Synthesis, characterization, DNA binding, cleavage activity, cytotoxicity and molecular docking of new nano water-soluble [M(5-CH2PPh3-3,4-salpyr)](ClO4)2 (M = Ni, Zn) complexes

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Synthesis, characterization, DNA binding, cleavage

activity, cytotoxicity and molecular docking of

new nano water-soluble [M(5-CH₂PPh₃-3,4-

salpyr)](ClO₄)₂(M = Ni, Zn) complexes†

Cite this: DOI: 10.1039/c5dt04788a

Zeinab Mandegani,^aZahra Asadi,*^aMozaﬀar Asadi,^aHamid Reza Karbalaei-Heidari^b

and Banafsheh Rastegari^b

Some new water soluble complexes [N,N’-bis{5-[(triphenyl phosphonium chloride)-methyl]salicylidine}-3,4-

diaminopyridine] M(^II), which are formulated as nano-[Zn(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂(1), [Zn(5-

CH₂PPh₃-3,4-salpyr)](ClO₄)₂(2), nano-[Ni(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂(3), [Ni(5-CH₂PPh₃-3,4-salpyr)]

(ClO₄)₂(4), and [N,N’-bis{5-[(triphenyl phosphonium chloride)-methyl]salicylidine}-2,3-diaminopyridine]Ni(II)

1

[Ni(5-CH₂PPh₃-2,3-salpyr)](ClO₄)₂(5) have been isolated and characterized by elemental analysis, FT-IR, H

NMR, ¹³C NMR, ³¹P NMR, and UV-vis spectroscopy. The morphology and size of the nano complexes were

determined using FE-SEM and TEM. In vitro DNA binding studies were investigated by UV-vis absorption

spectroscopy, viscosity measurements, CD spectroscopy, cyclic voltammetry, emission spectra and gel

electrophoresis, which suggest that the metal complexes act as eﬃcient DNA binders. The absorption spec-

troscopy of the compounds with DNA reveals that the DNA binding aﬃnity (K_b) has this order: 3 > 4 > 5 >

1 > 2 > Ligand. The metal complexes show DNA binding stronger than the ligand, which is expected due to

the nature of the metal. The nano complexes display DNA binding stronger than the other complexes which

is related to the eﬀect of size on binding aﬃnity and the Ni(^II) complexes reveal DNA binding stronger than

the corresponding Zn(^II) analogues, which is expected due to their z* eﬀect and geometry. The prominent

double strand DNA cleavage abilities of compound 3 are observed in the absence of H₂O₂with eﬃcien-

cies of more than 50% even at 70 µM complex concentration. Surprisingly, Zn(^II) complexes (compounds

1 & 2) exhibit a higher cytotoxicity (IC₅₀: 7.3 & 10.9 µM at 24 h; IC₅₀: 4.6 & 8.7 µM at 48 h) against human

hepatoma (HepG2) and HeLa cell lines than the Ni(^II) complexes (compounds 3, 4 & 5) and 5-ﬂuorouracil

as control in spite of their inability to cleave DNA. Finally, DNA binding interactions were performed by

docking studies. Density functional theory (DFT) studies were performed using the GAUSSIAN 03

program. The DFT method with B3LYP functional, LANL2DZ basis set for metal centers and 6-311g* for

other atoms was used. The synthesized compounds and DNA were simulated by molecular docking to

explore more details of the ligands conformation and their orientations in the active site of the receptor.

Received 8th December 2015,

Accepted 25th February 2016

DOI: 10.1039/c5dt04788a

www.rsc.org/dalton

indicative agents. The capability to interact with DNA has been

determined by numerous factors for example coordination geo-

Introduction

The interactions between small molecular ligands and deoxy- metry and the nature of the ligand. Additional necessary requi-

ribonucleic acid (DNA) are of great importance in biology and sites that such complexes should obviously retain are to be

medicine, due to their essential roles in biochemical, biological stable and inert in the biological environment and being water-

and medicinal processes. Meanwhile, DNA remains a biological soluble. Usually, water solubility has been increased by functio-

target of great interest for the design of therapeutic drugs and nalizing the ligand by charged or polar groups.^1–11

Currently one of the few most widely used antitumor drugs

is cisplatin for treating certain human cancers with incredible

^aDepartment of Chemistry, Faculty of Sciences, Shiraz University, Shiraz 71454, Iran.

success but the development of drug resistance and the dose-

limiting nephrotoxicity prevent its potential eﬃciency.^12,13So,

E-mail: Zasadi@shirazu.ac.ir

^bMolecular Biotechnology Laboratory, Department of Biology, Faculty of Sciences,

there is significant attention focused on the design of new

metal based anticancer drugs that display improved selectivity

Shiraz University, PO Box 71467–13565, Shiraz 71454, Iran

†Electronic supplementary information (ESI) available. See DOI: 10.1039/

and novel modes of DNA interaction similar to non-covalent

c5dt04788a

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interactions that mimic the mode of interaction of [M(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂(M = Zn 1, 2 and Ni 3, 4) and

biomolecules.¹⁴

[Ni(5-CH₂PPh₃-2,3-salpyr)](ClO₄)₂(5) were synthesized that the

Transition metal complexes that include multi dentate aro- complexes 1 and 3 were synthesized with nano size . The com-

matic ligands, with square planar N₄or N₂O₂coordination, plexes have been fully characterized by various physico-

have some suitable biological properties.^15–20

chemical techniques, namely, elemental analyses, spectral (FT-

Nickel(II)²¹and zinc(II)^22–24have a variety of biological acti- IR, ¹H, ¹³C ,³¹P NMR, UV/vis) and cyclic voltammetry (CV). The

vities. Ni(^II) complexes have shown antibacterial,^25,26anti- binding properties of the complexes with fish DNA under

fungal,²⁷antimicrobial²⁸and anticancer/antiproliferative^29–31physiological conditions have been studied using UV-Vis spec-

activities and the zinc complex is utilized for the treatment of trophotometry, DNA viscosity measurements, cyclic voltamme-

Alzheimer disease,^32,33and can act as radioprotective agents,³⁴try (CV), circular dichroism (CD), fluorescence spectroscopy,

tumor photosensitizers,³⁵anticonvulsants,^36,37antidiabetic agarose gel electrophoresis, anticancer activities of the com-

insulin-mimetics,^38–40anti-inflammatories^41,42and show cyto- plexes and molecular docking. The Ni(^II) complexes (3–5) show

toxicity against human cancer cell lines.^43,44

DNA binding aﬃnities higher than the Zn(^II) complexes (1, 2)

The application of nanoparticles (NPs) for biomedical and interestingly, complex 3 is higher than complex 5. The

usage is usual in the literature and continues to be a rapidly nano-sized complexes 1 and 3 display DNA binding stronger

growing research field, with great emphasis on imaging and than complexes 2 and 4 which are not nano-sized.

drug delivery.^45–48The unique properties of NP based systems

It is remarkable that all the complexes display cytotoxicity

give strategic advantages over genuine molecular thera- against human hepatoma (HepG2) and cervical cancer (HeLa)

peutics,⁴⁹but new complexities must be examined to make NP cell lines higher than the currently used chemotherapeutic

based applications a practical option in the clinic. Nano- agent, 5-fluorouracil. The Zn(^II) complexes exhibit higher cyto-

particles (NPs), as a subgroup of nanomaterials (NMs), may be toxicity than their Ni(^II) analogues in spite of their inability to

determined as particles with all three external dimensions in cleave DNA. Also, both 1 & 3 are remarkable in killing the

the range 1–100 nm although, variations on this definition cancer cell lines and are more eﬃcient than the classical com-

exist.⁵⁰The special physicochemical properties of NMs are plexes 2 & 4 through apoptosis which suggests the importance

suitable for a wide range of applications and due to their of the nano-sized form of the complexes.

small size and structure oﬀer novel capabilities to these

materials.^51–57The development of NMs is also motivated by

the confidence that these chemicals will deliver enhanced

Experimental section

Materials

performances and new functionalities leading, e.g., to smart

drugs and assisting in achieving sustainable development, e.g.,

by reducing the consumption of energy and materials and

reducing environmental pollution.⁵⁸

Zinc(^II) acetate dihydrate, nickel(II) acetate tetrahydrate, 2,3-

diaminopyridine, 3,4-diaminopyridine, salicylaldehyde, tri-

phenylphosphine, sodium perchlorate, paraformaldehyde,

conc. HCl, 0.5% NaHCO₃solution, acetonitrile, ethanol,

methanol, deuterated dimethyl sulfoxide (DMSO-d₆), ethylene

diamine tetra acetic acid (EDTA), 3-chloroacetylacetone,

ammonium acetate and potassium bromide (KBr) were pur-

chased from Merck, Fluka, Acros and Aldrich.

Agarose powder was purchased from Invitrogen. Fish de-

oxyribonucleic acid was obtained from New England Biolabs

(Beijing) Ltd. Tris-HCl-NaCl buﬀer solution (TBS, 1 mM Tris,

5 mM NaCl, pH 7.2) was used for FS-DNA binding experiments

and TAE buﬀer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA,

pH 8.0) was used for the gel electrophoresis experiments. All

reagents were used as received and solvents were purified by

the standard methods.

Double distilled deionized water was used to prepare

buﬀers, and the ionic strength in buﬀers was adjusted with

NaCl. The DNA concentration in base pairs was determined

using an extinction coeﬃcient of 6600 M⁻¹cm⁻¹at 260 nm.

The ratio A₂₆₀/A₂₈₀> 1.80 was used to indicate high DNA

purity. The DNA solution was stored for a short period of time

at 4 °C if not used immediately.

