LC-NMR and LC-MS analysis of 2,3,10,11-oxygenated protoberberine metabolites in Corydalis cell cultures

Article abstract of DOI:10.1016/S0031-9422(03)00497-7

The metabolism of 2,3,10,11-oxygenated protoberberine alkaloids was studied in cell cultures of Corydalis species. Without prior isolation, the structures of the metabolites were determined by LC-MS and LC-NMR analyses. Tetrahydropseudocoptisine α-N-metho salt, pseudoprotopine, and pseudomuramine were identified for the first time, and preliminary evidence for metabolic pathways to the formation of these alkaloids were obtained.

Full text of DOI:10.1016/S0031-9422(03)00497-7

Phytochemistry 64 (2003) 1229–1238
www.elsevier.com/locate/phytochem
LC–NMR and LC–MS analysis of
2,3,10,11-oxygenated protoberberine metabolites
in Corydalis cell cultures
Kinuko Iwasaa,*, Ayako Kuribayashia, Makiko Sugiuraa, Masataka Moriyasua,
Dong-Ung Leeb, Wolfgang Wiegrebec
aKobe Pharmaceutical University, 4-19-1 Motoyamakita, Higashinada-ku, Kobe 658-8558, Japan
bDepartment of Biochemistry, College of Natural Science, Dongguk University, Kyongju 780-714, South Korea
cInstitute of Pharmacy, Regensburg University, D-93040 Regensburg, Germany
Received 7 January 2003; accepted 8 July 2003
Abstract
The metabolism of 2,3,10,11-oxygenated protoberberine alkaloids was studied in cell cultures of Corydalis species. Without prior
isolation, the structures of the metabolites were determined by LC–MS and LC–NMR analyses. Tetrahydropseudocoptisine a-N-
metho salt, pseudoprotopine, and pseudomuramine were identified for the first time, and preliminary evidence for metabolic path-
ways to the formation of these alkaloids were obtained.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Corydalis platycarpa; Corydalis ochotensis var. raddeana; Fumariaceae; Cell cultures; Biotransformation; LC–MS; LC–NMR; Secondary
metabolism; Protoberberine alkaloids
1. Introduction
spirobenzylisoquinolines has been demonstrated
(Zenk, 1994; Iwasa, 1995), no studies on the bio-
synthesis of 2,3,10,11-oxygenated protoberberines have
been presented, in spite of their occurrence in some
plant species including Corydalis species (Preininger,
1986). This paper describes the structural analysis of the
metabolites obtained from cell cultures of Corydalis
species administered with 2,3,10,11-oxygenated proto-
berberines (pseudoprotoberberine). Metabolites were
identified by LC–MS and LC–NMR (Wolfender et al.,
2001). These complimentary techniques enabled the
rapid determination of their structures without the
isolation of individual metabolites. This analysis is
valuable to detect compounds with interesting structural
features and to target their isolation (by preparative
HPLC). There is no application thus far of LC–
NMR to biosynthetic studies. On the other hand,
application of LC–NMR to drug metabolism, natural
products identification and characterization of isomeric
mixtures produced by chemical reactions have been
reviewed (Hostettmann et al., 1997; Wolfender et al.,
1998, 2001).
Protoberberine alkaloids differ from each other in the
number and placement of various oxygen functions on
the aromatic rings. The two oxygenation patterns most
frequently are oxygen atoms at carbons 2,3,9,10 and
2,3,10,11. The former is the most commonly occurring
type, while the latter has been labeled ‘‘pseudoproto-
berberine’’, and is not as widespread in its occurrence
(Preininger, 1986). Some representatives of the
2,3,10,11-oxygenated alkaloids display higher activity in
some biological tests (e.g., antimalarial activity) than
the corresponding 2,3,9,10-substituted alkaloids (Iwasa
et al., 1999). While the biosynthetic conversion of the
2,3,9,10-oxygenated
alkaloidal types, such as the protopines, benzophenan-
thridines, rhoeadines, benzindanoazepines, and
protoberberines
into
other
* Corresponding author. Tel.: +81-078-453-0031; fax: +81-078-
435-2080.
