Wrightiamines A and B, two new cytotoxic pregnane alkaloids from Wrightia javanica

Article abstract of DOI:10.1248/cpb.51.737

Two new pregnane alkaloids, wrightiamines A (1) and B (2), were isolated from the extract of the tropical Apocynaceous plant Wrightia javanica collected in Thailand, and their structures were elucidated by spectral data. Wrightiamine B (2) was preparaed from 3β-hydroxy-5α-pregnan-20-one to establish the configuration of the C-20 position as S. Wrightiamine A (1) exhibited cytotoxic activity against vincristine-resistant murine leukemia P388 cells.

Full text of DOI:10.1248/cpb.51.737

June 2003
Notes
Chem. Pharm. Bull. 51(6) 737—739 (2003)
737
Wrightiamines A and B, Two New Cytotoxic Pregnane Alkaloids from
Wrightia javanica
Souichi KAWAMOTO,a Takashi KOYANO,b Thaworn KOWITHAYAKORN,c Haruhiro FUJIMOTO,a
a
d
d
,a
*
Emi OKUYAMA, Masahiko HAYASHI, Kanki KOMIYAMA, and Masami ISHIBASHI
a Graduate School of Pharmaceutical Sciences, Chiba University; 1–33 Yayoi-cho, Inage-ku, Chiba 263–8522, Japan:
b Temko Corporation; 4–27–4 Honcho, Nakano-ku, Tokyo 164–0012, Japan: c Department of Horticulture, Faculty of
Agriculture, Khon Kaen University; Khon Kaen 40002, Thailand: and d The Kitasato Institute; 5–9–1 Shirokane, Minato-
ku, Tokyo 108–8642, Japan. Received January 30, 2003; accepted March 7, 2003
Two new pregnane alkaloids, wrightiamines A (1) and B (2), were isolated from the extract of the tropical
Apocynaceous plant Wrightia javanica collected in Thailand, and their structures were elucidated by spectral
data. Wrightiamine B (2) was preparaed from 3b-hydroxy-5a-pregnan-20-one to establish the configuration of
the C-20 position as S. Wrightiamine A (1) exhibited cytotoxic activity against vincristine-resistant murine
leukemia P388 cells.
Key words Apocynaceae; Wrightia javanica; pregnane alkaloid; cytotoxicity
consistent with the pregnane skeleton. The H–1H COSY
spectrum suggested the presence of an amino group on C-3,
and the methine proton on C-3 (dH 3.58) was observed as a
broad triplet (Jϭ11 Hz), indicating that H-3 is a-axial and
that the C-3 amino group has b-equatorial orientation. The
imino proton (dH 7.67, H-18) showed HMBC correlations to
C-13, C-17, and C-20, thus suggesting that the imino carbon
(C-18) was attached at the C-13 position and the nitrogen of
the imino group was connected to the C-20 position to con-
struct a 1-pyrroline ring. Substantial nuclear Overhauser ef-
fect (NOE) correlations were observed for H-20/H-17 and
H3-21/H-16b, indicating that H-20 is a and the methyl group
(C-21) has b orientation. From these results, the structure of
wrightiamine A was concluded to be 1, which corresponds to
a 5,6-dihydro derivative of conkurchine (3).3) Comparison of
1
Wrightia javanica DC. (Apocynaceae) is a small tree
widely distributed over the northern Malayan peninsula and
other south Asian areas, and its milky lotion has been used as
a folk medicine.1) During our search for bioactive natural
products from tropical plants,2) we investigated the chemical
constituents of leaves of W. javanica collected in Thailand.
Here we describe the isolation and structure elucidation of
two new pregnane alkaloids, wrightiamines A (1) and B (2),
and preparation of compound 2 to establish the C-20 config-
uration. 1 exhibited cytotoxic activity against vincristine-
(VCR)-resistant murine leukemia P388 cells, while the cyto-
toxicity of 2 was weak.
The leaves of W. javanica, collected in Thailand, were ex-
tracted with MeOH, and the MeOH extract was subjected to
solvent partitioning to give hexane-, EtOAc-, n-BuOH-, and
water-soluble fractions. The n-BuOH-soluble fraction con-
taining Dragendorff reagent-positive spots on TLC examina-
tion was subjected to repeated chromatography on silica gel
and Sephadex LH-20, followed by further purification with
HPLC on ODS to give 1 and 2.
the H- and 13C-NMR data of 1 with those of 3 in the litera-
1
ture3) also supported the structure of 1.
