Synthesis, crystal structures, and biological evaluation of Cu(II) and Zn(II) complexes of 2-benzoylpyridine Schiff bases derived from S-methyl- and S-phenyldithiocarbazates

Article abstract of DOI:10.1016/j.jinorgbio.2011.09.034

Two NNS tridentate Schiff base ligands of 2-benzoylpyridine S-methyldithiocarbazate (HL¹) and 2-benzoylpyridine S-phenyldithiocarbazate (HL²) and their transition metal complexes [Cu₂(L¹)₂(CH₃

Full text of DOI:10.1016/j.jinorgbio.2011.09.034

Journal of Inorganic Biochemistry 106 (2012) 117–125
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis, crystal structures, and biological evaluation of Cu(II) and Zn(II)
complexes of 2-benzoylpyridine Schiff bases derived from
S-methyl- and S-phenyldithiocarbazates
b
Ming Xue Li a,b, Li Zhi Zhang a, Chun Ling Chen a, Jing Yang Niu a, , Bian Sheng Ji
⁎
a
Institute of Molecular and Crystal Engineering, College of Chemistry and Chemical Engineering, Henan University, Kaifeng 475004, PR China
Key Laboratory of Natural Medicines and Immune Engineering, Henan University, Kaifeng 475004, PR China
b
a r t i c l e i n f o
a b s t r a c t
Two NNS tridentate Schiff base ligands of 2-benzoylpyridine S-methyldithiocarbazate (HL1) and 2-
benzoylpyridine S-phenyldithiocarbazate (HL2) and their transition metal complexes [Cu2(L1)2(CH3COO)]
(ClO4) (1), [Zn2(L1)2(ClO4)2] (2), [Zn(L2)2](3) have been prepared and characterized by elemental analysis,
IR, MS, NMR and single-crystal X-ray diffraction studies. In the solid state, each of two Schiff bases remains
in its thione tautomeric form with the thione sulfur atom trans to the azomethine nitrogen atom. Under sim-
ilar prepared conditions, three new complexes showed distinctly different coordination modes depending on
their coordinating preferences. Each copper atom in S-bridged dinuclear complex [Cu2(L1)2(CH3COO)](ClO4)
(1) is surrounded by five donor atoms in a square-pyramidal fashion (4+1). [Zn2(L1)2(ClO4)2] (2) is a dimer
in which each zinc atom adopts a seven-coordinate distorted pentagonal bipyramidal geometry, while mono-
nuclear [Zn(L2)2] (3) has octahedral coordination geometry. Biological studies, carried out in vitro against se-
lected bacteria, fungi, and K562 leukaemia cell line, respectively, have shown that different substituted
groups attached at the dithiocarbazate moieties and metals showed distinctive differences in the biological
property. Zinc(II) complexes 2 and 3 could distinguish K562 leukaemia cell line from normal hepatocyte
QSG7701 cell line. Effect of the title compounds on Mitochondria membrane potential (MMP) and PI-
associated fluorescence intensity in K562 leukaemia cell line are also studied. The title compounds may
exert their cytotoxicity activity via induced loss of MMP.
Article history:
Received 16 July 2011
Received in revised form 6 September 2011
Accepted 29 September 2011
Available online 5 October 2011
Keywords:
2-benzoylpyridine
S-methyl- and S-phenyldithiocarbazates
Crystal structure
Cytotoxicity
MMP
© 2011 Elsevier Inc. All rights reserved.
1. Introduction
the specific targeting of hypoxic tissue [6], as new, effective drugs for
treatment of refractory neuroblastoma in children [7]. On the other
Sulfur–nitrogen chelating agents derived from S-alkyl/aryl esters
of dithiocarbazic acid are of particular importance due to varied coor-
dination environments which determine a variety of possible confor-
mations and marked biological activities [1-3]. In many cases, the
biological properties of S-alkyl/aryl dithiocarbazate are often related
to the donor sequence of the ligands with different substituted dithio-
carbazate esters showing widely different biological activities, al-
though they may vary only slightly in their molecular structure. In
addition, biological activities of the metal complexes differ from
those of either the ligand or the metal ion itself, and increased
and/or decreased biological activities are reported for several transi-
tion metal complexes such as copper(II) and cobalt(II) [4, 5].
hand, the zinc(II) complexes are important. Intracellular distribution
of several zinc(II) complexes have been tracked in different cancer
cell lines [8]. In particular, copper(II) and zinc(II) complexes are
well-known for their significant pharmaceutical properties [9-11]
and in most cases were found to be generally more active than their
free ligands [12, 13]. Thus, knowledge of the biological evaluation of
these complexes is crucial to develop new antimicrobial agents and
cytotoxic agents.
In recent years we have been working on the structural and bio-
logical properties of heterocyclic thiosemicarbazones and their
transition metal complexes [14-18]. These results reveal that thio-
semicarbazones derived from 2-benzoylpyridine and their transi-
tion metal complexes show significant cytotoxic activities. After a
careful literature search, we can affirm that the coordination chem-
istry and pharmacological studies of 2-benzoylpyridine dithiocarba-
zonates are scarce. Therefore, it seemed important for us to
combine copper/zinc with 2-benzoylpyridine dithiocarbazonates
and evaluate their coordination chemistry and their potential as
anti-microbial and anti-cancer agents.
