Substitution of PPh3+ as a lipophilic cation on new water-soluble Co(II) and Zn(II) Schiff base complexes: Effect of central metal and substitutional group of ligand on DNA-complex interaction

Article abstract of DOI:10.1016/j.jphotochem.2017.01.022

New series of water soluble Schiff base complexes by conjugating the lipophilic triphenylphosphonium cation to aromatic moiety of Schiff base ligand, aiming to increase the water solubility and passing through cell membrane were synthesized and characterized. Four new complexes; [N,N′-bis{5-[(triphenylphosphoniumchloride)-methyl]salicylidine}-phenylenediamine] with the formula [Zn(5-CH₂PPh₃-4-H-1,2-salphen)] (ClO₄)₂ (1), [Zn(5-CH₂PPh₃-4-CH₃-1,2-salphen)](ClO₄)₂ (2), [Zn(5-CH₂PPh₃-4-Cl-1,2-salphen)](ClO₄)₂ (3), [Co(5-CH₂PPh₃-4-CH₃-1,2-salphen)](ClO₄)₂ (4) were characterized by elemental analysis, FT-IR, ¹H NMR, ¹³C NMR, ³¹P NMR and UV–vis spectroscopy. Cobalt(II) complex also characterized by thermal gravimetry analysis (TG). DNA interaction studies were investigated by UV–vis absorption spectroscopy, viscosity measurements, circular dichroism (CD) spectroscopy and fluorescence spectroscopy. The DNA binding affinity (K_b) obtained from absorption spectroscopy had this order: 2?>?3?>?4?>?1?>?ligand(L) which revealed that metal complexes had more propensities to interact with DNA. In comparing the metal complexes; Zn(II) complexes revealed DNA binding stronger than the corresponding Co(II) analogue. All the complexes displayed cytotoxicity against Raji, Jurkat and A549 cell lines with potency more than that of cisplatin and thus they are good candidate to act as promising anticancer drug. Finally, docking studies were performed to further investigate the DNA binding interactions.

