Crystal structures and antimicrobial and cytotoxic activities of zinc(II), nickel(II) and copper(II) complexes of N-(piperidylthiocarbonyl)benzamide

Article abstract of DOI:10.1002/aoc.3262

A new zinc(II) complex of N-(piperidylthiocarbonyl)benzamide, [ZnL₂], has been synthesized and characterized using elemental analysis, Fourier transform infrared and ¹H NMR spectroscopies. X-ray diffraction indicates that [ZnL_2<

Full text of DOI:10.1002/aoc.3262

Full Paper
Received: 12 September 2014
Revised: 15 November 2014
Accepted: 16 November 2014
Published online in Wiley Online Library: 15 January 2015
(wileyonlinelibrary.com) DOI 10.1002/aoc.3262
Crystal structures and antimicrobial
and cytotoxic activities of zinc(II), nickel(II)
and copper(II) complexes of
N-(piperidylthiocarbonyl)benzamide
Juan Xiea, Zhongqin Chengb, Wen Yanga, Huanhuan Liua, Weiqun Zhoua*,
Mengying Lib and Yunlong Xua
A new zinc(II) complex of N-(piperidylthiocarbonyl)benzamide, [ZnL2], has been synthesized and characterized using elemental
1
analysis, Fourier transform infrared and H NMR spectroscopies. X-ray diffraction indicates that [ZnL2] presents a tetrahedral
structure within an O2S2 donor set, which is different from analogous square planar [NiL2] and [CuL2] available in the literature.
The antimicrobial activities of [ZnL2], [NiL2] and [CuL2] were evaluated against fungi and bacteria. The results show that [ZnL2]
is the best for control of the studied fungi and bacteria, and its antimicrobial activity is close to that exhibited by commercial prod-
ucts. The relationship between the structures and antimicrobial activities of the complexes was further investigated using density
functional theory calculations. It is elucidated that the increase of the polarity of carbonyl and thiocarbonyl groups determines
antifungal and antibacterial activities. Moreover, the cytotoxicity of the complexes was tested against human cancer cells
(hepatocellular carcinoma (SK-Hep-1) and breast carcinoma (MCF-7)). The [CuL2] complex is found to be the most cytotoxic.
Copyright © 2015 John Wiley & Sons, Ltd.
Additional supporting information may be found in the online version of this article at the publisher’s web site.
Keywords: metal complexes; crystal structures; antimicrobial activity; cytotoxic activity; DFT calculation
methods. The antifungal and antibacterial properties of the
complexes were also investigated. The relationship between the
Introduction
Platinum complexes have been extensively used as antitumor
agents following the success of cisplatin.[1,2] However, due to the
high cost of platinum, it is necessary to find other metal complexes
as replacements. Among the non-platinum compounds for cancer
treatment, copper(II), nickel(II) and zinc(II) complexes have been re-
ported to possess antitumor activity at least comparable to that of
cisplatin, while they exhibit less kidney toxicity.[3–5] Zinc and copper
complexes have received much attention due to the fact that they
are bio-essential elements responsible for numerous bioactivities in
living organisms.[1,3,5] The clinical success of platinum drugs has
stimulated extensive investigations in searching for new metallic
species with improved pharmacological properties.[6] Recently,
pyridyl-type ligands and copper(II) terpyridyl complexes have been
reported to show moderate anticancer activities.[7]
Thiourea and its derivatives are widely applied as antimicrobial
agents due to their diverse biological activities.[8,9] A series of
metal–thiourea complexes have been synthesized and confirmed
to have antimicrobial and antitumor properties.[10–13] Keeping in
view the biological and medicinal importance of thiourea and the
role of metal elements in biology, a novel Zn(II) complex, [ZnL2],
with N-(piperidylthiocarbonyl)benzamide as ligand (HL) was de-
signed and synthesized in the work reported here. The syntheses
of [NiL2] and [CuL2] complexes have been reported,[14–16] but the
crystal structures and bioactivities of these complexes have not
been described. In the work reported here, the crystal structures
of [ZnL2], [NiL2] and [CuL2] were determined using X-ray diffraction
structures of the complexes and their antimicrobial activity was
explored based on density functional theory (DFT) calculations.
Furthermore, in order to understand their potential as new metal-
based anticancer drugs, the cytotoxic effects towards cancer cells
and normal cells of [ZnL2], [NiL2] and [CuL2] were evaluated with
cisplatin as the reference drug.
