Antioxidant and antiproliferative activities of hydroxyl-substituted Schiff bases

Article abstract of DOI:10.1016/j.bmcl.2010.03.039

Eight hydroxyl-substituted Schiff bases with the different number and position of hydroxyl group on the two asymmetric aromatic rings (A and B rings) were prepared by the reaction between the corresponding aromatic aldehyde and aniline. Their antioxidant effects against the stable galvinoxyl radical (GO^{{radical dot}}) in ethyl acetate and methanol, and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced DNA strand breakage, and their antiproliferative effects on human hepatoma HepG2 cells, were investigated. Structure-activity relationship analysis demonstrates that o-dihydroxyl groups on the aromatic A ring and 4-hydroxyl group attached to the aromatic B ring contribute critically to the antioxidant and antiproliferative activities.

Full text of DOI:10.1016/j.bmcl.2010.03.039

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2417–2420
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antioxidant and antiproliferative activities of hydroxyl-substituted Schiff bases
a
a
b
a
a
a
a
Li-Xia Cheng , Jiang-Jiang Tang , Hui Luo , Xiao-Ling Jin , Fang Dai , Jie Yang , Yi-Ping Qian ,
a
a,
*
Xiu-Zhuang Li , Bo Zhou
State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, Gansu 730000, China
a
b
0
School of Medicine, Jinggangshan University, Ji an, Jiangxi 343009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight hydroxyl-substituted Schiff bases with the different number and position of hydroxyl group on the
two asymmetric aromatic rings (A and B rings) were prepared by the reaction between the corresponding
Received 28 November 2009
Revised 2 February 2010
Accepted 6 March 2010
Available online 11 March 2010
Å
aromatic aldehyde and aniline. Their antioxidant effects against the stable galvinoxyl radical (GO ) in
0
ethyl acetate and methanol, and 2,2 -azobis(2-amidinopropane hydrochloride) (AAPH)-induced DNA
strand breakage, and their antiproliferative effects on human hepatoma HepG2 cells, were investigated.
Structure–activity relationship analysis demonstrates that o-dihydroxyl groups on the aromatic A ring
and 4-hydroxyl group attached to the aromatic B ring contribute critically to the antioxidant and antipro-
liferative activities.
Keywords:
Schiff bases
Antioxidant
Galvinoxyl radical
DNA
Ó 2010 Elsevier Ltd. All rights reserved.
Antiproliferative effect
Structure–activity relationship
Reactive oxygen species (ROS) and free radicals such as super-
oxide anion, hydrogen peroxide and hydroxyl radicals are consid-
ered to be implicated in degenerative processes related to aging,
cancer and atherosclerosis, mainly because they can induce the
bases. On our ongoing research project on bioantioxidants, we pre-
viously found that simple structural modification of resveratrol
could significantly enhance its antioxidative activity. Encouraged
6
by the aforementioned information and in an attempt to better
understand the structure–activity relationship of hydroxyl-substi-
tuted Schiff bases as antioxidants and cancer chemopreventive
agents, we synthesized herein eight hydroxyl-substituted Schiff
bases (1–8) with the different number and position of hydroxyl
group on the two asymmetric aromatic rings (A and B rings), and
investigated their antioxidant effects against the stable galvinoxyl
1
oxidative damage of cell membranes, DNA, and proteins. Thus,
blocking the generation of ROS and free radicals by supplementa-
tion of antioxidants might have a beneficial role in preventing
these free radical-related diseases.2 The attachment of hydroxyl
groups on the aromatic ring makes hydroxyl-substituted Schiff
bases the effective antioxidants, and potential drugs to prevent dis-
ease related to free radical damage. Recently, Liu and co-workers
have reported the protective effects of hydroxyl-substituted Schiff
bases against free radical-induced peroxidation of triolein in
Å
0
radical (GO ) in ethyl acetate and methanol, and 2,2 -azobis(2-ami-
dinopropane hydrochloride) (AAPH)-induced DNA strand break-
age, and their antiproliferative effects on human hepatoma
HepG2 cells.
3
micelles, haemolysis of human red cells, and oxidation of DNA.
On the other hand, hydroxyl-substituted Schiff bases obtained
from the reaction between the corresponding aromatic aldehyde
and aniline, have a similar structure to trans-stilbene skeleton of
Eight hydroxyl-substituted Schiff bases (1–8) were easily syn-
thesized using the corresponding aromatic aldehyde and aniline
7
in a small amount of water or methanol (Scheme 1). The full de-
0
1
13
resveratrol (3,5,4 -trihydroxy-trans-stilbene), a well-characterized
tails for synthesis, characterization ( H NMR, C NMR and EI-MS)
and the subsequent activity tests of compounds (1–8) have been
described in the Supplementary data.
antioxidant and cancer chemopreventive molecule found in grapes
4
and a variety of medicinal plants. Their structural differences exist
only in the connection of two aromatic rings, one is carbon–nitro-
gen double bond, and the other is carbon–carbon double bond.
Although many studies have investigated the antioxidant proper-
It is well-known that one of the main characteristic responsible
for the antioxidant activity of a phenolic compound is its ability to
Å
scavenge free radicals. GO is a relatively stable oxygen radical and
5
8
ties of resveratrol, there have been only a few reports of antioxi-
has been widely used for evaluating antioxidant activities. Conse-
dant and antiproliferative effects of hydroxyl-substituted Schiff
quently, a quantitative kinetic study of the scavenging reaction of
Å
1
–8 toward GO at 25 °C was performed in ethyl acetate and meth-
Å
anol by UV–vis spectroscopy by recording the decay of the GO vis-
*
Corresponding author. Fax: +86 931 8915557.
E-mail address: bozhou@lzu.edu.cn (B. Zhou).
ible absorbance (kmax = 428 nm). If a large excess of compound 3
0
960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.039

