Phytochemistry 52 (1999) 959±963
Indole and monoterpene alkaloids from the leaves of Kopsia
dasyrachis
Toh-Seok Kam a,*, Yeun-Mun Choo a, Wei Chen a, Jia-Xing Yao b
a
Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia
Department of Chemistry, University of York, Heslington, York YO10 5DD, United Kingdom
b
Received 12 April 1999; accepted 28 April 1999
Abstract
A new monoterpene alkaloid, kinabalurine G, in addition to 11 indole alkaloids and the catechine±skytanthine compound,
kopsirachine, was obtained from the leaf extract of Kopsia dasyrachis. The structure of the novel alkaloid, danuphylline, was
con®rmed by an X-ray analysis. # 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Kopsia species; Apocynaceae; Indole and monoterpene alkaloids
1. Introduction
We recently reported the alkaloidal composition of
the stem bark extract of Kopsia dasyrachis Ridl., one
of about seventeen Malaysian Kopsia species, which is
found in Sabah, Malaysian Borneo (Kam,
Subramaniam & Chen, 1999). A previous study of the
leaf alkaloids of K. dasyrachis from Borneo yielded
three new indoles, viz., kopsidasine, kopsidasine Noxide, kopsidasinine (Homberger & Hesse, 1982) and
in addition, kopsirachine, which is constituted from
the union of catechine and two units of skytanthine
(Homberger & Hesse, 1984). We have also reported
the structure elucidation (Kam, Lim, Choo &
Subramaniam, 1998), as well as a biomimetic, electrochemically-mediated semisynthesis (Kam, Lim &
Choo, 1999) of the novel pentacyclic indole, danuphylline, which was obtained in minute amount from the
leaf extract of this plant. We now report the full alkaloidal composition of the leaf, including the isolation
of a new monoterpene alkaloid, as well as con®r-
* Corresponding author. Tel.: +603-759-4266; fax: +603-7594193.
E-mail address: tskam@kimia.um.edu.my (T.S. Kam).
mation of the structure of danuphylline by X-ray
analysis.
2. Results and discussion
The ethanol extract of the leaves furnished a basic
fraction which upon extensive chromatography yielded
a total of 13 alkaloids, viz., methyl chanofruticosinate
1 (Chen, Li, Kirfel, Will & Breitmaier, 1981), methyl
11,12-methylenedioxychanofruticosinate 2 (Chen et al.,
1981; Kam, Tan, Hoong & Chuah, 1993), methyl Ndecarbomethoxychanofruticosinate 3 (Chen et al.,
1981; Kam et al., 1993), methyl 11,12-methylenedioxyN-decarbomethoxychanofruticosinate 4 (Chen et al.,
1981; Kam et al., 1993), kopsamine 5 (Crow &
Michael, 1962; Feng, Kan, Potier, Kan & Lounasmaa,
1983; Gilbert, 1965; Kam & Sim, 1998; Zheng, Zhou
& Huang, 1989), 11,12-dimethoxykopsamine 6 (Kam
& Sim, 1998; Zheng et al., 1989), kopsamine N(4)oxide 7 (Kam & Sim, 1998; Zheng et al., 1989), pleiocarpine 8 (Homberger & Hesse, 1982), 12-methoxypleiocarpine 9 (Kam & Sim, 1998), kopsi®ne 10 (Kam
et al., 1999; Kam & Subramaniam, 1998), kopsirachine
11 (Homberger & Hesse, 1984), danuphylline 12 (Kam
0031-9422/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 1 - 9 4 2 2 ( 9 9 ) 0 0 2 6 5 - 4
960
T.-S. Kam et al. / Phytochemistry 52 (1999) 959±963
et al., 1998, 1999), and kinabalurine G 13, which is the
N-oxide of a hydroxyskytanthine. Compound 2 is the
major alkaloid found in the leaves, and as with compounds 1, 3 and 4, have been encountered previously
in other Kopsia species (Chen et al., 1981; Kam et al.,
1993). Danuphylline 12 represents a new indole alkaloid possessing a novel pentacyclic carbon skeleton
(Kam et al., 1998). We have carried out an electroche-
mically-mediated semisynthesis starting from 2 based
on the proposal that its probable origin is via unravelling, through a retro-aldol sequence, of an intermediate
carbinol amine 14, derived in turn from an iminium
ion precursor (Kam et al., 1999). We have now carried
out an X-ray diraction analysis which has con®rmed
the structure reported earlier based on spectral data.
