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DNA Binding Studies of New Ferrocene based Bimetallics.


Summary: Organotin (IV) ferrocenyl benzoates: [(e5-C5H5)Fe(e5-C5H5)C6H4COOSn(C7H7)3] (1) and [{(e5-C5H5)Fe(e5-C5H5)C6H4COO}2Sn(C7H7)2] (2) were synthesized and characterized by FT-IR, multinuclear (1H and 13C) NMR and elemental analysis. Cyclic voltammetric data were used to investigate the redox behavior and DNA binding parameters. The change in peak potential and peak current was used to probe the mode of interaction, binding constant and free binding energy. The DNA-binding potency of compound 1 (K1 = 5.66 x 103M-1) and 2 (K2 = 2.46 x 103 M-1) were calculated and found to be lower than 3 (K3 = 4.80 x 105 M-1). The negative value of [?]G signified the spontaneity and high conformational stability of the compounds with DNA.

Keywords: 4- ferrocenylbenzoic acid, organotin (IV), ferrocene based bimetallics, DNA binding.


Cancer is one of the deadliest diseases for humans. There are more than 100 types of cancer that differ in many characteristics including growth rate, causes, invasiveness and response to treatment. Cancer is a genetic disease characterized by uncontrolled cell division and metastasis. There are a number of factors that can transform normal cells to malignant ones. These include smoking, diet and ionising radiations such as ultraviolet and X-rays that cause damage to DNA and other biomolecules [1]. There are three main methods of cancer treatment: surgery, radiation therapy and chemotherapy, surgery in combination with radiation therapy and chemotherapy is still the best method of treatment in most of the cancer cases. Chemotherapy uses anti-cancer drugs (cytotoxic drugs) to destroy cancer cells [2]. Cisplatin {cis-diamminedichloro platinum(II)} was first synthesised in 1845, its antitumour activity was discovered much later and accidentally in 1965 by Rosenberg [3].

There are limitatins in its use including acquired resistance and toxic side effects [4, 5]. One of possible reason for Overcoming cisplatin resistance may lie in the difference in nature of interaction with the DNA [6]. As we know cisplatin binds covalently with the DNA [7], so there is need of drugs which binds with the DNA noncovalently. Ferrocene and its derivatives have been excessively studied as potential chemotherapeutics [8-10]. Ferrocene derivatives may bind with the DNA via both covalent and noncovalent modes of interaction. The anticancer activity of ferrocene derivatives is found to be dependent on the oxidation state of iron in the ferrocene moiety [11]. Organotin containing compounds are screened as potential anticancer agents and shows different mechanisms at the molecular level [12]. Herein we report synthesis, characterization and DNA binding studies of organotin incorporated ferrocene derivatives.

The electrochemical behaviour and DNA binding studies are carried out by using cyclic voltammetry [13].

Result and Discussion

Compounds (1-2) were synthesized in accordance to scheme 1. The compounds have been characterized by elemental analyses and spectroscopic techniques such as FT-IR, multinuclear (1H and 13C) NMR.

FT- IR spectrum showed absorption bands at (446, 459 cm-1) (1) and (427, 424 cm-1) (2) due to presence of Sn-C and Sn-O respectively which indicated formation of (1-2). The disappearance of characteristic peak for -OH moiety of carboxylic acid clearly justified the bonding with Sn. The 1H NMR spectra for 1-2 showed disappearance of -COOH peak and appearance of extra peaks for benzyl group. The statement is equally supported by 13C NMR data.

1H NMR spectra, ferrocenyl protons appear at 4-5 ppm whereas ferrocenyl carbons appear in the range of 60-90 ppm in 13C NMR spectra. The sp3 and sp2 protons and carbons appear in the usual regions in 1H and 13C NMR spectra however sp3 protons of benzyl moiety are shielded and appeared upfield. The remaining peaks were observed at appropriate chemical shifts and integral values, procedure and complete data is given in the experimental section [13, 14].

DNA Binding Studies

Investigations of interactions of Drug with DNA have enormous importance in life science [15]. Interest in understanding the association of Drug molecules with duplex DNA has been developed in the hope of understanding the mode of binding [16]. The non-covalent interactions of drug with DNA may involve three possible modes of interactions: intercalation, groove binding and electrostatic interactions [17]. There are different techniques which can be used to understand the mode of interaction and DNA binding parameters. One of the sophisticated and sensitive techniques is cyclic voltammetry. Voltammetric measurements were performed in a single compartment cell with a three electrode configuration with the objective of understanding the redox behavior and the DNA binding affinities of 1-2 [18-20]. The measurements were carried out with increasing concentration of calf thymus DNA (1 mL of 20 uM, 40 uM and 60 uM) against constant concentration (1 mM) of 1-2.

