Complexes of 2-hydroxyacetophenone semicarbazones: A novel series of superoxide dismutase mimetics

Article abstract of DOI:10.1016/j.bmcl.2010.03.099

A series of copper(II) and zinc complexes of 2-hydroxyacetophenone semicarbazones have been prepared and evaluated as superoxide dismutase (SOD) mimetics. The SOD-like activity of parent ligands and complexes were determined by the inhibition of nitroblue tetrazolium (NBT) reduction method, using xanthine/xanthine oxidase as the superoxide radical generator. The obtained results indicate that Cu(II) complexes exhibited the most potent SOD-like activities with the IC₅₀ values ranging from ca. 0.2 to 4 μM. Among copper complexes, 2-hydroxy-4-methoxyacetophenone semicarbazone analog was the most active compounds (IC₅₀ ≈0.2 μM).

Full text of DOI:10.1016/j.bmcl.2010.03.099

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3070–3073
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Complexes of 2-hydroxyacetophenone semicarbazones: A novel series
of superoxide dismutase mimetics
a
a
a
a
a
Maliheh Safavi , Alireza Foroumadi , Maryam Nakhjiri , Mohammad Abdollahi , Abbas Shafiee ,
b
c
d
d,
*
Hoda Ilkhani , Mohammad Reza Ganjali , Seyed Jalal Hosseinimehr , Saeed Emami
Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran 14174, Iran
Department of Chemistry, Faculty of Science, University of Guilan, PO Box 1914, Rasht, Iran
Center of Excellence in Electrochemistry, University of Tehran, Tehran, Iran
a
b
c
d
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of copper(II) and zinc complexes of 2-hydroxyacetophenone semicarbazones have been prepared
and evaluated as superoxide dismutase (SOD) mimetics. The SOD-like activity of parent ligands and com-
plexes were determined by the inhibition of nitroblue tetrazolium (NBT) reduction method, using xan-
thine/xanthine oxidase as the superoxide radical generator. The obtained results indicate that Cu(II)
complexes exhibited the most potent SOD-like activities with the IC50 values ranging from ca. 0.2 to
Received 20 October 2009
Revised 13 February 2010
Accepted 29 March 2010
Available online 1 April 2010
4 lM. Among copper complexes, 2-hydroxy-4-methoxyacetophenone semicarbazone analog was the
Keywords:
Superoxide dismutase activity
SOD-mimetics
most active compounds (IC50 ꢀ0.2
lM).
Ó 2010 Elsevier Ltd. All rights reserved.
Copper complexes
Zinc complexes
2
-Hydroxyacetophenone
Semicarbazone
ÁÀ
Reactive oxygen species (ROS) such as superoxide O2 and their
complexes of copper and other metals have also been reported to
8
,9
products are credited with important roles in the oxidative stress
of biological systems. Oxidative stress causes damage to lipid
membranes, proteins, lipoproteins, nucleic acids, and many other
biomolecules, and leads to the development of a wide range of path-
ophysiological conditions and serious diseases including cardiac
ischemic/reperfusion injury, inflammatory processes, neurodegen-
exhibit SOD mimic activities.
amino acid residues, peptides, and salicylate derivatives,
rocyclic and tetradentate Schiff-bases
SOD-like analogs.
Moreover, copper complexes of
1
10–13
mac-
1
4,15
are known to be useful
Studies of semicarbazones and their metal complexes have
been subject of interest because of their chemical and biological
properties. In particular, several copper(II) complexes of semicar-
bazones have been reported to exhibit SOD-like activity. For exam-
ple, the copper(II) complexes of the Schiff base salicylaldehyde
2
–4
erative disorders, atherosclerosis, cancer, or diabetes mellitus.
Accordingly, the use of superoxide dismutase (SOD) enzymes and
modified SODs have been suggested as a useful therapy in combating
the action of superoxide radicals in the pathogenesis of inflamma-
tory, degenerative, and metabolic diseases. However, application
of natural SODs is limited by their high cost and short plasma half-
life, and also some disadvantages in their oral bioavailability, chem-
1
6,17
semicarbazone has been reported as a SOD mimic.
In this
5
Letter, we present preparation and SOD-like activity evaluation of
copper(II) and zinc complexes of 2-hydroxyacetophenone
semicarbazones.
