Novel N-4-piperazinyl-ciprofloxacin-chalcone hybrids: Synthesis, physicochemical properties, anticancer and topoisomerase i and II inhibitory activity

Article abstract of DOI:10.1016/j.ejmech.2013.08.040

A group of novel N-4-piperazinyl-ciprofloxacin-chalcone hybrids was prepared. One-dose anticancer test results indicated that compounds 3a and 3g exhibited the highest ability to inhibit the proliferation of different cancer cell lines. Compound 3a exhibited a broad-spectrum of anti-tumor activity without pronounced selectivity while compound 3g revealed high selectivity toward the leukemia subpanel with selectivity ratio of 6.71 at GI₅₀ level. Moreover, compounds 3e and 3j have shown remarkable topo II inhibitory activity compared to etoposide at 100 μM and 20 μM concentrations. Compounds 3e and 3j exhibited comparably potent topo I inhibitory activity at 20 μM concentration compared to camptothecin. Compounds 3e and 3j exhibited strong topo II inhibitory activities compared to topo I at 20 μM concentration. Studying of the solubility and partition coefficient revealed higher lipophilicity of the hybrids 3a-j compared to the parent ciprofloxacin.

Full text of DOI:10.1016/j.ejmech.2013.08.040

Accepted Manuscript
Novel N4-Piperazinyl Ciprofloxacin Chalcone Hybrids: Synthesis, Physicochemical
Properties, Anticancer and Topoisomerase I and II Inhibitory Activity
Mohamed Abdel-Aziz, So-Eun Park, Gamal El-Din A.A. Abuo-Rahma, Mohamed A.
Sayed, Youngjoo Kwon
PII:
S0223-5234(13)00557-6
10.1016/j.ejmech.2013.08.040
EJMECH 6387
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 April 2013
Revised Date: 22 August 2013
Accepted Date: 25 August 2013
Please cite this article as: M. Abdel-Aziz, S.-E. Park, G.E.-D.A.A. Abuo-Rahma, M.A. Sayed, Y.
Kwon, Novel N4-Piperazinyl Ciprofloxacin Chalcone Hybrids: Synthesis, Physicochemical Properties,
Anticancer and Topoisomerase I and II Inhibitory Activity, European Journal of Medicinal Chemistry
(2013), doi: 10.1016/j.ejmech.2013.08.040.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Novel N4-Piperazinyl Ciprofloxacin Chalcone Hybrids: Synthesis,
Physicochemical Properties, Anticancer and Topoisomerase I and II
Inhibitory Activity
Mohamed Abdel-Aziza,, So-Eun Parkb, Gamal El-Din A.A. Abuo-Rahmaa*, Mohamed
A. Sayedc, Youngjoo Kwonb
aDepartment of Medicinal Chemistry, Faculty of Pharmacy, Minia University, 61519
Minia, Egypt.
bCollege of Pharmacy and Graduate School of Pharmaceutical Sciences, Global Top 5
Program, Ewha Womans University, Seoul 120-750, Republic of Korea.
cDepartment of Pharmaceutics, Faculty of Pharmacy, Minia University, 61519 Minia,
Egypt.
*Corresponding author. Tel.: +201003069431; fax: +20862369075
E-mail address: gamalaburahma@yahoo.com (Gamal El-Din A.A. Abuo-Rahma).
Abstract
A group of novel N-4-piperazinyl-ciprofloxacin chalcone hybrids was prepared. One-
dose anticancer test results indicated that compounds 3a and 3g exhibited the highest
ability to inhibit the proliferation of different cancer cell lines. Compound 3a
exhibited a broad-spectrum of antitumor activity without pronounced selectivity while
compound 3g revealed high selectivity toward the leukemia subpanel with selectivity
ratio of 6.71 at GI50 level. Moreover, compounds 3e and 3j have shown remarkable
topo II inhibitory activity compared to etoposide at 100 µM and 20 µM
concentrations. Compounds 3e and 3j exhibited comparably potent topo I inhibitory
activity at 20 µm concentration compared to camptothecin. Compounds 3e and 3j
exhibited strong topo II inhibitory activities compared to topo I at 20 µM
concentration. Studying of the solubility and partition coefficient revealed higher
lipophilicity of the hybrids 3a-j compared to the parent ciprofloxacin.
Keywords: Antitumor, Ciprofloxacin, Chalcone, Topoisomerase, Hybrids.
1

ACCEPTED MANUSCRIPT
_____________________________________________________________________
Graphical abstract
O
F
COOH
O
O
N
H3CO
H3CO
F
COOH
O
N
N
O
N
N
H
O
N
OCH3
N
N
H
3g
3a
GI50 ranging from 0.21 to 57.6 µM
GI50 ranging from 0.43 to 39.6 µM
Novel N-4-piperazinyl-ciprofloxacin chalcone hybrids were prepared and evaluated
for their anticancer and topoisomerase inhibitory activities.
_____________________________________________________________________
1. Introduction
Fluoroquinolones are known synthetic antibacterial agents which have been the
subject of many research interests [1]. Recent reports have shown the importance of
fluoroquinolones as antitumor agents [2]. DNA topoisomerases (topo) are ubiquitous
enzymes that perform essential cellular functions involved in replication,
recombination, and packaging and unfolding of DNA in chromatin [3]. Topo I and II
inhibitors which have various structures with different side chains have been reported
[4,5]. Clinically potent anticancer agents, topo inhibitors like camptothecin [6],
etoposide [7] and doxorubicin [8] bind to the cleavable complex formed between topo
and DNA, and keep it from going back to the original DNA. Now agents directly
inhibiting topo are urgently being requested. Additionally, topo II represents the
cellular target for quinolone antibacterial agents and a wide variety of anticancer
drugs that was attributed to the mechanistic similarities and sequence homologies of
their target topoisomerase II [9]. It is reported that ciprofloxacin inhibits
mitochondrial topoisomerase II and therefore affects cellular energy metabolism. In a
concentration exceeding 80 µg/mL, ciprofloxacin induces apoptosis while at 25
µg/mL concentration it inhibits proliferation of Jurkat cells without any symptoms of
cell death through inhibition of mitosis [10]. Moreover, it was reported that
ciprofloxacin could be used for the experimental adjunctive therapy of lung cancer
[11], ciprofloxacin derivative I (chart I) showed potent in vitro antitumor activity [2].
2

ACCEPTED MANUSCRIPT
Furthermore, in vitro evaluation of ciprofloxacin analogue, trovafloxacin showed its
high effectiveness in inhibition of the growth of P388 murine leukemia cells [12]. It is
well established that, the inhibition of topoisomerase II and physicochemical
properties of fluoroquinolones is greatly affected by the nature of the C-7 substituent
[13]. Studies demonstrated that the introduction of O-benzyl moiety on oxime group
of N-(2-oxyimino) piperazinyl quinolone series changes the biological profile of
piperazinyl quinolones from antibacterial to cytotoxic activity [9].
Naturally occurring and synthetic chalcone derivatives are of current interest
as cytotoxic agents [14,15]. Chalcones had been reported to inhibit cancer cell
proliferation, induce apoptosis in various cell types and exhibit remarkable effect
against skin carcinogenesis [16]. Several mechanisms have been reported for the
cytotoxic action of chalcone derivatives including; inhibiting tubulin polymerization
[17], inhibition of angiogenesis, induction of apoptosis, anti-estrogenic activity and
reversal of multidrug resistance or combination of these mechanisms [18]. A recent
report showed that the 2-phenylquinoline/chalcone hybrid (chalcone II, chart 1) is
highly active against the growth of MDA-MB-231cells with IC50 less than 0.10 µM in
addition to inhibition of H1299SKBR-3, MCF-7 and SKBR-3 cells with IC50 of 0.71,
0.91 and 0.52. µM, respectively [19].
Chart 1
Several reports mentioned that the introduction of a substituent on N-4 piprazinyl
moiety of quinolones altered physicochemical properties of quinolones that may
affect the cytotoxicity of these compounds and selectivity of these hybrids towards
topo I and topo II [20-23]. Based on the above mentioned studies, we report herein the
synthesis of certain C7-piperazinyl ciprofloxacin chalcone hybrids for the purpose of
improvement of the physicochemical properties of ciprofloxacin and/or synergistic
effect through combining ciprofloxacin and chalcone in one compact structure. The
designed compounds were evaluated for their in vitro anticancer activities using
different cancer cell lines in addition to evaluation of their inhibitory activity against
topoisomerase I and II enzymes and DNA unwinding assay. Also, the effect of
introducing the designed N4-piperazinyl substituents on the physicochemical
properties of ciprofloxacin has been studied.
3

