Synthesis, characterization and biological evaluation of ferrocene based poly(azomethene)esters

Article abstract of DOI:10.1016/j.jorganchem.2014.11.004

A new ferrocene containing poly(azomethine)ester (PFe) was synthesized by polycondensation of a preformed 4-((2-(4hydroxybenzylideneamino)ethylimino)methyl)phenol, (SB) and 1,1′-ferrocenyl dicarbonyl chloride. Various aliphatic and aromatic sequences (1,3

Full text of DOI:10.1016/j.jorganchem.2014.11.004

Journal of Organometallic Chemistry 779 (2015) 91e99
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis, characterization and biological evaluation of ferrocene
based poly(azomethene)esters
Asghari Gul a c, Sehrish Sarfraz , Zareen Akhter , Muhammad Siddiq , Saima Kalsoom ,
,
b
c
c
c
d
e
b, *
Fouzia Perveen , Farzana Latif Ansari , Bushra Mirza
Department of Physics, COMSATS IIT, Islamabad, Pakistan
Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Research Centre for Modeling and Simulations, National University of Science and Technology, Islamabad, Pakistan
Pakistan Council for Science and Technology, Shahra-i-jamhuriat, Islamabad 45320, Pakistan
a
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2014
Received in revised form
A new ferrocene containing poly(azomethine)ester (PFe) was synthesized by polycondensation of a
preformed 4-((2-(4hydroxybenzylideneamino)ethylimino)methyl)phenol, (SB) and 1,1 -ferrocenyl
0
dicarbonyl chloride. Various aliphatic and aromatic sequences (1,3-propan-diol, 1,6-hexan-diol,
poly(dimethylsiloxane), hydroxyl-terminated (n ¼ 550)), (1,1,1,3,3,3-hexaflouro)bisphenol propane and
bisphenol A) were incorporated in the parent chain to synthesize respective terpolymers. Spectroscopic
and elemental data were used to study structure of the prepared material which was then screened for
antibacterial, brine shrimp cytotoxic, free radical induced oxidative DNA damage and DPPH free radical
scavenging activity. Among all, PFeF showed growth inhibition of Staphylococcus aureus, Micrococcus
luteus, Bordetella bronchiseptica and Salmonella typhimurium. In brine shrimp cytotoxicity assay PFeF and
PFePr exhibited LD50 < 1 ppm showing highly cytotoxic behavior. Schiff base monomer (SB) significantly
inhibited the tumor (36.02%) and revealed higher radical scavenging activity (IC50 13.65 ppm) than
polymers. In general, all polymers (PAMEs) had good antioxidant and DNA protecting behavior. Molec-
ular docking and QSAR studies carried to investigate the interactions between polymers and double
stranded DNA showed good correlation of binding strength and activity of the material. The binding
2
2 October 2014
Accepted 6 November 2014
Available online 25 December 2014
Keywords:
Synthesis
Characterization
Poly(azomethine)esters
Biological evaluation
MEO docking
Binding constants
constant (K
b
) is indicative of the binding strength of the material with DNA. The high value of K
b
implied
that respective drugs bind to DNA most strongly.
©
2014 Elsevier B.V. All rights reserved.
Introduction
molecular modeling of ferrocene-poly(azomethine)ester is very
scarce. The stability of ferrocenyl group in aqueous, aerobic media
and its promising electrochemical properties make ferrocene and
its derivatives ideal for biological applications and conjugation
with biomolecules. Ferrocene is transformed into the ferrocenium
ion through one electron reversible oxidation however; sub-
stituents on the ferrocene moiety have the ability to influence this
redox behavior by altering level, HOMO [7e10]. So reversibility
may be lowered significantly. The low cytotoxity of ferrocene in
Poly(azomethine)esters have received considerable attention in
the recent years owing to their unusual properties such as thermal
stability, good mechanical strength, semiconducting behavior,
electronic, optoelectronic, nonlinear optical and electrochemical
properties [1]. They have potential applications in the field of
medicine due to their significant antifungal, antibacterial, anti-
tumor and antioxidant activities [1e6].
Moreover, assimilation of a ferrocenyl group into an organic
material often yields unexpected properties. An exhaustive survey
revealed that the literature on the biological evaluation and
biological system, its lipophillicity, the p-conjugated system and
the resulting exclusive electron-transfer ability make ferrocene-
polymer good candidate for investigation of their biological ap-
plications. Polycondensation is a straight forward procedure that
permits to use a large range of functionalized monomers resulting
in polymers with internal polar functions which could influence
the properties of material [11].
*
Corresponding author. Tel.: þ92 51 90643007; fax: þ92 51 9219888.
E-mail address: bushramirza@qau.edu.pk (B. Mirza).
http://dx.doi.org/10.1016/j.jorganchem.2014.11.004
022-328X/© 2014 Elsevier B.V. All rights reserved.
0

92
A. Gul et al. / Journal of Organometallic Chemistry 779 (2015) 91e99
The purpose of this study is to synthesize and characterize
The stoichiometry of the material was confirmed by
elemental (C, H, N) analysis which showed a good relation be-
tween the suggested structures and the experimental results.
