Tricalysionoside a, a megastigmane gentiobioside, sulfatricalysines a-F, and tricalysiosides X-Z, ent-kaurane glucosides, from the leaves of Tricalysia dubia

Article abstract of DOI:10.1248/cpb.59.72

Further isolation work on the water-soluble fraction of a MeOH extract of Tricalysia dubia afforded one new megastigmane gentiobioside, named tricalysionoside A (1), and three sulfates, named sulfatricalysines A-C (2-4). Extensive isolation work on the 1-BuOH-soluble fraction of a MeOH extract of T. dubia yielded sulfatricalysines D-F (5-7) and three new ent-kaurane glucosides, named tricalysiosides X-Z (8-10). The structures of the new compounds were elucidated by analyses of one- and two-dimensional NMR spectroscopic data. The absolute stereochemistry of tricalysionoside A (1) was established by modified Mosher's method.

Full text of DOI:10.1248/cpb.59.72

72
Regular Article
Chem. Pharm. Bull. 59(1) 72—77 (2011)
Vol. 59, No. 1
Tricalysionoside A, a Megastigmane Gentiobioside, Sulfatricalysines
A—F, and Tricalysiosides X—Z, ent-Kaurane Glucosides, from the
Leaves of Tricalysia dubia
a
a
a
,a
*
Junko SHITAMOTO, Sachiko SUGIMOTO, Katsuyoshi MATSUNAMI, Hideaki OTSUKA,
Takakazu SHINZATO,b and Yoshio TAKEDA
c
a Graduate School of Biomedical Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-ku, Hiroshima 734–8553,
Japan: b Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 1 Senbaru, Nishihara-cho,
c
Nakagami-gun, Okinawa 903–0213, Japan: and Faculty of Pharmacy, Yasuda Women’s University; 6–13–1 Yasuhigashi,
Asaminami-ku, Hiroshima 731–0153, Japan.
Received July 29, 2010; accepted October 8, 2010; published online November 1, 2010
Further isolation work on the water-soluble fraction of a MeOH extract of Tricalysia dubia afforded one new
megastigmane gentiobioside, named tricalysionoside A (1), and three sulfates, named sulfatricalysines A—C (2—
4). Extensive isolation work on the 1-BuOH-soluble fraction of a MeOH extract of T. dubia yielded sulfatri-
calysines D—F (5—7) and three new ent-kaurane glucosides, named tricalysiosides X—Z (8—10). The structures
of the new compounds were elucidated by analyses of one- and two-dimensional NMR spectroscopic data. The
absolute stereochemistry of tricalysionoside A (1) was established by modified Mosher’s method.
Key words Tricalysia dubia; Rubiaceae; megastigmane glucoside; sulfate; ent-kaurane glucoside
Tricalysia (Syn. Canthium), which comprises approxi- spectrum also suggested the presence of a conjugated system
1
mately 50 species, is distributed in subtropical and tropical (234 nm). In the H-NMR spectrum, the signals for two sin-
areas of Asia and Africa. Some species are used for medici- glet methyls, two doublet methyls (Jꢁ6 Hz and Jꢁ1 Hz), one
nal purposes, e.g. the roots, leaves and stem bark of Can- olefinic proton, and two anomeric protons [dH 4.32 (d,
thium subcordatum are used as folk medicines in Africa.1) Jꢁ8 Hz) and 4.39 (d, Jꢁ8 Hz)] were observed. The 13C-
Tricalysia dubia (LINDL.) OHWI (Syn. Canthium dubium NMR spectrum exhibited six signals assignable for a termi-
LINDL.) (Rubiaceae) is an evergreen shrub or tree that grows nal b-glucopyranose and further six signals for the sugar
to a height of about 2—4 m. It is distributed in the south of moiety. The remaining 13 signals comprised those of four
China, Taiwan and the southern part of Japan.2) In the course methyls, three methylenes, one trisubstituted double bond,
of our study of Okinawa’s promising resource plants, con- one oxymethine, two quaternary carbons, and a carbonyl car-
stituents of the title plant, T. dubia, were investigated. In pre- bon (Table 1). One of the quaternary carbons was expected to
vious studies, considerable numbers of rearranged ent-kau- possess an oxygen atom from its chemical shift (dC 79.4).
rane glucosides3) and ent-kaurane glucosides4,5) were isolated From the above evidence, 1 was assumed to be a megastig-
from the leaves of T. dubia and stems of the same plant also mane derivative with two sugar units. A set of 13C-NMR sig-
provided some ent-kaurane derivatives.6) Sulfates are fre- nals for the sugar moiety showed a close resemblance to that
quently found in sugar polymers, such as dermatane sulfate of gentiobiose,14) and detailed inspection of the one- and two-
and keratan sulfate. Bile acids are conjugated with sulfate. dimensional NMR spectra established the structure of the
Compounds are sometimes conjugated with sulfate in a aglycone to be 9-epi-blumenol B.15,16) Thus the structure of 1
metabolic system. Sulfuric acid esters are also found in plant was expected to be similar to that of blumenol B gentiobio-
products.7—13) However, the occurrence of sulfates in plants side, namely icariside B5 6ꢂ-O-b-D-glucopyranoside (Fig.
is relatively rare. From the leaves of T. dubia, one megastig- 1).17,18) Absolute configuration of glucose was determined to
mane glucoside (1) and six glucoside sulfates (2—7) were
Table 1. 13C-NMR Spectroscopic Data for Tricalysionoside
(CD3OD, 100 MHz)
A (1)
isolated together with three new ent-karurane glucosides
(8—10). This paper deals with their structural elucidation.
C
C
Results and Discussion
From the H2O- and 1-BuOH-soluble fractions of a MeOH
extract of the leaves of T. dubia, one megastigmane glucoside
(1), six glucoside sulfates (2—7), and three ent-kaurane glu-
cosides (8—10) were isolated by a combination of various
kinds of chromatography. Their structures were elucidated by
spectroscopic analyses and by modified Mosher’s method.
