Synthesis and biological evaluations of some Schiff-base esters of ferrocenyl aniline and simple aniline

Article abstract of DOI:10.1016/j.jorganchem.2009.02.032

The synthesis and biological studies of some long chain esters containing Schiff bases and their ferrocenyl analogues were carried out. The 4-amino ferrocene was prepared by the reported method. Long chain esters were synthesized by the condensation of di

Full text of DOI:10.1016/j.jorganchem.2009.02.032

Journal of Organometallic Chemistry 694 (2009) 2198–2203
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Synthesis and biological evaluations of some Schiff-base esters of ferrocenyl
aniline and simple aniline
a
a
b
b
Haq Nawaz a, Zareen Akhter a, , Sumaira Yameen , Huamaira M. Siddiqi , Bushra Mirza , Arshia Rifat
*
a Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological studies of some long chain esters containing Schiff bases and their ferrocenyl
analogues were carried out. The 4-amino ferrocene was prepared by the reported method. Long chain
esters were synthesized by the condensation of different aliphatic acids with the corresponding aldehyde.
The esters were then reacted with aniline as well as with 4-aminophenyl ferrocene to give corresponding
Schiff bases. All the synthesized compounds were analyzed by elemental, FTIR and proton NMR studies,
were also investigated for a range of biological activities. Determined by crown gall tumor inhibition
assay. Antioxidant and DNA protective activities were determined by DPPH free radical scavenging assay
and OH radical induced oxidative DNA damage assay, respectively. Among all test compounds, o-
hydroxy-p-n-octadecanoyloxy-benzylidine-p-ferrocenyl aniline (FA2.1: a ferrocene containing Schiff
base) showed highest antitumor, DPPH free radical scavenging and DNA protective activities.
Ó 2009 Elsevier B.V. All rights reserved.
Received 24 November 2008
Received in revised form 18 February 2009
Accepted 24 February 2009
Available online 9 March 2009
Keywords:
Schiff base
Schiff-base esters
Ferrocene
Ferrocenyl aniline
Biological activities
1. Introduction
Schiff bases. The purpose of DPPH scavenging assay was to evalu-
ate the antioxidative potential of test samples. While in vitro OH
Drug discovery process involves synthesis of novel bioactive
compounds which undergo different in vitro and in vivo testing be-
fore their clinical administration [1,2]. In last few years organome-
tallics have got great importance owing to their potential biological
activities [3,4]. Among organometallics, ferrocene is known as
chemically and thermally stable specie. The greater stability of fer-
rocene system led to its incorporation in many research activities.
Nowadays, ferrocene is no longer a chemical curiosity, rather it
provides a widely applicable platform for the preparation of func-
tional derivatives used in many areas like catalysis, material sci-
ence, crystal engineering and bio-organometallic chemistry [5–8].
The activities of certain drugs are reported to be enhanced by the
addition of ferrocene moiety to their structures; e.g., ferrocene
aspirin [4], antimalarial drug ferrocene quine [9], an anticancer
drug ferrocifen [10], ferrocene–hydroxytamoxifen [11] and ferr-
chloroquine [12]. The anticancer potential of ferrocene containing
compounds is also well reported [13–16].
radical induced DNA damage system served to evaluate DNA pro-
tection in oxidative stress.
2. Experimental
2.1. Materials
Ferrocene, 4-nitroaniline, sodium nitrate, hexadecyltrimethy-
lammonium bromide, hydrochloric acid, aniline, hydrazine, dim-
ethylaminopyridene, hexadecyltrimethyl ammonium bromide
(PTC) N,N0-dicyclohexylcarbodiimide and 2,4-dihydroxybenzalde-
hyde were obtained from Fluka, Switzerland. Solvents such as
dichloromethane, absolute ethanol, diethyl ether, hexane, ethyl
acetate and petroleum ether were obtained from Merck Germany
and freshly dried as per requirement using the standard methods.
2.2. Synthesis of 4-aminophenyl ferrocene
Keeping in view the reported biological studies, simple Schiff
bases (AN1, AN2, AN1.1 and AN2.1), Schiff bases with ferrocene
addition (FA1, FA2, FA1.1 and FA2.1) and their reactants (Z1,
Z1.1, A1, A2, A1.1 and A2.1) were investigated for their differential
biological activities towards antitumor, antioxidant and DNA
protecting effects. In the present study crown gall tumor inhibition
assay is used for verification of anticancerous activity of these
4-Ferrocenyl aniline was prepared in two steps by the reported
method [5].
