Synthesis and in vitro inhibitory activity of matrine derivatives towards pro-inflammatory cytokines

Article abstract of DOI:10.1016/j.bmcl.2010.09.075

Matrine, a sophora alkaloid, exhibited good anti-inflammation effects in our previous report. In the present study, a series of matrine derivatives were synthesized via classical Michael addition. Biological studies showed that the synthetic derivatives h

Full text of DOI:10.1016/j.bmcl.2010.09.075

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7537–7539
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro inhibitory activity of matrine derivatives towards
pro-inflammatory cytokines
Honggang Hu a, , Shaozhan Wang b, , Chunmei Zhang a, Liang Wang c, Li Ding b, Junping Zhang b,
,
⇑
Qiuye Wu a,
⇑
a Department of Organic Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, People’s Republic of China
b Department of Biochemical Pharmacy, School of Pharmacy, Second Military Medical University, Shanghai 200433, People’s Republic of China
c School of Pharmacy, Yantai University, Yantai 264005, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2010
Revised 2 September 2010
Accepted 14 September 2010
Available online 17 September 2010
Matrine, a sophora alkaloid, exhibited good anti-inflammation effects in our previous report. In the pres-
ent study, a series of matrine derivatives were synthesized via classical Michael addition. Biological stud-
ies showed that the synthetic derivatives had good inhibitory effect towards TNF-a production and NFjB
transcriptional activity. The introduction of various amino groups to the keto beta position could improve
the biochemical profile, resulting in the identification of more potent derivates, such as 1f, with higher
inhibitory activity than both matrine and sophoramine.
Keywords:
Matrine
Ó 2010 Published by Elsevier Ltd.
Pro-inflammatory cytokine
NFjB
Derivatives
SAR
Sophora alkaloids, isolated from Sophora flavescens Ait. (Kushen),
on TNF-a production and NFjB transcriptional activity in
have been found to possess a variety of pharmacological effects,
including anti-inflammation and immunity-regulation activity.1–5
The main active components are matrine, sophocarpine, and oxy-
matrine, whose structures are shown in Figure 1.6 Our previous
studies7–9 have found that matrine significantly inhibited the pro-
RAW264.7 cells were evaluated to investigate the structure–
activity relationship.
The synthesis of 1a–j, as shown in Scheme 1, started from soph-
ocarpine 2. First, 2 was transformed into 3 in high yield by treat-
ment with Lawesson’s reagent.12 The product was characterized
by 1H NMR, 13C NMR and MS. Then, reaction of 3 with different
amines afforded the target compounds 1a–j in almost quantitative
yields.13 All the derivatives were identified by 1H NMR, 13C NMR,
and MS.14
Furthermore, to verify the influence of the sulfur atom on
biological activity of the derivatives, compound 5 was synthesized
in two steps according to Tinarelli’s method.15 As shown in
Scheme 2, treating 2 with azidotrimethylsilane, AcOH, and DBU
duction of pro-inflammatory cytokines (TNF-a, IL-1, or IL-6) in lipo-
polysaccharide (LPS) stimulated murine peritoneal macrophages
and Kupffer cells as well as in several animal inflammation models.
Other groups also reported that matrine and oxymatrine exerted
anti-inflammation effects through inhibiting nuclear factor-kappa
B (NFj
B) activation.10,11
Considering the good pharmacological effects of matrine, we are
interested in developing a general and practical strategy for the
preparation of matrine derivates for structure–activity relationship
studies and for the exploration of new anti-inflammation drugs. To
explore the synthetic strategy, sophocarpine is a suitable starting
material because it contains an
a,b-unsaturated carbonyl group
that is reactive toward a variety of useful nucleophiles. Therefore,
in the present study, matrine derivatives 1a–j and 5 were designed
and synthesized by using sophocarpine (Fig. 2), and their influence
⇑
Corresponding authors. Tel./fax: +86 21 8187 1225 (Q.W.); tel./fax: +86 21 8187
1328 (J.Z.).
E-mail addresses: jpzhang08@hotmail.com (J. Zhang), wuqysmmu@sohu.com (Q.
Wu).
Honggang Hu and Shaozhan Wang contributed equally to this work.
Figure 1. Structures of sophora alkaloids.
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.09.075

