Synthesis and biological assessment of ciprofloxacin-derived 1,3,4-thiadiazoles as anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2020.104383

The quinolone-3-carboxylic acid scaffold is essential structure for antibacterial activity of fluoroquinolones such as ciprofloxacin. Modification of 3-carboxylic functionality in this structure can be used for switching its activity from antibacterial to anticancer. Accordingly, a series of C-3 modified ciprofloxacin derivatives containing N-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-carboxamide moiety was synthesized as novel anticancer agents. Most of compounds showed significant activity against MCF-7, A549 and SKOV-3 cancer cells in the MTT assay. In particular, compounds 13a-e and 13g were found to be as potent as standard drug doxorubicin against MCF-7 cell line (IC₅₀s = 3.26–3.90 μM). Furthermore, the 4-fluorobenzyl derivatives 13h and 14b with IC₅₀ values of 3.58 and 2.79 μM exhibited the highest activity against SKOV-3 and A549 cells, being as potent as doxorubicin. Two promising compounds 13e and 13g were further tested for their apoptosis inducing activity and cell cycle arrest. Both compounds could significantly induce apoptosis in MCF-7 cells, while compound 13e was more potent apoptosis inducer resulting in an 18-fold increase in the proportion of apoptotic cells at the IC₅₀ concentration in MCF-7 cells. The cell cycle analysis revealed that compounds 13e and 13g could increase cell portions in the sub-G1 phase, inducing oligonucleosomal DNA fragmentation and apoptosis confirmed by comet assay.

Full text of DOI:10.1016/j.bioorg.2020.104383

Journal Pre-proofs
Synthesis and biological assessment of ciprofloxacin-derived 1,3,4-thiadia-
zoles as anticancer agents
Hamideh Ahadi, Mohammad Shokrzadeh, Zahra Hosseini-khah, Nasrin
Ghassemi barghi, Majid Ghasemian, Elnaz Emadi, Mehryar Zargari, Nima
Razzaghi-Asl, Saeed Emami
PII:
S0045-2068(20)31681-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.104383
YBIOO 104383
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Bioorganic Chemistry
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13 October 2020
Please cite this article as: H. Ahadi, M. Shokrzadeh, Z. Hosseini-khah, N. Ghassemi barghi, M. Ghasemian, E.
Emadi, M. Zargari, N. Razzaghi-Asl, S. Emami, Synthesis and biological assessment of ciprofloxacin-derived
1,3,4-thiadiazoles as anticancer agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
2020.104383
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Synthesis and biological assessment of ciprofloxacin-derived 1,3,4-thiadiazoles
as anticancer agents
a
Hamideh Ahadi , Mohammad Shokrzadeh b, Zahra Hosseini-khah c, Nasrin Ghassemi barghi b,
Majid Ghasemian d, Elnaz Emadi e, Mehryar Zargari e, Nima Razzaghi-Asl f, Saeed Emami g,*
aDepartment of Medicinal Chemistry, Student Research Committee, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
b Department of Toxicology and Pharmacology, Faculty of Pharmacy, Mazandaran University of
Medical Sciences, Sari, Iran
c Diabetes Research Center, Mazandaran University of Medical Sciences, Sari, Iran
d Department of Clinical Biochemistry, School of Medicine, Shahid Beheshti University of Medical
Sciences (SBMU), Tehran, Iran
e Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari,
Iran
f
Department of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical
Sciences, Ardabil, Iran
g Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
*Corresponding Author
E-mail: sdemami12@gmail.com; semami@mazums.ac.ir
1

Abstract
The quinolone-3-carboxylic acid scaffold is essential structure for antibacterial activity of
fluoroquinolones such as ciprofloxacin. Modification of 3-carboxylic functionality in this structure
can be used for switching its activity from antibacterial to anticancer. Accordingly, a series of C-
3 modified ciprofloxacin derivatives containing N-(5-(benzylthio)-1,3,4-thiadiazol-2-yl)-
carboxamide moiety was synthesized as novel anticancer agents. Most of compounds showed
significant activity against MCF-7, A549 and SKOV-3 cancer cells in the MTT assay. In particular,
compounds 13a-e and 13g were found to be as potent as standard drug doxorubicin against MCF-7
cell line (IC50s = 3.26-3.90 µM). Furthermore, the 4-fluorobenzyl derivatives 13h and 14b with
IC50 values of 3.58 and 2.79 µM exhibited the highest activity against SKOV-3 and A549 cells,
being as potent as doxorubicin. Two promising compounds 13e and 13g were further tested for
their apoptosis inducing activity and cell cycle arrest. Both compounds could significantly induce
apoptosis in MCF-7 cells, while compound 13e was more potent apoptosis inducer resulting in an
18-fold increase in the proportion of apoptotic cells at the IC50 concentration in MCF-7 cells. The
cell cycle analyzing revealed that compounds 13e and 13g can increase cell portions in the sub-
G1 phase, inducing oligonucleosomal DNA fragmentation and apoptosis confirmed by comet
assay.
Keywords: Fluoroquinolones; Ciprofloxacin; 1,3,4-Thiadiazole; Anticancer activity; Apoptosis
inducer; Cytotoxicity.
2

1. Introduction
Currently, anticancer agents have important position in the chemotherapy of malignancies.
However, their potency and safety are not enough to overcome all kinds of cancers efficiently.
Furthermore, there are some difficulties in the cancer chemotherapy, mainly due to the special
characteristics of the disease such as resistance, diversity, different etiology and metastasis. This
situation along with increasing number of cancer patients around the world instigate medicinal
chemist to find new anticancer agents [1-3].
Fluoroquinolones are a large family of compounds with synthetic origin that are prominent in the
field of antibacterial chemotherapy. These antibacterial agents prevent replication and
transcription of DNA by targeting DNA gyrase and/or topoisomerase IV [4]. On the other hand,
there are many reports on ability of fluoroquinolones to inhibit the division of cancer cells with
diverse mechanisms including DNA intercalation, induction of apoptosis, and arresting of the cell
cycle. Ciprofloxacin (CF, 1a, Fig. 1), one of fluoroquinolone antibacterials that is used to treat
diverse kinds of bacterial infections, including skin, bone, joint, respiratory and urinary tracts
infections [5, 6]. Based on several reports, CF exhibits anticancer activity against some tumor cell
lines including U87MG (Glioblastoma), MDA MB-231 (Breast), Colo829 (Melanoma), H460
(Lung cancer), Panc-1 (Pancreatic cancer), LOVO (Colon cancer), PC3, MLC9981 (Prostate
cancer), HT-29 (Colorectal carcinoma), HTB9 (Bladder cancer), and K562 (Leukaemia cells) [4].
CF has good penetration into different tissues and glands. However, in vitro studies have
demonstrated that high concentration of CF is required for anticancer activity [7]. Thus CF could
be a candidate scaffold for structural modification in order to obtain potential anticancer agents.
From a structure-activity relationship point of view, the carboxylic acid group at 3-position of
fluoroquinolones is imperative for antibacterial activity and is a good choice for structural
modification to switch into the effective anticancer activity [8, 9]. Accordingly, several strategies
such as derivertization, bioisosteric replacement and cyclization of 3-carboxylic acid of quinolone
antibacterials were applied to design of quinolone-based anticancer agents. There have already
been several instances of conversion of 3-COOH in CF to hydrazide functionality. For instance,
3

