Pharmacological characteristics of some 3-salicylidenehydrazono-2- indolinone coordination compounds

Article abstract of DOI:10.1007/s00044-009-9229-1

1 3-Salicylidenehydrazono-2-indolinone (SHI) complexes with Co(II), Ni(II), Cu(II), Zn(II), and Pd(II) were synthesized. All compounds were evaluated for their antimicrobial activity against seven pathogenic bacteria and four fungi. The compounds also hav

Full text of DOI:10.1007/s00044-009-9229-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:771–781
DOI 10.1007/s00044-009-9229-1
ORIGINAL RESEARCH
Pharmacological characteristics of some
3-salicylidenehydrazono-2-indolinone coordination
compounds
Sandra S. Konstantinovi c´ Æ Vanja S. Caki c´
Received: 7 November 2008 / Accepted: 2 July 2009 / Published online: 29 July 2009
Birkh a¨ user Boston 2009
ꢀ
Abstract 3-Salicylidenehydrazono-2-indolinone (SHI) complexes with Co(II),
Ni(II), Cu(II), Zn(II), and Pd(II) were synthesized. All compounds were evaluated
for their antimicrobial activity against seven pathogenic bacteria and four fungi. The
compounds also have been screened for anti-inflammatory activity. The complexes
have better antimicrobial and anti-inflammatory than ligand itself. The in vitro
antiamoebic activity was evaluated against the HM1:IMSS strain of Entamoeba
histolytica, and the results were compared with the standard drug, metronidazole.
The preliminary test results showed that the complexes had better antiamoebic
activity than their respective ligands. SHI and its complexes were found to possess
potent antioxidant activity and be better than standard antioxidants, such as vitamin
C and mannitol. In particular, the Cu(II) complex displayed excellent activity on the
superoxide radical.
Keywords Isatin ꢀ Schiff base ꢀ Complex ꢀ Antibacterial activity ꢀ
Antifungal activity ꢀ Anti-inflammatory activity ꢀ Antiamoebic activity ꢀ
Antioxidant activity
Introduction
The synthetic versatility of isatin has led to an extensive use of this compound in
organic synthesis. It has stemmed from the interest in the biological and pharmaco-
logical properties of its derivatives (da Silva et al., 2001). Also, thiosemicarbazone and
mono and bis-hydrazone derivatives are of considerable interest because of their
S. S. Konstantinovi c´ (&)
Faculty of Technology, Bulevar Oslobodjenja 124, 16000 Leskovac, Serbia
e-mail: sakisandra12@yahoo.com
V. S. Cakic´
Institute of Public Health, Maksima Kova cˇ evi c´ a 11, 16000 Leskovac, Serbia

772
Med Chem Res (2010) 19:771–781
chemistry and potentially beneficial biological activities, such as antitumor, antibac-
terial, antiviral, and antimalarial activities (Jouad et al., 2001; Konstantinovi c´ et al.,
2
008). Many coordination compounds of transition metals with isatin derivatives
show greater activity than the ligands alone. In our previous work, we gave structure
and preliminary results on antibacterial activity of Co(II), Ni(II), Cu(II), and Zn(II)
complexes of SHI (Konstantinovi c´ et al., 2003). In the present study, our goal was to
achieve a better biological profile at lower concentrations by preparing and evaluating
SHI complexes with Co(II), Ni(II), Cu(II), Zn(II), and Pd(II). The antioxidant
ꢁꢂ
•
activities (scavenging effects on O2 and OH ) of the SHI and its complexes also have
been studied. The information obtained from this study will be helpful for developing
some new antioxidants.
Materials and methods
General procedure for 3-Salicylidenehydrazono-2-indolinone (SHI)
The hydrazono-2-indolinone synthesized by a standard procedure was dissolved in
the minimum amount of 95% ethanol (Konstantinovi c´ et al., 2003). To this solution,
salicylaldehyde was added in an equimolar amount under continuous refluxing for
approximately 45 min. The mixture was acidified with H SO to pH 4–4.5. The
2
4
colored product was filtered, washed with ethanol, and dried in vacuo over CaCl2.
