Tetrahedron Letters 55 (2014) 7203–7205
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Three new terpenoids, sterebins O, P1, and P2, isolated from Stevia
rebaudiana fermented by Saccharomyces cerevisiae
Hitoshi Kamauchi a, Tatsuhiko Kon b, Kaoru Kinoshita a, Kunio Takahashi a, Kiyotaka Koyama a,⇑
a
b
Department of Pharmacognosy and Phytochemistry, Meiji Pharmaceutical University, Noshio 2-522-1, Kiyose-shi, Tokyo 204-8588, Japan
Shalom Co., Ltd, Shiboukusa Aza Tachizawa 3041-6, Oshino-mura, Minamitsuru-gun, Yamanashi 401-0511, Japan
a r t i c l e
i n f o
Article history:
Received 23 August 2014
Revised 30 October 2014
Accepted 4 November 2014
Available online 15 November 2014
Keywords:
Fermentation
Diversity
Stevia rebaudiana
Saccharomyces cerevisiae
Melanogenesis
a b s t r a c t
The diversity of natural compounds is extensive but not unlimited. We therefore studied the feasibility of
fermenting plant extracts to increase the diversity of natural compounds for use as drug candidates.
Three new terpenoids, sterebins O (1), P1 (2), and P2 (3), were isolated from an extract of Stevia rebaudiana fermented by Saccharomyces cerevisiae. The structures of these compounds were established using
NMR, MS, and IR methods. The absolute configuration of 1 was determined by X-ray diffraction analysis,
and that of 2 and 3 from the ECD spectrum of their benzoate derivatives. These three compounds were
not observed in S. rebaudiana extracts by TLC, suggesting that the compounds were generated during
the fermentation process. Compounds 1, 2, and 3 all inhibited melanogenesis in theophylline-stimulated
B16 melanoma cells, with 1 exhibiting the lowest IC50 value (9.8 lM). The results indicate that fermentation of plant extracts may provide a route for generating many useful compounds.
Ó 2014 Elsevier Ltd. All rights reserved.
Introduction
The diversity of natural compounds plays a pivotal role in the
development of drugs. However, the contribution of naturally
occurring compounds to the development of drugs will likely
decrease because of the difficulty of finding novel compounds from
natural resources. Therefore, new approaches for achieving chemical diversity are anticipated. One of these approaches is Diversity
Oriented Synthesis.1 Diversity Oriented Synthesis leads to structurally complex and diverse compounds from simple compounds.
Diversity Enhanced Extracts are natural extracts subjected to
Diversity Oriented Synthesis2 and are another approach for
increasing chemical diversity. Diversity Enhanced Extracts
therefore provide an efficient way for obtaining novel naturalproduct-like compounds.
We recently focused on the fermentation of natural resources as
a novel approach for increasing chemical diversity. For example,
dicoumarol, isolated from sweet clover fermented by microbes,3
was the lead compound for the cardiovascular drug WarfarinÒ.
Therefore microbial fermentation of natural resources can induce
chemical diversity. In this letter, three new terpenoids, sterebins
O (1), P1 (2), and P2 (3), were isolated from Stevia rebaudiana fermented by Saccharomyces cerevisiae. The absolute configuration of
1 was determined by X-ray diffraction analysis, and those of 2 and
⇑ Corresponding author. Tel.: +81 42 495 8913; fax: +81 42 495 8912.
E-mail address: [email protected] (K. Koyama).
http://dx.doi.org/10.1016/j.tetlet.2014.11.012
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.
3 were determined from the ECD spectrum of their benzoate
derivatives.
Evaluation of the melanogenesis inhibitory activities of 1, 2, and
3 using B16 melanoma cells showed that all exhibit activity, with
IC50 values of 9.8 lM, 17 lM, and 75 lM, respectively.
Results and discussion
Leaves and stems of Stevia rebaudiana were fermented by Saccharomyces cerevisiae, extracted with EtOH/H2O (3:7), then the
extract was subjected to Diaion HP-20 column chromatography
(CC), silica gel CC, and octadecylsilyl silica gel CC. Three new terpenoids, sterebins O (1), P1 (2), and P2 (3), were obtained (Fig. 1).