Information about the mode and range of binding of

metal center complexes to DNA is important for understanding

the cleavage properties of metal complexes. Transition metal

complexes are known to bind to DNA via both covalent and/or

non-covalent interactions. In the covalent binding mode a

labile ligand of the complex can be exchanged by a nitrogen

base of DNA such as guanine N7, while the non-covalent DNA

interactions include intercalative, electrostatic and groove

binding of a metal complex outside of the DNA helix, along

the major or minor groove.^59,60

So in the DNA molecule some types of sites bind such as:

(i) between two base pairs, (ii) in the minor groove, (iii) in the

major groove, and (iv) on the outside of the helix. The inter-

action advantages depend on the structure of DNA-interacting

molecules and the nature of DNA. Small changes in the struc-

ture of a DNA-interacting molecule may influence the binding

types and stability of the molecule/DNA complex.⁶¹

Taking into consideration the biological role of nickel and

zinc and the fact that metal complexes with drugs may exhibit

more pronounced biological properties in comparison with

free metal complexes, herein, we report the synthesis of the

novel water soluble Ni(^II) and Zn(II) complexes with the ligand

Methods and instrumentations

[N,N′-bis{5-[(triphenyl phosphonium chloride)-methyl]salicyli- The H NMR and ¹³C NMR spectra in DMSO-d₆were recorded

1

dine}-3,4-diaminopyridine] (Scheme 1). The final product, on a Bruker Avance DPX 250 spectrometer (with TMS as the

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Scheme 1 Synthetic route for (a) (3-formyl-4-hydroxybenzyl)triphenylphosphonium chloride and (b) L (c) complexes 1–5.

reference). ³¹P NMR spectra in DMSO-d₆were recorded using a precipitate was collected by filtration, washed with 0.5% NaHCO₃

Bruker Ultrashield 400 spectrometer (with 85% H₃PO₄as the solution then with water until it became neutral, and dried in

reference). UV-vis measurements were carried out using vacuum. The product (Scheme 1) was recrystallized from aceto-

Perkin-Elmer (LAMBDA 2) UV-vis spectrophotometers. FT-IR nitrile and petroleum ether. A mixture of 5-(chloromethyl)

spectra were recorded by using a Shimadzu FTIR 8300 infrared salicylaldehyde (10.2 g, 60 mmol) and triphenyl phosphine

spectrophotometer. Elemental analysis was carried out by (15.7 g, 60 mmol) in 200 mL acetonitrile was refluxed for 4 h

using a Thermo Fininngan-Flash 1200. Melting points of com- and cooled. The precipitated phosphonium salt was filtered

pounds were determined using a BUCHI 535. All experiments oﬀ and washed with ether.

were carried out in triple distilled water at pH = 7.2, 1 mM Tris

Yield: 85%. M.p. 250–251 °C. Color: white. Anal. calcd for

buﬀer and 5 mM NaCl.

C₂₆H₂₂PO₂Cl: C, 72.22; H, 5.12%. Found: C, 72.37; H, 5.24%.

Fluorescence spectra were recorded on a Perkin-Elmer FT-IR (KBr, cm⁻¹): 3741.6 (ν_OH), 2869 (ν_C–H), 1674.1 (ν_CvO).

(LS45) spectrofluorimeter equipped with a Lauda-ecoline-RE ¹H NMR (250 MHz, DMSO-d₆) δ (ppm): 11.18 (s, 1H, OH),

104 thermostat at 37 °C. Transmission electron microscopy 10.14 (s, 1H, CHO), 7.87 (dd, J = 10.1, 4.5 Hz, 3H, ArH), 7.69

(TEM) images were obtained on a Zeiss EM10C transmission (dq, J = 12.5, 7.6 Hz, 12H, ArH), 7.26–7.15 (m, 1H, ArH),

electron microscope using an Acc voltage of 80 kV. Field Emis- 7.11–6.92 (m, 2H, ArH), 5.13 (d, J = 15.0 Hz, 2H, CH₂).

sion Scanning Electron Microscopy (FE-SEM) images were ¹³C NMR (63 MHz, DMSO-d₆) δ (ppm): 189.2 (CHO), 160.6,

obtained using a HITACHI S-4160 with a voltage of 20 kV. 137.9, 137.8, 135.0, 134.1, 134.0, 133.9, 133.8, 130.1, 130.1,

Electrochemistry studies were performed using an Auto lab 129.9, 129.8, 122.4, 122.4, 118.3, 118.0, 117.8, 116.9 (aromatic

302N. An incubator and an ELISA reader (Biotek Instruments, carbons), 34.2 (CH₂) (spectral data in Fig. S1–S3†).

Inc., USA) were used for anticancer studies.

[N,N′-Bis{5-[(triphenyl

salicylidine}310-3,4-diaminopyridine](L). To

stirred solution of (3-formyl-4-hydroxybenzyl)triphenylphos-

phosphonium

chloride)-methyl]

a

vigorously

Synthesis

(3-Formyl-4-hydroxybenzyl)triphenylphosphonium chloride. phonium chloride (0.6 g, 1.25 mmol) in 50 mL water, an etha-

A mixture of salicylaldehyde (8.5 mL, 80 mmol), paraformalde- nolic solution (20 mL) of 3,4-diaminopyridine (0.07 g,

hyde (1 mL, 50 mmol) and 50 mL of conc. HCl was stirred at 0.62 mmol) was added dropwise. The solution turned to light

room temperature for 48 h. The resulting dark red powder yellow and the mixture refluxed for 3 h. After that, NaClO₄·H₂O

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(0.15 g, 1.25 mmol) dissolved in a minimum amount of water

(5 mL) was added. The resulting yellow powder (Scheme 1) was

collected by filtration, washed with cold ethanol and ether and

dried in air.

[(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂. Yield: 85%. M.p. >310 °C.

Color: light yellow. Anal. calcd for C₅₇H₄₇N₃Cl₂O₁₀P₂: C, 62.08;

H, 4.66; N, 3.81%. Found: C, 62.30; H, 4.81; N, 3.57%. FT-IR

(KBr, cm⁻¹): 1650.9, (ν_CvN), 1487.8 (ν_CvC), 1087.8, 650.9

(ν_ClO

−

). UV-vis. (H₂O): λ_max(nm) = 387, 333. ¹HNMR (250 MHz,

4

DMSO-d₆) δ (ppm): 11.12 (s, 2H, OH), 8.72 (s, 1H, HCvN),

8.53 (s, 1H, HCvN), 7.87 (d, J = 7.1 Hz, 8H, ArH), 7.68 (dd, J =

17.8, 4.4 Hz, 23H, ArH), 7.23–7.16 (m, 3H, ArH), 7.08–6.98 (m,

2H, ArH), 6.82 (d, J = 8.5 Hz, 3H, ArH), 5.06 (dd, J = 15.1, 3.1

Hz, 4H, CH₂P). ¹³C NMR (63 MHz, DMSO-d₆) δ (ppm): 160.93,

160.88 (CvN), 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), 34.24, 33.98 (CH₂). ³¹P NMR (162 MHz, DMSO-d₆)

δ (ppm): 22.58 (spectral data in Fig. S4–S8†).

Nano [N,N′-bis{5-[(triphenyl phosphonium chloride)-methyl]

salicylidine}-3,4-diaminopyridine]zinc(^II) perchlorate. [Zn(5-

CH₂PPh₃-3,4-salpyr)](ClO₄)₂(1). The nano complex was syn-

thesized by the slow addition of a solution of (3-formyl-4-

hydroxybenzyl)triphenylphosphonium

chloride

(0.6

g,

1.25 mmol) in 50 mL water into a hot solution of Zn(aceta-

te)₂·2H₂O (0.14 g, 0.62 mmol) dissolved in water over 7 h and

then a solution of 3,4-diaminopyridine (0.07 g, 0.62 mmol) in

20 mL ethanol was added into this mixture for 30 min. The

solution turned to yellow and the mixture was refluxed for

40 h. After that, NaClO₄·H₂O (0.15 g, 1.25 mmol) dissolved in a

minimum amount of water was added to the reaction mixture.

The resulting yellow powder (Scheme 1) was collected by cen-

trifugation, washed three times (3 × 2 mL) with ether and two

times (2 × 1 mL) with cold ethanol and dried in vacuum.

Fig. 1 (a) TEM and (b) FE-SEM images of nano-particles of Zn(5-

CH₂PPh₃-3,4-salpyr)](ClO₄)₂.

A transmission electron microscopy (TEM) image showed

nano-particles with sizes between 40–75 nm (Fig. 1a). [Zn(5-

CH₂PPh₃-3,4-salpyr)](ClO₄)₂. Yield: 91%. M.p. >310 °C. Color:

yellow. Anal. calcd for C₅₇H₄₅N₃Cl₂O₁₀ZnP₂·H₂O: C, 59.62; H,

phonium chloride (0.6 g, 1.25 mmol) in 10 mL water and then

4.13; N, 3.66%. Found: C, 58.90; H, 4.00; N, 3.50%. FT-IR (KBr, an ethanolic solution (5 mL) of 3,4-diaminopyridine (0.07 g,

cm⁻¹): 3440.8 (ν_O–H), 1620.1 (ν_CvN), 1527.5 (ν_CvC), 1110.9,

0.62 mmol) were added dropwise. The solution turned to dark

yellow and the mixture refluxed for 3 h. After that, NaClO₄·H₂O

(nm) = 386, 332. HNMR (250 MHz, DMSO-d₆) δ (ppm): 10.31 (0.15 g, 1.25 mmol) dissolved in a minimum amount of water

686.6 (ν_ClO), 624.9 (ν_M–O), 501.5 (ν_M–N). UV-vis. (H₂O): λ_max

−

4

1

(5 mL) was added. The resulting yellow powder was collected

by filtration, washed with cold ethanol and ether and dried in

(s, 1H, HCvN), 10.10 (s, 1H, HCvN), 9.12–8.39 (m, 1H, ArH),

7.88 (t, J = 7.3 Hz, 7H, ArH), 7.80–7.42 (m, 23H, ArH), 7.38 (d,

J = 2.5 Hz, 1H, ArH), 7.27–7.06 (m, 2H, ArH), 6.99 (d, J = 4.7 air. The complex was dissolved in CH₂Cl₂(6 mL) and ethyl

acetate (3 mL) and the resulting solution was allowed to

evaporate slowly at room temperature. The solid product was

Hz, 2H, ArH), 6.85–6.56 (m, 2H, ArH), 6.47 (d, J = 7.1 Hz, 1H,

ArH), 5.03 (d, J = 14.7 Hz, 4H, CH₂). ¹³C NMR (63 MHz, DMSO-

d₆) δ (ppm): 162.1, 161.3 (CvN), 157.7, 149.7, 146.6, 144.7, filtered oﬀ and washed with ethyl acetate and ether.