Copper complexes, which are stable and relatively lipophilic, rep-
resent a class of compounds that have recently been the subject of in-
tense research because of their potential as radiopharmaceuticals for
⁎
Corresponding author. Tel./fax: +86 378 3886876.
E-mail addresses: jyniu@henu.edu.cn, limingxue@henu.edu.cn (J.Y. Niu).
0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2011.09.034

118
M.X. Li et al. / Journal of Inorganic Biochemistry 106 (2012) 117–125
In the present work, with the main aim of comparison, we have test-
76%. M.p. 176−177°C. Anal. calcd for C20H17N3S2 (%): C, 66.03; H,
4.68; N, 11.55. Found C, 66.10; H, 4.75; N, 11.36. 1H NMR (DMSO-d6,
δ ppm): 8.86 (s, 1H, NH), 8.05(s, 1H), 7.62(s, 1H), 7.50−7.41(m,
6H), 7.33−7.26(m, 6H), 4.49(s, 2H). 13 C NMR (DMSO-d6, δ ppm):
198.8, 151.3, 149.6, 147.7, 138.78, 136.96, 136.71, 130.34, 129.72,
129.18, 129.04, 128.95, 127.74, 127.02, 125.86, 38.14. ESI-MS (m/z):
386.1=[HL2 +Na+], calc. mass=386.1. Yellow crystals suitable for
X-ray studies were obtained by slow evaporation of its ethanol
solution.
ed the biological activity of 2-benzoylpyridine S-methyldithiocarbazate
HL1, 2-benzoylpyridine S-phenyldithiocarbazate HL2 (Scheme 1), and
their transition metal complexes formulated as [Cu2(L1)2(CH3COO)]
(ClO4) (1), [Zn2(L1)2(ClO4)2] (2), [Zn(L2)2] (3) against bacteria, fungi,
and K562 leukemic cell line, respectively. Our objective is to compare
the variation in biological activity by changing different substituted
groups attached at the dithiocarbazate moieties and metals. To explore
their biochemical mechanism of cytotoxicity initially, effect of the title
compounds on Mitochondria membrane potential (MMP) and Propi-
dine iodide (PI)-associated fluorescence intensity in K562 leukaemia
cell line are studied. In addition, we also describe the synthesis, charac-
terization and single crystal X-ray crystal structures of the title com-
pounds here.
2.2.3. Synthesis of complex 1
An ethanol solution (10 mL) containing Cu(ClO4)2∙6H2O (0.09 g,
0.25 mmol) was added to an ethanol solution (20 mL) of HL1
(0.14 g, 0.5 mmol) and NaOAc (0.04 g, 0.5 mmol). The resulting reac-
tion mixture was refluxed for 4 h. The solid product formed was iso-
lated and washed with ethanol. This crude product was further
recrystallized from ethanol to give green microcrystals which were
filtered off and dried over P4O10 in vacuo. Yield, 64%. M.p. 145−
147°C. Anal. calcd for C30H27ClCu2N6S4O6 (%): C, 41.94; H, 3.15; N,
9.79. Found C, 41.99; H, 3.11; N, 9.61. ESI-MS (m/z): 757.0=[HL2 +
Na+], calc. mass=757.0. Green crystals suitable for X-ray studies
were obtained by slow evaporation of its ethanol solution. Further-
more, in accordance with our findings, the reaction of one equivalent
metal ion with one equivalent of ligand also generates the desired
complex 1 indicating the 1:1 metal/ligand product formed readily.
2. Material and methods
2.1. General techniques and apparatus
All solvents and reagents are commercially available and were
used without further purification. Elemental analysis of C, H and N
was performed with a Perkin-Elmer 240 analyzer. The infrared spec-
tra were recorded from KBr discs with a Nicolet 170 FT infrared spec-
trophotometer. The mass spectra were carried out on an Esquire 3000
LC-MS mass spectrophotometer. Melting points were determined
with a X-5 Micro Processor Melting-point Apparatus. 1H and13
C
NMR spectra were recorded in DMSO–d6 using a BrukerAV-400
2.2.4. Synthesis of complex 2
spectrometer.
Complex 2 was prepared by a similar procedure to that of complex
1 using Zn(ClO4)2∙6H2O in place of Cu(ClO4)2∙6H2O. Yield, 58%. M.p.
261−263°C. Anal. calcd for C28H24Cl2Zn2N6S4O8 (%): C, 37.23; H,
2.66; N, 9.31. Found C, 37.29; H, 2.64; N, 9.08. ESI-MS (m/z):
988.9=[Zn2(HL1) (L1)(ClO4)2(H2O)5] +, calc. mass=988.9. Yellow
crystals suitable for X-ray studies were obtained by slow evaporation
of its ethanol solution.
