Facile synthetic access to and biological evaluation of the macrocyclic core of apoptolidin

Article abstract of DOI:10.1021/ol0346335

(Matrix presented) Oxidative cleavage of the C-20/C-21 bond in apoptolidin (1) provides two fragments of similar complexity, facilitating a divide-and-diversify strategy for the determination of the structural basis for apoptolidin's biological activity,

Full text of DOI:10.1021/ol0346335

ORGANIC
LETTERS
2003
Vol. 5, No. 13
2299-2302
Facile Synthetic Access to and
Biological Evaluation of the Macrocyclic
Core of Apoptolidin
,†
Paul A. Wender,* Orion D. Jankowski,† Elie A. Tabet,† and Haruo Seto‡
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080, and
Department of Applied Biology and Chemistry, Faculty of Applied Bio-Science Tokyo
UniVersity of Agriculture, Japan
wenderp@stanford.edu
Received April 11, 2003
ABSTRACT
Oxidative cleavage of the C-20/C-21 bond in apoptolidin (1) provides two fragments of similar complexity, facilitating a divide-and-diversify
strategy for the determination of the structural basis for apoptolidin’s biological activity, the remarkably selective induction of apoptosis in
sensitive cell lines. The ability of compounds derived from this cleavage to inhibit mitochondrial F F1-ATPase is reported.
0
In 1997, Seto and co-workers isolated the 20-membered
macrolide apoptolidin (1) in a screen for novel compounds
that selectively induce apoptosis in cells transformed with
the E1A oncogene.1 Apoptolidin was subsequently found to
be among the top 0.1% most selective of 37 000 compounds
assayed in the National Cancer Institute’s 60 cell line screen.2
Khosla and co-workers have demonstrated that apoptolidin
is an inhibitor of F0F1-ATPase and proposed that the
inhibition of this enzyme could be the basis for the reported
biological activity of 1.3 Because of the remarkably selective
activity of 1 and its potentially novel mode of action,
apoptolidin represents a promising lead compound for the
treatment of cancer. The medicinal potential and structural
complexity of apoptolidin has prompted considerable interest
in synthetic approaches to this target.4 A highly promising
alternative approach to accessing this compound, and more
importantly, analogues with clinical potential, arises from
the fact that 1 can be obtained in high yield from its natural
source (109 mg/L). Consequently, a key challenge for
advancing this therapeutic lead is the development of
methods for the selective modification of its diverse func-
tionality, as needed to access derivatives with improved
activity and stability.
† Stanford University.
‡ Tokyo University of Agriculture.
(1) (a) Kim, J. W.; Adachi, H.; Shin-ya, K.; Hayakawa, Y.; Seto, H. J.
Antibiotics 1997, 50, 628. (b) Hayakawa, Y.; Kim, J. W.; Adachi, H.; Shin-
ya, K.; Fujita, K.; Seto, H. J. Am. Chem. Soc. 1998, 120, 3524.
(2) Developmental Therapeutics Program NCI/NIH. http://dtp.nci.nih.gov
(accessed April 2003).
(3) (a) Salomon, A. R.; Voehringer, D. W.; Herzenberg, L. A.; Khosla,
C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14766. (b) Salomon, A. R.;
Voehringer, D. W.; Herzenberg, L. A.; Khosla, C. Chem. Biol. 2001, 8,
71. (c) Salomon, A. R.; Zhang, Y.; Seto, H.; Khosla, C. Org. Lett. 2001, 3,
57.
10.1021/ol0346335 CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/23/2003

