Kulokekahilide-2, a cytotoxic depsipeptide from a cephalaspidean mollusk Philinopsis Speciosa

Article abstract of DOI:10.1021/np049949f

A cytotoxic depsipeptide, kulokekahilide-2 (1), was isolated from a cephalaspidean mollusk, Philinopsis speciosa. The structure elucidation of kulokekahilide-2 was carried out by spectroscopic analysis and chemical degradation. Kulokekahilide-2 showed pot

Full text of DOI:10.1021/np049949f

1332
J . Nat. Prod. 2004, 67, 1332-1340
Ku lok ek a h ilid e-2, a Cytotoxic Dep sip ep tid e fr om a Cep h a la sp id ea n Mollu sk
P h ilin opsis speciosa †
Yoichi Nakao,*,‡ Wesley Y. Yoshida,‡ Yuuki Takada, J unji Kimura, Liu Yang,§ Susan L. Mooberry,§,| and
Paul J . Scheuer‡
Department of Chemistry, University of Hawaii at Manoa, 2545 The Mall, Honolulu, Hawaii 96822-2275, Natural Products
Program, Cancer Research Center of Hawaii, University of Hawaii at Manoa, 1236 Lauhala Street, Honolulu, Hawaii 96813,
and Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe,
Sagamihara, 229-8558, J apan
Received J anuary 24, 2004
A cytotoxic depsipeptide, kulokekahilide-2 (1), was isolated from a cephalaspidean mollusk, Philinopsis
speciosa. The structure elucidation of kulokekahilide-2 was carried out by spectroscopic analysis and
chemical degradation. Kulokekahilide-2 showed potent cytotoxicity against several cell lines (P388, SK-
OV-3, MDA-MB-435, and A-10 with IC50 values ranging from 4.2 to 59.1 nM) indicating cancer cell
selectivity.
The marine carnivorous mollusk Philinopsis speciosa is
a bountiful source of structurally and biologically unique
compounds.1 Among them, the most characteristic con-
stituents are depsipeptides,1c-f which are reminiscent of
those from other marine mollusks such as Dolabella
auricularia2 and Onchidium sp.3 The Philinopsis com-
pounds are thought to be sequestered by predation of
smaller sized mollusks such as the sea hare Stylocheilus
longicaudus, which feeds on cyanobacteria.1d Further
investigation of the cytotoxic fractions of P. speciosa led to
the isolation of a new depsipeptide, kulokekahilide-2 (1),1f
which is closely related to aurilide (2) isolated from D.
auricularia.2a
The organic extract of P. speciosa was evaporated and
separated by the modified Kupchan procedure4 to yield
n-hexane, CH2Cl2, and aqueous MeOH extracts. The CH2-
Cl2 extract was purified by a two-step ODS flash chroma-
tography process, followed by gel filtration, and amino
column chromatography. The fraction containing peptides
was further separated by sequential ODS HPLC to give
kulokekahilide-2 (1; 3.4 mg; 3.8 × 10-5 % yield based on
wet weight).
structural framework consisting of peptidal and polyketidal
moieties (substructures a and b, respectively). Substruc-
ture a was composed of five amino acids, Ile, Sar, MePhe,
and two Ala, and 2-hydroxyisocaproic acid (Hica). The
sequence of these residues was deduced from HMBC
correlations between H-21/C-14, H3-32/C-20, H3-35/C-23,
H-37 and NH-37/C-33, and H-43 and NH-43/C-36 to make
substructure a .
Substructure b was elucidated as follows: COSY analy-
sis connected proton signals from the olefinic proton H-3,
via the allyllic methylene protons H2-4 and H-5 oxymethine
signal, to the methine proton H-6, which showed correla-
tions to the methyl at C-12 and the oxymethine proton H-7.
The other spin system could be traced from CH3-10, via
an olefinic proton H-9, to the other methyl (CH3-13)
through an allyllic coupling (J ) 1.1 Hz). These two units
were connected by HMBC5 cross-peaks observed between
H-7/C-13, H3-13/C-7, and H-7/C-8. Further analysis of the
HMBC spectrum connected C-3 and C-2 (cross-peaks
between H2-4/C-2), which was also bearing a methyl group
(CH3-11) and a carbonyl carbon C-1 (cross-peaks between
H-3/C-11, H3-11/C-3, H-3/C-1, and H3-11/C-1) to furnish the
partial structure b.
The molecular formula of kulokekahilide-2 (1) was
established as C44H67N5O10 on the basis of HRFABMS [m/z
826.4942 (M + H)+ (∆ -2.4 mmu)]. In the 1H NMR
spectrum (CD2Cl2), 1 exhibited two sets of signals in a 1:1
ratio, which was later assigned to two conformers, 1cis and
1tr a n s, derived from the cis-trans isomerism at the amide
bond between N-methylphenylalanine (MePhe) and N-
methylglycine (Sar).
Substructures a and b were connected on the basis of
HMBC analysis. The R-proton (H-15) of Hica showed a
cross-peak to the C-1 carbonyl carbon of substructure b,
and H-7 of b correlated with the C-42 carbonyl carbon of
the C-terminal Ala-2 residue of substructure a to make a
26-membered ring.
NOESY analysis supported the sequence of substructure
a for both 1cis and 1tr a n s; however, differences in NOE
signals for these conformers were observed between the
MePhe and Sar residues. In 1tr a n s, NOEs were observed
between H-24 and H-27/Me-35; on the other hand, in 1cis,
no NOE was seen among these protons, but instead an
NOE was observed between H-24/Ha-34. These NOE
patterns suggested a trans-amide linkage between MePhe/
Sar for 1tr a n s and cis for 1cis.
Detailed analysis of the 2D NMR data enabled us to
assign all signals for both 1cis and 1tr a n s and revealed a
† Dedicated to the late Dr. D. J ohn Faulkner (Scripps) and the late Dr.
Paul J . Scheuer (Hawaii) for their pioneering work on bioactive marine
natural products.
* To whom correspondence should be addressed. Present address:
Laboratory of Aquatic Natural Products Chemistry, Graduate School of
Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657,
J apan. Tel: +81-3-5841-5299. Fax: +81-3-5841-8166. E-mail: ayocha@
mail.ecc.u-tokyo.ac.jp.
‡ Department of Chemistry, University of Hawaii at Manoa.
§ Natural Products Program, Cancer Research Center of Hawaii, Uni-
versity of Hawaii at Manoa.
Further NOE analysis enabled us to predict the relative
stereochemistry at three successive methines, C-5 to C-7.
Although rotation between C-3 and C-4 seemed different
between 1cis and 1tr a n s, NOEs around C-5 to C-7 were
well preserved. Diagnostic NOEs from Me-12 to H-5 and
| Present address: Department of Physiology and Medicine, Southwest
Foundation for Biomedical Research, PO Box 760549, San Antonio, TX
78245.
Aoyama Gakuin University.
10.1021/np049949f CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/31/2004

Cytotoxic Depsipeptide from Philinopsis speciosa
J ournal of Natural Products, 2004, Vol. 67, No. 8 1333
Ch a r t 1. Structures of Kulokekahilide-2 (1) and Aurilide (2)
Ta ble 1. NMR Data of Kulokekahilide-2 (1) in CD2Cl2
1tr a n s
1cis
atom no.
13C
1H (ppm, mult., Hz)
HMBC
13C
1H (ppm, mult., Hz)
HMBC
1
2
3
4a
4b
5
6
7
166.9
128.2
141.9
32.5
168.9
128.9
143.0
31.4
6.97 dt 8.4, 1.1
2.37 dd 14.3, 8.4
2.14 m
3.51 bdd 8.7, 5.6
2.11 m
1, 11
2, 3
2, 3, 5
6.92 ddd 9.0, 4.9, 1.3
2.14 m
2.30 ddd 16.1, 9.0, 8.5
3.67 m
1, 11
2, 3, 5
3
72.1
41.4
83.5
133.2
125.9
13.1
12.7
11.6
11.2
170.4
72.6
40.9
72.1
40.3
83.4
132.4
126.4
13.1
12.6
10.7
11.0
171.2
73.5
40.6
5, 7
5, 6, 8, 13, 42
2.05 m
5, 7
5.22 d 9.8
4.93 d 10.7
5, 6, 8, 9, 12, 13, 42
8
9
5.55 qd 6.6, 1.1
1.61 dq 6.6, 0.9
1.83 bs
0.79 d 6.9
1.64 bs
10, 13
8, 9
1, 2, 3
5, 6, 7
7, 8, 9
5.56 bqd 6.7, 1.1
1.60 dq 6.6, 1.1
1.86 bs
0.70 d 7.1
1.54 bs
7, 10, 13
8, 9
1, 2, 3
5, 6, 7
7, 8, 9
10
11
12
13
14
15
16a
16b
17
18
19
20
21
22
NH
23
24
25a
25b
26
27
28
29
30
31
32
33
34a
34b
35
36
37
38
39a
39b
40
41
NH
42
43
44
NH
5.14 dd 10.3, 5.8
1.77 m
1.71 m
1.69 m
0.91 d 6.3
0.91 d 6.3
1, 14, 16
4.83 dd 8.5, 3.3
1.83 m
1.54 m
1, 14, 16, 17
14, 15, 17, 18, 19
14, 15, 17, 18, 19
18, 19
14, 15, 17, 18, 19
15, 17, 18, 19
16, 18, 19
16, 17, 19
16, 17, 18
24.9
21.9
23.3
173.0
45.5
17.5
25.0
21.9
23.3
173.6
45.1
16.5
1.76 m
0.91 d 6.3
0.92 d 6.7
16, 17, 19
16, 17, 18
4.71 dq 7.8, 6.7
0.87 d 6.7
7.10 d 7.8
14, 20, 22
20, 21
4.56 dq 7.6, 7.1
0.78 d 7.1
6.53 d 7.6
14, 20, 22
20, 21
14
172.5
56.3
35.3
170.2
54.3
35.3
5.62 dd 9.1, 7.0
3.25 dd 14.1, 7.0
3.07 dd 14.1, 9.1
23, 25, 32
23, 24, 26, 27, 31
23, 24, 26, 27, 31
5.40 dd 10.3, 5.8
3.05 dd 14.5, 10.3
2.98 dd 14.5, 5.8
20, 25, 32
23, 24, 26, 27, 31
24, 26, 27, 31
136.9
129.9
128.6
127.2
128.6
129.9
31.2
137.4
129.8
128.4
126.8
128.4
129.8
30.4
7.32 bd 8.0
7.25 dd 8.0, 7.1
7.21 t 7.1
7.25 dd 8.0, 7.1
7.32 bd 8.0
2.95 s
25, 29, 31
26, 30
27, 31
26, 28
25, 27, 29
20, 24
7.14 bd 7.7
7.21 dd 7.7, 7.0
7.16 t 7.0
25, 29, 31
26, 30
27, 31
7.21 dd 7.7, 7.0
7.14 bd 7.7
2.97 s
26, 28
25, 27, 29
20, 24
169.7
52.9
169.5
51.5
4.28 bd 15.4
3.72 bd 15.4
2.71 s
33
3.99 d 17.9
3.35 d 17.9
2.91 s
33, 35
23, 33, 35
23, 34
23, 33, 35
23, 34
36.3
170.8
58.8
35.8
25.1
36.7
171.7
57.7
37.8
25.0
4.09 dd 8.2, 7.1
1.96 m
1.48 m
33, 36, 38, 39, 41
4.38 dd 9.2, 8.5
1.89 m
33, 36, 38, 39, 41
1.37 m
1.37 m
0.94 t 6.7
0.97 d 7.1
7.51 d 9.2
38, 40, 41
38, 40, 41
38, 39
37, 38
33
1.12 m
38, 40
38, 39
37, 38
33
11.3
16.0
0.88 t 7.5
0.92 d 5.7
6.95 d 8.2
10.9
15.60
171.6
48.7
18.5
170.6
50.1
17.4
4.44 dq 7.6, 7.1
1.35 d 7.1
6.32 d 7.6
36, 42, 44
42, 43
36
4.25 dq 6.7, 7.1
1.38 d 7.1
36, 42
42
6.36 d 6.7
36
H-7 were indicative of the relative stereochemistry as
To confirm the relative stereochemistry predicted above,
5S*,6S*,7S*.
