Tricalysiolides A-F, new rearranged ent-kaurane diterpenes from Tricalysia dubia

Article abstract of DOI:10.1016/j.tet.2005.11.024

Six rearranged ent-kaurane diterpenes, tricalysiolides A-F, having the cafestol-type carbon framework were isolated from the wood of Tricalysia dubia (Rubiaceae). Their absolute structures were determined on the basis of the 2D NMR spectroscopy, X-ray cry

Full text of DOI:10.1016/j.tet.2005.11.024

Tetrahedron 62 (2006) 1512–1519
Tricalysiolides A–F, new rearranged ent-kaurane
diterpenes from Tricalysia dubia
Koichi Nishimura,a Yukio Hitotsuyanagi,a Noriko Sugeta,a Kei-ichi Sakakura,a Kazuya Fujita,a
Haruhiko Fukaya,a Yutaka Aoyagi,a Tomoyo Hasuda,a Takeshi Kinoshita,b Dong-Hui He,c
Hideaki Otsuka,c Yoshio Takedad and Koichi Takeyaa,
*
aSchool of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
bFaculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko-machi, Tsukui-gun, Kanagawa 199-0195, Japan
cGraduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan
dFaculty of Integrated Arts and Sciences, The University of Tokushima, 1-1 Minamijosanjima-cho, Tokushima 770-8502, Japan
Received 29 September 2005; accepted 4 November 2005
Available online 15 December 2005
Abstract—Six rearranged ent-kaurane diterpenes, tricalysiolides A–F, having the cafestol-type carbon framework were isolated from the
wood of Tricalysia dubia (Rubiaceae). Their absolute structures were determined on the basis of the 2D NMR spectroscopy, X-ray
crystallographic analysis, and chemical methods.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
17
13
15
11
9
OH
OR2
OH
OH
R
20
C
1
14
7
16
Tricalysia dubia (Lindl.) Ohwi (Rubiaceae) is an evergreen
shrub or tree that is wildly distributed in Taiwan and the
southern parts of China and Japan. From the leaves of this
plant, unique rearranged ent-kaurane glycosides, tricalysio-
sides A–G, have been isolated.1 In the present study, from
the wood of this plant, we isolated six new rearranged ent-
kaurane diterpenes, tricalysiolides A–F (1–6), and deter-
mined their structures.
2
R1
O
H
A
4
HB
6
O
O
H
H
18
19
R1
R2
O
R
tricalysiolide A (1)
tricalysiolide B (2)
tricalysiolide C (3)
tricalysiolide D (4)
H
OH
H
H
H
Me
tricalysiolide E (5)
H
tricalysiolide F (6) OH
OMe
H
Figure 1. Structures of tricalysiolides A–F (1–6).
2. Results and discussion
(3365 cmK1) and lactone (1742 cmK1) groups. The 1H
NMR spectrum showed the presence of one tertiary
methyl (d 0.67) and one olefinic proton (d 5.73)
(Table 1). The 13C NMR spectrum suggested that 1
was a diterpenoid derivative with a total of 20 carbons,
consisting of one methyl, nine methylenes, four methines,
one trisubstituted double bond giving highly shielded (d
111.4) and deshielded (d 175.1) signals, one carbonyl,
and three quaternary carbons (Table 2). On the basis of
detailed analysis of the 1H–1H COSY, HMQC, and
HMBC spectra, 1 was shown to be a cafestol-type
rearranged kaurane diterpenoid with two hydroxyls at
C-16 and C-17. In the HMBC spectrum, H-18 was
correlated with C-3, C-4, C-5, and C-19, which implied
that 1 had an a,b-unsaturated-g-lactone group attached to
ring A (Fig. 2). From the NOESY correlations between
By a series of column chromatography on highly porous
synthetic resin (Diaion HP-20), silica gel, aminopropyl-
bonded silica gel, and ODS HPLC, a hot MeOH extract of
air-dried wood of T. dubia afforded six diterpenes named
tricalysiolides A–F (1–6) (Fig. 1).
Tricalysiolide A (1) was isolated as an amorphous solid,
whose molecular formula was determined to be
C20H28O4 from the [MCH]C peak at m/z 333.2074
(calcd for C20H29O4, 333.2066) in the HRESIMS. The
IR spectrum indicated that 1 possessed hydroxyl
Keywords: Tricalysiolide; Diterpene; Tricalysia dubia.