Experimental
Materials and Instrumentation
All solvents, chemicals and reagents were of analytical reagent
grade and used without further purification. Double-distilled water
was used throughout the experiments. Ciprofloxacin was pur-
chased from Xinyuanshun (Hubei, China). Ampicillin was purchased
from Traditional Chinese Medicine (China) Ltd. Benzoyl chloride,
*
Correspondence to: Weiqun Zhou, College of Chemistry, Chemical Engineering
and Materials Science, Soochow University, 199 Ren’ai Road, Suzhou, 215123,
People’s Republic of China. E-mail: wqzhou@suda.edu.cn
a College of Chemistry, Chemical Engineering and Materials Science, Soochow
University, 199 Ren’ai Road, Suzhou, 215123, People’s Republic of China
b School of Biology and Basic Medical Sciences, Soochow University, 199 Ren’ai
Road, Suzhou, 215123, People’s Republic of China
Appl. Organometal. Chem. 2015, 29, 157–164
Copyright © 2015 John Wiley & Sons, Ltd.

J. Xie et al.
KSCN, acetone, piperidine, dimethylsulfoxide (DMSO), Ni(NO3)2, Cu
(NO3)2 and Zn(NO3)2 were purchased from TCI (Shanghai) Develop-
ment Co. Ltd (China). Melting points were determined with a Kofler
melting point apparatus (uncorrected). IR spectra were obtained
using KBr discs with a Nicolet 170SX FT-IR spectrophotometer.
NMR spectra were recorded in deuterated chloroform (CDCl3)
solution at room temperature with an INOVA 400. Elemental analy-
sis (C, H, N and S) was performed using a Yannaco CHNSO Corder
MT-3 analyzer. Absorption spectra were recorded with a CARY50
UV–visible spectrophotometer.
piperidyl), 1584.4 (C–H, Ph), 1515.7 (C–H, Ph), 1481.0 (C–H, Ph),
1420.2 (C–H, piperidyl), 1244.0 (C–O), 1128.7 (C–N), 1025.3 (C–S),
780.9 (C–H, Ph), 715.6 (C–H, Ph). 1H NMR (CDCl3, 400 MHz, δ,
ppm): 8.13 (d, J = 7.6Hz, 4H, Ph, H2), 7.47 (t, J = 7.6 Hz, 2H, Ph, H4),
7.38 (dd, J1 = 7.6 Hz, J2 = 7.6 Hz, 4H, Ph, H3), 4.11 (dd, J1 = 5.6 Hz,
J2 = 4.8Hz, 8H, H1′), 1.65–1.69 (m, 12H, H2′ and H3′). 13C NMR (CDCl3,
100 MHz, δ, ppm): 207.12 (HN–CS), 171.21 (HN–CO), 128.94 (C1),
128.13 (C2), 126.25 (C4), 126.10 (C3), 49.13 (C1′), 25.82 (C2′), 24.31
(C3′). The labels of atoms are shown in Scheme 1.
X-ray diffraction experiments were carried out at room tempera-
ture with a Rigaku Mercury CCD diffractometer using graphite
monochromated Mo Kα radiation (λ = 0.71070/0.71073 Å). Crystals
suitable for X-ray diffraction determination were prepared using
the solvent evaporation method in dimethylformamide. Single
crystals were mounted with grease at the top of a glass fiber. Cell
parameters were refined on all observed reflections using the
program CrystalClear (Rigaku and MSC, version 1.3, 2001).[17] The
collected data were reduced using the CrystalClear program and
an absorption correction (multiscan) was applied. The structures
were solved by direct methods and refined by full-matrix least-
squares methods on F2 using the SHELXTL software package.[18,19]
The non-hydrogen atoms were refined anisotropically and the
hydrogen atoms were added in their idealized positions. The crys-
tallographic information files have been deposited in the
Cambridge Crystallographic Data Centre as 857864 ([NiL2]),
857865([CuL2]) and 916441 ([ZnL2]).
[CuL2]. Dark green; m.p. 167–168 °C. Anal. Calcd for
C26H30CuN4O2S2 (%): C, 55.94; H, 5.42; N, 10.04; S, 11.49. Found
(%): C, 56.00; H, 5.28; N, 10.14; S, 11.48. FT-IR (cmꢀ1): 2916.3 (C–H,
piperidyl), 2838.5 (C–H, piperidyl), 1579.1 (C–H, Ph), 1487.3 (C–H,
Ph), 1409.6 (C–H, piperidyl), 1239.3 (C–O), 1127.2 (C–N), 1006.5
(C–S), 798.6 (C–H, Ph), 710.4 (C–H, Ph), 678.8 (C–H, Ph).
