Rigenolides B and C, conjugates of norsecoiridoid and secoiridoid glucoside from Gentiana rigescens Franch.

Article abstract of DOI:10.1016/j.tetlet.2017.02.075

Two new conjugates of norsecoiridoid and secoiridoid glucoside, rigenolides B (1) and C (2), were isolated from a Chinese herbal medicine Gentiana rigescens Franch. Their structures were established by comprehensive spectroscopic analysis. The absolute configurations of 1 and 2 were elucidated by comparison of the ECD spectra with TDDFT calculated spectra.

Full text of DOI:10.1016/j.tetlet.2017.02.075

Tetrahedron Letters 58 (2017) 1459–1461
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Tetrahedron Letters
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Rigenolides B and C, conjugates of norsecoiridoid and secoiridoid
glucoside from Gentiana rigescens Franch.
Yoshihiro Suyama a, Naonobu Tanaka a,b, Kazuyoshi Kawazoe a, Kotaro Murakami c, Shun-Lin Li d,
Han-Dong Sun d, Yoshiki Kashiwada a,
⇑
a Graduate School of Pharmaceutical Sciences, Tokushima University, Tokushima 770-8505, Japan
b Graduate School of Bioscience and Bioindustry, Tokushima University, Tokushima 770-8513, Japan
c Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto 862-0082, Japan
d State Key Laboratory of Phytochemistry and Plant Resources in West China, Kumming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new conjugates of norsecoiridoid and secoiridoid glucoside, rigenolides B (1) and C (2), were isolated
from a Chinese herbal medicine Gentiana rigescens Franch. Their structures were established by compre-
hensive spectroscopic analysis. The absolute configurations of 1 and 2 were elucidated by comparison of
the ECD spectra with TDDFT calculated spectra.
Received 25 January 2017
Revised 21 February 2017
Accepted 24 February 2017
Available online 27 February 2017
Ó 2017 Elsevier Ltd. All rights reserved.
Keywords:
Norsecoiridoid
Secoiridoid
Rigenolides B and C
Gentiana rigescens
Gentianaceae
The genus Gentiana, the largest member of Gentianaceae,
consists of about 400 species. Various Gentiana plants have been
included in herbal remedies for poor appetite and digestive prob-
lems worldwide.1 A number of iridoids, secoiridoids, and their
glycosides have been isolated from Gentiana plants to date.
Gentiana rigescens Franch. is a perennial herb, whose roots are used
to treat hepatitis and cholecystis by the Yi ethnic minority group
living in Yunnan province, China. As part of our study for tradi-
tional herbal medicines used by ethnic minority groups in Yunnan
province,2 aimed at searching for new drug leads, we have investi-
gated the extract from the aerial parts of G. rigescens and reported
the isolation of secoiridoids and norsecoiridoids.3 Among others,
rigenolide A3 is a structurally unique secoiridoid glucoside possess-
ing a cyclobutane moiety. Further investigation of the extract
resulted in the isolation of two new conjugates of norsecoiridoid
and secoiridoid glucoside, rigenolides B (1) and C (2). In this paper,
we describe the isolation and structure elucidation of 1 and 2.
The MeOH extract from the aerial parts of G. rigescens (1.9 kg,
dry) was partitioned with EtOAc and H2O. Chromatographic sepa-
ration of the H2O-soluble materials using Diaion HP-20 column,
ODS column, silica gel column, and reversed phase HPLC afforded
rigenolides B (1, 3 mg) and C (2, 1 mg) (Chart 1).
Rigenolide B (1)4 was obtained as an optically active white
amorphous powder f½a D20
ꢁ 72:0ðc 0:31; MeOHÞg. The molecular
ꢀ
formula of C25H30O12 was established by the HRESIMS (m/z
545.1617 [M+Na]+,
D
ꢁ 1.8 mmu). Comparison of the 1H and 13C
NMR spectra (Table 1) with the literature data for known secoiri-
doids indicated that 1 had a secoiridoid glucoside moiety (unit A,
C-1–C-11 and C-10–C-60) and unit B (C-300–C-1100). Unit A of 1 was
deduced to be gentiopicroside5 by resemblance of their NMR data.
In contrast, the 1D NMR spectra suggested that unit B was com-
prised of nine carbons including one a,b-unsaturated carboxy moi-
ety, one acetal carbon, one oxygenated sp3 methine, three sp3
methylenes, including one oxygenated, and one secondary methyl
(Table 1). 1H-1H COSY cross-peaks of oxygenated sp3 methylene
(H2-700) with H2-600 and HMBC correlations for H2-700 with C-500
and C-1100 and for H2-600 with C-400 revealed the presence of an
a,
b-unsaturated-d-lactone moiety (C-400–C-700 and C-1100) (Fig. 1).
