Isolation and stereostructures of dolastatin G and nordolastatin G, cytotoxic 35-membered cyclodepsipeptides from the Japanese sea hare Dolabella auricularia

Article abstract of DOI:10.1021/jo9608228

A bioassay-directed fractionation of the cytotoxic constituents of the Japanese sea hare Dolabella auricularia resulted in the isolation of two 35-membered depsipeptides dolastatin G (1) and nordolastatin G (2), which showed cytotoxicity against HeLa S₃ cells with IC₅₀ values of 1.0 and 5.3 μg/mL, respectively. The gross structures of these substances were established by spectroscopic analysis including 2D NMR techniques. The absolute stereostructure of 1 was determined by chiral HPLC analysis of amino acid components obtained from acid hydrolysis of 1 and by the enantioselective syntheses of two degradation products arising from polyketide portions. Nordolastatin G (2) is a congener that has the same absolute stereochemistry as that of 1.

Full text of DOI:10.1021/jo9608228

6340
J . Org. Chem. 1996, 61, 6340-6345
Isola tion a n d Ster eostr u ctu r es of Dola sta tin G a n d Nor d ola sta tin
G, Cytotoxic 35-Mem ber ed Cyclod ep sip ep tid es fr om th e J a p a n ese
Sea Ha r e Dola bella a u r icu la r ia
Tsuyoshi Mutou, Takashi Kondo, Makoto Ojika, and Kiyoyuki Yamada*
Department of Chemistry, Faculty of Science, Nagoya University, Chikusa, Nagoya 464, J apan
Received May 6, 1996X
A bioassay-directed fractionation of the cytotoxic constituents of the J apanese sea hare Dolabella
auricularia resulted in the isolation of two 35-membered depsipeptides dolastatin G (1) and
nordolastatin G (2), which showed cytotoxicity against HeLa S3 cells with IC50 values of 1.0 and
5.3 µg/mL, respectively. The gross structures of these substances were established by spectroscopic
analysis including 2D NMR techniques. The absolute stereostructure of 1 was determined by chiral
HPLC analysis of amino acid components obtained from acid hydrolysis of 1 and by the
enantioselective syntheses of two degradation products arising from polyketide portions. Nordo-
lastatin G (2) is a congener that has the same absolute stereochemistry as that of 1.
The Western Indian Ocean sea hare Dolabella auricu-
fractionation using silica gel (i. toluene/EtOAc and
EtOAc/MeOH, step gradient; ii. 4:1 hexane/acetone to
acetone, linear gradient) and ODS silica gel (70% aqueous
MeOH to MeOH, linear gradient) successively. Further
separation by HPLC (ODS, 60% aqueous MeCN) afforded
dolastatin G (1) (35 mg) as colorless prisms and a fraction
containing nordolastatin G (2). The fraction containing
2 was purified by repeated preparative TLC with two
solvent systems (2.5:1 CHCl3/acetone and 2:1 benzene/
acetone) to afford 2 (0.5 mg) as a colorless powder.
Dolastatins G (1) and nordolastatin G (2) showed cyto-
toxicity against HeLa S3 cells with IC50 values of 1.0 and
5.3 µg/mL, respectively.
laria (Aplysiidae) is known to be a rich source of anti-
neoplastic and/or cytostatic peptides such as dolastatins
10 and 15.1 We have examined the constituents of
J apanese specimens of this animal and found several
cytotoxic depsipeptides2 and other unique metabolites.3
We now report the isolation and structural determination
of cytotoxic macrocyclic depsipeptides of mixed peptide-
polyketide biogenesis, dolastatin G (1) and nordolastatin
G (2), from the J apanese sea hare D. auricularia.
The molecular formula of 1 was established to be
C57H96N6O13 by combustion analysis and high-resolution
FABMS. 1H and 13C NMR data for 1 are summarized in
Table 1, in which all of the protonated carbons were
assigned by 1H-13C COSY data. The depsipeptide nature
of 1 was indicated by the presence of six nitrogen atoms
and nine sp2 carbon signals in the ester and amide region
(δ 166.8-175.5) of the 13C NMR spectrum as well as by
the absorption bands at 1735, 1635, and 1460 cm-1 in
the IR spectrum. DQF-COSY experiments and HMBC
data (Table 1) established the presence of six amino acid
residues (two prolines, two N-methylvalines, N,O-di-
methylserine, and N-methylisoleucine) and two hydroxy
acid portions (C35-C46 and C47-C57). One of the nine
sp2 carbon signals (δ 170.2) in the ester and amide region
was assigned to C37 of the enol ether group by the HMBC
correlations (C37/H36, C37/H39, and C37/H44). The
location of a hydroxyl group in 1 was determined to be
at C49 by the COSY correlation between the oxymethine
proton at δ 4.17 (H49) and the D2O-exchangeable proton
at δ 4.23. The low-field chemical shifts of H42 (δ 5.00)
and H53 (δ 5.10) suggest that the acyloxy groups are at
these positions. The E geometry of two olefinic bonds
was evidenced by difference NOE data (H36/H44 and
H40/H45). The degree of unsaturation in 1 suggests a
cyclic nature of 1. The HMBC correlations shown in
Table 1 enabled us to connect the partial structures
described above (six amino acids and two polyketide
units) and to assign all the carbonyl and quaternary
olefinic carbons, establishing the gross structure of 1.
The MeOH extract of the internal organs of the sea
hare D. auricularia (40 kg, wet wt), collected in Mie
Prefecture, J apan, was partitioned between EtOAc and
water. The EtOAc-soluble material, which exhibited
strong cytotoxicity against HeLa S3 cells with an IC50 of
1.2 µg/mL, was further partitioned between 90% aqueous
MeOH and hexane. The material obtained from the
aqueous MeOH portion was subjected to bioassay-guided
X Abstract published in Advance ACS Abstracts, August 15, 1996.
(1) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Fujii, Y.; Kizu, H.; Boyd,
M. R.; Boettner, F. E.; Doubek, D. L.; Schmidt, J . M.; Chapuis, J .-C.;
Michel, C. Tetrahedron 1993, 49, 9151-9170.
(2) (a) Sone, H.; Nemoto, T.; Ojika, M.; Yamada, K. Tetrahedron Lett.
1993, 34, 8445-8448. (b) Sone, H.; Nemoto, T.; Ishiwata, H.; Ojika,
M.; Yamada, K. Tetrahedron Lett. 1993, 34, 8449-8452. (c) Ishiwata,
H.; Nemoto, T.; Ojika, M.; Yamada, K. J . Org. Chem. 1994, 59, 4710-
4711. (d) Ojika, M.; Nemoto, T.; Nakamura, M.; Yamada, K. Tetrahe-
dron Lett. 1995, 36, 5057-5058. (e) Nakamura, M.; Shibata, T.;
Nakane, K.; Nemoto, T.; Ojika, M.; Yamada, K. Tetrahedron Lett. 1995,
36, 5059-5062. (f) Sone, H.; Shibata, T.; Fujita, T.; Ojika, M.; Yamada,
K. J . Am. Chem. Soc. 1996, 118, 1874-1880.
The molecular formula of the minor congener nordo-
(3) (a) Ojika, M.; Nemoto, T.; Yamada, K. Tetrahedron Lett. 1993,
34, 3461-3462. (b) Sone, H.; Kondo, T.; Kiryu, M.; Ishiwata, H.; Ojika,
M.; Yamada, K. J . Org. Chem. 1995, 60, 4774-4781. (c) Ojika, M.;
Nagoya, T.; Yamada, K. Tetrahedron Lett. 1995, 36, 7491-7494.