The crystals of 12 are orthorhombic belonging to
T.-S. Kam et al. / Phytochemistry 52 (1999) 959±963
the space group P212121, with a = 7.2558 (5) AÊ,
b = 16.0230 (10) AÊ, c = 18.798 (2) AÊ; a=b=g=908;
V = 2185.4 (3) AÊ3; DX=1.430 Mg mÿ3 and Z = 4.
The structure was solved by the direct method SAPI91 (Fan, Yao, Zheng, Gu & Qian, 1991), and re®ned
by the full matrix least squares method. The ®nal Rfactor was 0.0769. As shown in the perspective diagram of Fig. 1, compound 12 does indeed possess the
novel ring system proposed earlier based on spectral
analysis (Kam et al., 1998). Furthermore the results
also con®rm the trans disposition of the N(4) lone pair
and H-21, as well as the location of the formamide-H
within the anisotropic in¯uence of the aromatic ring,
resulting in the anomalously high ®eld resonance of
the formamide proton in 1H NMR.
Compound 13 was the most polar alkaloid isolated
and was also obtained in minute amount as a colourless oil. The mass spectrum showed a molecular ion at
m/z 199 which analyzed for C11H21NO2, with other
fragment peaks at m/z 183, 166, 110, 84 and 58. The
latter three fragments are characteristic of a skytanthine type alkaloid (Cordell, 1977; Kam,
Yoganathan & Chen, 1997). The IR spectrum showed
the presence of a hydoxyl group (3404 cmÿ1), while
the 1H NMR spectrum indicated the presence of two
CH3CH groups and an N-methyl group, which was
rather deshielded at d 3.17. This observation, coupled
with the polar nature of this compound, and the observation of a strong M-16 fragment in the mass spec-
961
trum, suggested that compound 13 is an N-oxide. This
was readily con®rmed by FeSO4 reduction of 13 which
yielded the parent monoterpene alkaloid 15. The Nmethyl signal is now shifted up®eld to d 2.30, while the
resonances of the two a-carbons, C-1 and C-3, are also
shifted up®eld from d 67.3 and 67.2 to d 62.5 and 57.7
respectively (Table 1). The presence of a low ®eld, quaternary carbon signal at dC 79.1, indicated that the hydroxyl function is attached to a quaternary carbon, i.e.
Fig. 1. X-Ray structure of 12.
962
T.-S. Kam et al. / Phytochemistry 52 (1999) 959±963
Table 1
1
H and 13C NMR spectral dataa for compounds 13, 15 and 16
Position
13b
dH
1a
1b
3a
3b
4a
5a
6
6
7
7
8b
9
N-Me
8-Me
4-Me
3.04
2.95
3.11
2.89
3.02
2.17
1.17
1.70
1.37
1.86
1.76
±
3.17
1.02
0.96
dt (12.5,2)
d (12.5)
ddd (11,4,2)
t (11)
m
m
tdd (13,11,8)
dtd (13,9,3)
dddd (14,10,8,3)
dddd (14,11,9,3)
m
s
d (7)
d (7)
15
16c
dC
dC
dC
67.3
62.5
57.1
67.2
57.7
57.9
24.0
44.3
20.3
29.1
44.4
19.9
46.2
40.2
31.3
28.8
29.7
22.2
39.6
79.1
61.0
16.6
15.9
39.1
78.2
45.9
17.1
16.8
36.5
30.8
46.5
22.7
17.5
a
CDCl3, 400 MHz.
assignments based on COSY, HMQC and HMBC.
c
From Ref. (Homberger & Hesse, 1984).
