Structures of amamistatins A and B, novel growth inhibitors of human tumor cell lines from Nocardia asteroides

Article abstract of DOI:10.1016/S0040-4020(00)00591-3

Two new lipopeptides, amamistatins A and B, were isolated from Nocardia asteroides SCRC-A2359, and their absolute stereostructures were determined by spectroscopic and chemical analyses. Amamistatin A inhibited the growth of human rumor cell lines. (C) 20

Full text of DOI:10.1016/S0040-4020(00)00591-3

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 6435±6440
Structures of Amamistatins A and B, Novel Growth Inhibitors of
Human Tumor Cell Lines from Nocardia asteroides
Susumu Kokubo,a Kiyotake Suenaga,a Chikara Shinohara,b Tomoko Tsujib and
Daisuke Uemuraa,
*
aDepartment of Chemistry, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan
bSagami Chemical Research Center, Nishi-Ohnuma 4-4-1, Sagamihara 229-0012, Japan
Received 11 May 2000; accepted 23 June 2000
AbstractÐTwo new lipopeptides, amamistatins A and B, were isolated from Nocardia asteroides SCRC-A2359, and their absolute
stereostructures were determined by spectroscopic and chemical analyses. Amamistatin A inhibited the growth of human tumor cell
lines. q 2000 Elsevier Science Ltd. All rights reserved.
Introduction
culture broth through a series of processes: EtOAc extrac-
tion, silica gel column chromatography, and reversed-phase
HPLC. The biological method for monitoring the biological
active components was used cytotoxicity against mouse
lymphocytic leukemia cells (P388). The molecular formula
of 1 was determined to be C37H55N5O11 by HRFABMS [m/z
746.3981, calcd for C37H56N5O11 (M1H)1 746.3976]. The
IR spectrum showed bands at 1740 and 1660 cm21 that were
assigned to ester and amide groups, respectively. A FeCl3
test of 1 indicated the presence of a phenolic functionality.
In our screening for compounds derived from microorganisms
that inhibited the growth of human tumor cell lines,
amamistatins A (1) and B (2) were isolated from Nocardia
asteroides SCRC-A2359, which was isolated from a soil
sample collected at Amami Island in Kagoshima Prefecture,
Japan. We have previously reported the isolation and struc-
ture of 1 and 2.1 The total synthesis of 1 has been already
achieved by Shioiri and co-workers.2 Recently, we also
determined the absolute structure of 2. We report here the
detailed isolation and structure determination of 1 and 2.
1
The NMR data for 1 are summarized in Table 1. The H
NMR spectrum of 1 showed the presence of two amide NH
groups (d 8.93 and 7.48), three hydroxyl groups (d 9.93,
9.77, and 9.50), a 1,2,4-trisubstituted benzene ring (d 7.25,
7.04, and 7.00) and a methoxy group (d 3.76). In the 13C
NMR and DEPT spectra, 37 carbon signals were observed,
including ®ve carbonyl carbons (d 173.3, 171.6, 168.6,
161.6, and 160.8), nine aromatic carbons (d 157.1, 152.4,
152.1, 150.2, 128.5, 120.0, 118.4, 110.1, and 109.3), one
oxymethine carbon (d 77.9), one methoxy carbon (d
55.6), and four nitrogen-bearing carbons (d 52.3, 52.0,
50.9, and 48.9). Duplicate signals due to a formyl group
(dC 161.6, 157.0; dH 8.22, 7.88) were also observed. Due
to restricted rotation about the formamide moiety, the NMR
signals for several protons and carbons were doubled at
238C, as shown in Table 1. The doubled NMR signals
broadened at 60 and 908C, and were superimposed at
1208C. The remaining carbon signals were assigned to
four methyls, 12 methylenes, and one quaternary carbon.
Results and Discussion
Structure of amamistatin A
Amamistatins were puri®ed from the mycelial cake of
A detailed analysis of the phase-sensitive DQF-COSY and
HOHAHA spectra of 1 gave three partial structures, two
lysine residues [NH(1)±C2±C6 and NH(2)±C20±C24]
and C9±C16 (Fig. 1). In addition, the presence of a
Keywords: peptides; oxazoles; degradation; stereochemistry.
