The synthesis and evaluation of novel hydroxyl substituted chalcone analogs with in vitro anti-free radicals pharmacological activity and in vivo anti-oxidation activity in a free radical-injury Alzheimer's model

Article abstract of DOI:10.3390/molecules18021693

Alzheimer's disease (AD) pathogenesis involves an imbalance between free radical formation and destruction. In order to obtain a novel preclinical anti-AD drug candidate, we synthesized a series of novel hydroxyl chalcone analogs which possessed anti-free radical activity, and screened their effects on scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH free radicals in vitro. Compound C7, 4,2'-dihydroxy-3,5-dimethoxychalcone was found to have potent activity in these anti-free radical activity tests. Further research revealed that C7 could elevate glutathione peroxidase (GSH-PX) and super oxide dismutase (SOD) levels and lower malonaldehyde (MDA) level in vivo in the Alzheimer's model. The indication of C7's effect on AD needs further study.

Full text of DOI:10.3390/molecules18021693

Molecules 2013, 18, 1693-1703; doi:10.3390/molecules18021693
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Synthesis and Evaluation of Novel Hydroxyl Substituted
Chalcone Analogs with in Vitro Anti-Free Radicals
Pharmacological Activity and in Vivo Anti-Oxidation Activity in
a Free Radical-Injury Alzheimer’s Model
Ying Pan 1,†, Yicun Chen 2,†, Qingnan Li 3, Xiaoyu Yu 1, Jinzhi Wang 1 and Jinhong Zheng 1,*
1
Department of Chemistry, Shantou University Medical College, Shantou 515041, Guangdong, China
2
Department of Pharmacology, Shantou University Medical College, Shantou 515041,
Guangdong, China
3
Shantou Central Hospital, Shantou 515041, Guangdong, China
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: jhzheng@stu.edu.cn;
Tel.: +86-754-8890-0499; Fax: +86-754-8855-7562.
Received: 29 November 2012; in revised form: 20 December 2012 / Accepted: 20 January 2013 /
Published: 28 January 2013
Abstract: Alzheimer’s disease (AD) pathogenesis involves an imbalance between free
radical formation and destruction. In order to obtain a novel preclinical anti-AD drug
candidate, we synthesized a series of novel hydroxyl chalcone analogs which
possessed anti-free radical activity, and screened their effects on scavenging
2,2-diphenyl-1-picrylhydrazyl (DPPH) and OH free radicals in vitro. Compound C7,
4,2'-dihydroxy-3,5-dimethoxychalcone was found to have potent activity in these anti-free
radical activity tests. Further research revealed that C7 could elevate glutathione peroxidase
(GSH-PX) and super oxide dismutase (SOD) levels and lower malonaldehyde (MDA) level
in vivo in the Alzheimer’s model. The indication of C7’s effect on AD needs further study.
Keywords: Alzheimer’s; free radical injury; polypheols; hydroxyl-substituted chalcones

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1. Introduction
Free radicals have been implicated in the etiology of several human diseases, as well as ageing. Many
studies indicate that mitochondrial reactive oxygen species (ROS) production and oxidative damage to
mitochondrial DNA results in ageing [1–3]. Alzheimer’s disease (AD) is the most common form of
neurodegenerative disease associated with dementia in elderly people. A growing body of evidence
suggests that AD pathogenesis involves an imbalance between free radical formation and destruction [4–8].
This concept originally derived from the free radical hypothesis of aging, with age-related accumulation
of free radicals resulting in damaged cell components. That age is a key risk factor in AD provides
support for this hypothesis [9,10].
A role for oxidative stress in the pathogenetic cascade of events in AD and other neurodegenerative
disorders is appealing because neurons are post-mitotic cells and gradually accumulate oxidative
damage over time, which would account for the late life onset and the slowly progressive nature of these
disorders [11]. Moreover, ‘antioxidants’ are substances that neutralize free radicals or their actions [12].
Epidemiologic studies show that higher intake of foods with functional attributes including polyphenol,
and flavonoid constituents or higher intake of some synthetic products such as vitamin E have a putative
antioxidant effects in reducing the risk of AD [13].
In the clinic, exifone (Figure 1A) is a classical anti-AD drug that treats cognitive decline associated
with aging and corrects memory dysfunction [14]. From the structure of exifone, we can see it is an
analogue of the polyhydroxyl chalcones. It was reported that chalcones have a wide spectrum of
biological activities, especial antioxidant activity. Hydroxyl chalcones embrace hydroxyl substitutions,
one of the key groups to greatly enhance the antioxidant activity of chalcones, mainly because of their
easy conversion to phenoxy radicals through the hydrogen atom transfer mechanism [15]. In order to
obtain a novel preclinical anti-AD drug candidate, we designed and synthesized the series of compounds
C1–7shown in Table 1 and screened these potential compounds for anti-free radical activity in vitro for
further study. A preliminary structure-activity relationship study of these compounds was carried out
meanwhile. Then, we test the compounds’ effect on SOD, MDA and GSH-PX levels to evaluate the
anti-oxidation activity in vivo in a free radical-injury Alzheimer’s model.
Figure 1. The structure of exifone (A); chalcones (B); C7: the
polyphenol-3,5-dimethoxy-4,2'-dihydroxychalcone (C).