The calculations were made on the basis of structure of repeat
unit present in the macro-chain. The data showed that in each
polymer, monomers were in equimolar proportion. However
somewhat higher contents of carbon found may be because of
ferrocenyl acid group which was not included in calculations. In
the case of condensation terpolymer comprising silanol (Si) in
chain, the elemental analysis was not possible because at higher
temperatures silicon interact with carbon to form ceramic ma-
terial [8,11].
poly(azomethine)esters with aliphatic/aromatic segments, to
examine these systems for biological activities and to study the
mechanism involved in the process by MOE docking.
Results and disscussion
The diol-terminated Schiff base (SB) was synthesized according
to the reported method, (Scheme 1) [8]. Poly(azomethine)ester, PFe
was prepared using low temperature solution phase poly-
condensation of monomer 4-((2-(4-hydroxybenzylideneamino)
ethylimino)methyl)phenol (SB) and diacid chloride (Fe), (Scheme
The UV/visible spectra were recorded qualitatively in ethanol
at room temperature to confirm the presence of different chro-
mophores in the PAMEs chain. The absorbance in the range
2
). The purpose of incorporation of different aliphatic/aromatic
diols in parent macromolecule is to study their effect on the prop-
erties of macro chains, (Scheme 3). The polymerization was carried
out using one-pot three-reactant reaction in an in situ process.
The reaction was carried out at atmospheric pressure to avoid
any side reaction and decomposition of thermally sensitive
monomers. The maintenance of low reflux temperature was
essential to assure reaction by dissolving diols completely in the
solvent [1,8,11]. The material was found soluble in common organic
solvents. FTIR, UV/vis and NMR techniques were used to confirm
the functional groups present in the monomer (SB), polymer (PFe)
and terpolymers (PFePr, PFeH, PFeSi, PFeB, PFeF).
3
20e392 nm showed pep* transition indicative of the presence
of PheN]CePh chromophore in the polymer backbone and the
bands appearing in region 418e435 nm are representative of
ded transition in the ferrocenyl group, in all PAMEs [8,11]. When
comparing UV/visible spectra of the polymers in each series a
hypochromic shift of the aromatic
pep* transition was noted,
possibly due to the formation of newly formed ester linkage. The
bathochromic shifting is owing to the extended conjugation,
which is possible in PFe, PFeB and PFeF while it is interrupted by
methylene and silyl spacers in PFeH, PFePr and PFeSi.
In FTIR spectra important changes were observed in the
spectra of initial and final products as some of the signals dis-
appeared and some new appeared. The IR spectrum of SB showed
Molecular weight determination by LLS technique
ꢀ1
a strong peak around 3400(s) cm indicating the presence of
hydroxyl group (hydroxybenzaldehyde) at the ends. The absence
Both static and dynamic laser light scattering analysis were
carried out with, a commercial LLS spectrometer Brookhaven
ꢀ
1
of sharp C]O peak in region 1700e1720(s) cm
coincidence
model BI-APD with HeeNe laser (
l
¼ 637 nm) and BI-9000AT
ꢀ1
with the presence of a band at 1620(s) cm showed that alde-
Digital Autocorrelator. All LLS measurements were carried out at
ꢁ
hydic group had been transformed to azomethine linkage (C]N)
25 C. The static LLS results are summarized in Table 1. The posi-
[
8,11,12].
The presence of absorption peak probable for the ester linkage
C]O), (CeO) along with the band for imine linkage appeared
tive values of A
the present polymers at 25 C. The A
2
indicate that the solvent used is good solvent for
ꢁ
2
value increases with the
(
decrease in the molecular weight, which indicates that the solvent
quality decreases with the increase in the molecular weight
[13,14].
It can be concluded from dynamic laser light scattering ex-
periments (performed at different angles) that different polymer
have different solvent contribution with their chains and thus
have various conformations due to different repeat units. The
hydrodynamic radius R
polymer chains, as it involves both the chain and the amount of
the solvent associated with, whereas the radius of gyration R is
related to the size of chain and in other words to the excluded
volume of polymer in the solvent and mostly depends on the
thermodynamic quality of the solvent [13e16]. It is known that
the ratio of the radius of gyration to the hydrodynamic radius, i.e.
ꢀ
1
ꢀ1
around
1720e1750(s)
cm
respectively confirmed the formation of
PAMEs/terpolymers [8,11,12]. In addition, the absence of broad peak
,
1101e1200(s)
cm
and
ꢀ
600e1645(s) cm
1
1
ꢀ
1
for OH group, around 3400 cm coincidence with the appearance
ꢀ
1
of a band for diacid chloride group in region 780e540 cm showed
the occurrence of CeCl group at the ends of macro chains. In
addition to the above peaks, absorption bands related to the
functional group present in the diols added to the chain were also
found in each spectrum in their respective region.
h
reflects the physical dimensions of the
g
1
H NMR spectrum of the SB recorded in deuterated DMSO,
exhibited all the characteristic signals confirming the presence of
azomethine linkage in the product [8,11,12].
