Sesquiterpene lactone glycosides from the roots of Ferula varia

Article abstract of DOI:10.1248/cpb.c12-00350

Seven new sesquiterpene lactone glycosides (1-7) were isolated from the H₂0-soluble fraction from the MeOH extract of the roots of Ferula varia. Their structures were elucidated by extensive spectroscopic analyses. The absolute configurations o

Full text of DOI:10.1248/cpb.c12-00350

July 2012
Regular Article
Chem. Pharm. Bull. 60(7) 913–919 (2012)
913
Sesquiterpene Lactone Glycosides from the Roots of Ferula varia
,a
Shin-ichiro Kurimoto,a Kyoko Suzuki,a Mamoru Okasaka,a Yoshiki Kashiwada,*
Olimjion Kakhkharovich Kodzhimatov,b and Yoshihisa Takaishia
a Graduate School of Pharmaceutical Sciences, University of Tokushima; 1–78 Shomachi, Tokushima 770–8505,
b
Japan: and Institute of Botany and Botanical Garden; F. Kodzhaev, st. 32, 700143, Tashkent, Uzbekistan.
Received April 17, 2012; accepted May 1, 2012
Seven new sesquiterpene lactone glycosides (1–7) were isolated from the H2O-soluble fraction from the
MeOH extract of the roots of Ferula varia. Their structures were elucidated by extensive spectroscopic analy-
’
ses. The absolute configurations of compounds 1 and 2 were determined by modified Mosher s method.
Key words Ferula varia; sesquiterpene lactone glycoside; Umbelliferae; Uzbekistan
We have been investigating medicinal plants of Uzbeki- spectroscopy (COSY) spectrum of 1 revealed the connectivity
stan in search of new drug leads.1) The roots of Ferula varia of C-1 to C-3, of C-5 to C-6, and of C-8 to C-9. The presence
(SCHRENK) TRAUTV. (Umbelliferae) have been used tradition- of an eudesmane skeleton with a γ-lactone between C-6 and
ally in Uzbekistan to treat fever and intestinal parasites and C-7 was confirmed from the heteronuclear multiple bond cor-
as a mouth rinse.2) We previously reported the isolation and relations (HMBC) of H-6 [δH 4.38 (1H, d, J=11.7Hz)] with
structure elucidation of six new sesquiterpene lactones from C-7 (δC 76.6) and C-12 (δC 180.4); Me-13 (δH 1.41) with C-7,
the EtOAc-soluble fraction of the roots of this plant. Some of C-11 (δC 76.6), and C-12 (δC 180.4); Me-14 (δH 0.95) with C-1
these lactones were found to enhance cytotoxicity against a (δC 85.4) and C-5 (δC 50.3), C-9 (δC 32.9), and C-10 (δC 39.8);
multidrug-resistant cancer cell line (KB-C2) in the presence and of Me-15 (δH 1.73) with C-3 (δC 123.9), C-4 (δC 133.8),
of 2.5µM (a nontoxic concentration) colchicine.3) Our continu- and C-5. The location of the glucosyl moiety was assigned
ing chemical study of this plant has resulted in the isolation to C-1 from the HMBC correlation of H-1′ [δH 4.25 (1H,
and characterization of seven new sesquiterpene lactone gly- d, J=7.8Hz)] with C-1, while its β-linkage was concluded
cosides (1–7) from the H2O-soluble fraction. In this paper, from the J-value (7.8Hz) of the anomeric proton signal. The
we describe the isolation and elucidate the structures of these nuclear Overhauser enhancement and exchange spectroscopy
compounds.
(NOESY) correlations of Me-14 with H-6 and H-8α [δH 1.79
The H2O-soluble fraction from the MeOH extract of the (1H, ddd, J=6.3, 13.7, 14.2Hz)] indicated that ring B adopted
roots of F. varia was separated by repeated column chroma- a chair conformation. The orientations of the hydroxyl groups
tography to give seven new sesquiterpene lactone glycosides at C-1, C-7, and C-11 were assigned as α in each case by the
(1–7).
NOESY correlations of H-1 with H-9β [δH 1.28 (1H, ddd,
Compound 1 was obtained as a white amorphous powder. J=5.4, 13.7, 13.7Hz)], and of Me-13 with H-5 [δH 2.23 (1H, d,
The molecular formula of 1 was assigned as C21H32O10 by J=11.7Hz)] and H-9β (Fig. 2B). The absolute configuration of
4)
’
high-resolution electrospray ionization (HR-ESI) MS (m/z 1 was determined by modified Mosher s method. Thus, the
467.1877 [M+Na]+). The 1H-NMR spectrum of 1 showed (S)-α-methoxy-α-trifluoromethyl phenylacetate (MTPA) ester
the presence of two tert-methyls (δH 0.95, 1.41), one vinyl of the aglycone (1a) was obtained by treatment of 1a with (R)-
methyl (δH 1.73), three methylenes [δH 1.28 (1H, ddd, J=5.4, MTPA chloride, while treatment of 1a with (S)-MTPA chlo-
13.7, 13.7Hz), 1.79 (1H, ddd, J=6.3, 13.7, 14.2Hz), 2.05, 2.09, ride gave (R)-MTPA ester of 1a. The 1S, 5R, 6S, 7S, 10S, 11S
Δ
2.21, and 2.49 (each 1H, m)], one methine [δH 2.23 (1H, d, absolute configuration could be assigned based on the δ val-
4)
Δ
J=11.7Hz)], two oxygenated methines [δH 3.54 (1H, dd, J=6.8, ues ( δ=δS−δR) illustrated in Fig. 3. On the basis of these
9.7Hz), 4.38 (1H, d, J=11.7Hz)], and one olefinic proton [δH findings, the structure of 1 was elucidated as shown in Fig. 1.
