Synthesis, characterization and biological studies of S-4-methylbenzyl-β-N-(2-furylmethylene)dithiocarbazate (S4MFuH) its Zn2+, Cu2+, Cd2+ and Ni2+ complexes

Article abstract of DOI:10.1016/j.ica.2015.08.029

S-4-methylbenzyl-β-N-(2-furylmethylene)dithiocarbazate (S4MFuH, 1) derived from the condensation reaction of furaldehyde (Fu) with S-4-methylbenzyldithiocarbazate (S4MBDTC) has been complexed with transition metal acetates to give Zn(S4MFu)₂ (2

Full text of DOI:10.1016/j.ica.2015.08.029

Accepted Manuscript
Synthesis, characterization and biological studies of S-4-Methylbenzyl-β-N-(2-
furylmethylene)dithiocarbazate (S4MFuH) its Zn2+, Cu2+, Cd2+ and Ni2+ com-
plexes
Enis Nadia Md Yusof, Thahira B.S.A. Ravoof, Junita Jamsari, Edward R.T.
Tiekink, Abhimanyu Veerakumarasivam, K.A. Crouse, M.Ibrahim M. Tahir,
Haslina Ahmad
PII:
S0020-1693(15)00435-1
http://dx.doi.org/10.1016/j.ica.2015.08.029
ICA 16672
DOI:
Reference:
Inorganica Chimica Acta
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 April 2015
3 June 2015
24 August 2015
Please cite this article as: E.N. Md Yusof, T.B.S. Ravoof, J. Jamsari, E.R.T. Tiekink, A. Veerakumarasivam, K.A.
Crouse, M.Ibrahim M. Tahir, H. Ahmad, Synthesis, characterization and biological studies of S-4-Methylbenzyl-
β-N-(2-furylmethylene)dithiocarbazate (S4MFuH) its Zn2+, Cu2+, Cd2+ and Ni2+ complexes, Inorganica Chimica
Acta (2015), doi: http://dx.doi.org/10.1016/j.ica.2015.08.029
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Synthesis, characterization and biological studies of S-4-Methylbenzyl-β-N-
(2-furylmethylene)dithiocarbazate (S4MFuH) its Zn2+, Cu2+, Cd2+ and Ni2+
complexes
Enis Nadia Md Yusofa, Thahira B.S.A. Ravoofa*, Junita Jamsaria, Edward R. T.
Tiekinkb, Abhimanyu Veerakumarasivama, K. A Crousea,c, M. Ibrahim M. Tahira and
Haslina Ahmada
aDepartment of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor
bDepartment of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia
cDepartment of Chemistry, Cape Breton University, Sydney, Nova Scotia, Canada B1P 6L2
*corresponding author: thahira@upm.edu.my
S-4-methylbenzyl-β-N-(2-furylmethylene)dithiocarbazate (S4MFuH, 1) derived from the
condensation reaction of furaldehyde (Fu) with S-4-methylbenzyldithiocarbazate
(S4MBDTC) has been complexed with transition metal acetates to give Zn(S4MFu)2 (2),
Cd(S4MFu)2 (3), Cu(S4MFu)2 (4) and Ni(S4MFu)2 (5). It is evident from the shift in
ν(C=N) and ν(N-N) in the IR spectra of the complexes that deprotonated 1 acts as a bidentate
ligand coordinating through the azomethine nitrogen and thiolato sulphur atoms. This was
confirmed by single crystal X-ray diffractometry. The U-shaped dithiocarbazate 1 exists in
the E configuration with the thione bond anti to the azo bond. A change in conformation is
noted in the transition metal complexes resulting from deprotonation and N-S-chelation. 2
and 3 display a distorted tetrahedral geometry with the major cause of the distortion being
two close intramolecular M…O interactions. Binding interaction studies with calf thymus
DNA demonstrated that 4 also had the strongest DNA binding affinity (Kb = 2.85 x 104 M-1)
1

among all compounds prepared in this work. The Cu(II) complex, 4, was also moderately
active against estrogen receptor-positive breast cancer cells, MCF-7 (IC50 = 3.02 µM) while
the remainder were inactive against MCF-7 and all showed no activity towards receptor
negative breast cancer cells, MDA-MB-231.
Keywords: S-4-methylbenzyldithiocarbazate; furaldehyde; transition metal complexes; NS
bidentate Schiff base; Single crystal X-ray diffraction; cytotoxic activity.
1. Introduction
Since the early investigations on substituted derivatives of dithiocarbazate, NH2NHCS2, and
their metal complexes in the 1970's [1], these compounds have continued to be of interest to
researchers because of the wide variation in their structures and properties, and especially
because of their bioactivity [1-3]. S-benzyl-β-N-(5-methyl-2-furylmethylene)dithiocarbazate,
which differs from the ligand in this study only in the para substituent in the benzyl ring, was
reported to be inactive against human T-lymphoblastic leukemia (CEM-SS), but chelation
with metal ions produced cytotoxic complexes which also showed inhibition of Aspergillus
ochraceous (398) [4].
Carcinostatic and fungitoxic activities in some related Schiff bases and metal complexes have
been reported previously [5]. The Schiff bases derived from 2-acetyl- and 2-benzoylpyridine
with S-methyl- and S-benzoyldithiocarbazate were [6] shown to be toxic towards A. solani,
F. equisetti and M. phaseolina. Cu(II), Ni(II), Zn(II) and Cd(II) octahedral complexes of
two 2-(6-methylpyridin-2-ylmethylene) hydrazine carbodithioate ligands were active against
estrogen receptor positive MCF-7 and estrogen receptor negative MDA-MB-231 breast
cancer cell lines. However, only the Cu(II) and Cd(II) complexes inhibited methicillin-
2

