SYNTHESIS, CHARACTERIZATION OF NOVEL FURAN BASED POLYMERIC

NANOPARTICLES AND THEIR BIOLOGICAL ACTIVITIES

Dakshayinichandrashekarachar1*, Chaitramallu M2,Rekha N D2, Devaraju kesagudu2 ,Ranjini P2
1

Deapratment of chemistry Government College for women, Mandya and Research scholar at yuvarajas college mysuru570005, India
2
Department of chemistry yuvarajas college, Department of biotechnology,JSS,ooty road,Mysuru,India

Abstract:
Objective: PEGylation( polymeric substance) is clinically proven and attract both scientific and
commercial interests, here we emphasize the overcome the drawback of solution phase methods, the five
membered ring, imidazolone moiety is present in a wide range of naturally occurring molecules, for
example furan is a five membered heterocyclic nucleus which contain oxygen atom as heteroatom having
a broad spectrum of biological activity and here we attempt PEGlated product, involved free carbonyl
terminal was used for conjugation via condensation. The solid matrix characterized by spectral studies
and biological activity.
Result: we prepared a series of PEGylated 3-(4-Acetyl-phenyl)-5-aryldine-2-furan-2-yl-3,5-dihydroimidazol-4-one with different aldehydes through Erlenmeyer reaction and condensation methods by
using PEG-aldehyde .These newly synthesized compounds were characterized by IR,
1
HNMR,MASS,SEM,DLS studies. All final compounds are screened for their antioxidant, antiinflammatory activities done through by DPPH, Nitric oxide radical scavenging, ferrous ion chelating,
Haemolytic assay and CAM assay.
Conclusion: furan based compounds having imidazolone moiety having greater importance in medicinal
chemistry. In this work we attempt to highlight those compounds which show good potential against
Biological activity. Of all the compounds 3c followed by 3b showed to be a potent in antioxidant and
Haemolytic activity. And in CAM assay, all the compounds are potent antiangiogenic molecules in vivo.
Keywords: PEGlyted 2-furyl-4-arylidine-5(4H)-oxazolones, celite-545, Erlenmeyer reaction,
condensation reaction, Diels order reaction, antioxidant, anti-inflammatory and Chorioallantoic
membrane assay.
Introduction
PEGylation of small organic molecule or drugs,
protein are conjugated to the distal end of PEG
carrier, here PEGs are used as conjugate agent.
This PEGylation is to avoid glycol and modified
polymer, which plays a vital role in drug
delivery, due to morphological behaviour shows
good potent against targeted one [1-4]. It
increases the solubility in water and chemical
stability.[5].Generally macromolecular PEGs
may block activity of small active agents at the
target dells via steric hindrance. Because of to
overcome, the low molecular weight PEGylation
(<10,000 Da) was employed, which is conjugate
chemically, enzymaticaly actively transferred
into their target sites, for reasonable attachments
are narmally called prodrug approach [6-9]. To
increase the drug load, different types of PEG
conjugations are employed such as branched ,
Forked and multi armed(star like ) PEGS for
examples like NKTR-102(PEG-irinotecon)EZN2208(PEG-SN38)&NKT R-105(PRG-docetaxel).
Branched or umbrella like structure, this
technology is preferred in protein or enzyme
PEGylation but is not applied as frequently with
small molecule [10-13].PEGylation of the most

commonly what we used for the antiinflammatory drug gentamycin, antimicrobial

drug amphotercin-B and curcumin which has
been used as anti-inflammatory, antioxidant and
anticancer effect, shows significant increase in
their activity at very lower concentrations
compared with their respective parent drugs [1417]. Here we synthesize some furan based
imidazolone compounds and adding some
melamine groups to increase the activity through
Erlenmeyer reaction, condensation reaction,
Diels order reaction and all the compounds are
converted in to nano size range from 1-360nm
using PEGylation and screened for antioxidant
activity using DPPH radical scavenging, Nitric
oxide, Ferrous ion chelating assay and
Haemolytic activity, CAM assay . The
compounds are confirmed through Mass, IR,
1HNMR, DLS and SEM.
Chemistry
Meterial and methods
The entire chemical purchased from Sigma and
SDfine chemicals, product obtained are
characterized by spectral studies and DLS, SEM,
which is obtained from IOE, University of

