Identification and biological activities of new taccalonolide microtubule stabilizers

Article abstract of DOI:10.1021/jm200757g

The taccalonolides are a unique class of microtubule stabilizers that do not bind directly to tubulin. Three new taccalonolides, Z, AA, and AB, along with two known compounds, taccalonolides R and T, were isolated from Tacca chantrieri and Tacca integrifolia. Taccalonolide structures were determined by 1D and 2D NMR methods. The biological activities of the new taccalonolides, as well as taccalonolides A, B, E, N, R, and T, were evaluated. All nine taccalonolides display microtubule stabilizing activity, but profound differences in antiproliferative potencies were noted, with IC₅₀ values ranging from the low nanomolar range for taccalonolide AA (32 nM) to the low micromolar range for taccalonolide R (13 μM). These studies demonstrate that diverse taccalonolides possess microtubule stabilizing properties and that significant structure-activity relationships exist. In vivo antitumor evaluations of taccalonolides A, E, and N show that each of these molecules has in vivo antitumor activity.

Full text of DOI:10.1021/jm200757g

ARTICLE
pubs.acs.org/jmc
Identification and Biological Activities of New Taccalonolide
Microtubule Stabilizers
Jiangnan Peng,*,† April L. Risinger,† Gary A. Fest,† Evelyn M. Jackson,† Gregory Helms,‡ Lisa A. Polin,§ and
Susan L. Mooberry*,†
†Department of Pharmacology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States
‡Nuclear Magnetic Resonance Center, Department of Chemistry, Washington State University, Pullman, Washington 99164,
United States
§Department of Oncology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit,
Michigan 48201, United States
S Supporting Information
b
ABSTRACT: The taccalonolides are a unique class of microtubule stabilizers that do not bind
directly to tubulin. Three new taccalonolides, Z, AA, and AB, along with two known compounds,
taccalonolides R and T, were isolated from Tacca chantrieri and Tacca integrifolia. Taccalonolide
structures were determined by 1D and 2D NMR methods. The biological activities of the new
taccalonolides, as well as taccalonolides A, B, E, N, R, and T, were evaluated. All nine taccalonolides
display microtubule stabilizing activity, but profound differences in antiproliferative potencies were
noted, with IC50 values ranging from the low nanomolar range for taccalonolide AA (32 nM) to the
low micromolar range for taccalonolide R (13 μM). These studies demonstrate that diverse
taccalonolides possess microtubule stabilizing properties and that significant structureÀactivity
relationships exist. In vivo antitumor evaluations of taccalonolides A, E, and N show that each of these molecules has in vivo
antitumor activity.
’ INTRODUCTION
biochemical assays.6 The ability of the taccalonolides to cause
microtubule stabilizing effects through a unique binding site and
an entirely distinct mechanism of action prompted our interest in
understanding this class of molecules.
Microtubule stabilizers are one of the most important classes
of anticancer therapeutics used in the clinic today. The taxoid
microtubule stabilizer paclitaxel has been widely used in the
treatment of solid tumors, including breast, ovarian, and lung
cancers for over a decade as a single agent and in combination
with targeted therapies. In spite of their clinical utility, the
shortcomings of paclitaxel and the second generation semisyn-
thetic taxoid, docetaxel, include innate and acquired drug resis-
tance and dose limiting toxicities.1 Two new microtubule
stabilizers have been approved for clinical use in the past 3 years:
the epothilone ixabepilone and the taxoid cabazitaxel, which
circumvent some but not all of the shortcomings of first and
second generation microtubule stabilizers.2,3 These microtubule
stabilizing drugs all bind to the interior lumen of the intact
microtubule at the taxoid binding site, which causes a stabiliza-
tion of microtubule protofilament interactions and thereby
decreases the dynamic nature of microtubules.4
Two additional classes of microtubule stabilizers that do not
bind within the taxoid site have been isolated from nature:
laulimalides/peloruside A and the taccalonolides. Laulimalide
and peloruside A have recently been shown to bind to the
exterior of the microtubule at a site distinct from the taxoid
binding site but result in microtubule stabilization effects nearly
identical to those of the taxoids.5 The taccalonolides are unique
in that they do not bind directly to microtubules/tubulin and do
not enhance the polymerization of purified tubulin in
Intense efforts over the past 3 decades have identified a
large variety of interesting chemical compounds from the roots
and rhizomes of Tacca species, including 25 taccalonolides,
denoted as taccalonolides AÀY.7À15 However, there have been
limited biological studies on the taccalonolides. In 2003, we first
reported the microtubule stabilizing activities of taccalonolides A
and E.16 Follow-up studies showed preliminary structureÀ
activity relationships (SAR) for the antiproliferative activities of
taccalonolides A, E, B, and N. The antiproliferative potencies of
these four taccalonolides in HeLa cells were all in the mid-
nanomolar range (190À644 nM).17
In this study we isolated three previously undescribed tacca-
lonolides designated: Z (5), AA (6), and AB (7). The biological
activities of these molecules, as well as of two previously isolated
but biologically uncharacterized taccalonolides, R (9) and T (8),
are presented. The mechanisms of action of all the taccalonolides
were evaluated and compared to taccalonolides A and E. Each
of these taccalonolides stabilizes cellular microtubules and
causes mitotic accumulation of cancer cells with multiple
abnormal mitotic spindles. The relative potencies of these
Received: June 13, 2011
Published: July 29, 2011
r
2011 American Chemical Society
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Figure 1. Structures of the taccalonolides.