*
Corresponding author. Tel.: C81 426 76 3007; fax: C81 426 77 1436;
e-mail: takeyak@ps.toyaku.ac.jp
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.11.024

K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519
Table 1. H NMR data for tricalysiolides A–F (1–6) in C5D5Na
1513
1
Proton
1b
2b
3b
4b
5c
6b
1a
1b
2a
2b
1.67 (m)
1.78 (m)
1.52 (m)
2.52 (d, 13.7)
2.00 (m)
1.68 (m)
1.27 (m)
2.40 (ddd, 14.0, 4.1, 2.3)
1.87 (dd, 14.0, 4.7)
1.66 (m)
2.32 (dd, 17.6, 6.7)
1.80 (m)
5.62 (dt, 6.7, 2.1)
4.40 (t, 6.1)
0.97 (td, 13.8, 3.7)
2.21 (m)
1.52 (m)
0.94 (td, 13.8, 3.6)
2.20 (m)
1.49 (m)
6.09 (dd, 6.4, 1.6)
3
5
6a
6b
7a
7b
9
11a
11b
12a
12b
13
14a
14b
15a
15b
17a
17b
18
4.75 (t, 8.9)
1.90 (m)
1.50 (m)
1.41 (m)
1.61 (m)
1.49 (m)
1.13 (d, 8.8)
1.69 (m)
1.47 (m)
1.86 (m)
1.42 (m)
2.43 (s-like)
2.02 (dd, 11.3, 4.1)
1.92 (d, 11.3)
1.82 (d, 14.2)
1.72 (d, 14.2)
4.11 (dd, 10.8, 4.6)
4.05 (dd, 10.8, 4.6)
5.73 (s)
4.75 (t, 9.3)
1.87 (m)
1.44 (m)
1.40 (m)
1.52 (m)
1.45 (m)
1.13 (d, 8.8)
1.69 (m)
1.46 (m)
1.85 (m)
1.45 (m)
2.48 (s-like)
1.82 (d, 11.0)
1.69 (m)
1.74 (d, 14.5)
1.48 (d, 14.5)
4.08 (dd, 12.3, 4.7)
4.00 (dd, 12.3, 4.7)
5.73 (s)
2.57 (d, 9.7)
1.59 (m)
1.38 (m)
1.65 (m)
1.56 (m)
1.29 (d, 8.6)
1.76 (m)
1.54 (m)
1.91 (m)
1.50 (m)
2.47 (s-like)
2.06 (dd, 11.1, 4.7)
1.97 (d, 11.1)
1.86 (d, 14.3)
1.77 (d, 14.3)
4.14 (dd, 10.8, 5.0)
4.06 (dd, 10.8, 5.0)
5.81 (s)
2.12 (m)
1.56 (m)
1.48 (m)
1.67 (m)
1.58 (m)
1.26 (d, 9.0)
1.76 (m)
1.52 (m)
1.92 (m)
1.50 (m)
2.17 (dd, 11.7, 2.4)
1.64 (m)
1.43 (m)
3.05 (dt, 11.8, 2.5)
1.78 (m)
1.56 (m)
1.65 (m)
1.53 (m)
1.68 (m)
1.62 (m)
1.21 (d, 8.7)
1.48 (m)
1.41 (m)
2.40 (d, 8.5)
1.92 (m)
1.72 (m)
1.88 (m)
1.42 (m)
1.95 (m)
1.59 (m)
2.50 (s-like)
2.10 (m)
2.46 (s-like)
2.04 (dd, 11.3, 4.4)
1.88 (d, 11.3)
1.84 (d, 14.1)
1.71 (dd, 14.1, 1.6)
4.14 (dd, 10.8, 4.0)
4.05 (dd, 10.8, 4.0)
5.91 (s-like)
0.84 (s, 3H)
2.51 (s-like)
2.10 (dd, 11.4, 4.4)
2.01 (d, 11.4)
1.88 (d, 14.2)
1.83 (d, 14.2)
4.14 (dd, 10.8, 5.2)
4.05 (dd, 10.8, 5.2)
5.97 (dd, 2.5, 1.6)
0.94 (s, 3H)
1.98 (d, 11.4)
1.89 (d, 14.2)
1.80 (dd, 14.2, 1.5)
4.18 (dd, 10.9, 4.7)
4.09 (dd, 10.9, 4.7)
6.00 (d, 1.7)
0.79 (s, 3H)
3.16 (s, 3H)
20
0.67 (s, 3H)
0.80 (s, 3H)
0.66 (s, 3H)
OMe-3
OMe-16
OH-1
OH-3
OH-16
OH-17
3.31 (s, 3H)
6.87 (d, 5.8)
9.49 (s)
5.22 (s)
6.16 (br s)
5.24 (s)
6.16 (t, 6.2)
5.22 (s)
6.16 (t, 5.1)
5.24 (s)
6.16 (br s)
5.21 (s)
6.12 (t, 5.3)
5.72 (br s)
1
a Assignments based on H–1H COSY, HMBC, and HMQC experiments. Multiplicity and J-values in Hz are given in parentheses.
b Recorded at 500 MHz.
c Recorded at 400 MHz.