ACCEPTED MANUSCRIPT
2. Results and discussion
2.1. Chemistry
The synthesis of designed compounds is outlined in Scheme 1. Chalcone
derivatives 1a-j were synthesized by a base catalyzed Claisen-Schmidt condensation
of 4-aminoacetophenone with different benzaldehyde derivatives [24-26]. Treatment
of the chalcone derivative intermediates 1a-j with bromoacetyl bromide in the
presence of potassium carbonate afforded the corresponding 2-bromo-N-{4-[3-
arylacryloyl]phenyl}acetamides 2a-j in high yields. Alkylation of ciprofloxacin with
the acylated chalcones 2a-j in acetonitril using triethylamine as a base gave the target
ciprofloxacine chalcone hybrids 3a-j in a good yield (Scheme 1) [27]. The prepared
compounds were identified by 1H-NMR, 13C-NMR and mass spectrometry. The purity
of the newly prepared compounds was checked by elemental analyses. The NMR data
for compounds 3a-j showed the known characteristic pattern for both the parent
ciprofloxacin and chalcone derivatives in addition to the characteristic singlet of -N-
CH2- moiety at δ = 4.1-4.4 ppm.
Scheme 1
2.2. Biological investigations
2.2.1. Screening of anticancer activity
Compounds 3a, 3d, 3e, 3g, and 3j were selected by the National Cancer Institute
(NCI) according to the protocol of the Drug Evaluation Branch of the National Cancer
Institute, Bethesda, USA for in vitro anticancer screening [28]. Primary in vitro one-
dose anticancer assay was performed in full NCI 60 cell lines derived from nine tumor
subpanels, including leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate,
and breast cancer cell lines. The selected compounds were added at a single
concentration (10-5 M) and the culture was incubated for 48 h. End point
determination was made with a protein binding dye sulforhodamine B (SRB). Results
for each compound were reported as a mean graph of the percent growth of the treated
cells when compared to the untreated control cells. Compound 3a achieved
remarkable cell growth inhibition activity against most of the tested cell lines
including leukemia, non-small cell lung cancer A549/ATCC, HOP-92, NCI-H23,
4

ACCEPTED MANUSCRIPT
NCI-H460, NCI-H522, colon cancer HT29, KM12, CNS cancer SF-295, SF-539, SF-
539, U251, melanoma LOX IMVI, MDA-MB-435, SK-MEL-5, ovarian cancer
OVCAR-8, renal cancer 786-0, SN12C, breast cancer MCF7, BT-549 cell lines. A
complete cell death was recorded for the colon cancer cell lines HCT-116 where the
growth percent was -83.61. Compound 3a revealed moderate cell growth inhibition
against leukemia HL-60 (TB), colon cancer SW-620, CNS cancer SF-268, SNB-19,
melanoma M14, ovarian cancer IGROV1, renal cancer ACHN, RXF-393, TK-10,
UO-31, prostate cancer DU-145, breast cancer MDA-MB-468 cell lines (Table 1).
Table 1
Compound 3g achieved remarkable cell growth inhibition activity against
most of the tested cell lines (Table 1). A complete cell death was recorded for
leukemia CCRF-CEM, HL-60 (TB), MOLT-4, RPMI-8226, non-small cell lung
cancer A549/ATCC, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-
H522, colon cancer COLO 205, HCC-2998, HCT-116, HT29, KM12, CNS cancer
SF-268, SF-295, SF-539, U251, melanoma LOX IMVI, M14, MDA-MB-435, SK-
MEL-5, UACC-62, ovarian cancer IGROV1, OVCAR-3, renal cancer 786-0, A498,
RXF-393, breast cancer BT-549 and MDA-MB-468 cell lines. Compound 3g
indicated a remarkable cell growth inhibition activity against most of the tested cell
lines including leukemia K-562, non-small cell lung cancer HOP-62, colon cancer
HCT-15, SW-620, ovarian cancer OVCAR-4, OVCAR-5, OVCAR-8, renal cancer
ACHN, SN12C, TK-10, UO-31, prostate cancer PC-3, DU-145, breast cancer MCF7,
MDA-MB-231/ATCC, T-47D cell lines. Compound 3g revealed moderate cell growth
inhibition against CNS cancer SNB-19, melanoma MALME-3M, SK-MEL-2, SK-
MEL-28, UACC-257 and ovarian cancer SK-OV-3 cell lines (Table 1). The results
indicated also that compound 3d exhibited moderate cell growth inhibition against
non-small cell lung cancer NCI-H522, colon cancer HCT-116 cell lines
(Supplementary material), while compound 3e revealed a remarkable cell growth
inhibition activity only against colon cancer HCT-116 cell line (Supplementary
material).
The obtained results indicate that compounds 3a and 3g exhibited the highest ability
to inhibit the proliferation of different cancer cell lines (Table 1) compared to
5

ACCEPTED MANUSCRIPT
compounds 3d, 3e and 3j derivatives. From the obtained results; several conclusions
could be deduced, a different chalcone substituent attached to the piperazinyl moiety
of ciprofloxacin might contribute to the activity of the synthesized compounds; the
presence of three OCH3 groups is preferable over the presence of one OCH3 groups
(compound 3g has superior anticancer activity against different cancer cell lines over
compound 3e). Also the presence of an electron donating group (OCH3) in
compounds 3e and 3g has better anticancer activity against different cancer cell lines
over the presence of an electron withdrawing group (Cl) in compound 3d. Meanwhile,
the unsubstituted derivative 3a exhibited better anticancer activity against different
cancer cell lines than the derivatives containing an electron withdrawing group
(compound 3d).
2.2.2. In vitro five-dose full NCI 60 cell panel assay
Compounds 3a and 3g were selected for advanced five-dose testing against the full
panel of 60 human tumor cell lines. All the 60 cell lines representing nine tumor
subpanels were incubated at five different concentrations (0.01, 0.1, 1, 10 and 100
µM). The outcomes were used to create log concentration versus% growth inhibition
curves and three response parameters (GI50, TGI, and LC50) were calculated for each
cell line. The GI50 value (growth inhibitory activity) corresponds to the concentration
of the compound causing 50% decrease in net cell growth, the TGI value (cytostatic
activity) is the concentration of the compound resulting in total growth inhibition
(TGI) and LC50 value (cytotoxic activity) is the concentration of the compound
causing net 50% loss of initial cells at the end of the incubation period of 48 h. From
the results in Table 2, it is clear that compound 3a exhibited remarkable anticancer
activity against most of the tested cell lines representing nine different subpanels.
Compound 3a showed high activity against most of the tested cell lines with GI50
ranging from 0.43 to 39.6 µM (Table 2). The criterion for selectivity of a compound
depends upon the ratio obtained by dividing the full panel MID (the average
sensitivity of all cell lines toward the test agent) (µM) by their individual subpanel
MID (µM). Ratios between 3 and 6 refer to moderate selectivity; ratios > 6 indicate
high selectivity toward the corresponding cell line, while compounds not meeting
either of these criteria rated nonselective [29]. In this context, compound 3a was
found to have broad-spectrum antitumor activity against the nine tumor subpanels
6

ACCEPTED MANUSCRIPT
tested with selectivity ratios ranging between 0.42 and 1.87 at the GI50 level.
Compound 3a was found to be broad-spectrum antitumor agent against different
tested tumor subpanels with no selectivity toward the tested cell lines.
Table 2
Furthermore, compound 3g exhibited remarkable anticancer activity against most of
the tested cell lines representing nine different subpanels and showed high activity
against most of the tested cell lines with GI50 ranging from 0.21 to 57.6 µM (Table 3).
Compound 3g was found to have broad-spectrum antitumor activity against the tested
nine tumor subpanels with selectivity ratios ranging between 0.29 and 6.71 at the GI50
level. Compound 3g exhibited high selectivity toward the leukemia subpanel with
selectivity ratio of 6.71 at GI50 level and moderate selectivity toward the non-small
cell lung cancer, colon cancer, renal cancer and prostate cancer subpanels with
selectivity ratio of 5.53, 3.17, 3.86 and 4.62 respectively, at GI50 level.
Table 3
2.2.3. Topoisomerase I and II inhibitory activities
The conversion of supercoiled plasmid DNA to relaxed DNA by topo I and II was
examined in the presence of the test compounds 3a-j as shown in Fig. 1 and Fig. 2.
Camptothecin and etoposide, well-known topo I and II inhibitors, respectively, were
used as positive controls. Inhibitory activities were evaluated both at 100 µM and 20
µm. As shown in Fig. 1, all of the tested compounds exhibited remarkable topo I
inhibitory activity at 100 µM concentration. Compounds 3f, 3h, 3i and 3j displayed
the most significant topo I inhibitory activity and they revealed stronger topo I
inhibitory activity than the positive control, camptothecin (Table 4). Compounds 3a,
3b, 3c, 3d, 3e and 3g displayed remarkable topo I inhibitory activity compared to the
positive control, camptothecin (Table 4) at 100 µM concentration. Moreover,
Compounds 3e and 3j exhibited significant topo I inhibitory activity at 20 µM
concentration compared to the positive control, camptothecin (Table 4).
7

