Kulokekahilide-1, a cytotoxic depsipeptide from the cephalaspidean mollusk Philinopsis speciosa

Article abstract of DOI:10.1021/jo010176z

The cytotoxic depsipeptide kulokekahilide-1, which contains two unusual amino acids, 4-phenylvaline and 3-amino-2-methylhexanoic acid, was isolated from the cephalaspidean mollusk Philinopsis speciosa. Structure elucidation of kulokekahilide-1 was carried out by spectroscopic analysis and chemical degradation. The absolute stereochemistry was determined by Marfey analysis for amino acids and chiral HPLC analysis for hydroxy acids. All four stereoisomers of 4-phenylvaline and 3-amino-2-methylhexanoic acid, which were necessary for Marfey analysis, were synthesized by use of the Heck reaction and Evans's method, respectively. Kulokekahilide-1 showed cytotoxicity against P388 murine leukemia cells with an IC₅₀ value of 2.1 μg/mL.

Full text of DOI:10.1021/jo010176z

1760
J . Org. Chem. 2002, 67, 1760-1767
Ku lok ek a h ilid e-1, a Cytotoxic Dep sip ep tid e fr om th e
Cep h a la sp id ea n Mollu sk P h ilin opsis speciosa
J unji Kimura, Yuuki Takada, Tomoko Inayoshi, Yoichi Nakao, Gilles Goetz,
Wesley Y. Yoshida, and Paul J . Scheuer*
Department of Chemistry, University of Hawaii at Manoa, 2545 The Mall, Honolulu, Hawaii 96822-2275,
and Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University,
6-16-1 Chitosedai, Setagaya, Tokyo 157-8572, J apan
office@gold.chem.hawaii.edu
Received February 14, 2001
The cytotoxic depsipeptide kulokekahilide-1, which contains two unusual amino acids, 4-phenyl-
valine and 3-amino-2-methylhexanoic acid, was isolated from the cephalaspidean mollusk Phili-
nopsis speciosa. Structure elucidation of kulokekahilide-1 was carried out by spectroscopic analysis
and chemical degradation. The absolute stereochemistry was determined by Marfey analysis for
amino acids and chiral HPLC analysis for hydroxy acids. All four stereoisomers of 4-phenylvaline
and 3-amino-2-methylhexanoic acid, which were necessary for Marfey analysis, were synthesized
by use of the Heck reaction and Evans’s method, respectively. Kulokekahilide-1 showed cytotoxicity
against P388 murine leukemia cells with an IC50 value of 2.1 µg/mL.
In tr od u ction
chemistry of unusual amino acids often requires a
synthetic approach when they are not commercially
available. In the case of dolastatin 16, neither dolaphen-
valine nor dolamethylleucine was determined for their
absolute stereochemistry.4a We have synthesized all four
stereoisomers of these two unusual amino acids 2 and 3
and thus were able to determine the absolute stereo-
chemistry of 1.
In this paper, we describe isolation, structure elucida-
tion, and biological activity of this compound, as well as
synthesis of two unusual amino acids, 4-phenylvaline (2,
dolaphenvaline)4a and 3-amino-2-methylhexanoic acid (3).
The marine mollusk Philinopsis speciosa harbors a
variety of chemical constituents, which include alkyl-
pyridines,1 polypropionates,2 and especially peptides.3 Its
peptidic constituents are, to some extent, similar to those
of Dolabella auricularia and Onchidium sp., which
suggests their origin from dietary cyanobacteria.4 In fact,
cyclic peptides lyngbyabellins isolated from the marine
cyanobacterium Lyngbya majuscula contain 7,7-dichloro-
2,2-dimethyl-3-hydroxyoctanoic acid,5 which is presumed
to be a precursor of 2,2-dimethyl-3-hydroxyoctynoic acid
in kulolide-1 from P. speciosa.3a,b Further investigation
of the cytotoxic fraction from P. speciosa has led to the
isolation of a new cyclic bidepsipeptide, kulokekahilide-1
(1).6 Kulokekahilide-1 is related to dolastatin 16 isolated
by Pettit et al. from D. auricularia,4a which contains two
unusual amino acids: 4-phenylvaline (2, dolaphenvaline)
and 3-amino-2,4-dimethylpentanoic acid (dolamethylleu-
cine, Dml). Kulokekahilide-1 also involves two unusual
amino acids: the same 4-phenylvaline (2, Pval) and
3-amino-2-methylhexanoic acid (3, Amha) instead of
dolamethylleucine. Determination of absolute stereo-
Resu lts a n d Discu ssion
The organic extract of P. speciosa was evaporated and
separated by modified Kupchan procedure7 to yield
(1) Coval, S. J .; Schulte, G. R.; Matsumoto, G. K.; Roll, D. M.;
Scheuer, P. J . Tetrahedron Lett. 1985, 26, 5359-5362.
(2) Coval, S. J .; Scheuer, P. J . J . Org. Chem. 1985, 50, 3024-3025.
(3) (a) Reese, M. T.; Gulavita, N. K.; Nakao, Y.; Hamann, M. T.;
Yoshida, W. Y.; Coval, S. J .; Scheuer, P. J . J . Am. Chem. Soc. 1996,
118, 11081-11084. (b) Nakao, Y.; Yoshida, W. Y.; Szabo, C. M.; Baker,
B. J .; Scheuer, P. J . J . Org. Chem. 1998, 63, 3272-3280. (c) Nakao,
Y.; Yoshida, W. Y.; Scheuer, P. J . Tetrahedron Lett. 1996, 37, 8993-
8996.
(4) (a) Pettit, G. R.; Xu, J -p.; Hogan, F.; Williams, M. D.; Doubek,
D. L.; Schmidt, J . M.; Cerny, R. L.; Boyd, M. R. J . Nat. Prod. 1997,
60, 752-754. (b) Ferna´ndez, R.; Rodr´ıguez, J .; Quin˜oa´, E.; Riguera,
R.; Mun˜oz, L.; Ferna´ndez-Sua´rez, M.; Debitus, C. J . Am. Chem. Soc.
1996, 118, 11635-11643 and references therein.
(5) (a) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J . Mooberry,
S. L. J . Nat. Prod. 2000, 63, 611-615. (b) Milligan, K. E.; Marquez, B.
L.; Williamson, R. T.; Gerwick, W. H. J . Nat. Prod. 2000, 63, 1440-
1443. (c) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J . J . Nat.
Prod. 2000, 63, 1437-1439.
(6) Kekahi means another in Hawaiian, thereby kulokekahilide
means another kulolide.
