Digipregnosides A–C, three novel rearranged 11,12-secopregnane glycosides, and digipregnosides D and E, 12,20-epoxypregnane glycosides from the seeds of Digitalis purpurea

Article abstract of DOI:10.1016/j.tetlet.2021.153020

Three novel rearranged 11,12-secopregnane glycosides—named, digipregnosides A (1), B (2), and C (3)—and two novel 12,20-epoxypregnane glycosides—named, digipregnosides D (4) and E (5)—were isolated from the seeds of Digitalis purpurea. Their structures were determined based on extensive spectroscopic analysis, including long-range heteronuclear single quantum multiple bond correlation (HSQMBC) spectral data, and the results of hydrolytic cleavage. Compounds 1 and 4 showed tumor selective cytotoxicity against SBC-3 small cell lung carcinoma cells at a sample concentration of 0.001 μM and induced apoptotic cell death in SBC-3 cells with IC₅₀ values of 1.3 ± 0.16 and 0.17 ± 0.072 μM respectively.

Full text of DOI:10.1016/j.tetlet.2021.153020

Tetrahedron Letters 70 (2021) 153020
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Digipregnosides A–C, three novel rearranged 11,12-secopregnane
glycosides, and digipregnosides D and E, 12,20-epoxypregnane
glycosides from the seeds of Digitalis purpurea
Kazuhiro Takatori , Minpei Kuroda , Masaki Mishima , Yukiko Matsuo , Yoshihiro Mimaki a
a
a
b
a,
⇑
a
Department of Medicinal Pharmacognosy, Tokyo University of Pharmacy and Life Sciences, School of Pharmacy, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minamiosawa, Hachioji 192-0397, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Three novel rearranged 11,12-secopregnane glycosides—named, digipregnosides A (1), B (2), and C
Received 19 February 2021
Revised 17 March 2021
Accepted 19 March 2021
Available online 24 March 2021
(
3)—and two novel 12,20-epoxypregnane glycosides—named, digipregnosides D (4) and E (5)—were
isolated from the seeds of Digitalis purpurea. Their structures were determined based on extensive spec-
troscopic analysis, including long-range heteronuclear single quantum multiple bond correlation
(
HSQMBC) spectral data, and the results of hydrolytic cleavage. Compounds 1 and 4 showed tumor selec-
tive cytotoxicity against SBC-3 small cell lung carcinoma cells at a sample concentration of 0.001 M and
induced apoptotic cell death in SBC-3 cells with IC50 values of 1.3 ± 0.16 and 0.17 ± 0.072 M respectively.
Ó 2021 Elsevier Ltd. All rights reserved.
l
Keywords:
Digitalis purpurea
Rearranged 11,12-secopregnane glycoside
l
1
2,20-epoxypregnane glycoside
SBC-3 cells
Introduction
performance liquid chromatography (HPLC) to obtain
1
(
12.8 mg), 2 (3.4 mg), 3 (23.5 mg), 4 (25.3 mg), and 5 (14.0 mg)
Our current phytochemical research has revealed that the seeds
of Digitalis purpurea contained various cytotoxic cardenolide glyco-
sides [1]. Of these, glucodigifucoside exhibited carcinoma-specific
cytotoxicity against ACHN human renal adenocarcinoma cells
associated with upregulation of p21/Cip1 protein levels [2]. Our
continuous phytochemical examination of the methanolic extract
of D. purpurea seeds resulted in the isolation of three novel rear-
ranged 11,12-secopregnane glycosides—named, digipregnosides A
(Fig. 1). Details about the extraction and isolation procedures are
provided in the Supporting Information document.
Results and discussion
Digipregnoside A (1) was isolated as an amorphous solid. Its
molecular formula, C35H O14, which requires 10 degrees of unsat-
52
uration, was determined by high-resolution electrospray ioniza-
(1), B (2), and C (3)—and two novel 12,20-epoxypregnane glyco-
sides—named, digipregnosides D (4) and E (5). The chemical struc-
tures of 1–5 were determined based on extensive spectroscopic
analysis, including long-range heteronuclear single quantum mul-
tiple bond correlation (HSQMBC) spectral data, and the results of
hydrolytic cleavage. The cytotoxic activities of the isolated com-
pounds were evaluated against SBC-3 small cell lung carcinoma
and TIG-3 normal human fibroblast cells.
