Isolation of labeled 9-dihydrobaccatin III and related taxoids from cell cultures of Taxus canadensis elicited with methyl jasmonate

Article abstract of DOI:10.1021/np990201k

Cell suspension cultures of Taxus canadensis rapidly produced paclitaxel (1) and other taxoids in response to elicitation with methyl jasmonate. Three of these taxoids, of potential value in the synthesis of taxoid analogues, have been isolated from cell

Full text of DOI:10.1021/np990201k

J . Nat. Prod. 1999, 62, 1395-1398
1395
Isola tion of La beled 9-Dih yd r oba cca tin III a n d Rela ted Ta xoid s fr om Cell
Cu ltu r es of Ta xu s ca n a d en sis Elicited w ith Meth yl J a sm on a te
Raymond E. B. Ketchum,†,‡ Manish Tandon,§, Donna M. Gibson,| Tadhg Begley,§ and M. L. Shuler*,†
School of Chemical Engineering, and Department of Chemistry and Chemical Biology, Cornell University,
Ithaca, New York 14853, and USDA, ARS, US Plant, Soil, and Nutrition Lab, Ithaca, New York 14853
Received April 28, 1999
Cell suspension cultures of Taxus canadensis rapidly produced paclitaxel (1) and other taxoids in response
to elicitation with methyl jasmonate. Three of these taxoids, of potential value in the synthesis of taxoid
analogues, have been isolated from cell cultures of Taxus canadensis and identified as 13-acetyl-9-
dihydrobaccatin III (2), baccatin VI (3), and 9-dihydrobaccatin III (4). Of these metabolites, 9-dihydro-
baccatin III (4) has not been isolated from any Taxus species, whereas 13-acetyl-9-dihydrobaccatin III
(2) and baccatin VI (3) have been isolated from a number of natural sources. 2D NMR techniques, mass
spectrometry, and partial synthesis were used to rigorously elucidate the structure and stereochemistry
of these natural products.
Paclitaxel (1), a diterpenoid extracted from the bark of
the pacific yew (Taxus brevifolia) and other yew species,
is one of the most recent and effective drugs in the
treatment of cancer (Figure 1). Its activity against ovarian
and breast cancer, its biology, and its biochemistry have
been thoroughly reviewed.1 It may also prove to be a useful
treatment against many other types of cancer, including
nonsmall-cell lung cancer and2 gastric carcinoma,3 and of
noncancerous conditions, such as polycystic kidney dis-
ease.4
The greatest concentrations of paclitaxel (1) are about
0.02% to 0.1% of the dry weight of T. brevifolia bark.5 The
low concentration of paclitaxel (1) in the bark, the lethal
nature of bark harvest, and the slow growth of the tree
have led to the development of alternative means of
paclitaxel (1) production. Semisynthetic methods have been
developed for the production of paclitaxel (1) (and its
analogue docetaxel or Taxotere) using advanced taxane
diterpenoid (taxoid) metabolites isolated from readily avail-
able natural sources, such as 10-deacetylbaccatin III
extracted from leaves of yew.6 However, with increasing
applications in medicine, especially in the treatment of
additional cancer types, and its use much earlier in the
F igu r e 1. Structure of paclitaxel (taxol) (1), 13-acetyl-9-dihydrobac-
catin III (2), baccatin VI (3), and 9-dihydrobaccatin III (4).
course of intervention, the supply and cost of these drugs
will remain important issues. Total synthesis of paclitaxel
(1) has been achieved by several elegant routes, but the
yields are too low to be commercially feasible.7,8
isolation and identification of several other taxoids present
in elicited cultures and accounting for as much as 60% of
the total taxoids in an elicited cell culture of Taxus
canadensis Marsh (Taxaceae).