Full text of DOI:10.1016/j.jphotochem.2017.01.022

Accepted Manuscript
Title: Substitution of PPh3+ as a lipophilic cation on new
water-soluble Co(II) and Zn(II) Schiff base complexes: effect
of central metal and substitutional group of ligand on
DNA-complex interaction
Authors: Zahra Asadi, Elahe Haddadi, Moslem Sedaghat
PII:
S1010-6030(16)31088-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2017.01.022
JPC 10511
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
18-11-2016
14-1-2017
17-1-2017
Please cite this article as: Zahra Asadi, Elahe Haddadi, Moslem Sedaghat, Substitution
of PPh3+ as a lipophilic cation on new water-soluble Co(II) and Zn(II) Schiff base
complexes: effect of central metal and substitutional group of ligand on DNA-
complex interaction, Journal of Photochemistry and Photobiology A: Chemistry
http://dx.doi.org/10.1016/j.jphotochem.2017.01.022
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Substitution of PPh3 as a lipophilic cation on new water-soluble Co(II) and Zn(II)
Schiff base complexes: effect of central metal and substitutional group of ligand on
DNA-complex interaction
Zahra Asadi, Elahe Haddadi, Moslem Sedaghat
Department of Chemistry, College of Sciences, Shiraz University, Shiraz 71454, Iran
* Corresponding author. E-mail address: zasadi@shirazu.ac.ir
Tel: +98 713 6137118; Fax: +98 713 646 0788
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Graphical abstract
Highlights
 New series of water soluble complexes were synthesized and characterized.
 DNA binding potency of these complexes was investigated with different methods.
 Cytotoxicity of these complexes against Raji, Jurkat and A549 cell lines were studied.
 Docking studies were performed to further investigate the DNA binding interactions
Abstract
New series of water soluble Schiff base complexes by conjugating the lipophilic
triphenylphosphonium cation to aromatic moiety of Schiff base ligand, aiming to increase the
water solubility and passing through cell membrane were synthesized and characterized. Four
new complexes; [N,Nʹ-bis{5-[(triphenylphosphoniumchloride)-methyl]salicylidine}-
phenylenediamine] with the formula [Zn(5-CH2PPh3-4-H-1,2-salphen)] (ClO4)2 (1), [Zn(5-
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CH2PPh3-4-CH3-1,2-salphen)](ClO4)2 (2), [Zn(5-CH2PPh3-4-Cl-1,2-salphen)](ClO4)2 (3), [Co(5-
CH2PPh3-4-CH3-1,2-salphen)](ClO4)2 (4) were characterized by elemental analysis, FT-IR, 1H
NMR, 13C NMR, 31P NMR and UV-vis spectroscopy. Cobalt(II) complex also characterized by
thermal gravimetry analysis (TG). DNA interaction studies were investigated by UV-vis
absorption spectroscopy, viscosity measurements, circular dichroism (CD) spectroscopy and
fluorescence spectroscopy. The DNA binding affinity (Kb) obtained from absorption
spectroscopy had this order: 2341ligand(L) which revealed that metal complexes had more
propensities to interact with DNA. In comparing the metal complexes; Zn(II) complexes revealed
DNA binding stronger than the corresponding Co(II) analogue. All the complexes displayed
cytotoxicity against Raji, Jurkat and A549 cell lines with potency more than that of cisplatin and
thus they are good candidate to act as promising anticancer drug. Finally, docking studies were
performed to further investigate the DNA binding interactions.
Keywords: water soluble, Schiff base complex, DNA, DFT, docking study, anticancer activity.
1. Introduction
Transition metal Schiff base complexes are important compounds due to their structural
similarities with natural biological molecules, like vitamin B12[1] and they have biological
activities including antifungal, antibacterial, anticancer and herbicidal applications[2-14]. Such
inorganic compounds prepare wide field for the design of metal based drugs. Among transition
metals, the first row transition metals have attracted special attention. Studies on their DNA
binding affinity and other biological activities have been done during recent years[15-17]. The
reasons of this attention are directly related to the coordination and redox properties of transition
metal compounds[18]. To design effective anticancer drugs, it is essential to assess the
interactions of metal complexes with DNA[19]. These kinds of studies are also important to
understand the toxicity of drugs [20-23].
Complexes bind to DNA via covalent and non-covalent interactions. Cisplatin; an important
anticancer drug; bind to DNA through covalent interaction but this kind of interaction has
significant side effects because of the formation of covalent cross-links. Drugs that interact
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through non-covalent modes are of great importance, this mode of binding contains electrostatic
interaction, groove binding and intercalation. Intercalation of drugs is related to the type of metal
and ligand[24]. Metal complexes with aromatic ligands intercalate between DNA base pairs
through the - interaction of the aromatic section with the stacked aromatic planes of the
nitrogen bases. Cationic molecules have electrostatic interactions with the negatively charged
phosphate groups of DNA. In groove binding, hydrogen bonding of metal complexes with
oxygen and /or nitrogen atoms of the DNA bases or with the sugar fragments, plays an important
role in interaction[25].
Transition metal compounds are of great interest as potential drugs because they are essential
for the normal functioning of living organisms. In many researches, zinc(II) complexes showed
biological activity such as antimicrobial efficacy, antibacterial and DNA interaction[26-29]. For
studying the biological activity of the metal complexes, it is important to select a green solvent
for example H2O, and provide natural biological condition. Most of the metal Schiff base
complexes are not soluble in water but with providing some ionic groups on the Schiff base
ligand the hydrophilicity of the complex can be increased. Recently, Murphy and Smith
published a review on mitochondria-targeted antioxidants which developed by conjugating the
lipophilic triphenylphosphonium cation which placed on an antioxidant moiety[30]. The main
characteristics of these compounds are their ability to pass easily through all biological
membranes.
In recent years, we have studied the interaction of some water soluble metal Schiff base
complexes with human serum albumin (HSA)[31] and bovine serum albumin (BSA)[32-34]. In
this paper, the interaction of some zinc(II) and cobalt(II) water soluble Schiff base complexes
with Herring Sperm DNA (HS-DNA) was studied. The zinc(II) complexes possessed different
substitutional groups on the amine bridge of the Schiff base (Scheme 1). The final product,
[M(5-CH2PPh3-4-R-1,2-salphen)](ClO4)2 (M=Zn, R=H (1), M=Zn, R=CH3 (2), M=Zn, R=Cl (3),
M=Co, R=CH3 (4), and the free ligand were fully characterized by various physicochemical
techniques, namely, elemental analyses and spectral (FT-IR, 1HNMR, 13CNMR, 31PNMR, UV-
vis). The binding property of the complexes and ligand with HS-DNA under physiological
conditions was studied using UV-vis spectrophotometry, DNA viscosity measurements,
fluorescence spectroscopy and circular dichroism (CD) spectroscopy. Also the anticancer
activities of the complexes and molecular docking studies were performed. The main goal of this
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study is investigation of the structural features on the DNA binding interactions. Two important
factors in the interaction of complexes with DNA (external binding and π–π stacking) were
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considered in the designing of complexes by putting CH2PPh3 substituent on the aromatic
moiety of the Schiff base ligand. These groups also increased the water solubility of the
complexes and their easily passing through all biological membrane.
2. Experimental
2.1. Reagents and instruments
All chemicals were purchased commercially. Experiments were performed in Tris-HCl buffer
at pH 7.2. Stock solution of FS-DNA was prepared by dissolving approximately 1-2 mg of FS-
DNA in 2 mL Tris-HCl buffer and stored for 24h at 4°C. The concentration of HS-DNA solution
was determined by spectrophotometry at 260 nm using an extinction coefficient (ε) of 6600 M-
1cm-1. DNA solutions were used no more than 4 days. A solution of FS-DNA in the buffer gave a
ratio of UV absorbance at 260 and 280 nm of 1.98, indicating that the DNA was sufficiently free
from protein[35]. The 1HNMR and 13CNMR spectra were recorded on Bruker Avance DPX
250MHz spectrometer. 31PNMR spectra were recorded on Bruker Ultrashield 400MHz
spectrometer. Elemental analysis for C, H and N were carried out by Thermo Fininngan-Flash
1200. FT-IR spectra were recorded as KBr pellets on a Shimadzu FTIR 8300 infrared
spectrophotometer in the range of 4000–400cm-1. Perkin–Elmer (LAMBDA 2) UV-vis.
spectrophotometer was used to measure the UV-vis. spectra and Perkin-Elmer (LS45)
spectrofluorimeter equipped with a Lauda-ecoline-RE 104 thermostat for emission spectra.
Thermal gravimetric analyses were recorded on a Perkin-Elmer Pyris Diamond model. Circular
dichroism spectra of DNA were obtained by using Aviv Model 215 spectropolarimetre equipped
with a peltier temperature control device.
2.2. Synthesis
2.2.1. Synthesis of (3-formyl-4-hydroxybenzyl)triphenylphosphoniumchloride
A mixture of salicylaldehyde (0.08mol), para-formaldehyde (0.05mol) and 50mL of conc.
HCl was stirred at room temperature for 48h. The resulting dark red powder precipitate was
collected by filtration, washed with 0.5% NaHCO3 solution then water, and dried in vacuum. The
product was crystallized from acetonitrile and petroleum ether. A mixture of 5-
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chloromethylsalicylaldehyde (0.06mol) and triphenylphosphine (0.06mol) in 200mL acetonitrile
was refluxed for 8h and cooled. The precipitated phosphonium salt was filtered off and washed
with ether.
Yield: 41%, m.p.=250-251°C, Color: white, Anal. Found(Calc.):C26H22PO2Cl: C, 72.27(72.14);
H, 5.14 (5.12). FT-IR (KBr, cm–1): 3741(νOH), 2869(νC-H), 1674(νC=O). 1H NMR (250 MHz,
DMSO-d6): δ (ppm) = 11.18 (s, 1H, OH),10.14 (s, 1H, CHO), 7.87 (dd, J = 10.1, 4.5 Hz, 3H,
ArH), 7.69 (dq, J = 12.5, 7.6 Hz, 12H, ArH), 7.26 – 7.15 (m, 1H, ArH), 7.11– 6.92 (m, 2H,
ArH), 5.13 (d, J = 15.0 Hz, 2H, CH2). (spectral data in Fig.S1)
Synthesis of [N,Nʹ-bis{5-[(triphenylphosphoniumchloride)-methyl]salicylidine}-
phenylenediamine] (L)
To a vigorously stirred solution of (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride
(1.25mmol) in 50 mL water, an ethanolic solution (20 mL) of pheneylenediamin (0.62 mmol)
was added dropwisely. The solution turned to light yellow and the mixture refluxed for 3 h. After
that, NaClO4 (1.25 mmol) dissolved in a minimum amount of water (5 mL) and was added. The
resulting yellow powder (Scheme 1) was collected by filtration, washed with cold ethanol and
ether and dried in the air.
Yield: 85%. m.p (>310°C), Color: light yellow. Anal. Found (Calc.) for C58H48Cl2N2O10P2: C,
65.55 (65.36); H, 4.57 (4.54); N, 2.50 (2.63). FT-IR (KBr, cm–1): 1650 (νC=N), 1487 (νC=C), 1087,
650 (νClO4-). 1HNMR (250 MHz, DMSO-d6) δ (ppm): 11.12 (s, 2H, OH), 8.53(s, 2H, HC=N),
7.87- 6.82 (m, 40H, ArH) 5.06 (dd, J = 15.1, 3.1Hz, 4H, CH2P). 13C NMR (250 MHz, DMSO-
d6) δ (ppm): 160.9, 160.8 (C=N), 138.0, 137.9, 135.0, 134.0, 133.9, 131.5, 130.4, 130.1, 129.9,
128.8, 122.4, 118.2, 118.0, 117.95, 116.9 (aromatic carbons), 34.2, 33.9 (CH2). 31P NMR (400
MHz, DMSO-d6) δ (ppm): 22.58. UV–vis. (H2O): λmax (nm) = 387, 333. (spectral data in Figs.
S2, S3, S4).
2.2.2. Synthesis of the complexes
To a vigorously stirred solution of (3-formyl-4-hydroxybenzyl)triphenylphosphonium
chloride (1.25mmol) in 50mL water, metal(II) acetate (0.62mmol) dissolved in water (l0mL) was
added. Then an ethanolic solution (20mL) of diamine (0.62 mmol) was added dropwisely to
synthesize the complexes. The solution turned to yellow and the mixture refluxed for 3h. After
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that, NaClO4 (1.25mmol) was dissolved in a minimum amount of water and added to the reaction
mixture. The resulting yellow powder was collected by filtration, washed with cold ethanol and
ether and dried in the air. All steps in synthesis of Co(II) complex were done under N2
atmosphere.
a)[N,Nʹ-bis{5-[(triphenylphosphoniumchloride)-methyl]salicylidine}-phenylenediaminato]
zinc(II)perchlorate (1), Yield: 75%, Color: Yellow, m.p.>250°C, Anal. Found (Calc.) for
C58H46Cl2N2O10P2Zn.H2O: C, 60.55 (60.78); H, 4.17 (4.13); N, 2.40 (2.44). FT-IR (KBr, cm–1):
3440(νO-H), 1620(νC=N), 1527(νC=C), 1103, 686(νClO4), 617 (νM-N), 493(νM-O).1H NMR (250 MHz,
DMSO-d6) δ (ppm) = 6.41-7.86 (m, 40H, ArH), 8.52 (d, 2H, HC=N), 4.90 (d, 4H,CH2P). 13C
NMR (250 MHz, DMSO-d6) δ (ppm): 162.1, 161.3 (C=N), 157.7, 149.7, 146.6, 144.7, 137.5,
135.0, 134.8, 134.0, 133.9, 130.2, 130.0, 129.6, 123.6, 123.2, 118.5, 117.7, 116.1, 113.7
(aromatic carbons), 34.6, 34.2 (CH2). 31P NMR (400 MHz, DMSO-d6) δ (ppm): Pa (21.77), Paʹ
(21.69). UV-vis.(H2O): λmax(nm) = 382, 268. (spectral data in Figs.S2, S3, S4)
b)[N,Nʹ-bis{5-[(triphenylphosphoniumchloride)-methyl]salicylidine}-4-methyl-phenylene
