Ferrocene-Based Aliphatic and Aromatic Poly(azomethine)esters

Mar 29, 2013 - Brianna M. Upton , Raymond M. Gipson , Selma Duhović , Brian R. Lydon , Nicholas M. Matsumoto , Heather D. Maynard , Paula L. Diacones...
0 downloads 0 Views 291KB Size
Download PDF
Article pubs.acs.org/Macromolecules

Ferrocene-Based Aliphatic and Aromatic Poly(azomethine)esters: Synthesis, Physicochemical Studies, and Biological Evaluation Asghari 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 potential therapeutic agents, a series of biologically significant poly(azomethine)esters (fe-PAME) were synthesized by the reaction of preformed (E)-4-((4-hydroxyphenylimino)methyl)phenol (SB) with 1,1-di(chlorocarboxyl)ferrocene, (PFe). Different aliphatic 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) were incorporated in the parent chain to study their effect on biological activity. The overall results led to the identification of some interesting polymers which seem to be potent antioxidants, highly cytotoxic, and more importantly DNA protecting and hence can be studied further for other pharmacological activities to be used as potential drug candidates. FTIR and 1H NMR spectroscopic studies and elemental analysis were used to establish structural elucidation and structure−property relations. Laser light scattering was used to determine molecular parameters.

■

INTRODUCTION Ferrocene macromolecules have drawn much attention because of their useful applications like chemical modification of electrodes, electrochemical sensors, charge dissipation material and therapeutic applications. The stability of the ferrocenyl group in aqueous, aerobic media and its promising electrochemical properties make ferrocene and its derivatives ideal for biological applications and conjugation with biomolecules.1 Assimilation of a ferrocenyl group into an organic material often yields unexpected biological activity.2 Ferrocene is transformed into the ferrocenium ion (Fc+) through oneelectron reversible oxidation; however, substituents on the ferrocene moiety have the capability to influence this redox behavior by altering the energy level of HOMO,3 so reversibility may be lowered significantly.4 The low cytotoxicity of ferrocene in biological system, its lipophillicity, the cytotoxicity of its metabolites toward tumors, the π-conjugated system and the resulting exclusive electron-transfer ability make its polymers good candidates for the investigation of their biological applications.2−4 An exhaustive literature survey revealed that in addition to these material, poly(azomethine)s have shown significant antifungal, antibacterial, antitumor and antioxidant activities.7,8 The literature on the synthesis of ferrocene-poly(azomethine)s by polycondensation is very scarce. Although this procedure is straightforward that does not require stringent reaction conditions and also permit to use a large range of functionalized monomers resulting in polymers with internal polar functions (esters, imide etc.) which could influence the properties of material.5,6 Macromolecular systems based on ferrocenyl units along with flexible aliphatic or more rigid aromatic organic sequences can induce properties like solubility and flexibility. © XXXX American Chemical Society

However these types of materials with ferrocene in their core chain, so far have reported show lower molecular weights.2,9,10 The efficient mean to improve the physical and chemical properties of material is the chemical modification of macro chains by introducing flexible aliphatic spacers in the main chain, pendent alkyl groups along the backbone, by the copolymerization of different soft groups, by forming composites or by dopant engineering.11−14 We recently addressed the molecular weight limitation and solubility issue encountered with previously investigated poly(azomethine)esters. This led to the variety of highmolecular-weight, soluble organometallic, biologically active poly(azomethine)esters and their terpolymers by using low temperature solution condensation technique.

■

MATERIALS AND METHODS

Materials. 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, SigmaAldrich), 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, 2013 Revised: March 19, 2013

A

dx.doi.org/10.1021/ma400192u | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