ACCEPTED MANUSCRIPT
Figure 1
Figure 2
The topo I inhibitory activities of the tested compounds are summarized in Table 4
and Fig.1. The results indicated that the substituted chalcone derivatives gave good
topo I inhibitory activity at 100 µM concentration than the unsubstituted one.
Moreover, compound 3j gave the best topo I inhibitory activity among the tested
group at 100 µM concentration.
Most of the tested compounds have shown significant topo II inhibitory activity at
100 µM as summarized in Table 4. Compounds 3e and 3j have shown stronger topo II
inhibitory activity than etoposide at 100 µM and 20 µM concentration. Compounds
3h and 3i displayed stronger topo II inhibitory activity than etoposide at 100 µM
concentration. Compounds 3b and 3c exhibited moderate topo II inhibitory activity at
100 µM and 20 µM concentration as shown in Fig. 2 and Table 4. Moreover,
compounds 3e, 3h, 3i and 3j showed both significant topo I and II inhibitory
activities. The results also indicated that compounds 3e and 3j exhibited strong topo II
inhibitory activities compared to topo I at 20 µM concentration. While compound 3e
showed remarkable topo II inhibitory activity compared to topo I at 100 µM
concentration.
The results of topo II inhibitory activity listed in (Table 4) indicated that the entire
compounds with dioxolo moiety (compound 3j) have shown stronger topo II
inhibitory activity among the tested compounds at 100 µM concentration while
compound 3e with p-methoxy substituent has shown stronger topo II inhibitory
activity among the tested compounds at 20 µM concentration. Moreover, increasing
the number of methoxy groups in compounds 3f and 3g have deleterious effect on the
topo II inhibitory activities of these compounds.
Table4
8

ACCEPTED MANUSCRIPT
The obtained results indicated that introduction of the chalcones derivatives into the
N4-piperazinly moiety of ciprofloxacin increases dramatically the anticancer activity
of the tested compounds relative to the very weak anticancer ciprofloxacin.2
Increasing the antiproliferative activity of the tested compounds may be attributed to
either synergistic effect of the chalcone derivatives and/or alteration of the
physicochemical properties of ciprofloxacin. Moreover it is obvious that the type of
substituent on chalcone moiety has a key role in the anticancer activity. Additionally,
there is no direct correlation between the anticancer activity of the tested compounds
and their inhibitory effect against topo I and topo II (c.f compound 3J against 3g).
2.2.4. DNA unwinding assay
DNA unwinding assay was carried out using a supercoiled pHOT1 DNA as a
substrate, since unwinding of the double strand of DNA helix is a practical
characteristic of intercalating drugs [30]. As shown in Fig. 3, amsacrine (m-AMSA), a
well-known DNA intercalator [31,32], blocked unwinding of pHOT1 DNA in the
presence of excess topo I in dose-dependent manner which is consistent with result
previously reported [33] while compound 3e and 3j did not block DNA unwinding at
high concentration up to 1000 µM treatment. Based on this result it is estimated that
compound 3e and 3j do not interact with DNA but interact with topoisomerases for
their inhibitory activity.
Figure 3
2.3. Physicochemical properties
2.3.1. Solubility determination
Solubility of ciprofloxacin and the synthesized hybrids 3a-j was determined at
different pH's of 6.9 and 7.8 at 37oC and the results are listed in Table 5. The pH
solubility profile of ciprofloxacin and its intrinsic solubility as a function of
temperature reported by Yu et al [34] showed that both have an endothermic heat of
solution with minimum solubility at pH~7. In agreement, Ross and Riley [35] found
o
that the intrinsic solubility of fluoroquinolones at 25 C ranged from 0.0297 to 2.75
mg/ml. The results of pH–solubility profiles of ciprofloxacin and hybrids 3a-j showed
a clear decrease in the solubility of the synthesized compounds 3a-j compared to
9

ACCEPTED MANUSCRIPT
ciprofloxacin at different pH's of 6.9 and 7.8 at 37oC. Additionally, ciprofloxacin and
the synthesized hybrids 3a-j showed higher solubility in pH 6.9 over that of pH 7.8.
From the above mentioned results, it is obvious that the introduction of N-4
piperazinyl chalcone substituents decreased the water solubility of ciprofloxacin by
alteration of the zwitter ionic nature of the parent ciprofloxacin that was attributed to
their effect on the N-4 protonation ability [27].
Table 5
2.3.2. Partition coefficient
The experimental partition coefficient (LogPexp) between n-octanol and buffer
for the newly synthesized hybrids 3a-j compared to the parent ciprofloxacin was
determined using traditional shake-flask method [35,36], the results are listed in Table
6. From these results, the experimental partition coefficient (LogPexp) of ciprofloxacin
was found to be -0.1432. Introduction of N-4 piperazinyl chalcone derivatives
significantly altered the partition coefficient pattern in a range from -.0812 to 1.4684
(Table 6). The increasing of LogPexp of the synthesized compounds 3a-j compared to
ciprofloxacin has a promising effect on increasing the lipophilicity of these
compounds that may effect cell penetration.
Table 6
3. Conclusion
A group of novel N-4-piperazinyl-ciprofloxacin chalcone derivatives were prepared
and characterized by their spectral data. One-dose anticancer test results indicated that
compounds 3a and 3g exhibited the highest ability to inhibit the proliferation of
different cancer cell lines. In vitro five-dose full NCI 60 cell panel assay revealed that
compound 3a exhibited a broad-spectrum anti-tumor activity against the nine tumor
subpanels tested without pronounced selectivity while compound 3g revealed high
selectivity toward the leukemia subpanel with selectivity ratio of 6.71 at GI50 level.
Most of the tested compounds have shown good topo I and topo II inhibitory activity
at 100 µM. Compounds 3e and 3j exhibited comparable topo I inhibitory activity at
20 µm concentration compared to the positive control, camptothecin. Additionally,
compounds 3e and 3j have shown remarkable topo II inhibitory activity compared to
10

ACCEPTED MANUSCRIPT
etoposide at 100 µM and 20 µM concentrations. The topo inhibitory activity of
compounds 3e and 3j might be an attribute of affecting to topo not binding to DNA.
Both solubility and partition coefficient determination revealed increase of the lipid
solubility of the prepared hybrids 3a-j compared to the parent ciprofloxacin that may
affect on the cell permeability of these compounds.
This preliminary study of the anti-tumor activity of the compounds 3a and 3g
represents a novel strategy for the discovery of promising lead anticancer compounds
which requires further investigations.
Acknowledgment
Authors thank the Development Therapeutics Program of the National Cancer
Institute, Bethesda, MD, USA, for in vitro evaluation of anticancer activity. This work
was funded by the Faculty of Pharmacy, Minia University, Minia, Egypt (Chemistry,
Physicochemical properties) and by the Basic Science Research Program through the
National Research Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (2010-0002646) (for topoisomerase I & II inhibitory
activities measurement).
4. Experimental section
4.1. Chemistry
Reactions were monitored by TLC: Pre-coated plastic sheets, 0.2 mm silica gel with
fluorescent indicator (Macherey-Nagel). Melting points were determined on Stuart
1
electrothermal melting point apparatus and were uncorrected. H NMR spectra were
recorded using a Bruker Advance 300 MHz NMR spectrometer. 13C NMR spectra
were recorded using a Bruker Advance (75 MHR) spectrometer. LC-MS
measurements were performed on an Agilent Technologies 1100 LC/MSD trap using
TMS as internal reference. Chemical shifts δ values are given in parts per million
(ppm) using CDCl3 (7.29), DMSO-d6 (2.5) or DMSO-d6+CF3COOH as solvents and
coupling constants (J) in Hertz. Splitting patterns are designated as follows: s, singlet;
d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m, multiplet; bs, broad singlet
instrument. Elemental analyses were performed at the analytical laboratory, institute
of Resource Development and analysis, Kumamoto University, Kumamoto, Japan.
11

ACCEPTED MANUSCRIPT
Chalcones 1a-j are synthesized according to a reported procedure [24-26]. The
compounds 2d, 2e and 2j were synthesized as reported [37].
4.1.1. General procedure for the synthesis of 2-bromo-N-{4-[3-arylacryloyl]-
phenyl}acetamides 2a-c, 2f-i.
To a stirred mixture of chalcones 1a-j (6.30 mmol) in dichloromethane (20 mL) and
potassium carbonate (1.30 mmol) in 100 mL water, cool in an ice bath, bromoacetyl
bromide (1.856 g, 9.20 mmol) in 30 mL dichloromethane was added in a dropwise
manner with stirring over 30 min. Stirring was continued for 2 h at 0oC, and at r. t.
overnight. The reaction mixture was extracted with dichloromethane (2 x 60 mL). The
organic layer was washed with distilled water (2 x 40 mL), dried over anhydrous
sodium sulphate, filtered, evaporated under vacuum and the residue was recrystallized
from ethanol.
4.1.1.1. 2-Bromo-N-(4-((E)-3-phenylacryloyl)phenyl)acetamide (2a)
o
1
Pale yellow powder; mp: 157-158 C ; Yield = 1.8 g, 83.72%; H-NMR (300 MHz,
DMSO-d6) δ 10.74 (s, 1H, -NH), 8.19 (d, 2H, J = 8.4 Hz, Ar-H), 7.90-7.72 (m, 6H, 5
13
Ar-H + =CH), 7.51-7.44 (m, 3H, 2 Ar-H + =CH), 4.11 (s, 2H, -CH2); C-NMR (75
MHz, DMSO-d6) δ 187.56, 165.34, 143.45, 143.10, 134.73, 132.83, 130.47, 129.91,
128.86, 128.76, 120.20, 118.68, 30.28; LC-MS: m/z (C17H14BrNO2) = 344 (M++1,
100%), 346.0 (92%).
4.1.1.2. 2-Bromo-N-(4-((E)-3-(2-chlorophenyl)acryloyl)phenyl)acetamide (2b)
o
1
Yellow powder; mp: 188-189 C; Yield = 1.7 g, 71.73%; H-NMR (300 MHz,
DMSO-d6) δ 10.81(s, 1H, -NH), 8.24-8.02 (m, 3H, 2Ar-H + =CH), 7.82 (d, 2H, J =
8.4 Hz, Ar-H), 7.66-7.55 (m, 2H, Ar-H), 7.52-7.44 (m, 3H, 2Ar-H + =CH), 4.14 (s,
2H, -CH2); 13C-NMR (75 MHz, DMSO-d6) δ 187.34, 165.35, 143.12, 138.04, 134.28,
132.52, 132.38, 131.76, 130.05, 130.01, 129.93, 128.46, 127.57, 124.68, 118.71,
118.66, 30.26; LC-MS: m/z (C17H13BrClNO2) = 378 (M++1, 92%), 380 (100%), 382
(30%).
4.1.1.3. 2-Bromo-N-(4-((E)-3-(3-chlorophenyl)acryloyl)phenyl)acetamide (2c)
12

