Microwave assisted synthesis of some hybrid molecules derived from norfloxacin and investigation of their biological activities

Article abstract of DOI:10.1016/j.ejmech.2013.06.045

Norfloxacin was converted to 7-(4-amino-2-fluorophenyl)piperazin derivative (2) via the formation of nitro compound. The synthesis of the norfloxacin derivatives containing 1,3-thiazole or 1,3-thiazolidin moiety was performed from the reaction of 4-chloro

Full text of DOI:10.1016/j.ejmech.2013.06.045

European Journal of Medicinal Chemistry 67 (2013) 230e242
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Microwave assisted synthesis of some hybrid molecules derived from
norfloxacin and investigation of their biological activities
Meltem Yolal Mentese a, Hacer Bayrak a, Yıldız Uygun a, Arif Mermer a, Serdar Ulker b,
,
Sengul Alpay Karaoglu b, Neslihan Demirbas a
*
a Karadeniz Technical University, Department of Chemistry, 61080 Trabzon, Turkey
b
ꢀ
Recep Tayyip Erdogan University, Department of Biology, 53100 Rize, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Norfloxacin was converted to 7-(4-amino-2-fluorophenyl)piperazin derivative (2) via the formation of
nitro compound. The synthesis of the norfloxacin derivatives containing 1,3-thiazole or 1,3-thiazolidin
moiety was performed from the reaction of 4-chlorophenacylbromide or ethyl bromoacetate with
compounds 4e7 obtained starting from 2. 3-Fluoro-4-[4-(2-methoxyphenyl)piperazin-1-yl]aniline (14),
5-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-4-phenyl-4H-1,2,4-triazole-3-thiol (18) and {[4-(2-
methoxy phenyl)piperazin-1-yl]methyl}-1,3,4-oxadiazol-2-thiol (19) were obtained starting from 1-(2-
methoxyphenyl)piperazine by several steps. The treatment of hydrazide (16) with several aldehydes
afforded N0-[(2-hydroxyphenyl)methylen]- (20), N0-[(3-hydroxy-4-methoxy phenyl)methylen]- (21) or
N0-[1H-indol-3-ylmethylene]-2-[4-(2-methoxyphenyl)piperazin-1-yl]acetohydrazide (22). Then, com-
pounds 14, 18, 19 and 22 were condensed with 7-[4-(chloroacetyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (3) that was obtained from norfloxacine.
Received 13 May 2013
Received in revised form
19 June 2013
Accepted 23 June 2013
Available online 29 June 2013
Keywords:
Norfloxacin
1,3-Thiazole
1,2,4-Triazole
Antimicrobial activity
Antiurease activity
All newly synthesized compounds were screened for their antimicrobial activities and some of them
exhibited excellent activity. Moreover, one compound was found to have antiurease activity.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
diseases by displaying excellent pharmacokinetic properties, high
antimicrobial activity, and low side effects [6e10].
Although significant progress has been made for the treatment
and control of microbial infections by introducing new strategies
and combinatorial therapy, antimicrobial resistance continues to be
one of major concerns to the public health and scientific commu-
nities worldwide. Because, infections caused by resistant pathogens
fail to response to treatment resulting in prolonged illness and
greater risk of death. The alarming rates of emerging and ree-
merging microbial threats coupled with increasing antibacterial
resistance have emphasized the urgent need for new and more
effective antibacterial agents with high safety profile [1e5].
These agents are known as specific inhibitors of the bacterial
DNA gyrase, an enzyme which is responsible for negatively super-
coiling covalently closed circular DNA, and also in catenation and
decatenation reactions [10]. These compounds have had good
success against Gram-negative bacteria, but they are resistant to
Gram-positive pathogens, such as Staphylococcus aureus. Although
they displays some adverse events such as CNS side effects,
phototoxicity, and arthropathy, the more serious events are rare,
and there exists a continuous need for novel quinolones to over-
come the limitations of existing drugs [9,11].
Since the introduction in use of nalidixic acid for the treatment
of bacterial infections in 1962, a number of derivatives have been
synthesized. Among them, fluoroquinolones such as norfloxacin,
pefloxacin, enoxacin, ofloxacin, and ciprofloxacin play a major role
in the treatment of bacterial community or hospital acquired
It was reported that, the fluorine atom and the 1-alkyl,
1,4-dihydro-4-oxo-quinoline-3-carboxylic acid skeleton of fluo-
roquinolones is essential for potency represented in binding with
type-II topoisomerase enzymes, DNA gyrase and topoisomerase.
The 6-fluoro and 7-piperazinyl groups were reported as responsible
for the broad spectrum and antipseudomonal activities of fluo-
roquinolones. Moreover, C-7 substituent is the most adaptable site
for chemical modifications. This area also controls the pharmaco-
kinetic properties of the drugs, with the basic nitrogen. Various
substitutions of piperazine nucleus at C-7 position has resulted in a
* Corresponding author. Tel.: þ90 462 3772600; fax: þ90 4623253196.
E-mail
addresses:
neslihan@ktu.edu.tr,
ndemirbas651@mynet.com
(N. Demirbas).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.045

M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
231
wide range of clinically useful fluoroquinolone antibacterials such
as norfloxacin, ciprofloxacin, perfloxacin, pefloxacin, ofloxacin,
amifloxacin, fleroxacin, lomefloxacin, sparfloxacin, difloxacin,
enoxacin, enrofloxacin, levofloxacin, marbofloxacin, and orbi-
floxacin [9,10,12e16]. Furthermore, N-1 alkylation is essential to
keep the molecule in tautomeric keto form, because keto form
possess antibacterial activity, while enol form is completely inac-
tive [17]. It is known that the molecule part linked the piperazine
nucleus at C-7 is regarded as the drugeenzyme interaction domain
[9,18e20]. In addition, C-7 substituent dominantly controls cell
permeability and inhibition of DNA gyrase and ultimately in-
fluences the bioactivity, spectrum, solubility and pharmacokinetics
[9,21]. In the 7-piperazinyl quinolone skeleton, piperazine ring
displays enough structural convenience to allow several molecular
modifications. Much efforts have been devoted on the structural
modification at C-7 position in quinolones by incorporating several
substituents containing some five- and six-membered heterocycles
especially N-containing heterocycles such as pyridine, thiadiazole,
piperidine etc [22].
1,2,4-Triazole ring is known as versatile lead molecule for
designing potential bioactive structures. Its derivatives have been
incorporated to yield a wide variety of therapeutically important
activities including antibacterial [23e25] anti-inflammatory [26],
CNS depressant [27], anti-tubercular [28] anti-HIV [29], and anti-
proliferative [30]. 1,2,4-Triazole system is a structural element of
many drugs that have anti-fungal activity such as fluconazole,
itraconazole, and voriconazole [31]. Also, there are other known
drugs containing 1,2,4-triazole nucleus, e.g., triazolam, a benzodi-
azepine class psychotropic drug, rizatriptan (antimigraine), nefa-
zodone (antidepressant), ribavirin (antiviral), alprazolam
(analgesic) and etizolam (hypnotic and sedative). Vorozole, letro-
zole and anastrozole are aromatase inhibitors and used especially
for the treatment of breast cancer, which interrupts synthesis of
estrogen in the body [32e36].
N-Mannich base derivatives of imides, amides, amines, hydan-
toin and urea derivatives have been used as potentially useful
prodrug candidates. The group linked to parent amine by Mannich
reaction is believed to increase the lipophilicity of molecule at
physiological pH values by decreasing their protonation, and this
restriction of protonation results in enhancement of absorption
through bio-membranes [51,52]. The lipophilicity of fluo-
roquinolones can influence their ability to cross bacterial mem-
branes [10]. Furthermore, the neutral species of fluoroquinolones
are more lipophilic than zwitterionic form. It was reported that
lipophilicity an important factor affecting the intestinal absorption
of quinolones, and steric and electronic effects or charge density,
can affect lipophilicity [10,53].
Bacterial urease enzymes, which accelerate hydrolysis of urea to
ammonia gas with the reaction rate at least 1014 over the sponta-
neous reaction, have been reported as important virulent factors
including several important pathogenesis such as pyelonephritis,
hepatic coma, peptic ulceration, injection-induced urinary stones
and stomach cancer [54e56]. The detrimental impact of ureases is
not only on human health. As a result of urease activity, the NH3 lost
from fertilizers is an economic impact for farmers. Moreover, the
interference of NH3 to the atmosphere from urea will subsequently
be deposited to land or water. The result of this is eutrophication
and acidification of natural ecosystems on a regional scale [57].
In the present study, as a part of our general program in
the continued research for new hybrid molecules with antimicro-
bial and antiurease activity, it has been planned to introduce bio-
logically active 1,2,4-triazole, 1,3-thiazole, 1,3-oksazole, 1,3-
thiazolidinone or 1,3-oksazole nucleus at position-4 of piperazine
ring of norfloxacine, that is a synthetic chemotherapeutic antibac-
terial drug occasionally used to treat common as well as compli-
cated urinary tract infections [58].
2. Results and discussion
Another new class of synthetic antimicrobial agents, oxazolidi-
nones, have been reported to possess activity against numerous
multidrug-resistant Gram-positive organisms [37]. Beside the
drugs linezolid and eperezolid, a number of various oxazolidinone
derivatives have been synthesized as antimicrobial compounds
[38e41].
Another privileged scaffold thiazolidinone constitute a very
attractive target for combinatorial synthesis due to its structure
activity relationship, and it belongs to an important class of N and S
containing heterocycles, which are widely used as key building
blocks in the field of design, synthesis of new pharmaceutical
agents [42,43]. Further, the presence of the NeCeS linkage in the
thiazolidines is also responsible for nematocidal, fungicidal, anti-
bacterial and antiviral activities [43].
2.1. Chemistry
The synthetic strategies adopted to obtain the target com-
pounds are depicted in Schemes 1, 2 and 3. In the present study, the
synthesis of compound 1 was performed by condensation of 3,4-
difluoronitrobenzene with norfloxacin and this compound was
characterized by the presence of two strong bands at 1475 and
1335 cmꢀ1 in the FT-IR spectrum due to nitro group. These signals
disappeared, when compound 1 was converted to the corre-
sponding amine (2) by the reduction of nitro group, instead, two
strong absorption bands was recorded at 3433 and 3332 cmꢀ1
derived from eNH2 function. In the 1H NMR spectrum, this group
resonated at 5.03 ppm as D2O exchangeable singlet.
It is well known that more efficacious antibacterial agents can
be designed by joining two or more biologically active heterocyclic
systems together in a single molecular framework [1,44e46]. These
synergistic antimicrobial combinations have several major advan-
tages, including the potential to slow down the development of
drug resistance, a broader antimicrobial spectrum, and a potential
reduction in the dose and undesired side effects of each drug [46].
An example of a combination treatment that has been used suc-
cessfully is the administration of amphotericin B and flucytosine for
the management of cryptococcal meningitis [47e49].
It has been reported that the combination of antibacterial quin-
olones with other pharmacophores or drug fragments might result
in pharmaceutically important hybrid molecules with difunctional
targets, drug synergism or new action mechanisms to overcome
drug resistance. In this connection, a number of studies have been
devoted for the synthesis of quinolone hybrids incorporating several
bioactive moieties at C-7 position of quinolone scaffold [50].
The treatment of norfloxacin with chloroethanoylchloride yiel-
ded compound 3 [59] that was used as an intermediate for further
condensations.
The treatment of norfloxacin with several iso(thio)cyanates
generated the corresponding carbono(thio)ylamino derivatives (4e
7). The FT-IR spectra of derivatives 4, 6 and 7 showed an absorption
band at 1245 (for 4), 1209 (for 6) or 1246 cmꢀ1 (for 7) indicating the
presence of C]S double bond in the structure. Moreover, additional
signals derived from benzyl, ethyl or 3-morpholin-4-ylpropylamino
moiety at the position 7 of norfloxacin skeleton observed at the
related chemical shift values in the 1H and 13C NMR spectra of
compounds 4e7. Furthermore, the FT-IR and 1H NMR spectra of
these compounds displayed signals derived from two NH protons
(exchangeable with D2O), while no signal derived from eNH2 group
was present.
The synthesis of 7-[4-(4-{[3-alkyl-4-oxo-1,3-thiazolidin-2-
ylidene]amino}-2-fluorophenyl)piperazin-1-yl]-1-ethyl-6-fluoro-