taccalonolides range from 32 nM to 13 μM, offering a broad
range of activity that provides an opportunity to explore SAR.
the hydroxyl proton signal at δ 3.64 and the carbonyl carbon at
δ 208.34 (C-6) suggested that the hydroxyl group is located at
C-5. The configuration of this hydroxyl group was determined as
R by the NOE correlations between 5-OH/H-7,9,4R. The other
1H and 13C NMR data for 5 are similar to those for 1; thus, 5 was
determined as 5R-hydroxytaccalonolide A, and this was con-
firmed by 2D NMR data. A trivial name taccalonolide Z was given
to 5, and its structure is shown in Figure 1.
Taccalonolide AA (6) was isolated as a white powder. The
proton NMR spectrum of 6 showed characteristics almost iden-
tical to those of 5, indicating a similar taccalonolide structure: five
acetyl signals at δ 2.20, 2.15, 2.14, 2.00, 1.98, five methyl signals at
δ 1.64 (s), 1.34 (s), 1.04 (s), 0.91 (d, J = 7.0 Hz), 0.72 (s), five
acetoxylated methine signals at δ 5.72 (d, J = 11.0 Hz), 5.55 (d,
J = 9.5 Hz), 5.25 (br), 5.23 (brd, J = 11.0 Hz), 4.91 (d, J = 5.0 Hz),
two epoxyl methine signals at δ 3.72 (t, J = 4.5 Hz) and 3.59 (br),
one olefinic signal at δ 5.09 (br). Taccalonolide AA (6) has one
more acetyl signal than taccalonolide Z (5). The chemical shift of
H-7 at δ 5.72 (d, J = 11.0 Hz) was ∼0.99 ppm downfield than
that of 5, suggesting that this additional acetyl group was located
at 7-OH. An HMBC correlation between H-7 and a carbonyl
carbon at δ 170.8 confirmed this assignment. The other 1H, 13C,
and 2D NMR data are similar to those for 5; thus, the structure
of 6 was determined as shown in Figure 1 and a trivial name
taccalonolide AA was assigned.
’ RESULTS AND DISCUSSION
Taccalonolide Isolation and Structure Elucidation. The roots
and rhizomes of T. chantrieri and T. integrifolia were extracted using
supercritical fluid CO2 with methanol. After separation by flash
chromatography using silica gel columns, normal and reverse phase
HPLC was employed to obtain purified taccalonolides. Taccalo-
nolides A (1), E(3), R(9), T(8), andAA(6) wereisolatedfromT.
chantrieri, while taccalonolide Z (5) was obtained from T. integri-
folia (Figure 1). Mild base hydrolysis of taccalonolides A, E, and Z
yielded taccalonolides B (2), N (4), and AB (7), respectively.
Taccalonolide Z (5) was obtained as a white powder. The
proton NMR spectrum showed four acetyl signals at δ 2.16, 2.13,
2.00, 1.97, five methyl signals at δ 1.64 (s), 1.34 (s), 0.98 (s), 0.89
(d, J = 7.2 Hz), 0.73 (s), five oxygenated methine signals at δ 5.53
(t, J = 10.2 Hz), 5.23 (br), 5.22 (dd, J = 9.6, 2.4 Hz), 4.85 (d, J =
5.4 Hz), 4.73 (dd, J = 10.2, 5.4 Hz), two epoxyl methine signals at
δ 3.74 (t, J = 4.5 Hz) and 3.61 (dt, J = 4.2, 1.8 Hz), one olefinic
signal at δ 5.06 (d, J = 1.2 Hz). All these proton NMR data are
similar to those of taccalonolide A and indicated that 5 is a
taccalonolide type steroid. The molecular formula of C36H46O15
was determined by HRMS of 719.2934 (calcd 719.2915),
suggesting that 5 has one more oxygen than taccalonolide
A (1). In addition, three signals for hydroxyl groups were
observed at δ 3.64 (s), 3.45 (d, J = 5.4 Hz), and 2.58 (s), which
is one more than taccalonolide A. The carbon-13 NMR showed
seven oxygenated carbon signals at δ 79.08, 78.74, 74.13, 74.06,
71.20, 71.17, 71.14 and confirmed one more hydroxyl group for 5
compared to taccalonolide A. The 3J HMBC correlation between
Taccalonolide AB (7) was obtained as white powder. The
LC/MS showed pseudomolecular ions at 677 [M + H]+, 694
[M + NH4]+, and 699 [M + Na]+, indicating the loss of an acetyl
group from taccalonolide Z (5). The proton NMR showed the
chemical shift of H-15 of 7 at δ 4.75 (ddd, J = 3.5, 9.0, 11.6 Hz),
which is shifted 0.78 ppm upfield compared with that of 5,
suggesting the loss of acetyl group at 15-OH. The HMBC
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Figure 2. Effects of the taccalonolides on interphase cells. HeLa cells were treated for 18 h with vehicle (A), 0.5 μM paclitaxel (B), 3.5 μM taccalonolide
A (C), 0.8 μM taccalonolide B (D), 2.5 μM taccalonolide E (E), 1.3 μM taccalonolide N (F), 57 μM taccalonolide R (G), 3.5 μM taccalonolide T (H),
0.6 μM taccalonolide Z (I), 0.32 μM taccalonolide AA (J), or 13.5 μM taccalonolide AB (K). Interphase microtubule structures were visualized by
indirect immunofluorescence using a β-tubulin antibody.