The 1H-1H COSY spectrum also suggested the presence of a 2-oxy-
genated propyl group (C-800–C-1000), while HMBC cross-peaks of
H-900/C-400, H-900/C-500, and H-300/C-400 indicated the connectivity of
C-500 to C-900 and of C-400 to the acetal carbon (C-300). The ether link-
age between C-300 and C-800 was disclosed by an HMBC correlation
for H-300 with C-800. The syn relationship for H-300 and H-800 was
⇑
Corresponding author.
E-mail address: kasiwada@tokushima-u.ac.jp (Y. Kashiwada).
http://dx.doi.org/10.1016/j.tetlet.2017.02.075
0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.

1460
Y. Suyama et al. / Tetrahedron Letters 58 (2017) 1459–1461
11
O
O
O
11
11
4
O
O
O
O
O
7
6
O
11"
O
11"
O
O
3
11"
9
9
O
O
6'
5
6'
S
S
7"
6"
O
10
3"
O
3"
R
R
1
1
O
6'
R
S
4"
9
O
O
O
O
O
HO
HO
3"
1
R O
1'
O
1'
8
S
HO
HO
5"
9"
8"
8"
O
O
OH
rigenolide B (1)
OH
rigenolide C (2)
HO
O
OH1'
HO
1H-1H COSY
HMBC
8"
10"
Chart 1. The structures of rigenolides B (1) and C (2).
NOESY
1 and 2
indicated by a NOESY correlation between these protons. The
structure of unit B, thus assigned (Fig. 1), corresponds to epi-
swercinctolide B,3 previously isolated from the same plant origin
by our group. These units were shown to be linked through an
ether linkage between C-300 (unit B) and C-60 (unit A) by an HMBC
cross-peak of H-300 to C-60. Therefore, the gross structure of rigeno-
lide B (1) was assigned as shown in Fig. 1.
Fig. 1. Key 2D NMR correlations of rigenolides B (1) and C (2).
relationships for 1 and 2 were indicated by resemblance of their
3JH-1/H-9 values (Table 1) with the literature value (J = 3.0 Hz).5
Therefore, taking the H-300/H-800-syn relationships in 1 and 2
assigned by NOESY analysis into account, the ECD spectra of their
four possible diastereomers (3a: 1S,9R,300R,800R; 3b: 1S,9R,300S,800S;
3c: 1R,9S,300R,800R; 3d: 1R,9S,300S,800S) were calculated11 to compare
with those experimental spectra of 1 and 2. The experimental ECD
spectrum of gentiopicroside was also compared with the calcu-
lated spectra (Fig. 2). In the calculated ECD spectra of 1S,9R isomers
(3a and 3b), negative Cotton effects were found around 270 nm,
whereas 1R,9S isomers (3c and 3d) showed positive Cotton effects
around 270 nm. These observations suggested that the sign of Cot-
ton effect around 270 nm reflects the absolute configuration at C-1
and C-9. A negative Cotton effect at 277 nm observed in gentiopi-
croside possessing the 1S,9R configuration was well consistent
with the observation described above. Rigenolides B (1) and C (2)
also showed negative Cotton effects at 278 and 273 nm, respec-
tively (Fig. 3), suggesting the 1S,9R configurations of 1 and 2.
In contrast, the 300R,800R configurations of 1 and the 300S,800S
Rigenolide C (2)6 was isolated as an optically active white amor-
phous powder f½a D20
ꢁ 96:6ðc 0:10; MeOHÞg, and its molecular for-
ꢀ
mula, C25H30O12, determined by the HRESIMS (m/z 545.1653 [M
+Na]+,
D
+ 1.8 mmu) was identical to that of 1. Though the 1H
and 13C NMR spectra of 2 and 1 were almost superimposable
(Table 1), subtle differences were found for the chemical shifts of
C-50 and C-300. These observations suggested their diastereomeric
relationship, whereas the H-300/H-800-syn relationship for 2 was
revealed by a NOESY correlation for H-300/H-800 (Fig. 1).
