Synthesis and protective effect of new ligustrazine-benzoic acid derivatives against CoCl2-induced neurotoxicity in differentiated PC12 cells

Article abstract of DOI:10.3390/molecules181013027

A series of novel ligustrazine-benzoic acid derivatives were synthesized and evaluated for their protective effect against cobalt chloride-induced neurotoxicity in differentiated PC12 cells. Combining hematoxylin and eosin staining, we found compound that (3,5,6-trimethylpyrazin-2-yl)methyl 3-methoxy-4-[(3,5,6-trimethylpyrazin-2- yl)methoxy]benzoate (4a) displayed promising protective effect on the proliferation of the injured PC12 cells (EC₅₀ = 4.249 μM). Structure-activity relationships are briefly discussed.

Full text of DOI:10.3390/molecules181013027

Molecules 2013, 18, 13027-13042; doi:10.3390/molecules181013027
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Protective Effect of New Ligustrazine-Benzoic
Acid Derivatives against CoCl2-Induced Neurotoxicity in
Differentiated PC12 Cells
Penglong Wang 1, Honggui Zhang 1, Fuhao Chu 1, Xin Xu 1, Jinxuan Lin 2, Chunxiao Chen 2,
Guoliang Li 1, Yatao Cheng 1, Lin Wang 1, Qiang Li 1, Yuzhong Zhang 2,* and Haimin Lei 1,*
1
School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing 100102, China;
E-Mails: wpl581@126.com (P.W.); bzy714@163.com (H.Z.); chufhao@163.com (F.C.);
xuxin_asia@163.com (X.X.); Liguoliangsx@163.com (G.L.); artery86@hotmail.com (Y.C.);
wanglin431@126.com (L.W.); lq_cn@126.com (Q.L.)
2
Department of Pathology, Beijing University of Chinese Medicine, Beijing 100102, China;
E-Mails: mmzmmk@163.com (J.L.); springxiao-85@163.com (C.C.)
* Authors to whom correspondence should be addressed; E-Mails: zyz100102@126.com (Y.Z.);
hm_lei@126.com (H.L.); Tel.: +86-10-6428-6964 (Y.Z.); +86-10-8473-8641 (H.L.);
Fax: +86-10-8268-6933 (Y.Z. & H.L.).
Received: 10 September 2013; in revised form: 30 September 2013 / Accepted: 9 October 2013 /
Published: 18 October 2013
Abstract: A series of novel ligustrazine-benzoic acid derivatives were synthesized
and evaluated for their protective effect against cobalt chloride-induced neurotoxicity
in differentiated PC12 cells. Combining hematoxylin and eosin staining, we found
compound that (3,5,6-trimethylpyrazin-2-yl)methyl 3-methoxy-4-[(3,5,6-trimethylpyrazin-2-
yl)methoxy]benzoate (4a) displayed promising protective effect on the proliferation of the
injured PC12 cells (EC50 = 4.249 µM). Structure-activity relationships are briefly discussed.
Keywords: ligustrazine; neuroprotective effect; cobalt chloride; PC12 cell; HE staining
1. Introduction
Stroke is one of the most devastating diseases in China as well as worldwide. Most strokes (80%)
are ischemic and the disease relates with both cerebrovascular system and cranial nerves [1].
Currently, treatments for ischemic stroke include the use of thrombolytic and neuroprotective agents;

Molecules 2013, 18
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thrombolytic drugs lyse blood clots to restore blood flow, and neuroprotective treatments prevent cell
death during and after ischemia and reperfusion [2,3]. 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 [1,4], so the development of new neuroprotective agents that inhibit neuronal damage
induced by cerebral ischemia is of great significance [4].
Ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), one of the major effective components of the
Chinese traditional medicinal herb Ligusticum Chuanxiong Hort, has been widely used for ischemic
stroke therapy in China for many years. TMP has multiple mechanisms of action, including
thrombolysis and neuroprotection, both of which were important for effectively protecting the brain
tissue from ischemic and reperfusion damage in ischemic stroke [5–8]. A series of previous studies
proved that ligustrazinyloxy-aromatic acid derivatives could inhibit platelet aggregation and protect
damaged endothelial cell proliferation, and that they exhibited much higher activities than TMP [9–11].
Many of these aromatic acids, such as protocatechuic acid, vanillic acid, salicylic acid, the parent
compounds of ligustrazine derivatives, also showed interesting neuroprotective activity [12–16]. To
further improve TMP’s neuroprotective effects, inspired by the biological characteristics of TMP and
aromatic acids, we integrated the ligustrazine ring and hydroxybenzoic acid fragments into one
molecule according to the combination principle in medicinal chemistry, constructing a series of new
ligustrazine-benzoic acid derivatives.
PC 12 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 [17,18]. 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 [19,20]. Herein, the novel
ligustrazine-benzoic acid derivatives’ neuroprotective activities were evaluated on CoCl2-induced damage
in differentiated PC12 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay and hematoxylin and eosin (HE) staining. Structure-activity relationships (SARs) of these new
compounds are also discussed.
2. Results and Discussion
2.1. Chemistry
All the designed derivatives were synthesized via the routes outlined in Scheme 1 and Scheme 2. As
the important intermediates, 2-(bromomethyl)-3,5,6-trimethylpyrazine (1) and 3,5,6-trimethylpyrazine-
2-carboxylic acid (2) were prepared according to our previous study [21,22]. In Scheme 1, typical
synthetic procedures for 1a–5a involved the combination of bromo TMP and other hydroxybenzoic
acids through the formation of ester and ether bonds under alkaline conditions, followed by hydrolysis
of the corresponding ligustrazinyloxybenzoic acid esters 1a–5a in 20% (w/v) KOH aqueous solution,
to obtain compounds 1b–5b.

