Biocatalyzed cross-coupling of sinomenine and guaiacol by Antrodiella semisupina

Article abstract of DOI:10.1021/ol800024u

In search of more potent derivatives of sinomenine (1), a clinically available natural alkaloid for the treatment of rheumatoid arthritis (RA), biocatalyzed cross-coupling of sinomenine and guaiacol (2) by Antrodiella semisupina, provided two unique C-C coupled (3 and 4) and one C-0 linked (5) novel metabolites. The structures of the metabolites were elucidated by means of MS, 2D NMR techniques and X-ray analysis. 4 exhibited more potent inhibitory activity on IL-6 production than 1 in human synovial sarcoma cell (SW982), and 5 stimulated IL-6 production.

Full text of DOI:10.1021/ol800024u

ORGANIC
LETTERS
2008
Vol. 10, No. 6
1119-1122
Biocatalyzed Cross-Coupling of
Sinomenine and Guaiacol by Antrodiella
semisupina
Zhang-Shuang Deng,† Jian-Xin Li,*,† Peng Teng,† Peng Li,† and Xiao-Ru Sun‡
Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical
Engineering, Nanjing UniVersity, Nanjing, 210093, P. R. China and Medical School,
Nanjing UniVersity, Nanjing, 210093, P. R. China
lijxnju@nju.edu.cn
Received January 5, 2008
ABSTRACT
In search of more potent derivatives of sinomenine (1), a clinically available natural alkaloid for the treatment of rheumatoid arthritis (RA),
biocatalyzed cross-coupling of sinomenine and guaiacol (2) by Antrodiella semisupina, provided two unique C C coupled (3 and 4) and one
O linked (5) novel metabolites. The structures of the metabolites were elucidated by means of MS, 2D NMR techniques and X-ray analysis.
4 exhibited more potent inhibitory activity on IL-6 production than 1 in human synovial sarcoma cell (SW982), and 5 stimulated IL-6 production.
−
C
−
Natural products continue to provide pharmaceutics with a
plethora of valuable candidates for drug discovery. Micro-
organisms and their enzymes have been proven to possess
versatile biocatalysts and are widely believed to be a
powerful tool not only for generation of new, active, and
less toxic derivatives from bioactive natural products but also
for effective synthesis of intermediates of new or improved
drugs.1,2 Sinomenine (7,8-didehydro-4-hydroxy-3,7-dimethoxy-
17-methylmorphinan-6-one, 1), a natural morphine-type
alkaloid, was isolated from the Chinese medicinal plant
Sinomenium acutum (Thunb.) Rehd. et Wils. with a high
content.3,4 It has been used for the treatment of rheumatic
disease including rheumatoid arthritis (RA) for a long time
in clinic,5 and a variety of bioactivities, such as anti-
inflammation, immuno-suppression, antiarthritis, and car-
dioprotection have also been reported.6-9 Furthermore, a
number of sinomenine derivatives have been chemically
synthesized for anti-inflammatory purposes.10
Our previous work has been focused on structural modi-
fication by a chemical method.11 As a part of a research
project to explore new and more bioactive derivatives from
sinomenine using microbial transformation for drug discov-
ery, we screened numbers of fungi and discovered 3 strains
(5) Liu, L.; Resch, K.; Kaever, V. Int. J. Immunopharmacol. 1994, 16,
685-691.
(6) Li, X. J.; Yue, P. Y. K.; Ha, W. Y.; Wong, D. Y. L.; Tin, M. M. Y.;
Wang, P. X.; Wong, R. N. S.; Liu, L. Life Sci. 2006, 79, 665-673.
(7) Yao, Y. M.; Tan, Z. R.; Hu, Z. Y.; Guo, X.; Cheng, Z. N.; Wang, L.
S.; Zhou, H. H. Clin. Chim. Acta 2005, 356, 212-217.
(8) Wang, Y.; Fang, Y. F.; Huang, W. H.; Zhou, X.; Wang, M. H.; Zhong,
B.; Peng, D. Z. J. Ethnopharmacol. 2005, 98, 37-43.
(9) Jiang, X. J.; Shi, E.; Nakajima, Y.; Sato, S. Life Sci. 2006, 78, 2543-
2549.
† School of Chemistry and Chemical Engineering.
‡ Medical School.
(1) Zhang, Q. B.; Rich, J. O.; Cotterill, I. C.; Pantaleone, D. P.; Michels,
P. C. J. Am. Chem. Soc. 2005, 127, 7286-7287.
(2) Cheng, Z. H.; Yu, B. Y.; Cordell, G. A.; Qiu, S. X. Org. Lett. 2004,
6, 3163-3165.
