Microwave-Assisted synthesis of some hybrid molecules containing penicillanic acid or cephalosporanic acid moieties and investigation of their biological activities

Article abstract of DOI:10.1007/s00044-013-0898-4

Ethyl 4-Amino-2-fluorophenylpiperazin-1-carboxylates containing a 1,3-oxazol(idin)e, 5-thioxo-1,2,4-triazole, 1,3,4-thiadiazole, 5-thioxo-1,3,4-oxadiazole, or 1,3-thiazole nucleus were obtained starting from ethyl piperazine-1-carboxylate (1) by several steps. The treatment of amine, 3 or hydrazide, 9 with several aromatic aldehydes generated the corresponding arylmethyleneamino (3a-f) or arylidenehydrazino (12a-c) compounds. The Mannich reaction between the 1,2,4-triazole or 1,3,4-oxadiazole compounds and 7-Aca produced cephalosporanic acid derivatives. Penicillanic acid derivatives were obtained when 6-Apa was used in the Mannich reactions. The synthesized compounds were screened for their antimicrobial, antilipase, and antiurease activities. Some of them were found to possess good-moderate antimicrobial activity against the test microorganisms. Two compounds exhibited antiurease activity, and four of them displayed antilipase activity.

Full text of DOI:10.1007/s00044-013-0898-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0898-4
ORIGINAL RESEARCH
Microwave-assisted synthesis of some hybrid molecules containing
penicillanic acid or cephalosporanic acid moieties
and investigation of their biological activities
•
•
•
Serap Basoglu Serdar Ulker Sengul Alpay-Karaoglu
Neslihan Demirbas
Received: 23 July 2013 / Accepted: 13 December 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Ethyl 4-amino-2-fluorophenylpiperazin-1-car-
boxylates containing a 1,3-oxazol(idin)e, 5-thioxo-1,2,4-
triazole, 1,3,4-thiadiazole, 5-thioxo-1,3,4-oxadiazole, or
1,3-thiazole nucleus were obtained starting from ethyl
piperazine-1-carboxylate (1) by several steps. The treat-
ment of amine, 3 or hydrazide, 9 with several aromatic
aldehydes generated the corresponding arylmethyleneami-
no (3a–f) or arylidenehydrazino (12a–c) compounds. The
Mannich reaction between the 1,2,4-triazole or 1,3,4-oxa-
diazole compounds and 7-aca produced cephalosporanic
acid derivatives. Penicillanic acid derivatives were
obtained when 6-apa was used in the Mannich reactions.
The synthesized compounds were screened for their anti-
microbial, antilipase, and antiurease activities. Some of
them were found to possess good-moderate antimicrobial
activity against the test microorganisms. Two compounds
exhibited antiurease activity, and four of them displayed
antilipase activity.
Introduction
The limitations of the existing antibacterial drugs caused
by various reasons including drug resistance, the serious
side effects, and/or lack of efficacy made infectious dis-
eases a vicious cycle. In addition, the treatment of resistant
strains requires a prolonged therapy containing the use of
more toxic drugs and increases the financial burden. The
rising prevalence of multi-drug resistant bacteria continues
to serve medicinal chemists to search and discove novel
antimicrobial agents effective against pathogenic micro-
organisms resistant to current treatment.
Among the strategies addressed to the synthesis of
compounds possessing antimicrobial activity, the syntheses
of hybrid molecules incorporating different heterocyclic
moieties have been attracting widespread attention (Mal-
likarjuna et al., 2009).
A number of N-containing heterocyclic compounds
constitute important building blocks in organic and
medicinal chemistry. For example, triazoles have been
shown to possess a number of desirable activities in the
context of medicinal chemistry. Ribavirin (antiviral), ri-
zatriptan (antimigraine), alprazolam (psychotropic), fluco-
nazole, and itraconazole (antifungal) are the best examples
for potent drugs possessing triazole nucleus (Holla et al.,
2006; Walczak et al., 2004; Jones et al., 1965; Ashok et al.,
2007). Tazobactam, a b-lactamase inhibitor is the other
best known example of triazole containing structures with
the broad spectrum antibiotic piperacillin (Kategaonkar
et al., 2010).
Keywords Piperazine Á 1,3-Oxa(thia)zole Á
5-Oxo-1,3-oxazolidine Á 1,2,4-Triazole Á
7-Aminocephalosporanic acid Á 6-Aminopenicillanic acid Á
Biological activity
S. Basoglu Á N. Demirbas (&)
Department of Chemistry, Faculty of Sciences, Karadeniz
Technical University, 61080 Trabzon, Turkey
e-mail: neslihan@ktu.edu.tr
Substituted piperazines constitute another class of
important pharmacophores, which are found in many
marketed drugs, such as the HIV protease inhibitor, Cri-
xivan (Chaudhary et al., 2006). Ciprofloxacin, norfloxacin,
pefloxacine, ofloxacin, and enoxacin are fluoroquinolone
S. Ulker Á S. Alpay-Karaoglu
Department of Biology, Recep Tayyip Erdogan University,
53100 Rize, Turkey
123

Med Chem Res
class antibacterial drugs characterized by having a piper-
azine moiety at C-7 of quinolone skeleton, and they have
been used for the treatment of bacterial infections (Fo-
roumadi et al., 2005).
the intermediate and target compounds was performed
according to the reactions outlined in Schemes 1, 2, and 3.
The starting compound ethyl 1-piperazinecarboxylate (1)
was provided commercially.
The compounds having a thiazolidinone nucleus are of
interest due to their broad spectrum of biological activities
such as bactericidal, fungicidal, antimicrobial, antiprolif-
erative, antiviral, anticonvulsant, anticancer, and anti-
inflammatory activities (Vicini et al., 2008; Wang et al.,
2011; Lv et al., 2010; Metwally et al., 2010; Balzarini
et al., 2009; Havrylyuk et al., 2009; Subtelna et al., 2010;
Mushtaque et al., 2012).
Ethyl 4-(4-amino-2-fluorophenyl)piperazine-1-carbox-
ylate (3), that was obtained starting from compound 1 by
two steps, was converted to the corresponding arylmethy-
lenamino derivatives (4a–f) by the treatment with several
1
aromatic aldehydes. In the FT-IR and H NMR spectra of
these compounds, no signal pointing the –NH2 group was
seen. Instead, additional signals derived from aldehyde
moiety were recorded at the related chemical shift values in
1
Mannich bases, which are known to be physiologically
reactive since their basic function rendering the molecule
soluble in aqueous solvents when it is transformed into
aminium salt, have been reported as potential biological
agents (Karthikeyan et al., 2006). N-Mannich bases have
been used successfully to obtain prodrugs of amine as well
as amide-containing drugs (Zhao et al., 2009). Some
Mannich bases derived from 1,2,4-triazole nucleus have
been reported to possess protozocidal and antibacterial
activity (Ashok et al., 2007; Almajan et al., 2009; Bayrak
et al., 2009, 2010; Demirbas et al., 2009; Bektas et al.,
2010; Patole et al., 2006).
the H NMR spectra.
The cyclocondensation of compound 5, that was
obtained from the reaction of 4 with benzylisocyanate, with
ethyl bromoacetate or 4-chlorophenacyl bromide produced
the corresponding hybrid molecules incorporating a 4-oxo-
1,3-oxazolidine (6) or 4-chlorophenyl)-1,3-oxazole (7)
nucleus in the 2-fluorophenylpiperazine-1-carboxylate
1
skeleton. The H and 13C NMR spectra of compound 7
exhibited additional signals at aromatic region originated
from 4-chlorophenyl nucleus as a result of condensation.
Moreover, the elemental analyses and mass spectral data of
derivatives 6 and 7 were compatible with the suggested
structures.
Schiff bases have gained importance in medicinal and
pharmaceutical fields due to their most versatile properties
as organic synthetic intermediates and also possessing a
broad range of biological activities, such as antitubercu-
losis, anticancer, analgesic and anti-inflammatory, anti-
convulsant, antibacterial, and antifungal activities (Patole
et al., 2006, Hearn and Cynamon, 2004; Ren et al., 2002;
Demirbas et al., 2002; Lohray et al., 2006).
The treatment of compound 3 with ethyl bromoacetate
at room temperature in the presence of triethylamine
resulted in the formation of compound 8. When compound
8 was converted to the corresponding hydrazide (9) by
refluxing with hydrazine hydrate, the signals originated
1
from ester function was disappeared in the H and 13C
NMR spectra. Instead, new signals due to –NHNH2 protons
were seen at 5.93 and 9.09 ppm. Meanwhile, the stretching
frequency band of this group was recorded at 3,313 cm-1
as a wide signal characteristic for the hydrazide structure.
Compounds 6 and 7 gave mass fragmentation confirming
the proposed structures.
We envisage that hybrid compound incorporating a
4-(2-fluorophenylene)-piperazine core with several het-
erocyclic moieties responsible for biological activity in a
single molecular frame could lead to the novel potent
antimicrobial and antiurease agents. Highly substituted
piperazines can be expected to increase antimicrobial
activity probably by enhancing lipophilicity of molecule.
In continuation of our research program on the synthesis
of hybrid molecules containing various heterocyclic moi-
eties, we planned the synthesis of 4-(2-fluoro-
The synthesis of compounds 10 and 11 was carried out
by the treatment of compound 7 with the corresponding
isothiocanates. These compounds displayed spectroscopic
data and elemental analysis results consistent with the
assigned structures.
phenyl)piperazine
derivatives
along
with
their
The intramolecular cyclization of compound 10 gener-
ated the corresponding 1,3,4-thiazole compound (12) in
acidic media. On the other hand, the basic treatment of
compounds 10 and 11 caused to the cyclization of the
(arylamino)carbonothioylhydrazino side change leading to
the formation of 5-thioxo-4,5-dihydro-1H-1,2,4-triazol
derivatives (13 and 14). With the conversion of compounds
10 and 11 to compounds 12–14, two of NH signals were
antimicrobial and antiurease activities.
Results and discussion
The main aim of the present study is the synthesis and
antimicrobial activity evaluation of new piperazine deriv-
atives incorporating several heterocyclic moieties includ-
ing 1,3-oxadiazole, 1,2,4-triazole, 1,3-oxa(thia)zole,
penicillanic acid, and/or cephalosporanic acid. Synthesis of
1
disappeared in the H NMR spectra. It is well-known that
type of compounds can stay in thioxo or mercapto tauto-
meric form. In the present study, compounds 13 and 14 are
123