Molecules 2013, 18
Scheme 1. Synthetic routes to ligustrazine derivatives 1a–5a, 1b–5b.
13029
O
O
N
N
N
O
Br
OH
O
ii
N
O
N
O
OH
N
1
i
HO
R
R
N
N
R
1a - 5a
1b - 5b
Reagents and Conditions: (i) DMF, K2CO3, 85 °C, 1.5 h; (ii) 20% KOH, 60 °C, 45 min.
Scheme 2. Synthetic routes to ligustrazine derivatives 1c−5c, 1d−5d.
O
O
N
N
O
O
Br
N
N
OH
O
N
N
N
O
O
O
OH
N
1
i
N
2
ii
HO
HO
R
N
R
R
1c - 5c
1d - 5d
Reagents and Conditions: (i) DMF, NaHCO3, r.t., 12 h; (ii) CH2Cl2, EDCI/DMAP, r.t., 16 h.
In Scheme 2, single ester combination of TMP with hydroxybenzoic acids 1c–5c was achieved
by controlling the reaction temperature and alkali catalyst strength. Compounds 1c–5c were
reacted with 3,5,6-trimethylpyrazine-2-carboxylic acid (2) catalyzed by 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) in CH2Cl2, respectively;
we thus successfully obtained compounds 1d–5d. The structures of all the target compounds (Table 1)
were confirmed by 1H-NMR, 13C-NMR, MS and high resolution mass (HRMS). Of all the compounds,
1a−5a, 2b, 5b, 1c−5c, 1d−5d were new compounds and 1b, 3b, 4b had been reported
previously [11,23], but none of the compounds’ neuroprotective activities had been explored.
Table 1. The structures of ligustrazine-benzoic acid derivatives.
Structure
Yield
Structure
Yield
61.1%
85.1%
1a
1b
64.0%
47.6%
1c
1d

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Table 1. Cont.
Structure
Yield
Structure
Yield
61.7%
64.4%
81.9%
2a
2b
52.6%
2c
3a
3c
2d
3b
3d
61.7%
63.2%
89.7%
60.1%
62.5%
67.5%
87.4%
54.7%
4a
4c
4b
4d
58.4%
87.1%
5a
5b

Molecules 2013, 18
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Table 1. Cont.
Structure
Yield
Structure
Yield
51.2%
54.7%
5c
5d
2.2. Biological Activities
2.2.1. Protective Effect on Injured Neuronal-like PC12 Cells
Using TMP as the positive control drug, all the synthesized compounds were tested for their
protective effects on neuronal-like PC12 cells damaged by CoCl2. This revealed the proliferation
rates (%) at different concentration and 50% effective concentrations (EC50) for protecting damaged
PC12 cells of the ligustrazine-benzoic acid derivatives in Table 2. 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 = 64.459 µM).
Table 2. The EC50 of the ligustrazine-benzoic acid derivatives for protecting damaged
PC12 cells.
Proliferation rate (%)
Compound
EC50 (µM)
60 µM
61.63
16.27
81.60
126.60
47.14
5.50
30 µM
58.87
25.49
77.58
117.10
36.44
32.37
27.96
46.91
10.84
0.47
15 µM
27.69
27.48
64.50
89.76
36.01
−1.76
6.99
7.5 µM
18.97
33.71
32.08
52.98
−7.22
−8.25
−22.43
11.21
−10.40
−3.71
−1.87
72.74
22.27
12.04
−7.01
13.55
36.63
36.13
30.06
14.19
10.60
3.75 µM
4.58
1a
2a
3a
4a
5a
1b
2b
3b
4b
5b
1c
27.359
40.715
11.467
4.249
15.65
30.07
31.12
−5.94
−16.00
−22.76
6.44
45.029
90.345
95.484
38.493
>100
16.91
27.30
6.29
33.84
−2.28
−0.08
19.32
96.07
9.20
−10.73
−5.67
−17.07
58.09
−1.86
7.31
27.14
21.88
55.78
57.72
9.18
85.723
73.801
5.828
21.67
90.74
18.75
12.54
27.93
41.08
44.19
40.13
62.41
37.43
12.11
2c
3c
43.909
66.230
68.265
37.398
33.245
22.210
29.683
46.678
64.459
4c
14.28
27.45
22.15
37.79
41.79
51.74
23.97
11.76
5c
15.68
50.28
29.07
46.79
15.27
11.50
14.71
−11.72
2.86
1d
2d
3d
4d
5d
TMP
1.163
33.47
1.16
11.90
9.44

