Taxines from the needles of Taxus wallichiana

Article abstract of DOI:10.1016/S0031-9422(01)00305-3

A taxine, 5αO-(3′-dimethylamino-3′-phenylpropionyl) taxinine M (1) together with two known compounds 7-O-acetyltaxine A (2) and 2α-acetoxy-2′ β-deacetylaustrospicatine (3) were isolated from the needles of the Himalayan yew, Taxus wallichiana Zucc. Their structures were elucidated on the basis of the NMR spectral data, ESI-MS/MS analysis and chemical methods. Compounds 1 and 3 showed moderate cytotoxic activity against the lung cancer cell line A549 in vitro.

Full text of DOI:10.1016/S0031-9422(01)00305-3

Phytochemistry 58 (2001) 1167–1170
www.elsevier.com/locate/phytochem
Taxines from the needles of Taxus wallichiana
Jeevan Kumar Prasain, Piotr Stefanowicz, Taira Kiyota, Farida Habeichi, Yasuo Konishi*
Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec, Canada H4P 2R2
Received 9 March 2001; received in revised form 6 June 2001
Abstract
A taxine, 5ꢀO-(30-dimethylamino-30-phenylpropionyl) taxinine M (1) together with two known compounds 7-O-acetyltaxine A (2)
and 2ꢀ-acetoxy-20ꢁ-deacetylaustrospicatine (3) were isolated from the needles of the Himalayan yew, Taxus wallichiana Zucc. Their
structures were elucidated on the basis of the NMRspectral data, ESI–MS/MS analysis and chemical methods. Compounds 1 and 3
showed moderate cytotoxic activity against the lung cancer cell line A549 in vitro. # 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Taxus wallichiana; Taxaceae; Taxine; Cytotoxic
1. Introduction
acid-base partition scheme, we isolated 1 (0.5 mg) along
with two other known taxines, 7-O-acetyltaxine A (2)
and 2ꢀ-acetoxy-2ꢁ-deacetylaustrospicatine (3). Purifica-
tion of these compounds was performed by semi-pre-
parative reversed phase HPLC.
The emergence of paclitaxel (taxol) as one of the most
useful anticancer natural products has stimulated great
interest in analysis of Taxus species with a view to find-
ing alternative sources of this compound. As a result,
numerous taxoids have been reported from various
Taxus species (Wani et al., 1971; Chen and Kingston,
1994; Zhang et al., 2000). The needles of the Himalayan
yew (Taxus wallichiana Zucc., Taxaceae) is one of the
valuable source of taxoids (Appendino et al., 1992;
Chattopadhyay et al., 1999). Recently, a new taxane
IDN5109, derived from 14ꢁ-hydroxy-10-deacetylbacca-
tin III, is reported to have a potent antitumor activity in
ovarian carcinoma (Nicoletti et al., 2000). In course of
our program to isolate bio-active taxoids from the nee-
dles of Taxus wallichiana, we isolated a new taxine 5ꢀ-O-
(30-dimethylamino-30-phenylpropionyl) taxinine M (1).
To the best of our knowledge, this is the first benzoate
containing cytotoxic taxine belonging to taxagifine type.
We report herein the structure elucidation of this minor
compound together with its cytotoxic activity against
the lung tumor cell line A549.
Compound 1 was obtained as a colorless solid having
25
[y]
2500 (0.001 M, MeOH). The HR–FAB–MS
308
showed an intense [M+H]+ ion at m/z 862.36481 which
corresponded to a molecular formula of C46H55NO15.
Low collision energy electrospray tandem mass spec-
trometric (ESI–MS/MS) analysis of this compound
provided a series of product ions representing diterpene
ring fragments and side chains. The product ion at m/z
194.1 corresponds to a loss of 193.1 Da from the m/z
861.8 to a product ion at m/z 668.7.
Further loss of three acetic acid molecules (3Â60 Da),
an acetyl group (42 Da), and a benzoic acid molecule (122
Da) produced an ion at m/z 324.9. There is an another
series of product ions due the loss three molecules of acetic
acid, an acetyl, and a benzoic acid molecule originating
from the ion at m/z 668.7. From these fragmentation
patterns, it was deduced that compound 1 has at least
four acetyl groups, one benzoate, and a nitrogen con-
taining side chain (193.2 Da).
2. Results and discussion
The product ion at m/z 194.1 was further character-
ized by the MS/MS/MS experiment. Four product ions
were observed at m/z 46, 79, 107 and 149 corresponding
to (CH3)2NH+2 , [C6H7]+, C7H6OH+ and C6H5C2H2
COOH+2 , respectively. Thus these data indicated the
presence of an N-dimethyl phenyl propionate side chain
in the molecule. Furthermore, the side chain was con-
sidered to be 3-(dimethylamino)-3-phenylpropionate
The air-dried needles (1 kg) of T. wallichiana were
extracted with EtOH, and the EtOH extract was dissolved
in EtOAc and extracted with 10% HCl. Following an
* Corresponding author. Tel.: +1-514-496-6339: Fax: +1-514-496-
5143.
E-mail address: yasuo.konishi@nrc.ca (Y. Konishi).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00305-3

