Biocatalyzed cross-coupling of sinomenine and 1,2-dihydroxybenzene by Coriolus unicolor

Article abstract of DOI:10.1016/j.cclet.2012.01.004

Sinomenine is a clinically available drug for the treatment of rheumatoid arthritis (RA). In a continuous research aiming at discovery of sinomenine derivates with better bioactivity, a cross-coupling reaction of sinomenine and 1,2-dihydroxybenzene catalyzed by a fungus Coriolus unicolor afforded an unique CC cross-coupled compound 2, together with (S)-disinomenine and (R)-disinomenine. The structure of 2 was elucidated by MS and NMR spectroscopy. Compound 2 was further assayed for the inhibitory activity on IL-6 overproduction in SW982 cells and exhibited a much more potent activity on IL-6 (96% inhibition) compared with those of (S)-disinomenine and sinomenine (17% and 12% inhibition, respectively).

Full text of DOI:10.1016/j.cclet.2012.01.004

Available online at www.sciencedirect.com
Chinese Chemical Letters 23 (2012) 321–324
www.elsevier.com/locate/cclet
Biocatalyzed cross-coupling of sinomenine and
1,2-dihydroxybenzene by Coriolus unicolor
b,c
d
Zhang Shuang Deng a, , Dan Zhao , Yi Hu , Jian Xin Li
,
*
b,c,**
Kun Zou a, Jun Zhi Wang a
a Hubei Key Laboratory of Natural Products Research and Development, College of Chemistry and Life Science,
China Three Gorges University, Yichang 443002, China
b Institute of Chemical Biology and Drug Innovation, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
c School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China
d State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing
University of Technology, Nanjing 210009, China
Received 21 September 2011
Available online 30 January 2012
Abstract
Sinomenine is a clinically available drug for the treatment of rheumatoid arthritis (RA). In a continuous research aiming at
discovery of sinomenine derivates with better bioactivity, a cross-coupling reaction of sinomenine and 1,2-dihydroxybenzene
catalyzed by a fungus Coriolus unicolor afforded an unique C–C cross-coupled compound 2, together with (S)-disinomenine and
(R)-disinomenine. The structure of 2 was elucidated by MS and NMR spectroscopy. Compound 2 was further assayed for the
inhibitory activity on IL-6 overproduction in SW982 cells and exhibited a much more potent activity on IL-6 (96% inhibition)
compared with those of (S)-disinomenine and sinomenine (17% and 12% inhibition, respectively).
# 2012 Zhang Shuang Deng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Sinomenine; 1,2-Dihydroxybenzene; Cross-coupling; Coriolus unicolor; IL-6
Sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-17-methylmorphinan-6-one, 1), a morphine-type alkaloid
isolated from the stems of a medicinal plant Sinomenium autumn (Thumb) Rehd. et Wils, has been clinically available
for the treatment of rheumatoid arthritis (RA) in China [1]. However, due to its weak efficacy [2], unstable molecular
structure under acidic, basic or high temperature conditions [3–5], and some adverse gastrointestinal effects [6], the
usage of sinomenine was quite limited. In order to overcome these defects, chemical modifications on sinomenine
were extensively performed [7], however, only a few improvements were achieved. Microbial transformation as an
effective tool for the structure modification of natural products has been widely used in medicinal chemistry owing to
its convenient operation, metabolite diversity and economical availability [8–10]. In our previous study, we have
demonstrated that a fungus Antrodiella semisupina was capable of converting sinomenine and guaiacol to produce a
* Corresponding author.
** Corresponding author at: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210093, China.
E-mail addresses: d.zhangshuang@gmail.com (Z.S. Deng), lijxnju@nju.edu.cn (J.X. Li).
1001-8417/$ – see front matter # 2012 Zhang Shuang Deng. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2012.01.004