Molecules 2013, 18
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The derivatives containing bis-ligustrazine substituents 1a–5a and 2d–5d exhibited high potency,
with EC50 values below 50 µM; among them 4a was the most active compound, with an EC50 value of
4.249 µM. We found that most of bis-ligustrazine derivatives’ neuroprotective activities were better
than that of the single-ligustrazine componds. This result revealed the combinational effects of two
ligustrazinyl pharmacophores. This structure-activity relationship analysis was in agreement with the
previously studied protective effects of ligustrazine derivatives on damaged ECV-304 cells [24].
However, this principle did not work in the case of 2c (EC50 = 5.828 µM) on the injured PC12 cells
model; this was possibly caused by the usage of different cell types.
In addition, it was observed that ether-joined derivatives’ protective effects were better than those of
the ester ones, as exemplified by comparison of the respective pairs of compounds such as 1a > 1d,
3a > 3d, 4a > 4d, 5a > 5d. Structure–activity relationship analysis among 1a–5a revealed that
compounds with substituents at the para-position of the benzoyloxy moiety seemed to be more active
than those with substituents at other positions in the same series, and approximately followed an
tendency in activity 4-OH, 3-OCH3 > 4-OH > 2-OH > 3-OH > 3, 4-OH. These findings may provide a
new framework for the design of new ligustrazine derivatives as neuroprotective drugs for treating
cerebral ischemic stroke.
Moreover, previous studies displayed that 3b could inhibit platelet aggregation properties [11,23];
while the current study also showed that 3b and its congener structures exhibited good neuroprotective
activities. Based on the above evidence, we reason that ligustrazine-benzoic acid derivatives possess
both thrombolytic and neuroprotective effects and may be more efficacious than either a thrombolytic
or neuroprotective agent alone.
2.2.2. Effect of 4a on CoCl2-Induced Cell Injury
Under optical microscopy, we found that undifferentiated PC12 cells proliferated to form
clone-like cell clusters without neural characteristics as shown in Figure 1-I-A; normal differentiated
PC12 cells showed round cell bodies with fine dendritic networks, and the cell edges were intact and
clear (Figure 1-I-B); moreover, the mean value expressed as percent of neurite-bearing cells in NGF
treated cells was 51.7% (Figure 1-II). In contrast, incubation of cells with 200 mM of CoCl2 for 12 h
induced shrinkage of the cell bodies, disappearance of cell reticular formation, and disruption of the
dendritic networks (Figure 1-I-C); the mean value of neurite-bearing cells (21.8%, Figure 1-II) showed
a significant decrease. Pretreatment with 4a dramatically alleviated morphological manifestations of
cell damage and led to a pronounced increase (65.8%, Figure 1-II) in neurite-bearing cells compared to
model cells (Figure 1-I-D).
3. Experimental Section
3.1. Chemistry
Reactions were monitored by TLC using silica gel coated aluminum sheets (Qingdao Haiyang
1
13
Chemical Co., Qingdao, China) and visualized in UV light (254 nm). H-NMR and C-NMR assays
were recorded on a Bruker AVANCE 500 NMR spectrometer (Fällanden, Switzerland) and chemical
shifts are reported in δ (ppm). Mass spectra were recorded with an LC Autosampler Device: Standard

Molecules 2013, 18
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G1313A instrument (Agilent, New York, NY, USA). HRMS spectra were obtained using a Thermo
ScientificTM LTQ Orbitrap XL hybrid FTMS instrument (Thermo Technologies, New York, NY,
USA). Melting points (uncorrected) were measured on an X-5 micro melting point apparatus (Beijing,
China). Flash column chromatography was performed using 200–300 mesh silica gel. The yields were
calculated based on the last step reaction. All chemicals and solvents used were analytical or
high-performance liquid chromatography grade.
Figure 1. Effect of 4a on differentiated PC12 cell injury induced by CoCl2.
Values are means ± SD; * p < 0.05, ** p < 0.01 compared to CoCl2 group.
2-(Bromomethyl)-3,5,6-trimethylpyrazine (1). Compound 1 was prepared according to our previously
reported method [21]. The crude product, with 70% purity, was not purified further as it caused a
strong mucous membrane irritation.

Molecules 2013, 18
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3,5,6-Trimethylpyrazine-2-carboxylic acid (2). Compound 2 was prepared according to the method
described by Wang et al. and Li et al., with minor modifications [22,25]. To a solution of
trimethylpyrazine (5.0 g, 36.8 mmol) in water (200 mL), aqueous potassium permanganate (KMnO4)
solution (8.6 g KMnO4:150 mL water) was added dropwise at room temperature over about 60 min.
Upon completion of the addition, the mixture was stirred at 50 °C for 12 h, then the warm reaction
mixture was filtered and washed with hot water (300 mL, 90 °C). The filtrate and washing liquor were
combined, cooled to 0–5 °C, and the pH adjusted to 2.0 with concentrated hydrochloric acid.
Extraction was performed with ethyl acetate (200 mL × 3), and the organic phase was dried with
anhydrous sodium sulfate. The solvent was removed by distillation under vacuum, and the residue was
recrystallized from acetone to produce a light yellow solid (2.39 g, yield: 47.8%), m.p.: 162–163 °C [26].
3.1.1. General Procedure for the Preparation of Ligustrazine Derivatives 1a–5a (Scheme 1)
Compound 1 (9.0 mmol) and the corresponding hydroxybenzoic acid (3.0 mmol) were dissolved in
dry DMF, then K2CO3 (6.0 mmol) was added and the mixture was kept at 85 °C for 1.5 h under a
nitrogen atmosphere. The warm reaction mixture was poured into ice-water and the crude product was
extracted with ethyl acetate. After drying the organic layer over anhydrous Na2SO4 and evaporating the
solvent under vacuum, the crude products were purified by flash chromatography and recrystallization
from acetone.
(3,5,6-Trimethylpyrazin-2-yl)methyl
2-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate
(1a).
1
White solid, m.p.: 76.2–76.9 °C. H-NMR (CDCl3) (ppm): 7.84 (m, 1H, Ar-H), 7.45 (m, 1H, Ar-H),
7.16 (m, 1H, Ar-H), 6.98 (m, 1H, Ar-H), 5.36 (d, J = 2 Hz, 2H, -CH2), 5.22 (d, J = 2 Hz, 2H, -CH2),
13
2.45–2.52 (m, 18H, -CH3). C-NMR (CDCl3) (ppm): 165.8, 158.0, 151.2, 151.2, 150.4, 149.4, 148.8,
148.3, 145.2, 144.9, 133.6, 132.0, 120.6, 120.1, 113.6, 70.7 (-CH2), 65.8 (-CH2), 21.7 (-CH3),
21.7 (-CH3), 21.4 (-CH3), 21.3 (-CH3), 20.6 (-CH3), 20.4 (-CH3). MS (ESI) m/z: 407.1 [M+H]+,
HRMS (ESI) m/z: 407.20633 [M+H]+, calcd. for C23H27N4O3 407.20049.
(3,5,6-Trimethylpyrazin-2-yl)methyl
3-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate
(2a).
1
White solid, m.p.: 135.1–135.7 °C. H-NMR (CDCl3) (ppm): 7.70 (brs, 1H, Ar-H), 7.67 (m, 1H, Ar-H),
7.35 (m, 1H, Ar-H), 7.21 (m, 1H, Ar-H), 5.45 (s, 2H, -CH2), 5.18 (s, 2H, -CH2), 2.51–2.60 (m, 18H,
-CH3). 13C-NMR (CDCl3) (ppm): 166.0, 158.6, 151.5, 151.5, 149.9, 149.4, 149.0, 148.8, 145.2,
144.8, 131.1, 129.5, 122.7, 120.2, 115.4, 70.0 (-CH2), 66.0 (-CH2), 21.7 (-CH3), 21.7 (-CH3),
21.5 (-CH3), 21.4 (-CH3), 20.6 (-CH3), 20.6 (-CH3). MS (ESI) m/z: 407.0 [M+H]+, HRMS (ESI)
m/z: 407.20618 [M+H]+, calcd. for C23H27N4O3 407.20049.
(3,5,6-Trimethylpyrazin-2-yl)methyl
4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate
(3a).
1
White solid, m.p.: 70.2–71.0 °C. H-NMR (CDCl3) (ppm): 8.00 (d, J = 8.5 Hz, 2H, Ar-H), 7.02 (d,
J = 9.0 Hz, 2H, Ar-H), 5.42 (s, 2H, -CH2), 5.21 (s, 2H, -CH2), 2.52-2.59 (m, 18H, -CH3).
13C-NMR (CDCl3) (ppm): 165.9, 162.5, 151.6, 151.3, 150.0, 149.4, 149.0, 148.7, 145.1, 145.0, 131.8,
122.6, 114.5, 70.0 (-CH2), 65.7 (-CH2), 21.7 (-CH3), 21.7 (-CH3), 21.5 (-CH3), 21.4 (-CH3),
20.6 (-CH3), 20.6 (-CH3). MS (ESI) m/z: 407.0 [M+H]+, HRMS (ESI) m/z: 407.20627 [M+H]+, calcd.
for C23H27N4O3 407.20049.