For the synthesis of the complexes, the 3-salicylidenehydrazono-2-indolinone of
isatin was dissolved in the minimum amount of 95% ethanol. To this refluxing
solution, metal(II) chloride was added in equimolar amounts. The refluxing was
continued for approximately 2 hr. The colored products were filtered, washed with
ethanol, and dried in vacuo over CaCl . The spectroscopic analysis and structure
2
(
Fig. 1) of those compounds were published previously (Konstantinovi c´ et al.,
2
003).
Microanalysis for carbon, hydrogen, and nitrogen was carried with a Carlo Erba
106 microanalyzer. The chloride content was determined potentiometric. The
1
metal contents were determined by using a Virial AA-457 double-beam spectrom-
eter. The melting points were determined by using Thomas-Hoover melting point
apparatus and are uncorrected. The molar conductivity of the complexes was
-
3
-3
measured with an Iskra Conductivity Meter 0.67 using 10 mol ꢀ dm solutions
in DMF. FTIR spectra were recorded using a Michaelson Bomen MB-series
Fig. 1 Complexes of 3-
salicylidenehydrazono-2-
indolinone
H
N
Cl
O
=
=
O
N
M
⋅Cl
N = CH
M = Co(II), Ni(II), Cu(II), Zn(II)

Med Chem Res (2010) 19:771–781
773
spectrophotometer, using KBr pellet (1 mg/100 mg) technique. The electronic
spectra were recorded on a Perkin/Elmer Lambda 15 UV/VIS spectrophotometer
-
3
-3
1
using 10 mol ꢀ dm solutions in DMF. H NMR spectra were obtained in DMSO
solution with a Gemini-200 ‘‘HF NMR’’ spectrometer. The magnetic susceptibility
measurements were made at room temperature using MSB-MKI magnetic balance
(
Sherwood Scientific Ltd.). The data were corrected for diamagnetism.
-Salicylidenehydrazono-2-indolinone. Yield: 89.65%; m.p. 175ꢁC. IR (KBr,
3
-
1
mmax/cm ): 3541 (OH), 3400 (NH), 1730 (C=O), 1605 (C=N), 1585(CH=N), 1359
(C-O); UV/VIS (DMF, m(10 cm /e): 36.8/1637, 30.8/1627; H-NMR (DMSO, d,
ppm) 6.85–7.16 (m, 4H, Ar), 7.18–7.39 (m, 4H, Ar), 8.35 (s, 1H, N-H), 8.59 (s, 1H,
3
-1
1
C=CH)) 12.56 (s, 1H, O-H). Anal. calcd. for C H O N (Mr = 265): C: 67.92;
1
5 11 2 3
H: 4.15; N: 15.84%; found: C: 67.90; H: 4.10; N: 15.80%.
-
1
Co (SHICl) Cl Yield: 35.12%; m.p. 190ꢁC. IR (KBr, mmax/cm ): 3400 (N-H),
3
-1
1
706 (C=O), 1587(C=N), 1545 (CH=N), 1359 (C-O); UV/VIS (DMF, m(10 cm
/
1
e): 37.7/751, 29.9/698, 16.4/391, 15.9/541, 15.5/381; H-NMR (DMSO, d, ppm)
6
C=CH). Anal. calcd. for C H O N CoCl (Mr = 393.8): C: 45.69; H: 2.54; N:
.85–7.18 (m, 4H, Ar), 7.19–7.39 (m, 4H, Ar), 8.25 (s, 1H, N-H), 8.58 (s, 1H,
1
5
10
2
3
2
1
1
0.66; Cl: 18.02; Co: 14.95%; found: C: 45.65; 2.50% H, N: 10.73; Cl: 18.09; Co:
4.85%.
