Design, synthesis and evaluation of new ligustrazine derivatives as potential plasma-stable neuroprotective agents

Article abstract of DOI:10.1039/c7md00003k

A series of ligustrazine-phenolic acid esters which exhibited promising neuroprotective activities have previously been reported. Nevertheless, we found that these ester compounds (like T-VA) were not stable in plasma by further in vivo studies. To investigate plasma-stable neuroprotective agents, a series of new ligustrazine derivatives were synthesized by conjoining ligustrazine and phenols with ester, ether and amide bonds. Most of the compounds exhibited higher protective effects against CoCl₂-induced neurotoxicity in differentiated PC12 cells than ligustrazine. Structure-activity relationships were also briefly discussed. We found that compound 2c (2-((2-methoxy-4-(((3,5,6-trimethylpyrazin-2-yl)methoxy) methyl)phenoxy)methyl)-3,5,6-trimethylpyrazine) displayed the highest protective effect on the PC12 cells damaged by CoCl₂ (EC₅₀ = 1.07 μM). Preliminary stability investigation in rat plasma was verified in vitro and better plasma stability was observed with 2c in comparison to T-VA.

Full text of DOI:10.1039/c7md00003k

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DOI: 10.1039/C7MD00003K
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Design, synthesis and evaluation of new ligustrazine derivatives as
potential plasma-stable neuroprotective agents†
Chenze Zhang a, Wenqiang Yan a, Rui Zhao a, Bing Xu a, Xiong Fang b, Mengmeng Yan a, Yuzhong
Zhang c, Penglong Wang a* and Haimin Lei a*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Abstract: A series of ligustrazine-phenolic acid esters which exhibited promising neuroprotective activities have previously
been reported. Nevertheless, we found that these ester compounds (like T-VA) were not stable in plasma by further in
vivo studies. To investigate plasma-stable neuroprotective agents, a series of new ligustrazine derivatives was synthesized
by conjoining ligustrazine and phenols with ester, ether and amide bonds. Most compounds exhibited higher protective
effect against CoCl2-induced neurotoxicity in differentiated PC12 cells than ligustrazine. Structure–activity relationships
were also briefly discussed. We found compound 2c (2-((2-methoxy-4-(((3,5,6-trimethylpyrazin-2-yl) methoxy) methyl)
phenoxy) methyl) -3,5,6-trimethylpyrazine) displayed the highest protective effect on the PC12 cells damaged by CoCl2
(EC50 = 1.07μM). Preliminary stability investigation in rat plasma was verified in vitro and better plasma stability was
observed with 2c in comparison to T-VA.
www.rsc.org/
results showed that ligustrazine derivatives presented
neuroprotective effects on injured differentiated PC12 cells, of
which, T-VA (C24H28N4O4) (Figure 1) displayed promising
protective effect (EC50 = 4.25 μM).12 In our previous study, T-
VA also exhibited neuroprotective effect in a rat model of
ischemic stroke with concomitant improvement of motor
functions.15
Introduction
Stroke is one of the most devastating diseases and a main
cause of mortality in China as well as worldwide.1 Most strokes
are ischemic and the disease relates with both cerebrovascular
system and cranial nerves.2 Despite the remarkable progress
achieved in the therapy of stroke during the last two decades,
there is no effective chemotherapy for cerebral ischemic
stroke and neuroprotective agents that could attenuate lots of
the clinical problems of ischemic stroke.3,4 This may explain a
growing interest in discovering drugs lead by traditional
medicine.5
However, because of carboxyl groups in phenolic acids
and cinnamic acids, all these ligustrazine derivatives have ester
bonds.16 To enrich the type of ligustrazine derivatives, twenty-
one compounds were synthesized by conjoining ligustrazine
and phenols with ester, ether and amide bonds. Their
neuroprotective activities were evaluated on injured PC12 cells
by thiazole blue (MTT) assay; and 2c (Figure 1) displayed the
highest protective effect. The structure–activity relationships
(SARs) of these novel compounds are also briefly discussed.