[ZnL2]. Colorless; m.p. 128–129 °C. Anal. Calcd for C26H30N4O2S2Zn
(%): C, 55.76; H, 5.40; N, 10.00; S, 11.45. Found (%): C, 55.75; H, 5.31;
N, 10.06; S, 11.40. FT-IR (cmꢀ1): 2942.7 (C–H, piperidyl), 2852.5 (C–H,
piperidyl), 1591.5 (C–H, Ph), 1501.3 (C–H, Ph), 1423.6 (C–H,
piperidyl), 1243.8 (C–O), 1118.6 (C–N), 1012.9 (C–S), 849.8 (C–H,
Ph), 792.4 (C–H, Ph), 706.2 (C–H, Ph), 650.6 (C–H, Ph). 1H NMR (CDCl3,
400 MHz, δ, ppm): 8.19 (d, J= 7.2Hz, 4H, Ph, H2), 7.48 (t, J = 7.2 Hz,
2H, Ph, H4), 7.39 (dd, J1 = 7.2Hz, J2 = 7.2Hz, 4H, Ph, H3), 4.16 (dd,
J1 = 15.6 Hz, J2 = 4.8Hz, 8H, H1′), 1.75–1.80 (m, 12H, H2′ and H3′). 13
C
NMR (CDCl3, 100 MHz, δ, ppm): 206.97 (HN–CS), 170.76 (HN–CO),
129.42 (C1), 128.63 (C2), 126.97 (C4), 126.64 (C3), 48.93 (C1′), 26.09
(C2′), 24.54 (C3′).
Synthesis and Characterization
The ligand and the complexes [ML2] were prepared as detailed
previously.[14,16,20] The synthetic route is shown in Scheme 1.
Benzoyl isothiocyanate (2) was obtained by the condensation of
benzoyl chloride (1) with KSCN in acetone solution. Afterwards,
the fresh solution was added dropwise to a stoichiometric amount
of piperidine (3) with stirring and refluxing for 1–2 h. The solids (4)
were filtered, washed with water and dried to yield the ligand. The
complexes were obtained by the reaction of metal ions with the
ligand (molar ratio of M2+:L = 1:2) in ethanol in the presence of
triethylamine.
Minimum Inhibitory Concentration (MIC) Assay
Based on the CLSI broth microdilution method,[21] the determina-
tion of MICs via microdilution assay was performed in sterilized
96-well polypropylene microtiter plates (Sigma-Aldrich) in a final
volume of 200μl. Bacteria were grown overnight in nutrient broth.
Mueller–Hinton (MH) broth (100 μl) containing fungi and bacteria
(5× 105 CFU mlꢀ1) was added to 100μl of the culture medium
containing the test compound (1 to 64μmol lꢀ1 in serial twofold
dilutions). The plates were incubated at 37°C for 20 h in an incuba-
tor. About 50 μl of 0.2% triphenyltetrazolium chloride (TTC),
a colorimetric indicator, was added to each well of microtiter
plates and incubated at 35 °C for 1.5 h. The TTC-based MIC was
determined as the lowest concentration of oxacillin that
showed no red color change indicating complete growth
inhibition.
[NiL2]. Purple; m.p. 204–206 °C. Anal. Calcd for C26H30N4NiO2S2 (%):
C, 56.43; H, 5.46; N, 10.12; S, 11.59. Found (%): C, 57.50; H, 5.63; N,
10.51; S, 12.1. FT-IR (cmꢀ1): 2939.0 (C–H, piperidyl), 2853.7 (C–H,
Bacterial Growth Inhibition
The synthetic complexes and antibiotics were added to strain
cultures to final concentrations of 2.0, 4.0, 8.0, 16.0 and
32.0 μmol lꢀ1 [22]
The strains were cultivated in an automated
.
Bioscreen C system (Lab Systems, Helsinki, Finland) using an MH
broth culture medium. The working volume in the wells of the
Bioscreen plate was 300μl, which comprised 150 μl of the MH broth
and 150μl of the test solution. The temperature was controlled at
35°C, and the optical density of the cell suspensions was measured
automatically at 600 nm in regular intervals of 10 min for 20 h.
Before each measurement, the culture wells were automatically
shaken for 60 s. Statistical data for each experiment were obtained
from at least two independent assays performed in duplicate.
Scheme 1. Synthetic route to ligand HL (5) and complexes [ML2] (6).
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Appl. Organometal. Chem. 2015, 29, 157–164

Crystal structures and bioactivities of Zn, Ni and Cu complexes
Evaluation of Cytotoxic Activity
at 4.17ppm in HL shifts downfield to 4.11 ppm in [NiL2] and
4.16 ppm in [ZnL2] due to the negative induction effect of N–C¼S
group. The 13C NMR spectra of both complexes show appreciable
differences with respect to that of the free ligand, especially in
the carbon atoms of the carbonyl and the thionyl groups, which
can be attributed to the resonance induced by the elimination of
proton in N–H. For example, the characteristic signal of the
thiocarbonyl carbon centered at 179.31 ppm in HL is shifted down-
field to 207.12 ppm for [NiL2] and 206.97 ppm for [ZnL2]. Similarly,
the (C¼S) resonance shifts from 138.52 ppm in HL to 171.21 ppm
in [NiL2] and 170.76ppm in [ZnL2]. These data corroborate the coor-
dination of the ligands to the metal through these groups as well as
the weakening of the double bonds.
Cell lines were placed in Dulbecco’s modified Eagle’s medium
which contained 10% heat-inactivated fetal bovine serum, penicil-
lin (100 μg mlꢀ1) and streptomycin (100 μg mlꢀ1), then incubated
in an incubator at 37 °C under a humidified 5% CO2 atmosphere.
