Mumic acids A-E: New diterpenoids from mumiyo

Article abstract of DOI:10.1007/s11418-013-0771-2

Five new diterpenoids belonging to labdane and isopimarane skeletons, mumic acids A-E (1-5), have been isolated from mumiyo. Their structures and absolute configurations were elucidated on the basis of spectroscopic data and chemical derivatization. The Japanese Society of Pharmacognosy and Springer Japan 2013.

Full text of DOI:10.1007/s11418-013-0771-2

J Nat Med
DOI 10.1007/s11418-013-0771-2
NOTE
Mumic acids A–E: new diterpenoids from mumiyo
•
Yuko Kiren Alfarius Eko Nugroho Yusuke Hirasawa Osamu Shirota
•
•
•
•
Myrzaim Bekenova Narbekov Omorbay Narbekovich Marina Shapilova
•
•
•
Hiromichi Maeno Hiroshi Morita
Received: 26 March 2013 / Accepted: 4 April 2013
Ó The Japanese Society of Pharmacognosy and Springer Japan 2013
Abstract Five new diterpenoids belonging to labdane
and isopimarane skeletons, mumic acids A–E (1–5), have
been isolated from mumiyo. Their structures and absolute
configurations were elucidated on the basis of spectro-
scopic data and chemical derivatization.
scientific evidences on the chemical components and bio-
activity are lacking [1]. In our search for new bioactive
compounds [2–11], the mumiyo in Kyrgyzstan was inves-
tigated, resulting in the isolation of five new diterpenoids,
mumic acids A–E (1–5) and agathic acid (6) [12]. The
structure elucidation of 1–5 are reported herein (Fig. 1).
Mumic acid A {1, [a]2D8 ?9 (c 0.4, MeOH)} was
isolated as a colorless oil, with molecular formula
C22H32O6, as determined by HRESITOFMS [m/z 415.2072
(M ? Na)?, D -2.5 mmu]. IR absorptions suggested the
presence of carbonyl (1737 and 1715 cm-1) and hydroxy
(3420 cm-1) groups. The 1H NMR data (Table 1) of 1
suggested the presence of an oxygenated methine (dH 5.30,
br s) and 4 methyls (dH 0.70, s; dH 1.18, s; dH 2.10, s; dH
2.12, s). The 13C NMR data (Table 2) revealed 22 carbon
resonances due to 3 carbonyls, 2 sp2 quaternary carbons, 2
sp3 quaternary carbons, 1 sp2 methines, 3 sp3 methines, an
Keywords Mumiyo ꢀ Mumic acids A–E ꢀ Diterpenoid ꢀ
Labdane
Introduction
Mumiyo, also known as mumijo, mumie, or shilajit, is a
material often found as crusts in rock cracks or interstices
in the alpine region of Central Asia. Mumiyo has been used
as a traditional medicine in the former Soviet Union, India,
and Tibet for more than 3000 years, and is currently
available in numerous countries as a food supplement [1].
Although there are many claims on the activity of mumiyo,
1
sp2 methylene, 6 sp3 methylenes, and 4 methyls. The H
and 13C NMR data (Tables 1 and 2) of 1 showed simi-
larities to those of agathic acid (6) isolated in this study,
suggesting the structure of 1 as a labdane type of diterp-
enoid related to 6.