the four possible diastereoisomers of triol, 3a , 3b, 3c, and

1334 J ournal of Natural Products, 2004, Vol. 67, No. 8
Nakao et al.
F igu r e 1. Key COSY and HMBC correlations for 1.
F igu r e 3. NOE patterns of substructure b for 1cis and 1tr a n s.
8a -d .9 Reduction of amides 8a -d to the aldehydes 9a -d
proceeded with diisobutylaluminum hydride (DIBAL) in
THF.10
To establish the C-5 stereocenter by the second coupling
reaction, the vinylogous Mukaiyama aldol reaction11 was
applied to aldehydes 9a -d and 1-methoxy-2-methyl-1-
trimethylsiloxy-1,3-butadiene,12 affording conjugated meth-
yl esters 10a -d , respectively. The relative stereochemistry
at C-5 through C-7 in 10a -d was assigned on the basis of
NMR analysis after the diols were derivatized to the
corresponding acetonides.13 All attempts to employ the
modified Mitsunobu reaction14 to prepare the desired 5S
configuration for both 3a and 3d by inverting the 5-OH in
both 10a and 10d were not successful. Therefore, 10a and
10d were subjected to Moffatt oxidation,15 which yielded
corresponding ketoesters 11a and 11d . Stereoselective
16
17
reduction of 11a with LiAlH4 and 11d with NaBH4
afforded the desired protected triol 13a and methyl ester
12d , respectively.
DIBAL reduction of methyl esters 10b, 10c, and 12d
afforded deprotected triol 3b as well as protected triols 13c
and 13d . Removal of TBS groups in 13a , 13c, and 13d
afforded 3a , 3c, and 3d , respectively. Thus, all four possible
diastereoisomers of 2,6,8-trimethyl-2,8-decadiene-1,5,7-triol
were successfully prepared.
F igu r e 2. NOE patterns of substructure a for 1cis and 1tr a n s.
3d , were prepared by diastereoselective synthesis. These
triols were prepared basically following the method em-
ployed for aurilide from Dolabella auricularia.2a The
synthetic strategy for 3a and 3c applied the syn-selective
aldol reaction by Evans,6 whereas the anti-selective aldol
reaction by Heathcock7 was used for 3b and 3d .
Comparison of 1H NMR spectra of synthetic triols 3a -d
with that obtained from natural 1 clearly indicated the
relative stereochemistry as 5S*,6S*,7S* (Figure 4).
To deduce the absolute stereochemistry of C-5 through
C-7, S- and R-MTPA esters (1a and 1b) were introduced
to the C-5 hydroxyl group of 1, respectively.18 Although
values for H-11 and H-12 (underlined) did not show the
expected sign, the ∆δ(S-R) values for H-3, -4, -7, -8, -9, -10,
and -13 were suggestive of 5S,6S,7S, which is identical to
that of aurilide (2). MM2 calculation suggested that
introduction of MTPA esters at C-5 causes a dramatic
change in ring conformation for both 1a and 1b. For both
models, rings were bent at C-4 and C-6 to make the bulky
MTPA ester groups protrude from the ring. As a result of
these conformational changes, methyl groups 11 and 12
might be located outside of the shielding area by phenyl
rings of the MTPA esters (Figure 5). Proof of the stereo-
chemistry obtained above by total synthesis of 1 is in
progress.
The syn-selective aldol reaction via a closed transition
state of N-propionyl oxazolidinone 5a with trans-2-methyl-
2-butenal, which used 1 equiv of Lewis acid, Bu2BOTf, gave
the aldol 6a with the 2R,3R configuration. Conversely, the
anti-selective aldol reaction via an open transition state of
5a with the same aldehyde, which used 2 equiv of Bu2BOTf,
provided the aldol 6b with the 2S,3R configuration. Like-
wise, the stereoselective aldol reaction of oxazolidinone 5b
with trans-2-methyl-2-butenal provided aldols 6c (2S,3S)
and 6d (2R,3S), respectively. Subsequent treatment of
6a -d with the aluminum amide reagent derived from N,O-
dimethylhydroxylamine hydrochloride and AlMe3, accord-
ing to the procedure of Weinreb,8 gave the desired tran-
samination product, the N-methoxy-N-methylamides 7a -
d . Protection of 7a -d with tert-butyldimethylsilyl (TBS)
chloride and imidazole afforded the corresponding amides
To assign the absolute configuration of Hica and the
amino acids, 1 was converted to fragment 4 (Scheme 2).

Cytotoxic Depsipeptide from Philinopsis speciosa
J ournal of Natural Products, 2004, Vol. 67, No. 8 1335
Sch em e 1. Synthetic Route to Triols 3a -d
One-half the quantity of 4 was acid hydrolyzed and
separated by ODS HPLC to yield Hica, Ala, Ile, and MePhe.
The absolute stereochemistry of Hica was determined as
D by chiral HPLC analysis. Marfey analysis19 of each amino
acid indicated L-Ile, L-MePhe, and both D- and L-Ala
residues were present in 1. To differentiate between the
configurations for Ala-1 and -2, the remaining quantity of
4 was subjected to hydrazinolysis,20 which yielded only
Ala-2 as an intact amino acid. Marfey analysis disclosed
the L-stereochemistry for Ala-2; therefore Ala-1 was de-
duced as having D-stereochemistry.
Kulokekahilide-2 (1) showed potent cytotoxicity against
the cell lines P388, SK-OV-3, MDA-MB-435, and A-10 with
IC50 values of 4.2, 7.5, 14.6, and 59.1 nM, respectively, and
it showed cancer cell selectivity, as the A-10 cell line is not
transformed. Kulokekahilide-2 was also tested for its effects
on microtubules, intermediate filaments, and actin fila-
ments, but it showed no effects on these cytoskeleton
networks. Recently, the combinatorial syntheses of aurilide
analogues were achieved.21 Further study with these
analogues will disclose the detailed structure-activity
relationship and their mode of action.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured on a digital spectropolarimeter. UV spectra
were measured with a diode array spectrophotometer. NMR
1
spectra were recorded at 500.115 MHz for H and 125.766 MHz
for 13C. Glycerol was used as a matrix for FABMS measure-
ments. Poly(ethylene glycol) was used as a marker for HR-
FABMS.
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 n-hexane, CH2Cl2, and aqueous
MeOH extracts. The CH2Cl2 extract was evaporated to dryness
and purified by a two-step ODS flash chromatography process
(first with aqueous MeOH as solvent, second with aqueous
MeCN), followed by gel filtration (Sephadex LH-20, MeOH)
and amino column chromatography [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
F igu r e 4. Comparison of 1H NMR spectra of triols.

1336 J ournal of Natural Products, 2004, Vol. 67, No. 8
Nakao et al.
F igu r e 5. ∆δ(S-R) values and models of MTPA esters (1a ,b).
Sch em e 2. Degradation Scheme of 1
[COSMOSIL 5C18-AR, MeCN/H2O (7:3)], giving nine fractions
(1-9). Fraction 2 was separated by sequential ODS HPLC
[COSMOSIL 5C18-AR, MeCN/H2O (1:1); 2-PrOH/H2O (1:1);
MeCN/H2O (55:45); 2-PrOH/H2O (47.5:52.5)] and finally puri-
fied again on an ODS column [COSMOSIL 5C18-MS, MeCN/
1.65 (d, 3H, J ) 6.9 Hz, H-6′), 1.63 (s, 3H, H-7′), 1.16 (d, 3H,
J ) 6.9 Hz, H-8′), 0.89 (d, 3H, J ) 6.4 Hz, H-6); 13C NMR
(CDCl3) δ 176.9, 152.6, 134.3, 133.1, 128.8, 128.7 (2C), 125.6
(2C), 120.5, 78.9, 75.5, 54.9, 40.6, 14.3, 13.1, 13.0, 10.4.