Drug effects on exponentially growing cancer cells were deter-
mined by a CCK-8 (Cell Counting Kit-8, Dojindo Molecular Technol-
ogies Inc.) assay. Cancer cells (hepatocellular carcinoma (SK-Hep-1)
and breast carcinoma (MCF-7)) and normal cells (hepatocytes (QSG-
7701)) were seeded at a density of 3000 cells/100 μl per well in 96-
well plates and incubated for 24 h. QSG-7701 was used as compar-
ison with the cancer cells. The samples (cisplatin (DDP), HL, [NiL2],
[CuL2] and [ZnL2]) were dissolved in 10% DMSO stock solutions
and serially diluted with DMSO to concentrations of 10 and
50 μmol lꢀ1. The samples were added to the corresponding wells.
The monolayer cells were incubated for 48 h. Then, 10 μl CCK-8 so-
lutions in culture medium were added to each well. The plates were
incubated for an additional 2 h, where DMSO and DDP were taken
as the blank and the positive control group, respectively. Each sam-
ple had five wells and each experiment was repeated three times.
UV–visible absorbance was measured at 570 nm using a microplate
reader (MK3 Thermo Scientific Microplate Reader, Thermo Fisher
Scientific). During these tests, no signs of decomposition were no-
ticed. The cytotoxicity was assessed as the percentage of inhibition
of cancer cell growth at 570 nm of the variable tested sample using
the following formula: inhibition (%) = [(A0 ꢀ At)/A0] × 100 (At is the
absorbance of the tested samples at 570 nm; A0 is the absorbance
in the absence of the tested samples at 570 nm). The IC50 value
(μmol lꢀ1), which represented the concentration of the extracts that
gave 50% growth inhibition relative to the control, was calculated
for each extract from the dose–response curves.[23,24] All experi-
ments were repeated three times and least significant difference
analysis was used to indicate a statistically significant difference.
FT-IR Spectral Analysis
The FT-IR spectra of the ligand and the complexes are shown in Fig.
S1. In the FT-IR spectra of the complexes, the stretching vibrations
of N–H (3161.7 cmꢀ1) of the ligands are absent, which indicates that
the ligands participate in the coordination reaction as the enamine
anions. The peaks at 1651.4 and 1018.9 cmꢀ1 in HL shift while the
peaks appear at about 1240 and 900 cmꢀ1 in the complexes, which
indicates that the formal C¼O and C¼S double bonds of HL are
weakened to partial double bonds upon coordination. Moreover,
coordination enhances the resonance delocalization of nitrogen
atom lone pair electrons, introducing more double bond character
for C–N–C. The vibrational bands at about 1128 cmꢀ1 are attributed
to the C¼N partial bonds in all complexes. Therefore, there is elec-
tron delocalization in the S1–C8–N1–C1–O1 fragment connecting
by resonance delocalized bonds.
X-ray Structures
A summary of selected bond lengths and bond angles for the
complexes and HL is given in Table 1; data for HL are derived from
Al-abbasi et al.[31]
DFT Calculations
Because Lꢀ participates in the coordination reaction as an
enamine anion, a reorganization of the donor atoms must be
observed in the three complexes. As listed in Table 1, the C–S bond
lengths for [NiL2], [CuL2] and [ZnL2] are longer than that for HL, and
the same trend is observed for C–O. By contrast, the amide C–N
bond lengths in [NiL2], [CuL2] and [ZnL2] are shorter than that
in HL. This indicates that, as a result of coordination, the C–O
and C–S bonds weaken but the C–N bond strengthens.
The initial molecular geometries of the complexes were obtained
from the crystal structures and were optimized using DFT calcula-
tions. Beck’s three-parameter hybrid (B3LYP)[25–27] method was
employed to optimize the ground structures of the complexes[28]
at the level of 6-31G (d). The Guassian-09[29] program package
was used for all calculations. The harmonic vibration calculations
showed positive harmonic vibrations and confirmed the stability
of the structures.
As shown in Fig. 1, there are discrete cis- and trans-[NiL2] in the
crystal structure of [NiL2]. In other words, the asymmetric cell is
composed of two independent molecules. For the cis-[NiL2]
molecule, the Ni atom lies on a center of inversion, while for
the trans-[NiL2] the molecule lies on a general position. The con-
formational isomerization that arises as a result of coordination
(see above) leads to planar C–N–C–S residues (torsion angles:
C1–N1–C8–S1= ꢀ13.5(5)°; C14–N3–C21–S2= ꢀ9.6(4)°; C27–N5–
C34–S3= ꢀ1.2(3)°) compared to ca 116.6(1)° in HL.[31] The
cis-[NiL2] molecule is similar to [Ni(N-(morpholinothiocarbonyl)
benzamide)2],[32] cis-bis(1,1-diethyl-3-benzoylthiourea)nickel(II)[33]
and cis-bis(1,1-diethyl-3-2-chlorobenzoylthiourea-O,S)nickel(II).[34]
The slightly distorted planar square geometry results from the
strained six-membered Ni–S1–C8–N1–C1–O1 chelate ring. Struc-
tures resembling the trans-Ni(II) complex have not been reported.
