Synthesis of cryptothilone 1, the first cryptophycin-epothilone hybrid

Article abstract of DOI:10.1021/ol0614020

A hybrid structure was synthesized in which one portion is characteristic of a cryptophycin and a second sector bears the signature of an epothilone. The hybrid, in contrast to parent cryptophycin and epothilone systems, showed no effect on the tubulin as

Full text of DOI:10.1021/ol0614020

ORGANIC
LETTERS
2006
Vol. 8, No. 18
3947-3950
Synthesis of Cryptothilone 1, the First
Cryptophycin Epothilone Hybrid
−
James D. White,*,† Helmars Smits,† and Ernest Hamel‡
Department of Chemistry, Oregon State UniVersity, CorVallis, Oregon 97331, and
Toxicology and Pharmacology Branch, DeVelopmental Therapeutics Program, DiVision
of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National
Institutes of Health, Maryland 21702
james.white@oregonstate.edu
Received June 8, 2006
ABSTRACT
A hybrid structure was synthesized in which one portion is characteristic of a cryptophycin and a second sector bears the signature of an
epothilone. The hybrid, in contrast to parent cryptophycin and epothilone systems, showed no effect on the tubulin assembly reaction.
Cryptophycins and epothilones are cytotoxic natural products
of widely different origin, the one emanating from a blue-
green alga1 and the other from a soil bacterium.2 Interestingly,
they both possess tubulin binding properties that inhibit cell
proliferation at mitosis.3,4 Cryptophycins are believed to bind
to the ends of microtubules and, like vinblastine and certain
other antimitotic agents, they disrupt the polymerization
process by which R,â-tubulin heterodimers condense into
aggregates.5
Epothilones, on the other hand, are known to bind to an
interior region of the microtubule at a site close to that which
complexes taxol.6 This site is believed to be located on the
â-tubulin subunit in a location adjacent to the neighboring
protofilament. The epothilones and taxol coordinate to
microtubules in a manner that reduces the rate of R/â-tubulin
dissociation by serving as a bracketing device. This arrange-
ment stabilizes and augments the proportion of tubulin
polymer and prevents nuclear division, leading in turn to
apoptosis (“programmed cell death”). Thus, although both
cryptophycins and epothilones are antimitotic agents which
block cell proliferation by interfering with mitotic spindle
division between the metaphase and anaphase, they operate
at different sites on tubulin.
† Oregon State University.
‡ National Cancer Institute at Frederick.
(1) (a) Schwartz, R. E.; Hirsch, C. F.; Sesin, D. F.; Flor, J. E.; Chartrain,
M.; Fromtling, R. E.; Harris, G. H.; Salvatore, M. J.; Liesch, J. M.; Yudin,
K. J. Ind. Microbiol. 1990, 5, 113. (b) Smith, C. D.; Zhang, X.; Mooberry,
S. L.; Patterson, G. M. L.; Moore, R. E. Cancer Res. 1994, 54, 3779. (c)
Barrow, R. A.; Hemscheidt, T.; Liang, J.; Paik, S.; Moore, R. E.; Tius, M.
A. J. Am. Chem. Soc. 1995, 117, 2479. (d) Golakoti, T.; Ogino, J.; Heltzel,
C. E.; Husebo, T. L.; Jensen, C. M.; Larsen, L. K.; Patterson, G. M. L.;
Moore, R. E.; Mooberry, S. L.; Corbett, T. H.; Valeriote, F. A. J. Am. Chem.
Soc. 1995, 117, 12030. (e) Subbaraju, G. V.; Galakoti, T.; Patterson, G. M.
L.; Moore, R. E. J. Nat. Prod. 1997, 60, 302.
A comparison of the structures of cryptophycins and
epothilones reveals intriguing similarities as well as one
significant difference. Figure 1 shows the structure of natural
cryptophycin 4 and trans-epothilone C, a synthetic analogue
known to have tubulin polymerization properties similar to
(2) (a) Ho¨fle, G.; Bedorf, N.; Gerth, H.; Reichenbach (GBF), DE-B
4138042, 1993; Chem. Abstr. 1993, 120, 52841. (b) Ho¨fle, G.; Bedorf, N.;
Steinmeth, H.; Schomburg, D.; Gerth, H.; Reichenbach, H. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1567.
(3) (a) Koiso, Y.; Morita, K.; Kobayashi, M.; Wang, W.; Ohyabu, N.;
Iwasaki, S. Chem. Biol. Interact. 1996, 102, 183. (b) Morita, K.; Koiso,
Y.; Hasimoto, Y.; Kobayashi, M.; Wang, W.; Ohyabu, N.; Iwasaki, I. Biol.
Pharm. Bull. (Jpn.) 1995, 43, 1598.
(4) (a) Bollag, D. M.; McQueney, P. A.; Zhu, J.; Hensens, O.; Koupal,
L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Cancer Res. 1995,
55, 2325. (b) For a recent review, see: Altmann, K.-H.; Wartmann, M.;
O’Reilly, T. Biochim. Biophys. Acta 2000, 1470, M79.
(5) Hamel, E. Med. Res. ReV. 1996, 16, 207.
(6) For a review of the chemical biology of epothilones, see: Nicolaou,
K. C.; Roschangar, F.; Vourloumis, F. Angew. Chem., Int. Ed. 1998, 37,
2014.
10.1021/ol0614020 CCC: $33.50
© 2006 American Chemical Society
Published on Web 08/12/2006

