Synthesis, molecular docking, antimicrobial evaluation, and DNA cleavage assay of new thiadiazole/oxadiazole ciprofloxacin derivatives

Article abstract of DOI:10.1007/s00706-019-02478-4

Abstract: Herein we report the synthesis of new N-4-piperazinyl thiadiazole and oxadiazole ciprofloxacin derivatives and their antibacterial and antimycobacterial activities. Although thiadiazole ciprofloxacin derivatives compound showed broad spectrum antibacterial activity against all the tested strains either Gram-positive or Gram-negative organisms, the oxadiazole derivatives exhibited weaker antibacterial and antimycobacterial activities than thiadiazole derivatives against most of the tested strains compared with the reference ciprofloxacin. Moreover, the antimycobacterial screening revealed that compounds which containing thiadiazole scaffold potently inhibited Mycobacterium smegmatis at MIC of 1.56 and 3.13, respectively, and modestly inhibited the drug-resistant strains. DNA cleavage assay revealed that thiadiazole ciprofloxacin derivatives inhibited supercoil relaxation, albeit to a lesser extent than ciprofloxacin, and it also increased the amount of nicked substrate produced. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-019-02478-4

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-019-02478-4
ORIGINAL PAPER
Synthesis, molecular docking, antimicrobial evaluation, and DNA
cleavage assay of new thiadiazole/oxadiazole ciprofoxacin derivatives
Hamada H. H. Mohammed1,2 · Samar H. Abbas1 · El‑Shimaa M. N. Abdelhafez1 · James M. Berger3 ·
Satoshi Mitarai4 · Masayoshi Arai5 · Gamal El‑Din A. A. Abuo‑Rahma1
Received: 28 March 2019 / Accepted: 2 July 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
Herein we report the synthesis of new N-4-piperazinyl thiadiazole and oxadiazole ciprofoxacin derivatives and their anti-
bacterial and antimycobacterial activities. Although thiadiazole ciprofoxacin derivatives compound showed broad spectrum
antibacterial activity against all the tested strains either Gram-positive or Gram-negative organisms, the oxadiazole deriva-
tives exhibited weaker antibacterial and antimycobacterial activities than thiadiazole derivatives against most of the tested
strains compared with the reference ciprofoxacin. Moreover, the antimycobacterial screening revealed that compounds which
containing thiadiazole scafold potently inhibited Mycobacterium smegmatis at MIC of 1.56 and 3.13, respectively, and mod-
estly inhibited the drug-resistant strains. DNA cleavage assay revealed that thiadiazole ciprofoxacin derivatives inhibited
supercoil relaxation, albeit to a lesser extent than ciprofoxacin, and it also increased the amount of nicked substrate produced.
Graphic abstract
Keywords Ciprofoxacin · Thiadiazole · Oxadiazole · Antimycobacterial · Antibacterial · DNA cleavage assay
Introduction
Tuberculosis (TB) represents one of the most serious micro-
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-019-02478-4) contains
supplementary material, which is available to authorized users.
bial infections all over the world [1, 2] which is caused by
mycobacterium tuberculosis [3, 4]. TB is considered the
4
* El-Shimaa M. N. Abdelhafez
Bacteriology Division, Department of Mycobacterium
shimaanaguib_80@mu.edu.eg
Reference and Research, Research Institute of Tuberculosis,
Japan Anti-Tuberculosis Association, Kiyose 204-8533,
Japan
1
Department of Medicinal Chemistry, Faculty of Pharmacy,
Minia University, El Minya 61519, Egypt
5
Graduate School of Pharmaceutical Sciences, Osaka
2
Department of Pharmaceutical Chemistry, Faculty
University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
of Pharmacy, Deraya University, New Minia 61519, Egypt
3
Department of Biophysics and Biophysical Chemistry, Johns
Hopkins University School of Medicine, Baltimore, MD,
USA
Vol.:(0123456789)
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H. H. H. Mohammed et al.
Fig. 1 Structures of some antitubercular fuoroquinolones
frst cause of death from infectious diseases ranking above
HIV/AIDS and the ninth leading cause of death worldwide
according to WHO report in 2016 [4]. This infection spreads
through the air and causes millions of deaths every year
[5]. According to ofcial estimates, one-third of world’s
population has been afected by latent tuberculosis [6]. Fur-
thermore, the most widely used chemotherapy for treatment
of uncomplicated TB cases is a combination of isoniazid,
rifampicin, pyrazinamide, and ethambutol [7, 8]. In the last
few years, the incidence of tuberculosis has remarkably
increased especially new cases of multidrug-resistant tuber-
culosis (MDR-TB) and extensively drug-resistant tuberculo-
sis (XDR-TB) [8–10]. The emergence of multidrug-resistant
tuberculosis (MDR-TB) and extensively drug-resistant tuber-
culosis (XDR-TB) strains which are virtually untreatable
using current medications has made the situation more and
more serious. Because of its ongoing impact on global health
system, there is an urgent need to develop novel antituber-
cular agents to combat this pathogen [8, 11].
small and lipophilic to enhance the activity. Substituents at
the C-7 are essential and are responsible for enhancement of
lipophilicity, bioavailability, and safety [17–19]. Lipophilic-
ity is an important parameter to be considered in the design
of novel antitubercular lead compounds [20] and it is an
essential component in mycobacterial cell wall involved in
pathogenicity and drug resistance [21].
On the other hand, the 1,3,4-oxadiazole and 1,3,5-thia-
diazole and their derivatives have considerable biological
importance and could be seen in diferent bioactive mol-
ecules [22]. In the last few years, they have received much
more attention in medicinal chemistry as surrogates for
carboxylic acids, esters and carboxamides and become an
important construction motif for the development of new
drugs [23, 24]. The 1,3,4-thiadiazole nucleus has been
observed as a common motif in several antimicrobial and
antitubercular drug leads [23, 25–27]. The presence of nitro-
gen atoms in these heterocyclic moieties is readily able to
bind with a variety of enzymes and receptors in biologi-
cal systems and thus display versatile biological activities
including antibacterial [25, 28, 29], antifungal [30, 31],
antitubercular [32, 33], anti-infammatory [34, 35], analgesic
[36] and anticancer activities [36–38]. Accordingly, several
compounds containing these heterocyclic moieties have been
evaluated for their diferent biological activities; As a result,
hybrids of fuoroquinolone and 1,3,4-thiadiazoles had been
considered as interesting targets for designing new antibac-
terial [39–42] and antitubercular candidates [21, 40]. For
example, fuoroquinolones/thiadiazole derivative I (Fig. 1)
showed signifcant activity against MB H37Rv comparable
to moxifoxacin [21]. Also, some oxadiazole derivatives dis-
played antitubercular activity such as compounds II and III
(Fig. 2) [41, 43].
Nowadays, antibacterial fluoroquinolones like cipro-
foxacin, lomefoxacin, ofoxacin and lomefoxacin (Fig. 1)
are used as second-line agents in combination with other
known antituberculosis drugs according to WHO guidelines
in treatment of MDR-TB patients [12]. Moreover, moxifoxa-
cin and levofoxacin are under phase II and III clinical trial
of several regimens for treatment of MDR-TB, respectively
[13].DNA gyrase enzyme is the target enzyme of fuoroqui-
nolones which is involved in DNA supercoiling and bacterial
replication. Fluoroquinolones can bind to the enzyme–DNA
binary complex leading to blocking DNA replication and
consequently cell death [14, 15]. Based on the reported SAR
studies of fuoroquinolones, the β-keto carboxylic acid moi-
ety is essential for hydrogen bonding interactions with DNA
bases [16]. The N-1 and C-8 substituents should be relatively
1 3