2
418
L.-X. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2417–2420
Scheme 1. Synthetic scheme for the production of hydroxyl-substituted Schiff bases (1–8) and chemical structures of compounds investigated.
Å
was employed, the decay of GO in ethyl acetate occurred with
pseudo-first-order kinetics (Fig. 1). Plotting this pseudo-first-order
rate constant (kobs) versus the concentration of compound 3 gave a
straight line (the inset of Fig. 1). The slope of the straight line read-
Table 1
Å
GO -scavenging activity and antiproliferative activity against HepG2 cells of hydroxyl-
substituted Schiff bases
ka (Mꢁ1
s
ꢁ1
)
IC50c
(lM)
Compounds
Å
ily gave access to the second-order rate constant (k) for the GO -
Ethyl acetate
Methanol
scavenging reaction by 3. The values of k for compounds (1–8) in
1
2
3
4
5
6
7
8
0.01 ± 0.001
0.11 ± 0.01
31.9 ± 2.8
0.64 ± 0.05
1.10 ± 0.10
0.19 ± 0.01
103 ± 3
0.04 ± 0.002
0.22 ± 0.02
120 ± 3
0.91 ± 0.053
2.48 ± 0.21
0.22 ± 0.02
2.19 ± 0.09 ꢂ 10
14.8 ± 0.09 ꢂ 10
42.0 ± 3.7
>300
>300
108 ± 1
>300
>300
>300
6.8 ± 0.1
5.6 ± 0.1
79.6 ± 8.1
4.1 ± 0.2
ethyl acetate and methanol were summarized in Table 1. It ap-
Å
peared that the rate constants for the reactions of GO with these
compounds in methanol were remarkably higher than that in ethyl
acetate. Litwinienko and Ingold have observed previously an
abnormal increase of rate constants of 2,2-diphenyl-1-pic-
3b
3b
Å
3b
rylhydrazyl (DPPH ) radical scavenging reaction in alcoholic media
1.08 ± 0.07 ꢂ 10
which was attributed to partial ionization of the phenolic and a
Resveratrol
VP-16
13.4 ± 1.2
Å 9
very fast electron transfer from phenolate anion to DPPH . These
a
studies, together with our recent results in resveratrol and its ana-
Data are expressed as the mean ± SD for three determinations.
6
a
10
b
logues and
a
-pyridoin and its derivatives, suggest that, in alco-
The rates were measured by the second-order kinetics with the ratio of [7 or 8]/
Å
[
GO ] being 1/1.
holic media, the sequential proton loss electron transfer (SPLET
mechanism) predominates over the direct hydrogen atom transfer
c
Antiproliferative activity is expressed as IC50 values, the concentration for the
compound to cause 50% inhibition of the cell viability. Data are expressed as the
mean ± SD for three determinations.
(
HAT mechanism) for hydroxyl-substituted Schiff bases. This ki-
netic results made apparent the influence of the different number
and position of hydroxyl group on the two asymmetric aromatic
rings, with the ranking activity order being 8 > 7 > 3 > 5 > 4 >
Å
2
ꢀ 6 > 1. Compound 3 was more active in the GO -scavenging
In addition, the ability of compounds (1–8) to inhibit AAPH-in-
duced oxidative damage of DNA was also assessed in vitro by mea-
suring the conversion of supercoiled pBR322 plasmid DNA to the
open circular and linear forms using agarose gel electrophoresis
reaction than compound 2, indicating that the lower O–H bond
dissociation enthalpy is obtained for 4-OH in B ring. Notably, com-
pounds (7 and 8) bearing o-diphenolic groups exhibited remark-
Å
13
ably higher GO -scavenging activity than those bearing no such
analysis. As shown in Figure 2A, the supercoiled DNA was grad-
groups and resveratrol, a well-known antioxidant. It is well-estab-
lished fact that the o-diphenolic group is one of the main contrib-
utors to the antioxidant activity of phenolic compounds, which is
mainly due to its low oxidative potential11 and resonance stabiliza-
tion of the resulting phenoxyl radical intermediate with
subsequent o-quinone formation.12
ually converted to open-circular DNA (indication of a single-strand
breakage) with the increase of concentration of AAPH, and that
open-circular DNA was gradually converted to linear DNA (indica-
tion of a double-strand breakage) with the further increase of con-
centration of AAPH. The inhibition effects produced by compounds
(1–8) depended on the specific compound used as exemplified in
Figure 2B. On the basis of the percentage of intact supercoiled
DNA, the relative activity order in inhibiting the DNA strand break-
age was as follows: 8 > 4 > 7 > 3 > 5 ꢀ 6 > 2 > 1 (Fig. 2C), which is
Å
similar to that obtained from kinetic measurement in GO -scaveng-
ing reaction.
Furthermore, the antiproliferative effect of compounds (1–8) on
HepG2 cells was assessed by sulforhodamine B assay,14 and the
IC50 values and their stand deviations are reported in Table 1.
The clinically used anticancer drug, etoposide (VP-16), and resve-
ratrol were used reference drugs. Compounds are mainly divided
into three different groups according to the IC50 values: com-
pounds (7 and 8) bearing o-diphenolic groups were most active,
followed by resveratrol and compound 4, while the other com-
pounds were inactive. Especially, the antiproliferative activity of
7
and 8 were comparable to that of VP-16, a positive control.
Intriguingly, compounds (7 and 8) with higher antioxidant activity
exhibited higher antiproliferative activity, which reinforces the
idea of designing antioxidant-based cancer chemoprevention
agents.
Because the most of Schiff bases can be hydrolyzed back to the
corresponding amines and aldehydes depending on the reaction
conditions, we also determined the stability of compound 8 in
Figure 1. Spectral changes observed upon addition of compound 3 (50
l
M) to an
Å
ethyl acetate solution of GO (5
l
M) at 298 K (interval: 10 min). Inset: plot of the
pseudo-first-order rate constant (kobs) versus the concentration of compound 3.

