Synthesis and biological evaluation of unique stereodimers of sinomenine analogues as potential inhibitors of NO production

Article abstract of DOI:10.1016/j.bmc.2011.04.006

Inhibition of the excessive NO production has been recognized as a potential means for the treatment of rheumatoid arthritis (RA). In order to discover more potent inhibitors and explore the preliminary structure activity relationship, a series of unique

Full text of DOI:10.1016/j.bmc.2011.04.006

Bioorganic & Medicinal Chemistry 19 (2011) 3096–3104
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of unique stereodimers of sinomenine
analogues as potential inhibitors of NO production
a
b
a
a
a
b
b
Peng Teng , Hai-Liang Liu , Zhang-Shuang Deng , Zhi-Bing Shi , Yun-Mian He , Li-Li Feng , Qiang Xu ,
Jian-Xin Li
a
a,
⇑
Key Lab of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing 210093, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of the excessive NO production has been recognized as a potential means for the treatment of
rheumatoid arthritis (RA). In order to discover more potent inhibitors and explore the preliminary struc-
ture activity relationship, a series of unique stereodimers of sinomenine analogues were designed and
synthesized. Their inhibitory activity on NO production and cytotoxicity were evaluated using LPS-acti-
vated murine macrophages RAW264.7 assay and MTT method, respectively. Among these compounds,
Received 24 February 2011
Revised 2 April 2011
Accepted 4 April 2011
Available online 7 April 2011
1
a, 2, 2a, 2b, and 4 showed potent inhibitory activity on NO production without obvious cytotoxicity. Fur-
Keywords:
Sinomenine
Stereodimer
Inhibitor
Nitric oxide
iNOS
thermore, 2, 2a, and 2b significantly suppressed mRNA expression of iNOS. Interestingly, (S)-dimers dis-
played a better bioactivity than (R)-dimers. These compounds may sever as lead candidates in the
development of novel therapeutic drugs for RA treatment.
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
tions.8,9 Abnormal or high expression of NO and pro-inflammation
cytokines play an important role in the pathogenesis of several
1
0,11
Nitric oxide (NO) is an endogenous free radical generated from
arthritis diseases.
Blocking the localized excess production of
1
2
an NO-liberating compound L-arginine catalyzed by a family of
NO was identified as a way of treating RA.
nitric oxide synthase (NOS) including endothelial NOS (eNOS),
neuronal NOS (nNOS), and inducible NOS (iNOS). The short-life
molecule NO has gained its reputation of being an important
signaling mediator of vasorelaxation, neurotransmission, and host
defence at low concentration.1 The readily diffusible NO is in-
volved in various signals, such as cardiovascular, pain, cancer,
Sinomenine (1), a natural alkaloid isolated from Chinese medic-
inal plant Sinomenium acutum, has been employed clinically for the
treatment of RA in China for a long time. Furthermore, a variety
of other bioactivities has also been reported, such as treating mes-
angial proliferative nephritis, adjuvant arthritis, osteoarthritis,
1
3
,2
inflammation mediated neurodegenerative disorders and other
3
,4
14–16
and inflammation. Among the nitric oxide synthase family, iNOS
can predominantly generate high expression of NO, which modu-
lates the inflammation signals through multiple pathways and
plays role of great importance in the regulation of immune
immune-related diseases.
However, the efficacy of sinome-
nine for the treatment of RA is quite weak. Additionally, certain ad-
1
7–19
verse effects have also been reported.
Therefore, to get novel
and better derivatives with sinomenine scaffold, a structure mod-
ification was conducted.
5
reactions. Aberrant NO synthesis catalyzed by iNOS, located in
macrophage, has been involved in a variety of pathological signal
pathways, such as stroke, hypertension, cancer, ischemia, inflam-
mation, colitis, and rheumatoid arthritis. Thus, blocking the exces-
Dimers of sinomenine by C
the past 80 years, including some biocatalytic products,
1 1
–C coupling have been studied in
20,21
how-
6
ever, so far neither synthesis of other dimer analogues, nor the bio-
activities of the dimers have been emanated. It is reported that
dimerization of two natural product moieties represents the prom-
ising and fundamental approaches in the filed of designing novel
drug leads with high efficacy and low toxicity in medicinal chem-
sive NO production has been recognized as a potential means for
the treatment of these diseases.7
Rheumatoid arthritis (RA), a chronic and systemic inflammation
disease has symptoms of progressive demolish of the articular car-
tilage, hyperplasia of the synovium, and damages to the joint tissue
of bones, accompanied by the disability and limitation of bones’ ac-
2
2,23
istry,
We have already identified the stereochemistry of the (S)- and
R)-disinomenine and discovered that (S)-disinomenine possessed
which inspired us to dimerize the sinomenine analogues.
(
⇑
Corresponding author. Tel./fax: +86 25 83686419.
E-mail address: lijxnju@nju.edu.cn (J.-X. Li).
a more potent inhibitory activity on IL-6 production compared
with sinomenine, while (R)-disinomenine exhibited a stimulative
0
968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.006

P. Teng et al. / Bioorg. Med. Chem. 19 (2011) 3096–3104
3097
effect.24,25 In the present study, as an effort to develop novel mol-
ecules with better bioactivity and understand the structure–activ-
ity relationships (SAR), a series of stereodimers of sinomenine
analogues were synthesized and their inhibitory activity on NO
production and cytotoxicity were evaluated using LPS-activated
murine macrophages RAW264.7 assay and MTT method, respec-
tively. Furthermore, the suppressive effect on mRNA expression
of iNOS of the compounds with potent activity was analyzed by re-
verse transcription-polymerase chain reaction (RT-PCR) analysis.
under the mild conditions (Scheme 1) and could easily be sepa-
rated by a silica gel column chromatography.
It should be noted that based on the results of single crystal X-
ray diffraction analyses, the stereostructures of 1a and 1b could
1
also be elucidated by circular dichroism (CD) spectra and
H
2
4
NMR data. In the CD spectrum, a positive Cotton effect was ob-
served in (S)-disinomenine (1a) at 296.7 nm, while no Cotton effect
was exhibited at same wavelength of the CD spectrum of (R)-disi-
nomenine (1b), indicating the difference on regiochemistry of
biphenyl linkage. Moreover, the protons signal at C-2 in 1a (d
.27, s, 2H) displayed an upfield shift versus that of 1b (d 6.45,
s, 2H).
H
6
H
2
2
. Results and discussion
.1. Syntheses of stereodimers of sinomenine
Dimerization of sinomenine was firstly conducted using potas-
2
.2. Syntheses of stereodimers of sinomenine analogues
The treatment of sinomenine (1) with zinc amalgam afforded
4
sium permanganate (KMnO ) oxidation by Goto in 1929 and the
2
7
2
0
the ring-C reduced cyclohexane structure (2). The dimerization
of 2 was also carried out followed the sinomenine’s dimerization,
existence of two dimers were confirmed. However, after which,
no reports were followed up on the stereostructures of the dimers
due to the difficulty of the crystallization of the dimers.2 Our pre-
vious work successfully identified the stereostructures of two sin-
omenine dimers by single crystal X-ray diffraction analysis
6
2
an oxidation with MnO in methanol solution followed a column
chromatography afforded the target dimers 2a and 2b.
The N-methyl group of sinomenine ring-D can be eliminated
2
8,29
both by chemical and metabolic approaches.
study, N-demethylsinomenine (3) was prepared in satisfactory
yield by allowing a mixture of sinomenine and H to stand
4 h at room temperature and followed by a reduction in the pres-
In the present
(
Scheme 1), (S)-disinomenine (1a) and (R)-disinomenine (1b) that
possessed different angle around C1–C1 bond, and also discovered
that in KMnO aqueous solution the two dimers could be pH con-
2 2
O
4
25
2
trolled stereoselectively synthesized. Additionally, (S)-disinome-
nine can also be stereoselectively afforded by biocatalyst with a
fungus Antrodiella semisupina.24 These results encouraged us to ex-
pand the diversity of these dimers and discovery new molecules
with better bioactivities, therefore, the other dimers were prepared
via similar approaches to allow access to versatile analogues to
investigate structure–activity relationship (SAR) of stereodimers
of sinomenine analogues.
3
0
ence of FeSO
4
.
Oxidation of N-methyl group in the ring-D could
facilely afford the intermediate oxynitride in an almost complete
conversion (98.7%), which was then subjected to methanol solu-
tion in the presence of reducing agent FeSO , affording target mol-
4
ecule (3) in moderate yield (71.5%, Scheme 2).
Carbonyl group of sinomenine was reacted with hydroxylamine
hydrochloride to afford oxime (7), followed by reduction with Pd/C
to provide the compound 6b-amino-7b-methoxy-7,8-dihydro
sinomenine (8). Due to the two additional chiral centers (C-6 and
C-7), compound 8 should have four stereoisomers, however, the
To more efficiently synthesize the dimers, an oxidation of sin-
omenine with manganese dioxide (MnO
was performed, as expected, compounds 1a and 1b were prepared
2
) in methanol solution
2
Scheme 1. Synthesis of 1a and 1b and X-ray crystal structures from Ref. 25. Reagent and conditions: (i) MnO , MeOH, rt, 24 h.