2.3. General procedure for the synthesis of long chain esters (A1, A1.1,
A2 and A2.1)
In a two neck round bottom flak equipped with magnetic stirrer
and reflux condenser, the corresponding aldehyde (10 mmol), long
chain aliphatic acid (10 mmol) and dimethylaminopyridene
* Corresponding author. Tel.: +92 51 90642111; fax: +92 51 2873869.
E-mail address: zareenakhter@yahoo.com (Z. Akhter).
0022-328X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jorganchem.2009.02.032

H. Nawaz et al. / Journal of Organometallic Chemistry 694 (2009) 2198–2203
2199
(1 mmol) were dissolved in 1:1 mixture of dimethylformamide and
dichloromethane and stirred at 0 °C. To this stirred solution, a solu-
tion of N,N0-dicyclohexycarbodiimide (10 mmol) was added drop
wise and stirred at 0 °C for 1 h and then stirred at room tempera-
ture for 3 h. The progress of the reaction was monitored by thin
layer chromatography. After the completion of the reaction, the
reaction mixture was filtered and DCM was removed from filtrate
by rotary evaporator. The remaining mixture was poured in water
and precipitated ester was filtered and recrystallized with n-hex-
ane to get pure product [17–19].
N, 2.28%. IR (cmꢀ1) 507, 1605, 1631, 1754, 2850, 2818. 1H NMR
(DMSO-d6,
d ppm): d d d
(–CH@N–) = 8.32s; (–CH3) = 1.03t;
(–CH2COO–) = 2.38t: d (–CH2–) = 1.76m, 1.36m; d (Ar–H) = 7.33m,
7.96m, d (–C5H5) = 4.04s: d (–C5H4–) = 4.27t; d (–C5H4–) = 4.65t.
2.4.7. O-hydroxy-p-n-hexadecanoyloxy-benzylidine-p-ferrocenyl
aniline (FA1.1)
Orange solid, yield (74%), m.p. 123–138 °C. Anal. Calc. for
C39H49O3NFe: C, 73.70; H, 7.72; N, 2.20. Found; C, 72.49; H, 7.49;
N, 2.45%. IR (cmꢀ1) 493, 1601, 1621, 1754, 2849, 2918. 1H NMR
(DMSO-d6,
d ppm): d d d
(–CH@N–) = 8.16s; (–CH3) = 1.01t;
2.4. General procedure for the synthesis of Schiff-bases esters (AN1,
AN1.1, AN2, AN2.1, FA1, FA1.1, FA2 and FA2.1 [20]
(–CH2COO–) = 2.34t: d (–CH2–) = 1.5m, 1.03m; d (Ar–H) = 7.48m, d
(Ar–OH) = 14.09s;
(–C5H4–) = 4.62t.
d
(–C5H5)2 = 3.98s:
d
(–C5H4–) = 4.27t;
d
In a pre-backed round flask (100 ml) equipped with stirrer and
reflux condenser, the solution of corresponding long chain aro-
matic ester (2.5 mmol) in absolute ethanol was added. To this,
the solution of corresponding aniline (2.5 mmol) in absolute etha-
nol was added drop wise with constant stirring. The reaction mix-
ture was refluxed for 4–5 h. After the completion of the reaction,
the ethanol was removed by rotary evaporator and the product
was recrystallized from absolute ethanol.
2.4.8. O-hydroxy-p-n-octadecanoyloxy-benzylidine-p-ferrocenyl
aniline (FA2.1)
Orange solid, yield (79%), m.p. 100–120 °C. Anal. Calc. for
C39H49O3NFe: C, 80.31; H, 7.84; N, 2.68. Found: C, 79.39; H, 7.60;
N, 2.34%. IR (cmꢀ1) 503, 1601, 1622, 1756, 2850, 2922. 1H NMR
(DMSO-d6,
d ppm): d d d
(–CH@N–) = 8.70s; (–CH3) = 0.91t;
(–CH2COO–) = 2.35t: d (–CH2–) = 2.58m, 1.14m; d (Ar–H) = 7.48m, d
(Ar–OH) = 13.80s;
(–C5H4–) = 4.25t.
d
(–C5H5) = 4.08s:
d
(–C5H4–) = 4.18t;
d
2.4.1. P-n-hexadecananoyloxy-benzaldehyde-p-aniline (AN1)
Off white solid, yield (74%), m.p. 57–76 °C. Anal. Calc. for
C29H41O2N: C, 80.00; H, 9.42; N, 3.21. Found: C, 79.69; H, 9.58; N,
3.69%. IR (cmꢀ1) 1625, 1656, 1746, 2920, 2850. 1H NMR (DMSO-
d6, d ppm): d (–CH@N–) = 8.16s; d (–CH3) = 1.03t; d (–CH2COO–) =
2.37t: d (–CH2–) = 1.75m, 1.35m; d (Ar–H) = 7.80m, 7.10m.
