New farnesane-type sesquiterpenes, hedychiols A and B 8,9-diacetate, and inhibitors of degranulation in RBL-2H3 cells from the rhizome of Hedychium coronarium

Article abstract of DOI:10.1248/cpb.50.1045

Two new farnesane-type sesquiterpenes, hedychiols A and B 8,9-diacetate, were isolated from the methanolic extract of the fresh rhizome of Hedychium coronarium Koen. cultivated in Japan. Their stereostructures were elucidated on the basis of chemical and

Full text of DOI:10.1248/cpb.50.1045

August 2002
Chem. Pharm. Bull. 50(8) 1045—1049 (2002)
1045
New Farnesane-Type Sesquiterpenes, Hedychiols A and B 8,9-Diacetate,
and Inhibitors of Degranulation in RBL-2H3 Cells from the Rhizome of
Hedychium coronarium
Toshio MORIKAWA, Hisashi MATSUDA, Yasuko SAKAMOTO, Kazuho UEDA, and Masayuki YOSHIKAWA*
Kyoto Pharmaceutical University; Misasagi, Yamashina-ku, Kyoto 607–8412, Japan.
Received February 25, 2002; accepted April 8, 2002
Two new farnesane-type sesquiterpenes, hedychiols A and B 8,9-diacetate, were isolated from the methano-
lic extract of the fresh rhizome of Hedychium coronarium KOEN. cultivated in Japan. Their stereostructures were
elucidated on the basis of chemical and physicochemical evidence. The inhibitory effects of isolated constituents
on the release of b-hexosaminidase from RBL-2H3 cells were examined, and hedychilactone A and coronarin D
were found to show the inhibitory activity.
Key words hedychiol; Hedychium coronarium; degranulation inhibitor; hedychilactone A; coronarin D; Zingiberaceae
Hedychium coronarium KOEN. (Zingiberaceae), which has Furthermore, we also examined the inhibitory effects of
many common names such as butterfly ginger, butterfly lily, the isolated constituents on nitric oxide (NO) production in
cinnamon jasmine, garland flower, and ginger lily, is widely lipopolysaccharide (LPS)-activated mouse peritoneal macro-
cultivated in India, Southeast Asian countries, South China, phages.1,2) Among them, labdane-type diterpenes, including
Japan, and Brazil. The rhizomes of H. coronarium (“土羌活” 3 and 6, showed potent inhibitory activity, and one of the
in Chinese, Hanashukusha in Japanese) have been used for mechanisms of action of NO production inhibitory activity
the treatment of headache, lancinating pain, contusion in- was suggested to be attributed to inhibitory activity against
flammation and sharp pain due to rheumatism in Chinese tra- the induction of inducible nitric oxide synthase (iNOS) in
ditional medicine. In the course of characterization studies LPS-activated macrophages.2)
on the bioactive constituents of Zingiberaceae natural medi-
As a continuing study of this natural medicine, we addi-
cines,1—12) we have already reported that three new labdane- tionally isolated two new farnesane-type sesquiterpenes,
type diterpene lactones, hedychilactones A (3), B (4), and hedychiols A (1) and B 8,9-diacetate (2), from the methano-
C (5), were isolated from the methanolic extract of the lic extract of the fresh rhizome of H. coronarium. This
fresh rhizome of Japanese H. coronarium.1,2) In addition, the paper deals with the structural elucidation of farnesane-type
methanolic extract, coronarin D (6), which was obtained as a sesquiterpenes (1, 2). In addition, we have described the in-
principal labdane-type diterpene, and its methyl ether (7) hibitory effect of isolated constituents from the fresh rhizome
were found to show an inhibitory effect on the increase in of H. coronarium on an immediate allergic reaction by moni-
vascular permeability induced by acetic acid in mice.1,2) toring the release of b-hexosaminidase from rat basophilic
Chart 1
To whom correspondence should be addressed. e-mail: shoyaku@mb.kyoto-phu.ac.jp
© 2002 Pharmaceutical Society of Japan

1046
Vol. 50, No. 8
leukemia (RBL-2H3) cells.
