Antineoplastic agents. 590. X-ray crystal structure of dolastatin 16 and syntheses of the dolamethylleuine and dolaphenvaline units

Article abstract of DOI:10.1021/np100877h

Three advances necessary to bring dolastatin 16 (1) into full-scale preclinical development as an anticancer drug have been accomplished. The X-ray crystal structure of dolastatin 16 has been solved, which allowed stereoselective syntheses of its two new

Full text of DOI:10.1021/np100877h

ARTICLE
pubs.acs.org/jnp
Antineoplastic Agents. 590. X-ray Crystal Structure of Dolastatin 16
and Syntheses of the Dolamethylleuine and Dolaphenvaline Units†
George R. Pettit,* Thomas H. Smith, Jun-Ping Xu, Delbert L. Herald, Erik J. Flahive, Collin R. Anderson,
Paul E. Belcher, and John C. Knight
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe,
Arizona 85287-1604, United States
S Supporting Information
b
ABSTRACT: Three advances necessary to bring dolastatin 16
(1) into full-scale preclinical development as an anticancer drug
have been accomplished. The X-ray crystal structure of dolas-
tatin 16 has been solved, which allowed stereoselective synth-
eses of its two new amino acid units, dolamethylleuine (Dml)
and dolaphenvaline (Dpv), to be completed. The X-ray crystal
structures of synthetic Z-Dml and TFA-Dpv have also been
completed.
ery early in the discovery of the biologically remarkable and
structurally unique peptides from the sea hare Dolabella
V
auricularia, which we designated dolastatins, it became clear that
certain members (e.g., 10ꢀ15) exhibited a variety of important
properties that include anticancer2 and antifungal activities.3
Indeed, dolastatin 10 and three structural modifications are
currently in human cancer phase II and phase III clinical trials.2a
Two derivatives of dolastatin 15 are also in cancer clinical trials
(phase IꢀII).2a
When we extended our field collections of D. auricularia from
the Indian Ocean to the Western Pacific (Papua New Guinea and
the Philippines), we were able to expand the dolastatin series to
16ꢀ19.4 Dolastatin 16 (1)4a especially proved to be an excep-
tionally potent inhibitor of cancer cell growth and a candidate for
further development. However, the latter important initiative has
been delayed by the need for unequivocal configurational assign-
ments and a practical total synthesis of dolastatin 16. We are
pleased to report herein the X-ray crystal structure of dolastatin
16 (1) and syntheses of the new amino acid units dolamethyl-
leuine (2) and dolaphenvaline (3).
will require a practical total synthesis for scale-up production.
However, the microalgae investigations continue to be very
productive and promising for the future.
Other options for obtaining certain dolastatin members
appeared likely some 35 years ago when we considered2d that
Dolabella species derived nutrition by consuming marine micro-
algae and that such exogenous sources might be providing the
dolastatins or intermediates. This expectation has been amply
realized over the past decade by the isolation of dolastatins
10ꢀ164a,5 or close analogues from the cyanobacterium Lyngbya
majuscula and other such microalgae. Thus, fermentation meth-
ods using marine cyanobacteria may eventually be competitive
with total syntheses for scale-up production of new anticancer
drugs in the family. At present, the yields from these initial
experiments remain very low, and for the foreseeable future the
provision of dolastatin 16 for cancer clinical trial development
The three most obvious challenges to finding a useful synth-
esis of dolastatin 16, namely, an X-ray crystal structure to confirm
the configuration and convenient stereoselective syntheses of the
new amino acid units 2 and 3, have been met as follows.
Dolastatin 16 was originally isolated (in 3.1 ꢁ 10ꢀ7% yield) as
an amorphous powder, and a long period of attempts at crystal-
lization were unsuccessful. Eventually, we found that very slow
(over three years) crystal formation from acetonitrile and water
provided X-ray quality crystals. Structurally, dolastatin 16 is a
Received: December 7, 2010
Published: May 03, 2011
r
Copyright
2011 American Chemical Society and
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Figure 1. X-ray structure of dolastatin 16 (1). The atoms of this cyclic depsipeptide and solvent (one acetonitrile and three water) molecules are
displayed as 30% probability thermal ellipsoids.