Hu et al., have showed that some hydrazide-hydrazone of CF (2, Fig. 1) can inhibit the growth of
cancer cell lines such as CHO (Chinese hamster ovary), HL60 and L1210 (murine leukemia) [10].
In a similar work by Shi et al., ciprofloxacin-piperonal hydrazone (3) has been described as
cytotoxic agent against SMMC-7721, MCF-7 and HCT-8 cancer cells [11].
O
N
O
F
OH
N
HN
Ciprofloxacin (CF, 1a)
O
O
O
N
2
O
O
N
3
O
F
N
F
N
N
N
R
H
H
N
N
HN
HN
Figure 1. Structure of ciprofloxacin and related derivatives as reported anticancer agents.
On the other hand, thiadiazole is a key five-membered heterocyclic core, found in many anticancer
compounds [12-15]. Sulfur containing heterocycles such as thiadiazoles have distinct chemical
features due to the presence of electron deficient, bivalent sulfur atom with two areas of positive
electrostatic potential, in which the low lying C−S σ* orbitals can interact with electron donors
(such as O and N atoms). The ability of thiadiazoles for non-covalent interactions involving sulfur
atom in the compound conformation and ligand−protein interactions make them as an interesting
scaffold in drug design and development [16,17]. Among the thiadiazole derivatives, the most
attractive ones are those containing alkyl(aryl)amido- and/or benzylthio moieties at positions 2
and 5, respectively [18]. In the search for new lead compounds targeting cancer cells including
chronic myeloid leukemia (CML), Radi group described thiadiazole derivatives 4 and 5 (Fig. 2) as
Bcr-Abl inhibitors [19]. Moreover, some analogs of 4 and 5 namely N-(5-(benzylthio)-1,3,4-
thiadiazol-2-yl)-2-phenylacetamides (6) were synthesized by Aliabadi and coworkers. The in vitro
4

results indicated that the breast cancer cell line MCF-7 had the highest susceptibility towards
compounds 6 [20].
N
O
N
N
N
O
F
NO2
S
S
S
N
S
N
H
H
F
Cl
5
BO1 (4)
R
F
N
N
O
S
S
N
H
6
N
O
N
O
N
F
S
R1
S
N
H
N
N
R
13:
14:
R= H
R= Et
R1= H, Cl, Cl2, Br, F, NO2
Figure 2. Structures of 1,3,4-thiadiazole-based compounds 4-6 with anticancer activity and designed compounds 13-
14 as ciprofloxacin-thiadiazole hybrids.
The above mentioned findings prompted us to design a novel series of quinolone-based
thiadiazoles derived from ciprofloxacin. Thus we report here, the synthesis and biological
evaluation of compounds 13-14 as anticancer agents (Fig. 2).
5

2. Results and discussion
2.1. Chemistry
The sequence of the reactions used for the synthesis of novel quinolone-based thiadiazoles is
illustrated in Scheme 1. Initially, 5-amino-1,3,4-thiadiazole-2-thiol (8) was synthesized by
heterocyclization of thiosemicarbazide (7) in the presence of carbon disulfide and KOH in absolute
ethanol. The compound 8 was subjected to the reaction with benzyl chloride or bromide derivatives
to afford S-benzyl derivatives 9a-j in good yields [21].
1) CS2, KOH, EtOH,
Benzyl halides,
N
S
N
N N
Reflux, 8 h
NaOH,
R
H2N
S
H2N
H2N
SH
N
NH2
S
2) HCl
S
EtOH, rt, 24 h
H
9a-j
8
7
O
O
O
O
F
O
F
Boc2O, NaHCO3
OH
OH
Cl
OEt
N
N
N
THF:H2O (1:1),
rt, 24 h
N
TEA, dry CH2Cl2, rt
HN
O
N
10
O
CF (1a)
O
N
O
O
N
N
O
N
O
F
F
S
O
OEt
Compounds
R
S
N
9a-j
H
N
N
O
N
O
N
12a-j
O
O
11
Trifluoroacetic acid,
CH2Cl2, 0 °C-rt, 4 h
N
N
O
O
F
S
S
N
R
H
R= H, 3-Cl, 4-Cl, 2,4-Cl2, 3,4-Cl2,
2,6-Cl2, 2-F, 4-F, 4-Br, 4-NO2
N
N
HN
13a-j
Scheme 1. Synthesis of compounds 13a-j.
6

On the other hand, in order to modify the 3-carboxylic acid functionality of CF, the NH of
piperazinyl group was protected with tert-butyloxycarbonyl (Boc) group. Thus, treatment of CF
with Boc2O in the presence of sodium bicarbonate in THF-H2O gave the N-Boc-CF (10). For
activation of 3-carboxilic acid, compound 10 was reacted with ethyl chloroformate and
triethylamine (TEA) as a base in dry dichloromethane (DCM) to obtain intermediate 11. The
reaction of compound 11 with aminothiadiazoles 9a-j afforded the corresponding amides 12a-j.
Finally, deprotection of the N-Boc-amides (12a-j) with trifluoroacetic acid (TFA) in DCM at room
temperature produced compounds 13a-j. Furthermore, for obtaining compounds 14, enrofloxacin
(1b) the commercially available derivative of CF, was subjected to coupling with appropriate
aminothiadiazole 9. Similarly, activation of COOH functional group was conducted by using ethyl
chloroformate, as described for the preparation of compounds 12 (Scheme 2).
The structures of the synthesized compounds 12-14 were confirmed on the basis of their spectral
data (IR, 1H-NMR, 13C-NMR, MS and elemental analyses). Particularly, in the 1H-NMR spectra
of compounds 12, the presence of singlet in the range of 13.99-14.01 ppm is related to the NH of
amide, indicating the involvement of NH in the hydrogen bonding with 4-oxo of quinolone (Fig.
3). The full interpretation of spectroscopic data could be found in Experimental section.
O
N
N
O
N
O
O
N
F
O
F
S
R
S
OH
N
Compound 9
Cl
OEt
H
N
N
TEA,
dry CH2Cl2, rt
N
N
14a: R
14b: R
= H
= 4-F
Enrofloxacin (1b)
Scheme 2. Synthesis of compounds 14a,b.
7

R1
N
N
O
H
S
S
O
N
5
8
4
F
2
N
N1
N
R
Figure 3. Presentation of the possible intramolecular hydrogen bond in compounds 12-14 and the atom numbering of
the core structure.
2.2. Effect of compounds 13-14 on the viability of cancer cells
All the synthesized ciprofloxacin-based compounds 13a-j, 14a,b and two N-Boc protected
compounds 12, were evaluated for their in vitro activity against three human cancerous cell lines;
breast MCF-7, lung A549 and ovarian SKOV-3 cancer cells. The cell viability was determined by
using MTT assay after 48 h incubation. The results were expressed by median growth inhibitory
concentration (IC50 in µM) as shown in Table 1. Ciprofloxacin (CF) and doxorubicin were included
in the bioassay as parent and reference drugs, respectively.
As seen in Table 1, while the parent quinolone (CF) showed no activity against tested cancer cells
(IC50 > 100 µM), all the quinolone-based compounds had significant activities at least against one
cell line, displaying IC50 values in the range of 2.79-26.28 µM. The obtained IC50 values for
standard drug doxorubicin were 2.25-3.48 µM.
Notably, the N-Boc derivatives 12b and 12h and N-ethyl analog 14a were found to be inactive
against MCF-7 and SKOV-3 cells (IC50 >50 µM). In the unsubstituted piperazine series 13a-g,
MCF-7 cells were more sensitive than A549 and SKOV-3 cells. In particular, compounds 13a-e
and 13g with IC50 values ≤3.90 µM were as potent as doxorubicin against MCF-7. Interestingly,
all tested compounds were active against A549 cells. While compounds 14a and 14b bearing N-
ethylpiperazine moiety were the most active compounds against A549, the N-Boc-piperazine
derivative 12b was the less potent one. The activity of compound 14b (IC50 A549= 2.79 µM) was
comparable to that of standard drug doxorubicin. In the case of SKOV-3 cancer cells, the
unsubstituted piperazine derivatives 13a-h showed potent activity (IC50 <10 µM). The most potent
compound was 13h (IC50 SKOV-3= 3.58 µM), being as potent as doxorubicin.
8