(4R,5S,2′S,3′R,4′E)-3-(2′,4′-Dim eth yl-3′-h yd r oxy-1′-oxo-
4′-h exen yl)-4-m eth yl-5-p h en yl-2-oxa zolid in on e (6b). A
stirred solution of N-propionyl oxazolidinone 5a (466 mg, 2.0
mmol) in dry CH2Cl2 (6 mL) under argon was treated with 1
M dibutylboron triflate in CH2Cl2 (4.0 mL, 4.0 mmol) and
diisopropylethylamine (440 µL, 2.4 mmol) at 0 °C. After 30
min, the reaction mixture was cooled to -78 °C and trans-2-
methyl-2-butenal (250 µL, 250 mmol) was added dropwise.
After 2 h at -78 °C, the reaction was quenched by addition of
pH 7 aqueous phosphate buffer (4 mL) and oxidized with 30%
hydrogen peroxide/methanol (1:1, 8 mL). The resulting solution
was allowed to slowly warm from -78 °C to 0 °C over a period
of 1 h. The solvent was evaporated under reduced pressure.
The residue was dissolved in water (10 mL) and extracted with
EtOAc (10 mL × 3). The combined organic layer was washed
with 5% NaHCO3 (10 mL) and brine (10 mL), dried over
MgSO4, and concentrated under reduced pressure, yielding a
viscous yellow oil. The crude oil was purified by PTLC (EtOAc/
n-hexane, 25:75), and the aldol 6b was obtained as a colorless
oil (383 mg, 1.2 mmol, 60%): [R]D +40° (c 0.18, CHCl3);
H2O (1:1)] to give kulokekahilide-2 (1; 3.4 mg; 3.8 × 10-5
%
yield based on wet weight).
Ku lok ek a h ilid e-2 (1): colorless amorphous solid; [R]D -15°
(c 0.04, MeOH); UV (MeOH) 205 nm (ꢀ 15 000); see Table 1
1
for H and 13C NMR data; HRFABMS m/z 826.4942 (M + H)+
(for C44H68N5O10, ∆ -2.4 mmu).
(4R,5S,2′R,3′R,4′E)-3-(2′,4′-Dim eth yl-3′-h yd r oxy-1′-oxo-
4′-h exen yl)-4-m et h yl-5-p h en yl-2-oxa zolid in on e (6a ). A
stirred solution of N-propionyl oxazolidinone 5a (1.0 mL, 5.0
mmol) in dry CH2Cl2 (15 mL) under argon was treated with 1
M dibutylboron triflate in CH2Cl2 (5.5 mL, 5.5 mmol) and
diisopropylethylamine (1.1 mL, 6.0 mmol) at 0 °C. After 30
min, the reaction mixture was cooled to -78 °C and trans-2-
methyl-2-butenal (530 µL, 5.5 mmol) 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 solution was stirred at 0 °C for 1 h. The solvent
was evaporated under reduced pressure. 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 preparative thin-layer
chromatography (PTLC) (EtOAc/n-hexane, 25:75), and the
aldol 6a was obtained as a colorless oil (1.48 g, 4.7 mmol,
94%): [R]D +27° (c 0.25, CHCl3); IR (KBr) 3449, 1780, 1699,
+
HREIMS m/z 300.1575 (M - OH) (for C18H22NO3, ∆ -2.4
mmu); IR (KBr) 3448, 1780, 1699, 1346, 1197, 767, 700 cm-1
;
1H NMR (CDCl3) δ 7.29-7.43 (m, 5H, Ar-H), 5.66 (d, 1H, J )
7.1 Hz, H-5), 5.52 (q, 1H, J ) 6.6 Hz, H-5′), 4.78 (dq, 1H, J )
7.1, 6.6 Hz, H-4), 4.13 (overlapping dq and d, 2H, H-2′, H-3′),
2.55 (brs, 1H, OH), 1.67 (s, 3H, H-7′), 1.62 (d, 3H, J ) 6.6 Hz,
H-6′), 1.06 (d, 3H, J ) 6.4 Hz, H-8′), 0.90 (d, 3H, J ) 6.6 Hz,
H-6); 13C NMR (CDCl3) δ 176.6, 153.4, 135.2, 133.2, 128.7 (3C),
125.6 (2C), 123.6, 81.2, 78.9, 55.2, 40.7, 14.8, 14.3, 13.1, 10.6.
(4S,2′S,3′S,4′E)-3-(2′,4′-Dim et h yl-3′-h yd r oxy-1′-oxo-4′-
h exen yl)-4-isop r op yl-2-oxa zolid in on e (6c). Using the
method described for the preparation of 6a , N-propionyl-
oxazolidinone 5b (500 mg, 2.7 mmol) was treated with 1 M
1
1363, 1195, 767, 700 cm-1; H NMR (CDCl3) δ 7.30-7.44 (m,
5H, Ar-H), 5.67 (d, 1H, J ) 7.3 Hz, H-5), 5.63 (q, 1H, J ) 6.9
Hz, H-5′), 4.77 (dq, 1H, J ) 6.9, 6.4 Hz, H-4), 4.37 (brs, 1H,
H-3′), 3.98 (dq, 1H, J ) 6.9, 3.7 Hz, H-2′), 2.74 (brs, 1H, OH),

Cytotoxic Depsipeptide from Philinopsis speciosa
J ournal of Natural Products, 2004, Vol. 67, No. 8 1337
dibutylboron triflate in CH2Cl2 (3.0 mL, 3.0 mmol) and
diisopropylethylamine (600 µL, 3.2 mmol). The resulting enol
borinate was allowed to react with trans-2-methyl-2-butenal
(300 µL, 3.0 mmol). After workup and purification, aldol 6c
was obtained as a colorless oil (686 mg, 2.6 mmol, 94%): [R]D
+70° (c 0.99, CHCl3); IR (KBr) 3449, 2926, 1778, 1703, 1384,
temperature. The reaction mixture was quenched with water
and extracted with EtOAc (15 mL × 3). The combined organic
layers were dried over anhydrous MgSO4 and concentrated
under reduced pressure, yielding a colorless oil. The resulting
oil was purified by column chromatography (EtOAc/n-hexane,
50:50), and protected amide 8a was obtained as a colorless oil
(415 mg, 1.3 mmol, 100%): [R]D -5° (c 0.27, CHCl3); IR (KBr)
1
1205 cm-1; H NMR (CDCl3) δ 5.60 (q, 1H, J ) 6.9 Hz, H-5′),
4.44 (ddd, 1H, J ) 9.2, 4.1, 2.8 Hz, H-4), 4.32 (brs, 1H, H-3′),
4.27 (dd, 1H, J ) 9.2, 9.2 Hz, H-5b), 4.21 (dd, 1H, J ) 9.2, 2.8
Hz, H-5a), 3.97 (dq, 1H, J ) 6.9, 3.7 Hz, H-2′), 2.90 (brs, 1H,
OH), 2.34 (dsept, 1H, J ) 6.9, 4.1 Hz, H-6), 1.62 (d, 3H, J )
6.9 Hz, H-6′), 1.58 (s, 3H, H-7′), 1.16 (d, 3H, J ) 7.3 Hz, H-8′),
2958, 2932, 2858, 1666, 1381, 1256, 1057, 876, 837, 775 cm-1
;
1H NMR (CDCl3) δ 5.36 (q, 1H, J ) 6.9 Hz, H-5), 4.11 (d, 1H,
J ) 9.2 Hz, H-3), 3.63 (s, 3H, Me-O), 3.11 (overlapping brs
and m, 4H, Me-N, H-2), 1.56 (s, 3H, H-7), 1.52 (d, 3H, J ) 7.3
Hz, H-8), 1.17 (d, 3H, J ) 6.9 Hz, H-6), 0.88 (s, 9H, (Me)3CSi),
0.04 (s, 3H, Me-Si), -0.04 (s, 3H, Me-Si); 13C NMR (CDCl3) δ
176.2, 136.4, 121.6, 80.2, 61.5, 40.4, 32.1, 25.8(3C), 18.2, 14.8,
13.0, 11.0, -4.8, -5.1.
0.91 (d, 3H,J ) 6.9 Hz, H-8), 0.87 (d, 3H, J ) 6.9 Hz, H-7); 13
C
NMR (CDCl3) δ 177.4, 153.5, 134.0, 120.5, 75.1, 63.3, 58.3, 40.4,
28.3, 17.9, 14.7, 13.1, 13.0, 11.0.
(4S,2′R,3′S,4′E)-3-(2′,4′-Dim et h yl-3′-h yd r oxy-1′-oxo-4′-
h exen yl)-4-isop r op yl-2-oxa zolid in on e (6d ). Using the
method described for the preparation of 6b , N-propionyl-
oxazolidinone 5b (185 mL, 1.0 mmol) was treated with 1 M
dibutylboron triflate in CH2Cl2 (2.0 mL, 2.0 mmol) and
diisopropylethylamine (220 µL, 1.2 mmol). The resulting enol
borinate was allowed to react with trans-2-methyl-2-butenal
(125 µL, 1.25 mmol). After workup and purification, aldol 6d
was obtained as a colorless oil (219 mg, 0.8 mmol, 81%): [R]D
+55° (c 0.18, CHCl3); HREIMS m/z 252.1604 (M+ - OH) + (for
(2S,3R,4E)-3-(ter t-Bu tyld im eth ylsilyloxy)-N-m eth oxy-
N,2,4-tr im eth yl-4-h exen a m id e (8b). To a stirred suspension
of N,O-dimethylhydroxylamine hydrochloride (216 mg, 2.2
mmol) in THF (2.2 mL) at 0 °C under argon was slowly added
1.0 M trimethylaluminum in n-hexane (2.2 mL, 2.2 mmol) with
concomitant evolution of gas. The resulting homogeneous
solution was stirred for 30 min at room temperature and then
recooled to 0 °C, and a solution of aldol 6b (351 mg 1.1 mmol)
in THF (2.2 mL) was added over a period of 5 min. The solution
was stirred for 2.5 h at 50 °C, and then ice-cooled 0.5 M
aqueous HCl (30 mL) and EtOAc (10 mL) were added. The
layers were separated, and the aqueous layer was extracted
with EtOAc (10 mL × 3). The combined organic layers were
dried over anhydrous MgSO4 and concentrated under reduced
pressure. To the residue were added TBSCl (500 mg, 3.3 mmol)
and imidazole (450 mg, 6.6 mmol) in DMF (6 mL) and stirred
overnight at room temperature. The reaction mixture was
quenched with water and extracted with EtOAc (15 mL × 3).
