Eight new diterpenoids and two new nor-diterpenoids from the stems of Croton cascarilloides

Article abstract of DOI:10.1248/cpb.c12-01002

From the stems of Croton cascarilloides, eight new diterpenoids, named crotocascarins A-H (1-8), having a crotofolane skeleton were isolated along with two new nor-diterpenoids (9 and 10), named crotocascarins α and β, derived through rearrangement of the crotofolane skeleton. The structures of these compounds were elucidated by means of extensive one- and two-dimensional NMR spectroscopic analyses. The absolute structures of the diterpene moiety were determined by application of the circular dichroism (CD) rule for the γ-lactone ring. The relative structures of the two crotofolanes (1 and 2) and one rearranged compound (9) were confirmed by X-ray crystallographic analyses. Compounds 1, 2 and 9 possessed 2-methylbutyric acid in their molecules, the absolute configuration of which was found to be 2S by comparison of its HPLC behavior with that of an authentic sample. Therefore, the absolute structures of these crotocascarins (1, 2 and 9) were unambiguously determined. The absolute structures of crotofolanes are reported for the first time in this paper.

Full text of DOI:10.1248/cpb.c12-01002

April 2013
Regular Article
Chem. Pharm. Bull. 61(4) 411–418 (2013)
411
Eight New Diterpenoids and Two New Nor-Diterpenoids from the Stems
of Croton cascarilloides
a
a
a
a
Susumu Kawakami, Hiroki Toyoda, Liva Harinantenaina, Katsuyoshi Matsunami,
,
a,b
c
b
d
Hideaki Otsuka,* Takakazu Shinzato, Yoshio Takeda, Masatoshi Kawahata, and
d
Kentaro Yamaguchi
a
Graduate School of Biomedical and Health Sciences, Hiroshima University; 1–2–3 Kasumi, Minami-ku, Hiroshima
b
7
7
34–8553, Japan: Faculty of Pharmacy, Yasuda Women’s University; 6–13–1 Yasuhigashi, Asaminami-ku, Hiroshima
c
31–0153, Japan: Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus; 1 Sembaru,
d
Nishihara-cho, Nakagami-gun, Okinawa 903–0213, Japan: and Faculty of Pharmaceutical Sciences, Tokushima
Bunri University, Kagawa Campus; 1314–1 Shido, Sanuki, Kawaga 769–2193, Japan.
Received November 16, 2012; accepted December 21, 2012; advance publication released online February 1, 2013
From the stems of Croton cascarilloides, eight new diterpenoids, named crotocascarins A–H (1–8), hav-
ing a crotofolane skeleton were isolated along with two new nor-diterpenoids (9 and 10), named crotocasca-
rins α and β, derived through rearrangement of the crotofolane skeleton. The structures of these compounds
were elucidated by means of extensive one- and two-dimensional NMR spectroscopic analyses. The absolute
structures of the diterpene moiety were determined by application of the circular dichroism (CD) rule for
the γ-lactone ring. The relative structures of the two crotofolanes (1 and 2) and one rearranged compound (9)
were confirmed by X-ray crystallographic analyses. Compounds 1, 2 and 9 possessed 2-methylbutyric acid in
their molecules, the absolute configuration of which was found to be 2S by comparison of its HPLC behavior
with that of an authentic sample. Therefore, the absolute structures of these crotocascarins (1, 2 and 9) were
unambiguously determined. The absolute structures of crotofolanes are reported for the first time in this
paper.
Key words Croton cascarilloides; Euphorbiaceae; crotocascarin; crotofolane; diterpenoid
2
6
Crotofolane-type diterpenoids have fused 5-, 6- and 7-mem-
Crotocascarin A (1), [α]D +16.4, was isolated as color-
bered rings, and are expected to be biosynthesized from less rods and its elemental composition was determined to
1
)
cembranes via lathyrane through cross annular cyclization.