Compounds with ester or amide bonds like T-VA, are
liable to enzymatic hydrolysis in vivo such as the action of
Ligustrazine, also known as 2,3,5,6-tetramethylpyrozine
(
TMP), is one of the most important active ingredients of the
traditional Chinese medicine herb Ligusticum wallichii.6,7
Previous studies have shown various pharmacological activities
of this compound, such as calcium antagonism, free radical-
scavenging and antioxidant.8–11 To further improve TMP’s
neuroprotective properties, a series of novel ligustrazine-
phenolic acids derivatives have been designed and synthesized
based on several neuroprotective ingredients from Chinese
traditional medicinal herbs as starting materials.12-14 The
plasma esterases.17-19 Hence, plasma stability is
a very
important issue in drug development. It has a direct impact on
drug performance in vivo and serves as the major limiting
factor for drug candidate screening at an early stage of
preclinical study.20,21 Therefore, to verify potential druggability
of 2c, compared with T-VA, its plasma stability in rat plasma
was preliminarily evaluated in vitro.
O
N
N
N
O
O
N
N
N
N
N
O
N
O
O
O
N
2c
TMP
T-VA
Figure 1. The structures of TMP,T-VA and 2c.
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Results and Discussion
Chemistry
As the important intermediates, 2-(chloromethyl)-3,5,6-
trimethylpyrazine ( ) and 3,5,6-trimethylpyrazine-2-carboxylic
acid ( ) were prepared according to Scheme 1 and Scheme 2
details showed in the ESI . All the designed derivatives
1
2
（
）
Scheme 3. Synthetic routes to ligustrazine derivatives 1a-1c, 2a-2c.
Reagents and conditions: (i) DMF, K2CO3, 85 °C, 4 h; (ii) DMF, NaH,
80 °C, 4h.
were synthesized via the routes outlined in Scheme 3, Scheme
4 and Scheme 5.
Table 1 The structures of ligustrazine derivatives
Compounds
R
H
n
1
2
1
1a
1b
1c
,
2a
2b
2c
,
3a
,
,
4a
4b
4c
,
5a
5b
5c
,
6a
6b
6c 7c
,
7a
,
,
3b
,
,
,
7b
H
,
,
3c
,
,
,
,
OCH3
As shown in Scheme 3, compounds (1a-1c
,
2a-2c) were
synthesized by alkylation of 4-Hydroxybenzyl alcohol, 4-
Hydroxyphenethyl alcohol and 4-Hydroxy-3-methoxybenzyl
alcohol with intermediate
coupling reaction between intermediate
amines were performed using 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI), 1-
1
, respectively. The single-step
Scheme 4. Synthetic routes to ligustrazine derivatives 3a-3c, 4a-4c, 5a-
5c. Reagents and conditions: (i) DMF, EDCI, HOBt, DIPEA, r.t., 12 h; (ii)
DMF, K2CO3, 85 °C, 4 h; (iii) DMF, EDCI, DMAP, r.t., 12 h.
2
and the phenol
Hydroxybenzotriazole (HOBT) and N, N-Diisopropylethylamine
(DIPEA) in anhydrous N, N-Dimethylformamide (DMF), then we
got the ligustrazine derivatives (3a-3c, as shown in Route i of
Scheme 4). Subsequently, typical synthetic procedures for 4a-
4c involved the combination of 3a-3c and chloro-TMP through
the formation of ether bonds under alkaline conditions.
Compounds 3a-3c were reacted with intermediate
2 under
Scheme 5. Synthetic routes to ligustrazine derivatives 6a-6c, 7a-7c.
Reagents and conditions: (i) CH2Cl2, EDCI, DMAP, r.t., 12 h.
catalysis of by EDCI and DMAP, respectively; we thus
successfully obtained compounds 5a-5c (Route iii of Scheme 4).
