Immunomodulatory constituents from an ascomycete, Eupenicillium crustaceum, and revised absolute structure of macrophorin D

Article abstract of DOI:10.1021/np010152n

Fractionation guided by immunomodulatory activity of the EtOAc extract of the Ascomycete Eupenicillium crustaceum has afforded two new naturally occurring products, 4′-oxomacrophorin D (1) and 4′-oxomacrophorin A (2), as the immunosuppressive components o

Full text of DOI:10.1021/np010152n

1
234
J . Nat. Prod. 2001, 64, 1234-1237
Im m u n om od u la tor y Con stitu en ts fr om a n Ascom ycete, Eu pen icilliu m
cr u sta ceu m , a n d Revised Absolu te Str u ctu r e of Ma cr op h or in D
,
†
†
†
†
†
Haruhiro Fujimoto,* Etsuko Nakamura, Yong-Pil Kim, Emi Okuyama, Masami Ishibashi, and
Takeshi Sassa
‡
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33, Yayoi-cho, Inage-ku, Chiba 263-8522, J apan, and
Department of Bioresources, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, J apan
Received March 23, 2001
Fractionation guided by immunomodulatory activity of the EtOAc extract of the Ascomycete Eupenicillium
crustaceum has afforded two new naturally occurring products, 4′-oxomacrophorin D (1) and 4′-
oxomacrophorin A (2), as the immunosuppressive components of this fungus [1: 3-hydroxy-3-methyl-
glutaryl (HMG) conjugate of 2]. The structures including the absolute configurations of 1 and 2 have
been determined on the basis of chemical correlation of 1 with macrophorin D (3). The absolute
configuration of the HMG moiety in 3 has been revised from 3R to 3S.
In our recent screening program on immunomodulatory
absorption of a conjugated >CdO system. The IR spectrum
constituents from fungi, nine 2-pyrones from Gelasinospora
of 1 showed the absorptions of -OH, -COOH, and
multiforis,1 G. heterospora, and G. longispora, a hexa-
,2
2
-1
R,â-unsaturated >CdO groups (3448, 1750, and 1688 cm
,
2
1
13
ketide from G. heterospora and G. longispora, a 2-pyrone
respectively). The H and C NMR data of 1 including two-
dimensional COSY, HMQC, and HMBC data suggested
that 1 was a new compound similar to a drimane-type
and two macrocyclic diesters from Diplogelasinospora
3
grovesii, a macrocyclic sesterterpenetriol and three 2-fura-
4
7
nones from Gelasinospora kobi, two anthraquinones, two
sesquiterpenyl metabolite, macrophorin D (3), which was
octaketides, and two dioxopiperazines from Microascus
isolated as a self-growth inhibitor, together with an anti-
microbial and cytotoxic congener, macrophorin A (4), from
5
8
tardifaciens, and four sesterterpenes and a sesquiterpen-
6
etriol diester from Emericella aurantio-brunnea have been
a Fungi Imperfecti Macrophoma sp. On oxidation with
MnO in CHCl , 3 was converted into a compound that was
isolated as immunosuppressive constituents. Herein, we
report that the EtOAc extract of an Ascomycete, Eupeni-
cillium crustaceum Ludwig, appreciably suppressed the
proliferation (blastogenesis) of mouse splenic lymphocytes
stimulated with mitogens, concanavalin A (Con A) and
lipopolysaccharide (LPS). Solvent partitions followed by
repeated chromatographic fractionations of the extract,
monitored by immunomodulatory activity, afforded two
compounds, tentatively named EC-A (1) and B (2), as the
immunosuppressive components of this fungus. This paper
deals with the structures and immunosuppressive activities
of these compounds.
