Novel liver-specific nitric oxide (NO) releasing drugs with bile acid as both NO carrier and targeting ligand

Article abstract of DOI:10.1016/j.cclet.2014.04.001

Novel liver-specific nitric oxide (NO) releasing drugs with bile acid as both the NO carrier and targeting ligand were designed and synthesized by direct nitration of the hydroxyl group in bile acids or the 3-O-hydroxyl alkyl derivatives, with the intact 24-COOH being preserved for hepatocyte specific recognition. Preliminary biological evaluation revealed that oral administrated targeted conjugates could protect mice against acute liver damage induced by acetaminophen or carbon tetrachloride. The nitrate level in the liver significantly increased after oral administration of 1e while nitrate level in the blood did not significantly change. Co-administration of ursodeoxycholic acid (UDCA) significantly antagonized the increase of nitrate in the liver resulted by administration of 1e.

Full text of DOI:10.1016/j.cclet.2014.04.001

Chinese Chemical Letters 25 (2014) 787–790
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Novel liver-specific nitric oxide (NO) releasing drugs with bile acid as
both NO carrier and targeting ligand
a
b
b
b
b
a,
Xue-Yuan Jin , Shi-Yong Fan , Hong-Wu Li , Wei-Guo Shi , Wei Chen , Hui-Fen Wang *,
b,
Bo-Hua Zhong *
a
Chinese PLA General Hospital & Chinese PLA Medical School, Beijing 100853, China
b
Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Novel liver-specific nitric oxide (NO) releasing drugs with bile acid as both the NO carrier and targeting
ligand were designed and synthesized by direct nitration of the hydroxyl group in bile acids or the 3-O-
hydroxyl alkyl derivatives, with the intact 24-COOH being preserved for hepatocyte specific recognition.
Preliminary biological evaluation revealed that oral administrated targeted conjugates could protect
mice against acute liver damage induced by acetaminophen or carbon tetrachloride. The nitrate level in
the liver significantly increased after oral administration of 1e while nitrate level in the blood did not
significantly change. Co-administration of ursodeoxycholic acid (UDCA) significantly antagonized the
increase of nitrate in the liver resulted by administration of 1e.
Received 6 January 2014
Received in revised form 13 March 2014
Accepted 24 March 2014
Available online 13 April 2014
Keywords:
Liver-specific
Nitric oxide releasing drugs
Hepatoprotective activity
Bile acid
ß 2014 Hui-Fen Wang and Bo-Hua Zhong. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
1
. Introduction
Nitric oxide (NO) mediates multiple physiological and patho-
However, NCX-1000 has not met the endpoints in phase 2 clinical
trials.
NCX-1000 was prepared by conjugating the NO releasing
moiety with the 24-COOH of UDCA by two ester bonds. However,
the intact 24-COOH of bile acid is essential for liver specificity [5].
Conjugating with the 24-COOH may abolish the targeting of UDCA,
and the ester bond in the conjugate is not stable in human blood.
Novel liver-specific nitric oxide (NO) releasing drugs were
designed and synthesized by use of the hydroxyl group on the
steroid nucleus at position 3, 7, or 12 as the conjugating group with
the preserved 24-COOH for hepatocyte specific recognition.
Compounds 1a–1d were nitrates of 3-OH, 7-OH, or 12-OH. In
these compounds, the bile acids are used as both the NO carrier and
logical processes [1]. Previous studies have demonstrated that NO
can protect the liver from injury and ameliorate the progress of
liver fibrosis or cirrhosis. NO-releasing drugs may represent an
important therapy for liver damage, fibrosis, and cirrhosis [2,3].
However, non-specific NO-releasing drugs such as isosorbide-5-
mononitrate induce significant reduction of arterial pressure,
which limits their clinical application [4].
Liver targeted therapy can deliver chemotherapeutic drugs
selectively to the liver to avoid side effects or toxicities. Bile acids
are the only small molecules with high oral availability that are
taken in specifically by the liver [5,6]. Conjugating NO releasing
components with bile acids such as cholic acid (CA) or
ursodeoxycholic acid (UDCA) can deliver NO selectively to the
liver for the treatment of liver diseases without significant
reduction of arterial pressure. NCX-1000 (Fig. 1) is the first
liver-specific NO donor drug that can protect mice against acute
liver injury induced by acetaminophen (APAP) or Con A [7,8].
targeting ligand. Compounds 1e and 1f werenitrates of 3-O(CH
2 4
) OH
and 3-O(CH OH derivatives of UDCA, respectively. The linkage
2 2
)
chain between nitrate and UDCA can modulate the release rate of NO
from the conjugates.
Preliminary biological evaluation revealed that the targeted
conjugates can protect mice against acute liver injury induced by
4
APAP or carbon tetrachloride (CCl ). Both the site and number of
nitration could affect the biological activity. Compounds 1e and 1f
are two most active compounds and show more potent protective
effects than NCX-1000. Further in vivo nitrate distribution
investigation revealed that the nitrate level in the liver signifi-
cantly increased after oral administration of 1e, while nitrate level
*
Corresponding authors.
E-mail addresses: wanghf0501@163.com (H.-F. Wang),
bohuazhong@yahoo.com (B.-H. Zhong).
1
001-8417/$ – see front matter ß 2014 Hui-Fen Wang and Bo-Hua Zhong. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2014.04.001

