Synthesis, characterization, in vitro antimicrobial and anticancer evaluation of random copolyesters bearing biscoumarin units in the main chains

Article abstract of DOI:10.1007/s11164-014-1806-3

A series of aliphatic-aromatic random copolyesters bearing a biscoumarin group were synthesized by phase-transfer-catalyzed interfacial polycondensation of 3,3′-methylene-bis(4-hydroxycoumarin)1 and aromatic diols such as hydroquinone, resorcinol, ethyl resorcinol, bisphenol-A, and curcumin with sebacoyl chloride. These copolyesters were obtained with yields in the range between 76 and 85 % and the inherent viscosity between 0.22 and 0.33 dl/g. All the copolyesters were found to be soluble in chlorinated and polar aprotic solvents. The chemical structures of the copolyesters were analyzed by Fourier transform infrared spectroscopy and proton nuclear magnetic resonance (¹H-NMR) spectroscopy. The physical properties of copolyesters obtained by altering the aromatic diols 3, were characterized by thermogravimetric analysis, differential scanning calorimetry, gel permeation chromatography, and X-ray diffraction (XRD) technique. Copolyesters exhibited good thermal stability having a decomposition temperature above 223 °C. Copolyesters with the bisphenol group as backbone exhibited higher thermal stability than others. Results revealed that the copolyesters exhibit a glass transition temperature in the range of -11 to 54 °C. It was found that copolyester with ethyl resorcinol and curcumin shown low and high T _g values, respectively. XRD measurement revealed the amorphous nature of copolyester with a low degree of crystallinity. Agar disc diffusion method was employed to study the antimicrobial activity of these random copolyesters. The synthesized copolyester was subjected to in vitro anticancer activity against lung cancer (Hep-2) cell line.

Full text of DOI:10.1007/s11164-014-1806-3

Res Chem Intermed
DOI 10.1007/s11164-014-1806-3
Synthesis, characterization, in vitro antimicrobial
and anticancer evaluation of random copolyesters
bearing biscoumarin units in the main chains
Narendran Kandaswamy Nanthini Raveendiran
•
Received: 9 June 2014 / Accepted: 2 September 2014
Ó Springer Science+Business Media Dordrecht 2014
Abstract A series of aliphatic–aromatic random copolyesters bearing a bis-
coumarin group were synthesized by phase-transfer-catalyzed interfacial polycon-
0
densation of 3,3 -methylene-bis(4-hydroxycoumarin)1 and aromatic diols such as
hydroquinone, resorcinol, ethyl resorcinol, bisphenol-A, and curcumin with seba-
coyl chloride. These copolyesters were obtained with yields in the range between 76
and 85 % and the inherent viscosity between 0.22 and 0.33 dl/g. All the copoly-
esters were found to be soluble in chlorinated and polar aprotic solvents. The
chemical structures of the copolyesters were analyzed by Fourier transform infrared
1
spectroscopy and proton nuclear magnetic resonance ( H-NMR) spectroscopy. The
physical properties of copolyesters obtained by altering the aromatic diols 3, were
characterized by thermogravimetric analysis, differential scanning calorimetry, gel
permeation chromatography, and X-ray diffraction (XRD) technique. Copolyesters
exhibited good thermal stability having a decomposition temperature above 223 °C.
Copolyesters with the bisphenol group as backbone exhibited higher thermal sta-
bility than others. Results revealed that the copolyesters exhibit a glass transition
temperature in the range of -11 to 54 °C. It was found that copolyester with ethyl
resorcinol and curcumin shown low and high T values, respectively. XRD mea-
g
surement revealed the amorphous nature of copolyester with a low degree of
crystallinity. Agar disc diffusion method was employed to study the antimicrobial
activity of these random copolyesters. The synthesized copolyester was subjected to
in vitro anticancer activity against lung cancer (Hep-2) cell line.
Keywords Biscoumarin copolyesters ꢀ Antimicrobial ꢀ Anticancer ꢀ Amorphous
N. Kandaswamy (&) ꢀ N. Raveendiran
Postgraduate and Research Department of Chemistry, Pachaiyappa’s College, Chennai 600 030,
India
e-mail: knarenchem@gmail.com
123