be C H O by high-resolution (HR)-electrospray ionization
25 32 7
In 1975, this type of diterpenoid was first found in Jamai- (ESI)-mass spectrometry. The IR spectrum showed absorption
can Croton species, C. corylifolius Lamarck as crotofolin bands for ester carbonyl and lactone carbonyl groups (1763,
1
,2)
−1
−1
1
A as shown in Fig. 1. Only two other species, Kenyan C. 1739 cm ), and double bonds (1650cm ). In the H-NMR
3)
dichogamus Pax and Congolese C. haumanianus J. Léo- spectrum, signals for two singlet methyls, two doublet meth-
nard, were also found to contain rare crotofolanes. Genus yls and one triplet methyl together with ones for two olefinic
Croton (Euphorbiaceae) comprises about 600 species and is protons (δH 5.06, 5.09) and two oxygenated methine protons
4)
1
1
distributed in tropical areas of both hemispheres. Croton oil (δ 3.17, 5.48) were observed (Table 1). The H– H correlation
H
obtained from seeds of C. tiglium Linné has a strong purga- spectroscopy (COSY) and heteronuclear multiple bond corre-
tive effect and the occurrence of cocarcinogenic agents; fatty lation (HMBC) spectra (Fig. 3) together with one-dimensional
acid esters of diterpene phorbol in this plant has attracted our ones indicated the presence of a 2-methylbutanoic acid moi-
5
)
13
interest to investigate this genus. C. cascarilloides räuscheL ety, the remaining 20 C-NMR signals comprising those of
is an evergreen shrubby tree that grows on elevated coral three methyls, three methylenes, five methines, one tetra- and
reefs of the Okinawa Islands, Taiwan, southern China, the one disubstituted double bonds, three oxygenated tertiary
Malay Peninsula and Malaysia. Leaves are oblong-lanceolate carbons, one carbonyl carbon and one hemiketal carbon (δC
to oblong-oval, and their undersurface is covered by shiny 107.5) (Table 2). The ten degrees of unsaturation, based on
6
)
white ramenta. Our phytochemical investigation of the stems the results of HR-ESI-MS, other than two carbonyl groups
14.5kg) of C. cascarilloides collected in the Okinawa Islands and two double bonds, required six ring systems in the skel-
(
led to the isolation of eight new crotofolane-type diterpenoids, eton. Precise inspection of two-dimensional NMR spectra led
given trivial names crotocascarins A–H (1–8), and two new to the conclusion that compound 1 was a diterpenoid with
nor-diterpenoids with a new skeleton, given trivial names
crotocascarins α and β (9, 10). The absolute configuration of
crotofolanes was first determined in this study.
Results and Discussion
Using several types of chromatography, diterpenoids (1–8)
and nor-diterpenoids (9, 10) were isolated from a CH Cl -
2
2
soluble fraction prepared from the MeOH extract of the stems
of C. cascarilloides (Fig. 2).
The authors declare no conflict of interest.
Fig. 1. Structure of Crotofolin A, Isolated from C. corylifolius
*
To whom correspondence should be addressed. e-mail: hotsuka@hiroshima-u.ac.jp
© 2013 The Pharmaceutical Society of Japan

4
12
Vol. 61, No. 4

April 2013
413
1
1
Fig. 3. H– H COSY and Selected HMBC Correlation of 1
Fig. 2. Structures of New Compounds Isolated
Fig. 4. ORTEP Drawing of 1
The crystal structure has crystallographic numbering.
an unusual carbon skeleton (Fig. 2). To confirm this, X-ray
crystallographic analysis of 1 was performed and the relative
stereostructure of 1 was established to be that of a derivative configuration at the 9-position of the α,β-unsaturated γ-lactone
of crotofolane-type diterpenoid (Fig. 4). The positive Cotton ring was also S, and chirality analysis of the 2-methylbutanoic
effect observable in the circular dichroism (CD) spectrum [Δε acid moiety by HPLC with an optical rotation detector estab-
+
3.41 (251)] empirically indicated the absolute configuration lished the absolute structure of 2 to be as shown in Fig. 2.
2
4
at the 9-position of the α,β-unsaturated γ-lactone ring was
Crotocascarin C (3), [α] +88.9, and crotocascarin D (4),
D
24
7
,8)
S, and chirality analysis of the 2-methylbutanoic acid moi- [α]D +2.6, were also similar compounds to the aforemen-
ety by HPLC with an optical rotation detector established the tioned ones with the respective elemental compositions of
absolute configuration of 1 to be shown in Fig. 2. This is the C H O and C H O . In the NMR spectra of 3 and 4, a
2
5
32
8
25 32
6
first report of the absolute structure of a crotofolane diterpene hemiketal carbon signal (C-9: δC 106.4) and an oxygenated
being disclosed and the absolute configuration of the α,β- carbon signal (C-11: δC 73.7) with δH 4.61 were observed in
unsaturated γ-lactone portion, presumed based on the empiri- the former, and an oxygenated carbon signal (C-9: δC 82.2)
cal rule for the CD spectra, was proved to be correct.