diaminato]zinc(II)perchlorate (2). Yield: 88%; m.p. >250°C, Color: Yellow, Anal. Found
(Calc.): C59H48Cl2N2O10P2Zn.H2O: C, 61.14 (61.02); H, 4.36 (4.34); N, 2.43 (2.41). FT-IR (KBr,
cm–1): 3463(νO-H), 1620(νC=N), 1473(νC=C), 1095, 694(νClO4), 624(νM-O), 493(νM-N). 1H NMR (250
MHz, DMSO-d6) δ (ppm) = 8.51(s, 1H, HC=N), 8.47(s, 1H, HC=N), 6.40-7.87 (m, 41H, ArH),
4.93 (d, 4H, CH2P), 2.37 (s, 3H, CH3).13C NMR (250 MHz, DMSO-d6) δ (ppm): 162.4,
161.6(C=N), 155.7, 150.5, 146.2, 142.2, 140.3, 139.5, 138.7, 135.0, 134.0, 133.9, 130.2, 130.0,
126.8, 126.0, 122.4, 122.0, 118.5, 117.3, 10.4.6 (aromatic carbons), 34.9, 34.1 (CH2), 30.2
(CH3). 31P NMR (400 MHz, DMSO-d6) δ (ppm): Pa (21.90), Paʹ (21.75). UV-vis. (H2O):
λmax(nm) = 460, 268. (spectral data in Figs. S2, S3, S4)
c)[N,Nʹ-bis{5-[(triphenylphosphoniumchloride)-methyl]salicylidine}-4-chloro-phenylene
diaminato]zinc(II)perchlorate (3), Yield: 61%, m.p>250◦C, Color: Yellow, Anal. Found
(Calc.):C58H45Cl3N2O10P2Zn: C,59.62(59.86); H,3.81(3.90); N, 2.50(2.41). FT-IR (KBr, cm–1):
3440 (νO-H), 1612 (νC=N), 1488 (νC=C), 1110, 686 (νClO4-), 624 (νM-N), 493 (νM-O), 1H NMR (250
MHz, DMSO-d6, room temperature) δ(ppm) =8.51 (d, 2H, HC=N), 6.54-7.19(m, 41H, ArH),
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4.95(d, 4H,CH2P). 13C NMR (250MHz, DMSO-d6) δ (ppm): 162.7, 161.5 (C=N), 155.2, 150.2,
146.3, 142.5, 140.7, 139.0, 138.0, 135.7, 134.2, 133.0, 130.8, 130.0, 126.4, 126.0, 122.5, 122.3,
118.0, 117.0, 104.5 (aromatic carbons), 34.8, 34.6 (CH2). 31P NMR (400 MHz, DMSO-d6) δ
(ppm): Pa (21.95), Paʹ (21.80). UV-vis. (H2O): λmax(nm) =380, 265. (spectral data in
Figs.S2,S3,S4)
d)[N,Nʹ-bis{5-[(triphenylphosphoniumchloride)-methyl]salicylidine}-4-methyl-phenylene
diaminato]cobalt(II) perchlorate (4), Yield: 64%, m.p >250◦C, Color: Brown, Anal. Found
(Calc.): C59H48Cl2N2O10P2Co.H2O: C, 61.49(61.36); H, 4.29(4.36); N, 2.51(2.43), FT-IR (KBr,
cm–1): 3448(νO-H), 1612(νC=N), 1488(νC=C), 1095, 709(νClO4-), 624(νM-N), 501(νM-O). UV-vis.
(H2O): λmax(nm) = 464, 265. TG/DTG: mass loss percent %, Found (Calc.) : H2O: 2 (1.5) ,
2ClO4: 21(20.4). (spectral data in Figs. S3, S4, S5)
CAUTION! The perchlorate salts could be potentially explosive. Therefore, only small
quantities of the sample were handled to avoid any possible explosion.
Solubility and stability
All the synthesized complexes were soluble in water, dimethylformamide (DMF) and
dimethylsulfoxide (DMSO). The complexes were stable in the solid phase at ambient
temperature in the light. The solutions were stable for a long period in light and were not air
sensitive.
2.3. DNA binding experiments
Absorption titration experiments were conducted by keeping the concentration of the
compounds at 50μM while varying the concentration of DNA from 0 to 260 μM. Absorption
values were recorded after each successive addition of DNA solution, followed by an
equilibration period. Fluorescence measurements of the metal complex at an excitation
wavelength of 320 nm, and emission spectra between 340-550 nm were recorded. Each spectrum
was recorded at 200nm/min scan speed and 10nm slit width for both the excitation and emission
monochromators. Fluorescence measurements were carried out by keeping the concentration of
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the complexes at 30μM while varying the DNA concentration from 0 to 260 μM. Viscosity
measurement was performed by using an Ostwald viscometer, immersed in a thermostated
water-bath maintained to 25.0 ± 0.1°C. The DNA concentration was fixed at 10μM, and flow
time was measured with a digital stopwatch. The measurements were carried out three times for
each sample for calculating the average flow time. The plots of relative specific viscosity
(η/ηo)1/3 (where ηo and η are the specific viscosity contributions of DNA in the absence (ηo) and
in the presence of the complex (η)) were drown against ri (ri=[complex]/[DNA]= 0, 0.05, 0.10,
0.15, 0.20, 0.23).
Circular dichroism spectrum of DNA was obtained by using JASCO J-716 spectropolarimeter
equipped with a peltier temperature control device. All experiments were done using a 1 or 0.2
cm path quartz cell. Each CD spectrum was collected after aveRajing over at least 2
accumulations using a scan speed of 100nmmin-1 and a 1s response time. Machine plus cuvette
baselines were subtracted and the resultant spectrum zeroed outside the absorption bands.
2.4. Computational details
The structure of complexes was optimized with density functional theory (DFT) by B3LYP
method and 6-311g basis set for all atoms. In this work we used gauss view 05 for drawing the
structure of these complexes and Gaussian 03 program to ground state geometry optimization
and other calculations.
2.5. Molecular docking
The molecular geometry of complexes was subjected to energy minimization by DFT
calculations at the B3LYP method and 6-311G basis set for all atoms.
The crystal structure of DNA (PDB code: 4HC9) was taken from the protein Data bank
(www.rsbs.org/pdb)[36]. Which had been equilibrated at a nuclei-like condition was used as the
receptor. Then, the optimized compounds were imported to Molegro Virtual Docker (MVD)[37]
to carry out the docking simulations. This software generates the best DNA-ligand
configurations according to several scoring criteria such as Moldock and Rerank scores. In MVD
the units are arbitrary, but an ideal hydrogen bond contributes to the overall energy[38]. We have
selected score as Moldock score [GRID], with GRID resolution of 0.3 Å. Algorithm selected for
docking was Moldock along with number of runs as 20 [39]. For each docking calculation, 10
different poses were done.
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2.6. Cell culture and MTT assay for analysis of anti-cancer properties of the complexes
The growth inhibitory effects of the complexes against three cell lines including Jurkat human
T cell leukemia, Raji Burkitt's lymphoma and A549 lung carcinoma were examined by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. The cell lines were cultured
in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (Gibco,
Berlin, Germany). Cells were maintained at 37ºC with 5% CO2 and 95% humidity. The cells
were fed until confluent and expanded by trypsinization (for A549), then subcultured at lower
numbers in new culture flasks. A predetermined number of cells (15×103 for Jurkat and Raji cells
and 7.5×103 for A549 cells) were seeded in the wells of a 96-well plate with varying
concentrations of the complexes (0.1–100 μg/mL) and incubated at 37ºC with 5% CO2 for 48 h.
Cells treated only with dimethylsulfoxide (solvent) at a concentration equal to the test wells was
used as negative control and those treated only with
cisplatin as positive control. 10 μL MTT solutions (5mg mL-1 in RPMI medium) were added to
each well and incubation was continued for 4 h. At the end, the produced insoluble formazan was
dissolved by adding DMSO and shaking for 10 min. Then, the optical density (OD) was read at
570nm with a reference wavelength of 630 nm in a microplate reader (Bio-Tek’s ELx808, VT).
The percentage of growth inhibition was determined as follows; [1–(test OD/negative control
OD)] × 100. The concentration of 50% cell inhibition (IC50) value was calculated from the graph
of inhibition percentage against different compounds concentrations.
3. Results and discussion
3.1. Spectral properties of the complexes
The ligands and complexes 1-4 were isolated using the procedure reported (Scheme 1). All
the complexes were characterized by elemental analysis, FT-IR, 1H NMR, 13C NMR, 31P NMR,
UV–vis. Spectroscopy. In the 1H NMR spectrum of the precursor aldehyde a single signal at
11.60 ppm was assigned to OH proton. Aldehydic proton was revealed at 10.63ppm and the
aromatic protons were revealed in the range 7.41-8.37 ppm. In the spectra of the ligand, the
singlet signal at 11.09 ppm is assigned to the OH group. However, in the spectra of the
complexes there was no resonance attributable to OH, indicating the coordination of ligand in the
anionic form upon deprotonation. In the spectrum of symmetrical complex 1 a single signal was
observed for the -C(H)=N proton[40, 41] which clearly indicated that the magnetic environment
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was equivalent for the imine protons. But in the 1H NMR spectra of the unsymmetrical
complexes 2, 3 two imine protons were revealed due to different chemical environments. Signals
due to OH of the aldehyde were absent in the spectra of the complexes revealing the
deprotonation of OH groups and coordination through the oxygen atoms[42]. The doublet at
about 4.90 ppm was associated to the CH2 group. 1H NMR spectrum of the cobalt(II) complex
gave no signal due to its paramagnetism.
In the 13C NMR spectra, resonances observed around 160.0 - 163.0 ppm were due to the
azomethine carbons and aromatic carbon atoms observed around 103.0 - 157.0 ppm. The CH2
carbon resonance is observed around 34.0 ppm.
31P NMR spectra were recorded to confirm the presence of triphenylphosphine. One or two
peaks observed at 20.0-23.0 ppm suggested the presence of two equivalent or non-equivalent
triphenylphosphines.
In the FT-IR spectrum of the aldehyde a broad medium intensity band at 3741cm-1 was
assigned to O-H vibration and medium-weak band at 1674cm-1 to the CHO vibration. The
presence of strong imine (C=N) bands at 1634cm-1 (L), 1620cm-1 (1,2) and 1612cm-1 (3,4)
confirmed the formation of the Schiff base ligand and Schiff base complexes. C=N vibration
frequency of 1 shifts toward lower frequencies in comparing with the ligand. This suggested the
coordination of ligand to metal ion through the nitrogen of azomethine which resulted in
decreasing the bond order of (C=N) linkage. Two peaks around 1110 and 700cm-1 were assigned
to the ClO4 groups. The vibration bands around 3440cm-1 was due to the presence of lattice and
coordinated water in the complexes[43]. In the lower frequency region medium-weak bands at
around 620 and 493cm-1 were assigned to (M–N) and (M–O) vibrations, respectively.
In the UV-vis absorption spectra of the compounds, the intensive absorption bands at 260-464
nm were assigned to the π→π* and n→ π* transitions of aromatic rings or azomethine groups of
the Schiff base.
The thermal decomposition of the complex 4 was studied to evaluate its thermal stability.
During the thermal analysis, heating rates were set to 20ºCmin-1 under nitrogen atmosphere, and
the weight loss was measured from the ambient temperature up to 800ºC. This method was also
used to establish that only one water molecule was coordinated to the central metal and the water
molecule did not coordinate strongly and was removed easier than the tetradentate ligand.
Complex 4 decomposed in three steps. The first step was related to dehydration that occurred
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between 240-280C. Dehydration process was associated with exothermic DTA peak. The
second step, which occurred in the range 280-350C, was related to the release of both ClO4
groups. The pyrolysis of remained part of the ligand took place as shown by an exothermic DTA
peak in the range 350-800C. (Fig.S5).
3.2. DNA binding studies
3.2.1. Absorption spectral studies
Generally, determination of the binding strength and the mode of DNA binding with small
molecules is done by using electronic absorption spectroscopy [44]. Two important evidences in
this method are: the change in absorbance and shift in wavelength upon addition of increasing
concentrations of DNA solution in a fixed concentration of metal complexes. These evidences
give valuable information on the mode of interaction. Hypochromism with or without a small red
or blue shift guides to intercalation mode of binding. The intercalative mode involves a strong
stacking interaction between the planar aromatic chromophore and the base pairs of DNA[45,
46]. The extent of hypochromism is commonly consistent with the strength of the intercalative
binding interaction[47-50].
In studying the interactions of the synthesized complexes, one of the important modes of
interactions was electrostatic attraction between the two positively charged residues of the metal
+
complexes (PPh3 ) and negatively charged phosphate groups of DNA. Also phenyl groups of
complexes can interact with DNA through intercalation.
The absorption spectra of the complexes in the absence and presence of FS-DNA are shown
in Fig. 1 and Fig. S6. It was observed that by addition of HS-DNA, the intensity of the
absorption band at around 300-420 nm was reduced (hypochromism) with no shift.
Hypochromism indicated the intercalative binding of the complexes between DNA duplex[51,
52]. In the intercalative binding mode, the π* orbital of the intercalated complex could interact
with the π orbital of the DNA base pairs, thus, the π→π* transition energy was decreased and the
coupling π orbital partially filled by electrons, so, decreasing the transition possibilities and
concomitantly resulting in the hypochromism[53]. In order to obtain a more quantitative
determination of the interaction strength, intrinsic-binding constant, Kb, was determined using
equation (1)[54]:
[DNA]/(εa-εf) = [DNA]/(εb-εf) +1/Kb(εb-εf)
(1)
12

