Dolastatin H and isodalastatin H, potent cytotoxic peptides from the sea hare Dolabella auricularia: Isolation, stereostructures, and synthesis

Full text of DOI:10.1021/ja9519086

1874
J. Am. Chem. Soc. 1996, 118, 1874-1880
Dolastatin H and Isodolastatin H, Potent Cytotoxic Peptides
from the Sea Hare Dolabella auricularia: Isolation,
Stereostructures, and Synthesis
Hiroki Sone, Takunobu Shibata, Tatsuya Fujita, Makoto Ojika, and
Kiyoyuki Yamada*
Contribution from the Department of Chemistry, Faculty of Science, Nagoya UniVersity,
Chikusa, Nagoya 464, Japan
ReceiVed June 12, 1995X
Abstract: A bioassay-directed investigation of the cytotoxic constituents of the Japanese sea hare Dolabella auricularia
resulted in the isolation of two cytotoxic compounds designated dolastatin H (2) and isodolastatin H (3) from the
wet animals at the yields of 9 × 10-7%. On the basis of spectroscopic analysis, these compounds were shown to
be new peptides that were closely related to dolastatin 10 (1) isolated from Western Indian Ocean specimens of this
animal. The notable structural feature of these new compounds, 2 and 3, is that 3-phenylpropane-1,2-diol is attached
through the ester linkage to the C-terminus of a tetrapeptide containing unusual amino acids. The absolute
stereostructures of 2 and 3 were unambiguously determined by enantioselective total synthesis. A cytotoxicity test
for synthetic 2, 3, and their C-2 epimers revealed that the stereochemistry of the 3-phenylpropane-1,2-diol moiety on
the C-terminus plays an important role in their cytotoxicity. In ViVo antitumor activity against murine P388 leukemia
was evaluated, and it was shown that isodolastatin H (3) exhibited antitumor activity a little weaker than that of
dolastatin 10 (1).
The Western Indian Ocean sea hare Dolabella auricularia is
report here the isolation of these new compounds and their
absolute stereostructures, which were determined using the
enantioselective synthesis of 2 and 3 and their stereoisomers.
Results on the biological evaluation of these synthetic com-
pounds are also described.
known to produce remarkably potent antitumor agents with
novel structures that have been referred to as dolastatins.1
Among these, dolastatin 10 (1) is a unique linear peptide
containing unusual amino acids and has been shown to possess
unprecedented potent antitumor activity.2 Due to its chemical,
biological, and clinical significance, extensive studies on the
total synthesis of 13 and the structure-activity relationships of
synthetic analogues3d,4 have been carried out. We recently found
that the Japanese specimens of this animal contained new
cytotoxic peptides and depsipeptides5 and other unique me-
tabolites,6 which have not been isolated from Western Indian
Ocean specimens. Further investigation of the cytotoxic
constituents of the Japanese sea hare D. auricularia has resulted
in the isolation of two potent cytotoxic peptides, dolastatin H
(2)7 and isodolastatin H (3),7 as very minor constituents. We
X Abstract published in AdVance ACS Abstracts, February 1, 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) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Tuinman, A. A.; Boettner,
F. E.; Kizu, H.; Schmidt, J. M.; Baczynskyj, L.; Tomer, K. B.; Bontems,
R. J. J. Am. Chem. Soc. 1987, 109, 6883-6885.
(3) (a) Pettit, G. R.; Singh, S. B.; Hogan, F.; Lloyd-Williams, P.; Herald,
D. L.; Burkett, D. D.; Clewlow, P. J. J. Am. Chem. Soc. 1989, 111, 5463-
5465. (b) Hamada, Y.; Hayashi, K.; Shioiri, T. Tetrahedron Lett. 1991,
32, 931-934. (c) Tomioka, K.; Kanai, M.; Koga, K. Tetrahedron Lett.
1991, 32, 2395-2398. (d) Shioiri, T.; Hayashi, K.; Hamada, Y. Tetrahedron
1993, 49, 1913-1924.
(4) (a) Pettit, G. R.; Singh, S. B.; Hogan, F.; Burkett, D. D. J. Med.
Chem. 1990, 33, 3132-3133. (b) Miyazaki, K.; Gondo, M.; Sakakibara,
K. Pept. Chem. 1993, 85-88. (c) Pettit, G. R.; Srirangam, J. K.; Herald,
D. L.; Hamel, E. J. Org. Chem. 1994, 59, 6127-6130.
(5) (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. Tetrahedron Lett.
1995, 36, 5057-5058. (e) Nakamura, M.; Shibata, T.; Nakane, K.; Nemoto,
T.; Ojika, M.; Yamada, K. Tetrahedron Lett. 1995, 36, 5059-5062.
(6) Ojika, M.; Nemoto, T.; Yamada, K. Tetrahedron Lett. 1993, 34,
3461-3462.
(7) Dolastatins A and B: Pettit, G. R. Eur. Patent EP 124 984, 1984;
Chem. Abstr. 1985, 102, 72870f. Dolastatins C (ref 5a), D (ref 5b), and E
(refs 5d,e). Dolastatins F and G: to be published. Professor Pettit and
one of the authors (K.Y.) agreed that the names of our peptides and
depsipeptides isolated from Japanese specimens of D. auricularia should
be termed dolastatins C, D, E, F, ... etc. (7th International Symposium on
Marine Natural Products, July, 1992; Capri, Italy).
0002-7863/96/1518-1874$12.00/0 © 1996 American Chemical Society

Dolastatin H and Isodolastatin H
J. Am. Chem. Soc., Vol. 118, No. 8, 1996 1875
Table 1. 1H NMR Data for Dolastatin H (2) and Isodolastatin
H (3) in C6D6
Results and Discussion
a
Isolation. The MeOH extract of the internal organs of the
sea hare D. auricularia, collected in Mie Prefecture, Japan, was
partitioned between EtOAc and H2O. The EtOAc-soluble
material, which exhibited cytotoxicity against HeLa-S3 cells with
an IC50 of 1.2 µg/mL, was further partitioned between 9:1
MeOH/H2O and hexane. Bioassay-guided separation of the
material obtained from the aqueous MeOH portion was per-
formed by repeated chromatography on silica gel [(1) benzene/
EtOAc, then EtOAc/MeOH, step gradient; (2) 80:16:4 to 0:80:
20 benzene/EtOAc/MeOH, linear gradient] and ODS silica gel
[(1) 4:1 MeOH/H2O, then MeOH; (2) 7:3 MeOH/H2O to MeOH,
linear gradient] to give a cytotoxic fraction (IC50 ) 0.036 µg/
mL). The fraction was further separated by reversed-phase
HPLC (5:5 to 6:4 MeCN/H2O, linear gradient) and subsequently
by silica gel TLC (20:7:3 CHCl3/acetone/MeOH) to give a
potent cytotoxic fraction (IC50 ) 0.0038 µg/mL), which
contained dolastatin H (2) and isodolastatin H (3) in a ratio of
1:1. This cytotoxic fraction was then purified by silica gel TLC
(12:1 CHCl3/MeOH) and by reversed-phase HPLC (7:3 MeOH/
0.01 M NH4OAc) to afford pure 2 (9 × 10-7% yield based on
wet weight) and 3 (9 × 10-7%) as a colorless powder. The
cytotoxicities of 2 and 3, while not tested with natural 2 and 3
because of the inadequate amounts of natural samples (0.3 mg
each), were demonstrated using synthetic samples of these
compounds as described below.
position
1
2
3
3.76 dd (11.0, 1.9)
4.60 dd (11.0, 9.5)
4.35 m
2.58 dd (13.5, 6.6)
2.84 dd (13.5, 6.6)
7.05-7.20 m
2.57 dq (10.3, 7.0)
1.30 d (7.0)
4.29 dd (10.3, 1.5)
3.27 s
4.28 ddd (6.3, 4.2, 1.5)
1.57, 1.90 m
1.21, 1.63 m
2.84, 3.00 br
1.91, 2.11 br
4.12 br
3.80 ddd (13.0, 7.0, 6.2)
3.90 ddd (13.0, 7.5, 2.4)
5.54 m
2.77 dd (14.0, 6.2)
2.93 dd (14.0, 7.5)
7.07-7.15
2.51 dq (10.6, 7.0)
1.22 d (7.0)
4.27 dd (10.6, 1.1)
3.27 sb
2
3
5, 6, 7
9
9a
10
10a,b
11
12
13
14
17
4.20 m
1.62, 1.92 m
1.20, 1.63 m
2.83, 3.01 br
1.90, 2.10 br
4.13 br
18
18a,b
19
3.25 s
4.99 br
1.48 br
3.26 sb
4.99 br
1.47 br
19a
19b
19c
19d
20a
22
22a
22b
22c
22d
23
25
25b,c
26
27
28
1.06, 1.41 m
0.88 t (7.7)
0.93 d (7.0)
2.73 s
4.99 dd (8.8, 8.1)
1.86 m
1.23, 1.71 m
0.86 t (7.7)
1.06 d (6.6)
6.72 d (8.8)
2.30 d (7.0)
2.19 s
1.07, 1.40 m
0.87 t (7.3)
0.94 d (7.0)
2.73 s
4.98 dd (8.8, 7.6)
1.86 m
1.22, 1.70 m
0.85 t (7.3)
1.05 d (6.6)
6.68 d (8.8)
2.29 d (7.0)
2.19 s
Gross Structures. Dolastatin H (2) has a molecular formula
of C41H70N4O8, which was determined by high-resolution
FABMS [m/z 747.5294 (MH+), ∆ + 2.2 mmu] and high-field
1H NMR data (Table 1). The IR spectrum showed bands at
3425, 1730, 1660, 1635, and 1260 cm-1 that were assigned to
hydroxyl, ester, and amide groups. The 1H NMR data showed
the presence of an amide NH group (δ 6.72) and an N-
methylamide group (δ 2.73), suggesting the peptidic nature of
2. Resonances in the 1H NMR spectrum were assigned by DQF-
COSY, HMBC, and decoupling experiments, as shown in Table
1.8 Although the 13C NMR spectrum could not be obtained
due to the scarcity of the sample, carbon chemical shifts were
partially determined by HMBC experiments (JCH ) 6 and 8
Hz). These spectroscopic data suggested the presence of five
components in 2:N,N-dimethylvaline (dolavaline, Dov), isoleu-
cine (Ile), dolaisoleuine (Dil), dolaproine (Dap), and 3-phenyl-
propane-1,2-diol (Figure 1). Three of them, Dov, Dil, and Dap,
are the unusual amino acid units of dolastatin 10 (1).2 It was
difficult to prove the presence of the Dil unit, since most proton
signals from this unit were extremely broad and no cross-peaks
were observed in the COSY spectrum regarding the connec-
tivities between H-19 and H-19a and between H-19a and H-19b.