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J.K. Prasain et al. / Phytochemistry 58 (2001) 1167–1170
(Winterstein ester) in view of the biosynthesis of taxoid
phenylisoserine side chain (Leete and Badem, 1966).
Table 1
1H and 13C NMRspectroscopic data of 1
a
Position
ꢂC
ꢂH
1
2
3
4
5
6
48.1
2.48 m
6.11 br d, 10.2
3.42 d, 10.2
69.5
40.0
140.0
76.0
40.0
5.23 br s
2.0 m
1.55 m
5.34 br d, 6.2
7
8
64.0
49.7
70.4
64.0
80.2
91.4
205.7
34.5
9
5.31 d, 3.0
5.29 d, 3.0
4.15 br s
10
11
12
13
14
3.10 dd, 11.5, 19.2
2.65 d, 19.2
15
16
17
49.7
15.4
82.2
1.26
4.10 d, 7.9
3.65 d, 8.0
1.15 s
18
19
11.9
60.8
5.19 d, 12.2
4.37 d, 12.2
5.49 s
20
116.7
4.75 s
N(Me)2
38.8
42.3
2.76 s
2.88 s
19-OCOC6H5
19-OBz (ipso)
19-OBz (ortho)
19-OBz (meta)
19-OBz (para)
10
167.5
130.1
130.1Â2
128.6Â2
133.7
Ob
8.12 d, 7.9
7.48 m
7.58 m
20
30
35.9
3.78 m
4.95 m
65.9
30-Ph (ipso)
30-Ph (ortho)
30-Ph (meta)
30-Ph (para)
Ac
141.0
130.1
128.6
128.6
168.0
168.0Â2
172.2
21.2Â2
20.5Â2
7.48 m
7.48 m
7.48 m
In order to confirm the structure of this side chain, 3-
dimethylamino-3-phenylpropionic acid was synthesized
by reacting 3-amino-3-phenylpropionic acid with formal-
dehyde and sodium borohydride. The MS/MS analysis of
which produced product ions identical to those of the
ion 194. Thus the presence of a Winterstein ester in 1
was concluded and remaining structure elucidation was
performed by its NMRspectral analysis.
1.97 s, 2.11 s
1.97 s, 2.06 s
a
The 13C NMRdata were deduced from the HMQC and HMBC spectra.
Direct measurement of 13C NMRwas not possible due to the meager quan-
tity of the sample. ꢂ in ppm and J in Hz. Ob=obscure. 1D and 2D NMR
experiments (500 MHz in CDCl3).
1
A part of the H NMRspectrum of 1 in MeOH was
bridge to C-12. The carbonyl carbon (ꢂ 205.7) showed a
long range correlation with the methyl group (ꢂ 1.15)
which showed correlations with both C-12 and C-11 in
the HMBC spectrum. Thus the carbonyl carbon was
assigned at C-13. The long range correlation observed in
the HMBC spectrum of 1 between H-19 and the benzo-
ate carbonyl clearly indicated the position of benzoate
group at C-19. Likewise, the H2-20 showed correlations
with both C-3 and C-5, indicating an exocyclic double
bond at C-4. The presence of a Winterstein ester in 1
was supported by the signals at 2.88 (3H, s), 2.76 (3H,
s), 3.78 (2H, m), 4.95 (1H, m), and 7.48 (5H, m) in the
1H NMRspectrum. Since the compound was purified
similar to that of taxinine M (Beutler et al., 1991), indi-
cating the presence of a tetracyclic structure with one
isolated double bond, four acetates and one benzoate
group (Table 1). The connectivities of the protons in the
taxane skeleton of 1 were determined by its 1H–1H
1
COSY spectrum. The H NMRspectrum of 1 showed
the presence of only two methyl groups (ꢂ 1.15 s and
1.26 s) instead of four, which is common in many tax-
oids, and two oxymethylenes (ꢂ 4.10, d, 7.9 Hz, 3.65, d,
8.0 Hz, and ꢂ 5.19, d, 12.2 Hz, 4.37, d, 12.2 Hz).
The HMQC and HMBC spectra of 1 indicated the
oxygenation at C-17, with the oxygen present as an oxido

