Design and synthesis of novel C14-hydroxyl substituted triptolide derivatives as potential selective antitumor agents

Article abstract of DOI:10.1021/jm900342g

It has long been considered that the free β hydroxyl group at C14 of triptolide (1) is essential to its potent anticancer activity. In this study, we synthesized novel derivatives of 1 with a hydroxyl group substituted by epoxy groups (4-8) or a five-memb

Full text of DOI:10.1021/jm900342g

J. Med. Chem. 2009, 52, 5115–5123 5115
DOI: 10.1021/jm900342g
Design and Synthesis of Novel C14-Hydroxyl Substituted Triptolide Derivatives as Potential Selective
Antitumor Agents
Zheng Li,†, Zhao-Li Zhou,§, Ze-Hong Miao,‡ Li-Ping Lin,‡ Hui-Jin Feng,† Lin-Jiang Tong,‡ Jian Ding,*,§,‡ and
Yuan-Chao Li*,†
†Department of Medicinal Chemistry and ‡Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, PR China, and §Department
of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang 110016, PR China. These authors
contributed equally.
Received March 17, 2009
It has long been considered that the free β hydroxyl group at C14 of triptolide (1) is essential to its potent
anticancer activity. In this study, we synthesized novel derivatives of 1 with a hydroxyl group substituted
by epoxy groups (4-8) or a five-membered ring (11-13). Compounds (4-8) showed significant in vitro
anticancer activity although less potent than 1. Although with an R oxygen configuration at the C14
position, (14S)-14,21-epoxytriptolide (4) exhibited the highest potency among all these derivatives,
clearly challenging the traditional viewpoint on the necessity of C14β-hydroxyl group of compound 1.
Further studies revealed that while displaying broad spectrum in vitro anticancer activity, compound
4 demonstrated prominent selective in vivo anticancer activity, particularly against human ovarian SK-
OV-3 and prostate PC-3 cancers with obviously lower toxicity than 1. Noticeably, compound 4 was also
highly effective against multidrug resistant cancer cells. Therefore, our study gives new insights into the
structure-activity relationship of 1 and also produces a promising anticancer drug candidate with
unique anticancer activities.
Introduction
some conventional chemotherapeutic drugs, 1 has similar or
even superior anticancer activity, especially over p53 mutated
or multidrug resistant cells.9 All of these antitumor properties
mentioned above suggest that 1 should be a promising anti-
cancer drug, however, its clinical development has been
discontinued. One major obstacle for this is the severe toxicity
of 1, and the tissues and organs being inflicted include
gastrointestinal tracts, liver, kidney, heart, blood cells, bone
marrow, testes, and ovariesaswell asskin.16,17 Inaddition, the
therapeutic window of 1 is so narrow that the toxic dose is just
about twice the effective dose6 or even equal in some reports.18
Previous studies on the structure-activity relationship of 1
indicated that the characteristic hydrogen-bonded C9,C11-
epoxy-C14β-hydroxy system may account for its antitumor
effect,19 meanwhile, the configuration of the hydroxyl group
should be the βorientation.2,20 Onthe basisoftheseprinciples,
for a long time the aim of C14 modification was just to
improve the lead compound’s water solubility by carboxyla-
tion of C14β-OH to introduce water solubility enhancing
moieties.21,22 However, most of these derivatives are enzyma-
tically converted into 1 in vivo, thus retaining many of its side
effects. For example, 14-succinyl triptolide sodium salt
(PG490-88)23 (Figure 1), a water-soluble prodrug converted
to 1 in serum, entered into phase I clinical trials in Europe for
the treatment of solid tumors in 2003,23 but later this clinical
trial was discontinued.24 On the other hand, some medicinal
chemists have found recently that substituting C14-hydroxyl
with other groups such as fluoride while keeping β orientation
of the substituents could retain the cytotoxicity of 1,25 and the
anticancer activity of compound 1 analogues may not be
Tripterygium wilfordii Hook. f. (TWHFa), commonly
known as Lei Gong Teng (Thunder God Vine), has been used
in Traditional Chinese Medicine to treat autoimmune and
inflammatory diseases such as rheumatoid arthritis for cen-
turies.1-3 Triptolide (1) (Figure 1), the major component
responsible for the clinical properties of TWHF, was first
isolated from TWHF extracts and characterized as a diterpe-
noid triepoxide lactone containing an 18 (4f3) abeo-abietane
skeleton in 1972.4 Right after its isolation, 1 was shown to
possess high antileukemic activity in murine models4 besides
its remarkable efficacy in rheumatoid arthritis,5 and this led to
extensive studies regarding its antitumor properties.
Compound 1 induces growth inhibition and apoptosis
against various cultured human cancer cell lines6-9 as well
as on primary cultures of human tumor samples.10,11 In
addition to its impressive in vivo activity against murine L-
1210 leukemia,4 1 could also inhibit the growth of xenografts
formed by different solid tumor cells6,9,12 and the experimen-
tal metastasis of B16F10 cells.9 Meanwhile, 1 was shown to
sensitize cells to Apo/Trail, tumor necrosis factor-R, or DNA-
damaging chemotherapeutic agents.13-15 Compared with
*To whom correspondence should be addressed. For Y.C.L.: phone,
þ86-21-50806600 ext 3502; fax, þ86-21-50807288; E-mail, ycli@mail.
shcnc.ac.cn. For J.D.: phone, þ86-21-50806600 ext 2424; fax, þ86-21-
50806722; E-mail, jding@mail.shcnc.ac.cn.
a Abbreviations: TWHF, Tripterygium wilfordii Hook. f.; SRB, sul-
forhodamine B; MDR, multidrug resistance; VER, verapamil; ADR,
adriamycin; VCR, vincristine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; RTV, relative tumor volume.
r
2009 American Chemical Society
Published on Web 07/28/2009
pubs.acs.org/jmc

5116 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Li et al.
explained as simply as before.19 Therefore, there is still big
confusion on the role of C14-hydroxyl and any derivative of 1
that retains its anticancer activity but lowers risk in toxicity is
still the concern of anticancer drug development.
focused our attention on introducing another epoxide group
at C14 position in order to obtain a series of 14,21-epoxy-
triptolide derivatives (Scheme 1). Quad-epoxy compound
4 was obtained from triptonide through a modified Corey-
Chaykovsky methylenation reaction.26 We found that treat-
ment of triptonide with NaH and Corey methylenation
reagent in anhydrous DMSO at room temperature cleanly
furnished 4 in 85% yield, while in the presence of t-BuOK
under the same reaction conditions provided a 61:39 mixture
of 4 and its diastereoisomer 5. Nucleophilic ring-opening at
the C12 position of compound 4 by 1.67% HCl in acetone
provided chloride 6. Alternatively, treatment of quad-epoxy 4
with SeO2 in 1,4-dioxane under reflux introduced a hydroxyl
group at C5 to give compound 7 as the main product (50%
yield) and a dehydration compound 8 in 10% yield. The
absolute stereochemistry of C14 in compound 4 was deter-
mined by X-ray crystallography (Figure 2).