However, the HMBC correlations shown in Figure 1 clarified
these connectivities. The HMBC correlation between the
oxymethylene protons (H-1) of 3-phenylpropane-1,2-diol and
the carbonyl carbon (C-8) of Dap indicates connectivity between
these two components, as shown in Figure 1. Although the
presence of three carbonyl groups (C-8, C-21, and C-24) was
disclosed by HMBC experiments, the molecular formula of 2
suggested the presence of an additional carbonyl group, which
must correspond to the C-16 carbonyl group of the Dil portion.
Further evidence for the connectivity of these partial structures
could not be obtained from either HMBC experiments or
NOESY data due to the scarcity of the sample. However,
considering that the Dov and Dap units are the N- and C-termini
of 2, respectively, the carbonyl carbon (C-24) of the Dov unit
must be bonded to the amino nitrogen (N-23) of Ile and the
2.02 m
2.01 m
0.96 d (7.0)
1.11 d (7.0)
5.26 d (3.4)
0.96 d (7.0)
1.11 d (7.0)
5.21 dd (7.5, 7.0)
OH
a Recorded at 600 MHz. Coupling constants (Hz) are in parentheses.
b Interchangeable signals.
Figure 1. Partial structures of 2 derived from 2D NMR experiments.
Important HMBC correlations are shown with arrows.
methine carbon (C-22) of Ile should be connected to the
carbonyl carbon (C-21) of the Dil unit. Thus, the gross structure
of dolastatin H is unequivocally shown as 2.
The gross structure of isodolastatin H (3) was elucidated by
comparison with the spectral data of 2. The molecular formula
of 3 was identical with that of 2. The 1H NMR data (Table 1)
were similar to those for 2 except for the chemical shifts of the
oxymethylene (H-1) and oxymethine (H-2) protons: the signals
due to H-1 were observed at δ 3.76 and 4.60 and the signal of
H-2 was at δ 4.35 in 2, whereas the signals arising from H-1
were observed at δ 3.80 and 3.90 and the signal of H-2 was at
δ 5.54 in 3. These data suggest that the Dap unit in 3 is
esterified by the secondary hydroxyl group of 3-phenylpropane-
1,2-diol. Thus, isodolastatin H (3) was shown to be a structural
isomer of 2 with regard to the 3-phenylpropane-1,2-diol moiety.
(8) The numbering adopted in this paper corresponds to that of dolastatin
10 (1) (ref 2).

1876 J. Am. Chem. Soc., Vol. 118, No. 8, 1996
Sone et al.
Scheme 1a
group in 16 gave (2S)-3. The epimer (2R)-3 was also
synthesized using ent-6 in the same manner. Of the two
stereoisomers, the spectral data for (2S)-3, [R]28 -47.6° (c
D
0.051, MeOH), were identical to those for natural 3, [R]24D -47°
(c 0.04, MeOH), thus establishing the absolute stereostructure
of 3.
Cytotoxicity of Dolastatin H (2), Isodolastatin H (3), and
Their C2-Epimers. Since the cytotoxicity of natural 2 and 3
could not be tested, as described above, synthetic 2 and 3, as
well as their epimers C2-epi-2 and C2-epi-3, were subjected to
cytotoxicity testing using the HeLa-S3 cell line. Both synthetic
2 and 3 showed potent cytotoxicity with IC50 values of 0.0022
and 0.0016 µg/mL, respectively, indicating that both natural 2
a (a) DIBAL, CH2Cl2, hexane, 0 °C. (b) t-BuPh2SiCl, imidazole,
DMF, rt. (c) BnBr, LiN(SiMe3)2, DMF, THF, rt. (d) HF, MeCN, H2O,
0 °C f rt.
and 3 are the active principles of the cytotoxic fraction [IC50
)
0.0038 µg/mL (see the section on isolation)] prepared from the
sea hare D. auricularia. It is noteworthy that the C2-epimers
of 2 and 3 are much less cytotoxic (IC50 ) 0.020 and 0.029
µg/mL, respectively) than 2 and 3 themselves. These findings
suggest that the cytotoxicity of 2 and 3 is quite sensitive to the
C-terminal structures of these peptides. It should be noted that
the phenyl group of the C-terminus of 1 was reported to be
essential for biological activity in a study of a structure-activity
relationships of dolastatin 10 (1).4b
Absolute Stereochemistry and Synthesis. The stereochem-
istries of dolastatin H (2) and isodolastatin H (3) were
determined by the synthesis of the possible stereoisomers,
assuming that the stereochemistries of the Dov, Dil, and Dap
units were identical to those of dolastatin 10 (1) and that
isoleucine has an L configuration. Since the absolute stereo-
chemistry of the 3-phenylpropane-1,2-diol unit is unknown, our
synthetic targets were (2S)-2 and (2S)-3 and their C2-epimers
(2R)-2 and (2R)-3.
Evaluation of in ViWo Antitumor Activity for Dolastatin
H (2) and Isodolastatin H (3). Dolastatin H (2) showed acute
toxicity against mice at doses above 10 mg/kg by intravenous
injection. On the basis of this toxicity test, in ViVo antitumor
activity of 2 and 3 was examined. While no significant activity
was shown for dolastatin H (2), isodolastatin H (3) exhibited
antitumor activity with a T/C of 141% at a dose of 6 mg/kg/
day against P388 leukemia (intraperitoneal tumor inoculation-
intravenous drug administration). This antitumor activity for
3 is a little weaker than that for dolastatin 10 (1) (T/C ) 155%
at 6.5 µg/kg/day against P388 leukemia)1 and 3 requires a higher
concentration than 1.
Conclusion. New potent cytotoxic peptides, dolastatin H (2)
and isodolastatin H (3), were isolated from Japanese specimens
of the sea hare D. auricularia. Their structures were deduced
and unambiguously established by total synthesis. The cyto-
toxicities of 2, 3, and their C-2 epimers were evaluated and
depended largely on the C-terminal stereostructures of these
peptides. The present investigation provides the first isolation
from the natural source of potent cytotoxic peptides, 2 and 3,
which are closely related to dolastatin 10 (1), a well-known
potent antitumor agent.2
The 3-phenylpropane-1,2-diol unit 8 was prepared from the
optically pure epoxy alcohol 49 (Scheme 1). Regioselective
reduction10 of 4 gave (S)-3-phenylpropane-1,2-diol (5),11 which
was converted to silyl ether 6. Benzylation of 6 followed by
deprotection of the silyl group of the resulting benzyl ether 7
gave the desired primary alcohol 8. The enantiomer of 8 was
also prepared from ent-4 in the same manner. The Dap and
Dil units, Boc-Dap (9) and Cbz-Dil-O-t-Bu (11), were synthe-
sized using the methods of Shioiri3d and Koga,3c respectively.