The combined organic layers were dried over anhydrous
MgSO4 and concentrated under reduced pressure, yielding a
yellow oil. The resulting oil was purified by PTLC (EtOAc/n-
hexane, 15:85), and a protected amide 8b was obtained as a
colorless solid (317 mg, 1.0 mmol, 92%): [R]D +30° (c 0.25,
CHCl3); IR (KBr) 2929, 2856, 1663, 1387, 1250, 1057, 862, 837,
C
14H22NO3, ∆ 0.5 mmu); IR (KBr) 3449, 2966, 1774, 1699,
1
1386, 1205, 748, 706 cm-1; H NMR (CDCl3) δ 5.52 (q, 1H, J
) 6.9 Hz, H-5′), 4.45 (dt, 1H, J ) 7.3, 3.2 Hz, H-4), 4.27 (dd,
1H, J ) 9.2, 7.3 Hz, H-5b), 4.21 (dd, 1H, J ) 9.2, 3.2 Hz, H-5a),
4.14 (dq, 1H, J ) 8.7, 6.4 Hz, H-2′), 4.06 (d, 1H, J ) 8.7 Hz,
H-3′), 2.63 (brs, 1H, OH), 2.39 (m, 1H, H-6), 1.65 (s, 3H, H-7′),
1.62 (d, 3H, J ) 6.9 Hz, H-6′), 1.03 (d, 3H, J ) 6.4 Hz, H-8′),
0.91 (d, 3H, J ) 6.9 Hz, H-8), 0.89 (d, 3H, J ) 6.9 Hz, H-7);
13C NMR (CDCl3) δ 176.7, 154.5, 135.3, 123.4, 81.3, 63.3, 58.9,
40.3, 28.4, 17.9, 14.7, 14.6, 13.1, 10.7.
(2R,3R,4E)-3-H yd r oxy-N-m et h oxy-N,2,4-t r im et h yl-4-
h exen a m id e (7a ). To a stirred suspension of N,O-dimethyl-
hydroxylamine hydrochloride (317.5 mg, 3.25 mmol) in CH2Cl2
(6 mL) at 0 °C under argon was slowly added 15% trimethyl-
aluminum in toluene (1.6 mL, 3.2 mmol) with concomitant
evolution of gas. The resulting homogeneous solution was
stirred for 40 min at room temperature and then recooled to
0 °C, and a solution of aldol 6a (516 mg, 1.6 mmol) in CH2Cl2
(5 mL) was added over a period of 5 min. The solution was
stirred for 1.5 h at 0 °C, and then ice-cooled 0.5 M aqueous
HCl (30 mL) and CH2Cl2 (10 mL) were added. The layers were
separated, and the aqueous layer was extracted with CH2Cl2
(10 mL × 3). The combined organic layers were dried over
anhydrous Na2SO4 and concentrated under reduced pressure.
The residue was purified by PTLC (EtOAc/n-hexane, 50:50),
and amide 7a was obtained as a colorless solid (265 mg, 1.32
mmol, 81%): [R]D -8° (c 0.16, CHCl3); IR (KBr) 3430, 2935,
1635, 1456, 1384, 991 cm-1; 1H NMR (CDCl3) δ 5.65 (q, 1H, J
) 6.9 Hz, H-5), 4.26 (brs, 1H, H-3), 3.71 (s, 3H, Me-O), 3.20
(brs, 3H, Me-N), 3.07 (m, 1H, H-2), 1.63 (d, 3H, J ) 6.9 Hz,
H-6), 1.59 (s, 3H, H-7), 1.09 (d, 3H, J ) 7.3 Hz, H-8); 13C NMR
(CDCl3) δ 178.0, 133.6, 120.4, 75.4, 61.5, 36.9, 32.0, 13.3, 13.0,
10.4.
(2S,3S,4E)-3-Hydr oxy-N-m eth oxy-N,2,4-tr im eth yl-4-h ex-
en a m id e (7c). Using the method described for the preparation
of 7a , N,O-dimethylhydroxylamine hydrochloride (254 mg, 2.6
mmol) was treated with 15% trimethylaluminum in toluene
(1.3 mL, 2.6 mmol). To the resulting solution was added a
solution of aldol 6c (350 mg, 1.3 mmol). After workup and
purification, amide 7c was obtained as a colorless solid (90.6
mg 0.45 mmol, 35%): [R]D +11° (c 0.31, CHCl3); 1H NMR
(CDCl3) δ 5.65 (q, 1H, J ) 6.9 Hz, H-5), 4.26 (brs, 1H, H-3),
3.71 (s, 3H, Me-O), 3.20 (brs, 3H, Me-N), 3.08 (m, 1H, H-2),
1.64 (d, 3H, J ) 6.9 Hz, H-6), 1.59 (s, 3H, H-7), 1.09 (d, 3H, J
) 6.9 Hz, H-8); 13C NMR (CDCl3) δ 178.0, 133.7, 120.3, 75.4,
61.5, 36.9, 31.9, 13.2, 12.9, 10.4.
1
777 cm-1; H NMR (CDCl3) δ 5.43 (q, 1H, J ) 6.4 Hz, H-5),
4.14 (d, 1H, J ) 10.1 Hz, H-3), 3.73 (s, 3H, Me-O), 3.14
(overlapping m and brs, 4H, H-2, Me-N), 1.60 (d, 3H, J ) 6.4
Hz, H-6), 1.56 (s, 3H, H-7), 0.83 (d, 3H, J ) 6.9 Hz, H-8), 0.79
(s, 9H, (Me)3CSi), -0.02 (s, 3H, Me-Si), -0.06 (s, 3H, Me-Si);
13C NMR (CDCl3) δ 176.5, 135.6, 123.3, 81.7, 61.3, 38.8, 31.8,
25.6 (3C), 18.0, 14.2, 13.0, 10.0, -5.0, -5.3.
(2S,3S,4E)-3-(ter t-Bu tyld im eth ylsilyloxy)-N-m eth oxy-
N,2,4-tr im eth yl-4-h exen a m id e (8c). Using the method de-
scribed for the preparation of 8a , amide 7c (265 mg, 1.3 mmol)
was silylated with TBSCl (600 mg, 4.0 mmol) and imidazole
(540 mg, 7.9 mmol) to give protected amide 8c as a colorless
oil (167 mg, 0.53 mmol, 84%): [R]D +3° (c 0.50, CHCl3); 1H
NMR (CDCl3) δ 5.35 (q, 1H, J ) 6.4 Hz, H-5), 4.09 (d, 1H, J )
9.1 Hz, H-3), 3.61 (s, 3H, Me-O), 3.07 (overlapping brs and m,
4H, Me-N, H-2), 1.55 (s, 3H, H-7), 1.50 (d, 3H, J ) 6.4 Hz,
H-6), 1.15 (d, 3H, J ) 6.9 Hz, H-8), 0.86 (s, 9H, (Me)3CSi),
0.02 (s, 3H, Me-Si), -0.06 (s, 3H, Me-Si); 13C NMR (CDCl3) δ
176.2, 136.3, 121.5, 80.2, 61.4, 40.4, 32.0, 25.8 (3C), 18.2, 14.7,
12.9, 10.9, -4.8, -5.1.
(2R,3S,4E)-3-(ter t-Bu tyld im eth ylsilyloxy)-N-m eth oxy-
N,2,4-tr im eth yl-4-h exen a m id e (8d ). To a stirred suspension
of N,O-dimethylhydroxylamine hydrochloride (302 mg, 3.1
mmol) in THF (3.0 mL) at 0 °C under argon was slowly added
1.0 M trimethylaluminum in n-hexane (3.0 mL, 3.0 mmol) with
concomitant evolution of gas. The resulting homogeneous
solution was stirred for 30 min at room temperature and then
recooled to 0 °C, and a solution of aldol 6d (166 mg, 0.62 mmol)
in THF (1.0 mL) was added over a period of 5 min. The solution
was stirred overnight at room temperature, and then ice-cooled
0.5 M aqueous HCl (30 mL) and EtOAc (10 mL) were added.
The layers were separated, and the aqueous layer was
extracted with EtOAc (10 mL × 3). The combined organic
layers were dried over anhydrous MgSO4 and concentrated
under reduced pressure. To the residue were added TBSCl (272
mg, 1.8 mmol) and imidazole (245 mg, 3.6 mmol) in DMF (3
(2R,3R,4E)-3-(ter t-Bu tyld im eth ylsilyloxy)-N-m eth oxy-
N,2,4-tr im eth yl-4-h exen a m id e (8a ). A mixture of 7a (265
mg, 1.3 mmol), TBSCl (600 mg, 4.0 mmol), and imidazole (540
mg, 7.9 mmol) in DMF (6 mL) was stirred overnight at room

1338 J ournal of Natural Products, 2004, Vol. 67, No. 8
Nakao et al.
mL) and stirred overnight at room temperature. The reaction
mixture was quenched with water and extracted with EtOAc
(10 mL × 3). The combined organic layers were dried over
anhydrous MgSO4 and concentrated under reduced pressure,
yielding a viscous yellow oil. The resulting oil was purified by
PTLC (EtOAc/n-hexane, 15:85), and protected 8d was obtained
as a colorless solid (127 mg, 0.4 mmol, 65%): [R]D -31° (c 0.23,
CHCl3); IR (KBr) 2930, 2858, 1662, 1386, 1250, 1057, 862, 837,
1.61 (d, 3H, J ) 6.4 Hz, H-6), 1.56 (s, 3H, H-7), 0.84
(overlapping s and d, 12H, (Me)3CSi, H-8), 0.01 (s, 3H, Me-
Si), -0.05 (s, 3H, Me-Si); 13C NMR (CDCl3) δ 205.4, 135.3,
123.1, 80.6, 50.2, 25.7 (3C), 18.0, 12.9, 10.9, 10.5, -4.6, -5.4.