The stereochemistry of rigenolides B (1) and C (2) were assigned
as follows. Acid hydrolysis7 of 1 gave a sugar moiety, which was
treated successively with
and phenylisothiocyanate.8,9 HPLC analysis of the reaction mixture
revealed the sugar moiety of 1 to be
-glucose.10 Similarly, the
-glucose. The H-1/H-9-anti
L-cysteine methyl ester hydrochloride
D
sugar moiety of 2 was assigned as
D
configuration of
2
were concluded by resemblance of the
Table 1
1H and 13C NMR data for rigenolides B (1) and C (2) in CD3OD.
Position
1
2
dH (J in Hz)
dC
98.8
150.7
104.9
127.1
117.2
70.9
dH (J in Hz)
dC
1
3
4
5
6
7
5.62 (1H, d, 3.1)
7.44 (1H, d, 1.1)
–
5.60 (1H, d, 3.1)
7.45 (1H, d, 1.1)
–
98.9
150.9
104.8
127.2
117.0
70.9
–
–
5.60 (1H, m)
5.59 (1H, m)
5.06 (1H, dd, 17.7, 1.3)
4.98 (1H, dd, 17.7, 3.5)
5.74 (1H, ddd, 17.2, 10.2, 6.9)
3.28 (1H, m)
5.06 (1H, dd, 17.7, 1.3)
4.98 (1H, dd, 17.7, 3.5)
5.71 (1H, ddd, 17.4, 10.4, 6.9)
3.28 (1H, m)
8
9
135.0
46.7
135.0
46.7
10
5.20 (1H, ddd, 17.2, 1.3, 1.3)
5.18 (1H, ddd, 10.2, 1.3, 1.3)
–
4.63 (1H, d, 8.0)
3.14 (1H, dd, 9.0, 8.0)
3.34 (1H, dd, 9.0, 9.0)
3.27 (1H, m)
3.48 (ddd, 9.0, 6.5, 1.7)
4.05 (1H, dd, 11.7, 1.7)
3.77 (1H, dd, 11.7, 6.5)
5.39 (1H, s)
118.5
5.22 (1H, ddd, 17.4, 1.3, 1.3)
5.19 (1H, ddd, 10.4, 1.3, 1.3)
–
118.6
11
10
20
30
40
50
60
166.3
100.5
74.5
77.9
71.5
77.7
68.6
166.3
100.6
74.5
78.2
71.9
76.6
68.6
4.63 (1H, d, 8.0)
3.17 (1H, dd, 9.0, 8.0)
3.35 (1H, dd, 9.0, 9.0)
3.24 (1H, dd, 9.0, 9.0)
3.54 (1H, ddd, 9.0, 6.8, 1.9)
4.01 (1H, dd, 11.5, 1.9)
3.81 (1H, dd, 11.5, 6.8)
5.36 (1H, s)
300
400
500
600
95.0
123.7
157.1
29.3
93.9
123.7
156.8
29.3
–
–
–
–
2.59 (1H, ddd, 18.0, 9.7, 6.0)
2.39 (1H, ddd, 18.0, 4.8, 4.8)
4.38 (2H, m)
2.60 (1H, ddd, 17.0, 10.0, 6.0)
2.36 (1H, ddd, 17.0, 4.6, 4.6)
4.38 (2H, m)
700
800
900
67.0
63.0
37.5
66.9
63.2
37.5
4.20 (1H, m)
4.21 (1H, m)
2.29 (1H, dd, 19.0, 3.9)
2.21 (1H, dd, 19.0, 10.6)
1.27 (d, 6.3)
2.27 (1H, dd, 18.9, 4.1)
2.21 (1H, dd, 18.9, 10.3)
1.26 (1H, d, 6.3)
–
1000
1100
21.0
165.0
21.0
165.0
–

Y. Suyama et al. / Tetrahedron Letters 58 (2017) 1459–1461
1461
Acknowledgments
This investigation was partly supported by Grant-in-Aid for Sci-
entific Research (B, No. 18406028) for the Japan Society for the Pro-
motion of Science.
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Fig. 2. Experimental ECD spectrum of gentiopicroside and calculated spectra of
possible diastereomers (3a–3d) of rigenolides B (1) and C (2).
4. Rigenolide B (1): white amorphous powder; f½a D20
ꢀ
–72.0 (c 0.31, MeOH);
HRESIMS: m/z 545.1617 [M+Na]+ (calcd for C25H30O12Na, 545.1635); IR (KBr)
m
max 3424, 2918, and 1700 cmꢁ1; UV (MeOH) kmax 206 (log
and 272 (3.23 sh) nm; ECD (MeOH)
(nm) ꢁ0.55 (278), ꢁ1.73 (227), and
+1.67 (201); 1H and 13C NMR (Table 1).
