RESEARCH ARTICLE International Research Journal of...

Venkatesan et al., 2016/ Biological activity of coumarin based methacrylate copolymer

International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 3 (4): 1-12 1

RESEARCH ARTICLE

International Research Journal of Pharmaceutical and Biosciences

Pri -ISSN: 2394 - 5826 http://www.irjpbs.com e-ISSN: 2394 - 5834

SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITY OF COUMARIN BASED METHACRYLATE

COPOLYMER

S. Venkatesan1, B. Ranjithkumar1, S. Rajeshkumar2, K. Anver Basha1*

1,*P.G. & Research Department of Chemistry, C. Abdul Hakeem College, Hakeem Nagar, Melvisharam-632 509,

Tamil Nadu, India.

1. 2School of Bio-Sciences and Technology, VIT University, Vellore, Tamil Nadu, India.

Article info Abstract

Article history: Received 12 APR 2016 Accepted 24 MAY 2016

* Corresponding author

[email protected];

Mobile: +91-9894066901; Fax: 04172 - 266487.

Copyright 2016 irjpbs

Our ongoing investigations on the synthesis, characterization, thermal stability and antibacterial activity of coumarin based copolymer. 7-Methacryloyloxy-4-methylcoumarin (MAOMC) was synthesized by reacting 7-hydroxy-4-methyl coumarin with Methacryloyl chloride. The monomer was characterized by Fourier transform infrared spectroscopy (FT-IR), 1H-NMR, 13C-NMR spectroscopy. Copolymers of MAOMC with Glycidylmethacrylate(GMA) at different feed composition was prepared by free radical solution polymerization at 70±1°C using Benzyl peroxide(BPO) as an initiator and Ethylmethyketone (EMK) as a solvent. The copolymers were characterized by FT-IR,1H-NMR spectroscopy. Thermogravimetric analysis (TGA) of polymers carried out in a nitrogen atmosphere showed moderate thermal stability in the region 30 to 300ºC. As, Antibacterial activity of the polymers prepared was also investigated against the selected pathogenic bacteria’s. The antibacterial activity of the copolymer increases as the MAOMC content increases in the copolymer. This shows that coumarin moiety place very important role as antimicrobial agent. Keywords:7-Methacryloyloxy-4-methylcoumarin, Copolymerization, Thermogravimetric analysis, Antibacterial activity.



INTRODUCTION Functional polymers are macromolecules to which chemically bound functional groups are attached which can be utilized as reagents, catalysts, protecting groups, etc. The polymer properties can be modified either by chemical reactions on pendant groups or by changing the physical nature of the polymers, such as their physical form, porosity and solvation behaviour. Such properties are importance for the functionalization reactions and eventual applications of the reactive polymers. Functionalised polymers can be prepared by chemical modification of polymers either under classical conditions or using phase transfer catalysis technique. A functional polymer possesses the combination of the physical properties of the polymer support and the chemical reactivities of the attached functional group. The use of a functional polymer depends on the physical properties and the chemical constitution of the polymers. Coumarins, known as benzopyran-2-ones, form an elite class of naturally occurring compounds that possess promising therapeutic perspectives. Due to diversity in their structural complexity, these range from simple substituted coumarins to polysubstituted polycyclic/fused coumarins. These belong to the flavonoid class of plant secondary metabolites and have a variety of biological activities, usually associated with low toxicity [1,2]. Coumarin and Coumarin-related compounds have proved for many years to have significant therapeutic potential and are a plentiful source of potential drugs candidate in relation to its safety and efficacy. Hydroxy derivatives of 4-methyl coumarin are important group of coumarin derivatives showing medicinal as well as other applications, as optical brightening agents, dispersed fluorescent and tunable dye lasers [3]. The 7-hydroxy-4-methyl coumarin is used as fluorescent brightener, and efficient dye lasers [4]. 7-Hydroxycoumarin is a widely used structure in introducing the coumarin unit into polymer [5,6]. Although there are a huge number of reports on monomeric coumarin derivatives, there are only a few reports on coumarin polymers [7, 8]. Coumarin polymers have not received much attention in the literature. But the reported coumarin polymers possess variety of functions and application in many fields, such as biochemicals, electro optical materials, organic-inorganic hybrid materials, liquid crystalline materials, and light harvesting/energy transferring materials [9-11]. These polymers were mostly linear polymers [12] and the coumarin functional group was introduced as a pendent group or terminal group of the polymer chain [13-16]. One method of achieving antimicrobial polymers is the preparation of polymerizable monomers containing biocide moieties and then polymerizing subsequently or copolymerizing with other monomers. There are reports on the antifungal activities on monomeric coumarin, although works on coumarin polymers are rare. Since, coumarin and its derivatives have attracted considerable interest because of various physiological and biochemical properties. Also the use of antimicrobial polymers offers promise for enhancing the efficacy of some existing antimicrobial agents and minimizing the environmental problems accompanying