Then, we switchedto the construction of the five-membered
ring derivatives of 1. Treatment of triptonide with (isopro-
poxy-dimethylsilyl)methylmagnesium chloride followed by
Tamao oxidation27 provided C14β-hydroxymethylepitripto-
lide 10. Modifying the two hydroxyl groups of 10 by cyclic
sulfitation with thionyl chloride resulted in two diastero-
isomers 11 and 12, which are different in the configuration
of sulfur atom. They can both be oxidized with NaIO4 and a
In this study, to explore whether the C14β-hydroxyl group
of 1 is faithfully critical to its anticancer activity and to find
novel C14 substituted compound 1 derivatives as more pro-
mising antitumor drug candidates, we designed two series of
C14-spiro-triptolide derivatives with the C14-hydroxyl sub-
stituted by a chiral epoxy group or five-membered ring, which
were impossible to form intramolecular hydrogen bond and
assessed their anticancer activity. Our results significantly
challenge the traditional learnings and, in particular, give
new insights into the structure-activity relationship of 1.
Moreover, we discovered that a novel derivative of 1, com-
pound 4, possesses unique selective in vivo anticancer activity
and potential low systematic toxicity, which favorably makes
it a very promising anticancer drug candidate.
Chemistry
The synthetic strategy followed for the preparation of the
analogues 4-8 and 10-13 of compound 1 is depicted in
Schemes 1 and 2. We used triptonide (3), which was extracted
from TWHF of our region, as starting material and initially
catalytic amount of RuCl3 3H2O to give sulfate 13 as the sole
3
Figure 1. Triptolide, epitriptolide, (14S)-14,21-epoxytriptolide
and 14-succinyl triptolide sodium salt.
Figure 2. X-ray single crystal structure of (14S)-14,21-epoxytrip-
tolide (4).
Scheme 1. Synthesis of Derivatives 4, 5, 6, 7, and 8a
a Reagents and conditions: (a) (CH3)3SOI, NaH, DMSO, rt, 20 min, 85% (exclusively 4); (b) (CH3)3SOI, t-BuOK, DMSO, rt, 20 min, 65%, (4/5 =
1.6:1); (c) 1.67% HCl aq, acetone, reflux 7 h, 39%; (d) SeO2, 1,4-dioxane, reflux 24 h, 60% (7/8 = 5:1).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5117
product. The absolute configuration of the sulfur atom in 11
was determined by X-ray crystallography (Figure 3). Herein,
we also found another stereospecific route to synthesize
compound 4 from dihydroxy 10 in two steps. Selective
mesylation of the primary alcohol in 10 followed by cycliza-
tion with K2CO3 in MeOH solely afforded quad-epoxy
compound 4 (Scheme 2).
examine whether the substitution affected their biological
activities, we first evaluated the in vitro anticancer effects of
those target compounds (4-8 and 11-13) against three
human tumor cell lines derived from ovary (SK-OV-3),
breast (MDA-MB-468), and prostate (PC-3) using sulfor-
hodamine B (SRB) assays.28 The result revealed that
although there is no free C14β-hydroxyl group in our new
derivatives, the series of 14,21-epoxytriptolide derivatives
were all effective against those three cell lines, with IC50
values ranging from 0.10 to 7.34 μM (Table 1). Among them,
compound 4 exhibited the highest potency, with the lowest
IC50 value (0.10 μM for SKOV-3 cells) (Table 1). Unexpect-
edly, compound 5, the oxygen’s direction of which was the
same with that of 1, exhibited much less potency than its
Results and Discussion
Substitution of the C14β-Hhydroxyl Group of 1 with a
Chiral Epoxy Group Retains Its in Vitro Anticancer Activity.
As shown above, we obtained two series of new compound
1 analogues by substituting its C14β-hydroxyl group. To
Table 1. In Vitro Anticancer Activity of Compound 1 Derivatives in
SK-OV-3, MDA-MB-468, and PC-3 Cells
IC50 (μM)a
compd
SK-OV-3
MDA-MB-468
PC-3
1
0.009
0.79
0.10
0.01
1.32
0.02
1.60
0.27
2.70
0.47
7.34
1.80
>100
20.01
6.52
13.93
2
4
0.18
1.92
5
1.56
0.14
6
0.21
2.37
7
1.86
0.57
8
1.14
10
11
12
13
>100
>100
5.10
>100
>100
>100
>100
>100
a IC50: The drug concentration required for 50% inhibition of cell
proliferation, while the maximum concentration used here was 100 μM.
Figure 3. X-ray single crystal structure of (14S,SS)-14-spiro-
14R,21-sulfinyldioxytriptolide (11).
Scheme 2. Synthesis of Derivatives 4, 10, 11, 12, 13, and 14a
a Reagents and conditions: (a) (isopropoxy-dimethylsilyl) methyl chloride, Mg, Br-(CH2)2-Br, THF, reflux 45 min; (b) KF, KHCO3, 30% H2O2, 0 °C,
2 h, 60% over 2 steps; (c) SOCl2, Et3N, CH2Cl2, 0 °C, 2 h, 79%, (11/12 = 1.2:1); (d) NaIO4, RuCl3 3H2O, CH3CN/H2O = 4:1, rt, 15 min, 86%; (e) MsCl,
3
Py, rt, 10 min, 84%; (f) K2CO3, CH3OH, rt, 20 min, 90%.

5118 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Li et al.
diastereoisomer compound 4 even though the latter pos-
sesses an oxygen atom of R direction on C14. Compound 6,
which could easily transform to compound 4, showed similar
but a little less cytotoxicity against those three cell lines. The
data indicates that the C14β-hydroxyl group of 1 is not
unchangeable even in order to generate analogues of potent
anticancer activity. Our result apparently challenges the
classical structure-activity relationship of 1 that considers
the C14β-hydroxyl group to be essential for its anticancer
activity.2,19,20 In the meantime, our result shows that intro-
duction of a chiral epoxy group to this important site of the
lead compound is an effective modification method to retain
the potent anticancer activity.
SKOV-3 and PC-3 cell lines. We presumed that the large
space taken by C14-five-membered ring might form a spatial
obstacle that prevented the interaction between these deri-
vatives and their target molecule(s) responsible for initiating
their cytotoxic effects.
