Ferrocene-Based Aliphatic and Aromatic Poly(azomethine)esters:Synthesis, Physicochemical Studies, and Biological EvaluationAsghari Gul,† Zareen Akhter,†,* Muhammad Siddiq,† Sehrish Sarfraz,‡ and Bushra Mirza‡

†Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan‡Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan

*S Supporting Information

ABSTRACT: In continuation to our efforts in finding potentialtherapeutic agents, a series of biologically significant poly-(azomethine)esters (fe-PAME) were synthesized by the reactionof preformed (E)-4-((4-hydroxyphenylimino)methyl)phenol(SB) with 1,1-di(chlorocarboxyl)ferrocene, (PFe). Differentaliphatic and aromatic sequences (1,3-propandiol, 1,6-hexandiol,and poly(dimethylsiloxane), hydroxyl-terminated (n = 550), 1,1,1,3,3,3-hexafluorobis(phenol)propane, and bisphenol A) wereincorporated in the parent chain to study their effect on biological activity. The overall results led to the identification of someinteresting polymers which seem to be potent antioxidants, highly cytotoxic, and more importantly DNA protecting and hencecan be studied further for other pharmacological activities to be used as potential drug candidates. FTIR and 1H NMRspectroscopic studies and elemental analysis were used to establish structural elucidation and structure−property relations. Laserlight scattering was used to determine molecular parameters.

■ INTRODUCTION

Ferrocene macromolecules have drawn much attention becauseof their useful applications like chemical modification ofelectrodes, electrochemical sensors, charge dissipation materialand therapeutic applications. The stability of the ferrocenylgroup in aqueous, aerobic media and its promising electro-chemical properties make ferrocene and its derivatives ideal forbiological applications and conjugation with biomolecules.1

Assimilation of a ferrocenyl group into an organic materialoften yields unexpected biological activity.2 Ferrocene istransformed into the ferrocenium ion (Fc+) through one-electron reversible oxidation; however, substituents on theferrocene moiety have the capability to influence this redoxbehavior by altering the energy level of HOMO,3 soreversibility may be lowered significantly.4 The low cytotoxicityof ferrocene in biological system, its lipophillicity, thecytotoxicity of its metabolites toward tumors, the π-conjugatedsystem and the resulting exclusive electron-transfer ability makeits polymers good candidates for the investigation of theirbiological applications.2−4

An exhaustive literature survey revealed that in addition tothese material, poly(azomethine)s have shown significantantifungal, antibacterial, antitumor and antioxidant activities.7,8

The literature on the synthesis of ferrocene-poly(azomethine)sby polycondensation is very scarce. Although this procedure isstraightforward that does not require stringent reactionconditions and also permit to use a large range of functionalizedmonomers resulting in polymers with internal polar functions(esters, imide etc.) which could influence the properties ofmaterial.5,6 Macromolecular systems based on ferrocenyl unitsalong with flexible aliphatic or more rigid aromatic organicsequences can induce properties like solubility and flexibility.

However these types of materials with ferrocene in their corechain, so far have reported show lower molecular weights.2,9,10

The efficient mean to improve the physical and chemicalproperties of material is the chemical modification of macrochains by introducing flexible aliphatic spacers in the mainchain, pendent alkyl groups along the backbone, by thecopolymerization of different soft groups, by formingcomposites or by dopant engineering.11−14

We recently addressed the molecular weight limitation andsolubility issue encountered with previously investigatedpoly(azomethine)esters. This led to the variety of high-molecular-weight, soluble organometallic, biologically activepoly(azomethine)esters and their terpolymers by using lowtemperature solution condensation technique.

■ MATERIALS AND METHODSMaterials. Ferrocene (mp = 172−174 °C, Fluka), acetyl chloride

(bp = 51−52 °C, Fluka), thionyl chloride (bp = 74.6 °C, Fluka),aluminum chloride (mp = 192.4 °C, Fluka), 4-aminophenol (mp =188−190 °C, Fluka), 4-hydroxybenzaldehyde (mp = 112−114 °C,Fluka), p-toluenesulfonic acid (monohydrated, mp = 98−102 °C,Fluka) 1,3-propandiol (211−217 °C, Sigma-Aldrich), 1,6-hexandiol(250 °C, Sigma-Aldrich), poly(dimethylsiloxane), hydroxyl-terminated(n = 550, Sigma-Aldrich), 1,1,1,3,3,3-hexafluorobis(phenol)propane(160−163 °C, Sigma-Aldrich), and bisphenol A (158−159 °C, Sigma-Aldrich), nutrient broth medium (b.p = 121 °C, dissolved in water,Merck), nutrient agar medium (bp = 121 °C, dissolved in water,Merck), 2,2-diphenyl-1-picrylhydrazyl (mp ∼ 135 °C, Sigma-Aldrich)were used as received. The chemicals from commercial sources were

Received: January 29, 2013Revised: March 19, 2013

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used as received whereas the solvents (dichloromethane, diethyl ether,and ethanol (Sigma-Aldrich) and dimethyl sulfoxide (Sigma-Aldrich)were dried according to the reported method.15