The copper(II) and zinc complexes of 2-hydroxyacetophenone
semicarbazones (3a–c and 4a–c, respectively) were prepared as
illustrated in Figure 1. The ligands 2-hydroxyacetophenone
semicarbazones 2a–c were synthesized by reaction of 2-hydroxy-
acetophenones 1a–c with semicarbazide hydrochloride.18 The cop-
per(II) and zinc complexes were prepared by refluxing equimolar
6
ical stability, cell permeability and immunogenicity. Therefore, low
molecular weight SOD mimics have been potentially studied to
overcome these limitations.7
Most SOD mimics have been designed with a redox active metal
center, similar to the active site metals of the natural SODs, that is,
copper(II), which allows the change of metal coordination occur-
ring during the catalytic process. Many low molecular weight
amounts of CuCl
EtOH for 2 h. Followed by cooling, a crystalline product was ob-
tained, which were collected, washed with chilled ethanol, and
2
Á6H
2 2
O or ZnCl and the corresponding ligand in
*
Corresponding author. Tel.: +98 151 3543082; fax: +98 151 3543084.
1
9
E-mail address: sd_emami@yahoo.com (S. Emami).
dried under vacuum.
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.099

M. Safavi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3070–3073
3071
O
NH2
Cl
O
NH
OH O
OH
O
M
NH2
N
a
b
N NH
CH3
CH3
CH3
1a
2a
3a : M = Cu (II)
a : M = Zn
4
O
NH2
Cl
O
NH
OH O
OH
M
O
NH2
N
a
b
N NH
CH3
CH3
HO
HO
HO
CH3
1b
2b
3
b : M = Cu (II)
4
b : M = Zn
c
O
NH2
Cl
O
NH
OH O
OH
O
M
NH2
N
a
b
N NH
CH3
CH3
MeO
CH3
MeO
MeO
1c
2c
3c : M = Cu (II)
c : M = Zn
4
Figure 1. Synthesis of ligands 2a–c, copper(II) complexes 3a–c, and zinc complexes 4a–c. Reagents and conditions: (a) semicarbazide hydrochloride, CH
CuCl O or ZnCl , EtOH, reflux, 2 h; (c) MeI, K CO , acetone, reflux.
Á6H
3
COONa, H
2
O, rt; (b)
2
2
2
2
3
The SOD activity of parent ligands and complexes were deter-
mined by the inhibition of nitroblue tetrazolium (NBT) reduction,
using xanthine/xanthine oxidase as the superoxide radical genera-
Table 1
SOD activity (NBT inhibition) of free ligands 2a–c and their Cu(II) and Zn complexes
(3a–c and 4a–c)
2
0
tor. Catalase was also added to remove H
2
O
2
from the system.
ÁÀ
The NBT reduction by O2 and formation of blue formazan was
spectrophotometrically monitored at 560 nm, estimating its initial
rate in the absence, and in the presence of different concentrations
of compounds. The IC50 values were determined by regression
analysis and interpolation of the percent of inhibition versus con-
centration. The IC50 value is defined as the concentration of the
compounds that is required to inhibit the reduction rate of NBT
by 50%.
O
NH2
Cl
NH
O
OH N
M
NH2
O
N NH
CH3
R
R
CH3
2
a-c
3a-c and 4a-c
The SOD activity of Cu(II) and Zn complexes were compared
with those of free ligands, copper(II) chloride and the native
Compound
R
M
IC50a
(lM)
2
2
2
3
a
b
c
H
OH
OMe
H
OH
OMe
H
OH
OMe
—
583.09 ± 31.01
>1000
>1000
4.06 ± 0.35
0.51 ± 0.24
0.2 ± 0.13
151.8 ± 5.05
90.7 ± 4.3
Cu
of the test derivatives indicate that free ligands 2b and 2c exhibited
no SOD activity (IC50 >1000 M), while remaining ligand 2a
2 2
Zn -SOD, as shown by the IC50 values in Table 1. The IC50 values
l
a
Cu(II)
Cu(II)
Cu(II)
Zn
Zn
Zn
showed some activity. All Cu and Zn complexes were SOD mimics,
and showed much greater SOD activity than their corresponding
parent ligands. The rage of IC50 values for Cu(II) complexes were
3b
3c
4a
4b
4c
from ca. 0.2 to 4
lM, while the IC50 values of Zn complexes were
527.01 ± 73.93
4.77 ± 1.34
5.2 units/mL
more than about 90
lM. These results supported the fact that cop-
CuCl
—
—
2
SODb
—
per is more suitable for the SOD activity. Among copper complexes,
methoxy analog of semicarbazone 3c was the most active com-
a
SOD activity was determined by indirect assay, nitroblue tetrazolium (NBT)
pound (IC50 value ꢀ0.2
l
M). The introduction of hydroxy or meth-
method. The IC50 value (means ± SD, n = 3) is defined as the concentration of test
oxy group at the 4-position of acetophenone system in the copper
complexes, increased the SOD activity. Also, the SOD activity of
methoxy analogs was higher than that of the hydroxy derivatives
in this series. In contrast, substitution of hydroxy or methoxy
group in free ligands decreased the SOD activity.
