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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Quinoline based polymeric drug for biological applications: synthesis, characterization, antimicrobial, and drug releasing studies a
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P. Uma , J. Suresh , Revathy Selvaraj , S. Karthik & A. Arun
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PG & Research Department of Chemistry, Government Arts College, Thiruvannamalai 606603, Tamil Nadu, India Published online: 27 Nov 2014.
Click for updates To cite this article: P. Uma, J. Suresh, Revathy Selvaraj, S. Karthik & A. Arun (2015) Quinoline based polymeric drug for biological applications: synthesis, characterization, antimicrobial, and drug releasing studies, Journal of Biomaterials Science, Polymer Edition, 26:2, 128-142, DOI: 10.1080/09205063.2014.985022 To link to this article: http://dx.doi.org/10.1080/09205063.2014.985022
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Journal of Biomaterials Science, Polymer Edition, 2015 Vol. 26, No. 2, 128–142, http://dx.doi.org/10.1080/09205063.2014.985022
Quinoline based polymeric drug for biological applications: synthesis, characterization, antimicrobial, and drug releasing studies P. Uma, J. Suresh, Revathy Selvaraj, S. Karthik and A. Arun* PG & Research Department of Chemistry, Government Arts College, Thiruvannamalai 606603, Tamil Nadu, India
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(Received 20 July 2014; accepted 4 November 2014) Novel acrylate monomer of quinoline-based chalcone 1-(4-(7-chloroquinolin-4-ylamino)phenyl) acrylate (CPA) was synthesized using (4-(2-chloroquinolin-5-ylamino) phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (CPE) and acryloyl chloride. CPA is characterized by different techniques like IR, 1H NMR and UV–visible spectrometry techniques. Poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA) are prepared by solution polymerization technique using CPA, acrylic acid (AA) and hydroxyethylacrylate (HEA), respectively. The antimicrobial activities of the compounds are tested using four different micro-organisms. In vitro cumulative drug release studies are done using UV visible spectroscopic technique. The molecular weights of these polymers are found to be around 5000 g/mol. The synthesized polymers showed two stages of thermal decomposition temperature centred around 220 and 350 °C, respectively. The antimicrobial activity of the polymer sample is found to be very high and especially for gram-negative bacteria with a minimum value of 3.91 μg/ mL. The in vitro drug-releasing rate is dependent on the comonomer, pH and temperature of the medium. Keywords: chalcone; acrylate; antimicrobial activity; drug releasing study
1. Introduction The uses of polymeric systems having pharmacological activity provide very good local activities, reducing the toxicological risks and in addition act as a release system of the pharmacological active residue which is controlled by chemical reactions, mainly hydrolytic under enzymatic and non-enzymatic processes. Generally, drug conjugates are usually prepared by esterification, acrylation or alkylation. By attaching bioactive substrates to the synthetic or naturally occurring macromolecules, it is expected to increase the therapeutic efficiency while lowering their toxicity. Generally speaking, the activity of the polymeric drug is related to the functional group and the nature of the polymeric substance. According to Vogl and Tirrell [1], the introduction of hydrophilic, hydrophobic or polyelectrolytic moieties will enhance the activity of the drug on micro-organisms. Also, it was observed that poly(methacrylic acid) has no effect on the micro-organisms like Escherichia coli. Incorporating hydroxyl or carboxylic groups in the polymer backbone could, in fact, increase the hydrophilic character of the polymeric drug. Most of the synthesized polymeric drugs having high-hydrophilic character were produced by copolymerising the drug conjugates with acrylic acid as a *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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hydrophilic part. The advantage of acrylic acid as a comonomer in a polymer chain bearing active drug moiety is its ability to assist the cleavage of amide or ester linkage through neighbouring group participation mechanism. These properties make such systems quite interesting and provide longer delivery times with lower dosages. Chalcones are condensed products of the substituted aromatic aldehydes with simple or various substituted acetophenones in the presence of alkali. Compounds with the backbone of chalcone possesses various biological activities such as anti-microbial, [2] anti-inflammatory,[3] analgesic,[4] anti-ulcerative,[5] immune-modulatory,[6] antimalarial,[7] anti-cancer, anti-viral,[8] anti-leishmanial,[9] anti-oxidant,[10] antiplatelet and[11] anti-hyperglycemic.[12] The presence of the reactive keto group and the vinylenic group in the chalcone and their analogues possesses the antioxidant activity.[13] Similar to chalcones, quinolines are also an important class of compounds because of their bioactive properties and medicinal uses such as anti-malarial,[14] anti-inflammatory [15] and tyrosine kinase inhibiting agents.[16] The synthesis of quinolinyl chalcones is scarily reported in literature [17,18] and is generally synthesized by ClaisenSchmidt condensation reaction. Dominguez et al. [19] have reported the synthesis of some quinolinyl chalcones and claimed that their compounds showed anti-malarial activity. Moussaoui et al. [20] have also described the synthesis and cytotoxicity effect of quinolinyl chalcones upon K 562 human leukaemia cell lines. Chloroquine and other quinolines had been served as anti-malarial chemotherapy for more than 40 years. The achievement of these drugs is based on excellent clinical efficacy, limited host toxicity, ease of use and simple cost-effective synthesis.[21] The 7-chloroquinoline moiety, a pharmacophore present in several established anti-malarials such as chloroquine and amodiaquine is effectively bound to heme and consequently inhibits the hemozoin formation. The present study is also aimed at exploiting this feature by synthesis chalcone using 7-chloroquinoline as a base compound. In general, polymeric drug based on acrylates has a higher activity against ATCC bacterial strains.[22] Therefore, it will be interesting to combine the effect of quinoline and acrylate in the same platform to see how it shows antibacterial properties. For that reason, we investigated the biological activity of the chalcone polymer containing chlorine as a biologically active site. In this article, we have reported a synthesis, antimicrobial and drug releasing behaviour of acryalated derivative (CPA) of chalcone (CPE) based on 4,7-dichloroquinoline and their polymers. Antimicrobial activities of CPE, CPA, Poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA) are tested on four different bacterial strains. Also, in vitro drug releasing behaviour of the polymer film is determined using UV–visible spectroscopic method. 2. Materials and methods 4-Hydroxybenzaldehyde and acrylic acid is used as such received from Aldrich Chemicals. 4,7-Dichloroquinoline and 1-4-aminophenylethanonen are received from Merck chemicals. Triethylamine (TEA) was received from SD Fine Chemicals. Ethylmethylketone (MEK), acrylic acid (AA), hydroxyethylacrylate (HEA) and acroloylchloride are received from Merck and used as such. ALPHA BRUKER FT-IR spectrophotometer is used for recording IR spectra using KBr pellet method. 1H NMR spectra of the samples were run on a Bruker FT-NMR spectrophotometer operating at 500 MHz’s using CDCl3 as a solvent and tetramethylsilane (TMS) as an internal reference. UV- absorption measurements were recorded using LABINDIA model UV 320 instrument by dissolving polymer samples in Tetrahydrafuran (THF). 1-(4-(2 Chloroquinolin-5-ylamino)
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phenyl)ethanone (CE) is synthesized using 4,7-dichloroquinoline and para-amino-acetophenone. Thermal analysis of the polymers is done using a Mettler 3000 thermal analyser at the heating rate of 10 °C/min in the air. The molecular weight of the polymer was obtained using a Schimadzu instrument with THF as an eluent at a flow rate of 0.3 mL/min. Muller-Hinton broth and Muller-Hinton agar were obtained from Himedia and used as such. MTCC stains of gram-negative bacteria (Escherichia coli (739), Pseudomonas aeruginosa (424)) and gram-positive bacteria (Staphylococcus aureus (3381), Bacillus cereus (430)) are obtained from the Christian Medical College (CMC), Vellore, India and used for culturing the bacteria.