232
M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
O
N
O
O
N
O
F
F
OH
OH
F
iv
N
F
N
N
N
X
2
H2N
N
H
RHN
ii
X= S
CH2C6H5,
CH2C6H5,
C2H5,
4: R=
O
O
F
X= O
X= S
X= S
5: R=
6: R=
7: R=
OH
F
N
N
(CH2)3
N
O,
N
1
O2N
i
vi
O
N
O
O
N
O
F
F
OH
OH
v
N
F
N
HN
N
O
S
Norfloxacine
N
CH2C6H5
C2H5
8: R=
9: R=
N
R
iii
O
O
F
O
N
O
OH
F
OH
N
N
F
O
N
N
Cl
N
3
Cl
S
N
N
CH2C6H5
11: R= C2H5
10: R=
R
Scheme 1. i: 3,4-Difluoronitrobenzene in acetonitrile, NaHCO3, reflux or MW irradiation; ii: Pd/C in n-butanol, hydrazine hydrate reflux or MW irradiation; iii: chloroethanoyl-
chloride, triethylamine in THF, room temperature; iv: benzylisothiocyanate (for 4), benzylisocyanate (for 5), ethylisothiocyanate (for 6) or 4-morpholinopropylisothiocyanate (for 7)
in ethanol, reflux or MW irradiation; v: ethyl bromoacetate in glacial acetic acid, dried sodium acetate, MW irradiation; vi: 4-chlorophenacylbromide in dry chloroform, dried
sodium acetate, reflux or MW irradiation.
4-oxo-1,4-dihydroquinoline-3-carboxylic acids (8 and 9) was car-
ried out by microwave irritation of the mixture of compound 4
or 6 and ethyl bromoacetate in glacial acetic acid at 150 ꢁC, 200 W
in the presence of dried sodium acetate. On the other
molecular masses; and these compounds have given elemental
analysis results consistent with the proposed structures.
Substituted hydrazides can be considered as useful tools for
the synthesis of nitrogen, sulfur or oxygen containing compounds
[25]. Compound 16 that was obtained starting from 1-
(2-methoxyphenyl)piperazine by two steps, was converted to 5-
{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}-4-phenyl-4H-1,2,4-
triazole-3-thiol (18) with the aim to merge two heterocyclic nuclei
responsible biological activity namely piperazine and 1,2,4-triazole
in a single molecule. Similarly, the synthesis of compound 19 was
performed by the reaction of the hydrazide (16) with CS2 in basic
media. This idea originated from the intent to introduce a 1,3,4-
oxadiazole nucleus to piperazine skeleton. A singlet characteristic
for the eSH group was recorded at 13.85 (for 18) or 14.35 (for 19)
ppm in the 1H NMR spectra of compounds 18 and 19. FT-IR spectra
of these compounds exhibited absorption bands originated from e
SH function at 2829 and 2848 cmꢀ1. Moreover compounds 18 and
19 gave reasonable mass fragmentation and elemental analysis
data consistent with the assigned structures.
hand, the condensation of compounds
4 and 6 with 4-
chlorophenacylbromide by microwave irradiation or refluxing in
chloroform produced 7-[4-(4-{[3-alkyl-5-(4-chlorophenyl)-1,3-
thiazol-2(3H)-ylidene]amino}-2-fluoro phenyl)piperazin-1-yl]-1-
ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acids (10
and 11). The disappearance of NH signals in the FT-IR and 1H NMR
supported the condensation leading to the formation of com-
pounds 8e11. Another evidence for the cyclocondensation between
compounds 4 or 6 and ethyl bromoacetate is the appearance of a
signal at 4.02 ppm (for 9) or 4.11 ppm (for 8) in the 1H NMR spectra,
which correspond the C5 protons of thiazolidinone nucleus. These
carbons resonated at 37.39 ppm (for 9) or 45.37 ppm (for 8) in the
13C NMR spectra. The additional support for the formation of the
targeted compounds, 8e11 was obtained by the appearance of
[M þ 1] ion peaks at corresponding m/z values confirming their

M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
233
N
N
NO2
N
N
NH2
ii
OCH3
F
OCH3
F
13
14
i
O
N
OCH3
12
NH
N
N
iii
O
OCH3
15
iv
O
N
N
N
N
NHNH2
N
N
i
SH
O
H
OCH3
16
19
vi
v
O
O
S
N
N
N
N
NHN
NHNH
N
H
Ar
H
H
17
20: Ar=
21: Ar=
vii
HO
OCH3
OH
N
N
N
N
SH
N
22: Ar=
OCH3
N
H
18
Scheme 2. i: 3,4-Difluoronitrobenzene in tetrahydrofuran, room temperature; ii: PdeC in n-butanol, hydrazine hydrate, reflux; iii: ethyl bromoacetate in tetrahydrofuran, trie-
thylamine, room temperature; iv: Hydrazine hydrate in absolute ethanol, reflux; v: phenylisothiocyanate in absolute ethanol, reflux; vi: NaOH in water, reflux; vii: KOH in water/
ethanol, CS2, reflux; viii: 2-hydroxybenzaldehyde (for 20), 3-hydroxy-4-methoxybenzaldehyde (for 21) or ındol-3-carbaldehyde (for 22) in ethanol, reflux.
The treatment of hydrazide, 16 with 2-hydroxybenzaldehyde, 3-
hydroxy-4-methoxybenzaldehyde or indol-3-carbaldehyde in
ethanolic solution generated the corresponding imine compounds,
N0-[(substituted phenyl)methylen]-2-[4-(2-methoxy phenyl)piper-
azin-1-yl]acetohydrazides (20e22). The condensation of com-
pound 21 with compound 3 resulted in the formation of 1-ethyl-6-
fluoro-7-[4-({2-methoxy-4-[({[4-(2-methoxyphenyl)piperazin-1-
yl]acetyl}hydrazono)methyl]phenoxy}acetyl)piperazin-1-yl]-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (23). The structures of
these compounds (20e23) were confirmed on the basis of spec-
troscopic methods and elemental analysis. It is interesting to note
that the compounds having arylidenehydrazide structure may exist
as E/Z geometrical isomers about C]N double bond and as cis/trans
amide conformers [60,61]. According to the literature [61], the
compounds containing imine bond are present in higher percent-
age in dimethyl-d6 sulfoxide solution in the form of geometrical E
isomer about C]N double bond. The Z isomer can be stabilized in
less polar solvents by an intramolecular hydrogen bond. In the
present study, the spectral data was obtained in dimethyl-d6 sulf-
oxide solution and no signal belonging to Z isomer has been
observed. On the other hand, the cis/trans conformers of E isomer
were present in the dimethyl-d6 sulfoxide solution of compounds
20e23.
The synthesis of compounds 24 and 25 was carried out by the
condensation of compound 3 with 18 and 19, respectively, with the
aim to introduce a 1,3,4-oxadiazole or 1,2,4-triazole nucleus to
norfloxacin skeleton. Thesecompoundsexhibited FT-IR,1HNMR and
13C NMR spectra consistent with the assigned structures. Moreover,
[M þ K] ion peaks have been included in their mass spectra, and
rational elemental analysis results have been obtained for these
compounds. The condensation of 14 and 16 with compound 3 yiel-
ded the corresponding norfloxacin derivatives (26 and27) contain-
ing at the position 7 a 3-fluoro-4-[4-(2-methoxyphenyl)piperazin-
1-yl]phenylglycyl (for 26) or 4-[(2-{[4-(2-methoxyphenyl)

234
M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
O
N
O
F
OH
OCH3
O
N
O
N
N
N
N
N
H
O
OCH3
23
O
N
O
F
OH
N
N
N
N
N
S
O
O
N
i
OCH3
24
25
O
N
O
F
OH
ii
N
N
N
N
N
S
N
iii
O
N
3
OCH3
O
O
iv
F
OH
N
N
v
F
N
HN
O
N
N
26
OCH3
O
N
O
F
OH
N
O
N
N
N
NH NH
O
OCH3
27
Scheme 3. i: NaOH and compound 21 in dimethyl sulfoxide, 80 ꢁC; ii: NaOH and compound 24 in dimethyl sulfoxide, 80 ꢁC; iii: NaOH and compound 18 in dimethyl sulfoxide,
80 ꢁC; iv: NaOH and compound 14 in dimethyl sulfoxide, 80 ꢁC; v: NaOH and compound 16 in dimethyl sulfoxide, 80 ꢁC.
piperazin-1-yl]acetyl}hydrazino)acetyl] (for 27) moiety, and their
structures have been elucidated by spectroscopic methods and
elementalanalysis.1H NMR spectrumof derivative 26displayed only
one signal due to NH group at 8.69 ppm integrating one proton as a
result of condensation, while 1H NMR spectrum of derivative 27
showed two signals due to two NH protons at 8.52 and 11.57 ppm.
The signal originated from methoxy group resonated at 3.96 (for 27)
or 3.97 (for 26) ppm in the 1H NMR spectra of these compounds.
These carbons were recorded at 55.40 (for 27) and 56.09 (for 26) in
the 13C NMR spectra. Other groups resonated in the related chemical
shift values in the 1H NMR and 13C NMR spectra. The additional
support for the formation of the targeted compounds (26 and 27)
was obtained by the appearance of molecular ion peaks at corre-
sponding m/z values confirming their molecular masses. Moreover,
compounds 26 and 27 gave reasonable elemental analysis results
consistent with the proposed structures.
2.2. Biological activity
2.2.1. Antimicrobial activity
The nitro compound (1) exhibited good antimicrobial activity
toward the test microorganisms except Candida albicans (Ca) and