resistant staphylococcus (MRSA), Bacillus subtilis wild type (B29), Pseudomonas
aeruginosa (60690), Salmonella choleraesuis (S.C.), Candida albicans (C.A.), Asper-
gillusochraceous (398) and Saccaromycesceciricaee (20341) [7].
The activity of some compounds can be enhanced when they are complexed to metals.
Sigman and co-workers reported the first copper(II) complexes containing 1, 10-
phenantroline ligands that showed good DNA cleavage activity. They were developed as
therapeutic agents [8]. Binuclear copper(II) complexes have been found to have better DNA
interaction than mononuclear complexes [9]. It has been reported that interaction of DNA
with metal complexes may be the result of electrostatic interactions with the negatively
charged nucleic sugar-phosphate, or through grooves of the DNA double helix, or by
intercalation between the stacked base pairs of native DNA [10]. Howe-Grant et al., in an
early study of the intercalation of DNA, reported that square planar Pt(II) complexes of
aromatic ligands, 2,2',2''-terpyridine, o-phenanthroline and 2,2’-bipyridine intercalated into
DNA sequences producing strong interactions [11]. It was determined that increasing the
concentration of calf thymus DNA increased the intensity of the π→π* and LMCT absorption
bands, but did not affect d-d transitions in the metal complexes reflecting the stacking
interaction of the planar aromatic rings of the complexed ligand with double-helix strand of
DNA base pairs [12]. The significant hypochromism and red shift were due to the strong
π→π* stacking interaction between the aromatic chromophore of the ligand in the copper(II)
complexes and the base pair of DNA [13, 14].
As part of our ongoing study of dithiocarbazate compounds that exhibit specific biological
profiles, we report herein the synthesis, characterization, DNA binding affinity studies and
3

cytotoxicity of a Schiff base, S-4-methylbenzyl-β-N-(2-furylmethylene) dithiocarbazate
(S4MFuH, 1) and its copper, nickel, zinc and cadmium complexes.
2. Experimental
2.1. Materials and reagents
All chemicals and solvents were of analytical grade and were used as received
Chemicals: 4-methylbenzyl chloride (ACROS), potassium hydroxide (HmbG),
hydrazine hydrate (Fluka), carbon disulphide (BDH), furaldehyde (Fluka), nickel(II) acetate
tetrahydrate (Fluka), zinc(II) acetate dihydrate(Fluka), cadmium(II) acetate dihydrate (Fluka),
copper(II) acetate (Univar) and nitric acid (65%) (Fisher).
Solvents: acetonitrile (Baker), absolute ethanol (99.8%, Scharlau), ethanol (95%, J.
Kollin Chemical), methanol (Fisher Scientific) and dimethylsulphoxide (Scharlau).
2.2. Synthesis of S-4-methylbenzyldithiocarbazate (S4MBDTC)
Following a procedure adapted from Ravoof et al. [15], potassium hydroxide (11.4 g, 0.2
mol) was dissolved in ethanol (70 ml, 90%). To this solution, hydrazine hydrate (10 g, 0.2
mol) was added and the mixture was maintained at 0°C in an ice-salt bath. Carbon disulphide
(15.2 g, 0.2 mol) was added drop-wise with vigorous stirring (750 rpm) over a period of 1 h.
The two layers that formed were separated using a separating funnel. The light-brown lower
layer was dissolved in 40% ethanol (60 mL) below 5°C. The mixture was kept in an ice-bath
and 4-methylbenzyl chloride (26.5 mL, 0.2 mol) was added drop-wise with vigorous stirring.
The sticky white product, S4MBDTC, was filtered and left to dry overnight in a desiccator
over anhydrous silica gel. Yield: 77%. m.p. 90-92 °C (lit. 86-89 °C).
4

2.3. Synthesis of S-4-methylbenzyl-β-N-(2-furylmethylene) dithiocarbazate, S4MFuH (1)
S-4-Methylbenzyldithiocarbazate (S4MBDTC) (2.12 g, 0.01 mol) was dissolved in hot
acetonitrile (150 mL) and added to an equimolar amount of furaldehyde (0.96g) in ethanol
(20 mL). The mixture was maintained at 70oC while stirring for 30 min. The product was
recrystallised from CH3CN:EtOH (1:1).Yield, 85%.m.p. 165-166 °C.
S
S
O
HN
O
NH2
stirred and heated (70 °C), 1 h
H
N
S
O
N
S
Scheme 1. Synthesis of S4MFuH (1) [4]
2.4. Synthesis of M(S4MFu)2 where M = Zn(II), Cd(II), Cu(II) and Ni(II)
Acetate salts of Zn(II), Cd(II), Cu(II) and Ni(II) (1 mmol) were dissolved in hot 95% ethanol
(10 ml) and added to a solution of S4MFuH (0.5 mmol) in hot acetonitrile (80 ml) and heated
for 30 min. Yield for each complex: ~80%.
5