10.1021/jo010176z CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/14/2002

Kulokekahilide-1
J . Org. Chem., Vol. 67, No. 6, 2002 1761
Ta ble 1. NMR Da ta of Ku lok ek a h ilid e-1 (1) in CD3CN
1H
1H
C
13C (ppm, mult, Hz)
HMBC
NOESY
C
13C (ppm, mult, Hz)
HMBC
NOESY
1
170.6
30a 26.1 2.28 m
30b 1.55 m
31 25.8 1.80 m
32a 47.2 3.09 m
2
3
4
5
6
7
8
9
77.3 5.38 d 2.7
29.1 2.17 m
20.5 0.93 d 6.3
16.5 0.97 d 6.7
170.4
60.2 5.07 d 11.1
26.5 2.24 m
19.9 0.85 d 6.5
C: 1, 3, 4, 5, 49 H: 5, 11; NH: 35
H: 32a,b
H: 18, 31
H: 18, 19, 31
C: 2, 3, 5
C: 2, 3, 4
C: 31
C: 31
32b
2.96 m
33 175.1
34 41.7 2.57 qd 7.2, 3.4 C: 33
35 50.8 3.88 m
36a 37.4 1.18 m
C: 6, 8, 9, 10, 11 H: 11, 16a,b
C: 9
C: 7, 8, 10
C: 7, 8, 9
H: 35
H: 34, 39
C: 34
10 18.2 0.78 d 6.7
11 30.1 2.99 s
12 173.4
36b
37a 20.2 1.13 m
37b 1.04 m
1.10 m
C: 1, 7
H: 7
13 62.4 4.37 dd 9.4, 2.2 C: 12
14a 31.3 2.20 m
38 14.3 0.71 t 6.9
39 15.1 0.87 d 7.2
C: 33, 34, 35
H: 35; NH: 35
H: 2, 29, 39
14b
15a 25.6 2.05 m
15b 1.98 m
2.01 m
NH
7.55 d 10.1
40 168.5
41 72.7 5.23 dd 9.0, 5.8 C: 33, 42, 43
H: 44, 48, 50
16a 48.5 3.79 ddd 9.1,
7.9, 2.4
H: 7, 16b; NH: 18
H: 7, 16a
42a 38.3 3.03 dd 13.7, 5.8 C: 40, 41, 43, 44 H: 44, 48, 50
16b
3.44 m
42b
2.96 dd 13.7, 9.0 C: 40, 41, 43, 44 H: 44, 48
17 172.1
43 137.4
18 52.5 4.92 bd 8.7
C: 17, 19, 20, 27 H: 19, 20a 22,
26, 32a,b
44 130.3 7.29
C: 46, 48
H: 41, 42a,b, 50
19 40.7 1.90 m
20a 41.8 2.61 dd 13.4, 7.5 C: 18, 19, 21,
22, 26, 27
H: 18, 22, 26, 32b
H: 18, 22, 26
45 129.5 7.31
46 128.0 7.25
C: 43, 47
C: 44, 48
20b
2.27 dd 13.4, 7.0 C: 18, 19, 21,
22, 26, 27
47 129.5 7.31
C: 43, 45
C: 44, 46
21 141.8
48 130.3 7.29
49 172.2
50 58.5 4.17 dd 8.7, 7.7 C: 49, 51, 52
51a 31.3 2.01 m
H: 41, 42a,b, 50
H: 41, 44, 48
22 130.5 7.26
23 129.1 7.27
24 127.0 7.19
25 129.1 7.27
26 130.5 7.26
27 14.8 0.62 d 6.7
C: 24, 26
C: 21, 25
C: 22, 26
C: 21, 23
C: 22, 24
C: 18
H: 18, 19, 20a
51b
52a 22.7 2.00 m
52b 1.83 m
1.92 m
H: 18, 19, 20a
H: 22, 26, 53a;
NH: 18
H: 53b
NH
28 171.7
6.70 d 8.9
C: 12
H: 27
53a 47.2 3.48 m
53b 3.26 m
H: 27, 53a
H: 52b, 53a
29 60.0 4.56 d 7.2
C: 17, 28, 30,
31, 32
NH: 35
hexane, CH2Cl2, and aqueous MeOH extracts. The CH2-
Cl2 extract was submitted to two-step ODS flash chro-
matography, followed by gel filtration, and amino short
column chromatography. The fraction containing pep-
tides was further separated by ODS HPLC yielding nine
fractions (fractions 1-9). From fractions 6 and 7, kulo-
kekahilide-1 (1; 1.4 mg; (1.6 × 10-5)% yield based on wet
weight) was obtained after repetitive ODS HPLC.
The molecular formula of kulokekahilide-1 (1) was
determined as C53H74N6O10 on the basis of HR-FABMS
[m/z 955.5550 (M + H)+ (∆ +0.5 mmu)] and NMR
spectral analysis (Table 1). NMR analysis revealed that
N-methylvaline (MeVal), phenyllactic acid (Plac), 2-hy-
droxy-3-methylbutyric acid (Hmba), and three residues
of Pro are present, as well as the two other unusual
amino acids 2 and 3.
The amino acid 2 contains a phenyl group at the
γ-position, which was supported by HMBC correlations
between H2-20/C-21 and 22. From CH2-20, COSY cor-
relations could be traced to the NH bonded to C-18 via
CH-19, which was also bearing a methyl group. The
R-methine proton of this unit also showed an HMBC8
correlation to a carbonyl carbon at δ 172.1, thereby
completing the substructure of 4-phenylvaline (2, Pval).
The last amino acid residue 3 was elucidated as
follows. Connecting COSY cross-peaks led to the two spin
systems of NH-CH-CH2-CH2-CH3 and CH3-CH. Although
no COSY cross-peak was seen between the two methine
protons, HMBC correlations between H-35/C-34 and H3-
39/C-35 confirmed the connectivity between C-34 and 35.
Further long-range CH coupling between H3-39 and
carbonyl carbon at δ 175.1 revealed the structure of
3-amino-2-methylhexanoic acid (3, Amha).
Sequencing of these units was accomplished by HMBC
and NOESY analyses. HMBC correlations revealed three
sets of fragments, i.e., Pro3-Hmba-MeVal (H-2/C-49 and
H3-11/C-1), Pro1-Pval-Pro2 (NH-18/C-12 and H-29/C-17),
and Amha-Plac (H-41/C-33). NOESY cross-peaks (H-7/
H2-16, H-29/NH-35, H-41/H-50, and H-42a/H-50) con-
nected these substructures to complete the planar struc-
ture of 1 (Figure 1). Syn th esis of Un u su a l Am in o
Acid s 2 a n d 3. As the planar structure of 1 was
determined, we next embarked on synthesis of all four
stereoisomers of 2 and 3, which are necessary for Marfey
analysis.9 The synthetic approach to 2 involved a Heck
reaction of 3,4-dehydrovaline with halobenzene,10 fol-
lowed by catalytic hydrogenation. The stereochemistry
was determined by NMR spectroscopy and X-ray analy-
(7) Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Sigel, C. W. J .
Org. Chem. 1973, 38, 178-179.
(8) Bax, A.; Azolos, A.; Dinya, Z.; Sudo, K. J . Am. Chem. Soc. 1986,
108, 8056-8063.
(9) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
(10) Browing, A. F.; Greeves, N. Transition Metal in Organic
Synthesis; Gibson, S. E., Ed.; Oxford University Press: New York,
1997; Chapter 2, pp 35-50.

1762 J . Org. Chem., Vol. 67, No. 6, 2002
Kimura et al.
To our surprise, the diastereomers (7a and 7b) gave
better separation in Si-HPLC with 20% ethyl acetate in
hexane than the diastereomers 6, which were barely
separated by HPLC. Purified 7a and 7b were hydrolyzed
by a 2:1 mixture of 6 M hydrochloric acid and acetic acid
under reflux to give the corresponding 4-phenylvaline
(2a , 2S3R) and 2b (2S3S), respectively. Similarly, (2R)-
N-phthaloyl-3,4-dehydrovaline methyl ester (8) obtained
from D-valine was reacted with iodobenzene in the
presence of palladium acetate and silver nitrate to afford
(2R)-N-phthaloyl-3,4-dehydro-4-phenylvaline methyl es-
ter (9). Meanwhile, compound 9 was converted into the
diastereomers (10c and 10d ) of (2R)-N-cyclohexanedi-
carboxy-4-phenylvaline methyl ester by complete cata-
lytic hydrogenation. The diastereomers of 10c and 10d
could also be separated by HPLC. Hydrolysis of 10c and
10d afforded the corresponding free 4-phenylvaline (2c;
F igu r e 1. Amino acid sequence of 1.
sis. However, 3 was diastereoselectively synthesized with
optically pure oxazolidinones and aldehydes by use of
Evans’s method.11
Heck reaction between 3,4-dehydrovaline and haloben-
zene was applied to the synthesis of 4-phenylvaline.