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2.1. Synthesis of 1-(4-(2chloroquinolin-5-ylamino)phenyl) ethanone (CE) In a 500 mL round-bottom flask fitted with a reflux condenser provided with a magnetic stirrer, 20 g (0.11 mol) of 4,7-dichloroquinoline and 14.8 g (0.11 mol) of paraamino acetophenone are added and dissolved completely. To the reaction mixture, methanol was added and the reaction mixture was stirred for 6 h at 65 °C. The solvent was evaporated under vacuum and solid mass was obtained. The solid mass was recrystalized from methanol to obtain the compound as a yellow solid. Yield: 85%. Molecular weight is 296 g/mol and melting point is 160–162 °C. 2.2. Synthesis of 1-(4-(2-chloroquinolin-5-ylamino) phenyl)-3-(4-hydroxyphenyl) prop-2-en-1-one (CPE) Into a conical flask containing 30 mL of ethanol, 0.04 mol (11 g) of substituted quinoline and 0.04 moles (5 g) of 4-hydroxybenzaldehyde are added and stirred. With this solution, 4 g of sodium hydroxide dissolved in 20 mL of distilled water is added drop wise at room temperature. The contents of the flask are stirred for 20 h at room temperature. The mixture is then neutralized using dilute hydrochloric acid and the formed precipitate is filtered out and recrystalized from methanol. Yield: 75%. Molecular weight is 400 g/mol and melting point is 168–170 °C. 1H NMR (δ, ppm): 9.0–7.7 (13H, m, aromatic CH) and 7.0 (1H, d, CH present in the chalcone), 6.9 (1H, d, CH present in the chalcone) and 3.9 (1H, b, NH proton). FT-IR (cm−1) 3184 (–OH), 3030 (aromatic CH-stretching), 2881 (aliphatic CH-stretching), 1672 and 1646 (cis and trans C=O) and 1609 (aliphatic CH=CH). 2.3. Synthesis of 1-(4-(7-chloroquinolin-4-ylamino) phenyl) acrylate (CPA) 0.0123 mol (5 g) of CPE dissolved in 500 mL of MEK is taken into a 500 mL roundbottom flask fitted with a dropping funnel and mechanical stirrer. 0.012 mol (0.92 g) of TEA is added into the content of the flask. 0.012 mol (1.25 mL) of acryloyl chloride in 20 mL of MEK is taken into a dropping funnel and added drop by drop with constant stirring into the round-bottom flask which is kept in an ice bath. The addition of acryloyl chloride is maintained in such a way that the temperature of the reaction is kept under 0–5 °C. The reaction mixture is stirred at room temperature for 3 h. The precipitate (triethyl amine hydrochloride) is filtered and washed thoroughly with MEK. The filtrate is then taken into a 500 mL separating funnel, washed with 100 mL of distilled water taken in three portions and the organic layer is dried using anhydrous sodium sulphate. The product thus obtained by evaporating the solvent. The crude solid product was recrystallised using methanol. Yield: 74%; Mol.wt: 455.5 g/mol; 1H NMR (δ, ppm):
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9.0–7.5 (13H, m, aromatic CH), 6.9 (2H, d, CH present in the chalcone), 6.3 (2H, d, CH2 of acrylate), 6.6 (1H, t, CH of acrylate) and 3.8 (1H, b, NH proton). FT-IR (cm−1): 3413 (Ar-NH-Ar), 3030 (aromatic CH-stretching), 2897 (aliphatic CH-stretching), 1725 (–C=O of ester), 1677 & 1646 (cis and trans C=O) and 1619 (vinylic).
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2.4. Synthesis of pol(CPA) CPA (1.0 g) and 0.03 g of BPO (3 wt.%) are placed in a polymerization tube containing 10 mL of MEK. The polymerization tube was degassed using nitrogen gas and heated at 70 ± 1 °C in a thermostatic water bath for 24 h. Poly(CPA) is precipitated by adding the reaction mixture to a large excess of methanol with rapid stirring. Yield = 0.9 g (90%). 1H NMR (δ ppm): 9.0–7.6 (13H, m, aromatic CH), 7.0 (2H, d, CH present in the chalcone), 3.7 (1H, b, NH proton) and 0.9–1.3 (3H, m, aliphatic CH). FT-IR(cm−1): 3413 (Ar-NH-Ar), 3030 (aromatic CH-stretching), 2962 (aliphatic CH-stretching), 1742 (–C=O of ester), 1697 and 1646 (cis and trans C=O) and 1619 (aliphatic CH=CH). 2.5. General procedure for the preparation of copolymer Copolymerization is done using a solution polymerization technique. Equimolar mixture of monomer 1 and monomer 2 were taken in a polymerization tube containing MEK as a solvent and BPO (2wt.%) as a free radical initiator. The reaction medium was made inert by passing the nitrogen gas through the inlet of the polymerization tube. The polymerization is then carried out at 70 ± 1 °C for 24 h. The polymer is then precipitated using methanol and the precipitate obtained was then filtered in sintered crucible, washed with methanol and weighed. This method is used for the preparation of the following copolymers. 2.5.1. Synthesis of poly (CPA-co-AA) (0.004 mol) 1.38 g of CPA and (0.004 mol) 0.28 g of AA, 10 mL of MEK and 0.02 g (2wt.%) of BPO are used for the synthesis of poly (CPA-co-AA). Yield = 0.88 g. 1 H NMR (δ, ppm): 8.9–7.4 (13H, m, aromatic CH), 7.1 (1H, d, CH present in the chalcone), 7.0 (1H, d, CH present in the chalcone), 3.7 (1H, b, NH proton) and 1.0–2.5 (6H, m, aliphatic). FT-IR (cm−1): 3413 (Ar-NH-Ar), 3030 (aromatic CH-stretching), 2962 (aliphatic CH-stretching), 1742 (–C=O of ester), 1649 (C=O) and 1618 (aliphatic CH=CH). 2.5.2. Synthesis of poly (CPA-co-HEA) (0.004 mol) 1.38 g of CPA and (0.004 mol) 0.46 g of HEA, 10 mL of MEK and 0.02 g (2wt.%) BPO are used for the synthesis of poly(CPA-co-HEA). Yield = 1.0 g. 1 H NMR (δ, ppm): 8.8–7.5 (13H, m, aromatic CH), 7.1 (1H, d, CH present in the chalcone), 7.0 (1H, d, CH present in the chalcone), 4.6 (1H, b, OH proton), 3.9 (1H, b, NH proton), 3.6 (2H, d, CH2 connected to OH), 3.4 (2H, d, CH2 connected to ester) and 1–2.5 (6H, m, aliphatic). FT-IR (cm−1): 3336 (–OH), 3036 (aromatic CH-stretching), 2986 (aliphatic CH-stretching), 1740 (–C=O of ester), 1670 (C=O) and 1618 (aliphatic CH=CH).
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2.6. Drug susceptibility test The drugs were tested by the disc–diffusion method. Diluted bacterial cultures (100 mL) were spread on sterile Mueller-Hinton agar plates, after which 8 mm diameter discs (sterile blank) impregnated with the drug to be tested, were placed on the plates. The plates were incubated for 24 h at 37 °C under aerobic conditions and the diameter of the inhibition zone around each disc was then measured and recorded. If the drugs were found to be active in the disc–diffusion test (inhibition zone > 10 mm), they were further evaluated for determining the minimum inhibitory concentration (MIC) values.
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2.7. Minimum inhibitory concentration (MIC) The drugs were screened for their anti-bacterial activities against S. aureus (MTCC3381), B. cereus (MTCC430), E. coli (MTCC739) and P. aeruginosa (MTCC424). MIC was evaluated by turbidity method. A loopful of bacteria was inoculated with 100 mL of nutrient broth at 37 °C for 20 h in a test tube shaker at 150 rpm. The test compounds were prepared by dissolving in a minimal volume of DMSO and were serially diluted in Mueller-Hinton broth at concentration ranging from 1 μg/mL to 100 μg/mL. The 24-h bacterial cultures were then transferred into 10 mL of MullerHinton broth and incubated at 37 °C for 24 h. The growth of the bacteria was determined by measuring the turbidity by optical density reading at 600 nm after 24 h. Thus, MIC was generally read as the smallest concentration of the drug in the series that prevents the development of growth of test organisms. All the experiments were done in triplicate. 2.8. Statistical analysis MIC value and the drug releasing rate were measured in triplicate. Statistical analysis of the MIC value was performed using the unpaired student’s t test. Differences were considered significant for the p values of < 0.01. 2.9. Preparation of film 200 mg of the drug sample was dissolved in a minimum quantity of the DMSO. The resulting solution was placed in a film-forming glass plates. This glass plate was dried in vacuo at 30°C for 24 h. The film was pulled out from the glass plate and the thickness of the film was measured using electronic screw gauge. The thickness of the film was in the range of 100–200 μm. This film was used for the controlled drug delivery studies. 2.10. Control drug release study In vitro drug release pattern was studied at different temperatures (37 and 40°C) and of different pH values (7.4 and 9.2) of the medium. Pieces of 11.5 cm2 of copolymer film (100–200 μm thickness) were taken and soaked in 10 mL of phosphate-buffered solutions having the pH 7.4 and 9.2. 5 mL of the solution were periodically collected for analysis and replaced with fresh medium (the same volume). The amount of quinoline in the medium was determined using UV spectroscopy absorption at λmax = 270 nm. These measurements were performed by a UV/Visible detector Labindia system. The cumulative drug released was plotted against the time intervals.