Equipments. Melting point was determined on a Mel-Temp.(mitamura Riken Rogyo, Inc.) by using open capillary tubes. FTIRspectra in KBR pellets were recorded in Perkin-Elmer 1600 seriesFTIR spectrophotometer. Nuclear magnetic resonance was carried outby using BrukerAvance 300 digital NMR in DMSO-d6 as solvent andtetramethylsilane as an internal standard. Elemental analyses wereobtained on a Vaio-EL instrument. UV−visible studies of dilutepolymer solutions in dried ethanol were performed on Shimadzu,1998, 2000. A commercial light-scattering spectrometer (BI-APDequipped with a BI9000AT digital Auto correlator) was used alongwith a He−Ne laser (output power ∼400 mW at λ = 638 nm) as alight source. Relevant measurements were carried out at 25 + 0.1 °C.The software used was BI-ISTW.Bio Screening. Antibacterial Assay. The antibacterial activities of

the SB and fe-PAMEs were checked by agar well diffusion method.8 A24-h incubated culture of each of six bacterial strains were usedincluding two Gram-positive ones, i.e., Staphylococcus aureus andMicrococcus luteus and four Gram-negative strains, i.e., Salmonellatyphimurium, Enterobacter aerogenes, Escherichia coli, and Bordetellabronchiseptica. Each sample was assayed at 1 mg/mL concentration.Roxithomycin, Cefixime USP, and DMSO were used as controls.Then, 100 μL of each test solution and controls were poured in thewells of culture plates. After incubation at 37 °C for 24 h, the clear(inhibition) zones were detected around wells of antibiotics and activepolymers with the help of vernier caliper. The polymers havingantibacterial activity were then subjected to determine minimuminhibitory concentration (MIC). It was checked at lower concen-trations of active test polymers (0.8, 0.6, 0.4, and 0.2 mg/mL).Brine Shrimp Cytotoxic Assay. Brine shrimp (Artemiasalina)

lethality assay was performed in triplicate to test the toxicity of SBand fe-PAMEs.9 The eggs of brine shrimps were hatched in artificialseawater solution for 48 h. The sample was prepared by dissolving 1mg of each material in 1 mL of solvent, methnol, or DMSO dependingon the solubility of the polymer. This stock solution of 1000 ppmconcentration was further diluted to 100, 10, and 1 ppm for testing.After 24 h of incubation at room temperature (25−28 °C) the aliveshrimps were counted with the help of magnifying glass. Thepercentage mortality rate formula in Table 4 was used and the datawere also analyzed with LD50 values.Antitumor Potato Disc Assay. The sample SB and fe-PAMEs were

screened for crown gall tumor inhibition by antitumor potato diskassay.10 48h old single colony culture of Agrobacterium tumefaciens (AT10) bacterial strain was used for induction of tumor on potato disks(0.5 cm thickness) which were prepared under complete asepticconditions using sterilized instruments (HgCl2 0.1%). Two potatodisks were used for each treatment. The activity of polymers waschecked at 1000 ppm, 100, 10, and 1 ppm in DMSO solvent. Aftertreatment with test agents and AT 10 strain, each disk was thenincubated at 28 °C for 21 days and then stained with the Lugol’ssolution (10% KI and 5% I2). After that number of tumor werecounted with the help of dissecting microscope. Each experiment wascarried out in triplicate and IC50 values were also calculated.

Percentage tumor inhibition was calculated by using formula givenin Table 5.

Determination of Antioxidant Activity. The free radical scavengingactivity was measured by using 2,2-diphenyl-1-picrylhydrazyl freeradical (DPPH) assay which was performed according to theprocedure described by Kulisicet al. modified by Obeid et al.1,10

DPPH solution was prepared by dissolving 3.2 mg of DPPH in 100mL of 82% methanol. The stock solution of the test polymers wasprepared in 1 mL of DMSO at concentration of 1000 ppm. This stocksolution was further diluted to 100 and 10 ppm. The reaction mixturewith a final volume of 3 mL was prepared by adding 2 mL of DPPHsolution, 0.9 mL of Tris HCL buffer, and 100 uL of the test polymer at1000, 100, and 10 ppm concentration to get the final concentration of33.33, 3.33, and 0.33 ppm in the reaction mixture. Negative controlwas prepared by adding 100 uL of DMSO and 2 mL of DPPHsolution. After incubation of reaction mixture at room temperature indark for 30 min, absorbance was calculated at 517 nm on a UV/visiblelight spectrophotometer against blank. Each experiment was carriedout in triplicate and results were evaluated by calculating IC50 value.

16

The percentage scavenging of DPPH free radical for eachconcentration of each test polymer was determined by the formulagiven in Table 5.