-
1
Ni (SHICl) Cl: Yield: 41.25%; m.p. 184ꢁC. IR (KBr, mmax/cm ): 3376 (N-H),
698 (C=O), 1579 (C=N), 1545 (CH=N), 1378 m(C-O). UV/VIS (DMF,
1
3
-1
1
m(10 cm /e): 37.7/761, 28.5/623, 18.9/596, 13.5/488. H NMR (DMSO,d ppm)
.84–7.18 (m, 4H, Ar), 7.18–7.39 (m, 4H, Ar), 8.26 (s, 1H, N-H), 8.58 (s, 1H,
C=CH). Anal. calcd. for C H O N NiCl (Mr = 393.7): C: 45.72; H: 2.54; N:
6
1
5
10
2
3
2
1
1
0.66; Cl: 14.95; Ni: 18.02%; found: C: 45.35; H: 2.35; N: 10.45; Cl: 18.10; Ni:
5.01%.
-
1
Cu (SHICl) Cl: Yield: 45.63%; m.p. 195ꢁC. IR (KBr, mmax/cm ): 3400 (N-H),
3
-1
/
1
704 (C=O), 1590 (C=N), 1560 (CH=N), 1367 (C-O); UV/VIS (DMF, m(10 cm
1
e): 37.7/732, 25.9/614, 21.4/412, 15.5/384; H-NMR (DMSO, d, ppm) 6.84–7.18
(
calcd. for C H O N CuCl (Mr = 398.5): C: 45.17; H: 2.51; N: 10.54; Cl: 17.82;
m, 4H, Ar), 7.19–7.39 (m, 4H, Ar), 8.25 (s, 1H, N-H), 8.58 (s, 1H, C=CH). Anal.
1
5
10
2
3
2
Cu: 15.93%; found: C: 45.00; H: 2.12; N; 10.23; Cl: 17.65; Cu: 15.69%.
-
1
Zn (SHICl) Cl: Yield: 36.06%; m.p. 203ꢁC. IR (KBr, mmax/cm ): 3361 (N-H),
3
-1
/
1
698 (C=O), 1592 (C=N), 1545 (CH=N), 1378 (C-O); UV/VIS (DMF, m(10 cm
1
e): 37.8/847, 29.5/573, 26.9/514; H-NMR (DMSO, d, ppm) 6.85–7.18 (m, 4H, Ar),
7
.19–7.39 (m, 4H, Ar), 8.25 (s, 1H, N-H), 8.58 (s, 1H, C=CH). Anal. calcd. for
C H O N ZnCl (Mr = 400.4): C: 44.95; H: 2.49; N: 10.48; Cl: 17.73; Zn:
1
5
10
2
3
2
1
6.33%; found: C: 45.02; H: 3.00; N: 10.40; Cl: 17.69; Zn: 16.69%.
-
1
Pd (SHICl) Cl: Yield: 61.05%; m.p. 193ꢁC. IR (KBr, mmax/cm ): 3400 (N-H),
3
-1
/
1
714 (C=O), 1590 (C=N), 1560 (CH=N), 1360 (C-O); UV/VIS (DMF, m(10 cm
1
e): 37.1/745, 26.8/627, 25.5/447; H-NMR (DMSO, d, ppm) 6.77–7.25 (m, 4H, Ar),
7
C H O N PdCl (Mr = 441.4): C: 40.78; H: 2.27; N: 7.25; Cl: 16.09; Pd:
.05–7.44 (m, 4H, Ar), 8.25 (s, 1H, N-H), 8.58 (s, 1H, C=CH). Anal. calcd. for
1
5
10
2
3
2
2
4.11%; found: C: 40.7; H: 2.22; N: 7.23; Cl: 16.11; Pd: 24.2%.

