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L.-S. Gan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3845–3849
R1 R2
Xyl α-OH OH =O
R3
R4
R5
H
H
H
H
close to those of 1 except signals for the xylose moiety. The molec-
ular formula of C30H48O6 and further 2D NMR experiments con-
firmed its structure as the aglycone of compound 1. In the acid
hydrolysis experiment of 1, compound 2 was also detected by
co-TLC with the reaction product. Compared to 2, compound 321
showed an additional acetyl group as indicated by a 1H NMR
methyl singlet at dH 2.21 (3H, s) and two 13C NMR carbon signals
at dC 170.9 and 21.4. The acetyl group was determined as 15-OAc
by a key HMBC correlation from H-15 to the ester carbonyl carbon.
Saponification of 3 by 1 M NaOMe in MeOH yielded compound 2,
which, in addition to 2D NMR experiments, further confirmed
1
2
3
4
5
6
7
8
H
H
H
α-OH OH =O
α-OH OAc =O
β-OH OH =O
HO
24
H
O
R5
R2
12
20
Xyl α-OH OH OH
Xyl β-OH OH OH
OH
H
H
H
H
H
21
R4
H
H
β-OH OAc OAc
β-OH OAc OH
H
15
16
R3
9
10
11
Xyl α-OH OAc OH
30
R1O
H
α-OH OAc OH
H
H
H
H
H
OH
OH
OAc OH
OH
OAc OAc
OAc OAc
29 28
12 Xyl
13 Xyl
the structure of
3 a-acetoxy-20,24-epoxy-
as (20S⁄,24R⁄)-15
H
H
3b,12a
,25-trihydroxy-9,19-cyclolanostan-16-one. Compound 422
14 Xyl α-OH OH
exhibited the same molecular formula as that of 2. The dd
Figure 1. Structures of cycloartane triterpenoids isolated from the rhizomes of
Beesia calthifolia.
(J = 9.0, 2.0 Hz) multiplicity of H-12 in the 1H NMR data of 4
(Table 1) suggested a b-OH instead of the
a-OH configuration in
compound 2. This deduction was then confirmed by 2D NMR
experiments, including HSQC, HMBC and NOESY. A key NOESY cor-
relation between H-12 and H3-30 clearly showed the b configura-
tion for 12-OH (Fig. 3).
H3-28 and H3-29 to C-3, C-4, and C-5, from H2-19 to C-1, C-5, C-9,
C-10, and C-11, from H3-18 to C-12, C-13, C-14, and C-17, from H3-
30 to C-8, C-13, C-14, and C-15, from H3-21 to C-20, C-22, and C-17,
and from H3-26 and H3-27 to C-24 and C-25. An HMBC correlation
of H-24/C-20 confirmed the 20,24-epoxy linkage. The carbonyl
group was located at C-16 by the correlation between H-17 and
C-16. An HMBC correlation from the anomeric hydrogen H-10 to
Compound 5 has a molecular formula of C35H58O11 as deter-
mined by HRESIMS.23 The 1H NMR data showed two typical signals
at dH 0.64 (1H, d, J = 4.0 Hz) and 0.43 (1H, d, J = 4.0 Hz) assignable
to H2-19, as well as six methyl singlets indicating that one of the
original methyl was modified. A total of 35 carbons were exhibited
in the 13C NMR spectrum, including an anomeric carbon at dC 107.4
and twelve signals in the range of dC 60–90. The above data
indicated the existence of a pentose moiety and eight other oxy-
gen-bearing carbons. The HSQC spectrum indicated that two of
the oxygen-bearing carbons were methylenes, including one
assignable to CH2-50 of the pentose and the other CH2OH-18
possibly derived from a methyl.11 HMBC data first allowed the
verification of the same carbon skeleton for 5 as those of 1–4.