Sterebin O (1)4 was obtained as colorless needles, and the
molecular formula was deduced to be C16H26O4 by HRFABMS
(m/z 283.1907 [M+H]+). The IR spectrum showed absorptions
12
1
20
2
10
3
5
4
19
18
1
13
O
8
H
H
14
11
9
7
6
OH
15
O
O
H
H
17
OH
OH
OH16
O
H
OH
OH
2
H
Figure 1. Structures of compounds 1–3.
OH
OH
3
7204
H. Kamauchi et al. / Tetrahedron Letters 55 (2014) 7203–7205
attributable to hydroxyl (3450 cm 1) and carbonyl (1740 cm 1)
groups. The 1H NMR spectrum showed signals attributable to
two oxymethines at H-6 (dH 3.73, 1H, m overlapped) and H-7 (dH
3.70, 1H, m overlapped). Furthermore, the 13C NMR spectrum
showed one oxygenated quaternary carbon C-8 (dC 87.7), two oxygenated carbons C-6 (dC 73.0) and C-7 (dC 81.5), three methylenes
C-1 (dC 39.1), C-2 (dC 17.9) and C-3 (dC 43.2), and four methyl
carbons C-17 (dC 17.9), C-18 (dC 35.7), C-19 (dC 22.1) and C-20 (dC
16.4) (Table 1). HMBC correlations were present from H-5 to C-6
and C-10, H-11a to C-8 and C-12, H-11b to C-9 and C-12, H-17
to C-7 and C-8, H-19 to C-3, C-4 and C-18, H-20 to C-1, C-5 and
C-9, and a 1H–1H COSY correlation was evident from H-5 to H-6
(Fig. 2). These spectroscopic data for 1 were similar to those for
sterebin A,5 except for the side chain. The HMBC correlations from
H-11b to C-9 and C-12, and the IR spectrum (1740 cm 1), showed
that sterebin O (1) contains a lactone ring (Fig. 2). The absolute
configuration of sterebin O (1) was determined by X-ray analysis
(deposited at the Cambridge Crystallographic Data Centre, reference number CCDC 989363) with a flack parameter x = 0.0(2)6
(Fig. 3).
Sterebin P1 (2)7 was obtained as a colorless oil. The molecular
formula, C20H34O5, was based on HRFABMS (m/z 339.2530
[M+H]+). The 1H NMR spectrum showed signals attributable to olefinic protons at H-14 (dH 5.81, 1H, dd, J = 17.4, 10.7), H-15a (dH
5.22, 1H, d, J = 17.4), and H-15b (dH 5.03, 1H, d, J = 10.7) and three
oxymethines at H-6 (dH 3.68, 1H, dd, J = 11.0, 8.8), H-7 (dH 3.49, 1H,
d, J = 8.8), H-12 (dH 3.98, 1H, dd, J = 9.3, 3.4). Furthermore, the 13C
NMR spectrum (Table 1) showed two olefinic carbons C-14 (dC
142.3) and C-15 (dC 113.8), two oxygenated quaternary carbons
C-8 (dC 83.7) and C-13 (dC 74.6), three oxygenated carbons C-6
(dC 73.0), C-7 (dC 82.2) and C-12 (dC 83.0), four methylenes C-1
(dC 39.5), C-2 (dC 18.1), C-3 (dC 43.5), and C-11 (dC 24.1), and five
methyl carbons C-16 (dC 21.7), C-17 (dC 17.6), C-18(dC 35.8), C-19
(dC 22.6), and C-20 (dC 16.1) (Table 1). Comparison of the NMR
spectra of sterebin P1 (2) and sterebin O (1) allowed assignment
of C-1 to C-10 and methyl signals (C-17, 18, 19 and 20). The HMBC
correlations from H-16 to C-12, C-13, and C-14, and the 1H–1H
COSY correlations from H-11 to H-12 and H-14 to H-15 allowed
OH
O
O
O
H
H
OH
O
H
OH
OH
1
H
H
OH
H
OH
2
OH
OH
3
Figure 2. 1H–1H COSY (bold lines) and key HMBC (arrows) correlations for
compounds 1–3.
Figure 3. ORTEP drawing of 1 obtained by X-ray analysis [Flack parameter:
x = 0.0(2)].
assignment of the side chain (Fig. 2). 1H and 13C NMR spectra of
this side chain showed close similarities to those of 12b,19dihydroxymanoyl oxide.8 The relative configuration of sterebin
P1 (2) was established by analysis of NOESY correlations (Fig. 4).
