synthesis, characterization, electrochemistry and evaluation of biological activities of some...

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Full PaperReceived: 25 February 2010 Revised: 22 April 2010 Accepted: 19 May 2010 Published online in Wiley Online Library: 2 July 2010

(wileyonlinelibrary.com) DOI 10.1002/aoc.1690

Synthesis, characterization, electrochemistryand evaluation of biological activities of someferrocenyl Schiff basesMuhammad Zaheera, Afzal Shaha, Zareen Akhtera∗, Rumana Qureshia,Bushra Mirzab, Misbah Tauseef b and Michael Boltec

Synthesis of ferrocenyl Schiff bases (1–6) was carried out by the condensation reaction of 4-ferrocenyl aniline with differentsubstituted aromatic aldehydes and acetyl acetone. Compounds were characterized by physical measurements, elementalanalysis, FT-IR, 1H-NMR and 13C-NMR spectroscopy. Single crystal X-ray analysis of compound 2 showed the co-planarity ofboth aromatic rings connected by a C–N double bond. Compounds demonstrated reversible one-electron redox behavior andtheir peak currents were found to increase linearly with the square root of the sweep rate ν1/2. The overall electrode processeswere found to be diffusion controlled. Compounds 1 and 4 showed low cytotoxicity and appreciable antifungal, antioxidantand DNA protection activities. Copyright c© 2010 John Wiley & Sons, Ltd.

Supporting information may be found in the online version of this article.

Keywords: ferrocene; Schiff bases; electrochemistry; biological applications

Introduction

Ferrocene and its derivatives find extensive applications inareas like homogeneous catalysis,[1 – 3] material science,[4] non-linear optics[5 – 7] and molecular sensors.[8 – 10] Study of thebioorganometallic chemistry of ferrocene compounds beganin the 1960s[11] and recently they have attracted much moreattention.[12 – 17] The stability of the ferrocenyl group in aqueous,aerobic media, the accessibility of a large variety of derivatives, andits favorable electrochemical properties make ferrocene and itsderivatives very popular molecules for biological applications andfor conjugation with biomolecules.[18] Incorporation of a ferrocenefragment into an organic compound often produces unexpectedbiological activity.[19] Ferrocenic analogs of chloroquine, meflo-quine and quinine, synthesized by various researchers,[20,21] havemanifested enhanced antimalarial activities. Replacing the aro-matic ring of the well-known anticancer drug tamoxifen withferrocene (called ferrocifen) produced a compound that exhib-ited a strong effect against breast cancer cells that were resistantto tamoxifen.[22] Similarly the antibiotic activity of penicillin andcephalosporin was enhanced many times upon the introductionof ferrocene moiety in these drugs.[23]

Ferrocene is readily converted into ferrocenium ion (Fc+)through one-electron reversible oxidation; however, substituentson ferrocene moiety influence this redox behavior by changingthe energy level of HOMO,[24] so reversibility may be significantlylowered.[25] The low cytotoxicity of ferrocene in biologicalsystems, its lipophillicity, the cytotoxicity of its metabolitestowards tumors,[26,27] the pi-conjugated system and the resultingexclusive electron-transfer ability[28] make its derivatives goodcandidates for the investigation of their biological applications.Schiff bases and their complexes are largely studied becauseof their interesting and important properties such as theirability to reversibly bind oxygen,[29] redox systems in biological

systems[30] and oxidation of DNA.[31] These compounds showgood antibacterial,[32] antitumor[33] and antifungal activities,[34]

especially those derived from p-substituted aniline and itscomplexes have a variety of applications in biology.[35] Schiffbases also find applications in radiopharmaceuticals,[36] and inthe treatment of various diseases, as pesticides and plant growthregulators.[37]

Thus introduction of ferrocene moiety into a Schiff base could beinteresting as far as its electrochemistry and biological applicationsare concerned. Here we report the synthesis, characterization,electrochemical properties and biological studies of some Schiffbases derived from phenyl ferrocene. Because of the lowsolubilities of the compounds in DMSO at desired concentrations,only two (1 and 4) of the synthesized compounds could beinvestigated for their biological applications.

Experimental

Materials and Physical Measurements

Ferrocene, 4-nitroaniline, sodium nitrite, hexadecyltrimethyl-ammonium bromide, 3- nitrobenzaldehyde, 2,4-dichlorobenzal-

∗ Correspondence to: Zareen Akhter, Department of Chemistry, Quaid-i-AzamUniversity, Islamabad 45320, Pakistan. E-mail: [email protected]

a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan

b Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320,Pakistan

c Institut fur Anorganische chemie, J. W. Goethe-Universitat Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt/Main, Germany

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M. Zaheer et al.

dehyde, 3-bromobenzaldehyde, 3,4- dimethoxybenzaldehyde, 3-fluorobenzaldehyde and acetylacetone were obtained from Fluka,Switzerland. Solvents like ethyl alcohol, ethyl acetate, diethyl ether,toluene and hexane were obtained from Merck Germany and werefreshly dried using standard methods. The elemental analysiswas performed using a CHNS-932 LECO instrument. Meltingtemperature was determined on a Mel-Temp. (Mitamura RikenKogyo) apparatus using open capillary tubes and is uncorrected.The solid-state Fourier transform infrared spectrum was recordedon Bio-Rad Excalibur FTIR, model 3000 MX using KBr pellets. 1H-and 13C-NMR spectra were obtained on a Bruker 300 MHz NMRspectrophotometer in CDCl3 using teteramethyl silane as internalreference. The data for the crystal structure analysis were collectedon a STOE IPDS-II diffractometer with monochromated Mo-Kα

radiation at 173.