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Med Chem Res (2010) 19:771–781
In vitro antimicrobial activity
The compounds were evaluated for their in vitro antibacterial activity against
Escherichia coli, Staphylococcus aureus, Enterobacter, Proteus mirabilis, Bacillus
anthracis, Pseudomonas aeruginosa, and Streptococcus faecalis. Minimum inhib-
itory concentration (MIC) was assessed by the agar dilution method according to
guidelines established by the NCCLS standard M7-A5 (NCCLS, 2000). Sulfameth-
oxazole and trimethoprim was used as standard drugs. A series of glass tubes
containing different concentrations of the compounds with Mueller-Hinton broth
was inoculated with the required amount of the inoculum to obtain a suspension of
5
microorganism, containing 10 colony-forming units per milliliter. One growth
control tube was prepared with the addition of the compound and one blank tube
was prepared without the addition of microorganism. The tubes were incubated at
3
7ꢁC for 24 hr. The turbidity produced in each tube was observed. The minimum
-3
inhibitory concentration (MIC in lg ꢀ cm ) was considered to be the lowest
concentration, which exhibited the same turbidity as the blank tube. The observed
MIC is presented in Table 1.
In vitro antifungal activity
The compounds were evaluated for their in vitro antifungal activity against
Microsporum gypsum, Epidermophyton floccosum, Histoplasma capsulatum, Can-
dida albicans, and Aspergillus niger following the guidelines in NCCLS document
M27-A using the microdilution broth method (NCCLS, 1997). Clotrimazole was
used as reference antifungal agent. Solutions of the test compounds and reference
drug were dissolved in DMF. Antifungal activities of the yeast were performed in
RPMI 1640 medium (Sigma), which had been buffered to pH 7.0 with 0.165 M
-3
Table 1 MIC values (lg ꢀ cm ) of the investigated compounds against tested bacteria
Compound
S. aureus B.
anthracis
Enterobacter E. coli
P.
P.
aeruginosa mirabilis faecalis
S.
SHI
625
1250
1250
625
1250
312.5
312.5
39.06
39.06
9.76
625
625
78.12
1250
78.12
Co(SHICl)Cl
Ni(SHICl)Cl
Cu(SHICl)Cl
Zn(SHICl)Cl
Pd(SHICl)Cl
156.25
156.25
39.06
9.76
156.25
156.25
39.06
39.06
9.76
156.25
156.25
39.06
39.06
39.06
625
156.25
39.06
39.06
9.76
78.12
39.06
9.76
156.25
39.06
39.06
625
9.76
9.76
Sulfamethoxazole 2500
2500
1250
[5000
2500
2500
78.12
[5000
Trimethoprim
CoCl
2500
[5000
[5000
[5000
[5000
2500
2500
156.25
19.53 [5000
156.25
2
ꢀ6H
ꢀ6H
ꢀ2H
2
O
[5000
[5000
[5000
[5000
[5000
[5000
[5000
5000
[5000
NiCl
2
2
O
[5000
[5000
[5000
5000
[5000
[5000
[5000
5000
[5000
[5000
[5000
2500
[5000
[5000
[5000
2500
[5000
[5000
[5000
2500
CuCl
2
2
O
ZnCl
2
PdCl
2

Med Chem Res (2010) 19:771–781
775
-3
Table 2 MIC values (lg ꢀ cm ) of the investigated compounds against tested fungi
Compound
M. gypsum
E. floccosum
H. capsulatum
C. albicans
A. niger
SHI
78.12
19.53
39.08
9.76
39.06
19.53
19.53
9.76
78.12
19.53
39.06
19.53
19.53
9.76
39.06
9.76
4.88
2.44
\0.3
\0.3
0.3
39.06
9.76
4.88
4.88
2.44
2.44
2.44
Co(SHICl)Cl
Ni(SHICl)Cl
Cu(SHICl)Cl
Zn(SHICl)Cl
Pd(SHICl)Cl
Clotrimazole
4.88
9.76
4.88
4.88
4.88
2.44
19.53
morpholinopropanesul-fonic acid (Sigma), as outlined in document M27-A. The
3
stock yeast inoculum suspensions were adjusted to concentration of (0.5–2.5) 9 10
-
1
cells ml by the spectrophotometric method and an aliquot of 100 ll was added to
each tube of the serial dilution. The chemical compounds-broth medium serial tube
dilutions inoculated with each yeast were incubated on a rotary shaker at 37ꢁC for
4
8–72 hr at 150 rpm. The minimum inhibitory concentrations of the chemical
compounds were recorded as the lowest concentration of each chemical compounds
in the tubes with no growth (i.e., no turbidity) of inoculated yeast. The observed
MIC is presented in Table 2.