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3. Results and discussion The parent compound chalcone-(4-(2-chloroquinolin-5-ylamino)-3-(4-hydroxyphenyl) prop-2en-1-one (CPE) and the monomer 1-(4-(7-chloroquinolin-4-ylamino)phenyl)acrylate) (CPA) were prepared according to Schemes 1 and 2, respectively, and characterized by IR and 1H NMR techniques. Poly(CPA) was synthesized using a novel CPA monomer according to Scheme 2. The poly(CPA-co-AA) and poly(CPA-co-HEA) (Scheme 3) were prepared in EMK by a free radical polymerization technique and characterized by FT-IR, 1H NMR and UV methods to confirm the structure. The NMR spectrum of the monomer CPA and poly(CPA) is shown in the Figure 1. The NMR spectrum of CPA showed characteristic peaks for the aromatic protons between 8.9 and 7.5 ppm. The polymerisable vinyl protons appear at 6.2 and 6.6 ppm. The disappearance of peak at 6.2 and 6.6 ppm in poly(CPA) confirms that the monomers are completely converted to polymers. Also, the appearance peak at 0.9–1.3 ppm confirms the polymer formation. Similar to poly(CPA), other copolymer showed expected peaks in the proton NMR spectrum and the values are presented in the experimental section. The IR spectrum of the monomer and the polymers are presented in Figure 2. The presence of peak at 1730 cm−1 confirms the formation of acrylated product from CPE. The aromatic and aliphatic CH stretching frequencies are seen around 3100 and 2900 cm−1, respectively. The thermal stability of the polymer is measured and reported in this paper. The molecular weight of the polymer was determined by GPC. The weight average molecular weight of polymers was around 5000 g/mol (Table 1). The polydispersity values of the all polymers are less than 2, quantitatively suggesting that the polymer was terminated by disproportion method, which was typical of acrylates. The antimicrobial activity of the polymer was evaluated using MIC method.
Scheme 1.
Synthesis of CPE.
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Scheme 2.
Synthesis of CPA and poly(CPA).
Scheme 3.
Synthesis of poly(CPA-co-AA) and poly(CPA-co-HEA).
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Figure 1.
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H NMR spectrum: (a) CPA, (b) poly(CPA).
Figure 2. FT-IR Spectrum: (a) CPA, (b) poly(CPA), (c) poly(CPA-co-AA), and (d) poly(CPAco-HEA).
3.1. Solubility The solubility data of the polymer samples are presented in Table 2. It shows that all the copolymers are soluble in DMSO, dimethylformamide, acetone and tetrahydrofuran, but insoluble in polar solvents like chloroform, water and methanol, and partially soluble in ethanol. The copolymer was not soluble in non-polar solvents like benzene, n-hexane and carbon tetrachloride.
136 Table 1.
P. Uma et al. TGA, molecular weight, UV data of the synthesized materials. TGA
Polymer
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UV data
1st decomposition temperature (°C)
2nd decomposition temperature (°C)
Mn × 103
Mw × 103
Mw/ Mn
Aromatic C=C
Vinylic C=C
220 225
350 335
2.88 3.10
4.85 5.40
1.68 1.74
263, 280 260, 277
332 330
220
355
2.65
4.95
1.87
263, 275
331
Poly(CPA) Poly(CPAco-AA) Poly(CPAco-HEA)
Table 2.
Molecular weight
Solubility data of monomer and polymers at 30 °C.
Polymer CPA Poly(CPA) Poly(CPAco-AA) Poly(CPAco-HEA)
nH2O MeOH EtOH CCl4 CHCl3 DMSO DMF Acetone THF Hexane − − −
+ − −
+ ± −
− − −
+ − −
+ + +
+ + +
+ + +
+ + +
− − −
−
−
−
−
−
+
+
+
+
−
Note: +, = Soluble; ± , = Partially soluble; − , = Insoluble.
3.2. UV studies UV–visible spectrum of the poly(CPA) is shown in Figure 3 as a representative of the polymer series. The absorption values are presented in Table 1. Figure 3 shows three characteristic peaks of absorption around 260, 270 and 320 nm. The peaks at 260 and
Figure 3.
UV spectrum of poly(CPA).
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3.3. Molecular weights Molecular weights of the polymers are determined using GPC method. The poly(CPA) showed the weight average molecular weight of Mw=4.85 × 103, the number average molecular weight of Mn = 2.88 × 103 and the polydispersity index value of Mw/ Mn = 1.65. A similar trend is observed for other polymer sample in this series and is presented in Table 1. The observed polydispersity values confirm that the termination of the polymer is by disproportionation method (Mw/Mn < 2). Acrylates generally undergo termination by disproportionation method. 3.4. Thermal analysis Thermogravimetric analysis is used to estimate the percentage weight loss of the copolymer against temperature. The TGA and differential thermo gravimetric curves of poly(CPA) are shown in Figure 4 and different decomposition temperatures are presented in Table 1. These results show that the poly(CPA) undergoes decomposition in two stages. The first decomposition temperature revolved around 220 °C and the second decomposition temperature centred around 350 °C. These two-stage decomposition temperatures of the copolymer clearly show that the copolymer has good thermal stability. Similar trends were observed for other polymer samples and the values were presented in Table 1. This type of two-stage decomposition is reported for similar types of polymeric systems.[22]
20
DTG( g/min)
Weight loss (%)
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270 nm correspond to aromatic double bond presents in the chalcone and quinoline, respectively. The absorption peak at 320 nm corresponds to the π–π* transition of > CH=CH < in the CPA system. Similar to the poly(CPA), UV–visible spectrum of the poly(CPA-co-AA) and poly(CPA-co-HEA) showed the peak of absorption for aromatic double bond and >CH=CH< peaks around 270 and 330 nm, respectively. The peak intensity at 270 nm is used for in vitro drug-releasing study.
120
220
320
Temperature (0C)
Figure 4.
TGA and DTG spectrum of poly(CPA).
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3.5. Antimicrobial activity The MIC values (average of triplicates) of the CPA monomer, poly(CPA), poly(CPA-coAA) and poly(CPA-co-HEA) are shown in Table 3. The effect of comonomer in the polymer chain on the antimicrobial activity is investigated by comparing the MIC values of poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA). The result is presented as a bar diagram in Figure 5. The results showed that the presence of hydrophilic comonomer in the polymer chain enhanced the antimicrobial activity on both gram-positive and gram-negative bacteria. However, more pronounced comonomer effect is witnessed in the case of gram-positive bacteria (Table 3). This may be due to the differences in the constituents present in the cell wall of the microbes which might have brought about such a variation in the comonomer effects between the two types of stains. In the case of gram-negative bacteria, the remarkable observation lies in the MIC value of P. aeruginosa. The antimicrobial activity of poly(CPA-co-AA) on the P. aeruginosa was found to be very high. The MIC value (3.91 μg/mL) was higher than the MIC values of linezolid (a new oxazolidinone class of compound) which is in the human clinical trial. Besides having a higher MIC value, the synthetic procedure of poly(CPA-co-AA) is very simple to become a good competitor for other commercially available compounds. The P. aeruginosais considered to be a highly resistant species among the gram-negative bacteria. Several studies have demonstrated a low susceptibility of P. aeruginosa towards both hydrophobic and hydrophilic antibiotics. The MIC value of poly(CPA-co-AA) over P. aeruginosa was though found to be high (3.91 μg/mL), considering the intrinsic resistance capability of the bacterium. Table 3.
MIC values of CPA, poly (CPA), poly (CPA-co-AA) and poly (CPA-co-HEA). Minimum inhibiting concentration (μg/mL)
Samples
S. aureus
B. cereus
E. coli
P. aeruginosa
CPA Poly(CPA) Poly(CPA-co-AA) Poly(CPA-co-HEA)
7.81 ± 0.5 7.81 ± 0.8 15.6 ± 0.5 7.81 ± 0.2
15.6 ± 0.8 15.6 ± 0.3 7.8 ± 0.2 7.8 ± 0.8
62.5 ± 1 31.5 ± 2.5 15.6 ± 0.5 15.6 ± 0.8
7.8 ± 0.7 31.2 ± 0.8 3.91 ± 0.2 31.2 ± 2.3
Figure 5. Comparative antimicrobial graph of CPA, poly(CPA), poly(CPA-co-AA) and poly (CPA-co-HEA).