N. Kandaswamy, N. Raveendiran
Introduction
Polyesters are of great importance because of their low glass transition temperature,
high thermal stability, and excellent mechanical property, which can withstand
extreme conditions. Of particular interest, aromatic aliphatic polymers find versatile
applications in various fields. A probe through the literature indicates that there has
been rising interest in synthesizing aromatic aliphatic copolyester [1–6] as
biomaterials [7, 8] in a variety of medical disciplines. In spite of the unique
properties such as thermal stability, good chemical resistance, and excellent
mechanical strength, polymers have been significantly limited by their fabrication
process due to the high glass transition temperature and poor solubility in common
organic solvents. Extensive studies have been made to sort out the problems of
solubility and processability of copolyesters. The main focus of research is to
develop polymers by using suitable monomers, stoichiometry, and experimental
conditions to improve the thermal properties, mechanical properties, and process-
ability. The incorporation of bulky monomers [3, 9] or the use of bent monomers [3,
0
1
0] containing biscoumarin units such as 3,3 -methylene-bis(4-hydroxycoumarin) 1,
containing polar carbonyl groups or the use of meta oriented aromatic units along
with introduction of flexible spacer [3, 10–15] in the polymer chain, tends to reduce
the interaction between the polymer chains and eventually leads to an increase in
free volume and solubility with enhance processability by retaining its thermal
stability. When considering a biomaterial for implantation, the first and foremost
requirement is nontoxic and chemically inert/active and sustainable. Biocompatible
means that the biomaterials should not form thrombi in the blood system, which
ultimately leads to tumor in surround tissues, instantly declined by the body.
Implantation of surgical devices such as screws, plates, sutures, prosthesis anchor,
staples, and valves has the potential to infect patients with various microbes. To
encounter this problem it is required to develop a drug-delivery device capable of
delivering an antimicrobial drug in the vicinity of surgical implants or the
implantation of antimicrobial polymer-coated medical devices has to be introduced.
Often, surgical implantation leads to adverse side effects such as pain and swelling.
Sometimes long-term inflammation leads to development of dysplasia. Coumarin
and its derivatives are one of the active classes of organic compounds possessing a
wide spectrum of biological activity including anticoagulant, estrogenic, antimi-
crobial, analgesic, antibacterial, antifungal, anti-inflammatory, antitumor, and anti-
0
HIV. Of particular interest 3,3 -methylene-bis(4-hydroxycoumarin) 1 exhibits anti-
proliferative activity with less toxicity.
By considering the above facts, it is required to develop a biomaterial possessing
both antimicrobial and anticancer activity. The aim of the present investigation is to
synthesize and characterize a series of random copolyesters containing a bioactive
biscoumarin group and different aromatic diols through aliphatic diacid chloride by
interfacial polycondensation technique [16]. The synthesis of proposed random
copolyesters is to investigate the influence of the nonlinear and bulky biscoumarin
group along with para or meta-oriented aromatic units on the properties such as
solubility and thermal behavior of the copolyesters. The derived copolyesters were
1
characterized by inherent viscosity measurements, solubility tests, FTIR, H-NMR
1
23

