Piperazine-azole-fluoroquinolone hybrids: Conventional and microwave irradiated synthesis, biological activity screening and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2019.01.009

A series of new 1,2,4-triazole and 1,3,4-oxadiazole derivatives was obtained via several steps sequential reactions of phenyl piperazine. Then, these compounds were converted to the corresponding fluoroquinolone hybrids via one pot three component Mannich

Full text of DOI:10.1016/j.bioorg.2019.01.009

Accepted Manuscript
Piperazine-Azole-Fluoroquinolone Hybrids: Conventional and Microwave Ir-
radiated Synthesis, Biological Activity Screening and Molecular Docking Stud-
ies
Arif Mermer, Ozlem Faiz, Ahmet Demirbas, Neslihan Demirbas, Manikandan
Alagumuthu, Sivakumar Arumugam
PII:
S0045-2068(18)30841-1
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.009
YBIOO 2713
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 August 2018
1 January 2019
6 January 2019
Please cite this article as: A. Mermer, O. Faiz, A. Demirbas, N. Demirbas, M. Alagumuthu, S. Arumugam,
Piperazine-Azole-Fluoroquinolone Hybrids: Conventional and Microwave Irradiated Synthesis, Biological Activity
Screening and Molecular Docking Studies, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.01.009
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Piperazine-Azole-Fluoroquinolone Hybrids: Conventional and Microwave
Irradiated Synthesis, Biological Activity Screening and Molecular Docking
Studies
Arif Mermera, Ozlem Faizb, Ahmet Demirbasa, Neslihan Demirbasa*, Manikandan
Alagumuthuc, Sivakumar Arumugamc
a) Karadeniz Technical University, Department of Chemistry 61080 Trabzon TURKEY
b) Recep Tayyip Erdogan University, Department of Chemistry, 53100 Rize TURKEY
c) Dept. of Biotechnology, School of Bio-Sciences and Technology, VIT, Vellore-632014,
India.
Abstract
A series of new 1,2,4-triazole and 1,3,4-oxadiazole derivatives was obtained via several steps
sequential reactions of phenyl piperazine. Then, these compounds were converted to the
corresponding fluoroquinolone hybrids via one pot three component Mannich reaction. All
the reactions were examined under conventional and microwave mediated conditions, and
optimum conditions were determined. The effect of different solvents and microwave power
on microwave prompted reactions was investigated as well. All the newly synthesized
1
13
compounds were characterized by FTIR, H NMR, C NMR and EI MS spectral techniques.
The antimicrobial activity, DNA gyrase and Topoisomerase IV inhibition potentials were
performed. The results obtained showed that fluoroquinolone hybrids possess good
antimicrobial activity. Moreover, Fluoroquinolone-azole-piperazine hybrids synthesized in the
present study displayed excellent DNA gyrase inhibition. To unveil the interaction mode of
compounds to receptor, a molecular docking study was performed. With an average least
binding energy of -9.5 kcal/mol, all compounds were found to have remarkable inhibitory
potentials against DNA gyrase (E. coli).
Keywords: Fluoroquinolone; 1,2,4-triazole; Microwave irradiation; Antimicrobial activity;
DNA gyrase; Topoisomerase IV; Molecular docking.
1. Introduction
The increasing multidrug resistance among bacteria, viruses and fungi to currently used
drugs has emerged an alarming and reemerging microbial threat and has become a major
public health concern worldwide with nearly 15 million deaths every year. Several pathogens
which appeared to be under control have once again been fatal since they have developed a
variety of strategies to enhance their survival skills in the presence of antimicrobial agents.
1

Estimates suggested that unless significant progress has been made, drug resistant infections
may result in ten million deaths annually by 2050. In order to manage drug resistance, the
development of novel chemotypes which act upon novel molecular targets, has emerged as an
urgent and crucial requirement. However, there have only been prepared a few molecules with
new mode of action to medical use. More recently, the molecular hybridization concept based
on the combination of structural units of two or more drug fragments through the fusion in
one molecular framework with improved properties has been adopted as a new and attractive
strategy [1-11]. The key advantage of hybrid molecules consisting of several pharmacophore
groups each with different mode of action is to inhibit bacterial targets via unique binding
sites or via novel modes of action or to exhibit dual mode of action [12-20]. Moreover,
molecular hybrids can display better pharmacokinetic profile, therapeutic index and more
importantly low tendency to resistance. Quinolones have been the centre of considerable
scientific and clinical interest since their discovery in the early 1960s. This is because they
potentially offer many of the attributes of an ideal antibiotic, combining high potency, a broad
spectrum of activity, good bioavailability, oral and intravenous formulations, high serum
levels, a large volume of distribution indicating concentration in tissues and a potentially low
incidence of side-effects. Much research has attempted to make these potential attributes real.
The first quinolone nalidixic acid was developed, but it took more than a decade before
additional compounds, such as flumequin, norfloxacin and enoxacin became available for
clinical use. The main use for all these agents was the treatment of urinary tract infection [21].
Since the discovery of norfloxacin (NFLX) by Koga et al. in the early 1980s, quinolones have
become one of the most attractive agents in the chemotherapy of both community-acquired
and serious hospital-acquired infections [21-23].
These drugs perform their activity by binding to two type II bacterial topoisomerase
enzymes, DNA gyrase and topoisomerase IV. This causes to permanent double stranded DNA
breaks, and results in cell death [24]. However, the rapidly increasing level of resistance
towards quinolone class antibacterial drugs has limited their therapeutic efficacy and served
the scientists to design either novel structural classes of inhibitors targeting gyrase and
topoisomerase IV; or to discover new quinolone hybrids [25, 26]. During the past 30 years,
hundreds of quinolone derivatives have been discovered, and a large body of structure–
activity relationship (SAR) has been accumulated. The activity of quinolone class
antibacterial agents arises from bicyclic heterocyclic ring and also the structure of peripheral
substituents and their spatial relationship. These groups can provide additional affinity
2

towards bacterial enzymes, enhance the cell penetration or alter pharmacokinetic properties
[27]. The in silico studies on quinolone class antibiotics have showed that the basic group at
C-7 position is the most suitable site for chemical modifications. it constitutes an area with a
great influence on potency, spectrum and safety and most of studies aiming the discovery of
new quinolone hybrids have been focused on the functionalization at C-7 in quinolone
skeleton [24, 28-30]. In general, 5- and 6-membered N-containing rings including piperazinyl,
pyrrolidinyl and piperidinyl groups have been proven as optimal substituents [24, 31].
N-Containing heterocycles which have been regarded as important tools medicinally, have
given a new aspect to new drug development studies. The therapeutic efficacy of 1,2,4-
triazole and 1,3,4-oxadiazole derivatives have been well studied in the several pathological
conditions including inflammation, cancer, pain, tuberculosis or hypertension [32–35]. The
compounds including these units have been reported to have several biological activities such
as antimicrobial, anticancer, antioxidant, analgesic, antihypertensive etc [36-38]. Microwave
irradiated syntheses have been proven as an effective technique in terms of environment,
reaction time, high yields, ease of work-up and isolation of products [39]. Moreover, with the
emergence of ‘green chemistry concept’, the scientists have shifted their focus to solvent-free
methods which provide facile reactions conditions with high yields of pure products and
eliminate or minimize the use of organic solvents which are often expensive, toxic and
difficult to remove [40-42]. In light of these aforementioned statements, we have focused on
the design, ecofriendly synthesis and antibacterial evaluation of new piperazine-azole-
fluoroquinolone conjugates (Fig. 1). Phenyl piperazine scaffold has been selected as the key
prototype structural unit and the combination of phenyl piperazine skeleton with azole and
fluoroquinolone units has been achieved by both the conventional and microwave irradiated
techniques.
Figure 1. General representation of the target compounds.
2. Results and Discussion
3

2.1. Chemistry
The designed fluoroquinolone-triazole hybrid compounds 7a, b and 8a-j have been
prepared according to the synthetic procedure presented in Scheme 1. In order to development
more efficient synthetic procedures for these reactions, microwave (MW) irradiation has also
been applied in addition to conventional methods. The compounds 2 and 3 have been
synthesized following the procedure reported earlier [43, 44]. Initially, the carbo(thio)amide
derivatives (5a-e) have been obtained from the nucleophilic attack of hydrazide-NH2 to
carbon atom of iso(thio)cyanate function. The evidence for the formation of compounds 5a-e
1
have been provided by FT IR, H NMR, 13C NMR and EI-MS. For the synthesis of 6a-e, two
methods have been used including conventional heating under reflux conditions (Method 1)
or microwave mediated conditions (Method 2). The latter method has provided more efficient
procedure with much shorter reaction times varying between 15-20 min, whereas 9-15 hours
have been needed for conventional method (Table 1). Moreover, MW irradiation has
promoted the reaction yields from 77%-84% to 84%-90%. In method 2, higher microwave
energy than 150 W has caused to a decrease in yields of compounds 6a-e.
Table 1. Time, power, yield data and melting point for compounds 4 and 5a-e
Compound
Microwave Irradiation
Method
Conventional
Method
Melting Point
(oC)
Time
(min)
15
15
15
15
15
20
Power
(W)
150
150
150
150
150
150
Yield
(%)
92
89
86
85
90
84
Time
(h)
10
10
13
11
9
Yield
(%)
84
81
77
80
84
78
201-203
231-232
207-208
203-204
223-224
202-203
4
5a
5b
5c
5d
5e
15
Time
(min)
Power
(W)
Yield
(%)
Catalyst
(10%)
Entry
Solvent
20
50
70
50
70
50
70
50
50
50
86
THF
THF
EtOH
EtOH
DMF
DMF
-
-
-
-
-
-
1
2
3
4
5
6
7
8
9
25
20
25
20
25
20
20
20
84
82
80
93
89
93
93
91
-
p-TSA
FeCl3
HCl
-
-
4