Synthesis, molecular docking, antimicrobial evaluation, and DNA cleavage assay of new…
Fig. 2 Design of the target compounds
Because of the fast development of resistance to fuo-
roquinolones and the demand of newer anti-bacterial and
anti-tubercular fuoroquinolone candidates with broad spec-
trum of activity and enhanced pharmacokinetic profle, we
decided to examine the impact of substitution at the piper-
azinyl nitrogen of the parent quinolones. The present work
involves the synthesis of new N-4-piperazinyl (1,3,4-thiadia-
zole)ciprofoxacin 3a–3e and 1,3,4-oxadiazole derivatives
8a–8e (Fig. 2) and screening of their antibacterial and anti-
mycobacterial activities. In addition, molecular modeling
studies and DNA cleavage assay have been carried out to
study the potential mechanism of action of the newly syn-
thesized compounds.
Results and discussion
Chemistry
The target ciprofoxacin/thiadiazole compounds 3a–3e and
their intermediates were prepared as outlined in Scheme 1.
2-Amino-1,3,4-thiadiazole derivatives 1a–1e were pre-
pared by refuxing a mixture of the appropriate benzoic acid
derivatives and thiosemicarbazide in presence of phosphorus
oxychloride according to the reported procedure [44, 45].
Acylation of thiadiazole derivatives 1a–1e with bromoacetyl
bromide using potassium carbonate as a base in dichlo-
romethane gives the corresponding acetamide derivatives
2a–2e. Alkylation of ciprofoxacin with acetamide deriva-
tives 2a–2e was achieved in acetonitrile in the presence of
triethylamine (TEA) to aford the target compounds 3a–3e
in a good yield.
1 3

H. H. H. Mohammed et al.
Scheme 1
The synthesis of oxadiazole/ciprofoxacin compounds
6a–6e is shown by Scheme 2. Heating at reflux of the
appropriate benzoic acid derivatives with absolute ethanol
in the presence of concentrated sulphuric acid as a dehy-
drating agent aforded the corresponding esters 4a–4e in
high yield. Consequently, hydrazinolysis of the ethyl ester
derivatives 4a–4e with hydrazine monohydrate in refux-
ing ethanol aforded the corresponding carbohydrazides
5a–5e in high yield. The formed hydrazides 5a–5e was
confrmed by their reported melting points [46]. Heating at
refux of equimolar amounts of the hydrazides 5a–5e and
carbon disulfde in ethanolic KOH aforded the correspond-
ing 1,3,4-oxadiazole-2-thiol derivatives 6a–6e. The forma-
tion of compounds 6a–6e was confrmed by their reported
melting points [47]. Amidation of ciprofoxacin to aford
compound 7 was carried out by bromoacetyl bromide in
basic condition which was further alkylated into compounds
8a–8e as described before for derivatives 3a–3e. The newly
synthesized compounds were identifed by 1H NMR, 13C
NMR and HRMS.
coli (ATCC 8739) and Candida albicans (ATCC 10231).
The tested compounds were assayed using the standard
agar cup difusion method and ciprofoxacin as a reference
[48]. Results of the antibacterial screening are outlined in
Table 1. According to the MICs in Table 1, it can be deduced
that most of the tested compounds showed a higher anti-
bacterial activity than the reference ciprofoxacin against
Gram-positive and Gram-negative bacteria. It was found
that compounds 3a–3e displayed potent activity against S.
aureus with MICs of 1.31, 1.85, 1.45, 6.42 and 2.7 µg/cm3,
respectively. Meanwhile, compounds 3–e displayed remark-
able antibacterial activity against E. coli, with MICs of 1.12,
1.63, 3.61, 6.14, and 7.15 µg/cm3, respectively. Moreover,
compounds 3d, 3e, and 8b revealed high potency against
Klebsiella pneumonia with MIC of 5.96, 3.13 and 2.17 µg/
cm3, respectively. On contrast, other compounds displayed
moderate to weak activity. Furthermore, ciprofloxacin
derivatives 3a, 3e, and 8b showed potent activity against
ps. aeruginosa with MICs of 7.4, 1.29 and 2.17 µg/cm3,
respectively.
Based on the above-mentioned results, it is obvious that
compounds 3e showed broad spectrum antibacterial activity
against all the tested strains either Gram-positive or Gram-
negative. In general, the oxadiazole derivatives 8a, 8b, and
8d exhibited weaker antibacterial activity than thiadiazole
derivatives against most of the tested strains. It can be con-
cluded that there is no specifc substituent that can determine
the activity of either electron donating or electron withdraw-
ing. The enhanced activity of some of the tested derivatives
Biological investigations
Screening of antibacterial activities
The antibacterial activity of compounds 3a–3e, 8a, 8b, and
8d was evaluated in vitro against Staphylococcus aureus
(ATCC 6538), Klebsiella pneumoniae (ATCC 10031),
Pseudomonas aeruginosa (ATCC 10145), Escherichia
1 3

Synthesis, molecular docking, antimicrobial evaluation, and DNA cleavage assay of new…
Scheme 2
may be due to improvement in the physicochemical proper-
ties and consequently enhancing permeability to microbial
cells.
Table 1 The MICs of antibacterial and antifungal activities of the
tested compounds, ciprofoxacin and ketoconazole (µg/cm3)
Screening of antimycobacterial activity
Compound
MIC/µg/cm3
The in vitro antimycobacterial activity of compounds 3a,
3c–3e, 8a, 8b, 8d against the non-pathogenic M. smegmatis
was initially evaluated using the MTT method [49] under
aerobic conditions using isonicotinic acid hydrazide (INH)
as a reference. The obtained MICs (µg/cm3) of the target
compounds against M. smegmatis are shown in Table 2.
By comparison with the reference INH, the thiadiazole
derivatives 3c and 3e displayed potent antitubercular activity
with MIC of 1.56 and 3.13 μg/cm3, respectively. Moreover,
compound 3d showed a comparable activity to INH with
MIC of 6.25 μg/cm3, however, ciprofoxacin–oxadiazole
derivatives 8a, 8b, and 8d exhibited no activity with MICs
(>200 μg/cm3). The above results indicate that ciprofoxa-
cin/thiadiazole derivatives exhibited better inhibition than
ciprofoxacin–oxadiazole derivatives and in turn the most
active derivatives 3a, 3d, and 3e were further screened
G+ve bac-
teria
G−ve bacteria
S. aureus
E. coli K. pneumo-
niae
P. aeruginosa
3a
3b
3c
3d
3e
8a
8b
8d
1.31
1.85
1.45
6.42
2.7
11.3
Na
1.12
1.63
3.61
6.14
7.15
35.43
12.57
10.13
5.96
7.4
9.11
15.23
28.75
1.29
89.23
2.17
Na
3.13
24.68 64.12
12.4
Na
9.28
2.17
Na
6.29
Na
Ciprofoxa- 8.62
8.62
cin
MIC minimum inhibitory concentration, G+ve bacteria Gram-posi-
tive bacteria, G−ve bacteria Gram-negative bacteria
1 3