The seeds of D. purpurea (2.0 kg) were extracted with MeOH.
The MeOH extract (140 g) was passed through a porous-polymer
polystyrene resin (Diaion HP-20) column, and the EtOH-eluted
fraction (80 g) was subjected to silica gel column chromatography
tion time-of-flight mass spectrometry (HRESITOFMS) (m/z
+
13
719.3235 [M + Na] , calcd for C35
H52NaO14 719.3255) and
C
NMR (35 carbon signals) spectral data (Table 1). The IR spectrum
ꢀ1
of 1 showed the presence of hydroxy group (3393 cm ) and car-
ꢀ1
1
bonyl groups (1769 and 1762 cm ). The H NMR spectrum dis-
played signals arising from two tertiary methyl groups at d 1.64
and 1.36 (each s), and a secondary methyl group at d 1.25 (d,
H
H
J = 6.5 Hz), which indicated that 1 possessed a steroid skeleton
(Table S1). In addition, signals for two anomeric protons were
observed at d
hydrolysis of 1 produced
the carbohydrate moieties, while the genuine aglycone was
decomposed. The identification of -diginose and -glucose,
H
5.19 (d, J = 7.7 Hz) and 4.80 (dd, J = 9.6, 1.7 Hz). Acid
D
-diginose (Dgn) and -glucose (Glc) as
D
(
CC), octadecylsilanized silica gel CC, and preparative high
D
D
including their absolute configurations, was carried out by direct
HPLC analysis of the hydrolysate using an optical rotation detector.
The b-orientations of the anomeric centers of the Dgn and Glc units
⇑
Corresponding author.
E-mail address: matsuoy@toyaku.ac.jp (Y. Matsuo).
https://doi.org/10.1016/j.tetlet.2021.153020
040-4039/Ó 2021 Elsevier Ltd. All rights reserved.
0

K. Takatori, M. Kuroda, M. Mishima et al.
Tetrahedron Letters 70 (2021) 153020
2
1
OMe
H
1
5
OH
O 1''
O
H
O
1
9
11
O
R O
2
O
HO
14
12
3
H
H
OH
MeO
O
1
8
O
O
4
6
1
'
R1
R1
H
R2
H
1
2
3
H
OH
-D-Glcp
2
1
H
O18 20
H
H
HO
1
2
O
HO
HO
1
7
1
9
1
1
O
H
O 1''
OH
15
HO
1
'''
O
O
HO
1
4
HO
O
OH
3
H
H
O
O
MeO
6
1
'
4
O
H
H
HO
O
H
O
H
H
HO
4
a
O
H
H
H
HO
HO
HO
O
O
O
HO
1
'''
O
OH HO
1
''
O
HO
O
H
H
OH
O
O
MeO
4
1
'
OH
5
Fig. 1. Structures of 1–5 and 4a.
were ascertained by the relatively large 3JH-1,H-2 (ax) values of their
and the molecular formula of the aglycone moiety of 1 was calcu-
lated as C22 , that is, eight degrees of unsaturation. Interpreta-
tion of the
anomeric protons (9.6 and 7.7 Hz, respectively). These data and the
30 6
H O
1
3
1
1
C NMR spectrum indicated that 1 was a pregnane diglycoside,
H– H correlated spectroscopy (COSY) and
2

K. Takatori, M. Kuroda, M. Mishima et al.
Tetrahedron Letters 70 (2021) 153020
Table 1
1
H and 13C NMR (500 and 125 MHz, respectively) spectroscopic data for 1–5 and 4a.