20
50
92
-
InCl3
10
Table 2. Optimization of the model reaction conditions for compound 8a.
Our previous studies have showed that 1,2,4-triazoles containing a C=S (SH) group rather
prefer the mercapto form, while the derivatives including C=O (OH) function are almost
present in keto form [45-47]. The treatment of hydrazide (3) with carbon disulfide in the
presence of KOH has yielded the corresponding 1,3,4-oxadiazole (4) under conventional
heating and microwave mediated conditions. MW irradiation has decreased the reaction time
from 10 h to 15 min and increased the reaction yield from 84% to 92%. The screening of the
reaction condition has showed that the presence of catalysts has no significant impact on the
yield of the desired compound. The best result has been obtained in DMF at 50 W maximum
power without any catalyst (Table 2, Entry 5) at a yield of 93%. The remaining hybrid
compounds, 7b, 8b-j have been obtained through applying the optimized conditions described
above (Table 3). The structures of compounds 7a,b; 8a-j has been identified on the basis of
FT IR, 1H NMR, 13C NMR and mass spectrometric data.
5

Scheme 1. Synthetic pathway for the preparation of new compounds. i: BrCH2COOEt, TEA,
THF; ii: NH2NH2.H2O, EtOH; iii: CS2, KOH(aq), EtOH; iv: R1NCX; v: NaOH(aq), EtOH; vi:
HCHO, DMF, Ciprofloxacin or Norfloxacin
Table 3. The results of reaction yield and time for the synthesized compounds 7a,b and 8a-j
in Microwave Irradiation and Conventional Method
Microwave Irradiation Method
Conventional Method
Time (h) Yield (%)
24 86
Compound
Time (min)
Yield (%)
20
20
20
20
20
20
20
20
20
20
20
20
91
93
93
89
91
94
96
90
95
92
89
88
7a
7b
8a
8b
8c
8d
8e
8f
24
24
24
24
24
24
24
24
24
24
24
86
86
82
85
88
86
81
89
82
79
81
8g
8h
8i
8j
2.2. Biological Activity
2.2.1. Antimicrobial activity
Antimicrobial activity of all compounds obtained were presented in Table 4 (Only positive
results were presented). Among these, carbo(thio)amide and triazole derivatives did not show
any activity against the test microorganism. The fluoroquinolone derivatives exhibited good,
moderate and excellent activity almost all microorganism with the MIC values between
0.125-65 µg/mL. Compare with the standard drug Ciprofloxacin, these compounds showed
good-moderate activity except for 7b, 8b and 8h which showed the same MIC values for K.
pneumoniae and only 8h showed 0.5 µg/mL MIC value against A. haemolyticus. 7b and 8b
showed excellent activity against S. aureus against ampicillin used as a standard drug for
gram positive microorganisms. At the same time, 7b, 8b, 8h, 8j exhibited better activity
against E. faecalis than the standard drug with the MIC value 8 µg/mL. On the other hand,
Gentamicin was used as standard drug for Gram-negative microorganism. For E. coli, all
compounds showed excellent activity against Gentamicin, also 7b, 8b and 8h were found to
have excellent activity for K. pneumoniae with the MIC value 0.25 µg/mL. On the other hand,
excellent activities with the MIC values varying 0.5–2 µg/mL were observed for compounds
7b, 8b and 8h for A. haemolyticus and P. aeruginosa. 7b, 8b and 8h showed the best
6

antimicrobial activity against test microorganisms when the results were evaluated
collectively.
Table 4. Screening for the activity of newly synthesized compounds 7a,b and 8a-j.
Microorganisms and minimal inhibition concentration (µg/mL)
Compound
Gram-positive
microorganism
Gram-negative microorganism
Sa
16
2
16
2
16
8
16
8
16
4
16
8
1
1.56
-
Ef
32
8
64
8
32
16
32
16
32
8
32
8
0.5
12.5
-
Ec
0.5
0.125
2
0,125
0.5
0.125
1
0.125
0.5
0.125
0.5
0.125
0.03
-
Pa
8
2
16
2
16
4
16
4
8
2
16
4
Kp
4
0.25
4
0.25
2
0.5
2
0.5
1
0.25
2
2
0.25
-
Ah
16
1
-
1
-
4
16
2
8
0.5
16
4
0.25
-
7a
7b
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
Cip.
Amp.
Gen.
0.25
-
1.56
0.78
0.39
0.78
Sa:Staphylococcus aureus ATCC 25923, Ef:Enterococcus faecalis ATCC 29212, Ec:Escherichia coli ATCC
25922, Pa: Pseudomonas aeruginosa ATCC 27853, Kp:Klebsiella pneumoniae ATCC 13883, Ah:Acinetobacter
haemolyticus ATCC 19002. Cip.: Ciprofloxacin, Amp.: Amphicilin, Gen.: Gentamicin
2.2.2. Results of DNA gyrase and Topo-IV inhibition studies
DNA gyrase and Topoisomerase IV are type-II topoisomerases enzymes, involving in the
negative supercoiling which is a vital part of bacterial survival. Thus, we have assessed the
inhibition potentials of present study compounds against these two enzymes. The results of
DNA gyrase and Topo-IV inhibition study is depicted in Table 6. As per our observations, the
activity has been totally a dose dependent (Fig. 2a). Compounds 5a to 6e have been found
totally inactive for the activity. On the other hand, fluoroquinolone-azole-piperazine hybrids
have showed excellent activity with lowest IC50 values. Remarkably, compounds 8c, 8f, 8i, 8j,
7a and 7b were the excellent group compounds for DNA gyrase inhibition (Fig. 2b). At this
point we have recognized the consistence between the molecular docking and the enzyme
inhibition study in which the result has favored DNA gyrase. Meanwhile, the low selective
TOPO-IV inhibition activity was also recognized from the obtained IC50 values. Among all
compounds, 8j was exhibited most potent activity with a calculated relative % activity of 85
% and IC50 of 0.75 µM. A maximum activity was found at the 10-6 concertation, which
indicates the necessity of low amount of target candidate drug to have a maximum activity.
7

The activity variation and druggability suggestions against the present study compound was
assessed by comparing the DNA gyrase and Topo-IV inhibition study results of the
compounds with the standard drug, Novobiocin’s (Novo) results. Novo exhibited a significant
DNA gyrase as well as TOPO-IV activities. A relative % activity of 82.52 % and an IC50
value of 1.0 µM was showed by Novo against DNA gyrase inhibition. In figure 2a&b, the
results of Novo and potentially identified compound 8j were compared. In the dose response
curve, even though the curve elevation started equally and slopped at same concentrations, a
slight variation could be seen between 8j and Novo. This activity equality or slightly elevated
activity of 8j indicate its druggability potentials as a future DNA gyrase inhibitor.
Table 5. Results of DNA gyrase and DNA Topo-IV inhibition studies.
Comp.
No.
IC50 (µM) DNA Gyrase
IC50 (µM) DNA Topo-IV
10.12 ± 0.14
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
4
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
8a
8b
8c
8d
8e
8f
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
˃ 25.00
2.25 ± 0.28
3.25 ± 0.34
5.24 ± 0.88
4.88 ± 0.76
1.25 ± 0.22
2.05 ± 0.38
1.84 ± 0.15
1.00 ± 0.18
0.88 ± 0.18
1.50 ± 0.12
0.95 ± 0.08
0.75 ± 0.07
15.24 ± 1.05
12.48 ± 1.25
˃ 25.00
˃ 25.00
14.36 ± 1.45
15.78 ± 1.57
˃ 25.00
˃ 25.00
16.46 ± 1.74
18.67 ± 1.68
6.78 ± 1.08
8.24 ± 1.64
8g
8h
8i
8j
8

Figure 2a. Dose response curve of 8j against DNA gyrase inhibition. Figure 2b. DNA gyrase
and TOPO-IV inhibition potential of compounds 7a, 7b and 8a-j. *Novobiocin.
2.2.3. Results of molecular docking studies
From the molecular docking study results, the attained free energy value reveals that the
compounds 8a-j having a fortunate selectivity towards DNA gyrase over TOPO-IV (ParC,
PDB ID: 1ZVU) (Fig. 3). Based on the molecular mechanistic values, we have categorized the
compounds as most active, intermediates and inactive groups. Mostly, the first category was
used to evaluate in vitro experiments. The most significant binding affinity of the docked
compounds was recognized through the obtained inhibitory constant (ki). Compounds 7a,b
and 8a-j fortunately have established with a significant binding affinity (ki values < 1 ± 0.5
µM). The average and sum of all docking molecular mechanics values have given a clear-cut
idea about the activity selectivity of compounds towards the DNA gyrase inhibition (average
least binding energy value of -11.214 kcal/mol). Meanwhile, TOPO-IV activity average was
also reliable (-8.24 kcal/mol). While discussing about the ligand efficiency, with a significant
average value of -0.32, fluoroquinolone-1,2,4-triazole hybrids were found to have efficiency
towards 2XCT (DNA gyrase). The average inhibitory constant (ki) indicates the possible
bioactivity potentials of the compounds in low molecular concentrations towards both DNA
gyrase (0.0035 µM) as well as the TOPO-IV (0.5 µM).
9