Y. Kiren ꢀ A. E. Nugroho ꢀ Y. Hirasawa ꢀ H. Morita (&)
Faculty of Pharmaceutical Sciences, Hoshi University, Ebara
2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan
e-mail: moritah@hoshi.ac.jp
1
Analysis of the H–1H COSY of 1 (Fig. 2) revealed 3
partial structures, a (C-1–C-3), b (C-5–C-7), and c (C-9,
C-11 and C-12). HMBC correlations of H3-20 to C-1, C-5,
C-9, and C-10 revealed the connectivity of partial struc-
tures a, b, c, and C-20 through C-10. The connectivity of
partial structures a, b, C-18, and C-19 through C-4 was
suggested by the HMBC correlations of H3-18 to C-3, C-4,
C-5, and C-19. HMBC correlations of H2-17 to C-7, C-8,
and C-9 indicated the connectivity of partial structures b, c,
and C-17 through C-8. The presence of an acetoxy group at
C-3 was deduced from the HMBC correlations of H-3 and
a methyl (dH 2.10, s) to a carbonyl (dC 172.4). Finally, the
HMBC correlations of H3-16 to C-12, C-13, and C-14 and
O. Shirota
Faculty of Pharmaceutical Sciences at Kagawa Campus,
Tokushima Bunri University, 1314-1 Shido, Sanuki,
Kagawa 769-2193, Japan
M. Bekenova ꢀ N. O. Narbekovich
Commonwealth of Independent States International Phytocenter,
20 Tynystanov Str., Bishkek 720005, Kyrgys Republic
M. Shapilova ꢀ H. Maeno
Cokey Systems, Sanbancho KS Bldg. 1F, 2, Sanbancho,
Chiyoda-ku, Tokyo 102-0075, Japan
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J Nat Med
Fig. 1 Structures of mumic acids A–E (1–5)
Table 1 13H NMR data of mumic acids A–E (1–5) in CD3OD at 300 K
1a
2b
3b
4a
5b
1a
1b
2a
1.43, m
1.65, m
1.71, m
2.25, m
5.30, br s
1.41, td, 13.7, 3.7
1.63, br d, 12.8
1.72, m
1.11, td, 13.4, 3.7
1.85, br d, 12.7
1.52, m
1.05, dd, 12.3, 4.3
2.15, t, 12.3
4.12, m
1.29, dt, 13.4, 3.2
1.78, m
1.60, m
2b
3a
2.22, br t, 14.6
5.33, br s
1.96, m
1.65, m
1.11, br d, 13.4
2.22, td, 13.4, 3.7
1.39, dd, 11.7, 4.4
1.93, m
1.02, d, 12.3
2.39, t, 12.3
1.35, d, 12.0
1.83, m
4.00, dd, 11.3, 3.1
3b
5
1.75, m
1.94, m
2.00, m
1.98, m
2.44, m
1.71, m
1.55, m
1.72, m
2.00, m
2.30, m
5.66, s
1.82, dd, 12.2, 2.2
1.93, m
1.82, br d, 12.2
1.25, m
6a
6b
7a
2.00, m
2.04, m
2.00, m
1.50, m
1.95, m
1.93, m
1.95, br t, 13.2
2.41, m
2.07, dd, 14.2, 3.8
2.24, br t, 14.2
1.76, d, 9.2
1.51, m
7b
9
2.44, m
2.43, dd, 10.7, 3.5
1.63, br d, 11.0
1.54, m
1.69, m
1.80, m
11a
11b
12a
12b
14
15a
15b
16a
16b
17a
17b
18
19
20
Ac
10
1.54, m
2.10, m
1.70, m
1.72, m
2.34, m
1.64, m
2.02, m
2.02, m
5.39, br t, 5.4
1.33, m
2.31, ddd, 13.6, 9.8, 4.2
5.62, s
2.30, ddd, 13.8, 9.6, 4.3
5.60, s
1.50, m
3.92, dd, 7.4, 4.4
3.44, dd, 11.3, 4.4
3.50, dd, 11.3, 7.4
1.64, s
5.32, s
3.30, dd, 8.8, 2.2
2.12, s
2.13, s
2.12, s
3.36, dd, 11.2, 8.8
3.70, dd, 11.2, 2.2
0.96, s
4.57, s
4.92, s
1.18, s
4.56, s
4.91, s
1.25, s
4.54, s
4.89, s
1.26, s
4.52, s
4.87, s
1.26, s
1.12, s
0.82, s
0.70, s
2.10, s
0.65, s
0.62, s
0.68, s
2.09, s
5.49, d, 7.7
3.40, dd, 8.9, 7.7
3.42, dd, 8.9, 8.7
3.51, dd, 9.3, 8.7
3.73, d, 9.3
5.47, d, 8.0
20
30
40
50
3.40, dd, 9.1, 8.0
3.42, dd, 9.3, 9.1
3.52, dd, 9.6, 9.3
3.77, d, 9.6
a
b
400 MHz; 700 MHz
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Table 2 13C NMR data of mumic acids A–E (1–5) in CD3OD at 300 K
No.
1a
2b
3b
4a
5b
No.