[Zn(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂.

Yield:

94%.

M.p.

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

>310 °C. Color: yellow. Anal. calcd for C₅₇H₄₅N₃Cl₂O₁₀ZnP₂·

(CH₂). ³¹P NMR (400 MHz, DMSO-d₆) δ (ppm): P_a(21.77), H₂O: C, 59.62; H, 4.13; N, 3.66%. Found: C, 59.40; H, 3.93;

N, 3.50%. Spectral data (FT-IR, UV-vis, H NMR, ¹³C NMR and

1

P_a′(21.69) (spectral data in Fig. S9–S14†).

[N,N′-Bis{5-[(triphenyl phosphonium chloride)-methyl]sali-

³¹P NMR) of complex (2) were the same as complex (1).

cylidine}-3,4-diaminopyridine]zinc(^II)

CH₂PPh₃-3,4-salpyr)](ClO₄)₂(2). To a vigorously stirred solu-

tion of Zn(acetate)₂·2H₂O (0.14 g, 0.62 mmol) in 25 mL water,

perchlorate.

[Zn(5-

Nano [N,N′-bis{5-[(triphenyl phosphonium chloride)-methyl]

salicylidine}-3,4-diaminopyridine]nickel(^II) perchlorate. [Ni(5-

CH₂PPh₃-3,4-salpyr)](ClO₄)₂(3). The nano complex was

a

solution

of

(3-formyl-4-hydroxybenzyl)triphenylphos- synthesized by slow addition of a solution of (3-formyl-4-

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hydroxybenzyl)triphenylphosphonium

1.25 mmol) in 50 mL water into

chloride

(0.6

g, (t, J = 5.5 Hz, 1H, ArH), 7.98–7.82 (m, 7H, ArH), 7.80–7.57 (m,

a

hot solution of 24H, ArH), 7.31 (s, 2H, ArH), 6.83 (dd, J = 8.4, 4.1 Hz, 2H, ArH),

Ni(acetate)₂·4H₂O (0.15 g, 0.62 mmol) dissolved in water over 6.64 (dd, J = 9.5, 3.9 Hz, 2H, ArH), 5.06 (d, J = 14.5 Hz, 4H,

7 h and then a solution of 3,4-diaminopyridine (0.07 g, CH₂). ¹³C NMR (63 MHz, DMSO-d₆) δ (ppm): 162.4, 161.6

0.62 mmol) in 20 mL ethanol was added to the mixture for (CvN), 155.7, 150.5, 146.2, 142.2, 140.3, 139.5, 138.7, 135.0,

30 min. The solution turned to yellow and the mixture was 134.0, 133.9, 130.2, 130.0, 126.8, 126.0, 122.4, 122.0, 118.5,

refluxed for 48 h. After that, NaClO₄·H₂O (0.15 g, 1.25 mmol) 117.3, 10.4.6 (aromatic carbons), 34.9, 34.1 (CH₂). ³¹P NMR

was dissolved in a minimum amount of water added to the (400 MHz, DMSO-d₆) δ (ppm): P_a(21.90), P_a′(21.75) (spectral

reaction mixture. The resulting red powder (Scheme 1) was data in Fig. S15–S20†).

collected by centrifugation, washed three times (3 × 2 mL) with

ether and two times (2 × 1 mL) with cold ethanol and dried in cylidine}-3,4-diaminopyridine]nickel(^II) perchlorate. [Ni(5-

vacuum. CH₂PPh₃-3,4-salpyr)](ClO₄)₂(4). To a vigorously stirred solu-

A transmission electron microscopy (TEM) image showed tion of Ni(acetate)₂·4H₂O (0.15 g, 0.62 mmol) in 25 mL water,

[N,N′-Bis{5-[(triphenyl phosphonium chloride)-methyl]sali-

nano-particles with sizes between 30–55 nm (Fig. 2a).

[Ni(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂. Yield: 92%.

a solution of (3-formyl-4-hydroxybenzyl)triphenylphosphonium

M.p. chloride (0.6 g, 1.25 mmol in 10 mL water) and then an etha-

>310 °C. Color: red. Anal. calcd for C₅₇H₄₅N₃Cl₂O₁₁NiP₂: C, nolic solution (5 mL) of 3,4-diaminopyridine (0.07 g,

59.97; H, 4.15; N, 3.68%. Found: C, 59.70; H, 3.95; N, 3.40%. 0.62 mmol) were added dropwise. The solution turned to dark

FT-IR (KBr, cm⁻¹): 1620.1 (ν_CvN), 1527.5 (ν_CvC), 1095.5, 694.9 red and the mixture refluxed for 3 h. After that, NaClO₄·H₂O

(ν_ClO), 624.9 (ν_M–O), 501.5 (ν_M–N). UV-vis (H₂O): λ_max(nm) = (0.15 g, 1.25 mmol) dissolved in a minimum amount of water

−

4

1

476, 375. H NMR (250 MHz, DMSO-d₆) δ (ppm): 9.45 (s, 1H, (5 mL) was added. The resulting red powder was collected by

HCvN), 9.30 (s, 1H, HCvN), 8.50 (t, J = 5.1 Hz, 1H, ArH), 8.05 filtration, washed with cold ethanol and ether and dried in air.

The complex was dissolved in CH₂Cl₂(6 mL) and ethyl acetate

(3 mL) and the resulting solution was allowed to evaporate

slowly at room temperature. The solid product was filtered oﬀ

and washed with ethyl acetate and ether.

[Ni(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂.

Yield:

90%.

M.p.

>310 °C. Color: red. Anal. calcd for C₅₇H₄₅N₃Cl₂O₁₁NiP₂: C,

59.97; H, 4.15; N, 3.68%. Found: C, 59.62; H, 3.95; N, 3.49%.

1

Spectral data (FT-IR, UV-vis, H NMR, ¹³C NMR and ³¹P NMR)

of complex (4) were the same as complex (3).

[N,N′-Bis{5-[(triphenyl phosphonium chloride)-methyl]sali-

cylidine}-2,3-diaminopyridine]nickel(^II) perchlorate. [Ni(5-

CH₂PPh₃-2,3-salpyr)](ClO₄)₂(5). To a vigorously stirred solu-

tion of Ni(acetate)₂·4H₂O (0.15 g, 0.62 mmol) in 25 mL water, a

solution of (3-formyl-4-hydroxybenzyl)triphenylphosphonium

chloride (0.6 g, 1.25 mmol) in 10 mL water and then an etha-

nolic solution (5 mL) of 2,3-diaminopyridine (0.07 g,

0.62 mmol) was added dropwise. The solution turned to dark

red and the mixture refluxed for 3 h. After that, NaClO₄·H₂O

(0.15 g, 1.25 mmol) dissolved in a minimum amount of water

(5 mL) was added. The resulting red powder was collected by

filtration, washed with cold ethanol and ether and dried in air.

The complex was dissolved in CH₂Cl₂(6 mL) and ethyl acetate

(3 mL) was added and the resulting solution was allowed to

evaporate slowly at room temperature. The solid product was

filtered oﬀ and washed with ethyl acetate and ether.

[Ni(5-CH₂PPh₃-2,3-salpyr)](ClO₄)₂.

Yield:

91%.

M.p.

>310 °C. Color: red. Anal. calcd for C₅₇H₄₅N₃Cl₂O₁₁NiP₂: C,

59.97; H, 4.15; N, 3.68%. Found: C, 60.09; H, 3.88; N, 4.00%.

FT-IR (KBr, cm⁻¹): 1620.1 (ν_CvN), 1527.5 (ν_CvC), 1095.5, 694.9

(ν_ClO), 624.9 (ν_M–O), 501.5 (ν_M–N). UV-vis. (H₂O): λ_max(nm) =

−

4

1

472, 375. H NMR (250 MHz, DMSO-d₆) δ (ppm): 9.28 (s, 1H,

HCvN), 9.14 (s, 1H, HCvN), 8.03 (s, 1H, ArH), 7.98–7.81 (m,

7H, ArH), 7.80–7.51 (m, 24H, ArH), 7.44–7.14 (m, 3H, ArH),

7.04 (d, J = 7.3 Hz, 1H, ArH), 6.83 (d, J = 7.3 Hz, 2H, ArH),

6.65–6.47 (m, 1H, ArH), 5.06 (d, J = 13.7 Hz, 4H, CH₂).

Fig. 2 (a) TEM and (b) FE-SEM images of nano-particles of Ni(5-

CH₂PPh₃-3,4-salpyr)](ClO₄)₂.

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¹³C NMR (63 MHz, DMSO-d₆) δ (ppm): 162.7, 161.5 (CvN), (1.0 × 10⁻³M) were carried out in the potential range from

155.2, 150.2, 146.3, 142.5, 140.7, 139.0, 138.0, 135.7, 134.2, −1.0 V to 1.5 V. Electrochemistry of these newly synthesized

133.0, 130.8, 130.0, 126.4, 126.0, 122.5, 122.3, 118.0, 117.0, Schiﬀ base metals was studied by cyclic voltammetry at a scan

10.4.5 (aromatic carbons), 34.7, 34.5 (CH₂). ³¹P NMR rate of 0.10 V s⁻¹in H₂O–DMSO solution.

