Synthesis, characterization of 1,2,4-triazole Schiff base derived 3d-metal complexes: Induces cytotoxicity in HepG2, MCF-7 cell line, BSA binding fluorescence and DFT study

Article abstract of DOI:10.1016/j.saa.2016.08.008

Two novel Schiff base ligands H₂L¹ and H₂L² have been synthesized by condensation reaction of amine derivative of 1,2,4-triazole moiety with 2-hydroxy-4-methoxybenzaldehyde. Co(II), Ni(II), Cu(II) and Zn(II) of the synthesized Schiff bases were prepared by using a molar ratio of ligand:metal as 1:1. The structure of the Schiff bases and synthesized metal complexes were established by ¹H NMR, UV–Vis, IR, Mass spectrometry and molar conductivity. The thermal stability of the complexes was study by TGA. Fluorescence quenching mechanism of metal complexes 1–4 show that Zn(II) and Cu(II) complex binds more strongly to BSA. In DFT studies the geometries of Schiff bases and metal complexes were fully optimized with respect to the energy using the 6-31?+?g(d,p) basis set. The spectral data shows that the ligands behaves as binegative tridentate. On the basis of the spectral studies, TGA and DFT data an octahedral geometry has been assigned for Co(II), Ni(II), square planar for Cu(II) and tetrahedral for Zn(II) complexes. The anticancer activity were screened against human breast cancer cell line (MCF-7) and human hepatocellular liver carcinoma cell line (Hep-G2). Result indicates that metal complexes shows increase cytotoxicity in proliferation to cell lines as compared to free ligand.

Full text of DOI:10.1016/j.saa.2016.08.008

Synthesis, characterization of 1,2,4-triazole Schiff base derived 3d-metal
complexes: Induces cytotoxicity in HepG2, MCF-7 cell line, BSA binding
fluorescence and DFT study
Prateek Tyagi, Monika Tyagi, Swati Agrawal, Sulekh Chandra, Himan-
shu Ojha, Mallika Pathak
PII:
DOI:
Reference:
S1386-1425(16)30461-9
doi: 10.1016/j.saa.2016.08.008
SAA 14594
To appear in:
Received date:
Revised date:
Accepted date:
21 April 2016
20 July 2016
7 August 2016
Please cite this article as: Prateek Tyagi, Monika Tyagi, Swati Agrawal, Sulekh Chan-
dra, Himanshu Ojha, Mallika Pathak, Synthesis, characterization of 1,2,4-triazole Schiff
base derived 3d-metal complexes: Induces cytotoxicity in HepG2, MCF-7 cell line, BSA
binding fluorescence and DFT study, (2016), doi: 10.1016/j.saa.2016.08.008
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ACCEPTED MANUSCRIPT
Synthesis, characterization of 1,2,4-triazole Schiff base derived 3d-metal complexes:
Induces cytotoxicity in HepG2, MCF-7 cell line, BSA binding Fluorescence and DFT
study
Prateek Tyagia, Monika Tyagia, Swati Agrawalb, Sulekh Chandraa,*, Himanshu Ojhac, Mallika
Pathakd
a
Department of Chemistry, Zakir Husain Delhi College, University of Delhi, JLN-Marg,
New Delhi 110002, India
b
Department of Chemistry, Moti Lal Nehru College, Benito Juarez Marg, New Delhi
110021, India
c
Institute of Nuclear Medicine & Allied Sciences, DRDO, Delhi 110054, India
d Department of Chemistry, Miranda House, University of Delhi, Delhi, 110007, India
*Corresponding author
Tel.: +91-11-22911267; Fax: +91-11-23215906
Email: schandra_00@yahoo.com
Abstract
Two novel Schiff base ligands H2L1 and H2L2 have been synthesized by condensation
reaction of amine derivative of 1,2,4-triazole moiety with 2-hydroxy-4-
methoxybenzaldehyde. Co(II), Ni(II), Cu(II) and Zn(II) of the synthesized Schiff bases were
prepared by using a molar ratio of ligand : metal as 1:1. The structure of the Schiff bases and
synthesized metal complexes were established by 1H NMR, UV–Vis, IR, Mass spectrometry
and molar conductivity. The thermal stability of the complexes was study by TGA.
Fluorescence quenching mechanism of metal complexes 1–4 show that Zn(II) and Cu(II)
complex binds more strongly to BSA. In DFT studies the geometries of Schiff bases and
metal complexes were fully optimized with respect to the energy using the 6-31+g(d,p) basis
set. The spectral data shows that the ligands behaves as binegative tridentate. On the basis of
the spectral studies, TGA and DFT data an octahedral geometry has been assigned for Co(II),
Ni(II), square planar for Cu(II) and tetrahedral for Zn(II) complexes. The anticancer activity
were screened against human breast cancer cell line (MCF-7) and human hepatocellular liver
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carcinoma cell line (Hep-G2). Result indicates that metal complexes shows increase
cytotoxicity in proliferation to cell lines as compared to free ligand.
