The new generation drug candidate molecules: Spectral, electrochemical, DNA-binding and anticancer activity properties

Accepted Manuscript The new generation drug candidate molecules: spectral, electrochemical, DNAbinding and anticancer activity properties Ayşegül Gölcü, Harun Muslu, Derya Kılıçaslan, Mustafa Çeşme, Özge Eren, Fatma Ataş, İbrahim Demirtaş PII:

S0022-2860(16)30276-9

DOI:

10.1016/j.molstruc.2016.03.078

Reference:

MOLSTR 22389

To appear in:

Journal of Molecular Structure

Received Date: 16 February 2016 Revised Date:

24 March 2016

Accepted Date: 24 March 2016

Please cite this article as: A. Gölcü, H. Muslu, D. Kılıçaslan, M. Çeşme, Ö. Eren, F. Ataş, İ. Demirtaş, The new generation drug candidate molecules: spectral, electrochemical, DNA-binding and anticancer activity properties, Journal of Molecular Structure (2016), doi: 10.1016/j.molstruc.2016.03.078. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

The new generation drug candidate molecules: spectral, electrochemical, DNA-binding and anticancer activity properties Ayşegül Gölcü*,a, Harun Muslub, Derya Kılıçaslanb, Mustafa Çeşmea, Özge Erena, Fatma Ataşa and İbrahim Demirtaşc Department of Chemistry, Kahramanmaraş Sütçü İmam University, 46100 Kahramanmaraş, Turkey; b

Afşin Vocational High School, Kahramanmaraş Sütçü İmam University, 46500, Kahramanmaraş, Turkey

Department of Chemistry, Çankırı Karatekin University, 18100 Çankırı, Turkey

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c

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a

ABSTRACT

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The new generation drug candidate molecules [Cu(5-Fu)2Cl2H2O] (NGDCM1) and [Zn(5-Fu)2(CH3COO)2] (NGDCM2) were obtained from the reaction of copper(II) and zinc(II) salts with the anticancer drug 5-fluoracil (5-Fu). These compounds have been characterized by spectroscopic and analytical techniques. Thermal behavior of the compounds were also investigated. The electrochemical properties of the compounds have been investigated by cyclic voltammetry (CV) using glassy carbon electrode.

The biological

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activity of the NGDCM1 and NGDCM2 has been evaluated by examining their ability to bind to fish sperm double strand DNA (FSdsDNA) with UV spectroscopy. UV studies of the interaction of the 5-Fu and metal derivatives with FSdsDNA have shown that these compounds can bind to FSdsDNA. The binding constants of the compounds with FSdsDNA

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have also been calculated. Thermal decomposition of the compounds lead to the formation of

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CuO and ZnO as final products.

The effect of proliferation 5-Fu, NGDCM1 and NGDCM2 were examined on the

HeLa cells using real-time cell analyzer with three different concentrations.

Keywords: metal based drugs, 5-Fluoracil, electroanalysis, DNA binding, xCELLigence

Introduction Many bioinorganic and bioanalytical research groups have focused on the synthesis of metal-based drug candidate molecules since it was proved to have anticancer activity of cis-

ACCEPTED MANUSCRIPT platinum. Especially in the last fifty years, many metal complex compounds of biological assays in-vitro and in-vivo were performed. The most commonly used metals are Pt(II/IV), Cu(II), Zn(II), Ru(II/III) and Au(I/III) [1]. Cancer, also known as a malignant tumor or malignant neoplasm, is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body [2,3]. Not all tumors are cancerous; benign

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tumors do not spread to other parts of the body. Possible signs and symptoms include: a new lump, abnormal bleeding, a prolonged cough, unexplained weight loss, and a change in bowel movements among others. While these symptoms may indicate cancer, they may also occur due to other issues. There are over 100 different known cancers that affect humans. The

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discovery and development of anticancer drugs, especially cytotoxic agents, differ significantly from the drug development process for any other indication. The unique challenges and opportunities in working with these agents are reflected in each stage of the

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drug development process. 5-Fu (5-Fluoro-1H,3H-pyrimidine-2,4-dione) (Scheme) sold as Adrucil among others, is a drug that is a pyrimidine analog which is used in the treatment of

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cancer.