The aromatic ring in the spectrum appeared in region
7
.8e6.8 ppm. The resonance for imine linkage appeared at 8.3 ppm
<R
<R
g
>/<R
h g
>, reflects the chain conformation. The ratio of <R >/
representing presence of two imine protons in the SB. The meth-
ylene group resonated at 1.2e0.9 ppm as triplet showing the
presence of four identical protons. The spectra of the soluble
organometallic material(PAMEs) was recorded under similar
conditions.
h
> ~1.0e2.0 indicates that the polymer have a coil chain
conformation in the solvent used [16].
The biological evaluation of material was carried out employ-
ing various essays and the mechanism was studied by MOE
docking.
Scheme 1. Synthesis of Schiff base (SB) [8].

A. Gul et al. / Journal of Organometallic Chemistry 779 (2015) 91e99
93
Scheme 2. Synthesis of poly(azomthine)esters, PFe.
Biological studies
polymers had LD50 greater than 1, showing less toxic behavior
Table 3). The results showed that all polymers expect PFeSi, had
(
PFeF was found most active antibacterial polymer tested
Table 2). It showed MIC 0.2 mg/ml for Staphylococcus aureus,
.4 mg/ml for both Enterobacter aerogenes and Micrococcus luteus,
which might be due to the presence of highly electronegative hexa
floro group in the macrochain [7,11,17,18]. Schiff base organome-
tallic polyesters, PFeB and PFeH showed significant antibacterial
behavior and resulted in formation of zones against Salmonella
typhimurium.
LD50 values lower than the value for Schiff base monomer.
Hence, it can be deduced that esterification of Schiff bases and
addition of certain groups has enhanced the cytotoxicity of
material in the dose dependent manner [7,17]. The test material
showed cytotoxic activity, hence, was tested for antitumor po-
tential. SB displayed 36% inhibition of tumors at 100 ppm which
was decreased by decreasing its concentration (Table 4), and
remained significant upto 0.1 ppm. From literature it is known
that Schiff bases and their derivatives are potential anticancer
agents [19].
(
0
In the case of Brine shrimp cytotoxic assay, PFeF and PFePr
were found highly cytotoxic with LD50 < 1 whereas the other
Scheme 3. Synthesis of terpolymers PFePr, PFeH, PFeSi, PFeB, PFeF.

94
A. Gul et al. / Journal of Organometallic Chemistry 779 (2015) 91e99
Table 1
Table 3
ꢁ
Laser light-scattering results for the polymers (DMSO and MeOH at 25 C).
Percentage mortality of brine shrimps at different concentrations of poly(-
azomethine)esters and Schiff base.
Polymers
M
W
(g/mol)a <R
g
> (nm)b
A
2
cm3 mol gꢀ2 c Rh (nm)
R
g
/R
h
D
p
S. No.
Polymer name
Percentage mortality
4
ꢀ2
ꢀ3
PFe
3.9 ꢂ 10
2.11 ꢂ 10
1.45 ꢂ 10
4.7 ꢂ 10
1.7 ꢂ 10
4.05 ꢂ 10
111
2.1 ꢂ 10
e
e
e
4
6
5
5
5
PFeH
PFeB
PFeSi
PFeF
PFePr
95
294
130
53
3.2 ꢂ 10
60
e
1.5
e
1.6
e
100 ppm
10 ppm
1 ppm
LD50
63.79
13.72
0.609
0.486
50.08
236.90
e
e
e
e
1
2
3
4
5
6
SB
71.42
80.1
28.57
42.2
0
e
e
e
PFeH
PFeF
PFePr
PFeB
PFeSi
14.28
55
50
7.14
10.71
e
e
e
100
77.77
82.14
28.571
17.85
83
65
1.3
1.3
82.14
85.71
28.57
*
The specific refractive index increment, dn/dC ¼ 0.097 mL/g for all the polyesters.
is the molecular weight.
is the degree of polymerization.
The relative errors are: dn/dC, ±1%, M
M
w
D
p
w
g 2
, ±5%, <R > & A , ±10%.
a
Weight average molecular weight of the polymers.
Radius of gyration.
Second virial coefficient.
b
c
Table 4
Percentage tumor inhibition and IC50 of poly(azomethine)esters and Schiff base.
S. No. Polymer name Percentage inhibitiona
00 ppm 10 ppm
IC50
1
1 ppm
24.257
7.893
12.073
15.77
0.1 ppm
PFeF had 30% tumor inhibition at 100 ppm that can be due to the
polarity created by highly electronegative hexa floro group in the
chain [20]. Kovjazin and coworker reported that polar, ferrocene
containing material exhibit inhibition of tumor activity [21],
whereas the activity of other polymers having non polar parts in
their structures was insignificant.
DPPH free radical scavenging assay represents that SB was more
radical scavenging with IC5013.65 (Table 5 and Fig. 1). The ter-
polymers were less oxidant than the Schiff base (SB).