(400 MHz, DMSO-d₆) δ (ppm): Pa, Pa′ (22.59) (Spectral data in

Fig. S21–S25†).

Circular dichroic spectra of DNA were obtained by using a

JASCO J-716 spectropolarimeter equipped with a Peltier temp-

CAUTION! The perchlorate salts could be potentially explosive. erature control device. All experiments were done using a 1 or

Therefore, only small quantities of the sample were handled to 0.2 cm path quartz cell. Each CD spectrum was collected after

avoid any possible explosion.

averaging over at least 2 accumulations using a scan speed of

100 nm min⁻¹and a 1 s response time. Machine plus cuvette

baselines were subtracted and the resultant spectrum zeroed

Solubility and stability

The complexes were soluble in water, dimethyl formamide outside the absorption bands.

(DMF) and dimethyl sulfoxide (DMSO). The complexes were

Emission intensity measurements were carried out using a

stable in the solid phase at ambient temperature in the light. Perkin-Elmer (LS45) spectrofluorimeter with 10 nm excitation

The solutions were stable on keeping for a long period in light. and 10 nm emission slit widths. The excitation wavelength was

fixed and the emission range was adjusted before measure-

DNA binding experiments

ments. The samples were placed in quartz cuvettes of 1 cm

Concentrated stock solutions of metal complexes were pre- optical path. In these experiments, 3 mL of the complex solu-

pared by dissolving them in 1 mM Tris HCl/5 mM NaCl buﬀer tion (5 μM) were poured into the cell. Emission spectra were

at pH 7.2 and diluting suitably with the corresponding buﬀer recorded after each addition (10 μL) of DNA solution (10³μM)

to the required concentrations for all the experiments. The into the same buﬀer at 310 K. The observed fluorescence

DNA concentration was measured from its absorption intensity intensities were also corrected for dilution. The reaction time

at 260 nm using a molar absorption coeﬃcient value of has been studied and the results showed that 4 min was

6600 dm³mol⁻¹cm^{−1 62}

.

enough for stabilization. So the change in fluorescence emis-

The absorption spectra were recorded on a Perkin-Elmer sion intensity was measured within 4 min after each addition.

lambda 2 UV-Vis spectrophotometer. A solution of DNA in the

DNA cleavage and ethidium bromide competitive studies

buﬀer gave a ratio 1.98, of UV absorbance at 260 and 280 nm,

A₂₆₀/A₂₈₀, indicating that the DNA was suﬃciently free from Gel electrophoresis experiments were performed using 500 ng

protein. In these experiments, 3 mL of the complex solutions supercoiled pBlu2KSM DNA in 10 mM Tris-HCl buﬀer pH 7.2

([Ni] = 20 μM, [Zn] = 90 μM) were poured into the cell. Absor- (in 1% DMSO) with the final concentrations of 10–100 µM

bance spectra were recorded after each successive addition metal complexes in a total volume of 10 µl. The samples were

(10 μL) of DNA solution and equilibration (ca. 4 min) at 310 K. then incubated for 1 h at 37 °C, followed by electrophoresis on

The binding of metal complexes to DNA has been studied 1% agarose gel in TAE buﬀer containing 1.0 µg ml⁻¹ethidium

through the changes in absorbances and shifts in wavelength. bromide at 60 V for 1 h and finally, photographed under UV

In order to obtain a more quantitative determination of the light. The eﬃciency of the cleavage activity of the complexes

interaction strength, the intrinsic-binding constant, K_b, was was determined by the ability of the complex to cause single or

determined using spectroscopic titration data at various wave- double strand breaks in DNA and form a linear form (LF) or

lengths where the diﬀerence in absorption was the maximum nicked circular (NC) DNA compared to its supercoiled (SC)

after equilibrium.

ones.

An Ostwald viscometer was used for viscosity measure-

ments. The temperature was constant at 25 0.5 °C by using a

temperature bath.

Anticancer activity studies

Cell viability tests were performed against two cell lines includ-

The concentration of DNA was 10 μM, and flow time was ing human hepatoma (HepG2) and cervical cancer cell (HeLa)

measured with a digital stopwatch. The mean values were used with MTT assay as described previously.⁶³The stock solutions

to evaluate the viscosity η of the samples. The values for rela- of the metal complexes were prepared in DMSO ranging from

tive specific viscosity (η/η_o)^1/3, where η_oand η are the specific 0.1 to 1% with regard to the fact that DMSO was found to be

viscosity contributions of DNA in the absence (η_o) and in the non-toxic to the cells until 1% concentration. Then the cells

presence of the complex (η), were plotted against r_i(r_i

[complex]/[DNA] = 0, 0.05, 0.10, 0.15, 0.20, 0.25).

=

were treated with series of diﬀerent concentrations of DMSO

dissolved ligand ranging from 1–100 μg ml⁻¹24 h after seeding

Cyclic voltammetry was carried out using a CH Instruments of 5 × 10³cells per well in 200 μl of fresh culture medium and

electrochemical analyser. All voltammetric experiments were DMSO was used as the negative control. After 24 and 48 h,

performed in a single compartmental cell of volume 10–15 mL 20 μl of MTT solution (5 mg ml⁻¹in PBS buﬀer) was added to

containing a three-electrode system comprising a Pt working each well and the plates were wrapped with aluminium foil

electrode, a reference electrode (Ag/Ag⁺in TBAP/acetonitrile and incubated for 4 h at 37 °C. The purple formazan product

solution), and a Pt auxiliary electrode. The measurements of formed was dissolved by the addition of 100 μl of 100% DMSO

CV for H₂O–DMSO solution containing Schiﬀ base complexes to each well. The absorbance was monitored at 570 nm

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(measurement) and 630 nm (reference) using a 96 well plate

All the complexes were characterized by elemental analysis,

reader (Biotek Instruments, Inc., USA).⁶⁴Data were collected FT-IR, ¹H NMR, ¹³C NMR,³¹P NMR, UV-vis spectroscopy and

for three replicates each and were used to calculate the mean. the morphology of the nano complexes were determined using

The percentage of inhibition was calculated from these data FE-SEM and TEM. ¹H NMR spectral data of the zinc and nickel

using the formula:

1

complexes are given in the Experimental section. The H NMR

Mean OD of untreated cells ðcontrolÞ ꢀ Mean OD of treated cells

ꢁ 100

Mean OD of untreated cells ðcontrolÞ

Theoretical calculations

spectra of L and complexes 1–5 showed a complex overlap of

signals in the δ 6.50–7.90 ppm range corresponding to aro-

matic protons of the ligand and coordinated triphenyl-

phosphine.⁷²A doublet corresponding to the azomethine

group was observed at the ∼8.50–9.50 ppm range.⁷³In the

spectra of the ligand, the singlet appearing at δ 11.09 ppm is

assigned to the OH group. However, in the spectra of the com-

plexes there was no resonance attributable to OH, indicating

the coordination of ligand in the anionic form upon deproto-

nation. A doublet corresponding to the CH₂P appeared at

δ 5.00–5.10 ppm in the compounds.

Geometry optimization was performed with the hybrid density

functional theory method by Becke’s hybrid three-parameter

exchange functional and the nonlocal correlation functional of

Lee, Yang, and Parr (B3LYP). The Hay and Wadt basis set

LANL2DZ was used for the metal centers while for the rest of

the atoms the 6-311G* standard basis set was employed.⁶⁵All

structures were fully optimized in the gas phase with default

convergence criteria. The Gaussian 03 software package was

used for all computations.⁶⁶

Molecular docking

In the ¹³C NMR spectra, the azomethine carbon resonance

is observed at 160.0–163.0 ppm. In all compounds, aromatic

carbon atoms were observed around 103.0–157.0 ppm. Three

signals corresponding to the presence of triphenylphosphine

observed at 138.0–116.9 ppm (L), 157.7–113.7 ppm (complexes

1 and 2) and 155.7–104.6 ppm (complexes 3–5) are in the

range of the reported values.⁷⁴The CH₂carbon resonance is

observed at 30.0–34.0 ppm.

In order to confirm the presence of triphenylphosphine,

³¹P NMR spectra were recorded. Two peaks observed at

20.0–23.0 ppm suggested the presence of two non-equivalent

triphenylphosphines.

The molecular geometry of L, complexes 1, 3 and 5 were sub-

jected to energy minimization by DFT calculations by the

B3LYP method using the LANL2DZ basis set for the central

atoms and 6-311G* for all the non-metal atoms for ground

state geometry optimization.

The crystal structures of 3US0 (pdb code d(AAACATGTTTA-

TAAACATGTTT) was selected from the Protein Data Bank

(http://www.pdb.org),⁶⁷the protein molecules were removed

from this structure before performing docking calculations

which had been equilibrated at a nuclei-like condition⁶⁸and

used as the receptor.

Electronic spectral data for the complexes in H₂O are given

in the Experimental section. These complexes show intensive

absorption bands at 220–380 nm, which are attributed to the

π → π* transition in aromatic rings or azomethine groups and

the n → π* transition in the pyridine ring.

Then, the optimized ligands were imported to the Molegro

Virtual Docker (MVD)⁶⁹to carry out the docking simulations.

This software generates the best DNA–ligand configurations

according to several scoring criteria such as Moldock and

Rerank scores.

The FT-IR spectrum of aldehyde exhibited a broad medium

intensity band at 3741 cm⁻¹due to the O–H. The medium–

weak band at 1674 cm⁻¹was assigned to the CHO bond. The

structure of the Schiﬀ base complexes was indicated by the

presence of strong imine (CvN) bands at 1620 cm⁻¹. Two

In MVD the units are arbitrary, but an ideal hydrogen bond

contributes to the overall energy.⁷⁰We have selected the score

as the Moldock score [GRID], with a GRID resolution of 0.3 Å.

