Facile Synthesis, Characterization, and In Vitro Antimicrobial and

Research ArticleFacile Synthesis Characterization and In Vitro Antimicrobialand Anticancer Activities of Biscoumarin Copolyester BearingPendant 3-(Trifluoromethyl)Styrene

Narendran Kandaswamy and Nanthini Raveendiran

Postgraduate and Research Department of Chemistry Pachaiyapparsquos College Chennai 600 030 India

Correspondence should be addressed to Narendran Kandaswamy knarenchemgmailcom

Received 20 May 2014 Revised 28 July 2014 Accepted 31 July 2014 Published 28 October 2014

Academic Editor Qingling Feng

Copyright copy 2014 N Kandaswamy and N Raveendiran This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Synthesis of random biscoumarin copolyester bearing pendant 3-(trifluoromethyl)styrene was prepared by the reaction ofbiscoumarin monomer 3 and hydroquinone 5 with azeloyl chloride The influence of pendant 3-(trifluoromethyl)styrene unit onthe properties of copolyester such as inherent viscosity solubility and thermal stability was investigated and compared in detailThe inherent viscosity and polydispersity index of the copolyester were found to be 015 dLg and 136 respectively The chemicalstructure of the copolyester was investigated by Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magneticresonance (1H-NMR) spectroscopy The physical properties of copolyester were characterized by thermogravimetric analysis(TGA) differential scanning calorimetry (DSC) gel permeation chromatography (GPC) and X-ray diffraction (XRD) techniqueAgar disc diffusion method was employed to study the antimicrobial activity of the random copolyester In vitro anticancer activityagainst lung cancer (Hep-2) cell line was also investigated

1 Introduction

Aliphatic aromatic copolyester [1ndash3] bears high thermal sta-bility and excellent mechanical strength and is being used invarious disciplines A probe through the literature indicatesthat there has been a rising interest in synthesizing aliphaticaromatic copolyester [4ndash6] as biomaterials [7] for a varietyof biomedical applications In spite of unique propertiessuch as thermal stability good chemical resistance andexcellent mechanical strength these polymers encounter theproblem of fabrication process due to high glass transitiontemperature and poor solubility in common organic solventsThe incorporation of bulky [3 8] or the use of pendantmonomers [3 9] such as biscoumarin 1 containing polarcarbonyl groups along with introduction of flexible spacer [310 11] in the polymer chain tends to reduce the inter- actionbetween polymer chains and eventually leads to increasein free volume and solubility with improvement in proces-sability [12ndash15] by maintaining its thermal stability When

considering the properties of biomaterial [16] the first andpremier requirement is chemically inertactive nontoxic andsustainable by the human body Implantation of surgicaldevices like sutures prosthesis anchor staples and valvesmay lead to incorporation of foreign object into the humanbody that has the potential to infect patients with variousmicrobes Sometimes this surgical implantation leads toharmful side effects like inflammation if it persists forlong time it ultimately leads to dysplasia Coumarin andits derivatives are one of the important classes of organiccompounds possessing intense biological activity includinganticoagulant estrogenic antimicrobial analgesic antibac-terial antifungal anti-inflammatory antitumor and anti-HIV Of particular interest coumarin derivative with styrenepharmacophore having trifluoromethyl group at the metaposition enhances its biological activity By compiling theabove facts it is necessary to develop polymer possessing easyfabrication process and fulfil the requirement of biomaterialby showing activities against microbial stains and some

Hindawi Publishing CorporationInternational Scholarly Research NoticesVolume 2014 Article ID 369473 9 pageshttpdxdoiorg1011552014369473

2 International Scholarly Research Notices

O

OH

OCF3

CF3

O

H2

+

O

OH OH

O

3

100∘C

1 2

OO

Scheme 1 Synthesis of monomer 3

cancers The aim of the present investigation is to synthesizeand characterize a random copolyester containing biologicalactive biscoumarin group bearing 3-(trifluoromethyl)styreneby inherent viscosity measurements solubility tests FTIR1H-NMR spectroscopy X-ray diffraction analysis (XRD)thermogravimetric analysis (TGA) and differential scanningcalorimetry (DSC) The synthesized copolyester was sub-jected to in vitro anticancer activity against lung cancer (Hep-2) cell line and antimicrobial studies

2 Experimental

21 Materials and Methods 4-Hydroxycoumarin (Aldrich)was used as such 3-(trifluoromethyl) Cinnamaldehyde hy-droquinone azeloyl chloride dimethylacetamide and pyri-dine (Aldrich) were used as received Solvents were purifiedaccording to the standard procedure in the literature [17]Hep-2 cell lines were obtained from National Centre for CellSciences Pune (NCCS) The cells were maintained in mini-mal essential medium (MEM) supplemented with 10 FBSpenicillin (100 120583gmL) and streptomycin (100 120583gmL) in ahumidified atmosphere of 50 120583gmL CO