E-mail address: k-iwasa@kobepharma-u.ac.jp (K. Iwasa).
0031-9422/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0031-9422(03)00497-7

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K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238

K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238
1231
2. Results and discussion
2.2. LC–MS and LC–NMR
2.1. Synthesis
The LC-APCI/MS were measured with SIM (selected
ion monitoring) and TIM (total ion monitoring) in the
positive ion mode. Molecular weight information was
obtained on the basis of a protonated ion [M+H]+, or
a cluster ion [M+HCF3]+ recorded in the LC–APCI/
MS. The LC–NMR spectra were measured by the
stopped-flow mode (Smallcombe et al., 1995).
The pseudoprotoberberine precursor, tetrahydro-
pseudocoptisine 1 (Lenz, 1977) was synthesized from
2,3,10,11-tetrademethyltetrahydropseudopalmatine. The
latter was prepared from tetrahydropseudopalmatine
(another pseudoprotoberberine precursor) 2 (Lenz,
1977), which, in turn, was derived from tetra-
hydropapaverine. The a-N-metho salts 3a and 4a were
obtained by treating 1 and 2 with methyl iodide, in
addition to the b-N-metho salts 3b and 4b. Pseudopro-
toberberines, 6 and 7 (Lenz, 1977) were prepared by
dehydrogenation of 1 and 2 with Pd/C, respectively. The
structures of synthetic compounds were determined by
2.3. Precursor administration
Callus tissues of Corydalis platycarpa Makino or
Corydalis ochotensis var. raddeana were incubated at
ꢀ
25 C on an agar medium for 1 and 2 or in a liquid
medium for 3aD and 4aD containing the substrate for
an appropriate time (Table 3). Following incubation,
media and cells were individually extracted according to
the procedure shown in Fig. 1. The alkaloid fractions,
E-1, E-2, C-1, and C-2 (Fig. 1), which are organic
soluble, were subjected to LC–MS and LC–NMR.
1
analysis MS, H NMR, and NOESY spectral data. The
deuterated N-metho salts (3aD and 4aD) were prepared
by treating 1 and 2 with deuterated methyl iodide. The
1H NMR and MS spectral data of the N-metho salts are
summarized in Tables 1 and 2, respectively.
Fig. 1. Preparation of samples for LC–NMR and LC–MS measurements.

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K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238
Scheme 2.
Scheme 1.
Table 1
1H NMR spectral dataa of synthetic pseudoprotoberberine alkaloids (CD3OD; d ppm, 500 MHz)
1-H 4-H 9-H 12-H OCH2O OMe
5-H
6-H
8-H
13-H
H-13a
N-Me
3a 6.79 6.80 6.70 6.72
3b 6.98 6.80 6.73 6.89
4a 6.88 6.90 6.80 6.82
4b 7.03 6.91 6.84 6.98
5.99
5.97
3.28 m
3.24 m
3.83 m
3.52 m
4.77b 3.44 dd (18; 5.5)
4.61b 3.09 dd (18; 10)
4.74 dd (10; 5.5) 3.23
6.00
5.99
3.39 m
3.14 m
3.91 m
3.84 m
4.74b 3.91 m
4.58b 3.03 dd (18; 12)
5.0 dd (12; 5.5)
2.94
c
3.851, 3.847 3.24–3.4 (3H)
3.83, 3.82
m
3.53 m
3.50 dd (18; 6)
4.65b 3.12 dd (18; 10.5)
4.77 dd (10.5; 6) 3.24
3.90, 3.88
3.87, 3.86
3.43 ddd
(17; 12, 6)
3.18 dm (17)
3.97 dd
(12; 6)
3.91 dd (12; 5)
4.80b 4.06 dd (17.5; 5)
4.64b 3.09 dd (17.5; 12)
5.07 dd (12; 5)
2.96
a
Coupling constant (Hz, in parentheses).
Doublet, J=14.5–15.5 Hz.