Scheme 1
Figure 2. X-ray structure of N-Z-dolamethylleuine (8). Atoms are
displayed as 30% probability thermal ellipsoids.
cyclodepsipeptide containing two new amino acids, dolamethyl-
the ArndtꢀEistert reaction6 followed by a Wolff rearrangement7
leuine (Dml, 2), a β-amino acid, and dolaphenvaline (Dpv, 3). As
reported previously,4a the structure of 1 without assignment of
the configuration of the novel amino acids was achieved by high-
field NMR and tandem MS/MS mass spectroscopic interpreta-
tions. X-ray crystallographic analysis of 1 has now confirmed its
cyclodepsipeptide structure and permitted the configurational
assignments of the novel amino acids as 2R,3R for 2 and 2S,3R for
3 (Figure 1).
Synthesis of the β-amino acid dolamethylleuine 2 as its Z-
protected synthon was carried out in four steps as outlined in
Scheme 1 (13% overall yield). With Z-R-valine (4) as substrate,
of the resulting diazoketone 5 afforded the protected β-amino
acid 6. Methylation at the R-position was accomplished stereo-
selectively with LDA and iodomethane to afford 7.6,8 Deprotec-
tion of the tert-butyl ester by use of trifluoroacetic acid (TFA)
and triethylsilane (TES)9 in DCM provided Z-Dml (8). This
crystalline acid was subjected to X-ray crystallography, which
confirmed the desired configuration (Figure 2).
Dolaphenvaline (3) was later reported by Scheuer10 as a
constituent of kulokekahilide-1, a cyclodepsipeptide from the
cephalaspidean mollusk Philinopsis speciosa. As part of the
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Scheme 2
Figure 3. X-ray structure of N-trifluoroacetyldolaphenvaline (20).
Atoms are displayed as 30% probability thermal ellipsoids.
that sequence, and 10 seemed to be a viable starting point for
the synthesis of N-trifluoroacetyldolaphenvaline, as outlined in
Scheme 2.
However, while this approach appeared feasible on a pilot
scale, it had deficiencies in terms of lack of convergence and
potential scale-up problems. Therefore, we explored the more
convergent approach outlined in Scheme 3, beginning with the
DCC/DMAP-mediated condensation of N-trifluoroacetylgly-
cine (11) with 1213 to provide allylic ester 13 in 87% yield.
Claisen rearrangement of 13 with LHMDS in the presence of
Al(O-i-Pr)3 and quinidine afforded the γ,δ-unsaturated amino
acid 14. After methylation with TMSCHN2, methyl ester 15 was
subjected to oxidative cleavage of the double bond by reaction
with OsO4 followed by NaIO4 to afford ketone 17 in reasonable
yield. However, this material was contaminated by a difficultly
separable byproduct tentatively identified as the intermediate
diol on the basis of its NMR spectrum. Despite extensive
experimentation, it was not feasible either to drive this reaction
to completion or to obtain a pure sample of 17. Attempts to
selectively remove the ketone group of 17 via the NaCNBH3/
ZnI214 procedure did not lead to the desired reduced product but
rather a mixture of lactones (19) that presumably arise from
cyclization of an intermediate alcohol.
Scheme 3
With the intent of avoiding lactone formation, the use of the
tert-butyl ester for carboxyl protection was explored. Reaction of
14 with tert-butyl acetoacetate and a catalytic amount of H2SO4
in a sealed vessel15 gave tert-butyl ester 16. An attempt to carry
out the oxidative cleavage reaction via the procedure used for 15
failed with 16 as the substrate. However, a two-step procedure
using N-methylmorpholine N-oxide (NMO) as co-oxidant16 was
successful and yielded 18 cleanly in 70% yield. An unexpected
bonus of the tert-butyl ester approach is that both 16 and 18
proved to be nicely crystalline solids, whereas methyl esters 15
and 17 were obtained as oils. Interestingly, the NMO-mediated
oxidative cleavage approach successful with 16 failed with the
methyl ester analogue 15. With the tert-butyl ester ketone 18 in
structure elucidation of kulokekahilide-1, all four diastereoi-
somers of dolaphenvaline were prepared via a non-stereospecific
approach. Since we required a stereocontrolled synthesis, an
attractive approach to inducing the required chirality appeared to
be the Claisen rearrangement of allylic esters of protected amino
acids in the presence of chiral ligands.11,12 The reported re-
arrangement of allyl ester 9 followed by methylation led to the
γ,δ-unsaturated amino acid methyl ester 10 in high yield, with
excellent stereoselectivity and reproducibility.12 We repeated
14
hand, the NaCNBH3/ZnI2 deoxygenation was attempted.