Figure 3. Results of molecular docking studies. 3A. Ligand efficiency values; 3B. Binding
energy values; 3C. Obtained inhibitory constant (ki) value for PDB ID: 2XCT; 3D. Obtained
inhibitory constant (ki) value for PDB ID: 1ZVU
2.2.4. Molecular assessments of Receptor-Ligand interactions of DNA gyrase and
TOPO-IV and compounds 7a, 7b and 8a-j
To unveil the mechanism of action and molecular interaction by means of hydrogen
bonding and non-covalent bonding (π-π interaction and π-cation interactions), the established
best docked poses have been analyzed. Various drug efficacy factors have been contributing
in these types’ communications/interactions between drug and receptor. These factors are
mainly controlling the strength, duration, and different types of the drug-receptor interaction.
In fact, these factors are decisively dictating the strength of the interaction established by a
drug (by constituting the complex) against its receptor. This has been known as the affinity.
Therefore, the binding affinity is considered as one of the chief in silico measurement to
adjudge on new chemical entity’s drug efficacy.
Various intermolecular forces are involved in the interaction establishment between drug
and binding sites such as Van der Waals forces (weak bonds and transient reversible effects),
Hydrogen bonds (intermediate bonds and transient reversible effects), Covalent bonds (strong
bonds and long-lasting or irreversible effects) and non-covalent bonding (electromagnetic
interactions among molecules). Fig. 4a-b has illustrated the interaction of top scored
10

compounds to DNA gyrase (PDB ID: 2XCT) and TOPO-IV (PDB ID: 1ZVU). Ser1173 was
the main amino acid residue to establish hydrogen bond and Arg1033to establish the non-
covalent cation-π interaction DNA gyrase (PDB ID: 2XCT). Docking results were also
suggested to evaluate further possible in vitro and in vivo analysis to unveil the drug ability of
the present study compounds.
Figure 4A. Interaction of compound 8a to DNA Gyrase (PDB: 2XCT). Figure 4B.
Interaction of compound 8a to DNA Gyrase (PDB: 2XCT) binding pocket.
3. Experimental
3.1. General
All the chemicals were purchased from Fluka Chemie AG Buchs and Sigma Aldrich and
used without further purification. Melting points of the synthesized compounds were
determined in open capillaries on a Büchi B-540 melting point apparatus and are uncorrected.
Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254
aluminium sheets. The mobile phase was ethyl acetate: diethyl ether (1:1), and detection was
made using UV light. FT-IR spectra were recorded using a Perkin Elmer 1600 series FTIR
1
spectrometer. H NMR and 13C NMR spectra were registered in DMSO-d6 on a BRUKER
1
13
AVENE II 400 MHz NMR spectrometer (400.13 MHz for H and 100.62 MHz for C). The
chemical shifts are given in ppm relative to Me4Si as an internal reference, J values are given
in Hz. The mass spectra were obtained on a Quattro LC-MS (70 eV) Instrument. Microwave
irradiated reactions were performed in a CEM Discovery monomod synthesis reactor.
3.1.1. 5-[(4-Phenylpiperazin-1-yl)methyl]-1,3,4-oxadiazole-2-thiol (4)
Method 1. Compound 3 (10 mmol) and CS2 (15 mmol) were added to a solution of KOH (10
mmol) in 10 mL H2O and 10 mL ethanol and the reaction mixture was refluxed for 10 h.
11

Then, the reaction content was acidified with conc. HCl to pH 6. The precipitate formed was
filtered off, washed with H2O and recrystallized from ethanol to afford the desired compound.
Yield: 84%.
Method 2. The solution of compound 3 (10 mmol), CS2 (15 mmol) and KOH (10 mmol) in
H2O+ethanol was irradiated in monomod microwave reactor in closed vessel with the
o
pressure control at 100 C, 150 W maximum power for 15 min (hold time). After the
completion of the reaction, (monitored by TLC), the mixture was acidified with conc. HCl to
pH 6. The precipitate formed was filtered off, washed with H2O and recrystallized from
ethanol to afford the desired compound. Yield: 92 %.
1
FT-IR (υmax, cm-1): 3011 (ar-CH), 2855 (-SH), 1602 (C=N). H NMR (DMSO-d6, δ ppm):
2.65 (d, 4H, J=4.0 Hz, 2CH2), 3.15 (s, 4H, 2CH2), 3.74 (s, 2H, CH2), 6.79 (t, 1H, J=12.0 Hz,
13
arH), 6.93 (d, 2H, J=8.0 Hz, arH), 7.21 (t, 2H, J=12.0 Hz, arH), 14.42 (s, 1H, SH). C NMR
(DMSO-d6, δ ppm): 48.52 (2CH2), 51.56 (CH2), 52.42 (2CH2), arC: [116.00 (2CH), 119.45
(CH), 129.37 (2CH), 151.30 (C)], 161.09 (oxadiazole C-5), 178.49 (oxadiazole C-2). EI MS
m/z (%): 106.04 (44), 163.08 (33), 175.16 (49), 277.23 ([M+1]+, 100), 315.22 ([M+K]+, 40).
3.1.2. General method for the synthesis of compounds 5a-e
A mixture of compound 3 (10 mmol) and the corresponding iso(thio)cyanate (15 mmol) in
dichloromethane was stirred at room temperature for 24 h (monitored by TLC). After
evaporating the solvent under reduced pressure, a solid obtained. The crude product was
purified by crystallization from appropriate solvents.
3.1.2.1. N-Phenyl-2-[(4-phenylpiperazin-1-yl)acetyl]hydrazincarbothioamide (5a)
Recrystallized from acetone. Yield: 95 %, Mp. 184-185 °C. FT-IR (υmax, cm-1): 3306, 3273 ve
1
3210 (3NH), 3112 (ar-CH), 1665 (C=O), 1235 (C=S). H NMR (DMSO-d6, δ ppm): 2.69 (s,
4H, 2CH2), 3.18 (s, 6H, 3CH2), 6.78 (t, 1H, J=16.0 Hz, arH), 7.17-7.24 (m, 3H, arH), 7.35 (t,
2H, J=16.0 Hz, arH), 7.45 (d, 2H, J=5.0 Hz, arH), 9.59 (bs, 2H, 2NH), 9.92 (bs, 1H, NH). 13C
NMR (DMSO-d6, δ ppm): 46.71 (2CH2), 52.71 (2CH2), 59.95 (CH2), arC: [113.11 (2CH),
115.15 (CH), 124.19 (2CH), 126.19 (3CH), 129.83 (2CH), 135.90 (C), 137.31 (C)], 155.15
(C=O), 181.69 (C=S). EI MS m/z (%): 173.27 (96), 208.31 (52), 220.26 (49), 294.40 (49),
369.44 ([M] +, 100).
3.1.2.2. N-Benzyl-2-[(4-phenylpiperazin-1-yl)acetyl]hydrazincarbothioamide (5b)
12

Recrystallized from acetone. Yield: 90 %, Mp. 193-194 °C. FT-IR (υmax, cm-1): 3204 (3NH),
1
3052 (ar-CH), 1671 (C=O), 1233 (C=S). H NMR (DMSO-d6, δ ppm): 2.64 (s, 4H, 2CH2),
3.15 (s, 2H, CH2), 3.38 (s, 2H, CH2 + su), 3.70 (s, 4H, 2CH2), 7.02 (d, 2H, J= 8.0 Hz, arH),
7.06-7.43 (m, 5H, arH), 8.05 (d, 2H, J=8.0 Hz, arH), 9.59 (bs, 2H, 2NH), 9.94 (bs, 1H, NH).
13C NMR (DMSO-d6, δ ppm): 46.69 (CH2), 51.82 (2CH2), 54.10 (2CH2), 59.93 (CH2), arC:
[113.13 (d, J=7.0 Hz, 2CH), 126.16 (d, J=5.0 Hz, 2CH), 127.47 (CH), 129.13 (CH), 129.57
(d, J=5.0 Hz, 2CH), 130.55 (2CH), 133.46 (C), 155.08 (C)], 169.52 (C=O), 192.95 (C=S). EI
MS m/z (%): 106 (100), 135 (42), 320 (53), 331 (48), 384 ([M+1] +, 48).
3.1.2.3. N-(4-Fluorophenyl)-2-[(4-phenylpiperazin-1-yl)acetyl]hydrazin carbothioamide
(5c)
Recrystallized from acetone. Yield: 92 %, Mp. 188-190 °C. FT-IR (υmax, cm-1): 3184 (3NH),
1
3032 (ar-CH), 1669 (C=O), 1226 (C=S). H NMR (DMSO-d6, δ ppm): 2.66 (s, 4H, 2CH2),
3.16 (d, 4H, J=16.0 Hz, 2CH2), 3.36 (s, H2O+CH2), 6.78 (t, 1H, J=12.0 Hz, arH), 6.94 (d, 2H,
J=8.0 Hz, arH), 7.16-7.23 (m, 4H, arH), 7.41 (s, 2H, arH), 9.63 (bs, 2H, 2NH), 9.90 (bs, 1H,
13
NH). C NMR (DMSO-d6, δ ppm): 48.48 (2CH2), 53.24 (2CH2), 60.28 (CH2), arC: [115.24
(d, J=22.0 Hz, 2CH), 115.83 (2CH), 119.23 (2CH), 123.99 (CH), 129.39 (2CH), 135.95 (C),
151.45 (C), 152.02-154.50 (dC-F, J=248.0 Hz, C)], 169.23 (C=O), 181.60 (C=S). EI MS m/z
(%): 106 (93), 388 ([M+1] +, 100), 389 ([M+2] +, 86).
3.1.2.4. N-(4-Ethyl)-2-[(4-phenylpiperazin-1-yl)acetyl]hydrazincarbothioamide (5d)
Recrystallized from acetone. Yield: 90 %, Mp. 175 °C. FT-IR (υmax, cm-1): 3232 (3NH), 3063
1
(ar-CH), 1670 (C=O), 1234 (C=S). H NMR (DMSO-d6, δ ppm): 1.08 (t, 3H, J=16.0 Hz,
CH3), 2.67 (s, 4H, 2CH2), 3.13 (s, 2H, 2CH2), 3.18 (s, 4H, 2CH2), 3.48 (s, 2H, CH2), 6.78 (t,
1H, J=16.0 Hz, arH), 6.94 (d, 2H, J=8.0 Hz, arH), 7.22 (t, 2H, J=16.0 Hz, arH),7.82 (bs, 1H,
NH), 9.12 (bs, 1H, NH), 9.65 (bs, 1H, NH). 13C NMR (DMSO-d6, δ ppm): 14.92 (CH3), 38.91
(CH2), 48.37 (2CH2), 53.19 (2CH2), 59.85 (CH2), arC: [115.84 (2CH), 119.26 (CH), 129.39
(2CH), 151.40 (C)], 176.89 (C=O), 190.63 (C=S). EI MS m/z (%): 322 ([M+1] +, 100), 323
([M+2] +, 85).
3.1.2.5. N-Benzyl-2-[(4-phenylpiperazin-1-yl)acetyl]hydrazincarboxamide (5e)
Recrystallized from acetone. Yield: 86 %, Mp. 211-212 °C. FT-IR (υmax, cm-1): 3380 ve 3304
1
(3NH), 3037 (ar-CH), 1713 (C=O), 1663 (C=O). H NMR (DMSO-d6, δ ppm): 2.62 (s, 4H,
13