Figure 3. Effects of the taccalonolides on cell cycle distribution. HeLa cells were treated with vehicle (A), 12 nM paclitaxel (B), 3.5 μM taccalonolide
A (C), 0.8 μM taccalonolide B (D), 2.5 μM taccalonolide E (E), 1.3 μM taccalonolide N (F), 111 μM taccalonolide R (G), 3.5 μM taccalonolide T (H),
0.6 μM taccalonolide Z (I), 0.32 μM taccalonolide AA (J), or 13.5 μM taccalonolide AB (K) for 18 h and stained with Kirshan’s reagent. Open and filled
arrowheads denote the populations of cells with 2N (G1) and 4N (G2/M) DNA content, respectively.
correlation between 15-OH (δ 4.94) and C-15 (δ 71.5) con-
firmed the assignment.
interrupt microtubule dynamics in mitosis, leading to mitotic
arrest. The effect of the taccalonolides on mitotic progression
was analyzed by flow cytometry. All nine taccalonolides caused
an accumulation of cells in the G2/M phase of the cell cycle with
4N DNA content (Figure 3). This accumulation is identical to
the mitotic arrest that is observed after treatment of HeLa cells
with paclitaxel (Figure 3).
The effects of the taccalonolides on mitotic spindle structures
were evaluated to test whether they caused mitotic spindle defects
leading to cell cycle arrest. β-Tubulin and DNA were visualized in
HeLa cells by indirect immunofluorescence and DAPI staining,
respectively. Mostof the cellstreated witheachtaccalonolideatthe
concentration that caused G2/Maccumulation werefoundtobein
Microtubule Stabilization and Mitotic Arrest. The ability of
the newly isolated taccalonolides to cause bundling of interphase
microtubules was evaluated in HeLa cells. Consistent with the
effects of taccalonolides A and E, which were shown to exert
interphase microtubule bundling in previous studies,16 taccalo-
nolides B, N, R, T, Z, AA, and AB each caused the formation of
thick bundled microtubule tufts typical of microtubule stabilizers
including paclitaxel (Figure 2). Although microtubule stabilizers
cause an increase in the density of interphase microtubules, the
mechanism by which these agents inhibit the proliferation of
cancer cells in vitro is widely accepted to be due to their ability to
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Figure 4. Effects of the taccalonolides on mitotic spindles. HeLa cells were treated for 18 h with vehicle (A), 12 nM paclitaxel (B), 3.5 μM taccalonolide
A (C), 0.8 μM taccalonolide B (D), 2.5 μM taccalonolide E (E), 1.3 μM taccalonolide N (F), 57 μM taccalonolide R (G), 3.5 μM taccalonolide T (H),
0.6 μM taccalonolide Z (I), 0.32 μM taccalonolide AA (J), or 13.5 μM taccalonolide AB (K). The microtubule structures in mitotic cells were visualized
by indirect immunofluorescence using a β-tubulin antibody.
Table 1. Antiproliferative Potency of Taccalonolidesa
Antiproliferative Activities of the Taccalonolides. The
antiproliferative potencies of the taccalonolides were evaluated
taccalonolide
IC50 (nM)
in HeLa cells using the SRB assay. The most potent taccalonolide
is the newly identified taccalonolide AA, with an IC50 of
32.3 nM (Table 1). This makes taccalonolide AA the most potent
taccalonolide identified thus far. This low nanomolar potency is
closer to other naturally occurring microtubule stabilizers, including
paclitaxel, the epothilones, laulimalide, and peloruside A, than the
initially studied taccalonolides A and E.17 Other taccalonolides that
had IC50s in the nanomolar range include taccalonolides Z (120
nM), B (190 nM), N (247 nM), T (335 nM), A (594 nM), and E
(644 nM) (Table 1). Taccalonolides AB and R were significantly less
potent, with IC50 values of 2.8 and 13.1 μM, respectively (Table 1).
The 400-fold difference in activity between the most and least potent
taccalonolides isolated provides the opportunity to explore the
structureÀactivity relationships among the taccalonolides.