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1
Table 1. Chemical structure, physical properties, H-NMR and MS spectral data of the
synthesized hydroxyl-substituted chalcones.
Melting
point (°C)
76–78
Physical properties/
Molecular formula
Yellow crystal/
C15H12O2
1H-NMR
No.
C1
R1
R2
MS(m/z)
(400 MHz, DMSO-d6)
6.89 (1H, d, J = 16, =CH)
7.29 (1H, d, J = 16, =HC)
6.89 ~ 8.10 (m, 9H, ArH)
10.31 (s, 1H, OH)
2-OH
H
223.0[M−H]+
223.0[M−H]+
223.0[M−H]+
239.0[M−H]+
C2
C3
C4
H
2'-OH
H
88–90
83–85
Yellow crystal/
C15H12O2
7.02 (1H, d, J = 16, =CH)
7.42 (1H,d, J = 16, =CH)
7.02 ~ 8.28 (m, 9H, ArH)
12.47(s, 1H, OH)
4-OH
4-OH
Yellow crystal/
C15H12O2
7.75 (1H, d, J = 15.6, =CH)
8.14 (1H, d, J = 15.6, HC=)
6.86 ~ 7.75 (m, 9H, ArH)
10.15 (s, 1H, OH)
2'-OH
193–195
Transparent crystal/
C15H12O3
7.43 (d, J = 15.2, 1H, CH= )
7.81 (d, J = 15.2, 1H, CH= )
6.79 ~ 7.80 (m, 8H, ArH)
9.58 (s, 1H, OH)
11.64 (s, 1H, OH)
C5
4-OH,
2'-OH
158–160
Transparent crystal/
C15H11NO5
3.77 (s, 3H, OCH3)
269.1[M−H]+
3-OCH3
7.75 (d, J = 16, 1H, CH= )
8.15 (d, J = 16, 1H, CH= )
6.78 ~ 7.77 (m, 7H, ArH)
9.14 (s, 1H, OH)
C6
C7
4-OH,
3-NO3
2'-OH
2'-OH
144–146
172–174
Yellow crystal/
C15H11NO5
7.56 (d, J = 16.4, 1H, CH= )
7.97 (d, J = 16.4, 1H, CH= )
7.02 ~ 8.10 (m, 7H, ArH)
11.18 (s, 1H, OH)
284.0[M−H]+
298.9[M−H]+
4-OH,
Red crystal/
C17H16O5
3.88 (s, 6H, OCH3)
3,5-2OCH3
7.62 (d, J = 16, 1H, CH=)
8.02 (d, J = 16, 1H, CH=)
7.02 ~ 8.35 (m, 6H, ArH)
9.20 (s, 1H, OH), 12.91
(s, 1H, OH)