2
3
identical with 1 including optical rotation. Comparison of
1
3
the H NMR spectrum of 1 (in CDCl ) with that of 3 showed
that the signal of H-4′ at δ 4.62 (d, J ) 1.5 Hz) disappeared
and the signal of H-5′ at δ 3.81 (d, 1.5) shifted to δ 3.77 (s),
indicating that the secondary OH at position 4′ in 3 was
oxidized to a ketone group to give 1 (Table 1). For the
structure of macrophorin D, the (3R)-3-hydroxy-3-methyl-
glutaryl [(3R)-HMG] conjugate of the drimane-type ses-
quiterpenyl cyclohexenone epoxide (3a ) has already been
7
proposed. Therefore, the structure of EC-A was considered
to be expressed as 1a including the absolute configuration
of (3R)-HMG (Figure 1). But, the (3R)-HMG group is rare
in naturally occurring products, because (3S)-HMG-CoA
gives (3R)-mevalonate, which is the biosynthetic precursor
of various isoprenoids, with the aid of HMG-CoA reductase
The EtOAc extract of E. crustaceum IFM42163 ()CBS-
44.61) cultivated on sterilized rice medium suppressed the
3
Con A-induced proliferation of mouse splenic lymphocytes
by 99% at 50 µg/mL. The concentrated solution of the
extract in MeOH was partitioned between n-hexane and
water into an n-hexane layer and an aqueous suspension.
The aqueous suspension was further partitioned with
EtOAc and water into an EtOAc layer and an aqueous layer
9
shown in Figure 2. Thus, we reinvestigated the absolute
configuration of the HMG moiety of 1.
To hydrolyze reductively the HMG ester in EC-A, lithium
triethylborohydride (super-hydride, LiEt
3
BH) seemed to be
[
yields (%) of the n-hexane, EtOAc, and aqueous layers
after evaporation of the solvents from the EtOAc extract:
7.8, 46.8, and 4.5, respectively]. The n-hexane, EtOAc, and
more suitable than borane, which was utilized formerly to
7
hydrolyze the HMG ester in macrophorin D. LiEt
reduces exclusively the ester group in the presence of both
ester and carboxyl groups.10 Reduction of 1 with LiEt
BH
3
BH
4
aqueous layers suppressed the Con A-induced proliferation
by 21, 46, and -19% at 12.5 µg/mL, respectively. Repeated
chromatographic fractionation of the EtOAc layer guided
by the immunomodulatory activity afforded two immuno-
suppressive components, EC-A (1) and B (2) [yields (%) of
3
in dry THF followed by treatment with dilute HCl gave a
lactone product (5), which was not identical with authentic
(
(
3S)-mevalonolactone, but was identical with authentic
3R)-mevalonolactone in chiral HPLC analysis. This iden-
1
and 2 from the EtOAc extract: 2.33 and 0.764, respec-
tively].
The molecular formula of 1 was determined to be
tification was further confirmed by the fact that 5-O-acetyl-
-[(S)-1-phenylethyl]amide derived from 5 (6) was identical
1
with authentic (3R)-5-O-acetyl-1-[(S)-1-phenylethyl]meva-
lonamide11 in reversed-phase HPLC analysis (Figure 2).
Therefore, the structure, including absolute configuration
of EC-A, is not 1a containing the (3R)-HMG group, but 1
containing the (3S)-HMG group. Accordingly, the absolute
C
28
H
38
O
8
by HRFABMS. The UV spectrum of 1 gave the
*
To whom correspondence should be addressed. Tel: +81-43-290-2914.
Fax: +81-43-290-3021. E-mail: fujimoto@p.chiba-u.ac.jp.
†
Chiba University.
Yamagata University.