lastatin G (2), C56H94N6O13, was determined by high-
1
resolution FABMS. Resonances in the H and 13C NMR
spectra of 2 were similar to those of 1, except for the
S0022-3263(96)00822-5 CCC: $12.00 © 1996 American Chemical Society

Isolation of Dolastatin G and Nordolastatin G
J . Org. Chem., Vol. 61, No. 18, 1996 6341
Ta ble 1. NMR Da ta a n d HMBC Cor r ela tion s for Dola sta tin G (1) in C6D6
position
1Ha
13Cb
HMBCc
H-2, 53
H-3 (1.53)
H-2
H-2, 3
H-2, 3 (1.91)
position
1Ha
13Cb
HMBCc
1
2
3
4
5
170.9 s
60.3 d
31.7 t
21.9 t
46.0 t
29
30
31
32
33
34
35
36
37
38
39
40
5.61 d (11.0)
2.39 m
1.15, 1.41 m
0.90 t (7.3)
0.84 d (6.2)
2.85 s
57.0 d
33.3 d
24.6 t
H-30, 33, 34
H-29, 32, 33
H-29, 32, 33
4.65 br d (8.1)
1.53, 1.91 m
1.22, 1.45 m
3.45 ddd (12.1, 8.8, 8.8)
3.67 ddd (12.1, 8.8, 2.2)
11.1 q
15.9 q
30.9 q
167.9 s
93.4 d
170.2 s
133.2 s
130.4 d
31.6 t
H-29, 31
H-29
H-29, 34, 36
6
7
8
169.2 s
58.2 d
27.9 d
19.0 q
18.8 q
29.6 q
171.2 s
58.4 d
27.7 t
24.2 t
47.3 t
H-7
5.18 d (11.0)
2.42 ddq (11.0, 6.6, 6.6)
0.77 d (6.6)
H-8, 9, 10, 11
H-7, 9, 10
H-7, 8, 10
H-7, 8, 9
H-7
H-7, 11, 13
H-14 (1.52)
H-13
4.89 s
H-36, 39, 44
H-36, 45
H-45
9
10
11
12
13
14
15
16
1.04 d (6.6)
5.73 m
1.84 m
3.07 s
H-39, 42
2.00 br dd (12.5, 5.9)
1.73 m
5.00 dq (4.4, 6.2)
1.11 d (6.2)
3.08 s
4.68 br d (8.8)
1.52, 1.90 m
1.65, 2.37 m
3.13 ddd (9.9, 9.9, 7.3)
3.80 br t (8.8)
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
OH
38.5 t
74.2 d
14.6 q
54.9 q
14.9 q
13.5 q
175.5 s
48.3 d
74.0 d
35.7 t
23.6 t
32.1 t
78.5 d
30.9 d
19.9 q
15.2 q
14.8 q
H-40, 42, 43
H-46
H-42
H-13, 14
H-13, 14 (1.90)
1.96 br s
H-39
17
18
19
166.8 s
54.8 d
68.6 t
H-18
H-19, 21
H-18, 20
1.12 d (6.6)
H-40 (2.00), 42
H-42, 48
H-56
5.94 dd (11.4, 4.8)
3.62 dd (11.4, 4.8)
3.92 dd (11.4, 11.4)
2.91 s
2.79 dq (9.5, 7.0)
4.17 m
OH, H-48, 56
20
21
22
23
24
25
26
27
28
58.3 q
29.8 q
170.7 s
58.9 d
27.4 d
17.7 q
20.4 q
30.8 q
170.4 s
H-19 (3.92)
H-18
1.75, 1.82 m
1.43, 2.07 m
1.89, 2.28 m
5.10 ddd (7.7, 5.5, 2.2)
1.77 m
0.83 d (6.6)
1.07 d (7.0)
0.85 d (6.6)
4.23 d (10.9)
2.90 s
H-18, 21, 23
H-24, 25, 26, 27
H-23, 25, 26
H-23, 24, 26
H-23, 24, 25
H-23
5.42 d (10.6)
2.67 dqq (10.6, 6.6, 6.6)
0.93 d (6.6)
H-55, 57
H-55, 57
H-53, 54, 57
H-48
1.01 d (6.6)
3.31 s
H-53, 54, 55
H-27, 29
a
b
Recorded at 600 MHz. Coupling constants in Hz are in parentheses. Recorded at 100 MHz. c Recorded at 400 MHz. Parameters
were optimized for J CH ) 8 Hz.
Sch em e 1a
Sch em e 2a
a
Key: (a) LiAlH4, THF, 0 °C; (b) t-BuPh2SiCl, imidazole, DMF,
a
Key: (a) O3, MeOH, -78 °C; (b) LiAlH4, Et2O, rt; (c)
0 °C; (c) DEAD, Ph3P, p-NO2C6H4CO2H, benzene, rt; (d) NaOMe,
MeOH, rt.
t-BuPh2SiCl, imidazole, DMF, 0 °C; (d) Bu4NF, THF, rt; (e)
(MeO)2CMe2, 10-camphorsulfonic acid (CSA), acetone, rt.
relative stereochemistry of C48-C49 was determined to
be anti from the spin-spin coupling constants J 47a,48
J 48,49 ) 11.5 Hz in 5.
)
observation of the signals due to the keto group (δC37
193.8) and a methylene group (δH36 3.41 and 3.60) in place
of those due to the methyl group (δH44 3.08) of the enol
ether part and an olefinic proton (δH36 4.89) in 1.
Interpretation of these NMR data coupled with the
molecular formula indicated that 2 was the 44-nor
derivative of 1.
The absolute stereostructures of 1 and 2 were eluci-
dated as follows. Acid hydrolysis of 1 (6 M HCl at 110
°C) followed by chiral HPLC analysis [CHRALPAK MA-
(+)] established the configurations of six amino acids to
be all L. The absolute stereochemistry of five asymmetric
carbons (C41, C42, C48, C49, and C53) in 1 was deter-
mined by the enantioselective syntheses of degradation
products of 1. Ozonolysis of 1 followed by LiAlH4
reduction and selective protection of primary hydroxyl
groups with tert-butyldiphenylsilyl chloride produced the
C39-C43 fragment 3 and the C47-C57 fragment 4
derived from two polyketide portions (Scheme 1). Diol 4
was further converted to hydroxy acetonide 5. Thus, the
To determine the absolute stereochemistry of 3, we
synthesized two possible diastereomeric silyl ethers, 3a
and 3b (Scheme 2). Reduction of lactone 64 followed by
selective protection of the resulting primary hydroxyl
group gave silyl ether 3a . The diastereomer 3b was
prepared by methanolysis of p-nitrobenzoate 7, which
was obtained by a modified Mitsunobu reaction5 of 3a .
On comparison of the spectroscopic data and specific
rotations, synthetic 3b, [R]25 +6 (c 0.12, CHCl3), was
D
found to be identical to natural 3, [R]30 -6 (c 0.11,
D
CHCl3), except for the sign of specific rotation, establish-
ing the 41S,42R configurations in 1.
The absolute stereochemistry of 5 was determined by
the syntheses of two possible diastereomeric hydroxy
(4) Bystro¨m, S.; Ho¨gberg, H.-E.; Norin, T. Tetrahedron 1981, 37,
2249-2254.
(5) Martin, S. F.; Dodge, J . A. Tetrahedron Lett. 1991, 32, 3017-
3020.

6342 J . Org. Chem., Vol. 61, No. 18, 1996
Mutou et al.
Sch em e 3a
and ether (Na-benzophenone ketyl), benzene (Na), triethyl-
amine (calcium hydride), DMSO (calcium hydride under
reduced pressure), CH2Cl2 (P2O5), acetone (anhydrous K2CO3),
and MeOH (Mg). All moisture-sensitive reactions were per-
formed under an atmosphere of nitrogen.
Isola tion of Dola sta tin G (1) a n d Nor d ola sta tin G (2).
