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Synthesis and Chemical Characterization and DNA Binding of Ferrocene Based Ureas.

Byline: Muhammad Ayaz, Amin Badshah, Bhajan Lal, Ataf Ali Altaf, Saqib Kamal, Shafqat Ali, Imtiaz ud-Din and Afzal Shah

Summary:This article reports synthesis and characterization of 1-(4-ferrocenylphenyl)-3-(6-(3-(4- ferrocenylphenyl)ureido)hexyl)urea (1), 1-(3-ferrocenylphenyl)-3-(6-(3-(3- ferrocenylphenyl)ureido)hexyl)urea (2) and 1-(3-ferrocenylphenyl)-3-(6-(3-(3- ferrocenylphenyl)ureido)-2-methylphenyl)urea (3) and their binding with DNA binding. Structural characterization was accomplished by FT-IR, multinuclear (1H and 13C) NMR spectroscopy and melting point.

Cyclic voltammetric measurements were used to probe electrochemical behavior, mode of interaction and DNA binding potency with parameters 1-3. In voltammogram, a shift in peak potential and drop in peak current was observed with increasing concentration of CT-DNA. The negative shift in peak potential indicated electrostatic interactions for 1-3 with negatively charged phosphate of DNA backbone. The binding constant, binding free energy, binding ratio werecalculated and found very impressive. The high negative value of -G signifies the spontaneity and high conformational stability of the compounds with DNA.

Keywords: Ferrocene, Urea, Cyclic Voltammetry, DNA Binding.

Introduction

The search for metal-based antitumor drugs started from the discovery of cisplatin by Rosenberg in 1965 that could effectively inhibit tumor growth [1]. It is currently used in 50-70% of all cancer patients showing good activity against testicular, ovarian, oropharyngeal, bronchogenic, cervical and bladder carcinomas, lymphoma, osteosarcoma, melanoma and neuroblastoma [2]. Although cisplatin is extensively used in cancer therapy yet it has high toxicity that leads to many side effects which limit the administered dose [3, 4] and also some tumors are resistant to it [5].

Apart from the development of platinum based drugs other metal-based anticancer agents have been developed which appear to exhibit fewer side effects [6, 7]. The ruthenium and iron based drugs are better alternatives to platinum drug and show good cytotoxic effects with reduced general toxicity and side effects [8, 9]. The antitumour potency is directly related to the DNA binding affinity of the drug. The development of DNA biosensors in recent times has played an important role toward diagnostic and biomedical as well as forensic applications. Electrochemical DNA studies are used to predict the drug DNA interaction [10].

Ferrocenyl derivatives with low oxidation potential are attracting increasing attention due to their ability to catalyze the production of reactive oxygenated species (ROS) under physiological conditions that generate cytotoxic effects [11]. Some ferrocene derivatives have shown cytotoxicity against lung tumours [12], breast cancer [13], antiproliferative effect, DNA detection [14]. Ferrocene incorporated thioureas are found to have potential antitumour activity against different parent and drug resistant human ovarian tumours [15]. Sorafenib is one of the a commercial drug which is being used clinically for the treatment of renal cell carcinoma (RCS) and hapatocellular carcinoma (HCC) [16] contains urea functionality in its structure.

As early reports [17, 18] had manifested that both urea and ferrocene based compounds have enormous applications in the development of bioactive compounds, here, the synthesis of compounds 1-3 was accomplished by combining these two moieties and screened for their potential DNA binding affinities by cyclic voltammetery.

Experimental

Materials and Methods

Ferrocene, p-nitroaniline, m-nitroaniline, di- isocyanates (1,6-hexamethylenediisocyanate, 2,6- toluenedi-isocyanate), sodium nitrite, HCl and all other chemicals were purchased from Sigma Aldrich and used as received. 4-ferrocenyl-aniline and 3-ferrocenyl- aniline were synthesized in accordance with a reported procedure [19] Solvents such as ethanol, methanol, acetone, diethyl ether, petroleum ether were purified before use according to the standard reported protocols [20]. Sodium salt of deoxyribonucleic acid was purchased from Acros organics. Melting points were measured with BIO COTE Model SMP10 melting point apparatus. FT-IR and multinuclear (1H and 13C) NMR spectra were obtained with Thermo Scientific Nicolet6700 FT-IR and BRUKER AVANCE 300 MHz NMR spectrometer.

General Synthetic Procedure

Di-isocynate was dissolved in dried acetone; to this solution ferrocenyl-aniline was added drop wise under nitrogen atmosphere and kept on stirring for four hours. The reaction mixture was then poured into the ice cold water and stirred well. The solid product (urea) was filtered off and washed with deionized water.

Synthesis of 1-(4-ferrocenylphenyl)-3-(6-(3-(4- ferrocenylphenyl)ureido)hexyl)urea (1)

Hexamethylene di-isocyanate (0.15 mL, 1.05 g/cm3) was dissolved in dried acetone; to this solution 4-ferrocenyl-aniline (0.5 g, 18 mmol) was added drop wise under nitrogen atmosphere and kept on stirring for four hours. The extent of reaction was continuously monitored by TLC. The reaction mixture was then poured into the ice cold water and stirred well.