Readily available starting (S)- or (R)-valine was trans-
formed into 3,4-dehydrovaline derivatives using known
procedures.12
The reaction of (2S)-N-phthaloyl-3,4-dehydrovaline
methyl ester (4) with iodobenzene in the presence of
palladium acetate and triethylamine afforded undesired
N-phthaloyl-2,3-dehydrovaline methyl ester, quantita-
tively. The product was formed as the result of hydrogen
transfer by triethylamine acting as base. The alternative
reaction of using silver nitrate instead of triethylamine
gave (2S)-N-phthaloyl-3,4-dehydro-4-phenylvaline methyl
ester (5) in 51% yield. Catalytic hydrogenation of 5
afforded diastereomeric mixtures of (2S)-N-phthaloyl-4-
phenylvaline methyl ester (6) and (2S)-N-1,2-cyclohex-
anedicarboxy-4-phenylvaline methyl ester (7) in an ap-
proximately 1:1 ratio. Generally, hydrogenation of an
aromatic ring needs high pressure or Raney Ni catalyst;
therefore the formation of compound 7 was not expected.
1
2R3S) and 2d (2R3R). The H NMR spectrum of 10c is
superimposable on that of 7a , and likewise, 10d has the
same spectrum as 7b; this is due to their enantiomeric
relationship (Scheme 1).
1
The H NMR spectrum of 7a was similar to that of 7b
except for two sets of signals. While one doublet of
doublets was observed at δ 3.15 (J ) 13.3, 3.8 Hz, Hb-4)
and one doublet at δ 0.76 (CH3-3) in 7a , the chemical
shifts of the corresponding protons in 7b were δ 2.67
(J ) 14.6, 6.7 Hz, Hb-4) and 1.00 (CH3-3). These results
suggested that Hb-4 in 7a and CH3-3 in 7b were
deshielded by the carboxyl group. Irradiation of δ 2.28
(Ha-4) increased intensity of the peaks at δ 7.17-7.32
(H-6, H-10), 4.55 (H-2), and 0.76 (CH3-3). Similarly,
NOEs between Ha-4/H-14 and H-2/CH3-3 in 7b were
observed (Figure 2). These coupling constants and NOEs
were reasonably explained by a Dreiding model; thus
crystallized 7a seems to be the 2S3R isomer, whereas
the oily 7b is the 2S3S isomer.
Sch em e 1a
a
(a) Idobenzene, AgNO3, Pd(OAc)2, MeCN; (b) H2, PtO2, EtOH; (c) 6 M HCl-AcOH (2:1), 120 °C.

Kulokekahilide-1
J . Org. Chem., Vol. 67, No. 6, 2002 1763
was recently applied by Riguera et al.14 The enol borinate
of optically pure N-propionyl oxazolidinones and alde-
hydes was diastereoselectively coupled to obtain enan-
tiomerically pure aldols, whose hydroxyl group was
converted to an amino group by an SN2 reaction (Scheme
2).
Reaction of N-propionyl oxazolidinone (11a ) with n-
butanal gives the aldol 12 with 2S3R configuration,
whereas the reaction of oxazolidinone 11b with the same
aldehyde provides aldol 13 with 2R3S configuration.
Compounds 12 and 13 were obtained optically pure since
no other diastereomers were detected in the 1H NMR
spectrum. Tosylation in pyridine afforded intermediates
14 and 15 with yields of 70% and 68%, respectively.
Reaction with sodium or tetramethylguanidium azide
provided the azido derivatives 16 and 17 with inversion
of configuration at C-3. Hydrogenation over palladium-
charcoal provided 3-amino-2-methylhexanoic acid (3c,
2S3S; 3d , 2R3R).
F igu r e 2. NOE correlations of 7a and 7b.
Auspiciously, 7a and 10c crystallized from ethanol, and
we could perform single-crystal X-ray analysis in order
to confirm the absolute configurations. Crystallographic
data, data collection, and reduction parameters are
summarized in Supporting Information. The molecular
structures of 7a and 10c are shown in Figure 3. Dihedral
angles (ø) of H(3)-C(3)-C(4)-Ha(4) and H(3)-C(3)-
C(4)-Hb(4) for 7a are 193° and 76°. The intramolecular
nonbonded distances of H(2)-Ha(4) and H(2)-Hb(4) are
2.61 and 2.52 Å, respectively. Similarly, Ha(4)-H(6) and
Hb(4)-H(10) are 2.52 and 2.38 Å, respectively. Analyses
of their crystal structures well agreed with NMR spectral
data. Also, the cis relationship between H-14 and H-19
in 7a was confirmed.
To prepare the two other stereoisomers (3a , 2S3R; 3b,
2R3S), inversion of one of the chiral centers of both 12
and 13 was needed. We decided to invert the configura-
tion at C-3 and tried a modified Mitsunobu reaction,15
which unfortunately did not work. Our next strategy was
to invert the configuration at C-3 of tosylate 14 and 15
using bromine, which successfully yielded the desired 18
(2S3R) and 19 (2R3S). To avoid decreasing yield, azido
20 and 21 were partially purified before being used for
the synthesis of 3a and 3b. Thus, all four theoretically
possible isomers of 4-phenylvaline and 3-amino-2-methyl-
hexanoic acid were successfully prepared.
The synthetic strategy for all four stereoisomers of 3
relied on the reaction developed by Evans et al.,13 which
F igu r e 3. Molecular structures for 7a and 10c.

1764 J . Org. Chem., Vol. 67, No. 6, 2002
Kimura et al.
Sch em e 2a
a
(a) (nBu)2BOTf, iPr2EtN, CH2Cl2, 0 °C and then 1-butanal, -78 °C; (b) p-TsCl, Py; (c) tetramethyl-guanidium azide, CH2Cl2 or NaN3,
15-crown-5, DMF; (d) H2O2, LiOH then HCl, and H2, Pd-C, AcOH-H2O; (e) LiBr, acetone reflux.
using a KBr disk. Melting points were determined in open
capillary tubes. NMR spectra were recorded at 500 or 300 MHz
for 1H and 125 and 75 MHz for 13C. Glycerol was used as
matrix for FAB-MS measurements.
Absolu te Ster eoch em istr y of 1. The absolute ster-
eochemistry of the proteinogenic amino acids was deter-
mined by Marfey analysis as D-MeVal and three L-Pro.
The stereochemistry of 4-phenylvaline was also deter-
mined as 2S3R by Marfey analysis by using all four
stereoisomers synthesized as above. All four synthetic
Amha were also used in Marfey analysis, which showed
2R3R-Amha is in 1. The absolute stereochemistry of the
hydroxy acids was determined by chiral HPLC analysis
as L-phenyllactic acid and S-2-hydroxy-3-methylbutyric
acid.
Isola tion . Philinopsis speciosa (300 animals, 9.0 kg wet
weight) collected on midsummer nights in 1994 at Shark’s
Cove, Pupukea, O’ahu, were extracted with EtOH (3 × 3 L)
and CHCl3/MeOH (1:1, 3 L). The combined extracts were
concentrated and extracted with CHCl3. The aqueous layer was
further extracted with n-BuOH, and the n-BuOH extract was
combined with the CHCl3 layer. The combined organic layers
were evaporated to dryness and separated by the modified
Kupchan procedure to yield hexane, CH2Cl2, and aqueous
MeOH extracts. The CH2Cl2 extract was evaporated to dryness
and submitted to two-step ODS flash chromatography (first
with aqueous MeOH as solvent, second with aqueous MeCN),
followed by gel filtration (Sephadex LH-20, MeOH) and amino
short column chromatography [Lichroprep NH2, φ 1.5 × 3.5
cm, CHCl3, CHCl3/MeOH (9:1), CHCl3/MeOH/H2O (7:3:0.5),
and MeOH]. The CHCl3/MeOH (9:1) fraction was separated
by ODS HPLC [COSMOSIL 5C18-AR, MeCN/H2O (7:3)], giving
nine fractions (fractions 1-9). Fractions 6 and 7 were sepa-
rated by the same scheme [COSMOSIL 5C18-AR, 2-PrOH/H2O
(1:1)], and fractions containing identical peaks were combined.
The combined fraction was purified by repetitive ODS HPLC
[COSMOSIL 5C18-AR, with MeCN/H2O (65:35) and then with
2-PrOH/H2O (47.5:52.5)], yielding kulokekahilide-1 (1; 1.4 mg;
1.6 × 10-5 % yield based on wet weight). Ku lok ek a h ilid e-1
(1): colorless amorphous solid; [R]D +22° (c 0.07, MeOH); UV
(MeOH) 235 nm (ꢀ 3,200), 205 (ꢀ 14,000); HR-FABMS (M +
H)+ m/z 955.5550 (calcd for C53H75N6O10 955.5545).
