Design, synthesis, and biological evaluation of potent discodermolide fluorescent and photoaffinity molecular probes

Article abstract of DOI:10.1021/ol0520166

(Chemical Equation Presented) The design, synthesis, and biological evaluation of a series of (+)-discodermolide molecular probes possessing photoaffinity and fluorescent appendages has been achieved. Stereoselective olefin cross-metathesis comprised a ke

Full text of DOI:10.1021/ol0520166

ORGANIC
LETTERS
2005
Vol. 7, No. 23
5199-5202
Design, Synthesis, and Biological
Evaluation of Potent Discodermolide
Fluorescent and Photoaffinity Molecular
Probes
Amos B. Smith, III,*,† Paul V. Rucker,† Ignacio Brouard,† B. Scott Freeze,†
Shujun Xia,‡ and Susan Band Horwitz‡
Department of Chemistry, UniVersity of PennsylVania, Philadelphia,
PennsylVania 19104, and Department of Molecular Pharmacology,
Albert Einstein College of Medicine, Bronx, New York 10461
smithab@sas.upenn.edu
Received August 21, 2005
ABSTRACT
The design, synthesis, and biological evaluation of a series of (+)-discodermolide molecular probes possessing photoaffinity and fluorescent
appendages has been achieved. Stereoselective olefin cross-metathesis comprised a key tactic for construction of two of the molecular
probes. Three photoaffinity probes were radiolabeled with tritium.
The mechanism of action of (+)-discodermolide (1, Figure
1), similar to that of the clinically proven anti-cancer agent
paclitaxel (Taxol, 2), entails the binding and stabilization of
microtubules,1 which results in potent inhibition of cell
proliferation in a number of human cancer cell lines.2,3a
Importantly, discodermolide retains activity against pacli-
taxel-resistant cell lines.2a
Despite the shared mechanism, the Horwitz, Smith, and
Danishefsky groups demonstrated that discodermolide and
† University of Pennsylvania.
‡ Albert Einstein College of Medicine.
Figure 1. (+)-Discodermolide and Taxol.
(1) ) Hung, D. T.; Chen, J.; Schreiber, S. L. Chem. Biol. 1996, 3, 287-
293. For the isolation of (+)-discodermolide, see: Gunasekera, S. P.;
Gunasekera, M.; Longley, R. E.; Schulte, G. K. J. Org. Chem. 1990, 55,
paclitaxel exhibit a highly synergistic antiproliferative co-
operativity.3 Similar synergism was not observed with other
4912-4915. Correction J. Org. Chem. 1991, 56, 1346.
(2) (a) Kowalski, R. J.; Giannakakou, P.; Gunasekera, S. P.; Longley,
R. E.; Day, B. W.; Hamel, E. Mol. Pharm. 1997, 52, 613-622. (b) ter
Haar, E.; Kowalski, R. J.; Hamel, E.; Lin, C. M.; Longley, R. E.;
Gunasekera, S. P.; Rosenkranz, H. S.; Day, B. W. Biochemistry 1996, 35,
243-250.
combinations of microtubule-stabilizing agents, such as
paclitaxel and epothilone B, which display only an additive
effect on tumor cell growth inhibition. Further collaboration
10.1021/ol0520166 CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/14/2005