2.5. Antitumor potato disc assay
Antitumor potato disc method was used for the determination
of antitumor activities [21]. Briefly, 48-h-old single colony culture
of Agrobactreium tumefaciens (At-10) was used as an agent for
tumor induction on potato discs. Rifampicine (10 mg/ml) was
used as a specificity drug for At-10. Each test sample was evalu-
ated for antitumor activity at three concentrations i.e., 1000, 100
2.4.2. O-hydroxy-p-n-hexadecananoyloxy-benzaldehyde-p-aniline
(AN1.1)
Yellow solid, yield (75%), m.p. 78–94 °C. Anal. Calc. for
C29H41O3N: C, 77.16; H, 9.09; N, 3.10. Found: C, 77.40; H, 9.83; N,
3.38%. IR (cmꢀ1) 1629, 1655, 1756, 2917, 2849, 3311. 1H NMR
(DMSO-d6, d ppm): d (–CH@N–) = 8.63s; d (Ar–OH) = 13.60s; d
(–CH3) = 0.92t; d (–CH2COO–) = 2.58t: d (–CH2–) = 2.30m, 1.27m; d
(Ar–H) = 7.47m, 7.28m.
and 10 lg/ml with DMF as negative control and vincristine as
standard drug. Under complete aseptic conditions sterilized
instruments were used to make 8 ꢁ 5 mm potato discs from sur-
face sterilized (HgCl2 0.1%) healthy potatoes tubers. Ten potato
discs were placed on each plate containing 1.5% agar–agar in dis-
tilled water. After treatment with test agents and At-10 strain on
each disc, plates were incubated (28 °C) for 21 days than stained
with Lugol’s solution (10% KI and 5% I2) and number of tumors
was counted with the help of dissecting microscope. Each
experiment was carried out in triplicate and data was statistically
analyzed using ANOVA and Duncan’s Multiple Range Test. Per-
centage tumor inhibition was calculated by using formula given
in Table 1.
2.4.3. P-n-octadecananoyloxy-benzaldehyde-p-aniline (AN2)
Light yellow solid, yield (73%), m.p. 62–78 °C. Anal. Calc. for
C31H45O2N: C, 80.34; H, 9.71; N, 3.02. Found: C, 79.02; H, 10.00; N,
3.16%. IR (cmꢀ1) 1560, 1657, 1749, 2918, 2850. 1H NMR (DMSO-d6,
d ppm): d (–CH@N–) = 8.17s; d (–CH3) = 1.02t; d (–CH2COO–) =
2.37t: d (–CH2–) = 1.75m, 1.20m; d (Ar–H) = 7.86m, 7.14m.
2.4.4. O-hydroxy-p-n-octadecananoyloxy-benzaldehyde-p-aniline
(AN2.1)
2.6. DPPH free radical scavenging assay
Light yellow solid, yield (75%), m.p. 62–80. Anal. Calc. for
C31H45O3N: C, 77.66; H, 9.39; N, 2.92. Found: C, 77.02; H, 9.89;
N, 3.04%. IR (cmꢀ1) 1576, 1691, 1759,2917, 2850, 3400. 1H NMR
DPPH free radical scavenging assay was used to determine the
free radical scavenging efficiency of test samples, as reported ear-
lier [22]. DPPH is a stable free radical present in crystallized form.
Reagents used for this assay were 0.1 mM DPPH in ethanol and
50 mM Tris–HCl in distilled water. Reaction mixture contained
DPPH, Tris–HCl and test sample in fixed volume ratio of 2 ml,
(DMSO-d6, d ppm): d (–CH@N–) = 8.67s; d (Ar–OH) = 13.62s
d
(–CH3) = 0.91t; d (–CH2COO–) = 2.37t: d (–CH2–) = 2.60m, 1.14m; d
(Ar–H) = 7.28m, 6.72m.
2.4.5. P-n-hexadecanoyloxy-benzylidine-p-ferrocenyl aniline (FA1)
Orange solid, yield (70%), m.p. 97–110 °C. Anal. Calc. for
C39H49O2NFe: C, 74.60; H, 7.91; N, 2.26. Found: C, 74.52; H, 8.44;
N, 2.62%. IR (cmꢀ1) 499, 1605, 1622, 1755, 2850, 2917. 1H NMR
900
glass vials and incubated at room temperature for half an hour.