1
2
3
positions
d
H
J (Hz)
d
C
d
H
J (Hz)
d
C
d
H
J (Hz)
d
C
1
2
ax
1.14
2.91
1.83
2.08
3.87
2.61
2.38
–
5.40
2.16
2.14
3.27
2.67
–
–
–
–
2.13
–
2.01
2.35
2.51
1.36
1.64
4.57
1.25
3.34
ddd
ddd
dddd
br d
m
13.5, 13.5, 3.2
13.5, 3.4, 3.2
13.5, 13.5, 11.2, 3.4
13.5
37.0
1.14
2.91
1.82
2.08
3.85
2.60
2.37
–
5.40
2.17
2.13
3.27
2.67
–
–
–
–
2.13
–
2.01
2.34
2.51
1.36
1.64
4.57
1.24
3.34
ddd
ddd
m
m
m
13.7, 13.7, 3.2
13.7, 3.2, 3.2
36.9
1.24
3.07
2.55
1.90
4.06
4.79
–
ddd
ddd
br ddd
br dd
ddd
d
13.5,13.5, 3.3
13.5, 3.3, 3.3
13.5, 12.8, 3.3
12.8, 3.3
11.8, 3.6, 2.3
2.3
37.3
eq
ax
eq
29.9
29.8
23.8
3
4
20.9 (W1/2
)
77.0
38.8
77.0
38.7
78.6
74.6
eq
ax
ddd
m
13.1, 4.4, 1.9
ddd
m
13.0, 4.7, 2.0
5
6
7
142.6
121.0
37.2
142.6
121.0
37.1
–
145.0
125.8
37.2
br s
m
m
dddd
d
br s
m
m
m
d
5.40
2.27
2.14
3.42
2.67
–
–
–
–
2.13
–
2.02
2.37
2.51
1.33
2.08
4.56
1.24
3.36
br d
ddd
m
m
d
3.3
a
b
17.5, 5.2, 5.2
8
9
1
1
1
1
1
1
1
11.2, 11.2, 5.7, 2.2
11.2
25.4
50.0
38.3
170.8
179.2
52.2
57.6
111.8
33.3
25.4
50.0
38.3
170.7
179.2
52.1
57.5
111.7
33.3
25.5
50.2
38.4
170.5
179.1
52.1
57.7
111.6
33.3
11.1
11.2
0
1
2
3
4
5
6
d
2.2
d
2.6
d
2.3
a
b
dd
m
ddd
s
s
m
d
14.6, 11.8
11.8, 5.8, 4.2
dd
m
ddd
s
s
dq
d
14.6, 11.9
12.0, 6.3, 4.1
dd
m
m
s
s
m
d
14.7, 11.9
1
1
1
2
2
7
8
9
0
1
46.8
20.7
18.4
74.7
16.5
51.3
46.8
20.7
18.4
74.6
16.5
51.2
46.8
20.7
20.5
74.5
16.4
51.2
6.3, 6.3
6.3
6.5
6.5
OMe
s
s
s
0
1
4.80
2.39
2.17
3.47
4.21
3.54
1.55
3.40
dd
9.6, 1.7
98.4
33.1
4.79
2.37
2.16
3.47
4.12
3.54
1.50
3.36
dd
9.6, 1.9
12.4, 12.2, 9.6
12.2
98.3
33.0
4.98
2.36
2.13
3.40
4.20
3.58
1.56
3.30
dd
9.6, 1.5
12.1, 11.7, 9.6
12.1
97.9
32.7
0
2
ax
eq
ddd
ddd
ddd
br s
q-like
d
12.2, 12.0, 9.6
12.0, 2.7, 1.7
12.2, 4.1, 2.7
ddd
br d
ddd
br s
q-like
d
ddd
br d
br d
br s
q-like
d
0
3
80.2
73.6
70.8
17.9
56.1
12.4, 4.3, 2.7
80.0
73.8
70.7
17.8
56.1
11.7
80.0
73.6
70.9
17.8
56.0
0
4
0
5
6.4
6.4
6.4
6.4
6.4
6.4
0
6
OMe
s
s
s
0
1
2
3
4
5
6
’
’
’
’
’
’
5.19
3.99
4.25
4.19
3.97
4.58
4.37
d
7.7
104.8
76.0
78.3
71.9
78.6
63.2
5.19
3.99
4.26
4.30
3.88
4.51
d
7.9
104.8
75.4
76.4
81.1
76.6
62.4
5.17
3.99
4.25
4.18
3.96
4.58
4.36
d
7.8
104.7
75.9
78.2
71.9
78.5
63.1
0
0
0
0
0
dd
dd
dd
ddd
dd
dd
8.8, 7.7
8.8, 8.8
8.8, 8.8
8.8, 5.7, 2.4
11.6, 2.4
11.6, 5.7
dd
dd
dd
m
s
8.8, 7.9
8.8, 8.8
8.8, 8.8
dd
dd
dd
ddd
dd
dd
8.7, 7.8
8.8, 8.7
8.8, 8.8
8.8, 5.7, 2.4
11.6, 2.4
11.6, 5.7
a
b
(2H)
0
0
0
0
0
0
1
2
3
4
5
6
’’
’’
’’
’’
’’
’’
5.13
4.10
4.20
4.22
3.99
4.51
4.32
d
7.7
104.5
74.6
78.4
71.4
78.2
62.3