DNA Protection Assay. The effects of SB and fe-PAMEs on plasmidDNA in vitro was studied by free radical induced oxidative DNAdamage analysis which was carried out by the procedure of Tian andHua, modified by Nawaz et al.1,10 The reaction was conducted in anEppendorf tube at a total volume of 15 μL containing followingcomponents; 0.5 μg pBR322 DNA suspended in 3 μL of 50 mMphosphate buffer (pH 7.4), 3 μL of 2 mM FeSO4, 5 μL of testedsamples (SB and fe-PAMEs) and 4 μL of 30% H2O2. Resulting mixturewas incubated at 37 °C for 1 h and was subjected to 1% agarose gelelectrophoresis for 1 h at 100 V. DNA bands (supercoiled, linear, andopen circular) were stained with ethidium bromide and were analyzedqualitatively by scanning with Doc-IT computer program (VWR).Evaluations of antioxidant or prooxidant effects on DNA were basedon the increase or loss percentage of supercoiled monomer, comparedwith the control value. To avoid the effects of photoexcitation ofsamples, experiments were done in the dark. Antioxidant activity of thetest polymers was examined by comparing the bands of samples withcontrols.

Polycondensation. Synthesis of Monomer: (E)-4-((4-hydroxypheny l im ino)methy l )pheno l (SB ) . (E) -4 - ( (4 -hydroxyphenylimino)methyl)phenol was synthesized according topreviously reported method.17

Synthesis of 1, 1′-Di(chlorocarboxyl)ferrocene (Fe). 1,1-Di-(chlorocarboxyl)ferrocene (mp = 95−100 °C) was prepared by thereported method.16,17

Synthesis of Polymer (PFe). The polymerization was carried out ina 250 mL three-necked round-bottom flask equipped with a refluxcondenser, a gas inlet and a magnetic stirrer. The monomers, SB anddiacid chloride (Fe) were added in a molar ratio 1:1 in 50 mL drieddichloromethane (CH2Cl2). The reaction mixture was maintained at 0°C by means of ice bath. Five mLtriethylamine (Et3N) was added tothe reaction mixture under inert nitrogen atmosphere, with constantstirring. After 24 h it was refluxed for 1 h and then the reaction

Scheme 1. Synthesis of Organometallic PAMEs

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contents were poured into water to eliminate triethylammoniumchloride salt (Et3NHCl) from organic layer and to precipitate polymer.The material obtained was filtered, washed several times with water,ethanol and then dried in air,16 Scheme 1.PFe: 453; dark orange; 89%. UV/vis (nm): 376, 430. FTIR (cm−1,

KBr): 3199 (aromatic C−H), 1709 (CO), 1623 (azomethine), 1033(C−O), 483 (ferrocenyl), 751 (C−Cl). 1H NMR [δ, deuterateddimethyl sulfoxide (DMSO-d6)]: δ (ppm): 8.42 (1H, s −CHN−),7.4−7.0 (m, aromatic H), 2.3 (3H, s methyl), 2.1 (1H, s OH), 2.02(1H, s methylene). Anal. Calcd: C, 66.14; H, 4.09; N, 3.0. Found: C,66.22; H, 4.19; N, 3.09.Synthesis of Terpolymers (PFePr, PFeH, PFeSi, PFeB, PFeF). The

synthesized monomer having Schiff base linkage and the commercialdiol (R = 1,3-propandiol, 1,6-hexandiol, poly(dimethylsiloxane),hydroxyl-terminated (n = 550), 1,1,1,3,3,3-hexafluorobis(phenol)-propane or bisphenol A), from scheme: 2 were taken into a two-necked round-bottom flask under inert atmosphere (N2). Then 50 mLof dried dichloromethane (CH2Cl2) followed by 5 mL of triethylamine(Et3N) was added at low temperature using an ice bath with constantstirring. Then the diacid (Fe) was added under same reactionconditions. The reactant ratio was 1:1:2 for the Schiff base (SB), thediol (R) used, and 1,1-di(chlorocarboxyl)ferrocene respectively. After24 h, the reaction mixture was refluxed for 1 h and then poured intowater for removing triethylammonium chloride salt (Et3NHCl) fromthe organic layer. Polymers were obtained as precipitates, which werethen washed several times with water and ethanol and then dried inair,16 Scheme 2.PFeB: yield 86%, dark orange powder. UV/vis (λmax) (C2H5OH):

380 and 425 nm. FTIR (cm−1, KBr): 3160 (aromatic C−H), 2994(aliphatic C−H), 1765, 1769 (CO), 1027, 1031 (C−O−C), 1693(azomethine), 762 (C−Cl), 499 (ferrocenyl). 1H NMR [δ, deuterateddimethyl sulfoxide (DMSO-d6)] δ (ppm): 8.46 (2H, s, azomethine),7.43−7.1 (aromatic), 4.9−4.4 (b, ferrocenyl), 2.33 (6H, s, methyl).Anal. Calcd: C, 72.39; H, 4.87; N, 3.24. Found; C, 73.39; H, 5.02; N,3.79.PFeF: 655; orange brown; 88%. UV/vis (λmax) (C2H5OH): 367 and

421 nm. FTIR (cm−1, KBr): 3178 (aromatic C−H), 1783, 1782 (CO), 1142, 1138 (C−O−C), 1689, 1683 (azomethine), 762 (C−Cl),521 (ferrocenyl). 1H NMR [δ, deuterated dimethyl sulfoxide (DMSO-

d6)] δ (ppm): 8.78 (2H, s, azomethine), 7.88−7.3 (aromatic), 5.1−4.5(b, ferrocenyl). Elemental analysis; Calcd: C, 73.4; H, 5.0; N, 2.11.Found; C, 73.14; H, 4.98; N, 2.21.

