Synthesis, structure-activity relationship and biological evaluation of novel N-substituted matrinic acid derivatives as host heat-stress cognate 70 (Hsc70) down-regulators

Article abstract of DOI:10.1016/j.bmcl.2011.06.071

Oxymatrine (1) is a natural anti-hepatitis B virus (HBV) drug that down-regulates host heat-stress cognate 70 (Hsc70) expression through a mechanism different from that of nucleosides. Taking Hsc70 as a target against HBV, 26 novel N-substituted matrinic acid analogs were designed, synthesized and evaluated for their regulation of Hsc70 mRNA expression with 1 as the lead. The SAR analysis revealed that (i) the carboxyl group at the 11-position was required for activity; (ii) introducing of a substituent on the nitrogen atom at the 12-position of 3, especially substituted benzyl, might significantly improve the activity. Among these analogs, compound 9p possessing N-p-methoxylbenzyl afforded an increased anti-HBV effect in comparison with 1. We consider 9p a promising anti-HBV candidate.

Full text of DOI:10.1016/j.bmcl.2011.06.071

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4732–4735
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, structureÀactivity relationship and biological evaluation of novel
N-substituted matrinic acid derivatives as host heat-stress cognate 70
(Hsc70) down-regulators
Na-Na Du , Xin Li , Yu-Ping Wang, Fei Liu, Yan-Xin Liu, Chun-Xin Li, Zong-Gen Peng, Li-Mei Gao,
⇑
⇑
Jian-Dong Jiang , Dan-Qing Song
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 February 2011
Revised 2 June 2011
Accepted 16 June 2011
Available online 23 June 2011
Oxymatrine (1) is a natural anti-hepatitis B virus (HBV) drug that down-regulates host heat-stress cog-
nate 70 (Hsc70) expression through a mechanism different from that of nucleosides. Taking Hsc70 as a
target against HBV, 26 novel N-substituted matrinic acid analogs were designed, synthesized and evalu-
ated for their regulation of Hsc70 mRNA expression with 1 as the lead. The SAR analysis revealed that (i)
the carboxyl group at the 11-position was required for activity; (ii) introducing of a substituent on the
nitrogen atom at the 12-position of 3, especially substituted benzyl, might significantly improve the
activity. Among these analogs, compound 9p possessing N-p-methoxylbenzyl afforded an increased
anti-HBV effect in comparison with 1. We consider 9p a promising anti-HBV candidate.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
N-Substituted matrinic acid
Heat-stress cognate 70 (Hsc70)
Structure–activity relationship
Mechanism
Down-regulators
Anti-HBV activity
Oxymatrine (1, Fig. 1), a natural drug extracted from the plant
Kushen (Sophora japonica), has been used to treat hepatitis B pa-
tients in China for decades with a confirmed safety.1 Its mode of ac-
tion against hepatitis B virus (HBV) is distinctly different from that
of nucleosides currently used in clinic such as lamivudine (3TC)
and adefovir.2–5 Recently, we have identified host heat-stress cog-
nate 70 (Hsc70) to be a key target of compound 1.6 Compound 1
significantly down-regulates host Hsc70 mRNA expression at the
post transcriptional level through destabilizing Hsc70 mRNA, and
then inhibits the replication of HBV and exhibits anti-HBV efficacy.
As this target is not a viral enzyme, antiviral agents working
through this mechanism not only inhibit the replication of wild-
type HBV but also the drug-resistant mutants.6,7 In addition,
Hsc70 gene knockout showed no abnormality in mice, indicating
a good safety after inhibition of Hsc70.8 Therefore, targeting host
Hsc70 is a novel and effective approach to discover highly potent
and safe anti-HBV drug candidates overcoming drug-resistance.6b
The structure–activity relationship (SAR) analysis for down-
regulation in Hsc70 was initiated with modifications of the
substituents at the 1-, 13- and 14-positions with 1 as the lead.6b
The primary SAR revealed that the oxygen atom at the 1-position
is not essential for the activity. In exploring the chemical mecha-
nism of this class of compounds and searching for potent anti-
HBV agents working through down-regulation of Hsc70 expres-
sion, SAR was developed by focusing our study on the function of
the ring D. Thus, the ring D of 1 or matrine (2) was opened, and
the resultant oxymatrinic acid (3) or matrinic acid (4, Fig. 1) still
retained moderate activity, similar to that of its parent 1 or 2
(Table 1). It was deduced that ring D might not be required for
activity. Thus, a novel class of N-substituted matrinic acid deriva-
tives against HBV could be obtained by introducing different
14
O
H
N12
13
D
N
12
COOH
11
11
H
H
5
7
5
7
4
3
4
3
H
H
H
H
N
X
N
X
10
2
10
2
⇑
Corresponding authors. Tel.: +86 10 63188423; fax: +86 10 63017302 (J.D.J.);
3: X=O
4: no X available
1: X=O
tel.: +86 10 63165268; fax: +86 10 63165268 (D.Q.S.).
E-mail addresses: jiang.jdong@163.com (J.-D. Jiang), songdanqingsdq@hotmail.
com (D.-Q. Song).
2: no X available
Figure 1. Structures of the compounds 1À4.
These authors made equal contribution to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.071