Scheme. The chemical structure of 5-Fu

It is a suicide inhibitor and works through irreversible inhibition of thymidylate

synthase. It belongs to the family of drugs called the antimetabolites [4]. It is on the World Health Organization's List of Essential Medicines, a list of the most important medications needed in a basic health system [5]. 5-Fu acts in several ways, but principally as a thymidylate synthase (TS) inhibitor. Interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, which is a nucleoside required for DNA replication. Thymidylate synthase

methylate’s

deoxyuridine

monophosphate

(dUMP)

to

form

thymidine

monophosphate (dTMP). Administration of 5-Fu causes a scarcity in dTMP, so rapidly

ACCEPTED MANUSCRIPT dividing cancerous cells undergo cell death via thymine less death [6]. Calcium folinate provides an exogenous source of reduced folinates and hence stabilizes the 5-Fu-TS complex, hence enhancing 5-Fu's cytotoxicity [7]. Several metal complexes of 5-Fu have been synthesized and characterized so far. Firstly, the Mn(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II) complexes have been synthesized

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and characterized by Singh and et al. [8]. Similarly, a green compound of 5-fluorouracil with copper has been prepared in aqueous alkaline solution by Allan and McCloy [9]. The palladium(II) complex of 5-fluorouracil-l-acetic acid has been prepared and characterized by means of elemental analysis, molar conductivity, IR, UV, H-NMR spectra and TG-DTA

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analysis by Huang at al. [10] and the antitumor activity (against HL-60 human leukemia cells in vitro) results of palladium(II) complex of Fu have been given through the text. In Tyagi’s text, the solution studies have been given pH-metrically to study the interaction of Co(II),

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Ni(II), Cu(II), Zn(II) and Cd(II) metal ions with 5-Fu and in the presence of each other (ternary) at 25±0.1 oC temperature and a constant ionic strength of 0.1 M NaNO3 in aqueous solution [11].

Our research group, since 2001, synthesize new metal-based drug candidate molecules and investigate certain structural characterization and analytical aspects. In our anticancer

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studies, we use the drug substances used in the treatment of cancer already on the market with the idea of reducing the toxic side effect of the molecule to be connected to the metal. In this study, the Cu(II) and Zn(II) complexes have been synthesized as the new generation drug candidate molecules of -commonly anal, breast, colorectal, esophageal,

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stomach, pancreatic and skin cancers (especially head and neck cancers) actinic keratosis and used in the treatment of Bowen disease- 5-Fu. The new structure of the synthesized molecules were characterized using spectroscopic and analytical methods as given in the abstract

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portion. Additionally, antiproliferative activity of 5-Fu and new synthesized compounds on HeLa cells (human uterus carcinoma) was investigated in vitro. Antiproliferative effect of the all compounds were tested at 10, 50 and 100 ppm using Real-Time Cell Analyzer (xCELLigence). The results obtained were worked in the same terms cis-platin, oxaliplatin and was comparable with the results of carbomaplatine. Experimental General 5-Fu was kindly provided by Koçak Farma (İstanbul, Turkey). FSdsDNA was purchased from Sigma, NaCl, Zn(CH3COO)2 and CuCl2 were purchased from Merck. All the chemicals and solvents were reagent grade and were used as purchased. FSdsDNA stock

ACCEPTED MANUSCRIPT solution was prepared by dilution of FSdsDNA to buffer solution (containing 150 mM NaCl and 15 mM tris-HCl at pH 7.0) followed by exhaustive stirring at 4 °C for three days [12], and kept at 4 0C for no longer than a week. The stock solution of FSdsDNA gave a ratio of UV absorbance at 260 and 280 nm (A260/A280) of 1.89, indicating that the DNA was sufficiently free of protein contamination [13]. The FSdsDNA concentration was determined by the UV

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absorbance at 260 nm after 1:20 dilution using e = 6600 M-1 cm-1[14]. Elemental analyses (C, H and N) were performed using a LECO CHNS 932 elemental analyzer. Infrared spectra of the compounds were obtained using KBr discs (4000-400 cm-1) with a Perkin Elmer spectrum 400 FT-IR spectrophotometer. The electronic spectra were obtained in the 200-900 nm range by a Perkin Elmer Lambda 45 spectrophotometer. Mass spectra of the ligands were recorded

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on a LC/MS APCI AGILENT 1100 MSD spectrophotometer. 1H NMR spectra were recorded on a Bruker 400 MHz instrument. TMS was used as internal standard and DMSO or acetone

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as solvents. The amount of metal in the complex was determined using ICP-OES techniques. (Perkin Elmer Optima 2100. Operating parameters; Nebulizer flow: 0.8L/min, auxiliary flow: 0.2 L/min, plasma flow: 1.7 L/min, Sample flow rate: 1.5 mL/min, equilibration time: 15sec., RF power: 1452 watts). The thermal analysis studies of the complex were performed on a Perkin Elmer STA 6000 simultaneous Thermal Analyzer under nitrogen atmosphere at a

Synthesis of NGDCM1

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heating rate of 10 °C/min.