* Corresponding author. Tel./fax: 181-52-789-3654;
e-mail: uemura@chem3.chem.nagoya-u.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00591-3

6436
S. Kokubo et al. / Tetrahedron 56 (2000) 6435±6440
Table 1. NMR Data for amamistatin A (1) in DMSO-d6 at 238C
Position
1H (ppm)
13C (ppm)
168.6a
50.9
30.0
27.0
25.5
52.3
Position
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
1H (ppm)
13C (ppm)
1
2
3
4
5
6
1.88 m
1.35 m
1.56 m
29.9
22.6
26.4
4.40 br d (11.5)b
1.74 m, 1.39 m
1.78 m, 1.63 m
1.66 m, 1.39 m
3.87 dd (15.8, 11.7)
3.47 dd (15.8, 3.8)
3.37 m [3.43 m]c
8.22 s [7.88 s]c
48.9 [45.4]c
161.6 [157.0]c
160.8
128.5
152.4
11.4
157.1
110.1
150.2
118.4
120.0
7
8
9
173.3
45.6
77.9
29.4
25.6
28.5
28.6
31.1
22.0
13.9
19.7 [19.6]c
22.9
171.6
52.0
2.65 s
5.09 d (9.0)
1.43 m, 1.37 m
1.18 m, 1.12 m
1.15 m
1.23 m
1.15 m
1.23 m
0.81 t (7.1)
1.09 s
1.04 s
10
11
12
13
14
15
16
17
18
19
20
7.00 d (9.0)
7.04 dd (9.0, 2.9)
152.1
109.3
55.6
7.25 d (2.9)
3.76 s
9.93 br s
37
C32±OH
OH
OH
NH(1)
NH(2)
9.77 br s
9.50 br s [9.93 br s]c
7.48 br d (6.2)
8.93 br d (8.2) [8.89 br d (8.2)]c
4.49 m [4.47 m]c
a Recorded at 150 MHz.
b Recorded at 600 MHz. Coupling constants (Hz) are in parentheses.
c Observed as doubled signals.
methyloxazole group in 1 was revealed by correlations in
the HMBC spectrum, H-29/C27 and H-29/C28, as well as
by characteristic NMR signals (dH-29 2.65, dC27 128.5, dC28
152.4, and dC30 157.1) that corresponded to those of the
methyloxazole group in nostocyclamide.3 The connections
among the aromatic partial structural units were clari®ed by
the HMBC correlations (H-37/C35, H-33/C31, C32-OH/
C32, C32-OH/C31, and H-36/C30). HMBC correlations
suggested the presence of a 2-aminocaprolactam structure
(H-2/C1 and H-6/C1), a 2,2-dimethyl-3-hydroxydecanoic
acid portion (H-9/C8, H-9/C7, H-18/C8, H-18/C9, and
H-18/C17), and an N-formyllysine residue (H-20/C19,
H-25/C24, and H-20/C26), which were connected by
HMBC correlations, H-2/C7 and H-9/C19.
indicated that 1 had two amide N±OH groups, which were
degraded to amide NH groups by hydrogenolysis. Although
a connection between C26 and C27 could not be established,
such connectivity was obvious based on a consideration of
the molecular formula of 1. Thus, the gross structure of
amamistatin A was clari®ed as shown in formula 1. The
1
chemical shifts of 13C and H NMR spectra of 1 were
considerably in accord with those of formobactin4 closely
related to 1, except positions 34, 35 and 36.
