Cytotoxic and Apoptosis-inducing Activities of Taraxastane-type Triterpenoid Derivatives in Human Cancer Cell Lines

Article abstract of DOI:10.1002/cbdv.201500356

Twenty-eight taraxastane-type triterpenoid derivatives 4?–?31 were prepared from the naturally occurring triterpenoids faradiol (1) and heliantriol C (3). The cytotoxic activities of these compounds and arnidiol (2) were evaluated in leukemia (HL60), lung (A549), duodenal (AZ521), and breast (SK-BR-3) cancer cell lines. 21-Oxoarnidiol (18) and faradiol 3,16-di-O-l-alaninate (31) exhibited potent cytotoxicity, with 50% inhibitory concentrations of 0.5?–?2.7?μm. In particular, flow cytometric analysis indicated that compound 31 induced typical apoptotic cell death in HL60 cells. These results suggested that taraxastane-type triterpenoid derivatives might provide useful antitumor agents with apoptosis-inducing activity.

Full text of DOI:10.1002/cbdv.201500356

Received Date: 18-Nov-2015
Accepted Date: 29-Feb-2016
Article Type: Full Paper
Cytotoxic and Apoptosis-inducing Activities of Taraxastane-type
Triterpenoid Derivatives in Human Cancer Cell Lines
by Motohiko Ukiya*a), Chika Ohkuboa), Masahiro Kuritab), Makoto Fukatsua), Takashi
Suzukib), and Toshihiro Akihisaa)c)
a) College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-
ku, Tokyo 101-8308, Japan (phone: +81-3-32590816; fax: +81-3-32937572; e-mail:
ukiya.motohiko@nihon-u.ac.jp)
b) College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi-shi, Chiba 274-
8555, Japan
c) Akihisa Medical Clinic, 1086-3 Kamo, Sanda-shi, Hyogo 669-1311, Japan
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdv.201500356
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Twenty-eight taraxastane-type triterpenoid derivatives 4 – 31 were prepared from the
naturally occurring triterpenoids faradiol (1) and heliantriol C (3). The cytotoxic activities of
these compounds and arnidiol (2) were evaluated in leukemia (HL60), lung (A549), duodenal
(AZ521), and breast (SK-BR-3) cancer cell lines. 21-Oxoarnidiol (18) and faradiol 3,16-di-O-
L-alaninate (31) exhibited potent cytotoxicity, with 50% inhibitory concentrations of 0.5 – 2.7
μM. In particular, flow-cytometric analysis indicated that compound 31 induced typical
apoptotic cell death in HL60 cells. These results suggested that taraxastane-type triterpenoid
derivatives might provide useful antitumor agents with apoptosis-inducing activity.
Keywords: Taraxastane triterpenoid derivative, Cytotoxic activity, Apoptosis-
inducing activity.
Introduction. - Natural products can provide important sources of new anticancer
drugs [1]. In recent decades, many natural products have been isolated from natural resources
and evaluated for their cytotoxic activities against human cancer cell lines. Among these,
triterpenoids provide a unique group of agents with potential antitumor activities [2][3].
We have previously studied the bioactivities of triterpenoids derived from Compositae
flowers and reported their anti-inflammatory [4 – 6], anti-tubercular [7], chemopreventive
[6][8][9], and cytotoxic activities [6][8]. Among the compounds reported in these previous
papers, the taraxastane-type triterpenoids, faradiol (1; taraxast-20-ene-3β,16β-diol), arnidiol
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(2; taraxast-20(30)-ene-3β,16β-diol), and heliantriol C (3; taraxast-20-ene-3β,16β,22α-diol)
exhibited potent antitumor promoting effects [10] and cytotoxic effects in a National Cancer
Institute screen of 60 human cancer cell lines (NCI 60) [8].
The taraxastane skeleton is similar to the ursane skeleton, except that it shows
■inversed configurations?■ at C(18), C(19), and C(20). The ursane skeleton is known to be
present in bioactive natural compounds because ursolic acid, an ursane-type triterpenoid, has
been reported to show a broad range of bioactivities, including anticancer effects [11][12].
On the other hand, the ring D/E junction of taraxastane (the trans-form) differs from
that of ursane (the cis-form), and taraxastane-type triterpenoids were thus expected to show
unique bioactivities. We previously reported that taraxastane-type triterpenoids showed more
potent cytotoxicity than ursane-type triterpenoids in the NCI 60 assay [8]. Although the
structure–activity relationship (SAR) of synthetic taraxastane-type triterpenoid derivatives
has been studied with respect to their anti-inflammatory effects [13], no systematic SAR
studies of the anticancer activities of these compounds have been reported.
We therefore prepared 28 derivatives (compounds 4 – 31) from the naturally
occurring compounds 1 and 3. Here, we describe the preparation of these taraxastane-type
triterpenoid derivatives and evaluation of their cytotoxic activities against four human cancer
cell lines.
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Results and Discussion. - The present study focused on the A, D, and E rings of the
taraxastane derivatives because these were considered to be important for the anti-
inflammatory effects of these compounds [13]. The ring E structure is particularly
characteristic of these taraxastane-type triterpenoids. Furthermore, acylation with an amino
acid was reported to provide a useful means of improving the solubility of betulinic acid
without reducing its cytotoxicity; this is a lupane-type triterpenoid that is a promising
anticancer drug candidate [14]. On the basis of this information, we prepared taraxastane-type
derivatives from 1 and 3 by i) modification of ring E (4 – 28) and ii) acylation of the OH
groups at C(3) and C(16) in rings A and D (29 – 31).
Modification of Ring E. Starting from faradiol (1), twenty-five compounds (4 – 28)
were synthesized as shown in Schemes 1 – 5. Firstly, the double bond in ring E of faradiol (1)
was oxidized to prepare epoxide; this was expected to increase polarity and the ability to act
as a H-bond acceptor, prior to further derivatization. Faradiol (1) was acetylated with Ac2O to
protect the OH groups, giving faradiol 3,16-di-O-acetate (4) in 89% yield. Epoxidation of the
C(20)=C(21) bond of acetate 4 was achieved by treatment with m-chloroperbenzoic acid (m-
CPBA) to give faradiol α-epoxide 3,16-di-O-acetate (5) in 78% yield. The O-acetate 5 was
hydrolyzed by treatment with KOH in MeOH to give the corresponding alcohol, faradiol α-
epoxide (6) in 89% yield (Scheme 1).
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For preparing the ketone and epimeric drivatives of faradiol (1), compound 1 was
oxidized with K2Cr2O7 to afford three compounds that were oxidized at C(3) and/or C(16); 7
– 9 were obtained in 18%, 5%, and 65% yield, respectively. Two C=O groups at C(3) and
C(16) of faradione (9) were reduced with NaBH4 to give 16-epifaradiol (10) in 25% yield. In
addition, faradione (9) was oxidized with m-CPBA to give the epoxide, faradione α-epoxide
(11) in 48% yield (Scheme 2).