2.5. Instrumentation
IR spectra were recorded using PerkinElmer Spectrum 100 with Universal ATR Polarization
in the range of 4000-280 cm-1. C, H, N and S elemental analyses were carried out using a
LECO CHNS-932 instrument. Metal determinations were carried out using a Perkin-Elmer
Plasma 1000 Emission Spectrometer. Molar conductivities of 10-3 M solutions of the metal
complexes in DMSO were measured at 27 °C using a Jenway 4310 conductivity meter fitted
with a dip-type cell with a platinised electrode. Electronic spectra were recorded on a
Shimadzu UV-1650 PC recording spectrophotometer (1000-200 nm). Magnetic
susceptibilities were measured with a Sherwood Scientific MSB-AUTO magnetic
susceptibility balance at 25 °C. Melting points were determined using an Electrothermal
digital melting point apparatus.
2.5.1. X-ray crystallography
X-ray diffraction measurements for 1–3 were performed at 100 K on an Oxford
Diffraction Gemini CCD diffractometer [16]. The structures were solved by direct methods
(SHELXS97 [17] through the WinGX Interface [18]) and refined (anisotropic displacement
parameters, C-bound H atoms in the riding model approximation and a weighting scheme of
2
2
2
the form w = 1/[σ2(Fo ) + aP2 + bP] where P = (Fo + 2Fc )/3) with SHELXL97 on F2 [17].
The N-bound hydrogen atoms in 1 were refined with N–H = 0.88 0.01 Å. For 3, the
maximum and minimum residual electron density peaks of 1.29 and 1.60 e Å-3, were located
0.98 and 0.84 Å, respectively, from the Cd atom. The molecular structures shown in Figs 1
and 2, were drawn with 50% displacement ellipsoids for 1 and 70% for 2 and 3 [18]. The
overlay diagram, Fig. 1c, was drawn with QMol [19] and the crystal packing diagrams with
DIAMOND [20].
6

2.6. DNA binding studies
The DNA binding experiments were carried out at 25°C. DNA concentration per nucleotide
(4.21 x 10-5M) was determined using the molar absorption coefficient (6.6 x 103 M-1 cm-1) at
260 nm [21]. Absorption titration experiments were performed maintaining the concentration
of the metal complex solution at 50 µM and gradually increasing the concentration of CT-
DNA. The compounds were dissolved in DMSO and Tris-HCI buffer (50:50) containing 5
mM Tris, pH 7.1 and 25 mM NaCl [22] at room temperature (25°C). The solutions were
scanned over the range 250-800 nm. Absorbance values were recorded 10 min after the
addition of DNA solution. The binding constant, Kb was determined, using the equation :
[DNA] / (εa- εf) = [DNA] / (εb- εf) + 1/Kb(εa- εf)
where [DNA] is the concentration of DNA in the base pairs, εa corresponds to the apparent
molar extinction coefficient Aabs/[M], εf is the extinction coefficient for the free metal [M]
complex and εb is the extinction coefficient for the fully bound metal complex [21-24].
2.7. Cytotoxic Assay
The MCF-7 (estrogen receptor positive human breast cancer) and MDA-MB-231 (estrogen
receptor negative human breast cancer) cell lines used in this study (ATCC, Virginia, USA)
were cultured in RPMI-1640 (High glucose) medium supplemented with 10% fetal calf
serum (Sigma). The cytotoxic activity was determined using the microtitration of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, USA) as
previously reported [25]. Untreated cells were included for each compound as negative
control. The standard breast cancer chemotherapeutic, Tamoxifen was used as the standard
7