The absolute stereostructure of 1 was elucidated as follows
(Scheme 1). Hydrogenolysis of 1 followed by methanolysis
gave methyl ester 5 and alcohol 7. Acidic hydrolysis of 5
and 7 (6 M HCl, 1108C, 24 h) followed by separation by
reversed-phase HPLC gave lysines, respectively. The
absolute con®gurations of the lysines in 5 and 7 were
respectively determined to be d and l by a chiral HPLC
analysis. The absolute stereochemistry of C9 in 7 was deter-
mined using a modi®ed Mosher's method.5 The alcohol 7
was transformed into (S)- and (R)-MTPA esters, 9a and 9b,
The locations of the two remaining hydroxyl groups in 1
were determined as follows. Hydrogenolysis (H2, Pd/C,
MeOH, 238C) of 1 gave 3, the molecular weight of which
was 32 MS units (2£O) less than that of 1. In addition, the
1H NMR spectrum of 3 showed the presence of two more
amide NH groups (d 6.53, 6.04) than in 1. This information
1
the H NMR signals of which were assigned based on the
Figure 1. Partial structures of amamistatin A (1) based on phase-sensitive DQF-COSY and HOHAHA spectra and selected HMBC correlations.

S. Kokubo et al. / Tetrahedron 56 (2000) 6435±6440
6437
Structure of amamistatin B
The molecular formula of 2 was determined to be
C36H53N5O10 by HRFABMS [m/z 716.3901, calcd for
C36H54N5O10 (M1H)1 716.3871], which is 30 MS units
(CH2O) less than that of 1. The NMR data for 2 are summar-
1
ized in Table 2. The H NMR spectrum of 2 showed the
presence of a 1,2-disubstituted benzene ring (d 7.85, 7.40,
7.03, and 6.99). In the 13C NMR and DEPT spectra, 36
carbon signals were observed, including ®ve carbonyl
carbons (d 177.3, 173.3, 171.1, 164.5, and 164.0), nine
aromatic carbons (d 160.3, 158.5, 154.9, 134.4, 130.3,
128.1, 121.4, 118.7, and 112.3), one oxymethine carbon
(d 80.9), and four nitrogen-bearing carbons (d 54.6, 54.4,
53.5, and 51.5), while that of a methoxy carbon was not
observed. The gross structure of 2 was determined by the
same 2D NMR technique as described above for 1. Thus,
amamistatin B was shown to be the demethoxy derivative of
1.
The absolute stereostructure of 2 was elucidated by a pro-
cedure similar to that for 1. Hydrogenolysis of 2 followed by
methanolysis gave methyl ester 6 and alcohol 8. The H
1
NMR spectrum of 8 was identical to that of 7 derived
from amamistatin A (1). Acidic hydrolysis of 6 and 8
(6 M HCl, 1108C, 24 h) followed by separation by
reversed-phase HPLC gave lysines. The absolute con®gura-
tions of the lysines in 6 and 8 were respectively determined
to be d and l by a chiral HPLC analysis. Since both of the
lysines from 7 and 8 were l, the absolute stereochemistry of
C9 in 8 should be the same as that in 7. Thus, the absolute
stereochemistry of amamistatin B was determined as shown
in formula 2.
Scheme 1. (a) H2, Pd/C, MeOH; (b) NaOMe, MeOH; (c) (R)- or (S)-
MTPACl, Py.
2D NMR spectra, and the Dd values (dS2dR, ppm) were
then calculated. The results established that the absolute
stereochemistry of C9 in 7 was S. Thus, the absolute stereo-
chemistry of amamistatin A was determined as depicted in
formula 1.