mysore,Mysuru. The PEG-CHO was prepared

according to procedure of Harris and et al.,
(l984)[18]. The products were monitored by TLC
technique and their melting point by an open
capillary method which is uncorrected.
Scheme-1
Preparation
nanoparticle

of

polymeric

Scheme-2
Preparation
of
polymeric
nanoparticle with melamine group

1. General procedure for the preparation of

the compounds (2a-d)
DMF/POCl3 mixture was prepared by addition of
DMF to POCl3 with constant stirring for 30
minutes maintaining 0-5 C. To this add
substituted
oxazolones,
Para
amino
acetophenone (1:1) ratio and catalytic amount of
Celite -545 were added and refluxed for 2-hours
for maintaining 0C. The solid was poured into
crushed ice allow to settle for few minutes filter
and purification done through column
chromatography [19].
1.1. General procedure for the preparation of
the compound (4a)
The substituted compound(2a)(0.01m)and 1,1(methylenedi-4,1-phenylene)
bismaleimide
(0.01m)in THF was refluxed for 4 hours at
60C,the un aromatized product called as DA
adduct(4a) and it is reversible in nature.
Therefore the product further refluxed by adding
2ml of acetic anhydride to offered aromatization.
The resultant product was poured in to crush icecold water washed, filter and air dried [20].
1.2. PEGylation of the compounds 2(a-d)
PEGylation of 3-(4-Acetyl-phenyl)-5-aryldine-2furan-2-yl-3,5-dihydro-imidazol-4-one
compounds were done through by precipitation
method. According to Raman Dhivya et al.,(2015)
[21]and

with

slight

modification,

all

the

substituted3-(4-Acetyl-phenyl)-5-aryldine-2furan-2-yl-3,5-dihydro-imidazol-4-one
compounds are dissolved in chloroform at lab
temperature with little amount of KOH pellets
and added drop wise into the PEG-CHO which is
dissolved in distilled water and stirred vigorously
for two hours, then kept it aside for 10 minutes
to separate the aqueous layer from organic layer .
Aqueous layer and excess of PEG-CHO removed
by washing organic layer with distilled water in

several times by centrifugation and made to boil

(40C) to evaporate CHCl3, which gave waxy
liquid, after cooling becomes a solid.

PEGylated compound of 3c ((Z)-1-(4acetylphenyl)-2-(furan-2y)-4(3,4,5-tri methoxy

benzylidene)-1H-imidizol-5(4H)-one-)
IR(KBr,cm-1);3160(Ar-HStr),
2882(aliphaCHstr),1649(C=O),
1063(CNStr)1HNMR
(400,MHz,CDCl3. /ppm); Peak at 3.648
(PEGbackbone),3.5(OCH3)2.34(CH2=CH2
-C=O),2,53(O-CH2-CH2),7.5(Ar-H);
ESIMS
+
(M/Z); 4916(M) .

Fig1. Graphical representation of formation

PEGylated compounds.
Preparation of (Z)-1-(4-acetylphenyl)-2(furan-2-yl)-4(furan-2-yl-methylene)-1Himidazol-5(4H)-one and 1, 1-(methylenedi-4,1phenylene)bismaleimide (A-B)adduct (4a)
Colour:Yellowsolid,Yield:90%MP>275C
.IR(KBr,cm-1);3167(Ar-HStr)
3030(aliphaCHstr),1709(C=O),1030(CN
Str);1HNMR
(400,MHz,CDCl3./ppm);2.5(CH3),3.9(CH2=CH
2-)6.9(H>C=C<H)
6.5-8.2(ArCH).
ESIMS
+
(M/Z); 996(M) .