Biologica l Activity. Although dolastatin 16 showed
marked cytotoxicity against several cancer cell lines,4a
cytotoxicity of kulokekahilide-1 (1) against P388 was only
moderate (IC50 2.1 µg/mL).
Exp er im en ta l Section
Gen er a l P r oced u r es. UV spectra were measured with a
diode array spectrophotometer. Optical rotations were mea-
sured on a digital spectropolarimeter. IR spectra were recorded
(11) Evans, D. A.; Bartroli, J .; Shih, T. L. J . Am. Chem. Soc. 1981,
103, 2127-2129.
(12) Sakai, R.; Stroh, J . G.; Sullins, D. W.; Reinhart, K. L. J . Am.
Chem. Soc. 1995, 117, 3734-3748.
(13) Evans, A. E.; Britton, T. C.; Elleman, J . A.; Dorow, R. L. J .
Am. Chem. Soc. 1990, 112, 2, 4011-4030.
(14) Fernandez, R.; Rodriguez, J .; Quinoa, E.; Riguera, R.; Munoz,
L.; Fernandez-Suarez, M.; Debitus, C. J . Am. Chem. Soc. 1996, 118,
11635-11643.
N-P h th a loyl-2,3-d eh yd r ova lin e Meth yl Ester . (2S)-N-
Phthaloyl-3,4-dehydrovaline methyl ester (4) was obtained
(15) (a) Mitsunobu, O. Synthesis 1981, 1-28. (b) Galynker, I.; Still,
W. C. Tetrahedron Lett. 1982, 23, 4461-4464.

Kulokekahilide-1
J . Org. Chem., Vol. 67, No. 6, 2002 1765
from (S)-valine by the method of Easton et al.16 Iodobenzene
(204 mg, 1.0 mmol), triethylamine (152 mg, 1.5 mmol), and
palladium acetate (2.3 mg, 0.01 mmol) were added with
stirring to a solution of 4 (329 mg, 1.3 mmol) in MeCN (1 mL)
under nitrogen. After reflux for 8 h, the reaction mixture was
hydrolyzed with water (20 mL) and extracted with ether (20
mL × 2). The organic layer was dried over sodium sulfate,
which was removed by filtration. The solvent was evaporated
under reduced pressure, and the oily residue was identified
to be undesired N-phthaloyl-2,3-dehydrovaline by NMR analy-
H-17), 1.86 (m, 4H,H-15, H-18), 2.28 (dd, 1H, J ) 13.2, 9.9
Hz, Ha-4) 2.82 (m, 1H, H-3), 2.93 (m, 2H, H-14, H-19), 3.16
(dd, 1H, J ) 13.2, 4.0 Hz, Hb-4), 3.73 (s, 3H, -OCH3), 4.55 (d,
1H, J ) 7.6 Hz, H-2), 7.17-7.32 (m, 5H, Ar-H). Anal. Calcd
for C20H25NO4: C, 69.95; H, 7.34; N, 4.08. Found: C, 69.54;
1
H, 7.33; N, 3.99. Com p ou n d 10d (2R,3R): H NMR (CDCl3)
δ 1.00 (d, 3H, J ) 6.8 Hz, CH3-3), 1.43 (m, 4H, H-16, H-17),
1.82 (m, 4H, H-15, H-18), 2.23 (dd, 1H, J ) 14.7, 11.0 Hz, Ha-
4), 2.68 (dd, 1H, J ) 14.5, 6.9 Hz, Hb- 4), 2.82 (m, 3H, H-3,
H-14, H-19), 3.72 (s, 3H, -OCH3), 4.55 (d, 1H, J ) 7.3 Hz, H-2),
7.13-7.29 (m, 5H, Ar-H). N-Cycloh exylca r boxy-4-cyclo-
h exylva lin e (d ia ster eom er ic m ixtu r e): colorless oil, 1H
NMR (CDCl3) δ 0.86, 1.00 (d, CH3-3), 2.58 (m, H-2), 2.90 (m,
H-3, H-14, H-19), 3.72 (s, -OCH3), 4.41 and 4.43 (d, H-2). This
product was not further investigated.
1
sis. H NMR (CDCl3) δ 1.83, 2.39 (s, CH3-3), 3.64 (s, -OCH3),
7.75, 7.88 (m, phthaloyl-H). The product was not further
investigated.
(2S)-N-P h th a loyl-4-p h en yl-3,4-d eh yd r ova lin e (5). The
reaction using silver nitrate instead of triethylamine proceeded
to give desired product 5. Iodobenzene (408 mg, 2.0 mmol),
palladium acetate (190 mg, 0.85 mmol), and silver nitrate (340
mg, 2.0 mmol) were added with stirring to a solution of 4 (442
mg, 1.7 mmol) in MeCN (4 mL) under nitrogen. After 6 h at
50 °C, the reaction mixture was hydrolyzed with water (20
mL) and extracted with ether (20 mL × 2). The organic extract
was separated by PLC (EtOAc/benzene 1:14) to give 5 in 51%
yield (colorless oil). 1H NMR (CDCl3) δ 2.04 (s, 3H, CH3-3),
3.81 (s, 3H, -OCH3), 5.51 (s, 1H, H-2), 6.63 (s, 1H, H-4), 7.30
(m, 5H, aromatic-H), 7.75, 7.89 (m, 4H, phthaloyl-H); 13C NMR
(CDCl3) δ 16.2 (q, C-11), 52.8 (q, C-12), 59.4 (d, C-2), 123.6 (d,
C-15, C-18), 127.0, 128.1, 129.1 (d, C- 6, C-7, C-8, C-9, C-10),
131.5 (s, C-3), 131.8 (s, C-14, C-19) 131.9 (d, C-4), 134.2 (d,
C-16, C-17), 136.6 (s, C-5), 167.3 (s, C-13, C-20), 168.4 (s, C-1).
(2S,3R)- a n d (2S,3S)-N-1,2-cycloh exa n ed ica r b oxy-4-
p h en ylva lin e Meth yl Ester s (7a ,b). Compound 5 (293 mg)
was hydrogenated in the presence of platinum oxide (60 mg)
in ethanol (7 mL). After removal of the catalyst, the reaction
mixture was separated by PLC by use of ethyl acetate and
benzene in a ratio of 1:14. The products were mixtures of
N-phthaloyl-4-phenylvaline methyl ester (6, 144 mg, 50%) and
the byproducts (7, 112 mg, 38%), which were unexpectedly
formed by hydrogenation of the phthaloyl group. Part of
byproducts 7 was purified by HPLC to afford 7a and 7b in a
1:1 ratio, but the diastereomer 6 could not be separated.