9-H2) and three quaternary carbons (3, 7, 11-C), which
Isolation of Chemical Constituents from the Fresh Rhi- were superimporsable on those of (ϩ)-nerolidol (12), expect
zome of H. coronarium The methanolic extract from the for a signal due to the secondary hydroxyl group. In addition,
1
fresh rhizome of H. coronarium was partitioned into a mix- the connectivity of the H–1H and the quaternary carbons
ture of ethyl acetate (AcOEt) and water to furnish the in 1 was clarified by 1H–1H correlation spectroscopy
AcOEt-soluble portion and H2O-soluble portion, as de- (H–H COSY) and heteronuclear multiple bond correlation
scribed.2) The AcOEt-soluble fraction was subjected to ordi- (HMBC) experiments. Thus, the H–H COSY experiment on
nary- and reversed-phase silica gel column chromatography, 1 indicated the presence of the partial structures shown as
and finally HPLC, to furnish two farnesane-type sesquiter- bold lines in Fig. 1 (1-C–2-C, 4-C–6-C, 8-C–10-C). In the
penes, hedychiols A (1, 0.00084% from the fresh rhizome) HMBC experiment, long-range correlations were observed
and B 8,9-diacetate (2, 0.000086%), together with 11 con- between the following protons and carbons of 1 (1-H2 and 2-
stituents including hedychilactones A—C (3—5).
C; 6-H and 8-C; 8-H and 6-C, 10-C; 12-H3 and 10-C, 11-C,
Stereostructures of Hedychiols A (1) and B 8,9-Diace- 13-C; 13-H3 and 10-C, 11-C, 12-C; 14-H3 and 6-C, 7-C, 8-C;
tate (2) Hedychiol A (1) was isolated as a colorless oil 15-H3 and 1-C, 3-C, 4-C), so that the connectivities of the
with negative optical rotation ([a]D25 Ϫ2.4°). The IR spec- quaternary carbons (3, 7, 11-C) in 1 were clarified. The
trum of 1 showed absorption bands due to hydroxyl and exo- above-mentioned evidence led us to confirm the planar struc-
methylene functions at 3372 and 1375 cmϪ1. The molecular ture of 1.
formula C15H26O2 of 1 was determined from the quasimolec-
Next, the absolute stereostructure of the 8-position of 1
ular ion peak at m/z 261 (MϩNa)ϩ in the positive-ion fast was determined by application of modified Mosher’s
atom bombardment (FAB)-MS and by high-resolution MS method13) to the 8-mono-(R)- and (S)-2-methoxy-2-trifluo-
1
measurement. The H-NMR (CDCl3) and 13C-NMR spectra romethylphenylacetate (MTPA esters, 1a, 1b), which were
(Table 1) of 1 showed signals assignable to four tertiary prepared by esterification of the 8-hydroxyl group in 1
methyls [d 1.29, 1.62, 1.63, 1.71 (all s, 15, 14, 13, 12-H3)], a with (R)- and (S)-2-methoxy-2-trifluoromethylphenylacetic
methine bearing a hydroxyl group [d 3.97 (dd, Jϭ7.3, acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-
7.3 Hz, 8-H)], an exo-methylene [d 5.06 (dd, Jϭ0.9, 10.7 carbodiimide (EDC·HCl) and 4-dimethylaminopyridine (4-
Hz), 5.22 (dd, Jϭ0.9, 17.4 Hz), 1-H2], and three olefins [d DMAP). As shown in Fig. 1, signals due to protons at C-4, 5,
5.08 (dd-like, 10-H), 5.40 (dd, Jϭ7.0, 7.3 Hz, 6-H), 5.91 (dd, 6, and 14 of the (R)-MTPA ester (1a) appeared at higher
Jϭ10.7, 17.4 Hz, 2-H)], together with three methylenes (4, 5, fields than those of the (S)-MTPA ester (1b) [Dd: positive],
while signals due to protons attached to C-9, 10, 12, and 13
of 1a were observed at a lower field as compared to those of
1b [Dd: negative]. Consequently, the absolute configuration
at the 8-position in 1 has been determined to be S.
Table 1. 13C-NMR Data for Hedychiols A (1) and B 8,9-Diacetate (2)
1
2
To elucidate the absolute stereostructure of the 3-position,
1 was chemically related to (ϩ)-nerolidol (12), whose ab-
solute configuration has been reported to be an S-configura-
tion.14) As shown in Fig. 2, ozone degradation of 12 in
MeOH followed by treatment with NaBH4 furnished 1,4-pen-
tandiol15) and the triol (12a).16) On the other hand, 12a was
also obtained from 1 by the same procedure. Consequently,
the absolute stereostructure of 1 was determined to be as
shown.