Compound 2 was suggested to have the molecular formula
C21H35NO based on the HR-FAB-MS data (m/z 318.2812,
MϩH, D ϩ1.5 mmu). The H-NMR spectrum of 2 showed
1
signals due to two tertiary methyl [dH 0.63, 3H, s (H3-18);
0.89, 3H, s (H3-19)] and one secondary methyl [dH 1.59, 3H,
d, Jϭ6.5 Hz (H3-21)] groups, and its 13C-NMR spectrum re-
vealed the presence of a ketone (dC 209.4) and a nitrogen-
bearing methine carbon (dC 50.4). The IR spectrum of 2
showed absorption bands due to an amino (3450 cmϪ1) and a
carbonyl (1690 cmϪ1) group. Since one of five unsaturation
degrees was due to the ketone group and no other sp2 carbon
1 was obtained as colorless amorphous solid and was sug-
gested to have the molecular formula C21H34N2 based on its
high resolution (HR)-FAB-MS data (m/z 315.2790, MϩH, D
ϩ1.1 mmu). The IR spectrum of 1 showed absorption bands
due to an amino (3400 cmϪ1) and an imino group (1650
cmϪ1). The presence of an imino group was also indicated
1
from the H- and 13C-NMR signals [dH 7.67, 1H, s (H-18);
1
dC 169.9 (C-18)]. The H-NMR spectrum of 1 showed sig-
nals due to one tertiary methyl [dH 0.83, 3H, s (H3-19)], one
secondary methyl [dH 1.39, 3H, d, Jϭ7.0 Hz (H3-21)], and
two nitrogen-bearing sp3 methines [dH 3.58, 1H, br t, Jϭ11.0
Hz (H-3) and 4.07, 1H, m (H-20)]. In addition to these
1
groups, the 13C-NMR spectrum aided by H-detected het-
eronuclear multiple quantum coherence (HMQC) experi-
ments revealed the presence of nine sp3 methylenes, five sp3
methines, and two sp3 quaternary carbons. Since one of six
unsaturation degrees was accounted for by the imino group
and no other sp2 carbon signals were observed in the 13C-
NMR spectrum, 1 was inferred to have five rings. The 1H–1H
correlation spectroscopy (COSY) and heteronuclear multiple
bond connectivity (HMBC) spectra of 1 showed correlations
* To whom correspondence should be addressed. e-mail: mish@p.chiba-u.ac.jp
© 2003 Pharmaceutical Society of Japan

738
Vol. 51, No. 6
20S-isomer (2) on the basis of the comparison of TLC and
1H-NMR spectral data. Thus the structure of wrightiamine B
was established to be 20S-amino-5a-pregnan-3-one (2).
The cytotoxic activities of 1 and 2 against VCR-resistant
murine leukemia P388 cells were examined, and the IC50 val-
ues in the presence and absence of VCR 12.5 ng/ml were 2.0
and 3.1 mg/ml, respectively, for 1, and 22 and ca. 25 mg/ml,
respectively, for 2. Thus compound 1 was cytotoxic but had
no reversal effect of multidrug resistance,8) while the cytotox-
icity of 2 was weak.
Experimental
General Optical rotations were recorded on a JASCO J-20. IR spectra
were measured on KBr disks in a Hitachi 260-10 infrared spectrophotome-
ter. NMR spectra were recorded on JEOL JNM GSX-A400, A500, and
ecp600 spectrometers. HR-FAB mass spectra were acquired on a JMS HX-
110 mass spectrometer.
Plant Materials Leaves of W. javanica were collected in Khon Kaen,
Thailand. A voucher specimen is maintained in the Department of Horticul-
ture, Faculty of Agriculture, Khon Kaen University.