PEGylated compound of 3d ((Z)-1-(4acetylphenyl)-2-(furan-2-yl)-4-(4-methoxy

bezylidene)-1H-imidazole-5(4H)-one)
IR (KBr, cm-1); 3160(Ar-H Str), 2882(aliphaCHstr),1649(C=O),
1063(CN
Str)1HNMR(400,MHz,CDCl3./ppm);Peak
at3.648 (PEG backbone),3.9(OCH3),2.34(CH2=CH2-C=O),2,53(O-CH2CH2),7.2(Ar-H);
ESIMS (M/Z); 4796(M) +.

2. Spectral studies of PEGylated compounds;

PEGylated compound of 3a ((Z)-1-(4acetylphenyl)-2-(furan-2-yl)-4(furan-2-ylmethylene)-1H-imidazol-5(4H)-one).
IR (KBr, cm-1); 3160(Ar-H Str), 2882 (aliphaCHstr), 1649(C=O), 1063 (CNStr). 1HNMR
(400, MHz,CDCl3 ./ppm); Peak at 3.648 (PEG
backbone) ,2.32(-CH2=CH2 -C=O),2,53(O-CH2CH2),7.2(Ar-H). ESIM (M/Z); 4717(M) +

PEGylated compound of 3b ((Z)-1-(4acetylphenyl)-4-benzylidene-2-(furan-yl)-1Himidazol-5(4H)-one-)

IR(KBr,cm-1);3160(Ar-HStr),
2882(aliphaCHstr),1649(C=O),1063(CN
Str).1HNMR
(400,MHz,CDCl3./ppm);Peak
at3.648
(PEGbackbone),2.32(-CH2=CH2-C=O) ,2,53(OCH2-CH2),7.2(Ar-H);ESIM(M/Z); 4737(M) +

DA adduct (A-B type)-PEG (4b)

IR(KBr,cm-1);3160(Ar-HStr),
2882(aliphaCHstr),1646(C=O),1063(CN Str ) 1HNMR
(400,MHz,CDCl3./ppm);Peak at 3.648 (PE G
backbone),2.34(-CH2=CH2-=C=O) 2.53(O-CH2CH2),7.2(Ar-H);

3. Biological activity
3.1Antioxidant assay

3.1.1. DPPH radical scavenging assay;

DPPH radical scavenging activity was done by
the method of Shone et al., (1998) with little
modifications. Briefly, one ml of DPPH solution
(0.1 mM in methanol) was incubated with
gradient concentrations (20g/ml to 100g/ml)
of the synthetic Imidazolone compounds, shaken
and incubated for 30 min at room temperature
and absorbance was read at 517nm against a
blank. BHT was used as reference compound.
The radical scavenging activity was measured as
decreases in the absorbance of DPPH and
calculated by using the following equation.
Radical scavenging potential was expressed
asIC50 value, which represents the sample
concentration at which 50% of the DPPH radical
were scavenged.
Scavenging effect= [1-sample absorbance
(517nm)/control absorbance (517nm) 100]
3.1.2. Nitric oxide radical scavenging assay;
Nitric oxide radical scavenging activity was
performed by the method of (Marcoci et.al,
1994), with minor modifications. Nitric oxide
was generated from Sodium nitroprusside, in
aqueous solution at 7.3 PH, spontaneously
generated nitric oxide reacts with oxygen to
produce nitrite ions that can be estimated by the
Griess reagent Scavengers of nitric oxide
compete with oxygen leading to reduced
production of nitric oxide.
Sodium
Nitroprusside (5mM) in Phosphate buffer saline
was mixed with the synthetic imidazolone
compounds are incubated at room temperature
for 60 min. The sample from the above was
reacted with Griess reagent (1% sulphanilamide,
2%H3PO4 and 1% naphthalene diamine
dihydrochloride). The absorbance of the
chromophore formed during the diazotization of
nitrite with sulphanilamide and coupling with
naphthalene diamine was read at 546 nm and
referred to the absorbance of BHT treated into
the same way with Griess reagent. The radical
scavenging activity was measured using the
equation described for DPPH radical scavenging
activity.
3.1.3. Ferrous ion chelating assay:
Ferrous ion chelating ability was measured
according to Gordon M.H. 1990 et al, method
29. For the assay, three sets of test tube were
taken. One tube was taken as control to this
FeCl3 (200 mM) and K3Fe (CN) 6 (400 mM)
were added and the volume was made up to 1 ml
by adding distilled water. For the second tube,
EDTA (40 mM), FeCl3 (200 mM) and K3Fe(CN)6