5.35 (1H, brs)], together with one anomeric proton [δH 4.25
A pseudomolecular ion peak at m/z 451.1951 [M+Na]+ was
(1H, d, J=7.8Hz)]. The 13C-NMR spectrum displayed 21 car- observed in the positive ion HR-ESI-MS of 2, indicating the
bon resonances including three methyl carbons (δC 11.1, 22.1, molecular formula of C21H32O9, which was 16 mass units
22.9), three sp3 methylene carbons (δC 28.8, 32.9×2), one sp3 smaller than that of 1. The 1H- and 13C-NMR spectra were cor-
methine carbon (δC 50.3), two oxygen-bearing sp3 methine related with those of 1, but differed in the appearance of a me-
carbons (δC 85.4, 87.4), one sp3 quaternary carbon (δC 39.8), thine [δH 2.61 (1H, m); δC 45.7] instead of an oxygen-bearing
two oxygen-bearing sp3 quaternary carbons (δC 76.6×2), one sp3 quaternary carbon seen in 1; this suggested that 2 was also
sp2 methine carbon (δC 123.9), one sp2 quaternary carbon a eudesmane-type sesquiterpene lactone glucoside. This was
(δC 133.8), and one ester carbonyl carbon (δC 180.4). It also further supported by the H–1H COSY correlations of H-5–H-
1
displayed six oxygenated sp3 carbon resonances (δC 106.1, 6–H-7 (δH 2.61)–H2–8-H2-9, coupled with the HMBC cross
75.6, 78.2, 71.7, 77.8, 62.8) assignable to a glucosyl moiety, peak of Me-13 with C-7 (δC 45.7). The enzymatic hydrolysis
which was confirmed by enzymatic hydrolysis to liberate of 2 gave, along with glucose, an aglycone, which was identi-
glucose and an aglycone (1a). These data suggested that 1 fied as 1α,11α-dihydroxy-3,4-dehydro-5βH,6αH,7αH,10αCH3-
was a sesquiterpene lactone glucoside. The H–1H correlation eudesmane-6,12-olides by comparing the physical and spectral
1
data with those described in the literature.5) The location of
the glucosyl moiety was assigned to C-1 from the HMBC
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: kasiwada@tokushima-u.ac.jp
© 2012 The Pharmaceutical Society of Japan

914
Vol. 60, No. 7
Fig. 1. New Sesquiterpene Lactone Glycosides from F. varia
Δ
Fig. 3.
δ Values of (S)- and (R)-MTPA Esters of 1a
1
Fig. 2A. Key HMBC and H–1H COSY Correlations of Compound 1
Fig. 4. Key NOESY Correlations of 2
Fig. 2B. Key NOESY Correlations of 1
correlation of H-1' [δH 4.28 (1H, d, J=7.8Hz)] with C-1 (δC
86.6), and its β-linkage was elucidated by the coupling con-
stant of the anomeric proton signal (J=7.8Hz). The boat con-
formation of ring B was suggested from the NOESY correla-
tions of H-5 with H-1, H-8β, and Me-13 (Fig. 4). The absolute
configuration of 2 was elucidated as 1S, 5R, 6S, 7R, 10S, 11S
Δ
Fig. 5.
δ Values of (S)- and (R)-MTPA Esters of 2a
’
by the modified Mosher s method (Fig. 5). From these obser-
vations, the structure of 2 was determined as shown (Fig. 1).
locations of the hydroxyl group and secondary methyl group
Compound 3 had the same molecular formula (C21H32O9) were concluded to be at C-7 and C-11 (δC 41.5), respectively,
1
as 2. The H- and 13C-NMR spectroscopic data were closely from the HMBC cross peaks of the secondary methyl signal
correlated with those of 2 except for the observation of a with C-7 (δC 78.4) and C-12, and of H-6 with C-7 and C-12,
secondary methyl signal [δH 1.07 (3H, d, J=7.1Hz); δC 22.9] respectively. The NOESY correlations of Me-14 with H-6 and
instead of one of the tert-methyl signals seen in 2. The H-8α suggested that ring B adopted a chair conformation. The

July 2012
915
β configurations of H-1, H-5, and H-11 were assigned by the
NOESY correlations of H-1 with H-9β, and of H-11 with H-5
and H-9β (Fig. 6). The location of the glucosyl moiety was
assigned to the C-1 hydroxyl group from the HMBC correla-
tions of H-1′ [δH 4.26 (1H, d, J=7.8Hz)] with C-1 (δC 85.1),
and its β-linkage was concluded from the J-value (7.8Hz) of
H-1′. Based on these data, the structure of 3 was elucidated as
shown in Fig. 1.
