Dichloro-4-quinolinol-3-carboxylic acid: Synthesis and antioxidant abilities to scavenge radicals and to protect methyl linoleate and DNA

Article abstract of DOI:10.1016/j.ejmech.2010.01.018

5,7-, 5,8-, 6,8-, 7,8-Dichloro-4-quinolinol-3-carboxylic acid (5,7-, 5,8-, 6,8-, 7,8-DCQA) together with 7-chloro-4-quinolinol-3-carboxylic acid (7-CQA) and 4-quinolinol-3-carboxylic acid (QA) were synthesized to investigate the antioxidant properties. 5,7-DCQA exhibited the highest ability to scavenge 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS^+.), 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and galvinoxyl radicals. 6,8-DCQA possessed the highest efficacy to protect methyl linoleate against 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced oxidation. 5,7-, 5,8-DCQA and QA were able to retard the β-carotene-bleaching in β-carotene-linoleic acid emulsion. In addition, 5,8- and 6,8-DCQA efficiently protected DNA against hydroxyl radical (.OH)-mediated oxidation, and 5,8-DCQA and 7-CQA were active to protect DNA against AAPH-induced oxidation. Furthermore, only 7-CQA can protect DNA against Cu²⁺/glutathione (GSH)-mediated oxidation. Dichloro-4-quinolinol-3-carboxylic acids were potent to be antiradical drugs, and were worthy to be researched pharmacologically.

Full text of DOI:10.1016/j.ejmech.2010.01.018

European Journal of Medicinal Chemistry 45 (2010) 1821–1827
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Dichloro-4-quinolinol-3-carboxylic acid: Synthesis and antioxidant abilities
to scavenge radicals and to protect methyl linoleate and DNA
*
Guo-Xiang Li, Zai-Qun Liu , Xu-Yang Luo
Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China
a r t i c l e i n f o
a b s t r a c t
Article history:
5,7-, 5,8-, 6,8-, 7,8-Dichloro-4-quinolinol-3-carboxylic acid (5,7-, 5,8-, 6,8-, 7,8-DCQA) together with
7-chloro-4-quinolinol-3-carboxylic acid (7-CQA) and 4-quinolinol-3-carboxylic acid (QA) were synthe-
sized to investigate the antioxidant properties. 5,7-DCQA exhibited the highest ability to scavenge 2,20-
azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTSD.), 2,20-diphenyl-1-picrylhydrazyl
(DPPH) and galvinoxyl radicals. 6,8-DCQA possessed the highest efficacy to protect methyl linoleate
against 2,20-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced oxidation. 5,7-, 5,8-DCQA and
Received 2 November 2009
Received in revised form
7 January 2010
Accepted 8 January 2010
Available online 20 January 2010
QA were able to retard the b-carotene-bleaching in b-carotene-linoleic acid emulsion. In addition, 5,8-
Keywords:
and 6,8-DCQA efficiently protected DNA against hydroxyl radical (.OH)-mediated oxidation, and 5,8-
DCQA and 7-CQA were active to protect DNA against AAPH-induced oxidation. Furthermore, only 7-CQA
can protect DNA against Cu2þ/glutathione (GSH)-mediated oxidation. Dichloro-4-quinolinol-3-carboxylic
acids were potent to be antiradical drugs, and were worthy to be researched pharmacologically.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antioxidant
4-Quinolinol
DNA
Free-radical
Methyl linoleate
1. Introduction
analogues as potential antiviral agents [13]. The antioxidant
capacities of 7-chloro (fluoro)-4-quinolinol, a clinic anticancer
Many theories are developed to explain the molecular mecha-
nisms of aging [1], among which the oxidative stress is widely
admitted [2]. The oxidative stresses of DNA, membrane, lipid and
protein vary cellular components, leading to aging and diseases.
The free radical-induced in vivo oxidation is regarded as the key to
the pathogenesis of cardiovascular diseases and cancer [3]. Thus,
the use of antioxidant to protect biological species against radical-
induced oxidation becomes a novel therapeutic and nutritional
idea [4,5]. Some components in diet and medicinal herbs attract
much attention because of free-radical-scavenging, antivirus,
antiinflammatory, wound healing, and antibacterial activities [6–9].
Meanwhile, design and synthesis of antioxidants with special
structural feature are of importance to develop novel drugs and to
extend usage of known drugs.
