Synthesis of the novel liqustrazine derivatives and their protective effect on injured vascular endothelial cell damaged by hydrogen peroxide

Article abstract of DOI:10.1016/S0960-894X(03)00359-7

A series of novel 2-acyloxymethyl-3,5,6-trimethylpyrazine derivatives was designed and synthesized. Most compounds were found to be 1.5-4.5-fold higher potency than tetramethylpyrazine (TMP) in stimulating the proliferation of normal vascular endothelial cells and in protecting against hyperoxic acute injury. The most active one is the 2-nicotinoyl ester 5a exhibiting the maximum proliferation rate (P_max) of 88.57% at the concentration of 0.1 mmol L^-1. Structure-activity relationships of these compounds were discussed.

Full text of DOI:10.1016/S0960-894X(03)00359-7

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2123–2126
Synthesis of the Novel Liqustrazine Derivatives and Their
Protective Effect on Injured Vascular Endothelial Cell
Damaged by Hydrogen Peroxide
Xinyong Liu,a,* Rui Zhang,a Wenfang Xu,a Chaowu Li,b
Quanqin Zhaoc and Xingpo Wangc
aInstitute of Medicinal Chemistry, School of Pharmacy, No. 44 Wenhuaxi Road, Jinan 250012, China
bQilu Hospital, Jinan 250012, China
cSchool of Chemistry and Engineer, Shandong University, Jinan 250012, China
Received 17 February 2003; revised 9 April 2003; accepted 9 April 2003
Abstract—A series of novel 2-acyloxymethyl-3,5,6-trimethylpyrazine derivatives was designed and synthesized. Most compounds
were found to be 1.5–4.5-fold higher potency than tetramethylpyrazine (TMP) in stimulating the proliferation of normal vascular
endothelial cells and in protecting against hyperoxic acute injury. The most active one is the 2-nicotinoyl ester 5a exhibiting the
maximum proliferation rate (Pmax) of 88.57% at the concentration of 0.1 mmol Lꢀ1. Structure–activity relationships of these
compounds were discussed.
# 2003 Elsevier Science Ltd. All rights reserved.
Endothelial cells play a critical physiological role in
maintaining normal vessel and organ function. Much
evidence show that vascular endothelial cells damage
that causes the alteration of endothelial permeability
barrier and vascular tone is a major promoter of both
atherogenesis and thrombosis and, consequently cardio-
vascular events. Oxidative stress is a cardiovascular risk
factor and contributes significantly to endothelial injury
during atherogenesis. Therefore, the protection of
endothelial cells against damage caused by oxidative
stress is a very important therapeutic strategy.1
disease and ischemic cerebrocardiac vascular disease.8,9
More recently, it has been found to be more effective in
protection against vascular endothelial cell injury.10
However, pharmacodynamics studies found that TMP
presented low bioavailability and to be metabolized fast
in vivo with short half-life of t1/2=2.89 h, so accumu-
lated toxicity often appeared in the patients for keeping
an effective plasma concentration by the frequent
administration.11 Therefore, it is necessary to develop
new generation of the cerebrocardiac vascular drugs
from molecular modification of TMP.
Liqustrazine (tetramethyl pyrazine, TMP), one major
efficient component from the Chinese traditional medi-
cine herb Chuanxiong (Ligusticum wallichii Franchat),
has long been reported to inhibit the platelet aggrega-
tion,2 to cause negative chronotropic and inotropic
responses on isolated atria,3 to inhibit vasoconstriction
in isolated vascular strips,4 and to act as a vasodilator, a
free radical scavenger, anti-thrombosis and anti-hyper-
tension agent (Fig. 1).5ꢀ7 Currently, TMP is widely used
in China as a new kind of calcium antagonist for the
treatment of coronary atherosclerotic cardiovascular
Metabolism investigation of TMP found that 2-
hydroxymethyl-3,5,6-trimethylpyrazine (HTMP) was
the major product in the metabolic process in vivo.12
HTMP had even been synthesized in our previous
work13 and found it possessed a better cardiovascular
activity than TMP.14 In the present paper, we will
describe the preparation and biological effects of the
acyl derivatives of the HTMP, in which some functional
groups, like nicotinoyl, acetylsalicyloyl, ferulolyl, 2-(4-
Cl-phenoxy)-2-methylpropionyl, cinnamoyl and 3,4,5-
trimethoxylbenzoyl, were connected to the hydroxyl
group for acquiring the pharmacologically combina-
tional effect. Other acyl groups were also introduced in
order to further expand our exploration and better
*Corresponding author. Tel.: +86-531-838-2005; fax: +86-531-838-
2731; e-mail: xinyongl@jn-public.sd.cninfo.net
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00359-7

2124
X. Liu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2123–2126
most active compound was the nicotinoyl ester 5a,
which displayed 4 times higher in proliferation rate at 6
times lower concentration than the control drug TMP.