Key Words: Schiff Base, Co(II), Ni(II),Cu(II) and Zn(II) complexes, DFT, Anticancer
activity, BSA binding
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1. Introduction
There is a considerable increase in the use of metal complexes for cancer treatment after the
accidental discovery of the biological activity of platinum complex, cisplatin, in 1965 by
Rosenburg [1]. In recent years, many studies associated with metal-based drugs show
promising biological activity and are of great interest in chemistry and biology [2]. Literature
survey revealed that substituted heterocyclic upon reaction with transition metal salts form
complexes that show enhanced physiochemical and pharmacological properties [3–6].
Heteroaromatic moiety on combination with a positive charged metal centre leads to
complexes that show well defined geometries, which can easily interact with biomolecules
[7].
Transition metal complexes of 1,2,4-triazole substituted moiety have acknowledged
considerable interest because of their brilliant coordination potential and diverse
pharmacological properties, notable for antibacterial, antifungal, antitumor activities [8–12].
A number of commercially available drugs having 1,2,4-triazole moiety like Vorozole,
Letrozole and Anastrozole (Fig. 1) are used for the treatment of breast cancer [13]. Other
includes, Fluconazole, a very well known antifungal drug and Trazodone is known for their
antidepressant properties (Fig. 1) [14, 15].
Similarly, the phenolic aldehyde compound like vanillin is very well known. Valen Schiff
bases are well known in literature and represents a class of molecule which show extensive
biological properties [16–18]. In contrary, a very little work has been published on Schiff
base derived from another isomer of vanillin i.e. 2-hydroxy-4-methoxybenzaldeyde [19]. An
added advantage of using 2-hydroxy-4-methoxybenzaldehyde over vanillin is the position of
–OH group. The –OH group present at the ortho position can easily deprotonate and
coordinate with metal centre.
Metals like cobalt, nickel and copper have great affinity for coordination because of their
smaller size, higher nuclear charge. To the best of our knowledge no work has been reported
on the synthesis of metal complexes of Schiff bases derived from 1,2,4-triazole amine
derivatives and 2-hydroxy-4-methoxybenzaldehyde. Two novel Schiff base ligands H2L1 and
1
H2L2 and their metal complexes were synthesized and characterized by H NMR, Mass, IR
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and analytical data. The synthesized complexes were screened for their in vitro anticancer
activity against cell line MCF-7 and HepG2.
Fig. 1.
2. Experimental
2.1 Materials and Methods
All the chemicals were used of Anala R grade and received from Sigma-Aldrich and Fluka.
Metal salts were purchased from E. Merck and used as received. MTT (3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide) and 0.25% trypsin and 0.02%
EDTA mixture was purchased from Himedia (India). Fetal bovine serum (FBS) was
purchased from Biowest (USA).
2.2 General procedure for the synthesis of Ligands H2L1 –H2L2
Intermediate 3a-3b were synthesized from starting compounds (1a-1b) (Scheme 1) as per the
method reported in literature [13–14]. First the hydrazides (1a-1b) were treated with carbon
disulphide and potassium hydroxide in absolute ethanol and are converted to their potassium
salts (2a-2b). On reaction with hydrazine hydrate the potassium salt undergoes ring closure to
yield the amine, 3a-3b. For the synthesis of ligand H2L1 equimolar quantity (10 mmol) of 3a
and 2-hydroxy-4-methoxybenzaldehyde (10 mmol) were dissolved in 30 mL of acetic acid.
The resultant solution was refluxed for 6 h under constant stirring. After completion of the
reaction the solution was poured into crushed ice.
Scheme 1
The separated product was filtered, washed with ice cold water, and recrystallized with a 1:1
solution of DMF & methanol. Similar method was used for the synthesis of ligand H2L2. The
structure of the synthesized Schiff based were supported by 1H NMR, IR and Mass spectra.
2.2.1 2-(((3-mercapto-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-yl)imino)methyl)-4-
methoxyphenol H2L1
o
Yield: 74(%). Color (pale-yellow). M.p. >260 C. IR (KBr, cm-1): 1598 (HC=N), 2704
(–SH), 3230 (–OH). 1H NMR (DMSO-d6, δ, ppm) 3.80 (s, 3H, –OCH3), 6.82–6.83 (m,
2H, Ar–H), 7.81–7.87 (m, 1H, Ar –H), 8.12 (d, 2H, J = 6.92 Hz, Ar–H), 8.75–8.82 (m, 2H,
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Ar –H), 9.79 (s, 1H, N=CH), 11.57 (s, 1H, –OH), 14.45 (s, 1H, triazole –SH). Anal. Calcd.
for C15H13N5O2S (327.36): C: 55.04; H: 4.00; N: 21.39; Found: C: 55.0; H: 4.02; N: 21.35%.
Mass spectrum (ESI) [M+H]+ = 328.01.
2.2.2 2-(((3-mercapto-5-(pyridin-3-yl)-4H-1,2,4-triazol-4-yl)imino)methyl)-4-
methoxyphenol H2L2
o
Yield: 69(%). Color (yellow). M.p. >260 C. IR (KBr, cm-1): 1598 (HC=N), 2698 (–SH),
1
3213 (–OH). H NMR (DMSO-d6, δ, ppm): 3.89 (s, 3H, –OCH3), 6.72 (d, 1H, J = 7.92 Hz,
Ar–H), 6.89–6.93 (m, 1H, Ar –H), 7.89–7.93 (m, 2H, Ar–H), 8.73–8.79 (m, 2H, Ar –H), 9.37
(d, 1H, J = 7.32 Hz, Ar–H), 9.93 (s, 1H, N=CH), 11.69 (s, 1H, –OH), 14.39 (s, 1H, triazole –
SH). Anal. Calcd. for C15H13N5O2S (327.36): C: 55.04; H: 4.00; N: 21.39; Found: C: 55.12;
H: 3.98; N: 21.36%. Mass spectrum (ESI) [M+H]+ = 328.13.
