Crotocascarins I-K: Crotofolane-type diterpenoids, crotocascarin γ, isocrotofolane glucoside and phenolic glycoside from the leaves of croton cascarilloides

Article abstract of DOI:10.1248/cpb.c15-00635

From the 1-BuOH-soluble fraction of a methanol (MeOH) extract of the leaves of Croton cascarilloides, crotofolanes: crotocascarins I-K, nor-crotofolane: crotocascarin γ, isocrotofolane glucoside and phenolic glycoside were isolated by a combination of various separation techniques. Their structures were elucidated mainly from the NMR spectroscopic evidence. The structure of crotocascarin K was first elucidated by spectroscopic analysis and then was confirmed by X-ray crystallographic analysis. Its absolute structure was finally determined by the modified Mosher's method. Isocrotofolane glucoside was found to possess a new skeleton, however, its absolute structure remains to be determined.

Full text of DOI:10.1248/cpb.c15-00635

Vol. 63, No. 12
Chem. Pharm. Bull. 63, 1047–1054 (2015)
1047
Regular Article
Crotocascarins I–K: Crotofolane-Type Diterpenoids, Crotocascarin γ,
Isocrotofolane Glucoside and Phenolic Glycoside from the Leaves of
Croton cascarilloides
a
b
,a,b
a
Susumu Kawakami, Katsuyoshi Matsunami, Hideaki Otsuka,* Masanori Inagaki,
a
c
c
Yoshio Takeda, Masatoshi Kawahata, and Kentaro Yamaguchi
a
Department of Natural Products Chemistry, Faculty of Pharmacy, Yasuda Women’s University; 6–13–1 Yasuhigashi,
b
Asaminami-ku, Hiroshima 731–0153, Japan: Department of Pharmacognosy, Graduate School of Biomedical and
Health Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-ku, Hiroshima 734–8553, Japan: and Department
c
of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University Kagawa Campus; 1314–1
Shido, Sanuki, Kagawa 769–2193, Japan.
Received August 16, 2015; accepted September 28, 2015
From the 1-BuOH-soluble fraction of a methanol (MeOH) extract of the leaves of Croton cascarilloides,
crotofolanes: crotocascarins I–K, nor-crotofolane: crotocascarin γ, isocrotofolane glucoside and phenolic
glycoside were isolated by a combination of various separation techniques. Their structures were elucidated
mainly from the NMR spectroscopic evidence. The structure of crotocascarin K was first elucidated by
spectroscopic analysis and then was confirmed by X-ray crystallographic analysis. Its absolute structure was
finally determined by the modified Mosher’s method. Isocrotofolane glucoside was found to possess a new
skeleton, however, its absolute structure remains to be determined.
Key words Croton cascarilloides; Euphorbiaceae; crotocascarin; crotofolane; nor-crotofolane; isocrotofolane
A diterpenoid, phorbol 12-myristate 13-acetate, isolated tion of various chromatographic techniques.
2
6
from Croton oil is known to have a potent tumor-promoting
Crotocascarin I (1), [α]D +32.0, was isolated as an amor-
1,2)
effect and is often employed in biomedical research to acti- phous powder and its elemental composition was determined
vate protein kinase C. Thus, Croton species plants attracted to be C H O by high-resolution (HR)-electrospray ioniza-
3)
2
0
24
5
our attention. C. cascarilloides RÄUSCHEL is an evergreen tion (ESI)-mass spectrometry (MS). The IR spectrum showed
shrubby tree that grows on elevated coral reefs of the Okina- absorption bands at 3478cm− and 1739cm assignable to
wa Islands, Taiwan, southern China, the Malay Peninsula and hydroxy and γ-lactone functional groups. The UV absorption
Malaysia. The leaves are oblong-lanceolate to oblong-oval, and band at 223nm indicated the presence of an α,β-unsaturated
1
−1
4
)
1
their undersurface is covered by shiny white ramenta. In a ketone moiety. In the H-NMR spectrum, signals for one
5
)
previous study, investigation of the constituents of the stems singlet methyl at δH 1.05, two doublet methyls at δH 1.02
of Croton cascarilloides provided eight rare crotofolane-type (J=7.2Hz) and 1.91 (J=1.2Hz), and two olefinic protons at δ
H
13
and two rearranged crotofolane-type diterpenoids. In this 5.06 (1H, s) and 5.07 (1H, s) were observed. The C-NMR
study, from the 1-BuOH-soluble fraction of a MeOH extract of spectrum together with distortionless enhancement polariza-
the leaves of C. cascarilloides, three crotofolane-type diterpe- tion transfer (DEPT) results revealed a total of 20 resonances,
noids, named crotocascarins I–K (1–3), a nor-diterpenoid, cro- which comprised those of three methyls, three methylenes,
tocascarin γ (4), a diterpenoid glucoside with a new skeleton, three oxygenated methines, three methines, three oxygenated
which was named isocrotofolane glucoside (5), and one pheno- tertiary carbons, tetrasubstituted and exo-cyclic double bonds,
lic glycoside (6) along with two known compounds (7, 8) were and one carbonyl carbon. Except for the absence of a 2-meth-
isolated. The structures of the new compounds isolated were ylbutyrate moiety, these functionalities were the same as those
elucidated from spectroscopic evidence and that of crotocasca- of crotocascarin D (9), which was isolated from the branches
5
)
rin K (3) was determined by X-ray crystallographic analysis, of the title plant. When compared the NMR spectra of 1 and
followed by application of the modified Mosher’s method to 9, the H-1 signal shifted up from δH 5.49 (crotocascarin D) to
1
clarify its absolute structure. The known compounds were δH 4.20 in the H-NMR spectrum, and the C-1 signal shifted
6
)
13
identified as angelicoidenol 2-O-β-D-glucopyranoside (7) and up from δ 74.5 (crotocascarin D) to δ 73.6 in the C-NMR
C
C
7
)
senecrassidiol (8) by comparison of the physico-chemical spectrum. Therefore, the structure of crotocascarin I (1) was
data with those reported in the literature.
