Discovery and synthesis of new immunosuppressive alkaloids from the stem of Fissistigma oldhamii (Hemsl.) Merr.

Article abstract of DOI:10.1016/j.bmc.2006.10.034

Three new alkaloids (1-3) and twenty-one known compounds were isolated from the stem of Fissistigma oldhamii (Hemsl.) Merr. which was the ruler herb in an approved Traditional Chinese herbal formula used for treatment of rheumatoid arthritis in China and synthesis of one new immunosuppressive alkaloid was achieved. These compounds, including the crude extracts of this herb, exhibited strong activities in the inhibition of T and B cell proliferation.

Full text of DOI:10.1016/j.bmc.2006.10.034

Bioorganic & Medicinal Chemistry 15 (2007) 988–996
Discovery and synthesis of new immunosuppressive alkaloids
from the stem of Fissistigma oldhamii (Hemsl.) Merr.
Yi-Nan Zhang, Xiang-Gen Zhong, Zong-Ping Zheng, Xu-Dong Hu,
Jian-Ping Zuo* and Li-Hong Hu*
Shanghai Research Center for Modernization of Traditional Chinese Medicine and First Department of Pharmacology,
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 201203, China
Received 5 September 2006; revised 16 October 2006; accepted 16 October 2006
Available online 19 October 2006
Abstract—Three new alkaloids (1–3) and twenty-one known compounds were isolated from the stem of Fissistigma oldhamii
(Hemsl.) Merr. which was the ruler herb in an approved Traditional Chinese herbal formula used for treatment of rheumatoid
arthritis in China and synthesis of one new immunosuppressive alkaloid was achieved. These compounds, including the crude
extracts of this herb, exhibited strong activities in the inhibition of T and B cell proliferation.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
ation (see Table 2). Fissistigma oldhamii was previously
reported to contain alkaloids,7 furanones,8 cyclopente-
nones,9 flavonoids, and chalcones.
Rheumatoid arthritis (RA) is a chronic inflammatory
disease of the joints. It is a disease that afflicts large pop-
ulation around the world with no effective treatment
available so far for its cure. Scientific evidence exists that
RA is a type of autoimmune disease1 and that it has
strong relevance with T and B cells.2–4 The evidences
provided prove that T and B lymphocytes play a vital
role in the pathogenesis of RA suggesting that blockade
of these cells may prove to be an effective therapeutic
mechanism in a variety of autoimmune disorders. Thus,
many immunosuppressive agents were employed for the
therapy of RA.
In order to identify the active constituents responsible
for inhibition of T and B lymphocyte proliferation from
this herb we systematically examined the ethanolic ex-
tract in the present study. Twenty-four compounds,
including 9 aporphine alkaloids, 8 aristolactams, 4
oxoaporphines, 2 dioxoaporphines, and 1 morphinandi-
ene, are being reported. The structures of three new
compounds 1–3 were mainly elucidated by NMR data.
The two oxoaporphine alkaloids exhibited stronger
activities against T and B cell inhibition than others.
Due to low yield of 1 from the plant material, we have
accomplished its total synthesis which enabled us to
synthesize sufficient compound for further biological
studies.
In search of new anti-RA agents with high efficacy and
low toxicity, attention to anti-RA herbs or formulas
from Traditional Chinese Medicine was an obvious
logical research option resulting in the findings we like
to report here. A SFDA (State Food and Drug Adminis-
tration in China) approved herbal formula, zuanshanf-
eng syrup,5 and its ruler herb, the stem of Fissistigma
oldhamii (Hemsl.) Merr.,6 were found to have strong
inhibitory activities against T and B lymphocyte prolifer-
2. Results and discussion
2.1. Structural elucidation of three new compounds (1–3)
2.1.1. Oxodiscoguattine (1). Compound 1, isolated as red
amorphous powder, showed a molecular ion peak at m/z
335.0788 in its HREI-MS, indicating a molecular
formula of C19H13NO5 (calcd for [M]+, 335.0794). Its
UV spectrum (absorption maxima at 230, 265 sh, 330,
*
Corresponding authors. Tel./fax: +86 21 50802221 (L.-H.H.); tel./fax:
+86 21 50806701 (J.-P.Z.); e-mail addresses: simmhulh@mail.shcnc.
ac.cn; jpzuo@mail.shcnc.ac.cn
The two authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.034

Y.-N. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 988–996
989
and 356 nm) and IR spectrum (a conjugated carbonyl
group absorption band at 1660 cmꢀ1) indicated 1 was
an oxoaporphine derivative.10 Its NMR spectra exhibit-
ed the presence of two methoxyl groups [dH 3.98 and dC
55.6; dH 3.97 and dC 55.8], one methylenedioxy group
[dH 6.22 (2H, s) and dC 101.7], one aromatic protons
[dH 7.10 (1H, s) and dC 101.7], two meta-coupled
aromatic protons [dH 7.65 (1H, d, J = 2.6) and dC
120.4; dH 6.83 (1H, d, J = 2.6) and dC 105.2], and two
ortho-coupled aromatic protons [dH 7.71 (1H, d,
J = 5.1) and dC 124.2; dH 8.82 (1H, d, J = 5.1) and dC
143.7]. In addition, one carbonyl group [dC 182.3] and
10 quaternary carbons were present in the molecule.