13
Where εa, εf and εb, are the apparent, free, and bound metal complex extinction coefficients,
respectively. In particular, εf was determined by a calibration curve of the isolated metal complex
in aqueous solution, following the Beer’s law. εa was determined as the ratio between the
measured absorbance and the complex concentration, Aobs/[complex].
A plot of [DNA]/(εb-εf ) versus [DNA] gave a slope of 1/(εb-εf ) and a Y intercept equal to
1/Kb(εb-εf); Kb is the ratio of the slope to the Y intercept.
Intrinsic binding constants for complexes 1-4 were in the 1.05×104M−1-2.54×102M−1 range
and for ligand (L) 1.22×101M−1. Kb for classical intercalators is in the order of 107 M−1[53]. The
values of intrinsic binding constants indicated the weaker intercalation of these complexes
compared to classical intercalators.
By comparing the intrinsic binding constants of the complexes and ligand, it was found that
Zn(II) complexes bound to DNA stronger than Co(II) complex and all the metal complexes
stronger than free ligand (L). The order of binding affinity is 2341>L.
Effective nuclear charge for Zn atom is greater than Co atom. So it is concluded that Zn
complexes had electrostatic interaction with DNA with more probability. From the UV
spectroscopic titration, the main mode of binding was suggested to be intercalation.
The DNA binding affinity of 2 was stronger than 3 and 1. Complex 2 contained methyl group
on the bridged amine, so van der Waals interactions had a noticeable portion in binding mode,
also hydrophobic interactions between the methyl groups in the complex and DNA interior was
probable.
A halogen atom (Cl) as an electron withdrawing group on complex 3 makes it to be a more
positive complex so the electrostatic portion of interaction increased. Complex 3 as a positively
charged moiety was assumed to directly affect the negatively charged oxygen of the phosphate
group of DNA. On the other hand organic halogen atoms are favorably involved in a wide
variety of non-covalent biomolecules-complex interactions, such as halogen bonds C-X...O and
hydrogen bonds C-X...H, so the affinity of 3 to interact with DNA increased.
In spite of the fact that effective charge of Zn atom is greater than Co atom but complex 4
containing methyl group on the ligand, so van der Waals interactions between methyl group and
thymine methyl group of DNA and also hydrophobic interactions between the methyl groups and
DNA interior, increased the binding affinity of 4 comparing to 1[54].
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14
However, the binding constants of complexes were much lower than the potential
intercalators like ethidiumbromide (7×107M-1)[55]. The Kb values (Table 1) were comparable to
that of transition metal complexes[56-59].
3.2.2. Viscosity measurements
Hydrodynamic measurement that is sensitive to length change (i.e. viscosity) is considered as
most critical test in predicting the nature of binding of the complexes to DNA. As in the
intercalation mode of binding the complex is inserted between DNA base pairs, the length of
DNA increases which leading to increase in DNA viscosity. In contrast bending (or kinking) of
the DNA helix in the case of nonclassical intercalation, reduce the effective length of DNA and
concomitantly its viscosity, while groove binding cause less pronounced changes (positive or
negative) or no changes in DNA viscosity [55,60]. The values of relative specific viscosity
(η/η0)1/3, here η and η0 are the specific viscosity contributions of DNA in the absence and in the
presence of the complex were plotted against ri(ri = [complex]/[DNA]) (Fig. 2). For 1-4 and L, at
lower concentration of the compound, the viscosity decreased or no changed and then increased
at higher concentration of the compound. It is concluded that at least in the lower concentration
of the complexes non classical intercalation (i.e. van der Waals interactions, groove binding or
external binding) was main mode of binding although at higher concentration intercalation was
the main mode of binding[54].
3.2.3. Fluorescence studies
A most commonly technique used to study the interaction between small molecules and DNA
is fluorescence spectroscopy. One of the advantages of this technique is its high sensitivity and
selectivity. Fluorescent spectroscopy can be used to determine the mode of binding of drugs to
DNA. The effective intercalators cause a significant enhancement of the fluorescence intensity.
But groove binding agents (agents that can have electrostatic, hydrogen bonding, or hydrophobic
interactions) cause a decrease in fluorescence intensity because, after interaction, these agents are
close to the sugar-phosphate backbone of DNA. Fluorescence emission is very sensitive to the
14

