Synthesis of some novel heterocyclic compounds derived from diflunisal hydrazide as potential anti-infective and anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2006.12.038

Three novel series of 2′,4′-difluoro-4-hydroxybiphenyl-3-carboxylic acid derivatives namely 4-substituted-1,2,4-triazoline-3-thiones (4a-g); 2-substituted-1,3,4-thiadiazoles (5a-g) and 2-substituted-1,3,4-oxadiazoles (6a-g) have been synthesized. Twenty-o

Full text of DOI:10.1016/j.ejmech.2006.12.038

European Journal of Medicinal Chemistry 42 (2007) 893e901
http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some novel heterocyclic compounds derived
from diflunisal hydrazide as potential anti-infective and
anti-inflammatory agents*
a
a
a
Sx. Guniz Kuc¸ukguzel a, , Ilkay Kuc¸ukguzel , Esra Tatar , Sevim Rollas ,
_
*
¨
¨ ¨ ¨ ¨ ¨
¨ ¨
¨
Fikrettin Sxahin b,c, Medine Gulluce b, Erik De Clercq d, Levent Kabasakal e
a
_
Marmara University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Tibbiye Cad. No. 49, Haydarpasa, Istanbul 34668, Turkey
b Ataturk University, Biotechnology Application and Research Center, Erzurum 25240, Turkey
c
_
Yeditepe University, Faculty of Engineering and Architecture, Department of Genetic and Bioengineering, Istanbul 34755, Turkey
d Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
e Marmara University, Faculty of Pharmacy, Department of Pharmacology, Tibbiyie Cad. No. 49, Haydarpasa, Istanbul 34668, Turkey
Received 20 June 2006; received in revised form 21 December 2006; accepted 21 December 2006
Available online 25 February 2007
Abstract
Three novel series of 20,40-difluoro-4-hydroxybiphenyl-3-carboxylic acid derivatives namely 4-substituted-1,2,4-triazoline-3-thiones (4aeg);
2-substituted-1,3,4-thiadiazoles (5aeg) and 2-substituted-1,3,4-oxadiazoles (6aeg) have been synthesized. Twenty-one of the newly synthesized
compounds were tested against various bacteria, fungi, yeast species and virus. In addition, we have replaced the carboxylic acid group of di-
flunisal with heterocycles and the anti-inflammatory activity of heterocycles reported here. Compound (5d) showed activity against Escherichia
coli A1 and Streptococcus pyogenes ATCC-176 at a concentration of 31.25 mg/mL, whereas cefepime, the drug used as standard, has been found
less active against the bacteria mentioned above. Compound (4b) has exhibited activity against Aspergillus variecolor and Trichophyton rubrum
at a concentration of 31.25 and 15.25 mg/mL, whereas Amphotericin B, the drug used as standard, has been found less active against the yeast
and fungi. The highest antiviral activity was found in the 1,3,4-thiadiazole derivative (5a) having a methyl group at 2nd position against Sindbis
virus at 9.6 mg/mL. Compound (4c) exhibited the highest anti-inflammatory activity (73.03%) whereas diflunisal, the drug used as standard, has
been found less active (24.16%). Compound (5f) presented similar antinociceptive activity with the standard drug (paw withdrawal latency was
19.21 s compared to that of diflunisal which was 19.14 s, in hot plate test).
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 1,2,4-Triazoline-3-thione; 1,3,4-Thiadiazole; 1,3,4-Oxadiazole; Diflunisal; Anti-inflammatory agents
1. Introduction
toxic antimicrobial agents. Ribavirin, fluconazole and cefazo-
lin are antiviral, antifungal and antibacterial drugs which
contain 1,2,4-triazole and 1,3,4-thiadiazole rings. Moreover,
owing to the increasing biological importance of 1,3,4-oxadia-
zoles, particularly in the field of chemotherapy and the existing
antimicrobial activity of 1,2,4-triazoles [1,2] and 1,3,4-thiadia-
zoles [3,4], it was considered of interest to link the 20,40-
difluoro-4-hydroxybiphenyl-3-carboxylic acid (Diflunisal )
backbone to some 1,3,4-oxadiazoles, 1,2,4-triazoles and 1,3,4-
thiadiazoles in order to investigate the effect of such struc-
tural variation on the anticipated antimicrobial and antiviral
As resistance to antimicrobial drugs is widespread, there is
an increasing need for identification of novel structure leads
that may be of use in designing new, potent and less
*
This work was partly presented at the Second International Meeting on
Medicinal and Pharmaceutical Chemistry, Antalya e Turkey P9, 10e14
October 2004.
* Corresponding author. Tel.: þ90216 349 12 16; fax: þ90216 345 29 52.
E-mail address: gkucukguzel@marmara.edu.tr (Sx.G. Kuc¸ukguzel).
¨ ¨
¨
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.12.038

894
Sx.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 42 (2007) 893e901
activities. The use of several drugs to treat inflammatory
conditions associated with infection is a problem especially in
case of patients with impaired liver or kidney functions or to
avoid drugedrug interaction. Morever, studies describe the
derivatization of the carboxylic acid function of representative
NSAIDs, which resulted in an increased anti-inflammatory
activity with reduced ulcerogenic effect [5]. In addition, 1,3,4-
oxadiazole [6], 1,2,4-triazole [7,8] and 1,3,4-thiadiazole
[9,10] derivatives were reported to possess anti-inflammatory
activities. In view of the observations, we aimed investigating
anti-infective and anti-inflammatory activities of these novel
compounds related to diflunisal.