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-11
C-12
C-13
C-14
C-15
CH3CO–
111.8
144.9
73.3
41.8
22.3
126.0
137.1
77.1
111.9
144.8
73.2
41.4
22.4
130.9
130.4
79.9
34.2
70.7
120.2
134.6
25.9
18.0
11.7
119.5
139.8
25.9
18.6
12.7
27.9
21.1
21.1
170.1
170.1
Hedychiol B 8,9-diacetate (2) was isolated as a colorless
oil with negative optical rotation ([a]D21 Ϫ18.8°). The molec-
ular formula C19H30O5 of 2 was determined from the quasi-
molecular ion peak at m/z 361 (MϩNa)ϩ in the positive-ion
FAB-MS and by high-resolution MS measurement. The IR
spectrum of 2 showed absorption bands of hydroxyl, olefin
and exo-methylene functions at 3459, 1740, and 1374 cmϪ1.
27.8
CH3CO–
1
The H-NMR (CDCl3) and 13C-NMR (Table 1) spectra of 2
Measured in CDCl3, at 125 MHz.
showed signals assignable to four tertiary methyls [d 1.27,
Fig. 1

August 2002
1047
Fig. 2
Fig. 3
1.59, 1.70, 1.74 (all s, 15, 14, 13, 12-H3)], two acetyl methyls 12a was also obtained by ozone oxidation of 2a, followed by
[d 2.01, 2.04 (both s)], three olefins [d 5.00 (br d, Jϭca. NaBH4 reduction. Consequently, the absolute stereostructure
10 Hz, 10-H), 5.49 (dd, Jϭ7.3, 7.3 Hz, 6-H), 5.89 (dd, of the 3-position of 2 was determined to be an S-configura-
Jϭ10.7, 17.4 Hz, 2-H)], an exo-methylene [d 5.06 (dd, tion.
Jϭ1.2, 10.7 Hz), 5.20 (dd, Jϭ1.2, 17.4 Hz), 1-H2)], and two
Inhibitory Effect of Constituents from H. coronarium
methines bearing an acetyl group [d 5.15 (d, Jϭ7.9 Hz, 8-H), on the Release of b-Hexosaminidase from RBL-2H3 Cells
5.72 (dd, Jϭ7.9, 9.5 Hz, 9-H)], together with two methylenes Histamine, which is released from mast cells stimulated by
(4, 5-H2) and three quaternary carbons (3, 7, 11-C). Next, the an antigen or a degranulation inducer, is usually determined
1
connectivities of the H–1H and the quaternary carbons in 2 as a degranulation marker in in vitro experiments on immedi-
were clarified by H–H COSY and HMBC experiments. As ate allergic reactions. b-Hexosaminidase is also stored in the
shown in Fig. 3, the H–H COSY experiment on 2 indicated secretory granules of mast cells, and is released concomi-
the presence of three partial structures shown as bold lines tantly with histamine when mast cells are immunologically
(1-C–2-C, 4-C–6-C, 8-C–10-C), whereas, in the HMBC ex- activated.18,19) Therefore, it is generally accepted that b-hex-
periment, long-range correlations were observed between the osaminidase is a degranulation marker of mast cells. Previ-
following protons and carbons (1-H2 and 2-C; 2-H2 and 3-C; ously, we reported inhibitors of the release of b-hex-
4-H2 and 3-C; 8-H and 7-C, 10-C, 14-C; 12-H3 and 10-C, 11- osaminidase isolated from Myrica rubra.20) In our continuous
C, 13-C; 13-H3 and 10-C, 11-C, 12-C; 14-H3 and 6-C, 7-C, search for antiallergic principles from natural sources, we ex-
8-C; 15-H3 and 3-C, 4-C). Thus, the connectivities of the amined the effects of constituents from the fresh rhizome of
quaternary carbons (3, 7, 11-C) in 2 were clarified.
H. coronarium on the release of b-hexosaminidase induced
Next, the relative stereostructure of the 8 and 9-hydroxyl by dinitrophenylated bovine serum albumin (DNP-BSA)
groups in 2 was clarified by nuclear Overhauser effect spec- from RBL-2H3 cells sensitized with anti-DNP IgE. Two
troscopy (NOESY) experiment on the 8,9-acetonide (2b). antiallergic compounds, tranilast and ketotifen fumarate,
Namely, treatment of 2 with 0.1% sodium methoxide showed weak inhibition with IC50 values of 0.49 and 0.22
(NaOMe) furnished the desacetyl derivative, hedychiol B mM, respectively. On the other hand, luteolin and curcumin,
(2a), which was subsequently treated with 2,2-dimethoxy- which were reported to inhibit degranulation in this experi-
propane to give the 8,9-acetonide (2b). The NOESY experi- mental model,20,21) showed relatively strong inhibition, with
ment on 2b showed NOE correlations between the 8,9-pro- IC50 values of 3.0 and 82 mM, respectively. As shown in Table
tons and acetonide methyls, as shown in Fig. 3, so that the 2, hedychilactone A (3) and coronarin D (6) showed in-
stereostructure of the 8 and 9-positions of 2 was deter- hibitory activity. Especially, 6 was found to show strong ac-
mined.17)
tivity (IC50ϭ57 mM). In addition, hedychenone (10) enhanced
The absolute stereostructure of the 3-position of 2 was de- the release of b-hexosaminidase from RBL-2H3 cells.