H. H. H. Mohammed et al.
Table 2 The MICs of the tested
compounds and the reference
INH against M. smegmatis (µg/
cm3)
they are less active than the reference drugs LVFX and
MFLX (MICs: 0.03–8 µg/cm3). Among of them, compound
3d displayed the highest activity (MICs: 2 µg/cm3) against
the drug-resistant strain R-2012-59 (MDR), M. fortuitum
ATCC06841 and M. tuberculosis R2012-123 (pansensitive).
Similarly, the ciprofoxacin derivative 3e exhibited activity
(MICs: 4 µg/cm3) against R-2012-59 (MDR) strain, M. for-
tuitum ATCC06841 and M. tuberculosis R2012-123 (pan-
sensitive). While derivative 3a exhibited the lowest activity
against R-2012-59 (MDR) and M. tuberculosis R2012-123
(pansensitive) (MICs: 8 µg/cm3).
Compound MICa against M.
smegmatis/μg/
cm3
3a
3c
3d
3e
8a
8b
8d
INH
12.5
1.56
6.25
3.13
>200
>200
>200
5
MIC minimum inhibitory con-
Cleavable complex DNA gyrase assay
centration
Fluoroquinolones act through the inhibition of type II bacte-
rial topoisomerase enzymes (DNA gyrase subunits encoded
by gyrA and gyrB) [50, 51] which transiently catalyzes
double-strand DNA breaks as it negatively supercoils DNA.
The resealing of the double-strand breaks that normally fol-
lows DNA strand passage is inhibited by fuoroquinolones
through generation of persistent covalent enzyme–DNA
adducts called cleaved complexes. The formed cleaved
complex disrupts normal DNA replication, inducing DNA
damage, and triggering cell death mechanisms [52]. In vitro
cleavable DNA gyrase complex assay is an excellent indictor
for DNA gyrase inhibition by furoquinolones. The ability
of the newly designed derivatives to generate cleaved DNA
complex was examined by linear DNA formation from a
starting supercoiled plasmid using ciprofoxacin as a refer-
ence compound.
Table 3 The MICs of the tested compounds and the reference levo-
foxacin and moxifoxacin against pathogenic drug-resistant and drug-
susceptible strains of M. tuberculosis (µg/cm3)
Strain
MIC/μg/cm3
3a
3d 3e
Levofoxacin Moxifoxacin
H37Rv ATCC27294
8
8
4
2
16
4
0.5
0.25
0.125
0.06
M. tuberculosis
R2012-123 (pan-
sensitive)
R-2012-59 (MDR)
R-2012-97 (XDR)
M. abscessus
ATCC19977
8
16
2
32
4
32
0.25
8
≤0.03
2
>32
>32 >32 >32 >32
M. chelonae
>32 4
8
4
0.5
0.25
0.06
ATCC35752
Highly potential active antimycobacterial compounds
3a, 3d, and 3e were selected to investigate their ability to
inhibit DNA gyrase. Inhibition of DNA gyrase activity of
the tested compounds was measured by the ability of DNA
gyrase to facilitate the formation of the cleavable complex
[53, 54]. The results of cleavable complex DNA gyrase assay
of compounds 3a, 3d, and 3e showed a good correlation
between the MTB cleaved DNA gyrase assay and in vitro
MTB MIC, Fig. 3.
M. fortuitum
ATCC06841
4
2
0.125
towards pathogenic drug-resistant and drug-susceptible
strains of M. tuberculosis, the results are shown in Table 3.
On the other hand, the results in Table 3 revealed that
the target compounds 3a, 3d, and 3e have potential activity
against the pathogenic drug-resistant and drug-susceptible
strains of M. tuberculosis (MICs: 2–32 µg/cm3), however,
Fig. 3 DNA cleavage of MTB
gyrase induced by compounds
3a, 3d, and 3e
1 3

Synthesis, molecular docking, antimicrobial evaluation, and DNA cleavage assay of new…
Table 4 Energy scores for the complexes formed by the tested com-
pounds 3a–3e, 8a–8e and the reference moxifoxacin in the active site
of topoisomerase II enzyme (PDB: 5bs8)
data bank (PDB: 5bs8) using Molecular Operating Environ-
ment (MOE®) version 2014.09 using moxifoxacin as refer-
ence for validation of the method [55].
Compound
dG/kJ/mol
Compound
dG/kJ/mol
The theoretical predictions from the molecular docking
study were found to be in agreement with the experimentally
observed antitubercular activity for the tested compounds
3a, 3c, 3d, 8a, 8b, and 8d. All highly active derivatives
were successfully docked into the active site of mycobac-
terial topoisomerase II enzyme. The binding free energies
from the major docked poses are listed in Table 4 and the
most favorable poses of the tested compounds are shown in
Figs. 4, 5, 6, 7, 8, 9, and 10. All of the tested compounds
have high binding afnity to the enzyme as the binding
free energy (dG) values of them range from − 94.6485 to
−126.3844 kJ/mol comparable to the reference moxifoxacin
(dG=−123.8058 kJ/mol).
Moxifoxacin
−123.8058
−99.70346
−129.8726
−100.2223
−126.3844
−123.1125
8a
8b
8c
8d
8e
−96.0071
−106.3955
−94.6485
−96.5505
−107.9679
3a
3b
3c
3d
3e
dG (kJ/mol) the binding free energies
Concerning the cleavable complex DNA gyrase assay, it
was found that compounds 3a and 3d induced DNA cleav-
age and inhibited supercoil relaxation at 5–500 μM. Moreo-
ver, compound 3d seems to act as an inhibitor of supercoil
relaxation, and 3d also was found to increase the amount of
nicked substrate produced. This could be due either to true
inhibition or to intercalation, while compound 3e induced
DNA cleavage at 50–500 µM and poison gyrase at 500 μM.
These results indicate true inhibition or intercalation of
gyrase enzyme.
The docking result of the reference compound moxifoxa-
cin is completely consistent with the mode of action of fuo-
roquinolones. The 2D diagram showed a crucial chelation
with magnesium ion (Mg 12:101) through the C-3 carbox-
ylic group and C-4 carbonyl functionality which is medi-
ated via water molecules [56, 57]. Moreover, stabilization
of the reference moxifoxacin within the active site occurred
through strong van der Waals interactions with the nucleo-
tide bases of DNA (DG H11, DA F11, DA E15, DA G15).
In addition, two pi–cation interactions with the nucleotide
bases of DNA (DT F10 and DT H10) were observed. Fur-
thermore, it forms an additional hydrogen bond with the
amino acid residue Arg A128. Also, several hydrophobic
interactions observed with active site of the enzyme docking
Results with topoisomerase II (Figs. 5, 6, 7) revealed that
most of the thiadiazole and oxadiazole tested compounds
Docking studies
Enzyme–ligand docking was carried out for all the synthe-
sized derivatives 3a–3e, 8a–8e and moxifoxacin as a refer-
ence to predict the binding interactions between the newly
synthesized ciprofoxacin derivatives and Mycobacterium
topoisomerase II (gyrase) enzyme obtained from the protein
Fig. 4 3D and 2D diagrams
illustrate the binding modes
of the reference moxifoxacin
interacted with the active site of
Mycobacterium gyrase enzyme
1 3

