Antimutagenic activity of ent-kaurane diterpenoids from the aerial parts of Isodon japonicus

Article abstract of DOI:10.1016/j.tetlet.2017.07.106

From the methanolic extract of the aerial parts of Isodon japonicus, two new ent-kaurane diterpenes, isodonterpenes I and II, were isolated together with 10 known diterpenes. Their chemical structures were elucidated on the basis of chemical and physicoch

Full text of DOI:10.1016/j.tetlet.2017.07.106

Accepted Manuscript
Antimutagenic activity of ent-kaurane diterpenoids from the aerial parts of Iso-
don japonicus
Takahiro Matsumoto, Seikou Nakamura, Naoto Kojima, Tomohiro Hasei,
Masayuki Yamashita, Tetsushi Watanabe, Hisashi Matsuda
PII:
S0040-4039(17)30975-9
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.07.106
TETL 49188
Reference:
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
9 June 2017
26 July 2017
31 July 2017
Please cite this article as: Matsumoto, T., Nakamura, S., Kojima, N., Hasei, T., Yamashita, M., Watanabe, T.,
Matsuda, H., Antimutagenic activity of ent-kaurane diterpenoids from the aerial parts of Isodon japonicus,
Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.2017.07.106
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Tetrahedron Letters
Antimutagenic activity of ent-kaurane diterpenoids from the aerial parts of
Isodon japonicus
Takahiro Matsumoto, Seikou Nakamura, Naoto Kojima, Tomohiro Hasei, Masayuki
Yamashita, Tetsushi Watanabe, Hisashi Matsuda*
Kyoto Pharmaceutical University, Misasagi-Shichono-cho 1, Yamashina-ku, Kyoto 607-8412,
Japan
Abstract: From the methanolic extract of the aerial parts of Isodon japonicus, two new ent-
kaurane diterpenes, isodonterpenes I and II, were isolated together with 10 known diterpenes.
Their chemical structures were elucidated on the basis of chemical and physicochemical
evidence. The absolute configuration of isodonterpene I was elucidated by Cu-Kα X-ray
crystallographic analysis. Antimutagenic activities of the major diterpenes were evaluated by
the Ames test. This study represents the first evaluation of the antimutagenic activities of ent-
kaurane diterpenes.
Keywords: Isodon japonicus; ent-kaurane diterpene; isodonterpene; antimutagenic activity
*
Corresponding author.
Tel.: +81-75-595-4634; fax: +81-75-595-4768; e-mail: matsuda@mb.kyoto-phu.ac.jp
1

Tetrahedron Letters
Isodon japonicus (Burm. f.) H. Hara (Lamiacaeae) is a perennial plant with a wide
1
distribution in China and Japan. The aerial parts of I. japonicus have been used as a traditional
2
medicine for the treatment of gastrointestinal disorders, tumors, and inflammatory diseases. As
part of our ongoing research program for the discovery of bioactive constituents of Asian
3−8
traditional medicines, we recently reported the isolation of six new neolignan glucosides,
named isodonosides I−VI, and a new megastigmane glucoside named isodonmegastigmane I,
from I. Japonicus, together with fifteen known constituents and evaluated their cell protective
9
effects against BaP (Benzo[a]pyrene)-induced cytotoxity. As a continuation of this study, we
found that the methanolic extracts of I. japonicus showed antimutagenic effects against 3-
amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), by the Ames test [inhibition: 60.7% at
1
25 µg/plate]. According to these findings, we pursued the isolation of the antimutagenic
constituents of the aerial parts of I. japonicus. This paper reports the structural elucidation of
new constituents, isodonterpenes I (1) and II (2), the structures of ten known constituents, and
the antimutagenic activities of selected ent-kaurane diterpenes.
O
1
2
O
R3
R2
1
3
1
6
HO
H14
17
1
O
R3
H
2
R4
O
5
7
R2
O
O
R1
H
OH
R1
1
OH
18
19
2
OH
1
3
4
2
3
1
3
4
5
6
: R = OH, R = H, R = H, R = H
7: R = OH, R = H, R = H
1
2
3
4
1
2
3
: R = H, R = H, R = H, R = OH
8: R = H, R = OH, R = H
1
2
3
4
1
2
3
: R = H, R = OH, R = H, R = OH
9: R = H, R = H, R = H
1
2
3
4
1
2
3
: R = H, R = H, R = OMe, R = OH
10: R = H, R = H, R = OMe
1
2
3
4
: R = H, R = OMe, R = H, R = OH
R1
HO
GlcO
H
OAc
H
R2
O
O
OH
H
OH
CHO O
OH
1
2
11
2: 1R, R = OH, R = H
Glc = -D-glucopyranosyl
1
2
1
2: 1S, R = H, R = OH
Figure 1. Chemical structures of 1–12
2