1
2
3
4
5
6
SB
36.093
29.527
10.76
21.743
15.79
21.474
6.137
3.410
7.19
215.69
PFeH
PFeF
PFePr
PFeB
PFeSi
15.073
30.490
16.66
576.42
193.72
868.60
23.68
19.735
24.843
19.297 1043.20
ꢀ9.023
ꢀ12.543 ꢀ16.870 ꢀ20.940 ꢀ115.44
a
More than 20% tumor inhibition is significant.
If the DNA-binding capability is considered from the point of
view of the chain types, it is possible to evidence that the intro-
duction of aromatic groups always gives rise to favorable in-
teractions [17,20,22]. It is evident from literature that most of the
Schiff bases bearing two hydroxyl groups on the aromatic ring are
antioxidant in nature [22]. In the present study poly(azomethine)
esters exhibited less antioxidant activity, the reason might be the
absence of free hydroxyl radicals on phenyl rings [22,23]. Therefore,
it can be concluded that the polymerization of monomer and
addition of blocks has reduced the antioxidant behavior of Schiff
base.
In Free radical induced oxidative DNA damage assay SB, PFeF,
PFePr, PFeB and PFeSi showed good protection at all concentrations
tested (Table 6 and Fig. 2). PFeH indicated slight protection at all
concentrations used. It means all the polymers and the monomer
were DNA protecting in nature due to their antioxidant behavior.
Zaheer and coworkers in 2011 have reported the DNA protecting
activity of ferrocenyl azomethine (Schiff bases) [17].
polymer. The material, therefore, is able to bind DNA tighter than
the reference drug (doxorubicin) [24], which is further testified by
the average values calculated by taking into account, the modula-
tion of the chain on the selected skeletons (Table 7, last row) and
the influence of the polycondensed system on each side chain.
By comparison of the behavior among the terpolymers, it was
found that the increase in the chain length and aliphatic moieties
b
enhances the binding capability since binding constants (K ) of
PFePr and PFeH are greater than PFe, PFeB and PFeF. A pictorial
representation of interacting modes of polymers with DNA was
acquired. PFePr interacted with DNA by mix mode of interactions.
Planar Ferrocene part of PFePr intercalates between the DNA base
pairs and its side chain developed interactions with the flanking
base pairs of major groove (Fig. 3). High K
b
values of PFePr
¼ 2.872 ꢂ 10 M ) are due to two modes of interactions which
can be related to the LD50 values in Table 3. PFePr has least values of
LD50 and highest value of K because the polymer which has high
7
ꢀ1
(K
b
b
binding strength its small concentration will cause suffice mortal-
ity. The polymer which has highest value of LD50 should have least
value of binding constant but this is not the case in the present
study. Reason behind this rationale may be the fact that while
Docking studies were carried out for antitumor materialeDNA
complex. Using MOE tool, binding free energy (DG) was calculated
on the basis of force field energy calculations (Table 7). The calcu-
lated free energy of binding the material with DNA was found in the
range, ꢀ7.0992 to ꢀ9.7228 kcal/mol for PFe to PFeF respectively.
The results immediately reveal that, in general, all the synthesized
material has high capacity to bind DNA and have stable interactions
b
calculating K values a polymer interacts only with DNA. Whereas
LD50 values are result of various interaction of a polymer with DNA,
enzymes or metabolites present in the cells. Binding constants and
free energies values of polymers are tabulated in Table 7. PFeB
develops electrostatic interactions with the negatively charged
with
DG values significantly more negative than the parent
Table 2
Antibacterial activity of poly(azomethine)esters and Schiff base SB at 1 mg/ml against six bacterial strains.
S. No.
Compound name
Mean zone of inhibition (mm) ± S.D
S. typhimurium
B. bronchiseptica
M. luteus
S. aureus
E. aerogens
E. coli
1
2
3
4
5
6
7
8
SB
PFeH
PFeF
PFePr
PFeB
PFeSi
Roxithromycin
Cefixime
NIL
9.7 ± 0.3
NIL
NIL
9.6 ± 0.6
NIL
NIL
NIL
12.1 ± 0.17
NIL
NIL
NIL
12.7 ± 0.64
36.83 ± 0.72
NIL
NIL
14.9 ± 0.21
NIL
NIL
NIL
25.6 ± 0.12
22.83 ± 0.72
NIL
NIL
15.5 ± 0.46
NIL
NIL
NIL
32.4 ± 0.4
27.5 ± 0.5
NIL
NIL
17.50 ± 0.44
NIL
NIL
NIL
16.17 ± 0.38
28.96 ± 0.55
NIL
NIL
NIL
NIL
NIL
NIL
15.2 ± 0.2
34.2 ± 0.26
21.3 ± 0.31
31 ± 0.9

A. Gul et al. / Journal of Organometallic Chemistry 779 (2015) 91e99
95
Table 5
Table 6
Percentage DPPH free radical scavenging exhibited by Schiff bases and
poly(azomethine)esters.
Comparison of DNA protection activity of Schiff base and poly(azomethine)esters.