The supply of paclitaxel (1) and semisynthetic precursors
must rely on biological methods of production, either from
plants in the genus Taxus or in cell cultures derived from
them.9 Inducible Taxus cell cultures, capable of producing
significant amounts of paclitaxel (1) and related taxoids,
provide an excellent tool for the study of this complex
biosynthetic pathway.10-12 We have previously reported the
production of up to 117 mg‚L-1 of paclitaxel (1) within 5
days of elicitation.10 However, paclitaxel (1) never ac-
counted for more than 20% of the total taxoids in the
culture, based on detector response. Here we report on the
Resu lts a n d Discu ssion
Measurable differences in paclitaxel (1) concentration
between elicited cultures and the control are detectable
within 8 h of elicitation and are apparent under normal
analytical conditions within 24 h.10 Paclitaxel (1) concen-
tration increases in the culture medium until 12 to 14 days
after elicitation and then begins to decline as the cells
senesce.10 However, paclitaxel (1) is only one of several
taxoids that accumulate in the medium and accounts for
only 13-20% of the total taxoids present in the elicited
culture.10 Two other taxoids, eluting at approximately 7
and 19 min, together account for 39-62% of the total
taxoids present in an elicited culture, depending on the
culture age (Figure 2). These compounds were isolated by
* To whom correspondence should be addressed. Tel.: (607) 255-7577.
Fax: (607) 255-9166. E-mail: mls@cheme.cornell.edu.
† School of Chemical Engineering.
‡ Present Address: Institute of Biological Chemistry, P.O. Box 646340,
Washington State University, Pullman, WA 99164-6340.
§ Department of Chemistry and Chemical Biology.
Present Address: ArQule, Inc., 200 Boston Avenue, Medford, MA 02155.
| USDA, ARS.
10.1021/np990201k CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/11/1999

1396 J ournal of Natural Products, 1999, Vol. 62, No. 10
Ketchum et al.
F igu r e 3. HPLC chromatograms of natural, synthetic, and natural
+ synthetic mixtures of (A) 9-dihydrobaccatin III (4) and (B) baccatin
VI (3).
F igu r e 2. HPLC chromatogram of taxoids produced in suspension
cultures of a 19 day-old suspension culture of T. canadensis. Culture
was elicited with 200 µM methyl jasmonate on day 7 of the culture
cycle and taxoids recovered from the medium 12 days later. Paclitaxel
(1) concentration in the culture medium is 50 mg‚L-1. Peak identity:
paclitaxel (1), 13-acetyl-9-dihydrobaccatin III (2), baccatin VI (3),
9-dihydrobaccatin III (4).
Sch em e 1. Semisynthesis of Baccatin VI (3) and
9-Dihydrobaccatin III (4) from 13-Acetyl-9-dihydrobaccatin III
(2)
multiple injections of a partially purified and concentrated
extract of the suspension medium on an analytical column
with subsequent fractionation. Although the method was
more time-consuming than separation using semiprepara-
tive HPLC, the use of an autosampler and fraction collector
allowed for unattended collection of 1- to 8-mg quantities
of as many as 25 peaks of interest in 48 h. The fractions
collected were often sufficiently pure for subsequent NMR
analysis, eliminating the need for additional purification.
To identify the structure of taxoid 2 (Figure 1), 2D NMR
techniques including DQ-COSY, HMQC, and HMBC were
used to establish unambiguously the chemical shifts of each
of the protons and carbons as well as their points of
attachment. The relative stereochemistry of the substitu-
ents on the carbon framework of the molecule was estab-
lished from a 2D ROESY experiment. The spectroscopic
data of taxoid 2 were found to be identical to those reported
in the literature for 13-acetyl-9-dihydrobaccatin III isolated
from various natural sources.13 Thus, it was established
unambiguously that taxoid 2 was 13-acetyl-9-dihydrobac-
catin III.