termined by a similar method used for 1. As shown in Fig. 2,

1048
Vol. 50, No. 8
Table 2. Inhibitory Effects of Constituents from H. coronarium on the Release of b-Hexosaminidase from RBL-2H3 Cells
Inhibition (%)
0 mM
10 mM
30 mM
100 mM
Hedychiol A (1)
0.0Ϯ3.2
0.0Ϯ1.7
0.0Ϯ1.9
0.0Ϯ5.8
0.0Ϯ2.3
0.0Ϯ1.9
0.0Ϯ2.9
0.0Ϯ2.6
0.0Ϯ7.3
0.0Ϯ2.1
0.0Ϯ3.4
0.0Ϯ3.5
Ϫ1.0Ϯ1.5
0.1Ϯ1.6
Ϫ0.1Ϯ1.7
14.1Ϯ2.2
Ϫ1.8Ϯ1.1
4.6Ϯ1.8
Ϫ3.0Ϯ3.8
Ϫ6.7Ϯ1.9
Ϫ6.4Ϯ4.1
Ϫ37.2Ϯ3.1**
Ϫ4.7Ϯ3.5
Ϫ2.1Ϯ3.5
Ϫ6.4Ϯ2.9
1.5Ϯ1.1
6.0Ϯ2.3
Ϫ3.5Ϯ2.8
11.4Ϯ1.2**
39.1Ϯ2.7**
Ϫ3.5Ϯ2.2
Hedychiol B 8,9-diacetate (2)
Hedychilactone A (3)
Hedychilactone B (4)
Hedychilactone C (5)
Coronarin D (6)
Coronarin D methyl ether (7)
Coronarin E (8)
Labda-8(17),13(14)-dien-15,16-olide (9)
Hedychenone (10)
27.2Ϯ4.2**
Ϫ2.3Ϯ2.7
23.0Ϯ2.7**
Ϫ14.5Ϯ3.3*
Ϫ9.8Ϯ1.4*
Ϫ25.2Ϯ3.9*
Ϫ51.7Ϯ0.2**
Ϫ11.2Ϯ3.4
2.2Ϯ2.6
Ϫ0.5Ϯ2.1
93.5Ϯ0.4**
Ϫ18.2Ϯ2.7**
Ϫ13.8Ϯ2.1**
Ϫ13.3Ϯ4.5
Ϫ45.7Ϯ1.8**
Ϫ17.7Ϯ3.1**
11.8Ϯ1.3*
7-Hydroxyhedychenone (11)
(ϩ)-Nerolidol (12)
Each value represents the meanϮS.E.M. (nϭ4). Significantly different from the control: * pϽ0.05, ** pϽ0.01.
Experimental
treated in the usual manner to give a residue which was purified by silica gel
The following instruments were used to obtain physical data: specific ro- column chromatography (0.2 g, n-hexane–AcOEtϭ10 : 1) to give 1a (3.0 mg,
tations, Horiba SEPA-300 digital polarimeter (lϭ5 cm); UV spectra, Shi- 70%). Through a similar procedure, 1b (3.1 mg, 53%) was prepared from 1
madzu UV-1200 spectrometer; IR spectra, Shimadzu FTIR-8100 spectrome- (3.0 mg, 13.2 mmol) using (S)-MTPA (9.2 mg, 39.5 mmol), EDC·HCl (8.1
ter; electron impact (EI)-MS and high-resolution MS, JEOL JMS-GCMATE mg, 39.5 mmol) and 4-DMAP (3.2 mg, 26.4 mmol).