Some novel pharmacological functions are even found in
small molecules. 8-Quinolinol used in combination with pacli-
taxel improves the treatment on breast cancer [10]. Clioquinol
(5-chloro-7-iodo-8-quinolinol) is a drug for the treatment of
aging and memory impairment [11]. 4-Quinolinol derivatives
were found to have various biological activities [12], and chloro-
substituted 4-quinolinols were used to prepare novel nucleoside
drug in China, as well as its structural analogues with carboxylic
and ester groups at 3-position were investigated in the experi-
mental system of erythrocyte hemolysis induced by 2,20-azo-
bis(2-amidinopropane) dihydrochloride (AAPH) [14]. The
antioxidant effectiveness of the complex formed between
7-chloro (fluoro)-4-quinolinol-related compounds and
b-cyclo-
dextrin was discussed as well [15]. Moreover, 5,7-dichloro-4-
quinolinol-3-carboxylic acid was reported to inhibit glycine
receptor [16], leading to the present study on the antioxidant
effectiveness of a series of dichloro-substituted 4-quinolinol-3-
carboxylic acid including 5,7-, 5,8-, 6,8-, and 7,8-dichloro-4-
quinolinol-3-carboxylic acid (5,7-, 5,8-, 6,8-, and 7,8-DCQA).
7-Chloro-4-quinolinol-3-carboxylic acid (7-CQA) and 4-quinoli-
nol-3-carboxylic acid (QA) were utilized as reference antio-
xidants in this work. These compounds were abbreviated as
4-quinolinols.
2. Chemistry
Skraup cyclization is a traditional method to introduce hydroxyl
group into benzene ring in quinoline. For example, as shown in Eq.
(1), clioquinol, a drug for the treatment of Alzheimer, Parkinson and
Huntington diseases [17], can be prepared by the corresponding
aminophenol and acrolein formed by the dehydration and oxida-
tion of glycerol in the presence of sulfuric acid.
* Corresponding author. Tel.: þ86 431 88499174; fax: þ86 431 88499159.
E-mail address: zaiqun-liu@jlu.edu.cn (Z.-Q. Liu).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.018

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G.-X. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1821–1827
The abundant components of polyunsaturated fatty acids
(PUFAs) in lipids and membranes, and the susceptibility to the
oxidation make linoleic acid a substrate usually employed to mimic
PUFAs undergoing the oxidation chemically [30]. Linoleic acid can
be oxidized by the ambient atmosphere automatically, and the
autoxidation of linoleic acid can be inhibited by
b
-carotene, called
b-carotene-bleaching test. If a compound can hinder the decrease
Gould–Jacobs [18] and Niementowski reactions [19], and many
of the absorbance for
b-carotene, it acts as an antioxidant to
other methods [20] have been developed to introduce hydroxyl
group into pyridine ring in quinoline. As shown in Eq. (2), the
condensation between an amine and triethyl orthoformate gener-
ates imine 1 that converts into imine 2 in boiling diethyl malonate.
Then, an isomerization transforms C]C in imide 2, leading to the
formation of alkenyl amine 3. An intramolecular cyclization
connects ortho-position of benzene ring with C]O in eCOOC2H5 to
form ethyl 4-quinolinol-3-carboxylate, the precursor of 4-quinoli-
nol-3-carboxylic acid [21].
prohibit the autoxidation of linoleic acid [31]. The oxidation of
methyl linoleate initiated by 2,20-azobis(2-amidinopropane)
dihydrochloride (AAPH, ReN]NeR, R]eCMe2C(]NH)NH2),
a water-soluble azo radical resource, is applied to mimic PUFA
undergoing radical-induced oxidation, and can be followed by
measuring the concentration of methyl linoleate via gas chroma-
tography (GC) [32].
Many experimental systems have been applied to explore the
mechanisms of radical-induced DNA oxidation [33]. AAPH- and
hydroxyl radical (.OH)-induced oxidative damages of DNA are
useful in vitro systems because the rate of peroxyl radical (ROO.)
generated from the decomposition of AAPH depends upon the
.
concentration of AAPH [34], and OH can be readily generated by
mixing tetrachlorohydroquinone and H2O2 [35]. Supercoiled strand
of DNA transforms into open circular and linear forms in the
process of radical-induced oxidation [36], and subsequently,
generates more than 20 carbonyl species that can be determined
spectophotometrically after reacting with thiobarbituric acid (TBA)
[37]. Thus, carbonyl species formed in the oxidation of DNA were
also called as TBA reactive substance (TBARS). Cu(II) plus gluta-
thione (GSH) generate GS. to oxidize DNA, progressing the forma-
tion of 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodG) [38] and
TBARS [39]. The aforementioned experimental systems were in
vitro methods to evaluate the antioxidant activity to protect DNA
against radical-induced oxidation.