2CH2), 3.09 (s, 4H, 2CH2), 3.48 (bs, H2O+2CH2), 4.25 (s, 2H, CH2), 7.01 (s, 3H, arH), 7.28
13
(s, 7H, arH), 7.91 (s, 1H, NH), 8.05 (s, 1H, NH), 9.53 (bs, 1H, NH). C NMR (DMSO-d6, δ
ppm): 43.14 (CH2), 46.68 (2CH2), 52.64 (2CH2), 59.89 (CH2), arC: [113.05 (2CH), 126.18
(CH), 126.94 (CH), 127.02 (2CH), 127.41 (CH), 127.48 (CH), 128.53 (CH), 128.59 (CH),
137.28 (C), 155.12 (C)], 158.65 (C=O), 169.53 (C=O). EI MS m/z (%): 113.02 (60), 368.36
([M+1] +, 100).
3.1.3. General method for the synthesis of compounds 6a-e
Method 1. A solution of corresponding carbo(thio)amide 5a-e (10 mmol) in ethanol/water
(1:1) was refluxed in the presence of 2 N NaOH for 10 h (for 6a), 13 h (for 6b), 11 h (for 6c),
9h (for 6d) and 15 h (for 6e) (the progress of the reaction was monitored by TLC). Then, the
resulting solution was cooled to room temperature and acidified to pH 5 with 37% HCl. The
precipitate formed was filtered off, washed with water, and recrystallized from an appropriate
solvent to give the target compound.
Method 2. The mixture of the corresponding compound 5 (10 mmol) and 2 N NaOH in
ethanol (10 mL) was irradiated in monomod microwave reactor in closed vessel (physical
parameters, reaction time and yields were given in Table 4). Then the resulting solution was
cooled to room temperature and acidified to pH 5 with 37% HCl. The precipitate formed was
filtered off, wash with water, and recrystallized from an appropriate solvent to give the target
compound.
3.1.3.1. 4-Phenyl-5-[(4-phenylpiperazin-1-yl)methyl]-4H-1,2,4-triazol-3-thiol (6a)
1
Recrystallized from ethanol. FT-IR (υmax, cm-1): 3095 (ar-CH), 2759 (-SH), 1598 (C=N). H
NMR (DMSO-d6, δ ppm): 2.50 (t, 4H, J=5.0 Hz, 2CH2), 3.19 (s, 4H, 2CH2), 3.40 (s, 2H,
CH2), 7.04 (d, 2H, J=8.0 Hz, arH), 7.43 (t, 4H, J=12.0 Hz, arH), 7.66-7.70 (m, 2H, arH), 8.06
13
(d, 2H, J=8.0 Hz, arH), 13.42 (s, 1H, SH). C NMR (DMSO-d6, δ ppm): 45.54 (CH2), 48.59
(2CH2), 51.45 (d, J=22.0 Hz, 2CH2), arC: [116.76 (2CH), 126.53 (CH), 128.39 (2CH), 129.61
(CH), 130.25 (2CH), 130.87 (CH), 134.08 (CH), 148.40 (C), 151.57 (C)], 150.20 (triazol C-
5), 169.61 (triazol C-3). EI MS m/z (%): 205.16 (100), 265.35 (42), 307.30 (35), 352.33
([M+1] +, 92), 374.28 ([M+Na] +, 30).
3.1.3.2 4-Benzyl-5-[(4-phenylpiperazin-1-yl)methyl]-4H-1,2,4-triazol-3-thiol (6b)
14

1
Recrystallized from ethanol. FT-IR (υmax, cm-1): 3090 (ar-CH), 2751 (-SH), 1597 (C=N). H
NMR (DMSO-d6, δ ppm): 2.38 (s, 4H, 2CH2), 3.27 (s, 4H, 2CH2), 3.54 (s, 2H, CH2), 4.89 (s,
2H, CH2), 6.96 (s, 1H, arH), 7.53 (s, 2H, arH), 7.58 (s, 4H, arH), 8.02 (s, 3H, arH), 13.89 (s,
1H, SH). 13C NMR (DMSO-d6, δ ppm): 46.67 (CH2), 51.42 (CH2), 51.88 (CH2), 51.97
(2CH2), arC: [117.72 (2CH), 126.13 (2CH), 127.61 (CH), 129.39 (2CH), 130.02 (CH), 130.78
(2CH), 139.14 (C), 148.40 (C), 150.79 (C)], 153.05 (triazol C-5), 174.54 (triazol C-3). EI MS
m/z (%): 134.98 (44), 208.18 (50), 288.46 (45), 388.32 ([M+Na] +, 100).
3.1.3.3. 4-(4-Fluorophenyl)-5-[(4-phenylpiperazin-1-yl)methyl]-4H-1,2,4-triazol-3-thiol
(6c)
Recrystallized from ethylacetate. FT-IR (υmax, cm-1): 3087 (ar-CH), 2830 (-SH), 1589 (C=N).
1H NMR (DMSO-d6, δ ppm): 2.37 (t, 4H, J=12.0 Hz, 2CH2), 3.28 (t, 4H, J=12.0 Hz, 2CH2),
3.42 (s, 2H, CH2), 6.97 (d, 2H, J=8.0 Hz, arH), 7.36 (s, 1H, arH), 7.40 (d, 2H, J=8.0 Hz, arH),
7.55 (q, 2H, J=16.0 Hz, arH), 8.03 (d, 2H, J=8.0 Hz, arH), 13.90 (s, 1H, SH). 13C NMR
(DMSO-d6, δ ppm): 46.66 (CH2), 50.58 (2CH2), 51.89 (2CH2), arC: [113.13 (2CH), 116.28
(d, J=23.0 Hz, 2CH), 121.22 (CH), 126.13 (2CH), 131.17 (d, J=9.0 Hz, 2CH), 137.41 (C),
149.59 (C), 161.29-163.74 (dC-F, J=245.0 Hz, C)], 155.05 (triazol C-5), 168.95 (triazol C-3).
EI MS m/z (%): 175 (100), 285 (64), 301 (63), 315 (62), 369 ([M] +, 46).
3.1.3.4. 4-Ethyl-5-[(4-phenylpiperazin-1-yl)methyl]-4H-1,2,4-triazol-3-thiol (6d)
1
Recrystallized from ethanol. FT-IR (υmax, cm-1): 3089 (ar-CH), 2762 (-SH), 1597 (C=N). H
NMR (DMSO-d6, δ ppm): 1.30 (t, 3H, J=16.0 Hz, CH3), 2.58 (s, 4H, 2CH2), 3.12 (t, 4H,
J=12.0 Hz, 2CH2), 3.64 (s, 2H, CH2), 4.04 (q, 2H, J=8.0 Hz, CH2), 6.78 (t, 1H, J=16.0 Hz,
13
arH), 6.93 (d, 2H, J=8.0 Hz, arH), 7.21 (q, 2H, J=8.0 Hz, arH), 13.63 (s, 1H, SH). C NMR
(DMSO-d6, δ ppm): 13.72 (CH3), 39.26 (CH2), 48.69 (2CH2), 52.33 (CH2), 52.85 (2CH2),
arC: [115.97 (2CH), 119.44 (CH), 129.38 (2CH), 149.47 (C)], 151.36 (triazol C-5), 167.36
(triazol C-3). EI MS m/z (%): 106 (100), 213 (41), 304 ([M+1] +, 24).
3.1.3.5. 4-Benzyl-5-[(4-phenylpiperazin-1-yl)methyl]-1H-1,2,4-triazol-5-one (6e)
1
Recrystallized from acetone. FT-IR (υmax, cm-1): 3083 (ar-CH), 1578 (C=N). H NMR
(DMSO-d6, δ ppm): 2.50 (t, 4H, J=4.0 Hz, 2CH2), 3.32 (s, 2H, CH2), 3.36 (s, H2O+CH2), 4.89
(s, 2H, CH2), 6.97 (d, 2H, J=8.0 Hz, arH), 7.26 (d, 3H, J=8.0 Hz, arH), 7.31 (s, 1H, arH), 7.34
13
(d, 2H, J=8.0 Hz, arH), 8.04 (d, 2H, J=12.0 Hz, arH), 11.75 (s, 1H, NH). C NMR (DMSO-
15

