Dolastatins. 26. Synthesis and stereochemistry of dolastatin 11

Article abstract of DOI:10.1021/ja963857y

The first synthesis of dolastatin 11, a potent antineoplastic agent from the sea hare Dolabella auricularia, confirmed the proposed structure and established the last configuration in this natural product and in dolastatin 12, majusculamide C, and 57-norm

Full text of DOI:10.1021/ja963857y

J. Am. Chem. Soc. 1997, 119, 2111-2113
2111
Dolastatins. 26. Synthesis and Stereochemistry of Dolastatin
111a
Robert B. Bates,*,† Kennard G. Brusoe,† Jennifer J. Burns,† Sriyani Caldera,†
Wei Cui,† Sanjeev Gangwar,† Michelle R. Gramme,† Kelly J. McClure,†
Gregory P. Rouen,† Heiko Schadow,† Chad C. Stessman,† Stuart R. Taylor,†
Vicky H. Vu,† Gayle V. Yarick,† Jianxing Zhang,† George R. Pettit,‡ and
Roger Bontems‡
Contribution from the Department of Chemistry, UniVersity of Arizona, Tucson, Arizona 85721,
and Cancer Research Institute and Department of Chemistry, Arizona State UniVersity,
Tempe, Arizona 87287-1604
ReceiVed NoVember 6, 1996X
Abstract: The first synthesis of dolastatin 11, a potent antineoplastic agent from the sea hare Dolabella auricularia,
confirmed the proposed structure and established the last configuration in this natural product and in dolastatin 12,
majusculamide C, and 57-normajusculamide C.
Dolastatins 11 (1) and 12 (2), isolated from the sea hare
Dolabella auricularia, inhibit growth of the murine P388
lymphocytic leukemia with ED50 2.7 × 10-3 and 7.5 × 10-2
µg/mL, respectively.1b They were characterized largely by
NMR and MS, with the configurations except in the Ibu and
Map units based on the striking similarities of their NMR
parameters to those of majusculamide C (3)2 and 57-normajus-
culamide C (4),3 closely-related antifungal agents from the blue-
green alga Lyngbya majuscula. The Map configurations were
determined to be 2S,3R in 1-4 by NMR and optical rotatory
dispersion (ORD) comparison of synthetic (2R,3S)-Map with
(2S,3R)-Map (5) from the degradation of majusculamide C (3).4
Until the present work, the stereochemistry was unknown at
the stereogenic center in Ibu, since the aminoketone obtained
from this segment of majusculamide C (3) was racemic after
hydrolysis with HCl.2 We herein report the first dolastatin 11
(1) synthesis. The strategic approach was similar to Shioiri’s
convergent syntheses of didemnins.5
Scheme 1
The synthesis of the natural (2S,3R)-Map stereoisomer (5)
has been reported by two routes.6,7 We first made it using the
five-step method described for its enantiomer4 with minor
changes. Later, we used the eight-step procedure of Jefford
and McNulty,7 because their synthesis, though longer, has
nonvolatile, nonazidic intermediates. Map (5) was readily
converted to Boc-Map (6) for incorporation of this valuable
material late in the synthesis.
The other new amino acid, Ibu (see structure 1), was prepared
in dipeptide 9 as shown in Scheme 1. Attempts to obtain Boc-
L-Ibu by having the acylimidazole prepared from Boc-L-Ala
react with the dianion from isobutyric acid8 failed, presumably
for steric reasons. In the successful route, the acylimidazole
derivative of Boc-L-Ala was allowed to react with benzyl
lithioacetate9 to give â-ketoester 7, which was hydrogenolyzed
at 0 °C to the â-ketoacid. This carboxylic acid was coupled
with L-Ala-O-Bn without isolation to give dipeptide 8, which
was dimethylated to complete the Ibu unit in dipeptide 9. The
dimethylation yield was much better (86 Vs 35%) when the two
equivalents of sodium hydride were added one at a time with a
45 min interval. Benzyl group removal gave highly crystalline
dipeptide 10 for incorporation late in the synthesis.
† University of Arizona.
(3) Mynderse, J. S.; Hunt, A. H.; Moore, R. E. J. Nat. Prod. 1988, 51,
1299.