S. No.
Polymer
name
Comparison of DNA protection activity of material
at respective concentration
S. No.
Polymer name
Percentage scavenging
IC50
33.33 ppm
3.33 ppm
0.33 ppm
1000 ppm
100 ppm
10 ppm
1
2
3
4
5
6
SB
82.653
41.550
24.150
45.623
45.320
29.487
33.513
28.713
22.817
17.267
27.743
14.990
27.257
22.713
11.203
19.850
20.197
5.367
13.65
1
2
3
4
5
6
SB
þþþ
þ
þþþ
þ
þþþ
þ
PFeH
PFeF
PFePr
PFeB
PFeSi
49.39
134.13
38.78
39.73
65.28
PFeH
PFeF
PFePr
PFeB
PFeSi
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
þþþ
Single negative (ꢀ) sign means no protection. In this case, there is no super-coiled
DNA.
sugar phosphate structure of DNA and flanking base pairs of groove
Fig. 4). Having a look over the molecular docked complex of PFeF
with DNA duplex it appears that planar aromatic part of polymer
intercalates between the base pairs. Aromatic side chain twists it-
self in such a way that it turns to groove and long aliphatic chain
spans maximum number of base pairs forming strong intrastrand
Single Positive (þ) sign means slight protection. In this case, open circular þ linear
band > super-coiled.
(
Two positive (þþ) sign means moderate protection. In this case, super-
coiled > open circular with a linear band.
Three positive (þþþ) sign means good protection. In this case, super-coiled > open
circular with no linear band.
crosslinking (Fig. 5). Negative values of DG depict that the process
on interaction of polymers with DNA is spontaneous [24].
Ligplots were generated to view 2-D interaction profiles of ter-
polymers with DNA. It is clear from Fig. 6 that Silanol develops
hydrophobic interactions with the Adenine A5 and thymine A7 &
A4 residues. Fig. 7 shows hydrogen bonding and hydrophobic in-
teractions of 1,1,1,3,3,3-hexaflouro) bisphenol propane with DNA
base pair residues.
pellets were recorded in Perkin Elmer 1600 series FTIR spectro-
photometer. Nuclear magnetic resonance was carried out by using
Bruker Avance 300 digital NMR in DMSO-d as solvent and tetra-
6
methylsilane as an internal standard. Elemental analyses were
performed on a Vaio-EL instrument. UV/visible spectra of dilute
polymer solutions in dried ethanol were recorded on Shimadzu,
1998, 2000. A commercial light-scattering spectrometer (BI-APD
equipped with a BI9000AT digital Autocorrelator) was used along
Material and methods
with a HeeNe laser (output power ~400 mW at
l
¼ 638 nm) as a
light source. Relevant measurements were carried out at
ꢁ
Materials
25 þ 0.1 C. The software used was BI-ISTW.
ꢁ
Ferrocene (m.p
¼
172e174 C, Fluka), acetyl chloride
ꢁ
ꢁ
(
b.p ¼ 51e52 C, Fluka), thionyl chloride (b.p ¼ 74.6 C, Fluka),
Synthesis
ꢁ
aluminum chloride (m.p ¼ 192.4 C, Fluka), 4-hydroxybenzaldehyde
ꢁ
ꢁ
(
m.p ¼ 112e114 C, Fluka), 1,3-propan-diol (211e217 C, Sigma
Synthesis of monomer: 4-((2-(4-hydroxybenzylideneamino)
ethylimino)methyl)phenol: (SB)
ꢁ
Aldrich), 1,6-hexan-diol (250
C, Sigma Aldrich), poly(-
dimethylsiloxane), hydroxyl-terminated (n ¼ 550, Sigma Aldrich),
0.25 g ethylene diamine was taken in two-necked round-bottom
flask fitted with reflux condenser, hot plate and magnetic stirrer
under inert nitrogen atmosphere. 1 g 4-hydroxybenzaldehyde,
along with 50 ml dried ethanol was added to the flask. The reaction
mixture was refluxed for 6 h with constant stirring and the progress
was monitored by TLC (n-hexane and ethanol, 3:1 respectively).
The yellow colored product was re-crystallized in dried ethanol,
Scheme. 1, Scheme 1 [8,11].
ꢁ
(
1,1,1,3,3,3hexaflouro)bisphenol propane (160e163 C, Sigma
ꢁ
Aldrich) and bisphenol A (158e159 C, Sigma Aldrich), nutrient
ꢁ
broth medium (b.p ¼ 121 C, dissolved in water, Merck), nutrient
ꢁ
agar medium (b.p ¼ 121 C, dissolved in water, Merck), 2, 2-
ꢁ
Diphenyl-1-picrylhydrazyl (m.p ~135 C, Sigma Aldrich) were used
as received. The solvents used i.e. dichloromethane, diethylether and
ethanol (Sigma Aldrich), dimethyl sulfoxide (Sigma Aldrich) were
dried according to the reported method [25].
(MW ¼ 268, yellow, yield ¼ 90%), FTIR (cmꢀ , KBr): 3401 (OH),
1
1
3
125 (aromatic-H), 2985 (aliphatic-H), 1609 (NeH). H NMR [
d
,
Equipments
deuterated dimethyl sulfoxide (DMSO-d
Ne), 7.86e6.80 (aromatic-H), 1.2e0.9 (eCH
nal-OH). Elemental analysis: calc. (C 73.87, H 6.15, N 10.70), found:
(C 73.90, H 6.12, N 10.83).