2at 37∘C MEM was

purchased from Hi Media Laboratories Fetal bovine serum(FBS) was purchased from Cistron laboratories Trypsinmethyl thiazolyl diphenyl-tetrazolium bromide (MTT) anddimethyl sulfoxide were purchased from Sisco Research Lab-oratory Chemicals Mumbai

Inherent viscosity was determined at a polymer con-centration of 05 gdL in NMP at 30∘C using an Ubbelohdesuspended level viscometer 3 (wv) solutions were taken asa standard for solubility of copolyester in various solvents FT-IR spectra were recorded on Perkin Elmer 883 spectropho-tometer 1H-NMR spectra for monomer and polymer wererecorded on a Bruker 400MHz spectrometer using CDCl

3as

a solventThermogravimetric analysis (TGA) was performedin nitrogen at 10∘Cmin with a TGA instrument SDT Q600Differential scanning calorimetry (DSC) was performed withDSC 200 F3 Maia instrument at a heating rate of 10∘Cminunder nitrogen atmosphereGel permeation chromatography(GPC) analysis was used to determine number averagemolecular weight (119872

119899) and weight average molecular weight

of the polymer (119872119908) by WATERS 501 equipped with three

ultrastyragel columns and a differential refractive indexdetector using THF as eluent and polystyrene standards wereemployed for calibration The flow rate was 1mLmin X-Ray

diffraction measurements were recorded on a Bruker XRDD8 FOCUS using Cu K120572 radiation source

Antibacterial activity of random copolyester was deter-mined by disc diffusion method on Muller Hinton agar(MHA) medium and Sabouraud Dextrose agar (SDA)medium respectively Both Muller Hinton agar and Sabour-aud Dextrose agar media were poured into the petri plateAfter the medium was solidified the inoculums were spreadon the solid plates with sterile swab moistened with thebacte- rial suspension The discs were placed in MHA andSDA plates and 20 120583L of sample concentration was addedEach sample was placed in the discThe plates were incubatedfor 24 h at 37∘C Then the antimicrobial activity was deter-mined by measuring the diameter of zone of inhibitionStandard antibiotic ampicillin (20 120583gdisc) was used asreference Fresh bacterial cultures of gram negative bacterianamely Escherichia coli (MTCC-729) and Salmonella typhi(MTCC-531) and gram positive bacteria Bacillus subtilis(MTCC-2274) and Staphylococcus aureus (MTCC-3160) wereused for the antibacterial test Antifungal activity of extractswas determined by disc diffusion method on SabouraudDextrose agar medium Disc diffusion assay (DDA) canalso be performed for screening by standard method Stockcultures weremaintained at 4∘C on nutrient agar slant Activecultures for experiments were prepared by transferring aloop full of culture from the stock cultures into the test tubescontaining Sabouraud Dextrose broth which were incubatedfor 24 h at 37∘C Aspergillus Niger and Aspergillus Flavuswerethe fungi which were used for the study

22 Synthesis of Monomer 3 Mixture of 4-hydroxycoumarin1 (2 g 4mmol) and 3-(trifluoromethyl) cinnamaldehyde 2(12 g 2mmol)wasmixed thoroughly andheated in oil bath at100∘C (Scheme 1) After 30min the sample was tested by TLCand it was found that there were nomonomers present whichindicates the completion of reaction The reaction mixturewas then cooled to room temperature Hot ethanol (10mL)was added to the solid mass The solid product obtained wasfiltered dried and recrystallized using ethanol to give yellowcolour powder (26 g 838) 119877

119891 025 (119899-hexane EtOAc-

1 1) 1H-NMR (400MHz CDCl3) 120575 (ppm) 1177 (s 1H)

1128 (s 1H) 804ndash802 (t 2H) 763ndash760 (m 2H) 758 (s 1H)756ndash754 (d 1H) 748ndash747 (d 1H) 743ndash737 (m 5H) 682ndash677 (dd 1H) 658ndash654 (dd 1H) 551ndash550 (m 1H)

International Scholarly Research Notices 3

O

OH

OOOH

O

CF3

CF3

OO

OO

O

O

O O

OO

n

+ CCH2C7

O

O

Cl Cl HOOH

PyridineDMAc3

5

6

4

+

Scheme 2 Synthesis of copolyester 6

23 Synthesis of Copolyester 6 A three-neck flask fittedwith mechanical stirrer was charged with monomer 3(0001mmol) and hydroquinone 5 (0001mmol) and freshdistilled DMAc (30mL) (Scheme 2) The mixture was cooledin an ice bath 0004mmol of pyridine was added and thetemperature was reduced to 0∘C Then azeloyl dichloride4 (0002mmol) was added to the suspended reaction massand the resulting mixture was stirred at room temperaturefor 1 h and then at 80∘C for 7 h The reaction mixture waspoured into 50mL methanol and the precipitated polymerwas collected by filtration washed thoroughlywithmethanoland dried at 80∘C under vacuum for 10 h to give pale yellowcolour amorphous powder (yield 718)