positive control. Cytotoxicity levels were expressed as IC50 values, i.e., the concentration of
compound that results in 50% cell death as compared to the negative controls in vitro.
3. Results and discussion
Table 1
Analytical and physical data for the Schiff base (1) and its metal complexes, (2-5).
Analytical (%) calculated
(found)
Melting
point
Molar
Compound
Formula
Color
conductance
(S cm2 mol-1)
(°C)
C
H
N
M
-
57.90
4.86
9.65
(10.03)
8.70
C14H14N2OS2
Yellow 165-166
-
1
2
3
4
5
(58.34) (4.78)
52.21 4.07
(50.71) (4.12)
48.56 3.79
(47.96) (3.72)
52.36 4.08
(50.48) (4.03)
52.75 4.11
(51.73) (3.87)
Light
10.15
(10.00)
16.17
(15.60)
9.89
C28H26N4O2S4Zn
182-183
brown
0.14
0.17
1.05
0.42
(9.28)
8.11
C28H26CdN4O2S4 Yellow 189-191
(7.15)
8.72
Dark-
C28H26CuN4O2S4
C28H26N4NiO2S4
171-172
brown
(8.75)
8.79
(9.59)
9.15
Brown 217-218
(8.03)
(9.25)
All compounds are non-hygroscopic and stable at room temperature. Whereas 1 is soluble in
common organic solvents, its metal complexes are insoluble in water and many organic
solvents other than DMSO and DMF. Analytical data for the synthesized compounds are
detailed in Table 1. The low molar conductance values show that the metal complexes are
non-electrolytes [15]. The Zn(II) and Cd(II) complexes, are diamagnetic and are expected to
have tetrahedral geometry. The magnetic moment of 1.88 B.M for the Cu(II) complex 4
indicates the presence of one unpaired electron, suggesting a square planar environment [4].
The Ni(II) complex is also diamagnetic, evidence of a square planar geometry.
8

2.1. IR spectral studies
Table 2
Important IR spectral bands (cm-1) of the Schiff base and its metal complexes
Compound
v(N–H)
v(C=N)
1611 (m)
1592 (m)
1587 (m)
1610 (m)
1610 (m)
v(N–N)
1016 (s)
1033 (s)
1031 (s)
1019 (s)
1015 (s)
v(CSS)
762(s)
755 (s)
760 (s)
754 (s)
751 (s)
v(COC)
1095 (m)
1088 (m)
1087 (m)
1100 (m)
1151 (m)
2956 (w)
1
2
3
4
5
-
-
-
-
Important characteristic absorptions are given in Table 2. Compound 1 contains a thioamide
group (-NH–C(=S)SR-) which is known to undergo thione-thiol tautomerism [1]. The
medium intensity peak at 2955 cm-1. consistent with v(N–H) shows that 1 exists
predominantly in the thione form in the solid-state. This assignment is further supported by
the absence of v(S-H) at ~2570 cm-1. v(N–H) band is absent in the spectra of the metal
complexes (2-5) indicating deprotonation of the N–H group on binding to the metal ion. A
sharp band at 1611 cm-1 in the spectrum of the free ligand (1) is attributed to the v (C=N)
stretch of the azomethine group. This band shifted to lower wavenumbers in all the
complexes confirming the involvement of nitrogen in coordination to the metal ions. The
shift in v(CSS) stretching to lower wavenumbers is expected on complexation [4]. The
stretching vibration, v(COC) of the furan ring shifted upon complexation (2-5) as a result of
interaction of the furanic oxygen atom with the central metal ion [26, 27].
3.2. NMR spectral studies
1
In the H NMR spectrum of S4MFuH (1), recorded in DMSO-d6,, the resonance for N–H
appears at 13.22 ppm. The absence of a signal at ~4 ppm (thiol H) indicates that 1 exists
9

predominantly in the thione form in DMSO-d6 solution as well as in the solid-state. The
remaining resonances observed were those expected for the molecular formula proposed.
The signals at 200.04 and 114.56 ppm in the 13C NMR spectrum are attributed to C=S and
C=N functionalities, respectively, in line with the greater electronegativity of the sulphur
atom.
3.3. Mass spectral studies
The mass spectrum for 1 is consistent with the proposed formula (molecular ion m/z 290).
•
The base peak observed at m/z 105 for 1 is assigned to the C8H9 fragment and peaks
•
observed at m/z 138, 91 and 77 correspond to fragments C8H9S•, C7H7 and C6H4••,
respectively.
3.4. Electronic spectral studies
Table 3
Electronic Spectral Data for S4MFuH (1) and its metal complexes, (2-5).
Electronic spectra (in DMSO)
Compound
aλmax(blog εmax
348 (4.80)
351 (5.09)
348 (3.60)
)
1
2
3
4
5
351 (4.98), 383 (4.46), 779 (2.36)
348 (4.93), 458 (3.68), 592 (2.19)
aλ max in nm
b log εmax = L mol-1 cm-1
10

The electronic spectral data for compounds 1-5 are tabulated (Table 3). Compounds 1-5 were
dissolved in DMSO at room temperature at three concentrations, i.e. 10-3, 10-4 and 10-5 M.
The π-π* transition associated with non-bonding electrons of the azomethine chromophore
[28] in the spectrum of 1 was recorded at 348 nm. The dark-brown copper complex (4)
→Cu(II) charge
showed a broad d-d absorption at 592-779 nm. A strong S
-transfer band at ca
→π*) at 256 nm were also observed. The occurrence of an
383 nm and an intraligand band (n
→Cu LMCT band is common in square planar Cu(II) complexes
S
of thiosemicarbazones and
dithiocarbazates [29, 30]. The diamagnetic Ni(II) complex (5) showed an absorption band at
1
1
592 nm consistent with the A1g → Eg transition of a square planar d8 metal complex. The
1
1
other two bands observed corresponded to the transitions 1A1g → A2g (348 nm) and A1g →
1B1g (458 nm) expected for a square planar complex. The absorption observed for the Zn
complex (2) at 407 nm is due to LMCT [10]. Absorptions consistent with ligand π → π* and
L → M charge transfer bands appeared in the spectrum of the cadmium complex (3).
3.5. Structural commentary
The crystallographic asymmetric unit of 1 comprises two independent molecules as
illustrated in Fig. 1; selected bond lengths (Å) and angles (°) are collected in Table 4 and
crystallographic and refinement details for 1–3 are given in Table 5.
11