Tricalysionoside A (1), [a]D24 ꢀ16.9, was isolated as an
amorphous powder and its elemental composition was deter-
mined to be C25H42O13 by high-resolution (HR)-electrospray
ionization (ESI)-mass spectrometry (MS). In the IR spec-
trum, absorption bands for hydroxyl groups (3388 cmꢀ1) and
a conjugated ketone (1712 cmꢀ1) were observed. The UV
1
2
3
4
5
6
7
8
9
10
11
12
13
43.1
51.1
201.1
126.7
171.7
79.4
34.7
33.1
78.3
22.3
24.5
24.1
22.0
Glc 1ꢂ
2ꢂ
104.3
75.3
78.1
71.7
77.1
70.0
104.9
75.1
78.1
71.6
78.2
62.8
3ꢂ
4ꢂ
5ꢂ
6ꢂ
Glc 1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp

January 2011
73
Fig. 2. Results with the Modified Mosher’s Method (dS–R
)
Table 2. 13C-NMR Spectroscopic Data for Sulfates (2—7) (CD3OD,
100 MHz)
C
2
3
4
5
6
7
1
2
3
4
5
6
7
8
9
122.6
158.5
119.2
135.2
123.9
132.1
168.6
135.1
106.6
154.5
138.6
154.5
106.6
71.7
70.6
28.8
125.9
134.6
21.6
136.4
105.0
154.3
135.3
154.3
105.0
132.0
126.5
18.4
139.2
129.32
129.27
128.7
129.27
129.32
71.9
112.1
146.3
73.8
43.0
23.5
130.3
132.9
76.1
27.7
14.1
102.9
75.1
76.9
77.9
76.3
62.6
14.6
10
1ꢂ
103.6
74.9
76.2
77.1
76.7
62.1
52.9
102.8
75.2
76.8
77.9
76.5
62.6
104.1
75.1
76.8
77.9
76.3
62.4
105.1
75.6
76.5
77.5
76.7
62.3
103.1
75.2
76.9
77.9
76.4
62.6
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
–OCH3
3,5-OCH3
4-OCH3
56.7
61.2
57.0
Fig. 1. Structures of Isolated Compounds
be of the D-series from its optical rotation value and that at were obviously shifted downfield due to esterification (dH of
the 6-position was confirmed to be S from the Cotton effects 2a: 3.46—3.65 and that of 2: 4.28, and dC of 2a: 71.5 and
observed in the circular dichroism (CD) spectrum.18) The ab- that of 2: 77.1).21) Thus the structure of 2 was elucidated to
solute configuration at the 9-position was independently de- be methyl salicylate b-D-glucopyranoside 4ꢂ-O-sulfate, as
termined to be S from the results with the modified Mosher’s shown in Fig. 1. There is the possibility that the methyl ester
method19) applied to the aglycone, tricalysionol A (1a) (Fig. was an artifact. Sulfate (m/z: 80.9646) and phosphate (m/z:
2). This was opposite to that of blumenol B and dihydrovom- 80.9742) are both present in living systems and the differ-
ifoliol b-D-glucopyranoside, and the same as that of icariside ence in mass between them on HR-MS is 10 millimass units.
B5.20)
From the following observations, the presence of the sulfate
Sulfatricalysine A (2), [a]D25 ꢀ23.3, was isolated as an was confirmed. At first, in the NMR spectra, no P–O–C or
amorphous powder and its elemental composition was deter- P–O–C–H coupling was observed. Second, when the aqueous
mined to be C14H18O11S by HR-ESI-MS. The IR and UV layer of the acid hydrolyzate was neutralized with Ba(OH)2, a
spectra indicated that 2 was a glycosidic compound white precipitate was obtained and this precipitate was not
(3416 cmꢀ1) with an aromatic ring (1600, 1489 cmꢀ1 and soluble in 2 M HCl.7) Finally, on negative-ion MS, 2 was
283 nm, respectively) and a carbonyl group (1712 cmꢀ1). In found to possess only three exchangeable protons on the ad-
the 1H-NMR spectrum, signals for sequentially arranged four dition of D2O.
aromatic protons, methylene protons in a primary alcohol, an
Sulfatricalysine B (3), [a]D24 ꢀ24.9, was isolated as an
anomeric proton and methoxy protons were observed. The amorphous powder and its elemental composition was deter-
coupling pattern of other protons that might comprise a sugar mined to be C16H24O12S by HR-ESI-MS. In the NMR spec-
moiety was exactly the same as that of b-glucopyranose. The trum, the signals for the sugar moiety were essentially the
13C-NMR spectrum exhibited six aromatic carbons, one car- same as those of 2. The aglycone comprised a symmetrically
bonyl, one methoxy carbon, and six carbon signals for the substituted aromatic ring, three methoxy groups, and a pri-
sugar moiety (Table 2). From the above evidence, 2 was ex- mary alcohol. Since the aromatic protons (dH 6.76) showed a
pected to be a methyl salicylate glucoside (2a) with a sulfate correlation cross peak with the methylene carbon (dC 71.7)
moiety. On acid hydrolysis of 2, salicylic acid was taken up in the heteronuclear multiple bond connectivity (HMBC)
in the organic layer and the aqueous layer was determined by spectrum, the structure of the aglycone was elucidated to be
1
HPLC to contain D-glucose. Detailed analysis of H–1H cor- a 4,5,6-trimethoxybenzyl alcohol. Therefore the structure of
relation spectroscopy (COSY) and heteronuclear single 3 is as shown in Fig. 1.
quantum coherence (HSQC) spectra indicated that the 1H and
Sulfatricalysine C (4), [a]D24 ꢀ4.6, was also isolated as an
13C signals for the 4-position of the glucopyranoside moiety amorphous powder and its elemental composition was deter-

74
Vol. 59, No. 1
Table 3. 13C-NMR Spectroscopic Data for Tricalysiosides X—Z (8—10),
and Tricalysiosides T (11) and H (12) (C5D5N, 100 MHz)
mined to be C12H22O9S. From the NMR spectroscopic data,
the sugar moiety was also expected to be a b-D-glucopyra-
nose 4ꢂ-O-sulfate and the signals assignable to the aglycone
were essentially the same as those reported for (Z)-hex-3-en-
1-ol.22) Therefore the structure of 4 was elucidated to be as
shown in Fig. 1.
C
8
9b)
10
11c)
12d)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
40.6 (41.8)a)
18.7 (19.4)
36.1 (37.6)
38.3 (40.7)
57.1 (58.3)
20.4 (21.0)
37.2 (37.4)
48.0 (48.7)
56.4 (57.5)
39.8 (39.1)
18.8 (19.5)
26.2 (26.8)
43.6 (44.1)
36.8 (36.6)
82.8 (83.6)
81.7 (82.6)
66.1 (66.5)
28.3 (28.4)
73.5 (74.1)
18.4 (18.9)
105.0 (104.8)
75.0 (75.2)
76.8 (77.0)
77.3 (77.8)
76.1 (76.0)
62.0 (62.4)
40.5
18.7
36.5
38.4
56.8
20.3
37.4
49.3
56.4
40.0
19.1
25.8
44.2
36.8
94.6
81.7
66.8
28.2
73.1
18.5
105.4
75.4
78.9
72.0
78.5
63.0
106.6
75.4
78.8
72.0
78.4
62.9
92.5
28.2
36.5
43.9
56.5
22.5
43.7
45.9
55.6
45.8
21.4
27.3
45.7
38.8
53.6
82.2
66.4
28.7
176.8
12.7
104.8
75.5
79.2
71.8
78.5
62.9
96.0
74.1
79.3
71.2
79.0
62.3
40.7
18.7
36.2
38.4
57.1
20.4
37.3
48.0
56.4
39.8
18.9
26.3
43.6
36.8
82.7
81.3
66.2
28.3
73.2
18.4
105.5
75.4
78.8
71.9
78.3
63.0
92.6
28.3
36.7
43.6
56.0
22.8
43.7
46.0
55.7
45.8
21.2
27.4
45.7
39.0
53.6
82.1
66.4
29.3
179.9
13.0
104.6
75.8
79.1
71.8
78.5
62.9
Sulfatricalysine D (5), [a]D24 ꢀ10.9, was isolated as an
amorphous powder and its elemental composition was deter-
mined to be C17H24O11S. From the NMR spectroscopic data,
the sugar moiety was expected to be the same as the preced-
ing sulfates, and the aglycone part comprised a symmetri-
cally substituted aromatic ring, two equivalent methoxy
groups, a trans double bond, and a terminal methyl group. In
the HMBC spectrum, correlation cross peaks between the
aromatic protons (dH 6.67) and an olefinic carbon (dC 132.0)
and anomeric proton (dH 4.88), and an aromatic carbon (dC
135.3) with an oxygen atom were observed. Thus the struc-
ture of 5 was elucidated to be 3,5-dimethoxy-1-(E)-pro-
penylphenol 4-O-b-D-glucopyranoside-4ꢂ-O-sulfate, as shown
in Fig. 1.