taccalonolide A
taccalonolide B
taccalonolide E
taccalonolide N
taccalonolide R
taccalonolide T
taccalonolide Z
taccalonolide AA
taccalonolide AB
paclitaxel
594 ( 43
190 ( 3
644 ( 10
247 ( 16
13144 ( 1390
335 ( 24
120 ( 7.5
32.3 ( 1.9
2767 ( 107
1.2 ( 0.1
a The concentration of each drug that causes 50% inhibition of cellular
proliferation (IC50) after 48 h of treatment was measured using the SRB
assay (n = 3À5). IC50 values for taccalonolides A, E, B, and N are from
Risinger et al.17
StructureÀActivity of the Taccalonolides. Our previous
work comparing the potency of taccalonolides A, B, E, and N
in various drug sensitive and drug resistant cell lines gave a
preliminary indication of the SAR of the taccalonolides, specifi-
cally the consequence of the presence or absence of an acetate
group at C11 and/or C15.17 Taccalonolides A and E differ only by
the respective presence or absence of an acetoxy group at the
C11 position and they did not show major differences in
potency, suggesting that this acetoxy functionality did not
influence potency or microtubule stabilizing activity. Similarly,
mitosis as evidenced by a “rounded up” cellular morphology and
condensed DNA. These mitotic cells contained multiple abnormal
mitotic spindles, which is another common effect of microtubule
stabilizing agents (Figure 4). These findings demonstrate that all
nine taccalonolides, A, B, E, N, R, T, Z, AA, and AB, are micro-
tubule stabilizers that cause mitotic arrest of cells with multiple
abnormal mitotic spindles.
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Table 2. Antitumor Effects of Taccalonolides A, E, and N in the Mammary 16/C Modela
total dose
(mg/kg)
gross log
cell kill
median (range) of tumor size (mg)
treatment
T/C (%)
T À C (day)
and number of tumor-free mice on day 12
mean body weight loss (%)
no treatment
paclitaxel
-
-
0
-
19
16
4
-
1029 (63À2827)
0 (all zero)
0 (all zero)
256 (108À756)
172 (0À947)
1/5 = 0
À2.2
À6.3
73.5
56
4.8
4.0
1.0
1.25
taccalonolide A
taccalonolide A
taccalonolide E
0
À16.7b
À6.3
40
24
17
90
5
À4.1
taccalonolide E
taccalonolide N
taccalonolide N
54
36
20
81
0
1
5
1
0.25
1.25
0.25
837 (0À1755)
1/5 = 0
À2.0
À8.2
À2.1
(0À126)
3/5 = 0
43
445 (0À2277)
1/5 = 0
a T/C is the median tumor mass of a given treated group (T) divided by the median tumor mass of the control (C) and expressed as a percent. The tumor
growth delay (T À C) is the median number of days between the time the treatment (T) and control (C) group tumors reach
the predetermined size of 1000 mg. The gross log cell kill is calculated by [(T À C)/3.32] Â Td, where Td is the tumor volume doubling time
(in days). b A 20% lethality (1/5 mice) was observed at this dose.
taccalonolides B and N also differ from one another only by the
presence or absence of an acetoxy group at C11 and showed
comparable activity to one another. As evidenced by these two
pairs of compounds, the presence or absence of the C11 acetoxy
group did not have a large effect on potency.17 Another SAR
evaluation made possible with these two pairs of compounds is
the contribution of the C15 acetate. Taccalonolides B and N are
produced by mild base hydrolysis of the C15 acetate of taccalo-
nolides A and E, respectively, resulting in a hydroxyl group at
this position. A consistent increase in potency was observed
upon hydrolysis of the C15 acetate as indicated by the 3.1-fold
greater potency of taccalonolide B compared to A and the 2.
6-fold greater potency of taccalonolide N compared to E in
HeLa cells.17
We now expanded the number of taccalonolides available for
SAR analysis from four to nine by adding three new taccalono-
lides as well as two others that have not yet been evaluated for
antiproliferative activities. Analysis of the potencies of these
taccalonolides provided another opportunity to examine the
effect of the C11 acetoxy group, since the only difference between
taccalonolides AA and R is the presence of this acetoxy sub-
stituent in taccalonolide AA. In contrast to the relative unim-
portance of the C11 acetoxy moiety on potency between the
taccalonolides A and E or B and N, this modification caused a
400-fold difference in potency between taccalonolides AA and R
(Table 1). The other structural differences between this new pair
of taccalonolides and taccalonolides A, E, B, N occur in the
southern part of the molecule where there is a hydroxyl group at
C5 and an acetate at 7-OH (Figure 1). Therefore, it appears that
these structural features in the southern portion of taccalonolides
AA and R confer sensitivities to the constituents present at C11.
These data suggest that interactions across the molecule can
influence the potency of a taccalonolide.
However, when this same acetate is hydrolyzed in taccalo-
nolide Z to yield taccalonolide AB, the potency is decreased by
23-fold (Table 1). Again, context is important because the only
difference between taccalonolides Z and A is a hydroxyl group
at the C5 position (Figure 1). Finally, taccalonolide T is unique
from the other taccalonolides evaluated in this study in that
it contains a bulky isovalerate substituent at the C1 position
(Figure 1). This is the only difference between taccalonolides R
and T and provides a dramatic 38-fold increase in potency
(Table 1). It will be interesting to see whether adding steric
bulk at this position has a consistent effect on potency in further
studies.