These data, together with comparison with NMR data
of the known aporphine alkaloid, dicentrinone from
the literature,11 established the structure of compound
1, as a new 9,11-dimethoxy-1,2-methylenedioxyoxoa-
porphine, named oxodiscoguattine,12 and its 1H and
13C NMR spectra (Table 1) were assigned unambigu-
ously by the long-range correlations in its HMBC,
HMQC, and NOESY spectra, as shown in Scheme 1.
The structure was also confirmed by our synthesis work
below (Scheme 2).
2.1.2. Oxocalycinine (2). Compound 2 was obtained
as red amorphous powder. The molecular formula of
C18H11NO5 was assigned to 2 on the basis of its
HREI-MS (found m/z 321.0623, calcd for [M]+ was
1
m/z 321.0637). The H and 13C NMR data of 2 were
very similar to those of compound 1 (see Table 1), which
was only lack of a methoxyl group. Cross peaks between
the methoxyl proton to H-8 and H-10 in its NOESY
spectrum (see Fig. 1) determined that the hydroxyl
group should be located at C11 and the residual methox-
yl group to C9. So, compound 2, named oxocalycinine,13
was determined as a new 11-hydroxy-9-methoxy-1,
2-methyl-enedioxyoxoaporphine.
2.1.3. Oldhamactam (3). Compound 3, mp 224–226 ꢁC,
was obtained as brownish-yellow powder. The HREI-
MS, exhibiting a molecular ion peak at m/z 325.0934,
indicated that the molecular formula was C18H15NO5
Table 1. NMR spectroscopic data of compounds 1a, 2, and 3
Position
1 (in CDCl3)
2 (in DMSO)
Position
3 (in DMSO)
dH
dC
dH
dC
dH
dC
1
1a
147.5
124.1
123.1
152.8
101.7
136.1
124.2
143.7
144.0
122.4
124.0
156.8
107.9
135.4
124.0
144.1
1
115.7
153.5
153.3
146.0
125.1
109.6
117.0
126.8
110.8
156.3
125.7
99.5
2
1b
2
3
4
4a
3
3a
7.10, s
7.41, s
4b
5
4
7.71, d, (5.1)
8.82, d, (5.1)
7.95, d, (5.1)
8.73, d, (5.1)
8.58, d, (8.1)
7.35, dd, (8.1, 7.5)
7.03, d, (7.5.)
5
6
6
6a
7
144.0
182.3
102.4
116.2
161.0
105.2
158.4
134.1
101.7
55.6
146.5
181.3
102.7
113.6
160.3
101.5
152.2
134.2
102.1
55.5
8
8a
7
8
8a
7.65, d, (2.6)
6.83, d, (2.6)
7.34, d, (2.7)
6.85, d, (2.7)
9
10
7.53, s
133.2
123.0
166.2
62.5
9
10
10a
11
11
11a
OMe, 2
OMe, 3
OMe, 4
OH, 8
NH
4.39, s
3.90, s
4.08, s
10.10, s
10.90, s
61.4
60.7
OCH2O
OMe, 9
OMe, 11
6.22, s
3.98, s
3.97, s
6.34, s
4.02, s
55.8
a 1H NMR (300 MHz) was referenced to d 2.50 (DMSO) and 13C NMR (75 MHz) to d 39.51 (DMSO); 1H NMR (300 MHz) was referenced to d 7.26
(CDCl3) and 13C NMR (75 MHz) to d 77.23 (CDCl3); chemical shifts are shown in the d scale with J values (Hz) in parentheses.
Scheme 1. Structure of compound 1 ,2, 3 and HMBC correlations of 1.

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Y.-N. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 988–996
Scheme 2.
1
(calcd for [M]+, 325.0951). The IR spectrum showed
group. In the H NMR spectrum of 3, an AB2 pattern
was observed at d 8.58 (1H, d, J = 8.1 Hz), d 7.35 (1H,
dd, J = 8.1, 7.5 Hz), and d 7.03 (1H, d, J = 7.5 Hz).
The NOESY correlations observed between d 4.39/
3.90, 3.90/4.08, 4.08/8.58, 8.58/7.35, 7.35/7.03, and 10.10
(–OH)/7.03 (see Fig. 1), and determined that the hydrox-
yl group was located at C8 and three methoxyl groups
were located at C2, C3, and C4. Thus, 3 was established
absorptions for NH (3378 cmꢀ1), C@O (1667 cmꢀ1
)
groups, and phenolic hydroxyl group (3439 cmꢀ1),
respectively. The UV spectrum also exhibited absorp-
tions at 252, 296, and 397 nm, which corresponded to a
phenanthrene chromophore. Comparing its H and 13C
1
NMR data (see Table 1) with that of the known com-
pound aristolactam F II (21),14 3 had an extra methoxyl

Y.-N. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 988–996
991
Figure 1. The NOESY correlations of compounds 1, 2, and 3.
as 10-amino-8-hydroxy-2,3,4-trimethoxyphen-anthrene-
1-carboxylic acid lactam, which was given a trivial name,
oldhamactam.
was far less than CsA. Compounds 1, 2, 4, 14, 17, 19,
20, 24, and 1k had relatively good activities (IC50
<
5 lM), among which compound 4 had the best SI value.