1ꢂ
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
1ꢃ
2ꢃ
3ꢃ
4ꢃ
5ꢃ
6ꢃ
Sulfatricalysine E (6), [a]D25 ꢀ11.8, was an amorphous
powder with an elemental composition of C13H18O9S. The
NMR spectroscopic data showed that 6 possessed a mono-
substituted benzene ring and a primary alcohol (Table 2).
Signals assignable to 4ꢂ-O-sulfated b-D-glucopyranose were
also observed and thus the structure of 6 was elucidated to
be benzyl alcohol O-b-D-glucopyranoside-4ꢂ-O-sulfate, as
shown in Fig. 1.
Sulfatricalysine F (7), [a]D24 ꢀ3.3, was an amorphous
compound with an elemental composition of C16H28O10S. As
shown by the spectroscopic data, 7 also possessed a O-b-D-
a) Data for CD3OD. b) At 150 MHz. c) Data from ref. 5. d) Data from ref. 4.
glucopyranoside-4ꢂ-O-sulfate moiety in its molecule and the phous powder and its elemental composition was determined
structure of the aglycone was expected to be an acyclic to be C32H52O15 by HR-ESI-MS. In the 13C-NMR spectrum,
monoterpene. The NMR data for the aglycone section were signal for a carbonyl carbon (dC 176.8), and similarly to in
essentially the same as those for betulabuside A isolated the case of 9, 12 signals assignable to two sets of b-glucopy-
from Erigeron breviscapus.23) Therefore the structure of 7 ranoses were observed. One of the anomeric carbon signals,
was betulabuside A 4ꢂ-O-sulfate, as shown in Fig. 1. The ab- however, appeared at dC 96.0, which implied that one of the
solute stereochemistries at the 3-positions of betulabuside A b-glucopyranosyl units was connected to the aglycone
and 7 remain to be determined.
through an ester bond. The 13C-NMR spectral signals for the
Tricalysioside X (8), [a]D24 ꢀ18.2, was isolated as an aglycone showed a close resemblance to those of 12,4) except
amorphous powder and its elemental composition was deter- for a significant upfield shift of the carbonyl carbon by
mined to be C26H44O12S by HR-ESI-MS. 13C-NMR spectro- 3.1 ppm (Table 3). To this carbonyl carbon, the anomeric pro-
scopic data were essentially the same as those reported for ton (dH 6.21) on dC 96.0 was correlated in the HMBC spec-
11,5) except for the sugar moiety, whose 13C-NMR data were trum. Therefore the structure of 10 was elucidated to be tri-
indistinguishable from those of the preceding sulfates (Table calysioside H 19-O-b-D-glucopyranosyl ester, as shown in
3). Therefore the structure of 8 was elucidated to be tricaly- Fig. 1.
sioside T (11) 4ꢂ-O-sulfate, as shown in Fig. 1.
It is noteworthy to mention that from the leaves of T.
Tricalysioside Y (9), [a]D23 ꢀ36.8, was isolated as an dubia, seven sulfated glucosides, sulfatricalysines A—F (2—
amorphous powder and its elemental composition was deter- 7) and tricalysioside X (8) were isolated. In these com-
mined to be C32H54O14 by HR-ESI-MS. Of 32 signals ob- pounds, only the 4-position of glucopyranose was sulfated.
served in the 13C-NMR spectrum, 12 were assignable to two Sulfated compounds were rarely found in some plants.
sets of terminal b-glucopyranosides. The remaining 20 sig- Saponins with 2-O-sulfated glucopyranoside and 4-O-sul-
nals for the aglycone portion showed strong similarity to fated xylopyranoside have been isolated from Eclipta alba24)
those of 11, except for an obvious downfield shift of C-15 by and Mollugo spergula,25) respectively. Phenolic compounds
11.9 ppm and a slight downfield shift of C-8 by 1.3 ppm with 6-O-sulfated glucopyranoside were found in bark of
(Table 3). In the HMBC spectrum, one (dH 5.04) of the Bursera simaruba.26) In T. dubia, highly site-specific sulfo-
anomeric protons showed a significant correlation cross peak transferase may operate to form 3-O-sulfated glucopyrano-
with the C-15 (dC 94.6) signal. Therefore the structure of 9 sides.
was elucidated to be 15-O-b-D-glycopyranoside of 11, as
shown in Fig. 1.
Experimental
Tricalysioside Z (10), [a]D23 ꢀ8.0, was isolated as an amor-
General Optical rotations were measured on a JASCO P-1030 digital

January 2011
75
polarimeter. IR and UV spectra were measured on Horiba FT-710 and
3.32 (2H, m, H-4ꢂ, 4ꢃ), 3.30 (2H, m, H-3ꢂ, 3ꢃ), 3.28 (1H, m, H-5ꢃ), 3.21 (1H,
dd, Jꢁ9, 8 Hz, H-2ꢃ), 3.16 (1H, dd, Jꢁ9, 8 Hz, H-2ꢂ), 2.65 (1H, d, Jꢁ18 Hz,
H-2a), 2.14 (1H, dd, Jꢁ8, 1 Hz, H-2b), 2.07 (1H, m, H-7a), 2.04 (3H, d,
Jꢁ1 Hz, H3-13), 1.85 (1H, m, H-7b), 1.80 (1H, m, H-8a), 1.50 (1H, m, H-
8b), 1.24 (3H, d, Jꢁ6 Hz, H3-10), 1.09 (3H, s, H3-12), 1.02 (3H, s, H3-11);
13C-NMR (CD3OD, 100 MHz): Table 1; CD De (nm): ꢄ0.65 (326), ꢀ2.57
(256), ꢄ4.52 (221) (cꢁ4.58ꢅ10ꢀ5 M); HR-ESI-MS (positive-ion mode) m/z:
573.2513 [MꢄNa]ꢄ (Calcd for C25H42O13Na: 573.2517).
1
JASCO V-520 UV/Vis spectrophotometers, respectively. H- and 13C-NMR
spectra were taken on JEOL JNM a-400 and ECA-600 spectrometers at 400
or 600 MHz and 100 or 150 MHz, respectively, with tetramethylsilane as in-
ternal standard. CD spectra were obtained with a JASCO J-720 spectropo-
larimeter. Positive-ion HR-ESI-MS was performed with an Applied Biosys-
tems QSTAR XL NanoSprayTM System.
A highly porous synthetic resin (Diaion HP-20) was purchased from Mi-
tsubishi Kagaku (Tokyo, Japan). Silica gel CC was performed on silica gel
60 (E. Merck, Darmstadt, Germany), and ODS open CC on Cosmosil 75C18-
OPN (Nacalai Tesque, Kyoto, Japan) [Fꢁ50 mm, Lꢁ25 cm, linear gradient:
MeOH–H2O (1 : 9, 1.5 l)→(7 : 3, 1.5 l), fractions of 10 g being collected].