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The enhanced antimicrobial property due to comonomer AA can be ruled out as poly(AA) showed absence of any antimicrobial activity.[1,23] This provides a clear picture that the antimicrobial activity enhancement of poly(CPA-co-AA) over the poly (CPA) on P. aeruginosa and E. coli is due to the availability of active drug moiety (CPE) adjacent to the cell wall of the micro-organism and/or the opening up of the cell wall to allow the active drug moiety to penetrate through the micro-organism. With regards to the first condition, we feel that the availability of the drug adjacent to the cell wall was increased due to the polymer chain forming the cage-like structure around the micro-organism. Such structures could be stabilized by both ionic and hydrogen bonding exhibited between the polymeric chain and the cell wall of the micro-organism.[24] As far as the second possibility, cell wall opening can be triggered by the ionic strength and the hydrophilic nature of the medium adjacent to the cell wall of the micro-organism.[1] In poly(CPA-co-AA), the comonomer AA was involved in increasing the ionic strength of the medium adjacent to the cell wall of the micro-organism. It appears from the results that these two factors operate simultaneously to increase the activity of poly(CPA-co-AA) over poly(CPA) and poly(CPA-co-HEA). The comonomer AA acts as an anchor to deliver the active drug moiety through the above-mentioned two mechanisms. Moreover, the AA present in the poly(CPA-co-AA) can engage in neighbouring group participation mechanism for the detachment of active drug moiety from that of the polymeric chain.[25] 3.6. Drug-releasing study The cumulative drug-releasing graph for the polymer carrying AA and HEA as a comonomer was shown in Figures 6 and 7. This graph shows that the pH of the medium plays a vital role in deciding the rate of release of the drug from the polymer backbone. As predicted and reported for the similar type of systems, the drug release rate is enhanced when the pH of the medium was changed from 7.4 to 9.2.[26] This might be
Figure 6. Effect of pH and temperature (▲, = pH 9.2 at 37 °C; ■ ,= pH 7.4 at 40 °C; and ♦, = pH 7.4 at 37 °C) on the drug releasing rate of copoly(DAA-AA) film.
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Figure 7. Effect of pH and temperature (▲, = pH 9.2 at 37 °C; ■, = pH 7.4 at 40 °C; and ♦, = pH 7.4 at 37 °C) on the drug releasing rate of copoly(DAA-HEA) film.
due to the fact that at pH 7.4, the ester linkage that connects the drug to the polymer backbone shows resistance to hydrolysis. The irregular releasing pattern observed in the studied system is due to the sampling technique employed in this study. Earlier studies have reported this type of behaviour and this can be overcome by changing the testing sample from the film to granular type. Since we are studying a releasing pattern at different temperatures, we intended to go for the film type, rather than the granular type of the testing sample. As the basicity of the medium increases, the ester linkage was more pronounced to hydrolysis and releases the active drug moiety to the surroundings at a faster rate. Apart from this, aromatic system is also responsible for the higher release rate. These sorts of aromatic system exhibiting faster release rates than the aliphatic system were observed by many previous research workers. The hydrolysis of ester bonds in the basic medium was reported earlier. Upon changing the temperature from 37 °C to 40 °C at pH 7.4, only a slight increase in releasing rate is observed. This was due to the fact that as the temperature increases the chain mobility increases, which in turn allows the water molecules to penetrate through the polymer matrix to enhance the hydrolysis rate of the system. This type of chain mobility responsible for the enhancement of releasing rate and the effect may be more pronounced when the polymer sample is in film form. This type of change in the releasing rate when we change the pH has got major advantages. The most important applications were sustained oral drug delivery system provided from the pH difference between the stomach and the intestine. 3.7. Effect of comonomer on drug releasing rate The effect of comonomer on the drug releasing rate between of poly(CPA-co-AA) and poly(CPA-co-HEA) is shown in Figure 8. This graph shows that the polymer-bearing AA as a comonomer releases the drug in much faster rate than its counterpart HEA. The reason may be due to the fact that the acrylic acid in the polymer backbone assists the hydrolysis rate through the neighbouring group participating mechanism and this
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Figure 8. Effect of comonomer type (♦, = AA; ■, = HEA) on the drug releasing rate from the polymer film (pH 9.2 at 37 °C).
type of assistance was absent in the case of polymer-bearing HEA as a comonomer. Though the HEA present in the polymer chain was involved in increasing the hydrophilic nature of the polymer matrix, but the effect was comparatively lower than that of AA which effectively participated in the neighbouring group participation mechanism. 4. Conclusion Novel chalcone-type monomer CPA was prepared from CPE and acryloyl chloride. Poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA) were prepared using solution polymerization technique. The synthesized polymers were characterized by IR and NMR techniques. The weight average molecular weight of the synthesized polymers was low around 5000 g/mol. Thermal stability of the polymers were very high, first and second decomposition temperature lies around 230 and 350 °C, respectively. The UV data showed the characteristic peaks for aromatic and vinylic absorption around 270 and 330 nm, respectively. Antimicrobial activity of the four compounds [CPA, poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA)] were tested in S. aureus, B. cereus, E. coli and P. aeruginosa microbes. The copolymers showed very high activity against tested bacteria. Remarkable values were obtained for P. aeruginosa. The drug releasing pattern of the polymer film was monitored for 4 weeks using UV–visible spectroscopic technique. Drug releasing graph suggested that both pH and the temperature of the medium affect the releasing of the drug from the polymer film. We also found that the rate of release increased upon increasing the pH and temperature of the medium. References [1] Vogl O, Tirrell D. Functional polymers with biologically active group. J. Macromol. Sci. Chem. 1979;13:415–439. [2] Mokle SS, Sayeed MA, Chopde K. Synthesis and antimicrobial activity of some chalcone derivatives. Int. J. Chem. Sci. 2004;2:96–100.
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[3] Ballesteros JF, Sanz MJ, Ubeda A, Miranda MA, Iborra S. Synthesis and pharmacological evaluation of 2′-hydroxychalcones and flavones as inhibitors of inflammatory mediators generation. J. Med. Chem. 1995;38:2794–2797. [4] Viana GS, Bandeira MA, Matos F. Analgesic and anti-inflammatory effects of chalcones isolated from myracrodruon urundeuva allemão. J. Phytomedicine. 2003;10:189–195. [5] Mukarami S, Muramatsu M, Aihara H, Otomo S. Inhibition of gastric H+, K(+) ATPase by the anti-ulcer agent, sofalcone. Biochem. Pharmacol. 1991;42:1447–1551. [6] Barfod L, Kemp K, Hansen M, Kharazmi A. Synthesis and antimicrobial activity of some chalcone derivatives. Int. J. Immunopharmacol. 2002;2:545–555. [7] Liu M, Wilairat P, Go ML. Antimalarial alkoxylated and hydroxylated chalcone. J. Med. Chem. 2001;44:4443–4452. [8] Nielsen SF, Chen M, Theander TG, Kharazmi A, Brøgger Christensen SB. Synthesis of antiparasitic licorice chalcones. Bioorg. Med. Chem. Lett. 1995;5:449–452. [9] Vogel S, Barbic M, Jürgenliemk G, Heilmann J. Synthesis, cytotoxicity, anti-inflammantory activity of chalcones and influence of A-ring modifications on the pharmacological effect. Eur. J. Med. Chem. 2010;45:2206–2213. [10] Zhao LM, Jin HS, Sun LP, Piao HR, Quan ZS. Synthesis and evaluation of antiplatelet activity of trihydroxychalcone derivatives. Bioorg. Med. Chem. Lett. 2005;15:5027–5029. [11] Satyanarayana M, Tiwari P, Tripathi BK, Srivastava AK, Pratap R. Synthesis and antihyperglycemic activity of chalcone based aryloxypropanolamines. Bioorg. Med. Chem. 2004;12:883–889. [12] Nowakowska Z, Kędzia B, Schroeder G. Synthesis, physicochemical properties and antimicrobial evaluation of new (E)-chalcones. Eur. J. Med. Chem. 2008;43:707–713. [13] Mayekar AN. Synthesis, characterization and antimicrobial study of some new cyclohexenone derivatives. Int. J. Chem. 2010;2:114–123. [14] Larsen RD, Corley EG, King AO, Carroll JD, Davis P. Practical route to a new class of LTD4 receptor antagonists. J. Org. Chem. 1996;61:3398–3405. [15] Chen YL, Fang KC, Sheu JY, Hsu SL, Tzeng CC. Synthesis and antibacterial evaluation of certain quinolone derivatives. J. Med. Chem. 2004;44:2374–2377. [16] Billker O, Lindo V, Panico M, Etiene AE, Paxton T. Identification of xanthurenic acids the putative inducer of malaria development in the mosquito. Nature. 1998;392:289–292. [17] Rezig R, Chebah M, Rhouati S, Ducki S, Lawrence NJ. Synthesis of some quinolinyl aryl unsaturated ketones. J. Soc. Alg. Chim. 2000;10:111–120. [18] Ducki S, Hadfield JA, Lawerence NJ, Liu CY. Isolation of E-1-(4-hydroxyphenyl)-but-1-en3-one from scutellaria barbata. Planta Med. 1996;62:185–186. [19] Domínguez JN, Charris JE, Lobo G, Dominguez NG, Moreno MM. Synthesis of quinolinyl chalcones and their evaluation of their antimalarial activity. Eur. J. Med. Chem. 2001;36:555–560. [20] Moussaoui F, Belfaitah A, Debache A, Rhouati S. Synthesis and characterization of some new aryl quinolinyl α, β unsaturated ketones. J. Soc. Alg. Chim. 2002;12:71–78. [21] Ruiz C, Haddad M, Alban J, Bourdy G, Reategui R, Castillo D, Sauvain M, Deharo E, Estevez Y, Arevalo J, Rojas R. Activity guided isolation of antileishmanial compounds from piper hispidum. Phytochem. Lett. 2011;4:363–366. [22] Ahmed MR, Sasttry VG, Bano N, Ravichantra S, Raghavendra M. Synthesis and cytotoxic, anti oxidant activities of new chalcone derivatives. Russ. J. Chem. 2011;4:289–294. [23] Arun A, Reddy BSR. Polymeric drug for antimicrobial activity studies: synthesis and characterization. J. Bioact. Compat. Polym. 2003;18:219–228. [24] Nagaini Z, Siti M, Fadzillah H, Hussain H, Kamaruddin K. Synthesis and antimicrobial studies of (E)-3-(4-Alkyloxyphenyl)1-(2-hydroxyphenyl)prop-2-ene-1-one, (E)-3-(4Alkyloxyphenyl)-1-(4-hydroxyphenyl)prop-2-ene-1-one and their analogues. World J. Chem. 2009;4:09–14. [25] Bolard J, Legrand P, Heitz F, Cybulska B. One sided action of amphotericin boncholestrolcontaining membrane is determined by its self-association in the medium. Biochemistry. 1991;30:5707–5715. [26] Davaran S, Hanaee J, Khosravi A. Release of 5-amino salicylic acid from acrylic type polymeric prodrugs designed for colon-specific drug delivery. J. Controlled Release. 1999;58:279–287.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Quinoline based polymeric drug for biological applications: synthesis, characterization, antimicrobial, and drug releasing studies a