As shown in Scheme 1, this method was improved. The imide 3
was formed directly by heating the reactant in diethyl ethox-
ymethylenemalonate [18]. In our work, dichloro-4-quinolinol-3-
carboxylic acids were synthesized following this method. Diphenyl
ether was used as a media for the intramolecular cyclization
occurring at high temperature. Meanwhile, the surplus diethyl
ethoxymethylenemalonate and amine were distilled from the
mixture with the temperature increasing. As a result, ethyl
dichloro-4-quinolinol-3-carboxylate was precipitated from
diphenyl ether. So, the addition of diphenyl ether combined the
isolation of surplus reagents with the cyclization reaction in one-
pot. Then, hydrolysis under basic condition and acidification
afforded dichloro-4-quinolinol-3-carboxylic acid. 7-CQA and QA
were prepared following this method as well [22–24].
4. Results and discussion
4.1. Scavenging ABTSþ., DPPH and galvinoxyl radicals
The reaction between an antioxidant and ABTSD. reflects the
ability of the antioxidant to reduce radicals since ABTSD. is gener-
ated from the oxidation of ABTS salt, and is widely used to assess
the antioxidative capacity of phenolics [40]. The reactions between
an antioxidant and DPPH and galvinoxyl radicals reveal the abilities
of the antioxidant to donate its hydrogen to N- and O-centered
radicals [41,29]. Therefore, the reactions with these radicals will
give direct evidence for 4-quinolinols to scavenge radicals. Fig. 1
illustrates the residual percentages of these radicals after 1.5 mM
4-quinolinols were incubated with these radicals for 30 min. Low
percentage implies that the 4-quinolinol has high free-radical-
scavenging activity.
3. Pharmacology
Free-radical-induced oxidative stress may modify the redox
status of tissues and the activation status of redox sensitive tran-
scription factors, and may be an important mechanism for aging
[25] and cancer [26]. Thus, the obtained compounds are first
screened by trapping 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) cationic radical (ABTSD.) [27], 2,20-diphenyl-1-picrylhy-
drazyl (DPPH) [28], and galvinoxyl radicals [29].
OH
NH2
OH
5
1. C2H5OCH=C(COOC2H5)2; 1000C,3 h
4
COOH
COOC2H5
1. OH-
6
3
2
7
R1
+
; 250 0C ,2 h, N2
N
2. H
2.
O
N
8
R1
R1
R1 = H
3-chloro
R1 = H; QA
7-chloro; 7-CQA
2,3-dichloro
2,4-dichloro
2,5-dichloro
3,5-dichloro
7,8-dichloro; 7,8-DCQA
6,8-dichloro; 6,8-DCQA
5,8-dichloro; 5,8-DCQA
5,7-dichloro; 5,7-DCQA
Scheme 1. Synthetic routine for dichloro-4-quinolinol-3-carboxylic acid.

G.-X. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1821–1827
1823
radicals are related to the stabilization of 5,7-DCQA radical. As
shown in Eq. (3), the resonance structure of 4-quinolinol radical
may help us to understand the effects of two chlorine atoms on the
stabilization of 4-quinolinol radical.
103.6
102.1
103
98
100
100.6
99.9
97.5
93
90
88
The hydrogen atom in the hydroxyl group of 4-quinolinol (I) is not
very easy to be abstracted by a radical (R.) because of the intra-
molecular hydrogen bond formed between eOH and eCOOH. Once
the hydrogen atom in hydroxyl group is abstracted by a radical to
form 4-quinolinol radical (II), the single electron may transfer to 3-,
N-, 6-, 8-position via resonance structures. As an electron-with-
drawing group, eCOOH in resonance structure III cannot supply the
electron to the radical, and is not beneficial for the stabilization of
the 4-quinolinol radical. As an electron-withdrawing group,
chlorine atom also cannot supply the electron to 4-quinolinol
radical when the single electron transfers to 6- and 8-pisition via
resonance structure. Chlorine atoms at 5- and 7-position avoid
direct encountering the single electron locating at 6- and 8-position
via resonance. In addition, as we all know that the stabilization of
104. 2
103. 5 103. 5
105
102
102
100
100
95
93.5
90
a-tocopherol radical makes a-tocopherol an effective antioxidant.
Two methyl groups at ortho-position (in the framework) stabilizes
the single electron in the oxygen-centered radical, viz., the steric
effects of two methyl groups largely contribute to increase the
102
98
100
antioxidant effectiveness of
a-tocopherol. Similarly, in the reso-
nance structure VI two chlorine atoms locate at ortho-position of
the radical (in the framework) when the single electron transfers to
6-position via resonance structure. The steric effects of chlorine
atom at 5- and 7-position may also stabilize the single electron, and
consequently, enhance the stabilization of the radical of 5,7-DCQA.
Thus, 5,7-DCQA exhibits higher antioxidant effectiveness than
other 4-quinolinols.
94.6
94.7
94.1
93.8
93.4
94
91.2
90
Fig. 1. Percentages of residual ABTSþ., DPPH and galvinoxyl radicals in the presence of
1.5 mM 4-quinolinols for 30 min.