Again, as with methyl ester 17, the lactone mixture 19 was the
main product. However, since the epimeric lactones (19) were
an intermediate reduction product, the reductive process was
completed via transfer hydrogenolysis of 19 with 1,4-cyclohex-
adiene and Pd/C to afford protected dolaphenvaline 20 (22.6%
overall yield via 16). This crystalline acid was subjected to X-ray
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crystallography, which confirmed the desired configuration
(Figure 3).
The unequivocally established configuration as well as the
preceding stereoselective syntheses of protected Dml and Dpv
have allowed our total synthetic approaches to scale-up prepara-
tion of dolastatin 16 (1) to proceed nicely, and this will be
reported when complete.
322.2041 [M þ H]þ (calcd for C18H28NO4, 322.2018); anal. C 67.05,
H 8.44, N 4.40%, calcd for C18H27NO4, C 67.26, H 8.47, N 4.36%.
tert-Butyl (2R,3R)-3-N-Z-amino-2,4-dimethylpentanoate (7). To a
stirred mixture of dipyridyl indicator, LiCl (0.77 g, 18 mmol), and
diisopropylamine (2.0 mL, 14 mmol) in THF (30 mL) at ꢀ78 °C under
N2 was added BuLi (1.6 M solution in hexane, 8.75 mL, 14 mmol) dropwise
until the mixture turned a wine-red color. The mixture was stirred
at ꢀ78 °C for 15 min, and 6 (1.90 g, 5.9 mmol) in THF (15 mL) was
added. The reaction mixture was stirred for 1 h, followed by the addition
of iodomethane (1.9 mL, 31 mmol). Stirring was continued for 21 h at
ambient temperature. The reaction was terminated by the addition of
saturated NH4Cl (30 mL), and the mixture was extracted with EtOAc
(150 mL). The extract was washed with 10% Na2S2O3 (30 mL), and the
washing was back-extracted with EtOAc (100 mL). The organic solu-
tions were combined, dried, and evaporated. The residue was further
separated by chromatography on silica gel (60 g, 9:1 hexaneꢀacetone)
to yield 1.60 g (80%) of 7 as a colorless oil: Rf 0.44 (9:1 hexaneꢀ
acetone); [R]23D þ22 (c 0.70, CHCl3); 1H NMR δ 7.33 (5H, m), 5.62
(1H, d, J = 7.2 Hz), 5.10 (2H, s), 3.44 (1H, m), 2.66 (1H, m), 1.71 (1H,
m), 1.42 (9H, s), 1.18 (3H, d, J = 7.2 Hz), 0.96 (3H, d, J = 6.6 Hz), 0.92
(3H, d, J = 6.6 Hz); 13C NMR δ 174.6, 156.4, 136.4, 127.9, 80.3, 65.9,
59.0, 40.7, 31.4, 27.5, 19.3, 18.7, 15.3; HRMS m/z 336.2155 [M þ H]þ
(calcd for C19H30NO4, 336.2175); anal. C 68.16, H 8.91, N 4.47%, calcd
for C19H29NO4, C 68.03, H 8.71, N 4.18%.
’ EXPERIMENTAL SECTION
General Experimental Procedures. All starting reagents were
used as purchased unless otherwise stated. Reactions were monitored by
TLC on Analtech silica gel GHLF uniplates visualized under long- and
short-wave UV irradiation and stained with H2SO4/heat, phosphomo-
lybdic acid/heat, or KMnO4/heat. Solvent extracts were dried over
anhydrous sodium sulfate. Where appropriate, the crude products were
separated by flash chromatography on silica gel (230ꢀ400 mesh ASTM)
from E. Merck.
Melting points are uncorrected and were determined employing an
1
Electrothermal Mel-Temp apparatus. The H and 13C NMR spectra
were recorded employing Varian Gemini 300, Varian Unity 400, or
Varian Unity 500 instruments in CDCl3 unless otherwise indicated.