(4) Bates, R. B.; Gangwar, S. Tetrahedron: Asymmetry 1993, 4, 69.
(5) Hamada, Y.; Kondo, Y.; Shibata, M.; Shioiri, T. J. Am. Chem. Soc.
1989, 111, 669.
(6) Davies, S. G.; Ichihara, O.; Walters, I. A. S. Synlett 1994, 117.
(7) Jefford, C. W.; McNulty, J. HelV. Chim. Acta 1994, 77, 2142.
(8) Pfeffer, P. E.; Silbert, L. S. Liebig’s Ann. Chem. 1970, 35, 262.
(9) Benzyl lithioacetate was prepared by the procedure used by Shioiri5
to make ethyl lithioacetate.
‡ Arizona State University.
X Abstract published in AdVance ACS Abstracts, February 15, 1997.
(1) (a) For the previous contribution in this series refer to Antineoplastic
Agents. 383: G. P. Kalemkerian, X. Ou, S. K. Madan, and G. R. Pettit,
manuscript in preparation. (b) Pettit, G. R.; Kamano, Y.; Kizu, H.; Dufresne,
C.; Herald, C. L.; Bontems, R.; Schmidt, J. M.; Boettner, F. E.; Nieman,
R. A. Heterocycles 1989, 28, 553.
(2) Carter, D. C.; Moore, R. E.; Mynderse, J. S.; Niemczura, W. P.; Todd,
J. S. J. Org. Chem. 1984, 49, 236.
S0002-7863(96)03857-7 CCC: $14.00 © 1997 American Chemical Society

2112 J. Am. Chem. Soc., Vol. 119, No. 9, 1997
Bates et al.
Scheme 2
Scheme 4
Scheme 3
Because its three methyl groups attached to heteroatoms could
be added simultaneously, dipeptide 14 was synthesized as a unit
as shown in Scheme 2. To provide convergence in the synthesis,
the Gly-N-Me-Leu-Gly tripeptide 19 was synthesized as shown
in Scheme 3. Scheme 4 shows the assembly of the units used
to synthesize dolastatin 11 (1). The hydroxyacid Hmp (22) was
prepared from L-Ile by treatment with nitrous acid under
conditions used on L-Leu10 followed by low-temperature re-
crystallization (a previous preparation2 gave a mixture of
diastereomers which was separated by HPLC).
The linear nonadepsipeptide 26 was first prepared from
hexadepsipeptide 23 and a tripeptide acid derived from Map
(5) and dipeptide 10. Since this first approach gave the ester
bond in only a 10% yield, it was replaced by the route in Scheme
4, which gave a 66% yield of ester 24 from acid 6 and alcohol
23 and was not so wasteful of the valuable Map and Ibu units.
The acid/base-sensitive ketone-containing Ibu unit was in-
corporated late in the synthesis to avoid epimerization. An
indication that its chiral center would survive the removal of
the Boc group in the penultimate reaction was that majuscula-
mide C (3) was recovered unchanged upon treatment with CF3-
COOD under the conditions of Boc deprotection.
in the closely related depsipeptides dolastatin 12 (2),1b majus-
culamide C (3),2 and 57-normajusculamide C (4).3
Experimental Section
Solvent and reagent abbreviations used are Bn ) benzyl, Boc )
tert-butyloxycarbonyl, BOP-Cl ) bis(2-oxo-3-oxazoladinyl)phosphinic
chloride,12 CDI ) carbonyldiimidazole, DCC ) N,N′-dicyclohexyl-
carbodiimide, DCU ) N,N′-dicyclohexylurea, EDC ) 1-[3-(dimethyl-
amino)propyl]-3-ethylcarbodiimide hydrochloride, HBTU ) O-benzo-
triazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate,11 Hmb
) (S)-2-hydroxy-3-methylbutanoic acid, Hmp ) (2S,3S)-2-hydroxy-
3-methylpentanoic acid, HOBT ) 1-hydroxybenzotrizole, IBCF )
isobutyl chloroformate, Ibu ) (S)-4-amino-2,2-dimethyl-3-oxopentanoic
acid, LDA ) lithium diisopropylamide, Map ) (2S,3R)-3-amino-2-
methylpentanoic acid, NMM ) N-methylmorpholine, TBTU ) O-ben-
zotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborate,14 TFA
) trifluoroacetic acid, and Z ) benzyloxycarbonyl.