3
098
P. Teng et al. / Bioorg. Med. Chem. 19 (2011) 3096–3104
Scheme 2. Synthesis of 2a, 2b, 3a, 3b, 4a, 4b, 5a and 5b. Reagent and conditions: (i) zinc amalgam, concentrated HCl, reflux, 24 h; (ii) 15% H
2 2 4
O , rt, 24 h; FeSO , MeOH, rt, 6 h;
(
iii) NH OH, EtOH, reflux, 8 h; (iv) NH HCO , Pd/C (10%), MeOH, reflux, 6 h; (v) MsCl or TsCl, Et N, CH Cl , rt, 5 h; (vi) MnO , MeOH, rt, 48–72 h.
2
4
3
3
2
2
2
reaction afforded only one pure compound rather than diastereo-
mers based on chiral HPLC analysis (see Supplementary data).
The configurations of C-6 and C-7 of 8 were determined based on
2
Reaction of 3, 4, and 5 with MnO in dried methanol afforded
corresponding dimers 3a, 3b, 4a, 4b, 5a, and 5b with different spa-
tial configuration, respectively, and was outlined in Scheme 2.
3
1–33
the comparisons of coupling constant (J) with reported data.
It has been reported that quinoxaline moiety could provide an
1
35,36
In the H NMR spectrum, the protons at C-5 (4.08, dd, J = 14.4,
.0 Hz; 1.25, dd, J = 14.4, 3.0 Hz) and the proton at C-6 (3.38, dd,
effective bioactivity.
Thus, a vicinal 6,7-diketone (9) and its
3
quinoxaline derivative (6) were prepared followed the reported
3
7,38
J = 6.9, 3.0 Hz) clearly revealed that the coupling constant of pro-
tons at C-6 and C-7 positions (J6,7) was 6.9 Hz although the split
pattern of 7-H was unreadable due to the signal overlapping. Be-
cause the preferential conformation of ring-C is a chair form, as
can be seen in Figure 1, the J6,7 at axial and equatorial bonds in four
isomers of 8 displayed different values. Due to the J6,7 was 6.9 Hz,
the configurations of C-6 and C-7 were determined to be both 6H
and 7H at axial bonds (Fig. 1).
protocols (Scheme 3).
For the dimerization of 6, although many
efforts with different oxidants and pH controlled stereoselective
syntheses, unfortunately, no targeted (S)- and (R)-dimers of 6 were
obtained, mostly probably due to the strong steric hindrance. How-
ever, when a biotransformation of 6 by a strain of fungi (A. semisup-
ina) was carried out, (S)-dimer (6a) was successfully afforded,
while corresponding (R)-dimer was still failed to obtain (Scheme
3).
Because the sulfonyl group manifests excellent effects in the
field of medicinal molecules,34 the amino group was further mod-
ified by several sulfonyl chlorides, aiming to get new compounds
owning better bioactivity. Therefore, p-toluene sulfonyl chloride
was firstly employed, compound 8 reacted with the sulfonyl chlo-
The stereostructures of (S)-dimers (2a, 3a, 4a, 5a, 6a) and (R)-di-
mers (2b, 3b, 4b, 5b) were determined by comparison the CD spec-
tra and H NMR data with those of 1a and 1b. In the CD spectra of
1
all (S)-dimers, a positive Cotton effect between 292 and 300 nm
was clearly observed, while no Cotton effect was displayed in the
same wavelength range of the CD spectra of all (R)-dimers (see
3
ride in the present of Et N gave the compound 4 with excellent
yield (96.1%). The same approach was also employed in the small
methyl sulfonyl group to afford corresponding substituted amino
compound (5).
Supplementary data). Additionally, the same as 1a and 1b, in 1
H
NMR spectra, the proton signal at C-2 of all (S)-dimers showed
an upfield shift compared with that of all (R)-dimers. Based on
Figure 1. The possible stereoisomers of 8.

P. Teng et al. / Bioorg. Med. Chem. 19 (2011) 3096–3104
3099
2
Scheme 3. Synthesis of 6a. Reagent and conditions: (i) 10% HCl, reflux, 10 h; (ii) o-phenylenediamine, MeOH, N , 6 h; (iii) Antrodiella semisupina, mineral medium, 25 °C,
incubation on rotary shaker.
those physiochemical and NMR data, the stereostructures of the
dimers were elucidated as shown in Schemes 2 and 3.
Table 2
The effect of compounds on LPS-induced NO production in RAW264.7 cells
Compound
Inhibition (%)
50 ( M)
2
.3. Bioactivity evaluation
100 (lM)
l
25 (lM)
1
1a
12.8 ± 2.3
23.7 ± 2.1
ꢀ2.1 ± 2.7
30.6 ± 4.2
10.7 ± 2.8
16.0 ± 2.2
ꢀ7.1 ± 4.0
39.8 ± 4.1
0.1 ± 2.9
15.0 ± 3.1
6.6 ± 3.2
ꢀ3.0 ± 5.5
ꢀ2.9 ± 5.4
ꢀ1.3 ± 3.1
12.1 ± 3.7
ꢀ6.1 ± 4.1
ꢀ9.3 ± 2.3
ꢀ11.6 ± 8.4
10.9 ± 6.7
ꢀ2.3 ± 1.5
ꢀ0.2 ± 8.0
ꢀ8.4 ± 6.6
55.4 ± 0.5
*
To evaluate the inhibitory effects on the excessive NO produc-
1
2
3
3
b
tion, all the compounds were assayed using LPS-activated murine
*
*
*
*
macrophage-like RAW264.7 cell culture systems.39 Because the
28.1 ± 0.6
ꢀ0.8 ± 1.6
ꢀ12.6 ± 5.4
ꢀ11.0 ± 3.8
30.0 ± 4.6
ꢀ3.7 ± 5.1
0.2 ± 7.6
8.6 ± 6.6
74.9 ± 2.5
0.0 ± 6.4
inhibitory activity of the compounds is sometimes a result of their
toxic effect and consequently might cause an erroneous conclu-
sion, prior to the following bioactivity analyses, the cytotoxicity
of the synthesized compounds was determined by MTT assay
and summarized in Table 1. The data showed that the most of
a
3b
4
5
5
5
*
ꢀ3.5 ± 14.4
a
b
9.3 ± 6.6
9.9 ± 4.4
86.0 ± 0.3
the compounds had cytotoxicity IC50 of more than 100
compounds 2a and 2b displayed cytotoxicity IC50 of 9.5 and
4.2 M, and 4a and 4b at 43.5 and 72.9 M, respectively. There-
lM, only
*
*
AG
Control
1
l
l
The cells of control group were treated with LPS (1 lg/mL) only. The data of control
fore, all the activity profiles were carried out below the toxic IC50
concentrations.
Sinomenine (1) showed a very weak activity on the production
of NO at 100 lM, although no cytotoxicity was detected at this
concentration (Tables 1 and 2). (S)-Disinomenine (1a) showed
obvious increase on the inhibitory activity of NO compared with
the parent compound 1, while interestingly, (R)-disinomenine
group was pegged as 0.0%, while other data were calculated relative to it. Cells were
treated with test compounds prior to LPS treatment. AG: aminoguanidine as a
positive control. Data are expressed as mean ± SD, n = 4.
‘
ꢀ’ A minus sign of the data means enhancement effect on NO production.
*
Significant differences compared with control group, p <0.01.
displayed a prodigious enhancement in activity and a dose-depen-
dence effect compared with parent compound 1, and its dimers (4a
and 4b) both exhibited good inhibitory effect, while (S)-dimer (4a)
was more potent than (R)-dimer (4b) (Table 3). Of all of com-
pounds, (S)-dimer (2a) of 2 showed the most potent efficacy, a
(1b) displayed no improvement of the activity. The same as 1a
and 1b, (R)-dimer (2b) also had a good activity, but weaker than
2
1
a, while, the monomer (2) could also inhibit NO production at
00 M. Compound 3 was an N-dimethyl group structure of 1
l
and exhibited a modest inhibitory effect, while, dimeric com-
pounds 3a and 3b showed the similar results as 3. Compound 4
4
1.6% inhibition at 6.25 lM, while no cytotoxicity was observed
at this concentration. These results suggested that the deoxygen-
ation and dimerization could elevate the activity.