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2. Results and Discussion
2.1. Synthesis of C1–7
It was reported that hydroxyl chalcones had a wide spectrum of biological activities, including
antioxidant activity and chalcone analogs such as curcumin, and exifone have anti-AD activity.
Hydroxyl-substituted chalcone analogs could develop into novel anti-AD drug candidates. The chalcone
analogs C1–7 were successfully synthesized via Claisen-Schmidt condensation using NaOH or
pyperidine as a catalyst in this study (Scheme 1). The chemical structures and physical data of the
1
synthesized hydroxyl-substituted chalcones C1–7 were listed in Table 1. H-NMR and mass
spectroscopy identifications of C1–7 were shown in Table 1. Reports indicated hydroxyl substitutions
on benzyl rings greatly affected the reactivity. When the total amount of hydroxyl-substitutions
(phenolic hydroxyls) on benzaldehydes and acetophenones exceeds one, i.e., easy to ionize in NaOH
solution and the carbonyl-C seemed to fail to retain its original polarity, the reaction would fail [16,17],
so NaOH is used as a catalyst in the syntheses of C1–3. In our study, we found piperidine catalysis seems
a highly effective and promising method in Claisen-Schmidt condensation for synthesizing
dihydroxy-substituted chalcones. Piperidine is successfully used as a catalyst in the syntheses of C4–7.
Scheme 1. The synthesis of the polyphenol-3, 5-dimethoxy-4, 2'-dihydroxychalcone (C7).
2.2. Activity of C1–7 in Scavenging DPPH and OH Free Radicals
ROS has been identified as an important mediator of cell structure damage to lipids, proteins, and
nucleic acids. ROS has been associated with aging, AD and other neurodegenerative disorders. Host
antioxidant defenses control the level of reactive free radicals, but cellular damage occurs when free
radicals have overwhelmed this defense. Thus, the antioxidant might be an important therapeutic
strategy in inhibiting development of AD and other neurodegenerative disorders.
Thus, a series hydroxyl substituted chalcone analogs C1–7 were synthesized and their anti-free
radical activities were evaluated. Vitamin C, whose anti-oxidation effects were demonstrated in many
in vitro experiments and in humans and must be ingested for nutrition, was selected as the positive
control. After the test processes and statistical calculations, we obtained concentration-clearance
(DPPH) curves (Figure 2A). In the same way, concentration-clearance (OH) curve was obtained as
follows (Figure 2B). The IC50 of VC and C1–7 were listed in Table 2. In this study, results indicated the
IC50 (OH) and (DPPH) values of C7 (0.441, 0.255 mM) were similar to that of VC (0.442, 0.241 mM)
and C7 showed the strongest anti-free radical activity in all compounds C1–7 in vitro (Figure 2, Table 2).

Molecules 2013, 18
Figure 2. Concentration-clearance on DPPH (A), Concentration-clearance on OH (B).
1697
(A)
(B)
Table 2. IC50 values for DPPH and OH· clearance of hydroxyl-substituted chalcones.
Compound
OH· clearance IC50 (mM)
DPPH· clearance IC50 (mM)
C1
C2
1.655
2.741
10.040
5.726
0.750
-----
0.836
1.688
0.769
3.65
C3
C4
C5
0.521
-----
C6
C7
0.441
0.442
0.255
0.241
Vit C
Tests of activities on DPPH and OH assay are one of the best-known and frequently used methods for
evaluating the free-radical-scavenging activity. They have been widely used in model systems to
investigate the scavenging activities of several natural compounds such as phenolic compounds,
anthocyanins, crude mixtures such as methanol extracts of plants, or some synthetic products [11,18].
The study of structure-activity relationships of C1–7 (Table 1) indicated that the effect of C5, C7 on
free radical scavenging properties are stronger than C1–4, and C6 shows low scavenging capability.
It is not hard to understand that phenolic groups on the A ring and B ring were key to the free-radical
scavenging activity. In this study, the results also demonstrated that the OCH3 group can increase the
free radical scavenging properties (C5 vs. C1–4). Furthermore, 3,5-dimethoxy can maximize the
anti-free radical activity (C7 vs. C5). Recently, Chan et al. demonstrated that the antioxidant activity of
phenolics, such as curcumin is due not only to the number of phenolic groups but also the
ortho-methoxyphenolic functionality [19]. The ortho-methoxy group can form an intramolecular
hydrogen bond with the phenolic hydroxy group, making the H-atom abstraction from the
ortho-methoxyphenols surprisingly easy. Furthermore, the electron withdrawing group NO2 in A ring
(C6) decreased the scavenging properties, but the electron donating group OCH3 increases those.
Our comparative study demonstrated that the antioxidant activities of C1–7 can arise from the
phenolic groups, ortho-methoxyphenol and the electron properties of substituent groups.