The molecular formula of compound 4 was assigned as
1
C21H30O9 by the HR-ESI-MS. The H- and 13C-NMR spectra
revealed the presence of two tert-methyls, one vinyl methyl,
two methylenes, one methine, two oxygenated methines, one
quaternary carbon, one oxygen-bearing quaternary carbon,
one disubstituted olefin, one trisubstituted olefin, one car-
bonyl group, and one glucosyl moiety, which implied that 4
also was a eudesmane-type sesquiterpene lactone glucoside.
This was further confirmed by the 2D-NMR experiments.
The presence of the double bonds at C-2 (C-3) was assigned
Fig. 6. Key NOESY Correlations of 3
1
from the H–1H COSY correlations of H-1–H-2 [δH 5.91 (1H,
dd, J=5.5, 10.0Hz)]–H-3 [δH 5.81 (1H, d, J=10.0Hz)], while
1
the H–1H COSY correlation of H2-8–H2-9, coupled with the
HMBC correlations of Me-13 (δH 1.75) with C-7 (δC 166.8)
and C-11 (δC 119.8), of Me-14 with C-1, and of Me-15 with
C-3 (δC 140.1), indicated the location of the double bond at
C-7 (C-11). The presence of the hydroxyl group at C-4 was
revealed by the HMBC cross peaks of H-3 with C-4 (δC 69.1),
and of Me-15 with C-4. The location and linkage of the glu-
cosyl moiety were elucidated from the analysis of the HMBC
1
Fig. 7A. Key HMBC and H–1H COSY Correlations of Compound 4
1
spectrum and from the H-NMR spectroscopic data (Fig. 7A).
The orientations of H-6 and Me-14 were elucidated as α by
the NOE correlations of Me-14 with H-6 and H-8α, while the
β-orientations of H-1, H-5, and Me-15 were deduced from the
NOESY cross peaks of H-1 with H-9β and of H-5 with Me-15
(Fig. 7B). Thus, the structure of 4 was elucidated as shown
(Fig. 1).
Compound 5 gave a pseudomolecular ion peak at m/z
Fig. 7B. Key NOESY Correlations of 4
565.2257 ([M+Na]+ Calcd for 565.2261) in the positive-ion
HR-ESI-MS, suggesting the molecular formula C26H38O12. to be attached at C-1 and C-11, respectively, by the HMBC
The 1H- and 13C-NMR spectra showed the signals due correlations of H-1 with C-1′ (δC 168.7), and of H-1′ [δH 4.64
to two tert-methyl groups, two vinyl methyl groups, two (1H, d, J=7.7Hz)] with C-11 (δC 81.7). The linkage of the glu-
methylenes, two methines, four oxygen-bearing methines, cosyl moiety was assigned as β from the coupling constant
one quaternary carbon, one oxygen-bearing quaternary car- value (J=7.7Hz) of the anomeric proton signal (Fig. 8A). The
bon, one exo-methylene, one sp2 methine, two sp2 quater- NOESY correlations of Me-14 with H-2, H-6, H-7, and H-8α,
nary carbons, two carbonyl carbons, and a glucosyl moiety. and of H-1 and H-9α indicated that ring B adopted a half-chair
The data for the aglycone moiety were similar to those of conformation, and they were α-oriented. The β configurations
a eudesmane-type sesquiterpene lactone with an angeloyl of H-3, H-5, and Me-13 were elucidated from the NOESY
group (1β-angeloyloxy-11β-hydroxy-5βH,6αH,7αH,10α-methyl- cross peaks of H-5 with H-3, H-9β, and Me-13 (Fig. 8B).
eudesm-2(3),5(15)-diene-6,12-olide, I) previously isolated Based on this evidence, the structure of 5 was characterized
from the EtOAc-soluble fraction of this plant,3) except for as shown in Fig. 1.
