First total synthesis of tubulysin B

Article abstract of DOI:10.1021/ol902320w

[Chemical Equation Presented] The first total synthesis of tubulysin B is described. The aziridine route to tubuphenylalanine (Tup) of the tubulysin D/U-series could not be transferred to the synthesis of tubutyrosine (blue moiety). Therefore, tubutyrosin

Full text of DOI:10.1021/ol902320w

ORGANIC
LETTERS
2009
Vol. 11, No. 24
5567-5569
First Total Synthesis of Tubulysin B
Orlando Pando,† Simon Do¨rner,† Rainer Preusentanz,† Annika Denkert,†
Andrea Porzel,† Wolfgang Richter,‡ and Ludger Wessjohann*,†
Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3,
D-06120 Halle (Saale), Germany, and R&D Biopharmaceuticals, Martinsried, Germany
wessjohann@ipb-halle.de
Received October 7, 2009
ABSTRACT
The first total synthesis of tubulysin B is described. The aziridine route to tubuphenylalanine (Tup) of the tubulysin D/U-series could not be
transferred to the synthesis of tubutyrosine (blue moiety). Therefore, tubutyrosine (Tut) was synthesized by a Wittig olefination/diastereoselective
catalytic reduction sequence. Interestingly, the C-2 epimer of tubulysin B has a cytotoxic activity almost identical to the natural diastereomer.
Tubulysins comprise a natural product family of highly active
antimitotic tetrapeptides isolated by Ho¨fle’s group from
myxobacteria culture broths.1 At the cell level, they inhibit
tubulin polymerization and induce apoptosis.1,2
Tubulysins are composed of four amino acids (Figure
1): D-N-methyl pipecolic acid (Mep), L-isoleucine (Ile),
tubuvaline (Tuv), which is itself based on two condensed
amino acids, and tubutyrosine (Tut) or tubuphenylalanine
(Tup). In addition, an unsual tertiary amide N,O-acetal ester
is present in the most active tubulysins.
Tubulysins A and D are representative for the basic
structures of the most potent members. Their cell growth
inhibitory activity exceeds that of well-known chemothera-
peutic agents like taxol or vinblastine by 10-fold to more
than 100-fold. Therefore, tubulysins are excellent candidates
for the development of novel anticancer drugs. A significant
drawback for further applications is the limited availability
by fermentation due to a very low production yield.
Although some tubulysins have recently been synthesized
by us and others, either all are simplified analogues or the
synthetic routes contain steps that proved difficult to repeat,
especially under scale up conditions. Most important, all belong
to the D-series with a C-terminal tubuphenylalanine.3,4 In
Figure 1.
Natural tubulysins A, B (with Tut, R1 ) OH), and D
(with Tup, R1 ) H).
contrast to that, so far only tubulysins A and B from the
Tut-series advanced to early preclinical use. Thus, only
tubulysins A and B have been used in various cancer cell
targeting bioconjugate approaches with very encouraging
results. They are also the only tubulysins carried on to animal
experiments.5
(3) (a) Do¨mling, A.; Beck, B.; Eichelberger, U.; Sakamuri, S.; Menon,
S.; Chen, Q. Z.; Lu, Y.; Wessjohann, L. A. Angew. Chem. 2006, 118, 7393.
Do¨mling, A.; Beck, B.; Eichelberger, U.; Sakamuri, S.; Menon, S.; Chen,
Q. Z.; Lu, Y.; Wessjohann, L. A. Angew. Chem., Int. Ed. 2006, 45, 7235.
(b) Do¨mling, A.; Beck, B.; Eichelberger, U.; Sakamuri, S.; Menon, S.; Chen,
Q. Z.; Lu, Y.; Wessjohann, L. A. Angew. Chem. 2007, 119, 2399. Do¨mling,
A.; Beck, B.; Eichelberger, U.; Sakamuri, S.; Menon, S.; Chen, Q. Z.; Lu,
Y.; Wessjohann, L. A. Angew. Chem., Int. Ed. 2007, 46, 2347.
† Leibniz Institute of Plant Biochemistry.
‡ R&D Biopharmaceuticals.
(1) Sasse, F.; Steinmetz, H.; Heil, J.; Ho¨fle, G.; Reichenbach, H. J.
Antibiot. 2000, 53, 879.
(2) Steinmetz, H.; Glaser, N.; Herdtweck, E.; Sasse, F.; Reichenbach,
H.; Ho¨fle, G. Angew. Chem., Int. Ed. 2004, 43, 4888
.
10.1021/ol902320w  2009 American Chemical Society
Published on Web 11/17/2009