ACCEPTED MANUSCRIPT
o
Pale yellow powder; mp: 157-158 C ; Yield = 1.9 g, 80.20%; 1H-NMR (300 MHz,
DMSO-d6)δ 10.76 (s, 1H, -NH), 8.22 (d, 2H, J = 8.4 Hz, Ar-H), 8.07-8.01 (m, 2H,
Ar-H), 7.83-7.68 (m, 4H, 3 Ar-H + =CH), 7.52-7.45 (m, 2H, 1Ar-H + =CH), 4.12 (s,
2H, -CH2); 13C-NMR (75 MHz, DMSO-d6) δ 187.43, 165.34, 143.05, 141.68, 137.01,
136.99, 133.78, 132.67, 132.61, 130.57, 130.06, 129.95, 127.84, 127.75, 123.55,
118.65, 30.24; LC-MS: m/z (C17H13BrClNO2) = 378 (M++1, 92%), 380.0 (100%),
382 (31%).
4.1.1.4. 2-Bromo-N-(4-((E)-3-(3,4-dimethoxyphenyl)acryloyl)phenyl)acetamide
(2f)
o
1
Yellow powder; mp: 149-150 C ; Yield = 2.0 g, 79.05%; H-NMR (300 MHz,
DMSO-d6)δ 10.75 (s, 1H, -NH), 8.19 (d, 2H, J = 8.6 Hz, 2H, Ar-H), 7.82-7.73 (m,
4H, 3Ar-H + =CH), 7.56-7.54 (m, 2H, 1Ar-H + =CH), 7.03 (d, 1H, J= 8.6 Hz, Ar-H),
13
4.12 (s, 2H, -CH2), 3.89 (s, 3H, -OCH3), 3.83 (s, 3H, -OCH3); C-NMR (75 MHz,
DMSO-d6) δ 187.40, 165.31, 151.00, 149.05, 143.98, 142.72, 133.11, 130.26, 129.76,
129.60, 127.62, 123.82, 119.50, 118.62, 55.70, 55.60, 30.57; LC-MS: m/z
(C19H18BrNO4) = 404 (M++1, 92%), 406 (100%).
4.1.1.5. 2-Bromo-N-(4-((E)-3-(3,4,5-trimethoxyphenyl)acryloyl)phenyl)acetamide
(2g)
o
1
Yellow powder; mp: 166-167 C ; Yield = 2.3 g, 84.56%; H-NMR (300 MHz,
DMSO-d6) δ 10.70 (s, 1H, -NH), 8.17 (d, 2H, J = 8.6 Hz, Ar-H), 7.84-7.65 (m, 4H,
2Ar-H+2 CH=), 7.21 (s, 2H, Ar-H), 4.10 (s, 2H, -CH2), 3.85 (s, 6H, 2-OCH3), 3.70 (s,
3H. –OCH3); 13C-NMR (75 MHz, DMSO-d6) δ 187.50, 165.34, 153.08, 152.90,
144.00, 142.86, 132.97, 130.26, 129.89, 121.10, 118.63, 106.50, 60.10, 56.12, 30.27;
LC-MS: m/z (C20H20BrNO5) = 434 (M++1, 92%), 436 (100%).
4.1.1.6. 2-Bromo-N-(4-((E)-3-(3-nitrophenyl)acryloyl)phenyl)acetamide (2h)
o
1
Yellow powder; mp: 190-191 C; Yield = 2.0 g, 81.97%; H-NMR (300 MHz,
DMSO-d6)δ 10.71(s, 1H, -NH), 8.68 (s, 1H, Ar-H), 8.26-8.05 (m, 5H, Ar-H), 7.78-
7.52 (m, 4H, 2Ar-H + -CH=CH), 4.09 (s, 2H, -CH2); 13C-NMR (75 MHz, DMSO-d6)
δ 187.31, 165.33, 148.32, 143.12, 140.78, 136.60, 134.89, 132.46, 130.20, 130.08,
13

ACCEPTED MANUSCRIPT
124.52, 124.41, 122.80, 118.62, 30.25; LC-MS: m/z (C17H13BrN2O4) = 389 (M++1,
100%), 391.0 (90%).
4.1.1.7. 2-Bromo-N-(4-((E)-3-(2,4-dimethylphenyl)acryloyl)phenyl)acetamide (2i)
o
Buff red powder; mp: 182-183 C; Yield = 1.90 g, 81.55%; 1H-NMR (300 MHz,
DMSO-d6)δ 10.81(s, 1H, -NH), 8.18 (d, J = 8.4 Hz, 2H, Ar-H), 8.00-7.36 (m, 5H, 3
Ar-H + -CH=CH), 7.18-7.09 (m, 2H, Ar-H), 4.13 (s, 2H, -CH2), 2.41(s, 3H, -CH3),
2.32 (s, 3H. -CH3); 13C-NMR (75 MHz, DMSO-d6) δ 187.59, 165.32, 142.84, 140.53,
140.15, 137.86, 132.99, 131.38, 130.53, 129.80, 127.04, 126.78, 121.66, 118.68,
30.27, 20.88, 19.20; LC-MS: m/z (C19H18BrNO2) = 372 (M++1, 92%), 374 (100%).
4.1.2. General Procedure for synthesis of compounds 3a-j
To a mixture of ciprofloxacin (0.662 g, 0.02 mol) and the respective chalcone
derivative (0.02 mol) in acetonitril (50 ml), TEA (0.04 M) was added. The mixture
was heated under reflux for 5 hr. The precipitate formed was filtered off while hot,
washed with acetonitril and dried under vacuum.
4.1.2.1. 7-(4-((4-((E)-3-(Phenylacryloyl)phenylcarbamoyl)methyl)piperazin-1-yl)-
1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic acid (3a)
1-
Pale yellow powder; m.p: 269-270°C; Yield = 0.80 g, 67.0%; NMR (300 MHz,
DMSO-d6) δ 11.20 (s, 1H, -NH), 8.78 (s, 1H, H-2), 8.23 (d, 2H, J = 8.4 Hz, Ar-H)
7.97-7.70 (m, 7H, 5 Ar-H + =CH + H-5), 7.64 (d, 2H, J = 7 Hz, H-8), 7.56-7.47 (m,
2H, Ar-H + =CH), 4.46 (s, 2H, -CH2), 4.24-3.71 (m, 9H, -N-CH cyclopropyl + 8H,
piperazinyl-H), 1.44-1.33 (m, 2H, cyclopropyl-H), 1.24-1.22 (m, 2H, cyclopropyl-H);
13C-NMR (75 MHz, DMSO-d6) δ 187.35, 176.22, 165.77, 163.30, 159.16, 158.65,
158.14, 157.63, 152.50, 146.50, 143.40, 142.01, 134.59, 133.00, 132.00, 128.54,
120.63, 118.82, 116.82, 113.00, 109.19, 106.81, 60.50, 51.48, 37.96, 33.20, 7.38; LC-
MS: m/z = 595.4, M+1 (100%) Anal. Calcd. C34H31FN4O5.0.25H2O (594.23): C,
68.16; H, 5.30; N, 9.35. Found: C, 68.22; H, 5.22; N, 9.44.
4.1.2.2. 7-(4-((4-((E)-3-(2-Chlorophenyl)acryloyl)phenylcarbamoyl)methyl)-
piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid (3b)
14