6
)]:
d
(ppm): 8.32 (eCH]
e), 9.80 (termi-
2
ꢀCH
2
Melting point was determined on a Mel-Temp. (mitamura Riken
Rogyo, Inc.) by using open capillary tubes. FTIR spectra in KBR
Fig. 1. Comparison of negative control of DPPH free radical scavenging assay and results of three concentrations of Schiff base (P-1).

96
A. Gul et al. / Journal of Organometallic Chemistry 779 (2015) 91e99
Fig. 2. Effect of poly(azomethine)ester PFePr and PFeB on plasmid DNA. L: DNA ladder
1 Kb); P: pBR322 plasmid; X: pBR322 plasmid treated with FeSO and H (positive
control): Lane 1: Control for the damage effect of polymer on DNA;
plasmid þ 1000 ppm Of PFePr; Lane 2: Plasmid þ 1000 ppm of PFePr þ FeSO þ H
Lane 3: Plasmid þ 100 ppm of PFePr þ FeSO þ H ; Lane 4: Plasmid þ10 ppm of
PFePr þ FeSO þ H ; Lane 5: Control for the damage effect of polymer on DNA;
plasmid þ 1000 ppm Of PFeB; Lane 6: Plasmid þ 1000 ppm of PFeB þ FeSO þ H
Lane 7: Plasmid þ 100 ppm of PFeB þ FeSO þ H ; Lane 8: Plasmid þ 10 ppm of
PFeB þ FeSO þ H
(
4
2 2
O
4
2 2
O ;
4
2 2
O
4
2 2
O
4
2 2
O ;
4
2 2
O
4
2 2
O .
0
Synthesis of 1,1 -di (chlorocarboxyl)ferrocene: (Fe)
ꢁ
1,1-Di(chlorocarboxyl)ferrocene (m.p ¼ 95e100 C) was pre-
pared by the reported method [8,11].
Polycondensation
Fig. 3. Molecular docked complex of PFePr (labeled yellow) with DNA. (For interpre-
tation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
Synthesis of polymer: (PFe)
5
0 mL dried dichloromethane (CH
2
Cl
2
) was taken in a 250 ml
three-necked round-bottom flask, equipped with
a reflux
condenser, a gas inlet and a magnetic stirrer. The monomers, SB and
2 2
dried dichloromethane (CH Cl ) followed by 5 ml triethylamine
diacid chloride (Fe) were added to the flask in a molar ratio 1:1. The
ꢁ
(Et N) was added at low temperature using ice bath with constant
reaction mixture was maintained at 0 C by using ice bath. 5 ml
triethylamine (Et N) was added to the reaction mixture under inert
3
stirring. Then the diacid (Fe) was added under same reaction con-
ditions. The reactant ratio was 1:1:2 for the SB, the diol (R) used,
and 1, 1-di(chlorocarboxyl) ferrocene respectively. After 24 h the
3
nitrogen atmosphere, with constant stirring. After 24 h it was
refluxed for 1 h and then the reaction contents were poured into
3
water to eliminate triethylammonium chloride salt (Et NHCl). The
polymer was obtained in the form of precipitates which were
filtered, washed several times with water, dichloromethane and
dried in air [11] Scheme 2.
PFe; yield 87%, Orange, UV/visi (nm) 382 (PheN]CePh), 431
ꢀ1
(
ferrocenyl). FTIR (cm
, KBr): 3099 (aromatic CeH), 2054
(
aliphatic), 1771 (C]O), 1631 (azomethine), 1073 (CeO), 490 (fer-
1
rocenyl), 751 (CeCl). H NMR [
d, deuterated dimethyl sulfoxide
(
DMSO-d )]: (ppm): 8.30 (2H,s eCH]Ne), 7.3e6.8 (m, aromatic
6
d
H), 4.8e5.0 (ferrocenyl) 1.7e1.1 (m, methylene). Elemental analysis:
calcd C, 67.14; H, 4.72; N, 5.51. Found; C, 67.20; H, 4.21; N, 5.43.
Synthesis of terpolymers (PFePr, PFeH, PFeSi, PFeB, PFeF):
The synthesized monomer (SB from Scheme 1) and the
commercial diol (R
¼
1, 3-propan-diol, 1,6-hexan-diol,
poly(dimethylsiloxane), hydroxyl-terminated (n ¼ 550, (1,1,1,3,3,3-
hexaflouro)bisphenol propane or bisphenol A), from Scheme 3
were taken in two-necked round-bottom flask, fitted with
2
condenser, magnetic stirrer and under inert atmosphere (N ). 50 ml
Table 7
Binding constants and free energy values of polyazo(methine)ester with DNA
calculated from molecular docking data.