Table 1
Cytotoxicity of the synthesized compounds
Table 3
Compound
Cytotoxicity IC50
(l
M)a
The effect of compounds 2a, 2b, 4a and 4b on LPS-induced NO production in
RAW264.7 cells
1
>100
>100
>100
>100
9.5
14.2
>100
>100
>100
>100
43.5
72.9
>100
>100
>100
57.9
6.3
1
1
2
2
2
3
3
3
4
4
4
5
5
5
6
6
a
b
Compound
Inhibition (%)
3.125 ( M)
6
.25 (
l
M)
l
1.5625 (lM)
*
a
b
2a
2b
6a
41.6 ± 1.3
35.2 ± 0.2
NT
17.0 ± 7.0
16.9 ± 2.4
NT
2.7 ± 3.1
ꢀ4.4 ± 3.9
8.4 ± 2.1
*
a
b
5
0 (lM)
25 (
lM)
12.5 (lM)
4a
4b
6
AG
NT
ꢀ2.2 ± 6.2
NT
4.9 ± 2.4
ꢀ2.9 ± 6.2
11.4 ± 3.8
52.5 ± 0.7
0.0 ± 6.6
2.6 ± 1.0
ꢀ4.0 ± 2.9
1.3 ± 3.4
ꢀ
a
b
*
*
76.5 ± 3.4
a
b
Control
Cells were treated with test compounds prior to LPS treatment. AG: aminoguani-
dine as a positive control. Data are expressed as mean ± SD, n = 4.
a
‘
ꢀ’ A minus sign of the data means enhancement effect on NO production.
a
Cytotoxicity IC50: the concentration of inhibition of 50%
cell growth with four experiments.
NT: not tested due to cytotoxicity.
*
Significant differences compared with control group (0.0%), p <0.01.

3
100
P. Teng et al. / Bioorg. Med. Chem. 19 (2011) 3096–3104
Figure 2. The effect of 1, 1a, 2, 2a, 2b and 4 on the mRNA expression of iNOS by RT-PCR.
Above results revealed that the dimerization and the stereo-
structure did gave the impact on the activity. It is reported that
phenolic compounds possess a capacity to trap free radicals.
4. Experimental section
4.1. Synthesis
4
0,41
Because the present dimers possess the same phenolic moiety (A
ring), it is feasible that the dimers inhibit the NO production via
scavenging NO directly. However, although (R)- and (S)-dimers
have the same phenolic moiety, it is clear that the (S)-dimers with
less steric hindrance possessed better inhibitory effect on NO pro-
duction than the (R)-dimers, which was extraordinarily interesting
for the further research on the SAR.
The excessive expression of NO is greatly dominated by iNOS,
which is one of the most pivotal signals during the process of
LPS-induced NO production. In an effort to study the preliminary
mechanism of the compounds with potent inhibitory activity, the
RT-PCR experiment was performed to assay the effect of five se-
lected compounds on mRNA expression of iNOS. The RT-PCR results
4.1.1. General
1H and 13C NMR spectra were measured on a Bruker Advance
II 300 spectrometer using tetramethylsilane as internal standard.
Chemical shift (d) are reported in parts per million (ppm) and
1
3
coupling constants J are reported in hertz (Hz), C NMR spectra
were fully decoupled, and the following abbreviations are used:
singlet (s), doublet (d), triplet (t), double-double (dd), double-
triplet (dt), triplet-double (td), broad (br) and multiplet (m).
The CD spectra were tested on a Model J-810 automatic record-
ing spectropolarimeter in methanol. The optical rotations were
recorded on Autopol I Automatic Polarimeter (Spectronic Cam-
spec Ltd, UK) in CH OH or CHCl . Chromatographic separations
3
3
(
Fig. 2) reveled that compounds 1a, 2, 2a, 2b, and 4 markedly inhib-
were performed on silica gel columns by column chromatogra-
phy (Kieselgel 300–400 mesh) with CH Cl /CH OH (15:1–9:1, v/
ited the mRNA expression of iNOS in a dose-dependent manner,
2
2
3
while the parent compound 1 only faintly restrained the expression
3 2
v) or EtOAc/CH OH/H O (37:2:1–17:2:1, v/v/v) as eluents. All
at 100
l
M. These results revealed that the sinomenine analogues
reactions were monitored by TLC on GF254 plates that were
visualized under a UV lamp (254 nm). Evaporation of solvent
was performed in vacuo with rotating evaporator. Commercially
available chemicals were purchased from Alfa Aesar. The purity
of the final compounds was determined by HPLC on an Agilent
1200 system using a Agilent Eclipse XPB-C18 column (4.6 ꢁ
might play crucial role in the pathway involved iNOS.
3
. Conclusion
We have designed, synthesized and biologically evaluated a ser-
ies of unique stereodimers of sinomenine analogues on the inhib-
itory effect of NO overproduction and mRNA overexpression of
iNOS using LPS-induced murine RAW264.7 cells. Among the syn-
thesized compounds, 1a, 2, 2a, 2b, and 4 displayed a potent inhib-
itory activity and also exerted the activity via suppressing mRNA
expression of iNOS in a dose-dependent manner. Reduction of
ring-C (2) could be beneficial to increase of the activity. The pres-
ent results unfolded that dimerization of sinomenine analogues
with weak steric hindrance (S-dimers) showed a better inhibitory
potency both on NO overproduction and mRNA expression of iNOS,
while those with a strong steric hindrance (R-dimers) showed
almost no benefit on the enhancement of the activity, some of
150 mm, 5
CH OH/H O (45:55, v/v) with 0.125% of ethylenediamine (EDA)
to CH OH/H O (80:20, v/v) with 0.125% of EDA to CH OH/H
lm particle size) with a gradient mobile phase of
3
2
3
2
3
2
O
(100:0, v/v) with 0.125% of EDA at a flow rate of 1 mL/min, with
UV monitoring at the wavelength of 230–300 nm with a runtime
of 35 min. The compound purity analysis data of final com-
pounds (greater than 95%) can be seen in the Supplementary
data.
4.1.2. General procedure for the synthesis of dimers
To a solution of mono compound in dried CH
added MnO and the mixture was stirred at room temperature
for 24–72 h. The solution was then filtered and washed by CH OH
(10 mL ꢁ 3). The filtrate and the washes were combined, dried over
anhydrous Na SO and concentrated. The resulting brown solid
was purified using silica gel column chromatography with
CH Cl /MeOH as eluent to yield the title compounds.
3
OH (50 mL) was
2
(
(
R)-dimers (3b, 4b) even promoted the secretion of NO. Why the
R, S)-dimers only with different angle around C1–C1 bond exhib-
3
ited reversed activity clearly warrant further studies, and now the
mechanism clarification and further expansion of the related com-
pounds are under way in our laboratory.
2
4
2
2

P. Teng et al. / Bioorg. Med. Chem. 19 (2011) 3096–3104
3101
4
.1.2.1. 7-Demethoxy-6-desoxo-7, 8-dihydro sinomenine (2).
A
solution (to pH 9–10) and then extracted with CHCl
The combined organic layers were dried over Na SO
3
(30 mL ꢁ 3).
mixture of zinc power (52 g, 0.8 mol) and mercury(II) chloride (4 g,
2
4
and concen-
1
4
5 mmol) in concentrated hydrochloric acid/distilled water (4 mL/
0 mL) was stirred at room temperature for 5 min. The aqueous
trated in vacuum to afford the intermediate oxynitride (1.37 g,
98.7%), which was used in the next step without purification. The
intermediate oxynitride (1.37 g, 3.95 mmol) was dissolved in
solution was decanted and the prepared amalgamated zinc was
covered with concentrated hydrochloric acid (80 mL) and distilled
water (40 mL) in sequence. Then the material sinomenine (13.2 g,
methanol (30 mL) and added with FeSO
4
ꢃ7H
2
O (2.22 g, 8 mmol)
under stirring at 0 °C and then warm to room temperature for
6 h. The solvent was removed and ethylenediaminetetraacetic acid
(EDTA) aqueous solution (0.6 M, 40 mL) was added to remove
4
0 mmol) was added immediately and the reaction mixture was
heated to 90 °C and stirred for 24 h. Concentrated hydrochloric
acid (20 mL) was added every 8 h. After 24 h, the reaction mixture
was cooled to room temperature, basified with concentrated
ammonia solution, and extracted with dichloromethane
2
+
3+
excessive Fe and Fe under stirring for 4 h. The reaction was
basified attentively with concentrated ammonia solution (to pH
9–10) and the extracted with CHCl
ganic layers were dried over Na
Purification by column chromatography (CH
v/v) afforded the title compound 3 as a gray solid (889 mg,
3
(50 mL ꢁ 4). The combined or-
SO , filtered and concentrated.
Cl /MeOH, 15:1–6:1,
(
50 mL ꢁ 3). The combined organic phase were dried over Na
and concentrated in vacuum. The crude residue were purified by
silica gel column chromatography (EtOAc/MeOH/NH OH,
70:20:10, v/v/v) to provide the target compound (5.16 g, 45%).