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P. Uma , J. Suresh , Revathy Selvaraj , S. Karthik & A. Arun
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PG & Research Department of Chemistry, Government Arts College, Thiruvannamalai 606603, Tamil Nadu, India Published online: 27 Nov 2014.
Click for updates To cite this article: P. Uma, J. Suresh, Revathy Selvaraj, S. Karthik & A. Arun (2015) Quinoline based polymeric drug for biological applications: synthesis, characterization, antimicrobial, and drug releasing studies, Journal of Biomaterials Science, Polymer Edition, 26:2, 128-142, DOI: 10.1080/09205063.2014.985022 To link to this article: http://dx.doi.org/10.1080/09205063.2014.985022
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Biomaterials Science, Polymer Edition, 2015 Vol. 26, No. 2, 128–142, http://dx.doi.org/10.1080/09205063.2014.985022
Quinoline based polymeric drug for biological applications: synthesis, characterization, antimicrobial, and drug releasing studies P. Uma, J. Suresh, Revathy Selvaraj, S. Karthik and A. Arun* PG & Research Department of Chemistry, Government Arts College, Thiruvannamalai 606603, Tamil Nadu, India
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(Received 20 July 2014; accepted 4 November 2014) Novel acrylate monomer of quinoline-based chalcone 1-(4-(7-chloroquinolin-4-ylamino)phenyl) acrylate (CPA) was synthesized using (4-(2-chloroquinolin-5-ylamino) phenyl)-3-(4-hydroxyphenyl)prop-2-en-1-one (CPE) and acryloyl chloride. CPA is characterized by different techniques like IR, 1H NMR and UV–visible spectrometry techniques. Poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA) are prepared by solution polymerization technique using CPA, acrylic acid (AA) and hydroxyethylacrylate (HEA), respectively. The antimicrobial activities of the compounds are tested using four different micro-organisms. In vitro cumulative drug release studies are done using UV visible spectroscopic technique. The molecular weights of these polymers are found to be around 5000 g/mol. The synthesized polymers showed two stages of thermal decomposition temperature centred around 220 and 350 °C, respectively. The antimicrobial activity of the polymer sample is found to be very high and especially for gram-negative bacteria with a minimum value of 3.91 μg/ mL. The in vitro drug-releasing rate is dependent on the comonomer, pH and temperature of the medium. Keywords: chalcone; acrylate; antimicrobial activity; drug releasing study
1. Introduction The uses of polymeric systems having pharmacological activity provide very good local activities, reducing the toxicological risks and in addition act as a release system of the pharmacological active residue which is controlled by chemical reactions, mainly hydrolytic under enzymatic and non-enzymatic processes. Generally, drug conjugates are usually prepared by esterification, acrylation or alkylation. By attaching bioactive substrates to the synthetic or naturally occurring macromolecules, it is expected to increase the therapeutic efficiency while lowering their toxicity. Generally speaking, the activity of the polymeric drug is related to the functional group and the nature of the polymeric substance. According to Vogl and Tirrell [1], the introduction of hydrophilic, hydrophobic or polyelectrolytic moieties will enhance the activity of the drug on micro-organisms. Also, it was observed that poly(methacrylic acid) has no effect on the micro-organisms like Escherichia coli. Incorporating hydroxyl or carboxylic groups in the polymer backbone could, in fact, increase the hydrophilic character of the polymeric drug. Most of the synthesized polymeric drugs having high-hydrophilic character were produced by copolymerising the drug conjugates with acrylic acid as a *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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hydrophilic part. The advantage of acrylic acid as a comonomer in a polymer chain bearing active drug moiety is its ability to assist the cleavage of amide or ester linkage through neighbouring group participation mechanism. These properties make such systems quite interesting and provide longer delivery times with lower dosages. Chalcones are condensed products of the substituted aromatic aldehydes with simple or various substituted acetophenones in the presence of alkali. Compounds with the backbone of chalcone possesses various biological activities such as anti-microbial, [2] anti-inflammatory,[3] analgesic,[4] anti-ulcerative,[5] immune-modulatory,[6] antimalarial,[7] anti-cancer, anti-viral,[8] anti-leishmanial,[9] anti-oxidant,[10] antiplatelet and[11] anti-hyperglycemic.[12] The presence of the reactive keto group and the vinylenic group in the chalcone and their analogues possesses the antioxidant activity.[13] Similar to chalcones, quinolines are also an important class of compounds because of their bioactive properties and medicinal uses such as anti-malarial,[14] anti-inflammatory [15] and tyrosine kinase inhibiting agents.[16] The synthesis of quinolinyl chalcones is scarily reported in literature [17,18] and is generally synthesized by ClaisenSchmidt condensation reaction. Dominguez et al. [19] have reported the synthesis of some quinolinyl chalcones and claimed that their compounds showed anti-malarial activity. Moussaoui et al. [20] have also described the synthesis and cytotoxicity effect of quinolinyl chalcones upon K 562 human leukaemia cell lines. Chloroquine and other quinolines had been served as anti-malarial chemotherapy for more than 40 years. The achievement of these drugs is based on excellent clinical efficacy, limited host toxicity, ease of use and simple cost-effective synthesis.[21] The 7-chloroquinoline moiety, a pharmacophore present in several established anti-malarials such as chloroquine and amodiaquine is effectively bound to heme and consequently inhibits the hemozoin formation. The present study is also aimed at exploiting this feature by synthesis chalcone using 7-chloroquinoline as a base compound. In general, polymeric drug based on acrylates has a higher activity against ATCC bacterial strains.[22] Therefore, it will be interesting to combine the effect of quinoline and acrylate in the same platform to see how it shows antibacterial properties. For that reason, we investigated the biological activity of the chalcone polymer containing chlorine as a biologically active site. In this article, we have reported a synthesis, antimicrobial and drug releasing behaviour of acryalated derivative (CPA) of chalcone (CPE) based on 4,7-dichloroquinoline and their polymers. Antimicrobial activities of CPE, CPA, Poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA) are tested on four different bacterial strains. Also, in vitro drug releasing behaviour of the polymer film is determined using UV–visible spectroscopic method. 2. Materials and methods 4-Hydroxybenzaldehyde and acrylic acid is used as such received from Aldrich Chemicals. 4,7-Dichloroquinoline and 1-4-aminophenylethanonen are received from Merck chemicals. Triethylamine (TEA) was received from SD Fine Chemicals. Ethylmethylketone (MEK), acrylic acid (AA), hydroxyethylacrylate (HEA) and acroloylchloride are received from Merck and used as such. ALPHA BRUKER FT-IR spectrophotometer is used for recording IR spectra using KBr pellet method. 1H NMR spectra of the samples were run on a Bruker FT-NMR spectrophotometer operating at 500 MHz’s using CDCl3 as a solvent and tetramethylsilane (TMS) as an internal reference. UV- absorption measurements were recorded using LABINDIA model UV 320 instrument by dissolving polymer samples in Tetrahydrafuran (THF). 1-(4-(2 Chloroquinolin-5-ylamino)
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phenyl)ethanone (CE) is synthesized using 4,7-dichloroquinoline and para-amino-acetophenone. Thermal analysis of the polymers is done using a Mettler 3000 thermal analyser at the heating rate of 10 °C/min in the air. The molecular weight of the polymer was obtained using a Schimadzu instrument with THF as an eluent at a flow rate of 0.3 mL/min. Muller-Hinton broth and Muller-Hinton agar were obtained from Himedia and used as such. MTCC stains of gram-negative bacteria (Escherichia coli (739), Pseudomonas aeruginosa (424)) and gram-positive bacteria (Staphylococcus aureus (3381), Bacillus cereus (430)) are obtained from the Christian Medical College (CMC), Vellore, India and used for culturing the bacteria.