N.-N. Du et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4732–4735
4733
Table 1
used as a protective agent and methanol and petroleum ether
(60À90 °C) as the mixture solvent with good yields of 90À95%.12
The intermediate 8 was obtained through the reaction of 7 with
various RX (R = alkyl or acyl) in CH2Cl2 in the presence of anhy-
drous K2CO3. In this procedure, N,N-dimethyl matrinic ester (11)
was acquired, owing to the effect of small methyl and reaction
activity of CH3I.13 Finally, m-cresol was used as a de-protective re-
agent as well as solvent to transform compound 8 or 11 into de-
sired products 9À10 or 12À13. In the course of de-protection of
9g, the m-cresol underwent condensation with intermediate 8,
and gave the product 9k. Similarly, during the preparation of de-
sired N-(2-bromopropionyl) matrinic acid, the same condensation
reaction took place to get the final product 9l.
Twenty-eight synthetic compounds were initially screened for
their regulating activity on Hsc70 mRNA expression using a spe-
cific real time RT-PCR assay in human liver HepG2 cells transfected
with the full genome of HBV (HepG2.2.15).14 Structures of the ana-
logs and their down-regulation in Hsc70 are shown in Table 1.
The SAR analysis was first concentrated on the variations of the
substituents at position 12 of 3 or 4. Attachment of different alkyl
groups including ethyl (9a), n-propyl (9b), cyclopropylmethyl (9c)
or di-methyl (12), all of them lost their activity on Hsc70 mRNA
expression completely. Compound 9d bearing b-hydroxyethyl at
the same position afforded moderate down-regulating expression
in Hsc70, similar to that of the lead 1.
Structures and down-regulating expression on Hsc70 of the N-substituted matrinic
acid derivatives
R1
N
CH3
N+
H
I-
H3C
H
R2
COOH
H
H
H
H
N
X
N
X
3-10
12 and 13
a
Compd
R1
R2
X
Hsc70 mRNA
1
2
3
4
52.0 0.10
52.4 0.17
45.5 0.05
46.2 0.05
<10
39.3 0.10
25.5 1.9
12.2 1.1
<10
H
H
H
H
COOH
COOH
CH2OH
CH2OH
CH2OH
CH2OH
COOH
COOH
O
/
O
/
/
/
5
6a
6b
6c
9a
9b
CH2Ph
CH2PhOCH3-p
CH2 CH3
/
/
CH2CH2CH3
<10
Acyl substituents including aliphatic and aromatic substitutents
were done at the 12-position as well, by which eight compounds
9eÀl were created. The results showed that all of them lost their
activity of Hsc70 down-regulation partially or completely,
regardless of their size of the side chains. Similarly, sulfonyl deriv-
atives 9m and 9n exhibited decreased activity of Hsc70 expression
in comparison with the parent 1. In another variation, benzyl,
p-methoxybenzyl or m-methoxybenzyl substituents on the nitro-
gen atom (compounds 9oÀq) exhibited activity more or similar
to the lead 1. In particular, compound 9p15 bearing p-methoxyben-
zyl afforded the most potent activity in Hsc70 down-regulatory
expression with about 1.3-fold increase over that of 1. We there-
fore deduced that substituted benzyl at the 12-position might be
helpful for the activity.
Next, SAR study was moved on the carboxyl group of compound
3 or 4. The carboxylic acid of compounds 3À4, 9o and 9p was re-
duced to alcohol, the resultant compounds 5 and 6aÀc exhibited
a decreased activity on Hsc70 expression in comparison with its
parent, respectively. Similarly, carboxyl was converted into ester
or amide, respectively, and the resultant products almost lost their
effect of down-regulating in Hsc70 (data not shown). It appeared
that the carboxyl might play an important role in binding to spec-
ulated biological targets.
In addition, the SAR analysis for the oxygen atom at the 1-posi-
tion was carried out as well. Compounds 5, 10c and 13 with oxygen
atom at the 1-position had no activity similar to that of their par-
ents 6a, 9c and 12, respectively. Similarly, the oxygen atom was
introduced into the nitrogen atom of compound 4, 9o or 9p,
respectively, the resultant 3, 10o and 10p also retained their activ-
ity on Hsc70 expression. It is deduced that the oxygen atom at the
1-position is not an essential element in keeping compounds with
potency of Hsc70 down-regulation. The results are basically consis-
tent with our previous SAR analysis.6b
Five compounds 3, 4, 9n, 9o and 9p with moderate down-regu-
lation in Hsc70 mRNA activity were further tested their effect
against HBV in HepG2.2.15 cells with a specific real-time PCR as-
say. As shown in Figure 2, their inhibition rate of intracellular
HBV DNA replication was basically consistent with activity in
Hsc70 down-regulation. Among these analogs, compound 9p
showed the most potent anti-HBV effect, higher than 1 (77% vs
56%). In this test, 3TC was used as a positive reference; therefore,
9c
COOH
/
<10
H2C
10c
COOH
O
<10
H2C
CH2CH2OH
CHO
COCH3
COCH2Cl
COCH2OH
COPh
COCH@CHPh
COCH2OPhCH3-m
COCH(CH3)OPhCH3-m
SO2Ph
SO2PhCH3-p
CH2Ph
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
9o
10o
9p
10p
9q
12
13
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
/
/
/
/
/
/
/
/
/
/
/
/
O
/
O
/
/
O
53.7 0.10
<10
29.8 0.05
<10
<10
33.6 0.11
<10
<10
<10
22.1 0.08
39.8 0.10
55.9 0.13
58.2 0.11
72.1 0.11
69.7 0.13
55.6 0.10
14.1 0.12
<10
CH2Ph
CH2PhOCH3-p
CH2PhOCH3-p
CH2PhOCH3-m
(CH3)2
(CH3)2
a
% of inhibition. Hep2.2.15 cells were cultured in the MEM medium, and incu-
bated respectively with 1 or its analogs (100 g/mL) for 24 h. Down-regulation of
Hsc70 mRNA expression was determined with real-time RT-PCR method. The data
shown were mean SD of three separate experiments for % of the inhibition of
Hsc70 expression.
l
groups into the nitrogen atom at the 12-position of 3 or 4. On the
basis of this strategy, a series of N-substituted matrinic acid ana-
logs were designed, synthesized and evaluated for their regulation
of Hsc70 mRNA expression in the present study.
Twenty-eight N-substituted matrinic acid analogs were semi-
synthesized as described in Scheme 1, that includes two synthetic
routes (Methods A and B) with commercially available 1 or 2 as
starting material. The desired products 3 and 4 were prepared
via hydrolysis of 1 and 2 in aqueous KOH9,10 and then acidification
using dilute HCl with a good yield of 90% and 87%, respectively. The
oxymatrinol (5) and matrinol (6) were obtained through a selective
reduction of 3, 4 or 9 with yields of 30À50%, in which LiAlH4 was
used as the reductive agent and THF as the solvent (Method A).11
The key intermediate 7 was synthesized by the carboxyl protec-
tion of 3 or 4 (Method B), in which diphenyldiazomethane was

