Synthesis and biological evaluation of some hydrazone derivatives as anti-inflammatory agents

Article abstract of DOI:10.2174/157018012799129828

In the present study, some hydrazone derivatives were synthesized via the reaction of 3-cyclohexylpropionic acid hydrazide with various benzaldehydes. The chemical structures of the compounds were elucidated by spectroscopic techniques such as IR, ¹H-NMR and FAB-MS and elemental analyses. The compounds were evaluated for their antiinflammatory and cytotoxic activities. Anti-inflammatory activity was determined in terms of inhibition of NF-κB, ROS generation and iNOS activity. Several derivatives inhibited NF-κB and iNOS, but no effect was observed on intracellular ROS generation. Furthermore no cytoxicity was observed. Biological activity compared with the chemical structural information suggests that different functional groups on the phenyl ring influence the physicochemical properties and thus modulate biological activity.

Full text of DOI:10.2174/157018012799129828

310
Letters in Drug Design & Discovery, 2012, 9, 310-315
Synthesis and Biological Evaluation of Some Hydrazone Derivatives as
Anti-inflammatory Agents
Zafer Asım Kaplancikli*,a, Mehlika Dilek Altintopa, Ahmet Özdemira, Gülhan Turan-Zitounia,
Shabana I. Khanb,c and Nurhayat Tabancad
aAnadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskisehir, Turkey
bNational Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of
Mississippi, University, MS 38677, USA
cDepartment of Pharmacognosy, School of Pharmacy, University of Mississippi University, MS 38677 USA
dNational Center for Natural Products Research, The University of Mississippi, University, MS 38677 USA
Received October 18, 2011: Revised November 30, 2011: Accepted December 01, 2011
Abstract: In the present study, some hydrazone derivatives were synthesized via the reaction of 3-cyclohexylpropionic
acid hydrazide with various benzaldehydes. The chemical structures of the compounds were elucidated by spectroscopic
1
techniques such as IR, H-NMR and FAB-MS and elemental analyses. The compounds were evaluated for their anti-
inflammatory and cytotoxic activities. Anti-inflammatory activity was determined in terms of inhibition of NF-ꢀB, ROS
generation and iNOS activity. Several derivatives inhibited NF-ꢀB and iNOS, but no effect was observed on intracellular
ROS generation. Furthermore no cytoxicity was observed. Biological activity compared with the chemical structural
information suggests that different functional groups on the phenyl ring influence the physicochemical properties and thus
modulate biological activity.
Keywords: Hydrazone, Anti-inflammatory activity, Cytotoxicity, Antioxidant activity.
INTRODUCTION
discovered new effective analgesic, anti-inflammatory and
antithrombotic agents bearing hydrazone moiety and carried
out considerable research for identifying the pharmacophoric
contribution of N-acylhydrazone moiety to modulation of
biological activity [25].
Inflammation, a biological stress response of vascular
tissues to harmful stimuli, such as pathogens, damaged cells,
or irritants, is a protective attempt by the organism to remove
the injurious stimuli and then initiate the healing response
[1,2].
In this study, we described the synthesis of some
hydrazone derivatives and focused on their anti-
inflammatory activity and cytotoxicity.
Nonsteroidal anti-inflammatory drugs (NSAIDs) act
primarily by inhibiting cyclooxygenase (COX) enzymes,
which catalyze the first step in the prostaglandin
biosynthesis. These agents are currently used as first-line
therapeutics in the treatment of osteoarthritis, rheumatoid
arthritis, systemic lupus erythematosis, and other
inflammatory syndromes to reduce inflammation and control
pain. In each case, the treatment is palliative rather than
disease modifying [3].
