydro acid carbonyl group), ∼1665 cm−1 (C=O stretch of amide),
quinoline-3-carboxylicacid (3b) IR (KBr): ∼3470 cm−1 ∼1540 cm−1 (N-H bending of amide), ∼1405 cm−1 (C-O
(O-H stretch), ∼3280 cm−1 (N-H stretch of amide), stretch of carbonyl group), ∼1270 cm−1 (O-H bending),
∼2970 cm−1 (alkeneandaromaticC-Hstretch), ∼2890 cm−1 ∼1150 cm−1 (C-O stretch of -OCH3), ∼1045 cm−1 (C-
(cyclopropyl C-H stretch), ∼1725 cm−1 (C=O stretch of F stretch), ∼770 cm−1 (C-H out-of-plane bending of meta
acid carbonyl group), ∼1670 cm−1 (C=O stretch of amide), disubstituted benzene). H NMR (400 MHz, CDCl3) δ 1.25
1
∼1580 cm−1 (N-H bending of amide), ∼1425 cm−1 (C-O (t, 2H, J = 7.2 Hz), 1.42 (t, 2H, J = 6.7 Hz), 2.88 (m,
stretch of carbonyl group), ∼1285 cm−1 (O-H bending), 4H), 3.26 (s, 2H), 3.43 (m, 4H), 3.56 (tt, 1H, J = 7.2 Hz,
∼1030 cm−1 (C-F stretch), ∼770 cm−1 (C-H out-of-plane
J = 4.0 Hz), 3.83 (s, 3H), 6.71 (d, 1H, J = 8.3 Hz), 7.06 (d,
bending of meta disubstituted benzene), ∼750 cm−1 (C- 1H, J = 9.0 Hz), 7.24 (d, 1H, JH−F = 8.3 Hz), 7.34 (t, 1H),
Cl stretch). 1H NMR (400 MHz, CDCl3) δ 1.22 (t, 2H, 7.4 (d, 1H, J = 7.5 Hz), 8.02 (d, 1H, JH−F = 12.8 Hz), 8.77
J = 7.2 Hz), 1.32 (t, 2H, J = 6.7 Hz), 2.89 (m, 4H), 3.28 (s, (s, 1H), 9.00 (s, 1H), 14.96 (s, 1H). 13C NMR (100.61 MHz,
2H), 3.40 (m, 4H), 3.60 (tt, 1H, J = 7.2 Hz, J = 4.0 Hz), CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56, 166.43, 153.21
7.23–7.25 (d, 1H, J = 8.6 Hz), 7.33 (t, 1H), 7.40 (d, 1H, (d, JC−F = 249.3 Hz), 147.23, 145.12 (d, JC−F = 10.3 Hz),
JH−F = 7.5 Hz), 7.45 (d, 1H, J = 8.6 Hz), 7.85 (s, 1H), 139.61, 134.21, 129.62, 127.9, 122.91, 120.42, 119.91 (d,
7.99 (d, 1H, JH−F = 13.2 Hz), 8.75 (s, 1H), 9.15 (s, 1H),
JC−F = 8.1 Hz), 116.7, 111.97 (d, JC−F = 24.14 Hz),
15.01 (s, 1H). 13C NMR (100.61 MHz, CDCl3) δ 176.82 107.12, 104.89 (d, JC−F = 3.7 Hz), 60.67, 54.8, 51.12, 49.16,
(d, JC−F = 2.2 Hz), 167.56, 166.43, 153.21 (d, JC−F
=
35.58, 8.12. ESI-MS (m/z): calcd. for C26H27FN4O5 494.19,
249.3 Hz), 147.23, 145.12 (d, JC−F = 10.3 Hz), 136.61, found 495.29 [M + H]+.
134.21, 133.31, 129.62, 127.90, 122.91, 120.42, 119.91 (d,
JC−F = 8.1 Hz), 111.97 (d, JC−F = 24.14 Hz), 107.12,
104.89 (d, JC−F = 3.7 Hz), 60.67, 51.12, 49.16, 35.58, 8.12.
2.2b51-Cyclopropyl-6-fluoro-7-(4-(2-(4-methoxypheny
ESI-MS (m/z): calcd. For C25H24ClFN4O4 498.14, found
lamino)-2-oxoethyl)piperazin-1-yl)-4-oxo-1,4-dihydroq
uinoline-3-carboxylicacid (3e) IR (KBr): ∼3460 cm−1
(O-H stretch), ∼3280 cm−1 (N-H stretch of amide),
499.28 [M + H]+.
2.2b3 7-(4-(2-(2-Chlorophenylamino)-2-oxoethyl) pip- ∼2975cm−1 (alkeneandaromaticC-Hstretch), ∼2880 cm−1
erazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro- (cyclopropyl C-H stretch), ∼1715 cm−1 (C=O stretch of
quinoline-3-carboxylicacid (3c) IR (KBr): ∼3455 cm−1 acid carbonyl group), ∼1665 cm−1 (C=O stretch of amide),
(O-H stretch), ∼3275 cm−1 (N-H stretch of amide), ∼1540 cm−1 (N-H bending of amide), ∼1400 cm−1 (C-O
∼2970 cm−1 (alkeneandaromaticC-Hstretch), ∼2890 cm−1 stretch of carbonyl group), ∼1270 cm−1 (O-H bending),
(cyclopropyl C-H stretch), ∼1725 cm−1 (C=O stretch of ∼1160 cm−1 (C-O stretch of -OCH3), ∼1040 cm−1 (C-
acid carbonyl group), ∼1675cm−1 (C=O stretch of amide), F stretch), ∼830 cm−1 (C-H out-of-plane bending of para
∼1590 cm−1 (N-H bending of amide), ∼1410 cm−1 (C-O disubstituted benzene). H NMR (400 MHz, CDCl3) δ 1.22
1
stretch of carbonyl group), ∼1285 cm−1 (O-H bending), (t, 2H, J = 7.2 Hz), 1.28 (t, 2H, J = 6.7 Hz), 2.88 (m,
∼1030 cm−1 (C-F stretch), ∼740 cm−1 (C-H out-of-plane 4H), 3.26 (s, 2H), 3.44 (m, 4H), 3.56 (tt, 1H, J = 7.0 Hz,
bending of ortho disubstituted benzene), ∼760 cm−1 (C-
J = 4.0 Hz), 3.8 (s, 3H), 6.89 (d, 2H, J = 8.3 Hz), 7.39
Cl stretch). 1H NMR (400 MHz, CDCl3) δ 1.22 (t, 2H, (d, 1H, JH−F = 7.5 Hz), 7.5 (d, 2H, J = 9.0 Hz), 8.02 (d,
J = 7.2 Hz), 1.32 (t, 2H, J = 6.7 Hz), 2.89 (m, 4H), 3.28 (s, 1H, JH−F = 12.8 Hz), 8.74 (s, 1H), 8.89 (s, 1H), 14.97 (s,
2H), 3.4 (m, 4H), 3.6 (tt, 1H, J = 7.2 Hz, J = 4.0 Hz), 7.35 1H). 13C NMR (100.61 MHz, CDCl3) δ 176.62 (d, JC−F
=
(t, 1H), 7.43 (t, 1H), 7.4 (d, 1H, JH−F = 7.5 Hz), 7.55 (d, 1H, 2.2 Hz), 167.36, 166.63, 153.31 (d, JC−F = 249.3 Hz),
J = 8.6 Hz), 7.75 (d, 1H, J = 8.6 Hz), 7.99 (d, 1H, JH−F
=
147.23, 145.12 (d, JC−F = 10.3 Hz), 139.65, 134.61, 132.34,
13.2 Hz), 8.75 (s, 1H), 9.15 (s, 1H), 15.01 (s, 1H). 13C NMR 122.62, 119.91 (d, JC−F = 8.1 Hz), 115.67, 111.57 (d,
(100.61 MHz, CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56,
JC−F = 24.14 Hz), 107.12, 104.89 (d, JC−F = 3.7 Hz),
166.43, 153.21 (d, JC−F = 249.3 Hz), 147.23, 145.12 (d, 60.67, 55.18, 51.12, 49.16, 35.58, 8.12. ESI-MS (m/z): calcd.
