Conformation-activity relationships in polyketide natural products: A new perspective on the rational design of epothilone analogues

Article abstract of DOI:10.1021/ja028196l

The syntheses of C14-methyl analogues of epothilone B and D are described. Conformational analysis using computational methods, X-ray crystallography, and NMR studies showed that the stereochemistry at C14 has a pronounced effect on the conformation of th

Full text of DOI:10.1021/ja028196l

Published on Web 11/27/2002
Conformation-Activity Relationships in Polyketide Natural Products: A New
Perspective on the Rational Design of Epothilone Analogues
Richard E. Taylor,* Yue Chen, and Alicia Beatty†
Department of Chemistry and Biochemistry and the Walther Center for Excellence in Cancer Research,
251 Nieuwland Science Hall, UniVersity of Notre Dame, Notre Dame, Indiana 46556-5670
David C. Myles and Yiqing Zhou
Kosan Biosciences Inc., 3832 Bay Center Place, Hayward, California 94545
Received August 19, 2002; E-mail: taylor.61@nd.edu
The promising activities of the epothilones in in vitro and in
vivo cancer models have attracted significant attention from the
synthetic, biological, and medicinal communities.1 Several epothilones
are currently in clinical trials against a range of cancer types. These
efforts have yielded hundreds of analogues in this exciting class
and have identified the critical structural features necessary for
biological activity. Despite these efforts, the role that the conforma-
tion of the epothilone macrocycle plays in displaying the pendant
functional groups has gone under-recognized.
It has been a goal of our program to design analogues of complex
polyketide macrolides to probe the effect of conformation on
biological activity. In this contribution, we report epothilone
analogues in which conformational families have been stabilized
through rational substitution. This approach provides insights into
the biologically active conformation of the epothilone class of
natural products.
Figure 1. Conformations of the C10-C15 epoxide region.
compounds 3 and 4. We rationalized that 3 and 4 would prefer-
entially exist in conformers I and II (R ) Me), respectively. In
addition, the presence of the C14-methyl substituent would
destabilize the alternative conformer. These preferences were
supported by computer modeling techniques similar to our previous
analysis of the natural products.2
The target compounds were prepared by a route based on our
previously reported synthesis of epothilone B.6 Thiazole aldehyde
5, an intermediate in several synthetic routes to these compounds,
is the point of divergence for the synthesis of 3 and 4, Scheme 1.
We have used a combination of computational methods and high-
field NMR experiments to study the conformational properties of
the epothilones.2 Our previous report concentrated on the C1-C8
polypropionate region and concluded that the epothilones populate
two major conformational families in solution, with the distribution
of conformers being controlled by syn-pentane interactions. We also
showed that the major contributor was related to the conformation
observed in the solid state.3
Scheme 1
Previously reported structure-activity relationships have shown
that neither the presence nor the stereochemistry of the epoxide is
essential to biological activity of these compounds.1c Conformational
analysis of this region of the macrolide suggested that accessible
conformations could be clustered into two families shown as I and
II (R ) H, Figure 1). Several groups have reported models of the
epothilone pharmacophore and its relationship to other classes of
microtubule-stabilizing natural products based on computational
methods and reported structure-activity relationships.4 In fact, two
of these studies have proposed that the bioactive conformation of
the epothilone epoxide region is similar to conformer II.4a,b,5 In an
effort to distinguish conformers I and II and to determine which is
more closely related to the biologically active conformation of the
epothilones, we have considered simple C14-methyl substituted
Brown asymmetric crotylboration7 efficiently controlled the enantio-
and diastereoselectivity of the C14, C15 stereogenic centers.
Protection of the secondary hydroxyl as a tert-butoxydiphenylsilyl
ether followed by oxidative cleavage provided aldehydes 6 and 7.
The conversion of each of aldehydes 6 and 7 to epo D analogues
8 and 9 proceeded efficiently though identical synthetic sequences.