24 In Vitro Assay for Anticancer Activity (MTT Assay) TheMTT assay was performed as first described by Mosmannwith the modifications suggested by Denizot and Lang Cells(1 times 105well) were plated in 24-well plates and incubatedat 37∘C with 5 CO

2condition After the cell reaches the

confluence the various concentrations of copolyester 6 wereadded and incubated for 24 h After incubation the samplewas removed from the well and washed with phosphate-buffered saline (pH 74) or MEM without serum 100 120583Lwell(5mgmL) of 05 3-(45-dimethyl-2-thiazolyl)-25-diphen-yl-tetrazolium bromide (MTT) was added and incubated for4 h After incubation 1mL of DMSO was added in all thewells The absorbance at 570 nm was measured with UV-spectrophotometer using DMSO as the blankMeasurementswere performed and the concentration required for a 50inhibition (IC

50) was determined graphically The cell

viability was calculated using the following formula

cell viability = (119860570

of treated cells119860570

of control cells) times 100 (1)

Graphs were plotted using the of cell viability in 119910-axis and concentration of the sample in 119909-axis Cell controland sample control were included in each assay to compare

3500 3000 2500 2000 1500 1000 500

Wave number (cmminus1)

Monomer 3

Copolyester 6

1726 1648 1104

Figure 1 FT-IR spectra of monomer and copolyester

the full cell viability in cytotoxicity and anticancer activityassessments

3 Results and Discussion

31 FTIR and 1H-NMRAnalysis The structures of the copol-yester were confirmed using FTIR 1H-NMR spectroscopythe representative FTIR spectrum of both monomer 3 andcopolyester 6 was shown in Figure 1 The polymer exhibitedcharacteristic absorption bands at 1726 cmminus1 this is attributedto the presence of carbonyl stretching of ester group Bothmonomer and polymer exhibited peak at 1648 cmminus1 this isdue to the stretching of carbonyl group of biscoumarin unitThe absorption band at 1104 cmminus1 is due to the stretchingvibration of C-F in bothmonomer 3 and polymer 6 TMS (te-tramethylsilane) as internal standard and CDCl

3as solvent at

room temperature were used to analyze the 1H-NMR spectraof monomer 3 and its respective copolyester 6 The 1H-NMR


OH OH

O OOO

CF3

102

100

680 675 670 665 660 655

091

090

203

213

104

107

103

520

102

100

102

13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

102

203

213

104

107

103

520

552 550 805 765 760 755 750 745 740

Figure 2 1H-NMR spectrum of monomer 3 in CDCl3

spectra of monomer 3 and copolyester 6 are presented inFigures 2 and 3 In 1H-NMR spectrum of monomer the twoprotons of cinnamyl group appeared at 682ndash677 ppm and658ndash654 ppmand it has been encircled distinctly in Figure 2The two hydroxyl groups seem to appear at two different

positions 1177 and 1128 ppm representing that these twogroups are nonequivalent In the proton NMR of copolyesterbridged carbon bearing pendant 3-(trifluoromethyl)styrene(peak b) appeared at 555 ppm thus confirming the incor-poration of biscoumarin monomer 3 in the polymeric chain


13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

OOO

O

OO

O O

OO

CF3

n

a

a

b

b

c

c

d

d

Figure 3 1H-NMR spectrum of copolyester 6 in CDCl3

The characteristic peak at 113ndash234 ppm is attributed to thealiphatic acid dichloride The aromatic protons related tobiscoumarin 3 and hydroquinone 5 in the polymer backboneappeared in the region of 822ndash710 ppm and two peaks inthe region of 666 and 591 ppm related to the alkene protons(ndashCH=CHndash peak c amp d) of the styrene group respectively

32 Polymer Solubility Solubility parameter is an importantcriterion which has to be considered for polymer process-ing The copolyester showed good solubility in chlorinatedsolvents and polar aprotic solvents this may be attributedto the presence of unsymmetric biscoumarin moiety whichleads to reduced rigidity of polymer chains This increase insolubility may be attributed to the incorporation of bulky3-(trifluoromethyl)styrene pendant unit which disrupts thepolymer chain and hinders close chain from packing thereby reducing chain interactions Thus it is inferred thatthe incorporation of 3-(trifluoromethyl)styrene pendant unitinfluence more in solubility