Tetrahedron Letters
A methanolic extract of the dried aerial parts (953.4 g, 8.36%) of I. japonicus (cultivated in
Tokushima, Japan) was partitioned into an EtOAc-H
soluble fraction (393.3 g, 3.45%) and an aqueous layer. The aqueous layer was further
extracted with 1-butanol (BuOH) to give 1-butanol (27.4 g, 0.24%) and H O (532.3 g, 4.67%)
2
O (1:1, v/v) mixture to furnish an EtOAc
2
soluble fractions. The EtOAc and 1-butanol soluble fractions showed antimutagenic effects
against Trp-P-1[inhibition: 74.3% and 18.0%, respectively, at 125 µg/plate]. On the other hand,
2
the H O soluble fraction showed no detectable effects. Therefore, the EtOAc and 1-butanol
soluble fractions were subjected to normal- and reversed-phase silica-gel column
chromatography and repeated HPLC. From the 1-butanol soluble fraction, two new diterpenes,
isodonterpene I (1, 5.0 mg, 0.000044%) and II (2, 19.5 mg, 0.00017%), were isolated
together with a known compound ent-7β,20-epoxy-kaur-16-ene-1β,6α,7α,14α,15α-pentanol-
1
0
1
-O-β-D-glucopyranoside (12, 23.0 mg, 0.00020%). From the EtOAc soluble fraction, nine
10
known compounds, oridorin (3, 107.0 mg, 0.078%), hebeirubescensin H (4, 105.7 mg,
1
1
12
.077%), rabdternin F (5, 22.1 mg, 0.016%), rabdternin E (6, 34.7 mg, 0.025%), enmein
12
0
1
3
14
7, 63.3 mg, 0.074%), nodosin (8, 110.0 mg, 0.13%), isodocarpin (9, 4.1 mg, 0.011%),
15
(
16
17
serrin C (10, 3.3 mg, 0.009%), and isodonal (11, 28.5 mg, 0.078%) were isolated (Fig. 1).
Isodonterpene I (1) was isolated as an amorphous powder. The IR spectrum of 1 suggested the
–
presence of hydroxy (3334 cm ), and ether (1066 cm ) groups. In the EI-MS of 1, a
1
–1
+
molecular ion peak [M] was observed at m/z 364 and the molecular formula C20
H O
28 6
was
) and
1
determined by HRMS measurement of the molecular ion peak. The H NMR (pyridine-d
5
13
C NMR (Table 1) spectra of 1, which were assigned by various NMR experiments, showed
signals assignable to an ent-kaurane diterpene {two methyl groups [δ 1.39 (s, H-18), 1.64 (s,
H-19)], five methylene groups [δ 2.29 (m, H-2α), 2.40 (m, H-2β), 2.51 (m, H-11α), 1.94 (m,
H-11β), 2.20 (m, H-12), 2.51 (m, H-14), 4.46 (d, J = 10.0 Hz, H-20), 4.77 (d, J = 10.0 Hz, H-
2
0)], six methine groups [δ 3.89 (m, H-1), 3.70 (app. d, H-3), 1.59 (d, J = 6.0 Hz, H-5), 4.84
(
d, J = 10.0 Hz, H-6), 1.78 (dd, J = 5.5, 12.5 Hz, H-9), 2.91 (app. d, J = 6.5 Hz, H-13)], and
an olefin [δ 5.27 (s, H-17), 5.97 (s, H-17)]}. As shown in Figure 2, the DQF COSY and
HMBC experiments of 1 indicated the presence of certain structures. The relative
configuration of 1 was determined as 1S', 3R', 5R', 6S', 7S', 8S', 9S', 10S', 13R' by the NOESY
experiment; correlations were observed between H-1 and H-2β, H-2β and H-3, H-3 and H-19,
3