Microorganisms were provided by the Department of Clinical
Microbiology, Faculty of Medicine; and Plant Diagnostic Lab-
oratory, Faculty of Agriculture at Ataturk University, Erzurum,
¨
Turkey. Identity of the microorganisms used in this study was
confirmed by Microbial Identification System in Biotechnol-
ogy Application and Research Center at Ataturk University.
¨
The minimal inhibition concentration (MIC) values were also
studied for the microorganisms which were determined as
sensitive to (4aec), (4g) and (5d) in disc diffusion assay. 5-
(20,40-Difluoro-4-hydroxybiphenyl-5-yl)-2-phenylamino-1,3,4-
thiadiazole (5d) has shown activity against Escherichia coli
A1 and Streptococcus pyogenes ATCC-176 at a concentration
of 31.25 mg/mL, whereas Cefepime, the drug used as standard,
has been found less active against the bacteria mentioned
above. 5-(20,40-Difluoro-4-hydroxybiphenyl-5-yl)-4-ethyl-1,2,
4-triazoline-3-thiones (4b) has exhibited activity against
Aspergillus variecolor and Trichophyton rubrum at a concen-
tration of 31.25 and 15.25 mg/mL, respectively, whereas
Amphotericin B, the drug used as standard, has been found
less active against the yeast and fungi mentioned above.
Some synthesized compounds demonstrated antifungal activ-
ity, whereas anticandidal activity was never observed.
2. Chemistry
20,40-Difluoro-4-hydroxybiphenyl-3-carboxylic acid (anal-
gesic and anti-inflammatory drug, diflunisal) and methanol
in the presence of a few drops of concentrated sulfuric acid
were heated and 20,40-difluoro-4-hydroxybiphenyl-3-carbox-
ylic acid methyl ester (1) was isolated. By heating this methyl
ester and hydrazine-hydrate in methanol 20,40-difluoro-4-
hydroxybiphenyl-3-carboxylic acid hydrazide (2) [11] was
obtained. Compound (2) and alkyl/aryl isothiocyanates were
heated in ethanol to yield original 1-(20,40-difluoro-4-hydroxy-
biphenyl-3-carbonyl)-4-alkyl/arylthiosemicarbazides (3aeg)
[12]. Alkaline cyclisation of the compounds (3aeg) using
sodium hydroxide afforded the corresponding 5-(20,40-di-
fluoro-4-hydroxybiphenyl-5-yl)-4-substituted-1,2,4-triazoline-
3-thiones (4aeg). Reacting thiosemicarbazides (3aeg) with
concentrated sulfuric acid at room temperature resulted in
the formation of the corresponding 5-(20,40-difluoro-4-hydrox-
ybiphenyl-5-yl)-2-alkyl/arylamino-1,3,4-thiadiazole (5aeg).
Cyclisation of (3aeg) using iodine and alkali gave the desired
products 5-(20,40-difluoro-4-hydroxybiphenyl-5-yl)-2-alkyl/ar-
ylamino-1,3,4-oxadiazole (6aeg). Synthetic route to com-
pounds (4aeg), (5aeg) and (6aeg) are shown in Scheme 1.
However, in one of our previous studies [12], formation of
desired 4-thiazolidinones from 1-(20,40-difluoro-4-hydroxybi-
phenyl-3-carbonyl)-4-cyclohexylthiosemicarbazide failed and
instead 5-(20,40-difluoro-4-hydroxybiphenyl-5-yl)-2-cyclohex-
ylamino-1,3,4-oxadiazole, which corresponds to compound
6g in the present study, was obtained (Scheme 2). Structures
3.2. Antiviral activity
Compounds (4aeg), (5aeg) and (6aeg) were tested for an-
tiviral activity and cytotoxicity in various viral test systems,
according to previously published procedures [16e20]. The
synthesized compounds were tested against vesicular stomati-
tis virus, coxsackie virus B4, respiratory syncytical virus, para-
influenza-3 virus, reo virus, Sindbis virus, Punto Toro virus,
herpes simplex virus type 1 and 2 and vaccinia virus-induced
cytopathicity at subtoxic concentrations in HeLa, Vero or Hel
cell culture. Compounds (4b), (4d), (4e) and (4g) which are
the 1,2,4-triazole derivatives of diflunisal exhibited antiviral
inhibitions against coxsackie virus B4, herpes simplex virus-
1 TK-KOS, vaccinia virus and Sindbis virus at 16 mg/mL,
respectively. Compounds (5a) and (5c) which are the 1,3,4-
thiadiazole derivatives of diflunisal having methyl and allyl
groups, respectively, at 2nd position exhibited antiviral inhibi-
tion against herpes simplex virus-1 TK-KOS and herpes sim-
plex virus-1 TK-KOS ACV, Sindbis virus, Coxsackie virus B4
and Punto Toro virus at 16 mg/mL, respectively. 5-(20,40-Di-
fluoro-4-hydroxybiphenyl-5-yl)-2-allylamino-1,3,4-oxadiazole
(6c) exhibited antiviral inhibition against herpes simplex
virus-2 at 16 mg/mL. The highest antiviral activity was found
in the 1,3,4-thiadiazole derivative (5a) having a methyl group
at 2nd position against Sindbis virus at 9.6 mg/mL.
1
of these compounds were characterized using H NMR and
HREI-MS spectral data. The physical and spectral data of
the synthesized compounds in this study are given in Tables
1 and 2.