Test samples were examined at 100, 50 and 25 g/ml as final
ll and 100 ll, respectively. Final volume was kept in caped
l
(DMSO-d6,
d ppm): d d d
(–CH@N–) = 8.32s; (–CH3) = 1.03t;
concentration from stock. Ascorbic acid was used as a positive
control and DMF as negative control. Change in DPPH purple col-
or was observed by spectrophotometric absorbance at 517 nm.
Each experiment was performed in triplicate and data was ana-
lyzed using ANOVA and Duncan’s Multiple Range test. Percentage
free radical scavenging was calculated by using formula given in
Table 2.
(–CH2COO–) = 2.38t: d (–CH2–) = 1.45m, 1.36m; d (Ar–H) = 7.95m,
7.55m, d (–C5H5) = 4.02s: d (–C5H4–) = 4.27t; d (–C5H4–) = 4.65t.
2.4.6. P-n-octadecanoyloxy-benzylidine-p-ferrocenyl aniline (FA2)
Orange solid, yield (72%), m.p. 114–125 °C. Anal. Calc. for
C41H53O2NFe: C, 76.04; H, 8.19; N, 2.16. Found: C, 75.48; H, 8.23;

2200
H. Nawaz et al. / Journal of Organometallic Chemistry 694 (2009) 2198–2203
Table 1.
Antitumor activity of tested compounds.
Organic compounds
Percentage tumor inhibition S.E.
IC50
(
l
g/ml)
Organic compounds
Percentage tumor inhibition S.E.
1000 ( g/ml) 100 ( g/ml) 10 (lg/ml)
IC50
900
>1000
>1000
>1000
>1000
563
(lg/ml)
1000 (lg/ml)
100 (lg/ml)
10 (
l
g/ml)
l
l
Z1CDE
29 3.9
27 3.5
19 7.8
55 12
38 8.6
19 5.3
38 5.1
–
25 1.2
22 3.2
12 2.6
27 11.4
9
1.2
>1000
>1000
>1000
868
>1000
>1000
>1000
–
AN2ABC
AN1.1CDE
AN2.1DE
FA1CDE
52 7.7
24 11.1
29 10
34 5.4
18 4.6
28 5.6
11 2.3
Z1.1CDE
A1DE
13 1.9
3
14
3
0.2
4
0.3
2.7
7
2.7
6
2
A2ABCDE
A1.1CDE
A2.1E
20 15.5
30 8.1
58 4.4
71 3.5
15 6.9
20 2.1
41 6.5
40 0.2
15 1.2
18 7.0
40 4.2
35 4.8
3
4
2.8
1.2
FA2CDE
1
FA1.1AB
FA2.1A
AN1BCDE
ꢀve Control
23 7.7
–
17 8.6
20
0.003
–
Vincristine
100
0
100
0
100
0
ꢀ
ꢁ
100ꢀAverage no of tumors in sample
Percentage tumor inhibition ¼
ꢁ 100.
Average no of tumors in negative control
ꢂ Standard drug vincristine was used as a positive control.
ꢂ More than 20% inhibition is significant.
ꢂ Data represents average percentage tumor inhibition of three replicates S.E.
ꢂ Letters ranging from A to E indicate least significant difference rank orders of percentage tumor inhibition. Values with similar letters are not significantly different from
each other at p < 0.05.
2.7. OH radical induced oxidative DNA damage analysis
hydrazine as reducing agents and reaction mixture was refluxed
for about 10–12 h (scheme 1) [5].
Study of protection of DNA by test samples was carried out as
reported in vitro OH radical induced oxidative DNA damage assay
[23]. Plasmid pBR322 DNA (Fermentas, Germany) was diluted by
using 50 mM phosphate buffer pH 7.4 to get a concentration of
Long chain esters were prepared by reacting different long
chain alkyl aliphatic acids with the corresponding aldehydes
0.5
ume of 15
(10 g/ml), 2 mM FeSO4 and 30% H2O2. For each experiment a
l
g/3
l
l. Reaction mixture (kept in eppendorf tube at a total vol-
l
l) contained diluted pBR322 (0.5 g/ml) test sample
l
NO2
Cl-N2+
Ph
NO2
l
1 Kb DNA ladder (L), a negative control (only plasmid DNA, P)
and plasmid DNA treated with H2O2 and FeSO4 as positive control
(Fenton reaction mixture, X) were also used. Reaction mixture was
incubated at 37 °C for 1 h in dark. Each test was subjected to 0.9%
agarose gel electrophoresis in 1X TBE gel running buffer. Ethedium
bromide stained gel was photographed under the UV light and ana-
lyzed by Lab Image 1D gel analysis software (2006).