Crotocascarin B (2), [α]D +81.8, was isolated as colorless coupling pattern fot H-11 in 3 to that of 2 placed the hydroxy
plates and its elemental composition was determined to be group at the 11-position in a β-orientation. Since the H– H
and a methylene signal (C-11: δ 36.2) in the latter. A similar
C
2
6
1
1
C H O by HR-ESI-MS. NMR spectroscopic data indicated COSY spectrum of the former exhibited the proton chain from
2
5
32
7
that 2 was a similar compound to 1, except for the disap- H -10 to H-11, along with the observation of a hemiketal car-
2
13
pearance of the hemiketal signal, instead of which, two oxy- bon in the C-NMR spectrum, and that of the latter one from
methine signals (δH 5.14 on δC 78.4 and 4.53 on 72.7) were H-9 to H -11 through H -10, their structures were established
2
2
observed. Since these oxymethine protons were correlated to be as shown in Fig. 2. A similar coupling pattern for H-11
1
1
in the H– H COSY spectrum through methylene protons on in 3 to that of 2 placed the hydroxy group at the 11-position
C-10, the planar structure of 2 was established to be as shown in a β-orientation and positive Cotton effects at 252nm (Δε
in Fig. 2. To confirm this, X-ray crystallographic analysis of +4.47, +1.17, respectively) substantiated that the absolute con-
2
was performed and the relative stereostructure of 2 was figurations of the diterpene moieties were the same as those
established to be that of a derivative of crotofolane-type of aforementioned compounds. The absolute configuration of
9)
diterpenoid (Fig. 5). The positive Cotton effect observable 2-methylbutanoic acid in 3 and 4 must also be the same as
13
in the CD spectrum [Δε +1.36 (249)] indicated the absolute that in 1 and 2, judging from the C-NMR chemical shifts of

4
14
Vol. 61, No. 4
13
Table 2.
C
C-NMR Spectroscopic Data for Crotocascarins A–H (1–8), and α and β (9, 10) (100MHz, CDCl3)
1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
74.6
33.3
36.9
60.4
57.9
56.5
44.3
159.0
107.5
41.9
34.8
146.8
39.5
68.9
130.4
170.8
9.6
115.1
12.7
20.2
175.4
41.2
26.7
11.7
17.2
75.8
32.7
36.4
60.1
57.8
55.9
44.4
162.0
78.4
44.1
72.7
148.9
31.7
68.8
128.2
173.4
9.7
115.2
12.3
19.3
178.0
41.2
26.6
11.4
16.2
76.6
32.5
36.3
60.2
57.9
56.5
44.4
158.6
106.4
45.7
73.7
148.4
31.8
68.8
129.8
170.3
9.6
116.3
12.2
19.7
178.8
41.4
26.6
11.5
16.6
74.5
33.2
36.9
60.5
57.6
56.1
44.1
161.7
82.2
37.7
36.2
146.0
40.7
68.7
128.5
173.0
9.6
115.2
12.7
19.5
175.2
41.2
26.8
11.7
17.1
75.4
32.6
36.4
60.2
58.0
56.4
44.7
159.0
109.5
40.4
73.7
149.3
31.4
68.8
129.9
170.7
9.7
115.9
12.3
20.1
178.1
41.5
26.5
11.5
16.5
52.8
74.6
33.4
36.9
60.4
57.8
56.4
44.2
158.7
107.2
42.0
34.8
146.6
39.5
68.8
130.6
170.5
9.7
115.1
12.5
20.3
175.6
34.2
19.3
19.4
76.7
32.8
36.3
60.1
57.7
55.9
44.2
161.9
78.3
44.1
72.6
148.7
31.7
68.8
128.2
173.3
9.7
115.2
12.1
19.3
178.1
34.3
19.3
18.7
76.3
32.5
36.3
60.2
57.9
56.5
44.4
158.5
106.4
45.8
73.7
148.3
31.6
68.7
129.9
170.2
9.6
116.3
12.1
19.7
179.0
34.4
19.2
18.9
75.5
34.4
34.3
64.8
75.4
88.0
48.0
60.1
174.7
35.8
67.9
146.1
32.4
66.0
202.0
75.7
34.5
34.3
65.2
75.6
87.9
47.4
62.2
175.1
27.1
27.2
141.0
34.7
65.9
201.7
1
1
1
1
1
1
1
1
1
1
2
26.0
113.8
12.6
22.0
175.5
41.2
26.6
11.8
16.9
25.4
110.4
12.7
22.1
175.0
41.6
26.6
11.7
17.3
1
2
3
4
5
′
′
′
′
′
–
OCH3
the acid moiety and the five-membered ring.
2
5
Crotocascarin E (5), [α]D +95.2, was isolated as an amor-
phous powder and its elemental composition was determined
to be C H O by HR-ESI-MS. The NMR spectroscopic data
2
6
34
8
were essentially the same as those of crotocascarin C (Tables
, 2), except for the presence of a methoxy signal [δH 3.57
1
(
3H, s)], which crossed the ketal carbon one (δ 109.5) in the
C
HMBC spectrum. Therefore, the structure of 5 was assigned,
as shown in Fig. 1. The methoxy derivative is probably an
artifact formed during the extraction and isolation processes.