1
mass spectrometer; FAB-MS and high-resolution MS, JEOL JMS-SX 102A
1a: Colorless oil. H-NMR (CDCl3) d: 1.29, 1.48 (3H each, both s, 15,
mass spectrometer; 1H-NMR spectra, JNM-LA500 (500 MHz) spectrometer; 14-H3), 1.52 (2H, m, 4-H2), 1.59, 1.69 (3H each, both s, 13, 12-H3), 2.04
13C-NMR spectra, JNM-LA500 (125 MHz) spectrometer with tetramethylsi- (2H, m, 5-H2), 2.27 (1H, ddd, Jϭ5.9, 8.0, 14.7 Hz, 9-H), 2.47 (1H, ddd,
lane as an internal standard; HPLC detector, Shimadzu RID-6A refractive Jϭ8.0, 8.2, 14.7 Hz, 9-H), 3.54 (3H, s, –OCH3), 5.03 (1H, dd, Jϭ8.0,
index detector; ozone generator, Nihon Ozone O-3-2 ozone generator.
8.0 Hz, 10-H), 5.08 (1H, dd, Jϭ1.2, 10.6 Hz, 1-H), 5.22 (1H, dd, Jϭ1.2,
The following experimental conditions were used for chromatography: 17.4 Hz, 1-H), 5.32 (1H, dd, Jϭ5.9, 8.2 Hz, 8-H), 5.48 (1H, dd, Jϭ7.9,
ordinary-phase silica gel column chromatography, Silica gel BW-200 7.9 Hz, 6-H), 5.90 (1H, dd, Jϭ10.6, 17.4 Hz, 2-H), 7.36—7.50 (5H, m, Ph).
1
(Fuji Silysia Chemical, Ltd., 150—350 mesh); reversed-phase silica gel col-
1b: Colorless oil. H-NMR (CDCl3) d: 1.29 (3H, s, 15-H3), 1.58 (2H, m,
umn chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, 4-H2), 1.54, 1.62, 1.63 (3H each, all s, 13, 14, 12-H3), 2.07 (2H, m, 5-H2),
Ltd., 100—200 mesh); TLC, pre-coated TLC plates with Silica gel 60F254 2.23, 2.40 (1H each, both m, 9-H2), 3.51 (3H, s, –OCH3), 4.92 (1H, dd,
(Merck, 0.25 mm) (ordinary phase) and Silica gel RP-18 F254S (Merck, Jϭ7.0, 7.3 Hz, 10-H), 5.07 (1H, dd, Jϭ1.2, 10.7 Hz, 1-H), 5.21 (1H, dd,
0.25 mm) (reversed phase); reversed-phase HPTLC, pre-coated TLC plates Jϭ1.2, 17.4 Hz, 1-H), 5.34 (1H, dd, Jϭ5.8, 8.0 Hz, 8-H), 5.55 (1H, dd,
with Silica gel RP-18 WF254S (Merck, 0.25 mm); detection was achieved by Jϭ7.3, 7.3 Hz, 6-H), 5.91 (1H, dd, Jϭ10.7, 17.4 Hz, 2-H), 7.37—7.50 (5H,
spraying with 1% Ce(SO4)2–10% aqueous H2SO4, followed by heating.
Isolation of Hedychiols A (1) and B 8,9-Diacetate (2) Fraction 7
(2.8 g), obtained from the AcOEt-soluble portion of the fresh rhizome of H.
m, Ph).
Deacetylation of Hedychiol B 8,9-Diacetate (2) A solution of 2
(2.9 mg, 8.6 mmol) in 0.1% NaOMe–MeOH (0.8 ml) was stirred at room
coronarium (cultivated in Kagawa Prefecture, Japan), and isolated hedychi- temperature for 4 h. The reaction mixture was neutralized with Dowex HCR-
lactones A—C (3—5), and coronarin D (6), as reported previously,2) were W2 (Hϩ form) and the resin was removed by filtration. Evaporation of the
further separated by reversed-phase silica gel chromatography [84 g, solvent from the filtrate under reduced pressure gave a residue which was
MeOH–H2O (70 : 30→80 : 20→90 : 10)→MeOH] and finally HPLC [YMC- purified by silica gel column chromatography (0.2 g, n-hexane–AcOEtϭ
pack ODS-A 250ϫ20 mm i.d., MeOH–H2O (65 : 35 or 80 : 20)] to furnish 5 : 1) to furnish hedychiol B (2a, 2.1 mg, 96%).
hedychiols A (1, 54 mg) and B 8,9-diacetate (2, 6 mg).
