New taxanes from the needles of Taxus canadensis

Article abstract of DOI:10.1021/np000053u

Nine minor taxanes were identified for the first time in the Canadian yew needles. Four of these metabolites are new: 5-epi-cinnamoylcanadensene (1), 2,9,10,13-tetraacetoxy-20-cinnamoyloxy-taxa-4(5),11(12)-diene (2), 2'-acetyl-epi-taxol (3), and 9-deacety

Full text of DOI:10.1021/np000053u

J . Nat. Prod. 2000, 63, 929-933
929
New Ta xa n es fr om th e Need les of Ta xu s ca n a d en sis
J unzeng Zhang,† Francoise Sauriol,‡ Orval Mamer,§ and Lolita O. Zamir*,†
Human Health Research Center, INRS-Institut Armand-Frappier, Universite du Quebec, 531 Boulevard des Prairies,
Laval, Quebec, Canada, H7V 1B7, Department of Chemistry, Queen’s University, Kingston, Ontario, Canada, K7L 3N6,
and Biomedical Mass Spectrometry Unit, McGill University, 1130 Pine Avenue West, Montreal, Quebec, Canada, H3A 1A3
Received February 4, 2000
Nine minor taxanes were identified for the first time in the Canadian yew needles. Four of these
metabolites are new: 5-epi-cinnamoylcanadensene (1), 2,9,10,13-tetraacetoxy-20-cinnamoyloxy-taxa-
4(5),11(12)-diene (2), 2′-acetyl-epi-taxol (3), and 9-deacetyltaxinine E (4).
The initial report of paclitaxel1 from the bark of Taxus
brevifolia has stimulated the isolation of almost 350
natural taxanes from yews.2-4 Taxus canadensis Marsh.
(Taxacae) differs from other species in this genus by its
modest appearance (low trailing bush) and by the taxanes
9-12
specific to this yew.5-8 We have therefore started
systematic analysis of the needles of this yew.
a
In the present work, the detailed structures of nine
minor taxanes isolated from the needles of Taxus canaden-
sis for the first time were characterized, and four of them
are new taxanes. One of them, 5-epi-cinnamoylcanadensene
(1), is the third bicyclic highly oxygenated taxane isolated
from the needles of the Canadian yew.13-15 In addition, a
highly oxygenated derivative of taxa-4(5),11(12)-diene,
2,9,10,13-tetraacetyl-20-cinnamoyloxy-taxa-4(5),11(12)-di-
ene (2), 2′-acetyl-7-epi-taxol (3), and 9-deacetyl-taxinine E
(4) are new taxanes reported here. Five other metabolites
found in other yews are characterized for the first time in
the needles of the Canadian yew.
Resu lts a n d Discu ssion
The NMR data of compound 1 are shown in Table 1. Its
1H NMR, COSY, and HMQC spectra (1H and 13C NMR
data, see Table 1) revealed the presence of five acetyl
groups, one cinnamoyl unit, and four methyls (on nonpro-
tonated carbons). Two of the methyl singlets (δ 1.11 and
1.25 ppm) were COSY-correlated peaks as geminal methyls
(Me-16 and Me-17). The presence of three aliphatic meth-
ylene groups (δ 2.78 and 2.05, H2-6; δ 2.58 and 2.08, H-14a
and H-14b, and δ 4.65 and 3.55, H-20a and H-20b) were
also observed from the COSY spectrum. The HMBC cor-
relations of H-2 (δ 5.73) to the olefinic C-3 (δ 121.7); Me-
19 to C-7 (δ 66.7) and to the olefinic C-8 and C-9 (δ 124.3
and 143.0); and the highly deshielded H-10 (δ 7.25) to
olefinic C-9, C-12 (δ 135.7), and C-15 (δ 36.1) revealed that
1 is a bicyclic taxane. The relative stereochemistry was
determined on the basis of the NOESY connectivities
(Table 1) and shown to be similar to 5-epi-canadensene,15
in particular around the two double bonds at C-3(C-4), C-8
(C-9), and H-5â. The positioning of the cinnamoyl group
on C-5 is delicate, as we did not observe a HMBC correla-
tion of H-5 to its carbonyl carbon. We observed a very weak
NOESY correlation of H-5 with the R-CH of the cinnamoyl
group, but due to the large distance involved between these
two protons we cannot take this observation as solid proof.
The best evidence that the cinnamoyl group is indeed
positioned at C-5 came from the influence this group had
on the NMR shifts of neighboring positions: H-5, C-5, C-4,
and C-6 relative to 5-epi-canadensene. It is interesting to
note that the C-5R-cinnamoyl unit influences the NMR
shift of H-7 and H-10, resulting in substantial deshielding
on these protons, and also causes H-3 and Me-18 to shift,
because of the U-shape of the molecule (H-3 is substantially
more shielded, while Me-18 experiences a large deshield-
* To whom correspondence should be addressed. Tel.: +1 (450) 687-5010,
ext. 4260. Fax: +1 (514) 481-2797. E-mail: Lolita.Zamir@iaf.uquebec.ca.
† Human Health Research Center.
‡ Department of Chemistry, Queen’s University.
§
Biomedical Mass Spectrometry Unit.