2
5
24
Crotocascarins F (6), [α]D +16.8, and G (7), [α]D +81.7,
were isolated as amorphous powders, and crotocascarin H (8),
2
4
[
α] +94.2, as colorless needles, and their elemental composi-
tions were determined to be C H O , C H O and C H O ,
D
2
4
30
7
24 30
7
24 30
8
13
respectively, by HR-ESI-MS. The C-NMR spectral data for
their diterpeneoid regions were essentially superimposable on
those of crotocascarins A (1), B (2), and C (3), respectively.
The common acyl moiety of these diterpenoids comprised
four carbons, i.e., two doublet methyls, one methine, whose
proton was coupled as a septet, and a carbonyl carbon. There- Fig. 5. ORTEP Drawing of 2
fore, the structure of the acyl moiety was expected to be
The crystallization solvent is omitted from the structure.
isobutanoic acid and the gross structures of 6–8 were shown
to be as in Fig. 1. The absolute configurations of 6, 7 and 8 indicated the presence of 2-methylbutanoic acid as an acyl
were expected to be the same as those of crotocascarins A (1), substituent, thus the terpenoid region comprised 19 carbons,
B (2), and C (3), respectively, from similar respective optical i.e., three methyls, two methylenes, three oxymethines, three
rotation values and similar positive Cotton effect, [6: +4.66 methines, three oxygenated tertiary carbons, one quaternary
(
251), 7: +1.41 (248) and 8: +8.73 (252), respectively].
carbon, an exomethylene moiety and two carbonyl carbons.
Crotocascarin α (9), [α]D +78.7, was isolated as colorless The presence of the 2-methylbutanoic acid moiety and other
plates and its elemental composition was determined to be functionalities, observed in one-dimensional NMR spectra,
2
6
C H O by HR-ESI-MS. In the IR spectrum, absorption implied that crotocascarin α (9) was a compound related to
2
4
32
8
−1
bands for carbonyl groups (1761, 1721cm ) and a double crotofolanes. Since extensive examination of two-dimensional
bond (1634cm ) were observed. The NMR spectroscopic data NMR data unfortunately did not lead to a structure which
−1

April 2013
415
satisfied all the spectroscopic data, an attempt was made to amorphous powder and its elemental composition was deter-
solve the structure by X-ray crystallographic analysis and an mined to be C H O , which was one oxygen atom less than
2
4
32
7
9)
ORTEP drawing of 9 is presented in Fig. 6. Crotocascarin α that of 9. The NMR spectroscopic data indicated that croto-
9) has a new skeleton and was probably derived from some cascarin β (10) was a congeneric compound to 9 with three
(
crotofolane, like crotocascarin B (2), through several steps, methylene carbons. One of the oxymethine protons at δ 4.17,
H
such as decarboxylation, C–C bond migration, oxidation, etc. which was observed in the NMR spectrum of 9 was obviously
(
Fig. 7). The absolute configuration of the 2′-position was replaced by methylene protons, with H-1 and H-5 remaining
determined to be S by the same method used for 1 and 2. intact. Therefore, the structure of 10 was elucidated to be as
Therefore, the structure of 9 is shown in Fig. 2, including the shown in Fig. 2, namely 11-deoxycrotocascarin α. The abso-
absolute one.
lute configuration of 2-methylbutanoic acid must also be the
2
3
13
Crotocascarin β (10), [α]D +35.0, was isolated as an same as that in 1, 2 and 8, judging from the C-NMR chemi-
cal shifts of the acid moiety and the five-membered ring.
Only seven crotofolane-type diterpenoids have been isolated
1,2)
so far, four from Jamaican C. corylifolius, two from Kenyan
3)
4)
C. dichogamus, and one from Congolese C. haumanianus.
In these studies, without exception, the authors used an
X-ray crystallographic method to come to a the final conclu-
sion as to the relative structure. In this investigation on C.
cascarilloides, a series of crotofolanes was isolated and two
nor-diterpenes having a new skeleton probably derived from
a crotofolane through the postulated biosynthetic scheme in
Fig. 7. The structures of 1 and 2 were solved by X-ray crystal-
lography using a direct method, and assignment of the abso-
lute configuration of the acyl moiety, 2-methylbutanoic acid,
obtained on chemical degradation of crotocascarins A (1) and
B (2) as S provided information on the absolute structure of a
crotofolane for the first time.
