Synthesis and biological activity of hydrazide-hydrazones and their corresponding 3-Acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles

Article abstract of DOI:10.1007/s00044-011-9882-z

Various 3-Acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles (11-20) were prepared by the reaction of aryl substituted hydrazones of 4-fluorobenzoic acid hydrazide (1-10) with acetic anhydride. The structures of the synthesized compounds 11-20, were c

Full text of DOI:10.1007/s00044-011-9882-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3499–3508
DOI 10.1007/s00044-011-9882-z
ORIGINAL RESEARCH
Synthesis and biological activity of hydrazide–hydrazones
and their corresponding 3-acetyl-2,5-disubstituted-2,3-dihydro-
1,3,4-oxadiazoles
¨
•
•
•
˘
˘
Bedia Koc¸yigit-Kaymakc¸ıoglu Emine Elc¸in Oruc¸-Emre Seda Unsalan
•
•
•
Nurhayat Tabanca Shabana Iqrar Khan David Earl Wedge
˙
•
Gokalp Is¸can Fatih Demirci Sevim Rollas
•
¨
Received: 25 August 2011 / Accepted: 4 November 2011 / Published online: 20 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Various 3-acetyl-2,5-disubstituted-2,3-dihydro-
1,3,4-oxadiazoles (11–20) were prepared by the reaction of
aryl substituted hydrazones of 4-fluorobenzoic acid hydra-
zide (1–10) with acetic anhydride. The structures of the
synthesized compounds 11–20, were confirmed by UV, IR,
1H-NMR and mass spectroscopic methods. Antifungal eval-
uation of the hydrazide–hydrazones 1–10 and corresponding
3-acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles 11–
20, against clinical and standard Candida pathogens have
been performed by using agar diffusion to indentify the active
compounds, which were later subjected to a broth microdi-
lution assay to justify the activity level in terms of minimum
inhibitory concentrations (MIC). 4-Fluorobenzoic acid
[(5-bromothiophen-2-yl)methylene]hydrazide, showed the
highest inhibitory activity against Candida albicans (MIC:
125 lg/ml), and when compared with ketoconazole. In
addition, bioauthographic antifungal activity against plant
pathogenic fungi such as Colletotrichum, Botrytis, Fusar-
ium, and Phomopsis was conducted. 4-Fluorobenzoic acid
[(5-bromothiophen-2-yl)methylene]hydrazide was the most
active analog against P. viticola with 91% inhibitionat 30 lM
after 144 h. Furthermore, known and the newly synthesized
compounds were also screened through a panel of bioassays
to determine their anti-inflammatory, cytotoxic, and antioxi-
dant activities in mammalian cells. 3-Acetyl-5-(4-fluoro-
phenyl)-2-(3-hydroxy-4-methoxyphenyl)-2,3-dihydro-1,3,
4-oxadiazole, showed a strong inhibition of NF-jB-depen-
dent transcription in SW1353 cells with IC50 value of 0.75
lg/ml. 4-Fluorobenzoic acid [(3-hydroxy-4-methoxyphenyl)
methylene]hydrazide, and 3-acetyl-5-(4-fluorophenyl)-2-(3-
hydroxy-4-methoxyphenyl)-2,3-dihydro-1,3,4-oxadiazole on
intracellular ROS generation in PMA induced HL-60 cells
demonstrated potent activity with IC50 values of 0.9 lg/ml. A
strong inhibition of the activity of iNOS activity in LPS
induced RAW 264.7 cells was observed for 3-acetyl-5-
(4-fluorophenyl)-2-(4-hydroxyphenyl)-2,3-dihydro-1,3,4-oxa-
diazole with IC50 value of 0.3 lg/ml.
¨
˘
˘
B. Koc¸yigit-Kaymakc¸ıoglu (&) ꢀ S. Unsalan ꢀ S. Rollas
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Marmara University, 34668 Istanbul, Turkey
e-mail: bkaymakcioglu@marmara.edu.tr
E. E. Oruc¸-Emre
Department of Chemistry, Faculty of Arts and Sciences,
Gaziantep University, 27310 Gaziantep, Turkey
N. Tabanca (&)
National Center for Natural Products Research, The University
of Mississippi, Mississippi, MS 38677, USA
e-mail: ntabanca@olemiss.edu
S. I. Khan
Keywords Hydrazide–hydrazones ꢀ 1,3,4-Oxadiazoles ꢀ
Anti-inflammatory ꢀ Cytotoxicity ꢀ Antioxidant ꢀ
Antifungal
National Center for Natural Products Research and Department
of Pharmacognosy, School of Pharmacy, University
of Mississippi, Mississippi, MS 38677, USA
D. E. Wedge
USDA-ARS- Natural Products Utilization Research Unit,
University of Mississippi, Mississippi, MS 38677, USA
Introduction
˙
G. Is¸can ꢀ F. Demirci
Looking at the importance of oxadiazole nucleus, it was
thought that it would be worthwhile to synthesize new
Department of Pharmacognosy, Faculty of Pharmacy, Anadolu
University, 26470 Eskis¸ehir, Turkey
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Med Chem Res (2012) 21:3499–3508
oxadiazole derivatives and screen them for potential bio-
logical activities. Owing to their diverse biological prop-
erties, their synthesis has increased noticeably in recent
years (Khalil et al., 2003; Jin et al., 2006; Hassan et al.,
2008; Shirote and Bhatia 2011).
All the other aromatic and aliphatic protons were observed at
the expected region in their NMR spectrum. In LC–MS,
molecular ion peaks were in agreement with proposed
molecular weight. All new compounds gave satisfactory
elemental analysis results.
