Cytotoxic and apoptosis-inducing activities of steviol and isosteviol derivatives against human cancer cell lines

Article abstract of DOI:10.1002/cbdv.201200406

Seventeen steviol derivatives, i.e., 2-18, and 19 isosteviol derivatives, i.e., 19-37, were prepared from a diterpenoid glycoside, stevioside (1). Upon evaluation of the cytotoxic activities of these compounds against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines, nine steviol derivatives, i.e., 5-9 and 11-14, and five isosteviol derivatives, i.e., 28-32, exhibited activities with single-digit micromolar IC₅₀ values against one or more cell lines. All of these active compounds possess C(19)-O-acyl group, and among which, ent-kaur-16-ene-13,19-diol 19-O-4′,4′,4′-trifluorocrotonate (14) exhibited potent cytotoxicities against four cell lines with IC₅₀ values in the range of 1.2-4.1 μM. Compound 14 induced typical apoptotic cell death in HL60 cells upon evaluation of the apoptosis-inducing activity by flow-cytometric analysis. These results suggested that acylation of the 19-OH group of kaurane- and beyerane-type diterpenoids might be useful for enhancement of their cytotoxicities with apoptosis-inducing activity. Copyright

Full text of DOI:10.1002/cbdv.201200406

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
177
Cytotoxic and Apoptosis-Inducing Activities of Steviol and Isosteviol
Derivatives against Human Cancer Cell Lines
by Motohiko Ukiya*, Shingo Sawada, Takashi Kikuchi, Yasunori Kushi, Makoto Fukatsu, and
Toshihiro Akihisa
College of Science and Technology, Nihon University, 1-8-14 Kanda Surugadai, Chiyoda-ku,
Tokyo 101-8308, Japan (phone: þ81-3-3259-0816; fax: þ81-3-3293-7572;
e-mail: ukiya.motohiko@nihon-u.ac.jp)
Seventeen steviol derivatives, i.e., 2–18, and 19 isosteviol derivatives, i.e., 19–37, were prepared from
a diterpenoid glycoside, stevioside (1). Upon evaluation of the cytotoxic activities of these compounds
against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines, nine
steviol derivatives, i.e., 5–9 and 11–14, and five isosteviol derivatives, i.e., 28–32, exhibited activities with
single-digit micromolar IC50 values against one or more cell lines. All of these active compounds possess
C(19)-O-acyl group, and among which, ent-kaur-16-ene-13,19-diol 19-O-4’,4’,4’-trifluorocrotonate (14)
exhibited potent cytotoxicities against four cell lines with IC50 values in the range of 1.2–4.1 mm.
Compound 14 induced typical apoptotic cell death in HL60 cells upon evaluation of the apoptosis-
inducing activity by flow-cytometric analysis. These results suggested that acylation of the 19-OH group
of kaurane- and beyerane-type diterpenoids might be useful for enhancement of their cytotoxicities with
apoptosis-inducing activity.
1. Introduction. – Stevioside (1) is a sweet-tasting diterpenoid glycoside occurring
abundantly in the leaves of Stevia rebaudiana Bertoni (Asteraceae), which has been
popularly used as a sugar substitute in Japan, Korea, and Brazil for decades [1][2].
Hydrolysis of 1 (cf. Scheme 1) with mineral acid affords isosteviol (19; 16-oxobeyeran-
19-oic acid; cf. Scheme 4), a beyerane-type diterpenoid [3]. On the other hand,
treatment of 1 with NaIO4 and KOH produces steviol (2; (5b,8a,9b,10a,13a)-13-
hydroxykaur-16-en-18-oic acid) [4], a kaurane-type diterpenoid which constitutes an
aglycone moiety of 1. Several studies have suggested that, besides sweetness, 1, along
with related compounds, which include rebaudioside A (second most abundant sweet
principle of S. rebaudiana leaf), 2, and 19, may also offer therapeutic benefits, as they
have antihyperglycemic, antihypertensive, anti-inflammatory, antitumor, antidiarrheal,
diuretic, and immunomodulatory properties [2]. In addition, compounds 1, 2, and 19
have been suggested to be valuable chemopreventive agents against chemical
carcinogenesis [5][6]. On the other hand, a number of derivatives of 2 and 19 have
been prepared and evaluated for their bioactivities which included anti-inflammatory
[7–9], cytotoxic [10–12], antibacterial [10][13], glycosidase-inhibitory [14][15], plant-
growth-regulation [16], antihyperglycemic [17], and glucocorticoid-agonist activities
[18]. We have reported the inhibitory effects of 2, 19, and some of their derivatives
against DNA polymerase, DNA topoisomerase, and EpsterinꢀBarr virus early antigen
(EBV-EA) acitivation [3][19]. In a continuing study on the evaluation of the biological
potential of kaurane- and beyerane-type diterpenoids, we have prepared 36 derivatives,
ꢀ 2013 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
i.e., 2–37, from the natural diterpenoid glycoside 1. Here, we describe the preparation
of these derivatives and evaluation of their cytotoxic activities against four human
cancer cell lines. Synthesis and induction of apoptosis-signaling pathway of the
derivatives of kaurenoic acid, a kaurane-type diterpenoid, has recently been reported
[20].
2. Results and Discussions. – Preparation of Steviol Derivatives. Starting from
stevioside (1), treatment with NaIO4 and KOH led to the aglycone steviol (2) [4].
Esterification of 2 with diazo(trimethylsilyl)methane (TMSꢀCHN2) provided steviol
methyl ester (3). Reduction of 3 with LiAlH4 gave the desired alcohol 4 (Scheme 1).
Compound 4 was acylated with di(pyridin-2-yl) thionocarbonate (DPTC) in the
presence of cinnamic acid analogs, crotonic acid analogs, and PhCOOH to yield a series
of 19-O-acylated derivatives, 5–14 (Scheme 2).
Scheme 1
i) NaIO4, KOH, H2O; 26%. ii) TMSꢀCHN2, benzene, MeOH; 96%. iii) LiAlH4, Et2O; 77%.
Scheme 2
i) RCOOH, di(pyridin-2-yl)thionocarbonate (DPTC), 4-(dimethylamino)pyridine (DMAP), toluene;
19–70% (for 5–12, 14). ii) Benzyl chloride (BnCl)/pyridine; 9% (13).
As will be discussed below, ent-kaur-16-ene-13,19-diol 19-O-cinnamate (5), a
steviol derivative, exhibited potent cytotoxicity against three cancer cell lines, A549,
AZ521, and SK-BR-3. To clarify the efficacy of the 13-OH group on the cytotoxicity, we
prepared ent-kaur-16-en-19-ol 19-O-cinnamate (18), the 13-deoxy analog of 5, from
ent-kaur-16-en-19-oic acid (15), which has been isolated previously from the pollen of
Helianthus annuus [21]. Thus, as shown in Scheme 3, compound 15 was converted to its
methyl ester with TMSꢀCHN2 (!16) and then reduced with LiAlH4 to yield 17. The
alcohol 17 was acylated with cinnamic acid to yield the cinnamate 18.