Biological activities
Amamistatins A (1) had anti-proliferative, but not cell-
killing, effects against several kinds of human tumor cell
lines. The IC50 values of 1 were 0.48, 0.56, and 0.24 mM
Table 2. NMR Data for amamistatin B (2) in CD3OD at 238C
Position
1H (ppm)
13C (ppm)
Position
1H (ppm)
13C (ppm)
1
171.1a
53.5
32.3
29.2
27.3
54.6 [54.5]b
19
20
173.3 [173.2]b
54.4 [54.5]b
2
3
4
5
6
4.52 br d (10.3)c
1.88 m, 1.55 m
1.91 m, 1.76 m
1.76 m, 1.56 m
3.92 dd (15.9, 11.8)
3.65 dd (15.9, 3.8)
4.67 dd (9.9, 5.1)
[4.64 dd (9.9, 5.1)]b
2.02 m, 1.93 m
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
32.2 [32.1]b
24.7 [24.6]b
28.1
1.56 m, 1.45 m
1.77 m, 1.70 m
3.52 t (6.2) [3.62 m, 3.54 m]b
8.27 s [7.93 s]b
51.5 [47.7]b
164.5 [159.9]b
164.0
7
8
9
177.3
48.0
5.17 dd (10.2, 2.1)
1.55 m, 1.48 m
1.28 m, 1.24 m
1.20 m
1.32 m
1.20 m
1.25 m
0.84 dd (7.1, 6.1)
1.18 s
1.18 s
80.9 [80.8]b
31.6 [31.5]b
27.7
130.3
154.9
12.2
160.3
112.3
158.5
118.7
134.4
10
11
12
13
14
15
16
17
18
2.71 s
30.7
30.8
33.3
24.1
14.8
22.2 [22.0]b
23.6 [23.3]b
7.03 dd (8.3, 0.7)
7.40 ddd (8.3, 7.3, 1.5)
6.99 ddd (7.9, 7.3, 0.7)
7.85 dd (7.9, 1.5)
121.4
128.1
a Recorded at 150 MHz.
b Observed as doubled signals.
c Recorded at 600 MHz. Coupling constants (Hz) are in parentheses.

6438
S. Kokubo et al. / Tetrahedron 56 (2000) 6435±6440
against MCF-7 breast, A549 lung, and MKN45 stomach
cancer cell lines, respectively. Amamistatins A (1) and B
(2) exhibited potent cytotoxicity against mouse lymphocytic
leukemia cells (P388), with IC50 values of 15 and 16 ng/mL,
respectively. This result may be meant that the methoxy
group of 1 is not essential for biological activity. The struc-
tures of 1 and 2 are closely related to those of formobactin4
and nocobactin NA.6 Mycobactins form extremely stable
hexadentate iron (III) complexes by binding the iron with
two hydroxamic acids and a 2-hydroxyphenyloxazoline
moiety.7 Therefore, it was presumed that the antitumor
activities of amamistatins are based on their properties as
siderophores. Further studies on the mode of action and the
selectivity for tumor cells are currently in progress.
National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Tsukuba,
Japan, under the name SCRC-A2359 and accession number
FERM P-16804.
N. asteroides SCRC-A2359 was added to medium
(100 mL) containing 3% soluble starch, 1% glucose,
0.5% peptone, 2% bean-peptide, 0.5% yeast extract
and 0.2% CaCO3 at pH 7.0 in a 500 mL ¯ask, and
pre-cultured at 26.58C and 160 rpm for 4 days. This
pre-culture was added to 45 units of same ¯ask and
cultured under the same conditions for 35 days. The
cultured broth (4.5 L) was centrifuged, and the mycelial
cake was extracted with methanol (900 mL). The extract
was concentrated to a volume of 50 mL by an evapora-
tor, adjusted to pH 3 with hydrochloric acid and
extracted with ethyl acetate. The organic layer was
concentrated to give an oil (450 mg). The materials
were dissolved with chloroform-methanol (2:1) and
absorbed with silica gel (0.9 g, Wako gel C-200). This
was applied to a silica gel column (260£12 mm, i.d.),
washed with 450 mL of hexane±acetone (4:1) and
eluted with 450 mL of hexane±acetone (3:2). The eluate
was concentrated in vacuo to give an oil (281 mg). This
oily material was separated by HPLC [CAPCELL PAK
C18 UG120 (250£20 mm, i.d.); ¯ow rate, 20 mL/min;
detection, UV 210 nm; solvent, acetonitrile±0.1%
aqueous phosphoric acid (3:2)]. The fractions at
retention times 11.6 and 12.6 min were concentrated,
and the aqueous residues were extracted with ethyl
acetate. The extracts were concentrated to give amami-
statin A (1) (21.8 mg) and amamistatin B (2) (6.2 mg)
as brown oils, respectively.