2
SO
4
2
4
2
2
4
1
4
3
71.5%). H NMR (CDCl , 300 MHz) d: 6.65 (d, J = 8.4 Hz, 1H), 6.54
1
H NMR (CDCl
J = 8.4 Hz, 1H), 3.86 (s, 3H), 3.36 (dd, J = 10.2 and 4.2 Hz, 1H),
.94 (d, J = 17.7 Hz, 1H), 2.78 (dd, J = 5.4 and 3.3 Hz, 1H), 2.67
3
, 300 MHz) d: 6.70 (d, J = 8.4 Hz, 1H), 6.62 (d,
(d, J = 8.4 Hz, 1H), 5.42 (d, J = 2.1 Hz, 1H), 4.33 (d, J = 15.6 Hz, 1H),
3.81 (s, 3H), 3.55 (t, J = 4.2 Hz, 1H), 3.48 (s, 3H), 3.18 (dd, J = 18.0
and 5.7 Hz, 1H), 3.02 (br s, 1H), 2.84 (dd, J = 14.1 and 3.9 Hz, 2H),
2.61 (dt, J = 12.6 and 3.6 Hz, 1H), 2.48 (d, J = 15.6 Hz, 1H), 1.93
2
(
(
5
1
3
dd, J = 18.3 and 6.3 Hz, 1H), 2.44–2.50 (m, 1H), 2.39 (s, 3H), 2.09
dt, J = 12.0 and 3.6 Hz, 1H), 1.84–1.93 (m, 1H), 1.55–1.78 (m,
1
3
3
(br d, J = 12.6 Hz, 1H). C NMR (CDCl , 75 MHz) d: 193.8, 152.4,
H), 1.07–1.45 (m, 1H). 13C NMR (CDCl
145.1, 144.8, 130.3, 122.7, 118.4, 114.7, 109.1, 56.0, 54.8, 50.0,
3
, 75 MHz) d: 144.7, 144.5,
+
32.2, 125.9, 118.5, 108.2, 58.1, 56.2, 47.8, 46.8, 42.8, 38.4, 38.1,
7.1, 27.4, 26.8, 24.3, 23.3. Positive ESI-MS m/z: 288 [M+H] , posi-
49.6, 45.9, 41.4, 38.9, 36.1, 33.7. Positive ESI-MS m/z: 316 [M+H] .
+
+
tive HRESIMS m/z found 288.1935 [M+H] (calcd for C18
2
H26NO
2
,
4.1.2.4. (S)-17-Demethyl disinomenine (3a) and (R)-17-demethyl
disinomenine (3b). The titled compounds 3a and 3b were pre-
pared according the procedure described above; 17-demethyl sin-
2
D
5
88.1964). ½
aꢂ
3
¼ þ38:33 (c 0.60, CH OH).
4
.1.2.2. (S)-7-Demethoxy-6-desoxo-7, 8-dihydro disinomenine
omenine (3, 630 mg, 2.0 mmol) was dissolved in dried CH
(50 mL) and MnO (3000 mg, 34.5 mmol) was added with magnetic
stirring, and the mixture was remained at room temperature for
3 days and then filtered and washed by CH
OH (10 mL ꢁ 3). The fil-
trate and the washes were combined, dried over anhydrous MgSO
and concentrated. The resulting brown solid was purified using sil-
3
OH
(
(
2a) and (R)-7-demethoxy-6-desoxo-7, 8-dihydro disinomenine
2b). The title compounds 2a and 2b were prepared according the
2
procedure described above; 7-demethoxy-6-desoxo-7, 8-dihydro
sinomenine (2, 574 mg, 2.0 mmol) was dissolved in dried CH OH
50 mL) and MnO (3000 mg, 34.5 mmol) was added with mag-
netic stirring, and the mixture was remained at room temperature
for 3 days, and then filtered and washed by CH
OH (10 mL ꢁ 3).
The filtrate and the washes were combined, dried over anhydrous
Na SO and concentrated. The resulting brown solid was purified
3
3
4
(
2
4
ica gel column chromatography with chloroform/methanol/NH OH
3
(60:40:1, v/v/v) as eluent to yield 3a (75%) and 3b (12%).
1
Compound 3a: H NMR (CDCl
3
, 300 MHz) d: 6.30 (s, 2H), 5.38 (d,
2
4
J = 2.4 Hz, 2H), 4.37 (d, J = 15.6 Hz, 2H), 3.75 (s, 6H), 3.49 (s, 6H),
3.36 (br t, J = 3.6 Hz, 2H), 2.90 (br s, 2H), 2.85 (dd, J = 13.0 and
3.9 Hz, 2H), 2.75 (dd, J = 18.4 and 5.6 Hz, 2H), 2.60 (dd, J = 13.0
and 3.9 Hz, 2H), 2.51 (d, J = 15.6 Hz, 2H), 2.19 (d, J = 18.4 Hz, 2H),
using silica gel column chromatography with chloroform/metha-
nol (15:1, v/v) as eluent to yield 2a (33%) and 2b (45%).
1
Compound 2a: H NMR (CDCl
3
, 300 MHz) d: 6.51 (s, 2H), 3.85 (s,
6
H), 3.44 (d, J = 12.3 Hz, 2H), 2.74 (t, J = 3.3 Hz, 2H), 2.56 (d, J = 10.5
2.02 (d, J = 12.2 Hz, 2H), 1.72 (td, J = 13.0 and 3.9 Hz, 2H). 13
C
and 3.9 Hz, 2H), 2.42 (dd, J = 14.1 and 6.0 Hz, 2H), 2.39 (d,
J = 6.3 Hz, 2H), 2.32 (s, 6H), 2.19–2.27 (m, 4H), 1.59–1.84 (m,
3
NMR (CDCl , 75 MHz) d: 193.8, 152.3, 145.1, 143.8, 131.0, 127.6,
122.9, 114.5, 110.4, 56.0, 54.8, 49.7, 49.4, 46.3, 41.6, 39.4, 36.3,
1
1
3
0H), 1.08–1.45 (m, 12H). 13C NMR (CDCl
3
, 75 MHz) d: 143.3,
32.4. Positive ESI-MS m/z: 629 [M+H] , HRESIMS m/z: found:
+
+
25
D
31.3, 129.2, 126.1, 109.8, 57.7, 56.2, 47.7, 46.6, 42.9, 38.5, 38.2,
629.2836 [M+H] (calcd for
C
36
H
41
N
2
O ,
8
629.2863).
½
a
ꢂ
¼
+
7.1, 27.3, 26.6, 23.7, 23.2. Positive ESI-MS m/z: 573 [M+H] , posi-
þ91:43 (c 0.53, CH OH). CD (c = 0.05 mM, CH
3
3
OH):
h
212
+
tive HRESIMS m/z found 573.3668 [M+H] (calcd for C36
49
H N
2
O
4
,
+12.91 mdeg, h243 +12.22 mdeg, h271 ꢀ5.32 mdeg, h293 +0.59 mdeg.
2
D
5
1
5
73.3692).
CH
OH): h207 +9.81 mdeg, h223 ꢀ10.39 mdeg, h255 +6.00 mdeg,
293 +3.62 mdeg.
Compound 2b: H NMR (CDCl
½
a
ꢂ
¼ þ65:00 (c 0.60, CH
3
OH). CD (c = 0.05 mM,
3
Compound 3b: H NMR (CDCl , 300 MHz) d: 6.45 (s, 2H), 5.27
3
(d, J = 1.8 Hz, 2H), 4.38 (d, J = 15.6 Hz, 2H), 3.81 (s, 6H), 3.54 (s,
6H), 3.24 (br t, J = 4.5 Hz, 2H), 2.94 (brs, 2H), 2.78 (dd, J = 12.6
and 4.8 Hz, 2H), 2.63 (dt, J = 12.9 and 3.0 Hz, 2H), 2.50 (d,
J = 15.3 Hz, 2H), 2.25–2.30 (m, 2H), 1.99–2.03 (m, 4H), 1.74 (dt,
h
1
3
, 300 MHz) d: 6.52 (s, 2H), 3.86 (s,
6
2
H), 3.44(dd, J = 11.7 and 1.5 Hz, 2H), 2.69 (dd, J = 5.4 and 3.0 Hz,
H), 2.57 (d, J = 18.9 Hz, 2H), 2.47–2.51 (m, 2H), 2.28 (s, 6H), 2.12
1
3
J = 12.6 and 5.1 Hz, 2H).
3
C NMR (CDCl , 75 MHz) d: 194.1,
(
dt, J = 12.6 and 3.3 Hz, 2H), 2.03 (d, J = 6.9 Hz, 2H), 1.86 (dd,
J = 18.6 and 6.0 Hz, 2H), 1.75–1.78 (m, 2H), 1.58–1.70 (m, 8H),
152.5, 145.4, 144.0, 131.0, 128.0, 123.1, 114.6, 110.0, 56.1, 54.6,
45.2, 41.5, 38.8, 35.5, 32.6, 31.0, 23.9. Negative ESI-MS m/z: 627
1
3
ꢀ
+
1
.32–1.42 (m, 4H), 1.26 (t, J = 7.2 Hz, 2H), 1.11–1.20 (m, 4H).