H-1a/H3-20, H-1b/H-9, H-3/H-5, H-5/H-9, H-9/H-15b,
H-11a/H-15b, H-11a/H-17a, H-11a/H-17b, H-12a/H-17a,
H-12a/H-17b, and H-14b/H3-20 (Fig. 3), its stereostruc-
ture was determined to be as shown in Figure 1.
Compound 1 corresponds to the aglycone moiety of
tricalysiosides B–E obtained previously from the leaves
of the same plant source.1
The absolute configuration of 1 was determined by the
X-ray crystallographic analysis of its p-bromobenzoate 7
Table 2. 13C NMR data for tricalysiolides A–F (1–6) in C5D5N
OH
Carbon
1a
2a
3a
4a
5b
6a
69.0
OH
1
35.8
36.1
35.6
35.9
39.6
H
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
OMe-3
OMe-16
29.9
81.4
175.1
48.7
22.0
40.2
44.5
53.5
42.9
19.4
26.3
45.8
38.0
53.8
81.5
66.4
111.4
173.7
14.7
35.0
105.7
173.7
47.4
22.1
40.3
44.6
53.7
43.8
19.5
26.4
45.9
38.1
53.8
81.5
66.4
112.6
171.5
14.4
33.6
107.3
171.2
47.5
21.8
40.1
44.5
53.6
43.6
19.5
26.3
45.9
38.0
53.7
81.5
66.3
115.2
170.5
14.5
49.9
29.9
81.4
175.0
48.7
22.0
40.2
44.2
53.5
42.9
19.5
26.0
42.0
37.7
49.8
87.3
60.9
111.4
173.7
14.7
107.4
149.7
159.9
45.5
21.8
39.9
44.3
52.8
41.9
18.8
26.3
45.9
37.6
53.5
81.5
66.3
110.5
170.6
15.3
109.3
151.4
160.3
39.2
21.8
40.0
44.3
44.4
44.3
18.4
26.4
46.0
37.6
53.9
81.5
66.4
111.7
170.3
16.1
O
H
HMBC
O
H
C
Figure 2. Selected HMBC correlations for 1.
49.3
a Recorded at 125 MHz.
b Recorded at 100 MHz.
Figure 3. Selected NOESY correlations for 1.

1514
K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519
(Scheme 1, Fig. 4). The value of the final refined Flack
parameter 0.012(7) confirmed that the absolute configu-
ration of 1 was as depicted in Figure 1.
Tricalysiolide B (2) was isolated as an amorphous powder.
Its molecular formula was determined to be C20H28O5 from
the [MCH]C peak at m/z 349.2033 (calcd for C20H29O5,
1
349.2015) in the HRESIMS. The H and 13C NMR spectra
of 2 were similar to those of 1. The differences noted
between 1 and 2 were that the C-3 signal, observed as a
methine carbon signal (d 81.4) in 1, was observed as a
quaternary carbon signal (d 105.7) in 2. The C-3 signal was
correlated with the hydroxyl proton (d 9.49) in the HMBC
spectrum in 2, showing the location of the hydroxyl group
was at C-3. The NOESY correlation detected between H-5
and OH-3 indicated that the hydroxyl group at C-3 was of
b-orientation. The structure and absolute stereochemistry of
2 were determined to be as shown in Figure 1, by the
preparation of 1 from 2 (Scheme 1). Reduction of 2 with
sodium borohydride afforded a product, which was shown to
be identical to natural 1 by comparison of their spectral data
and optical rotations.
Tricalysiolide C (3) was isolated as colorless prisms. Its
molecular formula was determined to be C21H30O5 from the
[MCH]C peak at m/z 363.2182 (calcd for C21H31O5,
1
363.2171) in the HRESIMS. The H and 13C NMR spectra
of 3 were very similar to those of 2, implying that 2 and 3
had the same basic structure. The only difference noted
between 2 and 3 was that the NMR spectra of 3 displayed
signals due to a methoxyl group (dH 3.16, dC 49.9), whose
methoxyl protons were correlated with the C-3 signal in the
HMBC spectrum, thus demonstrating the location of the
methoxyl group being at C-3 in 3. A NOESY correlation
detected between H-5 and OCH3-3 indicated that the
methoxyl group was of b-orientation. Thus, compound 3
was determined to be the 3-O-methyl analogue of 2. This
structure was confirmed by the X-ray analysis (Fig. 5).
Alkaline hydrolysis and subsequent acid treatment of 3
afforded 2 (Scheme 1), establishing that the absolute
structure of 3 was as shown in Figure 1.