Conclusion
Two new lipopeptides, amamistatins A (1) and B (2), were
isolated from N. asteroides. The absolute stereostructures of
1 and 2 were determined by spectroscopic and chemical
analyses. Amamistatin A (1) inhibited the growth of
human tumor cell lines such as MCF-7 breast, A549 lung,
and MKN45 stomach cancer cell lines. Further biological
studies of 1 and 2 are currently in progress.
Experimental
General aspects
Optical rotations were measured with a JASCO DIP-1000
polarimeter. NMR spectra were determined on a JEOL
JNM-A400 [400 MHz (1H)] or a JEOL JNM-A600
1
[600 MHz (1H) and 150 MHz (13C)] spectrometer. The H
Amamistatins A (1) and B (2). 1: [a]2D6ˆ29.8 (c 0.61,
MeOH); UV (MeOH) lmax 335 (e 11,000), 272 (e 22,000)
nm; FT/IR (CHCl3) 3340, 1740, 1660, 1500 cm21. 2:
[a]2D8ˆ28.2 (c 0.47, MeOH); UV (MeOH) lmax 307 (e
12,000), 266 (e 22,000) nm; FT/IR (CHCl3) 3350, 1740,
and 13C chemical shifts were referenced to the solvent peaks
[(dHˆ3.31 and dCˆ49.5 in methanol-d4), (dHˆ2.49 and
dCˆ39.5 in DMSO-d6), or (dHˆ7.26 in CDCl3)]. Infrared
(IR) spectra were measured with a JASCO FT/IR-230 spec-
trophotometer. UV/VIS spectra were recorded on a JASCO
V-550 spectrophotometer. High-resolution mass spectra
(HRMS) and FAB mass spectra (FAB-MS) were obtained
on a JEOL JMS-LG2000 mass spectrometer. Column chro-
matography was performed on silica gel (Wako gel C-200).
Reversed-phase high performance liquid chromatography
(HPLC) was carried out on CAPCELL PAK C18 UG120
(Shiseido Co., Ltd), Develosil ODS-HG-5, Develosil Ph-5
(Nomura Chemical Co., Ltd), or a chiral column CROWN-
PAK CR (1) (Daicel Chemical Ind., Ltd). Preparative TLC
was carried out using Merck precoated silica gel 60 F254
HPTLC plates. Absolute methanol was distilled from
Mg(OMe)2. Pyridine was distilled from calcium hydride.
1665, 1510 cm21
.
Hydrogenolysis of 1. To a solution of amamistatin A (1)
(1.6 mg, 2.1 mmol) in methanol (0.3 mL) was added 10%
palladium±carbon (3 mg). After being stirred at room
temperature for 8 h under a hydrogen atmosphere, the reac-
tion mixture was ®ltered through a pad of Celite, and the
residue was washed with methanol. The ®ltrate and wash-
ings were combined and concentrated. The residual oil was
puri®ed by HPLC [Develosil ODS-HG-5 (250£10 mm,
i.d.); ¯ow rate, 2 mL/min; detection, UV 254 nm; solvent,
methanol±H2O (4:1)] to give 3 (tRˆ43.7 min, 1.1 mg, 72%)
as a colorless oil: FT/IR (CHCl3) 3350, 1740, 1665,
1
1510 cm21; H NMR (600 MHz, CDCl3) d (ppm) 10.23
(s, 1H) [10.05 (s, 1H)], 8.31 (br d, Jˆ7.9 Hz, 1H) [8.16
(br d, Jˆ8.8 Hz, 1H)], 8.19 (br s, 1H), 7.68 (br d,
Jˆ6.2 Hz, 1H) [7.90 (br d, Jˆ5.3 Hz, 1H)], 7.30 (d,
Jˆ3.0 Hz, 1H), 6.98 (dd, Jˆ8.9, 3.0 Hz, 1H), 6.95 (d,
Jˆ8.9 Hz, 1H), 6.53 (br t, Jˆ6.3 Hz, 1H) [7.78 (br t,
Jˆ6.3 Hz, 1H)], 6.04 (m, 1H), 5.04 (dd, Jˆ10.5, 2.5 Hz,
1H) [4.97 (dd, Jˆ10.4, 2.6 Hz, 1H)], 4.71 (dd, Jˆ15.4,
7.9 Hz, 1H) [4.75 (ddd, Jˆ12.0, 8.8, 4.1 Hz, 1H)], 4.49
(ddd, Jˆ12.0, 6.2, 1.6 Hz, 1H) [4.47 (ddd, Jˆ11.7, 5.3,
1.6 Hz, 1H)], 3.84 (s, 3H) [3.83 (s, 3H)], 3.41 (m, 1H),
Isolation
The producing organism, strain SCRC-A2359, was isolated
from a soil sample collected at Amami Island in Kagoshima
Prefecture, Japan. Isolate was analyzed by MIDI's system
based on gas liquid chromatography analysis of fatty acids
produced from organism.8 A similarity index (SI) value of
0.667 with N. asteroides was obtained. This SI value is large
enough to suggest that isolate SCRC-A2359 is a strain of N.