M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
235
Saccharomyces cerevisiae (Sc), which are yeast like fungi. With the
reduction of nitro group to amine function, excellent antibacterial
activities were observed for compound 2 with the MIC values of
<1.9 or 7.8 mg/mL. Similarly, the chloroacetylation of norfloxacin
gave the compound 3 having excellent antimicrobial activity on test
urease with an IC50 value of 20.51 ꢂ 9.09
m
g/mL. The other com-
pounds have moderate or no inhibitory activity (Table 2). Dose
dependent inhibitory effect of compound 25 was depicted in Fig. 1.
At low concentrations of compound 25 caused urease inhibitory
activity than thiourea. This compound might be considered as po-
tential antibiotics to treat infections.
microorganisms with the MIC values varying between 0.21 and
15.6 mg/mL, except Ca. and Sc. Even this result is better than standard
drugs Ampicillin and Streptomycin. Among the carbon-
othioylamino derivatives (4, 6 and 7), compound 4 was found to
have excellent activity against the test microorganisms except Ca.
3. Experimental
3.1. General
and Sc. with the MIC values between 0.97 and 31.5 mg/mL. On the
other hand, the carbonylamino derivative (5) of 4 displayed mar-
ginal activities against Escherichia coli (Ec.), enteric bacteria, S.
aureus (Sa.) and Enterococcus faecalis (Ef.), which are Gram positive
cocci, Bacillus cereus (Bc.) that is Gram positive spore bacillus, C.
albicans (Ca.) and S. cerevisiae (Sc.). It can be speculated that this
lower activity is due to the slight solubility of compound 5 even in
dimethyl sulfoxide. The carbonthioylamino derivative containing a
morpholin-4-ylpropyl nucleus (7) demonstrated good-moderate
activities on the test microorganisms except Mycobacterium smeg-
matis (Ms.), a nonpigmented rapidly growing mycobacterium, Ca.
and Sc., while other carbonthioylamino derivative including an
ethyl group instead of morpholin-4-ylpropyl nucleus (6) was found
to have slight activity toward Ec., Ca. and Sc. with the MIC values of
All the chemicals were purchased from Fluka Chemie AG Buchs
(Switzerland) 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 aluminum sheets. The mobile phase was ethyl
acetate:ethyl ether (1:1), and detection was made using UV light.
FT-IR spectra were recorded using a Perkin Elmer 1600 series FTIR
spectrometer. 1H NMR and 13C NMR spectra were registered in
DMSO-d6 on a BRUKER AVENE II 400 MHz NMR Spectrometer
(400.13 MHz for 1H and 100.62 MHz for 13C) or Varian-Mercury
200 MHz NMR Spectrometer (200 MHz for 1H and 50 MHz for
13C). The chemical shifts are given in ppm relative to Me4Si as an
internal reference, J values are given in Hz. The elemental analysis
was performed on a Costech Elemental Combustion System CHNSeO
elemental analyzer. All the compounds gave C, H and N analysis
within ꢂ0.4% of the theoretical values. The Mass spectra were ob-
tained on a Quattro EI-MS (70 eV) Instrument.
250e500 mg/mL.
Among the imine compounds (20e22), compounds 20 and 21,
which contain a 2-hydroxyphenylmethylene or 4-(hydroxy-3-
methoxyphenyl)methylene moiety in the 4-(2-methoxyphenyl)
piperazin-1-yl]acetohydrazide skeleton, displayed good-moderate
activity against the test microorganisms with the MIC values be-
tween 250 and 31.5 mg/mL.
3.1.1. 1-Ethyl-6-fluoro-7-[4-(2-fluoro-4-nitrophenyl)piperazin-1-
yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (1)
According to the results obtained, it can be concluded that the
conversion of norfloxacin to the derivatives which contain a bulky
group in the position 7 (compounds 23e27) resulted in completely
inactivation of these compounds against the test microorganisms.
3.1.1.1. Method 1. The solution of norfloxacin (10 mmol) and 3,4-
difluoronitrobenzene (50 mmol) in acetonitrile was refluxed in
the presence of NaHCO3 (30 mmol) for 5 h. Then, the mixture was
poured into ice water. The yellow precipitated product was filtered
off and recrystallized from dimethyl sulfoxide. Yield 86%, m.p.
314 ꢁC.
2.2.2. Antiurease activity
The synthesized compounds were assayed for their in vitro
inhibitory activity against Jack bean urease. Three of those com-
pounds showed perfect urease inhibition. Thiourea with IC50 value
3.1.1.2. Method 2. The mixture of norfloxacin (10 mmol), 3,4-
difluoronitrobenzene (50 mmol) and NaHCO3 (30 mmol) was
irradiated in closed vessels with the pressure control at 100 ꢁC for
51.62 ꢂ 7.28
mg/mL was used as standard inhibitor. Among tested
compounds 25 was found to be the best inhibitory effect against
Table 1
Screening for antimicrobial activity of the compounds (
mg/mL).
Compound no
Microorganismsa and minimal inhibition concentration
Ec
Yp
Pa
Sa
Ef
Bc
Ms
Ca
Sc
1
2
3
4
5
6
7
8
31.5
<1.9
0.21
<1.9
250
500
3.4
31.5
<1.9
1.7
<1.9
e
e
e
e
e
e
e
e
7.81
15.6
31.5
e
<1.9
0.21
<1.9
500
e
<1.9
0.21
<1.9
500
e
<1.9
0.21
<1.9
500
e
1.95
0.42
0.97
e
e
e
e
e
e
e
500
250
e
250
250
e
e
e
e
62.5
62.5
e
500
e
15.6
125
250
e
31.25
125
250
250
e
125
125
250
e
e
15.6
125
e
62.5
e
250
250
250
e
250
250
250
e
9
e
10
11
20
21
Amp.
Srp.
Flu
e
e
e
e
e
e
e
1000
31.3
31.3
15
e
31.3
31.3
10
31.3
31.3
18
62.5
31.3
>128
31.3
31.3
35
15.6
15.6
10
31.3
31.3
250
62.5
62.5
31.3
4
<8
<8
a
Ec: Escherichia coli ATCC 25922, Yp: Yersinia pseudotuberculosis ATCC 911, Pa: Pseudomonas aeruginosa ATCC 43288, Sa: Staphylococcus aureus ATCC 25923, Ef: Enterococcus
faecalis ATCC 29212, Bc: Bacillus cereus 702 Roma, Ms: Mycobacterium smegmatis ATCC607, Ca: Candida albicans ATCC 60193, Sc: Saccharomyces cerevisiae RSKK 251, Amp.:
Ampicillin, Strep.: Streptomycin, Flu.: Fluconazole, (ꢀ): no activity.

236
M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
Table 2
maximum power (The progress of the reaction was monitored by
TLC). Then, the catalyst was separated by filtration and washed by
hot DMF. The combined filtrate poured into ice water The obtained
orange solid was recrystallized from dimethylformamide:water
(1:5). Yield 70%, m.p. 257 ꢁC. FT-IR (nmax, cmꢀ1): 3433 and 3332
(NH2), 3051 (areCH), 2955 and 2853 (aliphatic CeH), 1702 and
1627 (2C]O), 1254 (CeO). Elemental analysis for C22H22F2N4O3,
calculated (%), C, 61.68; H, 5.18; N, 13.08, found (%), C, 61.58; H, 5.21;
Inhibitory activities and IC50 values of the synthesized compounds against Jack Bean
urease.
Compound no
% Inhibition
100 g/mL
IC50 (mg/mL)
m
25
Thiourea
86.2%
92.2%
20.51 ꢂ 9.09
51.62 ꢂ 7.28
N, 13.15. 1H NMR (DMSO-d6,
d ppm): 1.41 (s, 3H, CH3), 3.03 (s, 4H,
2CH2), 3.42 (s, 4H, 2CH2), 4.58 (d, 2H, CH2, J ¼ 6.0 Hz), 5.03 (s, 2H,
NH2, exch. D2O), 6.33e6.39 (m, 2H, arH), 6.84 (t, 1H, arH), 7.21 (d,
1H, arH), 7.90 (d, 1H, arH, J ¼ 12.0 Hz), 8.94 (s, 1H, olefiniceCH),
4 min (hold time) at 200 W maximum power. After the completion
of the reaction, (monitored by LC), the mixture was poured into ice
water. The precipitate was collected by filtration and recrystallized
from dimethyl sulfoxide. Yield 90%, m.p. 314 ꢁC, FT-IR (nmax, cmꢀ1):
3051 (areCH), 1732 and 1615 (2C]O), 1475 and 1335 (NO2), 1237
(CeO). Elemental analysis for C22H20F2N4O5, calculated (%), C,
57.64; H, 4.40; N, 12.22, found (%), C, 57.79; H, 4.42; N, 12.03. 1H
15.30 (s, 1H, OH, exch. D2O). 13C NMR (DMSO-d6,
d ppm): 15.10
(CH3), 49.79 (CH2), 50.58 (2CH2), 51.75 (2CH2), arC: [106.80 (CH),
107.77 (C), 110.26 (CH), 112.02 (CH), 117.19 (CH), 119.36 (C), 119.66
(CH),120.12 (C),120.54 (C),137.85 (2C),145.78 (C),146.35 (C),150.51
(CH)], 166.85 and 176.90 (2C]O). EI MS m/z (%): 451.51 ([M þ Na]þ,
15), 429.54 ([M þ 1]þ, 28), 380.67 (56), 246.33 (43), 149.16 (100),
138.21 (98), 121.19 (68).
NMR (DMSO-d6,
4CH2), 4.61 (q, 2H, CH2, J ¼ 6.0 Hz), 7.26 (bs, 2H, arH), 7.93e8.10 (m,
3H, arH), 8.97 (s, 1H, olefiniceCH), 15.29 (s, 1H, OH, exch. D2O). 13
NMR (DMSO-d6, ppm): The spectrum could not be obtained, due
d
ppm): 1.43 (t, 3H, CH3, J ¼ 6.0 Hz), 3.56 (s, 8H,
C
3.1.3. 7-[4-(Chloroacetyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-oxo-
1,4-dihydro quinoline-3-carboxylic acid (3)
d
to the slight solubility in any NMR solvent. EI MS m/z (%): 481.42
([M þ Na]þ, 42), 460.39 ([M þ 2]þ, 25), 459.45 ([M þ 1]þ, 100),
441.43 (18), 213.17 (31), 183.07 (22), 155.91 (25), 153.85 (50), 148.97
(56), 134.95 (31), 118.93 (81), 105.98 (84).
Chloroethanoylchloride (15 mmol) was added to the mixture of
compound 2 (10 mmol) and triethylamine (30 mmol) in THF cooled
to ꢀ5 ꢁC drop wise over a 2-hour period. Then, the temperature was
allowed to reach to room temperature and the mixture was stirred
for 4 h. The precipitate was filtered off and washed by water. The
resulting white oily product was recrystallized form dimethyl
sulfoxide to afford the desired compound. Yield 90%, m.p. 241e
242ꢁ, FT-IR (nmax, cmꢀ1): 3055 (areCH), 2984 (aliphatic CeH), 1713,
3.1.2. 7-[4-(4-Amino-2-fluorophenyl)piperazin-1-yl]-1-ethyl-6-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2)
3.1.2.1. Method 1. Pd/C (5 mmol) catalyst was added to the solution
of compound 1 (10 mmol) in n-butanol, and the mixture was
refluxed in the presence of hydrazine hydrate (50 mmol) for 20 h
(The progress of the reaction was monitored by TLC). Then, the
catalyst was separated by filtration and washed with hot dime-
thylformamide. The combined filtrate poured into ice water and an
orange solid was obtained. This was recrystallized from dime-
thylformamide:water (1:5). Yield 30%, m.p. 257 ꢁC.
1656 and 1623 (C]O). 1H NMR (DMSO-d6)
d ppm: 1.42 (t, 3H, CH3,
J ¼ 8.0 Hz), 3.35e3.38 (m, 4H, 2CH2), 3.58e3.70 (m, 4H, 2CH2), 4.48
(s, 2H, CH2), 4.59 (q, 2H, CH2, J ¼ 8.0 Hz), 7.20 (d, 1H, arH, J ¼ 8.0 Hz),
7.92 (d, 1H, arH, J ¼ 12.0 Hz), 8.96 (s, 1H, olefiniceCH), 15.31 (s, 1H,
OH, exch. D2O). 13C NMR (DMSO-d6)
d ppm: 15.06 (CH3), 42.03
(CH2), 42.54 (2CH2), 45.71 (CH2), 49.76 (CH2), 50.14 (CH2), arC:
[106.95 (d, CH, J ¼ 2.0 Hz), 107.78 (C), 111.92 (d, CH, J ¼ 22.0 Hz),
120.35 (d, C, J ¼ 7.0 Hz), 137.80 (C), 145.76 (d, C, J ¼ 9.0 Hz), 149.29
(CH), 153.49 (d, C, J ¼ 248.0 Hz)], 165.51 (C]O), 166.85 (C]O),
176.90 (C]O). EI MS m/z (%): 418.08 ([M]þ, 100), 381.29 (48), 342.11
(24).
3.1.2.2. Method 2. Pd/C (5 mmol) catalyst and hydrazine hydrate
were added to the solution of compound 1 (10 mmol) in n-butanol,
and the reaction mixture was irradiated in closed vessels with the
pressure control at 150 ꢁC for 50 min (hold time) at 200 W
3.1.4. 7-[4-(4-{[(Benzylamino)carbonothioyl]amino}-2-
fluorophenyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (4)
120
3.1.4.1. Method 1. The solution of compound 2 (10 mmol) in abso-
lute ethanol was refluxed with benzylisothiocyanate (20 mmol) for
40 h. On cooling the reaction mixture to room temperature, a solid
formed. This crude product was collected by filtration and recrys-
tallized from acetone. Yield 84%, m.p. 199e200 ꢁC.
Compound 25
Thiourea
100
80
60
40
20
0
3.1.4.2. Method 2. The mixture of compound 2 (10 mmol) and
benzylisothiocyanate (20 mmol) in absolute ethanol was irradiated
in open vessels under reflux condition at 78 ꢁC for 1 h (hold time) at
200 W maximum power. The white solid formed was recrystallized
from acetone. Yield 90%, m.p. 199e200 ꢁC. FT-IR (nmax, cmꢀ1): 3248
and 3186 (NH), 3045 (areCH), 2949 and 2847 (aliphatic CeH), 1731
and 1615 (C]O), 1245 (CeO and C]S). Elemental analysis for
C
30H29F2N5O3S, calculated (%), C, 62.38; H, 5.06; N, 12.12, found (%),
C, 62.18; H, 5.19; N, 12.12. 1H NMR (DMSO-d6,
d
ppm): 1.42 (s, 3H,
1
10
100
Concentration (µg/mL)
CH3), 3.03 (s, 2H, CH2), 3.20 (s, 2H, CH2), 3.37 (s, 4H, 2CH2 þ H2O),
4.60e4.73 (m, 4H, 2CH2), 7.08 (s, 2H, CH), 7.23e7.43 (m, 7H, arH),
7.92e7.92 (d, 1H, arH, J ¼ 7.0 Hz), 8.20 (s, H, NH, exch. D2O), 8.96 (s,
1H, olefiniceCH), 9.61 (s, H, NH, exch. D2O), 15.33 (s, 1H, OH, exch.
Fig. 1. Dose-dependent inhibitory effect of compound 25. Thiourea was used as
standard inhibitor. Inhibitory effect of compound 25 and Thiourea were measured at
mg/mL concentrations. Residual activities of compounds are
expressed as the mean ꢂ S.D. in triplicate.
the range of 250 to 0.114
D2O). 13C NMR (DMSO-d6,
d ppm): 15.10 (CH3), 49.79 (CH2), 50.57