JC−F = 10.3 Hz), 136.61, 134.21, 131.31, 126.42, 122.49, for C26H27FN4O5 494.19, found 495.23 [M + H]+.

121 Page 4 of 11
2.2b6
J. Chem. Sci. (2018) 130:121
7-(4-(2-(3-Chloro-4-fluorophenylamino)-2 stretch of carbonyl group), ∼1285 cm−1 (O-H bending),
1
-oxoethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo ∼1035 cm−1 (C-F stretch). H NMR (400 MHz, CDCl3) δ
-1,4-dihydroquinoline-3-carboxylicacid (3f) IR (KBr): 1.22 (t, 2H, J = 7.2 Hz), 1.38 (t, 2H, J = 6.7 Hz), 2.89 (m,
∼3470 cm−1 (O-H stretch), ∼3280 cm−1 (N-H stretch of 4H), 3.27 (s, 2H), 3.44 (m, 4H), 3.56 (tt, 1H, J = 7.0 Hz,
amide), ∼2970 cm−1 (alkene and aromatic C-H stretch),
J = 4.0 Hz), 7.13 (t, 1H), 7.33 (t, 2H), 7.37 (d, 1H, JH−F
=
=
∼2890 cm−1 (cyclopropyl C-H stretch), ∼1725 cm−1 7.0 Hz), 7.59 (d, 2H, J = 7.6 Hz), 8.01 (d, 1H, JH−F
(C=O stretch of acid carbonyl group), ∼1670 cm−1 12.7 Hz), 8.72 (s, 1H), 9.02 (s, 1H), 14.96 (s, 1H). 13C NMR
(C=O stretch of amide), ∼1590 cm−1 (N-H bending of (100.61 MHz, CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56,
amide), ∼1420 cm−1 (C-O stretch of carbonyl group), 166.43, 153.21 (d, JC−F = 249.3 Hz), 147.23, 145.12 (d,
∼1285 cm−1 (O-H bending), ∼1025 cm−1 (C-F stretch),
JC−F = 10.3 Hz), 138.50, 134.51, 129.02, 128.42, 122.45,
∼1130 cm−1 (C-F stretch), ∼750 cm−1 (C-Cl stretch).1H 119.91 (d, JC−F = 8.1 Hz), 117.83, 111.97 (d, JC−F
=
NMR (400 MHz, DMSO-D6) δ 1.22 (t, 2H, J = 7.2 Hz), 24.14 Hz), 104.89 (d, JC−F = 3.7 Hz), 60.67, 51.12, 49.16,
1.28 (t, 2H, J = 6.7 Hz), 2.89 (m, 4H), 3.27 (s, 2H), 3.44 35.58, 8.12. ESI-MS (m/z): calcd. for C25H25FN4O4 464.18,
(m, 4H), 3.56 (tt, 1H, J = 7.0 Hz, J = 4.0 Hz), 7.19 (t, 1H), found 464.29 [M + H]+.
7.54 (d, 1H), 7.89 (s,1H), 7.93 (d, 1H, JH−F = 12.7 Hz), 8.06
(s, 1H), 8.72 (s, 1H), 9.08 (s, 1H), 15.15 (s, 1H). 13C NMR
2.2b9 1-Cyclopropyl-6-fluoro-7-(4-(2-(methyl(phenyl)
amino)-2-oxoethyl) piperazin-1-yl)-4-oxo-1,4-dihydro
(100.61 MHz, CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56,
quinoline-3-carboxylicacid (3i) IR (KBr): ∼3475 cm−1
(O-H stretch), ∼2975 cm−1 (alkene and aromatic C-H
stretch), ∼2885 cm−1 (cyclopropyl C-H stretch),
∼1720 cm−1 (C=O stretch of acid carbonyl group),
∼1670 cm−1 (C=O stretch of amide), ∼1420 cm−1 (C-O
stretch of carbonyl group), ∼1285 cm−1 (O-H bending),
∼1035cm−1 (C-F stretch).1H NMR (400 MHz, CDCl3) δ
166.43, 153.21 (d, JC−F = 249.3 Hz), 147.23, 145.12 (d,
JC−F = 10.3 Hz), 148.95, 134.61, 133.21, 124.32, 123.42,
120.09, 119.91 (d, JC−F = 8.1 Hz), 113.56, 111.97 (d,
JC−F = 24.14 Hz), 107.12, 104.89 (d, JC−F = 3.7 Hz),
60.67, 51.12, 49.16, 35.58, 8.12. ESI-MS (m/z): calcd. for
C25H23ClF2N4O4 516.13, found 517.32 [M + H]+.