asteroides. Strain SCRC-A2359 has been deposited in the

S. Kokubo et al. / Tetrahedron 56 (2000) 6435±6440
6439
3.24 (m, 1H), 3.22 (m, 2H) [3.26 (m, 2H)], 2.74 (s, 3H), 1.98
(m, 2H) [2.05 (m, 2H)], 1.98 (m, 1H), 1.86 (m, 1H) [1.82 (m,
1H)], 1.74 (m, 1H), 1.73 (m, 1H), 1.71 (m, 1H) [1.79 (m,
1H)], 1.61 (m, 1H), 1.59 (m, 1H), 1.58 (m, 2H) [1.61 (m,
2H)], 1.54 (m, 1H), 1.37 (m, 2H) [1.41 (m, 2H)], 1.25 (m,
1H), 1.23 (m, 2H) [1.21 (m, 2H)], 1.22 (m, 2H), 1.18 (m,
2H), 1.16 (s, 3H), 1.15 (s, 3H) [1.14 (s, 3H)], 1.12 (m, 2H),
1.11 (m, 1H), 0.82 (t, Jˆ7.1 Hz, 3H). The counterparts of
the doubled signals in brackets; FAB-MS m/z 714 [M1H]1.
form±methanol (49:1)] to give (S)-MTPA ester 9a. By the
same procedure as described above, (R)-MTPA ester 9b was
obtained by reacting alcohol 7 and (1)-MTPA chloride. 9a:
1H NMR (600 MHz, CDCl3) d (ppm) 7.58 (m, 2H), 7.39 (m,
3H), 7.17 (m, 1H), 5.87 (m, 1H), 5.41 (t, Jˆ6.0 Hz, 1H),
4.43 (dd, Jˆ11.1, 6.3 Hz, 1H), 3.53 (s, 3H), 3.25 (m, 2H),
2.00 (m, 1H), 1.99 (m, 1H), 1.85 (m, 1H), 1.80 (m, 1H), 1.49
(m, 2H), 1.42 (m, 1H), 1.38 (m, 1H), 1.32±1.18 (m, 6H),
1.26 (m, 2H), 1.25 (s, 3H), 1.19 (m, 2H), 1.17 (s, 3H), 0.86
(t, Jˆ7.0 Hz, 3H). 9b: 1H NMR (600 MHz, CDCl3) d (ppm)
7.60 (m, 2H), 7.39 (m, 3H), 7.17 (m, 1H), 5.86 (m, 1H), 5.41
(t, Jˆ6.3 Hz, 1H), 4.39 (dd, Jˆ10.4, 6.0 Hz, 1H), 3.55 (s,
3H), 3.24 (m, 2H), 2.02 (m, 1H), 2.00 (m, 1H), 1.86 (m, 1H),
1.79 (m, 1H), 1.53 (m, 2H), 1.43 (m, 1H), 1.39 (m, 1H),
1.33±1.25 (m, 4H), 1.30 (m, 2H), 1.26 (m, 2H), 1.23 (m,
2H), 1.20 (s, 3H), 1.13 (s, 3H), 0.88 (m, 3H). The Dd values
(dS2dR) in ppm: 20.07 (H-11), 20.04 (H-10), 0 (H-9),
10.05 (H-17), 10.04 (H-18), 10.04 (H-2).