Molecules 2013, 18
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(3,5,6-Trimethylpyrazin-2-yl)methyl 3-methoxy-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy] benzoate
(4a). White solid, m.p.: 108.8–109.6 °C. 1H-NMR (CDCl3) (ppm): 7.64 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H),
7.55 (d, J = 1.5 Hz, 1H, Ar-H), 7.06 (d, J = 8.5 Hz, 1H, Ar-H), 5.43 (s, 2H, -CH2), 5.27 (s, 2H, -CH2),
3.88 (s, 3H, -OCH3), 2.52-2.62 (m, 18H, -CH3). 13C-NMR (CDCl3) (ppm): 166.0, 152.1, 151.3, 151.2,
150.0, 149.2, 149.2, 149.1, 148.7, 145.2, 145.2, 123.6, 122.8, 112.7, 112.6, 70.7 (-CH2), 65.7 (-CH2),
56.0 (-OCH3), 21.6 (-CH3), 21.6 (-CH3), 21.5 (-CH3), 21.4 (-CH3), 20.6 (-CH3), 20.6 (-CH3). MS (ESI)
m/z: 437.2 [M+H]+, HRMS (ESI) m/z: 437.21692 [M+H]+, calcd. for C24H29N4O4 437.21106.
(3,5,6-Trimethylpyrazin-2-yl)methyl
3,4-bis[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoate
(5a).
White solid, m.p.: 132.9–133.7 °C. 1H-NMR (CDCl3) (ppm): 7.76 (brs, 1H, Ar-H), 7.69 (d, J = 8.5 Hz,
1H, Ar-H), 7.05 (d, J = 8.5 Hz, 1H, Ar-H), 5.43 (s, 2H, -CH2), 5.23 (s, 2H, -CH2), 5.17 (s, 2H, -CH2),
13
2.47–2.60 (m, 27H, -CH3). C-NMR (CDCl3) (ppm): 165.8, 152.8, 151.3, 151.3, 151.1, 150.1, 150.0,
149.3, 149.0, 148.6, 148.6, 148.1, 145.4, 145.1, 145.1, 124.5, 122.8, 115.4, 113.1, 71.1 (-CH2),
70.8 (-CH2), 65.8 (-CH2), 21.7 (-CH3), 21.7 (-CH3), 21.6 (-CH3), 21.5 (-CH3), 21.3 (-CH3),
21.3 (-CH3), 20.6 (-CH3), 20.6 (-CH3), 20.5 (-CH3). MS (ESI) m/z: 557.2 [M+H]+, HRMS (ESI)
m/z: 557.28503 [M+H]+, calcd. for C31H37N6O4 557.27980.
3.1.2. General Procedure for the Preparation of Ligustrazine Derivatives 1b–5b (Scheme 1)
An aqueous solution of KOH (4.0 mmol) was added to a solution of ligustrazine derivatives 1a–5a
(2.0 mmol) in ethanol (30 mL). The mixture was stirred at 60 °C for 40 min (checked by TLC). Upon
completion of the reaction, pH was adjusted to 3 with 10% hydrochloric acid, and then the solution
was extracted with ethyl acetate (100 mL). The organic layer was washed with water
(100 mL × 2), dried over anhydrous Na2SO4 and evaporated. The solids were dried under vacuum and
purified by recrystallization from acetone to give pure target compounds.
2-[(3,5,6-Trimethylpyrazin-2-yl)methoxy]benzoic acid (1b). White solid, m.p.: 172.2–172.9 °C.
1H-NMR (CDCl3) (ppm): 8.10 (d, J = 7.5 Hz, 1H, Ar-H), 7.53 (t, J = 8.0 Hz, 1H, Ar-H), 7.16 (d,
J = 8.5 Hz, 1H, Ar-H), 7.12 (t, J = 7.5 Hz, 1H, Ar-H), 5.40 (s, 2H, -CH2), 2.53 (brs, 6H, -CH3),
13
2.52 (s, 3H, -CH3). C-NMR (CDCl3) (ppm): 166.2, 157.0, 151.6, 149.1, 146.9, 143.6, 134.4, 133.6,
122.6, 122.6, 113.6, 68.1 (-CH2), 21.5 (-CH3), 20.7 (-CH3), 20.0 (-CH3). MS (ESI) m/z: 273.0 [M+H]+,
HRMS (ESI) m/z: 273.12250 [M+H]+, calcd. for C15H17N2O3 273.11609.
3-[(3,5,6-Trimethylpyrazin-2-yl)methoxy]benzoic acid (2b). White solid, m.p.: 139.2–139.9 °C.
1H-NMR (CDCl3) (ppm): 7.79 (brs, 1H, Ar-H), 7.74 (m, 1H, Ar-H), 7.39 (m, 1H, Ar-H), 7.25 (m, 1H,
13
Ar-H), 5.25 (s, 2H, -CH2), 2.64 (s, 3H, -CH3), 2.57 (brs, 6H, -CH3). C-NMR (CDCl3) (ppm): 170.7,
158.5, 151.5, 149.9, 149.0, 145.4, 131.2, 129.6, 123.1, 120.7, 115.6, 69.7 (-CH2), 21.5 (-CH3),
21.3 (-CH3), 20.4 (-CH3). MS (ESI) m/z: 273.0 [M+H]+, HRMS (ESI) m/z: 273.12234 [M+H]+, calcd.
for C15H17N2O3 273.11609.
4-[(3,5,6-Trimethylpyrazin-2-yl)methoxy]benzoic acid (3b). White solid, m.p.: 160.7–161.4 °C.
1H-NMR (CDCl3) (ppm): 8.07 (d, J = 8.5 Hz, 2H, Ar-H), 7.07 (d, J = 8.5 Hz, 2H, Ar-H), 5.26 (s, 2H,
13
-CH2), 2.63 (s, 3H, -CH3), 2.56 (s, 3H, -CH3), 2.56 (s, 3H, -CH3). C-NMR (CDCl3) (ppm): 171.2,