However, trans-[NiL2] shows some similarities to copper complexes
such as trans-bis[N,N-diethyl-N-(p-nitrobenzoyl)thioureato]copper
(II),[35] trans-[Cu(SON(CNiPr2)2)2][36] and trans-[Cu(3-OHFT)2],[37]
The DFT calculated results were mainly used for the analysis of
structure–activity relationships. The reliability can be validated by
the agreement between the crystallographic parameters (such as
bond lengths, angles and torsion angles) and the calculated
geometries.
Results and Discussion
NMR Spectral Analysis
1
The H NMR spectral data of HL[30] (the NMR data of HL can be
found in the supporting information) and its nickel(II) and zinc(II)
complexes are recorded in the Experimental section. The signal at
8.52 ppm corresponding to the proton of the NH group in HL is ab-
sent for the metal complexes, where the ligands participate in the
coordination reaction as the enamine anions with electron delocal-
ization in the S1–C8–N1–C1–O1 fragment. The N–CH2 proton signal
Appl. Organometal. Chem. 2015, 29, 157–164
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J. Xie et al.
Table 1. Key geometric parameters of the determined crystals and optimized structures
HLa
cis-[NiL2] trans-[NiL2]
X-ray Calc.
[CuL2]
[ZnL2]
X-ray
Calc.
X-ray
Calc.
X-ray
Calc.
X-ray
Calc.
Bonds (Å)
M–O1
1.853(2)
1.861(2)
2.133(1)
2.140(1)
1.728(4)
1.719(4)
1.264(4)
1.257(4)
1.320(4)
1.325(4)
1.336(4)
1.342(4)
1.339(4)
1.340(4)
1.844
1.835(2)
2.172(1)
1.716(4)
1.268(4)
1.316(4)
1.341(4)
1.344(4)
1.821
1.905(3)
2.244(8)
1.732(4)
1.268(5)
1.313(5)
1.349(5)
1.339(5)
1.895
1.949(2)
1.968(2)
2.291(1)
2.286(1)
1.742(3)
1.743(3)
1.277(3)
1.271(3)
1.305(4)
1.310(3)
1.341(4)
1.344(4)
1.329(4)
1.326(4)
1.948
M–O2
1.844
2.184
2.184
1.757
1.757
1.272
1.272
1.333
1.333
1.335
1.335
1.357
1.357
1.949
2.318
2.318
1.763
1.763
1.276
1.276
1.328
1.328
1.334
1.334
1.356
1.356
M–S1
2.206
1.745
1.277
1.328
1.340
1.359
2.295
1.749
1.277
1.329
1.341
1.360
M–S2
S1–C8
1.673(2)
1.225(2)
1.369(2)
1.419(2)
1.328(2)
1.683
1.222
1.396
1.419
1.345
S2–C21
O1–C1
O2–C14
N1–C1
N3–C14
N1–C8
N3–C21
N2–C8
N4–C21
Angles (°)
O1–M–O2
O1–M–S1
O1–M–S2
O2–M–S1
O2–M–S2
S1–M–S2
85.4(1)
95.6(8)
168.9(1)
169.9(9)
94.9(8)
86.0(4)
85.0
108.4(9)
98.7(7)
108.6
93.3
176.1
176.1
93.3
94.4(8)
85.6(8)
94.3
93.8(1)
86.2(1)
95.4
117.0
99.3
117.8(7)
118.1(7)
97.7(6)
93.8
117.0
99.3
85.9
85.0
118.1(4)
116.4
Dihedral angles (°)
S1–C8–N1–C1
ꢀ116.6(1)
126.5
ꢀ9.6(4)
ꢀ13.3
ꢀ1.2(3)
ꢀ13.2
ꢀ12.6(8)
ꢀ19.0
45.0(3)
ꢀ30.2
aData are taken from Al-abbasi et al.[31]
structure of cis complexes with copper(II),[33,38] but similar to trans-
bis[N,N-diethyl-N-(p-nitrobenzoyl)thioureato]copper(II),[39] trans-[Cu
(SON(CNiPr2)2)2][34] and trans-[Cu(3-hydroxyflavothione)2].[35] The
Cu–O and Cu–S bond lengths are consistent with those in trans-
bis[N,N-diethyl-N-(p-nitrobenzoyl)thioureato]copper(II).[33] The Cu–S
and Cu–O bond lengths are longer than Ni–S and Ni–O in [NiL2] due
to the Jahn–Teller effect[40] and the greater electronegativity of Cu.