2.2. Synthesis
2.2.1. Synthesis of HL1
An ethanol solution (15 mL) containing 2-benzoylpyridine
(0.73 g, 4.0 mmol) was added dropwise to an ethanol solution
(20 mL) of S-methyldithiocarbazate (0.49 g, 4.0 mmol) with five
drops of acetic acid as catalyst. After refluxed for 2 h, the resultant so-
lution was filtered. Products separated were recrystallised from hot
ethanol and dried over P4O10 in vacuo. Yield, 81%. M.p. 113−114°C.
Anal. calcd for C14H13N3S2 (%): C, 58.46; H, 4.52; N, 14.61. Found C,
58.53; H, 4.60; N, 14.41. 1H NMR (DMSO-d6, δppm): 8.86 (s, 1H,
NH), 8.02(s, 1H), 7.63(s, 1H), 7.55–7.42 (m, 6H), 7.30 (s, 1H), 2.53
(s, 3H). 13 C NMR (DMSO-d6, δ ppm): 200.8, 151.4, 149.6, 147.2,
138.8, 136.8, 130.3, 129.6, 129.0, 127.0, 125.8, 17.34. ESI-MS (m/z):
288.2=[HL1 +H+], calc. mass=288.1. Yellow crystals suitable for
X-ray studies were obtained by slow evaporation of its ethanol
solution.
2.2.5. Synthesis of complex 3
Complex 3 was prepared by a similar procedure to that of complex
2 using 2-benzoylpyridine S-phenyldithiocarbazate in place of 2-
benzoylpyridine S-methyldithiocarbazate. Yield, 62%. M.p. 211−
213°C. Anal. calcd for C40H32ZnN6S4 (%): C, 60.73; H, 4.05; N, 10.63.
Found C, 60.81; H, 4.01; N, 10.44. ESI-MS (m/z): 789.2=[Zn(L2)
(HL2)] +, calc. mass=789.1. Yellow crystals suitable for X-ray studies
were obtained by slow evaporation of its ethanol solution.
2.3. X-ray crystallography
Crystallographic data were collected with a Siemens SMART-
CCD diffractometer with graphite-monochromated MoKα radiation
(λ=0.71073 Å). The structure were solved by Direct Methods and re-
fined by full-matrix least-squares on F2 with anisotropic displacement
2.2.2. Synthesis of HL2
HL2 was prepared by a similar procedure to that of HL1 using S-
phenyldithiocarbazate in place of S-methyldithiocarbazate. Yield,
Scheme 1. Dithiocarbazates ligands (HL1 and HL2).

M.X. Li et al. / Journal of Inorganic Biochemistry 106 (2012) 117–125
119
Table 1
Summary of crystal data and refinement results for HL1, HL2 and complexes 1−3.
Crystal Data
HL1
HL2
1
2
3
Formula
C14H13N3S2
287.39
Monoclinic
P21/n
12.5242(16)
5.5115(7)
20.520(3)
95.440(2)
1410.1(3)
4
C20H17N3S2
363.49
Monoclinic
P21/c
12.3607(19)
12.599(2)
11.7952(19)
90.588(3)
1836.9(5)
4
C30H27ClCu2N6S4O6
858.35
Monoclinic
P21/c
12.1408(12)
24.357(3)
13.1986(13)
116.5580(10)
3491.1(6)
4
C28H24Cl2Zn2N6S4O8
902.41
Monoclinic
P21/c
10.7126(9)
7.6265(7)
22.338(2)
103.4400(10)
1775.1(3)
2
C40H32ZnN6S4
790.33
Monoclinic
P21/c
14.5341(8)
15.9047(9)
17.2705(9)
94.1880(10)
3981.6(4)
4
Formula weight
Crystal system
Space group
a/Å
b/Å
c/Å
β/°
Volume/Å3
Z
Dcalcd/g cm−3
Crystal habit
Crystal color
μ/mm−1
1.354
cuboid
yellow
0.366
1.314
stick
yellow
0.297
1.633
rectangle
green
1.688
block
yellow
1.794
1.318
block
yellow
0.863
1.586
Crystal size/mm
θ range/°
No. reflns collected
Data/restrnts/params
GOOF
0.32×0.21×0.15
1.83−25.00
2487
1915/0/173
0.961
0.29×0.23×0.18
1.65–26.00
3592
2240/0/226
1.090
0.32×0.23×0.16
1.67–26.00
6863
5198/34/442
1.105
0.29×0.23×0.18
1.87–25.00
3092
2527/40/227
1.023
0.34×0.26×0.23
1.74–25.00
6695
5327/0/460
0.986
Rint
0.0426
0.0388
0.0382
0.0393
0.0200
R1, wR2 (IN2σ(I))
R1, wR2 (all data)
0.0326, 0.0821
0.0442, 0.0870
0.0504, 0.1570
0.0797, 0.1648
0.0507, 0.1531
0.0702, 0.1633
0.0981, 0.3128
0.1083, 0.3303
0.0581, 0.1921
0.0719, 0.2074
parameters for all non-hydrogen atoms using SHELXTL [19]. The hy-
drogen atoms were added in idealized geometrical positions. Table 1
summarizes crystal and refinement data for the five compounds.