Experimental
Fig. 6. ORTEP Drawing of 9
General Melting points were measured on a Yanagimoto
Fig. 7. Possible Biosynthetic Pathway from 2 to 9

4
16
Vol. 61, No. 4
micro melting point apparatus and are uncorrected. Optical From fractions 140–151, 10.5mg of 8 was obtained in a crys-
rotations were measured on a JASCO P-1030 digital polar- talline state. The residue (12.2mg) in fractions 152–151 was
imeter. IR and UV spectra were measured on Horiba FT-710 purified by HPLC [Inertsil (Ph-3), H O–MeOH, 1:1; 1.6mL/
2
and JASCO V-520 UV/Vis spectrophotometers, respectively. min] to afford a further amount (5.1mg) of 8 from the peak at
1
13
H- and C-NMR spectra were taken on a JEOL JNM α-400 41 min.
at 400MHz and 100MHz with tetramethylsilane as an inter-
The residue (155mg) in fractions 73–85 obtained on the
nal standard. CD spectra were obtained with a JASCO J-720 second run of ODS open CC was again subjected to silica
spectropolarimeter. Positive-ion HR-ESI-MS was performed gel CC (Φ=10mm, L=40cm) with n-hexane (250mL)→n-
with an Applied Biosystems QSTAR XL NanoSpray™ Sys- hexane–EtOAc (1:1, 250mL), and then n-hexane–EtOAc
tem. Silica gel column chromatography (CC) was performed (1:1, 250mL). Fractions of 2mL were collected. The residue
on Kiesel Gel (silica gel 60) (70–230 mesh) (E. Merck, (2.3mg) in fractions 106–113 was purified by HPLC (H O–
2
Darmstadt, Germany) and reversed-phase octadecylsilanized MeOH, 2:3) to give 0.7mg of 10 from the peak at 21min. The
(
ODS) open CC on Cosmosil 75C -OPN (Nacalai Tesque, residue (64.9mg) in fractions 124–134 was purified by HPLC
18
Kyoto, Japan) (Φ=50mm, L=25cm). HPLC was performed (H O–MeOH, 3:7) to yield 8.6mg of 6 and 5.4mg of 2 from
2
on an ODS column (Inertsil ODS-3; GL Science, Tokyo, the peaks at 10min and 11min. respectively. From fractions
Japan; Φ=6mm, L=25cm, 1.6mL/min), and the eluate was 135–147, 2.5mg of 3 was obtained in a crystalline state.
monitored with UV (210nm) and refractive index monitors.
The residue (126mg) in fractions 86–95 obtained on the
(
S)-(+)-2-Methylbutanoic acid was purchased from Wako Pure second run of ODS open CC was again subjected to silica
gel CC (Φ=10mm, L=40cm) with n-hexane (250mL)→n-
Plant Material Stems of C. cascarilloides were collected hexane–EtOAc (1:1, 250mL), and then n-hexane–EtOAc
Chemical Industries, Ltd. (Osaka, Japan).
at Okinawa in June 2004, and a voucher specimen was depos- (1:1, 250mL), fractions of 2mL being collected. The residue
ited in the Herbarium of the Department of Pharmacognosy, (13.1mg) in fractions 114–123 was purified by HPLC (H O–
2
Graduate School of Biomedical and Health Sciences, Hiro- MeOH, 3:7) to give 6.5mg of 1 and 2.9mg of 4 from the
shima University (04-CC-Okinawa-0628).
Extraction and Isolation Stems (14.5kg) of C. cascaril-
peaks at 14min and 15min, respectively.
Crotocascarin (1): Colorless rods (MeOH), mp
A
2
6
−1
loides were extracted with MeOH (15L×3) for a week at 220–221°C, [α]D +16.4 (c=0.95, CHCl ); IR ν
(KBr) cm :
3
max
2
5°C. The combined extract was concentrated to 6L and then 3399, 2966, 2930, 1763, 1739, 1650, 1456, 1180, 1146, 1018,
partitioned with n-hexane (6L, n-hexane extract: 92.1g). The 802; UV λ
(MeOH) nm (logε): 223sh (3.87), 208 (4.21);
max
1
13
methanolic layer was concentrated and the resulting residue
H-NMR (400MHz, CDCl ): Table 1; C-NMR (100MHz,
3
was suspended in 6L of H O. The H O layer was partitioned CDCl ): Table 1; CD Δε (nm): +3.41 (251), −8.39 (224)
2
2
3
–
5
with 6L each of CH Cl , EtOAc and 1-BuOH to give 39.1g, (c=2.02×10 m, MeOH); HR-ESI-MS (positive-ion mode) m/z:
0.5g and 52.2g of the respective residues.