In contrast to the molecular structures of [NiL2] and [CuL2], [ZnL2]
presents a tetrahedral structure (Fig. 2(b)). The tetrahedral angles
around Zn are in the range 97.7(6)–118.1(4)°. The narrower angles
(<109.5°) for S1–Zn1–O1 and S2–Zn1–O2 may be attributed to
geometric constraints of the six-membered chelate rings. The same
trends in the C–S, C–O and C–N bond lengths observed for the Ni
and Cu analogs pertain, a process that may be compared to
imine–enamine tautomerism.[41,42]
Figure 1. Molecular structures of two isomers of [NiL2] (40% probability
displacement ellipsoids). The cis-[NiL2] molecule is located on a center of
inversion while the trans-[NiL2] molecule has no crystallographic symmetry.
Antimicrobial Activities
Table 2 summarizes the data for the antimicrobial study of the com-
mercial products fluconazole and ampicillin, as well as of HL and
the three metal complexes. The antimicrobial activities are evalu-
ated by measuring the average values of MIC. The studied com-
plexes exhibit varying degrees of inhibitory effects on the growth
of the tested fungal and bacterial strains (MIC for the com-
plexes = 2–32μmol lꢀ1). In antifungal assay, the MIC values of
[ZnL2] are 8 μmol lꢀ1 for all the studied fungi, which is the same
as that of fluconazole. The MIC values of HL, [NiL2] and [CuL2] are
higher than that of [ZnL2]. In the antibacterial investigation, [ZnL2]
which also adopt a planar square geometry. The Ni–O and Ni–S
bond lengths in trans-[NiL2] are shorter and longer, respectively,
than the equivalent bonds in cis-[NiL2], reflecting the different trans
effect of the donor atoms. Other bond lengths and angles are in
good agreement.
The molecular structure of [CuL2] is shown in Fig. 2(a). [CuL2], with
crystallographic twofold symmetry, adopts a square planar geome-
try with a trans configuration, which is different from the reported
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Crystal structures and bioactivities of Zn, Ni and Cu complexes
three complexes. The energies of HOMO decrease in the following
order: [NiL2] > HL> [CuL2] > [ZnL2], which is the same order as the
MIC values. These results indicate that the lower the EHOMO value,
the smaller the MIC value. For example, the MIC values of [NiL2],
HL, [CuL2] and [ZnL2] against the fungus Botrytis cinerea are 32.0,
16.0, 16.0 and 8.0μmol lꢀ1, respectively, and the corresponding
values against the bacterium Bacillus subtilis are 16.0, 8.0, 4.0 and
2.0 μmol lꢀ1, respectively. While the EHOMO values of [NiL2], HL,
[CuL2] and [ZnL2] are ꢀ0.1892, ꢀ0.2065, ꢀ0.2097 and ꢀ0.2167 a.
u., respectively. It seems that a lower HOMO energy is helpful in
the interaction with the active part of the microorganism. Further
analyses of structure–activity relationships illuminate the impor-
tance of the charge population in the complexes. Negative charges
on oxygen and sulfur can offer electrons during interactions with a
microorganism.
Figure 2. Molecular structures of (a) [CuL2] (with crystallographic twofold
symmetry) and (b) [ZnL2] (40% probability displacement ellipsoids).
shows the lowest MIC values against all the studied bacteria. In
particular, the MIC value of [ZnL2] is 2.0μmol lꢀ1 against Bacillus
subtilis, which is even smaller than that of ampicillin. In general,
the antimicrobial activity of [ZnL2] is the best among the test
compounds. More importantly, [ZnL2] shows similar antimicrobial
activity, both antibacterial and antifungal, to that of the
commercial drugs.
The negative charges on O of the carbonyls in the complexes are
greater than that of HL due to coordination. The order of the nega-
tive charge on oxygen is Q[ZnL2] > Q[CuL2] > Qtrans-[NiL2] > Qcis-[NiL2]
and the order of the negative charge on sulfur is Q[ZnL2]
[CuL2] > Qcis-[NiL2] > Qtrans-[NiL2]. For the studied complexes, the
,
>
Q
increase of the negative charges on O of the carbonyl enhances
the antimicrobial activity. The negative charge on oxygen is
inversely proportional to the values of MIC. As a result, the enhance-
ment of the polarity shows the order [ZnL2] > [CuL2] > [NiL2], which
is inversely proportional to the values of MIC.[28]
Structure–Activity Relationship
On the other hand, the thiocarbonyl could be considered as
another active site. The enhancement of the polarity of the
thiocarbonyl enhances antimicrobial activity. However, in all
complexes, the negative charges on S atoms of thiocarbonyls de-
crease compared with that of HL, which results in the reduction
of the polarity of thiocarbonyls. The decrease of the negative
charges on S atoms in [NiL2] is the greatest. The enhancement of
the polarity of the carbonyls is partially offset by the large decrease
of the polarity of the thiocarbonyls, which results in the reduction of
the antimicrobial activity. For [CuL2] and [ZnL2], the large enhance-
ment of polarity of the carbonyl exceeds the small reduction of
polarity of the C–S bonds, and causes an enhancement of
antimicrobial activity.