In this study, known hydrazide-hydrazone and newly
synthesized 1,3,4-oxadiazole derivatives were investigated
in a panel of bioassays that include, anti-inflammatory,
antioxidant, cytotoxic and antimicrobial activities against
human pathogenic bacteria or fungi. All the compounds
were also tested for growth inhibition of several plant
pathogenic fungi from the genera Colletotrichum, Botrytis,
Fusarium, and Phomopsis. To the best of our knowledge
we are reporting for the first time the biological activity of
new 1,3,4-oxadiazole derivatives in this present study.
Antifungal activity against human pathogens
The antifungal activity of the compounds was studied with
eight pathogenic Candida sp. Ketoconazole, was used as
reference agents or inhibitory activity against the tested
fungi. Minimal inhibitory concentrations (MIC) were recor-
ded as the minimum concentration of a compound that
inhibits the growth of tested microorganisms. All of the
compounds tested illustrated medium to very good antican-
didal inhibitory activity when compared with the reference
agents. The MIC values were found within the range of
125–1,000 lg/ml against all evaluated strains. The results are
summarized in Table 1. In comparing MIC values with the
standard reference agent, compound 9, 4-fluorobenzoic
acid [(5-bromothiophen-2-yl)methylene]hydrazide, showed
equal activity (125 lg/ml) against C. albicans (NRRL
Y-12983). Compound 18, 3-acetyl-5-(4-fluorophenyl)-2-
[2-(4-(dimethylamino)phenyl) ethenyl]-2,3-dihydro-1,3,4-
oxadiazole, showed moderate inhibitory activity (125 lg/ml)
against C. tropicalis (NRRL Y-12968) and C. krusei (NRRL
Y-7179). Compound 7, 4-fluorobenzoic acid [(4-dimethy-
laminocinnamyl)methylene]hydrazide, and compound 14,
3-acetyl-5-(4-fluorophenyl)-2-(3-hydroxy-4-methoxyphenyl)-
2,3-dihydro-1,3,4-oxadiazole, showed moderate inhibition
with the 125 lg/mL MIC value towards C. glabrata (clin-
ical isolate) and C. utilis (NRRL Y-900), respectively. The
other compounds were found less active when compared
with ketoconazole against the tested microorganisms.
Results and discussion
Chemistry
General procedures for the preparation of target compounds
11–20 are represented in the Scheme 1. Hydrazide–hydra-
zones derivatives 1–10 were prepared by condensation of
4-fluorobenzoic acid hydrazide with appropriate aldehydes
(Koc¸yigit-Kaymakcıoglu et al., 2006; Kaymakcıoglu et al.,
2009). Physicochemical and spectroscopic characteriza-
tion of the hyrazide-hydrazones 1–10 have been previ-
ously described (Koc¸yigit-Kaymakcıoglu et al., 2006).
3-Acetyl-2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles
11–20 were synthesized by reacting compounds (1–10) with
Ac2O (Rollas et al., 2002). All 3-acetyl-2,5-disubstituted-
2,3-dihydro-1,3,4-oxadiazoles, except compound 11 (Li
et al., 2010) are new compounds. Compounds 11–20 were
isolated in satisfactory yields (49–90%) and purified by
recrystallisation, using ethanol. The purities of the synthe-
sized compounds were checked by reversed phase HPLC
(Chromasil C18 3.6 9 150 mm column using acetonitrile
and water (50:50 v/v) as the eluent). All compounds showed
a single peak with a retention time of 3.219–4.693 min. The
structures of 11–20 were supported by elemental analysis
Antifungal activity against plant pathogens
Biological activity of a natural product involves several
key characteristics that apply regardless of whether the
activity is for pharmaceutical or agrochemical application
(Wedge and Camper, 2000). Several 1,3,4-oxadiazole
derivatives were chosen for further investigation in a
research program at the United States Department of
Agriculture aimed at identifying natural fungicides and
biopesticides. A total of 20 compounds were subsequently
evaluated in a 96-well micro-dilution broth assay for
antifungal activity against Colletrichum acutatum, C. fra-
gariae, C. gloeosporioides, Botrytis cinerea, Phomopsis
obscurans, P. viticola and Fusarium oxysporum. This
96-well microtiter assay has been extensively used to
determine and compare the sensitivity of fungal plant
pathogens to natural and synthetic compounds with known
commercial fungicides (Sobolev et al., 2011). Oxadiazole
1
and by the spectral data achieved from UV, IR, H-NMR,
and mass spectroscopy, which were in agreement with the
proposed structures.
The IR spectra of 11–20 had different characteristics from
those of the hydrazone derivatives as they showed no N–H
stretching bands in the 3248–3450 cm-1 region and only
C=O bands in the 1,673–1,685 cm-1 region, which were
1
attributed to the C=O stretching of acetyl group. The H
NMR data were also consistent with the assigned structures.
The spectra of 11–20 displayed the O-CHR-N resonance
of the oxadiazoline ring at d 6.68–7.26 ppm in accordance
with the literature (Rollas et al., 2002; Ergenc¸ et al., 1989).
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Med Chem Res (2012) 21:3499–3508
3501
Scheme 1 Synthetic pathways
used for the preparation of 1–20.
Reagents and conditions:
(a) Ar–CHO, C2H5OH, D;
(b) Ac2O, D
a
NH
F
NH
F
NH2
Ar
N
O
O
1-10
b
O
N
CH3
N
F
H
O
Ar
11-20
Compound
1, 11
Ar
C6H5 -
Compound
6, 16
Ar
3-OH-4-OCH3-C6H4-
(CH3)2N-C6H4-
2, 12
4-BrC6H4-
4-F-C6H4-
7, 17
3, 13
8, 18
(CH3)2N-C6H4CH=CH-
5-Bromo-thiophen-2-yl-
Furan-2-yl
4, 14
4-OH-C6H4-
9, 19
5, 15
4-OCH3-C6H4-
10, 20
Reagents and conditions: (a) Ar-CHO, C2H5OH, Δ; (b) Ac2O, Δ.
derivatives were found to be active only against five spe-
cies: C. acutatum, C. gloeosporioides, F. oxysporum,
P. obscurans and P. viticola, (Figs. 1, 2, 3, 4, 5). No
antifungal activity was observed against C. fragariae and
B. cinerea. In the microtiter broth bioassays, compound 19
also showed 49% inhibition against C. acutatum at 30 lM
after 72 h (Fig. 1). The most active compounds 2, 9, and 19
inhibited growth of C. gloeosporioides 22, 32, and 66% at a
dose of 30 lM, after 72 h treatment, respectively (Fig. 2).