‡
1
0.1021/np010152n CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/22/2001

Notes
J ournal of Natural Products, 2001, Vol. 64, No. 9 1235
Ta ble 1. NMR Data for 4′-Oxomacrophorin D (1), 4′-Oxomacrophorin A (2), and Macrophorin D (3) in CDCl3, δ (ppm), from TMS as
an Internal Standard, [coupling constants (Hz) in parentheses]
1
2
3
position
δH
δC
δH
δC
δH
1
2
3
4
5
6
7
8
9
1.19 (m), 1.75 (m)
1.55 (2H, m)
1.19 (m), 1.41 (m)
38.85 (t)
19.27 (t)
41.94 (t)
33.55 (s)
55.44 (d)
24.28 (t)
37.93 (t)
148.66 (s)
51.28 (d)
39.70 (s)
20.01 (t)
1.24 (m), 1.75 (m)
1.53 (2H, m)
1.15 (m), 1.38 (m)
38.89 (t)
19.32 (t)
41.99 (t)
33.60 (s)
55.50 (d)
24.35 (t)
38.02 (t)
148.74 (s)
51.41 (d)
39.76 (s)
20.24 (t)
1.25 (m), 1.75 (m)
1.55 (2H, m)
1.18 (m), 1.40 (m)
1.11 (dd, 12.7, 2.4)
1.31 (m), 1.75 (m)
1.95 (m), 2.36 (br d, 12.0)
1.10 (br d, 14.4)
1.28 (m), 1.70 (m)
1.94 (m), 2.35 (br d, 12.3)
1.68 (d, 11.2)
1.12 (br d, 12.5)
1.32 (m), 1.75 (m)
1.88 (m), 2.36 (m)
1.68 (d, 11.0)
1.71 (d, 12.5)
1
1
0
1
2.05 (dd, 14.1, 11.0),
2.02 (dd, 14.8, 11.2),
2.49 (d,14.8)
4.50 (s), 4.81 (s)
0.85 (3H, s)
0.78 (3H, s)
0.69 (3H, s)
1.97 (m)
2
.46 (d, 14.1)
2.38 (d, 14.3)
4.54 (br s), 4.82 (br s)
0.87 (3H, s)
0.80 (3H, s)
0.70 (3H, s)
1
1
1
1
2
3
4
5
1
2
3
4
5
6
7
1
2
3
4
5
6
4.51 (s), 4.82 (s)
0.87 (3H, s)
0.80 (3H, s)
0.71 (3H, s)
106.85 (t)
33.49 (q)
21.60 (q)
14.42 (q)
191.67 (s)
132.87 (d)
142.11 (s)
192.12 (s)
58.80 (d)
62.56 (s)
59.62 (t)
170.68 (s)
44.49 (t)
69.81 (s)
44.48 (t)
170.68 (s)
27.26 (q)
106.87 (t)
33.52 (q)
21.64 (q)
14.46 (q)
192.02 (s)
132.06 (d)
146.84 (s)
193.55 (s)
59.06 (d)
62.50 (s)
59.17 (t)
′
′
′
′
′
′
′
′′
′′
′′
′′
′′
′′
6.57 (s)
6.62 (s)
5.92(br s)
4.62 (d, 1.5)
3.81 (d, 1.5)
3.77 (s)
3.72 (s)
4.83 (d, 16.3), 4.98 (d, 16.3)
2.73 (d, 14.4), 2.77 (d, 14.4)
2.67 (d, 16.4), 2.70 (d, 16.4)
1.41 (3H, s)
4.33 (d, 16.8), 4.52 (d, 16.8)
4.74 (d, 15.7), 4.95 (d, 15.7)
2.67 (d, 14.3), 2.74 (d, 14.3)
2.64 (d, 15.5), 2.66(d, 15.5)
1.40 (3H, s)
F igu r e 1.
structure of macrophorin D has been revised from 3a to 3.
Thus, the structure of EC-A, a new immunosuppressant,
has been verified as 4′-oxomacrophorin D (1) (Figure 1).
The molecular formula of 2 was determined to be
F igu r e 2.
absorption of a conjugated >CdO system, the same as that
of 1. The IR spectrum of 2 showed the absorptions of -OH
22 30 4
C H O by HRFABMS. The UV spectrum of 2 gave the

1
236 J ournal of Natural Products, 2001, Vol. 64, No. 9
Notes
Ta ble 2. Immunosuppressive Effects of 4′-Oxomacrophorin D
1) and 4′-Oxomacrophorin A (2) and Some Compounds on Con
A-Induced and LPS-Induced Proliferations of Mouse Splenic
Lymphocytes
UV/VIS, and J ASCO 970 UV detectors at 220 nm. Evaluation
of suppressive activity (IC50 values) of samples against the
proliferation of mouse (BALB/c, male, 7-11 weeks) splenic
lymphocytes stimulated with Con A and LPS was executed
with the same method as that described in our previous report
(
IC50 (µg/mL)
[
this method is based on the formation ratio of MTT-formazan
compound
Con A-induced
LPS-induced
from exogenous 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-
1
2
4.5
0.5
2.7
2.0
0.25
2.7
tetrazolium bromide (MTT) in lymphocytes].2
F u n ga l Ma ter ia l. Eupenicillium crustaceum IFM42163
azathioprine
cyclosporin A
FK506 (tacrolimus)
()CBS344.61) was deposited earlier at Research Institute for
0.04
1.5 × 10
0.07
1.6 × 10
-
5
-3
Chemobiodynamics, Chiba University (present name: Re-
search Center for Pathogenic Fungi and Microbial Toxicoses,
Chiba University). The voucher specimen was also deposited
in Laboratory of Natural Products Chemistry, Graduate School
of Pharmaceutical Sciences, Chiba University. This fungus was
cultivated on sterilized moistened rice in Roux flasks (200
g/flask × 26) at 25 °C for 24 days to give reddish brown moldy
rice.