2.2b7 7-(4-(2-(2-Bromophenylamino)-2-oxoethyl)pipe- 1.22 (t, 2H, J = 7.2 Hz), 1.28 (t, 2H, J = 6.7 Hz), 2.89
razin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1, 4-dihydroq (m, 4H), 3.27 (s, 2H), 3.44 (m, 4H), 3.49 (s, 3H), 3.56 (tt, 1H,
uinoline-3-carboxylicacid (3g) IR (KBr): ∼3460 cm−1
J = 7.0 Hz, J = 4.0 Hz), 7.27 (d, 2H, J = 7.6 Hz), 7.39
(O-H stretch), ∼3280 cm−1 (N-H stretch of amide), (d, 1H, JH−F = 7.0 Hz), 7.43 (t, 1H), 7.53 (t, 2H), 8.01 (d,
∼2970 cm−1 (alkeneandaromaticC-Hstretch), ∼2890 cm−1 1H, JH−F = 12.7 Hz), 8.72 (s, 1H), 14.96 (s, 1H). 13C NMR
(cyclopropyl C-H stretch), ∼1725 cm−1 (C=O stretch of (100.61 MHz, CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56,
acid carbonyl group), ∼1670 cm−1 (C=O stretch of amide), 166.43, 153.21 (d, JC−F = 249.3 Hz), 147.23, 145.12 (d,
∼1590 cm−1 (N-H bending of amide), ∼1420 cm−1 (C-O
JC−F = 10.3 Hz), 138.50, 134.51, 129.82, 127.42, 122.45,
stretch of carbonyl group), ∼1285 cm−1 (O-H bending), 119.91 (d, JC−F = 8.1 Hz), 117.83, 111.97 (d, JC−F
=
∼1035 cm−1 (C-F stretch), ∼750 cm−1 (C-H out-of-plane 24.14 Hz), 104.89 (d, JC−F = 3.7 Hz), 60.67, 51.12, 49.16,
bending of ortho disubstituted benzene), ∼560 cm−1 (C- 35.58, 34.67, 8.12. ESI-MS (m/z): calcd. for C26H27FN4O4
Br stretch).1H NMR (400 MHz, CDCl3) δ 1.22 (t, 2H, J = 478.2, found 479.36 [M + H]+.
7.2 Hz), 1.28 (t, 2H, J = 6.7 Hz), 2.89 (m, 4H), 3.27 (s, 2H),
3.44 (m, 4H), 3.56 (tt, 1H, J = 7.0 Hz, J = 4.0 Hz), 7.1 (t,
2.2b10 1-Cyclopropyl-6-fluoro-7-(4-(2-(3-nitropheny
lamino)-2-oxoethyl)piperazin-1-yl)-4-oxo-1,4-dihydroq
1H), 7.31 (t, 1H), 7.4 (d, 1H, JH−F = 7.0 Hz), 7.71 (d, 1H,
uinoline-3-carboxylicacid (3j) IR (KBr): ∼3470 cm−1
(O-H stretch), ∼3285 cm−1 (N-H stretch of amide),
∼2975cm−1 (alkeneandaromaticC-Hstretch), ∼2880 cm−1
(cyclopropyl C-H stretch), ∼1715 cm−1 (C=O stretch of
acid carbonyl group), ∼1665 cm−1 (C=O stretch of amide),
∼1540 cm−1 (N-H bending of amide), ∼1520 cm−1 (N-O
asymmetric stretch), ∼1405 cm−1 (C-O stretch of carbonyl
group), ∼1360 cm−1 (N-O symmetric stretch), ∼1270 cm−1
(O-H bending), ∼1150 cm−1 (C-O stretch of -OCH3),
∼1045 cm−1 (C-F stretch), ∼780 cm−1 (C-H out-of-plane
J = 8.9 Hz), 7.86 (d, 1H, J = 8.9 Hz), 8.02 (d, 1H, JH−F
=
12.7 Hz), 8.74 (s, 1H), 9.07 (s, 1H), 14.97 (s, 1H). 13C NMR
(100.61 MHz, CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56,
166.43, 153.21 (d, JC−F = 249.3 Hz), 147.23, 145.12 (d,
JC−F = 10.3 Hz), 138.05, 134.61, 132.31, 131.65, 127.62,
125.42, 119.91 (d, JC−F = 8.1 Hz), 117.23, 111.97 (d,
JC−F = 24.14 Hz), 107.12, 104.89 (d, JC−F = 3.7 Hz),
60.67, 51.12, 49.16, 35.58, 8.12. ESI-MS (m/z): calcd. for
C25H24BrFN4O4 542.09, found 543.23 [M + H]+.
2.2b8
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-oxo-2- bending of meta disubstituted benzene). 1H NMR (400 MHz,
(phenylamino) ethyl)piperazin-1-yl)-1,4-dihydroquino CDCl3) δ 1.22 (t, 2H, J = 7.2 Hz), 1.32 (t, 2H, J = 6.7 Hz),
line-3-carboxylicacid (3h) IR (KBr): ∼3465 cm−1 (O- 2.89 (m, 4H), 3.28 (s, 2H), 3.40 (m, 4H), 3.60 (tt, 1H,
H stretch), ∼3285 cm−1 (N-H stretch of amide), ∼2970 cm−1
J = 7.2 Hz, J = 4.0 Hz), 7.15 (d, 1H, J = 8.6 Hz), 7.33 (t,
(alkene and aromatic C-H stretch), ∼2890 cm−1 (cyclo- 1H), 7.4 (d, 1H, JH−F = 7.5 Hz), 7.55 (d, 1H, J = 8.6 Hz),
propyl C-H stretch), ∼1725 cm−1 (C=O stretch of acid 7.85 (s, 1H), 7.99 (d, 1H, JH−F = 13.2 Hz), 8.75 (s, 1H), 9.15
carbonyl group), ∼1670 cm−1 (C=O stretch of amide), (s, 1H), 15.01 (s, 1H). 13C NMR (100.61 MHz, CDCl3) δ
∼1590 cm−1 (N-H bending of amide), ∼1420 cm−1 (C-O 176.82 (d, JC−F = 2.2 Hz), 167.56, 166.43, 153.21 (d,

J. Chem. Sci. (2018) 130:121
Page 5 of 11 121
JC−F = 249.3 Hz), 147.23, 145.12 (d, JC−F = 10.3 Hz), 2.2b13 1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(2-oxo-2-(3
137.61, 135.21, 133.31, 128.62, 126.60, 122.91, 120.42, -(trifluoromethyl) phenylamino) ethyl) piperazin-1-yl)-
119.91 (d, JC−F = 8.1 Hz), 111.97 (d, JC−F = 24.14 Hz), 1,4-dihydroquinoline-3-carboxylic acid (3m) IR (KBr):
107.12, 104.89 (d, JC−F = 3.7 Hz), 60.67, 51.12, 49.16, ∼3470 cm−1(O-H stretch), ∼3280 cm−1 (N-H stretch of
35.58, 8.12. ESI-MS (m/z): calcd. for C25H24FN5O6 509.18, amide), ∼2970 cm−1 (alkene and aromatic C-H stretch),
found 510.32 [M + H]+.