The NGDCM1 was obtained according to a general procedure: A solution of a metal salt

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(1 mmol) dissolved in 5 ml of MeOH was added to a solution of 5-Fu ligand (1 mmol) in 5 ml of distilled water and finally 15 ml of MeOH was added to mixture and the mixture was heated under reflux for 1 day. At the end of the reaction, determined by TLC, the precipitate

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was filtered off, washed with distilled water, hexane and dried under vacuum. Physical properties and other spectroscopic data are given below (Fig.1): [Cu(5-Fu)2Cl]H2O: (C8H8N4O5F2CuCl2) Yield: 60%, color: green m.p:245 °C. Elemental analysis, found (calcd. %): C:27.95(27.38); H:3.88(2.86); N:10.86(12.75). Mass spectrum (LC/MS): m/z 412, [C8H8F2N4O5CuCl2+H+] (%6.7), m/z 393, [C8H6F2N4O4CuCl2] (%1.5), m/z 319 [C8H6N4O4F2Cu+2H+] (%76.8), m/z 239.1, [C6H4N2O4Cu] (%100), m/z 175.1 [C4H3O4Cu] (%81.1), m/z 101 [CuO2H2+3H+] FT-IR: (ATR, cm-1) 3031; υ(-OH), 2974; υ(-NH) in phase, 2823; υ(-N-H) out of phase, 1660; υ(-C=O), 1587; υ(-C=O), (C=C) in phase,

ACCEPTED MANUSCRIPT 1431; υ(-C-F), 437; υ(M-O), 342; υ(M-Cl). UV-vis: λmax nm, (ε m-1cm-1) DMSO as solvent: 345.1, Conductivity: 33.9µS.

Synthesis of NGDCM2

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The NGDCM2 was obtained according to a general procedure: A solution of a metal salt (1 mmol) dissolved in 5 ml of MeOH was added to a solution of 5-Fu ligand (2 mmol) in 5 ml of distilled water and finally 15 ml of MeOH was added to mixture and the mixture was heated under reflux for 1 day. At the end of the reaction, determined by TLC, the precipitate

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was filtered off, washed with distilled water, EtOH and dried under vacuum. Physical properties and other spectroscopic data are given below(Fig. 2):

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[Zn(5-Fu)(CH3COO)2]: (C14H14N4O6F2Zn) Yield: 60%, color: white m.p: 290 °C. Elemental analysis, found (calcd. %): C:27.95(27.38); H:3.88(2.86); N:10.86(12.75). Mass spectrum (LC/MS):m/z 441.1, [C14H14F2N4O6Zn-4H+] (%6.4), m/z 386, [C10H10F2N4O6Zn] (%4.5), m/z 301 [C6H12N4O6Zn] (%9.8), m/z 228.1 [C4H8N2O5Zn] (%100), m/z 101.1 [ZnO2H2] (%8.7), FT-IR: (ATR, cm-1) 3037; υ(-OH), 2987; υ(-N-H) in phase, 2887; υ(-N-H) out of phase, 1635; υ(-C=O), 1619; υ(-C=O), (C=C) in phase, 1414; υ(-C-F), 437; υ(M-O). UV-vis: λmax nm, (ε

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m-1cm-1) DMSO as solvent: 323.2, Conductivity: 33.9µS. Electrochemical measurements

Voltammetric measurements at the glassy carbon working electrode was performed using

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a BAS 100W (Bioanalytical System, USA) electrochemical analyzer. Glassy carbon working electrode (BAS; Φ: 3mm diameter), an Ag+/AgCl reference electrode (BAS; 3M KCl) and

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platinum wire counter electrode and a standard one-compartment three electrode cell of 10 mL capacity were used in all experiments. Glassy carbon working electrode was polished manually with aqueous slurry of alumina powder (Φ: 0.01 µm) on a damp smooth polishing cloth (BAS velvet polishing pad), before each measurement. All measurements were realized at room temperature. Mettler Toledo MP 220 pH meters was used for the pH measurements using a combined electrode (glass electrode reference electrode) with an accuracy of ±0.05 pH. Spectrophotometric DNA-binding studies study