Tricalysiolide D (4) was isolated as colorless prisms. Its
molecular formula, C21H30O4, determined from the [MC
H]C peak at m/z 347.2232 (calcd for C21H31O4, 347.2222)
in the HRESIMS showed that it was higher than that of 1 by
one methylene unit. The 1H and 13C NMR spectra of 4 were
similar to those of 1, except that a singlet methoxyl proton
signal (d 3.31) and one carbon signal (d 49.3), which were
not observed in the spectra of 1, were observed in the spectra
of 4 and that the C-16 signal (d 87.3) of 4 was in a lower
field than the corresponding signal of 1 (d 81.5). The HMBC
correlation between the methoxyl protons and C-16
indicated that the location of the methoxyl group was at
C-16 to show that 4 was the 16-O-methyl ether of 1. The
NOESY correlation between H-14b and OCH3-16 indicated
that the methoxyl group at C-16 was of a-orientation. This
structure was confirmed by the X-ray analysis (Fig. 6). Its
absolute configuration was established by the preparation of
4 from 1 (Scheme 1). Tosylate 8, produced by treatment of 1
with p-toluenesulfonyl chloride, gave, on treatment with
sodium carbonate, epoxide 9. Methanolysis of 9 in the
presence of boron trifluoride diethyl etherate afforded a

K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519
1515
Figure 4. ORTEP representation of 7 as determined by single-crystal X-ray analysis.
1
product, which was shown to be identical to natural 4 by
comparison of their spectral data and optical rotations. Thus,
the absolute structure of 4 was determined to be as shown in
Figure 1.
was less than that of 1 by two hydrogen atoms. The H
and 13C NMR spectra of 5 were generally similar to
those of 1, except for the C-2 and C-3 signals. The C-2
methylene (d 29.9) and C-3 methine (d 81.4) signals in 1
were of olefinic methine (d 107.4) and quaternary
(d 149.7) carbons, respectively, in 5. When 2 was
treated with (trimethylsilyl)diazomethane2 in chloroform–
methanol (10/1), the product was shown to be identical
to natural 5 by their spectral data and optical rotations.
Tricalysiolide E (5) was obtained as an amorphous
powder. Its molecular formula was determined to be
C20H26O4 from the [MCH]C peak at m/z 331.1901
(calcd for C20H27O4, 331.1909) in the HRESIMS, which
Figure 5. ORTEP representation of tricalysiolide C (3) as determined by single-crystal X-ray analysis.

1516
K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519
Figure 6. ORTEP representation of tricalysiolide D (4) as determined by single-crystal X-ray analysis.
Thus, the structure of 5 was determined to be as shown
in Figure 1.
Tricalysiolides A–F (1–6) showed a weak cytotoxic activity
on P-388 murine leukemia cells with IC50 values of 40, 40,
50, 40, 17, and 30 mg/mL, respectively.
Tricalysiolide F (6) was isolated as an amorphous powder.
Its molecular formula was determined to be C20H26O5 from
the [MCH]C peak at m/z 347.1882 (calcd for C20H27O5,
347.1858) in the HRESIMS, which was higher than that of 5
3. Experimental
3.1. General experimental procedures
1
by one oxygen atom. The H and 13C NMR spectra of 6
resembled those of 5, demonstrating that they were of the
same basic structure. The differences noted between them
were that the C-1 methylene signal (d 39.6) in 5 was
observed as an oxymethine carbon signal (d 69.0) in 6. The
NOESY correlation between H-1 and H3-20 indicated that
the hydroxyl group at C-1 was of b-orientation. Treatment
of 6 with triethylsilane and boron trifluoride diethyl
etherate3 afforded a reduction product, which was shown
to be identical to natural 5 by comparison of their spectral
data and optical rotations. Accordingly, the absolute
structure of 6 was determined to be as shown in Figure 1.
Optical rotations were measured on a JASCO P1030 digital
polarimeter. IR spectra were recorded on a JASCO FT/IR 620
spectrophotometer. UV spectra were obtained using a JASCO
V-530 spectrophotometer. NMR spectra were measured on
Bruker DRX-500 and DPX-400 spectrometers at 300 K. The
1H chemical shifts in C5D5N were referenced to the residual
C5D4HNresonanceat7.21 ppm, andthe13C chemicalshiftsto
the solvent resonance at 135.5 ppm. Mass spectra were
obtained using a Micromass LCT spectrometer. Preparative
HPLC was carried out on a JASCO PU-986 pump unit
equipped with a UV-970 UV detector (l 220 nm) and an
Inertsil PREP-ODScolumn (10 mm, 20!250 mm), byusing a
MeOH–H2O or a MeCN–H2O solvent system at a flow rate of
10 mL/min. X-ray single crystal analysis was taken on a
Mac Science DIP diffractometer with Mo Ka radiation
Tricalysiolides A–F (1–6) and tricalysiosides A–G1 are
unusual compounds in which one of the geminal methyls is
rearranged to the other methyl to form an ethyl unit at C-4
of the ent-kaurane skeleton. So far, the only known
compounds with this unusual carbon framework are
cafestol4,5 and several related furokauranes6–10 from Coffea
plants (Rubiaceae). Tricalysiolides A (1), D (4) and
tricalysiosides A–G are characterized by an a,b-unsatu-
rated-g-lactone ring fused to the ring A, and tricalysiolides
B (2), C (3), E (5), and F (6) by a further oxidized ring A
structure.
