Synthesis of the tubuvaline-tubuphenylalanine (Tuv-Tup) fragment of tubulysin

Article abstract of DOI:10.1021/ol048252i

(Chemical Equation Presented) Advanced intermediates and analogues of tubulysins were prepared in a convergent strategy.

Full text of DOI:10.1021/ol048252i

ORGANIC
LETTERS
2004
Vol. 6, No. 22
4057-4060
Synthesis of the
Tubuvaline-Tubuphenylalanine (Tuv-Tup)
Fragment of Tubulysin
Peter Wipf,* Takeshi Takada,† and Michael J. Rishel
Department of Chemistry, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260
pwipf@pitt.edu
Received August 31, 2004
ABSTRACT
Advanced intermediates and analogues of tubulysins were prepared in a convergent strategy.
A new family of cytostatic peptides, the tubulysins, were
isolated in 2000 by Sasse et al. from the myxobacterial strains
Archangium gephyra and Angiococcus disciformis (Figure
1).1 The tubulysin sequence is related to metabolites of the
compounds for the development of the anticancer drug LU-
103793, which has entered Phase II clinical investigations.4
Tubulysins have also proven to be extraordinarily potent
microtubule-perturbing agents. All tubulysins are highly
active in mammalian cell cultures. Tubulysin D showed an
average IC50 of 0.04 ng/mL in these growth inhibition assays,
compared to 0.4 ng/mL for dolastatin 10.
Recently, Ho¨fle’s group reported the hydrolysis of tubu-
lysin D by hydrochloric acid,5 yielding 3-methylbutyric acid,
formaldehyde, N-methyl-D-pipecolic acid, L-isoleucine, and
the novel amino acids tubuvaline (Tuv) and tubuphenyl-
alanine (Tup) (Figure 2).6
Although the tubulysins boast impressive biological activ-
ity, the lack of water-solubility of the natural products likely
represents a major detriment in their development toward
clinically useful anticancer agents.7 Therefore, we intend to
(1) Sasse, F.; Steinmetz, H.; Heil, J.; Ho¨fle, G.; Reichenbach, H. J.
Antibiot. 2000, 53, 879.
(2) Pettit, G. R. Pure Appl. Chem. 1994, 66, 2271.
(3) Vedejs, E.; Kongkittingam, C. J. Org. Chem. 2001, 7355.
(4) de Arruda, M.; Cocchiaro, C. A.; Nelson, C. M.; Grinnell, C. M.;
Janssen, B.; Haupt, A.; Barlozzari, D. Cancer Res. 1995, 55, 3085.
(5) Ho¨fle, G.; Glaser, N.; Leibold, T.; Karama, U.; Sasse, F.; Steinmetz,
H. Pure Appl. Chem. 2003, 75, 167.
Figure 1. Structures of tubulysins and related peptide antimitotics.
(6) For recent synthetic studies, see: Friestad, G. K.; Marie, J.-C.;
Deveau, A. M. Org. Lett. 2004, 6, 3249.
(7) QikProp (v2.1, Schro¨dinger Inc.: New York, 2003) analysis of
tubulysin D predicts a log S of -7.8 for aqueous solubility, an apparent
Caco-2 permeability of 1.6 nm/s, and a total solvent-accessible surface area
(SASA) of 1324 Å2. These values are considerably outside the 95% range
of drugs.
cyanobacterium Lyngbya majuscula, the dolastatins,2 and
hemiasterlin.3 Dolastatin 10 and dolastatin 15 served as lead
† On sabbatical leave from Sankyo Agro Co., Ltd., Yasu, Shiga, Japan,
2002-2003.
10.1021/ol048252i CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/25/2004