M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
237
(CH2), 50.80 (2CH2), 51.70 (2CH2), arC: [102.74 (CH), 106.73 (CH),
107.74 (C), 110.27 (CH), 111.84 (d, CH J ¼ 23.5 Hz), 120.09 (C), 121.52
(CH), 127.58 (CH), 128.00 (CH), 128.09 (CH), 128.97 (2CH), 129.37 (d,
C, J ¼ 9.5 Hz), 137.85 (C), 139.66 (C), 146.08 (d, C, J ¼ 6.0 Hz), 146.44
(d, C, J ¼ 10.0 Hz), 149.20 (CH), 153.65 (d, C, J ¼ 251.5 Hz), 155.85 (d,
C, J ¼ 365.0 Hz)], 166.85 and 176.86 (2C]O), 181.40 (C]S). EI MS m/
z (%): 609.81 (100), 578.20 ([M þ 1]þ, 55), 517.17 (75), 101.00 (70).
(d, 1H, arH, J ¼ 14.0 Hz), 8.95 (s, 1H, olefiniceCH), 9.41 (s, 1H, NH,
exch. D2O),15.33 (s,1H, OH, exch. D2O). 13C NMR (DMSO-d6,
ppm):
d
14.14 (CH3), 14.38 (CH3), 38.65e40.10 (DMSO þ CH2), 49.05 (CH2),
50.09 (CH2), arC: [106.09 (CH), 107.07 (C), 111.00 (CH), 111.22 (CH),
119.17 (CH), 119.38 (CH), 119.45 (C), 134.38 (C), 135.81 (d, C,
J ¼ 9.3 Hz), 137.10 (C), 145.36 (d, C, J ¼ 10.4 Hz), 148.43 (CH), 152.88
(d, C, J ¼ 248.0 Hz), 154.26 (d, C, J ¼ 242.0 Hz)], 166.07 and 176.09
(2C]O),179.97 (C]S). EI MS m/z (%): 538.10 ([M þ Na]þ, 30), 516.12
([M þ 1]þ, 100), 389.16 (46), 102.10 (57).
3.1.5. 7-[4-(4-{[(Benzylamino)carbonyl]amino}-2-fluorophenyl)
piperazin-1-yl]-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (5)
3.1.5.1. Method 1. The solution of compound (2) (10 mmol) in ab-
solute ethanol was refluxed with benzylisocyanate (20 mmol) for
48 h. On cooling the reaction mixture to room temperature, a solid
was formed. This crude product was collected by filtration and
recrystallized from dimethyl sulfoxide:water (1:5). Yield 82%, m.p.
319e320 ꢁC.
3.1.7. 1-Ethyl-6-fluoro-7-{4-[2-fluoro-4-({[(3-morpholin-4-
ylpropyl)amino]carbonothioyl}amino)phenyl]piperazin-1-yl}-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (7)
3.1.7.1. Method 1. The solution of compound 2 (10 mmol) in abso-
lute ethanol was refluxed with 4-morpholinopropylisothiocyanate
(20 mmol) for 80 h. On cooling the reaction mixture to room
temperature, a solid formed. This crude product was collected by
filtration and recrystallized from acetone. Yield 70%, m.p. 215e
216 ꢁC.
3.1.5.2. Method 2. The mixture of compound 2 (10 mmol) and
benzylisocyanate (20 mmol) in absolute ethanol was irradiated in
open vessels under reflux condition at 78 ꢁC for 1 h (hold time) at
200 W maximum power. The yellow solid formed was recrystal-
lized from dimethyl sulfoxide:water (1:5). Yield 95%, m.p. 319e
320 ꢁC. FT-IR (nmax, cmꢀ1): 3325 and 3292 (NH), 3047 (areCH),
2956 and 2839 (aliphatic CeH), 1730 and 1616 (C]O), 1245 (CeO),
1223 (CeO). Elemental analysis for C30H29F2N5O4, calculated (%), C,
64.16; H, 5.21; N, 12.47, found (%), C, 64.32; H, 5.01; N, 12.56. 1H
3.1.7.2. Method 2. The mixture of compound 2 (10 mmol) and 4-
morpholinopropylisothiocyanate (20 mmol) in absolute ethanol
was irradiated in open vessels under reflux condition at 76e78 ꢁC
for 1 h (hold time) at 200 W maximum power. The yellow solid
formed was recrystallized from acetone. Yield 70%, m.p. 215e
216 ꢁC. FT-IR (nmax, cmꢀ1): 3342 and 3169 (NH), 3048 (areCH), 2936
(aliphatic CeH), 1727 and 1614 (2C]O), 1246 (CeO). Elemental
analysis for C30H36F2N6O4S, calculated (%), C, 58.62; H, 5.90; N,
13.67, found (%), C, 58.32; H, 5.72; N, 13.70. 1H NMR (DMSO-d6,
NMR (DMSO-d6,
d
ppm): 1.40 (t, 3H, CH3, J ¼ 6.0 Hz), 3.11 (s, 4H,
2CH2), 3.55 (s, 4H, 2CH2), 4.22 (d, 2H, CH2, J ¼ 4.0 Hz), 4.60 (q, 2H,
CH2, J ¼ 6.0 Hz), 6.33 (s, H, NH, exch. D2O), 6.99 (s, 2H, arH), 7.24e
7.29 (m, 6H, arH), 7.44 (d, 1H, arH, J ¼ 15.2 Hz), 7.87 (d, 1H, arH,
J ¼ 13.4 Hz), 8.63 (s, 1H, NH, exch. D2O), 8.93 (s, 1H, olefiniceCH),
d
ppm): 1.43 (t, 3H, CH3, J ¼ 8.3 Hz), 1.72 (s, 2H, CH2), 3.21 (s, 4H,
2CH2), 3.21 (s, 2H, CH2), 3.34 (s, 2H, CH2), 3.50 (d, 12H, 6CH2), 4.62
(q, 2H, CH2 J ¼ 8.3 Hz), 7.07e7.11 (m, 1H, CH), 7.27 (s, 1H, CH), 7.36
(d, 1H, arH, J ¼ 12.0 Hz), 7.80 (bs, 1H, NH, exch. D2O), 7.96 (d, 1H,
arH, J ¼ 16.0 Hz), 8.04e8.09 (m, 1H, CH), 8.98 (s, 1H, olefiniceCH),
9.53 (bs, 1H, NH, exch. D2O), 15.36 (s, 1H, OH, exch. D2O). 13C NMR
15.36 (s, 1H, OH, exch. D2O). 13C NMR (DMSO-d6,
d ppm): 14.36
(CH3), 42.70 (CH2), 49.05 (CH2), 49.68 (2CH2), 50.39 (2CH2), arC:
[106.02 (d, CH, J ¼ 14.6 Hz), 106.20 (CH), 107.08 (C), 111.14 (d, C,
J ¼ 23.3 Hz), 113.53 (CH), 119.37 (CH), 126.68 (CH), 127.00 (2CH),
128.34 (2CH), 133.13 (d, C, J ¼ 9.4 Hz), 136.27 (d, C, J ¼ 11.0 Hz),
137.15 (C), 140.26 (C), 145.42 (d, C, J ¼ 11.1 Hz), 148.49 (CH), 152.91
(d, C, J ¼ 248.0 Hz), 154.95 (d, C, J ¼ 241.0 Hz)], 155.11, 166.08 and
176.12 (3C]O). EI MS m/z (%): 561.215 ([M þ 1]þ, 100), 471.42 (50),
328.16 (75), 150.07 (33).
(DMSO-d6,
d ppm): 14.38 (CH3), 24.92 (CH2), 42.43 (CH2), 48.87
(CH2), 49.05 (CH2), 49.61 (CH2), 50.08 (CH2), 53.08 (2CH2), 55.78
(CH2), 65.81 (2CH2), arC: [106.58 (d, CH, J ¼ 9.8 Hz), 107.06 (C),
111.11 (d, CH, J ¼ 23.0 Hz), 112.23 (d, CH, J ¼ 26.0 Hz), 118.68 (d, CH,
J ¼ 119.0 Hz), 119.42 (d, C, J ¼ 8.8 Hz), 121.24 (CH), 137.10 (C), 139.67
(d, C, J ¼ 8.6 Hz), 145.37 (d, C, J ¼ 10.2 Hz), 148.46 (CH), 152.83 (d, C,
J ¼ 248.0 Hz), 154.36 (d, C, J ¼ 230.0 Hz), 156.74 (C)], 166.06 and
176.08 (2C]O), 180.13 (C]S). EI MS m/z (%): 614.24 ([M þ 1]þ, 100),
586.21 (35), 381.48 (25), 203.10 (10).
3.1.6. 1-Ethyl-7-[4-(4-{[(ethylamino)carbonothioyl]amino}-2-
fluorophenyl)piperazin-1-yl]-6-fluoro-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid (6)
3.1.6.1. Method 1. The solution of compound 2 (10 mmol) in abso-
lute ethanol was refluxed with ethylisothiocyanate (20 mmol) for
65 h. On cooling the reaction mixture to room temperature, a solid
formed. This crude product was collected by filtration and recrys-
tallized from dimethyl sulfoxide:water (1:5). Yield 91%, m.p. 241e
242 ꢁC.
3.1.8. 7-[4-(4-{[3-Benzyl-4-oxo-1,3-thiazolidin-2-ylidene]amino}-
2-fluorophenyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (8)
3.1.8.1. Method 1. The mixture of compound 3 (10 mmol) and ethyl
bromoacetate (10 mmol) in glacial acetic acid was irradiated in
closed vessels with the pressure control at 150 ꢁC for 75 min (hold
time) at 200 W maximum power in the presence of dried sodium
acetate (10 mmol). Then, the solution was poured into ice water and
a white solid formed. This was recrystallized from acetone. Yield
75%, m.p. 159e160 ꢁC.
3.1.6.2. Method 2. The mixture of compound 2 (10 mmol) and
ethylisothiocyanate (20 mmol) in absolute ethanol was irradiated
in open vessels under reflux condition at 78 ꢁC for 1 h (hold time) at
200 W maximum power. The yellow solid formed was recrystal-
lized from dimethyl sulfoxide:water (1:5). Yield 95%, m.p. 241e
242 ꢁC. FT-IR (nmax, cmꢀ1): 3352 and 3149 (NH), 3047 (areCH), 2985
and 2813 (aliphatic CeH), 1713 and 1626 (2C]O), 1263, 1248, 1228
and 1209 (CeO and C]S). Elemental analysis for C25H27F2N5O3S,
calculated (%), C, 58.24; H, 5.28; N, 13.58, found (%), C, 58.29; H,
3.1.8.2. Method 2. The mixture of compound 3 (10 mmol) and
dried sodium acetate (20 mmol) in glacial acetic acid was stirred at
room temperature for 15 min. After that ethyl bromoacetate
(15 mmol) was added into it and irradiated in closed vessels with
the pressure control at 160 ꢁC for 60 min (hold time) at 200 W
maximum power. Then, the solution was poured into ice water and
a white solid formed. This was recrystallized from acetone. Yield
90%, m.p. 159e160 ꢁC. FT-IR (nmax, cmꢀ1): 3060 (areCH), 2927 and
2834 (aliphatic CeH), 1720, 1624 and 1612 (3C]O), 1247 (CeO).
5.21; N, 13.56. 1H NMR (DMSO-d6,
d ppm): 1.09 (t, 3H, CH3,
J ¼ 7.0 Hz), 1.42 (s, 3H, CH3), 3.19 (s, 4H, 2CH2), 3.36 (s, 2H,
CH2 þ H2O), 3.45 (s, 4H, 2CH2), 4.61 (s, 2H, CH2), 7.04 (s, 2H, arH),
7.32e7.38 (m, 2H, arH þ NH, exch. D2O), 7.75 (s, 1H, arH), 7.92