The DCCC (Tokyo Rikakikai, Tokyo, Japan) was equipped with 500 glass
columns (Fꢁ2 mm, Lꢁ40 cm), and the lower and upper layers of a solvent
mixture of CHCl3–MeOH–H2O–n-PrOH (9 : 12 : 8 : 2) were used as the sta-
tionary and mobile phases, respectively. Five-gram fractions were collected
and numbered according to their order of elution with the mobile phase.
HPLC was performed on an ODS column (Inertsil; GL Science, Tokyo,
Japan; Fꢁ6 mm, Lꢁ250 mm, 1.6 ml/min), and the eluate was monitored by
UV detector at 254 nm, and a refractive index monitor. Crude hesperidinase
was a generous gift from Tanabe Pharmaceutical Co., Ltd. (Tokyo, Japan).
The (R)-(ꢄ)- and (S)-(ꢀ)-a-methoxy-a-trifluoromethylpheylacetic acids
(MTPA) were purchased from Nacalai Tesque Inc. (Kyoto, Japan).
Sulfatricalysine
A
(2): Amorphous powder, [a]D25 ꢀ23.3 (cꢁ0.49,
MeOH); IR nmax (film) cmꢀ1: 3416, 2949, 1712, 1600, 1489, 1451, 1242,
1079, 1046, 986; UV lmax (MeOH) nm (log e): 283 (3.35), 227 (3.81), 208
(3.94); 1H-NMR (CH3OD, 400 MHz) d: 7.76 (1H, dd, Jꢁ8, 1 Hz, H-6), 7.54
(1H, ddd, Jꢁ8, 8, 1 Hz, H-4), 7.39 (1H, dd, Jꢁ8, 1 Hz, H-3), 7.14 (1H, ddd,
Jꢁ8, 8, 1 Hz, H-5), 4.96 (1H, d, Jꢁ8 Hz, H-1ꢂ), 4.28 (1H, dd, Jꢁ10, 9 Hz,
H-4ꢂ), 3.94 (1H, dd, Jꢁ12, 2 Hz, H-6ꢂa), 3.89 (3H, s, –OCH3), 3.81 (1H, dd,
Jꢁ9, 9 Hz, H-3ꢂ), 3.82 (1H, dd, Jꢁ12, 5 Hz, H-6ꢂb), 3.64 (1H, dd, Jꢁ9,
8 Hz, H-2ꢂ), 3.61 (1H, ddd, Jꢁ10, 5, 2 Hz, H-5ꢂ); 13C-NMR (CD3OD,
100 MHz): Table 2; HR-ESI-MS (negative-ion mode) m/z: 393.0494
[MꢀH]ꢀ (Calcd for C14H17O11S: 393.0497); HR-ESI-MS (on addition of
D2O) (negative-ion mode) m/z: 396.0679 [MꢀH]ꢀ (Calcd for
C14H14D3O13S: 396.0685).
Sulfatricalysine
B
(3): Amorphous powder, [a]D24 ꢀ24.9 (cꢁ0.37,
MeOH); IR nmax (film) cmꢀ1: 3432, 2939, 1592, 1507, 1460, 1422, 1237,
1124, 1079, 984; UV lmax (MeOH) nm (log e): 230sh (3.80), 211 (4.13); 1H-
NMR (CH3OD, 400 MHz) d: 6.76 (2H, s, H-2, 6), 4.85 (1H, d, Jꢁ12 Hz, H-
7a), 4.65 (1H, d, Jꢁ12 Hz, H-7b), 4.37 (1H, d, Jꢁ8 Hz, H-1ꢂ), 4.14 (1H, dd,
Jꢁ10, 9 Hz, H-4ꢂ), 3.93 (1H, dd, Jꢁ12, 2 Hz, H-6ꢂa), 3.85 (6H, s, –OCH3 at
C-3, C-5), 3.77 (1H, dd, Jꢁ12, 6 Hz, H-6ꢂb), 3.75 (3H, s, –OCH3 at C-4),
3.66 (1H, dd, Jꢁ9, 9 Hz, H-3ꢂ), 3.40 (1H, ddd, Jꢁ10, 6, 2 Hz, H-5ꢂ), 3.37
(1H, dd, Jꢁ9, 8 Hz, H-2ꢂ); 13C-NMR (CD3OD, 100 MHz): Table 2; HR-ESI-
MS (negative-ion mode) m/z: 439.0920 [MꢀH]ꢀ (Calcd for C16H23O12S:
439.0915); HR-ESI-MS (on addition of D2O) (negative-ion mode) m/z:
442.1105 [MꢀH]ꢀ (Calcd for C16H20D3O12S: 442.1104).
Plant Material Leaves of T. dubia (LINDL.) OHWI (Rubiaceae) were col-
lected in Okinawa, Japan, in August 1990, and a voucher specimen was de-
posited in the Herbarium of the Department of Pharmaceutical Sciences,
Graduate School of Biomedical Sciences, Hiroshima University (90-TD-
Okinawa-0822).
Extraction and Isolation The H2O-soluble fraction (324 g)3) was sub-
jected to Diaion HP-20 CC (Fꢁ60 mm, Lꢁ60 cm), using H2O–MeOH
(9 : 1, 8 l), (7 : 3, 3 l), (1 : 1, 8 l), and (3 : 7, 8 l), and MeOH (3 l), 2 l fractions
being collected. The residue (13.0 g in fractions 6—9) of the 30—50%
MeOH eluate obtained on HP-20 CC was subjected to silica gel (250 g) CC
with increasing amounts of MeOH in CHCl3 [CHCl3 (2 l), and CHCl3–
MeOH (99 : 1, 3 l), (97 : 3, 3 l), (19 : 1, 3 l), (37 : 3, 3 l), (9 : 1, 3 l), (7 : 1, 3 l),
(17 : 3, 3 l), (33 : 7, 3 l), (4 : 1, 3 l), (3 : 1, 3 l) and (7 : 3, 3 l)], and CHCl3–
MeOH–H2O (70 : 30 : 4, 3 l), 500 ml fractions being collected. The residue
(1.88 g) in fractions 35—42 was separated by octadecyl silica (ODS) open
CC then the residue (364 mg) in fractions 29—42 was purified by DCCC.
This residue (169 mg) was further separated by Sephadex LH-20 CC
(Fꢁ25 mm, Lꢁ120 cm, MeOH, 10 g fraction being collected). Compounds
4 (22.5 mg), 3 (10.3 mg), and 2 (45.0 mg) were obtained as precipitates from
fractions 51—53, 54—58, and 59—63, respectively. The residue (1.97 g) in
fractions 43—52 obtained on silica gel CC was separated by ODS open CC,
and the residue (218 mg) in fractions 68—78 was purified by DCCC. This
residue (76.7 mg) was separated by Sephadex LH-20 CC to yield 20.0 mg of
1 in fractions 35—37.