Random copolyesters bearing biscoumarin units
spectroscopy, X-ray diffraction analysis (XRD), gel permeation chromatography
(
(
GPC), thermogravimetric analysis (TGA), and differential scanning calorimetry
DSC). The synthesized copolyester was subjected to in vitro anticancer activity
against lung cancer (Hep-2) cell line and antimicrobial studies.
Experimental
Materials and methods
4-Hydroxycoamarin (Aldrich) was used as such. Formaldehyde, curcumin, resor-
cinol, ethylresorcinol, hydroquinone, bisphenol-A, sebacoyl chloride (Aldrich) were
used as received. Tetra-n-butylammonium bromide (TBAB) and sodium hydroxide
(
Merck, India) were used as received. Solvents were purified according to the
standard procedure in the literature [17]. Solvents like chloroform (CHCl3),
dichloromethane (DCM), tetrahydrofuran (THF), dimethylformamide (DMF),
dimethylsulfoxide (DMSO), dimethylacetamide (DMAc) and N-methylpyrrolidone
(
NMP) were used for determining the solubility behavior of copolyesters. The
0
melting point of 3,3 -methylene-bis(4-hydroxycoumarin) was determined by open
capillary method. Inherent viscosity was measured at a polymer concentration of
0
.5 g/dl in NMP at 30 °C using an Ubbelohde suspended-level viscometer. 5 %
w/v) solutions were taken as a criterion for solubility of copolyesters in various
(
solvents at room temperature or upon heating. FTIR spectra were recorded on
1
PerkinElmer 883 spectrophotometer. H-NMR spectra for monomer and polymers
were recorded on a Bruker 400-MHz spectrometer using CDCl or DMSO-d as a
3
6
solvent at room temperature. Molecular weight of copolyesters was measured on a
Waters 501 GPC equipped with a polystyrene-divinylbenzene column and a
differential refractive index detector. Polystyrene was employed as calibration
-
1
standard and chloroform as solvent. TGA was performed in nitrogen at 10 °C min
with a TGA instrument SDT Q600. DSC was performed with a DSC 200 F3 Maia
-
1
instrument at a heating rate of 10 °C min under a nitrogen atmosphere. XRD
measurements were recorded on a Bruker XRD D8 FOCUS using Cu-Ka radiation
source.
Antibacterial activity of random copolyesters was determined by disc diffusion
method on Mueller-Hinton agar (MHA) medium and Sabouraud dextrose agar
(
SDA) medium, respectively. Both MHA and SDA media were poured into the Petri
plate. After solidification, the inoculums were spread on the solid plates with a
sterile swab moistened with the bacterial suspension. The discs were placed in MHA
and SDA plates and 20 ll of sample concentration was added. Each sample was
placed in the disc. The plates were incubated for 24 h at 37 °C. The antimicrobial
activity was then determined by measuring the diameter of zone of inhibition.
Standard antibiotic ampicillin was used as a reference. Fresh bacterial cultures of
Gram-negative bacteria namely Escherichia coli and Salmonella typhi and Gram-
positive bacteria Bacillus subtilis and Staphylococcus aureus were used for the
antibacterial test. Antifungal activity of extracts was determined by disc diffusion
method on SDA medium. Disc diffusion assay (DDA) can also be performed for
123

N. Kandaswamy, N. Raveendiran
screening by standard method. Stock cultures were maintained at 4 °C on Nutrient
agar Slant. Active cultures for experiments were prepared by transferring a loop full
of culture from the stock cultures into the test-tubes containing Sabouraud dextrose
broth, which were incubated at 24 h at 37 °C. Aspergillus niger and Aspergillus
flavus were the fungi that were used for the study. Amphotericin-B was used as
standard. Hep-2 cell lines were obtained from National Centre for Cell Sciences, Pune
(
NCCS). The cells were maintained in Minimal Essential Medium (MEM) supple-
mented with 10 % FBS, penicillin (100 lg/ml), and streptomycin (100 lg/ml) in a
humidified atmosphere of 50 lg/ml CO at 37 °C. MEM was purchased from Hi
2
Media Laboratories. Fetal bovine serum (FBS) was purchased from Cistron
Laboratories. Trypsin, methylthiazolyl diphenyl-tetrazolium bromide (MTT), and
DMSO were purchased from Sisco research laboratory chemicals Mumbai.
0
Synthesis of 3,3 -methylene-bis(4-hydroxycoumarin)1
A mixture of 40 % formaldehyde solution (0.8 g, 1.0 mmol) and 4-hydroxycoum-
arin (3 g, 2.0 mmol) was taken in 100 ml of distilled water in a round-bottomed
flask. With vigorous stirring, the reaction temperature was maintained at 70–75 °C.
After 1 h, the aliquot was tested by TLC and found that there were no monomers
present, which indicates the completion of the reaction. The solid obtained was
filtered, dried, and recrystallized using DCM/methanol (25:75) to give a white
1
powder (2.6 g, 83.8 %). R : 0.25 (n-hexane: EtOAc-1:1). mp: 272–275 °C. H-NMR
f
(
400 MHz, CDCl ): d (ppm): 11.32 (s, 2H); 7.988–8.00 (d, 2H); 7.57–7.61 (t, 2H);
.34–7.39 (t, 4H); 3.84 (s, 2H). FTIR: c (cm ) 3,647, 3,064, 1,650, 1,596, 1,565,
,453, 1,344, 765.
3
-1
7
1
Copolymerization procedure
3
In a three-neck round-bottomed flask (500 cm in volume) equipped with a
0
mechanical stirrer, dry nitrogen inlet, outlet and dropper, a mixture of 3,3 -
methylene-bis(4-hydroxycoumarin) 1 and aromatic diol 3 each (0.002 mol) was
taken. Sodium hydroxide (0.008 mol) dissolved in 80 ml of distilled water was
added and vigorously stirred for 5 min until the appearance of clear solution and
then 2 ml of 2 % TBAB was added. An amount of 0.004 mol of appropriate acid
dichloride, dissolved in 30 ml of dry methylene chloride was slowly added for
2
0 min at room temperature with vigorous stirring and the stirring was continued for
a further period of 1.5 h at 2,000 rpm. The reaction mixture was then poured into
methanol; the precipitated polymer was filtered and washed several times with
water. The polymer was dissolved in chloroform and reprecipitated in methanol to
remove methylated ester impurity and dried under reduced pressure at 80 °C for
2
4 h.
In vitro assay for anticancer activity (MTT assay)
The MTT assay was performed as first described by Mosmann with the
5
modifications suggested by Denizot and Lang. Cells (1 9 10 /well) were plated
1
23