In the reaction of 4-quinolinols with ABTSD., only 5,7-DCQA
decreases the percentage of ABTSD. to 90%, and other 4-quinolinols
4.2. Protecting methyl linoleate and bleaching
acid emulsion
b-carotene in linoleic
almost cannot influence the percentage of ABTSD.
. Similar
phenomenon is also found in the reaction between 4-quinolinols
and galvinoxyl radical. 5,7-DCQA is still able to decrease the
percentage of galvinoxyl radical to 93.5%, while the percentages of
galvinoxyl radical are close to the control experiment in the pres-
ence of other 4-quinolinols, indicating that the hydroxyl group in
5,7-DCQA can donate its hydrogen atom to O-centered radical, and
can reduce ABTSD. radical. On the other hand, all the 4-quinolinols
employed herein are able to decrease the percentage of DPPH
radical to w94%, demonstrating that hydroxyl group in 4-quinoli-
nols are able to donate the hydrogen atom to N-centered radical.
5,7-DCQA can decrease the percentage of DPPH radical to 91.2% in
this case. The strongest abilities of 5,7-DCQA to trap and to reduce
The abundant composition of polyunsaturated fatty acids
(PUFAs) such as linoleic, linolenic, and arachidonic acids makes
low-density lipoprotein and membrane phospholipids susceptible
to be attacked by ROS, leading to atherothrombotic cardiovascular
diseases eventually [42]. Thus, radical-induced oxidation of linoleic
acid usually acts as an in vitro experimental system to mimic PUFA
undergoing ROS-induced oxidative stress. The oxidation of linoleic
acid can be followed by measuring the formation of peroxide of
linoleic acid via high performance liquid chromatography (HPLC)
[30]. The decay of the concentration of linoleic acid can also be
detected by GC with methyl linoleate applied [32]. In the control
experiment, the concentration of methyl linoleate decreases from

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G.-X. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1821–1827
13.3 to 11.8 mM after 4 h, indicating that 1.5 mM methyl linoleate
was exhausted by 40 mM AAPH-induced oxidation during this
period. The concentrations of methyl linoleate were detected at 4 h
in the presence of 2.0 mM 4-quinolinols. Thus, the concentration of
the exhausted methyl linoleate was calculated and compared with
that in the control experiment (1.5 mM). The obtained percentages
of the exhausted concentration for methyl linoleate are outlined in
Fig. 2. Low percentage indicates that the 4-quinolinol possesses
high activity in this case.
base II [43]. Similarly, in the resonance structure IV the sp2 orbital of
N atom with a single electron is overlapped by the p orbital of
chlorine atom at 8-position. This may contribute to the stabilization
of the radical with chlorine atom at 8-position.
Fig. 2 shows that the percentages of the exhausted concentra-
tion of methyl linoleate in the presence of 4-quinolinols are even
higher than that in the control experiment, indicating that QA,
7-CQA and 5,7-DCQA accelerate the exhaustion of methyl linoleate
and function as prooxidants to improve AAPH-induced oxidation of
methyl linoleate. On the other hand, the additions of 5,8-, 7,8- and
6,8-DCQA decrease the percentage of the exhausted concentration
of methyl linoleate, indicating that these 4-quinolinols are able to
protect methyl linoleate against AAPH-induced oxidation. Espe-
cially, 6,8-DCQA decreases the percentage of the exhausted
concentration of methyl linoleate even to 7.3%, revealing that the
antioxidant activity of 6,8-DCQA is much higher than other 4-qui-
nolinols in this case. A common character in the structure of 5,8-,
7,8- and 6,8-DCQA is a chlorine atom attaching to 8-position.
We have compared the antioxidant abilities of 2-((4-N,N-dime-
thylaminobenzylidene)amino)phenol (Schiff base I) and 4-((4-N,N-
dimethylaminobenzylidene)amino)phenol (Schiff base II), and
found that the antioxidant effectiveness of Schiff base I is higher
than that of Schiff base II in AAPH-induced hemolysis of erythro-
cytes. The sp2 orbital of N atom with electron pair may overlap the
p orbital of oxygen atom with the single electron, leading to the
stabilizationof the radical ofSchiff base I is higher thanthatof Schiff
Linoleic acid and b-carotene form a water-soluble emulsion with
Triton X-100 as the surfactant. The oxygen dissolved in water
initiates the autoxidation of linoleic acid and generates peroxyl
radical of linoleic acid (LOO.). LOO. is able to bleach
b
-carotene. In
this work the absorbance of the emulsion decreases from 1.00 to
0.58 after 100 min, indicating that
-carotene is bleached by LOO.
during this period. The amount of the exhausted -carotene is
b
b
related to the decrease of the absorbance, 1.00 ꢀ 0.58 ¼ 0.42.