The first target (2S)-2 was synthesized as follows (Scheme
2). Condensation of primary alcohol 8 and Boc-Dap (9) under
the Keck conditions12 gave ester 10. Deprotection of the Cbz
group of Cbz-Dil-O-t-Bu (11) followed by coupling with Cbz-
L-isoleucine using (benzotriazol-1-yloxy)tripyrrolidinophospho-
nium hexafluorophosphate (PyBOP)13 gave dipeptide 12, which
was converted to tripeptide 13 by condensation with N,N-
dimethylvaline (Dov)14 using DEPC.15 After deprotection,
tripeptide 13 and ester 10 were coupled using DEPC to provide
tetrapeptide 14, debenzylation of which yielded (2S)-2. The
epimer (2R)-2 was also synthesized using ent-8 by the same
sequence of reactions employed for the synthesis of (2S)-2. Of
the two synthetic stereoisomers, (2S)-2 was found to be identical
to natural 2 in all respects, including specific rotation [synthetic
2 [R]28 -48.0° (c 0.061, MeOH); natural 2, [R]25 -56°
Experimental Section
General. Melting points are uncorrected. Optical rotations were
measured with a JASCO DIP-181 polarimeter. UV and IR spectra were
recorded on a JASCO UVIDEC-510 spectrophotometer and a JASCO
IR-810 spectrophotometer, respectively. NMR spectra were recorded
on a JEOL JNM EX270 (270 MHz for 1H), a JEOL ALPHA400 (100
MHz for 13C), or a JEOL ALPHA600 (600 MHz for 1H). NMR
chemical shifts were referenced to solvent peaks: δH 7.26 (residucal
CHCl3) for CDCl3, δH 7.16 (residual C6HD5) and δC 128.0 for benzene-
d6. Mass spectra were determined on a JEOL JMS LG2000 spectrom-
eter operating in the FAB mode (m-nitrobenzyl alcohol as a matrix).
Elemental analyses were performed with a LECO CHN-900 elemental
analyzer. Both TLC analysis and preparative TLC were conducted on
0.25 mm E. Merck precoated silica gel 60 F254. Fuji Silysia silica gel
BW-820 MH was used for column chromatography unless otherwise
noted. Preparative HPLC and medium-pressure liquid chromatography
(MPLC) were performed using a JASCO TRI ROTAR pump and
JASCO 880 pumps, respectively. Unless otherwise stated, materials
were obtained from commercial suppliers and used without further
purification. Organic solvents for anhydrous reactions were distilled
from the following drying agents: triethylamine and diisopropylethy-
lamine (CaH2), DMF (CaH2 under reduced pressure), and CH2Cl2
D
D
(c 0.04, MeOH)], thus establishing the absolute stereostructure
of 2.
The synthesis of (2S)-3 was performed by a sequence similar
to that used for the synthesis of (2S)-2 (Scheme 3). Thus,
esterification of Boc-Dap (9) with silyl ether 6 gave ester 15.
Ester 15 and tripeptide 13 were deprotected, respectively, and
condensed to provide tetrapeptide 16. Removal of the silyl
(9) Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.; Masamune, H.;
Sharpless, K. B. J. Am. Chem. Soc. 1987, 109, 5765-5780.
(10) Suzuki, T.; Saimoto, H.; Tomioka, H.; Oshima, K.; Nozaki, H.
Tetrahedron Lett. 1982, 23, 3597-3600.
(11) (a) Bergstein, W.; Kleemann, A.; Martens, J. Synthesis 1981, 76-
78. (b) Cardillo, G.; Orena, M.; Romero, M.; Sandri, S. Tetrahedron 1989,
45, 1501-1508.
(12) Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394-2395.
(13) Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31,
205-208.
(14) Bowman, R. E.; Stroud, H. H. J. Chem. Soc. 1950, 1342-1345.
(15) Shioiri, T.; Yokoyama, Y.; Kasai, Y.; Yamada, S. Tetrahedron 1976,
32, 2211-2217.

Dolastatin H and Isodolastatin H
J. Am. Chem. Soc., Vol. 118, No. 8, 1996 1877
Scheme 2a
a (a) DCC, 4-(dimethylamino)pyridine (DMAP), 10-camphorsulfonic acid (CSA), CH2Cl2, 0 °C. (b) H2, 10% Pd-C, MeOH, rt. (c) Cbz-Ile,
PyBOP, i-Pr2EtN, CH2Cl2, 0 °C. (d) DEPC, Et3N, DMF, 0 °C. (e) CF3CO2H, CH2Cl2, 0 °C. (f) CF3CO2H, CH2Cl2, rt. (g) H2, 10% Pd-C,
MeOH, H2O, AcOH, rt.
Scheme 3a
a (a) DCC, DMAP, CSA, CH2Cl2, 0 °C. (b) CF3CO2H, CH2Cl2, 0 °C. (c) CF3CO2H, CH2Cl2, 0 °C f rt. (d) DEPC, Et3N, DMF, 0 °C. (e) HF,
MeCN, H2O, 0 °C.
(P2O5). All moisture-sensitive reactions were performed under an
atmosphere of nitrogen. Organic extracts were dried over anhydrous
Na2SO4.
µg/mL) as an oil. The active fraction was further purified by preparative
TLC (12:1 CHCl3/MeOH, Rf ) 0.52) followed by preparative HPLC
[Develosil ODS-10 (20 × 250 mm), 7:3 MeCN/0.01 M NH4OAc, flow
rate 5.0 mL/min, detection at 215 nm] to give pure 2 (0.3 mg, tR ) 42
min) and 3 (0.3 mg, tR ) 46 min) as colorless powders, respectively.
Isolation of Dolastatin H (2) and Isodolastatin H (3). D.
auricularia (33 kg, wet wt) were collected by hand at a depth of 0-1
m off the coast of the Shima Peninsula, Mie Prefecture, Japan, in April
1993. The specimens, which were stored at -20 °C for 4 months,
were divided into the internal organs and thick outer skin, and the former
(20 kg, wet wt) were extracted with MeOH (40 L). The methanolic
extract was concentrated to ca. 2 L in vacuo and extracted with EtOAc
(3 × 2 L). After concentration in vacuo, the EtOAc extract (91.4 g,
IC50 against HeLa-S3 cells ) 1.2 µg/mL) was dissolved in 9:1 MeOH/
H2O (1 L), and the solution was washed with hexane (2 × 1 L).
Evaporation of the aqueous MeOH portion gave a dark brown oil (30.8
g), which was chromatographed on silica gel (590 g), eluting with 1:1
benzene/EtOAc, EtOAc, 95:5, 9:1, and 8:2 EtOAc/MeOH, and MeOH,
successively. The fraction (1.46 g, IC50 ) 0.1 µg/mL) eluted with 9:1
EtOAc/MeOH was subjected to MPLC [Fuji Silysia Micro Bead Silica
Gel 4B (65 g), linear gradient from 80:16:4 to 0:80:20 benzene/EtOAc/
MeOH, flow rate 6.0 mL/min]. The fraction eluted between 28:58:14
and 18:66:16 benzene/EtOAc/MeOH (371 mg, IC50 ) 0.044 µg/mL)
was subjected to chromatographic filtration through a pad of ODS
(Nacalai Tesque Cosmosil 75C18-OPN, 7.2 g), eluting with 4:1 MeOH/
H2O and then MeOH. The fraction eluted with 4:1 MeOH/H2O (300
mg) was subjected to MPLC [Nomura Chemical Develosil ODS 30/
60 (70 g), linear gradient from 7:3 MeOH/H2O to MeOH, flow rate
5.0 mL/min]. The fraction eluted between 92:8 MeOH/H2O and MeOH
(46 mg, IC50 ) 0.036 µg/mL) was separated by preparative HPLC
[Develosil ODS-10/20 (20 × 250 mm), linear gradient from 5:5 to 6:4
MeCN/H2O, flow rate 5.0 mL/min, detection at 215 nm] to afford an
oil (16 mg, tR ) 50-110 min, IC50 ) 0.019 µg/mL). This oil was
separated by preparative TLC (20:7:3 CHCl3/acetone/MeOH) to give
a potent cytotoxic fraction (4.0 mg, Rf ) 0.53-0.87, IC50 ) 0.0038
Dolastatin H (2): Rf ) 0.60 (10:1 CHCl3/MeOH); [R]25 -56° (c
D
0.04, MeOH); UV (MeOH) λmax 208 (ꢀ 23 000) nm; IR (CHCl3) 3425,
1730, 1660, 1635, 1495, 1455, 1260, 1095, 1060 cm-1; 1H NMR data,
see Table 1; HRFABMS calcd for C41H71N4O8 m/z 747.5272 (MH+),
found 747.5294.
Isodolastatin H (3): Rf ) 0.60 (10:1 CHCl3/MeOH); [R]25 -47°
D
(c 0.04, MeOH); UV (MeOH) λmax 208 (ꢀ 23 000) nm; IR (CHCl3)
3425, 1725, 1660, 1635, 1495, 1455, 1260, 1095, 1060 cm-1; 1H NMR
data, see Table 1; HRFABMS calcd for C41H71N4O8 m/z 747.5272
(MH+), found 747.5309.
(S)-3-Phenylpropane-1,2-diol (5). To a solution of epoxy alcohol
49 (492 mg, 3.28 mmol) in CH2Cl2 (7.0 mL) was added a solution of
diisobutylaluminum hydride (DIBAL) in hexane (1.0 M, 9.85 mL) over
7 min with stirring at 0 °C. The solution was stirred at 0 °C for 30
min, the reaction was quenched by the addition of ethyl acetate (5.0
mL), and the solution was warmed to ambient temperature. The mixture
was diluted with 1 M HCl (30 mL) and ether (30 mL) and stirred at
ambient temperature for 30 min. The organic layer was separated, and
the aqueous layer was extracted with ether (2 × 50 mL). The organic
layer and the extracts were combined, washed with saturated aqueous
NaCl (20 mL), dried, and concentrated. The residual oil was purified
by column chromatography (1:1 and then 1:2 hexane/EtOAc) to give
511 (307 mg, 62%) as crystals: colorless needles; mp 46-47 °C
(hexane/CH2Cl2); Rf ) 0.19 (1:1 hexane/EtOAc); [R]18D -35.4° (c 1.00,
EtOH) [lit.11a [R]20 -36° (c 1, EtOH)].