a
The IC50 value of each sample was calculated from the
correlation curve between the sample concentration (horizontal
axis) and the cell proliferation (vertical axis). The curve of each
sample was drawn with 7 points, each of which represented the
mean of 3 experiments on each correlation between 7 different
concentrations and cell proliferations.
Isola tion of EC-A (1) a n d B (2) fr om E. cr u sta ceu m .
The moldy rice was extracted with EtOAc (7.8 L) with shaking
at room temperature for 6 h two times to give an EtOAc
solution (15.6 L), which gave, after evaporation in vacuo, a
wine-red EtOAc extract (43.6 g). A portion of the EtOAc extract
(31.2 g) was dissolved in MeOH (60 mL). The MeOH solution
and R,â-unsaturated >CdO groups (3448 and 1685 cm-1,
1
respectively). Comparison of the H NMR spectrum of 2
with that of 1 showed that 2 was similar to 1 except that
2
lacked the signals due to the (3S)-HMG moiety. This
1
3
observation was also supported from comparison of the
C
2
was suspended in H O (1.2 L). The suspension was partitioned
NMR spectrum of 2 with that of 1 (Table 1). These spectral
data, and the biosynthetic consideration that EC-B may
be biosynthesized via a pathway similar to that of 1,
indicated that the structure including absolute configura-
tion of EC-B was 2 (4′-oxomacrophorin A) (Figure 1). Ayer
et al. have isolated macrophorin A (4) from Penicillium
brevi-compactum and prepared the 4′-oxo derivative of 4
with n-hexane (1.2 L) two times into an n-hexane layer (after
evaporation in vacuo, 14.9 g) and an aqueous suspension. The
aqueous suspension was further partitioned with EtOAc (1.2
L) two times into an EtOAc layer (14.6 g) and an aqueous layer
(1.4 g). A part of the EtOAc layer (7.80 g) was subjected to
chromatography on a Si gel column (Wako, C-200) with CHCl
3
,
CHCl -MeOH (100:1, v/v, 50:1, 50:1, 50:1), and MeOH to give
3
(7) from 4 with MnO
2
oxidation.12 It was considered that 2
fractions 1-6 (0.49, 1.61, 1.66, 1.15, 0.58, 3.14 g), respectively.
The IC50 values of the fractions 1-6 against the Con A-induced
proliferation of the lymphocytes were >50, <10, <10, 25-35,
might be identical with 7, although 2 was not directly
compared with 7 described in the literature.12 Macrophorin
A (4) has also been isolated from E. crustaceum NR-
RL22307 ()CBS457.72) by Wang et al.13 However, to the
best of our knowledge, this is the first time that 4′-
oxomacrophorin A (2) has been isolated as an immunosup-
pressant from a natural source.
3
5-45, and 25-35 µg/mL, respectively. A portion of fraction
3 (620 mg) was chromatographed on an ODS column (Waters
18 Sep-Pak cartridge, 16 × 50 mm) with 90% MeOH to give
a fraction that was further chromatographed on an HPLC ODS
CN
C
column (Senshu Pegasil, 20 × 250 mm) with 70% CH
3
containing 0.1% TFA at a flow rate of 8.0 mL/min to afford
EC-A (1) (145 mg). A part of fraction 2 (101 mg) was
chromatographed on a Si gel column (Wako, C-200) with
n-hexane-EtOAc (8:1) to give EC-B (2) (8.0 mg).
The immunosuppressive activities (IC50 values) of 1 and
2
were calculated against Con A- (T-cells) and LPS-induced
(B-cells) proliferation of mouse splenic lymphocytes, as
2
5
shown in Table 2. Both 1 and 2 suppressed about two times
higher the proliferation of B-cells than that of T-cells.