Compound 4 Elicits Broad-Spectrum in Vitro Antitumor
Effects. To characterize the anticancer activity of compound
4, we further extended its in vitro anticancer evaluation to a
panel of 21 human tumor cell lines, which come from distinct
human tissues, including leukemia, lung cancer, gastric
cancer, hepatoma, colon cancer, breast cancer, ovarian
cancer, prostate cancer, renal cancer, cervical cancer, rhab-
domyosarcoma, and glioma cells. The data showed that
compound 4 exhibited a similar in vitro anticancer spectrum
but reduced anticancer activities when compared to com-
pound 1 (Table 2). Compound 4 displayed apparent cyto-
toxicity with IC50 values ranging from 0.18 to 0.91 μM
against almost all of the above human tumor cell lines,
except that this compound appeared to be relatively less
effective against MKN-28 with an IC50 value of 2.54 μM.
The results indicate that compound 4 possesses a broad
anticancer spectrum of in vitro anticancer actions.
Compound 4 Produces Direct Cytotoxic Effects on Multi-
drug Resistant Cancer Cell Lines. Multidrug resistance
(MDR), especially to drugs of natural origin, is an important
impediment to the effective chemotherapy of cancer. It has
always been the focused area to develop new agents to
circumvent MDR. To examine the anticancer activity of
compound 4 against MDR tumor cells, we used three
classical MDR cell lines (K562/A02, KB/VCR, and MCF-
7/ADR) that were significantly resistant to the correspond-
ing drugs used to establish these cell lines. The IC50 values of
compound 4 in these MDR cell lines were 0.74, 0.47, and 0.91
μM, while in their corresponding sensitive cell lines (K562,
KB, and MCF-7), its IC50 values were 0.42, 0.61, and
0.36 μM, respectively. The results indicated that compound
4 showed approximately equivalent cytotoxicity against each
MDR subline as compared with their parental cell lines, with
resistance factors of 1.76, 0.77, and 2.53, respectively
(Figure 4A). However, the metabolism of compound 4 was
not seen via the p-glycoprotein transportation pathway
because verapamil (VER), a well-characterized MDR rever-
sal agent, did not increase its cytotoxic effect on KB/VCR
cells but enhanced the cytotoxicity of VCR approximately
10-fold as expected (Figure 4B).
On the other hand, the series of analogues (11, 12, 13) with
five-membered spiro sulfate or sulfite substituents on C14
nearly completely lost their cytotoxicity against MDA-MB-
468, and only compound 12 retained weak potency against
Table 2. IC50 Calues of Compound 4 and 1 in a Panel of Human Tumor
Cell Lines
IC50(mean ( SD)a, μM
origins
blood
cell lines
Molt-4
K562
compd 4
compd 1
0.251 ( 0.077
0.421 ( 0.125
0.702 ( 0.228
0.175 ( 0.032
2.538 ( 0.215
0.219 ( 0.037
0.907 ( 0.301
0.648 ( 0.218
0.350 ( 0.020
0.332 ( 0.069
0.386 ( 0.200
0.280 ( 0.067
0.360 ( 0.076
0.270 ( 0.023
0.538 ( 0.059
0.637 ( 0.218
0.256 ( 0.024
0.615 ( 0.120
0.293 ( 0.064
0.507 ( 0.195
0.657 ( 0.235
0.017 ( 0.002
0.050 ( 0.010
0.059 ( 0.012
0.015 ( 0.0004
0.200 ( 0.042
0.010 ( 0.003
0.052 ( 0.023
0.029 ( 0.006
0.018 ( 0.004
0.020 ( 0.005
0.022 ( 0.009
0.024 ( 0.002
0.019 ( 0.002
0.010 ( 0.001
0.028 ( 0.007
0.043 ( 0.012
0.024 ( 0.009
0.043 ( 0.019
0.014 ( 0.001
0.047 ( 0.040
0.049 ( 0.015
lung
stomach
A549
SGC-7901
MKN-28
HCT-116
SW1116
HCT-15
SMMC-7721
BEL-7402
786-O
colon
liver
kidney
breast
MDA-MB-231
MCF-7
SK-OV-3
HO-8910
PC-3
ovary
prostate
others
DU-145
KB
Rh30
Hela
U251
a IC50 (mean ( SD): Cells in 96-well plates were treated with various
concentrations of compound 4 for 72 h to get the IC50 values which were
determined from dose-response curves, using MTT or SRB assays; IC50
values were presented as mean ( SD of three independent experiments.
Figure 4. Anti-MDR activity of compound 4. (A) Resistance factors of compound 4 and reference drugs. K562/A02, KB/VCR, and MCF/
ADR cell lines are MDR cell lines derived from K562, KB, and MCF-7 cells, respectively. (B) The effect of VER on the cytotoxicity of
compound 4 and VCR in KB/VCR cells. The inhibition rate was assessed by SRB assays, and each data point represents the mean of three
independent experiments.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5119
Figure 5. Antitumor effect of compound 4 in nude mouse models. The models were generated as described in the Experimental Section.
Compound 4 displayed selective in vivo antitumor activity, as indicated by its special potent effect against ovarian SK-OV-3 (A) and prostate
PC-3(B) cancers but moderate action in the SGC-7901 model (C) and no effect in the SMMC-7721 model (D) and in the MDA-MB-468 model
(E). No body weight difference was seen between compound 4 and vehicle treated groups and the body weight curves were illustrated in
(F). Significant differences between compound 4 treatment groups and control were determined using a Student t test. * P < 0.05; ** P < 0.01.
Compound 4 Exerts Selective in Vivo Anticancer Effects.
Compound 1 has been reported to have a wide in vivo
anticancer spectrum with apparent effects against human
liver,29 gastric,9 and breast cancers6,9 but to reveal a marginal
effect against ovarian cancer in vivo.30 Here we investigated
the in vivo anticancer effects of compound 4 against human
breast (MDA-MB-468), liver (SMMC-7721), stomach
(SGC-7901), ovary (SK-OV-3), and prostate (PC-3) cancer
xenografts in nude mice. Surprisingly, the results showed
that compound 4, orally administered, was highly effective
against human ovarian and prostate cancer xenografts in
contrast to its modest to minimal activity in the models of
human gastric, hepatic, and breast cancer xenografts at the
same dosage (Figure 5). The treatment with compound 4
resulted in a marked, even total, regression of tumors in the
models of SK-OV-3 and PC-3 xenografts on the 21st day
post the first drug administration. In the SK-OV-3 model,
compound 4 at 2 and 4 mg/kg achieved a tumor growth
inhibition rate of 87.5% and 97.4%, respectively. Moreover,
tumor xenografts totally disappeared in 3 mice out of 6 at 4
mg/kg and in 1 mouse out of 6 at 2 mg/kg. The similar result
was seen in the PC-3 models. However, compound 4 demon-
strated relatively weak antitumor effect in the model of
gastric cancer SGC-7901 xenografts, with a growth inhibi-
tion rate of only 75% even at the maximal dose used (4 mg/
kg). More prominently, compound 4 led to no anticancer
activity in the models of hepatic cancer SMMC-7721 and
breast cancer MDA-MB-468 xenografts. These data indicate
that compound 4 elicits selective anticancer effects, specifi-
cally against ovarian and prostrate cancers, although the
mechanism(s) are still open to be clarified.