Specimens of D. auricularia (40 kg, wet wt) were collected by
hand at a depth of 0-1 m off the coast of the Shima Peninsula,
Mie Prefecture, J apan, in May 1993 and stored at -20 °C for
several months until extraction. The specimens were sepa-
rated into the internal organs and the thick outer skin, and
the former (23 kg) was extracted with MeOH (48 L). This
methanolic extract was concentrated in vacuo to ca. 1/12 of
its volume and extracted with EtOAc (6 × 2 L). The EtOAc
extracts were concentrated in vacuo, the residue (135.5 g, IC50
against HeLa S3 cells ) 1.2 µg/mL) was dissolved in 9:1 MeOH/
H2O (2 L), and the solution was washed with hexane (2 × 2
L). Evaporation of the aqueous MeOH portion gave a dark
brown oil (40.3 g), which was chromatographed on silica gel,
eluting with 1:1 toluene/EtOAc, EtOAc, 95:5, 90:10, and 80:
20 EtOAc/MeOH, and MeOH, successively. The fraction eluted
with 95:5 EtOAc/MeOH (2.36 g) was subjected to silica gel
MPLC [Micro Bead Silica Gel 4B (Fuji Silysia, 64 g), linear
gradient from 80:20 hexane/acetone to acetone, flow rate 6.0
mL/min]. The fraction eluted between 7:93 and 5:95 hexane/
acetone (516 mg) was subjected to reversed-phase MPLC
[Develosil ODS 30/60 (Nomura Chemical, 70 g), linear gradient
from 70:30 MeOH/H2O to MeOH, flow rate 5.0 mL/min]. The
fraction eluted between 94:6 MeOH/H2O to MeOH (151 mg)
was further separated by preparative HPLC [Develosil ODS
10 column (10 × 250 mm), 60:40 MeCN/H2O, flow rate 5.0 mL/
min] to afford pure dolastatin G (1) (35 mg) and a fraction
containing nordolastatin G (2) (19.9 mg).
a
Key: (a) (i-PrO)2P(O)CH2CO2Et, t-BuOK, THF, -78 f 0 °C;
(b) NiCl2, NaBH4, MeOH, rt; (c) LiAlH4, THF, -78 f 0 °C; (d)
(COCl)2, DMSO, CH2Cl2, -78 °C; Et3N, -78 f 0 °C; (e) (4R,5S)-
4-methyl-5-phenyl-3-propionyl-2-oxazolidinone, Bu2BOTf, Et3N,
CH2Cl2, -78 f 0 °C; (f) DEAD, Ph3P, p-NO2C6H4CO2H, benzene,
rt; (g) LiOH, H2O2, aqueous THF, rt; (h) LiAlH4, THF, rt; (i)
(MeO)2CMe2, CSA, acetone, rt; (j) H2, 20% Pd(OH)2/C, dioxane,
40 °C; (k) NaOMe, MeOH, rt.
The fraction containing 2 (19.9 mg) was chromatographed
on alumina (2 g) with 100:0, 98:2, and 95:5 EtOAc/MeOH to
afford crude 2 (2.5 mg), which was purified by repeated
preparative TLC with two solvent systems (2.5:1 CHCl3/
acetone and 2:1 benzene/acetone) to afford pure 2 (0.5 mg) as
a colorless amorphous powder. Using the same procedure as
described above, the sea hares (146 kg wet wt) collected in
1993 and 1994 were extracted and separated to yield an
additional sample of 2 (0.7 mg). Thus, a total amount of 2
obtained from 186 kg of wet animals was 1.2 mg.
acetonides, 5a and 5b (Scheme 3). The homologation of
aldehyde 86 by the Horner-Emmons reaction gave
conjugated ester 9, which was converted to alcohol 10
by sequential reduction. The Swern oxidation of 10 gave
aldehyde 11, which was converted to aldol 12 with 96%
de by the Evans aldol reaction.7 Inversion of the hydroxyl
group in 12 afforded p-nitrobenzoate 13, which was
converted to acetonide 14 by a sequence of reactions.
Debenzylation of 14 afforded hydroxy acetonide 5a . The
diastereomeric hydroxy acetonide 5b was obtained by
inversion at C53 of 5a followed by methanolysis of the
resulting p-nitrobenzoate 15. Of the two synthetic dia-
stereomers of hydroxy acetonide, 5a and 5b, the spectral
Dola sta tin G (1): colorless prisms; mp 138-139 °C (hex-
ane/benzene); tR ) 11.0 min [Develosil ODS-HG-5 column (4.6
× 250 mm), 85:15 MeOH/H2O, flow rate 1.0 mL/min, detection
at 215 nm]; Rf ) 0.32 (5:1 CHCl3/acetone); [R]25D -211 (c 0.40,
MeOH); UV (MeOH) λmax 205 (ꢀ 38 200), 250 nm (9600); IR
(CHCl3) 3450 (br), 1735, 1635, 1460, 1195 cm-1 1H and 13C
;
NMR data, see Table 1; HRMS (FAB) calcd for C57H97N6O13
(MH+) 1073.7113, found 1073.7070. Anal. Calcd for C57
-
data for 5a , [R]26 +20 (c 0.10, CHCl3), was identical to
D
H96N6O13: C, 63.78; H, 9.01; N, 7.83. Found: C, 63.61; H, 8.92;
N, 7.59.
that of natural 5, [R]26 +19 (c 0.07, CHCl3), in all
D
respects, establishing the 48R,49R,53S configurations in
1. On the basis of these findings the complete stereo-
structure of dolastatins G was determined as shown in
1. The absolute stereochemistry of nordolastatin G (2)
was shown to be identical to that of dolastatin G (1), since
1 was converted to 2 on acidic hydrolysis (HCl, aqueous
dioxane, rt, 98%).
Nor d ola sta tin G (2): colorless amorphous powder; tR
)
17.2 min [Develosil ODS-HG-5 column (4.6 × 250 mm), 80:20
MeOH/H2O, flow rate 1.0 mL/min, detection at 215 nm]; Rf )
0.23 (5:1 CHCl3/acetone); [R]25 -183 (c 0.11, MeOH); UV
D
(MeOH) λmax 205 (ꢀ 35 900), 225 (sh, 22 700), 291 nm (5000);
IR (CHCl3) 3450 (br), 1730, 1635, 1460, 1195, 1100 cm-1; H
1
NMR (600 MHz, C6D6) δ 0.75 (d, J ) 7.0 Hz, 3 H, H-25), 0.76
(d, J ) 6.2 Hz, 6 H, H-9 and H-33), 0.81 (t, J ) 7.3 Hz, 3 H,
H-32), 0.82 (d, J ) 7.3 Hz, 3 H, H-46), 0.84 (d, J ) 7.3 Hz, 6
H, H-55 and H-57), 0.95 (d, J ) 6.2 Hz, 3 H, H-26), 1.01 (m, 1
H, H-31), 1.01 (d, J ) 6.6 Hz, 3 H, H-10), 1.07 (d, J ) 6.4 Hz,
3 H, H-56), 1.08 (d, J ) 6.4 Hz, 3 H, H-43), 1.21 (m, 2 H, H-4
and H-31), 1.40 (m, 1 H, H-4), 1.44 (m, 1 H, H-51), 1.52 (m, 1
H, H-14), 1.56 (m, 1 H, H-3), 1.62 (m, 1 H, H-15), 1.72 (m, 1
H, H-50), 1.73 (m, 1 H, H-40), 1.74 (m, 2 H, H-41 and H-54),
1.77 (br s, 3 H, H-45), 1.88 (m, 1 H, H-14), 1.90 (m, 3 H, H-3,
H-50, and H-52), 2.04 (m, 1 H, H-51), 2.22 (m, 2 H, H-30 and
H-40), 2.25 (m, 1 H, H-52), 2.36 (m, 1 H, H-15), 2.40 (m, 1 H,
H-8), 2.51 (m, 1 H, H-24), 2.68 (s, 3 H, H-34), 2.70 (m, 1 H,
H-48), 2.83 (s, 3 H, H-21), 2.86 (s, 3 H, H-20), 3.06 (s, 3 H,
H-11), 3.08 (s, 3 H, H-27), 3.12 (m, 1 H, H-16), 3.41 (d, J )
14.3 Hz, 1 H, H-36), 3.45 (m, 1 H, H-5), 3.60 (d, J ) 14.3 Hz,
1 H, H-36), 3.60 (dd, J ) 11.4, 4.7 Hz, 1 H, H-19), 3.66 (m, 1
H, H-5), 3.77 (br t, J ) 8.8 Hz, 1 H, H-16), 3.84 (dd, J ) 11.4,
Exp er im en ta l Section
Gen er a l Meth od s. Melting points are uncorrected. NMR
spectra were measured at 270, 400, or 600 MHz for 1H and
100 MHz or 150 MHz for 13C. J values are given in Hz. Both
TLC analysis and preparative TLC were conducted on E.
Merck precoated silica gel 60 F254 (0.25 mm layer thickness).
Fuji Silysia silica gel BW-820 MH and E. Merck aluminum
oxide 90 (activity II-III) were used for column chromatography
unless otherwise noted. Organic solvents for anhydrous
reactions were distilled from the following drying agents: THF
(6) Ishiwata, H.; Sone, H.; Kigoshi, H.; Yamada, K. J . Org. Chem.
1994, 59, 4712-4713.
(7) Gage, J . R.; Evans D. A. Org. Synth. 1989, 68, 83-91.