the observation of the signals due to two additional oxygen-
Compound 6 was obtained as a pale yellow amorphous pow-
bearing methines and a sugar moiety, as well as the ab- der. The molecular formula of 6 was established as C26H38O13
1
sence of a disubstituted olefinic signal. The existence of the by HR-ESI-MS. The H and 13C NMR spectra were correlated
eudesmane-type sesquiterpene lactone structure with three with those of I,3) except for the observation of signals due to
oxygen-bearing functions and an angeloyl group was also two sugar moieties and the absence of signals arising from an
supported by 2D-NMR spectral analysis. The locations of the angeloyl group. The two sugar moieties were confirmed as
1
oxygen-bearing methines were determined to be C-1, C-2, and glucose and apiose by acid hydrolysis of 6. The H–1H COSY
1
C-3 from the H–1H COSY correlations of H-1 [δH 5.04 (1H, and HMBC experiments revealed that the aglycone of 6 had
d, J=3.1Hz)]–H-2 [δH 3.64 (1H, dd, J=3.1, 9.9Hz)]–H-3 [δH the same planar structure as I.3) The presence of β-apiosyl-(1
→
4.04 (1H, d, J=9.9Hz)], together with the HMBC correlations 6)-β-glucosyl moiety at C-1 was confirmed from the HMBC
of Me-14 with C-1 (δC 80.0), and of H2-15 with C-3 (δC 74.5). correlations of H-1′ [δH 4.37 (1H, d, J=7.7Hz)] with C-1 (δC
The angeloyl group and the glucosyl moiety were confirmed 77.5), and of H2-6′ [δH 3.57 (1H, dd, J=6.4, 11.3Hz), 3.93

916
Vol. 60, No. 7
Fig. 9B. Key NOESY Correlations of 6
1
Fig. 8A. Key HMBC and H–1H COSY Correlations of Compound 5
Fig. 10. Key NOESY Correlations of 7
moieties in 7, similar to those found in 6, were in good agree-
ment with the presence of the β-apiosyl-(1 6)-β-glucosyl moi-
→
ety. In contrast, the signals arising from the aglycone moiety
were similar to those found in 2 except for the observation
of signals due to one oxygenated methylene [δH 3.30 (1H, d,
J=9.5Hz), 3.40 (1H, d, J=9.5Hz); δH 73.5] and one disubsti-
tuted olefin [δH 5.23 (1H, d, J=9.4Hz), 5.94 (1H, dd, J=4.0,
9.4Hz); δC 128.1, 130.0], along with the absence of one tert-
methyl signal. The HMBC correlations of Me-15 with C-3,
Fig. 8B. Key NOESY Correlations of 5
1
C-4, and C-5, together with the H–1H COSY correlations of
H-1 [δH 5.23 (1H, d, J=9.4Hz)]–H-2 [δH 5.94 (1H, dd, J=4.0,
9.4Hz)]–H-3 [δH 5.72 (1H, d, J=4.0Hz)] indicated the presence
of the double bonds at C-1(2) and C-3(4). In addition, the oxy-
genated methylene could be assigned to C-14 from the HMBC
correlations of the oxygenated methylene signal with C-1,
1
C-5, C-9, and C-10. The H–1H COSY and HMBC correla-
tions revealed that 7 had the same partial structure on ring B
and the γ-lactone unit as 2. The location of the β-apiosyl-(1
→
6)-β-glucosyl moiety at C-14 was elucidated by the HMBC
correlations of H-1′ [δH 4.06 (1H, d, J=7.8Hz)] with C-1. The
NOESY correlations of H-5 with Me-13 and H2-14, and those
of Me-13 with H-9β, revealed that H-5, Me-13, and H2-14
1
Fig. 9A. Key HMBC and H–1H COSY Correlations of Compound 6
”
(1H, dd, J=1.6, 11.3Hz)] with C-1 (δC 110.9) (Fig. 9A). The α were β-oriented. The α configurations of H-6 and H-7 were
configurations of H-1, H-6, H-7, and Me-14 were indicated by determined from the NOESY cross peaks of H-6 with H-7 and
the NOE correlations of Me-14 with H-1, H-6, H-7, and H-8α, H-8α (Fig. 10). On the basis of these observations, the struc-
and of H-1 and H-9α. The β configurations of H-5 and Me-13 ture of 7 was assigned as shown in Fig. 1.
were elucidated by the NOE correlations of H-9β with H-5 and
Me-13 (Fig. 9B). From these observations, the structure of 6
Experimental
was assigned as shown (Fig. 1).
