Design, synthesis, and biological evaluation of EF- and ABEF- analogues of (+)-Spongistatin 1

Article abstract of DOI:10.1021/ol100418n

The design, synthesis, and biological evaluation of two potential (+)-spongistatin 1 analogues have been achieved. The analogues, incorporating tethers (red) in place of the ABCD and the CD components of the (+)-spongistatin 1 macrolide, were designed suc

Full text of DOI:10.1021/ol100418n

ORGANIC
LETTERS
2010
Vol. 12, No. 8
1792-1795
Design, Synthesis, and Biological
Evaluation of EF- and ABEF- Analogues
of (+)-Spongistatin 1
Amos B. Smith III,*,† Christina A. Risatti,† Onur Atasoylu,† Clay S. Bennett,†
Karen TenDyke,‡ and Qunli Xu§
Department of Chemistry, Monell Chemical Senses Center and Laboratory for
Research on the Structure of Matter, UniVersity of PennsylVania, Philadelphia,
PennsylVania 19104, and Next Generation Systems and Oncology Product Creation
Unit, Eisai, Inc., 4 Corporate DriVe, AndoVer, Massachusetts 01810
smithab@sas.upenn.edu
Received February 18, 2010
ABSTRACT
The design, synthesis, and biological evaluation of two potential (+)-spongistatin 1 analogues have been achieved. The analogues, incorporating
tethers (red) in place of the ABCD and the CD components of the (+)-spongistatin 1 macrolide, were designed such that the conformations
of the retained skeleton (blue) would mimic the assigned major solution conformation of the natural product The nanomolar cytotoxicity
observed for the ABEF analogue provides strong support for the assigned solution conformation.
Members of the spongistatin family of natural products were
independently isolated by three research groups, Pettit1 (spong-
istatins), Kitigawa2 (altohyrtins), and Fusetani3 (cinachyrolide),
from the marine sponges Spongia, Spirastrella, and Cinachyra,
respectively. Spongistatin 1 [(+)-1, Figure 1], the most abundant
congener, has been characterized as an antimitotic agent that
† University of Pennsylvania.
‡ Next Generation Systems, Eisai, Inc.
§ Oncology Product Creation Unit, Eisai, Inc.
(1) (a) Pettit, G. R.; Cichacz, Z. A.; Gao, F.; Herald, C. L.; Boyd, M. R.
J. Chem. Soc., Chem. Commun. 1993, 1166. (b) Pettit, G. R.; Cichacz, Z. A.;
Gao, F.; Herald, C. L.; Boyd, M. R. J. Chem. Soc., Chem. Commun. 1993,
1805.
(2) (a) Kobayashi, M.; Aoki, S.; Sakai, H.; Kawazoe, K.; Kihara, N.;
Sasaki, T.; Kitagawa, I. Tetrahedron Lett. 1993, 34, 2795. (b) Kobayashi,
M.; Aoki, S.; Sakai, H.; Kihara, N.; Sasaki, T.; Kitigawa, I. Chem. Pharm.
Figure 1
. (a) Spongistatins 1 and 2. (b) Calculated hydrogen
Bull. 1993, 41, 989.
bonding network of (+)-spongistatin 1.
(3) Fusetani, N.; Shinoda, K.; Matsunaga, S. J. Am. Chem. Soc. 1993,
115, 3977.
10.1021/ol100418n  2010 American Chemical Society
Published on Web 03/18/2010

inhibits tubulin polymerization by binding in the tubulin vinca
alkaloid domain.4 Importantly, subnanomolar cytotoxicity against
several chemoresistant cancer cell lines has been demonstrated,
making (+)-spongistatin 1 and congeners thereof important
antitumor lead compounds.5 From a structural perspective, the
spongistatins also display significant architectural complexity;
common features include the polyether backbone with 24
stereocenters, two spiroketals, a bis-pyran unit embedded within
a 42-membered macrolide, and a triene side chain bearing a
vinyl chloride. The first total syntheses of (+)-spongistatin 2
by Evans6 and (+)-spongistatin 1 by Kishi,7 confirming each
structure, have been followed by syntheses from the Smith,8
Paterson,9 Crimmins,10 Heathcock,11 and Ley12 laboratories.
