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;