In order to introduce a conjugated enone structure as the Michael reaction acceptor in
ring E, which was expected to increase cytotoxicity, compound 5 was treated with HCl in
CHCl3 to yield arnitriol A 3,16-di-O-acetate (12) and heliantriol F 3,16-di-O-acetate (15) in
41% and 28% yield, respectively. A previous study [15] showed that the HCl treatment of the
epoxide at C(20) and C(21) of taraxastane-type triterpenoids produced a 20(30)-en-21-ol
structure. However, we found that the treatment of epoxide 5 with HCl afforded both the
expected compound (12) and an unexpected compound (15), which had a 20-en-30-ol
structure. Compound 12 was acetylated or hydrolyzed to afford the corresponding
peracetylated compound (13) and alcohol (14) in 80% and 77% yield, respectively.
Compound 15 was acetylated or hydrolyzed to afford the corresponding peracetylated
compound (16) and alcohol (17) in 80% and 85% yield, respectively. Selective oxidation of
the OH group at C(21) of arnitriol A (14) with MnO2 afforded 21-oxoarnidiol (18). Arnitriol
3,16-di-O-acetate (12) was oxidized with K2Cr2O7 to afford 30-oxofaradiol 3,16-di-O-acetate
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(19), which has an CHO group at C(20). A similar reaction yielding an α,β-unsaturated
aldehyde from unsaturated alcohol was reported by Gollnick and Schade [16] for the
preparation of 7,7-dimethylbicyclo[4.1.10]hept-3-ene-3-carboaldehyde. Compound 19 was
hydrolyzed to give the corresponding alcohol 20 in 79% yield (Scheme 3).
In order to cleave the epoxy ring at C(20) and C(21) of compound 5, thus increasing
its polarity, the compound was treated with HClO4, followed by acetylation (owing to partial
hydrolysis of the products) to afford the unexpected ketones, 21 and 23 in 39% and 28%
yield, respectively. Their structures were elucidated by spectral analyses, including HR-ESI-
MS, IR, 1H- and 13C-NMR, DEPT, HMQC, HMBC, 1H,1H-COSY, and NOESY spectra.
Briefly, compounds 21 and 23 were determined as C34H54O5 using HR-ESI-MS (m/z
565.3874 and 565.3873 ([M + Na]+), resp.) and 13C-DEPT-NMR. The close similarity
between their 1H-NMR spectra and that of the parent compound 5, with the exception of ring
E, indicated that they had 3β,16β-diacetoxytaraxastane structures. HMBC analysis of 21
identified a correlation between the Me singlet at δ(H) 1.12 (Me(30)) and the ketone C-atom
signal at δ(C) 211.2, indicating that this compound had a ketone group adjacent to C(20)
bearing Me(30). Thus, the structure of 21 was determined to be 3β,16β-diacetoxytaraxastane-
21-one. This conclusion was supported by comprehensive spectral analysis. The structure of
23 was also determined to be the epimer of 21 at C(20) by NOESY experiments. In the
NOESY analysis of 21, a correlation was observed between δ(H) 1.12 (Me(30)) and δ(H)
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0.73 (Me(28)). However, no correlation between δ(H) 0.97 (Me(30)) and δ(H) 1.21
(MeC(28)) was observed in the NOESY analysis of 23. These results indicated that the
MeC(30) of 21 was β-oriented, while that of 23 was α-oriented. Interestingly, alkaline
hydrolysis of 21 afforded a mixture of 22 and 24. The production of 24 was thought to be due
to a base-catalyzed enolization during hydrolysis (Scheme 4).
Heliantriol C (3), which was previously isolated from a chrysanthemum flower extract
[4], was selected to probe the effect of the oxygen function at C(22). Compound 3 was
acetylated to give heliantriol C 3,16,22-tri-O-acetate (25) in 75% yield. To prepare
conjugated enone compound as a Michael reaction acceptor, oxidation of the OH group at the
allylic position C(22) of compound 3 was achieved using MnO2 to afford 22-oxofaradiol (26)
in 30% yield. The C(20)=C(21) bond of compound 25 was oxidized by m-CPBA to afford
heliantriol C α-epoxide 3,16,22-tri-O-acetate (27) in 66% yield, and compound 27 was
hydrolyzed to the corresponding alcohol (28) in 51% yield (Scheme 5).
Acylation of the OH Groups at C(3) and C(16) in Rings A and D. In order to evaluate
the influence of the acyl group, introduced at the OH groups at C(3) and C(16), on
cytotoxicity, faradiol (1) was acylated with succinic anhydride and dimethylsuccinic
anhydride to afford the corresponding compounds, 29 and 30, in yields of 62% and 45%,
respectively. In addition, acylation with tert-butoxycarbonyl (Boc)-protected L-alanine was
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carried out, followed by deprotection of the Boc group using HCl to afford faradiol di-O-L-
alaninate (31) in 38% yield (Scheme 6).
Cytotoxic Activity. The cytotoxic activities of compounds 1 – 31 against leukemia
(HL60), lung (A549), duodenal (AZ521), and breast (SK-BR-3) cancer cell lines were
determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-3H-tetrazolium bromide
(MTT) assay. Cisplatin, which is one of the most effective and widely used chemotherapeutic
drugs employed in the treatment of human cancers, was selected as a positive control. The
growth inhibition in the presence of each compound was expressed as the 50% inhibitory
concentration (IC50) value, and these results are compiled in the Table.
Compounds with IC50 values that were lower than that of cisplatin were considered
active. In particular, 18 and 31 showed potent cytotoxicities, with IC50 values of 0.5 – 2.7 μM
against the cancer cell lines used in this study. On the other hand, 26 showed potent selective
cytotoxicity against the A549 lung cancer cell line (IC50 1.0 μM).
Comparison of compounds 2 and 18 indicated that the presence of a ketone group
beside the C(20)=C(30) bond substantially enhanced cytotoxicity against all four cancer cell
lines. This was the expected result for the conjugated enone structure. In addition, the
presence of a ketone group neighboring the C(20)=C(21) bond considerably enhanced
selective cytotoxicity against the A549 lung cancer cell line, as shown by compounds 1 and
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26. The structural difference between 18 and 26 related to the orientation of the double bonds
that interacted with their ketone groups. Compound 18 has an exocyclic double bond,
whereas 26 has an endocyclic double bond. The selective cytotoxicity of 26 against A549
lung cancer cells may therefore be attributable to the position of the double bond. In addition,
compounds 22 and 24, which have the same structures as hydrogenated structure of
compound 18 C(20)=C(30) bond, were much less cytotoxic than 18, as expected.
Furthermore, conjugated aldehyde 20 was more cytotoxic than the corresponding alcohol 17.
These results indicated that the conjugated enone structure was essential for cytotoxicity.