General Experimental Procedures Optical rotations
The molecular formula of 7 was assigned as C26H38O13 were measured with a JASCO DIP-370 digital polarimeter. MS
by the HR-ESI-MS experiment. The presence of two sugar were obtained on a Waters LCT PREMIER 2695. NMR spec-
moieties was indicated by two anomeric resonances. In addi- tra were measured on Bruker AVANCE-400 Fourier transform
1
tion, the H- and 13C-NMR spectroscopic data for the sugar spectrometers (1H-NMR: 400MHz, 13C-NMR: 100MHz)

July 2012
917
Table 1. 1H- and 13C-NMR Data for Compounds 1–4 in CD3OD
1
2
3
4
Position
1Ha)
13Cb)
85.4
1Ha)
13Cb)
86.6
1Ha)
13Cb)
85.1
1Ha)
13Cb)
75.9
1
2
3.54 (1H, dd,
6.8, 9.7)
3.51 (1H, dd,
6.5, 10.2)
3.51 (1H, dd,
6.8, 9.1)
3.84 (1H, d,
5.5)
2.49 (1H, m)
32.9
2.47 (1H, m)
32.8
2.49 (1H, m)
33.0
5.91 (1H, dd,
5.5, 10.0)
123.7
140.1
2.21 (1H, m)
2.08 (1H, m)
2.08 (1H, m)
3
5.35 (1H, brs)
123.9
5.36 (1H, brs)
—
123.4
5.34 (1H, brs)
123.9
5.81 (1H, d,
10.0)
—
—
—
4
5
133.8
50.3
134.1
48.8
133.8
50.4
69.1
52.0
2.23 (1H, d,
11.7)
2.15 (1H, d,
10.2)
2.05 (1H, m)
1.95 (1H, d,
11.1)
6
4.38 (1H, d,
11.7)
87.4
4.70 (1H, dd,
7.2, 10.2)
80.2
4.29 (1H, d,
11.0)
87.8
5.24 (1H, d,
11.1)
82.0
—
—
—
7
8
76.6
28.8
2.61 (1H, m)
1.50 (1H, m)
45.7
19.5
78.4
29.7
166.8
23.3
2.05 (1H, m)
1.92 (1H, m)
2.76 (1H, dd,
4.7, 14.2)
1.79 (ddd, 6.3,
13.7, 14.2)
1.81 (1H, dd,
4.8, 14.1)
2.46 (1H, ddd,
4.3, 12.7,
14.2)
23.3
36.3
9
2.09 (1H, m)
32.9
1.87 (1H, m)
1.55 (1H, m)
33.1
2.19 (1H, m)
31.9
2.03 (1H, ddd,
4.7, 12.7,
13.7)
1.28 (ddd, 5.4,
13.7, 13.7)
1.21 (1H, ddd,
4.1, 13.8,
14.1)
1.49 (1H, dd,
4.3, 13.7)
—
—
—
—
—
—
—
10
11
39.8
76.6
38.4
75.7
40.0
41.5
38.9
2.81 (1H, q,
119.8
7.1)
—
—
—
—
12
13
180.4
22.1
180.2
20.8
180.7
6.7
177.4
8.1
1.41 (3H, s)
1.32 (3H, s)
1.07 (3H, d,
7.1)
1.75 (3H, s)
14
15
0.95 (3H, s)
1.73 (3H, s)
11.1
22.9
0.91 (3H, s)
1.75 (3H, s)
14.6
22.4
0.90 (3H, s)
1.73 (3H, s)
10.7
22.9
1.08 (3H, s)
1.39 (3H, s)
19.4
31.4
1′
4.25 (1H, d,
7.8)
106.1
4.28 (1H, d,
7.8)
106.2
4.46 (1H, d,
7.8)
106.0
4.32 (1H, d,
7.7)
100.7
2′
3.12 (1H, t,
7.8)
75.6
3.14 (1H, t,
7.8)
76.0
3.13 (1H, t,
7.8)
75.5
3.05 (1H, t,
7.7)
75.2
3′
4′
5′
6′
78.2
71.7
77.8
62.8
78.2
71.7
77.7
62.8
78.2
71.7
77.7
62.8
78.1
71.8
77.9
62.9
3.78 (1H, d,
11.5)
3.80 (1H, dd,
2.1, 11.8)
3.79 (1H, dd,
2.0, 11.9)
3.76 (1H, m)
3.59 (1H, dd,
4.9, 11.5)
3.61 (1H, dd,
5.1, 11.8)
3.60 (1H, dd,
5.3, 11.9)
3.60 (1H, dd,
5.6, 11.8)
a) δ ppm (mult., J in Hz), 400MHz. b) δ ppm, 100MHz.
