7
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X.-Y. Jin et al. / Chinese Chemical Letters 25 (2014) 787–790
2.2. Pharmacology
O
O
2.2.1. Protective effect against acute liver injury induced by CCl
4
or
H
APAP
H
Baclb/c mice were randomly divided (n = 10, half male and half
female). Compounds were suspended in 1% sodium methoxycel-
lulose and administered by intragastric at dosage of 50 mg/kg.
Sixty minutes after administration of the test compounds, 0.1%
H
H
OH
HO
H
NCX-1000
4
CCl in olive oil at dose of 10 mL/kg or APAP at dose of 200 mg/kg
Fig. 1. Structure of NCX1000 and targeted compounds.
was administrated by i.p. injection to induce the acute liver injury
model. Saline was subcutaneous injected as normal control.
Twelve hours after administration of carbon tetrachloride or
APAP, same amount of the compounds was administered by
intragastric. Twenty-four hours after the second administration of
compounds, whole blood samples were taken from the suborbital
sinus and the samples were centrifuged, the plasma was frozen and
stored immediately at À70 8C. Aspartate aminotransferase (AST)
and alanine aminotransferase (ALT) was determined with the
Olympus AU5400 automated biochemistry analyzer (Olympus
Corporation, Tokyo, Japan).
in the blood did not significantly changed. Co-administration of
UDCA significantly antagonized the increase of nitrate in the liver
resulted by 1e, which demonstrated that the delivery of 1e to the
liver was mediated via the bile acid transport system.
2
. Experimental
2.1. Chemistry
2.2.2. NO distribution in vivo
After overnight fasting, male Baclb/c mice were randomly
The synthesis of compounds 1a and 1b is outlined in Scheme 1.
divided into 4 groups (n = 10). Compounds were suspended in 1%
sodium methoxycellulose and administered by intragastric injec-
tion. The mice in the first group were treated with saline as control;
the mice in the second group were treated with 1e (10 mg/mL,
The 24-COOH of bile acids was first protected by methylation in
HCl-methanol solution to give 2a. Target compound 1a was
obtained by nitration of all three hydroxyl groups of 2a with
2
fuming nitric acid in Ac O and then deprotection with 5% KOH
2
0 mL/kg); in the third group, mice were treated with UDCA
menthol solution. The 3-OH of 2a was selectively reacted with
acetyl chloride to give the 3-OH protected derivative 3b.
Compound 1b was obtained by nitration of 3b followed by
deprotection of 4b, similar to 1a.
(
50 mg/mL, 20 mL/kg); in the fourth group, mice were treated with
UDCA (50 mg/mL, 20 mL/kg) and 1e (10 mg/mL, 20 mL/kg). Five
mice from each group were sacrificed in 2 h or 4 h after
administration of the compounds. Whole blood samples were
taken from the suborbital sinus, and the samples were centrifuged.
The plasma was frozen and stored immediately at À70 8C. The
livers were collected at the same time and rapidly frozen in liquid
nitrogen and maintained at À80 8C until analysis. Liver lysates
were prepared by homogenization in Tris–HCl buffer (0.01 mol/
L + NaCl 0.1 mol/L). The homogenates were centrifuged at
Compounds 1c and 1d were synthesized as depicted in Scheme
2
. The methylated derivatives (2a and 2d) of bile acids were
reacted with Ac O to give the acetate derivatives 3c and 3d; 3c and
d were selectively deprotected to give the 3-OH free derivative 4c
2
3
and 4d. The target compounds 1c and 1d were obtained by
nitration and then deprotection as that of 1a.
Compounds 1e and 1f were prepared as outlined in Scheme 3.
1
5,000 rpm for 15 min and the supernatant was taken for nitrate
4
d was reacted with methanesulfonyl chloride to obtain the
determination.
methanesulfonate derivative 6. Compound 6 was then reacted
with 1, 4-butanediol or ethylene glycol to give the hydroxyl
alkylated derivatives 7e and 7f, respectively. Compounds 7e and 7f
were nitrated and then deprotected to afford target compounds 1e
and 1f, respectively. NCX-1000 was synthesized according to the
literature [9].
The nitrate concentration was measured by Griess assay using a
colorimetric assay kit (Beyotime, China) [11]. Briefly, 50
sample solutions were mixed with 50 L Griess reagent I and
L Griess reagent II, and the mixture was then placed at room
mL of the
m
50 m
temperature for 10 min. The absorbance was measured at 540 nm,
and sodium nitrate solutions at different concentrations were used
as positive controls for the standard curve.
The structures of compounds 1a–1f were characterized [10].
Scheme 1. Synthesis route of compounds 1a–1b. (a) HCl–methanol, r.t., 12 h; (b, e) acetic anhydride, fuming nitric acid, À5 8C, 1 h; (c, f) 5% KOH menthol solution, reflux, 1 h;
d) acetyl chloride, pyridine, 0 8C, 1 h, then r.t., 2 h.
(
Jin, Xue-Yuan