2.3 General procedure for the synthesis of metal complexes 1–8
Schiff base metal(II) complexes (1–4) of ligand H2L1 were synthesized from H2L1 (0.34 g, 1
mmol) in (DMF : Methanol in 1:1) (20 mL) with corresponding metal(II) salts {chloride of
Co(II) (0.24g, 1mmol), Ni(II) (0.24 g, 1 mmol), Cu(II) (0.17 g, 1 mmol) and Zn(II) (0.14 g, 1
mmol)} in methanol (20 mL) in 1:1 ratio (Scheme 1). The solution was refluxed for 10–16
hrs. The resulting solution was reduced to half volume on a water bath and kept aside
overnight. The solid product separated out, which was vacuum filtered, washed with cold
ethanol, diethyl ether and dried under vacuum over anhydrous CaCl2 (Yield: 58–72%).
Similarly, Schiff base metal(II) (5–8) complexes of Schiff base H2L2 were prepared.
Physical, analytical and spectral data of ligands and metal complexes are given in Table 1.
Attempt to grow the single crystal of the metal complexes was not successful. To have a
understanding of the molecular and electronic structure of the synthesized metal complexes,
their DFT study is done. The stability of the proposed geometry is also confirmed by the
binding energies values.
2.4 Analysis
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The carbon and hydrogen were analyzed on Carlo-Erba 1106 elemental analyzer. The
nitrogen content of the complexes was determined using Kjeldahl’s method. Molar
conductance was measured on the ELICO (CM82T) conductivity bridge. ESI-MS spectra
were obtained using a VG Biotech Quattrro mass spectrometer equipped with an elctrospray
ionisation source in the mass range of m/z 100 to m/z 1000. IR spectra (CsBr) were recorded
on FTIR BX-II spectrophotometer. NMR spectra were recorded with a model Bruker
Advance DPX-300 spectrometer operating at 400 MHz using DMSO-d6 as a solvent and
TMS as internal standard. The electronic spectra were recorded in DMSO on Shimadzu UV
mini-1240 spectrophotometer. Thermogravimetric analysis (TGA) was carried out in
o
dynamic nitrogen atmosphere (30 ml/min) with a heating rate of 10 C/min using a
Schimadzu TGA-50H thermal analyzer. EPR spectra of the Cu(II) complexes were recorded
as polycrystalline sample at room temperature on E4-EPR spectrometer using the DPPH as
the g-marker. Fluorescence spectra was recorded on FS920 Edinburgh Instruments, UK, with
standard 3.5 ml quartz cell. The excitation source was xenon lamp (450 W) and sample
chamber was equipped with peltier accessory.
2.5 DFT Calculations
The theoretical calculations were performed using the 6-31+g(d,p) basis set as incorporated
in Gaussian 09W program [20]. The geometries were optimized with respect to energy using
the B3LYP three parameter density functional, which includes Becke’s gradient exchange
correction [21], the Lee, Yang, Parr correlation functional [22] and the Vosko, Wilk, Nusair
correlation functional [23].
2.6 In vitro studies (Cell culturing)
The cell lines HepG2 (liver hepatocellular carcinoma) and MCF-7 (human breast adreno
carcinoma) were cultured as monolayers and maintained in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine,
100 U/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere with 5% CO2 at
o
37 C in T-75 flasks and were sub cultured twice a week. For the assays, cells (2 x 103
cells/well in 200 µl of complete DMEM) were placed in each well of a 96 well flat bottom
plate. Cells were allowed to adhere for overnight, and then treated with 5 µM and 10 µM
concentration of each H2L1, H2L2 and metal complexes (1–8) in DMSO for 48 hrs. The cell
proliferation of the control (untreated cells) was fixed to 100%. After completion of
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incubation period, 20 µl MTT (5mg/ml) was added to each well for 2 hrs. Following which
media was removed and 100 µl of DMSO was added to each well in order to solubilize the
formazan. The plate was read using the ELISA reader at a wavelength of 540 nm.
3. Results and discussion
3.1 Micro analysis and molar conductance
The synthesized metal(II) complexes are stable in air, microcrystalline solid, partial soluble in
o
hot methanol, soluble in DMF and DMSO and decompose above 260 C. The molar
conductance of the metal complexes 1–8 in DMF showed values in range 5.4–11.6 ohm-1
cm2 mol-1, which indicates that synthesized complexes are non-electrolytes [24]. Physical
measurements and analytical data of the ligands and complexes 1–8 are given in Table 1.
Table 1
3.2 Mass Spectra
In the ESI mass spectrum of ligand H2L1 peak corresponding to [M+H]+ ion appear as
molecular ion peak at m/z = 328.01 amu. The molecular ion peak confirms the proposed
formula [C15H13N5O2S].+, however the base peak of H2L1 was observed at m/z 178.01 amu.