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2.1. Synthesis of 1-(4-(2chloroquinolin-5-ylamino)phenyl) ethanone (CE) In a 500 mL round-bottom flask fitted with a reflux condenser provided with a magnetic stirrer, 20 g (0.11 mol) of 4,7-dichloroquinoline and 14.8 g (0.11 mol) of paraamino acetophenone are added and dissolved completely. To the reaction mixture, methanol was added and the reaction mixture was stirred for 6 h at 65 °C. The solvent was evaporated under vacuum and solid mass was obtained. The solid mass was recrystalized from methanol to obtain the compound as a yellow solid. Yield: 85%. Molecular weight is 296 g/mol and melting point is 160–162 °C. 2.2. Synthesis of 1-(4-(2-chloroquinolin-5-ylamino) phenyl)-3-(4-hydroxyphenyl) prop-2-en-1-one (CPE) Into a conical flask containing 30 mL of ethanol, 0.04 mol (11 g) of substituted quinoline and 0.04 moles (5 g) of 4-hydroxybenzaldehyde are added and stirred. With this solution, 4 g of sodium hydroxide dissolved in 20 mL of distilled water is added drop wise at room temperature. The contents of the flask are stirred for 20 h at room temperature. The mixture is then neutralized using dilute hydrochloric acid and the formed precipitate is filtered out and recrystalized from methanol. Yield: 75%. Molecular weight is 400 g/mol and melting point is 168–170 °C. 1H NMR (δ, ppm): 9.0–7.7 (13H, m, aromatic CH) and 7.0 (1H, d, CH present in the chalcone), 6.9 (1H, d, CH present in the chalcone) and 3.9 (1H, b, NH proton). FT-IR (cm−1) 3184 (–OH), 3030 (aromatic CH-stretching), 2881 (aliphatic CH-stretching), 1672 and 1646 (cis and trans C=O) and 1609 (aliphatic CH=CH). 2.3. Synthesis of 1-(4-(7-chloroquinolin-4-ylamino) phenyl) acrylate (CPA) 0.0123 mol (5 g) of CPE dissolved in 500 mL of MEK is taken into a 500 mL roundbottom flask fitted with a dropping funnel and mechanical stirrer. 0.012 mol (0.92 g) of TEA is added into the content of the flask. 0.012 mol (1.25 mL) of acryloyl chloride in 20 mL of MEK is taken into a dropping funnel and added drop by drop with constant stirring into the round-bottom flask which is kept in an ice bath. The addition of acryloyl chloride is maintained in such a way that the temperature of the reaction is kept under 0–5 °C. The reaction mixture is stirred at room temperature for 3 h. The precipitate (triethyl amine hydrochloride) is filtered and washed thoroughly with MEK. The filtrate is then taken into a 500 mL separating funnel, washed with 100 mL of distilled water taken in three portions and the organic layer is dried using anhydrous sodium sulphate. The product thus obtained by evaporating the solvent. The crude solid product was recrystallised using methanol. Yield: 74%; Mol.wt: 455.5 g/mol; 1H NMR (δ, ppm):
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9.0–7.5 (13H, m, aromatic CH), 6.9 (2H, d, CH present in the chalcone), 6.3 (2H, d, CH2 of acrylate), 6.6 (1H, t, CH of acrylate) and 3.8 (1H, b, NH proton). FT-IR (cm−1): 3413 (Ar-NH-Ar), 3030 (aromatic CH-stretching), 2897 (aliphatic CH-stretching), 1725 (–C=O of ester), 1677 & 1646 (cis and trans C=O) and 1619 (vinylic).
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2.4. Synthesis of pol(CPA) CPA (1.0 g) and 0.03 g of BPO (3 wt.%) are placed in a polymerization tube containing 10 mL of MEK. The polymerization tube was degassed using nitrogen gas and heated at 70 ± 1 °C in a thermostatic water bath for 24 h. Poly(CPA) is precipitated by adding the reaction mixture to a large excess of methanol with rapid stirring. Yield = 0.9 g (90%). 1H NMR (δ ppm): 9.0–7.6 (13H, m, aromatic CH), 7.0 (2H, d, CH present in the chalcone), 3.7 (1H, b, NH proton) and 0.9–1.3 (3H, m, aliphatic CH). FT-IR(cm−1): 3413 (Ar-NH-Ar), 3030 (aromatic CH-stretching), 2962 (aliphatic CH-stretching), 1742 (–C=O of ester), 1697 and 1646 (cis and trans C=O) and 1619 (aliphatic CH=CH). 2.5. General procedure for the preparation of copolymer Copolymerization is done using a solution polymerization technique. Equimolar mixture of monomer 1 and monomer 2 were taken in a polymerization tube containing MEK as a solvent and BPO (2wt.%) as a free radical initiator. The reaction medium was made inert by passing the nitrogen gas through the inlet of the polymerization tube. The polymerization is then carried out at 70 ± 1 °C for 24 h. The polymer is then precipitated using methanol and the precipitate obtained was then filtered in sintered crucible, washed with methanol and weighed. This method is used for the preparation of the following copolymers. 2.5.1. Synthesis of poly (CPA-co-AA) (0.004 mol) 1.38 g of CPA and (0.004 mol) 0.28 g of AA, 10 mL of MEK and 0.02 g (2wt.%) of BPO are used for the synthesis of poly (CPA-co-AA). Yield = 0.88 g. 1 H NMR (δ, ppm): 8.9–7.4 (13H, m, aromatic CH), 7.1 (1H, d, CH present in the chalcone), 7.0 (1H, d, CH present in the chalcone), 3.7 (1H, b, NH proton) and 1.0–2.5 (6H, m, aliphatic). FT-IR (cm−1): 3413 (Ar-NH-Ar), 3030 (aromatic CH-stretching), 2962 (aliphatic CH-stretching), 1742 (–C=O of ester), 1649 (C=O) and 1618 (aliphatic CH=CH). 2.5.2. Synthesis of poly (CPA-co-HEA) (0.004 mol) 1.38 g of CPA and (0.004 mol) 0.46 g of HEA, 10 mL of MEK and 0.02 g (2wt.%) BPO are used for the synthesis of poly(CPA-co-HEA). Yield = 1.0 g. 1 H NMR (δ, ppm): 8.8–7.5 (13H, m, aromatic CH), 7.1 (1H, d, CH present in the chalcone), 7.0 (1H, d, CH present in the chalcone), 4.6 (1H, b, OH proton), 3.9 (1H, b, NH proton), 3.6 (2H, d, CH2 connected to OH), 3.4 (2H, d, CH2 connected to ester) and 1–2.5 (6H, m, aliphatic). FT-IR (cm−1): 3336 (–OH), 3036 (aromatic CH-stretching), 2986 (aliphatic CH-stretching), 1740 (–C=O of ester), 1670 (C=O) and 1618 (aliphatic CH=CH).
