Biologically active meta-substituted ferrocenyl nitro and amino complexes: Synthesis, structural elucidation, and DFT calculations

Accepted Manuscript Biologically active meta-substituted ferrocenyl nitro and amino complexes: Synthesis, structural elucidation, and DFT calculations Faiza Asghar, Amin Badshah, Saira Fatima, Shumaila Zubair, Ian S. Butler, Muhammad Nawaz Tahir PII:

S0022-328X(17)30329-7

DOI:

10.1016/j.jorganchem.2017.05.033

Reference:

JOM 19959

To appear in:

Journal of Organometallic Chemistry

Received Date: 25 March 2017 Revised Date:

13 May 2017

Accepted Date: 15 May 2017

Please cite this article as: F. Asghar, A. Badshah, S. Fatima, S. Zubair, I.S. Butler, M.N. Tahir, Biologically active meta-substituted ferrocenyl nitro and amino complexes: Synthesis, structural elucidation, and DFT calculations, Journal of Organometallic Chemistry (2017), doi: 10.1016/ j.jorganchem.2017.05.033. 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.

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Biologically active meta-substituted ferrocenyl nitro and amino complexes: Synthesis, structural elucidation, and DFT calculations Faiza Asghara, b, c, Amin Badshaha*, Saira Fatimaa, Shumaila Zubaira,

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Ian S. Butlerb, Muhammad Nawaz Tahird

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a*. Corresponding Author: Coordination Chemistry Laboratory, Department of Chemistry, Quaid- i-Azam University (45320) Islamabad, Pakistan. [email protected] a. Coordination Chemistry Laboratory, Department of Chemistry, Quaid-i-Azam University (45320) Islamabad, Pakistan. b. Department of Chemistry, McGill University, Montreal, QC, Canada H3A 2K6. c. Department of Chemistry, University of Wah, Quaid Avenue, Wah (47000), Pakistan. d. Department of Physics, University of Sargodha, Sargodha, Pakistan.

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Abstract

In our search for new anticancer and DNA interacting drugs, ferrocene-incorporated nitro 1a-1b and aniline compounds 2a-2b were successfully synthesized and characterized by various physicochemical and spectroscopic methods. The proposed nitrophenylferrocenes were prepared by the coupling reaction between ferrocene and the diazonium salts of different nitroanilines using a phase transfer catalyst. In the subsequent reactions, these nitro compounds were reduced

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to the corresponding anilines using zinc dust/ammonium formate. Charge distribution and the HOMO/LUMO energies of the optimized structures that were calculated using the DFT/B3LYP method correlate well with the experimentally determined redox potential values. The nature and extent of binding of these complexes with the biomolecule, SS-DNA was examined by cyclic

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voltammetry, UV-Vis spectroscopy, and viscometry; the complexes exhibited good binding strengths to DNA. The diffusion coefficients of the compound-DNA adducts for all the

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complexes are lower than that of the free compound and the small values of the binding site sizes also indicate the dominance of electrostatic interactions. These compounds have also been demonstrated to be decent candidates in terms of free radical scavenging, protein kinase inhibition, and cytotoxicity.

Keywords: Anticancer activity; Free-radical scavenging; Ferrocene; DFT; Spectroscopic study. 1.

Introduction Cancer is the foremost cause of death globally [1]. Regardless of the dramatic advances

in the development of anticancer agents, the cancer death ratio has remained constant over the 1

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past 35 years (ca. 200 deaths for every 100,000 people). In USA, cancer overtook cardiac disorder in 2005 but, even so, every fifth death is instigated by cancer [2]. Efficient treatment of cancer is of paramount importance. Although metals have long been used for therapeutic purposes in a more or less empirical manner [3], the prospective of metal-based anticancer drugs

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has only been recognized and explored only relatively recently following the discoveries of the pharmacological action of cisplatin (cis-PtCl2(NH3)2) and carboplatin (C6H12N2O4Pt) [4-5]. Despite,

the

broad

spectrum

of

efficacy

of

these

drugs

against

different

tumors (testicular, ovarian, breast, bladder, lung, cervical, and brain tumors), they suffer from

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numerous shortcomings, viz. neurotoxicity, nephrotoxicity, gastrointestinal issues, and bone marrow toxicity, and develop acquired resistance after sustained treatment [6-9].

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Some of the most promising novel, non-platinum metal antineoplastic agents have evolved from the arena of bioorganometallic chemistry [10-15]. In this regard, a distinctive effort has been devoted regarding ferrocene-incorporated anticancer agents, possibly owing to the reversible oxidation and reduction of the iron center, which could produce hydroxyl radicals that damage DNA [16]. In 1978, Brynes et al. demonstrated the antitumor action of ferrocenyl derivatives possessing amine or amide functionality towards the lymphocytic leukemia P-388

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cell line [17]. Meanwhile, numerous different categories of ferrocenyl compounds have been synthesized and evaluated in terms of anticancer properties. For instance, Kraatz and coworkers have prepared ferrocene-pyrazole conjugates, which presented virtuous antitumor activities against human adenocarcinoma MCF-7 cells [18]. In recent years, the anticancer action of

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ferrocene derivatives toward A549 human lung carcinoma cells, HepG2 human hepatocellular liver carcinoma cells, and melanoma cells has also been reported for both in vitro and in vivo

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studies [19]. It is alleged that the ferrocene labelled organic frameworks can offer a new incentive for building structures with unparalleled traits to review and modify the anticancer potential [20-21].

The nitro group relative to a hydrogen substituent accelerates the mutagenic and

carcinogenic strength of an aromatic compound. The chemotherapeutic potential of the compounds possessing a nitro aromatic group is chiefly attributed to strong electron-withdrawing character, polarity, small size and ability of the nitro group to form hydrogen bonds [22-23]. Nitroaromatic compounds can exert their biological effects by binding to one or more molecular targets (usually proteins) [24-25]. In addition, the enzymatic reduction of nitro group to reactive 2

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species such as nitroso and N-hydroxyl amine is ultimately responsible for bio-activation of these nitro drugs [26-27]. We report here, the synthesis of biologically active meta-substituted nitrophenyl ferrocene and ferrocenyl aniline derivatives. Bio-assays (cytotoxicity, protein kinase inhibition) and a study of the interaction of the synthesized compounds with SS-DNA were

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carried out for the purpose of increasing our knowledge on the mechanism of action of these potential drugs. Density functional theory (DFT) calculations were performed on the molecular

2.