(fungi), respectively. The minimal inhibitory concentration (MIC)
was detected as the lowest concentration of drug in plate for which
no visible growth took place by macroscopic evaluation. All determi-
nations were performed in triplicate and confirmed by three separate
experiments.
2.4. Antimicrobial activity
2.5. cytotoxicity assay
Gram-positive, Gram-negative bacteria, moulds and yeast were
used for antimicrobial studies. Gram positive bacteria: Bacillus subtilis,
Staphylococcus aureus, Agrobacterium tumefaciens. Gram negative bac-
teria: Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimur-
ium. Moulds: Aspergillus niger, Mucor mucedo, Penicillium oxalicum.
Yeast: Candida lusitaniae. All microorganisms were provided by
China General Microbiological Culture Collection Center (CGMCC).
The bacterial strains were grown in Mueller-Hinton Agar (MHA)
plates at 37 °C, while the moulds and yeast were grown in Potato
Dextrose Agar (PDA) and Sabouraud Dextrose Agar (SDA)media, re-
spectively, at 28 °C. The minimal inhibitory concentrations (MIC,
μg/mL) were estimated by the disk diffusion method [20]. The final
concentration of all cultures in Mueller-Hinon agar (MHA) for bacte-
ria and potato dextrose agar (PDA) and Sabouraud dextrose agar
(SDA) for mould spores and yeast cells was adjusted to 106 CFU/mL
(bacteria) or 2×105 CFU/mL (mould spores and yeast cells) and
used for inoculation in the MIC test. Serial dilutions of the test com-
pounds (dissolved in 10% DMSO in PBS) were prepared at concentra-
tions of 0–2000 μg/mL. To each plate was inoculated with 0.1 mL of
the prepared bacterial and fungi cultures. Similarly, each plate carried
a blank disc, with medium solvent containing 10% DMSO only in the
center to serve as a control, as well as control antibiotics ampicillin
(Amp), streptomycin (Str), kanamycin sulfate (Kan) (for bacteria)
and nystatin (Nys) (for fungi). The inoculated plates were then incu-
bated at either 37 °C for 18–20 h (bacteria) or 28 °C for 48–96 h
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay was carried out to evaluate cytotoxicity in K562 leukae-
mia cell line and QSG7701 cell line. Cells were plated into 96-well
plates at a cell density of 1×104 cells per well and allowed to grow
in a CO2 incubator. After 24 h, the medium was removed and replaced
by fresh medium containing the tested compounds which were dis-
solved in DMSO at 0.01 M and diluted to various concentrations with
phosphate-buffered saline (PBS) before the experiment, the final con-
centration of DMSO is lower than 1%. After 24 h incubation, cultures
were incubated in 100 μL of medium with 10 μL of 5 mg/mL MTT solu-
tion for 4 h at 37 °C. The medium with MTT was removed, and 100 μL
of DMSO was added to each well to dissolve the formazan. The absor-
bance at 570 nm was measured with microplate reader (Bio-Tek
ELX800, USA). The inhibitory percentage of each compound at various
concentrations was calculated, and the IC50 value was determined.
Table 2
IR spectral assignments (cm−1) of HL1, HL2 and complexes 1−3.
Compounds
ν(C=N)
ν(N–N)
ν(N–H)
ν(C=S)
ν(C−S)
(py)
HL1
1
1583
1539
1545
1585
1542
1119
1163
1155
1119
1155
3058
1055
607
622
641
607
633
997
974
2
HL2
3
3058
1052
965
Fig. 1. Molecular structure of HL1.

120
M.X. Li et al. / Journal of Inorganic Biochemistry 106 (2012) 117–125
Table 4
Selected bond lengths (Å) and angles (°) of HL2 and complex 3.
HL2
3
N(2)–C(9)
S(2)–C(8)
N(1)–N(2)
N(1)–C(8)
S(1)–C(7)
S(1)–C(8)
C(8)–N(1)–N(2)
C(9)–N(2)–N(1)
N(1)–C(8)–S(2)
N(1)–C(8)–S(1)
1.302(4)
1.652(3)
1.366(3)
1.334(4)
1.806(3)
1.764(3)
120.4(2)
118.4(2)
121.1(2)
112.2(2)
Zn(1)–S(2)
Zn(1)–N(2)
Zn(1)–N(3)
S(2)–C(8)
N(2)–C(9)
S(4)–C(28)
N(5)–Zn(1)–S(2)
N(5)–Zn(1)–N(3)
N(2)–Zn(1)–S(4)
N(6)–Zn(1)–S(4)
2.4171(1)
2.145(3)
2.242(3)
1.715(4)
1.285(5)
1.721(4)
114.44(9)
90.26(1)
119.54(9)
153.58(8)
Table 2. The infrared spectral bands most useful for determining the
mode of coordination of the ligands are the ν(C=N), ν(N−N) and
ν(C=S) vibrations. The IR spectra of HL1 and HL2 do not display
ν(S–H) at around 2700 cm−1 indicating that in the solid state two li-
gands remain in the thione form [21]. A comparison of the IR spectra
of the ligands with their complexes indicate that the ν(N–H ) bands of
the free ligands are not present in the spectra of the complexes, sup-
porting deprotonation of the ligands during coordination. The shift of
the ν(C=N) bands of the free ligands to lower wavenumbers and the
shift of the ν(N–N) bands to higher wavenumbers in the IR spectra of
the complexes support coordination of the ligands to the metal atom
via the azomethine nitrogen atom [22]. The HL1 and HL2 showed
ν(C=S) modes at 1055 and 1052 cm−1, respectively. In the spectra
of the complexes, the ν(C=S) modes observed disappeared and con-
sequently the ν(C−S) modes are observed confirming that complex-
ation occurred [23]. The breathing motion of the pyridine ring is
shifted to a higher frequency upon complexation and is consistent
with pyridine ring nitrogen coordination. The ν(ClO4) bands in com-
plexes 1 and 2 are found at 1089 and 1088 cm−1, respectively. Based
on the above spectral evidences, it is confirmed that the free ligand is
tridentate, coordinating via pyridyl nitrogen, azomethine nitrogen
and the thiolato sulfur atoms. These observations have also been con-
firmed by X-ray single crystal structure analysis.