As expected, the introduction of some functional
groups, such as nicotinoyl 5a, acetylsalicyloyl 5b, fer-
ulolyl 5c and 2-(4-chlorophenoxyl)-2-methyl propionyl
5d, which are active in cerebrocardiac vascular profiles,
produced the remarkable combinational effects.
Figure 1. Structures of Liqustrazine (tetramethyl pyrazine, TMP) (a),
HTMP (b) and newly synthesized derivatives (c).
understand the structure–activity relationships of TMP
derivatives.
In the cinnamoyl substituted series, compounds 5c, 5h–j
are potentially active but in decreasing order with
increase of the substitution number of methoxy groups
at the phenyl moiety. Replacement of the cinnamoyl
group (5h) with dihydrocinnamoyl group (5g) improved
the activity, and the values of Pmax increased from 77.32
to 80.54%. In the substituted or unsubstituted benzoic
acid ester series (5k–t), compounds with methoxy sub-
stituents are moderately active and with chloro sub-
stituents seriously impaired the activity except for
4-chlorobenzoic acid ester (5q) presenting a particular
high potency (Pmax=82.55%, C=0.1 mmol Lꢀ1).
2-Iodobenzoic acid ester (5t) possessed a better activity
than the corresponding 2-chloro compound (5o). Bro-
mobenzoic acid esters 5r–s completely lost the activity. In
other analogues, dihydrocinnamic acid ester (5g) was
more active than phenylacetic acid ester (5f), which might
reflect the importance of alkyl chain length of the aralkyl
carboxylic acid. All features described above should be
considered in the design of novel TMP derivatives.
The titled compounds were prepared starting from
TMP via four steps as shown in Scheme 1. The impor-
tant intermediate HTMP 4 was prepared according to
our previous work,13 but with some modification in the
workup, in which the crude compound of 2-acetyloxy-
methyl-3,5,6-trimethylpyrazine 3 was synthesized by the
acetylation and rearrangement reaction of compound 2
with acetic anhydride, and directly hydrolyzed with
sodium hydroxide water solution in one-pot reaction.
HTMP was recrystallized from petroleum ether (30–
60 ꢁC) to obtain the pure material, 64% yield from
TMP, mp 88–89 ꢁC. Compounds 5 were prepared by the
acylation of HTMP in the following two methods. In
method (a), the corresponding carboxylic acid was
straightly reacted with HTMP in the presence of DCC
and DMAP. In method (b), the carboxylic acid was
firstly transformed to acyl chloride, and then reacted
with HTMP in the presence of pyridine as a base. Total
21 compounds derived from TMP (5a–u) were synthe-
sized. All compounds were not reported in the literature
before. Their chemical structures were confirmed by IR,
1H NMR and MS spectrometries, and microanalysis.15
Protective effects of some active Liqustrazine derivatives
were also tested on acute injured HUVECs damaged by
hydrogen peroxide. The concentration of 75 mmol Lꢀ1
of hydrogen peroxide was selected to induce cells
damage identified by cells morphological examination.10
The results listed in Table 2 showed that compounds 5a,
5f, 5g, 5i and 5q were the better inhibitors than TMP
against HUVECs proliferation suppression caused by
hydrogen peroxide injury. At the concentration of 1.2
mmol Lꢀ1, compounds 5f and 5g remarkably improved
the proliferation rates of injured HUVECs exceeding to
the normal rates (0.00%) of HUVECs viability, and at
the concentration of 1.5 mmol Lꢀ1 compound 5a gave
the similar result. The protective effects of the active
compounds 5a, 5f, 5g, 5i and 5q were in concentration-
dependent at the tested concentration range of 0.1–1.5
mmol Lꢀ1, which was paralleled with their properties of
stimulating HUVECs proliferation. Interestingly, com-
pounds 5b and 5d active in stimulating normal
HUVECs proliferation, seemed to show no activity in
the tested concentration for protecting the injured
All compounds were tested for stimulating the pro-
liferation of cultured human umbilical vascular endo-
thelial cells (HUVECs) and for protecting against acute
injury damaged by hydrogen peroxide. TMP was used
as the control drug. The viability of normal and injured
HUVECs was assessed by methyl thiazolyl tetrazolium
(MTT) assay according to ref 10. The structures and the
maximum percentage of stimulating proliferation
(Pmax%) corresponding to the concentrations of the
compounds were outlined in Table 1.