Overlapped with H2O.
b
c
2.3.1. Administration of tetrahydropseudocoptisine 1
Tetrahydropseudocoptisine 1 was administered to the
cultured cells of C. platycarpa and C. ochotensis var.
raddeana. Fr. E-2 (see Fig. 1) from each culture both
d 4.65 and 3.13. These data indicate that the structure
represented by peak 2 is the trifluoroacetate of pseudo-
coptisine 6. This assignment was supported by the
APCI/MS data which showed a cluster ion at m/z 390
[M+HCF3]+. Comparison of the stopped-flow 1H
afforded peaks 1, 2, and
3 in the LC which
displayed a protonated ion, or a corresponding cluster
ion, at m/z 354 ([M+H]+), 390 ([H+HCF3]+), and 324
([M+H]+), respectively, in the APCI/MS.
Table 2
Mass spectral data of synthetic pseudoprotoberberine alkaloids
1
The stopped-flow H NMR spectra of peak 1 showed
formula
LSIMSa m/z [MꢁX]+ HR-LSIMS
two N-methyl groups at d 2.91 and 3.00 (ratio ca. 2:1)
revealing N-protonation, two methylendioxy groups at d
5.97 and 5.94, and four aromatic protons at d 7.04 and 7.08
(1H), 6.80 and 6.75 (2H), and 6.71 and 6.66 (1H each)
(Fig. 2). Peak 1 had a protonated ion at m/z 354 in the
APCI/MS. On the basis of this evidence, peak 1 was
recognized to be the trifluoroacetate of pseudoprotopine 5.
The stopped-flow 1H NMR spectra of peak 2 dis-
played six aromatic protons as singlets at d 9.10, 8.36,
7.51, 7.47, 7.41, and 6.92, two methylenedioxy groups at
d6.25 and 6.05, and two methylene protons as triplets at
Calc.
Found
3a
C20H20NO4 338
20H17D3NO4 341
338.1391 338.1387
341.1580 341.1574
338.1391 338.1372
341.1580 341.1582
370.2014 370.2020
373.2205 373.2213
370.2014 370.2031
373.2206 373.2208
3aD
3b
C
C20H20NO4
338
20H17 D3NO4 341
370
3bD
4a
C
C22H28NO4
4aD
4b
C
C
C
22H25 D3NO4 373
22H28NO4 370
22H25 D3NO4 373
4bD
a
X: Clꢁ ; Iꢁ ; CF3COOꢁ.

K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238
1233
NMR spectral data with those of synthetic 6 measured
under the same conditions confirmed the identification.
The stopped-flow H NMR spectra of peak 3 exhib-
ited four aromatic protons, two methylenedioxy pro-
tons, and nine aliphatic protons in the region of d 3.7
and 2.7. The APCI/MS analysis of peak 3 showed a
protonated ion at m/z 324. These data identify peak 3 as
the trifluoroacetate of tetrahydropseudocoptisine 1.
Fr. C-1 (see Fig. 1) obtained from each culture both
afforded peaks 4 and 5 in the LC, which displayed a
protonated ion and a cluster ion at m/z 338 and 390,
respectively, in the APCI/MS. The stopped-flow 1H
NMR spectrum of peak 4 show four aromatic protons
at d 6.78, 6.75, 6.70, and 6.67, two methylenedioxy pro-
tons at d 5.95 and 5.93, one methyl signal at d 3.13, and
nine aliphatic protons between d 4.8 and 2.9 (Fig. 3).
The structure of peak 4 was deduced to be the a N-
metho salt of tetrahydropseudocoptisine 1. Its stopped-
1
1
flow H NMR spectrum was identical to that of tetra-
hydropseudocoptisine a-N-metho salt 3a and not that of
Table 3
Administration of tetrahydropseudoprotoberberines 1, 2, 3aD, and 4aD to cell cultures of Corydalis platycarpa and Corydalis ochotensis var.