Med Chem Res
Scheme 1 i 3,4-
Difluoronitrobenzene in ethanol,
reflux for 6 h. ii Pd–C,
O
O
N
NH
hydrazine hydrate in n-butanol,
reflux for 7 h. iii Indole-3-
carboxaldehyde in absolute
ethanol, irradiation by MW at
150 W, 110 °C for 30 min. iv
Benzylisothiocyanate in
absolute ethanol, reflux for
10 h. v Ethyl bromoacetate in
absolute ethanol, dried sodium
acetate, reflux for 13 h. vi
4-Chlorophenacylbromide in
absolute ethanol, dried sodium
acetate, reflux for 11 h
1
i
O
O
ii
O
N
N
NO2
O
N
N
NH2
F
F
2
3
iii
O
iv
O
N
N
N
R
F
O
O
4
N
H
O
N
N
N
4a:
4b:
N
H
F
NO2
Br
5
4c:
4d:
v
O
O
O
N
N
N
HO
O
N
vi
F
4e:
4f:
OCH3
6
Cl
N
H
O
O
N
O
N
N
N
F
7
present predominately in the thioxo form as it was shown
by the C=S band at 1,244–1,250 cm-1 in the FT-IR spectra
1
of these compounds. Furthermore, the H NMR spectra of
and 13C NMR spectra, additional signal corresponding to
the 6-apa or 7-aca-ammonium salt was recorded at the
related chemical shift value.
compounds 13 and 14 revealed clearly the absence of the
signal originated from SH proton, instead of that, two
signals due to NH proton on 1,2,4-triazol ring was recorded
at 10.45 (for 13) or 11.27 (for 14), that is characteristic for
4,5-dihydro-1H-1,2,4-triazoles.
The conversion of arylcarbonothioylhydrazino side
change to 4-chlorophenyl-3-phenyl-1,3-thiazole ring (18)
was accomplished with the treatment of 4-chlorophenacyl
bromide. This compound was characterized by spectro-
1
scopic techniques including H NMR, 13C NMR, FT-IR,
The synthesis of Mannich bases (15–17) was performed
by the reaction of compounds 13 and 14 with 6-aminope-
nicillanic acid, 6-apa (for 17) or 7-aminocephalosporanic
acid, 7-aca (for 15 and 16) in tetrahydrofuran at room
temperature in the presence of triethylamine and formal-
dehyde. The occurrence of the alkylaminomethylation was
provided by the disappearance of signal for the proton at
the N-1 nitrogen of the 1,2,4-triazole ring. Moreover, in 1H
EI-MS, and elemental analysis.
The synthesis of ethyl arylidenehydrazino-piperazine-1-
carboxylate derivatives (19a–c) was performed by micro-
wave irradiation of compound 9 with several aromatic
aldehydes namely 3-hydroxy-4-methoxybenzaldehyde,
pyridine-4-carbaldehyde, and 2-hydroxybenzaldehyde. In
the FT-IR spectra of these arylidenehydrazino compounds,
absorptionbands characteristic for NH groups werevisible in
123

Med Chem Res
O
O
N
N
NH2
O
O
N
N
NHCH2COOEt
i
F
F
3
8
ii
O
N N
S
H
O
O
N
N
N
NH
O
N
N
NHCH2CONHNH2
F
12
F
9
iv
iii
O
N NH
N
O
H
O
H
H
H
O
N
N
N
S
N
N
O
N
N
N
N
F
H
S
X
F
v
10: X= F
11: X= H
13: X= F
14: X=H
F
vi
O
N N
NH
S
H
N
N
N
O
S
vii
viii
N
N
OAc
O
F
CO2HN
15: X= F
16: X= H
X
O
N N
NH
H
S
N
N
N
O
S
N
N
O
F
CO2HN
17
Cl
O
O
S
H
N
N
N
F
N
O
N
N
H
18
Scheme 2 i Ethyl bromoacetate, Et3N, THF, rt for 14 h. ii Hydrazine
hydrate in ethanol, reflux for 14 h. iii 4-Fluorophenylisothiocyanate
or phenylisothiocyanate in absolute ethanol, reflux for 10 h. iv H2SO4,
rt for 2 h. v NaOH in water, reflux for 3 h. vi 7-Aca, HCHO, Et3N in
THF, rt, for 4 h. vii 6-Apa, HCHO, Et3N in THF, rt, for 4 h. vii
4-Chlorophenacylbromide in absolute ethanol, dried sodium acetate,
reflux for 12 h
the ranges of 3,357–3,181 cm-1. Another piece of evidence
for condensation was the appearance of a signal as singlet
1
integrating for one proton in the H NMR spectra, which
corresponds to the N=CH proton of azomethyne group.
Moreover, these compounds gave mass fragmentation and
elemental analysis confirming the proposed structures.
123

Med Chem Res
O
Ar
O
19a: R=
OCH3
OH
O
N
N
NH
N
N
F
H
19
19b: R=
19c: R=
N
i
HO
N NH
O
O
O
S
O
N
N
NHCH2CONHNH2
N
O
N
NH
F
ii
F
9
20
iii
NH
N N
O
S
O
S
iv
NH
N
OAc
N
N
O
O
F
COO-HN+
21
NH
S
N N
O
S
N
O
NH
N
N
O
O
COO-HN+
F
22
Scheme 3 i 3-Hydroxy-4-phenoxybenzaldehyde, pyridine-4-carbal-
dehyde, 2-hydroxybenzaldehyde in absolute ethanol, irradiation by
MW at 200 W, 140 °C for 30 min. ii CS2 and KOH in ethanol, reflux
for 13 h. iii 7-Aca, HCHO, Et3N in THF, rt, for 4 h. iv 6-Apa, HCHO,
Et3N in THF, rt, for 4 h
Ethyl 4-(2-fluoro-4-{[(5-thioxo-4,5-dihydro-1,3,4-oxa-
diazol-2-yl)methyl]amino} phenyl)piperazine-1-carboxyl-
ate (20) was prepared from the reaction of compound 9
with CS2 in the basic media. The attempts for amin-
oalkylations of compound (20) by Mannich reaction
allowed the isolation of the corresponding products (21 and
22) after 4 (for 21) or 6 h (for 22) at room temperature.
This idea originated from the intent to introduce the pen-
icillanic acid or cephalosporanic acid nucleus to (piperazin-
1-yl)-2-thioxo-1,3,4-oxadiazole skeleton. As different from
20, the NMR spectra of the obtained Mannich bases (21
and 22) displayed additional signals derived from penicil-
lanic- or cephalosporanic-acid moiety and –CH2—linkage
at the related shift and integral values as D2O nonex-
changeable signals.
activity on Bacillus cereus (Bc), that is Gram positive
spore bacillus. With the reduction of nitro group of 2 to
amine (compound 3), additional activities towards
Staphylococcus aureus (Sa), that is Gram positive coccus,
Candida albicans (Ca), and Saccharomyces cerevisiae
(Sc), which are yeast like fungi. For the imine com-
pounds (4a–f), the highest activity was observed against
Mycobacterium smegmatis (Ms) that is an atypical
tuberculosis factor leading mortality, with the inhibition
zone varying between 10 and 25 mm. The compounds
containing 1,2,4-triazole and cephalosporanic- or peni-
cillanic-acid moiety (compounds 15–17) displayed good-
moderate activity on some of the test microorganisms.
The highest activity was observed for compound 17 on
Bc with the inhibition zone of 16 mm. This result is
better than standard drug ampicillin. Other compounds
containing penicillanic acid or cephalosporanic acid core
Among the synthesized compounds ethyl 4-(2-fluoro-
4-nitrophenyl)piperazine-1-carboxylate
(2)
exhibited
123

Med Chem Res
(21 and 22) displayed good-moderate activity against the
test microorganisms.
Experimental
The synthesized compounds were assayed for their
in vitro urease inhibitory activity against Jack bean urease.
Two of those compounds showed perfect urease inhibition.
No inhibitory effect was detected for other compounds.
Thiourea with IC50 value 54.56 4.17 lg mL-1 was used
as standard inhibitor. Among tested compounds, compound
15 was found to be the best inhibitory effect against urease
with an IC50 value of 4.67 0.53 lg mL-1. At the various
final concentrations the compound 15 showed more
inhibitory effect than standard urease inhibitor thiourea.
Also, compound 17 has the highest inhibitory activity than
thiourea. These compounds might be considered as
potential antibiotics to treat infections.
Chemistry
General information for chemicals
All the chemicals were purchased from Fluka Chemie AG
Buchs (Switzerland) and used without further purification.
Melting points of the synthesized compounds were deter-
mined in open capillaries on a Bu
¨chi 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 ace-
tate:diethyl ether, 1:1, and detection was made using UV
light. FT-IR spectra were recorded as potassium bromide
pellets using a Perkin Elmer 1600 series FT-IR spectrom-
All compounds were evaluated with regard to pan-
creatic lipase activity and compounds 12, 13, 14, and 15,
which are 1,3,4-thiadizole or 1,2,4-triazole derivatives
including also 4-fluorophenylpiperazine nucleus, showed
moderate anti-lipase activities at final concentration of
6.25 lg mL-1. No inhibitory effect was detected for
other compounds. Orlistat, known pancreatic lipase
inhibitor used as anti-obesity drug, showed inhibitory
effect by 99 % at the same concentration.
1
eter. H NMR and 13C NMR spectra were registered in
DMSO-d6 on a BRUKER AVANCE II 400 MHz NMR
Spectrometer (400.13 MHz for 1H and 100.62 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 Ele-
mental Combustion System CHNS–O elemental analyzer.
All the compounds gave C, H, and N analysis within
0.4 % of the theoretical values. The mass spectra were
obtained on a Quattro LC–MS (70 eV) instrument.
Conclusion
Ethyl 4-(2-fluoro-4-nitrophenyl)piperazine-1-carboxylate
(2) The solution of 3,4-difluoronitrobenzene (10 mmol)
in excess amount of ethyl 1-piperazinecarboxylate
(40 mmol) was allowed to reflux for 6 h (the progress of
the reaction was monitored by TLC). Then, the mixture
was poured into ice-water. The precipitated product was
filtered off and recrystallized from ethanol. Yield 97 %, m.
p: 90–93 °C. FT-IR (KBr, m, cm-1): 3099 (ar–CH), 1509,
and 1354 (NO2). Elemental analysis for C13H16FN3O4
calculated (%): C, 52.52; H, 5.42; N, 14.13. Found (%): C,
This study reports microwave-assisted synthesis of some
new hybrid molecules containing penicillanic acid or
cephalosporanic acid moieties with some other phar-
macophore heterocycles in a single structure. Hence
herein we combined all these potential chemothera-
peutic units, namely 1,2,4-triazole, 1,3-thiazole, 1,3-
oxazole, 1,3,4-oxadiazole, piperazine, penicillanic acid,
cephalosporanic acid moieties. The antimicrobial, anti-
urease, and antilipase screening studies were also per-
formed in the study.
1
52.64; H, 5.70; N, 14.00. H NMR (DMSO-d6, d ppm): 1.
Among the synthesized compounds, the compounds
containing 1,2,4-triazole and cephalosporanic- or penicil-
lanic-acid moiety (15–17) displayed good-moderate activ-
ity on some of the test microorganisms. The highest
activity was observed for compound 17 on Bc with the
inhibition zone of 16 mm. This result is better than stan-
dard drug ampicillin. Moreover, compounds 15 and 17
exhibited an inhibitory effect against urease. Other com-
pounds containing penicillanic acid or cephalosporanic
acid core (21 and 22) displayed good-moderate activity
against the test microorganisms. Furthermore, compounds
12, 13, 14, and 15, which are 1,3,4-thiadizole or 1,2,4-
triazole derivatives including also 4-fluorophenylpipera-
zine nucleus, showed moderate anti-lipase activities at final
19 (t, 3H, CH3, J = 7.0 Hz), 3.26 (s, 4H, 2CH2), 3.51 (s,
4H, 2CH2), 4.06 (q, 2H, CH2, J = 6.6 Hz), 7.16 (t, 1H,
arH, J = 7.8 Hz), 8.00 (d, 2H, arH, J = 7.8 Hz). 13C NMR
(DMSO-d6, d ppm): 11.47 (CH3), 40.46 (2CH2), 45.81
(2CH2), 57.92 (CH2), arC: [105.00 (CH), 109.09 (d, CH,
JC–F = 26.0 Hz), 116.54 (d, CH, JC–F = 154.0 Hz), 136.
43 (C), 142.01 (C), 146.05 (C)], 151.46 (C=O). MS m/z
(%): 301.29 (32), 167.01 (18), 159.03 (19), 148.96 (100),
113.05 (34).
Ethyl 4-(4-amino-2-fluorophenyl)piperazine-1-carboxylate
(3) Pd–C (5 mmol) catalyst was added to the solution of
compound (2) (10 mmol) in n-butanol, and the mixture was
refluxed in the presence of hydrazine hydrate (50 mmol)
for 7 h. The progress of the reaction was monitored by
concentration of 6.25 lg mL-1
.
123