General Procedure for the Synthesis of Schiff Bases 1–6

4-Ferrocenyl aniline was synthesized following a reportedprocedure.[38] In a pre-backed two-necked flask supplied withmagnetic stirrer, 0.2 g (0.72 mmol) of 4-ferrocenyl aniline in 30 mlof dried ethanol was mixed with the equimolar amount of substi-tuted aromatic aldehydes in 30 ml of dried ethanol. The mixturewas heated under reflux and progress of reaction was monitoredby TLC. The required product was formed in 5–6 h. The solventwas removed under vacuum; solid was recrystallized from hotethanol and characterized using spectroscopic techniques.

N-(3-Nitrobenzylidene)-4-ferrocenyl aniline (1)

Rusty orange solid; m.p. 41 ◦C; yield (0.26 g, 89%). Anal. calcd forFeC23H18N2O2: C, 67.3; H, 4.3; N, 6.8. Found: C, 67.4; H, 4.3; N, 6.8%;νmax/cm−1: 1656 (CH[ ]N), 3156 (Ar[–]H), 1531, 1385 (NO2), 1134,1011 (Cp), 479 (Fc[–]Cp); δH (300 MHz, CDCl3, Me4Si): 8.8 (1H, t,J = 8.1 Hz Ph), 8.6 (1H, s, CH[ ]N), 8.2–8.3 (3H, m, Ph), 7.5–7.6(2H, m, Ph), 7.2–7.3 (2H, m, Ph), 4.7 (2H, t, J = 1.8 Hz, C5H4), 4.4 (2H,t, J = 1.8 Hz, C5H4), 4.0 (5H, s, C5H4); δC (75 MHz, CDCl3, Me4Si):155.8 (CHN), 148.4 (Ar[–]NO2), 138.3 (C6H4), 133.9 (C6H4), 129.8(C6H4), 126.8 (C6H4), 125.4 (C6H4), 123.4 (C6H4), 121.2 (C6H4), 69.2(C5H5), 69.6 (C5H4),66.4 (C5H4).

N-(2,4-Dichlorobenzylidene)-4-ferrocenyl aniline (2)

Dark brown solid; m.p. 150 ◦C; yield (0.24 g, 78%). Anal. calcd forFeC23H17NCl2: C, 63.5; H, 3.9; N, 3.2. Found: C, 63.6; H, 3.9; N,3.2%; νmax/cm−1: 1662 (C[ ]N), 3144 (Ar[–]H), 1613, 1459 (Ar[–]Hbending), 562 (C[–]Cl), 1101, 1042 (Cp), 497 (Fe[–]Cp); δH (300 MHz,CDCl3, Me4Si): 8.9 (1H, s, CH[ ]N), 8.2 (2H, d, J = 8.7, Ph), 7.5 (2H,d, J = 2.1 Hz, Ph), 7.2–7.3 (2H, m, Ph), 7.3 (1H, s, Ph), 4.7 (2H, t,J = 1.8 Hz, C5H4), 4.3 (2H, t, J = 1.8 Hz, C5H4), 4.0 (5H, s, C5H4); δC

(75 MHz, CDCl3, Me4Si): 154.3 (CHN), 138.3 (C[–]Cl), 133.9 (C[–]Cl),148.9 (C[–]N), 132.0 (C6H4), 129.7 (C6H4), 129.4 (C6H4), 127.7 (C6H4),69.68 (C5H5), 69.1 (C5H4), 66.4 (C5H4).

N-(4-Bromobenzylidene)-4-ferrocenyl aniline (3)

Dark brown solid; m.p. 209 ◦C; yield (0.27 g, 85%). Anal. calcdfor FeC23H18NBr: C, 62.1; H, 4.0; N, 3.1. Found: C, 62.0; H, 4.0; N,3.1%; νmax/cm−1: 1657 (C[ ]N), 3149 (Ar[–]H), 1603, 1400 (Ar[–]Hbending), 626 (C[–]Br), 1127, 1016 (Cp), 467 (Fe[–]Cp);δH (300 MHz,CDCl3, Me4Si): 8.5 (1H, s, CHN), 7.8 (2H, d, J = 8.4 Hz, C6H4), 7.6(2H, d, J = 8.4 Hz, C6H4), 7.5 (2H, d, J = 8.4 Hz, C6H4), 7.2 (2H, d,

J = 8.4 Hz, C6H4), 4.6 (2H, t, J = 1.8 Hz, C5H4), 4.3 (2H, t, J = 1.8 Hz,C5H4), 4.0 (5H, s, C5H4); δC (75 MHz, CDCl3, Me4Si): 157.7 (CHN),149.1 (Ar[–]Br),137.7 (C6H4), 130–125 (C6H4), 69.6 (C5H5), 69.0(C5H5), 66.4 (C5H5).