compound that is required to inhibit the reduction rate of NBT by 50%.
b
2 2
Native Cu Zn -SOD enzyme as positive control.
set while the fourth position is being occupied by the chloride ion.
Our knowledge about the redox properties of copper complexes of
2-hydroxyacetophenone semicarbazones as N,O,O donor ligands
are limited. Generally, the redox properties of the complexes de-
pend on several factors such as the chelate ring size, axial ligation,
The proposed structure of complexes 3a–c consists of a neutral,
mononuclear four-coordinate copper(II) complex with 2-hydroxy-
acetophenone semicarbazone ligands providing N,O,O donor atom

3
072
M. Safavi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3070–3073
degree and distribution of unsaturation and substitution pattern in
the chelate ring.
CuIIL
H O
O2
2
2
2
1
In order to understanding of the redox properties of these
copper(II) complexes, cyclic voltammetric measurements of com-
pounds 2a–c and 3a–c were carried out.22 Cyclic voltammograms
CuIL
O2
O2
(
CV) of the complexes were recorded and were compared with
Figure 4. Proposed mechanism for the dismutation of superoxide radicals by
copper(II) complexes.
those obtained with the respective free ligands, in identical exper-
imental conditions. The compounds were scanned at three scan
rates. All the cyclic voltammograms are more or less similar with
minor variation in the position and appearance of some peaks.
1/2) is practically constant for all the compounds
p
The ratio (I /m
The CV obtained for representative ligand 2a, between À1.2 V
which indicated that is diffusion controlled. For all complexes the
couple corresponding to Cu(I/II) redox process can be inferred as
quasi-reversible one-electrode processes on the basis of the above
observation.
À1
and 1.8 V at
v
= 100 mV s , is presented in Figure 2. The complete
scan shows only one redox process at Epa = 0.99 V that can be re-
lated to the oxidation of the phenolic group of the ligand. It is pos-
sible to infer that the oxidized species changes through this
reaction into a different and electrochemically active compound
which is reduced at À0.87 V. Figure 3 shows CV for the copper(II)
complex 3a. In this voltammogram, the redox phenol couple was
decreased and a new and high redox peak was appeared at
Proposed mechanism for the dismutation of superoxide radicals
by copper(II) complexes of 2-hydroxyacetophenone semicarba-
zones could be explained by the redox cycling of the complexes
II
as shown in Figure 4, in which Cu L is used as an example for sim-
II
I
plicity. The complex Cu L could be reduced to Cu L complexes by
E
pa = 0.49 and Epc = À0.41 for copper center. This shows that the
I
superoxide radicals. The resulting Cu L complexes are readily oxi-
dized to Cu L complexes, while the superoxide radicals are con-
phenol group of ligand is binding with central Cu. The copper cen-
ter is reduced in one successive two electron transfer reactions
II
verted to hydrogen peroxide. On the other hand, the coordination
geometry is an important factor in characterizing SOD-mimetic
complexes. The catalytic cycle of the native Cu Zn -SOD enzyme,
2 2
(Fig. 3). The first reduction can be attributed to the Cu(II)/Cu(I) pro-
cess. This couple is found to be quasi-reversible.
requires the reversible conformational change of the active site
from a square planar arrangement of Cu(II) into a distorted tetra-
2
3
hedral form, more adequate to Cu(I). This considerable distortion
of the geometry of copper(II) in native Cu Zn -SOD enzyme is one
2
2
of the features that enhances the catalytic activity of this enzyme.
Therefore, it could be expected that copper(II) complexes exhibit-
ing appreciable square planar distortion should also show high
2
4
SOD-like activity.