Table 1. The IC50 values (μM) of final compounds 13 and 14, and intermediate compounds 12b and 12h against three
different cell lines.
N
O
N
O
N
F
S
R1
S
N
H
N
N
R
Compound
R
H
H
H
H
H
H
H
H
H
H
Et
Et
R1
MCF-7
3.84
3.58
3.90
3.31
3.26
5.71
3.34
9.48
7.71
15.79
>50
A549
10.21
9.97
SKOV-3
9.66
7.17
8.50
7.6
13a
H
13b
3-Cl
4-Cl
2,4-Cl2
3,4-Cl2
2,6-Cl2
2-F
13c
6.49
13d
8.52
13e
10.53
14.8
5.08
4.14
5.43
3.58
10.57
16.58
>50
13f
13g
9.69
13h
4-F
6.95
13i
4-Br
4-NO2
H
5.50
13j
23.51
4.02
14a
14b
4-F
4.99
>50
2.79
10.29
>50
12b
Boc
3-Cl
4-F
26.28
11.09
>100
2.25
12h
Boc
>50
>50
Ciprofloxacin
Doxorubicin
-
-
-
>100
3.48
>100
3.47
-
Each experiment was performed in triplicate and the results were showed as mean values
9

By comparing IC50 values of the series, some structure-activity relationships could be concluded.
The simplest compound 13a (R= R1= H) showed good activity against all cell lines especially
MCF-7 cells. While the introduction of halogens (Cl or F) on benzyl group could not improved
the activity against MCF-7, but significantly increased the inhibitory potency against SKOV-3.
Particularly, the better result on SKOV-3 was obtained with 4-fluoro- substitution. Conversely,
this substituent (R1= 4-F) decreased the activity against MCF-7 (compare 13h with 13a). The
comparison of 2-fluorobenzyl derivative 13g with the unsubstituted benzyl analog 13a revealed
that the 2-fluoro substitution can improve the activity against SKOV-3 while serving the level of
activity on MCF-7 cells. Among the halobenzyl compounds (13b-i), the 4-bromo- analog showed
the greater activity against A549. These findings indicate that the effect of substituent on the benzyl
group depends on the type of cell line. The introduction of strong electron-withdrawing NO2 group
on the para-position of benzyl group resulted in compound 13j with diminished activity against
all cell lines.
The comparison of N-ethylpiperazine derivatives (14a and 14b) with their parent counterparts (13a
and 13h, respectively) indicates that the ethyl group has positive effect on cytotoxic activity against
A549. Indeed, the N-ethylpiperazine derivatives 14b was the most potent compound against this
cell line. However, the introduction of ethyl on piperazine ring remarkably diminished the activity
on SKOV-3 as observed in compounds 14a and 14b.
2.3. Apoptosis inducing activity of compounds 13e and 13g
Actually, many of the anticancer drugs commonly treat malignancies by induction of apoptosis in
cancer cells. Nowadays, apoptotic pathways have currently been important targets for development
of new anticancer agents [22]. In order to check the potential of our compounds for induction of
apoptosis in cancer cells, we use Annexin V/PI double staining technique for analyzing of MCF-
7 treated with selected compounds 13e and 13g. It should be noted that these two compounds were
the most active ones against MCF-7 cells thus we selected them for further studies on MCF-7 cells.
As shown in Fig. 4, the results demonstrated that the percentages of the cell in regions Q3 and Q2
for treated cells significantly increased in comparison with those of control cells. After 48 h of
treatment with 13e and 13g the number of early apoptotic cells increased from 1.82 to 53.2 and
10

18.6%, respectively. Also the percentage of cell in the late apoptotic region reached from 2.14 to
21.3 and 13%, respectively. Overall, both compounds could significantly induce apoptosis in
MCF-7 cells, while compound 13e was more potent apoptosis inducer. An 18-fold increment in
the proportion of apoptotic cells occurred after treating with the IC50 concentration (3.26 µM) of
13e in MCF-7 cells.
Figure 4. Induction of apoptosis in MCF-7 cells after 48 h treatment with 13e and 13g (at IC50 concentrations).
Quadrant panels have been separated into four gates that labeled Q1 (necrotic cells, AnnexinV−/PI+), Q2 (late apoptotic
cells, Annexin V+/PI+), Q3 (early apoptotic cells, Annexin V+/PI−) and Q4 (viable cells, Annexin V−/PI−). The control
group was untreated cells.
2.4. Cell cycle arrest by compounds 13e and 13g
The cell cycle regulation is an important way for the control of cell growth and anticancer therapy
[23]. Therefore, study of the effect of new cytotoxic agents on the proportion of cells in the cell
cycle phases can help to highlight the anticancer properties and involved mechanisms.
The effect of representative compounds 13e and 13g on the cell cycle of MCF-7 cells was analyzed
by using flow cytometric method and the distribution of cells in different phases was determined.
As presented in Fig. 5, after treating with 13e and 13g, the percentages of cells in the sub-G1 phase
11

remarkably elevated from 0.94 to 34.68 and 35.72%, respectively. On the other hand, the obtained
results showed that the percentage of cells in G1, S and G2/M phases decreased when comparing
with control cells. These results suggested that compounds 13e and 13g (at the concentration of
IC50 values) can increase the proportion of sub-G1 phase, inducing oligonucleosomal DNA
fragmentation and apoptosis.
Control
13g
13e
Sub-G1:
35.72
Sub-G1: 34.68
G1: 40.12
Sub-G1: 0.94
G1: 50.54
G1: 40.79
S: 15.18
S:12.79
S: 34.73
G2/M: 12.41
G2/M: 13.79
G2/M: 8.31
Figure 5. The cell cycle analyzes of MCF-7 cells after treating with compounds 13e and 13g (IC50 concentration, 48
h). The untreated cells were as control group.
2.5. Evaluation of compounds 13e and 13g for DNA damaging by comet assay
The most important cytotoxic mechanisms of quinolones could be the conversion of transient DNA
double-strand breaks into permanent lesions followed by cell-cycle arrest and apoptosis in cancer
cells [24]. The comet assay is known as a facile and delicate in which single cell gel electrophoresis
is used to detecting DNA damage of individual cells [25].
The possible ability of compounds 13e, 13g and CF (as parent drug) on the DNA damage was
investigated by the alkaline comet assay in cancer cell MCF-7. Doxorubicin was also included as
standard drug with well-known interfering action on the DNA function and structure. Precisely,
compounds 13e, 13g and CF were tested at the concentrations of 1, 5 and 10 µM and compared to
12

doxorubicin (1, 5 and 10 µM) as positive control. Different parameters including tail length, %
DNA in tail and tail moment was used to scoring of DNA damage as shown in Fig. 6. Doxorubicin
at the lower concentration of 1 µM showed higher potentials of DNA damage in MCF-7 cell. Also
both compounds 13e and 13g even at low concentration of 1 µM displayed a significant increase
in the DNA damage in comparison with control group. Furthermore, all parameters including tail
length, % DNA in tail and tail moment, in compounds 13e- or 13g-treated cells were significantly
higher than those of parent quinolone CF. However, the effect of both compounds was less than
that of doxorubicin as compared by all parameters.
DOX
CF
DOX
CF
DOX
CF
13e
13e
13e
80
60
40
20
0
80
60
40
20
0
100
80
60
40
20
0
^^^^
^^^^
****
****
****
****
****
****
****
****
*
10M
5M
Control
1M
10M
5M
Control
1M
10M
5M
Control
1M
B
C
A
DOX
CF
DOX
DOX
CF
CF
13g
13g
13g
80
60
40
20
0
80
60
40
20
0
100
80
^^^^
^^^^
****
^^^^
****
****
****
****
****
****
****
60
40
20
0
****
10M
5M
Control
10M
5M
1M
Control
1M
10M
5M
Control
1M
A
C
B
Figure 6. DNA damage in MCF-7 cells evaluated by the comet assay and scored as tail parameters: (A) Tail length;
(B) % DNA in tail and (C) tail moment. The cells were treated with the compounds 13e and 13g (1, 5 and 10 µM) and
compared to doxorubicin (Dox) and ciprofloxacin (CF) (same concentrations), and negative control (untreated).
13