H. H. H. Mohammed et al.
Fig. 5 3D and 2D diagrams
illustrate the binding modes
of compounds 3a and 3b
interacted with the active site of
Mycobacterium gyrase enzyme
Fig. 6 3D and 2D diagrams
illustrate the binding modes of
compounds 3c interacted with
the active site of Mycobacte-
rium gyrase enzyme
1 3

Synthesis, molecular docking, antimicrobial evaluation, and DNA cleavage assay of new…
Fig. 7 3D and 2D diagrams
illustrate the binding modes of
compounds 3e interacted with
the active site of Mycobacte-
rium gyrase enzyme
Fig. 8 3D and 2D diagrams
illustrate the binding modes
of compounds 8a and 8b
interacted with the active site of
Mycobacterium gyrase enzyme
showed good binding with the enzyme and made several
interactions comparable to that of the reference moxifoxa-
cin. Compound 3b (Fig. 5) exhibited the same interactions
with the conserved amino acids as moxifoxacin, while com-
pound 3a lacked only two van der Waals interactions with
the nucleotide bases of DNA (DA E15, DA G15) than that
of the interactions that was shown by moxifoxacin.
topoisomerase II as moxifoxacin, but the pi–cation interac-
tions were not found except in compound 3c.
Concerning oxadiazole–ciprofoxacin derivatives 8a–8e,
compound 8b showed typical interactions as the reference
(Fig. 8), however, compound 8a dismissed only one binding
van der Waals interactions with the nucleotide base DNA
(DA E15) and hydrogen bonding with the amino acid residue
Arg A128 (Fig. 8).
On the other hand, compounds 3c–3e (Figs. 6, 7)
showed the same interactions within the binding site of
1 3

H. H. H. Mohammed et al.
Fig. 9 3D and 2D diagrams
illustrate the binding modes of
compounds 8c and 8d interacted
with the active site of Mycobac-
terium gyrase enzyme
Fig. 10 3D and 2D diagrams
illustrate the binding modes of
compounds 8e interacted with
the active site of Mycobacte-
rium gyrase enzyme
Compound 8c–8e kept most of the interaction modes,
however, they lack pi–cation interactions except in com-
pound 8d (Fig. 9). Hydrogen bond with the amino acid resi-
due Arg A128 was not found in both compounds 8e and 8c
(Figs. 9, 10).
potent activity against both G+ve or G−ve bacteria strains
than oxadiazole/ciprofoxacin derivatives 8a, 8b, 8d, and
8e. it is worth to mention that compounds 3e showed broad
spectrum antibacterial activity against all the tested strains
either G + ve or G−ve. Results indicated that compounds
3a and 3c–3e displayed considerable antimycobacterial
activity compared to the reference isoniazid against M.
smegmatis, while compounds 8a, 8b, and 8d showed no
activity with MICs (>200 μg/cm3). Moreover, compounds
3a, 3d, and 3e showed relatively promising activity against
the drug-resistant strain R-2012-59 (MDR), M. fortuitum
ATCC06841 and M. tuberculosis R2012-123 (pansensi-
tive). Furthermore, docking studies showed comparable
binding scores and similar binding interactions as that of
Conclusions
New N-4-piperazinyl ciprofoxacin thiadiazole derivatives
3a–3e and oxadiazole derivatives 8a–8e were prepared
and characterized by diferent spectroscopic techniques.
Results of antibacterial assay declared that thiadiazole/
ciprofloxacin derivatives 3a–3e showed generally more
1 3