conventional antimicrobial agents by reducing the residual toxicity of the agents, increasing their efficiency and selectivity, and prolonging the lifetime of the antimicrobial agents. Kenawy and coworkers discussed on the requirements of antimicrobial polymers, factors affecting the antimicrobial activities, methods of synthesizing antimicrobial polymers, major field of application, and future and perspectives in the field of antimicrobial polymers in their review article [17]. Brahmbhatt et al. prepared poly (3-phenoxycoumarin ethylene) s and determined their toxicity effect on various fungal and bacterial strains [18]. Polymer supports based on glycidyl methacrylate (GMA) are mainly used as excellent thermosetting adhesives that have gained popularity because of their superior performance in many publications such as binding of drugs and bio molecules [18] and in electronic industries as negative electron beam resists [19]. The presence of the epoxy group in GMA allows modification of the copolymers for various applications. They serve as basic materials for the formulations of the base and topcoats in the leather industry [20,21].GMA basedpolymers have drawn special interest due to their superiorperformance in more specific applications, like drug delivery and biomolecular binding [22]. Thus, the copolymers ofGMA with other acrylates are useful materials for differentspecialty applications. Due to various physiological and biochemical properties of coumarin, our interest was to synthesize and developed polymer containing coumarin moiety. With this view, the synthesis of MAOMC and itscopolymerization with GMA by free radical solution polymerization was under taken.The prepared monomer and polymers were characterized by FT-IR and 1H-NMR and 13C-NMRspectroscopy. The thermal properties of the polymers were analyzed by TGA. Antibacterial activity of the different copolymers was carried out against selectedbacteria stain likeBacillus subtilis, Klebsiella pneumoniaeand Klebsiella planticola. MATERIALS AND METHODS Methacryloyl Chloride was prepared from methacrylic acid and benzoyl chloride using the procedure of Stempel et al[23].Glycidylmethacrylate(Sigma-Aldrich)was then distilled in an atmosphere of nitrogen and reduced pressure. It was then collected in a clean dry amber colored bottle and kept in the refrigerator at 5oC. Benzoyl peroxide (BPO) was recrystallized from chloroform-methanol (1:1). The AR grade MEK, hexane, chloroform, and methanol were purified by the conventional methods.Triethylamine was dried using sodium metal. Bacterial medium Muller Hinton Agar was purchased from Himedia, Mumbai. Measurements FT-IR spectrum of the intermediates and copolymer were recorded on Perkin-Elmer FT-IR spectrophotometer with KBr pellets. 1H NMR spectra and 13C NMR spectra were recorded in a BRUKER 500 MHz using d6-DMSO solvent. The number average and weight avarage (Mn and Mw) of the copolymer were measured by gel permeation chromatography (GPC) using liquid chromatograph Waters 2690 on THF as on eluent, which was calibrated with polystyrene standard. Thermogravimetric analysis (TGA) was performed with TA



instruments His Res TGA 2950 thermal analyzer at a heating rate of 10°C min-1 under static air atmosphere. Synthesis of 7-Methacryloyloxy-4-methylcoumarin (MAOMC) Monomer The monomer, 7-Methacryloyloxy-4-methylcoumarin (MAOMC), was synthesized through the following reaction procedure. 7-Hydroxy 4-methyl Coumarin (5g, 0.033 mol) and triethylamine (6.9 ml, 0.049 mol) were taken in a three-necked RB flask fitted with a magnetic stirrer and a dropping funnel. To this mixture, 300 ml of MEK was added and placed in an ice bath. Methacryloyl chloride (3.87 ml, 0.039 mol) dissolved in MEK (25 ml) was added dropwise into the flask maintaining the temperature below 0- 5 oC. The reaction was allowed to proceed for 1h at 0- 5 oC with constant stirring. Then the ice bath was removed and the reaction mixture was stirred for another 2h at room temperature. The reaction mixture was washed with water and followed by washing with 1N NaOH solution for removal of the traces of methacryloyl chloride. The organic layer was passed through anhydrous Na2SO4 and dried. The crude product was purified by recrystallization from methanol. The product was found to be white crystals (Scheme II).The yield was 93%.