PFeH: 533; orange brown; yield 89%. UV/vis (λmax) (C2H5OH):330 and 415 nm. FTIR (cm−1, KBr): 3153 (aromatic C−H), 2905(aliphatic C−H), 1735, 1742 (CO), 1075, 1081 (C−O−C), 1637(azomethine), 722 (C−Cl), 481 (ferrocenyl). 1H NMR [δ, deuterateddimethyl sulfoxide (DMSO-d6)] δ (ppm): 8.23 (2H, s, azomethine),7.5−6.8 (aromatic), 4.8−4.5 (b, ferrocenyl), 2.6−2.1 (12H, m,methylene). Anal. Calcd: C, 68.27; H, 5.60; N, 2.53. Found; C,68.0; H, 5.43; N, 2.43.

PFePr: 511; yield 88%; orange brown. UV/vis (λmax) (C2H5OH):345 and 425 nm. FTIR (cm−1, KBr): 3193 (aromatic C−H), 2943(aliphatic C−H), 1709, 1713 (CO), 1102, 1107 (C−O−C), 1609,azomethine, 736 (C−Cl), 503 (ferrocenyl). 1H NMR [δ, deuterateddimethyl sulfoxide (DMSO-d6)] δ (ppm): 8.21 (2H, s, azomethine),7.48−7.1 (aromatic), 4.8−4.7 (b, ferrocenyl), 2.5−2.2 (6H, m,methylene). Anal. Calcd: C, 66.75; H, 5.0; N, 3.0. Found: C, 69.90;H, 4.31; N, 3.01.

PFeSi: yield 87%; orange brown. UV/vis (λmax) (C2H5OH): 321and 417 nm. FTIR (cm−1, KBr): 3157 (aromatic C−H), 2945(aliphatic C−H), 1702, 1709 (CO), 1073, 1079 (C−O−C), 1651,(azomethine), 701 (C−Cl), 497 (ferrocenyl). 1H NMR [δ, deuterateddimethyl sulfoxide (DMSO-d6)] δ (ppm): 8.4 (2H, s, azomethine),7.2−6.7 (aromatic), 4.8−4.3 (b, ferrocenyl), 2.1−1.6 (m, methyl).

■ RESULTS AND DISCUSSIONThe diol-terminated Schiff base monomer (SB) required forpreparing targeted material was synthesized by a reportedmethod as outlined in the Supporting Information.16,17

Poly(azomethine)ester PFe was synthesized using low temper-ature solution condensation polymerization of monomer (E)-4-((4-hydroxyphenylimino)methyl)phenol (SB) and diacid chlor-ides (Fe), Scheme 1. The purpose of incorporation of differentaliphatic and aromatic diols in parent macromolecules is toensure better solubility and to study structure−propertyrelation, Scheme 2. The targeted material was prepared bycombining stoichiometric amount of the monomers in one-pot

Scheme 2. Synthesis of Organometallic Terpolymers

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three-reactants employing in situ process. The reaction wascarried out at atmospheric pressure to avoid any side reactionand decomposition of thermally sensitive monomers. Themaintainess of low reflux temperature was essential to ensurereaction by dissolving diols completely in the solvent.5,17−19 Allpolymers were found soluble in common organic solvents.FTIR and NMR spectroscopy were used to confirm thefunctionalities present in the monomer (SB), polymer (PFe)and terpolymers (PFePr, PFeH, PFeSi, PFeB, PFeF).In FTIR spectra significant changes were observed in the

spectral properties of initial and final products as some of thesignals disappeared and some new appeared. The IR spectrumof SB showed a strong peak around 3400(s) cm−1 indicatingthe presence of hydroxyl group (hydroxybenzaldehyde) at theterminals. The absence of a sharp CO peak in the region1700−1720 (s) cm−1 along with the presence of a peak at 1620(s) cm−1 showed that aldehydic group had been transformed toazomethine linkage(CN).16,19,20 The spectra of poly-(azomethine)esters (PAMEs) and their condensation terpol-ymers exhibited all the characteristic bands expected forpresumed structures. The presence of absorption bandsexpected for the ester linkage (CO), (C−O) along withthe peak for imine group appeared in the ranges 1720−1750(s), 1101−1200 (s), and 1600−1645 (s) cm−1, respectively,showed the formation of PAMEs/terpolymers.16,19 In addition,the absence of a broad peak for the OH group, around 3400cm−1 coincident with the appearance of a band for diacidchloride group in the region of 780−540 cm−1 confirmed thepresence of C−Cl group at the terminal of macrochains. In thecase of terpolymers, the above-mentioned peaks for ester andimine appeared as symmetric doublet peaks representingsuccessful incorporation of diols in the parent chain. Additionalabsorption bands related to the functional group present in thediols added to the chain were found in each spectrum in theirrespective regions, e.g., C−H aliphatic appeared around 3000−2900 cm−1 in the spectra of material having diols H, Pr, and Sias a part of their chain. Characteristic FTIR absorption peaksfor the Si−O−Si group present in PFeSi appeared as a doubletaround 1020 and 2900 cm−1. Aromatic C−H appeared around3100 cm−1in polymers PFeB and PFeF. It is known that theSi−CCO group is very sensitive toward both hydrolysis andalcoholysis occurring readily in the absence of acid/basecatalysts. In case of terpolymers based on Si unit thedecomposition of material with time was expected as reportedearlier. Therefore, these terpolymers have potential asbiodegradable.19 There are many factors influencing thehydrolytical stability of PFeSi in the macro chain like stericbulk around the Si or electronegativity of the substituentsattached to the Si atoms. An advanced study on the kinetics ofhydrolytically degradability will be described in future article.