ACCEPTED MANUSCRIPT The interaction of the 5-Fu, NGDCM1 and NGDCM2 with FSdsDNA has been studied with UV spectroscopy in order to investigate the possible binding modes to FSdsDNA and to calculate the binding constants to FSdsDNA (Kb). In UV titration experiments, the spectra of FSdsDNA in the presence of NGDCM1 has been recorded for a constant FSdsDNA concentration in diverse [Compound]/[FSdsDNA] mixing ratios (r). The intrinsic binding

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constant, Kb, of the with FSdsDNA has been determined through the UV spectra for a constant NGDCM1 and NGDCM2 concentration (1.62x10-4 M) in the absence and presence of FSdsDNA for diverse r values. [15] Anticancer activity studies of the compounds

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Preparation of samples

Stock solutions of the samples were prepared in DMSO and diluted with Dulbecco’s

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modified eagle medium (DMEM). DMSO final concentration is below 1% in all tests.

Cell lines and cell culture

HeLa cancer cell line was grown in Dulbecco’s modified eagle medium (DMEM)

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supplemented with 10% fetal bovine serum (FBS), 2% penicillin streptomycin. The medium was changed twice a week. Anticancer assay

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Anticancer effects of the compounds were investigated on C6 cells (Rat Brain tumor cells) and HeLa cells (human uterus carcinoma) using impedance-based real time detection of

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cellular viability was conducted using the xCELLigence system Real-Time Cell Analyzer RTCA-MP (Roche Diagnostics, Penzberg, Germany). Recording of cell index values (CI) and normalization was performed using the RTCA Software 1.2 (Roche Diagnostics, Penzberg, Germany). A self-check using RTCA Resistor Plate 96 was conducted prior to any experiment. Impedance measurements were carried out in designated 96 well E-plates (Roche, Penzberg, Germany). The impedance readout as recorded by the xCELLigence system is expressed as arbitrary cell index-values. Cultured cells were grown in 96-well plates (COSTAR, Corning, USA) at a density of 3104 cells/ well. In each experimental set, cells were plated in triplicates and replicated twice. The cell lines were exposed to three concentrations (10, 50 and 100 ppm) of all compounds, for 48 h at 37 °C in a humidified atmosphere of 5% CO2. Oxaliplatin, carboplatin and cisplatin were used as standard

ACCEPTED MANUSCRIPT compounds. Cells were than incubated for vernight before applying the xCELLigence assay reagent (Roche, Germany) according to manufacturer’s procedure. Results were reported as percentage of the inhibition of cell proliferation, where the optical density measured from vehicle-treated cells was considered to be 100% of proliferation. All assays were repeated at

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least third using HeLa cell [14].

Results and discussion

The elemental analysis and physical characteristics of compounds were discussed in

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experimental section. Compounds melt at higher points, are insoluble in water and most organic solvents except DMSO and DMF. The elemental analysis data of the compounds indicate 2:1 (ligand: metal) stoichiometry for both NGDCM1 and NGDCM2.

The

molar

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conductivity measurement was performed for both compounds are in methanol/DMSO (v/v; 1/9) mixture solution. The molar conductance values were found identical in both compounds, and indicating the nature of the electrolyte weak compounds. The complexes are very stable solids at room temperature without decomposition for a long time. The FT-IR spectrum of 5-Fu and NGDCM1 are given in Figure 3. 5-Fu shows

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characteristic bands of the υ(C2=O) and υ(C4=O) at 1682 cm-1 and 1619 cm-1, respectively. And also there are two characteristic bands 2971 cm-1 and 2802 cm-1, which are belong to υ(N-H) stretching. In NGDCM1 and NGDCM 2 compounds, υ(C2=O) stretching was shifted to 1660 cm-1 for NGDCM1 and 1635 cm-1 for NGDCM2. It results from one of the

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coordination side of the 5-Fu is υ(C2=O) group in both NGDCM1 and NGDCM2 compounds. The υ(N-H) stretching of 5-Fu was shifted to 2987 cm-1 and 2887 cm-1 for NGDCM2

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compound. It results from the one of the coordination side of the 5-Fu is υ(N-H) group in NGDCM2.