Meanwhile, the decrease of the absorbance in the presence of
0.3 mM 4-quinolinols is also measured and compared with that in
the control experiment (0.42). The percentages of the decrease of
the absorbance are outlined in Fig. 2. Low percentage indicates that
the 4-quinolinol has high ability to protect linoleic acid against the
autoxidation. It can be found in Fig. 2 that 7,8- and 6,8-DCQA behave
as prooxidants herein. On the contrary, the percentages of the
decrease of the absorbance are relative low in the presence of QA, 7-
CQA, 5,7- and 5,8-DCQA, indicating that QA, 7-CQA, 5,7- and 5,8-
DCQA act as antioxidants to inhibit the autoxidation of linoleic acid.
.
4.3. Protecting DNA against OH-, AAPH-, and Cu2þ/GSH-induced
oxidation
.OH, one of the in vivo ROS, is usually used as a radical resource
to evaluate antioxidant activity in vitro. Recently, it was reported
that .OH can be generated by Fenton reaction between tetra-
chlorohydroquinone and H2O2 in a metal-free media [35]. Deoxy-
ribose in DNA is the main target to the attack of .OH, and
malondialdehyde is formed as the final product that can be
measured after reacting with TBA. Some researchers regarded
TBARS as the contribution from the heating in the post-treatment
other than from the oxidation of DNA. Actually, in the blank
experiment, the absorbance at 535 nm did not vary in the absence
113. 3
111. 3
106. 7
120
80
40
0
100
62.7
41.9
7. 3
.
of OH, AAPH and Cu2þ/GSH (data not shown). To take AAPH-
induced oxidation of DNA as an example, in the control experiment
the absorbance at 535 nm was 0.60 before the oxidation of DNA,
and increased to 0.87 after 4 h. The increase of the absorbance is
related to the formation of TBARS during the oxidation of DNA. In
the presence of 4-quinolinols the absorbance is measured and
compared with that in the control experiment (0.87). The
percentages of TBARS generated in the oxidation of DNA mediated
by various radicals are shown in Fig. 3. Low percentage indicates
that the 4-quinolinol has relative high efficacy to protect DNA
against radical-induced oxidation. All the 4-quinolinols except 7-
CQA can protect DNA against .OH-induce oxidation. Especially, the
additions of 5,8- and 6,8-DCQA even decrease the percentages of
128.7
130
110
90
107.9
100
91.1
77.3
77.3
77.3
.
TBARS lower than 85%. OH is able to react with benzene to form
70
phenol. For example, 2,3- and 2,5-dihydroxylbenzoic acid were
.
detected in OH-induced oxidation of salicylic acid [35]. Thus, the
antioxidant behaviors of 4-quinolinols may also ascribe to the
.
direct interactions between OH and 4-quinolinols.
The decomposition of AAPH generates peroxyl radical (ROO.) to
abstract H atom from the C-40 atom of DNA, causing strand breaks
and generating TBARS eventually [44]. The protective effects of
4-quinolinols on DNA against AAPH-induced oxidation are not as
Fig. 2. Percentages of the exhausted methyl linoleate (13.3 mM as the beginning
concentration) induced by 35.6 mM AAPH after 4 h in the presence of 2.0 mM 4-qui-
nolinols, and percentages of the decrease of the absorbance at 460 nm after 100 min in
the presence of 0.3 mM 4-quinolinols.

G.-X. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1821–1827
1825
higher than that in the control experiment, indicating that 5,8-
and 7,8-DCQA behave as prooxidants in this case. The different
effects of 4-quinolinols on DNA depend on the radical-generating
methods since the oxidation mechanisms of DNA are different in
101. 6
100
100
90
92. 7
.
the case of OH, ROO. or GS. as initiators. The present findings can
90.4
88.7
be summed up and illustrated in Table 1. 7-CQA, 5,8- and 6,8-
DCQA exhibit relative high inhibition activities to radical-induced
oxidation of DNA.
84.6
83.3
80
It can be also found in Fig. 3 that the percentage of TBARS
generated is even higher than 100% when 7-CQA and QA are used
in .OH- and AAPH-induced oxidation of DNA, respectively, and 5,8-
DCQA and 7,8-DCQA are applied in Cu2þ/GSH-mediated oxidation
of DNA. These results imply that much more TBARS is generated in
the aforementioned experiments, demonstrating that these
compounds act as prooxidants under these experimental condi-
tions. Hence, the antioxidant and prooxidant mechanisms of
4-quinolinols are worthy to be explored in the further research
work.