1H NMR spectrum of the SB recorded in DMSO exhibitedall the characteristic signals confirming the presence ofazomethine linkage in the product.16 The spectra of the fe-PAMEs were recorded under similar conditions. The cyclo-pentadienyl protons appear in the range 4.30−4.39 ppm and4.75−4.90 ppm. Some signal broadening in 1H NMR spectraindicated the presence of paramagnetic impurities as a result ofoxidation of ferrocene into the ferrocenium ion as reported inliterature.16,19

All 1H NMR spectra also exhibited a signal in the range 8.3−8.7 ppm indicating the presence of HCN proton whereas theresonance in the range 6.9−7.9 ppm showed aromatic protons.In addition to these signals, which were common in all the

spectra, terpolymers having aliphatic diols (Pr and H) showedmultiplet signal in the range 2.1 to 2.6 ppm (aliphatic −CH).The terpolymer having diol “B” showed additional signals in therange 7.0−8.0 ppm due to additional aromatic rings present inchain and at 2.3 ppm (−CH3 present in bisphenol). PFeFshowed resonance around 7.4 to 8.1 (aromatic rings), slightlydeshielded owing to the presence of electronegative F (CF3) inthe unit added whereas PSi exhibited additional resonancepeaks at 1.6−2.1 due to CH3 attached to PFeSi.The stoichiometery of the SB and PAMEs was confirmed by

elemental (C, H, N) analysis which showed a good correlationbetween the proposed structures and the experimental results.The calculations were made based on the structure of repeatunit present in the polymer chain. The elemental analysisshowed that in each polymer, monomers were in equimolarproportion. However somewhat higher contents of carbonfound may be because of ferrocenyl acid group which was notincluded in calculations. In the case of condensation terpolymercontaining silanol(Si) in chain, the elemental analysis was notpossible because at higher temperatures silicon interact withcarbon to form ceramic material.16,19,20

The UV/visible spectra were registered qualitatively inethanol at room temperature to confirm the presence ofdifferent groups in the fe-PAMEs chain as they were soluble inmost organic solvents. The material was dissolved in ethanol tomake dilute solutions for the study. The absorbance in therange 320−392 nm showed π−π* transition indicative of thepresence of Ph−NC−Ph chromophore in the polymerbackbone and the bands appearing in the region 418−435 nmare representative of d−d transition in the ferrocenyl group, inall fe-PAMEs.16,21 The bathochromic shifting is due to theextended conjugation, which is possible in PFe, PFeB, andPFeF while it is interrupted by methylene and silyl spacers inPFeH, PFePr, and PFeSi.

Molecular Parameter Determination by LLS Techni-que. Both static and dynamic laser light (LLS) scatteringanalysis were carried out with, a commercial LLS spectrometer.All LLS measurements were carried out at 25 °C. Fiveconcentrations ranging from 1.20 × 10−3 to 5.12 × 10−3 g/mLwere prepared by dilution for the measurements. All polymersolutions were clarified by using a 0.45-m Whatman filter inorder to remove dust completely. The angular dependence ofthe excess absolute time-averaged scattered light intensity,known as the Rayleigh ratio (Rvv(q)) of dilute solution ofdifferent concentrations C (g/mL) at different scattering angleswere measured.22 By measuring Rvv(θ) at different C and θ, wewere able to determine Mw, z-average radius of gyration (Rg)and the A2 from the Zimm plot that incorporate C and θ in asingle grid. The static LLS results are summarized in Table 1.

Table 1. Laser light-Scattering Results for the Polymers(DMSO and MeOH at 25 °C)a

polymers Mw (g/mol) ⟨Rg⟩ (nm) A2 (cm3 mol g−2) Rh (nm) Rg/Rh

PFe 1.67 × 104 80 3.0 × 10−3 50 1.60PFeH 2.1 × 106 134 2.0 × 10−4 75 1.78PFeB 5.0 × 104 75 2.50 × 10−3 55 1.40PFeSi 3.67 × 106 145 1.50 × 10−4 85 1.70PFeF 5.2 × 104 85 2.40 × 10−3 60 1.33PFePr 2.1 × 105 80 7.70 × 10−4 50 1.60

aEstimated uncertainties: 10% in Rg; 5% in Rh, 10% in Mw and 10% inA2.