using tetramethylsilane (TMS) as an internal standard. Col-
Extraction and Isolation The dried roots of F. varia
umn chromatography: silica gel 60N (63–210µm, Kanto (1.4kg) were crushed and extracted with MeOH for 4h at
°
Kagaku, Japan), Diaion HP-20 (Mitsubishi Chemical, Japan), 60 C three times. The MeOH extracts were concentrated in
Sephadex LH-20 (25–100µm; GE Health Care, U.K.), MCI-gel vacuo to give a gum (227g), which was partitioned between
CHP 20P (75–150µm; Mitsubishi Chemical, Japan), YMC- EtOAc and H2O. The H2O-soluble fraction (115g) was subject-
pack ODS-A (S-50µm; YMC Co., Ltd., Japan). Preparative ed to chromatography over Diaion HP-20 [MeOH–H2O (0:1
→
HPLC: CAPCELL PACK C18 SG120 (250×20mm; 5µm; Shi- 1:0)] to give 6 fractions. Fr. 2 was fractionated by Sephadex
seido, Japan), GPC (Gel-Permeation Chromatography) [Asahi LH-20 (H2O) to afford fractions 2.1–2.8. Fr. 2.3 was applied
pack GS-310 2G (MeOH, SHOWA DENKO)]. TLC: silica gel to a YMC ODS-A column [MeOH–H2O (0:1
60 F254 (Merck, Germany). fraction 2.3.1–2.3.13. Compoud 4 (2mg) was isolated from
Plant Material The roots of Ferula varia were collected fr. 2.3.5 by MCI gel CHP 20P CC [MeOH–H2O (1:4 1:0)],
in Kyzylkum, Uzbekistan, in April 2001. Herbarium speci- and then GPC on HPLC (MeOH). Fr. 2.3.9 was subjected to
mens (ESM-01ky-10) are deposited in the herbarium of the a MCI gel CHP 20 column [MeOH–H2O (1:1 1:0)] to afford
Graduate School of Pharmaceutical Sciences, Kyoto Univer- 5 fractions (2.3.9.1–2.3.9.5). Fr. 2.3.9.2 was further purified by
sity. GPC on HPLC (MeOH), followed by ODS HPLC (CAPCELL
→1:0)] to yield
→
→

918
Vol. 60, No. 7
Table 2. 1H- and 13C-NMR Data for Compounds 5–7 in CD3OD
5
6
7
Position
1Ha)
13Cb)
1Ha)
13Cb)
1Ha)
13Cb)
1
2
5.04 (1H, d, 3.1)
80.0
73.6
3.74 (1H, d, 4.6)
77.5
5.23 (1H, d, 9.4)
128.1
130.0
3.64 (1H, dd, 3.1,
9.9)
5.91 (1H, d, 4.6,
9.7)
125.3
5.94 (1H, 4.0, 9.4)
3
4
5
6
4.04 (1H, d, 9.9)
—
74.5
146.4
45.2
6.29 (1H, d, 9.7)
—
135.1
144.7
42.3
5.72 (1H, d, 4.0)
—
141.3
119.7
43.4
2.32 (1H, d, 10.3)
2.69 (1H, d, 9.8)
2.33 (1H, d, 9.7)
4.46 (1H, t, 9.7)
5.07 (1H, dd, 1.9,
10.3)
77.5
4.88 (1H, dd, 8.6,
9.8)
79.6
84.1
7
8
3.00 (1H, dd, 6.2,
13.9)
42.9
19.5
2.62 (1H, dd, 5.7,
13.9)
45.5
19.2
2.47 (1H, m)
45.1
19.7
1.78 (1H, m)
1.69 (1H, m)
1.59 (1H, m)
1.77 (2H, m)
1.86 (1H, m)
1.78 (1H, m)
9
31.6
2.36 (1H, m)
30.8
2.03 (1H, ddd, 4.7,
4.7, 13.8)
29.0
1.21 (1H, m)
1.14 (1H, dt, 5.4,
1.38 (1H, m)
14.3)
—
—
—
—
—
10
11
12
13
14
39.5
81.7
38.1
75.7
41.0
75.2
—
—
—
—
178.5
19.6
181.1
22.1
181.6
22.5
1.44 (3H, s)
0.90 (3H, s)
1.41 (3H, s)
0.78 (3H, s)
1.43 (3H, s)
3.40 (1H, d, 9.5)
3.30 (1H, d, 9.5)
1.95 (3H, s)
19.6
19.7
73.5
15
5.49 (1H, s)
5.11 (1H, s)
—
108.9
5.32 (1H, s)
116.5
24.1
5.15 (1H, s)
1′
168.7
129.1
139.7
16.1
21.0
—
4.37 (1H, d, 7.7)
3.07 (1H, t, 7.7)
3.29 (1H, m)
3.20 (1H, t, 9.2)
3.34 (1H, m)
101.3
75.3
78.2
71.9
77.0
68.6
4.06 (1H, d, 7.8)
3.09 (1H, t, 7.8)
104.8
75.3
78.4
71.7
76.9
68.6
—
2′
3′
4′
5′
6′
6.11 (1H, q, 7.1)
1.95 (3H, d, 7.1)
1.88 (3H, s)
—
3.93 (1H, dd, 1.6,
11.3)
3.90 (1H, m)
3.54 (1H, m)
4.94 (1H, d, 2.3)
3.57 (1H, dd, 6.4,
11.3)
1″
2″
3″
4″
4.64 (1H, d, 7.7)
99.1
75.0
77.9
71.5
5.00 (1H, d, 2.2)
3.85 (1H, d, 2.2)
—
110.9
78.0
80.5
75.0
111.0
78.0
78.4
75.0
3.11 (1H, d, t, 7.7)
3.91 (1H, d, 9.3)
3.72 (1H, d, 9.3)
3.53 (1H, m)
—
5″
6″
77.8
62.7
65.7
—
65.7
—
—
3.59 (1H, dd, 4.7,
10.8)
3.78 (1H, d, 10.8)
a) δ ppm (mult., J in Hz), 400MHz. b) δ ppm, 100MHz.