Far-IR region also scanned for the investigation of metal-oxygen and metal-nitrogen

vibrations. The υ(M-O) stretching were observed at 437 cm-1 for both NGDCM2 and NGDCM2 compounds. The υ(M-N) stretching was observed at 347 cm-1 for NGDCM2 compound. This vibration band did not occur in the 5-Fu spectrum. When we compare the 1H-NMR spectra of 5-Fu and NGDCM2, there are some differences because of the complexation (Fig. 4. and Fig.5.). The peak at 4 ppm in 5-Fu spectrum was shifted to 3.6 ppm in NGDCM2 spectrum. And the peak at 7.4 ppm in 5-Fu

ACCEPTED MANUSCRIPT spectrum almost disappeared in NGDCM2 spectrum. Because of the limited H atom in the 5Fu molecule, there is not much information about complexation with the 1H-NMR spectrum. The LC/MS spectrum of the NGDCM2 compound shows peak at m/z 413. This peak can be attributed to the molecular ion peak [M]+. Alike the molecular ion peak of NGDCM2 compound showed at m/z 439 ([M-2]) (Fig. 6.). The mass fragmentation of NGDCM2 was

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given in Figure 7.

In order to give more detail about the structure of compounds, thermal behavior of the NGDCM1 and NGDCM2 were investigated (Table 1). The thermo-gravimetric analysis for the compounds were carried out within the temperature range from ambient temperature up to

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1000 °C. The thermal results of the NGDCM1 is given in Figure 8. It can be easily understood from the thermal curves of compounds that in NGDCM1 there is a weight lose

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between 30-120 °C, because of the removal of coordinated water molecule. The examination of TG curves showed that compound decompose in four steps between 300-900 °C, temperature ranges. Which can be attributed to organic parts of compound. The last product of the thermos-gravimetric analysis are metal oxides for both compounds. The photoluminescence properties of the 5-Fu, NGDCM1 and NGDCM2 were

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investigated in DMF solution and different concentrations (1.0x10-3 -1.0x10-7 M) at room temperature. The emission and excitation spectra of the compounds 5-Fu and NGDCM1 were shown in Figure 9 and Figure 10, respectively. The intensity of the emission and excitation peak decreased toward the lower concentration, which results from lesser substance quantity

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in lower concentrations [16].

DNA association interactions are of interest for chemistry, molecular biology, and

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medicine particularly for drug discovery and environmental flash medical processes. They concern association with both inorganic and organic compounds as well as various types of assisted interactions such as metal and metal based drug-DNA chemistry. DNA-based biosensor serves as effective screening tools for in vitro tests of this large group of DNA interactions. Due to the preconcentration determination of a trace low molecular mass analyst or group of analyst could also be a result of the study. These noncovalent host-guest interactions are represented mainly by [17,18]. i)

Intercalation between the stucked base pairs of dsDNA

ii)

Binding at major or minor grooves of the dsDNA, and

iii)

Electrostatic interactions.

ACCEPTED MANUSCRIPT In UV titration experiments, the spectra of FSdsDNA in the presence of each compounds have been recorded for a constant FSdsDNA concentration in diverse [compound]/ [FSdsDNA] mixing ratios (r). The intrinsic binding constants, Kb, of the compounds with FSdsDNA have been determined through the UV spectra of the compounds recorded for a constant compound 1.78x10-4 M DNA concentration in the absence and presence of

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FSdsDNA for diverse r values. Figure 11 and Figure 12 illustrates the spectral changes occurred in 1x10-5 M methanolic solution of 5-Fu and NGDCM1 upon addition of increasing amounts of FSdsDNA. Even though no appreciable change in the position of the intraligand band of compounds are

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observed by addition of FSdsDNA, the intensity of the band centered at 267 nm for NGDCM1 (from 267 to 265 for NGDCM1 and from 264 to 260 for NGDCM2) is increased in

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the presence of DNA up to r = 9 and a blue shift of 2 or 4 nm is observed for higher amounts of DNA. The hypsochromic effect observed might be ascribed to external contact (electrostatic binding) or that both compounds could uncoil the helix structure of DNA and made more bases embedding in DNA exposed [19,20]. The intrinsic binding constant Kb of 5Fu, NGDCM1 and NGDCM2 with FSdsDNA represents the binding constant per DNA base pair, can be obtained by monitoring the changes in absorbances between 267 and 264 nm with

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increasing concentrations of FSdsDNA from plots [DNA]/ εa –εf versus [DNA] and is given by the ratio of slope to the y intercept, according to the following equation. [DNA] / (εa - εf) = [DNA] / (εb -εf) + 1 / Kb(εa -εf) where εa = Aobs / [Compound], εa = extinction coefficient for the free compound and εb=

