Taxoids from Taxus chinensis

Article abstract of DOI:10.1021/np060345g

Two new taxoids, 13,15-epoxy-13-epi-taxayunnasin A (1) and taxchinin N (2), were isolated from the leaves and stems of Taxus chinensis. Compound 2 is the first taxoid to be reported with an α,β-unsaturated lactone at C-4, C-5, C-20, and C-2. The structure of 1 was elucidated by spectroscopic analysis and confirmed by semisynthesis from taxayunnasin A, while 2 was determined structurally using spectroscopic methods and by single-crystal X-ray diffraction.

Full text of DOI:10.1021/np060345g

J. Nat. Prod. 2006, 69, 1813-1815
1813
Taxoids from Taxus chinensis
Yu Zhao,†,‡ Fu-Sheng Wang,§ Li-Yan Peng,† Xiao-Li Li,† Gang Xu,† Xiao-Xin Luo,† Yang Lu, Li Wu, Qi-Tai Zheng, and
Qin-Shi Zhao*,†
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650204, People’s Republic of China, Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of
China, Pharmaceutical Department of Dali UniVersity, Dali 671000, People’s Republic of China, and Beijing Institute of Materia Medica,
Chinese Academy of Medical Sciences, Beijing 100050, People’s Republic of China
ReceiVed July 16, 2006
Two new taxoids, 13,15-epoxy-13-epi-taxayunnasin A (1) and taxchinin N (2), were isolated from the leaves and stems
of Taxus chinensis. Compound 2 is the first taxoid to be reported with an R,â-unsaturated lactone at C-4, C-5, C-20,
and C-2. The structure of 1 was elucidated by spectroscopic analysis and confirmed by semisynthesis from taxayunnasin
A, while 2 was determined structurally using spectroscopic methods and by single-crystal X-ray diffraction.
The unique structure, novel mechanism of action, and clinical
importance of the antitumor agent paclitaxel continues to stimulate
a great deal of interest in the isolation of further taxoids from plants
in the genus Taxus.1-6 In a continuing search for new taxoids, we
have previously isolated several new taxoids from Taxus chinensis
(Pilger) Rehd. (Taxaceae).7-9 On further investigation of an extract
of the leaves and stems of T. chinensis, two minor new taxoids,
13,15-epoxy-13-epi-taxayunnasin A (1) and taxchinin N (2), were
isolated. The isolation and structure elucidation of 1 and 2 are the
subject of this report.
was confirmed by a ROESY NMR experiment, in which correla-
tions of H-2/H-9, Me-17, and Me-19 indicated that H-2 and H-9
are â-oriented, while correlations of H-3/H-7, H-14R, H-10/Me-
18, and H-5/H-7 confirmed the R-configuration of H-3, H-7, H-10,
and H-5 (Figure 1). A ROESY correlation between H-20â and Me-
19 supported the â-configuration of the oxetane ring. ROESY
correlations were observed for H-13/H-14R and H-14R/H-3, while
there was no correlation between H-13 and H-14â, which suggested
H-13 is in the R-orientation. Compound 1 resembles structurally
13-deacetoxy-13,15-epoxy-11(15f1)-abeo-13-epi-baccatin IV,13 in
which a benzoxy group is at C-2 while an acetyl group occurs at
C-10. In order to confirm the structure of 1, the chemical
transformation from taxayunnansin A to 1 was conducted success-
fully and employed methanesulfonyl chloride in pyridine in
excellent yield (99%). The mechanism of this transformation could
be explained as an intramolecular SN2 nucleophilic substitution
involving OH-15 and C-13, which is accordance with an in-
version of the configuration of H-13 in 1 (Scheme 1). Moreover,
this transformation might also be the biosynthetic route of
compound 1.
Compound 2 was obtained as a colorless, amorphous powder.
Its molecular formula, C30H38O12, was determined by HRESIMS
([M + Na]+, m/z 613.2260, calcd 613.2261). The IR spectrum
showed strong absorptions at 1745, 1692, and 1631 cm-1, corre-
sponding to ester carbonyl groups and an R,â-unsaturated lactone,
respectively. The UV spectrum showed an absorption at λmax 241
Compound 1 was obtained as a colorless, amorphous powder.