Scheme 1. Synthesis of Organometallic PAMEs

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. FTIR spectra in KBR pellets were recorded in Perkin-Elmer 1600 series FTIR spectrophotometer. Nuclear magnetic resonance was carried out by using BrukerAvance 300 digital NMR in DMSO-d6 as solvent and tetramethylsilane as an internal standard. Elemental analyses were obtained on a Vaio-EL instrument. UV−visible studies of dilute polymer solutions in dried ethanol were performed on Shimadzu, 1998, 2000. A commercial light-scattering spectrometer (BI-APD equipped with a BI9000AT digital Auto correlator) was used along with a He−Ne laser (output power ∼400 mW at λ = 638 nm) as a light 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 A 24-h incubated culture of each of six bacterial strains were used including two Gram-positive ones, i.e., Staphylococcus aureus and Micrococcus luteus and four Gram-negative strains, i.e., Salmonella typhimurium, Enterobacter aerogenes, Escherichia coli, and Bordetella bronchiseptica. 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 the wells of culture plates. After incubation at 37 °C for 24 h, the clear (inhibition) zones were detected around wells of antibiotics and active polymers with the help of vernier caliper. The polymers having antibacterial activity were then subjected to determine minimum inhibitory concentration (MIC). It was checked at lower concentrations 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 SB and fe-PAMEs.9 The eggs of brine shrimps were hatched in artificial seawater solution for 48 h. The sample was prepared by dissolving 1 mg of each material in 1 mL of solvent, methnol, or DMSO depending on the solubility of the polymer. This stock solution of 1000 ppm concentration was further diluted to 100, 10, and 1 ppm for testing. After 24 h of incubation at room temperature (25−28 °C) the alive shrimps were counted with the help of magnifying glass. The percentage mortality rate formula in Table 4 was used and the data were 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 disk assay.10 48h old single colony culture of Agrobacterium tumefaciens (AT 10) bacterial strain was used for induction of tumor on potato disks (0.5 cm thickness) which were prepared under complete aseptic conditions using sterilized instruments (HgCl2 0.1%). Two potato disks were used for each treatment. The activity of polymers was checked at 1000 ppm, 100, 10, and 1 ppm in DMSO solvent. After treatment with test agents and AT 10 strain, each disk was then incubated at 28 °C for 21 days and then stained with the Lugol’s solution (10% KI and 5% I2). After that number of tumor were counted with the help of dissecting microscope. Each experiment was carried out in triplicate and IC50 values were also calculated.

Percentage tumor inhibition was calculated by using formula given in Table 5. Determination of Antioxidant Activity. The free radical scavenging activity was measured by using 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH) assay which was performed according to the procedure described by Kulisicet al. modified by Obeid et al.1,10 DPPH solution was prepared by dissolving 3.2 mg of DPPH in 100 mL of 82% methanol. The stock solution of the test polymers was prepared in 1 mL of DMSO at concentration of 1000 ppm. This stock solution was further diluted to 100 and 10 ppm. The reaction mixture with a final volume of 3 mL was prepared by adding 2 mL of DPPH solution, 0.9 mL of Tris HCL buffer, and 100 uL of the test polymer at 1000, 100, and 10 ppm concentration to get the final concentration of 33.33, 3.33, and 0.33 ppm in the reaction mixture. Negative control was prepared by adding 100 uL of DMSO and 2 mL of DPPH solution. After incubation of reaction mixture at room temperature in dark for 30 min, absorbance was calculated at 517 nm on a UV/visible light spectrophotometer against blank. Each experiment was carried out in triplicate and results were evaluated by calculating IC50 value.16 The percentage scavenging of DPPH free radical for each concentration of each test polymer was determined by the formula given in Table 5. DNA Protection Assay. The effects of SB and fe-PAMEs on plasmid DNA in vitro was studied by free radical induced oxidative DNA damage analysis which was carried out by the procedure of Tian and Hua, modified by Nawaz et al.1,10 The reaction was conducted in an Eppendorf tube at a total volume of 15 μL containing following components; 0.5 μg pBR322 DNA suspended in 3 μL of 50 mM phosphate buffer (pH 7.4), 3 μL of 2 mM FeSO4, 5 μL of tested samples (SB and fe-PAMEs) and 4 μL of 30% H2O2. Resulting mixture was incubated at 37 °C for 1 h and was subjected to 1% agarose gel electrophoresis for 1 h at 100 V. DNA bands (supercoiled, linear, and open circular) were stained with ethidium bromide and were analyzed qualitatively by scanning with Doc-IT computer program (VWR). Evaluations of antioxidant or prooxidant effects on DNA were based on the increase or loss percentage of supercoiled monomer, compared with the control value. To avoid the effects of photoexcitation of samples, experiments were done in the dark. Antioxidant activity of the test polymers was examined by comparing the bands of samples with controls. Polycondensation. Synthesis of Monomer: (E)-4-((4hydroxyphenylimino)methyl)phenol (SB). (E)-4-((4hydroxyphenylimino)methyl)phenol was synthesized according to previously reported method.17 Synthesis of 1, 1′-Di(chlorocarboxyl)ferrocene (Fe). 1,1-Di(chlorocarboxyl)ferrocene (mp = 95−100 °C) was prepared by the reported method.16,17 Synthesis of Polymer (PFe). The polymerization was carried out in a 250 mL three-necked round-bottom flask equipped with a reflux condenser, a gas inlet and a magnetic stirrer. The monomers, SB and diacid chloride (Fe) were added in a molar ratio 1:1 in 50 mL dried dichloromethane (CH2Cl2). The reaction mixture was maintained at 0 °C by means of ice bath. Five mLtriethylamine (Et3N) was added to the reaction mixture under inert nitrogen atmosphere, with constant stirring. After 24 h it was refluxed for 1 h and then the reaction B

dx.doi.org/10.1021/ma400192u | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