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.
(

X.-Y. Jin et al. / Chinese Chemical Letters 25 (2014) 787–790
789
Scheme 2. Synthesis route of compounds 1c–1d. (a) acetic anhydride, pyridine, DMAP, r.t., 6 h; (b) HCl–methanol, r.t., 1–3 h; (c) acetic anhydride, fuming nitric acid, À5 8C,
h; (d) 5% KOH menthol solution, reflux, 1 h.
1
Scheme 3. Synthesis route of compounds 1e–1f. (a) methanesulfonyl chloride, pyridine, 0 8C, 1 h, then r.t.,2 h; (b) butanediol or ethylene glycol, pyridine, 100 8C, 2 h; (c) acetic
anhydride, fuming nitric acid, À5 8C, 1 h; (d) 5% KOH menthol solution, reflux, 1 h.
All results are expressed as mean Æ SD. For multiple comparisons
statistical analysis was performed by one-way ANOVA followed by
Tukey multiple comparison tests with SPSS 20.0 software. P < 0.05
was considered to be statistically significant.
4
either plasma ALT or AST in the CCl or the APAP model. All the
active compounds except 1c showed greater inhibitory effects on
plasma ALT than on plasma AST.
Compounds 1e and 1f exhibited much more profound
4
protective effects against both CCl and APAP induced hepatocyte
injury than NCX-1000, UDCA, and 1a–1d. The difference in the
activity between 1e or 1f and other target compounds may result
from the difference in NO releasing rate from these compounds.
Nitrate of the axial hydroxyl group on the steroid nucleus may not
be easily reached by cytochrome P450 or glutathione-S-transfer-
ase, enzymes that are responsible for the hydrolysis of organic
nitrate. In vitro NO releasing experiments revealed that 1a–1d
release NO much more slowly than 1e or 1f after incubation with
HepG2 cells (Fig. 2). Ursodeoxycholic acid is a hepatocyte
protective agent [12]. The nitrates of ursodeoxycholic acid (1d–1f)
showed better hepatocyte protective effects than the nitrates of
cholic acid (1a–1c). This may be explained by the synergistic effect
of UDCA and NO released from the target compounds 1d–1f.
3
. Results and discussion
3.1. Protective effect against acute liver injury induced by CCl
4
or
APAP
4
Both CCl and APAP treatment resulted in significant increase of
ALT and AST in the blood. As shown in Table 1, at doses of 50 mg/kg,
compounds 1b, 1d, 1e, 1f and NCX-1000 significantly inhibited the
rise of both plasma ALT and AST in both the CCl
compound 1a and UDCA showed some effects on both plasma ALT
and AST in the CCl model but only plasma ALT in the APAP model;
compound 1c significantly inhibited the rise of plasma AST in the
CCl model and plasma ALT in the APAP model; CA has no effect on
4
and APAP models;
4
4
Table 1
*
Protective activity against CCl
4
and APAP induced acute liver-injury.
CCl model
Group
4
APAP model
ALT (U/L)
AST (U/L)
ALT (U/L)
AST (U/L)
NC
377 Æ 113
2855 Æ 765
1703 Æ 618
2150 Æ 577
2184 Æ 564
2261 Æ 597
2061 Æ 560
818 Æ 527
489 Æ 146
3647 Æ 861
2633 Æ 608
2410 Æ 796
2549 Æ 675
2601 Æ 745
2088 Æ 866
2354 Æ 457
2206 Æ 594
3378 Æ 756
2965 Æ 451
391 Æ 159
3416 Æ 575
1774 Æ 339
1948 Æ 661
306 Æ 215
3048 Æ 722
1843 Æ 462
2626 Æ 459
2377 Æ 493
2667 Æ 606
2348 Æ 590
1698 Æ 480
1920 Æ 423
3154 Æ 809
2805 Æ 620
*
*
**
**
**
MC
#
#
#
#
#
##
##
##
NCX-1000
##
#
1
1
1
1
1
1
a
b
c
d
e
f
##
#
2071 Æ 1024
#
#
2307 Æ 751
#
#
#
##
#
##
#
1558 Æ 565
1100 Æ 746
1167 Æ 466
3243 Æ 797
2240 Æ 731
#
#
##
##
##
##
##
#
1020 Æ 582
2521 Æ 627
1940 Æ 336
CA
#
#
#
UDCA
NC: normal control, MC: model control.
*
Values are mean Æ SD; n = 10.
**
P < 0.01 vs. NC group.
P < 0.05 vs. MC group.
P < 0.01 vs. MC.
#
#
#