Random copolyesters bearing biscoumarin units
in 24-well plates and incubated in 37 °C with 5 % CO condition. After the cell
2
reaches the confluence, the various concentrations of copolyester were added and
incubated for 24 h. After incubation, the sample was removed from the well and
washed with phosphate-buffered saline (pH 7.4) or MEM without serum. An
amount of 100 ll/well (5 mg/ml) of 0.5 % 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl–tetrazolium bromide (MTT) was added and incubated for 4 h. After
incubation, 1 ml of DMSO was added in all the wells. The absorbance at 570 nm
was measured with a UV spectrophotometer using DMSO as the blank. Measure-
ments were performed and the concentration required for a 50 % inhibition (IC )
5
0
was determined graphically. The % cell viability was calculated using the following
formula:
%
cell viability ¼ ðA570 of treated cells=A570 of control cellsÞ ꢁ 100
Graphs were plotted using the % of cell viability in the Y-axis and concentration
of the sample in the X-axis. Cell control and sample control were included in each
assay to compare the full cell viability in cytotoxicity and anticancer activity
assessments.
Results and discussion
Synthesis of copolyesters
A new series of copolyesters containing biscoumarin units was synthesized by
phase-transfer catalyzed interfacial polycondensation technique as shown in
0
Scheme 1. This involves a reaction of one equivalent of 3,3 -methylene-bis(4-
hydroxycoumarin) 1 and one equivalent of aromatic diol 3 with two equivalents of
aliphatic acid dichloride in dichloromethane-aqueous sodium hydroxide system
using TBAB as a phase-transfer catalyst. During the progress of reaction, the
reaction mixture became hazy and the system was temporarily homogeneous and
the polymer became precipitated during the polycondensation process. The yield of
the copolyesters was found to be quantitative (76–85 %) as shown in Table 1. From
GPC that the number average molecular weight (M ) of the copolyester 4a and 4d
n
are 10,400 and 11,912 and weight average molecular weight (M ) are 12,012 and
w
1
2,824 with 1.15 and 1.07 as polydispersity index values, respectively. This
interfacial technique was chosen rather than a homogeneous system because it has
marked advantages such as easy operation and simplicity in product isolation.
Polymer characterization
FTIR analysis
Structural assignments of the peaks coincide with the spectral data of the FTIR
analysis. All the copolyesters show characteristic absorption bands due to carbonyl
123