(400 mM) were added and the volume was made

up to 1 ml by adding distilled water. For the third
one, test compounds
imidazolone with
concentrations 20, 40, 60, 80 and 100 g, FeCl3
(200 mM) and K3Fe(CN)6 (400 mM) were added
and the volume was made up to 1 ml by adding
distilled water. The tube was incubated for 10
min at 20 C and the absorbance was read at 700
nm and ion chelating ability was calculated. The
anti-oxidant activity of all the compounds was
compared with that of BHT. Radical scavenging
activity was expressed as percentage;
3.2 Indirect study of haemolytic assay
A semi quantitative indirect haemolytic assay
(BOMAN and KALETTA, 1957) was employed.
Briefly, packed human erythrocytes, egg yolk
and phosphate buffer saline were mixed (1:1:8
v/v).one ml of this suspension was incubated
with 20g enzyme for 10 min at 37C.The
reaction was stopped by adding 10 ml of ice cold
phosphate buffer saline and centrifuged at 4C
for 10 min at 800xg.The amount of haemoglobin
released in the supernatant was measured at
540nm.
3.3. CAM assay
The fertilized eggs were incubated for 5 days at
37C in a humidified atmosphere. A window was
made under aseptic conditions on the eggshell to
check for proper development of the embryo.
The window was resealed and the embryo was
allowed to develop further. On the 11th day,
PEGylated of 3a, 3b, 3c, 3d, 4b was applied on
the Whatman filter disc, air dried and placed
over the CAM. The window was closed again
and the eggs were incubated for another 2 days.
The window was opened on the 13th day and
inspected for changes in the micro vessel density
in the area under the cover slip and photographed
[22-23].
4. Result and discussion
The compounds 2(a-d) are synthesized through
Erlenmeyer reaction, condensation and DA (AB)
reaction, made to react with PEG-CHO. These
are waxy solid matrix and easily soluble in water
and organic solvents, the size of these substances
from 1nm to 99 nm range.
The spectral data of IR absorption band
between,
1020-1090cm-1,
1709cm-1,27702900cm-1,3130-3314cm-1 due to C=N ,C=O ,CH
aliphatic and Aromatic C-H stretching, and of
PEGlyted compound the -C=0 stretching appear

at lower frequency due to

1649cm-1, instead of 1709cm-1.

at

Fig2. IR spectrum of 4a(a) and 3b(b)

1
HNMR shows that the shift 2.2- 2.5,-CH3,
3.2-3.9(-OCH3), 6.5-8.1( Ar-H, FuranH)
PEGylated compound of 1H NMR spectrum
shows peak at3.648 (PEG backbone) ,2.32(CH2=CH2-C=O),2,53(O-CH2-CH2), 7.2(Ar-H).
For the indication of the end group attachment of
drug to the polymer linearly.

Fig3. 1HNMR spectrum of 3a

Fig4. Mass Spectrum of compound 3a.

Fig5. 1HNMR spectrum of compounds 4a (a)

and 4b (b).
Mass spectra showed agreeable value for
proposed structures. I.e. the mass of the original
one is increased by PEGylation which was the
presence of end group attachment of the drug to
the polymer linearly.
4.1. Surface morphology
SEM images and histogram of the particle size
distributions of the PEGylated compound are
shown in fig.5 the average particle size obtained
1 to 300 nm respectively.
(a)

4.2Biological activity
Antioxidant assay:
3a
4a

3b
4b

3c
BHT

3d

100

% DPPH scavenging

50
0
20 40 60 80 100

Concentration in g/mL
Fig.7.DPPH scavenging assay of compounds 3a4b
250
200
150
100
% Nitric oxide scave n ging
50
3a
3b
3c
3d
0

4a

4b

BHT

C on ce ntration in g/m L

Fig.8.Nitric Oxide radical scavenging assay of

3a-4b
3a

3b

3c

3d

4a

4b

BHT
200
100
% Fe rrou s ion scave ngin g

Concentration in g/mL

Fig.9. Ferrous ion scavenging assay of

compounds 3a-4b
4.3. Haemolytic assay:

Fig6. SEM image (a) and (b) DLS of PEGylated

compounds.