Faradiol (1), faradiol α-epoxide (6), arnitriol A (14), heliantriol F (17), 30-oxofaradiol
(20), 3β,16β-dihydroxytaraxastan-21-one (22), and its 20-epimer (24) tended to show smaller
IC50 values than their acetyl derivatives against one or more cancer cell lines. These
compounds have taraxastane-type skeletons bearing OH groups at C(3) and C(6). In contrast,
the cytotoxicities of heliantriol C (3) and heliantriol C α-epoxide (28), which have
taraxastane-type skeletons bearing OH groups at C(3), C(16), and C(22), tended to show less
cytotoxicity than their acetyl derivatives, 25 and 27. These results suggested that the OH
groups at C(3) and C(16) and the AcO group at C(22) played important roles, enhancing the
cytotoxicity of these taraxastane-type triterpenoids against cancer cell lines.
The dihydroxy compounds, faradiol (1) and arnidiol (2), showed more potent
cytotoxicities than their corresponding trihydroxy forms, heliantriol C (3) and arnitriol A (14).
In addition, arnidiol (2) exhibited more potent cytotoxicity than faradiol (1). These results
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were consistent with those of a previous study where the dihdyroxytaraxastane-type
triterpenoids, faradiol (1) and arnidiol (2), showed marked cytotoxicities in the NCI 60 screen
[8]. The number of OH groups and the position of the double bond were identified as
important determinants of cytotoxicity against the human cancer cell lines used in the present
study.
Although acetylation of faradiol (1) decreased cytotoxicity, faradiol 3,16-di-O-
succinate (29), 22-dimethylsuccinate (30), and di-O-L-alaninate (31) showed marked
cytotoxicities against human cancer cell lines. Since compounds 29, 30, and 31 have highly
polar moieties at the ends of their acyl groups (COOH and NH2 groups), acylation of
taraxastane-type triterpenoids with a highly polar acyl group substantially enhanced their
cytotoxicity against human cancer cell lines.
Apoptosis-Inducing Activity of Compounds 18 and 31. Compounds 18 and 31
exhibited potent cytotoxic activities against HL60 (IC50 0.3 and 0.5 μM), and these were
evaluated for their effects on early apoptosis in this cell line. HL60 cells were incubated with
these test compounds for 24 and 48 h, and then the cells were analyzed using flow cytometry
with annexin V-propidium iodide (PI) double staining. Exposure of the membrane
phospholipid, phosphatidylserine, to the external cellular environment is one of the earliest
markers of apoptotic cell death [17]. Annexin V is a calcium-dependent phospholipid-binding
protein with a high affinity for cell surface phosphatidylserine. PI does not enter whole cells
with intact membranes and was therefore used to differentiate between early apoptotic
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(annexin V-positive, PI-negative), late apoptotic (annexin V-positive, PI-positive), or necrotic
(annexin V-negative, PI-positive) cell death. As shown in the Figure, the proportion of early
apoptotic cells (lower right) was increased from 1.7% to 5.7% after treatment of HL60 with 5
μM 18 for 24 h and reached 13.6% at 48 h; the proportion of late apoptotic cells (upper right)
was also increased from 1.0% to 5.4% after 24 h and reached 41.0% at 48 h. However, the
proportion of necrotic cells (upper left) had also increased after 24 h (from 0.8% to 9.9%) and
48 h (18.0%). This observation of fewer early apoptotic cells, as compared to the number of
necrotic cells, indicated that this α,β-unsaturated ketone (18) mainly induced HL60 cell death
via necrotic pathways. In contrast, treatment of HL60 with 5 μM 31 increased the proportion
of early apoptotic cells from 3.1% to 19.4% at 24 h and to 39.4% at 48 h; the proportion of
late apoptotic cells was also increased after 24 h (from 0.9% to 19.7%) and 48 h (22.2%),
while that of necrotic cells was 9.5% after 24 h and 6.5% after 48 h. This observation of more
early apoptotic cells than necrotic cells indicated that compound 31, a conjugate of a
taraxastane-type triterpenoid and an amino acid, mainly induced HL60 cell death via the
apoptotic pathway.
Conclusions. – In summary, 31 taraxastane-type triterpenoids, comprising 28
derivatives (4 – 31) prepared from faradiol (1) and heliantriol C (3), along with arnidiol (2),
were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), duodenal
(AZ521), and breast (SK-BR-3) cancer cell lines. 21-Oxoarnidiol (18) and faradiol di-O-L-
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alaninate (31) exhibited potent cytotoxicity against the cancer cell lines used in this study and
31 was shown to induce apoptotic cell death in HL60 cells. These results suggested that the
acylation of the OH groups at C(3) and C(16) of taraxastane-type triterpenoids with amino
acids might provide a useful enhancement of their cytotoxic and pro-apoptotic activities.
Further studies are underway in our laboratory to clarify the SAR of triterpenoid-amino acid
conjugates.
We thank Mr. Akira Takekawa for his technical assistance and Prof. Atsuyoshi Nishina
for his helpful advice. This work was partly supported by JSPS KAKENHI Grant Number
15K08004. We would like to thank Editage (www. editage.jp) for English language editing.
Experimental Part
General
Thin-layer chromatography (TLC): silica gel 60G (Merck & Co. Inc.). Column
chromatography (CC): silica gel 60 (SiO2, 230 – 400 mesh; Merck & Co. Inc., Darmstadt,
Germany). Reversed-phase (RP) prep. HPLC: octadecyl silica column (Pegasil ODSII
column, 25 cm × 10 cm i.d.; Senshu Scientific Co., Ltd., Tokyo, Japan) at 25° with a mobile
phase of MeOH (4 ml/min; HPLC system I) or with MeOH/H2O 95:5 (4 ml/min; HPLC
system II). Normal-phase HPLC: silica column (Silica 4251-N column, 25 cm × 10 mm i.d.;
Senshu Scientific Co., Ltd.) at 25° with a mobile phase of hexane/AcOEt 6:4 (4 ml/min;
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HPLC system III) or hexane/AcOEt 85:15 (2 ml/min; HPLC system IV). IR Spectra:
PerkinElmer Spectrum One FT-IR spectrophotometer in KBr disks; ෤ in cm–1. 1H-NMR
Spectra: JEOL ECX-400 spectrometer (at 400 (1H) and 100 MHz (13C)) at r.t.; CDCl3 or
CDCl3/CD3OD soln.; δ in ppm rel. to Me4Si as the internal standard, J in Hz. HR-ESI-MS:
Agilent 1100 LC/MSD TOF (time-of-flight) system (ionization mode: positive or negative;
cap. voltage: 3000 V; fragmentor voltage: 225 V). HR-APCI-MS: Agilent 1100 LC/MSD TOF
system (ionization mode: positive; cap. voltage: 3000 V; fragmentor voltage: 40 V; corona
current: 2.0 μA). Microplate reader: Sunrise-Basic (Tecan Japan Co., Ltd., Kawasaki, Japan).