Med Chem Res
TLC. Then, the catalyst was separated by filtration and the
solvent was evaporated under reduced pressure. The solid
obtained was recrystallized from ethanol. Yield 65 %. M.p:
116–119 °C. FT-IR (KBr, m, cm-1): 3,423 and 3,341
(NH2), 1682 (C=O). Elemental analysis for C13H18FN3O2
calculated (%): C, 58.41; H, 6.79; N, 15.72. Found (%): C,
7.09–7.37 (m, 3H, arH), 8.14 (d, 2H, arH, J = 7.8 Hz), 8.
35 (d, 2H, arH, J = 7.8 Hz), 8.84 (s, 1H, N=CH). 13C
NMR (DMSO-d6, d ppm): 15.16 (CH3), 43.39 (CH2), 50.64
(CH2), 55.44 (2CH2), 61.57 (CH2), arC: [110.57 (d, CH,
JC–F = 32.7 Hz), 117.30 (CH), 120.24 (d, CH, JC–F = 41.
0 Hz), 123.62 (d, C, JC–F = 42.5 Hz), 124.76 (2CH), 130.
18 (2CH), 139.53 (C), 142.26 (C), 146.09 (d, C, JC–F = 51.
0 Hz), 150.35 (d, C, JC–F = 96.0 Hz)], 153.46 (C=O), 160.
32 (N=CH).
1
58.31; H, 6.87; N, 15.78. H NMR (DMSO-d6, d ppm): 1.
18 (t, 3H, CH3, J = 7.0 Hz), 2.76 (s, 4H, 2CH2), 3.45 (s,
4H, 2CH2), 4.04 (q, 2H, CH2, J = 7.4 Hz), 5.03 (s, 2H,
NH2), 6.33 (d, 2H, arH, J = 12.4 Hz), 6.76 (t, 1H, arH,
J = 9.0 Hz). 13C NMR (DMSO-d6, d ppm): 14.53 (CH3),
43.56 (2CH2), 51.07 (2CH2), 60.75 (CH2), arC: [101.66 (d,
CH, JC–F = 23.0 Hz), 109.39 (CH), 120.92 (d, CH,
JC–F = 4.05 Hz), 128.70 (d, C, JC–F = 9.5 Hz), 145.72 (d,
C, J = 10.6 Hz), 154.18 (d, C, JC–F = 34.5 Hz)], 158.65
(C=O). MS m/z (%): 268.10 ([M?1]?,100).
Ethyl 4-(4-{[(4-bromophenyl)methylene]amino}-2-fluoro-
phenyl)piperazine-1-carboxylate (4c) The mixture of
compound
3
(10 mmol) and 4-bromobenzaldehyde
(10 mmol) in absolute ethanol was irradiated at 150 W and
150 °C for 30 min. The yellow solid obtained was re-
crystallized ethanol. Yield: 84 %, M.p: 124–126 °C. FT-IR
(KBr, m, cm-1): 3053 (ar–CH), 1671 (C=O), 1434 (C=N),
1210 (C–O). Elemental analysis for C20H21BrFN3O2 cal-
culated (%): C, 55.31; H, 4.87; N, 9.68. Found (%): C, 55.
71; H, 4.90; N, 9.79. 1H NMR (DMSO-d6, d ppm): 1.19 (t,
3H, CH3, J = 7.0 Hz), 2.98 (s, 4H, 2CH2), 3.51 (s, 4H,
2CH2), 4.05 (q, 2H, CH2, J = 7.0 Hz), 6.93–7.27 (m, 3H,
arH), 7.71 (d, 2H, arH, J = 7.8 Hz), 7.84 (d, 2H, arH, J =
8.2 Hz), 8.65 (s, 1H, N=CH). 13C NMR (DMSO-d6, d
ppm): 15.26 (CH3), 41.40 (CH2), 44.04 (CH2), 50.78
(2CH2), 61.56 (CH2), arC: [105.00 (CH), 109.44 (d, CH,
JC–F = 22.5 Hz), 119.80 (d, CH, JC–F = 58.2 Hz), 125.61
(C), 131.05 (2CH), 132.57 (2CH), 135.83 (C), 138.83 (d,
C, JC–F = 8.75 Hz), 146.26 (d, C, JC–F = 8.5 Hz), 153.39
(C)], 155.27 (C=O), 159.44 (N=CH).
Ethyl 4-(2-fluoro-4-{[pyridin-4-ylmethylene]amino}phenyl)
piperazine-1-carboxylate (4a) Indole-3-carboxaldehyde
(10 mmol) was added to the solution of compound 3
(10 mmol) in absolute ethanol and the reaction mixture
was irradiated by microwave at 150 W and 110 °C for
30 min. After removing in the solvent under reduced
pressure, an oily product obtained. This was recrystallized
from butyl acetate and diethyl ether (1:2). Yield: 81 %, M.
p: 162–163 °C. FT-IR (KBr, m, cm-1): 1686 (C=O), 1508
(C=N), 1224 (C–O). Elemental analysis for C19H21FN4O2
calculated (%): C, 64.03; H, 5.94; N, 15.72. Found (%): C,
1
64.18; H, 6.14; N, 15.78. H NMR (DMSO-d6, d ppm): 1.
19 (t, 3H, CH3, J = 6.6 Hz), 3.00 (s, 4H, 2CH2), 3.51 (s,
4H, 2CH2 ? H2O), 4.04–4.11 (m, 2H, CH2), 7.04–7.34 (m,
3H, arH), 7.80 (d, 2H, arH, J = 4.2 Hz), 8.71 (s, 3H,
arH ? N=CH). 13C NMR (DMSO-d6, d ppm): 15.26
(CH3), 44.01 (CH2), 50.69 (CH2), 51.83 (2CH2), 61.57
(CH2), arC: [102.18 (CH), 109.63 (d, CH, JC–F = 21.
0 Hz), 120.05 (d, CH, JC–F = 31.5 Hz), 121.37 (C), 122.77
Ethyl 4-{2-fluoro-4-[(2-hydroxybenzylidene)amino]phenyl}
piperazine-1-carboxylate (4d) The solution of compound
3 (10 mmol) in absolute ethanol was refluxed with
2-hydroxybenzaldehyde (10 mmol) for 7 h. On cooling the
reaction content to room temperature, a solid appeared. This
crude product was filtered off and recrystallized from acetone.
Yield: 83 %. M.p: 136–137 °C. FT-IR (KBr, m, cm-1):1697
(C=O), 1510 (C=N), 1225 (C–O). Elemental analysis for
C20H22FN3O3 calculated (%): C, 64.68; H, 5.97; N, 11.31.
Found (%): C: 64.31; H: 5.78; N: 11.48. 1H NMR (DMSO-d6,
d ppm): 1.21 (brs, 3H, CH3), 3.00 (s, 4H, 2CH2), 3.52 (s, 4H,
2CH2), 4.06 (brs, 2H, CH2), 6.97–7.59 (m, 7H, arH), 8.95 (s,
1H, N=CH), 13.02 (s, 1H, OH). 13C NMR (DMSO-d6, d ppm):
15.26 (CH3), 44.40 (2CH2), 50.66 (2CH2), 61.59 (CH2), arC:
[109.50 (d, CH, JC–F = 22.0 Hz), 117.24 (2CH), 119.33
(CH), 119.87 (C), 120.22 (d, CH, JC–F = 28.5 Hz), 133.18
(CH), 133.86 (CH), 139.28 (d, C, JC–F = 9.0 Hz), 143.26 (d,
C, JC–F = 8.5 Hz), 153.32 (C), 156.74 (d, C, JC–F = 145.
5 Hz)], 160.82 (C=O), 163.17 (N=CH).
(2CH), 139.48 (d, C, JC–F = 9.0 Hz), 144.37 (d, C, JC–F
=
120.0 Hz), 151.14 (2CH), 154.23 (d, C, JC–F = 103.
2 Hz)], 158.09 (N=CH), 158.90 (C=O). MS m/z (%): 357.
11 ([M?1]?, 64), 302.10 (100), 342.24 (80).
Ethyl 4-(2-fluoro-4-{[(4-nitrophenyl)methylene]amino}phenyl)
piperazine-1-carboxylate (4b) The mixture of compound 3
(10 mmol) and 4-nitrobenzaldehyde (10 mmol) in absolute
ethanol was irradiated by microwave at 150 W and 110 °C
for 10 min. The solid obtained was recrystallized from
ethyl acetate:petroleum ether (1:2). Yield: 58 %, M.p:
164–166 °C. FT-IR (KBr, m, cm-1): 3074 (ar–CH), 1696
(C=O), 1510, and 1341 (NO2), 1433 (C=N), 1215 (C–O).
Elemental analysis for C20H21FN4O4 calculated (%): C, 59.
99; H, 5.29; N, 13.99. Found (%): C, 60.12; H, 5.45; N, 14.
19. 1H NMR (DMSO-d6, d ppm): 1.19 (brs, 3H, CH3), 3.10
(s, 4H, 2CH2), 3.51 (s, 4H, 2CH2), 4.04–4.17 (m, 2H, CH2),
Ethyl 4-(2-fluoro-4-{[(4-methoxyphenyl)methylene]amino}
phenyl)piperazine-1-carboxylate (4e) The solution of
123