Any test compound possessing \50% growth inhibition at
30 lM in this bioassay is considered to have weak anti-
fungal activity. The same compounds 2, 9, and 19 dem-
onstrated 35, 40, and 67% growth inhibition against
F. oxysporum at 30 lM after 72 h (Fig. 3). Of the twenty
compounds tested, compound 19 demonstrated the greatest
level of antifunal activity against P. obscurans with 65%
inhibition at 30 lM after 144 treatments (Fig. 4). Com-
pound 9 was the most active analog against P. viticola with
91% inhibition at 30 lM after 144 h (Fig. 5). The struc-
tural modification of hydrazone derivatives, i.e., replace-
ment of bromo at C4 of phenyl ring with C5 of thiophene
moiety resulted in an increase in the inhibition of
C. gloeosporioides. The comparison of inhibibition of
C. gloeosporioides of hydrazone 9 and oxadiazole deriva-
tive 19 demonstrated that the cyclization of hydrazones
increased the activity profile.
Furthermore, it was thought that the antifungal activity
influenced by the aromatic ring substituted by a bromo
atom due was to the increase in lipophilicity. This change
may allow for great transport across the lipid bilayer of the
fungal membrane and hence achieve a higher concentration
of antifungal molecules inside the fungal mycelia.
Cytotoxic, anti-inflammatory, and antioxidant activity
All synthesized compounds 1–20 were screened in a panel
of bioassays to evaluate their antioxidant, anti-inflamma-
tory and cytotoxic activities. The transcription factor
NF-jB plays a key role in regulation of genes involved in
immune and inflammatory responses and apoptosis. The
activation of NF-jB has been implicated in different types
of cancers and in many human chronic inflammatory dis-
eases. Therefore, NF-jB is a central target for a variety of
anti-inflammatory agents (Ankisetty et al., 2010). A large
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Med Chem Res (2012) 21:3499–3508
Table 1 Anticandidal evaluation of hydrazide-hydrazones (1–10) and 1,3,4-oxadiazole derivatives (11–20) as MIC values in lg/ml
Compounds
A*
B
C
D
E
F
G
H*
1
nt
500
500
500
500
500
500
500
500
250
500
1000
1000
500
500
500
500
500
500
500
1000
500
500
500
500
500
500
250
500
1000
500
500
125
250
250
500
250
nt
nt
500
500
500
500
500
500
500
500
250
500
1000
500
500
250
500
500
500
125
250
1000
500
500
500
500
500
500
250
500
1000
500
500
250
500
500
1000
250
500
500
500
500
500
500
500
500
125
500
500
500
500
500
500
500
500
500
250
250
nt
2
500
250
nt
3
500
500
4
nt
5
nt
1000
250
250
250
125
500
250
250
6
nt
7
nt
500
8
500
250
250
500
250
500
250
250
250
250
500
[1000
500
16
500
9
250
10
500
11
1000
250
1000
500
12
13
500
500
14
250
500
15
500
250
16
500
250
17
500
500
18
125
500
19
[1000
1000
62
[1000
500
[1000
500
[1000
500
[1000
1000
125
[1000
500
[1000
500
20
Ketoconazole
62
62
62
125
31
A*, Candida albicans (clinical isolate); B, Candida albicans (ATCC 90028); C, Candida utilis (NRRL Y-900); D, Candida tropicalis (NRRL
Y-12968); E, Candida krusei (NRRL Y-7179); F, Candida parapsilosis (NRRL Y-12696); G, Candida albicans (NRRL Y-12983); H*, Candida
glabrata (clinical isolate); nt, not determined
Fig. 1 Growth inhibition of Colletotrichum acutatum (Ca) after 72 h
Fig. 2 Growth inhibition of Colletotrichum gloeosporioides (Cg)
using 96-well microdilution broth assay in a dose response and the
commercial fungicide standard captan
after 72 h using 96-well microdilution broth assay in a dose response
and the commercial fungicide standard captan
number of natural and synthetic compounds are currently
being investigated for their effect on NF-jB activity. Of
the tested compounds, 16 showed a strong inhibition of
NF-jB-dependent transcription in SW1353 cells induced
by phorbol myristate acetate (PMA) with IC50 values
of 0.75 lg/ml, which was comparable with the activity
of positive control, parthenolide (IC50 of 0.68 lg/ml)
(Table 2). Compounds 1 and 14 also showed considerable
activity with IC50 values of 2.8 and 5.5 lg/ml, respectively.