raddeana
Substrates (mg)
Callusa
Weight of dry cells (g)
Medium (ml)
Incubation time (days)
Weight of fractions (mg)
Fr. E-1
Fr. E-2
Fr. C-1
Fr. C-2
1
60d
60d
48
Ab
Bb
Ac
Bc
Ab
Bb
Ac
Bc
1.58
2.16
5.02
5.70
2.51
2.04
4.19
4.76
200
200
800
800
200
200
800
800
10
10
28
28
10
10
28
28
9.6
7.2
14.6
/
5.4
6.7
8.0
4.6
6.7
6.4
15.5
11.9
9.0
3.4
4.5
2.0
18.1
8.8
12.0
9.1
1
3aD
3aD
2
22.2
6.5
14.1
3.8
/
48
60d
60d
30
12.1
10.8
20.4
13.6
2
/
4aD
4aD
8.1
12.7
30
a
b
c
Callus-derived plants A: C.platycarpa; B: C. ochotensis var. raddeana.
Suspension cultures.
Static cultures.
d
Addition of S-adenosyl-l-methionine (60 mg).
Fig. 2. Stopped-flow 1H NMR spectra of peak 1 of Fr. E-2 obtained from feeding experiment of tetrahydropseudocoptisine 1.

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K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238
1
b-N-metho salt 3b. Finally, peak 5 was found to repre-
sent the trifluoroacetate of pseudocoptisine 6 by LC–
NMR and LC–MS analyses.
As a result of feeding experiments using tetra-
hydropseudocoptisine 1 as a precursor, in cultured cells
of C. platycarpa and C. ochotensis var. raddeana it was
determined that 1 was oxidized to produce pseudo-
coptisine 6 and was stereospecifically N-methylated to
give rise to the a-N-metho salt 3a, which could be oxi-
dized to afford pseudoprotopine 5 (Scheme 1).
N-methyl group. The stopped-flow H-NMRspectrum
of peak 7 was identical with that of the precursor 3aD.
Alkaloid 3aD therefore underwent oxidation to produce
[N-CD3]-pseudoprotopine 5D.The metabolic conver-
sions 1!3a!5 and 1!6 were thus demonstrated in the
cultured cells of C. platycarpa and C. ochotensis var.
raddeana (Scheme 1).
2.3.3. Administration of tetrahydropseudopalmatine 2
Administration of tetrahydropseudopalmatine
2
afforded peaks 8 and 9 in the LC of Fr. E-1 from each
culture. Peaks 8 and 9 yielded a cluster ion and a pro-
tonated ion at m/z 422 and 356, respectively, in the
2.3.2. Biotransformation of 3a to 5
Administration experiments using [N-CD3]-tetra-
hydropseudocoptisine a-N-metho salt 3aD were con-
ducted using cultured cells of C. platycarpa and C.
ochotensis var. raddeana in order to clarify stereospecific
conversion of 3a to 5 (Table 3). An agar medium was
employed in these administration experiments in order
to extend the incubation period from 10 to 28 days. Fr.
C-1 (see Fig. 1) obtained from each culture both affor-
ded peaks 6 and 7 in the LC, which displayed proto-
nated ions at m/z 357 and 341, respectively, in the
APCI/MS. It was, therefore, likely that the ions at m/z
357 and 341 were due to deuterated pseudoprotopine
5D and tetrahydropseudocoptisine a-N-metho salt 3aD,
1
APCI/MS. The stopped-flow H NMR spectra corre-
sponding to peak 8 displayed six aromatic protons as
singlets at d 9.17, 8.48, 7.58, 7.55, 7.48, and 7.04, four
methoxyl groups at d 4.06, 3.99, 3.93, and 3.88, and two
methylene protons as triplets at d 4.70 and 3.18. These
data suggested that the structure of the metabolite in
peak 8 was pseudopalmatine 7. This suggestion was
supported by the APCI/MS data (a cluster ion at m/z
422 [M+HCF3]+), and was confirmed by comparison
of the stopped-flow 1H NMR data with that of synthetic
7 measured under the same conditions. The stopped-
1
flow H NMR spectra derived from peak 9 in the LC
1
respectively. The stopped-flow H NMR spectrum cor-
responding to peak 6 was identical with that of proto-
nated pseudoprotopine 5 (Section 2.3.1), except for the
exhibited four aromatic protons, four methoxyl groups,
and nine aliphatic protons. APCIMS of peak 9 showed
a protonated ion at m/z 356. Thus, peak 9 proved to be
Fig. 3. Stopped-flow 1H NMR spectra of peak 4 of Fr. C-1 obtained from feeding experiment of tetrahydropseudocoptisine 1 and synthetic a- and
b-N-metho salts of 1 (3a and 3b).