3. Conclusion
We have synthesized a series of C-3 modified ciprofloxacin derivatives containing N-(5-
(benzylthio)-1,3,4-thiadiazol-2-yl)-carboxamide moiety as novel anticancer agents. The MTT
assay of synthesized compounds against three different cancer cells including MCF-7, A549 and
SKOV-3 indicated that most of them have remarkable activity (IC50 values ≤ 26.28 µM), in
contrast to ciprofloxacin with no anticancer activity (IC50 values > 100 µM). Certainly, the
inhibitory activity of compounds 13a-e and 13g against MCF-7 cell line (IC50s = 3.31-3.90 µM)
was comparable to that of standard drug doxorubicin. Although the IC50 values of compounds 13a-
e and 13g against other cells (A549 and SKOV-3) was ≤ 10.53 µM, but the 4-fluorobenzyl
derivatives 13h and 14b with IC50 values of 3.58 and 2.79 µM displayed superior activity on
SKOV-3 and A549 cells, being as potent as doxorubicin. The flow cytometric analysis of two
representative compounds 13e and 13g in MCF-7 cells demonstrated that both compounds could
significantly induce apoptosis in MCF-7 cells, cell cycle arrest and increment of cells in the sub-
G1 phase, which further confirmed by comet assay, indicating the potential of compounds in DNA
damage and oligonucleosomal DNA fragmentation in MCF-7 cells. The results of this study
revealed the C-3 conjugated quinolone antibacterial drug ciprofloxacin with 5-(benzylthio)-1,3,4-
thiadiazol-2-amine derivatives can be considered as new lead for development of anticancer
agents.
4. Experimental
4.1. General chemistry
All solvents and reagents were obtained from Merck Company and used without further
purification. The required benzyl halides were also commercially available (Merck or Sigma-
Aldrich). Ciprofloxacin hydrochloride was production of Temad Co. (Tehran, Iran). The
intermediate compound 8 was prepared from thiosemicarbazide and CS2 based on the reported
method [26].
4.2. General procedure for the preparation of S-benzyl derivatives 9a-j
14

A solution of 5-amino-1,3,4-thiadiazole-2-thiol (8, 5 mmol), in ethanol 80% (75 mL) was stirred
at room temperature. Then, a 0.1 N NaOH solution (25 mL) was added dropwise and the stirring
was continued. After 0.5 h, the appropriate benzyl halide (5 mmol) was added, and the reaction
mixture was stirred at r.t. for 24 h. The resulting white precipitate was filtered and washed with
water to give corresponding product 9 which was used for next step without further purification.
4.3. Synthesis of N-Boc-CF (10)
To a mixture of CF (1a, 5 mmol) in THF-H2O (1:1) was added NaHCO3 (5 mmol) and stirred for
30 min. Then, the reaction mixture was cooled down to 0-5 ºC and di-tert-butyl dicarbonate (5
mmol) was added over 10 min and subsequently stirred at ambient temperature overnight. The
reaction progression was monitored by TLC using CHCl3-CH3OH (9:1) as mobile phase. Upon
completion of the reaction, ice-cold water was poured onto the reaction mixture and the white
participated solid was filtered, washed with water and dried to give compound 10. Yield 93%, m.p.
240-245 °C, 1H NMR (400 MHz, DMSO-d6) δ: 1.10-1.40 (m, 4H, c-Pr), 1.46 (s, 9H, t-Bu), 2.10-
2.67 (m, 4H, Pip), 3.5-3.58 (m, 4H, Pip), 3.7-3.9 (m, 1H, c-Pr), 7.61 (d, 1H, J = 7.2 Hz, H-8 Quin),
7.95 (d, 1H, J = 13.2 Hz, H-5 Quin), 8.69 (s, 1H, H-2 Quin), 15.21 (s, 1H, OH). Anal. Calcd for
C22H26FN3O5: C, 61.24; H, 6.07; N, 9.74. Found: C, 61.20; H, 6.21; N, 9.76.
4.4. General procedure for the synthesis of ciprofloxacin amides (12 and 13)
A mixture of N-Boc-CF (10, 1 mmol) and triethylamine (2 mmol) in dry CH2Cl2 (5 mL) was stirred
at room temperature for 30 min. Then the mixture was cooled to 0ºC, ethyl chloroformate (1 mmol)
was added dropwise and stirring was continued for 1 h while the temperature reached to the room
temperature gradually. After getting a solution, the appropriate thiadiazole-amine 9 (1 mmol) was
added and the reaction mixture was stirred overnight. Finally, the obtained mixture was
concentrated under reduced pressure and the residue was washed with ethyl acetate (for removing
the unreacted intermediate 9) to give crude products 12a-j. In the case of 12b and 12h, crude
product was purified by flash column chromatography eluting with chloroform-methanol (98:2) to
afford pure compounds.
15