Compound 2 displayed about eight times higher immuno-
suppressive activity than 1, indicating that the presence
EC-A (4′-Oxom a cr op h or in D) (1): yellow viscous oil; [R]
35.5° (c 0.30, MeOH); UV (MeOH) λmax (log ꢀ) 221 (3.97), 260
sh, 3.47) nm; IR νmax (KBr) 3448, 2939, 2860, 1750, 1688, 1647,
D
+
(
-1
1
446, 1388, 1201, 971, 896 cm ; positive FABMS m/z 525 [(M
+
-
+
Na) ]; negative FABMS m/z 501 [(M - H) ]; HRFABMS m/z
of a free -CH
2
OH group at position 3′ in 2 might be very
+
5
25.2464 (calcd for C28
38 8
H O Na [(M + Na) ], 525.2464); CD
important for the immunosuppressive activity of 2. The
immunosuppressive activity of 2 was about five to 10 times
(
(
0.797 mM, MeOH) ∆ꢀ(nm) -1.05 (336), 0.00 (297), -2.65
1
13
266), +5.01 (237), +3.69 (228), +6.79 (213); H NMR and
C
2
higher than that of azathioprine, although lower than
NMR, see Table 1.
2
2
EC-B (4′-Oxom a cr op h or in A) (2): yellow viscous oil; [R]D25
10.2° (c 0.30, MeOH); UV (MeOH) λmax (log ꢀ) 219 (3.87), 268
those of cyclosporin A and FK506 (taclolimus).
+
(
1
1
sh, 3.33) nm; IR νmax (KBr) 3448, 2927, 2858, 1685, 1647, 1459,
389, 1203, 1093, 892 cm (lit. 3440, 2960, 1710 (sh), 1687,
460, 1440, 1385, 1200, 885); EIMS m/z (%) 358 [M , 29] (lit.
m/z (%) 358 [M , 4]); HRFABMS m/z 359.2225 (calcd for
Exp er im en ta l Section
-
1
12
+
12
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
and CD spectra were measured with a J ASCO DIP 140 digital
polarimeter and a J ASCO J -500 spectropolarimeter, respec-
+
+
12
C
22
H
31
O
4
[(M + H) ], 359.2223) (lit. HREIMS m/z 358.2155
+
[
-
(
M ]); CD (1.12 mM, MeOH) ∆ꢀ(nm) -0.30 (341), 0.01 (297),
tively. UV and IR spectra were recorded on Hitachi U-3200
1
13
2.41 (263), +7.88 (220). H NMR and C NMR, see Table 1
1
and J ASCO FT/IR-230 spectrophotometers, respectively.
H
1
the H NMR data were very similar to those of compound 7
1
3
and C NMR spectra were measured with J EOL J NM-A400
12
described in the literature ).
(
1H, 399.65; C, 100.40 MHz) and -A500 ( H, 500.00; C,
13
1
13
Con ver sion of Ma cr op h or in D (3) to EC-A (1). Activated
1
25.65 MHz) spectrometers using chemical shift, δ (ppm),
MnO
macrophorin D (3), which was isolated by one of us, T.S., et
al.,7 (2.5 mg) in CHCl
(600 µL). The reaction mixture was
2
(50 mg) (Wako) was added to a solution of authentic
values from TMS as an internal standard. EIMS, FABMS, and
HRFABMS spectra were recorded on a J EOL AUTO MS-20,
a J EOL J MS-AX505 HA using m-nitrobenzyl alcohol (m-NBA)
as a matrix, and a J EOL J MS-HX110A spectrometer using
m-NBA as a matrix, respectively. Column chromatography was
performed with Wako Si gel C-200. HPLC was performed using
Senshu SSC-3100, Waters 600-E, and J ASCO PU-980 flow
systems equipped with Senshu SSC-5200, Waters 486 Tunable
3
stirred at room temperature for 20 min and filtered to remove
insoluble substances. The filtrate was evaporated in vacuo to
furnish 4′-oxomacrophorin D (0.8 mg), yellow viscous oil, which
was identical with EC-A (1) by 1H NMR (CDCl ) and CD
3
spectra (MeOH) and TLC behavior [Merck Kieselgel 60F254
CHCl -MeOH (9:1), R 0.42].