10.1021/np000053u CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/06/2000

930 J ournal of Natural Products, 2000, Vol. 63, No. 7
Ta ble 1. 1H and 13C NMR Data for Taxane 1 in CDCl3
Zhang et al.
position
δ 1H mult.a (J in Hz)
δ
13Cb
HMBC
NOESY
1
2
3
4
1.73 o dd (7.4; 4.0)
5.73 o dd (11.6; 4.0)
5.71 o d (11.6)
46.8
70.3
121.7
137.4
69.5
2/3, 14a, Me-16, Me-17
1, 5, 20a, Me-17
see H-2
3
5
5.91 br s
2.78 ddd (15.9; 12.8; 2.8)
2.05 o m
6a, CHdR
5, 6b, 20b, Me-19
5, 6a, 7
6a
6b
7
8
9
35.3
4, 7, 8
5, 9, 19, 169.2
5.45 d (9.9)
66.7
124.3
143.0
68.4
136.3
135.7
70.0
2/3, 20a, Me-18
10
11
12
13
14a
14b
15
16
17
18
19
20a
20b
OAc
7.25 o s
9, 12, 15, 167.6
11, 12
7, Me-18
5.27 d (9.5)
2.58 ddd (17.4; 9.4; 7.4)
2.08 o m
14a, Me-16, Me-18
13, Me-16
13, 14a
26.0
36.1
33.1
24.9
16.8
12.1
57.4
1.11 s
1.25 s
2.24 s
1.62 s
4.65 d (12.9)
3.55 br d (12.9)
2.19 s
1, 11, 15, 17
1, 11, 15, 16
11, 12, 13
7, 8, 9
5
3, 4
167.6
169.2, 170.4
167.6
1, 13, 14a
2/3, 7, 13, CHdR
6a, 20b, Me-17
2/3, 20b
6a, 20a, Me-19
20.3
21.1
20.6
21.1
2.00 s × 2
1.94 s
1.82 s
169.2
OCinn
CdO
CHdR
CHdâ
Ph-1
o
165.4
117.9
145.7
134.2
127.8
129.0
130.4
6.57 d (16.1)
7.87 d (16.1)
Ph-1, CdO
C R, CdO, Ph-o
7.53 m
7.40 m
7.40 m
Ph-1, Ph-m
m
p
a
Mult. ) multiplicity: br, broad; d, doublet; m, multiplet; o, overlapping; s, singlet; t, triplet. The precision of the coupling constants
is (0.5 Hz. The 13C NMR chemical shifts were extracted from the HMQC and HMBC (for quaternary carbons) experiments ((0.2 ppm).
b
ing). To confirm these observations, a small amount of
5-epi-canadensene was esterified with cinnamic acid with-
out prior protection of the C-20 hydroxyl group. The
expected 20-cinnamoyl-5-epi-canadensene (1a ) and 5,20-
biscinnamoyl-epi-canadensene (1b) were obtained. Their
NMR data were in accord with the structure of 1 being
5-epi-cinnamoylcanadensene. HRFABMS confirmed the
elemental composition of the sodiated quasimolecular ions
of 1, 20-cinnamoyl-5-epi-canadensene (1a ) and 5,20-bis-
cinnamoyl-epi-canadensene (1b).
ring A and a cinnamoyl substituent on C-13, 13-cinnamoyl-
baccatin III, 17,18 exhibits a chemical shift of δ 6.21 for H-13.
On the other hand, for C-13-acetoxy taxanes, the H-13
chemical shift is in the range of δ 5.2-5.8. It was concluded,
therefore, that the structure of taxane 2 (H-13 δ 5.6, Table
2) is 2,9,10,13-tetraacetoxy-20-cinnamoyloxy-taxa-4(5),11(12)-
diene. This compound is a highly oxygenated derivative of
taxa-4(5),11(12)-diene, the proven precursor of taxol in T.
brevifolia.19,20 Compound 2 is the second oxygenated de-
rivative of taxa-4(5),11(12)-diene isolated as a natural
product. The first one, 2R,20-dihydroxy-9R-acetoxytaxa-
4,11-dien-13-one, was isolated from the needles and bark
of T. chinensis var. mairei.21 The relative stereochemistries
of the different groups in 2 were derived from the NOESY
correlations shown in Table 2. HRFABMS confirmed the
elemental composition of the sodiated quasimolecular ion
of 2. The co-metabolites 1 and 2 suggest that in T.
canadensis there might be two alternate biosynthetic
pathways of taxanes: (a) formation of the tricyclic hydro-
carbon taxa-4(5),11(12)-diene, which is then oxygenated to
give taxane 2, and (b) formation of a yet unidentified
bicyclic hydrocarbon, which is oxygenated to give the
canadensene family, including 1. The canadensenes might
be dead-end metabolites or may be further cyclized to
tricyclic taxanes.