Anti-inflammatory activity
The anti-inflammatory activity was determined (Laurence and Bacharach, 1964) by
formalin induced paw endema method in Wister albino rat by using Plethysmo-
-
1
graph. Diclofenac sodium (50 mg ꢀ kg ) was taken as standard drug. The test
compounds were administrated orally at three dose levels (100, 200, and 400 mg)
3
intraperitoneally 30 min before administration of formalin (0.1 cm of 1% w/v) in
the plantar region of the paw. The paw volumes were measured at 15, 30, 60, and
20 min after formalin administration. These results are presented in Table 3.
1
Antiamoebic activity
Preliminary experiments were performed to determine the antiamoebic activities of
the in vitro culture against HM1:IMSS strain of E. histolytica as previously
described (Wright et al., 1998). The E. histolytica strain HM1:IMSS was cultured in
3
tubes (15 cm ) by using Diamond TYIS-33 medium (Diamond et al., 1978). All the
compounds were dissolved in DMF (40 ll). The maximum concentration of DMF
did not exceed 0.1% at which level no inhibition of amoebal growth occurred and is
further confirmed by the experiments performed in our laboratory, followed by
-
3
adding enough culture medium to obtain concentration of 1 mg ꢀ cm (Rodrigues
Pereira et al., 1996; Jiang et al., 2003). Stock solutions of the compounds were
-
3
prepared freshly before use at a concentration of 0.1 mg ꢀ cm . Twofold serial
dilutions were made in the wells of 96-well microtiter plate (Costar). Each test
includes metronidazole as a standard amoebicidal drug, with control wells (culture

776
Med Chem Res (2010) 19:771–781
Table 3 Anti-inflammatory activity of investigated compounds
Compound
Reduction of edema (%)
-1
Dose (mg ꢀ kg
)
15 min
30 min
45 min
60 min
120 min
?
?
?
SHI
100
13.3***
13.3***
13.3***
23.3***
23.3***
33.3***
13.3***
33.3***
33.3***
13.3***
13.3***
23.3***
23.3***
33.3***
33.3***
23.3***
33.3***
33.3***
23.3***
?
40
40
44.4
48.9
?
?
51.1
?
55.5
2
4
00
00
44.4
?
40
?
?
55.6
?
51.1
56.6
?
60
?
40
?
?
51.1
Co(SHICl)Cl
Ni(SHICl)Cl
Cu(SHICl)Cl
Zn(SHICl)Cl
Pd(SHICl)Cl
Diclofenac
100
44.4
?
42.2
?
?
55.5
?
64.4
2
4
00
00
51.1
?
51.5
?
?
64.4
?
55.6
66.7
?
60
?
?
51.5
100
23.3***
?
40
44.4
?
?
?
64.4
2
4
00
00
51.1
55.6
?
60
?
40
?
?
66.7
55.6
?
40
?
?
51.1
?
55.5
100
48.9
?
44.4
?
?
55.5
?
55.6
2
4
00
00
51.5
?
60
?
51.1
?
?
64.4
64.4
?
60
?
44.4
?
?
64.4
100
51.5
?
60
?
51.5
?
64.4
?
66.7
2
4
00
00
?
64.4
?
?
?
71.1
66.7
66.7
?
60
?
51.5
?
?
64.4
100
55.5
?
60
?
55.5
?
64.4
?
66.7
2
4
00
00
?
?
?
?
64.4
?
75
66.7
?
75
66.7
?
75
71.1
?
75
?
50
33
?