The synthesis of Tut-containing tubulysins like tubulysin
A and B has not been accomplished so far. Herein, the first
total synthesis of tubulysin B and its C2-epimer is described.
The Tut subunit turned out to be more challenging than
anticipated. Despite the structural analogy with Tup, the Tut
synthesis could not be accomplished in sufficient yield following
the aziridine route as described previously for the synthesis of
Tup.3 This is quite surprising since the p-hydroxy group is
electronically decoupled; i.e., the phenol is benzylic, not
phenylic. Neither the free hydroxy nor protected forms were
efficient in the aziridine opening reaction. In addition, the Tut
intermediates and the product form the corresponding γ-lactams
more easily than the Tup-analogues. This unexpectedly different
reactivity of phenylethyl (homobenzylic) substituents vs
p-hydroxyphenylethyl was also observed by others.6
The synthesis of the key intermediate, the R,ꢀ-unsaturated
ester 5, was achieved in 44% overall yield by oxidation of the
commercially available N,O-protected tyrosinol 4 to the alde-
hyde followed by a Wittig olefination. Complete epimerization
was observed when a Swern oxidation was carried out instead
of the TEMPO protocol. Ethyl ester cleavage, subsequent
menthyl ester formation, and catalytic hydrogenation (e.g., with
Pd/C, or Noyori catalyst) rendered a mixture of diastereoisomers
that was difficult to separate by flash column chromatography.
Instead, a direct catalytic reduction of the Wittig product over
Pd on charcoal7 affords a mixture of C2-diastereomers (2.5:1
ratio) that is easily resolved by preparative HPLC. Despite
several attemps, crystals suitable for X-ray analysis could not
be obtained, and the unambiguous stereochemical assigment
of the diastereoisomers could not be accomplished at this stage.
Thus, both compounds were submitted to basic hydrolysis directly
followed by removal of the Boc protecting group to afford the
corresponding amino acid salts 7a and 7b (Scheme 1). This
protocol avoids the formation of undesirable γ-lactams.8 The
diastereomeric building blocks were separately carried on
to tubulysin B and its C2-epimer. Gratifyingly, the diaste-
reomer with the natural stereochemistry turned out to be the
major one (in 53% yield), which is in agreement with
previous results in similar systems.6
The convergent synthesis of the tripeptide 13 (Scheme
2) was accomplished starting from the uncommon amino
Scheme 2. Synthesis of Tripeptide 13
Scheme 1
.
Synthesis of the Tubutyrosine (Tut) Hydrochloride
Salts 7a and 7b
acid tubuvaline, which was synthesized featuring a three-
component approach.3 The introduction of the rare N,O-acetal
proved to be a difficult task even following the route
described by Ellman and co-workers.4a In our hands, an
undesired cyclization to the six-membered acetal took place
(4) (a) Peltier, H. M.; McMahon, J. P.; Patterson, A. W.; Ellman, J. A.
J. Am. Chem. Soc. 2006, 128, 16019. (b) Patterson, A. W.; Peltier, H. M.;
Sasse, F.; Ellman, J. A. Chem.sEur. J. 2007, 13, 9534. (c) Patterson, A. W.;
Peltier, H. M.; Ellman, J. A. J. Org. Chem. 2008, 73, 4362. (d) Wipf, P.;
Takada, T.; Rishel, M. J. Org. Lett. 2004, 6, 4057. (e) Raghavan, B.;
Balasubramanian, R.; Steele, J. C.; Sackett, D. L.; Fecik, R. A. J. Med.
Chem. 2008, 51, 1530. (f) Balasubramanian, R.; Raghavan, B.; Begaye,
A.; Sackett, D. L.; Fecik, R. A. J. Med. Chem. 2009, 52, 238. (g) Sani, M.;
Fossati, G.; Huguenot, F.; Zanda, M. Angew. Chem., Int. Ed. 2007, 119,
3596. (h) Ulrich, A.; Chai, Yi.; Pistorius, D.; Elnakady, Y. A.; Herrmann,
J. E.; Weissman, K. J.; Kazmaier, U.; Mu¨ller, R. Angew. Chem., Int. Ed
2009, 48, 4422.
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Org. Lett., Vol. 11, No. 24, 2009