Moderate activity was observed for compound 3 with IC50
value of 15 lg/ml. A luciferase construct with binding sites
for Sp-1 was used as a control because this transcription
factor is relatively unresponsive to inflammatory media-
tors. Measurement of Sp-1-mediated luciferase expression
in parallel to NF-kB mediated luciferase expression is
employed for detecting agents that non-specifically inhibit
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Med Chem Res (2012) 21:3499–3508
3503
Fig. 3 Growth inhibition of Fusarium oxysporum (Fo) after 72 h
using 96-well microdilution broth assay in a dose response and the
commercial fungicide standard captan
Fig. 5 Growth inhibition of Phomopsis viticola (Pv) after 144 h
using 96 well microdilution broth assay in a dose response and the
commercial fungicide standard captan
controls. A strong inhibition of the activity of iNOS
activity in LPS induced RAW 264.7 cells was observed for
compound 14 with IC50 value of 0.3 lg/ml. The effect was
comparable with the positive control parthenolide (IC50
=
0.4 lg/ml). Compound 16 also inhibited iNOS activity
with IC50 value of 4.0 lg/ml. The other compounds, 4, 6,
9–12, 17 were moderately active (inhibition of[30% at the
highest concentration), while 1–3, 5, 7–8, 13, 15, 18–20
were inactive. Cytotoxicity of test samples to macrophages
was also determined in parallel to determine if the inhibi-
tion of iNOS is due to cytotoxic effects. None of the
compounds were cytotoxic to RAW 264.7 cells. The
comparison of iNOS activity of hydrazone and oxadiazole
derivatives showed that oxadiazoles bearing a phenyl,
4-bromophenyl, 4-hydroxyphenyl, 3-hydroxy-4-methoxy-
phenyl, 4-dimethyl aminophenyl were active compounds.
Especially, the substitution of a hydroxyl to a phenyl
group in the 4-position (compound 14) resulted activity
comparable to parthenolide. The inhibitory activity of
compound 16 which had hydroxyl and methoxyl group at 3-
and 4-position of phenyl ring was also significant (Table 2).
Since reactive oxygen species (ROS) and oxidative
stress play an important role in major human degenerative
diseases such as cancer, inflammation, atherosclerosis, and
aging, we investigated the effect of these compounds on
intracellular ROS generation in PMA induced HL-60 cells.
A strong antioxidant effect was demonstrated by, 6, 10, 16
with IC50 values in range of 0.7–0.9 lg/ml. Compounds 5
and 20 were also effective with IC50 of 2.5 and 5.2 lg/ml,
respectively. Compounds 3 and 7 showed mild activity
(IC50 = 20 and 23 lg/ml, respectively). Moderate cyto-
toxicity was observed towards HL-60 cells by several of
them (see Table 2). Among the most active compounds
(6, 10, and 16) 6 was noncytotoxic while 10 and 16 showed
cytotoxicity at much higher concentrations than the effec-
tive concentrations for antioxidative activity (Table 2)
indicating that the antioxidant effect of these compounds is
Fig. 4 Growth inhibition of P. obscurans (Po) after 144 h using
96-well microdilution broth assay in a dose response and the
commercial fungicide standard captan
luciferase expression due to cytotoxicity, inhibition of
luciferase enzyme activity or light output (Tabanca et al.,
2007a, b). None of the compounds inhibited Sp-1 depen-
dent luciferase expression except 1. Compound 1 inhibited
Sp-1-mediated activity at much higher concentration
(IC50 = 14 lg/ml) than the concentration responsible for
inhibiting NF-jB activity (IC50 = 2.8 lg/ml).
The introduction of methoxy group at 4-position of
phenyl ring in oxadiazole moiety and also replacement of
the hydroxyl group at 4-position to 3-position of phenyl
ring (compound 16) resulted stonger inhibition of NF-jB
activity compared to compound 14 which only had a
hydroxyl group at 4-position of phenyl ring.
Inducible nitric oxide synthase (iNOS) plays a key role
in the development of inflammatory diseases. Thus an
iNOS inhibitor could be considered a potential anti-
inflammatory agent (Ankisetty et al., 2010) Inhibition of
lipopolysaccharide (LPS)-induced nitric oxide synthase
activity in RAW 264.7 cells was determined (Table 2) by
measuring the decrease in nitrite production in cells treated
with compounds 1–20 in comparison to untreated vehicle
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Med Chem Res (2012) 21:3499–3508
Table 2 Anti-inflammatory and antioxidant activities of tested compounds
Compounds Inhibition of NF-jB activity in Antioxidant Activity (inhibition of ROS
Inhibition of iNOS activity (NO production)
in RAW 264.7 cells
SW1353 cells
generation) in HL-60 cells
IC50 (lg/ml)
IC50 (lg/ml)
IC50 (lg/ml)
NF-jB activity
SP-1
Antioxidant activity
Cytotoxicity
iNOS activity
Cytotoxicity
1
2.8
14
NA
NA
20
NA
NA
NA
NA
20
NA
NA
NA
[25
NA
22
NA
NA
NA
[25
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
2
NA
15
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
3
4
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
5.5
NA
2.5
0.9
23
5
6
NA
[31.25
NA
16
7
NA
NA
[25
[25
[25
[25
NA
0.3
8
NA
NA
0.7
NA
NA
NA
NA
NA
0.9
NA
NA
NA
5.2
9
10
27
11
31.3
NA
NA
NA
NA
20
12
13
14
15
NA
0.75
NA
NA
NA
NA
NA
4
16
17
NA
NA
5.9
20
18
NA
NA
NA
19
20
Doxorubicina
24
0.16
Parthenolidea 0.68
Troloxa
7.5
0.4
13
0.3
a
Standard compounds with known biological activities. NA no activity up to IC50 = 25 lg/ml
not related to their effect on cell viability. Out of the
hydrazone and oxadiazole compounds, the antioxidant
activity of compound 6 may be explained due to the
presence of hydroxyl and methoxyl groups.
activity. Compound 9 showed equal activity with keto-
conazole against Candida albicans. The same compound
also demonstrated potential for further development for
control of Phomopsis viticola. Phomopsis cane and leaf
spot, caused by the fungus P. viticola, can cause significant
losses to the vine grape industry in the worldwide. Evalu-
ation of the biological activity of some of these compounds
in the present study indicated their potential as anti-
inflammatory agents through their effects on various tar-
gets. Some compounds displayed remarkable antioxidant
activity. Hydrazide-hydrazone compounds 1, 6, 10 and
1,3,4-oxadiazole compounds 14, 16, 20 can be interesting
source for lead compounds for anti-inflammatory research.