The algorithm selected for docking was Moldock along with a

number of runs as 20.⁷¹For each docking calculation, 10

diﬀerent poses were requested. All other parameters were kept

at their default values. Parameter settings, pose generation

and simplex evolution were selected as default settings. After

docking energy minimization is an essential parameter along

with optimized hydrogen bonding.

peaks around 1110 and 700 cm⁻¹were related to the ClO₄

−

group. The vibration band for the complexes around

3440 cm⁻¹was attributed to the presence of lattice and co-

ordinated water.⁷⁵The medium-weak bands at 2990 and

3050 cm⁻¹are observed for the aromatic (C–H) stretching

bands. The phenolic (C–O) stretching bands, because of the

participation of oxygen in the C–O–M bond, are observed in

the region of 1200–1220 cm⁻¹. The ring skeletal vibrations

(CvC) were consistent in the region of 1440–1550 in all com-

plexes.⁷⁶In the lower frequency region, the medium–weak

bands observed at 551–563 and 435–458 cm⁻¹have been

assigned respectively to the (M–N) and (M–O) vibrations.^77,78

The SEM and TEM images of nano complexes (1, 3) are

shown in Fig. 1 and 2. The TEM image of complex 1 shows

Results and discussion

Syntheses and spectral properties of complexes

The complexes [M(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂(M = Zn (1, 2)

Ni (3, 4) show that the complexes 1 and 3 were synthesized

with nano size) and [Ni(5-CH₂PPh₃-2,3-salpyr)](ClO₄)₂(5) were

isolated using the procedure reported (Scheme 1).

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ꢀ

ꢁ

1=2

2K_b²C_t½DNAꢂ

that the average size of the nano Zn(II) complex was around

40–75 nm (Fig. 1a). The morphology and size of nano complex

1 were studied by FE-SEM and it seems that the particles are

semispherical and confirmed the result of TEM (Fig. 1b).

The average size of the nano Ni(^II) complex 3 was detected

by TEM as around 30–55 nm (Fig. 2a). The FE-SEM of nano

complex 3 shows semispherical morphology for nano Ni(^II)

complex 3 and the average size was shown to be the same as in

the TEM study (Fig. 2b).

b ꢀ b²

ε_aꢀ ε_f

ε_bꢀ ε_f

s

¼

ð1Þ

ð2Þ

2K_bC_t

K_b½DNAꢂ

b ¼ 1 þ k_bC_tþ

2s

where ε_ais the extinction coeﬃcient observed for the absorp-

tion band at a given DNA concentration, ε_fis the extinction

coeﬃcient of the complex free in solution, and ε_bis the extinc-

tion coeﬃcient of the complex when fully bound to DNA. The

intrinsic binding constants K_bof the complexes 3–5 were in

the 1.70 × 10⁶–1.38 × 10⁷range. These values are comparable

to that observed for classical intercalators whose K_bvalues are

DNA binding studies

Absorption spectral studies. Electronic absorption spectro-

scopy is one of the most common methods to explore the

interaction of compounds with DNA. The change in absor-

bance and shift in wavelength upon addition of DNA solution

in a fixed concentration of metal complexes gives important

information on the type of interaction. A compound binding

to DNA through intercalation usually results in hypochromism

with or without a red or blue shift, because of the intercalative

mode concerning a strong stacking interaction between the

planar aromatic chromophore and the base pairs of DNA.^79,80

in the order of 10⁷M^{−1 89}

The intrinsic binding constants of L

.

and complexes 1, 2 with DNA were obtained by monitoring the

changes in the absorbance of the LMCT band of the complexes

with the increasing concentration of DNA using functional

eqn (3):⁹⁰

½DNAꢂ=ðε_aꢀ ε_fÞ ¼ ½DNAꢂ=ðε_bꢀ ε_fÞ þ 1=k_bðε_bꢀ ε_fÞ

ð3Þ

where [DNA] is the concentration of DNA in base pairs, ε_a, ε_f

The range of hypochromism is usually consistent with the and ε_bcorrespond to A_obs/[complex], the extinction coeﬃcient

strength of the intercalative binding interaction.⁸¹The hypo- of the free complex, and the extinction coeﬃcient of the

chromicity is specific to the interaction between the electronic complex in the fully bound form, respectively. K_bwas obtained

states of the compound chromophores and those of the DNA from the ratio of the slope to intercept by using the plot of

bases, meanwhile the red shift is concomitant with the [DNA]/(ε_a− ε_f) vs. [DNA] (Table 1).

decrease in the energy gap between the highest occupied and

The intrinsic binding constants K_bof the Zn(II) complexes

lowest unoccupied molecular orbitals (HOMO and LUMO) were in the range 3.3 × 10³–2.2 × 10³

M

⁻¹. However, the

after binding of the complex to DNA.^82,83The absorption observed binding constant is smaller than the classical inter-

spectra of Ni(^II) complexes with DNA are shown in Fig. 3. Upon calators and metallointercalators where the binding constant

increasing the concentration of DNA in the solution of the was reported to be in the order of 10⁷M^{−1 91}

,

but they are

Ni complexes, the bands at 375 nm showed hypochromism and comparable to the observed value for some complexes like

a red-shift, indicating that the interaction with DNA results in [Co(phen)₃]³⁺(1.6 × 10^{4 −1});⁹²[Ru(phen)₃]²⁺(0.55 × 10⁴

⁻¹);⁹³[Zn(flmq)₂(bipy)] (4.71 × 10⁴M⁻¹);⁹⁴[Zn(flmq)₂(H₂O)₂]

M

the direct formation of a new complex with double-helical

M

DNA resulting in stabilization of the DNA duplex, while Zn(^II) (7.93 × 10⁴M⁻¹);⁹³[Zn(erx)₂(bipy)] (k_b= 2.61 × 10⁴M⁻¹);⁹⁵

complexes exhibit both hypochromism and hyperchromism [NiL¹(NH₃)] (k_b= 2.3 × 10⁴M⁻¹);⁹⁶[NiL⁵(H₂O)]·H₂O (k_b= 1.99 ×

for the 386 nm and 330 nm bands, respectively without shifts 10⁴

M

⁻¹);⁹⁶tricationic Co(III) complexes with an asymmetric

in the band position. The overall spectral changes with hypo- ligand, [Co (phen)₂(pdta)]³⁺(k_b= 2.8 × 10⁴);⁹⁷[Co (bpy)₂-

chromicity and isosbestic point induced by binding planar (CNOIP)]³⁺(k_b= 5 × 10⁴M⁻¹).⁹⁸

polyaromatic molecules to DNA are suggestive of strong inter-

The achieved values indicate that the Zn(II) complexes are

actions. Also the hyperchromic eﬀect ascends mainly because moderately bound to FS-DNA⁸⁹and electrostatic interactions

of the presence of charged cations which bind to DNA via cannot be ruled out.

electrostatic attraction to the phosphate group of the DNA

From the results obtained, it has been found that the com-

backbone and hence causing a contraction and overall damage plexes strongly bound with DNA relative to the ligand and

to the secondary structure of DNA.⁸⁴The hyperchromic eﬀect Ni(^II) complexes are better relative to Zn(II) complexes. Further-

may also be ascribed to external contact (electrostatic more the size of particles influences the interaction. The order

binding)⁸⁵or to partial untwisting of the helix structure of of binding aﬃnity is 3 > 4 > 5 > 1 > 2 > L.

DNA, exposing more bases of the DNA.⁸⁶These spectroscopic

The eﬀective nuclear charge for the Zn atom is more than

characteristics suggest that the complexes had some inter- the Ni atom so it seems that the electrostatic interaction with

action with DNA. The basic binding constant K_bis a useful DNA is more likely for the Zn complexes; on the other hand

tool to define the magnitude of the binding strength of com- the square planer structure for Ni (d⁸complexes) is more

pounds with DNA. For Ni complexes the intrinsic binding con- responsible for the strong intercalative interaction with respect

stant of the complex, K_b, was determined by using the to the Zn complexes (d¹⁰) with the tetrahedral structure which

nonlinear least-squares fitting analyses.^87,88

correlates well with their strong DNA binding aﬃnity.

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Fig. 3 Absorption spectra of (a) L (50 μM), (b, c) complexes 1, 2 (90 μM) and (d–f) complexes 3–5 (20 μM) in 1 mM Tris HCl buﬀer at pH 7.2, in the

absence and presence of increasing amounts of DNA (0–260 μM in (a–c) and 0–146 μM in (d–f). Insets: plot of [DNA]/(ε_a− ε_f) vs. [DNA] for L, 1, 2

and the least squares ﬁt of (ε_a− ε_f)/(ε_b− ε_f) vs.[DNA] using the MvH-equation for 3–5.

The results derived from the UV titration experiments ted to the z* eﬀect and geometry. The complexes exhibited

propose that all complexes can bind to DNA although the exact more hypochromicity than the ligand, signifying that the

mode of binding cannot be only suggested by UV spectro- binding strength of the complexes is much stronger than that

scopic titration studies.⁹⁹Nonetheless, the existence of hypo- of the free ligands. Intercalation is the most possible binding

chromism for the complexes could be considered as the first mode between Ni(^II) complexes and DNA because of the more

evidence that the binding of the complexes involving inter- planar aromatic structure of these complexes, but the inter-

calation between the base pairs of DNA cannot be ruled out. calative ability of Zn(^II) complexes to DNA appears weak and

The diﬀerent behaviors between the complexes may be attribu- suggests the involvement of L and 1, 2 in DNA groove binding.

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concentration of Zn(^II) complexes, indicating that the binding

mode of DNA with 1, 2 may be the groove binding.

