Design, total synthesis, and evaluation of novel open-chain epothilone analogues

Article abstract of DOI:10.1021/ol0528787

The design, total synthesis, and biological evaluation of two open-chain analogues of epothilone incorporating the critical C1-C8 fragment and the aromatic side chain held together by a small molecular scaffold have been achieved. Biological evaluation revealed that further restraint between the flexible C1-C8 region and the molecular scaffold may be necessary for potent inhibition of cell proliferation.

Full text of DOI:10.1021/ol0528787

ORGANIC
LETTERS
2006
Vol. 8, No. 4
685-688
Design, Total Synthesis, and Evaluation
of Novel Open-Chain Epothilone
Analogues
Mamoun M. Alhamadsheh,† Richard A. Hudson,†,‡ and
L. M. Viranga Tillekeratne*,†,‡
Department of Medicinal & Biological Chemistry, College of Pharmacy, and
Department of Chemistry, College of Arts & Sciences,
UniVersity of Toledo, Toledo, Ohio 43606
ltillek@utnet.utoledo.edu
Received November 28, 2005
ABSTRACT
The design, total synthesis, and biological evaluation of two open-chain analogues of epothilone incorporating the critical C1
and the aromatic side chain held together by a small molecular scaffold have been achieved. Biological evaluation revealed that further
restraint between the flexible C1 C8 region and the molecular scaffold may be necessary for potent inhibition of cell proliferation.
−C8 fragment
−
Discovery of the anticancer agent paclitaxel1 and the elucida-
tion of its unique mechanism of action as a promoter of
tubulin polymerization and microtubule stabilization2 opened
a new era in anticancer drug discovery research. Although
clinically used for treatment of ovarian and breast cancers,3
poor water solubility and susceptibility to multi-drug-resistant
(MDR) cancer cells4 limited its clinical use. In adddition,
paclitaxel has a complex molecular architecture, which is
intolerant to chemical modification, and attempts to synthe-
size active analogues of simpler structure have not been
productive.5
A number of other natural products with a paclitaxel-like
mechanism of action have been reported.6-10 Prominent
among them are the epothilones 1-4 (Figure 1), initially
† College of Pharmacy.
‡ College of Arts & Sciences.
(1) Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggan, P.; McPhail, A.
T. J. Am. Chem. Soc. 1971, 93, 2325.
(2) (a) Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277, 665. (b)
Schiff, P. B.; Horwitz, S. B. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 1561.
(c) Rowinsky, E. K.; Cazenave, L. A.; Donehower, R. C. J. Natl. Cancer
Inst. 1990, 82, 1247.
Figure 1. Structures of some natural epothilones.
isolated from the cellulose-degrading myxobacterium Sor-
angium cellulosum.6,10,11 Improved water solubility and
demonstrated activity against MDR cell lines made them
attractive potential alternatives to paclitaxel in cancer treat-
ment.11 The simpler molecular structure of the epothilones
has facilitated the synthesis of new drug candidates.11
(3) (a) Rowinsky, E. K.; Donehower, R. C. New Engl. J. Med. 1995,
332, 1004. (b) Cortes, J. E.; Pazdur, R. J. Clin. Oncol. 1995, 13, 2643. (c)
Rowinsky, E. K. Annu. ReV. Med. 1997, 48, 353.
(4) (a) Horwitz, S. B.; Cohen, D.; Rao, S.; Ringel, I.; Shen, H. J.; Yang,
C. P. Monogr. Natl. Cancer Inst. 1993, 15, 55. (b) Gupta, R. S. J. Cell.
Physiol. 1983, 114, 137.
(5) Wang, M.; Cornett, B.; Nettles, J.; Liotta, D. C.; Snyder, J. P. J.
Org. Chem. 2000, 65, 1059.