33 Inherent Viscosity The inherent viscosity of the copolyes-ter was determined in NMP at 30∘C at the concentrationof 05 gdL using an Ubbelohde suspended level viscome-ter by calculating the values of flow time of pure solventand copolyester The inherent viscosity of the copolyester

was found to be 015 dLg In general inherent viscosi-ties increase with increase in molecular weight It is evi-dent that copolyester exhibits lower viscosity value becausethe presence of unsymmetrical bulky monomer with 3-(trifluoromethyl)styrene pendant unit may tend to reducethe polymer chain length and hence inherent viscosity valuedecreases It is also reflected in the GPC data The numberaveragemolecular weight (119872

119899) andweight averagemolecular

weight (119872119908) of the copolyester 6 are 4489 and 6138 with

136 as polydispersity index value respectively It is inferredthat the copolyester formed in a random manner and lowermolecular weight of the polymer is suitable for the biologicalapplication

34 DSC and TGA Analysis The 119879119892of the copolyester was

determined by DSC at a heating rate of 10∘Cminminus1 under ni-trogen atmosphere and the DSC thermograms of copolyes-ter 6 shows glass transition temperature (119879

119892) at minus37∘C as

presented in Figure 4 The convincing explanation for thislow glass transition temperature can be attributed to the factthat the pendant 3-(trifluoromethyl)styrene in the polymericstructurewas connected to the biscoumarin unit whichmadethe polymer chains unable to array regular and hinders theclose chain packing thus reduce the rigidity of the polymericstructure As expected 119879

119892was dependant on the structure of

diacid chloride component Thus it is evident that pendant


Hea

t flow

endo

Temperature (∘C)minus50 0 50 100

Tg = minus37∘C

Figure 4 DSC thermogram of random copolyester 6

100 200 300 400 500 600

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)

Figure 5 TGA curve of copolyester 6

group and flexible spacer decreases rigidity ultimately lead tolow glass transition temperature

The influence of polymer structure on the thermal prop-erties of copolyester was investigated by thermogravimetricanalysis (TGA) at a heating rate of 10∘C minminus1 in nitrogenatmosphere and the TGA curve was shown in Figure 5 Theinitial decomposition temperature (IDT) the temperature for10weight loss (119879

10) and themaximumdecomposition tem-

perature (119879max) of copolyesters are 251∘C 305∘C and 397∘C

Initial decomposition temperature and the temperature at10 w loss (119879

10) indicate its good thermal stability

35 XRD Analysis X-ray diffraction patterns of copolyester6 exhibit the essential amorphous nature of this randomcopolyester Copolyester exhibits some diffused degree ofcrystallinity at 2120579 range of about 7∘ 31∘ and 45∘ This obser-vation could be explained that the introduction of flexiblespacer is responsible for the least degree of crystallinityshown in X-ray diffraction pattern In addition the presenceof pendant 3-(trifluoromethyl)styrene reduced coplanarityhindered the dense packing of polymer chains and destroyed

10 20 30 40 50 60 70 80

0

50

100

150

200

250

300

350

Inte

nsity

(au

)

2120579

Figure 6 X-ray diffractogram of copolyester 6

0

5

10

15

20

25

Inhi

bito

ry zo

nes (

mm

)

Monomer 3Copolyester 6

E co

li

P ae

rugi

nosa

B su

btili

s

S a

ureu

s

A n

iger

A fl

avus

Monomer and copolyester

Figure 7 Comparative activities of monomer 3 and copolyester 6against various microbes

the ordered arrangement resulting in amorphous nature ofthese polyesters which is also reflected in their improved sol-ubility Copolyester 6 exhibits with conspicuous amorphoushalo at 2120579 range of about 15∘ to 30∘ as shown in Figure 6

36 Antibacterial and Antifungal Studies The purpose of an-tibacterial studies was to find out the efficacy of copolyester6 to inhibit the growth of microorganisms in the nearvicinity of surgical implantation The antibacterial activitiesof the synthesized monomer 3 [18 19] and the copolyester6 were tested against gram positive and gram negativebacteria and their results for the antibacterial zone they in-hibit are presented in Table 1 The comparative activities ofmonomer 3 and copolyester 6 against microbes are shownin Figure 7 Copolyester 6 with pendant 3-(trifluorometh-yl)styrene exhibited moderate to good activity against gram


Table 1 Zones of inhibition (in mm) of the compounds against various bacteria

Compound E coli P aeruginosa B subtilis S aureus20120583g 40 120583g 60120583L 20 120583g 40 120583g 60120583g 20 120583g 40 120583g 60 120583g 20 120583g 40 120583g 60120583g

Monomer 3 16 16 18 15 16 16 17 17 18 16 16 19Copolyester 6 18 18 19 17 18 18 18 19 20 19 19 20

Table 2 Zones of inhibition (in mm) of the compounds against fungus

Compound A niger A flavus20120583g 40 120583g 60 120583g 20120583g 40 120583g 60 120583g