Isolation of Dolastatin G and Nordolastatin G
J . Org. Chem., Vol. 61, No. 18, 1996 6343
11.4 Hz, 1 H, H-19), 4.19 (m, 1 H, H-49), 4.20 (m, 1 H, OH),
4.65 (br d, J ) 7.7 Hz, 1 H, H-2), 4.67 (br d, J ) 8.8 Hz, 1 H,
H-13), 4.92 (m, 1 H, H-42), 5.07 (m, 1 H, H-53), 5.16 (d, J )
11.0 Hz, 1 H, H-7), 5.30 (d, J ) 10.6 Hz, 1 H, H-23), 5.44 (d,
J ) 10.6 Hz, 1 H, H-29), 5.90 (dd, J ) 11.4, 4.7 Hz, 1 H, H-18),
6.95 (m, 1 H, H-39); 13C NMR (150 MHz, C6D6) δ 11.0 (q, C-32),
11.8 (q, C-45), 13.7 (q, C-46), 14.9 (q, C-57), 15.1 (q, C-56), 15.2
(q, C-43), 15.7 (q, C-33), 17.5 (q, C-25), 18.8 (q, C-10), 18.9 (q,
C-9), 19.8 (q, C-55), 20.2 (q, C-26), 21.9 (t, C-4), 23.5 (t, C-51),
24.1 (t, C-31), 24.2 (t, C-15), 27.2 (d, C-24), 27.7 (t, C-14), 27.8
(d, C-8), 29.6 (q, C-11), 29.8 (q, C-21), 30.0 (d, C-54), 30.2 (q,
C-27), 30.6 (q, C-34), 31.7 (t, C-3), 32.1 (t, C-52), 32.7 (d, C-30),
33.5 (t, C-40), 35.5 (t, C-50), 38.5 (d, C-41), 46.0 (t, C-5), 47.2
(t, C-16), 48.4 (d, C-48), 48.5 (t, C-36), 55.0 (d, C-18), 57.4 (d,
C-29), 58.2 (d, C-7), 58.3 (q, C-20), 58.5 (d, C-13), 58.8 (d, C-23),
60.3 (d, C-2), 68.7 (t, C-19), 73.9 (d, C-49), 74.0 (d, C-42), 78.5
(d, C-53), 138.3 (s, C-38), 144.7 (d, C-39), 166.8 (s, C-17), 168.0
(s, C-35), 169.1 (s, C-6), 169.6 (s, C-28), 170.5 (s, C-22), 170.9
(s, C-1), 171.1 (s, C-12), 175.6 (s, C-47), 193.8 (s, C-37); MS
(FAB) m/z 1059 (MH+); HRMS (FAB) calcd for C56H95N6O13
(MH+) 1059.6956, found 1059.6940.
H), 7.65-7.70 (m, 4 H); MS (FAB) m/z 357 (MH+); HRMS
(FAB) calcd for C22H33O2Si (MH+) 357.2250, found 357.2263.
(2S,3R,7S)-1-(tert-Butyldiphenylsiloxy)-2,8-dimethyl-3,7-
nonanediol (4): colorless oil; Rf ) 0.13 (4:1 hexane/EtOAc);
[R]26 +8 (c 0.14, CHCl3); IR (CHCl3) 3470 (br), 1425, 1110,
D
1055 cm-1; H NMR (270 MHz, CDCl3) δ 0.81 (d, J ) 7.3 Hz,
1
3 H), 0.91 (d, J ) 6.6 Hz, 3 H), 0.92 (d, J ) 6.9 Hz, 3 H), 1.05
(s, 9 H), 1.35-1.84 (m, 10 H), 3.40 (m, 1 H), 3.62 (dd, J ) 10.2,
7.6 Hz, 1 H), 3.65 (m, 1 H), 3.76 (dd, J ) 10.2, 4.0 Hz, 1 H),
7.36-7.48 (m, 6 H), 7.64-7.70 (m, 4 H); MS (FAB) m/z 443
(MH+); HRMS (FAB) calcd for C27H43O3Si (MH+) 443.2982,
found 443.2998. (4R,5S)-4-[(S)-4-Hydroxy-5-methylhexyl]-
2,2,5-trimethyl-1,3-dioxane (5): colorless oil; Rf ) 0.35 (3:1
hexane/acetone); [R]26 +19 (c 0.07, CHCl3); IR (CHCl3) 3610,
D
3460 (br), 1460, 1390, 1370, 1200, 1060 cm-1; H NMR (400
1
MHz, C6D6) δ 0.41 (d, J ) 6.3 Hz, 3 H), 0.84 (d, J ) 6.9 Hz, 3
H), 0.86 (d, J ) 6.8 Hz, 3 H), 1.32 (s, 3 H), 1.54 (s, 3 H), 1.24-
1.66 (m, 8 H), 1.70-1.82 (m, 1 H), 3.18 (br ddd, J ) 8.3, 4.3,
4.3 Hz, 1 H), 3.30 (dd, J ) 11.5, 11.5 Hz, 1 H), 3.32 (m, 1 H),
3.59 (dd, J ) 11.5, 4.9 Hz, 1 H); MS (FAB) m/z 245 (MH+);
HRMS (FAB) calcd for C14H29O3 (MH+) 245.2117, found
245.2104.
Degr a d a tion of Dola sta tin G (1). A stream of ozone gas
was passed through a solution of 1 (4.8 mg, 0.0045 mmol) in
MeOH (0.5 mL) at -78 °C for 15 min. The solution was then
flushed with nitrogen, and dimethyl sulfide (0.025 mL) was
added. The mixture was warmed to room temperature and
concentrated to give an oil (4.0 mg). To a stirred solution of
the oil (4.0 mg) in Et2O (0.75 mL) at 0 °C was added a 1.0 M
solution of lithium aluminum hydride in Et2O (0.045 mL, 0.045
mmol). The solution was stirred at room temperature for 1.5
h, and then a small amount of ice (ca. 1 g) and 0.2 M aqueous
HCl (1 mL) were added. After being stirred at room temper-
ature for 30 min, the mixture was extracted with EtOAc (8 ×
2 mL). The combined extracts were washed with H2O (1 mL)
and saturated aqueous NaCl (1 mL), dried (Na2SO4), and
concentrated. The residual oil was dissolved in DMF (0.3 mL),
and the solution was cooled to 0 °C. To the solution were
added imidazole (13.1 mg, 1.92 mmol) and tert-butyldiphenyl-
silyl chloride (0.01 mL, 0.385 mmol) with stirring. The
reaction mixture was stirred at 0 °C for 20 min, and a small
amount of ice (ca. 1 g) was added. The resulting mixture was
stirred at room temperature for 15 min and extracted with
EtOAc (5 × 3 mL). The combined extracts were washed with
saturated aqueous NaCl (1 mL), dried (Na2SO4), and concen-
trated. The residual oil was purified by column chromatog-
raphy on silica gel (1 g, step gradient from 8:1 to 2:1 hexane/
EtOAc) to give crude 3 (7.1 mg) and crude 4 (2.3 mg). Further
purification of crude 3 by column chromatography on silica
gel (0.5 g, 5:1 and then 3:1 hexane/Et2O) to give pure 3 (1.3
mg, 82%) as a colorless oil and further purification of crude 4
by column chromatography on silica gel (0.5 g, 3:1 and then
2:1 hexane/EtOAc) to give pure 4 (1.7 mg, 86%) as a colorless
oil.