The NMR data of the second new taxane (2) are shown
1
in Table 2. The H NMR spectrum showed signals corre-
sponding to four skeleton methyls, four acetyl groups, and
one cinnamoyl substituent. The spectra resembled taxinine
E11,16 except for the presence of an AB system (δ 4.98 and
4.78) with a very large coupling constant (J ) 14.0 Hz) that
could only be assigned to the methylene H-20. COSY and
HMQC experiments revealed that H-5 (δ 5.79) was an
olefinic proton, suggesting the presence of a double bond
at C-4 and C-5 (we could not obtain the chemical shift of
C-4 from the HMBC spectrum due to insufficient correla-
tions). The positions of the acetyl groups at C-2, C-9, and
C-10 were secured by the appropriate HMBC correlations
between H-2 (δ 5.56), H-9 (δ 5.94), and H-10 (δ 5.91) with
the carbonyl groups at 169.1, 169.6, and 169.9 ppm,
respectively. The only other groups requiring placement
were an acetyl and a cinnamoyl group. Unfortunately,
HMBC correlations, which would rigorously prove their
location, could not be observed. Comparative analysis
between the observed NMR shifts of 2 with various taxanes
having acetyls or cinnamoyls at C-13 or C-20 was therefore
used. The only known natural taxane with a six-membered
Taxane 3 was isolated as an analogue of 7-epi-taxol,22
and the 1H NMR data of the two compounds were very
similar. The only difference was at C-2′; H-2′ had a
chemical shift at δ 5.55 (J ) 3.3 Hz) in 3, while in 7-epi-
taxol it occurred at δ 4.81 (d, J ) 2.6 Hz). The downfield
shift of this proton signal suggested the location of an
acetate at C-2′. This was confirmed by the HMBC correla-

New Taxanes from T. Canadensis
J ournal of Natural Products, 2000, Vol. 63, No. 7 931
Ta ble 2. 1H and 13C NMR Data for Taxane 2 in CDCl3
position
δ 1H mult.a (J in Hz)
δ
13Cb
HMBC
11, 15
NOESY
2, 14a, Me-16
3, 9, Me-17, Me-19
2, 7b, 10, 14b, 20b, Me-18
1
2
3
1.79 br d (8.5)
5.56 br d (4.0)
3.35 m
46.1
71.9
44.4
3, 8, 14, 169.1
4
5
5.79 m (3.4)
2.12 m
2.01m
125.1
22.5
28.2
6, 20a
6a, 6b
7a
7b
8
5, 7b, Me-19
7b, Me-19
3, 6, 7a, 10
1.42 dt (11.9; 6.9)
42.0
76.1
72.2
135.8
137.9
69.1
9
5.94 d (10.7)
5.91 d (10.7)
7, 8, 10, 19, 169.6
9, 12, 15, 169.9
2, Me-17, Me-19
3, 7b, Me-18
10
11
12
13
14a
14b
15
16
17
18
19
20a
20b
OAc
5.61 br d (10.1)
2.78 ddd (16.1; 10.1; 8.5)
1.88 dd (16.1; 3.5)
14a, Me-16
1, 13, 14b, Me-16
3, 14a, 20b
28.9
38.0
33.0
25.5
15.6
17.6
67.0
1.03 s
1.76 s
1.97 s
0.96 s
4.98 d (14.0)
4.78 d (14.0)
2.09 s
2.06 s
2.06 s
1, 11, 15, 17
1, 11, 15, 16
11, 12, 13
3, 7, 8, 9
13, 14a, Me-17
2, 9, Me-16
3, 10, 13
2, 6, 7a, 9
5, 20b
3, 14b, 20a
20.9
20.9
21.7
21.1
170.5
169.6
169.1
169.9
2.01 s
OCinn
CdO
CHdR
CHdâ
Ph-1
o
166.6
117.8
144.8
134.4
128.1
129.1
130.3
6.46 d (16.0)
7.70 d (16.0)
Ph-1, CdO
C R, CdO, Ph-o
7.53 m
7.38 m
7.38 m
Ph-p
Ph-1, Ph-o, Ph-p
m
p
a
Mult. ) multiplicity: br, broad; d, doublet; m, multiplet; o, overlapping; s, singlet; t, triplet. The precision of the coupling constants
is (0.5 Hz. The 13C NMR chemical shifts were extracted from the HMQC and HMBC (for quaternary carbons) experiments ((0.2 ppm).
b
tions of H-2′ to its geminal acetyl (δ 169.8) as well as to
the C-1′ carboxylate (δ 167.8). The stereochemistry of
compound 3 as determined by NOESY correlations was the
same as that for 7-epi-taxol. Natural taxanes with a C-2′-
acetyl have not been reported among the yews. However,
these derivatives are easily prepared by acetylation.23
Indeed, acetylation of 7-epi-taxol yielded a compound
identical to 3, whose structure was, therefore, 2′-acetyl-7-
epi-taxol. HRFABMS confirmed the elemental composition
of its quasimolecular ion.
E,26 1â,7â-dihydroxy-4â,20-epoxy-2R,5R,9R,10â,13R-penta-
acetoxytax-11-ene,27 1â,9R-dihydroxy-4â,20-epoxy-2R,5R,
7â,10â,13R-pentaacetoxytax-11-ene,27 and 1â-hydroxy-10-
deacetyl-baccatin I,28 based on the NMR and HRFABMS
data. The compounds 1â,7â-dihydroxy-4â,20-epoxy-2R,
5R,9R,10â,13R-pentaacetoxytax-11-ene and 1â,9R-dihydroxy-
4â,20-epoxy-2R,5R,7â,10â,13R-pentaacetoxytax-11-ene were
isolated as a mixture. Indeed, the facile intramolecular acyl
migration among C-7, C-9, and C-10 made their separation
very difficult. This observation has been made by other
researchers.28
Taxane 4 was identified as 9-deacetyl-taxinine E from
its NMR data, which were very different from the NMR
data reported for 10-deacetyl-taxinine E, a compound
isolated from the leaves and stems of T. chinensis.24 The
HMBC correlations confirmed the assignments of H-9 and
H-10: the Me-19 protons showed the expected four cor-
relations (2J and 3J ) to C-3, C-7, C-8, and C-9; H-9 was
correlated to C-7, C-8, C-10, and C-19, whereas H-10 was
correlated to C-9, C-11, C-12, and C-15 and to the acetyl
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were recorded on a J ASCO DIP-370 digital polarimeter. All
NMR and HRFABMS data were obtained with the same
conditions and instruments reported previously.11 Similarly,
liquid column chromatography and preparative TLC were
performed on Si gel and precoated Si gel plates, respectively.11
Analytical HPLC and preparative HPLC were carried out on
the same instruments and conditions published,11 except that
only a 50-min linear gradient method (25% to 100% of CH3CN
in H2O, flow rate: 18 mL/min) was used. Semipreparative
HPLC was performed on the same system as the preparative
HPLC but using two Partisil 10 ODS-2 MAG-9 semiprepara-
tive columns (Whatman) connected in series (9.4 × 500 mm),
and a 50-min gradient method was used with a flow rate at 3
mL/min.