A variety of synthetic approaches to various fragments of the
spongistatins have also been reported,13 as well as the synthesis
of 1 g of (+)-spongistatin 1 (1) reported by this laboratory.14
The design and synthesis of spongistatin analogues has been
impeded by the molecular complexity, with only a few studies
reported. Early on, Kishi and co-workers7 reported that epimer-
ization of the CD spiroketal at C(23) affords an analogue
possessing cytotoxicity similar to that of (+)-spongistatin 1,
while our laboratory reported that side-chain analogues based
on the R-D-glucose scaffold maintain modest micromolar
activity, albeit most likely via a different mechanism.15 Sub-
sequently, Paterson et al.16 disclosed that unsaturation of the
E-ring, achieved by elimination of water at C(35,36) and
formation of a double bond, affords an analogue with increased
cytotoxicity relative to (+)-spongistatin 1; a similar observation
was made for spongistatin 2 by Heathcock and co-workers,11
while truncation of the triene side chain by Paterson resulted
in a dramatic loss of activity.16
More recently, Heathcock reported that spongistatin ana-
logues, including acyclic congeners having only the E and
F rings, as well as cyclic EF, ABEF, and ABCD analogues,
where at least one ring had been replaced with a polyethylene
linker,17 do not maintain significant cytotoxicity.
Taken together, the available SAR data suggests that the
E and F rings, as well as the triene side chain, are critical
for biological activity. The question thus becomes: How
important are the AB and CD spiroketals? Are these
structural units required for potent cyctotoxicity or simply
to enforce the bioactive conformation and, in particular, the
nearly linear conformation of the western perimeter as
observed in the assigned solution conformation.18
To achieve answers, we chose a minimalist design strategy,
specifically to maintain a macrolide structure, first with excision
of the AB and CD spiroketal units and then with an analogue
lacking only the CD unit. Two design criteria were foremost: (1)
Analogue construction would take advantage of advanced inter-
mediates readily available either from our 1-g synthesis and/or
commercially, and (2) we would select tethers that would enforce
the western perimeter to maintain the same low energy linear
conformation as defined by our solution conformation.18
We first targeted macrolide 3, which maintained the EF bis-
pyran system but lacked the AB and CD spiroketals (Figure 2).
(4) (a) Bai, R.; Cichacz, Z. A.; Herald, C. L.; Pettit, G. R.; Hamel, E.
Mol. Pharmacol. 1993, 44, 757. (b) Bai, R.; Taylor, G. F.; Cichacz, Z. A.;
Herald, C. L.; Kepler, J. A.; Pettit, G. R.; Hamel, E. Biochemistry 1995,
34, 9714.
(5) (a) Pettit, G. R.; Cichacz, Z. A.; Gao, F.; Herald, C. L.; Boyd, M. R.;
Schmidt, J. M.; Hooper, J. N. J. Org. Chem. 1993, 58, 1302. (b) Pettit,
G. R. Pure Appl. Chem. 1994, 66, 2271.
(6) (a) Evans, D. A.; Coleman, P. J.; Dias, L. C. Angew. Chem., Int. Ed.
1997, 36, 2738. (b) Evans, D. A.; Trotter, B. W.; Coˆte´, B.; Coleman, P. J.
Angew. Chem., Int. Ed. 1997, 36, 2741. (c) Evans, D. A.; Trotter, B. W.;
Coˆte´, B.; Coleman, P. J.; Dias, L. C.; Tyler, A. N. Angew. Chem., Int. Ed.