The results showed that all of the newly synthesized
compounds significantly increased the normal HUVECs
viability in comparison with the positive stimulator of
TMP. Among the compounds, 5a, 5c, 5d, 5g and 5q
exhibited extraordinary high potency in stimulating
HUVECs proliferation. All of their maximum values of
proliferation rate (Pmax%) were revealed over 80%. The
Scheme 1. Reagents: (i) 30% H2O2, AcOH; (ii) Ac2O; (iii) 20% NaOH; (iv) (a) RCOOH/DCC, DMAP or (b) RCOCl/Py.

X. Liu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2123–2126
2125
Table 1. The structures and maximum values of stimulating HUVECs proliferation (Pmax%) of the novel Liqustrazine derivatives 5a–u
a
c
Compd
RCO–
Method
A0(570nm)
A570nm
Pmax%
C (mM)
5a
5b
5c
5d
5e
Nicotinoyl
Acetylsalicyloyl
Ferulolyl
a
b
a
a
b
b
a
b
a
a
a
a
a
a
b
b
b
a
a
a
a
0.831ꢂ0.05
0.831ꢂ0.05
0.831ꢂ0.05
0.831ꢂ0.05
0.629ꢂ0.02
0.745ꢂ0.04
0.745ꢂ0.04
0.882ꢂ0.05
0.831ꢂ0.05
0.831ꢂ0.05
0.745ꢂ0.04
0.745ꢂ0.04
0.882ꢂ0.05
0.882ꢂ0.05
0.629ꢂ0.02
0.745ꢂ0.04
0.745ꢂ0.04
0.629ꢂ0.02
0.629ꢂ0.02
0.629ꢂ0.02
0.882ꢂ0.05
1.433ꢂ0.06
1.433ꢂ0.06
1.56ꢂ0.03**b
1.420ꢂ0.03**
1.528ꢂ0.05**
1.561ꢂ0.06**
0.859ꢂ0.05*
1.312ꢂ0.05**
1.345ꢂ0.02**
1.564ꢂ0.04**
1.458ꢂ0.05**
1.414ꢂ0.06**
1.281ꢂ0.05**
1.237ꢂ0.04**
1.349ꢂ0.03**
1.286ꢂ0.03**
0.831ꢂ0.07 *
1.187ꢂ0.03**
1.36ꢂ0.112**
0.565ꢂ0.03
88.57
70.88
83.87
87.85
37.00
76.11
80.54
77.32
75.45
70.16
71.95
66.04
52.95
45.80
32.10
59.32
82.55
Nod
0.1
0.1
0.1
0.1
0.6
0.1
0.1
0.6
0.1
0.1
0.1
0.1
0.1
0.1
0.6
0.1
2-(4-Cl-phenoxy)-2-methylpropionyl
Benzoyl
Penylacetyl
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
5t
5u
TMP
HTMP
Dihydrocinnamoyl
Cinnamoyl
4-Methoxylcinnamoyl
2,4-Dimethoxylcinnamoyl
2-Methoxylbenzoyl
4-Methoxylbenzoyl
3,4-Dimethoxylbenzoyl
3,4,5-Trimethoxylbenzoyl
2-Chlorobenzoyl
3-Chlorobenzoyl
4-Chlorobenzoyl
4-Bromobenzoyl
2,4-Dibromobenzoyl
2-Iodobenzoyl
0.1
0.1–1.5
0.1–1.5
1.5
0.6
0.6
0.561ꢂ0.01
No
0.858ꢂ0.08**
1.494ꢂ0.04*
1.729ꢂ0.06*
1.835ꢂ0.06**
61.36
69.39
20.66
28.03
2-Furoyl
2-Hydrogen
0.6
*p<0.01,**p<0.05 compared with corresponding control value.
aThe absorption values at 570nm were measured using an ELISA plate reader, A
absorption values of compounds.