Noticeably, compound 4 was well tolerated, not causing
death or serious systematic toxicity in mice. The change in
the body weight of the tested mice in the groups treated with

5120 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Li et al.
compound 4 or with vehicle was basically identical
(Figure 5F). There was no distinguishable gross anatomy
differences observed in livers or kidneys between the com-
pound 4 treated groups and the vehicle groups. In contrast,
the lead compound 1 was reported to cause animal death
within doses 4 times9,31 or twice6 over the effective doses, and
the severe toxicity and narrow therapeutic window limited its
clinical development as a anticancer agent.16,17 Considering
that there was a 4-time distance between the lowest effective
dosage in SK-OV-3 xenografts model with T/C (%) value of
50.2 (1 mg/kg) and the highest dosage we used (4 mg/kg) in
our experiments, compound 4 appears to have a larger safety
window than compound 1. Detailed toxicity evaluations on
compound 4 are underway that will be helpful to precisely
reveal its safety at least in animals.
XDB-C18; 5 μm; 4.6 mm ꢀ 250 mm) with an Agilent 1100 series
system attached to a Hewlett-Packard chromatograph manager.
(14S)-14,21-Epoxytriptolide (4) and (14R)-14,21-epoxytripto-
lide (5). Method A. (CH3)3SOI (374 mg, 1.7 mmol) and t-BuOK
(174 mg, 1.5 mmol) were dissolved in dry DMSO (6.0 mL) under
Ar atmosphere. The mixture was stirred for 20 min at room
temperature. An anhydrous DMSO (6.0 mL) solution of tripto-
nide (3) (360 mg, 1.0 mmol) was added all at once to the above
mixture. The mixture was stirred at room temperature until TLC
analysis revealed the absence of the starting material. H2O
(10.0 mL) was added to the mixture and then it was extracted
with ethyl acetate. The organic layer was washed with water and
brine, dried over anhydrous Na2SO4, and concentrated. The crude
product was purified first by chromatography on silica gel using
ethyl acetate/cyclohexane (1/5). Then we use HPLC to separate
the couple of diastereomers. Finally, we gained 4 (0.149 g,
0.40 mmol, 40%) and 5 (0.093 g, 0.25 mmol, 25%) separately.
Method B. (CH3)3SOI (74.8 mg, 0.34 mmol) and NaH (60%)
(12 mg, 0.3 mmol) were dissolved in dry DMSO (2.5 mL) under
Ar atmosphere. The mixture was stirred for 20 min at room
temperature. A solution of triptonide (71.6 mg, 0.2 mmol) in dry
DMSO (2 mL) was added to the above mixture. The mixture was
stirred at room temperature until starting material disappeared.
H2O (4.0 mL) was added to the mixture and then it was extracted
with ethyl acetate. The organic layer was washed with water and
brine, dried over anhydrous Na2SO4, and concentrated. The
crude product was purified by chromatography on silica gel
using ethyl acetate/cyclohexane (1/5) to give exclusively 4 as
white solide in 85% yield (63.2 mg, 0.17 mmol).
Conclusions
A series of novel derivatives of 1 as potential anticancer
agents were synthesized and tested for their cytotoxicity
against three human tumor cell lines. Previous studies have
indicated that the characteristic hydrogen-bonded C9,C11-
epoxy-C14β-hydroxy system may account for the antitumor
effect of compound 1 by selectively alkylating the thiol groups
of key enzymes regulating tumor growth.19 But in our study,
the series of 14,21-epoxytriptolide derivatives (4-8) with C14-
hydroxyl substituted by a chiral epoxy group which were
impossible to form intramolecular hydrogen bond still re-
tained moderate to good in vitro anticancer activity, especially
compound 4, with a different oxygen direction at C14 position
from that of 1, exhibited much higher potency than its
diastereoisomer compound 5. On the other hand, the cyto-
toxicity of C14 five-membered ring substituents analogues
(11, 12, 13) reduced greatly against all three cell lines. On the
basis of the above results, we presumed that the size and stereo
configuration of functional groups at the C14 position may
exert an important influence on interaction between the
derivatives of compound 1 and their target molecule(s) re-
sponsible for initiating their cytotoxic effects although the
exact mechanism is still unclear. Further studies revealed that
although demonstrating broad -spectrum in vitro anticancer
activity, compound 4 exhibited significant selectivity in its in
vivo anticancer activity, as indicated by its specially highly
effectiveness against human ovarian and prostate cancers.
Moreover, compound 4 showed remarkable cell killing effect
on MDR tumor cells. More importantly, its larger safety
window seems to make it more applicable. Therefore, this
study gives new insights into the structure-activity relation-
ship of 1 and generates a new compound (compound 4) as a
promising anticancer drug candidate.
Method C. To a solution of 14 (504 mg, 1.1 mmol) in CH3OH
(20.0 mL) was added K2CO3 (1.33 g, 9.6 mmol). The mixture
was stirred for 20 min under room temperature. After removal
of the solvent under reduced pressure, the residue was diluted
with water and then extracted with ethyl acetate, washed
with brine, and dried with anhydrous Na2SO4. The crude
product was purified via chromatography on silica gel using
ethyl acetate/cyclohexane (1/5) to give 4 in 90% yield (368 mg,
0.99 mmol).
1
Compound 4. H NMR(CDCl3, 300 MHz) δ 4.67 (s, 2 H), 3.88
(d, J =2.9 Hz, 1 H), 3.52 (d, J=3.1 Hz, 1 H), 3.40 (d, J=5.5 Hz,
1 H), 2.84 (d, J=5.2 Hz, 1 H), 2.80-2.69 (m, 2 H), 2.37-2.25 (m,
1 H), 2.21-2.05 (m, 2 H), 1.91-1.80 (m, 2 H), 1.57 (dd, J =12.4,
4.9 Hz, 1 H), 1.29-1.17 (m, 1 H), 1.06 (s, 3 H), 0.86 (d, J =6.8
Hz, 3 H), 0.80 (d, J=6.8 Hz, 3 H). 13C NMR (CDCl3, 100 MHz)
δ 173.2 (C), 160.2 (C), 125.2 (C), 69.9 (CH2), 65.2 (C), 65.0 (C),
58.4 (C), 56.8 (CH), 55.9 (CH), 55.6 (C), 54.1 (CH), 47.9 (CH2),
40.4 (CH), 35.6 (C), 30.2 (CH2), 23.4 (CH2), 23.2 (CH), 19.8
(CH3), 17.6 (CH3), 17.0 (CH2), 13.5 (CH3). IR (KBr) 3427, 3016,
2981, 2929, 1745, 1674, 1442, 1022 cm-1. MS (EI, 70 eV) m/z (%)
373 ([M þ 1]þ, 2), 372 (Mþ, 1), 357 (5), 343 (21), 91 (100). HRMS
(EI) calcd for C21H24O6 372.1573, found 372.1578. Anal.