HRMS data were recorded with a JEOL LCmate or JEOL GCmate mass
spectrometer. Elemental analyses were determined by Galbraith La-
boratories, Inc., Knoxville, TN. X-ray structure analyses were performed
on a Bruker AXS Smart 600 diffractometer. The X-ray data have been
submitted as Supporting Information.17 Descriptions of the X-ray
techniques utilized in our laboratory have been previously described.18
1-Diazo-2-oxo-(3R)-3-benzyloxycarbonylamino-4-methylpentane
(5). A solution of Z-R-valine (4, 1.01 g, 3.98 mmol) and TEA (0.57 mL,
415 mg, 4.11 mmol) in THF (20 mL) under N2 was cooled to ꢀ15 °C.
Ethyl chloroformate (0.39 mL, 446 mg, 4.11 mmol) in THF (4 mL) was
added, and the solution stirred at ꢀ15 °C for 30 min. The solution was
filtered and the precipitate washed with THF (10 mL). The combined
filtrate and washings were diluted with acetonitrile (20 mL) and cooled
to 0 °C under N2. Trimethylsilyldiazomethane (4 mL of a 2 M solution
in hexane, 8 mmol) was added, and the solution stirred at ambient
temperature for 18 h. The reaction mixture was diluted with ether
(80 mL), washed successively with 10% citric acid (50 mL), saturated
NaHCO3 (50 mL), and 5 M NaCl (20 mL), dried, evaporated, and co-
evaporated with toluene (15 mL). The residue was separated by
chromatography on silica gel (30 g, 7:3 hexaneꢀEtOAc) to afford
0.37 g (34%) of 5 as a pale yellow solid: mp 68ꢀ69 °C; Rf 0.21 (4:1
hexaneꢀEtOAc); [R]23D þ25 (c 1.10, CHCl3); 1H NMR δ 7.33 (5H,
m), 5.39 (2H, br s), 5.11 (2H, s), 4.13 (1H, m), 2.09 (1H, heptet), 0.99
(3H, d), 0.89 (3H, d); anal. C 61.31, H 6.56, N 14.93%, calcd for
C14H17N3O3, C 61.08, H 6.22, N 15.26%.
tert-Butyl (3S)-3-Z-amino-4-methylpentanoate (6). Diazo deriva-
tive 5 (0.602 g, 2.19 mmol) was dissolved in t-BuOH (9 mL) under N2 at
70 °C. Silver benzoate (80.2 mg, 0.35 mmol) in TEA (0.94 mL, 685 mg,
6.70 mmol) was added dropwise, and the mixture stirred at 70 °C in the
dark for 4 h. The mixture was allowed to cool and was filtered through
Celite, and the solvent was evaporated. The residue was partitioned
between EtOAc (100 mL) and saturated NaHCO3 (20 mL). The
organic phase was separated, washed with saturated NaHCO3
(20 mL), H2O (20 mL), and 5 M NaCl (20 mL), and dried, and the
solvent was evaporated. The residue was chromatographed (silica gel, 23
g; 9:1 hexaneꢀacetone) to provide 0.443 g (63%) of 6 as a colorless oil:
Rf 0.46 (5:1 hexaneꢀacetone); [R]23D þ22 (c 1.20, CHCl3); 1H NMR δ
7.34 (5H, m), 5.12 (1H, d), 5.09 (2H, s), 3.81 (1H, qt), 2.45 (1H, dd, J =
5, 15 Hz), 2.37 (1H, dd, J = 7, 15 Hz), 1.81 (1H, m), 1.42 (9H, s), 0.93
(3H, d, J = 3 Hz), 0.91 (3H, d, J = 3 Hz); 13C NMR δ 170.5, 155.5, 136.2,
127.9, 127.5, 80.4, 66.0, 53.3, 37.9, 31.5, 27.5, 18.7, 18.0; HRMS m/z
(2R,3R)-3-Z-Amino-2,4-dimethylpentanoic Acid (8). To a solution of
7 (1.6 g, 4.8 mmol) in DCM (9.9 mL) under N2 was added a mixture of
trifluoroacetic acid (4.6 mL, 62 mmol) and triethylsilane (1.9 mL, 12
mmol). Stirring was continued for 4 h at ambient temperature. Solvents
were removed and the residue co-evaporated with toluene (2 ꢁ 30 mL).