Fe
PTC, Et2O
Fe
Pd-C
Hydrizine
EtOH
3. Results and discussion
3.1. Synthesis
NH2
To prepare 4-aminophenyl ferrocene, 4-nitrophenyl ferrocene
was first prepared through the arylation of ferrocene by the diazo-
nium salt of 4-nitroaniline in the presence of phase transfer cata-
lyst using ethanol as solvent. The 4-nitrophenyl ferrocene was
then reduced to 4-aminophenyl ferrocene using 5% Pd/C and
Fe
Scheme 1. Synthesis of ferrocenyl aniline.
Table 2.
DPPH free radical scavenging activity of tested compounds.
Organic compounds
Percentage scavenging S.E.
100 ( g/ml) 50 ( g/ml)
ꢀ5 2.7
0.7
IC50
(lg/ml)
Organic compounds
Percentage scavenging S.E.
IC50 (lg/ml)
l
l
25 (
l
g/ml)
100 (lg/ml)
50 (lg/ml)
25 (
l
g/ml)
Z1EF
ꢀ6 1.2
ꢀ2 2.2
ꢀ1 0.7
ꢀ3 0.9
ꢀ6 0.7
ꢀ11 0.3
8
2
2
0.6
0.7
0.5
–
–
–
–
–
–
AN2D
16 0.1
77 2.2
13 1.6
12 0.3
52 1.4
11 0.5
36 2.6
44 0.6
30 6.2
56 1.3
8
0.2
>100
49.3
>100
>100
>100
174.1
45.7
8.2
Z1.1E
1
AN1.1A
AN2.1D
FA1C
36 0.3
11 0.5
11 2.6
30 1.2
26 1.2
41 0.2
A1E
ꢀ1 1.4
ꢀ5 0.3
ꢀ8 2.6
ꢀ4 0.6
A2FG
ꢀ1 5.8
ꢀ7 0.5
ꢀ5 0.4
42
44
0
0
A1.1H
A2.1G
AN1E
FA2B
FA1.1C
FA2.1A
Ascorbic acid
38 1.8
65 2.8
1
0.1
5
0.4
4
–
0.2
>100
–
ꢀve Control
–
–
70.3
0
74
0
81
0
ꢂ Absorbance was taken at 517 nm spectrophotometrically.
ꢂ Tested samples were tested at final dilution of 100, 50 and 25
lg/ml.
ꢂ Commercial antioxidant ascorbic acid was used as a positive control.
ꢂ ‘‘–” represents pro-oxidant activities.
ꢀ
ꢁ
absorbance of negative controll-absorbance of sample
Absorbance of negative control
ꢂ Percentage tumor inhibition ¼
ꢁ 100.
ꢂ Data represents average percentage scavenging of three replicates S.E.
ꢂ Least significant difference rank order on the basis of DPPH percentage scavenging is represented by letters ranging from A to H at p < 0.05.

H. Nawaz et al. / Journal of Organometallic Chemistry 694 (2009) 2198–2203
2201
(4-hydroxy benzaldehyde, 2,4-dihydroxybenzaldehyde) in the
presence of DCC and DMAP (Scheme 2) using DCM and DMF as sol-
vents. The products obtained on evaporating the solvent were
recrystallized with n-hexane to get pure product.
Schiff-bases esters were synthesized according to the Scheme 2
given below in which corresponding aniline was refluxed with long
chain esters in inert atmosphere using dry nitrogen and absolute
ethanol as solvent.
cene. The unsubstituted cyclopentadienyl ring of ferrocene gave a
singlet in the range 3.98–4.04 ppm for the five hydrogens and
the substituted one gave the triplets at 4.27 ppm for the two
hydrogens and at 4.62–4.65 ppm for the other two hydrogens in
agreement with the literature values [24]. All the Schiff bases
showed a sharp singlet around 8.16–8.63 ppm for azomethine pro-
ton in accordance with literature [24].
4-Nitrophenyl ferrrocene and 4-aminophenyl ferrocene as well
as all esters and ester containing Schiff bases were characterized by
their characteristic color, melting points, CHNS, FTIR and proton
NMR spectral data. The elemental analyses of all the products are
in agreement with the calculated values. The I.R spectra of these
products show all the characteristics peaks. Asymmetric and sym-
metric stretches of NO2 in case of 4-aminophenyl ferrocene were
observed at 1508 and 1339 cmꢀ1, respectively, which were absent
3.2. Biological activities
The biological study reported herein is accessed by using se-
lected biological assays. This is an attempt to investigate possible
biological activities of synthesized compounds and any possible
change in activities followed by ferrocene addition.