D
ent-5. Using the same procedure as described for the preparation
of 5, ent-4 was converted to ent-511b: mp 45-46 °C (hexane/CH2Cl2);
[R]22 +29.9° (c 1.05, EtOH).
D

1878 J. Am. Chem. Soc., Vol. 118, No. 8, 1996
Sone et al.
(S)-1-(tert-Butyldiphenylsiloxy)-2-hydroxy-3-phenylpropane (6).
To a solution of alcohol 5 (184 mg, 1.22 mmol) and imidazole (182
mg, 2.68 mmol) in DMF (1.0 mL) was added tert-butyldiphenylsilyl
chloride (0.35 mL, 1.3 mmol) with stirring at 0 °C. After being stirred
at ambient temperature for 20 min, the solution was diluted with 1:1
benzene/EtOAc (50 mL), and the mixture was washed with 10%
aqueous citric acid (5 mL), H2O (5 mL), saturated aqueous NaHCO3
(5 mL), H2O (5 mL), and saturated aqueous NaCl (5 mL) successively,
dried, and concentrated. The residual oil was purified by column
chromatography (20:1 and then 10:1 hexane/EtOAc) to give silyl ether
6 (368 mg, 78%) as a colorless oil: Rf ) 0.28 (10:1 hexane/EtOAc);
mg, 86%) as a colorless oil: Rf ) 0.68 (2:1 hexane/EtOAc); [R]30
D
-40.0° (c 1.34, CHCl3); IR (CHCl3) 1730, 1680, 1495, 1475, 1455,
1
1400, 1365, 1245, 1160, 1095, 1025, 900, 865 cm-1; H NMR (270
MHz, CDCl3) δ 1.27 (d, J ) 6.9 Hz, 3 H), 1.47 (s, 9 H), 1.55-1.74
(m, 1 H), 1.74-2.05 (m, 3 H), 2.47-2.63 (m, 1 H), 2.79-2.99 (m, 2
H), 3.13-3.29 (m, 1 H), 3.32-3.62 (m, 1 H), 3.41 (s, 3 H), 3.65-
4.18 (m, 5 H), 4.45 (d, J ) 11.6 Hz, 1 H), 4.56 (br d, J ) 11.6 Hz, 1
H), 7.16-7.33 (m, 10 H); FABMS m/z (relative intensity) 534 (MNa+,
7), 512 (MH+, 11), 456 (7), 412 (100), 380 (6), 316 (6), 170 (66), 138
(37), 114 (82); HRFABMS (addition of NaI) calcd for C30H41NO6Na
m/z 534.2831 (MNa+), found 534.2853.
[R]28 -0.30° (c 1.0, CHCl3); IR (CHCl3) 3580, 1600, 1585, 1495,
D
C2-epi-10. Using the same procedure as described for the prepara-
tion of 10, ent-8 was coupled with 9 to give C2-epi-10 in 75% yield:
1
1470, 1425, 1115, 1080, 820 cm-1; H NMR (270 MHz, CDCl3) δ
colorless oil; Rf ) 0.68 (2:1 hexane/EtOAc); [R]30 -20.9° (c 0.86,
1.08 (s, 9 H), 2.43 (d, J ) 4.3 Hz, 1 H), 2.77 (d, J ) 6.6 Hz, 2 H),
3.57 (dd, J ) 10.0, 6.4 Hz, 1 H), 3.67 (dd, J ) 10.0, 4.0 Hz, 1 H),
3.94 (m, 1 H), 7.12-7.31 (m, 5 H), 7.32-7.48 (m, 6 H), 7.60-7.75
(m, 4 H); FABMS (addition of NaI) m/z (relative intensity) 413 (MNa+,
22), 199 (46), 135 (100), 117 (53); HRFABMS calcd for C25H30O2-
SiNa m/z 413.1913 (MNa+), found 413.1928.
D
CHCl3); IR (CHCl3) 1730, 1680, 1495, 1475, 1455, 1400, 1365, 1245,
1160, 1095, 1025, 900, 865 cm-1; 1H NMR (270 MHz, CDCl3) δ 1.25
(d, J ) 6.3 Hz, 3 H), 1.46 (s, 9 H), 1.56-2.04 (m, 4 H), 2.46-2.66
(m, 1 H), 2.76-2.98 (m, 2 H), 3.14-3.29 (m, 1 H), 3.40-3.62 (m, 1
H), 3.41 (s, 3 H), 3.65-4.36 (m, 5 H), 4.41-4.66 (m, 2 H), 7.16-
7.34 (m, 10 H); FABMS m/z (relative intensity) 534 (MNa+, 6), 512
(MH+, 9), 456 (7), 412 (100), 170 (70), 138 (26), 114 (86); HRFABMS
calcd for C30H41NO6Na m/z 534.2831 (MNa+), found 534.2838.
Cbz-Ile-Dil-O-t-Bu (12). A mixture of Cbz-Dil-O-t-Bu (11)3c (518
mg, 1.32 mmol) and 10% Pd on carbon (134 mg) in MeOH (6.6 mL)
was stirred at ambient temperature under 1 atm of H2 gas for 70 min.
The reaction mixture was filtered through a membrane filter (pore size
0.50 µm), and the catalyst was washed with MeOH (15 mL). The
filtrate and the washing were combined and concentrated. The residue
and Cbz-L-isoleucine (525 mg, 1.98 mmol) were dissolved in CH2Cl2
(5.0 mL), and the solution was cooled to 0 °C. To the solution were
added (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluoro-
phosphate (PyBOP)13 (1.04 g, 2.01 mmol) and diisopropylethylamine
(0.70 mL, 4.0 mmol) with stirring. The mixture was slowly warmed
to ambient temperature over 11 h with stirring and further stirred for
6.5 h at ambient temperature. The mixture was diluted with 1:3
benzene/EtOAc (200 mL) and washed with 10% aqueous citric acid
(20 mL), H2O (20 mL), saturated aqueous NaHCO3 (20 mL), H2O (20
mL), and saturated aqueous NaCl (20 mL) successively, dried, and
concentrated. The residual oil was purified repeatedly by column
chromatography [(1) silica gel, 9:1 and then 8:1 hexane/EtOAc, (2)
flash chromatography using silica gel FL60D, step gradient from 20:1
ent-6. Using the same procedure as described for the preparation
of 6, ent-5 was converted to ent-6: [R]25 +0.25° (c 1.7, CHCl3).
D
(S)-2-(Benzyloxy)-1-(tert-butyldiphenylsiloxy)-3-phenylpropane
(7). To a solution of silyl ether 6 (1.88 g, 4.82 mmol) and benzyl
bromide (2.90 mL, 24.4 mmol) in DMF (7.5 mL) was added a solution
of LiN(SiMe3)2 in THF (1.0 M, 7.20 mL) with stirring at ambient
temperature. After being stirred at ambient temperature for 1.5 h, the
reaction mixture was cooled to 0 °C and diluted with saturated aqueous
NH4Cl (30 mL). The mixture was extracted with 1:1 hexane/benzene
(200 mL, 2 × 100 mL). The combined organic extracts were washed
with H2O (30 mL) and saturated aqueous NaCl (30 mL), dried, and
concentrated. The residual oil was purified by flash chromatography
twice (Fuji Silysia silica gel FL60D, 90:1 hexane/ether) to give benzyl
ether 7 (1.74 g, 75%) as a colorless oil: Rf ) 0.27 (10:1 hexane/EtOAc);
[R]30 -34.1° (c 1.19, CHCl3); IR (CHCl3) 1600, 1585, 1495, 1475,
D
1
1455, 1425, 1115, 1080, 820 cm-1; H NMR (270 MHz, CDCl3) δ
1.09 (s, 9 H), 2.81 (m, 1 H), 2.98 (m, 1 H), 3.63-3.80 (m, 3 H), 4.36
(d, J ) 11.6 Hz, 1 H), 4.52 (d, J ) 11.6 Hz, 1 H), 7.10-7.45 (m, 16
H), 7.63-7.73 (m, 4 H); FABMS (addition of NaI) m/z (relative
intensity) 503 (MNa+, 54), 197 (57), 135 (100), 105 (45); HRFABMS
calcd for C32H36O2SiNa m/z 503.2383 (MNa+), found 503.2410.
ent-7. Using the same procedure as described for the preparation
of 7, ent-6 was converted to ent-7: [R]25 +34.8° (c 0.74, CHCl3).