3. Biological studies
3.1. Antimicrobial activity
3.3. Anti-inflammatory activity
The antimicrobial activity of the synthesized compounds
(4aeg), (5aeg) and (6aeg) against laboratory strains totally
54 microbial cultures’ isolates of 35 bacteria and 19 fungi
and yeast species, were used in this study by using microwell
dilution assay [13,14] and MIC agar dilution assay [15]. The
list of microorganisms used is given in Tables 3 and 4.
The anti-inflammatory activity of the synthesized com-
pounds (4aeg), (5aeg) and (6aeg) was evaluated by carra-
geenan induced paw edema method of Kasahara et al. [21].
The compounds were tested at 50 mg/kg oral dose and were
compared with the reference drug diflunisal. The tested
compounds showed anti-inflammatory activity ranging from

xS.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 42 (2007) 893e901
895
O
NH2
H
CO2H
OH
CO2CH3
N
a
b
F
F
OH
F
OH
F
F
F
1
2
c
H
H
N
N
S
R
O
N
H
F
OH
F
3a-g
d
H
N
f
R
N
N
S
N
N
e
H
O
N
R
R
N
F
OH
F
OH
N
N
H
F
S
F
6a-g
4a-g
F
OH
F
5a-g
Scheme 1. Synthetic route to compounds 4aeg, 5aeg and 6aeg. Reagents and conditions: (a) CH3OH/H2SO4, reflux; (b) NH2NH2$H2O, reflux; (c) R-NCS/EtOH,
reflux; (d) NaOH (2 N), reflux; (e) H2SO4; (f) I2/KIeNaOH.
23.85 to 73.03% inhibition (Fig. 1 and Table 5) whereas the
reference drug diflunisal showed 24.16% inhibition after 4 h.
The anti-inflammatory activity of 1,2,4-triazole derivatives
(4aeg), 1,3,4-thiadiazole derivatives (5aeg) and 1,3,4-oxadia-
zole derivatives (6aeg) was in the range from 36.34%
to 73.03%, from 41.36% to 57.25% and from 23.85%
to 41.36%, respectively. 5-(20,40-Difluoro-4-hydroxybiphenyl-
5-yl)-4-allyl-1,2,4-triazoline-3-thiones (4c) was found to pos-
sess highest anti-inflammatory activity by 73.03% inhibition
of against the carrageenan induced paw edema. We have
replaced the carboxylic acid group of diflunisal with additional
heterocycles, which have been found to possess an interesting
profile of anti-inflammatory activity.
For determination of antinociceptive activity conventional
hot plate testing according to the method of Woolfe and Mc
Donald [22] was used. The latency to jumping/licking of
a hind paw was measured once before (control) and at
60 min after drug administration of the tested compounds.
The tested compounds showed antinociceptive activity result-
ing in paw withdrawal latency ranging from 9.81 s to 19.21 s
whereas the same values for the reference drug diflunisal
remained at 19.14 s after 1 h of drug administration (Fig. 2
and Table 5).
The antinociceptive activity results using hot plate test
model showed that 1,2,4-triazole derivatives (4aeg), 1,3,4-
thiadiazole derivatives (5aeg) and 1,3,4-oxadiazole deriva-
tives (6aeg) exhibited paw withdrawal latency values ranging
from 13.25 s to 17.07 s, from 12.50 s to 19.21 s and from
9.81 s to 12.06 s, respectively. 5-(20,40-Difluoro-4-hydroxybi-
phenyl-5-yl)-4-(4-methoxyphenyl)-1,3,4-thiadiazole (5f) pre-
sented similar antinociceptive activity with the standard drug
(paw withdrawal latency was 19.21 s compared to that of
diflunisal which was 19.14 s, in hot plate test).
As most of the designed compounds of the present study
showed higher anti-inflammatory activity and some of them
showed comparable antinociceptive properties compared to di-
flunisal, oral bioavailability was considered to play an impor-
tant role for the development of bioactive molecules as
therapeutic agents. Therefore, a computational study for pre-
diction of ADME properties of the molecules was performed
by determination of lipophilicity, topological polar surface
area (TPSA), absorption (% ABS) and simple molecular de-
scriptors used by Lipinski in formulating his ‘‘rule of five’’.
Calculations were performed using Molinspiration online
property calculation toolkit (http://www.molinspiration.com)
[24]. Table 6 represents a calculated percentage of absorption

896
Sx.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 42 (2007) 893e901
R
H
N
S
N
NH
OEt
N
O
N
H
OEt
Br
O
N
R
S
OH
O
H
OH
F
OH
F
F
3a-g
F
_
EtOH
OEt
_
HS
O
R
S
N
N
O
N
H
N
H
N
O
N
O
R
OH
F
OH
F
F
F
4-thiazolidinone
6g
R : C6H11
R : CH3, C2H5, CH2CH=CH2,
C6H4CH3 (4), C6H4OCH3 (4)
Scheme 2. Mechanism of 4-thiazolidinone or 1,3,4-oxadiazole ring closure from 1-acyl-4-alkyl/aryl thiosemicarbazides.