1a
2b
3b
4a
65.9
5b
80.7
1
34.2
34.0
40.4
49.1
38.35
15
172.1
18.9
170.3
18.9
170.3
18.9
2
25.6
75.8
47.0
51.3
27.1
39.8
149.3
56.5
41.2
23.0
40.6
158.8
118.7
b
25.5
74.8
48.4
51.6
26.7
39.7
148.9
56.4
41.2
22.9
40.8
161.8
116.8
c
21.2
39.1
45.7
57.9
27.3
39.9
149.3
56.6
41.6
22.9
40.8
162.0
116.7
65.5
47.6
46.0
56.6
26.8
39.4
148.9
57.6
42.2
23.8
128.7
135.1
78.7
27.9
76.6
54.9
51.4
25.4
36.5
138.6
52.2
38.39
19.5
31.1
39.1
129.4
16
12.7
108.6
29.6
63.9
23.1
3
17
107.0
24.6
107.3
24.4
107.0
29.3
4
18
182.5
12.1
5
19
180.3
13.1
175.5
13.6
177.2
13.8
180.7
12.3
6
20
15.5
7
COMe
COMe
10
20
30
40
50
60
172.4
21.2
172.1
21.1
8
9
95.4
95.3
73.8
10
11
12
13
14
a
73.8
78.2
78.1
73.3
73.2
78.0
174.0c
77.5
173.7c
100 MHz; 175 MHz; assigned from HMBC correlations
presence of a sugar moiety. Except for the signals assigned
1
to the sugar moiety, the H and 13C NMR data (Tables 1
and 2) of 2 are highly similar to those of 1, suggesting 2 to
be a glycoside derivative of 1. The sugar moiety was
identified as D-glucuronic acid on the basis of HPLC
analysis with chiral detector of the acid hydrolysate of 2.
The HMBC correlation of H-10 to C-19 and C-50 suggested
the pyranose form of the sugar moiety and the connectivity
of C-19 and C-10 through an oxygen atom. The ROESY
1
correlations for H-10/H-30 and H-50, and the H–1H cou-
pling constant value of H-10/H-20 (7.7 Hz) indicated the
a-orientation of H-10. Further analysis of the 2D NMR data
confirmed the structure of 2 as 19-O-b-D-glucuronic acid-1.
Mumic acid C {3, [a]3D0 ?5 (c 0.2, MeOH)} was isolated
as a colorless oil and had molecular formula C26H38O10, as
determined by HRESITOFMS [m/z 533.2398 (M ? Na)?,
D ?3.5 mmu]. IR absorptions suggested the presence of
Fig. 2 Selected 2D NMR correlations for mumic acid A (1)
H-14 to C-15 completed the structure of 1. Thus, 1 was
deduced to be a new labdane diterpenoid with an acetoxy
group at C-3 and carboxylic acids at C-4 and C-14.
The relative configuration of 1 was determined by
analyses of the NOESY correlations (Fig. 3) and H–1H
1
carbonyl (1732 and 1716 cm-1) and hydroxy (3420 cm-1
)
1
groups. The H NMR data (Table 1) of 3 suggested the
coupling constant data. The a-orientation of H-5, H-9, and
C-18 was deduced from the NOESY correlations of H-5/H-
9 and H3-18, and those of H3-20/H-2b and H2-11 suggested
the b-orientation of C-20. The small 1H–1H coupling
constant value of H-3/H-2a and H-2b indicated H-3 to be
b-oriented. Finally, the C-13–C-14 double bond was
deduced to be of the E configuration based on the NOESY
correlation of H-12a/H-14. Thus, the relative configuration
of 1 was elucidated to be as shown in Fig. 1.
1
presence of a sugar moiety. The differences in the H and
13C NMR data (Tables 1 and 2) of 3 and 6 are reminiscent
to the differences observed between 1 and 2. Thus, 3 was
assumed to be a 19-O-b-glucuronic acid derivative of
agathic acid (6). Acid hydrolysis of 3 gave 6 and a sugar,
which was identified as D-glucuronic acid on the basis of
HPLC analysis with chiral detector.