elucidated to be as shown in Fig. 1, namely the deacyl form of
crotocascarin D (9). The absolute structure of 1 was the same
as that of crotocascarin D (9), as judged from a similar posi-
Results and Discussion
From the 1-(butanol) BuOH-soluble fraction of a MeOH ex- tive Cotton effect at 252nm (Δε +1.42) in the circular dichro-
5
)
tract of the leaves of C. cascarilloides, three crotofolane-type ism (CD) spectrum to that observed in crotocascarin D.
diterpenoids, named crotocascarins I–K (1–3), a nor-diterpe-
2
4
Crotocascarin J (2), [α]D +9.1, was isolated as an amor-
noid, crotocascarin γ (4), a diterpenoid glucoside with a new phous powder and its elemental composition was determined
skeleton, which was named isocrotofolane glucoside (5), and to be C H O , which is one oxygen more than in the case of
2
0
24
6
1
3
a phenolic glycoside (6) were isolated by means of a combina- 1. The C-NMR spectrum was similar to that of 1, and this
*
To whom correspondence should be addressed. e-mail: otsuka-h@yasuda-u.ac.jp
©
2015 The Pharmaceutical Society of Japan

1
048
Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
Fig. 1. The Structures of Compounds Isolated and Reference Compounds
together with the absence of an oxygenated methine proton at
the 9-position means the 9-position must have been replaced
by a hydroxy functional group to form a ketal, which was
supported by the appearance of δC 108.0, with the simultane-
ous disappearance of the oxygenated methine signal at δ 82.3
C
observed for 1. Therefore, the structure of crotocascarin J (2)
was elucidated to be as shown in Fig. 1, namely the deacyl
form of crotocascarin A (10). The absolute structure of 2 was
the same as that of crotocascarin A, as judged from a similar
5
)
positive Cotton effect at 255nm (Δε +2.54).
2
4
Crotocascarin K (3), [α]D +96.7, was isolated as colorless
plates and its elemental composition was determined to be
C H O by HR-ESI-MS. The C-NMR spectrum displayed
13
2
0
24
5
2
0 carbon signals, which showed close resemblance to those
Fig. 2. Important HMBC Correlations of Crotocascarin K (3)
of crotocascarin I (1), except for the presence of one more
exo-cyclic double bond and a secondary alcohol, and the dis-
1
appearance of an epoxy ring at C-5 and C-6. In the H-NMR hydroxy group at C-5 (Fig. 2). The CD spectrum of 3 exhib-
spectrum, although exo-cyclic methylene protons resonated at ited a strong Cotton effect at 226nm (Δε +16.6), however, the
the same frequencies, H-18a and 20a at δ 5.24, and H-18b and spectrum profile was different from those of 1 and 2, probably
H
2
0b at δH 5.09, significant heteronuclear multiple-bond con- due to the closeness of the new exo-cyclic double bond to
8
,9)
nectivity (HMBC) cross peaks between these exo-methylene another double bond between C-8 and C-15. Thus, applica-
protons and C-5, C-6 and C-7 suggested that the position of tion of the CD spectrum rule to the stereochemistry of the
the new exo-cyclic double bond was at C-6 and the secondary 9-position was too ambiguous to draw a correct conclusion.

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1049
1
1
Fig. 5. H– H COSY and Diagnostic HMBC Correlations of 5
Fig. 3. ORTEP Drawing of Crotocascarin K (3)
Dual arrow curves denote HMBC correlations were observed in both directions.
The structure has crystallographic numbering.
ing those of four methyls, three methylenes, three methines,
one oxygenated methine, two oxygenated tertiary carbons,
tetrasubstituted, trisubstituted and exo-cyclic double bonds,
1
1
and a carbonyl carbon signals. On H– H correlation spectros-
copy (COSY), the presence of three proton chains, i.e., CH3–
CH–CH –, –CH–CH–CH= and –CH –CH –, was confirmed.
2
2
2
From the degree of unsaturation, 5 must have a tetracyclic
structure, and to establish the structure HMBC correlations
were precisely inspected. Correlations between H -19 (δ
3
H
1.11) and C-1 (δC 213.3), C-3 (δC 38.1), and δH 2.35 and 3.16
on C-3 and C-4 (δC 174.1) and C-14 (δC 139.4) established a
five membered-ring, which must involve an α,β-unsaturated
ketone (Fig. 5). Further correlations H-5 and C-4, C-14, H-7
and C-5, C-6, C-13, and H-13 and C-4 and C-14 established
Fig. 4. Results with the Modified Mosher’s Method for Crotocascarin
K (3)
Therefore, X-ray crystallographic analysis was performed to a six-membered ring fused at the C-4 and C-14 tetrasubsti-
confirm the structure, Fig. 3 showing an ORTEP drawing of tuted double bond. When C-18, isopropyl (C-15, -16, -17) and
3
, followed by application of the modified Mosher’s method methyl (C-20) groups were excluded as exo-cyclic carbons, the
10)
to establish the absolute structure (Fig. 4). As a result, the remaining four carbons must form a third seven-membered
spectroscopically deduced structure of 3 was proved to be ring in crotofolanes. The HMBC correlations H-7 and C-9,
correct and the absolute stereochemistry was found to be the C-12, C-13, H-8 and C-6, C-10, H-13 and C-11, C-12, C-18,
same as that of other crotofolanes isolated from C. cascaril- H -18 and C-11, C-12 and C-13, and H -20 and C-5 and C-7
2
3
loides (Fig. 1).