Molecules 2013, 18
13036
162.9, 151.6, 150.0, 148.9, 145.1, 132.3, 122.4, 114.6, 69.9 (-CH2), 21.7 (-CH3), 21.4 (-CH3),
20.5 (-CH3). MS (ESI) m/z: 273.0 [M+H]+, HRMS (ESI) m/z: 273.12234 [M+H]+, calcd. for
C15H17N2O3 273.11609.
3-Methoxy-4-[(3,5,6-trimethylpyrazin-2-yl)methoxy]benzoic acid (4b). White solid, m.p.: 190.1–190.9 °C.
1H-NMR (CDCl3) (ppm): 7.74 (dd, J = 8.5, 1.5 Hz, 1H, Ar-H), 7.61 (d, J = 1.5 Hz, 1H, Ar-H),
7.12 (d, J = 8.5 Hz, 1H, Ar-H), 5.31 (s, 2H, -CH2), 3.92 (s, 3H, -OCH3), 2.65 (s, 3H, -CH3),
13
2.54 (s, 3H, -CH3), 2.55 (s, 3H, -CH3). C-NMR (CDCl3) (ppm): 171.2, 152.5, 151.5, 150.1, 149.2,
148.8, 145.1, 124.2, 122.5, 112.7, 112.6, 70.6 (-CH2), 56.0 (-OCH3), 21.6 (-CH3), 21.3 (-CH3),
20.5 (-CH3). MS (ESI) m/z: 303.2 [M+H]+, HRMS (ESI) m/z: 303.13272 [M+H]+, calcd. for
C16H19N2O4 303.12666.
3,4-bis[(3,5,6-Trimethylpyrazin-2-yl)methoxy]benzoic acid (5b). White solid, m.p.: 183.8–184.5 °C.
1H-NMR (CDCl3) (ppm): 7.76 (d, J = 1.7 Hz, 1H, Ar-H), 7.69 (dd, J = 8.5, 1.7 Hz, 1H, Ar-H),
7.13 (d, J = 8.5 Hz, 1H, Ar-H), 5.27 (s, 2H, -CH2), 5.24 (s, 2H, -CH2), 2.53–2.58 (m, 18H, -CH3).
13C-NMR (CDCl3) (ppm): 170.4, 153.1, 151.5, 151.4, 150.2, 150.2, 148.7, 148.6, 148.0, 145.4, 145.1,
124.9, 122.8, 115.7, 113.0, 70.9 (-CH2), 70.7 (-CH2), 21.6 (-CH3), 21.6 (-CH3), 21.3 (-CH3),
21.2 (-CH3), 20.5 (-CH3), 20.5 (-CH3). MS (ESI) m/z: 423.0 [M+H]+, HRMS (ESI) m/z: 423.20093
[M+H]+, calcd. for C23H27N4O4 423.19541.
3.1.3. General Procedure for the Preparation of Ligustrazine Derivatives 1c–5c (Scheme 2)
Compound 1 (3.0 mmol) and hydroxybenzoic acid (3.0 mmol) were dissolved in dry DMF (25 mL),
then NaHCO3 (4.0 mmol) was added and the mixture was kept at room temperature for
12 h under nitrogen atmosphere. Then reaction mixture was poured into ice-water and the crude
product was extracted with ethyl acetate. After drying the organic layer over anhydrous Na2SO4 and
evaporating the solvent under vacuum, the crude products were purified by flash chromatography and
recrystallization from acetone.
(3,5,6-Trimethylpyrazin-2-yl)methyl 2-hydroxybenzoate (1c). White solid, m.p.: 84.4–85.1 °C.
1H-NMR (CDCl3) (ppm): 10.66 (s, 1H, -OH), 7.85 (dd, J = 8.0, 1.5 Hz, 1H, Ar-H), 7.47 (td, J = 8.0,
1.5 Hz, 1H, Ar-H), 7.01 (d, J = 8.0 Hz, 1H, Ar-H), 6.87 (t, J = 8.0 Hz, 1H, Ar-H), 5.49 (s, 2H, -CH2),
13
2.61 (s, 3H, -CH3), 2.56 (s, 3H, -CH3), 2.54 (s, 3H, -CH3). C-NMR (CDCl3) (ppm): 169.5, 161.6,
151.7, 149.2, 149.1, 144.2, 135.9, 130.1, 119.2, 117.7, 112.3, 65.8 (-CH2), 21.7 (-CH3), 21.4 (-CH3),
20.6 (-CH3). MS (ESI) m/z: 273.2 [M+H]+, HRMS (ESI) m/z: 273.12241 [M+H]+, calcd. for
C15H17N2O3 273.11609.
(3,5,6-Trimethylpyrazin-2-yl)methyl 3-hydroxybenzoate (2c). White solid, m.p.: 110.1–110.8 °C.
1H-NMR (CDCl3) (ppm): 7.53 (m, 1H, Ar-H), 7.46 (brs, 1H, Ar-H), 7.26 (m, 1H, Ar-H), 7.04 (m, 1H,
Ar-H), 5.42 (s, 2H, -CH2), 2.59 (s, 3H, -CH3), 2.55 (s, 3H, -CH3), 2.53 (s, 3H, -CH3).
13C-NMR (CDCl3) (ppm): 166.2, 156.6, 151.7, 149.5, 149.2, 144.9, 129.7, 121.6, 121.6, 120.9, 116.3,
65.6 (-CH2), 21.4 (-CH3), 21.2 (-CH3), 20.3 (-CH3). MS (ESI) m/z: 273.0 [M+H]+, HRMS (ESI)
m/z: 273.12201 [M+H]+, calcd. for C15H17N2O3 273.11609.