N-(3,4-Dimethoxybenzylidene)-4-ferrocenyl aniline (4)

Orange solid; m.p. 151 ◦C; yield (0.25 g, 82%). Anal. calcd forFeC25H23O2N: C, 70.6; H, 5.4; N, 3.3. Found: C, 70.5; H, 5.4; N, 3.3%);νmax/cm−1: 1662 (C[ ]N), 3144 (Ar[–]H) 1618, 1141, 1019 (Cp),487 (Fe[–]Cp); δH (300 MHz, CDCl3, Me4Si): 8.5 (1H, s, CH[ ]N),7.6 (1H, s, C6H3), 7.4–7.6 (2H, m, C6H4), 7.1–7.2 (2H, m, C6H4),6.9 (2H, d, J = 8.1 Hz, C6H3), 4.6 (2H, t, J = 1.8 Hz, C5H4), 4.3(2H, t, J = 1.8 Hz, C5H4), 4.0 (5H, s, C5H4), 4.0 (3H, s, OCH3), 3.9(3H, s, OCH3); δC (75 MHz, CDCl3, Me4Si): 158.8 (CH[ ]N), 151.9(ArC[–]N), 149.8 (ArC[–]OCH3), 149.4 (ArC[–]OCH3), 136.9 (C6H4),129.7 (C6H4), 126.7 (C6H3), 124.3 (C6H3), 121.0 (C6H4), 110.5 (C6H3),108.8 (C6H3), 69.6 (C5H4), 68.9 (C5H4), 66.3 (C5H5), 56.0 (OCH3).

N-(4-Fluorobenzylidene)-4-ferrocenyl aniline (5)

Orange solid; m.p. 201 ◦C; yield (0.23 g, 86%). Anal. calcd forFeC23H18 NF: C, 72.0; H, 4.7; N, 3.6. Found: C, 71.8; H, 4.7; N, 3.6%;νmax/cm−1: 1662 (C[ ]N), 3143 (Ar[–]H) 1145, 1020 (Cp), 471(Fe[–]Cp); δH (300 MHz, CDCl3, Me4Si): 8.5 (1H, s, H3), 7.9–8.0 (2H,m, C6H4), 7.5–7.6 (2H, m, C6H4), 7.1–7.3 (4H, m, C6H4), 4.6 (2H, t,J = 1.8 Hz, C5H4), 4.3 (2H, t, J = 1.8 Hz, C5H4),4.0 (5H, s, C5H5);δC (75 MHz, CDCl3, Me4Si): 160.2 (CHN), 136.4 (C6H4), 126.5 (C6H4),124.6 (C6H4), 69.6 (C5H5), 69.0 (C5H4).

N-4-(Phenylimino)pent-2-en-2-ol ferrocene (6)

A 0.6 g (2.15 mmol) aliquot of 4-ferrocenylaniline was mixed with1 ml (973 mmol) of freshly distilled acetyl acetone in 60 ml ofdried toluene in a pre-backed two-neck flask supplied with amagnetic stirrer. The mixture was refluxed and water formedwas separated azeotropically using Dean and Stark apparatus.Monitoring of the reaction with TLC showed the completion ofreaction after 100 h reflux. The solvent was rotary evaporated andsolid obtained was washed with n-pentane and recrystallized fromethanol. Dark orange solid; m.p. 107 ◦C; yield (0.77 g, 87%). Anal.calcd for FeC21H21NO: C,70.2; H, 5.8; N, 3.9. found C, 69.9; H, 5.9;N, 3.8%); νmax/cm−1: 1609 (C[ ]N), 3135 (Ar[–]H), 2925 (aliphaticC[–]H), 1110, 1015 (Cp), 486 (Fe[–]Cp), 3498 (O[–]H); δH (300 MHz,CDCl3, Me4Si): 12.4 (1H, s, OH), 7.4 (2H, d, J = 8.4 Hz, C6H4),7.0 (2H,d, J = 8.4 Hz, C6H4), 5.2 (1H, s, [–]CH[ ]C[–]), 4.3 (2H, t, J = 1.8 Hz,C5H4), 4.0 (5H, s, C5H4), 2.1 (3H, s, CH3), 2.0 (3H, s, CH3); δC (75 MHz,CDCl3, Me4Si): 160.3 (C[ ]N), 136.8 (C6H4), 136.5 (C6H4), 126.6(C6H4), 124.6 (C6H4), 97.5 ([–]CH[ ]), 69.6 (C5H5), 69.0 (C5H4), 66.4(C5H5), 29.2 (CH3), 19.5 (CH3).

Electrochemistry

Voltammetric experiments were performed using PGSTAT 302 withAutolab GPES version 4.9 Eco Chemie, Utrecht, the Netherlands.Measurements were carried out in a conventional three-electrodecell with saturated calomel electrode (SCE) as reference electrode,a thin Pt wire as counter electrode and a bare glassy carbonelectrode (GCE) with a geometric area of 0.071 cm2 as the workingelectrode. Prior to experiments, the GCE was polished with 0.25 µmdiamond paste on a nylon buffing pad, followed by washing withwater. For electrochemical measurements the test solution waskept in an electrochemical cell (model K64 PARC) connected tothe circulating thermostat LAUDA model K-4R.

