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parison of the 13C-NMR data with those of the ent-kaurane
glycosides tricalysiolides A—C, E, and F2) that possess such
a system. A 16b,17-dihydroxy system in the ent-kaurane
diterpenoids such as cussovantoside A13) exhibits distinctly
Extraction and Isolation The powdered, air-dried roots of T. okelensis
was extracted with CH2Cl2–MeOH (1 : 1) under the standard extraction pro-
tocol at NCI. The crude extract (7.1 g) was subjected to silica gel chro-
matography (4.5ꢆ45 cm) using stepwise gradient elution CHCl3/MeOH at
20 : 1 (1000 ml), 10 : 1 (900 ml), 8 : 1 (900 ml), 6 : 1 (700 ml), 4 : 1 (800 ml),
different chemical shifts for C-13, C-16, and C-17. The 2 : 1 (600 ml), 1 : 1 (300 ml), and finally with MeOH (500 ml) to afford 11
pooled fractions (A—L) according to TLC. Fr. F (900 mg) was chro-
matographed on a C18 reversed-phase column (3.0ꢆ35 cm) using CH3CN/
H2O at 40 : 60 (1800 ml), 50 : 50 (2000 ml), 60 : 40 (1000 ml), 70 : 30 (600
ml), and finally with MeOH (400 ml) to afford 8 pooled fractions according
to TLC. Fr. 3 (102 mg) was further purified on a semi-preparative C18 re-
versed-phase HPLC column using 24% CH3CN/H2O (0.1% trifluoroacetic
acid (TFA)), at a flow rate of 4 ml/min, to afford compound 2 (7.1 mg, tR
12.1 min). Fr. G (750 mg) was similarly chromatographed on a C18 reversed-
ROESY data of 1 also provided stereochemical information
regarding the ent-kaurane skeleton: NOE correlations were
observed between H-3b and H-5b, between Me-20a and 19-
CHO, H-14a, H-2a, and H-6a, and between H-9b and H-
15b. Other key NOE and HMBC correlations of 1 are shown
in Table 1 and fully supports the structure ent-kauran-
3a,16a,17-triol-19-al 3-O-[5-O-vanilloyl-b-D-apiopyranosyl
(1→6)]-b-D-glucopyranoside.
phase column (3.0ꢆ30 cm) using a stepwise gradient elution of MeOH/H2O
at 30 : 70 (1800 ml), 40 : 60 (2100 ml), and 50 : 50 (1000 ml) to give 7 pooled
Tricalysioside W (2) possesses a molecular formula of
C42H60O17 that was determined by the high-resolution ESI-
MS (m/z 859.3777 Calcd for [C42H60O17ꢀNa]ꢀ, 859.3723)
and 13C-NMR data. The NMR data of 2 due to the aglycone
and sugar moieties were almost identical to those of 1 (Table
1). It appears that the only differences between 1 and 2 are
the acyl moieties substituted at the C-5 hydroxy group of the
apiosyl unit. Compared to 1, the additional resonances in the
1H-NMR spectrum at dH 7.94 and 6.62 (1H each, d, Jꢁ
16.0 Hz) that correlated with the carbon resonances at dC
fractions. Fr. 5 (48 mg) was further purified on a semi-preparative C18 re-
versed-phase HPLC column using 22% CH3CN/H2O (0.1% TFA), at a flow
rate of 4 ml/min, to afford compound 1 (17.6 mg, tR 12.4 min).
Compound 1: Amorphous pale yellow powder; [a]D25 ꢃ42.4 (cꢁ0.03,
MeOH); UV (MeOH) lmax (e) 219 (741), 255 (524), 295 (229) nm; IR
(neat) nmax 3379, 2937, 1677, 1594, 1428, 1283, 1201, 1134, 799, 722 cmꢃ1
;
NMR data (pyridine-d5), see Table 1; HR-ESI-MS m/z 803.3474 (Calcd for
[C39H56O16ꢀNa]ꢀ, 803.3461).
Compound 2: Amorphous pale yellow powder; [a]D25 ꢃ20.0 (cꢁ0.04,
MeOH); UV (MeOH) lmax (e) 240 (7943), 330 (5248), nm; IR (neat)
n
max 3346, 2934, 1673, 1514, 1458, 1280, 1123, 1040, 800, 722 cmꢃ1; NMR
data (pyridine-d5), see Table 1; HR-ESI-MS m/z 859.3777 (Calcd for
146.4 (d) and 115.3 (d), respectively, in the HMQC spectrum [C42H60O17ꢀNa]ꢀ, 859.3723).