Experimental

2.1

Materials and methods

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structures to determine the energy of frontier molecular orbitals and the charge distribution.

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Ferrocene, 2-methoxy-5-nitroaniline, 2-methyl-5-nitroaniline, hydrochloric acid, sodium nitrite, ammonium formate, Zn dust, and all the other chemicals were purchased from Sigma Aldrich/Fluka and used as received. The organic solvents, such as diethyl ether, petroleum ether, acetone, ethanol and ethyl acetate, were dried and purified before use according to conventional methods [28]. Melting points were determined in a capillary tube using an electro-thermal melting point apparatus model MP-D Mitamura Riken Kogyo (Japan) and are reported without 400 cm-1 range. 1H- and

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correction. FT-IR data were obtained on a Nicolet 6700 Diamond ATR spectrometer in the 400013

C-NMR measurements were recorded on a Bruker AV500 MHz

spectrometer using deuterated solvent (DMSO) and TMS (Si(CH3)4) as a reference. Raman

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spectra (±1 cm-1) were measured on an InVia Renishaw spectrometer, using argon-ion (514.5 nm) and near-infrared diode (785 nm) lasers. The Renishaw WiRE 2.0 software was used for the Raman data acquisition and spectral manipulation. Elemental analyses were performed using a

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LECO-932 CHNS analyzer, while the Fe concentrations were determined on Perkin-Elmer atomic absorption spectrophotometer model 2380. 2.2

Synthesis of nitrophenylferrocenes 1a-1b The synthesis of the nitrophenylferrocenes 1a-1b was accomplished by adopting the

method reported previously by our group with some modifications [29]. Nitroanilines (a-b) (100 mmol) were added in small portions to 60 mL of 18% aqueous hydrochloric acid solution to form a slurry, while maintaining the temperature at 0-5 °C using a salt water-ice bath. A solution

3

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of sodium nitrite (100 mmol) in 25 mL of water was introduced dropwise to the slurry. After complete addition, the reaction mixture was stirred for 45 min at low temperature (< 5 °C) in order to form the diazonium salt. Ferrocene (50 mmol) and hexadecyltrimethylammonium bromide (CTAB) (1.37 mmol) in 200 mL diethyl ether were cooled to 0-5 °C. The diazonium

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salt solution was then added dropwise to the ferrocene solution containing a phase-transfer catalyst, under constant magnetic stirring and the mixture was maintained below 5 °C. The reaction mixture was stirred overnight at room temperature after which it was concentrated under vacuum and the residue was washed thoroughly with deionized water. The crude product was

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then steam distilled to recover the unreacted ferrocene. The residual crude solid was recrystallized from petroleum ether to give the nitrophenylferrocenes 1a-1b as dark red needles

2.2.1

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(Scheme 1). 2-methoxy-5-nitrophenylferrocene (1a)

Quantities used were 16.8 g (100 mmol) 2-methoxy-5-nitroaniline (a), 6.91 g (100 mmol) sodium nitrite, 60 mL concentrated HCl, 0.5 g (1.37 mmol) hexadecyltrimethylammonium bromide (CTAB) and 9.2 g (50 mmol) ferrocene. Yield 68%; Dark red crystals; m.p. 173°C; FT-

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IR and Raman (powder, cm-1): 3083, 3057 (C-Haromatic), 1595, 1600 (C=C), 1555, 1548 (NOasym), 1340, 1344 (N-Osym), 479, 485 (Fe-Cp); 1H-NMR (500 MHz, DMSO-d6, ppm) δ 8.14 (s, 1H, ArH), 7.84 (d, 1H, J = 7.5 Hz, ArH), 7.64 (d, 1H, J = 8.0 Hz, ArH), 4.93 (s, 2H, C5H4), 4.48 (s, 2H, C5H4), 4.12 (s, 5H, C5H5), 3.97 (s, 3H, OCH3);

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C-NMR (125.81, DMSO-d6, ppm) δ

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148.7, 141.6, 131.8, 130.9, 122.1, 121.4, 86.3, 70.5, 70.1, 67.3; Elemental anal. Calcd. (%) for

16.63. 2.2.2

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C17H15FeNO3: C, 60.57; H, 4.46; N, 4.13; Fe, 16.52. Found (%): C, 60.51; H, 4.57; N, 4.04; Fe,

2-methyl-5-nitrophenylferrocene (1b) Quantities used were 15.2 g (100 mmol) 2-methyl-5-nitroaniline (b), 6.91 g (100 mmol)

sodium nitrite, 60 mL concentrated HCl, 0.5 g (1.37 mmol) hexadecyltrimethylammonium bromide (CTAB), and 9.2 g (50 mmol) ferrocene. Yield 75%; Dark red solid; m.p. 163 °C; FTIR and Raman (powder, cm-1): 3063, 3020 (C-Haromatic), 2931, 2857 (C-Haliphatic), 1541, 1553 (NOasym), 1340, 1349 (N-Osym), 1602, 1591 (C=C), 478, 482 (Fe-Cp); 1H-NMR (500 MHz, DMSOd6, ppm) δ 8.03 (s, 1H, ArH), 7.89-7.55 (m, 2H, ArH), 4.70 (s, 2H, C5H4), 4.44 (s, 2H, C5H4), 4

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4.08 (s, 5H, C5H5), 2.49 (s, 3H, CH3); 13C-NMR (125.81 MHz, DMSO-d6, ppm) δ 146.1, 137.4, 132.7, 131.2, 125.4, 120.8, 81.5, 70.8, 69.6, 68.2, 21.7; Elemental anal. Calcd. (%) for C17H15FeNO2: C, 63.57; H, 4.72; N, 4.35; Fe, 17.38. Found (%): C, 63.51; H, 4.65; N, 4.41; Fe,

2.3

Synthesis of ferrocenylanilines 2a-2b

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17.32.