Chemicals and Materials. Compounds were purchased from the following sources:
dimethylsuccinic anhydride, MTT, and DMSO from Sigma-Aldrich Japan Co. (Tokyo,
Japan); 4-(dimethylamino)pyridine (DMAP), NaBH4, succinic anhydride, and m-CPBA from
Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); water-soluble carbodiimide (WSCD) from
Peptide Institute Inc. (Osaka, Japan); RPMI-1640 medium, fetal bovine serum (FBS), and
penicillin-streptomycin from Invitrogen Co. (Auckland, NZ); rh annexin V/fluorescein
isothiocyanate (FITC) kit (Bender MedSystems®) from Cosmo Bio Co., Ltd. (Tokyo, Japan);
1-hydroxybenzotriazole monohydrate (HOBt) from Dojindo Molecular Technologies Inc.
(Kumamoto, Japan); and K2Cr2O7, MnO2, and cisplatin from Wako Pure Chemical Industries
Ltd. (Osaka, Japan). All other chemicals and reagents were of anal. grade. Faradiol (1),
arnidiol (2), and heliantriol C (3) were isolated from the compositae flower, as described
previously [4].
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Faradiol 3,16-Di-O-acetate (= (3β,16β,18α,19α)-Urs-20-ene-3,16-diyl Diacetate;
4). To a soln. of 1 (700 mg, 1.6 mmol) in pyridine (12 ml), Ac2O (12 ml, 128.2 mmol) was
added and stirred at r.t. for 16 h. The mixture was then added to H2O and extracted with Et2O
(3 × 100 ml). The Et2O fraction was washed with H2O, dried (Na2SO4), and evaporated under
reduced pressure to yield 4 (686 mg, 82%) which was identified by spectral comparison with
an authentic sample [4].
Faradiol α-Epoxide 3,16-Di-O-acetate (= (3β,16β,18α,19α,20β,21α)-20,21-
Epoxyursane-3,16-diyl Diacetate; 5). To a soln. of 4 (670 mg, 1.3 mmol) in CH2Cl2 (75 ml),
NaHCO3 (104 mg) and m-CPBA (219 mg, 1.3 mmol) were added and stirred at r.t. for 20 h.
After this period, the mixture was washed with 1M aq. NaOH and H2O, dried (Na2SO4), and
evaporated under reduced pressure to yield crude products (829 mg). The crude products
were purified with Si-gel TLC (hexane/AcOEt 4:1) to give 5 (540 mg, 78%) which was
identified by spectral comparison with an authentic sample [4].
Faradiol α-Epoxide (= (3β,16β,18α,19α,20β,21α)-20,21-Epoxyursane-3,16-diol;
6). Compound 5 (20 mg, 0.04 mmol) was dissolved in 1M KOH/MeOH (5 ml) and allowed to
stand at r.t. overnight. After this period, the mixture was poured into H2O and extracted with
Et2O (2 × 30 ml), dried (Na2SO4), and evaporated under reduced pressure to yield 6 (15 mg,
89%) which was identified by spectral comparison with an authentic sample [4].
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3β-Hydroxytaraxast-20-en-16-one (= (3β,18α,19α)-3-Hydroxyurs-20-en-16-one;
7), 16β-Hydroxytaraxast-20-en-3-one (= (16β,18α,19α)-16-Hydroxyurs-20-en-3-one; 8),
and Faradione (= (18α,19α)-Urs-20-ene-3,16-dione; 9). A soln. of K2Cr2O7 (234 mg, 0.8
mmol) and conc. H2SO4 (3.25 ml) in H2O (16.3 ml) was added to 1 (500 mg, 1.1 mmol)
dissolved in Et2O (65 ml). The mixture was stirred at r.t. for 7 h, then the Et2O fraction was
washed with sat. NaHCO3 aq. and H2O, dried (Na2SO4), and evaporated under reduced
pressure to yield crude products (536.0 mg). These were purified with Si-gel TLC
(hexane/AcOEt 4:1) to give 9 (322 mg, 65%) and a mixture of 7 and 8, which was separated
by HPLC (system I) to yield 7 (tR 4.2 min; 89 mg, 18%) and 8 (tR 2.7 min; 25 mg, 5%). These
compounds were identified by spectral comparison with published data [18].
16-Epifaradiol (= (3β,16α,18α,19α)-Urs-20-ene-3,16-diol; 10). To a soln. of 9 (84
mg, 0.2 mmol) in benzene (1.5 ml) and MeOH (3 ml), NaBH4 (132 mg, 3.5 mmol) was added,
and the mixture was stirred at r.t. for 5 h. After this period, 1M HCl (5 ml) was added to the
mixture and stirred for a few min prior to extraction with Et2O (2 × 20 ml). The Et2O fraction
was washed with H2O, dried (Na2SO4), and concentrated under reduced pressure to furnish
10 (21 mg, 25%), which was identified by spectral comparison with published data [18].
Faradione α-Epoxide (= (18α,19α,20β,21α)-20,21-Epoxyursane-3,16-dione; 11).
To a soln. of 9 (30 mg, 0.07 mmol) in CH2Cl2 (15 ml), NaHCO3 (12 mg) and m-CPBA (24
mg, 0.14 mmol) were added and stirred at r.t. for 3 days. After this period, the mixture was
washed with 1M aq. NaOH and H2O, dried (Na2SO4), and evaporated under reduced pressure
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to yield crude products (39.1 mg). These were purified with Si-gel TLC (hexane/AcOEt 4:1)
to give 11 (15 mg, 48%).
Amorphous solid. IR (KBr): 752, 1118, 1384, 1459, 1701, 2952. 1H-NMR (400
MHz): 0.83 (s, 3 H); 0.95 (s, 3 H); 1.03 (s, 3 H); 1.08 (s, 3 H); 1.09 (s, 3 H); 1.12 (s, 3 H);
1.18 (d, J = 6.4, 3 H); 1.36 (s, 3 H); 3.09 (d, J = 6.0, 1 H). HR-APCIMS (pos.): 455.3504 ([M
+
+ H]+, C30H47O3 ; calc. 455.3519).
Arnitriol A 3,16-Di-O-acetate (= (3β,16β,18α,19α,21α)-21-Hydroxyurs-20(30)-
ene-3,16-diyl Diacetate; 12) and Heliantriol F 3,16-Di-O-acetate (= (3β,16β,18α,19α)-30-
Hydroxyurs-20-ene-3,16-diyl Diacetate; 15). To a soln. of 5 (203 mg, 0.4 mmol) in CHCl3
(120 ml), conc. HCl (0.2 ml) was added and stirred at r.t. for 1 day. After this period, the
CHCl3 fraction was washed with sat. aq. NaHSO4, dried (Na2SO4), and evaporated under
reduced pressure to yield a crude product (209 mg). This product was purified with Si-gel
TLC (hexane/AcOEt 4:1) to furnish 12 (84 mg, 41%) and 15 (56 mg, 28%). Compound 15
was identified by spectral comparison with published values [19].