10.1021/ol0528787 CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/26/2006

Extensive SAR studies have shed light on key function-
alities of the molecule critical for biological activity.11-13
These studies have shown that the C1-C8 sector constitutes
a critical part of the molecule for biological activity and is
not amenable to change.11-13 The aromatic side chain at C15,
endo-orientation, with the C10-C15 fragment folded beneath
the macrocycle.21 Danishefsky’s group has shown that the
presence of a C-9-C10 trans double bond is well tolerated,22
whereas the corresponding cis-isomer had diminished activ-
ity.23 These findings are consistent with the tubulin-bound
conformation of epothilone A reported by Carlomagno et
al.,15 in which the C8-C10 fragment adopts an antiperiplanar
conformation, analogous to that in the X-ray crystal structure.6b
Thus, the C9-C15 region of the molecule may play an
important role, inter alia, in conformational stabilization
necessary for receptor binding. This may be mainly in setting
the critical relative geometry between the aromatic side chain
and the C1-C8 region. Recent studies on the bioactive
conformation of epothilone underscore the importance of
conformation-activity relationships, in addition to classical
SAR.15,16,20-24
though necessary, can tolerate some modification;11-13
a
nitrogen heterocycle connected to the macrolactone ring by
an olefinic spacer at the position ortho to the nitrogen atom
is the minimum requirement. Nicolaou et al. showed that
replacing the thiazole ring with methylpyridines generated
potent analogues.14 In the tubulin-bound conformation of
epothilone A, as determined by NMR studies in aqueous
solution, the 16-Me and 19-H have been shown to adopt a
syn orientation as in the crystal structure obtained from
methanol/water.15,16 This makes the nitrogen atom of the
aromatic ring more accessible for hydrogen bonding with
receptor functional groups. Active analogues incorporating
substantial modification in the C9-C15 sector have been
synthesized. Analogues with cis and trans C12/C13 olefinic
bonds17,18 as well as C12/C13 cyclopropyl moieties19 retain
substantial activity, pointing to a role for oxirane function
in 1 and 2 as a stabilizer of the bioactive conformation, rather
than serving as an electrophilic center or a hydrogen bond
acceptor.20 Contrary to earlier suggestions of an exo-
oreintation of the epoxide ring, Nettles et al., by a combina-
tion of NMR, electron crystallography, and molecular
modeling studies, proposed that tubulin-bound epothilone A
adopts a conformation in which the epoxide ring is in an
In view of the presumptive role played by the C9-C15
segment in maintaining the conformational stability necessary
for receptor binding, we speculated that a molecule in which
the critical C1-C8 region and the aromatic side chain of
epothilones are held in position by a small molecular scaffold
may satisfy such conformational requirements for receptor
binding and would also add to the repository of molecules
available for SAR comparison.
Accordingly, we designed compound 5a (Scheme 1),
which embodies the critical C1-C8 fragment of natural
Scheme 1
(6) (a) Hofle, G.; Bedorf, N.; Gerth, K.; Reichenbach, H. (GFE), DE-B
4138042, 1993 [Chem. Abstr. 1993, 120, 52841]. (b) Hofle, G.; Bedorf,
N.; Steinmetz, H.; Schomburg, D.; Girth, K.; Reichenbach, H. Angew.
Chem., Int. Ed. Engl. 1996, 35, 1567. (c) Gerth, K.; Bedorf, N.; Hofle, G.;
Irschik, H.; Reichenbach, H. J. Antibiot. 1996, 49, 560. (d) 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.
(7) (a) Gunasekera, S. P.; Gunasekera, M.; Longley, R. E.; Schulte, G.
K. J. Org. Chem. 1990, 55, 4912. (b) Longley, E.; Caddigan, D.; Harmody,
D.; Gunasekera, M.; Gunasekera, S. Transplantation 1991, 52, 650. (c) Ter
Haar, E.; Kowalski, R.; Hamel, E.; Lin, C.; Longley, R.; Gunasekera, S.;
Rosenkranz, H.; Day, B. Biochemistry 1996, 35, 243.
(8) (a) D’Ambrosio, M.; Guerriero, A.; Pietra, F. HelV. Chim. Acta 1987,
70, 2019. (b) D’Ambrosio, M.; Guerriero, A.; Pietra, F. HelV. Chim. Acta
1988, 71, 964. (c) Ketzinel, S.; Rudi, A.; Schleyer, M.; Benayahu, Y.;
Kashman, Y. J. Nat. Prod. 1996, 59, 873. (d) Lindel, T.; Jensen, P.; Fenical,
W.; Long, B.; Casazza, A.; Carboni, J.; Fairchild, C. J. Am. Chem. Soc.
1997, 119, 8744.