(% ABS), topological polar surface area (TPSA) and Lipinski
parameters of the synthesized compounds 4aeg, 5aeg and
6aeg. Percentage of absorption (% ABS) was estimated using
the equation: % ABS ¼ 109 e 0.345 ꢀ TPSA, according to
Zhao et al. [23]. TPSAwas also calculated using Molinspiration
online property calculation toolkit [24] according to the
fragment-based method of Ertl et al. [25]. Polar surface area,
together with lipophilicity, is an important property of a mole-
cule in transport across biological membranes. Too high
TPSA values give rise to a poor bioavailability and absorption
Table 1
Physical and spectral data for 4aeg, 5aeg, 6aeg
Compound
R
Molecular formula
M.p (ꢁC)
Yield (%)
HREI-MS (m/z) calculated/found
4a
4b
4c
4d
4e
4f
CH3
C2H5
C15H11F2N3OS
C16H13F2N3OS
C17H13F2N3OS
C20H15F2N3OS
C21H15F2N3OS
C21H15F2N3OS
C20H19F2N3OS
C15H11F2N3OS
C16H13F2N3OS
C17H13F2N3OS
C20H15F2N3OS
C21H15F2N3OS
C21H15F2N3OS
C20H19F2N3OS
C15H11F2N3O2
C16H13F2N3O2
C17H15F2N3O2
C20H13F2N3O2
C21H15F2N3O2
C21H15F2N3O3
C20H19F2N3O2
238e240
180e182
192e194
272e274
258e262
244
90.54
71.06
82.79
67.97
83.62
72.43
79.36
68.44
84.07
61.50
93.98
55.72
68.90
45.98
75.45
56.43
43.65
73.21
67.41
62.15
67.45
319.0524/319.0589
333.0780/333.0730
345.0781/345.0752
381.0781/381.0745
395.0937/395.0911
411.0886/411.0858
387.1251/387.1219
319.0624/319.0613
333.0780/333.0736
345.0781/345.0749
381.0781/381.0766
395.0937/395.0885
411.0886/411.0869
387.1250/387.1224
303.0819/303.0811
317.0976/317.0974
329.0973/329.1008
365.0975/365.0990
379.1165/379.1140
395.1114/395.1096
371.1478/371.1447
CHeCH]CH2
C6H5
C6H4CH3(4)
C6H4OCH3(4)
C6H11
4g
5a
5b
5c
5d
5e
5f
242e244
292 (dec.)
222e224
171e180
174
CH3
C2H5
CHeCH]CH2
C6H5
C6H4CH3(4)
C6H4OCH3(4)
C6H11
246e260
224e226
178e180
239e240
176e177
142e144
224e227
238e240
248e250
194e195
5g
6a
6b
6c
6d
6e
6f
CH3
C2H5
CHeCH]CH2
C6H5
C6H4CH3(4)
C6H4OCH3(4)
C6H11
a6g
a
Ref. [12].

xS.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 42 (2007) 893e901
897
Table 2
1H NMR spectral data of 4aeg, 5aeg, 6aeg
Compound
1H NMR
4a
4b
4c
3.25 (s, 3H, NeCH3); 6.75e7.52 (m, 6H, AreH); 9.88, 10.61 (2s, 1H, AreOH); 13.78 (2s, 1H, NH).
0.99 (t, 3H, NeCH2eCH3); 3.78 (q, 2H, NeCH2eCH3); 7.01e7.51 (m, 6H, AreH); 10.29e10.77 (br, 1H, AreOH); 13.54 (s, 1H, NH).
4.47 (d, 2H, NeCH2eCH]); 4.65 (d, 1H, eCH]CH2, J ¼ 17.2 Hz, trans); 4.88 (d, 1H, eCH]CH2, J ¼ 10.3 Hz, cis); 5.49e5.68 (m, 1H,
eCH]CH2), 6.99e7.49 (m, 6H, AreH); 10.56 (s, 1H, AreOH); 13.84 (s, 1H, NH).
4d
4e
4f
6.89e8.12 (m, 11H, AreH); 10.30 (s, 1H, AreOH); 11.07 (s, 1H, NH).
2.29 (s, 3H, AreCH3); 6.83e7.46 (m, 10H, AreH); 9.48e11.35 (br, 1H, AreOH); 12.93e14.89 (br, 1H, NH).
3.63 (s, 3H, OCH3); 6.73e7.40 (m, 10H, AreH); 9.97 (s, 1H, AreOH); 13.66 (s, 1H, NH).
4g
5a
5b
5c
0.80e1.66 (m, 10H, cyclohexyl CH2); 2.06 (s, 1H, NeCHecyclohexyl); 6.97e7.45 (m, 6H, AreH); 10.43 (s, 1H, AreOH); 13.71 (s, 1H, NH).
2.89 (s, 3H, NeCH3); 6.97e8.00 (m, 7H, AreH and NHeCH3); 11.10 (s, 1H, AreOH).
1.17 (t, 3H, NeCH2eCH3); 3.31 (q, 2H, NeCH2eCH3); 7.02e7.98 (m, 7H, AreH and NHe CH2eCH3), 11.10 (s, 1H, AreOH).
3.97 (d, 2H, NeCH2eCH]); 5.07 (d, 1H, eCH]CH2, J ¼ 17.0 Hz, trans); 5.25 (d, 1H, eCH]CH2, J ¼ 10.2 Hz, cis); 5.75e5.94 (m, 1H,
eCH]CH2), 7.01e8.34 (m, 7H, AreH and NHeAr); 11.09, 12.02 (2 s, 1H, AreOH).
5d
5e
5f
6.97e8.18 (m, 11H, AreH); 9.85 (s, 1H, NHeC6H5); 12.00 (s, 1H, AreOH).