These findings strongly suggest that the SAR for the taccalo-
nolides is not simple and instead suggest that there are complex
relationships among multiple sites on the taccalonolide back-
bone. On the basis of the limited data with these taccalonolides,
we can categorize the taccalonolides into two groups, those with
the 5-hydroxy group and those without the 5-hydroxy group. For
taccalonolides without the 5-hydroxyl group, which include the
taccalonolides A, B, E, and N, hydrolysis of the C15 acetate
resulted in 2- to 3-fold increase in potency, and the C11 acetoxy
group did not affect the activity. For taccalonolides with the
5-hydroxyl group, taccalonolides Z, AA, AB, T, and R, the
presence of the C11 acetoxy group dramatically increased the
activity (taccalonolide AA vs R), while hydrolysis of the C15
acetate decreased the activity (taccalonolide Z vs AB). Finally,
adding bulk to the acetate at C1 also increased potency
(taccalonolide T vs R). Although there does not appear to be a
clear link between potency and any particular chemical substi-
tuent on the taccalonolide backbone, these data highlight the
importance of isolating additional taccalonolides and making
directed chemical modifications to further probe the complex
interactions across the molecule. In future studies we will probe
the effects of introducing different bulky groups on C1 together
with acetoxy groups at C11 to find the best combination of
substituents at these sites. For example, the addition of a bulky
substituent at the C1 of taccalonolide AA may further improve
the potency. Other studies planned will further evaluate the roles
of the different acetylating groups at C7 and C15.
Another indication that the individual chemical substituents
on the taccalonolide backbone interact in a complex manner to
influence activity is shown by the effects of hydrolysis of the C15
acetate. As mentioned above, when this acetate is hydrolyzed in
taccalonolide A or E, the resulting products, taccalonolides B and
N, show a 2.6- to 3.1-fold increase in potency (Table 1).17
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In Vivo Antitumor Activity. Antitumor studies were con-
ducted to evaluate the in vivo activity of taccalonolides A, E, and
N. This evaluation is important since in vitro activity is not neces-
sarily retained in vivo because of pharmacokinetic properties and
drug metabolism. The syngeneic murine mammary carcinoma
16/C model was used because it is an incurable, rapidly growing
tumor that provides a rigorous test of new agents.18,19 A total
dose of 73.5 mg/kg paclitaxel was used as a positive control,
and as expected, it provided excellent antitumor effects with
a 0% T/C, 19 day tumor growth delay (T À C), and 4.8 gross log
cell kill (Table 2). In comparison, a total dose of 56 mg/kg
taccalonolide A provided excellent antitumor activity with
a 0% T/C, 16 day tumor growth delay (T À C), and 4.0 gross
log cell kill (Table 2). However, with this dose and schedule,
taccalonolide A also produced a 16.7% mean body weight loss
and delayed toxicity with one lethality occurring 16 days after
the final dose was administered. A lower dose of taccalonolide A
(40 mg/kg total) was better tolerated but less effective, yielding a
24% T/C and 1.0 gross log cell kill (Table 2).
Taccalonolide E at a total dose of 90 mg/kg provided a 17%
T/C and 1.25 gross log cell kill with a well-tolerated maximal
4.1% body weight loss. At a lower total dose of 54 mg/kg,
taccalonolide E yielded an 81% T/C. Similarly, taccalonolide N at
a total dose of 36 mg/kg generated a T/C of 0% and a 1.25 gross
log cell kill, while the 20 mg/kg total dose was less effective with a
T/C of 43% and a 0.25 gross log kill (Table 2). These data
indicate that 56 mg/kg taccalonolide A provided the longest
tumor growth delay (T À C) and the highest gross log cell kill of
the taccalonolides tested in this trial. However, at this dose
taccalonolide A was above the maximum tolerated dose (MTD)
because it caused substantial weight loss and 20% lethality.
Antitumor effects at doses over the MTD are difficult to interpret
because they cannot be clearly separated from the toxic effects on
the whole animal. However, a slightly lower total dose of
taccalonolide A, 40 mg/kg, showed antitumor activity with low
toxicity (Table 2). Additionally, in a previous study a 38 mg/kg
total dose of taccalonolide A was highly effective against a drug
resistant tumor and caused no drug deaths,17 suggesting that
taccalonolide A has a narrow therapeutic window. At the highest
nontoxic doses tested, all the taccalonolides showed comparable
antitumor activity, suggesting that the core structure of this class
of molecules possesses antitumor activity that may be amenable
to refinement and improvement through the isolation of addi-
tional taccalonolides and/or analogue development. Pharmaco-
kinetic and metabolism studies are planned for the future to
further understand the factors that affect in vivo efficacy of the
taccalonolides.
spectrometer equipped with ESI. The purities of all compounds were
determined to be greater than 95% by LC/MS and NMR.
Plant Material. Tacca chantreiri and T. integrifolia plants were
purchased from a commercial grower. The roots and rhizomes were
collected from living plants and stored at À80 °C until lyophilized.
Extraction and Isolation of the Taccalonolides. Dried and
pulverized rhizomes (2.3 kg) of T. chantrieri were extracted in several
batches using supercritical CO2 with MeOH. The crude extracts were
washed with hexanes and extracted with CH2Cl2. The CH2Cl2 extracts
were subjected to silica gel flash chromatography and eluted with
hexances/isopropanol (82:18) to obtain the taccalonolide enriched
fraction. This fraction (1.4 g) was further purified on a silica gel HPLC
column and eluted with isooctane/isopropanol (81:19) to yield frac-
tions 1À8. Taccalonolides A (1) and E (3) were obtained from fractions
2 and 4, respectively. Fraction 1 (33 mg) was separated on a C-18 HPLC
column, eluting with a gradient of acetonitrile/H2O from 30% to 80%
over 40 min to yield 1.2 mg of taccalonolide AA (6) and 0.8 mg of
taccalonolide T (8). Fraction 3 was purified on a silica gel flash column
and eluted with CH2Cl2/acetone 85:15 to yield taccalonolide R (9).