Significance levels: * P \ 0.1, ** P \ 0.01, *** P \ 0.05, and P \ 0.001 compared with control
The significance levels P refer to comparisons made between OA-challenged rats and saline-treated
controls (t-test)
medium plus amoebae) and a blank (culture medium only). All the experiments
were performed in triplicate at each concentration level and repeated thrice. The
amoebae suspension was prepared from a confluent culture by pouring off the
medium at 37ꢁC and adding 5 ml of fresh medium, chilling the culture tube on ice
to detach the organisms from the side of the flask. The number of amoeba per
milliliter was estimated with a hemocytometer, using trypan blue exclusion to
confirm the viability. The suspension was diluted to 105 organisms per milliliter by
adding fresh medium, and 170 ll of this suspension was added to the test and
control wells in the plate so that the wells were completely filled (total volume,
4
3
40 ll). An inoculum of 1.7 ꢀ 10 organisms per well was chosen so that confluent,
but not excessive, growth took place in control wells. Plates were sealed and gassed
for 10 min with nitrogen before incubation at 37ꢁC for 72 hr. After incubation, the
growth of amoebae in the plate was checked with a low power microscope. By
inverting the plate, the culture media was removed with gentle shaking and then
immediately washed with NaCl solution (0.9%) at 37ꢁC. This procedure was
completed quickly and the plate was not allowed to cool to prevent the detachment
of amoebae. It was allowed to dry at room temperature. After drying, the amoeba
was fixed with chilled methanol by keeping it in ice bath for 15 min, drying and

Med Chem Res (2010) 19:771–781
777
Table 4 In vitro antiamoebic
Compound
IC50 (lM)
SD
activities of tested compounds
against (HM1: IMSS) strain of
E. histolytica
SHI
3.9
0.14
0.05
0.05
0.06
0.06
0.05
0.1
Co(SHICl)Cl
Ni(SHICl)Cl
Cu(SHICl)Cl
Zn(SHICl)Cl
Pd(SHICl)Cl
Metronidazole
1.44
2.23
1.44
0.9
0.9
1.8
SD standard deviation
staining with (0.5%) aqueous eosin for 15 min. The stained plate was washed once
with tap water and then twice with distilled water and allowed to dry. A 200 ll
-
3
portion of 0.1 mol ꢀ dm NaOH solution was added to each well to dissolve the
protein and release the dye. The optical density of the resulting solution in each well
was determined at 490 nm with a microplate reader. The % inhibition of amoebal
growth was calculated from the optical densities of the control and test wells and
plotted against the logarithm of the dose of the drug tested. Linear regression
analysis was used to determine the best fitting straight line from which the IC50
value was found. These results are presented in Table 4.
Superoxide radical scavenging assay
Nitroblue tetrazolium (NBT), methionine (MET), and vitamin B (VitB ) were
2
2
purchased from Sigma Chemical Co. Vitamin C, safranin, and ethylenediaminete-
traacetic acid disodium salt (EDTA) were purchased from Merck. All materials and
solvents were of analytical grade. EDTA–Fe(II) and Na HPO –KH PO buffers
2
4
2
4
were prepared with twice distilled water.
ꢁꢂ
The superoxide radicals O2 were generated in vitro by nonenzymatic system
and determined spectrophotometrically by nitro blue tetrazolium (NBT) photo
reduction method with a little modification in the method adopted elsewhere
ꢁꢂ
(
for O2 can be calculated by measuring the absorbance at 560 nm. Solution of
Winterbourn, 1979; Sharma et al., 2005). The amount of O2 and suppression ratio
ꢁ
ꢂ
MET, VitB , and NBT were prepared at avoiding light. The tested compounds were
2
dissolved in DMF (N,N-dimethylformamide). The assay mixture, in a total volume
of 5 ml, contained MET (10 mM), NBT (46 mM), VitB (3.3 mM), the tested
2
compound (2–20 mM), and a phosphate buffer (67 mM, pH 7.8). After illuminating
with a fluorescent lamp at 30ꢁC for 10 min, the absorbance of the samples (A ) was
i
measured at 560 nm. The sample without the tested compound was used as control
and its absorbance was A . All experimental results were expressed as
0
mean ± standard deviation (SD) of triplicate determinations. The suppression ratio
ꢁ
ꢂ
for O2 was calculated from the following expression:
A ꢂ A
0
i
Suppression ratio ð%Þ ¼
ꢃ 100
A
0

778
Med Chem Res (2010) 19:771–781
Drug activity was expressed as the 50% inhibitory concentration (IC ). IC
50
5
0
values were calculated from regression lines where: x was the tested compound
concentration in mM and y was percent inhibition of the tested compounds.