(9) Corley, D.; Herb, R.; Moore, R.; Scheuer, P.; Paul, V. J. Org. Chem.
1988, 53, 3644.
(10) Altmann, K.-H. Curr. Opin. Chem. Biol. 2001, 5, 424.
(11) Wartmann, M.; Altmann, K.-H. Curr. Med. Chem.: Anti-Cancer
Agents 2002, 2, 123.
(12) Nicolaou, K. C.; Roschanger, F.; Vourloumis, D. Angew. Chem.,
Int. Ed. 1998, 37, 2014.
(13) Su, D. S.; Balog, A.; Meng, D.; Bertinato, P.; Danishefsky, S. J.;
Zheng, Y. H.; Chou, T. C.; He, L.; Horwitz, S. B. Angew. Chem., Int. Ed.
Engl. 1997, 36, 2093.
(14) Nicolaou, K. C.; Scarpelli, R.; Bollbuck, B.; Werschkun, B.; Pereira,
M.; Wartmann, M.; Altmann, K.-H.; Zaharevtiz, D.; Gussio, R.; Gianna-
kakou, P. Chem. Biol. 2000, 7, 593.
(15) Carlomagno, T.; Blommers, M. J. J.; Meiler, J.; Jahnke, W.; Schupp,
T.; Petersen, F.; Schinzer, D.; Altmann, K.-H.; Griesinger, C. Angew. Chem.,
Int. Ed. Engl. 2003, 42, 2511.
(16) Heinz, D. W.; Schubert, W.-D.; Hofle, G. Angew. Chem., Int. Ed.
Engl. 2005, 44,1298.
(17) (a) Meng, D.; Su, D. S.; Balog, A.; Bertinato, P.; Sorensen, E. J.;
Danishefsky, S. J.; Zheng, Y. H.; Chou, T. C.; He, L.; Horwitz, S. B. J.
Am. Chem. Soc. 1997, 119, 2733. (b) Danishefsky, S. J.; Zheng, Y. H.;
Chou, T. C.; He, L.; Horwitz, S. B. Angew. Chem., Int. Ed. Engl. 1997, 36,
757.
epothilone, including all crucial stereocenters. The methyl-
substituted olefinic spacer was incorporated in a cyclopen-
686
Org. Lett., Vol. 8, No. 4, 2006

tenyl molecular scaffold, which carried a 2-pyridyl substit-
uent as the aromatic moiety, in compliance with the
requirement of an aromatic ring with a nitrogen atom ortho
to the point of attachment of the ring to the macrocycle for
maximal activity.14
The retrosynthesis for compound 5a is shown in Scheme
1. We reasoned that the chiral alcohol 6 should be accessible
via stereoselective reduction of ketone 7, which in turn is
easily derived from diketone 8. The carboxylic acid 9a can
be synthesized from aldehyde 10 by Brown’s allylation25-
oxidation protocol. Stereoselective aldol coupling between
the Mori’s ketone 1226 and isobutyraldehyde 13 can be used
to generate 11 as the precursor of 10.
tivity (84% ee). The absolute stereochemistry and enantio-
meric excess of alcohol 6 was confirmed by a modified
Mosher ester method.29,30
The synthesis of carboxylic acid 9a was undertaken as
shown in Schemes 3 and 4. Mori’s ketone 12 was prepared
in two steps in 43% overall yield as reported.26,31 By
kinetically controlling the aldol reaction between the Z-
enolate of 12 and isobutyraldehyde using LDA at -78 °C,
we were able to form the desired syn aldol enantiomers (()-
16 diastereoselectively (syn:anti 24:1)26,32 (Scheme 3). No
Scheme 3
Thiazolium salt-catalyzed addition of 2-pyridinecarbox-
aldehyde 15 to the activated double bond of ethyl vinyl
ketone 14 furnished the diketone 8,27 which in turn underwent
an intramolecular aldol reaction to the cyclopentenone 7
(Scheme 2). Enantioselective reduction of 7 with BH3-THF,
Scheme 2
attempt was made to resolve the racemic syn diols (()-16
at this stage.