Table 1 Absorption spectral properties of compounds bound to DNA,

concentrations of solution L and complexes at pH 7.2 in buﬀer solutions

are 50 μM (L), 90 μM (1, 2), 20 μM (3–5)

Circular dichroism spectroscopy

λ

Change in

Red shift

(nm)

K_b(M⁻¹

)

s_opt

Circular dichroic spectroscopy is a useful method for monitor-

ing changes in the DNA structure in solution and provides

information about binding interactions with 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 helicity¹⁰²and

these bands are quite sensitive to the mode of DNA inter-

actions 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, while

the classical intercalation increases the base stacking and

stabilizes helicity, and so enhances the intensity of the positive

band.¹⁰³

Fig. 5 depicts the CD spectra of DNA with increasing con-

centrations of metal complexes. The addition of complex 1 to

the solution of DNA induced a decrease in intensity for the

negative band at ∼245 nm and the positive band at ∼275 nm

with a small blue shift of about 4 nm (Fig. 5a), suggesting that

the stacking mode and the orientation of base pairs in DNA

were disturbed. The same result for complex 2 was observed

(Fig. S26a†).

The phenomenon for complex 3 was similar to that for

complex 1 at ∼275 nm bands, while a much larger decrease in

intensity for the positive band at ∼275 nm and a new small

positive band at ∼244 nm were observed as shown in Fig. 5b.

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

forms.¹⁰⁴The same result for complex 4 was observed but with

a lower decrease in intensity (Fig. S26b†). For complex 5

(Fig. 5c), there was a decrease in intensity for the negative

band at ∼245 nm and for the positive band at ∼275 nm

without a shift in the band positions. This showed that the

DNA would interact with these complexes and might be dis-

torted into other structures.¹⁰⁵

Compound (nm) absorbance

L

1

2

3

4

5

387

386

375

Hypochromism

0

8

7

6

0.25 × 10³

3.33 × 10³

2.22 × 10³

13.84 × 10⁶1.0

9.99 × 10⁶

1.70 × 10⁶

1.3

1.1

Viscosity measurements

Viscosity measurement is often considered as an eﬀective

mode to determine the binding mode between small mole-

cules and DNA. Intercalating agents are expected to lengthen

the double helix to accommodate the ligands in between the

bases, leading to an increase in the viscosity of DNA.¹⁰⁰

However, for the electrostatic or groove binding, there is a

slight eﬀect on the viscosity of DNA.¹⁰¹The values of relative

specific viscosity (η/η_o), where η and η_oare the specific viscos-

ities of DNA in the presence and absence of the complex, are

plotted against r (= [Complex]/[DNA]) (Fig. 4). When small

planar aromatic molecules intercalate between the neighbor-

ing base pairs of DNA, the double helix loosens to accommo-

date the intercalation, which increases the length of the DNA

helix. Since the viscosity of DNA solution is very sensitive to

the changes of DNA length, the increased viscosity of DNA

solution can be associated with the specific intercalation

binding mode. The results reveal that the presence of the three

Ni(^II) complexes has a significant eﬀect on the viscosity of

FS-DNA solution. The increased degree of viscosity, which may

depend on its aﬃnity to DNA, follows the order of 3 > 4 > 5,

which parallels the hypochromism and DNA binding aﬃnities.

As shown in Fig. 4, the viscosities of DNA in the Tris-HCl

buﬀer solution almost do not change with the increasing

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,¹⁰⁶so the conformation of DNA had partly changed due

to the binding interaction between these complexes and DNA.

Electrochemical studies

Cyclic voltammetry has demonstrated to be a very sensitive

analytical method to determine changes in the redox behav-

iour of metallic species in the presence of biologically impor-

tant molecules.^107,108The electrochemical examinations of

metal–DNA interactions can provide a valuable complement to

spectroscopic methods, e.g., for non-absorbing types, and yield

information about interactions with both the reduced and oxi-

dized forms of the metal.¹⁰⁹Cyclic voltammetry of complexes

(1–4) in H₂O/DMSO showed oxidation waves in the sweep

range from −1 V to +1.5 V (Fig. 6). In the absence of DNA,

Fig. 4 Eﬀects of increasing amounts of

(0–2.34 μM) on the viscosity of DNA (10 μM) in 1 mM Tris-HCl buﬀer (r =

0.0–0.25) at 25 °C.

L and complexes (1–5)

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Fig. 6 Cyclic voltammograms of the complex: (a) complex 1 (1000 μM),

(b) complex 2 (1000 μM) in the absence (1) and in the presence of (2–5)

of DNA (10 μL, 10⁴μM in each injection) at 25.0 °C at 50 mV s⁻¹scan

rate in 2% DMSO/1 mM Tris HCl/5 mM NaCl at pH 7.2.

shift while the anodic potential Epa shifts to negative values.

These shifts of the potentials display that complex 1 can bind

to DNA by both intercalation and electrostatic interactions.¹¹⁰

The observed decrease in both the anodic and cathodic cur-

rents, suggest the binding of the complexes to the large gradu-

ally diﬀusing DNA molecule.¹¹¹Further addition of FS-DNA

(0.2–1.0 μM) led to the disappearance of the oxidative waves.

The same result for complex 2 was observed.

In the absence of DNA, complex (3) shows an anodic peak

at 1.136 V and a cathodic peak at −0.599 V (Fig. 6b). With the

increase in concentration of DNA (10 μ, 10⁴μM in each injec-

tion) in a constant amount of the complex (10 mL, 10³μM),

the voltammetric response of the compound changed as is evi-

denced by the sequential drop in peak current and gradual

Fig. 5 CD spectra of DNA (200 μM) at increasing complexes concen-

tration (0–40 μM) in buﬀer (1.0 mM Tris-HCl, 5 mM NaCl); (a) complex 1

(1000 μM); (b) complex 3 (1000 μM), (c) complex 5 (100 μM).

complex (1) shows an anodic peak at 0.987 V and a cathodic peak potential shift in the positive direction at both cathodic

peak at −0.641 V. With the increase in concentration of DNA and anodic peaks that suggests the intercalation of complex 3

(10 μL, 10⁴μM in each injection) in a constant amount of the

into DNA. The observed decrease in both the anodic and

complex (10 mL, 10⁻³M), the voltammetric response of the cathodic currents, suggests the binding of the complexes to

compound changed as is evidenced by the sequential drop in the large slowly diﬀusing DNA molecule.¹¹¹The same result

peak current and, the cathodic potential Epc shows a positive

for complex 4 was observed.

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Fluorescence studies

Table 2 The Stern–Volmer quenching constant for the interactions of

L and complexes 1, 2 and K_fof them with DNA at 37 °C

To further confirm the interaction between the compounds

and DNA, emission experiments were carried out. Ni and Zn

complexes and the ligand show fluorescence at room tempera-

ture in solution or in the presence of FS-DNA, and their

binding to DNA can be directly predicted through the emission

spectra.

a

Compound

K_sv

k_q

L

1

2

1434.3

4040.6

409.0

1.4 × 10¹¹

4.0 × 10¹¹

4.0 × 10¹⁰

^aK_SVis obtained from the slope of the straight line.

Thus, competitive EB binding studies can be ruled out.¹¹²

In the case of intercalating drugs, the molecules are inserted

into the base stack of the helix. The rotation of the free mole-

cules prefers the radiationless deactivation of the excited

states, but if the drugs are bound to DNA the deactivation

through fluorescence emission is favored, and a significant

increase in the fluorescence emission is generally observed. In

the case of groove binding factors, electrostatic, hydrogen

bonding or hydrophobic interactions are engaged and the

molecules are close to the sugar-phosphate backbone, it being

possible to detect a decrease in the fluorescence intensity in

the presence of DNA.¹¹³Fluorescence emission is very sensitive

to the situation, and therefore the fluorophore transfer from

high to low polarity environments usually causes spectral

shifts (10–20 nm) in the excitation and emission spectra of

drugs.¹¹⁴

For Zn(II) complexes and the ligand with increasing concen-

trations of DNA, the intensity of the fluorescence spectra emis-

sion band at a maximum wavelength of about 498 nm

obviously decreased and at about 445 nm slightly enhanced.

Complex 1, which bound to DNA more strongly, is more

eﬃcient than complex 2 in quenching emission because of its

size.

about 450 nm. As shown in Fig. 7, the fluorescence intensities

of the complexes are increased with increasing concentration

of FS-DNA, which agrees with those observed for other inter-

calators¹⁰¹and confirms their interaction with FS-DNA.

This hints that the complexes can be inserted between DNA

base pairs deeply and that they can bind to DNA. The binding

of the complexes to DNA leads to an obvious increase in emis-

sion intensity which is also observed with complexes contain-

ing ligands bearing NH and OH groups.^100,116This is due to

the hydrophobic environment inside the DNA helix reducing

the approachability of the solvent molecules as well as limiting

the mobility of the complex at the binding site, which causes

a reduction of the vibrational modes leading to a higher emis-

sion intensity.¹¹⁷

These fluorescence improvements exhibit that the complex

interacted with DNA and their quantum eﬃciency was

increased.

Like the quenching process, the enhancement constant can

be obtained by eqn (5) ¹¹⁸

F₀

F

¼ 1 ꢀ K_E½Eꢂ

ð5Þ

Quenching of ligand and Zn(^II) complexes bound to DNA is

in good agreement with the linear Stern–Volmer equation.¹¹⁵

The enhancement constants of Ni(^II) complex were calcu-

lated using eqn (5) (Fig. 7 and Table 3).

To determine the strength of the interaction of complexes

with DNA, the value of the binding constant (K_f) resulted from

the Scatchard equation:¹¹⁹

F₀

F

¼ 1 þ K_SV½Qꢂ ¼ 1 þ k_qτ₀½Qꢂ

ð4Þ

where F₀and F are the steady-state fluorescence intensities in

the absence and presence of a quencher, respectively. K_SVis

the Stern–Volmer quenching constant and [Q] is the concen-

tration of the quencher (DNA). The bimolecular quenching

rate constant is k_qand the lifetime of the fluorophore is τ_oin

the absence of the quencher (DNA). Since the fluorescence life-

time is naturally near 10⁻⁸s, the bimolecular quenching con-

stant (k_q) is calculated from K_sv= k_qτ_o.¹¹⁵The fluorescence

quenching curve of the complex by FS-DNA (Fig. 6) illustrates

good agreement with the linear curve of the Stern–Volmer

equation and K_svis calculated by the ratio of the slope to the

intercept (Table 2). The moderately large value of K_svindicates

that the L, complexes 1 and 2 are bound to DNA. According to

eqn (4), k_qis greater than the limiting diﬀusion rate constant

(2.0 × 10¹⁰M⁻¹s⁻¹) for a biomacromolecule, indicating the

existence of a static quenching mechanism. In the static

quenching mechanism, the fluorophore and the quencher

form a complex in the ground state.