ꢀ
1
Polymers
Binding constants K
b
/M
Free energy values DG/kcal/mol
5
PFe
1.60 ꢂ 10
ꢀ7.0992
ꢀ8.7272
ꢀ10.1692
ꢀ5.464
6
PFePr
PFeH
PFeB
PFeF
2.52 ꢂ 10
7
2.87 ꢂ 10
4
Fig. 4. Molecular docked complex of PFeB (labeled purple) with DNA. (For interpre-
tation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
1.01 ꢂ 10
7
1.33 ꢂ 10
ꢀ9.7228

A. Gul et al. / Journal of Organometallic Chemistry 779 (2015) 91e99
97
7.3e6.8 (m, aromatic), 4.8e4.9 (b, ferrocenyl), 2.5e2.2 (16H, m,
methylene). Elemental analysis calcd. C 67.10 H 6.0 N 4.0. Found; C
67.0 H 5.87 N 4.18.
2 5
PFePr; yield 89%, Light yellow powder UV/vis (lmax) (C H OH);
ꢀ1
3
73 and 435 nm. FTIR (cm , KBr): 3156 (aromatic CeH), 2904
(
(
aliphatic CeH), 1707, 1713 (C]O), 1053, 1057 (CeOeC), 1639
1
azomethine), 736 (CeCl), 502 (ferrocenyl). H NMR [
)] (ppm): 8.14 (2H, s, azomethine),
.2e6.8 (aromatic), 4.9e5.1 (b, ferrocenyl), 2.4e2.2 (10H, m,
d, deuterated
dimethyl sulfoxide (DMSO-d
7
6
d
methylene). Elemental analysis calcd. C, 66.72; H, 5.30; N 4.30.
Found; C, 66.53; H, 5.0; N, 4.40.
PFeSi; yield 86%, Light powder, UV/vis (
l
max) (C
2
H
5
OH); 327 and
35 nm. FTIR (cm , KBr): 3117 (aromatic CeH), 2934 (aliphatic
CeH), 1700, 1709 (C]O), 1071, 1075 (CeOeC), 1611 (azomethine),
ꢀ1
4
1
7
01 (CeCl), 499 (ferrocenyl). H NMR [
sulfoxide (DMSO-d )] (ppm): 8.1 (2H, s, azomethine), 7.0e6.4
aromatic, m), 4.6e4.2 (b, ferrocenyl), 2.0e1.4 (m, methyl/
d, deuterated dimethyl
6
d
(
methylene).
Bioassays
Antibacterial assay
The antibacterial activities of the Schiff base and its terpolymers
was checked by agar well diffusion method, using bacterial strains
of S. aureus, M. luteus, S. typhimurium, E. aerogenes, Escherichia coli
and Bordetella bronchiseptica. Preparation of the media and all in-
oculations were performed as described earlier [23]. The antibac-
terial activity was determined on the basis of size of zone of
bacterial growth inhibition.
Fig. 5. Molecular docked complex of PFeF (labeled yellow) with DNA. (For interpre-
tation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
reaction mixture was refluxed for 1 h and then poured into water
for removing triethylammonium chloride salt (Et NHCl) and to
3
precipitate PAME. The polymer was filtered, washed several time
with water, ethanol and then dried under air [8,11], Scheme 3.
Brine shrimp cytotoxic assay
PFeB; yield 88%, Light yellow powder, UV/vis (
l
max) (C
2
H
5
OH);
30 and 421 nm. FTIR (cm , KBr): 3109 (aromatic CeH), 2949
aliphatic CeH), 1709, 1714 (C]O), 1037, 1040 (CeOeC), 1670
Brine shrimp (Artemia salina) lethality assay was utilized for
preliminary assessment of toxicity of terpolymers and their
monomer [10]. The hatching of brine shrimp eggs and the proce-
dure of the assay was followed as reported [10]. Methanol or DMSO
was used as solvent depending on solubility of the polymer. In
order to determine the percentage mortality rate following formula
was used and the data was analyzed with LD50 values.
ꢀ1
3
(
(
1
azomethine), 762 (CeCl), 511 (ferrocenyl). H NMR [
)] (ppm): 8.5(2H, s, azomethine),
.6e7.1 (aromatic), 5.0e4.6 (b, ferrocenyl), 2.4e2.0 (10H, m, methyl/
d, deuterated
dimethyl sulfoxide (DMSO-d
6
d
7
methylene). Elemental analysis calcd. C, 72.66; H, 5.55; N, 5.00.
ðNumber of alive shrimpsÞControl ꢀ Sample
Percentage mortality ¼
ꢂ 100
ðNumber of alive shrimpsÞControl
Found; C, 72.34; H, 5.21; N, 5.27.
Antitumor potato disc assay
PFeF; yield 89%, Light yellow powder, UV/vis (
l
max) (C
2
H
5
OH);
21 and 428 nm. FTIR (cm , KBr): 3199 (aromatic CeH), 2017
aliphatic CeH), 1786, 1791 (C]O), 1091, 1097 (CeOeC), 1633 (azo-
The test samples were screened for tumor inhibition by
antitumor potato disc assay as performed earlier [20]. Tumor
inhibiting activity of polymers was checked at the concentration
of 100 ppm, 10 ppm, 1 ppm and 0.1 ppm of the test polymer
using AT10 bacterial strain under complete aseptic conditions.