∼2890 cm−1 (cyclopropyl C-H stretch), ∼1725 cm−1
(C=O stretch of acid carbonyl group), ∼1670 cm−1
(C=O stretch of amide), ∼1590 cm−1 (N-H bending of
amide), ∼1420 cm−1 (C-O stretch of carbonyl group),
∼1285 cm−1 (O-H bending), ∼1210 cm−1 (C-F stretch),
∼1030 cm−1 (C-F stretch), ∼770 cm−1 (C-H out-of-plane
bending of meta disubstituted benzene). 1H NMR (400 MHz,
CDCl3) δ 1.22 (t, 2H, J = 7.2 Hz), 1.32 (t, 2H, J = 6.7 Hz),
2.89 (m, 4H), 3.28 (s, 2H), 3.40 (m, 4H), 3.60 (tt, 1H,
J = 7.2 Hz, J = 4.0 Hz), 7.15 (d, 1H, J = 8.6 Hz),
7.33 (t, 1H), 7.4 (d, 1H, JH−F = 7.5 Hz), 7.55 (d, 1H,
J = 8.6 Hz), 7.85 (s, 1H), 7.99 (d, 1H, JH−F = 13.2 Hz),
8.75 (s, 1H), 9.15 (s, 1H), 15.01 (s, 1H). 13C NMR (100.61
MHz, CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56, 166.43,
2.2b11 1-Cyclopropyl-7-(4-(2-(ethyl(phenyl)amino)-2-
oxoethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroqu
inoline-3-carboxylicacid (3k) IR (KBr): ∼3475 cm−1 (O-
H stretch), ∼3000 cm−1 (alkane C-H stretch), ∼2900 cm−1
(alkene and aromatic C-H stretch), ∼2885 cm−1 (cyclopropyl
C-H stretch), ∼1720 cm−1 (C=O stretch of acid carbonyl
group), ∼1670 cm−1 (C=O stretch of amide), ∼1420 cm−1
(C-O stretch of carbonyl group), ∼1285 cm−1 (O-H bending),
∼1035 cm−1 (C-F stretch). H NMR (400 MHz, CDCl3) δ
1
1.22 (t, 2H, J = 7.2 Hz), 1.28 (t, 2H, J = 6.7 Hz), 1.35
(t, 3H), 2.89 (m, 4H), 3.27 (s, 2H), 3.44 (m, 4H), 3.56 (tt,
1H, J = 7.0 Hz, J = 4.0 Hz), 3.86 (q, 2H), 7.27 (d, 2H,
J = 7.6 Hz), 7.39 (d, 1H, JH−F = 7.0 Hz), 7.43 (t, 1H),
7.53 (t,2H), 8.01 (d, 1H, JH−F = 12.7 Hz), 8.72 (s, 1H),
14.96 (s, 1H). 13C NMR (100.61 MHz, CDCl3) δ 176.82
153.21 (d, JC−F = 249.3 Hz), 147.23, 145.12 (d, JC−F
=
10.3 Hz), 137.61, 135.21, 133.31, 128.62, 126.60, 125.34,
122.91, 120.42, 119.91 (d, JC−F = 8.1 Hz), 111.97 (d,
JC−F = 24.14 Hz), 107.12, 104.89 (d, JC−F = 3.7 Hz),
60.67, 51.12, 49.16, 35.58, 8.12. ESI-MS (m/z): calcd. for
C26H24F4N4O4 532.18, found 533.32 [M + H]+.
(d, JC−F = 2.2 Hz), 167.56, 166.43, 153.21 (d, JC−F
=
249.3 Hz), 147.23, 145.12 (d, JC−F = 10.3 Hz), 138.50,
134.51, 129.82, 127.42, 122.45, 119.91 (d, JC−F = 8.1 Hz),
117.83, 111.97 (d, JC−F = 24.14 Hz), 104.89 (d, JC−F
=
2.2b14
7-(4-(2-(3-Chloro-2-methylphenylamino)-2-
3.7 Hz), 60.67, 51.12, 49.16, 35.58, 34.67, 13.56, 8.12. ESI-
MS (m/z): calcd. for C27H29FN4O4 492.20, found 493.36
[M + H]+.
oxoethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylicacid (3n) IR (KBr):
∼3465 cm−1 (O-H stretch), ∼3285 cm−1 (N-H stretch of
amide), ∼3000 cm−1 (alkane C-H stretch), ∼2970 cm−1
(alkene and aromatic C-H stretch), ∼2890 cm−1 (cyclo-
propyl C-H stretch), ∼1725 cm−1 (C=O stretch of acid
carbonyl group), ∼1670 cm−1 (C=O stretch of amide),
∼1590 cm−1 (N-H bending of amide), ∼1420 cm−1 (C-O
stretch of carbonyl group), ∼1285 cm−1 (O-H bending),
2.2b12 1-Cyclopropyl-6-fluoro-7-(4-(2-(4-nitrophenyla
mino)-2-oxoethyl)piperazin-1-yl)-4-oxo-1,4-dihydroqu
inoline-3-carboxylicacid (3l) IR (KBr): ∼3470 cm−1(O-
H stretch), ∼3285 cm−1 (N-H stretch of amide), ∼2975 cm−1
(alkene and aromatic C-H stretch), ∼2880 cm−1 (cyclo-
propyl C-H stretch), ∼1715 cm−1 (C=O stretch of acid
carbonyl group), ∼1665 cm−1 (C=O stretch of amide),
∼1540 cm−1 (N-H bending of amide), ∼1530 cm−1 (N-O
asymmetric stretch), ∼1405 cm−1 (C-O stretch of carbonyl
group), ∼1350 cm−1 (N-O symmetric stretch), ∼1270 cm−1
(O-H bending), ∼1150 cm−1 (C-O stretch of -OCH3),
∼1045 cm−1 (C-F stretch), ∼830 cm−1 (C-H out-of-plane
bending of para disubstituted benzene). 1H NMR (400 MHz,
CDCl3) δ 1.22 (t, 2H, J = 7.2 Hz), 1.28 (t, 2H, J = 6.7 Hz),
2.89 (m, 4H), 3.27 (s, 2H), 3.44 (m, 4H), 3.56 (tt, 1H,
J = 7.0 Hz, J = 4.0 Hz), 7.31 (d, 2H, J = 8.7 Hz),
7.39 (d, 1H, JH−F = 7.0 Hz), 7.56 (d, 2H, J = 8.7 Hz),
8.02 (d, 1H, JH−F = 12.7 Hz), 8.74 (s, 1H), 9.05 (s,
1H), 14.97 (s, 1H). 13C NMR (100.61 MHz, CDCl3) δ
176.82 (d, JC−F = 2.2 Hz), 167.56, 166.43, 153.21 (d,
1
∼1035 cm−1 (C-F stretch), ∼740 cm−1 (C-Cl stretch). H
NMR (400 MHz, CDCl3) δ 1.22 (t, 2H, J = 7.2 Hz), 1.32 (t,
2H, J = 6.7 Hz), 2.21 (s, 3H), 2.89 (m, 4H), 3.28 (s, 2H), 3.4
(m, 4H), 3.6 (tt, 1H, J = 7.2 Hz, J = 4.0 Hz), 7.16 (d, 1H,
J = 8.6 Hz), 7.23 (t, 1H), 7.4 (d, 1H, JH−F = 7.5 Hz), 7.45
(d, 1H, J = 8.6 Hz), 7.99 (d, 1H, JH−F = 13.2 Hz), 8.75
(s, 1H), 9.15 (s, 1H), 15.01 (s, 1H). 13C NMR (100.61 MHz,
CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56, 166.43, 153.21
(d, JC−F = 249.3 Hz), 147.23, 145.12 (d, JC−F = 10.3 Hz),
137.61, 135.21, 134.31, 132.24, 127.62, 124.6, 119.91 (d,
JC−F = 8.1 Hz), 115.67, 111.97 (d, JC−F = 24.14 Hz),
107.12, 104.89 (d, JC−F = 3.7 Hz), 60.67, 51.12, 49.16,
35.58, 13.56, 8.12. ESI-MS (m/z): calcd. for C26H26ClFN4O4
512.16, found 512.28, [M + H]+.