1997, 36, 2744. (d) Evans, D. A.; Trotter, B. W.; Coleman, P. J.; Coˆte´, B.;
Dias, L. C.; Rajapakse, H. A.; Tyler, A. N. Tetrahedron 1999, 55, 8671.
(7) (a) Guo, J.; Duffy, K. J.; Stevens, K. L.; Dalko, P. I.; Roth, R. M.;
Hayward, M. M.; Kishi, Y. Angew. Chem., Int. Ed. 1998, 37, 187. (b)
Hayward, M. M.; Roth, R. M.; Duffy, K. J.; Dalko, P. I.; Stevens, K. L.;
Guo, J.; Kishi, Y. Angew. Chem., Int. Ed. 1998, 37, 192.
(8) (a) Smith, A. B., III; Lin, Q.; Doughty, V. A.; Zhuang, L.; McBriar,
M. D.; Kerns, J. K.; Brook, C. S.; Murase, N.; Nakayama, K. Angew Chem.,
Int. Ed. 2001, 40, 196. (b) Smith, A. B., III; Zhu, W.; Shirakami, S.;
Sfouggatakis, C.; Doughty, V. A.; Bennett, C. S.; Sakamoto, Y. Org. Lett.
2003, 5, 761.
(9) Paterson, I.; Chen, D. Y.-K.; Coster, M. J.; Acena, J. L.; Bach, J.;
Gibson, K. R.; Keown, L. E.; Oballa, R. M.; Trieselmann, T.; Wallace,
D. J.; Hodgson, A. P.; Norcross, R. D. Angew. Chem., Int. Ed. 2001, 40,
4055.
(10) Crimmins, M. T.; Katz, J. D.; Washburn, D. G.; Allwein, S. P.;
McAtee, L. F. J. Am. Chem. Soc. 2002, 124, 5661.
(11) Heathcock, C. H.; McLaughlin, M.; Medina, J.; Hubbs, J. L.;
Wallace, G. A.; Scott, R.; Claffey, M. M.; Hayes, C. J.; Ott, G. R. J. Am.
Chem. Soc. 2003, 125, 12844.
(12) Ball, M.; Gaunt, M. J.; Hook, D. F.; Jessiman, A. S.; Kawahara,
S.; Orsini, P.; Scolaro, A.; Talbot, A. C.; Tanner, H. R.; Yamanoi, S.; Ley,
S. V. Angew Chem., Int. Ed. 2005, 44, 5433.
Figure 2. (a) EF analogue and (b) overlay of EF analogue and
(13) Recent reviews with references: (a) Yeung, K.-S.; Paterson, I. Chem.
ReV. 2005, 105, 4237. (b) Pietruszka, J. Angew. Chem., Int. Ed. 1998, 37,
2629.
predicted solution conformation of (+)-spongistatin 1.
(14) Smith, A. B., III; Tomioka, T.; Risatti, C. A.; Sperry, J. B.;
Sfouggatakis, C. Org. Lett. 2008, 10, 4359.
Possible C(1)-C(28) tethers were selected on the basis of length,
orientation, and flexibility after in silico screening of known
fragment libraries. We then performed Monte Carlo conformational
searches (5000 steps) on analogues featuring these linkers. Ana-
(15) Smith, A. B., III; Lin, Q.; Pettit, G. R.; Chapuis, J.-C.; Schmidt,
J. M. Bioorg. Med. Chem. Lett. 1998, 8, 567.
(16) (a) Paterson, I.; Chen, D. Y.-K.; Coster, M. J.; Acena, J. L.; Bach,
J.; Wallace, D. J. Org. Biomol. Chem. 2005, 3, 2431. (b) Paterson, I.; Acena,
J. L.; Bach, J.; Chen, D. Y.-K.; Coster, M. J. Chem. Commun. 2003, 462.
Org. Lett., Vol. 12, No. 8, 2010
1793

logues preserving the same low energy conformation were
subjected to full conformational searches. These studies resulted
in selection of the biaryl ether tether (Figure 2). Overlay of the
resulting analogue 3 with the twisted or “infinity”-shaped major
solution conformation of spongistatin in water revealed excellent
overlap along the western perimeter specifically along the E, F,
and AB rings.
opened congener of the desired EF analogue 3, as evidenced by
the presence of a 13C NMR signal at 211.4 ppm. Presumably, the
ring strain of the tether is sufficient to drive the equilibrium toward
the open δ-hydroxy ketone congener of 3.