Reduction of nitrophenylferrocenes 1a-1b to ferrocenylanilines 2a-2b was carried out by mixing the different nitrophenylferrocenes (10 mmol) with Zn dust (40 mmol) in 100 mL ethanol

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at room temperature [30]. To this suspension, a solution of ammonium formate (40 mmol) in ethanol (20 mL) was added dropwise with constant magnetic stirring. After 30 min of stirring, the reaction mixture was heated under reflux for 10 h, and the progress of the reaction was

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monitored by TLC at regular intervals. On completion, the reaction mixture was filtered off and the filtrate was evaporated under reduced pressure. The resulting crude solid was recrystallized from ethyl acetate and the desired anilines 2a-2b were obtained as orange-red crystalline solids (Scheme 1). 2.3.1

4-methoxy-3-ferrocenylaniline (2a)

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Quantities used were 3.37 g (10 mmol) 2-methoxy-5-nitrophenylferrocene (1a), 2.62 g (40 mmol) Zn dust and 2.52 g (40 mmol) ammonium formate. Yield 79%; Orange-red solid; m.p. 184 °C; FT-IR and Raman (powder, cm-1): 3466, 3384 (NH2), 3095, 3043 (C-Haromatic), 1604, 1597 (C=C), 481, 474 (Fe-Cp); 1H-NMR (500 MHz, DMSO-d6, ppm) δ 7.09 (d, 1H, J =

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8.0 Hz, ArH), 6.97 (s, 1H, ArH), 6.74 (dd, 1H, 1J = 8.0 Hz, 2J = 8.5 Hz, ArH), 5.09 (s, 2H, NH2), 4.64 (s, 2H, C5H4), 4.32 (s, 2H, C5H4), 4.01 (s, 5H, C5H5), 3.85 (s, 3H, OCH3);

13

C-NMR

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(125.81 MHz, DMSO-d6, ppm) δ 144.8, 138.8, 129.2, 115.6, 115.3, 113.2, 85.1, 69.8, 69.2, 66.7; Elemental anal. Calcd. (%) for C17H17FeNO: C, 66.45; H, 5.58; N, 4.55; Fe, 18.19. Found (%): C, 66.37; H, 5.50; N, 4.66; Fe, 18.11. 2.3.2

4-methyl-3-ferrocenylaniline (2b) Quantities used were 3.21 g (10 mmol) 2-methyl-5-nitrophenylferrocene (1b), 2.62 g (40

mmol) Zn dust and 2.52 g (40 mmol) ammonium formate. Yield 75%; Orange solid; m.p. 168 °C; FT-IR and Raman (powder, cm-1): 3404, 3351 (NH2), 3074, 3088 (C-Haromatic), 2943, 2894 (C-Haliphatic), 1601, 1590 (C=C), 472, 482 (Fe-Cp); 1H-NMR (500 MHz, DMSO-d6, ppm) δ 7.44 5

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(d, 1H, J = 8.0 Hz, ArH), 6.98 (s, 1H, ArH), 6.77 (d, 1H, J = 7.5 Hz, ArH), 4.85 (s, 2H, NH2), 4.57 (s, 2H, C5H4), 4.25 (s, 2H, C5H4), 3.99 (s, 5H, C5H5), 2.37 (s, 3H, CH3); 13C-NMR (125.81 MHz, DMSO-d6, ppm) δ 139.3, 134.6, 131.7, 125.4, 121.2, 117.9, 84.5, 69.8, 69.3, 66.7, 21.6; Elemental anal. Calcd. (%) for C17H17FeN; C, 70.14; H, 5.86; N, 4.80; Fe, 19.19. Found (%): C,

2.4

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70.21; H, 5.88; N, 4.75; Fe, 19.24. Crystallographic analysis

Diffraction data for the crystallized compound (1a) were collected on a Bruker Kappa

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APEXII CCD Diffractometer equipped with a graphite-monochromated Mo-Kα (λ = 0.71073 Å) radiation source. Data collection used ω scans, and a multi-scan absorption correction was

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applied. The structure was solved using SHELXS-97 program. Final refinement on F2 was carried out by full-matrix least squares using SHELXL-97 software [31]. 2.5

DNA binding studies

2.5.1

Cyclic voltammetry

Voltammetric experiments were performed using a Biologic SP-300 voltammeter running

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on EC-Lab Express V 5.40 software, Japan. Analytical grade TBAP (tertiarybutylammonium perchlorate) was used as supporting electrolyte and N2 gas (99.9 %) was purged through the mixture to avoid any interference from oxygen. Commercial salmon sperm DNA obtained from Sigma Aldrich (Cat. No. 74782) was solubilized in doubly distilled water to prepare a stock

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solution of 6 x 10-4 M from which working concentrations of DNA were prepared. The concentration of the stock solution was measured by UV absorbance at 260 nm using an epsilon

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value of 6600 M-1 cm-1. For electrochemical measurements, a known concentration of the test solution in ethanol was kept in an electrochemical cell and the voltammogram was recorded in the absence of DNA. The procedure was then repeated for systems with a constant concentration of the drug and varying concentrations of DNA. The working electrode was polished with alumina powder and rinsed with distilled water before each measurement [32]. 2.5.2

UV-Vis spectrophotometry Absorption spectra were recorded on a Shimadzu 1800 UV-Vis spectrophotometer. The

absorption spectrum of a known concentration of the synthesized compound in ethanol was 6

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recorded without DNA. The spectroscopic response was then monitored for the same amount of complex on addition of small aliquots of DNA solution. All samples were allowed to equilibrate for 15 min prior to each spectroscopic measurement [32]. Based on the variation in absorption maxima (λmax) in the presence of DNA, the binding constant “K” of compound-DNA complex

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was calculated using the Benesi-Hildebrand equation given below [33]:

Eq 1

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where Ao and A are the absorbance of the free compound and of the compound-DNA complex, ƐG and ƐH-G corresponds to the molar extinction coefficient of the free compound, and the

2.5.3

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extinction coefficient of the compound in the fully bound form, respectively. Viscometry