As shown in Scheme 5, 4-Hydroxybenzyl alcohol, 4-
Hydroxyphenethyl alcohol and 4-Hydroxy-3-methoxybenzyl
alcohol were treated with different proportions of
Protective effect on injured neuronal-like PC12 cells
PC12 is a cell line derived from a rat adrenal medulla
pheochromocytoma. The differentiated PC12 cells induced by
nerve growth factor (NGF) have the typical characteristic of
the neurons in form and function; therefore it is widely used as
a model for in vitro neuron research.22 Cobalt chloride (CoCl2),
a water-soluble compound, was used in this investigation
because it is one of the best-known chemical inducer of PC12
injury; CoCl2-induced neurotoxicity in differentiated PC12 cells
is commonly used to screen new candidates for the
intervention of stroke.23-25 All the synthesized compounds
were tested for their protective effects on the neuronal-like
PC12 cells damaged by CoCl2, and TMP was used as the
positive control drug. This revealed the proliferation rates (%)
at different concentration and 50% effective concentrations
(EC50) for protecting damaged PC12 cells of the ligustrazine-
phenols derivatives in Figure 2.
intermediate
2
and catalyzed by EDCI and DMAP, in CH2Cl2 to
7a-7c).
afford the corresponding ligustrazine derivatives (6a-6c
,
The structures of the entire target compounds (Table 1) were
confirmed by 1H-NMR, 13C-NMR and high resolution mass
(HRMS). All of the compounds were new compounds and none
of the compounds’ neuroprotective activities had been
explored.
Scheme 1. Synthesis of 2-(chloromethyl)-3,5,6-trimethylpyrazine.
Reagents and conditions: (i) AcOH, H2O2(30%), 90 °C, 4 h; (ii) Ac2O,
reflux, 105 °C，2.5 h; (iii) NaOH, THF: MeOH: H2O, r.t., 1 h; (iv) SOCl2,
CH2Cl2, 0 °C, 2.5 h; KOH C2H5OH.
Scheme 2. Synthesis of 3,5,6-trimethylpyrazine-2-carboxylic acid.
Reagents and conditions: (i) KMnO4, 55 °C, 24 h
2 | J. Name., 2012, 00, 1-3
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bodies with fine dendritic networks similar to those nervecells,
and the cell edges were intact and clear (Figure 3-B).
Figure 2. EC50 value of ligustrazine derivatives. The results showed that
ether bonds may contribute to enhance the efficacy of such newly
ligustrazine derivatives
The results showed that TMP and its derivatives
presented protective effects on injured differentiated PC12
cells and most of the ligustrazine derivatives were more active
(with lower EC50 values) than TMP (EC50 = 64.46 µM).
Figure 3. Protective effects of compound 2c against CoCl2-induced
injury in differentiated PC12 cells (
×
200). The most representative
: Undifferentiated PC12 cells. : Differentiated
: CoCl2-induced neurotoxicity of differentiated
To enrich the type of ligustrazine derivatives, 21 new
ligustrazine derivatives were synthesized by conjoining
ligustrazine and phenols with ester, ether and amide bonds.
Interestingly, derivatives containing ether bonds like 1a-1c and
2a-2c exhibited high potency, with EC50 values below 15 µM;
among them 2c was the most active compound, with an EC50
value of 1.07 µM. Furthermore, we found these ether
conjuncted derivatives which containing bis-ligustrazine
substituents showed better neuroprotective activities than the
fields were shown.
PC12 cells by NGF.
A
C
B
PC12 cells. D: CoCl2-induced neurotoxicity + 2c (30 μM).
Moreover, the mean value expressed as percentage of
neurite-bearing cells in NGF treated cells was 58.5% (Figure 4).
single-ligustrazine componds, such as 2a > 1a
1c Base on the above evidence, the additive methoxy moiety
in 1c and 2c might enhance the protective activity, such as 1c >
1a > 1b 2c > 2a > 2b. This structure-activity relationship
, 2b > 1b, 2c >
.