ACCEPTED MANUSCRIPT
Pale yellow powder; m.p: 248-250 °C; Yield = 0.88 g, 70.40%; H-NMR (300 MHz,
DMSO-d6) δ 11.10 (s, 1H, -NH), 8.68 (s, 1H, H-2), 8.24 (d, 2H, J = 8.4 Hz, Ar-H),
8.19-8.08 (m, 4H, 4 Ar-H), 7.98-7.86 (m, 3H, Ar-H + =CH + H-5), 7.65 (d, 1H, J = 9
Hz, H-8), 7.57-7.42 (m, 2 H, Ar-H + =CH), 4.46 (s, 2H, -CH2), 3.89-3.46 (m, 9H, -N-
CH cyclopropyl + piperazinyl 8H), 1.45-1.38 (m, 2H, cyclopropyl H), 1.24-1.20 (m,
2H, cyclopropyl H); 13C-NMR (75 MHz, DMSO-d6) δ 187.35, 175.00, 163.29,
161.66, 159.15, 158.63, 158.12, 157.60, 152.50, 142.50, 134.40, 132.28, 131.37,
129.20, 123.00, 120.56, 118.81, 118.75, 116.75, 112.93, 109.12, 106.90, 61.10, 51.80,
38.04, 34.00, 7.40; LC-MS: m/z = 629.4, M+1 (100%), 631.4 (32%). Anal. Calcd.
C34H30ClFN4O5 (628.19): C, 64.91; H, 4.81; N, 8.91. Found: C, 64.63; H, 4.74; N,
8.88.
4.1.2.3. 7-(4-((4-((E)-3-(3-Chlorophenyl)acryloyl)phenylcarbamoyl)methyl)-
piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid (3c)
1
Pale yellow powder; m.p: 290-292 °C; Yield = 0.6 g, 48%; H-NMR (300 MHz,
DMSO-d6) δ 10.90 (s, 1H, -NH), 8.69 (s, 1H, H-2), 8.20 (d, 2 H, J = 8.4 Hz, Ar-H),
7.95-7.60 (m, 8H, 4 Ar-H, H-5 + H-8 + 2 =CH), 7.46-7.44 (m, 2H, Ar-H), 4.41 (s, 2H,
-CH2), 3.89-3.60 (m, 8 H, piperazinyl-H), 3.43-3.39 (m, 1H, -N-CH cyclopropyl)
13
1.44-1.35 (m, 2H, cyclopropyl-H), 1.24-1.19 (m, 2H, cyclopropyl- H); C-NMR (75
MHz, DMSO-d6) δ 187.35, 175.00, 165.62, 163.08, 158.47, 157.96, 157.44, 147.50,
143.48, 141.95, 140.10, 138.20, 136.93, 133.88, 130.70, 129.68, 127.63, 120.65,
119.51, 116.83, 113.02, 109.21, 107.50, 58.80, 52.10, 38.20, 34.20, 7.50; LC-MS:
m/z = 629.4, M+1 (100%), 631.4 (32%). Anal. Calcd. C34H30ClFN4O5 (628.19): C,
64.91; H, 4.81; N, 8.91. Found: C, 64.65; H, 4.76; N, 9.31.
4.1.2.4. 7-(4-((4-((E)-3-(4-Chlorophenyl)acryloyl)phenylcarbamoyl)methyl)-
piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid (3d)
1
Pale yellow powder; m.p: >300°C; Yield = 0.85 g, 68%; H-NMR (300 MHz,
DMSO-d6) δ 11.09 (s, 1H, -NH), 8.70 (s, 1H, H-2), 8.25 (d, 2H, J = 8.4 Hz, Ar-H)
8.01-7.84 (m, 6H, 4 Ar-H + H-5 + =CH), 7.78-7.64 (m, 2H, H-8 + =CH), 7.53 (d, 2H,
Ar-H, J = 8.4 Hz, Ar-H), 4.45 (s, 2H, -CH2), 3.89-3.50 (m, 9H, -N-CH cyclopropyl +
15

ACCEPTED MANUSCRIPT
piperazinyl H), 1.43-1.36 (m, 2H, cyclopropyl H), 1.25-1.20 (m, 2H, cyclopropyl H);
13C-NMR (75 MHz, DMSO-d6) δ 187.35, 175.50, 165.78, 163.20, 159.15, 158.64,
158.12, 157.61, 152.10, 146.20, 142.02, 135.01, 133.57, 132.10, 130.27, 129.93,
128.76, 122.42, 120.65, 118.85, 116.83, 113.01, 109.20, 106.85, 66.33, 52.50, 38.15,
35.71, 7.40. LC-MS: m/z = 629.4, M+1 (100%), 631.4 (32%). Anal. Calcd.
C34H30ClFN4O5 (628.19): C, 64.91; H, 4.81; N, 8.91. Found: C, 64.65; H, 4.64; N,
9.31.
4.1.2.5. 7-(4-((4-((E)-3-(4-Methoxyphenyl)acryloyl)phenylcarbamoyl)methyl)-
piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid (3e)
o
1
Pale yellow powder; m.p: 286-288 C; Yield = 0.90 g, 72.10%; H-NMR (300 MHz,
DMSO-d6)δ 11.10 (s, 1H, -NH), 8.75 s, 1H, H-2), 8.20 (d, 2H, J = 8.4 Hz, Ar-H)
7.87-7.75 (m, 7 H, 4 Ar-H, H-5 + 2 =CH), 7.65 (d, 1H, J = 8 Hz, H-8), 7.02 (d, 2H, J
= 8.4 Hz, Ar-H), 4.45 (s, 2H, -CH2), 3.87 (s, 3H, -OCH3), 3.85-3.52 (m, 9H, -N-CH
cyclopropyl H, piperazinyl-H), 1.42-1.37 (m, 2H, Cyclopropyl H), 1.25-1.22 (m, 2H,
Cyclopropyl H); 13C-NMR (75 MHz, DMSO-d6) δ 187.34, 175.20, 165.80, 161.25,
159.15, 158.63, 158.11, 157.60, 143.51, 141.30, 139.40, 133.46, 130.80, 129.56,
127.18, 120.54, 118.77, 116.73, 114.09, 112.92, 109.11, 106.86, 59.50, 55.50, 51.51,
38.03, 29.91, 7.80; LC-MS: m/z = 625.5, M+1 (100%) Anal. Calcd. for
C35H33FN4O6.0.25H2O (624.24): C, 66.81; H, 5.37; N, 8.91. Found: C, 66.61; H, 5.15;
N, 8.83.
4.1.2.6. 7-(4-((4-((E)-3-(3,4-Dimethoxyphenyl)acryloyl)phenylcarbamoyl)methyl)-
piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid (3f)
1
Yellow powder; m.p: 236-237 °C; Yield = 0.77 g, 59.20%; H-NMR (300 MHz,
CDCl3) δ 14.92 (s, 1H, -COOH), 9.28 (s, 1H, -NH), 8.64 (s, 1H, H-2), 7.99 (d, 2H, J =
8.4 Hz, Ar-H) 7.89 (d, 1H, JHF = 12.8 Hz, H-5), 7.75-7.68 (m, 3 H, 2 Ar-H + =CH),
7.39-7.15 (m, 4H, 2 Ar-H, H-8, =CH) 6.89 (d, 1H, J = 8.4 Hz, Ar-H), 4.00 (s, 2H, -
CH2), 3.97 (s, 3H, -OCH3), 3.92 (s, 3H, -OCH3) 3.57-3.29 (m, 5H, -N-CH
cyclopropyl-H + piperazinyl-H), 3.10-2.85 (m, 4H, piperazinyl-H), 2.91-2.68 (m, 4H,
piperazinyl H), 1.43-1.37 (m, 2H, Cyclopropyl H), 1.22-1.17 (m, 2H, Cyclopropyl H);
16

ACCEPTED MANUSCRIPT
13C-NMR (75 MHz, DMSO-d6) 188.77, 175.60, 166.80, 161.25, 159.40, 158.10,
157.60, 155.23, 151.5, 147.31, 145.45, 144.40, 141.41, 138.98, 134.16, 129.80,
127.82, 124.60, 123.50, 119.20, 118.88, 112.60, 107.94, 104.95, 62.01, 57.50, 56.02,
53.18, 49.81, 35.37, 8.20; LC-MS: m/z = 655.4, M+1 (100%); Anal. Calcd. for
C36H35FN4O7 (654.25): C, 66.04; H, 5.39; N, 8.56. Found: C, 65.76; H, 5.23; N, 8.44.
4.1.2.7. 7-(4-((4-((E)-3-(3,4,5-Trimethoxyphenyl)acryloyl)phenylcarbamoyl)-
methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3g)
1
Pale yellow powder; m.p: 271-273 °C. Yield = 0.72 g (53.0%); H-NMR (300 MHz,
DMSO-d6 + CF3COOH) δ 11.10 (s, 1H, -NH), 8.71 (s, 1H, H-2), 8.26 (d, 2H, J = 8.4
Hz, Ar-H), 8.00-7.95 (m, 4 H, 2 Ar-H, + H-5 + =CH), 7.75-7.65 (m, 2H, H-8 + =CH),
7.24 (s, 2H, Ar-H), 4.44 (s, 2H, -CH2), 3.90 (s, 6H, 2-OCH3); 3.76 (s, 3H, -OCH3),
3.85-3.50 (m, 8H, piperazinyl-H), 3.25-3.10 (m, 1H, -N-CH Cyclopropyl), 1.42-1.37
(m, 2H, Cyclopropyl-H), 1.25-1.19 (m, 2H, Cyclopropyl-H); 13C-NMR (75 MHz,
DMSO-d6) δ 187.60, 175.40, 165.80, 161.30, 159.14, 158.63, 158.12, 157.61, 153.05,
149.20, 147.50, 142.20, 136.10, 132.00, 130.30, 120.66, 118.84, 116.84, 113.03,
109.21, 106.86, 106.31, 79.00, 55.87, 55.20, 53.20, 52.50, 34.50, 8.30. LC-MS: m/z =
685.5, M+1 (100%); Anal. Calcd. for C37H37FN4O8.0.25 H2O (684.3): C, 64.48; H,
5.48; N, 8.13. Found: C, 64.43; H, 5.36; N, 8.15.
4.1.2.8. 7-(4-((4-(( E)-3-(3-Nitrophenyl)acryloyl)phenylcarbamoyl)methyl)-
piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylic
acid (3h)
1
Pale yellow powder; m.p: > 300 °C; Yield = 0.88 g, 70.40%; H-NMR (300 MHz,
DMSO-d6 + CF3COOH) δ 11.13 (s, 1H, -NH), 8.79 (s, 1H, H-2), 8.71 (s, 1H, Ar-H)
8.39-8.17 (m, 5 H, 4 Ar-H + =CH)), 8.01 (d, 1H, J = 13 Hz, H-5), 7.91-7.70 (m, 4 H,
2 Ar-H + H-8 + =CH), 7.65 (d, 1H, J = 8.4 Hz, Ar-H), 4.44 (s, 2H, -CH2), 3.92-3.39
(m, 9H, -N-CH cyclopropyl-H + 8 piperazinyl-H), 1.42-1.32 (m, 2H, Cyclopropyl-H),
1.23-1.20 (m, 2H, Cyclopropyl-H); 13C-NMR (75 MHz, DMSO-d6) 187.34, 175.50,
167.00, 165.20, 159.15, 158.64, 158.13, 157.61, 142.50, 141.30, 140.70, 139.30,
138.60, 135.00, 130.20, 126.10, 124.48, 120.71, 118.87, 116.89, 113.07, 112.50,
109.25, 106.84, 61.20, 51.51, 46.30, 34.50, 9.10; LC-MS: m/z = 640.50, M+1 (100%)
17