(5R,6S,7R,2E,8E)-7-(ter t-Bu tyld im eth ylsilyloxy)-5-h y-
d r oxy-2,6,8-tr im eth yl-2,8-d eca d ien oic Acid Meth yl Ester
(10a ). Boron trifluoride etherate (128 µL, 1.02 mmol) was
added dropwise to a solution of aldehyde 9a (262 mg, 1.02
mmol) and 1-methoxy-2-methyl-1-trimethylsiloxy-1,3-butadi-
ene (209 mg, 1.12 mmol) in CH2Cl2 (10 mL) at -78 °C under
argon. The reaction mixture was stirred for 1 h at -78 °C and
quenched by addition of 5% NaHCO3 (10 mL). The aqueous
layer was extracted with CH2Cl2 (10 mL × 3). The combined
organic layers were dried over anhydrous Na2SO4 and con-
centrated under reduced pressure. The residue was purified
by PTLC (EtOAc/n-hexane, 20:80), and methyl ester 10a was
obtained as a colorless oil (304 mg, 0.82 mmol, 81%): [R]D +23°
(c 0.27, CHCl3); IR (KBr) 3440, 2929, 2858, 1718, 1256, 1055,
1
777 cm-1; H NMR (CDCl3) δ 5.41 (q, 1H, J ) 6.9 Hz, H-5),
4.12 (d, 1H, J ) 10.1 Hz, H-3), 3.71 (s, 3H, Me-O), 3.14
(overlapping brs and m, 4H, Me-N, H-2), 1.58 (d, 3H, J ) 6.9
Hz, H-6), 1.54 (s, 3H, H-7), 0.81 (d, 3H, J ) 6.9 Hz, H-8), 0.78
(s, 9H, (Me)3CSi), -0.04 (s, 3H, Me-Si), -0.08 (s, 3H, Me-Si);
13C NMR (CDCl3) δ 176.4, 135.5, 123.3, 81.6, 61.3, 38.7, 31.8,
25.6 (3C), 18.0, 14.1, 12.9, 10.0, -5.0, -5.4.
(2R,3R,4E)-3-(ter t-Bu tyld im eth ylsilyloxy)-2,4-d im eth -
yl-4-h exen a l (9a ). To a solution of amide 8a (407 mg, 1.29
mmol) in THF (5 mL) at -78 °C was added 1 M DIBAL in
THF (3.9 mL, 3.9 mmol) under argon. After 1.5 h the reaction
mixture was quenched with saturated aqueous Na2SO4 (10
mL) and EtOAc (10 mL) and the solution stirred vigorously.
After 10 min, anhydrous Na2SO4 (ca. 5 g) was added and the
reaction mixture stirred vigorously for a further 30 min. The
mixture was filtered through a pad of anhydrous Na2SO4 in a
funnel. The solvents were removed under reduced pressure.
The residue was purified by column chromatography (EtOAc/
n-hexane, 2.5:97.5), and aldehyde 9a was obtained as a
colorless oil (262 mg, 1.02 mmol, 79%): [R]D -1.9° (c 0.16,
CHCl3); IR (KBr) 3440, 2929, 2858, 1728, 1251, 1058, 837, 775
869, 837, 773 cm-1 1H NMR (CDCl3) δ 6.76 (t, 1H, J ) 6.9
;
Hz, H-3), 5.44 (q, 1H, J ) 6.4 Hz, H-9), 3.98 (d, 1H, J ) 6.9
Hz, H-7), 3.75 (m, 1H, H-5), 3.73 (s, 3H, Me-O), 2.42 (m, 1H,
H-4b), 2.25 (m, 1H, H-4a), 1.85 (s, 3H, H-13), 1.60 (overlapping
d and m, 4H, H-10, H-6), 1.52 (s, 3H, H-11), 0.91 (d, 3H, J )
6.9 Hz, H-12), 0.89 (s, 9H, (Me)3CSi), 0.05 (s, 3H, Me-Si), -0.04
(s, 3H, Me-Si); 13C NMR (CDCl3) δ 168.5, 138.8, 136.7, 129.2,
121.4, 81.8, 72.2, 51.7, 41.0, 34.6, 25.9 (3C), 18.1, 12.9, 12.7,
12.0, 7.5, -4.5, -5.2; anal. calcd for C20H38O4Si, C, 64.82; H,
10.34; found, C, 64.51; H, 10.23.
(5S,6R,7R,2E,8E)-7-(ter t-Bu tyld im eth ylsilyloxy)-5-h y-
d r oxy-2,6,8-tr im eth yl-2,8-d eca d ien oic Acid Meth yl Ester
(10b). Using the method described for the preparation of 10a ,
aldehyde 9b (262 mg, 1.02 mmol) and 1-methoxy-2-methyl-1-
trimethylsiloxy-1,3-butadiene (209 mg, 1.12 mmol) were treated
with boron trifluoride etherate (128 µL, 1.02 mmol) and yielded
methyl ester 10b as a colorless oil (111 mg, 0.3 mmol, 63%):
[R]D -10° (c 0.14, CHCl3); IR (KBr) 3505, 2929, 2858, 1716,
1
cm-1; H NMR (CDCl3) δ 9.60 (d, 1H, J ) 1.8 Hz, H-1), 5.41
(q, 1H, J ) 6.9 Hz, H-5), 4.21 (d, 1H, J ) 6.4 Hz, H-3), 2.46
(ddq, 1H, J ) 6.9, 6.4, 1.8 Hz, H-2), 1.54 (d, 3H, J ) 6.9 Hz,
H-6), 1.52 (s, 3H, H-7), 0.98 (d, 3H, J ) 6.9 Hz, H-8), 0.83 (s,
9H, (Me)3CSi), -0.02 (s, 3H, Me-Si), -0.07 (s, 3H, Me-Si); 13
C
NMR (CDCl3) δ 204.2, 135.5, 121.7, 77.9, 51.0, 25.7 (3C), 18.0,
12.8, 11.9, 9.2, -4.7, -5.4.
(2S,3R,4E)-3-(ter t-Bu tyld im eth ylsilyloxy)-2,4-d im eth -
yl-4-h exen a l (9b). To a solution of LiAlH4 (190 mg, 4.0 mmol)
in THF (4 mL) was added amide 8b (317 mg, 1.0 mmol) in
THF (10 mL) at -40 °C, and the mixture was stirred for 1 h.
The reaction mixture was quenched with 1 M aqueous HCl
(10 mL) and then extracted with EtOAc (10 mL × 3). The
combined organic layers were dried over anhydrous Na2SO4
and concentrated under reduced pressure. The residue was
purified by column chromatography (EtOAc/n-hexane, 2.5:
97.5), and aldehyde 9b was obtained as a colorless oil (124
mg, 0.48 mmol, 48%): [R]D +28° (c 0.45, CHCl3); IR (KBr) 3440,
1258, 1049, 862, 837, 775 cm-1 1H NMR (CDCl3) δ 6.78 (t,
;
1H, J ) 7.3 Hz, H-3), 5.51 (q, 1H, J ) 6.4 Hz, H-9), 4.00
(overlapping m and d, 2H, H-5, H-7), 3.70 (s, 3H, Me-O), 2.38
(m, 1H, H-4b), 2.19 (m, 1H, H-4a), 1.83 (s, 3H, H-13), 1.68 (m,
1H, H-6), 1.61, (d, 3H, J ) 6.4 Hz, H-10), 1.51 (s, 3H, H-11),
0.89 (overlapping s and d, 12H, (Me)3CSi, H-12), 0.06 (s, 3H,
Me-Si), -0.03 (s, 3H, Me-Si); 13C NMR (CDCl3) δ 168.4, 139.4,
135.4, 128.8, 121.2, 82.4, 70.6, 51.6, 39.1, 33.7, 25.8 (3C), 18.0,
12.9, 12.6, 12.5, 11.4, -4.6,-5.3; anal. calcd for C20H38O4Si,
C, 64.82; H, 10.34; found, C, 64.88; H, 10.24.
(5S,6R,7S,2E,8E)-7-(ter t-Bu t yld im et h ylsilyloxy)-5-h y-
d r oxy-2,6,8-tr im eth yl-2,8-d eca d ien oic Acid Meth yl Ester
(10c). Using the method described for the preparation of 10a ,
aldehyde 9c (70.6 mg, 0.28 mmol) and 1-methoxy-2-methyl-
1-trimethylsiloxy-1,3-butadiene (65 mg, 0.33 mmol) were
treated with boron trifluoride etherate (35 µL, 0.28 mmol) and
yielded methyl ester 10c as a colorless oil (47.8 mg, 0.13 mmol,
2930, 2858, 1730, 1251, 1053, 858, 837, 775 cm-1 1H NMR
;
(CDCl3) δ 9.74 (d, 1H, J ) 2.8 Hz, H-1), 5.44 (q, 1H, J ) 6.4
Hz, H-5), 4.06 (d, 1H, J ) 9.2 Hz, H-3), 2.55 (m, 1H, H-2),
1.62 (d, 3H, J ) 6.4 Hz, H-6), 1.56 (s, 3H, H-7), 0.84
(overlapping s and d, 12H, (Me)3CSi, H-8), 0.01 (s, 3H, Me-
Si), -0.04 (s, 3H, Me-Si); 13C NMR (CDCl3) δ 205.4, 135.3,
123.1, 80.6, 50.2, 25.7 (3C), 18.0, 13.0, 10.9, 10.5, -4.6, -5.4.
(2S,3S,4E)-3-(ter t-Bu tyld im eth ylsilyloxy)-2,4-d im eth yl-
4-h exen a l (9c). Using the method described for the prepara-
tion of 9a , amide 8c (132 mg, 0.42 mmol) in THF (2 mL) was
treated with 1 M DIBAL in THF (1.25 mL, 1.25 mmol) and
yielded aldehyde 9c as a colorless oil (86.5 mg, 0.34 mmol,
1
49%): [R]D -21° (c 0.30, CHCl3); H NMR (CDCl3) δ 6.75 (t,
1H, J ) 7.3 Hz, H-3), 5.43 (q, 1H, J ) 6.9 Hz, H-9), 3.97 (d,
1H, J ) 6.9 Hz, H-7), 3.73 (s, 3H, Me-O), 2.41 (m, 1H, H-4b),
2.25 (m, 1H, H-4a), 1.85 (s, 3H, H-13), 1.60 (overlapping d and
m, 4H, H-10, H-6), 1.52 (s, 3H, H-11), 0.91 (d, 3H, J ) 6.9 Hz,
H-12), 0.88 (s, 9H, (Me)3CSi), 0.05 (s, 3H, Me-Si), -0.05 (s,
3H, Me-Si); 13C NMR (CDCl3) δ 168.5, 138.8, 136.7, 129.2,
121.4, 81.8, 72.3, 51.7, 41.0, 34.6, 25.9 (3C), 18.1, 12.9, 12.7,
12.0, 7.5, -4.5, -5.2; anal. calcd for C20H38O4Si, C, 64.82; H,
10.34; found, C, 64.63; H, 10.28.