Its molecular formula, C35H42O12, was deduced from the HR-
FABMS ([M + H]+, m/z 655.2754, calcd 655.2755). Analysis of
the 1H and 13C NMR spectroscopic data (Table 1) and the HMQC
spectrum provided evidence that 1 possesses four acetyl groups,
one tetrasubstituted olefin, six oxymethines, three methylenes
(including one oxymethylene), four quaternary carbons (including
two oxygenated ones), and four methyl groups. The signals at δC
82.3 (s, C-4), δC 77.9 (t, C-20), δH 4.54 (d, J ) 4.8 Hz, H-20R),
and δH 4.52 (d, J ) 4.8 Hz, H-20â) indicated the presence of an
1
nm, suggesting the presence of an R,â-unsaturated ester. The H
and 13C NMR spectra of 2 showed the presence of a taxoid having
nine methyls (including five acetyl methyls), two methylenes, seven
methines (five oxygenated and one olefinic), and 12 quaternary
carbons (including three olefinic carbons) (Table 1). The charac-
teristic signals at δC 89.1 (s, C-1), 46.3 (d, C-3), 44.1 (s, C-8),
136.8 (s, C-11), 141.5 (s, C-12), and 43.7 (s, C-15) indicated that
2 has a 6/8/6 ring-system skeleton.10 On the basis of an analysis of
the 1H-1H COSY spectrum, the signals at δH 5.43 (1H, t, J ) 7.9
Hz), 6.01 (1H, d, J ) 11.0 Hz), 6.14 (1H, d, J ) 11.0 Hz), and
5.79 (1H, m) were assigned to H-7, H-9, H-10, and H-13,
respectively. The downfield chemical shift of H-7, H-9, H-10, and
H-13 implied that four acetoxy groups were attached to C-7, C-9,
C-10, and C-13, respectively, as confirmed by HMBC correlations
of H-7, H-9, H-10, and H-13 with the corresponding carbonyl
carbons of these acetyl groups. The remaining acetoxy group was
assigned to C-1, as deduced from the relative downfield chemical
shift (δC 89.1, s) of C-1. There were two double bonds [δC 136.8
(s, C-11), 141.5 (s, C-12), 128.7 (s, C-4), 134.7 (d, C-5)] in 2.
Except for the double bond between C-11 and C-12, another double
bond was assigned to C-4 and C-5 on the basis of the HMBC
1
oxetane moiety.10,11 Comparison of the H and 13C NMR spectro-
scopic data of 1 with those of taxayunnansin A showed that 1 is
very similar to taxayunansin A12 except for C-13 and C-15. The
HMBC correlation between H-13 (δH 4.32, d, J ) 0.6 Hz) and
C-15 (δC 79.0, s) suggested the presence of an oxygen bridge
between C-13 and C-15, forming a C-13(15)-tetrahydrofuran ring
moiety in 1. Three acetoxy groups were placed at C-2, C-7, and
C-9, while a benzoyloxy group was positioned at C-10 on the basis
of HMBC correlations. The remaining acetoxy group was placed
at C-4. Accordingly, compound 1 could be proposed as 13,15-
epoxy-13-epi-taxayunnansin A. The relative stereochemistry of 1
* To whom correspondence should be addressed. Tel: 86-871-5223058.
Fax: 86-871-5215783. E-mail: qinshizhaosp@yahoo.com.
† Kunming Institute of Botany, Chinese Academy of Sciences.
‡ Graduate School of the Chinese Academy of Sciences.
§ Pharmaceutical Department of Dali University.
Beijing Institute of Materia Medica.