ꢀ

ꢁ

F₀ꢀ F

log

¼ log K_fþ n log½DNAꢂ

ð6Þ

F

where F₀and F are the fluorescence intensities of the fluoro-

phore in the absence and in the presence of diﬀerent concen-

trations of DNA, respectively and n is the number of binding

sites.

The values of K_ffor L, 1, 2 were found to be 1.89 × 10³M⁻¹

,

36.90 × 10³M⁻¹and 3.78 × 10²M⁻¹, and n = 1.02, 1.25, 0.99

respectively.

In the case of Ni(^II) complexes, the emission intensity was

enhanced, that is F₀< F, and the titled equation becomes:¹¹⁸

ꢀ

ꢁ

F ꢀ F₀

log

¼ log K_fþ n log½DNAꢂ

ð7Þ

F

The values of K_ffor 3, 4, 5 were found to be 3.26 × 10²M⁻¹

,

The Ni(^II) complexes can emit fluorescence in Tris-HCl 2.91 × 10²M⁻¹and 4.59 × 10²M⁻¹and n = 0.76, 0.81, 0.81

buﬀer at ambient temperature with maxima appearing at respectively.

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Fig. 7 (a–f) Eﬀect of FS-DNA (0–260 μM in 1, 2, 4 and 0–65.8 μM in 3 and 0–132 μM in L, 5) on the emission intensity of the L and 1–5 solution

(1 μM). (Embedded) Stern–Volmer plot for the observed ﬂuorescence of L and complexes upon the addition of DNA.

system of the intercalated ligand due to the coordination with

the M(^II) ion. Since the complexes have a greater planar area

than that of the free ligand, the complexes penetrate more

deeply into and stack more strongly with the base pairs of the

DNA.

Table 3 Enhancement constants of complexes 3–5 with DNA at 37 °C

Complex

K_E

3

4

5

2645.5

1377.2

2480.7

K_Eis obtained from the slope of the straight line.

Gel electrophoresis study

Ligands with DNA interaction ability are always attractive due

to their ability to cause numerous eﬀects like gene expression

modification, cell cycle arrest and DNA cleavage activity.

Together or alone, these modifications change the fate of the

cell to the special programmed cell death called apoptosis.

DNA cleavage including single or double strand breaks are

mainly caused by ligands independently or with the contri-

The value of K_fclearly emphasizes the high aﬃnity of com-

pounds for DNA.

These results showed that the complexes bound more

strongly than the free ligands. The higher binding aﬃnity of

the M(^II) complexes is attributed to the extension of the π

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Fig. 8 Gel electrophoresis diagram of pBlu2KSM DNA in the presence

of increasing amounts of (a) complex 3 and (b) complex 5 (lanes 1–7).

Lane 1, DNA; lane 2, DNA + 30 µM ligand; lane 3, DNA + 40 µM ligand;

lane 4, DNA + 50 µM ligand; lane 5, DNA + 60 µM ligand; lane 6, DNA +

70 µM ligand; lane 7, DNA + 80 µM ligand, lane 8; DNA ladder. Linear

form (LF), nicked circular (NC) and positive and negative supercoiled

(SC) of DNA are labeled, respectively.

Fig. 10 Gel electrophoresis diagram of pBlu2KSM DNA in the presence

of increasing amounts of (a) complex 1, (b) complex 1 + H₂O₂, (c)

complex 2 and (d) complex 2 + H₂O₂. (a and c): Lane 1, DNA Ladder;

lane 2, DNA; lane 3, DNA + 30 µM ligand; lane 4, DNA + 40 µM ligand;

lane 5, DNA + 50 µM ligand; lane 6, DNA + 60 µM ligand; lane 7, DNA +

70 µM ligand, lane 8; DNA + 80 µM ligand. (b and d): Lane 1, DNA;

Lane 2, DNA + H₂O₂; lane 3, DNA + 30 µM ligand + H₂O₂; lane 3, DNA +

40 µM ligand + H₂O₂; lane 4, DNA + 50 µM ligand + H₂O₂; lane 5, DNA

+ 60 µM ligand + H₂O₂; lane 6, DNA + 70 µM ligand + H₂O₂; lane 7,

DNA + 80 µM ligand + H₂O₂, lane 8; DNA ladder. Linear form (LF),

nicked circular (NC) and positive and negative supercoiled (SC) of DNA

are labeled, respectively.

Fig. 9 Ethidium bromide displacement assay. Gel electrophoresis

diagram of HindIII digested pBlu2KSM DNA in the presence of increasing

amounts of (a) complex 3 and (b) complex 5. Lane 1, DNA ladder; lane 2,

DNA; lane 3, DNA + 50 µM ligand; lane 4, DNA + 60 µM ligand; lane 5,

DNA + 70 µM ligand; lane 6, DNA + 80 µM ligand; lane 7, DNA + 90 µM

ligand.

According to Fig. 9, at concentrations higher than 50 µM,

ethidium bromide displacement is started by complex 3 and

the DNA band disappeared with no significant migration of

the band. These results suggested the fact that the ethidium

bromide replacement ability of the ligand is stronger than the

twisting ability. The results may predict the complex 3 mode of

action that like ethidium bromide could intercalate between

DNA double strands causing insertion mutations and sub-

sequently aﬀect DNA replication and transcription.⁹²

Although compound 1 has strong DNA binding aﬃnity

based on fluorescence studies, diﬀerent concentrations of

compounds 1 and 2 alone and in the presence of H₂O₂doesn’t

show any intercalatory eﬀects and single or double strand

breaks (Fig. 10).

bution of oxidant factors like H₂O₂and some intermediate

elements like Cu²⁺that are increased in the malignancy pro-

cedure. Among them double strand breaks cause the most

harmful damage to the cells because cellular repair systems

like NHEJ (Non-Homologous End Joining) almost always repair

the strands with very low accuracy and several strand insertion/

deletions will occur that causes genome instability. Finally this

phenomenon drives cells to apoptosis. In this manner, super-

coiled (SC) plasmid pBlu2KSM DNA was incubated with

varying concentrations of complexes to investigate their DNA

cleavage ability. As shown in Fig. 8, although complexes 3 & 4

caused significant intercalatory eﬀects and also site-specific

double strand break in concentrations higher than 50 µM,

only complex 5 showed a slight intercalatory eﬀect. This

phenomenon is related to the nature of nickel as a successful

ligand in the interaction with DNA. Moreover at higher concen-

trations, there is a change in the electrophoretic mobility of

the NC form eventually leading to condensation of DNA. As

shown in Fig. 9, the process of DNA condensation seems to

start above 50 μM in 3 & 4 and 60 µM of 5 ligand concen-

trations. Ethidium bromide is introduced as a classical double

strand DNA intercalator to investigate the aﬃnity of ligands to

attach to the DNA. To further confirm the interaction modes

and aﬃnity of the complex 3 with DNA, experiments on the

competitive binding behavior of EB and compound 3 to DNA

were carried out. HindIII digested plasmids in the presence of

an increasing amount of complex 3 (lanes 3–7) were prepared

and EB introduced as a control for classic intercalative

interaction.

Cell viability

The ability of the drugs to induce either apoptosis or necrosis

seems to be a primary factor in determining their anticancer

eﬃcacy. Successful anticancer drugs usually trigger several cel-

lular mechanisms for killing the cells. These include DNA

replication interfering (alkylating, antimetabolites and DNA-

binding) agents, protein mimetics, cytostatic behaviour and

energy producing starvation.^120,121Additionally, the other

pharmacodynamic factors like solubility, interactions with

other biomolecules can aﬀect the therapeutic properties of the

drug.¹²²

According to Table 4, the anticancer activities of Ni(II) and

Zn(^II) complexes toward human HepG2 and HeLa cancer cell

lines have been examined in comparison with the currently

used drug 5-fluorouracil under similar conditions by using the

MTT assay. It is found that the compounds exhibit significant

cytotoxic activities in a time-dependent manner in the range of

0–26 μM in 24 hours of incubation compared to the generally

used 5-fluorouracil. Moreover, both nano-sized complexes

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tronic properties of all compounds including: total energy

(TE), dipole moment (DM), atomic charge of transition metal,

energies of frontier orbital (HOMO and LUMO) and the energy

gap between HOMO and LUMO orbital were calculated. The

optimized geometries of the ligands and complexes are shown

in Fig. 11. Selected geometrical parameters including bond

lengths, bond angles and other parameters for all compounds

are listed in Tables 5 and 6. Results show that all of the bond

lengths and bond angles are in the normal range.

Table 4 Comparative IC₅₀values of Ni(II) and Zn(II) complexes when

tested on HepG2 and HeLa cell lines after 24 and 48 hours

HepG2

24 h

HeLa

24 h

Complexes

48 h

4.6 0.4

8.7 0.2

11.7 0.5

18.9 1.8

21.1 1.4

25 2.7

48 h

4.7 0.3

9.1 0.4

11.2 1.6

20.6 0.7

21.9 2.2

15.8 2.1

1

2

3

4

7.3 0.9^a

10.9 1.8

16.3 0.6

23.6 0.4

25.9 1.3

33.5 2.9

6.8 0.7

10.3 1.5

15 1.7

24.7 1.1

22.4 0.5

20.4 1.2

5

5FU^b

Molecular docking simulation

^aIC₅₀= the concentration of drug required to inhibit the growth of

Molecular docking simulations of the synthesized compounds

and DNA were performed to explore more details on the ligand

conformation and their orientations in the active site of the

receptor.