were supportive of the structure shown in Fig. 1. The hydroxy
2
3
1
1
Crotocascarin γ (4), [α]D +21.8, was isolated as an amor- isopropyl group was placed at C-9 based on the H– H COSY
phous powder and its elemental composition was determined correlation from H-7 to H-8, and the HMBC correlations be-
to be C H O by HR-ESI-MS. The NMR spectroscopic tween H-7 and C-9, H-8 and C-15, and H -16 (or 17) and C-9
19
24
6
3
data were similar to those of crotocascarin β, except for the (Fig. 5), thus, a different skeleton from the crotofolane was
absence of a 2-methylbutyric acid moiety. Namely, 4 was as- formed, i.e., a new one named the isocrotofolane. The posi-
signed as deacyl crotocascarin β, as shown in Fig. 1.
tion of the sugar linkage was also confirmed by the HMBC
1
-Oxo-5,6,15-trihydroxyisocrotofola-4 (14), 8,12(18)-triene 5- spectrum and the mode of linkage was β as judged from the
2
4
O-β-D-glucopyranoside (isocrotofolane glucoside) (5), [α]D
63.6, was isolated as an amorphous powder and its elemen- analysis revealed that the glucose is in the D-series. Although
tal composition was determined to be C H O by HR-ESI- compound 5 was enzymatically hydrolyzed to give 5a, to
coupling constant of the anomeric proton (J=7.3Hz). HPLC
−
2
6
38
9
MS. The IR spectrum exhibited absorptions assignable to which the β-D-glucopyranosylation-induced shift-trend rule
−
1
−1
11) 13
hydroxy (3378cm ) and carbonyl (1690cm ) groups, and could be applied,
C-NMR chemical shift changes of C-4
−1
double bonds (1646cm ), and the UV spectrum showed an and C-6 from 5 to 5a did not observe the typical rule (C-4:
absorption for an α,β-unsaturated ketone at 234nm. In the Δδ −0.9ppm and C-6: Δδ +0.3) (Table 1). Furthermore, an
H-NMR spectrum, signals for three singlet methyls, one attempt to prepare α-methoxy-α-trifluoromethylphenylacetic
1
doublet methyl, three olefinic protons [δH 4.28 (s), 4.62 (s), acid (MTPA) esters was not successful, probably due to steric
6
.29 (d, J=6.0Hz)], one doublet methine proton [δH 4.60 (d, hindrance around the C-5 region. Assuming that the absolute
J=1.1Hz)] with an oxygen atom and an anomeric proton [δ stereochemistries of the ring junctures at the C-7 and C-13
.49 (d, J=7.3Hz)] were observed. In the C-NMR spectrum positions were the same as those of other crotofolanes isolated
H
13
4
together with DEPT revealed six typical signals assignable to from the title plant, phase-sensitive (PS) nuclear Overhauser
terminal glucopyranoside, the remaining 20 signals compris- enhancement (NOESY) spectroscopic correlation H-5 and H-7

1
050
Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
−1
placed the hydroxy group at the 5-position in a β-orientation, ring (1503cm ), and the UV absorption bands also supported
1
and those between H-7 and H -20, and H-5 and H -20 the the presence of the aromatic ring. The H-NMR spectrum
3
3
1
1
hydroxy group at the 5-position also in a β-orientation and along with H– H COSY and heteronuclear single quantum
the 20-methyl group in an α-orientation, as shown in Fig. 6. coherence spectra showed four aromatic proton signals in
Those between H-5 and H-3a (δH 3.16), H-3b (δH 2.35) and series, two anomeric protons at δH 5.52 and 5.69 on δC 102.8
H -19 and then H-2 (δH 2.45) and H-3a placed the 19-methyl and 111.2, respectively and a methoxy signal (Table 2). The
3
13
group in a β-orientation. Therefore, the structure of 5 was ten-
C-NMR spectrum showed five signals assignable to a termi-
nal apiofuranose and six for a hexopyranose unit, and HPLC
Compound 6, [α]D −93.6, was isolated as an amorphous analysis of the hydrolyzate of 6 gave two peaks for D-apiose
powder and its elemental composition was determined to be and D-glucose. In difference NOE experiments, on irradiation
C H O by HR-ESI-MS. The IR spectrum showed absorp- of the methoxy signal, significant signal enhancement was
tatively elucidated to be as shown in Fig. 1.
2
5
1
8
26 11
−1
tion bands for hydroxy groups (3364cm ) and an aromatic observed at H-3 [δH 6.90 (dd, J=8.1, 2.0Hz)], and those on
anomeric protons, δH 5.52 and 5.69, NOE enhancement was
observed at H-6 and H -6′ protons, respectively. Therefore,
2
the structure of compound 6 was elucidated to be 2-me-
thoxyphenol β-D-(6′-O-β-D-apiofuranosyl)glucopyranoside, as
shown in Fig. 1.