Hedychiol B (2a): Colorless oil. 1H-NMR (CDCl3) d: 1.25 (3H, s, 15-H3),
Hedychiol A (1): Colorless oil, [a]D26 Ϫ2.4° (cϭ0.800, CHCl3). High-res- 1.56 (2H, m, 4-H2), 1.60, 1.68, 1.72 (3H each, all s, 14, 13, 12-H3), 2.07
olution positive-ion FAB-MS: Calcd for C15H26O2Na (MϩNa)ϩ: 261.1839. (2H, m, 5-H2), 3.82 (1H, d, Jϭ7.2 Hz, 8-H), 4.30 (1H, dd, Jϭ7.2, 8.7 Hz, 9-
Found: 261.1830. IR (film): 3372, 2971, 2859, 1375 cmϪ1 1H-NMR H), 5.07 (1H, dd, Jϭ1.2, 10.8 Hz, 1-H), 5.14 (1H, d, Jϭ8.7 Hz, 10-H), 5.21
.
(CDCl3) d: 1.29 (3H, s, 15-H3), 1.59 (2H, m, 4-H2), 1.62, 1.63, 1.71 (3H
each, all s, 14, 13, 12-H3), 2.07 (2H, m, 5-H2), 2.23 (2H, m, 9-H2), 3.97 (1H,
dd, Jϭ7.3, 7.3 Hz, 8-H), 5.06 (1H, dd, Jϭ0.9, 10.7 Hz, 1-H), 5.08 (1H, dd-
(1H, dd, Jϭ1.2, 17.2 Hz, 1-H), 5.46 (1H, dd, Jϭ7.2, 7.2 Hz, 6-H), 5.90 (1H,
dd, Jϭ10.8, 17.2 Hz, 2-H). Positive-ion FAB-MS m/z: 277 (MϩNa)ϩ.
Preparation of the Acetonide (2b) from Hedychiol B (2a) A solution
like, 10-H), 5.22 (1H, dd, Jϭ0.9, 17.4 Hz, 1-H), 5.40 (1H, dd, Jϭ7.0, 7.3 Hz, of 2a (0.8 mg, 3.1 mmol) in 2,2-dimethoxypropane (0.5 ml) was treated with
6-H), 5.91 (1H, dd, Jϭ10.7, 17.4 Hz, 2-H). 13C-NMR (CDCl3) dC: given in Dowex HCR-W2 (Hϩ form, 10 mg), and the mixture was stirred at room
Table 1. Positive-ion FAB-MS m/z: 261 (MϩNa)ϩ.
temperature for 2 h. The resin was removed by filtration. Removal of the sol-
vent from the filtrate under reduced pressure yielded 2b (1.1 mg, quant.).
2b: Colorless oil. 1H-NMR (CDCl3) d: 1.25 (3H, s, 15-H3), 1.43, 1.45 (3H
each, both s, (CH3)2–C–), 1.52 (2H, m, 4-H2), 1.64, 1.65, 1.75 (3H each, all
s, 14, 13, 12-H3), 2.05 (2H, m, 5-H2), 3.97 (1H, d, Jϭ8.5 Hz, 8-H), 4.44 (1H,
Hedychiol B 8,9-Diacetate (2): Colorless oil, [a]D21 Ϫ18.8° (cϭ0.300,
CHCl3). High-resolution EI-MS: Calcd for C19H30O5Na (MϩNa)ϩ:
361.1991. Found : 361.1996. IR (film) 3459, 2971, 2929, 1740, 1374 cmϪ1
.
1H-NMR (CDCl3) d: 1.27 (3H, s, 15-H3), 1.54 (2H, m, 4-H2), 1.59, 1.70,
1.74 (3H each, all s, 14, 13, 12-H3), 2.01, 2.04 (3H each, both s, CH3CO–), dd, Jϭ8.5, 8.9 Hz, 9-H), 5.07 (1H, dd, Jϭ1.2, 10.7 Hz, 1-H), 5.14 (1H, d,
2.03 (2H, m, 5-H2), 5.00 (1H, br d, Jϭca. 10 Hz, 10-H), 5.06 (1H, dd, Jϭ1.2, Jϭ8.9 Hz, 10-H), 5.22 (1H, dd, Jϭ1.2, 17.4 Hz, 1-H), 5.50 (1H, dd, Jϭ7.0,
10.7 Hz, 1-H), 5.15 (1H, d, Jϭ7.9 Hz, 8-H), 5.20 (1H, dd, Jϭ1.2, 17.4 Hz, 1- 7.0 Hz, 6-H), 5.91 (1H, dd, Jϭ10.7, 17.4 Hz, 2-H). Positive-ion FAB-MS
H), 5.49 (1H, dd, Jϭ7.3, 7.3 Hz, 6-H), 5.72 (1H, dd, Jϭ7.9, 9.5 Hz, 9-H), m/z: 317 (MϩNa)ϩ.