10.1021/np060345g CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/18/2006

1814 Journal of Natural Products, 2006, Vol. 69, No. 12
Notes
Table 1. 1H and 13C NMR Spectroscopic Data for 1 and 2a
1
2
position
δC
δH
δC
δH
1
2
3
4
5
6
68.0 s
66.3 d
45.8 d
82.3 s
84.5 d
32.8 t
89.1 s
79.8 d
46.3 d
128.7 s
134.7 d
33.2 t
6.61 (1H, d, 7.5, H-2â)
3.23 (1H, d, 7.5, H-3R)
5.54 (1H, dd, 7.6, 1.2, H-2â)
3.53 (1H, m, H-3R)
4.91 (1H, dd, 9.6, 5.0, H-5R)
2.40 (1H, m, H-6â)
1.93 (1H, m, H-6R)
5.37 (1H, dd, 12.4, 6.0, H-7R)
6.70 (1H, dd, 3.8, 7.6, H-5)
2.34 (1H, m, H-6â)
2.79 (1H, m, H-6R)
5.43 (1H, t, 7.9, H-7R)
7
8
9
10
11
12
13
14
71.6 d
44.2 s
74.6 d
67.3 d
134.5 s
149.2 s
82.1 d
46.8 t
71.2 d
44.1 s
75.8 d
73.2 d
136.8 s
141.5 s
70.7 d
34.4 t
5.01 (1H, d, 5.7, H-9â)
6.31 (1H, d, 5.7, H-10R)
6.01 (1H, d, 11.0, H-9â)
6.14 (1H, d, 11.0, H-10R)
4.32 (1H, d, 0.6, H-13R)
1.60 (1H, brd, 8.3, H-14R)
2.14 (1H, overlap, H-14â)
5.79 (1H, m, H-13R)
3.03 (1H, m, H-14R)
2.07 (1H, m, H-14â)
15
16
17
18
19
20
79.0 s
26.8 q
28.8 q
11.9 q
13.8 q
77.9 t
43.7 s
30.7 q
22.9 q
16.3 q
13.5 q
166.9 s
0.73 (3H, s, Me-16)
1.16 (3H, s, Me-17)
1.74 (3H, s, Me-18)
1.79 (3H, s, Me-19)
4.54 (1H, d, 4.8, H-20R)
4.52 (1H, d, 4.8, H-20â)
1.16 (3H, s, Me-16)
1.67 (3H, s, Me-17)
2.07 (3H, s, Me-18)
1.01 (3H, s, Me-19)
OBz-10
i
o
m
p
165.4 s
130.5 s
130.8 d
129.7 d
134.5 d
8.22 (2H, dd, 8.3, 1.2)
7.59 (2H, t, 7.4)
7.69 (1H, t, 7.4)
OAc-1
OAc-2
OAc-4
OAc-7
OAc-9
OAc-10
OAc-13
OAc
OAc
OAc
OAc
OAc
169.6 s
172.2 s
169.9 s s
170.9 s
170.3 s
170.6 s
170.6 s
169.9 s
170.3 s
22.8 q
22.7 q
21.4 q
20.8 q
20.8 q
22.0 q
21.7 q
21.0 q
20.7 q
2.14 (3H, s)
2.12 (3H, s)
1.93 (3H, s)
1.87 (3H, s)
2.09 (3H, s)
2.06 (3H, s)
2.02 (3H, s)
2.01 (3H, s)
1.96 (3H, s)
a Data were recorded in acetone-d6 on a Bruker AM-400 MHz spectrometer; δ in ppm and J in Hz.
Figure 2. Selected ROESY correlations of 2.
Figure 1. Selected ROESY correlations of 1.
was established by a ROESY NMR experiment (Figure 2). The
â-orientations of H-2, H-9, and H-13 were apparent by the
correlations of H-2/H-9, Me-17, and Me-19, and H-13/Me-16, and
the R-orientations of H-3, H-7, and H-10 were evident from the
correlations of H-3/H-7 and H-14R, H-7/H-10, and H-10/Me-18
and H-7. Therefore, taxchinin N was determined as 2. X-ray
crystallographic analysis (Figure 3) of 2 confirmed the structure
deduced above and the relative configuration of 2.
correlations of H-3 with C-4 and C-5. The signal at δH 5.54 (1H,
dd, J ) 7.6, 1.2 Hz) was ascribed to H-2, as judged from the HMBC
correlations of H-2/C-1, C-8, and C-3. In turn, the signal at δC
166.9 (s) was assigned to C-20 from the HMBC correlations
between H-2 and C-20, which suggested the presence of a lactone
moiety between C-20 and C-2. The relative stereochemistry of 2
Scheme 1. Chemical Transformation from Taxayunnasin A to 1

Notes
Journal of Natural Products, 2006, Vol. 69, No. 12 1815
Taxchinin N (2): colorless, amorphous powder; [R]29 +40.0 (c
D
0.05, CHCl3); UV (CHCl3) λmax (log ꢀ) 241 (4.44) nm; IR (KBr) νmax
2933, 1745, 1692, 1631,1429, 1374, 1324, 1233, 1145 cm-1 13C NMR
;
(100 MHz, acetone-d6) and 1H NMR (400 MHz, acetone-d6) data, see
Table 1; ESIMS m/z 613 [M + Na]+; HRESIMS m/z 613.2260 [M +
Na]+ (calcd for C30H38O12Na, 613.2261).