˚
(lZ0.71073 A).
3.2. Plant material
Wood of T. dubia was collected in Iriomote Island, Okinawa,
in March 2003, and the plant origin was identified by Dr.
T. Kinoshita (Teikyo University, Japan). A voucher specimen
has been deposited at the Herbarium of Teikyo University.

K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519
1517
3.3. Extraction and isolation
3.4.5. Tricalysiolide E (5). Amorphous powder; [a]D25
K203 (c 0.26, pyridine); UV (MeOH) lmax nm (log 3) 276
(3.99); IR (film) nmax 3389, 2925, 2852, 1746, 1669, 1605,
Cut and air-dried wood (1.44 kg) of T. dubia was extracted
with hot MeOH (3!6 L). After removal of MeOH under
reduced pressure, the residue (104 g) was placed on a
column of HP-20 (DIAION, 600 g) and eluted with H2O,
H2O–MeOH (1/1), H2O–MeOH (1/4), MeOH, and acetone
(each 3 L) sequentially to give five fractions. After removal
of the solvent, the residue of the H2O–MeOH (1/4) fraction
(17 g) was subjected to silica gel (Merck Kieselgel 60,
230–400 mesh, 200 g) column chromatography
eluting sequentially with EtOAc, CHCl3–MeOH (10/1),
CHCl3–MeOH (3/1), and MeOH (each 500 mL). After
evaporation the CHCl3–MeOH (10/1) fraction (1.2 g) was
subjected to aminopropyl-bonded silica gel (Chromatorex,
200–350 mesh, 40 g) column chromatography eluting
sequentially with CHCl3–MeOH (50/1, then 30/1, then 10/1),
and MeOH (each 100 mL) to give fractions 1–5. Fraction 2
(16.2 mg) was further applied to ODS HPLC eluting with
MeOH–H2O (43/57) to afford 4 (5.6 mg). Fraction 3
(465.0 mg) was further separated by ODS HPLC eluting
with MeOH–H2O (44/56) to afford 1 (141.5 mg), 3
(50.8 mg), and 5 (6.8 mg). The CHCl3–MeOH (3/1) fraction
(7.15 g) was subjected to aminopropyl-bonded silica gel
(35 g) column chromatography eluting sequentially with
CHCl3–MeOH (30/1, then 10/1, then 5/1, then 3/1), and
MeOH (each 100 mL) to give fractions A–G. Fraction B
(276.7 mg) was further purified by repeated ODS HPLC
using MeOH–H2O (40/60) and then MeCN–H2O (18/82) to
afford 6 (14.8 mg). Fraction E (551.7 mg) was further
purified by repeated ODS HPLC using MeOH–H2O (40/60)
and then MeCN–H2O (17/83) to afford 2 (7.7 mg).
1
1464, 1455, 1445, 1259, 1213, 1055, 1021 cmK1; H and
13C NMR data, Tables 1 and 2; HRESIMS m/z 331.1901
[MCH]C (calcd for C20H27O4, 331.1909).
3.4.6. Tricalysiolide
F (6). Amorphous powder;
[a]2D5 K218 (c 0.14, pyridine); UV (MeOH) lmax nm
(log 3) 270 (3.89); IR (film) nmax 3419, 2927, 2867, 1781,
1747, 1672, 1608, 1454, 1395, 1209, 1017 cmK1; H and
1
13C NMR data, Tables 1 and 2; HRESIMS m/z 347.1882
[MCH]C (calcd for C20H27O5, 347.1858).