b
C-5 or C-9, based on a skytanthine-type carbon skeleton. Detailed analysis of the 1H and 13C NMR spectral data (COSY, HMQC, HMBC, NOE) and
comparison with d-skytanthine 16 (Homberger &
Hesse, 1984), enabled placement of the OH function
on C-9 and allowed full assignment of the NMR spectral data. For instance, the COSY spectrum revealed
the partial structure CH3CHCH2CH2CH corresponding to the Me(8)-C(8)-C(7)-C(6)-C(5) fragment, while
the HMBC spectrum showed two-bond correlations
from C-9 to H-5, H-1, and H-8, and three-bond correlations to H-4, H-6, H-7, and 8-Me which are consistent with the proposed structure. The COSY spectrum
showed long range W-coupling between H-1 and H-3
(2 Hz, see Table 1), which is only possible between H1a and H-3a (Kam et al., 1997). A similar W-coupling
was also observed between H-1a and H-5 (2 Hz),
which is possible only if H-5 is also a, and the ring
junction stereochemistry is cis. The stereochemistry of
the 4-methyl group is deduced to be b from the
observed J3b-4 value of 11 Hz, requiring H-4 and H-3b
to be in a trans-diaxial arrangement (Kam et al.,
1997). Finally, irradiation of H-1b causes NOE
enhancement of H-8 and vice versa, which establishes
the stereochemistry of the 8-methyl group as a. Based
on these results, the structure of kinabalurine G is as
shown in 13. The parent monoterpene, 9-hydroxy-dskytanthine 15, is unknown, and was not detected in
the present study, although a 9-hydroxyskytanthine of
unknown stereochemistry as well as a b-skytanthine Noxide, have been previously reported from Tecoma
stans (Dickinson & Jones, 1969) and Skytanthus acutus
(Streeter, Adolphen & Appel, 1969) respectively. The
occurrence of monoterpene alkaloids has also been
previously observed in two other Kopsia species, viz.,
K. pauci¯ora (Kam et al., 1997) and K. macrophylla
(Kan et al., 1995).
3. Experimental
3.1. Plant material
Plant material was collected from Sabah, Malaysia
and was identi®ed by Dr. K. M. Wong. Voucher specimens are deposited at the Herbarium of the Sabah
Forest Department, Sandakan, Sabah, Malaysia.
3.2. Extraction and Isolation
Extraction of alkaloids was carried out in the usual
manner as described in detail elsewhere (Kam & Tan,
1990). Essentially, the ground leaf material was
exhaustively extracted with 95% EtOH at ambient
temperature. The EtOH extract was then concentrated
under reduced pressure, partitioned into dilute HCl,
basi®ed with concentrated ammonia solution, and the
liberated alkaloids were then taken into chloroform to
give a basic fraction. The alkaloids were isolated by
repeated fractionation using CC and centrifugal TLC
on SiO2. Solvent systems used for chromatography
were CHCl3 with increasing proportions of MeOH
(CC) and Et2O, Et2O-hexane, CHCl3, CHCl3-MeOH
(Centrifugal TLC). The yields (g kgÿ1) of the alkaloids
(1±13) from the leaf extract were: 1 (0.032), 2 (0.281),
3 (0.004), 4 (0.004), 5 (0.046), 6 (0.028), 7 (0.056), 8
(0.003), 9 (0.006), 10 (0.002), 11 (0.313), 12 (0.004) and
13 (0.004).
3.3. Kinabalurine G (13)
[a]D+98 (CHCl3, c 0.129). EIMS, m/z (rel. int.): 199
[M+,C11H21NO2] (16), 183 (35), 166 (36), 156 (19), 150
(11), 139 (25), 126 (21), 110 (24), 96 (24), 84 (17) and
58 (100). HREIMS, [M+], found 199.1574, calcd for
C11H21NO2, 199.1572. 1H and 13C NMR: see Table 1.
3.4. Reduction of kinabalurine G (13)
Compound 13 (4 mg) was stirred in aqueous ferrous
sulfate (2.5%, 2 mL) at 808C for 0.5 h. The mixture
was then extracted with CHCl3 and chromatography
over SiO2 gave the parent monoterpene, 9-hydroxy-dskytanthine (15) (2 mg, 56%), API-LCMS, MH+, m/z
184 (C11H21NO+H). 1H NMR (400 MHz, CDCl3): d
0.87 (3H, d, J = 7 Hz, 4-Me), 1.00 (3H, d, J = 7 Hz,
8-Me), 1.84 (1H, t, J = 11.5 Hz, H-3), 2.04 (1H, d,
J = 12 Hz, H-1), and 2.30 (3H, s, N-Me). 13C NMR:
see Table 1.