Scheme 1a
Figure 1. Structure of isoapoptolidin.
a Reagents and conditions: (a) NaIO4, MeOH, H2O (1:1) (87%
To improve access to the core macrocycle and preserve
the 6-deoxy-4-O-methyl-L-glucose residue, protection of the
C-2′ or C-3′ alcohols prior to cleavage was explored.
Exposure of 1 to triethylsilyl triflate under high dilution
conditions, as reported previously, gave monosilylated apo-
ptolidin 5 in 36% yield along with recovered starting material
and bis-silylated apoptolidin (Scheme 2).6 With the C-2′
4).
It has been shown that apoptolidin isomerizes in aqueous
solution or slightly basic methanol to its ring-expanded
isomer, isoapoptolidin (2, Figure 1).5 This isomer is a less
potent inhibitor of mitochondrial F0F1-ATPase and is cer-
tainly present under the conditions used in cell-based assays.
As part of our efforts to elucidate the structural basis of
apoptolidin’s biological activity and access better and more
stable leads, we previously reported a procedure by which
the hydroxyl group array of apoptolidin can be selectively
functionalized while keeping the core structure intact.6 We
report herein an efficient procedure for readily accessing the
macrocyclic subunit of apoptolidin, thereby providing the
basis for exploring the activity of this subunit, and for
accessing derivatives in which this core is attached to diverse
components.
Scheme 2a
The C-20/C-21 diol functionality in 1 provides an ideal
site for oxidative cleavage to produce the corresponding
macrocyclic aldehyde 3 and δ-lactone 4. Toward this end,
treatment of 1 with sodium periodate in a 1:1 mixture of
methanol/water provided δ-lactone 4 in excellent yield but
only trace amounts of macrolide fragment 3 (Scheme 1).
(4) (a) Schuppan, J.; Wehlan, H.; Keiper, S.; Koert, U. Angew. Chem.,
Int. Ed. 2001, 40, 2063. (b) Toshima, K.; Arita, T.; Kato, K.; Tanaka, D.;
Matsumura, S. Tetrahedron Lett. 2001, 42, 8873. (c) Nicolaou, K. C.; Li,
Y.; Fylaktakidou, K. C.; Mitchell, H. J.; Sugita, K. Angew. Chem., Int. Ed.
2001, 40, 3854. (d) Nicolaou, K. C.; Li, Y.; Fylaktakidou, K. C.; Mitchell,
H. J.; Wei, H. X.; Weyershausen, B. Angew. Chem., Int. Ed. 2001, 40,
3849. (e) Schuppan, J.; Ziemer, B.; Koert, U. Tetrahedron Lett. 2000, 41,
621. (f) Sulikowski, G. A.; Jin, B. H.; Lee, W. M.; Wu, B. Org. Lett. 2000,
2, 1439. (g) Paquette, W. D.; Taylor, R. E. Abstracts of Papers of the Am.
Chem. Soc. 2003, 225, 241-ORGN. (h) Wu, B.; Jin, B.; Qu, T.; Liu, Q.;
Sulikowski, G. A. Abstracts of Papers of the Am. Chem. Soc. 2003, 225,
247-ORGN. (i) Chen, Y. Z.; Fuchs, P. L. Abstracts of Papers, 224th National
Meeting of the American Chemical Society, Boston, MA, Aug 18-22, 2002;
American Chemical Society: Washington, DC, 2002; 640-ORGN. (j)
Chaudhary, K.; Crimmins, M. T. Abstracts of Papers, 224th National
Meeting of the American Chemical Society, Boston, MA, Aug 18-22, 2002;
American Chemical Society: Washington, DC, 2002; 803-ORGN. (k)
Junker, B.; Pauette, B.; Guseilla, D.; Taylor, R. E. Abstracts of Papers,
223rd National Meeting of the American Chemical Society, Orlando, FL,
April 7-11, 2002; American Chemical Society: Washington, DC, 2002;
397-ORGN. (l) Xu, J.; Loh, T. P. Abstracts of Papers, 219th National
Meeting of the American Chemical Society, San Francisco, Mar 26-30,
2000; American Chemical Society: Washington, DC, 2000; 816-ORGN.
(5) (a) Wender, P. A.; Gulledge, A. V.; Jankowski; O. D.; Seto, H. Org.
Lett. 2002, 4, 3819. (b) Pennington, J. D.; Williams, H. J.; Salomon, A. R.;
Sulikowski, G. A. Org. Lett. 2002, 4, 3823.
a Reagents and conditions: (a) TESOTf, DCM, THF, pyridine
or 2,6-lutidine (ref 6).
alcohol suitably protected, NaIO4 mediated cleavage of 5
was investigated. Unfortunately, the only product isolated
from the reaction mixture in synthetically useful amounts
was again δ-lactone 4. This result suggests that the macrolide
portion of apoptolidin is not stable to the relatively acidic
conditions and long reaction times encountered in the
aqueous sodium periodate-mediated oxidation. Attempts to
perform this transformation under neutral or basic aqueous
conditions resulted in no observed reaction.7 Similarly, the
use of either sodium periodate supported on silica gel8 or
(7) Schmid, C. R.; Bryant, J. D.; Dowlatzedah, M.; Phillips, J. L.; Prather,
D. E.; Schantz, R. D.; Sear, N. L.; Vianco, C. S. J. Org. Chem. 1991, 56,
4056.
(6) Wender, P. A.; Jankowski, O. D.; Tabet, E. A.; Seto, H. Org. Lett.
2003, 5, 487.
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Org. Lett., Vol. 5, No. 13, 2003