238
M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
Elemental analysis for C32H29F2N5O4S, calculated (%), C, 62.23; H,
4.73; N, 11.34, found (%), C, 62.18; H, 4.89; N, 11.21. 1H NMR (DMSO-
maximum power. The solution poured into ice water. The white
solid formed and recrystallized from acetone. Yield 93%, m.p. 226e
227 ꢁC. FT-IR (nmax, cmꢀ1): 3098 and 3060 (areCH), 2980 and 2830
(aliphatic CeH), 1713, 1624 and 1625 (2C]O), 1245 (CeO).
Elemental analysis for C38H32ClF2N5O3S, calculated (%), C, 64.08; H,
4.53; N, 9.83, found (%), C, 64.36; H, 4.63; N, 9.69. 1H NMR (DMSO-
d6,
d ppm): 1.42 (s, 3H, CH3), 3.20 (s, 4H, 2CH2), 3.47 (s, 4H, 2CH2),
4.11 (s, 2H, CH2), 4.62 (s, 2H, CH2), 4.90 (s, 2H, CH2), 6.73e6.80 (m,
2H, arH), 7.10 (t, 1H, arH), 7.20e7.34 (m, 6H, arH), 7.92 (d, 1H, arH,
J ¼ 14.0 Hz), 8.95 (s, 1H, olefiniceCH), 15.35 (s, 1H, OH, exch. D2O).
13C NMR (DMSO-d6,
d
ppm): 14.37 (CH3), 32.54 (CH2), 45.37 (CH2),
d6, d ppm): 1.43 (s, 3H, CH3), 3.20 (s, 4H, 2CH2), 3.48 (s, 4H, 2CH2),
49.05 (CH2), 49.58 (CH2), 50.12 (2CH2), arC: [106.13 (CH), 107.09 (C),
109.34 (d, C, J ¼ 22.0 Hz), 116.99 (C), 119.41 (C), 120.02 (CH), 127.42
(CH), 128.10 (2CH), 128.97 (2CH), 135.91 (C), 136.04 (d, C,
J ¼ 8.0 Hz), 137.16 (C), 142.96 (C), 148.52 (CH), 153.61 (d, C,
J ¼ 387.0 Hz), 155.12 (d, C, J ¼ 227.0 Hz)], 166.07, 171.91 and 176.14
(3C]O). EI MS m/z (%): 640.12 ([M þ Na]þ, 19), 618.14 ([M þ 1]þ,
100), 389.17 (35), 212.08 (10).
4.60 (s, 2H, CH2), 5.04 (s, 2H, CH2), 6.37 (s, 1H, CH), 6.77e7.48 (m,
13H, arH), 7.94 (d, 1H, arH, J ¼ 12.0 Hz), 8.94 (s, 1H, olefiniceCH),
15.34 (bs, 1H, OH). 13C NMR (DMSO-d6,
d ppm): 15.09 (CH3), 48.43
(CH2), 49.67 (CH2), 50.38 (2CH2), 51.01 (2CH2), 98.06 (CH), arC:
[106.79 (CH), 109.78 (d, CH, J ¼ 21.0 Hz), 111.89 (d, CH, J ¼ 22.0 Hz),
117.47 (CH), 120.38 (C), 120.95 (CH), 127.14 (2CH), 127.76 (CH),
129.12 (2CH), 129.39 (2CH), 130.42 (C), 131.16 (2CH), 134.69 (2C),
135.29 (d, C, J ¼ 9.0 Hz), 137.77 (2C), 139.21 (C), 146.48 (d, C,
J ¼ 102.0 Hz), 147.07 (C), 149.17 (CH), 153.67 (d, C, J ¼ 264.0 Hz),
156.13 (d, C, J ¼ 262.0 Hz)], 159.82 (C]N), 166.86 and 176.75 (2C]
O). EI MS m/z (%): 712.16 ([M þ 1]þ, 100), 609.78 (20), 242.27 (13),
100.99 (13).
3.1.9. 1-Ethyl-7-[4-(4-{3-ethyl-4-oxo-1,3-thiazolidin-2-ylidene]
amino}-2-fluoro phenyl)piperazin-1-yl]-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (9)
3.1.9.1. Method 1. The mixture of compound 5 (10 mmol) and ethyl
bromoacetate (15 mmol) in glacial acetic acid was refluxed in the
presence of dried sodium acetate (20 mmol) for 60 h. The solution
poured into ice water. The white solid formed recrystallized from
acetone. Yield 77%, m.p. 260e261 ꢁC.
3.1.11. 7-[4-(4-{[5-(4-Chlorophenyl)-3-ethyl-1,3-thiazol-2(3H)-
ylidene]amino}-2-fluorophenyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (11)
3.1.11.1. Method 1. The mixture of compound 5 (10 mmol) and 4-
chlorophenacylbromide (10 mmol) in dry chloroform was
refluxed in the presence of dried sodium acetate (50 mmol) for
58 h. Then, the reaction mixture was allowed to reach room
temperature and the precipitated salt was separated by filtration.
After removing the solvent under reduced pressure, an oily
product was recrystallized from acetone. Yield 70%, m.p. 280e
281 ꢁC.
3.1.9.2. Method 2. The mixture of compound 5 (15 mmol) and dried
sodium acetate (20 mmol) in glacial acetic acid was stirred at room
temperature for 15 min. After that ethyl bromoacetate (15 mmol)
was added and irradiated in closed vessels with the pressure con-
trol at 160 ꢁC for 60 min (hold time) at 200 W maximum power. The
solution poured into ice water. The white solid formed recrystal-
lized from acetone. Yield 95%, m.p. 260e261 ꢁC. FT-IR (nmax, cmꢀ1):
3053 (areCH), 2961 and 2845 (aliphatic CeH), 1728 and 1625 (3C]
O), 1249 (CeO). Elemental analysis for C27H27F2N5O4S, calculated
(%), C, 58.37; H, 4.90; N, 12.61, found (%), C, 58.46; H, 4.98; N, 12.55.
3.1.11.2. Method 2. The mixture of compound 5 (15 mmol) and
dried sodium acetate (20 mmol) in glacial acetic acid was stirred at
room temperature for 15 min. After that 4-chlorophenacylbromide
(15 mmol) was added and irradiated in closed vessels with the
pressure control at 160 ꢁC for 60 min (hold time) at 200 W
maximum power. The solution poured into ice water. The white
solid formed and recrystallized from acetone. Yield 90%, m.p. 280e
281 ꢁC. FT-IR (nmax, cmꢀ1): 3044 (areCH), 2921 and 2849 (aliphatic
CeH), 1731 and 1614 (C]O), 1245 (CeO). Elemental analysis for
1H NMR (DMSO-d6,
d
ppm): 1.16 (t, 3H, CH3, J ¼ 4.0 Hz), 1.42 (s, 3H,
CH3), 3.20 (s, 4H, 2CH2), 3.46 (s, 4H, 2CH2), 3.72 (q, 3H, CH3,
J ¼ 4.0 Hz), 4.02 (s, 2H, CH2), 4.62 (s, 2H, CH2), 6.77e6.84 (m, 1H,
arH), 7.11e7.26 (m, 3H, arH), 7.91 (d, 1H, arH, J ¼ 14.0 Hz), 8.96 (s,
1H, olefiniceCH), 15.36 (s, 1H, OH, exch. D2O). 13C NMR (DMSO-d6,
d
ppm): 12.23 (CH3), 14.38 (CH3), 32.55 (CH2), 37.39 (CH2), 49.05
(CH2), 49.59 (2CH2), 50.13 (2CH2), arC: [106.12 (CH), 107.08 (C),
109.39 (d, CH J ¼ 21.6 Hz), 111.15 (d, CH, J ¼ 22.3 Hz), 117.06 (CH),
119.43 (d, C, J ¼ 8.2 Hz), 119.95 (CH), 135.85 (d, C, J ¼ 8.2 Hz), 137.15
(C), 143.25 (d, C, J ¼ 9.0 Hz), 145.37 (d, C, J ¼ 11.5 Hz), 148.49 (CH),
153.55 (d, C, J ¼ 379.0 Hz),154.14 (C]N),155.04 (d, C, J ¼ 245.0 Hz)],
166.06, 171.62 and 176.14 (3C]O). EI MS m/z (%): 578.09
([M þ Na]þ, 20), 556.11 ([M þ 1]þ, 65), 389.16 (100), 242.24 (25),
212.07 (30), 102.10 (72).
C
33H30ClF2N5O3S, calculated (%), C, 60.96; H, 4.65; N, 10.77, found
(%), C, 61.06; H, 4.78; N, 10.57. 1H NMR (DMSO-d6,
d
ppm): 1.08 (s,
3H, CH3), 1.42 (s, 3H, CH3), 3.21 (s, 4H, 2CH2), 3.48 (s, 4H, 2CH2), 3.77
(s, 2H, CH2), 4.63 (s, 2H, CH2), 6.28 (s, 1H, CH), 6.70e6.86 (m, 2H,
arH), 7.10 (s, 1H, arH), 7.28 (s, 1H, arH), 7.55 (s, 4H, arH), 7.95 (d, 1H,
arH, J ¼ 14.0 Hz), 8.97 (s, 1H, olefiniceCH), 15.32 (s, 1H, OH, exch.
D2O). 13C NMR (DMSO-d6,
d ppm): 13.11 (CH3), 14.39 (CH3), 30.66
(CH), 38.84e40.09 (DMSO þ CH2), 49.06 (CH2), 49.65 (CH2), 49.70
(CH2), 50.29 (2CH2), arC: [106.17 (CH), 107.07 (C), 109.16 (d, CH,
J ¼ 21.0 Hz), 111.16 (d, CH, J ¼ 23.0 Hz), 116.85 (CH), 119.40 (C),
120.21 (CH), 128.86 (2CH), 129.94 (C), 130.62 (2 CH), 134.03 (2C),
134.41 (C), 137.16 (C), 138.35 (C), 145.44 (d, C, J ¼ 10.0 Hz), 148.54
(CH), 154.21 (d, C, J ¼ 262.0 Hz), 154.17 (C), 156.74 (C]N)], 166.10
(C]O), 176.16 (C]O). EI MS m/z (%): 649. 17 ([M þ 1]þ, 100), 621.14
(60), 416.91 (44), 238.72 (25).
3.1.10. 7-[4-(4-{[3-Benzyl-5-(4-chlorophenyl)-1,3-thiazol-2(3H)-
ylidene]amino}-2-fluorophenyl)piperazin-1-yl]-1-ethyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (10)
3.1.10.1. Method 1. The mixture of compound (3) (10 mmol) and 4-
chlorophenacylbromide (10 mmol) in dry chloroform was refluxed
in the presence of dried sodium acetate (50 mmol) for 40 h. Then,
the reaction mixture was allowed to reach room temperature and
the precipitated salt was separated by filtration. After removing the
solvent under reduced pressure, an oily product was recrystallized
from acetone. Yield 93%, m.p. 226e227 ꢁC.
3.1.12. 1-(2-Fluoro-4-nitrophenyl)-4-(2-methoxyphenyl)piperazine
(13)
The mixture of 1-(2-methoxyphenyl)piperazine (12) (20 mmol)
and 3,4-difluoronitrobenzene (10 mmol) in tetrahydrofuran was
stirred at room temperature for 1 h. Then, ice water was poured
into the reaction mixture and was stirred for 10 min. The red solid
formed and recrystallized from ethyl acetate. Yield 61%, m.p. 101e
102 ꢁC. FT-IR (nmax, cmꢀ1): 3193 (areCH and NH), 1247 (OeC).
3.1.10.2. Method 2. The mixture of compound 3 (10 mmol) and
dried sodium acetate (20 mmol) in glacial acetic acid was stirred at
room temperature for 15 min. After that 4-chlorophenacylbromide
(15 mmol) was added and irradiated in closed vessels with the
pressure control at 160 ꢁC for 60 min (hold time) at 200 W