50% of the cancer cells (μM). ^b5-Fluorouracil.

The lowest-energy conformations show that all compounds

exhibited strong DNA binding and slightly more eﬀective anti- approach the gap between DNA minor grooves mainly through

cancer activity compared to classical ones. Although the DNA the metal centers and oxygen groups on the Schiﬀ base struc-

binding activity might be due to the enhanced interaction tures (Fig. 12, S27 and S29†). While no hydrogen bonding was

eﬃciency of the nano-sized complexes, the slight cellular tox- detected for any of the ligands, steric interaction was deter-

icity diﬀerences are related to quite complicated factors that mined to play a dominant role in binding of the ligands to the

should be investigated. The cell viability assay of the com- DNA. The steric interactions arise from the (3-formyl-4-

plexes showed that the Zn(^II) complexes are more eﬀective than hydroxybenzyl)triphenylphosphonium chloride template and

Ni(^II) complexes, despite the weakness of compound 1 to break specifically the phenyl rings. The ligands stretch inside the

DNA strands. The putative mechanism of these anticancer minor groove so that the positively charged atom i.e. the tran-

Zn(^II) complexes remains unclear and further analysis should sition metal and phosphor atoms are located between the

be performed. It could be emphasized that these compounds negative phosphate backbones of DNA to stabilize the ligands

probably activate diﬀerent and various pathways to induce through electrostatic potentials.

apoptosis processes in cells. The mechanism of apoptosis is

From a detailed analysis of the docked structures, it

complex and cells can lead to apoptosis due to various physio- appears that the metal complexes are close to the DNA struc-

logical and pathological conditions. Since the cell is a complex ture from the metal center and the oxygen groups of the Schiﬀ

unit and has complexity in its responses to various signals or base ligand and as mentioned in the Experimental section the

compounds, the lack of logical relationship between the strength nature of the metal has a great influence on interaction mode.

of DNA binding and cytotoxicity potentials of the complexes Furthermore, it has been observed that most of the minor

would be explainable. According to the Ni(^II) double strand groove binding docked compounds prefer AT DNA

breaking ability and knowing the fact that double strand breaks sequences rather than GC DNA sequences and this preferential

are very eﬃcient in triggering specifically the mitochondria binding leads to a better van der Waals’ interaction between

related apoptosis pathway, the appropriate cytotoxicity mechan- the drug molecules and DNA functional groups.¹²⁴It appears

ism of compound 3 might be the main intrinsic pathway of that the Ni compounds approach the thymine base while

apoptosis induced by DNA damage.¹²³When the DNA damage the Zn compound is close to the adenine base in the DNA

is recognized by the repaired protein complexes, the fate of the structure.

cell is survival or death. In this regard the cell cycle should be

The DNA binding aﬃnity of the complexes may be pre-

arrested to repair the damage or programmed cell death includ- dicted from the binding scores. The calculated binding scores

ing apoptosis, necrosis and autophagy will occur if the damage for all optimized compounds are in the range of −95.232 to

is strong enough. Although the Classical Non Homologous End −115.373 (Table 7). It should be pointed out that the above

Joining (C-NHEJ) repair system is introduced as the most docking calculation did not take the intercalation into

eﬀective and fast mechanism for double strand break repair, the account, and the results only allow a prediction of the binding

mechanism is error prone with minimum end processing and aﬃnity and the sterically acceptable conformations of the com-

cell death is the fate of most damaged cells.⁶²

plexes to DNA.

The modes of binding of the complexes with DNA obtained

from molecular docking studies correlate well with the experi-

mental findings.

DFT study

The geometries of ligands and complexes involved in this

Therefore, on the basis of Rerank Score results, complex 3

study were optimized by using the GAUSSIAN 03 program. The is found to be more eﬃcient towards the DNA target as com-

DFT method with B3LYP functional, LANL2DZ basis set for pared to the other complexes. The DNA binding potency

metal centers and 6-311g* for other atoms was used. The elec- follows the order of: 3 > 5 > 1 > L as supported by the experi-

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Fig. 11 The optimized geometrical structures of compounds: (a) free ligand, (b) [Zn(5-CH₂PPh₃-3,4-salpyr)](ClO₄)₂(1, 2), (c) [Ni(5-CH₂PPh₃-3,4-

salpyr)](ClO₄)₂(3, 4), (d) [Ni(5-CH₂PPh₃-2,3-salpyr)](ClO₄)₂(5).

Table 5 Selected bond lengths in Å and angle (°) by theoretical calcu-

lation by the B3LYP method

Complex 5

Complex 1

Complex 3

Bond length

Ni–N1

Ni–N2

Ni–O3

Ni–O4

1.879

1.877

1.851

1.849

Zn–N2

Zn–N3

Zn–O4

Zn–O23

2.126

2.120

1.951

1.954

Ni–N1

Ni–N2

Ni–O18

Ni–O3

1.878

1.863

1.849

1.848

Bond angles

N1–Ni–N2 86.699

N1–Ni–O3 94.008

N2–Ni–O4 94.151

O3–Ni–O4 85.141

N2–Zn–N3

N3–Zn–O4

N2–Zn–O23 88.532

78.897

88.923

N1–Ni–N2

86.320

N1–Ni–O18 94.410

N2–Ni–O3 93.852

O4–Zn–O23 103.384 O3–Ni–O18 85.421

167.267 N2–Ni–O18 179.159

N2–Ni–O3 179.291 N3–Zn–O23 167.034 N1–Ni–O3 179.581

N1–Ni–O4 179.144 N2–Zn–O4

Table 6 The computed electronic properties of complexes by using

theB3LYP method

Fig. 12 Computational docking models (using the MVD software) illus-

trating the interactions between DNA and complex 3.

Complex

5

Free

ligand

Complex

1

Complex

3

E_B3LYP(a.u.)

µ (Debye)

HOMO (a.u.)

LUMO (a.u.)

HOMO–LUMO

gap (eV)

−6226.00

10.125

−0.234

−0.126

2.936

−6227.182 −4783.298 −6227.182

4.567

−0.227

−0.112

3.135

1.021

−0.245

−0.125

3.253

4.567

−0.227

−0.112

3.135

mental finding. The results show the metal complexes act

better than free ligand and Ni complexes, because of more pla-

narity of their structures, are better than Zn complexes in their

interaction with DNA.

Metal charge

(mu)

1.206

1.032

1.241

1.032

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through the metal center and oxygen groups of the Schiﬀ base

ligand and prefer to bind to the AT DNA sequences rather than

GC DNA sequences and this preferential binding leads to

better van der Waals’ interaction between the drug molecules

and DNA functional groups.

The docking result also reveals the higher binding aﬃnity

of complex 3 towards DNA receptors in comparison with com-

plexes 1 and the free ligand.

Table 7 Docking results for binding scores of all the compounds to the

DNA structures

Complex

Rerank score

Free ligand

−95.232

−100.827

−115.373

−105.471

1

3

5

The main results of the present investigation confirmed

that the introduction of various metal eﬀects and the size of

particles on these complexes can influence the DNA binding

events and the 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 com-

pounds, which is useful for the design and synthesis of new

metal-based drugs. The inherent properties of inorganic nano-

particles such as their platform-like surface modifications,

surface to volume ratios, and unique optical and biological

properties make them excellent candidates for future bio-

medical applications.

Conclusions

The ligand (L) and new complexes have been synthesized and

well characterized. The DNA binding properties of L and com-

plexes 1–5 were examined by UV-Vis absorption spectra, emis-

sion spectra, viscosity, voltammetric techniques, and gel

electrophoresis. The DNA binding properties of the free

ligands and Zn(^II) and Ni(II) complexes were investigated by

absorption and fluorescence measurements. While L and com-

plexes 1, 2 interact with DNA, presumably by the groove

binding mechanism, complexes 3–5 interacted with DNA

through intercalation which was also confirmed by viscosity

measurements and voltammetric techniques of DNA solutions

in the presence of the complexes. The interaction occurrence

is supported by the following findings:

Acknowledgements

We are grateful to Shiraz University Research Council for its

financial support.

I. Metal complexes strongly bind with DNA relative to the

ligand.

II. Ni(^II) complexes are better relative to Zn(II) complexes

that may be attributed either to the z* eﬀect or geometry.

III. The size of particles influences interaction, so nano

complexes bound to DNA are much stronger.

XI. Among the Ni(^II) complexes: 3 > 4 > 5 and so complexes

3 & 4 containing 3,4-diaminopyridine as the bridging ligand

interacted strongly with DNA relative to 2,3-diaminopyridine

ones.

In the experiment on the DNA cleavage study by gel electro-

phoresis, the results showed that the double strand cleavage

ability of compound 3 is viable for some time, while com-

pounds 1 and 2 are able to cleave the DNA double strand helix

in the presence and absence of H₂O₂. The cell viability assay

showed the eﬀective anticancer activity of all complexes,

although the most eﬀective complexes were of the Zn(^II)

complex family.

These nanocompounds are active for several reasons: it is a

well-known fact that as their size gets smaller, their surface

area-to-volume ratio increases. Therefore, as the nanoparticles

are very small in size, they have an enormous surface area-to-

volume ratio. The available surface areas of the active com-

ponent of the nanoparticles are significant, which increase

contact between the biomolecule such as DNA and these nano-

compounds appreciably and helps to achieve a better reaction

rate and higher eﬃciency.

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Article Doi

Synthesis, characterization, DNA binding, cleavage activity, cytotoxicity and molecular docking of new nano water-soluble [M(5-CH2PPh3-3,4-salpyr)](ClO4)2 (M = Ni, Zn) complexes

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