Hydroxyl radical scavenging assay
ꢁ
The hydroxyl radicals OH in aqueous media were generated through the Fenton
system (Winterbourn, 1981). The solution of the tested compound was prepared with
3
DMF. The 5 cm assay mixture contained following reagents: safranin (11.4 mM),
EDTA–Fe(II) (40 mM), H O (1.76 mM), the tested compound (2–20 mM), and a
2
2
phosphate buffer (67 mM, pH 7.4). The assay mixtures were incubated at 37ꢁC for
0 min in a water bath, after which the absorbance was measured at 520 nm. All the
tests were run in triplicate and expressed as mean ± SD. The suppression ratio for
3
ꢀ
OH was calculated from the following expression:
Ai ꢂ A0
Suppression ratio ð%Þ ¼
ꢃ 100; where
Ac ꢂ A0
Ai = the absorbance in the presence of the tested compound
A0 = the absorbance in the absence of the tested compound, and
Ac = the absorbance in the absence of the tested compound, EDTA–Fe(II), H O
2
2
Results and discussion
Compounds were evaluated for their in vitro antimicrobial activity (Tables 1 and 2)
against seven pathogenic bacteria and four fungi (Rodrigues Pereira et al., 1996;
Jiang et al., 2003). The data for metal(II)chloride, sulfamethoxazole, trimethoprim,
and clotrimazole are included for comparison. As shown, all compounds have
different and very good antibacterial activity in vitro against the tested Gram-
positive, Gram-negative bacteria, and fungi. The results show that the coordination
compounds have enhanced activity compared with the ligand, which indicates that
the coordinated bimetals have an influence on the antimicrobial effects. Because all
complexes are soluble in DMF, different activity cannot be explained with different
solubility. However, the higher activity of the complexes, compared with the free
ligand, can be understood in terms of the chelating theory. This theory explains that
a decrease in the polarizability of the metal could enhance the lipophilicity of the
complexes (Podunavac-Kuzmanovi c´ et al., 1999). Of the SHI complexes tested,
Pd(II), Zn(II), and Co(II) complexes were the most active against all tested bacteria
and fungi. All of the complexes showed higher activity than sulfamethoxazole and
trimethoprim against S. aureus, B. anthracis, Enterobacter, P. aeruginosa, and P.
mirabilis. The antifungal activity of the compounds was studied with four
pathogenic fungi. The results are summarized in Table 3. Clotrimazole has been
used as reference for inhibitory activity against fungi. All of the compounds showed
a significant antifungal activity. When SHI complexes were compared with

Med Chem Res (2010) 19:771–781
779
clotrimazole, Pd(II) and Zn(II) showed higher activity against C. albicans and H.
capsulatum than standard drug, and equipotent against M. gypsum and A. niger.
Antimicrobial activity in all tested complexes decreases following order:
Pd [ Zn [ Cu [ Co [ Ni
Anti-inflammatory activity was exhibited by all tested compounds (Table 3). The
activity compared with the commercial diclofenac are less, but yet highly significant
especially for Pd(II) and Zn(II) complexes with SHI. Although the antimicrobial
activity was performed in vitro, the following mechanisms can be proposed:
1
2
.
.
The ligand is active, whereas metal ensures the suitable transport form.
Metal ion is active, whereas complex ensures its transport through cell
membrane.
3
.
Active metal ion can react with important cell center.