Sulfatricalysine C (4): Amorphous powder, [a]D24 ꢀ4.6 (cꢁ0.35, MeOH);
IR nmax (film) cmꢀ1: 3395, 2931, 16332, 1455, 1419, 1377, 1236, 1076,
1028, 982; 1H-NMR (CH3OD, 400 MHz) d: 5.46 (1H, dtd, Jꢁ11, 7, 1 Hz, H-
4), 5.38 (1H, ddt, Jꢁ11, 7, 1 Hz, H-3), 4.31 (1H, d, Jꢁ8 Hz, H-1ꢂ), 4.14
(1H, dd, Jꢁ10, 9 Hz, H-4ꢂ), 3.87 (1H, dd, Jꢁ12, 2 Hz, H-6ꢂa), 3.86 (1H, dt,
Jꢁ9, 7 Hz, H-1a), 3.76 (1H, dd, Jꢁ12, 5 Hz, H-6ꢂb), 3.66 (1H, dd, Jꢁ9,
9 Hz, H-3ꢂ), 3.56 (1H, dt, Jꢁ9, 7 Hz, H-1b), 3.40 (1H, ddd, Jꢁ10, 5, 2 Hz,
H-5ꢂ), 3.28 (1H, dd, Jꢁ9, 8 Hz, H-2ꢂ), 2.38 (2H, dddd, Jꢁ7, 7, 7, 1 Hz, H2-
2), 2.08 (2H, qdd, Jꢁ7, 7, 1 Hz, H2-5), 0.97 (3H, t, Jꢁ7 Hz, H3-6); 13C-
NMR (CD3OD, 100 MHz): Table 2; HR-ESI-MS (negative-ion mode) m/z:
341.0916 [MꢀH]ꢀ (Calcd for C12H21O9S: 341.0911); HR-ESI-MS (on addi-
tion of D2O) (negative-ion mode) m/z: 344.1105 [MꢀH]ꢀ (Calcd for
C12H18D3O9S: 442.1104).
Sulfatricalysine
D
(5): Amorphous powder, [a]D24 ꢀ10.9 (cꢁ0.19,
MeOH); IR nmax (film) cmꢀ1: 3289, 2961, 1580, 1506, 1462, 1414, 1240,
Separation of the 1-BuOH-soluble fraction (324 g) with Diaion HP-20 and
the residue (55.9 g) by silica gel CC have been described previously.3) An
aliquot (1.79 g) of the residue (5.16 g) in fractions 35—39 was separated by
ODS open CC and 17.5 mg of 5 was obtained from fractions 76—81 as a
precipitate. An aliquot (2.13 g) of the residue (3.70 g) in fractions 54—59
was separated by ODS open CC and the residue (186 mg) in fractions 31—
41 was purified by DCCC. The residue (31.1 mg) in fractions 15–18 was fi-
nally purified by HPLC (MeOH–H2O, 1 : 9) to give 3.9 mg of 6 from the
peak at 9 min. The residue (105 mg) obtained on ODS open CC was sepa-
rated by DCCC, then the residue (58.3 mg) in fractions 12—15 was sepa-
rated by Sephadex LH-20 CC. The residue (44.8 mg) in fractions 48—53
was purified by HPLC (MeOH–H2O, 3 : 17) to give 7.1 mg of 7 from the
peak at 17 min. The residue (30.1 mg) in fractions 219—223 obtained on
ODS open CC was purified by HPLC (MeOH–H2O, 1 : 1) to afford 3.8 mg of
9 from the peak at 15 min.
1
1132, 981; UV lmax (MeOH) nm (log e): 260 (4.05), 220 (4.25); H-NMR
(CH3OD, 400 MHz) d: 6.67 (2H, s, H-2, 6), 6.34 (1H, dd, Jꢁ16, 1 Hz, H-7),
6.22 (1H, dd, Jꢁ16, 6 Hz, H-8), 4.88 (1H, d, Jꢁ8 Hz, H-1ꢂ), 4.26 (1H, dd,
Jꢁ10, 9 Hz, H-4ꢂ), 3.85 (6H, s, –OCH3 at C-3, C-5), 3.81 (1H, dd, Jꢁ12,
2 Hz, H-6ꢂa), 3.75 (1H, dd, Jꢁ12, 5 Hz, H-6ꢂb), 3.73 (1H, dd, Jꢁ9, 9 Hz, H-
3ꢂ), 3.59 (1H, dd, Jꢁ9, 8 Hz, H-2ꢂ), 3.37 (1H, ddd, Jꢁ10, 5, 2 Hz, H-5ꢂ),
1.86 (3H, dd, Jꢁ6, 1 Hz, H3-9); 13C-NMR (CD3OD, 100 MHz): Table 2; HR-
ESI-MS (negative-ion mode) m/z: 435.0969 [MꢀH]ꢀ (Calcd for
C17H23O11S: 435.0966); HR-ESI-MS (on addition of D2O) (negative-ion
mode) m/z: 438.1158 [MꢀH]ꢀ (Calcd for C17H20D3O11S: 438.1154).
Sulfatricalysine
E
(6): Amorphous powder, [a]D25 ꢀ11.8 (cꢁ0.22,
MeOH); IR nmax (film) cmꢀ1: 3407, 2926, 1617, 1454, 1416, 1236, 1077,
1026, 981; UV lmax (MeOH) nm (log e): 209 (3.59); 1H-NMR (CH3OD,
400 MHz) d: 7.42 (2H, br d, Jꢁ7 Hz, H-2, 6), 7.32 (2H, br dd, Jꢁ7, 7 Hz, H-
3, 5), 7.27 (1H, dr dd, Jꢁ7, 7 Hz, H-4), 4.93 (1H, d, Jꢁ12 Hz, H-7a), 4.67
(1H, d, Jꢁ12 Hz, H-7b), 4.39 (1H, d, Jꢁ9 Hz, H-1ꢂ), 4.14 (1H, dd, Jꢁ10,
9 Hz, H-4ꢂ), 3.93 (1H, dd, Jꢁ12, 2 Hz, H-6ꢂa), 3.77 (1H, dd, Jꢁ12, 6 Hz, H-
6ꢂb), 3.65 (1H, dd, Jꢁ9, 9 Hz, H-3ꢂ), 3.40 (1H, ddd, Jꢁ10, 6, 2 Hz, H-5ꢂ),
3.36 (1H, dd, Jꢁ9, 8 Hz, H-2ꢂ); 13C-NMR (CD3OD, 100 MHz): Table 2; HR-
ESI-MS (negative-ion mode) m/z: 349.0604 [MꢀH]ꢀ (Calcd for C13H17O9S:
349.0598); HR-ESI-MS (on addition of D2O) (negative-ion mode) m/z:
352.0791 [MꢀH]ꢀ (Calcd for C13H14D3O9S: 352.0787).
The residue (2.24 g) in fractions 60—65 obtained on silica gel CC was
separated by ODS open CC, then the residue (82.2 mg) in fractions 116—
121 was purified by HPLC (MeOH–H2O, 2 : 3) to give 7.1 mg of 10 from the
peak at 11 min. The residue (116 mg) in fraction 142—155 was purified by
DCCC to give 19.7 mg 8 in fractions 10—12.