The solid product 1-(4-ferrocenylphenyl)-3-(6-(3-(4ferrocenylphenyl)ureido)hexyl)urea (1) was filtered off and washed with deionized water.

Yield: 73%, m.p = 289 oC. FT-IR ( cm-1): NH (3387, 3319 ), sp2-C-H (3084), C=O (1653), C=C Ar (1587, 1557), C-N (1240), Fe-cp (483): 1H-NMR (300 MHz, DMSO) ppm J (1H1H): 8.38 (s, 1H, CONH), 7.38 (d, 2H (8.7Hz) C6H4), 7.31 (d, 2H (8.7Hz) C6H4), 6.61 (s, 1H, CONH), 4.68 (t, 2H, (1.80Hz) C5H4), 4.27 (t, 2H, (1.65Hz) C5H4), 3.99 (s, 5H, C5H5), 3.07 (t, 2H, (6Hz) C3H6), 1.44 (m, 4H, C3H6); 13C-NMR (75 MHz, DMSO) ppm: 155.65 139.10, 131.5, 126.6, 118.1, 85.9, 69.7, 68.8, 66.1, 42.5, 30.2, 26.6.

Synthesis of 1-(3-ferrocenylphenyl)-3-(6-(3-(3- ferrocenylphenyl)ureido)hexyl)urea (2)

The methodology used for the the synthesis of (2) is the same as described for the compound 1 except using 3-ferrocenyl-aniline in place of 4-ferrocenyl- aniline.

Yield: 72%, m.p = 296 oC. FTIR (cm-1 ): NH (3380, 3316), sp2-CH (3033), C=O (1651), C=C Ar (1590, 1556), C-N (1245), Fe-cp (486): 1H-NMR (300 MHz, DMSO) ppm J (1H-1H): 8.40 (s, 1H, CONH), 7.55 (s, 1H (8.7Hz) C6H4), 7.23 (d, 1H (7.5Hz) C6H4), 7.13 (t, 2H (7.65Hz) C5H4), 7.06 (d, 1H (7.2Hz) C5H4), 6.16 (s, 1H, CONH), 4.66 (s, 2H, C5H4), 4.32 (s, 2H, C5H4), 4.02 (s, 5H, C5H5), 3.11 (t, 2H, J=5.7Hz), 1.40 (m, 4H, C3H6); 13C-NMR (75 MHz, DMSO) ppm: 155.4, 139.11, 131.5, 127.9, 126.6, 123.6, 118.1, 85.9, 69.7, 68.8, 66.1, 41.1, 29.3, 27.4.

Synthesis of 1-(3-ferrocenylphenyl)-3-(6-(3-(3- ferrocenylphenyl)ureido)-2-methylphenyl)urea (3)

The methodology used for the the synthesis of (2) is the same as described for the compound 2 except using 2,6-toluene di-isocyanate in place of hexamethylene di-isocyanate.

Yield: 72%, m.p., (315 oC). FTIR (cm-1 ): NH (3317 and 3299), sp2 CH (3029), C=O (1645), C=C Ar (1588, 1471), C-N (1217), Fe-cp (484): 1H-NMR (300 MHz, DMSO) ppm J (1H1H): 9.09 (s, 1H, CONH), 8.69 (s, 1H, CONH), 8.53 (s, 2H, C6H4), 7.95 (d, 1H, (12.6Hz) C6H3), 7.61 (t, 1H, (9.15Hz) C6H3), 7.25 (m, 4H, C6H4), 4.71 (s, 2H, C5H4), 4.34 (s, 2H, C5H4), 4.04 (s, 5H, C5H5), 2.29 (s, 3H); 13C- NMR (75 MHz, DMSO) d (ppm):153.1, 140.3, 140.2, 139.9, 138.3, 130.7, 129.2, 120.2, 116.4, 38.5, 59.9, 69.3, 66.8, 17.8.

DNA binding studies

Voltammetric 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; Standard Calomel Electrode SCE (Fisher Scientific Company cat # 136395), 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 drugs (1 mM) and varying concentration of CT-DNA (20 M to 60 M).

The working electrode was cleaned after every electrochemical assay. Sodium salt of calf thymus DNA was purchased from (Acros) and used as received. All sample solutions were buffered at pH 6 using phosphate buffer (0.1 M NaH2PO4 + 0.1 M NaOH) and were prepared in 20% aqueous DMSO (20% H2O : 80% DMSO). Tetrabutylammonium perchlorate (TBAP) was used as supporting electrolyte [21].

The stock solution of CT-DNA (200 M) was prepared by using doubly distilled water and stored at E O C. The concentration of CT-DNA was determined by UV absorbance at 260 nm (molar coefficient 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 [28, 32].

The electronic spectra of a known concentration of the compounds were obtained without DNA. The max obtained were in the range of 250-270 nm which is also the absorption region for DNA so it becomes very difficult to conclude weather the changes are due to increase in concentration of DNA or drug-DNA adduct formation.