Com p ou n d 6 (d ia ster eom er ic m ixtu r e): 1H NMR (CDCl3)
δ 0.79, 1.00 (d, CH3-3), 2.24, 2.31 (dd, Ha -4), 2.88 (m, H-3,
and one diastereomer Hb-4), 3.12 (dd, the other Hb-4), 4.71,
4.72 (d, H-2), 7.12 (m, aromatic-H), 7.55, 7.70, 7.80 (m,
phthaloyl-H). Com p ou n d 7a (2S,3R): crystallized from EtOH,
mp 68-70 °C; LREIMS m/z 343 [M+, C20H25NO4]; HREIMS
m/z 312.1563 [M - OCH3]+ (calcd for C19H22NO3 312.1594);
1H NMR (CDCl3) δ 0.76 (d, 3H, J ) 6.8 Hz, CH3-3), 1.46 (m,
4H, H-16, H-17), 1.87 (m, 4H,H-15, H-18), 2.28 (dd, 1H, J )
13.2, 9.8 Hz, Ha-4) 2.81 (m, 1H, H-3), 2.93 (m, 2H, H-14, H-19),
3.15 (dd, 1H, J ) 13.3, 3.8 Hz, Hb-4), 3.73 (s, 3H, -OCH3), 4.55
(d, 1H, J ) 7.9 Hz, H-2), 7.17-7.32 (m, 5H, Ar-H); 13C NMR
(CDCl3) δ 16.1 (q, C-11), 22.2 (t, C-16, C-17), 24.0, 24.4 (t, C-15,
C-18), 35.6 (d, C-3), 40.3, 40.1 (d, C-14, C-19), 41.6 (t, C-4),
52.9 (q, C-12), 56.3 (d, C -2), 126.6, 128.8, 129.8 (d, C- 6,
C-7, C-8, C-9, C-10), 140.4 (s, C-5), 169.6 (s, C-1), 179.5, 179.6
(s, C-13, C-20). Com p ou n d 7b (2S,3S): colorless oil, LREIMS
m/z 343 [M+, C20H25NO4]; 1H NMR (CDCl3) δ 1.00 (d, 3H, J )
6.7 Hz, CH3-3), 1.44 (m, 4H, H-16, H-17), 1.83 (m, 4H, H-15,
H-18), 2.23 (dd, 1H, J ) 14.6, 11.0 Hz, Ha-4), 2.67 (dd, 1H, J
) 14.6, 6.7 Hz, Hb- 4), 2.82 (m, 3H, H-3, H-14, H-19), 3.72 (s,
3H, -OCH3), 4.55 (d, 1H, J ) 7.5 Hz, H-2), 7.13-7.29 (m, 5H,
Ar-H).
Isola t ion of 4-P h en ylva lin e. (2S,3R)-4-P h en ylva lin e
(2a ). Compound 7a (18.7 mg) was treated with 6 M hydro-
chloric and glacial acetic acids (2:1, 1 mL) at 120 °C for 8 h.
The reaction mixture was evaporated by reduced pressure, and
the residue was applied to ODS-HPLC using MeOH/H2O (30:
70). Compounds 2a was obtained in 42% yield: [R]D +25° (c
0.11, H2O); mp 210-220 °C (dec); HRFABMS m/z 194.1182
+
1
[M + H] (calcd for C11H16NO2 194.1181); H NMR (D2O) δ
0.80 (d, J ) 6.7 Hz, CH3-3), 2.42 (m, H-3), 2.51 (dd, J ) 13.3,
9.2 Hz, H-4a), 2.71 (dd, J ) 12.9, 5.7 Hz, H-4b), 3.56 (d, J )
1.5 Hz, H-2), 7.19-7.31 (Ar-H); 13C NMR (D2O) δ 13.3 (q, C-11),
36.2 (d, C-3), 38.7 (t, C-4), 58.7 (d, C-2), 126.7, 128.9, 129.2 (d,
C-6, C-7, C-8, C-9, C-10), 139.7 (s, C-5), 174.2 (s, C-1).
(2S,3S)-4-P h en ylva lin e (2b). Similarly, 2b was obtained
by hydrolysis of 7b in 54% yield: [R]D +9° (c 0.12, H2O); mp
1
185-187 °C (dec); H NMR (D2O) δ 0.81 (d, J ) 6.8 Hz, CH3-
3), 2.25 (m, H-3), 2.41 (dd, J ) 13.3, 10.1 Hz, H-4a), 2.74 (dd,
J ) 13.0, 4.6 Hz, H-4b), 3.61 (d, J ) 1.5 Hz, H-2), 7.17-7.29
(Ar-H); 13C NMR (D2O) δ 14.4 (q, C-11), 36.5 (d, C-3), 38.0 (t,
C-4), 59.3 (d, C-2), 126.6, 128.7, 129.4 (d, C-6, C-7, C-8, C-9,
C-10), 140.0 (s, C-5), 173.8 (s, C-1).
(2R,3S)-4-p h en ylva lin e (2c). Compound 10c was hydro-
lyzed with 6 M hydrochloric and glacial acids to afford 2c: [R]D
-34° (c 0.11, H2O); mp 235-239 °C (dec); 1H NMR (D2O) δ
0.80 (d, J ) 6.7 Hz, CH3-3), 2.42 (m, H-3), 2.51 (dd, J ) 13.3,
9.2 Hz, H-4a), 2.71 (dd, J ) 12.9, 5.7 Hz, H-4b), 3.56 (d, J )
1.5 Hz, H-2), 7.19-7.31 (Ar-H); 13C NMR (D2O) δ 13.3 (q,
C-11), 36.2 (d, C-3), 38.7 (t, C-4), 58.7 (d, C-2), 126.7, 128.9,
129.2 (d, C-6, C-7, C-8, C-9, C-10), 139.7 (s, C-5), 174.2 (s, C-1).
(2R,3R)-4-p h en ylva lin e (2d ). Using the above method for
hydrolysis, 2d was obtained from 10d : [R]D -13° (c 0.11, H2O);
mp 193-195 °C (dec, recrystallized from aqueous EtOH); IR
(KBr) 3431, 3032, 2978, 2933, 1591, 1512, 1402, 1335, 733,
1
702 cm-1; H NMR (D2O) δ 0.81 (d, J ) 6.8 Hz, CH3-3), 2.25
(m, H-3), 2.41 (dd, J ) 13.3, 10.1 Hz, H-4a), 2.74 (dd, J ) 13.0,
4.6 Hz, H-4b), 3.61 (d, J ) 1.5 Hz, H-2), 7.17-7.29 (Ar-H); 13
C
NMR (D2O) δ 14.4 (q, C-11), 36.5 (d, C-3), 38.0 (t, C-4), 59.3
(d, C-2), 126.6, 128.7, 129.4 (d, C-6, C-7, C-8, C-9, C-10), 140.0
(s, C-5), 173.8 (s, C-1). Anal. Calcd for C11H15NO2: C, 68.37;
H, 7.82; N, 7.25. Found: C, 68.86; H, 7.76; N, 7.12.
(4S,2′S,3′R)-3-(3′-Hydr oxy-2′-m eth ylpen tyl)-4-isopr opyl-
2-oxa zolid in on e (12). A stirred solution of N-propionyl
oxazolidinone 11a (1.0 g, 5.4 mmol) in dry CH2Cl2 (15 mL)
under argon was treated with 1 M dibutylboron triflate (6.0
mL, 6.0 mmol, 1.1 equiv in CH2Cl2) and diisopropylmethy-
lamine (1.15 mL, 6.5 mmol, 1.2 equiv) at 0 °C. After 30 min,
the reaction mixture was cooled to -78 °C, and n-butanal (0.55
mL, 6.0 mmol, 1.1 equiv) was added dropwise. The resulting
mixture was stirred at -78 °C for 30 min and then 90 min at
room temperature. The reaction was quenched by addition of
pH 7 aqueous phosphate buffer (10 mL) and oxidized with 30%
hydrogen peroxide/methanol (1:1, 20 mL). The resulting solu-
tion was stirred at 0 °C for 1 h. The solvent was evaporated
under reduced pressure, and the residue was freeze-dried. The
residue was dissolved in water (30 mL) and extracted with
EtOAc (25 mL × 3). The combined organic layer was washed
with 5% NaHCO3 (25 mL) and brine (25 mL), dried over
MgSO4, and concentrated under reduced pressure, yielding a
viscous yellow oil. The crude oil was purified by flash chro-
matography (EtOAc/hexane 25:75), and the aldol 12 was
(2R,3S)- a n d (2R,3R)-N-Cycloh exa n ed ica r b oxy-4-
p h en ylva lin e Meth yl Ester (10c,d ). Similarly, (2R)-N-
phthaloyl-3,4-dehydro-4-phenylvaline methyl ester (9) obtained
from D-valine was completely hydrogenated by use of PtO2.