with the Horwitz group revealed that discodermolide, but
not paclitaxel, possesses another mechanism of tumor cell
growth inhibition, specifically the powerful induction of an
accelerated senescence phenotype,4 which may play a role
in the observed synergy.
Scheme 1
That paclitaxel and discodermolide share a common or
overlapping tubulin binding site was shown early on by
Schreiber et al.1 via displacement studies. However, unlike
the definitive structural information on the Taxol-tubulin
binding site, defined by Horwitz and Orr in an elegant series
of photoaffinity labeling studies beginning in 19945 and sub-
sequently shown by Nogales and co-workers by electron dif-
fraction studies,6 the precise binding site and orientation of
discodermolide at the molecular level remains largely spec-
ulative, despite extensive NMR and computational studies.7a,8
To address this issue, we have prepared five discodermolide
photoaffinity probes possessing benzophenone9 appendages.
At the outset of probe design and synthesis, we set four
goals. First, the labeled probe must possess potent biological
activity similar to the natural product; second, the number
of chemical transformations after incorporation of the label
should be minimal; third, the synthetic transformations must
be efficient; and finally, a single radiolabeled precursor, in
this case a benzophenone moiety possessing tritium, would
be ideal. Based on these criteria, and earlier structure-
activity relationship studies, we selected three sites for
benzophenone incorporation: the C(24) terminus, the C(19)
carbamate, and the C(1)-C(7) lactone.
a mixture of several cross-metathesis adducts. Analysis of
the 1H NMR spectrum revealed that not only was the nascent
olefin generated as a mixture of cis and trans isomers, but
that the C(21)-C(22) cis olefin had also undergone signifi-
cant isomerization.
Seeking to remediate the olefin geometry problem, we
examined the cross-metathesis tactic employing protected
butenediol derivatives 6 and 7,11 as well as R,â-unsaturated
ketone 8 (Scheme 2). Again, only complex mixtures of
isomers resulted.
To attach a benzophenone moiety at the C(24) terminus,
we initially explored an olefin cross-metathesis tactic with
readily available, protected discodermolide congeners (Scheme
1). Unfortunately, treatment of either (+)-3 or (+)-4, late-
stage intermediates in our gram-scale synthesis of discoder-
molide,10 with the benzophenone dimer 5, in the presence
of the Grubbs second-generation ruthenium catalyst, afforded
Scheme 2
(3) (a) Martello, L. A.; McDaid, H. M.; Regl, D. L.; Yang, C. H.; Meng,
D.; Pettus, T. R.; Kaufman, M. D.; Arimoto, H.; Danishefsky, S. J.; Smith,
A. B., III; Horwitz, S. B. Clin. Cancer Res. 2000, 6, 1978-1987. (b) Honore,
S.; Kamath, K.; Braguer, D.; Horwitz, S. B.; Wilson, L.; Briand, C.; Jordan,
M. A. Cancer Res. 2004, 64, 4957-4964.
(4) Klein, L. E.; Freeze, B. S.; Smith, A. B., III; Horwitz, S. B. Cell
Cycle 2005, 4, 501-507.
(5) (a) Rao, S.; Krauss, N. E.; Heerding, J. M.; Swindell, C. S.; Ringel,
I.; Orr, G. A.; Horwitz, S. B. J. Biol. Chem. 1994, 269, 3132-3134. (b)
Rao, S.; Orr, G. A.; Chaudhary, A. G.; Kingston, D. G. I.; Horwitz, S. B.
J. Biol. Chem. 1995, 270, 20235-20238. (c) Rao, S.; He, L.; Chakravarty,
S.; Ojima, I.; Orr, G. A.; Horwitz, S. B. J. Biol. Chem. 1999, 274, 37990-
37994.
(6) (a) Nogales, E.; Wolf, S. G.; Downing, K. H. Nature 1998, 391,
199-203. (b) Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K.
H. Cell 1999, 96, 79-88.
Undeterred, we next evaluated 2-butenediol as a cross-
metathesis coupling partner. In the event, treatment of trans-
2-butenediol and alcohol (+)-3 as a mixture (25:1 molar
ratio) with the Grubbs second-generation catalyst in benzene
at 45 °C furnished the desired diol (+)-9 in 71% yield as a
single trans isomer (Scheme 3). A mechanistic rationale for
this stereochemical outcome is under active investigation in
our laboratory.
(7) (a) Martello, L. A.; LaMarche, M. J.; He, L.; Beauchamp, T. J.; Smith,
A. B., III; Horwitz, S. B. Chem. Biol. 2001, 8, 843-855. (b) Burlingame,
M. A.; Shaw, S. J.; Sundermann, K. F.; Zhang, D.; Petryka, J.; Mendoza,
E.; Liu, F.; Myles, D. C.; LaMarche, M. J.; Hirose, T.; Freeze, B. S.; Smith,
A. B., III. Bioorg. Med. Chem. Lett. 2004, 14, 2335-2338. (c) Shaw, S. J.;
Sundermann, K. F.; Burlingame, M. A.; Myles, D. C.; Freeze, B. S.; Xian,
M.; Brouard, I.; Smith, A. B., III. J. Am. Chem. Soc. 2005, 127, 6532-
6533. (d) Smith, A. B., III; Freeze, B. S.; LaMarche, M. J.; Hirose, T.;
Brouard, I.; Xian, M.; Sundermann, K. F.; Shaw, S. J.; Burlingame, M. A.;
Horwitz, S. B.; Myles, D. C. Org. Lett. 2005, 7, 315-318. (e) Smith, A.
B., III; Freeze, B. S.; LaMarche, M. J.; Hirose, T.; Brouard, I.; Rucker, P.
V.; Xian, M.; Sundermann, K. F.; Shaw, S. J.; Burlingame, M. A.; Horwitz,
S. B.; Myles, D. C. Org. Lett. 2005, 7, 311-314.
(10) (a) Smith, A. B., III; Kaufman, M. D.; Beauchamp, T. J.; LaMarche,
M. J.; Arimoto, H. Org. Lett. 1999, 1, 1823-1826. (b) Smith, A. B., III;
Beauchamp, T. J.; LaMarche, M. J.; Kaufman, M. D.; Qiu, Y.; Arimoto,
H.; Jones, D. R.; Kobayashi, K. J. Am. Chem. Soc. 2000, 122, 8654-8664.
(11) Blackwell, H. E.; O’Leary, D. J.; Chatterjee, A. K.; Washenfelder,
R. A.; Bussmann, D. A.; Grubbs, R. H. J. Am. Chem. Soc. 2000, 122, 58-
71.
(8) Monteagudo, E.; Cicero, D. O.; Cornett, B.; Myles, D. C.; Snyder,
J. P. J. Am. Chem. Soc. 2001, 123, 6929-6930.
(9) Dorman, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661-5673.
5200
Org. Lett., Vol. 7, No. 23, 2005