1
carbonyl at δ 170.0. The H NMR data obtained were very
similar to those of taxinine E,11,16 except for H-9 (δ 4.34),
which was shielded, indicating the presence of a hydroxyl
group. This was further confirmed by a COSY correlation
of a hydroxyl proton to H-9. The stereochemistry obtained
from the NOESY spectrum also showed the same relative
stereochemistry as taxinine E. HRFABMS confirmed the
elemental composition of the sodiated quasimolecular ion
of 4. Proof of this structure was given by acetylation of
taxane 4, which gave a product identical to taxinine E.11,16
In addition, five known taxanes were isolated for the first
time from this plant. Their structures were determined as
2-deacetyl-taxinine J ,25 2-deacetyl-5-decinnamoyl-taxinine
P la n t Ma ter ia l. T. canadensis Marsh. was collected in
September 1997, at St-J ean, Quebec, Canada, and stored at 4
°C before drying when needed. Several specimens representing
this collection have been deposited in the herbarium of the
Montreal Botanical Gardens.

932 J ournal of Natural Products, 2000, Vol. 63, No. 7
Zhang et al.
E xt r a ct ion a n d Isola t ion . Ground, dried needles of T.
canadensis (4.7 kg) were extracted and treated as described
previously11 to yield 119 g of a dark brown residue. This extract
was separated using dry-column chromatography on Si gel (Si
gel 60, 70-230 mesh, Selecto Science, Norcross, GA, 1.5 kg, 8
× 83 cm) eluted with CH2Cl2-i-PrOH (9:1, 3.5 L). After elution,
the Si gel was cut into 19 equal bands, and each band was
individually eluted with EtOAc-MeOH (1:1, 600 mL). The
eluents of the columns from bands 5 through 8 were combined
and evaporated to yield 38 g of residue A, which was then
subjected to Si gel column chromatography (840 g, 9.5 × 22
cm) with hexane (1 L), hexane-CH2Cl2 (3:1 and 1:1, each 2
L), CH2Cl2 (2 L), CH2Cl2-EtOAc (4:1, 3:2, 2:3, and 1:4, each 2
L), EtOAc (2 L), and EtOAc-MeOH [4:1 (2 L), 3:2 (4 L)], to
yield fractions B (7.0-8.2 L), C (9.0-10.0 L), and D (10.0-
11.2 L).
Fraction B (4.1 g) was applied to a Si gel column (140 g, 3.5
× 39 cm), eluted with hexane (200 mL), hexane-EtOAc (9:1,
8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, and 1:9, each 200 mL), EtOAc
(200 mL), and EtOAc-MeOH (9:1, 200 mL), to yield fractions
B1 (1440-1520 mL, 353 mg) and B2 (1660-1820 mL, 81 mg).
B1 was further purified by preparative HPLC to afford
2-deacetyl-taxinine J 25 (6.2 mg). Similarly, purification of B2
by preparative HPLC was followed by preparative TLC
(CH2Cl2-MeOH, 95:5) to give 1 (1.1 mg).
(3H, s, OAc), 2.13 (4H, m, H-14b and OAc), 1.90 (3H, s, H-18),
1.66 (3H, s, H-19), 1.17 (3H, s, H-16), 1.13 (3H, s, H-17); 13C
NMR (CDCl3) δ 207.4 (C-9), 171.7 (OCOCH3), 169.8 (C-2′ and
OCOCH3), 169.1 (OCOCH3), 167.8 (C-1′), 167.3 (OCOPh), 167.0
(C-5′), 140.2 (C-12), 138.0-128.0 (3′-Ph, not assigned), 133.7
(OBz-p), 132.9 (C-11), 132.4 (5′-Ph-p), 130.3 (OBz-o), 129.5
(OBz-1), 129.1 (OBz-m and 5′-Ph-m), 127.4 (5′-Ph-o), 83.0 (C-
5), 82.1 (C-4), 79.1 (C-1), 78.0 (C-10), 77.9 (C-20), 75.7 (C-7),
75.6 (C-2), 74.0 (C-2′), 71.8 (C-13), 57.6 (C-8), 52.9 (C-3′), 42.5
(C-15), 40.6 (C-3), 35.8 (C-14), 35.2 (C-6), 25.9 (C-16), 22.5
(OCOCH3), 21.3 (C-17), 20.9, 20.7 (both OCOCH3), 16.2 (C-
19), 14.6 (C-18); HMBC correlations H-2/C-1, -3, -8, and OBz;
OH-7/C-6 and -7; H-10/C-9, -11, -12, -15, and OAc; H-16/C-1,
-11, -15, and -17; H-17/C-1, -11, -15, and -16; H-18/C-11, -12,
and -13; H-19/C-3, -7, -8, and -9; H-20/C-3 and -4; H-2′/C-1′
and OAc; H-3′/C-5′; H-4′/C-5′; NOESY correlations H-2/H-20,
-17, and -19; H-3/H-6a, OH-7, -10, -14a, and -18; H-5/H-6a,
-6b, OH-7 and -20; H-6a/H-3, -5, and OH-7; H-6b/H-5 and -7;
OH-7/H-3, -5, -6a, -6b, -7, -10, and -18; H-10/H-3, OH-7, and
-18; H-13/H-14b, -16 and -18; H-16/H-13; H-17/H-2; H-18/H-
3, OH-7, -10, -13, and -2′; H-19/H-2, -7, and -20; H-20/H-2, -5,
and -19; H-2′/H-18, -3′, and OAc; H-3′/H-2′, -4′, and OAc; H-4′/
H-3′; HRFABMS m/z 918.33123 (calcd for C49H53NO15Na,
918.33129); HPLC, tR ) 43.12 min; visualized as a black spot
on TLC plate with Rf ) 0.70 (CH2Cl2-Me2CO, 8:2).