J.K. Prasain et al. / Phytochemistry 58 (2001) 1167–1170
1169
by HPLC using acetonitrile/water/TFA system, the
dimethylamino group was protonated and the 1H NMR
signals were found to be shifted more downfield than
those of similar basic taxoids.
with ED50 10.1Æ2.4 and 28.3Æ3.8 mM, respectively,
while 2 was inactive.
Cytotoxicity of many taxines against cancer cells has
not been fully investigated. Taxinine M is reported to
show a weak toxicity in the brine shrimp assay (Beutler et
al., 1991). The co-occurrence of an aromatic group and
an alkaloid side chain in 1 may be responsible for its
cytotoxicity against A549 cells by the fact that similar
activity was observed with taxuspine N (Kobayashi et al.,
1996). Further elaboration on structure–activity relation-
ships of 1 is underway.
The stereochemistry of 1 around the diterpene skeleton
was deduced by comparison with taxinine M, in view the
similarity in splitting pattern (coupling constants) in the
1H NMR, and NOESY correlations. As in the case of
taxinine M, NOESY correlations were observed between
the proton pairs H-3/H-10, H-9/Me-17, H-2/Me-17, OH-
11/Hb-16, and Ha-16/Me-17. Moreover, transannular
interactions between the pairs H-3/H-10, H-9/Me-17,
H-2/Me-17 may occur because the internal space may
be inadequate for all the quasi-axial hydrogen atoms to
fit without interacting. In this situation, some of the
carbon bonds adopt eclipsed or partially eclipsed con-
formations (March, 1992).
The small value (ca. 3 Hz) of J9,10 is due to an eclipsed
conformation around the C-9, C-10 bond causing H-9
and H-10 to adopt an anticlinal position (Barboni et al.,
1995a,b). The conformation of ring B is in the tax-
agifine-type owing to the presence of the C-12, C-17
oxygen bridge and the large coupling constant between
H-2 and H-3 (Table 1). The signal at ꢂ 5.23 (1H, br s)
was characteristic of H-5ꢀ (Shi et al., 1999). The cou-
pling constant of H-7 (ꢂ 5.34, br d, 6.2 Hz) was similar
to that of taxinine M. The stereochemistry at the chiral
centre C-30 was assigned as R from the biosynthesis of
Winterstein’s acid (Leete and Badem, 1966). Based on
these evidences, the sterochemistry of 1 was elucidated,
and the isolate was determined to be 5ꢀ- O-(30-dime-
thylamino-30-phenylpropionyl) taxinine M.
Compounds 2 and 3 were identified as 7-O-acetyltaxine
A and 2ꢀ-acetoxy-20ꢁ-deacetylaustrospicatine, respec-
tively by comparison of their spectral data with published
ones (Ettouati et al., 1988; Barboni et al., 1995a,b). Com-
pound 1 is a minor constituent of the needles of the
Himalayan yew in which taxine’s concentration might
be much lower than in the European or the Japanese
yew (Barboni et al., 1995a,b).
We, for the first time, have attempted to elucidate the
structure of a taxine through MS/MS analyses. The MS/
MS analyses revealed that compound 1 is composed of
four major segments, i.e. a core diterpene ring, one benzo-
ate, four acetate, and one Winterstein ester side chain. It is
interesting to note that no taxines are reported to be of the
taxagifine type and compound 1 represents the first exam-
ple of the taxine with a taxagifine core structure and is the
second example of a taxoid involving both a benzoate and
the Winterstein ester (Kobayashi et al., 1996). This type of
compound possesses a different diterpene ring conforma-
tion than those of the taxol type. The unusual conforma-
tion may be responsible for diminution in biological
activity compared to taxol (Chauviere et al., 1982).
3. Experimental
3.1. General
CD spectra were recorded with a JASCO J-710 spectro-
polarimeter. Mass spectrometry measurements were per-
formed by using a Sciex API III tandem quadrupole mass
spectrometer. Full-scan mass spectra were obtained
while continuously scanning from m/z 300 to 1000 with
a step size of 0.1 m/z and with a dwell time of 2.0 ms.
MS/MS spectrum of 3-dimethylamino-3-phenylpro-
pionic acid was obtained by selecting the protonated ion
at m/z 194 at the first quadrupole (Q1), followed by
collision-induced dissociation (cid) with argon gas at the
second quadrupole (Q2). The resulting fragments were
analyzed by the third quadrupole (Q3).
MS/MS/MS experiment of compound 1 was performed
by accelerating 1 at the orifice with a high orifice potential
of 150 V and by fragmenting it at Q0. The fragment with
m/z 194 was selected at Q1 and was further fragmented at
Q2 with argon collision gas. The fragments were analyzed
by Q3. The parent scan of compound 1 in MS/MS/MS
was carried out under the same condition. After frag-
menting compound 1 at Q0 using orifice potential 150,
the parent fragment for the ion at m/z 445 was found in
the parent scan experiment. A high resolution FAB mass
spectrometry was VG Micromass ZAB 2F HS. 1H and 2D
NMRspectra were recorded on a Bruker AMX2 500
NMRspectrometer. HPLC was performed using a Waters
Delta Prep 4000 with a semi preparative RP-Vydac C-8.
The eluant was monitored by the absorbance at 254 nm.
Chemicals used for the synthesis of 3-dimethylamino-3-
phenylpropionic acid were purchased from Aldrich.
3.2. Plant material
The needles of T. wallichiana were obtained from
plants grown in the garden of Trichandra college, Trib-
huvan University, Nepal.
3.3. Extraction and isolation
Compounds 1–3 were evaluated for their cytotoxicity
against A549 cell line and 1 and 3 showed a cytotoxicity
Air-dried needles (1 kg) were extracted with EtOH (4
lit Â3) at room temperature. The EtOH solvent was