dd
dd
dd
ddd
m
8.5, 7.7
8.5, 8.5
8.5, 8.5
8.5, 5.7, 2.6
a
b
br d
11.5
4
4a
5
positions
d
H
J (Hz)
d
C
d
H
J (Hz)
d
C
d
H
J (Hz)
d
C
1
2
ax
eq
ax
eq
1.36
2.64
1.65
1.99
3.81
2.58
2.33
–
5.49
3.19
2.08
2.68
2.38
–
ddd
ddd
m
m
m
13.8, 13.8, 3.7
13.8, 3.4, 3.4
38.6
30.0
1.43
2.68
1.95
1.71
3.80
2.63
2.55
–
5.47
3.19
2.08
2.73
2.41
–
ddd
m
br d
br d
m
14.0, 14.0, 3.5
38.9
32.2
1.53
2.69
2.37
1.86
4.01
4.76
–
ddd
ddd
m
m
m
14.1, 14.1, 3.4
14.1, 3.6, 3.6
39.1
24.0
11.1
13.0
3
4
19.5 (W1/2
)
77.2
39.1
21.2 (W1/2
)
71.0
43.2
78.9
75.1
eq
ax
br dd
m
13.0, 2.6
ddd
dd
13.1, 4.9, 2.1
7.3, 7.3
d
2.3
5
6
7
140.7
122.0
29.4
141.6
121.3
29.4
–
143.5
126.7
29.5
br d
m
m
m
d
5.4
br d
m
m
m
d
5.3
5.89
3.25
2.22
2.86
2.43
–
dd
m
m
m
d
5.0, 1.9
a
b
8
9
1
1
1
29.7
57.2
39.9
207.6
102.0
29.7
57.3
39.8
207.7
102.0
29.7
57.8
39.9
207.4
102.0
13.2
13.3
13.3
0
1
2
–
–
–
–
–
–
(
continued on next page)
3

K. Takatori, M. Kuroda, M. Mishima et al.
Tetrahedron Letters 70 (2021) 153020
Table 1 (continued)
4
4a
5
positions
d
H
J (Hz)
d
C
d
H
J (Hz)
d
C
d
H
J (Hz)
d
C
1
1
1
1
3
4
5
6
–
2.44
–
2.38
2.19
2.37
1.63
1.29
4.66
1.14
55.5
57.4
218.1
39.8
–
2.44
–
2.34
2.20
2.37
1.63
1.35
4.66
1.14
55.4
57.4
218.5
39.8
–
2.46
–
2.38
2.19
2.38
1.61
1.79
4.66
1.14
55.5
57.3
218.2
39.8
br d
1.4
br s
br d
1.8
a
b
m
m
m
s
s
dq
d
dd
dd
br s
s
s
m
d
8.9, 1.8
8.9, 8.9
m
m
m
s
s
dq
d
1
1
1
2
2
7
8
9
0
1
50.5
21.1
20.3
73.0
16.7
50.5
21.0
20.4
73.0
16.7
50.5
21.1
22.9
73.0
16.7
6.3, 3.8
6.3
6.2, 3.7
6.2
6.3
0
0
1
2
4.75
2.33
2.09
3.45
4.10
3.53
1.47
3.35
dd
9.6, 1.9
12.0, 12.0, 9.6
12.0
98.6
33.0
4.89
2.30
2.05
3.35
4.38
3.57
1.67
3.28
dd
9.7, 1.7
98.2
32.8
ax
eq
ddd
br d
ddd
br s
q-like
d
ddd
ddd
ddd
br s
q-like
d
12.0, 12.0, 9.7
12.0, 2.6, 1.7
12.0, 4.2, 2.6
0
0
0
0
3
4
5
6
12.0, 4.3, 2.7
80.1
74.7
70.7
17.8
56.0
80.1
72.9
71.0
18.1
56.1
6.4
6.4
6.4
6.4
OMe
s
s
0
1
2
3
4
5
6
’
’
’
’
’
’
5.10
3.95
4.25
4.31
3.88
4.51
d
7.9
104.6
75.4
76.4
81.2
76.6
62.5
5.13
3.92
4.16
4.00
4.08
4.82
4.31
d
7.8
104.5
75.6
78.3
71.9
77.6
70.5
0
0
0
0
0
dd
dd
dd
ddd
s
8.8, 7.9
8.8, 8.8
8.8, 8.8
8.8, 3.4, 3.4
(2H)
dd
dd
dd
ddd
dd
dd
8.8, 7.8
8.8, 8.8
8.8, 8.8
8.8, 7.1, 1.6
11.8, 1.6
11.8, 7.1
a
b
0
0
0
0
0
0
1
2
3
4
5
6
’’
’’
’’
’’
’’
’’
5.18
4.1
d
7.9
104.8
73.9
78.2
71.5
78.5
62.4
5.19
4.05
4.23
4.26
3.96
4.54
4.40
d
7.8
105.6
75.2
71.7
78.5
78.5
62.8
dd
dd
dd
ddd
dd
m
8.6, 7.9
8.6, 8.6
8.6, 8.6
8.6, 6.0, 2.4
11.5, 2.4
dd
dd
dd
ddd
dd
dd
8.8, 7.8
8.8, 8.8
8.8, 8.8
8.8, 5.2, 2.4
11.8, 2.4
11.8, 5.2