3.2.1. Antitumor activity
in case of ferrocenyl aniline. The aromatic
t
C–H stretch in the ferro-
Potato disc tumor induction assay is a general reliable screen for
detecting antitumor agents [25–29]. Mechanisms for tumor induc-
tion are reported to be similar in plants and animals and potato
disc assay had shown a good correlation in results to other most
commonly used antitumor screening assays [30]. In present study
Agrobacterium tumefaciens (At-10) was used to induce tumors on
potato discs [31]. In each experiment vincristine as positive control
and DMF as negative control were used for reference. As depicted
by results in Table 1 DMF (negative control) have no interference
with the activity of bacterium to induce tumors whereas Vincris-
tine (positive control) showed 100% tumor inhibition at all concen-
trations tested. Potato disc tumor inhibition analysis of
cene derivatives appeared at 2924 and 3092 cmꢀ1. Esters gave a
strong band around 1700 cmꢀ1 representing the stretching vibra-
tions of the carbonyl group where as the esters containing hydro-
xyl group gave a broad band at 3327–3450 cmꢀ1 due to tO–H
stretch.
In case of schiff bases, the C@N stretch of azomethine group was
observed at 1560–1627 cmꢀ1 and in ferrocene based Schiff bases
the Fe–Cp vibrations were observed at 493–507 and 924–
1148 cmꢀ1. All the characteristics signals were observed in the
1H NMR spectra of the synthesized Schiff bases. The incorporation
of the ferrocene is indicated by the signals observed due to ferro-
R
O
C
O
DCC, DMAP
DCM, DMF
O
CHO
HO
CHO
C
R
OH
R
R'
R=
R' =
1) H
2) OH A 1.1
Reflux
Ethanol
1) CH3(CH2)13CH2-
A1
Y-NH 2
2) CH3(CH2)15CH2-
1) H A2
2) OH A2. 1
R
O
Z-1 = 4-hydroxybenzaldehyde
Z11 = 2,4-dihydroxybenzladehyde
O
C
N
Y
CH
R'
R'=
1) H AN1
2) OH AN1.1
R=
Y =
1)
1) CH3(CH2)13CH2
-
2)
R'=
1) H FA1
2) OH FA1.1
Fe
R'=
1) H AN2
2) OH AN2. 1
2)
CH3(CH2)15CH2-
1)
2)
R'=
1) H FA2
2) OH AN2 .1
Fe
Scheme 2. Synthesis of ferrocene containing Schiff bases.

2202
H. Nawaz et al. / Journal of Organometallic Chemistry 694 (2009) 2198–2203
synthesized organic compounds showed that all the test com-
pounds inhibit tumor production for treatment of 1000, 100 and
45.7 lg/ml IC 50 value DPPH scavenging was observed in an order
of ferrocene Schiff bases > Schiff bases, while no scavenging or pro-
oxidant activities were shown by their reacting compounds (Z1,
Z1.1, A1, A2, A1.1 and A2.1). Thus it could be assumed that turning
of reacting compounds into Schiff bases and ferrocene addition to
Schiff bases had resulted in increase in scavenging of free radicals.
Similar enhancing free radical quenching activity by ferrocene
addition was earlier reported [34].
10
bition was observed in dose dependant manner with highest inhi-
bition at 1000 g/ml concentration. Ferrocene Schiff base
lg/ml concentration at p < 0.05 (Table 1). Furthermore, the inhi-
l
(ferrocene being reported as anticrcinogen) FA2.1 exhibited the
best activity with 71% followed by FA1.1 with 58% inhibition at
1000 lg/ml. Antitumor activity can also be judged by 50% inhibi-
tory concentration (calculated by inhibition curves of three con-
centrations tested for each compound). Lower the IC 50 value
more effective is the tested compound. An IC 50 calculation also fa-
vours FA2.1 and FA 1.1 (ferrocene based Schiff bases) to be more
active as compared to simple Schiff bases and reactive compounds.
By analyzing structure effectiveness on activities it could be in-
ferred from these results that the ferrocene presence as well as
OH group substitution to benzene ring might had resulted in
enhancing antitumor activity of FA2.1 and FA1.1. This observed re-
sult was consistent with a previously reported study [32].