N. Kandaswamy, N. Raveendiran
OH
O
OH
O
O
C
O
+
Cl
CH2 C Cl
8
+
HO Ar' OH
OO
1
2
3a - 3e
NaOH
Dichloromethane
TBAB
O
O
O
O
O C
CH2 C O Ar' O
8
n
OO
O
4
a - 4e
CH3
Ar' :
,
,
,
CH3
a
b
c
d
O
O
e
O
O
Scheme 1 Synthesis of aliphatic aromatic random copolyesters 4a–e
-
1
stretching of the ester group at around 1,722–1,725 cm . However, it is interesting
-
to find that all the spectra show the absorption band at 1,648 cm , corresponding to
1
0
the stretching of the carbonyl group of 3,3 -methylene-bis(4-hydroxycoumarin) 1
molecule. Therefore it can be inferred that the biscoumarin molecule is an integral
part of the polymer backbone. Stretching vibration of ester C–O appears at
-
,080–1,083 cm . All the copolyesters show absorption around 2,850–2,950 cm
1
-1
1
due to the presence of the alkyl group in the polymer chain. No significant
absorption band in the OH stretching region was detected, confirming that the
dihydroxy monomers effectively got involved in polymerization.
1
23

Random copolyesters bearing biscoumarin units
0
Table 1 Physical properties of aliphatic aromatic copolyesters derived from 3,3 -methylene-bis(4-hy-
droxycoumarin) 1 with various aromatic diols 3a–e
Copolyester Aromatic
diols
Yield
(%)
g
g)
inh (dl/
IDT
T
10
T
max
T
g
T
m
a
b
c
d
e
e
(°C)
(°C)
(°C)
(°C)
(°C)
4
4
4
4
4
a
a
b
c
d
e
3a
3b
3c
3d
3e
85
78
72
82
76
0.26
0.24
0.22
0.33
0.30
231
229
223
263
221
275
262
267
292
259
321
371
355
407
310
17.8
63
61
43
64
-0.6
-11
18.1
54.3 110
g
inh of the copolyesters were measured in NMP at 30 °C at the concentration of 0.5 g/dl
b
-1
Initial decomposition temperature determined by TGA in nitrogen at heating rate 10 °C min
Temperature of 10 % weight loss
c
d
Maximum polymer decomposition temperature
e
Glass transition and melting temperature measured by DSC in nitrogen atmosphere at heating rate
-
1
1
0 °C min
1
Fig. 1 H-NMR spectrum of copolyester 4d in CDCl
3
1
H-NMR analysis
1
The H-NMR spectrum of the copolyester 4d and 4e is highlighted in Figs. 1 and 2.
The 4e spectrum shows characteristic signals in the range of 8.02–7.06 ppm due to
the aromatic protons of biscoumarin and curcumin. The a, b unsaturated protons of
the curcumin group expected to appear at about 7.53 ppm, buried along with the
aromatic region corresponds to the b proton of the unsaturated carbon (signal b) and
a signal at 6.85–6.90 ppm corresponds to a proton attached to the carbonyl group
123

N. Kandaswamy, N. Raveendiran
1
Fig. 2 H-NMR spectrum of copolyester 4e in DMSO
(
signal c). The signal exceptionally appeared at 6.17 ppm (signal e) correspond to
methyne proton of the enol structure. The methoxy protons (signal f) of the
curcumin group and the bridged carbon bearing two protons (signal d) of the
biscoumarin group appeared at 3.88 and 3.85 ppm, respectively, thus confirming
that both curcumin and biscoumarin effectively get involved in polymerization. The
signal characterizing the methylene groups of suberoyl unit (CH , signal a) appears
2
at 2.39–1.27 ppm. The characteristic signals regarding the chemical structure of
1
copolyester 4a–d are as follows. 4a: H-NMR (400 MHz, CDCl , ppm): d 7.23–7.52
3
(
m, Ar–H), 7.08 (s, Ar–H), 3.82 (s, biscoumarin bridged –CH –), 1.25–2.76 (m,
2
1
sebacoyl unit). 4b: H-NMR (400 MHz, CDCl , ppm): d 6.90–7.52 (m, Ar–H), 3.82
3
1
s, biscoumarin bridged –CH –), 1.25–2.76 (m, sebacoyl unit). 4c: H-NMR
(
2
(
400 MHz, CDCl , ppm): d 6.90–7.52 (m, Ar–H), 3.82 (s, biscoumarin bridged
3
–
CH –), 2.32–2.36 (q, ethyl –CH –), 1.17 (t, ethyl –CH ), 1.34–2.78 (m, sebacoyl
2 2 3
1
unit). 4d: H-NMR (400 MHz, CDCl , ppm): d 6.75–6.95 (d, Ar–H), 7.22–7.26 (d,
3
Ar–H), 7.30–7.53 (m, biscoumarin Ar–H), 3.83 (s, biscoumarin bridged –CH –),
1
2
.65 (s, gem dimethyl unit), 1.25–2.76 (m, sebacoyl unit).
Polymer solubility
The solubility parameter is an important criterion that has to be considered for
polymer processing. The solubility data (Table 2) shows that the copolyester 4a–
1
23