Subsequent ozonolysis of the silylated alcohol (()-11 gave
the aldehyde (()-10, which was stereoselectively converted
to the homoallylic alcohols 17a and 17b (84% de) by reaction
with (+)-allyldiisopinocampheylborane (Scheme 4) prepared
Scheme 4
using (R)-2-methyloxazaborolidine (CBS) catalyst,28 gave the
desired (S)-alcohol 6 in good yield (86%) and enantioselec-
(18) (a) Nicolaou, K. C.; Winssinger, N.; Pastor, J.; Ninkovic, S.; Sarabai,
F.; He, Y.; Vourloumis, D.; Yang, Z.; Li, T.; Giiannakakou, P.; Hamel, E.
Nature 1997, 387, 268. (b) Nicolaou, K. C.; Vourloumis, D.; Li, T.; Pastor,
J.; Winssinger, N.; He, Y.; Ninkovic, S.; Sarabia, F.; Vallberg, H.;
Roschangar, F.; King, N. P.; Finlay, M. R.; Giannakakou, P.; Verdier-Pinard,
P.; Hamel, E. Angew. Chem., Int. Ed. Engl. 1997, 36, 2097.
(19) (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) Nicolaou, K. C.; Namoto, K.; Li, J.; Ritzen, A.; Ulven, T.; Shoji,
M.; Zaharevitz, D.; Gussio, R.; Sackett, D. L.; Ward, R. D.; Hensler, A.;
Fojo, T.; Giannakakou, P. ChemBioChem. 2001, 2, 69. (c) Nicolaou, K.
C.; Namoto, K.; Ritzen, 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.
from (-)-B-methoxydiisopinocampheylborane and allylmag-
nesium bromide.25 The (S)-configuration at the homoallylic
(20) Altmann, K.-H. Curr. Pharm. Des. 2005, 11, 1595.
(21) Nettles, J. H.; Li, H.; Krahn, J. M.; Snyder, J. P.; Downing, K. H.
Science 2004, 305, 866.
(26) Mori, I.; Ishihara, K.; Heathcock, C. J. Org. Chem. 1990, 55, 1114.
(27) Stetter, H.; Krasselt, J. J. Heterocycl. Chem. 1977, 14, 573.
(28) Corey, E. J.; Helal, C. Angew. Chem., Int. Ed. 1998, 37, 1986.
(29) (a) Dale, J.; Mosher, H. J. Am. Chem. Soc. 1973, 95, 512. (b) Ohtani,
I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092.
(30) ∆δ ) (δS - δR) values for the MTPA esters of compound 6: H3-6
(+0.155), H2-5 (-0.14, -0.028), H2-4 (-0.029, -0.068).
(31) Mayr, H.; Klein, H.; Sippel, E. Chem. Ber. 1983, 116, 3624.
(32) Heathcock, C.; Buse, C.; Kleschick, W.; Pirrung, M.; Sohn, J.;
Lampe, J. J. Org. Chem. 1980, 45, 1066.
(22) Rivkin, A.; Yoshimura, F.; Gabarda, A. E.; Chou, T.-C.; Dong, H.;
Tong, W. P.; Danishefsky, S. J. J. Am. Chem. Soc. 2003, 125, 2899.
(23) (a) White, J. D.; Carter, R. G.; Sundermann, K. F. J. Am. Chem.
Soc. 2001, 123, 5407. (b) White, J. D.; Carter, R. G.; Sundermann, K. F.;
Waterman, M. [Erratum for J. Am. Chem. Soc. 2001, 123, 5407]. J. Am.
Chem. Soc. 2003, 125, 3190.
(24) Taylor, R. E.; Chen, Y.; Galvin, G. M.; Pabba, P. K. Org. Biomol.
Chem. 2004, 2, 127.
(25) Jadhav, P.; Bhat, K.; Perumal, P.; Brown, H. C. J. Org. Chem. 1986,
51, 432.
Org. Lett., Vol. 8, No. 4, 2006
687

alcohol carbon was assigned from the discernible proton
NMR signals of the MPTA esters and confirmed subse-
quently by Mosher ester analysis of the desilylated benzyl
esters of 9a and 9b (vide infra).29,34 Attempts to separate
the two diastereomers by chromatography were unsuccessful.