M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
239
Elemental analysis for C17H18N3O3F, calculated (%), C, 61.62; H, 5.48;
J ¼ 7.5 Hz), 10.04 (s, 1H, NH, exch. D2O). 13C NMR (DMSO-d6,
d
ppm):
N, 12.68, found (%), C, 61.55; H, 5.52; N, 12.61. 1H NMR (DMSO-d6,
50.76 (CH2), 50.92 (CH2), 53.69 (2CH2), 55.96 (OCH3), 58.34 (CH2),
arC: [112.47 (CH), 118.65 (CH), 121.48 (CH), 123.18 (CH), 141.85 (C),
152.62 (CeOCH3)], 171.48 (C]O).
d
ppm): 3.11 (s, 4H, 2CH2), 3.41 (s, 4H, 2CH2), 3.79 (s, 3H, CH3), 6.92
(d, 4H, arH, J ¼ 9.0 Hz), 7.21 (t, 1H, arH, J ¼ 9.0 Hz), 8.05 (2H, d, arH,
10.2 Hz). 13C NMR (DMSO-d6,
ppm): 38.91e40.99
J
¼
d
(DMSO þ CH2), 41.41 (CH2), 50.08 (CH2), 50.17 (CH2), 50.57 (CH2),
56.00 (OCH3), arC: [112.50 (CH), 113.17 (CH), 118.73 (CH), 118.82
(CH), 121.52 (CH), 121.98 (CH), 123.57 (CH), 140.01 (C), 141.34 (C),
146.15 (C), 150.34 (CeF), 155.26 (CeOCH3)].
3.1.16. {[4-(2-Metoxyphenyl)piperazin-1-yl]acetyl}-N-
phenylhydrazincarbothioamide (17)
Phenylisothiocyanate (10 mmol) was added to the solution of
compound 16 (10 mmol) in absolute ethanol and the mixture was
refluxed for 6 h. On cooling the mixture in cold overnight, a solid
obtained. This was recrystallized from ethanol to afford the
desired compound. Yield 51%, m.p. 177e180 ꢁC. FT-IR (nmax, cmꢀ1):
3562 and 3456 (3NH), 3158 (areCH), 1734 (C]O), 1227 (C]S).
Elemental analysis for C20H25N5O2S, calculated (%), C, 60.13; H,
6.31; N, 17.53, found (%), C, 60.29; H, 6.21; N, 17.68. 1H NMR
3.1.13. 3-Fluoro-4-[4-(2-methoxyphenyl)piperazin-1-yl]aniline (14)
PdeC (20 mmol) catalyst was added to the solution of com-
pound 13 (10 mmol) in n-butanol, and the mixture was allowed to
reflux in the presence of hydrazine hydrate (60 mmol) for 3 h (the
progress of the reaction was monitored by TLC). The catalyst was
removed by filtration. After evaporating the solvent under
reduced pressure, a white solid appeared. This crude product was
recrystallized from ethyl acetate to afford the desired compound.
Yield 65%, m.p. 78e79 ꢁC. FT-IR (nmax, cmꢀ1): 3306 and 3229 (NH
and NH2), 3066 (areCH), 1197 (OeC). Elemental analysis for
(DMSO-d6,
d ppm): 2.65 (s, 4H, 2CH2), 2.98 (s, 4H, 2CH2), 3.13 (s,
2H, CH2), 3.75 (s, 3H, OCH3), 6.89 (d, 4H, arH J ¼ 7.0 Hz), 7.16 (bs,
1H, arH), 7.33e7.41 (m, 4H, arH), 9.62 and 9.78 (bs, 3H, 3NH, exch.
D2O). 13C NMR (DMSO-d6,
d
ppm): 32.62e41.14 (DMSO þ CH2),
50.48 (CH2), 53.69 (CH2), 55.96 (OCH3), 56.63 (CH2), 60.43 (CH2),
arC: [112.43 (CH), 118.62 (CH), 121.52 (2CH), 123.22 (CH), 124.43
(CH), 128.90 (2CH), 139.53 (C), 141.75 (C), 152.58 (CeOCH3)],
171.39 (C]O), 181.14 (C]S). EI MS m/z (%): 120.81 (49), 135.89
(85), 149.90 (42), 150.78 (32), 161.92 (21), 163.98 (17), 189.95 (15),
204.96 (37), 254.96 (17), 279.18 (51), 339.18 (13), 384.29
C
17H20N3OF, calculated (%), C, 67.75; H, 6.69; N, 13.94, found (%), C,
67.77; H, 6.57; N, 13.89. 1H NMR (DMSO-d6,
d
ppm): 3.05 (s, 4H,
2CH2), 3.36 (s, 4H, 2CH2), 3.78 (s, 3H, CH3), 5.01 (s, 2H, NH2, exch.
D2O), 6.32 (brs, 2H, arH), 6.94 (brs, 5H, arH). 13C NMR (DMSO-d6,
d
ppm): 39.32e40.98 (DMSO þ CH2), 41.39 (CH2), 50.05 (CH2),
50.16 (CH2), 50.56 (CH2), 56.00 (OCH3), arC: [112.50 (CH), 112.64
(CH), 118.72 (CH), 118.81 (CH), 121.51 (CH), 121.96 (CH), 123.56
(CH), 140.00 (C), 141.18 (C), 146.02 (C), 152.66 (CeF), 155.22 (Ce
OCH3)].
(13), 385.29 (26), 423.40 ([M
þ
1
þ
Na]þ, 100), 424.40
([M þ 2 þ Na]þ, 37).
3.1.17. 5-{[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-4-phenyl-
4H-1,2,4-triazole-3-thiol (18)
3.1.14. Ethyl [4-(2-methoxyphenyl)piperazin-1-yl]acetate (15)
The solution of ethyl bromoacetate (10 mmol) in
tetrahydrofuran was added to the solution of 1-(2-methoxyphenyl)
piperazine (10 mmol) in tetrahydrofuran drop wise and the
mixture was stirred at room temperature in the presence of trie-
thylamine (10 mmol) for 2 h. The solid formed was removed by
filtration, and the solvent was evaporated under reduced pressure.
The oily crude product was recrystallized from ethanol to afford the
desired compound. Yield 99%, m.p. 66e68 ꢁC. FT-IR (nmax, cmꢀ1):
3062 (areCH), 1745 (C]O), 1197 (OeC). Elemental analysis for
A solution of compound 17 (10 mmol) in water was refluxed in
the presence of 2 N NaOH (20 mmol) for 1 h; then, the resulting
solution was cooled to room temperature and acidified to pH 4 with
37% HCl. The precipitate formed was filtered off, washed with water
and recrystallized from ethanol to afford the desired compound.
Yield 66%, m.p. 244e246 ꢁC. FT-IR (nmax, cmꢀ1): 3054 (areCH), 2829
(SH), 1593 (C]N). Elemental analysis for C20H23N5OS, calculated
(%), C, 62.97; H, 6.08; N, 18.36, found (%), C, 62.81; H, 6.28; N, 18.42.
1H NMR (DMSO-d6,
d ppm): 2.37 (s, 2H, CH2), 2.77 (s, 4H, 2CH2),
3.40 (s, 4H, 2CH2), 3.73 (s, 3H, OCH3), 6.80e6.93 (m, 4H, arH), 7.47
C
15H22N2O3, calculated (%), C, 64.73; H, 7.97; N, 10.06, found (%), C,
(bs, 5H, arH), 13.85 (s, 1H, SH, exch. D2O). 13C NMR (DMSO-d6,
64.59; H, 8.17; N, 10.26. 1H NMR (DMSO-d6,
d
ppm): 1.19 (t, 3H, CH3,
d ppm): 49.81 (2CH2), 51.63 (CH2), 52.18 (2CH2), 55.25 (CH3), arC:
J ¼ 7.0 Hz), 2.64 (s, 4H, 2CH2), 2.94 (s, 4H, 2CH2), 3.20 (s, 2H, CH2),
3.75 (s, 3H, OCH3), 4.10 (q, 2H, OCH2, J ¼ 7.0 Hz), 6.87e6.91 (m, 4H,
[111.85 (CH), 117.86 (CH), 120.74 (C), 122.39 (CH), 128.22 (2CH),
128.86 (2CH), 129.11 (CH), 134.00 (C), 140.98 (C), 149.23 (C)], 151.86
(triazole C-3), 168.14 (C]S).
arH). 13C NMR (DMSO-d6,
d ppm): 14.85 (CH3), 50.67 (2CH2), 52.92
(2CH2), 55.95 (OCH3), 59.20 (CH2), 60.54 (OCH2), arC: [112.48 (CH),
118.64 (CH), 121.48 (CH), 123.11 (CH), 141.85 (C), 152.63 (CeOCH3)],
170.61 (C]O); EI MS m/z (%): 106.01 (53), 149.97 (23), 151.0 (13),
205.1 (16), 279.3 ([M þ 1]þ, 100), 280.29 ([M þ 2]þ, 20), 301.29
([M þ Na]þ, 16), 365.5 (26).
3.1.18. {[4-(2-Methoxyphenyl)piperazin-1-yl]methyl}-1,3,4-
oxadiazol-2-thiol (19)
The solution of KOH (10 mmol) in water was added to the
solution of compound 16 in ethanol and the mixture was refluxed
7 h in the presence of CS2 (0.19 mL, 10 mmol). Then, it was cooled
to room temperature and acidified to pH 4 with 37% HCl. On
cooling the mixture in cold overnight, a solid obtained. This was
recrystallized from ethanol to give the target compound. Yield
22%, m.p. 125 ꢁC. FT-IR (nmax, cmꢀ1): 3050 (areCH), 2848 (SH),
1454 (C]N). Elemental analysis for C14H18N4O2S, calculated (%), C,
54.88; H, 5.92; N, 18.29, found (%), C, 54.71; H, 5.86; N, 18.40. 1H
3.1.15. [4-(2-Methoxyphenyl)piperazin-1-yl]acetohydrazide (16)
Hydrazine hydrate (30 mmol) was added to the solution of
compound 15 (10 mmol) in absolute ethanol and the mixture was
refluxed for 9 h. Then, the solvent was removed under reduced
pressure and the obtained oily mass was extracted with 5 mL of
dichloromethane three times. The combined organic layer was
dried on MgSO4 and the solvent was removed under reduced
pressure. The solid obtained was recrystallized from ethanol to give
the target compound. Yield 77%, m.p. 97e100 ꢁC. FT-IR (nmax, cmꢀ1):
3306 and 3229 (eNH and NH2), 3037 (areCH), 1645 (C]O).
Elemental analysis for C13H20N4O2, calculated (%), C, 59.07; H, 7.63;
N, 21.20, found (%), C, 59.30; H, 7.56; N, 21.15. 1H NMR (DMSO-d6,
NMR (DMSO-d6,
3.75 (s, 5H, OCH3 þ CH2), 6.86e6.91 (m, 4H, arH), 14.35 (bs, 1H,
SH, exch. D2O). 13C NMR (DMSO-d6, ppm): 50.62 (CH2), 51.93
d ppm): 2.63 (s, 4H, 2CH2), 2.95 (s, 4H, 2CH2),
d
(CH2), 52.92 (2CH2), 55.91 (OCH3), 55.98 (CH2), arC: [112.39 (CH),
118.68 (CH), 121.44 (CH), 123.26 (CH), 141.59 (C]N), 152.63 (Ce
OCH3)], 161.46 (triazole C), 178.73 (triazole CeS). EI MS m/z (%):
205.19 (46), 278.3 (13), 279.28 (30), 305.38 ([M ꢀ 1]þ, 100),
306.45 ([M]þ, 22).
d
ppm): 2.63 (s, 4H, 2CH2), 2.96 (s, 4H, 2CH2), 3.10 (s, 2H, NH2, exch.
D2O), 3.38 (s, 2H, CH2), 3.75 (s, 3H, OCH3), 6.91 (d, 4H, arH,