We next turned our attention to an analogue lacking only the
CD spiroketal unit (e.g., ABEF analogue 10, Figure 3). Confor-
From the synthetic perspective, the requisite biaryl ether tether
was anticipated to be available via a modified Ullmann ether
synthesis,19 employing the TIPS ester of 6 and 4-formylphenyl
boronic acid. With aldehyde 5 in hand, analogue 3 would then
be constructed using the same endgame strategy as in our gram
synthesis of (+)-spongistatin 1, employing EF Wittig salt (+)-4
(Scheme 1).
Scheme 1. Synthesis of EF Analogue 3
Figure 3. Retrosynthetic Analysis for ABEF analogue 10 and
overlay with (+)-spongistatin 1.
Toward this end, deprotonation of (+)-4 was achieved with
MeLi·LiBr followed by union with 5 to lead to a 4:1 mixture of Z
and E olefin isomers which could not be readily separated. The
poor selectivity is in contrast to that observed in our (+)-
spongistatin 1 synthesis, which produced exclusively the Z-olefin
isomer. The mixture of olefin isomers was next subjected to 3 equiv
of TBAF to remove the TIPS group, as well as the two F-ring
TES groups; the seco-acid olefin isomers (8) still could not be
separated. Pleasingly, however, macrocyclization occurred only
with the Z olefin isomer to furnish the desired product in 62%
yield. Presumably, the strain associated with incorporating an
E-olefin prevented macrocyclization. Global deprotection led to
an 11:1 mixture of products. However, upon detailed 1- and 2-D
NMR analysis, the major product, (+)-9, proved to be the ring-
mational searches performed on the Heathcock ABEF spongistatin
2 analogues indicated that the linkers selected were not capable of
preserving the nearly linear western perimeter of the spongistatins.
We therefore undertook calculations that eventually led to a range
of potential B to E tethers which were able to maintain the assigned
solution conformation of (+)-spongistatin 1 (i.e., linear). Selection
of the B to F tether illustrated in Figure 3 was based both on the
ease of synthesis and the ability to engage in hydrogen bonding.
That is, the length of the linker, as well as placement and orientation
of the ester bond, were optimized to increase the probability of a
hydrogen bond between the ester carbonyl and the C(42) hydroxyl
group on the F ring, which would maintain the desired linear
western perimeter conformation. The position of a Z-olefin adjacent
to the B ring was also selected both to rigidify the linker by allylic
strain and to simplify the synthesis.
(17) Wagner, C. E.; Wang, Q.; Melamed, A.; Fairchild, C. R.; Wild,
R.; Heathcock, C. H. Tetrahedron 2008, 64, 124.
(18) See the preceding article in this issue: Atasoylu, O.; Furst, G.;
Risatti, C.; Smith, A. B., III. Org. Lett. 2010, 12, 10.1021/ol100417d.
(19) Evans, D. A.; Katz, J. L.; West, T. R. Tetrahedron Lett. 1998, 39,
2937.