: absorption values of the blank control, A570nm :
0(570nm)
bMeanꢂSEM, n=6 (well).
cResults were expressed as percentage of absorption value in vehicle-treated control culture well, P(%)=(AꢀA0)/A0.
dNot active at the concentrations of 0.1–1.5 mmol Lꢀ1
.
Table 2. The protective effects of Liqustrazine derivatives on injured
HUVECs proliferation
88.57% at the concentration of 0.1 mmol Lꢀ1 in stimu-
lating the normal HUVECs proliferation, and Pmax value
of 3.79% at 1.5 mmol Lꢀ1 in protecting the damaged
HUVECs viability. Structure–activity relationships were
briefly discussed. Further bioassay of these compounds
on cerebrocardiac vascular activity in cell culture and
on animal models is underway.
Compd
Concentrations of compounds (mmol Lꢀ1
)
0a
0.1
0.3
0.6
1.2
1.5
5a
5b
5c
5d
5f
5g
5i
5q
ꢀ49.8
ꢀ45.0
ꢀ49.8
ꢀ49.8
ꢀ45.0
ꢀ45.0
ꢀ45.0
ꢀ45.0
ꢀ49.8
ꢀ49.8
ꢀ46.7
ꢀ34.2
ꢀ43.2
ꢀ51.2
ꢀ29.1
ꢀ29.7
ꢀ37.6
ꢀ40.5
ꢀ48.7
ꢀ43.6
ꢀ36.4
ꢀ46.4
ꢀ76.7
ꢀ40.4
ꢀ24.4
ꢀ23.5
ꢀ33.3
ꢀ28.6
ꢀ47.5
ꢀ40.5
ꢀ13.9
ꢀ53.8
ꢀ79.7
ꢀ72.3
ꢀ12.2
ꢀ11.5
ꢀ21.2
ꢀ28.3
ꢀ41.0
ꢀ38.2
ꢀ12.03.79
ꢀ63.4
ꢀ60.6
ꢀ83.4
6.01
NDb
ꢀ15.6
ꢀ69.0
4.43
5.70
Acknowledgements
5.62
ꢀ25.1
ꢀ2.46
ꢀ44.2
ꢀ36.4
ꢀ28.5
The financial supports of this work by Shandong Nat-
ural Science Foundation and Shandong Science &
Technology Bureau Foundation are gratefully
acknowledged.
ꢀ1.20
ND
TMP
HTMP
ꢀ35.8
aBlank control: The proliferation rate (P%) of HUVECs injured by 75
mmol Lꢀ1 of hydrogen peroxide without any compound addition. The
proliferation rate (%) of the nomal HUVECs was designated as
0.00%.
bND, not determined.
References and Notes
HUVECs. Compound 5c displayed observable protec-
tive action while the concentration reached to 1.5 mmol
L
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ꢀ1. Further studies for the structure–activity relation-
ships of these compounds on the profiles are needed.