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D
e
5. (a) Kromeyer A. Arch Pharm. 1862;110:27–39;
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6. Rigenolide C (2): white amorphous powder; f½a D23
ꢀ
–96.6 (c 0.10, MeOH);
HRESIMS: m/z 545.1653 [M+Na]+ (calcd for C25H30O12Na, 545.1635); IR (KBr)
mmax 3415, 2921, and 1702 cmꢁ1; UV (MeOH) kmax 204 (log
e
3.62), 235 (3.47
sh), and 270 (3.34) nm; ECD (MeOH)
De (nm) ꢁ4.30 (273), +2.40 (236), and
ꢁ3.40 (202); 1H and 13C NMR (Table 1).
7. Rigenolides B (1, 1.0 mg) and C (2, 0.8 mg) was separately treated with 5%
H2SO4 (2 mL) at 70 °C for 3 h. After neutralization by anion-exchange resin
(IRA-400, ORGANO Co.), solvent was evaporated to give the residue containing
the sugar moiety of 1 and 2, respectively.
8. Tanaka T, Nakashima T, Ueda T, Tomii K, Kouno I. Chem Pharm Bull.
2007;55:899–901.
9. The residue containing the sugar moiety of rigenolide B (1) and
methyl ester hydrochloride (0.5 mg) was dissolved in pyridine (100
mixture was heated at 60 °C for 1 h, and then phenylisothiocyanate (10
L
-cysteine
L) and the
L) was
l
l
added to the mixture and heated at 60 °C for 1h.
10. HPLC analysis of the reaction mixture Cosmosil 5C18 AR-II, Nacalai Tesque,
4.6 ꢂ 250 mm, flow rate 0.8 mL/min, UV detection at 250 nm, eluent CH3CN/50
mM H3PO4 aq. 1:3, column temperature 35 °C gave a peak at 17.2 min. The
retention time was identical to that of the derivative from authentic D-glucose.
11. Conformational searches and DFT calculations were carried out on Spartan 14
(Wavefunction, Irvine, CA) and Gaussian 0912 programs, respectively. Possible
four diastereomers [3a: 1S,9R,3‘‘R,8”R; 3b: 1S,9R,3‘‘S,8”S; 3c: 1R,9S,3‘‘R,8”R; 3d:
1R,9S,3‘‘S,8”S] of rigenolides B (1) and C (2) were subjected to conformational
search using MMFF94s as the force field. The initial stable conformers with
Boltzmann distributions over 1% (6, 8, 9, and 11 conformers, respectively) were
further optimized by DFT calculations at the B3LYP/6-31G(d) level in the
presence of MeOH with a polarizable continuum model (PCM). The stable
conformers with Boltzmann distributions over 1% were subjected to TDDFT
calculations at the B3LYP/6-31G(d) level in the presence of MeOH with a PCM.
The resultant rotatory strengths of the lowest 30 excited states for each
conformer were converted into Gaussian-type curves with half-bands (0.3 eV
for 3a and 3b; 0.2 eV for 3c and 3d) using SpecDis v1.61.13 The calculated CD
spectra were composed after correction based on the Boltzmann distributions
of the stable conformers. The calculated ECD spectrum of 3a was red-shifted by
10 nm.
Fig. 3. Experimental ECD spectra of rigenolides B (1) and C (2) and calculated
spectra of their possible diastereomers (3a–3d).
experimental ECD spectra of 1 and 2 with those calculated spectra
of 3a and 3b, respectively (Fig. 3). Consequently, the absolute
stereochemistry of rigenolides B (1) and C (2) were assigned as
shown in Chart 1.
The investigation of the aerial parts of Gentiana rigescens
resulted in the isolation of two new conjugates of norsecoiridoid
and secoiridoid glucoside, regenolides B (1) and C (2). Although
some conjugates structurally similar to 1 and 2 have been reported
to date,14 their absolute stereochemistry remains to be assigned. In
this study, the structures of rigenolides B (1) and C (2) including
the absolute stereochemistry were elucidated on the basis spectro-
scopic analysis, chemical conversion, and TDDFT ECD calculation.
12. Frisch MJ, Trucks GW, Schlegel HB, et al. Gaussian 09, Revision C. 01. Wallingford,
CT: Gaussian; 2010.
13. Schaumlöffel T, Hemberger A, Bringmann Y, SpecDis G. Version
1.61. Germany: University of Wuerzburg; 2013.
14. Geng C-A, Zhang X-M, Ma Y-B, et al. J Asian Nat Prod Res. 2010;12:542–548.