Synthesis, molecular docking, antimicrobial evaluation, and DNA cleavage assay of new…
the reference moxifoxacin. However, thiadiazole derivatives
3a–3e showed a more favorable interaction than oxadiazoles
ones 8a–8e. Additionally, cleavable complex DNA gyrase
assay results confrm inhibition or intercalation of gyrase
enzyme by the tested compounds. In summary, introduction
of the 1,3,4-thiadiazole moiety into the N4-piperazinyl of
ciprofoxacin enhanced antitubercular activity more than if
hybridized with oxadiazole moiety where compounds 3a,
3d, and 3e are promising antitubercular agents that are rec-
ommended to be further studied.
Bromo‑N‑(5‑(3,4‑dimethoxyphenyl)‑1,3,4‑thiadiazol‑2‑yl)‑
acetamide (2d) Yellow powder; 2.50 g, 69% yield; mp:
247–249 °C [58].
Bromo‑N‑(5‑(3,4,5‑trimethoxyphenyl)‑1,3,4‑thiadiazol‑2‑yl)‑
acetamide (2e) Yellow powder; 3.10 g, 84.00% yield; mp:
274–276 °C [58].
General procedure for synthesis of compounds 3a–3e
An equimolar mixture of 2a–2e (0.0008 mol) and ciprofoxa-
cin HCl (0.30 g, 0.0008 mol) in acetonitrile (50 cm3) and
TEA (0.002 g, 0.0016 mol) was heated at refux for 8-10 h.
Filter while hot. The residue obtained was washed with hot
acetonitrile to aford the target compounds 3a–3e.
Experimental section
Chemistry
Reactions were monitored by TLC, using Merck 9385
pre-coated aluminum plate silica gel (Kiesel gel 60)
5 cm×20 cm plates with a layer thickness of 0.2 mm. The
spots were detected by exposure to UV lamp at λ=254 nm.
Melting points were determined on Stuart electrothermal
melting point apparatus and were uncorrected. IR spectra
were recorded as KBr disks on a Bruker Vector 22 IR spec-
1‑Cyclopropyl‑6‑fuoro‑4‑oxo‑7‑(4‑(2‑oxo‑2‑((5‑phenyl‑1,3,4‑
thiadiazol‑2‑yl)amino)ethyl)piperazin‑1‑yl)‑1,4‑dihydroquin‑
oline‑3‑carboxylic acid (3a, C27H24FN6O4S) Pale yellow pow-
der; 0.35 g, 78% yield; Rf =0.1; mp: 296–297 °C; 1H-NMR
(400 MHz, DMSO-d6) δ 1.19–1.21 (2H, m, cyclopropyl–H),
1.34–1.35 (2H, m, cyclopropyl–H), 2.82–2.84 (4H, m, pip-
erazinyl–H), 3.41–3.43 (4H, m, piperazinyl–H), 3.52 (2H, s,
N–CH2–CO), 3.82–3.85 (1H, m, cyclopropyl–H), 7.54–7.56
(3H, m, Ar–H), 7.60 (1H, d, J = 8 Hz, H-8), 7.90 (1H, d,
J=12 Hz, H-5), 7.92–7.95 (2H, m, Ar–H), 8.68 (1H, s, H-
2), 15.12 (1H, brs, COOH); 13C-NMR (100 MHz, DMSO-
d6) δ 8.0, 36.3, 49.9, 52.6, 60.3, 106.9, 107.4, 111.6, 119.6,
127.4, 129.8, 130.73, 131.1, 139.7, 145.7, 148.2, 152.5,
158.5, 161.2, 166.5, 169.3, 176.7; ESI–MS (m/z): calcd.
For C27H24FN6O4S 547.15693, found 547.15648 [M−H]−.
1
trophotometer. NMR spectra (400 MHz for H, 100 MHz
for 13C) were observed in DMSO-d6 on Bruker AM400
spectrometer with tetramethylsilane as the internal stand-
ard. Chemical shifts (δ) values are given in parts per million
(ppm) using DMSO-d6 as solvent and coupling constants
are designated as (J) in Hz. Splitting patterns are designated
as follows: s, singlet; d, doublet; dd, doublet of doublet; t,
triplet; q, quartet; m, multiplet; brs, broad singlet. High-
resolution mass spectra (HRMS) were recorded on Thermo
Scientifc Q Exactive™ Orbitrap mass spectrometer and
reported as mass/charge (m/z) with percent relative abun-
dance. The intermediates 1a–1e were prepared according to
the reported procedures [39, 44, 45].
7‑(4‑(2‑((5‑(4‑Chlorophenyl)‑1,3,4‑thiadiazol‑2‑yl)‑
a m i n o ) ‑ 2 ‑ oxo e t hy l ) p i p e ra z i n ‑ 1 ‑ y l ) ‑ 1 ‑ c yc l o p ro ‑
pyl‑6‑fuoro‑4‑oxo‑1,4‑dihydro quinoline‑3‑carboxylic acid
(3b, C27H23ClFN6O4S) Pale yellow powder 0.35 g, 73% yield;
1
Rf = 0.2; mp: 295–296 °C; H-NMR (400 MHz, DMSO-
General procedure for synthesis of compounds 2a–2e
d6) δ 1.19–1.21 (2H, m, cyclopropyl–H), 1.33–1.35 (2H,
m, cyclopropyl–H), 2.82–2.85(4H, m, piperazinyl–H),
3.41–3.43 (4H, m, piperazinyl–H), 3.52 (2H, s, N–CH2–
CO), 3.81–3.85 (1H, m, cyclopropyl–H), 7.58–7.60 (3H,
m, 2Ar–H and H-8), 7.90 (1H, d, J = 13 Hz, H-5), 7.97
(2H, d, J=8 Hz, Ar–H), 8.67 (1H, s, H-2), 15.09 (1H, brs,
COOH); 13C-NMR (100 MHz, DMSO-d6) δ 8.0, 36.3, 49.9,
52.5, 60.3, 106.8, 107.4, 111.3, 119.2, 129.1, 129.6, 129.9,
135.6, 139.7, 145.4, 148.4, 153.6, 158.8, 161.3, 166.3,
169.3, 176.9; ESI–MS (m/z): calcd. For C27H23ClFN6O4S
581.11795, found 581.11810 [M−H]−.
Compounds 2a–2e were prepared as reported in literature
[58].
Bromo‑N‑(5‑phenyl‑1,3,4‑thiadiazol‑2‑yl)acetamide
(2a) Yellow powder; 2.31 g, 77% yield; mp: 201–202 °C
[58].
2‑Bromo‑N‑(5‑(4‑chlorophenyl)‑1,3,4‑thiadiazol‑2‑yl)aceta‑
mide (2b) Yellow powder; 2.70 g, 81% yield; mp: 236–
238 °C [58].
2‑Bromo‑N‑(5‑(4‑methoxyphenyl)‑1,3,4‑thiadiazol‑2‑yl)‑
acetamide (2c) Yellow powder; 2.60 g, 79% yield; mp:
189–191 °C [58].
1 3