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Biological activities of some ferrocenyl Schiff bases

Biological Studies

Antibacterial assay

Schiff bases 1 and4 were tested for their antibacterial activity.Antibacterial activities were studied against six bacterial strains:three Gram-negative (E. coli, ATCC no. 15224; Samonella setubal,ATCC no. 19196; and E. aerogenes, ATCC no. 13048) and three Gram-positive (S. aureus, ATCC no. 6538; B. subtillus, ATCC no. 6633; andM. leuteus, ATCC no. 10240. The agar well diffusion method[39]

was used for the determination of inhibition zone. A single colonyfrom each bacterial culture plate was transferred to nutrient broth(pH 7) and incubated at 37 ◦C for 24 h. Concentrations of eachcompound (1 mg ml−1) were prepared. Briefly, 0.75 ml of brothculture containing ca. 106 colony forming units per milliliter oftest strain was added to the 75 ml of nutrient agar medium, mixedwell, and than poured into a 14 cm sterile agar plate. Reactionwas performed in triplicate. Wells were prepared using an 8 mmsterilized metallic borer, sealed with media and filled with 100 µlof respective concentration of each compound. Roxythromycine(1 mg ml−1) and Cefixime (1 mg ml−1) were used as standard drugswhile DMSO was used as negative control. Plates were incubatedat 37 ◦C aerobically and the zone of inhibition was measured after24 h. Experiments were run in triplicate.

Antifungal assay

The agar tube dilution method[40] was used for the antifungalactivity of test compounds with some modification as reportedpreviously.[41] Activity was tested against five fungal strainsincluding Mucor species (0300), A. flavus (0064), A. fumigatus(66), F. solanni (0291) and A. niger (0198). Screw-capped testtubes containing sabouraud dextrose agar medium (SDA) wereautoclaved at 121 ◦C for 20 min. The tubes were allowed to cool to50 ◦C and non-solidified SDA was loaded with 66.6 µl of compoundfrom stock solution 12 mg ml−1 in DMSO. The tubes were thenallowed to solidify in a slanting position at room temperature. Thetubes were prepared in triplicate for each fungus species. The tubescontaining solidified media and test compound were inoculatedwith a 4 mm diameter piece of inoculum from a 7-day-old fungalculture. Media supplemented with DMSO and reference antifungaldrug were used as negative and positive controls, respectively.The tubes were incubated at 28 ◦C for 7 days. Growth in media wasdetermined by measuring linear growth (millimeters) and growthinhibition was calculated with reference to the negative control.

DPPH free radical scavenging assay

Radical scavenging activity of test compounds was measuredspectrophotometrically using a modified protocol as reportedearlier.[42] The antioxidant activity of one compound (1) wastested at three concentrations, 1000, 100 and 10 ppm, while of theother compound (4) was tested at two additional concentrationsof 10 and 5 ppm in order to get data for the IC25 calculation.A 100 µl aliquot of each test compound was added to 2 ml of0.1 mM DPPH solution in ethanol and 0.9 ml of 0.1 mM Tris–HClbuffer in reaction mixture. Reaction was performed in triplicate.Vials were capped and reaction mixture was incubated for 30 minat room temperature in the dark. Absorbance of the reactionmixture was measured at 517 nm. In the negative control 100 µl ofDMSO was added to the reaction mixture. Blank was preparedby mixing distilled water, ethanol and DMSO in a ratio of10 : 1:9. The percentage scavenging of DPPH free radicals foreach concentration of compound was calculated with referenceto absorbance of negative control.

Brine shrimp lethality assay

Test compounds were subjected to the brine shrimp lethalityassay[43] for cytotoxic activity. Artificial seawater was prepared bydissolving 20 g commercial sea salt (sigma) in 0.5 l distilled waterand aerated for 2 h with continuous stirring. Brine shrimp (Artemiasalina) eggs (sera, Heidelberg Germany) were hatched in a narrowrectangular dish filled with artificial seawater at 37 ◦C. After 48 hincubation, larvae were collected and transferred 10 larvae per vialcontaining 50 µl of test compound (10 000, 1000 and 100 ppm)and then the volume was made up to 5 ml. In the final reactionmixture the concentration was 1000, 100 and 10 ppm. Negativecontrol was run with 50 µl of DMSO instead of the test sample.Vials were placed uncovered at room temperature and illuminatedfor 24 h. After 24 h of incubation survivors were counted. Resultswere analyzed using the statistical computer program Finney.

OH radical induced oxidative DNA Damage analysis

Determination of antioxidant (protective) or pro-oxidant (dam-aging) activity of test compounds was conducted according toTian and Hua[44] the reaction was conducted in PCR microtubesin a total volume of 15 µl; 3 µl of 2-fold diluted plasmid DNA wastransferred to each micro tube followed by 5 µl of stock solutionof test compound at three different concentrations, 3000, 300 and30 ppm, to make final concentrations of reaction mixtures of 1000,100 and 10 ppm. Then, 3 µl of 2 mM FeSO4 and 4 µl of 30% H2O2

were added successfully. Four different controls are used in thisassay including positive control (X), containing 3 µl of phosphatebuffer instead of the test sample, negative control (P), with plasmidDNA and phosphate buffer with no treatment, a third control withhydrogen peroxide treatment (H) and a fourth control with Fe (II)treatment (F). Reaction mixtures were incubated at 37 ◦C in thedark for 1 h. Each mixture was run in 1× TBE buffer at 60 V for1 h in horizontal electrophoresis apparatus. For each run, a 1 kbladder and four controls, P, F, H and Xm were run simultaneously.The gels were photographed under UV light.