Construction of the ABEF analogue (10) was again envi-
sioned to take advantage of EF Wittig salt (+)-4, now with
AB aldehyde 11 (Figure 3). The tether would be incorporated
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Org. Lett., Vol. 12, No. 8, 2010

into 11 in two segments; first, a three-carbon unit would be
introduced by Wittig olefination of known AB aldehyde (-)-
15,20 followed by deprotection and esterification of 12 with
known acid 13,21 available from δ-valerolactone. Toward this
end, Parikh-Doering oxidation22 of alcohol (-)-17 (Scheme
2), followed by olefination with Wittig salt 16, produced (-)-
of the reaction mixture to KF, and (3) conversion to the TIPS-
ester by treatment with TIPSCl and Et3N provided alcohol (-)-12
in 96% yield. For chain extension, alcohol (-)-12 was esterified
with carboxylic acid 13 (88%), the latter available in one step via
hydrolysis of δ-valerolactone in the presence of PMBCl.21b
Oxidative removal of the PMB ether from (-)-12, followed by
Parikh-Doering oxidation,22 then provided (-)-11.
With aldehyde (-)-11 in hand, union with EF Wittig salt (+)-4
furnished the full seco ABEF carbon skeleton, possessing exclu-
sively the Z-olefin adjacent to the AB ring, which upon treatment
with 3 equiv of TBAF, Yamaguchi macrocyclization of the derived
seco-acid (-)-22, and global deprotection at low temperature
afforded ABEF analogue (-)-10. The structure, and in particular
the integrity of the E ring hemiketal, was confirmed by NMR
spectroscopy. Importantly, assignment of the solution conformation
of analogue (-)-10, defined exploiting NMR methods, and
deconvolution tactics employed in our solution assignment of (+)-
spongistatin 1 are in accordance with the initially calculated
conformations.
Scheme 2. Synthesis of ABEF Analogue (-)-10
Biological evaulation revealed that analogue (+)-9 (Table 1),
not surprisingly given the missing E ring hemiketal structural
component, was inactive against all four cell lines tested. However,
analogue (-)-10, wherein the AB spiroketal is retained but lacks
the CD spiroketal, displayed nanomolar activities against the tested
cancer cell lines. Studies demonstrating that the mode of action of
(-)-10 is similar to that of (+)-spongistatin 1 (1) will be published
in due course.
Table 1. IC50 (nM) Values of Spongistatin 1 and Analogues in the
Cell Growth Inhibition Assay with Different Cancer Cell Lines
MDA-MB-435 HT-29 H522-T1 U937
(+)-spongistatin 1
(+)-9
(-)-10
0.0225
>1000
82.8
0.058
>1000
161.2
0.16
>1000
297.2
0.059
>1000
60.5
In summary, the design and synthesis of a (+)-spongistatin 1
ABEF analogue (-)-10, based on the assigned solution conforma-
tion of (+)-spongistatin 1 (1), possessing nanomolar cell growth
inhibitory activity against several human tumor cell lines, has been
achieved and as such provides circumstantial evidence for the
assigned solution conformation of spongistatin 1 (1). Or as often
stated by Ralph Hirschmann: “Let the receptor be the judge.”
Acknowledgment. Financial support was provided by the
NIH (NCI) through Grant No. CA-70329. C.A.R. was
supported by an American Cancer Society-Michael Schmidt
Postdoctoral Fellowship.
Supporting Information Available: Spectroscopic and
analytical data and selected experimental procedures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
18 in 51% yield, along with deacylated (-)-19, which was
reacetylated to furnish an overall yield of 63%.
OL100418N
At this juncture, the tert-butyl ester was converted to a TIPS-
ester, which was envisioned to be more compatible with our
endgame strategy. A three-step sequence was required: (1) treat-
ment with TMSOTf, in the presence of 2,6-lutidine, (2) exposure
(20) Hubbs, J. L.; Heathcock, C. H. J. Am. Chem. Soc. 2003, 125, 12836.
(21) (a) Hoye, T.; Kurth, M. J.; Lo, V. Tetrahedron Lett. 1981, 22, 815.
(b) Jacobi, P. A.; Li, Y. Org. Lett. 2003, 5, 701.
(22) Parikh, J. P.; Doering, W. E. J. Am. Chem. Soc. 1967, 89, 5505.
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