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The positive values of A2 indicate that the solvent used isgood solvent for the present polymers at 25 °C. The A2 valueincreases with the decrease in the molecular weight, whichindicates that the solvent quality decreases with the increase inthe molecular weight.23,24 The correlation functions obtainedfrom dynamic light scattering were analyzed by the constrainedregularized CONTIN method.23−27 Distributions of decay rateΓ, converted to distributions of translational diffusioncoefficient; D = Γ/q2, where q = (4πn/λ) sin(θ/2), n is therefractive index of the solvent and θ is the scattering angle.Hence, distributions of hydrodynamic radius (Rh) wereevaluated using the Stokes−Einstein relationship Rh, = kT/(6πηD), where k is the Boltzmann constant, T is the absolutetemperature, and η is the viscosity of the solvent. Thehydrodynamic radius Rh reflects the physical dimensions ofthe polymer chains, as it involves both the chain and theamount of the solvent associated with, whereas the radius ofgyration Rg is related to the size of chain and, in other words, tothe excluded volume of polymer in the solvent and mostlydepends on the thermodynamic quality of the solvent. It isknown that the ratio of the radius of gyration to thehydrodynamic radius, i.e., ⟨Rg⟩/⟨Rh⟩, reflects the chainconformation. The ratio of ⟨Rg⟩/⟨Rh⟩ ∼ 1.0−2.0 indicatesthat the polymers have a coil chain conformation in the solventused.22−28

Bio Screening. The therapeutic potential of the materialreported herein is accessed by selected biological assays. This isan attempt to investigate possible biological activities ofsynthesized fe-PAMEs which were soluble in common organicsolvent. SB and fe-PAMEs comprising of different aliphatic,

aromatic and imine linkage were selected for biologicalscreening.

Antibacterial Assay. The bacterial growth inhibition effectof SB and fe-PAMEs was determined by agar well diffusionmethod. The results obtained from the antibacterial activity forSB and the polymers are summarized in Table 2. The SBshowed maximum zone of inhibition against four bacterialstrains namely S. typhimurium, B. bronchiseptica, M. leuteus, andS. aureus. PFeF was found to have highest antibacterial activityamong all the tested polymers against all the bacterial strainsused that might be due to the presence of fluorine based1,1,1,3,3,3-hexafluorobis(phenol)propane group.1,10,29 It hasbeen reported previously that fluorinated 4-thiazolidinoneshave good antibacterial activity. PFe showed some activityagainst one strain used i.e, S. typhimurium; however, PFePr andPFeSi based on flexible spacers were found to be inactive. MICwas determined for the active material, and the data are given inTable 3. On the basis of structure it was found that SB wasfairly active antibacterial agent that can be due to the presenceof free hydroxyl groups on the aromatic rings. The activity isstill enhanced when fluorinated moiety is added to ferrocenatedShiff base. PFe also showed antibacterial activity whichcontained Schiff base (SB) estirified with ferrocene. Theinactivity of PFePr and PFeSi can be due to introduction ofnonpolar block.

Cytotoxity. The data in case of Brine shrimp (Artemiasa-lina) lethality assay, tabulated in Table 4 showed that the SBexhibited cytotoxic nature with LD50 69 ppm. PFePr and PFeFhaving LD50 < 20 shows highly toxic behavior whereas PFeSishowed LD50 in range of 66.66. The material based on

Table 2. Antibacterial Activity of Poly(azomethine)esters and Their Monomers

mean zone of inhibition (mm) ± SD

S. no. codes S. typhimurium B. bronchiseptica M. leuteus S. aureus E. aerogens E. coli

1 SB 8.50 ± 0.1 9.4 ± 0.01 9.9 ± 0.1 10.03 ± 0.15 nil nil2 PFePr nil nil nil Nil nil nil3 PFe 8.03 ± 0.1 nil nil nil nil nil4 PFeH nil 11.1 ± 0.1 nil nil nil nil5 PFeB 11.2 ± 0.26 9.4 ± 0.5 nil nil nil nil6 PFeSi nil nil nil nil nil nil7 PFeF nil 10.1 ± 0.1 15.5 ± 0.15 18.30 ± 0.26 26 ± 0.5 nil9 Cefixime 31 ± 0.9 36.83 ± 0.72 22.83 ± 0.72 27.5 ± 0.5 28.96 ± 0.55 34.2 ± 0.3

Table 3. Minimum Inhibitory Concentration (MIC) of Active Polymers

minimum inhibitory concentration (MIC) mg/mL

S. no. codes M. leuteus E. aerogenes E. coli B. bronchiseptica S. aureus S. typhimurium

1 PFe − − − − − 0.82 PFeH − − − 1 − −3 PFeB − − − 1 − 0.84 PFeF 0.2 0.4 − 1 0.4 −

Table 4. Cytotoxic Activity

percentage mortality = (number of live shrimp) control − sample × 100/(numberof live shrimp) control

percentage mortality = (number of live shrimp) control − sample × 100/(number of live shrimp) control

percentage mortality percentage mortality

S. no codes 100 ppm 10 ppm 1 ppm LD50 S. no codes 100 ppm 10 ppm 1 ppm LD50

1 SB 67.8 17.85 7.14 69.27 5 PFeB 42.85 3.57 25 129.262 PFe 37.03 33.33 25.92 241.06 6 PFeSi 71.42 14.28 7.14 66.663 PFePr 80.74 77.77 62.16 0.017 7 PFeF 96.42 53.57 17.85 7.064 PFeH 29.62 19.51 14.81