(2R,3R)-5-(ter t-Bu t yld ip h en ylsiloxy)-3-m et h yl-2-p en -
ta n ol (3a ). To a stirred solution of (3R,4R)-3,4-dimethyl-4-
butanolide (6)4 (22.6 mg, 0.198 mmol) in THF (1.0 mL) at 0
°C was added a 1.0 M solution of lithium aluminum hydride
in THF (0.4 mL, 0.4 mmol) dropwise. The solution was stirred
at room temperature for 1.5 h, and then NaF (0.15 g) and a
mixture of 9:1 THF/H2O (2 mL) were added. After being
stirred at room temperature for 30 min, the mixture was
filtered through a pad of Celite. The residue was washed with
THF (20 mL), and the filtrate and the washings were combined
and concentrated. The residual oil was dissolved in DMF (1.0
mL), and the solution was cooled to 0 °C. To the stirred
solution were added imidazole (42 mg, 0.617 mmol) and tert-
butyldiphenylsilyl chloride (0.06 mL, 0.231 mmol). The reac-
tion mixture was stirred at 0 °C for 30 min, and a small
amount of ice (ca. 3 g) was added. The resulting mixture was
stirred at room temperature for 15 min and extracted with
EtOAc (8 × 3 mL). The combined extracts were washed with
saturated aqueous NaCl (2 mL), dried (Na2SO4), and concen-
trated. The residual oil was purified by column chromatog-
raphy on silica gel (5 g, step gradient from 5:1 to 3:1 hexane/
Et2O) to give silyl ether 3a (63.1 mg, 89%) as a colorless oil:
Rf ) 0.47 (4:1 hexane/EtOAc); [R]30 +8 (c 0.12, CHCl3); IR
D
(CHCl3) 3400 (br), 1470, 1430, 1110, 995 cm-1; H NMR (400
1
MHz, CDCl3) δ 0.86 (d, J ) 6.8 Hz, 3 H), 1.05 (s, 9 H), 1.15 (d,
J ) 6.3 Hz, 3 H), 1.42 (m, 1 H), 1.67-1.78 (m, 2 H), 2.20 (d, J
) 4.9 Hz, 1 H), 3.68 (ddd, J ) 10.2, 7.3, 4.9 Hz, 1 H), 3.76
(ddd, J ) 10.2, 5.4, 5.4 Hz, 1 H), 3.77 (m, 1 H), 7.36-7.46 (m,
6 H), 7.64-7.70 (m, 4 H); 13C NMR (100 MHz, CDCl3) δ 14.3
(q), 19.1 (s), 19.8 (q), 26.8 (q, 3 C), 35.5 (t), 37.1 (d), 62.4 (t),
70.7 (d), 127.6 (d, 4 C), 129.6 (d, 2 C), 133.5 (s, 2 C), 135.5 (d,
4 C); MS (FAB) m/z 357 (MH+); HRMS (FAB) calcd for
C22H33O2Si (MH+) 357.2250, found 357.2246.
To a stirred solution of 4 (1.6 mg, 0.0036 mmol) in THF (0.3
mL) at 0 °C was added a 1.0 M solution of tetrabutylammo-
nium fluoride in THF (0.02 mL, 0.02 mmol). The solution was
stirred at room temperature for 15 min and diluted with
saturated aqueous NH4Cl (1 mL). The mixture was extracted
with EtOAc (5 × 2 mL). The combined extracts were washed
with saturated aqueous NaCl (1 mL), dried (Na2SO4), and
concentrated. The residual oil was dissolved in acetone (0.4
mL), and 2,2-dimethoxypropane (0.1 mL, 0.81 mmol) and 10-
camphorsulfonic acid (2.1 mg, 0.0086 mmol) were added. The
mixture was stirred at room temperature for 30 min, diluted
with saturated aqueous NaHCO3 (1 mL), and extracted with
EtOAc (4 × 2 mL). The combined extracts were washed with
saturated aqueous NaCl (1 mL), dried (Na2SO4), and concen-
trated. The residual oil was purified by column chromatog-
raphy on silica gel (1 g, step gradient from 5:1 to 1:1 hexane/
EtOAc) to give 5 (0.8 mg, 90%) as a colorless oil. (2R,3S)-5-
(tert-Butyldiphenylsiloxy)-3-methyl-2-pentanol (3): colorless oil;
(2S,3R)-5-(ter t-Bu t yld ip h en ylsiloxy)-3-m et h yl-2-p en -
tyl 4-Nitr oben zoa te (7). To a stirred solution of silyl ether
3a (28.5 mg, 0.08 mmol), triphenylphosphine (148 mg, 0.564
mmol), and p-nitrobenzoic acid (93 mg, 0.556 mmol) in benzene
(2.0 mL) was added a 1.0 M solution of diethyl azodicarboxy-
late in benzene (0.56 mL, 0.56 mmol) at room temperature.
After being stirred at room temperature for 2.5 h, the reaction
mixture was concentrated. The residual oil was purified twice
by column chromatography [(1) silica gel 5 g, step gradient
from 7:1 to 1:1 hexane/benzene; (2) silica gel 5 g, step gradient
from 20:1 to 10:1 hexane/Et2O] to give p-nitrobenzoate 7 (34.5
mg, 76%) as a colorless oil: Rf ) 0.49 (5:1 hexane/Et2O); [R]25
D
+26.0 (c 1.11, CHCl3); IR (CHCl3) 1720, 1610, 1530, 1350, 1280,
1
1105 cm-1; H NMR (270 MHz, CDCl3) δ 0.95 (d, J ) 6.8 Hz,
3 H), 1.05 (s, 9 H), 1.30 (d, J ) 6.3 Hz, 3 H), 1.38 (m, 1 H),
1.83 (m, 1 H), 2.04 (m, 1 H), 3.66-3.83 (m, 2 H), 5.10 (dq, J )
6.3, 6.3 Hz, 1 H), 7.32-7.47 (m, 6 H), 7.62-7.69 (m, 4 H), 8.16
(m, 2H), 8.26 (m, 2 H); MS (FAB) m/z 528 (MNa+); HRMS
(FAB) calcd for C29H35NO5SiNa (MNa+) 528.2182, found
528.2174.
Rf ) 0.47 (4:1 hexane/EtOAc); [R]30 -6 (c 0.11, CHCl3); IR
D
(CHCl3) 3400 (br), 1475, 1425, 1115, 1085, 900 cm-1; 1H NMR
(400 MHz, CDCl3) δ 0.86 (d, J ) 6.8 Hz, 3 H), 1.05 (s, 9 H),
1.15 (d, J ) 6.3 Hz, 3 H), 1.53 (m, 1 H), 1.63-1.73 (m, 2 H),
2.37 (br s, 1 H), 3.63 (m, 1 H), 3.67 (ddd, J ) 10.3, 7.3, 4.9 Hz,
1 H), 3.76 (ddd, J ) 10.3, 4.9, 4.9 Hz, 1 H), 7.38-7.45 (m, 6
Syn th esis of Silyl Eth er 3b () en t-3). To a stirred
solution of p-nitrobenzoate 7 (33.2 mg, 0.065 mmol) in MeOH

6344 J . Org. Chem., Vol. 61, No. 18, 1996
Mutou et al.
(0.5 mL) at 0 °C was added a 0.5 M solution of NaOMe in
MeOH (0.15 mL, 0.75 mmol). The mixture was stirred at 0
°C for 30 min and at room temperature for 4.5 h. After
addition of ion-exchange resin (Amberlite IRC-50, H+ form,
120 mg), the mixture was stirred at room temperature for 20
min and then poured on a pad of the same resin (160 mg).
The resin was washed with MeOH (15 mL), and the filtrate
and the washings were combined and concentrated. The
residual oil was purified by column chromatography on silica
gel (2 g, step gradient from 4:1 to 2:1 hexane/Et2O) to give 3b
(21.6 mg, 92%) as a colorless oil: Rf ) 0.47 (4:1 hexane/EtOAc);
7.24-7.36 (m, 5 H); 13C NMR (100 MHz, CDCl3) δ 17.7 (q),
18.2 (q), 21.7 (t), 29.7 (t), 30.3 (d), 32.6 (t), 62.2 (t), 71.6 (t),
84.1 (d), 127.2 (d), 127.6 (d, 2 C), 128.1 (d, 2 C), 138.8 (s); MS
(EI) m/z (relative intensity) 236 (M+, 6), 193 (6), 128 (3), 91
(100); HRMS (EI) calcd for C15H24O2 (M+) 236.1776, found
236.1778.