(3) Bao, G. H.; Qin, G. W.; Wang, R.; Tong, X. C. J. Nat. Prod. 2005,
68, 1128-1130.
(4) Liu, B.; Jiang, H. L.; Shen, B.; Chang, Y. L. J. Chromatogr. A 2005,
1075, 213-215.
(10) Ye, X. R.; Yan, K. X.; Wu, K. M.; Feng, X. Z.; Huang, Y. M.;
Chou, P. Acta Pharm. Sinica. 2004, 39, 180-183.
(11) (a) Pan, Y.; Li, Y. F.; Bu, Q. M.; Huang, L. Q.; Wang, J.; Li, J. X.
CN Patent, 1785976A, 2006. (b) Pan, Y.; Li, Y. F.; Bu, Q. M.; Huang,
L. Q.; Wang, J.; Li, J. X. CN Patent, 1785977A, 2006. (c) Pan, Y.; Li, Y.
F.; Bu, Q. M.; Huang, L. Q.; Wang, J.; Li, J. X. CN Patent, 1962638A,
2006.
10.1021/ol800024u CCC: $40.75
© 2008 American Chemical Society
Published on Web 02/20/2008

to produce chemically, sinomenine (1) and guaiacol (2-meth-
oxyphenol, 2) were subjected synchronously to A. semisu-
pina, and three novel compounds including two C-C cou-
pled metabolites were obtained. This is the first report that
biocatalyzed coupling on multi-substrates has been con-
ducted.
Scheme 1. Cross-Coupling of Sinomenine 1 and Guaiacol 2
Biotransformed by Antrodiella semisupina
When 1 was incubated with A. semisupina under liquid
conditions for 96 h, a single product 3 with about 20% yield
based on the starting material (1) was obtained. This result
revealed that A. semisupina possessed a high capacity of
microbial transformation and provided an encouraging clue
to further introduce a small molecule into the sinomenine
skeleton. In our previous research, introduction of a bioactive
molecule with anti-inflammatory effect at N-atom of the
sinomenine skeleton provided a great enhancement of the
activity.11 Guaiacol (2) has been widely used for medicinal
practice as an anti-inflammatory and antibacterial agent;13,14
therefore, it was incorporated into the research. As expected,
coincubation of 1 and 2 with A. semisupina provided two
C-C coupled metabolites (3, 20% yield and 4, 13% yield)
and a C-O linked one (5, 5% yield) (Scheme 1).
that are capable of converting sinomenine to metabolites,
one of them, Antrodiella semisupina (Berk. and Curt.) Ryv.,
showed the highest transformation capacity.
There are several reports on peroxidase-catalyzed aryl
coupling.12 To the best of our knowledge, no work has been
performed on two or more substrates. In this paper, aimed
at the discovery of novel structural products that are difficult
Compound 3 was obtained as a colorless hexahedral crystal
(CHCl3/CH3OH/EtOAc ) 80:15:5), mp. 220-222 °C, and
showed [R]25 ) +12.9° (c ) 0.45, CH3OH). EI-MS and
D
Table 1. 1H NMR and 13C NMR Spectral Data (δ in ppm, J in Hz) of Compounds 3-5a
3
4
5
nob
H
C
H
C
H
C
1
2
3
130.7
110.7
144.8
56.0
131.8
111.0
144.5
56.0
144.1
103.1
147.5
56.1
6.27 (s)
3.75 (s)
6.56 (s)
3.80 (s)
6.44 (s)
3.73 (s)
3-OCH3
4
5
143.8
49.3
143.8
49.1
141.7
49.0
2.49 (d, 15.6)
4.41 (d, 15.6)
2.49 (d, 15.6)
4.42 (d, 15.6)
2.47 (d, 15.7)
4.37 (d, 15.7)
6
7
193.8
152.5
54.8
194.2
152.3
54.9
193.7
152.4
54.8
7-OCH3
3.51 (s)
3.52 (s)
3.49 (s)
8
9
10
5.43 (d, 2.4)
3.08 (brt, 4.0)
2.42 (dd, 18.6, 4.0)
2.32 (brd, 18.6)
115.0
56.4
23.9
5.46 (d, 1.9)
3.12 (brt, 4.0)
2.75 (brd, 18.6)
115.2
56.6
23.4
5.39 (d, 1.8)
3.19 (brt, 5.8)
3.00 (d, 18.5)
114.8
55.8
18.5
2.41 (dd, 18.6, 4.0)
2.36 (dd, 18.5, 5.8)
11
12
13
14
15
127.8
123.3
40.9
45.8
35.9
127.7
122.5
40.8
45.6
35.8
122.5
123.4
40.4
45.6
35.6
2.99 (brs)
3.01 (dd, 4.0, 1.9)
2.02 (dt, 12.4, 3.2)
1.89 (td, 12.4, 4.4)
2.10 (td, 12.2, 3.2)
2.57 (ddd, 12.2, 4.4, 1.5)
2.31 (s)
3.03 (brd, 5.8)
2.02 (dt, 12.2, 3.4)
1.91 (td, 12.2, 4.3,)
2.16 (td, 12.0, 3.4)
2.57 (ddd, 12.0, 4.3, 2.1)