H. H. H. Mohammed et al.
1‑Cyclopropyl‑6‑fluoro‑7‑(4‑(2‑((5‑(4‑methoxyphenyl)‑
1,3,4‑thiadiazol‑2‑yl)amino)‑2‑oxoethyl)piperazin‑
1‑yl)‑4‑oxo‑1,4‑dihydroquinoline‑3‑carboxylic acid (3c,
C28H27FN6O5S) Pale yellow powder; 0.33 g, 69% yield;
Rf =0.1; mp 298–299 °C; 1H-NMR (400 MHz, DMSO-d6)
δ 1.19–1.21 (2H, m, cyclopropyl–H), 1.33–1.35 (2H, m,
cyclopropyl–H), 2.82–2.84 (4H, m, piperazinyl–H), 3.41–
3.44 (4H, m, piperazinyl–H), 3.50 (2H, s, N–CH2–CO),
3.82–3.85 (1H, m, cyclopropyl–H), 3.86 (3H, s, OCH3),
7.09 (2H, d, J=8 Hz, Ar–H), 7.59 (1H, d, J =8 Hz, H-8),
7.87–7.92 (3H, m, H-5 and 2Ar–H), 8.67 (1H, s, H-2), 15.12
(1H, brs, COOH); 13C-NMR (100 MHz, DMSO-d6) δ 8.0,
36.3, 49.9, 52.6, 55.9, 60.28, 106.8, 107.4, 111.6, 115.3,
119.4, 123.3, 128.9, 139.7, 145.5, 148.4, 152.9, 157.8,
161.7, 162.37, 166.4, 169.1, 176.9. ESI–MS (m/z): calcd.
For C28H27FN6O5S 579.18204, found 579.35065 [M+H].+
General procedure for the synthesis of substituted ethyl
benzoates 4a–4e
Compounds 4a–4e were prepared as reported in literature
and used as crude product in the following step without char-
acterization [59].
General procedure for synthesis of substituted benzohy‑
drazides 5a–5e
Compounds 5a–5e were prepared as reported in literature
and recrystallized from ethanol [60].
Benzohydrazide (5a) 10.20 g, 75% yield, mp: 109–112 °C
(reported 112–114 °C) [60].
4‑Chlorobenzohydrazide (5b) 12.30 g, 77% yield, mp: 195–
1‑Cyclopropyl‑7‑(4‑(2‑((5‑(3,4‑dimethoxyphenyl)‑
1,3,4‑thiadiazol‑2‑yl)amino)‑2‑ oxoethyl)pipera‑
zin‑1‑yl)‑6‑fuoro‑4‑oxo‑1,4‑dihydroquinoline‑3‑carboxylic
acid (3d, C29H29FN6O6S) Pale yellow powder; 0.37 g, 74%
197 °C (reported 189–191 °C) [61].
4‑Methoxybenzohydrazide (5c) 12.30 g, 74% yield, mp:
134–137 °C (reported 136–140 °C) [60].
1
yield; Rf = 0.1; mp: 292–293 °C; H-NMR (400 MHz,
DMSO-d6) δ 1.19–1.21 (2H, m, cyclopropyl–H), 1.32–1.35
(2H, m, cyclopropyl–H), 2.81–2.84 (4H, m, piperazinyl–H),
3.40–3.42 (4H, m, piperazinyl–H), 3.50 (2H, s, N–CH2–CO),
3.81–3.83 (1H, m, cyclopropyl–H), 3.84 (3H, s, OCH3), 3.87
(3H, s, OCH3), 7.09–7.11 (1H, m, Ar–H), 7.45–7.49 (1H, m,
Ar–H), 7.51 (1H, s, Ar–H), 7.59 (1H, J=8 Hz d, H-8), 7.92
(1H, d, J=12 Hz, H-5), 8.67 (1H, s, H-2), 15.13 (1H, brs,
COOH); 13C-NMR (100 MHz, DMSO-d6) δ 8.0, 36.3, 49.9,
52.5, 56.2, 60.3, 106.5, 107.6, 110.16, 111.6, 112.7, 120.9,
123.7, 128.2, 139.7, 145.2, 148.4, 150.4, 151.5, 158.3,
161.6, 162,3, 166.4, 168.9, 176.5; ESI–MS (m/z): calcd.
For C29H29FN6O6S 607.17805, found 607.17822 [M−H]−.
3,4‑Dimethoxybenzohydrazide (5d) 10 g, 51% yield, mp:
145–146 °C as reported [60].
3,4,5‑Trimethoxybenzohydrazide (5e) 16.10 g, 71% yield,
mp: 155–160 °C (reported 158–160 °C) [59].
General procedure for synthesis of 5‑phenyl‑1,3,4‑oxadia‑
zole‑2‑thiol derivatives 6a–6e
A mixture of (0.01 mol) of the appropriate benzohydrazides
5a–5e, (0.56 g, 0.01 mol) of potassium hydroxide, and
(1.14 g, 0.90 cm3, 0.015 mol) of carbon disulfde in 50 cm3
of absolute ethanol was heated under refux for 8 h. The
solvent was evaporated under vacuum. The residue was
dissolved in ice-cold water and acidifed with dilute hydro-
chloric acid. The formed precipitate was fltered of, washed
with water, dried, and recrystallized from ethanol to give the
desired oxadiazole derivatives [47].
1‑Cyclopropyl‑6‑fluoro‑4‑oxo‑7‑(4‑(2‑oxo‑2‑((5‑(3,4,5‑
trimethoxyphenyl)‑1,3,4‑thiadiazol‑2‑yl)amino)ethyl)pip‑
erazin‑1‑yl)‑1,4‑dihydroquinoline‑3‑carboxylic acid (3e,
C
30H31FN6O7S) Pale yellow powder; 0.34 g, 65% yield;
Rf =0.2; mp: 284–285 °C; 1H-NMR (400 MHz, DMSO-d6)
δ 1.19–1.21 (2H, m, cyclopropyl–H), 1.33–1.35 (2H, m,
cyclopropyl–H), 2.82–2.84 (4H, m, piperazinyl–H), 3.41–
3.43 (4H, m, piperazinyl–H), 3.51 (2H, s, N–CH2–CO), 3.75
(3H, s, OCH3), 3.83–3.86 (1H, m, cyclopropyl–H), 3.89 (6H,
s, 2OCH3), 7.21 (2H, s, Ar–H), 7.58 (1H, d, J = 8 Hz, H-
8), 7.90 (1H, d, J = 13 Hz, H-5), 8.66 (1H, s, H-2), 15.09
(1H, brs, COOH); 13C-NMR (100 MHz, DMSO-d6) δ 8.0,
36.3, 49.9, 52.5, 56.7, 60.3, 60.7, 105.1, 106.8, 107.4, 111.3,
119.1, 126.0, 139.7, 145.7, 148.4, 152.2, 153.9, 154.7,
158.4, 162.4, 166.3, 169.2, 176.9; ESI–MS (m/z): calcd.
For C30H31FN6O7S 637.18862, found 637.18866 [M−H]−.
5‑Phenyl‑1,3,4‑oxadiazole‑2‑thiol (6a) White solid; 1.30 g,
72% yield; mp: 224–225 °C (reported 218–220 °C) [62].
5‑(4‑Chlorophenyl)‑1,3,4‑oxadiazole‑2‑thiol (6b) Yellowish
white solid; 1.53 g, 71% yield; mp: 176–177 °C (reported
176–178 °C) [62].
5‑(4‑Methoxyphenyl)‑1,3,4‑oxadiazole‑2‑thiol (6c) White
solid; 1.80 g, 86% yield; mp: 207–208 °C (reported 198–
200 °C) [62].
1 3