Notably, as mentioned above, due to the decrease of conjuga-
tion between the two ligands, the tetrahedral geometry of [ZnL2]
makes the C–O and C–S bonds in [ZnL2] longer than in [NiL2] and
[CuL2]. In addition, the tetrahedral geometry of [ZnL2] also makes
it easier for the carbonyl and the thiocarbonyl to contact with a
microorganism and inhibit the microorganism quickly, due to the
reduction of steric hindrance from the ligand on the opposite
side.[43,44]
The trends in antimicrobial activity are correlated with the elonga-
tion of the C–O and C–S bond lengths in the complexes. Notably,
the antimicrobial activity is evaluated in 10% DMSO solution to pro-
mote the solubility of the metal complexes in water. Considering the
low concentration of DMSO, the influence of solvent is neglected in
the following discussion of the structure–activity relationship.
Some of the geometric parameters of the determined crystals
and HL are listed in Table 1. The order of the C–O bond length is
[ZnL2] > [CuL2] = trans-[NiL2] > cis-[NiL2], and the order of the C–S
bond length is [ZnL2] > [CuL2] > cis-[NiL2] > trans-[NiL2]. The
elongation of the bond increases both the polarity and reactive
activation of C–O and C–S bonds.
On the other hand, the differences in the structures may result
in changes in the charge population of the complexes and
accordingly induce different antimicrobial activities. The geometry
optimization calculations of the three complexes [NiL2], [CuL2]
and [ZnL2] together with HL were performed at the level of 6-31G
(d) using the DFT (B3LYP) method; key derived data are given in
Table 3. Table 4 compiles the energies of the frontier molecular
orbitals (EHOMO and ELUMO), the transition energies of the frontier
molecular orbitals (ΔEH-L) and the charges on O, S atoms in the
Table 2. MIC values of ligand and complexes (concentration of samples: 250 μmol lꢀ1; SD: standard deviation)
Compound
MIC SD (μmol lꢀ1
)
Fungi
Bacteria
Botrytis
cinerea
Trichoderma Myrothecium Verticillium Shewanella
Pseudomonas
aeruginosa
Escherichia
coli
Staphylococcus
aureus
Bacillus
subtilis
spp.
spp.
Fluconazole 8.0 0.1
Ampicillin
8.0 0.1
8.0 0.1
8.0 0.1
8.0 0.1
4.0 0.1
8.0 0.1
16.0 0.1
8.0 0.1
4.0 0.1
4.0 0.1
8.0 0.1
16.0 0.1
4.0 0.1
4.0 0.1
8.0 0.1
16.0 0.1
32.0 0.1
16.0 0.1
8.0 0.1
4.0 0.1
8.0 0.1
16.0 0.1
4.0 0.1
2.0 0.1
HL
16.0 0.1
32.0 0.1
16.0 0.1
8.0 0.1
16.0 0.1
32.0 0.1
8.0 0.1
8.0 0.1
16.0 0.1
32.0 0.1
16.0 0.1
8.0 0.1
16.0 0.1 16.0 0.1
32.0 0.1 32.0 0.1
8.0 0.1 16.0 0.1
[NiL2]
[CuL2]
[ZnL2]
8.0 0.1
8.0 0.1
Appl. Organometal. Chem. 2015, 29, 157–164
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

J. Xie et al.
Table 3. Summary of X-ray crystallographic data and refinement details
[NiL2]
[CuL2]
[ZnL2]
Empirical formula
C26H30N4NiO2S2
553.37
Triclinic
P-1
C26H30CuN4O2S2
558.20
Monoclinic
C2/c
C26H30N4O2S2Zn
560.03
Monoclinic
P21/c
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
9.2705(6)
9.2566(5)
25.495(2)
74.483(8)
75.898(8)
68.581(7)
3
21.342(9)
8.601(3)
15.206(6)
90
14.575(3)
14.676(3)
12.385(3)
90
α (°)
β (°)
γ (°)
113.798(9)
90
94.51(3)
90
Z
4
4
D
calc (g cmꢀ3
)
1.424
1.452
1.409
μ(Mo Kα) (mmꢀ1
θ range (°)
)
0.944
1.050
1.119
3.1–25.4
18168
3.2–25.4
11464
2332
3.1–25.0
24711
4641
Reflections collected
Independent reflections
Rint
6980
0.040
0.068
0.034
Data with I > 2σ(I)
Parameters
GOF on F2
5204
1973
4095
476
161
316
1.09
1.14
1.05
R1 (I > 2σ(I))
0.055
0.060
0.045
wR2 (I > 2σ(I))
0.103
0.144
0.088
R1 (all data)
0.082
0.075
0.054
wR2 (all data)
Largest difference peaks (e Aꢀ3
0.115
0.152
0.09q
)
0.45 and ꢀ0.31
0.70 and ꢀ0.49
0.32 and ꢀ0.23
studied complexes, [CuL2] has the largest values of I at concentra-
tions of 10 and 50 μmol lꢀ1, which are close to those of DDP. For
example, for SK-Hep-1 cells, at a concentration of 10 μmol lꢀ1, the
inhibition ratio of [CuL2] is 56.1%, which is a little less than that of
DDP (62.8%). By contrast, I for [ZnL2] is less than that for [CuL2],
but greater than that for HL and [NiL2]. Similar conclusions apply
at concentrations of 50 μmol lꢀ1, as shown in Fig. 3.