DNA interaction

DNA was extracted from chicken blood by the method reported inthe literature.[45] Its stock solution was prepared in double-distilledwater and stored at 4 ◦C. The concentration of the stock solutionof DNA (200 µM in nucleotide phosphate, NP) was determined byUV absorbance at 260 nm using the molar extinction coefficient(ε) of 6600 M−1 cm−1.[46] Protein-free DNA was evidenced fromthe ratio of absorbance at 260 and 280 nm (A260/A280 = 1.85).[47]

Voltammograms of 0.5 mM solution of compounds were taken inthe presence of 20, 30 and 40 µM DNA.

Results and Discussion

Synthesis

Ferrocenyl aniline was prepared following Scheme 1, which wascondensed with the corresponding aldehydes and 2,4-butadione(Schemes 2 and 3), producing ferrocenyl Schiff bases (1–6). Thesynthesized compounds were characterized by melting points,CHN, FT-IR, 1H and 13C NMR spectral studies. Elemental analysisdata of all products were in good agreement with the calculatedvalues. IR spectra of these compounds showed all characteristicpeaks. Absorption bands in the range of 1640–1690 cm−1 were

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M. Zaheer et al.

Fe

O2N

PTCFe

NO2

Fe

NH2

N2Cl

Hydrazine

Pd-C

Scheme 1. Synthesis of 4-ferrocenyl aniline.

CHO

X

Y

Z

Fe

N CH X

Y

Z

1: X, Z = H, Y = NO22: X, Z = Cl, Y = H3: X, Y = H, Z = Br4: Y, Z = OCH3, X = H5: X, Y = H, Z = F

TolueneReflux

Fe

NH2

4-Ferrocenyl aniline aromatic aldehydes

Scheme 2. Synthesis of Schiff bases 1–5.

assigned to the stretching vibration of C[ ]N bond while those inthe range 3000–3150 cm−1 were possibly due to aromatic C[–]Hstretch. The absence of any peak at 3500–3300 cm−1 confirmedthe formation of Schiff bases. 1H NMR and 13C NMR spectra of thesynthesized Schiff bases were recorded in CDCl3 relative to TMS asreference. In 1H-NMR spectra of all compounds azomethine proton(CH[ ]N) was the most deshielded proton and gave a singletat 8.5–8.9 ppm while aromatic protons showed their signalsin the range 7.0–8.0 ppm. The benzene ring directly attachedto ferrocene contained two types of protons which appearedas a doublet with coupling constant of 8.4 Hz. Substitutedcyclopentadiene contained two types of protons, which appearedas two triplets at 4.3 and 4.7 ppm (J = 1.8 Hz) respectively. Allfive protons of unsubstituted cyclopentadiene were chemicallyequivalent and appeared in NMR spectra as singlets at 4.0 ppm.13C NMR spectra showed azomethine carbon to be the mostdeshielded, appearing at 155–166 ppm. Aromatic carbon atomsshowed their signals at 122,128, 134 and 153 ppm, respectively.Carbon atoms of the ferrocenyl moiety appeared at 66.4, 69.2, 69.6and 84.0 ppm, respectively.

Single Crystal X-ray Analysis of 2

Crystals of Schiff base 2, suitable for crystallographic analysis,were grown from the ethanol solution and molecular structuredetermination was carried out using X-ray crystallographicanalyses (Fig. 1). Product 2 grew in a monoclinic system withthe 21/c space group. Geometric parameters were unexceptionaland are summarized in Table 1. The two aromatic rings connectedby the C[–]N double were almost coplanar, making the dihedralangle 5.08(14)◦. The cyclopentadienyl ring was coplanar with the

adjacent phenyl ring [dihedral angle 6.90(14)◦]. Selected bondlengths and bond angles are summarized in Table 2.

Experimental crystallographic data for C23 H17 Cl2 Fe N (2)

M = 434.13, monoclinic, space group P21/c, a = 8.8383(6) Å,b = 16.7716(11) Å, c = 12.7984(8) Å, β = 96.335(5)◦, V =1885.6(2) Å3, Z = 4, Dc = 1.529 mg m−3, µ = 1.091 mm−1,F(000) = 888, γ = 0.71073 Å, T = 173(2) K, 10 987 reflectionscollected (±hh, ±k, ±l), 3535 independent (Rint = 0.0422), refinedparameters R1 = 0.0291, wR2 = 0.0730, goodness of fit S = 0.988.Crystallographic experimental data is given in Tables 1 and 2.

Cyclic Voltammetry of Compounds 1–6

The redox behavior of compounds 1–6 on bare glassy carbonelectrode was studied in absolute ethanol at 25 ◦C. The voltam-mograms of these compounds showed a pair of robust redoxwaves with �Ep = 70–78 mV. The CV behavior of compounds 4and 6 at various scan rates is shown in Fig. 2(a, b). The voltam-mogram featured a couple of well-defined and stable redox peaksin the potential range 0.0–1.0 V. The voltammetric response isattributed to the 1e redox process of Fe2+/Fe3+ couple. Figure 2(a,b) further reflects the reversibility of the redox processes as thepeak potentials are not significantly affected by the variation inscan rate. Moreover, the ferrocenyl group of compounds 4 and6 retained its simple one-electron ferrocene/ferrocenium redoxbehavior but with different peak potentials. The tunable oxida-tion potential and peak current of the ferrocenyl group by thevariation of substituents at the cyclopentadienyl ring/rings can beexploited for the estimation of useful electrochemical parametersand substituent effect.