D
to 10:1 hexane/EtOAc, (3) Cosmosil 75C18-OPN, 85:15 and then 9:1
(S)-2-(Benzyloxy)-1-hydroxy-3-phenylpropane (8). To a solution
of benzyl ether 7 (1.73 g, 3.60 mmol) in acetonitrile (60 mL) was added
47% hydrofluoric acid (30 mL, 810 mmol) with stirring at 0 °C. After
being stirred at 0 °C for 30 min and at ambient temperature for 2 h,
the reaction mixture was poured into a mixture of ice (1000 g) and
saturated aqueous NaHCO3 (1000 mL). The mixture was extracted
with ether (800 mL, 2 × 400 mL), and the combined organic extracts
were washed with saturated aqueous NaCl (200 mL), dried, and
concentrated. The residual oil was purified by column chromatography
(3:1 hexane/EtOAc) to give 8 (848 mg, 97%) as a colorless oil: Rf )
0.40 (2:1 hexane/EtOAc); [R]29D -14.5° (c 1.77, CHCl3); IR (CHCl3)
3580, 3460 (br), 1600, 1585, 1495, 1455, 1395, 1350, 1100, 1085, 1070,
MeOH/H2O, (4) Cosmosil 75C18-OPN, 4:1 MeOH/H2O, (5) silica gel,
3:1 hexane/EtOAc] to give dipeptide 12 (550 mg, 83%) as a colorless
oil: Rf ) 0.71 (2:1 hexane/EtOAc); [R]30D -8.75° (c 1.30, CHCl3); IR
(CHCl3) 3430, 1715, 1640, 1505, 1455, 1410, 1365, 1295, 1235, 1155,
1
1095, 1040, 1025, 840 cm-1; H NMR (270 MHz, CDCl3) (rotamer
ratio 5:1) δ 0.83 (t, J ) 7.3 Hz, 3 H), 0.88 (t, J ) 7.4 Hz, 3 H), 0.95-
1.17 (m, 2 H), 0.96 (t, J ) 6.9 Hz, 3 H), 0.98 (t, J ) 7.6 Hz, 3 H),
1.26-1.82 (m, 4 H), 1.44 (s, 1.5 H), 1.45 (s, 7.5 Hz), 2.30 (dd, J )
15.5, 8.9 Hz, 1 H), 2.41 (dd, J ) 15.8, 3.0 Hz, 0.17 H), 2.44 (dd, J )
15.5, 2.6 Hz, 0.83 H), 2.76 (s, 0.5 H), 2.97 (s, 2.5 H), 3.33 (s, 0.5 H),
3.34 (s, 2.5 H), 3.68 (dd, J ) 9.2, 5.0 Hz, 0.17 H), 3.88 (m, 0.83 H),
3.98 (m, 0.17 H), 4.52 (dd, J ) 9.2, 6.6 Hz, 0.83 H), 4.57-4.84 (m,
1 H), 5.09 (s, 2 H), 5.44 (d, J ) 9.2 Hz, 0.83 H), 5.60 (d, J ) 9.2 Hz,
0.17 H), 7.28-7.40 (m, 5 H); FABMS m/z (relative intensity) 529
(MNa+, 2), 507 (MH+, 22), 451 (41), 419 (26), 311 (11), 204 (28),
186 (15), 176 (21), 146 (12), 100 (100); HRFABMS (addition of NaI)
calcd for C28H46N2O6Na m/z 529.3254 (MNa+), found 529.3275.
1
1040, 1030, 915 cm-1; H NMR (270 MHz, CDCl3) δ 2.08 (br t, J )
4.6 Hz, 1 H), 2.81 (dd, J ) 13.5, 6.9 Hz, 1 H), 2.94 (dd, J ) 13.5, 6.3
Hz, 1 H), 3.49 (m, 1 H), 3.58-3.76 (m, 2 H), 4.49 (d, J ) 11.6 Hz, 1
H), 4.55 (d, J ) 11.6 Hz, 1 H), 7.16-7.38 (m, 10 H); FABMS m/z
(relative intensity) 265 (MNa+, 8), 243 (MH+, 16), 207 (22), 181 (18),
165 (12), 117 (100); HRFABMS (addition of NaI) calcd for C16H18O2-
Na m/z 265.1205 (MNa+), found 265.1208.
Dov-Ile-Dil-O-t-Bu (13). A mixture of dipeptide 12 (354 mg, 0.700
mmol) and 10% Pd on carbon (70 mg) in MeOH (3.5 mL) was stirred
at ambient temperature under 1 atm of H2 gas for 30 min. The reaction
mixture was filtered through a membrane filter (pore size 0.50 µm),
and the catalyst was washed with MeOH (8 mL). The filtrate and
washings were combined and concentrated. The residue and N,N-
dimethyl-L-valine14 (125 mg, 0.866 mmol) were dissolved in DMF (2.0
mL), and the solution was cooled to 0 °C. To the solution were added
DEPC (0.13 mL, 0.86 mmol) and triethylamine (0.12 mL, 0.86 mmol)
with stirring. After being stirred at 0 °C for 2 h, the mixture was diluted
with 1:2 benzene/EtOAc (100 mL) and extracted with 10% aqueous
citric acid (10 mL). The aqueous extract was made basic (ca. pH 9)
with NaHCO3 and extracted with CHCl3 (3 × 80 mL). The extracts
were combined, washed with saturated aqueous NaCl (20 mL), dried,
ent-8. Using the same procedure as described for the preparation
of 8, ent-7 was converted to ent-8: [R]25 +13.5° (c 0.65, CHCl3).
D
(S)-2-(Benzyloxy)-1-((N-(tert-butoxycarbonyl)dolaproyl)oxy)-3-
phenylpropane (10). DCC (639 mg, 3.10 mmol) was added to a stirred
solution of benzyl ether 8 (515 mg, 2.13 mmol), Boc-Dap (9)3d (732
mg, 2.55 mmol), 4-(dimethylamino)pyridine (DMAP) (62.8 mg, 0.514
mmol), and 10-camphorsulfonic acid (CSA) (60.2 mg, 0.259 mmol)
in CH2Cl2 (15 mL) at 0 °C, and the mixture was stirred at 0 °C for 12
h. The mixture was filtered through a plug of cotton, and the solid
was washed with 1:1 hexane/benzene (15 mL). The filtrate and the
washing were combined and concentrated. The residual oil was purified
by column chromatography (10:1 hexane/EtOAc) to give ester 10 (940

Dolastatin H and Isodolastatin H
J. Am. Chem. Soc., Vol. 118, No. 8, 1996 1879
and concentrated. The residual oil was chromatographed on ODS
(Cosmosil 75C18-OPN, 4:1 MeOH/H2O) to give 13 (331 mg, 95%) as
crystals: colorless prisms; mp 58-59 °C (pentane); Rf ) 0.50 (2:1
MeOH (5 mL). The filtrate and washings were combined, and to the
resulting solution was added 10% Pd on carbon (197 mg). The mixture
was stirred for 75 min under the same conditions as described above.
Since the hydrogenolysis reaction of 14 was slow, the catalyst was
further added to the mixture three times at intervals [weight of the
catalyst and time for stirring: (1) 109 mg for 140 min, (2) 152 mg for
17 h 40 min, (3) 120 mg for 3 h 45 min]. The reaction mixture was
filtered through a membrane filter (pore size 0.50 µm), and the catalyst
was washed with MeOH (20 mL). The combined filtrate and washings
were concentrated, and the residual oil was chromatographed on alumina
[aluminum oxide 90 (activity II-III), 2:1 and then 1:1 hexane/EtOAc]
to provide (2S)-2 (308 mg, 77%) as a colorless powder: [R]28D -48.0°
(c 0.061, MeOH); 13C NMR (100 MHz, C6D6) δ 11.0 (q, 2 C, C-19c
and C-22c), 14.9 (q, C-9a), 15.6 (q, C-22d), 16.1 (q, C-19d), 18.4 (q,
C-27), 20.3 (q, C-28), 24.3 (t, C-12), 25.0 (t, C-13), 25.0 (t, C-22b),
26.1 (t, C-19b), 27.9 (d, C-26), 32.0 (q, C-20a), 33.4 (d, C-19a), 37.7
(d, C-22a), 38.0 (t, C-17), 40.1 (t, C-3), 42.7 (q, 2 C, C-25bc), 45.5 (d,
C-9), 47.9 (t, C-14), 53.1 (d, C-22), 57.0 (d, C-19), 57.7 (q, C-18ab),
60.3 (d, C-11), 61.1 (q, C-10ab), 69.5 (t, C-1), 70.2 (d, C-2), 76.2 (d,
C-25), 78.8 (d, C-18), 81.6 (d, C-10), 126.4 (d, C-7), 128.5 (d, 2 C,
C-6), 129.8 (d, 2 C, C-5), 138.9 (s, C-4), 170.7 (s, C-16), 171.1 (s,
C-24), 173.4 (s, C-8), 174.0 (s, C-21). Anal. Calcd for C41H70N4O8:
C, 65.92; H, 9.44; N, 7.50. Found: C, 65.80; H, 9.59; N, 7.45.