Mumic acid D {4, [a]2D5 ?28 (c 2.0, MeOH)} was isolated
as a colorless oil, with molecular formula C20H32O5, as
determined by HRESITOFMS [m/z 375.2151 (M ? Na)?, D
?0.4 mmu]. IR absorptions suggested the presence of car-
bonyl (1694 cm-1) and hydroxy (3370 cm-1) groups. The
13C NMR data (Table 2) revealed 20 carbon resonances due
to 1 carbonyl, 2 sp2 quaternary carbons, 2 sp3 quaternary
Mumic acid B {2, [a]2D3 -7 (c 0.3, MeOH)} was isolated
as a colorless oil and had molecular formula C28H40O12, as
determined by HRESITOFMS [m/z 591.2438 (M ? Na)?,
D ?2.1 mmu]. IR absorptions suggested the presence of
carbonyl (1743 and 1721 cm-1) and hydroxy (3393 cm-1
)
1
groups. The H NMR data (Table 1) of 2 suggested the
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J Nat Med
Fig. 5 Selected 2D NMR correlations for mumic acid E (5)
determined by HRESITOFMS [m/z 375.2131 (M ? Na)?,
D -1.6 mmu]. IR absorptions suggested the presence of
carbonyl (1710 cm-1) and hydroxy (3370 cm-1) groups.
The 13C NMR data (Table 2) revealed 20 carbon reso-
nances due to 1 carbonyl, 1 sp2 quaternary carbons, 3 sp3
quaternary carbons, 1 sp2 methines, 4 sp3 methines, 7 sp3
Fig. 3 Selected NOESY correlations for mumic acid A (1)
1
methylenes, and 3 methyls. Analysis of the H–1H COSY
of 5 (Fig. 5) revealed 4 partial structures, a (C-1–C-3),
b (C-5–C-7), c (C-9, C-11 and C-12), and d (C-14 and
C-15). HMBC correlations of H3-20 to C-1, C-5, C-9, and
C-10 revealed the connectivity of partial structures a, b, c,
and C-20 through C-10. The connectivity of partial struc-
tures a, b, C-18, and C-19 through C-4 was suggested by
the HMBC correlations of H3-19 to C-3, C-4, C-5, and
C-18. HMBC correlations of H-14 to C-7, C-8, and C-9
indicated the connectivity of partial structures b, c, and
C-17 through C-8. Finally, the HMBC correlations of H3-
17 to C-12, C-13, C-14, and C-15 revealed the connectivity
of c, d, C-14, and C-17 through C-13.
Fig. 4 Selected 2D NMR correlations for mumic acid D (4)
carbons, 1 sp2 methines, 4 sp3 methines, an sp2 methylene, 6
sp3 methylenes, and 3 methyls. Analysis of the 2D NMR
correlations of 4 (Fig. 4) revealed the structure of 4 to be a
new labdane diterpenoid with hydroxyl groups at C-2, C-14,
and C-15, and carboxylic acids at C-19.
The configuration of 5 was determined as follows. The
b-orientation of C-17, C-19, and C-20 was deduced from
the ROESY correlations of H3-20/H3-17 and H3-19. The
1H–1H coupling constant value of H-3/H-2a and H-2b (11.3
and 3.1 Hz) indicated H-3 to be a-oriented. C-3 was
determined to be of S configuration based on the advanced
Mosher’s method [13]. The vicinal coupling constant value
of H-14/H-15a and H-15b (Hz, respectively) and the CE
signs in the CD spectrum [238 (De -12.5), 229 (0), and
221 (?6.9) nm] of the 3,15,16-tribenzoyl-18-methyl-deri-
vative of 5 [14] indicated the absolute configuration of
C-15 of the terminal 1,2-diol to be R. Thus, 5 was deduced
to be a new isopimarane diterpenoid with hydroxyl group
at C-3, C-15, and C-16, a carboxylic acid at C-18, and C-8–
C-14 double bond.