The voltammogram was recorded in the absence and presence of CT-DNA in sample solutions. On addition of increasing concentration of CT-DNA into1 mM solution of 1-2, drop in current ipa and shift in anodic potential were observed (as shown in figure 1 and figure 2).

The shift in peak potential is used to investigate mode of interaction between 1-2 and DNA. The negative shift in the peak potential is indicative of the presence of electrostatic interaction of the cationic drug with the anionic phosphate of DNA backbone whereas positive shift in the peak potential is characteristic for the intercalation of drug into double helical structure of DNA.

However ferrocene moiety because of its size cannot intercalate into the double helix DNA thus it is expected that 1-2 undergo only electrostatic interactions. As expected the negative shift is observed for 1-2 hence supporting the said statement. The shift in formal potential may also be used to find out which form: Fe2+ or Fe3+ interacts strongly with DNA. The binding ratio of reduced and oxidized species calculated according to the following equation (1) [21, 22].

Ebdeg and Efdeg are the formal potentials of the free and bound forms of drug respectively. The negative shift indicates that oxidized form interacts strongly with DNA for 1-2. The drop in current is attributed to diffusion of drug into double helical DNA resulting in the formation of supramolecular complex. As the supramolecular complex is formed, number of electron transference is decreased hence drop off in current is obvious. The increase in molecular weight of compound (due to adduct formation with DNA) also justifies the idea that heavy molecules migrate slowly to the electrode hence decrease in current is observed [23].The binding constant is determined using the following equation (2) [24]. where K is the binding constant, i and i0 are the peak currents with and without CT-DNA and A is the proportionality constant.

The plot of 1/[DNA] versus 1/(1[?]i/ i) yields binding constants and are listed inTable 1.

The DNA binding affinity of 4- ferrocenylbenzoic acid (3) is already reported by our research group. The DNA binding affinity of 3 is greater than 1-2 that may be attributed to the mishmash of binding modes that is, ferrocenyl moiety binds electrostatically with negatively charged phosphate of DNA backbone and intercalation of the planner benzoate moiety into the base pair pockets. After complexation with organotin, steric hindrance is increased due to which planner moiety fail to intercalate into double helix DNA [25].

1-2 only show electrostatic interactions as a result binding affinity is decreased as reflected by binding constant values. The binding constants of 1-3, protonated ferrocene [26] and 4-nitrophenyl ferrocene [27] are listed in Table 1.

The free binding energy is calculated as applied to equation (-[?]G = RT lnK). The negative value of free binding energy of 1-3 protonated ferrocene and 4-nitrophenyl ferrocenein kJ/mol at 25 degC shows the spontaneity of compound-DNA interaction [28] as listed in Table 1.



All the chemicals, including 4-aminobenzoic acid, ferrocene, hexadecyltrimethyl ammonium bromide and sodium nitrite were purchased form Sigma Aldrich and were used without purification while tribenzyltin chloride and dibenzyltin chloride was prepared by the literature method [29]. Solvents were distilled prior to use. Elemental analyses were carried out at the University of Manchester Micro- Analytical lab. FT-IR spectra were recorded by Specac Single Reflectance ATR Instrument (4000- 400cm-1).

BRUKER AVANCE 400 MHz NMR spectrometer was used for recording multinuclear (1H and 13C) NMR spectra. Synthesis of [(e5-C5H5)Fe(e5-C5H5)C6H4COOSn (C7H7)3] (1)

Tribenzyltin chloride (1.06 g, 2.41 mmol) was added to a 250 mL round bottom flask charged with 4- ferrocenylbenzoic acid (0.73 g, 2.4 mmol) and triethyl amine (0.33 g, 2.4 mmol) in 50 mL dry toluene with constant stirring and was refluxed for 6-8 hours. The progress of the reaction was monitored by TLC. After the completion of reaction, the mixture was cool to room temperature and filtered off.

The filtrate was vacuum evaporated. Yield: 57%: m.p = 140-142 oC. FTIR (u cm-1); sp2-(C-H) 3088, sp3-(C-H) 2918, (C=O asym) 1739, (C=O sym) 1539,(Fe-cp) 478, (Sn-C) 446, (Sn-O) 427: 1HNMR (400 MHz) (DMSO); 9.03(d = 2H, J = 8Hz), 8.45 (d = 2H, J = 8Hz), 7.96-7.41 (m = 15 H), 4.29 (s = 2H, e 5- C5H4), 4.74 (s = 2H, e5-C5H4 ), 3.94 (s = 5H, e 5- C5H5), 0.40 (s = 6H); 13CNMR (100MHZ) (DMSO):170.9, 144.82, 138.50, 137.62, 137.30, 136.15,130.51, 129.34, 128.65, 127.70, 83.50, 69.82, 69.72,66.88, -2.21: Elemental analysis Cal. (%) forC38H34FeSnO2: C, 65.46; H, .92; Fe, 8.01; Sn,17.03; O, 4.59. Found (%): C, 64.89; H, 4.34; Fe,8.71; Sn, 16.63; O, 5.43.