Tricalysionoside A (1): Amorphous powder, [a]D24 ꢀ16.9 (cꢁ0.36,
MeOH); IR nmax (film) cmꢀ1: 3388, 2926, 2886, 1712, 1634, 1376, 1072,
1043; UV lmax (MeOH) nm (log e): 250sh (3.57), 234 (3.66); 1H-NMR
(CH3OD, 400 MHz) d: 5.83 (1H, qd, Jꢁ1, 1 Hz, H-4), 4.39 (1H, d, Jꢁ8 Hz,
H-1ꢃ), 4.32 (1H, d, Jꢁ8 Hz, H-1ꢂ), 4.11 (1H, dd, Jꢁ12, 2 Hz, H-6ꢂa), 3.86
(1H, dd, Jꢁ12, 2 Hz, H-6ꢃa), 3.86 (1H, overlapped, H-9), 3.79 (1H, dd,
Jꢁ12, 6 Hz, H-6ꢂb), 3.67 (1H, dd, Jꢁ12, 6 Hz, H-6ꢃb), 3.43 (1H, m, H-5ꢂ),
Sulfatricalysine F (7): Amorphous powder, [a]D24 ꢀ3.3 (cꢁ0.49); IR nmax
(film) cmꢀ1: 3398, 2927, 1639, 1410, 1373, 1239, 1077, 984; 1H-NMR
(CH3OD, 400 MHz) d: 5.91 (1H, dd, Jꢁ17, 11 Hz, H-2), 5.48 (1H, ddd,
Jꢁ7, 7, 1 Hz, H-6), 5.20 (1H, dd, Jꢁ17, 2 Hz, H-1a), 5.03 (1H, dd, Jꢁ11,

76
Vol. 59, No. 1
evaporated to dryness, then the methanolic solution was absorbed on silica
gel and subjected to silica gel CC (20 g, Fꢁ18 mm, Lꢁ18 cm) with CHCl3
(150 ml) and CHCl3–MeOH (19 : 1, 150 ml, 9 : 1, 150 ml, 17 : 3, 150 ml and
7 : 3, 450 ml), 12-ml fractions being collected. An aglycone, tricalysionol A
(1a) (1.4 mg), and D-glucose (6.0 mg) were recovered in fractions 23—28
and 54—62, respectively.
2 Hz, H-1b), 4.27 (1H, d, Jꢁ8 Hz, H-1ꢂ), 4.21 (1H, dd, Jꢁ12, 1 Hz, H-8a),
4.12 (1H, dd, Jꢁ10, 9 Hz, H-4ꢂ), 4.04 (1H, dd, Jꢁ12, 1 Hz, H-8b), 3.89 (1H,
dd, Jꢁ12, 2 Hz, H-6ꢂa), 3.75 (1H, dd, Jꢁ12, 6 Hz, H-6ꢂb), 3.64 (1H, dd,
Jꢁ9, 9 Hz, H-3ꢂ), 3.36 (1H, ddd, Jꢁ10, 6, 2 Hz, H-5ꢂ), 3.30 (1H, dd, Jꢁ9,
8 Hz, H-2ꢂ), 2.09 (2H, m, H2-5), 1.68 (3H, d, Jꢁ1 Hz, H3-10), 1.55 (2H, m,
H2-4), 1.25 (3H, s, H3-9); 13C-NMR (CD3OD, 100 MHz): Table 2; HR-ESI-
MS (negative-ion mode) m/z: 411.1331 [MꢀH]ꢀ (Calcd for C16H27O10S:
411.1330); HR-ESI-MS (negative-ion mode) m/z: 415.1578 [MꢀH]ꢀ (Calcd
for C16H23D4O10S: 415.1581).
Tricalysionol A (1a): Amorphous powder, [a]D24 ꢄ61.7 (cꢁ0.12, MeOH);
1H-NMR (CD3OD, 400 MHz) d: 5.83 (1H, dq, Jꢁ1, 1 Hz, H-4), 3.66 (1H,
dqd, Jꢁ8, 6, 5 Hz, H-9), 2.59 (1H, d, Jꢁ18 Hz, H-2a), 2.16 (1H, d, Jꢁ18 Hz,
H-2b), 2.04 (3H, d, Jꢁ1 Hz, H3-13), 1.98 (1H, ddd, Jꢁ13, 13, 5 Hz, H-7a),
1.79 (1H, ddd, Jꢁ13, 13 4, Hz, H-7b), 1.68 (1H, dddd, Jꢁ13, 13, 4, 4 Hz, H-
8a), 1.39 (1H, dddd, Jꢁ13, 13, 8, 5 Hz, H-8b), 1.16 (3H, d, Jꢁ6 Hz, H3-10),
1.09 (3H, s, H3-12), 1.02 (3H, s, H3-11); 13C-NMR (CD3OD, 100 MHz) d:
201.0 (C-3), 171.8 (C5), 126.7 (C-4), 79.2 (C-6), 69.3 (C-9), 51.2 (C-2),
43.0 (C-1), 35.7 (C-7), 35.4 (C-8), 24.5 (C-11), 24.1 (C-12), 23.7 (C-10),
21.8 (C-13); HR-ESI-MS (positive-ion mode) m/z: 249.1458 [MꢄNa]ꢄ
(Calcd for C13H22O3Na: 249.1461). D-Glucose: [a]D23 ꢄ34.1 (cꢁ0.25, H2O,
24 h after being dissolved in the solvent).
Preparation of (R)- and (S)-MTPA Esters (1b and 1c, Respectively)
from 1a A solution of 1a (0.7 mg) in 1 ml of dehydrated CH2Cl2 was re-
acted with (R)-MTPA (48 mg) in the presence of 1-ethyl-3-(3-dimethyl-
aminopropyl)cardodiimide hydrochloride (EDC) (13 mg) and N,N-dimethyl-
4-aminopyridine (4-DMAP) (9 mg), then the mixture was occasionally
stirred at 25 °C for 30 min. After the addition of 1 ml of CH2Cl2, the solution
was washed with H2O (1 ml), 5% HCl (1 ml), NaHCO3–saturated H2O, then
brine (1 ml), successively. The organic layer was dried over Na2SO4 then
evaporated under reduced pressure. The residue was purified by preparative
TLC [silica gel (0.25 mm thickness), being applied for 18 cm, with develop-
ment with CHCl3–(CH3)2CO (19 : 1) for 9 cm, then elution with
CHCl3–MeOH (9 : 1)] to furnish an ester, 1b (0.5 mg). Through a similar
procedure, 1c (0.5 mg) was prepared from 1a (0.7 mg) using (S)-MTPA
(25 mg), EDC (14 mg), and 4-DMAP (11 mg).