,
3
f

Notes
Red u ctive Hyd r olysis of EC-A (1). A solution of LiEt
BH (1.0 M) in dry THF (20 µL) (Aldrich) was added to a
solution of EC-A (1) (5.0 mg) in dry THF (80 µL) under ice-
cooling in an Ar stream. The reaction mixture was stirred
J ournal of Natural Products, 2001, Vol. 64, No. 9 1237
3
-
less amorphous residue, which gave one peak (t
an HPLC ODS column (ODS-AM, 10 × 250 mm) (YMC) with
MeOH-H O (2:3) at a flow rate of 2.0 mL/min.
(3R)-5-O-Acetyl-1-[(S)-1-ph en yleth yl]m evalon am ide: 1H
NMR (CDCl ) δ 1.23 (3H, s, H -6), 1.51 (3H, d, J ) 6.8 Hz,
-2′), 1.82, 1.88 (each 1H, dd, 14.1, 6.8, H -, H -4), 2.04 (3H,
s, Ac), 2.28, 2.41 (each 1H, d, 14.8, H -, H -2), 4.23 (2H, t, 6.8,
-5), 4.51 (1H, br s, OH), 5.15 (1H, m, H-1′), 6.11 (1H, br peak,
R
84.8 min) on
2
under ice-cooling in Ar gas for 15 min. After addition of H
2
O
3
3
(100 µL) to the reaction mixture, 0.1 N HCl (500 µL) was added
H
3
a
b
to adjust its pH to 3-4. The reaction mixture was then stirred
at room temperature in Ar gas for 42 h to allow lactone
formation. The reaction mixture was partitioned with EtOAc
a
b
H
2
1
NH), 7.27-7.37 (5H, m, Ph) (these H NMR data were identical
(1.0 mL) three times into an EtOAc layer (yellow) and an
with those of (3R)-5-O-acetyl-1-[(S)-1-phenylethyl]mevalon-
1
1
+
aqueous layer (colorless). The aqueous layer which contained
a compound equal to mevalonolactone on TLC analysis was
evaporated in vacuo to give a resinous residue, which was
extracted with EtOAc (2.0 mL) two times to furnish an EtOAc
solution. The EtOAc solution was evaporated in vacuo to afford
compound 5 (0.8 mg), a colorless amorphous residue, which
amide described in the literature ); EIMS m/z (%) 293 (M ,
11).
P a r t 3. (S)-1-Phenylethylamine (2.0 mg) was added to a
solution of compound 5 (0.7 mg) in dry THF (100 µL) at room
temperature in an Ar stream. After stirring at room temper-
ature under Ar gas for 17 h, the reaction mixture was
evaporated in vacuo to give a residue, which was extracted
with EtOAc (2.0 mL) two times to afford an EtOAc solution.
was not identical with authentic (3S)-mevalonolactone (t
R
12.8
min), but identical with authentic (3R)-mevalonolactone
(
t
R
13.6 min) in HPLC analysis on a chiral CD-Ph column (4.6
250 mm) (Shiseido) with n-hexane-EtOH (2:3) at a flow rate
After washing with 0.1 N HCl followed by H
solution was evaporated in vacuo to furnish a colorless oily
residue. This residue was then acetylated with Ac O (100 µL)
2
O, the EtOAc
×
of 0.5 mL/min.
2
For m ation of 5-O-Acetyl-1-[(S)-1-P h en yleth yl]m evalon -
a m id e, P a r t 1. (S)-1-Phenylethylamine (60 mg) (Aldrich) was
added to a solution of (3RS)-mevalonolactone (20 mg) (Aldrich)
in dry THF (1.0 mL) at room temperature in an Ar stream.
After stirring at room temperature under Ar gas for 18 h, the
solvent was evaporated in vacuo to give a residue, which was
extracted with EtOAc (20 mL) to afford an EtOAc solution.