Fe

NH2O O

Fe

N

HO

TolueneReflux

4-Ferrocenyl aniline acac 6

Scheme 3. Synthesis of Schiff base 6 from acetylacetone.


65


Figure 1. ORTEP diagram of compound 2.

Table 1. Crystal data and structure refinement for compound 2

Empirical formula C23 H 17 C12 Fe N

Formula weight 434.13

Temperature 173(2) K

Wavelength 0.71073 Å

Crystal system Monoclinic

Space group P21/c

Unit cell dimensions a = 8.8383(6) Å α = 90◦

b = 16.7716(11) Åβ = 96.335(5)◦

c = 12.7984(8) Å γ = 90◦

Volume 1885.6(2) Å3

Z 4

Density (calculated) 1.529 mg m−3

Absorption coefficient 1.091 mm−1

F (000) 888

Crystal size 0.32 × 0.28 × 0.14 mm3

θ range for data collection 3.36–25.66◦

Index ranges −10 ≤ h ≤ 10, −20 ≤ k ≤20, −15 ≤ l ≤ 15

Reflections collected 10987

Independent reflections 3535 [R(int) = 0.0422]

Completeness to θ = 25.00◦ 99.7%

Absorption correction Semi-empirical from equivalents

Max. and min. transmission 0.8623 and 0.7217

Refinement method Full-matrix least-squares on F2

Data/restraints/parameters 3535/0/245

Goodness-of-fit on F2 0.988

Final R indices [I > 2σ (I)] R1 = 0.0291, wR2 = 0.0730

R indices (all data) R1 = 0.0375, wR2 = 0.0756

Extinction coefficient 0.0072(7)

Largest difference peak and hole 0.303 and −0.358 e Å−3

The electrochemical parameters of compounds 1–6 as ob-tained from CV are listed in Table 3. The electron-donating groupsaccelerate oxidation by lowering Epa and electron-withdrawinggroups make the oxidation difficult by increasing Epa. The resultsreveal that the formal potential varies in the sequence 5 < 3 < 2 <

6 < 4 < 1, suggesting the idea that the electrochemical oxidationbehavior of the oxidizing moiety ferrocene can be modulatedby changing the electronic properties of the substituents nearto the cyclopentadienyl ring. The strong electron-withdrawingnitro group in 1 makes the oxidation of the iron (of the ferrocenylgroup) difficult by pronouncedly shifting the oxidation potentialin more positive direction.

Table 2. Selected bond lengths (Å) and angles (deg) for compound2 (za25)

Fe(1)[–]C(6) 2.042(2)

Fe(1)[–]C(10) 2.046(2)

Fe(1)[–]C(1) 2.0469(19)

Fe(1)[–]C(5) 2.0488(19)

Fe(1)[–]C(4) 2.054(2)

N(1)[–]C(17) 1.270(3)

N(1)[–]C(14) 1.411(2)

C(1)[–]C(5) 1.428(3)

C(1)[–]Fe(1)[–]C(5) 40.81(8)

C(17)[–]N(1)[–]C(14) 122.03(18)

C(2)[–]C(1)[–]Fe(1) 68.78(11)

C(5)[–]C(1)[–]C(2) 107.24(16)

C(2)[–]Fe(1)[–]C(8) 128.41(9)

C(2)[–]Fe(1)[–]C(3) 41.01(8)

C(2)[–]Fe(1)[–]C(9) 167.60(10)

C(2)[–]Fe(1)[–]C(7) 106.84(9)

The diffusion coefficients were determined by the applicationof the following expression:[48]

i = 2.69 × 105n3/2ACo∗ D ν1/2 (1)

where i is the peak current (A), A is the surface area of theelectrode (cm2), Co

∗ is the bulk concentration (mol cm−3) of theelectroactive species, D is the diffusion coefficient (cm2 s−1) andν is the scan rate (V s−1).

The linearity of i vs ν1/2 plots (Fig. 3a, 3b) demonstrates that themain mass transport of these compounds to the electrode surfaceis controlled by the diffusion step. The values of the diffusioncoefficient (D) of compounds 1–6 shown in Table 3 are in goodagreement with the previously reported Schiff-based compoundsbearing ferrocene.[49] The decreasing trend in the values of D withthe increase in molecular weights of the compounds confirms theidea that a heavy molecule diffuses slowly to the electrode surface.However, the lowest D of comparatively lighter compound 6indicates that molecular weight is not the sole factor to decide thetrend in diffusion coefficient. The lower D of compound 6 may beattributed to the formation of hydrogen bonding of its hydroxylgroup with the solvent.