(a)
(b)
(c)
Figure 1: (a) and (b) The molecular structures of the two independent molecules comprising
the asymmetric unit of 1, showing atom-labeling scheme. (c) Overlay diagram of the two
independent molecules of 1, S1 (red image) and S2 (green), and of a closely related literature
precedent, benzyl 3-((E)-furfurylidene)dithiocarbazate [30] (blue). The molecules have been
superimposed so that the central CS2 residues are coincident.
The configuration about the imine bond is E and the overall shape of the molecule is that of a
flattened "U" with the aromatic residues being syn, the furanyl-O orientated toward the
12

concave region of the molecule, and with the thione-S directed to the outside of the molecule
and anti to the azo bond. The seven atoms comprising the central residue, i.e.,CSC(=S)NNC,
are coplanar with a r.m.s. deviation 0.0326 Å (the equivalent value for the second
independent molecule is 0.0405 Å). While to a first approximation the two independent
molecules have similar conformations, as highlighted in the overlay diagram in Fig. 1(c),
some differences are noted in the orientations of the terminal residues. While the difference
in the dihedral angles between the central residual and furanyl ring are not great, i.e., 8.55(16)
and 6.80 (17)º, for the two independent molecules, respectively, greater differences are seen
in the dihedral angles between the central residue and the tolyl rings, 62.53(6) and 80.69(7)º,
respectively, and between the furanyl and tolyl rings of 61.83(8) and 79.97(9)º. The relevant
structure in the literature most appropriate for comparison is that of benzyl 3-((E)-
furfurylidene)dithiocarbazate [30], i.e., the unsubstituted benzyl derivative of 1.
A
comparable overall conformation is found along with similar geometric parameters, but the
relative disposition of the terminal rings is almost orthogonal with a dihedral angle of
87.82(6)º, Fig. 1(c). The most significant intermolecular interactions in the crystal structure
of 1 are N–H…S(thione) hydrogen bonds [31] whereby the two independent molecules
associate via an eight-membered {…HNCS}2synthon to form dimeric aggregates. These
assemble into columns along the a-axis with no specific interactions between them. See Fig.
2 below for a view of unit cell contents.
13

Figure 2. A view in projection down the a-axis of the unit cell contents of 1. The N–H…S
hydrogen bonds are shown as orange dashed lines.
In the structure of 3 (Fig. 2(a) and Table 4), the zinc atom is N-,S-chelated by two
dithiocarbazate anions forming an N2S2 donor set. The resulting five-membered chelate rings
have different configurations with that containing the S1 atom being twisted about the Zn–S1
bond, and the second ring being an envelope with the Zn atom being the flap. The dihedral
angle between the two chelate rings of 83.13(5)ºis consistent with a tetrahedral geometry.
However, the range of tetrahedral angles is relatively wide, i.e., from an acute 86.10(5)º for
the S1–Zn–N2 chelate angle, consistent with the restricted bite angle of the ligand, to a wide
129.78(5)º for S1–Zn–N4. These deviations are ascribed to the close approach of the furanyl-
O atoms, Table 4. If the latter interactions were considered significant, the coordination
geometry might be considered as comprising two N-,O-,S-coordinating anions each
occupying mer-positions in a heavily distorted octahedral coordination geometry. The key
difference in geometric parameters as a result of deprotonation and coordination of 1 in 2 is
14

the elongation of the formally thione bond by 0.06-0.07 Å and the contraction of the
quaternary-C–N bond, by 0.03-0.04 Å, consistent with the formation of a thiolate-S and an
adjacent imine bond; the other bond lengths are remarkably similar, Table 4. The major
conformational change associated with coordination of 1 is the syn disposition of the thiolate-
C‒S and azo-N‒N bonds which arises owing to chelation and accompanying change in
conformation. As a result, some significant changes in bond angles ensue. Referring to
Table 4, the most notable changes are seen about the quaternary-C atoms where the S–C–S
angle has contracted by over 10° with a concomitant increase in the coordinated-S–C–N
angles by nearly the same amount in 2 compared with 1. The other significant change is
found in the narrower angles, i.e., by 6-7°, subtended at the non-coordinating-N1, N3 atoms
compared to 1.
In the structure of 3, Fig. 3(b) and Table 4, the cadmium atom exhibits essentially the same
features as just described for 2. The five-membered chelate rings have the comparable
configurations as for 2, i.e., that containing the S1 atom being twisted about the Cd–S1 bond
and the second ring being an envelope with the flap atom being Cd. The dihedral angle
between the two chelate rings is 81.74(7)º. Reflecting the larger size of the cadmium atom
and its propensity to expand its coordination number, the intramolecular Cd…O interactions
are shorter and therefore stronger than the comparable interactions in 2. As a consequence of
the close approach of the furyl-O atoms, the range of tetrahedral angles is wider in 3 than for
2, i.e., 78.49(8)º, for the S1–Zn–N2 chelate angle, to 134.40(8)º for S1–Zn–N4. If the Cd…O
interactions were considered significant, the resulting N2O2S2 donor set defines a polyhedron
best described as being based on a twisted trigonal prism in which each triangular face is
occupied by a single oxygen, nitrogen and sulphur atom. (See the caption to Fig. 3(b) below
for more details.)
15