The residue was dissolved in EtOAc (100 mL) and extracted with 6%
NaHCO3 (4 ꢁ 40 mL). The aqueous extracts were combined, acidified
(pH 2) with 6 N HCl, and extracted with EtOAc (3 ꢁ 40 mL). The
organic extracts were combined, washed with 5 M NaCl (20 mL), dried,
and evaporated to provide a colorless solid that crystallized from
2-propanolꢀwater to provide Z-Dml (8, 1.0 g, 77%) as colorless crystals:
mp 135 °C; Rf 0.59 (50:50:1 hexaneꢀacetoneꢀHOAc); [R]25D þ35 (c
0.86, CHCl3); 1H NMR δ 7.34 (5H, m), 5.61 (1H, d, J = 10.5 Hz), 5.11
(2H, s), 3.46 (1H, m), 2.83 (1H, m), 1.77 (1H, m), 1.25 (3H, d, J = 7.2
Hz), 0.96 (3H, d, J = 6.6 Hz), 0.93 (3H, d, J = 6.6 Hz); 13C NMR δ 179.4,
156.6, 136.2, 127.9, 127.5, 127.4, 66.1, 58.8, 39.8, 19.4, 18.8, 15.3; HRMS
m/z 280.1558 [M þ H]þ (calcd for C15H22NO4, 280.1549); anal. C
64.69, H 7.73, N 4.96%, calcd for C15H21NO4, C 64.50, H 7.58, N 5.01%.
(E)-2-Phenylbut-2-enyl 2-(2,2,2-Trifluoroacetamido)acetate (13).
To a suspension of N-trifluoroacetylglycine (11, 3.68 g, 21.50 mmol)
and (E)-2-phenyl-2-buten-1-ol (12, 2.68 g, 19.32 mmol) in DCM
(60 mL) at ꢀ40 °C under N2 was added via cannula a solution of
dicyclohexylcarbodiimide (4.43 g, 21.50 mmol) and 4-dimethylamino-
pyridine (0.269 g, 2.15 mmol) in DCM (60 mL). The solution was
stirred at ambient temperature for 18 h and filtered, and the precipitate
was washed with DCM (2 ꢁ 40 mL). The combined filtrate and washing
was washed with 10% citric acid (2 ꢁ 25 mL), H2O (10 mL), 6%
NaHCO3 (2 ꢁ 25 mL), and 5 M NaCl (20 mL) and dried, and the
solvent was evaporated. The residue was chromatographed (silica gel,
150 g; 4:1 hexaneꢀEtOAc) to afford 5.06 g (87%) of 13 as a pale yellow
oil that solidified on standing: mp 49ꢀ50 °C; Rf 0.66 (4:1
1
hexaneꢀEtOAc); H NMR δ 7.37 (2H, t, J = 7.5 Hz), 7.30 (1H, t,
J = 7.2 Hz), 7.18 (2H, d, J = 7.6 Hz), 6.75 (1H, br s), 5.95 (1H, q, J = 6.9
Hz), 4.91 (2H, s), 4.06 (2H, d, J = 4.9 Hz), 1.66 (3H, d, J = 6.9 Hz); 13C
NMR δ 167.9, 156.7 (m), 137.3, 135.4, 135.4, 128.5, 128.4, 127.4, 116.9,
70.8, 41.4, 14.6; MS APCIþ m/z 302.1026 [M þ H]þ (calcd for
C14H15F3NO3, 302.1004); anal. C 55.73, H 4.93, N 4.67%, calcd for
C14H14F3NO3, C 55.82, H 4.68, N 4.65%.