The configuration of 4 was determined as follows. The
a-orientation of H-5, H-9, and C-18 was deduced from the
NOESY correlations of H-5/H-9 and H3-18, and the RO-
ESY correlation of H3-20/H-2 and H2-11 suggested the
b-orientation of H-2 and C-20. The double bond of C-12–
C-13 was deduced to be of the E configuration based on the
NOESY correlation of H-12/H-14. C-2 was determined to
be of the R configuration based on the advanced Mosher’s
method [13]. The absolute configuration of C-14 of the
terminal 1,2-diol was deduced to be R based on the vicinal
coupling constant value of H-14/H2-15 (5.5 Hz) and the
Cotton effect (CE) signs [237 (De ?13.7), 229 (0), and 222
(-6.8) nm] of the 2,14,15-tribenzoyl-19-methyl-derivative
of 4 [14]. Thus, the structure of 4 was deduced to be
(2R, 4S, 5R, 9S, 10R, 14R, E)-2,14,15-trihydroxy-8(17),
12-labdadien-19-oic acid.
Compounds 1–5 were tested for cytotoxic activity
against the HL-60 (human promyelocytic leukemia) cell
line, LPS-induced NO production inhibitory activity on the
RAW264.7 (murine leukemic monocyte macrophage) cell
line, melanin-production inhibitory activity on the B16F10
(murine melanoma) cell line, lipid-droplet accumulation
inhibitory activity on the MC3T3-G2/PA6 (mouse pre-
Mumic acid E {5, [a]2D5 ?9 (c 0.3, MeOH)} was isolated
as a colorless oil, with molecular formula C20H32O5, as
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J Nat Med
adipocyte) cell line, and vasorelaxant activity on rat aortic
artery. All compounds gave negative results for these
bioactivity assays.
(H2O/MeOH, 4:1 ? 0:1). Further separation of the frac-
tion eluted with H2O/MeOH (4:1) using silica gel column
chromatography (CHCl3/MeOH, 1:0 ? 0:1, with 0.1 %
HCO2H) yielded 5 (24.8 mg, 0.053 %), and separation of
the fraction eluted with H2O/MeOH (1:1) using silica gel
column chromatography (CHCl3/MeOH, 1:0 ? 0:1, with
0.1 % HCO2H) and ODS HPLC (C-18 Capcell Pak MG-
III, 250 9 10 mm; 53 % MeOH aq. with 0.1 % HCO2H;
flow rate 2.0 mL/min; UV detection at 210 nm) yielded 4
(11.7 mg, 0.025 %).
Experimental section
General experimental procedures
Optical rotations were measured on a JASCO DIP-1000
polarimeter. UV spectra were recorded on a Shimadzu
UVmini-1240 spectrophotometer and IR spectra on a
JASCO FT/IR-4100 spectrophotometer. CD spectra were
recorded on a JASCO J-820 polarimeter. High-resolution
ESI MS were obtained on an LTQ Orbitrap XL (Thermo
On the other hand, a part of the water-soluble materials
(1.6 g) were separated by DIAION HP-20 column chro-
matography (H2O/MeOH, 1:0 ? 3:2) and the fraction
eluted with H2O/MeOH (3:2) was subjected to ODS silica
gel column chromatography (H2O/MeOH, 7:3 ? 3:7) to
obtain 2 (9.9 mg, 0.099 %) and 3 (15.0 mg, 0.15 %).
Mumic acid A (1): colorless oil; [a]2D8 ?9 (c 0.4,
1
Scientific). H and 2D NMR spectra were measured on a
400- or 700-MHz spectrometer at 300 K, while 13C NMR
spectra were measured on a 125- or 175-MHz spectrome-
ter. The residual CD3OD chemical shift used as an internal
standard are dH 3.31 and dC 49.0 and for CDCl3 are dH 7.26
and dC 77.0. Standard pulse sequences were used for the
2D NMR experiments.
MeOH); IR (ZnSe) mmax 3420, 2943, 1737, and 1715 cm-1
;
1H NMR data (Table 1) and 13C NMR data (Table 2);
ESIMS m/z 415 (M ? Na)?; HRESIMS m/z 415.2072
(M ? Na)?; calcd. for C22H32O6Na, 415.2097).
Mumic acid B (2): colorless oil; [a]2D3 -7 (c 0.3,
MeOH); IR (ZnSe) mmax 3393, 2943, 1743, and 1721 cm-1
;
Material
1H NMR data (Table 1) and 13C NMR data (Table 2);
ESIMS m/z 591 (M ? Na)?; HRESIMS m/z 591.2438
(M ? Na)?; calcd. for C28H40O12Na, 591.2417).
The natural mumiyo samples were collected from Kyrgys.