Monomer 3 14 14 15 15 16 17Copolyester 6 15 15 16 17 18 19

Table 3 Anticancer activities of copolyester 6 on Hep-2 cell line

Serial number Concentration (120583gmL) Dilution Absorbance (OD) Cell viability ()1 1000 Neat 005 9612 500 1 1 010 19233 250 1 2 014 26924 125 1 4 019 36535 625 1 8 023 44236 312 1 16 027 51927 156 1 32 033 63468 78 1 64 039 75009 Cell control mdash 052 100

positive and gramnegative bacteria comparedwithmonomer3 this may be due to the enhanced lipophilicity characterof the copolyester From the data of antifungal activity itis observed that the compounds are highly active againstA flavus compared with A niger which exhibits moderateactivity as shown in Table 2 The cell wall contains manyphosphates carbonyl and cysteinyl ligands which maintainthe integrity of the membrane by acting as a diffusionbarrier Furthermore increased lipophilicity enhances thepenetration of the copolyester into lipid membrane anddisturbs the respiration process of the cell and thus blocks thesynthesis of the proteins which restricts further growth of theorganism

The results towards these microbes revealed that the in-corporation of pendant 3-(trifluoromethyl)styrene into thebiscoumarin monomer 3 of particular that the presence oftrifluoromethyl group at the meta position of styrene en-hanced its activity apart from this the polymerised productstill enhanced its overall activity due to the lipophilic charac-ter

37 Anticancer Evaluation of Copolyester Viable cells weredetermined by the absorbance Concentration required for a50 inhibition of viability (IC

50) was determined graphically

from Figure 9 The absorbance was measured with a UV-spectrophotometer using wells without sample containingcells as blanks The effect of the copolyester 6 on the prolif-eration ofHep-2 cell line was expressed as the cell viability

The affected Hep-2 cell line at different concentration wasshown in Figure 8 The covalent conjugation of biscoumarinbearing pendant 3-(trifluoromethyl)styrene through esterlinkage increases its molecular size and steric hindrancemay improve its cellular permeability and restricts its cancerprogression [20 21] It is explicit from Table 3 that low con-centration of copolyester induced greater anticarcinogenicactivity effects on Hep-2 cell line than the individual agents

4 Conclusion

In summary the present study allows the conclusions thatthe copolyester showed good solubility in various organic sol-vents and it is explicit from the DSC data that the copolyesterexhibits low glass transition temperature by maintaining itsthermal stability this is attributed to the incorporation ofpendant 3-(trifluoromethyl)styrene group in the biscoumarinunit The structures of the random copolyester were con-firmed by FT-IR and NMR spectroscopy And incorpo-ration of pendant 3-(trifluoromethyl)styrene decreases thepercentage of crystallinity which confirms the tendenciestowards amorphous nature of copolyester exhibited by X-ray diffraction measurements The cytotoxic assay showsthat the copolyester is toxic to the Hep-2 cell line in lowerconcentration It is explicit from the antimicrobial datathat the copolyester exhibits significant biological activityagainst Escherichia coli salmonella typhi Bacillus subtilisand Staphylococcus aureus These lines of evidence show that


(a) (b) (c)

(d) (e)

Figure 8 Anticancer evaluation of copolyester 6 onHep-2 cell line at various concentrations (a) normalHep-2 cell line (b) 1000 120583gmL (c)250 120583gmL (d) 125 120583gmL and (e) 625 120583gmL

0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)

Concentration (120583gmL)

Figure 9 Graphical representation of activities of copolyester 6 inthe MTT Assay

the copolyester can be preferred as biomaterial in various bio-medical applications

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors would like to thank CLRI technical persons fortheir analytical support

References

[1] H Ben Abderrazak A Fildier S Marque et al ldquoCyclic andnon cyclic aliphatic-aromatic polyesters derived from biomass

study of structures by MALDI-ToF and NMRrdquo EuropeanPolymer Journal vol 47 no 11 pp 2097ndash2110 2011

[2] Y Tsai C Fan C Hung and F Tsai ldquoSynthesis and character-ization of amorphous poly(ethylene terephthalate) copolymerscontaining bis[4-(2-hydroxyethoxy)phenyl]sulfonerdquo EuropeanPolymer Journal vol 45 no 1 pp 115ndash122 2009

[3] T Vlad-Bubulac and C Hamciuc ldquoAliphatic-aromatic copoly-esters containing phosphorous cyclic bulky groupsrdquo Polymervol 50 no 10 pp 2220ndash2227 2009

[4] Y Chen Y Yang J Su L Tan and Y Wang ldquoPreparation andcharacterization of aliphaticaromatic copolyesters based onbisphenol-A terephthalate hexylene terephthalate and lactidemiotiesrdquo Reactive and Functional Polymers vol 67 no 5 pp396ndash407 2007