Compounds 10c and 10d were obtained in 34% and 27% yields,
respectively, and N-cyclohexylcarboxy-4-cyclohexylvaline was
also obtained in 22% yield. Compound 10c was recrystallized
from EtOH (mp 74-76 °C). Com p ou n d 10c (2R,3S): 1H NMR
(CDCl3) δ 0.76 (d, 3H, J ) 6.9 Hz, CH3-3), 1.46 (m, 4H, H-16,
(16) Easton, C. J .; Merrett, M. C. Tetrahedron 1997, 53, 1151-1156.

1766 J . Org. Chem., Vol. 67, No. 6, 2002
Kimura et al.
obtained as colorless oil (1.17 g, 4.55 mmol, 84%): [R]D +38°
(c 1.0, CHCl3); HREIMS m/z 239.1525 [M+ - H2O] (calcd for
C13H21NO3 239.1522); IR (neat) 3426, 2960, 1780, 1749, 1686,
(4S,2′S,3′S)-3-(3′-Azid o-2′-m eth ylp en tyl)-4-isop r op yl-2-
oxa zolid in on e (16). To a solution of tosylate 14 (207 mg, 0.50
mmol) in DMF (2 mL) under nitrogen was added sodium azide
(98 mg, 1.5 mmol) and 15-crown-5 (10 mg, 0.1 equiv). The
solution was stirred at 70 °C for 4.5 h, allowed to cool to room
temperature, then quenched with water (15 mL), and extracted
with EtOAc (10 mL × 4). The combined organic layer was dried
over MgSO4 and concentrated under reduced pressure, yielding
a viscous yellow oil. The crude oil was purified by PLC (EtOAc/
hexane 1:3), and azide 16 was obtained as a colorless oil (77
mg, 0.27 mmol, 54%): [R]D +114° (c 1.0, CHCl3); HREIMS m/z
254.1641 [M - N2] + (calcd for C13H22N2O3 254.1625); IR (neat)
2963, 2936, 2876, 2103, 1782, 1698, 1456, 1398, 1456, 1386,
1
1386, 1203 cm-1; H NMR (300 MHz, CDCl3) δ 0.86 (d, 3H, J
) 7.1 Hz, H-7), 0.90 (d, 3H, J ) 7.1 Hz, H-8), 0.92 (t, 3H, J )
5.9 Hz, H-6′), 1.23 (d, 3H, J ) 6.6 Hz, H-7), 1.27-1.40 (m, 2H,
H-5′), 1.41-1.58 (m, 2H, H-4′), 2.33 (dsept, 1H, J ) 4.0, 7.1
Hz, H-6), 2.92 (s, br, 1H, OH), 3.74 (dq, 1H, J ) 2.5, 7.1 Hz,
H-2′), 3.93 (m, 1H, H-3′), 4.20 (dd, 1H, J ) 3.0, 9.1 Hz, H-5a),
4.27 (t, 1H, J ) 3.0, 9.1 Hz, H-5b), 4.46 (dt, 1H, J ) 3.5, 8.1
Hz, H-4); 13C NMR (CDCl3) δ 10.6, 13.9, 14.5, 17.8, 19.1, 28.2,
35.8, 41.9, 58.1, 63.2, 70.8, 153.4, 177.8.
(4R,5S,2′R,3′S)-3-(3′-Hyd r oxy-2′-m eth ylp en tyl)-5-p h en -
yl-4-m eth yl-2-oxa zolid in on e (13). Using the method de-
scribed for the preparation of 12, N-propionyloxazolidinone
11b (1.0 mL, 5.0 mmol) was treated with 1 M dibutylboron
triflate (5.5 mL, 5.5 mmol, 1.1 equiv in CH2Cl2) and diisoprop-
ylethylamine (1.1 mL, 6.0 mmol, 1.2 equiv). The resulting enol
borinate was allowed to react with n-butanal (0.5 mL, 5.5
mmol, 1.1 equiv). After workup and purification, aldol 13 was
obtained as a colorless oil (1.31 g, 4.32 mmol, 86%): [R]D +1.7°
(c 2.0, CHCl3); HREIMS m/z 287.1523 [M - H2O]+ (calcd for
1
1246, 1204 cm-1; H NMR (CDCl3) δ 0.88 (d, 3H, J ) 6.8 Hz,
H-7), 0.93 (d, 3H, J ) 6.6 Hz, H-8), 0.95 (t, 3H, J ) 6.7 Hz,
H-6′), 1.20 (d, 3H, J ) 6.8 Hz, H-7′), 1.36-1.53 (m, 2H, H-5′),
1.53-1.70 (m, 2H, H-4′), 2.37 (m, 1H, H-6), 3.67 (m, 1H, H-3′),
3.86 (dq, 1H, J ) 7.1, 9.3 Hz, H-2′), 4.22 (dd, 1H, J ) 2.6, 9.2
Hz, Ha-5), 4.31 (dd, 1H, J ) 8.3, 9.0, Hz, Hb-5), 4.49 (m, 1H,
H-4); 13C NMR (CDCl3) δ 13.8, 14.7, 15.0, 17.9, 18.9, 28.4, 33.4,
42.1, 58.4, 63.4, 64.4, 153.5, 174.8.
(4R,5S,2′R,3′R)-3-(3′-Azid o-2′-m eth ylp en tyl)-5-p h en yl-
4-m eth yl-2-oxa zolid in on e (17). Using the method described
for the preparation of 16, tosylate 15 (146 mg, 0.32 mmol) in
CH2Cl2 (2 mL) was treated with tetramethyl guanidinium
azide (63 mg, 0.40 mmol, 1.25 equiv)17 in refluxing CH2Cl2 (2
mL) and yielded the azide 17 as a colorless oil (32 mg, 0.10
mmol, 30%): [R]D +7.0° (c 1.0, CHCl3); HREIMS m/z 302.1650
C
17H21NO3 287.1516); IR (neat) 3418, 1779, 1646, 1456, 1345,
1197 cm-1; H NMR (CDCl3) δ 0.89 (d, 3H, J ) 6.6 Hz, 6-H),
0.95 (t, 3H, J ) 7.1 Hz, H-6′), 1.24 (d, 3H, J ) 6.8 Hz, H-7′),
1.34-1.44 (m, 2H, H-5′), 1.45-1.61 (m, 2H, H-4′), 3.77 (ddq,
1H, J ) 1.2, 2.7, 7.1 Hz, H-2′), 3.97 (m, 1H, H-3′), 4.80 (dq,
1H, J ) 6.1, 7.1 Hz, H-4), 5.69 (d, 1H, J ) 7.1 Hz, H-5), 7.29-
7.46 (m, 5H, Ar-H); 13C NMR (CDCl3) δ 10.1, 13.9, 14.1, 19.0,
35.9, 42.1, 54.5, 71.1, 78.7, 125.4 (2C), 128.4 (2C), 128.5, 133.0,
152.5, 176.9.
1
+
[M - N2] (calcd for C17H22N2O3 302.1625); IR (neat) 2957,
2928, 2875, 2101, 1783, 1694, 1455, 1344, 1195 cm-1; 1H NMR
(CDCl3) δ 0.88 (d, 3H, J ) 7.6 Hz, H-6), 0.98 (t, 3H, J ) 6.8
Hz, H-6′), 1.21 (d, 3H, J ) 6.8 Hz, H-7′), 1.38-1.53 (m, 2H,
H-5′), 1.54-0.171 (m, 2H, H-4′), 3.71 (dt, 1H, J ) 2.7, 8.8 Hz,
H-3′), 3.84 (dq, 1H, J ) 6.8, 9.3 Hz, H-2′), 4.83 (dq, 1H, J )
6.6, 6.8 Hz, H-4), 5.72 (d, 1H, J ) 7.1 Hz, H-5), 7.30-7.46 (m,
5H, Ar-H); 13C NMR (CDCl3) δ 13.8, 14.3, 14.6, 18.9, 33.4, 42.3,
55.0, 64.6, 79.0, 125.6 (2C), 128.8 (2C), 128.8, 133.1, 152.6,
174.7.