In conclusion, a series of copper(II) and zinc complexes of 2-
hydroxyacetophenone semicarbazones have been prepared and
evaluated as superoxide dismutase (SOD) mimetics. The catalytic
SOD activity of parent ligands and complexes were compared by
the NBT reduction bioassay. The obtained results indicate that
Cu(II) complexes exhibited the most potent SOD-like activities
with the IC50 values ranging from ca. 0.2 to 4
complexes, 2-hydroxy-4-methoxyacetophenone semicarbazone
M).
lM. Among copper
analog was the most active compounds (IC50 value ꢀ0.2
l
These bioactive compounds have the potential to modulate the
biological properties of superoxide anion, thus it is interesting to
further investigate their therapeutic efficacy in different biological
models of diseases where the overproduction of superoxide radical
and/or SOD dysfunction have been implicated.
À1
Figure 2. Cyclic voltammogram of free ligand 2a, 0.01 M at 100 mV s scan rate.
Acknowledgments
This work was supported by a Grant from Pharmaceutical Sci-
ences Research Center, Tehran University of Medical Sciences.
References and notes
1
2
.
.
Halliwell, B.; Gutteridge, J. M. C. Free Radicals in Biology and Medicine, third ed.;
Oxford University Press: London, 2002.
Reilly, M. P.; Delanty, N.; Roy, L.; Rokach, J.; Callaghan, P. O.; Crean, P.; Lawson,
J. A.; FitzGerald, G. A. Circulation 1997, 96, 3314.
3.
4.
5.
6.
7.
8.
Emerit, J.; Edeas, M.; Bricaire, F. Biomed. Pharmacother. 2004, 58, 39.
Cathcart, M. K. Arterioscler. Thromb. Vasc. Biol. 2004, 24, 23.
Henke, S. L. Expert Opin. Ther. Patents 1999, 9, 169.
Riley, D. P. Chem. Rev. 1999, 99, 2573.
Salvemini, D.; Riley, D. P.; Cuzzocrea, S. Nat. Rev. Drug Disc. 2002, 1, 367.
Mizushima, Y.; Igarshi, R.; Wermuth, C. G.; Koga, N.; Kinig, H.; Metcalf, B. W.
Medicinal Chemistry for the 21st Century; Blackwell Scientific Publications:
Oxford, 1992.
9
.
Arslantas, A. Met-Based Drugs 2002, 9, 9.
À1
10. Brigelius, R.; Spottl, R.; Bors, W.; Lengfelder, E.; Saran, M.; Weser, U. FEBS Lett.
974, 47, 72.
Figure 3. Cyclic voltammogram of copper(II) complex 3a, 0.01 M at 100 mV s
scan rate.
1

M. Safavi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3070–3073
3073
1
1
1. Weser, U.; Richter, C.; Wendel, A.; Younes, M. Bioinorg. Chem. 1978, 8, 21.
2. Younes, M.; Lengfelder, E.; Zienau, S.; Weser, U. Biochem. Biophys. Res. Commun.
19. The structures of complexes were identified by their melting points, infrared
spectroscopy and elemental analysis. By coordination with copper or zinc,
1
978, 81, 576.
shifts of the carbonyl m(C@O), azomethyne m(C@N) and the phenolic m(C–O)
1
1
3. Younes, M.; Weser, U. FEBS Lett. 1976, 61, 209.
4. Durackova, Z.; Felix, K.; Fenikova, L.; Kepstova, I.; Labuda, J.; Weser, U.
Biometals 1995, 8, 183.
5. Miiller, J.; Felix, K.; Maichle, C.; Lengfelder, E.; Strahle, J.; Weser, U. Inorg. Chim.
Acta 1995, 233, 111.