[5] G Rabani H Luftmann andAKraft ldquoSynthesis and propertiesof segmented copolymers containing short aramid hard seg-ments and aliphatic polyester or polycarbonate soft segmentsrdquoPolymer vol 46 no 1 pp 27ndash35 2005

[6] M Bagheri K Didehban Z Rezvani and A A EntezamildquoThermotropic polyesters 1 synthesis characterization andthermal transition of poly[441015840-bis(120596-alkoxy)biphenyl isoph-thalate]rdquo European Polymer Journal vol 40 no 4 pp 865ndash8712004

[7] H Montes de Oca J E Wilson A Penrose et al ldquoLiquid-crys-talline aromatic-aliphatic copolyester bioresorbable polymersrdquoBiomaterials vol 31 no 30 pp 7599ndash7605 2010

[8] D Acierno R Fresa P Iannelli and P Vacca ldquoSegmentedliquid-crystalline polyesters with allyl groups as lateral sub-stituents Synthesis and characterizationrdquo Polymer vol 41 no11 pp 4179ndash4187 2000

[9] L-L Lin and J-L Hong ldquoMultiple melting behavior of athermotropic copolyester containing spirobicromane moietyrdquoPolymer vol 41 no 20 pp 7471ndash7481 2000

[10] T Ranganathan C Ramesh and A Kumar ldquoThermotropicliquid-crystalline polyesters containing biphenyl mesogens inthe main chain the effect of connectivityrdquo Journal of Polymer


Science A Polymer Chemistry vol 42 no 11 pp 2734ndash27462004

[11] R M Tejedor L Oriol M Pinol et al ldquoPhotoreactive main-chain liquid-crystalline polyesters Synthesis characterizationand photochemistryrdquo Journal of Polymer Science A PolymerChemistry vol 43 no 20 pp 4907ndash4921 2005

[12] G Chen and R W Lenz ldquoLiquid crystal polymers XVIIIComparison of the properties of aromatic polyesters with dif-ferent mesogenic units and a common flexible spacerrdquo Journalof Polymer Science A-1 Polymer Chemistry vol 22 no 11 pp3189ndash3201 1984

[13] M Bagheri K Didehban and A A Entezami ldquoThermotropicpolyesters (part 3) synthesis characterization and thermaltransition of random copolyesters containing terephthalate andisophthalate unitsrdquo Iranian Polymer Journal (English Edition)vol 13 no 4 pp 327ndash334 2004

[14] L-L Lin and J-L Hong ldquoSemi-rigid thermotrophic polyestercontaining a rigid bent spirobicromanemoieties-primary char-acterizations and the thermal behaviorrdquo Polymer vol 41 no 12pp 4501ndash4512 2000

[15] L Lin and J Hong ldquoEffect of isotropization on the thermalbehavior of two thermotropic copolyesters containing spirobi-cromane moietiesrdquo Polymer vol 42 no 3 pp 1009ndash1016 2001

[16] Y Chen R Wombacher J H Wendorff J Visjager P Smithand A Greiner ldquoDesign synthesis and properties of new bi-odegradable aromaticaliphatic liquid crystalline copolyestersrdquoBiomacromolecules vol 4 no 4 pp 974ndash980 2003

[17] D D Perrin and W L F Armarego Purification of LaboratoryChemicals Pergamon Press New York NY USA 1989

[18] A A Al-Rifai M T Ayoub A K Shakya K A Abu Safieh andM S Mubarak ldquoSynthesis characterization and antimicrobialactivity of some new coumarin derivativesrdquo Medicinal Chem-istry Research vol 21 no 4 pp 468ndash476 2012

[19] S RehmanM Ikram R J Baker et al ldquoSynthesis characteriza-tion in vitro antimicrobial and U2OS tumoricidal activities ofdifferent coumarin derivativesrdquo Chemistry Central Journal vol7 no 1 article 68 2013

[20] A Slusarz N S Shenouda M S Sakla et al ldquoCommonbotanical compounds inhibit the hedgehog signaling pathwayin prostate cancerrdquo Cancer Research vol 70 no 8 pp 3382ndash3390 2010

[21] A Barve T O Khor XHao et al ldquoMurine prostate cancer inhi-bition by dietary phytochemicalsmdashcurcumin and phenyethyli-sothiocyanaterdquo Pharmaceutical Research vol 25 no 9 pp 2181ndash2189 2008

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O

OH

OCF3

CF3

O

H2

+

O

OH OH

O

3

100∘C

1 2

OO

Scheme 1 Synthesis of monomer 3

cancers The aim of the present investigation is to synthesizeand characterize a random copolyester containing biologicalactive biscoumarin group bearing 3-(trifluoromethyl)styreneby inherent viscosity measurements solubility tests FTIR1H-NMR spectroscopy X-ray diffraction analysis (XRD)thermogravimetric analysis (TGA) and differential scanningcalorimetry (DSC) The synthesized copolyester was sub-jected to in vitro anticancer activity against lung cancer (Hep-2) cell line and antimicrobial studies

