Benzylisoquinoline alkaloids from Gnetum parvifolium

Article abstract of DOI:10.1021/np980472f

The investigation of the chemical constituents from the lianas of Gnetum parvifolium resulted in the isolation of three new benzylisoquinoline alkaloids, (±)-N-methylhigenamine (1), (-)-N-methylhigenamine N-oxide (2), and (±)-8-(p-hydroxybenzyl)-2,3,10,11-tetrahydroxyprotoberberine (3), together with two known alkaloids, higenamine and trigonelline. The structures of 1-3 were determined by chemical methods and spectral analysis including 2D NMR and NOE studies.

Full text of DOI:10.1021/np980472f

J . Nat. Prod. 1999, 62, 1025-1027
1025
Ben zylisoqu in olin e Alk a loid s fr om Gn etu m pa r vifoliu m
Qun Xu and Mao Lin*
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, People’s Republic of China
Received October 23, 1998
The investigation of the chemical constituents from the lianas of Gnetum parvifolium resulted in the
isolation of three new benzylisoquinoline alkaloids, (()-N-methylhigenamine (1), (-)-N-methylhigenamine
N-oxide (2), and (()-8-(p-hydroxybenzyl)-2,3,10,11-tetrahydroxyprotoberberine (3), together with two
known alkaloids, higenamine and trigonelline. The structures of 1-3 were determined by chemical
methods and spectral analysis including 2D NMR and NOE studies.
Gnetum parvifolium (Warb.) C. Y. Cheng (Gnetaceae) is
distributed in the southern part of the People’s Republic
of China and is used as a Chinese herbal medicine for the
treatment of bronchitis, rheumatoid arthritis and muscular
strain.1 Previous phytochemical investigations on plants
in this family have revealed an abundance of flavonoids
and stilbenoids.2 In 1980, a benzylisoquinoline alkaloid,
higenamine, which showed a strong cardiotonic effect, was
isolated from G. parvifolium.3 In recent years, a number
of stilbenoids have been described from this species.4 The
present study on G. parvifolium in our laboratory has
afforded three new benzylisoquinoline alkaloids (1-3)
together with two known alkaloids, higenamine and trigo-
nelline.
isoquinoline alkaloids (1, 2, and higenamine). An additional
crude alkaloid 3 was obtained on repeated chromatography,
but further purification of 3 was difficult due to its failure
to crystallize. Therefore, the pentaacetate derivative (4) of
3 was prepared in the normal manner.
Compound 1 was obtained as colorless crystals, [R]25
D
0° (c 0.24, DMSO). A molecular formula of C17H19O3N was
determined from a combination of the elemental analysis,
FABMS, and 13C NMR data. FABMS showed quasimolecu-
lar ion peaks at m/z 308 [M + Na]+ and 286 [M + 1]+,
indicating a molecular ion that is 14 mass units heavier
than that of higenamine. The IR and UV absorptions were
very similar to those of higenamine, suggesting the pres-
ence of a tetrahydrobenzylisoquinoline skeleton. The EIMS
gave a weak molecular ion peak at m/z 285 as well as two
fragment ions at m/z 178 (base peak, 6,7-dihydroxy-N-
methylisoquinoline fragment ion) and m/z 107 (p-hydroxy-
benzyl fragment ion), both characteristic of tetrahydroben-
zylisoquinoline alkaloids.5 Comparison of the 1H NMR and
13C NMR spectral data for 1 and higenamine demonstrated
the presence of an N-Me unit (δH 2.31, δC 42.0) in the
molecule of 1. Hence, 1 was assigned as (()-N-methylhi-
genamine.
Compound 2 was obtained as colorless needles, [R]25
D
-9.6° (c 0.12, DMSO). This compound was deduced to have
the same skeleton as compound 1 and higenamine based
on comparison of their spectral data (UV, 1H- and 13C
NMR). EIMS gave a weak molecular ion peak at m/z 301
and a fragment ion peak at m/z 285 [M - 16]+, which is
typical of an N-oxide.6 In addition, the C-1 signal appeared
at very low field (δC 77.5) in the 13C NMR spectrum,
consistent with the presence of an adjacent N-oxide group.
Analysis of all the obtained spectral data led to the
assignment of 2 as (-)-N-methylhigenamine N-oxide. The
relative stereochemistry between the N-methyl and p-
hydroxybenzyl groups was established to be syn based on
the chemical shifts of H-1.7 The absolute configuration of
2 was not determined.