The cyclization of depsipeptide 28 to dolastatin 11 (1) went
best (20-24%) employing HBTU as the peptide bond-forming
reagent;11 with BOP-Cl12 at high dilution, the yield was only
13%.
The synthetic procedure developed to obtain dolastatin 11
(1) offers the first practical route to this important anticancer
drug prospect;13 the yield from sea hares was only (3 ×
10-6)%,1b compared to 0.7-1.7% from the starting materials
in Schemes 1-3. The synthesis required no chromatography
in the early stages; only dolastatin 11 (1) and late intermediates
24 and 26 were purified by chromatography (HPLC). Key
intermediates 6, 10, 14, 19, and 22 were readily purified by
crystallization. The synthesis confirmed the proposed structure
of dolastatin 11 (1)1b and established for the first time the S
configuration for the Ibu unit in this natural product as well as
Reagents were purchased from Sigma-Aldrich Co. except for BOP-
Cl, which was from TCI America, Portland, OR. Most reactions were
carried out under argon, and solvent extracts of aqueous solutions were
dried over anhydrous magnesium sulfate. Column chromatography
employed silica gel (70-230 mesh) from E. Merck (Darmstadt). HPLC
purifications used a 21.4 mm i.d. × 25 cm reversed-phase C-18 column
and elution with water-acetonitrile mixtures of from 0 to 100%
acetonitrile over 30 min, except for the purification of dolastatin 11
(1), for which a 50 min protocol was used. Melting points were
uncorrected. NMR spectra were obtained at 250-500 MHz in CDCl3
unless otherwise noted, with TMS as an internal standard.
(10) Scheibler, H.; Wheeler, A. S. Ber. Dtsch. Chem. Ges. 1911, 44,
2684.
(11) Dourtoglou, V.; Gross, B.; Lambropoulou, V.; Zioudrou, C. Synthesis
1984, 572.
(12) Tung, R. D.; Rich, D. H. J. Am. Chem. Soc. 1985, 107, 4342.
(13) Dolastatin 11 (1) was recently placed in preclinical development
by the National Cancer Institute (Pettit, G. R. J. Nat. Prod. 1996, 59, 812).
(2S,3R)-Map (5). Method A. (3S,4R)-4-Methyl-5-hexen-3-ol4 was
converted to (2S,3R)-Map (5) by the method used to make its
enantiomer4 in comparable yields with the following changes. In the
(14) Erlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.; Carpino,
L. A.; Bienert, M. Tetrahedron Lett. 1993, 34, 4781.

Synthesis and Stereochemistry of Dolastatin 11
J. Am. Chem. Soc., Vol. 119, No. 9, 1997 2113
preparation of (2S,3R)-3-azido-2-methylpentanoic acid, the aqueous
layer was saturated with NaCl before extraction with ether. The
hydrogenation which gave Map (5) was conducted in absolute ethanol
rather than ethyl acetate. After the reaction, the solution was filtered
and the filter paper was washed with ethanol (5 mL) and water (50
mL). Concentration to 0.5 mL by rotary evaporation led (3 d) to
crystals. Washing (2 × 3 mL) with hot absolute ethanol gave colorless
crystals, mp 206-209 °C dec. Anal. Calcd for C6H13NO2: C, 54.94;
H, 9.99; N, 10.68. Found: C, 55.12; H, 10.20; N, 10.71. The
NMR δ 1.31 (3H, d, J ) 7.2 Hz), 1.44 (9H, s), 1.44 (3H, d, J ) 7.2
Hz, Ala-Me), 3.51 and 3.54 (2H, d, J ) 16.0 Hz), 4.30 (1H, p, J ) 7.2
Hz), 4.61 (1H, p, J ) 7.2 Hz, Ala-CH), 5.17 (1H, m), 5.17 and 5.18
(2H, d, J ) 12.5 Hz), 7.35 (5H, m), 7.49 (1H, m). Anal. Calcd for
C20H28N2O6: C, 61.21; H, 7.19; N, 7.14. Found: C, 61.44; H, 7.28;
N, 7.32.