The SI value of inhibition on LPS-induced B cell prolif-
eration indicated the fraction 0 (SI > 93) had more potent
value than CsA (SI = 8), although it also showed rela-
tively low activity. Particularly, compounds 1, 2, 4, 5,
14, 19, 21, 24, and 1k had much higher activities
(IC50 < 5 lM) than others, among which compound 4
(SI = 108) showed the best SI value.
2.2. Known compound isolated from the bark of
F. oldhamii (Hemsl.) Merr.
The other known compounds (see Scheme 2) obtained in
this study, calycinine (4),13 N-methyl-2,3,6-trim-
ethoxymorphinandien-7-one (5),7a aristololactam A IIIa
(6),15 xylopine (7),16 crebanine (8),13 isolaureline (9),13
asimilobine (10),13 duguevanine (11),13 corytuberine
(12),17 aristololactam A II (13),18 4,5-dioxodehydro asi-
milobine (14),19 isoboldine (15),14 goniothalactam
(16),14 aristolactam F I (17),20 glaucine (18),17 norcepha-
radione B (19),19 oxoxylopine (20),16 aristolactam F II
(21),14 O-methyl-moschatoline (22),21 aristololactam B
II (23),18 and aristololactam B III (24),18 were identified
by comparing their spectroscopic data (1H NMR, 13C
NMR, DEPT, NOESY, and MS) with published values.
From the preliminary immunosuppressive evaluation
in vitro, we could find the following. (i) This syrup
and the ruler herb both showed strong activities in
inhibiting the T or B cell proliferation, and their action
mechanism in treatment of the RA might be attributed
to inhibition of T and B cell functions. (ii) The major
effective components of this herb were alkaloids, such
as aporphines, oxoaporphines, and aristolactams. Fur-
thermore, the aporphines (4, 7, 10, 11, 15, and 1k) and
oxoaporphines (1, 2, 20, and 22) were more potent than
aristolactams (3, 6, 13, 16, 21, and 23) except 17 and 24.
For the toxicity of dione groups,24 dioxoaporphines (14,
19), which were not suitable for further research, dis-
played similar cytotoxicities to their immunosuppressive
activities. (iii) Interestingly in aporphines, secondary
amine (4, 7, 10, 11, 15, and 1k) exhibited a much more
potent inhibitory effect than the methyl substituted ter-
nary amine (8, 9, 12, 18), and 1,2 methylene group (4,
7, 11, 1k) was more active than the methoxy groups
(10, 15) at C1 and C2 position.
2.3. Biological activity evaluation and discussion
Extract from herbal formula syrup (sample 1), 95% eth-
anol extract of F. oldhamii, (fraction 0), and all isolated
compounds were tested in vitro for their cytotoxicity on
murine spleen cells, inhibition activity on concanavalin
A (ConA)-induced T cell proliferation, and lipopolysac-
charide (LPS)-induced B cell proliferation, with CsA
(see Experimental section) as control. The pharmacolog-
ical results of these compounds are summarized in
Table 2. The cytotoxicity activity of each compound
was expressed as the concentration of compound that
reduced cell viability to 50% (CC50). The immunosup-
pressive activity of each compound was expressed as
the concentration of compound that inhibited T and B
cell proliferation to 50% (IC50) of the control value.
The selective index (SI) value was used to evaluate the
bioactivity of compounds. In the experiments for testing
bioactivity of each compound, results were expressed as
means standard error. Student’s t-test was used to
determine variances between groups where appropriate,
although these data are not shown in Table 2.
2.4. Synthesis of oxodiscoguattine
As described above, compounds 1, 2, and 4 showed bet-
ter characteristics both in activity and in SI value, how-
ever, the isolated yield of 1 (0.014%) and 2 (0.012%)
from the title plant was relatively low. Considering that
these three compounds’ only difference in synthetic work
was the different starting substituted benzonate ester, we
chose the new compound oxodiscoguattine as our target
to probe the synthesis of this kind compound. And in
the next further biological study, this synthetic work