4.20
4.22
3.99
4.52
4.31
a
b
heteronuclear multiple quantum coherence (HMQC) spectra indi-
cated that three structural fragments, a, b, and c, made up the agly-
cone moiety of 1 (Fig. 2a). In the heteronuclear multiple bond
more than 3
JC,H coupling correlations (Fig. 2c). Other HMBC and
HSQMBC correlations were consistent with the rearranged 11,12-
secopregnane structure with a d -lactone group between C-11
correlation (HMBC) spectrum of 1, Me-19 at d
long-range correlations with C-1 at d 37.0, C-5 at d
at d 50.0, and C-10 at d 38.3, and the olefinic proton assigned
to H-6 at d 5.40 was correlated with C-4 at d 38.8 (Fig. 2b). These
correlations provided evidence that the structural fragments a and
b were connected via the C-5 and C-10 quaternary carbons and
H
1.64 showed
and C-15 and a
Overhauser effect (NOE) correlations between Me-19 (d
and H-1b(d 2.91)/H-4 b (d 2.38)/H-8 (d 3.27), between H-14
(d 2.13) and H-8 (d 3.27)/Me-18 (d 1.36), between H-17 (d
2.51) and Me-18 (d 1.36)/Me-21 (d
(d 3.34) and H-9 (d 2.67)/H-16 (d
c
-lactone ring between C-12 and C-20. Nuclear
C
C
142.6, C-9
H
1.64)
C
C
H
H
H
H
C
H
H
H
H
H
H
1.25), and between OMe
H
a 2.01), were indicative of
H
H
that Me-19 was attached to C-10. Next, the methyl singlet at d
.34 exhibited an HMBC correlation with C-15 at d 111.8, which
was assignable to the methyl acetal carbon. HMBC correlations
H
the B/C trans, C/D cis, and D/E cis ring connections [3] as well as
21 configuration, as shown in Fig. 3. The orientation of the oxy-
gen atom at C-3 was determined to beb based on the H-3 coupling
constants (m, W1/2 = 20.9 Hz) and NOEs between H-3 (d 3.87) and
3
C
a
from H-14 at d
H
2.13, H
2
-16 at d
H
2.35 and 2.01, and H-17 at d
H
H
2
.51 to the C-15 indicated that fragments b and c were linked
H-2
a
(d
H
2.08)/H-4
a
(d
H
2.61). In the HMBC spectrum of 1, long-
4
00
through the C-15 methyl acetal carbon. Furthermore, the
long-range correlation between the H-16 at d
170.8 and the correlation between H-9 at d 2.67 and C-11 con-
firmed that a d-lactone group was present in the C-ring moiety of
the steroid skeleton. In contrast, Me-18 at d 1.36 showed HMBC
cross peaks with C-13 at d 52.2, C-14 at d 57.6, and C-17 at d
6.8, implying that the steroid -ring unit formed a cyclopentane
J
C,H
range correlations were observed between H-1 of Glc at d
and C-4 of Dgn at d 73.6, and between H-1 of Dgn at d
C
and C-3 of aglycone at d 77.0. Thus, 1 was determined as shown
H
in Fig. 1.