2.47 (s, 3H, AreCH3); 7.08e8.17 (m, 11H, AreH); 10.26 (s, 1H, NHeC6H4eCH3); 11.15 (s, 1H, AreOH).
3.72 (s, 3H, OCH3); 6.91e8.14 (m, 11H, AreH); 10.15 (s, 1H, AreOH).
5g
6a
6b
6c
1.14e1.99 (m, 10H, cyclohexyl CH2); 2.24 (s, 1H, NeCHecyclohexyl); 7.11e7.78 (m, 6H, AreH); 11.12 (s, 1H, NH cyclohexyl);
2.49 (s, 3H, NeCH3); 7.10e7.78 (m, 7H, AreH and NHeCH3).
1.16 (t, 3H, NeCH2eCH3); 3.25 (q, 2H, NeCH2eCH3); 7.10e7.90 (m, 7H, AreH and NHeCH2eCH3).
3.86 (d, 2H, NeCH2eCH]); 5.12 (d, 1H, eCH]CH2, J ¼ 17.3 Hz, trans); 5.21 (d, 1H, eCH]CH2, J ¼ 10.4 Hz, cis); 5.84e5.96 (m, 1H,
eCH]CH2), 7.05e8.13 (m, 7H, AreH and NH); 10.28 (s, 1H, AreOH).
6d
6e
6f
a6g
a
6.97e7.79 (m, 11H, AreH); 10.41 (s, 1H, NHeC6H5); 10.73 (s, 1H, AreOH).
2.23 (s, 3H, AreCH3); 7.05e7.78 (m, 10H, AreH); 10.38 (s, 1H, NHeC6H4eCH3); 10.61 (s, 1H, AreOH).
3.63 (s, 3H, OCH3); 6.91e7.76 (m, 10H, AreH); 10.37 (s, 1H, NHeC6H4eOCH3); 10.51 (s, 1H, AreOH).
1.15e1.93 (m, 10H, cyclohexyl CH2); 2.24 (s, 1H, NeCHecyclohexyl); 7.10e7.88 (m, 6H, AreH); 10.33 (s, 1H, NH cyclohexyl).
Ref. [12].
of a drug. According to the above criterions, calculated percent-
ages of absorption for compounds 4aeg, 5aegand 6aeg ranged
between 81 and 90%.
heterocyclic classes (4c, 5c and 6a) with allyl or methyl substit-
uent at R position had zero violations to Lipinski rule of five.
According to these results, it has been understood that het-
erocyclic derivatives of diflunisal exhibited higher anti-inflam-
matory activity than diflunisal and compound 5f presented
similar antinociceptive activity.
In conclusion, a functional group transformation in a well-
known anti-inflammatory drug diflunisal was performed to
observe whether replacement of carboxylic acid function with
several heterocyclic rings (1,2,4-triazole; 1,3,4-thiadiazole and
1,3,4-oxadiazole) leads to an increase in biological activity, or
a different pharmacological profile. There were no direct corre-
lations observed between simple molecular properties such as
log P and anti-inflammatory or antinociceptive activity. Never-
theless, it was notable that three most active derivatives in each
4. Experimental
All chemical compounds were purchased from Fluka.
Diflunisal was provided by Sanovel Pharmaceuticals. Melting
points were taken on Buchi-530 apparatus and are uncorrected.
Table 3
Antimicrobial activities of new synthesized compounds against the bacterial strains tested based on disc diffusion method and microwell dilution method
Microorganisms e Code (Source)a
4a
4b
4c
4f
4g
5d
Antibioticsb
DDc
DD MIC DD MIC DD MIC
DD MIC
DD MIC DD MIC
MICd (max)
Enterococcus faecalis e ATCC-29122 (1)
Escherichia coli e A1 (1)
Proteus vulgaris e A161 (1)
Proteus vulgaris e KUKEM1329 (1)
Streptococcus pyogenes e ATCC-176 (1)
S. pyogenes e KUKEM-676 (1)
e
e
10
8
e
e
e
9
e
10
12
10
9
250
62.50
12
e
125
e
e
e
e
e
31.25
18 (SCF)
e(OFX)
31.25
62.50
e
e
e
e
16
14
16
18
e
125
125
e
125
125
e
125
250
e
10
e
125
10
12
e
125
125
e
62.50 12 (OFX) 125
62.50 13 (OFX) 125
9
e
e
e
e
12
14
62.25 10
62.25
62.25
125
31.25 10 (OFX)
e 13 (OFX)
62.50
31.25
e
e
9
e
e
Bacteria: Acinetobacter baumanii A8 (1), Acinetobacter lwoffi F1 (3), Bacillus macerans A199 (1), Bacillus megaterium A59 (2), Bacillus subtilis ATCC 6633 (1),
B. subtilis A57 (1), Brucella abortus A77 (1), Burkholdria cepacia A225 (2), Clavibacter michiganens A227 (2), Cedecea davisae F2 (3), Enterobacter cloacae
A135 (2), Klebsiella pneumoniae F3 (3), K. pneumoniae A137 (2), Morganella morganii F4 (3), Pseudomonas aeruginosa ATCC9027 (1), P. aeruginosa
ATCC27859 (1), P. aeruginosa F5 (3), Pseudomonas pseudoalkaligenes F6 (3), Pseudomonas syringae pv. tomato A35 (2), Salmonella cholerasuis arizonae
F7 (3), Salmonella enteritidis ATCC13076 (1), Serratia plymuthica F8 (3), Shigella sonnei F9 (3), Staphylococcus aureus A215 (1), S. aureus ATCC 29213
(1), Staphylococcus epidermis A233 (1), Staphylococcus hominis F10 (3), Xanthomonas campestris A235 (2), Yersinia enterocolitica F11 (3).