Med Chem Res
compound 3 (10 mmol) in absolute ethanol was refluxed
with 4-methoxybenzaldehyde (10 mmol) for 7 h. On
cooling the reaction content to room temperature, a solid
appeared. This crude product was filtered off and recrys-
tallized from ethanol. Yield: 42 %. M.p: 122–124 °C. FT-
IR (KBr, m, cm-1): 1688 (C=O), 1509 (C=N), 1225 (C–O).
Elemental analysis for C21H24FN3O3 calculated (%): C, 65.
44; H, 6.28; N, 10.90. Found (%): C, 65.56; H, 6.52; N, 11.
12. 1H NMR (DMSO-d6, d ppm): 1.19 (t, 3H, CH3, J = 6.
6 Hz), 2.96 (s, 4H, 2CH2), 3.49 (s, 4H, 2CH2), 3.82 (s, 3H,
O–CH3), 4.06 (q, 2H, CH2, J = 6.8 Hz), 6.71–6.78 (m, 1H,
arH), 7.04–7.22 (m, 5H, arH), 7.86 (d, 1H, arH, J = 8.
2 Hz), 8.58 (s, 1H, N=CH). 13C NMR (DMSO-d6, d ppm):
15.27 (CH3), 44.13 (CH2), 50.85 (CH2), 51.35 (2CH2), 56.
10 (O–CH3) 61.53 (CH2), arC: [109.73 (d, CH, JC–F = 38.
4H, 2CH2 ? H2O), 4.02 (q, 2H, CH2, J = 7.0 Hz), 4.26 (d,
2H, CH2, J = 6.0 Hz), 6.61 (brs, 1H, NH), 6.95 (s, 2H,
arH), 7.21–7.31 (m, 6H, arH), 8.62 (s, 1H, NH). 13C NMR
(DMSO-d6, d ppm): 15.27 (CH3), 41.39 (CH2), 43.39
(CH2), 44.15 (CH2), 51.23 (CH2), 60.45 (CH2), 61.52
(CH2), arC: [106.69 (d, CH, JC–F = 25.6 Hz), 114.19
(CH), 120.59 (CH), 127.42 (CH), 127.79 (2CH), 128.99
(2CH), 133.98 (d, C, JC–F = 9.55 Hz), 137.02 (d, C,
JC–F = 9.85 Hz), 140.98 (C), 156.65 (d, C, JC–F = 137.
5 Hz)], 155.83 (2C=O).
Ethyl 4-(4-{[3-benzyl-4-oxo-1,3-oxazolidin-2-ylidene]amino}-
2-fluorophenyl)piperazine-1-carboxylate (6) The mixture
of compound 5 (10 mmol) and ethyl bromoacetate in
absolute ethanol was refluxed in the presence of dried
sodium acetate (50 mmol) for 13 h. After removing the
solvent under reduced pressure, a solid appeared. This
crude product was washed water and the precipitated solid
was recrystallized from ethanol:water (1:2). Yield: 64 %.
M.p: 158–159 °C. FT-IR (KBr, m, cm-1): 1696, 1638 (2C=
O), 1429 (C=N), 1210 (C–O). Elemental analysis for
C23H25FN4O4 calculated (%): C, 62.72; H, 5.72; N, 12.72.
Found (%): C, 62.87; H, 5.98; N, 12.88. 1H NMR (DMSO-
d6, d ppm): 1.35 (t, 3H, CH3, J = 8.0 Hz), 3.02 (brs, 4H,
2CH2), 3.53 (s, 4H, 2CH2 ? H2O), 3.65 (brs, 2H, CH2), 4.
22 (q, 2H, CH2, J = 7.0 Hz), 4.44 (d, 2H, CH2, J = 5.
8 Hz), 7.08–7.12 (m, 3H, arH), 7.43–7.49 (m, 5H, arH).
13C NMR (DMSO-d6, d ppm): 15.26 (CH3), 43.37 (CH2),
44.16 (CH2), 51.24 (2CH2), 54.37 (CH2), 61.54 (CH2), 62.
49 (CH2), arC: [105.9 (d, CH, JC–F = 95.7 Hz), 114.21
(CH), 119.98 (d, CH, JC–F = 61.1 Hz), 127.38 (CH), 127.
78 (2CH), 128.97 (2CH), 133.72 (d, C, JC–F = 30.1 Hz),
136.95 (d, C, JC–F = 36.5 Hz), 142.15 (C), 143.15 (d, C,
JC–F = 211.6 Hz)], 155.30 (C=O), 155.92 (C=N), 161.28
(C=O). MS m/z (%): 479.16 ([M?K]?, 100).
9 Hz), 114.98 (2CH), 118.72 (CH), 121.90 (d, CH, JC–F
=
66.3 Hz), 129.12 (C), 131.24 (2CH), 132.53 (C), 138.35 (d,
C, JC–F = 21.0 Hz), 147.24 (C), 154.40 (d, C, JC–F = 94.
5 Hz), 160.10 (N=CH), 162.24 (C=O).
Ethyl 4-(2-fluoro-4-{[1H-indol-3-ylmethylene]amino}phenyl)
piperazine-1-carboxylate (4f) The solution of compound 3
(10 mmol) in absolute ethanol was refluxed with indol-3-
carbaldehyde (10 mmol) for 6 h. On cooling the reaction
content to room temperature, a solid appeared. This crude
product was filtered off and recrystallized from acetone.
Yield: 82 %. M.p: 184–186 °C. FT-IR (KBr, m, cm-1): 3484
(NH), 1678 (C=O), 1439 (C=N), 1220 (C–O). Elemental
analysis for C22H23FN4O2 calculated (%): C, 66.99; H, 5.88;
N, 14.20. Found (%): C, 66.76; H, 6.02; N, 14.01. 1H NMR
(DMSO-d6, d ppm): 1.20 (brs, 3H, CH3), 3.01 (s, 4H, 2CH2),
3.53 (s, 4H, 2CH2), 4.06 (brs, 2H, CH2), 7.29 (brs, 5H, arH),
8.08 (s, 1H, arH), 8.38 (s, 2H, arH), 9.06 (s, 1H, N=CH), 9.29
(s, 1H, NH). 13C NMR (DMSO-d6, d ppm): 15.21 (CH3), 44.
18 (CH2), 50.76 (CH2), 51.51 (2CH2), 62.46 (CH2), arC:
[108.93 (d, CH, JC–F = 23.4 Hz), 113.47 (d, CH, JC–F = 34.
4 Hz), 117.88 (CH), 118.82 (C), 120.71 (CH), 121.51 (CH),
121.84 (CH), 122.84 (CH), 123.76 (d, CH, JC–F = 41.0 Hz),
124.87 (C), 137.91 (d, C, J = 19.8 Hz), 139.24 (2C) 155.26
(d, C, JC–F = 4.0 Hz)], 153.18 (N=CH), 185.74 (C=O).
4-(4-{[3-Benzyl-5-(4-chlorophenyl)-1,3-oxazol-2(3H)-ylidene]
amino}-2-fluorophenyl) piperazine-1-carboxylate (7) The
mixture of compound
5 (10 mmol) and 4-chloro-
phenacylbromide (10 mmol) in absolute ethanol was
refluxed in the presence of dried sodium acetate (50 mmol)
for 11 h. Then, the reaction mixture was cooled to room
temperature and the precipitated salt was removed by fil-
tration. After evaporating the solvent under reduced pres-
sure, a solid appeared. This crude product recrystallized
with ethyl acetate: petroleum ether (1:2). Yield: 40 %, M.
p: 162–163 °C. FT-IR (KBr, m, cm-1): 1697 (C=O), 1429
Ethyl 4-(4-{[(benzylamino)carbonyl]amino}-2-fluorophenyl)
piperazine-1-carboxylate (5) The mixture of compound 3
(10 mmol) and benzylisothiocyanate (10 mmol) in abso-
lute ethanol was refluxed for 10 h. On cooling the reaction
mixture to room temperature, a solid formed. This crude
product was collected by filtration and recrystallized from
ethanol. Yield: 93 %. M.p: 153–155 °C. FT-IR (KBr, m,
cm-1): 3346, 3284 (2NH), 3063 (ar–CH), 1694, 1638 (2C=
O), 1236 (C–O). Elemental analysis for C21H25FN4O3
calculated (%): C, 62.99, H, 6.29; N, 13.99. Found (%): C,
(C=N),
1209
(C–O).
Elemental
analysis
for
C23H28ClFN4O3 calculated (%): C, 65.10, H, 5.28; N, 10.
47. Found (%): C, 65.14; H, 5.39; N, 10.49. 1H NMR
(DMSO-d6, d ppm): 1.17 (t, 3H, CH3, J = 7.6 Hz), 2.85 (s,
4H, 2CH2), 3.47 (s, 4H, 2CH2), 4.04 (q, 2H, CH2, J = 6.
2 Hz), 4.26 (brs, 2H, CH2), 6.85–6.94 (m, 4H, arH ? CH),
1
62.78; H, 6.07; N, 14.04. H NMR (DMSO-d6, d ppm): 1.
17 (t, 3H, CH3, J = 7.6 Hz), 2.85 (s, 4H, 2CH2), 3.40 (s,
123