Data for 12. Amorphous solid. IR (KBr): 754, 1026, 1247, 1371, 1735, 2945, 3436.
1H-NMR (400 MHz): 0.83 (s, 6 H); 0.85 (s, 3 H); 0.87 (s, 3 H); 1.02 (s, 3 H); 1.07 (s, 3 H);
1.23 (d J = 7.1, 3 H); 2.01 (s, 3 H); 2.04 (s, 3 H); 4.38 (dd, J = 5.4, 9.0, 1 H); 4.48 (dd, J = 5.1,
10.7, 1 H); 4.72 (dd, J = 4.6, 11.7, 1 H); 4.92 (br. s, 1 H); 5.00 (br. s, 1 H). HR-ESI-MS
+
(pos.): 565.3847 ([M + Na]+, C34H54NaO5 ; calc. 565.3868).
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Arnitriol A 3,16,21-Tri-O-acetate (= (3β,16β,18α,19α,21α)-Urs-20(30)-ene-
3,16,21-triyl Triacetate; 13). To a soln. of 12 (15 mg, 0.03 mmol) in pyridine (2 ml), Ac2O
(2 ml, 21.2 mmol) was added and stirred at r.t. for 16 h. After this period, the mixture was
added to H2O and extracted with Et2O (2 × 30 ml). The Et2O fraction was washed with H2O,
dried (Na2SO4), and evaporated under reduced pressure to yield 13 (13 mg, 80%).
Amorphous solid. IR (KBr): 755, 979, 1023, 1247, 1370, 1735, 2944. 1H-NMR (400
MHz): 0.84 (s, 3 H); 0.85 (s, 3 H); 0.87 (s, 3 H); 0.88 (s, 3 H); 1.02 (s, 3 H); 1.07 (s, 3 H);
1.16 (d, J = 6.9, 3 H); 2.00 (s, 3 H); 2.01 (s, 3 H); 2.04 (s, 3 H); 4.48 (dd, J = 4.8, 11.4, 1 H);
4.70 (dd, J = 4.5, 11.7, 1 H); 4.99 (br. s, 1 H); 5.10 (br. s, 1 H); 5.45 (dd, J = 4.8, 9.6, 1 H).
+
HR-ESI-MS (pos.): 607.3966 ([M + Na]+, C36H56NaO6 ; calc. 607.3969).
Arnitriol A (= (3β,16β,18α,19α,21α)-Urs-20(30)-ene-3,16,21-triol; 14). Compound
12 (26 mg, 0.05 mmol) was dissolved in 1M KOH/MeOH (5 ml) and allowed to stand at r.t.
overnight. After this period, the mixture was poured into H2O, extracted with Et2O (2 × 30
ml), dried (Na2SO4), and evaporated under reduced pressure to yield 14 (17 mg, 77%).
Amorphous solid. IR (KBr): 915, 986, 1031, 1136, 1389, 1418, 1446, 2937, 3399. 1H-NMR
(CDCl3/CD3OD 8:2, 400 MHz): 0.75 (s, 3 H); 0.76 (s, 3 H); 0.85 (s, 3 H); 0.96 (s, 3 H); 1.01
(s, 3 H); 1.03 (s, 3 H); 1.21 (d, J = 6.8, 3 H); 3.18 (dd, J = 7.0, 9.0, 1 H); 3.39 (dd, J = 4.1,
11.7, 1 H); 4.37 (dd, J = 5.4, 9.0, 1 H); 4.92 (br. s, 1 H); 5.00 (br. s, 1 H). HR-APCIMS
+
(pos.): 441.3709 ([M – H2O + H]+, C30H49O2 ; calc. 441.3732).
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Heliantriol F 3,16,30-Tri-O-acetate (= (3β,16β,18α,19α)-Urs-20-ene-3,16,30-triyl
Triacetate; 16). To a soln. of 15 (7 mg, 0.01 mmol) in pyridine (2 ml), Ac2O (2 ml, 21.2
mmol) was added and stirred at r.t. for 16 h. After this period, the mixture was added to H2O
and extracted with Et2O (2 × 30 ml). The Et2O fraction was washed with H2O, dried
(Na2SO4), and evaporated under reduced pressure to yield 16 (6 mg, 80%), which was
identified by spectral comparison with published data [19].
Heliantriol F (= (3β,16β,18α,19α)-Urs-20-ene-3,16,30-triol; 17). Compound 15 (7
mg, 0.01 mmol) was dissolved in 1M KOH/MeOH (5 ml) and allowed to stand at r.t.
overnight. After this period, the mixture was poured into H2O, extracted with Et2O (2 × 30
ml), dried (Na2SO4), and evaporated under reduced pressure to yield 17 (5 mg, 85%), which
was identified by spectral comparison with published data [13].
21-Oxoarnidiol (= (3β,16β,18α,19α)-3,16-Dihydroxyurs-20(30)-en-21-one; 18). To
a soln. of 14 (7 mg, 0.02 mmol) in dried CHCl3 (3 ml), MnO2 (98 mg, 1.1 mmol) was added
and stirred at r.t. for 1 h. After this period, the mixture was filtered and evaporated under
reduced pressure to yield a crude product (6 mg). This product was purified with Si-gel TLC
(hexane/AcOEt 1:1) to furnish 18 (2 mg, 29%), which was identified by spectral comparison
with published data [13].
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30-Oxofaradiol 3,16-Di-O-acetate (= (3β,16β,18α,19α)-30-Oxours-20-ene-3,16-
diyl Diacetate; 19). To a soln. of 12 (40 mg, 0.07 mmol) in Et2O (14 ml), K2Cr2O7 (22 mg,
0.07 mmol) in 3M H2SO4 (1.5 ml) was added and stirred at r.t. for 7 h. After this period, the
Et2O fraction was washed with H2O, dried (Na2SO4), and evaporated under reduced pressure
to yield a crude product (35 mg). This product was purified by HPLC (system III) to furnish
19 (tR 7.8 min; 14 mg, 35%), which was identified by spectral comparison with published
data [18].
30-Oxofaradiol (= (3β,16β,18α,19α)-3,16-Dihydroxyurs-20-en-30-al; 20).
Compound 19 (9 mg, 0.02 mmol) was dissolved in 1M KOH/MeOH (5 ml) and allowed to
stand at r.t. overnight. After this period, the mixture was poured into H2O and extracted with
Et2O (2 × 30 ml), dried (Na2SO4), and evaporated under reduced pressure to yield 20 (6 mg,
79%), which was identified by spectral comparison with published data [13].