Chemical Transformation of Taxayunnasin A to 1. Taxayunnasin
A (80 mg, 0.119 mmol) was dissolved in dry pyridine (3 mL). To this
solution was added MsCl (27.25 mg, 0.238 mmol). The reaction was
stirred at room temperature and monitored by TLC until all starting
material was consumed. The reaction mixture was diluted with EtOAc
and washed with diluted hydrochloric acid (10%), water, and brine.
The organic layer was then dried (Na2SO4) and concentrated. The
residue was chromatographed (10% ethyl acetate in chloroform) to
afford 1 (77.3 mg, 99%) as a colorless, amorphous powder; [R]27
D
+31.8 (c 0.67, CHCl3); 13C NMR (100 MHz, acetone-d6) and 1H NMR
(400 MHz, acetone-d6) data, see Table 1.
X-ray Structural Determination of Taxchinin N (2). Crystal-
lographic data for 2: C30H38O12, M ) 590.62, monoclinic, space group
P32 (or P31), a ) b ) 8.978(1) Å, c ) 33.622(2) Å, V ) 2347.0(4)
Å3, Z ) 3, d ) 1.292 g/cm3, crystal dimensions 0.20 × 0.20 × 0.50
mm was used for measurements on a MAC DIP-2030K diffractometer
with a graphite monochromator (ω scans, 2θmax ) 50.0°), Mo KR
radiation. The total number of reflections measured was 13 263, which
yielded an average number of reflections of 3342, of which 3076 were
observed (|F| g 2σ|F| ). Final indices: R1 ) 0.067, wR2 ) 0.219,
and the goodness-of-fit is 1.197. The crystal structure (2) was solved
by the direct method SHELXS-9714 and expanded using difference
Fourier techniques, refined by the program and method SHELXL-9714
and the full-matrix least-squares calculations. In the absence of
significant anomalous scattering, Friedel pairs were merged, and the
absolute configuration of this compound was not determined. Crystal-
lographic data for the structure of 2 have been deposited in the
Cambridge Crystallographic Data Centre (deposition number CCDC
613734). Copies of these data can be obtained free of charge via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB21EZ,
UK; fax (+44) 1223-336-033; or deposit@ ccdc.cam.ac.uk).
Figure 3. X-ray crystallographic structure of 2.
Experimental Section
General Experimental Procedures. Melting points were determined
on an XRC-1 micro melting point apparatus and are uncorrected. Optical
rotations were measured with a Horiba SEPA-300 polarimeter. UV
spectra were obtained on a UV 2401 PC spectrometer. IR spectra were
recorded on a Bio-Rad FTS-135 spectrometer with KBr pellets. 1H and
13C NMR experiments were performed on a Bruker AM-400 spec-
trometer, while 2D NMR spectra were recorded using a Bruker DRX-
500 NMR instrument. FABMS and HRFABMS were taken on a VG
Auto Spec-3000 or on a Finnigan-MAT 90 instrument. Column
chromatography was performed using silica gel (Qingdao Marine
Chemical Inc., Qingdao, People’s Republic of China), Lichroprep RP-
18 (Merck, Darmstadt, Germany), and Sephadex LH-20 (Pharmacia
Fine Chemical Co. Ltd.). Fractions were monitored by TLC, and spots
were visualized by heating silica gel plates sprayed with 10% H2SO4
in EtOH.
2
2
Supporting Information Available: Crystallographic data in cif
format. This material is available free of charge via the Internet at http://
pubs.acs.org.
Plant Material. The leaves and stems of Taxus chinensis (Taxaceae)
were collected in Liangshang of Sichuan Province, People’s Republic
of China, in March 2000, and identified by Prof. Lin Zhongwen. A
voucher specimen (No. 20012) has been deposited at the Kunming
Institute of Botany, Chinese Academy of Sciences, People’s Republic
of China.