,
analysis was in agreement with the previous studythat the
ether derivatives showed better protective effects than the
ester derivatives.12,13
Figure 4. Protective effects of compound 2c (30 μM) against CoCl2-
induced injury in differentiated PC12 cells. The neurite-bearing ration
are shown as means ± S.D. of at least three independent microscopic
field. ## means p < 0.01 compared with NGF group, ** means p < 0.01
compared with CoCl2 group.
However, most of the ligustrazine-phenols ester and
amide derivatives (3b-3c, 4a-4c, 5a-5c, 6b, and 7a-7c) showed
lower neuroprotective activity than the ligustrazine- phenols
ether derivatives. Meanwhile, it was observed that amide-
joined single-ligustrazine derivatives’ protective effects were
better than those bis-ligustrazine substituents ones, as
exemplified by comparison of the respective pairs of
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 (Figure 3-C); the mean value of neurite-bearing cells
(18.9%, Figure 4) showed a significant decrease. Pretreatment
with 2c (30 μM) dramatically alleviated morphological
manifestations of cell damage and led to an increase (77.4%,
Figure 4) in neurite-bearing cells compared to model cells
(Figure 3-D).
compounds such as 3a > 4a > 5a
addition, as for ligustrazine-phenols ester derivatives, the
additive ester bonds weakened the protective activity (6a > 7a
6b > 7b 6c > 7c). These findings may provide a new framework
, 3b > 4b > 5a, 3c > 4c > 5c. In
,
,
for the design of new ligustrazine derivatives as
neuroprotective drugs for treating cerebral ischemic stroke.
Plasma stability of 2c in vitro
Effect of 2c on CoCl2-induced cell injury
Plasma stability is
a
n
important issue in drug
development and serves as the major limiting factor for drug
candidate screening at an early stage of preclinical study.
Although 2c exhibits beneficial effects on CoCl2-induced cell
injury, its stability in biological matrix has not been studied. In
addition, our studies showed that T-VA was liable to enzymatic
hydrolysis in vivo such as the action of plasma esterases. Since
Under optical microscopy, as shown in Figure 3-A, we
found that undifferentiated PC12 cells that maintained under
normal conditions were small and proliferated to form clone-
like cell clusters without neural characteristics. By exposure to
NGF, normal differentiated PC12 cells showed round cell
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this structural modification may result in different stability, 2c Therefore, to verify potential druggability of 2c, compared
may have some advantages over T-VA. Therefore, to verify with T-VA, its plasma stability in rat plasma was preliminarily
potential druggability of 2c, compared with T-VA, its plasma evaluated by HPLC. These results indicated that ligustrazine-
stability in rat plasma was preliminarily evaluated by high phenolic acid esters (like T-VA) degradation in rat plasma may
performance liquid chromatography (HPLC). 26
Figure 5 indicates the residual 2c and T-VA percentages of can observably improve the plasma stability. This study
the initial concentration t0 after 240 min incubation in rat provides useful information for future design of analogous
caused by enzymatic hydrolysis, and structural modification
（
）
plasma. After incubated at 37 °C for 240 min, the residual ligustrazine derivatives and the metabolic study of 2c. Further
percentages of 2c (t240) were greater than 90%. There was studies of the mechanisms of 2c neuroprotective effects are
practically no degradation for 240 min at 37°C. In contrast, the currently underway. The results suggest that the attempt to
curve of residual T-VA indicates that more than 50 % of the apply structure combination to discover more efficient and
substrate was degraded within 60 min. These results indicated plasma-stable lead compounds from nature products is viable.
that T-VA degradation in rat plasma may caused by enzymatic
hydrolysis, and structural modification can improve the plasma
Acknowledgements
stability. This study provides useful information for future
design of analogous ligustrazine derivatives and the
This study was financially supported by the National Natural
Science Foundation of China (No. 81173519), Beijing Key
Laboratory for Basic and Development Research on Chinese
Medicine, the Innovation Team Project Foundation of Beijing
University of Chinese Medicine (Lead Compound Discovering
and Developing Innovation Team Project Foundation, No.
2011-CXTD-15).
pharmacokinetic study of 2c
.
Conflicts of Interest
The authors declare no conflict of interest.
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