The values of standard rate constant (ks) of the electrontransfer reaction of these compounds at the electrode surface


66

M. Zaheer et al.

i /A

i /A

4.00E-06

3.00E-06

2.00E-06

1.00E-06

0.00E+00

-1.00E-06

-2.00E-06

-3.00E-06

-4.00E-060 0.2

(a)

(b)

0.4 0.6 0.8 1

E (V) vs. SCE

2.50E-05

2.00E-05

1.50E-05

1.00E-05

5.00E-06

0.00E+00

-5.00E-06

-1.00E-05

-1.50E-05

-2.00E-050 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

E (V) vs. SCE

Figure 2. (a) CV behavior of 5 × 10−4 M compound 4 at GC electrode in0.1 M TBAFB at various scans rates ranging from 0.02 to 0.1 V s−1 witha difference of 0.01 V s−1. The arrowhead indicates increasing scan rate.(b) CV behavior of 1 mM compound 6 at GC electrode using 0.1 M TBAFBat various scans rates ranging from 0.1 to 0.6 V s−1 with a difference of0.1 V s−1. The arrowhead indicates increasing scan rate.

were obtained from Nicholson’s equation:[50]

ψ = ks[πD

nfυ

RT

]1/2 (2)

An examination of Table 3 reflects that the values of ks increasein the same order, 6<3<2<4<1<5, as followed by the values ofD, thus validating the Nicholson formula. The values of ks agree wellwith those reported for Schiff-based compounds bearing phenylferrocene.[49,51] Assuming Eo = E1/2 (for reversible reaction), thevalues of charge transfer coefficient (α) were determined by the

i/µA

i/µA

9

85

7

6

51

4

3 4

22 3

1

00.15 0.19 0.23 0.27 0.31 0.35

υ1/2(Vs-1)

υ1/2(V1)1/2

(a)

(b) 23

21y = 27.297x - 0.7664

R2 = 0.999719

17

15

13

11

9

7

50.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Figure 3. (a) Plots of i vs ν1/2 for the determination of the diffusioncoefficients of 5 × 10−4 M 1–5. Scan rates 0.02–0.1 V s−1 with a differenceof 0.01 V s−1. (b) Plots of i vs ν1/2, for the determination of the diffusioncoefficients of 1 mM 6 in absolute ethanol at 0.1–0.6 V s−1.

application of Kochi’s formula.[52]

α =[

(E1/2 − Ecp)

(Epa − Ec

p)

](3)

where α with a value of ∼0.5 indicates the reversibility of theredox process of all the compounds 1–6.

Biological Studies of Compounds 1 and 4

Ferrocene-based derivatives have a broad range of biologicalapplications. They are extensively used for medical purposes,electrocatalysis and the design of new signaling ion sensors.They have well-established spectroscopy and redox chemistry. A

Table 3. CV data of compounds 1–6

CV data Kinetic data

Compound Epa (V) Epc (V) �Ep (mV) Eo (V) D × 105 (cm2 s−1) ks ×102 (cm s−1) A

1 0.601 0.526 75 0.564 2.66 2.73 0.51

2 0.456 0.386 70 0.421 0.41 1.60 0.50

3 0.456 0.378 78 0.417 0.26 0.68 0.50

4 0.582 0.509 73 0.546 0.76 1.74 0.51

5 0.251 0.329 78 0.290 3.96 8.40 0.50

6 0.478 0.400 78 0.439 0.20 0.64 0.50

All potentials were measured vs SCE in ethanol at 0.1 V s−1. �Ep = (Epa − Epc). Eo = (Epa + Epc)/2.


67


literature review revealed that the biological action of ferrocenesis due to their electron transfer rates and redox potentials.Moreover, it has been established that ferrocenes have appreciableantineoplastic activity but the mechanism of their effectivenessin eliciting an anti-tumor effect is yet to be explored.[48] Thebiological study reported herein is accessed by using selectedbiological assays. Because of the low solubility of Schiff basesin DMSO, only two of the compounds 1,4 were tested for theirantibacterial, antifungal, antioxidant and DNA protection andcytotoxic activity.

Antibacterial assay

The antibacterial activity of Schiff bases (1,4) was tested against sixbacterial strains: three Gram-positive [B. subtilis (ATCC no. 6633),S. aureus (ATCC no. 6538) and Micrococcus luteus (ATCC no. 10240)]and three Gram-negative [E. coli (ATCC no. 15224), E. aerogenes(ATCC no. 13048) and S. setubal (ATCC no. 19196)]. Test compoundsshowed no significant activity against all the six bacterial strainstested. Test compounds showed no significant activity against allthe six bacterial strains tested. This may be attributed to the lesslipophilic character of Schiff bases, which does not favor theirpermeation through the lipoid layer of microbial membranes.

Antifungal assay

Compounds 1 and4 were subjected to antifungal activity againstfive fungal strains: Mucor species (0300), Aspergillus flavus (0064),Aspergillus fumigatus (66), Fusarium solanni (0291) and Aspergillusniger. The results as shown in Table 4 indicate effective interactionwith all the tested fungal species. The growth inhibition activitywas found to be maximum against Mucor species, i.e, >60%.However, interestingly, both the tested compounds showedgrowth-promoting effects on A. niger, as indicated by negativevalues of percentage of inhibition.