3β,16β-Diacetoxytaraxastan-21-one (= (3β,16β,18α,19α,20β)-21-Oxoursane-3,16-
diyl Diacetate; 21) and 3β,16β-Diacetoxy-20-epitaraxastan-21-one (=
(3β,16β,18α,19α,20α)-21-Oxoursane-3,16-diyl Diacetate; 23). To a soln. of 5 (300 mg,
0.55 mmol) in THF (48 ml), 3M aq. HClO4 (24 ml) was added and stirred at r.t. for 3 h. After
this period, the mixture was poured into 4M aq. NaOH (24 ml) and extracted with CHCl3 (2 ×
30 ml). The CHCl3 fraction was washed with H2O, dried (Na2SO4), and evaporated under
reduced pressure to yield a crude product. This product was partly hydrolyzed under acidic
conditions and was therefore acetylated again to produce a mixture of 21 and 23 (290 mg).
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This mixture was separated by HPLC (system IV) to furnish 21 (tR 38.6 min; 118 mg, 39%)
and 23 (tR 27.0 min; 85 mg, 28%).
Data of 21. Amorphous solid. IR (KBr): 755, 979, 1022, 1243, 1372, 1391, 1458,
1715, 1732, 2945. 1H-NMR (400 MHz): 0.73 (s, 3 H); 0.84 (s, 3 H); 0.86 (s, 3 H); 0.88 (s, 3
H); 1.05 (s, 3 H); 1.10 (s, 3 H); 1.12 (d, J = 6.5, 3 H); 1.25 (d, J = 5.8, 3 H); 1.86 (m, 1 H);
2.02 (s, 3 H); 2.04 (s, 3 H); 2.15 (d, J = 12.4, 1 H); 2.22 (d, J = 12.4, 1 H); 4.48 (dd, J = 4.8,
11.3, 1 H); 4.85 (dd, J = 4.8, 11.3, 1 H). 13C-NMR (100 MHz): 13.4 (C(28)); 14.3 (C(30));
16.0 (C(26)); 16.1 (C(27)); 16.3 (C(25)); 16.5 (C(23)); 18.1 (C(6)); 21.2 (COMe); 21.3
(COMe); 21.4 (C(11)); 23.6 (C(2)); 26.3 (C(29)); 27.9 (C(24)); 28.2 (C(12)); 32.5 (C(15));
34.3 (C(7)); 36.9 (C(10)); 37.8 (C(4)); 38.4 (C(1)); 39.1 (C(13)); 41.0 (C(19)); 41.3 (C(8));
42.6 (C(14)); 43.4 (C(17)); 49.2 (C(18)); 49.4 (C(9)); 51.1 (C(22)); 52.4 (C(20)); 55.3 (C(5));
77.0 (C(16)); 80.8 (C(3)); 170.6 (COMe); 170.9 (COMe); 211.2 (C(21)). HR-ESI-MS (pos.):
+
565.3874 ([M + Na]+, C34H54NaO5 ; calc. 565.3868).
Data of 23. Amorphous solid. IR (KBr): 756, 979, 1022, 1242, 1370, 1453, 1734,
2943, 2974, 3436. 1H-NMR (400 MHz): 0.75 (d, J = 6.8, 3 H); 0.85 (s, 3 H); 0.86 (s, 3 H);
0.89 (s, 3 H); 0.97 (d, J = 6.5, 3 H); 1.05 (s, 3 H); 1.08 (s, 3 H); 1.21 (s, 3 H); 2.00 (s, 3 H);
2.04 (s, 3 H); 4.48 (dd, J = 4.9, 11.7, 1 H); 4.70 (dd, J = 4.4, 11.3, 1 H). 13C-NMR (100
MHz): 11.1 (C(30)); 16.0 (C(26)); 16.2 (C(27)); 16.3 (C(25)); 16.5 (C(23)); 16.7 (C(28));
18.1 (C(6)); 19.7 (C(29)); 21.2 (COMe); 21.3 (COMe); 21.4 (C(11)); 23.7 (C(2)); 25.6
(C(12)); 27.9 (C(24)); 32.8 (C(15)); 34.0 (C(7)); 34.4 (C(19)); 37.0 (C(10)); 37.8 (C(4)); 38.5
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(C(1)); 38.6 (C(13)); 39.2 (C(17)); 41.l (C(8)); 42.6 (C(14)); 44.8 (C(20)); 48.5 (C(18)); 50.0
(C(9)); 51.7 (C(22)); 55.4 (C(5)); 79.4 (C(16)); 80.8 (C(3)); 170.4 (COMe); 170.9 (COMe);
+
213.6 (C(21)). HR-ESI-MS (pos.): 565.3873 ([M + Na]+, C34H54NaO5 ; calc. 565.3868).
3β,16β-Dihydroxytaraxastan-21-one (= (3β,16β,18α,19α,20β)-3,16-
Dihydroxyursan-21-one; 22) and 3β,16β-Dihydroxy-20-epitaraxastan-21-one (=
(3β,16β,18α,19α,20α)-3,16-Dihydroxyursan-21-one; 24). Compound 21 (42 mg, 0.08
mmol) was dissolved in 1M KOH/MeOH (10 ml) and allowed to stand at r.t. overnight. After
this period, the mixture was poured into H2O and extracted with Et2O (2 × 30 ml), dried
(Na2SO4), and evaporated under reduced pressure to furnish a mixture of 22 and 24 (36 mg).
This mixture was separated by HPLC (system III) to furnish 22 (tR 11.7 min; 9 mg, 25%) and
24 (tR 12.8 min; 12 mg, 34%).
Data of 22. Amorphous solid. IR (KBr): 754, 1388, 1455, 1705, 2874, 2935, 3418.
1H-NMR (400 MHz): 0.59 (s, 3 H); 0.73 (s, 3 H); 0.81 (s, 3 H); 0.93 (s, 3 H); 1.01 (s, 6 H);
1.08 (d, J = 6.8, 3 H); 1.21 (d, J = 5.5, 3 H); 1.83 (quint., J = 6.9, 1 H); 2.08 (d, J = 11.9, 1
H); 2.52 (d, J = 11.9, 1 H); 3.16 (dd, J = 5.5, 10.5, 1 H); 3.49 (dd, J = 5.0, 11.5, 1 H). HR-
+
ESI-MS (pos.): 459.3814 ([M + H]+, C30H51O3 ; calc. 459.3838).
Data of 24. Amorphous solid. IR (KBr): 755, 1036, 1387, 1459, 1704, 2873, 2946,
3400. 1H-NMR (400 MHz): 0.68 (d, J = 6.9, 3 H); 0.73 (s, 3 H); 0.82 (s, 3H); 0.92 (d, J = 7.3,
3 H); 0.93 (s, 3 H); 0.96 (s, 3 H); 1.01 (s, 3 H); 1.10 (s, 3 H); 1.87 (d, J = 19.2, 1 H); 2.59 (d,
J = 19.2, 1 H); 3.02 (quint., J = 6.9, 1 H); 3.15 (dd, J = 5.7, 10.8, 1 H); 3.36 (dd, J = 4.6, 11.5,
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+
1 H). HR-ESI-MS (pos.): 459.3811 ([M + H]+, C30H51O3 ; calc. 459.3838).
Heliantriol C 3,16,22-Tri-O-acetate (= (3β,16β,18α,19α,22α)-Urs-20-ene-3,16,22-
triyl Triacetate; 25). To a soln. of 3 (230 mg, 0.5 mmol) in pyridine (10 ml), Ac2O (10 ml,
105 mmol) was added and stirred at r.t. for 16 h. After this period, the mixture was added to
H2O and extracted with Et2O (2 × 100 ml). The Et2O fraction was washed with H2O, dried
(Na2SO4), and evaporated under reduced pressure to yield 25 (220 mg, 75%), which was
identified by spectral comparison with published data [4].