MATERIALS AND METHODS
Chemistry
All reagents were purchased from commercial suppliers
and were used without further purification. Melting points
(m.p.) were determined on a Electrothermal 9100 melting
point apparatus (Weiss-Gallenkamp, Loughborough, UK)
and are uncorrected. Proton nuclear magnetic resonance (1H-
NMR) spectra were recorded on Bruker 500 MHz
spectrometer (Bruker, Billerica, MA, USA). Chemical shifts
were expressed in parts per million (ppm) and
tetramethylsilane was used as an internal standard. Mass
spectra were recorded on a VG Quattro Mass spectrometer
(Agilent, Minnesota, USA). Elemental analyses were
performed on a Perkin Elmer EAL 240 elemental analyzer
(Perkin-Elmer, Norwalk, CT, USA).
The frequent use of NSAIDs as over-the-counter (OTC)
drugs has been rising in recent years and therefore the
adverse effects associated with the widespread use of these
agents have become an inevitable problem. In order to
overcome this serious problem, the search for new effective
anti-inflammatory agents has gained great importance [4-6].
Hydrazones have received considerable attention due to
their biological importance in medicinal chemistry. Many
studies have confirmed that hydrazone derivatives exhibit a
wide spectrum of biological effects including anti-
inflammatory activity [7-25]. Fraga and co-workers
General Procedure for Synthesis of the Compounds
3-cyclohexylpropionic acid hydrazide (1)
*Address correspondence to this author at the Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University,
Eskiꢀehir 26470, Turkey; Tel: +90-222-3350580/3776; Fax: +90-222-
3350750; E-mail: zakaplan@anadolu.edu.tr
The compound was prepared by reacting ethyl 3-
cyclohexylpropionate with hydrazine hydrate according to
the literature [26].
1875-628X/12 $58.00+.00
© 2012 Bentham Science Publishers

Some Hydrazone Derivatives as Anti-inflammatory Agents
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3 311
N-(4-florobenzylidene)-3-cyclohexylpropionic acid
hydrazide (2e)
N-(benzylidene)-3-cyclohexylpropionic acid hydrazide
derivatives (2a-j)
IR (KBr) ꢁmax (cm-1): 3472-3225 (NH), 1679 (C=O),
1588-1502 (C=N, C=C).
A mixture of 3-cyclohexylpropionic acid hydrazide (1)
(30 mmol) and appropriate benzaldehydes in absolute
ethanol (25 mL) was refluxed for 3-5 hours. The resulting
solid was filtered and crystallized from ethanol.
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.87-1.74
(11H, m, cyclohexane), 2.19-2.22 (2H, m, cyclohexane-
CH2), 2.60-2.63 (2H, t, CH2-CO), 7.26-8.17 (5H, m, phenyl
and CH=N protons), 11.21-11.35 (1H, two s, N-H).
N-(benzylidene)-3-cyclohexylpropionic acid hydrazide (2a)
IR (KBr) ꢁmax (cm-1): 3450-3246 (NH), 1680 (C=O),
MS (FAB); m/z: 277 [M+1]
1600-1542 (C=N, C=C).
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.87-1.75
(11H, m, cyclohexane), 2.19-2.22 (2H, cyclohexane-CH2),
2.60-2.65 (2H, t, CH2-CO), 7.40-8.18 (6H, m, phenyl and
CH=N protons), 11.19-11.35 (1H, two s, N-H).
Anal. Calc. for C16H21FN2O: C, 69.54; H, 7.66; N, 10.14.
Found: C, 69.54; H, 7.68; N, 10.11.
N-(4-hydroxybenzylidene)-3-cyclohexylpropionic acid
hydrazide (2f)
IR (KBr) ꢁmax (cm-1): 3458-3228 (NH), 1681 (C=O),
1595-1508 (C=N, C=C).
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.85-1.74
(11H, m, cyclohexane), 2.15-2.19 (2H, m, cyclohexane-
CH2), 2.57-2.60 (2H, t, CH2-CO), 6.80-8.04 (5H, m, phenyl
and CH=N protons), 9.86-9.89 (1H, O-H), 10.98-11.12 (1H,
two s, N-H).
MS (FAB); m/z: 259 [M + 1]
Anal. Calc. for C16H22N2O: C, 74.38; H, 8.58; N, 10.84.