An Ubbelohde viscometer was used for viscosity measurements at room temperature (25 ± 1 °C). Flow time was measured with a digital stopwatch and the measurements were made in triplicate to obtain an average flow time. The data are presented as relative specific viscosity

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(η/ηo), vs. binding ratio ([Complex]/[DNA]), where η is the viscosity of DNA in the presence of complex and ηo is the viscosity of DNA alone [34]. 2.6

DPPH scavenging activity

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The reducing abilities of the compounds were determined with the help of 1,1-diphenyl2-picrylhydrazyl (DPPH) in DMSO to produce 1,1-diphenyl-2-picrylhydrazine. The decrease in

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the absorption of DPPH was monitored to calculate the % age scavenging according to the following formula [35-36]:

Scavenging Activity (%) = Aₒ-A/Aₒ × 100

Eq 2

where Aₒ is the absorbance of free DPPH and A is the absorption of DPPH-compound mixture with an increasing concentration of compound. To a solution of DPPH (3.9 mg of DPPH in 100 ml DMSO) were added the increasing concentrations (12.5 µg/mL) of drug. The decrease in the absorption of DPPH was monitored spectrophotometrically after 30 min at a wavelength of 517 nm. All the readings were taken in triplicate and the average of the readings was used. 7

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2.7

DFT study Computational studies were carried out in order to calculate the energies of the frontier

molecular orbitals (EHOMO and ELUMO) and the charge distributions on the molecular structures

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using the DFT/B3LYP method combined with a 6-31G basis set. The density functional method (DFT) was used because of its simplicity and less time consumption compared to more sophisticated calculations [37]. The structures of the molecules were first optimized using DFT and then energy calculations were performed on the optimized structures. Gaussian 03W

2.8

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software was used for calculations. Protein kinase inhibition assay

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The kinase inhibition assays were performed using Streptomyces 85E strain according to the previously described protocol [38] with a slight modification. The microorganisms under examination were first refreshed in a sterile Trypton soy broth (Merck, Germany) for 24-48 h and then applied to petri plates containing ISP4 minimal medium. Then, 6-mm Whatman filter paper discs soaked with 5 µL of test sample (20 mg/mL DMSO) were employed on freshly seeded plates. Incubating the plates at 28 oC for 72 h was done to permit the growth of hyphae.

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The clear or bald zones around the disc, which indicate hyphae formation inhibition, were measured to the nearest mm with the help of a Vernier caliper. Surfactin served as the positive control, while DMSO impregnated discs were set as the negative control in order to confirm the

2.9

MTT assay

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non-toxic effect of DMSO.

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The growth inhibitory effect of the synthesized complexes on human leukemia THP-1

(ATCC# TIB-202), and breast cancer MCF-7 (ATCC# HTB-22) cell lines, and breast noncancerous cells MCF-10A (ATCC# CRL-10317) was assessed by use of the MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay, in which the metabolically active cells reduce MTT to yield a DMSO soluble formazan product that can be analyzed calorimetrically [39]. The cells were maintained and cultured in RPMI 1640 culture medium supplemented with 10% FBS (fetal bovine serum). Cisplatin was used as a control. In brief, 1 x 104 cells were seeded in triplicate into the wells of the flat bottomed 96-well culture plates. The 8

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plates were incubated at 37 °C for 24 h in a humidified atmosphere (5% carbon dioxide in air, pH 7.4) to permit the cells to attach. The samples were then added at different concentrations (0100 µM) to triplicate wells. After 72 h of incubation, the cells were exposed to 20 µL of MTT (5 mg/mL) and incubated for 4 h at 37 °C for further cultivation. The yellow formazan crystals thus

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produced in each well were solubilized in 200 µL of DMSO and the absorbance of the resulting solution was read at 570 nm using a microplate reader (Labsystems Multiskan MS). The sensitivity of the compounds 1a, 1b, 2a and 2b against the subjected cell lines was expressed in terms of IC50 values (drug concentration that resulted in 50% reduction of cell growth),

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calculated on the regression line where the absorbance values at 570 nm were plotted against

3.

Results and discussion

3.1

Chemistry

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logarithm of drug concentration. The experiment was performed in triplicate.

Compounds 1a-1b were synthesized by the coupling reaction between ferrocene and the diazonium salts of nitroanilines to form the respective nitrophenylferrocenes. The reactions were carried out in water-ether mixtures using a phase-transfer catalyst (CTAB). The presence of a

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phase-transfer catalyst increased the yield of the compounds from 25 to 65%. Reduction of the nitrophenylferrocenes 1a-1b to the corresponding ferrocenylanilines 2a-2b was performed using zinc dust/ammonium formate, thereby affording the products in high yield. Spectroscopic studies

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3.2

The FT-IR and Raman spectra of the compounds 1a-1b showed no bands above 3200

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cm-1, confirming the coupling of nitroanilines (a-b) with ferrocene. In the FT-IR, the nitro (NO2) moiety of the compounds 1a-1b exhibited two bands, i.e., the antisymmetric vibration in the 1550-1542 cm-1 region and the symmetric vibration in the range of 1351-1344 cm-1. The stretch due to aromatic-H groups was evident just above 3000 cm-1, while the characteristic peak for FeC associated with ferrocene group was observed in the range of 485-472 cm-1. All the other bands are visible in their usual region. The conversion of the nitro group (NO2) to an amino group (NH2) is justified by the absence of the N-O asymmetric and symmetric stretching bands in the region of 1550 cm-1 and 1350 cm-1 respectively, and the appearance of N-H stretching 9

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bands at 3466-3351 cm-1 supports the formation of the anilines 2a-2b, which confirms the reduction process. The 1H-NMR spectra of 1a-1b show that the unsubstituted cyclopentadienyl (η5-C5H5)

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ring of ferrocene yielded an intense singlet at ~4 ppm. In the case of the substituted Cp (η5-C5H4) ring, the ortho protons appear in the region of 4.95-4.53 ppm, while the meta protons occur in the range of 4.45-4.27 ppm. Aromatic protons were visible between 8-7 ppm. In compounds 2a2b, the appearance of a singlet at about 5.09-4.85 ppm due to the two NH protons, provides evidence for the successful reduction of the nitro group to an amino group. The signals due to

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aromatic and ferrocenyl protons for the compounds 2a-2b appear upfield when compared to the respective nitro derivatives 1a-1b, which is in accordance with the inductive and resonance 13

C-NMR data for 1a-1b revealed an intense singlet at 71-69 ppm for the

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effects. The

unsubstituted Cp carbons of ferrocene, whereas the substituted Cp ring provided three peaks, i.e., the ipso-carbon appeared between 87-81 ppm and the other two signals were apparent between 70-65 ppm. In the case of compounds 2a-2b, the signals due to the aromatic and ferrocenyl carbon atoms appear to be more shielded in comparison to the corresponding nitro analogues 1a-

amino functionality.