ACCEPTED MANUSCRIPT
Anal. Calcd. for C34H30FN5O7.H2O (639.63): C, 62.09; H, 4.90; N, 10.65. Found: C,
61,93; H, 4.52; N, 10.55.
4.1.2.9. 7-(4-((4-((E)-3-(2,4-Dimethylphenyl)acryloyl)phenylcarbamoyl)-
methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3i)
1
Pale yellow powder; m.p: 270-272 °C; Yield = 0.85 g, 68.50%; H-NMR (300 MHz,
DMSO-d6 + CF3COOH) δ 11.14 (s, 1H, -NH), 8.60 (s, 1H, H-2), 8.13 (d, 2H, J = 8.4
Hz, Ar-H) 7.96-7.71 (m, 7 H, 3 Ar-H + H-5 + H-8 + 2 =CH), 7.03 (d, 2 H, J= 6.0 Hz,
Ar-H), 4.37 (s, 2H, -CH2), 3.96-3.64 (m, 9H, -N-CH cyclopropyl-H, 8 piperazinyl-H),
2.38 (s, 3H, -CH3), 2.32 (s, 3H, -CH3), 1.47-1.29 (m, 2H, Cyclopropyl-H), 1.24-1.14
(m, 2H, Cyclopropyl-H); 13C-NMR (75 MHz, DMSO-d6) δ 187.34, 174.90, 165.78,
162.50, 159.15, 158.64, 158.13, 157.61, 142.50, 140.70, 139.30, 138.60, 128.50,
126.10, 122.50, 120.61, 118.80, 118.20, 116.80, 114.90, 112.98, 109.17, 106.60,
105.40, 61.20, 51.51, 46.30, 34.50, 28.91, 28.60, 9.10. LC-MS: m/z = 623.4, M+1
(100%) Anal. Calcd. for C36H35FN4O5.0.5 H2O (622.26): C, 68.45; H, 5.74; N, 8.87.
Found: C, 68.34; H, 5.45; N, 8.81.
4.1.2.10. 7-(4-((4-((E)-3-(Benzo[d][1,3]dioxol-5-yl)acryloyl)phenylcarbamoyl)-
methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-
carboxylic acid (3j)
1
Pale yellow powder; m.p: 296-298 °C; Yield = 1.0 g, 78.40%; H-NMR (300 MHz,
DMSO-d6 + CF3COOH) δ 11.10 (s, 1H, -NH), 8.73 (s, 1H, H-2), 8.18 (d, 2H, J = 8.4
Hz, Ar-H), 8.02-7.53 (m, 6 H, 3 Ar-H, H-5 + 2 =CH), 7.28 (d, 1 H, J = 8.4 Hz, H-8),
6.94 (d, 2H, J = 8.4 Hz, Ar-H), 6.10 (s, 2H, -OCH2O), 4.42 (s, 2H, -CH2), 4.05-3.40
(m, 9H, -N-CH cyclopropyl-H + 8 piperazinyl-H), 1.44-1.37 (m, 2H, cyclopropyl-H),
13
1.24-1.19 (m, 2H, cyclopropyl-H); C-NMR (75 MHz, DMSO-d6) δ 187.35, 176.22,
165.62, 163.13, 158.50, 157.90, 156.50, 152.50, 149.46, 148.09, 146.50, 143.40,
141.73, 138.99, 134.50, 129.55, 129.26, 120.76, 119.15, 116.94, 113.12, 109.31,
106.81, 101.45, 64.8, 62.50, 51.48, 46.30, 35.50, 7.38. LC-MS: m/z = 639.40, M+1
(100%) Anal. Calcd. C34H30ClFN4O5.H2O (638.22): C, 64.02; H, 5.07; N, 8.53.
Found: C, 63.80; H, 4.64; N, 8.38.
18

ACCEPTED MANUSCRIPT
4.2. Biological assays
4.2.1. Anticancer activity
The methodology of the NCI anticancer screening has been described in detail
elsewhere (http://www.dtp.nci.nih.gov) [27]. Briefly, the primary anticancer assay
was performed at approximately 60 human tumor cell lines panel derived from nine
neoplastic diseases, in accordance with the protocol of the Drug Evaluation Branch,
National Cancer Institute, Bethesda. Tested compounds were added to the culture at a
single concentration (10-5 M) and the cultures were incubated for 48 h. End point
determinations were made with a protein binding dye, SRB. Results for each tested
compound were reported as the percent of growth of the treated cells when compared
to the untreated control cells. The percentage growth was evaluated
spectrophotometrically versus controls not treated with test agents. The cytotoxic
and/or growth inhibitory effects of the most active selected compound were tested in
vitro against the full panel of about 60 human tumor cell lines at 10-fold dilutions of
five concentrations ranging from 10-4 to 10-8 M. A 48-h continuous drug exposure
protocol was followed and an SRB protein assay was used to estimate cell viability or
growth. Using the seven absorbance measurements [time zero (Tz), control growth in
the absence of drug (C), and test growth in the presence of drug at the five
concentration levels (Ti)], the percentage growth was calculated at each of the drug
concentrations levels. Percentage growth inhibition was calculated as: [(Ti - Tz)/(C -
Tz)] -100 for concentrations for which Ti > Tz, and [(Ti - Tz)/Tz] - 100 for
concentrations for which Ti < Tz. Three-dose response parameters were calculated for
each compound. Growth inhibition of 50% (GI50) was calculated from [(Ti -Tz)/(C -
Tz)] - 100 = 50, which is the drug concentration resulting in a 50% lower net protein
increase in the treated cells (measured by SRB staining) as compared to the net
protein increase seen in the control cells. The drug concentration resulting in TGI was
calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction
in the measured protein at the end of the drug treatment as compared to that at the
beginning) indicating a net loss of cells following treatment was calculated from [(Ti -
Tz)/Tz] - 100 = - 50. Values were calculated for each of these three parameters if the
level of activity is reached; however, if the effect was not reached or was exceeded,
the value for that parameter was expressed as more or less than the maximum or
19

ACCEPTED MANUSCRIPT
minimum concentration tested. The log GI50, log TGI, and log LC50 were then
determined, defined as the mean of the logs of the individual GI50, TGI, and LC50
values. The lowest values are obtained with the most sensitive cell lines. Compound
having logGI50 values -4 and <-4 was declared to be active.
4.2.2. Topo I Topo II inhibitory activity
DNA topo I inhibition assay was determined following the previously reported
method [38]. The prepared compounds were dissolved in DMSO at 20 mM as stock
solution. The activity of DNA topo I was determined by assessing the relaxation of
supercoiled DNA pBR322. The mixture of 100 ng of plasmid pBR322 DNA and 0.4
units of recombinant human DNA topo I (TopoGEN INC., USA) was incubated
without and with the prepared compounds at 37 oC for 30 min in the relaxation buffer
(10 mM Tris-HCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM
spermidine, 5% glycerol). The reaction in the final volume of 10 mL was terminated
by adding 2.5 mL of the stop solution containing 5% sarcosyl, 0.0025% bromophenol
blue, and 25% glycerol. DNA samples were then electrophoresed on a 1% agarose gel
at 15 V for 7 h with a running buffer of TAE. Gels were stained for 30 min in an
aqueous solution of ethidium bromide (0.5 mg/mL). DNA bands were visualized by
transillumination with UV light and were quantitated using AlphaImagerTM (Alpha
Innotech Corporation). DNA topo II inhibitory activity of compounds was measured
as follows [39]. The mixture of 200 ng of supercoiled pBR322 plasmid DNA and 1
unit of human DNA topo IIα (TopoGEN INC., USA) was incubated without and with
the prepared compounds in the assay buffer (10 mM Tris-HCl (pH 7.9) containing 50
mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15 mg/mL bovine serum
albumin) for 30 min at 30 oC. The reaction in a final volume of 20 mL was terminated
by the addition of 3 mL of 7 mM EDTA. Reaction products were analyzed on 1%
agarose gel at 25 V for 4 h with a running buffer of TAE. Gels were stained for 30
min in an aqueous solution of ethidium bromide (0.5 mg/mL). DNA bands were
visualized by transillumination with UV light and supercoiled DNA was quantitated
using AlphaImagerTM (Alpha Innotech Corporation). The added amount of DMSO for
each reaction mixture was same in both of enzyme only and enzyme with designated
compound for topo I and II assays. Etoposide, and camptothecin were purchased from
Sigma and used as positive controls.
20