Silylation of the hindered secondary hydroxyl group of 17a/
17b was achieved with TBSOTf and 2,6-lutidine to give a
mixture of 18a/18b in excellent yield. After conversion to
carboxylic acids 9a and 9b by ozonolysis followed by
Pinnick oxidation,33 the two products were conveniently
separated by column chromatography. By converting the
benzyl esters of desilylated 9a and 9b to the corresponding
Mosher diesters, the stereochemistry at C7 was established
as (S) and (R), respectively, while at the same time
reconfirming the stereochemistry at C3 of both diastereomers
as (S), thereby establishing the absolute configuration of 9a
and 9b as shown.34
Scheme 5
Coupling between 9a and alcohol 6 gave the corresponding
ester. However, attempts to desilylate it to obtain the final
product 5a proved problematic. Stronger conditions such as
20% TFA in methylene chloride accomplished desilylation,
but with concurrent cleavage of ester functionality giving
5a in only 20% yield, along with the desilylated carboxylic
acid 19a. Milder conditions resulted in partial or no desily-
lation. As the difficulty in removing the protecting groups
may be attributed to their highly hindered environment, we
considered direct esterification of the desilylated carboxylic
acids 19a and 19b with alcohol 6 (Scheme 5).
stereochemistry (3S,6R,7S), of natural epothilones, whereas
5b was the 6S,7R-diastereomer. It should be noted that a
meaningful conclusion about the level of cytotoxicity of 5a
cannot be drawn in the absence of a direct comparison with
a natural epothilone. Nevertheless, these results suggest a
modest level of selective activity and can be used as the basis
for further exploration of this class of open-chain analogues,
especially with emphasis on further conformational constraint
with respect to the aromatic residue. In 5a the methyl-
substituted olefinic spacer of epothilone was incorporated
in the cyclopentenyl molecular scaffold while the heteroaro-
matic ring was represented by a 2-pyridyl moiety, mimicking
that of active pyridine epothilone analogues.14 Low, but
selective, in vitro cell antiproliferative activity of compound
5a against CNS and ovarian cancer cell lines and the lack
of activity of compound 5b show that, while the molecular
scaffold may establish some conformational restriction
between the pyridyl group and the C1-C8 region, further
restraint between the flexible elements of the C1-C8 region
and the molecular scaffold may be necessary in establishing
active analogues. Efforts in this direction are in progress in
our laboratory.
Treating compounds 9a or 9b with 20% trifluoroacetic
acid in methylene chloride resulted in the formation of the
fully deprotected intermediates 19a or 19b, respectively
(Scheme 5). As expected, the carboxylic acids 19a or 19b
reacted with alcohol 6 to give the target molecules 5a or
5b, respectively, as the sole esterification product in good
yield (64% and 68%, respectively, over two steps). Neither
of the hindered alcohols reacted either intramolecularly or
intermolecularly with the carboxylic acid groups.
In preliminary cytotoxicity studies compound 5a showed
GI50 values of 21.9 and 45.1 µM against CNS cancer (SNB-
75) and ovarian cancer (OVCAR-4) cell lines, respectively,
in the NCI in vitro 60 cell line human tumor screen, but no
activity was observed against any of the other cell lines.
Compound 5b, on the other hand, was not active against
any of the cell lines. Interestingly, the C1-C8 fragment of
5a mimicked the corresponding region, including absolute
Acknowledgment. This work was partly supported by a
grant from the Elsa U. Pardee Foundation. We thank the
NCI’s Developmental Therapeutics Program for evaluation
of the compounds.
Supporting Information Available: Experimental de-
(33) Bal, B.; Childers, W. E.; Pinnick, H. Tetrahedron 1981, 37, 2091.
(34) ∆δ ) (δS - δR) values for the MTPA diesters of (a) 9a benzyl
ester: H2-Bn (-0.01), H2-2 (-0.07, -0.016), H1-3 (-0.031), H6-10,10′
(+0.076, +0.082), H1-6 (+0.021), H3-11 (+0.283), H1-8 (+0.016), H6-
9,9′ (-0.014); (b) 9b benzyl ester: H2-Bn (-0.023), H2-2 (-0.014, -0.036),
H1-3 (+0.011), H6-10,10′ (+0.087, +0.108), H1-6 (+0.189), H3-11
(-0.224), H1-8 (+0.03), H6-9,9′ (+0.082)
tails, characterization data of all compounds, and 1H and 13
C
NMR spectra of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL0528787
688
Org. Lett., Vol. 8, No. 4, 2006