15
environment and spectral shifts (10-20nm) in the excitation and emission spectra have been
observed during transferring the fluorophore between high and low polarity environment[60].
All synthesized complexes and ligand had emission spectra at room temperature thus binding
to DNA can be study by titration of the compounds with various concentration of DNA. Figs.3,
S7 show the emission spectra of the compounds in the absence and presence of HS-DNA.
For 1-4 and L with increasing the concentration of DNA the intensity of emission spectra
obviously decreased. Fluorescence quenching was described by the linear Stern-Volmer equation
(2)[61]:
F
0
[ ]
[ ]
= 1 + kSV Q = 1 + kqτ0 Q
(2)
F
where F and F0 are the fluorescence intensities in the presence and the absence of quencher,
respectively. KSV is the Stern-Volmer quenching constant, [Q] is the total concentration of
quencher, kq was the bimolecular quenching constant, and τ0 is the average lifetime of
biomolecule in the absence of quencher, and its value is 10-8s. Since fluorescence lifetime is
naturally near 10-8s, the bimolecular quenching constant (kq) is calculated from Ksv=kqτo[61].
The fluorescence quenching curve of the complex by DNA (Fig.4) illustrates a good agreement
with the linear curve of the Stern–Volmer equation and Ksv is calculated by the ratio of the slope
to the intercept (Table 2). The moderately large value of Ksv indicated that the complexes 1-4
were bound to DNA. According to equation (2), kq is greater than the limiting diffusion rate
constant (2.0×1010M-1s-1) for a biomacromolecule, indicating the existence of a static quenching
mechanism. In a static quenching mechanism, the fluorophore and the quencher form a complex
in the ground state[62, 63].
To determine the strength of the interaction of complexes with DNA, the value of the binding
constant (Kf) was resulted from a Scatchard equation (3)[64-67].
log (F −F) = log Kf + n log DNA
[
(3)
0
]
F
F0 and F are the fluorescence intensities of the fluorophore in the absence and in the presence
of different concentrations of DNA, respectively and n is the number of binding sites.
The values of Kf for 1-4 were found to be 3.68×103M-1, 23.90×103M-1, 3.78×103M-1, and
4.16×103M-1 and n=1.02, 1.25, 0.99, 1.1 respectively.
3.2.4. Anticancer studies
15