The absolute configuration of sterebin P1 (2) was determined using
the non-empirical exciton chirality CD method.9 The benzoate
derivative (4)10 was obtained from sterebin P1 (2) by treatment
with DMAP and benzoyl chloride in pyridine (Scheme 1). The
ECD spectrum of 4 had a Cotton effect at 239 nm ([h] 7332) and
Table 1
NMR spectroscopic data (400 MHz, CDCl3, d in ppm) for compounds 1–3
Position
Sterebin O (1)
dC
1
39.1
2
17.9
3
43.2
4
5
6
7
8
9
10
11a
b
12
13
14
15
33.9
58.9
73.0
81.5
87.7
56.2
35.4
28.4
16
17
18
19
20
dH (J in Hz)
1.07
1.42
1.25
1.48
1.24
1.47
(1H,
(1H,
(1H,
(1H,
(1H,
(1H,
m)
m)
m)
m)
m)
m)
1.28 (1H, m)
3.73 (1H, m)
3.70 (1H, m)
2.01 (1H, dd, 14.7, 6.7)
2.29 (1H, dd, 16.1, 6.7)
2.49 (1H, dd, 16.1, 14.7)
176.3
17.9
35.7
22.1
16.4
Sterebin P1 (2)
dC
39.5
18.1
43.5
35.3
59.1
73.0
82.2
83.7
57.7
33.7
24.1
83.0
74.6
142.3
113.8
1.35
1.17
1.00
0.97
(3H,
(3H,
(3H,
(3H,
s)
s)
s)
s)
21.7
17.6
35.8
22.6
16.1
dH (J in Hz)
1.01
1.42
1.38
1.68
1.20
1.41
(1H,
(1H,
(1H,
(1H,
(1H,
(1H,
m)
m)
m)
m)
m)
m)
1.17 (1H, m)
3.68 (1H, dd, 11.0, 8.8)
3.49 (1H, d, 8.8)
1.45 (1H, dd, 13.1, 7.3)
1.65 (1H, m)
1.76 (1H, m)
3.98 (1H, dd, 9.3, 3.4)
5.81
5.22
5.03
1.22
1.17
1.15
0.98
0.88
(1H,
(1H,
(1H,
(3H,
(3H,
(3H,
(3H,
(3H,
dd, 17.4, 10.7)
d, 17.4)
d, 10.7)
s)
s)
s)
s)
s)
Sterebin P2 (3)
dC
38.4
18.2
43.2
33.8
57.8
71.5
85.5
77.1
46.6
36.7
22.7
70.1
77.6
142.5
115.5
27.4
21.9
35.9
22.1
17.3
dH (J in Hz)
0.93
1.50
1.42
1.71
1.20
1.35
(1H,
(1H,
(1H,
(1H,
(1H,
(1H,
m)
m)
m)
m)
m)
m)
1.21 (1H, d, 10.7)
3.68 (1H, m)
3.52 (1H, d, 9.2)
1.84 (1H, m)
1.76 (1H, m)
1.86 (1H, m)
3.71 (1H, m)
5.82
5.39
5.23
1.33
1.31
1.16
1.00
0.86
(1H,
(1H,
(1H,
(3H,
(3H,
(3H,
(3H,
(3H,
dd, 17.1, 10.7)
dd, 17.1, 1.5)
dd, 10.7, 1.5)
s)
s)
s)
s)
s)
H. Kamauchi et al. / Tetrahedron Letters 55 (2014) 7203–7205
CH3
H3C
H
CH3
H
HO
CH3
CH3
H
OH
CH3
H
O
OH
H
H
H
CH3
H
H3C
HO
H
CH3
OH
O
OH
H
H
H
CH3
H
2
3
7205
The isolated compounds were tested for their inhibitory activity
against melanin synthesis in theophylline-stimulated B16 melanoma cells.13 Sterebins O (1), P1 (2), and P2 (3) showed melanin
synthesis inhibitory activity (IC50 = 9.8 lM, 17 lM, and 75 lM,
respectively). The activities of the isolated compounds were higher
than those of arbutin (IC50 = 408 lM) which was used as a positive
control. Under these conditions, the isolated compounds did not
decrease cell numbers compared with the control.
Supplementary data
Figure 4. NOESY correlations for compounds 2, 3.