The peak corresponding to various fragments in H2L1 appears at 311.18, 287.97, 281.18,
203.98, 180.81, 179.68, 177.95, 101.98, and 73.99 amu, respectively.
Similarly, for ligand H2L2 the molecular ion peak appears at 328.13 amu, while its base peak
was observed at m/z value of 178.14. The m/z peak of the metal complexes 1–8 are given in
Table 1. The mass spectrum of the metal complex 1 is displayed in Fig. 2 and the mass
spectrum of metal complexes 2–8 are given in supplementary data.
Fig. 2
1
3.3 H NMR Spectra
The 1H NMR spectra have been recorded for ligands H2L1 and H2L2 in DMSO. Appearance
of important singlet at δ 9.79 ppm, confirms the synthesis of the hybrid coupled Schiff-base
ligand HL1 (Fig. 3), was assigned to imine (CH=N) proton. A set of doublet and multiplet in
the region 6.82–8.82 ppm was assigned to the aromatic protons, whereas a weak singlet at
14.45 ppm corresponds to –SH proton of triazole ring. The proton of the –OH group appears
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as a singlet at 11.57 ppm. Similarly, in the spectrum of the ligand H2L2, the peak
corresponding to azomethine (–HC=N), –OH and–SH protons appear at 9.93, 11.69 and
14.39 ppm, respectively.
In 1H NMR spectrum of Zn(II) complex (4) of ligand H2L1 and complex (8) of ligand H2L2 a
shift of electron density from ligand to metal ion has been observed. The signal for the
azomethine proton is slightly deshielded and appeared at δ 10.01–10.12 ppm. This might be
due to the donation of electron density from nitrogen to the Zinc ion. Because of this electron
transfer from nitrogen to zinc, a coordinate bond is formed between them (Zn←N) [25]. The
characteristic signal for the –SH and –OH protons disappeared in the NMR of zinc complex,
which indicates the deprotonation of –SH and –OH protons during complex formation. All
other aromatic protons in the NMR spectrum of zinc complex appeared at almost same
chemical shift, as they appear in the spectrum on ligand. The signal for coordinated water in
appeared as a broad singlet at δ 3.95 and 3.97 ppm, respectively.
Fig. 3
3.4 IR Spectra
In order to understand the binding mode of synthesized ligands to the corresponding metal
ions the IR spectra of ligands H2L1 , H2L2 and their metal(II) complexes (1–8) are discussed.
The potential donor sites like azomethine nitrogen (–C=N), triazole (–SH) and phenol oxygen
(–OH), can easily coordinate with metal ions (Table 2). Band corresponding to (C=O)
stretching vibrations was not observed in the IR spectra of ligand H2L1. The band at 1598
cm-1 was assigned to (HC=N) linkage confirms the formation of Schiff base. Similarly, band
corresponding to (HC=N) was observed at 1601 cm-1 in ligand H2L2 [24]. The particular
bands for (S–H) vibrations was observed at 2704 cm-1 for ligand H2L1, while for ligand
H2L2 it appeared at 2698 cm–1 [27].
In the IR spectra of the metal complexes, it was observed that band corresponding to
(HC=N) group shifts to lower side by 15–19 cm-1, which indicates a bond is formed
between azomethine nitrogen and metal ion [28]. The band in the region of 1586–1579 cm-1
in the IR spectra of metal complexes 1–8 corresponds to (HC=N) group. The same is also
supported by the presence of band corresponding to (M–N) bond in the range of 423–449
cm-1.
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No band related to (S–H) stretching vibrations was observed in the IR spectra of metal
complexes, which indicates the deprotonation of the thiol group and coordination of thiol
sulphur to metal ion [28]. A lower frequency shift due to (C–S) in the IR spectra of metal
complexes around 660–672 cm-1, further confirms this. This is supported by the lower
frequency shift which appears in the metal complexes. A new band in range of 342–366 cm-1
in far IR spectra was assigned to (M–S) vibrations. The broad band corresponding to (–
OH) stretching vibrations, which appeared at 3230 cm–1 in H2L1 and 3213 cm–1 in H2L2
disappeared after complex formation, point up the deprotonation of phenol –OH during
complex formation. This is further supported by the appearance of new band due to metal–
oxygen bond formation in region 510–534 cm-1 [29].
Table 2
Broad band in the region 3290–3315 cm-1 point toward the presence of coordinated water
molecules in the complexes 1–8 [29]. This is further supported by the presence of band
corresponding to (–OH ) rocking and wagging mode of vibrations 765–793 and 710–723
cm-1.
The comparison of the IR data of the ligand and the corresponding metal ion enlighten the
tridentate nature of the synthesized Schiff base ligands H2L1and H2L2, which coordinates
through azomethine nitrogen, thiol sulphur and oxygen of phenol group. Based on above
spectral observations, we conclude that both ligands coordinate binegative tridentately around
the Co(II), Ni(II), Cu(II) and Zn(II) centre.