Acetyla tion of 7-epi-ta xol. 7-epi-Taxol7 (6 mg) was treated
with 0.5 mL of acetic anhydride and 0.5 mL of pyridine at room
temperature for 24 h and yielded a major product identical to
3: [R]21D -27° (c 0.34, CHCl3); (NMR, HRFABMS, HPLC, and
TLC).
Fraction C (5.7 g) was applied to a Si gel column (126 g, 4.5
× 21 cm) and eluted with hexane-CH2Cl2 [1:1 (300 mL), 4:6,
3:7, 2:8, and 1:9, each 200 mL], CH2Cl2 (200 mL), CH2Cl2-
EtOAc (9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, and 2:8, each 200 mL),
EtOAc (200 mL), and EtOAc-MeOH (8:2, 400 mL) to afford
fraction C1 (2480-3060 mL). Fraction C1 (449.5 mg) was
further purified by preparative HPLC, followed by preparative
TLC (CH2Cl2-Me2CO, 8:2 and hexane-n-BuOH, 8:2) and
semipreparative HPLC, to finally yield 1â,7â-dihydroxy-4â,20-
epoxy-2R,5R,9R,10â,13R-pentaacetoxytax-11-ene27 and 1â,9R-
dihydroxy-4â,20-epoxy-2R,5R,7â,10â,13R-pentaacetoxytax-11-
ene27 as a mixture (1.4 mg) and 1â-hydroxy-10-deacetyl-
baccatin I28 (0.5 mg).
9-Dea cetyl-ta xin in e E (4): [R]23D +85° (c 0.11, CHCl3); 1H
NMR (CDCl3) δ (1H, d, J ) 16.0 Hz, CHdâ OCinn) 7.49 (2H,
m, Ph-m OCinn), 7.40 (3H, m, Ph-m, p-OCinn), 6.67 (1H, d, J
) 16.0 Hz, CHdR OCinn), 5.86 (1H, d, J ) 9.7 Hz, H-10), 5.78
(1H, br t, J ) 8.0 Hz, H-13), 5.47 (1H, t, J ) 3.0 Hz, H-5), 5.42
(1H, dd, J ) 6.0, 1.9 Hz, H-2), 5.38, 5.01 (each 1H, s, H-20a
and 20b), 4.34 (1H, dd, J ) 9.7, 2.4 Hz, H-9), 3.34 (1H, d, J )
6.0 Hz, H-3), 2.65 (1H, m, H-14a), 2.31 (3H, s, H-18), 2.17 (1H,
o m, OH-9), 2.11 (3H, s, OCOCH3), 2.03 (3H, s, OCOCH3), 1.97
(1H, m, H-7a), 1.86 (1H, m, H-6a), 1.82 (1H, br d, J ) 8.8 Hz),
1.79 (3H, s, OCOCH3), 1.73 (1H, m, H-6b), 1.66 (1H, m, H-7b),
1.62 (1H, s, H-17), 1.47 (1H, dd, J ) 15.3, 7.6 Hz, H-14b), 1.10
(6H, s, H-16 and H-19); 13C NMR (CDCl3) δ 170.4, 170.0, 169.2
(3 × OCOCH3), 166.0 (OCinn), 145.0 (CHdâ OCinn), 136.4 (C-
12), 134.1 (Ph-1 OCinn), 134.0 (C-11), 130.4 (Ph-p OCinn),
129.1 (Ph-m OCinn), 127.9 (Ph-o OCinn), 118.7 (CHdR OCinn),
117.7 (C-20), 78.8 (C-5), 75.8 (C-9), 75.6 (C-10), 72.2 (C-2), 70.2
(C-13), 47.9 (C-1), 44.2 (C-8), 43.7 (C-3), 37.3 (C-15), 31.4 (C-
16), 28.7 (C-6), 28.4 (C-14), 27.1 (C-17), 25.7 (C-7), 21.5, 21.4,
21.0 (3 × OCOCH3), 15.2 (C-19), 18.2 (C-19); HMBC correla-
tions H-3/C-2, -8, and -19; H-9/C-7, -8, -10, and -19; H-10/C-9,
-11, -12, -15, and OAc; H-14a/C-2, -12, and -13; H-16/C-1, -11,
-15, and -17; H-17/C-1, -11, -15, and -16; H-18/C-11, -12, and
-13; H-19/C-3, -7, -8, and -9; NOESY correlations H-1/H-2, -14a,
-16, and -17; H-2/H-1, -9, -17, and -19; H-3/H-7b, -14b, and
-18; H-5/H-6a, -6b, and -20a; H-6a/H-5 and -6b; H-6b/H-5, -6a,
and -19; H-7a/H-7b and -19; H-7b/H-3, -7a, -10, and -18; H-9/
H-2, -17, and -19; H-10/H-7b and -18; H-13/H-14a and -16;
H-14a/H-1, -13, -14b, and -16; H-14b/H-3 and -14a; H-16/H-1,
-13, -14a, and -17; H-17/H-2, -9, -16, and -19; H-18/H-3, -7,
-10, -13, and CHdR; H-19/H-2, -6b, -7a, -9, -17, and -20b;
H-20a/H-20b; H-20b/H-20a; HRFABMS m/z 631.28824 (calcd
for C35H44O9Na, 631.28830); HPLC, tR ) 47.27 min; visualized
as a black spot on TLC plate with Rf ) 0.60 (CH2Cl2-Me2CO,
8:2).