Found: C, 74.40; H, 8.57; N, 10.88.
N-(4-nitrobenzylidene)-3-cyclohexylpropionic acid
hydrazide (2b)
IR (KBr) ꢁmax (cm-1): 3468-3255 (NH), 1685 (C=O),
1605-1530 (C=N, C=C).
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.88-1.75
(11H, m, cyclohexane), 2.24-2.27 (2H, m, cyclohexane-
CH2), 2.65-2.68 (2H, t, CH2-CO), 7.91-8.30 (5H, m, phenyl
and CH=N protons), 11.53-11.66 (1H, two s, N-H).
MS (FAB); m/z: 275 [M+1]
Anal. Calc. for C16H22N2O2: C, 70.04; H, 8.08; N, 10.21.
Found: 70.01; H, 8.11; N, 10.24.
N-(4-methoxybenzylidene)-3-cyclohexylpropionic acid
hydrazide (2g)
IR (KBr) ꢁmax (cm-1): 3468-3215 (NH), 1688 (C=O),
1612-1535 (C=N, C=C).
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.87-1.74
(11H, m, cyclohexane), 2.17-2.20 (2H, m, cyclohexane-
CH2), 2.59-2.62 (2H, t, CH2-CO), 3.79-3.80 (3H, OCH3),
6.99-8.10 (5H, m, phenyl and CH=N protons), 11.07-11.20
(1H, two s, N-H).
MS (FAB); m/z: 304 [M+1]
Anal. Calc. for C16H21N3O3: C, 63.35; H, 6.98; N, 13.85.
Found: C, 63.39; H, 6.95; N, 13.83.
N-(4-methylbenzylidene)-3-cyclohexylpropionic acid
hydrazide (2c)
IR (KBr) ꢁmax (cm-1): 3475-3239 (NH), 1684 (C=O),
1625-1510 (C=N, C=C).
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.85-1.75
(11H, m, cyclohexane), 2.18-2.21 (2H, m, cyclohexane-
CH2), 2.33-2.34 (3H, CH3), 2.60-2.63 (2H, t, CH2-CO), 7.24-
8.12 (5H, m, phenyl and CH=N protons), 11.13-11.27 (1H,
two s, N-H).
MS (FAB); m/z: 289 [M+1]
Anal. Calc. for C17H24N2O2: C, 70.80; H, 8.39; N, 9.71.
Found: C, 70.84; H, 8.41; N, 9.73.
N-(4-chlorobenzylidene)-3-cyclohexylpropionic acid
hydrazide (2h)
IR (KBr) ꢁmax (cm-1): 3442-3216 (NH), 1686 (C=O),
1590-1526 (C=N, C=C).
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.85-1.74
(11H, m, cyclohexane), 2.19-2.23 (2H, m, cyclohexane-
CH2), 2.61-2.65 (2H, t, CH2-CO), 7.49-8.15 (5H, m, phenyl
and CH=N protons), 11.27-11.40 (1H, two s, N-H).
MS (FAB); m/z: 273 [M+1]
Anal. Calc. for C17H24N2O: C, 74.96; H, 8.88; N, 10.28.
Found: C, 74.99; H, 8.90; N, 10.27.
N-(4-bromobenzylidene)-3-cyclohexylpropionic acid
hydrazide (2d)
IR (KBr) ꢁmax (cm-1): 3440-3222 (NH), 1688 (C=O),
1621-1511 (C=N, C=C).
MS (FAB); m/z: 293 [M+1]
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.87-1.74
(11H, m, cyclohexane), 2.19-2.23 (2H, m, cyclohexane-
CH2), 2.60-2.65 (2H, t, CH2-CO), 7.59-8.14 (5H, m, phenyl
and CH=N protons), 11.28-11.41 (1H, two s, N-H).
Anal. Calc. for C16H21ClN2O: C, 65.63; H, 7.23; N, 9.57.