(C21H24O6) C, H.
Compound 5. 1H NMR (CDCl3, 300 MHz) δ 4.79 (dt, J =
16.8, 2.6 Hz, 1 H), 4.60 (dd, J=16.8, 2.8 Hz, 1 H), 3.81 (d, J=3.1
Hz, 1 H), 3.52 (d, J =3.1 Hz, 1 H), 3.38 (d, J =4.2 Hz, 1 H), 2.89
(d, J=4.7 Hz, 1 H), 2.85 (d, J=4.7 Hz, 1 H), 2.81-2.72 (m, 1 H),
2.39-2.27 (m, 1 H), 2.17 (dt, J =14.3, 4.2 Hz, 1 H), 2.11-1.94
(m, 1 H), 1.94-1.81 (m, 2 H), 1.47-1.29 (m, 2 H), 0.96 (s, 3 H),
0.89 (d, J =6.8 Hz, 3 H), 0.77 (d, J =6.8 Hz, 3 H). 13C NMR
(CDCl3, 100 MHz) δ 173.2 (C), 161.6 (C), 125.4 (C), 70.8 (CH2),
65.9 (C), 64.6 (C), 58.3 (C), 57.2 (CH), 56.2 (CH), 55.9 (C), 54.7
(CH), 48.2 (CH2), 36.9 (CH), 36.1 (C), 28.4 (CH2), 26.9 (CH2),
23.0 (CH), 21.7 (CH3), 20.2 (CH3), 17.3 (CH3), 16.6 (CH2). IR
(KBr) 3433, 2947, 2929, 1768, 1755, 1689, 1442 cm-1. MS (EI, 70
eV) m/z (%) 372 (Mþ, 1), 357 (9), 343 (18), 325 (34), 259 (100).
HRMS (EI) calcd for C21H24O6 372.1573, found 372.1586.
Anal. (C21H24O6) C, H.
Experimental Section
Part of Chemistry. Mass spectra and high-resolution mass
spectra were measured on a Finnigan MAT-95 mass spectro-
meter. Elemental analysis was performed on a Carlo Erba 1106
instrument. IR spectra were recorded on a Nicollet Magna
FTIR-750 spectrometer using KBr pellets in cm-1 1H and
.
13C NMR spectra were determined on Bruker AM-300 and
Bruker AM-400 instruments using tetramethylsilane as internal
reference. The X-ray single crystal diffraction experiment was
carried out by the Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences. Silica gel 60H (200-300 mesh),
manufactured by Qingdao Haiyang Chemical Group Co.
(China), was used for general chromatography. HPLC analy-
sis was used a C18 reverse phase column (Agilent Eclipse
(12R,13R,14S)-12β-Chloro-13r-hydroxy-14,21-epoxytripto-
lide (6). To a solution of compound 4 (40 mg, 0.11 mmol) in
acetone (6.0 mL) was added aq HCl (1.67%, 6.0 mL, 2.7 mmol).

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16 5121
The mixture was gently refluxed for 7 h. After removal of most
of the solvent under reduced pressure, the residue was extracted
with ethyl acetate, washed with brine, and dried over anhydrous
Na2SO4. The crude product was purified via chromatography
on silica gel using ethyl acetate/cyclohexane (1/3) to provide 6 in
39% yield (17.5 mg, 0.043 mmol). 1H NMR (CDCl3, 300 MHz)
δ 4.69 (s, 2 H), 4.16 (d, J=5.7 Hz, 1 H), 3.75 (d, J=5.7 Hz, 1 H),
3.46 (d, J=6.0 Hz, 1 H), 2.93-2.83 (m, 2 H), 2.78 (d, J=5.1 Hz,
1 H), 2.36-2.25 (m, 1 H), 2.21-2.04 (m, 2 H), 1.94-1.74 (m, 2
H), 1.59 (dd, J=12.3, 4.7 Hz, 1 H), 1.35-1.21 (m, 1 H), 1.03 (s, 3
H), 0.99 (d, J =2.2 Hz, 3 H), 0.96 (d, J =2.2 Hz, 3 H). 13C NMR
(acetone-d6, 100 MHz) δ 173.7 (C), 162.2 (C), 124.6 (C), 76.6 (C),
70.7 (CH2), 69.9 (C), 60.3 (CH), 59.8 (C), 58.3 (CH), 58.2 (C),
58.2 (CH), 48.0 (CH2), 40.2 (CH), 35.8 (C), 31.3 (CH2), 28.8
(CH), 23.1 (CH2), 18.0 (CH3), 17.4 (CH2), 16.3 (CH3), 13.9
same temperature over a few minutes. After stirring at 0 °C for
2 h, the mixture was quenched with a saturated NH4Cl solution
and extracted with EtOAc. The organic layer was washed with
brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure to give a single adduct. To a stirred mixture of
colorless crude adduct, MeOH (5.0 mL), THF (8.0 mL),
KHCO3 (416 mg, 4.2 mmol), and KF (464 mg, 4.9 mmol) was
added H2O2 (30%, 1.1 mL, 9.71 mmol) dropwise at room
temperature. The mixture was stirred at room temperature until
starting material disappeared. Aqueous sat. Na2S2O3 solution
(50%) was added slowly to the mixture and stirred until a
negative starch/iodide test was observed. The mixture was
extracted with EtOAc. The organic layer was washed with brine,
dried over Na2SO4, and concentrated. The crude product was
purified by chromatography on silica gel using ethyl acetate/
cyclohexane (1/3) to provide 10 in 60% yield (234 mg,
0.6 mmol). 1H NMR (CDCl3, 300 MHz) δ 4.67 (s, 2 H), 4.26 (d,
J =11.8 Hz, 1 H), 3.87-3.80 (m, 2 H), 3.64 (d, J=11.5 Hz, 1 H),
3.46 (d, J =3.3 Hz, 1 H), 2.76-2.64 (m, 1 H), 2.45 (sept, J =
6.9 Hz, 1 H), 2.37-2.25 (m, 1 H), 2.23-2.04 (m, 2 H), 1.89 (t, J=
14.1 Hz, 1 H), 1.55 (dd, J =12.6, 5.2 Hz, 1 H), 1.25-1.13 (m,
1 H), 1.07 (s, 3 H), 0.91 (d, J =6.9 Hz, 3 H), 0.89 (d, J =6.9 Hz,
3 H). 13C NMR (CDCl3, 100 MHz) δ 173.2 (C), 160.2 (C), 125.4
(C), 74.4 (C), 70.0 (CH2), 67.5 (C), 65.3 (C), 65.2 (CH2), 65.0 (C),
56.5 (CH), 56.1 (CH), 54.4 (CH), 40.3 (CH), 36.0 (C), 30.1
(CH2), 25.5 (CH), 23.4 (CH2), 20.9 (CH3), 18.6 (CH3), 17.1
(CH2), 13.7 (CH3). IR (KBr) 3415, 3361, 2966, 2927, 2875, 1755,
1724, 1672, 1439, 1074, 1018 cm-1. MS (EI, 70 eV) m/z (%) 391
([M þ 1]þ, 2), 372 (1), 71 (100). HRMS (EI) calcd for C21H27O7
(M þ H)þ 391.1757, found 391.1752. Anal. (C21H26O7) C, H.