would give us a chance of looking for the proper targets
in vitro and for further structure–activity relationship
study. The approach of synthesis is outlined in
Scheme 3, starting with methylation of methyl 3,5-
On the basis of the inhibition of ConA-induced T lym-
phocyte proliferation, sample 1 and fraction 0 both
exhibited a satisfactory SI value although their activity

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Y.-N. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 988–996
Table 2. Inhibitory effects of CsA (positive control), syrup, crude extract, and compounds 1–24, 1k on spleen lymphocyte proliferation induced by
mitogens in vitro
Compounda
Cytotoxicity CC50 (M)
IC50 (M) [SIb]
Inhibition of ConA-induced T cell proliferation
Inhibition of LPS-induced B cell proliferation
1
3.23 · 10ꢀ5
4.39 · 10ꢀ6
2.01 · 10ꢀ4
3.16 · 10ꢀ4
5.89 · 10ꢀ5
>1 · 10ꢀ4
6.46 · 1ꢀ04ꢀ5
>1 · 10ꢀ4
>1 · 10ꢀ4
>1 · 10ꢀ4
>1 · 10
3.58 · 10ꢀ6 [9]
8.10 · 10ꢀ7 [5]
2.28 · 10ꢀ5 [9]
4.86 · 10ꢀ6 [65]
3.93 · 10ꢀ5 [1]
1.85 · 10ꢀ5 [>5]
5.69 · 1ꢀ04ꢀ6 [11]
>1 · 10
2.15 · 10ꢀ6 [15]
5.60 · 10ꢀ7 [8]
4.17 · 10ꢀ5 [5]
2.93 · 10ꢀ6 [108]
3.97 · 10ꢀ6 [15]
1.92 · 10ꢀ5 [>5]
8.10 · 1ꢀ04ꢀ6 [8]
>1 · 10
2
3
4
5
6
7
8
9
10
>1 · 10ꢀ4
>1 · 10ꢀ4
4.48 · 10ꢀ5 [>2]
6.86 · 10ꢀ6 [>14]
2.32 · 10ꢀ5 [>4]
>1 · 10ꢀ4
7.96 · 10ꢀ5 [>1]
5.54 · 10ꢀ6 [>18]
2.13 · 10ꢀ5 [>4]
>1 · 10ꢀ4
11
12
>1 · 10ꢀ4
>1 · 10ꢀ4
5.15 · 1ꢀ04ꢀ6
>1 · 10
13
14
4.93 · 10ꢀ6 [1]
2.40 · 10ꢀ5 [>4]
>1 · 10ꢀ4
3.12 · 10ꢀ7 [17]
2.96 · 10ꢀ5 [>3]
>1 · 10ꢀ4
15
16
>1 · 10ꢀ4
8.12 · 10ꢀ6
>1 · 10ꢀ4
3.91 · 10ꢀ6
1.62 · 10ꢀ5
2.47 · 10ꢀ5
>1 · 10ꢀ4
1.11 · 10ꢀ5
9.41 · 10ꢀ6
1.56 · 10ꢀ5
>500 lg/ml
>500 lg/ml
1.35 · 10ꢀ6
17
18
7.98 · 10ꢀ7 [10]
>1 · 10ꢀ4
1.30 · 10ꢀ6 [6]
>1 · 10ꢀ4
19
20
7.95 · 10ꢀ7 [5]
4.16 · 10ꢀ6 [3]
1.00 · 10ꢀ5 [2]
>1 · 10ꢀ4
6.72 · 10ꢀ6 [0.5]
7.29 · 10ꢀ6 [2]
3.96 · 10ꢀ6 [6]
>1 · 10ꢀ4
21
22
23
24
6.96 · 10ꢀ6 [1]
2.29 · 10ꢀ6 [4]
2.43 · 10ꢀ6 [6]
21.5 lg/ml [>23]
6.0 lg/ml [>83]
1.37 · 10ꢀ9 [985]
7.11 · 10ꢀ6 [1]
1.98 · 10ꢀ6 [4]
1.69 · 10ꢀ6 [9]
9.8 lg/ml [>51]
5.4 lg/ml [>93]
1.59 · 10ꢀ7 [8]
1k
Sample 1
Fraction 0
CsA
a The compounds tested for immunosuppressive activity were consistent with the description in the Experimental Section.
b Selectivity index [SI] is determined as the ratio of the concentration of the compound that reduced cell viability to 50% (CC50) to the concentration
of the compound needed to inhibit the proliferation to 50% (IC50) of the control value.
dihydroxybenzonate to form methyl 3,5-dimethoxy-
benzonate, followed by reduction of the benzonate
group and substitution by N-bromosuccinimide. After
the general method22 to get the 2-bromo-3,5-dimethoxy
phenylacetic acid (1f) through the hydrolyzation of
corresponding nitrile, 1f was coupled with 3,4-methylen-
edioxy-phenethylamine catalyzed by N-ethyl-N0-(3-dim-
ethylaminopropyl)carbodiimide hydrochloride (EDC)
and 1-hydroxybenzotriazole (HOBt). The amide 1g was
subject to a Bischler-Napieralski cyclization upon treat-
ment with phosphorous oxychloride, then sodium boro-
hydride reduction and di-tert-butyl dicarbonate (Boc2O)
protection to give the amine 1j. Oxodiscoguattine was
finally accomplished with 6.7% total yield through intra-
molecular phenol ortho-arylation23 using Pd-mediated
catalysis, which followed oxidation by I2 in refluxing
ethanol.
the most outstanding character after considering the com-
prehensive evaluation between SI value and activity.
According to observations and analyses above, we pre-
sumed that this TCM syrup is a very useful resource
to find good anti-RA leads. Isolation on the other six
auxiliary herbs in this syrup,5 further modification of
compounds 1 and 4 and evaluation in more vitro or vivo
models are now in progress. The qualitative structure–
activity relationship was expected to be useful in guiding
the design of new immunosuppressive agents with high
efficacy and low toxicity.