3.5 Conductance and magnetic susceptibility measurements
The molar conductance values of metal complexes were calculated at room temperature
taking DMF as a solvent and the results are displayed in Table 3. The molar conductance
value are generally used to predict the electrolytic or non-electrolytic nature of the metal
complexes. The obtained value of molar conductance (5.4–11.6 ohm-1 cm2 mol-1) for metal
complexes 1–8 hint their non-electrolytic behavior. The magnetic moment values of 4.98–
5.03 B.M. for Co(II) complexes undoubtedly suggest high spin octahedral geometry with
three unpaired electron for Co(II) complexes. The magnetic moment value of 2.94–2.98 B.M.
for Ni(II) complexes corresponds to two unpaired electrons and octahedral geometry [30].
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Similarly, Cu(II) complexes show magnetic moment values of 1.81–1.83 B.M., which
indicates one unpaired electron.
Table 3
3.6 Electronic spectra
The electronic absorption spectra of the Co(II), Ni(II) and Cu(II) complexes were recorded in
DMSO at room temperature and the result are listed in Table 3. The electronic spectra of
Co(II) complexes two transitions corresponding 10112–10749 cm–1 and 19668–19998 cm–1
to 4T1g(F)→4T2g(F) (ν1); 4T1g(F)→4T1g(P) (ν3), ν2 which is very close to (ν3) transition is not
observed, but it can be calculated [31] by using relation ν2 = ν1+10Dq. The ligand field
parameters (Dq, B, β, β%) have also been calculated for Co(II) complexes by using Band-
fitting equations [32]. The Racah parameter (B) is found to be 671-737 cm-1 (< 971 cm-1),
suggesting an overlapping of ligand metal orbital's. The nephelauxetic ratio (β) for cobalt
complexes is less than one suggesting partial covalency in the metal ligand bond. These
ligand field parameters suggest octahedral geometry for Co(II) complexes [32].
The electronic spectral data of Ni(II) complexes showed d-d bands in the region 10,713–
11,044, 15,656–16,415 and 24,125–25,675 cm-1, respectively, assigned to the transitions
3
3
3A2g(F) →3T2g(F), A2g(F) →3T1g(F) and A2g(F) →3T1g(P). The position of these bands
indicates that the complexes 2 and 6 have octahedral geometry around the Ni(II) centre. The
ligand field parameters (Dq, B, ) have also been calculated for Ni(II) complexes using
the Band-fitting equations. Racah parameters B is less than the free ion value i.e. 1041 cm–1,
which indicates covalent character of the metal ligand bond and overlapping of the ligand
metal orbitals. The crystal field splitting energy (Dq) value were found to be in the range
1071–1104 cm–1 suggested octahedral geometry for Ni(II) complexes. The ratio of the
 and further supported the octahedral geometry around the Ni(II) centre
[26,32].
Electronic absorption spectra of the copper complexes 3 and 7 display a transition in the
2
2
region 18978-20012 cm-1, which can be assigned as the B1g – A1g transition, revealing that
the Cu(II) complexes exist in the square planar geometry [33,34].
3.7 Thermal Analysis
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o
Thermogravimetric study of the synthesized metal complexes was done up to 1000 C at a
heating rate of 10 oC/min in nitrogen atmosphere. Metal complexes 1–4 and 5–8 show similar
decomposition pattern as evident from their TGA graphs. Complex 1 ([Co(L1)(H2O)3]) show
o
three step decomposition (Fig. 4) within temperature range of 140–660 C. First step
corresponds to the loss of three coordinated water molecules (Found 12.34%, calcd. 12.32%)
o
in temperature range of 140–170 C. The second decomposition step in temperature range
o
170–390 C with mass loss of 30.28% (calcd. 30.37%) corresponding to loss of C8H7NO. A
peak corresponding to mass loss of 40.24% (calcd. 40.20%) at 390–660 oC was due to the of
loss of triazole moiety in the third step and as a final product, it leaves CoO as residue (Found
1710%, calcd. 17.12%). The thermal decomposition for this complex can be formulated as
follows:
C15H17CoN5O5S)
[Co(L1)(H2O)3]
140–170 oC
(C15H11CoN5O2S) 170–390 oC
(C7H4CoN4OS)
( -3H2O)
- C8H7NO
390–660 oC
CoO
C7H4N4S
Similar behaviour was observed in the TGA curve of metal complexes 2–3, while the metal
complex 4 display two steps dissociation. Typical TGA curve for metal complexes 1–4 is
presented in Fig. 4. The temperature ranges and percentage mass losses of the decomposition
reactions for the metal complexes 1–4 are given in Table 4 together with evolved moiety and
the theoretical percentage mass losses.
fig. 4
Table 4
3.8 EPR spectra
EPR spectra of Cu(II) complexes were recorded at room temperature as polycrystalline
sample, on X band at frequency of 9.1 GHz under the magnetic-field strength of 3000G. The
analysis of Cu complex (3) gives g∥ = 2.148, g⊥ = 2.067, gav= 2.094 and G = 2.20 (Table 5).