(14S,SS)-14-Spiro-14r,21-sulfinyldioxytriptolide (11) and
(14S,SR)-14-Spiro-14R,21-sulfinyldioxytriptolide (12). To a so-
lution of compound 9 (78 mg, 0.2 mmol) in anhydrous CH2Cl2
(6.0 mL) was added dry Et3N (0.21 mL, 1.6 mmol) dropwise.
The mixture was then cooled to 0 °C. Under Ar atmosphere,
SOCl2 (0.3 mL, 1.2 mmol) was added to the mixture carefully.
After being stirred for 2 h, the mixture was quenched with water
and extracted with CH2Cl2. The organic layer was washed with
water and brine and dried over anhydrous Na2SO4. After
removal of the solvent under reduced pressure, the crude
product was purified by chromatography on silica gel using
acetate/cyclohexane (1/5) to give 11 in 43% yield (39.2 mg, 0.09
mmol) and 12 in 36% yield (30.5 mg, 0.07 mmol).
(CH3). IR (KBr) 3462, 2933, 2252, 1743, 1674, 1439, 1003 cm-1
.
MS (EI, 70 eV) m/z (%) 408 (Mþ, 9), 390 (3), 373 (8), 365 (100).
HRMS (EI) calcd for C21H25ClO6 408.1339, found 408.1339.
(5R,14S)-5r-Hydroxy-14,21-epoxytriptolide (7) and (14S)-
Δ5,6-dehydro-14,21-epoxytriptolide (8). To a solution of com-
pound 4 (74.4 mg, 0.20 mmol) in 1,4-dioxane (8.0 mL) was
added SeO2 (111 mg, 1.0 mmol). The mixture was gently
refluxed for 24 h. Then the mixture was cooled down to room
temperature, filtered through a short pad of silicon gel, and
rinsed with ethyl acetate. The solvent was removed under
reduced pressure. To the residue was added ethyl acetate and
saturated Na2CO3. After vigorous extraction, the organic layer
was washed with water and brine and dried over anhydrous
Na2SO4. After concentration, the residue was purified by chro-
matography on silica gel using CH2Cl2 to provide 7 in 50% yield
(38.8 mg, 0.1 mmol) and 8 in 10% yield (7.4 mg, 0.02 mmol).
Compound 7. 1H NMR (CDCl3, 300 MHz) δ 4.92 (dt, J =
17.1, 3.1 Hz, 1 H), 4.71 (dd, J=17.1, 3.9 Hz, 1 H), 3.92 (d, J=3.3
Hz, 1 H), 3.63 (d, J =3.3 Hz,1 H), 3.40 (d, J =4.8 Hz, 1 H), 2.86
(d, J=5.1 Hz, 1 H), 2.79 (d, J=5.1 Hz, 1 H), 2.41-2.04 (m, 4 H),
1.95-1.74 (m, 2 H), 1.29 (dd, J=12.7, 4.9 Hz, 1 H), 1.12 (s, 3 H),
0.87 (d, J =6.8 Hz, 3 H), 0.84 (d, J =6.8 Hz, 3 H). 13C NMR
(CDCl3, 100 MHz) δ 173.2 (C), 159.0 (C), 128.0 (C), 72.3 (C),
68.7 (CH2), 65.8 (C), 63.2 (C), 58.4 (C), 56.4 (CH), 55.5 (C), 55.0
(CH), 54.5 (CH), 48.0 (CH2), 40.6 (C), 31.0 (CH2), 24.6 (CH2),
23.4 (CH), 19.7 (CH3), 17.8 (CH3), 17.4 (CH2), 16.2 (CH3). IR
(KBr) 3479, 2956, 1736, 1668, 1442, 1037 cm-1. MS (EI, 70 eV)
m/z (%) 389 ([M þ 1]þ, 2), 373 (2), 359 (100), 341 (18). HRMS
(EI) calcd for C21H25O7 (M þ H)þ 389.1601, found 389.1606.
Anal. (C21H24O7) C, H.
1
Compound 11. H NMR (CDCl3, 300 MHz) δ 4.93 (d, J =9.6
Compound 8. 1H NMR (CDCl3, 300 MHz) δ 6.03 (d, J =3.7
Hz, 1 H), 4.95 (d, J =16.0 Hz, 1 H), 4.83 (dd, J =16.0, 2.5 Hz, 1
H), 3.86 (d, J =3.0 Hz, 1 H), 3.56-3.48 (m, 2 H), 2.92 (d, J =4.9
Hz, 1 H), 2.86 (d, J =4.9 Hz, 1 H), 2.51-2.40 (m, 1 H), 2.36-
2.21 (m, 1 H), 1.90 (sept, J =6.8 Hz, 1 H), 1.52-1.34 (m, 2 H),
1.29 (s, 3 H), 0.92 (d, J =6.8 Hz, 3 H), 0.78 (d, J =6.8 Hz, 3 H).