Isocrotofolane glucopyranoside (5) is a diterpene, having a
new carbon skeleton. Biosynthesis of crotofolane is expected
to start from geranylgeranylpyrophosphate and then proceed
via cembrane, casbane, lathyrane and then jatropholane (Fig.
7
). The final step is opening of the cyclopropane ring. In
pathway 1, the cleavage of the C-9 and C-15 bond forms croto-
folane, however, that in pathway 2 between the C-8 and C-15
bond may lead to the formation of a new skeleton, named the
isocrotofolane. This is the second example of a new diterpe-
noid skeleton, isolated from C. cascarilloides.
Fig. 6. Important PS NOESY Correlations of 5
Experimental
Glucose is omitted for clarity.
General Experimental Procedure Melting point (mp)
13
Table 1.
C
C-NMR Spectroscopic Data for Crotocascarins I–K (1–3), Crotocascarin γ (4), and Isocrotofolane Glucoside (5) (100MHz, CDCl3)
a)
a)
a)
1
2
3
4
5
5a
1
2
3
4
5
6
7
8
9
73.6
33.7
36.2
60.2
57.9
56.1
43.8
162.2
82.3
37.7
36.3
147.1
40.2
69.9
128.3
173.2
9.6
73.7
33.7
36.2
60.4
58.2
56.6
44.1
159.7
108.0
41.8
35.2
147.7
39.3
70.0
130.1
171.3
9.6
73.1
33.2
76.0
35.9
34.7
67.8
75.8
89.8
49.3
63.8
177.3
28.3
28.4
145.2
35.5
67.8
204.2
—
213.3
42.1
38.1
174.1
82.3
76.1
50.5
125.4
151.3
30.5
39.6
154.6
43.7
139.4
74.3
28.7
28.7
108.2
15.8
21.1
105.9
75.8
78.3
72.0
77.7
63.1
213.0
42.1
35.8
36.7
b)
b)
b)
67.6
175.0 (−0.9)
75.1 (+7.2)
75.8 (+0.3)
51.1
71.1
145.1
39.5
163.1
82.6
125.8
151.2
30.6
10
11
12
13
14
15
16
17
18
19
20
37.2
37.0
39.7
147.2
43.3
154.8
43.9
73.0
139.3
74.3
127.2
173.6
9.3
28.7
25.5
109.2
12.7
22.9
28.8
114.5
12.1
19.5
114.4
12.2
20.3
114.3
12.2
108.0
15.8
114.1
24.1
1
2
3
4
5
6
′
′
′
′
′
′
a) Data for CD OD. b) Δδ5–δ5b.
3

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1051
was measured with a Yanagimoto micro melting point appa-
A highly-porous synthetic resin (Diaion HP-20) was pur-
ratus and is uncorrected. Optical rotations were measured on chased from Mitsubishi Kagaku (Tokyo, Japan). Silica gel
a JASCO P-1030 digital polarimeter. IR and UV spectra were column chromatography (CC) was performed on silica gel
measured on Horiba FT-710 and JASCO V-520 UV/Vis spec- 60 (E. Merck, Darmstadt, Germany), and octadecyl silanized
1
13
trophotometers, respectively. H- and C-NMR spectra were silica gel (ODS) open CC on Cosmosil 75C -OPN (Nacalai
18
taken on a JEOL JNM α-400 spectrometer at 400MHz and Tesque, Kyoto, Japan) [Φ=50mm, L=25cm, linear gradient:
1
00MHz, respectively, with tetramethylsilane as an internal MeOH–H O (1:9, 1L)→(1:1, 1L), fractions of 10g being col-
2
standard. CD spectra were obtained with a JASCO J-720 spec- lected]. The droplet counter-current chromatograph (DCCC)
tropolarimeter. Positive-ion HR-ESI-MS was performed with (Tokyo Rikakikai, Tokyo, Japan) was equipped with 500
®
an Applied Biosystems QSTAR XL NanoSpray™ System.
glass columns (Φ=2mm, L=40cm), the lower and upper lay-
ers of a solvent mixture of CHCl –MeOH–H O–n-propanol
3
2
(
9:12:8:2) being used as the stationary and mobile phases,
1
3
1
Table 2. NMR Spectroscopic Data for Compound 6 ( C: 100MHz, H:
00MHz; C D N)
respectively. Five-gram fractions were collected and numbered
according to their order of elution with the mobile phase.
HPLC was performed on an ODS column (Inertsil; GL Sci-
ence, Tokyo, Japan; Φ=20mm, L=250mm, 6mL/min), and
the eluate was monitored with a UV detector at 254nm and
4
5
5
C
H
1
150.3
148.3
113.3
122.7
121.7
117.4
56.0
—
2
—
2
3
a refractive index monitor. Authentic D-apiose {[α] +9.4
3
6.90 (dd, J=8.1, 2.0Hz)
6.94 (ddd, J=8.1, 7.9, 2.0Hz)
7.06 (ddd, J=7.9, 7.9, 2.0Hz)
7.69 (dd, J=7.9, 2.0Hz)
3.67 (3H, s)
D
(
c=0.84, H O)} was obtained by chromatographic separation
4
2
of a hydrolyzate of apiin, isolated from commercial parsley
5
6
(Petroselinum crispum). D-Apiose was identified by NMR
spectroscopy. (R)- and (S)-α-MPTAs were purchased from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
12)
–
OCH3
1
2
3
4
5
6
′
′
′
′
′
′
102.8
74.9
5.52 (d, J=7.3Hz)
4.29 (overlapped)
Plant Material Leaves of C. cascarilloides RÄUSCHEL
(Euphorbiaceae) were collected in Kunigami-son, Kunigami-
gun, Okinawa, Japan, in July 2004, and a voucher specimen
was deposited in the Herbarium of Pharmaceutical Sciences,
Graduate School of Biomedical and Health Sciences, Hiro-
shima University (04-CC-Okinawa-0628).