103
104
100
96
100
99. 4
98.6
97. 9
95.4
95
5. Conclusion
92
The antioxidant properties of the obtained compounds are
screened in chemical and biological experimental systems,
resulting in complicated results of dichloro-substituted 4-quino-
linols to be antioxidants or prooxidants. It can be confirmed that
introducing two chlorine atoms into benzene ring greatly changes
the antioxidant property of 4-quinolinols in comparison with QA
and 7-CQA. 5,7-DCQA has the highest ability to scavenge free
radicals. 5,8-, 6,8- and 7,8-DCQA protect methyl linoleate against
AAPH-induced oxidation with 6,8-DCQA having the highest ability.
5,7- and 5,8-DCQA together with QA are able to protect linoleic
acid against the autoxidation. Except 7-CQA all the 4-quinolinols
115
111.3
103.4
105
95
100
98.3
96. 6
95.4
.
can protect DNA against OH-induced oxidation, while only 7-CQA
88.9
and 5,8-DCQA play antioxidant role in AAPH-induced oxidation of
DNA. In addition, 7-CQA acts as an antioxidant in Cu2þ/GSH-
induced oxidation of DNA. The present work gives some in vitro
results of dichloro-4-quinolinol to be antioxidants, and exhibits
that it is worthy to screen the pharmacology of these compounds
in vivo.
85
Fig. 3. Percentages of TBARS generated in the oxidation of 2.0 mg/mL DNA induced by
2.0 mM tetrachlorohydroquinone and 4.0 mM H2O2 for 30 min in the presence of
6. Experimental protocols
400
mM 4-quinolinols; percentages of TBARS generated in the oxidation of 2.0 mg/mL
DNA induced by 40 mM AAPH for 4 h in the presence of 200
m
M 4-quinolinols; and
6.1. Materials
percentages of TBARS generated in the oxidation of 2.0 mg/mL DNA induced by 5.0 mM
Cu2þ and 4.0 mM GSH for 4 h in the presence of 200
mM 4-quinolinols.
Diammonium salt of 2,20-azinobis(3-ethylbenzothiazoline-6-
sulfonate) (ABTS), DPPH and galvinoxyl radicals were purchased
from Fluka Chemie GmbH, Switzerland. AAPH, the naked DNA
sodium salt, methyl linoleate, linoleic acid, diethyl ethox-
ymethylenemalonate were purchased from ACROS ORGANICS,
Belgium. Other agents were of analytical grade and used directly.
The structures of the obtained compounds were identified by 1H
and 13C NMR (Varian Mercury 300 NMR spectrometer).
.
good as in OH-induced oxidation of DNA. Only 7-CQA and 5,8-
DCQA can decrease the percentages of TBARS to w95%. The addi-
tion of QA even generates more TBARS than that in the control
experiment, indicating that 4-quinolinol without chlorine as sub-
situent even promotes AAPH-induced oxidation of DNA. Thus,
introducing chlorine atoms is beneficial for 4-quinolinols to protect
DNA against ROO.-induced oxidation. The results for AAPH-induced
oxidation of DNA show that 7-CQA and 5,8-DCQA present the best
protective effects, and are not in agreement with that found for
protecting methyl linoleate. It is difficult to clarify the interaction
style between the antioxidants and DNA. But the linkage of the
antioxidant with DNA may influence the antioxidant behavior. So,
an antioxidant usually gives different results in biological and
chemical experimental systems [45].
Table 1
A summarization of 4-quinolinol to protect DNA against different radical-induced
oxidation.
4-Quinolinol
.OH
ROO.
GS.
QA
7-CQA
O
O
O
5,7-DCQA
5,8-DCQA
6,8-DCQA
7,8-DCQA
Cu(II) can react with GSH to form radical (GS.) via
Cu(II) þ GSH / Cu(I) þ GS., where GS. degrades DNA to produce
TBARS [38,39]. Fig. 3 shows that 7-CQA decreases TBARS to 88.9%,
and QA, 5,7- and 6,8-DCQA decrease TBARS to 95–98%. The addi-
tions of 5,8- and 7,8-DCQA even make the percentage of TBARS
O
O
O Indicates the corresponding 4-quinolinol has the relative high ability to protect
DNA against the radical-induced oxidation.

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G.-X. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1821–1827
6.2. Synthesis of 4-quinolinols and identification of structures
CH]CHe in phenyl), 12.98 (s, 1H, eOH), 14.60 (s, 1H, eCOOH); 13
NMR (75 MHz, DMSO-d6) : 177.7, 165.4, 146.2, 137.4, 137.2, 127.1,
C
d
A mixture of a corresponding amine (0.02 mol) and 4.76 g
(0.022 mol) of diethyl ethoxymethylenemalonate were heated in
a boiling water bath for 3 h and cooled to ambient temperature, fol-
lowed by 50 mL diphenyl ether added. The mixture was heated at
250 ꢁC in N2 for 2 h under stirring. A large amount of white deposit
were precipitated and cooled to ambient temperature, then diluted
with petroleum ether. The deposit was filtered, washed with petro-
leum ether, and dried at vacuum pressure. Then, the solid was
refluxed in 50 mL of 30% KOH aqueous solution with thin layer
chromatography (TLC) inspections identifying the end of the hydro-
lysis. The solution was cooled to ambient temperature and acidified
with 10% HCl aqueous solution to pH ¼ 2. The crude product was
precipitated and recrystallized with water and acetic acid (10:1) [18].