PAK C18 SG120) [MeOH:H2O=1:1] to give 5 (2mg) and 6 7:3:0.5)] and GPC (MeOH) to obtain 3 (14mg). Fr. 2.4.8 was
(18mg). Fr. 2.3.9.3 was separated by GPC on HPLC (MeOH), repeatedly chromatographed over sphadex LH-20 (EtOH),
and then silica gel CC [CHCl3–MeOH–H2O (8:2:0.2→6:4:1)] silica gel (CHCl3–MeOH–H2O=6:1:0.1) and YMC ODS-A
to yield 7 (6mg). Fr. 2.4 was applied to a YMC ODS-A col- (MeOH–H2O=4:1) to afford 2 (26mg).
umn [MeOH–H2O (0:1
→
1:0)] to give 9 fractions (2.4.1–2.4.9).
Compound 1: White amorphous powder; [α]1D9 −17.5 (c=0.4,
1
Fr. 2.4.7 was fractionated by Sephadex LH-20 CC [CHCl3– MeOH); H- and 13C-NMR data (CD3OD) see Table 1; HR-
MeOH (1:1)] and MCI gel CHP 20P CC [MeOH–H2O (1:1
1:0)] to yield fractions 2.4.7.1–2.4.7.6. Fr. 2.4.7.3 was sepa- 467.1893).
rated by YMC ODS-A CC [MeOH–H2O (1:1 1:0)] and silica Enzymatic Hydrolysis of 1 A solution of compound 1
gel CC [CHCl3–MeOH–H2O (6:1:0.1 7:3:0.5)] to give 3 (10mg) in water (2mL) was treated with Cellulase from Trich-
fractions (2.4.7.3.1–2.4.7.3.3). Purification of fr. 2.4.7.3.2 by oderma viride (3–10units/mg solid, Sigma) (20mg) at 37 C for
GPC (MeOH) and ODS HPLC (CAPCELL PAK C18 SG120) 3 weeks. The reaction mixture was diluted with MeOH, and
[MeOH–H2O=1:1] gave compound 1 (5mg). Fr. 2.4.7.4 was the resulting precipitates were filtrated off. The filtrate was
repeatedly fractionated by YMC ODS-A CC [MeOH–H2O purified by silica gel column chromatography [benzene–iso-
→
ESI-MS m/z 467.1877 [M+Na]+ (Calcd for C21H32O10Na,
→
→
°
(1:1
→
1:0)], silica gel CC [CHCl3–MeOH–H2O (6:1:0.1
→
propanol (15:1→8:1)] to give an aglycone (1a) (1mg) and a

July 2012
919
sugar (0.7mg). The sugar moiety was identified as glucose by 1.577 (3H, s, H3-13), 0.891 (3H, s, H3-14).
1
the TLC analysis [Rf: 0.33, n-BuOH–pyridine–H2O (6:4:3) on
Avicel SF cellulose].
(S)-MTPA ester of 2a (2as): H-NMR (pyridine-d5) δ: 5.199
(1H, brs, H-3), 5.131 (1H, dd, J=6.4, 10.0Hz, H-1), 4.967 (1H,
Preparation of (S)-MTPA or (R)-MTPA Esters of dd, J=6.6, 9.7Hz, H-6), 2.887 (1H, m, H-7), 2.492 (1H, brd,
1a Compound 1a (0.5mg) was dissolved with pyridine-d5 J=17.3Hz, H-2a), 2.327 (1H, d, J=9.7Hz, H-5), 1.937 (1H, m,
(0.4mL) in a NMR tube. The solution of (S)-MTPA or (R)- H-2b), 1.801 (3H, s, H3-15), 1.604 (3H, s, H3-13), 1.583–1.357
MTPA chloride (2 drops) was added to the NMR tube to kept (4H, m, H2-8, H2-9), 0.891 (3H, s, H3-14).
for 12h.
Compound 3: Pale yellow amorphous powder; [α]1D8 −9.6
1
(R)-MTPA ester of 1a (1ar): H-NMR (pyridine-d5) δ: 5.289 (c=1.6, MeOH); 1H- and 13C-NMR (CD3OD) see Table 1;
(1H, m, H-3), 5.269 (1H, m, H-1), 4.849 (1H, d, J=11.0Hz, HR-ESI-MS m/z 451.1954 [M+Na]+ (Calcd for C21H32O9Na,
H-6), 2.642 (1H, brd, J=17.6Hz, H-2a), 2.538 (1H, d, 451.1944).
J=11.0Hz, H-5), 2.214 (1H, m, H-2b), 2.167 (1H, dt, J=5.4,
Compound 4: White amorphous powder; [α]D20 −88.6 (c=1.2,
15.3Hz, H-9a), 2.004 (1H, m, H-8a), 1.864 (3H, s, H3-15), MeOH); H- and 13C-NMR (CD3OD) see Table 1; HR-ESI-MS
1
1.799 (1H, m, H-8b), 1.707 (3H, s, H3-13), 1.540 (1H, m, H-9b), m/z 449.1794 [M+Na]+ (Calcd for C21H30O9Na, 449.1788).