22-Oxofaradiol (= (3β,16β,18α,19α)-3,16-Dihydroxyurs-20-en-22-one; 26). To a
soln. of 3 (10 mg, 0.02 mmol) in CHCl3 (1 ml), MnO2 (95 mg, 1.1 mmol) was added and
stirred at r.t. for 12 h. After this period, the mixture was filtered and evaporated under reduced
pressure to give a crude product (10 mg). This product was purified with Si-gel TLC
(hexane/AcOEt 1:1) to give 26 (3 mg, 30%), which was identified by spectral comparison
with published data [19].
Heliantriol C Epoxide 3,16,22-Tri-O-acetate (= (3β,16β,18α,19α,20β,21α,22α)-
20,21-Epoxyursane-3,16,22-triyl Triacetate; 27). To a soln. of 25 (31 mg, 0.05 mmol) in
CH2Cl2 (10 ml), NaHCO3 (5 mg) and m-CPBA (168 mg, 1.0 mmol) were added and stirred at
r.t. for 19 h. After this period, the mixture was washed with 1M aq. NaOH and H2O, dried
(Na2SO4), and evaporated under reduced pressure to yield crude products (76 mg). These
crude products were purified with Si-gel TLC (hexane/AcOEt 4:1) to give 27 (21 mg, 66%).
Amorphous solid. IR (KBr): 978, 1023, 1240, 1375, 1449, 1739, 2947. 1H-NMR (400
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MHz): 0.84 (s, 3 H); 0.85 (s, 3 H); 0.87 (s, 3 H); 0.88 (s, 3 H); 1.01 (s, 3 H); 1.03 (s, 3 H);
1.13 (d, J = 6.0, 3 H); 1.33 (s, 3 H); 1.98 (s, 3 H); 2.05 (s, 3 H); 2.08 (s, 3 H); 3.29 (d, J = 6.0,
1 H); 4.48 (dd, J = 5.0, 11.0, 1 H); 4.67 (d, J = 6.0, 1 H); 5.05 (dd, J = 5.0, 11.9, 1 H). HR-
+
ESI-MS (pos.): 623.3849 ([M + Na]+, C36H56NaO7 ; calc. 623.3918).
Heliantriol C Epoxide (= (3β,16β,18α,19α,20β,21α,22α)-20,21-Epoxyursane-
3,16,22-triol; 28). Compound 27 (10 mg, 0.02 mmol) was dissolved in 1M KOH/MeOH (5
ml) and allowed to stand at r.t. overnight. After this period, the mixture was poured into H2O
and extracted with Et2O (2 × 30 ml), dried (Na2SO4), and evaporated under reduced pressure
to give a crude product (7 mg). This product was purified with Si-gel TLC (hexane/AcOEt
1:1) to yield 28 (4 mg, 51%).
Amorphous solid. IR (KBr): 806, 1040, 1263, 1379, 1456, 1715, 2929, 3448. 1H-
NMR (400 MHz): 0.69 (s, 3 H); 0.76 (s, 3 H); 0.85 (s, 3 H); 0.94 (s, 3 H); 1.03 (s, 3 H); 1.11
(s, 3 H); 1.12 (d, J = 6.0, 3 H); 1.38 (s, 3 H); 3.20 (dd, J = 5.0, 11.4, 1 H); 3.36 (d, J = 6.2, 1
H); 3.99 (dd, J = 4.5, 11.4, 1 H); 4.00 (d, J = 6.2, 1 H). HR-ESI-MS (pos.): 497.3637 ([M +
Na]+, C30H50NaO4 ; calc. 497.3601).
+
Faradiol 3,16-Di-O-succinate (= 4,4'-[(3β,16β,18α,19α)-Urs-20-ene-3,16-
diylbis(oxy)]bis(4-oxobutanoic Acid); 29). To a soln. of 1 (20 mg, 0.05 mmol) in pyridine (2
ml), succinic anhydride (21 mg, 0.2 mmol) and DMAP (10 mg, 0.08 mmol) were added and
refluxed for 3 h. After this period, the mixture was poured into H2O and extracted with Et2O
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(2 × 30 ml). The Et2O fraction was washed with 1M aq. HCl and H2O, dried (Na2SO4), and
evaporated under reduced pressure to yield a crude product (20 mg). This product was
purified with Si-gel TLC (hexane/AcOEt 7:3) to furnish 29 (18 mg, 62%). Amorphous solid.
IR (KBr): 757, 967, 1168, 1228, 1386, 1714, 1730, 2874, 2943. 1H-NMR (400 MHz): 0.79 (s,
3 H); 0.83 (s, 3 H); 0.84 (s, 3 H); 0.87 (s, 3 H); 1.00 (d, J = 6.3, 3 H); 1.04 (s, 3 H); 1.05 (s, 3
H); 1.64 (br. s, 3 H); 4.51 (dd, J = 6.1, 10.5, 1 H); 4.76 (dd, J = 4.9, 11.7, 1 H); 5.24 (d, J =
+
6.8, 1 H). HR-ESI-MS (pos.): 665.4029 ([M + Na]+, C38H58NaO8 ; calc. 665.4029).
Faradiol 3,16-Di-O-dimethylsuccinate (= 4,4'-[(3β,16β,18α,19α)-Urs-20-ene-3,16-
diylbis(oxy)]bis(2,2-dimethyl-4-oxobutanoic Acid); 30). To a soln. of 1 (20 mg, 0.05 mmol)
in pyridine (2 ml), dimethylsuccinic anhydride (50 mg, 0.4 mmol) and DMAP (10 mg, 0.08
mmol) were added and refluxed for 12 h. After this period, the mixture was poured into H2O
and extracted with Et2O (2 × 30 ml). The Et2O fraction was washed with 1M aq. HCl and H2O,
dried (Na2SO4), and evaporated under reduced pressure to yield a crude product (20 mg).
This product was purified by HPLC (system II) to furnish 30 (tR 8.0 min; 14.3 mg, 45%).