ACCEPTED MANUSCRIPT
4.2.3. DNA unwinding assay
One hundred ng of pHOT1 plasmid DNA (TopoGEN INC., USA) was relaxed by
incubation with 3 units of human DNA topo I (TopoGEN INC., USA) in topo I
reaction buffer (10 mM Tris-HCl pH 7.9) for 30 min at 37 °C. Then variable amounts
of the testing compounds were added and a total 10 ꢀL of reaction mixture was
continued for additional incubation for 30 min at 37 °C. The reaction was terminated
by the addition of 3 ꢀL of 7 mM EDTA. Reaction products were analyzed on 1%
agarose gel at 15 V for 15 h with a running buffer of TAE. Gels were stained for 30
min in an aqueous solution of ethidium bromide (0.5 ꢀg/ml). DNA bands were
visualized by transillumination with UV light and supercoiled DNA was quantitated
using AlphaImagerTM (Alpha Innotech Corporation). m-AMSA was used as a positive
control.
4.3. Physicochemical Properties
4.3.1. Solubility determination
The aqueous solubility of ciprofloxacin and their hybrids 3a-j in a
buffer of pH's of 6.9 and 7.8, at 37 ºC was measured. A known excess (10
mg) of the prepared compounds 3a-j or ciprofloxacin were placed into
suitable stopper sealed Erlenmeyer flasks containing fixed volume of
buffer of pH's of 6.9 and 7.8. Flasks were equilibrated for 48 hours, in a
temperature controlled shaking water bath maintained at 37 ºC and
shaked at rate of 100 rpm. Preliminary experiments indicated that after 48
hrs all solutions were saturated. After the equilibrium was reached, the
excess solid was allowed to settle down and filtered. Aliquots of the
filtrate properly diluted with the corresponding solvents to obtain
absorbance in the linear calibration range for each system and the
composition of the liquid phase was determined by UV spectroscopy at
corresponding wave length, λmax of 272 nm. All the experiment results
were an average of at least three agreeing independent measurements.
21

ACCEPTED MANUSCRIPT
4.3.2. Determination of partition coefficient
The experimental partition coefficient (Pexp) between n-octanol and phosphate buffer
was determined by a slight modification of the method earlier described by Vazquez
et al [35, 40]. Briefly, 100 µL of a stock solution (0.2 mg/mL) was diluted with 1.9
mL of appropriate phosphate buffer solution (pH 7.4) and mixed with 2 mL of octanol
(the organic and aqueous phase was mutually saturated), the vials were protected from
light by wrapping in aluminum foil. The two phases were vortexed for 3 min and
agitated for 5 h in a shaking water bath at 25 + 0.1 oC. After equilibration, the octanol
phase was removed with a Pasteur pipette and both phases were assayed
spectrophotometrically (λmax =272 nm) to determine drug concentration. The partition
coefficient was calculated as the ratio between molar concentration in n-octanol and
aqueous phase. The total concentration in both phases was measured by
spectrophotometry and the experimental partition coefficients (Log pexp) was
calculated by the following equation: Log p o/w = log [C] octanol / [C] aqueous
All partition coefficient determinations were made in triplicate. Spectronic Genesys,
connected to an IBM computer loaded with the Winspec application software (Milton
Roy, USA) and Jenway 6505 (Jenway LTD., UK) ultraviolet–visible
spectrophotometers with matched 1 cm quartz cells were used throughout this study
for all measurements.
References
[1] H. Koga, A. Itoh, S. Murayama, S. Suzue, T. Irikura, Structure-activity
relationships of antibacterial 6,7- and 7,8-disubstituted 1-alkyl-1,4-dihydro-4-
oxoquinoline-3-carboxylic acids, J. Med. Chem. 23 (1980) 1358-1363.
[2] J. Azéma, B. Guidetti, J. Dewelle, B. Le Calve, T. Mijatovic, A. Korolyov, J.
Vaysse, M. Malet-Martino, R. Martino, R. Kiss, R. 7-((4-Substituted)piperazin-
1-yl) derivatives of ciprofloxacin: synthesis and in vitro biological evaluation as
potential antitumor agents, Bioorg. Med. Chem. 17 (2009) 5396-5407.
[3] J.C. Wang, DNA topoisomerases, Ann. Rev. Biochem. 65 (1996) 635-692.
[4] S. Matsui, W. Endo, C. Wrzosek, K. Haridas, P. Seetharamulu, F. Hausheer, Y.
Rustum, Characterisation of a synergistic interaction between a thymidylate
22

ACCEPTED MANUSCRIPT
synthase inhibitor, ZD1694, and a novel lipophilic topoisomerase I inhibitor
karenitecin, BNP1100: mechanisms and clinical implications, Eur. J. Cancer 35
(1999) 984-993.
[5] S. Alper, T. Arpaci, E. Aki, I. Yalcin, Some new bi- and ter-benzimidazole
derivatives as topoisomerase I inhibitors, IL Farmaco 58 (2003) 497-507.
[6] Y.H. Hsiang, R. Hertzberg, S. Hecht, L.F. Liu, Camptothecin induces protein-
linked DNA breaks via mammalian DNA topoisomerase I, J. Biol. Chem. 260
(1985) 14873-14878.
[7] K.C. Chow, T.L. MacDonald, W.E. Ross, DNA binding by epipodophyllotoxins
and N-acyl anthracyclines: implications for mechanism of topoisomerase II
inhibition, Mol. Pharmacol. 34 (1988) 467-473.
[8] K.M. Tewey, T.C. Rowe, L. Yang, B.D. Halligan, L.F. Liu, Adriamycin-induced
DNA damage mediated by mammalian DNA topoisomerase II, Science 226
(1984) 466-468.
[9] S. Rajabalian, A. Foroumadi, A. hafiee, S. Emami, Functionalized N(2-
oxyiminoethyl)piperazinyl quinolones as new cytotoxic agents, Pharm
Pharmaceut Sci. 10 (2007) 153-158.
[10] R. Koziel, J. Szczepanowska, A. Magalska, K. Piwocka, J. Duszynski, K.
Zablocki, Ciprofloxacin inhibits proliferation and promotes generation of
aneuploidy in Jurkat cells, J. Physiol. Pharmacol. 61 (2010) 233-239.
[11] T. Kloskowski, N. Gurtowska, M. Nowak, R. Joachimiak, A. Bajek, J.
Olkowska, T. Drewa, The influence of ciprofloxacin on viability of A549,
HepG2, A375.S2, B16 and C6 cell lines in vitro, Acta Pol. Pharm. 68 (2011)
859-865.
[12] H. Thadepalli, F. Salem, S.K. Chuah, S. Gollapudi, Antitumor activity of
trovafloxacin in an animal model, In Vivo. 19 (2005) 269-276.
[13] J.M. Domagala, L.D. Hanna, C.L. Heifetz, M.P. Hutt, T.F. Mich, J.P. Sanchez,
M. Solomon, New structure-activity relationships of the quinolone antibacterials
using the target enzyme. The development and application of a DNA gyrase
assay, J. Med. Chem. 29 (1986) 394-404.
[14] S.J. Won, C.T. Liu, L.T. Tsao, J.R. Weng, H.H. Ko, J.P. Wang, C.N. Lin,
Synthetic chalcones as potential anti-inflammatory and cancer chemopreventive
agents, Eur. J. Med .Chem. 40 (2005) 103-112.
23