Scheme 2. Synthesis of Organometallic Terpolymers

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 (CO), 1075, 1081 (C−O−C), 1637 (azomethine), 722 (C−Cl), 481 (ferrocenyl). 1H NMR [δ, deuterated dimethyl 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 (CO), 1102, 1107 (C−O−C), 1609, azomethine, 736 (C−Cl), 503 (ferrocenyl). 1H NMR [δ, deuterated dimethyl 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): 321 and 417 nm. FTIR (cm−1, KBr): 3157 (aromatic C−H), 2945 (aliphatic C−H), 1702, 1709 (CO), 1073, 1079 (C−O−C), 1651, (azomethine), 701 (C−Cl), 497 (ferrocenyl). 1H NMR [δ, deuterated dimethyl 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).

contents were poured into water to eliminate triethylammonium chloride 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 (CO), 1623 (azomethine), 1033 (C−O), 483 (ferrocenyl), 751 (C−Cl). 1H NMR [δ, deuterated dimethyl sulfoxide (DMSO-d6)]: δ (ppm): 8.42 (1H, s −CHN−), 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 commercial diol (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 twonecked round-bottom flask under inert atmosphere (N2). Then 50 mL of dried dichloromethane (CH2Cl2) followed by 5 mL of triethylamine (Et3N) was added at low temperature using an ice bath with constant stirring. Then the diacid (Fe) was added under same reaction conditions. The reactant ratio was 1:1:2 for the Schiff base (SB), the diol (R) used, and 1,1-di(chlorocarboxyl)ferrocene respectively. After 24 h, the reaction mixture was refluxed for 1 h and then poured into water for removing triethylammonium chloride salt (Et3NHCl) from the organic layer. Polymers were obtained as precipitates, which were then washed several times with water and ethanol and then dried in air,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 (CO), 1027, 1031 (C−O−C), 1693 (azomethine), 762 (C−Cl), 499 (ferrocenyl). 1H NMR [δ, deuterated dimethyl 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 (C O), 1142, 1138 (C−O−C), 1689, 1683 (azomethine), 762 (C−Cl), 521 (ferrocenyl). 1H NMR [δ, deuterated dimethyl sulfoxide (DMSO-

■

RESULTS AND DISCUSSION The diol-terminated Schiff base monomer (SB) required for preparing targeted material was synthesized by a reported method as outlined in the Supporting Information.16,17 Poly(azomethine)ester PFe was synthesized using low temperature solution condensation polymerization of monomer (E)-4((4-hydroxyphenylimino)methyl)phenol (SB) and diacid chlorides (Fe), Scheme 1. The purpose of incorporation of different aliphatic and aromatic diols in parent macromolecules is to ensure better solubility and to study structure−property relation, Scheme 2. The targeted material was prepared by combining stoichiometric amount of the monomers in one-pot C

dx.doi.org/10.1021/ma400192u | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