In order to confirm the structures of 1 and 2, a chemical
transformation was carried out. Compound 1 was prepared
from higenamine through condensation with 37% formalin
and successive reduction by sodium borohydride at room
temperature.8 Oxidation of 1 using m-chloroperbenzoic acid
was successful in producing compound 2.9
Compound 3 was derivatized as its pentaacetate (4),
which was isolated as colorless needles from MeOH/H2O.
A molecular formula of C34H33O10N was established from
its elemental analysis, EIMS, and NMR data (Table 1). The
UV spectrum showed an isoquinoline absorption band at
The lianas of G. parvifolium were extracted with 60%
ethanol and the ethanolic extract was worked up using
conventional methods. The total alkaloid extracts were
combined and further separated over polyamide, followed
by Si gel column chromatography, yielding three benzyl-
* To whom correspondence should be addressed. Phone: +86 10 63165326.
Fax: +86 10 63017757. E-mail: Lin.Mao@public.gb.com.cn.
10.1021/np980472f CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/12/1999

1026 J ournal of Natural Products, 1999, Vol. 62, No. 7
Notes
Ta ble 1. 1H and 13C NMR Data for Compound 4 (Acetone-d6)a
position
δH
J (in Hz)
δC
HMBC
NOE
1a
1
2
3
4
133.7 (C)
7.16 s
122.1 (CH)
141.7 (C)b
141.5 (C)b
124.0 (CH)
138.5 (C)
C-1a
6.98 s
C-5, C-4
C-6
4a
5
2.90 m
2.77 m
3.12 m
2.77 m
4.12 dd
29.8 (CH2)
6
46.7 (CH2)
C-14, C-5
8
8a
9
10
11
12
12a
13
8.5, 5.5
66.8 (CH)
133.0 (C)
C-14, C-12a, C-15
C-8, C-8a, C-10/C-11
H-6, H-9, H-2′, H-6′,
6.82 s
7.00 s
123.1 (CH)
141.0 (C)b
141.2 (C)b
124.2 (CH)
136.5 (C)
C-13, C-12a, C-10/C-11
C-14, C-1a, C-12a
3.02 dd
2.90 m
4.58 dd
3.28 dd
2.95 dd
17.1, 5.0
32.1 (CH2)
H-1, H-12
H-1, H-13, H-15â
14
11.5, 5.0
14.0, 8.5
14.0, 5.5
50.7 (CH)
41.5 (CH2)
C-1, C-6, C-1a, C-4a
C-8, C-8a, C-1′
15R
15â
1′
138.4 (C)
2′, 6′
3′, 5′
4′
7.29 d
6.98 d
8.0
8.0
131.2 (CH)
122.0 (CH)
150.2 (C)
C-4′, C-15, C-3′, C-5′
C-4′
COCH3
2.03-2.08 5s
168.4-169.4 (C)
a
Assignments were confirmed by DEPT, 1H-1H COSY, and 1H-13C COSY experiments. bInterchangeable assignments.
275 nm (log ꢀ 3.92).10 The EIMS did not show a parent ion
but gave fragment ions at m/z 242, 164, and 136 resulting
from retro-Diels-Alder fragmentation, characteristic of a
compound based on the protoberberine skeleton.11 A promi-
nent peak at m/z 298, resulting from successive acetyl
losses at m/z 466, 424, 382, and 340, confirmed the presence
of four hydroxy groups from the protoberberine moiety. The
13C NMR and DEPT spectra displayed two methine, four
methylene, and eighteen aromatic carbons, of which ten
were quaternary. One of the methylenes (δ 46.8) and the
two methine carbons (δ 50.7 and 66.8) were shifted down-
field along with their corresponding protons because they
were all attached to a nitrogen atom. The 1H NMR
spectrum showed two doublets centered at δ 7.29 (2H) and
6.98 (2H), representing the four protons of a parasubsti-
tuted benzyl group. Four proton singlets between δ 6.82
and 7.16 were attributed to the aromatic protons of the
protoberberine moiety, suggesting a C-2, C-3, C-10, and
C-11 tetrahydroxy substitution pattern. The alphatic hy-
drogens of 4 comprised two ABX spin systems and one A2X2
system. Further structural features were determined from
NOE difference and HMBC data (Table 1). In the NOE
experiment, the effect observed between H-1 and H-13
helped to differentiate between the two ABX spin systems.