OH
O
H
H
OH
H
OH
OH
3
O
H
H
OH
2
OH
O
H
OBz
Bz-Cl
DMAP
70°C
48 h
Bz-Cl
DMAP
70°C
48 h
OBz
OBz
4
OBz
O
H
H
OBz
OBz
5
Scheme 1. Benzoylation of 2 and 3.
225 nm ([h] +7896). The negative Cotton effect at the longer
wavelength indicates negative chirality between the two benzoyl
groups at C-6 and C-7. Therefore, the absolute configurations of
C-6 and C-7 were determined to be R and R, respectively.
Sterebin P2 (3)11 was obtained as a colorless oil, and the
molecular formula was deduced to be C20H34O5 by HRFABMS
(m/z 339.2543 [M+H]+). 1H and 13C NMR spectra of sterebin P2
(3) were similar to those of sterebin P1 (2) (Table 1), indicating that
sterebin P2 (3) was likely a stereoisomer of sterebin P1 (2). The
major differences were the splitting pattern and chemical shift at
C-9 (dH 1.84, m; dC 46.6 in 3; dH 1.45, dd, J = 13.1, 7.3 Hz; dC 57.7
in 2), at C-12 (dH 3.71, m; dC 70.1 in 3; dH 3.98, dd, J = 9.3, 3.4 Hz;
dC 83.0 in 2), C-16 (dH 1.33, s; dC 27.4 in 3; dH 1.22, s; dC 21.7 in
2) and at C-17 (dH 1.31, s; dC 21.9 in 3; dH 1.17, s; dC 17.6 in 2).
The relative configuration of sterebin P2 (3) was established by
analyzing its NOESY correlations, except for C-12 (Fig. 4). The
relative configuration of C-12 was established from the benzoate
derivative (5)12 obtained from sterebin P2 (3) (Scheme 1). The
splitting pattern of H-12 (dH 5.20, 1H, t, J = 4.4) showed that the
12-hydroxyl group was in the axial orientation. The ECD spectrum
of 5 had a Cotton effect at 240 nm ([h] 3166) and 226 nm ([h]
+6533). Therefore, the absolute configurations of C-6 and C-7 were
determined to be R and R, respectively.
Supplementary data (experiment procedures, NMR spectra for
compounds 1–3) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.tetlet.2014.11.012.
References and notes
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Schreiber, S. L. Science 2000, 287, 1964–1969.
Kikuchi, H.; Sakurai, K.; Oshima, Y. Org. Lett. 2014, 16, 1916–1919.
Hollman, A. Br. Heart J. 1991, 66, 181.
Data for 1: Colorless needles (n-hexane–benzene); mp 158–159 °C; [a]19
D +36 (c
0.80, MeOH); IR (KBr) mmax 3450, 2940, 1740, 1560, 1460, 1100 cm 1; 13C NMR
1
(CDCl3) see Table 1; H NMR (CDCl3) see Table 1; FABMS (pos.) m/z (%): 283
[M+H]+ (100), 247(32); HRFABMS m/z 283.1907 [M+H]+ (calcd for C16H27O4,
283.1831).
Oshima, Y.; Saito, J.; Hikino, H. Tetrahedron 1986, 42, 6443–6446.
Flack, H. D. Acta Crystallogr. 1983, A39, 876–881.
Data for 2: Colorless oil; [a]18
D +16 (c 0.95, MeOH); IR (KBr) mmax 3450, 2940,
1700, 1060 cm 1; 13C NMR (CDCl3) see Table 1; 1H NMR (CDCl3) see Table 1;
+
FABMS m/z 339 [M+H] (8), 321 (100), 303 (19), 285 (20), 205 (23), 177 (18);
HRFABMS m/z 339.2530 [M+H]+ (calcd for C20H35O4, 344.2535).
Li, Y.; Kuo, Y. Chem. Pharm. Bull. 2002, 50, 498–500.
Nakanishi, K.; Harada, N. J. Am. Chem. Soc. 1969, 91, 3989–3991.
Data for 4: Colorless oil; [a]20
D +52 (c 0.07, MeOH); UV kmax (MeOH) nm (log e):
274 (3.41), 230 (4.11); IR (ATR) mmax 2961, 1722, 1260 cm 1; ECD kmax (MeOH)
25
nm ([h] ): 239 ( 7332), 225 (+7896); 13C NMR (CDCl3, 400 MHz) d: 16.1 (C20), 18.0 (C-2), 18.5 (C-17), 21.4 (C-19), 21.6 (C-16), 24.0 (C-11), 33.7 (C-4),
35.6 (C-10), 36.7 (C-18), 39.8 (C-1), 43.8 (C-3), 57.6 (C-9), 59.4 (C-5), 72.9 (C-6),
80.7 (C-13), 81.5 (C-7), 82.0 (C-12), 84.3 (C-8), 115.3 (C-15), 137.8 (C-14)
128.1–128.3, 129.5–129.9, 131.5, 132.5–132.8, 165.0–166.1 (each Bz); 1H NMR
(CDCl3 400 MHz) d: 0.91 (3H, s, H-20), 0.98 (3H, s, H-19), 1.09 (3H, s, H-18),
1.44 (3H, s, H-17), 1.69 (1H, d, J = 11.5 Hz, H-5), 1.73 (3H, s, H-16), 4.47 (1H, dd,
J = 10.4, 3.4 Hz, H-12), 5.35 (1H, d, J = 11.2 Hz, H-15b), 5.37 (1H, d, J = 17.6 Hz,
H-15a), 5.45 (1H, d, J = 9.3 Hz, H-7), 5.79 (1H, dd, J = 11.5, 9.3 Hz, H-6), 6.13
(1H, dd, J = 17.6, 11.2 Hz, H-14), 7.37–8.03 (15H, m, each Bz); FABMS m/z 651
[M+H]+ (7), 529 (20), 419 (38), 353 (38), 321 (43), 285 (38), 219 (100), 207
(79); HRFABMS m/z 673.3134 [M+Na]+ (calcd for C41H46O7Na, 673.3141).
1
Data for 3: [a]23
;
D +9 (c 0.28, MeOH); IR (KBr) mmax 3450, 2940, 1700, 1060 cm
13
C NMR (CDCl3) see Table 1; 1H NMR (CDCl3) see Table 1; FABMS m/z 339
+
[M+H] (20), 321 (100), 303 (28), 285 (28), 205 (37), 177 (28); HRFABMS m/z
339.2543 [M+H]+ (calcd for C20H35O4, 339.2535).
Data for 5: Colorless oil; [a]19
D +59 (c 0.13, MeOH); UV kmax (MeOH) nm (log e):
274 (3.18), 230 (3.86); IR (ATR) mmax 2959, 1721, 1264 cm 1; ECD kmax (MeOH)
25
nm ([h] ): 240 ( 3166), 226 (+6533); 13C NMR (CDCl3, 400 MHz) d: 16.7 (C20), 18.0 (C-2), 21.8 (C-17), 21.8 (C-19), 22.6 (C-11), 28.1 (C-16), 33.6 (C-4),
35.8 (C-10), 37.3 (C-18), 38.7 (C-1), 43.4 (C-3), 48.3 (C-9), 57.9 (C-5), 72.0 (C12), 73.1 (C-6), 75.7 (C-8), 77.3 (C-13), 83.4 (C-7), 113.9 (C-15), 141.5 (C-14)
128.0-128.5, 129.5–130.3, 132.4–133.1, 165.8–166.1 (each Bz); 1H NMR (CDCl3
400 MHz) d: 0.93 (3H, s, H-20), 0.97 (3H, s, H-19), 1.07 (3H, s, H-18), 1.23 (3H, s,
H-17), 1.62 (3H, s, H-16), 1.70 (1H, d, J = 11.7 Hz, H-5), 5.04 (1H, dd, J = 10.7,
2.0 Hz, H-15b), 5.20 (1H, t, J = 4.4 Hz, H-12), 5.54 (1H, dd, J = 16.8, 2.0 Hz, H15a), 5.60 (1H, d, J = 10.0 Hz, H-7), 5.78 (1H, dd, J = 11.7, 10.0 Hz, H-6), 5.85
(1H, dd, J = 16.8, 10.7 Hz, H-14), 7.39–8.08 (15H, m, each Bz); FABMS m/z 651
[M+H]+ (100), 529 (65), 407 (46), 391 (37); HRFABMS m/z 651.3323 [M+H]+
(calcd for C41H47O7, 651.3244).
Yoon, N.; Eom, T.; Kim, M.; Kim, S. J. Agric. Food Chem. 2009, 57, 4124–4129.