Scheme 3. Synthesis of Tubulysin B (2) and Its C-2 Epimer
after removal of the TES protecting group under acidic
conditions. A mixture of compounds 12a and 12b was
obtained. Several attempts to avoid or separate this byproduct
were not successful. Preparative HPLC allowed some
separation but with a low R-value. Eventually, the crude
mixture of compounds 12 was treated with Me3SnOH to
cleave the methyl ester. Acetylation of the secondary alcohol
was carried out following a standard procedure,4b to afford
the separable tripeptide 13 in 54% overall yield from 12a
after HPLC purification.
Finally, the hydrochloride salts of tubutyrosine and its
epimer (7a and 7b) were coupled to the free acid 13 through
pentafluorophenyl ester formation, to render synthetic tubu-
lysin B (2) and the tubulysin B (2R)-epimer (14) in 67%
and 51% yield, respectively (Scheme 3).
compounds 2 and 14 was evaluated against human cancer
cell lines, using taxol as a reference compound. Interestingly,
there are no significant differences between the GI50 values
of natural, synthetic, and epimeric tubulysin B (Table 1). A
Table 1. Cytotoxic Activity (GI50 Values [nM])
compound
PC-3a
HT-29b
nat. tub B (2)
synt. tub B (2)
(2R)-tub B (14)
taxol
0.3
1.1
0.8
7.2
0.5
1.0
1.4
-
a Human prostate cancer cell line. b Human colon cancer cell line.
Interestingly, the differences of tubulysin B and its C2-
epimer are minimal in almost all aspects. Rf values in TLC
and biological activity (v.i.) are identical within error limits,
and even analytical HPLC retention times and NMR spectra
are very similar. Only a comparative look at the NMR spectra
reveals the differences (see Supporting Information for
details), and a comparison of lists of shift values is not
conclusivessimilar to earlier observations we made in the
tubulysin U/V-series.3
relative importance of the stereocenter at C-2 might be
expected considering earlier reports in another series.9 Thus,
the measurements were repeated in an independent lab which
verified that the activity of both compounds is comparable
to that of the isolated natural metabolite. This fact reveals
that the stereochemistry of the methyl group at C-2 in
tubulysin B is of minor importance for the cytotoxic activity.
In summary, a short, convergent, and stereoselective
synthesis of tubulysin B and its equally active non-natural
C-2 epimer has been performed.
The biological activity of natural tubulysin B (provided
by R&D Biopharmaceuticals) along with that of the synthetic
Acknowledgment. The authors thank Anett Werner for
support with the HPLC analyses and Prof. Bernhard
Westermann for fruitful suggestions (both from the Leibniz
Institute of Plant Biochemistry). We thank Ontochem GmbH
for an independent measurement of HT-29 activities.
Supporting Information Available: Experimental pro-
cedures and spectral data of all new compounds, including
selected copies of 1H and 13C NMR spectra, HPLC
chromatograms, and HRMS (ESI-FT-ICR) spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(5) (a) Schluep, T.; Gunavan, P.; Ma, L.; Jensen, G. S.; Duringer, J.;
Hinton, S.; Richter, W.; Hwang, J. Clin. Cancer Res. 2009, 15, 181. (b)
Leamon, C. P.; Reddy, J. A.; Vetzel, M.; Dorton, R.; Westrick, E.; Parker,
N.; Wang, Yu.; Vlahov, I. Cancer Res. 2008, 68, 9839. (c) Reddy, J. A.;
Dorton, R.; Dawson, A.; Vetzel, M.; Parker, N.; Nicoson, J. S.; Westrick,
E.; Klein, P. J.; Wang, Y.; Vlahov, I. R.; Leamon, C. P. Mol. Pharmaceutics
2009, 6, 1518.
(6) Sherrill, R. G.; Andrews, C. W.; Bock, W. J.; Davis-Ward, R. G.;
Furfine, E. S.; Hazen, R. J.; Rutkowske, R. D.; Spaltenstein, A.; Wright,
L. L. Bioorg. Med. Chem. Lett. 2005, 15, 81.
(7) Ho¨fle, G.; Glasser, N.; Leibold, T.; Karana, U.; Sasse, F.; Steinmetz,
H. Pure Appl. Chem. 2003, 75, 167.
(8) Wipf, P.; Wang, Z. Org. Lett. 2007, 9, 1605.
(9) Balasubramanian, R.; Raghavan, B.; Steele, J. C.; Sackett, D. L.;
Fecik, R. A. Bioorg. Med. Chem. Lett. 2008, 18, 2996.
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