The roots and rhizomes of T. integrifolia (1445 g) were extracted to
yield 11.7 g of CH2Cl2 extract using the same method as T. chantrieri.
The CH2Cl2 extract was purified by silica gel flash chromatography
followed by repeated normal phase HPLC to yield 13.1 mg of taccalo-
nolide Z (5).
Hydrolysis of the Taccalonolides. Taccalonolide A (40 mg) was
dissolved in 4 mL of methanol, and to this solution 8 mL of 0.05 M
sodium bicarbonate was added. The solution was stirred at room
temperature for 44 h. The reaction solution was extracted with EtOAc
and purified on HPLC to yield 25.8 mg of taccalonolide B (2).
Taccalonolides N (4) and AB (7) were produced by hydrolysis of
taccalonolides E (3) and Z (5), respectively, using the same method.
Taccalonolide Z (5). White powder. HRESIMS: m/z 719.2934 (calcd
for C36H47O15 719.2915). ESIMS: m/z 719.4 [M + H]+, 736.4 [M +
NH4]+, 731.5 [M + Na]+. 1H NMR: δ (ppm) 5.53 (t, J = 9.8 Hz, H-15),
5.23 (br, H-12), 5.22 (dd, J = 9.6, 2.4 Hz, H-11), 5.06 (d, J = 1.5 Hz,
H-22), 4.85 (d, J = 5.4 Hz, H-1), 4.73 (dd, J = 10.2, 5.1 Hz, H-7), 3.74 (t,
J = 4.5 Hz, H-2), 3.64 (s, 5-OH), 3.61 (m, H-3), 3.45 (d, J = 5.2 Hz,
7-OH), 3.17 (t, J = 11.6 Hz, H-9), 2.58 (s, 25-OH), 2.57 (dd, J = 15.0, 1.6
Hz, H-4a), 2.52 (t, J = 10.1 Hz, H-14), 2.42 (dd, J = 13.4, 10.2 Hz, H-16),
2.23 (d, J = 16.7 Hz, H-4b), 2.16 (s, 3H, 1-OAc), 2.15 (m, H-20), 2.13
(s, 3H, 12-OAc), 2.00 (s, 3H, 15-OAc), 1.97 (s, 3H, 11-OAc), 1.81 (dd,
J = 13.4, 9.8 Hz, H-17), 1.64 (s, 3H, H-27), 1.56 (q, J = 10.8 Hz, H-8),
1.34 (s, 3H, H-28), 0.98 (s, 3H, H-18), 0.89 (d, 3H, J = 7.2 Hz, H-21),
0.73 (s, 3H, H-19). 13C NMR: δ (ppm) 208.34 (C-6), 178.10 (C-26),
172.07 (15-OAc), 170.85 (11-OAc), 169.40(1-OAc), 169.25 (12-OAc),
154.50 (C-23), 111.07 (C-22), 79.08 (C-5), 78.74 (C-25), 74.13 (C-12),
74.06 (C-1), 71.20 (C-15), 71.17 (C-7), 71.14 (C-11), 54.16 (C-14),
54.06 (C-3), 50.97 (C-16), 50.60 (C-2), 50.07 (C-24), 48.85 (C-17),
45.86 (C-10), 44.19 (C-8), 43.15 (C-13), 37.13 (C-9), 30.61 (C-20),
26.94 (C-4), 25.32 (C-28), 22.36 (15-OAc), 21.16 (11-OAc), 21.02
(12-OAc), 20.72 (1-OAc), 20.61 (C-27), 20.08 (C-21), 14.61 (C-19),
13.37 (C-18).
’ EXPERIMENTAL SECTION
Taccalonolide AA (6). White powder. HRESIMS: m/z 761.3032
(calcd for C38H49O16 761.3021). ESIMS: m/z 761.4 [M + H]+, 778.4
[M + NH4]+, 783.5 [M + Na]+, 701.3 [M À OAc]+. 1H NMR: δ (ppm)
5.73 (d, J = 11.0 Hz, H-7), 5.55 (t, J = 9.4 Hz, H-15), 5.25 (d, J = 2.6 Hz,
H-12), 5.23(dd, J = 11.7, 2.6 Hz, H-11), 5.09 (d, J = 1.4 Hz, H-21), 4.91
(d, J = 5.5 Hz, H-1), 3.72 (t, J = 4.5 Hz, H-2), 3.61 (s, 5-OH), 3.59 (m,
H-3), 3.30 (t, J = 11.4 Hz, H-9), 2.63 (t, J = 10.0 Hz, H-14), 2.62 (s, 25-
OH), 2.56 (brd, J = 14.5 Hz, H-4a), 2.43 (dd, J = 13.4, 9.8 Hz, H-16),
2.20 (s, 3H, 1-OAc), 2.19 (m, H-4b), 2.17 (m, H-20), 2.16 (s, 3H, 11-
OAc), 2.15 (s, 3H, 12-OAc), 2.03 (q, J = 11.0 Hz, H-8), 2.00 (s, 3H,
7-OAc), 1.98 (s, 3H, 15-OAc), 1.65 (s, 3H, H-27), 1.33 (s, 3H, H-28),
1.04 (s, 3H, H-18), 0.92 (s, 3H, H-21), 0.73(s, 3H, H-18). 13C NMR:
Chemistry. NMR spectra were recorded on a Bruker Avance 500,
600, or 700 MHz instrument equipped with CryoProbe and a Varian
VNMRS 600 MHz instrument. All spectra were measured and reported
in ppm using the residual solvent (CDCl3) as an internal standard. The
HR mass spectra were obtained using a Thermo Scientific LTQ Orbitrap
mass spectrometer. IR data were obtained on a Bruker Vector 22 with a
Specac Golden Gate ATR sampler. The UV spectra were measured on a
Varian Cary 5000 UVÀvis NIR spectrophotometer. TLC was per-
formed on aluminum sheets (silica gel 60 F254, Merck KGaA, Germany).