From the 1H and 13C NMR spectra of taxoid 3, it was
deduced that it was very similar to that of 13-acetyl-9-
dihydrobaccatin III (2) and that it also possessed two extra
acetyl groups compared to 2. Thus, taxoid 3 was tentatively
identified as baccatin VI. After a careful comparison of the
spectroscopic data of taxoid 3 with literature values for
baccatin VI, it was observed that they were identical.14-16
However, to confirm unambiguously the structure of taxoid
3, its partial synthesis was carried out by acetylating the
free alcoholic group at C-7 and C-9 of 13-acetyl-9-dihydro-
baccatin III (2) with acetic anhydride and (dimethylamino)-
pyridine (Scheme 1). The acetylated compound obtained
from the reaction and the naturally isolated 3 coeluted on
the naturally isolated taxoid coeluted on the HPLC column
(Figure 3). The H NMR spectrum of 9-dihydrobaccatin III
1
(4) reported in the literature was identical to that of the
synthetic compound and that of the taxoid isolated from
Taxus canadensis.17,18 Thus, taxoid 4 was identified as
9-dihydrobaccatin III. This is the first report of the isolation
of this compound from a natural source, and of any of these
three 9-dihydrobaccatin III derivatives from a Taxus cell
suspension culture.
Compounds 2-4 share the same basic structure, with
the only differences being the degree to which the hydroxyl
groups have been acylated. The difference between baccatin
III and 9-dihydrobaccatin III (4) is the presence of the
hydroxyl group at C-9, instead of the carbonyl found in
paclitaxel and the more common taxoids (Figure 1).
1
the HPLC and had identical H NMR spectra14-16 (Figure
3). Thus taxoid 3 was identified as baccatin VI.
Similarly, based on the 1H and 13C NMR spectra and MS
data, taxoid 4 (Figure 1) was tentatively assigned as
9-dihydrobaccatin III. To assign the structure unambigu-
ously, its partial synthesis was carried out from 13-acetyl-
9-dihydrobaccatin III (2) as reported in the literature.17,18
It was synthesized by reacting taxoid 2 with methyllithium,
resulting in the deacetylation of the acetyl group at C-13
(Scheme 1). The compound obtained from the reaction and
Although the role of the 9-dihydrobaccatin III (4) taxoid
derivatives is not clear, we have previously suggested that
the immediate precursor to paclitaxel (1) is 9-dihydrobac-
catin III (4), rather than baccatin III.10 Experiments are
currently planned to feed Taxus suspension cultures with
radiolabeled 9-dihydrobaccatin III to see if the label is
incorporated into more highly acylated taxoids.

Taxoids from Taxus canadensis
J ournal of Natural Products, 1999, Vol. 62, No. 10 1397
of filter-sterilized methyl jasmonate was added to the flasks.
Cells and medium were harvested at day 14, 7 days after
elicitation.
Extr a ction . The extracellular medium (1200 mL), contain-
ing the majority of the taxoids in the culture, was frozen in a
-58 °C shell freezer, and reduced to dryness in vacuo on a
Labconco lyophilizer. The dried residue was extracted four
times with Me2CO and reduced to dryness in vacuo on a Bu¨chi
Rotovapor and yielded 412 mg of dried residue.
Solid -P h a se Extr a ction P u r ifica tion . The dried residue
from the acetone extract was dissolved in 25 mL MeOH,
diluted with 25 mL H2O, and loaded onto a 10-g C18 (Varian
Mega Bond Elut) solid-phase extraction (SPE) column. The
column was washed with 50 mL of MeOH-H2O (40:60), and
taxoids eluted with 50 mL of MeOH-H2O (80:20). The taxoid
extract was evaporated to dryness in vacuo, first on a Bu¨chi
Rotovapor followed by freeze-drying on a Labconco lyophilizer.
A solution containing 40 mg‚mL-1 of this dried extract in
MeOH was used for purification by HPLC.
An a lysis a n d P u r ifica tion of Ta xoid s. Paclitaxel (1) and
other taxoids were extracted, analyzed, and verified using
HPLC methods developed in this laboratory and previously
described,25,26 with minor modifications. The column used was
a 250 × 4.6 mm Metachem Taxsil column, 5 µm particle size,
with an Upchurch ODS guard column. For purification of
individual taxoids, multiple 30-µL injections of the 40 mg‚mL-1
methanolic SPE-purified suspension extract were made on the
analytical column. Fractions were collected and evaporated to
dryness for subsequent analysis by HPLC, NMR, and mass
spectrometry.