125.3, 124.6, 121.5, 108.7. Calculation of elements for C10H5Cl2NO3:
C, 46.54; H, 1.95; N, 5.43; found C, 46.44; H, 1.91; N, 5.48.
6.3. Interactions of 4-quinolinols with ABTSþ., DPPH and galvinoxyl
radicals
The reactions between 4-quinolinols and ABTSD., DPPH and
galvinoxyl radicals were carried out as previous descriptions
[29,32]. DPPH and galvinoxyl radicals were dissolved in ethanol to
make the absorbance around 1.00 at 517 nm and 428 nm, respec-
tively. Two milliliter of 4.0 mM ABTS aqueous solution was oxidized
by 1.41 mM K2S2O8 for 16 h, then 100 mL of ethanol was added to
make the absorbance of ABTSD. around 0.70 at 734 nm. 4-Quino-
linols were dissolved in dimethyl sulfoxide (DMSO) as the stock
solution, and 0.1 mL was added to 1.9 mL of radical solution to keep
the final concentration of 4-quinolinols at 1.5 mM. The absorbance
(Adetect) was measured after 30 min. The absorbance of the control
experiment (Aref) containing 0.1 mL DMSO was also measured at
30 min. The percentages of residual radicals were calculated by
(Adetect/Aref) ꢂ 100.
6.2.1. 4-Quinolinol-3-carboxylic acid (QA) yield 48%
1H NMR (300 MHz, DMSO-d6)
d: 8.91 (s, 1H, CH]N), 8.30
(d, J ¼ 8.4 Hz, 1H, CH]CHe in phenyl), 7.90 (t, J ¼ 8.1 Hz and 7.2 Hz,
1H, CH]CHe in phenyl), 7.83 (d, J ¼ 8.1 Hz, 1H, CH]CHe in
phenyl), 7.61 (t, J ¼ 7.5 Hz and 7.5 Hz,1H, CH]CHe in phenyl),13.43
(s, 1H, eOH), 15.36 (s, 1H, eCOOH); 13C NMR (75 MHz, DMSO-d6)
d:
178.3, 166.3, 145.2, 139.4, 133.9, 126.2, 125.0, 124.4, 119.6, 107.6.
Calculation of elements for C10H7NO3: C, 63.49; H, 3.73; N, 7.40;
found C, 63.60; H, 3.58; N, 7.31.
6.4. Effects of 4-quinolinols on the oxidations of linoleic acid and
methyl linoleate
6.2.2. 7-Chloro-4-quinolinol-3-carboxylic acid (7-CQA) yield 56%
Methyl linoleate, AAPH, and 4-quinolinols were dissolved in
t-butanol/H2O (2:1, v:v) to 13.3 mM, 35.6 mM, and 2.0 mM as the
final concentration (C0), respectively. To quantitate the concentra-
tion of methyl linoleate, methyl palmitate was added to a final
concentration of 8.9 mM as an internal standard because, as an
ester of saturated fatty acid, it cannot be oxidized during the
oxidation of methyl linoleate. Then, the above solution was incu-
bated at 37 ꢁC to initiate the oxidation. After 4 h the concentrations
of methyl linoleate in the presence of 4-quinolinols and in the
control experiment were determined by GC and recorded as Cdetect
and Cref. The percentage of the concentration of the exhausted
methyl linoleate was calculated by (C0 ꢀ Cdetect)/(C0 ꢀ Cref) ꢂ 100.
GC analysis was performed on a Hewlett-Packard 1890 GC equip-
1H NMR (300 MHz, DMSO-d6)
d: 8.94 (s, 1H, CH]N), 8.27 (d,
J ¼ 8.7 Hz,1H, CH]CHe in phenyl), 7.84 (s,1H, CH]CHe in phenyl),
7.60 (d, J ¼ 8.7 Hz,1H, CH]CHe in phenyl), 15.2 (s, 1H, eCOOH); 13C
NMR (75 MHz, DMSO-d6) d: 177.5, 166.2, 146.5, 141.0, 138.1, 127.1,
126.2, 123.2, 119.4, 108.0. Calculation of elements for C10H6ClNO3: C,
53.71; H, 2.70; N, 6.26; found C, 53.83; H, 2.79; N, 6.31.