Similarly, the g-tensor values observed for complex 7 are g∥ = 2.241, g⊥ = 2.074, gav= 2.12
and G = 3.25. These g-values have been used to derive the ground state. In square planar
complexes, the unpaired electron lies in dx2-y2 orbital giving 2B1g as the ground state with g|| >
g⊥ > 2, while the unpaired electron lies in the dz2 orbital giving 2A1g as the ground state with
g⊥ > g|| > 2. In the present cases, the g|| > g⊥ > 2, therefore the unpaired electron is likely to
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be in the dx2-y2 orbital indicates square planar geometry around the copper(II) ion [35]. The
possibility of the dimeric form was ruled out as no signal was observed in the spectrum at
half field [36]. This is also supported by the magnetic moments of Cu complexes (1.81–1.83
BM) which confirm the mononuclear nature of the complexes.
Orbital reduction factors, K∥ and K⊥ values which are the measure of the spin-orbit coupling
constant λo =823 cm-1 for free copper(II) are obtained using the expression reported by
Hathaway et. al. [37] and Stevens et. al. [38]. The K∥ and K⊥ parameters (Table 5) are
calculated by using the relation K∥ = ca . and K⊥ = ca . where and  are the
coefficients of dx2 – y2, dxy and dxz, dyz orbitals in the MO's to which they contribute; thus  β1
and β measures σ-bonding, in-plane π-bonding and out-of-plane π bonding coefficients,
respectively. By assuming  = 1 (since the ligand has no bond pair available on the N atom
for bonding with dxy) the values of and  are calculated for copper(II) complexes. Both
values indicate covalent character in metal–ligand bond.
Table 5
3.9 Protein binding studies
Fluorescence quenching during intermolecular interaction is a common phenomenon that
occurs due to various processes such as excited-state reactions, molecular rearrangements,
energy transfer, ground-state complex formation and collision quenching [39]. The
fluorescence spectra of BSA in presence of metal complexes 1–4 has a strong emission
band (em) at 327 nm due to the tryptophan residues of BSA . The metal complexes 1–4 used
in the present study are expected to interact with the tryptophan residue of the BSA. Owe to
this, fluorescence quenching experiments were performed with BSA in the presence of
different concentrations to assess binding constants for binding of complexes 1–4 with BSA.
The fluorescence quenching experiment of BSA with metal complex 1 (Fig. 5(a)) clearly
elucidate that on keeping BSA concentration constant and increasing the metal complex
concentration from 6.62–33.2 decrease the emission intensity of BSA considerably. The
quenching observed may possibly be due to complexation of BSA with complexes [40].
Similar shift in fluorescence spectra of BSA in presence of different concentration of metal
complex was reported by Li et. al. [41]. The red shift observed was because of increase in
polarity of the microenvironment around the tryptophan residues after binding of complex 1
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with BSA. Possibly, it was due to loss of the compact structure of hydrophobic sub domain
IIA where tryptophan-214 is placed [42]. Similarly, fluorescence quenching experiment of
BSA with metal complex 2 (Fig. 5(b)) clearly explain that on keeping BSA concentration
constant and increasing the metal complex concentration from 6.62–33.2 decrease the
emission intensity of BSA considerably. The maximum intensity of BSA in absence of Ni
complex (2) was observed at 328 nm and addition of complex 2 with increasing concentration
causes a concentration dependent quenching of intrinsic fluorescence of BSA accompanied
with a red shift in the maxima from 327 to 334 nm (Fig. 5(b)).
In the fluorescence quenching experiment of copper complex (3) with BSA, the maximum
intensity of BSA in absence of complex (27) was observed at 327 nm and addition of
complex 27 cause a red shift in the maxima from 327 to 333 nm. (Fig. 5(c)). In fluorescence
quenching mechanism of zinc complex (4), addition of complex 4 quenched the emission
intensity of BSA at 336 nm. This quenching in intensity was accompanied with a blue shift
from 336 to 324 nm (Fig. 5(d)). It has been reported in literature that interaction between
some small molecules viz. PAAB, vitamin B12 or cyanocobalamin and colchicines with
BSA, blue shift of around 5–8.5 nm was observed in the emission spectra [43–45]. The blue
spectral shift indicates increase in hydrophobicity and decrease in polarity around the
tryptophan residues. Similar result was observed in fluorescence quenching of metal complex
4 with BSA. A blue shift of 12 nm in our result is comparable to blue shift values reported in
literature. This suggests that the binding interaction between the complex 4 and BSA result in
enhancement of hydrophobicity around the tryptophan residues of BSA.
Fig. 5
To investigate the fluorescence quenching mechanism for metal complexes 1–4 with BSA,
the fluorescence data were further analyzed with Stern-Volmer equation.
F0/F =1+Ksv [Q]
Where, F0 and F are the fluorescence intensities of BSA in the absence and presence of the
quencher molecule, Ksv is the Stern-Volmer quenching constant and [Q] is the concentration
of the quencher metal complex. The Stern-Volmer plots of metal complexes 1–4 with BSA
are given in Fig. 6.
Fig. 6
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The value of bimolecular quenching rate constant (kq) was obtained according to Equation
F0/F =1+Ksv [Q] = 1+kq0 [Q]
Where, kq is bimolecular quenching rate constant; o is the lifetime of BSA in the absence of
quencher and Ksv is the Stern Volmer constant. The value of o for BSA has been reported in
many studies and was found to be around 10-9 s. From the findings of the fluorescence
quenching study, the value of kq for metal complexes 1–4 (3.50–3.78 x 1013 M-1 s-1) was
much greater than the maximum scattering collision quenching constant (2 x 1010 M-1 s-1)
[46,47] which indicates that the probable quenching mechanism is static quenching. The
values of Ksv and Kq for the metal complexes 1–4 are given in Table 6.