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extinction coefficient for 5-Fu, NGDCM1 and NGDCM2 in the fully bound form, respectively. In plots [DNA] / (εb -εf) versus [DNA], Kb is given by the ratio of slope to the y

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intercept. The high value of Kb obtained for NGDCM1 and NGDCM2 suggest a strong binding of compound to FSdsDNA. Indeed, it is much higher than Kb calculated for 5-Fu (6.6x104 M-1), indicating that the coordination of 5-Fu drug to M(II) ion enhance significantly the ability to bind to FSdsDNA. This is an important point Kb of NGDCM1 and NGDCM2 is higher than the EB binding affinity for DNA (Kb = 1.23 ± 0.07x105) suggesting that electrostatic and intercalative interaction may affect EB displacement [21]. The Kb values of other compounds have been given in Table 2. Cyclic voltammetry (CV) is a type of potentiometric electrochemical measurements. The analyte must be redox active within the experimental potential window. In this study, 5Fu, NGDCM1 and NGDCM2 compounds were subjected to a cyclic voltammetry study in

ACCEPTED MANUSCRIPT order to characterize their electrochemical behavior on glassy carbon electrode (GCE), within the pH range of 2-12 in buffered aqueous media. In Cv studies, 5-Fu yielded one well-defined oxidation peak was observed at 1309 mV for 1x10-4 M 5-Fu solution (pH=5, BR buffer, abbreviation of the BR= Britton- Robinson). The effect of pH on peak potential (Ep) and peak current (Ip) of 5-Fu and NGDCM2 were

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studied via CV technique, respectively (Fig.13 and Fig. 14.). The plot Ep vs pH for the oxidation peak shifted to lower positive potentials as pH increased between pH 2-12 in BR buffer. The results are given in the following equation (r is the correlation coefficient):

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Epa = 1630.2 – 66.034 pH (r: 0.9982) (between pH 2-12)

When CV behavior of NGDCM1 and NGDCM2 in the same medium were analyzed, there is another oxidation peak occurs both NGDCM1 and NGDCM2 compounds, which

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result from the complexation of 5-Fu with Cu(II) and Zn(II) ions. For NGDCM1, new oxidation peak occurred at 33 mV and for NGDCM2, new oxidation peak occurred at -1087 mV at pH=5 in BR buffer. And the peak which belongs to the 5-Fu molecule at 1309 mV shifted to 1298 mV both in NGDCM1 and NGDCM2. The change in the potential values of NGDCM1 and NGDCM2 compounds are given in the following equations:

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Epa = 53.47 – 3.81 pH (r=0.9990) (pH=2-7) for NGDCM1

Epa = 1640.8 – 69.31 pH (r= 0.9984) (pH= 5-12) for NGDCM2 Typical comperative CV voltammograms of 5-Fu and NGDCM2 compounds are given

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Figure 15 and Figure 16, respectively.

The effect of pH on peak current was also examined for 5-Fu, NGDCM1 and

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NGDCM2 compounds within a range of pH = 2-12 in BR buffer solution. The best and sharpest peak and reproducible results were obtained at pH = 5 in BR buffer for both NGDCM1 and NGDCM2. Therefore, this medium was selected for the scan rate studies. Scan rate studies were carried out to analyze whether the process was under diffusion or absorption controlled at GCE. Scan rate studies were analyzed between 50-1000 mV/s. The typical CV scan rate voltammograms are given in Figure 17 and Figure 18 for 5-Fu and NGDCM1, respectively. The equations between peak current (Ip) vs square root of scan rate (v1/2) demonstrates the diffusional behavior. The equations are given below in pH = 5 BRB (n= 10 in all studies): Ipa (µA) = 3.0899 v1/2 (mVs-1) – 8.3464 (r = 0.9930) for 5-Fu

ACCEPTED MANUSCRIPT Ipa (µA) = 2.0584 v1/2 (mVs-1)-17.878 (r = 0.9995) for NGDCM1 Ipa (µA) = 1.7313 v1/2(mVs-1) +1.2347 (r= 0.9979) for NGDCM2 The effect of scan rate on peak current was also analyzed under same conditions with a plot of logarithm of peak current vs logarithm of scan rate, in the same scan rates values. The

logIp = 0.3904 logv (mVs-1) – 0.8218 (r= 0.9954) for 5-Fu logIp = 0.1882 logv (mVs-1) – 0.0804 (r= 0.9977) for NGDCM1

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logIp = 0.4219 logv (mVs-1) + 0.4187 (r= 0.9931) for NGDCM2

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equations are given below:

These slopes of the equations (0.3904, 0.1882, 0.4219) are close to the theoretical expected

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(0.5) value for an ideal reaction of diffusion controlled [22].