T.-S. Kam et al. / Phytochemistry 52 (1999) 959±963
963
3.5. X-ray diraction analysis of danuphylline 12
References
A total of 2333 re¯ections were collected by the o
scan method up to ymax of 25.478 on a CAD4 diractometer at 278C using MoKa(l=0.71073 AÊ) radiation.
The crystal dimensions are 0.4 0.1 0.1 mm. A total
of 985 re¯ections with I> 2s(I ) were observed and
were corrected for the Lorentz-polarization eect, but
not for absorption. The structure was solved by using
the direct method SAPI-91 (Fan et al., 1991). All nonhydrogen atoms were re®ned anisotropically by full
matrix least squares re®nement on an IBM 486 PC to
R = 0.0769, wR = 0.1268 for the observed re¯ections,
w=[s2(F2o)+(0.0563P )2]ÿ1, where P = (F2o+2 F2c )/3.
Hydrogen atoms were generated geometrically and
were allowed to ride on their respective parent atoms.
The atomic coordinates for the non-hydrogen atoms
and their equivalent isotropic displacement parameters,
calculated coordinates for the hydrogen atoms, anisotropic displacement parameters for the non-hydrogen
atoms, a full list of bond distances and angles, and the
structure factor table are deposited as supplementary
material at the Cambridge Crystallographic Data
Centre.
Chen, W.S., Li, S.H., Kirfel, A., Will, G., & Breitmaier, E. (1981).
Liebigs Ann. Chem., 1886.
Cordell, G. A. (1977). In R. H. F. Manske, The Alkaloids, Vol. XVI
(p. 435). New York: Academic Press.
Crow, W. D., & Michael, M. (1962). Australian Journal of
Chemistry, 15, 130.
Dickinson, E. M., & Jones, G. (1969). Tetrahedron, 25, 1523.
Fan, H. F., Yao, J. X., Zheng, C. D., Gu, Y. X., & Qian, J. Z.
(1991). SAPI-91, a computer program for automatic solution of
crystal structures from X-ray diraction data. Beijing, P. R.
China: Institute of Physics, Chinese Academy of Sciences.
Feng, X. Z., Kan, C., Potier, P., Kan, S. K., & Lounasmaa, M.
(1983). Planta Medica, 48, 280.
Gilbert, B. (1965). In R. H. F. Manske, The Alkaloids, Vol. VIII (p.
439). New York: Academic Press.
Homberger, K., & Hesse, M. (1982). Helvetica Chimica Acta, 65,
2548.
Homberger, K., & Hesse, M. (1984). Helvetica Chimica Acta, 67,
237.
Kam, T. S., & Sim, K. M. (1998). Phytochemistry, 47, 145.
Kam, T. S., & Subramaniam, G. (1998). Natural Product Letters, 11,
131.
Kam, T. S., & Tan, P. S. (1990). Phytochemistry, 29, 2321.
Kam, T. S., Lim, T. M., Choo, Y. M., & Subramaniam, G. (1998).
Tetrahedron Letters, 39, 5823.
Kam, T. S., Lim, T. M., & Choo, Y. M. (1999). Tetrahedron, 55,
1457.
Kam, T. S., Subramaniam, G., & Chen, W. (1999). Phytochemistry,
in press.
Kam, T. S., Tan, P. S., Hoong, P. Y., & Chuah, C. H. (1993).
Phytochemistry, 32, 489.
Kam, T. S., Yoganathan, K., & Chen, W. (1997). Journal of Natural
Products, 60, 673.
Kan, C., Sevenet, T., Hadi, A. H. A., Bonin, M., Quirion, J. C., &
Husson, H. P. (1995). Natural Product Letters, 7, 283.
Streeter, M., Adolphen, G., & Appel, H. H. (1969). Chemistry and
Industry (London), 1631.
Zheng, J. J., Zhou, Y. L., & Huang, Z. H. (1989). Acta Chimica
Sinica, 2, 168 (English Edn.).
Acknowledgements
We thank the University of Malaya and IRPA for
®nancial support of this work, and Dr. J. K.
MacLeod, Research School of Chemistry, Australian
National University for mass spectra.