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Z.S. Deng et al. / Chinese Chemical Letters 23 (2012) 321–324
Fig. 1. Cross-coupling of sinomenine and 1,2-dihydroxybenzene catalyzed by Coriolus unicolor.
C–C cross-coupled compound that exhibited more potent inhibitory activity on IL-1b-induced overproduction of IL-6
in human synovial sarcoma cell SW982 culture compared with sinomenine [11]. In this paper, as a successive study on
biocatalyzed cross-coupling of sinomenine and hydroxybenzene type molecules aiming at discovery of sinomenine
derivates with more potent bioactivity, we report a novel compound 2 catalyzed by Coriolus unicolor via linkage of
sinomenine and 1,2-dihydroxybenzene at C-1 position (Fig. 1) and its inhibitory on IL-6 production.
1. Results and discussion
Microbial transformation of sinomenine (1200 mg) and 1,2-dihydroxybenzene (350 mg) catalyzed by C. unicolor
was performed in 200 mL of potassium phosphate buffer (50 mmol/L) with 60 g wet mycelia on a rotary shaker at
160 rpm at 25 8C. After 24 h of incubation, the products were separated with silica gel chromatography to give (S)-
disinomenine (3, 500 mg, 42% yield based on weight relative to sinomenine), (R)-disinomenine (4, 195 mg, 16%
yield) and a mixed fraction (310 mg). The further purification of this fraction by HPTLC with EtOAc–CH3OH–
NH3ꢀH2O (17:2:1, v/v/v) as developing solution afforded compound 2 (221 mg) in 18% yield. The structures of (S)-
disinomenine (3) and (R)-disinomenine (4) were determined by comparison chemophysical data with those of
authentic compounds that elucidated by X-ray analysis in our previous studies [12].
25
Compound 2 was obtained as a white powder and showed mp 222–225 8C and ½aꢁD +21.8 (c 0.4, CH3OH). The
positive ion ESI-MS exhibited an ion peak at m/z [M+H]+ 438.1946 (calcd. for C25H28NO6 438.1917) indicating a
109 amu mass increase compared with that of sinomenine, which was equal to molecular weight of 1,2-
dihydroxybenzene moiety. Based on 1H NMR spectral data (Table 1), three new proton signals displayed at d 6.66 (s,
1H), 6.53 (dd, 1H, J = 8.0) and 6.86 (dd, 1H, J = 8.0) and the other signals were quite similar to those of sinomenine
except that only one aromatic proton signal was observed. These spectral data suggested that a single substitution at
aromatic ring of sinomenine was occurred. In the 13C NMR spectrum (Table 1), besides 19 carbon signals of
sinomenine, six additional carbon signals at d 115.1, 116.1, 120.8, 134.0, 144.3 and 145.0 were exhibited. The data
above revealed that a set of 1,2,4-trisubstituted phenyl group assignable to 1,2-dihydroxybenzene moiety was
connected with sinomenine, which was further supported by detailed analysis of 1H–1H COSY and HMQC spectral
data. In order to determine the connection position of two moieties, a HMBC spectrum was measured. Evidently, long-
range correlations between C-40 and H-2, C-1 and H-50 confirmed that C-40 of 1,2-dihydroxybenzene attached C-1 of
sinomenine to form a C–C bond. In view of all evidences, the structure of compound 2 was elucidated as shown in
Fig. 1.