Fig. 2. Molecular structure of HL2.
2.5.1. Measurement of mitochondrial membrane potential (MMP)
After incubation with the tested compounds with different con-
centration for 48 h, the K562/DOX cells were preloaded with Rhoda-
mine 123(Rh123) (2 μM) and Hoechst 33342 (3 mg/ml) for 30 min
at 37 °C, and then rinsed with freshly prepared phosphate-buffered
saline (PBS). The fluorescence intensity was measured at emission
wavelength of 530 nm and excitation wavelengths of 480 nm by
High-Content Screening Reader (Arrary Scan VTI 600, USA).
Statistical analysis: All the data were expressed as mean S.D. and
analyzed using analysis of variance (ANOVA) followed by Student's
t-test. Differences were considered statistically significant at pb0.05.
2.5.2. PI staining
After incubation with the tested compounds with different concen-
tration for 48 h, the K562/DOX cells were preloaded with Propidine io-
dide (PI)(100 μg/ml) and Hoechst 33342 (3 mg/ml) for 30 min at 37 °C,
and then rinsed with freshly prepared phosphate-buffered saline (PBS).
The fluorescence intensity was measured at emission wavelength of
620 nm and excitation wavelengths of 488 nm by High-Content Screen-
ing Reader (Arrary Scan VTI 600, USA).
3.2. X-ray crystallography
Statistical analysis: All the data were expressed as mean S.D.
and analyzed using analysis of variance (ANOVA) followed by Stu-
dent's t-test. Differences were considered statistically significant at
pb0.05.
In view of the structural similarity of HL1 and HL2 (see Figs. 1 and
2, and Tables 3–5), only HL1 was described in some detail. As shown
in Fig. 1, in the solid state, the free ligand HL1 remains in its thione
tautomeric form (supported by the presence of hydrazinic N−H
and C−S distance of 1.653(2) Å, which is much shorter than a single
C−S bond) with the thione sulfur atom trans to the azomethine nitro-
gen atom. A comparison of the N(1)–N(2) distance of 1.372(2) Å with
that in S-methyldithiocarbazate [24] shows that the bond is shorter
than a single N–N bond (1.44 Å) indicating that a significant π-
charge delocalization occurs along the C–N–N–C moiety. Unlike the
3. Results and discussion
3.1. Spectral studies
The tentative assignments of the significant IR spectral bands of
the free ligands HL1, HL2 and their complexes 1−3 are presented in
Table 3
Selected bond lengths (Å) and angles (°) of HL1, 1 and 2.
HL1
1
2
S(2)–C(2)
N(2)–C(3)
N(1)–N(2)
N(1)–C(2)
S(1)–C(2)
S(1)–C(1)
1.653(2)
1.300(2)
1.372 (2)
1.350(2)
1.738(2)
1.796(2)
Cu(1)–O(2)
Cu(1)–N(2)
Cu(1)–N(3)
Cu(1)–S(2)
S(2)–C(2)
S(4)–C(16)
N(1)–C(2)
O(2)–Cu(1)–S(2)
N(3)–Cu(1)–S(2)
O(2)–Cu(1)–N(2)
N(2)–Cu(1)–N(3)
N(2)–Cu(1)–S(2)
1.930(3)
1.950(4)
2.006(4)
2.278 (1)
1.749(5)
1.755(5)
1.293(6)
99.67(11)
163.54(12)
171.41(16)
80.50(15)
84.87(110
Zn(1)–O(1)
Zn(1)–N(3)
Zn(1)–S(2)
Zn(1)–N(2)
Zn(1)–O(4)
S(2)–C(2)
N(2)–C(3)
O(1)–Zn(1)–N(3)
N(3)–Zn(1)–S(2)
N(2)–Zn(1)–O(4)
N(2)–Zn(1)–O(1)
S(2)–Zn(1)–O(4)
2.072(1)
2.163(6)
2.390(2)
2.104(6)
2.163(12)
1.724(7)
1.287(9)
99.8(4)
154.31(2)
109.4(3)
142.5(3)
97.6(4)
C(2)–N(1)–N(2)
C(3)–N(2)–N(1)
N(1)–C(2)–S(2)
N(1)–C(2)–S(1)
S(2)–C(2)–S(1)
120.33(14)
118.09(14)
119.27(13)
114.74(12)
125.98(10)

M.X. Li et al. / Journal of Inorganic Biochemistry 106 (2012) 117–125
121
Table 5
Hydrogen bond lengths (Å) and bond angles (°) of HL1 and HL2.