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
179
Scheme 3
i) TMSꢀCHN2, benzene, MeOH; 97%. ii) LiAlH4, Et2O; 88%. iii) Cinnamic acid, DPTC, DMAP,
toluene; 32%.
Preparation of Isosteviol Derivatives. Isosteviol (19) was prepared by the treatment
of 1 with HCl. Eighteen derivatives, i.e., 20–37, were prepared from 19 as outlined in
Scheme 4. The C(16)¼O group of 19 was reduced with NaBH4 to afford the alcohol 20
[22]. Compound 20 was then acylated with five carboxylic acids, i.e., cinnamic acid, 2-,
3-, and 4-methylcinnamic acids, and 4,4,4-trifluorocrotonic acid, to yield the corre-
sponding acyl derivatives 21–25, respectively. In addition, 19 was converted to its (!
26) methyl ester and reduced with LiAlH4 to afford 27. The alcohol 27 was acylated
with five carboxylic acids to yield the mixtures of 16-O-monoacylated and 16-O,19-O-
Scheme 4
i) HCl, H2O; 98%. ii) NaBH4, benzene, MeOH; 39%. iii) RCOOH, DPTC, DMAP, toluene; 14–88%.
iv) TMSꢀCHN2, benzene, MeOH; 99%. v) LiAlH4, Et2O; 82%.

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
diacylated derivatives. Each mixture of acylated derivatives was separated by TLC on
1
SiO2 to afford 28–37. Their structures were confirmed by H-NMR, high-resolution
(HR) mass, and IR spectra.
Cytotoxic Activity. The cytotoxic activities of compounds 2–37 against leukemia
(HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines were
determined by means of 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 IC50 values were used to determine the growth inhibition in the
presence of test compounds, and the results are compiled in the Table.
Nine steviol, i.e., 5–9 and 11–14, and five isosteviol derivatives, i.e., 28–32,
exhibited potent activities with single-digit micromolar IC50 values against one or more
cell lines. Among the steviol derivatives, whereas the 19-OH compound 4 exhibited no
activity, its 19-O-acyl derivatives exhibited cytotoxicities against four cancer cell lines.
The cytotoxicities of Me cinnamates 6–8 were almost same as that of cinnamate 5,
which implied that Me substitution of the Ph group of cinnamate 5 has no influence on
the activity. Me or CN substitution at the prop-2-enoyl C¼C bond, and hydrogenation
of the prop-2-enoyl C¼C bond of cinnamate 5 decreased the activity against A549,
AZ521, and SK-BR-3 (compounds 9, 10, and 12 vs. 5). On the other hand, replacement
of the Ph group of cinnamate 5 by a CF3 group, leading to compound 14, enhanced
cytotoxicity substantially (IC50 2.9, 4.1, 1.7, and 1.2 mm against HL60, A549, AZ521 and
SK-BR-3, resp.). Furthermore, since the cinnamate 18, prepared from ent-kaur-16-en-
19-oic acid (15), exhibited no cytotoxicity against four cancer cell lines, the OH group
at C(13) seems to play an important role for the 19-O-acylated kaurane-type
diterpenoids to exhibit potent cytotoxicity against the cancer cell lines used in this
study.
Among the isosteviol derivatives, acylation of the 16-hydroxybeyerane 20 (!21–
25) increased cytotoxicity moderately against HL60, AZ521, and SK-BR-3 cells. On the
other hand, acylation of the 16,19-dihydroxybeyerane 27 was effective only in the case
of the 19-O-monocinnamoylation, which yielded 28 (IC50 8.2, 1.2, and 2.4 mm against
A549, AZ521, and SK-BR-3, resp.), for the enhancement of cytotoxicity. As has been
observed on the steviol derivatives, the 19-O-trifluorocrotonyl derivative isosteviol 32
exhibited potent cytotoxicity (IC50 6.6, 5.7, and 4.2 mm against HL60, A549, and AZ521,
resp.).
Apoptosis-Inducing Activity of Compound 14. Compound 14, the potent cytotoxic
compound against four human cancer cell lines, was then evaluated for its induction of
early apoptosis in HL60 cells. Exposure of the membrane phospholipids, phosphati-
dylserine, to the external cellular environment is one of the earliest markers of
apoptotic cell death [23]. Annexin V is a calcium-dependent phospholipid-binding
protein with high affinity for phosphatidylserine expressed on the cell surface.
Propidium iodide (PI) does not enter whole cells with intact membranes, and was used
to differentiate between early apoptotic (annexin V positive, PI negative), late
apoptotic (annexine V, PI double positive), or necrotic cell death (annexin V negative,
PI positive). The ratio of early apoptotic cells (Fig., lower right) was increased after
treatment with 6 mm of 14 in HL60 for 24 h (from 4.4 to 33.7%), and that of late
apoptotic cells (upper right) was increased after 24 h (from 1.0 to 33.7%) and 48 h

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
181
Table. Cytotoxic Activities (IC50 ꢁS. D. [mm]) of Steviol and Isosteviol Derivatives 2–37 against Four
Human Cancer Cell Linesa)
Compound
HL60 (Leukemia)
A549 (Lung)
AZ521 (Stomach)
SK-BR-3 (Breast)
2
3
>100
>100
>100
>100
>100
>100
>100
>100
4
>100
>100
>100
>100
5
6
7
8
64.2ꢁ1.1
5.3ꢁ2.3
15.9ꢁ4.4
8.8ꢁ0.5
56.2ꢁ4.3
27.7ꢁ0.3
>100
12.9ꢁ0.2
41.3ꢁ0.4
2.9ꢁ0.4
53.7ꢁ5.6
80.1ꢁ4.3
31.3ꢁ3.4
>100
10.5ꢁ1.0
7.2ꢁ1.0
6.0ꢁ2.0
2.9ꢁ0.6
17.0ꢁ3.2
>100
2.5ꢁ0.4
22.1ꢁ5.2
4.3ꢁ2.0
4.1ꢁ1.2
>100
48.9ꢁ5.8
14.7ꢁ3.6
>100
>100
>100
9.0ꢁ0.1
3.1ꢁ0.2
2.9ꢁ0.2
1.1ꢁ0.1
29.0ꢁ2.7
>100
27.2ꢁ3.7
38.0ꢁ1.0
2.5ꢁ1.0
1.7 ꢁ1.4
>100
47.4ꢁ1.8
63.0ꢁ3.9
>100
>100
>100
2.4ꢁ0.2
1.7ꢁ0.3
4.7ꢁ0.8
2.2ꢁ0.4
9.4ꢁ1.2
37.6ꢁ3.8
1.6ꢁ0.1
5.8ꢁ0.5
2.2ꢁ0.8
1.2 ꢁ0.1
11.0ꢁ1.0
15.5ꢁ0.6
16.4ꢁ4.0
>100
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Cisplatinb)
75.0ꢁ4.7
>100
>100
>100
22.7ꢁ5.7
81.6ꢁ5.0
57.5ꢁ4.6
32.8ꢁ6.6
71.5ꢁ5.7
>100
>100
>100
>100
>100
77.6ꢁ4.1
27.1ꢁ1.2
91.0ꢁ1.7
12.0ꢁ1.2
74.6ꢁ2.8
>100
15.2ꢁ2.7
72.6ꢁ4.6
31.0 ꢁ3.6
20.6ꢁ4.3
15.8ꢁ3.1
>100
38.7ꢁ1.7
>100
>100
>100
>100
>100
24.0ꢁ2.7
64.4ꢁ3.6
20.3ꢁ2.5
13.2ꢁ4.9
6.6ꢁ2.1
>100
>100
>100
>100
8.2ꢁ6.3
1.2ꢁ0.3
9.0ꢁ2.1
19.8ꢁ2.2
2.8ꢁ0.8
4.2ꢁ0.6
>100
2.4ꢁ0.7
25.0ꢁ4.2
2.9ꢁ0.6
3.9ꢁ0.8
25.2ꢁ0.5
>100
>100
>100
>100
>100
>100
20.9ꢁ3.3
5.7ꢁ1.6
>100
>100
>100
47.8ꢁ3.7
>100
>100
>100
>100
82.8ꢁ1.6
39.4ꢁ5.3
23.1ꢁ1.4
1.9ꢁ0.3
24.9ꢁ4.3
7.9ꢁ0.6
13.7ꢁ1.7
a) Cells were treated with compounds (1ꢂ10ꢀ4 –1ꢂ10ꢀ6 m) for 48 h, and cell viability was analyzed by
the MTT assay. IC50 Values based on triplicate experiments. b) Reference compound.