Extraction and Isolation Air-dried leaves of C. cas-
carilloides (6.53kg) were extracted with MeOH (45L) three
times. The MeOH extract was concentrated to 6L and then
washed with n-hexane (6L, 59.1g). The methanolic layer
was concentrated to a viscous gum. The gummy mass was
78.4
4.29 (overlapped)
71.4
4.07 (dd, J=9.2, 9.0Hz)
4.22 (ddd, J=9.2, 7.0, 1.3Hz)
4.79 (dd, J=11.0, 1.3Hz)
77.4
69.0
4
.13 (dd, J=11.0, 7.0Hz)
1″
2″
3″
4″
111.2
77.7
80.4
75.1
5.69 (d, J=2.4Hz)
4.72 (d, J=2.4Hz)
—
4.55 (d, J=9.3Hz)
.31 (d, J=0.3Hz)
4
5
″
65.7
4.16 (2H, s)
suspended in H O (6L), and then partitioned with EtOAc
2
Fig. 7. Possible Biosynthetic Pathway for Isocrotofolane

1
052
Chem. Pharm. Bull.
Vol. 63, No. 12 (2015)
(
6L) and 1-BuOH (6L), successively, to give 100g and 126g
Crotocascarin J (2)
Amorphous powder, [α] +9.1 (c=0.64, CHCl ); IR ν
max
2
4
of EtOAc and 1-BuOH-soluble fractions, respectively. The
remaining water-layer was concentrated to give a H O- (KBr) cm : 3433, 2929, 2874, 1745, 1645, 1415, 1384, 907;
soluble-fraction (263g). The 1-BuOH-soluble fraction was UV λmax (MeOH) nm (logε): 220 (3.54); H-NMR (400MHz,
D
3
−1
2
1
subjected to a Diaion HP-20 column (Φ=80mm, L=57cm), CDCl ) δ: 5.06 (1H, s, H-18a), 5.04 (1H, s, H-18b), 4.18 (1H,
3
and eluted with H O–MeOH (4:1, 6L), (3:2, 6L), (2:3, 6L), d, J=5.1Hz, H-1), 3.35 (1H, d, J=12.6Hz, H-13), 3.11 (1H, s,
2
and (1:4, 6L), and MeOH (6L), 1L-fractions being collected. H-5), 2.97 (1H, dd, J=12.6, 1.1Hz, H-7), 2.53 (1H, m, H-10a),
The residue (6.38g) in fractions 10–14 was subjected to silica 2.41 (1H, m, H-11a), 2.39 (1H, dd, J=13.5, 7.0Hz, H-3a), 2.33
gel CC (Φ=36mm, L=57cm), and eluted with CHCl (1.5L), (1H, ddd, J=14.8, 13.4, 6.4Hz, H-11b), 2.04 (1H, m, H-2),
3
CHCl –MeOH (99:1, 1.5L), (97:3, 1.5L), (19:1, 1.5L), (37:3, 1.89 (3H, d, J=0.9Hz, H -17), 1.68 (1H, dd, J=13.5, 10.2Hz,
3
3
1
.5L), (9:1, 1.5L), (7:1, 1.5L), (17:3, 1.5L), (33:7, 1.5L), H-3b), 1.56 (1H, ddd, J=13.6, 13.2, 5.7Hz, H-10b), 1.14 (3H,
13
(
2
4:1, 1.5L), (3:1, 1.5L), and (7:3, 1.5L), and MeOH (1.5L), s, H -20), 1.03 (3H, d, J=7.2Hz, H -19); C-NMR (100MHz,
50mL-fractions being collected. The residue (1.61g) in frac- CDCl ): Table 1; CD Δε (nm): +2.54 (255), −2.71 (223)
3
5
3
3
−
tions 59–79 was separated by ODS open CC to give 50.6mg (c=1.78×10 M, MeOH); HR-ESI-MS (positive-ion mode) m/z:
+
of crude 6 in fractions 58–64, which was finally purified by 383.1464 [M+Na] (Cacld for C H O Na: 383.1465).
HPLC (H O–MeOH, 3:2) to afford 2.9mg of pure 6 from the
peak at 5.4min.