The antiproliferative activities of the 5-Fu and other compounds were investigated against HeLa cell line. The cell line was exposed to three different concentrations (10,50,100 µg/mL) of all compounds. The antiproliferative activities of the compounds against HeLa cell line are given in Fig. 19. The activity of the 5-Fu has begun to show the effect, 15 hours after treatment. 50 µg/mL and 100 µg/mL concentrations showed close activity to each other.

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Therefore, cell death is not depending on concentration for 5-Fu. The activities of the NGDCM1, highest cell death occurred at 100 µg/mL. The activities of the NGDCM2 have increased to depending increase of doses against cell line. The antiproliferative activities of the compounds showed the following order: 5-Fu< NGDCM1< NGDCM2. According to the

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obtained results; NGDCM2 was found to exhibit the highest activity. The results are similar to our previous studies when the antiproliferative activities results are compared with the

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oxaliplatin, carboplatin and cisplatin [14].

Conclusion

The anticancer drug 5-Fu has been reacted with Cu(II) and Zn(II) salts and new generation drug candidate molecules have been synthesized and characterized with spectroscopic and electrochemical methods. 5-Fu behaves like a bidentate ligand through carbonyl oxygen’s in NGDCM1 and through one carbonyl oxygen and one deprotonated nitrogen atom in NGDCM2. The interaction of new molecules have been investigated with the interaction of FSdsDNA by UV titration method, these studies shows that NGDCM1 and

ACCEPTED MANUSCRIPT NGDCM2 can bind to FSdsDNA stronger than free ligand, 5-Fu. In order to compare the binding ability to FSdsDNA the intrinsic binding constants, Kb, have been calculated for all molecules. NGDCM1 exhibits much higher intrinsic binding constant to FSdsDNA than the NGDCM2 and 5-Fu. The results show that the metal environment can modulate the binding of new molecules with DNA. The antiproliferative effect of NGDCM2 is much more than 5-Fu.

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The results pave the way for future of clinical trials.

Acknowledgements

References

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(Project No: 2013/1-5 YLS) for the financial supports.

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The authors wish to thank TUBITAK (Project No: 112T721, COST/CM1105) and KSU

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Publishers B.V.. Amsterdam [10] J.Q.Qu, J. Huang, L.F. Wang, G.C. Sun, Chem. Pap. 55 (2001) 319-322. [11] S. Tyagi, S.M. Singh, S. Gencaslan, W.S. Sheldrick, U.P. Singh, Metal Based Drugs. 8 (2002) 6. [12] N. Demirezen, D. Tarınç, D. Polat, M. Cesme, A. Gölcü, M. Tümer, Spectrochim. Acta Part A Mol. Biomol. Spectrosc. 94 (2012) 243–255.

ACCEPTED MANUSCRIPT [13] G. Psomas, J. Inorg. Biochem. 102 (2008) 1798–1811. [14] M. Çesme, A. Gölcü and I. Demirtas, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 135 (2015) 887–906. [15] B. Dogan-Topal, S.A. Ozkan, Talanta. 83 (2011) 780–788.

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[16] S. Purtas, M. Kose, F. Tumer, M. Tumer, A. Golcu, G. Ceyhan, Journal of Molecular Structure. 1105 (2016) 293-307.

[17] J. Labuda, M. Fojta, F. Jelen, E. Palecek, American Scientific Publishers,

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Stevenson Ranch, CA, USA, (2006) 201-228.

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Chim.Acta 409 (2014) 379–389.

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[19] F. Darabi, H. Hadadzadeh, M. Ebrahimi, T. Khayamian, H.A. Rudbari, , Inorg.

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[21] http://en.wikipedia.org/wiki/Ethidium_bromide

[22] V. M. Avcıoglu,, A. Golcu, Synthesis and Reactivity in Inorganic, Metal-Organic,

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and Nano-Metal Chemistry. 45 (2015) 581–590.