2 Experimental

21 Materials and Methods 4-Hydroxycoumarin (Aldrich)was used as such 3-(trifluoromethyl) Cinnamaldehyde hy-droquinone azeloyl chloride dimethylacetamide and pyri-dine (Aldrich) were used as received Solvents were purifiedaccording to the standard procedure in the literature [17]Hep-2 cell lines were obtained from National Centre for CellSciences Pune (NCCS) The cells were maintained in mini-mal essential medium (MEM) supplemented with 10 FBSpenicillin (100 120583gmL) and streptomycin (100 120583gmL) in ahumidified atmosphere of 50 120583gmL CO

2at 37∘C MEM was

purchased from Hi Media Laboratories Fetal bovine serum(FBS) was purchased from Cistron laboratories Trypsinmethyl thiazolyl diphenyl-tetrazolium bromide (MTT) anddimethyl sulfoxide were purchased from Sisco Research Lab-oratory Chemicals Mumbai

Inherent viscosity was determined at a polymer con-centration of 05 gdL in NMP at 30∘C using an Ubbelohdesuspended level viscometer 3 (wv) solutions were taken asa standard for solubility of copolyester in various solvents FT-IR spectra were recorded on Perkin Elmer 883 spectropho-tometer 1H-NMR spectra for monomer and polymer wererecorded on a Bruker 400MHz spectrometer using CDCl

3as

a solventThermogravimetric analysis (TGA) was performedin nitrogen at 10∘Cmin with a TGA instrument SDT Q600Differential scanning calorimetry (DSC) was performed withDSC 200 F3 Maia instrument at a heating rate of 10∘Cminunder nitrogen atmosphereGel permeation chromatography(GPC) analysis was used to determine number averagemolecular weight (119872

119899) and weight average molecular weight

of the polymer (119872119908) by WATERS 501 equipped with three

ultrastyragel columns and a differential refractive indexdetector using THF as eluent and polystyrene standards wereemployed for calibration The flow rate was 1mLmin X-Ray

diffraction measurements were recorded on a Bruker XRDD8 FOCUS using Cu K120572 radiation source

Antibacterial activity of random copolyester was deter-mined by disc diffusion method on Muller Hinton agar(MHA) medium and Sabouraud Dextrose agar (SDA)medium respectively Both Muller Hinton agar and Sabour-aud Dextrose agar media were poured into the petri plateAfter the medium was solidified the inoculums were spreadon the solid plates with sterile swab moistened with thebacte- rial suspension The discs were placed in MHA andSDA plates and 20 120583L of sample concentration was addedEach sample was placed in the discThe plates were incubatedfor 24 h at 37∘C Then the antimicrobial activity was deter-mined by measuring the diameter of zone of inhibitionStandard antibiotic ampicillin (20 120583gdisc) was used asreference Fresh bacterial cultures of gram negative bacterianamely Escherichia coli (MTCC-729) and Salmonella typhi(MTCC-531) and gram positive bacteria Bacillus subtilis(MTCC-2274) and Staphylococcus aureus (MTCC-3160) wereused for the antibacterial test Antifungal activity of extractswas determined by disc diffusion method on SabouraudDextrose agar medium Disc diffusion assay (DDA) canalso be performed for screening by standard method Stockcultures weremaintained at 4∘C on nutrient agar slant Activecultures for experiments were prepared by transferring aloop full of culture from the stock cultures into the test tubescontaining Sabouraud Dextrose broth which were incubatedfor 24 h at 37∘C Aspergillus Niger and Aspergillus Flavuswerethe fungi which were used for the study

22 Synthesis of Monomer 3 Mixture of 4-hydroxycoumarin1 (2 g 4mmol) and 3-(trifluoromethyl) cinnamaldehyde 2(12 g 2mmol)wasmixed thoroughly andheated in oil bath at100∘C (Scheme 1) After 30min the sample was tested by TLCand it was found that there were nomonomers present whichindicates the completion of reaction The reaction mixturewas then cooled to room temperature Hot ethanol (10mL)was added to the solid mass The solid product obtained wasfiltered dried and recrystallized using ethanol to give yellowcolour powder (26 g 838) 119877

119891 025 (119899-hexane EtOAc-

1 1) 1H-NMR (400MHz CDCl3) 120575 (ppm) 1177 (s 1H)

1128 (s 1H) 804ndash802 (t 2H) 763ndash760 (m 2H) 758 (s 1H)756ndash754 (d 1H) 748ndash747 (d 1H) 743ndash737 (m 5H) 682ndash677 (dd 1H) 658ndash654 (dd 1H) 551ndash550 (m 1H)