3.2.3. OH radical induced oxidative DNA damage analysis
In a Fenton reaction, Fe2+ reacts with H2O2 and form OH radi-
cals. OH radical is considered to be the most damaging to biomol-
ecules [14,15]). DNA protecting activity of ferrocene sciff bases and
other synthesized compounds were investigated by in vitro OH
radical induced DNA damage system at 10 lg/ml final concentra-
tion. Normally pBR322 DNA exists in super-coiled form (SC). With
the attack of OH radical generated from Fenton reaction SC was
broken into open circular (OC). Data recorded in Fig. 1 depicts %
SC form as DNA protecting activity and % OC form as DNA damag-
ing activity of test agents.% SC form and % OC form were calculated
by Lab Image ID gel analysis software (2006) for each compound.
Negative control i.e., only pBR322 DNA showed given % SC form
used in the test, while there is total DNA damage in positive control
(X i.e., DNA + FeSO4 + H2O2). Some reacting compounds like Z1.1,
A1.1 and A2.1 showed greater than 60% DNA protecting activity.
Among all the test compounds, FA2.1 had shown maximum DNA
protection by% SC form retention of 64%.
3.2.2. DPPH free radical scavenging (Antioxidant activity)5
DPPH is a stable free radical. Antioxidants react with DPPH by
donating electron or hydrogen thus neutralizing it to yellow color
diphenyl picrylhydrazine [33]. This reduction of DPPH radical by
antioxidants can be determined by the decrease in absorbance at
517 nm spectrophotometrically, however, an increase in absor-
bance will show pro-oxidant activities. DPPH free radical scaveng-
ing activities of Schiff bases were assayed at three concentrations
100, 50 and 25 lg/ml as shown in Table 2. Ascorbic acid was used
as a positive control that showed DPPH discoloration. DPPH free
radical scavenging activity of tested compound was concentration
dependant. Higher radical scavenging was observed at higher con-
4. Conclusion
Long chain aliphatic esters as well as organic and ferrocene con-
taining Schiff bases were successfully synthesized. Results of dif-
ferent in vitro biocidal testing of Schiff bases and ferrocene Schiff
bases exhibit that ferrocene Schiff bases had shown good antitu-
mor (antimitotic), DPPH scavenging (antioxidant) and DNA
centration i.e. 100 lg/ml. DPPH scavenging analysis of compounds
showed AN1.1 and FA2.1 are the best scavengers of DPPH among
test agents by 77% and 65% scavenging activity respectively at
100 lg/ml final concentration. IC 50 (50% inhibitory concentration)
calculations support FA2.1 to be best DPPH radical quencher with
90
80
70
60
50
40
30
20
10
0
AN AN
1.1 2.1
FA1. FA2.
P
X
Z1 Z1.1 A1
A2 A1.1 A2.1 AN1 AN2
FA1 FA2
1
1
19.3
80.7
0
0
52.1 39.8 43.2 48.7 38.1 39.6 64.9 53.5 69.4 53.5 58.4 45.4 45.6 35.8
47.9 60.2 55.9 51.2 61.9 60.4 35.1 46.7 30.6 46.5 41.6 54.6 54.4 64.2
Open circular %
Superoiled %
Synthesized organic compounds
Fig. 1. DNA protective activity of synthesized organic compounds from oxidative damage. ꢂ All the synthesized organic compounds were tested at 10
lg/ml final
concentration. ꢂ Plasmid pBR322 (P) was used as a DNA source mostly present in super-coiled form. ꢂ FeSO4 and H2O2 generated OH radical by Fenton reaction in each test.
ꢂ Total DNA damage was observed in case of positive control (X) where there was only the plasmid DNA in interaction with the Fenton reaction. ꢂ % super-coiled form of
pBR322 DNA retained indicated protection while open-circular form represents damage in each treatment.

H. Nawaz et al. / Journal of Organometallic Chemistry 694 (2009) 2198–2203
2203
[14] V. Vrchovska, C. Sousa, P. Valenta, F. Ferreres, J.A. Pereira, Seabra, P.B. Andrade,
Food. Chem. 98 (2006) 416.
[15] C. Behl, B. Moosmann, Free. Radic. Biol. Med. 33 (2002) 182.
[16] P.S. Coker, J. Radecke, C. Guy, N.D. Camper, Phytomedicine 10 (2003)
133.
[17] G.Y. Yeap, S.T. Ha, P.L. Lim, P.L. Boey, W.A.K. Mahmood, Mol. Cryst. 423 (2004)
73.
[18] G.Y. Yeap, S.T. Ha, P.L. Boey, W.A.K. Mahmood, Mol. Cryst. 452 (2006) 73.
[19] G.Y. Yeap, W.S. Ooi, Mol. Cryst. 381 (2002) 169.
[20] S. Sudhakar, T. Narasimhaswamy, K.S.V. Srinivasan, Liq. Cryst. 27 (2000)
1525.
protecting activity as compared to other compound of same series.