HPLC was performed on a Waters Breeze HPLC system. LC/MS was
conducted on a Waters Alliance 2695 HPLC module, 996 photodiode
array detector, and Micromass Quattro triple quadrupole mass
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δ (ppm) 201.65 (C-6), 178.04 (C-25), 172.10 (15-OAc), 170.88
(11-OAc), 170.76 (7-OAc), 169.51 (1-OAc), 169.33 (12-OAc),
154.34 (C-23), 111.33 (C-22), 79.76 (C-5), 79.10 (C-26), 74.31
(C-7), 74.26 (C-1), 73.99 (C-12), 71.54 (11), 71.22 (C-15), 54.34
(14), 54.22 (C-3), 51.60 (C-16), 50.60 (C-2), 50.26 (C-24), 48.66 (C-
17), 45.64 (C-10), 43.61 (C-13), 39.48 (C-8), 38.57 (C-9), 30.75 (C-
20), 26.78 (C-4), 25.37 (C-28), 22.79 (15-OAc), 21.27 (7-OAc), 21.23
(12-OAc), 21.19 (11-OAc), 20.97 (1-OAc), 20.68 (C-21), 20.21 (C-
27), 14.88 (C-19), 13.74 (C-18).
In Vivo Testing. The antitumor efficacies of taccalonolides A, E,
and N were evaluated in the murine syngeneic Mammary 16/C model.18
The average mouse weight was 19.3 ( 1.0 g at the start of treatment.
Tumor fragments were bilaterally implanted subcutaneously in female
B6C3F1 (C57BL6 Â C3H) mice on day 0, then nonselectively dis-
tributed to the various treatment and control groups (n = 5 mice/group).
All drugs were administered by iv in a 0.2 mL volume. The taccalonolides
were solubilized in 50% DMSO/50% Cremophor to generate stocks of
10.0À12.1 mg/mL and then diluted with sterile water for injections.
Paclitaxel (Mayne Pharma; Salisbury South, SA, Australia) was diluted
with water from clinical grade stocks to a final concentration of 6 mg/mL.
The protocol design and antitumor efficacy analyses were performed
as described previously.19 The scheduling was based on our prior studies
to optimize antitumor activity and reduce toxicity. Each taccalonolide was
administered intravenously on days 1, 4, and 6 with an additional dose
2À3 days later for taccalonolides A and N. Taccalonolide E treatments
were also administered on days 8, 9, and 11 because the weight loss was
least severe in this treatment group. Mice were weighed and observed
daily, and tumor size was measured two to three times weekly. Tumor
mass was calculated using the followng formula: mass (mg) = length (mm) Â
[width (mm)]2/2. Antitumor efficacy was evaluated by the following
standard calculations. (1) % T/C: The median tumor mass of a given
treated group (T) divided by the median tumor mass of the control (C),
expressed as percent. The % T/C determination includes “zeros” and is
made when the control group tumors reach exponential growth at
approximately 700À1200 mg in size. (2) Tumor growth delay (T À C)
and tumor cell kill: These measurements are quantitative determinations
of antitumor activity. (T À C) is the median number of days between the
time the treatment (T) and control (C) group tumors reach the predeter-
mined size of 1000 mg. Tumor free survivors are excluded from this
calculation and are tabulated separately. Tumor cell kill is determined
using tumor growth delay (T À C) as described above in the following
formula: gross log cell kill = [(T À C)/3.32] Â Td, where Td is the
tumor volume doubling time (in days) estimated from the best fit
straight line from a log-linear growth plot of control group tumors in the
exponential growth phase.