(58.0%; Fig.). A small number of necrotic cells (upper left) also were detected after
24 h (from 0.4 to 4.6%). These results revealed that most of the cytotoxicity of 14 to
HL60 cells is due to inducing apoptotic cell death.
Conclusions. – In summary, 36 derivatives, i.e., 2–37, were prepared from stevioside
1, and their cytotoxic activities were evaluated against leukemia (HL60), lung (A549),

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Figure. Detection of 14-induced early and late apoptotic cells by annexin V/propidium iodide double
staining in HL60 cell. The HL60 cells were cultured with 6 mm of 14 for 24 and 48 h. Each value is the
mean of three experiments.
stomach (AZ521), and breast (SK-BR-3) cancer cell lines. Among the derivatives, the
19-O-acylated derivatives, especially ent-kaur-16-ene-13,19-diol 19-O-4’,4’,4’-trifluoro-
crotonate (14), exhibited potent cytotoxicity against the cancer cell lines used.
Furthermore, 14 was revealed to induce apoptotic cell death in HL60 cells. These
results suggested that acylation of the 19-OH group of kaurane- and beyerane-type
diterpenoids might be useful for the enhancement of their cytotoxicities with apoptosis-
inducing activity. The results of this study will be of value for further utilization of
stevioside in the product application in pharmaceutical field in the future.
This work was supported, in part, by a grant ꢂNihon University College of Science and Technology
Grant-in-Aid for Basic Science Researchꢃ, 2011.
Experimental Part
General. Column chromatography (CC): silica gel 60 (SiO2, 230–400 mesh, Merck & Co. Inc., D-
Darmstadt). TLC: silica gel 60G (for TLC, Merck & Co. Inc.). LC: Reversed-phase (RP) prep. high-
performance liquid chromatography (HPLC): octadecyl silica column (Pegasil ODS II column, 25 cmꢂ
10 mm i.d.; Senshu Scientific Co., Ltd., Tokyo, Japan) at 258 with MeOH/H2O 19 :1 (flow rate, 2.0 ml/
min). Crystallizations were performed in MeOH. M.p.: Yanagimoto Micro Mp apparatus; uncorrected.
1
IR Spectra: Perkin-Elmer Spectrum One FT-IR spectrophotometer in KBr disks; ˜n in cmꢀ1. H-NMR
Spectra: JEOL ECX-400 spectrometer, CDCl3; d in ppm, 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 mA). Microplate reader: Sunrise-Basic
(Tecan Japan Co., Ltd., Kawasaki, Japan).
Chemicals and Materials. Compounds were purchased as follows: MTT, Dulbeccoꢃs modified Eagleꢃs
medium (D-MEM), Eagleꢃs minimal essential medium (MEM), and DMSO from Sigma-Aldrich Japan
Co. (Tokyo, Japan), stevioside, carboxylic acids, TMSꢀCHN2 (0.6 mol/l hexane soln.), 4-(dimethylami-
no)pyridine (DMAP), DPTC, NaBH4, and LiAlH4 from Tokyo Chemical Industry Co., Ltd. (Tokyo,
Japan), RPMI-1640 medium, fetal bovine serum (FBS), antibiotics (100 units/ml penicillin and 100 mg/ml
streptomycin), and non-essential amino acid (NEAA) from Invitrogen Co. (Auckland, NZ), rh annexin
V/FITC kit (Bender MedSystemsꢀ) from Cosmo Bio Co. Ltd. (Tokyo, Japan), and cisplatin from Wako
Pure Chemical Industries Ltd. (Osaka, Japan). All other chemicals and reagents were of anal. grade. ent-

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
183
Kaur-16-en-19-oic acid (¼(5b,8a,9b,10a,13a)-kaur-16-en-18-oic acid; 15) was isolated from the pollen of
Helianthus annuus L. [21].
Cell Cultures. Cell lines HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast)
were obtained from Riken Cell Bank (Tsukuba, Ibaraki, Japan). Two cell lines, HL60 and SK-BR-3, were
grown in RPMI-1640 medium, while A549 and AZ521 cell lines were grown in D-MEM and in 90% D-
MEMþ10% MEMþ0.1 mm NEAA, resp. The medium was supplemented with 10% FBS and
antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Cells were incubataed at 378 in a 5%
CO2 humidified incubator.
Preparation of Steviol Derivatives. Steviol (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-18-oic
Acid; 2). To a soln. of stevioside (1; 4.1 g, 5.1 mmol) in H2O (300 ml), NaIO4 (5.9 g, 27.6 mmol) was
added and stirred at r. t. for 16 h. Then, to the mixture was added KOH (3.0 g, 53.6 mmol) and refluxed
for 1 h. H2O (300 ml) was added and pH was adjusted to 3–4 with AcOH; then the mixture was extracted
with AcOEt (2ꢂ200 ml). The AcOEt fraction was washed with H2O (2ꢂ ), dried (Na2SO4), and
evaporated under reduced pressure to yield 2 (415 mg, 26%), which was identified by spectral
comparison with literature data [19].