L.-X. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2417–2420
2419
Figure 2. Agarose gel electrophoresis pattern of pBR322 DNA strand breakage induced by AAPH and inhibited by compounds (1–8). Supercoiled plasmid DNA (100 ng) was
incubated with AAPH and/or compounds (1–8) in 25 L phosphate-buffered saline (PBS, pH 7.4) at 37 °C for 60 min. (A) DNA strand breakage induced by the indicated
concentration of AAPH: (lane 1) control; (lanes 2–7) 1.25, 2.5, 5, 10, 20, and 40 mM AAPH, respectively. (B) Inhibitory effects of compounds (1–8) (10 M) against AAPH
10 mM)-induced DNA strand breakage: (lane 1) control; (lane 2) 10 mM AAPH alone; (lanes 3–11) resveratrol, 1, 2, 3, 4, 5, 6, 7, and 8, respectively. (C) Quantitative analysis of
protective effects of compounds (1–8) against AAPH-induced DNA strand breakage. DNA damage is represented by the percentage of supercoiled DNA to native DNA.
l
l
(
phosphate-buffered saline (PBS) (pH 7.4) at 37 °C by UV–vis spec-
troscopy. As shown in Figure 3A, the rapid disappearance of the
absorptions of 8 centered at 340 and 428 nm suggested that it
undergoes degradation in PBS by hydrolysis. However, the degra-
dation was considerably inhibited by the plasma protein, human
serum albumin (HSA), and the UV–vis spectra of 8 bound to HSA
had a broadening weak in the range of 400–475 nm (Fig. 3B). These
results imply that HSA could stabilize compound 8 to maintain its
biological activities in vivo. Similarly, curcumin, a well-known can-
cer preventive agent with low stability in aqueous solution, has
also been confirmed to be well stabilized by the plasma proteins
1
5
HSA and fibrinogen.
In conclusion, eight hydroxyl-substituted Schiff bases were syn-
thesized and bio-evaluated for their antioxidant and antiprolifera-
tive activities in pursuit of more active antioxidant and cancer
chemopreventive agents. Structure–activity relationship analysis
Figure 3. UV–vis absorption spectra of compound 8 (20
lM) at 37 °C in (A) pH 7.4 PBS (interval: 5 min) and (B) HSA (20 lM) (interval: 5 min). Dot line shows UV–vis
spectrum of HSA, and arrows show the time-related absorbance changes.

2
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L.-X. Cheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2417–2420
indicates that o-diphenolic groups and 4-hydroxyl group attached
to the aromatic B ring are vital for their antioxidant and antiprolif-
erative activities.
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Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (Grant Nos. 20972063 and 20621091), the 111 Pro-
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