The residue (39.1g) of the CH Cl -soluble fraction was
subjected to silica gel CC (400g) (Φ=60mm, L=30cm) with 152–153°C, [α] +81.8 (c=1.52, CHCl ); IR (KBr) ν
CHCl3 (5L), CHCl –MeOH (15:1, 7L and 12:1, 5L), and cm : 3478, 2972, 2929, 1769, 1739, 1659, 1457, 1185, 1143,
MeOH (2L). Fractions of 500mL were collected. The residue 1014, 804; UV (MeOH) λmax nm (logε): 218 (4.00); H-NMR
3.11g) in fraction 14 was separated by two runs of ODS open (CDCl , 400MHz): Table 1; C-NMR (CDCl , 100MHz):
CC [H O–MeOH (1:1, 1L)→(1:9, 1L) and then H O–MeOH Table 2; CD Δε (nm): +1.36 (249), −1.27 (210) (c 4.31×10 m,
1:9, 250mL)→MeOH (250mL)], fractions of 10g being col- MeOH); HR-ESI-MS (positive-ion mode) m/z: 467.2017 [M+
lected. The residue (116mg) in fractions 137–152 obtained on Na] (Calcd for C H O Na: 467.2040).
2
2
+
1
467.2046 [M+Na] (Calcd for C H O Na: 467.2040).
Crotocascarin
2
5
32
7
B (2): Colorless plates (2-PrOH), mp
2
2
2
6
D
3
max
−
1
3
1
13
(
3
3
–
5
2
2
(
+
2
5
32
7
the first run of ODS open CC was again subjected to silica
Crotocascarin
C
(3): Colorless plates (MeOH), mp
2
4
gel CC (Φ=10mm, L=40cm) with n-hexane–EtOAc [(9:1, 203–205°C, [α] +88.9 (c=0.82, CHCl ); IR (KBr) ν
D
3
max
–1
100mL), (17:3, 100mL), (4:1, 100mL), (7:3, 100mL), (3:2, cm : 3448, 2974, 2938, 1759, 1734, 1649, 1140, 1081, 877;
1
1
00mL) and (1:1, 100mL)], and EtOAc (100mL). Fractions of UV (MeOH) λmax nm (logε): 214 (3.85); H-NMR (CDCl ,
3
13
2
mL were collected, and 3.1mg of 5 was obtained in fractions 400MHz): Table 1; C-NMR (CDCl , 100MHz): Table 2; CD
3
–
5
135–144.
Δε (nm): +4.47 (252), −10.61 (226) (c=1.78×10 m, MeOH);
+
The residue (166mg) in fractions 45–58 obtained on the HR-ESI-MS (positive-ion mode) m/z: 483.1985 [M+Na]
second run of ODS open CC was again subjected to silica (Calcd for C H O Na: 483.1989).
2
5
32
8
2
4
gel CC (Φ=10mm, L=40cm) with n-hexane (250mL) →
Crotocascarin D (4): Amorphous powder, [α]
+2.6
D
−1
n-hexane–EtOAc (1:1, 250mL), and then n-hexane–EtOAc (c=0.19, CHCl ); IR (KBr) νmax cm : 3463, 2972, 2932,
3
(
1:1, 250mL). Fractions of 2mL were collected. The residue 1794, 1748, 1651, 1457, 1161, 1112, 1062, 903; UV (MeOH)
1
(
16.4mg) in fractions 270–290 was finally purified by HPLC λmax nm (logε): 219 (4.00); H-NMR (CDCl , 400MHz):
3
13
(
3
H O–MeOH, 1:1) to give 3.5mg of 9 from the peak at Table 1; C-NMR (CDCl , 100MHz): Table 2; CD Δε (nm):
2
3
–
5
5 min.
+1.17 (252), −3.21 (212) (c=2.26×10 m, MeOH); HR-ESI-
+
The residue (101mg) in fractions 64–72 obtained on the MS (positive-ion mode) m/z: 451.2085 [M+Na] (Calcd for
second run of ODS open CC was again subjected to silica C H O Na: 451.2091).
2
5
32
6
2
5
gel CC (Φ=10mm, L=40cm) with n-hexane (250mL)→n-
Crotocascarin E (5): Amorphous powder, [α]
+95.2
D
−1
hexane–EtOAc (1:1, 250mL), and then n-hexane–EtOAc (c=0.15, CHCl ); IR (KBr) νmax cm : 3480, 2970, 2934,
3
(1:1, 250mL). Fractions of 2mL were collected. The residue 1767, 1739, 1457, 1190, 1139, 1085, 802; UV (MeOH) λmax
1
(
22.2mg) in fractions 123–139 was purified by HPLC (H O– nm (logε): 216 (3.92); H-NMR (CDCl , 400MHz): Table 1;
2
3
13
MeOH, 1:1) to give 12.3mg of 7 from the peak at 76min.
C-NMR (CDCl , 100MHz): Table 2; CD Δε (nm): +4.23
3

April 2013
417
–
5
(
(
251), −12.68 (224) (c=3.06×10 m, MeOH); HR-ESI-MS group: orthorhombic, P2 2 2 , T=120K, a=10.1775(10) Å,
1
1 1
+
3
positive-ion mode) m/z: 497.2129 [M+Na] (Calcd for b=10.4348(10)Å, c=25.908(3)Å, V=2751.5(5) Å , Z=4, D =
c
3
C H O Na: 497.2145).