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1b, which validates the reduction of an electron-withdrawing nitro group to an electron-donating

Elemental analyses (CHNS) of all the compounds showed that the calculated and found values for carbon, hydrogen, nitrogen, and iron are in good agreement with each other, which

3.3

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establishes that the compounds are amply pure in bulk. Structural studies

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Crystals of compound 1a appropriate for crystallographic exploration were grown from

petroleum ether by slow evaporation. The results pertaining to data collection and structure refinement demonstrate that 1a crystallizes in a monoclinic system with P21/n space group. An ORTEP diagram of 1a with atomic numbering scheme is presented in Figure 1. The basic crystal data and description of the diffraction experiment are summarized in Table 1, whereas the selected bond lengths and bond angles have been given in Table S1 (Supplementary Material). The structural parameters for the ferrocenyl moiety in compound 1a are within the customary ranges, and the iron atom is sandwiched almost perfectly centrally between the two

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cyclopentadienyl centroids. Furthermore, the cyclopentadienyl rings (Cp) of ferrocene are almost eclipsed with a tilt of only 3.17° between them. The phenyl ring attached with the Cp of ferrocene is not in plane with the Cp moiety. Intermolecular H-bond interactions are summarized in Table 2 and presented in Figure S2 (Supplementary Material). There are eight molecules per

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unit cell, in which the ferrocene moieties of two immediate molecules are held are at a maximum distance from each other in order to minimize the repulsion and this arrangement is extended throughout the lattice via non-bonding interactions to mediate a supramolecular structure as

DNA interaction study

3.4.1

Cyclic voltammetry

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3.4

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shown in Figure S1 and S2 (Supplementary Material).

A cyclic voltammetric study was executed with the aim of understanding the redox behavior and the DNA binding affinities of the synthesized compounds. Electrochemical measurements were undertaken using a three-electrode system, i.e., working (platinum disc electrode with a geometric area of 0.071 cm2), reference (saturated calomel electrode i.e. SCE) and auxiliary electrodes (platinum electrode with geometric area much greater than the working

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electrode). Variation in the peak current provided information about the DNA binding constants, whereas the mode of interaction of compound with DNA can be judged from the shift in the peak potential. Compound-DNA binding constants were determined with the help of following

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equation [40]:

log (1/[DNA]) = log K + log {I/(Io - I)}

Eq 3

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where K is the binding constant, I and Io are the peak currents with and without DNA. For the determination of binding site size the following equation was used [41]: Cb/Cf = K [free base pairs]/s

Eq 4

where s is the binding site size in terms of base pair, K is the binding constant, Cf represents the concentration of free species and Cb denotes the concentration of compound-DNA bound species. Considering the concentration of DNA in terms of nucleotide phosphate, the concentration of DNA base pair will be taken as [DNA base pair]/2 and Eq 4 can be written as [41]: 11

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Cb/Cf = K [DNA base pairs]/2s

Eq 5

and the value of Cb/Cf is equal to (Io-I)/I, which are the values of experimental peak currents. The diffusion coefficient of free drug and DNA-bound drug provides the best information about the

equation [42-43] was used for calculating the diffusion coefficients: 5

1/2

1/2 1/2

Ipa = 2.99 × 10 n (α n) A Co* Do

ʋ

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molecular mass of the compound-DNA adduct. The following form of the Randles-Sevcik

Eq 6

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where Ipa is the anodic peak current, Co* is the concentration of the reductant in mol L-1, A is the geometric area of the electrode in cm2, ʋ is the scan rate in V/s, α is the transfer coefficient, n is

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the number of electrons involved in the process, Do is the diffusion coefficient in cm2 s-1. The synthesized ferrocene-based compounds display similar electrochemical behavior with two well-defined and stable redox peaks in the potential range of -0.2-1.0 V. Figure 2 shows the DNA binding study of a representative compound (1a) with CV. The consistency of the voltage at different scan rates from the plots of current (mA) vs. potential (E/V vs. SCE) for the compound favors a quasi-reversible electrochemical process (Figure 2a). The voltammogram of

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1a indicates an oxidation maximum at 0.549 V and a reduction maximum at 0.423 V. With the addition of 2-10 µM DNA, a negative shift in the peak potential and a drop in the peak current is observed. This negative shift in potential justifies the probability of an electrostatic mode of interaction of the positively charged 1a with the anionic phosphate backbone of DNA. The

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decrease in current is attributed to the formation of a high molecular weight 1a-DNA adduct, which diffuses comparatively slowly, thus causing a reduction in peak current (Figure 2b). The diffusion coefficient of the 1a-DNA adduct is 7.50 x 10-7 cm2 s-1, and this is far less than the

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diffusion coefficient of the free 1a (8.31 x 10-7 cm2 s-1). This result indicates the slow diffusion of the high molecular weight 1a-DNA adduct as compared to the free 1a (Figure 2c). The binding constant (3.681 x 104 M-1), and binding site size (0.418 bp) were calculated using Eqs. 3 and 4 (Figure 2d and 2e).