3.5. Identification of structural relations between the
terpenoids by synthetic procedures (Scheme 1)
3.5.1. Preparation of compound 7. To a solution of 1
(20.0 mg, 0.060 mmol) in pyridine–CH2Cl2 (1/1, 1 mL)
were added p-bromobenzoyl chloride (66.0 mg,
0.301 mmol) and 4-(dimethylamino)pyridine (8.0 mg,
0.065 mmol) in one portion. After stirring at room
temperature for 4 h, the mixture was diluted with
EtOAc. The extract was washed successively with 5%
aqueous HCl and brine, dried (MgSO4), filtered, and
concentrated to give a residue, which was then purified
by silica gel column chromatography with CHCl3–MeOH
(50/1) to afford p-bromobenzoyl ester 7 (29.0 mg, 94%) as
colorless prisms (pyridine); mp 230–232 8C; [a]2D5 K92 (c
0.11, CHCl3); IR (film) nmax 3442, 2924, 2852, 1721, 1679,
1
1647, 1452, 1396, 1266, 1013 cmK1; H NMR (500 MHz,
pyridine-d5) d 8.07 (d, 2H, JZ8.4 Hz), 7.61 (d, 2H, JZ
8.4 Hz), 5.75 (s, 1H), 4.93 (d, 1H, JZ11.3 Hz), 4.76 (t, 1H,
JZ9.3 Hz), 4.71 (d, 1H, JZ11.3 Hz), 2.49 (br s, 1H), 2.12
(m, 1H), 2.06 (m, 1H), 1.97–1.92 (m, 2H), 1.89 (d, 1H, JZ
14.4 Hz), 1.84 (d, 1H, JZ14.4 Hz), 1.71–1.63 (m, 4H),
1.59–1.43 (m, 6H), 1.16 (d, 1H, JZ7.9 Hz), 0.98 (td, 1H,
JZ13.8, 3.3 Hz), 0.69 (s, 3H); 13C NMR (125 MHz,
pyridine-d5) d 174.9, 173.7, 166.2, 132.0 (2C), 131.7 (2C),
130.1, 128.1, 111.5, 81.4, 79.3, 70.1, 53.8, 53.4, 48.7, 46.4,
44.7, 42.9, 40.0, 37.9, 35.8, 29.9, 26.2, 22.0, 19.3, 14.7;
HRESIMS m/z 515.1410 [MCH]C (calcd for C27H32O5Br,
515.1433).
3.4. Characteristics of each terpenoid
3.4.1. Tricalysiolide A (1). Amorphous solid; [a]2D5 K215
(c 0.11, CHCl3); UV (MeOH) lmax nm (log 3) 217 (4.29),
276 (1.25); IR (film) nmax 3365, 2932, 2865, 1775, 1742,
1
1645, 1044, 1021 cmK1; H and 13C NMR data, Tables 1
and 2; HRESIMS m/z 333.2074 [MCH]C (calcd for
C20H29O4, 333.2066).
3.4.2. Tricalysiolide B (2). Amorphous powder; [a]D25
K160 (c 0.33, pyridine); UV (MeOH) lmax nm (log 3) 215
(3.93); IR (film) nmax 3394, 2929, 2866, 1754, 1735, 1657,
1
1451, 1220, 1038 cmK1; H and 13C NMR data, Tables 1
3.5.2. Reduction of compound 2. Sodium borohydride
(2.5 mg, 0.066 mmol) was added to a solution of 2 (5.0 mg,
0.014 mmol) in EtOH (1.5 mL), and then the mixture was
stirred at room temperature for 5 min. After evaporation of
the solvent, the residue was purified by ODS HPLC with
MeCN–H2O (23/77) to give a compound [1.7 mg, 36%,
[a]2D5 K186 (c 0.09, CHCl3)], which was shown to be
identified to the natural product 1, by comparison of their 1H
NMR and mass spectra, and optical rotations.
and 2; HRESIMS m/z 349.2033 [MCH]C (calcd for
C20H29O5, 349.2015).
3.4.3. Tricalysiolide C (3). Colorless prisms (CHCl3–
MeOH); mp 243–246 8C; [a]2D5 K243 (c 0.11, CHCl3); UV
(MeOH) lmax nm (log 3) 217 (4.46); IR (film) nmax 3419,
2933, 2865, 1762, 1656, 1452, 1194, 1170, 1053 cmK1; 1H
and 13C NMR data, Tables 1 and 2; HRESIMS m/z 363.2182
[MCH]C (calcd for C21H31O5, 363.2171).
3.5.3. Treatment of compound 3 with lithium hydroxide.
To a solution of 3 (5.0 mg, 0.014 mmol) in THF–H2O (10/1,
1.5 mL) was added lithium hydroxide monohydrate
(3.0 mg, 0.071 mmol), and the mixture was stirred at room
temperature for 48 h. After addition of acetic acid (1 mL) to
the mixture, it was evaporated to dryness. The residue was
subjected to ODS HPLC with MeCN–H2O (23/77) to give a
compound [4.2 mg, 87%, [a]2D5 K173 (c 0.11, pyridine)],
3.4.4. Tricalysiolide D (4). Colorless prisms (CHCl3–
MeOH); mp 251–253 8C; [a]2D5 K198 (c 0.25, pyridine);
UV (MeOH) lmax nm (log 3) 217 (4.13); IR (film) nmax
3398, 2937, 2864, 1750, 1646, 1448, 1159, 1069, 1047,
1014 cmK1 1H and 13C NMR data, Tables 1 and 2;
;
HRESIMS m/z 347.2232 [MCH]C (calcd for C21H31O4,
347.2222).