Found: C, 65.65; H, 7.25; N, 9.56.
N-(4-isopropylbenzylidene)-3-cyclohexylpropionic acid
hydrazide (2i)
IR (KBr) ꢁmax (cm-1): 3452-3240 (NH), 1688 (C=O),
1635-1555 (C=N, C=C).
MS (FAB); m/z: 339 [M+2]
Anal. Calc. for C16H21BrN2O: C, 56.98; H, 6.28; N, 8.31.
Found: C, 56.95; H, 6.25; N, 8.31.
1H NMR (500 MHz) (DMSO-d6) ꢂ (ppm): 0.85-1.74
(17H, m, cyclohexane, isopropyl (CH3)2, 2.18-2.21 (2H, m,

312 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3
Kaplancikli et al.
cyclohexane-CH2), 2.60-2.65 (2H, t, CH2-CO), 2.89-2.95
(1H, m, isopropyl CH), 7.30-8.12 (5H, m, phenyl and CH=N
protons), 11.15-11.27 (1H, two s, N-H).
added at various concentrations and after 30 minutes LPS (5
ꢂg/mL) was added and cells were further incubated for 24 h.
NO concentration 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 parallel to check if the
inhibition of iNOS is due to cytotoxic effects. Parthenolide
was used in each assay as the positive control.
MS (FAB); m/z: 301 [M+1]
Anal. Calc. for C19H28N2O: C, 75.96; H, 9.39; N, 9.32.
Found: C, 75.97; H, 9.41; N, 9.34.
N-(4-dimethylaminobenzylidene)-3-cyclohexylpropionic
acid hydrazide (2j)
IR (KBr) ꢀmax (cm-1): 3440-3215 (NH), 1685 (C=O),
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 [27]. DCFH-DA is
1610-1500 (C=N, C=C).
1H NMR (500 MHz) (DMSO-d6) ꢁ (ppm): 0.84-1.75
(11H, m, cyclohexane), 2.15-2.18 (2H, m, cyclohexane-
CH2), 2.57-2.59 (2H, t, CH2-CO), 2.92-3.05 (6H, N(CH3)2),
6.73-8.00 (5H, m, phenyl and CH=N protons), 10.90-11.02
(1H, two s, N-H).
a
non-fluorescent probe that diffuses into the cells.
MS (FAB); m/z: 302 [M+1]
Cytoplasmic esterases hydrolyze the DCFH-DA to 2',7'-
dichlorofluorescein (DCFH). The reactive oxygen species
(ROS) generated within cells oxidize DCFH to 2',7'-
dichlorofluorescein (DCF) that fluoresces. The ability of the
test compounds to inhibit production of DCF in PMA treated
HL-60 cells was measured in comparison to the vehicle
control. The IC50 values were calculated from dose curves of
the % DCF production versus test concentrations. Trolox
was used as positive control.
Anal. Calc. for C18H27N3O: C, 71.72; H, 9.03; N, 13.94.
Found: C, 71.74; H, 9.05; N, 13.97.
Biological Assays
In vitro cytotoxicity and anti-inflammatory activities
were determined at National Center for Natural Products
Research, Research Institute of Pharmaceutical Sciences,
The University of Mississippi.
Cytotoxicity Assay
Anti-Inflammatory 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
carcinoma); and SK-OV-3 (ovary carcinoma)]; and two
noncancerous cell lines Vero (African green monkey kidney
fibroblasts) and LLC-PK1 (pig kidney epithelial cells)] as
described earlier [27]. Cells were seeded at a density of
25,000 cells/well in 96-well plates and grown for 24 hours.
Samples were added and plates were incubated for 48 hours.
Cell viability was determined by using Neutral Red. IC50
values were obtained from dose response curves of percent
viability versus test concentrations. Doxorubicin was used as
a positive control.