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Table 1. Thermo analytical data of 5-Fu, NGDCM1 and NGDCM2

III Residue I NGDCM2 II III Residue

DTG results temp. (C˚) Weight loss Found (%) 21.74 390 8,87 450

570-780 790-990 28.92-110 120-500 510-830 835-990

660 900 156 370 650 900

Table 2. The intrinsic binding constants (Kb) of complexes with FSdsDNA

NGDCM1 NGDCM2

1.0x106 2.9x105

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6.6x104

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5-Fu

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Compounds Kb (±0.03)

evolved moiety H2O+2Cl N2H2

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TG results temp. Peak (C˚) 100-400 410-560

21.37 16.87 7.142 39.7 25.35 17.80

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stage I NGDCM1 II

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samples

C+2F+NH2 Cu 1.75 H2O 2(CH3COO)+2F+NH2 3C+N2H2 Zn

ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Proposed structure of NGDCM1 Fig. 2. Proposed structure of NGDCM2 Fig. 3. The infrared spectra (MIR) of the 5-Fu and NGDCM1

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Fig. 4. 1H NMR spectrum of 5-Fu Fig. 5. 1H NMR spectrum of NGDCM2

Fig. 7. Mass fragmentation of the NGDCM2

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Fig. 8. The thermal curves of the NGDCM1

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Fig. 6. Mass spectrum of the NGDCM2

Fig. 9. Photoluminescence spectrum of 5-Fu at different concentrations Fig. 10. Photoluminescence spectrum of NGDCM1 at different concentrations Fig. 11. Electronic absorption spectrum of 1x10-5 M 5-Fu in buffer solution in the presence of

FSdsDNA at increasing amounts. The arrow shows the intensity changes upon increasing

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concentration of FSdsDNA

Fig. 12. Electronic absorption spectrum of 1x10-5 M NGDCM1 in buffer solution in the

presence of FSdsDNA at increasing amounts. The arrow shows the intensity changes upon

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increasing concentration of FSdsDNA.

Fig. 13. Effects of pH on 5-Fu cathodic peak potential (a) and peak current (b) 5-Fu

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concentration 3x10-3M

Fig. 14. Effects of pH on NGDCM2 cathodic peak potential (a) and peak current (b) NGDCM2 concentration 3x10-3M Fig. 15. Cyclic voltammograms in the range +0 to +2 V of 5-Fu at Britton–Robinson buffer solution (pH: 2(a), pH: 5(b), pH: 7(c), pH: 9(d), pH: 12(e)). Scan rate=100 mVs-1. Supporting electrolyte = buffer solution (color figure available online). Fig. 16. Cyclic voltammograms in the range -2.0 to +1.5 V of NGDCM2 at Britton-Robinson buffer solution (pH: 2 (a), pH: 5 (b), pH: 7 (c), pH: 9 (d), pH: 12 (e)). Scan rate=100 mVs-1. Supporting electrolyte = buffer solution (color figure available online).

ACCEPTED MANUSCRIPT Fig. 17. The scan rate effect on peak current of 3x10-3M 5-Fu at Britton-Robinson buffer solution (pH:2) at different scan rates (Scan rate=100-1000 mVs-1) (color figure available online). Fig. 18. The scan rate effect on peak current of 3x10-3 M NGDCM1 at Britton-Robinson buffer solution (pH:2) at different scan rates (Scan rate=100-1000 mVs-1) (color figure

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available online).

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Fig. 19. Anticancer activities of the 5-Fu, NGDCM1 and NGDCM2

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Fig. 2.

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Fig. 1.

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5-Fu

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%T

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NGDCM 1

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Fig. 3.

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4000.0 3600 3200 2800 2400 2000 1800 -1 1600 1400 1200 1000 800 cm

600 450.0

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Fig. 4.

Fig. 5.

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Fig. 6.

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m/z: 441 f: 439.1 (%5)

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m/z :395 f:391(%24.5)

+2

m/z:303 f:303.0 (%58)

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-65

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m/z:238,9 f:238.1 f(12.3)

-88

m/z:172 f:175.1 (%3.4)

Fig. 7.

-63.1

m/z:99.8 f:101.0 (%57.4) -74

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Fig. 8.

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Fig. 11.

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Fig. 13.

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Fig. 12.

Fig. 14.

b a

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Fig. 15.

b

c

d

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a

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e

Fig. 16.

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Fig. 18.

Fig. 19.

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The aim of this study, is to synthesize new generation drug candidate molecules as alternatives to the metal based drugs used in treatment of diseases.

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