Table 6
Number of binding sites can be calculated from fluorescence titration data using the
following equation:
log (F0–F)/F = log Kb + n log [Q]
where Kb and n are the binding constant and binding site for metal complexes 1–4. On
plotting log[Fo/F] verses log[Q] (Fig. 7), a linear graph was obtained. The value of Kb and n
were calculated from the slopes and intercepts of the linear plots. The binding constant and
number of binding sites (n) are given in Table 6. The binding constant (Kb) value indicates
Cu and Zn complexes strongly bind to BSA. Again, the value of n is nearly 1 for binding of
both complexes to the protein used, which indicates the high affinity binding sites of
albumins. The value of n suggested that there were single class of independent binding sites
[48]. The corresponding Kb and n values were evaluated from the slopes and intercepts of the
linear plots, respectively. The binding constant and number of binding sites (n) are given in
Table 7.
Fig. 7
Table 7
3.10
Geometry optimization
Geometry Optimization was done using B3LYP functional with 6-31+g(d,p) basis sets as
incorporated in the Gaussian 09W programme in gas phase. The fully optimized geometries
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of the ligand H2L1 and complexes 1–4 are shown in Fig. 8. The numbering scheme for ligand
H2L1, and complexes 1–4 are given in Scheme 2. The values of the bond length and bond
angles calculated for metal complex 1 shows octahedral geometry around the Co(II) ion
(Table 8). A slight elongation in bond lengths C1–N1 , N2–C8, N2–C9 was noted in complexes
1 as ligand H2L1 coordinates via azomethine nitrogen. The –OH and –SH group deprotonate
and coordinated to Co(II) ion and no appreciable change was noted in bond lengths C3– O1
and C9–S1 after metal coordination. The C3– O1 and C9–S1 bond lengths was found to be
1.464 and 1.787 Ao, respectively. The four equatorial positions are occupied by azomethine
nitrogen, thiol sulphur and two water molecules, while two axial positions are occupied by
water molecule and oxygen of phenol group. The bond angles in the coordination sphere of
Co(II) complexes are found approximately near to the perpendicular value. The value of bond
angle NMS1MN1, O1MO11, O1MS1 and O11MO12 was found to be 85.46, 86.12,
87.23 and 94.12o, respectively. No appreciable change in bond lengths and bond angle was
seen in 1,2,4-triazole moiety and benzene ring.
Fig. 8
Scheme 2
Table 8
Metal complex 2 having Ni(II) as metal centre display similar behaviour and exist in
octahedral environment. The calculated bond lengths and bond angles lie in same range of the
calculated bond lengths and bong angles value of complex 1. However, complex 3 having
Cu(II) metal centre shows square planar arrangement around the metal centre. We also try to
optimize Cu(II) complex in octahedral arrangement by adding two more water molecule to
the metal centre. But the attempt to optimize the complex was failed as the Cu–S1 bond
length elongates to 4.2 Ao in the final structure, which causes bond to break and the five
membered ring of the 1,2,4- moiety flip out of the plane. Thus the square planar geometry for
Cu(II) is stable which also correlate our experimental results. A noteworthy point is that after
complexation bond length around the metal centres becomes slightly smaller. The bond
length C1–N1 , N1–N2, N2–C8 and N2–C9 becomes shorter by ~ 0.1 Ao. This clearly predicts
stronger complexation between ligand and metal centre. All the bond lengths and bond angles
were found to be in their expected range.
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Similar results was noted in complex 4 having zinc metal centre. All the computed bond
lengths and bond angles are listed in Table 5. To evaluate the extent of complexation and to
find the stability order, we have calculated the binding energy of the four complexes and the
result found was: (Binding Energy)3 > (Binding Energy)4 > (Binding Energy)1 > (Binding
Energy)2. On the basis of the above discussion following (Fig. 9) structures can be proposed
for the synthesized complexes.
Fig. 9
3.11
Cell viability determination
The in vitro cytotoxicity of the ligand H2L1, H2L2 and metal complexes (1–8) on human cell
lines HepG2 and MCF-7 was determined by a MTT based assay. The results are expressed as
the percentage of cell toxicity with respect to the control and are represented in Table 8. From
the MTT assay data, it can be concluded that control shows a toxicity of 0.25–0.50 % on cell
line HepG2 and MCF-7. Ligand H2L2 i.e. Schiff base derived from nicotinic acid hydrazide
display better activity in comparison to Schiff base derived from isonicotinic hydrazide
(H2L1). Ligand H2L2 show a cytotoxicity of 10 % and 9% against cell line HepG2 and MCF-
7 at concentration of 5 µM in comparison to 8% and 5% for ligand H2L1. Also, metal
complexes show higher cytotoxicity in comparison to free ligands. Metal complexes 4, 7 and
8 display cytotoxicity of 13, 20 and 15% against cell line HepG2, whereas 14, 18 and 19%
cytotoxicity was revealed against cell line MCF-7. On increasing the concentration from 5
µM to 10 µM no appreciable change in cytotoxicity against cell line HepG2 and MCF-7 was
observed for ligand H2L1 and H2L2. But at concentration of 10 µM complexes 3, 4, 7 and 8
show a significant increase in % inhibition on cell proliferation on cell line HepG2 (26, 22,
39 and 31%). Similarly, the complexes 3, 4, 7 and 8 shows a significant increase in %
inhibition on cell proliferation on cell line MCF-7 (22, 19, 30 and 35%). Cu(II) complexes
show best cytotoxicity to cancerous cells, followed by Zn(II) complexes and Co(II)
complexes. Ni(II) complexes show the least activity. Graphical representation of the
anticancer cell line viability of ligand H2L1, HL2 and their metal complexes 1–8 against cell
line Hep G2 and MCF-7 is shown in Fig. 10 and tabulated in Table 9.