Tricalysioside X (8): Amorphous powder, [a]D24 ꢀ18.2 (cꢁ0.28, pyri-
dine); IR nmax (film) cmꢀ1: 3354, 2930, 2871, 1634, 1443, 1375, 1242, 1071,
1024, 983; 1H-NMR (C5D5N, 400 MHz) d: 4.72 (1H, d, Jꢁ8 Hz, H-1ꢂ), 4.52
(1H, dd, Jꢁ12, 3 Hz, H-6ꢂa), 4.45 (1H, dd, Jꢁ8, 8 Hz, H-3ꢂ), 4.44 (1H, dd,
Jꢁ8, 8 Hz, H-4ꢂ), 4.43 (1H, dd, Jꢁ12, 5 Hz, H-6ꢂb), 4.22 (1H, d, Jꢁ10 Hz,
H-19a), 4.12 (2H, s, H2-17), 4.04 (1H, dd, Jꢁ8, 8 Hz, H-2ꢂ), 3.85 (1H, m, H-
5ꢂ), 3.83 (1H, s, H-15), 3.53 (1H, d, Jꢁ10 Hz, H-19b), 2.50 (1H, br s, H-13),
2.00 (1H, m, H-14a), 1.99 (1H, m, H-7a), 1.95 (1H, m, H-6a), 1.90 (1H, m,
H-3a), 1.79 (2H, m, H-6b and 14b), 1.76 (1H, m, H-12a), 1.70 (1H, m, H-
7b), 1.69 (1H, d, Jꢁ12 Hz, H-1a), 1.52 (1H, m, H-12b), 1.51 (2H, m, H2-
11), 1.30 (2H, m, H2-2), 1.14 (3H, s, H3-18), 1.11 (1H, br d, Jꢁ6 Hz, H-9),
1.10 (1H, m, H-3b), 1.00 (3H, s, H3-20), 0.91 (1H, d, Jꢁ12 Hz, H-5), 0.67
1
(1H, br dd, Jꢁ12, 12 Hz, H-1b); H-NMR (CD3OD, 400 MHz) d: 4.24 (1H,
d, Jꢁ8 Hz, H-1ꢂ), 4.17 (1H, dd, Jꢁ10, 10 Hz, H-4ꢂ), 4.05 (1H, d, Jꢁ10 Hz,
H-19a), 3.89 (1H, dd, Jꢁ12, 2 Hz, H-6ꢂa), 3.79 (1H, dd, Jꢁ12, 5 Hz, H-6ꢂb),
3.67 (1H, d, Jꢁ11 Hz, H-17a), 3.66 (1H, dd, Jꢁ10, 9 Hz, H-3ꢂ), 3.65 (1H, d,
Jꢁ11 Hz, H-17b), 3.44 (1H, br s, H-15), 3.40 (1H, ddd, Jꢁ10, 5, 2 Hz, H-
5ꢂ), 3.31 (1H, d, Jꢁ10 Hz, H-19b), 3.30 (1H, dd, Jꢁ9, 8 Hz, H-2ꢂ), 2.10
(1H, br s, H-13), 1.86 (1H, d, Jꢁ12 Hz, H-1a), 1.86 (1H, m, H-3a), 1.85 (1H,
d, Jꢁ10 Hz, H-14a), 1.74 (1H, m, H-6a), 1.64 (2H, m, H-2a, 11a), 1.60 (3H,
m, H-3b, 7a, 14b), 1.57 (2H, m, H2-12), 1.38 (4H, m, H-2b, 6b, 7b, 11b),
1.10 (1H, br d, Jꢁ9 Hz, H-9), 1.06 (3H, s, H3-20), 1.02 (3H, s, H3-18), 0.95
(1H, br d, Jꢁ11 Hz, H-5), 0.81 (1H, br dd, Jꢁ12, 12 Hz, H-1b); 13C-NMR
(C5D5N and CD3OD, 100 MHz): Table 3; HR-ESI-MS (negative-ion mode)
m/z: 579.2484 [MꢀH]ꢀ (Calcd for C26H43O12S: 579.2480); HR-ESI-MS (on
addition of D2O) (negative-ion mode) m/z: 585.2853 [MꢀH]ꢀ (Calcd for
C26H37D6O12S: 585.2857).
Tricalysionol A 9-O-(R)-MTPA ester (1b): Amorphous powder; 1H-NMR
(CDCl3, 400 MHz) d: 7.53—7.50 (2H, m, aromatic protons), 7.41—7.36
(3H, m, aromatic protons), 5.80 (1H, qd, Jꢁ1, 1 Hz, H-4), 5.13 (1H, m, H-
9), 3.55 (3H, q, Jꢁ1 Hz, –OCH3), 2.27 (1H, d, Jꢁ18 Hz, H-2a), 2.15 (1H, d,
Jꢁ18 Hz, H-2b), 1.93 (3H, d, Jꢁ1 Hz, H3-13), 1.84 (1H, m, H-7a), 1.82
(1H, m, H-8a), 1.59 (1H, m, H-8b), 1.56 (1H, m, H-7b), 1.35 (3H, d,
Jꢁ6 Hz, H3-10), 1.00 (3H, s, H3-11), 0.95 (3H, s, H3-12); HR-ESI-MS m/z:
465.1863 [MꢄNa]ꢄ (Calcd for C23H29O5F3Na: 465.1859). Tricalysionol A
9-O-(S)-MTPA ester (1c): Amorphous powder; 1H-NMR (CDCl3, 400 MHz)
d: 7.51—7.49 (2H, m, aromatic protons), 7.42—7.36 (3H, m, aromatic pro-
tons), 5.85 (1H, qd, Jꢁ1, 1 Hz, H-4), 5.15 (1H, m, H-9), 3.49 (3H, q,
Jꢁ1 Hz, –OCH3), 2.38 (1H, d, Jꢁ18 Hz, H-2a), 2.21 (1H, d, Jꢁ18 Hz, H-
2b), 1.98 (3H, d, Jꢁ1 Hz, H3-13), 1.91 (1H, m, H-7a), 1.88 (1H, m, H-8a),
1.77 (1H, m, H-7b), 1.64 (1H, m, H-8b), 1.29 (3H, d, Jꢁ6 Hz, H3-10), 1.034
(3H, s, H3-11), 1.029 (3H, s, H3-12); HR-ESI-MS m/z: 465.1850 [MꢄNa]ꢄ
(Calcd for C23H29O5F3Na: 465.1859).
Tricalysioside Y (9): Amorphous powder, [a]D23 ꢀ36.8 (cꢁ0.21, pyri-
1
dine); IR nmax (film) cmꢀ1: 3367, 2929, 2872, 1446, 1415, 1074, 1030; H-
NMR (C5D5N, 600 MHz) d: 5.04 (1H, d, Jꢁ8 Hz, H-1ꢃ), 4.84 (1H, d,
Jꢁ8 Hz, H-1ꢂ), 4.58 (1H, dd, Jꢁ11, 2 Hz, H-6ꢃa), 4.55 (1H, dd, Jꢁ11, 2 Hz,
H-6ꢂa), 4.38 (1H, dd, Jꢁ11, 5 Hz, H-6ꢂb), 4.33 (1H, d, Jꢁ10 Hz, H-19a),
4.25 (2H, br dd, Jꢁ8, 8 Hz, H-3ꢂ and 3ꢃ), 4.23 (1H, d, Jꢁ12 Hz, H-17a),
4.22 (1H, dd, Jꢁ8, 8 Hz, H-4ꢂ), 4.20 (1H, dd, Jꢁ8, 8 Hz, H-4ꢃ), 4.16 (1H,
dd, Jꢁ11, 6 Hz, H-6ꢃb), 4.08 (1H, d, Jꢁ12 Hz, H-17b), 4.03 (2H, br dd,
Jꢁ8, 8 Hz, H-2ꢂ, 2ꢃ), 4.02 (1H, m, H-5ꢂ), 3.98 (1H, s, H-15), 3.96 (1H, m,
H-5ꢃ), 3.51 (1H, d, J ꢁ10 Hz, H-19b), 2.40 (1H, br s, H-13), 2.08 (1H, m, H-
14a), 2.07 (1H, m, H-7a), 2.06 (1H, m, H-3a), 2.03 (1H, m, H-7b), 1.88 (1H,
d, Jꢁ11 Hz, H-14b), 1.73 (1H, m, H-12a), 1.68 (1H, d, Jꢁ12 Hz, H-1a),
1.68 (1H, m, H-6a), 1.52 (2H, br s, H2-11), 1.52 (1H, m, H-12b), 1.40 (1H,
m, H-6b), 1.32 (2H, m, H2-2), 1.26 (1H, br s, H-9), 1.10 (3H, s, H3-18), 0.98
(3H, s, H3-20), 0.97 (1H, br d, Jꢁ12 Hz, H-5), 0.91 (1H, m, H-3b), 0.71 (1H,
br dd, Jꢁ12, 12 Hz, H-1b); HR-ESI-MS (positive-ion mode) m/z: 685.3397
[M + Na]+ (Calcd for C32H54O14Na: 685.3405).