(a)
(b)
Figure 3: The molecular structures of (a) 2 and (b) 3. The intramolecular M…O interactions
are highlighted as dashed lines. If these interactions were considered significant, the
coordination geometry for the zinc atom is distorted octahedral with one triangular face being
defined by the S1, S3 and N4 atoms. In the case of cadmium, where the coordination
geometry would be considered twisted trigonal prismatic, one triangular face is occupied by
the S1, O2 and N2 atoms.
The most closely related structure in the literature available for comparison with 1 and 2 is a
cadmium complex with an unsubstituted benzyl group and with the furanyl ring bearing a
methyl group adjacent to the oxygen atom, i.e., bis[S-benzyl-β-N-(5-methyl-2-
furylmethylene) dithiocarbazato]cadmium(II) [2]. This structure adopts an arrangement very
similar to 3. Other structures worth mentioning are thienyl analogues of 1 but with
unsubstituted benzyl residues [32]. When complexed to nickel(II) and copper(II), the thienyl-
S atoms do not form intramolecular M…S interactions owing to the steric constraints
imposed by the square planar geometry about each metal center.
16

In the crystal packing of 2, the molecules self-assemble into double layers in the ac-plane via
π…π interactions between O2-furanyl and C3-tolyl rings. Layers are connected along the b-
axis by O1-furanyl-C-H…S3 interactions which by themselves assemble molecules into
helical chains. A view of the crystal packing is given as Fig. 4, and geometric details given
below, ref. [33].
Figure 4. A view in projection down the c-axis of the unit cell contents of 2. The C–H…S
and π…π interactions are shown as orange and blue dashed lines, respectively.
For 3, double layers are formed parallel to (101), see Fig. 5, and the only recognisable
interactions are of the type π…π again occurring between O2-furanyl and C3-tolyl rings [34].
17

Figure 5.A view of the unit cell contents of 3. The π…π interactions are shown as blue
dashed lines.
Table 4
Selected bond lengths and angles (Å, º) for 1–3.
___________________________________________________________________________
Compound
1
2 (M = Zn)
3 (M = Cd)
___________________________________________________________________________
Parameter
M–S1
–
2.2896(6)
2.2949(6)
2.0627(18)
2.052(2)
2.4825(11)
2.4923(10)
2.314(3)
2.269(3)
2.932(3)
2.793(3)
1.737(4)
1.753(4)
1.823(4)
1.746(4)
M–S3
–
M–N2
M–N4
M…O1
M…O2
C1–S1
C1–S2
C2–S2
C15–S3
–
–
–
2.9313(15)
2.8454(16)
1.742(2)
–
1.672(2)
1.746(2)
1.828(3)
1.682(2)
1.749(2)
1.826(2)
1.743(3)
18

C15–S4
1.751(2)
1.756(2)
1.762(4)
1.820(4)
1.406(4)
1.396(4)
1.306(5)
1.294(5)
1.292(5)
1.296(5)
111.8(2)
131.0(3)
117.3(3)
112.2(2)
131.4(3)
116.3(3)
114.8(3)
115.3(3)
C16–S4
1.816(3)
1.821(3)
N1–N2
1.384(3)
1.398(2)
N3–N4
1.385(3)
1.404(3)
C1–N1
1.334(3)
1.304(3)
C10–N2
1.285(3)
1.297(3)
C15–N3
1.327(3)
1.301(3)
C24–N4
1.280(3)
1.297(3)
S1–C1–S2
S1–C1–N1
S2–C1–N1
S3–C15–S4
S3–C15–N3
S4–C15–N3
C1–N1–N2
C15–N3–N4
124.04(15)
121.30(16)
114.66(17)
125.41(14)
120.24(17)
114.35(17)
121.33(18)
121.12(19)
113.09(12)
128.84(17)
118.08(17)
111.78(13)
130.25(18)
117.96(18)
114.33(18)
113.85(19)
___________________________________________________________________________
Table 5
Crystallographic and refinement details for 1–3.
___________________________________________________________________________
Compound
1
2
3
Formula
C14H14N2OS2
290.39
C28H26N4O2S4Zn
644.14
C28H26CdN4O2S4
691.17
Formula weight
Crystal color/habit
Crystal dimensions/mm
Crystal system
Space group
Yellow prism
0.06 x 0.12 x 0.33
triclinic
Light-brown prism
0.05 x 0.15 x 0.25
monoclinic
Yellow prism
0.05 x 0.13 x 0.25
triclinic
¯
P1
¯
P1
P21/n
19