Table 9
Fig. 10
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4. Conclusion
The newly synthesized Schiff base ligands H2L1, H2L2 coordinates in a tridentate fashion
around the metal (II) centre. Based on various physio-chemical techniques, Co(II) and Ni(II)
complexes possessed an octahedral geometry, while Cu(II) complexes exist in square planer
and Zn(II) form tetrahedral geometry. The presence of coordinated water was confirmed by
the TGA and IR data. The reasonable agreement between the theoretical and experimental
data reflects to the great extent the suitability of the applied basis set, 6-31+g(d,p) for this
type of work and confirms the suggested structure. The result of interaction of metal
complexes with BSA reveal that Zn complex (4) binds more strongly to BSA, followed by Cu
(3), Co(1) and Ni(2). Results of MTT assay revealed all metal complexes show moderate to
significant % inhibition on the cell proliferation on cell line HepG2 and MCF-7. Schiff base
derived from nicotinic acid hydrazide display better activity in comparison to Schiff base
derived from isonicotinic hydrazide. This inhibition on cell proliferation might be due to
azomethine (-HC=N-) linkage and/or hetero atoms present in these compounds.
5. Acknowledgements
We thank the Principal, Zakir Husain Delhi College for providing lab facilities. Authors are
thankful to DRDO, for financial assistance.
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Figure Captions
Fig. 1. Some triazole based market available drugs
Scheme 1. Preparation of the ligands H2L1, H2L2 and their metal complexes 1–8
Fig. 2. Mass Spectrum of metal complex 2
Fig. 3. 1H NMR spectra of ligand H2L1
Fig. 4. Thermogravimetric curves of metal complexes (1) [Co(L1)(H2O)3]; (2)
[Ni(L1)(H2O)3]; (3) [Cu(L1)(H2O)]; (4) [Zn(L1)(H2O)]
Fig. 5. Fluorescence quenching spectra of BSA at different concentrations of complexes 1(a),
2(b), 3(c), 4(d); [BSA] = 15 μM; [Complex 1–4] varied from 6.62–32.2 μM.
Fig. 6. Stern–Volmer Plot F0/F versus [Q] for the Binding of BSA with metal complexes 1–
4
Fig. 7. Stern–Volmer Plot log [F0-F /F] versus log [Q] for the Binding of BSA with metal
complexes 1–4
Fig. 8. Geometry Optimized structures of (a) Ligand H2L1, (b) Complex 1, (c) Complex 2,
(d) Complex 3, (d) Complex 4 (Colour Code: H=White, C=Grey, N= Blue, O=red, Co=
Grey, Ni= Silver Grey, Cu=Pink, Zn=grey)
Scheme 2. : Numbering Scheme of the optimized structures (a) Ligand H2L1, (b) Complexes
1–2; 5–6 (c) 3–4; 7–8
Fig. 9. Proposed Structure of the newly obtained metal complexes
Fig. 10. Ligand H2L1, H2L2 and metal complexes (1–8) inhibited the cell viability of HepG2
& MCF-7 cells (Data were assayed by ANOVA and Student’s t-test. Differences between
means were considered significant when yielding a P < 0.05. Results are presented as means
± S.D.
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Fig. 1. Some triazole based market available drugs
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Fig. 2. Mass Spectrum of metal complex 1
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Fig. 3. 1H NMR spectra of ligand H2L1
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Fig. 4. Thermogravimetric curves of metal complexes (1) [Co(L1)(H2O)3]; (2)
[Ni(L1)(H2O)3]; (3) [Cu(L1)(H2O)]; (4) [Zn(L1)(H2O)]
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Fig. 5. Fluorescence quenching spectra of BSA at different concentrations of complexes 1(a),
2(b), 3(c), 4(d); [BSA] = 15 μM; [Complex 1–4] varied from 6.62–32.2 μM.
26

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Fig. 6: Stern–Volmer Plot F0/F versus [Q] for the Binding of BSA with metal complexes
1–4
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Fig. 7 : Stern–Volmer Plot log [F0-F /F] versus log [Q] for the Binding of BSA with metal
complexes 1–4
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Fig. 8. Geometry Optimized structures of (a) Ligand H2L1, (b) Complex 1, (c) Complex 2,
(d) Complex 3, (d) Complex 4 (Colour Code: H=White, C=Grey, N= Blue, O=red, Co=
Grey, Ni= Silver Grey, Cu=Pink, Zn=grey)
29