Compared to compound 1, positions of the pentose and the other
hydroxyl groups remained the same. The carbonyl group in 1
was saturated to form a hydroxyl at C-16. The pentose was also
C-3 placed the pentose on C-3. The pentose was assigned as b-D-xy-
lose based on the NMR data and comparison with those derivatives
isolated from this plant, as well as further acid hydrolysis and co-
TLC with an authentic D
-xylose sample.11,12 As concluded from the
literature,3–14 relative configuration at C-12 in these 12-hydroxy-
20,24-epoxy-cycloartane triterpenoids can be assigned by the mul-
tiplicity of H-12 in the 1H NMR spectra, a t-like dd peak (both
J > 7.0 Hz) for the 12
(J1 P 8.0, J2 < 3.0 Hz) for the 12b-OH configuration. For compound
1, the configuration of OH-12 was assigned as by the dd (J = 8.2,
a-OH configuration and a broad d or dd peak
a
7.8 Hz) multiplicity of H-12, which was further confirmed by a
ROESY correlation of H-12/H3-18. ROESY correlations of H-3/H3-
28, H3-28/H-5, H-5/H-7
a, H-7a/H3-30, and H3-30/H-17showed
confirmed to be b-
as described above. Relative configuration at C-12 was determined
as -OH by the t-like dd (J = 8.2, 7.9 Hz) peak of H-12 and all the
D-xylose by the same acid hydrolysis protocol
their -orientation, while the b-oriented hydrogens exhibited cor-
a
relations of H3-29/H-19b, H-19b/H-8, H-8/H3-18, and H3-18/H-15
(Fig. 2). The aglycone of 1, compound 2, was chosen as a model
compound and studied theoretically on the rotation of the C-17–
C-20 single bond of these 20,24-epoxy-cycloartane triterpenoids.
A Monte Carlo conformational searching with MMFF94 force field
and an 1D Potential Energy Surface (PES) scan on the dihedral
angle of 21-20-17-16 by modredundant optimization at the
HF/6-31G(d,p) level showed that free rotation of the C-17–C-20
single bond was restricted to some extent (Supplementary data
S8). The configurations at C-20 and C-24 were then determined
as 20S⁄ and 24R⁄ by ROESY correlations of H-17/H3-21 and H3-
21/H-24, respectively. The structure of compound 1 was thus
a
relative configurations were verified by NOESY correlations as
shown in Figure 3. The structure of 5 were then determined to
be (20S⁄,24R⁄)-20,24-epoxy-3b-(b-
D
-xylopyranosyloxy)-9,19-cy-
clolanostane-12a,15a,16b,18,25-pentol.
Comparing to that of 5, the molecular formula of 6 showed loss
of one oxygen atom.24 In the 1H NMR spectrum, seven methyl sin-
glets indicated that the CH2OH-18 group in 5 was converted back
to a methyl group in 6. A broad d (J = 8.2 Hz) multiplicity for H-
12 in the 1H NMR spectrum indicated a b-OH configuration at C-
12. All the assignments were confirmed by 2D NMR experiments,
including HMQC, HMBC, and NOESY. Comparing to 13C NMR chem-
assigned as (20S⁄,24R⁄)-20,24-epoxy-12
(b- -xylopyranosyloxy)-9,19-cyclolanostan-16-one.
a,15a,25-trihydroxy-3b-
ical shifts of the corresponding known 12a-OH configuration
structure, beesioside N (14),12 chemical shifts for C-17 and C-18
of compound 6 showed the most differences (57.6 and 14.9 in 6,
and 48.5 and 20.7 in beesioside N, respectively), which may be
D
(20S⁄,24R⁄)-20,24-Epoxy-3b,12
a,15a,25-tetrahydroxy-9,19-
cyclolanostan-16-one (2)20 showed 1H and 13C NMR spectra very
HO
H
H3C
CH3
OH
H
O
H3C
CH3
H
H
H
H
O
H
H
HO
H
H
CH3
OH
H
H
H
O
H
O
XylO
OH
CH3
H
H
H
OH
H3C
H
H
O
OH
H
O
HO
HO
Figure 2. Key HMBC (left, H ? C) and ROESY (right, H M H) correlations of compound 1.