C
[MꢀH] , positive HRESIMS m/z: found: 629.2836 [M+H] (calcd
25
D
NMR (CDCl
5
3
, 75 MHz) d: 144.5, 143.4, 131.6, 129.9, 126.1, 108.8,
for C36
H
41
N
2
O
8
, 629.2863). ½
a
ꢂ
¼ þ6:49 (c 0.15, CH
3
OH). CD
7.7, 56.0, 47.9, 46.7, 43.0, 38.3, 38.2, 37.2, 27.3, 26.8, 23.3, 22.5.
(c = 0.1 mM, CH
ꢀ14.62 mdeg.
3
OH): h214 +24.39 mdeg, h233 +23.23 mdeg, h271
+
Positive ESI-MS m/z: 573 [M+H] , positive HRESIMS m/z: found:
5
73.3668 [M+H]+ (calcd for
OH). CD (c = 0.05 mM, CH
22.47 mdeg, h234 +6.00 mdeg.
C
36
H
49
N
2
O
4
,
573.3692).
½
aꢂ
25
D
¼
þ57:10 (c 0.72, CH
3
3
OH):
h
208
4.1.2.5. 6-Hydroxyimino sinomenine (7). Sinomenine (329 mg,
1 mmol), hydroxylamine hydrochloride (700 mg, 1 mmol), and
anhydrous NaOAc (410 mg, 5 mmol) were dissolved in absolute
ethanol (20 mL), and the mixture was heated at reflux for 8 h
and then concentrated to dryness. Water (20 mL) was added, and
the mixture was made base with 15% ammonia water and
+
4
4
.1.2.3. 17-Demethyl sinomenine (3). A solution of 1 (1.32 g,
mmol) in 15% H (20 mL) was allowed to warm to room tem-
2 2
O
perature stirred for 24 h, basified with concentrated ammonia

3
102
P. Teng et al. / Bioorg. Med. Chem. 19 (2011) 3096–3104
extracted with CH
with brine, dried over Na
lized from CH Cl /PE to give a white solid (327 mg, 95%). H NMR
CDCl , 300 MHz) d: 6.58 (d, J = 9.0 Hz, 1H), 6.49 (d, J = 9.0 Hz, 1H),
.10 (d, J = 15.0 Hz, 1H), 4.84 (s, 1H), 3.75 (s, 3H), 3.47 (s, 1H), 3.09
t, J = 3.0 Hz, 1H), 2.92 (d, J = 18.0 Hz, 1H), 2.77 (br s, 1H), 2.72 (dd,
J = 18.0 and 6.0 Hz, 1H), 2.53 (dd, J = 12.0 and 3.0 Hz, 1H), 2.39 (s,
2
Cl
2
(50 mL ꢁ 3). The CH
2
Cl
2
extract was washed
with magnetic stirring, and the mixture was remained at room
temperature for 3 days and then filtered and washed by CH OH
(10 mL ꢁ 3). The filtrate and the washes were combined, dried over
anhydrous MgSO and concentrated. The resulting yellow solid
was purified using silica gel column chromatography with chloro-
form/methanol (15:1–10:1, v/v) as eluent to yield 4a (160 mg, 33%)
2
SO , filtered, concentrated and recrystal-
4
3
1
2
2
(
5
(
3
4
and 4b (204 mg, 42%).
1
3
1
1
H), 1.91–2.08 (m, 3H), 1.79 (td, J = 12.0 and 3.0 Hz, 1H).
C
Compound 4a: H NMR (CDCl
3
, 300 MHz) d: 7.74 (dd, J = 6.6 and
NMR (CDCl
3
, 75 MHz) d: 150.9, 150.6, 144.8, 144.8, 130.9, 123.6,
1.8 Hz, 4H), 7.30 (dd, J = 8.4 and 0.2 Hz, 4H), 6.66 (s, 2H), 4.24 (d,
J = 9.0 Hz, 2H), 4.00 (td, J = 7.5 and 4.5 Hz, 2H), 3.94 (s, 6H), 3.88
(d, J = 3.3 Hz, 2H), 3.26 (td, J = 7.5 and 3.6 Hz, 2H), 3.04 (s, 6H),
1
3
17.9, 108.4, 104.4, 57.0, 55.9, 54.6, 47.7, 44.2, 42.6, 37.2, 35.8,
3.3, 24.1. Positive ESI-MS m/z: 345 [M+H] .
+
2
.72 (br d, J = 4.2 Hz, 2H), 2.43 (s, 6H), 2.32–2.41 (m, 2H), 2.25 (s,
4
.1.2.6. 6b-Amino-7b-methoxy-7,8-dihydro sinomenine (8). The
6H), 2.09–2.15 (m, 4H), 2.03 (d, J = 7.2 Hz, 4H), 1.90 (d,
J = 12.6 Hz, 2H), 1.76 (d, J = 12.0 Hz, 2H), 1.57–1.62 (m, 4H), 1.49
(dd, J = 13.2 and 4.8 Hz, 2H), 1.42 (d, J = 12.3 Hz, 2H). 13C NMR
oxime, 7 (344 mg, 1 mmol), and ammonium formate (630 mg,
0 mmol) was heated at reflux under magnetic stirring in the pres-
1
ence of 10% Pd/C (200 mg) and absolute methanol (20 mL) for 6 h,
filtered through a pad of Celite (eluted with MeOH, 50 mL), and
concentrated in vacuum to dryness. The resulting residue was dis-
solved in water (20 mL), made base (pH 9–10) with 15% ammonia
3
(CDCl , 75 MHz) d: 145.2, 143.8, 142.7, 138.9, 131.5, 129.2 (2C),
127.8, 127.1 (2C), 124.7, 112.0, 79.4, 57.0, 56.1, 51.0, 47.0, 44.4,
42.6, 38.0, 37.8, 35.8, 31.9, 29.7, 23.7, 22.7, 21.5. Positive ESI-MS
+
+
m/z: 971 [M+H] , positive HRESIMS m/z: found: 971.4275[M+H]
2
5
water, extracted with CH
tract was washed with brine, dried over Na
centrated. The resulting residue was purified by chromatography
on silica gel (CH Cl /MeOH, 9:1, v/v) providing 8 (305 mg, 92%) as
a white solid. H NMR (CDCl , 300 MHz) d: 6.59 (d, J = 8.1 Hz, 1H),
.52 (d, J = 8.1 Hz, 1H), 4.08 (dd, J = 14.4 and 3.0 Hz, 1H), 3.57 (s,
H), 3.38 (dd, J = 6.9 and 3.0 Hz, 6 -H), 3.32 (s, 3H), 3.26–3.32
-H), 2.82–2.93 (m, 2H), 2.72 (dd, J = 17.7 and
Cl
2 2
(50 mL ꢁ 3). The combined CH
2
Cl
2
ex-
(calcd for C52
CH OH). CD (c = 0.1 mM, CH
+4.17 mdeg, h300 +0.87 mdeg.
H
67
N
4
O
10
S
2
, 971.4299). ½
a
ꢂ
¼ þ121:67 (c 0.60,
D
2
SO , filtered and con-
4
3
3
OH):
h213 +10.68 mdeg,
h
258
1
2
2
3
Compound 4b: H NMR (CDCl , 300 MHz) d: 7.67 (d, J = 8.4 Hz,
1
3
4H), 7.27 (d, J = 8.4 Hz, 4H), 6.60 (s, 2H), 4.12 (d, J = 6.6 Hz, 2H),
3.94 (dd, J = 15.3 and 3.6 Hz, 2H), 3.89 (s, 6H), 3.76 (dd, J = 6.3
and 3.0 Hz, 2H), 3.37 (br t, J = 3.6 Hz, 2H), 3.21 (td, J = 8.1 and
3.6 Hz, 2H), 2.94 (br d, J = 13.5 Hz, 2H), 2.86 (s, 6H), 2.66 (br d,
J = 18.6 Hz, 2H), 2.53 (s, 6H), 2.41 (s, 6H), 2.32 (br t, J = 6.0 Hz,
6
3
a
(
overlapped, 7a
5
.1 Hz, 1H), 2.42 (dd, J = 11.4 and 3.3 Hz, 1H), 2.35 (s, 3H), 1.95–
2
.03 (m, 2H), 1.71–1.77 (m, 1H), 1.43–1.64 (m, 3H), 1.29 (dd,
4H), 2.10 (d, J = 13.5 Hz, 2H), 1.77 (td, J = 12.9 and 3.9 Hz, 2H),
1.65 (d, J = 12.9 Hz, 2H), 1.18–1.34 (m, 4H). C NMR (CDCl ,
3
13
13
J = 14.4 and 3.0 Hz, 1H); C NMR (CDCl
3
, 75 MHz) d: 146.0, 145.7,
1
4
30.3, 126.2, 118.8, 108.9, 81.6, 57.8 (2C), 48.9, 47.6, 45.2, 42.8,
75 MHz) d: 146.9, 144.6, 143.4, 140.3, 131.6, 129.7 (2C), 129.0,
127.4 (2C), 126.1, 123.8, 111.0, 58.9, 56.9, 56.0, 50.4, 48.7, 41.3,
40.8, 36.1, 34.4, 29.8, 27.9, 22.8, 21.7, 19.3. Positive ESI-MS m/z:
+
2.7, 38.3, 37.6, 35.3, 27.3, 24.2. Positive ESI-MS m/z: 333 [M+H] ,
+
positive HRESIMS m/z: found: 333.2157 [M+H] (calcd for
C
2
D
5
+
+
19 29 2 3
H N O , 333.2178). ½
a
ꢂ
3
¼ þ14:75 (c 0.61, CH OH).