Chart 1
Table 1. 13C-NMR Spectral Data of Compounds 1 and 2 in C5D5N
1
2
Position
Extraction and Isolation The air-dried leaves (200 g) were extracted
with MeOH. The MeOH extract (68 g) was partitioned between hexane
(200 mlϫ3) and 10% aqueous MeOH (200 ml), and the aqueous phase was
further extracted with EtOAc (200 mlϫ3) and n-BuOH (200 mlϫ2) to give
four corresponding fractions (4.7, 8.9, 17.9, 25.5 g, respectively). Part of the
n-BuOH-soluble fraction (8.3 g) was subjected to silica gel column chro-
matography (3.8ϫ33 cm) eluted with 0—100% MeOH/CHCl3. The fraction
eluted with MeOH/CHCl3 (1 : 1) was further separated by gel filtration with
Sephadex LH-20 (2.4ϫ33 cm) eluted with MeOH, followed by purification
on a second silica gel column (1.4ϫ13 cm; eluent: CHCl3/n-BuOH/AcOH/
H2O, 1.5 : 6 : 1 : 1) and a second Sephadex LH-20 column (1.4ϫ38 cm; elu-
ent: MeOH) to afford 1 (29.2 mg). Another part of the n-BuOH-soluble frac-
tion (9.6 g) was subjected to silica gel column chromatography (3.6ϫ25 cm)
eluted with 0—100% MeOH/CHCl3. The fraction eluted with MeOH/CHCl3
(1 : 1) was further separated by gel filtration on Sephadex LH-20 (1.5ϫ27
cm) eluted with MeOH, followed by purification with a second silica gel
column (1.5ϫ15 cm; eluent: CHCl3/n-BuOH/AcOH/H2O, 1.5 : 6 : 1 : 1) and
ODS flash column chromatography (1.5ϫ15 cm; eluent: 60—100% MeOH
with 0.1% trifluoroacetic acid (TFA)), and finally with ODS HPLC (Devel-
osil ODS UG-5, 10ϫ250 mm; eluent: 70% MeOH with 0.1% TFA) to give 2
(3.4 mg).
dC
dC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
37.4
27.6
51.3
33.9
45.4
28.7
32.9
37.6
54.4
36.1
23.4
33.7
66.9
55.2
29.4
24.7
49.3
169.9
17.7
69.2
12.5
37.7
37.4
209.4
43.9
45.7
28.0
30.8
34.2
52.6
34.2
20.5
38.3
41.7
55.0
23.4
26.5
54.7
11.2
10.3
50.4
19.1
Wrightiamine A (1): Colorless amorphous solid; [a]D25 Ϫ14° (cϭ0.2,
MeOH); IR (KBr) nmax 3400 and 1650 cmϪ1 1H-NMR (C5D5N) dH 7.67
;
(1H, s; H-18), 4.07 (1H, m; H-20), 3.58 (1H, br t, Jϭ11.0 Hz; H-3), 2.31
(1H, br t, Jϭ11.0 Hz; H-2a), 2.10 (2H, m; H-2b and H-4a), 1.95 (1H, t,
Jϭ11.0 Hz; H-4b), 1.39 (3H, d, Jϭ7.0 Hz; H3-21), and 0.83 (3H, s; H3-19);
13C-NMR (Table 1); electron impact (EI)-MS m/z 314 (Mϩ); FAB-MS m/z
315 (MϩH)ϩ; HR-FAB-MS m/z 315.2790 [Calcd. for C21H34N2, (MϩH)
315.2779].
signals were observed, 2 was suggested to have four rings.
The HMQC and distortionless enhancement by polarization
transfer (DEPT) data revealed that 2 has three methyls, nine
methylenes, six methines, and three quaternary carbons. The
HMBC spectral data of 2 were indicative of the pregnane
skeleton, and the ketone group was placed on C-3 (HMBC
Wrightiamine B (2): Colorless amorphous solid; [a]D25 ϩ5° (cϭ0.04,
MeOH); CD (MeOH) lext 289 (De 0.42), 237 (Ϫ0.053), 222 (0.17), and
1
209 nm (Ϫ0.19); IR (KBr) nmax 3450 and 1685 cmϪ1; H-NMR (C5D5N) dH
3.48 (1H, m; H-20), 2.35 (1H, m; H-2a), 2.34 (1H, m; H-2b), 2.24 (1H, m;
H-4a), 2.07 (1H, m; H-4b), 1.78 (1H, m; H-17), 1.59 (3H, d, Jϭ6.5 Hz; H3-
correlations: H2-1/C-3, H2-2/C-3, H2-4/C-3, and H2-5/C-3), 21), 0.86 (3H, s; H3-19), and 0.63 (3H, s; H3-18); 13C-NMR (Table 1); EI-
MS m/z 317 (Mϩ); FAB-MS m/z 318 (MϩH)ϩ; HR-FAB-MS m/z 318.2812
and the amino group on C-20 (HMBC correlations: H3-21/C-
[Calcd. for C21H36NO, (MϩH) 318.2797].