The value of I for [CuL2] against MCF-7 cells is relatively low. At
a concentration of 10 μmol lꢀ1, the value of I (38.9%) of [CuL2] is
about half that of DDP (79%). [ZnL2] shows a relatively low I, but
HL and [NiL2] show almost no inhibition ability. At a concentra-
tion of 50 μmol lꢀ1, I of [CuL2] is 40%. So, the cytotoxic effects
of [CuL2] are especially strong for the human breast cancer cells,
and are close to that exhibited by DDP. Additionally, as evident
from Table 5, HL and its metal complexes display different cyto-
toxic effects with different IC50 values. [CuL2] shows the lowest
IC50 values that are close to those of DDP, which has the greatest
cytotoxicity to SK-Hep-1 and MCF-7 cells. While [ZnL2] shows
higher IC50 values, which represents low cytotoxicity. The
comparison results of IC50 values are in accordance with the
cytotoxic inhibition ratio results.
The cytotoxic tests on QSG-7701 demonstrate that [CuL2] has
reduced cytotoxicity to normal cells compared to DDP, while HL,
[NiL2] and [ZnL2] show little cytotoxicity to the normal cells.
The cytotoxicity of copper(II) complexes may be caused by
their ability to bind and cleave DNA which leads to the arrest of
the cell cycle and apoptosis, or the generation of reactive oxygen
species, followed by cell death.[45,46] In light of the fact that copper
is a bio-essential element responsible for numerous bioactivities in
living organism, [CuL2] should be an excellent antitumor agent
for applications.
Table 4. Some calculated HOMO and LUMO energies, and atom
charges of HL and three complexes
HLa
cis-[NiL2]
trans-[NiL2]
[CuL2]
[ZnL2]
Energy (a.u.)
EHOMO
ELUMO
ΔEH-L
ꢀ0.2065
ꢀ0.0535
0.1530
ꢀ0.1932
ꢀ0.0529
0.1403
ꢀ0.1892
ꢀ0.0530
0.1361
ꢀ0.2097
ꢀ0.0780
0.1317
ꢀ0.2167
ꢀ0.0557
0.1610
Charge (e)
O1
C1
N1
C8
S1
ꢀ0.4946
0.5413
ꢀ0.5673
0.5683
ꢀ0.5887
0.5671
ꢀ0.6025
0.5692
ꢀ0.6190
0.5642
ꢀ0.6314
0.2951
ꢀ0.5505
0.3284
ꢀ0.5578
0.3295
ꢀ0.5425
0.3291
ꢀ0.5328
0.3211
ꢀ0.2844
ꢀ0.1263
ꢀ0.1042
ꢀ0.1961
ꢀ0.2356
aData are taken from Al-abbasi et al.[31]
Overall, the increase of polarity of the carbonyl and the
thiocarbonyl is caused by the increase of charge on the O atoms
and the S atoms as well as the elongation of the C–O and C–S
bonds. The increase of polarity and bond length of the carbonyl
and the thiocarbonyl will increase reactive activity towards microor-
ganisms and result in a strong antimicrobial activity.
Cytotoxic Activity
The cytotoxic inhibition ratio (I) of SK-Hep-1, MCF-7 and QSG-7701
was measured and the results are shown in Fig. 3. Among the
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Copyright © 2015 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2015, 29, 157–164

Crystal structures and bioactivities of Zn, Ni and Cu complexes
Conclusions
We successfully synthesized a new complex of zinc(II) with
N-(piperidylthiocarbonyl)benzamide. Its crystal structure was
determined using X-ray diffraction and it was compared with the
previously reported [NiL2] and [CuL2]. The results show that [ZnL2]
has a tetrahedral structure, while [NiL2] and [CuL2] have square
planar structures. The antimicrobial experiments reveal that [ZnL2]
has the greatest antifungal and antibacterial activities, which are
close to those of commercial products. A structure–activity relation-
ship explains the increase of the antimicrobial activities of [ZnL2]
and [CuL2] in terms of the increase of polarity of carbonyl and
thiocarbonyl groups. Furthermore, [ZnL2] is unsuitable as an
antitumor agent because of its low cytotoxicity to cancer cells,
while [CuL2] is found to have effective cytotoxic activity.
Acknowledgments
This work was supported by the National Natural Science Founda-
tion of China (no. 51079094) and the Natural Science Foundation
of Jiangsu Province (no. BK2010215).
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