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ferrocenyl and imine linkage is known to be more active ascompared to those based simple Schiff bases. There was nogeneral trend of cytotoxicity observed by varying the additionalgroup except that the fluorine-containing group showed themore cytotoxic behavior. Moreover the polymeric material wasfound more active as compare to SB. It means the addition ofblock esterified ferrocene can enhance the cytotoxic behavior tovarying level. The test polymers were most toxic at higherconcentrations and are less cytotoxic at lower concentrations;i.e., activity was dose dependent as reported previously.1,10,29−33

Tumor Inhibition. Potato disk tumor induction is a reliablescreen for detecting antitumor agents. As the mechanisms forthe assay is quite similar in plants and animals therefore thepotato disks exhibit a good correlation in results as compare tothe other most commonly used antitumor screening assays.Herein A. tumefaciens, AT-10, was used to induce tumors onpotato disks in order to screen soluble SB and fe-PAMEs(Figure 1). SB showed significant antitumor activity 33%

(Table 5). It is reported earlier that ferrocene exhibitsinhibition of tumor activity by several mechanisms includingthe one that is mediated by its effect on immune stimulation, byactivating T-lymphocytes.30−35 PFe showed highest tumorinhibition with IC50 7.15 ppm. PFeF having fluorine containinggroup showed less inhibition. PFeH and PFeSi are tumorinducing at lower concentrations. By varying the thirdadditional moiety “Pr” in the poly(azomethine)esters, therewas no trend of variation in inhibition observed. Thepercentage inhibition of PFe at 100 ppm is 60.76% (Table5).It is also reported in similar findings that Schiff base esters offerrocenyl aniline and simple aniline could be regarded aspotential anticancer drug candidate. If PFe is comparedstructurally with Schiff bases then it is observed that the Schiffbase activity is enhanced when it is esterified with ferrocenering containing acid chloride. It is also reported thatpolyaspartamide-based conjugates featuring ester-linked ferro-cene showed active inhibitory role against tumors.31−35 Thegeneral tumor inhibition behavior of test compounds based ontheir structures was SB >PFe > PFeF > PFePr > PFeH > PFeSi.

It can be deduced that the addition of blocks containing nonpolar part moieties reduce the activity against AT-10.

Antioxidant Activity (DPPH Free Radical Scavenging).Antioxidants react with DPPH (a stable free radical) bydonating electron or hydrogen thus neutralizing it to yellowcolored diphenylpicrylhydrazine. This reduction of DPPHradical by antioxidants can be determined by the decrease inabsorbance at 517 nm spectrophotometrically, however, anincrease in absorbance will show pro-oxidant activities. SB (IC503.15 ppm) has highest antioxidant activity among all testedmaterials whereas all fe-PAMEs showed good antioxidantbehavior as shown in Table 5.34,36 It is evident from literaturethat most of the Schiff bases exhibited antioxidant activity andalso that bearing two hydroxyl groups on the phenyl ringshowed excellent antioxidant activity in comparison withascorbic acid. Overall fe-PAMEs exhibited less antioxidantactivity; the reason can be the absence of free hydroxyl radicalson phenyl rings. The activity in fe-PAMEs is due to theferrocene ring as reported in the literature that poly(Schiffbases) when complexed with ferrocene exhibit antioxidantactivity. The results of the DPPH test (Figure 2) showed that

the activity depends strongly upon the presence of phenolicgroup but is improved by the influence of ferrocenyl fragment,as reviewed through literature. It is evident that ferrocene had apromising behavior as an antioxidant. The general behavior ofscavenging activity was dominant at highest concentration, i.e.,33.33 ppm (Table 5) in dose dependent manner.29−35

DNA Damage Analysis Assay. DNA protecting activity ofSB and other synthesized material was investigated in vitro byOH radical induced DNA damage system at 10, 100, and 1000ppm concentration (Figure 3). Normally pBR322 DNA existsin supercoiled form (SC). With the attack of OH radical(known as most damaging radical for biomolecules) generatedfrom Fenton, SC was broken into open circular (OC).Theability of the test polymers to cleave or protect the super coilingsubstrate such as plasmid DNA was examined in the DNAdamage analysis assay. All of the material showed excellentDNA protecting activity at all the three concentrations tested,i.e., 1000, 100, and 10 ppm none of them exhibited pro-oxidant

Figure 1. Effect of polymer PFe at different concentrations, oninhibition of tumor formation along with control for comparison.

Table 5. Tumor Inhibition and Antioxidant Activity (DPPH Free Radical Scavenging)

percent tumor inhibition = 100 − average number of sample × 100/average number of tumors ofcontrol

percentage scavenging = absorbance of control - absorbanceof test sample × 100/absorbance of control

percentage inhibition percentage scavenging

S. no. codes 100 ppm 10 ppm 1 ppm 0.1 ppm IC50 33.33 ppm 3.33 ppm 0.333 ppm IC50

1 SB 37.580 32.48 32.55 30.20 296.82 60.247 26.097 16.877 24.872 PFePr 27.63 17.10 12.5 9.61 242.70 30.790 23.68 12.75 77.863 PFe 60.757 52.917 47.263 21.73 7.15 56.327 19.200 5.367 28.554 PFeH 15.043 13.983 −8.473 −12.12 268.52 46.78 15.287 23.417 37.415 PFeB 39.223 33.683 11.323 11.157 140.90 29.150 8.397 20.637 82.886 PFeSi 8.030 3.620 −3.623 −7.082 459.36 42.953 26.380 16.063 42.727 PFeF 43.607 28.9 12.857 7.110 118.05 60.247 26.097 16.877 24.87

Figure 2. Results of DPPH free radical scavenging assay of FePr atthree concentrations of 33.3, 3.33, and 0.33 ppm.