Synthesis, molecular docking, antimicrobial evaluation, and DNA cleavage assay of new…
5‑(3,4‑Dimethoxyphenyl)‑1,3,4‑oxadiazole‑2‑thiol (6d) Yel-
lowish white solid; 1.95 g, 81% yield; mp: 254–255 °C
(reported: 237–238 °C) [63].
J=8.4 Hz, Ar–H), 8.68 (1H, s, H-2), 15.19 (1H, brs, COOH);
13C-NMR (100 MHz, DMSO-d6) δ 8.1, 36.4, 37.1, 41.9, 45.5,
49.5, 49.9, 107.2, 107.3, 111.7, 119.3, 122.4, 128.7, 130.1,
137.2, 139.6, 145.3, 148.6, 155.1, 164.34 164.8, 165.3,
166.4, 176.9; ESI–MS (m/z): calcd. For C27H23ClFN5O5S
582.10197, found 582.10236 [M−H]−.
5‑(3,4,5‑Trimethoxyphenyl)‑1,3,4‑oxadiazole‑2‑thiol
(6e) Yellowish white solid; 2.10 g, 78% yield; mp: 193–
194 °C (reported 186–188 °C) [47].
1‑Cyclopropyl‑6‑fuoro‑7‑(4‑(2‑((5‑(4‑methoxyphenyl)‑1,3,4‑
oxadiazol‑2‑yl)thio) acetyl)piperazin‑1‑yl)‑4‑oxo‑1,4‑dihyd‑
roquinoline‑3‑carboxylic acid (8c, C28H26FN5O6S) Yellow
powder; 0.46 g, 89% yield; Rf =0.2; mp: 247–248 °C; 1H-
NMR (400 MHz, DMSO-d6) δ 1.19–1.21 (2H, m, cyclopro-
pyl–H), 1.33–1.35 (2H, m, cyclopropyl–H), 3.39–3.47 (4H,
m, piperazinyl–H), 3.76–3.81 (5H, m, piperazinyl–H and
cyclopropyl–H), 3.86 (3H, s, OCH3), 4.58 (2H, s, S–CH2),
7.13 (2H, d, J=8 Hz, Ar–H), 7.58 (1H, d, J =8 Hz, H-8),
7.90–7.93 (3H, m, 2Ar–H and H-5), 8.66 (1H, s, H-2), 15.06
(1H, brs, COOH); 13C-NMR (100 MHz, DMSO-d6) δ 8.1,
36.3, 36.9, 41.9, 45.5, 49.5, 49.8, 55.9, 107.1, 107.2, 111.6,
115.3, 115.8, 119.3, 128.7, 139.6, 145.3, 148.5, 152.1,
154.6, 162.5, 163.2, 165.4, 166.4, 176.8; ESI–MS (m/z):
calcd. For C28H26FN5O6S 578.15151, found 578.15210
[M−H]−.
Synthesis of 7‑(4‑(2‑bromoacetyl)piperazin‑1‑yl)‑1‑cyclopro‑
pyl‑6‑fuoro‑4‑oxo‑1,4‑dihydroquinoline‑3‑carboxylic acid
(7) Compound 7 was synthesized as mentioned in literature
used without further purifcation to produce yellow powder;
4.88 g, 79% yield; Rf =0.2; mp: 273–274 °C [2].
General procedure for synthesis of 1‑cyclopropyl‑6‑fuoro‑4‑
oxo‑7‑(4‑(2‑((5‑aryl‑1,3,4‑oxadiazol‑2‑yl)thio)acetyl)piperazin‑
1‑yl)‑1,4‑dihydroquinoline‑3‑carboxylic acid derivatives 8a–
8e
An equimolar mixture of 6a–6e (0.0009 mol) and compound
7 (0.40 g, 0.0009 mol) in acetonitrile (50 cm3), TEA (0.48 g,
0.25 cm3, 0.0018 mol) was added. The reaction mixture was
stirred for 0.5 h. The formed precipitate was fltrated of. The
residue was washed with hot acetonitrile to aford the target
compounds 8a–8e [58].
1‑Cyclopropyl‑7‑(4‑(2‑((5‑(3,4‑dimethoxyphenyl)‑1,3,4‑oxa‑
diazol‑2‑yl)thio)acetyl) piperazin‑1‑yl)‑6‑fuoro‑4‑oxo‑1,4‑di‑
hydroquinoline‑3‑carboxylic acid (8d, C29H28FN5O7S) Yel-
low powder; 0.44 g, 81% yield; Rf =0.1; mp: 231–232 °C;
1H-NMR (400 MHz, CDCl3) δ 1.19–1.21 (2H, m, cyclo-
propyl–H), 1.31–1.33 (2H, m, cyclopropyl–H), 3.38–3.40
(2H, m, piperazinyl–H), 3.44–3.46 (2H, m, piperazinyl–H),
3.72–3.75 (4H, m, piperazinyl–H), 3.78–3.80 (1H, m, cyclo-
propyl–H), 3.83 (3H, s, OCH3), 3.86 (3H, s, OCH3), 4.64
(2H, s, S–CH2), 6.97 (1H, d, J=8.4 Hz, Ar–H), 7.42 (1H, d,
J=8.4 Hz, Ar–H), 7.52 (1H, s, Ar–H), 7.59 (1H, d, J=8 Hz,
H-8), 8.08 (1H, d, J=13 Hz, H-5), 8.79 (1H, s, H-2), 15.20
(1H, brs, COOH); ESI–MS (m/z): calcd. For C29H28FN5O7S
608.16207, found 608.16260 [M−H]−.
1‑Cyclopropyl‑6‑fuoro‑4‑oxo‑7‑(4‑(2‑((5‑phenyl‑1,3,4‑oxa‑
diazol‑2‑yl)thio)acetyl)piperazin‑1‑yl)‑1,4‑dihydroquino‑
line‑3‑carboxylic acid (8a, C27H24FN5O5S) Yellow powder;
1
0.45 g, 92% yield; Rf = 0.3; mp: 268–269 °C; H-NMR
(400 MHz, DMSO-d6) δ 1.20–1.22 (2H, m, cyclopropyl–
H), 1.33–1.35 (2H, m, cyclopropyl–H), 3.40–3.48 (4H,
m, piperazinyl–H), 3.77–3.84 (5H, m, piperazinyl–4H and
cyclopropyl–H), 4.62 (2H, s, S–CH2), 7.58–7.64 (4H, m,
3Ar–H and H-8), 7.93 (1H, d, J = 12 Hz, H-5), 7.98–8.00
(2H, m, Ar–H), 8.68 (1H, s, H-2), 15.08 (1H, brs, COOH);
13C-NMR (100 MHz, DMSO-d6) δ 8.1, 36.3, 36.8, 41.9,
45.6, 49.5, 107.03, 107.5 111.7, 120.2, 123.6, 126.9, 129.9,
132.5, 139.7, 146.2, 148.5, 152.2, 157.5, 163.5, 165.5,
166.3, 176.9; ESI–MS (m/z): calcd. For C27H24FN5O5S
548.14094, found 548.14136 [M−H]−.
1‑Cyclopropyl‑6‑fluoro‑4‑oxo‑7‑(4‑(2‑((5‑(3,4,5‑trimeth‑
oxyphenyl)‑1,3,4‑oxadiazol‑2‑yl)thio)acetyl)pipera‑
zin‑1‑yl)‑1,4‑dihydroquinoline‑3‑carboxylic acid (8e,
C30H30FN5O8S) Yellow powder; 0.50 g, 88% yield; Rf =0.2;
mp: 255–256 °C; 1H-NMR (400 MHz, DMSO-d6) δ 1.19–
1.21 (2H, m, cyclopropyl–H), 1.31–1.33 (2H, m, cyclo-
propyl–H), 3.43–3.46 (4H, m, piperazinyl–H), 3.74 (3H, s,
OCH3), 3.75–3.78 (4H, m, piperazinyl–H), 3.80–3.84 (1H,
m, cyclopropyl–H), 3.89 (6H, s, 2OCH3), 4.66 (2H, s, S–
CH2), 7.24 (2H, s, Ar–H), 7.59 (1H, d, J = 8.4 Hz, H-8),
7.95 (1H, d, J=13 Hz, H-5), 8.68 (1H, s, H-2), 15.20 (1H,
brs, COOH); 13C-NMR (100 MHz, DMSO-d6) δ 5.9, 34.2,
34.9, 39.8, 43.4, 47.4, 47.7, 54.5, 58.6, 102.1, 105.1, 109.5,
116.5, 117.3, 137.5, 138.9, 143.2, 146.5, 150.0 151.8, 152.5,
7‑(4‑(2‑((5‑(4‑Chlorophenyl)‑1,3,4‑oxadiazol‑2‑yl)thio)acetyl)‑
piperazin‑1‑yl)‑1‑cyclopropyl‑6‑fuoro‑4‑oxo‑1,4‑dihydro‑
quinoline‑3‑carboxylic acid (8b, C27H23ClFN5O5S) Yellow
powder; 0.43 g, 83% yield; Rf =0.3; mp: 265–566 °C; 1H-
NMR (400 MHz, DMSO-d6) δ 1.18–1.19 (2H, m, cyclo-
propyl–H), 1.31–1.33 (2H, m, cyclopropyl–H), 3.17–3.18
(1H, m, piperazinyl–H), 3.38–3.40 (1H, m, piperazinyl–
H), 3.43–3.45 (2H, m, piperazinyl–H), 3.74–3.79 (4H, m,
piperazinyl–H), 3.81–3.84 (1H, m, cyclopropyl–H), 4.67
(2H, s, S–CH2), 7.59 (1H, d, J=8 Hz, H-8), 7.66 (2H, d,
J=8.4 Hz, Ar–H), 7.94 (1H, d, J=12 Hz, H-5), 8.01 (2H, d,
1 3