3b

3c

3d

4b

3a

80
60
40

Fig.10. Haemolytic
compounds.

assay

of

PEGylated

4.4 Chorioallantoic membrane (CAM) assay:

%Haemolytic Inhibition 20
0

C on ce n tration in g/m L

Fig. 11. Angioinhibitory effect of 3a, 3b, 3c, 3d and 4b (100g) was dried on Whatmann discs and applied
onto the CAM of the developing chick embryo through a cut window. The CAM was observed for
inhibition of neovascularization. The data shown represents the result of an experiment which was done
using a minimum of six eggs in each group.
In the figure.6, 7, 8 the antioxidant activity of
PEGylated compounds 3c, 3d, 4b and followed
by 4a, were showing significant scavenging
activity indicating the potency of the molecules.
Some of the PEGylated compounds of nano size
above 90-399 nm are in lyophilic in nature
compared to the size below which is lyophobic
accelerate effectively on the biological activity
due to blockage of targeted moiety which poorly
executes the biological activity or there is an
increase in the activity due to the dosage or of
the proper orientation of targeted groups of the
compounds. Electron donating group like OCH3
and steric hindrance of trimethoxy groups
present in 3d and 3c were affected for the good
activity .This could be purely depending on the
morphological behaviour of the compounds. But
in the case of 4a the melamine group responsible
for the better antioxidant activity.

Among the five PEGylated nano compounds of

Haemolytic assay, 3c was found to be more
potent in inhibiting Phospholipase A2 enzyme
from the Russels viper snake venom with an
IC50 value of 30.66gs. followed by 3b with an
IC50 value of 85.34 g.3d and3a are showing
almost same activity with an IC50 value of
111.66gs and 113.31gs.Among all the
compounds 4b was found to be less potent with
an IC50 value of 139.48gs.
In Fig.11 which exhibit reduction of
angiogenesis in the CAM at the site of
application of compounds as compared to the
extensive angiogenesis seen in the control of
CAM. The data shown represents the results
using a minimum of six eggs in each group.
These results indicate that the five compounds,
3a,3b, 3c, 3dand 4b are potent antiangiogenic
molecules in vivo.

5. Conclusion
All synthesized compounds are screened for anti
oxidant and Haemolytic assay. In that the
compound 3c become good potent molecule,
because of having furan and imidazolone moeity
and steric hindrance of tri methoxy groups
present in that molecule, followed by 3b were
showed to be potency in the above Biological
activity but in the CAM assay all the compounds
are potent antiangiogenic molecules in vivo.
Moreover size of 99 -350 nm size and targeted
orientation of the groups are affected for the
biological activity.
Authorss contributions
Dakshayini and devaraju designed research
dakshayini
performed
the
research;
dakshayini,mallu and Devaraju analysed spectral
data; Rekha and Ranjini analyzed biological
data. Dakshayini and Rekha wrote the paper. All
the authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing
interests
Author details
1
Department of chemistry and Faculty of
Government College for women, Mandya.
Research scholar at Yuvarajas college, Mysuru,
University of Mysore, Mysuru-5.
2
Department of chemistry and research scholar at
yuvarajas college, Mysuru-5.
Department of Biotechnology, Faculty of JSS
College, Ooty road, Mysuru
Department of chemistry, Faculty of Yuvarajas
college, University of Mysore, Mysuru-5.
Department of Biotechnology, Maharani,s
science college,Mysuru-5
Acknowledgement
The author thankful to UGC to providing
opportunity for FIP facility to do this work and
thanks to IOE, university of Mysore,Mysuru for
the spectral studies, JSS college OOty Road for
Biological activity.
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