Scheme 1. R-Hydroxy-γ-amino Acid Synthesis
Figure 2. Acidic hydrolysis of tubulysin D.
3 (Scheme 1). The R,â-unsaturated ester derivative 2 was
readily obtained from N-Cbz-(S)-valinol (1) by TEMPO
oxidation9 followed by Wittig condensation. Enoate 2 was
also prepared in moderate yield from N-Cbz-(S)-valine
methyl ester by a one-pot reaction developed by the Knaus
group.10 Hydrogenation of 2 under various conditions,
including 3% Pd/C in ethyl acetate,11 did not afford the
γ-amino acid derivative 3 in good yield but rather led to
lactam formation. To achieve a selective reduction of the
R,â-unsaturated ester without deprotecting the N-Cbz group,
we employed the copper-catalyzed reduction conditions
developed by the Buchwald group.12 Commercially available
rac-BINAP was used in place of the chiral ligand in our
chemoselective reduction. In the presence of rac-BINAP,
tBuONa, CuCl, and PHMS, the γ-amino acid derivative 3
was obtained in good yield (80%). This selective conjugate
reduction represents a convenient method for the synthesis
of various N-Cbz-protected γ-amino acids.
use the natural product mainly as a lead structure for the
design of a focused library of analogues with improved
physicochemical properties.
On the basis of the precedence of the dolastatin congeners,
we also anticipate that the replacement of the N-methyl
pipecolate in tubulysins with an N,N-dimethylvaline group
should provide compounds of comparable activity, further
simplifying the construction of analogues. Our general
synthetic strategy for tubulysin library synthesis is demon-
strated by the preparation of oxazoline, thiazoline, oxazole,
and thiazole derivatives shown in Figure 3. Segments A and
Our approach required the R-hydroxylation of 3. The
Hanessian group has reported a diastereoselective alkylation
of similar γ-amino acid derivatives.13 Presumably, a highly
chelated dianionic species was involved in this conversion.
Accordingly, we investigated analogous reaction conditions
in the enolate hydroxylation. Treatment of 3 with NaHMDS
in THF at -78 °C, followed by the achiral Davis reagent,
gave R-hydroxy derivative 414 as a single diasteromer in 66%
yield. The use of KHMDS, LiHMDS, and LDA gave lower
yields of diastereomerically pure 4 (37-50%). The corre-
sponding N-Boc and N-trifluoroacetyl derivatives were also
subjected to the hydroxylation reaction, but product yields
remained low (30-50%). The diastereomeric purity of 4 was
determined by 1H and 13C NMR analyses. Finally, the
hydroxyl group was protected as the TBDPS ether 5 in good
(8) Davis, F. A.; Chen, B.-C. Chem. ReV. 1992, 92, 919.
(9) Leanna, M. R.; Sowin, T. J.; Morton, H. E. Tetrahedron Lett. 1992,
33, 5029.
(10) (a) Wei, Z. Y.; Knaus, E. E. Org. Prep. Proc. Int. 1994, 26, 243.
(b) Wei, Z. Y.; Knaus, E. E. Tetrahedron Lett. 1994, 35, 2305.
(11) Misiti, D.; Zappia, G.; Monache, G. D. Synthesis 1999, 873.
(12) Appella, D. H.; Moritani, Y.; Shintani, R.; Ferreira, E. M.; Buchwald,
S. L. J. Am. Chem. Soc. 1999, 121, 9473.
Figure 3. Retrosynthetic analysis for tubulysin D analogues.
B are prepared by diastereoselective hydroxylation or
hydrogenation of the corresponding γ-amino acid derivatives.
We envisioned preparing the R-hydroxy-γ-amino acid
derivative 4 by Davis oxidation8 of γ-amino acid derivative
(13) Hanessian, S.; Schaum, R. Tetrahedron Lett. 1997, 38, 163.
(14) All spectral data were in good agreement with the reported data:
Sutherland, A.; Willis, C. L. J. Org. Chem. 1998, 63, 7764.
4058
Org. Lett., Vol. 6, No. 22, 2004