a/Å
9.4658(8)
12.4477(7)
13.8832(9)
64.342(6)
76.011(6)
74.180(6)
1404.17(17)
4
11.0684(4)
15.5959(5)
17.1770(6)
90
10.7805(7)
11.0505(6)
12.8127(7)
91.758(4)
95.217(5)
107.662(6)
1445.67(15)
2
b/Å
c/Å
a/°
β/°
99.864(3)
90
γ/°
V/Å3
2921.29(17)
4
Z
Dc/g cm-3
1.374
1.465
1.588
F(000)
608
1328
700
Radiation, λ/Å
µ/mm-1
CuKα, 1.54180
3.378
MoKα, 0.71073
1.160
MoKα, 0.71073
1.078
Measured data
θ range/°
18488
12834
11904
3.6–71.6
5403
2.3–26.5
6040
2.3–26.5
5988
Unique data
Observed data (I≥2.0σ(I))
R, obs. data; all data
a, b in weighting scheme
Rw, obs. data; all data
Residual electron density
peaks/e Å3
4589
4906
4936
0.058; 0.066
0.114, 0.514
0.156; 0.166
0.035; 0.049
0.034, 0.987
0.077; 0.084
0.048; 0.063
0.060, 0
0.111; 0.123
0.95, -0.31
0.46, -0.53
1.29, -1.60
___________________________________________________________________________
3.6. DNA binding studies
Electronic absorption titrations are usually used to determine the binding affinity of
complexes to the calf-thymus DNA (CT-DNA) helix. The absorption spectra are recorded in
20

the absence and presence of CT-DNA (at a constant concentration of complexes, with
increasing concentration of CT-DNA). Hypochromism and red-shifts indicate strong stacking
interactions between the DNA double helix strand and an aromatic chromophore [35-37].
The electronic absorption spectra for the titrations of complexes (2-5) are reproduced in Fig.
6. Each spectrum displays two intense absorption bands attributed to the LMCT and intra-
ligand (π→π*) transitions of the aromatic chromophore in the regions 322-392 nm and 263-
282 nm, respectively. The decreasing energies of π→π* indicate that the π* orbital of the
ligands on the complexes are coupled with a π orbital of the DNA base pairs. This interaction
is manifested by hypochromism and red-shifts [22]. The hypochromism observed were 22%,
23%, 52%, and 25%, for complexes 2–5 respectively. In order to compare the binding
strengths of the complexes to CT-DNA, the binding constants were quantitatively
determined. The Kb obtained for complexes 2-5 are 1.42 x 104, 1.71 x 103, 2.85 x 104 and
3.42 x 103 M-1, evidence of good binding affinity due to π→π* stacking interactions between
the respective complex and the DNA base pairs for all complexes [22, 37-39]. The copper
complex (4) has the highest binding constant consistent with it having the lowest IC50 value
among the compounds assayed. The greater potency of 4 is expected to relate to its ability to
block the enzymatic binding to the nitrogen bases of DNA or RNA [40]. The data obtained
was comparable to N,N′-bis{5-[(triphenylphosphonium chloride)-methyl] salicylidine}-o-
phenylenediamine) Schiff base, which was revealed to have intercalative binding modes with
the value of intrinsic binding constant being 8.5 × 105 M−1 which was greater that its
cobalt(II) complex (Kb= 5 × 104 M−1). The cobalt(II) complex most likely interacted with
DNA in an electrostatic binding mode [41]. However, the values obtained were much lower
than the potential intercalators like ethidium bromide (kb = 7 × 107 M−1) [42]. The data in this
work was also comparable to [Co (phen)3]3+ (1.6 × 104 M−1) [42]; Co(III) complexes with
21

asymmetric ligand, [Co (phen)2(pdta)]3+ (kb = 2.8 × 104) [43]; [Co (bpy)2(CNOIP)]3+ (kb = 5
× 104 M−1) [44].
1.2
1.2
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
300
400
500
600
700
800
300
400
500
600
700
800
wavelength (nm)
wavelength (nm)
(a)
(b)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.2
1.0
0.8
0.6
0.4
0.2
0.0
300
400
500
600
700
800
300
400
500
600
700
800
wavelength(nm)
wavelength(nm)
(c)
(d)
Figure 6. Electronic absorption spectra of (a) 2, (b) 3, (c) 4 and (d) 5, (50 µM) recorded in
the absence and presence of increasing amounts of CT-DNA. Arrows show the absorption
changes upon increasing the concentration of CT-DNA.
3.7. Cytotoxicity Assay
Several factors are known to affect the biological activity of a given molecule. These
factors include lipophilicity, chemical structure, resonance, and thermodynamic stability [1,
22