Fraction D (5.6 g) was applied to a Si gel column (125 g, 4.5
× 20 cm) and eluted with CH2Cl2 (200 mL), CH2Cl2-EtOAc
(9:1, 8:2, 7:3, 6:4, 1:1, 4:6, 3:7, 2:8, and 1:9, each 200 mL),
EtOAc (200 mL), and EtOAc-MeOH (9:1 and 8:2, each 200
mL), to obtain fractions D1 (500-700 mL), D2 (700-1000 mL),
and D3 (1740-1840 mL). Fraction D1 (130 mg) was further
purified by preparative HPLC, followed by semipreparative
HPLC, to yield 2 (2.5 mg). Fraction D2 (206 mg) was also
subjected to preparative HPLC to give 3 (1.8 mg). The other
fractions from this HPLC column were further purified by
preparative TLC (hexane-n-BuOH, 8:2, CH2Cl2-MeOH, 9:1,
and CH2Cl2-Me2CO, 8:2) to afford 4 (4.9 mg). Fraction D3 (225
mg) was purified on a preparative HPLC column, followed by
preparative TLC (EtOAc and CH2Cl2-Me2CO, 8:2), to produce
2-deacetyl-5-decinnamoyl-taxinine E26 (2.3 mg).
5-epi-Cin n a m oylca n a d en sen e (1): [R]23 +110° (c 0.01,
D
CHCl3); 1H and 13C NMR, see Table 1; HRFABMS m/z
747.29889 (calcd for C39H48O13Na, 747.29926); HPLC, tR
)
42.68 min; visualized as a brown spot on TLC plate with Rf )
0.45 (CH2Cl2-MeOH, 95:5).
2,9,10,13-Te t r a a ce t oxy-20-cin n a m oyloxy-t a xa -4(5),
11(12)-d ien e (2): [R]23 +53° (c 0.06, CHCl3); 1H and 13C
D
NMR, see Table 2; HRFABMS m/z 673.29904 (calcd for
C
37H46O10Na, 673.29887); HPLC, tR ) 53.82 min; visualized
as a black spot on TLC plate with Rf ) 0.85 (CH2Cl2-MeOH,
95:5).
1
2′-Acetyl-7-epi-ta xol (3): [R]22 -30° (c 0.05, CHCl3); H
D
NMR (CDCl3) δ 8.16 (2H, d, J ) 7.8 Hz, OBz-o), 7.72 (2H, d,
J ) 7.7 Hz, 5′-Ph-o), 7.60 (1H, t, J ) 7.4 Hz, OBz-p), 7.52 (2H,
t, J ) 7.2 Hz, OBz-m), 7.49-7.30 (8H, m, 3′-Ph and 5′-Ph-m,
p), 6.87 (1H, d, J ) 9.1 Hz, H-4′), 6.82 (1H, s, H-10), 6.22 (1H,
t, J ) 9.0 Hz, H-13), 5.97 (1H, dd, J ) 9.1, 3.3 Hz, H-3′), 5.74
(1H, d, J ) 7.1 Hz, H-2), 5.55 (1H, d, J ) 3.3 Hz, H-2′), 4.93
(1H, dd, J ) 8.6, 3.4 Hz, H-5), 4.67 (1H, d, J ) 11.4 Hz, 7-OH),
4.38 (2H, br s, H-20a and b), 3.93 (1H, d, J ) 7.1 Hz, H-3),
3.70 (1H, br ddd, J ) 11.4, 4.7, 1.7 Hz, H-7), 2.53 (3H, s, OAc),
2.36 (1H, m, H-14a), 2.33, 2.28 (each, 1H, m, H-6a and b), 2.18
Acet yla t ion of 4. Compound 4 (2 mg) was treated with
acetic anhydride (0.5 mL) in pyridine (0.5 mL) at room
temperature for 22 h, and the product was determined by [R]21
D
+47° (c 0.12, CHCl3), 1H NMR, and HPLC data to be identical
to taxinine E.11,16
2-Dea cetyl-ta xin in e J : [R]23 +17° (c 0.15, CHCl3);25
D
HRFABMS m/z 689.29406 (calcd for C37H46O11Na, 689.29378);
HPLC, tR ) 43.89 min; visualized as a black spot on TLC plate
with Rf ) 0.75 (CH2Cl2-Me2CO, 8:2).

New Taxanes from T. Canadensis
J ournal of Natural Products, 2000, Vol. 63, No. 7 933
Refer en ces a n d Notes
Acetyla tion of 2-d ea cetyl-ta xin in e J . This compound (1.7
mg) was treated with acetic anhydride (0.5 mL) in pyridine
(0.5 mL) at room temperature for 22 h. It gave a product with
[R]24D +34° (c 0.21, CHCl3), 1H NMR, and HPLC data identical
to literature values for taxinine J .2,25
(1) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J .
Am. Chem. Soc. 1971, 93, 2325-2327.
(2) Kingston, D. G. I.; Molinero, A. A.; Rimoldi, J . M. In Progress in the
Chemistry of Organic Natural Products; Hertz, W., Kirby, G. W.,
Moore, R. E., Steglich, W., Tamm, C., Eds.; Springer-Verlag: New
York, 1993; Vol. 61, pp 1-206.