1.168 (3H, s, H3-14).
Compound 5: White amorphous powder; [α]D20 −15.0 (c=0.3,
(S)-MTPA ester of 1a (1as): H-NMR (pyridine-d5) δ: 5.252 MeOH); H- and 13C-NMR (CD3OD) see Table 2; HR-ESI-MS
1
1
(1H, m, H-3), 5.247 (1H, m, H-1), 4.891 (1H, d, J=11.0Hz, m/z 565.2257 [M+Na]+ (Calcd for C26H38O12Na, 565.2261).
H-6), 2.629 (1H, brd, J=17.5Hz, H-2a), 2.551 (1H, d,
Compound 6: Pale yellow amorphous powder; [α]D20 −177.6
J=11.0Hz, H-5), 2.250 (1H, dt, J=5.1, 14.2Hz, H-9a), 2.114 (c=1.4, MeOH); 1H- and 13C-NMR (CD3OD) see Table 2;
(1H, m, H-8a), 2.015 (1H, m, H-8b), 2.014 (1H, m, H-2b), 1.847 HR-ESI-MS m/z 557.2233 [M−H]− (Calcd for C26H38O13Na,
(3H, s, H3-15), 1.738 (3H, s, H3-13), 1.658 (1H, m, H-9b), 1.175 557.2234).
(3H, s, H3-14).
Acid Hydrolysis of 6 Compound 6 (1mg) was hydrolyzed
Compound 2: White amorphous powder; [α]1D8 −66.2 (c=2.5, with 1M HCl for 12h at 80 C. The reaction mixture was di-
°
1
MeOH); H- and 13C-NMR (CD3OD) see Table 1; HR-ESI-MS luted with H2O, and extracted with EtOAc. The H2O layer was
m/z 451.1951 [M+Na]+ (Calcd for C21H32O9Na, 451.1944).
neutralized with Amberlite IRA-400 resin and evaporated.
Enzymatic Hydrolysis of 2 A solution of compound 2 The residue was directly analyzed by TLC [Rf: 0.33 (glucose),
(10mg) in water (2mL) was treated with β-glucosidase from 0.55 (apiose), respectively, n-BuOH–pyridine–H2O (6:4:3) on
°
almonds (4.8units/mg solid, Sigma) (10mg) at 37 C for 9d. Avicel SF cellulose] to detect glucose and apiose.
The reaction mixture was worked up as for 1, and was puri-
Compound 7: White amorphous powder; [α]D20 −142.0
fied by silica gel column chromatography [CHCl3–MeOH (c=0.6, MeOH); 1H- and 13C-NMR (CD3OD) see Table 2;
–H2O (20:1:0
7:3:0.5)] to give an aglycone (2a) (6mg) and HR-ESI-MS m/z 581.2213 [M+Na]+ (Calcd for C26H38O13Na,
a glucose (3mg). 2a was identified as 1β,11α-dihydroxy-3,4- 581.2210).
dehydro-5βH,6αH,7αH,10αCH3-eudesmane-6,12-olides by
→
comparison of the physical and spectral data with described
in the literature.5) Glucose was identified by the TLC analysis
[Rf: 0.33, n-BuOH–pyridine–H2O (6:4:3) on Avicel SF cel-
lulose].
References
1) Kurimoto S., Okasaka M., Kashiwada Y., Kodzhimatov O. K.,
Takaishi Y., J. Nat. Med., 65, 180–185 (2011), and literatures cited
therein.
“
”
2) Wild Medicinal Plants of Central Asia, ed. by Sakhobiddinov S.
Preparation of (S)- or (R)-MTPA Ester of 2a 2a was
treated with (S)-MTPA or (R)-MTPA chloride in a NMR tube
as for 1a.
S., Gosizdat, Tashkent, 1948, p. 216.
3) Suzuki K., Okasaka M., Kashiwada Y., Takaishi Y., Honda G., Ito
M., Takeda Y., Kodzhimatov O. K., Ashurmetov O., Sekiya M.,
Ikeshiro Y., J. Nat. Prod., 70, 1915–1918 (2007).
1
(R)-MTPA ester of 2a (2ar): H-NMR (pyridine-d5) δ: 5.224
(1H, brs, H-3), 5.145 (1H, dd, J=6.5, 10.2Hz, H-1), 4.955 (1H,
dd, J=6.0, 9.7Hz, H-6), 2.836 (1H, m, H-7), 2.508 (1H, brd,
J=17.2Hz, H-2a), 2.304 (1H, d, J=9.7Hz, H-5), 2.127 (1H, m,
H-2b), 1.811 (3H, s, H3-15), 1.725–1.328 (4H, m, H2-8, H2-9),
4) Ohtani I., Kusumi T., Kashman H., Kakisawa H., J. Am. Chem.
Soc., 113, 4092–4096 (1991).
5) Holub M., Budĕšínský M., Smítalová Z., Šaman D., Rychlewska U.,
Tetrahedron Lett., 23, 4853–4856 (1982).