Z.S. Deng et al. / Chinese Chemical Letters 23 (2012) 321–324
323
Table 1
1H and 13C NMR spectral data (d in ppm, J in Hz) of compound 2.a
No.
dH
dC
No.
dH
dC
No.
16
dH
dC
1
132.3
111.5
143.8
55.0
8
9
5.48 (s)
114.6
57.0
23.8
2.23 (td, 11.4, 3.0)
2.74 (overlapped)
2.40 (s)
47.5
2
6.58 (s)
3.79 (s)
3.26–3.19 (overlapped)
2.77 (brd, 19.0)
2.63 (dd, 19.0, 5.2)
3
10
17
10
20
30
40
50
60
42.1
144.3
145.0
116.1
134.0
120.8
115.1
3-OCH3
4
5
143.6
48.5
11
12
13
14
15
126.1
121.5
40.4
2.55 (d, 15.6)
4.42 (d, 15.6)
6.66 (s)
6
194.5
152.4
56.0
3.12 (brs)
44.2
6.53 (d, 8.0)
6.86 (d, 8.0)
7
1.92–2.10 (overlapped)
1.92–2.10 (overlapped)
34.6
7-OCH3
3.52 (s)
a
Recorded in chloroform-d/methanol-d4 (5:1, v/v), 300 MHz.
The inhibitory activity of all the compounds on IL-1b-induced overproduction of IL-6 was evaluated in SW982
cells. As shown in Fig. 2, the same as our previous results [12], (S)-disinomenine displayed more potent IL-6 inhibitory
activity than sinomenine, while (R)-disinomenine stimulated the IL-6 production. Excitedly, compound 2 exhibited
much more potent inhibitory activity compared with that of (S)-disinomenine and sinomenine at all three
concentrations and the inhibitory rates of 2 were 83%, 95% and 96% at 50 mmol/L, 100 mmol/L and 200 mmol/L,
respectively.
The formation of C–C bond in modification of natural products is one of the key steps to generate derivates and
pivotal intermediates with potential bioactivity. In our previous study, we discovered that the fungus Antrodiella
semisupina could catalyze dimerization of sinomenine to form (S)-disinomenine stereoselectively and C–C cross-
coupling of sinomenine and guaiacol to give jointed product at C-1 position. As a successive study to explore
biocatalyzed cross-coupling reactions, sinomenine and another hydroxybenzene substrate, 1,2-dihydroxybenzene,
was subjected to same protocol using A. semisupina. However, no desired C–C cross coupled product was obtained.
Further screening resulted in a fungus C. unicolor that was capable to carry out C–C cross-coupling of sinomenine and
1,2-dihydroxybenzene to yield target compound 2. The present result provided another successful example of
biocatalyzed cross-coupling reaction. Moreover, the compound 2 showed a potent IL-6 inhibitory activity in vitro.
Microbial transformation of natural products is a powerful tool to produce novel drug candidates and key
intermediates that are difficult to be synthesized chemically, and has been widely used in pharmaceutical industry [14].
To our knowledge, bioconversion of natural products is generally limited to a single substrate. In the present study,
introduction of a second substrate, 1,2-dihydroxybenzene, in the culture system, sinomenine was modified through
linkage with second substrate at C-1 position. The possible mechanism of the formation of the C–C bond might be via
oxygen radicals that generated by oxidation of at phenyl OH groups in 1 and 2, and then the oxygen radicals were
delocalized to form carbon-located radicals, subsequently, coupling of these radicals produced C–C metabolites. This
hypothesis was supported by the fact that when the phenyl OH groups were protected, no targeted metabolites were
yielded. It is reported that in a culture system, a small molecule could act as an enzyme inducer and increase its
Fig. 2. Inhibitory effects of sinomenine, (S)-disinomenine, (R)-disinomenine and compound 2 on IL-6 production in SW982 cells. SW982 cells were
cultured for 2 days with 1 ng/mL of IL-1b [13]. The cells in control group were cultured without compounds. IL-6 concentrations in culture
supernatants were measured by ELISA kit. Each value represents the mean ꢂ S.D. from triplicate cultures. All compounds showed no significant
inhibitory effects on cell growth or morphology at 200 mmol/L.

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Z.S. Deng et al. / Chinese Chemical Letters 23 (2012) 321–324
metabolic activity to generate novel metabolites. It could also be as an enzyme inhibitor and block some metabolic
pathways to enhance the production of major metabolite, while the small molecule was not involved in molecular
assemble during the whole culture process [15]. In the current biotransformation of multi-substrates, clarifications of
which kinds of functions that 1,2-dihydroxybenzene acts as and what kinds of enzymes involve in the cross coupling
reaction are undergoing in our lab.
Acknowledgments
This work was supported by National Natural Science Foundation of China (Nos. 20872060, 20936002 and
31100080) and the Fundamental Research Funds for Central University (No. 1082020502). We are grateful to Dr. Yin
Ding (Nanjing University, China) for HRMS measurements.
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