Figure 2. Hybrid structures of a cryptophycin and an epothilone.
1 (3) contains the upper half of cryptophycin 4 and a lower
half common to most epothilones. Cryptothilone 2 (4) would
reverse this architecture by connecting the lower half of
cryptophycin 4 (1) with an upper sector found in natural
epothilones. Herein, we report the synthesis of 3 and the
results of initial experiments designed to test its effect on
tubulin assembly.
Figure 1. Structural homology of a cryptophycin and an epothilone.
the natural 12,13-cis isomer. Common to both structures are
(i) a 16-membered ring, (ii) an aryl substituent attached to a
conjugated double bond (which is epoxidized in some
cryptophycins), (iii) a methyl substituent at or in close
proximity to the conjugated double bond, (iv) (S) configu-
ration at the oxygen substituent to which the lactone carbonyl
is attached, and (v) an alkene (which is epoxidized in
epothilones A and B) separated from the acyloxy carbon by
one methylene unit. On the other hand, there is one region
of the macrocycle perimeter that is different in these two
structures. The C8-C11 segment of epothilones is relatively
flexible, whereas the cryptophycin sector that would super-
impose on this set of four atoms is quite rigid due to the
two amide linkages. Hydrogen bonding in this peptidic
section of the cryptophycin perimeter imposes a conformation
that is not matched in epothilones.
A hybrid structure that incorporates selected features of 1
and 2 and which also complies with the homologies (i-v)
noted above appeared to be a possible probe for investigating
the different tubulin binding modes characteristic of each of
the parent structures. Issues that could be addressed by this
approach include a better definition of the particular structural
motifs responsible for binding of cryptophycins and
epothilones to different regions of tubulin and whether the
distinction is due to recognition by the tubulin receptor of a
peptidic domain in one case and a lipophilic sector in the
other. Other questions such as whether cryptophycins and
epothilones can be made to exchange binding sites on tubulin
by structural modification or whether structures can be
generated which endow a hybrid molecule with cytotoxic
properties found in both parents (or perhaps in neither
ancestor) can also be addressed, in principle, by this strategy.
In a first attempt to answer these questions, two “cryp-
tothilone” hybrids were conceived (Figure 2). Cryptothilone
Our previous studies on the synthesis of cryptophycins7,8
and epothilones9-12 laid the groundwork for our approach
to the hybrid system 3. Alcohol 5 was prepared in five steps
from the known aldehyde 613 and was oxidized to aldehyde
7 with Dess-Martin periodinane (Scheme 1). A Wittig
reaction of 7 with phosphorane 8 afforded (E)-R,â-unsatur-
ated ester 9 in near quantitative yield, but attempts to remove
the tert-butyl ester from 9 with trifluoroacetic acid produced
an intractable mixture. The use of trimethylsilyl triflate for
this ester cleavage was more successful and gave carboxylic
acid 10 in excellent yield. However, this left us with the
problem of cleaving the C5 secondary TBS ether selectively
in the presence of two additional secondary TBS ethers
located in the epothilone sector after the coupling to produce
our cryptophilone precursor (vida infra). Although this had
been accomplished in high yield with TBAF in our route to
epothilone D,11 the strategy had failed completely in our
approach to certain epothilone analogues.12
Therefore, an alternative route from 9 was pursued in
which the silyl ether was first cleaved with TBAF to give
hydroxy ester 11 (Scheme 2). Subsequent treatment with ice-
cold trifluoroacetic acid then gave hydroxy acid 12. This
(7) White, J. D.; Hong, J.; Robarge, L. A. Tetrahedron Lett. 1998, 39,
8779.
(8) White, J. D.; Hong, J.; Robarge, L. A. J. Org. Chem. 1999, 64, 6206.
(9) White, J. D.; Carter, R. G.; Sundermann, K. F. J. Org. Chem. 1999,
64, 684.
(10) White, J. D.; Sundermann, K. F.; Carter, R. G. Org. Lett. 1999, 1,
1431.
(11) White, J. D.; Carter, R. G.; Sundermann, K. F.; Wartmann, M. J.
Am. Chem. Soc. 2001, 123, 5407.
(12) White, J. D.; Sundermann, K. F.; Wartmann, M. Org. Lett. 2002,
4, 995.
(13) Kozikowski, A. P.; Stein, P. D. J. Org. Chem. 1984, 49, 2301.
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Org. Lett., Vol. 8, No. 18, 2006