ACCEPTED MANUSCRIPT
[15] G. Achanta, A. Modzelewska, L. Feng, S.R. Khan, P. Huang, A boronic-
chalcone derivative exhibits potent anticancer activity through inhibition of the
proteasome, Mol. Pharmacol. 70 (2006) 426-433.
[16] N.J. Lawrence, R.P. Patterson, L.L. Ooi, D. Cook, S. Ducki, Effects of alpha-
substitutions on structure and biological activity of anticancer chalcones, Bioorg.
Med. Chem. Lett. 16 (2006) 5844-5848.
[17] R.J. Anto, K. Sukumaran, G. Kuttan, M.N. Rao, V. Subbaraju, R. Kuttan,
Anticancer and antioxidant activity of synthetic chalcones and related
compounds, Cancer Lett. 97 (1995) 33-37.
[18] S. Ducki, The development of chalcones as promising anticancer agents, IDrugs
10 (2007) 42-46.
[19] C.H. Tseng, Y.L. Chen, C.Y. Hsu, T.C. Chen, C.M. Cheng, H.C. Tso, Y.J. Lu,
C.C. Tzeng, Synthesis and antiproliferative evaluation of 3-
phenylquinolinylchalcone derivatives against non-small cell lung cancers and
breast cancers, Eur. J. Med. Chem. 59 (2013) 274-282.
[20] P. Yogeeswari, D. Sriram, R. Kavya, S. Tiwari, Synthesis and in-vitro
cytotoxicity evaluation of gatifloxacin Mannich bases, Biomed. Pharmacother.
59 (2005) 501–510.
[21] M.J. Nieto, F.L. Alovero, R.H. Manzo, M.R. Mazzieri, Benzenesulfonamide
analogs of fluoroquinolones. Antibacterial activity and QSAR studies, Eur. J.
Med. Chem. 40 (2005) 361–369.
[22] T.D. Gootz, P.R. Mcguirk, M.S. Moynihan, S.L. Haskell, Placement of alkyl
substituents on the C-7 piperazine ring of fluoroquinolones: dramatic differential
effects on mammalian topoisomerase II and DNA gyrase, Antimicrob. Agents
Chemother. 38 (1994) 130-133.
[23] F.L. Alovero, X.S. Pan, J.E. Morris, R.H. Manzo, L.M. Fisher, 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. 44 (2000)
320-325.
24

ACCEPTED MANUSCRIPT
[24] Y. Fengping, P. Yangping, S. Gonghua, L. Jizong, Solid phase synthesis of
amino-chalcones, J. Chem. Res. (2005) 311-312.
[25] K. Nakaya, K. Funabiki, K. Shibata, H. Muramatsu, M. M. Matsul, Flouresent
α,β-unsaturated carbonyl compounds and 2-methoxypyridines. Their application
to a quantitative analysis of carnitines, Bull. Chem. Soc. Jpn. 69 (1996) 2961-
2966.
[26] F.L. Ansari, S. Umbreen, L. Hussain, T. Makhmoor, S.A. Nawaz, M.A. Lodhi,
S.N. Khan, F. Shaheen, M.I. Choudhary, M.I. Atta-ur-Rahman, Syntheses and
biological activities of chalcone and 1,5-benzothiazepine derivatives: promising
new free-radical scavengers, and esterase, urease, and alpha-glucosidase
inhibitors, Chem. Biodivers. 2 (2005) 487-496.
[27] G.A. Abuo-Rahma, H.A. Sarhan, G.F.M. Gad, Design, synthesis, antibacterial
activity and physicochemical parameters of novel N-4-piperazinyl derivatives of
norfloxacin. Bioorg. Med. Chem. 17 (2009) 3879-3886.
[28] The methodology of the NCI anticancer screening has been described in detail
elsewhere (http://www.dtp.nci.nih.gov).
[29] S.A. Rostom, Synthesis and in vitro antitumor evaluation of some indeno[1,2-
c]pyrazol(in)es substituted with sulfonamide, sulfonylurea(-thiourea)
pharmacophores, and some derived thiazole ring systems, Bioorg. Med. Chem.
14 (2006) 6475-6485.
[30] B.A. D. Neto, A.A. M. Lapis, Recent developments in the chemistry of
deoxyribonucleic acid (DNA) intercalators: principles, design, synthesis,
applications and trends. Molecules 14 (2009) 1725-1746.
[31] J.L. Nitiss, Targeting DNA topoisomerase II in cancer chemotherapy. Nat. Rev.
Cancer, 9 (2009) 338-350.
[32] J.L. Nitiss, DNA topoisomerase II and its growing repertoire of biological
functions. Nat. Rev. Cancer, 9 (2009) 327-337.
[33] J.R. Vance, K.F. Bastow, Inhibition of DNA topoisomerase II catalytic activity
by the antiviral agents 7-chloro-1,3-dihydroxyacridone and 1,3,7-trihydroxy-
acridone. Biochem. Pharmacol. 58 (1999) 703-708.
[34] X. Yu, G. Zipp, G. Ray Davison, The effect of temperature and pH on the
25

ACCEPTED MANUSCRIPT
solubility of quinolone compounds: estimation of heat of fusion. Pharm. Res. 11
(1994) 522–527.
[35] D.L. Ross, C.M. Riley, Aqueous solubilities of some variously substituted
quinolone antimicrobials. Int. J. Pharm. 63 (1990) 237–250.
[36] S.A. Breda, A.F. Jimenez-Kairuz, R.H. Manzo, M.E. Olivera, solubility behavior
and biopharmaceutical classification of novel high-solubility ciprofloxacin and
norfloxacin pharmaceutical derivatives. Int. J. Pharm. 371 (2009) 106–113.
[37] G.A. Abuo-Rahma, M. Abdel-Aziz, M. Mourad, H. Farag, Synthesis, anti-
inflammatory activity and ulcerogenic liability of novel nitric oxide
donating/chalcone hybrids, Bioorg. Med. Chem. 20 (2012) 195-206.
[38] M. Fukuda, K. Nishio, F. Kanzawa, H. Ogasawara, T. Ishida, H. Arioka, K.
Bojanowski, M. Oka, N. Saijo, Synergism between cisplatin and topoisomerase I
inhibitors, NB-506 and SN-38, in human small cell lung cancer cells, Cancer
Res. 56 (1996) 789-793.
[39] D.H. Kang, J.S. Kim, M.J. Jung, E.S. Lee, Y. Jahng, Y. Kwon, Y. Na, New
insight for fluoroquinophenoxazine derivatives as possibly new potent
topoisomerase I inhibitor, Bioorg. Med. Chem. Lett. 18 (2008) 1520-1524.
[40] J.L. Vazquez, S. Merino, O. Domeinech, M. Berlanga, M. Vinas, M.T. Montero,
J. Hernandez-Borrell, Determination of the partition coefficients of a homologous
series of ciprofloxacin: influence of the N-4 piperazinyl alkylation on the
antimicrobial activity. Int. J. Pharm. 220 (2001) 53-62.
26

ACCEPTED MANUSCRIPT
Figure Captions
Chart 1: The structures of ciprofloxacin analogue I and II.
Fig. 1. Topoisomerase I inhibitory activity of compounds 3a-j. The compounds were
examined in a final concentration of 20 and 100 ꢀM, respectively, as
designated. Lane D: pBR322 only, Lane T: pBR322 + topo I, Lane C: pBR322
+ topo I + camptothecin, Lanes 3a-j: pBR322 + topo I + compounds in
designated concentrations.
Fig. 2. Topoisomerase II inhibitory activity of compounds 3a-j. Lane D: pBR322
only, Lane T: pBR322 + topo IIα, Lane E: pBR322 + topo IIα + etoposide,
Lanes 3a-j: pBR322 + topo IIα + compounds in designated concentrations.
Fig. 3. DNA unwinding activity of compounds 3a (A) and 3j (B). The supercoiled
DNA pHOT1 was incubated with excess amount of topo I at the 37℃ for
30min followed by addition of each compound in designated concentrations
and additional incubation at 37℃ for 30 min.
27

ACCEPTED MANUSCRIPT
Table 1. One-dose growth (%) of nine different cancer cell types for compounds 3a
and 3g.
Growth (%)
compound 3a compound 3g
Panel
Cell line
CCRF-CEM
15.18
-13.74
HL-60(TB)
K-562
MOLT-4
RPMI-8226
A549/ATCC
HOP-62
40.91
14.15
18.99
2.45
-33.71
1.66
-1.10
-18.77
-19.23
4.38
-1.75
-9.81
-35.60
-7.05
Leukemia
4.12
59.41
14.94
57.39
23.99
73.70
7.12
HOP-92
Non-small cell
lung cancer
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
-19.97
-71.54
-12.83
-11.57
-88.19
18.22
-3.37
-65.07
6.38
-1.11
-27.16
-15.75
34.93
76.59
-55.79
-54.74
31.44
-14.11
-1.52
30.03
34.04
-42.87
30.77
-23.47
-1.19
-25.75
16.43
16.88
4.74
98.19
33.28
-67.76
10.59
107.97
70.06
-83.61
56.81
11.39
25.29
41.03
36.95
15.17
26.19
40.27
61.80
14.91
5.67
51.04
31.85
19.16
53.73
85.67
13.64
61.13
35.47
42.88
54.88
86.41
82.60
6.30
Colon cancer
CNS cancer
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
Melanoma
Ovarian cancer
Renal cancer
93.47
93.58
23.23
28

ACCEPTED MANUSCRIPT
Growth (%)
compound 3a compound 3g
Panel
Cell line
A498
ACHN
CAKI-1
RXF-393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
57.71
40.05
73.07
41.72
11.31
39.90
44.99
55.18
43.12
12.50
55.15
51.91
9.61
-43.84
10.80
57.23
-87.64
10.33
19.07
2.96
21.51
2.17
Prostate cancer
Breast cancer
26.46
27.55
76.78
-86.69
17.55
-1.82
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
85.08
33.76
29