Full details of their syntheses are included in the Supporting
Information. (S)-14-methyl epothilone D 8 underwent a highly
selective epoxidation with mCPBA to provide (R)-14-methyl
epothilone B 3 in 55% yield, Scheme 2.
Epoxidation of (R)-14-methyl epothilone D 9 also proceeded in
a highly selective fashion, Scheme 3. We believe that 9 will exist
primarily in conformer II due to the conformational constraints
imposed by the C14-methyl substituent (vide infra). Therefore, it
†To whom questions regarding the X-ray diffraction studies should be addressed.
9
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J. AM. CHEM. SOC. 2003, 125, 26-27
10.1021/ja028196l CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2
marked contrast, analogues 9 and 10 (conformer II preference)
showed no measurable cytotoxicity.
The conformation-activity relationships presented herein strongly
support the importance of conformer I as the bioactive conformation
of the 12,13-epoxide (olefin) region of the epothilones. The
approach presented here offers a new perspective on rational design
of modified biologically active polyketide macrolides. The recent
advances in genetic engineering of polyketide synthases (PKS)10
may provide an alternative synthetic route to these and related
conformationally restricted analogues through manipulation of the
epothilone PKS gene cluster.11 Efforts along these lines are currently
being pursued. Additional conformational analogues of epothilone
have been prepared in our laboratory and the results will be reported
in due course.
Scheme 3
is expected that the stereochemistry of the epoxide is epimeric to
epothilone B and analogues 3 and 4. We formulate this epoxide as
10.
X-ray diffraction studies of single crystals of 3 and 8‚H2O (not
shown) showed that the conformation of each in the solid state
was quite similar to that reported for epothilone B and thus
conformer I in the epoxide region,8 Figure 2. Proton NMR coupling
Acknowledgment. Dr. Jaroslav Zajicek is thanked for his
assistance with NMR experiments. Support provided by the National
Cancer Institute/National Institutes of Health (CA85499). Y.C.
thanks the University of Notre Dame for support through a Reilly
Fellowship. R.E.T. acknowledges support from Bristol-Myers
Squibb, Kosan Biosciences, and Eli Lilly through the 2000 Lilly
Grantee award program.
Supporting Information Available: Full experimental and char-
acterization data for the preparation of 3, 8, 9, and 10 (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org
References
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C.; Roschanger, F.; Vourloumis, D. Angew. Chem., Int. Ed. 1998, 37,
2014.
(2) Taylor, R. E.; Zajicek, J. J. Org. Chem. 1999, 64, 7224.
(3) Hoefle, G.; Bedorf, N.; Steinmetz, H.; Schomburg, D.; Gerth, K.;
Reichenbach, H. Angew. Chem. 1996, 35, 1567.
Figure 2. Solid-state structure of 3.
constants (J14-15 ) 9.9, 10.5 Hz respectively) also supported this
preference in solution. In contrast, proton NMR coupling constants
of analogues 9 and 10 had the expected values for conformer II
(J14-15 ) 3.3, <2.0 Hz respectively). Computational models
suggested a H-C14-C15-H dihedral angle of 175° (Jcalc ) 10.9
Hz) in 3 (conformer I) and 62° (Jcalc ) 3.1 Hz) in 4 (conformer
II). Additional NMR evidence for these conformational differences
were observed in ROESY experiments with 8, 3, 9, and 10.9 An
NOE enhancement between H13 and the C16-CH3 was only
observed in analogues 9 and 10 but not in 8 and 3. These data not
only support the preference for conformer II but also the proposed
configuration of epoxide 10.
(4) (a) Giannakakou, P.; Gussio, R.; Nogales, E.; Downing, K. H.; Zaharevitz,
D.; Bollbuck, B.; Poy, G.; Sackett, D.; Nicolaou, K. C.; Fojo, T. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 2904. (b) Wang, M.; Xia, X.; Kim, Y.;
Hwang, D.; Jansen, J. M.; Botta, M.; Liotta, D. C.; Snyder, J. P. Org.