+

HO O

CH3

TEA/EMK

0-50C

O O

CH3

O

O

Cl

O

Methacryloyl choloride 7-Hydroxy-4-methyl coumarin

7-Methacryloxy-4-methyl coumarin(MAOMC)

O

Scheme 1. Synthesis of 7-Methacryloyloxy-4-methylCoumarin (MAOMC) Monomer

IR spectrum of MAOMC ( KBr, cm-1): 3433(-CH stretching vibration of the aromatic ring), 3061 (-CH3), 1726 (broad, C=O of acrylate and of coumarin moiety), 1618 (C=C), 1265 (asymmetric C-O-C), 1141 (symmetric C-O-C), 890 (-CH bending mode of vinyl group), 749 (rocking mode of vinyl group). 1H NMR (δ ppm) (60MHz): 2.17 (1H, -CH=), 1.75 (3H, CH3), 5.8 and6.3 (2H, non-equivalent methylene protons), 7.08-7.65 (3H, aromatic protons). Copolymerization of (MAOMC-Co-GMA): Copolymers of MAOMC with GMA having different compositions were synthesized by free radical polymerization in EMK solvent using BPO as a free radical initiator were taken in a polymerization tube and the reaction was carried out under nitrogen atmosphere at 70±1ºC with stirring. After desired time (<10% conversion), the polymer was precipitated by pouring the contents into an excess of methanol. The precipitated polymer was filtered and washed with methanol and were further purified by repeated precipitation by hexane from the CHCl3 solution and finally dried. (Scheme III (b))



OO

CH3

O O

n

OO

O

m

O O

CH3

O

7-Methacryloxy-4-methyl coumarin(MAOMC)

OBPO/EMK

70±10C

(b)

+

poly(MAOMC-CO-GMA)

OO

O

Glycidylmethacrylate

Scheme 2. Synthesis of Copoly(MAOMC-Co-GMA)

Solubility studies Solubility of the copolymer was tested in various polar and non-polar solvents. About 5-10 mg of the copolymer was added to about 2ml of different solvents in a test tube and kept overnight. The solubility of the polymers was noted after 24 hrs. Antibacterial Activity The antibacterial activity of isolated MAOMC-Co-GMA Polymer was tested by agar disc diffusion method. In the Muller Hinton Agar medium the test bacterial Bacillus subtilis, Klebsiella pneumoniae and Klebsiella planticola were included in this study to assess the susceptibility pattern of the compounds. Different ratio of MAOMC-Co-GMA polymers (20:80, 50:50 and 80:20) and different concentrations of the polymers like 10 μl of the diluted compounds were pipette out and immerse the disc respectively, allow to wet-dry and placed disc over the medium, then the plates were incubated at 37ºC for 24 -48 hours for observing zone of inhibition rate. RESULTS AND DISCUSSION Solubility The solubility of the newly prepared copolymers in various polar and non-polar solvents was tested at room temperature. The copolymers were easily soluble in various solvents such as chloroform, dichloromethane, dimethylformamide, dimethylsulfoxide, ethylmethylketone, toluene and acetone etc., Spectroscopic Analysis Spectroscopic Analysis of Monomer -7-Methacryloyloxy-4-methylcoumarin (MAOMC) FT-IR spectrum of MAOMC is shown in Figure 1. The absorption at 3061(-CH stretching vibration of the aromatic ring), 2982(-CH3), 1726 (broad, C=O of methacrylate and of coumarin moiety), 1618 (C=C), 1265 (asymmetric C-O-C), 1141 (symmetric C-O-C), 880 (-CH bending mode of vinyl group), 749 (rocking mode of vinyl group).



Figure 1 FT-IR Spectrum of 7-Methacryloyloxy-4-methylcoumarin (MAOMC). 1H NMR of MAOMC is shown in Figure 2. (δ ppm) (500MHz): 2.17 (3H, -CH3in methacrylate unit), 1.75 (3H, CH3in coumarin moiety), 6.3 and 5.8 (2H, non-equivalent methylene protons), 7.08-7.65 (3H, aromatic protons).

Figure 2 1H-NMR Spectrum of 7-Methacryloyloxy-4-methylcoumarin (MAOMC).

13C-NMR of MAOMC is shown in Figure 3 (δ ppm) (500MHz): 160.5(C=O of ester group of coumarin), 165.1 (C=O of ester of methacrylate), 135.3(CH2=), 114.5-155.2(benzene ring carbons), 18.7(CH3- in coumarin moiety), 18.3(CH3- in methacrylate)



Figure 3 13C-NMR Spectrum of 7-Methacryloyloxy-4-methylcoumarin (MAOMC).