three-reactants employing in situ process. The reaction was carried out at atmospheric pressure to avoid any side reaction and decomposition of thermally sensitive monomers. The maintainess of low reflux temperature was essential to ensure reaction by dissolving diols completely in the solvent.5,17−19 All polymers were found soluble in common organic solvents. FTIR and NMR spectroscopy were used to confirm the functionalities 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 the signals disappeared and some new appeared. The IR spectrum of SB showed a strong peak around 3400(s) cm−1 indicating the presence of hydroxyl group (hydroxybenzaldehyde) at the terminals. The absence of a sharp CO peak in the region 1700−1720 (s) cm−1 along with the presence of a peak at 1620 (s) cm−1 showed that aldehydic group had been transformed to azomethine linkage(CN).16,19,20 The spectra of poly(azomethine)esters (PAMEs) and their condensation terpolymers exhibited all the characteristic bands expected for presumed structures. The presence of absorption bands expected for the ester linkage (CO), (C−O) along with the 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 3400 cm−1 coincident with the appearance of a band for diacid chloride group in the region of 780−540 cm−1 confirmed the presence of C−Cl group at the terminal of macrochains. In the case of terpolymers, the above-mentioned peaks for ester and imine appeared as symmetric doublet peaks representing successful incorporation of diols in the parent chain. Additional absorption bands related to the functional group present in the diols added to the chain were found in each spectrum in their respective regions, e.g., C−H aliphatic appeared around 3000− 2900 cm−1 in the spectra of material having diols H, Pr, and Si as a part of their chain. Characteristic FTIR absorption peaks for the Si−O−Si group present in PFeSi appeared as a doublet around 1020 and 2900 cm−1. Aromatic C−H appeared around 3100 cm−1in polymers PFeB and PFeF. It is known that the Si−CCO group is very sensitive toward both hydrolysis and alcoholysis occurring readily in the absence of acid/base catalysts. In case of terpolymers based on Si unit the decomposition of material with time was expected as reported earlier. Therefore, these terpolymers have potential as biodegradable.19 There are many factors influencing the hydrolytical stability of PFeSi in the macro chain like steric bulk around the Si or electronegativity of the substituents attached to the Si atoms. An advanced study on the kinetics of hydrolytically degradability will be described in future article. 1 H NMR spectrum of the SB recorded in DMSO exhibited all the characteristic signals confirming the presence of azomethine linkage in the product.16 The spectra of the fePAMEs were recorded under similar conditions. The cyclopentadienyl protons appear in the range 4.30−4.39 ppm and 4.75−4.90 ppm. Some signal broadening in 1H NMR spectra indicated the presence of paramagnetic impurities as a result of oxidation of ferrocene into the ferrocenium ion as reported in literature.16,19 All 1H NMR spectra also exhibited a signal in the range 8.3− 8.7 ppm indicating the presence of HCN proton whereas the resonance 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) showed multiplet signal in the range 2.1 to 2.6 ppm (aliphatic −CH). The terpolymer having diol “B” showed additional signals in the range 7.0−8.0 ppm due to additional aromatic rings present in chain and at 2.3 ppm (−CH3 present in bisphenol). PFeF showed resonance around 7.4 to 8.1 (aromatic rings), slightly deshielded owing to the presence of electronegative F (CF3) in the unit added whereas PSi exhibited additional resonance peaks 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 correlation between the proposed structures and the experimental results. The calculations were made based on the structure of repeat unit present in the polymer chain. The elemental analysis showed that in each polymer, monomers were in equimolar proportion. However somewhat higher contents of carbon found may be because of ferrocenyl acid group which was not included in calculations. In the case of condensation terpolymer containing silanol(Si) in chain, the elemental analysis was not possible because at higher temperatures silicon interact with carbon to form ceramic material.16,19,20 The UV/visible spectra were registered qualitatively in ethanol at room temperature to confirm the presence of different groups in the fe-PAMEs chain as they were soluble in most organic solvents. The material was dissolved in ethanol to make dilute solutions for the study. The absorbance in the range 320−392 nm showed π−π* transition indicative of the presence of Ph−NC−Ph chromophore in the polymer backbone and the bands appearing in the region 418−435 nm are representative of d−d transition in the ferrocenyl group, in all fe-PAMEs.16,21 The bathochromic shifting is due to the extended conjugation, which is possible in PFe, PFeB, and PFeF while it is interrupted by methylene and silyl spacers in PFeH, PFePr, and PFeSi. Molecular Parameter Determination by LLS Technique. Both static and dynamic laser light (LLS) scattering analysis were carried out with, a commercial LLS spectrometer. All LLS measurements were carried out at 25 °C. Five concentrations ranging from 1.20 × 10−3 to 5.12 × 10−3 g/mL were prepared by dilution for the measurements. All polymer solutions were clarified by using a 0.45-m Whatman filter in order to remove dust completely. The angular dependence of the excess absolute time-averaged scattered light intensity, known as the Rayleigh ratio (Rvv(q)) of dilute solution of different concentrations C (g/mL) at different scattering angles were measured.22 By measuring Rvv(θ) at different C and θ, we were able to determine Mw, z-average radius of gyration (Rg) and the A2 from the Zimm plot that incorporate C and θ in a single 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 PFe PFeH PFeB PFeSi PFeF PFePr

Mw (g/mol)

⟨Rg⟩ (nm)

× × × × × ×

80 134 75 145 85 80

1.67 2.1 5.0 3.67 5.2 2.1

104 106 104 106 104 105

A2 (cm3 mol g−2) 3.0 2.0 2.50 1.50 2.40 7.70

× × × × × ×

10−3 10−4 10−3 10−4 10−3 10−4

Rh (nm)