Anti-inflammatory activity was determined in terms of
inhibition of NF-ꢀB-mediated transcription and inhibition of
intracellular generation of reactive oxygen species (ROS)
and nitric oxide (NO). Free radicals are formed in
pathological events, such as oxidative stress during
inflammatory processes, which are often correlated with
increased levels of inducible nitric oxide synthase (iNOS)-
derived nitric oxide. Nitrite and nitrate, nitric oxide stable
metabolites, can react with superoxide radical, thus
generating peroxynitrites which, in turn, may activate NF-
ꢀB. Inhibition of NF-ꢀB mediated transcription was
determined in human chondrosarcoma (SW1353) cells by a
reporter gene assay as described earlier [27]. In brief, at
about 75% confluency, cells were harvested and transfected
with NF-ꢀB reporter luciferase plasmid construct at 160 V
and one 70-ms pulse in a BTX Electro Square Porator T 820.
Transfected cells were plated in 96-well plates (1x105
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-ꢀB. After
removing medium, cells were lysed by adding 40 ꢂL 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 transcription factor to evaluate the toxicity of tested
compounds 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 [27]. 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
RESULTS AND DISCUSSION
Chemistry
Initially, 3-cyclohexylpropionic acid hydrazide (1) was
obtained by the reaction of ethyl 3-cyclohexylpropionate
with hydrazine hydrate [26]. The target compounds (2a-j)
were synthesized via the nucleophilic addition-elimination
reaction of 3-cyclohexylpropionic acid hydrazide with
various benzaldehydes. These reactions are summarized in
Scheme 1 and some properties of the compounds are listed in
Table 1.
The structures of the compounds (2a-j) were confirmed
by IR, 1H- NMR and mass spectral data and elemental
analyses.
In the IR spectra, all derivatives (2a-j) had a strong,
characteristic band in the region 1700-1650 cm-1 due to the
C=O stretching vibration. The N-H stretching vibration of

Some Hydrazone Derivatives as Anti-inflammatory Agents
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3 313
O
O
NH2NH2. H2O
NHNH2
OC2H5
1
CHO
R
O
N
N
H
R
2a-j
Fig. (1). The synthesis of hydrazone derivatives (2a-2j).
Table 1. Some Properties of Hydrazone Derivatives (2a-2j)
Compound
R
Yield (%)
M.p. (°C)
Molecular formula
Molecular weight
2a
2b
2c
2d
2e
2f
H
NO2
85
95
75
85
85
80
80
85
80
80
109-112
164-168
113-116
180-183
110-113,5
174-178
122-125
149-152
83-86
C16H22N2O
C16H21N3O3
C17H24N2O
C16H21BrN2O
C16H21FN2O
C16H22N2O2
C17H24N2O2
C16H21ClN2O
C19H28N2O
C18H27N3O
258
303
272
337
276
274
288
292
300
301
CH3
Br
F
OH
2g
2h
2i
OCH3
Cl
CH(CH3)2
N(CH3)2
2j
116-118
the compounds (2a-j) gave rise to a band at 3475-3215 cm-1.
The stretching bands for C=C and C=N groups were
observed at 1635-1500 cm-1.
In the 1H-NMR spectra of the compounds (2a-j), the
cyclohexyl protons were observed at 0.84-1.75 ppm. The
CH2-cyclohexyl protons were observed at 2.15-2.27 ppm.
The signal due to CH2-CO protons appeared at 2.57-2.68
ppm as a triplet. The N=CH and phenyl protons were
observed at 6.73-8.30 ppm. The hydrazone proton signal
appeared as two singlet peaks at 10.90-11.66 ppm.
Anti-inflammatory activity was determined by
monitoring the effects of these compounds on cellular targets
for inflammation. Three targets were selected for this study
which includes NF-ꢀB, reactive oxygen species (ROS) and
inducible nitric oxide synthase (iNOS).