Med Chem Res
7.28 (brs, 8H, arH), 7.45 (s, 1H, arH). 13C NMR (DMSO-
d6, d ppm): 15.27 (CH3), 43.36 (2CH2), 44.14 (2CH2), 51.
21 (CH2), 61.52 (CH2), 96.76 (CH), arC: [106.66 (d, CH,
JC–F = 25.6 Hz), 114.13 (CH), 120.50 (CH), 124.20
(2CH), 124.97 (2CH), 127.38 (CH), 127.78 (2CH), 128.97
(2CH), 133.90 (d, C, JC–F = 21.9 Hz), 137.14 (d, C,
JC–F = 11.0 Hz), 141.05 (2C), 155.28 (C), 155.63 (d, C,
JC–F = 240.5 Hz)], 155.91 (C ? C=O), 162.27 (C=N). MS
m/z (%): 535.12 ([M]?, 14), 479.16 (100), 423.16 (97),
138.12 (50).
arH), 6.83 (t, 1H, arH, J = 9.8 Hz), 9.09 (s, 2H, 2NH). 13C
NMR (DMSO-d6, d ppm): 15.27 (CH3), 43.09 (CH2), 44.30
(CH2), 46.04 (CH2), 51.78 (2CH2), 61.48(CH2), arC: [101.
10 (d, CH, J = 24.1 Hz), 108.53 (CH), 121.70 (CH), 130.
00 (d, C, JC–F = 9.5 Hz), 146.18 (d, C, JC–F = 10.0 Hz),
157.03 (d, C, JC–F = 240.9 Hz)], 155.26 (C=O), 169.97
(C=O). MS m/z (%): 380.47 ([M?2?K]?,100), 379.41
([M?1 ? K]?, 30), 267.22 ([M–CH2CONHNH2]?, 33),
234.18 (28).
Ethyl
4-(2-fluoro-4-{[2-(2-{[(4-fluorophenyl)amino]car-
Ethyl 4-{4-[(2-ethoxy-2-oxoethyl)amino]-2-fluorophenyl}
piperazine-1-carboxylate (8) To the mixture of com-
pound 3 (10 mmol) and triethylamine (10 mmol) in dry
tetrahydrofurane, ethylbromoacetate (10 mmol) was added
drop by drop at 0–5 °C. Then, the reaction mixture was
allowed to reach room temperature and stirred for 14 h (the
progress of the reaction was monitored by TLC). The
precipitated triethylammonium salt was removed by fil-
tration and the resulting solution was evaporated under
reduced pressure to dryness. The obtained yellow solid was
recrystallized from ethanol:water (1:2). Yield: 50.2 %. M.
p: 71–73 °C. FT-IR (KBr, m, cm-1): 3383 (NH), 1719 (C=
O), 1697 (C=O), 1220 (C–O). Elemental analysis for
C17H24FN3O4 calculated (%): C, 57.78; H, 6.85; N, 11.89.
Found (%): C, 57.74; H, 6.77; N, 11.97. 1H NMR (DMSO-
d6, d ppm): 1.35 (t, 6H, 2CH3, J = 7.0 Hz), 2.95 (s, 4H,
2CH2), 3.60 (s, 6H, 3CH2), 4.24 (q, 4H, 2CH2, J = 7.
0 Hz), 5.24 (s, 1H, NH), 6.44–6.59 (m, 2H, arH), 6.94–7.05
(m, 1H, arH). 13C NMR (DMSO-d6, d ppm): 14.80 (CH3),
15.24 (CH3), 44.23 (CH2), 45.49 (2CH2), 51.33 (CH2), 51.
75 (CH2), 61.01 (CH2), 61.52 (CH2), arC: [101.06 (d, CH,
JC–F = 24.1 Hz), 121.47 (d, CH, JC–F = 4.0 Hz), 121.67
(d, CH, JC–F = 4.0 Hz), 129.97 (d, C, JC–F = 9.9 Hz),
145.96 (d, C, JC–F = 10.6 Hz), 157.02 (d, C, JC–F = 240.
9 Hz)], 155.29 (C=O), 171.90 (C=O). MS m/z(%): 376.34
([M?Na]?, 75), 354.38 ([M?1]?,100), 222.17 (22), 149.
03 (49).
bonothioyl}hydrazino)-2-oxoethyl]amino}phenyl)piper-
azine-1-carboxylate (10) The solution of compound 9
(10 mmol) in absolute ethanol was refluxed with 4-fluor-
ophenylisothiocyanate (10 mmol) for 10 h. On cooling the
reaction mixture to room temperature, an oily product
appeared. This was recrystallized from butyl acetate: ethyl
ether (1:2). Yield: 50 %. M.p: 78–80 °C. FT-IR (KBr, m,
cm-1): 3225 (2NH ? NH2), 1671 (2C=O), 1210 (C–O).
Elemental analysis for C22H26F2N6O3S calculated (%): C,
53.66; H, 5.32; N, 17.06. Found (%): C, 53.78; H, 5.47; N,
17.14. 1H NMR (DMSO-d6, d ppm): 1.19 (brs, 3H, CH3), 2.
78 (s, 4H, 2CH2), 3.35 (s, 4H, 2CH2), 3.77 (brs, 2H, CH2), 4.
06 (brs, 2H, CH2), 5.91 (brs, 2H, 2NH), 6.35 (brs, 2H, arH),
6.83 (brs, 1H, arH), 7.17 (brs, 2H, arH), 7.39 (brs, 2H, arH),
9.56 (brs, 1H, NH), 9.69 (brs, 1H, NH), 10.08 (brs, 1H, NH).
13C NMR (DMSO-d6, d ppm): 17.45 (CH3), 43.56 (CH2),
46.49 (CH2), 53.96 (2CH2), 63.67 (CH2), 67.10 (CH2), arC:
[105.40 (d, CH, JC–F = 40.1 Hz), 114.19 (CH), 118.62 (d,
CH, JC–F = 36.6 Hz), 121.70 (2CH), 124.54 (2CH), 128.55
(d, C, JC–F = 36.4 Hz), 140.19 (d, CH, JC–F = 37.0 Hz),
150.36 (d, C, JC–F = 184.7 Hz), 157.43 (2C)], 168.24 (C=
O), 172.66 (C=O), 190.04 (C=S).
Ethyl 4-[4-({2-[2-(anilinocarbonothioyl)hydrazino]-2-oxo-
ethyl}amino)-2-fluorophenyl]
piperazine-1-carboxylate
(11) The mixture of compound 9 (10 mmol) and phen-
ylisothiocyanate (10 mmol) in absolute ethanol was heated
under reflux for 10 h. On cooling the reaction mixture to
room temperature, a white solid appeared. This crude
product was filtered off and recrystallized from ethanol.
Yield: 85 %, M.p: 160–163 °C. FT-IR (KBr, m, cm-1):
3340, 3256, 3193 (4NH), 1697 (C=O), 1633 (C=O), 1286
(C=S). Elemental analysis for C22H27FN6O3S calculated
(%): C, 55.68; H, 5.73, N, 17.71. Found (%): C, 55.98; H,
Ethyl 4-{2-fluoro-4-[(2-hydrazinyl-2-oxoethyl)amino]
phenyl}piperazine-1-carboxylate (9) Hydrazine hydrate
(25 mmol) was added to the solution of compound 8
(10 mmol) in ethanol and the mixture was heated under
reflux for 14 h. On cooling the mixture in cold overnight, a
white solid appeared. The crude product was filtered off
and recrystallized from ethyl acetate. Yield: 54 %. M.p:
153–155 °C. FT-IR (KBr, m, cm-1): 3313 (2NH ? NH2),
1675 (C=O), 1653 (C=O). Elemental analysis for
C15H22FN5O3 calculated (%): C, 53.09; H, 6.53; N, 20.64.
Found (%): C, 53.18; H, 6.79; N, 20.44. 1H NMR (DMSO-
d6, d ppm): 1.18 (t, 3H, CH3, J = 6.2 Hz), 2.77 (s, 4H,
2CH2), 3.37 (s, 4H, 2CH2), 4.05 (d, 2H, CH2, J = 7.0 Hz),
4.24 (s, 2H, CH2), 5.93 (brs, 2H, NH2), 6.25–6.39 (m, 2H,
1
5.78; N, 17.87. H NMR (DMSO-d6, d ppm): 1.19 (t, 3H,
CH3, J = 7.0 Hz), 2.78 (s, 4H, 2CH2), 3.47 (s, 4H, 2CH2),
3.77 (s, 2H, CH2), 4.04 (q, 2H, CH2, J = 7.2 Hz), 6.34–6.
51 (m, 2H, arH), 6.80–6.85 (m, 1H, arH), 7.17 (s, 1H, arH),
7.34–7.38 (d, 4H, arH, J = 8.2 Hz), 9.56 (s, 1H, NH), 9.69
(s, 1H, NH), 10.12 (s, 2H, 2NH). 13C NMR (DMSO-d6, d
ppm): 15.29 (CH3), 44.25 (CH2), 45.92 (CH2), 51.83
(2CH2), 61.51 (2CH2), arC: [101.29 (d, CH, JC–F = 24.
123

Med Chem Res
1 Hz), 108.72 (CH), 121.68 (CH), 125.92 (2CH), 126.48
(CH), 128.82 (2CH), 139.70 (C), 146.20 (d, C, JC–F = 10.
0 Hz), 154.00 (d, C, JC–F = 63.3 Hz), 157.35 (d, C,
JC–F = 209.8 Hz)], 168.64 (C=O), 170.64 (C=O), 181.58
(C=S). MS m/z (%): 475.41 ([M?1]?, 32), 414.53 (26),
413.53 (100), 149.03 (32).
[101.18 (d, CH, JC–F = 9.5 Hz), 108.66 (CH), 116.98 (d,
CH, JC–F = 23.0 Hz), 121.53 (C), 130.31 (d, C, JC–F = 10.
2 Hz), 131.07 (2CH), 131.25 (2CH), 145.33 (d, C, JC–F
=
10.6 Hz), 153.16 (d, C, JC–F = 213.8 Hz), 159.87 (d, C,
JC–F = 57.2 Hz)], 151.02 (C), 165.34 (C=O), 168.98
(C=S).
Ethyl 4-(4-{[(5-anilino-1,3,4-thiadiazol-2-yl)methyl]amino}-
2-fluorophenyl)piperazine-1-carboxylate (12) Concentrated
sulfuric acid (64 mmol) was added to compound 11
(10 mmol) dropwise while stirring, and the reaction mix-
ture was stirred in an ice bath for 15 min. Then, the mixture
was allowed to reach room temperature and stirred for
additional 2 h. The resulting solution was poured into ice
cold water and made alkaline (pH 8) with ammonia. The
precipitated product was filtered, washed with water, and
recrystallized from dimethysulfoxide:water (1:3). Yield
74 %. M.p: 93–95 °C. FT-IR (KBr, m, cm-1): 3257 (2NH),
1677 (C=O), 1433 (C=N). Elemental analysis for
C22H25F2N6O2S calculated (%): C, 57.88; H, 5.52; N, 18.
41. Found (%): C, 58.08; H, 5.75; N, 18.78. 1H NMR
(DMSO-d6, d ppm): 1.17 (t, 3H, CH3, J = 7.0 Hz), 2.78 (s,
4H, 2CH2), 3.46 (s, 6H, 3CH2 ? H2O), 4.03 (q, 2H, CH2,
J = 7.2 Hz), 4.48 (s, 1H, NH), 6.37–6.51 (m, 2H, arH), 6.
80–6.99 (m, 2H, arH), 7.17 (brs, 1H, NH), 7.27–7.33 (m,
3H, arH), 7.56 (d, 1H, arH, J = 7.8 Hz). 13C NMR
(DMSO-d6, d ppm): 14.47 (CH3), 42.36 (CH2), 43.37
(CH2), 45.14 (CH2), 50.03 (CH2), 50.92 (CH2), 60.72
(CH2), arC: [108.11 (d, CH, JC–F = 12.4 Hz), 116.95 (d,
CH, JC–F = 19.4 Hz), 121.30 (d, CH, JC–F = 33.3 Hz),
128.03 (CH), 128.75 (2CH), 128.96 (2CH), 129.53 (d, C,
JC–F = 9.5 Hz), 140.52 (C), 144.63 (d, C, JC–F = 10.
6 Hz), 156.51 (d, C, JC–F = 204.2 Hz)], 160.77 (C), 164.
32 (C), 169.87 (C=O). MS m/z (%): 458.16 ([M?2]?, 27),
457.16 ([M?1]?, 100).
Ethyl 4-(2-fluoro-4-{[(4-phenyl-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-3-yl)methyl] amino}phenyl)piperazine-1-car-
boxylate (14) A solution of compound 11 (10 mmol) in
ethanol water (1:1) was refluxed in the presence of 2 N
NaOH for 3 h. Then, the resulting solution was cooled to
room temperature and acidified to pH 7 with 37 % HCl.
The precipitate formed was filtered off, washed with water,
and recrystallized from ethyl acetate. Yield 70 %. M.p:
206–208 °C. FT-IR (KBr, m, cm-1): 3248, 3117 (2NH),
3049 (ar CH), 1660 (C=O), 1250 (C=S). Elemental analysis
for C22H25FN6O2S calculated (%): C, 57.88; H, 5.52; N,
1
18.41. Found (%): C, 57.51; H, 5.45; N, 18.49. H NMR
(DMSO-d6, d ppm): 1.13 (t, 3H, CH3, J = 7.4 Hz), 2.73 (s,
4H, 2CH2), 3.42 (s, 4H, 2CH2), 3.99 (s, 4H, 2CH2), 6.25–6.
32 (m, 2H, arH ? NH), 6.76–6.80 (m, 1H, arH), 7.36 (s,
2H, ar–H), 7.49 (brs, 4H, ar–H), 10.45 (s, 1H, NH). 13C
NMR (DMSO-d6, d ppm): 15.25 (CH3), 31.39 (CH2), 44.27
(2CH2), 51.68 (2CH2), 61.49 (CH2), arC: [101.27 (d, CH,
JC–F = 24 Hz), 108.63 (CH), 121.59 (CH), 128.76 (CH),
130.05 (2CH), 130.15 (2CH), 134.09 (2C), 145.50 (C),
150.92 (C)], 155.25 (C), 168.75 (C=S ? C=O). MS m/z
(%): 480.48 ([M?1 ? Na]?, 29), 479.54 ([M?Na]?, 100),
457.41 ([M?1]?, 85).
({[(6R,7R)-3-[(Acetyloxy)methyl]-7-({[3-[({4-[4-(ethoxy-
carbonyl)piperazin-1-yl]-3-fluorophenyl}amino)methyl]-
4-(4-fluorophenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-
1-yl]methyl}amino)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-
en-2-yl]carbonyl}oxy)triethyl ammonium (15) 7-Aca
(10 mmol) was added to the mixture of compound 13
(10 mmol), triethylamine (20 mmol), and formaldehyde
(50 mmol) in tetrahydrofurane, and the mixture was stirred
at room temperature 4 h. After removing the solvent under
reduced pressure, an oily product appeared. This was re-
crystallized from ethanol:water (1:2). Yield: 43 %. M.p:
68–70 °C. FT-IR (KBr, m, cm-1): 3359, 3263 (2NH), 3075
(ar–CH), 2988, 2973 (aliphatic CH), 1680, 1629 (4C=O),
1228 (C=S). Elemental analysis for C39H51F2N9O7S2 cal-
culated (%): C, 54.47; H, 5.98; N, 14.66. Found (%): C, 54.
Ethyl 4-[2-fluoro-4-({[4-(4-fluorophenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-3-yl]methyl}amino)phenyl]piper-
azine-1-carboxylate (13) A solution of compound 10
(10 mmol) in water was refluxed in the presence of 2 N
NaOH for 3 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. Yield: 61 %. M.p:
100–101 °C. FT-IR (KBr, m, cm-1): 1675 (C=O), 1244 (C=
1
S). H NMR (DMSO-d6, d ppm): elemental analysis for
1
C22H24F2N6O2S calculated (%): C, 55.68; H, 5.10; N, 17.
71. Found (%): C, 55.54; H, 5.28; N, 17.89. 1H NMR
(DMSO-d6, d ppm): 1.18 (brs, 3H, CH3) 2.78 (brs, 4H,
2CH2), 3.41 (brs, 4H, 2CH2), 4.08 (brs, 4H, 2CH2), 5.87
(brs, 2H, 2NH), 6.27 (brs, 2H, arH), 6.79 (brs, 1H, arH), 7.
45 (brs, 4H, arH). 13C NMR (DMSO-d6, d ppm): 15.26
(CH3), 44.25 (2CH2), 51.67 (2CH2), 61.50 (2CH2), arC:
70; H, 5.74; N, 14.55. H NMR (DMSO-d6, d ppm): 1.10
(brs, 12H, 4CH3) 1.74 (s, 3H, CH3), 2.86 (brs, 4H, 2CH2),
3.20 (s, 6H, 3CH2), 3.58 (brs, 6H, 3CH2), 4.04 (brs, 2H,
CH2), 4.52 (brs, 2H, CH2), 4.67 (s, 4H, 2CH2), 4.89 (s, 2H,
2CH), 5.42 (s, 2H, 2NH), 6.51 (brs, 2H, arH), 6.89 (brs,
1H, arH), 7.35–7.44 (m, 4H, arH). 13C NMR (DMSO-d6, d
ppm): 9.01 (3CH3), 15.04 (CH3), 23.44 (CH3), 25.69
123