3-Methyl-4-phenyl-2-(2,2,2-trifluoroacetamido)-(2S,3R)-pent-4-
enoic Acid (14). To a solution of hexamethyldisilazane (6.19 g, 8.0 mL,
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38.5 mmol) in THF (20 mL at ꢀ20 °C under N2) was added BuLi (1.6
M solution in hexane, 20 mL, 32 mmol). The solution was stirred
at ꢀ20 °C for 20 min and added via cannula to a suspension of 13 (2.00
g, 6.64 mmol), quinidine (4.30 g, 13.27 mmol), and aluminum isoprop-
oxide (2.04 g, 10.0 mmol) in THF (70 mL) at ꢀ78 °C. The solution was
allowed to come to ambient temperature, and stirring was continued for
18 h. The mixture was next diluted with EtOAc (250 mL) and washed
with 1 N HCl (3 ꢁ 75 mL). The combined washings were extracted with
EtOAc (50 mL). The organic solutions were combined and extracted
with 6% NaHCO3 (7 ꢁ 50 mL). The aqueous extracts were combined,
cooled in an ice bath, acidified (pH 1) with 6 N HCl, and extracted with
EtOAc (4 ꢁ 50 mL). The organic extracts were combined, washed with
5 M NaCl (20 mL), dried, and evaporated to give 1.54 g (77%) of 14 as a
led to 0.632 g (70%) of 18 as a colorless solid: mp 127ꢀ128 °C; Rf 0.19
1
(95:5 hexaneꢀEtOAc); [R]23 ꢀ39.0 (c 1.05, CH3OH); H NMR δ
D
7.92 (2H, dd, J = 7.5 and 1.3 Hz), 7.60 (1H, tt, J = 7.5 and 1.3 Hz), 7.49
(2H, t, J = 7.6 Hz), 7.03 (1H, br d, J = 6.7 Hz), 4.72 (1H, dd, J = 7.4 and
4.8 Hz), 4.13 (1H, m), 1.49 (9H, s), 1.36 (3H, d, J = 7.2 Hz); 13C NMR δ
200.0, 168.3, 156.9 (q), 135.5, 133.6, 128.8, 128.4, 83.8, 55.0, 42.9, 27.8,
14.2; MS APCIþ m/z 360.1417 [M þ H]þ (calcd for C17H21F3NO4,
360.1423); anal. C 56.39, H 5.60, N 3.96%, calcd for C17H20F3NO4, C
56.82, H 5.61, N 3.90%.
3-N-(20,20,20-Trifluoroacetamido)-4-methyl-2-oxo-5-phenyl-(3S,4S)-
tetrahydrofuran (19). To ketone 18 (331.2 mg, 0.92 mmol) in 1,2-
dichloroethane (5.0 mL) under N2 were added ZnI2 (440.2 mg, 1.38
mmol) and NaCNBH3 (434.7 mg, 6.90 mmol). The mixture was stirred
at 75 °C for 16 h, quenched with 9:1 saturated NH4Clꢀ6 N HCl
(20 mL), and extracted with EtOAc (3 ꢁ 15 mL). The extracts were
combined, washed successively with 6% NaHCO3 (2 ꢁ 15 mL) and 5 M
NaCl (10 mL), and dried. After evaporation of solvent, the residue was
chromatographed (silica gel, 10 g; 4:1 hexaneꢀEtOAc) to afford 0.123 g
1
pale yellow semisolid: Rf 0.76 (95:5:1 DCMꢀCH3OHꢀHOAc); H
NMR δ 9.10 (1H, br), 7.33 (5H, m), 6.42 (1H, d, J = 8.2 Hz), 5.37 (1H,
m), 5.15 (1H, s), 4.68 (1H, dd, J = 8.6 and 3.4 Hz), 3.57 (1H, m), 1.31
(3H, d, J = 7.2 Hz); 13C NMR δ 174.8, 156.6 (m), 148.6, 140.8, 128.6,
128.2, 126.7, 115.1, 55.0, 39.5, 14.0; MS APCIþ m/z 302.1010 [M þ
H]þ, (calcd for C14H15F3NO3, 302.1004).
1
(47%) of 19 as a white, waxy solid: Rf 0.32 (4:1 hexaneꢀEtOAc); H
Methyl 3-Methyl-4-phenyl-2-(2,2,2-trifluoroacetamido)-(2S,3R)-
pent-4-enoate (15). Carboxylic acid 14 (544.4 mg, 1.81 mmol) was
placed in 1:1 CH3OHꢀtoluene (6 mL) under N2, and trimethylsilyl-
diazomethane (2 M solution in hexane, 4.0 mL, 8.0 mmol) was added.