Steviol Methyl Ester (¼ Methyl (5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-18-oate; 3). To a soln. of 2
(202 mg, 0.6 mmol) in dried benzene (20 ml) and MeOH (10 ml), TMSꢀCHN2 (1.2 ml) was added and
stirred at r.t. for 1 h. Then, the mixture was evaporated to give 3 (203 mg, 96%), which was identified by
spectral comparison with literature data [24].
ent-Kaur-16-ene-13,19-diol (¼(5b,8a,9b,10a,13a)-Kaur-16-ene-13,18-diol; 4). To a soln. of 3
(204 mg, 0.6 mmol) in dried Et2O (20 ml), LiAlH4 (126 mg, 3.3 mmol) was added, and the mixture
was refluxed for 6 h under N2. To the mixture was added 1m HCl (5 ml) and H2O (5 ml), and stirred for a
few min, then extracted with Et2O (2ꢂ20 ml). The Et2O extract was washed with aq. NaHCO3 and H2O,
dried (Na2SO4), and concentrated under reduced pressure to furnish 4 (143 mg, 77%), which was
identified by spectral comparison with literature data [25].
ent-Kaur-16-ene-13,19-diol 19-O-Cinnamate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-18-yl
(2E)-3-Phenylprop-2-enoate; 5). To the soln. of 4 (22 mg, 0.07 mmol) in dried toluene (1 ml), DMAP
(20 mg, 0.2 mmol), DPTC (30 mg, 0.1 mmol), and cinnamic acid (26 mg, 0.2 mmol) were added. After
24 h, H2O was added (30 ml), and the mixture was extracted with Et2O (2ꢂ30 ml). The Et2O fraction
was washed with H2O (2ꢂ ), dried (Na2SO4), and evaporated under reduced pressure to afford a mixture
(36 mg), which was subjected to TLC (hexane/AcOEt 7:3) to furnish 5 (14 mg, 45%). White solid. M.p.
119–1238. IR (KBr): 3403, 2929, 2854, 1709, 1663, 1612, 1450, 1330, 1271, 1181, 1091. 1H-NMR
(400 MHz): 1.02 (s, 3 H); 1.07 (s, 3 H); 4.01 (d, J¼11.4, 1 H); 4.37 (d, J¼11.4, 1 H); 4.82 (br. s, 1 H); 4.98
(br. s, 1 H); 6.43 (d, J¼16.0, 1 H); 7.37–7.39 (m, 3 H); 7.52–7.54 (m, 2 H); 7.66 (d, J¼16.0, 1 H). HR-
ESI-MS (pos.-ion mode): 457.2707 ([MþNa]þ , C29H38NaO3þ ; calc. 457.2719).
Twenty-five acyl derivatives, i.e., 6–14, 18, 21–25, and 28–37, described below were prepared from
the corresponding alcohols by similar ways as described above for the preparation of 5.
ent-Kaur-16-ene-13,19-diol 19-O-2-Methylcinnamate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-
18-yl (2E)-3-(2-Methylphenyl)prop-2-enoate; 6). Treatment of 4 (31 mg, 0.1 mmol) with 2-methylcin-
namic acid (35 mg, 0.2 mmol) gave 6 (16 mg, 35%). White solid. M.p. 151–1558. IR (KBr): 3487, 2929,
2858, 1694, 1634, 1600, 1458, 1392, 1323, 1187, 1106. 1H-NMR (400 MHz): 1.02 (s, 3 H); 1.08 (s, 3 H); 2.43
(s, 3 H); 4.04 (d, J¼11.0, 1 H); 4.35 (d, J¼11.0, 1 H); 4.82 (br. s, 1 H); 4.98 (br. s, 1 H); 6.35 (d, J¼16.0,
1 H); 7.19 (d, J¼7.3, 1 H); 7.20 (t, J¼7.3, 1 H); 7.25–7.29 (m, 1 H); 7.56 (d, J¼7.8, 1 H); 7.97 (d, J¼16.0,
1 H). HR-ESI-MS (pos.): 471.2867 ([MþNa]þ , C30H40NaO3þ ; calc. 471.2875).
ent-Kaur-16-ene-13,19-diol 19-O-3-Methylcinnamate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-
18-yl (2E)-3-(3-Methylphenyl)prop-2-enoate; 7). Ttreatment of 4 (40 mg, 0.1 mmol) with 3-methylcin-
namic acid (46 mg, 0.3 mmol) yielded 7 (18 mg, 30%). White solid. M.p. 149–1558. IR (KBr): 3401, 2928,
2849, 1709, 1639, 1605, 1446, 1368, 1320, 1273, 1236, 1179, 1091. 1H-NMR (400 MHz): 1.02 (s, 3 H); 1.07 (s,
3 H); 2.37 (s, 3 H); 4.01 (d, J¼11.0, 1 H); 4.36 (d, J¼11.0, 1 H); 4.82 (br. s, 1 H); 4.98 (br. s, 1 H); 6.42 (d,
J¼16.0, 1 H); 7.19 (br. d, J¼7.3, 1 H); 7.27 (br. t, J¼7.8, 1 H); 7.33 (br. d, J¼6.9, 1 H); 7.34 (s, 1 H); 7.63
(d, J¼16.0, 1 H). HR-ESI-MS (pos.): 471.2885 ([MþNa]þ , C30H40NaO3þ ; calc. 471.2875).
ent-Kaur-16-ene-13,19-diol 19-O-4-Methylcinnamate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-
18-yl (2E)-3-(4-Methylphenyl)prop-2-enoate; 8). Treatment of 4 (31 mg, 0.1 mmol) with 4-methylcin-

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
namic acid (32 mg, 0.2 mmol) furnished 8 (17 mg, 37%). White solid. M.p. 145–1508. IR (KBr): 3393,
2925, 2853, 1708, 1637, 1608, 1457, 1386, 1309, 1272, 1179. 1H-NMR (400 MHz): 1.01 (s, 3 H); 1.07 (s, 3 H);
2.37 (s, 3 H); 4.00 (d, J¼11.0, 1 H); 4.36 (d, J¼11.0, 1 H); 4.81 (br. s, 1 H); 4.98 (br. s, 1 H); 6.38 (d, J¼
16.0, 1 H); 7.18 (d, J¼8.2, 2 H); 7.42 (d, J¼8.2, 2 H); 7.63 (d, J¼16.0, 1 H). HR-ESI-MS (pos.): 471.2881
([MþNa]þ , C30H40NaOþ3 ; calc. 471.2875).
ent-Kaur-16-ene-13,19-diol 19-O-a-Methylcinnamate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-
18-yl (2E)-2-Methyl-3-phenylprop-2-enoate; 9). Treatment of 4 (38 mg, 0.1 mmol) with a-methylcin-
namic acid (39 mg, 0.2 mmol) afforded 9 (39 mg, 70%). Amorphous solid. IR (KBr): 3339, 2925, 2849,
2627, 1706, 1682, 1634, 1575, 1449, 1261, 1203, 1122. 1H-NMR (400 MHz): 1.04 (s, 3 H); 1.08 (s, 3 H); 2.13
(br. s, 3 H); 4.00 (d, J¼11.0, 1 H); 4.37 (d, J¼11.0, 1 H); 4.82 (br. s, 1 H); 4.99 (br. s, 1 H); 7.31–7.35 (m,
3 H); 7.39–7.43 (m, 2 H); 7.67 (br. s, 1 H). HR-ESI-MS (pos.): 471.2853 ([MþNa]þ , C30H40NaO3þ ; calc.
471.2875).