d6, δ ppm): 44.25 (CH2), 46.41 (2CH2), 52.13 (2CH2), 53.28 (CH2), arC: [113.00 (2CH),
121.38 (CH), 126.13 (2CH), 127.48 (CH), 128.50 (2CH), 128.87 (2CH), 137.40 (C), 144.65
(C)], 154.96 (triazol C-3), 156.06 (triazol C-5). EI MS m/z (%): 168.93 (68), 205.22 (42),
372.33 ([M+Na]+, 100).
3.1.4. General method for the synthesis of compounds 7a,b and 8a-j
Method 1. The solution of ciprofloxacin (for 7b, 8b, 8d, 8f, 8h, 8j) or norfloxacin (for 7a, 8a,
8c, 8e, 8g, 8i), (10 mmol) in dimethyl formamide was stirred at room temperature in the
presence of formaldehyde (37%, 30 mmol) for 15 min. Then, the corresponding compound 4
or 6 (10 mmol) was added into it and stirred for additional 24 h. The reaction mixture was
poured to ice-water and a solid obtained. This crude product was recrystallized from an
appropriate solvent to give the desired compound.
Method 2. A mixture of ciprofloxacin or norfloxacin (10 mmol), the corresponding
compound 7 or 8 (10 mmol) and formaldehyde (37%, 30 mmol) in DMF was irradiated in a
monomod microwave reactor in a closed vessel under pressure control at 50 W for 20 min.
The solid obtained was purified by recrystallization from an appropriate solvent.
3.1.4.1. 1-Ethyl-6-fluoro-4-oxo-7-(4-{[5-[(4-phenylpiperazin-1-yl)methyl]-2-tioxo-1,3,4-
oxadiazole-3(2H)-yl]methyl}piperazin-1-yl)-1,4-dihydroquinolin-3-carboxylic acid (7a)
Recrystallized from ethyl acetate:ethanol (3:1). Mp. 153-154 °C. FT-IR (υmax, cm-1): 3065 (ar-
1
CH), 1721 (C=O), 1520 (C=N), 1238 (C=S). H NMR (DMSO-d6, δ ppm): 1.41 (t, 3H,
J=12.0 Hz, CH3), 2.66 (s, 4H, 2CH2), 2.74 (s, 2H, CH2), 2.90 (s, 4H, 2CH2), 2.94 (s, 4H,
2CH2), 3.13 (s, 4H, 2CH2), 3.78 (s, 2H, CH2), 4.58 (d, 2H, J=5.0 Hz, CH2), 5.07 (s, 2H, CH2),
6.67 (t, 1H, J=16.0 Hz, arH), 6.91 (d, 2H, J=8.0 Hz, arH), 7.19 (t, 3H, J=12.0 Hz, arH), 7.96
(s, 1H, arH), 8.95 (s, 1H, CH), 15.34 (s, 1H, OH). 13C NMR (DMSO-d6, δ ppm): 14.81 (CH3),
48.80 (2CH2), 45.92 (2CH2), 49.81 (2CH2), 51.66 (2CH2), 52.46 (2CH2), 69.93 (CH2), arC:
[106.47 (CH), 107.55 (C), 111.66 (d, J=23.0 Hz, CH), 115.98 (2CH), 119.44 (CH), 119.71
(C), 129.36 (2CH), 137.63 (C), 145.76 (d, J=11.0 Hz, C), 151.33 (C), 152.05-154.53 (dC-F,
J=248.0 Hz, C)], 148.98 (CH), 159.47 (oxadiazole C-5), 166.56 (C=O), 176.63 (C=O), 178.48
(oxadiazole C-2). EI MS m/z (%): 205.21 (100), 361.27 (50), 517.26 (45), 630.28 ([M+Na] +,
40).
16

3.1.4.2. 1-Cyclopropyl-6-fluoro-4-oxo-7-(4-{[5-[(4-phenylpiperazin-1-yl)methyl]-2-tioxo-
1,3,4-oxadiazole-3(2H)-yl]methyl}piperazin-1-yl)-1,4-dihydroquinolin-3-carboxylic acid
(7b)
Recrystallized from ethyl acetate:ethanol (3:1). Mp. 176 °C. FT-IR (υmax, cm-1): 3014 (ar-
CH), 1724 (C=O), 1598 (C=N), 1261 (C=S). 1H NMR (DMSO-d6, δ ppm): 1.17 (s, 2H, CH2),
1.32 (d, 2H, J=8.0 Hz, CH2), 2.66 (s, 4H, 2CH2), 2.74 (s, 2H, CH2), 2.90 (s, 2H, CH2), 2.96 (s,
4H, 2CH2), 3.13 (s, 4H, 2CH2), 3.79 (s, 2H, CH2), 3.96 (s, 1H, CH), 5.07 (s, 2H, CH2), 6.77
(t, 1H, J=16.0 Hz, arH), 6.90 (d, 2H, J=8.0 Hz, arH), 7.19 (t, 2H, J=16.0 Hz, arH), 7.55 (d,
13
1H, J=8.0 Hz, arH), 7.87 (d, 1H, J=12.0 Hz, arH), 8.66 (s, 1H, CH), 15.18 (s, 1H, OH). C
NMR (DMSO-d6, δ ppm): 8.04 (2CH2), 36.30 (CH), 48.60 (2CH2), 49.74 (CH2), 49.82
(2CH2), 51.66 (2CH2), 52.46 (2CH2), 69.91 (CH2), arC: [107.02 (CH), 107.20 (C), 111.42 (d,
J=23.0 Hz, CH), 115.97 (2CH), 119.04 (C), 119.28 (d, J=32.0 Hz, CH), 129.37 (2CH),
134.69 (C), 139.58 (C), 145.44 (d, J=10.0 Hz, C), 151.32 (C), 152.18-154.66 (dC-F, J=248.0
Hz, C)], 148.43 (CH), 159.46 (oxadiazole C-5), 166.38 (C=O), 176.80 (C=O), 178.49
(oxadiazole C-2). EI MS m/z (%): 619.02 ([M] +, 100).
3.1.4.3.
1-Ethyl-6-fluoro-4-oxo-7-[4-({4-phenyl-3-[(4-phenylpiperazin-1-yl)methyl]-5-
tioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}methyl)piperazin-1-yl]-1,4-dihydroquinolin-3-
carboxylic acid (8a)
Recrystallized from ethyl acetate:ethanol (3:1). Mp. 203-205 °C. FT-IR (υmax, cm-1): 3021 (ar-
CH), 1721 (C=O), 1548 (C=N), 1261 (C=S). 1H NMR (DMSO-d6, δ ppm): 1.44 (s, 3H, CH3),
2.40 (s, 4H, 2CH2), 2.92 (s, 4H, 2CH2), 3.02 (s, 4H, 2CH2), 3.39 (s, 4H, 2CH2), 3.48 (s, 2H,
CH2), 4.58 (s, 2H, CH2), 5.22 (s, 2H, CH2), 6.75 (s, 1H, arH), 6.82 (s, 1H, arH), 7.17 (s, 3H,
arH), 7.52 (d, 5H, J=16.0 Hz, arH), 7.91 (s, 1H, arH), 8.91 (s, 1H, CH), 15.26 (bs, 1H, OH).
13C NMR (DMSO-d6, δ ppm): 14.80 (CH3), 46.63 (2CH2), 49.56 (2CH2), 49.97 (CH2), 50.10
(2CH2), 51.67 (CH2), 51.89 (2CH2), 69.02 (CH2), arC: [106.41 (CH), 107.54 (C), 113.12
(2CH), 116.35 (d, J=23.0 Hz, 2CH), 119.76 (C), 122.45 (2CH), 124.50 (2CH), 126.11 (2CH),
131.28 (CH), 137.54 (d, J=23.0 Hz, 2C), 139.50 (C), 142.33 (C), 148.40 (C), 152.30-155.02
(dC-F, J=272.0 Hz, C)], 148.94 (CH), 158.65 (triazol C-5), 166.60 (C=O), 170.01 (triazol C-3),
176.63 (C=O). EI MS m/z (%): 205.18 (100), 460.27 (26), 682.14 ([M] +, 34).
3.1.4.4.
1-Cyclopropyl-6-fluoro-4-oxo-7-[4-({4-phenyl-3-[(4-phenylpiperazin-1-
yl)methyl]-5-tioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}methyl)piperazin-1-yl]-1,4-
dihydroquinolin-3-carboxylic acid (8b)
17