D–H…A
d(H…A )
d(D…A )
∠(DHA)
HL1
HL2
N(1)–H(1A)–N(3)
N(1)–H(1A)–N(3)
1.95
1.97
2.634(2)
2.650(4)
135.6
135.2
2-acetylpyrazine Schiff base of S-benzyldithiocarbazate [25], the pyr-
idine nitrogen atom N(3) is not trans to the azomethine nitrogen
atom N(2). The conformation of the molecule is mainly determined
by the intramolecular hydrogen bonds between the hydrazine nitro-
gen atom N(1) and the pyridine nitrogen atom N(3) (N(1)−H(1)…
N(3) 2.634(2) Å) (see Figs. 3 and 4, and Table 5).
The molecular structure of complex 1 contains dinuclear [Cu2(L1)2
(CH3COO)]+ cation in which sulfur atom bridges the two copper(II)
ions (Fig. 5) and free perchlorate anion. Each of the two copper
atoms in [Cu2(L1)2(CH3COO)]+ adopts
a five-coordinate near
square-pyramidal geometry with an N2S2O coordination environ-
ment. The pyridine nitrogen (N(3)/(N6)), the azomethine nitrogen
atom (N(2)/N(5)) and the thiolate sulfur atom (S(2)/S(4)) together
with the carboxyl oxygen atom (O(2)/O(1)) from acetate group li-
gand comprise the basal plane of the square-pyramid whereas
the thiolate sulfur atom (S(4)/S(2))of another ligand occupies the
apical position. The large difference between the two Cu–S distances
(Cu(1)−S(2) 2.278(1) Å/ Cu(2)−S(4) 2.264(1) Å in the basal plane
and Cu(1)−S(4)2.878 Å/Cu(2)−S(2) 2.838 Å in the apical position)
can be ascribed to a Jahn-Teller distortion [26]. Copper(II) dimers
with such a thiolate sulfur bridging have also been observed in
other dithiocarbazate copper(II) complexes [27].
The C–S bond length increases from 1.653(2) Å to 1.749(5) and
1.755(5) Å, respectively. Similarly, C(2)–N(1) suffers a significant de-
crease from 1.350(2) Å to 1.293(6) Å. These changes with enhanced
single and double bond characters indicate the ligand in the present
complex is coordinated in its deprotonated thiolate form as observed
in most complexes derived from S-alkyl/aryl dithiocarbazates [28].
The two dithiocarbazate ligands have slightly different Cu–N(pyri-
dine) bond distances and they are longer than the Cu–N(azomethine)
distances, this may be attributed to the fact that the azomethine ni-
trogen is a stronger base compared with the pyridine nitrogen [29].
Single crystal X-ray studies show that complex 2 is a centrosym-
metric dimer in which each zinc atom adopts a seven-coordinate dis-
torted pentagonal bipyramidal geometry with an N2SO4 coordination
environment, the Schiff base coordinating as a uninegatively charged
tridentate ligand chelating through the pyridine and azomethine ni-
trogen atoms and the thiolate, four oxygen atoms from two perchlorate
Fig. 4. Hydrogen bond in dashed lines in HL2.
groups completing the coordination sphere (Fig. 6). Zn(II) ion usually
favors tetrahedral coordination [23]. Zinc ion in complex 2 forms an
irregular seven-coordinate polyhedron regarded as a special mode of
bonding.
Complex 3 (Fig. 7) is a mononuclear six-coordinate complex with
the basic formula [Zn(L2)2]. The octahedrally hexa coordinate Zn(II)
center is coordinated in an N4S2 manner by two monodeprotonated
ligand moieties and form four five membered chelate rings. One sul-
fur atom, one imine nitrogen atom and one pyridine nitrogen atom
from one ligand and one imine nitrogen atom from another ligand oc-
cupy the basal positions, the two remaining positions in the octahe-
dral geometry are the axial ones which are occupied by one sulfur
atom and one pyridine nitrogen atom from the second ligand. The
measured C–S bond distances ca. 1.715(4) and 1.721(4) Å are within
the normal range of C–S single bonds, indicating that the dithiocarba-
zate moieties adopt the thiol tautomeric form and acted as a monone-
gative ligand.