(4S,2′S,3′R)-3-[3′-(4′′-Tolu en esu lfon yl)-2′-m eth ylpen tyl]-
4-isop r op yl-2-oxa zolid in on e (14). A stirred solution of aldol
12 (642 mg, 2.5 mmol) in dry pyridine (5 mL) under argon
was treated at 0 °C with p-toluenesulfonyl chloride (525 mg,
2.75 mmol, 1.1 equiv). After 70 h at room temperature, the
reaction mixture was poured over a chilled 1 M HCl solution
(75 mL). After extraction with CHCl3 (20 mL × 3), the
combined organic layer was washed with brine (20 mL × 2),
dried over MgSO4, and concentrated under reduced pressure,
yielding a viscous yellow oil. The crude oil was purified by flash
chromatography (EtOAc/hexane 10:90), and the tosylate 14
was obtained as colorless oil (720 mg, 1.75 mmol, 70%): [R]D
+77° (c 1.0, CHCl3); HREIMS m/z 411.1707 [M]+ (calcd for
(2S,3S)-3-Am in o-2-m eth ylh exa n oic Acid (3c). A stirred
solution of 16 (20 mg, 0.071 mmol) in THF/H2O (3:1, 1 mL),
cooled to 0 °C, was treated with 28 µL (0.28 mmol, 4 equiv) of
30% H2O2 followed by 6 mg (0.14 mmol, 2 equiv) of solid LiOH
H2O. After stirring at 0 °C for 30 min, the reaction mixture
was treated with a solution of 210 µL of 1.5 M Na2SO3, followed
by 700 µL of 0.5 M NaHCO3. Following removal of THF under
nitrogen, the residue was diluted to 10 mL with H2O and
extracted with CH2Cl2 (6 mL × 4). The aqueous layer was
acidified to pH 1-2 with 5 M HCl and extracted with EtOAc
(13 mL × 4). The organic layer was combined, dried over Na2-
SO4, and dried under nitrogen, yielding azido acid as colorless
oil. Then, the crude azido acid was hydrogenated by use of
10% Pd-C (10 mg) in AcOH/H2O (3:1, 5 mL). After removal
of the catalyst, the reaction contents were applied to ODS-
HPLC using MeOH/H2O/TFA (20:80:0.05). Compound 3c was
obtained as colorless oil (7.6 mg, 0.052 mmol, 74%): [R]D +0.5°
(c 0.19, H2O); IR (KBr) 3435, 2968, 2885, 1676, 1464, 1196,
1140 cm-1; 1H NMR (D2O) δ 0.87 (t, J ) 7.3 Hz, H-6), 1.23 (d,
J ) 7.3 Hz, CH3-2), 1.32, 1.38 (apparently sextet, but J was
unclear, H-5a, H-5b), 1.61 (apparently quartet, J was unclear,
H-4a, H-4b), 2.85 (apparently quintet, but J was unclear, H-2),
3.48 (q, J ) 6.2, 12.6 Hz, H-3); 13C NMR (D2O) δ 13.0 (q, CH3-
2), 13.2 (q, C-6), 18.0 (t, C-5), 32.3 (t, C-4), 41.4 (d, C-2), 53.3
(d, C-3), 178.7 (s, C-1). Anal. Calcd for C7H15NO2‚CF3COOH‚
H2O: C, 38.99; H, 6.45; N, 5.05. Found: C, 38.95; H, 6.41; N,
5.57.
C
20H29NO6S 411.1708); IR (neat) 2964, 2876, 1777, 1704, 1463,
1356, 1366, 1208, 1189, 1176, 1096, 911, 888 cm-1; H NMR
(CDCl3) δ 0.83 (t, 3H, J ) 7.3 Hz, H-6′), 0.88 (d, 3H, J ) 6.8
Hz, H-8), 0.92 (d, 3H, J ) 6.8 Hz, H-7), 1.16 (d, 3H, J ) 6.8
Hz, CH3-2), 1.20-1.35 (m, 2H, H-5′), 1.53-1.61 (m, 2H, H-4′),
2.39 (dsept, 1H, J ) 3.6, 7.1 Hz, H-6), 2.44 (s, 3H, Ar-CH3),
4.01 (dt, 1H, J ) 4.1, 6.8 Hz, H-2′), 4.21 (dd, 1H, J ) 2.2, 8.5
Hz, Ha-5), 4.34 (dd, 1H, J 1 ) J 2 ) 8.4 Hz, Hb-5), 4.40-4.45
(m, 1H, H-4), 5.03 (dt, 1H, J ) 3.9, 6.6 Hz, H-3′), 7.72 (d, 2H,
J ) 8.3 Hz, Ar-H), 7.77 (d, 2H, J ) 8.3 Hz, Ar-H); 13C NMR
(CDCl3) δ 10.3, 13.7, 14.8, 18.0, 18.2, 21.6, 28.5, 34.9, 41.6,
59.3, 63.8, 81.8, 121.8 (2C), 129.6 (2C), 134.1, 144.6, 154.4,
172.8.
1
(4R,5S,2′R,3′S)-3-{3′-[(4′′-Tolu en esu lfon yl)oxy]-2′-m eth -
ylp en tyl}-5-p h en yl-4-m eth yl-2-oxa zolid in on e (15). Using
the method described for the preparation of 14, aldol 13 (540
mg, 1.8 mmol) was treated with p-toluenesulfonyl chloride (380
mg, 2.0 mmol, 1.1 equiv) in dry pyridine (5 mL) and yielded
tosylate 15 as a colorless oil (560 mg, 1.22 mmol, 68%): [R]D
+
-29° (c 2.0, CHCl3); HREIMS m/z 287.1581 [M - TsOH]
(calcd for C17H21NO3 287.1516); IR (neat) 2964, 2935, 2876,
1777, 1705, 1456, 1365, 1350, 1176, 910, 888 cm-1; H NMR
1
(2R,3R)-3-Am in o-2-m eth ylh exa n oic Acid (3d ). Using the
same method employed in the preparation of 3c, 3d was
(CDCl3) δ 0.87 (t, 3H, J ) 7.3 Hz, H-6′), 0.92 (d, 3H, J ) 6.6
Hz, H-6), 1.17 (d, 3H, J ) 7.1 Hz, H-7′), 1.23-1.38 (m, 2H,
H-5′), 1.58-1.65 (m, 2H, H-4′), 2.45 (s, 3H, Ar-CH3), 4.02 (dq,
1H, J ) 3.4, 7.0 Hz. H-2′), 4.75 (dq, 1H, J ) 6.8 Hz, H-4), 5.10
(dt, 1H, J ) 3.2, 6.6 Hz, H-3′), 5.78 (d, 1H, J ) 7.1 Hz, H-5),
obtained from 17 in 95% yield: [R]D -3.1° (c 0.23, H2O);
+
HRFABMS m/z 146.1184 [M + H]
(calcd for C7H16NO2
146.1181); 1H NMR (D2O) δ 0.87 (t, J ) 7.3 Hz, H-6), 1.23 (d,
J ) 7.3 Hz, CH3-2), 1.32, 1.38 (apparently sextet, but J was
unclear, H-5a, H-5b), 1.61 (apparently quartet, J was unclear,
7.30-7.44 (m, 7H, Ar-H), 7.80 (d, 2H, J ) 8.1 Hz, Ar-H); 13
C
NMR (CDCl3) δ 9.4, 13.7, 14.4, 18.4, 21.7, 34.9, 41.6, 55.6, 79.4,
81.9, 125.6 (2C), 127.8 (2C), 128.6 (3C), 129.7 (2C), 133.3,
134.1, 144.7, 153.4, 172.5.
(17) Papa, A. J . J . Org. Chem. 1966, 31, 1426-1430.