Tetrahedron Letters
H-19 and H-5, H-18 and H-6, H-1 and H-9 (Fig. 2). Crystals of 1 were obtained from an
EtOH-H O solution; therefore Cu-Kα X-ray diffraction (CCDC 1562797) was employed to
2
determine the absolute configuration of 1 (Fig. 3). Namely, the absolute configuration was
determined to be 1S, 3R, 5R, 6S, 7S, 8S, 9S, 10S, 13R from the Flack parameter [absolute
structure parameter = -0.01(15)].
Figure 2. Important 2D NMR correlations of 1 and 2
4

Tetrahedron Letters
1 13
Table 1. H (500 MHz) and C NMR (125 MHz) data for 1 and 2.
Position
1
2
1
δ C
3
1
δ H (J in Hz)
13
δ C
1
δ H (J in Hz)
1
2
71.7
3.90 (m)
83.2
4.79 (dd, 5.5, 10.0)
α 2.34 (app. d, 15.0)
β 2.03 (app. d, 15.0)
α 1.21 (app. t, 13.5)
β 1.36 (app. t, 12.0)
α 2.29 (m)
β 2.40 (m)
39.6
74.6
27.1
38.9
3
3.70 (app. d, 11.0)
4
5
6
7
8
9
40.7
60.7
74.3
96.1
60.4
51.7
42.0
33.7
59.7
74.7
97.1
53.3
50.5
42.1
1.59 (d, 6.0)
1.71 (d, 4.5)
4.19 (d, 4.5)
4.84 (d, 10.0)
1.78 (dd, 5.5, 12.5)
3.00 (app. d, 8.5)
3.85 (m)
1
0
α 2.51 (m)
β 1.94 (m)
11
20.2
61.2
α 2.79 (m)
β 2.00 (m)
2.67 (dd, 4.5, 7.3)
1
1
1
2
3
4
30.1
35.2
26.6
2.20 (m)
43.8
37.9
28.7
2.91 (app. d, 6.5)
2.51 (m)
2
1
.13 (dd, 4.5, 16.5)
.96 (m)
1
1
5
6
211.1
154.4
75.1
5.33 (s)
161.9
5
5
.27 (s)
.97 (s)
1
7
115.7
106.2 4.87 (d, 7.5)
1
1
8
9
16.0
29.0
1.39 (s)
1.64 (s)
22.0
32.7
0.97 (s)
1.08 (s)
4
4
.77 (d, 10.0)
.46 (d, 10.0)
4.37 (d, 10.0)
4.77 (d, 10.0)
2
0
63.8
64.8
1
2
3
4
5
6
'
'
'
'
'
'
103.3 4.87 (d, 7.5)
75.8
79.3
71.5
78.3
62.8
4.19 (m)
4.21 (m)
4.23 (m)
3.85 (m)
4.48 (d, 11.0)
Measured in pyridine-d
5
Isodonterpene II (2) was isolated as an amorphous powder with a negative optical rotation
2
5
D
(
[α]
-18.0 c 0.65, MeOH). The IR spectrum of 2 suggested the presence of hydroxy (3365
–
1
–1
40
cm ), and ether (1076 cm ) groups and its molecular formula (C26H O11) was determined
5