Scheme 3a
Scheme 4a
a Reagents and conditions: (a) TEA, CD3OD.
salt or benzoyl anhydride followed by deprotection gave the
functionalized macrocycles 13 and 14, respectively. Acyl
migration was not observed under the conditions of these
transformations.
The functionalized macrolide and δ-lactone fragments
were assayed for inhibitory activity against mitochondrial
F0F1-ATPase (Table 1). While the δ-lactone fragment, 4, has
a Reagents and conditions: (a) Pb(OAc)4, DCM, PhMe, TEA
(87% 7, 89% 8); (b) HF‚pyridine, pyridine, THF; (c) NaBH4, THF;
(d) Me3OBF4, 2,6-di-tert-butyl-4-methylpyridine, DCM (41%); (e)
Bz2O, TEA, DMAP, DCM (73%).
Table 1. Activity of Apoptolidin Oxidative Cleavage Productsa
compound
IC50 (µM)
apoptolidin (1)6
0.7 ( 0.5
17 ( 5.0
190 ( 50.0
13 ( 5.0
16 ( 5.0
32 ( 5.0
34 ( 5.0
isoapoptolidin (2)6
4
tetrabutylammonium periodate9 in anhydrous solvents also
failed to achieve the desired oxidation.
12
13
14
15
Screening of other oxidants led to the finding that lead
tetraacetate effects diol cleavage in substrates derived from
1 with the C-20/C-21 diol exposed. After some experimenta-
tion, this reagent was found to work most effectively on
pentatriethylsilyl protected apoptolidin, 6. This reaction
reaches completion rapidly and under mild anhydrous
conditions, thus allowing for the isolation of both δ-lactone
7 and macrocycle 8 in excellent yields (Scheme 3; 87 and
89%, respectively) on up to a 220 mg scale.
With efficient and facile access to quantities of the
macrocyclic core in hand, attention was directed at evaluating
the stabilities and activities of derivatives. For this purpose,
the fully protected macrocyclic aldehyde 8 was smoothly
reduced using sodium borohydride in THF to produce the
primary alcohol 9. It was expected that 9 would be
susceptible to ring-expansion via acyl migration, as has been
observed with apoptolidin.5 Despite this possibility, 9 can
be fully desilylated using buffered HF‚pyridine to produce
macrolide 12 with no evidence of isomerization. In contrast,
when dissolved in a dilute solution of triethylamine in
methanol, macrolide 12 was observed to convert entirely to
its ring-expanded isomer 15 over a period of 72 h (Scheme
4).
a Assay conducted according to the procedure described in ref 5a.
greatly reduced potency as compared with apoptolidin,
macrolide fragments 12-15 retain significant activity in this
assay. Furthermore, alcohol 12 and the corresponding methyl
ether 13 both have a potency that is comparable to that of
isoapoptolidin in this assay system. Benzoate derivative 14,
in contrast, is approximately two times less active than 12
and 13. This trend parallels the slight decrease in potency
that is observed when apoptolidin is functionalized at C-20,
either as the acetate or the methyl ether.6 Ring expansion of
macrolide 12 to isomeric 15, which could be viewed as
functionalization at C-20, also results in a 50% decrease in
potency. The assay results for compounds 12-15 are
consistent with the reported activity of the methanolysis
product 16, as described by Khosla and co-workers (Figure
2).3c Compound 16 is reported to inhibit F0F1-ATPase with
an IC50 of 10 µM but is 2 orders of magnitude less active in
cells that are sensitive to 1. Experiments to correlate the
ability of these compounds to inhibit mitochondrial F0F1-
ATPase with their desired biological activity, the selective
induction of apoptosis, are currently underway.
The primary alcohol in 9 can be efficiently converted to
the methyl ether or benzoate ester as a means of preventing
macrolide ring expansion. Treatment of 9 with Meerwein’s
In summary, a procedure has been developed that allows
for facile access to the macrolide subunit of 1 and to new
derivatives of apoptolidin. The activities of these derivatives
in the F0F1-ATPase assay have been determined. While less
(8) Zhong, Y.-L.; Shing, T. K. M. J. Org. Chem. 1997, 62, 2622.
(9) Santaniello, E.; Manzocchi, A.; Farachi, C. Synthesis 1980, 563.
Org. Lett., Vol. 5, No. 13, 2003
2301

these derivatives, readily available in terms of both speed
and quantity, serve as superb advanced intermediates for
accessing and evaluating the activity of diverse analogues.
Further synthetic and assay studies in this and related series
are in progress.
Acknowledgment. This research was supported by a
grant (CA31845) from the National Cancer Institute. Fel-
lowship support to E.A.T. (American Cancer Society) and
O.D.J. (Eli Lilly) is gratefully acknowledged.
Supporting Information Available: Experimental pro-
cedures and characterization data for compound 4 and
compounds 7-15. This material is available free of charge
via the Internet at http://pubs.acs.org.
Figure 2. Acidic methanolysis product of 1 reported by Khosla
and co-workers.
active than 1, these derivatives do exhibit activity even
though they are greatly simplified structurally. Importantly,
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Org. Lett., Vol. 5, No. 13, 2003