4734
N.-N. Du et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4732–4735
O
H
R
H
N
N
H
N
H
OH
COOH
a
b
H
85-90%
30-40%
Method A
H
H
H
H
H
N
N
N
X
X
3 or 4
X
1 or 2
5 or 6
b
45-50%
R
N
H
R
N
H
c
90-95%
COOH
COOCHPh2
e
d
5-35%
H
H
H
H
N
N
X
H
N
X
COOCHPh2
9 or 10
H
8
H
H
CH3
N+
CH3
N+
N
X
7
H3C
H
H3C
I-
I-
e
COOH
COOCHPh2
d
H
H
5-15%
H
H
H
N
X
N
X
Method B
12 or 13
11
R = H, Acyl or Alkyl
X = O or no X available
Scheme 1. Synthesis of the N-substituted matrinic acid derivatives. Reagents and conditions for the chemical synthesis: (a) KOH/H2O, reflux, 9 h; then 3 N HCl; (b) LiAlH4,
THF, reflux, 3 h; (c) diphenyldiazomethane, MeOH/petroleum ether (boiling range 60À90 °C), overnight; (d) RX, K2CO3/CH2Cl2, rt, 1À24 h; (e) m-cresol, 80 °C, 8À9 h.
the figure also hints that the anti-HBV activity of compound 9p is
comparable to that of 3TC.
After overall evaluation, compound 9p showed the best activity
of down-regulating Hsc70 mRNA expression. Therefore, it was se-
lected to measure down-regulating Hsc70 expression by protein
level in HepG2.2.15 cells. As shown in Figure 3, compound 9p
(100
Hsc70 expression by protein level, stronger than that of the lead
1 (100 g/mL) as well. Compound 9p exhibited the best activity
of down-regulating Hsc70 protein at the concentration of 500 g/
lg/mL) showed a significant activity in down-regulating
l
l
mL. It should be mentioned here that as decrease in protein often
occurs after mRNA reduction, the 24 h result for protein reduction
was not as obvious as that at mRNA level.
In conclusion, by using host Hsc70 as a drug target against HBV,
28 novel N-substituted matrinic acid derivatives were semi-
synthesized and evaluated for their biological effect on Hsc70
mRNA down-regulation. The SAR analysis reveals that (i) the
Figure 2. Anti-HBV activity in compounds 1, 3, 4, 9o, 9p, 9q and 3TC in vitro.
HepG2.2.15 cells were untreated or treated with study compounds (100 g/mL) or
l
3TC (30 l
g/mL) as indicated for 24 h. ⁄p <0.05 as compared to that of untreated
control; ⁄⁄p <0.01 as compared to that of untreated control.
Hsc70
Actin
Control
1 (100)
9p (500)
9p (250)
9p (100)
9p (40)
(µg/mL)
Figure 3. The intracellular Hsc70 protein decreased dose-dependently in the HepG2.2.15 cells untreated or treated with 1 (100 lg/mL) and 9p (40, 100, 250 and 500 lg/mL,
respectively) for 24 h.