5.89 (1H, dd, Jϭ10.7, 17.4 Hz, 2-H). 13C-NMR (CDCl3) dC: given in Table
Conversion from (؉)-Nerolidol (12), and Hedychiols A (1) and B (2a)
to 12a A solution of 12 (25.0 mg, 0.11 mmol) in MeOH (2.0 ml) was oxi-
1. Positive-ion FAB-MS m/z: 361 (MϩNa)ϩ.
Preparation of the (R)-MTPA Ester (1a) and the (S)-MTPA Ester (1b) dized with O3 gas (3.0 g/h) at Ϫ78 °C for 2 h to give ozonide. Successively,
from Hedychiol A (1) A solution of 1 (2.2 mg, 9.7 mmol) in CH2Cl2 the reaction mixture was treated with NaBH4 (5.0 mg) and the mixture was
(1.0 ml) was treated with (R)-MTPA (6.8 mg, 29 mmol) in the presence of stirred at 0 °C for 30 min. The reaction mixture was quenched with acetone
EDC·HCl (6.0 mg, 29 mmol) and 4-DMAP (2.4 mg, 19 mmol), and the mix- (2.0 ml). Removal of the solvent under reduced pressure furnished a residue
ture was stirred under reflux for 6 h. The reaction mixture was poured into which was purified by silica gel column chromatography [1.0 g, CHCl3–
ice-water and the whole was extracted with AcOEt. The AcOEt extract was MeOH–H2Oϭ10 : 3 : 1 (lower layer)] to give 1,4-pentandiol (6.4 mg, 55%)

August 2002
1049
and triol (12a, 2.8 mg, 19%). Compound 12a was identified by comparison
of its physical data ([a]D, MS, 1H-NMR) with reported values.16) Through a
similar procedure, 1 (5.0 mg, 0.021 mmol) and 2a (1.2 mg, 0.005 mmol) in
MeOH (1.0 ml) were treated with O3 gas (3.0 g/h) at Ϫ78 °C for 2 h, and
successively treated with NaBH4 (2.0 mg), then the mixtures were stirred at
0 °C for 30 min, respectively. Work-up of the reaction mixtures, as described
above, gave products which were purified by silica gel column chromatogra-
phy [500 mg, CHCl3–MeOH–H2Oϭ10 : 3 : 1 (lower layer)] to furnish 12a
(2.2 mg from 1, 43%; 0.3 mg from 2a, 47%).
In this condition, it was calculated that 40—50% of b-hexosaminidase
was released from the cells in the control groups by determination of the
total b-hexosaminidase activity after sonication of the cell suspension.
Statistics Values are expressed as meansϮS.E.M. One-way analysis of
variance followed by Dunnett’s test was used for statistical analysis.
References and Notes
1) Matsuda H., Morikawa T., Sakamoto Y., Toguchida I., Yoshikawa M.,
Heterocycles, 56, 45—50 (2002).
Bioassay. Inhibitory Effect on the Release of b-Hexosaminidase
from RBL-2H3 Cells Inhibitory effects of test samples on the release of
b-hexosaminidase from RBL-2H3 cells were evaluated by the following pro-
cedure.21) RBL-2H3 cells were grown in Minimum Essential Medium Eagle
(MEM, Sigma Co., Ltd.) containing fetal calf serum (10%), penicillin (100
units/ml), and streptomycin (100 mg/ml). Before the experiment, cells were
dispensed into 24-well plates at the concentration of 2ϫ105 cells/well using
a medium containing 0.45 mg/ml of anti-DNP IgE, and these were incubated
overnight at 37 °C in 5% CO2 for sensitization of the cells. Then, cells were
washed twice with 500 ml of siraganian buffer [119 mM NaCl, 5 mM KCl,
0.4 mM MgCl2, 25 mM piperazine-N,NЈ-bis(2-ethanesulfonic acid) (PIPES),
40 mM NaOH, pH 7.2], then incubated in 160 ml of siraganian buffer (5.6 mM
glucose, 1 mM CaCl2, 0.1% BSA were added) for an additional 10 min at
37 °C. Next, 20 ml of test sample solution was added to each well and incu-
bated for 10 min, followed by the addition of 20 ml of antigen (DNP-BSA,
final concentration was 10 mg/ml) at 37 °C for 10 min to stimulate the cells
to evoke allergic reactions (degranulation). The reaction was stopped by
cooling in an ice bath for 10 min. The supernatant (50 ml) was transferred
into a 96-well microplate and incubated with 50 ml of substrate (1 mM p-ni-
trophenyl-N-acetyl-b-D-glucosaminide) in 0.1 M citrate buffer (pH 4.5) at
37 °C for 1 h. The reaction was stopped by adding 200 ml of stop solution
(0.1 M Na2CO3/NaHCO3, pH 10.0). The absorbance was measured by a mi-
croplate reader at 405 nm. The test sample was dissolved in dimethylsulfox-
ide (DMSO), and the solution was added to siraganian buffer (final DMSO
concentration was 0.1%).