Rg/Rh

50 75 55 85 60 50

1.60 1.78 1.40 1.70 1.33 1.60

a

Estimated uncertainties: 10% in Rg; 5% in Rh, 10% in Mw and 10% in A2. D

dx.doi.org/10.1021/ma400192u | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

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 2 3 4 5 6 7 9

SB PFePr PFe PFeH PFeB PFeSi PFeF Cefixime

8.50 ± 0.1 nil 8.03 ± 0.1 nil 11.2 ± 0.26 nil nil 31 ± 0.9

9.4 ± 0.01 nil nil 11.1 ± 0.1 9.4 ± 0.5 nil 10.1 ± 0.1 36.83 ± 0.72

9.9 ± 0.1 nil nil nil nil nil 15.5 ± 0.15 22.83 ± 0.72

10.03 ± 0.15 Nil nil nil nil nil 18.30 ± 0.26 27.5 ± 0.5

nil nil nil nil nil nil 26 ± 0.5 28.96 ± 0.55

nil nil nil nil nil nil nil 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 2 3 4

PFe PFeH PFeB PFeF

− − − 0.2

− − − 0.4

− − − −

− 1 1 1

− − − 0.4

0.8 − 0.8 −

Table 4. Cytotoxic Activity percentage mortality = (number of live shrimp) control − sample × 100/(number of 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 2 3 4

SB PFe PFePr PFeH

67.8 37.03 80.74 29.62

17.85 33.33 77.77 19.51

7.14 25.92 62.16 14.81

69.27 241.06 0.017

5 6 7

PFeB PFeSi PFeF

42.85 71.42 96.42

3.57 14.28 53.57

25 7.14 17.85

129.26 66.66 7.06

The positive values of A2 indicate that the solvent used is good solvent for the present polymers at 25 °C. The A2 value increases with the decrease in the molecular weight, which indicates that the solvent quality decreases with the increase in the molecular weight.23,24 The correlation functions obtained from dynamic light scattering were analyzed by the constrained regularized CONTIN method.23−27 Distributions of decay rate Γ, converted to distributions of translational diffusion coefficient; D = Γ/q2, where q = (4πn/λ) sin(θ/2), n is the refractive index of the solvent and θ is the scattering angle. Hence, distributions of hydrodynamic radius (Rh) were evaluated using the Stokes−Einstein relationship Rh, = kT/ (6πηD), where k is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent. The hydrodynamic radius Rh reflects the physical dimensions of the polymer chains, as it involves both the chain and the amount of the solvent associated with, whereas the radius of gyration Rg is related to the size of chain and, in other words, to the excluded volume of polymer in the solvent and mostly depends on the thermodynamic quality of the solvent. It is known that the ratio of the radius of gyration to the hydrodynamic radius, i.e., ⟨Rg⟩/⟨Rh⟩, reflects the chain conformation. The ratio of ⟨Rg⟩/⟨Rh⟩ ∼ 1.0−2.0 indicates that the polymers have a coil chain conformation in the solvent used.22−28 Bio Screening. The therapeutic potential of the material reported herein is accessed by selected biological assays. This is an attempt to investigate possible biological activities of synthesized fe-PAMEs which were soluble in common organic solvent. SB and fe-PAMEs comprising of different aliphatic,

aromatic and imine linkage were selected for biological screening. Antibacterial Assay. The bacterial growth inhibition effect of SB and fe-PAMEs was determined by agar well diffusion method. The results obtained from the antibacterial activity for SB and the polymers are summarized in Table 2. The SB showed maximum zone of inhibition against four bacterial strains namely S. typhimurium, B. bronchiseptica, M. leuteus, and S. aureus. PFeF was found to have highest antibacterial activity among all the tested polymers against all the bacterial strains used that might be due to the presence of fluorine based 1,1,1,3,3,3-hexafluorobis(phenol)propane group.1,10,29 It has been reported previously that fluorinated 4-thiazolidinones have good antibacterial activity. PFe showed some activity against one strain used i.e, S. typhimurium; however, PFePr and PFeSi based on flexible spacers were found to be inactive. MIC was determined for the active material, and the data are given in Table 3. On the basis of structure it was found that SB was fairly active antibacterial agent that can be due to the presence of free hydroxyl groups on the aromatic rings. The activity is still enhanced when fluorinated moiety is added to ferrocenated Shiff base. PFe also showed antibacterial activity which contained Schiff base (SB) estirified with ferrocene. The inactivity of PFePr and PFeSi can be due to introduction of nonpolar block. Cytotoxity. The data in case of Brine shrimp (Artemiasalina) lethality assay, tabulated in Table 4 showed that the SB exhibited cytotoxic nature with LD50 69 ppm. PFePr and PFeF having LD50 < 20 shows highly toxic behavior whereas PFeSi showed LD50 in range of 66.66. The material based on E

dx.doi.org/10.1021/ma400192u | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