Tetrahedron Letters
from positive-ion FABMS and HRMS measurements. Acidic hydrolysis of 2 with 5% aqueous
H
2 4
SO -1,4-dioxane (1:1, v/v) yielded D-glucose which was identified by HPLC of the
1
8
1
corresponding tolylthiocarbamoyl thiazolidine derivative. The H NMR (pyridine-d
13
) and C
5
NMR (Table 1) spectra of 2, which were assigned by various NMR experiments, showed
signals assignable to an ent-kaurane diterpene moiety {two methyl groups [δ 0.97 (s, H-18),
1
1
.08 (s, H-19)], five methylene groups [δ 2.34 (app. d, J = 15.0 Hz, H-2α), 2.03 (app. d, J =
5.0 Hz, H-2β), 1.21 (app. t, J = 13.5 Hz, H-3α), 1.36 (app. d, J = 12.0 Hz, H-3β), 2.79 (m,
H-12α), 2.00 (m, H-12β), 2.13 (dd, J = 4.5, 16.5 Hz, H-14α), 1.96 (m, H-14 β), 4.37 (d, J =
0.0 Hz, H-20), 4.77 (d, J = 10.0 Hz, H-20)], seven methine groups [δ 4.79 (dd, J = 5.5, 10.0
1
Hz, H-1), 1.71 (d, J = 4.5, 10.0 Hz, H-5), 4.19 (d, J = 4.5 Hz, H-6), 3.00 (app. d, J = 8.5 Hz,
H-9), 3.85 (m, H-11), 2.67 (dd, J = 4.5, 7.3 Hz, H-13), 5.33 (s, H-15)], an olefin [δ 4.87 (d, J
=
7.5 Hz, H-17)]} and a glucose moiety {δ 4.87 (d, J = 7.5 Hz, H-1'), 4.19 (m, H-2'), 4.21 (m,
H-3'), 4.23 (m, H-4'), 3.85 (m, H-5'), 4.48 (d, J = 11.0 Hz, H-6')}. As shown in Figure 2, the
D-NMR experiments of 2 indicated the presence of certain structures. The relative
2
configuration of 2 was determined as 1R', 5R', 6S', 7S', 8S', 9S', 10S', 11S', 13S', 15R' by the
NOESY experiment (Figure 2). Because of the other compounds isolated from this plant, 2
was assumed to have an ent-kaurane skeleton. Therefore, the absolute configuration was
deduced to be 1R, 5R, 6S, 7S, 8S, 9S, 10S, 11S, 13S, 15R.
Figure 3. ORTEP drawing of 1 (CCDC 1562797)
6

Tetrahedron Letters
19
Previously, we have reported the antimutagenic activities of coumarins, furanocoumarins,
20
and methoxyflavonoids. In the course of our studies on antimutagenic compounds, the
activities of diterpenes (3, 4, 7, 8, and 11) against Trp-P-1 and 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine (PhIP) were evaluated by the Ames test using the S.
typhimurium TA98 strain. This short-term assay has been extensively used to survey a large
variety of substances for mutagenic activity. Trp-P-1 and PhIP are well known mutagenic and
carcinogenic heterocyclic amines found in cooked meat. All tested constituents showed
antimutagenic effects against Trp-P-1 (Table 2) and PhIP (Table 3) without antimicrobial
activity at the examined concentrations. This study represents the first evaluation of the
antimutagenic activities of ent-kaurane diterpenes.
Table 2. Antimutagenic activity of major constituents against Trp-P-1 (0.04 µg/plate)
Revertants / plate
(
Inhibition %)
Dose (nmol/plate)
control
25
50
100
200
400
2
10±21
206±33
(0.7%)
156±5
137±2
(34.6%)
141±1
(32.9%)
165±1
(21.0%)
114±5
(46.0%)
124±2
(41.0%)
20
90±3.5
(57.3%)
96±16
(54.4%)
110±9
(47.5%)
91±5
97±7
73±9
3
(
0.0%)
(53.7%)
82±1
(65.2%)
68±4
2
10±21
0.0%)
10±21
0.0%)
10±21
0.0%)
10±21
0.0%)
4
7
8
(
(25.5%)
144±13
(31.5%)
139±17
(33.9%)
149±18
(28.9%)
10
(60.9%)
101±5
(52.0%)
57±4
(67.8%)
84±8
2
(
(59.9%)
56±6
2
(
(56.8%)
95±6
(72.8%)
67±11
(68.3%)
80
(73.3%)
2
11
(
(54.9%)
40
control
(0%)
20
Nobiletin
(4%)
(39%)
(52%)
(56%)
7