Amorphous solid. IR (KBr): 757, 968, 991, 1136, 1202, 1311, 1365, 1388, 1448, 1475, 1705,
1730, 2875, 2945, 2974. 1H-NMR (400 MHz): 0.77 (s, 3 H); 0.81 (s, 3 H); 0.84 (s, 3 H); 0.87
(s, 3 H); 1.00 (d, J = 6.0, 3 H); 1.04 (s, 3 H); 1.05 (s, 3 H); 1.28 (s, 3 H); 1.29 (s, 6 H); 1.30 (s,
3 H); 1.63 (br. s, 3 H); 4.51 (dd, J = 10.5, 5.5, 1 H); 4.75 (dd, J = 11.7, 4.3, 1 H); 5.25 (d, J =
+
6.4, 1 H). HR-ESI-MS (pos.): 721.4620 ([M + Na]+, C42H66NaO8 ; calc. 721.4650).
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Faradiol 3,16-Di-O-alaninate (= (3β,16β,18α,19α)-Urs-20-ene-3,16-diyl Bis(2-
aminopropanoate); 31). To a soln. of 1 (20 mg, 0.05 mmol), Boc-protected L-alanine (63 mg,
0.33 mmol), 1-hydroxybenzotriazole monohydrate (56 mg, 0.37 mmol) in DMF (420 μl), and
WSCD (140 μl, 0.8 mmol) were added at –20°, and the mixture was allowed to stand for 48 h.
at r.t. The mixture was diluted with H2O and extracted with AcOEt (2 × 5 ml). The AcOEt
fraction was washed with H2O, 1M HCl, and sat. aq. NaHCO3, dried (Na2SO4), and
concentrated under reduced pressure to afford a Boc-protected 31. Removal of the Boc group
was achieved by treating with 3M HCl in 1,4-dioxane (200 μl) for 30 min at r.t. After
deprotection, the mixture was suspended in Et2O (800 μl) and filtered to remove unreacted
Boc-protected 31. The precipitate was added to CHCl3 (300 μl) with Et3N (20 μl), washed
with H2O, and evaporated under reduced pressure to give 31 (10 mg, 38%) without further
purification. Amorphous solid. IR (KBr): 1052, 1455, 1628, 1733, 2347, 2943, 3448. 1H-
NMR (400 MHz): 0.82 (s, 3 H); 0.86 (s, 6 H); 0.89 (s, 3 H); 1.02 (d, J = 6.4, 3 H); 1.06 (s, 3
H); 1.08 (s, 3 H); 1.35 (d, J = 6.8, 1 H); 1.36 (d, J = 6.9, 1 H); 1.65 (br. s, 3 H); 3.53 (m, 2 H);
4.52 (m, 1 H); 4.77 (m, 1 H); 5.25 (d, J = 6.9, 1 H). HR-ESI-MS (pos.): 585.4623 ([M + H]+,
+
C36H61N2O4 ; calc. 585.4625).
Cytotoxicity Assay. Cytotoxicity assays were performed as described previously [20].
Briefly, 3 × 103 cells/well of HL60 (leukemia), A549 (lung), AZ521 (duodenum), and SK-
BR-3 (breast) cell lines were treated with the indicated compounds for 48 h prior to adding
MTT solution to each well. After incubation for 3 h, the generated blue formazan was
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solubilized using 0.04M HCl in i-PrOH. The absorbances at 570 nm (top) and 630 nm
(bottom) were measured by a microplate reader.
Annexin V-PI Double Staining. Apoptosis was detected in HL60 cells (1.5 × 105)
exposed to test compound (final concentration: 5 μM) using an rh Annexin V/FITC kit [20].
Cells were washed with annexin binding buffer, stained for 10 min, and analyzed in a flow
cytometer (Cell Lab Quanta SC; Beckman Coulter K.K., Tokyo, Japan) using the FL1 and
FL2 ranges for annexin V-FITC and PI, resp.
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[18] J. S. Pyrek, Rocz. Chem. 1977, 51, 2331.
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Received November 18, 2015
Accepted February 29, 2016
Table. Cytotoxic activities (IC50 S.D. [mM]) of taraxastane-type triterpenoid derivatives 1 –
31 against four human cancer cell linesa).
Compound
HL60
A549
(lung)
> 100
AZ521
SK-BR-3
(breast)
(leukemia)
11.7 8.7
10.1 1.0
> 100
(duodenum)
52.4 4.8
13.8 2.6
> 100
83.2 4.9
25.8 0.2
> 100
1
2
15.5 2.2
> 100
> 100
> 100
> 100
3
> 100
> 100
> 100
> 100
4
39.6 2.9
57.9 6.6
81.9 0.9
31.9 1.5
95.4 3.3
35.8 0.4
> 100
> 100
5
7.8 0.4
75.0 3.8
59.9 5.5
68.9 5.4
38.7 5.8
> 100
64.3 5.8
6
19.6 3.6
53.7 12.5
> 100
45.9 0.1
> 100
12.7 0.06 > 100
90.4 2.9
34.3 4.4
7
8
> 100
18.6 0.9
> 100
9
10
11
12
13
14
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
64.6 0.6
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> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
15
16
65.1 7.9
0.3 0.0
> 100
6.4 0.7
> 100
18.1 8.1
> 100
42.2 5.0
31.5 3.1
17
1.0 0.1
32.0 1.9
14.6 3.4
0.7 0.0
16.1 2.7
13.2 1.0
45.0 5.4
0.8 0.1
29.0 3.5
> 100
> 100
38.4 8.8
> 100
74.8 1.8
> 100
45.2 8.3
18
19
20
> 100
21
> 100
> 100
> 100
> 100
> 100
57.2 4.4
> 100
22
23
83.1 4.3
15.9 3.6
63.9 9.2
24
44.3 4.1
46.3 3.8
25
1.0 0.2
56.7 5.3
> 100
26
> 100
> 100
> 100
63.2 4.9
> 100
> 100
27
31.4 7.9
35.4 2.2
2.1 0.5
0.5 0.05
4.2 1.1
28
46.7 4.1
6.6 1.3
2.7 0.1
18.4 1.9
35.2 3.1
29
79.0 6.7
1.4 0.1
9.5 0.5
30.9 0.1
0.7 0.3
9.7 0.6
30
31
Cisplatinb)
a) Cells were treated with compounds (1 × 10–4 – 1 × 10–6 M) for 48 h, and cell
viability was analyzed by the MTT assay. IC50 Values based on triplicate five point.
b) Reference compound.
Caption:
Figure. Detection of early and late apoptosis. HL60 cells were cultured with 5 μM 18 or 31
for 24 or 48 h prior to annexin V-fluorescein isothiocyanate/propidium iodide (PI) double
staining and flow cytometry analysis. The lower left quadrant includes live cells (annexin V–
/PI–), the lower right quadrant shows early apoptotic cells (annexin V+/PI–), the upper right
quadrant shows late apoptotic cells (annexin V+/PI+), and the upper left quadrant shows
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necrotic cells (annexin V–/PI+). Percentage values indicate the proportion of cells in the
relevant quadrant. Each value is the mean of three experiments.
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