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(3,5,6-Trimethylpyrazin-2-yl)methyl 4-hydroxybenzoate (3c). White solid, m.p.: 183.2–184.0 °C.
1H-NMR (DMSO-d6) (ppm): 10.39 (s, 1H, -OH), 7.81 (d, J = 8.5 Hz, 2H, Ar-H), 6.84 (d, J = 8.5 Hz,
13
2H, Ar-H), 5.34 (s, 2H, -CH2), 2.50 (s, 3H, -CH3), 2.47 (s, 3H, -CH3), 2.45 (s, 3H, -CH3). C-NMR
(DMSO-d6) (ppm): 165.7, 162.6, 151.3, 149.2, 148.9, 145.4, 132.1, 120.3, 115.9, 65.3 (-CH2),
21.7 (-CH3), 21.5 (-CH3), 20.6 (-CH3). MS (ESI) m/z: 273.1 [M+H]+, HRMS (ESI) m/z: 273.12231
[M+H]+, calcd. for C15H17N2O3 273.11609.
(3,5,6-Trimethylpyrazin-2-yl)methyl 4-hydroxy-3-methoxybenzoate (4c). White solid, m.p.:
147.8–148.4 °C. 1H-NMR (CDCl3) (ppm): 7.62 (dd, J = 8.4, 1.5 Hz, 1H, Ar-H), 7.54 (d, J = 1.5 Hz, 1H,
Ar-H), 6.90 (d, J = 8.4 Hz, 1H, Ar-H), 5.41 (s, 2H, -CH2), 3.91 (s, 3H, -OCH3), 2.58 (s, 3H, -CH3),
13
2.52 (s, 3H, -CH3), 2.51 (s, 3H, -CH3). C-NMR (CDCl3) (ppm): 166.0, 151.3, 150.3, 149.3, 149.0,
146.2, 145.1, 124.4, 121.7, 114.1, 111.8, 65.6 (-CH2), 56.1 (-OCH3), 21.6 (-CH3), 21.4 (-CH3),
20.6 (-CH3). MS (ESI) m/z: 303.1 [M+H]+, HRMS (ESI) m/z: 303.13297 [M+H]+, calcd. for
C16H19N2O4 303.12666.
(3,5,6-Trimethylpyrazin-2-yl)methyl 3,4-dihydroxybenzoate (5c). White solid, m.p.: 184.8–185.5 °C.
1H-NMR (DMSO-d6) (ppm): 9.86 (s, 1H, -OH), 9.42 (s, 1H, -OH), 7.35 (brs, 1H, Ar-H), 7.31 (m, 1H,
Ar-H), 6.81 (d, J = 8.0 Hz, 1H, Ar-H), 5.32 (s, 2H, -CH2), 2.50 (s, 3H, -CH3), 2.46 (s, 3H, -CH3),
2.43 (s, 3H, -CH3). 13C-NMR (DMSO-d6) (ppm): 165.8, 151.4, 151.1, 149.2, 148.9, 145.6, 145.4,
122.5, 120.6, 116.7, 115.9, 65.3 (-CH2), 21.7 (-CH3), 21.5 (-CH3), 20.6 (-CH3). MS (ESI)
m/z: 289.1 [M+H]+, HRMS (ESI) m/z: 289.11725 [M+H]+, calcd. for C15H17N2O4 289.11101.
3.1.4. General Procedure for the Preparation of Ligustrazine Derivatives 1d–5d (Scheme 2)
To a solution of ligustrazine derivatives 1c–5c (1.0 mmol) in CH2Cl2 (30 mL) were successively
added compound 2 (1.2 mmol), EDCI (1.5 mmol), and DMAP (catalytic amount), and the mixture was
stirred at room temperature for 16 h under nitrogen atmosphere. The organic layer was washed with
brine and water, respectively. After drying the organic layer over anhydrous Na2SO4 and evaporating
the solvent under vacuum, the crude products were purified by flash chromatography and the target
compound was recrystallized from acetone.
2-[[(3,5,6-Trimethylpyrazin-2-yl)methoxy]carbonyl]phenyl 3,5,6-trimethylpyrazine-2-carboxylate (1d).
1
White solid, m.p.: 89.4–90.1 °C. H-NMR (CDCl3) (ppm): 8.16 (m, 1H, Ar-H), 7.63 (m, 1H, Ar-H),
7.39 (m, 1H, Ar-H), 7.26 (m, 1H, Ar-H), 5.30 (s, 2H, -CH2), 2.68 (s, 3H, -CH3), 2.63 (brs, 6H, -CH3),
2.45 (brs, 6H, -CH3), 2.42 (s, 3H, -CH3). 13C-NMR (CDCl3) (ppm): 164.3, 164.1, 155.2, 153.0, 151.3,
151.0, 149.5, 149.5, 148.7, 144.2, 137.3, 134.2, 132.4, 126.4, 124.2, 123.2, 66.3 (-CH2), 23.0 (-CH3),
22.5 (-CH3), 21.7 (-CH3), 21.7 (-CH3), 21.3 (-CH3), 20.4 (-CH3). MS (ESI) m/z: 421.0 [M+H]+,
HRMS (ESI) m/z: 421.18494 [M+H]+, calcd. for C23H25N4O4 421.17976.
3-[[(3,5,6-Trimethylpyrazin-2-yl)methoxy]carbonyl]phenyl
3,5,6-trimethylpyrazine-2-carboxylate
(2d). White solid, m.p.: 135.1–135.7 °C. 1H-NMR (CDCl3) (ppm): 7.97 (m, 1H, Ar-H), 7.91 (brs, 1H,
Ar-H), 7.50 (m, 1H, Ar-H), 7.45 (m, 1H, Ar-H), 5.44 (s, 2H, -CH2), 2.81 (s, 3H, -CH3), 2.61 (s, 3H, -CH3),
2.61 (s, 3H, -CH3), 2.58 (s, 3H, -CH3), 2.51 (s, 3H, -CH3), 2.50 (s, 3H, -CH3). 13C-NMR (CDCl3)