Similarly, NOEs between H-8 and H-9 and between H-13
and H-12 confirmed the C-10, C-11 substitution pattern
in ring D. In addition, a series of HMBC correlations for
H-1/C-1a, H-14/C-1, H-4/C-5, H-9/C-8, C-8a, and H-12/C-
13 confirmed the assignments of the aromatic protons
(Table 1). Other HMBC correlations for H-14/C-1, C-1a,
C-4a, C-6 and for H-8/C-14, C-12a were consistent with the
proposed protoberberine skeleton of 4.
years.12 The quinolizidine nucleus can assume a half-chair
conformation with one trans and two cis configurations
being possible. The trans-quinolizidine conformation is
associated with a high-field H-14 signal at 3.6 ( 0.2 ppm
and a low-field C-14 signal at 58.4 ( 0.3 ppm, while in cis-
quinolizidines H-14 is shifted to low field at 4.3 ( 0.2 ppm
and C-14 to high field at about 49-52 ppm. Compound 4
exhibited high-field C-14 (δC 50.0) and low-field H-14 (δH
4.58) signals, indicating a cis-quinolizidine conformation.
An observed NOE between methine H-14 and the benzylic
methylene H-15â proton indicated that H-14 and the
p-hydroxybenzyl group were oriented on the same side of
ring C, thus fixing the relative stereochemistry. Accord-
ingly, compound 4 was assigned as (()-8-(p-hydroxybenzyl)-
2,3,10,11-tetrahydroxyprotoberberine pentaaceate.
Protoberberine alkaloids with a C-8 substituent are quite
unusual, especially in the case of 8-benzylberberine-type
alkaloids.13,14 A distinguishing characteristic of such com-
pounds is their nonmethylated hydroxyl functions. Gottlieb
and Kubitzki have indicated that absence of methylation
is widespread in the Gnetaceae and other gymnosperms
and have provided an interesting insight into some of the
biogenetic sequences that occur within these plants.2,15 The
biogenesis of 3 may result from the condensation of
norlaudanosine with 4-hydroxyphenylpyruric acid (derived
from tyrosine) followed by cyclization.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a XT4-100X micromelting point apparatus and
are uncorrected. Optical rotations were measured on a Perkin-
Elmer 241 polarimeter, and IR spectra were obtained on
Perkin-Elmer 683 infrared spectrophotometer. NMR spectra
were taken on a Bruker AM 500 spectrophotometer using TMS
as an internal standard. EIMS were obtained on a ZAB-2F
mass spectrometer. Elemental analysis was carried out on a
MOD1106 elemental analyzer. Column chromatography was
performed using Si gel (Qing Dao Hai Yang Chemical Group
Co., Qing Dao, People’s Republic of China), polyamide (Li Xian
Chemical Plant, Li Xian, People’s Republic of China) and 001
The structure of the C-8 p-hydroxylbenzyl moiety in 4
1
was determined by H, 13C, 2D NMR, and by EIMS data.
In the COSY spectrum, H-8 correlated with the two
methylene protons at C-15. HMBC correlations between
H-15R/C-8, C-8a, C-1′ established the nature of this C-8
substitutent.
The stereochemistry of quinolizidine and protoberberine
nuclei have been extensively investigated in the past

Notes
J ournal of Natural Products, 1999, Vol. 62, No. 7 1027
× 7 polyvinylsulfonic ion-exchange resin (Tian J in Chemical
Plant, Tian J in, People’s Republic of China). TLC was con-
ducted on Si gel 60 F254 (Qing Dao Hai Yang Chemical Group
Co.) and detection was achieved by exposure to I2 vapor or
spraying with Dragendorff’s reagent.
(C-1), 59.6 (C-3), 55.5 (Me), 37.1 (C-R), 25.4 (C-4); EIMS m/z
301 [M]+ (4), 285 (8), 283 (22), 242 (97), 194 (16), 178 (70),
107 (23); anal. C 67.54%, H 6.35%, N 4.34%, calcd for
C
17H19O4N, C 67.78%, H 6.31%, N 4.65%.
(()-8-(p-Hyd r oxyben zyl)-2,3,10,11-tetr a h yd r oxyp r oto-
ber ber in e p en ta a cea te (4): colorless needles (MeOH/H2O);
mp 166-168 °C; [R]20D 0° (c 0.25, MeOH); UV (EtOH) λmax (log
ꢀ) 275 (3.92) nm; IR (KBr) νmax 3449, 1765, 1506, 1373, 1213
cm-1; 1H NMR and 13C NMR, see Table 1; EIMS m/z 466 (14),
424 (25), 382 (30), 340 (44), 298 (82), 242 (18), 164 (16), 136
(20), 107 (40), 77 (20), 42 (100); anal. C 66.43%, H 5.45%, N
2.21%, calcd for C34H33O10N, C 66.34%, H 5.37%, N 2.28%.
Higen a m in e: colorless plates (EtOH); mp 256-258 °C (lit.