yield by treatment with TBDPSCl and imidazole in DMF at
60 °C. Thus, the R-silyloxy-γ-amino acid methyl ester 5 was
obtained in five steps and 31% yield from valinol 1.
Saponification of 5 in 1 M LiOH in THF/H2O (3:1)15 and
immediate coupling with (S)-serine methyl ester and Good-
man reagent, DEPBT,16 provided dipeptide 6 in 62% yield.
The introduction of the oxazoline moiety was achieved by
cyclodehydration with DAST17 and led to the oxazoline
derivative 7 in 72% yield (Scheme 2).
possessing oxazoline (7), oxazole (8), thiazoline (10), and
thiazole (11) rings were readily accessible from the R-silyl-
oxy-γ-amino acid methyl ester 5. Although this cyclodehy-
dration methodology was well-suited for the synthesis of
tubuvaline analogues, we also sought a more direct method
for the synthesis of authentic tubuvaline. Conversion of
intermediates 4 and 5 to the terminal amides 12a and 12c
proceeded smoothly with anhydrous NH3 in MeOH (Scheme
4). Protection of 12a as the acetate by treatment with acetic
Scheme 2. Cyclodehydration of a Serine Residue Provides
Oxazoline Precursor for Divergent Heterocycle Synthesis
Scheme 4. Modified Hantzsch Approach to Thiazoles 14a and
14b
Alternatively, we explored the thiolysis of 7 to access
sulfur-containing heterocyclic analogues. Exposure to satu-
rated hydrogen sulfide solution in MeOH/Et3N (2:1)18 gave
thioamide 9 in good yield but as a mixture of diastereomers.
In contrast, thiolysis with H2S in MeOH/Et3N (10:1)
produced 9 without epimerization after 3 d at room temper-
ature. Cyclization of 9 with DAST at -78 °C provided
thiazoline 10 in 96% yield. Subsequent dehydrogenation with
BrCCl3 and DBU19 analogous to the synthesis of oxazole 8
gave thiazole 11. Thus, four tubuvaline building blocks
anhydride and pyridine in CH2Cl2 led to 12b, and treatment
with Belleau reagent20 generated thioamides 13a and 13b.
1
No epimerization was detected by H and 13C NMR. With
thioamides 13a and 13b in hand, conversion to thiazoles 14a
and 14b was accomplished in 78% and 70% yield, respec-
tively, by a modified Hantzsch protocol.21 Compared to the
iterative route shown in Scheme 3, this approach reduces
the number of steps for the synthesis of thiazole 11 (i.e., the
methyl ester of 14b) from 5 from five to three and increases
the overall yield from 24% to 41%.
Scheme 3. Conversion of Oxazoline 7 to Oxazole 8,
Thiazoline 10, and Thiazole 11
For the preparation of the tubuphenylalanine building
block 17, N-Boc-(S)-phenylalaninol was oxidized to the
aldehyde with catalytic TEMPO and chain-extended under
Wittig conditions (Scheme 5). Several attempts to hydro-
genate 15 diastereoselectively by Ru-BINAP catalysis22
failed. Thus, saponification of 15, hydrogenation over 10%
Pd/C, and reduction of the mixed anhydride with NaBH4
(15) Wipf, P.; Miller, C. P.; Grant, C. M. Tetrahedron 2000, 56, 9143.
(16) Li, H.; Jiang, X.; Ye, Y.; Fan, C.; Romoff, T.; Goodman, M. Org.
Lett. 1999, 1, 91.
(17) Phillips, A. J.; Uto, Y.; Wipf, P.; Reno, M. J.; Williams, D. R. Org.
Lett. 2000, 2, 1165.
(18) Wipf, P.; Uto, Y. J. Org. Chem. 2000, 65, 1037.
(19) Williams, D. R.; Lowder P. D.; Gu, Y,-G., Brooks, D. A.
Tetrahedron Lett. 1997, 38, 331.
(20) Belleau reagent, 2,4-bis(4-phenoxyphenyl)1,3-dithia-2,4-phosphe-
tane-2,4-disulfide, is a more soluble version of Lawesson reagent: (a) Lajoie,
G.; Le´pine, F.; Maziak, L.; Belleau, B. Tetrahedron Lett. 1983, 24, 3815.
(b) Sone, H.; Kondo, T.; Kiryu, M.; Ishiwata, H.; Ojika, M.; Yamada, K.
J. Org. Chem. 1995, 60, 4774.
(21) Schmidt, U.; Gleich, P.; Griesser, H.; Utz, R. Synthesis 1986, 992.
(22) Kitamura, M.; Tokunaga, M.; Noyori, R. J. Org. Chem. 1992, 57,
4053.
Org. Lett., Vol. 6, No. 22, 2004
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Scheme 5. Preparation of Tubuphenylalanine Segment
Scheme 6. Segment Condensations
gave 16 as a mixture of diastereomers (anti:syn ) 3:1). The
spectral data for the minor isomer were in good agreement
with an authentic sample of this isomer prepared by a known
method.13 After separation by chromatography on SiO2, the
anti-isomer 16a was silylated and N-deprotected to give the
primary amine 17.
Completion of the synthesis of Tuv-Tup analogues 22 and
23 and the authentic Tuv-Tub subunit 25 of tubulysin D
followed a parallel reaction scheme. Saponification and
DEPBT coupling with 17 led to amides 18-20 (Scheme 6).
The secondary alcohol in 20 was reacetylated in high yield.
Cleavage of the silyl ethers with HF/pyridine led to the
oxazole and thiazole building blocks 22 and 23. Oxidation
of thiazole acetate 24 with TEMPO and NaOCl2/NaOCl23
led to the N-protected Tuv-Tup segment 25. This acid was
isolated in 60% yield as a 9:1 mixture, and further separation
by reverse-phase HPLC provided pure 25 in 23% yield.
In conclusion, we have developed a diastereoselective
hydroxylation of γ-amino acid ester 3 with Davis reagent
that provides access to interesting γ-amino-R-hydroxy acid
derivatives.24 Tubuvaline and Tuv-Tup segments possessing
oxazoline (7), oxazole (22), thiazoline (10), and thiazole
(23-25) rings were readily prepared from the key intermedi-
ates 5 and 17. Further work toward N-terminal extended
analogues of tubulysin D and biological evaluation of these
derivatives is in progress.
Acknowledgment. This work has been supported by a
grant from NIH (GM-55433).
Supporting Information Available: Experimental pro-
cedures and spectral data for all new compounds, including
copies of 1H and 13C NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
(23) Zhao, M.; Li, J.; Mano, E.; Song, Z.; Tschaen, D. M.; Grabowski,
E. J. J.; Reider, P. J. J. Org. Chem. 1999, 64, 2564.
(24) (a) Honore, T.; Hjeds, H.; Krogsgaard-Larsen, P.; Christiansen, T.
R. Eur. J. Med. Chem. 1978, 13, 429. (b) Yokomatsu, T.; Yuasa, Y.;
Shibuya, S. Heterocycles 1992, 33, 1051. (c) Brenner, M.; Seebach, D.
HelV. Chim. Acta 2001, 84, 1181.
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