References and Notes
(1) Baloglu, E.; Kingston, D. G. I. J. Nat. Prod. 1999, 62, 1448-1472.
(2) Shi, W. Q.; Zhao, Y. M.; Si, X. T.; Li, Z. P.; Yamada, T.; Kiyota,
H. J. Nat. Prod. 2006, 69, 280-283.
Extraction and Isolation. The dried leaves and stems (15 kg) of T.
chinensis were extracted three times with 95% ethanol to give a crude
extract after concentrating under a vacuum. The residue was dissolved
in MeOH-H2O (9:1) and divided into MeOH-soluble and -insoluble
parts. The MeOH-soluble part was further extracted with chloroform
to give 250 g of extract. This was chromatographed over a silica gel
column employing solvents of increasing polarity (petroleum-EtOAc,
9:1-1:9, and acetone) to give 10 fractions, of which three fractions
(20 g) (petroleum-EtOAc, 9:1, 8:2, 7:3) were further chromatographed
over a silica gel column eluted by CHCl3-MeOH (100:1-50:1) to
afford two subfractions, 1-10 (5 g) and 11-25 (12.5 g). Subfraction
11-25 (12.5 g) was chromatographed on a silica gel column eluted
with cyclohexane-EtOAc (7:3) to give subfractions 1′-30′. Fractions
20′-30′ were combined and chromatographed on Sephadex LH-20
eluted with MeOH, and a mixture of compound 1 and 2 was obtained.
The mixture was purified employing Lichroprep RP-18 eluted by
MeOH-H2O (5.5:4.5) to give compounds 1 (7 mg) and 2 (6 mg).
13,15-Epoxy-13-epi-taxayunnasin A (1): colorless, amorphous
(3) Morita, H.; Machida, I.; Hirasawa, Y.; Kobayashi, J. J. Nat. Prod.
2005, 68, 935-937.
(4) Petzke, T.; Shi, Q. W.; Sauriol, F.; Mamer, O.; Zamir, L. O. J. Nat.
Prod. 2004, 67, 1864-1869.
(5) Shen, W. S.; Sauriol, F.; Mamer, O.; Zamir, L. O. Chem. Commun.
2003, 68-69.
(6) Shigemori, H.; Kobayashi, J. J. Nat. Prod. 2004, 67, 245-256.
(7) Wang, F. S.; Pen, L. Y.; Zhao, Y.; Xu, G.; Zhao, Q. S.; Sun, H. D.
J. Nat. Prod. 2004, 67, 905-907.
(8) Xia, Z. H.; Peng, L. Y.; Xu, G.; Zhao, Q.-S.; Sun, H. D. Chem.
BiodiVersity 2005, 2, 1316-1319.
(9) Xia, Z. H.; Peng, L. Y.; Li, R. T.; Zhao, Q. S.; Sun, H. D.
Heterocycles 2005, 65, 1403-1408.
(10) Zhou, J. Y.; Cheng, W. M.; Fang, Q. C. Acta Bot. Sin. 2000, 42,
1-9.
(11) Zhou, J. Y.; Fang, Q. C. Acta Bot. Sin. 1997, 39, 467-476.
(12) Liu, X. K.; Wu, D. G.; Wang, Z. Y. Chin. Sci. Bull. 1992, 23, 2186-
2189.
powder; [R]29 +25.0 (c 0.26, CHCl3); UV λmax (log ꢀ) (CHCl3) 241
(13) Barboni, L.; Gariboldi, P.; Torregiani, E.; Appendino, G.; Cravotto,
G.; Bombardelli, E.; Gabetta, B.; Viterbo, D. J. Chem. Soc., Perkin
Trans. I 1994, 3233-3238.
(14) Sheldrick, G. M. SHELX-97, Program for Crystal Structure Refine-
ment; University of Gottingen: Gottingen, Germany, 1997.
D
(3.91), 275 (3.14) nm; IR (KBr) νmax 2959, 2925, 2853, 1734, 1631,
1450, 1371, 1229 cm-1 13C NMR (100 MHz, acetone-d6) and 1H NMR
;
(400 MHz, acetone-d6) data, see Table 1; positive FABMS m/z 655
[M + H]+; positive HRFABMS m/z 655.2754 [M + H]+ (calcd for
C35H43O12, 655.2755).
NP060345G