The compound 1b displays a quasi-reversible electrochemical behavior, which is evident by a change in the peak potential at different scan rates (Figure 3a). The anodic and cathodic peaks appeared at 0.522 and 0.398 V, respectively, vs. SCE. In the presence of increasing concentration (2-10 µM) of DNA, the oxidation and reduction peak potentials of 1b shifted 12

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cathodically with the decay in peak currents (Figure 3b), which indicates an electrostatic interaction and provides 1b-DNA binding constant of 2.623 x 104 M-1 (Figure 3d). The diffusion coefficient of 1b-DNA (7.70 x 10-7 cm2 s-1) is less than the diffusion coefficient of free 1b (5.32 x 10-7 cm2 s-1). This may be due to the formation of slowly diffusing heavy 1b-DNA adduct

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(Figure 3c). The binding site size of 0.319 bp also illustrates that 1b interacts electrostatically with DNA (Figure 3e).

The cyclic voltammetric behavior of the compounds 2a and 2b in the absence and presence of DNA is shown in Figure 4 and 5. The voltammogram without DNA featured a well-

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defined redox pair at the potential values 0.575/0.602 and 0.426/0.437 V for oxidation and reduction, respectively. A negative shift in potential and a decrease in peak current on successive

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addition of DNA suggests an electrostatic interaction of 2a and 2b with DNA. The compoundDNA adduct has lower diffusion coefficient than that of the free compound and the small binding site size values are also indicative of an electrostatic mode of interaction. The important DNA binding parameters of the compounds studied are listed in Table 3. These binding constant values are far better than for protonated ferrocene (3.45 x 102 M-1) and are comparable with the results for many of the recently reported ferrocene derivatives (Scheme

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2) [43-46]. The synthesized ferrocenyl derivatives are believed to undergo electrostatic interaction of positively charged ferrocenium state with the negatively charged oxygen of DNA. The phenyl group present in the structure is also capable of forming π-H-bonding (π-stacking) with DNA bases. This may be due to the existence of amide in the structure that could interact

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with DNA bases via hydrogen-bonding, as previously reported for benzamides [47], ferrocenyl urea, thiourea and guanidine derivatives [48-50]. The binding constant results reveal that those

3.4.2

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compounds containing nitro substituent seem to be more promising DNA binders. UV-Vis spectroscopy The results obtained from CV were equally supported by UV-Vis spectroscopy

confirming the electrostatic interactions of the compounds with DNA. The characteristic UV spectrum of the compound 1a gave two peaks in the UV region. One at 238 nm is due to a π-π* transition in the cyclopentadienyl ring (Cp) of ferrocene and the other at 284 nm is attributed to the π-π* transition of electrons in the phenyl chromophore. Following the substantial addition of DNA, the UV-Vis spectra of the compounds showed a decrease in absorption with a slight blue 13

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shift (hypochromism) (Figure 6a). The hypochromic effect is thought to be due to the interaction between the electronic states of the binding chromophore and those of the DNA bases [32, 51]. It is likely that the strength of this electronic interaction would decline as the cube of the distance of separation between the chromophore and the DNA bases [52]. So, the noticeable

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hypochromism observed in our experiments proposed the close vicinity of the chromophore of synthesized complexes to the DNA bases. At the closest approach to DNA, the π* orbital of the binding moiety of compounds could couple with π orbital of purine or pyrimidine. The coupling π* orbital may get partially filled by electrons, thus decreasing the transition probabilities, and

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hence result in hypochromism [53]. A binding constant of 4.158 x 104 M-1 for 1a was determined from the Figure 6b. Viscometry

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3.4.3

The mode and the extent of interaction of the synthesized ferrocene analogues with DNA was further confirmed with the help of viscometry. The plot of relative viscosity (η/ηo) against [Complex]/[DNA] concentrations is presented in Figure 7. A decrease in the relative viscosity with the increasing concentrations of the complexes 1a, 1b, 2a, and 2b for a constant

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concentration of DNA is an indicative of electrostatic interactions [54]. The complex-DNA binding constants determined using viscometric measurements are in agreement with the values obtained from CV and UV (Table 3). DFT study

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Computational calculations were performed for these electroactive compounds in order to

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complement the experimental outcomes of cyclic voltammetry. For this reason, DFT calculations were undertaken using a 6-31G basis set for optimizing the molecular geometries. The redox potentials of the compounds measured from cyclic voltammetry revealed an analogous trend to that anticipated from the DFT work. The ease of reduction of the compounds was found to vary as: 2a > 2b > 1b > 1a. A

similar trend was acquired from the ELUMO values, i.e., the highest reduction potential and most negative ELUMO value of 2a corresponds to easiest reduction [55-57]. A more negative ELUMO favors addition of electrons as the energies of the orbitals are reduced. Figure 8 and 9 shows the representative graphical demonstration of the HOMO and LUMO orbitals of 1a and 2a, 14

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respectively. The HOMO-LUMO energy gaps were observed to be in close agreement with the electrochemical band gaps computed from the difference between the oxidation and reduction potential of the species (Eredox = Eoxi - Ered) (Table 5). DFT based measurements also facilitated us to determine the Mulliken charges on these molecular structures (Figure 10). The EHOMO values

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acquired from DFT were compared with the oxidation potentials obtained from the CV measurements. The oxidation potentials observed experimentally for compounds, 1a, 1b, 2a, and 2b fluctuate as: 1a > 1b > 2b > 2a. This observation is supported from the DFT study by comparing the EHOMO values, which is less negative for 1a, representing its ease of oxidation as

DPPH scavenging activity

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3.6

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compared to other three compounds (Figure 11 and Table 4).

The antioxidant activities of the tested compounds have been analyzed by the DPPH assay using ascorbic acid (AA) as a control and is stated in terms of IC50 values (Figure 12). Generally, the compounds 1a-1b revealed somewhat greater scavenging potential as compared to the compounds 2a-2b. The improved activity of 1a-1b can be accredited to the presence of strong electron-withdrawing nitro group, which can stabilize the resulting free radicals more than

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the compounds having electron-donating amino substituent. So, the structure-activity relationship is quite justified in that the electron-withdrawing substituents make the compounds more stable resonantly and polarized to ideally bind with the DNA electrostatically. Protein kinase inhibition assay

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3.7

The ferrocene-based compounds displayed varying degree of inhibition with the zones of

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inhibition ranging from 17.3 ± 0.42 to 21.6 ± 97 mm. Compound 2a was found to be most effective as it produced the maximum zone of inhibition on the culture plates. The significant kinase inhibition activity of these complexes may perhaps be related to their ability to form hydrogen bonds with the hydrophobic pocket of the enzyme. The results of the inhibition assay revealed that these compounds can be considered as potential candidates to inhibit tumor initiation. The data from the inhibition study are given in Table 5.