971 [M+H] , positive HRESIMS m/z: found: 971.4289 [M+H] (calcd
2
D
5
for C52
(c = 0.1 mM, CH
H
67
N
4
O
10
S
2
, 971.4299). ½
a
ꢂ
¼ þ120:51 (c 0.72, CH
3
OH). CD
4
.1.2.7. 6b-NH-Ts-7b-Methoxy-7,8-dihydro sinomenine (4). To
3
OH): h229 +18.19 mdeg, h293 ꢀ1.71 mdeg.
a solution of 8 (332 mg, 1 mmol) in dried CH
2
Cl (20 mL) was
added p-toluene sulfonyl chloride (228 mg, 1.2 mmol) and Et
0.21 mL, 1.5 mmol) at 0 °C. The reaction mixture was allowed to
warm to room temperature and after 5 h was quenched with water
5 mL), added more CH Cl (50 mL) into the system. Then the or-
ganic layer was separated, washed with brine (20 mL ꢁ 5), dried
over Na SO , filtered and concentrated. Purification by column
chromatography (CH Cl /MeOH, 9:1, v/v) afforded the titled com-
pound as white solid (467 mg, 96.1%).
00 MHz) d: 7.66 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.1 Hz, 2H), 6.73
d, J = 8.4 Hz, 1H), 6.62 (d, J = 8.4 Hz, 1H), 5.85 (br s, 1H), 4.11 (d,
2
3
N
4.1.2.9. 6b-NH-Ms-7b-Methoxyl-7,8-dihydro-sinomenine (5). To
a solution of 8 (332 mg, 1 mmol) in dried CH Cl (20 mL) was added
mesyl chloride (137 mg, 1.2 mmol) and Et N (0.21 mL, 1.5 mmol) at
0 °C. The reaction mixture was allowed to warm to room tempera-
ture and quenched with water (5 mL), added more CH Cl (50 mL)
into the system 5 h later. Then the organic layer was separated,
washed with brine (20 mL ꢁ 5), dried over Na SO , filtered and con-
centrated. Purification by silica gel column chromatography
(CH Cl /MeOH, 10:1, v/v) afforded the title compound as a white
3
solid (377 mg, 92%). H NMR (CDCl , 300 MHz) d: 6.75 (d,
(
2
2
3
(
2
2
2
2
2
4
2
2
2
4
1
a
3
H NMR (CDCl ,
3
(
2
2
1
J = 8.7 Hz, 1H), 3.93–3.97 (m, 1H), 3.88 (s, 3H), 3.79 (dd, J = 14.7
and 3.3 Hz, 1H), 3.21 (dt, J = 12.0 and 4.2 Hz, 1H), 2.97 (s, 3H),
J = 8.4 Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 6.32 (s, 1H), 4.08 (br s, 2H),
3.83–3.89 (m, 1H), 3.86 (s, 3H), 3.37 (s, 3H), 3.27–3.33 (m, 1H),
2.95 (d, J = 18.3 Hz, 1H), 2.86 (s, 3H), 2.70 (dd, J = 18.3 and 5.4 Hz,
1H), 2.45 (dd, J = 12.3 and 3.0 Hz, 1H), 2.37 (s, 3H), 1.99 (td,
J = 16.1 and 3.0 Hz, 1H), 1.87–1.93 (m, 1H), 1.67–1.80 (m, 2H),
1.51 (td, J = 12.6 and 4.5 Hz, 1H), 1.31–1.43 (m, 2H). 13C NMR
2
5
1
1
3
1
1
3
.91 (d, J = 18.3 Hz, 1H), 2.82–2.85 (m, 1H), 2.67 (dd, J = 18.3 and
.1 Hz, 1H), 2.40–2.44 (overlapped, 1H), 2.40 (s, 3H), 2.34 (s, 3H),
.97 (td, J = 12.3 and 3.0 Hz, 1H), 1.85 (dt, J = 12.9 and 2.4 Hz,
H), 1.73 (dt, J = 12.9 and 2.4 Hz, 1H), 1.59 (dt, J = 12.9 and
.0 Hz, 1H), 1.22–1.50 (m, 3H). 13C NMR (CDCl
3
, 75 MHz) d:
3
(CDCl , 75 MHz) d: 145.1, 144.6, 130.6, 124.2, 119.5, 110.0, 79.9,
45.0, 144.5, 142.4, 139.0, 130.5, 129.0 (2C), 126.9 (2C), 124.3,
19.3, 109.7, 79.2, 57.1, 56.4, 55.7, 51.0, 47.1, 44.6, 42.4, 37.8,
57.3, 56.7, 56.2, 52.1, 47.2, 44.7, 42.5, 42.1, 38.0, 37.9, 35.5, 28.2,
+
23.9. Positive ESI-MS m/z: 411 [M+H] , positive HRESIMS m/z:
+
+
7.4, 35.3, 28.9, 23.7, 21.4. Positive ESI-MS (+) m/z: 487 [M+H] , po-
found: 411.1922 [M+H] (calcd for
20 31 2 5
C H N O S, 411.1954).
+
25
sitive HRESIMS m/z: found: 487.2229 [M+H] (calcd for
C
½
aꢂ
¼ þ27:91 (c 0.43, CHCl
3
).
D
25
26
H
35
N
2
5
O S, 487.2267). ½
aꢂ
D
3
¼ þ67:21 (c 0.60, CH OH).
4
.1.2.10. (S )-6b-NH-Ms-7b-Methoxy-7,8-dihydro disinomenine
4
.1.2.8. (S )-6b-NH-Ts-7b-Methoxy-7,8-dihydro disinomenine
(5a) and (R )-6b-NH-Ms-7b-methoxy-7,8-dihydro disinomenine
(5b). 6b-NH-Ms-7b-Methoxy-7,8-dihydro sinomenine (8, 410 mg,
1.0 mmol) was dissolved in dried CH OH (40 mL) and MnO
3 2
(4a) and (R )-6b-NH-Ts-7b-methoxy-7,8-dihydro disinomenine
4b). The title compounds 4a and 4b were prepared according the
(
procedure described above. Specifically, 6b-NH-Ts-7b-methoxy-7,
-dihydro sinomenine (4, 486 mg, 1.0 mmol) was dissolved in
dried CH OH (40 mL) and MnO (1500 mg, 17.2 mmol) was added
(1500 mg, 17.2 mmol) was added with magnetic stirring, and the
mixture was remained at room temperature for 3 days and then fil-
8
3
2
tered and washed by CH
3
OH (10 mL ꢁ 3). The filtrate and the

P. Teng et al. / Bioorg. Med. Chem. 19 (2011) 3096–3104
3103
washes were combined, dried over anhydrous MgSO
4
and concen-
with chloroform/methanol (15:1, v/v) as eluent to yield 6a
trated. The resulting yellow solid was purified using silica gel col-
umn chromatography with chloroform/methanol (15:1, v/v) as
(243 mg, 63%).
Compound 6a: 1H NMR (CDCl
3
, 300 MHz) d: 8.00 (br t,
eluent to afford 9a (147 mg, 36%) and 9b (164 mg, 40%).