Methanolysis of 3. Sodium methoxide solution was
prepared by adding sodium metal (46 mg, 2.0 mmol) to
absolute methanol (2 mL). To a solution of 3 (1.7 mg,
2.4 mmol) in absolute methanol (0.3 mL) was added sodium
methoxide solution (0.3 mL) with stirring. After being
stirred at room temperature for 4 h under a nitrogen
atmosphere, amberlite IRC-50 (H1 form, 150 mg) was
added to the solution and stirred for 20 min. The solution
was ®ltered through a cotton plug, and the residue was
washed with methanol. The ®ltrate and washings were
combined and concentrated. The residual oil was separated
by HPLC [Develosil Ph-5 (250£20 mm, i.d.); ¯ow rate,
5 mL/min; detection, UV 215 nm; solvent, gradation
(30 min) methanol±H2O (1:1!1:0)] to give methyl ester 5
(tRˆ30.9 min, 0.9 mg, 72%) and alcohol 7 (tRˆ32.9 min,
0.8 mg, quant.) as colorless oils, respectively. Methyl ester
Hydrogenolysis of 2. The experimental procedure was the
same as that described for compound 3. 4 (A colorless oil):
1H NMR (400 MHz, CD3OD) d (ppm) 8.17 (br s, 1H), 7.84
(dd, Jˆ8.3, 1.5 Hz, 1H), 7.40 (dt, Jˆ8.3, 8.3, 1.5 Hz, 1H),
7.04 (d, Jˆ8.3 Hz, 1H), 6.99 (t, Jˆ8.3 Hz, 1H), 5.11 (dd,
Jˆ10.2, 2.4 Hz, 1H), 4.64 (dd, Jˆ9.5, 5.6 Hz, 1H), 4.48 (d,
Jˆ10.9 Hz, 1H), 3.82±3.45 (m, 3H), 3.28±3.12 (m, 3H),
2.71 (s, 3H), 2.01±1.85 (m, 4H), 1.80±1.70 (m, 2H),
1.63±1.38 (m, 6H), 1.34±1.17 (m, 10H), 1.16 (s, 6H),
0.83 (t, Jˆ6.6 Hz, 3H); FAB-MS m/z 684 [M1H]1.
1
5: H NMR (600 MHz, CDCl3) d (ppm) 9.96 (s, 1H), 8.20
(d, Jˆ1.3 Hz, 1H), 7.31 (m, 1H), 7.29 (d, Jˆ2.8 Hz, 1H),
7.01 (m, 1H), 7.00 (m, 1H), 5.69 (br s, 1H), 4.72 (td, Jˆ8.2,
4.7 Hz, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.37 (m, 1H), 3.31
(m, 1H), 2.75 (s, 3H), 1.98 (m, 1H), 1.89 (m, 1H), 1.62 (m,
2H), 1.47 (m, 2H); FAB-MS m/z 420 [M1H]1. Alcohol 7:
1H NMR (400 MHz, CDCl3) d (ppm) 7.25 (br d, Jˆ6.1 Hz,
1H), 5.94 (br dd, Jˆ5.9, 4.6 Hz, 1H), 4.50 (ddd, Jˆ10.0,
6.1, 1.1 Hz, 1H), 3.59 (d, Jˆ6.3 Hz, 1H), 3.46 (ddd, Jˆ10.3,
6.3, 2.1 Hz, 1H), 3.33 (m, 1H), 3.27 (m, 1H), 2.04 (m, 1H),
2.04 (m, 1H), 1.88 (m, 1H), 1.80 (m, 1H), 1.49 (m, 1H), 1.41
(m, 1H), 1.31±1.25 (m, 10H), 1.25 (m, 2H), 1.24 (s, 3H),
1.18 (s, 3H), 0.87 (t, Jˆ6.8 Hz, 3H); FAB-MS m/z 327
[M1H]1.