Inhibition of NF-ꢀB mediated transcription and
inhibition of intracellular generation of ROS and nitric oxide
(NO) were determined in cell based assays. Inhibition of NF-
ꢀB mediated transcription was seen in SW1353 cells with
IC50 values in the range of 6-14 ꢀg/mL (Table 2). The
considerable inhibition of NF-ꢀB activity in SW1353 cells
was observed for compounds 2a, 2c and 2h with IC50 values
of 6.9, 7.7 and 6.4 ꢀg/mL, respectively. This outcome
confirms that methyl and chloro groups have a considerable
influence on the inhibition of NF-ꢀB mediated transcription.
Other compounds (2d, 2e, 2g, 2i) inhibited the NF-ꢀB
activity to a much lesser extent, and their IC50 values were in
the range 10-14 ꢀg/mL.
The mass spectra of all compounds (2a-j) showed M+1
peaks, in agreement with their molecular formula. All
compounds (2a-j) gave satisfactory elemental analysis.
Biological Activity
All synthesized compounds (2a-j) were evaluated for
their anti-inflammatory effects and cytotoxicity in
mammalian cells.

314 Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3
Kaplancikli et al.
Table 2. Anti-Inflammatory and Antioxidant Activities of the Target Compounds (2a-2j)
Inhibition of
NF-ꢀB activity
in SW1353 cells
Antioxidant Activity
(inhibition of ROS
generation) in HL-60 cells
Inhibition of iNOS
activity (NO production)
in RAW 264.7 cells
Cytotoxicity (anti cell proliferation activity)
in a panel of cell lines
IC50 (ꢀg/mL)
SK-
IC50 (ꢀg/mL)
IC50 (ꢀg/mL)
IC50 (ꢀg/mL)
SK-
BT-
LLC-
PK12
Antioxidant
Cytotoxicity
activity
Compound
KB1
VERO2
NF-ꢀB
SP-1
iNOS
Cytotoxicity
MEL1
5491
OV-
31
2a
NA
NA
17
NA
NA
18
NA
NA
18.5
NA
NA
NA
NA
NA
NA
NA
NA
NA
12
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
>5
NC
NC
25
6.9
NA
7.7
14
NA
NA
19
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.24
11
>25
8.9
>25
9.5
12
NC
NC
NC
NC
NC
8.6
2b
2c
2d
NA
NA
NA
NA
NA
NA
NA
0.8
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.3
NC
NC
NC
NC
NC
17.5
NC
0.9
NA
NA
NA
NA
NA
14
2e
12
2f
NA
10
2g
12
6.7
2h
2i
6.4
10
10
>25
NC
NC
9.5
20
2j
NA
NA
Doxorubicin3
Parthenolide3
Trolox3
1.45 1.35
1.3
9
0.4
13.5
0.12
1Cancer cells. 2Noncancer cells. SK-MEL – Human malignant melanoma; KB – Human oral epidermal carcinoma; BT-549 – Human Breast ductal Carcinoma; SK-OV-3 – Human
ovary carcinoma; Vero – Monkey Kidney Fibroblasts; LLC-PK1 – Pig kidney epithelial cells. 3Standard compounds with known biological activities. NA – no activity up to 25
ꢂg/mL. NC – no cytotoxicity up to 25 ꢂg/mL.
Compounds 2c and 2i affected SP-1 activity as well,
indicating that their effect on NF-ꢀB was not specific. It can
be concluded that compounds bearing aliphatic groups
(methyl and isopropyl) on phenyl ring show anti-
inflammatory activity via non-selective inhibition of NF-ꢀB
mediated transcription.
dichlorofluorescin (DCF) fluorescence of which is measured
as an indicator of ROS generation. None of the compounds
showed any inhibition of intracellular ROS generation
indicating that they did not reduce the oxidative stress. None
of the compounds showed any cytotoxicity to the panel of
tested HL-60 cells.