2
0
24
6
Crotocascarin K (3)
Colorless plates, mp 244–245°C; [α]
2
2
4
+96.7 (c=0.15,
(KBr) cm : 3435, 2924, 2857, 1739, 1634,
max
D
−1
The residue (6.90g) in fractions 15–18 obtained on Diaion CHCl ); IR ν
3
HP-20 CC was subjected to silica gel CC (Φ=36mm, L=57 cm), 1406, 1384, 904; UV λ
(MeOH) nm (logε): 218 (3.99);
H-NMR (400MHz, CDCl ) δ: 5.24 (2H, s, H-18a, 20a), 5.09
max
1
and eluted with CHCl3 (1.5L), CHCl –MeOH (99:1, 1.5L),
3
3
(97:3, 1.5L), (19:1, 1.5L), (37:3, 1.5L), (9:1, 1.5L), (7:1, (2H, s, H-18b, 20b), 5.06 (1H, m, H-9), 4.42 (1H, s, H-5), 4.15
1.5L), (17:3, 1.5L), (33:7, 1.5L), (4:1, 1.5L), (3:1, 1.5L), (1H, d, J=5.2Hz, H-1), 3.55 (1H, dd, J=11.7, 0.7Hz, H-7),
and (7:3, 1.5L), and MeOH (1.5L), 250mL-fractions being 2.88 (1H, d, J=11.7Hz, H-13), 2.60 (2H, m, H-10a, 11a), 2.41
collected. The residue (921mg) in fractions 42–52 was sepa- (1H, dd, J=13.7, 7.2Hz, H-3a), 2.27 (1H, m, H-11b), 2.03 (1H,
rated by ODS open CC and the residue (45.7mg) in fractions m, H-2), 1.69 (3H, d, J=1.1Hz, H -17), 1.53 (1H, dd, J=13.7,
3
1
5
88–193 was then purified by DCCC to give 9.6mg of crude 10.2Hz, H-3b), 1.27 (1H, m, H-10b), 1.00 (3H, d, J=7.1Hz,
13
in fractions 87–97, which was finally purified by HPLC H -19); C-NMR (100MHz, CDCl ): Table 1; CD Δε (nm):
3
3
−5
(
H O–MeOH, 3:2) to afford 5.4mg of pure 5 from the peak +16.6 (226) (c=4.45×10 M, MeOH); HR-ESI-MS (positive-
2
at 20.0min.
ion mode) m/z: 367.1516 (Calcd for C H O Na: 367.1515).
20 24 5
The residue (11.7g) in 19–24 fractions obtained on Diaion
HP-20 CC was subjected to silica gel CC (Φ=50mm, L=57cm),
Crotocascarin γ (4)
Amorphous powder, [α] +21.8 (c=0.07, CHCl ); IR ν
max
2
3
D
3
−1
and eluted with CHCl (3L), CHCl –MeOH (99:1, 3L), (97:3, (film) cm : 3420, 2957, 2930, 1757, 1711, 1454, 1150, 891;
3
3
1
3
L), (19:1, 3L), (37:3, 3L), (9:1, 3L), (7:1, 3L), (17:3, 3L),
H-NMR (400MHz, CD OD) δ: 5.19 (1H, brs, H-18a), 4.91
3
(
33:7, 3L), (4:1, 3L), (3:1, 3L), and (7:3, 3L), and MeOH (1H, s, H-18b), 4.39 (1H, d, J=5.0Hz, H-1), 4.28 (1H, s, H-5),
(3L), 500mL-fractions being collected. The residue (598mg) 3.15 (1H, brd, J=13.6Hz, H-13), 2.85 (1H, d, J=13.6Hz, H-7),
in fractions 19–24 was subjected to ODS open CC and the 2.50 (1H, m, H-11a), 2.36 (3H, s, H -17), 2.20 (3H, overlapped,
3
residue (32.5mg) in fractions 118–124 was purified by HPLC H-10a, b, 11b), 2.19 (1H, dd, J=13.8, 7.3Hz, H-3a), 1.95 (1H,
(
2
H O–MeOH, 3:2) to afford the residue from the peak at m, H-2), 1.51 (1H, dd, J=13.8, 10.6Hz, H-3b), 1.20 (3H, s,
2
13
2min, which was then purified again by HPLC (Inertsil Ph-3, H -20), 1.00 (3H, d, J=7.2Hz, H -19); C-NMR (100MHz,
3
3
H O–MeOH, 13:7) to give 1.3mg of 4 and 3.4mg of 3 from CD OD): Table 1; HR-ESI-MS (positive-ion mode) m/z:
2
3
the peaks at 22min and 24min, respectively. Crotocascarins J 371.1475 (Calcd for C H O Na: 371.1465).
19
24
6
(
1
2) (59.8mg) and I (1) (6.3mg) were obtained from fractions
47–155 and 155–162, respectively.
Crotocascarin I (1)
Amorphous powder, [α] +32.0 (c=0.65, CHCl ); IR ν
Isocrotofolane Glucoside (5)
2
4
Amorphous powder, [α] −63.6 (c=0.36, MeOH); IR νmax
D
1
−
(film) cm : 3378, 2930, 2876, 1690, 1646, 1457, 1380, 1077,
897; UV λ (MeOH) nm (logε): 234 (3.84), 211 (3.79);
2
6
D
3
max
max
−1
1
(
KBr) cm : 3478, 2939, 2873, 1739, 1644, 1452, 1014, 904;
H-NMR (400MHz, CD OD) δ: 6.29 (1H, d, J=6.0Hz, H-8),
3
1
UV λmax (MeOH) nm (logε): 223 (3.75); H-NMR (400MHz, 4.62 (1H, s, H-18a), 4.60 (1H, d, J=1.1Hz, H-5), 4.49 (1H,
CDCl ) δ: 5.07 (1H, s, H-18a), 5.06 (1H, s, H-18b), 4.92 (1H, d, J=7.3Hz, H-1′), 4.28 (1H, s, H-18b), 3.93 (1H, dd, J=11.9.