Med Chem Res
(CH2), 44.05 (2CH2), 46.25 (CH2), 49.16 (3CH2), 51.29
(CH2), 51.56 (2CH2), 54.70 (2CH), 61.89 (CH2), 67.78
(CH2), arC: [103.99 (d, CH, JC–F = 12.45 Hz), 110.89
(CH), 117.08 (d, CH, JC–F = 23.45 Hz), 120.97 (2CH),
131.04 (2CH), 131.69 (C), 131.88 (C), 143.85 (d, C,
JC–F = 9.85 Hz), 154.78 (d, C, JC–F = 92.61 Hz), 162.96
(d, C, JC–F = 246.0 Hz)], 130.41 (C), 130.49 (C), 150.18
(triazole-C), 165.79 (C=O), 168.64 (C=O), 168.86 (C=S),
171.93 (C=O), 175.76 (C=O).
1764 (C=O), 1692 (C=O), 1609 (C=O), 1230 (C–O). Ele-
mental analysis for C37H52FN9O7S2 calculated (%): C, 56.
54; H, 6.67; N, 16.04. Found (%): C, 56.65; H, 6.79; N, 16.
1
87. H NMR (DMSO-d6, d ppm): 1.13 (t, 12H, 4CH3, J =
6.2 Hz), 1.39 (brs, 3H, CH3), 1.42 (brs, 3H, CH3), 3.02 (q,
8H, 4CH2, J = 7.0 Hz), 3.43 (s, 8H, 4CH2), 3.73 (brs, 2H,
CH2), 4.56 (s, 2H, 2CH), 5.41 (s, 2H, CH2), 6.24 (s, 1H,
CH), 6.77 (brs, 1H, NH), 7.36 (brs, 3H, ar–H), 7.50 (s, 5H,
ar–H). 13C-NMR (DMSO-d6, d ppm): 8.99 (3CH3), 14.53
(CH3), 27.13 (2CH3), 43.49 (2CH2), 44.96 (2CH2), 50.58
(CH2), 50.70 (3CH2), 50.94 (2CH2), 60.75 (C-(CH3)2), 70.
39 (CH), 73.89 (CH), 81.90 (CH), arC: [100.44 (d, CH,
JC–F = 24.1 Hz), 108.87 (d, CH, JC–F = 213.1 Hz), 120.
53 (d, CH, JC–F = 60.2 Hz), 128.18 (CH), 129.57 (2CH),
129.64 (2CH), 133.79 (d, C, JC–F = 14.9 Hz), 144.08 (d,
C, JC–F = 99.5 Hz), 146.84 (d, C, JC–F = 442.1 Hz)] 149.
26 (C), 154.53 (C), 156.88 (C=S), 167.90 (C=O), 168.09
(C=O), 170.16 (C=O).
[({(6R,7R)-3-[(Acetyloxy)methyl]-7-[({3-[({4-[4-(ethoxy-
carbonyl)piperazin-1-yl]-3-fluorophenyl}amino)methyl]-
4-phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-1-yl}me-
thyl)amino]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-2-
yl}carbonyl)oxy](triethyl)ammonium (16) To the mix-
ture of compound 14 (10 mmol), triethylamine (20 mmol)
and formaldehyde (50 mmol) in tetrahydrofurane, 7-aca
(10 mmol) was added. The mixture was stirred at room
temperature 4 h. After removing the solvent under reduced
pressure, an oily product appeared. This product recrystal-
lized ethyl acetate:hexane (1:2). Yield: 47 %. M.p:
64–66 °C. FT-IR (KBr, m, cm-1): 3662 (OH), 3374 (NH),
2988, 2901 (aliphatic CH), 1762 (C=O), 1687 (2C=O), 1629
(C=O), 1227 (C=S). Elemental analysis for C39H52FN9O7S2
calculated (%): C, 55.63; H, 6.22; N, 14.97. Found (%): C,
55.87; H, 6.33; N, 15.05. 1H NMR (DMSO-d6, d ppm): 1.11
(t, 12H, 4CH3, J = 7.0 Hz), 1.99 (s, 3H, CH3), 2.99 (q, 8H,
4CH2, J = 8.0 Hz), 3.87 (brs, 10H, 5CH2), 4.55 (s, 2H,
CH2), 4.68–4.80 (m, 4H, 2CH2), 5.40 (s, 2H, CH), 6.22 (brs,
2H, 2NH), 7.33 (brs, 3H, ar–H), 7.50–7.75 (m, 5H, ar–
H).13C-NMR (DMSO-d6, d ppm): 9.31 (3CH3), 15.22 (CH3),
21.38 (CH3), 25.79 (CH2), 41.30 (2CH2), 44.17 (2CH2), 45.
79 (3CH2), 51.40 (CH2), 51.64 (CH2), 61.49 (CH2), 66.68
Ethyl 4-[4-(3-{2-[5-(4-chlorophenyl)-3-phenyl-1,3-thiazol-
2(3H)-ylidene]hydrazino}-3-oxoethyl)-2-fluorophenylamino]
piperazine-1-carboxylate (18) The mixture of compound
11 (10 mmol) and 4-chlorophenacylbromide (10 mmol) in
absolute ethanol was refluxed in the presence of dried
sodium acetate (50 mmol) for 12 h. After removing the
solvent under reduced pressure, an orange solid appeared.
This product washed water and recrystallized ethanol.
Yield: 45 %. M.p: 60–62 °C. FT-IR (KBr, m, cm-1): 3345,
3259 (2NH), 3054 (ar–CH), 1677 (C=O), 1628 (C=O).
Elemental analysis for C30H30ClFN6O3S calculated (%): C,
59.15; H, 4.96; N, 13.80. Found (%): C, 59.05; H, 5.06; N,
1
13.87. H NMR (DMSO-d6, d ppm): 1.15 (brs, 3H, CH3),
2.76 (s, 4H, 2CH2), 3.61 (s, 6H, 3CH2 ? H2O), 4.03 (brs,
2H, CH2), 5.40 (s, 1H, NH), 6.44–6.54 (m, 1H, arH), 6.
84–6.96 (m, 2H, arH ? CH), 7.29–7.52 (m, 9H, arH), 7.95
(s, 1H, arH), 10.45 (s, 1H, NH). 13C NMR (DMSO-d6, d
ppm): 15.24 (CH3), 41.37 (CH2), 44.26 (CH2), 51.68
(CH2), 52.46 (2CH2), 61.48 (CH2), arC: [101.24 (d, CH,
J = 24.5 Hz), 108.66 (CH), 117.54 (2CH), 120.12 (C),
121.75 (2CH), 122.41 (2CH), 128.76 (CH), 129.71 (2CH),
130.37 (2CH), 130.76 (2C), 131.82 (C), 139.37 (2C), 146.
87 (d, C, JC–F = 133.95 Hz)], 155.26 (C=N), 158.92 (C=
O), 160.62 (C=O). MS m/z (%): 631.64 ([M-1 ? Na]?,
25), 464.59 (26), 463.58 (83), 441.62 (26), 360.57 (61),
267.31 (29), 195.00 (40), 149.00 (100), 135.03 (50), 121.06
(65).
(CH2), 67.69 (CH), 71.09 (CH), arC: [110.41 (d, CH, JC–F
=
34.2 Hz), 118.31 (d, CH, JC–F = 18.7 Hz), 123.22 (d, C,
JC–F = 22.1 Hz), 126.01 (CH), 128.74 (CH), 130.14 (2CH),
130.33 (2CH), 134.47 (d, C, JC–F = 7.3 Hz), 148.01 (d, C,
JC–F = 172.2 Hz), 149.99 (C)], 138.25 (2C), 155.28 (C),
166.21 (C=S), 169.90 (C=O), 170.92 (C=O), 171.19 (C=O),
172.95 (C=O).
[({(5R,6R)-6-[({3-[({4-[4-(Ethoxycarbonyl)piperazin-1-yl]-
3-fluorophenyl}amino)methyl]-4-phenyl-5-thioxo-4,5-dihy-
dro-1H-1,2,4-triazol-1-yl}methyl)amino]-3,3-dimethyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]hept-2-yl}carbonyl)oxy](tri-
ethyl)ammonium (17) To the mixture of compound 14
(10 mmol), triethylamine (20 mmol), and formaldehyde
(50 mmol) in tetrahydrofurane, 6-apa (10 mmol) was
added. The mixture was stirred at room temperature 4 h.
After removing the solvent under reduced pressure, an oily
product appeared. This product recrystallized ethyl acetate:
hexane (1:2). Yield: 41 %, M.p: 64–66 °C. FT-IR (KBr, m,
cm-1): 3393 (NH), 3073 (ar–CH), 2980 (aliphatic CH),
Ethyl 4-[2-fluoro-4-({2-[2-(3-hydroxy-4-methoxybenzyli-
dene)hydrazino]-2-oxoethyl} amino)phenyl]piperazine-1-
carboxylate (19a) The mixture of solution of compound
9 (10 mmol) and 3-hydroxy-4-methoxybenzaldehyde
(10 mmol) in absolute ethanol was irradiated by micro-
wave at 200 W and 140 °C for 30 min. On cooling the
123