To 1 kg of natural mumiyo, 4 L of water was added, and
the mixture was then stirred and heated to boiling for about
40 min. The mixture was cooled, and the precipitates were
separated from the supernatant. The precipitates were then
extracted with water repeatedly to obtain the water extract.
The supernatant and the water extract were combined and
subjected to centrifugation for 10 min. The supernatant
was collected and concentrated by an evaporator. The
concentrated solution was again subjected to centrifuga-
tion, and the resulting supernatant was concentrated by an
evaporator (until a water content of 30 %). The final con-
centrated solution (50 g) was cooled and then packed as
commercial mumiyo. The mumiyo sample used in this study
is stored at the Department of Pharmacognosy, Hoshi
University, as sample HOSHI12001.
Mumic acid C (3): colorless oil; [a]3D0 ?5 (c 0.2,
1
MeOH); IR (ZnSe) mmax 3420, 1732, and 1716 cm-1; H
NMR data (Table 1) and 13C NMR data (Table 2); ESIMS
m/z 533 (M ? Na)?; HRESIMS m/z 533.2398 (M ? Na)?;
calcd. for C26H38O10Na, 533.2363).
Mumic acid D (4): colorless oil; [a]2D5 ?28 (c 2.0,
1
MeOH); IR (ZnSe) mmax 3370, 2942, and 1694 cm-1; H
NMR data (Table 1) and 13C NMR data (Table 2); ESIMS
m/z 375 (M ? Na)?; HRESIMS m/z 375.2151 (M ? Na)?;
calcd. for C20H32O5Na, 375.2147).
Mumic acid E (5): colorless oil; [a]2D5 ?9 (c 0.3,
1
MeOH); IR (ZnSe) mmax 3370, 2938, and 1710 cm-1; H
NMR data (Table 1) and 13C NMR data (Table 2); ESIMS
m/z 375 (M ? Na)?; HRESIMS m/z 375.2131 (M ? Na)?;
calcd. for C20H32O5Na, 375.2147).
Extraction and isolation
Acid hydrolysis of 2 and 3
Mumiyo (50 g) was dissolved in water successively parti-
tioned with EtOAc and n-BuOH, and a part of the n-BuOH-
soluble materials (540 mg) were further separated with silica
gel column chromatography (CHCl3/MeOH, 1:0 ? 0:1). The
fractions eluted with CHCl3/MeOH (20:1) were combined
and subjected to ODS silica gel column chromatography
(H2O/MeOH, 3:2 ? 0:1) to obtain 1 (7.0 mg, 0.22 %).
The rest of the n-BuOH-soluble materials (7.5 g) were
subjected to ODS silica gel column chromatography
2 (2.0 mg) was treated with 2 M aqueous HCl (400 lL) at
100 °C for 1 h. After neutralization with 2 M aqueous
NaOH, the mixture was extracted with CHCl3. The aqueous
layer was submitted to HPLC analysis (GL science NH2
column u 4.6 9 250 mm, eluent: 70 % aqueous MeCN,
flow rate 1.0 mL/min, JASCO OR-1590 chiral detector).
Retention times of authentic L- and D-glucuronic acid
were as follows: L (4.9 min with negative intensity) and
D (4.9 min with positive intensity). The retention time of
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J Nat Med
glucuronic acid in the aqueous layer of hydrolysate of 2 was
4.9 min, with positive intensity. 3 (1.0 mg) was subjected to
a similar treatment as 3, and the retention time of glucose in
the aqueous layer of hydrolysate of 3 was 4.9 min, with
positive intensity.
4.50 (s; H-17a), 4.82 (s; H-17b), 1.29 (s; H3-18), 0.65 (s;
H3-20), and 3.66 (s; 19-OMe).
3,14,15-tri-O-[(R)-MTPA]-18-methyl-5
1H NMR (CDCl3, 400 MHz) d 1.43 (dd, 13.4, 3.2; H-1a),
1.78 (m; H-1b), 1.72 (m; H-2a), 2.01 (m; H-2b), 5.46 (dd,
11.3, 3.1; H-3), 1.80 (m; H-5), 1.10 (m; H-6a), 1.50 (m;
H-6b), 1.93 (m; H-7a), 2.14 (br d, 14.2; H-7b), 1.73 (m;
H-9), 5.21 (s; H-14), 5.22 (d, 8.8; H-15), 4.19 (dd, 11.2,
8.8; H-16a), 4.80 (br d, 11.2; H-16b), 1.00 (s; H3-17), 1.17
(s; H3-19), 0.80 (s; H3-20), and 3.58 (s; 18-OMe).