Synthesis of [{(e5-C5H5)Fe(e5- C5H5)C6H4COO}2Sn(C7H7)2] (2)

Complex 2 was prepared following the same protocol given for compound 1 but the ratio of tribenzyltin chloride and 4- ferrocenylbenzoic acid was changed to (2 : 1). The orange red solid was obtained after filtration. Yield: 48%: m.p = 268oC. FTIR (u cm-1); sp2-(C-H) 3046, sp3-(C-H) 2973, (C=O asym) 1596, (C=O sym) 1348, (Sn-C) 459, (Fe-cp) 480, (Sn-O) 424: 1HNMR (400 MHz) (CDCl3); 8.41(d = 4H, J = 8Hz), 7.48 (d = 4H, J = 8Hz), 7.45 - 7.37 (m = 10H), 4.73 (s = 4H, e5-C5H4 ), 4.40 (s = 4H, e5-C5H4 ), 4.04 (m = 10H, e5-C5H4 ), 0.58 (s = 4H); 13CNMR (100MHZ) (CDCl3): 173.80, 143.82, 142.50, 139.30, 137.15, 130.51, 129.34, 128.65, 127.70, 83.50, 69.82, 68.72, 65.88, -2.18: Elemental analysis Cal. (%) for C48H40FeSnO4: C, 67.40; H, 4.71; Fe, 6.53; Sn, 13.88; O, 7.48. Found (%): C, 66.38; H, 4.87; Fe, 7.01; Sn, 12.91; O, 8.83.

DNA binding studies by Cyclic Voltammetry

Cyclic voltammetric (CV) measurements were performed in a single compartment cell with a three electrode configuration using Eco Chemie Auto lab PGSTAT 12 potentiostat/galvanostat (Utrecht, The Netharlands) equipped with the electrochemical software package GPES 4.9. The three electrode system consisted of reference electrode; RE-1B silver-silver chloride (Ag/AgCl) saturated with sodium chloride (NaCl) of length 70 mm and outer diameter of 6.0 mm (ALS cat # 012167), a Beckman platinum wire of thickness 0.5 mm with an exposed end of 10 mm as the counter electrode and a bare glassy carbon electrode (surface area of 0.071 cm2) as working electrode. The voltammogram of a known volume of the test solution was recorded in the absence of calf thymus DNA (CT-DNA) after flushing out oxygen by purging with argon gas for 10 min just prior to each experiment.

The procedure was then repeated for systems with constant concentration of the drug (1 mM) and increasing concentration of CT-DNA (1 mL of 20 to 60 uM).

All the sample solutions were prepared in 80 % DMSO (80 % DMSO : 20 % H2O) and buffered at pH 6 by phosphate buffer (0.1 M NaH2PO4 + 0.1 M NaOH), 0.12 mM potassium chloride (KCl) was used as supporting electrolyte. The working electrode was cleaned after every electrochemical assay [30]. Methanol recrystallized tetrabutylammonium perchlorate (TBAP) was used as supporting electrolyte. The stock solution of CT-DNA (200 uM) was prepared by using doubly distilled water and stored at 4 degC. The concentration of CT-DNA was determined by UV absorbance at 260 nm (molar coefficient Ie of CT-DNA was taken as 6600 M-1 cm-1).

The nucleotide to protein (N/P) ratio of 1.85 was obtained from the ratio of absorbance at 260 and 280 nm (A260/A280 = 1.85), evidenced for protein free DNA [31].


Ferrocene incorporated bimetallics (1-2) have been synthesized and successfully characterized. The DNA binding potency was examined by using voltammetric measurements. The shift in formal potential reveals the mode of interaction between drug and DNA. The compounds (1-2) found to undergo an electrostatic interaction with the negatively charged phosphate of double helix DNA backbone


The authors are grateful to Quaid-i-Azam University and Higher Education Commission Islamabad, Pakistan for their financial support in the promotion of scientific research.


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Author:Khan, Nasir; Lal, Bhajan; Badshah, Amin; Altaf, Ataf Ali; Kamal, Shafqat Ali Saqib; Zia-Ur-Rehman
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9PAKI
Date:Jun 30, 2013
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