7538
H. Hu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7537–7539
Table 1
Effects of sophora alkaloids and derivatives on TNF-
a production by LPS-stimulated
RAW264.7 cells
Treatment
TNF activity
M)
Inhibition (%, 10
l
IC50 (lM)
Matrine
Sophoramine
5
—
—
—
9.1
41.5
9.6
23.99
16.17
52.5
33.9
29.3
22.8
8.4
>200
>200
>200
93.2
23.5
93.5
52.3
119.4
9.4
62.0
39.2
53.8
57.5
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
Figure 2. Target matrine derivatives.
Compound 1f showed the strongest inhibitory effect with an IC50
value of 9.4 M. It was clear that introduction of small substituents
l
to the amine group could improve the biochemical profile. More-
over, the sulfur atom had good contribution to the inhibitory effect.
The mis-regulation of the nuclear factor-kappa B (NFjB) signal
pathway is involved in a variety of inflammatory diseases that
leads to the production of inflammatory mediators.17 To further
determine the possible molecular mechanisms involved in the
action of sophora alkaloids on TNF-
a
production, we assessed the
effects of sophora alkaloids on NF
jB transcriptional activity in
LPS-stimulated RAW264.7 cells using a reporter gene assay.18
RAW264.7 cells (2 Â 105) co-transfected for 24 h with the mixture
Scheme 1. Synthesis of 1a–j. Reagents and conditions: (a) Lawesson’s reagent,
toluene, reflux, 2 h, in 85% yield; (b) RNH2, Et3N, CH2Cl2–MeOH (1:1), rt, overnight.
of pGL3.5XjB-luciferase, and pRL-TK-Renilla-luciferase were pre-
treated with sophora alkaloids (100
lM) for 30 min and then stim-
ulated with LPS (1 g/ml) for 6 h. NF
l
j
B luciferase activities were
in anhydrous toluene gave 4 in good yield, which was followed by
reduction of the azide group by catalytic hydrogenation to afford 5
in excellent yield (95%). Both 4 and 5 were characterized by 1H
NMR, 13C NMR, and MS.16
measured using the Dual-Luciferase Reporter Assay System (Pro-
mega) according to the manufacturer’s instructions. Data are nor-
malized for transfection efficiency by dividing firefly luciferase
activity with that of Renilla luciferase. As shown in Table 2, sopho-
The comparison of inhibitory effects of sophora alkaloids and
ra alkaloids significantly inhibited NFjB transcriptional activity.
compounds 1a–j on TNF-a production by LPS-stimulated macro-
The inhibitory effects of the derivatives with liner substituents
on amine group were better than those with branched and ring
substituents. Compound 1f which contained an allylamino group
was identified as the most potent inhibitor.
In summary, a highly efficient and versatile synthetic method
was developed for the synthesis of matrine derivatives. Biological
studies of the synthetic compounds suggest that introducing small
phages were determined by the method described in our previous
report.9 RAW264.7 cells (5 Â 105 cells/ml) were incubated with LPS
(Sigma, St. Louis, MO, USA) (1 lg/ml) for 6 h in the presence or ab-
sence of matrine, sophocarpine, 5, and 1a–j. The culture superna-
tants were collected, and the concentrations of TNF- were
a
measured by a commercially available ELISA kit (eBioscience, Cal-
ifornia, CA, USA). Data was analyzed by logarithm fit to generate a
dose–response curve using CurveExpert 1.3 Software (Daniel
J. Hyams, Hixson, TN). The calculated IC50 value is the concentra-
tion of the test compound that caused a 50% decrease in the max-
Table 2
Effects of sophora alkaloids on NF
RAW264.7 cells
jB transcriptional activity in LPS-stimulated
imal TNF-
From the results, TNF-
cells were significantly inhibited by matrine derivatives in a con-
centration-dependent manner. These inhibitory effects were not
attributed to their unspecific cytotoxic effects (data not shown).
a
production. The results are summarized in Table 1.
production by LPS-stimulated RAW264.7
Treatment
Relative NF
jB activity (NFjB/TK, fold)
a
Control
LPS
Matrine
Sophoramine
1.18 0.37
11.26 2.52**
5.11 1.24##
3.47 0.47##
6.54 0.06##
3.69 1.07##
1.96 0.53##
3.40 0.16##
6.56 0.47##
7.83 1.38##
1.47 0.33##
8.39 3.89##
4.85 1.97##
3.14 1.90##
4.75 1.23##
5
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
N = 3.
**
P <0.01 versus control.
P <0.01 versus LPS.
Scheme 2. Synthesis of 5. Reagents and conditions: (a) Me3SiN3, PhMe, AcOH, DBU,
0 °C to rt, 24 h, in 95% yield; (b) H2, 10% Pd/C, MeOH, rt, 2 h, in 98% yield.
##

H. Hu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7537–7539
7539
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on its anti-inflammatory activity. This discovery, as well as further
studies on the structures and activities of matrine derivatives
which should be significantly facilitated by the method presented
here, can be generally useful for understanding the functions of
substituent replacements in matrine and for the development of
new anti-inflammation agents.
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Acknowledgment
14. Representative analytical data for compound 1a: yield 92.3%, 1H NMR (600 MHz,
CDCl3, TMS): d 5.46 (dd, 1H, J = 12.0 Hz, 3.6 Hz), 4.29 (m, 1H), 3.55 (t, 1H,
J = 12.6 Hz), 3.26 (m, 1H), 3.00 (m, 1H), 2.89–2.78 (m, 3H), 2.46 (s, 3H), 2.18 (m,
1H), 2.08–1.86 (m, 7H), 1.80–1.68 (m, 2H), 1.67–1.52 (m, 3H), 1.51–1.39 (m,
3H). 13C NMR (150 MHz, CDCl3) d 195.42, 63.44, 56.86, 56.84, 55.42, 50.74,
49.30, 47.79, 42.62, 35.52, 33.52, 30.31, 27.54, 26.44, 21.02, 20.49. HR-Q-TOF-
MS, calcd for C16H28N3S+, M+H+, 294.1998; found, 294.2003.
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This work was supported by a grant from National Nature Sci-
ence Foundation of China (No. 30783087).
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J = 12.0 Hz), 2.80–2.70 (m, 2H), 2.62–2.57 (m, 1H), 2.40–2.35 (m, 1H), 2.15–
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