In this study a difference in biological activities was also noted be-
tween Schiff-base series and their reacting compounds. Consider-
ing significant antitumor and antioxidant activities of ferrocene
Schiff bases they could be regarded as potential anticancer drug
candidate with further advanced testing and optimization.
References
[21] N.R. Ferrigni, J.E. Putnam, B. Anderson, L.B. Jacobsen, D.E. Nichols, D.S. Moore,
J.L. McLaughlin, P.G. Powell, C.R.J. Smith, J. Nat. Prod. 45 (1982) 679.
[22] C.Y. Hsu, Biol. Res. 39 (2006) 281.
[23] Y.H. Tian, Food. Chem. 91 (2005) 413.
[24] D.L. Pavia, G.S. Lampman, G.S. Kriz, Introduction to Spectroscopy, Thomson
Learning, 2001.
[25] A. Ullah, F.L. Ansari, I.U. Haq, S. Nazir, B. Mirza, Chem. Biodiv. (2007) 4203.
[26] S. Inaytullah, R. Irum, A.U. Rehman, M.F. Chaudhry, B. Mirza, Pharm. Biol. 45
(2007) 397.
[27] M.S. Ahmad, M. Hussain, M. Hanif, S. Ali, B. Mirza, Molecules 12 (2007) 2348.
[28] M. Hanif, M. Hussain, S. Ali, M.H. Bhatti, M.S. Ahmad, B. Mirza, H.S. Evans, Turk.
J. Chem. 31 (2007) 349.
[29] M.S. Ahmad, M. Hussain, M. Hanif, S. Ali, B. Mirza, Chem. Biol. Drug Des, doi:
10.1111/j.1747-0285.2008.00668.x.
[30] A.C. Braun, Prog. Exp. Tumor. Res. 15 (1972) 165.
[31] A.G. Galsky, J.P. Wilsey, R.G. Powell, Plant Physiol. 65 (1980) 184.
[32] R. Kovjazin, T. Eldar, M. Patya, A. Vanichicin, H.M. Lander, A. Novogrodsky,
FASEB J. 17 (2003) 467.
[1] B. Patwardhan, A.D.B. Vaidya, M. Chorghade, Curr. Sci. India 86 (2004) 789.
[2] M. MacCoss, T.A. Baillie, Science 303 (2004) 1810.
[3] K.E. Dombowski, W. Baldwin, J.E. Sheats, J. Organomet. Chem. 302 (1986) 281.
[4] R. Epton, G. Marr, G.K. Rogers, J. Organomet. Chem. 150 (1978) 93.
[5] P. Hu, K. Zhao, Hong-Bo Xu, Molecules (2001) M251.
[6] A. Togni, R.L. Halterman (Eds.), Metallocenes, Wiley-VCH Verlag GmbH,
Weinheim, Germany, 1998.
[7] A. Togni, T. Hayashi (Eds.), Ferrocene: Homogenous Catalysis, Organic
Synthesis, Material VCH, Wenheim, 1995.
[8] P. Stepnicka (Ed.), Ferrocenes: Ligands, Material and Biomolecules, John Wiley
and Sons, 2008.
[9] O. Domarle, G. Blampain, H. Agnaniet, T. Nzadiyabi, J. Lebibi, J. Brocard, L.
Maciejewski, C. Biot, A.J. Georges, P. Millet, Antimicrob. Agents Chemother. 42
(1998) 540.
[10] S. Top, A. Vessieres, C. Cabestaing, I. Laios, G. Leclecq, C. Provot, G. Jaouen, J.
Organomet. Chem. 637 (2001) 500.
[11] S. Top, J. Tang, A. Vessieoes, D. Carrez, C. Provot, G. Jaouen, Chem. Commun.
(1996) 955.
[12] C. Biot, G. Glorian, L. Maciejewski, J. Brocard, O. Domarle, G. Blampain, P. Millet,
A.J. George, H. Abessolo, D. Dive, J. Lebibi, J. Med. Chem. 40 (1997) 3715.
[13] B. Halliwell, Ann. Rev. Nutr. 16 (1996) 33.
[33] G.H. Naik, K.I. Priyadarsini, J.G. Satav, M.M. Banavalikar, D.P. Sohoni, M.K.
Biyani, H. Mohan, Phytochemistry 63 (2003) 97.
[34] X. Wu, E.R.T. Tiekink, L. Kostetski, N. Kocherginsky, A.L.C. Tan, S.B. Khoo, P.
Wilairat, M. Go, Eur. J. Pharma. Sci. 27 (2006) 175.