JC−F = 249.3 Hz), 147.23, 145.12 (d, JC−F = 10.3 Hz), 2.2b15
1-Cyclopropyl-7-(4-(2-(2,4-dimethylpheny
138.95, 136.61, 133.31, 129.62, 120.42, 119.91 (d, JC−F lamino)-2-oxoethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
=
8.1 Hz), 111.97 (d, JC−F = 24.14 Hz), 107.12, 104.89 (d, dihydroquinoline-3-carboxylicacid (3o) IR (KBr):
JC−F = 3.7 Hz), 60.67, 51.12, 49.16, 35.58, 8.12. ESI- ∼3465 cm−1 (O-H stretch), ∼3285 cm−1 (N-H stretch of
MS (m/z): calcd. for C25H24FN5O6 501.16, found 502.28 amide), ∼3000 cm−1 (alkane C-H stretch), ∼2970 cm−1
[M + H]+.
(alkene and aromatic C-H stretch), ∼2890 cm−1

121 Page 6 of 11
J. Chem. Sci. (2018) 130:121
(Cyclopropyl C-H stretch), ∼1725 cm−1 (C=O stretch of 4H), 2.89 (m, 4H), 3.27 (s, 2H), 3.44 (m, 4H), 3.56 (tt, 1H,
acid carbonyl group), ∼1670 cm−1 (C=O stretch of amide),
J = 7.0 Hz, J = 4.0 Hz), 7.11 (d,2H, J = 8.6 Hz), 7.37
∼1590 cm−1 (N-H bending of amide), ∼1420 cm−1 (C-O (t, 1H), 7.93 (d, 1H, JH−F = 12.7 Hz), 8.06 (s, 1H), 8.72
stretch of carbonyl group), ∼1285 cm−1 (O-H bending), (s, 1H), 9.8 (s, 1H), 15.15 (s, 1H). 13C NMR (100.61 MHz,
1
∼1035 cm−1 (C-F stretch). H NMR (400 MHz, CDCl3) δ CDCl3) δ 176.98 (d, JC−F = 2.2 Hz), 168.69, 166.9, 154.89
1.22 (t, 2H, J = 7.2 Hz), 1.39 (t, 2H, J = 6.7 Hz), 2.29 (d, JC−F = 249.3 Hz), 147.44, 145.57 (d, JC−F = 10.3 Hz),
(s, 6H), 2.91 (m, 4H), 3.29 (s, 2H), 3.44 (m, 4H), 3.56 141.08, 139.01, 132.24, 128.06, 126.52, 120.01 (d, JC−F
=
(tt, 1H, J = 7.0 Hz, J = 4.0 Hz), 7.00 (s, 1H), 7.04 8.1 Hz), 112.31 (d, JC−F = 24.14 Hz), 108.03, 104.96 (d,
(d, 1H, J = 8.6 Hz), 7.37 (d,1H, JH−F = 7.5 Hz), 7.91
JC−F = 3.7 Hz), 61.69, 53.55, 49.95, 35.33, 25.11, 14.57,
(d, 1H, JH−F = 12.7 Hz), 7.98 (d, 1H, J = 8.6 Hz), 8.24. ESI-MS (m/z): calcd. for C29H33FN4O4 520.24, found
8.7 (s, 1H), 9.03 (s, 1H), 14.95 (s, 1H). 13C NMR (100.61 521.34 [M + H]+.
MHz, CDCl3) δ 176.99 (d, JC−F = 2.2 Hz), 167.37, 166.87,
154.87 (d, JC−F = 249.3 Hz), 152.35, 147.44, 145.47 (d,
2.2b18
1-Cyclopropyl-7-(4-(2-(3,4-dichloropheny
JC−F = 10.3 Hz), 139.03, 134.4, 132.95, 131.08, 127.51,
lamino)-2-oxoethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylicacid (3r) IR (KBr):
∼3465 cm−1 (O-H stretch), ∼3285 cm−1 (N-H stretch of
amide), ∼2970 cm−1 (alkene and aromatic C-H stretch),
∼2890 cm−1 (cyclopropyl C-H stretch), ∼1725 cm−1
(C=O stretch of acid carbonyl group), ∼1670 cm−1
(C=O stretch of amide), ∼1590 cm−1 (N-H bending of
amide), ∼1420 cm−1 (C-O stretch of carbonyl group),
∼1285 cm−1 (O-H bending), ∼1035 cm−1 (C-F stretch),
121.5, 120.01 (d, JC−F = 8.1 Hz), 112.61 (d, JC−F
=
24.14 Hz), 108.1, 104.93 (d, JC−F = 3.7 Hz), 62.07, 53.22,
50.04, 35.31, 20.85, 17.83, 8.24. ESI-MS (m/z): calcd. for
C27H29FN4O4 492.21, found 493.32 [M + H]+.
2.2b16
1-Cyclopropyl-7-(4-(2-(2,5-dimethylpheny
lamino)-2-oxoethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylicacid (3p) IR (KBr):
∼3465 cm−1 (O-H stretch), ∼3285 cm−1 (N-H stretch of
amide), ∼3000 cm−1 (alkane C-H stretch), ∼2970 cm−1
(alkene and aromatic C-H stretch), ∼2890 cm−1 (cyclo-
propyl C-H stretch), ∼1725 cm−1 (C=O stretch of acid
carbonyl group), ∼1670 cm−1 (C=O stretch of amide),
∼1590 cm−1 (N-H bending of amide), ∼1420 cm−1 (C-O
stretch of carbonyl group), ∼1285 cm−1 (O-H bending),
1
∼780 cm−1 (C-Cl stretch). H NMR (400 MHz, CDCl3) δ
1.22 (t, 2H, J = 7.2 Hz), 1.28 (t, 2H, J = 6.7 Hz), 2.89 (m,
4H), 3.27 (s, 2H), 3.44 (m, 4H), 3.56 (tt, 1H, J = 7.0 Hz,
J = 4.0 Hz), 7.54 (d, 1H, J = 8.8 Hz), 7.64 (d, 1H,
J = 8.8 Hz),7.90 (s, 1H), 7.93 (d, 1H, JH−F = 12.7 Hz),
8.06 (s, 1H), 8.72 (s, 1H), 9.8 (s, 1H), 15.15 (s, 1H). 13C NMR
(100.61 MHz, CDCl3) δ 176.82 (d, JC−F = 2.2 Hz), 167.56,
166.43, 153.21 (d, JC−F = 249.3 Hz), 147.23, 145.12 (d,
JC−F = 10.3 Hz), 138.05, 134.61, 131.21, 130.32, 129.42,
124.09, 121.23, 119.91 (d, JC−F = 8.1Hz), 111.97 (d,
JC−F = 24.14 Hz), 107.12, 104.89 (d, JC−F = 3.7 Hz),
60.67, 51.12, 49.16, 35.58, 8.12. ESI-MS (m/z): calcd. for
C25H23Cl2FN4O4 532.10, found 533.22 [M + H]+.