Acid Hydrolysis of Compound 1 A solution of compound 1 (2.2 mg)
indicated the presence of a typical trans-double bond system
in the acyl moiety of 2. In conjunction with other resonances
at dH 7.02 (s, 2H) and 3.85 (s, 6H) and dC 167.5 (s), 149.4 (s,
2C), 146.4 (d), 140.9 (s), 125.3 (s), 115.3 (d), 107.0 (d), and
56.6 (q, OMeꢆ2), the presence of a trans-sinapoyl moiety14)
in 2 was confirmed. Thus, the structure of 2 was established
as ent-kauran-3a,16a,17-triol-19-al 3-O-[5-O-E-sinapoyl-b-
D-apiopyranosyl(1→6)]-b-D-glucopyranoside. The assigment
of its NMR data (Table 1) was facilitated by the HMQC
and HMBC experiments and comparison with those of com-
pound 1.
ent-Kaurane diterpenoids are particularly rich in Labiatae
plants,15) with more than 500 compounds from the single
Isodon genus.16) However, there are only a few ent-kaurane
3-O-glycosides reported in the literature.17—23) This is the
first report of such compounds from the Tricalysia genus.
in 1 M HCl/dioxane (1 : 1, 2 ml) was refluxed at 95 °C for 1.5 h. After cool-
ing, the reaction mixture was diluted with H2O (2 ml) and extracted with
CHCl3 (3 mlꢆ3). The aqueous layer was neutralized by passing through an
Amberlite MB-150 column eluting with H2O. The eluent was concentrated
to dryness to yield a sugar residue, which was analyzed by TLC (silica gel,
CHCl3–MeOH–AcOH–H2O, 70 : 30 : 10 : 5) in comparison with standard
samples. Glucose (Rfꢁ0.29) and apiose (Rfꢁ0.47) were detected. Determi-
nation of the absolute configuration of the sugars followed a recently re-
ported procedure.24) Briefly, the sugar residue (about 0.7 mg) and L-cysteine
methyl ester (1 mg) was dissolved in pyridine (0.2 ml) and heated at 60 °C
for 1 h, and then o-tolyl isothiocyanate (1 mg) was added to the mixture and
heated at 60 °C for another 1 h. The reaction mixture (10 ml) was analyzed
by analytical HPLC eluting with 20% aqueous CH3CN containing 0.1% TFA
at a flow rate of 1 ml/min over 35 min-run. D-Glucose (tRꢁ17.63 min) and D-
apiose (tRꢁ27.02 min) were identified by comparing their retention times
with those of the authentic samples, while L-glucose showed a different re-
tention time at 16.21 min. L-Apiose was not included in the experiment due
to unavailability.
Acknowledgments The authors thank the Natural Products Branch
Repository Program at the National Cancer Institute for providing the plant
extract, Dr. B. Avula for recording HR-ESI-MS spectra, and Mr. F. T. Wig-
gers for obtaining NMR spectra. This work was supported by the NIH,
NIAID, Division of AIDS, Grant No. AI027094, the USDA Agricultural Re-
search Service Specific Cooperative Agreement No. 58-6408-2-0009, and
the China Scholarship Council.
Experimental
General Experimental Procedures Optical rotations were measured
with an Autopol IV polarimeter. UV was obtained from an HP 8453 diode
array spectrophotometer. IR spectra were recorded using a Thermo Nicolet
IR 300 FT/IR spectrometer. The 1D and 2D NMR (COSY, HMQC, HMBC
and ROESY) spectra using standard pulse programs were recorded at room
temperature in pyridine-d5 on a Bruker Avance DRX 500 FT spectrometer
operating at 500 (1H) or 125 (13C) MHz. The chemical shift values are rela- References
tive to the NMR solvent residue (dH/C 8.73/149.9). ESI-MS data were ob-
tained on an Agilent Series 1100 SL mass spectrometer. Column chromatog-
raphy was performed using normal phase silica gel (J. T. Baker, 40 mm) and
reversed-phase silica gel (RP-18, J. T. Baker, 40 mm). Semi-preparative
HPLC was conducted on a C18 column (Gemini, 250ꢆ10 mm, 5 mm) with
UV detection at 254 nm. Analytical HPLC was performed on a C18 column
(Gemini, 4.6ꢆ150 mm, 5 mm) with UV detection at 250 nm. TLC was car-
ried out on silica gel sheets (Alugram® Sil G/UV254, Macherey-Nagel, Ger-
many) and reversed-phase glass plates (RP-18 F254S, Merck, Germany) with
visualization by UV at 254 nm or spraying with 10% H2SO4 followed by
heating.
Plant Material The roots of Tricalysia okelensis was collected in South
Sandwick Islands Park Manovo-Gounda-St. Floris, Central Africa Republic
(coordinates: 21°12ꢅ00ꢄE, 08°29ꢅ00ꢄN) on May 25, 1987, and identified by
J. M. Fay. A voucher specimen is stored at National Smithsonian Institute in
Washington D.C., U.S.A.
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Xu, Wen-Hui