2.35 (s)
2.00 (dt, 12.4, 3.4)
1.93 (td, 12.4, 4.4)
2.14 (td, 12.0, 3.4)
2.58 (ddd, 12.0, 4.4, 1.8)
2.36 (s)
16
47.2
43.0
47.4
47.0
17
1′
2′
43.8
144.6
146.3
55.9
111.7
134.3
121.9
114.3
42.7
147.5
149.1
56.0
112.2
122.3
120.7
114.6
2′-OCH3
3.89 (s)
6.68 (s)
3.95 (s)
3′
4′
5′
6′
7.00-6.96 (overlapped)
7.00-6.96 (overlapped)
6.75 (m)
6.67 (dd, 8.4, 1.8)
6.94 (d, 8.4)
6.36 (dd, 8.5, 1.1)
a Recorded in Chloroform-d. b δ of 1′-17′ of compound 3 are the same as δ of 1-17, respectively.
1120
Org. Lett., Vol. 10, No. 6, 2008

high resolution (HR) EI-MS exhibited an ion peak at m/z
656 [M]+ and 656.3099 (calcd for 656.3098), indicating the
molecular formula was C38H44N2O8. Extensive analyses of
1H and 13C NMR spectra (Table 1) together with the use of
1H-1H COSY and HMQC data of 3 revealed that the signal
patterns of 3 were quite similar to those of 1 except that
only one aromatic proton signal was observed instead of the
characteristic AB pair of 1. However, the even molecular
weight of 656 implied the presence of an even number of
nitrogen atoms. These results suggested 3 might be a sym-
metrical dimer of 1, in which the connection position should
be at the aromatic ring. Further HMBC experiment confirmed
the substitution point was at C-1. Thus, the planar structure
of 3 was deduced; hereto, the stereochemistry of 3 was still
unclear. Unfortunately, the NOESY and CD data could not
provide any available clues. In addition, several papers have
reported the disinomenine, but no reliable information on
its exact structure, especially stereo-structure, was provided.15
Therefore, much effort was made to successfully produce
fine crystals to determine the structure directly by an X-ray
crystallographic analysis. Fortunately, the result clearly
confirmed the structure of 3 including stereo-structure (Figure
1), a dimer of 1 as shown in Scheme 1.
its molecular formula was determined to be C26H29NO6 [(M)+
451.1971, calcd for 451.1995] in HR-ESI-MS, which meant
the existence of an odd number of nitrogen atoms. The H
1
NMR spectrum clearly showed signals including a single
aromatic proton as 3 from sinomenine skeleton (Table 1).
Moreover, a set of 1,3,4-trisubstituted phenyl group and a
methoxyl group assignable to guaiacol were observed.
1
Detailed analyses of H-1H COSY and HMQC spectra
suggested that 4 should be composed of sinomenine and
guaiacol moieties. To determine the connectivity of the two
moieties, HMBC was measured. Clear long-range correlation
between C-4′ and H-2, C-1 and H-5′ proposed that C-4′ of
guaiacol attached to C-1 of sinomenine via a C-C bond.
From forenamed findings, the structure of 4 was deduced as
shown in Scheme 1.
Compound 5 was isolated as a slightly yellow powder
with a molecular ion peak at m/z 451.1962 [M]+ (calcd for
C26H29NO6, 451.1995) in HR-ESI-MS spectrum and [R]25
D
) +1.8° (c ) 0.45, CH3OH). Detailed analysis of 1H NMR
1
spectrum with H-1H COSY and HMQC revealed that 5
possessed the same sinomenine moiety as 4; however,
aromatic protons assignable to guaiacol moiety displayed 1,2-
disubstutited pattern which is the same as those of guaiacol.
Moreover, in 13C NMR spectrum of 5 together with analysis
of HMBC spectrum, C-1 (δ 144.1) displayed a marked
downfield shift compared with that (δ 131.8) of 4. This
evidence led us to suppose that sinomenine and guaiacol
moieties were connected via a ether bond, which was further
supported by the result of acetylation of 5, a monoacetate
of OH at C-4 was obtained. On the basis of these data,
compound 5 was elucidated as shown.