In addition, these 1–20 synthesized compounds were
also tested in a panel of mammalian kidney cells (Vero and
LLC-PK11) and cancer cells (SK-MEL, KB, BT-549, and
SK-OV-3) up to a concentration of 25 lg/ml to see their
cytotoxic and anti-cell proliferative effects but none
showed any effect (data not shown).
Conclusions
In conclusion, a detailed study concerning synthesis,
structure and biological activity of known hydrazide–
hydrazones and newly synthesized 1,3,4-oxadiazole deriv-
atives is reported. The in vitro antifungal activity against
human pathogenic fungi and plant pathogens were investi-
gated and some of the compounds have showed remarkable
Experimental
Chemistry
All chemicals and solvents were procured from Merck and
Aldrich (both Germany). Reactions were monitored by thin
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Med Chem Res (2012) 21:3499–3508
3505
layer chromatography (TLC) and purity of the products
was checked by high performance liquid chromatography
(HPLC). TLC was performed on Merck 60 F-254 silica gel
plates with visualization by UV-light using chloroform and
methanol as solvent system. HPLC (Agilent, Palo Alto,
CA, USA) was performed using a Chromasil C18
3.9 9 150 mm column. The eluent was acetonitrile and
water (50:50 v/v) and the flow rate was 1 ml/min, diode
array detection at 254 nm.
735. 1H-NMR (400 MHz, DMSO-d6) d (ppm): 2.26 (s, 3H,
–COCH3); 7.20 (s, 1H, -OCHR); 7.28-7.42 (t, 2H, ortho-
protons to F), 7.46 (d, 2H, ortho-protons to Br, J: 8.46 Hz);
7.66 (d, 2H, meta-protons to Br, J: 8.43 Hz); 7.84–7.94 (m,
2H, meta-protons to F). Anal. calcd for C16H12BrFN2O2
(363.18) : C, 52.91; H, 3.33; N, 7.71. Found: C, 52.75; H,
3.36; N, 7.69. MS-ES (m/z): 364.18 (MH?).
3-Acetyl-2,5-bis(4-fluorophenyl)-2,3-dihydro-1,3,4-oxadi-
azole (13) Yield: 87%. M.p. 126-128°C. Rt(acetonitrile
and water, 50:50 v/v) 3.219 min. IR (tmax, cm-1): 2970,
Melting points were determined on a SMP II apparatus
(Gehrden, Germany). The IR spectra were recorded on a
1
Schimadzu FTIR 8400S spectrometer (Kyoto, Japan). H
1
2860, 1675, 1570, 1475, 1420, 1210, 1040, 733. H-NMR
NMR spectra were recorded on Bruker Avance-DPX-400
spectrometer (Brillerica, MA, USA) in d6-DMSO. Chemical
shifts were recorded in parts per million downfield from
(400 MHz, DMSO-d6) d (ppm): 2.35 (s, 3H, –COCH3);
7.16–7.52 (m, 5H, –OCHR and ortho-protons to F),
7.84–8.11 (m, 4H, meta-protons to F). Anal. calcd for
C16H12F2N2O2 (302.27): C, 63.57; H, 4.00; N, 9.27. Found:
C, 63.56; H, 4.09; N, 9.35. MS-ES (m/z): 303.27 (MH?).
1
TMS. The splitting patterns of H-NMR were designed as
follows: s: singlet, d: doublet, t: triplet, q: quartet, m: mul-
tiplet. Mass spectra were recorded on LC–MS-Agilent 1100
(Palo Alto, CA, USA) series in the electrospray mode. Ele-
mental analysis was performed on Leco CHNS-932 analyzer
(Michigan, USA). (1H-NMR, mass and elemental analyses
were provided by the Scientific and Technical Research
Council of Turkey, TUBITAK).
3-Acetyl-5-(4-fluorophenyl)-2-(4-hydroxyphenyl)-2,3-dihy-
dro-1,3,4-oxadiazole (14) Yield: 49%. M.p. 137–139°C.
Rt(acetonitrile and water, 50:50 v/v) 3.970 min. IR (tmax
,
cm-1): 2960, 2850, 1680, 1565, 1475, 1410, 1230, 1045,
1
732. H-NMR (400 MHz, DMSO-d6) d (ppm) 2.28 (s, 3H,
–COCH3); 7.20-7.22 (m, 3H, –OCHR and ortho-protons to
OH); 7.36–7.41 (t, 2H, ortho-protons to F), 7.53 (d, 2H,
meta-protons to OH, J: 8.53 Hz); 7.89–7.93 (m, 2H, meta-
protons to F), 10.60 (s, 1H, OH). Anal. calcd for
C16H13FN2O3 (300.28): C, 64.00; H, 4.36; N, 9.33. Found:
C, 64.02; H, 4.38; N, 9.29. MS-ES (m/z): 301.28 (MH?).
Procedure for the preparation of the 3-acetyl-2,5-
disubstituted-2,3-dihydro-1,3,4-oxadiazole derivatives
11–20
Physicochemical and spectroscopic characterization of the
initial hydrazide–hydrazones, 4-fluorobenzoic acid (sub-
stitutedmethylene)hydrazides (1–10), have been described
in our previous study (Koc¸yigit-Kaymakcıoglu et al.,
2006). A mixture of an appropriate hydrazide-hydrazone
(1 mmol) and Ac2O (5 ml) was heated under reflux for 2 h.
After the mixture was cooled to room temperature, excess
Ac2O was decomposed by addition of H2O and the mixture
was stirred for 30 min. The separated product was filtered
and crystallized from EtOH.
3-Acetyl-5-(4-fluorophenyl)-2-(4-methoxyphenyl)-2,3-dihy-
dro-1,3,4-oxadiazole (15) Yield: 85%. M.p. 165–167°C.