The solution was stirred at ambient temperature for 16 h. The solvent
was evaporated, and the residue was chromatographed (silica gel, 17 g;
9:1 hexaneꢀEtOAc) to yield 0.46 g (81%) of 15 as a colorless oil: Rf 0.54
(4:1 hexaneꢀEtOAc); 1H NMR δ 7.33 (5H, m), 6.48 (1H, br d), 5.34
(1H, s), 5.11 (1H, d, J = 0.8 Hz), 4.64 (1H, dd, J = 8.7 and 4.0 Hz), 3.76
(3H, s), 3.48 (1H, m), 1.26 (3H, d, J = 7.1 Hz).
tert-Butyl 3-Methyl-4-phenyl-2-(2,2,2-trifluoroacetamido)-(2S,3R)-
pent-4-enoate (16). To carboxylic acid 14 (1.54 g, 5.12 mmol) in a
50 mL round-bottom flask were added tert-butyl acetoacetate (5.8 mL,
5.53 g, 34.97 mmol) and H2SO4 (43.1 mg, 0.44 mmol). The flask was
tightly stoppered, and the solution was stirred at ambient temperature
under N2 for 20 h. The mixture was cooled (ice) before dilution with
EtOAc (100 mL). The organic solution was washed with 6% NaHCO3
(4 ꢁ 20 mL) and 5 M NaCl (10 mL) and dried, and the solvent was
evaporated. The NaHCO3 washings were combined, acidified (pH 1)
with 6 N HCl, and extracted with EtOAc (3 ꢁ 15 mL). The extracts were
combined, washed with 5 M NaCl (10 mL), and dried, and the solvent
was evaporated to afford 0.65 g (42%) of 14. The neutral residue was
chromatographed (silica gel, 30 g, 95:5 hexaneꢀEtOAc) and led to 1.06
g (58%, 100% based on recovered starting material) of 16 as a colorless
solid: mp 108 °C; Rf 0.50 (95:5 hexaneꢀEtOAc); [R]23D 25.2 (c 1.04,
CH3OH); 1H NMR δ 7.34 (5H, m), 6.48 (1H, br d, J = 6.8 Hz), 5.33
(1H, s), 5.10 (1H, s), 4.52 (1H, dd, J = 8.4 and 3.5 Hz), 3.47 (1H, m),
1.49 (9H, s), 1.27 (3H, d, J = 7.0 Hz); 13C NMR δ 168.8, 156.5 (m),
149.0, 141.3, 128.5, 127.9, 126.8, 114.7, 83.3, 55.6, 39.9, 28.0, 14.3; MS
APCIþ m/z 358.1681 (0.6) [M þ H]þ (calcd for C18H23F3NO3,
358.1630), 302.0990 (100) [M þ H ꢀ C4H8]þ (calcd for
C14H15F3NO3, 302.1004); anal. C 60.14, H 6.41, N 3.96%, calcd for
C18H22F3NO3, C 60.50, H 6.21, N 3.92%.
NMR δ 7.37 (5H, m), 7.14 (1H, br d), 5.61 (0.33H, d, J = 8.4 Hz), 4.99
(0.67H, d, J = 10.2 Hz), 4.68 (1H, m), 2.99 (0.33 H, m), 2.59 (0.67H, m),
1.22 (0.67H, d, J = 6.6 Hz), 0.87 (0.33H, d, J = 6.9 Hz); HRMS (APCIþ)
m/z 288.0851 [M þ H]þ (calcd for C13H13F3NO3, 288.0848).