The possible mechanism in the formation of C-C bonds
to give unique structures is of great interest. From the
structures of compounds 3-5, most probably, oxidation of
1 or 2 generated oxygen radicals at OH group, and then the
oxygen radicals were delocalized to form carbon-located
radicals. Subsequent coupling of these radicals produced
C-C or C-O metabolites. It is reasonable that some
enzymes presented in A. semisupina might catalyze this type
of reaction.12 To verify the role of the OH group in the
formation of the metabolites, 4-acetyl sinomenine was
subjected to biotransformation followed the same protocol.
The result showed that no 3, 4, and 5 were detected by high-
performance thin layer chromatography (HPTLC) analysis.
This experiment strongly suggested that the 4-OH is a crucial
group during the formation the metabolites. However, as
mentioned previously,15 sinomenine could be oxidized by
silver nitrate to form two 1,1′-disinomenines (ratio ) 1:1)
although the structures were not determined. Our experiments
revealed that the MnO2 catalyzed reaction of 1 and 2
Figure 1. X-ray crystal structure of the compound 3 showing the
crystallographic numbering.
Compound 4 was obtained as a white powder, showed
mp. 151-153 °C, [R]25 ) +14.5° (c ) 0.4, CH3OH), and
D
(12) (a) Stabler, P. J.; Bruce, N. C. Appl. EnViron. Microbiol. 1998, 64,
4106-4108. (b) Stabler, P. J.; Holt, P. J.; Bruce, N. C. Appl. EnViron.
Microbiol. 2001, 67, 3716-3719. (c) Pezzella, A.; Lista, L.; Napolitano,
A.; d’Ischia, M. Chem. Res. Toxicol. 2005, 18, 1413-1419. (d) Pezzella,
A.; Lista, L.; Napolitano, A.; d’Ischia, M. J. Org. Chem. 2004, 69, 5652-
5659. (e) Aoyagi, N.; Ogawa, N.; Izumi, T. Tetrahedron Lett. 2006, 47,
4797-4801. (f) Nicotra, S.; Intra, A.; Ottolina, G.; Riva, S.; Danieli, B.
Tetrahedron: Asymmetry 2004, 15, 2927-2931. (g) Takemoto, M.; Suzuki,
Y.; Tanaka, K. Tetrahedron Lett. 2002, 43, 8499-8501. (h) Sridhar, M.;
Vadivel, S. K.; Bhalerao, U. T. Tetrahedron Lett. 1997, 38, 5695-5696.
(i) Tonami, H.; Uyama, H.; Nagahata, R.; Kobayashi, S. Chem. Lett. 2004,
33, 796-797. (j) Kobayashi, A.; Koguchi, Y.; Kanzaki, H.; Kajiyama, S.
I.; Kawazu, K. Biosci. Biotech. Biochem. 1994, 58, 133-134.
(14) He, W. Y.; Yao, X. J.; Liu, P. J. Sci. China, Ser. B: Chem. 2007,
37, 54-63.
(15) (a) Goto, K.; Sudzuki, H. Bull. Chem. Soc. Jpn. 1929, 4, 107-111.
(b) Goto, K. Bull. Chem. Soc. Jpn. 1929, 4, 129-132. (c) Goto, K.; Mitsui,
S. Bull. Chem. Soc. Jpn. 1931, 6, 33-39. (d) Goto, K.; Shishido, H. Bull.
Chem. Soc. Jpn. 1931, 6, 79-87. (e) Goto, K.; Yamamoto, I.; Matsumoto,
S. Proc. Jpn. Acad. 1954, 30, 883-886. (f) Okamoto, Y.; Yuge, E.; Nagai,
Y.; Katsuta, R.; Kishimoto, A.; Kobayashi, Y.; Kikuchi, T.; Tomita, M.
Tetrahedron Lett. 1969, 24, 1933-1935. (g) Minamikawa, J.; Iijima, I.;
Brossi, A. Heterocycles 1978, 10, 79-84.
(13) Ruley, A. T.; Sharma, N. C.; Sahi, S. V. Plant. Physiol. Biochem.
2004, 42, 899-906.
Org. Lett., Vol. 10, No. 6, 2008
1121

As shown in Figure 2, 4 displayed much more potent activity
than 1; however, 5 stimulated the IL-6 production.