Rt(acetonitrile and water, 50:50 v/v) 4.525 min. IR (tmax
,
cm-1): 2955, 2880, 1673, 1590, 1480, 1425, 1230, 1035,
1
735. H-NMR (400 MHz, DMSO-d6) d (ppm) 2.06 (s, 3H,
–COCH3); 3.76 (s, 3H, –OCH3); 6.76-7.10 (m, 7H,
–OCHR, ortho-protons to OCH3, ortho-protons to F and
meta-protons to OCH3); 7.54–7.68 (m, 2H, meta-protons to
F). Anal. calcd for C17H15FN2O3 (314.31): C, 64.96; H,
4.81; N, 8.91. Found: C, 64.84; H, 4.79; N, 8.95. MS-ES
(m/z): 315.31 (MH?).
3-Acetyl-5-(4-fluorophenyl)-2-phenyl-2,3-dihydro-1,3,4-oxa-
diazole (11) Yield: 76%. M.p. 103–105°C. Rt(acetonitrile
and water, 50:50 v/v) 4.555 min. IR (tmax, cm-1): 2950,
1
2860, 1678, 1580, 1470, 1420, 1220, 1030, 730. H-NMR
3-Acetyl-5-(4-fluorophenyl)-2-(3-hydroxy-4-methoxyphenyl)
-2,3-dihydro-1,3,4-oxadiazole (16) Yield: 82%. M.p.
135–137°C. Rt(acetonitrile and water, 50:50 v/v)
3.913 min. IR (tmax, cm-1): 2990, 2870, 1675, 1590, 1465,
(400 MHz, DMSO-d6) d (ppm): 2.28 (s, 3H, –COCH3);
6.96-8.01 (m, 10H, –OCHR and Ar–H). Anal. calc for
C16H13FN2O2 (284.28): C, 67.60; H, 4.61; N, 9.85.
Found: C, 67.65; H, 4.66; N, 9.83. MS-ES (m/z): 285.28
(MH?).
1
1428, 1225, 1040, 733. H-NMR (400 MHz, DMSO-d6) d
(ppm): 1.96 (s, 3H, -COCH3); 3.82 (s, 3H, -OCH3); 7.03-
7.47 (m, 6H, –OCHR, aromatic protons and ortho-protons
to F); 7.90–7.93 (m, 2H, meta-protons to F). Anal. calcd for
C17H15FN2O4 (330.31): C, 61.82; H, 4.58; N, 8.48. Found:
C, 61.88; H, 4.59; N, 8.47. MS-ES (m/z): 331.31 (MH?).
3-Acetyl-5-(4-fluorophenyl)-2-(4-bromophenyl)-2,3-dihy-
dro-1,3,4-oxadiazole (12) Yield: 90%. M.p. 126–128°C.
Rt(acetonitrile and water, 50:50 v/v) 4.693 min. IR (tmax
,
cm-1): 2948, 2850, 1685, 1580, 1465, 1420, 1225, 1040,
123

3506
Med Chem Res (2012) 21:3499–3508
3-Acetyl-5-(4-fluorophenyl)-2-(4-(dimethylamino)phenyl)-
2,3-dihydro-1,3,4-oxadiazole (17) Yield: 65%. M.p. 119–
121°C. Rt(acetonitrile and water, 50:50 v/v) 3.650 min. IR
(tmax, cm-1): 2960, 2870, 1674,1580, 1475, 1410, 1220,1035,
modified adaptation of the NCCLS reference document
¨
M27-A2 (NCCLS, 2002; Ozdemir et al., 2010) against
Candida albicans (clinical isolate), Candida albicans
(ATCC 90028), Candida glabrata (clinical isolate), Can-
dida utilis (NRRL Y-900), Candida tropicalis (NRRL
Y-12968), Candida parapsilosis (NRRL Y-12696), Can-
dida albicans (NRRL Y-12983). Antifungal evaluation of
the hydrazide–hydrazones 1–10 and corresponding 3-acetyl-
2,5-disubstituted-2,3-dihydro-1,3,4-oxadiazoles 11–20 against
clinical and standard Candida pathogenic strains have been
performed by using agar diffusion to the indentify the active
compounds which were later subjected to a broth microdi-
lution assay to justify the activity level in terms of MIC
1
730. H-NMR (400 MHz, DMSO-d6) d (ppm): 1.92 (s, 3H,
–COCH3); 3.05 (s, 6H, –N(CH3)2); 6.72–6.87 (m, 3H,
–OCHR and ortho-protons to ter.amine); 7.25–7.45 (m, 2H,
ortho-protons to F), 7.70 (d, 2H, meta-protons to ter. amine J:
8.95 Hz); 7.86–8.09 (m, 2H, meta-protons to F). Anal. calcd
for C18H18FN3O2 (327.35):C,66.04;H,5.54;N,12.84.Found:
C, 66.08; H, 5.52; N, 12.85. MS-ES (m/z): 328.35 (MH?).
Yield: 54%. M.p. 153–155°C. Rt(acetonitrile and water,
50:50 v/v) 3.840 min. IR (tmax, cm-1): 2965, 2870, 1678,
1
¨
(NCCLS, 2002; Ozdemir et al., 2010). Ketoconazole was
used as positive control.
1585, 1460, 1430, 1225, 1032, 736. H-NMR (400 MHz,
DMSO-d6) d (ppm): 1.92 (s, 3H, –COCH3); 2.78 (s, 6H,
-N(CH3)2); 3.00–3.10 (m, 2H, –CH2–); 6.74-6.77 (m, 3H,
–OCHR, ortho-protons to ter. amine); 7.02–7.06 (m, 2H,
–CH=CH–); 7.46-7.51 (m, 2H, ortho-protons to F),
7.56–7.73 (m, 3H, meta-protons to ter.amine, –CH=);
8.13-8.16 (m, 2H, meta-protons to F). Anal. calcd for
C20H20FN3O2 (353.39): C, 67.97; H, 5.70; N, 11.89. Found:
C, 67.96; H, 5.72; N, 11.85. MS-ES (m/z): 354.39 (MH?).