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activity (Table 5). SB fully protected the plasmid DNA fromdamage. fe-PAMEs also protected the plasmid DNA most ofthe time at all three concentrations 1000, 100, and 10 ppm(Table 6). Cytotoxic and tumor inhibitory activities might becorrelated with the DNA protection activities of the polymers.Available literature regarding the DNA protecting activities ofpoly(azomethine)esters is scarce.29−41

Elecrochemical investigations for organometallic material(PFeSi, PFeF, PFePr, PFeH, and PFeB) were also carried out.The dereviatives were found electroactive in positive potentialwindow showing only anodic peak. These ferrocenyl polymersare found to behave irreversibly in their electrochemicalresponses scan as reported by our group previously.16

■ CONCLUSION

A series of soluble ferrocene-poly(azomethine)ester consistingof aromatic and aliphatic spacers were successfully synthesized.Different flexible aromatic and aliphatic segments wereincorporated into the fe-PAMEs backbone to prepare theirrespective organometallic condensation terpolymers to studystructure−property relation. A combination of static anddynamic LLS showed that the poly(azomethine)esters andtheir condensation terpolymers has a coil chain conformation.In biological studies it was found that the fluorinated materialwas antibacterial with activity comparable to antibiotics used ascontrol. Significant antibacterial, cytotoxic and antitumoractivities indicated their potential to be used as antibioticsand anticancerous agents. All polymers were found highlypotent antioxidant and more importantly DNA protecting.These polymers can be studied further for pharmacologicalactivities to be used as potential drug candidates. Moreovertheir mechanism of action and structure activity relationship

(SAR) can also be studied. From all the studies mentioned, itwas concluded that the incorporation of different units is veryuseful factor that has influence on the solubility and otherproperties of the polymeric material.

■ ASSOCIATED CONTENT*S Supporting InformationSynthesis of Schiff base, 1H NMR spectrum of terpolymerPFeF, and representative 1H NMR spectrum of terpolymerPFeB. This material is available free of charge via the Internet athttp://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*(Z.A.) E-mail: zareenakhter@yahoo.com. Telephone:+925190642111.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe Department of Chemistry, Quaid-i-Azam UniversityIslamabad, is gratefully acknowledged for laboratory andtechnical facilities.

■ REFERENCES(1) Nawaz, H.; Akhter, Z.; Yameen, S.; Siddiqi, A. M.; Mirza, B.;Rifat, A. J. Organomet. Chem. 2009, 694 (14), 2198−2203.(2) Coker, P. S.; Radecke, J.; Guy, C.; Camper, N. D. Phytomedicine2003, 10, 133−138.(3) Kaya, I.; Koca̧, S. Int. J. Polym. Mater. 2007, 56, 197−206.(4) Kumar, L. R.; Sengodan, V.; Prasad, M. B.; Gopalakrishnan, K.;Sethupathi, K. Mater. Lett. 2002, 167−174.

Figure 3. Effect of polymerPFe and FeB on plasmid DNA. Key: L, DNA ladder (1 Kb); P, pBR322 plasmid; X, pBR322plasmid treated with FeSO4and H2O2 (positive control); lane 1, control for the damage effect of polymer on DNA; plasmid +1000 ppm of PFe; lane 2, plasmid + 1000 ppm ofPFe + FeSO4 + H2O2; lane 3, plasmid +100 ppm of PFe + FeSO4 + H2O2; lane 4, plasmid + 10 ppm of PFe + FeSO4 + H2O2; lane 5, control for thedamage effect of polymer on DNA; plasmid + 1000 ppm of PFeB; lane 6, plasmid +1000 ppm of PFeB + FeSO4 + H2O2; lane 7, plasmid +100 ppmof PFeB + FeSO4 + H2O2; lane 8, plasmid +10 ppm of PFeB + FeSO4 + H2O2.

Table 6. DNA Damage Analysis Assay

comparison of DNA damage/protection activity of polymers at respective concentrations

name 1000 ppm 100 ppm 10 ppm name 1000 ppm 100 ppm 10 ppm

1 SB +++ +++ +++ 5 PFeB +++ +++ +++2 PFePr + + + 6 PFeSi +++ +++ -3 PFe +++ +++ +++ 7 PFeF +++ +++ -4 PFeH ++ ++ ++

aSingle negative (−) sign means no protection. In this case, there is no supercoiled DNA. bSingle Positive (+) sign means slight protection. In thiscase, open circular + linear band > supercoiled. cTwo positive (++) means moderate protection. In this case, supercoiled > open circular with a linearband. dThree positive (+++) means good protection. In this case, supercoiled > open. eCircular with no linear band.

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