K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238
1235
tetrahydropseudopalmatine 2. Interestingly, N-methyl-
ated derivatives and protopine-type alkaloids were not
detected in these experiments.
domuramine 8 were demonstrated (Scheme 2). However,
N-methylation, as found for tetrahydropseudocoptisine
1 was not detected for tetrahydropseudopalmatine 2.
This may be due to the high substrate specificity of the
N-methyltransferase for pseudo-type protoberberines.
2.3.4. Biotransformation of 4a
Administration
of
[N-CD3]-tetrahydropseudo-
palmatine a-N-metho salt 4aD was undertaken analo-
gously as in Section 2.3.2. In the APCI/MS, peaks 10
and 11 in the LC of Fr C-1 displayed protonated ions at
m/z 389 and 373, respectively. The stopped-flow 1H
NMR spectra of peak 10 in the LC showed a set of sig-
nals indicating the formation of two salts as in the case
of pseudoprotopine 5.
The structure related to peak 10 was recognized to be
[N-CD3]-pseudomuramine 8D on the basis of the stop-
ped-flow 1H NMR (Fig. 4) and APCI/MS (m/z 389)
data. The stopped-flow 1H NMR spectra associated
with peak 11 in the LC exhibited four aromatic protons
at d 6.87, 6.81, 6.79, and 6.74, four methoxyl groups at d
3.75 (6H) and 3.74 (6H), and nine aliphatic protons at d
3.0 and 4.7.
From these stopped-flow 1H NMR and APCI/MS
(m/z 373) data, the structure responsible for peak 11
was deduced to be [N-CD3]-tetrahydropseudopalmatine
a-N-metho salt 4aD.
As a result of these administration experiments, the
oxidation of 2 to pseudopalmatine 7 and of 4a to pseu-
3. Concluding remarks
A metabolic pathway to 2,3,10,11-oxygenated tetra-
hydroprotoberberines in cultured cells of Corydalis spe-
cies was demonstrated for the first time by application
of LC–MS and LC–NMR techniques. Tetra-
hydropseudoprotoberberines (1 and 2) were oxidized to
pseudoprotoberberines (6 and 7) and were also N-
methylated to stereospecifically afford the a-N-metho
salts. The a-N-metho salts (3a and 4a) were oxidized to
produce pseudoprotopine-type alkaloids (5 and 8).
These transformations are similar to those of 2,3,9,10-
oxygenated protoberberines both in cultured cells and in
living whole plants of Corydalis species (Iwasa, 1995).
Tetrahydropseudocoptisine a-N-metho salt 3a, pseudo-
protopine 5, and psedomuramine 8 are new alkaloids,
first identified in these biotransformation studies. The
ability of Corydalis cultures to biotransform pseudo-
protoberberines suggests the existence of metabolic
pathways leading to the formation of new pseudo-
Fig. 4. Stopped-flow 1H NMR spectra of peak 10 of Fr. C-1 obtained from feeding experiment of the a-N-metho salt of tetrahydropseudopalmatine
(4aD).

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K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238
protoberberines in some plant species, since several
known pseudoprotoberberines occur in some plant spe-
cies including Corydalis species (Preininger, 1986). The
combination of HPLC with MS and NMR spectro-
scopy provides a powerful tool for the identification of
metabolites in biotransformation in cultured cells of
Corydalis species, and illustrates a useful approach for
the structural analysis of metabolites in crude extracts
of culture media and cells.