H. H. H. Mohammed et al.
161.8, 163.2, 163.3, 164.3, 174.7; ESI–MS (m/z): calcd. For
with DMSO starting from each original solution. In short,
0.2 cm3 of one solution was transferred to the 0.2 cm3 of
second solution, which was performed ten times, fnally
making 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125,
0.006 mg/cm3 of solutions. 0.05 cm3 of each solution was
diluted by 5 cm3 of Middlebrook 7H9-OADC and Muel-
ler–Hinton broth, respectively (1/100 dilution). 0.1 cm3 of
each serial solution was dispensed into one line of 96-wells
microplate. The last well was used as drug-free control. This
plate was prepared for each testing medium. The remaining
solutions were stored at − 80 °C. Each isolate strain was
inoculated into each plate in duplication. The inoculation
size was 0.1 cm3 of 105 CFU/cm3 (104 CFU for each well).
The fnal testing concentrations were as follows; 32, 16, 8,
4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03 µg/cm3. The inoculated
Middlebrook 7H9+OADC and Mueller–Hinton broth media
were incubated at 37 °C with 5% CO2 and 30 °C in room
air, respectively. When sufcient growth was observed in
the control well, the well with the minimum concentration
that showed growth suppression was considered as the MIC.
C30H30FN5O8S 638.17263, found 638.17322 [M−H]−.
Biological investigations
Screening of antimycobactrial activity
General Middlebrook 7H9 broth, Middlebrook 7H10 agar,
Middlebrook 7H11 agar, and Luria–Bertani (LB) broth were
obtained from BD (Franklin Lakes, NJ or Sparks, MD). Iso-
niazid, kanamycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) were purchased from
Sigma (St. Louis, MO).
Mycobacterial species strains and culture
Pathogenic M. smegmatis was grown in LB liquid medium
with 0.05% Tween 80 for competent cell preparation. Kana-
mycin was added into the medium at the following concen-
tration as required: kanamycin 40 mg/cm3 for E. coli and
20 mg/cm3 for Mycobacterium strains. Species and strains
including H37Rv (ATCC27293, pan-susceptible clinical
M. tuberculosis isolate (ID: MirokuRyoken 123), multid-
rug-resistant M. tuberculosis isolate (ID: FukujujiRyoken
59), extensively drug-resistant M. tuberculosis isolate (ID:
FukujujiRyoken 97) were tested in Middlebrook7H9 supple-
mented with OADC with 0.05% of Tween80 and M. absces-
sus standard strain ATCC19977, M. chelonae standard strain
ATCC35752 and M. fortuitum standard strain ATCC06841
were tested in Mueller–Hinton broth (pH 7.4).
Cleavable complex DNA gyrase assay
Reaction mixtures (50 μl) contained 50 mM Tris–HCl, pH
8.0 at 23 °C, 10 mM MgCl2, 50 mg/l bovine serum albu-
min, 1 mM ATP, 5 mg/l tRNA, 0.3 μg of plasmid pBR322
DNA, the indicated amounts of either the wild-type gyrase,
GyrA-G81C gyrase, or topoisomerase IV, and the indicated
concentrations of fuoroquinolone. Mixtures were incubated
at 37 °C for 15 min and then split into two samples. SDS was
added at a concentration of 1% (w/v) to one half of the reac-
tion mixtures, and the reaction mixtures were further incu-
bated at 37 °C for 10 min. EDTA and proteinase K were then
added to 50 mM and 100 mg/l, respectively, and incubation
was continued for an additional 15 min at 37 °C. The other
half of the reaction mixtures was incubated for 10 min at
37 °C in the presence of 50 mM EDTA before the proteinase
K treatment in the presence of SDS as described above. The
DNA products were purifed by extraction with phenol/chlo-
roform/isoamyl alcohol and then analyzed by electrophoresis
through vertical 1.2% (w/v) agarose gels at 2 V/cm for 12 hin
TAE bufer that contained 0.5 mg/l ethidium bromide. After
destaining in water, gels were photographed and quantifed
using an Eagle Eye II system (Agilent Technologies).
Determination of MIC against M. smegmatis
Mycobacterium smegmatis was grown in LB (Luria–Ber-
tani) liquid medium with 0.05% Tween 80 for competent cell
preparation at the faculty of pharmaceutical sciences, Osaka
University, Japan. The determination of MIC against M.
smegmatis was performed by the established MTT method
[53]. Briefy, M. smegmatis in the mid-logarithmic phase
(1 × 104 CFU per 0.1 cm3) was inoculated into each well
of a 96-well plate, and then the serially diluted sample was
added to each well. After 48 h incubation at 37 °C, 50 cm3
of MTT solution (0.5 mg/cm3) was added into each well
and incubation was carried out at 37 °C for an additional
6 h. The optical density (OD) at 560 nm was measured to
determine MICs.
Docking study
Determination of MIC against pathogenic M. tuber-
culosis strains
Docking simulation study is performed using Molecular
Operating Environment (MOE®) version 2014.09, Chemi-
cal Computing Group Inc., Montreal, Canada. The computa-
tional software operated under “Windows XP” was installed
on an Intel Pentium IV PC with a 1.6 GHz processor and
512 MB memory. The target compounds were constructed
All compounds (3.2 mg) were dissolved in 0.5 cm3 of
DMSO and prepared in 6.4 mg/cm3 (original solution).
Twofold serial dilutions were prepared for each compound
1 3

Synthesis, molecular docking, antimicrobial evaluation, and DNA cleavage assay of new…
Egypt. Also, Dr. Safwat M Rabea, is greatly acknowledged for meas-
into a 3D model using the builder interface of the MOE pro-
gram. After checking their structures and the formal charges
on atoms by 2D depiction, the following steps were carried
out:
uring NMR data of the synthesized compounds at British Colombia
University, Canada.
Compliance with ethical standards
•
All conformers were subjected to energy minimization,
all the minimizations were performed with MOE until
a RMSD gradient of 0.01 kJ/mol and RMS (root mean
square) distance of 0.1 Å with MMFF94X force feld and
the partial charges were automatically calculated.
Conflict of interest No fnancial or commercial conficts of interest
were declared by any of the authors.
References
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The obtained database was then saved as Molecular Data
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The co-crystallized ligand, moxifoxacin was deleted.
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