(3) Parmar, V. S.; J ha, A.; Bisht, K. S.; Taneja, P.; Singh, S. K.; Kumar,
A.; Poonam; J ain, R.; Olsen, C. E. Phytochemistry 1999, 50, 1267-
1304.
(4) Baloglu, E.; Kingston, D. G. I. J . Nat. Prod. 1999, 62, 1448-1472.
(5) Zamir, L. O.; Nedea, M. E.; Be´lair, S.; Sauriol, F.; Mamer, O.;
J acqmain, E.; J ean, F. I.; Garneau, F. X. Tetrahedron Lett. 1992, 33,
5173-5176.
(6) Gunawardana, G. P.; Premachandran, U.; Burres, M. S.; Whittern,
D. N.; Henry, R.; Spanton, S.; McAlpine, J . B. J . Nat. Prod. 1992, 55,
1686-1689.
(7) Zamir, L. O.; Nedea, M. E.; Zhou, Z. H.; Be´lair, S.; Sauriol, F.;
J acqmain, E.; J ean, F. I.; Garneau, F. X.; Mamer, O. Can. J . Chem.
1995, 73, 655-665.
(8) Nikolakakis, A.; Caron, G.; Cherestes, A.; Sauriol, F.; Mamer, O.;
Zamir, L. O. Bioorg. Med. Chem. 2000, 8 (6), 1269-1280.
(9) Zamir, L. O.; Nedea, M. E.; Zhou, Z.-H.; Caron, G.; Sauriol, F.; Mamer,
O. Phytochemistry 1996, 41, 803-805.
2-Dea cetyl-5-d ecin n a m oyl-ta xin in e E:26 [R]23 +20° (c
D
0.26, CHCl3); HRFABMS m/z 501.24648 (calcd for C26H38O8-
Na, 501.24644); HPLC, tR ) 30.61 min; visualized as a brown
spot on TLC plate with Rf ) 0.45 (CH2Cl2-Me2CO, 8:2).
1â,7â-Dih yd r oxy-4â,20-ep oxy-2r,5r,9r,10â,13r-p en t a -
a cetoxyta x-11-en e a n d 1â,9r-Dih yd r oxy-4â,20-ep oxy-2r,
5r,7â,10â,13r-p en ta a cetoxyta x-11-en e:27 HRFABMS m/z
633.25234 (calcd for C30H42O13Na, 633.25231); HPLC, tR
)
27.26 min; visualized as a green spot on TLC plate with Rf )
0.60 (CH2Cl2-Me2CO, 8:2).
1â-Hyd r oxy-10-d ea cetyl-ba cca tin I:28 [R]23D +60° (c 0.01,
CHCl3); HRFABMS m/z 633.25221 (calcd for C30H42O13Na,
633.25231); HPLC, tR ) 26.17 min; visualized as a green spot
on TLC plate with Rf ) 0.35 (CH2Cl2-Me2CO, 8:2).
Ester ifica tion of 5-epi-ca n a d en sen e. To a solution of
5-epi-canadensene15 (7 mg, 0.013 mmoL) in dry toluene was
added 10 mg of cinnamic acid (0.065 mmoL, 5 equiv), 14 mg
of DCC (dicyclohexylcarbodiimide, 0.065 mmoL, 5 equiv), and
8 mg of DMAP [4-(dimethylamino)pyridine, 0.065 mmoL, 5
equiv]. The mixture was stirred at 90 °C under nitrogen for 5
h (TLC showed no starting material left) and then evaporated
to dryness. This residue was then purified by preparative
HPLC and yielded 20-cinnamoyl-5-epi-canadensene (1a , 2.1
mg, 22% yield) and 5,20-biscinnamoyl-epi-canadensene (1b, 5.3
mg, 48% yield).
(10) Zamir, L. O.; Balachandran, S.; Zheng, Y. F.; Nedea, M. E.; Caron,
G.; Nikolakakis, A.; Vishwakarma, R. A.; Sauriol, F.; Mamer, O.
Tetrahedron 1997, 53, 15991-16008.
(11) Zamir, L. O.; Zhang, J .; Wu. J . H.; Sauriol, F.; Mamer, O. Tetrahedron
1999, 55, 14323-14340.
(12) Zamir, L. O.; Zhang, J .; Wu, J .; Sauriol, F.; Mamer, O. J . Nat. Prod.
1999, 62, 1268-1273.
(13) Zamir, L. O.; Zhou, Z, H.; Caron, G.; Sauriol, F.; Mamer O. J . Chem.
Soc., Chem. Commun. 1995, 529-530.
(14) Boulanger, Y.; Khiat, A.; Zhou, Z.-H.; Caron, G.; Zamir, L. O.
Tetrahedron 1996, 52, 8957-8968.
(15) Zamir, L. O.; Zhang, J .; Kutterer, K.; Sauriol, F.; Mamer O.; Khiat,
A.; Boulanger, Y. Tetrahedron 1998, 54, 15845-15860.
(16) Woods, M. C.; Chiang, H. C.; Nakadaira, Y.; Nakanishi, K. J . Am.
Chem. Soc. 1968, 90, 522-523.