For deprotection of 12a-j, a mixture of crude product N-Boc-ciprofloxacin amide 12a-j in
dichloromethane (DCM) was treated with trifluoroacetic acid (TFA) in an ice bath. After 0.5 h
stirring, the mixture was stirred at room temperature for 4 h. At the end, the obtained solution was
concentrated under reduced pressure and the residue was participated between CH2Cl2 and
saturated NaHCO3 solution in water. The separated organic layer was washed with water, dried
over Na2SO4. The solvent was evaporated under reduced pressure to give target compounds 13a-
j.
4.4.1. tert-Butyl 4-(3-((5-((3-chlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)carbamoyl)-1-cyclopropyl-
6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)piperazine-1-carboxylate (12b)
Yield 51%, m.p. 227-230 °C, IR (KBr, cm−1): 3435, 2918, 2849, 1696, 1676, 1614, 1520, 1482,
1379, 1243, 1164. 1H NMR (400 MHz, DMSO-d6) δ: 1.15-1.40 (m, 4H, c-Pr), 1.45 (s, 9H, 3×CH3),
3.27-3.34 (m, 4H, Pip), 3.53-3.59 (m, 4H, Pip), 3.78-3.90 (m, 1H, c-Pr), 4.54 (s, 2H, CH2), 7.30-
7.46 (m, 3H, H-4, H-5, H-6 Ph), 7.54 (s, 1H, H-2 Ph), 7.59 (d, 1H, J= 8.0 Hz, H-8 Quin), 7.99 (d,
1H, J=13.2 Hz, H-5 Quin) and 8.78 (s, 1H, H-2 Quin), 14.01 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 8.04, 28.52, 36.07, 37.09, 49.79, 79.65, 107.17, 111.98, 120.55, 127.99, 128.21,
129.30, 130.83, 133.46, 138.98, 140.03, 148.54, 153.52 (d, J= 247.5 Hz), 154.18, 158.53, 162.46,
174.99. MS (m/z, %): 670 (M+ <1), 429 (2), 382 (3), 355 (5), 282 (20), 266 (38), 207 (100), 191
(11), 149 (11), 125 (34), 91 (28). Anal. Calcd for C31H32ClFN6O4S2: C, 55.47; H, 4.81; N, 12.52.
Found: C, 55.56; H, 4.80; N, 12.33.
4.4.2.
tert-Butyl
4-(1-cyclopropyl-6-fluoro-3-((5-((4-fluorobenzyl)thio)-1,3,4-thiadiazol-2-
yl)carbamoyl)-4-oxo-1,4-dihydroquinolin-7-yl)piperazine-1-carboxylate (12h)
Yield 56%, m.p. 227-230 °C, IR (KBr, cm−1): 3434, 3073, 2921, 2853, 1690, 1669, 1607, 1575,
1506, 1479, 1420, 1389, 1339, 1334, 1245, 1214, 1156, 1129, 1033, 1001, 948, 897, 847, 801,
761, 730. 1H NMR (400 MHz, DMSO-d6) δ: 1.23-1.40 (m, 4H, c-Pr), 1.46 (s, 9H, 3×CH3), 3.27-
3.48 (m, 6H, Pip), 3.54-3.62 (m, 2H, Pip), 3.79-3.86 (m, 1H, c-Pr), 4.52 (s, 2H, CH2), 7.19 (t, 2H,
J= 8.8 Hz, H-3, H-5 Ph), 7.49 (dd, 2H, J= 8.4 and 5.6 Hz, H-2 and H-6 Ph), 7.59 (d, 1H, J= 8.0
Hz, H-8 Quin), 7.98 (d, 1H, J=13.2 Hz, H-5 Quin) and 8.77 (s, 1H, H-2 Quin), 13.99 (s, 1H, NH).
13C NMR (100 MHz, DMSO-d6) δ: 8.03, 28.51, 29.79, 36.27, 37.12, 49.77, 79.65, 107.94, 111.99,
16

115.82 (d, J= 21.4 Hz), 120.59, 131.56 (d, J= 8.2 Hz), 133.57, 138.97, 145.18, 148.43, 153.70 (d,
J= 247.3 Hz), 154.18, 158.47, 158.71, 162.16 (d, J= 245.0 Hz), 162.54, 174.98. MS (m/z, %): 654
(M+, 2), 362 (2), 308 (3), 265 (3), 203 (4), 191 (5), 148 (22), 137 (7), 123 (9), 111 (12), 97 (19),
85 (35), 69 (76), 57 (76), 43 (100). Anal. Calcd for C31H32F2N6O4S2: 56.87; H, 4.93; N, 12.84.
Found: 57.01; H, 4.99; N, 12.81.
4.4.3. N-(5-(Benzylthio)-1,3,4-thiadiazol-2-yl)-1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-
1,4-dihydroquinoline-3-carboxamide (13a)
Yield 33%; m.p. 176-179 °C; IR (KBr, cm−1): 3436, 2917, 2849, 1670, 1610, 1523, 1483, 1337,
1
1271, 1257, 1185, 799, 720. H NMR (400 MHz, CDCl3) δ: 1.10-1.43 (m, 4H, c-Pr), 3.08-3.17
(m, 4H, Pip), 3.27-3.36 (m, 4H, Pip), 3.53-3.59 (m, 1H, c-Pr), 4.52 (s, 2H, CH2 benzyl), 7.25-7.38
(m, 5H, Ph), 7.43 (d, 1H, J= 6.8 Hz, H-8 Quin), 8.08 (d, 1H, J= 13.2 Hz, H-5 Quin), 8.84 (s, 1H,
H-2 Quin). 13C NMR (100 MHz, DMSO-d6) δ: 8.02, 36.17, 37.98, 44.65, 49.34, 106.85, 107.18,
111.87 (d, J= 22.5 Hz), 120.48, 128.08, 129.03, 129.50, 137.15, 138.98, 145.11 (d, J= 10.1 Hz),
148.37, 153.32 (d, J= 247.3 Hz), 158.38, 158.94, 162.49, 174.92. MS (m/z, %): 536 (M+, <1), 524
(4), 495 (2), 368 (13), 313 (6), 239 (7), 95 (16), 69 (62), 43 (100). Anal. Calcd for C26H25FN6O2S2:
C, 58.19; H, 4.70; N, 15.66. Found: C, 58.33; H, 4.62; N, 15.63.
4.4.4.
N-(5-((3-Chlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)-1-cyclopropyl-6-fluoro-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13b)
Yield 36%; m.p. 187-191°C; IR (KBr, cm−1): 3435, 2921, 2848, 1674, 1625, 1612, 1518, 1481,
1336, 1257, 798, 679.1H NMR (400 MHz, CDCl3) δ: 1.20-1.48 (m, 4H, c-Pr), 2.00 (brs, 1H, N-H
Pip), 3.01-3.20 (m, 4H, Pip), 3.20-3.38 (m, 4H, Pip) 3.51-3.62 (m, 1H, c-Pr), 4.47 (s, 2H, CH2
benzyl), 7.21-7.37 (m, 4H, H-8 Quin, H-4, H-5 and H-6 Ph), 7.43 (s, 1H, H-2 Ph), 8.00 (d, 1H, J=
13.2 Hz, H-5 Quin), 8.79 (s, 1H, H-2 Quin). 13C NMR (100 MHz, CDCl3) δ: 8.21, 35.25, 37.58,
45.86, 50.96, 104.76, 108.23, 112.72, 112.96, 127.38, 127.95, 129.19, 129.91, 134.41, 138.48,
145.88, 147.35, 149.1, 152.5, 155.1, 158.62, 158.85, 162.72, 170.42, 175.08. MS (m/z, %): 570
(M+, <1), 329 (2), 314 (2), 257 (4), 155 (3), 125 (100), 102 (12), 89 (42), 41 (28). Anal. Calcd for
C26H24ClFN6O2S2: C, 54.68; H, 4.24; N, 14.72. Found: C, 54.44; H, 4.32; N, 14.71.
17