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2.3. Impact of C7 on SOD, MDA and GSH-PX Levels in Scopolamine-Induced AD Model
C7 showed potent anti-oxidation effects, so it was elected for further study on the anti-oxidation
activity in AD model. From the oxidative-stress etiological theory of AD, we administrated scopolamine
to stimulate AD, with chemical injuries to cholinergic nerve cells [20,21]. We measured SOD, MDA and
GSH-PX levels to assess the potential of three levels of C7 (low, medium, high) anti-oxidation activity
in AD [22]. Piracetam, a classical AchE inhibitor for AD, was selected as a positive control, because
many former studies of AD involving similar models (scopolamine administration) adopted piracetam
as a positive control [23,24]. From Figure 3A, we can see the SOD level was significantly lower in
model mice than in non-model mice (p < 0.01) and significantly higher in mice treated with piracetam or
C7 than in model mice (p < 0.01). From Figure 3B we can also see MDA level was significantly higher
in model mice than in non-model mice (p < 0.01) and piracetam and C7 can significantly down-regulate
the MDA level in model mice (p < 0.01). Figure 3C shows levels of GSH-PX. GSH-PX level was
significantly lower in model mice than in non-model mice (p < 0.01), and was significantly higher with
piracetam or C7 treatment mice than in model mice (p < 0.01).
Figure 3. Global cerebral SOD (A) MDA (B) GSH-PX (C) levels for all.
*, p < 0.05 vs. blank group, **, p < 0.01 vs. blank group; ##, p < 0.01 vs. model group.

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The increase in brain oxidative damage might be facilitated by a failure of antioxidant defense
mechanisms. Antioxidant enzymes that are important in preventing an excessive accumulation of ROS
include Cu/Zn- and Mn-SOD, GSH-Px, GSSG-R [25,26]. Membrane lipids present in subcellular
organelles are highly susceptible to free radical damage. Malondialdehyde (MDA) is a by-product of
lipid peroxidation induced by free radicals and is also widely used as a biomarker of oxidative stress [27].
We measured SOD, MDA and GSH-PX levels to assess the potential of three levels of C7
(low, medium, high) against oxidative damage in AD [20,22]. The results showed C7 was a potential
antioxidant in Alzheimer’s models in vivo.
3. Experimental
3.1. General
All synthetic reagents were from Shanghai Jingchun Reagents Co. (Shanghai, China); SOD, MDA
and GSH-PX testing kits were from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
3.2. Chemistry
Synthesis of Hydroxyl-substituted Chalcone Analogs
C1–3 were successfully synthesized via Claisen-Schmidt condensation using NaOH as a catalyst.
Substituted benzaldehyde (0.2 mol), substituted acetophenone (0.22 mol) and NaOH (30 g) were
dissolved in a mixed solvent of H2O (100 mL) and ethanol (50 mL) in a 250-mL pear-shaped flask. The
mixtures were stirred for 1–24 h in room temperature. Then, the mixtures turned into yellow or a dark
red sticky liquids and saturated hydrochloric acid was added until the pH value was about 1. Then yellow
solids were obtained and the products were purified by recrystallization from acetone and H2O. The
products were obtained in yields of 60–75%. For analytical data see Table 1.
C4–7 wer also successfully synthesized via Claisen-Schmidt condensation using pyperidine as a
catalyst. A 250-mL pear-shaped flask equipped with a effective magnetic stirring and a reflux condenser,
contaning a mixture of hydroxyl-substituted benzaldehyde (0.2 mol), 2-hydroxylacetophenone (0.22 mol)
and piperidine (10 mL), was put into an oil bath (160 °C). After 10 min, the mixture turned into a dark red
sticky liquid and was poured, with three ethanol absolute-rinses, into a 500-mL beaker with sufficient
10% NaOH in an ice-bath. With vigorous stirring, the mixture turned into an orange-red, non-sticky and
uniform solution. Then, saturated hydrochloric acid was added until the pH value reached 1–2, and
absolute ethanol was added to form a mixture of a turbid yellow solution and a dark-brown floating oily
substance. The mixture was cooled and kept at 4–5 °C for one day. The yellow solid crystallized from
the solution was filtered with adequate ethanol-absolute washing until it appeared as bright-yellow
crystals and was dried. The products were obtained in yields of 60–75%. For analytical data see Table 1.