255-256 °C);3a UV, IR, and NMR data are consistent with an
authentic sample and literature values.3a
P la n t Ma ter ia l. Lianas of G. parvifolium were collected
in Guangxi Province of the People’s Republic of China in March
1986 and identified by Professor W. Z. Song of our institute,
where a voucher sample has been deposited (No. 860321).
Extr a ction a n d Isola tion . Powered lianas (20 kg) of G.
parvifolium were extracted with 60% EtOH. The resultant
extract was concentrated to yield 1.6 kg of gum which was
then divided into acetone-soluble and acetone-insoluble frac-
tions. The latter fraction (513 g) was exhaustively extracted
with 1% HCl, and then basified with saturated Na2CO3 to pH
9. The total alkaloid extract was subjected to polyamide
column chromatography and eluted with (CH3)2CO-MeOH
(90:24) into eight fractions. Fraction IV was further chromato-
graphed over Si gel using (CH3)2CO-MeOH-HOAc (25:25:1)
as eluent to furnish compound 1 (410 mg). Fraction VI was
separated on Si gel with (CH3)2CO-EtOH mixtures of increas-
ing polarity to give four fractions. The fourth fraction was
eluted with (CH3)2CO-MeOH (1:1), and subjected to further
purification over Si gel, by eluting with (CH3)2CO-MeOH-
HOAc (25:25:1), to afford higenamine (257 mg) and 2 (14 mg),
which were finally purified by preparative TLC with (CH3)2-
CO-MeOH-HOAc (50:25:1) and crystallized from EtOH and
H2O, respectively. Fraction V was chromatographed on Si gel
with Me2CO-MeOH-EtOAc-H2O (2:2:4:1) as eluent, and
furnished a crude yellow gum (87 mg) which was acetylated
with acetic anhydride and pyridine. Flash chromatography
with CHCl3-Me2CO (30:2) yielded 4 (21 mg).
Tr igon ellin e: colorless plates (EtOH); mp 212-214 °C (lit.
218 °C, dec);16 UV, IR, and NMR data are consistent with
literature values.16
N-Meth yla tion of Higen a m in e. A solution of higenamine
(20 mg) in methanol (2 mL) was stirred with 37% formalin
(0.5 mL) at room temperature for 30 min. Sodium borohydride
(25 mg) was added and the solution was stirred for an
additional 30 min. After the solvent was removed in vacuo,
the residue was separated by preparative TLC with EtOH-
Me2CO (3:1), producing 12 mg of compound 1, identical with
the natural product (IR, EIMS, TLC).
Oxid a tion of 1. A solution of m-chloroperbenzoic acid in
methanol (17 mg in 2 mL) was added dropwise to compound
1 (28 mg) at 0-5 °C. The mixture was stirred for 2 days and
then the purified by preparative TLC using EtOH-Me2CO (3:
1). The product (16 mg) was identical with compound 2 (IR,
EIMS, TLC).
The alkaline aqueous extract was reacidified to pH 2 and
chromatographed on an ion-exchange resin column. The resin
was then extracted with methanol. Further purification of the
methanolic extract on a Si gel column eluted with (CH3)2CO-
H2O (30:2) afforded trigonelline (508 mg).
Ack n ow led gm en t . This work was supported by the
National Natural Science Foundation of China (No. 39570836).
Refer en ces a n d Notes
(1) Flora Republicae Popularis Sinicae; Academic Press: Beijing, 1990;
Vol. VII, pp 490-504.
(()-N-Meth ylh igen a m in e (1): colorless crystals (EtOH);
mp 148-150 °C; [R]25D 0° (c 0.24, DMSO); UV (EtOH) λmax (log
ꢀ) 225 (4.60, sh), 285 (4.08) nm; IR (KBr) νmax 3446, 3028, 2690,
(2) Gottlieb, O. R.; Kubitzki, K. Planta Med. 1984, 380-385.
(3) (a) Fujian Institute of Medica and Medicine. Acta Pharm. Sin. 1980,
15, 434-435. (b) Koshiyama, H.; Ohkuma, H.; Kawaguchi, H.; Hsu,
H. Y.; Chen, Y. P. Chem. Pharm. Bull. 1970, 18, 2564-2568.
(4) (a) Li, J . B.; Lin, M.; Li, S. Z.; Song, W. Z. Acta Pharm. Sin. 1991, 26,
437-440. (b) Lin, M.; Li, J . B.; Li, S. Z.; Yu, D. Q.; Liang, X. T.
Phytochemistry 1992, 31, 633-638. (c) Lin, M.; Li, J . B.; Wu, B.;
Zheng, Q. T. Phytochemistry 1991, 30, 4201-4203.