3.3. Antimicrobial activity
Taking into account that S-alkyl/aryl esters of dithiocarbazate
show antimicrobial activity [30], we have tested the ability of the
title compounds against both bacteria and fungi. Based on the mini-
mum inhibitory concentrations (Table 6), the synthesized com-
pounds showed varying degrees of inhibition against the tested
microorganisms. In general, the inhibitory activity against the
Fig. 3. Hydrogen bond in dashed lines in HL1.
Fig. 5. Molecular structure of complex 1.

122
M.X. Li et al. / Journal of Inorganic Biochemistry 106 (2012) 117–125
Fig. 6. Molecular structure of complex 2.
Fig. 7. Molecular structure of complex 3.
Gram-positive bacteria was higher than that of the Gram-negative
bacteria. HL1 and 1 showed excellent activity against Gram positive
bacteria B. subtilis, S. aureus and yeast C. lusitaniae with MIC values
of 1−5 μg/mL. The comparison of antimicrobial activity of the free li-
gands indicates that 2-benzoylpyridine S-methyldithiocarbazate HL1
is more potent than 2-benzoylpyridine S-phenyldithiocarbazate HL2
towards the tested microorganisms, which indicate that the structure
factors which govern antimicrobial activities are strongly dependent
on their substitutents. And at the same time, it is clearly observed
that complexation with metals has a synergetic effect on the antimi-
crobial activity of these compounds and the antimicrobial activity de-
pends upon the type of metal ion. Coupling of HL1 and HL2 to Zn(II),
respectively, decrease the antimicrobial activity of their parent li-
gands. As can be expected, copper(II) complex 1 results in enhanced
antimicrobial activity consistent with the previously reported
data [31].
Table 6
Minimal inhibitory concentration (μg/mL) of HL1, HL2 and complexes 1−3.
MIC (μg/mL)
microorganisms HL1
1
2
HL2
3
Solvent Amp Str Kan Nys
B. subtilis
S. aureus
1
5
1
1
100 100 125
–
–
–
–
–
–
–
–
–
–
5
5
10
–
20 10
20 10
20 20
–
–
–
–
–
–
–
–
–
–
–
–
A. tumefaciens
P. aeruginosa
E. coli
S. typhimurium
A. niger
M. mucedo
P. oxalicum
C. lusitaniae
100 12.5 125
100 12.5 125 125
–
–
–
–
40
10 20
40
a
–
–
–
–
–
–
–
–
–
–
–
–
–
5
20
–
10
20
10
20
100 62.5
–
–
1
1
125 125
a
No inhibition or MICN200 μg/mL, Solvent: 10% DMSO in PBS (No inhibitory against
the microorganisms).

M.X. Li et al. / Journal of Inorganic Biochemistry 106 (2012) 117–125
123
Fig. 8. The cytotoxic activities of the free ligands and the title complexes against K562 leukaemia cell line and normal hepatocyte QSG7701 cell line.
3.4. Antimour activity
increase the biological activity of their parent ligands, consistent with
other copper and zinc compounds [37, 38]. This confirms the conclu-
sion that the cytotoxic activities of Schiff base can be enhanced by co-
ordinating the ligand to Cu(II) and Zn(II) cations [39]. The better
activities of these metal complexes compared to their respective li-
gands may be due to chelation, which reduces the polarity of the cen-
tral metal atom because of partial sharing of its positive charge with
the ligand [40]. It should be emphasized that Zn(II) complex 3 effec-
tively inhibit K562 leukaemia cell line at concentrations more than
61-fold lower than HL2 and Cu(II) complex 1 effectively inhibit
K562 leukaemia cell line at concentrations more than 8-fold lower
than HL1. Importantly, IC50 values of zinc(II) complexes 2 and 3 are
obviously higher in QSG7701 cell line than in K562 leukaemia cell
line, indicating both of them have tumor cells selectivity. Therefore,
the complexes studied in the present work may be endowed with im-
portant cytotoxic properties and would be good candidates to be used
for medical practice as metal-based drugs.
In view of the cytotoxic activity of sulfur–nitrogen chelating
agents [32, 33], we have tested the ability of the title ligands and
their complexes to inhibit tumor cell growth against K562 leukaemia
cell line. To explore the toxicity of these compounds, their effect on
normal QSG7701 cell line is also described. In our experiments, IC50
values (compound concentration that produces 50% of cell death) in
micro molar units were calculated (see Fig. 8). Although a clear
structure-activity relationship cannot be deduced from the limited
number of compounds investigated, several preliminary conclusions
may be drawn.