Recrystallized from ethyl acetate:ethanol (3:1). Mp. 243-244 °C. FT-IR (υmax, cm-1): 3018 (ar-
CH), 1728 (C=O), 1599 (C=N), 1255 (C=S). 1H NMR (DMSO-d6, δ ppm): 1.18 (s, 2H, CH2),
1.33 (s, 2H, CH2), 2.40 (s, 4H, 2CH2), 2.92 (s, 4H, 2CH2), 3.03 (s, 4H, 2CH2), 3.27 (s, 4H,
2CH2), 3.48 (s, 2H, CH2), 3.82 (s, 1H, CH), 5.22 (s, 2H, CH2), 6.75 (d, 1H, J=5.0 Hz, arH),
6.82 (d, 2H, J=8.0 Hz, arH), 7.17 (d, 2H, J=5.0 Hz, arH), 7.50 (d, 2H, J=8.0 Hz, arH), 7.55 (d,
13
4H, J=8.0 Hz, arH), 7.89 (t, 1H, J=12.0 Hz, arH), 8.65 (s, 1H, CH), 15.13 (s, 1H, OH). C
NMR (DMSO-d6, δ ppm): 8.07 (2CH2), 36.31 (CH), 48.46 (2CH2), 49.90 (CH2), 50.09
(2CH2), 51.90 (2CH2), 52.42 (2CH2), 68.95 (CH2), arC: [106.93 (CH), 107.21 (C), 111.40 (d,
J=24.0 Hz, CH), 115.80 (2CH), 118.50 (CH), 119.03 (d, J=8.0 Hz, C), 119.34 (2CH), 128.73
(CH), 129.33 (CH), 129.41 (2CH), 129.78 (CH), 134.97 (C), 139.59 (C), 145.58 (C), 148.49
(C), 152.19-154.67 (dC-F, J=248.0 Hz, C)], 148.49 (CH), 151.23 (triazol C-5), 166.37 (C=O),
169.80 (triazol C-3), 176.79 (C=O). EI MS m/z (%): 205.15 (100), 696.11 ([M+2] +, 83).
3.1.4.5.
1-Ethyl-6-fluoro-4-oxo-7-[4-({4-benzyl-3-[(4-phenylpiperazin-1-yl)methyl]-5-
tioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}methyl)piperazin-1-yl]-1,4-dihydroquinolin-3-
carboxylic acid (8c)
Recrystallized from ethyl acetate:acetone (1:1). Mp. 248-249 °C. FT-IR (υmax, cm-1): 3063
1
(ar-CH), 1719 (C=O), 1546 (C=N), 1259 (C=S). H NMR (DMSO-d6, δ ppm): 1.41 (s, 3H,
CH3), 2.44 (s, 4H, 2CH2), 2.87 (s, 4H, 2CH2), 2.94 (s, 4H, 2CH2), 3.33 (s, 4H, 2CH2), 3.52 (s,
2H, CH2), 4.58 (s, 2H, CH2), 5.17 (s, 2H, CH2), 5.43 (s, 2H, CH2), 6.75 (s, 1H, arH), 6.82 (s,
2H, arH), 7.17 (s, 3H, arH), 7.27 (s, 2H, arH), 7.30 (s, 2H, arH), 7.90 (d, 1H, J=8.0 Hz, arH),
8.94 (s, 1H, CH), 15.33 (s, 1H, OH). 13C NMR (DMSO-d6, δ ppm): 14.81 (CH3), 46.74 (CH2),
47.87 (CH2), 48.20 (2CH2), 49.52 (CH2), 49.91 (CH2), 50.16 (CH2), 52.33 (2CH2), 52.63
(2CH2), 69.02 (CH2), arC: [106.38 (CH), 107.54 (C), 115.84 (2CH), 119.33 (2CH), 119.64
(C), 127.29 (2CH), 127.43 (CH), 128.87 (2CH), 129.36 (2CH), 136.44 (C), 137.63 (C),
145.72 (C), 148.61 (C), 152.03-154.50 (dC-F, J=247.0 Hz, C)], 148.97 (CH), 151.24 (triazol
C-5), 166.57 (C=O), 169.85 (triazol C-3), 176.61 (C=O). EI MS m/z (%): 694.29 (92), 696.23
([M] +, 100).
3.1.4.6.
1-Cyclopropyl-6-fluoro-4-oxo-7-[4-({4-benzyl-3-[(4-phenylpiperazin-1-
yl)methyl]-5-tioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}methyl)piperazin-1-yl]-1,4-
dihydroquinolin-3-carboxylic acid (8d)
18

Recrystallized from ethyl acetate:petroleum ether (3:1). Mp. 176-178 °C. FT-IR (υmax, cm-1):
1
3028 (ar-CH), 1728 (C=O), 1542 (C=N), 1256 (C=S). H NMR (DMSO-d6, δ ppm): 1.13 (s,
2H, CH2), 1.33 (s, 2H, CH2), 2.45 (s, 4H, 2CH2), 2.92 (s, 6H, 3CH2), 2.96 (s, 4H, 2CH2), 3.30
(s, 4H, 2CH2), 3.53 (s, 2H, CH2), 3.81 (s, 1H, CH), 5.21 (s, 2H, CH2), 5.41 (s, 2H, CH2), 6.76
(s, 1H, arH), 6.83 (s, 2H, arH), 7.18 (d, 2H, J=6.0 Hz, arH), 7.28 (s, 3H, arH), 7.30 (d, 2H,
J=5.0 Hz, arH), 7.57 (s, 1H, arH), 7.90 (d, 1H, J=12.0 Hz, arH), 8.66 (s, 1H, CH), 15.17 (s,
1H, OH). 13C NMR (DMSO-d6, δ ppm): 8.06 (2CH2), 36.30 (CH), 47.72 (2CH2), 47.88
(CH2), 48.24 (2CH2), 49.83 (CH2), 50.14 (2CH2), 52.34 (2CH2), 52.65 (2CH2), 69.03 (CH2),
arC: [106.96 (CH), 107.21 (C), 111.43 (d, J=23.0 Hz, CH), 115.87 (d, J=4.0 Hz, 2CH),
119.09 (C), 119.34 (CH), 127.30 (CH), 127.43 (CH), 127.86 (CH), 128.86 (CH), 128.88
(CH), 129.36 (2CH), 136.43 (C), 139.59 (C), 148.67 (d, J=10.0 Hz, C), 151.24 (C), 152.18-
154.66 (dC-F, J=248.0 Hz, C)], 148.43 (CH), 156.07 (triazol C-5), 166.39 (C=O), 169.86
(triazol C-3), 176.81 (C=O). EI MS m/z (%): 205.21 (100), 644.26 (32), 710.23 ([M+2] +, 73).
3.1.4.7. 1-Ethyl-6-fluoro-4-oxo-7-[4-({4-fluorophenyl-3-[(4-phenylpiperazin-1-yl)methyl]-
5-tioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}methyl)piperazin-1-yl]-1,4-dihydroquinolin-3-
carboxylic acid (8e)
Recrystallized from ethyl acetate:ethyl acetate (1:1). Mp. 164-165 °C. FT-IR (υmax, cm-1):
1
3063 (ar-CH), 1724 (C=O), 1599 (C=N), 1258 (C=S). H NMR (DMSO-d6, δ ppm): 1.42 (t,
3H, J=12.0 Hz, CH3), 2.39 (s, 8H, 4CH2), 2.95 (s, 4H, 2CH2), 3.00 (s, 4H, 2CH2), 3.47 (s, 2H,
CH2), 4.59 (d, 2H, J=8.0 Hz, CH2), 5.21 (s, 2H, CH2), 6.76 (s, 1H, arH), 6.85 (q, 2H, J=8.0
Hz, arH), 7.18 (d, 3H, J=8.0 Hz, arH), 7.40 (d, 3H, J=8.0 Hz, arH), 7.55 (d, 2H, J=5.0 Hz,
13
arH), 8.95 (s, 1H, CH), 15.35 (s, 1H, OH). C NMR (DMSO-d6, δ ppm): 14.83 (CH3), 48.49
(2CH2), 49.53 (CH2), 49.92 (CH2), 50.11 (2CH2), 51.94 (2CH2), 52.43 (2CH2), 70.97 (CH2),
arC: [106.40 (CH), 107.55 (C), 111.62 (d, J=23.0 Hz, CH), 115.70 (d, J=6.0 Hz, CH), 115.83
(d, J=22.0 Hz, 2CH), 119.35 (2CH), 119.66 (C), 129.35 (2CH), 131.12 (d, J=9.0 Hz, 2CH),
137.64 (C), 145.79 (C), 148.63 (d, J=17.0 Hz, C), 151.29 (C), 152.06-154.54 (dC-F, J=248.0
Hz, C), 163.79-166.58 (dC-F, J=279.0 Hz, C)], 148.97 (CH), 161.35 (triazol C-5), 168.62
(C=O), 169.95 (triazol C-3), 176.61 (C=O). EI MS m/z (%): 205.21 (100), 524.29 (27),
700.88 ([M] +, 70).
3.1.4.8.
1-Cyclopropyl-6-fluoro-4-oxo-7-[4-({4-fluorophenyl-3-[(4-phenylpiperazin-1-
yl)methyl]-5-tioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}methyl)piperazin-1-yl]-1,4-
dihydroquinolin-3-carboxylic acid (8f)
19