Lett. 1999, 1, 43. (c) Ojima, I.; Chakravarty, S.; Inoue, T.; Lin, S.; He,
L.; Horwitz, S. B.; Kuduk, S. D.; Danishefsky, S. J. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 4256. (d) Baloone, P.; Marchi, M. J. Phys. Chem.
A 1999, 103, 3097.
(5) The biological activity of 12,13-cyclopropyl and 12,13-aziridinyl epothilones
strongly support the importance of conformer I. (a) Johnson, J.; Kim, S.-
H.; Bifano, M.; DiMarco, J.; Fairchild, C.; Gougoutas, J.; Lee, F.; Long,
B.; Tokarski, J.; Vite, G. Org. Lett. 2000, 2, 1537. (b) Regueiro-Ren, A.;
Borzilleri, R. M.; Zheng, X.; Kim, S.-H.; Johnson, J. A.; Fairchild, C. R.;
Lee, F. Y. F.; Long, B. H.; Vite, G. D. Org. Lett. 2001, 3, 2693. (c)
Nicolaou, K. C.; Namoto, K.; Ritze´n, A.; Ulven, T.; Shoji, M.; Li, J.;
D’Amico, G.; Liotta, D.; French, C. T.; Wartmann, M.; Altmann, K.-H.;
Giannakakou, P. J. Am. Chem. Soc. 2001, 123, 9313.
(6) Taylor, R. E.; Chen, Y. Org. Lett. 2001, 3, 2221.
The biological activities of epothilone analogues 3, 8, 9, and 10
were evaluated against a panel of human tumor cell lines (Table
1). From this study, it is clear that the stereochemistry of the newly
(7) Brown, H. C.; Bhat, K. S. J. Am. Chem. Soc. 1986, 108, 5919.
(8) The authors have deposited the crystallographic data for 3 and 8‚H2O
with the Cambridge Crystallographic Data Center (CCDC 191723 and
191724). The data can be obtained, on request, from the Director,
Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2
1EZ, U.K or www.ccdc.cam.ac.uk/conts/retrieving.html.
Table 1. Cytotoxicity of Epothilone Analogues (IC50 nM)
(9) A strong NOE enhancement was observed between the H14 and one of
the C11 protons in both 8 and 9. In addition a strong NOE enhancement
was observed between H15 and H13 in epoxide 3 but not in epoxide 10.
An NOE enhancement between H14 and H15 was only observed in
analogues 9 and 10 but not in 8 and 3. The observed coupling constants
between H13 and H14, in all four analogues, were similar (J13-14 ) 8.1-
9.9 Hz). Attempts to obtain single crystals of 9 and 10 were unsuccessful.
(10) Khosla, C. J. Org. Chem. 2000, 65, 8127.
(11) (a) Julien, B.; Shah, S.; Ziermann, R.; Goldman, R.; Katz, L.; Khosla, C.
Gene 2000, 249, 153. (b) Tang, L.; Shah, S.; Chung, L.; Carney, J.; Katz,
L.; Khosla, C.; Julien, B. Science 2000, 287, 640. (c) Molnar, I.; Schupp,
T.; Ono, M.; Zirkle, R. E.; Milnamow, M.; Nowak-Thompson, B.; Engel,
N.; Toupet, C.; Stratmann, A.; Cyr, D. D.; Gorlach, J.; Mayo, J. M.; Hu,
A.; Goff, S.; Schmid, J.; Ligon, J. M. Chem. Biol. 2000, 7, 97.
compound
MCF-7
NCI/ADR
H460
SF
2a
2b
3
8
9
1.5
5
3
3.6
26
23
1.7
20
3
0.7
7
3
35
>1000
>1000
238
>1000
>1000
42
42
>1000
>1000
>1000
>1000
10
introduced methyl group at C14 has a significant impact on the
biological activity of the epothilone analogues. Compounds 3 and
8 (conformer I preference) maintain significant cytotoxicity. In
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