1
81%): [R]D +2.8° (c 0.54, CHCl3); H NMR (CDCl3) δ 9.63 (d,
1H, J ) 1.4 Hz, H-1), 5.44 (q, 1H, J ) 6.4 Hz, H-5), 4.23 (d,
1H, J ) 6.4 Hz, H-3), 2.50 (ddq, 1H, J ) 6.9, 6.4, 1.4 Hz, H-2),
1.58 (d, 3H, J ) 6.4 Hz, H-6), 1.55 (s, 3H, H-7), 1.01 (d, 3H, J
) 6.9 Hz, H-8), 0.86 (s, 9H, (Me)3CSi), 0.02 (s, 3H, Me-Si),
-0.04 (s, 3H, Me-Si); 13C NMR (CDCl3) δ 204.6, 135.6, 121.8,
78.0, 51.1, 25.7 (3C), 18.1, 12.9, 11.9, 9.3, -4.6, -5.3.
(5R,6S,7S,2E,8E)-7-(ter t-Bu t yld im et h ylsilyloxy)-5-h y-
d r oxy-2,6,8-tr im eth yl-2,8-d eca d ien oic Acid Meth yl Ester
(10d ). Using the method described for the preparation of 10a ,
aldehyde 9d (80.9 mg, 0.31 mmol) and 1-methoxy-2-methyl-
1-trimethylsiloxy-1,3-butadiene (70 mg, 0.37 mmol) were
treated with boron trifluoride etherate (40 µL, 0.34 mmol) and
yielded methyl ester 10d as a colorless oil (72.6 mg, 0.2 mmol,
63%): [R]D +10° (c 0.15, CHCl3); IR (KBr) 3440, 2929, 2858,
1717, 1256, 1049, 862, 837, 777 cm-1; 1H NMR (CDCl3) δ 6.78
(t, 1H, J ) 7.3 Hz, H-3), 5.50 (q, 1H, J ) 6.9 Hz, H-9), 4.00
(overlapping m and d, 2H, H-5, H-7), 3.70 (s, 3H, Me-O), 2.37
(m, 1H, H-4b), 2.19 (m, 1H, H-4a), 1.83 (s, 3H, H-13), 1.67 (m,
(2R,3S,4E)-3-(ter t-Bu tyld im eth ylsilyloxy)-2,4-d im eth -
yl-4-h exen a l (9d ). Using the method described for the
preparation of 9a , amide 8d (109 mg, 0.35 mmol) in THF (1
mL) was treated with 0.93 M DIBAL in n-hexane (560 µL, 0.52
mmol) and yielded aldehyde 9d as a colorless oil (80.9 mg, 0.32
mmol, 90%): [R]D -28° (c 0.49, CHCl3); IR (KBr) 3440, 2956,
2929, 2858, 1728, 1251, 1053, 860, 837, 775 cm-1 1H NMR
;
(CDCl3) δ 9.74 (d, 1H, J ) 3.2 Hz, H-1), 5.44 (q, 1H, J ) 6.9
Hz, H-5), 4.06 (d, 1H, J ) 8.7 Hz, H-3), 2.54 (m, 1H, H-2),

Cytotoxic Depsipeptide from Philinopsis speciosa
J ournal of Natural Products, 2004, Vol. 67, No. 8 1339
775 cm-1 1H NMR (CDCl3) δ 5.53 (dt, 1H, J ) 6.4, 1.4 Hz,
1H, H-6), 1.60 (d, 3H, J ) 6.9 Hz, H-10), 1.50 (s, 3H, H-11),
0.88 (overlapping s and d, 12H, (Me)3CSi, H-12), 0.05 (s, 3H,
Me-Si), -0.04 (s, 3H, Me-Si); 13C NMR (CDCl3) δ 168.4, 139.4,
135.4, 128.8, 121.2, 82.3, 70.6, 51.6, 39.1, 33.7, 25.8 (3C), 18.0,
12.9, 12.6, 12.5, 11.4, -4.6, -5.3; anal. calcd for C20H38O4Si,
C, 64.82; H, 10.34; found, C, 64.22; H, 10.24.
;
H-3), 5.41 (q, 1H, J ) 6.9 Hz, H-9), 4.12 (d, 1H, J ) 4.6 Hz,
H-7), 4.04 (s, 2H, H-1), 3.58 (dt, 1H, J ) 8.2, 3.2 Hz, H-5),
2.25 (m, 1H, H-4b), 2.15 (m, 1H, H-4a), 1.76 (m, 1H, H-6), 1.69
(s, 3H, H-13), 1.61 (overlapping s and d, 6H, H-11, H-10), 0.89
(s, 9H, (Me)3CSi), 0.80 (d, 3H, J ) 6.9 Hz, H-12), 0.05 (s, 3H,
Me-Si), -0.03 (s, 3H, Me-Si); 13C NMR (CDCl3) δ 137.1, 126.9,
122.3, 121.5, 80.4, 73.2, 68.9, 42.7, 32.7, 25.9 (3C), 18.1, 14.0,
13.0, 12.9, 11.8, -4.6, -5.2.
(6R,7R,2E,8E)-7-(ter t-Bu tyldim eth ylsilyloxy)-5-oxo-2,6,8-
tr im eth yl-2,8-d eca d ien oic Acid Meth yl Ester (11a ). To a
solution of 10a (80.0 mg, 0.22 mmol) in DMSO/diethyl ether
(1:1, 3 mL) were added pyridinium trifluoroacetate (21 mg,
0.11 mmol) and DCC (134 mg, 0.65 mmol). After stirring at
room temperature for 1.5 h, the reaction mixture was diluted
with EtOAc (10 mL), and the insolubles were removed by
filtration. The filtrate was washed with 0.5 M aqueous HCl
(10 mL) and saturated NaHCO3 (10 mL). The organic layer
was dried over MgSO4 and concentrated under reduced pres-
sure. The residue was purified by PTLC (EtOAc/n-hexane, 10:
90), and keto ester 11a was obtained as a colorless oil (54.2
mg, 0.15 mmol, 68%): 1H NMR (CDCl3) δ 6.85 (t, 1H, J ) 6.9
Hz, H-3), 5.32 (q, 1H, J ) 6.4 Hz, H-9), 4.04 (d, 1H, J ) 8.3
Hz, H-7), 3.71 (s, 3H, Me-O), 3.24 (m, 2H, H-4), 2.80 (dq, 1H,
J ) 8.3, 6.9 Hz, H-6), 1.79 (s, 3H, H-13), 1.55 (s, 3H, H-11),
1.51 (d, 3H, J ) 6.4 Hz, H-10), 1.08 (d, 3H, J ) 6.9 Hz, H-12),
0.85 (s, 9H, (Me)3CSi), 0.01 (s, 3H, Me-Si), -0.06 (s, 3H, Me-
Si); 13C NMR (CDCl3) δ 209.2, 167.9, 135.9, 133.2, 130.3, 122.5,
80.1, 51.8, 51.5, 42.6, 25.8 (3C), 18.1, 13.6, 12.9, 12.8, 11.1,
-4.7, -5.2.
(6R,7S,2E,8E)-7-(ter t-Bu tyldim eth ylsilyloxy)-5-oxo-2,6,8-
tr im eth yl-2,8-d eca d ien oic Acid Meth yl Ester (11d ). Using
the method described for the preparation of 11a , methyl ester
10d (160.8 mg, 0.43 mmol) was treated with pyridinium
trifluoroacetate (83 mg, 0.43 mmol) and DCC (266 mg, 0.51
mmol) in DMSO/Et2O (1:1, 6 mL) and yielded keto ester 11d
as a colorless oil (126 mg, 0.34 mmol, 80%): 1H NMR (CDCl3)
δ 6.98 (t, 1H, J ) 6.9 Hz, H-3), 5.40 (q, 1H, J ) 6.4 Hz, H-9),
4.04 (d, 1H, J ) 9.6 Hz, H-7), 3.71 (s, 3H, Me-O), 3.39 (m, 2H,
H-4), 2.80 (m, 1H, H-6), 1.82 (s, 3H, H-13), 1.57 (d, 3H, J )
6.9 Hz, H-10), 1.52 (s, 3H, H-11), 0.86 (d, 3H, J ) 13.3 Hz,
H-12), 0.77 (s, 9H, (Me)3CSi), -0.08 (s, 3H, Me-Si), -0.10 (s,
3H, Me-Si); 13C NMR (CDCl3) δ 210.3, 168.0, 135.2, 133.3,
130.0, 123.6, 82.3, 51.7, 49.4, 44.3, 25.7 (3C), 17.9, 13.7, 12.9
(2C), 10.0, -4.8, -5.5.