Acid Hydrolysis of Sulfatricalysine A (2) Sulfatricalysine A (2)
(12.1 mg) was hydrolyzed in 1 ml of 2 M HCl at 60 °C for 3 h. After cooling,
the reaction mixture was extracted with 1.5 ml of CHCl3 three times to give
1.1 mg of salicylicacid (2a). The aqueous layer was neutralized by addition
of Ba(OH)2 solution to give 4.1 mg of BaSO4 as a precipitate. The precipi-
tate was not soluble in 2 M HCl.
Tricalysioside Z (10): Amorphous powder, [a]D23 ꢀ8.0 (cꢁ0.50, pyri-
dine); IR nmax (film) cmꢀ1: 3367, 2928, 1729, 1593, 1446, 1371, 1073, 1025;
1H-NMR (C5D5N, 600 MHz) d: 6.21 (1H, d, Jꢁ8 Hz, H-1ꢃ), 4.97 (1H, d,
Jꢁ8 Hz, H-1ꢂ), 4.52 (1H, dd, Jꢁ12, 3 Hz, H-6ꢂa), 4.42 (1H, dd, Jꢁ12, 3 Hz,
H-6ꢃa), 4.38 (1H, dd, Jꢁ12, 5 Hz, H-6ꢂb), 4.34 (1H, dd, Jꢁ12, 5 Hz, H-6ꢃb),
4.30 (1H, dd, Jꢁ9, 9 Hz, H-4ꢃ), 4.23 (1H, br dd, Jꢁ8, 8 Hz, H-3ꢂ, 3ꢃ), 4.21
(1H, dd, Jꢁ8, 8 Hz, H-4ꢂ), 4.17 (1H, dd, Jꢁ8, 8 Hz, H-2ꢃ), 4.10 (1H, d,
Jꢁ11 Hz, H-17a), 4.00 (1H, dd, Jꢁ8, 8 Hz, H-2ꢂ), 4.00 (1H, m, H-5ꢂ, 5ꢃ),
3.98 (1H, d, Jꢁ11 Hz, H-17b), 3.72 (1H, dd, Jꢁ12, 4 Hz, H-1), 3.54 (1H, dd,
Jꢁ16, 7 Hz, H-11a), 2.64 (1H, m, H-2a), 2.55 (1H, ddd, Jꢁ14, 14, 4 Hz, H-
6a), 2.44 (1H, m, H-2b), 2.36 (1H, m, H-3a), 2.34 (1H, br s, H-13), 2.29
(1H, d, Jꢁ13 Hz, H-14a), 2.10 (1H, m, H-11b), 2.05 (1H, m, H-14b), 2.01
(1H, dd, Jꢁ14, 2 Hz, H-6b), 1.91 (1H, m, H-12a), 1.85 (2H, s, H2-15), 1.79
(1H, br d, Jꢁ14 Hz, H-7a), 1.65 (3H, s, H3-20), 1.62 (1H, br d, Jꢁ9 Hz, H-
9), 1.61 (1H, m, H-12b), 1.52 (1H, m, H-7b), 1.25 (3H, s, H3-18), 1.19 (1H,
ddd, Jꢁ14, 14, 4 Hz, H-3b), 1.11 (1H, br d, Jꢁ14 Hz, H-5); HR-ESI-MS
(positive-ion mode) m/z: 699.3194 [MꢄNa]ꢄ (Calcd for C32H52O15Na:
699.3198).
Salicylic acid (2a): Amorphous powder; IR nmax (film) cmꢀ1: 3192, 3065,
2926, 2854, 1663, 1610, 1485, 1464, 1241, 1217; UV lmax (MeOH) nm
(log e): 299 (3.26), 232sh (3.52) 209 (3.77); 1H-NMR (CDCl3, 400 MHz) d:
10.5 (1H, br s, –OH), 7.91 (1H, dd, Jꢁ8, 1 Hz, H-6), 7.51 (1H, ddd, Jꢁ8, 8,
2 Hz, H-5), 7.01 (1H, dd, Jꢁ8, 1 Hz, H-3), 6.92 (1H, ddd, Jꢁ8, 8, 2 Hz, H-
4); 13C-NMR (CDCl3, 100 MHz) d: 174.8 (C-7), 162.2 (C-2), 137.0 (C-4),
130.9 (C-6), 119.6 (C-5), 117.8 (C-3), 111.3 (C-1); HR-ESI-MS (negative-
ion mode) m/z: 137.0237 [MꢀH]ꢀ (Calcd for C7H5O3: 137.0233).
Sugar Analysis All the glucosides (3—10) (500 mg each) were hy-
drolyzed with 100 ml of 1 M HCl at 90 °C for 2 h. The reaction mixtures were
washed with 100 ml of EtOAc then the aqueous layers were analyzed by
HPLC with a chiral detector (JASCO OR-2090plus) on an amino column
[Asahipak NH2P-50 (4.6 mmꢅ250 mm), CH3CN–H2O (3 : 1), 1 ml/min] to
give peaks at 9.0 min with positive optical rotation signs. The peaks were
identified by co-chromatography with authentic D-glucose. Neutralized
aqueous layer of hydrolysate of 2, obtained in the above experiment, was
treated with Amberlite MB-3 then sugar was also analyzed to be D-glucose.
Enzymatic Hydrolysis of Tricalysionoside A (1) Tricalysionoside A
(1) (11.6 mg) in 2 ml of H2O was hydrolyzed with emulsin (19.8 mg) and
crude hesperidinase (6.0 mg) at 37 °C for 15 h. The reaction mixture was
Acknowledgements The authors are grateful for access to the supercon-
ducting NMR instrument at the Analytical Center of Molecular Medicine of

January 2011
77
Hiroshima University Faculty of Medicine and an Applied Biosystem
QSTAR XL system ESI (Nano Spray)-MS at the Analysis Center of Life
Science of the Graduate School of Biomedical Sciences, Hiroshima Univer-
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sity. The authors are also grateful for the use of the NMR instrument (JEOL 12) Inada A., Yamada M., Murata H., Kobayashi M., Toya H., Kato Y.,
ECA-600) at the Natural Science Center for Basic Research and Develop-
ment, Hiroshima University. This work was supported in part by Grants-in-
Aid from the Ministry of Education, Culture, Sports, Science and Technol-
ogy of Japan, the Japan Society for the Promotion of Science, and the Min-
istry of Health, Labour and Welfare. Thanks are also due to the Takeda Sci-
ence Foundation for financial support.
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