It can be deduced that the addition of blocks containing non polar part moieties reduce the activity against AT-10. Antioxidant Activity (DPPH Free Radical Scavenging). Antioxidants react with DPPH (a stable free radical) by donating electron or hydrogen thus neutralizing it to yellow colored diphenylpicrylhydrazine. This reduction of DPPH radical by antioxidants can be determined by the decrease in absorbance at 517 nm spectrophotometrically, however, an increase in absorbance will show pro-oxidant activities. SB (IC50 3.15 ppm) has highest antioxidant activity among all tested materials whereas all fe-PAMEs showed good antioxidant behavior as shown in Table 5.34,36 It is evident from literature that most of the Schiff bases exhibited antioxidant activity and also that bearing two hydroxyl groups on the phenyl ring showed excellent antioxidant activity in comparison with ascorbic acid. Overall fe-PAMEs exhibited less antioxidant activity; the reason can be the absence of free hydroxyl radicals on phenyl rings. The activity in fe-PAMEs is due to the ferrocene ring as reported in the literature that poly(Schiff bases) when complexed with ferrocene exhibit antioxidant activity. The results of the DPPH test (Figure 2) showed that

ferrocenyl and imine linkage is known to be more active as compared to those based simple Schiff bases. There was no general trend of cytotoxicity observed by varying the additional group except that the fluorine-containing group showed the more cytotoxic behavior. Moreover the polymeric material was found more active as compare to SB. It means the addition of block esterified ferrocene can enhance the cytotoxic behavior to varying level. The test polymers were most toxic at higher concentrations 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 reliable screen for detecting antitumor agents. As the mechanisms for the assay is quite similar in plants and animals therefore the potato disks exhibit a good correlation in results as compare to the other most commonly used antitumor screening assays. Herein A. tumefaciens, AT-10, was used to induce tumors on potato disks in order to screen soluble SB and fe-PAMEs (Figure 1). SB showed significant antitumor activity 33%

Figure 1. Effect of polymer PFe at different concentrations, on inhibition of tumor formation along with control for comparison. Figure 2. Results of DPPH free radical scavenging assay of FePr at three concentrations of 33.3, 3.33, and 0.33 ppm.

(Table 5). It is reported earlier that ferrocene exhibits inhibition of tumor activity by several mechanisms including the one that is mediated by its effect on immune stimulation, by activating T-lymphocytes.30−35 PFe showed highest tumor inhibition with IC50 7.15 ppm. PFeF having fluorine containing group showed less inhibition. PFeH and PFeSi are tumor inducing at lower concentrations. By varying the third additional moiety “Pr” in the poly(azomethine)esters, there was no trend of variation in inhibition observed. The percentage inhibition of PFe at 100 ppm is 60.76% (Table 5).It is also reported in similar findings that Schiff base esters of ferrocenyl aniline and simple aniline could be regarded as potential anticancer drug candidate. If PFe is compared structurally with Schiff bases then it is observed that the Schiff base activity is enhanced when it is esterified with ferrocene ring containing acid chloride. It is also reported that polyaspartamide-based conjugates featuring ester-linked ferrocene showed active inhibitory role against tumors.31−35 The general tumor inhibition behavior of test compounds based on their structures was SB >PFe > PFeF > PFePr > PFeH > PFeSi.

the activity depends strongly upon the presence of phenolic group but is improved by the influence of ferrocenyl fragment, as reviewed through literature. It is evident that ferrocene had a promising behavior as an antioxidant. The general behavior of scavenging 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 of SB and other synthesized material was investigated in vitro by OH radical induced DNA damage system at 10, 100, and 1000 ppm concentration (Figure 3). Normally pBR322 DNA exists in supercoiled form (SC). With the attack of OH radical (known as most damaging radical for biomolecules) generated from Fenton, SC was broken into open circular (OC).The ability of the test polymers to cleave or protect the super coiling substrate such as plasmid DNA was examined in the DNA damage analysis assay. All of the material showed excellent DNA protecting activity at all the three concentrations tested, i.e., 1000, 100, and 10 ppm none of them exhibited pro-oxidant