15

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3.8

Cytotoxicity The anticancer activity of the synthesized compounds along with that for cisplatin (used

as control) were determined against THP-1 and MCF-7 carcinoma cells and also towards the

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non-cancerous cell line (MCF-10A), using the MTT reduction assay. From the 50% inhibitory concentration (IC50) data listed in Table 6, it can be seen that the screened compounds exhibit significant activity against cancerous cells, although much less than cisplatin. However, these ferrocenyl complexes exert fewer toxic effects in normal cells. One probable cause for the

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variance in action as related to that for cisplatin may lie in the difference in the approach of interaction with the DNA [58]. Cisplatin binds covalently with nitrogenous bases in the DNA, while the synthesized complexes are assumed to experience electrostatic interactions as

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designated by electrochemical and spectroscopic studies. It is obvious from the Figure 13 that the tested compounds inhibited the growth of tumor cells in a dose-dependent fashion. 4.

Conclusions

Several nitro and amino substituted ferrocenyl derivatives were synthesized in good yields. The complexes were of high purity as indicated by various spectroscopic methods in solid

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state as well as in the solution phase. The complexes demonstrated good binding strengths with the DNA through an electrostatic mode of interaction. The presence of non-covalent interactions motivated us to explore the electrochemical and pharmacological potential of the complexes.

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This research has afforded more valuable insight into DNA-metal complex interactions and cytotoxicity. This knowledge is important for the rational design of new anticancer drugs.

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Supplementary Material

Single crystal X-ray diffraction data for the structural analysis has been deposited with the Cambridge Crystallographic Data Center, CCDC No. 1527391. The copy of this information may be obtained free of charge from [email protected] or http://www.ccdc.cam.ac.uk. Acknowledgements Faiza Asghar is highly obliged to the Higher Education Commission (HEC) of Pakistan for providing a scholarship under the program 5000 Indigenous PhD Scholarships at Quaid-i-Azam 16

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University, Islamabad and for a 6-month scholarship under the International Research Support Initiative Program (IRSIP) as a Graduate Research Trainee in the Department of Chemistry, McGill University, Montreal, Quebec, Canada.

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[58]

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Scheme 1. Synthetic scheme for nitrophenylferrocenes 1a-1b and ferrocenylanilines 2a-2b.

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Scheme 2. Drug-DNA binding constants for some recently reported ferrocene derivatives [43-

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46].

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Figure 1. ORTEP diagram of 1a with ellipsoid displacement, non-hydrogen atoms represented by 55% probability boundary spheres and hydrogen atoms are sphere of arbitrary size. The bond distance between: C(10)-C(11) = 1.477(4) Å, Fe(1)-C(5) = 2.037(3) Å, C(11)-C(16) = 1.417(4)

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= 1.216(4) Å.

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Å, O(3)-C(17) = 1.427(4) Å, C(12)-C(13) = 1.385(4) Å, N(1)-C(13) = 1.469(4) Å, and N(1)-O(1)

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Figure 2. (a) Plots of current vs. potential/V (SCE) at different scan rates for 1a. (b) Cyclic

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voltammogram of 1 mM 1a in ethanol with 1 mL of 0.5 M TBAP as supporting electrolyte in the absence and presence of 2-10 µM DNA showing a decrease in I from Io and a concentration dependent -ve shift in potential showing electrostatic interactions. (c) Plot of current vs. (V/s)1/2,

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for the determination of diffusion coefficient of free 1a and 1a-2 µM DNA. (d) Plot of log (1/[DNA]) vs. log {I/(Io-I)} for the determination of binding constant of 1a. (e) Plot of Cb/Cf vs. [DNA]/µM for the determination of binding site size of 2-10 µM DNA concentrations (1a).

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Figure 3. (a) Plots of current vs. potential/V (SCE) at different scan rates for 1b. (b) Cyclic

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voltammogram of 1 mM 1b in ethanol with 1 mL of 0.5 M TBAP as supporting electrolyte in the absence and presence of 2-10 µM DNA showing a decrease in I from Io and a concentration dependent -ve shift in potential showing electrostatic interactions. (c) Plot of current vs. (V/s)1/2,

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for the determination of diffusion coefficient of free 1b and 1b-2 µM DNA. (d) Plot of log (1/[DNA]) vs. log {I/(Io-I)} for the determination of binding constant of 1b. (e) Plot of Cb/Cf vs. [DNA]/µM for the determination of binding site size of 2-10 µM DNA concentrations (1b).

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Figure 4. Plots of current vs. potential/V (SCE) at different scan rates for 2a. (b) Cyclic

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voltammogram of 1 mM 2a in ethanol with 1 mL of 0.5 M TBAP as supporting electrolyte in the absence and presence of 2-10 µM DNA showing a decrease in I from Io and a concentration dependent -ve shift in potential showing electrostatic interactions. (c) Plot of current vs. (V/s)1/2,

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for the determination of diffusion coefficient of free 2a and 2a-2 µM DNA. (d) Plot of log (1/[DNA]) vs. log {I/(Io-I)} for the determination of binding constant of 2a. (e) Plot of Cb/Cf vs. [DNA]/µM for the determination of binding site size of 2-10 µM DNA concentrations (2a).

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Figure 5. Plots of current vs. potential/V (SCE) at different scan rates for 2b. (b) Cyclic voltammogram of 1 mM 2b in ethanol with 1 mL of 0.5 M TBAP as supporting electrolyte in the

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absence and presence of 2-10 µM DNA showing a decrease in I from Io and a concentration dependent -ve shift in potential showing electrostatic interactions. (c) Plot of current vs. (V/s)1/2,

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for the determination of diffusion coefficient of free 2b and 2b-2 µM DNA. (d) Plot of log (1/[DNA]) vs. log {I/(Io-I)} for the determination of binding constant of 2b. (e) Plot of Cb/Cf vs. [DNA]/µM for the determination of binding site size of 2-10 µM DNA concentrations (2b).