4. Experimental
4.1. General experimental procedure
1H (300 MHz) and 13C (75 MHz) NMR spectra were
recorded on a Varian Mercury-VX300 Fourier trans-
form spectrometer. The chemical shifts were reported
in d (ppm) using the d 7.26 signal of CDCl3 (1H
NMR) and the d 77.23 signal of CDCl3 (13C NMR) as
internal standards. Low resolution mass spectra were
obtained on a Shimadzu GCMS-QP5050A and high res-
olution mass spectra on a Finnigan MAT-95 spectrom-
eter. The IR and UV spectra were run on a Nicolet
3. Conclusion
In summary, the ruler herb of a certificated syrup treated
for RA has been studied. Twenty-four compounds includ-
ing three new alkaloids were isolated and identified,
among which one new compound was synthesized. All
of them as well as the crude extract were assessed for their
cytotoxicity of lymphocyte and activities on the mitogen-
induced T and B cell proliferation in comparison of CsA
in vitro. It is worth noting that compounds 1 and 4 had
Impact 410 spectrophotometer and a
UV2501PC spectrophotometer, respectively.
Shimadzu

Y.-N. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 988–996
993
Scheme 3. Reagents and conditions: (a) 2 equiv CH3I K2CO3, acetone, reflux, 6 h; (b) LiAIH4 THF, rt, 4 h; (c) NBS, CHCl3 rt, overnight; (d) MsCl,
CH2Cl2, 0 ꢁC, 2 h; (e) NaCN, DMF, rt, overnight; (f) 5 N NaOH, MeOH, reflux, overnight; (g) EDC, HOBt, CH2Cl2, 4 A molecular sieves, rt,
overnight; (h) 8 equiv POCl3, CH3CN, reflux, 3 h; then, NaBH4, MeOH; (i) Boc2O, 2 N NaOH (aq)/1,4-Dioxane (v/v, 1/2), rt, 2 h; (j) Pd(OAc)2,
Cy3P, Cs2CO3, 4 A molecular sieves, Argon, 100 ꢁC, overnight; (k) TFA, CH2Cl2 rt, 1.5 h; (l) l2, NaOAc, EtOH, reflux, 6 h.
All commercially available reagents were used without
further purification. The solvents used were all AR
grade and were redistilled under positive pressure of
dry nitrogen atmosphere in the presence of proper desic-
cant when necessary. The progress of the reactions was
monitored by analytical thin-layer chromatography
(TLC) on HSGF254 precoated silica gel plates.
from Sigma. [3H]Thymidine (1 mCi/mL) was purchased
from Shanghai Institute of Atomic Energy (SIAE). The
related in vitro experimental procedure was performed
according to our previous work.25
4.3.2. Animals. BALB/c mice, used at 6–8 weeks of age,
were purchased from Shanghai Experimental Animal
Center and were housed in a controlled environment
(12 h of light/12 h of dark photoperiod, 22 1 ꢁC,
55% 5% relative humidity). All husbandry and exper-
imental contacts made with the mice were conducted un-
der specific pathogen-free conditions. All mice were
allowed to acclimatize in our facility for 1 week before
any experiment started. All experiments were carried
out according to the NIH Guide for Care and Use of
Laboratory Animals and were approved by the Bioeth-
ics Committee of the Shanghai Institute of Materia
Medica.
4.2. Plant material
The stem of F. oldhamii (Hemsl.) Merr. was collected in
Jiangxi Province, People’s Republic of China, in March
2004, and identified by Xinhua Long. A voucher
specimen of the plant (No. 2004134) was deposited at
the herbarium of Shanghai Institute of Materia Medica,
Shanghai, P.R. China.
4.3. Biological assay
4.3.1. Materials. Stock solutions of compounds were pre-
pared with 100% dimethylsulfoxide (DMSO, Sigma) and
diluted with RPMI 1640 medium containing 10% fetal
bovine serum (FBS). MTT (3-(4,5-dimethylthylthiazol-
2-yl)-2,5-diphenyl-tetrazolium bromide), Concanavalin
A (ConA), and lipopolysaccharide (LPS) were purchased
4.3.3. Preparation of spleen cell from mice. BALB/c mice
were sacrificed, and spleens were removed aseptically. A
single cell suspension was prepared after cell debris, and
clumps were removed. Erythrocytes were depleted with
ammonium chloride buffer solution. Lymphocytes were
washed three times with PBS containing 2% FBS and

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Y.-N. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 988–996
were resuspended in RPMI 1640 medium at the indicat-
ed concentration.
The CHCl3 fraction (Fr. 1) was chromatographed over a
silica gel column and eluted with petroleum ether/CHCl3
(3:2–1:1), to give two subfractions (Fr. 1.1, Fr. 1.2). Sub-
fraction Fr. 1.1 was purified using petroleum ether/
CHCl3 (2:1) as solvents, to give 1 (12 mg) and 22
(25 mg). The other subfraction Fr. 1.2 was subjected
to chromatography (petroleum ether/CHCl3 1:1) on Sil-
ica gel to yield 23 (9 mg) and 24 (8 mg).
4.3.4. Cytotoxicity assay. Fresh spleen cells were ob-
tained from BALB/c mice (male, 7–9 weeks old). Spleen
cells (5 · 105 cells) were cultured in 96-well flat plates
with 200 lL RPMI 1640 media containing 10% FBS,
100 U/mL penicillin, and 100 lg/mL streptomycin in a
humidified, 37 ꢁC, 5% CO2-containing incubator for
48 h in the presence or absence of various concentra-
tions of compounds. An amount of 18 lL MTT (5 mg/
mL) was added to each well at the final 5 h of culture.