Tetrahedron Letters
Table 3. Antimutagenic activity of major constituents against PhIP (1.0 µg/plate)
Revertants / plate
(
Inhibition %)
Dose (nmol/plate)
control
25
50
100
200
400
4
79±0
330±43
(31.2%)
288±36
(39.9%)
425±31
(11.3%)
305±6
212±3
178±13
(62.8%)
134±26
(72.0%)
272±18
(43.2%)
169±11
(64.8%)
98±1
126±13
(73.7%)
98±13
(79.5%)
181±7
(62.3%)
89±2
93±7
3
(
0.0%)
(55.8%)
219±4
(80.7%)
70±4
4
79±0
0.0%)
79±0
0.0%)
79±0
0.0%)
79±0
0.0%)
4
7
8
(
(54.3%)
364±28
(24.0%)
234±60
(51.0%)
137±18
(71.0%)
5
(85.5%)
124±3
(74.2%)
70±1
4
(
4
(
(36.3%)
247±2
(81.4%)
94±14
(80.4%)
20
(85.5%)
4
11
(
(48.5%)
2.5
(79.5%)
10
control
(0%)
20
Nobiletin
(11%)
(35%)
(44%)
(56%)
In conclusion two new ent-kaurane diterpenes, isodonterpene I (1) and II (2) were isolated
together with 10 known diterpenoids from the methanolic extract of the aerial parts of Isodon
japonicus. The absolute configuration of isodonoside I (1) was elucidated by Cu-Kα X-ray
crystallographic analysis. Additionally, diterpenes (3, 4, 7, 8, and 11) showed antimutagenic
effects against Trp-P-1 and PhIP by the Ames test using the S. typhimurium TA98 strain
without antimicrobial activity at the examined concentrations.
Acknowledgments
We are grateful to Dr. Takashi Matsumoto (Rigaku Corp.) for X-ray structural analysis of
compound 1. This research was supported by MEXT (Ministry of Education, Culture, Sports,
Scientific and Technology) - Supported Program for the Strategic Research Foundation at
Private Universities, 2015–2017.
A. Supplementary material
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/
References and notes
8

Tetrahedron Letters
1
2
3
4
5
6
7
8
9
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Lett. 2016, 16, 3076.
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1
1
1
1
2. Takeda, Y.; Takeda, K.; Fujita, T.; Sun, H.D.; Minami, Y, Phytochemistry 1994, 35, 1513.
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1
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1
8
9

Tetrahedron Letters
1
2
2
9. Matsumoto, T.; Takahashi, K.; Kanayama, S.; Nakano, Y.; Imai, H.; Kibi, M.; Imahori, D.;
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Nat. Prod. Commun. 2017, 12, 23.
25
1. Isodonterpene I (1): amorphous powder; [α]
D
-40.3 (c 0.5, MeOH); IR (film) vmax 3334,
-
1
1
066 cm ; H NMR (pyridine-d
13
, 500 MHz,) and C NMR (125 MHz) δ given in Table 1;
1
5
+
EIMS m/z 364 [M] ; HREIMS m/z 364.1892 (calcd for C20
+
[M] , 364.1886).
H
28
O
6
2
5
D
2
2. Isodonterpene II (2): amorphous powder; [α]
-18.0 (c 0.65, MeOH); IR (film) vmax 3365,
-
1
1
854, 2923, 1076 cm ; H NMR (pyridine-d
13
, 500 MHz,) and C NMR (125 MHz) δ
2
5
+
given in Table 1; positive-ion FABMS m/z 551 [M + Na] ; HRFABMS m/z 551.2471
+
11Na [M + Na] , 551.2468).
(
calcd for C26
H
40
O
1
0

Tetrahedron Letters
Graphical Abstract
1
1

Tetrahedron Letters
Highlights
Antimutagenic activity of ent-kaurane diterpenoids from
the aerial parts of Isodon japonicus
Takahiro Matsumoto, Seikou Nakamura, Naoto Kojima, Tomohiro Hasei, Masayuki
Yamashita, Tetsushi Watanabe, Hisashi Matsuda*
ꢀ
ꢀ
ꢀ
Two new ent-kaurane diterpenes were isolated from the aerial parts of I. japonicus.
Absolute configuration was elucidated by X-ray crystallographic analysis.
Antimutagenic effects of the ent-kaurane diterpenes were evaluated.
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