1
∼1035 cm−1 (C-F stretch). H NMR (400 MHz, CDCl3) δ
1.22 (t, 2H, J = 7.2 Hz), 1.28 (t, 2H, J = 6.7 Hz), 2.12
(s, 3H), 2.34 (s, 3H), 2.89 (m, 4H), 3.27 (s, 2H), 3.44 (m,
4H), 3.56 (tt, 1H, J = 7.0 Hz, J = 4.0 Hz), 6.83 (d,1H,
J = 8.6 Hz), 7.07 (d, 1H, J = 8.6 Hz), 7.29 (s,1H), 7.93
(d, 1H, JH−F = 12.7 Hz), 8.06 (s, 1H), 8.72 (s, 1H), 9.8 (s,
1H), 15.15 (s, 1H). 13C NMR (100.61 MHz, CDCl3) δ 177.02
(d, JC−F = 2.2 Hz), 167.56, 166.43, 154.21 (d, JC−F
=
249.3 Hz), 147.23, 145.12 (d, JC−F = 10.3 Hz), 139.03,
136.87, 135.36, 130.19, 125.56, 123.32, 121.42, 119.94 (d,
JC−F = 8.1 Hz), 112.62 (d, JC−F = 24.14 Hz), 108.12,
104.93 (d, JC−F = 3.7 Hz), 62.13, 53.21, 50.05, 35.32, 21.24,
17.47, 8.24. ESI-MS (m/z): calcd. for C27H29FN4O4 492.21,
found 493.34 [M + H]+.
2.3 Cell culture and cell proliferation assay
Cell viability was determined by (4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) assay. Human lung
cancer cell line (A549), human cervical cancer cell line
(HeLa), Human Breast carcinoma cell lines MDA MB-231,
MCF7, Human Pancreatic Cancer line MiaPaca-2 and Human
2.2b17
1-Cyclopropyl-7-(4-(2-(2,6-diethylpheny Embryonic Kidney Cell lines (HEK) were employed in the
lamino)-2-oxoethyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4- currentstudy. Cells(1×104 cells/well)wereseededto96-well
dihydroquinoline-3-carboxylicacid (3q) IR (KBr): culture plate and cultured with or without different concen-
∼3465 cm−1 (O-H stretch), ∼3285 cm−1 (N-H stretch of trations of compounds for 48 h in a final volume of 200 μL.
amide), ∼3000 cm−1 (alkane C-H stretch), ∼2970 cm−1 After treatment, the medium was removed and 10 μL of MTT
(alkene and aromatic C-H stretch), ∼2890 cm−1 (cyclo- (10 mg/mL in PBS) was added to the fresh medium. After 2 h
propyl C-H stretch), ∼1725 cm−1 (C=O stretch of acid incubation at 37 ◦C, 100 μL extraction buffer was added to
carbonyl group), ∼1670 cm−1 (C=O stretch of amide), each well and plates were agitated for 1 min. The optical
∼1590 cm−1 (N-H bending of amide), ∼1420 cm−1 (C- density (O.D) was read at 570 using microplate reader (Mul-
O stretch of carbonyl group), ∼1285 cm−1 (O-H bend- timode Varioskan Flash Instrument-Thermo Scientific Ltd).
ing), ∼1230 cm−1 (C-C stretch of ethyl), ∼1035 cm−1 (C- Per cent inhibition of proliferation was calculated as a frac-
1
F stretch). H NMR (400 MHz, CDCl3) δ 1.22 (t, 2H, J = tion of control (without compound). All the experiments were
7.2 Hz), 1.28 (t, 2H, J = 6.7 Hz), 1.35 (t, 6H), 2.6 (q, carried out in triplicates. The results were represented as a

J. Chem. Sci. (2018) 130:121
Page 7 of 11 121
Scheme 1. Synthesis of ciprofloxacin derivatives (3 a–r).
percentage of cytotoxicity/viability. From the percentage of measuring the spectra to eliminate the absorbance of the
cytotoxicity, the IC50 values are calculated.
CtDNA itself.
2.4 Molecular docking studies
2.5b Fluorescence measurements: The fluorescence
spectral titrations were performed on a spectrofluorometer
(JASCO model FP-6300). The fluorescence emission spectra
were measured at 300 K over a wavelength range of 520–
740 nm with an exciting wavelength at 500 nm. Before mea-
suring fluorescence spectra, all solutions were stirred and
allowed to equilibrate for 5 min. For correct background flu-
orescence blank of Tris–HCl buffer was subtracted.
Further, the molecular docking studies of 3a–r were
performed using human topo-II isomerase being the tar-
get enzyme of CP using Schrödinger suite 2013. Crystal
co-ordinates for DNA topo-II isomerase was taken from
the Protein Data Bank (PDB ID: 4G0U). The multi-step
Schrödinger’s protein preparation tool (PPrep) has been used
for the final preparation of receptor model. Hydrogen was
added to the model automatically via the maestro inter-
face. PPrep neutralizes side chains and residues which are
not involving in salt bridges. This step is then followed by
restrained minimization using the OPLS 2005 force field to
RMSD of 0.3 A0. The 2D structure of 3a–r were sketched
and converted to 3D using maestro interface. Ligands were
prepared for docking using Ligprep, a module of Schrodinger.
A total of 10 conformations were generated for all the
compounds. Grid box was generated with coordinates of
X:26.2195; Y:99.8115; Z:32.8506 by considering co-crystal
ligand i.e., amsacrine. Docking studies were performed using
GLIDE, a module of Schrödinger.
3. Results and Discussion
3.1 Chemistry
In the current study, we synthesized a total of eighteen
(3a–r) 7-(substituted piperazin-1-yl) derivatives of CP
in a two-step process. The first step includes prepara-
tion of 2-chloro-N-(substitutedphenyl) acetamides (2a–
r) by coupling substituted anilines with chloroacetyl
chloride.27 In the second step, 2-chloro-N-(substituted
phenyl) acetamides were coupled with CP yielding the
title compounds (Scheme 1 and Table 1).
1
2.5 DNA binding
In general H NMR of all the title compounds dis-
played two triplets in the range 1.10–1.39 ppm and a
multiplet in the range 3.56–3.6 ppm corresponding to
the protons of cyclopropyl ring. Two multiplets of piper-
azine protons resonated in the range 2.88–3.44 ppm.