(5S,6S,7S,2E,8E)-7-(ter t-Bu t yld im et h ylsilyloxy)-5-h y-
d r oxy-2,6,8-tr im eth yl-2,8-d eca d ien oic Acid Meth yl Ester
(12d ). To a solution of 11d (108 mg, 0.29 mmol) in MeOH (1.5
mL) was added NaBH4 (37 mg, 0.88 mmol) at -40 °C. After 2
h, the reaction mixture was quenched with saturated NaHCO3
(10 mL) and extracted with EtOAc (10 mL × 3). The combined
organic layers were dried over MgSO4 and concentrated
reduced pressure. The residue was purified by PTLC (EtOAc/
n-hexane, 20:80), and ester 12d was obtained as a colorless
oil (53.5 mg, 0.15 mmol, 85%): [R]D -31° (c 0.30, CHCl3); IR
(KBr) 3440, 2954, 2929, 2858, 1717, 1256, 1047, 860, 837, 775
cm-1; 1H NMR (CDCl3) δ 6.94 (t, 1H, J ) 6.4 Hz, H-3), 5.36 (q,
1H, J ) 6.9 Hz, H-9), 3.82 (d, 1H, J ) 9.1 Hz, H-7), 3.79 (dt,
1H, J ) 7.3, 3.7 Hz, H-5), 3.70 (s, 3H, Me-O), 2.40 (m, 1H,
H-4b), 2.30 (m, 1H, H-4a), 1.83 (s, 3H, H-13), 1.75 (m, 1H, H-6),
1.57 (d, 3H, J ) 6.9 Hz, H-10), 1.53 (s, 3H, H-11), 0.86 (s, 9H,
(Me)3CSi), 0.62 (d, 3H, J ) 6.9 Hz, H-12), 0.06 (s, 3H, Me-Si),
-0.03 (s, 3H, Me-Si); 13C NMR (CDCl3) δ 168.4, 139.2, 136.3,
128.6, 123.3, 86.2, 74.1, 51.6, 41.0, 33.5, 25.8 (3C), 18.0, 13.0,
12.9, 12.6, 10.6, -4.4, -5.3.
(5S,6S,7R,2E,8E)-7-(ter t-Bu tyld im eth ylsilyloxy)-2,6,8-
tr im eth yl-2,8-d eca d ien -1,5-d iol (13a ). To a solution of Li-
AlH4 (28.5 mg, 0.75 mmol) in THF (750 µL) at -78 °C was
added ester 11a (27.6 mg, 0.075 mmol) in THF (500 µL). The
solution was stirred for 1 h at -78 °C and then warmed to 0
°C for 1 h. The reaction mixture was quenched with 1 M
aqueous HCl (10 mL) and extracted with EtOAc (15 mL × 3).
The combined organic layers were washed with saturated
NaHCO3 (10 mL) and dried over MgSO4. Removal of the
solvent gave a residue, which was purified by SIL-HPLC
(EtOAc/n-hexane, 45:55), and protected triol 13a was obtained
as a colorless oil (2.2 mg, 0.0064 mmol, 8.6%): [R]D +14° (c
0.21, CHCl3); IR (KBr) 3422, 2927, 1385, 1249, 1049, 870, 837,
(5S,6R,7S,2E,8E)-7-(ter t-Bu tyld im eth ylsilyloxy)-2,6,8-
tr im eth yl-2,8-d eca d ien -1,5-d iol (13c). To a solution of 10c
(35.0 mg, 0.095 mmol) in THF (2.0 mL) at -78 °C was added
0.93 M DIBAL in n-hexane (300 µL, 0.28 mmol). After the
reaction mixture was stirred for 1 h and warmed to 0 °C for 1
h, saturated aqueous Na2SO4 (10 mL) and EtOAc (10 mL) were
added and the solution was stirred vigorously. After 10 min,
anhydrous Na2SO4 (ca. 5 g) was added and the reaction
mixture stirred vigorously for 30 min. The mixture was filtered
through a pad of anhydrous Na2SO4 by vacuum filtration. The
solvents were removed under reduced pressure. The residue
was purified by SIL-HPLC (EtOAc/n-hexane, 45:55), and
protected triol 13c was obtained as a colorless oil (13.1 mg,
0.057 mmol, 60%): [R]D -17° (c 0.21, CHCl3); IR (KBr) 3421,
2927, 1385, 1256, 1057, 870, 835, 773 cm-1; 1H NMR (CDCl3)
δ 5.43 (overlapping t and q, 2H, H-3, H-9), 4.02 (s, 2H, H-1),
3.97 (d, 1H, J ) 6.9 Hz, H-7), 3.64 (ddd, 1H, J ) 6.4, 6.4, 1.8
Hz, H-5), 2.30 (m, 1H, H-4b), 2.13 (m, 1H, H-4a), 1.68 (s, 3H,
H-13), 1.62 (overlapping d and m, 4H, H-10, H-6), 1.52 (s, 6H,
H-11), 0.91 (d, 3H, J ) 6.9 Hz, H-12), 0.89 (s, 9H, (Me)3CSi),
0.06 (s, 3H, Me-Si), -0.04 (s, 3H, Me-Si); 13C NMR (CDCl3) δ
137.0, 136.8, 122.2, 121.3, 81.9, 72.8, 68.7, 40.7, 33.6, 25.9 (3C),
18.1, 13.9, 12.9, 11.9, 7.7, -4.5, -5.1.
(5S,6S,7S,2E,8E)-7-(ter t-Bu t yld im et h ylsilyloxy)-2,6,8-
tr im eth yl-2,8-d eca d ien -1,5-d iol (13d ). Using the method
described for the preparation of 13c, ester 12d (42.5 mg, 0.12
mmol) in THF (2.0 mL) was treated with 1 M DIBAL in THF
(570 µL, 0.57 mmol) and yielded protected triol 13d as a
colorless oil (35.5 mg). The resultant oil was used without
purification in the subsequent reaction.
(5S,6S,7R,2E,8E)-2,6,8-Tr im et h yl-2,8-d eca d ien -1,5,7-
tr iol (3a ). To a solution of 13a (2.1 mg, 0.006 mmol) in MeOH
(500 µL) was added pyridinium p-toluene sulfonate (PPTS) (9.5
mg, 0.038 mmol) overnight at room temperature. After con-
centration, removal of the salt by Waters Sep-Pak Vac 12 cm3
Silica-2g gave a residue, which was purified by ODS-HPLC
(MeCN/H2O, 40:60). Triol 3a was obtained as a colorless oil
(1.3 mg, 0.0057 mmol, 95%): [R]D +3° (c 0.14, CHCl3); 1H NMR
(CDCl3) δ 5.53 (overlapping t and q, 2H, H-3, H-9), 4.36 (brs,
1H, H-7), 4.05 (s, 2H, H-1), 3.70 (m, 1H, H-5), 2.35 (m, 2H,
H-4), 1.77 (m, 1H, H-6), 1.71 (s, 3H, H-13), 1.65 (d, 3H, J )
6.9 Hz, H-10), 1.56 (s, 3H, H-11), 0.88 (d, 3H, J ) 7.1 Hz, H-12);
13C NMR (CDCl3) δ 137.7, 136.1, 121.9, 118.8, 75.7, 74.9, 68.6,
39.6, 33.5, 14.0, 13.6, 13.0, 10.6.
(5S,6S,7R,2E,8E)-2,6,8-Tr im et h yl-2,8-d eca d ien -1,5,7-
tr iol (3b). To a solution of 10b (35.8 mg, 0.097 mmol) in THF
(1.0 mL) at -78 °C was added 1 M DIBAL in THF (2.4 mL,
2.4 mmol). After the reaction mixture was stirred for 30 min
and warmed to 0 °C for 1 h, saturated aqueous Na2SO4 (10
mL) and EtOAc (10 mL) were added and the solution was
stirred vigorously. After 10 min, anhydrous Na2SO4 (ca. 5 g)
was added and the reaction mixture stirred vigorously for 30
min. The mixture was filtered through a pad of anhydrous Na2-
SO4 in a funnel. The solvents were removed under reduced
pressure. The residue was purified by ODS-HPLC (MeCN/H2O,
40:60), and triol 3b was obtained as a colorless oil (11.0 mg,
0.048 mmol, 50%): [R]D -21° (c 0.31, CHCl3); HRFABMS m/z
457.3521 (2M + H)+ (for C26H49O6, ∆ -0.8 mmu); IR (KBr)
1
3390, 2918, 1418, 1383, 1009 cm-1; H NMR (CDCl3) δ 5.49
(overlapping t and q, 2H, H-3, H-9), 3.98 (overlapping s and
d, 3H, H-1, H-7), 3.87 (m, 1H, H-5), 2.33 (m, 1H, H-4b), 2.12
(m, 1H, H-4a), 1.85 (m, 1H, H-6), 1.68 (s, 3H, H-13), 1.62 (d,
3H, J ) 6.4 Hz, H-10), 1.57 (s, 3H, H-11), 0.80 (d, 3H, J ) 6.9
Hz, H-12),; 13C NMR (CDCl3) δ 137.3, 136.5, 122.6, 121.9, 80.9,
72.8, 68.6, 39.0, 31.7, 14.0, 13.0, 11.7, 11.1.

1340 J ournal of Natural Products, 2004, Vol. 67, No. 8
Nakao et al.
(5S,6R,7S,2E,8E)-2,6,8-Tr im et h yl-2,8-d eca d ien -1,5,7-
tr iol (3c). Using the method described for the preparation of
3a , protected alcohol 13c (9.1 mg, 0.027 mmol) in MeOH (1.0
mL) was treated with PPTS (20.0 mg 0.08 mmol) and yielded
triol 3c as a colorless oil (4.1 mg, 0.018 mmol, 68%): [R]D -6°
Amberlite GC50, followed by 400 µL of anhydrous hydrazine.
The reaction mixture was heated under argon for 60 h at 80
°C. After cooling to room temperature the reaction mixture
was frozen and lyophilized, suspended in water (1.2 mL),
filtered, and again frozen and freeze-dried. To the residue was
added 50 µL of 2.9 mM FDAA solution in acetone and 100 µL
1
(c 0.40, CHCl3); H NMR (CDCl3) δ 5.54 (q, 1H, J ) 6.9 Hz,
H-9), 5.46 (t, 1H, J ) 6.9 Hz, H-3), 4.18 (brs, 1H, H-7), 4.03 (s,
2H, H-1), 3.87 (ddd, 1H, J ) 8.2, 5.5, 1.8 Hz, H-5), 2.35 (m,
1H, H-4b), 2.15 (m, 1H, H-4a), 1.69 (overlapping s and m, 4H,
H-13, H-6), 1.64 (d, 3H, J ) 6.9 Hz, H-10), 1.55 (s, 3H, H-11),
0.85 (d, 3H, J ) 6.9 Hz, H-12); 13C NMR (CDCl3) δ 137.5, 136.1,
121.8, 119.1, 80.6, 75.4, 68.7, 38.8, 33.7, 14.0, 13.3, 13.0, 5.1;
anal. calcd for C13H24O3, C, 68.38; H, 10.59; found, C, 68.01;
H, 10.44.