1.218 Mg/m , F(000)=1088. Of the 13566 reflections col-
2
6
34
8
2
5
Crotocascarin F (6): Amorphous powder, [α]
c=0.22, CHCl ); IR (KBr) νmax cm : 3466, 2973, 2932, 1765, 0.0224), data/restraints/parameters 3212/0/334. The structure
739, 1651, 1459, 1337, 1191, 1154, 1066, 895; UV (MeOH) was solved in a similar manner to as that for compound 1.
λmax nm (logε): 219 (3.88); H-NMR (CDCl , 400MHz): Final goodness-of-fit on F =1.048, R =0.0344, wR =0.0868
Table 1; C-NMR (CDCl , 100MHz): Table 2; CD Δε (nm): based on I>2σ(I), and R =0.0378, wR =0.0891 based on all
4.66 (251), −10.18 (225) (c=2.56×10 m, MeOH); HR-ESI- data. The largest difference peak and hole were 0.335 and
+16.8 lected in the range of 3.14<2θ<53.4, 3212 were unique (R =
D
int
−
1
(
1
3
1
2
3
1
2
13
3
1
2
–
5
+
+ −3
MS (positive-ion mode) m/z: 453.1897 [M+Na] (Calcd for −0.228eÅ , respectively.
C H O Na: 453.1889). Alkaline Hydrolysis of Crotocascarins A (1), B (2) and
+81.7 α (8) Crotocascarins A (1) (2.3mg), B (2) (2.5mg) and α
c=0.82, CHCl ); IR (KBr) νmax cm : 3436, 1741, 1634, 1459, (2.0mg), and authentic (S)-(+)-2-methylbutyric acid were
2
4
30
7
2
4
Crotocascarin G (7): Amorphous powder, [α]
D
−
1
(
3
1
195, 1157, 1072, 887; UV (MeOH) λmax nm (logε): 220 (4.08); (500 µL) each dissolved in 1mL of a 1:1 mixture of 10%
H-NMR (CDCl , 400MHz): Table 1; C-NMR (CDCl3, KOH in H O and 50% aqueous dioxane, and then heated for
1
13
3
2
1
00MHz): Table 2; CD Δε (nm): +1.41 (248), −4.22 (212) 3h at 100°C. The cooled reaction mixtures were neutralized
−
5
+
(
4
c=1.91×10 m, MeOH); HR-ESI-MS (positive-ion mode) m/z: with Amberlite IR-120B (H ) and then the filtrates were evap-
53.1888 [M+Na] (Calcd for C H O Na: 453.1883).
+
orated. The four residues were analyzed by HPLC (column:
2
4
30
7
Crotocascarin
H (8): Colorless needles (CHCl3), mp Inertsil ODS-3, 6mm×250mm; solvent: 20% acetonitrile in
2
4
−1
2
3
8
4
42–244°C, [α] +94.2 (c=0.33, CHCl ); IR (KBr) νmax cm : H O containing 0.5% trifluoroacetic acid; flow rate: 1.6mL/
D 3 2
440, 2978, 2938, 1760, 1739, 1648, 1444, 1183, 1141, 1082, min) with a chiral detector (JASCO OR-2090plus) to give a
80; UV (MeOH) λ nm (logε): 214 (4.16); H-NMR (CDCl , peak of (S)-(+)-2-methylbutyric acid at 17.5min with a posi-
1
max
3
1
3
00MHz): Table 1; C-NMR (CDCl , 100MHz): Table 2; CD tive optical rotation sign.
3
–
5
Δε (nm): +8.73 (252), −23.36 (226) (c=1.47×10 m, MeOH);
X-Ray Crystallographic Analysis of Crotocascarin α
+
3
HR-ESI-MS (positive-ion mode) m/z: 469.1831 [M+Na]
(10) C H O , M=448.50, crystal size: 0.30×0.15×0.15 mm ,
2
4
32
8
(Calcd for C H O Na: 469.1832).
space group: monoclinic, P2 , T=120K, a=9.9294(12)Å,
2
4
30
8
1
Crotocascarin
α
(9): Colorless plates (CHCl3), mp b=9.1267(11)Å, c=12.5443(15)Å, β=98.650(1)°, V=1123.9(2)
2
6
−1
3
3
2
02–203°C, [α] +78.7 (c=0.13, CHCl ); IR (KBr) ν
cm : Å , Z=2, D =1.325 Mg/m , F(000)=1088. Of the 5560 reflec-
D
3
max c
1
3
(
479, 2968, 2926, 1761, 1721, 1634, 1461, 1193, 804; H-NMR tions collected in the range of 3.28<2θ<54.1, 2416 were
13
CDCl , 400MHz): Table 1; C-NMR (CDCl , 100MHz): unique (R =0.0154), data/restraints/parameters 2416/1/296.