a
Source of microorganisms: 1 ¼ Clinic human pathogenic organism, 2 ¼ Plant pathogenic, 3 ¼ Food pathogenic.
b
OFX ¼ Ofloxacin (10 mg/disc); SCF ¼ sulbactam (30 mg) þ cefoperazona (75 mg) (105 mg/disc) and NET ¼ Netilmicin (30 mg/disc), were used as positive ref-
erence standards antibiotic discs (Oxoid); Maxipime (mg/mL) was used as reference antibiotic in microwell dilution assay (Sigma).
c
Inhibition zone in diameter (mm) around the discs impregnated with 10 mL of the synthesized compounds.
Minimal inhibitory concentrations as mg/mL.
d

898
Sx.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 42 (2007) 893e901
4.1.2. 5-(20,40-Difluoro-4-hydroxybiphenyl-5-yl)-2-
alkyl/arylamino-1,3,4-thiadiazole (5aeg)
Compounds (3aeg) (0.01 mol) were dissolved in concen-
trated sulfuric acid. The mixture was stirred at room tempera-
ture for 30 min. It was poured into ice-cold cooling water and
washed with distilled water. The precipitate was recrystallized
from ethanol [27].
Table 4
Anticandidal and antifungal activities of 4b and 4c against the yeast and fungi
isolates tested based on disc diffusion method and MIC agar dilution assay
Test fungi (Source)a
4b
4c
Antibioticsb
DD MIC DD MIC DD
MIC
Fungi
Aspergillus flavus (3)
Aspergillus variecolor (3)
Trichophyton rubrum (1)
Trichophyton mentagrophytes (1)
16 31.25 14 31.25 e(NET) 15.62
14 31.25 10 62.50 e(NET) 62.50
16 15.25
e
e
e
e(NET) 15.62
14 31.25 e(NET) 31.25
e
4.1.3. 5-(20,40-Difluoro-4-hydroxybiphenyl-5-yl)-2-
Yeast: Candida albicans A117 (1), *Fungi: Alternaria solani, Aspergillus
niger, Fusarium acuminatum, Fusarium oxysporum, Fusarium solani, Fusa-
rium tabacinum, Microsporum canis, Monilinia fructicola, Mortierella alpina,
Penicillium spp., Rhizopus spp., Rhizoctonia solani, Sclorotinia minor, Scloro-
tinia sclerotiorum. (*These fungi were isolated from foodstuff).
alkyl/arylamino-1,3,4-oxadiazole (6aeg)
The appropriate 3aeg (0.01 mol) were suspended in etha-
nol (30 mL), aqueous NaOH (0.16 mol, 2 N) and iodine in
KI (aqueous 5%) were added with shaking at room tempera-
ture until the color of iodine persisted. The excess solvent
was removed under reduced pressure. The contents were
cooled and acidified with acetic acid (10%) to get the corre-
sponding free oxadiazoles. The solid was filtered, washed
with water, dried and recrystallized from ethanol [6].
a
Source of microorganisms: 1 ¼ Clinic human pathogenic organism,
2 ¼ Plant pathogenic, 3 ¼ Food pathogenic.
b
DD ¼ Inhibition zone in diameter (mm) around the discs impregnated with
the synthesized compounds (300 mg/disc); NET ¼ Netilmicin (30 mg/disc) was
used as positive reference standard antibiotic discs (Oxoid); MIC ¼ Mınımal
inhibitory concentrations (mg/mL) of standard antibiotic, AmpB ¼
Amphotericin B (mg/mL) (Sigma).
4.2. Microbiological studies
4.2.1. Antimicrobial activity
1H NMR spectra were obtained on a Bruker AVANC-DPX 400
instrument. High resolution 70 eV EI mass spectra were ob-
tained from a JEOL JMS-700 magnetic sector instrument.
4.2.1.1. Microbial strains. New synthesized compounds were
individually tested against laboratory strains totally 54 microbial
culture isolates of 35 bacteria and 19 fungi and yeast species. Mi-
croorganisms were provided by the Department of Biology,
Faculty of Art and Science, and at Ataturk University, Erzurum,
Turkey. The identity of the microorganisms used in this study
was confirmed by Microbial Identification System in Biotech-
nology Application and Research Center at Ataturk University.
4.1. Chemistry
4.1.1. 5-(20,40-Difluoro-4-hydroxybiphenyl-5-yl)-4-substituted-
1,2,4-triazoline-3-thiones (4aeg)
A solution of 0.01 mol (3aeg) in 2 N sodium hydroxide so-
lution (25 mL) was heated under reflux for 4 h. After cooling
to room temperature, concentrated hydrochloric acid was
added. The precipitate was filtered and washed several times
with distilled water. The compounds were recrystallized
from ethanol [26].
4.2.1.2. Disc-diffusion assay. Antimicrobial tests were carried
out by disc diffusion method using 100 mL of suspension
+++
***
75
50
25
0
+++
***
+++
***
+++
+++
+++
***
***
***
+++
***
+++
***
++
**
+++
***
++
+
++
++
***
***
*** ***
***
***
***
***
***
***
***
***
C
St 4a 4b 4c 4d 4e 4f 4g 5a 5b 5c 5d 5e 5f 5g 6a 6b 6c 6d 6e 6f 6g
Compounds
Fig. 1. Anti-inflammatory activity of compounds 4aeg, 5aeg and 6aeg; ***p < 0.001, **p < 0.01, *p < 0.05: compared with vehicle (control); þþþp < 0.001,
þþp < 0.01, þp < 0.05: compared with diflunisal (as standard drug).