Table 5. Tumor Inhibition and Antioxidant Activity (DPPH Free Radical Scavenging) percent tumor inhibition = 100 − average number of sample × 100/average number of tumors of control

percentage scavenging = absorbance of control - absorbance of 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 2 3 4 5 6 7

SB PFePr PFe PFeH PFeB PFeSi PFeF

37.580 27.63 60.757 15.043 39.223 8.030 43.607

32.48 17.10 52.917 13.983 33.683 3.620 28.9

32.55 12.5 47.263 −8.473 11.323 −3.623 12.857

30.20 9.61 21.73 −12.12 11.157 −7.082 7.110

296.82 242.70 7.15 268.52 140.90 459.36 118.05

60.247 30.790 56.327 46.78 29.150 42.953 60.247

26.097 23.68 19.200 15.287 8.397 26.380 26.097

16.877 12.75 5.367 23.417 20.637 16.063 16.877

24.87 77.86 28.55 37.41 82.88 42.72 24.87

F

dx.doi.org/10.1021/ma400192u | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

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

name

1000 ppm

100 ppm

10 ppm

SB PFePr PFe PFeH

+++ + +++ ++

+++ + +++ ++

+++ + +++ ++

5 6 7

name

1000 ppm

100 ppm

10 ppm

PFeB PFeSi PFeF

+++ +++ +++

+++ +++ +++

+++ -

a

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

(SAR) can also be studied. From all the studies mentioned, it was concluded that the incorporation of different units is very useful factor that has influence on the solubility and other properties of the polymeric material.

activity (Table 5). SB fully protected the plasmid DNA from damage. fe-PAMEs also protected the plasmid DNA most of the time at all three concentrations 1000, 100, and 10 ppm (Table 6). Cytotoxic and tumor inhibitory activities might be correlated with the DNA protection activities of the polymers. Available literature regarding the DNA protecting activities of poly(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 potential window showing only anodic peak. These ferrocenyl polymers are found to behave irreversibly in their electrochemical responses scan as reported by our group previously.16

■

ASSOCIATED CONTENT

S Supporting Information *

Synthesis of Schiff base, 1H NMR spectrum of terpolymer PFeF, and representative 1H NMR spectrum of terpolymer PFeB. This material is available free of charge via the Internet at http://pubs.acs.org.

■

■

AUTHOR INFORMATION

Corresponding Author

CONCLUSION A series of soluble ferrocene-poly(azomethine)ester consisting of aromatic and aliphatic spacers were successfully synthesized. Different flexible aromatic and aliphatic segments were incorporated into the fe-PAMEs backbone to prepare their respective organometallic condensation terpolymers to study structure−property relation. A combination of static and dynamic LLS showed that the poly(azomethine)esters and their condensation terpolymers has a coil chain conformation. In biological studies it was found that the fluorinated material was antibacterial with activity comparable to antibiotics used as control. Significant antibacterial, cytotoxic and antitumor activities indicated their potential to be used as antibiotics and anticancerous agents. All polymers were found highly potent antioxidant and more importantly DNA protecting. These polymers can be studied further for pharmacological activities to be used as potential drug candidates. Moreover their mechanism of action and structure activity relationship

*(Z.A.) E-mail: [email protected]. Telephone: +925190642111. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The Department of Chemistry, Quaid-i-Azam University Islamabad, is gratefully acknowledged for laboratory and technical 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. Phytomedicine 2003, 10, 133−138. (3) Kaya, I.; Koça, 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.

G

dx.doi.org/10.1021/ma400192u | Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