variant (+)-11 were separately subjected to isocyanate 12,
readily available in one step from 4-benzoylbenzoic acid.
Global desilylation furnished benzophenone carbamate (+)-
13 and the 14-normethyl counterpart (+)-14,7e respectively.
Two additional discodermolide photoaffinity probes, com-
prising replacement of the C(1)-C(7) lactone with a ben-
zophenone moiety, were designed and synthesized. For this
effort, Wittig union of known phosphonium salt (+)-1510a
with m- or p-benzoylbenzaldehyde (16 and 17, respectively)
exploiting zinc-oxide chelation13 efficiently furnished the
desired olefins (+)-18 and (+)-19 both in good yield and
with a high degree of cis selectivity (Scheme 6). Removal
of the PMB group under oxidative conditions, followed by
carbamate installation and global desilylation, led to the m-
and p-benzophenone analogues (+)-20 and (+)-21, respec-
tively.
Scheme 3
With an efficient method for diene functionalization in
hand, we turned to incorporation of a prospective benzophe-
none photoaffinity tag. Toward this end, chemoselective
esterification of the primary hydroxyl in diol (+)-9 with
commercially available 4-benzoylbenzoic acid in the presence
of 1-hydroxylbenzotriazole (HOBT) and 1-ethyl-3-(3′-di-
methylaminopropyl)carbodiimide methyl iodide salt (EDCI-
MeI) furnished the corresponding ester in excellent yield
(Scheme 4). Introduction of the requisite discodermolide
Scheme 6
Scheme 4
carbamate moiety via the Kocovsky protocol,12 followed by
global deprotection, then furnished the target ester (+)-10.
We next examined substitution of the discodermolide
C(19) carbamate with a carbamate bearing a benzophenone
moiety (Scheme 5). Here, alcohol (+)-3 and the 14-normethyl
Evaluation of the antiproliferative activity as well as the
tubulin polymerization profile (as measured by a turbidity
assay)14 for each of the potential photoaffinity probes was
carried out (Table 1). Based on these results, three com-
Scheme 5
Table 1. Bioassay Data for (+)-Discodermolide (1) and for the
Designed Molecular Probes
tubulin
A549
tubulin
A549
polymerization IC50
polymerization IC50
(%)
(nM)
(%)
(nM)
(+)-1
100
52
70
5
19.7
18
7.2
40
(+)-20
(+)-21
(+)-28
(+)-31
10
8
94
58
259
92
16
(+)-10
(+)-13
(+)-14
6.2
pounds [(+)-10, (+)-13, and (+)-14] were selected as
prospective photoaffinity probes, requiring incorporation of
a radiolabel.
(12) Kocovsky, P. Tetrahedron Lett. 1986, 27, 5521-5524.
(13) Moison, H.; Texier-Boullet, F.; Foucaud, A. Tetrahedron 1987, 43,
537-542.
Org. Lett., Vol. 7, No. 23, 2005
5201