Spectroscopic Analysis of Copolymer (7-Methacryloyloxy-4-methylcoumarin-Co-Glycidylmethacrylate): The FT-IR spectrum of the copolymer, poly (MAOMC-co-GMA) is shown in Figure 4. The spectrum confirms the copolymer in all aspects. poly (MAOMC-co-GMA) shows bands at 2956 and 2934 cm-1 may be attributed to the asymmetric and symmetric νC-H stretching vibration of methylene group, whereas the absorption at 1448 cm-1 is due to the bending vibration of ν-CH group. The strong absorptions at 1368 cm-1 and 1328 cm-1 related to the νC-H

stretching vibrations of CH3 and CH2 groups in both the GMA an MAOMC units. The broad band at 1732 cm-1 is assigned to νC=O stretching vibration of the ester group of the copolymer. A sharp band at 1261 cm-1 and a broad band at 1130 cm-1 are assigned νC-O-C

symmetric and asymmetric stretching vibration of the ester group.

Figure 4.FTIR Spectrum of Copoly(MAOMC-Co-GMA)



1H-NMR Spectrum of poly (MAOMC-co-GMA) is shown in Figure5. (δ ppm) (500 MHz): The signals at 7.2-7.6ppm are due to aromatic protons and the signal at 7.54ppm is for non equivalent proton of aromatic ring. The signals of –CH2 protons of GMA unit appeared at 3.4ppm. The two signal at3.1 and 2.8pm due to epoxy group CH2. The –CH3 proton of MAOMC and GMA shows signal at 1.4ppm. The formation of copolymer was evident from the disappearance of signals at 6.3 (2H, CH2=) and appearance of broad signals at 1.5ppm, due to CH2 backbone chain (2H, -CH2), its conformed formation of copolymer.

Thermal degradation of copolymers The thermal behaviour of copolymers was characterized using TG traces. The TGA curves (Figure. 6) clearly indicates that all the copolymers undergo two step degradation pattern. The first degradation is below 100oC which suggests thatit’shave some impurity or moisture. Different copolymer ratio (20:80, 80:20) exhibit excellent thermal stability with decomposition temperatures varying from 60 to 290oC. Thermal stability of the copolymer increases as the coumarin moiety increased[24].



Figure 6. TGA Spectrum of Copoly(MAOMC-Co-GMA) a) 20:80 b) 80:20.

The Figure 7 shows the antibacterial activity of different ratio of Copoly(MAOMC-Co-GMA) on pathogenic and opprtuneic bacteria. The inhibition zone was observed against Bacillus subtilis, Klebsiella pneumoniaeand Klebsiella planticola. In the quantitative assay 10μl of 20:80, 50:50 and 80:20 ratio of Copolymer were added in three respective wells. The zone of inhibition was increased when increasing the polymer concentration. The different ratio was applied for the antibacterial activity in that, 80:20 ratio giving the good results when compare to others shown in Figure 8. The increasing of polymer concentration may augment the killing of microorganisms [25]. In the all bacterial strains the maximum inibitiory activity was observed on the pathogenic bacteria Bacillus subtilisK. planticola and thenK. pneumoniae. Finally the pneumonial fever causing K. pneumoniae having the minium level of zone of inhibition. The results suggests that the Coumarin based copolymer was vigorously involved for the antibacterial activity will used for the antimicrobial based applications.

Figure 7. Zone of inhibitionCopoly(MAOMC-Co-GMA)



Figure 8. Antibacterial activityof Copoly(MAOMC-Co-GMA)

CONCLUSION The Coumarin methacrylate based copolymer has been successfully synthesized by free radical polymerization. The synthesised monomer and copolymer were characterizedby FT-IR, 1H NMR, and 13CNMR. The TGA analysis results support the copolymer havinggood thermal stability,the results show that 80:20 ratio is thermally more stable than the otherThe antibacterial activity studies against different bacterial stains were reported. The good zone of inhibition was observed for the copolymer of ratio 80:20, which exhibited remarkable antibacterial activities against disease causing bacterial isolates such as B. subtilis, K. pneumoniae, and K. planticola.The result implies increases inhibition with the increase of MAOMC content in the copolymer. REFERENCES [1] Borges, F., Roleira, F., Milhazes, N., Santana, L. and Uriarte, E. 2005.Simple coumarins

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http://pubs.acs.org/doi/abs/10.1021/ma060269o





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