Boc-Ibu-L-Ala-O-Bn (9). To a stirred solution of dipeptide 8 (5.5
g, 14 mmol), iodomethane (60 mL, 420 mmol), and THF (500 mL) at
0 °C was added with gas evolution 60% sodium hydride in mineral oil
(562 mg, 14 mmol). After 45 min at 0 °C, an additional 562 mg portion
of NaH was added. After stirring at 0 °C for 6 h and 25 °C for 16 h,
the solvent was evaporated and the residue dissolved in ethyl acetate
(300 mL). Washing with brine (2 × 100 mL) and evaporating gave
gem-dimethyl derivative 9 (5.05 g, 86%) contaminated with 0.45 g of
mineral oil which was not removed until the next step: 1H NMR δ
1.24 (3H, d, J ) 7.1 Hz), 1.40 (3H, d, J ) 7.2 Hz, Ala-Me), 1.42 (9H,
s), 1.45 (6H, s), 4.60 (1H, p, J ) 7.1 Hz), 4.71 (1H, p, J ) 7.2 Hz,
Ala-CH), 5.10 (1H, m), 5.14 and 5.20 (2H, d, J ) 12.1 Hz), 6.78 (1H,
m), 7.35 (5H, m). MS(EI), m/z 347 (M - t-BuO).
hydrochloride had [R]25 +14.1 (c 0.064, H2O).
D
Method B. The procedure of Jefford and McNulty7 was used but
on a 10-20 times larger scale with the following changes. The (2S,3S)-
2-methyl-3-(tosylamino)butano-4-lactone was purified by recrystalli-
zation from methanol rather than by chromatography. Ethyl (2S,3S)-
4-iodo-2-methyl-3-(tosylamino)butanoate was purified by recrystallization
from ethyl acetate-hexanes rather than by chromatography, but the
overall yield was better if this material was used without either
purification. The tosyl group was removed from (2S,3R)-2-methyl-3-
(tosylamino)pentanoic acid in 90% yield by heating in a pressure bottle
at 65 °C with 33% HBr in acetic acid for 3 d rather than by refluxing
with 48% aqueous HBr at atmospheric pressure for 90 min. (2S,3R)-
Map (5) was obtained from its hydrobromide not with propylene oxide,
but by adjusting the pH to 6.6 with 4 N NaOH, evaporating the water,
triturating the residue with ethanol, and evaporating.
Benzyl (4S)-N-Boc-4-amino-3-oxopentanoate (7). To a cooled (0
°C), stirred solution of Boc-L-Ala (2.0 g, 11 mmol) in THF (12 mL)
was added CDI (1.95 g, 12 mmol), and the solution was stirred at 0 °C
for 30 min and at 25 °C for 2 h. An LDA solution was prepared by
slow addition at -78 °C of 1.6 M n-BuLi (20.7 mL, 33 mmol) to dry
THF (30 mL) and diisopropylamine (4.62 mL, 33 mmol, dried over
molecular sieves). The mixture was warmed to 0 °C for 15 min and
recooled to -78 °C. Benzyl acetate (4.77 mL, 33 mmol, dried over
molecular sieves) was added, the solution was stirred for 1.25 h at -78
°C, and the acylimidazole solution prepared above was cannulated
dropwise into this solution. After stirring at -78 °C for an additional
15 min, 1 N hydrochloric acid (33 mL) was added and the mixture
was warmed to 0 °C, acidified to pH 3 with citric acid, and extracted
with ethyl acetate (3 × 100 mL). The combined solvent extract was
washed successively with 100 mL portions of 5% NaHCO3 and brine.
Evaporation of solvent and recrystallization in a freezer from ethyl
acetate-hexanes (2.5:100 mL) gave ketoester 7 (2.08 g, 58%): mp
51-53 °C: [R]20D -9.3° (c 0.4, CHCl3); 1H NMR δ 1.33 (3H, d, J )
7.2 Hz), 1.44 (9H, s), 3.60 and 3.62 (2H, d, J ) 16.0 Hz), 4.35 (1H,
p, J ) 7.2 Hz), 5.1 (1H, m), 5.18 (2H, br s), 7.36 (5H, m). Anal.