2) Matsuda H., Morikawa T., Sakamoto Y., Toguchida I., Yoshikawa M.,
Bioorg. Med. Chem., 10, 2527—2534 (2002).
3) Yoshikawa M., Yamaguchi S., Kunimi K., Matsuda H., Okuno Y.,
Yamahara J., Murakami N., Chem. Pharm. Bull., 42, 1226—1230
(1994).
4) Yamahara J., Matsuda H., Yamaguchi S., Shimoda H., Murakami N.,
Yoshikawa M., Natural Medicines, 49, 76—83 (1995).
5) Matsuda H., Ninomiya K., Morikawa T., Yoshikawa M., Bioorg. Med.
Chem. Lett., 8, 339—344 (1998).
6) Yoshikawa M., Murakami T., Morikawa T., Matsuda H., Chem. Pharm.
Bull., 46, 1186—1188 (1998).
7) Matsuda H., Morikawa T., Ninomiya K., Yoshikawa M., Bioorg. Med.
Chem., 9, 909—916 (2001).
8) Matsuda H., Morikawa T., Toguchida I., Ninomiya K., Yoshikawa M.,
Heterocycles, 55, 841—846 (2001).
9) Matsuda H., Morikawa T., Ninomiya K., Yoshikawa M., Tetrahedron,
57, 8443—8453 (2001).
10) Muraoka O., Fujimoto M., Tanabe G., Kubo M., Minematsu T., Ma-
tsuda H., Morikawa T., Toguchida I., Yoshikawa M., Bioorg. Med.
Chem. Lett., 11, 2217—2220 (2001).
11) Matsuda H., Morikawa T., Toguchida I., Ninomiya K., Yoshikawa M.,
Chem. Pharm. Bull., 49, 1558—1566 (2001).
12) Morikawa T., Matsuda H., Ninomiya K., Yoshikawa M., Biol. Pharm.
Bull., 25, 627—631 (2002).
13) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem. Soc.,
113, 4092—4096 (1991).
14) Vlad P., Soucek M., Collection Czechoslov. Chem. Commun., 27,
1726—1729 (1962).
15) 1,4-Pentandiol was identified by comparison of its physical data with
that of commercial reagent (Aldrich Chem. Co.).
16) Mori K., Tetrahedron, 31, 1381—1384 (1975).
17) Since there was very little of 2, we could not chracterize the absolute
stereostructure of the 8,9-positions of 2.
For estimating the spontaneous release of b-hexosaminidase from cells,
exactly the same procedure was followed (Normal), but without adding anti-
gen. Blank absorbance of the test material was measured to eliminate inter-
ference caused by the color of the test material itself. For this, only test ma-
terial and substrate were added without adding the cell extract (Blank).
Thus, the inhibition % of the release of b-hexosaminidase by the test mater-
ial was calculated by the following equation.
TϪBϪN
18) Schwartz L. B., Lewis R. A., Seldin D., Austen K. F., J. Immunol., 126,
1290—1294 (1981).
inhibition (%)ϭ 1Ϫ
ϫ100
CϪN
19) Marquardt D. L., Wasserman S. I., J. Immunol., 131, 934—939 (1983).
20) Matsuda H., Morikawa M., Tao J., Ueda K., Yoshikawa M., Chem.
Pharm. Bull., 50, 208—215 (2002).
21) Cheong H., Choi E. J., Yoo G. S., Kim K. M., Ryu S. Y., Planta Med.,
64, 577—578 (1998).
Control (C): antigen-IgE response was evoked without test sample; Test (T):
antigen-IgE response was evoked in the presence of test sample; Blank (B):
only test sample and substrate were added; Normal (N): antigen-IgE re-
sponse was not evoked, test sample was not added.