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X.-Y. Jin et al. / Chinese Chemical Letters 25 (2014) 787–790
10
were designed and synthesized. Compounds 1a–1d were
synthesized by selective nitration of the 3-OH, 7-OH, or 12-
1
a
OH on the steroid nucleus, while 1e and 1f were nitrates of 3-
O(CH ) OH and 3-O(CH ) OH derivatives of ursodeoxycholic
2 4 2 2
acid. The intact 24-COOH is preserved in all the targeted
compounds for hepatocyte specific recognition. All compounds
8
6
4
2
1
1
1
b
c
4
showed protective effects against both CCl and APAP induced
d
acute liver injury in mice models. Compounds 1e and 1f
exhibited much more profound protective effects against both
1e
1f
4
CCl and APAP induced hepatocyte injury than NCX-1000, UDCA,
and 1a–1d. The nitrate level in the liver is significantly increased
after administration of 1e while the change of nitrate level in the
blood is not significant. Co-administration of UDCA significantly
antagonized the increase of nitrate in the liver resulted by
administration of 1e, indicating that the NO-releasing com-
pounds were delivered specifically into the liver by the bile acid
mediated transportation system.
0
30 60 90 120 150 180 210 240 270
Time (min)
Acknowledgments
Fig. 2. NO release assay in vitro of target compounds. HepG2 cells were incubated in
triplicate with each compound. The levels of nitrate produced in the lysates of
HepG2 cells were determined using a calorimetric Griess assay [11].
This work was supported by the National High Technology
Research and Development (863) Project (No. 2006AA02A4C6) and
National Natural Science Foundation of China (Nos. 30572220 and
Blood
30972626).
Liver
4
3
2
1
00
00
00
00
0
*
*
*
*
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1
mediated route, the liver specific NO releasing compounds will
release NO by cytochrome P450 or glutathione-S-transferase
catalyzed hydrolysis, and the NO released is then transformed
into nitrate. So the concentration of nitrate in liver and in blood
was determined to evaluate the specificity of the target compound.
As shown in Fig. 3, the nitrate level in the liver is significantly
increased at 2 h and 4 h after administration of 1e, while the
change of nitrate level in the blood is not significant. UDCA has no
effect on the nitrate levels in the liver. Co-administration of UDCA
significantly antagonized the increase of nitrate in the liver
resulted by 1e. These results indicated that the NO-releasing
compounds we synthesized were delivered into the liver via the
bile acid mediated transportation system route and released NO
specifically in the liver.
1
[
6
1
6
1b: Yield 76%. Mp: 191–193 8C; H NMR (400 MHz, DMSO-d ): d 11.98 (s, 1H),
5
.39 (s, 1H), 5.09 (s, 1H), 3.24 (s, 1H), 1.98–0.97 (m, 23H), 0.99–0.78 (m, 11H). MS
1
(
FAB): m/z 497.7 (M-1). 1c: Yield 67%. Mp: 218–220 8C; H NMR (400 MHz,
DMSO-d ): d 11.93 (s, 1H), 4.85 (s, 1H), 4.20–4.18 (d, 2H), 3.79 (s, 1H), 3.63 (s,
H), 1.99–0.97 (m, 30H), 0.59 (s, 3H). MS (FAB): m/z 452.5 (M-1). 1d: Yield 62%.
6
1
1
Mp: 186–188 8C; H NMR (400 MHz, DMSO-d
6
): d 11.84 (s, 1H), 4.85 (s, 1H), 3.60–
3
.57 (d, 2H), 2.24 (s, 1H), 2.02–0.97 (m, 25H), 0.94 (d, 3H, J = 6.5 Hz), 0.85 (s, 3H),
1
0.63 (s, 3H). MS (FAB): m/z 436.4 (M-1). 1e: Yield 47%. Mp: 185–187 8C; H NMR
400 MHz, CDCl3): d 11.96 (s, 1H), 4.56–4.52 (t, 2H), 3.86–3.84 (d, 1H, J = 7.0 Hz),
(
3
5
.48 (s, 1H), 3.34–3.32 (t, 2H), 2.25–0.87 (m, 37H), 0.62 (s, 3H). MS (FAB): m/z
08.8 (M-1). 1f: Yield 43%. Mp: 174–176 8C; H NMR (400 MHz, CDCl3): d 12.05
1
(s, 1H), 4.56–4.54 (t, 2H), 3.90–3.87 (d, 1H, J = 6.8 Hz), 3.46 (s, 1H), 3.31–3.28 (t,
H), 2.26–1.04 (m, 27H), 0.97 (s, 3H), 0.89 (d, 3H, J = 6.4 Hz), 0.61 (s, 3H). MS
FAB): m/z 480.5 (M-1).
2
(
[
11] M.D. Lu, X. Zhou, Y.J. Yu, et al., Synthesis and in vitro biological evaluation of nitric
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4
. Conclusion
[12] Z. Xiang, Y.P. Chen, K.F. Ma, et al., The role of Ursodeoxycholic acid in non-
alcoholic steatohepatitis: a systematic review, BMC Gastroenterol. 13 (2013)
In conclusion, six novel liver specific NO releasing com-
1
40.
pounds with bile acid as both the NO carrier and targeting ligand