By design, the radiolabeled compounds were each con-
structed employing the same [3H]-4-benzoylbenzoic acid
intermediate 26, the synthesis of which is detailed in Scheme
7. The iodine/tritium exchange was outsourced to Perkin-
Elmer Life Sciences, New England Nuclear Division.
For the second fluorescent probe, attachment via the C(19)
carbamate was selected (Scheme 9). To this end, desymme-
trization of carbonyl diimidazole (CDI)17 by treatment with
alcohol (+)-3 in CH2Cl2 provided imidazole carbamate (+)-
29. Subsequent displacement of imidazole by dansyl sulfon-
amide 30, followed by global desilylation, furnished the
dansyl-substituted carbamate derivative (+)-31. Pleasingly,
Scheme 7
Scheme 9
The [3H]-4-benzoylbenzoic acid thus obtained was em-
ployed, after dilution with nonradiolabeled material, exactly
as described above for production of radiolabeled photoaf-
finity probes [3H]-10, [3H]-13, and [3H]-14.15 Importantly,
only two transformations were required with the radiolabeled
material. Studies directed toward tubulin photoincorporation,
digestion, and analysis are ongoing in the Horwitz Laboratory
and will be reported in due course.
In addition to the discodermolide photoaffinity probes
outlined above, two molecular probes each incorporating a
fluorescent dansyl tag were designed and synthesized. Such
probes hold the promise of localization of discodermolide in
treated cells and in turn the possibility of more precise in-
formation on the cellular mechanism(s) of action and possibly
on the nature of the synergistic relationship with paclitaxel.
For the first fluorescent probe we opted to append the
diene, exploiting the same protocol that proved successful
for the benzophenone counterpart (Scheme 8). Thus, chemose-
fluorescent probes (+)-28 and (+)-31 each exhibited potent
cell growth inhibitory activity and tubulin binding properties
(Table 1).
In conclusion, five prospective discodermolide photoaf-
finity probes possessing a benzophenone moiety have been
designed, synthesized, and evaluated for tubulin polymeri-
zation and cytotoxicity. Three of these probes, (+)-10, (+)-13,
and (+)-14, were then prepared in tritiated form and sub-
mitted for tubulin photoincorporation, digestion, and analysis
experiments in the Horwitz Laboratory. In addition, two dis-
codermolide fluorescent probes that hold the promise for elu-
cidation of the cellular location and the mechanism of action
of (+)-discodermolide have been designed and synthesized.
The results of biological investigations exploiting both the
photoaffinity and the fluorescent probes will be reported.
Scheme 8
Acknowledgment. Financial support was provided by the
National Institutes of Health (Institute of General Medical
Sciences) through Grant No. GM-29028 and the Department
of the Army through Grant No. DAMD 17-00-1-0404. We
also thank the American Cancer Society for a Postdoctoral
Fellowship (PF-99-353-01-CDD) to P.V.R. and the Minis-
terio de Educacio´n, Cultura y Deportes (Spain), for a
Postdoctoral Fellowship to I.B.
Supporting Information Available: Representative pro-
cedures, spectral data, and analytical data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
lective esterification of the primary hydroxyl in diol (+)-9
with acid 27,16 followed by carbamate installation and global
deprotection, furnished (+)-28.
OL0520166
(14) Gaskin, F.; Cantor, C. R.; Shelanski, M. L. J. Mol. Biol. 1974, 89,
737-758.
(15) Detailed experimental procedures for the production of the radio-
labeled compounds are presented in the Supporting Information.
(16) Acid 27 is generated via amination of dansyl chloride with
6-aminohexanoic acid.
(17) D’Addona, D.; Bochet, C. G. Tetrahedron Lett. 2001, 42, 5227-
5229.
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