Random copolyesters bearing biscoumarin units
Table 2 Solubility behavior of random copolyesters
Polymer
DMSO
DMF
DCM
CHCl
3
THF
Dioxane
DMAc
Acetone
NMP
4
4
4
4
4
a
b
c
d
e
??
??
??
??
??
??
??
??
??
??
??
??
??
??
??
??
??
??
??
??
?
?
??
??
??
??
??
-
-
-
-
-
?
?
?
??
??
??
??
?
?
? -
? -
? -
? -
Solubility: ??, soluble at room temperature; ?, soluble on heating; ? -, partial soluble or swollen on
heating; -, insoluble even on heating
e exhibited good solubility in chlorinated and polar aprotic solvents, which is
attributed to the presence of non-linear biscoumarin moiety along with different
flexible spacer leads to reduced rigidity of polymer chains [6]. All the copolyesters
are readily soluble in most of the polar solvents, which might be due to the presence
of the carbonyl group of biscoumarin unit in the polymer chain.
Inherent viscosity
The inherent viscosity of the copolyesters was determined in NMP at 30 °C at the
concentration of 0.5 g/dl using a Ubbelohde suspended-level viscometer. Polymers
with high molecular weights show higher viscosity values. In polymers with
comparable molecular weight, the rigid polymers exhibit relatively higher viscosity
values than flexible ones. From the values, it is evident that the polymer with the
bisphenol backbone shows comparatively high ginh than other polymers. From
Table 1, it is evident that the copolyesters with para-oriented aromatic diol 3a show
higher viscosity values than the meta-oriented one 3b, which is attributed to the fact
that meta orientation of the benzene ring reduces the rigidity, and hence effective
volume, leading to lower viscosity values. The effect of introduction of flexible
alkyl groups into the polymer backbone is very little, but measurable.
Thermal properties
The thermal properties of copolyesters were investigated by TGA at a heating rate
-
1
of 10 °C min in nitrogen atmosphere and the thermograms of the copolyesters
a–e are shown in Fig. 3. TGA data of all the copolyesters are summarized in
4
Table 1. The initial decomposition temperature (IDT) of all the copolyesters ranged
from 221 to 263 °C and the T10 values were in the range of 259–292 °C. Generally,
copolyesters with para-oriented monomeric diol are more thermally stable than
their respective meta-oriented types, which may be attributed to the compact
packing of polymer chains [18]. In addition, the length of the flexible spacer also has
a significant effect in deciding the thermal stability of copolyesters and it is explicit
from Table 1. Copolyester 4a is thermally more stable than 4b, which is attributed
to the difference in the orientation of the aromatic unit. It is observed that the
random copolyester 4d bearing bisphenol group shows higher thermal stability than
the others.
123

N. Kandaswamy, N. Raveendiran
4
4
4
4
4
a
b
c
d
e
1
00
8
6
4
2
0
0
0
0
0
100
200
300
400
500
600
Temperature (°C)
Fig. 3 TGA curves of biscoumarin copolyesters 4a–e
Glass transition temperature
The glass transition temperature (T ) of random copolyesters is determined by
g
-
1
DSC at a heating rate of 10 °C min under nitrogen atmosphere and presented
in Table 1. The thermograms are shown in Fig. 4. The glass transition temperature
is in the range of -11 to 54 °C. In general, incorporation of a bulky unit in the
polymer chain restricts free rotation and enhances the T value, but the presence of a
g
flexible spacer reduces the rigidity of the polymer chain and ultimately leads to
lower T values, which is explicit from Table 1. The bulky biscoumarin unit 1 and
g
the various aromatic diols 3a–e forming copolyesters along with longer flexible
spacers result in lower T values [1, 19]. Copolyester 4a containing hydroquinone
g
unit shows highest T value than its corresponding copolyester 4b bearing resorcinol
g
unit and this could be attributed to the difference in stereoregularity along the
polymer segments. Thus, the copolyester 4a maintains its segmental symmetry
(
para orientation) there by enhancing its rigidity and forms a closed-pack
arrangement [20, 21] and ultimately leads to a higher Tg value than its
unsymmetrical segmented (meta orientation) [22, 23] copolyester 4b. It is
interesting to observe that among the series of random copolyesters, the polymer
having a pendant ethyl unit in the resorcinol group exhibited lower T values,
g
whereas polymers with curcumin groups at the backbone show higher T values.
g
X-ray diffraction
X-ray diffraction patterns of copolyesters corroborated the semicrystalline and
amorphous character of random copolyesters. All the copolyesters showed wide
1
23