ent-Kaur-16-ene-13,19-diol 19-O-2-Cyanocinnamate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-
18-yl (2E)-2-Cyano-3- phenylprop-2-enoate; 10). Treatment of 4 (50 mg, 0.2 mmol) with 2-cyano-3-
phenylacrylic acid (50 mg, 0.3 mmol) yielded 10 (14 mg, 19%). Amorphous solid. IR (KBr): 3414, 2927,
2851, 2223, 1727, 1606, 1450, 1270, 1205, 1189, 1108, 1092. 1H-NMR (400 MHz): 1.07 (s, 3 H); 1.08 (s, 3 H);
4.12 (d, J¼11.0, 1 H); 4.47 (d, J¼11.0, 1 H); 4.82 (br. s, 1 H); 4.99 (br. s, 1 H); 7.49–7.53 (m, 2 H); 7.55–
7.57 (m, 1 H); 8.00 (br. d, J¼7.3, 2 H); 8.24 (s, 1 H). HR-APCI-MS (pos.): 460.2878 ([MþH]þ
,
C30H38NOþ3 ; calc. 460.2852).
ent-Kaur-16-ene-13,19-diol 19-O-3-(Pyridin-3-yl)acrylate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-
16-en-18-yl (2E)-3-(Pyridin-3-yl)prop-2-enoate; 11). Treatment of 4 (20 mg, 0.07 mmol) with 3-
(pyridin-3-yl)acrylic acid (20 mg, 0.1 mmol) afforded 11 (17 mg, 59%)). White solid. M.p. 132–1388.
IR (KBr): 3352, 2924, 2853, 1710, 1646, 1613, 1587, 1457, 1419, 1288, 1193, 1090, 1025. 1H-NMR
(400 MHz): 1.02 (s, 3 H); 1.07 (s, 3 H); 4.03 (d, J¼11.0, 1 H); 4.39 (d, J¼11.0, 1 H); 4.82 (br. s, 1 H); 4.99
(br. s, 1 H); 6.51 (d, J¼16.1, 1 H); 7.35 (dd, J¼4.9, 7.7, 1 H); 7.65 (d, J¼16.1, 1 H); 7.86 (dt, J¼1.5, 7.7,
1 H); 8.61 (dd, J¼1.5, 4.9, 1 H); 8.76 (d, J¼1.5, 1 H). HR-APCI-MS (pos.): 436.2879 ([MþH]þ
,
C28H38NOþ3 ; calc. 436.2852).
ent-Kaur-16-ene-13,19-diol 19-O-3-Phenylpropionate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-
18-yl 3-Phenylpropanoate; 12). Treatment of 4 (31 mg, 0.1 mmol) with 3-phenylpropanoic acid (34 mg,
0.2 mmol) afforded 12 (25 mg, 56%). Amorphous solid. IR (KBr): 3524, 2931, 2853, 1724, 1664, 1444,
1394, 1373, 1292, 1248, 1176, 1106. 1H-NMR (400 MHz): 0.90 (s, 3 H); 1.01 (s, 3 H); 2.63 (t, J¼7.8, 2 H);
2.95 (t, J¼7.8, 2 H); 3.86 (d, J¼11.0, 1 H); 4.22 (d, J¼11.0, 1 H); 4.81 (br. s, 1 H); 4.97 (br. s, 1 H); 7.18–
7.21 (m, 3 H); 7.26–7.28 (m, 2 H). HR-ESI-MS (pos.): 459.2854 ([MþNa]þ , C29H40NaO3þ ; calc.
459.2875).
ent-Kaur-16-ene-13,19-diol 19-O-Benzoate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-16-en-18-yl Ben-
zoate; 13). To a soln. of 4 (50 mg, 0.2 mmol) in dried pyridine (5 ml), PhCOCl (54mg, 0.4 mmol) was
added. After 24 h, H2O (30 ml) was added, and the mixture was extracted with Et2O (2ꢂ30 ml). The
Et2O fraction was washed with H2O (2ꢂ ), dried (Na2SO4), and evaporated under reduced pressure to
give a mixture (139 mg), which was subjected to TLC (hexane/AcOEt 7:3) and HPLC (tR 10.4 min) to
furnish 13 (6 mg, 9%). White solid. M.p. 117–1228. IR (KBr): 3339, 2928, 2850, 1715, 1663, 1603, 1451,
1282, 1121. 1H-NMR (400 MHz): 1.08 (s, 3 H); 1.09 (s, 3 H); 4.12 (d, J¼11.0, 1 H); 4.49 (d, J¼11.0, 1 H);
4.82 (br. s, 1 H); 4.99 (br. s, 1 H); 7.45 (br. t, J¼7.8, 2 H); 7.56 (br. t, J¼7.4, 1 H); 8.04 (br. d, J¼7.1, 2 H ) .
HR-ESI-MS (pos.): 431.2554 ([MþNa]þ , C27H36NaO3þ ; calc. 431.2562).
ent-Kaur-16-ene-13,19-diol 19-O-4’,4’,4’-Trifluorocrotonate (¼(5b,8a,9b,10a,13a)-13-Hydroxykaur-
16-en-18-yl (2E)-4,4,4-Trifluorobut-2-enoate; 14). Treatment of 4 (31 mg, 0.1 mmol) with 4,4,4-
trifluorocrotonic acid (44 mg, 0.3 mmol) yielded 14 (16 mg, 37%). White solid. M.p. 95–998. IR
1
(KBr): 3401, 2929, 2854, 1731, 1458, 1330, 1268, 1136, 1120. H-NMR (400 MHz): 0.98 (s, 3 H); 1.04 (s,
3 H); 4.01 (d, J¼11.0, 1 H); 4.39 (d, J¼11.0, 1 H); 4.82 (br. s, 1 H); 4.98 (br. s, 1 H); 6.49 (dq, J¼1.8, 15.6,
1 H); 6.75 (dq, J¼6.4, 15.6, 1 H). HR-APCI-MS (pos.): 409.2346 ([MþHꢀH2O]þ , C24H32F3O2þ ; calc.
409.2354).
Methyl ent-Kaur-16-en-19-oate (¼ Methyl (5b,8a,9b,10a,13a)-Kaur-16-en-18-oate; 16). As described
for the preparation. of 3, treatment of 15 (119 mg, 0.4 mmol) with TMSꢀCHN2 (1.2 ml, 0.7 mmol) gave 16
(121 mg, 97%), which was identified by spectral comparison with literature data [26].

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
185
ent-Kaur-16-en-19-ol (¼(5b,8a,9b,10a,13a)-Kaur-16-en-18-ol; 17). As described for the preparation
of 4, treatment of 16 (100 mg, 0.3 mmol) with LiAlH4 (210 mg, 5.5 mmol) afforded 17 (80 mg, 88%),
which was identified by spectral comparison with literature data [27].
ent-Kaur-16-en-19-ol 19-O-Cinnamate (¼(5b,8a,9b,10a,13a)-Kaur-16-en-18-yl (2E)-3-Phenylprop-
2-enoate; 18). Treatment of 17 (52 mg, 0.2 mmol) with cinnamic acid (58 mg, 0.4 mmol) afforded 18
(24 mg, 32%). White solid. M.p. 133–1458. IR (KBr): 2923, 2869, 1717, 1660, 1642, 1449, 1383, 1328, 1288,
1254, 1205, 1178, 1035, 1008. 1H-NMR (400 MHz): 1.02 (s, 3 H); 1.07 (s, 3 H); 4.02 (br. d, J¼11.0, 1 H);
4.38 (d, J¼11.0, 1 H); 4.74 (br. s, 1 H); 4.80 (br. s, 1 H); 6.44 (d, J¼16.0, 1 H); 7.37–7.39 (m, 3 H); 7.52–
7.54 (m, 2 H); 7.66 (d, J¼16.0, 1 H). HR-ESI-MS (pos.): 441.2760 ([MþNa]þ , C29H38NaO2þ ; calc.
