H.-h. Zhu et al. / Bioorg. Med. Chem. Lett. 26 (2016) 882–884
883
O
such as arrhythmia, indicating a substantial decline in the potency
of liguzinediol by metabolism and highlighting the safety of
administration of liguzinediol.
O
N
N
N
OH
N
a
O
b
O
HO
HO
Cl
N
N
A practical route for preparation of 1 was not immediately appar-
ent. Firstly, we attempted to protect one hydroxymethyl and then
oxidize the other hydroxymethyl, but failed. It was probably due
to the existence of the aldehyde during the oxidation process leading
to the difficulty of purification. Then the following synthesis route
was selected. 3,6-Dimethylpyrazine-2,5-dicarboxylic acid (5) was
obtained by oxidizing the two hydroxymethyl groups in neutral
aqueous solution of potassium permanganate.22–24 Though esterifi-
cation to give compound 6, and compound 7 was obtained from 6 by
reduction under the condition of NaBH4, and metabolite 1 was
obtained after hydrolysis (Scheme 1).
11
12
N
COOH
NH2
S
N
COOH
NH2
d
c
S
O
N
HO
N
O
13
3
Scheme 3. Synthesis of metabolite 3. Reagents and conditions: (a) CH3COCl,
33.05%; (b) SOCl2, 37.64%; (c) -cysteine, 67.63%; (d) 40% NaOH, 75.6%.
L
N
COOH
NH2
N
COOH
O
Commercially available glucurone was used for preparing the
metabolite 2. Anhydrous condition was indispensable for the syn-
thesis of the bromo-glycoside donor and molecular sieves were
needed to ensure the reaction smoothly. It has been described by
previous published literature that the catalyst TMSOTF and
BF3ÁOEt2 could quickly connect the glycoside with the parent
compound.25–27 However, due to the alkalescence of liguzinediol,
the activity of these acidic catalysts was reduced, leading to a
lower yield of compound 10. Metabolite 2 can be obtained via
alkali-promoted deprotection of the acetyl group28 (Scheme 2).
Metabolite 3 was synthesized as illustrated in Scheme 3. The
parent compound (liguzinediol) was selectively protected with
acetyl chloride at 0 °C, and then reacted with thionyl chloride to
a
S
S
HO
HO
HN
N
N
3
4
Scheme 4. Synthesis of metabolite 4. Reagents and conditions: (a) CH3COCl, 34.6%.
Table 1
HPLC retention time of metabolites 1–4
1
2
3
4
HPLC retention time
(min)
Metabolite
Synthetic
sample
3.637 6.687 3.000 5.705
3.675 6.710 2.988 5.709
afford compound 12. L-Cysteine was slowly added to the mixture
of 12 to yield 13 in saturated NaHCO3 solution. Alkaline hydrolysis
of compound 13 and subsequently acidizing the solution with HCl
provided the desired metabolite 3. After acetylation, metabolite 4
can be obtained from metabolite 3 (Scheme 4).
The synthetic metabolites were identified by comparing HPLC
retention time and MS/MS spectra data with those of the in vivo
metabolites of liguzinediol (Table 1). To investigate whether the
positive inotropic activity of liguzinediol was attributed to the
metabolites in vivo, the biological evaluations were performed in
this work.
experiments were performed in accordance with the Animal Care
and Committee of Nanjing University of Chinese Medicine and
the committee outline of the National Institutes of Health of USA
Guide for the Care and Use of Laboratory Animals.29 The rats were
anesthetized by 20% urethane (1.2 gÁkgÀ1), and their hearts were
rapidly excised followed by immersion in perfusion fluid at 0 °C.
After pruning the isolated aorta, the hearts were perfused in the
Langendorff apparatus, with perfusion solution (37 °C) containing
(in mmolÁLÀ1): NaCl 117, KCl 5.7, CaCl2 1.8, NaHCO3 4.4, NaH2PO4-
Á2H2O 1.5, MgCl2Á6H2O 1.7, HEPES 20, Glucose 11, and gassed with
95% O2 plus 5% CO2 (pH 7.23 with NaOH). During perfusion, the
intubation method was used to ensure that the pressure sensor
probe was inserted through the left atrial into the left ventricle.
Left ventricular contraction curve was recorded by the pressure
transducer, which connects with a RM6240 type multi-channel
physiological signal acquisition processing system. Liguzinediol
and its metabolites were added in the perfusion solution and
infused through retrograde perfusion of the coronary aorta. The
indices of left ventricular cardiac function were measured, includ-
ing left ventricular systolic pressure (LVSP), left ventricular
diastolic pressure (LVDP), peak rise rate of left ventricular pressure
(+dp/dtmax), peak decrease rate of left ventricular pressure
(Àdp/dtmax) and heart rate (HR).30
Male Sprague–Dawley (SD) rats (280 20 g) (Shanghai SLAC
Laboratory Animal Co. Ltd.) were used in this study. Animal
O
O
N
N
N
N
a
OH
OH
b
O
HO
HO
O
N
N
O
O
5
6
O
O
N
N
c
O
d
OH
HO
HO
N
N
7
1
Scheme 1. Synthesis of metabolite 1. Reagents and conditions: (a) KMnO4, 86.7%;
(b) H2SO4, 70 °C, 88.2%; (c) NaBH4, 30.9%; (d) 5% NaOH, 56%.
The baseline values of LVSP, LVDP, HR, +dp/dtmax and Àdp/dtmax
were summarized in Table 2 (n = 6, respectively). In liguzinediol
HO
MeOOC
O
MeOOC
MeOOC
a
b
O
c
O
O
O
HO
OH
OH
AcO
AcO
OAc
O
HO
AcO
AcO
OH
OAc
8
OAc
Br
OH
9
N
N
N
MeOOC
AcO
AcO
OH
HOOC
HO
HO
OH
e
d
O
O
O
O
N
OAc
OH
2
10
Scheme 2. Synthesis of metabolite 2. Reagents and conditions: (a) NaOH; (b) Ac2O, 68.91%; (c) 33% HBr, 96.98%; (d) liguzinediol, Ag2CO3, reflux, 30.0%; (e) 20% NaOH, 81.39%.