(S)-5-(Ben zyloxy)-6-m eth ylh ep ta n a l (11). To a stirred
solution of oxalyl chloride (0.08 mL, 0.92 mmol) in CH2Cl2 (2.0
mL) at -78 °C was added a 2.1 M solution of DMSO in CH2-
Cl2 (0.84 mL, 1.76 mmol) dropwise. The resulting solution was
stirred at -78 °C for 7 min, and a solution of alcohol 10 (105
mg, 0.445 mmol) in CH2Cl2 (1.5 mL, 2 × 0.75 mL rinse) was
added dropwise. The mixture was stirred at -78 °C for 10
min, and triethylamine (0.64 mL, 4.59 mmol) was added. The
resulting mixture was stirred at -78 °C for 10 min and at 0
°C for 15 min. H2O (8 mL) was added, and the mixture was
extracted with 4:1 benzene/Et2O (3 × 15 mL). The combined
extracts were washed with saturated aqueous NaCl (8 mL),
dried (Na2SO4), and concentrated. The residual oil was
purified by column chromatography on silica gel (3 g, 8:1 and
then 6:1 hexane/Et2O) to give aldehyde 11 (97 mg, 93%) as a
colorless oil: Rf ) 0.49 (5:1 hexane/EtOAc); [R]26D -20.4 (c 1.02,
CHCl3); IR (CHCl3) 2730, 1720, 1600, 1500, 1455, 1390, 1365
[R]25 +6 (c 0.12, CHCl3); 13C NMR (100 MHz, CDCl3) δ 15.8
D
(q), 19.1 (s), 20.1 (q), 26.8 (q, 3 C), 35.5 (t), 37.9 (d), 62.2 (t),
71.7 (d), 127.7 (d, 4 C), 129.7 (d, 2 C), 133.5 (s, 2 C), 135.6 (d,
4 C); MS (FAB) m/z 357 (MH+); HRMS (FAB) calcd for
1
C22H33O2Si (MH+) 357.2250, found 357.2273. IR and H NMR
(400 MHz, CDCl3) spectra were identical with those of the
C39-C43 fragment 3.
Eth yl (S)-5-(Ben zyloxy)-6-m eth yl-2-h ep ten oa te (9). To
a stirred solution of diisopropyl [(ethoxycarbonyl)methyl]-
phosphonate (1.8 mL, 7.6 mmol) in THF (3 mL, 2 × 1 mL rinse)
at 0 °C was added potassium tert-butoxide (670 mg, 6.0 mmol),
and the resulting solution was stirred at 0 °C for 1 h. The
solution was cooled to -78 °C, and a solution of (S)-3-
(benzyloxy)-4-methylpentanal (8)6 (360 mg, 1.75 mmol) in THF
(5 mL) was added. After being stirred at -78 °C for 1 h and
at 0 °C for 1 h, the reaction mixture was diluted with Et2O
(20 mL), saturated aqueous NH4Cl (20 mL), and H2O (20 mL),
successively. The organic layer was separated, and the
aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The
organic layer and the extracts were combined, washed with
saturated aqueous NaCl (25 mL), dried (Na2SO4), and concen-
trated. The residual oil was purified by column chromatog-
raphy on silica gel (30 g, 12:1 hexane/Et2O) to give conjugated
ester 9 (355 mg, 94%) as a colorless oil: Rf ) 0.58 (5:1 hexane/
1
cm-1; H NMR (400 MHz, CDCl3) δ 0.91 (d, J ) 6.6 Hz, 3 H),
0.93 (d, J ) 5.9 Hz, 3 H), 1.44-1.87 (m, 4 H), 1.95 (dqq, J )
5.3, 6.6, 5.9 Hz, 1 H), 2.41 (m, 2 H), 3.16 (ddd, J ) 5.9, 5.9, 5.3
Hz, 1 H), 4.47 (d, J ) 11.5 Hz, 1 H), 4.55 (d, J ) 11.5 Hz, 1 H),
7.24-7.36 (m, 5 H), 9.74 (t, J ) 1.7 Hz, 1 H); 13C NMR (100
MHz, CDCl3) δ 17.6 (q), 18.2 (t), 18.4 (q), 29.4 (t), 30.2 (d), 43.9
(t), 71.6 (t), 83.7 (d), 127.4 (d), 127.7 (d, 2 C), 128.2 (d, 2 C),
138.9 (s), 202.5 (d); MS (EI) m/z (relative intensity) 234 (M+,
1), 191 (9), 128 (8), 91 (100); HRMS (EI) calcd for C15H22O2
(M+) 234.1620, found 234.1593.
(4R,5S)-3-[(2R,3S,7S)-7-(Ben zyloxy)-2,8-d im eth yl-3-h y-
d r oxyn on a n oyl]-4-m eth yl-5-p h en yl-2-oxa zolid in on e (12).
To a stirred solution of (4R,5S)-4-methyl-5-phenyl-3-propionyl-
2-oxazolidinone (195 mg, 0.836 mmol) in CH2Cl2 (1.7 mL) at 0
°C were added a 1.0 M solution of dibutylboron triflate in CH2-
Cl2 (0.92 mL, 0.92 mmol) and triethylamine (0.16 mL, 1.15
mmol), successively. The reaction mixture was stirred at 0
°C for 30 min and cooled to -78 °C. A solution of aldehyde 11
(96 mg, 0.41 mmol) in CH2Cl2 (1.0 mL, 2 × 0.75 mL rinse)
was added, and the reaction mixture was stirred at -78 °C
for 1.5 h and at 0 °C for 15 min. The mixture was diluted
with 0.5 M phosphate buffer (pH 7) (1 mL). MeOH (9 mL)
and 30% aqueous H2O2 (1 mL) were added, and the mixture
was stirred at 0 °C for 1 h. The organic layer was separated,
and the aqueous layer was extracted with CH2Cl2 (2 × 20 mL).
The organic layer and the extracts were combined, washed
with 5% aqueous NaHCO3 (15 mL) and saturated aqueous
NaCl (15 mL), dried (Na2SO4), and concentrated. The residual
oil was purified by column chromatography on silica gel (10
g, 3:1 and then 2.5:1 hexane/Et2O) to give aldol 12 (189 mg,
99%) as a colorless oil. The diastereomeric ratio of the product
was shown to be 98:2 by 13C NMR analysis. 12: Rf ) 0.16
EtOAc); [R]26 -0.5 (c 0.49, CHCl3); IR (CHCl3) 1710, 1660,
D
1470, 1460, 1370 cm-1; H NMR (270 MHz, CDCl3) δ 0.93 (d,
1
J ) 6.9 Hz, 3 H), 0.95 (d, J ) 6.9 Hz, 3 H), 1.29 (t, J ) 6.9 Hz,
3 H), 1.88 (dqq, J ) 5.9, 6.9, 6.9 Hz, 1 H), 2.42 (ddd, J ) 7.3,
5.6, 1.6 Hz, 2 H), 3.27 (ddd, J ) 5.9, 5.6, 5.6 Hz, 1 H), 4.18 (q,
J ) 6.9 Hz, 2 H), 4.49 (d, J ) 11.5 Hz, 1 H), 4.55 (d, J ) 11.5
Hz, 1 H), 5.88 (dt, J ) 15.5, 1.6 Hz, 1 H), 7.01 (dt, J ) 15.5,
7.3 Hz, 1 H), 7.25-7.35 (m, 5 H); 13C NMR (100 MHz, CDCl3)
δ 14.2 (q), 18.0 (q), 18.1 (q), 31.0 (d), 33.5 (t), 60.0 (t), 71.8 (t),
82.9 (d), 123.0 (d), 127.4 (d), 127.6 (d), 128.2 (d), 138.4 (s), 146.0
(d), 166.2 (s); MS (EI) m/z (relative intensity) 276 (M+, 2), 163
(15), 91 (100); HRMS (EI) calcd for C17H24O3 (M+) 276.1725,
found 276.1717.
(S)-5-(Ben zyloxy)-6-m eth ylh ep ta n ol (10). To a stirred
solution of conjugated ester 9 (440 mg, 1.59 mmol) in MeOH
(16 mL) at 0 °C were added nickel(II) chloride (34.7 mg, 0.27
mmol) and sodium borohydride (184 mg, 4.86 mmol). After
being stirred at room temperature for 2 h, the mixture was
diluted with CH2Cl2 (15 mL), 6 M aqueous HCl (1 mL), and
H2O (6 mL), successively. The organic layer was separated,
and the aqueous layer was extracted with CH2Cl2 (2 × 20 mL).
The organic layer and the extracts were combined, washed
with saturated aqueous NaHCO3 (15 mL), H2O (15 mL), and
saturated aqueous NaCl (15 mL), dried (Na2SO4), and concen-
trated. The residual oil was dissolved in THF (5.4 mL). To
the solution cooled at -78 °C was added a 1.0 M solution of
lithium aluminum hydride in THF (3.2 mL, 3.2 mmol). The
mixture was stirred at -78 °C for 3.5 h and at room temper-
ature for 30 min, and then NaF (0.65 g) and a 9:1 mixture of
THF and H2O (20 mL) were added. After being stirred at room
temperature for 1 h, the mixture was filtered through a pad
of Celite. The residue was washed with THF (40 mL), and
the filtrate and the washings were combined and concentrated.