1995) and related sequences were used to suppress the
peaks of CH3CN, its C-13 satellites, and the residual
HOD in D2O. FIDs were collected with 16 K data
points, a spectral width, 9000 Hz, a 3 ms 90ꢀ pulse, a
1.82 s acquisition time, and a 0.08 s pulse delay
depending on the sample concentrations, 24–4016 scans
were accumulated (1 min–2 h and 15 min). Prior to
Fourier transformation, an exponential appotization
function was applied to the FID corresponding to a line
broadening of 1 Hz. The HPLC system consisted of
Varian Pro Star model-230 solvent delivery system and
Varian Pro Star Model-310 variable-wavelength UV–vis
detector. The outlet of the UV detector was connected
to the LC–NMR probe via a sampling unit (Rheodyne).
The separation was performed on a Cosmosil 5 C18-AR
(4.6 i.d. ꢂ 150 mm) reversed phase column. The mobile
phase was 0.1 M NH4OAc in D2O (0.05% TFA, A), to
which MeCN (B) was added by a linear gradient: initial
20% of B, 10 min, 30% of B, 20 min, 30% of B, 30
min, 100% of B. The flow rate was 1 ml/min (detec-
tion: 280 nm).
4. Experimental
4.1. General
Conventional 1H NMR and NOESY spectra were
obtained on a Varian VXR-500S spectrometer (1H: 500
MHz) in CD3OD. Mass spectra were determined on a
Hitachi M 80 instrument at 75 eV.
4.2. Materials
In 1989 and 1981, respectively, the calli of C. platy-
carpa Makino and C. ochotensis var. raddeana were
derived from the stems of wild plants grown in Kobe
(Japan) on Murashige and Skoog’s medium containing
2,4-dichloro phenoxyacetic acid (1 mg/l), kinetin (0.1
mg/l), yeast extract (0.1%), and agar (1%). Callus tis-
sues were subcultured every 3 or 4 weeks on the same
fresh medium at 25ꢀ in the dark. S-Adenosyl-l-methio-
nine was purchased from Sigma (USA).
4.5. Syntheses
4.5.1. Tetrahydropseudopalmatine 2
To a solution of tetrahydropapaverine hydrochloride
(Sigma) (9.5 g) in hot H2O (80 ml) was added dropwise
37% formaldehyde (20 ml) for 20 min. After further
refluxing for 30 min, the mixture was cooled, basified
with 10% KOH, and the liberated pale yellow powder
was ꢀcrystallized from EtOH to yield 2 (8.2 g), mp 155–
157 C. The free base was dissolved in methanolic HCl
solution and the solvent was evaporated to give 2
4.3. LC–APCI/MS
ꢀ
hydrochloride, mp 195–197 C (dec).
LC–APCI/MS was carried out using a Hitachi M-
1000H connected to a Hitachi L-6200 intelligent pump
and a Hitachi L-4000 UV detector. LC was performed
on a Cosmosil 5 C18-AR (4.6 i.d. ꢂ 150 mm) reversed
phase column. The mobile phase was 0.1 M NH4OAc
(0.05% TFA, A), to which MeCN (B) was added by a
linear gradient: initial 20% of B, 10 min, 30% of B, 20
min, 30% of B, 30 min, 100% of B. The flow rate was 1
ml/min (detection: 280 nm). APCI/MS conditions:
nebulizer and vaporizer temperatures were 320 and
4.5.2. 2,3,10,11-Tetrademethyltetrahydropseudopalmatine
A solution of 2 (2 g) in 47% HBr (20 ml) was heated
until reflux began, this being continued for 7 h. Following
which the hydrobromic acid was evaporated in vacuo.
Water was added to the residue and the crystalline
product was collected by filtration to afford
2,3,10,11-tetrademethyltetrahydropseudopalmatine
hydrobromide (1.85 g), mp 262–268 C (dec). H NMR
(CD3OD) d 6.78 (1H, s, 1-H), 6.70 (1H, s, 12-H), 6.64
(1H, s, 4-H), 6.61 (1H, s, 9-H), 4.65 (1H, dd, J=12.0,
4.5 Hz, 13a-H), 4.43 (2H,br s, 8-H), 3.77 (1H, br s, 6-H),
3.61 (1H, br d, J=17.0 Hz, 13-H), 3.47 (1H, td, J=12.0,
4.5 Hz, 6-H), 3.18 (1H, m, 5-H), 2.99 (1H, dd, J=17.0,
12.0 Hz, 13-H), 2.93 (1H, m, 5-H).
ꢀ
1
ꢀ
399 C, respectively. The drift voltage was 20 V. The
quasi-molecular ions were monitored using the SIM
method.