13C NMR (CDCl3, 100 MHz) δ 173.0 (C), 153.1 (C), 140.4 (C),
126.9 (C), 121.8 (CH), 68.9 (CH2), 65.1 (C), 64.4 (C), 61.3 (C),
56.6 (CH), 55.8 (C), 54.2 (CH), 53.7 (CH), 48.4 (CH2), 37.2 (C),
30.3 (CH2), 23.1 (CH), 22.8 (CH3), 20.3 (CH3), 17.2 (CH3), 17.1
Hz, 1 H), 4.68 (s, 2 H), 4.58 (d, J =9.6 Hz, 1 H), 3.84 (d, J =3.0
Hz, 1 H), 3.71 (d, J =5.7 Hz, 1 H), 3.61 (d, J =2.9 Hz, 1 H),
2.78-2.67 (m, 1 H), 2.61 (sept, J =6.9 Hz, 1 H), 2.38-2.26 (m, 1
H), 2.25-2.05 (m, 2 H), 1.91 (t, J =14.1 Hz, 1 H), 1.54 (dd, J =
12.6, 4.5 Hz, 1 H), 1.26-1.14 (m, 1 H), 1.07 (s, 3 H), 0.95 (d, J =
6.9 Hz, 3 H), 0.93 (d, J =6.9 Hz, 3 H). 13C NMR (CDCl3, 100
MHz) δ 173.1 (C), 159.8 (C), 125.5 (C), 91.9 (C), 74.1 (CH2), 69.9
(CH2), 65.1 (C), 64.9 (C), 61.7 (C), 56.5 (CH), 55.6 (CH), 55.5
(CH), 40.4 (CH), 35.8 (C), 30.0 (CH2), 25.7 (CH), 23.3 (CH2),
20.6 (CH3), 18.7 (CH3), 17.1 (CH2), 13.5 (CH3). IR (KBr) 3475,
2972, 2933, 2875, 1745, 1680, 1441, 1219, 1018 cm-1. MS (EI, 70
eV) m/z (%) 436 (Mþ, 2), 407 (1), 393 (6), 241 (100). HRMS (EI)
calcd for C21H24SO8 436.1192, found 436.1199.
(CH2). IR (KBr) 3433, 2979, 2927, 1747, 1657, 1358, 1024 cm-1
.
MS (EI, 70 eV) m/z (%) 370 (Mþ, 13), 355 (22), 341 (91), 327
(95), 115 (100). HRMS (EI) calcd for C21H22O6 370.1416, found
370.1404. Anal. (C21H22O6) C, H.
1
Compound 12. H NMR (CDCl3, 300 MHz) δ 4.80 (d, J =9.6
Hz, 1 H), 4.68 (s, 2 H), 4.58 (d, J =9.6 Hz, 1 H), 3.84 (d, J =3.0
Hz, 1 H), 3.79 (d, J =5.6 Hz, 1 H), 3.53 (d, J =3.0 Hz, 1 H),
2.80-2.68 (m, 1 H), 2.38-2.26 (m, 1 H), 2.24-2.02 (m, 3 H),
1.96 (t, J =14.1 Hz, 1 H), 1.54 (dd, J =12.6, 5.1 Hz, 1 H), 1.28-
1.15 (m, 1 H), 1.10 (s, 3 H), 0.92 (d, J =6.8 Hz, 3 H), 0.90 (d, J =
6.8 Hz, 3 H). 13C NMR (CDCl3, 100 MHz) δ 173.1 (C), 159.9
(C), 125.3 (C), 92.5 (C), 74.1 (CH2), 69.9 (CH2), 66.5 (C), 65.0
(C), 61.0 (C), 58.3 (CH), 55.4 (CH), 54.7 (CH), 40.4 (CH), 35.7
(C), 30.2 (CH2), 25.7 (CH), 23.3 (CH2), 20.5 (CH3), 18.2 (CH3),
17.0 (CH2), 13.4 (CH3). IR (KBr) 3435, 2970, 2933, 2877, 2254,
1755, 1674, 1444, 1346, 1223, 1020 cm-1. MS (EI, 70 eV) m/z (%)
(14S)-14β-Hydroxymethylepitriptolide (10). Under Ar atmo-
sphere, a portion (1.0 mL) of a solution of (isopropoxy-
dimethylsilyl) methyl chloride (0.72 mL, 4.0 mmol) in anhy-
drous THF (7.0 mL) was added to Mg turnings (108 mg,
4.5 mmol). To the stirred mixture was added a few drops of
1,2-dibromoethane at 60 °C and an exothermic reaction started
in several minutes. The remaining solution was added dropwise
over 5 min. After the addition was completed, the gray mixture
was refluxed for 45 min and then cooled to 0 °C. A solution of
triptonide (3) (358 mg, 1.0 mmol) in anhydrous THF (10.0 mL)
was added to the Grignard reagent (freshly prepared) at the

5122 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 16
Li et al.
436 (Mþ, 7), 421 (9), 71 (100). HRMS (EI) calcd for C21H24SO8
436.1192, found 436.1182.
(Tianjin, China). The vincristine-selected MDR subline
KB/VCR33 and doxorubicin-selected MDR subline MCF-7/
ADR34,35 was obtained from Zhongshan University of Medical
Sciences (Guangzhou, China). All these cell lines except MCF-7
and MCF-7/Adr were maintained in RPMI 1640 medium
(Gibco, Grand Island, NE) supplemented with 10% (v/v)
heat-inactivated fetal bovine serum (Gibco), 2 mmol/L gluta-
mine, 100 IU/mL penicillin, 100 μg/mL streptomycin, and 10
mmol/L HEPS (pH 7.4) in a humidified atmosphere of 95% air
with 5% CO2 at 37 °C. MCF-7 and the MCF-7/ADR cells were
grown in the same medium supplemented with 1 mmol/L
sodium pyruvate and 0.01 mg/mL bovine insulin.
Cytotoxicity Assays. The cytotoxicity of compound 1 deriva-
tives and the reference drugs was examined using a panel of
human tumor cell lines with the methods described before.36
Briefly, cells in 96-well plates were treated in triplicate with
gradient concentrations of tested agents at 37 °C for 72 h, then
assessed by the microculture tetrazolium [3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide, MTT] (Sigma, Saint
Louis, MO) assay for leukemia cell lines, or by the sulforhoda-
min B (Sigma) assay for solid tumor cell lines. The cytotoxicity
was expressed as an IC50, defined as the concentration required
for 50% inhibition of cell growth compared with control cells
and calculated with the logit method. Resistance factor was
calculated as the ratio of IC50 in MDR cells over IC50 in the
corresponding parental cells. The reference drugs ADR, VCR,
and the MDR reversal agent VER were all purchased from
Sigma with purity over 95%.
In Vivo Antitumor Activity Assays. BALB/cA nu/nu mice
aged 4-5 weeks were bred in the Shanghai Institute of Materia
Medica (Shanghai, China), and all experiments were done
according to the institutional ethical guidelines on animal care.
Under sterile conditions, well developed tumors were cut into
1 mm3 fragments and transplanted sc into the right flank of nude
mice using a trocar. When the tumor reached a volume of 100-
200 mm3, the mice were randomly assigned into control and
treatment groups. Treatment groups receiving compound 4 (po)
dissolved in vehicles and control groups were given vehicles
alone. Vehicles and compound 4 were administered daily, and
each experimental group contained six animals. The sizes of the
tumors were measured twice a week using microcalipers. The
tumor volume (V) was calculated as (length ꢀ width2)/2. The
individual relative tumor volume (RTV) was calculated as
follows: RTV =Vt/V0, where Vt is the measured volume each
day, and V0 is the volume at the beginning of the treatment. The
therapeutic effect of the compound was expressed as the volume
ratio of treatment to control (T/C):37 T/C (%) =100% ꢀ (mean
RTV of the treated group/mean RTV of the control group).