In conclusion, a series of novel TMP derivatives was syn-
thesized by way of the acylation reaction of HTMP. Some
compounds were found better activity than TMP in stim-
ulating the proliferation of normal vascular endothelial
cells and in protecting against hyperoxic acute injury, as
seen in 2-nicotinoyl ester 5a, which exhibited Pmax value of
8. Sun, B. L.; Pract, J. Integ. Chin. Mod. Med. 1992, 5, 562.

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J=9.0Hz), 5.26 (s, 2H, CH 2), 2.49 (s, 3H, 6-CH3), 2.45 (s, 3H,
5-CH3), 2.41(s, 3H, 3-CH3), 1.60(s, 6H, CH 3–C–CH3); IR
(nujol, cmꢀ1): 2992, 2939, 2924, 2857 (nCH), 1739 (nC¼O),
1594, 1489 (nC¼C,), 1282, 1093 (nC-O-C, CH2OCO), 1238, 1139
(nC-O-C, CO–Ar). 5f: 1H NMR: 7.32–7.27 (m, 5H, Ar–H), 5.21
(s, 2H, CH2O), 3.68 (s, 2H, CH2–Ar), 2.50(s, 3H, 6-CH 3), 2.49
(s, 3H, 5-CH3), 2.42 (s, 3H, 3-CH3); IR (KBr, cmꢀ1): 3027,
2943, 2932, 2850( nCH), 1736 (nC¼O), 1495, 1412 (nC¼C), 1245
(nC-O-C), 1128; MS (m/z): M+1: 271.4. 5g: 1H NMR: 7.27–
7.16 (m, 5H, Ar–H), 5.19 (s, 2H, CH2O), 2.97 (t, 2H, CH2–
Ar), 2.70(t, 2H, CO–CH 2), 2.51 (s, 3H, 6-CH3), 2.49 (s, 3H, 5-
CH3), 2.46 (s, 3H, 3-CH3); IR (nujol, cmꢀ1): 3027, 2924, 2862
(nCH), 1739 (nC¼O), 1496, 1415 (nC¼C), 1158 (nC-O-C); MS (m/
14. Chen, X. M.; Xu, S. J.; Ma, Y. W. Chin. Med. Chem.
1996, 6, 254.
15. 1H NMR (CDCl3, d ppm), IR and MS(EI) data for
representative compounds: 5a: 1H NMR: 9.22–7.36 (m, 4H,
Ar–H), 5.45 (s, 2H, CH2), 2.58 (s, 3H, 6-CH3), 2.53 (s, 3H,
5-CH3), 2.47 (s, 3H, 3-CH3); IR (KBr, cmꢀ1): 3085, 3068, 3044
(nPyꢀH), 2983, 2949, 2854 (nCH), 1706 (nC¼O), 1587, 1416
(nC¼C,), 1276, 1144 (nC-O-C), 745 (gArꢀH); MS (m/z): M+1:
258.9. 5b: 1H NMR: 7.58–6.94 (m, 4H, Ar–H), 5.38 (s, 2H,
CH2), 2.56 (s, 3H, 6-CH3), 2.52 (s, 3H, 5-CH3), 2.51 (s, 3H,
CH3COO–Ar), 2.31 (s, 3H, 3-CH3). IR (nujol, cmꢀ1): 2991,
2952, 2923 (nCH), 1753 (nC¼O), 1615, 1469 (nC¼C), 1308, 1253
(nC-O-C); MS (m/z): M+1: 314.1. 5c: IR (KBr, cmꢀ1): 3326
(nNH), 2923, 2850( nCH), 1762, 1700 (nC¼O), 1629, 1509, 1423
1
z): M+1: 285.2. 5i: H NMR: 7.67 (d, 1H, ¼CH–Ar, J=16.2
0
0
0
Hz), 70.41 (d, 2H, C2 -H, C6 -H, J=11.7 Hz), 6.89 (d, 2H, C3 -
H, C5 -H, J=9.0Hz), 6.35 (d, 1H, CO–CH ¼, J=16.2 Hz),
5.31 (s, 2H, CH2), 3.83 (s, 3H, OCH3), 2.57 (s, 3H, 5-CH3),
2.52 (s, 6H, 5,6-CH3); IR (KBr, cmꢀ1): 2972, 2960, 2923, 2836
(vCH), 1704 (nC¼O), 1636 (nC¼C), 1602, 1512, 1412 (nC¼C),
1289, 1166 (nC-O-C, CH2OCO), 1248, 1026 (vC-O-C, Ar-O-
1
CH3), 927 (g¼CH); MS (m/z): M+1: 313.4. 5q: H NMR: 7.95
(d, 2H, C2 -H, C6 -H), 7.36 (d, 2H, C30 -H, C50-H), 5.40(s,
2H, CH2), 2.54 (s, 3H, 6-CH3), 2.52 (s, 3H, 5-CH3), 2.48 (s,
3H, 3-CH3); IR (KBr, cmꢀ1): 3056, 2985, 2956, 2920 (vCH),
1724 (nC¼O), 1596, 1415 (nC¼C), 1274 (nC-O-C); MS (m/z):
M+1: 291.
0
0
(nC¼C), 1274, 1218 (nC-O-C, CH2OCO), 1235, 1155 (nC-O-C
,
Ar-O-CH3); M0S (m/z): M+1: 371.9. 5d: 1H NMR: 7.11 (d,
0
0
0
2H, C2 -H, C6 -H, J=8.7 Hz), 6.76 (d, 2H, C3 -H, C5 -H,