240
M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
3.1.19. N0-[(2-Hydroxyphenyl)methylen]-2-[4-(2-methoxyphenyl)
piperazin-1-yl]acetohydrazide (20)
dimethyl sulfoxide was stirred at 80 ꢁC in the presence of sodium
hydroxide (50 mmol) for 12 h (for 24), 48 h (for 23), 52 h (for 25),
62 h (for 26) or 73 h (for 27). Then, the reaction mixture was
allowed to reach room temperature and neutralized with dilute
HCl. The solution poured into ice water. The white solid formed was
recrystallized from acetone.
2-Hydroxybenzaldehyde (10 mmol) was added to the solution
of compound 16 (10 mmol) in ethanol, and the mixture was
refluxed in the presence of one drop of H2SO4 for 3 h. On cooling the
mixture in cold overnight, a solid formed. This was recrystallized
from ethanol to afford the desired compound. Yield 86%, m.p. 164e
165 ꢁC. FT-IR (nmax, cmꢀ1): 3169 (OH and NH), 3058 (areCH), 1447
(C]N), 1239 (OeC). Elemental analysis for C20H24N4O3, calculated
(%), C, 65.20; H, 6.57; N, 15.21, found (%), C, 65.40; H, 6.47; N, 15.36.
3.1.22.1. 1-Ethyl-6-fluoro-7-[4-({2-methoxy-4-[({[4-(2-
methoxyphenyl)piperazin-1-yl]acetyl}hydrazono)methyl]phenoxy}
acetyl)piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(23). Yield 57%, m.p. 195e197 ꢁC. FT-IR (nmax, cmꢀ1): 3426 (OH),
3056 (areCH), 1725 and 1699 (4C]O). Elemental analysis for
1H NMR (DMSO-d6,
d ppm): 2.81 (s, 4H, 2CH2), 3.06 (s, 4H, 2CH2),
3.77 (s, 5H, OCH3 þ CH2), 6.90e7.49 (m, 6H, arH), 7.26 (bs, 1H, arH),
7.51 and 7.71 (s, 1H, arH, cis/trans conformers), 8.36 and 8.54 (1H,
N]CH, cis/trans conformers), 10.15 and 11.10 (1H, brs, OH, cis/trans
conformers exch. D2O), 11.63 and 11.85 (1H, s, NH, cis/trans con-
C
39H44FN7O8, calculated (%), C, 61.81; H, 5.85; N, 12.94, found (%), C,
61.68; H, 5.92; N, 12.86. 1H NMR (DMSO-d6,
d
ppm): 1.42 (t, 3H, CH3
J ¼ 8.0 Hz), 3.26e3.69 (m, 8H, 4CH2 þ H2O), 3.76e3.83 (m, 12H,
6CH2), 4.43e4.46 (m, 3H, OCH3), 4.60 (q, 2H, CH2 J ¼ 8.0 Hz), 5.01 (s,
4H, 2CH2), 7.21 (bs, 3H, arH), 7.23 (bs, 2H, arH), 7.93 (d, 2H, arH
J ¼ 6.0 Hz), 7.97 (d, 1H, arH J ¼ 2.0 Hz), 8.70 (s, 1H, arH), 8.96 (s, 1H,
olefiniceCH), 8.90 and 9.40 (1H, s, NH, cis/trans conformers exch.
formers, exch. D2O). 13C NMR (DMSO-d6,
d ppm): 47.66 (CH2), 49.36
(CH2), 52.80 (CH2), 55.56 (OCH3), 56.21 (CH2), 59.34 (CH2), arC:
[112.08 (CH), 116.50 (CH), 118.24 (CH), 118.45 (CH), 119.72 (CH),
121.06 (CH), 122.99 (CH), 129.56 (CH), 139.88 (C), 140.90 (C), 148.11
(CH), 152.11 (CeOCH3), 157.29 (CeOH)], 166.98 (C]O).
D2O), 15.33 (1H, bs, OH, exch. D2O). 13C NMR (DMSO-d6,
d ppm):
15.02 (CH3), 41.75 (2CH2), 42.48 (CH2), 44.41 (CH2), 49.76 (2CH2),
50.27 (2CH2), 51.34 (CH2), 57.20 (2OCH3), 61.89 (2CH2), arC: [106.92
(CH), 107.84 (C), 109.43 (C), 111.82 (CH), 112.06 (CH), 112.51 (CH),
112.74 (CH), 118.90 (2CH), 121.52 (2CH), 123.54 (C), 136.83 (C),
137.84 (C), 144.65 (C), 149.24 (CH), 151.90 (C), 152.27 (C), 153.32 (d,
C, J ¼ 284.0 Hz), 154.36 (C)], 149.71 (CH), 164.50, 166.05, 166.73 and
172.20 (4C]O). EI MS m/z (%): 758.32 ([M þ 1]þ, 89), 701.24 (25),
570.22 (40), 473.34 (43), 381.29 (100).
3.1.20. N0-[(3-Hydroxy-4-methoxyphenyl)methylen]-2-[4-(2-
methoxyphenyl)piperazin-1-yl]acetohydrazide (21)
3-Hydroxy-4-methoxybenzaldehyde (10 mmol) was added to
the solution of compound 16 (10 mmol) in ethanol, and the mixture
was refluxed in the presence of one drop of H2SO4 for 2 h. On cooling
the mixture in cold overnight, a solid formed. This was recrystallized
from dimethyl sulfoxide to afford the desired compound. Yield 82%,
m.p. 114e116 ꢁC. FT-IR (nmax, cmꢀ1): 3387 (OH), 3194 (areCH and
NH), 1687 (C]O), 1247 (OeC). Elemental analysis for C21H26N4O4,
calculated (%), C, 63.30; H, 6.58; N,14.06, found (%), C, 63.33; H, 6.41;
3.1.22.2. 1-Ethyl-6-fluoro-7-{4-[({5-[4-(2-methoxyphenyl)piperazin-
1-yl]-1,3,4-oxadiazol-2-yl}thio)acetyl]piperazin-1-yl}-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (24). Yield 43%, m.p. 77e79 ꢁC.
FT-IR (nmax, cmꢀ1): 3054 (areCH), 1720 and 1623 (C]O). Elemental
analysis for C31H34FN7O6S, calculated (%), C, 57.13; H, 5.26; N, 15.04,
N,14.26. 1H NMR (DMSO-d6,
d ppm): 3.25 (s, 3H, OCH3), 2.78 (bs, 4H,
2CH2), 3.19 (bs, 4H, 2CH2), 3.95 (s, 5H, OCH3 þ CH2), 6.89e6.96 (m,
6H, arH), 7.17e7.21 (m,1H, arH), 7.82 and 8.12 (s,1H, N]CH, cis/trans
conformers), 9.50 (1H, s, OH, exch. D2O), 11.20 and 11.40 (1H, s, NH,
cis/trans conformers, exch. D2O). 13C NMR could not been obtained
due to the reason that the solubility of this compound in any NMR
solvent. EI MS m/z (%): 149.9 (14), 189.95 (38), 204.96 (100), 206.03
(23), 399.31 ([M þ 1]þ, 94), 400.01 ([M þ 2]þ, 41).
found (%), C, 56.95; H, 5.38; N, 15.24. 1H NMR (DMSO-d6,
d ppm):
1.41 (s, 3H, CH3), 3.38 (bs, 16H, 2CH2), 3.71e3.78 (m, 6H, 8CH2), 4.60
(bs, 3H, OCH3), 6.90e7.19 (m, 4H, arH), 7.91 (bs, 2H, arH), 8.96 (bs,
1H, olefiniceCH), 15.32 (bs, 1H, OH, exch. D2O). 13C NMR (DMSO-d6,
d
ppm): 14.75 (CH3), 33.91 (CH2), 37.19 (CH2), 41.77 (CH2), 42.16
(CH2), 45.78 (CH2), 45.96 (CH2), 49.75 (2CH2), 50.15 (CH2), 52.62
(2CH2), 56.06 (OCH3), arC: [106.77 (CH), 107.84 (C), 111.81 (CH),
112.61 (d, CH, J ¼ 67.0 Hz), 118.97 (CH), 120.16 (C), 121.51 (2CH),
137.84 (2C), 149.22 (C), 152.25 (2CH), 152.60 (oxadiazole C-2),
153.48 (d, C, J ¼ 247.0 Hz), 165.47 (oxadiazole C-5)], 166.71 and
176.84 (3C]O). EI MS m/z (%): 690.22 ([M þ K]þ, 20), 666.24 (100),
413.26 (40), 381.29 (72), 344.61 (38).
3.1.21. N0-[1H-Indol-3-ylmethylene]-2-[4-(2-methoxyphenyl)
piperazin-1-yl]acetohydrazide (22)
Indole-3-carbaldehyde (10 mmol) was added to the solution of
compound 16 (10 mmol) in ethanol, and the mixture was refluxed
in the presence of one drop of H2SO4 for 2 h. On cooling the mixture
in cold overnight, a solid formed. This was recrystallized from
ethanol to afford the desired compound. Yield 82%, m.p.114e116 ꢁC.
FT-IR (nmax, cmꢀ1): 3190 (areCH and NH), 1689 (C]O), 1242 (OeC).
Elemental analysis for C22H25N5O2, calculated (%), C, 67.50; H, 6.44;
N, 17.89, found (%), C, 67.56; H, 6.35; N, 17.92. 1H NMR (DMSO-d6,
3.1.22.3. 1-Ethyl-6-fluoro-7-(4-{[(5-{[4-(2-methoxyphenyl)piper-
azin-1-yl]methyl}-4-phenyl-4H-1,2,4-triazol-3-yl)thio]acetyl}piper-
azin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid
(25).
d
ppm): 2.77 (s, 4H, 2CH2), 3.05 (s, 4H, 2CH2), 3.77 (s, 5H,
Yield 68%, m.p. 70e71 ꢁC. FT-IR (nmax, cmꢀ1): 3423 (OH), 3056 (are
CH), 1715 and 1626 (C]O). Elemental analysis for C38H41FN8O5S,
calculated (%), C, 61.61; H, 5.58; N, 15.13, found (%), C, 61.49; H, 5.66;
OCH3 þ CH2), 6.92 (d, 3H, arH, J ¼ 8.2 Hz), 7.18 (brs, 3H, arH), 7.42
(brs, 2H, arH), 7.83 (s, 1H, NH ¼ CH), 8.19 and 8.43 (1H, N]CH, cis/
trans conformers), 11.00 and 11.62 (1H, s, NH, cis/trans conformers,
exch. D2O), indoleNH was not observed. 13C NMR (DMSO-d6,
N, 14.89. 1H NMR (DMSO-d6,
d
ppm): 1.42 (t, 3H, CH3, J ¼ 8.0 Hz),
2.79 (bs, 4H, 2CH2), 3.40e3.55 (m, 8H, 4CH2 þ H2O), 3.67e3.78 (m,
7H, OCH3 þ 2CH2), 4.35 (s, 2H, CH2), 4.60 (q, 2H, CH2, J ¼ 8.0 Hz),
6.80e6.93 (m, 4H, arH), 7.20 (d, 1H, arH, J ¼ 8.0 Hz), 7.52e7.58 (m,
5H, arH), 7.96 (d, 1H, arH, J ¼ 6.0 Hz), 8.97 (s, 1H, olefiniceCH), 15.33
d
ppm): 48.59 (CH2), 50.01 (CH2), 53.17 (CH2), 53.56 (CH2), 55.99
(OCH3), 56.66 (CH2), arC: [112.49 (CH), 118.70 (CH), 118.88 (CH),
121.17 (CH), 121.52 (CH), 122.58 (CH), 123.39 (CH), 123.88 (CH),
124.53 (C), 124.62 (CH), 137.59 (C), 140.61 (C), 141.49 (C), 152.54 (Ce
OCH3)], 148.53 (CH), 169.99 (C]O). EI MS m/z (%): 190.07 (18),
205.15 (38), 392.42 ([M þ 1]þ, 100), 393.43 ([M þ 2]þ, 19).
(s, 1H, OH, exch. D2O). 13C NMR (DMSO-d6,
d ppm): 14.21 (CH3),
36.01 (2CH2), 41.28 (2CH2), 45.11 (2CH2), 49.05 (2CH2), 49.47 (CH2),
52.25 (2CH2), 55.27 (OCH3), arC: [106.14 (CH), 107.10 (CH), 111.36 (d,
CH, J ¼ 23.0 Hz), 117.88 (2CH), 119.14 (CH), 119.52 (C), 120.74 (CH),
122.45 (CH), 127.19 (2CH), 129.73 (CH), 137.13 (C), 145.12 (C), 148.58
(CH),151.52 (2C),152.76 (d, C, J ¼ 248.0 Hz),151.88 (C and triazole C-
3)], 154.00 (C), 165.43 (C]O), 166.04 (C]O and triazole C-5), 176.16
3.1.22. General method for the synthesis of compound 23e27
The mixture of compound 3 (10 mmol) and compound 14 (for
26), 16 (for 27), 18 (for 25), 19 (for 24) or 21 (for 23) (10 mmol) in