Inhibition of iNOS activity was also observed in LPS-
induced RAW 264.7 cells with IC50 values in the range of
9.5 to 25 ꢂg/mL (Table 2). Compounds 2c, 2e and 2i were
more active than the other compounds. It is apparent that
there is a positive correlation between the inhibition of iNOS
activity and three functional groups, namely methyl, floro,
and isopropyl substituents on phenyl ring. Compounds 2b,
2d and 2j were comparatively less active than other
compounds or not effective in the inhibition of NF-ꢀB and
iNOS activities. Cytotoxicity to RAW cells was also
measured as part of iNOS assay to determine if the cell
viability was affected by test samples during the assay and if
the inhibition of iNOS could be as a result of toxicity.
Compounds 2f, 2g and 2h were toxic to RAW cells. Toxicity
could result from the effects of hydroxy, methoxy, and
chloro groups, respectively.
The cytotoxic effects of the compounds (2a-2j) were
determined against selected human cancer cell lines (SK-
MEL, KB, BT-549, SKOV-3, VERO, and LLC-PK1). No
cytotoxicity was observed for any of the samples up to 25
ꢂg/mL except compound 2c. The compound bearing methyl
substituent (2c) was weakly active towards to all cancer cell
lines with IC50 values of 12 to 18.5 ꢂg/mL and was inactive
against mammalian Vero (monkey kidney fibroblast) cell.
However compound 2c was toxic to LLC-PK1 (pig kidney
epithelial) cell (IC50: 25 ꢂg/mL). Although compound 2i was
inactive to all these cancer lines, it showed toxicity to LLC-
PK1 (IC50: 17.5 ꢂg/mL) cell. Toxicity could be due to
increased lipophilicity associated with isopropyl group.
CONCLUSION
In the present paper, we synthesized a series of
hydrazone derivatives and evaluated their anti-inflammatory
activity and cytotoxicity. These observations clearly
indicated that functional groups at the para position on the
phenyl ring have a crucial influence on anti-inflammatory
activity and toxicity. In particular, compound 2a, which is
the unsubstituted derivative, can be identified as the most
promising anti-inflammatory agent due to its inhibitory
Inhibition of reactive oxygen species (ROS) was
determined in HL-60 cells by DCFH-DA method to examine
antioxidant activity of these compounds. DCFH-DA is a
non-fluorescent probe that diffuses into the cells.
Cytoplasmic esterases hydrolyse the DCFH-DA to 2',7'-
dichlorofluorescin (DCFH). The reactive oxygen species
(ROS) generated within HL-60 cells oxidize DCFH to 2',7'-

Some Hydrazone Derivatives as Anti-inflammatory Agents
Letters in Drug Design & Discovery, 2012, Vol. 9, No. 3 315
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Cacic, M.; Trkovnik, M.; Cacic, F.; Has-Schon, E. Synthesis and
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effect on NF-ꢀB mediated transcription with an IC50 value of
6.9 ꢀg/mL. In addition, no effect was seen on the growth of
solid tumor cell lines and kidney epithelial and kidney
fibroblast cells. In the view of this study, further research
can be carried out on the development of new effective anti-
inflammatory agents bearing hydrazone moiety by the
modification of compound 2a.
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CONFLICT OF INTEREST
The authors have reported no conflict of interest.
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Vicini, P.; Incerti, M.; La Colla, P.; Loddo, R. Anti-HIV evaluation
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ACKNOWLEDGEMENTS
Gemma, S.; Kukreja, G.; Fattorusso, C.; Persico, M.; Romano, M.
P.; Altarelli, M.; Savini, L.; Campiani, G.; Fattorusso, E.; Basilico,
N.; Taramelli, D.; Yardley, V.; Butini, S. Synthesis of N1-
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antimalarial agents active against CQ-resistant P. falciparum
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Ragavendran, J.V.; Sriram, D.; Patel, S.K.; Reddy, I.V.;
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of anticonvulsants from a combined phthalimide-GABA-anilide
and hydrazone pharmacophore. Eur. J. Med. Chem., 2007, 42, 146-
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The authors also thank Mr. John Trott, Mr. Paul Bates,
Ms. Katherine Martin for great assistance with biological
assays. The authors express their gratitude for USDA, ARS,
NPURU financial support.
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