Then 90 lL of lysis buffer (10% SDS, 50% DMF, pH
7.2) was added to each well for 6–7 h and the absor-
bance value at 570 nm was collected by microplate read-
er. The percentage of cell death was determined using
the following formula:
The CHCl3–MeOH (50:1) fraction (Fr. 2) was crude sep-
arated through Sephadex LH-20 column (25–100 lm,
Merck, Darmstadt, Germany; CHCl3/MeOH 1:1) to
get two subfractions (Fr. 2.1, Fr. 2.2). The simple sub-
fraction (Fr. 2.1) was further purified on silica gel
(CHCl3/MeOH 50:1) to obtain 5 (28 mg) and 11
(13 mg). The relative complex subfraction (Fr. 2.2) was
chromatographed over MCI gel CHP 20P (75–150 lm,
Mitsubishi Kasei Industry Co., Ltd., Tokyo, Japan,
MeOH–water, 50%, 60%, and 80%) to give three further
subfractions (Fr. 2.2.1–Fr. 2.2.3). The Fr. 2.2.1 and Fr.
2.2.3, both advanced on purified Sephadex LH-20 col-
umn (MeOH), gave 16 (6 mg) and 19 (6 mg), respective-
ly. The residue subfraction (Fr. 2.2.2) was subjected to
silica gel (CHCl3/MeOH 100:1–50:1) to obtain 17 (61
mg), 2 (10 mg) and a subfraction (Fr. 2.2.2.1). Interest-
ingly, the unsolvable solid in MeOH, when we prepared
to concentrated Fr. 2.2.2.1 in vacuum, was filtered and
washed to obtain 3 (20 mg).
cytotoxicityð%Þ
¼ ½compoundsðOD570
Þ
ꢀ backgroundðOD570Þꢁ=½controlðOD570
ꢀ backgroundðOD570Þꢁ ꢂ 100
Þ
4.3.5. T Cell and B cell function assay. Fresh spleen cells
were obtained from BALB/c mice (male, 7–9 weeks old).
The 5 · 105 spleen cells were cultured at the same condi-
tions as those mentioned above. The cultures were
unstimulated or stimulated with 5 lg/mL ConA or
10 lg/mL LPS to induce T cell or B cell proliferative
responses, respectively. The compounds were added to
cultures with indicated concentrations to test their bio-
activities. Proliferation was assessed in terms of uptake
of [3H]thymidine during 8 h of pulsing with 20 kBq
[3H]thymidine for each well, and then cells will be har-
vested onto glass fiber filters by a Basic 96 harvester.
The incorporated radioactivity was counted by a liquid
scintillation counter.
The other four fractions (CHCl3/MeOH, 40:1, 20:1,
10:1, and 5:1), separated through repeated Silica gel,
MCI, and Sephadex LH-20 following the procedure
described as above, were got as 15 (25 mg), 18 (7 mg),
20 (6 mg), and 21 (9 mg) in Fr. 3; 8 (12 mg), 9 (5 mg),
and 13 (18 mg) in Fr. 4; 4 (85 mg), 7 (12 mg), and 10
(15 mg) in Fr. 5 and 6 (32 mg), 12 (12 mg), and 14
(33 mg), in Fr. 6, respectively.
4.5. Oxodiscoguattine (1)
1
Red amorphous powder; H and 13C NMR spectrum,
4.4. Extraction and isolation
see Table 1; HREI-MS m/z 335.0788 (calcd for [M]+,
m/z 335.0794); EI-MS m/z 335 (100), 334 (58), 319 (9),
305 (13), 292 (15), 264 (23), 249 (20), 221 (18), 44 (30);
UV (MeOH) kmax (loge) 230 (4.62), 265 (4.45), 330
(4.06), 356 (4.03) nm; IR tmax (KBr) 3047, 2958, 1660,
The commercial syrup (produced by Jiangxi Jinding
Pharmceutical Co. Ltd., China) 100 mL was primarily
purified by D-101 porous resin using H2O as eluant to
wash the superfluous sugar and other additives away.
The active component of syrup (sample 1, 1.4 g) got
from the concentration of 95% EtOH eluant was initial-
ly tested in the inhibition of T and B lymphocytes. (see
Table 2)
1601, 1457, 1413, 1282, 1043, 939 cmꢀ1
.
4.6. Preparation of compound 1b–1
4.6.1. 2-(2-bromo-3,5-dimethoxyphenyl)acetonitrile (1e).
To the dry CH2Cl2 (30 mL) solution of 1d (2.50 g,
10 mmol) in the presence of triethyl amine (2.1 mL,
15.0 mmol), the solution of methanesulfonyl chloride
(0.85 mL, 11.0 mmol) in CH2Cl2 (20 mL) was added
dropwise in 1 h under ice bath. Finishing the addition,
the reaction mixture was still stirred for further 2 h at
room temperature. The mixture was then separated by
CH2Cl2 (2 · 100 mL) and saturated NaHCO3 solution
(50 mL). The collected organic layers were washed by
water (50 mL), and brine, dried over Na2SO4, and con-
centrated. Without further purification, the crude prod-
uct was directly added to the solution of DMF (30 mL)
The dried and milled stem of F. oldhamii (Hemsl.) Merr
(4.0 kg) was extracted using 95% ethanol (3 · 10 L) at
reflux temperature. After filteration and evaporation of
the solvent under reduced pressure, the combined ex-
tracts (86.0 g) were subjected to D-101 porous resin,
eluted with H2O, then 95% EtOH. After that, only the
95% ethanol parts (fraction 0) exhibited the activities
in T and B lymphocyte inhibition, which was collected,
concentrated (19.0 g), and primarily separated by
column chromatography on silica gel 200–300 mesh
eluted with CHCl3/MeOH (CHCl3, 50:1, 40:1, 20:1,
10:1, and 5:1) to afford six parts (Fr. 1–Fr. 6).