Med Chem Res
Table 1 Screening for antimicrobial activity of the compounds
(50 lL)
Table 2 Inhibitory activities of the synthesized compounds against
Jack Bean urease
Comp. no Microorganisms and inhibition zone (mm)
Compound
% Inhibition S.D.
100 0.1
IC50 S.D.
54.56 4.17
Ec
Yp
Pa
Sa
Ef
Bc
Ms
Ca
Sc
Thiourea
2
a
b
-
–
2
–
–
–
–
8
–
–
6
–
8
–
–
–
–
–
–
–
–
–
–
6
–
9
–
–
–
–
–
8
8
18
–
–
8
–
–
–
–
6
–
–
–
–
–
–
–
–
6
–
6
–
8
6
6
–
6
–
–
15
18
–
–
6
6
–
–
–
8
–
–
–
3
11 3.3
–
3
11
–
–
–
15
8
15
8
4a
4b
4d
4e
4f
N.s.
N.s.
-
–
4a
4b
4c
4d
4e
4f
–
10
–
–
–
–
6
–
8
–
–
–
–
–
–
–
–
–
–
–
8
9
–
–
–
–
–
8
9
10
–
–
–
–
–
–
–
–
8
8
1
0.2
3.0
–
–
–
20
20
25
–
15
10
20
6
15
10
10
7
-
–
–
–
5
-
–
6
–
6
6
3
–
5
–
–
–
7
N.s.
7
–
6
–
–
–
–
–
–
8
3.1
3.0
1
–
7
–
–
–
–
–
–
9
7
–
8
–
–
6
–
–
–
10
12
14
15
17
18
19a
19b
19c
20
4
–
9
–
–
6
–
7
–
56
-
4
–
10
11
12
13
14
15
16
17
18
19a
19b
19c
20
21
22
Amp.
Strep.
Flu.
–
–
6
–
–
–
–
10
–
–
6
–
–
–
100 1.5
4.67 0.53
–
–
–
6
6
100 2.1
45.37 0.78
–
–
–
–
8
10
–
-
–
–
–
–
–
6
6
–
–
8
-
6
–
–
–
10
6
–
47 0.1
6
10
–
–
–
10
12
12
6
-
13
10
–
16
6
14
–
6
N.s.
–
8
8
–
–
9
N.s. Not soluble
a
No inhibition
–
–
–
–
8
–
b
–
8
–
–
8
6
Not determined
10
6
6
6
15
20
18
8
12
8
10
9
10
10
15
10
8
65; H, 6.06; N, 14.98. 1H NMR (DMSO-d6, d ppm): 1.17 (t,
3H, CH3, J = 6.8 Hz), 2.77 (s, 4H, 2CH2), 3.36 (s, 6H,
3CH2), 3.78 (s, 3H, O–CH3), 3.99 (q, 2H, CH2, J = 6.
6 Hz), 5.80 (brs, 1H, NH), 6.04 (brs, 1H, NH), 6.32–6.37
(m, 3H, arH), 6.84–6.98 (m, 3H, arH), 9.27 (s, 1H, N=CH),
11.35 (s, 1H, OH). 13C NMR (DMSO-d6, d ppm): 15.26
(CH3), 44.29 (CH2), 44.62 (2CH2), 51.78 (2CH2), 56.22
(OCH3), 61.48 (CH2), arC: [101.23 (d, CH, JC–F = 22.
0 Hz), 108.47 (CH), 112.58 (d, CH, JC–F = 15.0 Hz), 120.
73 (CH), 120.96 (CH), 121.72 (CH), 127.64 (C), 129.83 (d,
C, JC–F = 9.1 Hz), 146.25 (C), 146.46 (C), 150.34 (d, C,
JC–F = 6.5 Hz), 151.36 (d, C, JC–F = 388.7 Hz)], 144.44
(N=CH), 167.17 (C=O), 171.66 (C=O). MS m/z (%): 497.
56 ([M?1 ? Na]?, 31) 496.56 ([M?Na]?,100), 370.41
(19), 360.65 (22).
12
35
10
35
25 [25
(–), no activity
Ec, Escherichia coli ATCC 25922; Yp, Yersinia pseudotuberculosis
ATCC 911; Pa, Pseudomonas aeruginosa ATCC 43288; Sa, Staph-
ylococcus aureus ATCC 25923; Ef, Enterococcus faecalis ATCC
29212; Bc, Bacillus cereus 702 Roma; Ms, M. smegmatis ATCC607;
Ca, Candida albicans ATCC 60193; Sc, Saccharomyces cerevisiae
RSKK 251; Amp., Ampicillin; Strep., Streptomycin; Flu.,
Fluconazole
reaction mixture to room temperature a solid was appeared.
This crude product was recrystallized from ethanol. Yield:
72 %. M.p: 183–185 °C. FT-IR (KBr, m, cm-1): 3342,
3181 (2NH), 3096 (ar–CH), 1678 (2C=O), 1437 (C=N),
1211 (C–O). Elemental analysis for C23H28FN5O5 calcu-
lated (%): C, 58.34; H, 5.96; N, 14.79. Found (%): C, 58.
Ethyl 4-[2-fluoro-4-({2-oxo-2-[2-(pyridin-4-ylmethylene)
hydrazino]ethyl}amino)phenyl] piperazine-1-carboxylate
(19b) The mixture of compound 9 (10 mmol) and
123

Med Chem Res
Table 3 Porcine pancreatic
lipase inhibitory activity of
synthesized compounds
2-hydroxybenzaldehyde (10 mmol) in absolute ethanol was
irradiated by microwave at 200 W and 140 °C for 30 min.
On cooling the reaction mixture to room temperature a
solid was appeared. This crude product was recrystallized
from ethanol. Yield: 50 %. M.p: 155–157 °C. FT-IR (KBr,
m, cm-1): 3675 (OH), 3357, 3270 (2NH), 3059 (ar–CH),
1707, 1676 (2C=O), 1428 (C=N), 1230 (C–O). Elemental
analysis for C22H26FN5O4 calculated (%): C, 59.58; H, 5.
Compound no.
% Inhibition
2
–
3
–
5
–
6
16
33
22
20
–
7
8
1
9
91; N, 15.79. Found (%): C, 59.72; H, 6.16; N, 15.77. H
10
NMR (DMSO-d6, d ppm): 1.17 (brs, 3H, CH3), 2.78 (s, 4H,
2CH2), 3.45 (s, 6H, 3CH2), 4.02–4.03 (m, 2H, CH2), 6.39
(brs, 2H, 2NH), 6.85 (brs, 4H, arH), 7.41 (brs, 3H, arH), 8.
70 (s, 1H, N=CH), 10.56 (brs, 1H, OH). 13C NMR (DMSO-
d6, d ppm): 15.25 (CH3), 41.29 (CH2), 44.18 (2CH2), 51.51
(2CH2), 61.52 (CH2), arC: [108.24 (CH), 116.79 (d, CH,
JC–F = 36.2 Hz), 119.18 (C), 120.18 (CH), 122.19 (d, CH,
JC–F = 53.4 Hz), 126.61 (CH), 131.22 (CH), 132.68 (CH),
137.00 (C), 141.26 (d, C, JC–F = 10.6 Hz), 152.71 (d, C,
JC–F = 252.9 Hz), 157.86 (C)], 146.15 (N=CH), 159.33
(C=O), 163.12 (C=O). MS m/z (%): 466.51 ([M?1?Na]?,
16), 444.55 ([M?1]?, 25), 249.20 (19), 241.19 (18), 149.
03 (100), 135.07 (33), 121.06 (45), 103.04 (40).
11
–
12
68
63
75
73
6
13
14
15
16
17
–
18
1
19a
–
19b
–
19c
–
20
33
99
–
Orlistat
Ethyl 4-(2-fluoro-4-{[(5-thioxo-4,5-dihydro-1,3,4-oxadia-
zol-2-yl)methyl]amino}phenyl) piperazine-1-carboxylate
(20) The mixture of compound 9 (10 mmol) and carbon
disulfide (20 mmol) in absolute ethanol was refluxed in the
presence of dried potassium hydroxide (10 mmol) for 13 h.
Then, the resulting solution was cooled to room tempera-
ture and acidified with acetic acid. The precipitate formed
was filtered off, washed with water, and recrystallized from
ethyl acetate:petroleum ether (1:3) Yield 68 %. M.p:
210–212 °C. FT-IR (KBr, m, cm-1): 3300 (2NH), 1675 (C=
O), 1428 (C=N), 1249 (C=S). Elemental analysis for
C16H20FN5O3S calculated (%): C, 50.38; H, 5.29; N, 18.36.
Found (%): C, 50.51; H, 5.66; N, 18.74. 1H NMR (DMSO-
d6, d ppm): 1.17 (t, 3H, CH3, J = 6.6 Hz), 2.77 (s, 4H,
2CH2), 3.47 (s, 2H, CH2), 4.03 (q, 2H, CH2, J = 7.0 Hz),
4.34 (d, 2H, CH2, J = 5.0 Hz), 6.33–6.52 (m, 4H, ar-
2H ? 2NH), 6.85 (t, 1H, arH, J = 8.6 Hz). 13C NMR
(DMSO-d6, d ppm): 15.25 (CH3), 41.37 (2CH2), 44.25
(2CH2), 51.64 (CH2), 61.50 (CH2), arC: [101.41 (d, CH,
JC–F = 24.1 Hz), 108.78 (CH), 121.78 (CH), 130.67 (d, C,
JC–F = 9.9 Hz), 144.97 (d, C, JC–F = 10.6 Hz), 156.95 (d,
C, JC–F = 241.9 Hz)], 155.28 (C=O), 163.00 (C), 185 (C=
S).
DMSO control
Positive control
All compounds were screened at
concentration of 6.25 lg mL-1
–
pyridine-4-carbaldehyde (10 mmol) in absolute ethanol was
irradiated by microwave at 200 W and 140 °C for 30 min.
On cooling the reaction mixture to room temperature a solid
was appeared. This crude product was recrystallized from
ethanol. Yield: 85 %. M.p: 184–185 °C. FT-IR (KBr, m,
cm-1): 3356, 3269 (2NH), 3057 (ar–CH), 1707, 1679 (2C=
O), 1428 (C=N), 1230 (C–O). Elemental analysis for
C21H25FN6O3 calculated (%): C, 58.87; H, 5.88; N, 19.61.
Found (%): C, 58.97; H, 6.00; N, 19.97. 1H NMR (DMSO-d6,
d ppm): 1.16 (brs, 3H, CH3), 2.76 (s, 4H, 2CH2), 3.41 (s, 4H,
2CH2), 4.02–4.03 (m, 2H, CH2), 4.21 (s, 2H, CH2), 6.35–6.
51 (m, 2H, arH), 6.83 (brs, 1H, arH), 7.69 (brs, 2H, arH), 8.63
(s, 3H, 2arH ? CH), 11.80 (s, 2H, 2NH). 13C NMR (DMSO-
d6, d ppm): 15.26 (CH3), 47.25 (CH2), 51.79 (2CH2), 52.85
(2CH2), 61.37 (CH2), arC: [107.70 (d, CH, JC–F = 45.1 Hz),
114.07 (C), 118.26 (d, CH, JC–F = 29.3 Hz), 120.15 (CH),
124.56 (2CH), 137.02 (C), 141.37 (d, C, JC–F = 50.6 Hz),
146.20 (2CH), 152.26 (d, C, JC–F = 161.2 Hz)], 150.31 (N=
CH), 160.00 (C=O), 166.71 (C=O). MS m/z (%): 467.51
([M?K]?, 19) 451.55 ([M?Na]?,65), 429.53 ([M?1]?, 76),
267.35 (45), 201.02 (23), 162.98 (25), 160.98 (30), 149.03
(100), 135.01 (72), 118.99 (71).
({[(6R,7R)-3-[(Acetyloxy)methyl]-7-({[5-[({4-[4-(ethoxy-
carbonyl)piperazin-1-yl]-3-fluorophenyl}amino)methyl]-
2-thioxo-1,3,4-oxadiazol-3(2H)-yl]methyl}amino)-8-oxo-5-
thia-1-azabicyclo[4.2.0]oct-2-en-2-yl]carbonyl}oxy)(triethyl)
ammonium (21) To the mixture of compound 20
(10 mmol), triethylamine (20 mmol) and formaldehyde
Ethyl 4-[2-fluoro-4-({2-[2-(2-hydroxybenzylidene)hydra-
zino]-2-oxoethyl}amino)phenyl] piperazine-1-carboxylate
(19c) The mixture of compound 9 (10 mmol) and
123