The cyclic voltammetric measurements were carried out by using constant concentrations of compounds i.e. 1 mM and increasing concentration of calf thymus DNA (1 mL of 20 M, 40 M and 60 M). The voltammogram was recorded in the absence and presence of CT-DNA in sample solutions

Results and Discussion

Compounds (1-3) were synthesized in accordance to Scheme-1. The compounds have been characterized by spectroscopic techniques such as FT- IR and multinuclear (1H and 13C) NMR.

Equation

Scheme-1: Synthesis of ferrocene based ureas.

In FT-IR spectra, two peaks appeared in the range of 3200-3400 cm-1 for N-H stretching, Sp2 (C-H stretching) appeared in usual range 3000-3100 cm-1 and the diagnostic peak for (C=O) appeared in the range of 1640-1660 cm-1. The characteristic peak of the Fe - cap appeared at 482-484 cm-1. 1H-NMR spectra, ferrocenyl protons appeared at 4-5 ppm giving three different signals; one signal for unsubstituted cp ring and two for substituted cp ring. Ferrocenyl carbons appear in the range of 60-90 ppm, displaying four signals for non- equivalent carbons that is, one signal for unsubstituted cp ring and three for substituted cp ring in the 13C-NMR spectra. The sp3 and sp2 protons and carbons appear in the usual regions in 1H and 13C-NMR spectra [20, 21].

Cyclic Voltammetric Measurements

Cyclic voltammetric (CV) measurements were performed with the objective of understanding the redox behaviour and the DNA binding affinities of 1-3 in a single compartment cell with a three electrode configuration [22-24] The voltamometric behavior of into 1 mM compound 3 is shown in Fig. 1 that shows a shift in anodic potential and drop in current ipa on addition of increasing concentration of CT-DNA. 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 a decrease in electron transfer takes place causing reduction in intensiy of current. The increase in molecular weight of compound (due to adduct formation with DNA) confirms the idea that heavy molecules migrate slowly to the electrode, hence decrease in current is obvious. The shift in formal potential reveals the mode of interaction between drug and DNA.

A negative shift in peak potential is indicative of the presence of electrostatic interaction of the cationic drug with the anionic phosphate of the DNA backbone whereas intercalation of drug into the double helical structure of DNA causes a positive shift in the peak potential. All the three 1-3 have shown a negative shift in peak potential (see supplementary data for 1-2) which is attributed to electrostatic interactions between the drug and DNA. The shift in formal potential can be used to find which form is interacting strongly with DNA i.e. ferrocene or ferrocinium. The binding ratio of reduced and oxidized species was calculated by the following equation (1) [25].

Equation

Eb and Ef are the formal potentials of the bound and free forms of drug respectively. The mechanism of interaction with DNA may involve; Fe2+ may bind first with DNA followed by oxidation of Fe2+ to Fe3+ which may generate reactive oxygenated species (ROS) in vivo that cause oxidative stress and damage to DNA, However, at the same time other possibility of binding with DNA is that first oxidation of Fe2+ to Fe3+ takes place and then oxidized form may be responsible for the interaction with negatively charged phosphate of double helix DNA backbone as shown in Scheme-2 [26, 27].

The binding parameters were calculated according to the equation (2) [28]. The plot of 1/[DNA] versus 1/(1-i/io) (figure 1) yielded binding constants. The binding affinity of 1 and 3 is greater than that of the protonated ferrocene and 4-nitrophenyl ferrocene [29, 30] and are summarized in Table-1.

Equation

were K is the binding constant, i and io are the peak currents with and without DNA and A is the proportionality constant. The negative value of binding energy (-G = RT lnK) of 1-3 in kJ/mol at 25 C shows the spontaneity of compound-DNA interaction [31] as listed in Table-1.

Table-1: Binding constant and binding energy values of 1-3, protonated ferrocene and 4-nitrophenyl ferrocene.

###Compound###Binding Constant (M -1)###-G ( kJ/mol)

###1###3.75 x 104###26.10

###2###2.00 x 103###18.83

###3###7.42 x 104###27.78

Protonated ferrocene###3.45 x 102###13.29

4-nitrophenyl ferrocene###3.85 x 103###20.45

Electronic Absorption Studies

The electronic spectrum of 1-3 without DNA has max value in the range 250-270 nm which is also the absorption range of DNA (260 nm) [32]. On addition of increasing concentration of DNA hyperchromic effect was observed but at the same time it could not be conclusively decided that the change in absorption in a particular region was due to addition of DNA or due the interaction of DNA with compounds 1-3. Cyclic voltammetric measurements thus were used to probe mode of interaction and binding parameters of the synthesized compounds with DNA.

Conclusions

Three ferrocene incorporated N, N'- disubstituted ureas (1-3) were synthesized and successfully characterized. The compounds (1-3) undergo an electrostatic interaction with the negatively charged phosphate of DNA backbone. The compound 1 and 3 have shown greater DNA binding affinities compared to simple ferrocene and 4-nitrophenyl ferrocene.

Acknowledgement

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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