Calcd for C17H23NO5: C, 63.54; H, 7.21; N, 4.36. Found: C, 63.60;
H, 7.28; N, 4.33.
Boc-Ibu-L-Ala-Map-Hmp-Gly-N-Me-L-Leu-Gly-N-Me-L-Val-O,N-
diMe-L-Tyr-O-Bn (26). A solution of TFA salt 25 (70 mg, 0.0796
mmol), dipeptide 10 (26 mg, 0.0796 mmol), TBTU (127 mg, 0.398
mmol), and N,N-diisopropylethylamine (69 µL, 0.40 mmol) in DMF
(2 mL) was stirred for 20 h. Product separation by HPLC gave
depsipeptide 26 (43 mg, 45%): 1H NMR (16 rotamers) δ 0.21, 0.55,
0.71 (d, J ) 6.0-6.3 Hz, Val Me’s), 1.42 (9H, s), 2.37-3.05 (9H, s),
3.75 (3H, s), 5.13 and 5.21 (2H, d, J ) 12.5 Hz), 6.73, 6.84, 7.07, and
7.12 (4H, m), 7.34 (5H, br s), FABMS m/z 1193 [M + 1]+.
Dolastatin 11 (1). A mixture of the salt 28 (141 mg, 0.125 mmol),
DMF (60 mL), HBTU (213 mg, 0.623 mmol), and triethylamine (34
µL, 0.249 mmol) was stirred for 7 h at 25 °C under argon. Evaporation
of the solvent and separation of the residue by HPLC gave dolastatin
11 (1; 25 mg, 20% yield, retention time 45 min): [R]25D -143 (c 0.33,
CH2Cl2); FABMS m/z 985 [M + 1]+. The synthetic dolastatin 11 (1)
1
was identical with the natural product by comparison of H and 13C
NMR spectra, HPLC, tlc, and cancer cell line activity.
Acknowledgment. The University of Arizona group are very
grateful to the Division of Cancer Treatment, Diagnosis and
Centers, NCI, DHHS, the Research Corp., and Bristol-Myers
Squibb Co. for financial aid, to Drs. S. G. Davies, C. W. Jefford,
and J. McNulty for generous gifts of Map (5), to Dr. R. E. Moore
for a sample of majusculamide C (3), and to Dr. T. J. Siahaan
for mass spectra. The Arizona State University-Cancer Research
Institute group are pleased to acknowledge the financial
assistance provided by the Division of Cancer Treatment,
Diagnosis and Centers, NCI, DDHS, the Arizona Disease
Control Research Commission, and the Robert Dalton Endow-
ment Fund as well as other assistance provided by Drs. C. L.
Herald and J. M. Schmidt.
[(4S)-N-Boc-4-amino-3-oxopentanoyl]-L-Ala-O-Bn (8). A solution
of ketoester 7 (6.0 g, 18.7 mmol) in CH2Cl2 (200 mL) was stirred with
5% Pd/C (2 g) under hydrogen (1 atm) at -5 to 0 °C for 3 h (reaction
appeared complete by TLC). The cold mixture was quickly (to avoid
decarboxylation of the intermediate â-ketoacid) filtered through Celite
into a stirred mixture of alanine benzyl ester hydrochloride (4.03 g,
18.7 mmol) and NMM (2.05 mL, 18.7 mmol) in CH2Cl2 (200 mL) at
0 °C. To this mixture was added EDC (3.95 g, 20.6 mmol). After
stirring at 0 °C for 3 h and 25 °C for 16 h, the solvent was evaporated,
the residue was dissolved in ethyl acetate (300 mL), and the solution
was washed successively with 100 mL portions of 10% citric acid,
water, saturated NaHCO3, and brine. Filtration, evaporation, and one
Supporting Information Available: Preparations of com-
pounds 6, 10-25, 27, and 28, proton NMR spectra of natural
and synthetic dolastatin 11 (1) and intermediates 5-28, and
carbon NMR shifts of natural and synthetic 1 (39 pages). See
any current masthead page for ordering and Internet access
instructions.
recrystallization from ethyl acetate-hexanes (25:500 mL) gave dipep-
1
tide 8 (4.65 g, 63%): mp 82-85 °C; [R]20 -4.6° (c 2.0 CHCl3); H
JA963857Y
D