Two sharp doublets resonated in the range 7.2–8.02 ppm
due to C-5 and C-8 protons of the FQ moiety. The C-2
protons of FQ showed a sharp singlet in the range 8.64–
8.77 ppm. One of the singlet peak due to the proton of
amide in the range of 8.7–9.15 ppm. Broad peak owing
2.5a UV- Visible measurement: The DNA binding
experiments were carried in Tris–HCl buffer solution (5 mM,
pH 7.4) using the compound solution in DMSO. In UV–
visible measurements, a constant concentration of compound
3o was treated with different concentrations of the CtDNA.
The DNA solutions of equivalent concentrations were mea-
sure as reference solutions in the experiment. The absorbance
(A)wasrecordedaftersuccessiveadditionsofvariousconcen-
trations of CtDNA. An equal amount of CtDNA was added
to the compound solution and the reference solution while to the proton of carboxylic acid functional group in the

121 Page 8 of 11
J. Chem. Sci. (2018) 130:121
Table 1. Synthesized compounds: structure, m.p, yield and docking score (3a–r).
Entry
R
R1
M.p (◦C) Yield (%) Docking Score (SP)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
4-Cl
3-Cl
2-Cl
3-OCH3
4-OCH3
3-Cl-4-F
2-Br
H
H
3-NO2
H
4-NO2
3-CF3
H H
H
H
H
H
H
H
H
CH3
H
222–224
249–251
289–290
219–221
208–210
245–246
284–285
259–261
189–190
265–266
73
67
63
77
67
64
65
82
85
82
64
60
65
63
65
62
67
71
−7.963
−7.743
−7.78
−7.694
−7.95
−7.848
−7.892
−7.831
−7.206
−7.521
−5.382
−7.924
−8.048
−7.847
−7.446
−7.68
CH2CH3 180–182
H
H
H
H
H
H
H
257–258
222–223
268–269
256–258
244–245
261–262
214–216
3-Cl-2-CH3
2,4-diCH3
2,5-diCH3
2,6-diC2H5
3,4-diCl
3q
3r
−7.459
−7.729
−7.577
Ciprofloxacin
range 14.96–15.15 ppm. The acetyl link protons showed cancer) cell line. Substitution with 2,4-dimethylphenyl
multiplet in the range 3.26–3.3. Further, the structure of at C7 position (3o) exhibited promising anticancer activ-
the title compounds was substantiated from 13C NMR ity against Miapaca, HeLa, MDA MB-231 cancer cell
and ESI-MS respectively.
lines. Nevertheless, 3o was found to be potent than CP
against MCF7 cell line with IC50 value 26.45 μM.
From the IC50 values, it is clear that most of the
active compounds exhibited less cytotoxicity towards
the normal embryonic kidney cell line (HEK) compared
to their anti-cancer potential against tested cancer cell
lines, which justifies the role of the novel synthesized
compounds as anti-cancer agents.
3.2 Antiproliferative activity
The antiproliferative activity of 3a–r on human can-
cer cell lines such as lung cancer (A-549), pancreatic
cancer (Mia Paca-2), cervical cancer (HeLa), metastatic
breast cancer (MDA MB-231), breast cancer (MCF-7)
cell lines along with a non-cancerous human embryonic
kidney cell line (HEK) was carried using MTT assay,28
and the results are tabulated in Table 2. CP and doxoru-
bicin are used as reference standards.
3.3 Molecular docking studies
All the compounds 3a–r showed significant growth Further, the molecular docking studies of 3a–r were per-
inhibition on A549 cell line with IC50 values ranging formed using human topo-II isomerase being the target
from 11.69 0.26 to 15.27 1.68 μM. Substitution enzyme of CP. Docking scores by standard precision
of chloro, methoxy at various positions (3a, 3b, 3c (Glide-SP) and shown in Table 1. Amino acid interac-
and 3d) demonstrated lowest IC50 and better growth tion pattern of a few active compounds 3d, 3o and 3n
inhibition whereas compounds bearing electron with- are shown in Figure S11 (Supplementary Information)
drawing groups (3j and 3l) like nitro at meta and para along with amsacrine (topoisomerase inhibitor) as stan-
position exhibited moderate activity against A549 (lung dard. Amsacrine has shown docking score of −5.86.

J. Chem. Sci. (2018) 130:121
Page 9 of 11 121
Table 2. IC50 values (μM) for compounds (3a–r) in five human cancer cell lines (A549, MiaPaca,
HeLa, MDA MB-231, MCF-7) as well as normal cell line HEK.
Entry
A549a
MiaPacab
HeLac
MDA MB 231d
MCF7d
HEKe
3a
3b
3c
3d
3e
3f
3g
3h
3i
13.29 0.44 45.4 0.52
11.69 0.26 37.02 0.47 76.3 0.76
>100
34.3 0.41
32.79 0.4
>100
>100
>100
72.41 1.69
73.63 1.08
>100
11.71 0.18 35.57 0.48 91.95 0.2 49.77 0.42
12.64 0.37 70.23 1.69 49.08 0.88 30.76 0.82
12.72 0.83 36.92 0.74 51.77 0.48
12.36 0.35 33.22 0.32 >100
13.1 1.78 41.14 0.96 77.89 1.13
13.83 1.61 59.22 0.33 79.76 1.76 78.08 0.31
13.25 0.27 59.35 1.66 75.18 0.92 >100
12.99 0.67 69.01 0.99 60.23 0.57 41.53 0.57
12.71 0.85 30.04 0.9 83.02 1.65 42.05 1.25
13.84 0.94 40.55 1.94 78.57
>100
>100
>100
>100
>100
86.37 0.61
62.85 1.05 76.8 0.89
>100
>100
>100
>100
>100
>100
64.54 0.66
>100
>100
>100
>100
>100
3j
3k
3l
1
44.93 0.22 75.55 1.25
3m
3n
3o
3p
3q
3r
15.27 1.68 40.25 1.94 77.54 0.26 54.47 0.62
13.74 0.84 29.12 0.34 61.57 0.52 33.44 0.44
14.21 0.66 26.6 0.05 29.28 0.11 25.34 0.25 26.45 0.33
14.09 0.89 48.94 0.77 68.62 0.25 45.65 0.86 >100
13.88 0.09 35.39 0.22 >100 70.62 1.47 63.92 0.35
13.32 0.17 36.93 0.31 85.09 0.46 84.34 2.69
19.31 0.58 21.62 0.28 65.82 0.74 25.47 0.37
>100
>100
>100
>100
>100
76.58 1.67
>100
>100
96.72 1.23
>100
>100
CP
Doxo 4.45 0.02 4.25 0.03 4.29 0.006 4.16 0.01
4.92 0.01 64.58 1.92
IC50 isconcentrationatwhich50%ofcellsundergocytotoxiccelldeathduetocompoundtreatment.
IC50 values are indicated as the mean SD of three independent experiments.
CP = Ciprofloxacin, Doxo = Doxorubicin.
aLung cancer, bpancreatic cancer, ccervical cancer, dbreast cancer, enormal embryonic kidney cells.