441.2769).
Isosteviol (¼16-Oxobeyeran-19-oic Acid; 19). To the soln. of 1 (8.0 g, 10.0 mmol) in H2O (300 ml),
12m HCl (6 ml) was added, and the mixture was refluxed for 2 h. Then, the mixture was extracted with
AcOEt (2ꢂ200 ml). The AcOEt fraction was washed with H2O (2ꢂ ), dried (Na2SO4), and evaporated
under reduced pressure to yield 19 (3.1 g, 98%), which was identified by spectral comparison with
literature data [19].
(16a)-16-Hydroxybeyeran-19-oic Acid (20). To a soln. of 19 (103 mg, 0.3 mmol) in benzene (5 ml)
and MeOH (2.5 ml), NaBH4 (219 mg, 5.8 mmol) was added, and the mixture was stirred at r.t. for 5 h.
Then, 1m HCl (5 ml) was added, and the mixture was stirred for few min, then extracted with Et2O (2ꢂ
20 ml). The Et2O fraction was washed with H2O, dried (Na2SO4), and concentrated under reduced
pressure to furnish 20 (40 mg, 39%), which was identified by spectral comparison with literature data
[15][22].
(16a)-16-Hydroxybeyeran-19-oic Acid 16-O-Cinnamate (¼(16a)-16-{[(2E)-3-Phenylprop-2-eno-
yl]oxy}beyeran-19-oic Acid; 21). Treatment of 20 (30 mg, 0.1 mmol) with cinnamic acid (61 mg,
0.4 mmol) gave 21 (37 mg, 88%). White solid. M.p. 100–1068. IR (KBr): 2947, 2848, 1711, 1694, 1638,
1451, 1311, 1172. 1H-NMR (400 MHz): 0.80 (s, 3 H); 0.95 (s, 3 H); 1.22 (s, 3 H); 4.86 (dd, J¼5.0, 9.6, 1 H);
6.44 (d, J¼16.0, 1 H); 7.35–7.36 (m, 3 H); 7.51–7.53 (m, 2 H); 7.64 (d, J¼16.0, 1 H). HR-ESI-MS (pos.):
473.2677 ([MþNa]þ , C29H38NaOþ4 ; calc. 473.2668).
(16a)-16-Hydroxybeyeran-19-oic Acid 16-O-2-Methylcinnamate (¼(16a)-16-{[(2E)-3-(2-Methyl-
phenyl)prop-2-enoyl]oxy}beyeran-19-oic Acid; 22). Treatment of 20 (19 mg, 0.06 mmol) with 2-
methylcinnamic acid (21 mg, 0.1 mmol) afforded 22 (9.2 mg, 33%). White solid. M.p. 89–958. IR
(KBr): 2926, 2851, 1714, 1635, 1456, 1313, 1262, 1172. 1H-NMR (400 MHz): 0.81 (s, 3 H); 0.96 (s, 3 H);
1.23 (s, 3 H); 2.43 (s, 3 H); 4.87 (dd, J¼5.5, 9.6, 1 H); 6.36 (d, J¼15.8, 1 H); 7.17–7.20 (m, 2 H); 7.24–7.27
(m, 1 H); 7.56 (br. d, J¼7.3, 1 H); 7.97 (d, J¼15.8, 1 H). HR-ESI-MS (pos.): 487.2798 ([MþNa]þ
,
C30H40NaOþ4 ; calc. 487.2824).
(16a)-16-Hydroxybeyeran-19-oic Acid 16-O-3-Methylcinnamate (¼(16a)-16-{[(2E)-3-(3-Methyl-
phenyl)prop-2-enoyl]oxy}beyeran-19-oic Acid; 23). Treatment of 20 (21 mg, 0.07 mmol) with 3-
methylcinnamic acid (21 mg, 0.1 mmol) furnished 23 (14 mg, 46%). White solid. M.p. 98–1058. IR
(KBr): 2928, 2849, 1713, 1695, 1635, 1456, 1312, 1269, 1236, 1176. 1H-NMR (400 MHz): 0.81 (s, 3 H); 0.95
(s, 3 H); 1.22 (s, 3 H); 2.36 (s, 3 H); 4.85 (dd, J¼5.0, 9.6, 1 H); 6.43 (d, J¼16.0, 1 H); 7.17 (br. d, J¼7.8,
1 H); 7.26 (br. t, J¼9.1, 1 H); 7.32 (br. d, J¼8.7, 1 H); 7.34 (br. s, 1 H); 7.62 (d, J¼16.0, 1 H). HR-ESI-MS
(pos.): 487.2824 ([MþNa]þ , C30H40NaO4þ ; calc. 487.2824).
(16a)-16-Hydroxybeyeran-19-oic Acid 16-O-4-Methylcinnamate (¼(16a)-16-{[(2E)-3-(4-Methyl-
phenyl)prop-2-enoyl]oxy}beyeran-19-oic Acid; 24). Treatment of 20 (20 mg, 0.06 mmol) with 4-
methylcinnamic acid (22 mg, 0.1 mmol) afforded 24 (9 mg, 31%). White solid. M.p. 95–1008. IR
(KBr): 2926, 2850, 1710, 1695, 1635, 1456, 1310, 1270, 1168. 1H-NMR (400 MHz): 0.80 (s, 3 H); 0.95 (s,
3 H); 1.22 (s, 3 H); 2.37 (s, 3 H); 4.85 (dd, J¼5.0, 9.6, 1 H); 6.40 (d, J¼15.8, 1 H); 7.17 (d, J¼7.8, 2 H);
7.42 (d, J¼7.8, 2 H); 7.62 (d, J¼15.8, 1 H). HR-ESI-MS (pos.): 487.2827 ([MþNa]þ , C30H40NaO4þ ; calc.
487.2824).
(16a)-16-Hydroxybeyeran-19-oic Acid 16-O-Trifluorocrotonate (¼(16a)-16-{[(2E)-4,4,4-Trifluoro-
but-2-enoyl]oxy}beyeran-19-oic acid; 25). Treatment of 20 (30 mg, 0.09 mmol) with 4,4,4-trifluorocro-
tonic acid (31 mg, 0.2 mmol) afforded 25 (11 mg, 27%). White solid. M.p. 90–968. IR (KBr): 3448, 3020,
2929, 2852, 1724, 1693, 1666, 1519, 1467, 1440, 1308, 1261, 1215, 1137, 1052. 1H-NMR (400 MHz): 0.79 (s,

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CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
3 H); 0.93 (s, 3 H); 1.22 (s, 3 H); 4.84 (dd, J¼5.0, 10.1, 1 H); 6.51 (dq, J¼1.8, 15.8, 1 H); 6.74 (dq, J¼6.4,
15.8, 1 H). HR-ESI-MS (neg.): 441.2235 ([MꢀH]ꢀ , C24H32O4F3ꢀ ; calc. 441.2253).