, followed by heating at 80 °C for 3 min. After
of 1 M NaHCO3
being cooled to room temperature, the reaction mixture was
neutralized with 50 µL of 2 M HCl and diluted with 100 µL of
MeCN/H2O/TFA (50:50:0.05). This solution was analyzed by
reversed-phase HPLC [Inertsil ODS-2, MeCN/H2O/TFA (25:
75:0.05)]. The only unmodified residue, Ala-2, was analyzed
to show L-Ala.
(5S,6S,7S,2E,8E)-2,6,8-Tr im eth yl-2,8-d eca d ien -1,5,7-tr i-
ol (3d ). Using the method described for the preparation of 3a ,
a mixture of 13d (35.5 mg) in MeOH (2.0 mL) was treated
with PPTS (75 mg 0.3 mmol) and yielded triol 3d as a white
solid (14.3 mg, 0.063 mmol, 55% from 12d ): mp 91-92 °C;
[R]D -23° (c 0.18, CHCl3); HRFABMS m/z 457.3513 (2M + H)+
(for C26H49O6, ∆ -1.7 mmu); IR (KBr) 3275, 2918, 1418, 1386,
Ack n ow led gm en t. We thank N. Fusetani, S. Fukuzawa,
and T. Wakimoto at the University of Tokyo for bioassay and
mass spectral measurements. Financial assistance by the
National Science Foundation, 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.
1
1014 cm-1; H NMR (CDCl3) δ 5.52 (t, 1H, J ) 6.4 Hz, H-3),
5.43(q, 1H, J ) 6.0 Hz, H-9), 3.99 (s, 1H, H-1), 3.88 (d, 1H, J
) 9.1 Hz, H-7), 3.67 (dt, 1H, J ) 8.7, 3.2 Hz, H-5), 2.34 (m,
1H, H-4b), 2.18 (m, 1H, H-4a), 1.73 (m, 1H, H-6), 1.67 (s, 3H,
H-13), 1.60 (overlapping s and d, 6H, H-11, H-10), 0.64 (d, 3H,
J ) 6.9 Hz, H-12); 13C NMR (CDCl3) δ 137.7, 136.6, 123.5,
121.5, 85.0, 76.8, 68.6, 40.2, 33.1, 14.0, 13.4, 13.0, 10.3.
P r ep a r a tion of 3d fr om 1. To the solution of Kulokeka-
hilide-2 (1, 0.3 mg in 1 mL of Et2O) was added 25 µL of 1 M
LiAlH4 in Et2O. After stirring for 30 min at room temperature,
the reaction mixture was partitioned between H2O and Et2O.
The organic layer was concentrated and then separated by
ODS HPLC with 60% MeCN to yield 3d : 1H NMR (CD3OD),
see Figure 4.
Su p p or tin g In for m a tion Ava ila ble: NMR spectra of kulokeka-
hilide-2 (1). This material is available free of charge via the Internet
at http://pubs.acs.org.
Refer en ces a n d Notes
(1) (a) Coval, S. J .; Schulte, G. R.; Matsumoto, G. K.; Roll, D. M.; Scheuer,
P. J . Tetrahedron Lett. 1985, 26, 5359-5362. (b) Coval, S. J .; Scheuer,
P. J . J . Org. Chem. 1985, 50, 3024-3025. (c) 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. (d) Nakao,
Y.; Yoshida, W. Y.; Szabo, C. M.; Baker, B. J .; Scheuer, P. J . J . Org.
Chem. 1998, 63, 3272-3280. (e) Nakao, Y.; Yoshida, W. Y.; Scheuer,
P. J . Tetrahedron Lett. 1996, 37, 8993-8996. (f) For the origin of the
name “kulokekahilide-1”, refer to: Kimura, J .; Takada, Y.; Inayoshi,
T.; Nakao, Y.; Goetz, G.; Yoshida, W. Y.; Scheuer, P. J . J . Org. Chem.
2002, 67, 1760-1767.
MTP A Ester s of 1. Kulokekahilide-2 (1, 0.3 mg each) was
reacted with R- or S-MTPACl (10 µL) in 300 µL of CH2Cl2
containing 10 mg of DMAP. The reaction mixtures were
partitioned with EtOAc/0.1 M NaHCO3, and the EtOAc layers
were washed with 0.1 M HCl and H2O. The obtained EtOAc
layers were evaporated and then separated by ODS HPLC
[COSMOSIL 5C18-AR II, MeCN/H2O (7:3 and 8:2)] to yield S-
and R-MTPA esters (1a and 1b, respectively).
(2) (a) Suenaga, K.; Mutou, T.; Shibata, T.; Itoh, T.; Kigoshi, H.; Yamada,
K. Tetrahedron Lett. 1996, 37, 6771-6774. (b) 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. (c) Pettit, G. R.;
Xu, J .-P.; Hogan, F.; Cerny, R. L. Heterocycles 1998, 47, 491-496.
(3) (a) Rodr´ıguez, J .; Ferna´ndez, R.; Quin˜oa´, E.; Riguera, R.; Debitus,
C.; Borchet, P. Tetrahedron Lett. 1994, 35, 9239-9242. (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.
(4) Kupchan, S. M.; Britton, R. W.; Ziegler, M. F.; Sigel, C. W. J . Org.
Chem. 1973, 38, 178-179.
1
1a : H NMR (CD3CN) δ 6.585 (H-3), 2.521 (H-4a), 2.2076
(H-4b), 4.582 (H-5), 2.300 (H-6), 5.316 (H-7), 5.703 (H-9), 1.627
(H-10), 1.892 (H3-11), 0.734 (H3-12), 1.580 (H-13); FABMS m/z
1042 (M + H)+.
(5) Bax, A.; Azolos, A.; Dinya, Z.; Sudo, K. J . Am. Chem. Soc. 1986, 108,
8056-8063.
1b: 1H NMR (CD3CN) δ 6.608 (H-3), 2.558 (H-4a), 2.276 (H-
4b), 4.825 (H-5), 2.298 (H-6), 5.269 (H-7), 5.571 (H-9), 1.574
(H-10), 1.790 (H3-11), 0.776 (H3-12), 1.337 (H-13); FABMS m/z
1042 (M + H)+.
(6) (a) Evans, D. A.; Bartroli, J .; Shih, T. L. J . Am. Chem. Soc. 1981,
103, 2127-2129. (b) Pettit, G. R.; Burkett, D. D.; Barko´czy, J .;
Breneman, G. L.; Pettit, W. E. Synthesis 1996, 719-725.
(7) (a) Danda, H.; Hansen, M. M.; Heathcock, C. H. J . Org. Chem. 1990,
55, 173-181. (b) Raimundo B. C.; Heathcock, C. H. Synlett 1995,
1213-1214.
Meth a n olysis of 1. Kulokekahilide-2 (1, 0.3 mg) was
treated with 0.1 M MeONa (0.5 mL) overnight, then parti-
tioned between H2O and CHCl3. The organic layer was
(8) Basha, A.; Lipton, M.; Weinreb, S. M. Tetrahedron Lett. 1977, 22,
3815-3818.
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Am. Chem. Soc. 1996, 118, 4322-4343.
concentrated and separated by ODS HPLC [COSMOSIL 5C18
-
MS, MeOH/H2O (8:2 and 19:1)] to yield fragment 4.
Absolu te Ster eoch em istr y of Am in o a n d Hyd r oxyl
Acid Resid u es. A half portion of fragment 4 was hydrolyzed
(6 M HCl, 105 °C, 18 h) and dried under N2. The residue was
dissolved in MeOH and separated on reversed-phase HPLC
(Inertsil prep-ODS) using a gradient of MeCN/H2O/TFA from
1:99:0.05 to 23:77:0.05 to yield three amino acids and Hica.
Hica was analyzed by chiral HPLC [Chiralpak MA(+), MeCN/
H2O (15:85) with 2 mM CuSO4], confirming D-Hica.
To each of the isolated amino acids were added 50 µL of 2.9
mM FDAA solution in acetone and 100 µL of 1 M NaHCO3,
followed by heating at 80 °C for 3 min. After being cooled to
room temperature, the reaction mixtures were neutralized
with 50 µL of 2 M HCl and diluted with 100 µL of MeCN/H2O/
TFA (50:50:0.05). These solutions were analyzed by reversed-
phase HPLC [Inertsil ODS-2, MeCN/H2O/TFA (25:75:0.05)] to
furnish D- and L-Ala, N-Me-L-Phe, and L-Ile.
(12) Hoffman R. V.; Kim, H. J . Org. Chem. 1991, 56, 1014-1019.
(13) (a) Rychnovsky, S. D.; Rogers, B.; Yang, G. J . Org. Chem. 1993, 58,
3511-3515. (b) Rychnovsky S. D.; Skalitzky, D. J . Tetrahedron Lett.
1990, 31, 945-948. (c) Evans, D. A.; Rieger, D. L.; Gage, J . R.
Tetrahedron Lett. 1990, 31, 7099-7100.
(14) Mitsunobu, O. Synthesis 1981, 1-28.
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5670.
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1024.
(17) Mutou, T.; Suenaga, K.; Fujita, T.; Itoh, T.; Takada, N.; Hayamizu,
K.; Kigoshi, H.; Yamada, K. Synlett 1977, 199-201.
(18) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J . Am. Chem.
Soc. 1991, 113, 4092-4096.
(19) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
(20) (a) Goetz, G.; Nakao, Y.; Scheuer, P. J . J . Nat. Prod. 1997, 60, 562-
567. (b) Schroeder, W. A. Methods Enzymol. 1972, 25, 138-143.
(21) Takahashi, T.; Nagamiya, H.; Doi, T.; Griffiths, P. G.; Bray, A. M. J .
Comb. Chem. 2003, 5, 414-428.
Hyd r a zin olysis of F r a gm en t 4 fr om Ku lok ek a h ilid e-
2. The remaining half of 4 was added with 10 mg of dry
NP049949F