M.Y. Mentese et al. / European Journal of Medicinal Chemistry 67 (2013) 230e242
241
(C]O). EI MS m/z (%): 765.27 ([M þ K]þ, 28), 741.29 (100), 413.26
24 h at 35 ꢁC. Brain Heart Infusion broth (BHI) (Difco, Detriot, MI)
(28), 382.29 (63).
was used for M. smegmatis, and incubated for 48e72 h at 35 ꢁC [62].
Ampicillin (10 mg) and fluconazole (5 mg) were used as standard
3.1.22.4. 1-Ethyl-6-fluoro-7-[4-(N-{3-fluoro-4-[4-(2-methoxyphenyl)
piperazin-1-yl]phenyl}glycyl)piperazin-1-yl]-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (26). Yield 33%, m.p. 177e
178 ꢁC. FT-IR (nmax, cmꢀ1): 3100 (NH), 3057 (areCH), 1720 and 1623
(C]O). Elemental analysis for C35H39FN6O5, calculated (%), C, 65.41;
H, 6.12; N, 13.08, found (%), C, 65.56; H, 6.32; N, 12.98. 1H NMR
antibacterial and antifungal drugs, respectively. Dimethylsulph-
oxide with dilution of 1:10 was used as solvent control. Only pos-
itive results were presented in Table 1.
3.2.2. Urease inhibition assay
Reaction mixtures comprising 25
buffer (100 mM urea, 0.01 M K2HPO4, 1 mM EDTA and 0.01 M LiCl,
pH 8.2) and 100 mM urea were incubated with 5 L of the test
mL of Jack Bean Urease, 55 mL of
(DMSO-d6)
d ppm: 1.42 (bs, 3H, CH3), 3.47 (bs, 8H, 4CH2), 3.79 (bs,
8H, 4CH2), 3.97 (s, 3H, OCH3), 4.60 (bs, 2H, CH2), 5.01 (bs, 2H, CH2),
6.99 (bs, 3H, arH), 7.08 (bs, 2H, arH), 7.20 (bs, 2H, arH), 7.82 (bs, 1H,
arH), 7.92 (bs, 1H, arH), 8.69 (s, 1H, NH, exch. D2O), 8.96 (s, 1H,
olefiniceCH), 15.33 (bs, 1H, OH, exch. D2O). 13C NMR (DMSO-d6)
m
compounds at room temperature for 15 min in microtiter plates.
The production of ammonia was measured by indophenol method
and used to determine the urease inhibitory activity. The phenol
d
ppm: 15.02 (CH3), 43.17 (2CH2), 44.40 (2CH2), 49.76 (2CH2), 50.98
reagent (45
mL, 1% w/v phenol and 0.005% w/v sodium nitroprus-
(2CH2), 56.09 (OCH3), 61.89 (2CH2), arC: [106.92 (2CH), 107.84 (C),
111.83 (CH), 112.94 (d, CH, J ¼ 22.0 Hz), 112.75 (2CH), 118.87 (CH),
121.55 (2CH), 136.84 (C), 137.84 (C), 144.64 (C), 145.77 (C), 149.26
(CH), 152.27 (C), 153.33 (d, C, J ¼ 282.0 Hz), 154.38 (C), 164.51 (C),
166.05 (C)], 166.77, 172.18 and 176.86 (3C]O).
side) and alkali reagent (70
m
L, 0.5% w/v sodium hydroxide and 0.1%
v/v NaOCl) were added to each well and the increasing absorbance
at 625 nm was measured after 20 min, using a microplate reader
(Molecular Device, USA). The percentage inhibition was calculated
from the formula 100 ꢀ (ODtestwell/ODcontrol) ꢃ 100. Thiourea was
used as the standard inhibitor. In order to calculate IC50 values,
different concentrations of synthesized compounds and standard
were assayed at the same reaction conditions [63].
3.1.22.5. 1-Ethyl-6-fluoro-7-{4-[(2-{[4-(2-methoxyphenyl)piperazin-
1-yl]acetyl}hydrazino)acetyl]piperazin-1-yl}-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (27). Yield 49%, m.p. 192e
195 ꢁC. FT-IR (nmax, cmꢀ1): 3410 (OH), 3058 (areCH), 1707 (C]O).
Elemental analysis for C31H38FN7O6, calculated (%), C, 59.70; H, 6.14;
N, 15.72, found (%), C, 59.56; H, 5.94; N, 15.92. 1H NMR (DMSO-d6,
Acknowledgments
The support provided by Scientific and Technological Research
Council of Turkey (TUBITAK, Project no: 111T427).
d
ppm): 1.42 (s, 3H, CH3), 3.14e3.50 (m, 8H, 4CH2 þ H2O), 3.68e3.96
(m, 15H, OCH3 þ 6CH2), 4.58 (s, 2H, CH2), 6.88e7.18 (m, 4H, arH),
7.85e7.91 (m, 2H, arH), 8.52 (s, 1H, NH, exch. D2O), 8.94 (s, 1H,
olefiniceCH), 11.57 (s, 1H, NH, exch. D2O), 15.30 (bs, 1H, OH, exch.
Appendix A. Supplementary data
D2O), NH was not observed. 13C NMR (DMSO-d6,
d ppm): 14.04
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.06.045.
(CH3), 40.85 (2CH2), 49.05 (2CH2), 49.45 (2CH2), 52.14 (CH2), 53.25
(CH2), 55.40 (OCH3), 57.93 (CH2), 59.71 (2CH2), arC: [105.94 (CH),
107.07 (C), 111.18 (d, CH, J ¼ 23.0 Hz), 111.91 (CH), 111.95 (CH), 118.23
(CH), 119.46 (C), 120.79 (CH), 137.07 (2C), 145.08 (C), 148.50 (CH),
151.83 (C), 152.72 (d, C, J ¼ 248.0 Hz)], 166.01 (C]O), 167.18 (C]O),
172.20 (C), 176.06 (C]O). EI MS m/z (%): 623.27 ([M]þ, 26), 503.20
(30), 381.29 (100), 353.26 (16).
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