(14S)-14-Spiro-14r,21-sulfonyldioxytriptolide (13). To a solu-
tion of compound 11 (40 mg, 0.092 mmol) in CH3CN (4.0 mL)
was added NaIO4 (31 mg, 0.14 mmol), RuCl3 3H2O (6 mg,
3
0.028 mmol), and H2O (1.0 mL) in sequence. The mixture was
stirred at room temperature for 15 min. After removal of the
solvent under reduced pressure, the residue was diluted with
ethyl acetate and washed with water and brine and dried over
anhydrous Na2SO4. The crude product was purified by chro-
matography on silica gel using acetate/cyclohexane (1/4) to
provide 13 in 86% yield (36 mg, 0.079 mmol). 1H NMR
(CDCl3, 300 MHz) δ 4.92 (d, J = 10.0 Hz, 1 H), 4.72-4.63
(m, 3 H), 3.87 (d, J =3.0 Hz, 1 H), 3.83 (d, J =5.6 Hz, 1 H), 3.65
(d, J =3.0 Hz, 1 H), 2.79-2.68 (m, 1 H), 2.51 (sept, J =6.8 Hz,
1 H), 2.39-2.07 (m, 3 H), 1.98 (t, J =14.2 Hz, 1 H), 1.53 (dd, J =
12.6, 5.2 Hz, 1 H), 1.29-1.14 (m, 1 H), 1.09 (s, 3 H), 0.99 (d, J =
6.8 Hz, 3 H), 0.97 (d, J =6.8 Hz, 3 H). 13C NMR (CDCl3, 100
MHz) δ 172.9 (C), 159.4 (C), 125.6 (C), 91.3 (C), 73.4 (CH2), 69.9
(CH2), 66.3 (C), 65.2 (C), 61.9 (C), 58.3 (CH), 55.7 (CH), 55.5
(CH), 40.3 (CH), 35.7 (C), 30.2 (CH2), 25.5 (CH), 23.2 (CH2),
20.4 (CH3), 18.3 (CH3), 17.0 (CH2), 13.4 (CH3). IR (KBr) 3442,
2970, 2941, 1749, 1676, 1441, 1394, 1217, 1001 cm-1. MS (EI, 70
eV) m/z (%) 452 (Mþ, 8), 437 (10), 423 (10), 111 (100). HRMS
(EI) calcd for C21H24SO9 452.1141, found 452.1169.
(14S)-14β-(Hydroxymethyl)epitriptolide (14). Compound 10
(0.50 g, 1.3 mmol) was dissolved in dry pyridine (4.0 mL,
49.3 mmol). The solution was cooled to 0 °C, then MsCl
(0.61 mL, 7.7 mmol) was added to the solution dropwise and
stirred for 10 min at room temperature. After removal of the
solvent under reduced pressure, the residue was diluted with
water, then extracted with ethyl acetate, washed with brine, and
dried with anhydrous Na2SO4. After concentration, the residue
was purified by chromatography by ethyl acetate/cyclohexane
(1/2) to provide 14 as colorless oil. Yield: 0.504 g (84%).
1H NMR (CDCl3, 300 MHz) δ 4.70-4.57 (m, 4 H), 3.80 (d,
J =3.2 Hz, 1 H), 3.77 (d, J =5.6 Hz, 1 H), 3.47 (d, J =3.2 Hz,
1 H), 3.10 (s, 3 H), 2.77-2.67 (m, 1 H), 2.54 (sept, J =6.9 Hz,
1 H), 2.37-2.26 (m, 1 H), 2.25-2.06 (m, 1 H), 1.91 (dd, J =14.8,
13.4 Hz, 1 H), 1.54 (dd, J =12.9, 5.1 Hz, 1 H), 1.28-1.13 (m,
2 H), 1.07 (s, 3 H), 0.92 (d, J =7.7 Hz, 3 H), 0.89 (d, J =7.7 Hz,
3 H). 13C NMR (CDCl3, 100 MHz) δ 173.2 (C), 160.1 (C), 125.4
(C), 74.5 (C), 72.0 (CH2), 69.9 (CH2), 67.8 (C), 65.1 (C), 63.3 (C),
55.9 (CH), 55.4 (CH), 54.0 (CH), 40.5 (CH), 37.5 (CH), 36.0 (C),
29.7 (CH2), 25.6 (CH3), 23.5 (CH2), 20.5 (CH3), 18.5 (CH3), 17.1
(CH2), 13.5 (CH3). IR (KBr) 3483, 3024, 2972, 2937, 1749, 1674,
1446, 1354, 1174 cm-1. MS (EI, 70 eV) m/z (%) 468 (Mþ, 1), 450
(3), 432 (1), 407 (1), 354 (17), 111 (100). HRMS (EI) calcd for
C22H28SO9 468.1454, found 468.1457.
Biology. Cell Lines and Cell Culture. Human gastric adeno-
carcinoma SGC-7901, hepatocellular carcinoma BEL-7402,
renal cell carcinoma 786-O, and ovarian epitheloid carcinoma
HO-8910 cell lines were obtained from the cell bank of the
Shanghai Institute of Materia Medica, Chinese Academy of
Sciences. Human chronic myelogenous leukemia K562, lung
adenocarcinoma A549, breast cancer MCF-7, MDA-MB-231,
and MDA-MB-468, colorectal cancer HCT-116 and HCT-15,
oral squamous cell carcinoma KB, prostate cancer DU-145, and
cervical cancer Hela cell lines were purchased from the Amer-
ican Type Culture Collection (Manassas, VA). Human acute
lymphoblastic leukemia Molt-4, gastric adenocarcinoma
MKN-28, prostate cancer PC-3, and ovarian cancer SK-OV-3
cell lines were from Japanese Foundation of Cancer Research
(Tokyo, Japan). Human hepatocellular carcinoma SMMC-
7721 was a gift from the Second Military Medical School
(Shanghai, China). The rhabdomyosarcoma cell line Rh30
was a gift from St. Jude Children’s Research Hospital
(Memphis, TN). The doxorubicin-selected multidrug resi-
stant (MDR) cell subline K562/A0232 was purchased from the
Institute of Hematology, Chinese Academy of Medical Science
Acknowledgment. We thank Li-Juan Lu and Yong Xi for
the animal experiments. This work was supported by a grant
(no. 30721005) from the Natural Science Foundation of
China (NSFC).
Supporting Information Available: Elemental analysis data
for 1, 2, and compounds 4, 5, 7, 8, 10, HPLC results of
compounds 6, 11, 12, 13, and two crystallographic files of
compounds 4 and 11 in CIF format. This material is available
free of charge via the Internet at http://pubs.acs.org.
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