4.4.5.
N-(5-((4-Chlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)-1-cyclopropyl-6-fluoro-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13c)
Yield 34%; m.p. 116-119°C; IR (KBr, cm−1): 3436, 2919, 2849, 1671, 1624, 1611, 1522, 1482,
1337, 1256, 798, 761. 1H NMR (400 MHz, CDCl3) δ: 1.20-1.44 (m, 4H, c-Pr), 1.73 (brs, 1H, N-H
Pip), 3.07-3.15 (m, 4H, Pip), 3.26-3.34 (m, 4H, Pip) 3.52-3.60 (m, 1H, c-Pr), 4.47 (s, 2H, CH2
benzyl), 7.29 (d, 2H, J= 8.4 Hz, H-2 and H-6 Ph), 7.33 (d, 1H, J= 7.2 Hz, H-8 Quin), 7.37 (d, 2H,
13
J= 8.4 Hz, H-3 and H-5 Ph), 8.04 (d, 1H, J= 13.2 Hz, H-5 Quin), 8.82 (s, 1H, H-2 Quin). C
NMR (100 MHz, DMSO-d6) δ: 7.98, 36.09, 37.11, 45.81, 51.13, 106.39, 107.06, 111.70 (d, J=
23.0 Hz), 120.03 (d, J= 7.5 Hz), 128.96, 131.35, 132.63, 136.49, 138.98, 145.78 (d, J= 9.7 Hz),
148.18, 153.36 (d, J= 247.4 Hz), 158.47, 158.53, 162.49, 174.85. MS (m/z, %): 570 (M+, <1), 287
(4), 245 (5), 161 (7), 125 (100), 89 (52), 57 (36), 43 (53). Anal. Calcd for C26H24ClFN6O2S2: C,
54.68; H, 4.24; N, 14.72. Found: C, 54.89; H, 4.20; N, 14.83.
4.4.6.
1-Cyclopropyl-N-(5-((2,4-dichlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)-6-fluoro-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13d)
Yield 41%; m.p 185-188 °C; IR (KBr, cm−1): 3435, 2922, 2850, 1674, 1624, 1613, 1517, 1481,
1337, 1256, 798, 761. 1H NMR (400 MHz, DMSO-d6) δ: 1.11-1.38 (m, 4H, c-Pr), 2.86-2.93 (m,
4H, Pip), 3.15-3.21 (m, 4H, Pip), 3.37 (brs, 1H, NH), 3.61-3.83 (m, 1H, c-Pr), 4.54 (s, 2H, CH2
benzyl), 7.40 (dd, 1H, J= 8.2 and 2.0 Hz, H-5 Ph), 7.44 (d, 1H, J= 7.6 Hz, H-6 Ph), 7.53 (d, 1H,
J= 8.4 Hz, H-8 Quin) 7.66 (d, 1H, J= 2.4 Hz, H-3 Ph), 7.78 (d, 1H, J=13.2 Hz, H-5 Quin), 8.65
13
(s, 1H, H-2 Quin). C NMR (100 MHz, DMSO-d6) δ: 7.98, 35.58, 36.15, 45.79, 51.13, 106.42,
107.03, 111.70 (d, J= 23.2 Hz), 120.03 (d, J= 7.2 Hz), 128.00, 129.52, 133.12, 133.73, 133.98,
134.76, 138.98, 145.78 (d, J= 9.9 Hz), 148.19, 153.36 (d, J= 247.3 Hz), 157.73, 158.86, 162.53,
174.86. MS (m/z, %): 604 (M+, 10), 515 (4), 401 (9), 347 (5), 302 (5), 245 (9), 217 (8), 180 (10),
160 (53), 144 (10), 125 (100), 108 (12), 89 (57), 76 (58), 59 (39), 44 (66). Anal. Calcd for
C26H23Cl2FN6O2S2: C, 51.57; H, 3.83; N, 13.88. Found: C, 51.55; H, 3.71; N, 14.01.
4.4.7.
1-Cyclopropyl-N-(5-((3,4-dichlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)-6-fluoro-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13e)
18

Yield 31%; m.p. 104-107°C; IR (KBr, cm−1): 3421, 2918, 2849, 1671, 1624, 1611, 1522, 1482,
1
1338, 1256, 798. H NMR (300 MHz, DMSO-d6) δ: 1.08-1.43 (m, 4H, c-Pr), 2.80-3.89 (m, 9H,
Pip and c-Pr), 4.52 (s, 2H, CH2 benzyl), 7.43 (d, 1H, J= 7.5 Hz, H-6 Ph), 7.48 (d, 1H, J= 7.2 Hz,
H-5 Ph), 7.61 (d, 1H, J=8.1 Hz, H-8 Quin), 7.72 (s, 1H, H-2 Ph), 7.82 (d, 1H, J= 13.5 Hz, H-5
Quin), 8.67 (s, 1H, H-2 Quin). 13C NMR (75 MHz, DMSO-d6) δ: 8.03, 36.19, 36.50, 45.06, 49.94,
106.67, 107.13, 111.82 (d, J= 22.7 Hz), 120.39, 129.84, 130.60, 131.11, 131.44, 138.92, 138.98,
145.24, 148.26, 153.30 (d, J= 250 Hz), 158.25, 158.61, 162.48, 174.86 (d, J= 2.3 Hz). MS (m/z,
%): 604 (M+, 2), 532 (2), 451 (4), 377 (5), 281 (6), 239 (6), 224 (5), 209 (2), 193 (2), 162 (5), 149
(12), 137 (8), 124 (8), 112 (9), 101 (9), 83 (20), 69 (26), 57 (41), 44 (100). Anal. Calcd for
C26H23Cl2FN6O2S2: C, 51.57; H, 3.83; N, 13.88. Found: C, 51.65; H, 3.70; N, 13.73.
4.4.8.
1-Cyclopropyl-N-(5-((2,6-dichlorobenzyl)thio)-1,3,4-thiadiazol-2-yl)-6-fluoro-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13f)
Yield 35%; m.p. 240-244°C; IR (KBr, cm−1): 3436, 2920, 2841, 1677, 1626, 1508, 1480, 1335,
1285, 1258, 796, 761. 1H NMR (400 MHz, CDCl3) δ: 1.20-1.29 (m, 2H, c-Pr), 1.37-1.45 (m, 2H,
c-Pr), 1.71 (brs, 1H, NH Pip), 3.09-3.17 (m, 4H, Pip), 3.27-3.35 (m, 4H, Pip), 3.53-3.61 (m, 1H,
c-Pr), 4.82 (s, 2H, CH2), 7.19 (t, 1H, J= 8.0 Hz, H-4 Ph), 7.31-7.38 (m, 3H, H-3, H-5 Ph, H-8
Quin), 8.07 (d, 1H, J=13.2 Hz, H-5 Quin), 8.84 (s, 1H, H-2 Quin). 13C NMR (100 MHz, CDCl3)
δ: 8.21, 34.96, 45.92, 51.06, 104.69, 108.40, 112.97 (d, J= 23.5 Hz), 121.12, 128.45, 129.48,
132.52, 134.19, 136.14, 138.50, 140.72, 145.83, 147.35, 152.45, 153.8 (d, J= 235 Hz), 158.43,
159.29, 162.84, 175.17. MS (m/z, %): 604 (M+, <1), 488 (2), 416 (4), 352 (71), 330 (9), 314 (9),
288 (11), 256 (5), 192 (8), 159 (100), 125 (53), 104 (12), 89 (33), 63 (23), 44 (12). Anal. Calcd for
C26H23Cl2FN6O2S2: C, 51.57; H, 3.83; N, 13.88. Found: C, 51.66; H, 3.89; N, 13.80.
4.4.9.
1-Cyclopropyl-6-fluoro-N-(5-((2-fluorobenzyl)thio)-1,3,4-thiadiazol-2-yl)-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13g)
Yield 38%; m.p. 201-203 °C; IR (KBr, cm−1): 3412, 2919, 2848, 1669, 1624, 1611, 1523, 1482,
1338, 1256, 798, 760. 1H NMR (400 MHz, CDCl3) δ: 1.19-1.45 (m, 4H, c-Pr), 1.79 (brs, 1H, N-
H, Pip), 3.08-3.18 (m, 4H, Pip), 3.25-3.39 (m, 4H, Pip), 3.45-3.61 (m, 1H, c-Pr), 4.55 (s, 2H, CH2
benzyl), 7.03-7.14 (m, 2H, H-5, H-6 Ph), 7.23-7.31 (m, 1H, H-3 Ph), 7.34 (d, 1H, J= 6.8 Hz, H-8
19