3
ddq, J=12.0, 3.6, 1.2Hz, H-9), 4.20 (1H, d, J=5.4Hz, H-1), 1.8Hz, H-6′a), 3.69 (1H, dd, J=11.9, 5.9Hz, H-6′b), 3.33
3.11 (1H, s, H-5), 3.08 (1H, d, J=12.6Hz, H-7), 2.90 (1H, d, (4H, m, H-2′, 3′, 4′, 5′), 3.16 (1H, m, H-3a), 2.65 (1H, brd,
J=12.6Hz, H-13), 2.58 (1H, dddd, J=12.6, 4.2, 3.6, 3.6Hz, J=8.8Hz, H-13), 2.56 (1H, m, H-11a), 2.51 (1H, m, H-10a),
H -10a), 2.55 (1H, ddd, J=13.2, 3.6, 3.6Hz, H-11a), 2.41 (1H, 2.45 (1H, m, H-2), 2.35 (1H, m, H-3b), 2.27 (1H, dd, J=8.8,
2
dd, J=13.8, 7.2Hz, H-3a), 2.19 (1H, ddd, J=13.2, 12.6, 4.2Hz, 6.0Hz, H-7), 2.00 (2H, m, H-10b, 11b), 1.39 (3H, s, H -20),
3
H-11b), 2.02 (1H, ddqd, J=10.6, 7.2, 7.2, 5.4Hz, H-2), 1.91 1.35 (3H, s, H -17), 1.34 (3H, s, H -16), 1.11 (3H, d, J=7.3Hz,
3
3
1
3
(
1
3H, d, J=1.2Hz, H -17), 1.65 (1H, dd, J=13.8, 10.6Hz, H-3b), H -19); C-NMR (100MHz, CD OD): Table 1; HR-ESI-
3
3
3
+
.21 (1H, dddd, J=13.2, 12.6, 12.0, 4.2Hz, H -10b), 1.05 (3H, MS (positive-ion mode) m/z: 517.2385 [M+Na] (Calcd for
2
13
s, H -20), 1.02 (3H, d, J=7.2Hz, H -19); C-NMR (100MHz, C H O Na: 517.2408).
3
3
26 38
9
−5
CDCl ): Table 1; CD Δε (nm): +1.42 (252) (c=1.91×10 M,
MeOH); HR-ESI-MS (positive-ion mode) m/z: 367.1514
[
Compound 6 (6)
Amorphous powder, [α] −93.6 (c 0.19, MeOH); IR νmax
(film) cm : 3364, 2928, 1503, 1255, 1069; UV λ
3
2
5
D
+
−1
M+Na] (Cacld for C H O Na: 367.1515).
(MeOH)
2
0
24
5
max
1
nm (logε): 333 (2.74), 269 (3.25), 220 (3.57); H-NMR

Vol. 63, No. 12 (2015)
Chem. Pharm. Bull.
1053
1
3
(
400MHz, C D N): Table 2; C-NMR (100MHz, C D N): (3H, d, J=6.8Hz, H -19); HR-ESI-MS (positive-ion mode) m/z:
5 5 5 5 3
+
Table 2; HR-ESI-MS (positive-ion mode) m/z: 441.1363 583.1904 [M+Na] (Calcd for C H O F Na, 583.1920).
M+Na] (Calcd for C H O Na: 441.1367).
X-Ray Crystallographic Analysis of Crotocascarin K (4.1mg) in H O (1mL) was hydrolyzed with β-glucosidase
3
0
31
7 3
+
[
Enzymatic Hydrolysis of 5 Isocrotofolane glucoside (5)
18
26 11
2
3
(
3) C H O5 , M=344.39, crystal size: 0.23×0.20×0.12 mm , (4.0mg) at 37°C for 4d. The reaction mixture was evaporated
20 24 7
space group: orthorhombic, P2 2 2 , T=150K, a=10.2738(14)Å, to dryness, and then the remaining residue was subjected to
1
1 1
3
b=11.3380(15)Å, c=14.764(2)Å, V=1719.8(4) Å , Z=4, D = silica gel CC (Φ=2cm, L=15cm) with increasing amounts of
c
3
1
.330Mg/m , F(000)=736. The data were measured using a MeOH in CHCl [CHCl –MeOH (9:1; 50mL), (7:3; 100mL)
3 3
Bruker SMART 1000 CCD diffractometer, using MoKα and (1:1; 100mL)] 5mL-fractions being collected. An agly-
graphite-monochromated radiation (λ=0.71073Å) in the range cone (5a) (2.5mg) was obtained in fractions 17–24 and the
of 4.52<2θ<54.3. Of 8399 reflections collected, 2019 were sugar (1.2mg) in 51–70. Compound 5a: Amorphous powder,
2
4
−1
unique (R =0.0204, data/restraints/parameters 2019/0/2368). [α] −115.0 (c 0.17, CDCl ); IR ν (film) cm : 3321, 2927,
int
D
3
max
The structure was solved by a direct method using the pro- 2871, 1683, 1650, 1455, 1373, 1054, 827; UV λ (MeOH) nm
gram SHELXTL-97. The refinement and all further calcula- (logε): 231 (3.98), 214 (3.93); H-NMR (400MHz, CD OD)
max
13)
1
3
tions were carried out using SHELXTL-97. The absorption δ: 6.29 (1H, d, J=6.0Hz, H-8), 4.61 (1H, s, H-18a), 4.30 (1H,
14)
correction was carried out utilizing the SADABS routine.