1518
K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519
which was shown to be identical to natural 2 by comparison
of their H NMR and mass spectra, and optical rotations.
material 2 (1.0 mg, 9%) and 3 (3.8 mg, 33%), another
compound [1.6 mg, 15%, [a]2D5 K136 (c 0.06, pyridine)]
was obtained. On the basis of the 1H NMR and mass spectra,
and optical rotations, the third compound was shown to be
identical to the natural product 5.
1
3.5.4. Tosylation of compound 1. To a solution of 1
(6.4 mg, 0.019 mmol) in pyridine (1 mL) was added
p-toluenesulfonyl chloride (20 mg, 0.10 mmol) at 0 8C.
The mixture was kept at room temperature for 18 h, and
then was diluted with EtOAc. The solution was washed
successively with aqueous Na2CO3 and brine, dried
(MgSO4), filtered, and concentrated to give a residue,
which, by ODS HPLC with MeCN–H2O (50/50),
gave compound 8 (7.0 mg, 75%) as an amorphous solid:
[a]2D5 K152 (c 0.17, CHCl3); IR (film) nmax 3445, 2930,
3.5.8. Reduction of compound 6. Triethylsilane (0.5 mL,
6.1 mmol) and BF3$OEt2 (5 mL, 0.039 mmol) were added to
a solution of compound 6 (5.0 mg, 0.014 mmol) in CH2Cl2
(2 mL). The mixture was stirred at room temperature for
4 h. After neutralization with sodium acetate (10 mg), the
solvent was removed by evaporation and the residue was
separated by ODS HPLC with MeCN–H2O (28/72) to give a
compound [1.7 mg, 36%, [a]2D5 K101 (c 0.05, pyridine)],
which was shown to be identical to the natural product 5, by
comparison of their 1H NMR and mass spectra, and optical
rotations.
1
1746, 1644, 1357, 1189, 1175 cmK1; H NMR (500 MHz,
pyridine-d5) d 8.06 (d, 2H, JZ8.0 Hz), 7.27 (d, 2H, JZ
8.0 Hz), 5.72 (s, 1H), 4.74 (t, 1H, JZ9.0 Hz), 4.58 (d, 1H,
JZ9.6 Hz), 4.55 (d, 1H, JZ9.6 Hz), 2.40 (br s, 1H), 2.22 (s,
3H), 2.19 (m, 1H), 1.98 (m, 1H), 1.86–1.84 (m, 2H), 1.79 (d,
1H, JZ14.3 Hz), 1.71 (d, 1H, JZ14.3 Hz), 1.64–1.55 (m,
3H), 1.51–1.32 (m, 7H), 1.07 (d, 1H, JZ8.5 Hz), 0.95 (td,
1H, JZ13.6, 3.6 Hz), 0.62 (s, 3H); 13C NMR (125 MHz,
pyridine-d5) d 174.8, 173.6, 145.1, 133.6, 130.3 (2C), 128.4
(2C), 111.5, 81.3, 78.8, 75.5, 53.3, 53.2, 48.6, 45.9, 44.5,
42.8, 39.9, 37.8, 35.8, 29.8, 25.8, 21.9, 21.3, 19.1, 14.6;
HRESIMS m/z 487.2130 [MCH]C (calcd for C27H35O6S,
487.2154).
3.6. X-ray single crystallographic analysis
The structures of compounds 3, 4, and 7 were determined by
the direct method using the maXus crystallographic soft-
ware package11 and the refinement was carried out by the
program SHELXL-97.12
Crystal data for 3. C21H30O5; MZ362.45; orthorhombic;
˚
˚
space group P212121; aZ11.155(3) A; bZ13.087(3) A; cZ
K3
3.5.5. Epoxidation of compound 8. A solution of 8
(6.1 mg, 0.013 mmol) in EtOH (3 mL) was stirred with
Na2CO3 (15 mg, 0.14 mmol) at room temperature for 48 h.
The mixture was filtered and the filtrate was evaporated. By
ODS HPLC with MeCN–H2O (50/50), the residue gave
compound 9 (2.9 mg, 74%) as an amorphous powder: [a]D25
K223 (c 0.06, CHCl3); IR (film) nmax 2929, 2861, 1778,
1749, 1645, 1456, 1258, 1180, 1160, 1127, 1048, 1037,
˚
˚
12.476(3) A; VZ1821.31(8) A; ZZ4; DXZ1.322 Mg m
m(Mo Ka)Z0.093 mmK1; RZ0.0349; RwZ0.0974.
;
Crystal data for 4. C21H30O4; MZ346.45; orthorhombic;
˚
˚
space group P212121; aZ6.899(10) A; bZ11.322(5) A; cZ
˚
˚
22.828(10) A;
VZ1783.11(11) A;
ZZ4;
DXZ
1.291 Mg mK3; m(Mo Ka)Z0.088 mmK1; RZ0.0403;
RwZ0.1064.