6.2.3. 5,7-Dichloro-4-quinolinol-3-carboxylic acid (5,7-DCQA) yield
60%
1H NMR (300 MHz, DMSO-d6)
d
: 8.92 (s, 1H, CH]N), 7.78 (s, 1H,
CH]CHe in phenyl), 7.71 (s, 1H, CH]CHe in phenyl), 13.40 (s, 1H,
eOH), 15.02 (s, 1H, eCOOH); 13C NMR (75 MHz, DMSO-d6)
: 177.9,
d
165.8, 145.8, 142.3, 137.4, 134.3, 128.3, 119.9, 118.4, 109.3. Calculation
of elements for C10H5Cl2NO3: C, 46.54; H, 1.95; N, 5.43; found C,
46.69; H, 1.87; N, 5.61.
ped with a SE-54 30 m ꢂ 0.25 mm capillary column, 0.25
mm film
thickness, N2. The temperature in chamber and injector was 260 ꢁC
and 280 ꢁC, respectively, and the temperature in the hydrogen
flame ionization detector was 300 ꢁC [32]. The same volume of
DMSO was involved in the control experiment.
6.2.4. 5,8-Dichloro-4-quinolinol-3-carboxylic acid (5,8-DCQA) yield
38%
An emulsion was prepared by dissolving 5.0 mg of b-carotene,
1H NMR (300 MHz, DMSO-d6)
d
: 8.60 (s, 1H, CH]N), 8.00
40 mg of linoleic acid and 400 mg of Triton X-100 in 5.0 mL of
CHCl3. After CHCl3 was evaporated under vacuum pressure, 100 mL
of oxygen-saturated water was added and then shaken under
(d, J ¼ 8.4 Hz, 1H, CH]CHe in phenyl), 7.58 (d, J ¼ 8.4 Hz, 1H,
CH]CHe in phenyl), 12.75 (s, 1H, eOH), 14.82 (s, 1H, eCOOH); 13C
NMR (75 MHz, DMSO-d6)
d: 178.2, 165.5, 145.3, 138.2, 133.5, 131.9,
ultrasonic vibration to form homogeneous b-carotene-linoleic acid
128.7, 122.4, 122.1, 109.6. Calculation of elements for C10H5Cl2NO3:
C, 46.54; H, 1.95; N, 5.43; found C, 46.38; H, 2.01; N, 5.55.
emulsion (lmax ¼ 460 nm) with the absorbance recorded as A0 [31].
DMSO solutions of 4-quinolinols (0.3 mL) were mixed with 2.7 mL
of the emulsion with the final concentration of 4-quinolinols being
0.3 mM. The absorbance of the mixture was measured after
100 min and recorded as Adetect. The same volume of DMSO was
involved in the control experiment, and the absorbance was also
measured at 100 min and recorded as Aref. The percentage of the
decrease for the absorbance was calculated by (A0 ꢀ Adetect)/
(A0 ꢀ Aref) ꢂ 100.
6.2.5. 6,8-Dichloro-4-quinolinol-3-carboxylic acid (6,8-DCQA) yield
45%
1H NMR (300 MHz, DMSO-d6)
d: 8.60 (s, 1H, CH]N), 8.25
(d, J ¼ 1.5 Hz, 1H, CH]CHe in phenyl), 8.13 (d, J ¼ 1.5 Hz, 1H,
CH]CHe in phenyl), 13.04 (s, 1H, eOH), 14.75 (s, 1H, eCOOH); 13C
NMR (75 MHz, DMSO-d6) d: 176.9, 165.4, 145.9, 135.2, 133.5, 130.5,
126.8, 124.7, 123.5, 108.7. Calculation of elements for C10H5Cl2NO3:
C, 46.54; H, 1.95; N, 5.43; found C, 46.51; H, 2.05; N, 5.37.
6.5. Effects of 4-quinolinols on the oxidations of DNA
6.2.6. 7,8-Dichloro-4-quinolinol-3-carboxylic acid (7,8-DCQA) yield
40%
.OH-mediated oxidation of DNA was performed as previous
description [37]. DNA sodium salt was dissolved in phosphate
buffered solution (PBS: 6.1 mM Na2HPO4, 3.9 mM NaH2PO4), and
13.4 mL of DNA solution was mixed with 0.1 mL of DMSO solution
1H NMR (300 MHz, DMSO-d6)
d
: 8.60 (s, 1H, CH]N), 8.23
(d, J ¼ 8.7 Hz, 1H, CH]CHe in phenyl), 7.80 (d, J ¼ 8.7 Hz, 1H,

G.-X. Li et al. / European Journal of Medicinal Chemistry 45 (2010) 1821–1827
1827
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chlorohydroquinone. Finally, 1.0 mL of 60 mM PBS solution of
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the same as above mentioned. The same volume DMSO was
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