Preparation of 20S- and 20R-Amino-5a-pregnan-3-one (2, 7) Com-
mercially available 3b-hydroxy-5a-pregnan-20-one (4, 721 mg) was con-
20 and H-17/C-20). The 13C-NMR chemical shift of the C-19
methyl carbon (dC 10.3) suggested that 2 has the 5a-H con-
figuration, since the C-19 of 5a- and 5b-pregnan-3,20-dione
resonated at dC 10.3 and 22.6, respectively.4) To determine
the configuration of the C-20 amino-bearing methine carbon,
compound 2 and its 20-epimer were prepared as shown in
Chart 1. 3b-Hydroxy-5a-pregnan-20-one (4) was converted
into the known 20S- and 20R-amino-5a-pregnan-3b-ol (5, 6)
using procedures reported in the literature.5,6) The absolute
configurations of the C-20 of 5 and 6 have been firmly estab-
lished based on chemical evidence.7) Jones oxidation of 5 and
6 afforded 20S- and 20R-amino-5a-pregnan-3-one (2, 7), re-
spectively, and wrightiamine B proved to be identical to the
verted into known 20S-amino-5a-pregnan-3b-ol (5, 326 mg) and 20R-
amino-5a-pregnan-3b-ol (6, 104 mg) using the procedures reported in the
literature5,6) [(i) NH2OH.HCl, pyridine, EtOH, reflux, 5 h, 92%; (ii) H2, PtO2,
AcOH, 16 h; (iii) silica gel column, ether saturated with NH3/MeOH
(85 : 15), 5: 63%; 6: 20%]. 20S-aminoalcohol (5, 6.6 mg) dissolved in ace-
tone (1.0 ml) was treated with 6 ml of Jones reagent (CrO3 2.67 g, conc.
H2SO4 2.3 ml, and H2O ca. 7.7 ml) for 5 min at room temperature. After
addition of 4 N NaOH (10 ml), the reaction mixture was extracted with
CHCl3 (3 mlϫ10), dried over MgSO4, and purified with silica gel column
chromatography (9ϫ35 mm; 5—10% MeOH/CHCl3) to give 20S-amino-5a-
pregnan-3-one (2, 5.1 mg). 20R-Aminoalcohol (6, 6.4 mg) was converted
into 20R-amino-5a-pregnan-3-one (7, 5.0 mg). 7: [a]D25 Ϫ3° (cϭ0.2, MeOH);
1
IR (KBr) nmax 3450 and 1685 cmϪ1; H-NMR (C5D5N) dH 3.54 (1H, m; H-

June 2003
739
20), 2.49 (1H, m; H-2a), 2.07 (1H, m; H-2b), 2.20 (1H, m; H-4a), 2.06 (1H,
m; H-4b), 1.70 (1H, m; H-17), 1.49 (3H, d, Jϭ6.5 Hz; H3-21), 0.87 (3H, s;
H3-18), and 0.78 (3H, s; H3-19); EI-MS m/z 317 (Mϩ); FAB-MS m/z 318
(MϩH)ϩ; HR-FAB-MS m/z 318.2796 [Calcd. for C21H36NO, (MϩH)
318.2797].
2) For a previous report in this series, see: Tanaka T., Ishibashi M., Fuji-
moto H., Okuyama E., Koyano T., Kowithayakorn T., Hayashi M.,
Komiyama K., J. Nat. Prod., 65, 1709—1711 (2002).
3) Janot M. M., Jarreau F. X., Minh T.-H., Qui K.-H., Goutarel R., Bull.
Soc. Chim. France, 1964, 1555—1563 (1964).
4) Blunt J. W., Stothers J. B., Org. Mag. Res., 9, 439—464 (1977).
5) van de Woude G., van Hove L., Bull. Soc. Chim. Berg., 76, 566—578
(1967).
6) Biesemans M., van de Woude G., van Hove L., Bull. Soc. Chim. Berg.,
94, 59—68 (1985).
7) Fieser L. F., Fieser M., “Steroids,” Reihold, New York, 1959, p. 861.
8) Kam T. S., Subramaniam G., Sim K.-M., Yoganathan K., Koyano T.,
Toyoshima M., Rho M.-C., Hayashi M., Komiyama K., Bioorg. Med.
Chem. Lett., 8, 2769—2772 (1998).
Acknowledgments This work was partly supported by a Grant-in-Aid
from the Ministry of Education, Science, Sport, and Culture of Japan, and
by Grants-in-Aid from the Shorai Foundation for Science and Technology,
Mochida Memorial Foundation for Medical and Pharmaceutical Research,
and Suzuken Memorial Foundation.
References and Notes
1) Corner E. J. H., Watanabe K., “Illustrated Guide to Tropical Plants,”
Hirokawa Publishing Company, Tokyo, 1969, p. 660.