Synthesis of 2,14,15-tri-O-acyl-19-methyl-4
and 3,14,15-tri-O-acyl-18-methyl-5
To a solution of 4 (0.8 mg in 100 lL MeOH), 20 lL of
TMS-diazomethane (10 % in n-hexane) was added and left
at room temperature. After 10 min, the reaction mixture
was dried under an N2 stream, and the resulting residue
(0.8 mg) was dissolved in 150 lL of CH2Cl2. To the
CH2Cl2 solution, a catalytic amount of 4-(dimethyl-
amino)pyridine and 2 lL of triethylamine were added, and
the mixture was then separated into three containers (50 lL
each). Into the container, (R)-MTPA chloride, (S)-MTPA
chloride, or benzoyl chloride was added, and the solutions
were allowed to stand at room temperature overnight. The
residue obtained under an N2 stream was subjected to SiO2
column chromatography (CHCl3) to obtain the tri-(S)-
MTPA, tri-(R)-MTPA, and tri-benzoyl derivatives of
19-methyl-4. The same procedure was used to obtain tri-
(S)-MTPA, tri-(R)-MTPA, and tri-benzoyl derivatives of
19-methyl-5.
3,14,15-tri-O-[(S)-MTPA]-18-methyl-5
1H NMR (CDCl3, 400 MHz) d 1.42 (dd, 13.4, 3.2; H-1a),
1.76 (m; H-1b), 1.60 (m; H-2a), 1.93 (m; H-2b), 5.46 (dd,
11.3, 3.1; H-3), 1.82 (m; H-5), 1.12 (m; H-6a), 1.51 (m;
H-6b), 1.95 (m; H-7a), 2.19 (br d, 14.2; H-7b), 1.73 (m;
H-9), 5.26 (s; H-14), 5.22 (d, 8.8; H-15), 4.07 (dd, 11.2,
8.8; H-16a), 4.81 (br d, 11.2; H-16b), 1.05 (s; H3-17), 1.19
(s; H3-19), 0.80 (s; H3-20), and 3.66 (s; 18-OMe).
3,14,15-tri-O-benzoyl-18-methyl-5
UV (MeOH) kmax (log e) 228 (4.84) nm; CD (MeOH) kmax
1
(De) 238 (-12.5), 229 (0), and 221 (?6.9) nm. H NMR
2,14,15-tri-O-[(R)-MTPA]-19-methyl-4
(CD3OD, 400 MHz) 5.39 (br d 11.0; H-3), 5.50 (s; H-14),
5.35 (dd, 8.4, 2.2; H-14), 4.47 (dd, 11.0, 8.4; H-15a), 4.77
(br d, 11.0; H-15b), 0.94 (s; H3-17), 1.09 (s; H3-19), 0.78
(s; H3-20), and 3.67 (s; 18-OMe).
1H NMR (CDCl3, 400 MHz) d 1.11 (dd, 12.3, 4.3; H-1a),
2.20 (t, 12.3; H-1b), 5.57 (m; H-2), 1.30 (d, 12.3; H-3a),
2.55 (t, 12.3; H-3b), 1.86 (m; H-7a), 2.38 (m; H-7b), 2.08
(m, H2-11), 5.51 (br t, 5.4; H-12), 5.60 (dd, 7.4, 4.4; H-14),
4.27 (dd, 11.3, 7.4; H-15a), 4.49 (dd, 11.3, 4.4; H-15b),
1.63 (s; H3-16), 4.38 (s; H-17a), 4.84 (s; H-17b), 1.29 (s;
H3-18), 0.62 (s; H3-20), and 3.68 (s; 19-OMe).
Acknowledgments This work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan (MEXT) and a grant from the Open
Research Center Project of Hoshi University.
2,14,15-tri-O-[(S)-MTPA]-19-methyl-4
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1H NMR (CDCl3, 400 MHz) d 1.20 (dd, 12.3, 4.3; H-1a),
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