Kulokekahilide-1
J . Org. Chem., Vol. 67, No. 6, 2002 1767
H-4a, H-4b), 2.85 (apparently quintet, but J was unclear, H-2),
3.48 (q, J ) 6.2, 12.6 Hz, H-3); 13C NMR (D2O) δ 13.0 (q, CH3-
2), 13.2 (q, C-6), 18.0 (t, C-5), 32.3 (t, C-4), 41.4 (d, C-2), 53.3
(d, C-3), 178.7 (s, C-1).
nitrogen was added sodium azide (59 mg, 0.90 mmol) and 15-
crown-5 (6 mg, 0.1 equiv). After stirring at 70 °C for 3 h, the
solution was quenched with water (15 mL) and extracted with
EtOAc (10 mL × 4). The extract was roughly separated by PLC
(EtOAc/hexane 1:4) to afford crude azido-2′-methylpentyl)-4-
isopropyl-2-oxazolidinone (20, 58 mg) as a yellow oil. Using
the above hydrolysis and hydrogenation methods for prepara-
tion of 3c, a stirred solution of crude 20 in THF/H2O was
treated with H2O2 followed by LiOH H2O. Then, the crude
azido acid was hydrogenated by the use of 10% Pd-C in AcOH/
H2O. Compound 3a was purified by ODS-HPLC using MeOH/
H2O/TFA (20:80:0.05) to give a colorless oil (6.6 mg, 0.046
(4S,2′S,3′S)-3-(3′-Br om o-2′-m eth ylp en tyl)-4-isop r op yl-
2-oxa zolid in on e (18).18 To a solution of the tosylate 14 (300
mg, 0.73 mmol) in dry acetone (2.5 mL) was added anhydrous
lithium bromide (300 mg, 3.4 mmol, 4.7 equiv) at room
temperature under argon. After 4 h reflux, the reaction
mixture was allowed to cool to room temperature, and water
was added (15 mL). The resulting mixture was extracted with
EtOAc (10 mL × 4) and CH2Cl2 (10 mL × 1), the combined
organic layer was dried over MgSO4 and concentrated under
reduced pressure, yielding a viscous yellow oil. The crude oil
was purified by flash chromatography (EtOAc/hexane 5:95),
and bromide 18 was obtained as a colorless oil (170 mg, 0.50
mmol, 75%): [R]D +138° (c 1.0, CHCl3); HREIMS m/z 319.0782
[M]+ (calcd for C13H22NO379Br 319.0777); IR (neat) 2963, 2935,-
1875, 1782, 1700, 1464, 1386, 1300, 1251, 1204 cm-1; 1H NMR
(CDCl3) δ 0.84 (d, 3H J ) 6.8 Hz, H-7), 0.88 (d, 3H, J ) 7.1
Hz, H-8), 1.17 (t, 3H, J ) 7.3 Hz, H-6′), 1.38-1.49 (m, 1H,
5′-H), 1.55-1.76 (m, 2H, H-4′, H-5′), 1.78-1.90 (m, 1H, H-4′),
2.32 (dsept, 1H, J ) 4.0, 7.1 Hz, H-6), 4.18 (dd, 1H, J ) 2.8,
9.2 Hz, Ha-5), 4.27 (t, 1H, J ) 8.7 Hz, Hb-5), 4.22-4.40 (m,
2H, H-2′, H-3′), 4.44 (m, 1H, H-4); 13C NMR (CDCl3) δ 13.3,
14.7, 16.1, 17.8, 20.1, 28.4, 36.4, 45.0, 56.8, 58.5, 63.4, 153.5,
174.5.
1
mmol, 62%): [R]D +7.4° (c 0.13, H2O); H NMR (D2O) δ 0.87
(t, J ) 7.2 Hz, H-6), 1.18 (d, J ) 7.3 Hz, CH3-2), 1.32, 1.37 (m,
H-5a, H-5b), 1.50-1.65 (m, H-4a, H-4b), 2.84-2.89 (m, H-2),
3.52 (m, H-3);13C NMR (D2O) δ 11.1 (q, C-6), 12.9 (q, CH3-2),
18.3 (t, C-5), 31.7 (t, C-4), 40.8 (d, C-2), 52.6 (d, C-3), 178.4 (s,
C-1). Anal. Calcd for C7H15NO2‚CF3COOH‚H2O: C, 38.99; H,
6.45; N, 5.05. Found: C, 38.38; H, 6.09; N, 5.04.
(2R,3S)-Am in o-2-m eth ylh exa n oic Acid (3b). Azidation
of 19 by use of sodium azide afforded 21, and subsequent
hydrolysis followed by hydrogenation of 21 (the same prepara-
tion for 3a ) gave 3b quantitatively: [R]D -9.7° (c 0.15, H2O);
1H NMR (D2O) δ 0.87 (t, J ) 7.2 Hz, H-6), 1.18 (d, J ) 7.3 Hz,
CH3-2), 1.32, 1.37 (m, H-5a, H-5b), 1.50-1.65 (m, H-4a, H-4b),
2.84-2.89 (m, H-2), 3.52 (m, H-3);13C NMR (D2O) δ 11.1 (q,
C-6), 12.9 (q, CH3-2), 18.3 (t, C-5), 31.7 (t, C-4), 40.8 (d, C-2),
52.6 (d, C-3), 178.4 (s, C-1).
(4R,5S,2′R,3′R)-3-(3′-Br om o-2′-m eth ylp en tyl)-5-p h en yl-
4-m eth yl-2-oxa zolid in on e (19). Using the method described
for the preparation of 18, tosylate 15 (676 mg, 1.47 mmol) was
treated with lithium bromide (640 mg, 7.4 mmol, 5 equiv) in
refluxing acetone (5.0 mL) and yielded bromide 19 as a
Ack n ow led gm en t . We thank Professor Nobuhiro
Fusetani, Dr. Seketsu Fukuzawa, and Mr. Toshiyuki
Wakimoto at the University of Tokyo for bioassay and
mass spectral measurements and Mr. Gen Unoki at
Aoyama Gakuin University for valuable discussions.
Financial assistance by the National Science Founda-
tion, the Sea Grant College Program, and Pharma Mar.
S.A. is gratefully acknowledged. Y. N. was financially
supported by J apan Society for the Promotion of Science
Postdoctoral Fellowships for Research Abroad.
colorless oil (377 mg, 1.03 mmol, 70%): [R]D -40° (c 1.0,
CHCl3); HREIMS m/z 367.0875 [M]+ (calcd for C17H22NO3
-
79
Br 367.0777); IR (neat) 2960, 2936, 2876, 1783, 1699, 1456,
1
1386, 1343, 1196 cm-1; H NMR (CDCl3) δ 0.90 (d, 3H, J )
6.6 Hz, H-6), 0.91 (t, 3H, J ) 7.2 Hz, H-6′), 1.26 (d, 3H, J )
6.4 Hz, H-7′), 1.42-1.48 (m, 1H, H-5′), 1.60-1.81 (m, 2H, H-4′,
H-5′), 1.82-1.96 (m, 1H, H-4′), 4.32 (m, 1H, H-3′), 4.35 (m,
1H, H-2′), 4.81 (dq, 1H, J ) 6.8, 6.9 Hz, H-4), 5.70 (d, 1H, J )
7.1 Hz, H-5), 7.30-7.45 (m, 5H, Ar-H); 13C NMR (CDCl3) δ
13.3, 14.2, 15.8, 20.1, 36.5, 45.4, 55.0, 57.1, 79.0, 125.5 (2C),
128.6 (2C), 128.7, 133.1, 152.6, 174.3.
Su p p or tin g In for m a tion Ava ila ble: Tables of crystal
data for 7a and 10c, Marfey analysis of amino acids, chiral
HPLC analysis of R-hydroxy acids, and NMR spectra for 1.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(2S,3R)-3-Am in o-2-m eth ylh exa n oic Acid (3a ). To a solu-
tion of bromide 18 (98 mg, 0.30 mmol) in DMF (1.5 mL) under
(18) (a) Wiberg, K. B.; Lowry, B. R. J . Am. Chem. Soc. 1963, 85,
3188-3193. (b) Howden, M. E. H.; Maercker, A.; Burdon, J .; Roberts,
J . D. J . Am. Chem. Soc. 1966, 88, 1732-1737.
J O010176Z