16
In the current study the synthetic Schiff base complexes were also screened for their
anticancer activities on A549, Jurkat and Raji cell lines as the target. The cell lines were
incubated in the presence of increasing concentrations of the complexes for 48 h, and then
cytotoxicity was measured. Generally, all the compounds showed strong growth inhibitory
activity against the cell lines. In order to compare the cytotoxic effects of the complexes, the IC50
values were determined. IC50 value is defined as the concentration of a compound where 50% of
the cell growth is inhibited. As shown in Table 3, based on the IC50 values, the order of cytotoxic
activity of complexes for A549 was 2 > 4 > 3 > 1. The sequence of cytotoxic activity of
complexes for Jurkat was 2> 3> 1> 4 and for Raji cell line was 2, 3>1> 4. As these results show
complex 2 was more effective on A549 cells which are a solid tumor cell line (IC50, 13.6
μg/mL). In contrast, complex 3 had a stronger activity against leukemia lymphoma cells (IC50,7.6
μg/mL for Jurkat and 9.1 μg/mL for Raji) compared to A549 (IC50, 19.5 μg/mL). The IC50 values
of cis-platin for A549 cell line was 79.4 μg/mL, 25.0 μg/mL for Jurkat and 30.1 μg/mL for Raji
cells (Table 3).
3.3. DFT study
The geometries of complexes were optimized by using the GAUSSIAN 03 program. DFT
calculations at the B3LYP method and 6-311G basis set for all atoms were done. The optimized
geometries of the complexes are shown in Fig.5. Selected geometrical parameters including bond
lengths, bond angles for all compounds are listed in Tables 4. Results showed that all of the bond
lengths and bond angles are in the normal range.
According to data reported in Table 4, the water molecule was coordinated to the central
metal of the complexes. Optimized structure suggested a square planar geometry around central
atom with two "N" atoms from diamine and two "O" atoms of phenolic group. Therefore, axial
position occupies by water molecule. Also results showed that two perchlorate groups bind to the
complexes as ion pairs.
3.4. Docking investigations
The design of molecules that can recognize specific structures of nucleic acid play an
important role to development of new chemotherapeutic drugs[68]. Molecular docking technique
is a useful method to understand the drug-DNA interaction especially in the mechanistic study by
16