4.4. LC–NMR
LC–NMR data were acquired using
a
Varian
4.5.3. Tetrahydropseudocoptisine 1
UNITY-INOVA-500 spectrometer (1H: 500 MHz)
equipped with a PFG indirect-detection LC–NMR
probe with a 60 ml flow-cell (active volume). 1H–1D
NMR spectra were obtained in stopped-flow mode.
Varian WET solvent suppression (Smallcombe et al.,
To a solution of 2,3,10,11 -tetrademethyltetrahydro-
pseudopalmatine (200 mg) in DMSO (2 ml), were added
NaOH (250 mg) and CH2Cl2 (20 ml). The mixture was
refluxed under N2 atmosphere for 4.5 h and was allowed
to stand overnight under N2 atmosphere. The residue

K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238
1237
was triturated with a mixture of H2O and CH2Cl2 and
then extracted with dilute HCl. The acidic solution was
basified with NH4OH and extracted with CH2Cl2. The
combined organic extracts were dried and evaporated
to furnish crude 1 which was purified by prep. HPLC
[0.1 M NH4OAc (0.05% TFA)–MeOH (0.05% TFA) ].
The eluent was evaporated and the residue was further
purified by HPLC [H2O (0.05% TFA)–MeOH
(0.05%TFA)ꢀ] to give the trifluoroacetate of 1 (70 mg),
mp 171–174 C (dec.).
NH4OAc (0.05% TFA)-MeOH (0.05% TFA)]. The
eluent was evaporated and the residue was further pur-
ified by HPLC [H2O (0.05% TFA)-MeOH (0.05%
TFA)] to give the b-N-methyl-D3 trifluoroacetate 3bD,
ꢀ
mp 230–231 C (dec.).
The mother liquor of the iodide mixture (250 mg) was
evaporated and after several recrystallizations in
MeOH, the methiodide was further purified by prep.
HPLC [0.1 M NH4OAc (0.05% TFA)–MeOH (0.05%
TFA)] and the eluent was purified with HPLC [H2O
(0.05% TFA)–MeOH (0.05% TFA)] to yield amor-
phous a-N-methyl-D3 trifluoroacetate 3aD (100 mg). 1H
NMR data of 3aD and 3bD were identical with those of
3a and 3b, respectively, except for the N-methyl group.
4.5.4. Pseudocoptisine 6
Tetrahydropseudocoptisine (1) (200 mg) was refluxed
with HOAc (50 ml) containing 5% Pd/C (80 mg) for 10
h. The mixture was then filtered through Celite 545. The
filtrate was basified with NH4OH, extracted with
CHCl3, and the organic solubles were evaporated in
vacuo to dryness. The residue was purified by prep.
HPLC [0.1 M NH4OAc (0.05% TFA)–MeOH (0.05%
TFA)]. The eluent was evaporated and the residue was
further purified by HPLC [H2O (0.05% TFA)–MeOH
(0.05% TFAꢀ)] to give the trifluoroacetate of 6 (90 mg),
mp 215–218 C (dec.).
4.5.7. Tetrahydropseudopalmatine ꢀ- and ꢁ-N-metho
salts 4a and 4b
Tetrahydropseudopalmatine 2 (110 mg) was dissolved
in Me2CO (5 ml) and CHCl3 (1 ml). CH3I (1 ml) was
added and the reaction mixture was allowed to stand at
room temp. for 1 h. The resulting crystals were filtered
to produce the b-N-methyl iodide 4b (122 mg), mp 251–
ꢀ
252 C (dec.). The filtrate was evaporated to afford a
mixture of the a- and b-N-methyl iodides (40 mg,
48:43). After several recrystallizations, the iodides were
further purified by prep. HPLC [0.1 M NH4OAc
(0.05% TFA)–MeOH (0.05% TFA)] and subsequently
with HPLC [H2O (0.05% TFA)–MeOH (0.05% TFA)]
to yield the amorphous a-N-methyl trifluoroacetate 4a.