The residual oil was purified twice by column chromatography
[(1) silica gel 14 g, 2:1 hexane/Et2O; (2) silica gel 14 g, 2.5:1
and then 2:1 hexane/Et2O] to give alcohol 10 (362 mg, 97%)
(5:1 hexane/EtOAc); [R]25 +2.5 (c 1.06, CHCl3); IR (CHCl3)
D
3540 (br), 1780, 1690, 1605, 1500, 1460, 1380, 1365, 1345,
1
1240, 1200 cm-1; H NMR (400 MHz, CDCl3) δ 0.89 (d, J )
6.8 Hz, 3 H), 0.92 (d, J ) 6.8 Hz, 3 H), 0.93 (d, J ) 6.8 Hz, 3
H), 1.23 (d, J ) 6.8 Hz, 3 H), 1.38-1.60 (m, 6 H), 1.94 (dqq, J
) 5.3, 6.8, 6.8 Hz, 1 H), 2.86 (br s, 1 H), 3.18 (m, 1 H), 3.76
(dq, J ) 2.9, 6.8 Hz, 1 H), 3.96 (m, 1 H), 4.50 (d, J ) 11.7 Hz,
1 H), 4.54 (d, J ) 11.7 Hz, 1 H), 4.79 (dq, J ) 7.3, 6.8 Hz, 1
H), 5.67 (d, J ) 7.3 Hz, 1 H), 7.24-7.44 (m, 10 H); 13C NMR
(100 MHz, CDCl3) δ 10.3 (q), 14.2 (q), 17.8 (q), 18.3 (q), 22.3
(t), 30.0 (t), 30.4 (d), 34.1 (t), 42.2 (d), 54.6 (d), 71.4 (d), 71.7
(t), 78.7 (d), 84.1 (d), 125.5 (d, 2 C), 127.2 (d), 127.6 (d, 4 C),
128.1 (d, 2 C), 128.6 (d), 133.0 (s), 139.0 (s), 152.5 (s), 177.1
(s); MS (FAB) m/z 490 (MNa+), 468 (MH+); HRMS (FAB) calcd
for C28H38NO5 (MH+) 468.2750, found 468.2781.
(4R,5S)-3-[(2R,3S,7S)-7-(Ben zyloxy)-2,8-d im eth yl-3-[(4-
n itr oben zoyl)oxy]n on a n oyl]-4-m eth yl-5-p h en yl-2-oxa zo-
lid in on e (13). To a stirred solution of aldol 12 (188 mg, 0.402
mmol), triphenylphosphine (737 mg, 2.8 mmol), and p-ni-
trobenzoic acid (468 mg, 2.8 mmol) in benzene (8.0 mL) was
added diethyl azodicarboxylate (0.44 mL, 2.8 mmol) at room
temperature. After being stirred at room temperature for 33
as a colorless oil: Rf ) 0.14 (5:1 hexane/EtOAc); [R]26 -16.9
D
(c 0.91, CHCl3); IR (CHCl3) 3620, 3450 (br), 1600, 1500, 1460,
1
1390, 1370, 1350 cm-1; H NMR (400 MHz, CDCl3) δ 0.91 (d,
J ) 7.2 Hz, 3 H), 0.93 (d, J ) 6.8 Hz, 3 H), 1.34-1.60 (m, 7
H), 1.93 (dqq, J ) 4.8, 7.2, 6.8 Hz, 1 H), 3.16 (m, 1 H), 3.62
(m, 2 H), 4.48 (d, J ) 11.6 Hz, 1 H), 4.54 (d, J ) 11.6 Hz, 1 H),

Isolation of Dolastatin G and Nordolastatin G
J . Org. Chem., Vol. 61, No. 18, 1996 6345
h, the reaction mixture was concentrated. The residual oil was
purified twice by column chromatography [(1) silica gel 30 g,
step gradient from 15:1 to 8:1 hexane/acetone; (2) silica gel
FL60D (Fuji Silysia) 30 g, step gradient from 20:1 to 13:1
hexane/acetone] to give p-nitrobenzoate 13 (190 mg, 77%) as
(3.0 mg, 0.012 mmol), triphenylphosphine (24.3 mg, 0.092
mmol), and p-nitrobenzoic acid (15.1 mg, 0.090 mmol) in
benzene (0.4 mL) was added a 0.64 M solution of diethyl
azodicarboxylate in benzene (0.14 mL, 0.089 mmol) at room
temperature. After being stirred at room temperature for 2.5
h, the reaction mixture was concentrated. The residual oil was
purified by column chromatography on silica gel (2 g, 15:1 and
then 5:1 hexane/Et2O) and preparative TLC (30:1 benzene/
acetone) to give p-nitrobenzoate 15 (2.8 mg, 58%) as a colorless
oil: Rf ) 0.28 (10:1 hexane/acetone); [R]25D +35 (c 0.15, CHCl3);
IR (CHCl3) 1720, 1610, 1530, 1460, 1365, 1280, 1120, 1100
a colorless oil: Rf ) 0.30 (4:1 hexane/acetone); [R]25 -24.7 (c
D
0.16, CHCl3); IR (CHCl3) 1780, 1725, 1700, 1605, 1530, 1460,
1
1385, 1345, 1275 cm-1; H NMR (270 MHz, CDCl3) δ 0.85 (d,
J ) 6.6 Hz, 3 H), 0.88 (d, J ) 6.6 Hz, 3 H), 0.91 (d, J ) 6.3 Hz,
3 H), 1.28 (d, J ) 6.9 Hz, 3 H), 1.36-1.98 (m, 7 H), 3.13 (m, 1
H), 4.28 (dq, J ) 8.3, 6.9 Hz, 1 H), 4.38 (d, J ) 11.5 Hz, 1 H),
4.49 (d, J ) 11.5 Hz, 1 H), 4.65 (dq, J ) 7.3, 6.6 Hz, 1 H), 5.42
(d, J ) 7.3 Hz, 1 H), 5.53 (ddd, J ) 8.3, 8.3, 3.3 Hz, 1 H), 7.20-
7.44 (m, 10 H), 8.19 (br d, J ) 6.9 Hz, 2 H), 8.26 (br d, J ) 6.9
Hz, 2 H); 13C NMR (100 MHz, CDCl3) δ 14.0 (q), 14.3 (q), 17.7
(q), 18.4 (q), 21.1 (t), 29.9 (t), 30.4 (d), 31.7 (t), 41.4 (d), 54.9
(d), 71.7 (t), 76.2 (d), 78.8 (d), 83.9 (d), 123.5 (d, 2 C), 125.5 (d,
2 C), 127.3 (d), 127.5 (d, 2 C), 128.2 (d, 2 C), 128.7 (d, 2 C),
128.8 (d), 130.7 (d, 2 C), 132.9 (s), 135.5 (s), 138.9 (s), 150.5
(s), 152.6 (s), 163.7 (s), 173.8 (s); MS (FAB) m/z 617 (MH+);
HRMS (FAB) calcd for C35H41N2O8 (MH+) 617.2863, found
617.2887.
1
cm-1; H NMR (270 MHz, CDCl3) δ 0.70 (d, J ) 6.6 Hz, 3 H),
0.97 (d, J ) 6.6 Hz, 3 H), 0.98 (d, J ) 6.6 Hz, 3 H), 1.33 (s, 3
H), 1.39 (s, 3 H), 1.30-1.80 (m, 7 H), 2.00 (ddd, J ) 6.6, 6.6,
5.0 Hz, 1 H), 3.41 (m, 1 H), 3.46 (dd, J ) 11.5, 11.2 Hz, 1 H),
3.65 (dd, J ) 11.5, 5.3 Hz, 1 H), 5.04 (ddd, J ) 7.6, 5.0, 5.0
Hz, 1 H), 8.21 (br d, J ) 8.9 Hz, 2 H), 8.29 (br d, J ) 8.9 Hz,
2 H); MS (FAB) m/z 394 (MH+); HRMS (FAB) calcd for C21H32
-
NO6 (MH+) 394.2230, found 394.2224.