Methanolysis of 4. The experimental procedure was the
same as that described for compounds 5 and 7. Methyl
ester 6 (a colorless oil): H NMR (400 MHz, CD3OD) d
1
(ppm) 8.02 (s, 1H), 7.85 (dd, Jˆ7.8, 1.5 Hz, 1H), 7.39
(ddd, Jˆ8.3, 7.2, 1.5 Hz, 1H), 7.03 (dd, Jˆ8.3, 1.2 Hz,
1H), 6.98 (ddd, Jˆ7.8, 7.2, 1.2 Hz, 1H), 4.58 (dd, Jˆ9.6,
5.5 Hz, 1H), 3.74 (s, 3H), 3.23 (m, 2H), 2.70 (s, 3H),
2.05±1.90 (m, 2H), 1.62±1.43 (m, 3H), 1.34 (m, 1H);
FAB-MS m/z 390 [M1H]1.
Acidic hydrolysis of 6 and 8. A solution of methyl ester 6
(50 mg, 0.1 mmol) in 6 M hydrochloric acid (0.1 mL) was
stirred at 1108C in a sealed tube for 1 day. The reaction
mixture was concentrated under reduced pressure. The
residue was separated by HPLC [Develosil ODS-HG-5
(250£10 mm, i.d.); ¯ow rate, 2 mL/min; detection, UV
205 nm; solvent 0.05% aqueous TFA] to give lysine
(tRˆ7.3 min). The stereochemistry of lysine from 6 was
determined to be d by chiral HPLC analysis [CROWNPAK
CR (1) (150£4.0 mm, i.d.); ¯ow rate, 0.4 mL/min; detec-
tion, UV 200 nm; solvent, 0.13% aqueous HClO4]. The
stereochemistry of lysine in 8 was determined to be l by
the procedure described above. The retention times of d and
l lysines were 7.0 and 8.2 min, respectively.
Acidic hydrolysis of 5 and 7. A solution of methyl ester 5
(0.5 mg, 1.2 mmol) in 6 M hydrochloric acid (0.1 mL) was
stirred at 1108C in a sealed tube for 1 day. The reaction
mixture was concentrated under reduced pressure. The
residue was separated by HPLC [Develosil ODS-HG-5
(250£4.6 mm, i.d.) £2; ¯ow rate, 1 mL/min; detection,
UV 205 nm; solvent 0.05% aqueous TFA] to give lysine
(tRˆ5.6 min). The stereochemistry of lysine from 5 was
determined to be d by chiral HPLC analysis [CROWNPAK
CR (1) (150£4.0 mm, i.d.); ¯ow rate, 0.4 mL/min; detec-
tion, UV 200 nm; solvent, 0.13% aqueous HClO4]. The
stereochemistry of lysine in alcohol 7 was determined to
be l by the same procedure as described above. The reten-
tion times of d and l lysines were 5.4 and 5.9 min,
respectively.
Esteri®cation of alcohol 7. To a solution of alcohol
7 (0.2 mg, 0.61 mmol) in pyridine (0.1 mL) was added
(2)-MTPA chloride (50 mg, 0.20 mmol). After being stir-
red at room temperature for 2 days, these reaction mixtures
were concentrated under blowing nitrogen gas. The residual
oil was separated by preparative TLC, Rfˆ0.44 [chloro-
Acknowledgements
This work was supported in part by Wako Pure Chemical
Industries Ltd and by Grants-in-Aid (Nos. 11175101 and
12045235) for Scienti®c Research from the Ministry of
Education, Science, Sports, and Culture, Japan.

6440
S. Kokubo et al. / Tetrahedron 56 (2000) 6435±6440
4. Murakami, Y.; Kato, S.; Nakajima, M.; Matsuoka, M.; Kawai,
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