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2.6. Drug susceptibility test The drugs were tested by the disc–diffusion method. Diluted bacterial cultures (100 mL) were spread on sterile Mueller-Hinton agar plates, after which 8 mm diameter discs (sterile blank) impregnated with the drug to be tested, were placed on the plates. The plates were incubated for 24 h at 37 °C under aerobic conditions and the diameter of the inhibition zone around each disc was then measured and recorded. If the drugs were found to be active in the disc–diffusion test (inhibition zone > 10 mm), they were further evaluated for determining the minimum inhibitory concentration (MIC) values.
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2.7. Minimum inhibitory concentration (MIC) The drugs were screened for their anti-bacterial activities against S. aureus (MTCC3381), B. cereus (MTCC430), E. coli (MTCC739) and P. aeruginosa (MTCC424). MIC was evaluated by turbidity method. A loopful of bacteria was inoculated with 100 mL of nutrient broth at 37 °C for 20 h in a test tube shaker at 150 rpm. The test compounds were prepared by dissolving in a minimal volume of DMSO and were serially diluted in Mueller-Hinton broth at concentration ranging from 1 μg/mL to 100 μg/mL. The 24-h bacterial cultures were then transferred into 10 mL of MullerHinton broth and incubated at 37 °C for 24 h. The growth of the bacteria was determined by measuring the turbidity by optical density reading at 600 nm after 24 h. Thus, MIC was generally read as the smallest concentration of the drug in the series that prevents the development of growth of test organisms. All the experiments were done in triplicate. 2.8. Statistical analysis MIC value and the drug releasing rate were measured in triplicate. Statistical analysis of the MIC value was performed using the unpaired student’s t test. Differences were considered significant for the p values of < 0.01. 2.9. Preparation of film 200 mg of the drug sample was dissolved in a minimum quantity of the DMSO. The resulting solution was placed in a film-forming glass plates. This glass plate was dried in vacuo at 30°C for 24 h. The film was pulled out from the glass plate and the thickness of the film was measured using electronic screw gauge. The thickness of the film was in the range of 100–200 μm. This film was used for the controlled drug delivery studies. 2.10. Control drug release study In vitro drug release pattern was studied at different temperatures (37 and 40°C) and of different pH values (7.4 and 9.2) of the medium. Pieces of 11.5 cm2 of copolymer film (100–200 μm thickness) were taken and soaked in 10 mL of phosphate-buffered solutions having the pH 7.4 and 9.2. 5 mL of the solution were periodically collected for analysis and replaced with fresh medium (the same volume). The amount of quinoline in the medium was determined using UV spectroscopy absorption at λmax = 270 nm. These measurements were performed by a UV/Visible detector Labindia system. The cumulative drug released was plotted against the time intervals.
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3. Results and discussion The parent compound chalcone-(4-(2-chloroquinolin-5-ylamino)-3-(4-hydroxyphenyl) prop-2en-1-one (CPE) and the monomer 1-(4-(7-chloroquinolin-4-ylamino)phenyl)acrylate) (CPA) were prepared according to Schemes 1 and 2, respectively, and characterized by IR and 1H NMR techniques. Poly(CPA) was synthesized using a novel CPA monomer according to Scheme 2. The poly(CPA-co-AA) and poly(CPA-co-HEA) (Scheme 3) were prepared in EMK by a free radical polymerization technique and characterized by FT-IR, 1H NMR and UV methods to confirm the structure. The NMR spectrum of the monomer CPA and poly(CPA) is shown in the Figure 1. The NMR spectrum of CPA showed characteristic peaks for the aromatic protons between 8.9 and 7.5 ppm. The polymerisable vinyl protons appear at 6.2 and 6.6 ppm. The disappearance of peak at 6.2 and 6.6 ppm in poly(CPA) confirms that the monomers are completely converted to polymers. Also, the appearance peak at 0.9–1.3 ppm confirms the polymer formation. Similar to poly(CPA), other copolymer showed expected peaks in the proton NMR spectrum and the values are presented in the experimental section. The IR spectrum of the monomer and the polymers are presented in Figure 2. The presence of peak at 1730 cm−1 confirms the formation of acrylated product from CPE. The aromatic and aliphatic CH stretching frequencies are seen around 3100 and 2900 cm−1, respectively. The thermal stability of the polymer is measured and reported in this paper. The molecular weight of the polymer was determined by GPC. The weight average molecular weight of polymers was around 5000 g/mol (Table 1). The polydispersity values of the all polymers are less than 2, quantitatively suggesting that the polymer was terminated by disproportion method, which was typical of acrylates. The antimicrobial activity of the polymer was evaluated using MIC method.
Scheme 1.
Synthesis of CPE.
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Scheme 2.
Synthesis of CPA and poly(CPA).
Scheme 3.
Synthesis of poly(CPA-co-AA) and poly(CPA-co-HEA).
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Figure 1.
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H NMR spectrum: (a) CPA, (b) poly(CPA).
Figure 2. FT-IR Spectrum: (a) CPA, (b) poly(CPA), (c) poly(CPA-co-AA), and (d) poly(CPAco-HEA).
3.1. Solubility The solubility data of the polymer samples are presented in Table 2. It shows that all the copolymers are soluble in DMSO, dimethylformamide, acetone and tetrahydrofuran, but insoluble in polar solvents like chloroform, water and methanol, and partially soluble in ethanol. The copolymer was not soluble in non-polar solvents like benzene, n-hexane and carbon tetrachloride.
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Polymer
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UV data
1st decomposition temperature (°C)
2nd decomposition temperature (°C)
Mn × 103
Mw × 103
Mw/ Mn
Aromatic C=C
Vinylic C=C
220 225
350 335
2.88 3.10
4.85 5.40
1.68 1.74
263, 280 260, 277
332 330
220
355
2.65
4.95
1.87
263, 275
331
Poly(CPA) Poly(CPAco-AA) Poly(CPAco-HEA)
Table 2.
Molecular weight
Solubility data of monomer and polymers at 30 °C.
Polymer CPA Poly(CPA) Poly(CPAco-AA) Poly(CPAco-HEA)
nH2O MeOH EtOH CCl4 CHCl3 DMSO DMF Acetone THF Hexane − − −
+ − −
+ ± −
− − −
+ − −
+ + +
+ + +
+ + +
+ + +
− − −
−
−
−
−
−
+
+
+
+
−
Note: +, = Soluble; ± , = Partially soluble; − , = Insoluble.
3.2. UV studies UV–visible spectrum of the poly(CPA) is shown in Figure 3 as a representative of the polymer series. The absorption values are presented in Table 1. Figure 3 shows three characteristic peaks of absorption around 260, 270 and 320 nm. The peaks at 260 and
Figure 3.
UV spectrum of poly(CPA).
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3.3. Molecular weights Molecular weights of the polymers are determined using GPC method. The poly(CPA) showed the weight average molecular weight of Mw=4.85 × 103, the number average molecular weight of Mn = 2.88 × 103 and the polydispersity index value of Mw/ Mn = 1.65. A similar trend is observed for other polymer sample in this series and is presented in Table 1. The observed polydispersity values confirm that the termination of the polymer is by disproportionation method (Mw/Mn < 2). Acrylates generally undergo termination by disproportionation method. 3.4. Thermal analysis Thermogravimetric analysis is used to estimate the percentage weight loss of the copolymer against temperature. The TGA and differential thermo gravimetric curves of poly(CPA) are shown in Figure 4 and different decomposition temperatures are presented in Table 1. These results show that the poly(CPA) undergoes decomposition in two stages. The first decomposition temperature revolved around 220 °C and the second decomposition temperature centred around 350 °C. These two-stage decomposition temperatures of the copolymer clearly show that the copolymer has good thermal stability. Similar trends were observed for other polymer samples and the values were presented in Table 1. This type of two-stage decomposition is reported for similar types of polymeric systems.[22]
20
DTG( g/min)
Weight loss (%)
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270 nm correspond to aromatic double bond presents in the chalcone and quinoline, respectively. The absorption peak at 320 nm corresponds to the π–π* transition of > CH=CH < in the CPA system. Similar to the poly(CPA), UV–visible spectrum of the poly(CPA-co-AA) and poly(CPA-co-HEA) showed the peak of absorption for aromatic double bond and >CH=CH< peaks around 270 and 330 nm, respectively. The peak intensity at 270 nm is used for in vitro drug-releasing study.