Digipregnoside B (2) had the molecular formula C41
which was larger than that of 1 by C , corresponding to
one hexosyl unit. Enzymatic hydrolysis of 2 with naringinase
yielded 1 and -glucose. In the HMBC spectrum of 2, long-range
H
5.19
4.80
0
0
a
H
2.01 and C-11 at
H
d
C
H
H
62 19
H O ,
C
C
C
6 10 5
H O
4
D
ring and that Me-18 was linked to C-13. The steroid A–D-ring units,
including a pair of olefinic carbons and one ester carbonyl carbon,
accounted for six of the eight degrees of unsaturation required by
the molecular formula of the aglycone moiety of 1. Additional
D
0
00
correlations were observed between H-1 of Glc at d
H
5.13 and
5.19 and C-4 of
4.79 and C-3 of
0
0
00
0
C-4 of Glc at d
Dgn at d
C
81.1, between H-1 of Glc at d
H
0
C
73.8, and between H-1 of Dgn at d
H
HMBC correlations from H-14, H-17, and Me-18 to C-12 at d
C
C
the aglycone at d 77.0. Thus, 2 was characterized as shown in
1
79.2 and C-13, as well as the remaining two degrees of unsatura-
Fig. 1.
tion suggested that the steroid E-ring formed a
was confirmed by long-range correlations from H-20 at d
c
-lactone ring. This
4.57 and
1.25 to C-12 in the HSQMBC spectrum, which provides
Digipregnoside C (3) had the molecular formula C35
52 15
H O ,
H
which was larger than that of 1 by one oxygen atom. Comparison
1
3
Me-21 at d
H
of the C NMR spectrum of 3 with that of 1 showed significant dif-
4

K. Takatori, M. Kuroda, M. Mishima et al.
Tetrahedron Letters 70 (2021) 153020
1
4
21
9
(
a)
2
3
14
CH3
1
6
8
1
7
2
0
1
1
7
O
H- H COSY
O
6
fragment c
fragment a
fragment b
OMe
21
20
(
b)
1
6
OH
O
11
O
15
17
O
1
9
1
4
O
12
HO
O
9
1
''
10
HO
13
18
8
14
HO
4'
3
O
O
5
O
MeO
6
1
1
1
'
H- H COSY
HMBC
OMe
21
20
(
c)
1
1
O
15
O
1
1
1
9
H- H COSY
O
12
1
9
1
4
13
HSQMBC
8
1
8
O
5
O
6
Fig. 2. (a) Structural fragments composed of the aglycone moiety of 1, (b) key HMBC correlations of 1, and (c) key HSQMBC correlations of 1.
1
9
H
O
H
O
4
a
O
H
H
H
O
H
H
H
2
1
1
8
O
O
3
1
4
9
8
2
17
6
1
4
O
O
O
1
6
H
H
H
H
HO
HO
HO
OH
O
O
H
H
O
H
H
H
O
HO
O
Compound 1
O
H
1
9
O
O
H
H
H
H
H
1
8
HO
HO
1
9
O
O
H
H
4
OMe
8
14
17
O
H
6
O
O
H
O
H
O
1
6
H
H
H
O
2
1
HO
Fig. 4. A possible biosynthetic pathway of the aglycone moiety of 1 from 4a.
Compound 4a
Fig. 3. Key NOE correlations of the aglycone moiety of 1 and 4a.
spin-coupling constant of 3JH-3,H-4 = 2.3 Hz allowed the configura-
tion at C-4 to be determined as b in 3. The diglycoside moiety
attached to C-3 of the aglycone of 3 was ascertained to be the same
as that of 1 by analysis of the HMBC spectra and the results of the
acid hydrolysis of 3. Thus, 3 was characterized as shown in Fig. 1.