J = 3.0 Hz, 2H), 7.87 (br t, J = 3.0 Hz, 2H), 7.61 (dd, J = 6.0 and
3.0 Hz, 4H), 6.33 (s, 2H), 5.13 (dd, J = 15.0 Hz, 2H), 3.69 (s, 6H),
3.21 (dd, J = 18.0 and 6.0 Hz, 2H), 2.96–3.07(m, 6H), 2.53–2.65
(m, 6H), 2.38 (s, 6H), 2.21–2.33 (m, 2H), 2.19 (d, J = 12.0 Hz, 2H),
1
Compound 5a: H NMR (CDCl
3
, 300 MHz) d: 6.64 (s, 2H), 4.20 (d,
J = 12.0 Hz, 2H), 4.10 (br t, J = 4.2 Hz, 2H), 3.97 (d, J = 14.8 and
3
6
.3 Hz, 2H), 3.90 (s, 6H), 3.41 (s, 6H), 3.33–3.39 (m, 2H), 2.91 (s,
H), 2.79 (s, 2H), 2.39–2.48 (m, 4H), 2.29 (s, 6H), 2.15 (t,
1
3
3
2.00 (td, J = 12.0 and 6.0 Hz, 2H). C NMR (CDCl , 75 MHz) d:
J = 9.3 Hz, 2H), 1.95 (d, J = 12.9 Hz, 2H), 1.83 (d, J = 12.6 Hz, 2H),
154.0, 153.3, 145.0, 143.8, 141.6, 141.4, 131.2, 128.9, 128.8 (2C),
128.4, 128.1, 123.6, 110.4, 56.3 (2C), 47.1, 44.9, 43.3, 42.8, 38.7,
1
1
1
4
.72 (d, J = 12.9 Hz, 2H), 1.59 (td, J = 12.9 and 4.2 Hz, 2H), 1.35–
1
3
+
.43 (m, 4H). C NMR (CDCl
3
, 75 MHz) d: 145.5, 143.7, 132.1,
36.7, 33.9, 23.0. Positive ESI-MS m/z: 773 [M+H] , HRESIMS m/z:
+
27.7, 124.7, 110.6, 79.9, 57.2, 56.6, 56.5, 52.0, 47.5, 43.9, 42.4,
found: 773.3788 [M+H] (calcd for
C
48
H
49
N
6
O
4
,
773.3815).
2
5
2.1, 37.7, 37.4, 35.8, 28.1, 22.2. Positive ESI-MS m/z: 819
½
aꢂ
¼ þ359:22 (c 0.42, CHCl
). CD (c = 0.05 mM, CH OH): h208
3 3
D
+
+
[
M+H] , positive HRESIMS m/z: found: 819.3644 [M+H] (calcd
+17.46 mdeg, h244 +20.00 mdeg, h292 +4.15 mdeg.
25
for C40
H
59
N
4
O
10
S
2
, 819.3673). ½
aꢂ
¼ þ116:13 (c 0.43, CH
3
OH).
D
CD (c = 0.1 mM, CH
3
OH): h205 +14.98 mdeg, h257 +10.27 mdeg,
4.2. Biological assay
h
297 +12.48 mdeg.
Compound 5b: 1H NMR (CDCl
, 300 MHz) d: 13C NMR (CDCl
4.2.1. RAW264.7 cell culture
3
3
,
75 MHz) d: 6.57 (s, 2H), 4.09 (br s, 4H), 3.94 (d, J = 14.4 Hz, 2H),
3.87 (s, 6H), 3.39 (s, 6H), 3.33 (d, J = 12.0 Hz, 2H), 2.91 (s, 2H),
2.87 (s, 6H), 2.57–2.63 (m, 4H), 2.29(s, 6H), 2.08–2.15 (m, 2H),
1.99 (br d, J = 14.4 Hz, 2H), 1.89 (d, J = 12.6 Hz, 2H), 1.72 (d,
RAW264.7 cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 5%
fetal bovine serum (FBS) (Invitrogen), 100 U/mL penicillin, and
100
lg/mL streptomycin and incubated at 37 °C in a humidified
J = 12.6 Hz, 2H), 1.62 (td, J = 12.2 and 4.2 Hz, 2H), 1.43 (d,
atmosphere containing 5% CO .
2
13
3
J = 13.8 Hz, 2H), 1.24–1.36 (m, 2H). C NMR (CDCl , 75 MHz) d:
In all cell cultures, each compound was prepared in dimethyl
sulphoxide (DMSO) followed by dilution with culture medium to
desired concentrations, and DMSO final concentration was 0.1%.
DMSO at 0.1% was added into control (RAW264.7 cells were trea-
ted with LPS only) and blank (RAW264.7 cells only, without any
treatments) groups and showed no effects on cells.
1
5
45.3, 144.0, 131.4, 127.7, 124.5, 112.1, 79.6, 59.6, 57.1, 56.5,
2.0, 47.0, 43.8, 42.5, 42.0, 38.1, 37.7, 35.8, 28.4, 23.9. Positive
+
+
ESI-MS m/z: 819 [M+H] , HRESIMS m/z: found: 819.3652 [M+H]
2
D
5
(
calcd for C40
CH OH). CD (c = 0.1 mM, CH
1.67 mdeg.
H
59
N
4
O
10
S
2
, 819.3673). ½
a
h
ꢂ
¼ þ101:38 (c 0.61,
3
3
OH):
211 +23.00 mdeg,
h
290
ꢀ
4
.2.2. Measurement of nitrite levels in the culture supernatants
4
4
.1.2.11. 6,7-Quinoxaline sinomenine (6). Sinomenine (9.9 g,
RAW264.7 cells were plated at a density of 5 ꢁ 10 cells in a 96-
0
.03 mol) was dissolved in 10% HCl (50 mL) under N and stirred
2
well plate, and compounds at various concentrations (0.5–200 lM)
at reflux. After 10 h, the mixture was cooled to ambient tempera-
ture followed by base workup (pH 10) with 15% ammonia water
were added to each plate with or without LPS (1
lg/mL). After a
24 h incubation period, 100 L of culture medium was mixed with
l
and then extracted with CH
was washed with brine, dried over Na
and recrystallized from MeOH to give a flavescent solid 9 (7.65 g,
1%). A solution of compound 9 (315 mg, 1 mmol) and o-phenyl-
enediamine (130 mg, 1.2 mmol) in MeOH (30 mL) was stirred un-
der N atmosphere for 12 h. The mixture was filtered and
washed with MeOH (10 mL ꢁ 3), and the filter cake was collected
as a white solid (329 mg, 85%). 1H NMR (CDCl
, 300 MHz) d:
.98–8.01 (m, 1H), 8.84–8.87 (m, 1H), 7.56–7.63 (m, 2H), 6.59 (s,
2
Cl
2
(80 mL ꢁ 3). The CH
2
Cl
2
extract
an equal volume of Griess reagent (0.2% naphthylethylenediamine
dihydrochloride and 2% sulfanilamide in 5% phosphoric acid). After
10 min incubation at room temperature with protection from light,
the absorbance value was measured at 540 nm. Aminoguanidine
(AG) was used as positive control.
2
SO , filtered, concentrated
4
8
2
4.2.3. Cell viability assay
4
3
Cells were plated at a density of 5 ꢁ 10 cells in a 96-well plate,
7
1
3
1
and compounds were added to each plate at the indicated concen-
H), 6.58 (s, 1H), 6.07 (s, 1H), 5.02 (dd, J = 15.0 Hz, 1H), 3.70 (s,
H), 3.25 (dd, J = 6.0 Hz, 1H), 3.19 (dd, J = 6.0 Hz, 1H), 3.08 (brs,
H), 2.88–3.02 (m, 3H), 2.58–2.62 (m, 2H), 2.48 (s, 3H), 2.20 (td,
trations. After a 24 h incubation period, 20 lL MTT reagent (5 mg/
mL) was added, and the cells were incubated for 4 h. The superna-
tants were aspirated, and the formazan crystals in each well were
J = 12.0 and 3.0 Hz, 1H), 2.10 (d, J = 12.0 Hz, 1H), 1.94 (td, J = 12.0,
dissolved in 200 lL of dimethyl sulfoxide for 30 min at 37 °C. The
1
3
and 6.0 Hz, 1H). C NMR (CDCl
3
, 75 MHz) d: 153.9, 153.5. 144.7,
44.5, 141.5, 131.1, 128.8, 128.8 (2C), 128.3, 123.1, 118.8, 109.0,
6.7, 56.0, 47.0. 44.6, 43.1, 42.8, 38.2, 36.3, 33.7, 23.4. Positive
absorbance value was monitored by microplate reader at 570 nm
and the cytotoxicity (%) was calculated using the following
formula:
1
5
+
ESI-MS m/z: 388 [M+H] , positive HRESIMS-ESI m/z: found:
The cytotoxicity ð%Þ ¼ ½1 ꢀ ðCompounds ðOD570 nm
Background ðOD570 nmÞÞ=ðControl ðOD570 nm
ꢀ Background ðOD570 nmÞÞꢂ ꢁ 100
Þ
+
3
88.2000 [M+H] (calcd for C24
26 3 2
H N O , 388.2025).
ꢀ
Þ
4
.1.2.12. (S)-6,7-Quinoxaline disinomenine (6a). After 4 days of
incubation of mycelia in mineral medium (50 mL, consists of pota-
to extract 200 g, glucose 20 g, KH PO 3.0 g, MgSO O 1.5 g,
ꢃ7H
trace vitamin B , per liter, and with pH 6.0) on a rotary shaker at
60 rpm at 25 °C, 6,7-quinoxaline sinomenine (6, 387 mg,
.0 mmol) was added into the fermentation flask, and the mixture
2
4
4
2
1
The potency results were expressed as the 50% inhibitory con-
centration (IC50).