17
placing a small molecule in the binding site of DNA mainly in a non-covalent fashion.
The lowest-energy conformations show that all compounds occupied the minor grooves of
DNA (Fig. 6 and Fig. S8). The ligands stretch inside the minor groove through the bridging
phenyl group and the atoms surrounding it. By considering the ligand structure it is obvious that
steric interactions played the dominant role in binding of the ligand to the DNA. The steric
interactions arise from the (3-formyl-4-hydroxybenzyl) triphenylphosphonium chloride template
and specifically its phenyl rings. From a detailed analysis of the docked structures, it appears that
the metal complexes closed to the DNA structure from the metal center and oxygen groups of
Schiff base ligand. Also it confirms the experimental finding that the nature of metal has high
influence on interaction mode. By comparing the optimized structures of ligand and complexes,
it is clear that the ligand is more folded than the complexes thus stretching of the complexes to
the minor groove of DNA is more effective than the ligand.
The DNA binding affinity of the complexes may be predicted from the binding scores. The
calculated binding scores for all optimized compounds are in the range of -96.953 – -114.267
(Table 5). It should be pointed out that the above docking calculations only allow a prediction to
the binding affinity and the sterically acceptable conformations of the complexes to DNA.
The modes of binding of the complexes with DNA obtained from molecular docking studies
correlate well with the experimental findings. Therefore, on the basis of Rerank Scores results,
complex 2 has more DNA binding propensity as compared to the other complexes. The DNA
binding affinity follows the order of: 2>3>4>1>L as supported by the experimental findings. The
results show that metal complexes act better than ligand and Zn complexes are better than Co
complex in their interaction with DNA.
3.5. Circular dichroism spectroscopy
During the drug-DNA interactions the DNA morphology changes, a useful technique to
monitor these changes is circular dichroism (CD), since CD signals are sensitive to small
variations in the chiral conformation of DNA. The CD spectrum of free DNA is of the typical B-
form, with a positive Cotton effect near 275 nm due to base stacking and a negative Cotton effect
near 245 nm due to right-handed helicity[69] and these bands are quite sensitive to the mode of
DNA interactions with small molecules. Thus simple groove binding and electrostatic interaction
of small molecules display small or no perturbation on the base-stacking and helicity bands,
17