benzene/acetone); [R]27 -43.4° (c 0.58, MeOH); IR (CHCl3) 3430,
D
3370 (br), 1720, 1660, 1635, 1500, 1460, 1410, 1370, 1300, 1240, 1150,
1095, 1035, 840 cm-1; 1H NMR (270 MHz, CDCl3) (rotamer ratio 15:
1) δ 0.80 (t, J ) 7.4 Hz, 3 H), 0.89 (t, J ) 6.9 Hz, 3 H), 0.90 (d, J )
6.6 Hz, 3 H), 0.95 (d, J ) 6.6 Hz, 3 H), 0.95-1.42 (m, 3 H), 0.99 (d,
J ) 6.6 Hz, 3 H), 1.00 (d, J ) 6.6 Hz, 3 H), 1.44 (s, 0.56 H), 1.46 (s,
8.44 H), 1.52-1.85 (m, 3 H), 2.07 (dqq, J ) 6.0, 6.0, 6.0 Hz, 1 H),
2.20-2.37 (m, 1 H) [for major rotamer: 2.29 (dd, J ) 15.5, 9.6 Hz,
0.94 H)], 2.24 (s, 5.63 H), 2.27 (s, 0.37 H), 2.38-2.51 (m, 1 H) [for
major rotamer: 2.45 (dd, J ) 15.5, 2.3 Hz, 0.94 H)], 2.42 (d, J ) 6.6
Hz, 1 H), 2.75 (s, 0.19 H), 3.01 (s, 2.81 H), 3.34 (s, 2.81 H), 3.35 (s,
0.19 H), 3.68 (dd, J ) 10.1, 5.0 Hz, 0.83 H), 3.88 (m, 0.94 H), 3.96
(m, 0.06 H), 4.75 (m, 0.94 H), 4.81 (dd, J ) 9.2, 7.3 Hz, 0.94 H), 5.09
(dd, J ) 9.2, 3.7 Hz, 0.06 H), 6.81 (d, J ) 9.2 Hz, 0.94 H), 7.04 (d,
J ) 9.2 Hz, 0.06 H); FABMS m/z (relative intensity) 500 (MH+, 12),
444 (3), 241 (2), 204 (3), 169 (2), 100 (100). Anal. Calcd for
C27H53N3O5: C, 64.89; H, 10.69; N, 8.41. Found: C, 64.72; H, 10.67;
N, 8.36.
2-O-Benzyldolastatin H (14). To a solution of tripeptide 13 (607
mg, 1.22 mmol) in CH2Cl2 (3.0 mL) was added CF3CO2H (3.0 mL)
with stirring at 0 °C, and the solution was stirred at ambient temperature
for 1.5 h to give a solution (solution A). To a solution of ester 10
(650 mg, 1.27 mmol) in CH2Cl2 (3.0 mL) was added CF3CO2H (3.0
mL) with stirring at 0 °C, and the solution was stirred at 0 °C for 40
min to give a solution (solution B). The solutions A and B were
combined and concentrated to give an oil, which was dissolved in DMF
(5.0 mL). To this solution were added DEPC (0.25 mL, 1.5 mmol)
and triethylamine (0.95 mL, 6.8 mmol) with stirring at 0 °C. After
being stirred at 0 °C for 2 h, the mixture was diluted with benzene
(200 mL) and H2O (50 mL), and the aqueous layer was made basic
(ca. pH 11) with Na2CO3. The organic layer was separated, and the
aqueous layer was extracted with benzene (2 × 100 mL). The organic
layers were combined, washed with H2O (50 mL) and saturated aqueous
NaCl (50 mL), dried, and concentrated. The residual oil was purified
by flash chromatography (silica gel FL60D, step gradient from 2:1 to
0:1 hexane/EtOAc) followed by column chromatography on alumina
[E. Merck aluminum oxide 90 (Activity II-III), step gradient from 3:1
to 1:1 hexane/EtOAc] to give 14 (888 mg, 87%) as a colorless oil; Rf
) 0.51 (9:1 CHCl3/MeOH); [R]28D -49.5° (c 0.79, MeOH); IR (CHCl3)
3430, 3370 (br), 1730, 1655, 1635, 1495, 1455, 1425, 1415, 1240, 1165,
1095 cm-1; 1H NMR (270 MHz, CDCl3) (rotamer ratio 4:1) δ 0.81 (t,
J ) 7.3 Hz, 3 H), 0.86 (t, J ) 7.3 Hz, 3 H), 0.90 (d, J ) 6.9 Hz, 3 H),
0.90-1.23 (m, 2 H), 0.96 (d, J ) 6.3 Hz, 3 H), 0.99 (d, J ) 6.3 Hz,
3 H), 1.00 (d, J ) 6.9 Hz, 3 H), 1.23-1.44 (m, 1 H), 1.29 (d, J ) 6.9
Hz, 2.4 H), 1.30 (d, J ) 6.9 Hz, 0.6 H), 1.50-1.88 (m, 5 H), 1.89-
2.66 (m, 7 H), 2.24 (s, 4.8 H), 2.25 (s, 1.2 H), 2.90 (d, J ) 6.6 Hz, 2
H), 3.02 (s, 2.4 H), 3.09 (s, 0.6 H), 3.26-3.52 (m, 2 H), 3.29 (s, 2.4
H), 3.32 (s, 0.6 H), 3.39 (s, 2.4 H), 3.40 (s, 0.6 H), 3.68-5.08 (m, 10
H), 6.84 (d, J ) 9.2 Hz, 1 H), 7.15-7.34 (m, 10 H); FABMS m/z
(relative intensity) 859 (MNa+, 2), 837 (MH+, 5), 805 (2), 241 (3),
170 (2), 117 (3), 100 (100); HRFABMS (addition of NaI) calcd for
C48H76N4O8Na m/z 859.5561 (MNa+), found 859.5569.
(2R)-2 [) 2-epi-Dolastatin H]. Using the same procedure as
described for the preparation of (2S)-2, C2-epi-14 was converted to
(2R)-2 in 73% yield: colorless powder; Rf ) 0.35 (2:1 benzene/acetone);
[R]26 -52.1° (c 0.065, MeOH); IR (CHCl3) 3430 (br), 1730, 1665,
D
1635, 1495, 1455, 1410, 1245, 1095, 1060 cm-1; 1H NMR (600 MHz,
C6D6) δ 0.86 (t, J ) 7.5 Hz, 3 H, H-22c), 0.89 (t, J ) 7.5 Hz, 3 H,
H-19c), 0.97 (d, J ) 6.8 Hz, 6 H, H-27 and H-19d), 1.04 (d, J ) 6.6
Hz, 3 H, H-22d), 1.06 (m, 1 H, H-19b), 1.12 (d, J ) 6.6 Hz, 3 H,
H-28), 1.23 (m, 2 H, H-13 and H-19b), 1.34 (d, J ) 7.0 Hz, 3 H,
H-9a), 1.43 (m, 1 H, H-19b), 1.47 (m, 1 H, H-19a), 1.59 (m, 1 H,
H-12), 1.66 (m, 1 H, H-13), 1.71 (m, 1 H, H-22b), 1.87 (m, 2 H, H-12
and H-22a), 1.91 (m, 1 H, H-17), 2.02 (dqq, J ) 7.0, 6.8, 6.6 Hz, 1 H,
H-26), 2.10 (br, 1 H, H-17), 2.20 (s, 6 H, H-25bc), 2.30 (d, J ) 7.0
Hz, 1 H, H-25), 2.58 (dq, J ) 10.6, 7.0 Hz, 1 H, H-9), 2.72 (s, 3 H,
H-20a), 2.78 (dd, J ) 13.7, 5.5 Hz, 1 H, H-3), 2.83 (br, 1 H, H-14),
3.01 (br, 1 H, H-14), 3.03 (dd, J ) 13.7, 8.1 Hz, 1 H, H-3), 3.26 (s, 3
H, H-10ab), 3.29 (s, 3 H, H-18ab), 3.68 (dd, J ) 11.0, 6.4 Hz, 1 H,
H-1), 4.14 (m, 1 H, H-2), 4.14 (br, 1 H, H-18), 4.18 (m, 1 H, H-11),
4.27 (dd, J ) 10.6, 1.1 Hz, 1 H, H-10), 4.82 (dd, J ) 11.0, 2.2 Hz, 1
H, H-1), 4.98 (br, 1 H, H-22), 5.00 (br, 1 H, H-19), 5.21 (d, J ) 6.2
Hz, 1 H, OH), 6.72 (d, J ) 8.8 Hz, 1 H, NH), 7.11 (m, 1 H, H-7), 7.20
(m, 2 H, H-6), 7.31 (m, 2 H, H-5); 13C NMR (100 MHz, C6D6) δ 11.0
(q, C-19c), 11.1 (q, C-22c), 14.7 (q, 9a), 15.6 (q, C-22d), 16.1 (q,
C-19d), 18.3 (q, C-27), 20.4 (q, C-28), 24.3 (t, C-12), 24.9 (t, C-22b),
25.0 (t, C-13), 26.1 (t, C-19b), 27.9 (d, C-26), 32.0 (q, C-20a), 33.4
(d, C-19a), 37.7 (d, C-22a), 38.0 (t, C-17), 40.3 (t, C-3), 42.8 (q, 2 C,
C-25bc), 45.6 (d, C-9), 47.9 (t, C-14), 53.1 (d, C-22), 57.1 (d, C-19),
57.9 (q, C-18ab), 60.2 (d, C-11), 61.1 (q, C-10ab), 69.1 (t, C-1), 70.8
(d, C-2), 76.3 (d, C-25), 78.8 (d, C-18), 81.7 (d, C-10), 126.4 (d, C-7),
128.5 (d, 2 C, C-6), 129.9 (d, 2 C, C-5), 139.3 (s, C-4), 170.3 (s, C-16),
171.1 (s, C-24), 173.4 (s, C-8), 173.9 (s, C-21); FABMS m/z (relative
intensity) 769 (MNa+, 3), 747 (MH+, 5), 100 (100). Anal. Calcd for
C41H70N4O8: C, 65.92; H, 9.44; N, 7.50. Found: C, 65.79; H, 9.70;
N, 7.56.
C2-epi-14. Using the same procedure as described for the prepara-
tion of 14, C2-epi-10 was coupled with 13 to give C2-epi-14 in 89%
yield: colorless oil; Rf ) 0.51 (9:1 CHCl3/MeOH); [R]26 -39.8° (c
D
(S)-2-((N-(tert-Butoxycarbonyl)dolaproyl)oxy)-1-(tert-butyldiphe-
nylsiloxy)-3-phenylpropane (15). DCC (480 mg, 2.33 mmol) was
added to a solution of silyl ether 6 (756 mg, 1.94 mmol), Boc-Dap
(9)3d (672 mg, 2.34 mmol), DMAP (118 mg, 0.966 mmol), and 10-
camphorsulfonic acid (135 mg, 0.581 mmol) in CH2Cl2 (20 mL) with
stirring at 0 °C, and the mixture was stirred at 0 °C for 11 h. The
mixture was filtered through a plug of cotton, and the solid was washed
with 1:1 hexane/benzene (30 mL). The filtrate and washings were
combined and concentrated. The residual oil was purified by column
chromatography (step gradient from 15:1 to 8:1 hexane/EtOAc) to give
ester 15 (1.09 g, 85%) as a colorless oil: Rf ) 0.40 (5:1 hexane/EtOAc);
0.904, MeOH); IR (CHCl3) 3430, 3370 (br), 1730, 1655, 1635, 1495,
1455, 1425, 1415, 1240, 1165, 1095 cm-1; 1H NMR (270 MHz, CDCl3)
(rotamer ratio 4:1) δ 0.76-1.22 (m, 20 H), 1.24-1.43 (m, 1 H), 1.27
(d, J ) 6.9 Hz, 0.6 H), 1.28 (d, J ) 6.9 Hz, 2.4 H), 1.50-1.85 (m, 5
H), 1.85-2.68 (m, 7 H), 2.24 (s, 4.8 H), 2.25 (s, 1.2 H), 2.68-2.97
(m, 2 H), 3.02 (s, 2.4 H), 3.08 (s, 0.6 H), 3.23-3.52 (m, 2 H), 3.29 (s,
2.4 H), 3.32 (s, 0.6 H), 3.39 (s, 2.4 H), 3.41 (s, 0.6 H), 3.60-5.09 (m,
10 H), 6.86 (d, J ) 9.2 Hz, 1 H), 7.15-7.34 (m, 10 H); FABMS m/z
(relative intensity) 837 (MH+, 5), 100 (100); HRFABMS calcd for
C48H76N4O8Na m/z 859.5561 (MNa+), found 859.5549.
(2S)-2 [) Dolastatin H (2)]. A mixture of tetrapeptide 14 (451
mg, 0.540 mmol) and 10% Pd on carbon (101 mg) in 12:5:3.5 MeOH/
H2O/AcOH (20.5 mL) was stirred at ambient temperature under 1 atm
of H2 gas for 1 h. The reaction mixture was filtered through a
membrane filter (pore size 0.50 µm), and the catalyst was washed with
[R]30 -41.9° (c 1.3, CHCl3); IR (CHCl3) 1725, 1680, 1600, 1590,
D
1500, 1475, 1455, 1430, 1400, 1395, 1365, 1255, 1160, 1115, 820 cm-1
;
1H NMR (270 MHz, CDCl3) δ 1.07 (s, 9 H), 1.13-1.22 (m, 3 H), 1.47
(s, 9 H), 1.55-1.74 (m, 2 H), 1.74-1.97 (m, 2 H), 2.30-2.54 (m, 1
H), 2.82-2.99 (m, 1 H), 3.05 (dd, J ) 13.9, 5.9 Hz, 1 H), 3.12-3.26

1880 J. Am. Chem. Soc., Vol. 118, No. 8, 1996
Sone et al.
(m, 1 H), 3.30 (s, 3 H), 3.34-3.90 (m, 5 H), 5.16-5.32 (m, 1 H),
7.13-7.30 (m, 5 H), 7.31-7.47 (m, 6 H), 7.61-7.76 (m, 4 H); FABMS
(addition of NaI) m/z (relative intensity) 682 (MNa+, 16), 660 (MH+,
4), 560 (49), 239 (15), 197 (37), 170 (36), 135 (100), 114 (40);
HRFABMS calcd for C39H53NO6SiNa m/z 682.3539 (MNa+), found
682.3519.
extracted with CHCl3 (100 mL, 2 × 50 mL). The combined organic
layers were washed with saturated aqueous NaCl (20 mL), dried, and
concentrated. The residual oil was purified by column chromatography
(10:10:1 hexane/EtOAc/MeOH) to provide (2S)-3 (274 mg, 87%) as
crystals: colorless prisms; mp 84-85 °C (hexane/CH2Cl2); [R]28
D
-47.6° (c 0.051, MeOH); 13C NMR (100 MHz, C6D6) δ 10.9 (q, C-19c),
11.0 (q, C-22c), 14.8 (q, C-9a), 15.6 (q, C-22d), 16.1 (q, C-19d), 18.4
(q, C-27), 20.3 (q, C-28), 24.3 (t, C-12), 25.0 (t, C-13), 25.0 (t, C-22b),
26.1 (t, C-19b), 27.9 (d, C-26), 32.0 (q, C-20a), 33.2 (d, C-19a), 37.6
(d, C-22a), 37.1 (t, C-3), 37.9 (t, C-17), 42.7 (q, 2 C, C-25bc), 46.2 (d,
C-9), 48.0 (t, C-14), 53.1 (d, C-22), 56.9 (d, C-19), 57.8 (q, C-18ab),
60.2 (d, C-11), 61.2 (q, C-10ab), 63.4 (t, C-1), 76.1 (d, C-25), 76.4 (d,
C-2), 79.0 (d, C-18), 81.8 (d, C-10), 126.6 (d, C-7), 128.5 (d, 2 C,
C-6), 129.8 (d, 2 C, C-5), 137.7 (s, C-4), 170.7 (s, C-16), 171.1 (s,
C-24), 172.9 (s, C-8), 174.0 (s, C-21). Anal. Calcd for C41H70N4O8:
C, 65.92; H, 9.44; N, 7.50. Found: C, 65.71; H, 9.43; N, 7.45.
C2-epi-15. Using the same procedure as described for the prepara-
tion of 15, ent-6 was coupled with 9 to give C2-epi-15 in 77% yield:
colorless oil; Rf ) 0.40 (5:1 hexane/EtOAc); [R]30 -8.71° (c 1.0,
D
CHCl3); IR (CHCl3) 1725, 1680, 1600, 1590, 1500, 1475, 1455, 1430,
1
1400, 1395, 1365, 1255, 1160, 1115, 820 cm-1; H NMR (270 MHz,
CDCl3) δ 1.06 (s, 9 H), 1.07-1.15 (m, 3 H), 1.46 (s, 9 H), 1.53-1.96
(m, 4 H), 2.34-2.52 (m, 1 H), 2.85-3.05 (m, 2 H), 3.13-3.25 (m, 1
H), 3.34 (s, 3 H), 3.46-3.95 (m, 5 H), 5.14-5.25 (m, 1 H), 7.12-
7.48 (m, 11 H), 7.55-7.74 (m, 4 H); FABMS m/z (relative intensity)
660 (MH+, 11), 560 (93), 239 (17), 199 (39), 170 (65), 135 (100), 114
(70); HRFABMS calcd for C39H54NO6Si m/z 660.3720 (MH+), found
660.3741.