Recrystallized from ethyl acetate:ethyl acetate (1:1). Mp. 145-146 °C. FT-IR (υmax, cm-1):
1
3070 (ar-CH), 1723 (C=O), 1599 (C=N), 1257 (C=S). H NMR (DMSO-d6, δ ppm): 1.18 (s,
2H, CH2), 1.33 (d, 2H, J=8.0 Hz, CH2), 2.40 (s, 8H, 4CH2), 2.48 (s, 2H, CH2), 2.94 (s, 8H,
4CH2), 3.82 (s, 1H, CH), 5.21 (s, 2H, CH2), 6.76 (t, 1H, J=12.0 Hz, arH), 6.86 (q, 2H, J=6.0
Hz, arH), 7.18 (q, 2H, J=8.0 Hz, arH), 7.40 (t, 2H, J=20.0 Hz, arH), 7.53-7.59 (m, 3H, arH),
13
7.91 (d, 1H, J=12.0 Hz, arH), 8.66 (s, 1H, CH), 15.20 (s, 1H, OH). C NMR (DMSO-d6, δ
ppm): 8.07 (2CH2), 36.32 (CH), 48.49 (2CH2), 49.91 (CH2), 50.06 (2CH2), 51.94 (2CH2),
52.43 (2CH2), 70.97 (CH2), arC: [106.99 (CH), 107.22 (C), 111.43 (d, J=22.0 Hz, CH),
115.84 (d, J=5.0 Hz, 2CH), 116.21 (d, J=6.0 Hz, CH), 119.04 (C), 119.35 (CH), 129.35
(2CH), 131.12 (d, J=9.0 Hz, 2CH), 139.62 (C), 145.56 (d, J=9.0 Hz, C), 148.62 (d, J=16.0
Hz, C), 151.27 (d, J=4.0 Hz, C), 152.21-154.69 (dC-F, J=248.0 Hz, C), 166.39-168.62 (dC-F,
J=223.0 Hz, C)], 148.45 (CH), 161.35 (triazol C-5), 163.79 (C=O), 169.97 (triazol C-3),
176.80 (C=O). EI MS m/z (%): 205.15 (48), 380.35 (67), 522.29 (44), 609.17 (49), 611.17
(50), 708.16 (82), 710.17 (85),712.12 ([M]+, 100), 704.12 ([M+2] +, 96).
3.1.4.9. 1-Ethyl-6-fluoro-4-oxo-7-[4-({4-ethyl-3-[(4-phenylpiperazin-1-yl)methyl]-5-tioxo-
4,5-dihydro-1H-1,2,4-triazol-1-yl}methyl)piperazin-1-yl]-1,4-dihydroquinolin-3-
carboxylic acid (8g)
Recrystallized from ethyl acetate:ethanol (3:1). Mp. 193-194 °C. FT-IR (υmax, cm-1): 3095 (ar-
CH), 1718 (C=O), 1600 (C=N), 1243 (C=S). 1H NMR (DMSO-d6, δ ppm): 1.16 (s, 3H, CH3),
1.33 (d, 3H, J=8.0 Hz, CH3), 2.60 (s, 4H, 2CH2), 2.74 (s, 4H, 2CH2), 2.92 (s, 4H, 2CH2), 3.27
(s, 4H, 2CH2), 3.70 (s, 2H, CH2), 3.81 (s, 2H, CH2), 4.11 (d, 2H, J=5.0 Hz, CH2), 5.13 (s, 2H,
CH2), 6.77 (t, 1H, J=12.0 Hz, arH), 6.89 (d, 2H, J=8.0 Hz, arH), 7.19 (t, 2H, J=12.0 Hz, arH),
7.54 (d, 1H, J=5.0 Hz, arH), 7.87 (d, 1H, J=16.0 Hz, arH), 8.64 (s, 1H, CH), 15.13 (s, 1H,
13
OH). C NMR (DMSO-d6, δ ppm): 13.50 (CH3), 14.74 (CH3), 48.65 (2CH2), 49.51 (2CH2),
49.87 (CH2), 50.11 (2CH2), 52.15 (2CH2), 52.88 (2CH2), 68.53 (CH2), arC: [106.38 (CH),
107.54 (C), 111.66 (d, J=23.0 Hz, CH), 115.93 (2CH), 119.54 (d, J=15.0 Hz, CH), 119.68
(C), 129.43 (2CH), 137.62 (C), 145.75 (d, J=10.0 Hz, C), 148.36 (C), 152.02-154.50 (dC-F,
J=248.0 Hz, C)], 148.96 (CH), 151.28 (triazol C-5), 166.55 (C=O), 168.46 (triazol C-3),
176.59 (C=O). EI MS m/z (%): 205.21 (100), 517.26 (35), 658.54 ([M+1+Na] +, 38).
20

3.1.4.10.
1-Cyclopropyl-6-fluoro-4-oxo-7-[4-({4-ethyl-3-[(4-phenylpiperazin-1-
yl)methyl]-5-tioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}methyl)piperazin-1-yl]-1,4-
dihydroquinolin-3-carboxylic acid (8h)
Recrystallized from ethyl acetate:ethanol (3:1). Mp. 229-230 °C. FT-IR (υmax, cm-1): 3073 (ar-
1
CH), 1726 (C=O), 1543 (C=N), 1252 (C=S). H NMR (DMSO-d6, δ ppm): 1.33 (t, 4H,
J=12.0 Hz, 2CH2), 1.41 (t, 3H, J=12.0 Hz, CH2), 2.58 (s, 4H, 2CH2), 2.74 (s, 4H, 2CH2), 2.90
(s, 4H, 2CH2), 3.34 (s, H2O+2CH2), 3.69 (s, 1H, CH), 4.10 (d, 2H, J=8.0 Hz, CH2), 4.58 (d,
2H, J=8.0 Hz, CH2), 5.12 (s, 2H, CH2), 6.78 (d, 1H, J=8.0 Hz, arH), 6.88 (d, 2H, J=8.0 Hz,
arH), 7.15-7.21 (m, 3H, arH), 7.89 (d, 1H, J=16.0 Hz arH), 8.94 (s, 1H, CH), 15.33 (s, 1H,
OH). 13C NMR (DMSO-d6, δ ppm): 8.04 (2CH2), 13.51 (CH3), 36.28 (CH), 44.07 (CH2),
48.65 (2CH2), 49.80 (2CH2), 50.07 (2CH2), 52.16 (2CH2), 52.89 (2CH2), 68.53 (CH2), arC:
[106.92 (CH), 107.20 (C), 111.39 (d, J=23.0 Hz, CH), 115.93 (2CH), 119.01 (d, J=7.0 Hz, C),
119.46 (CH), 129.37 (2CH), 139.57 (C), 145.47 (d, J=10.0 Hz, C), 148.38 (C), 152.16-154.64
(dC-F, J=248.0 Hz, C)], 148.40 (CH), 151.28 (triazol C-5), 166.35 (C=O), 168.47 (triazol C-3),
176.76 (C=O). EI MS m/z (%): 205.15 (49), 646.26 ([M] +, 90), 648.27 ([M+2] +, 100).
3.1.4.11. 7-[4-({4-Benzyl-5-oxo-3-[(4-phenylpiperazin-1-yl)methyl]-4,5-dihydro-1H-1,2,4-
triazol-1-yl}methyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-3-
carboxylic acid (8i)
Recrystallized from ethyl acetate:ethanol (3:1). Mp. 253-255 °C. FT-IR (υmax, cm-1): 3059 (ar-
1
CH), 1703 (C=O), 1677 (C=O), 1600 (C=N). H NMR (DMSO-d6, δ ppm): 1.41 (t, 3H,
J=12.0 Hz, CH3), 2.44 (s, 4H, 2CH2), 2.82 (s, 4H, 2CH2), 3.23 (s, 4H, 2CH2), 3.39 (s, 6H,
3CH2), 4.58 (d, 2H, J=5.0 Hz, CH2), 4.96 (s, 2H, CH2), 6.94 (d, 2H, J=8.0 Hz, arH), 7.17 (d,
1H, J=8.0 Hz, arH), 7.25-7.33 (m, 6H, arH), 7.91 (d, 1H, J=16.0 Hz, arH), 8.02 (d, 2H, J=8.0
13
Hz, arH), 8.94 (s, 1H, CH), 15.33 (s, 1H, OH). C NMR (DMSO-d6, δ ppm): 14.78 (CH3),
44.87 (CH2), 46.38 (2CH2), 49.53 (2CH2), 49.78 (2CH2), 52.12 (2CH2), 52.97 (2CH2), 66.05
(CH2), arC: [106.35 (CH), 107.52 (C), 112.92 (2CH), 116.04 (2CH), 119.80 (C), 126.13
(2CH), 127.41 (2CH), 127.80 (CH), 128.92 (2CH), 137.38 (C), 137.58 (d, J=9.0 Hz, C),
143.57 (C), 145.82 (d, J=10.0 Hz, C), 152.04-154.96 (dC-F, J=292.0 Hz, C)], 148.91 (CH),
155.23 (triazol C-3), 161.87 (triazol C-5), 166.59 (C=O), 176.58 (C=O). EI MS m/z (%):
488.32 (47), 575.20 (88), 577.15 (53), 680.16 ([M] +, 100).
21

3.1.4.12. 7-[4-({4-Benzyl-5-oxo-3-[(4-phenylpiperazin-1-yl)methyl]-4,5-dihydro-1H-1,2,4-
triazol-1-yl}methyl)piperazin-1-yl]-1-Cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-3-
carboxylic acid (8j)
Recrystallized from ethyl acetate:ethanol (3:1). Mp. 199-200 °C. FT-IR (υmax, cm-1): 3059 (ar-
CH), 1710 (C=O), 1670 (C=O), 1600 (C=N). 1H NMR (DMSO-d6, δ ppm): 1.20 (s, 2H, CH2),
1.33 (s, 2H, CH2), 2.44 (s, 4H, 2CH2), 2.73 (s, 2H, CH2), 3.25 (s, 4H, 2CH2), 3.34 (s,
H2O+4CH2), 3.39 (s, 2H, CH2), 3.89 (s, 1H, CH), 5.03 (s, 2H, CH2), 6.97 (d, 2H, J=8.0 Hz,
arH), 7.28-7.35 (m, 6H, arH), 7.58 (t, 1H, J=16.0 Hz, arH), 7.91 (d, 1H, J=12.0 Hz, arH), 8.04
13
(d, 2H, J=8.0 Hz, arH), 8.66 (s, 1H, CH), 15.22 (s, 1H, OH). C NMR (DMSO-d6, δ ppm):
8.04 (2CH2), 36.30 (CH), 44.81 (CH2), 46.42 (CH2), 48.39 (CH2), 50.36 (2CH2), 51.10 (CH2),
52.16 (2CH2), 53.04 (2CH2), 67.53 (CH2), arC: [106.92 (CH), 107.23 (C), 111.42 (d, J=26.0
Hz, CH), 113.10 (2CH), 119.02 (C), 126.15 (2CH), 127.44 (CH), 127.53 (CH), 127.80 (2CH),
128.91 (2CH), 137.43 (d, J=6.0 Hz, C), 139.57 (C), 143.58 (C), 152.12-154.08 (dC-F, J=194.0
Hz, C)], 145.63 (triazol C-3), 148.44 (CH), 155.02 (triazol C-5), 166.39 (C=O), 176.83
(C=O). EI MS m/z (%): 581.10 (92), 583.11 (100), 694.16 ([M] +, 36).
3.2. Antimicrobial activity
The test microorganisms were obtained from the Hifzissihha Institute of Refik Saydam
(Ankara, Turkey) and were as follows: Staphylococcus aureus ATCC 25923, Enterococcus
faecalis ATCC 29212, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC
27853, Klebsiella pneumoniae ATCC 13883, Acinetobacter haemolyticus ATCC 19002. All
the newly synthesized compounds were weighed and dissolved in DMSO to prepare extract
stock solution of 10.000 µg/mL. The antimicrobial effects of the substances were tested
quantitatively in respective broth media by using double micro-dilution and the minimal
inhibition concentration (MIC) values (µg/mL) were determined. The antibacterial were
performed in Mueller-Hinton broth (MH) (Difco, Detroit, MI) at pH.7.3 and buffered Yeast
Nitrogen Base (Difco, Detroit, MI) at pH 7.0, respectively. The micro dilution test plates were
o
incubated for 18–24 h at 35 C. Brain Heart Infusion broth (BHI) (Difco, Detriot, MI) was
o
used for M. smegmatis, and incubated for 48–72 h at 35 C [48]. Ampicillin (10 µg),
gentamicin (10 µg) and ciprofloxacin (10 µg) were used as standard antibacterial respectively.
Dimethylsulphoxide with dilution of 1:10 was used as solvent control. All results were
presented in Table 3.
22