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J.K. Prasain et al. / Phytochemistry 58 (2001) 1167–1170
evaporated under reduced pressure to obtain a semi-
solid EtOH extract (110 g). A fraction of EtOH extract
(50 g) was dissolved in EtOAc and extracted with 10%
HCl. The acidic phase was washed with EtOAc (2 Â 200
ml) and neutralized with Na2CO3, followed by extraction
with EtOAc. After drying (Na2SO4), a semi-solid residue
(0.5 g) was obtained which was further fractionated by the
reversed-phase HPLC [a linear gradient of acetonitrile
from 0 to 40% (0.6%/min) in 0.1% TFA; flow rate, 13 ml/
min)]. Compounds 1, 2 and 3 were eluted at the 40, 36 and
29% CH3CN in the gradient system, respectively. All
collected fractions were lyophilized immediately, and
tested for cytotoxic activity. Those found inactive and
complicated mixture were discarded. After lyophilization,
0.9 mg of 1, 1.0 mg of 2 and 1.5 mg of 3 were obtained.
same condition for 48 h. The medium was removed by
aspiration, and crystals of viable cells were dissolved in
DMSO (100 ml/well) and the plate was read immediately
on a microtitrer plate reader (SOFTmax, Molecular
Device) at the wavelength of 540 nm. Absorbance taken
from cell grown in the absence of test compound was
considered as a negative control.
Acknowledgements
We thank Dr. Justin Powlowski (Concordia University,
Montreal) for his help in recording CD spectrum.
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1
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1
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0
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