1
1
was maintained under the same conditions as described above for
an additional 4 days. Mycelia were harvested by filtration, and
4.2.4. Reverse transcriptase-polymerase chain reaction (RT-
PCR) analysis of iNOS mRNA expression
then the filtrate was adjusted to pH 9 by NH
CH Cl
then concentrated under vacuum to afford the crude yellow prod-
uct, which was purified using silica gel column chromatography
4
OH, extracted with
SO , filtrated and
To examine iNOS mRNA expression levels in RAW264.7 cells,
total RNA was extracted using the Trizol reagent, and cDNA was
2
2
(50 mL ꢁ 3), dried over anhydrous Na
2
4
synthesized from 1
lg RNA utilizing M-MLV reverse transcriptase
according to manufacturer’s instruction (TOYOBO, JAPAN). The

3
104
P. Teng et al. / Bioorg. Med. Chem. 19 (2011) 3096–3104
9. Strand, V.; Kimberly, R.; Isaacs, J. D. Nat. Rev. Drug Disc. 2007, 6, 75.
PCR reaction was performed by the following reaction conditions:
9
7
PCR products were run on a 1% agarose gel and were visualized
by ethidium bromide staining. The produced bands in the gel were
then photographed. The primer sequences used in this study were
as follows: iNOS: forward, 5 -CAACATCAGGTCGGCCATCACT-3 ;
reverse, 5 -ACCAGAG GCAGCACATCAAAGC-3 ; b-actin: forward,
1
0. Ohtsuka, M.; Konno, F.; Honda, H.; Oikawa, T.; Ishikawa, M.; Iwase, N.; Isomae,
K.; Ishii, F.; Hemmi, H.; Sato, S. J. Pharmacol. Exp. Ther. 2002, 303, 52.
1. Hu, X. D.; Yang, Y.; Zhong, X. G.; Zhang, X. H.; Zhang, Y. N.; Zheng, Z. P.; Zhou,
Y.; Tang, W.; Yang, Y. F.; Hu, L. H.; Zuo, J. P. J. Ethnopharmacol. 2008, 120, 447.
2. Van’t Hof, R. J.; Ralston, S. H. Immunology 2001, 103, 255.
3. Xu, M.; Liu, L.; Qi, C.; Deng, B.; Cai, X. Planta Med. 2008, 74, 1423.
4. Wang, Y.; Fang, Y. F.; Huang, W. H.; Zhou, X.; Wang, M. H.; Zhong, B.; Peng, D. Z.
J. Ethnopharmacol. 2005, 98, 37.
4 °C for 5 min and 30 cycles at 94 °C for 30 s, 55 °C for 30 s and
2 °C for 30 s with a final elongation step of 72 °C for 5 min. The
1
1
1
1
0
0
0
0
15. Huang, B. D.; He, A. S.; Fu, M.; Sheng, P. Y.; Liao, W. M. J. Med. Plants Res. 2010, 4,
830.
1
0
0
0
5
-TGCTGTCCCTGTATGCCTCT-3 ; reverse, 5 -TTTGATGTCACG CAC
1
1
1
6. Liu, Z. Q.; Chan, K.; Zhou, H.; Jiang, Z. H.; Wong, Y. F.; Xu, H. X.; Liu, L. Life Sci.
0
GATTT-3 .
2005, 77, 3197.
7. Song, S. H.; Shen, X. Y.; Tang, Y.; Wang, Z. X.; Guo, W. Y.; Ding, G. S.; Wang, Q.
X.; Fu, Z. R. Int. Immunopharmacol. 2010, 10, 679.
8. Huang, G. D.; Li, J. B.; Huang, Y. H.; Huang, D. F.; Tang, L. J.; Zhang, S.; Li, Q. J.
Emerg. Tradit. Chin. Med. 2007, 16, 416.
Acknowledgments
1
2
2
9. Yamasaki, H. Acta Med. Okayama 1976, 30, 1.
0. Goto, K. S. H. Bull. Chem. Soc. Jpn. 1929, 4, 107.
1. Goto, K. Bull. Chem. Soc. Jpn. 1929, 4, 129.
This work was supported by National Natural Science Founda-
tion of China
2007CB714504) and the National Natural Science Fund for Crea-
tive Research Groups of China (20821063). We thank Dr. Yin Ding
Nanjing University, China) for HRMS measurements.
(20872060, 90913023), the 973 Program
(
22. Zuo, Z.; Jimenez, X.; Witte, L.; Zhu, Z. P. Protein Eng. 2000, 13, 361.
23. Amara, J. F.; Clackson, T.; Rivera, V. M.; Guo, T.; Keenan, T.; Natesan, S.; Pollock,
R.; Yang, W.; Courage, N. L.; Holt, D. A.; Gilman, M. P. Natl. Acad. Sci. U.S.A. 1997,
(
9
4, 10618.
2
2
2
2
4. Deng, Z. S.; Li, J. X.; Teng, P.; Li, P.; Sun, X. R. Org. Lett. 2008, 10, 1119.
5. Deng, Z. S.; Zhao, Y.; He, C. C.; Jin, J.; He, Y. M.; Li, J. X. Org. Lett. 2008, 10, 3879.
6. Minamikawa, J. I.; Tijima, I.; Brossi, A. Heterocycles 1978, 10, 79.
7. Goto, K. I. R. Chem. Pharm. Bull. 1931, 93.
Supplementary data
1
Supplementary data (CD spectra of dimers, H NMR spectra and
HPLC analysis results of the compounds) associated with this
article can be found, in the online version, at doi:10.1016/
j.bmc.2011.04.006. These data include MOL files and InChiKeys of
the most important compounds described in this article.
28. Christians, U. K., V. W. U.S. Patent, 6372756B1, 2002.
2
3
9. Cheng, W. M.; Qiu, F.; Yao, X. S. J. Asian Nat. Prod. Res. 2007, 9, 13.
0. McCamley, K.; Ripper, J. A.; Singer, R. D.; Scammells, P. J. J. Org. Chem. 2003, 68,
9847.
31. Iijima, I.; Minamikawa, J. I.; Jacobson, A. E.; Brossi, A.; Rice, K. C. J. Org. Chem.
1978, 43, 1462.
3
3
3
2. Sayre, L. M.; Portoghese, P. S. J. Org. Chem. 1980, 45, 3366.
3. Weller, D. D.; Rapoport, H. J. Med. Chem. 1976, 19, 1171.
4. Bachi, M. D.; Korshin, E. E.; Hoos, R.; Szpilman, A. M.; Ploypradith, P.; Xie, S. J.;
Shapiro, T. A.; Posner, G. H. J. Med. Chem. 2003, 46, 2516.
References and notes
1
2
3
.
.
.
Bruckdorfer, R. Mol. Aspects Med. 2005, 26, 3.
Bruene, B. Pharmacol. Res. 2010, 61, 265.
Huang, Z. H.; Huang, P. L.; Panahian, N.; Dalkara, T.; Fishman, M. C.; Moskowitz,
M. A. Science 1994, 265, 1883.
Huang, P. L.; Huang, Z. H.; Mashimo, H.; Bloch, K. D.; Moskowitz, M. A.; Bevan, J.
A.; Fishman, M. C. Nature 1995, 377, 239.
35. Portoghese, P. S.; Nagase, H.; Maloneyhuss, K. E.; Lin, C. E.; Takemori, A. E. J.
Med. Chem. 1991, 34, 1715.
36. Li, J. F.; Zhao, Y.; Cai, M. M.; Li, X. F.; Li, J. X. Eur. J. Med. Chem. 2009, 44, 2796.
37. Goto, K. S. H. Bull. Chem. Soc. Jpn. 1935, 10, 597.
38. Lou, Y. T.; Zhou, H. B.; Zou, J.; Yan, L. C.; Bi, E. G.; Sun, B.; Yao, Z. J. Tetrahedron
Lett. 2010, 51, 485.
4
.
5
6
.
.
Groves, J. T.; Wang, C. C. Y. Curr. Opin. Chem. Biol. 2000, 4, 687.
Maccallini, C.; Patruno, A.; Besker, N.; Ali, J. I.; Ammazzalorso, A.; De Filippis, B.;
Franceschelli, S.; Giampietro, L.; Pesce, M.; Reale, M.; Tricca, M. L.; Re, N.;
Felaco, M.; Amoroso, R. J. Med. Chem. 2009, 52, 1481.
Vallance, P. Fund. Clin. Pharmacol. 2003, 17, 1.
39. Xie, Q. W.; Whisnant, R.; Nathan, C. J. Exp. Med. 1993, 177, 1779.
40. Ciz, M.; Pavelkova, M.; Gallova, L.; Kralova, J.; Kubala, L.; Lojek, A. Physiol. Res.
2008, 57, 393.
41. Kang, K. S.; Yamabe, N.; Kim, H. Y.; Okamoto, T.; Sei, Y.; Yokozawa, T. J.
Ethnopharmacol. 2007, 113, 225.
7
8
.
.
Smolen, J. S.; Steiner, G. Nat. Rev. Drug Disc. 2003, 2, 473.