Syn th esis of Hyd r oxy Aceton id e 5b. To a stirred solu-
tion of p-nitrobenzoate 15 (2.5 mg, 0.006 mmol) in MeOH (0.8
mL) at 0 °C was added a 1.0 M solution of NaOMe in MeOH
(0.2 mL, 0.2 mmol). The mixture was stirred at room tem-
perature for 4.5 h. After addition of ion-exchange resin
(Amberlite IRC-50, H+ form, 500 mg), the mixture was stirred
at room temperature for 1 min and then poured on a pad of
the same resin (1 g). The resin was washed with MeOH (15
mL), and the filtrate and the washings were combined and
concentrated. The residual oil was purified twice by column
chromatography [(1) silica gel 1 g, 8:1 and then 1.5:1 hexane/
Et2O; (2) silica gel 1 g, 1.5:1 hexane/Et2O] to give 5b (1.4 mg,
(4R,5S)-4-[(S)-4-(Ben zyloxy)-5-m et h ylh exyl]-2,2,5-t r i-
m eth yl-1,3-d ioxa n e (14). To a stirred solution of p-nitroben-
zoate 13 (9.6 mg, 0.015 mmol) in 4:1 THF/H2O (0.5 mL) at 0
°C were added 30% aqueous H2O2 (0.03 mL, 0.26 mmol) and
2.5 M aqueous LiOH (0.035 mL, 0.09 mmol). After the reaction
mixture was stirred at 0 °C for 2.5 h and at room temperature
for 15 h, 1.5 M aqueous Na2SO3 (0.2 mL) was added. The
resulting mixture was acidified (ca. pH 1) with 6 M aqueous
HCl and extracted with CH2Cl2 (5 × 6 mL). The combined
extracts were dried (Na2SO4) and concentrated to give a crude
carboxylic acid (8.8 mg). To a stirred solution of the crude
carboxylic acid (8.8 mg) in THF (0.4 mL) at 0 °C was added a
1.0 M solution of lithium aluminum hydride in THF (0.12 mL,
0.12 mmol) dropwise. The solution was stirred at room
temperature for 1.3 h, and then NaF (25 mg) and a mixture
of 9:1 THF/H2O (1.5 mL) were added. After being stirred at
room temperature for 30 min, the mixture was filtered through
a pad of Celite. The residue was washed with THF (10 mL),
and the filtrate and the washings were combined and concen-
trated. The residual oil was dissolved in acetone (1.0 mL),
and then 2,2-dimethoxypropane (0.5 mL, 4.0 mmol) and 10-
camphorsulfonic acid (2.2 mg, 0.01 mmol) were added. The
mixture was stirred at room temperature for 3 h, diluted with
saturated aqueous NaHCO3 (1.5 mL), and extracted with Et2O
(3 × 5 mL). The combined extracts were washed with
saturated aqueous NaCl (2 mL), dried (Na2SO4), and concen-
trated. The residual oil was purified by column chromatog-
raphy on silica gel (1 g, 10:1 hexane/Et2O) and preparative
TLC (25:1 hexane/acetone) to give acetonide 14 (3.0 mg, 60%)
90%) as a colorless oil: Rf ) 0.35 (3:1 hexane/acetone); [R]25
D
+53 (c 0.10, CHCl3); IR (CHCl3) 3610, 3460 (br), 1460, 1390,
1370, 1200, 1060 cm-1; 1H NMR (400 MHz, C6D6) δ 0.41 (d, J
) 6.8 Hz, 3 H), 0.84 (d, J ) 6.8 Hz, 3 H), 0.87 (d, J ) 6.8 Hz,
3 H), 1.00 (br s, 1 H), 1.32 (s, 3 H), 1.55 (s, 3 H), 1.26-1.66 (m,
8 H), 3.17 (br ddd, J ) 7.3, 4.9, 4.9 Hz, 1 H), 3.29 (m, 1 H),
3.29 (dd, J ) 11.7, 11.2 Hz, 1 H), 3.59 (dd, J ) 11.7, 5.3 Hz, 1
H); MS (FAB) m/z 245 (MH+); HRMS (FAB) calcd for C14H29O3
(MH+) 245.2117, found 245.2105.
Con ver sion of Dola sta tin G (1) in to Nor d ola sta tin G
(2). To a stirred solution of 1 (9.1 mg, 0.008 mmol) in dioxane
(0.6 mL) at room temperature was added 2 M aqueous HCl
(0.2 mL). The mixture was stirred at room temperature for
50 min and then poured into saturated aqueous NaHCO3 (2
mL) at 0 °C. The resulting mixture was extracted with CH2-
Cl2 (3 × 3 mL). The combined extracts were washed with
saturated aqueous NaCl (2 mL), dried (Na2SO4), and concen-
trated. The residual oil was purified by column chromatog-
raphy on silica gel (1 g, 4:1 and then 3:1 benzene/acetone) to
give 2 (8.7 mg, 98%) as a colorless amorphous powder: Rf )
as a colorless oil: Rf ) 0.49 (4:1 hexane/acetone); [R]25 +13
D
(c 0.16, CHCl3); IR (CHCl3) 1605, 1500, 1460, 1385, 1370, 1270,
1
910, 700 cm-1; H NMR (270 MHz, CDCl3) δ 0.72 (d, J ) 6.6
0.23 (5:1 CHCl3/acetone); [R]27 -176 (c 0.09, CHCl3); HRMS
Hz, 3 H), 0.92 (d, J ) 6.9 Hz, 3 H), 0.93 (d, J ) 6.6 Hz, 3 H),
1.38 (s, 3 H), 1.42 (s, 3 H), 1.28-1.71 (m, 7 H), 1.91 (dqq, J )
5.3, 6.9, 6.6 Hz, 1 H), 3.15 (ddd, J ) 5.9, 5.3, 5.3 Hz, 1 H),
3.43 (m, 1 H), 3.48 (dd, J ) 11.6, 11.2 Hz, 1 H), 3.68 (dd, J )
11.6, 5.3 Hz, 1 H), 4.51 (s, 2 H), 7.22-7.38 (m, 5 H); MS (EI)
m/z (relative intensity) 334 (M+, 3), 319 (8), 291 (7), 276 (11),
91 (100); HRMS (EI) calcd for C21H34O3 (M+) 334.2508, found
334.2518.
D
(FAB) calcd for C56H95N6O13 (MH+) 1059.6956, found 1059.6960.
The IR and 1H NMR (600 MHz, C6D6) spectra were identical
with those of natural 2.
Ack n ow led gm en t. This work was supported in part
by Grants-in-Aid for Scientific Research (Grants.
06680557 and 07680626) and for Scientific Research on
Priority Areas (Natural Supramolecules No. 06240103)
from the Ministry of Education, Science, and Culture,
J apan.
Syn th esis of Hyd r oxy Aceton id e 5a () 5). A mixture
of acetonide 14 (5.5 mg, 0.0165 mmol) and 20% Pd(OH)2 on
carbon (2.0 mg) in dioxane (0.8 mL) was stirred under an
atmosphere of hydrogen at 40 °C for 1.8 h. The mixture was
filtered through a pad of Celite, and the residue was washed
with EtOAc (10 mL). The filtrate and the washings were
combined and concentrated. The residual oil was purified by
column chromatography on silica gel (1 g, 2.5:1 hexane/Et2O)
to give 5a (3.8 mg, 94%) as a colorless oil: Rf ) 0.35 (3:1
hexane/acetone); [R]26D +20 (c 0.10, CHCl3); MS (FAB) m/z 245
(MH+); HRMS (FAB) calcd for C14H29O3 (MH+) 245.2117, found
245.2098. IR and 1H NMR (400 MHz, C6D6) spectra were
identical with those of 5.
Su p p or tin g In for m a tion Ava ila ble: 1D and 2D NMR
spectra and HPLC traces of 1 and 2; table of 1H and 13C NMR
1
assignments for 2; H NMR spectra of 3, 3a ,b, 4, 5, 5a ,b, 7,
and 9-15 (29 pages). This material is contained in libraries
on microfiche, immediately follows this article in the microfilm
version of the journal, and can be ordered from the ACS; see
any current masthead page for ordering information.
(4R,5S)-4-[(R)-4-[(4-Nitr oben zoyl)oxy]-5-m eth ylh exyl]-
2,2,5-tr im eth yl-1,3-d ioxa n e (15). To a stirred solution of 5a
J O9608228