Methyl Isosteviol (¼ Methyl 16-Oxobeyeran-19-oate; 26). As described for the preparation of 3,
treatment of 19 (145 mg, 0.5 mmol) with TMSꢀCHN2 (2 ml, 1.2 mmol) gave 26 (150 mg, 99%), which was
identified by spectral comparison with literature data [19].
(16a)-Beyerane-16,19-diol (27). As described for the preparation of 4, treatment of 26 (148 mg,
0.4 mmol) with LiAlH4 (374 mg, 9.8 mmol) yielded 27 (112 mg, 82%), which was identified by spectral
comparison with literature data [16].
16a,19-Dihydroxybeyerane 19-O-Cinnamate (¼(16a)-16-Hydroxybeyeran-19-yl (2E)-3-Phenyl-
prop-2-enoate; 28) and 16a,19-Dihydroxybeyerane Di-O-cinnamate (¼(16a)-Beyerane-16,19-diyl
(2E,2’E)-Bis(3-phenylprop-2-enoate); 33). Treatment of 27 (32 mg, 0.1 mmol) with cinnamic acid
(66 mg, 0.4 mmol) afforded a crude product (74 mg), which was subjected to TLC (hexane/AcOEt 7:3)
to furnish 28 (22 mg, 48%) and 33 (16 mg, 27%).
Data for 28. White solid. M.p. 135–1408. IR (KBr): 3436, 2931, 2845, 1704, 1640, 1450, 1387, 1284,
1
1253, 1205, 1182. H-NMR (400 MHz): 0.91 (s, 3 H); 0.95 (s, 3 H); 1.01 (s, 3 H); 3.86 (dd, J¼4.6, 11.0,
1 H); 3.98 (d, J¼11.0, 1 H); 4.39 (d, J¼11.0, 1 H); 6.43 (br. d, J¼16.0, 1 H); 7.36–7.38 (m, 3 H); 7.51–
7.53 (m, 2 H); 7.65 (d, J¼16.0, 1 H). HR-ESI-MS (pos.): 459.2879 ([MþNa]þ , C29H40NaO3þ ; calc.
459.2875).
1
Data for 33. Amorphous solid. IR (KBr): 2929, 2849, 1711, 1638, 1450, 1310, 1203, 1173. H-NMR
(400 MHz): 0.94 (s, 3 H); 0.96 (s, 3 H); 1.02 (s, 3 H); 3.99 (d, J¼11.0, 1 H); 4.38 (d, J¼11.0, 1 H); 4.88
(dd, J¼5.0, 9.6, 1 H); 6.42 (br. d, J¼15.6, 1 H); 6.47 (br. d, J¼15.6, 1 H); 7.37–7.39 (m, 6 H); 7.51–7.56
(m, 4 H); 7.65 (d, J¼15.6, 1 H); 7.67 (d, J¼15.6, 1 H). HR-ESI-MS (pos.): 589.3299 ([MþNa]þ
,
C38H46NaOþ4 ; calc. 589.3294).
16a,19-Dihydroxybeyerane 19-O-2-Methylcinnamate (¼(16a)-16-Hydroxybeyeran-19-yl (2E)-3-(2-
Methylphenyl)prop-2-enoate; 29) and 16a,19-Dihydroxybeyerane Di-O-2-methylcinnamate (¼(16a)-
Beyerane-16,19-diyl (2E,2’E)-Bis[3-(2-methylphenyl)prop-2-enoate]; 34). Treatment of 27 (31 mg,
0.1 mmol) with 2-methylcinnamic acid (61 mg, 0.4 mmol) gave 29 (8.7 mg, 19%) and 34 (9.3 mg, 15%).
Data for 29. White solid. M.p. 133–1428. IR (KBr): 3488, 2929, 2847, 1700, 1632, 1458, 1321, 1276,
1180, 1163, 1001. 1H-NMR (400 MHz): 0.91 (s, 3 H); 0.96 (s, 3 H); 1.01 (s, 3 H); 2.43 (s, 3 H); 3.86 (dd, J¼
4.6, 10.5, 1 H); 4.00 (d, J¼11.0, 1 H); 4.38 (d, J¼11.0, 1 H); 6.34 (d, J¼15.6, 1 H); 7.18–7.22 (m, 2 H);
7.24–7.28 (m, 1 H); 7.56 (br. d, J¼7.3, 1 H); 7.96 (d, J¼15.6, 1 H). HR-ESI-MS (pos.): 473.3020 ([Mþ
Na]þ , C30H42NaOþ3 ; calc. 473.3032).
Data for 34. Amorphous solid. IR (KBr): 2925, 2850, 1713, 1635, 1456, 1312, 1264, 1173, 1033.
1H-NMR (400 MHz): 0.95 (s, 3 H); 0.98 (s, 3 H); 1.03 (s, 3 H); 2.44 (s, 3 H); 2.45 (s, 3 H); 4.01 (d, J¼11.0,
1 H); 4.37 (d, J¼11.0, 1 H); 4.90 (dd, J¼5.0, 10.1, 1 H); 6.35 (d, J¼16.0, 1 H); 6.39 (d, J¼16.0, 1 H);
7.12–7.22 (m, 6 H); 7.49 (d, J¼7.8, 1 H); 7.52 (d, J¼7.3, 1 H); 7.96 (d, J¼16.0, 1 H); 7.99 (d, J¼16.0, 1 H).
HR-ESI-MS (pos.): 617.3606 ([MþNa]þ , C40H50NaO4þ ; calc. 617.3607).
16a,19-Dihydroxybeyerane 19-O-3-Methycinnamate (¼(16a)-16-Hydroxybeyeran-19-yl (2E)-3-(3-
Methylphenyl)prop-2-enoate; 30) and 16a,19-Dihydroxybeyerane Di-O-3-methylcinnamate (¼(16a)-
Beyerane-16,19-diyl (2E,2’E)-Bis[3-(3-methylphenyl)prop-2-enoate]; 35). Treatment of 27 (29 mg,
0.09 mmol) with 3-methylcinnamic acid (62 mg, 0.4 mmol) afforded 30 (19 mg, 45%) and 35 (13 mg,
23%).
Data for 30. White solid. M.p. 125–1328. IR (KBr): 3436, 2930, 2846, 1706, 1628, 1452, 1309, 1284,
1253, 1205, 1181. 1H-NMR (400 MHz): 0.91 (s, 3 H); 0.95 (s, 3 H); 1.01 (s, 3 H); 2.37 (s, 3 H); 3.86 (dd, J¼
4.6, 10.5, 1 H); 3.97 (d, J¼11.0, 1 H); 4.38 (d, J¼11.0, 1 H); 6.41 (d, J¼16.0, 1 H); 7.18 (br. d, J¼6.9,
1 H); 7.27 (br. t, J¼7.8, 1 H); 7.33 (br. d, J¼6.9, 1 H); 7.34 (br. s, 1 H); 7.62 (d, J¼16.0, 1 H). HR-ESI-MS
(pos.): 473.3016 ([MþNa]þ , C30H42NaO3þ ; calc. 473.3032).