3
3
i
n
t
Table 2; HR-ESI-MS (positive-ion mode) m/z: 471.1973 [M+ The structure was solved in a similar manner to as that for
+
2
Na] (Calcd for C H O Na: 471.1989).
compound 1. Final goodness-of-fit on F =1.056, R =0.0315,
2
4
32
8
1
2
3
Crotocascarin β (10): Amorphous powder, [α] +35.0 wR =0.0794 based on I>2σ(I), and R =0.0335, wR =0.0809
c=0.04, CHCl ); IR (KBr) ν
654, 1460, 1162, 889; H-NMR (CDCl , 400MHz): Table 1; 0.285 and −0.208eÅ , respectively.
C-NMR (CDCl , 100MHz): Table 2; HR-ESI-MS (positive-
ion mode) m/z: 455.2044 [M+Na] (Calcd for C H O Na: graphic data for 1 (CCDC 894968), 2 (CCDC 761004), and
55.2040).
D
2
1
2
−
1
(
cm : 3467, 2926, 1741, 1714, based on all data. The largest difference peak and hole were
3
max
1
−3
1
3
13
Supplementary Data Supplementary X-ray crystallo-
3
+
2
4
32
7
4
10 (CCDC 761005) can be obtained free of charge via www.
X-Ray Ccrystallographic Analysis of Crotocascarin A ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
3
(
1) C H O , M=444.51, crystal size: 0.38×0.20×0.10 mm , Crystallographic Data Centre, 12 Union Road, Cambridge
25 32 7
space group: orthorhombic, P2 2 2 , T=90K, a=6.1970(15)Å, CB2 1EZ, U.K.; fax: (+44) 1223–336–033; or deposit@ccdc.
1
1 1
3
b=15.124(4)Å,
c=25.145(6)Å,
V=2356.6(10)Å ,
Z=4, cam.ac.uk).
3
D =1.253 Mg/m , F(000)=952. The data were measured using
c
a Bruker SMART 1000 CCD diffractometer, using MoKα
Acknowledgements The authors are grateful for access to
graphite-monochromated radiation (λ=0.71073Å) in the the superconducting NMR instrument (JEOL JNM α-400) at
range of 3.04<2θ<56.7. Of the 14241 reflections collected, the Analytical Center of Molecular Medicine of the Hiroshima
5
548 were unique (R =0.0421, data/restraints/parameters University Faculty of Medicine, and an Applied Biosystem
int
5548/0/298. The structure was solved by a direct method using QSTAR XL system ESI (Nano Spray)-MS at the Analysis
10)
the program SHELXTL-97. The refinement and all further Center of Life Science of the Graduate School of Biomedical
calculations were carried out using SHELXTL-97. The Sciences, Hiroshima University. This work was supported in
11)
absorption correction was carried out utilizing the SADABS part by Grants-in-Aid from the Ministry of Education, Cul-
10)
routine. The H atoms were included at the calculated posi- ture, Sports, Science and Technology of Japan, and the Japan
tions and treated as riding atoms using the SHELXTL default Society for the Promotion of Science. Thanks are also due to
parameters. The non-H atoms were refined anisotropically the Research Foundation for Pharmaceutical Sciences and the
2
using weighted full-matrix least-squares on F . Final good- Takeda Science Foundation for the financial support.
2
ness-of-fit on F =1.078, R =0.0408, wR =0.0997 based on
1
2
I>2σ(I), and R =0.0496, wR =0.1114 based on all data. The References
1
2
−3
largest difference peak and hole were 0.645 and −0.402eÅ ,
respectively.
1) Chan W. R., Prince E. C., Manchand P. S., Springer J. P., Clardy J.,
J. Am. Chem. Soc., 97, 4437–4439 (1975).
2)
Burke B. A., Chan W. R., Pascoe K. O., Blout J. F., Manchand P. S.,
Tetrahedron Lett., 20, 3345–3348 (1979).
X-Ray Crystallographic Analysis of Crotocascarin B (2)
3
C H O , M=504.60, crystal size: 0.50×0.30×0.15 mm , space
2
8
40
8

4
18
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3
)
Jogia M. K., Andersen R. A., Párkányi L., Clardy J., Dublin H. T.,
Sinclair A. R. E., J. Org. Chem., 54, 1654–1657 (1989).
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Snatzke G., Angew. Chem., 7, 14–25 (1968).
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Kawahata M., Yamaguchi K., Tetrahedron Lett., 51, 4320–4322
(2010).
10) Sheldrick G. M., Acta Crystallogr. A, 64, 112–122 (2008).
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1996.
4)
5
6
7
)
)
)