Synthesis and protective effect of new ligustrazine derivatives against CoCl2-induced neurotoxicity in differentiated PC12 cells. Part 2

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Synthesis and protective effect of new ligustrazine
derivatives against CoCl2-induced neurotoxicity in
differentiated PC12 cells. Part 2†
Cite this: Med. Chem. Commun.,
2015, 6, 806
Bing Xu,‡a Yan Gong,‡a Xin Xu,a Chenze Zhang,a Yuzhong Zhang,b Fuhao Chu,a
a
a
Hongbing Liu,ac Penglong Wang and Haimin Lei
*
*
Ligustrazine-benzoic acid derivatives which exhibited promising neuroprotective activities have previously
been reported. To further improve the neuroprotective effect of ligustrazine, a series of new ligustrazine
derivatives was synthesized and evaluated for their protective effect against cobalt chloride-induced neuro-
toxicity in differentiated PC12 cells. Most compounds exhibited higher activity than ligustrazine, of which,
we found compound IJE)-IJ3,5,6-trimethylpyrazin-2-yl)methyl 3-IJ2,3,4-trimethoxyphenyl)acrylate (9)
displayed the highest protective effect on the PC12 cells damaged by CoCl2 (EC50 = 0.719 μM). Structure–
activity relationships are briefly discussed.
Received 9th December 2014,
Accepted 1st February 2015
DOI: 10.1039/c4md00552j
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treating ischemic stroke, exhibiting anti-apoptotic and antiox-
idant roles.12–14 Moreover, it has previously been reported
Introduction
Based on the active ingredient of traditional Chinese medi-
cine, the application of the “combination principle” for
designing lead compounds has drawn considerable
attention.1–7 To date, despite the remarkable progress
achieved in theory, effective approaches to regenerate injured
nerves, for example due to Stroke, Alzheimer's disease (AD)
and Parkinson's disease (PD), have not yet been found.8–10 To
discover lead compounds with neural repair-promoting effect
is of great significance.11 In our previous work in this field, a
series of novel ligustrazine-benzoic acid derivatives have been
designed and synthesized using several neuroprotective ingre-
dients from Chinese traditional medicinal herb as starting
materials.7 The results showed that ligustrazine-benzoic acid
derivatives presented protective effects on injured differenti-
ated PC12 cells, of which, IJ3,5,6-trimethylpyrazin-2-yl)methyl
that ligustrazine derivatives exerted synergistic potential
vasculoprotective effects.15,16 As part of our continuing
research,
a series of novel ligustrazine derivatives was
designed and synthesized inspired by the potent bioactivities
of ligustrazine-benzoic acid derivatives and biological charac-
teristics of phenolic acids on neuroprotection.17–21 All new
compounds were fully characterized. Their neuroprotective
activities were evaluated on damaged PC12 cells by 3-IJ4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay;22,23 and the structure–activity relationships (SARs) of
these novel compounds are also discussed.
Results and discussion
Chemistry
3-methoxy-4-ij(3,5,6-trimethylpyrazin-2-yl)methoxy]
IJC24H29N4O4, 437.21106) displayed promising protective effect
on the injured PC12 cells (EC50 = 4.249 μM).7
Ligustrazine IJ2,3,5,6-tetramethylpyrazine, TMP), one of the
major efficient components from the Chinese traditional
medicine herb Ligusticum chuanxiong Hort, has multiple
neuroprotective effects, such as improving memory deficits,
benzoate
All the target derivatives were synthesized via the routes
outlined in Schemes 1–2. The important intermediates
2-bromomethyl-3,5,6-trimethylpyrazine (1) and 3,5,6-tri-
methylpyrazine-2-carboxylic acid (2) were prepared according
to our previous study.1,7 As shown in Scheme 1, various
substituted cinnamic acids underwent alkylation with the
intermediate 2-bromomethyl-3,5,6-rimethylpyrazine, to afford
a School of Chinese Pharmacy, Beijing University of Chinese Medicine,
Beijing 100102, China. E-mail: hm_lei@126.com, wpl581@126.com
b Department of Pathology, Beijing University of Chinese Medicine,
Beijing 100102, China
c College of TCM, Xinjiang Medical University, Xinjiang 830011, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c4md00552j
Scheme 1 Synthesis of ligustrazine derivatives (1–12). Reagents and
conditions: (i) DMF, K2CO3, 85 °C, 1.5 h.
‡ These authors contributed equally to this work.
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Table 2 The structures of ligustrazine derivatives 13a–16a, 13b–16b
Structure
Yield
Structure
Yield
52.6%
52.1%
13a
14a
13b
14b
42.6%
61.3%
30.2%
48.2%
Scheme
2 Synthesis of ligustrazine derivatives (13–16). Reagents
and conditions: (i) DMF, K2CO3, 85 °C, 1.5 h. (ii) CH2Cl2, EDCI/DMAP,
r.t., 16 h.
15a
16a
15b
16b
ligustrazine-cinnamic
acid
derivatives
(1–10).
The
59.1%
54.7%
ligustrazinyloxy-benzoates (11 and 12) were synthesized
(Scheme 1) starting from benzoic acids, followed by alkyl-
ation with 2-bromomethyl-3,5,6-trimethylpyrazine. Likewise,
ligustrazinyloxy-phenol derivatives (13a–16a) were synthesized
(Scheme 2) by alkylation of paeonol, eugenol and phenolics
at their phenolic hydroxyl groups with 2-bromomethyl-3,5,6-
trimethylpyrazine, respectively.
As shown in Scheme 2, paeonol, eugenol and phenolics
were treated with 3,5,6-trimethylpyrazine-2-carboxylic acid (2),
catalyzed by 1-IJ3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCI) and 4-dimethylamino pyridine (DMAP),
in CH2Cl2 to afford the corresponding ligustrazine derivatives
(13b–16b). All the target compounds (Tables 1 and 2) were
structurally confirmed by 1H NMR, 13C NMR, and high resolu-
tion mass (HRMS).
effects on the neuronal-like PC12 cells damaged by CoCl2,
and TMP was used as the positive control drug. The results,
proliferation rate (%) at different concentration and 50%
effective concentration (EC50) for protecting damaged PC12
cells of ligustrazine derivatives, are summarized in Table S3
(see ESI†) and Fig. 1. As shown in Table S3,† compounds
derived from cinnamic acids (3, 4, 5, 6, 7, 9, 10), paeonol
(13a) and eugenol (16a) displayed good protective effects on
the injured differentiated PC12 cells (EC50 < 15.0 μM).
All the compounds derived from methoxy cinnamates (3,
4, 5, 6, 7, 8, 9 and 10) exhibited high potency with EC50
values below 24 μM, among which 9 was the most active one
with an EC50 value at 0.719 μM. We found that the methoxy
substituent at the ortho position exhibited much better
Biological activities
Protective effect on injured neuronal-like PC12 cells. All
the synthesized compounds were tested for their protective
Table 1 The structures of ligustrazine derivatives 1–12
Compd
n
R1
R2
R3
R4
Yield
1
2
3
4
5
6
7
8
1
1
1
1
1
1
1
1
1
1
0
0
H
H
OCH3
H
H
OCH3
H
OCH3
OCH3
H
H
H
H
OCH3
H
OCH3
OCH3
H
OCH3
OCH3
H
H
CH3
H
H
OCH3
H
OCH3
H
OCH3
OCH3
H
H
H
H
H
H
H
H
OCH3
H
61.3%
57.2%
53.7%
48.6%
51.1%
47.6%
46.9%
51.0%
48.3%
50.3%
58.3%
21.6%
9
10
11
12
OCH3
H
OOCCH3
H
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suggesting that they may be more effective on Stroke, AD and
PD than either a neuroprotective agent or vascular protective
drug alone. These findings will be likely to provide a new
framework for the design of new ligustrazine derivatives with
neuroprotective drugs for treating neurological diseases.
Effect of 9 on CoCl2-induced cell injury. Under an optical
microscopy, undifferentiated PC12 cells proliferated to form
clone-like cell clusters without neural characteristics as
shown in Fig. 2A; normal differentiated PC12 cells showed
round cell bodies with fine dendritic networks, and the cell
edges were intact and clear (Fig. 2B). Moreover, the mean
value expressed as percentage of neurite-bearing cells in NGF
treated cells was 58.3% (Fig. 3). In contrast, the incubation of
cells with 200 mM of CoCl2 for 12 h induced shrinkage of the
cell bodies, disappearance in cell reticular formation, and
disruption of the dendritic networks (Fig. 2C); the mean
value of neurite-bearing cells (13.1%, Fig. 3) showed a signifi-
cant decrease. While pretreatment with 9 dramatically allevi-
ated morphological manifestations of cell damage and
exerted a clear increase (50.8%, Fig. 3) in neurite-bearing
cells compared to model cells (Fig. 2D).
Fig. 1 Structure–activity relationships of ligustrazine derivatives. The
results showed that both olefinic group and o-methoxy substituent
may contribute to enhance the efficacy of such newly ligustrazine
derivatives.
neuroprotective activities than other methoxy-cinnamic acid
derivatives, such as (3 > 5 > 4), (6 > 7), (9 > 10) (Fig. 1). And
on this basis, the protective effects of ligustrazine-cinnamic
acid derivatives were enhanced as the methoxy moiety
increase (9 > 6 > 3 > 1). Compared to 1, the additive methyl
in compound 2 weakened the protective activity. Structure–
activity relationship analysis among 1–10 has an approximate
tendency in activity: 2,3,4-OCH3 > 3,4,5-OCH3 > 2,3-OCH3 >
2-OCH3 > 3,4-OCH3 > 4-OCH3 > 3-OCH3 > 2,5-OCH3 > no
substituent > 4-CH3.
Moreover, most of the ligustrazine-cinnamic acid deriva-
tives (1, 3–10) showed better neuroprotective activity than the
ligustrazine-benzoic acids (11, 12); compound 12 derived
from gallic acid showed no measurable protective activity.
Ligustrazinyl eugenol ether (16a), containing an olefinic
group in the eugenol fragment, proved to have a beneficial
effect on damaged PC12 cells with an EC50 value of 4.615
μM. All of these results disclosed that a trans olefinic bond
group may be favorable for the protective activity.
Conclusions
In conclusion, 20 ligustrazine derivatives were designed, syn-
thesized and biologically evaluated for their protective effects
on damaged PC12 cells. The preliminary biological results
have demonstrated that most of ligustrazine-cinnamic acid
derivatives exhibited good protective effects in comparison
with TMP. Among the active compounds, 9 is the most active
congener with EC50 0.719 μM, which is much higher than
that of TMP and compounds synthesized in our previous
study, presenting a most promising lead for further investiga-
tion. Furthermore, structure–activity relationship was
discussed briefly. It was observed that the ether derivatives
In addition, from the obtained results, it was observed that
the ether derivatives showed better protective effects than the
ester derivatives, such as 13a > 13b, 14a > 14b, 15a > 15b,
16a > 16b (Fig. 1). This structure–activity relationship analysis
was in agreement with our previous study.7
Furthermore, previous studies proved that 1 and 11 could
stimulate the proliferation of cultured human umbilical vas-
cular endothelial cells (HUVECs);24 16a exhibited inhibitory
effect on adenosine diphosphate (ADP)-induced platelet
aggregation and protective effect against hydrogen peroxide
IJH2O2)-induced oxidative damage on ECV-304 cells;25 the cur-
rent study also showed that 1, 11 and 16a and its congener
structures exhibited good neuroprotective activities. Based on
the above evidences, we reason that the new synthetic
ligustrazinylated derivatives possess multiple pharmacologi-
cal activities such as neuroprotection, anti-platelet aggrega-
tion, and protection against vascular endothelial cell injury,
Fig. 2 Protective effects of compound 9 against CoCl2-induced injury
in differentiated PC12 cells (×200). The most representative fields are
shown. A: Undifferentiated PC12 cells. B: Differentiated PC12 cells by
NGF. C: CoCl2-induced neurotoxicity of differentiated PC12 cells. D:
CoCl2-induced neurotoxicity + 9 (60 μM).
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