Quin), 7.47 (t, 1H, J= 7.6 Hz, H-4 Ph), 8.05 (d, 1H, J= 13.2 Hz, H-5 Quin), 8.82 (s, 1H, H-2 Quin).
13C NMR (100 MHz, CDCl3) δ: 8.21, 29.71, 31.51, 35.22, 45.78, 50.86, 104.78, 108.36, 112.90
(d, J= 22.8 Hz), 115.55 (d, J= 20.8 Hz), 121.14, 124.22, 129.61, 129.69, 131.40, 138.49, 145.84,
147.36, 153.80 (d, J= 249.0 Hz), 158.71, 159.07, 160.05 (d, J= 220.0 Hz), 162.75, 175.16. MS
(m/z, %): 554 (M+, <1), 438 (2), 349 (6), 288 (3), 240 (3), 142 (4), 109 (100), 83 (20), 57 (8), 45
(8). Anal. Calcd for C26H24F2N6O2S2: C, 56.30; H, 4.36; N, 15.15. Found: C, 56.36; H, 4.27; N,
15.18.
4.4.10.
1-Cyclopropyl-6-fluoro-N-(5-((4-fluorobenzyl)thio)-1,3,4-thiadiazol-2-yl)-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13h)
Yield 33%; m.p 189-191 °C; IR (KBr, cm−1): 3434, 2921, 2849, 1675, 1625, 1610, 1508, 1481,
1258, 841, 798, 761. 1H NMR (400 MHz, CDCl3) δ: 1.21-1.28 (m, 2H, c-Pr), 1.35-1.43 (m, 2H, c-
Pr), 3.06-3.14 (m, 4H, Pip), 3.22-3.29 (m, 4H, Pip), 3.52-3.59 (m, 1H, c-Pr), 4.45 (s, 2H, CH2
benzyl), 7.00 (t, 2H, J= 8.4 Hz, H-3, H-5 Ph), 7.29 (d, 1H, J=7.2 Hz, H-8 Quin), 7.39 (dd, 2H, J=
8.8 Hz, J= 5.6 Hz, H-2, H-6 Ph), 7.92 (d, 1H, J=13.2 Hz, H-5 Quin), 8.75 (s, 1H, H-2 Quin). 13C
NMR (100 MHz, CDCl3) δ: 8.20, 35.25, 37.59, 45.85, 50.92, 104.76, 108.10, 112.68 (d, J= 23.3
Hz), 115.58 (d, J= 21.4 Hz), 120.89 (d, J= 7.4 Hz), 130.85 (d, J= 8.2 Hz), 132.16 (d, J= 2.9 Hz),
138.45, 145.78 (d, J= 10.4 Hz), 147.35, 153.57 (d, J= 249.3 Hz), 158.55, 159.05, 162.26 (d, J=
245.0 Hz), 162.61, 174.99. MS (m/z, %): 554 (M+, <1), 438 (2), 349 (3), 245 (2), 164 (2), 142 (7),
109 (100), 83 (10), 59 (6), 42 (6). Anal. Calcd for C26H24F2N6O2S2: C, 56.30; H, 4.36; N, 15.15.
Found: C, 56.48; H, 4.35; N, 15.06.
4.4.11.
N-(5-((4-Bromobenzyl)thio)-1,3,4-thiadiazol-2-yl)-1-cyclopropyl-6-fluoro-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13i)
Yield 21%; m.p. 123-125°C; IR (KBr, cm−1): 3425, 2919, 2848, 1671, 1624, 1611, 1522, 1482,
1
1337, 1256, 798, 761. H NMR (500 MHz, DMSO-d6) δ: 1.16-1.50 (m, 4H, c-Pr), 2.92 (m, 4H,
Pip), 3.23 (m, 4H, Pip), 3.33 (m, 1H, c-Pr), 4.48 (s, 2H, CH2 benzyl), 7.35-7.58 (m, 5H, Ph, and
13
H-8 Quin), 7.89 (d, 1H, J= 13.0 Hz, H-5 Quin), 8.72 (s, 1H, H-2 Quin). C NMR (100 MHz,
DMSO-d6) δ: 8.01, 36.15, 37.16, 44.86, 49.65, 106.71, 107.14, 111.85 (d, J= 22.6 Hz), 120.38,
121.180, 131.68, 131.89, 136.93, 138.97, 145.23 (d, J= 10.0 Hz), 148.33, 153.33 (d, J= 247.5 Hz),
20

158.47, 158.58, 162.49, 174.88. MS (m/z, %): 614 (M+, <1), 471 (4), 204 (8), 184 (4), 171 (100),
121 (5), 106 (11), 91 (62), 76 (13), 63 (31), 45 (13). Anal. Calcd for C26H24BrFN6O2S2: C, 50.73;
H, 3.93; N, 13.65. Found: C, 50.61; H, 4.09; N, 13.88.
4.4.12.
1-Cyclopropyl-6-fluoro-N-(5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)-4-oxo-7-
(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxamide (13j)
Yield 28%; m.p. 193-196°C; IR (KBr, cm−1): 3435, 2917, 2849, 1668, 1625, 1611, 1515, 1482,
1351, 1257, 798, 761. 1H NMR (400 MHz, DMSO-d6) δ: 1.16-1.37 (m, 4H, c-Pr), 2.88-2.98 (m,
4H, Pip), 3.20-3.27 (m, 4H, Pip), 3.78-3.88 (m, 1H, c-Pr), 4.65 (s, 2H, CH2 benzyl), 7.50 (d, 1H,
J=7.6 Hz, H-8 Quin), 7.70 (d, 2H, J= 8.4 Hz, H-2, H-6 Ph), 7.89 (d, 2H, J=13.6 Hz, H-5 Quin)
8.20 (d, 2H, J=8.8 Hz, H-3, H-5, Ph), 8.72 (s, 1H, H-2 Quin). 13C NMR (100 MHz, DMSO-d6) δ:
7.99, 36.17, 36.96, 45.65, 50.93, 106.63, 107.11, 111.82 (d, J= 23.5 Hz), 120.16, 124.10, 130.76,
139.09, 145.78, 147.21, 148.38, 152.20, 153.8 (d, J= 245.0 Hz), 158.12, 158.72, 162.67, 175.01.
MS (m/z, %): 581 (M+, 6), 513 (5), 366 (6), 356 (4), 314 (9), 287 (17), 269 (9), 245 (22), 202 (25),
186 (10), 168 (11), 157 (10), 137 (72), 118 (48), 106 (45), 91 (46), 77 (59), 65 (65), 44 (100).
Anal. Calcd for C26H24FN7O4S2: C, 53.69; H, 4.16; N, 16.86. Found: C, 53.70; H, 4.29; N, 16.82.
4.5. Preparation of enrofloxacin derivatives 14a and 14b
To a mixture of enrofloxacin (1b, 1.0 mmol) in dry CH2Cl2 (3 mL) was added triethylamine (2
mmol), and stirred at room temperature for 30 min. Then, ethyl chloroformate (1mmol) was added
dropwise at 0 ºC and the stirring was continued for 1 h and allowed the reaction mixture to reach
room temperature to get a solution. After addition of the corresponding thiadizole-amine 9, the
reaction was stirred overnight. Finally, the precipitated solid was filtered and washed carefully
with cold dichloromethane to obtain desired amide 14.
4.5.1. N-(5-(Benzylthio)-1,3,4-thiadiazol-2-yl)-1-cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxamide (14a)
Yield 35%; m.p. 236-240°C; IR (KBr, cm−1): 3467, 2925, 2821, 1674, 1626, 1610, 1523, 1481,
1
1380, 1334, 1253, 1126, 1049, 1024, 762, 677. H NMR (400 MHz, DMSO-d6) δ: 1.07 (t, 3H,
21