1
1022 cmK1; H NMR (500 MHz, pyridine-d5) d 5.75 (s,
1H), 4.75 (t, 1H, JZ8.7 Hz), 2.89 (d, 1H, JZ5.0 Hz), 2.82
(d, 1H, JZ5.0 Hz), 2.21 (m, 1H), 1.92–1.83 (m, 4H),
1.71–1.56 (m, 4H), 1.55–1.30 (m, 8H), 1.11 (d, 1H, JZ
8.0 Hz), 0.95 (td, 1H, JZ13.8, 3.7 Hz), 0.64 (s, 3H); 13C
NMR (125 MHz, pyridine-d5) d 174.8, 173.6, 111.7, 81.3,
65.7, 52.2, 49.9, 49.0, 48.7, 45.1, 42.7, 42.6, 39.2, 38.9,
35.9, 29.8, 28.7, 21.6, 20.3, 14.9; HRESIMS m/z 315.1949
[MCH]C (calcd for C20H27O3, 315.1960).
Crystal data for 7. C27H31BrO5$C5H5N; MZ594.52;
˚
orthorhombic; space group P212121; aZ6.259(10) A; bZ
˚
˚
˚
19.99(10) A; cZ22.348(10) A; VZ2796.12(19) A; ZZ4;
DXZ1.412 Mg mK3 m(Mo Ka)Z1.512 mmK1
RZ
0.0501; RwZ0.067; Flack parameterZ0.012(7).
;
;
Crystallographic data for 3, 4, and 7 reported in this paper
have been deposited at the Cambridge Crystallographic
Data Centre, under the reference numbers CCDC 280148,
280149, and 280147, respectively. Copies of the data can be
obtained, free of charge, on application to the Director,
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
C44 1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
3.5.6. Methanolysis of compound 9. To a solution of 9
(2.6 mg, 0.0083 mmol) in MeOH (1.5 mL) was added
BF3$OEt2 (3 mL, 0.024 mmol). The mixture was stirred at
room temperature for 20 min. After neutralization with
sodium acetate (5 mg), the solvent was evaporated under
reduced pressure. By ODS HPLC with MeCN–H2O (40/60)
the residue gave a compound [0.9 mg, 31%, [a]2D5 K241 (c
0.05, pyridine)], which was shown to be identical to the
1
References and notes
natural product 4 by comparison of their H NMR spectra
and optical rotations.
1. He, D.-H.; Otsuka, H.; Hirata, E.; Shinzato, T.; Bando, M.;
Takeda, Y. J. Nat. Prod. 2002, 65, 685–688.
2. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull.
1981, 29, 1475–1478.
3.5.7. Treatment of compound 2 with (trimethylsilyl)-
diazomethane. To a solution of 2 (11.0 mg, 0.032 mmol) in
CHCl3–MeOH (10/1, 3 mL) was added (trimethylsilyl)dia-
zomethane (2.0 M in hexanes, 1 mL, 2.0 mmol). The
mixture was stirred at room temperature for 1 h. After
removal of the solvent and subsequent ODS HPLC of the
residue with MeCN–H2O (22/78), along with the starting
3. Adlington, M. G.; Orfanopoulos, M.; Fry, J. L. Tetrahedron
Lett. 1976, 2955–2958.
4. Wettstein, A.; Hunziker, F.; Miescher, K. Helv. Chim. Acta
1943, 26, 1197–1218.

K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519
1519
5. Djerassi, C.; Cais, M.; Mitscher, L. A. J. Am. Chem. Soc. 1959,
81, 2386–2398.
10. Prewo, R.; Guggisberg, A.; Lorenzi-Riatsch, A.; Baumann,
¨
T. W.; Wettstein-Battig, M. Phytochemistry 1990, 29, 990–992.
6. Kaufmann, H. P.; Sen Gupta, A. K. Chem. Ber. 1963, 96,
2489–2498.
´ ´
7. De Rostolan, J.; Poisson, J. Cafe, Cacao, The 1970, 14,
11. Mackay, S.; Gilmore, C. J.; Edwards, C.; Stewart, N.; Shankland,
K. maXus Computer Program for the Solution and Refinement of
Crystal Structures; MacScience, Japan and The University of
Glasgow: Bruker Nonius, The Netherlands, 1999.
47–49.
8. Ducruix, A.; Pascard-Billy, C.; Hamonniere, M.; Poisson, J.
`
12. Sheldrick, G. M. SHELXL-97: Program for the Refinement of
¨ ¨
Crystal Structures; University of Gottingen: Gottingen,
J. Chem. Soc., Chem. Commun. 1975, 396–397.
9. Richter, H.; Spiteller, G. Chem. Ber. 1979, 112, 1088–1092.
Germany, 1997.