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This work was supported by the Ph.D. Programs Foundation of
Ministry of Education of China (20101106110035) and the Na-
tional S&T Major Special Project on Major New Drug Innovation
(2009ZX09301-003) and the Student Fund of Innovation Project
of Peking Union Medical College and the IMB Research Foundation.
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15. Analytical data for selected final compound 9p. Mp 78.8À80.7 °C. 1H NMR
(CD3OD, 500 MHz): d 7.39À7.40 (d, J = 8.5 Hz, 2H), 6.95À6.96 (d, J = 8.5 Hz, 2H),
4.66À4.69 (d, J = 12.5 Hz, 1H), 3.91À3.94 (d, J = 12.5 Hz, 1H), 3.74 (s, 3H),
3.36À3.41 (m, 1H), 3.12À3.19 (m, 2H), 2.99À3.02 (m, 1H), 2.82À2.86 (m, 1H),
2.61 (s, 2H), 2.36À2.45 (m, 2H), 2.16À2.22 (m, 2H), 2.03À2.06 (m, 2H),
References and notes
1.57À1.91 (m, 10H), 1.49À1.51 (d, J = 13.0 Hz, 1H); IR:
m 3394 (OH), 2945, 2873
(CH2), 1715 (C@O), 1612, 1515 (C@C), 1062 (CÀO) cmÀ1; HRMS: calcd for
1. (a) Cai, X.; Wang, G. J.; Qu, Y.; Fan, C. H.; Zhang, R. Q.; Xu, W. S. J. Sec. Mil. Med.
Univ. 1997, 18, 47; (b) Chen, Y. X.; Mao, B. Y. J.; Jiang, J. Zhong Guo Zhong Xi Yi Jie
He Za Zhi 2002, 22, 335.
C
23H34N2O3 (M+H)+ 387.2648, found 387.2646.