DPPH free radical scavenging assay

DPPH is a stable free radical. Antioxidants react with DPPH bydonating electron or hydrogen, thus neutralizing it to diphenyl

picrylhydrazine.[52] This reduction of DPPH radical by antioxidantcan be determined by the decrease in absorbance at 517 nmspectrophotometrically; however a decrease in absorbance willshow no pro-oxidant activities. Compounds 1 and 4 were subjectedto DPPH assay to determine their antioxidant or prooxidantbehavior. Compounds were tested at three concentrations, 100,50 and 25 ppm. Test was performed in triplicate and the resultsare shown in Table 5. Table 5 indicates the mean percentagescavenging at all concentrations and IC25 values. Compound 4manifested significant activity with an IC25 of 6.21. These resultssupport our earlier report about ferrocene-containing Schiff basesshowing good antioxidant potentials.[53]

Brine shrimp lethality assay

Test compounds were also screened for cytotoxicity by brineshrimp lethality assay. The test was performed at 1000, 100 and10 ppm. The reaction was performed in triplicate. Criteria forcytotoxicity are lowest lethality dose for killing 50% shrimp larvae.The test compounds had LD50 values of 18.22 and 292.95 as shownin Table 6.

OH radical-induced oxidative DNA Damage analysis

These synthesized ferrocenyl Schiff bases were subjected to H2O2-induced DNA damage assay as a test of the DNA protection activityof these test compounds. The results are shown in Fig. 4. Schiffbases 1 and 4 were found to be effective DNA-protecting agents athigher concentrations (1000 and 100 ppm) but could not protectDNA at lower concentrations (10 ppm).

DNA interaction

Ferrocene compounds have been documented well for theirDNA cleavage activities through intercalation or electrostaticinteractions.[54,55] Cyclic voltammetry is a reliable analytical tool forthe study of these interactions. Voltammograms of the compoundsare recorded and any shift in peak currents after the addition ofDNA reveals any kind of interaction between the compound and

Table 4. Antifungal activities of test compounds

Percentage of growth inhibition + STDEV

Compound A. fumigatus A. flavus F. solanii Mucour A. niger

1 12.19 ± 8.44 39.99 ± 20.78 9.9 ± 1.8 71.42 ± 1.78 −0.0042 ± 2.12

4 4.87 ± 4.22 17.99 ± 4 65.76 ± 1.8 63.57 ± 7.42 −6.38 ± 4.25

Terbinafine 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0

(−ve) control – – – – –

Test compounds showed significant growth inhibition against Mucor species while having a growth-promoting effect on A. niger species.

Table 5. Percentage scavanging and IC25 data for test compounds

Percentage scavanging

Compound 100 ppm 50 ppm 25 ppm 10 ppm 5 ppm IC25 Remarks

1 8.468468 13.91892 13.33333 – – >100 Antioxidant

4 40.05022 37.09981 38.10421 37 23.1 6.218407 Antioxidant

Test compounds reduce DPPH free radical to neutral species which is determined spectrophotometrically at 517 nm.


68

M. Zaheer et al.

Table 6. Cytotoxicity data for test compounds

Number of shrimps Number of shrimps killedused at each LD50 value

Compound dose level 1000 ppm 100 ppm 10 ppm (ppm)

1 30 22 9 1 292.95

4 30 30 25 11 18.22

Test compounds are most toxic at higher concentrations, i.e. 1000 ppm, and are less cytotoxic at lower concentrations.

P F H X 1 2 3 4 5 6 7 8 9 L

L 1 KB ladder

P pBR322 plasmid

F pBR322 plasmid treated with FeSO4

H pBR322 plasmid treated with H2O2

X (positive control) pBR322 plasmid treated with FeSO4 and H2O2

Lane 1 pBR322 plasmid treated with FeSO4 and H2O2 + compound 1 (10 ppm)






Lane 7 pBR322 plasmid treated with FeSO4 and H2O2 + compound Fc (10 ppm)



Figure 4. Effect of compounds 1, 4 and ferrocene on pBR322 plasmid DNA.

DNA. The interaction of compounds with different concentrations(20, 30 and 40 µM) of DNA was studied using cyclic voltammetry. Noprominent shift in peak potential and peak current was observedin the absence and presence of DNA, proving that compounds donot interact with DNA (Fig. 5).

Conclusion

Ferrocenyl Schiff bases 1–6 were successfully synthesized andcharacterized using spectroscopic techniques. Their redox prop-erties were studied using cyclic voltammetry. These compoundswere synthesized in good yield and were in highly pure form.From the variation in the values of the formal potential ofcompounds 1–6 it can be concluded that the oxidation potential

of the oxidizing moiety ferrocene can be modulated by changingthe electronic properties of the substituents attached to thecyclopentadienyl ring. The values of the diffusion coefficient ofcompounds 1–5 were found to decrease with the increase inmolecular weight. The ks varied in the same sequence, whichwas followed by the values of D. The charge transfer coefficient(α = ∼0.5) indicated the reversibility of the redox processes.Compounds did not interact with DNA at different concentrations.Compounds 1 and4 showed appreciable antifungal, antioxidantand DNA protection activities and possessed low cytotoxicity.

Acknowledgment

The authors are grateful to the Department of Chemistry,Quaid-I-Azam University, Islamabad, Pakistan and the Institute


69


-3.00E-06

-2.00E-06

-1.00E-06

0.00E+00

1.00E-06

2.00E-06

3.00E-06

4.00E-06

0 0.2 0.4 0.6 0.8 1 1.2

E

I

Series2Series3Series4Series5

Figure 5. Voltammetric behavior of 0.5 mM compound 4 in the absence(series 2) and presence of 20 (series 3), 30 (series 4) and 40 (series 5) µMDNA.

for Inorganic Chemistry, University of Frankfurt, Germany, forproviding laboratory and analytical facilities.

Supporting information

Supporting information may be found in the online version of thisarticle.

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