3.4 DNA binding
point toward that compound 3o binding strength is good
through the intercalate mode.
3.4a UV- Visible spectra: We explore the absorbance
spectra of 3o-CtDNA interaction with UV-Visible spec-
tra. We observed compound 3o peak was at near
220nm. Although on the following addition of CtDNA
to compound 3o, the compound absorbance was slowly
decreasing, that means hypochromic effect (Figure S12,
Supplementary Information). This hypochromic result
is owing to the overlap of electron cloud of the com-
pound 3o with the CtDNA base pairs and it is a feature of
anintercalatingbindingmode.29–32 Theintrinsicbinding
constant (Kb) was found out from following equation.33
3.4b Fluorescence spectra: The synthesized com-
pound can replace EB from EB-DNA, the fluorescence
of the solution will be quenched owing to the free
EB molecules are readily quenched by the adjacent
water molecules.36,37 The quenching fluorescence of
EB-CtDNA by the compound (3o) is shown in Figure
S14 (Supplementary Information).
The compound 3o had no fluorescence, so the com-
pound binding with CtDNA can’t be predicted directly
by emission spectra. The spectroscopic changes of
EB-CtDNA are often utilized to study the interaction
between CtDNA and newly synthesized molecule.38–40
The quenching of EB-CtDNA by the synthesized
compound3oisingoodagreementwiththelinearStern–
Volmer equation, which gives additional evidence that
3o binds to DNA.
ε
ε
ε
ε
ε
ε
f
[DNA] /| a − | = [DNA]/| b − | + 1/Kb|
−
f
f
b
(1)
Here [DNA] represents the concentration of DNA in
ε
ε
f
base pairs, is apparent extinction coefficient, is the
a
extinction coefficient of the compound in the absence of
I0
ε
DNA and b is extinction coefficient of compound when
= 1 + Ksv [Q]
(2)
I
fully bound to DNA.34,35 Kb is the equilibrium binding
constant (in M–1) of compound binding to DNA.
In the above equation I0 is the emission intensity in the
Kb was calculated by the ratio of slope to inter- absence of quencher, I is the emission intensity in the
cept (Figure S13, Supplementary Information). The Kb presence of quencher, Ksv is the Stern–Volmer quench-
for compound 3o is 9.312 × 104M−1. These outcome ing constant, and [Q] is the quencher concentration.

121 Page 10 of 11
J. Chem. Sci. (2018) 130:121
The linear relationship of I0/I versus [Q] recommend
that the quenching result for this system is a static type,
means non-fluorescence complex is formed between
compound 3o and CtDNA. Stern–Volmer quenching
constant (Ksv) is given by the ratio of the slope to
the intercept (Figure S15, Supplementary Information),
value of Ksv is 5.1 × 104M−1. This statistics obviously
indicate the interaction of 3o with CtDNA.
4. El-Rayes B F, Grignon R, Aslam N, Aranha O and Sarkar
F H 2002 Ciprofloxacin inhibits cell growth and syner-
gises the effect of etoposide in hormone resistant prostate
cancer cells Int. J. Oncol. 21 207
5. Ebisuno S, Inagaki T, Kohjimoto Y and Ohkawa T 1997
The cytotoxic effect of fleroxacin and ciprofloxacin on
transitional cell carcinoma in vitro Cancer 15 2263
6. Herold C, Ocker M, Ganslmayer M, Gerauer H, Hahn E
G and Schuppan D 2002 Ciprofloxacin induces apoptosis
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7. Shaharyar M, Ashraf Ali
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2007 Synthesis and antiproliferative activity
4. Conclusion
of 1-[(sub)]-6-fluoro-3-[(sub)]-1,3,4-oxadiazole-2-yl-7-
piperazino-1,4-dihydro-4-quinolinone derivatives Med.
Chem. Res. 16 292
In summary, we conclude that electron donating sub-
stitution affects the anti-proliferative activity of CP
derivatives. These results reveal the importance of
chloro, methoxy, methyl substituents at dissimilar posi-
tions and further modification on the CP derivatives
could lead to the synthesis of a promising aspirant to
develop potential anti-proliferative agent. Many of the
synthesized compounds do not exhibit toxic effect on
normal human embryonic kidney cell line (HEK) com-
pared with doxorubicin. DNA-binding properties of the
synthesized compounds investigated by fluorescence
clearly denote that the compound can bind to DNA
through intercalation mode.
8. Kozeil R, Szczepanoswska J, Magalska A, Piwocka K,
Duszynski J and Zablock K 2010 Ciprofloxacin inhibits
proliferation and promotes generation of aneuploidy in
Jurkat cells J. Physiol. Pharmacol. 61 233
9. Mondal E R, Das S K and Mukherjee P 2004 Compara-
tive evaluation of antiproliferative activity and induction
of apoptosis by some fluoroquinolones with a human
non-small cell lung cancer cell line in culture Asian Pac.
J. Cancer Prev. 5 196
10. KloskowskiT, GurtowskaN, OlkowskaJ, MarcinNowak
J, Adamowicz J, Tworkiewicz J, Debski R, Grzanka A
and Drewa T 2012 Ciprofloxacin is a potential topoiso-
merase II inhibitor for the treatment of NSCLC Int. J.
Oncol. 41 1943
11. Kloskowski T, Olkowska J, Nazlica A and Drewa T 2010
The influence of ciprofloxacin on hamster ovarian cancer
cell line CHO AA8 Acta Pol. Pharm. 67 345
Supplementary Information (SI)
12. Esmaeilzadeh A, Ebtekar M, Biglar A and Mohammad
Hassan Z 2012 Influence of ciprofloxacin on glioma cell
line GL26: A new application for an old antibiotic Afr.
J. Microbiol. Res. 6 4891
Spectral data for the characterization of compounds are given
in the supplementary information. Supplementary Informa-
tion is available at www.ias.ac.in/chemsci.
13. Aranha O, Wood Jr D P and Sarkar F H 2000
Ciprofloxacin mediated cell growth inhibition, S/G2-M
cell cycle arrest, and apoptosis in a human transitional
cell carcinoma of the bladder cell line Clin. Cancer. Res.
6 891
14. Kloskowski T, Gurtowska N, Nowak M, Joachimiak R,
Bajek A, Olkowska J and Drewa T 2011 The influence
of ciprofloxacin on viability of A549, HepG2, A375.S2,
B16 and C6 cell lines in vitro Acta Pol. Pharm. 68 859
15. YogeeswariP, SriramD, KavyaRandTiwariS2005Syn-
thesis and in-vitro cytotoxicity evaluation of gatifloxacin
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2005 Benzenesulfonamide analogs of fluoroquinolones.
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Acknowledgements
Sincere thanks to the Department of Biotechnology (No.BT/
PR4801/MED/29/370/2012), Government of India, New
Delhi for funding the project. The author NS thanks UGC,
New Delhi for the award of senior research fellowship; we
also thank Devendar for maintaining cell culture.
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