epothilone segment, the hydroxyl group of 12 was masked
as its triethylsilyl ether 14.
Scheme 1. Synthesis of a Cryptophycin Sector of
The epothilone sector required for assembling 3 was
derived from 15, prepared from keto aldehyde 16 in six steps
as described previously.11 Hydrogenolysis of the p-meth-
oxybenzyl ether of 15 yielded alcohol 17 (Scheme 3), but
Cryptothilone 1
Scheme 3. Coupling of Cryptophycin and Epothilone Sectors
of Cryptothilone 1
highly polar substance was unstable under a variety of
conditions, including those contemplated for coupling of 12
with a segment corresponding to C1-C9 of the epothilone
nucleus. One of the substances formed from 12 was δ-lactone
13 resulting from (E)-to-(Z) isomerization of the conjugated
olefin. In light of this problem, a milder route to hydroxy
acid 12 was sought, and it was found that exposure of 9 to
acetic acid in aqueous 2-propanol resulted in cleavage of
both the silyl ether and the tert-butyl ester to give 12. To
carry this carboxylic acid forward for coupling with an
Scheme 2. Coupling Precursor for a Cryptophycin Sector of
Cryptothilone 1
attempts to esterify this alcohol with carboxylic acid 14
employing carbodiimide activation gave a low yield of the
desired product. Finally, the Yamaguchi protocol,14 in which
14 was activated with 2,4,6-trichlorobenzoyl chloride and
the resultant anhydride treated with 17 in the presence of
DMAP and hot toluene, afforded ester 18 in good yield.
Previous experience gleaned from our studies on epothilones
had demonstrated that tert-butyldimethylsilyl ethers at C3
and C7 are not cleaved with TBAF, and it was therefore
possible to remove both the TES ether and the trimethylsi-
lylethyl ester from 18 with this reagent.
It was assumed that hydroxy acid 19 would lactonize to
20 under the same conditions we had used to close the 16-
(14) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. Jpn. 1979, 52, 1989.
Org. Lett., Vol. 8, No. 18, 2006
3949

membered lactone of epothilone D and various analogues,
but this proved to be a false hope. Exposure of 19 to the
Yamaguchi protocol14 led to an intractable mixture from
which no lactone could be isolated. Fortunately, Keck-
Steglich macrolactonization15 of 19 was successful and
delivered 20 in acceptable yield. Final cleavage of the pair
of TBS ethers from 20 with trifluoroacetic acid gave
cryptothilone 1 (3).
up to 40 µM implies that this substance has no affinity for
either the cryptophycin or the taxol/epothilone binding site
on tubulin. However, further experiments including cell line
assays are necessary before the concept of a structural motif
based around these hybrid molecules as cytotoxic agents can
be dismissed. One of those experiments will entail the
synthesis of a second cryptothilone 4.
Preliminary experiments with cryptothilone 1 (3) in which
crytophycin 1 and taxol (substituted for epothilone A) were
used as controls showed that hybrid molecule 3 had no effect
on the rate of tubulin assembly into microtubules. Both
cryptophycin 1 and taxol were active under the experimental
conditions used. The fact that 3 showed no acceleration of
tubulin polymerization or depolymerization at concentrations
Acknowledgment. Financial support was provided by the
National Institute of General Medical Sciences (GM-50574).
Supporting Information Available: Experimental pro-
cedures, characterization data, and NMR spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(15) Boden, E. P.; Keck, G. E. J. Org. Chem. 1985, 50, 2394.
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