Due to our interest in the biosynthesis of taxol, feeding
studies with labeled acetate were performed on these
suspension cell cultures. Biosynthetic studies on plants or
needles of Taxus species suffer from a number of disad-
vantages, namely: (a) very low levels of incorporation of
the label in the natural product occur, (b) low levels of
production of the natural product are evident, and (c) there
are difficulties in the isolation of the natural product itself.
Carrying out biosynthetic studies in cell cultures is one
possible method to circumvent some of these problems.
Feeding studies were conducted by adding [1,2-13C2]acetate
simultaneously with methyl jasmonate to Taxus canadensis
suspension cultures. Paclitaxel (1) and 13-acetyl-9-dihy-
drobaccatin III (2) were isolated, purified, and analyzed
by 13C NMR spectroscopy. It was observed that the labeled
acetate was incorporated intact into the acetyl groups at
C-4, C-10, and C-13 (J ) 30 Hz). No incorporation was
observed in the benzoate moiety or in the terpene skeleton,
thus supporting the recently proposed hypothesis that
geranylgeranyl diphosphate required in the biosynthesis
of paclitaxel (1) in Taxus canadensis is not derived from
the mevalonate pathway requiring two acetates.19 This
observation is also consistent with an earlier report that
[3H]sodium acetate labeled similar positions of taxoids in
stems and needles of T. canadensis.20
Because paclitaxel (1) is almost insoluble in water, a
combination of polyethoxylated castor oil and ethanol
(Cremophor EL-EtOH) is used to dilute the drug before
administering it to patients. Cremophor is toxic, and the
use of this delivery vehicle led to some extreme hypersen-
sitivity reactions in some patients in early clinical trials.
Now, patients are usually treated with a variety of drugs
to block histaminic responses prior to treatment with
paclitaxel (1).21
13C La belin g. Six flasks containing 200 mL of a 7-day-old
culture were fed a filter-sterilized aqueous solution of [1,2-
13C2]sodium acetate to give a final concentration of 2 mM.
Cultures were elicited with 200 µM of methyl jasmonate at
the same time that the labeled sodium acetate was added.
Cultures were harvested at day 16 (9 days after elicitation),
and the taxoids were extracted, purified, and analyzed by 13C
NMR.
Consequently, there has been a great deal of interest in
the synthesis of promising new taxoid analogues with
greater anticancer activity or increased aqueous solubility.
One such analogue, 9-dihydrotaxol, synthesized from 13-
acetyl-9-dihydrobaccatin III (2) isolated from leaves of T.
canadensis, has recently shown promise as an anti-cancer
drug.17,22 Relative to paclitaxel (1), 9-dihydrotaxol has
greater stability, is 20 times more soluble in water;
demonstrates less toxicity to mice, which allows for a four-
fold increase in the daily dosage; and yields a six-fold
greater cure rate against human MX-1 solid tumor xe-
nografts in mice.17,22 Should 9-dihydrotaxol become an
important chemotherapeutic agent, Taxus cell suspension
cultures developed in our laboratory provide a convenient,
reliable, renewable, and rapidly produced source of the
semisynthetic starting material, 13-acetyl-9-dihydrobac-
catin III (2).
NMR Meth od s. Nuclear magnetic resonance (NMR) spec-
tra (1H and 13C) were recorded on a Bruker AF-300, a Varian
VXR-400S, or a Varian Unity 500 instrument in the specified
deuterated solvent with tetramethylsilane (TMS) as an inter-
1
nal standard for H spectra and CDCl3 as an internal standard
for the 13C spectra. 2D homonuclear 1H NMR, DQCOSY,
gradient-enhanced 1H-13C HMQC, and HMBC spectroscopy
were performed on a Varian Unity 500 spectrometer operating
at 499.93 MHz for proton and 125.72 MHz for carbon.27,28
ROESY was performed on a Varian Unity 500 MHz spectrom-
eter.