Data for 35. Amorphous solid. IR (KBr): 2926, 2850, 1714, 1635, 1456, 1372, 1312, 1265, 1236, 1176.
1H-NMR (400 MHz): 0.93 (s, 3 H); 0.96 (s, 3 H); 1.01 (s, 3 H); 2.37 (s, 3 H); 2.38 (s, 3 H); 3.98 (d, J¼11.0,
1 H); 4.37 (d, J¼11.0, 1 H); 4.87 (dd, J¼5.5, 9.6, 1 H); 6.40 (d, J¼16.0, 1 H); 6.45 (d, J¼16.0, 1 H); 7.18–
7.20 (m, 2 H); 7.25–7.27 (m, 2 H); 7.32–7.36 (m, 4 H); 7.62 (d, J¼16.0, 1 H); 7.63 (d, J¼16.0, 1 H). HR-
ESI-MS (pos.): 617.3580 ([MþNa]þ , C40H50NaO4þ ; calc. 617.3607).

CHEMISTRY & BIODIVERSITY – Vol. 10 (2013)
187
16a,19-Dihydroxybeyerane 19-O-4-Methycinnamate (¼(16a)-16-Hydroxybeyeran-19-yl (2E)-3-(4-
Methylphenyl)prop-2-enoate; 31) and 16a,19-Dihydroxybeyerane Di-O-4-methylcinnamate (¼(16a)-
Beyerane-16,19-diyl (2E,2’E)-Bis[3-(4-methylphenyl)prop-2-enoate]; 36). Treatment of 27 (30 mg,
0.1 mmol) with 4-methylcinnamic acid (61 mg, 0.4 mmol) gave 31 (19 mg, 43%) and 36 (9 mg, 15%).
Data for 31. White solid. M.p. 113–1188. IR (KBr): 3421, 2932, 2843, 1705, 1636, 1453, 1254, 1181,
1026. 1H-NMR (400 MHz): 0.91 (s, 3 H); 0.95 (s, 3 H); 1.00 (s, 3 H); 2.37 (s, 3 H); 3.86 (dd, J¼4.6, 10.5,
1 H); 3.97 (d, J¼11.0, 1 H); 4.38 (d, J¼11.0, 1 H); 6.38 (d, J¼16.0, 1 H); 7.18 (d, J¼8.0, 2 H); 7.42 (d, J¼
8.0, 2 H); 7.62 (d, J¼16.0, 1 H). HR-ESI-MS (pos.): 473.3011 ([MþNa]þ , C30H42NaO3þ ; calc. 473.3032).
1
Data for 36. Amorphous solid. IR (KBr): 2925, 2850, 1714, 1635, 1456, 1310, 1257, 1168. H-NMR
(400 MHz): 0.94 (s, 3 H); 0.96 (s, 3 H); 1.02 (s, 3 H); 2.37 (s, 3 H); 2.38 (s, 3 H); 3.98 (d, J¼11.0, 1 H); 4.38
(d, J¼11.0, 1 H); 4.88 (dd, J¼5.0, 9.6, 1 H); 6.38 (d, J¼16.0, 1 H); 6.43 (d, J¼16.0, 1 H); 7.18 (d, J¼7.7,
2 H); 7.19 (d, J¼8.2, 2 H); 7.43 (d, J¼8.2, 2 H); 7.45 (d, J¼7.7, 2 H); 7.63 (d, J¼16.0, 1 H); 7.64 (d, J¼
16.0, 1 H). HR-ESI-MS (pos.): 617.3564 ([MþNa]þ , C40H50NaO4þ ; calc. 617.3607).
16a,19-Dihydroxybeyerane 19-O-Trifluorocrotonate (¼(16a)-16-Hydroxybeyeran-19-yl (2E)-4,4,4-
Trifluorobut-2-enoate; 32) and 16a,19-Dihydroxybeyerane Di-O-trifluorocrotonate (¼(16a)-Beyerane-
16,19-diyl (2E,2’E)-Bis(4,4,4-trifluorobut-2-enoate); 37). Treatment of 27 (31 mg, 0.1 mmol) with 4,4,4-
trifluorocrotonic acid (65 mg, 0.5 mmol) yielded 32 (18 mg, 41%) and 37 (8 mg, 14%).
Data for 32. White solid. M.p. 112–1198. IR (KBr): 3443, 3019, 2929, 2851, 1727, 1667, 1455, 1391,
1372, 1308, 1261, 1215, 1138, 1053. 1H-NMR (400 MHz): 0.91 (s, 3 H); 0.93 (s, 3 H); 0.97 (s, 3 H); 3.86 (dd,
J¼4.6, 10.5, 1 H); 3.98 (d, J¼11.0, 1 H); 4.41 (d, J¼11.0, 1 H); 6.49 (dq, J¼1.8, 15.6, 1 H); 6.74 (dq, J¼
6.4, 15.6, 1 H). HR-APCI-MS (pos.): 411.2551 ([MþHꢀH2O]þ , C24H34F3O2þ ; calc. 411.2511).
Data of 37. Amorphous solid. IR (KBr): 3020, 2928, 2854, 1726, 1667, 1458, 1442, 1371, 1308, 1262,
1
1215, 1139, 1050. H-NMR (400 MHz): 0.89 (s, 3 H); 0.94 (s, 3 H); 0.98 (s, 3 H); 3.98 (d, J¼11.0, 1 H);
4.38 (d, J¼11.0, 1 H); 4.86 (t, J¼7.5, 1 H); 6.46–6.55 (m, 2 H); 6.70–6.80 (m, 2 H). HR-ESI-MS (pos.):
573.2412 ([MþNa]þ , C28H36F6NaOþ4 ; calc. 573.2415).
Cytotoxicity Assay. Cytotoxicity assay was performed according to the method described in [28].
Briefly, HL60 (leukemia), A549 (lung), AZ521 (stomach), and SK-BR-3 (breast) cell lines (each 3ꢂ103
cells/well) were treated with compounds for 48 h, and then MTT (¼ 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium bromide) soln. was added to the well. After incubation for 3 h, the generated
i
blue formazan was solubilized with 0.04m HCl in PrOH. The absorbances at 570 nm (top) and 630 nm
(bottom) were measured with a microplate reader.
Annexin V/Propidium Iodide Double Staining. Apotosis was detected using an rh Annexin V/FITC
kit [29], and HL60 (1.5ꢂ105 cells) was exposed to test compound (final concentration: 6 mm). To prepare
the cell sample for flow cytometry, cells were washed with annexin-binding buffer and stained with
annexin Vꢀfluorecein isothiocyanate (FITC) and propidium iodide (PI) for 10 min. The cell samples
were analyzed by 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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Received November 30, 2012