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Figure 6. (a) Representative plots of absorbance vs. wavelength of 25 µM 1a in ethanol with increasing concentration of DNA (3-18 µM). (b) Plot of Ao/A-Ao vs. 1/[DNA] for the

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determination of DNA binding constant of 1a.

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Figure 7. Effect of increasing concentrations of complexes 1a, 1b, 2a, and 2b on relative

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specific viscosity at 25 ± 1 ⁰C. [DNA] = 60 µM and [Complex] = 5-25 µM in ethanol.

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graphical demonstration of LUMO of 1a.

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Figure 8. (a) Representative graphical demonstration of HOMO of 1a. (b) Representative

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graphical demonstration of LUMO of 2a.

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Figure 9. (a) Representative graphical demonstration of HOMO of 2a. (b) Representative

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2b.

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Figure 10. Mulliken charge distribution on the molecular structures (a) 1a, (b) 1b, (c) 2a, and (d)

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Figure 11. Comparison of oxidation and reduction potentials of 1 mM ferrocenyl derivatives 1a,

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1b, 2a, and 2b in DMSO recorded at a scan rate of 100 mV/s.

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Figure 12. In vitro antioxidant activity data of synthesized ferrocenyl analogs with a comparison

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to ascorbic acid (AA).

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Figure 13. Cell viability (%) of cancerous and non-cancerous cells at various concentrations of

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(a) 1a, (b) 1b, (c) 2a, and (d) 2b, after 72 h of incubation.

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Table 1. Crystal data of 1a.

3

Volume Å Crystal System Space group Z Density (calculated)

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Index Ranges

Absorption coefficient (µ) F(000) 2 Goodness-of-fit on F (S)

Largest diff. peak and hole Final R indices [1>2sigma(I)] R indices (all data) θ range for data collection (°)

3

1.540 g/cm -9<=h<=5, -12<=k<=12, -44<=l<=44 1.049 1392 1.060 0.27 and -0.39 eÅ-3 R1 = 0.0437, wR2 = 0.0921 R1 = 0.0630, wR2 = 0.1007 2.02-25.50

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1527391 C17H15FeO3 337.15 296(2) 0.71073 7.5145(6) 10.5007(8) 36.919(3) 90° 93.451(4) 90° 2907.9(4) Monoclinic P21/n 8

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CCDC Number Empirical formula Formula weight Temperature (K) Wavelength Å a [Å] b [Å] c [Å] α [deg] β [deg] γ [deg]

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1a

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Table 2. Intermolecular hydrogen bonds, distance (Å) and angles (°) in 1a.

H

A

d(D-H)/Å

d(H-A)/Å

d(D-A)/Å

D-H-A/°

C15

H15

O5

0.93

2.59

3.256(2)

129.1

C17

H17B

O1

0.96

2.43

3.290(2)

149.2

C32

H32

O2

0.93

2.53

3.448(4)

168.0

C34

H34B

O4

0.96

2.55

3.505(4)

173.9

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H-Bonding

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Table 3. Important parameters for redox behavior and DNA binding studies (CV, UV and Viscometry). CV Do [cm2s-1] drug-DNA

Binding site Binding Constant size ‘s’ [bp] K [M-1]

3.68 x 104

8.31 x 10-7

7.50 x 10-7

0.418

1b

2.62 x 104

7.70 x 10-7

5.32 x 10-7

0.319

2a

9.69 x 103

2.87 x 10-7

2.15 x 10-7

0.229

2b

1.46 x 104

6.13 x 10-7

3.19 x 10-7

0.262

Binding Constant K [M-1]

4.16 x 104

4.76 x 104

3.01 x 104

3.88 x 104

1.40 x 104

1.05 x 104

2.39 x 104

1.96 x 104

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Viscometry

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Do [cm2s-1] Free drug

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Code Binding Constant K [M-1]

UV

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Table 4. EHOMO and ELUMO values obtained through DFT calculations. EHOMO

ELUMO

HOMO-LUMO

Electrochemical

Code

(eV)

(eV)

Band Gap (eV)

Band Gap (V)

1a

-0.3129

-0.2037

0.1092

1b

-0.3618

-0.2305

0.1213

2a

-0.4493

-0.2912

0.1581

2b

-0.4017

-0.2740

0.1277

RI PT

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0.1261 0.1309 0.1455

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0.1387

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Zone of Inhibition (mm)

1a

18.5 ± 0.65

1b

20.1 ± 1.13

2a

21.6 ± 0.97

2b

17.3 ± 0.42

PC

25

SC

Sample Code

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Table 5. Protein kinase inhibition assay of synthesized ferrocenyl derivatives.

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PC = Surfactin was used as standard drug (positive control), while DMSO was used as negative control.

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Table 6. Cytotoxicity against human tumor and normal cells, after 72 h of incubation.

IC50 (µM) MCF-7

Cisplatin

1.97 ± 0.78

1.55 ± 1.82

1a

2.61 ± 1.01

3.24 ± 0.64

1b

2.93 ± 0.88

2.56 ± 1.07

2a

3.86 ± 1.25

4.72 ± 0.96

2b

3.49 ± 0.73

4.02 ± 1.25

MCF-10A

RI PT

THP-1

17.3 ± 0.59 14.9 ± 1.27

17.6 ± 1.62 16.4 ± 0.74

SC

Sample Code

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16.8 ± 0.95

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The DNA binding proficiency of ferrocenyl complexes has been investigated by electrochemical, spectroscopic, and viscometric methods and show that they are excellent DNA binders through an electrostatic association. These new complexes also demonstrated good anticancer and DPPH scavenging potential. The HOMO-LUMO energies and the charge distributions was determined

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through DFT analysis.

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New ferrocene-based nitro and amino complexes.

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Structural interpretation by FT-IR, Raman, NMR, AAS, CHNS, single crystal XRD.

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Good DNA binders and free radical scavengers.

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Theoretical measurements were made using DFT/B3LYP method.

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Decent candidates in terms of cytotoxicity and protein kinase inhibition.

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•