13-Acetyl-9-d ih yd r oba cca tin III (2): 1H NMR (CDCl3, 500
MHz) δ 8.10 (2H, d, J ) 7.1 Hz, C6H5COO), 7.62 (1H, t, J )
7.4 Hz, C6H5COO), 7.48 (2H, t, J ) 8.2 Hz, C6H5COO), 6.18
(1H, d, J ) 10.9 Hz, H-10), 6.17 (1H, t, J ) 9.2 Hz, H-13), 5.76
(1H, d, J ) 6.0 Hz, H-2), 4.96 (1H, d, J ) 8.7 Hz, H-5), 4.44
(2H, m, H-7, H-9), 4.32 (A of AB, 1H, d, J ) 8.2 Hz, H-20A),
4.17 (B of AB, 1H, d, J ) 8.2 Hz, H-20B), 3.06 (1H, d, J ) 5.5
Hz, H-3), 2.55 (1H, m, H-6A), 2.29 (3H, s, CH3COO), 2.22-
1.90 (2H, m, H-14), 2.20 (3H, s, CH3COO), 2.15 (3H, s, CH3-
COO), 1.95 (3H, d, J ) 1.1 Hz, H-18), 1.97-1.82 (1H, m, H-6B),
1.82 (3H, s, H-19), 1.78-1.66 (2H, m, C-OH), 1.68 (3H, s,
H-17), 1.25 (3H, s, H-16); 13C NMR (CDCl3, 75 MHz) δ 170.45
(s, CH3COO), 170.23 (s, CH3COO), 169.3 (s, CH3COO), 167.01
(s, C6H5COO), 139.63 (s, C-12), 134.84 (s, C-11), 133.71 (s,
C6H5COO), 130.07 (s, C6H5COO), 129.18 (s, C6H5COO), 128.63
(s, C6H5COO), 84.0 (s, C-5), 82.05 (s, C-4), 78.47 (s, C-1), 76.79
(s, C-9), 76.56 (s, C-20), 73.98 (s, C-7), 73.47 (s, C-2), 73.20 (s,
C-10), 69.74 (s, C-13), 47.04 (s, C-3), 44.89 (s, C-8), 43.0 (s,
C-15), 37.94 (s, C-6), 35.30 (s, C-14), 28.27 (s, C-17), 22.86 (s,
CH3COO), 22.56 (s, C-16), 21.34 (s, CH3COO), 21.25 (s, CH3-
COO), 14.87 (s, C-18), 12.48 (s, C-19); FABMS m/z 631 [(M +
H)+].
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. All reactions requir-
ing nonaqueous conditions were performed in oven-dried
glassware under a positive pressure of N2. All solvents were
distilled. The term in vacuo refers to the removal of solvent to
constant sample weight (<0.05 mmHg). Commercial TLC
plates were Merck 60F254. Flash column chromatography was
performed according to the method of Still23 on Merck type 60
Si gel, 230-240 mesh.
Gr ow th Con d ition s. Cell line CO93P, initiated from T.
canadensis embryos and maintained as previously described,9,24
was used for these experiments. A 40-mL culture of 14-day-
old cells was used to inoculate 160 mL of fresh B5NB medium,
Ba cca tin VI (3). Acetic anhydride (0.048 g, 0.48 mmol) was
added to a solution of 13-acetyl-9-dihydrobaccatin III (2)
(0.0038 g, 4.8 µmol) and (dimethylamino)pyridine (0.058 g, 0.48
mmol) in dichloromethane (3 mL). The reaction was allowed
in
a 500-mL Erlenmeyer flask with Bellco silicone/foam
closures. Cultures were elicited as previously described.10
Briefly, 7 days after inoculation, 200 µM (final concentration)

1398 J ournal of Natural Products, 1999, Vol. 62, No. 10
Ketchum et al.