(2R)-3 [) 2-epi-Isodolastatin H]. Using the same procedure as
described for the preparation of (2S)-3, C2-epi-16 was converted to
(2R)-3 in 70% yield: colorless powder; Rf ) 0.55 (3:3:1 hexane/EtOAc/
MeOH); [R]26D -53.8° (c 0.052, MeOH); IR (CHCl3) 3430 (br), 1725,
1665, 1635, 1495, 1455, 1410, 1095 cm-1; 1H NMR (600 MHz, C6D6)
δ 0.88 (t, J ) 7.3 Hz, 6 H, H-19c and H-22c), 0.97 (d, J ) 7.0 Hz, 3
H, H-27), 0.98 (d, J ) 6.6 Hz, 3 H, H-19d), 1.07 (m, 1 H, H-19b),
1.08 (d, J ) 7.0 Hz, 3 H, H-22d), 1.12 (d, J ) 6.6 Hz, 3 H, H-28),
1.25 (m, 1 H, H-22b), 1.30 (d, J ) 7.0 Hz, 3 H, H-9a), 1.37 (m, 1 H,
H-13), 1.43 (m, 1 H, H-19b), 1.57 (br, 1 H, H-19a), 1.60 (m, 1 H,
H-12), 1.74 (m, 1 H, H-22b), 1.77 (m, 1 H, H-13), 1.89 (m, 1 H, H-22a),
1.91 (m, 1 H, H-12), 2.04 (dqq, J ) 7.0, 7.0, 7.0 Hz, 1 H, H-26), 2.21
(s, 6 H, H-25bc), 2.22 (br, 2 H, H-17), 2.33 (d, J ) 7.0 Hz, 1 H, H-25),
2.56 (dq, J ) 8.6, 7.0 Hz, 1 H, H-9), 2.78 (s, 3 H, H-20a), 2.92 (br, 1
H, H-14), 3.01 (dd, J ) 13.5, 7.7 Hz, 1 H, H-3), 3.07 (dd, J ) 13.5,
6.6 Hz, 1 H, H-3), 3.10 (br, 1 H, H-14), 3.20 (s, 3 H, H-10ab), 3.30 (s,
3 H, H-18ab), 3.64 (dd, J ) 12.6, 4.9 Hz, 1 H, H-1), 3.92 (dd, J )
12.6, 2.4 Hz, 1 H, H-1), 4.10 (dd, J ) 8.6, 2.0 Hz, 1 H, H-10), 4.16
(br, 1 H, H-18), 4.57 (ddd, J ) 8.6, 4.0, 2.0 Hz, 1 H, H-11), 5.00 (br,
1 H, H-19), 5.02 (br, 1 H, H-22), 5.24 (m, 1 H, H-2), 6.79 (d, J ) 9.2
Hz, 1 H, NH), 7.04 (m, 1 H, H-7), 7.12 (m, 2 H, H-6), 7.22 (m, 2 H,
H-5); 13C NMR (100 MHz, C6D6) δ 11.0 (q, C-19c or C-22c), 11.1 (q,
C-22c or C-19c), 13.1 (q, C-9a), 15.7 (q, C-22d), 16.2 (q, C-19d), 18.4
(q, C-27), 20.3 (q, C-28), 24.7 (t, C-12), 25.1 (t, C-13), 24.9 (t, C-22b),
26.1 (t, C-19b), 27.9 (d, C-26), 31.8 (q, C-20a), 33.2 (d, C-19a), 36.6
(t, C-3), 37.6 (d, C-22a), 38.1 (t, C-17), 42.7 (q, 2 C, C-25bc), 43.4 (d,
C-9), 47.8 (t, C-14), 53.1 (d, C-22), 57.4 (d, C-19), 57.9 (q, C-18ab),
60.6 (d, C-11), 60.8 (q, C-10ab), 62.5 (t, C-1), 76.3 (d, C-25), 77.2 (d,
C-2), 79.2 (d, C-18), 82.0 (d, C-10), 126.7 (d, C-7), 128.7 (d, 2 C,
C-6), 129.9 (d, 2 C, C-5), 137.9 (s, C-4), 170.3 (s, C-16), 171.1 (s,
C-24), 173.8 (s, C-21), 174.0 (s, C-8); FABMS m/z (relative intensity)
747 (MH+, 20), 100 (100). Anal. Calcd for C41H70N4O8: C, 65.92;
H, 9.44; N, 7.50. Found: C, 65.80; H, 9.58; N, 7.50.
1-O-(tert-Butyldiphenylsilyl)isodolastatin H (16). To a solution
of tripeptide 13 (506 mg, 1.01 mmol) in CH2Cl2 (5.0 mL) was added
CF3CO2H (5.0 mL) with stirring at 0 °C, and the solution was stirred
at 0 °C for 1.5 h and at ambient temperature for 30 min to give a
solution (solution A). To a solution of ester 15 (637 mg, 0.967 mmol)
in CH2Cl2 (5.0 mL) was added CF3CO2H (5.0 mL) with stirring at 0
°C, and the solution was stirred at 0 °C for 70 min to give a solution
(solution B). The solutions A and B were combined and concentrated
to give an oil, which was dissolved in DMF (3.0 mL). To this solution
were added triethylamine (0.40 mL, 2.9 mmol) and DEPC (0.17 mL,
1.0 mmol) with stirring at 0 °C. After being stirred at 0 °C for 10 h,
triethylamine (0.40 mL, 2.9 mmol) was added and the mixture was
stirred at 0 °C for 70 min. The mixture was diluted with CHCl3 (200
mL) and saturated aqueous NaHCO3 (20 mL), and the aqueous layer
was made basic (ca. pH 10) with Na2CO3. The organic layer was
separated, and the aqueous layer was extracted with CHCl3 (2 × 100
mL). The organic layers were combined, washed with saturated
aqueous NaCl (20 mL), dried, and concentrated. The residual oil was
purified by column chromatography [(1) silica gel, step gradient from
30:30:1 to 10:10:1 hexane/EtOAc/MeOH); (2) Sephadex LH-20, 1:1
CH2Cl2/MeOH, twice] to give 16 (491 mg, 52%) as a colorless oil; Rf
) 0.65 (2:1 benzene/acetone); [R]27 -45.9° (c 0.32, MeOH); IR
D
(CHCl3) 3430, 3360 (br), 1725, 1655, 1635, 1500, 1455, 1415, 1260,
1
1165, 1095 cm-1; H NMR (270 MHz, CDCl3) (rotamer ratio 3:1) δ
0.77-1.22 (m, 20 H), 1.06 (s, 6.8 H), 1.08 (s, 2.2 H), 1.19 (d, J ) 6.9
Hz, 0.7 H), 1.20 (d, J ) 6.9 Hz, 2.3 H), 1.27-1.47 (m, 1 H), 1.50-
2.17 (m, 8 H), 2.20-2.54 (m, 4 H), 2.25 (s, 4.5 H), 2.27 (s, 1.5 H),
2.82-3.14 (m, 2 H), 3.02 (s, 2.3 H), 3.08 (s, 0.7 H), 3.19-3.50 (m, 2
H), 3.29 (s, 4.6 H), 3.32 (s, 0.7 H), 3.35 (s, 0.7 H), 3.63-4.30 (m, 5
H), 4.64-5.11 (m, 2 H), 5.13-5.34 (m, 1 H), 6.87 (d, J ) 9.2 Hz, 1
H), 7.12-7.30 (m, 5 H), 7.31-7.49 (m, 6 H), 7.60-7.72 (m, 4 H);
FABMS m/z (relative intensity) 985 (MH+, 5), 669 (13), 431 (41), 401
(15), 343 (100), 327 (47), 311 (75), 242 (34), 163 (36), 133 (48), 100
(65); HRFABMS (addition of NaI) calcd for C57H88N4O8SiNa m/z
1007.6270 (MNa+), found 1007.6300.
Acknowledgment. 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, Japan. We thank Dr. Y. Kakui (Nippon
Kayaku Co. Ltd.) for biological testings. A fellowship of Japan
Society for the Promotion of Science for Japanese Junior
Scientists to H.S. is gratefully acknowledged.
C2-epi-16. Using the same procedure as described for the prepara-
tion of 16, C2-epi-15 was coupled with 13 to give C2-epi-16 in 60%
yield: colorless oil; Rf ) 0.65 (2:1 benzene/acetone); [R]27D -29.9° (c
0.91, MeOH); IR (CHCl3) 3430, 3360 (br), 1725, 1655, 1635, 1500,
1455, 1415, 1260, 1165, 1095 cm-1 1H NMR (270 MHz, CDCl3)
;
(rotamer ratio 3:1) δ 0.75-1.24 (m, 20 H), 1.05 (s, 6.8 H), 1.07 (s, 2.2
H), 1.19 (d, J ) 6.9 Hz, 3 H), 1.26-1.44 (m, 1 H), 1.48-2.16 (m, 8
H), 2.20-2.63 (m, 4 H), 2.24 (s, 4.5 H), 2.26 (s, 1.5 H), 2.80-3.08
(m, 2 H), 3.02 (s, 2.3 H), 3.10 (s, 0.7 H), 3.24-3.48 (m, 2 H), 3.28 (s,
2.3 H), 3.31 (s, 2.3 H), 3.32 (s, 0.7 H), 3.33 (s, 0.7 H), 3.60-4.22 (m,
5 H), 4.62-4.96 (m, 2 H), 5.11-5.26 (m, 1 H), 6.74-6.92 (m, 1 H),
7.12-7.47 (m, 11 H), 7.56-7.70 (m, 4 H); FABMS m/z (relative
intensity) 985 (MH+, 8), 100 (100); HRFABMS calcd for C57H89N4O8-
Si m/z 985.6450 (MH+), found 985.6468.
Supporting Information Available: 1H NMR spectra of
1
natural 2 and 3; H and 13C NMR spectra of synthetic 2, 3,
C2-epi-2, and C2-epi-3 (10 pages). This material is contained
in many libraries on microfiche, immediately follows this article
in the microfilm version of the journal, can be ordered from
the ACS, and can be downloaded from the Internet; see any
current masthead page for ordering information and Internet
access instructions.
(2S)-3 [) Isodolastatin H (3)]. To a solution of tetrapeptide 16
(417 mg, 0.424 mmol) in acetonitrile (4.2 mL) was added 47%
hydrofluoric acid (7.5 mL, 240 mmol) with stirring at 0 °C. After
being stirred at 0 °C for 30 min, the reaction mixture was poured into
a mixture of ice (25 g) and saturated aqueous NaHCO3 (25 mL), and
the resulting mixture was made basic (ca. pH 11) with Na2CO3 and
JA9519086