ꢀ
1
3
(
1
methine), 762 (CeCl), 523 (ferrocenyl). H NMR [
dimethyl sulfoxide (DMSO-d )] (ppm): 8.78 (2H, s, azomethine),
.9e7.4 (aromatic), 5.3e4.8 (b, ferrocenyl), 2.6e2.2 (4H, methy-
lene). Elemental analysis calcd: C, 73.27; H, 4.76; N, 4.0. Found; C,
d, deuterated
6
d
ꢁ
7
After incubation period of 21 days at 28 C, number of tumors
was counted after staining with the Lugol's solution (10% KI and
73.10; H, 4.58; N, 3.9.
2
5% I in 100 ml distilled water). Results were recorded in terms of
PFeH; yield 87%, Light yellow powder, UV/vis (
76 and 419 nm. FTIR (cm , KBr): 3173 (aromatic CeH), 2899
l
max) (C
2
H
5
OH);
IC50values. Percentage tumor inhibition was calculated by using
formula given below.
ꢀ
1
3
Percent tumor inhibition ¼ 1
00 ꢀ Average number of tumors of sample
ꢂ 100
Average number of tumors of control
(
(
aliphatic CeH), 1734, 1740 (C]O), 1071, 1075 (CeOeC), 1654
DPPH free radical scavenging assay
The capability of Schiff base and its terpolymers to act as a free
radical scavenger was assessed by DPPH (2,2-diphenyl-1-
1
azomethine), 722 (CeCl), 510 (ferrocenyl). H NMR [
d, deuterated
dimethyl sulfoxide (DMSO-d
6
)] d (ppm) 8.3 (2H, s, azomethine),

98
A. Gul et al. / Journal of Organometallic Chemistry 779 (2015) 91e99
Free radical induced oxidative DNA damage assay
Antioxidant effects of Schiff base and terpolymers were also
checked in vitro by free radical induced oxidative DNA damage
2
þ
analysis using Fenton reaction [7]. In this reaction, Fe reacts with
resulting in the production of hydroxyl radical which is
2 2
H O
considered to be most harmful to biomolecules [21,22].
Plasmid pBR322 DNA (Fermentas, Germany) was diluted by
using 50 mM phosphate buffer pH 7.4 to get a concentration of
0
.5
at various concentrations, 3
were prepared. For each experiment, a 1 Kb DNA ladder (L), plasmid
pBR322 DNA treated with H and FeSO as positive control and a
m
g/3
m
l. Reaction mixtures (diluted pBR322, 5
m
l of test samples
m
l of 2 mM FeSO and 4
4
ml of 30% H
2 2
O )
2
O
2
4
negative control (only plasmid pBR322 DNA) were used. After in-
ꢁ
cubation at 37 C for 1 h in the dark, reaction mixtures were sub-
jected to 1% agarose gel electrophoresis in 1X TBE gel running
buffer. The gel (stained with ethidium bromide) picture was taken
under UV light. Antioxidant activity of the test polymers was
examined by comparing the bands of samples with controls.
Fig. 6. Ligplot showing 2-D interactions of Silanol with DNA.
Docking procedure
picrylhydrazyl) assay [17]. DPPH solution was prepared as
described earlier [5]. The solutions of the test polymers in DMSO
were of 1000 ppm, 100 ppm and 10 ppm concentration. The reac-
tion mixture (3 ml) was prepared by adding 2 ml of DPPH solution,
MOE-dock by Chemical Computing Group Inc. was used for the
molecular modeling. All the studies were carried out on a
Pentium1.6 GHz workstation, 512 MB memory with the
Windows Operating System. MOE 2008 was employed to draw the
ligand and to optimize geometry, the molecular dynamic
AMBER and semi-empirical PM3 methods were used. The DNA
duplex receptor structure (having the base pair sequence CCA-
0
.9 ml of trisHCl buffer and 100
concentration of 33.33 ppm, 3.33 ppm and 0.33 ppm in the reaction
mixture. Negative control consisting of 100 l of DMSO and 2 ml of
ml of the test polymer to get the final
m
DPPH solution, was used. Absorbance at 517 nm on a UV/Visible
light spectrophotometer against blank was calculated to interpret
results in terms of IC50values. The percentage scavenging of DPPH
free radical for each concentration of each test polymer was
determined by the following formula.
0
0
TAATTTACC:CCTATGAAATCC with 12 base pairs running in 3 -5
direction.) was obtained from Protein Data Bank (1t69 dna). The
edited structure was imported into MOE workstation. All hydrogen
atoms were added to the structure with their standard geometry
Absorbance of control ꢀ Absorbance of test sample
Percentage scavenging ¼
ꢂ 100
Absorbance of control
Fig. 7. Ligplot showing 2-D interactions of (1,1,1,3,3,3-hexaflouro) bisphenol propane with DNA.

A. Gul et al. / Journal of Organometallic Chemistry 779 (2015) 91e99
99
followed by their energy optimization tool using the MOPAC 7.0.
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