45]. Many dithiocarbazate Schiff bases and their metal complexes have been shown to exhibit
a wide range of cytotoxic activities, with small changes in the chemical composition often
leading to vast differences in selectivity and activity [45-49]. The complexation of Schiff
bases with metal ions reduces the polarity of the metal ions through the partial sharing of
positive charge with the donor atoms and π-electron delocalization upon chelation [50]. This
results in the enhancement of the lipophilic characteristics of the central metal atom allowing
the blocking of cellular enzymatic activity [51].
Compound 1 was inactive against the two breast cancer cell lines assayed, [MCF-7 (estrogen
receptor positive human breast cancer) and MDA-MB-231 (estrogen receptor negative human
breast cancer)]. Consistent with previously published findings, the cytotoxicity of 1
improved with the complexation to a metal[1]. The Cu(II) complex, 4, showed moderate
cytotoxic activity against the MCF-7 cell line (IC50= 3.02µM) but was inactive against MDA-
MB-231, however. complexes 2, 3 and 5 were inactive against both. This order of potency is
consistent with an earlier investigation [11] and is related to both the ionic radii and the
ligand field stabilization energies. The Cu(II) ion has the smallest radius of the first-row
transition metals. This results in the strongest binding of the ligand that plays an important
role in ligation and transport upon complexation. In addition, the activity of the Cu(II)
complex could be due to other factors such as redox activity, ligand exchange reactions, the
existence of metabolic pathways for transport and excretion, and activation of metal-
dependent enzymes all of which, could play vital roles in determining the most cytotoxic
metal [52, 53]. The cytotoxic data were also comparable to the copper complexes of
quinoline-2-carboxaldehyde, which showed dose-dependent cell growth inhibition in LNCaP
and PC-3 cell (prostate cancer cell lines) at IC50 of 5 µM and 7 µM after 72 h of treatment.
23

These complexes were expected to be used for targeting the ubiquitin-proteasome pathway
for the treatment of prostate cancer [54].
Copper complexes with a CH3 moiety, an electron donating group, at the para position of the
benzene ring have been shown to have higher cytotoxic activity than those with a methyl
group at the ortho position [15]. A methyl group in the ortho position is affected by steric
interference and interactions with the lone pair from the neighbouring sulphur atom. In
addition, the presence of methyl groups prevents metabolic hydroxylation and subsequently
makes the copper complexes less cytotoxic towards cancer cells [55].
4. Conclusion
The synthesis of S-4-methylbenzyl-β-N-(2-furylmethylene)dithiocarbazate (S4MFuH), a new
Schiff base and its Zn2+, Cu2+, Cd2+ and Ni2+ complexes is reported. S4MFuH, 1, has an E
configuration, features the thione anti to the azo bond and has a U-shape overall. When
deprotonated, it chelates through nitrogen and sulfur to Zn2+ (2) and Cd2+ (3), resulting in a
change in conformation (syn) as revealed by X-ray crystallography. A distorted tetrahedral
geometry with two bidentate NS ligands was found for complexes 2 and 3, with two close
intramolecular M…O interactions being largely responsible for the distortions. The Cu2+
complex (4) is paramagnetic indicating that it has tetrahedral geometry while the diamagnetic
character of 5 shows that it is likely to be square planar. The biological activity of the Schiff
base and its metal complexes was evaluated against MCF-7 (estrogen receptor positive
human breast cancer) and MDA-MB-231 (estrogen receptor negative human breast cancer)
cell lines. Complex 4 was found to be moderately active against MCF-7 cells but inactive
towards MDA-MB-231 while all other compounds were inactive against both cell lines. All
complexes bound to CT-DNA with a moderate binding affinity, Kb, = 104-103 M-1. As
24

expected, the most cytotoxic compound, complex 4, showed the strongest CT-DNA binding
affinity among them.
Acknowledgements
We thank the Department of Chemistry, the Molecular Genetics Laboratory and the
Department of Obstetrics and Gynaecology, Universiti Putra Malaysia, and the Malaysian
Genome Institute for the use of their facilities. This research was funded by Universiti Putra
Malaysia (UPM) and the Malaysian Government under the Research University Grant
Scheme (RUGS No. 9419400), the Malaysian Fundamental Research Grant Scheme (FRGS
No. 01–02-13-1344FR) and the Science Fund under the Ministry of Science, Technology and
Innovation (MOSTI) (06-01-04-SF1810). E.N.M.Y and J.J. wish to thank UPM for the award
of Graduate Research Fellowships. Support from the High Impact Research Ministry of
Education Grant UM.C/625/1/HIR/MoE/SC/12 from the Ministry of Higher Education
Malaysia and the University of Malaya is also gratefully acknowledged.
Supplementary Material
The crystallographic data for the structural analysis of compounds C14H14N2OS2,
C28H26N4O2S4Zn and C28H26CdN4O2S4, 1–3, have been deposited with the Cambridge
Crystallographic Data Centre, CCDC No. for 1 is 1004617, for 2 is1004618 and for 3 is
1004619. A copy of this information may be obtained free of charge from the Director,
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Tel.: +44 (0) 1223 762911; E-
mail: deposit@ccdc.cam.ac.uk).
25

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