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3.3. Pharmacology
3.3.1. Tests of Activity of C1–7 in Scavenging DPPH-free Radicals
The antioxidant activity of the C1-7 on DPPH-free radicals was estimated according to the procedure
reported by Molyneux, Goupy et al. [28,29]. C1–7 and Vc (positive control) were prepared as
ethanol-absolute solutions of 0.19, 0.38, 0.5, 1.5, 3, 6, and 12 mmol/L. All absorption values were
measured in 517 nm. The antioxidant activity (AA) of the sample was expressed as percentage
disappearance of DPPH:
AA (%) = [1 − (A control − A sample)/A blank] × 100%
where A blank is absorption values of DPPH in the presence of water instead of compounds.
3.3.2. Tests of Activity of C1–7 in Scavenging OH Free Radicals
The antioxidant activity of the C1–7 on OH free radicals was estimated according to the procedure
reported by Yu et al. [30]. 0.1% H2O2 (29.5 mmol/L, calibrated by potassium permanganate titration),
7.5 mmol/L ferrous sulfate (freshly prepared), 7.5 mmol/L phen (dissolved in ethanol), 0.1 mmol/L
acetic acid (pH = 3.0), C1–7 and Vc (positive control) were prepared as ethanol-absolute solutions of
0.19, 0.38, 0.75, 1.5, 3, 6, 12 mmol/L. All absorption values were measured in 536 nm. The antioxidant
activity (AA) of the sample was expressed as percentage disappearance of OH:
AA (%) = [(A sample − A control) − (A injured − A blank)]/(A uninjured − A injured) × 100%
3.3.3. Tests of C7 on SOD, MDA and GSH-PX Levels on Scopolamine-Induced Alzheimer’s Models
We obtained 60 Kunming mice, weighing 22–25 g, from the barrier unit at the Laboratory Animal
Center of Southern Medical University (Guangzhou, China). Animals were subjected to alternate 12 h
periods of dark and light (lights on at 6:00 to 18:00) with temperature about 25 °C and humidity
40%–60%. Mice were randomly divided into six groups (10 rats in each group): control group,
scopolamine group (model group), piracetam group, different dose of C7 group (low dose, medium
dose, high dose). Mice in piracetam group and C7 group were intragastrically (i.g.) administered
piracetam (500 mg/kg), C7 (125 mg/kg, 250 mg/kg, 500 mg/kg), respectively, for 14 consecutive days.
Mice in the control and model groups were treated similarly with corresponding volumes of saline. All
mice except control group received scopolamine (5 mg/kg by intraperitoneal injection) after the last
treatment of drugs or saline on day 14. Thirty min later, animals were sacrificed, and whole brain tissues
were removed. Homogenates were made of 10% (w/v) in cold normal saline, and supernatants were
collected immediately after 12,000 r/min centrifugation for 15 min and stored at −20 °C following the
instructions of Nanjing Jiancheng Bioengineering Institute.
3.4. Statistical Analysis
Data are shown as mean+/−SD, and analysis involved use of SPSS 10.0 (SPSS Inc., Chicago, IL,
USA) by one-way ANOVA followed by SNK-LSD test.

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4. Conclusions
A growing body of evidence indicates that increased oxidative stress resulting from free radical
damage to cellular functions is associated with a number of age-related disorders including
atherosclerosis and arthritis. In recent years, considerable data have indicated that free radicals play a
significant role in the pathogenesis of neurodegenerative disorders and promote the progression of AD [24].
So anti-free radical and anti-oxidation could be a potential therapeutic target to prevent and treat AD.
C7, a novel chalcone analoge was confirmed to be highly potent in scavenging DPPH and OH free
radicals in vitro. Tests of anti-free radical activity in response to oxidative stress in mice revealed that C7
could elevate glutathione peroxidase (GSH-PX) and super oxide dismutase (SOD) levels and lower
malonaldehyde (MDA) level in a free-radical–injured scopolamine-induced Alzheimer’s model. Further
behavioral tests would be applied to examine the C7 effect on AD in future studies. The preliminary
study of structure-activity relationships of C7 indicated that its activity maybe be related to its structure
which comprised both a 4-hydroxyl substituted chalcone and 3,5 dimethoxy-substitution. In summary,
C7, a novel hydroxyl-substituted chalcone analogs possesses ree radical scavenging properties in vitro
and anti-oxidation properties in a free radical-injury Alzheimer’s model in vivo. It can be a preclinical
anti-AD drug candidate for the next steps of research.
Acknowledgements
This work was supported by a grant from Guangdong province Natural Science Foundation of China
(No. 10151503102000048), and the teamwork projects funded by Guangdong Natural Science
Foundation of China (No.9351503102000001), Doctoral Fund of Ministry of Education of China
(No. 20104402120006). Science and Technology Planning Project of Guangdong Province in China
(No. 2010B030700043).
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Sample Availability: Samples of the compounds are available from the authors.
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