4.5.5. Tetrahydropseudocoptisine ꢀ- and ꢁ-N-metho
salts 3a and 3b
CH3I (1 ml) was added to a solution of tetra-
hydropseudocoptisine 1 (50 mg) in a mixture of Me2CO
(4 ml) and CHCl3 (2 ml). After standing at room tem-
perature for 1 h, the resulting precipitates (57 mg) were
collected by decantation to afford a mixture of the a-
and b-N-methyl iodides (16:84), which were separated
by recrystallization from Me2CO/MeOH. The b-N-
methyl iodide was treated with AgCl in MeOH to con-
1
For H NMR and MS data, see Tables 1 and 2.
4.5.8. [N-CD3]-Tetrahydropseudopalmatine ꢀ- and
ꢁ-N-metho salts 4aD and 4bD
CD3I (1 g) in Me2CO (2 ml) was added to 2 (500 mg)
in Me2CO (20 ml) and CHCl3 (4 ml). The mixture was
allowed to stand at room temp. for 2.5 h, the resulting
crystals were collected by decantation to givꢀe the b-N-
methyl-D3 iodide 4bD (490 mg), mp 259-260 C (dec.).
The mother liquor was evaporated, and the crystals
were recrystallized from MeOH to give a-N-methyl-D3
iodide 4aD (71 mg, mp 228–230ꢀ), which was converted
to the a-N-methyl chloride, mp 218–223 ꢀC. For MS
ꢀ
vert it into the b-N-methyl chloride 3b, mp 245–248 C
(dec.). For H NMR and MS data, see Tables 1 and 2.
1
The mother liquor of the iodide mixture (57 mg) was
allowed to stand at room temperature overnight, the
solution was evaporated to yield crystals (21 mg), which
consisted of a mixture of the a- and b-N-methyl iodides
(88:12). This mixture was separated by prep. HPLC [0.1
M NH4OAc (0.05% TFA)–MeOH (0.05% TFA)] and
further purified by HPLC [H2O (0.05% TFA)–MeOH
(0.05% TFA)] to give the amorphous a-N-methyl tri-
fluoroacetate (3a). For 1H NMR and MS data, see
Tables 1 and 2.
1
data, see Table 2. H NMR data of 4aD and 4bD were
identical with those of 4a and 4b, respectively, except
for the N-methyl group.
4.5.9. Pseudopalmatine 7
4.5.6. [N-CD3]-Tetrahydropseudocoptisine ꢀ- and
ꢁ-N-metho salts 3aD and 3bD
Tetrahydropseudopalmatine 2 hydrochloride (520
mg) in HOAc (100 ml) containing 5% Pd/C (180 mg)
was heated until reflux began, this being maintained
for 28 h. After addition of 5% Pd/C (100 mg), the
mixture was then reflux maintained at temperature
for additional 20 h. The mixture was filtered through
Celite 545, and the HOAc was evaporated in vacuo.
The residue was dissolved in EtOH, NaI was added,
and the precipitated iodide (426 mg) was collected by
CD3I (1 g, Aldrich) in Me2CO (5 ml) was added to a
solution of 1 (340 mg) in a mixture of Me2CO (50 ml)
and CHCl3 (50 ml). After standing at room temp., the
resulting crystals were collected by decantation to afford
a mixture of the a- and b-N-methyl-D3 iodides (250 mg,
1:4). After several recrystallizations from MeOH, the
iodides were further purified by prep. HPLC [0.1 M

1238
K. Iwasa et al. / Phytochemistry 64 (2003) 1229–1238
filtration. This iodide was treated with AgClꢀin MeOH
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Acknowledgements
Kinuko Iwasa thanks the Alexander von Humboldt
Foundation, Bonn, Germany, for a scholarship. Dong-
Ung Lee appreciates a research grant from Dongguk
University.