Molecules 2013, 18
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(ppm): 165.2, 164.2, 155.5, 152.7, 151.5, 150.8, 149.7, 149.3, 149.1, 144.6, 137.9, 131.4, 129.5, 127.5,
126.8, 123.3, 66.1 (-CH2), 22.9 (-CH3), 22.3 (-CH3), 21.7 (-CH3), 21.6 (-CH3), 21.5 (-CH3),
20.6 (-CH3). MS (ESI) m/z: 421.0 [M+H]+, HRMS (ESI) m/z: 421.18512 [M+H]+, calcd. for
C23H25N4O4 421.17976.
4-[[(3,5,6-Trimethylpyrazin-2-yl)methoxy]carbonyl]phenyl 3,5,6-trimethylpyrazine-2-carboxylate (3d).
1
White solid, m.p.: 89.5–90.2 °C. H-NMR (CDCl3) (ppm): 8.14 (d, J = 8.5 Hz, 2H, Ar-H),
7.32 (d, J = 9.0 Hz, 2H, Ar-H), 5.46 (s, 2H, -CH2), 2.83 (s, 3H, -CH3), 2.63 (s, 3H, -CH3), 2.63 (s, 3H,
13
-CH3), 2.60 (s, 3H, -CH3), 2.54 (s, 3H, -CH3), 2.52 (s, 3H, -CH3). C-NMR (CDCl3) (ppm): 165.4,
163.9, 155.7, 154.7, 152.8, 151.5, 149.7, 149.3, 149.1, 144.7, 137.8, 131.4, 127.6, 122.0, 66.0 (-CH2),
22.9 (-CH3), 22.4 (-CH3), 21.7 (-CH3), 21.7 (-CH3), 21.5 (-CH3), 20.6 (-CH3). MS (ESI) m/z: 421.0
[M+H]+, HRMS (ESI) m/z: 421.18521 [M+H]+, calcd. for C23H25N4O4 421.17976.
2-Methoxy-4-[[(3,5,6-trimethylpyrazin-2-yl)methoxy]carbonyl]phenyl
3,5,6-trimethylpyrazine-2-
1
carboxylate (4d). White solid, m.p.: 113.2–113.8 °C. H-NMR (CDCl3) (ppm): 7.73 (m, 1H, Ar-H),
7.72 (brs, 1H, Ar-H), 7.26 (d, J = 8.0 Hz, 1H, Ar-H), 5.46 (s, 2H, -CH2), 3.88 (s, 3H, -OCH3),
2.84 (s, 3H, -CH3), 2.64 (s, 3H, -CH3), 2.62 (s, 3H, -CH3), 2.61 (s, 3H, -CH3), 2.54 (s, 3H, -CH3),
13
2.53 (s, 3H, -CH3). C-NMR (CDCl3) (ppm): 165.6, 163.4, 155.4, 152.7, 151.5, 151.1, 149.7, 149.3,
149.1, 144.7, 144.0, 138.0, 128.7, 123.0, 122.9, 113.6, 66.0 (-CH2), 56.1 (-OCH3), 22.8 (-CH3),
22.4 (-CH3), 21.7 (-CH3), 21.7 (-CH3), 21.5 (-CH3), 20.6 (-CH3). MS (ESI) m/z: 451.2 [M+H]+,
HRMS (ESI) m/z: 451.19550 [M+H]+, calcd. for C24H27N4O5 451.19032.
2-Hydroxy-4-[[(3,5,6-trimethylpyrazin-2-yl)methoxy]carbonyl]phenyl
3,5,6-trimethylpyrazine-2-
1
carboxylate (5d). White solid, m.p.: 182.2–182.9 °C. H-NMR (CDCl3) (ppm): 9.70 (brs, 1H, -OH),
7.88 (s, 1H, Ar-H), 7.83 (m, 1H, Ar-H), 7.01 (d, J = 8.5 Hz, 1H, Ar-H), 5.40 (s, 2H, -CH2),
2.72 (s, 3H, -CH3), 2.58 (brs, 6H, -CH3), 2.53 (s, 3H, -CH3), 2.51 (s, 3H, -CH3), 2.50 (s, 3H, -CH3).
13C-NMR (CDCl3) (ppm): 165.3, 163.2, 156.2, 153.2, 152.8, 151.3, 149.6, 149.3, 149.1, 145.1, 137.9,
137.2, 129.6, 125.1, 121.7, 117.4, 65.5 (-CH2), 22.5 (-CH3), 22.2 (-CH3), 21.5 (-CH3), 21.3 (-CH3),
21.2 (-CH3), 20.5 (-CH3). MS (ESI) m/z: 437.2 [M+H]+, HRMS (ESI) m/z: 437.18036 [M+H]+, calcd.
for C23H25N4O5 437.17467.
3.2. Bio-Evaluation Methods
3.2.1. Protective Effect on Damaged Differentiated PC12 Cells [9,24–26]
PC12 cells were cultured in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum,
10% (v/v) horse serum and 100 U/mL penicillin-streptomycin (Thermo Technologies, New York, NY,
USA) at 37 °C in a humidified atmosphere of 5% CO2. When cells achieved the desired density of
>80% confluency original medium was removed and cells were cultured with the serum-free
medium for 14 h. Then the cells were suspended in 1640 medium supplemented with
10% (v/v) fetal bovine serum, and seeded into poly-L-lysine-coated 96-well culture plates at 7 × 103
cells/well, differentiated by treated with 50 ng/mL NGF for 48 h. After these, the differentiated PC12
cells were pretreated with various concentrations (60, 30, 15, 7.5, 3.75 µM) of ligustrazine derivatives