Random copolyesters bearing biscoumarin units
4
e
Tg=54.3
4
d
Tg=18.1
4
c
Tg=-11
4
a
Tg=17.8
-20
0
20
40
Temperature (°C)
60
80
100
120
Fig. 4 DSC thermograms of random copolyesters
4
a
4
b
d
4
4
e
1
0
20
30
40
50
2θ
Fig. 5 X-ray diffractograms of copolyesters
123

N. Kandaswamy, N. Raveendiran
1
23

Random copolyesters bearing biscoumarin units
halos over the range of 2h = 10–40 (Fig. 5), suggesting essentially the amorphous
nature of the derived polyesters. This could be attributed to the presence of bulky
biscoumarin group into the polymer backbone, which hinders the dense chain
packing of polymer chains, resulting in the amorphous nature of these polyesters,
which is also reflected in their improved solubility. However, the copolyesters 4a,
4
2
b, and 4d exhibit some degree of crystallinity, showing peaks at 2h & 17.5° and
0.1°.
Antibacterial activity
The results of antimicrobial activity are presented in Table 3 and the comparative
activities of monomer 1 and copolyesters against various microbes are shown in
Figs. 6, 7, 8, and 9. The monomer and the copolyesters are active against microbes
such as Gram-negative species, viz, E. coli and S. typhi, Gram-positive species viz,
B. subtilis and S. aureus with inhibitory zone ranges of 10.0–17.0 and
1
0.0–22.0 mm, respectively. All the copolyester exhibiting enhanced antibacterial
activity compared with the monomer 1 and this is due to the lipophilicity of the
copolyesters. In general, more lipophilic compounds can penetrate greater the
lipophilic cell membranes of Gram-positive bacteria, while less lipophilic
compound are more liable to penetrate the cell wall of Gram-negative bacteria. It
is explicit from Table 3 that of all the copolyesters, 4e exhibits extreme antibacterial
activity and this is attributed to the ability of a,b-unsaturated ketone from curcumin,
undergo a conjugated addition to a nucleophilic group like thiol group in an
essential protein of the microorganism. The presence of more lipophilic methoxy
0
moiety at the position 4,4 of curcumin and small percentage of end phenolic and
hydroxyl group of coumarin has contributed wide spectrum of antibacterial activity.
From the above discussion, it is clear that the structure of the copolyester has a
distinct effect on the antibacterial activities, thus proving that these could act as
efficient antibacterial polymers.
Fig. 6 Antibacterial activity against E. coli
123

N. Kandaswamy, N. Raveendiran
Fig. 7 Antibacterial activity against S. typi
Fig. 8 Antibacterial activity against B. subtilis
Antifungal activity
The antifungal activity of copolyesters was studied against two fungal species, viz.,
A. niger and A. flavus, and is graphically depicted in Figs. 10 and 11. Antifungal
activity of both monomer and copolyesters at various concentrations is shown in
Table 4. Both the copolyesters 4d and 4e exhibited very good activity against A.
niger, and A. flavus. Increased lipophilicity enhances the penetration of the
copolyester into lipid membrane and these copolyesters also disturb the respiration
process of the cell and thus block protein synthesis, which restricts further growth of
the organism. It is inferred that the lipophilicity character of copolyester enhances
the antifungal activity of the copolyesters.
1
23