3.3. In vitro inhibitory activity screening and determination of IC50 values on E. coli
gyrase
The assay for determining IC50 values (Inspiralis) was performed on black streptavidin-
coated 96-well microtiter plates (Thermo Scientific Pierce). The plate was first rehydrated
with the wash buffer supplied (20mMTris-HCl (pH 7.6),137mM NaCl, 0.01% (w/v) BSA,
0.05% (v/v) Tween 20). Biotinylated oligonucelotide was immobilized onto the wells. The
excess of oligonucleotide was then washed off and the enzyme assay carried out in the wells.
The final reaction volume of 30 mL in buffer (35 mM Tris HCl pH 7.5; 24 mM KCl; 4 mM
MgCl2; 2 mM DTT; 1.8 mM spermidine; 1 mM ATP; 6.5% (w/v) glycerol; 0.1 mg/mL
albumin) contained 1.5 U of DNA gyrase from E. coli 0.75 mg of relaxed pNO1 plasmid, and
3 mL of inhibitors solution in 10% DMSO and 0.008% Tween® 20. Reactions were
incubated for 30 min at 37 oC and, after addition of the TF buffer (50 mM NaOAc pH 5.0, 50
mM NaCl and 50 mM MgCl2), which terminated the enzymatic reaction, for another 30 min
at room temperature to allow triplex formation (biotin-oligonucleotide-plasmid). The unbound
plasmid was then washed off using TF buffer, and a solution of SybrGOLD stain in T10
buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA) was added. After mixing, the fluorescence
(excitation, 485 nm; emission, 535 nm) was read using a BioTek's Synergy H4 microplate
reader. Preliminary screening was performed at inhibitor concentrations of 100 mM and 10
mM. For the most potent compounds IC50 was determined with 7 concentrations of the
inhibitors. IC50 values were calculated using GraphPad Prism software and represent the
concentration of inhibitor where the residual activity of the enzyme is 50 % in three
independent measurements; the final result is given as their average value. Novobiocin (IC50
¼ 0.17 mM (lit. 0.08 mM) for E. coli DNA gyrase was used as a positive control [49].
3.4. In vitro inhibitory activity screening and determination of IC50 values on E. coli
topoisomerase IV
The assay for the determination of IC50 values (Inspiralis) was performed on the black
streptavidin-coated 96-well microtiter plates (Thermo Scientific Pierce). The plate was first
rehydrated with the supplied wash buffer (20 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.01%
(w/v) BSA, 0.05% (v/v) Tween® 20) and biotinylated oligonucelotide was immobilized onto
the wells. The excess of oligonucleotide was then washed off, and the enzyme assay carried
out in the wells. The final reaction volume of 30 mL in buffer (40 mM HEPES KOH (pH 7.6),
100 mM potassium glutamate, 10 mM magnesium acetate, 10 mM DTT, 1 mM ATP, 0.05
23

mg/mL albumin) contained 1.5 U of topoisomerase IV from E. coli or S. aureus, 0.75 mg of
supercoiled pNO1 plasmid, and 3 mL of inhibitors solution in 10% DMSO and 0.008%
Tween® 20. Reactions were incubated for 30 min at 37 oC, and after addition of the TF buffer
(50 mM NaOAc pH 5.0, 50 mM NaCl and 50 mM MgCl2), which terminated the enzymatic
reaction, for another 30 min at room temperature to allow triplex formation (biotin-
oligonucleotide-plasmid). The unbound plasmid was then washed off using TF buffer and the
solution of SybrGOLD stain in T10 buffer (10 mM Tris-HCl pH 8.0 and 1mM EDTA) was
added. After mixing, fluorescence (excitation, 485 nm; emission, 535 nm) was read using a
BioTek's Synergy H4 microplate reader. Preliminary screening was performed at inhibitor
concentrations of 100 mM and 10 mM. For most potent compounds IC50 was determined with
7 concentrations of the inhibitors. IC50 values were calculated using GraphPad Prism software
and represent the concentration of inhibitor where the residual activity of the enzyme is 50%
in three independent measurements; the final result is given as their average value.
Novobiocin (IC50 ¼ 11 mM (lit. 10 mM) for E. coli topoisomerase IV was used as a positive
control.
3.5. Molecular docking studies
Docking studies of most active (determined through enzyme inhibition studies) new
fluoroquinolone-1,2,4-triazole hybrids (only 7a, 7b and 8a-j) into the binding pockets of
DNA gyrase (PDB ID: 1SUU) and TOPO-IV (PDB ID: 1S16) was carried out using
Autodock4.2.6, Autodock Tools 1.5.6 and the Arguslab version 4.0.1. 3D crystal structures
were retrieved from the protein data bank (PDB) (Source: www.rcsb.org/pdb/). All molecular
docking tactics and parameters were referred from our previous reports [50-52]. Statistical
mechanical analysis for the ligands and the lowest binding energy, ligand efficiency (it is used
in drug discovery investigations to support in narrowing focus to candidate drugs with
optimum amalgamations of physicochemical and pharmacological potentials [53]) and the
inhibitory constant (ki) values were extracted. Molecular interaction (hydrogen bonding, π-π
interaction, and π-cation interaction) results were validated.
4. Discussion
In our initial effort, to synthesis medicinally important small molecules, compound 8a, has
been selected as a model product to determine the optimum reaction conditions. In this trial,
the model reaction has been carried out in polar solvent including ethanol, tetrahydrofurane
and DMF in the presence of different Lewis and Bronsted acid catalysts, such as p-TSA,
24

FeCl3, InCl3 and HCl under the microwave irradiated conditions. In all cases, progress of
reaction has been monitored by TLC analysis. The condensation of the azoles, 4 and 6a-e with
fluoroquinolone class antibacterial drugs namely norfloxacin and ciprofloxacin has been
carried out under the Mannich reaction conditions. A Mannich reaction which leads to the
formation of aminoalkylated products has been descript as a one pot condensation between
three components consisting of a substrate containing at least one active hydrogen, an
aldehyde component and an amine reagent [54, 55]. N-Mannich bases constitute important
tools to development of new drugs containing amine or amide function [56]. It has been
believed that the group linked to parent amine through a Mannich reaction cause to increase
the absorption of molecules through bio-membrans as a result of their diminished protonation
causing to increase the lipophilicity of molecules at physiological pH values [57].
Furthermore, the transformation into iminium salts render the Mannich bases to soluble in
aqueous solvents [58].
Bringing the achieved compounds to medicinal applications, these compounds were
initially tested for their antimicrobial effects and DNA gyrase and TOPO-IV inhibitions. DNA
gyrase and TOPO-IV inhibition result of all compounds was compared to the standard drug,
Novobiocin. The obtained potentially good DNA gyrase and TOPO-IV inhibition by the
compounds was further validated through binding affinity calculations and molecular
interaction validations. DNA gyrase and TOPO-IV are the enzymes that are highly involved
in the synthesis of bacterial genetic material, especially in the DNA supercoiling processes
[59, 60]. Due to this reason these two enzymes are being the antibacterial drug targets. In the
present study, compounds 7a, 7b and 8a-j were showed remarkable antibacterial as well as
effective DNA gyrase inhibition activities. Compound 8j can be a future antibiotic.
4. Conclusion
We have designed the Microwave irradiated and conventional synthesis, antibacterial
activity, DNA gyrase and topoisomerase IV inhibition of new piperazine functionalized
fluoroquinolone hybrids. Compared to conventional method, by the microwave irradiation
method, reaction yield and time was significantly decreased. At the same time, as a result of
optimization studies for the synthesis of fluoroquinolone derivatives as target molecules, it
was determined that Lewis acids and solvent were not affected. The antibacterial activity
screening studies have revealed that new hybrid molecules consisting of fluoroquinolone-
azole-piperazine conjugation (7a,b and 8a-j) have good-excellent antibacterial activity against
25

the test microorganisms. Furthermore, these hybrids have exhibited good DNA gyrase
inhibition with the IC50 values varying between 0.134-1.84 µg/mL. To unveil the interaction
mode of compounds to receptor, a molecular docking study has been performed. With an
average least binding energy of -9.5 kcal/mol, all compounds have been found to possess
remarkable inhibitory potentials against DNA gyrase (E. coli). Conclusively, considering their
bio-activity potentials, perhaps highly substitute (8a-j) piperazine-azole functionalized
fluoroquinolone hybrids (7a,b and 8a-j) could be the future antibiotics.
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