Article

(5) Simionescu, M.; Marcu, M.; Cazacu, M.; Racles, C. Eur. Polym. J. 2002, 38, 229−233. (6) Stepnicka, P. Ferrocenes: Ligands, Materials and Bio-molecules: John Wiley and Sons: London, 2008; pp 393−401. (7) Whittell, G. R.; Hager, M. D.; Schubert, U. S.; Manners, I. Nat. Mater. 2011, 10, 176−188. (8) Duru, C. M.; Onyedineke, N. E. J. Am. Sci. 2010, 6 (6), 119−122. (9) Zaheer, M.; Shah, A.; Akhter, Z.; Qureshi, R.; Mirza, B.; Tauseef, M.; Bolte, M. Appl. Organomet. Chem. 2011, 25 (1), 61−69. (10) Sultan, M. T.; Butt, M. S.; Anjum, F. M.; Jamil, A.; Akhter, S.; Nasir, M. Pak. J. Bot. 2009, 41 (3), 1321−1330. (11) Wenz, G.; Steinbrunn, M. B.; Landfester, K. Tetrahedron. 1997, 53, 15575−15592. (12) Haraha, A. Adv. Polym. Sci. 1997, 133, 92−141. (13) Whang, D.; Kim, K. J. Am. Chem. Soc. 1997, 119, 451−452. (14) Grigoras, M.; Farcas, A. J. Optoelectron. Adv. Mater. 2000, 525− 530. (15) Armarego, W. L. F.; Chai, C. L.; Purification of Laboratory Chemicals; Butterworth Heinenann: London. 2003. (16) Gul, A.; Akhter, Z.; Bhatti, A.; Siddiq, M.; Khan, A.; Siddiqe, H. M.; Janjua, N. K.; Shaheen, A.; Sarfraz, S.; Mirza, B. J. Organomet. Chem. 2012, 719, 41−53. (17) But, M. S.; Akhter, Z.; Zafar-ul-zaman, M.; Siddiqe, H. M. Colloid polym. Sci. 2008, 287, 1455−1461. (18) Iwan, A.; Sek, D. Prog. Polym. Sci. 2008, 33, 289−345. (19) Cazacu, M.; Munteanu, G.; Racles, C.; Vlad, A.; Marcu, M. J. Organomet. Chem. 2006, 691 (17), 3700−3707. (20) Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Introduction to Spectroscopic, 3rd ed.; Thomson Learning Inc.: New York, 2001. (21) Akhter, Z.; Khan, M. S.; Bashir, M. A. Appl. Organometal. Chem. 2005, 19, 848−853. (22) Chu, B., Laser Light Scattering, 2nd ed., Academic Press: New York, 1991. (23) Zimm, B. H. J. Chem. Phys. 1948, 16, 1099. (24) Siddiq, M.; Wu, C.; Shuqin, B.; Chen, T. Macromolecules 1996, 29, 3157−3160. (25) Pecora, R.; Berne, J. Dynamic Light Scattering: Plenum Press: NY, 1976. (26) Khan, A.; Farooqi, Z. H.; Siddiq, M. J. Appl. Polym. Sci. 2012, 124, 951−957. (27) Stockmayer, W. H.; Schmidt, M. Pure Appl. Chem. 1982, 54, 407. (28) Stockmayer, W. H.; Schmidt, M. Macromolecules 1948, 17, 509. (29) Kazakov, S. V.; Galaev, I. Yu.; Mattiasson, B. Int. J. Thermophys. 2002, 23. (30) Racles, C.; Cozan, V.; Sajo, I. High Perform. Polym. 2007, 19, 541−552. (31) De Souza, A. C.; Pires, A. T.; Soldi, V. J. Therm. Anal. Calorim. 2002, 70, 405. (32) Eldes, I. High Perform. Polym. 2002, 14, 397. (33) Kannan, P.; Raja, S.; Sakthive, P. Polymer 2004, 45, 7895−7902. (34) Shah, A.; Zaheer, M.; Qureshi, R.; Akhter, Z.; Nazar, M. F. Spectrochim. Acta, Part A 2010, 75, 1082−1087. (35) Shah, T. J.; Desai, V. A. ARKIVOC xiv 2007, 218−228. (36) Chen, A. S.; Taguchi, T.; Aoyama, S.; Sugiura, M.; Haruna, M.; Wang, M. W. Free Radical Biol. Med. 2003, 35 (11), 1392−1403. (37) Aanandhi, M. V.; Mansoori, M. H.; Shanmugapriya, S.; George, S.; Shanmugasundaram, P. Res. J. Pharm. Biol. Chem. Sci. 2010, 1 (4), 1083−1089. (38) Cacic, M.; Molnar, M.; Sarkanj, B.; Schon, E. H.; Rajkovic, V. Molecules 2010, 15, 6795−6809. (39) Milaeva, E. R.; Filimonova, S. I.; Meleshonkova, N. N.; Dubova, L. G.; Shevtsova, E. F.; Bachurin, S. O.; Zefirov, N. S. Bioinorg.Chem. Appl. 2010, 2010, 165482. (40) Kovjazin, R.; Eldar, T.; Patya, M.; Vanichkin, A.; Lander, H. M.; Novogrodsky, A. Fed. Am. Soc. Exp. Biol. J. 2010, 17, 467−469. (41) Top, S.; Dauer, B.; Vaissermann, J.; Jaouen, G. J. Organomet. Chem. 1997, 541, 355−361.

H

dx.doi.org/10.1021/ma400192u | Macromolecules XXXX, XXX, XXX−XXX