to stir for 4 h at room temperature. The reaction was quenched
by the addition of 5% HCl (1 mL) and H2O (5 mL). The organic
layer was separated, and the aqueous layer once again was
extracted with dichloromethane. The organic extracts were
combined, dried over Na2SO4, and concentrated. Flash column
chromatography (Si gel, MeOH-CH2Cl2, 2:98) afforded 3 mg
(70%) of pure synthetic baccatin VI (3) as a white solid: 1H
NMR (CDCl3, 500 MHz) δ 8.10 (2H, d, J ) 6.8 Hz, C6H5COO),
7.6 (1H, t, J ) 7.3 Hz, C6H5COO), 7.49 (2H, d, J ) 7.8 Hz,
C6H5COO), 6.22 (1H, d, J ) 11.2 Hz, H-10), 6.18 (1H, t, J )
11.2 Hz, H-13), 6.0 (1H, d, J ) 11.2 Hz, H-9), 5.88 (1H, d, J )
6.4 Hz, H-2), 5.58 (1H, m, H-7), 4.98 (1H, d, J ) 8.9 Hz, H-5),
4.34 (A of AB, 1H, d, J ) 8.3 Hz, H-20A), 4.13 (B of AB, 1H,
d, J ) 8.3 Hz, H-20B), 3.19 (1H, d, J ) 5.9 Hz, H-3), 2.49 (1H,
m, H-6A), 2.30 (3H, s, CH3COO), 2.28-2.10 (2H, m, H-14), 2.2
(3H, s, CH3COO), 2.12 (3H, s, CH3COO), 2.11 (3H, s, CH3COO),
2.04 (3H, s, CH3COO), 2.00 (3H, s, H-18), 1.9-1.82 (2H, m,
H-6B, C-OH), 1.80 (3H, s, H-16), 1.58 (3H, s, H-19), 1.23 (3H,
s, H-17); FABMS m/z 715 [(M + H)+]; HRMS m/z 715.2970
(calcd for C37H47O14, 715.2966).
Refer en ces a n d Notes
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9-Dih yd r oba cca tin III (4). Methyllithium (60 µL, 1.4 M
solution in ethyl ether, 87 µmol) was added in a dropwise
manner to a solution of 13-acetyl-9-dihydrobaccatin III (2)
(0.0084 g, 13.3 µmol) in THF (1.5 mL), which had been cooled
to -78 °C. The reaction was then stirred for 2 h at -78 °C.
The reaction was quenched by addition of H2O (2 mL) followed
by EtOAc (3 mL). The organic layer was separated and the
aqueous layer once again extracted with EtOAc. The organic
extracts were combined, dried over Na2SO4, and concentrated.
Flash column chromatography (Si gel, MeOH-CH2Cl2, 4:96)
afforded 3.4 mg (43%) of pure synthetic 9-dihydrobaccatin III
(4) as a white solid: 1H NMR (CDCl3, 300 MHz) δ 8.10 (2H,
m, C6H5COO), 7.6 (1H, m, C6H5COO), 7.48 (2H, m, C6H5COO),
6.13 (1H, d, J ) 10.74 Hz, H-10), 5.72 (1H, d, J ) 5.91 Hz,
H-2), 4.92 (1H, d, J ) 9.1 Hz, H-5), 4.80 (1H, br t, H-13), 4.44
(2H, m, H-7, H-9), 4.36 (A of AB, 1H, d, J ) 8.1 Hz, H-20A),
4.16 (B of AB, 1H, d, J ) 8.1 Hz, H-20B), 3.10 (1H, d, J ) 5.91
Hz, H-3), 2.53 (1H, m, H-6A), 2.31-2.17 (2H, m, H-14), 2.28
(3H, s, CH3COO), 2.17-2.03 (2H, m, C-OH), 2.13 (3H, s, CH3-
COO), 2.11 (3H, d, J ) 1.1 Hz, H-18), 2.00-1.79 (2H, m, H-6,
C-OH), 1.82 (3H, s, H-19), 1.65 (3H, s, H-17), 1.40-1.15 (1H,
m, C-OH), 1.10 (3H, s, H-16); 13C NMR (CDCl3, 75 MHz) δ
171.6, 170.4, 167.0, 142.8, 134.3, 133.7, 130.1, 129.3, 128.6,
84.2, 82.2, 78.6, 77.2, 73.9, 73.8, 73.4, 68.5, 46.8, 45.1, 42.6,
40.8, 38.7, 37.9, 28.3, 23.1, 22.1, 21.4, 15.2, 12.5; FABMS m/z
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