ferences in the signals attributable to the A-ring portion; the C-4
carbon at d
at d 74.6 (CH) in 3, indicating that a hydroxy group was located at
C-4 of the aglycone of 3. The H– H COSY spectrum and HMBC cor-
relations from H-3 at d 4.06 and H-6 at d 5.40 to C-4 at d 74.6
supported the presence of a hydroxy group at C-4. NOE correla-
tions between H-4 (d 4.79) and H-3 (d 4.06), and the proton
C 2
38.8 (CH ) in 1 was replaced by an oxymethine carbon
C
1
13
1
1
The H and C NMR spectral data of 4 (C40H60O18) were essen-
1
tially analogous to those of diginigenin glycoside [4]. The H NMR
H
H
C
spectrum of 4 displayed three anomeric proton signals at d
H
5.18
(
d, J = 7.9 Hz), 5.10 (d, J = 7.9 Hz), and 4.75 (dd, J = 9.6, 1.9 Hz),
H
H
5

K. Takatori, M. Kuroda, M. Mishima et al.
Tetrahedron Letters 70 (2021) 153020
1
00
TIG-3
1
00
SBC-3
8
6
4
2
0
0
0
0
0
8
0
60
40
20
0
*
0
.001
0.01
0.1
1
10
0.001
0.01
0.1
1
10
Compound 1 ( M)
Compound 3 ( M)
1
1
20
00
1
00
8
6
4
2
0
0
0
0
0
8
6
4
2
0
0
0
0
0
*
*
*
0
.001
0.01
0.1
1
10
0
.001
0.01
0.1
1
10
Compound 5 ( M)
Compound 4 ( M)
Fig. 5. Cell viability of 1 and 3–5 in SBC-3 and TIG-3 cells Cell viability was determined by a modified MTT assay after incubation with 0.001, 0.01, 0.1, 1.0, and 10
–5 for 24 h. Data represent the mean value ± standard error of the mean (S.E.M.) of three experiments performed in triplicate; **p < 0.001 vs. cell viability of TIG-3 cells,
p < 0.01 vs. cell viability of TIG-3 cells.
lM of 1 and
3
*
and acid hydrolysis of 4 gave 4a,
D
-glucose, and
the C NMR spectrum of 4a was compared to that of diginigenin
5], the C-12 carbon at d 90.0 (CH) in diginigenin was replaced
by the downfield-shifted quaternary carbon at d 102.0 (C) in 4a.
Furthermore, an HMBC correlation was observed between Me-18
at d 1.63 and C-12 at d 102.0 in 4a. These data indicated that
a was the C-12 hydroxy derivative of diginigenin. NOE correla-
tions between H-1 (d 1.43) and H-9 (d 2.41), between Me-19
1.35) and H-8 (d 2.73)/H-4b (d 2.55), between Me-18 (d
2.44)/H-17 (d 2.37), and between H-17 and
D
-diginose. When
The MeOH extract of Digitalis purpurea seeds showed potent
cytotoxicity against SBC-3 cells (IC50 1.5 g/mL). In this study, 1
and 3–5 were evaluated for their cytotoxic activities against SBC-
3 small cell lung carcinoma and TIG-3 normal human fibroblast
cells, using an modified MTT assay (Fig. 5). Therefore, 1, 4, and 5
displayed cytotoxic activities against SBC-3 cells with IC50 values
1
3
l
[
C
C
H
C
4
of 1.3 ± 0.16, 0.17 ± 0.072, and 2.1 ± 0.52
ically applied anticancer agent etoposide was used as a positive
control (IC50 1.0 ± 0.036 M against SBC-3 cells; IC50 greater
than 10 M against TIG-3 cells). Compounds 1 and 4 showed
tumor selective cytotoxicity against SBC-3 small cell lung carci-
noma cells at a sample concentration of 0.001 M. When SBC-3
lM, respectively. The clin-
a
H
H
(d
H
H
H
H
l
1
.63) and H-14 (d
H
H
l
Me-21 showed that the stereochemistry of 4a was the same as that
of diginigenin; thus, the configuration of the C-12 hydroxy group
l
1
13
was b (Fig. 3). Analysis of the H and C NMR spectra of 4 con-
firmed that the sugar moiety attached to C-3 of the aglycone was
coincident with that of 3. Accordingly, 4 was elucidated as 12
cells were treated with either 1 or 4 and stained with caspase-
3/7 green detection reagent and Hoechst, fragmented and con-
densed nuclear chromatins and activated caspase-3/7 signals were
observed (Fig. S1 in Supporting Information). Compounds 1 and 4
were shown to induce apoptotic cell death in SBC-3 cells.
a,20
a
-epoxy-3 b -[(O- b -
D
-glucopyranosyl-(1 ? 4)-O- b -
D-glu-
copyranosyl-(1 ? 4)- b -
D-diginopyranosyl)oxy]-12 b -hydrox-
ypregn-5-ene-11,15-dione.