xS.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 42 (2007) 893e901
899
Table 5
Pharmacological data for 4aeg, 5aeg, 6aeg
impregnated with 15 mL of each compounds (300 mg/disc) at
the concentration of 20 mg/mL and placed on the inoculated
agar. DMSO impregnated discs were used as negative con-
trols. Ofloxacin (10 mg/disc), sulbactam (30 mg) þ cefoperazon
(75 mg) (105 mg/disc) and/or netilmicin (30 mg/disc) were used
as positive reference standards to determine the sensitivity of
one strain/isolate in each microbial species tested. The inocu-
lated plates were incubated at 37 ꢁC for 24 h for clinical
bacterial strains, 48 h for yeast and 72 h for fungi isolates. Plant
associated microorganisms were incubated at 27 ꢁC. Antimicro-
bial activity was evaluated by measuring the zone of inhibition
against the test organisms. Each assay in this experiment was
repeated twice.
Compound
Anti-inflammatory
activity
Antinociceptive
activity (paw withdrawal
latency in seconds ꢂ S.E.M)
(%inhibition ꢂ S.E.M)
Control
Diflunisal
4a
e
9.6 ꢂ 0.99
24.2 ꢂ 1.83***
56.9 ꢂ 3.92***
46.2 ꢂ 2.92***
73.0 ꢂ 1.97***
36.3 ꢂ 2.63***
51.6 ꢂ 4.69***
53.5 ꢂ 3.65***
64.4 ꢂ 6.72***
45.4 ꢂ 3.63***
50.2 ꢂ 2.63***
57.2 ꢂ 3.24***
43.0 ꢂ 2.71*
43.5 ꢂ 2.57**
55.3 ꢂ 3.76***
41.4 ꢂ 3.35*
41.4 ꢂ 3.35*
36.8 ꢂ 1.66
19.1 ꢂ 1.18***
16.4 ꢂ 1.44*
17.1 ꢂ 1.81**
16.8 ꢂ 1.34**
16.0 ꢂ 1.15*
14.2 ꢂ 1.02
4b
4c
4d
4e
4f
4g
15.0 ꢂ 1.47
13.2 ꢂ 0.29
5a
5b
13.5 ꢂ 0.38
13.5 ꢂ 0.38
5c
5d
13.9 ꢂ 0.42
4.2.1.3. Microwell dilution assay. The minimal inhibition con-
centration (MIC) values were also studied for the microorgan-
isms which were determined as sensitive to (4aec), (4feg)
and (5d) in disc diffusion assay. The inocula of microorgan-
isms were prepared from 12 h broth cultures and suspensions
were adjusted to 0.5 McFarland standard turbidity. Com-
pounds (4aec), (4feg) and (5d) dissolved in 10% dime-
thylsulfoxide (DMSO) were first diluted to the highest
concentration (500 mg/mL) to be tested, and then serial two-
fold dilutions were made in a concentration range from 7.8
to 500 mg/mL in 10 mL sterile test tubes containing nutrient
broth. MIC values of (4aec), (4feg) and (5d) against bacterial
strains and Candida albicans isolates were determined based
on a microwell dilution method [13,14].
16.5 ꢂ 1.30**
14.5 ꢂ 0.89
5e
5f
5g
19.2 ꢂ 0.91***
12.5 ꢂ 0.54þ
10.9 ꢂ 0.97þþþ
11.0 ꢂ 0.76þþþ
9.8 ꢂ 1.07þþþ
12.0 ꢂ 1.30þþ
11.1 ꢂ 0.94þþþ
12.0 ꢂ 1.79þþ
12.0 ꢂ 1.79þþ
6a
6b
6c
6d
36.6 ꢂ 3.33
39.5 ꢂ 2.73
6e
33.8 ꢂ 1.93
6f
6g
35.3 ꢂ 2,94
23.8 ꢂ 1.72
The results were given as means ꢂ SEM. Analysis of variance ANOVA,
followed by Turkey’s multiple comparisons test.
***p < 0.001, **p < 0.01, *p < 0.05: compared with vehicle (control);
þþþp < 0.001, þþp < 0.01, þp < 0.05: compared with diflunisal (as standard
drug).
The 96-well plates were prepared by dispensing into each
well 95 mL of nutrient broth and 5 mL of the inoculum.
A 100 mL (4aec), (4feg) and (5d) initially prepared at the
concentration of 600 mg/mL was added into the first wells.
Then, 100 mL from their serial dilutions was transferred into
six consecutive wells. The last well containing 195 mL of
containing 108 CFU/mL of bacteria, 106 CFU/mL of yeast and
104 spore/mL of fungi spread on MuellereHinton agar, sabo-
urand dextrose agar (SDA), and potato dextrose agar (PDA)
medium, respectively. The discs (6 mm in diameter) were
25
***
20
***
**
**
**
*
*
15
10
5
++
++
++
+
+++
+++
+++
+++
0
C
St 4a 4b 4c 4d 4e 4f 4g 5a 5b 5c 5d 5e 5f 5g 6a 6b 6c 6d 6e 6f 6g
Compounds
Fig. 2. Antinociceptive activity of compounds 4aeg, 5aeg and 6aeg; ***p < 0.001, **p < 0.01, *p < 0.05: compared with vehicle (control); þþþp < 0.001,
þþP < 0.01, þp < 0.05: compared with diflunisal (as standard drug: St).