As shown in Fig. 8, heterocyclic substituted dithiocarbazates and
their metal complexes show particularly effective cytotoxic activity
against K562 leukaemia cell line, due to the NNS tridentate system
[34]. The comparison of cytotoxic activity of the free ligands and
their metal complexes indicates that HL2 shows slightly lower IC50
value (35 μM) than the HL1 (37 μM), complex 3 (IC50 =0.6 μM) show
remarkably higher activity than complex 2 (IC50 =22 μM), which in-
dicate that the presence of bulky nonpolar S-substituents at dithio-
carbazates moiety enhanced the cytotoxic activities very similar to
the previously reported N(4)-substituted thiosemicarbazones cases
[35, 36]. And at the same time, it is clearly observed that complexa-
tion with metals has a synergetic effect on the biological activity of
these compounds and the biological activity depends upon the type
of metal ion. As can be expected, the title Cu(II) and Zn(II) complexes
3.5. Effect of the title compounds on mitochondria membrane potential
(MMP) and PI-associated fluorescence intensity in K562 leukaemia cell
line
Mitochondria was considered to be a major site of cytotoxic agents
through electron leakage from the electron transport chain [41, 42],
the decreased MMP may open the mitochondrial permeability transi-
tion (MPT) and trigger the release of cytochrome c which activate
Fig. 9. Effect of HL1, 1 and 2 on MMP in K562 leukaemia cell line. Each value represents the mean S.D. from four experiments. #: pb0.05 vs control.

124
M.X. Li et al. / Journal of Inorganic Biochemistry 106 (2012) 117–125
Fig. 10. Effect of HL2and 3 on MMP in K562 leukaemia cell line. Each value represents the mean S.D. from four experiments. #: pb0.05 vs control.
caspase cascade, causing the cell death. In the present report, rhoda-
mine 123 (Rh123), a mitochondrial specific stain that is dependent
on the transmembrane potential, and another one, Propidine iodide
(PI), were applied for the determination of MMP and apoptosis cells
as well as necrosis cells, respectively. As shown in Figs. 9–12, the PI-
associated fluorescence intensity in K562 leukaemia cells was in-
creased in concentration-dependent manner companying de-
creased MMP after incubation with the tested compounds for 48 h,
indicating the remarkable cytotoxic activity in vitro. The present re-
sults also revealed that the complexes 1 and 3 exhibited more potent
a
Fig. 11. Effect of HL1, 1 and 2 on PI-associated fluorescence intensity in K562 leukaemia line. Each value represents the mean S.D. from four experiments. #: pb0.05 vs control.
Fig. 12. Effect of HL2 and 3 on PI-associated fluorescence intensity in K562 leukaemia line. Each value represents the mean S.D. from four experiments. #: pb0.05 vs control.

M.X. Li et al. / Journal of Inorganic Biochemistry 106 (2012) 117–125
125
[11] S.C. Lim, K.A. Price, S.F. Chong, B.M. Paterson, A. Caragounis, K.J. Barnham, P.J.
Crouch, J.M. Peach, J.R. Dilworth, A.R. White, P.S. Donnelly, Journal of Biological
Inorganic Chemistry 15 (2010) 225–235.
effects than their parent ligand, which was consistent with their ac-
tions in MTT assay.
[12] T. Rosu, E. Pahontu, S. Pasculescu, R. Georgescu, N. Stanica, A. Curaj, A. Popescu, M.
Leabu, European Journal of Medicinal Chemistry 45 (2010) 1627–1634.
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Abbreviations
HL1
2-benzoylpyridine S-methyldithiocarbazate
2-benzoylpyridine S-phenyldithiocarbazate
Mitochondria membrane potential
HL2
MMP
[15] M.X. Li, C.L. Chen, D. Zhang, J.Y. Niu, B.S. Ji, European Journal of Medicinal Chemistry
45 (2010) 3169–3177.
DMSO-d6 Deuterated dimethyl sulphoxide
[16] M.X. Li, D. Zhang, L.Z. Zhang, J.Y. Niu, Inorganic Chemistry Communications 13
(2010) 1268–1271.
[17] M.X. Li, D. Zhang, L.Z. Zhang, J.Y. Niu, B.S. Ji, Inorganic Chemistry Communications
13 (2010) 1572–1575.
[18] M.X. Li, D. Zhang, L.Z. Zhang, J.Y. Niu, B.S. Ji, Journal of Organometallic Chemistry
696 (2011) 852–858.
[19] G.M. Sheldrick, SHELXTL (Version 5.1), Bruker AXS Inc, Madison, Wisconsin (USA),
1997.
MIC
DMSO
MTT
Minimal inhibitory concentrations
Dimethyl sulphoxide
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
Phosphate-buffered saline
Propidine iodide
PBS
PI
MPT
Rh123
Mitochondrial permeability transition
Rhodamine 123
[20] S.A. Khan, K. Saleem, Z. Khan, European Journal of Medicinal Chemistry 42 (2007)
103–108.
[21] S. Singh, N. Bharti, F. Naqvi, A. Azam, European Journal of Medicinal Chemistry 39
(2004) 459–465.
[22] M.A. Ali, A.H. Mirza, M.H.S.A. Hamid, P.V. Bernhardt, O. Atchade, X. Song, G. Eng, L.
May, Polyhedron 27 (2008) 977–984.
[23] M.T.H. Tarafder, K. Chew, K.A. Crouse, A.M. Ali, B.M. Yamin, H.K. Fun, Polyhedron
21 (2002) 2683–2690.
Acknowledgements
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This work was financially supported by the National Natural Sci-
ence Foundation of China (21071043), the China Postdoctoral Science
Foundation (20090460847) and the Natural Science Foundation of
the Educational Department of Henan Province (2010B150003).
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