The H and 13C NMR spectral features of 5 (C40
1
H
60
O19) were clo-
sely related to those of 4. However, the molecular formula of 5 was
higher by one oxygen atom than that of 4, and the existence of a
hydroxy group at C-4 of the aglycone moiety was suggested by
Conclusion
1
13
In conclusion, three novel rearranged 11,12-secopregnane gly-
cosides, digipregnosides A (1), B (2), and C (3), and two novel
the H and C signals at d
H
4.76 (d, J = 2.3 Hz) and d
as in 3. The H– H COSY spectrum and HMBC correlations from
H-3 at d 4.01 and H-6 at d 5.89 to C-4 supported the presence
of a hydroxy group at C-4. NOE correlations between H-4 and H-
and the proton spin-coupling constant of 3
JH-3,H-4 = 2.3 Hz
C
75.1 (CH),
1
1
1
2,20-epoxypregnane glycosides, digipregnosides D (4) and E (5),
were isolated from the seeds of Digitalis purpurea. Compounds 1,
, and 5 displayed cytotoxic activities against SBC-3 cells, and 1
and 4 showed tumor selective cytotoxicity at a sample concentra-
tion of 0.001 M and induced apoptotic cell death in SBC-3 cells.
H
H
4
3
allowed the configuration at C-4 to be determined as b. In the
HMBC spectrum of 5, long-range correlations were observed
l
0
00
00
between H-1 of Glc at d
H
5.19 (d, J = 7.8 Hz) and C-6 of Glc at
0
0
0
d
C
70.5, between H-1 of Glc at d
H
5.13 (d, J = 7.8 Hz) and C-4 of
0
Dgn at d
C
72.9, and between H-1 of Dgn at d
H
4.89 (dd, J = 9.7,
78.9. Thus, 5 was identified
-glucopyranosyl-(1 ? 4)-O- b -
-diginopyranosyl)oxy]-4 b,12 b -dihy-
Declaration of Competing Interest
1
.7 Hz) and C-3 of the aglycone at d
as 12 ,20 -epoxy-3b-[(O- b -
glucopyranosyl-(1 ? 4)- b -
C
a
a
D
D-
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
D
droxypregn-5-ene-11,15-dione.
A plausible biogenetic pathway of the novel rearranged 11,12-
secopregnane skeleton is proposed as depicted in Fig. 4. The agly-
cone moiety of 1 is presumed to be biosynthesized from the agly-
cone of 4. After the C-11/C-12 bond of ring C was oxidatively
cleaved, the hydroxy group of the C-11 carboxy functionality
attacks the C-15 carbonyl group, resulting in the formation of the
Acknowledgments
We thank Tomomi Atou and Ginta Igarashi for their technical
support.
c- and d-lactone rings.
6

K. Takatori, M. Kuroda, M. Mishima et al.
Tetrahedron Letters 70 (2021) 153020
[
2] T. Fujino, M. Kuroda, Y. Matsuo, S. Kubo, C. Tamura, N. Sakamoto, Y. Mimaki, M.
Hayakawa, Cardenolide glycosides from the seeds of Digitalis purpurea exhibit
carcinoma-specific cytotoxicity toward renal adenocarcinoma and
hepatocellular carcinoma cells, Biosci. Biotechnol. Biochem. 79 (2015) 177–184.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2021.153020.
[3] M. Kuroda, S. Kubo, S. Uchida, H. Sakagami, Y. Mimaki, Amurensiosides A–K, 11
new pregnane glycosides from the roots of Adonis amurensis, Steroids 75 (1)
(
2010) 83–94.
[
4] A. Perrone, A. Capasso, M. Festa, E. Kemertelidze, C. Pizza, A. Skhirtladze, S.
Piacente, Antiproliferative steroidal glycosides from Digitalis ciliata, Fitoterapia
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7