CH3), 1.12-1.43 (m, 4H, c-Pr) 2.61 (m, 4H, Pip), 3.20-3.73 (m, 7H, Pip and NCH2Me), 3.80-3.95
(m, 1H, c-Pr), 4.52 (s, 2H, CH2 benzyl), 7.30 (t, 1H, J= 8.0 Hz, H-4 Ph), 7.36 (t, 2H, J= 7.6 Hz,
H-3, H-5 Ph), 7.44 (d, 2H, J= 7.2 Hz, H-2, H-6 Ph), 7.55 (d, 1H, J= 7.2 Hz, H-8 Quin), 7.93 (d,
1H, J= 12.0 Hz, H-5 Quin), 8.75 (s, 1H, H-2 Quin), 13.97 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 7.99, 12.39, 36.13, 37.97, 49.82, 51.98, 52.42, 55.39, 106.56, 107.09, 111.72 (d, J=
23.6 Hz), 120.18 (d, J= 7.2 Hz), 128.08, 129.03, 129.50, 137.11, 138.92, 145.21 (d, J= 10.1 Hz),
148.21, 153.29 (d, J= 247.4 Hz), 158.32, 158.89, 162.42, 174.83. MS (m/z, %): 564 (M+, 6), 383
(4), 368 (8), 342 (27), 286 (6), 257 (8), 121 (4), 91 (100), 70 (8), 57 (33), 42 (36). Anal. Calcd for
C28H29FN6O2S2: C, 59.56; H, 5.18; N, 14.88. Found: C, 59.71; H, 5.32; N, 14.69.
4.5.2.
1-Cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluoro-N-(5-((4-fluorobenzyl)thio)-1,3,4-
thiadiazol-2-yl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (14b)
Yield 33%; m.p. > 300°C; IR (KBr, cm−1): 3469, 3401, 2918, 1667, 1627, 1608, 1504, 1482, 1278,
1217, 1031, 921, 779, 676. 1H NMR (500 MHz, DMSO-d6) δ: 1.15-1.40 (m, 7H, c-Pr and CH3),
3.10-3.50 (m, 8H, Pip), 3.55-3.67 (m, 2H, NCH2Me), 3.81-3.93 (m, 1H, c-Pr), 4.50 (s, 2H, CH2
benzyl), 7.16 (t, 2H, J= 8.5 Hz, H-3, H-5 Ph), 7.42-7.50 (m, 2H, H-2, H-6 Ph), 7.59 (d, 1H, J= 6.5
Hz, H-8 Quin), 7.98 (d, 1H, J= 12.5 Hz, H-5 Quin), 8.76 (s, 1H, H-2 Quin), 13.90 (s, 1H, NH).
13C NMR (100 MHz, DMSO-d6) δ: 8.06, 12.29, 36.15, 37.12, 46.77, 48.99, 50.36, 51.16, 107.33,
111.89 (d, J= 23.0 Hz), 115.82 (d, J= 20.7 Hz), 127.18, 131.57 (d, J= 7.9 Hz), 138.97, 138.45,
143.94, 148.87, 153.39 (d, J= 247.8 Hz), 158.67, 158.78, 162.12 (d, J= 245.5 Hz), 162.57, 174.84.
MS (m/z, %): 582 (M+, 4), 368 (3), 342 (14), 257 (3), 139 (5), 109 (100), 83 (14), 70 (5), 57 (21),
42 (20). Anal. Calcd for C28H28F2N6O2S2: C, 57.72; H, 4.84; N, 14.42. Found: C, 57.65; H, 4.82;
N, 14.49.
4.6. MTT assay
The MTT (3-(4,5-dimethyl thiadiazole-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay was
performed to evaluate the cytotoxic effect of synthesized compounds (12b, 12h, 13a-j, 14a and
14b) against three different cancerous cell lines MCF-7, A549 and SKOV-3. The cell lines were
obtained from Pasture Institute, Iran. The procedures used for cell culture, treatment and viability
22

assay were based on reported methods in literatures [27,28]. The reference drug doxorubicin and
DMSO 1% was used as positive and negative controls, respectively. The quinolone drug CF was
included in the cell viability assay as parent compound for more comparisons. The IC50 values
were determined from curves constructed by plotting cell viability (%) versus concentration, using
Prism 6, GraphPad Software.
4.7. Cell cycle analysis
The cell cycle analysis was performed in MCF-7 cells, treated with compounds 13e and 13g, using
flow cytometric method. Briefly, MCF-7 cells (3×105 cells/well) were seeded in 6-well plate and
incubated with IC50 concentration of 13e and 13g for 48 h. Subsequently, both treated and
untreated cells were collected and washed with PBS (phosphate buffered saline) twice, and fixed
with 70% ethanol for 3 hours. Then, RNase A (5 µL, 10 mg/mL concentration) was added to each
sample and after 1 h at r.t, mixed with 10 μL of PI (50 μg/mL final concentration). At the end, 30
min after addition of PI, the cell distribution was determined by FACS Calibur flow cytometer and
utilizing FlowJo software ver. X.0.7 [28].
4.8. Annexin-V/PI double staining assay
For analyzing apoptotic and necrotic cells after treatment with the selected compounds 13e and
13g, annexin-V/PI double staining method was carried out followed by flow cytometry [29].
Briefly, MCF-7 cells with optimum density (3 ×105) were cultured in 6-well plates and after 24 h
of incubation; the cells were treated with IC50 concentration of 13e and 13g. After 48 h, the treated
and untreated cells were harvested and washed twice with cold PBS. Then, the cells were
resuspended in binding buffer (500 µL) containing 10 µL of Annexin V-fluorescein isothiocyanate
(FITC). Subsequently, propidium iodide (PI) solution (10 μL) was added to each sample and the
stained cells were kept in the dark at r.t for 15 min and subjected to flow cytometry. For
determining the percentage of apoptotic cells in each quadrant, output data were analyzed using
FlowJo software ver. X.0.7.
4.8. Comet assay
23

The DNA damage in MCF-7 cells, exposed to 13e and 13g was measured by alkaline single cell
gel electrophoresis (SCGE) method as reported previously [30]. The MCF-7 cells were treated
with the compounds 13e and 13g at the concentrations of 1, 5 and 10 µM for 1 h. Doxorubicin and
CF were included for comparison. The untreated cells were used as negative control. The DNA
damage parameters were estimated by using Comet Score software project (CASP).
Acknowledgment
This work was partly the Ph.D. thesis of HA and supported by a grant from the Research Council
of Mazandaran University of Medical Sciences (Grant No. 2777).
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26

Graphical Abstract
Anticancer Agents
Antibacterial drug
N
O
O
O
O
N
F
S
F
OH
S
N
R1
H
N
N
N
N
HN
HN
Ciprofloxacin
13e:
R1= 3,4-Cl2
MCF-7: IC= 3.26 
SKOV-3: IC= 5.08 
13g:
R1= 2-F

IC50 against cancer cells >100
(MCF-7, A549, SCOV-3)
M
IC= 3.34 
IC= 5.43 
As potent as
apoptosis
Doxorubicin
Sub-G1
DNA
population
damage
27

Research Highlights
 C-3 modified ciprofloxacin analogs bearing thiadiazole-carboxamide were synthesized as anticancer
agents.
 MTT assay showed that most of them have significant activity against MCF-7, A549 and SKOV-3.
 Compounds 13a-e and 13g were as potent as doxorubicin against MCF-7 cell line (IC50s = 3.26-3.90
µM).
 Promising compounds 13e and 13g induced apoptosis and increased sub-G1 cell population in MCF-7
cells.
 Comet assay demonstrated the potential of compounds in DNA damage and fragmentation.
28