1548, 1518, 1456, 1408, 1277, 1109, 839 cm-1 1H NMR
;
(DMSO-d6, 500 MHz) δ 6.89 (2H, d, J ) 8.3 Hz, H-2′, H-6′),
6.58 (2H, d, J ) 8.3 Hz, H-3′, H-5′), 6.38 (1H, s, H-5), 6.34
(1H, s, H-8), 3.48 (1H, t, J ) 5.1 Hz, H-1), 2.81 (1H, dd, J )
14.2, 5.1 Hz, H-R), 2.71 (1H, dd, J ) 14.2, 5.1 Hz, H-R), 2.57-
2.99 (1Η, m, H-3), 2.48-2.55 (1H, m, H-4), 2.31 (3H, s, Me);
13C NMR (DMSO-d6, 125 HMz) δ 155.1 (C-4′), 143.2 (C-6),
142.9 (C-7), 130.2 (C-2′, C-6′), 128.1 (C-8a), 124.4 (C-4a), 114.9
(C-5), 114.5 (C-3′, C-5′), 114.4 (C-8), 114.4 (C-1′), 64.0 (C-1),
46.9 (C-3), 42.0 (Me), 39.7 (C-R), 22.4 (C-4); EIMS m/z 285 [M]+
(1), 178 (100), 107 (8), 77 (5), 44 (40); FABMS m/z 308 [M +
Na]+, 286 [M + 1]+; anal. C 71.94%, H 6.60%, N 4.87%, calcd
for C17H19O3N, C 71.58%, H 6.67%, N 4.91%.
(5) Ohashi, M.; Wilson, J . M.; Budzikiewicz, H.; Shamma, M.; Slusarchyk,
W. A.; Djerassi, C. J . Am. Chem. Soc. 1963, 85, 2807-2810.
(6) Bild, N.; Hesse, M. Helv. Chim. Acta 1967, 50, 1885-1892.
(7) Bremner, J . B.; Thuc, L. V. Aust. J . Chem. 1980, 33, 379-394.
(8) Bick, I. R. C.; Sevenet, T.; Sinchai, W.; Skelton, B. W.; White, A. H.
Aust. J . Chem. 1981, 34, 195-207.
(9) Craig, J . C.; Purushothaman, K. K. J . Org. Chem. 1970, 35, 1721-
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(10) Huang, L.; Yu, D. Q. Application of the Ultraviolet Absorption
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(11) Chen, C. Y.; Maclean, D. B. Can. J . Chem. 1968, 46, 2501-2506.
(12) (a) Iwasa, K.; Cushman, M. J . Org. Chem. 1982, 47, 545-552. (b)
Suan, R.; Silva, M. V.; Valpuesta, M. Tetrahedron 1990, 46, 4421-
4428. (c) J ahangir; Maclean, D. B.; Holland, H. L. Can. J . Chem. 1987,
65, 727-733. (d) Tourwe, D.; Binst, G. V.; Kametani, T. Org. Magn.
Reson. 1977, 9, 341-346. (e) Baird, P. D.; Blagg, J .; Davies, S. G.;
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(13) Lopes, L. M. X. Phytochemistry 1992, 31, 4005-4009.
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Planta Med. 1973, 380-385.
(-)-N-Meth ylh igen a m in e N-oxid e (2): colorless needles
(H2O); mp 156-159 °C; [R]25D -9.6° (c 0.12, DMSO); UV (H2O)
λmax 225 (4.35, sh), 284 (3.64) nm; IR (KBr) νmax 3473, 1623,
1515, 1251, 841 cm-1; 1H NMR (D2O, 500 HMz) δ 6.80 (2H, d,
J ) 8.4 Hz, H-2′, H-6′), 6.79 (1H, s, H-5), 6.71 (2H, d, J ) 8.4
Hz, H-3′, H-5′), 6.66 (1H, s, H-8), 5.53 (1H, br s, H-1), 3.78
(1H, dd, J ) 11.0, 3.2 Hz, H-R), 3.75 (1H, dd, J ) 11.0, 2.8 Hz,
H-R), 3.15-4.13 (1H, m, H-3), 3.07 (3H, s, Me), 2.55-3.01 (1H,
m, H-3); 13C NMR (DMSO-d6, 125 HMz) δ 155.8 (C-4′), 145.1
(C-6), 143.3 (C-7) 130.5 (C-2′, C-6′), 127.9 (C-8a), 124.7 (C-4a),
119.1 (C-1′), 115.2 (C-5), 115.1 (C-3′, C-5′), 114.6 (C-8), 77.5
NP980472F