in pyridine (200 µL) at room temperature for 24 h to afford
compound 6 (0.2 mg), colorless amorphous residue, which was
not identical with authentic (3S)-5-O-acetyl-1-[(S)-1-phenyl-
ethyl]mevalonamide (t
(3R)-5-O-acetyl-1-[(S)-1-phenylethyl]mevalonamide (t
R
83.2 min), but identical with authentic
84.8
R
min) in HPLC analysis using an ODS column (ODS-AM, 10 ×
2
250 mm) (YMC) with MeOH-H O (2:3) at a flow rate of 2.0
After washing with 0.1 N HCl followed by H
solution was evaporated in vacuo to give a colorless oily
residue. This residue was then acetylated with Ac O (0.5 mL)
in pyridine (1.0 mL) at room temperature for 8 h to afford
3RS)-5-O-acetyl-1-[(S)-1-phenylethyl]mevalonamide (3.8 mg),
a colorless amorphous residue, which gave two peaks (t 83.2
and 84.8 min, integral intensity 1:1) on an HPLC ODS column
ODS-AM, 10 × 250 mm) (YMC) with MeOH-H O (2:3) at a
flow rate of 2.0 mL/min [the peak, t 84.8 min, was verified to
2
O, the EtOAc
mL/min.
Ack n ow led gm en t. We are grateful to Dr. T. Uegaki of
Fujisawa Pharmaceutical Co., Ltd. for the gift of FK506.
2
(
Refer en ces a n d Notes
R
(1) Fujimoto, H.; Satoh, Y.; Nakayama, M.; Takayama, T.; Yamazaki,
(
2
M. Chem. Pharm. Bull. 1995, 43, 547-552.
(
2) Fujimoto, H.; Sumino, M.; Nagano, J .; Natori, H.; Okuyama, E.;
Yamazaki, M. Chem. Pharm. Bull. 1999, 47, 71-76.
3) Fujimoto, H.; Nagano, J .; Yamaguchi, K.; Yamazaki, M. Chem.
Pharm. Bull. 1998, 46, 423-429.
R
be that of (3R)-5-O-acetyl-1-[(S)-1-phenylethyl]mevalonamide
(
by co-chromatography of both samples of (3RS)- and (3R)-5-
O-acetyl-1-[(S)-1-phenylethyl]mevalonamides]. The 1H NMR
(4) Fujimoto, H.; Satoh, Y.; Yamazaki, M. Chem. Pharm. Bull. 1998, 46,
2
11-216.
and EIMS spectra of (3RS)-5-O-acetyl-1-[(S)-1-phenylethyl]-
mevalonamide were identical with those of (3R)-5-O-acetyl-1-
(
5) Fujimoto, H.; Fujimaki, T.; Okuyama, E.; Yamazaki, M. Chem.
Pharm. Bull. 1999, 47, 1426-1432.
(6) Fujimoto, H.; Nakamura, E.; Okuyama, E.; Ishibashi, M. Chem.
Pharm. Bull. 2000, 48, 1436-1441.
[
(S)-1-phenylethyl]mevalonamide formed from (3R)-mevalono-
lactone.
P a r t 2. (S)-1-Phenylethylamine (35 mg) was added to a
solution of (3R)-mevalonolactone (26 mg) (Wako) in dry THF
1.0 mL) at room temperature in an Ar stream. After stirring
(7) Sassa, T.; Nukina, M. Agric. Biol. Chem. 1984, 48, 1923-1925.
(8) Sassa, T.; Yoshikoshi, H. Agric. Biol. Chem. 1983, 47, 187-189.
(9) In Dictionary of Biochemistry, Version 3 (in J apanese); Imahori, K.,
Yamakawa, T., Eds.; Tokyo-Kagakudojin: Tokyo, 1998; p 1411.
(
(
10) Brown, H. C.; Kim, S. C.; Krishnamurthy, S. J . Org. Chem. 1980,
5, 1-12.
at room temperature under Ar gas for 18 h, the reaction
mixture was treated in the same way as described for the
formation of (3RS)-5-O-acetyl-1-[(S)-1-phenylethyl]mevalona-
mide from (3RS)-mevalonolactone to give a colorless oily
4
(
11) Hirai, N.; Koshimizu, K. Phytochemistry 1981, 20, 1867-1869.
(12) Ayer, W. A.; Altena, I. V.; Browne, L. M. Phytochemistry 1990, 29,
1661-1665.
(
13) Wang, H.-J .; Gloer, J . B.; Wicklow, D. T.; Dowd, P. F. Appl. Environ.
residue. This residue was then acetylated with Ac
in pyridine (0.5 mL) at room temperature for 8 h to afford (3R)-
-O-acetyl-1-[(S)-1-phenylethyl]mevalonamide (5.0 mg), color-
2
O (0.25 mL)
Microbiol. 1995, 61, 4429-4435.
5
NP010152N