18
while the classical intercalation increases the base stacking and stabilizes helicity, and so
enhances the intensity of the positive band[70]. Circular dichroism spectra were recorded at 25οC
with increasing amount of complexes (10µM to 40 µM) at constant concentration of the FS-
DNA (200 µM). As shown in Fig. 7, by addition the complexes to the DNA solution a decrease
in the intensity of positive band at 275nm was observed which confirm the interaction of
complex with DNA through groove and/or surface bonding. Also, the decrease of the intensity of
the positive band (275 nm) was likely to be because of a transition from the extended nucleic
acid right-handed double helix to a more compact form[71] so the conformation of DNA had
partly changed due to the binding interaction between these complexes and DNA. In the case of
complex 2&3 (Fig. 7) a much larger decrease in intensity for the positive band at 275 nm and a
new small positive band at 234 nm were observed. Since such a band and the shift of the main
positive band toward shorter waves were characteristic of Z-DNA, it may mean that the DNA
strands were locally converted into Z-DNA form[72] this showed that the DNA would interact
with these complexes and might be distorted into other structures[73].
4. Conclusion
Some new Zn(II) and Co(II) Schiff base complexes have been synthesized and fully
characterized. The DNA binding properties of complexes 1–4 and ligand (L) were examined by
UV-vis absorption spectra, emission spectra, viscosity techniques and circular dichroism (CD).
From these findings, it is concluded that, ligand had lower binding affinity than the complexes
and groove binding was the main mode of interaction for complexes 1-4. The binding of Zn(II)
complexes are better relative to Co(II) complex that may be attributed either to the z* effect or
geometry. Among the Zn(II) complexes: 2>3>1 and so complexes 2&3 containing CH3 & Cl
groups on the bridging ligand interacted strongly with DNA relative to complex 1, because van
der Waals interactions and hydrophobic interactions involve in the mode of interaction.
As easily passing through cell membrane is important in studying anticancer activity of the
complexes
+
Moreover, the DNA docking studies revealed that the -CH2-PPh3 groups on the ligand play
an important role in binding with the nucleotide phosphate unit of the DNA backbone and all the
metal complexes (Zn, Co) interacted in the minor groove of DNA through the metal centre and
oxygen groups of Schiff base ligand. The docking result also reveals the higher binding affinity
18

19
of complex 2 towards DNA receptors in comparison to complexes 1, 3, 4 and the free ligand.
The present study revealed that various metal effect and different Schiff base ligand on these
complexes can influence the DNA binding events and in vitro anticancer activities, thus
suggesting that the DNA binding ability and the anticancer activities may possibly be tuned
through varying these factors in these compounds, which is useful for the design and synthesis of
new metal-based drugs.
Acknowledgements
We are grateful to Shiraz University Research Council for its financial support.
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λ(nm)
Fig.1. Absorption spectra of complex 2 (50 μM) in 1mM Tris-HCl buffer at pH 7.2, in the absence and
presence of increasing amounts of DNA (0-260 μM )
1.035
1.03
1.025
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1.015
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1
1
2
3
4
L
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0
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0.2
0.25
Fig. 2. Effects of increasing amounts of complexes (1-4)(0-2.34 μM) on the viscosity of
DNA(10μM) in 1 mM Tris–HCl buffer. (r = 0.0–0.23) at 25 ℃
22

23
380
330
280
230
180
130
80
950
850
750
650
550
450
350
250
150
2
1
30
340
390
440
490
540
590
340
390
440
490
540
λ(nm)
λ(nm)
500
700
400
300
200
100
0
600
500
400
300
200
100
0
3
4
346
396
446
496
546
596
340
390
440
490
540
590
λ (nm)
λ(nm)
500
400
300
200
100
0
5
ligand
340
390
440
490
540
590
λ (nm)
Fig. 3. Effect of F-DNA (in each inject 5 μL 10-2 molL-1) on the emission intensity of the
compound solution
23

24
1.4
1.3
1.2
1.1
1
1
2
3
4
L
0.9
0.8
0.00E+00 5.00E-06 1.00E-05 1.50E-05 2.00E-05 2.50E-05 3.00E-05
[Q]
Fig. 4. Stern–Volmer plots for the observed fluorescence of complexes upon addition of DNA.
24

25
1
2
3
4
ligand
Fig. 5: The optimized geometrical structure of the complexes and ligand.
25

26
Fig. 6. Computational docking models illustrating the interactions between DNA and complex 4.
26

27
10
5
10
5
b
a
DNA
DNA+10μM complex
DNA+20μM complex
DNA+40μM complex
0
0
DNA
DNA+10μM complex
DNA+20μM complex
DNA+40μM complex
-5
-10
-5
-10
230
250
270
290
310
330
230
250
270
290
310
330
Wavelength/nm
Wavelength/nm
10
10
5
DNA
d
DNA+10μM complex
DNA+20μM complex
DNA+40μM complex
c
5
0
0
DNA
-5
DNA+10μM complex
DNA+20μM complex
DNA+40μM complex
-5
-10
-10
230
250
270
290
310
330
230
250
270
290
310
330
Wavelength/nm
Wavelength/nm
Fig.7. CD spectra of DNA (200 μM) at increasing complex concentration (0- 40 μM) in Buffer
(1.0 mM Tris–HCl, 5 mM NaCl); [complex]= 1000 μM, a)complex 1, b) complex 2, c) complex
3, d) complex 4.
27

28
a)
b)
Cl
Cl-
+
P
PPh3
OH
H
HCl
OH
H
+ OH(CH2O)nH
OH
H
acetonitrile
room temperature
48 h
O
O
O
R
N
-
ClO4
-
R
ClO4
ClPh3P
2
water
ethanol
OH
H
N
+
P
+
P
reflux
3h
OH HO
H2N
NH2
O
c)
ClPh3P
R
OH
H
-
ClO4
-
ClO4
2
1) M(acetate)2 . nH2O
water + ethanol
reflux
O
N
O
N
O
P
M
P
2) NaClO4. H2O + water
R
(1) M= Zn(II) , R = H
(2) M= Zn(II) , R = CH3
(3) M= Zn(II) , R = Cl
(4) M= Co(II) , R = CH3
H2N
NH2
Scheme 1: Synthetic route for a) (3-formyl-4-hydroxybenzyl)triphenylphosphoniumchloride b)
ligand c) complexes 1-4.
28

29
Table 1: Absorption spectral properties of compounds bound to DNA
Compound
Change in
Kb(M-1)
λ (nm)
absorbance
L
1
2
3
4
384
360
320
385
420
Hypochromism 1.22×101
Hypochromism 2.54×102
Hypochromism 1.05×104
Hypochromism 5.13×103
Hypochromism 1.15×103
Table 2: The Stern-Volmer quenching constant for the interaction of complexes with DNA.
complex
Ksv (M−1)
kq (M−1s-1)
L
1
1124
4285
1.1×1011
4.2×1011
11.3×1011
2
11314
3
4
10743
7657
10.7×1011
7.6×1011
29