This is the first demonstration that multi-substrate cou-
plings were generated by fungi, and sinomenine, a clinically
available drug, was modified to generate unique C-C
coupled derivatives. Because microbial transformations of
natural products is a powerful tool to produce novel drugs
and key intermediates that are difficult to be synthesized
chemically, our results provide an efficient means by which
the substrate can be selectively modified by a second one.
Further expansion of the unique C-C cross-coupling reac-
tions, enzymological research, and bioassays are currently
in progress.
Acknowledgment. This work was supported by the
National Natural Science Funds for Creative Research
Groups of China (20521503) and 973 Program (2007CB71-
4504). We thank for Prof. Ren-Gen Xiong and Dr. Heng-
Yun Ye (Southeast University, China) for X-ray analysis and
Prof. Yong-Hua Yang (Nanjing University, China) for the
microbial strain identification.
Figure 2. Inhibitory effects of sinomenine (1) and the metabolites
(3, 4, and 5) on IL-6 production in SW982 cells. Control: no added
compounds; 1, 3-5: added each compound, respectively. SW982
cells were cultured for 2 d with 1 ng/mL of IL-1â in the presence
or absence of compounds. IL-6 concentrations in culture superna-
tants were measured by ELISA. Each value represents the mean (
S.D. from triplicate cultures. All compounds showed no significant
inhibitory effects on cell growth or morphology at 200 µM.18
Supporting Information Available: Experimental details
for the incubation, separation of metabolites from incubation
mixture, spectroscopic data for metabolites, and bioactivity
for related compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
provided main compound 5 and minor two 1,1′-disinome-
nines; no 4 was detected. Furthermore, horseradish peroxi-
dase (HRP) catalyzed reaction of 1 or 1 and 2 provided the
same results, only two 1,1′-disinomenines were obtained.
These results implied that there should be an enzyme system
presented in A. semisupina responsible for the unique C-C
couplings with stereoselectivity and enzymatic exclusivity,
which was different from the conversions via HRP and
chemical reaction. Further research is essential to clarify it.
Human synovial sarcoma cells (SW982) are characterized
by high levels of expression of proinflammatory cytokines,
cyclooxygenase (COX)-2, rostaglandin (PG)E2, interleukin
(IL)-6, and matrix metalloproteinases (MMPs) genes in the
presence of IL-1â stimulation.16 The cytokines are thought
to participate in the pathogenesis of numerous inflammatory
diseases, including rheumatoid arthritis (RA).17 Therefore,
the metabolites together with 1 were tested for their inhibitory
activity on IL-1-induced IL-6 production in SW982 cells.
OL800024U
(16) (a) Niwa, M.; Fukuoka, K.; Fujimoto, T.; Maruyama, I. N. J.
Biotechnol. 2004, 114, 55-58. (b) Joyner, D. E.; Bastar, D.; Randall, R. L.
J. Orthopaed. Res. 2006, 24, 1163-1169. (c) Fumio, T.; Kenji, O.;
Toshihiko, S.; Masato, H.; Shiro, M. Immunol. Lett. 1999, 68, 275-279.
(d) He, X. B.; Wang, J. L.; Guo, Z. H.; Liu, Q. Y.; Chen, T. Y.; Wang, X.
J.; Cao, X. T. Immunol. Lett. 2005, 98, 91-96.
(17) (a) Jani, M.; Tordai, H.; Trexler, M.; Ba´nyai, L.; Patthy, L. Biochimie
2005, 87, 385-392. (b) Hashimoto, Y.; Kakegawa, H.; Narita, Y.; Hachiya,
Y.; Hayakawa, T.; Kos, J.; Turk, V.; Katunuma, N. Biochem. Biophys. Res.
Commun. 2001, 283, 334-339. (c) Chen, Y. W.; Li, J. Y.; Zhang, J. B.;
Zhao, T. T.; Zou, L. Y.; Tang, Y.; Zhang, X. P.; Wu, Y. Z. Int.
Immunopharmacol. 2005, 5, 1446-1457. (d) Mavunkel, B. J.; Chakravarty,
S.; Perumattam, J. J.; Luedtke, G. R.; Liang, X.; Lim, D.; Xu, Y. J.; Laney,
M.; Liu, D. Y.; Schreiner, G. F.; Lewicki, J. A.; Dugar, S. Bioorg. Med.
Chem. Lett. 2003, 13, 3087-3090.
(18) Yamazaki, T.; Shimosaka, S.; Sasaki, H.; Matsumura, T.; Tukiyama,
T.; Tokiwa, T. Toxicol. in Vitro 2007, 21, 1530-1537.
1122
Org. Lett., Vol. 10, No. 6, 2008