bands, in comparison to the values found in the free 2-hydroxyacetophenone
semicarbazone ligands, were observed for the complexes. They were
consistent with the tridentate coordination of the semicarbazone derivative
ligand through the enolic oxygen atom, the azomethynic nitrogen atom and
the phenolic oxygen atom and were in accordance with the published
data.17
1
1
1
1
6. Beraldo, H.; Gambino, D. Mini-Rev. Med. Chem. 2004, 4, 159.
7. Patole, J.; Dutta, S.; Padhye, S.; Sinn, E. Inorg. Chim. Acta 2001, 318, 207.
8. General procedure for synthesis of compounds 2a–c: To an aqueous solution of
semicarbazide hydrochloride (20.0 mmol) and sodium acetate (25.0 mmol), 2-
hydroxyacetophenone derivative (10.0 mmol) was added with stirring
resulting in a precipitate. The stirring was continued for 2 h at ambient
temperature, after which the precipitate was filtered, washed with distilled
water and dried in vacuum to give 2a–c. Compound 2a: yield 68%; mp 218–
20. SOD activity: To
a mixture of xanthine oxidase (56 mUnit/mL), catalase
(6 units/mL), bovine serum albumin (15% W/V), EDTA (3 mM) and nitroblue
tetrazolium (75 mM) in phosphate buffer (10 mM, pH 7), the required
concentration of test compound were added. After 30 s, the reaction was
started by adding xanthine solution (3 mM) in phosphate buffer. The
absorbance of the reaction mixture was monitored spectrophotometrically at
560 nm for 10 min. The SOD activity was calculated base on the rate of
À1
1
2
20 °C; IR (KBr, cm ):
mmax 3344, 3235, 1625, 1597; H NMR (400 MHz,
absorbance change per minute (DA/min). SOD activity was expressed in terms
DMSO-d ) d 12.75 (s, 1H, OH), 9.64 (s, 1H, NNH), 7.49 (d, 1H, H-6, J = 6.8 Hz),
6
of the IC50 of NBT reduction obtained from a linear regression plot of inhibition
percent versus concentration.
21. John, R. P.; Sreekanth, A.; Rajakannan, V.; Ajith, T. A.; Prathapachandra Kurup,
M. R. Polyhedron 2004, 23, 2549.
22. Cyclic voltammograms were recorded by means of a palmsens PC running from
À1.2 V to 1.8 V. Experiments was performed using different one compartment
electrochemical cells with three electrode system. A 5 mm electrochemical cell
was used with a glassy carbon electrode as working electrode, a platinum wire
auxiliary electrode, and Ag/AgCl reference electrode. The initial potential was
À1.2 V, vertex potential was 1.8 V and E step was 5 mV in different scan rates.
23. Fielden, E. M.; Rotilio, G.. In Copper Proteins and Copper Enzymes; Lontie, R., Ed.;
CRC Press: Boca Raton, 1984; Vol. II, p 27. Chapter 2.
7
.20 (t, 1H, H-4, J = 8.4 Hz), 6.84 (t, 1H, H-5, J = 6.4 Hz), 6.83 (d, 1H, H-3,
+
J = 8.0 Hz), 6.21 (s, 2H, NH
2
), 2.22 (s, 3H, CH
3
); MS: m/z (%) 193 (M , 91), 149
À1
(
70), 134 (100), 92 (97). Compound 2b: yield 75%; mp 227–228 °C; (KBr, cm ):
1
m
1
6
max 3483, 3263, 3206, 1680, 1593; H NMR (400 MHz, DMSO-d
6
) d 12.99 (s,
H, 2-OH), 9.66 (s, 1H, NNH), 9.47 (s, 1H, 4-OH), 7.30 (d, 1H, H-6, J = 8.4 Hz),
.27 (dd, 1H, H-5, J = 8.8 Hz and 2.4 Hz), 6.22 (d, 1H, H-3, J = 2.4 Hz), 6.12 (s, 2H,
+
NH
2 3
), 2.17 (s, 3H, CH ); MS: m/z (%) 209 (M , 85), 192 (80), 165 (72), 137 (100),
1
35 (79), 108 (74), 79 (35). Compound 2c: yield 24%; mp 224–225 °C ; (KBr,
À1
1
cm ):
H, OH), 9.55 (s, 1H, NNH), 7.41 (d, 1H, H-6, J = 8.8 Hz), 6.43 (dd, 1H, H-5,
J = 8.8 Hz and 2.6 Hz), 6.39 (d, 1H, H-3, J = 2.4 Hz), 6.15 (s, 2H, NH ), 3.74 (s, 3H,
); MS: m/z (%) 223 (M , 57), 206 (37), 164 (50), 150
100), 148 (46), 119 (29), 77 (36).
6
mmax 3503, 3170, 1700, 1593; H NMR (400 MHz, DMSO-d ) d 13.18 (s,
1
2
+
OCH
(
3
), 2.19 (s, 3H, CH
3
24. Díaz, A.; Cao, R.; Fragoso, A.; Sánchez, I. Inorg. Chem. Commun. 1999, 2,
358.