Med Chem Res
Antimicrobial activity assessment
(50 mmol) in tetrahydrofurane, 7-aca (10 mmol) was
added. The mixture was stirred at room temperature 4 h.
After removing the solvent under reduced pressure, a liquid
product appeared. This was recrystallized by column
chromatography (n-hexane:ethyl acetate, 4:1). Yield 58 %.
FT-IR (KBr, m, cm-1): 3373 (OH ? NH), 2980, 2974
(aliphatic CH), 1676 (4C=O), 1432 (C=N), 1232 (C=S).
Elemental analysis for C33H47FN8O8S2 calculated (%) C:
51.68; H: 6.18; N: 14.61. Found (%): C: 51.47; H: 6.00; N:
All bacterial and yeast strains were obtained from the Hi-
fzissihha Institute of Refik Saydam (Ankara, Turkey) and
were as follows: Pseudomonas aeruginosa ATCC 27853,
Enterococcus faecalis ATCC 29212, S. aureus ATCC
25923, B. cereus 709 ROMA, Ms: M. smegmatis
ATCC607, C. albicans ATCC 60193, Sc: S. cerevisiae
RSKK 251. All the newly synthesized compounds were
dissolved in dimethyl sulfoxide (DMSO) and ethanol to
1
14.67. H-NMR (DMSO-d6) d ppm: 1.12 (t, 12H, 4CH3,
prepare chemicals of stock solution of 10 mg mL-1
.
J = 7.0 Hz), 1.99 (s, 3H, CH3), 2.98–3.18 (m, 12H, 6CH2),
3.82 (brs, 8H, 4CH2), 4.00 (s, 2H, CH2), 4.56 (s, 2H, CH2),
4.65 (s, 1H, CH), 5.19 (s, 1H, CH), 6.40 (brs, 2H, 2NH), 6.
90 (brs, 1H, ar–H), 6.94 (brs, 2H, ar–H). 13C-NMR
(DMSO-d6) d ppm: 9.33 (3CH3), 15.15 (CH3), 21.39
(CH3), 25.75 (CH2), 40.94 (CH2), 43.66 (CH2), 44.04
(CH2), 46.26 (2CH2), 48.64 (CH2), 50.95 (3CH2), 61.71
(CH2), 67.38 (CH2), 67.73 (CH), 70.89 (CH), arC: [107.63
(d, CH, JC–F = 11.8 Hz), 113.45 (CH), 115.47 (CH), 120.
42 (d, C, JC–F = 34.7 Hz), 122.05 (C), 150.83 (d, C,
JC–F = 273.3 Hz)], 130.04 (C), 134.26 (C), 155.50 (C=O),
155.65 (C=O), 162.28 (C), 175.25 (2C=O), 189.74 (C=S).
Agar-well diffusion method
Simple susceptibility screening test using agar-well diffu-
sion method as adapted earlier (Ahmad et al., 1998) was
used. Each microorganism was suspended in Mueller–
Hinton (MH) (Difco, Detroit, MI, USA) broth and diluted
approximately to 106 colony forming unit (cfu) mL-1
.
They were ‘‘flood-inoculated’’ onto the surface of MH agar
and Sabouraud dextrose agar (SDA) (Difco, Detriot, MI,
USA) and then dried. For C. albicans and C. tropicalis,
SDA were used. Five-millimeter diameter wells were cut
from the agar using a sterile cork-borer, and 50 mL of the
extract substances was delivered into the wells. The plates
were incubated for 18 h at 35 °C. Antimicrobial activity
was evaluated by measuring the zone of inhibition against
the test organism. Ampicillin (10 mg) and Fluconazole
(5 mg) were used as standard drugs. Dimethyl sulfoxide
and ethanol were used as solvent controls. The antimicro-
bial activity results are summarized in Table 1.
({[(5R,6R)-6-({[5-[({4-[4-(Ethoxycarbonyl)piperazin-1-yl]-
3-fluorophenyl}amino)methyl]-2-thioxo-1,3,4-oxadiazol-
3(2H)-yl]methyl}amino)-3,3-dimethyl-7-oxo-4-thia-1-aza
bicyclo[3.2.0]hept-2-yl]carbonyl}oxy)(triethyl)ammonium
(22) To the mixture of compound 20 (10 mmol), trieth-
ylamine (20 mmol), and formaldehyde (50 mmol) in tet-
rahydrofurane, 6-apa (10 mmol) was added. The mixture
was stirred at room temperature 6 h. After removing the
solvent under reduced pressure, a liquid product appeared.
This was recrystallized by column chromatography
(n-hexane:ethyl acetate, 4:1). Yield 66 %. FT-IR (KBr, m,
cm-1): 3676 (OH), 2901, 2987 (aliphatic CH), 1768 (C=
O), 1683 (2 C=O), 1431 (C=N), 1231 (C=S). Elemental
analysis for C31H47FN8O6S2 calculated (%): C, 52.38; H, 6.
Urease inhibition assay
Reaction mixtures comprising 25 lL of Jack bean urease,
55 lL of 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 lL of the test compounds at room temper-
ature 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 reagent
(45 lL, 1 % w/v phenol, and 0.005 % w/v sodium nitro-
prusside) and alkali reagent (70 lL, 0.5 % w/v sodium
hydroxide, and0.1 %v/vNaOCl)wereaddedtoeachwell 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 for-
mula 100 - (OD test well/OD control) 9 100. Thiourea was
usedasthestandardinhibitor. InordertocalculateIC50 values,
different concentrations of synthesized compounds and stan-
dard were assayed at the same reaction conditions (Weather-
burn, 1967). The obtained results are presented in Table 2.
1
66; N, 15.76. Found (%): C, 52.18; H, 6.79; N, 15.55. H-
NMR (DMSO-d6, d ppm): 0.99–1.21 (m, 18H, 6CH3), 2.90
(q, 8H, 4CH2, J = 7.0 Hz), 3.38 (q, 8H, 4CH2, J = 7.
2 Hz), 3.98–4.08 (m, 4H, 2CH2), 4.55 (s, 1H, CH), 5.26 (s,
1H, CH), 5.30 (s, 1H, CH), 5.38, 5.45 (brs, 2H, 2NH), 6.80
(brs, 1H, ar–H), 6.94 (brs, 2H, ar–H). 13C-NMR (DMSO-
d6, d ppm): 9.32 (3CH3), 15.25 (CH3), 27.77 (CH3), 32.62
(CH3), 44.13 (CH2), 45.67 (2CH2), 51.09 (CH2), 51.50
(CH2), 52.61 (CH2), 56.73 (C–(CH3)2), 61.52 (CH2), 62.23
(CH2), 62.99 (CH2), 63.59 (CH2), 65.39 (CH), 67.00 (CH),
73.68 (CH), arC: [107.41 (d, CH, JC–F = 9.8 Hz),113.72
(d, CH, JC–F = 33.0 Hz), 120.07 (CH), 134,64 (d, C,
JC–F = 9.1 Hz), 143.12 (d, C, JC–F = 9.5 Hz), 154.47 (d,
C, JC–F = 81.2 Hz)], 163.63 (C), 170.45 (C=O), 170.91
(C=O), 172.13 (C=O), 175.29 (C=S).
123

Med Chem Res
Bektas H, Karaali N, Sahin D, Demirbas A, Alpay Karaoglu S,
Demirbas N (2010) Synthesis and antimicrobial activities of
some new 1,2,4-triazole derivatives. Molecules 15:2427–2438
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Med Chem 14:1819–1826
Anti-lipase activity assay
The inhibitory effects of those compounds were evaluated
against porcine pancreatic lipase (PPL) (15 ng mL-1).
Lipase activity assay was done according to Verger et al.,
(Woods et al., 2003). Microtiter plates were coated with
purified tung oil TAGs. Compounds were mixed with PPL
1:2 (v/v) and incubated for 30 min. The microtiter plates
containing purified tung oil, lipase solution, and assay
buffer (10 mM Tris–HCl buffer, pH 8.0, containing
150 mM NaCl, 6 mM CaCl2, 1 mM EDTA, and
3 mg mL-1b-cyclodextrin) were recorded continuously for
40 min against the buffer alone by using microplate reader
(SpectraMax M5, Molecular Devices) at 272 nm. The
inhibitory activity of those compounds and Orlistat, a
positive control against pancreatic lipase, were measured at
concentration of 6.25, 2.08, and 1.04 lg mL-1. Residual
activities were calculated by comparing to control without
inhibitor (T?). The assays were done in triplicate. The IC50
value was determined as the concentration of compound
that give 50 % inhibition of maximal activity. The results
are presented in Table 3.
˘
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Acknowledgments This Project was supported by Scientific and
Technological Research Council of Turkey (TUBITAK, Project No:
107T333) and Karadeniz Technical University, BAP, Turkey (Ref.
No. 8623) and is gratefully acknowledged.
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
Kategaonkar AH, Shinde PV, Kategaonkar AH, Pasale SK, Shingate
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new 2-chloro-3-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)quino-
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