Y.-N. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 988–996
995
with sodium cyanide (740 mg, 15.0 mmol) followed. The
mixture was stirred overnight and then extracted by
ether (3 · 50 mL) after diluted with water (50 mL).
The combined organic layer were evaporated in vacuum,
and the residue was subjected to column chromatogra-
phy on silica gel using 5% ethyl acetate in petroleum
ether as eluant to give 2.08 g 1e, yield 81.2%. 1H
NMR (CDCl3, 300 MHz): d 6.53 (d, J = 2.8 Hz, 1H),
6.48 (d, J = 2.8, 1H), 3.91 (s, 2H), 3.86 (s, 3H), 3.78
(s, 3H). EI-MS m/z 255.
dry DMF (40 mL) under an argon atmosphere was heat-
ed at 100 ꢁC overnight. The reaction mixture was al-
lowed to cool and then carefully diluted with 1 N HCl
(20 mL), then extracted with ethyl acetate (2 · 50 mL).
The organic extracts were combined, washed with brine,
dried over by Na2SO4, and concentrated. The residue
was subject to column chromatography on silica gel
using petroleum ether/ethyl acetate (10/1) to give 1j
(600 mg, 1.4 mmol) with moderate yield 71.7% from
1
1h. H NMR (CDCl3, 300 MHz): d 6.56 (s, 1H), 6.47
(m, 1H), 6.46 (m, 1H), 6.03 (d, J = 1.5 Hz, 1H), 5.86
(d, J = 1.5 Hz, 1H), 4.63 (m, 1H), 4.38 (m, 1H), 3.88
(s, 3H), 3.84 (s, 3H), 2.57–2.93 (m, 6H).
4.6.2. N-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-2-(2-bromo-
3,5-dimethoxyphenyl)acetamide (1g). A solution of 1f
(1.95 g, 7.1 mmol), HOBt (1.08 g, 8.0 mmol), 3,4-methy-
˚
lenedioxyphenethylamine (1.40 g, 8.5 mmol), and 4 A
A mixture of TFA/CH2Cl2 (2 mL/10 mL) was added to
2 h, and the reaction mixture was stirred for 1.5 h at
room temperature. The volatiles were removed in vacu-
um and the residue was diluted with 5% NaHCO3
(5 mL). The aqueous mixture was extracted with ethyl
acetate (2 · 25 mL) and the combined organic extracts
were dried (Na2SO4) and concentrated to afford 1k
(400 mg, 1.2 mmol, 87.2%) as white solid. 1H NMR
molecular sieves (1.00 g) in dry CH2Cl2 (35 mL) was
treated with EDC (1.50 g, 8.0 mmol) and the resulting
reaction mixture was stirred at room temperature over-
night. The mixture was partitioned by CH2Cl2 (2 ·
100 mL) and water (50 mL), washed by 1 N HCl
(25 mL), saturated NaHCO3 (50 mL), and brine, dried
over (Na2SO4), and concentrated. The residue was puri-
fied by column chromatography on silica gel using
petroleum ether/acetone (1:1) as eluant to give 2.00 g
product 1g (66.9%). 1H NMR (CDCl3, 300 MHz): d
6.64 (d, J = 7.5 Hz, 1H), 6.55 (d, J = 1.8 Hz, 1H), 6.48
(dd, J = 7.5, 1.8 Hz, 1H), 6.45 (d, J = 2.8 Hz, 1H), 6.42
(d, J = 2.8 Hz, 1H), 5.91 (s, 2H), 5.44 (br s, 1H), 3.88
(s, 3H), 3.78 (s, 2H), 3.68 (s, 3H), 3.40 (q, J = 6.6 Hz),
2.66 (t, J = 6.6 Hz); EI-MS m/z 421.
(CDCl3, 300 MHz):
d
6.55 (s, 1H), 6.47 (d,
J = 2.0 Hz, 1H), 6.44 (d, J = 2.0 Hz, 1H), 6.00 (d,
J = 1.5 Hz, 1H), 5.84 (d, J = 1.5 Hz, 1H), 3.87 (s, 3H),
3.83 (s, 3H), 3.76 (dd, J = 13.0 Hz, 4.2 Hz, 1H), 3.31
(m, 2H), 2.94 (m, 2H), 2.59–2.79 (m, 2H). 13C NMR
(CDCl3, 75 MHz): d 29.45 (CH2), 38.94 (CH2), 43.34
(CH2), 54.02 (CH), 55.59 (CH3), 55.94 (CH3), 97.84
(CH), 100.25 (CH2), 105.15 (CH), 107.05 (CH), 113.91
(C), 126.17 (C), 126.18 (C), 129.51 (C), 139.61 (C),
139.62 (C), 146.93 (C), 157.79 (C), 160.46 (C); EI-MS
m/z 325.