Anticandidal assay
The activity of the compounds 1–20 were first screened
using an agar diffusion method for C. albicans (clinical
isolate) and C. tropicalis and all active compounds (inhi-
bition zones [9–11 mm, at 2,000 lg/ml concentration)
were further evaluated using the microdilution broth
method to identify the MIC against all Candida spp.
3-Acetyl-2-(5-bromothiophen-2-yl)-5-(4-fluorophenyl)-2,3-
dihydro-1,3,4-oxadiazole (19) Yield: 65%. M.p. 145–
147°C. Rt(acetonitrile and water, 50:50 v/v) 4.150 min. IR
(tmax, cm-1): 2955, 2860, 1680, 1580, 1475, 1425, 1220,
Broth microdilution assay
1
1033, 730. H-NMR (400 MHz, DMSO-d6) d (ppm): 2.20
The test compounds and the antimicrobial standards were
first dissolved in dimethyl sulfoxide (DMSO) which was
used to prepare the stock solutions at an initial concentra-
tion of 2,000 lg/ml. Serial dilution series were prepared in
100 ll MHB with an equal amount of the test samples. The
last row was filled only with water as growth control for the
microorganism. Overnight grown microorganism suspen-
sions were first diluted in double strength MHB and stan-
dardized to 108 CFU/ml (using McFarland No: 0.5) under
sterile conditions. Then each microorganism suspension
was pippetted into each well and incubated at 37°C for
24 h. Ketoconazole was used as a standard antifungal agent
against Candida spp. Sterile distilled water and medium
served as a positive growth control. The first well without
turbidity was assigned as the minimum inhibitory con-
centration (MIC, in lg/ml).
(s, 3H, –COCH3); 6.82 (s, 1H, C3-H proton of thiophen);
7.16 (s, 1H, –OCHR); 7.30–7.45 (m, 3H, ortho-protons to F
and C4-H proton of thiophen), 7.75–7.97 (m, 2H, meta-
protons to F). Anal. calcd for C14H10BrFN2O2S (369.20) :
C, 45.54; H, 2.73; N, 7.59; S, 8.68. Found: C, 45.50; H,
2.74; N, 8.66; S, 8.68. MS-ES (m/z): 370.20 (MH?).
3-Acetyl-5-(4-fluorophenyl)-2-(furan-2-yl)-2,3-dihydro-1,3,
4-oxadiazole (20) Yield: 67%. M.p. 130–132°C. Rt(ace-
tonitrile and water, 50:50 v/v) 3.835 min. IR (tmax, cm-1):
1
2950, 2870, 1685, 1560, 1450, 1430, 1230, 1040, 732. H-
NMR (400 MHz, DMSO-d6) d (ppm): 2.25 (s, 3H, –COCH3);
6.52 (d, 1H, J: 3.25 Hz, C3-H proton of furan); 6.78 (t, 1H, C4-
H proton of furan); 7.26 (s, 1H, –OCHR); 7.29–7.44 (m, 2H,
ortho-protons to F), 7.73 (s, 1H, C5-H proton of furan),
7.88–8.00 (m, 2H, meta-protons to F). Anal. calcd for
C14H11FN2O3 (274.24): C, 61.61; H, 4.04; N, 10.21. Found:
C, 61.60; H, 4.05; N, 10.19. MS-ES (m/z): 275.24 (MH?).
Antifungal assay against plant pathogens
A standardized 96-well micro-dilution broth assay devel-
oped by Wedge and Kuhajek (1998) for the discovery of
natural fungicidal agents was used to evaluate the anti-
fungal activity of test compounds (Wedge and Camper
2000). Isolates of Colletotrichum acutatum Simmonds,
Colletotrichum fragariae Brooks, Colletotrichum gloeo-
sporioides (Penz.) Penz & Sacc. In Penz, Botrytis cinerea
Biological assays
Antifungal activity against human pathogens
The antifungal properties of compounds 1–20 were evalu-
ated by the broth microdilution method according to a
123

Med Chem Res (2012) 21:3499–3508
3507
Pers.:Fr, Fusarium oxysporum Schlechtend:Fr, Phomopsis
obscurans (Ellis and Everh.) B. sutton, and P. viticola
Sacc., were used to evaluate the antifungal activity of the
test compounds using in vitro micro-dilution broth assay.
Each microtiter test well received 80 ll of RPMI 1640
(Roswell Park Memorial Institute mycological broth
1640, Life Technologies, Grand Island, New York) and
3[N-morpholino]propanesulfonic acid (MOPS, Sigma
Chemical Co., St. Louis, Missouri) buffered broth, 100 ll
of conidial suspension at 1.0 9 104 conidia/ml, and 20 ll
of test compound solution. The commercial fungicide
captan was used as an internal fungicide standard in all
assays. Each fungus was challenged in a dose–response
format using test compounds, with final treatment con-
centrations of 0.3, 3.0, and 30.0 lM. Microtiter plates
(Nunc MicroWell, untreated; Roskilde, Denmark) were
covered with a plastic lid and incubated in a growth
chamber at 24 1°C and a 12-h photoperiod under a light
intensity of 60 5 lmol/m2/s. Growth was then evaluated
by measuring absorbance (620 nm) of each well using a
microplate reader (model SpectraCount; Packard Instru-
ment Company, Meriden, Connecticut).
lysed by adding 40 ll of a 1:1 mixture of LucLite reagent
and PBS containing 1 mM calcium and magnesium.
Luciferase activity was measured as light output on a
SpectraMax plate reader. IC50 values were obtained from
dose response curves. Sp-1 was used as a control tran-
scription factor to evaluate the toxicity of tested com-
pounds in the same assay. Parthenolide was used as the
positive control.