Taccalonolide AB (7). White powder. HRESIMS: m/z 677.2814
(calcd for C34H45O14 677.2809). ESIMS: 677.4 [M + H]+, 694.4
[M + NH4]+, and 699.4 [M + Na]+. 1H NMR: δ (ppm) 5.27 (dd, J =
11.9, 2.1 Hz, H-11), 5.22 (d, J = 2.1 Hz, H-12), 5.01 (br, H-21), 4.93 (d,
J = 3.6 Hz, 15-OH), 4.91 (dd, J = 10.8, 4.6 Hz, H-7), 4.83 (d, J = 5.4 Hz,
H-1), 4.62 (br, 25-OH), 4.47 (ddd, J = 11.1, 9.0, 3.4 Hz, H-15), 4.05 (d,
J = 4.5 Hz, 7-OH), 3.76 (t, J = 4.5 Hz, H-2), 3.69 (s, 5-OH), 3.63 (m,
H-3), 3.17 (t, J = 11.6 Hz, H-9), 2.56 (brd, J = 15.7 Hz, H-4a), 2.43 (dd,
J = 13.0, 11.0 Hz, H-16), 2.26 (m, J = 16.8 Hz, H-4b), 2.24 (m,
H-14),2.17 (s, 3H, 1-OAc), 2.15 (m, H-20), 2.14 (s, 3H, 12-OAc), 1.99
(s, 3H, 11-OAc), 1.86 (dd, J = 13.2, 9.9 Hz, H-17), 1.69 (s, 3H, H-27),
1.64 (q, J = 10.9 Hz, H-8), 1.37 (s, 3H, H-28), 0.97 (s, 3H, H-18), 0.89
(d, 3H, J = 7.0 Hz, H-21), 0.78 (s, 3H, H-19). 13C NMR: δ (ppm) 207.23
(C-6), 175.35 (C-26), 171.12 (12-OAc), 169.64 9 (1-OAc), 169.51 (12-
OAc), 154.90 (C-22), 110.43 (C-21), 79.10 (C-25), 78.75 (C-5), 74.41
(C-12), 74.12 (C-1), 72.04 (C-7), 71.46 (C-15), 70.89 (C-11), 57.57
(C-14), 54.12 (C-3), 51.04 (C-24), 50.79 (C-2), 50.28 (C-16),
48.19 (C-17), 46.06 (C-10), 44.06 (C-14), 43.82 (C-8), 36.66 (C-9),
31.17 (C-20), 27.07 (C-4), 25.62 (C-28), 21.99 (C-27), 21.35 (12-OAc),
21.14 (11-OAc), 20.83 (1-OAc), 20.30 (C-21), 14.70 (C-19), 13.44 (C-18).
Cell Culture. The HeLa cervical cancer cell line was obtained
from American Type Tissue Culture Collection (Manassas, VA) and
grown in Basal Media Eagle (BME) medium (Invitrogen; Carlsbad, CA)
supplemented with 10% fetal bovine serum (Hyclone; Logan, UT)
and 50 μg/mL gentamicin sulfate (Invitrogen).
Inhibition of Cellular Proliferation. The antiproliferative effects
of the taccalonolides were evaluated using the SRB assay20 as previously
described.16 The concentration of drug that caused a 50% inhibition of
cellular proliferation (IC50) was calculated from the linear portion of the
log of the dose response curve. Each IC50 represents the mean and
standard deviation of three to five independent experiments, each
performed in triplicate. Paclitaxel is included as a reference compound.
The determination of IC50 values was performed on taccalonolide
material after NMR analysis and subsequent lyophilization. Ethanol
was used as the vehicle for all cellular studies.
Immunofluorescence. Cellular microtubules in interphase or
mitotic HeLa cells were visualized using indirect immunofluorescence
techniques as previously described.16 Cells were treated for 18 h with
vehicle, a taccalonolide, or the positive control paclitaxel, fixed with
methanol and microtubules visualized with a β-tubulin antibody.
Representative images of interphase and mitotic cells were acquired
using a Nikon Eclipse 80i fluorescence microscope and compiled using
NIS Elements AR 3.0 software. Concentrations of taccalonolides that
caused similar levels of mitotic arrest at 18 h were used (5À10Â their
IC50 values). Paclitaxel requires a substantially higher concentration,
400Â the IC50, to initiate interphase bundling.
The mice were purchased from the NCI—Frederick Animal Produc-
tion Program (Frederick, MD), housed in an AALAC-approved facility
under fully licensed veterinary care (Wayne State University, Detroit, MI),
and provided water and food ad libitum.
’ ASSOCIATED CONTENT
S
Supporting Information. 1D and 2D NMR spectra of
b
compounds 5, 6, and 7. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For S.L.M: Phone: (210) 567-4788. Fax: (210) 567-4300. E-mail:
Mooberry@uthscsa.edu. For J.P.: Phone: (210) 567-6267. Fax:
(210) 567-4300. E-mail: Pengj2@uthscsa.edu.
’ ACKNOWLEDGMENT
Flow Cytometry. HeLa cells were incubated for 18 h with vehicle,
each taccalonolide or paclitaxel as a positive control. The cells were
harvested, and the DNA was stained with propidium iodide using
Krishan’s reagent.21 Cellular DNA content was analyzed using a FACS
Calibur flow cytometer (BD Biosciences). Data were plotted as propi-
dium iodide intensity versus the number of events using ModFit LT 3.0
software (Verity Software, Topsham, ME). Concentrations of paclitaxel
or taccalonolide that caused similar levels of mitotic arrest at 18 h were
used (5À10Â their IC50 values).
This work was supported by NCI Grant CA121138 (S.L.M.),
DOD-CDMRP Postdoctoral Award BC087466 (A.L.R.), and
NCI Grant P30 CA054174 (S.L.M.).
’ ABBREVIATIONS USED
HSQC, heteronuclear single quantum coherence; HMBC, hetero-
nuclear multibond correlation spectroscopy; SAR, structureÀactivity
relationship
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ARTICLE
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