(2S,3R)-2-(2,2,2-Trifluoroacetamido)-3-methyl-4-phenyl-2-butanoic
Acid (20). To lactone 19 (74.7 mg, 0.26 mmol) in CH3OH (2.0 mL)
under N2 (cooled to 0 °C) was added 10% Pd/C (75 mg), followed by
1,4-cyclohexadiene (0.21 g, 2.60 mmol), and the mixture was stirred at
ambient temperature under N2 for 4 h. The reaction mixture was diluted
with CH3OH (10 mL), and the solution was filtered through Celite. The
filter cakewas washedwith CH3OH(10mL). The solvent was evaporated
from the combined filtrate and washings, and the residue was dissolved in
EtOAc (20 mL) and extracted with 6% NaHCO3 (3 ꢁ 15 mL). The
organicphase was washed with 5 M NaCl(10 mL) and dried, and removal
of solvent gave 48.5 mg of recovered lactone 19. The NaHCO3 extracts
were combined, acidified (pH 1) with 6 N HCl, and extracted with EtOAc
(3 ꢁ 15 mL). The extracts were combined, washed with 5 M NaCl
(10 mL), and dried, and the solvent was evaporated to afford 22.7 mg
(86% based on recovered starting material) of 20 as a white solid: mp
122 °C; Rf 0.38 (97.5:2.5:0.5 DCMꢀCH3OHꢀHOAc); [R]25D þ24.2 (c
1.49, CH3OH); 1H NMR δ 9.78 (1H, br), 7.31 (2H, t, J = 7.3 Hz), 7.23
(1H, t, J = 7.3 Hz), 7.17 (2H, d, J = 7.1 Hz), 6.64 (1H, d, J = 8.4 Hz), 4.75
(1H, dd, J = 8.8 and 3.2 Hz), 2.76 (1H, dd, J = 12.9 and 6.0 Hz), 2.55 (1H,
m), 2.49 (1H, dd, J = 13.1 and 8.0 Hz), 0.99 (3H, d, J = 6.6 Hz); 13C NMR
δ 175.23, 157.17 (m), 138.58, 128.96, 128.64, 126.71, 115.60 (m), 55.73,
39.64, 37.85, 14.84; MS APCIꢀ m/z 288.0835 [M ꢀ H]ꢀ (calcd for
C13H13F3NO3, 288.0845); anal. C 53.15, H 4.69, N 4.70%, calcd for
C13H14F3NO3 0.2H2O, C 53.32, H 4.96, N 4.78%.
3
’ ASSOCIATED CONTENT
S
Supporting Information. X-ray crystal structure data for
b
dolastatin 16 (1), Z-Dml (8), and trifluoroacetyl-Dpv (20). This
tert-Butyl 3-Methyl-4-oxo-4-phenyl-2-(2,2,2-trifluoroacetamido)-
(2S,3R)-butanoate (18). To olefin 16 (903.4 mg, 2.53 mmol) in THF
(25 mL) under N2 were added NMO (60 wt % solution in H2O,
0.90 mL, 5.06 mmol) and OsO4 (4 wt % solution in H2O, 1.50 mL, 0.25
mmol). The solution was stirred at ambient temperature for 16 h, and
NaIO4 (2.16 g, 10.12 mmol) was then added, followed by H2O
(2.7 mL). Stirring was continued for 4 h, and the reaction mixture was
then diluted with EtOAc (200 mL) and washed with 10% Na2S2O3 (4 ꢁ
50 mL). The combined washings were extracted with EtOAc (50 mL).
The organic solutions were combined and washed with 5 M NaCl
material is available free of charge via theInternetathttp://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@asu.
edu.
’ ACKNOWLEDGMENT
(20 mL) and dried, and the solvent was evaporated. The residue was
We are pleased to acknowledge the very necessary financial
separated by chromatography (silica gel, 30 g; 9:1 hexaneꢀEtOAc) and
support provided by grants RO1 CA 90441-02-05, 2R56-CA
1007
dx.doi.org/10.1021/np100877h |J. Nat. Prod. 2011, 74, 1003–1008

Journal of Natural Products
ARTICLE
090441-06A1, and 5RO1 CA 090441-07 from the Division of
Cancer Treatment and Diagnosis, NCI, DHHS; The Arizona
Disease Control Research Commission; Dr. Alec D. Keith;
J. W. Kieckhefer Foundation; Margaret T. Morris Foundation;
the Robert B. Dalton Endowment Fund; and Dr. William Crisp
and Mrs. Anita Crisp. For other very helpful assistance, we thank
Dr. Fiona Hogan.
(16) Keck, G. E.; Giles, R. L.; Cee, V. J.; Wager, C. A.; Yu, T.; Kraft,
M. B. J. Org. Chem. 2008, 73, 9675–9691.
(17) CCDC 801992 (1), 801990 (8), and 801991 (20) contain the
supplementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
(18) Pettit, G. R.; Knight, J. C.; Herald, D. L.; Pettit, R. K.; Hogan, F.;
Mukku, V. J. R. V.; Hamblin, J. S.; Dodson, M. J., II; Chapuis, J.-C. J. Nat.
Prod. 2009, 72, 366–371.
DEDICATION
†In memory of Academician Georgy B. Elyakov (1929ꢀ2005), a
pioneering expert in the chemistry of marine organism constitu-
ents, who is deeply missed.
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