20-Cin n a m oyl-5-epi-ca n a d en sen e (1a ): 1H NMR δ 7.46
(1H, d, J ) 16.0 Hz, CHdR), 7.49 (2H, m, Ph-H), 7.38 (3H, m,
Ph-H), 6.92 (1H, s, H-10), 6.51 (1H, d, J ) 11.6 Hz, H-3), 6.37
(1H, d, J ) 16.0 Hz, CHdâ), 5.84 (1H, dd, J ) 11.6, 4.3 Hz,
H-2), 5.28 (1H, d, J ) 9.2 Hz, H-7), 5.08 (1H, d, J ) 7.7 Hz,
H-13), 4.93 (1H, d, J ) 12.8 Hz, H-20a), 4.55 (1H, o d, J )
12.8 Hz, H-20b), 4.54 (1H, br s, H-5), 3.93 (1H, d, J ) 3.9 Hz,
OH-5), 2.63 (1H, dd, J ) 16.1, 7.8 Hz, H-6a), 2.53 (1H, m,
H-14a), 2.21 (3H, s, Ac), 2.18 (1H, o m, H-14b), 2.17 (3H, s,
Ac), 2.09 (3H, s, Ac), 2.03 (1H, dd, J ) 16.1, 4.5 Hz, H-6b),
1.97 (3H, s, Ac), 1.96 (3H, s, Ac), 1.94 (3H, s, Me-18), 1.82 (1H,
t, J ) 5.2 Hz, H-1), 1.67 (3H, s, Me-19), 1.29 (3H, s, Me-17),
1.12 (3H, s, Me-16); HRFABMS m/z 747.29889 (calcd for
(17) Morita, H.; Gonda, A.; Wei, L.; Yamamura, Y.; Wakabayashi, H.;
Takeya, K.; Itokawa, H. Phytochemistry 1998, 48, 857-862.
(18) Yang, S.-J .; Fang, J .-M.; Cheng, Y.-S. Phytochemistry 1999, 50, 127-
130.
(19) Koepp, A. E.; Hezari, M.; Zajicek, J .; Vogel, B. S.; LaFever, R. E.;
Lewis, N. G.; Croteau, R. J . Biol. Chem. 1995, 270, 8686-8690.
(20) Hefner, J .; Rubenstein, S. M.; Ketchum, R. E.; Gibson, D. M.;
Williams, R. M.; Croteau, R. Chem. Biol. 1996, 3, 479-489.
(21) Shi, Q. W.; Oritani, T.; Sugiyama, T.; Murakami, R.; Wei, H.-Q. J .
Nat. Prod. 1999, 62, 1114-1118.
(22) Huang, C. H. O.; Kingston, D. G. I.; Magri, N. F.; Samaranayake, G.
J . Nat. Prod. 1986, 49, 665-669.
(23) Mellado, W.; Magri, N. F.; Kingston, D. G. I.; Garcia-Arenas, R.; Orr,
G. A.; Horwitz, S. B. Biochem. Biophys. Res. Commun. 1984, 124,
329-336.
C
39H48O13Na, 747.29926).
5,20-Biscin n a m oyl-epi-ca n a d en sen e (1b): 1H NMR δ
7.89 (1H, d, J ) 16.0 Hz, CHdR1), 7.69 (1H, d, J ) 16.1 Hz,
CHdR2), 7.52 (4H, m, Ph-H), 7.39 (6H, m, Ph-H), 7.28 (1H, s,
H-10), 6.59 (1H, d, J ) 16.0 Hz, CHdâ1), 6.43 (1H, d, J )
16.1 Hz, CHdâ2), 5.99 (1H, d, J ) 11.0 Hz, H-3), 5.84 (1H, br
s, H-5), 5.84 (1H, m, H-2), 5.49 (1H, d, J ) 9.1 Hz, H-7), 5.27
(1H, d, J ) 9.4 Hz, H-13), 5.03 (1H, d, J ) 13.0 Hz, H-20a),
4.57 (1H, d, J ) 13.0 Hz, H-20b), 2.58 (2H, m, H-6a and -14a),
2.27 (3H, s, Me-18), 2.21 (3H, s, Ac), 2.12 (1H, m, H-6b), 2.02
(1H, m, H-14b), 1.98 (3H, s, Ac), 1.97 (3H, s, Ac), 1.95 (3H, s,
Ac), 1.86 (3H, s, Ac), 1.82 (1H, t, J ) 5.6 Hz, H-1), 1.66 (3H, s,
Me-19), 1.34 (3H, s, Me-17), 1.12 (3H, s, Me-16); HRFABMS
m/z 877.34148 (calcd for C48H54O14Na, 877.34113).
(24) Zhang, Z.; J ia, Z. Phytochemistry 1991, 30, 2345-2348.
(25) Morita, H.; Gonda, A.; Wei, L.; Yamamura, Y.; Wakabayashi, H.;
Takeya, K.; Itokawa, H. Planta Med. 1998, 64, 183-186. In this
publication, the [R]D -54.9° (c 0.23, CHCl3) reported for 2-deacetyl-
taxinine J differed from ours: [R]23 +17° (c 0.15, CHCl3). The NMR
D
(1H and 13C) data of our 2-deacetyltaxinine J and the reported data
were very similar. As additional confirmation of the structure of
2-deacetyltaxinine J , it was acetylated. The NMR data of the resulting
taxinine J , as well as the optical rotation obtained, agree with the
literature values. (Min, Z.-D.; J iang, H.; Liang, J .-Y. Acta Pharm. Sin.
1989, 24, 673-677.)
(26) Barboni, L.; Gariboldi, P.; Appendino, G.; Enriu, R.; Gabetta, B.;
Bombardelli, E. Liebigs Ann. 1995, 345-349.
(27) Chu, A.; Davin, L. B.; Zajicek, J .; Lewis, N. G.; Croteau, R. Phyto-
chemistry 1993, 34, 473-476.
(28) Zhang, H.; Mu, Q.; Xiang, W.; Yao, P.; Sun, H.; Takeda, Y. Phyto-
chemistry 1997, 44, 911-915.
Ack n ow led gm en t. We thank the Natural Science and
Engineering Research Council of Canada for support via an
operating grant to L.O.Z.
NP000053U