The in vitro antiamoebic activities of compounds were performed by using the
HM1:IMSS strain of E. histolytica to ascertain the effectiveness of metal complexes
compared with ligand. Metronidazole was used as the reference drug. The IC50
value for the metronidazole 1.8 lM, as estimated after logarithmic transformation
of the curve shown in Fig. 2, with IC50 and its dose response curve are given in
Fig. 2. The IC50 values for the tested compounds are given in Table 4. The results
were estimated as the percentage of growth inhibition compared with the inhibited
controls and plotted as probity values as a function of the drug concentration. IC50
and 95% confidence limits were interpolated in the corresponding dose response
curve. The complexation enhances the activity of the ligand; it may be due to
chelating, which reduces the polarity of the central metal atom because of partial
sharing of its positive charge with the ligand or, as previously mentioned, because
the complexes are mostly cationic lipophilic species. The complexes of Pd(II) and
Zn(II) complexes with SHI were more active then standard with less IC50 values
Fig. 2 Dose-response curve for standard drug metronidazole

780
Med Chem Res (2010) 19:771–781
(
0.90 lM, respectively). The results were statistically evaluated by analysis of
variance. The null hypothesis was tested using t-test. The significance of the
difference between the IC50 values of metronidazole and the compounds was
evaluated by t-test. The values of the calculated t were found higher than table value
of t at 5% level, thus concluding that the character under study is significantly
influenced by the treatment. Detailed studies of the toxicity of these compounds,
mechanism of action, and in vivo studies are in progress.
Superoxide radical scavenging activity
Considering the structure of investigated ligands, we selected SHI and its complexes
to test on superoxide and hydroxy scavenging activity. It can be seen that the
ꢁꢂ
inhibitory effect of the tested compounds on O2 are concentration related and the
suppression ratio increases with the increasing of sample concentration in the range
of the tested concentration (Table 5). The antioxidant activities of these compounds
are expressed as 50% inhibitory concentration (IC50 in lM). IC50 values of ligand,
Co(SHICl)Cl, Ni(SHICl)Cl, Cu(SHICl)Cl, Zn(SHICl)Cl, and Pd(SHICl)Cl are 15,
2
1, 19, 1.3, 14, and 2.6 lM, respectively. These compounds clearly show higher
superoxide dismutase activity compared with that of standard antioxidant, such as
vitamin C (IC50 = 852 lM). As it can be seen, Cu(II) complex shows higher
several-fold potent activity, and SOD-like activity decreases in order:
CuðSHIClÞCl [ PdðSHIClÞCl [ SHI [ ZnðSHIClÞCl [
NiðSHIClÞCl [ CoðSHIClÞCl
Hydroxy radical scavenging activity
ꢀ
We can find that all compounds scavenge OH also in a concentration-dependent
manner (Table 5). Usually, mannitol and vitamin C are used as standard hydroxy
radical scavenging reagent. When arriving at a similar inhibitory value, it can be
seen IC50 values of the ligand and its complexes are far less than that of mannitol
and vitamin C. The ligand and its complexes show a highly active scavenging
•
effect on OH . Moreover, it indicates that, as a standard hydroxy radical
scavenging reagent, mannitol is better than vitamin C. Among these compounds,
•
the order of the suppression ratio for OH Cu(SHICl)Cl [ Pd(SHICl)Cl [ SHI [
Zn(SHICl)Cl [ Ni(SHICl)Cl [ Co(SHICl)Cl at different concentrations. Taken
Table 5 Influence of
investigated compounds for
O2 and OH
Compound
IC50 (lM)
ꢁ
ꢂ
•
ꢁꢂ
ꢁ
OH
O2
SHI
15
21
19
1.3
14
2.6
7.29
9.05
8.12
0.95
6.51
1.78
Co(SHICl)Cl
Ni(SHICl)Cl
Cu(SHICl)Cl
Zn(SHICl)Cl
Pd(SHICl)Cl

Med Chem Res (2010) 19:771–781
781
together, these compounds are possess potent antioxidant activity and are better
than standard antioxidants, such as vitamin C and mannitol.
The results show that Cu(II) complex displays excellent activity on the
superoxide and hydroxy radicals and deserves to be further researched.
Acknowledgments This work was supported by the Ministry of Science and Environmental Protection
of the Republic of Serbia.
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