Random copolyesters bearing biscoumarin units
Fig. 9 Antibacterial activity against S. aureus
Fig. 10 Antifungal activity against A. niger
Anticancer evaluation of copolyesters against lung cancer (Hep-2) cell line
Viable cells were determined by absorbance. The copolyester concentration
required for a 50 % inhibition of viability (IC ) was determined graphically from
5
0
Fig. 12. The effect of the copolyesters on the proliferation of Hep-2 cell line was
expressed as the % cell viability. The affected Hep-2 cell line at different
concentrations is shown in Table 5. The anticancer activity results indicated that
most of the copolyesters exhibited inhibition activity against with varying intensities
on cancer cell line. Copolyesters 4c and 4e exhibited significant antitumor activity
(IC50 = 64, 34.4 lg/ml) and possess ethyl resorcinol and curcumin moiety, which is
known to exhibit antitumor activity along with the biscoumarin unit. The covalent
conjugation of curcumin and biscoumarin through ester linkage increases its
123

N. Kandaswamy, N. Raveendiran
Fig. 11 Antifungal activity against A. flavus
Table 4 Inhibition zone (mm) of the compounds against fungus
Compound
A. niger
A. flavus
50 lg
100 lg
150 lg
50 lg
100 lg
150 lg
1
–
9
9
9
9
11
11
12
15
17
–
10
9
–
10
10
10
13
15
16
10
12
10
12
13
18
4
4
4
4
4
a
b
c
d
e
10
11
13
15
10
11
13
15
18
10
12
14
a
Std
a
Amphotericin (20 lg/ml) as standard
Fig. 12 Inhibitory effect of copolyesters concentration on Hep-2 cell line
1
23

Random copolyesters bearing biscoumarin units
Table 5 In vitro anticancer activity of synthesized compounds
Compound Viability %
a
IC50 (lbg/ml)
Hep-2
Sample concentration (lg/ml)
1,000 500
250
125
62.5
31.2
15.6
7.8
Cell
control
1
4
4
4
4
4
a
11.53 23.07 36.53 46.15 61.53 69.23 82.69 92.30 100
13.46 26.92 38.46 50.00 63.46 82.69 90.38 94.23 100
13.46 25.00 36.53 50.00 61.53 73.07 82.69 90.38 100
7.69 13.46 25.00 38.46 50.00 63.46 75.00 86.53 100
11.53 21.15 36.53 48.07 61.53 73.07 82.69 92.30 100
5.76 15.38 26.92 40.38 46.15 51.92 61.53 73.07 100
108.2
128.9
118.2
67.4
a
b
c
d
e
120.3
34.4
‘
‘IC50, compound concentration required to inhibit tumor cell proliferation by 50 %’’
b
Lung cancer cell line (Hep-2)
molecular size and steric hindrance may improve its cellular permeability and
restrict its cancer progression [24, 25]. It is explicit from Table 5 that a low
concentration of copolyester 4e and 4c induced greater anticarcinogenic activity
effects on the Hep-2 cell line than the individual agents.
Conclusions
In summary, aliphatic aromatic biscoumarin random copolyesters have been
0
synthesized via phase transfer-catalyzed interfacial polycondensation of 3,3 -
methylene-bis(4-hydroxycoumarin) and various aromatic diols with sebacoyl
chloride. All the copolyesters exhibited very good solubility in various organic
solvents and this is attributed to the incorporation of the bulky biscoumarin unit.
Inherent viscosities were in the range of 0.22–0.33 dl/g. The structures of the
1
random copolyesters were confirmed by FTIR and H-NMR spectroscopy. Low
glass transition temperatures observed from DSC and XRD patterns of the
copolyesters reveal that all these copolyesters are found to be amorphous in nature.
0
The copolyesters derived from 3,3 -methylene-bis(4-hydroxycoumarin) show no
weight loss below 223 °C, indicating its good thermal stability, and have a low glass
transition temperature value in the range -11 to 54 °C. Of particular interest,
copolyester 4d with stereoregularity and low glass transition temperature there by
maintaining good thermal stability are preferable as biomaterials in various
biomedical applications. On the basis of above-mentioned physical properties and
antimicrobial and anticancer results, these copolyesters exhibited promising results
in the field of biomaterials for further development.
Acknowledgments The authors would like to thank CLRI technical staff for their analytical support.
123

N. Kandaswamy, N. Raveendiran
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