120
220
320
Temperature (0C)
Figure 4.
TGA and DTG spectrum of poly(CPA).
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3.5. Antimicrobial activity The MIC values (average of triplicates) of the CPA monomer, poly(CPA), poly(CPA-coAA) and poly(CPA-co-HEA) are shown in Table 3. The effect of comonomer in the polymer chain on the antimicrobial activity is investigated by comparing the MIC values of poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA). The result is presented as a bar diagram in Figure 5. The results showed that the presence of hydrophilic comonomer in the polymer chain enhanced the antimicrobial activity on both gram-positive and gram-negative bacteria. However, more pronounced comonomer effect is witnessed in the case of gram-positive bacteria (Table 3). This may be due to the differences in the constituents present in the cell wall of the microbes which might have brought about such a variation in the comonomer effects between the two types of stains. In the case of gram-negative bacteria, the remarkable observation lies in the MIC value of P. aeruginosa. The antimicrobial activity of poly(CPA-co-AA) on the P. aeruginosa was found to be very high. The MIC value (3.91 μg/mL) was higher than the MIC values of linezolid (a new oxazolidinone class of compound) which is in the human clinical trial. Besides having a higher MIC value, the synthetic procedure of poly(CPA-co-AA) is very simple to become a good competitor for other commercially available compounds. The P. aeruginosais considered to be a highly resistant species among the gram-negative bacteria. Several studies have demonstrated a low susceptibility of P. aeruginosa towards both hydrophobic and hydrophilic antibiotics. The MIC value of poly(CPA-co-AA) over P. aeruginosa was though found to be high (3.91 μg/mL), considering the intrinsic resistance capability of the bacterium. Table 3.
MIC values of CPA, poly (CPA), poly (CPA-co-AA) and poly (CPA-co-HEA). Minimum inhibiting concentration (μg/mL)
Samples
S. aureus
B. cereus
E. coli
P. aeruginosa
CPA Poly(CPA) Poly(CPA-co-AA) Poly(CPA-co-HEA)
7.81 ± 0.5 7.81 ± 0.8 15.6 ± 0.5 7.81 ± 0.2
15.6 ± 0.8 15.6 ± 0.3 7.8 ± 0.2 7.8 ± 0.8
62.5 ± 1 31.5 ± 2.5 15.6 ± 0.5 15.6 ± 0.8
7.8 ± 0.7 31.2 ± 0.8 3.91 ± 0.2 31.2 ± 2.3
Figure 5. Comparative antimicrobial graph of CPA, poly(CPA), poly(CPA-co-AA) and poly (CPA-co-HEA).
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The enhanced antimicrobial property due to comonomer AA can be ruled out as poly(AA) showed absence of any antimicrobial activity.[1,23] This provides a clear picture that the antimicrobial activity enhancement of poly(CPA-co-AA) over the poly (CPA) on P. aeruginosa and E. coli is due to the availability of active drug moiety (CPE) adjacent to the cell wall of the micro-organism and/or the opening up of the cell wall to allow the active drug moiety to penetrate through the micro-organism. With regards to the first condition, we feel that the availability of the drug adjacent to the cell wall was increased due to the polymer chain forming the cage-like structure around the micro-organism. Such structures could be stabilized by both ionic and hydrogen bonding exhibited between the polymeric chain and the cell wall of the micro-organism.[24] As far as the second possibility, cell wall opening can be triggered by the ionic strength and the hydrophilic nature of the medium adjacent to the cell wall of the micro-organism.[1] In poly(CPA-co-AA), the comonomer AA was involved in increasing the ionic strength of the medium adjacent to the cell wall of the micro-organism. It appears from the results that these two factors operate simultaneously to increase the activity of poly(CPA-co-AA) over poly(CPA) and poly(CPA-co-HEA). The comonomer AA acts as an anchor to deliver the active drug moiety through the above-mentioned two mechanisms. Moreover, the AA present in the poly(CPA-co-AA) can engage in neighbouring group participation mechanism for the detachment of active drug moiety from that of the polymeric chain.[25] 3.6. Drug-releasing study The cumulative drug-releasing graph for the polymer carrying AA and HEA as a comonomer was shown in Figures 6 and 7. This graph shows that the pH of the medium plays a vital role in deciding the rate of release of the drug from the polymer backbone. As predicted and reported for the similar type of systems, the drug release rate is enhanced when the pH of the medium was changed from 7.4 to 9.2.[26] This might be
Figure 6. Effect of pH and temperature (▲, = pH 9.2 at 37 °C; ■ ,= pH 7.4 at 40 °C; and ♦, = pH 7.4 at 37 °C) on the drug releasing rate of copoly(DAA-AA) film.
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Figure 7. Effect of pH and temperature (▲, = pH 9.2 at 37 °C; ■, = pH 7.4 at 40 °C; and ♦, = pH 7.4 at 37 °C) on the drug releasing rate of copoly(DAA-HEA) film.
due to the fact that at pH 7.4, the ester linkage that connects the drug to the polymer backbone shows resistance to hydrolysis. The irregular releasing pattern observed in the studied system is due to the sampling technique employed in this study. Earlier studies have reported this type of behaviour and this can be overcome by changing the testing sample from the film to granular type. Since we are studying a releasing pattern at different temperatures, we intended to go for the film type, rather than the granular type of the testing sample. As the basicity of the medium increases, the ester linkage was more pronounced to hydrolysis and releases the active drug moiety to the surroundings at a faster rate. Apart from this, aromatic system is also responsible for the higher release rate. These sorts of aromatic system exhibiting faster release rates than the aliphatic system were observed by many previous research workers. The hydrolysis of ester bonds in the basic medium was reported earlier. Upon changing the temperature from 37 °C to 40 °C at pH 7.4, only a slight increase in releasing rate is observed. This was due to the fact that as the temperature increases the chain mobility increases, which in turn allows the water molecules to penetrate through the polymer matrix to enhance the hydrolysis rate of the system. This type of chain mobility responsible for the enhancement of releasing rate and the effect may be more pronounced when the polymer sample is in film form. This type of change in the releasing rate when we change the pH has got major advantages. The most important applications were sustained oral drug delivery system provided from the pH difference between the stomach and the intestine. 3.7. Effect of comonomer on drug releasing rate The effect of comonomer on the drug releasing rate between of poly(CPA-co-AA) and poly(CPA-co-HEA) is shown in Figure 8. This graph shows that the polymer-bearing AA as a comonomer releases the drug in much faster rate than its counterpart HEA. The reason may be due to the fact that the acrylic acid in the polymer backbone assists the hydrolysis rate through the neighbouring group participating mechanism and this
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Figure 8. Effect of comonomer type (♦, = AA; ■, = HEA) on the drug releasing rate from the polymer film (pH 9.2 at 37 °C).
type of assistance was absent in the case of polymer-bearing HEA as a comonomer. Though the HEA present in the polymer chain was involved in increasing the hydrophilic nature of the polymer matrix, but the effect was comparatively lower than that of AA which effectively participated in the neighbouring group participation mechanism. 4. Conclusion Novel chalcone-type monomer CPA was prepared from CPE and acryloyl chloride. Poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA) were prepared using solution polymerization technique. The synthesized polymers were characterized by IR and NMR techniques. The weight average molecular weight of the synthesized polymers was low around 5000 g/mol. Thermal stability of the polymers were very high, first and second decomposition temperature lies around 230 and 350 °C, respectively. The UV data showed the characteristic peaks for aromatic and vinylic absorption around 270 and 330 nm, respectively. Antimicrobial activity of the four compounds [CPA, poly(CPA), poly(CPA-co-AA) and poly(CPA-co-HEA)] were tested in S. aureus, B. cereus, E. coli and P. aeruginosa microbes. The copolymers showed very high activity against tested bacteria. Remarkable values were obtained for P. aeruginosa. The drug releasing pattern of the polymer film was monitored for 4 weeks using UV–visible spectroscopic technique. Drug releasing graph suggested that both pH and the temperature of the medium affect the releasing of the drug from the polymer film. We also found that the rate of release increased upon increasing the pH and temperature of the medium. References [1] Vogl O, Tirrell D. Functional polymers with biologically active group. J. Macromol. Sci. Chem. 1979;13:415–439. [2] Mokle SS, Sayeed MA, Chopde K. Synthesis and antimicrobial activity of some chalcone derivatives. Int. J. Chem. Sci. 2004;2:96–100.
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