Inhibition of intracellular NO production as a result of
iNOS activity was assayed in mouse macrophages (RAW
264.7 cells) as described (Ankisetty et al., 2010; Quang
et al., 2006). Cells were seeded in 96-well plates at a
density of 50,000 cells/well and grown for 24 h for a
confluency of 75% or more. Test samples were added at
various concentrations and after 30 min LPS (5 lg/ml) was
added and cells were further incubated for 24 h. The
concentration of NO was determined by measuring the
level of nitrite in the cell culture supernatant with Griess
reagent. The degree of inhibition of nitrite production was
calculated in comparison to the vehicle control. IC50 values
were obtained from dose response curves. Cytotoxicity of
test samples to macrophages was also determined in par-
allel to check if the inhibition of iNOS is due to cytotoxic
effects. Parthenolide was included in each assay as the
positive control.
Using the 96-well plate micro-bioassay format, each
chemical was evaluated in duplicate at three concentra-
tions. The experiments were repeated three times over
time. Mean absorbance values and standard errors were
used to evaluate fungal growth at 48 h and 72 h, except for
P. obscurans and P. viticola the data were recorded at
144 h. Analysis of variance of means for percent inhibi-
tion/stimulation of each fungal species at each dose of test
compound relative to the untreated positive growth con-
trols was used to evaluate fungal growth. Treatments were
arranged as a split-plot design repeated four times. Whole-
plots were fungal isolates and sub-plots were chemicals.
Each dose level and response time was analyzed separately.
The SAS system analysis of variance procedure (Statistical
Analysis System, Cary, North Carolina) was used to
identify significant factors, and Fisher’s protected LSD was
used to separate means (Steel and Torrie, 1980).
Inhibition of intracellular ROS generation (antioxidant
activity) was assayed in human promyelocytic leukemia
(HL-60) cells. Cells were seeded in 96-well plates (100,000
cells/well) and treated with different concentrations of test
samples for 30 min. Cells were then stimulated with PMA
(100 ng/ml) for 30 min. ROS generation is determined by
using DCFH-DA as described previously (Tabanca et al.,
2007a, b). DCFH-DA is a non-fluorescent probe that dif-
fuses into the cells. Cytoplasmic esterases hydrolyze the
DCFH-DA to 20,70-dichlorofluorescin (DCFH). The reac-
tive oxygen species (ROS) generated within cells oxidize
DCFH to 20,7’-dichlorofluorescin (DCF) that fluoresces.
The ability of the test compounds to inhibit production of
DCF in PMA treated HL-60 cells was measured in com-
parison to the vehicle control. The IC50 values were cal-
culated from dose curves of the % DCF production versus
test concentrations. Trolox was used as positive control.
Anti-inflammatory assays
Inhibition of NF-jB mediated transcription was determined
in human chondrosarcoma (SW1353) cells by a reporter
gene assay as described earlier (Tabanca et al., 2007a, b).
In brief, at about 75% confluency, cells were harvested and
transfected with NF-jB reporter luciferase plasmid con-
struct at 160 V and one 70-ms pulse in a BTX Electro
Square Porator T 820. Transfected cells were plated in
96-well plates (1 9 105 cells/well) and incubated for 24 h.
After 24 h, cells were exposed to test samples for 30 min
and then incubated for 8 h with PMA (70 ng/ml) for the
activation of NF-jB. After removing medium, cells were
Cytotoxicity assay
Cytotoxicity was determined against a panel of four human
tumor cell lines [SK-MEL (malignant melanoma); KB
(oral epidermal carcinoma); BT-549 (breast ductal carci-
noma); SK-OV-3 (ovary carcinoma)] and two noncancer-
ous cell lines [Vero (African green monkey kidney
fibroblasts) and LLC-PK11 (pig kidney epithelial cells)] as
described earlier (Tabanca et al., 2003). Cells were seeded
at a density of 25,000 cells/well in 96-well plates and
123

3508
Med Chem Res (2012) 21:3499–3508
novel hydrazide-hydrazones and the study of their structure-
antituberculosis activity. Eur J Med Chem 41:1253–1261
Li S, Luo Y, Wang L, Li D (2010) Synthesis of 1,3,4-oxazolines and
1,3,4-oxadiazole containing fluorine and their anticancer activity.
Hecheng Huaxue 18:611–613 (Chem. Abst. 2010, 154:336040)
NCCLS, Reference Method for Broth Dilution Antifungal Suscepti-
bility Testing of Yeasts, Approved Standard, second ed., NCCLS
document M27-A2 [ISBN 1-56238-469-4], 2002
grown for 24 h. Samples were added and plates were
incubated for 48 h. Cell viability was determined by using
Neutral Red. Doxorubicin was used as a positive control.
Acknowledgments This project was financially supported by the
Marmara University Research Center (Project number SAG-060-
210). The authors express their gratitude for USDA, ARS, NPURU
financial support. Also Ms. J. Linda Robertson, Ms. Ramona Pace,
and Ms. Xiaoning Wang for assistance with the antifungal assay,
Mr. John Trott, Mr. Paul Bates, and Ms. Katherine Martin for bio-
assays for anti-inflammatory, antioxidant, and cytotoxic activities are
acknowledged.
¨
˙
Ozdemir A, Turan-Zitouni G, Kaplancıklı ZA, Is¸can G, Khan S,
Demirci F (2010) Synthesis and the selective antifungal activity
of 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine derivatives. Eur J
Med Chem 45:2080–2084
Quang DN, Harinantenaina L, Nishizawa T, Hashimoto T, Kohchi C,
Soma G, Asakawa Y (2006) Inhibition of nitric oxide production
in RAW 264.7 cells by azaphilones from Xylariaceous fungi.
Biol Pharm Bull 29:34–37
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