900
Sx.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 42 (2007) 893e901
Table 6
Predicted ADME, Lipinski parameters and molecular properties of the synthesized compounds 4aeg, 5aeg and 6aega
Compound
% ABS
TPSA
n-ROTB
n-ON acceptors
n-OHNH donors
mi log P
Formula weight
n violations
4a
4b
4c
90
90
90
90
90
87
90
89
89
89
89
89
86
89
84
84
84
84
84
81
84
89
53.85
53.85
53.85
53.85
53.85
63.08
53.85
58.04
58.04
58.04
58.04
58.04
67.27
58.04
71.18
71.18
71.18
71.18
71.18
80.41
71.18
57.53
2
3
4
3
3
4
3
3
4
5
4
4
5
4
3
4
5
4
4
5
4
2
4
4
4
4
4
5
4
4
4
4
4
4
5
4
5
5
5
5
5
6
5
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3.32
3.70
3.97
4.60
5.04
4.65
5.23
4.19
4.57
4.83
6.32
6.77
6.38
6.10
3.55
3.92
4.19
5.68
6.13
5.74
5.45
3.90
319.34
333.36
345.37
381.41
395.43
411.43
387.46
319.34
333.36
345.37
381.41
395.43
411.43
387.46
303.27
333.36
329.31
365.34
379.37
395.37
371.39
250.20
0
0
0
0
1
0
1
0
0
0
1
1
1
1
0
0
0
1
1
1
1
0
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
5g
6a
6b
6c
6d
6e
6f
6g
Diflunisal
a
% ABS: Percentage of absorption, TPSA: topological polar surface area, n-ON: number of hydrogen bond acceptors, n-OHNH: number of hydrogen bond
donors, n-ROTB: number of rotatable bonds. Calculations were performed using Molinspiration online property calculation toolkit (http://www.molinspira-
tion.com) [24].
nutrient broth without compound and 5 mL of the inoculum on
each strip was used as negative control. The final volume in
each well was 200 mL. Maxipime (BristoleMyers Squibb) at
the concentration range of 500e7.8 mg/mL was prepared in
nutrient broth and used as standard drug for positive control.
The plate was covered with a sterile plate sealer. Contents of
each well were mixed on plate shaker at 300 rpm for 20 s
and then incubated at appropriate temperatures for 24 h. Mi-
crobial growth was determined by plating 5 mL samples
from clear wells on nutrient agar medium. The compounds
tested in this study was screened two times against each organ-
ism. The MIC was defined as the lowest concentration of the
compounds to inhibit the growth of microorganisms.
compounds (4b) and (4c) where absence of growth was
recorded. Each test repeated at least twice.
4.2.2. Antiviral activity
Compounds (4aeg), (5aeg) and (6aeg) were tested for an-
tiviral activity and cytotoxicity in various viral test systems,
according to previously published procedures [16e20]. The
synthesized compounds tested vesicular stomatitis virus, Cox-
sackie virus B4, respiratory syncytical virus, parainfluenza-3
virus, reo virus, Sindbis virus, Punto Toro virus, herpes simplex
virus type 1 and 2 and vaccinia virus-induced cytopathicity at
subtoxic concentrations in HeLa, Vero or Hel cell culture.
4.3. Pharmacological activity
4.2.1.4. MIC agar dilution assay. MIC values were studied for
the fungi isolates which were determined as sensitive to (4b)
and (4c) in disc diffusion assay, as described previously
[15]. Compounds (4b) and (4c) dissolved in 10% dimethyl-
sulfoxide (DMSO) were added aseptically to sterile PDA
medium, containing Tween 20 (Sigma 0.5%, v/v), at the
appropriate volume to produce the concentration range of
7.8e500 mg/mL. The resulting PDA solutions were immedi-
ately poured into Petri plates after vortexing. The plates
were spot inoculated with 5 mL (104 spore/mL) of each fungal
isolate. Amphotericin B (Sigma A 4888) was used as a refer-
ence antifungal drug. The inoculated plates were incubated at
27 ꢁC and 37 ꢁC for 72 h for plant and clinical fungi isolates,
respectively. At the end of incubation period, the plates
were evaluated for the presence or absence of growth. MIC
values were determined as the lowest concentration of the
4.3.1. Anti-inflammatory activity
Balb/c mice of both sexes weighing approximately 20e25 g
were used. All the animals were left two days in the laboratory
conditions for aclimatization and maintained on standard pellet
diet and water ad libitum before the day of the experiment. The
last day food was withdrawn and they were given water only.
Test samples and reference compound were suspended in
0.5% methyl cellulose and administered to each mouse by us-
ing gastric gavage needle, in 50 mg/kg. The control group an-
imals, however, received the same volume of dosing vehicle,
each group consisted of six mice. The method of Kasahara
et al. was employed for anti-inflammatory activity testing
with some modifications. Diflunisal was used as reference
compound. Test samples were administered orally 60 min be-
fore the injection on 25 mL of freshly prepared solution of g

xS.G. Ku¨c¸u¨kgu¨zel et al. / European Journal of Medicinal Chemistry 42 (2007) 893e901
901
carrageenan (0.5 mg/mL) in physiological saline into subplan-
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This work was supported by the Research Fund of Marmara
˘
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