O

OH

OOOH

O

CF3

CF3

OO

OO

O

O

O O

OO

n

+ CCH2C7

O

O

Cl Cl HOOH

PyridineDMAc3

5

6

4

+












3500 3000 2500 2000 1500 1000 500


Monomer 3

Copolyester 6

1726 1648 1104





3as solvent at



OH OH

O OOO

CF3

102

100

680 675 670 665 660 655

091

090

203

213

104

107

103

520

102

100

102

13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

102

203

213

104

107

103

520

552 550 805 765 760 755 750 745 740





13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

OOO

O

OO

O O

OO

CF3

n

a

a

b

b

c

c

d

d
















Hea

t flow

endo


Tg = minus37∘C


100 200 300 400 500 600

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)









10 20 30 40 50 60 70 80

0

50

100

150

200

250

300

350

Inte

nsity

(au

)

2120579


0

5

10

15

20

25

Inhi

bito

ry zo

nes (

mm

)


E co

li

P ae

rugi

nosa

B su

btili

s

S a

ureu

s

A n

iger

A fl

avus




















4 Conclusion



(a) (b) (c)

(d) (e)


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)






Acknowledgment


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




O

OH

OOOH

O

CF3

CF3

OO

OO

O

O

O O

OO

n

+ CCH2C7

O

O

Cl Cl HOOH

PyridineDMAc3

5

6

4

+












3500 3000 2500 2000 1500 1000 500


Monomer 3

Copolyester 6

1726 1648 1104





3as solvent at



OH OH

O OOO

CF3

102

100

680 675 670 665 660 655

091

090

203

213

104

107

103

520

102

100

102

13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

102

203

213

104

107

103

520

552 550 805 765 760 755 750 745 740





13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

OOO

O

OO

O O

OO

CF3

n

a

a

b

b

c

c

d

d
















Hea

t flow

endo


Tg = minus37∘C


100 200 300 400 500 600

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)









10 20 30 40 50 60 70 80

0

50

100

150

200

250

300

350

Inte

nsity

(au

)

2120579


0

5

10

15

20

25

Inhi

bito

ry zo

nes (

mm

)


E co

li

P ae

rugi

nosa

B su

btili

s

S a

ureu

s

A n

iger

A fl

avus




















4 Conclusion



(a) (b) (c)

(d) (e)


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)






Acknowledgment


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




OH OH

O OOO

CF3

102

100

680 675 670 665 660 655

091

090

203

213

104

107

103

520

102

100

102

13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

102

203

213

104

107

103

520

552 550 805 765 760 755 750 745 740





13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

OOO

O

OO

O O

OO

CF3

n

a

a

b

b

c

c

d

d
















Hea

t flow

endo


Tg = minus37∘C


100 200 300 400 500 600

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)









10 20 30 40 50 60 70 80

0

50

100

150

200

250

300

350

Inte

nsity

(au

)

2120579


0

5

10

15

20

25

Inhi

bito

ry zo

nes (

mm

)


E co

li

P ae

rugi

nosa

B su

btili

s

S a

ureu

s

A n

iger

A fl

avus




















4 Conclusion



(a) (b) (c)

(d) (e)


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)






Acknowledgment


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




13 12 11 10 9 8 7 6 5 4 3 2 1 0

(ppm)

OOO

O

OO

O O

OO

CF3

n

a

a

b

b

c

c

d

d
















Hea

t flow

endo


Tg = minus37∘C


100 200 300 400 500 600

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)









10 20 30 40 50 60 70 80

0

50

100

150

200

250

300

350

Inte

nsity

(au

)

2120579


0

5

10

15

20

25

Inhi

bito

ry zo

nes (

mm

)


E co

li

P ae

rugi

nosa

B su

btili

s

S a

ureu

s

A n

iger

A fl

avus




















4 Conclusion



(a) (b) (c)

(d) (e)


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)






Acknowledgment


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




Hea

t flow

endo


Tg = minus37∘C


100 200 300 400 500 600

0

20

40

60

80

100

Wei

ght (

)

Temperature (∘C)









10 20 30 40 50 60 70 80

0

50

100

150

200

250

300

350

Inte

nsity

(au

)

2120579


0

5

10

15

20

25

Inhi

bito

ry zo

nes (

mm

)


E co

li

P ae

rugi

nosa

B su

btili

s

S a

ureu

s

A n

iger

A fl

avus




















4 Conclusion



(a) (b) (c)

(d) (e)


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)






Acknowledgment


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials


















4 Conclusion



(a) (b) (c)

(d) (e)


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)






Acknowledgment


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b) (c)

(d) (e)


0

20

40

60

80

100

120

1000 500 250 125 625 312 156 78 Cellcontrol

Cel

l via

bilit

y (

)






Acknowledgment


References
































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Documents

Facile Synthesis, Characterization, and In Vitro Antimicrobial and