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for 36 h. All measurements were performed after the cells were induced by CoCl2 (final concentration,
200 mM) for 12 h. Control differentiated cells were not treated with ligustrazine derivatives and CoCl2.
CoCl2 was dissolved in RPMI 1640 medium. ligustrazine derivatives were dissolved in DMSO. The
final concentration of DMSO was less than 0.1% (v/v).
After MTT solution (20 µL, 5 mg/mL) was added to each well, the plate was incubated for a further
4 h at 37 °C. The supernatant was removed carefully by pipetting from wells without disturbing the
attached cells and formazan crystals were solubilized by adding 200 µL of DMSO to each well and
shaken for 15 min. The absorbance at 490 nm was measured with a BIORAD 550 spectrophotometer
(Bio-rad, California, CA, USA). The proliferation rates of damaged PC12 cells were calculated by the
formula [OD490 (Compd) − OD490 (CoCl2)]/[OD490 (NGF) − OD490 (CoCl2)] × 100%; the EC50 values were
defined as the concentration of compounds that produced a 50% proliferation of surviving cells and
calculated using the following equation: −pEC50 = log Cmax − log 2 × (∑P − 0.75 + 0.25Pmax + 0.25Pmin),
Where Cmax = maximum concentration, ∑P = sum of proliferation rates, Pmax = maximum value of
proliferation rate and Pmin = minimum value of proliferation rate.
3.2.2. Observation of Morphologic Changes [26,27]
The PC12 cells culture procedure was similar to that described in section 3.2.1. After pretreatment
with the serum-free medium for 14 h, cells were seeded at a concentration of 7 × 104 cell/mL in a
volume of 0.8 mL on a poly-L-lysine-coated sterile cover slip in 6-well tissue culture plates, and
differentiated by treating with NGF (50 ng/mL) for 48 h. Then the differentiated PC12 cells were
pretreated with compound 4a (60 µM) for 36 h prior to exposure to CoCl2. After induction by CoCl2
for 12 h, culture medium was removed and the cells were fixed in 95% ethyl alcohol for 15 min. The
cells were washed twice with PBS and stained with hematoxylin for 8 min. The stained nuclei were
washed twice with diluted PBS, and consequently stained with eosin for 8 min. Next, H&E stained
sections were dehydrated through gradient ethanol and cleared in xylene. The residual procedure was
processed in accordance with the hematoxylin and eosin (HE) staining protocol. The cellular
morphology and percentage of cells showing neurite outgrowth was determined by light microscopy
(Nikon, Kobe, Japan). Cells with one or more neurites whose lengths were at least twice the diameter
of the cell body were scored as differentiated cells. Cell differentiation rate was calculated as the
number of differentiated cells/total cells. Neurite outgrowth was determined from at least three
different regions of interest in three independent experiments. All data are expressed as mean ± SD.
Data analysis was carried out using SAS software, version 8.1 (SAS Institute, Cary, NC, USA).
Statistically significant differences between the samples were evaluated by Student’s t-test and p <
0.05 was considered significant.
4. Conclusions
In conclusion, a series of novel ligustrazine-benzoic acid derivatives was designed, synthesized and
biologically evaluated for their protective effects on the damaged differentiated PC12 cell
proliferation. The preliminary biological results have demonstrated that most of ligustrazine-benzoic
acid derivatives exhibited good protective effects in comparison with TMP. Among the active
compounds, 4a is the most active congener, with EC50 4.249 µM, which is much higher than that of

Molecules 2013, 18
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TMP, and representing a most promising lead for further investigation. Studies of the anti-experimental
stroke effects and mechanism of 4a in a rat middle cerebral artery occlusion (MCAO) stroke model are
in progress and will be reported in the near future.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/10/13027/s1.
Acknowledgments
This study was financially supported by the National Natural Science Foundation of China
(No. 81173519), the Innovation Team Project Foundation of Beijing University of Chinese Medicine
(Lead Compound Discovering and Developing Innovation Team Project Foundation, No. 2011-CXTD-15)
and “Nautical Tradional Chinese Medicine” discipline (522/0100604054).
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1a–5a, 1b–5b, 1c–5c, 1d–5d are available from
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