4.6.3. ( ) 5-(2-Bromo-3,5-dimethoxybenzyl)-5,6,7,8-tet-
rahydro-[1,3]dioxolo[4,5-g] isoquinoline (1h). A solution
of 1g (2.00 g, 4.8 mmol) and POCl3 (3.5 mL, 38.0 mmol)
in dry acetonitrile was refluxed for 3 h, and its color
turned to dark red. After working up, the reaction mix-
ture was concentrated in vacuum and then dissolved in
MeOH (20 mL). Excess amount of sodium borohydride
was added to the mixture by portion under ice bath until
the color turned to pale yellow. The mixture was separat-
ed by ethyl acetate (2 · 50 mL) and saturated NaHCO3
(30 mL), washed by water (50 mL) and brine, then dried
by Na2SO4 and concentrated in vacuum. The residue was
chromatographed over silica gel using CHCl3/MeOH
(100:1) to get 800 mg 1h (41.6%). 1H NMR (CDCl3,
300 MHz): d 6.87 (s, 1H), 6.57 (s, 1H), 6.46 (d,
J = 2.8 Hz, 1H), 6.42 (d, J = 2.8 Hz, 1H), 5.91 (s, 2H),
4.25 (dd, J = 10.5 Hz, 2.7 Hz, 1H), 3.89 (s, 3H), 3.81 (s,
3H), 3.38 (dd, J = 13.5 Hz, 2.9 Hz, 1H), 3.22 (m, 1H),
2.91 (m, 2H), 2.72 (m, 2H); EI-MS m/z 405.
4.6.5. Oxodiscoguattine (1). To a refluxing solution of 1k
(100 mg, 0.3 mmol) and sodium acetate (100 mg,
1.2 mmol) in 10 mL ethanol, a solution of iodine
(198 mg, 0.8 mmol) in 10 mL ethanol was added drop-
wise during 15 min. After refluxing for 6 h, the solution
was dissolved in CHCl3 and washed by aqueous 5%
Na2SO3 (30 mL), saturated NaHCO3 (30 mL), and
brine. Removing the volatiles under vacuum, the dark
green solid was subject to column chromatography on
silica gel using CHCl3/MeOH (100:1) as eluant to get
1
67 mg final product (64.7%) as red powder. H NMR
(CDCl3, 300 MHz): d 8.83 (d, J = 5.1 Hz, 1H), 7.73 (d,
J = 5.1 Hz, 1H), 7.68 (d, J = 2.5 Hz, 1H), 7.11 (s, 3H),
6.84 (d, J = 2.5 Hz, 1H), 6.24 (s, 2H), 4.00 (s, 3H),
3.98 (s, 3H). 13C NMR (CDCl3, 75 MHz): d 55.59
(CH3), 55.84 (CH3), 101.70 (CH), 101.73 (CH2), 102.53
(CH), 105.20 (CH), 116.15 (C), 123.09 (C), 124.16 (C),
124.20 (CH), 134.06 (C), 136.15 (C), 143.64 (CH),
143.97 (C), 147.4 (C), 152.82 (C), 158.39 (C), 161.02
(C), 182.29 (C); EI-MS m/z 335.
4.6.4. ( ) Discoguattine (1k). To a mixture of aqueous 2 N
NaOH/1,4-dioxane (5 mL/5 mL) with 1h (800 mg,
2.0 mmol), Boc2O (475 mg, 2.2 mmol) in 5 mL 1,4-diox-
ane was added and stirred for 2 h at room temperature.
The reaction mixture was extracted by ethyl acetate
(2 · 50 mL), washed by water (2 · 30 mL) and brine,
dried (Na2SO4), and concentrated in vacuum to obtain
the crude product 1i.
1
4.6.6. Oxocalycinine (2). Red amorphous powder; H
and 13C NMR spectrum, see Table 1; HREI-MS m/z
321.0623 (calcd for [M]+, m/z 321.0637); EI-MS m/z
321 (100), 320 (55), 295 (38), 292 (20), 277 (11), 265
(50), 250 (42), 234 (13), 222 (23), 164 (27), 84 (52), 66
(56); UV (MeOH) kmax (log e) 225 (4.50), 267 (4.28),
356 (4.01) nm, IR tmax (KBr) 3425, 2951, 1660, 1601,
A
mixture of 1i, tricyclohexylphosphine (230 mg,
0.8 mmol), anhydrous cesium carbonate (1.95 g,
6.0 mmol), and palladium acetate (93 mg, 0.4 mmol) in
1450, 1283 cmꢀ1
.

996
Y.-N. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 988–996
9. Chia, Y. C.; Wu, J. B.; Wu, Y. C. Tetrahedron Lett. 2000,
41, 2199.
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156.
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1983, 46, 761.
4.6.7. Oldhamactam (3). Brownish-yellow amorphous
powder; 1H and 13C NMR spectrum, see Table 1;
HREI-MS m/z 325.0951 (calcd for [M]+, m/z
325.0934); EI-MS m/z 325 (100), 310 (42), 296 (25),
252 (22), 196 (10); UV (CHCl3) kmax (log e) 252 (4.24),
296 (4.82), 397 (4.92) nm; IR tmax (KBr) 3439, 3378,
1667, 1461, 1407, 1245 cmꢀ1
.
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