brs, H-5), 4.25 (1H, s, H-18b), 2.95 (1H, dd, J=18.7, 6.1Hz,
The H atoms were included at calculated positions and treated H-3a), 2.65 (1H, brd, J=9.0Hz, H-13), 2.59 (1H, m, H-10a),
as riding atoms using the SHELXTL default parameters. The 2.51 (1H, m, H-11a), 2.47 (1H, m, H-2), 2.42 (1H, dd, J=18.7,
non-H atoms were refined anisotropically using weighted full- 3.6Hz, H-3b), 2.21 (1H, dd, J=9.0, 6.0Hz, H-7), 2.00 (2H, m,
2
2
matrix least-squares on F . Final goodness-of-fit on F =1.047, H-10b, 11b), 1.35 (3H, s, H -16), 1.34 (3H, s, H -17), 1.32 (3H,
3
3
13
R =0.0288, wR =0.0737 based on I>2σ(I), and R =0.0323, s, H -20), 1.16 (3H, d, J=7.3Hz, H -19); C-NMR (100MHz,
1
2
1
3
3
wR =0.0757 based on all data. The largest difference peak CD OD): Table 1; HR-ESI-MS (positive-ion mode) m/z:
2
3
−3
+
and hole were 0.158 and −0.157 eÅ , respectively. Supple- 355.1887 [M+Na] (Calcd for C H O Na: 355.1879).
mentary X-ray crystallographic data for 3 (CCDC 1417185)
2
0
28
4
Sugar Analysis About 500µg each of 5 and 6 was hy-
can be obtained free of charge via www.ccdc.cam.ac.uk/conts/ drolyzed with 1M HCl (0.1mL) at 90°C for 2h. The reaction
retrieving.html (or from the Cambridge Crystallographic Data mixtures were partitioned with an equal amount of EtOAc
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) (0.1mL), and the water layers were analyzed by HPLC with
1223-336-033; or deposit@ccdc.cam.ac.uk).
a chiral detector (JASCO OR-2090plus) on an amino column
Preparation of (R)- and (S)-MTPA Esters (3a, b) from [Asahipak NH P-50 4E, CH CN–H O (4:1), 1mL/min]. A
2
3
2
3
A solution of 3 (0.55mg) in 0.5mL of dry CH Cl was hydrolyzate of 5 gave a peak for D-glucose at 20.0min, and
2 2
reacted with (R)-MTPA (25.4mg) in the presence of 1-ethyl-3- one of 6 gave peaks for D-apiose and D-glucose at 6.4min and
3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) 20.0min, respectively, with positive optical rotation signs. The
15.8mg) and N,N-dimethyl-4-aminopyridine (4-DMAP) peaks were identified by co-chromatography with authentic
15.5mg). The mixture was then occasionally stirred at 37°C samples.
(
(
(
for 24h. After the addition of CHCl3 (1.5mL), the reaction
mixture was successively washed with H O (1mL), 1M HCl
Acknowledgments The authors are grateful for access to
2
(
1mL), NaHCO -saturated H O (1mL), and brine (1mL). The the superconducting NMR instrument (JEOL JNM α-400) at
3 2
organic layer was dried with Na SO and evaporated under re- the Analytical Center of Molecular Medicine of the Hiroshima
2
4
duced pressure. The residue was purified by preparative TLC University Faculty of Medicine, and an Applied Biosystem
silica gel (0.25mm thickness), being applied for 8cm width, QSTAR XL system ESI (Nano Spray)-MS at the Analysis
[
with development with n-hexane–EtOAc (1:1) for 9cm and Center of Life Science of the Graduate School of Biomedi-
then eluting with CHCl –MeOH (9:1)] to furnish an ester 3a cal Sciences, Hiroshima University. This work was supported
3
(0.2mg) at Rf=0.38. Through the same procedure, 3b (0.3mg, in part by Grants-in-Aid from the Ministry of Education,
Rf=0.37) was prepared from 3 (0.55mg) using (S)-MTPA Culture, Sports, Science and Technology of Japan, and the
(
21.5mg), EDC (13.4mg), and 4-DMAP (16.1mg). (R)-MTPA Japan Society for the Promotion of Science (Nos. 22590006,
1
ester (3a): amorphous powder; H-NMR (400MHz, CDCl3) 23590130 and 15H04651). Thanks are also due to the Research
δ: 7.38–7.57 (5H, m, aromatic protons), 5.77 (1H, s, H-5), 5.31 Foundation for Pharmaceutical Sciences and the Takeda Sci-
(
1H, s, H-20b), 5.22 (1H, s, H-18b), 5.08 (1H, s, H-18a), 5.04 ence Foundation for the financial support.
1H, m, H-9), 4.78 (1H, s, H-20a), 4.13 (1H, m, H-1), 3.58 (1H,
d, J=11.2Hz, H-7), 3.54 (3H, s, -OMe), 2.88 (1H, d, J=11.2Hz,
H-13), 2.42 (1H, dd, J=13.7, 6.8Hz, H-3b), 2.00 (1H, m, H-2), interest.
.62 (1H, dd, J=13.7, 10.2Hz, H-3a), 1.26 (3H, s, H -17), 0.99
(
Conflict of Interest The authors declare no conflict of
1
3
(
3H, d, J=7.3Hz, H -19); HR-ESI-MS (positive-ion mode) References
3
+
m/z: 583.1912 [M+Na] (Calcd for C H O F Na, 583.1920).
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3
0
31
1
7 3
zuka Y., J. Biol. Chem., 257, 7847–7851 (1982).
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2
)
Hecker E., Naturwiss., 54, 282–284 (1967).
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3
3
)
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2
4)
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J=11.7Hz, H-7), 3.66 (3H, s, -OMe), 2.88 (1H, d, J=11.7Hz,
H-13), 2.42 (1H, dd, J=13.7, 7.3Hz, H-3b), 2.02 (1H, m, H-2),
5
)
1
.65 (1H, dd, J=13.7, 10.3Hz, H-3a), 1.26 (3H, s, H -17), 0.99
3

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0) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem.