Role of the A-ring of bryostatin analogues in PKC binding: Synthesis and initial biological evaluation of new A-ring-modified bryologs

Article abstract of DOI:10.1021/ol0504650

(Chemical Equation Presented) The syntheses of three newly designed bryostatin analogues are reported. These simplified analogues, which lack the A-ring present in the natural product but possess differing groups at C9, were obtained using a divergent app

Full text of DOI:10.1021/ol0504650

ORGANIC
LETTERS
2005
Vol. 7, No. 10
1995-1998
Role of the A-Ring of Bryostatin
Analogues in PKC Binding: Synthesis
and Initial Biological Evaluation of New
A-Ring-Modified Bryologs
Paul A. Wender,*,†,‡ Michael O. Clarke,† and Joshua C. Horan†
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080, and
Department of Molecular Pharmacology, Stanford UniVersity School of Medicine,
Stanford UniVersity, Stanford, California 94305-5080
wenderp@stanford.edu
Received March 3, 2005
ABSTRACT
The syntheses of three newly designed bryostatin analogues are reported. These simplified analogues, which lack the A-ring present in the
natural product but possess differing groups at C9, were obtained using a divergent approach from a common intermediate. All three analogues
exhibit potent, single-digit nanomolar affinity to protein kinase C.
The bryostatins are a family of structurally complex, marine-
derived macrocyclic polyketides1 that exhibit a unique range
of significant biological activities, including induction of
apoptosis, reversal of multidrug resistance, immune system
modulation, and efficacy against Alzheimer’s disease.2 Of
special therapeutic importance, bryostatin 1 has been shown
to enhance the overall efficacy of other oncolytic agents,
suggesting its potential use in combination therapy.3 Bryo-
statin 1 is currently in phase I and II clinical trials, as both
a single agent and in combination with other therapeutics.4
As a natural product, bryostatin is neither produced nor
selected by nature for human therapeutic performance.
Consequently, its advancement in clinical trials has been
slowed by both its scarcity and the associated difficulties in
accessing derivatives of this complex system. Harvesting of
bryostatin from marine sources is unlikely to provide a
sustainable supply due to justifiable concerns about the
delicate marine ecosystem. Aquaculture, genetic, and other
biosynthetic approaches offer alternative sources but would
still provide only bryostatin or its readily accessible deriva-
tives.5 Total synthesis provides more flexible access to
analogues, but its use is limited by the length of existing
syntheses (>70 steps).1 On the positive side, and pertinent
to these supply issues, is the exceptional potency of bryo-
statin: a full multiweek treatment only requires about 1.2
mg per patient.
† Stanford University.
‡ Stanford University School of Medicine.
(1) Hale, K. J.; Hummersone, M. G.; Manaviazar, S.; Frigerio, M. Nat.
Prod. Rep. 2002, 19, 413-453.
(2) (a) Kortmansky, J.; Schwartz, G. K. Cancer InVest. 2003, 21, 924-
936. (b) Mutter, R.; Wills, M. Bioorg. Med. Chem. 2000, 8, 1841-1860.
(c) A leading reference on Alzheimer’s research: Etcheberrigaray, R.; Tan,
M.; Dewachter, I.; Kuiperi, C.; Van der Auwera, I.; Wera, S.; Qiao, L. X.;
Bank, B.; Nelson, T. J.; Kozikowski, A. P.; Van Leuven, F.; Alkon, D. L.
Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11141-11146.
(3) (a) Mohammad, R. M.; Wall, N. R.; Dutcher, J. A.; Al-Katib, A. M.
Clin. Cancer Res. 2000, 6, 4950-4956. (b) Wang, S.; Wang, Z.; Boise, L.
H.; Dent, P.; Grant, S. Leukemia 1999, 13, 1564-1573.
(4) For current information, see: http://clinicaltrials.gov.
(5) (a) Aquaculture: Mendola, D. Biomol. Eng. 2003, 20, 441-458. (b)
Biosynthesis: Hildebrand, M.; Waggoner, L. E.; Liu, H.; Sudek, S.; Allen,
S.; Anderson, C.; Sherman, D. H.; Haygood, M. Chem. Biol. 2004, 11,
1543-1552.
10.1021/ol0504650 CCC: $30.25
© 2005 American Chemical Society
Published on Web 04/19/2005

Prompted by these considerations and the unique activity
profile of bryostatin, we set out to design simplified
analogues of bryostatin that could be produced in a practical
fashion through total synthesis and tuned for optimal
performance in the clinic. Toward this end, we proposed a
pharmacophore hypothesis for how bryostatin contacts its
putative receptor, protein kinase C (PKC), and designed
analogues that incorporate these features into a simplified
scaffold.6 Representative of this effort, analogue 1 (Figure
1), which is a more potent ligand for PKC than bryostatin,
affinity for the rat brain PKC isozyme mixture. Comparison
of analogues 1 and 2 suggests that the introduction of a
substituent at C9 could improve binding affinity by confer-
ring a higher level of conformational rigidity to the macro-
cycle.10 At the same time, this new substituent introduces
structural elements that could control target selectivity, PK,
and ADME characteristics for downstream preclinical evalu-
ation. In this paper, the synthesis and initial biological assay
of three A-ring-modified bryostatin analogues, 3, 4, and 5,
are reported.
The sterically demanding tert-butyl10a and phenyl substit-
uents in 3 and 4 were chosen to increase the conformational
rigidity in the former A-ring region of the molecule. At the
same time, the p-bromo-phenylpropyl substituent in 5 was
selected to examine the effect of a less sterically demanding
substituent and to provide a convenient handle (the aryl
bromide) for later diversification.
The new analogues were synthesized in a convergent
manner by coupling a top “spacer domain” with a bottom
“recognition domain” employing what has proven to be an
exceptionally effective two-step macrolactonization se-
quence.7 As a consequence of this late-stage convergence
of fragments, the generation of new A-ring-modified ana-
logues required only the synthesis of new spacer domains.
Scheme 1. Synthesis of Intermediates 8, 10, and 12a
Figure 1. Bryostatin 1 and synthetic analogues.
is more effective at inhibiting the growth of a variety of
human cancer cell types than the natural product. Compound
1 can be synthesized in less than half the number of steps
required to synthesize bryostatin and is available at a fraction
of the cost required to obtain the natural product from marine
sources.7
With the demonstration that simplified analogues retain
PKC binding as well as human cancer cell growth inhibitory
potency and can be prepared in a practical fashion, the next
goal of this program was to refine these new leads8 to
enhance beneficial function, minimize side effects, and
explore the mode of action.9 Toward these goals, a new
analogue 2 was recently disclosed wherein the A-ring of the
bryostatin analogue skeleton had been removed.6 Interest-
ingly, this new analogue exhibited single-digit nanomolar
(6) Wender, P. A.; Baryza, J. L.; Brenner, S. E.; Clarke, M. O.; Craske,
M. L.; Horan, J. C.; Meyer, T. Curr. Drug DiscoVery Technol. 2004, 1,
1-11.
(7) Wender, P. A.; Baryza, J. L.; Bennett, C. E.; Bi, F. C.; Brenner, S.
E.; Clarke, M. O.; Horan, J. C.; Kan, C.; LaCote, E.; Lippa, B. S.; Nell, P.
G.; Turner, T. M. J. Am. Chem. Soc. 2002, 124, 13648-13649.
(8) (a) Wender, P. A.; Baryza, J. L. Org. Lett. 2005, 7, 1177-1180. (b)
Wender, P. A.; Hilinski, M. K.; Mayweg, A. V. W. Org. Lett. 2005, 7,
79-82.
a DMP ) Dess-Martin periodinane. TPAP ) tetrapropyl-
ammonium perruthenate.
(9) Baryza, J. L.; Brenner, S. E.; Craske, M. L.; Meyer, T.; Wender, P.
A. Chem. Biol. 2004. 11, 1261-1267.
1996
Org. Lett., Vol. 7, No. 10, 2005

These were then coupled with the preexisting recognition
domain for which we have reported a practical synthesis.
The synthesis of these new spacer domains began with
installation of the appropriate “R” group into known aldehyde
6 (Scheme 1).7 In the cases where R ) t-Bu or Ph, the group
is installed via addition of the corresponding carbanion to
generate a mixture of secondary alcohols. Alcohols 7 and 8
were easily separated, and the undesired epimer was recycled
through a two-step oxidation/reduction procedure. For R )
Ph, the diastereomers were not easily separable and the
mixture was subjected to the same two-step oxidation/
reduction sequence to generate diastereomerically pure
alcohol 10. Preparation of the more elaborate intermediate
12 started with an asymmetric Brown’s allylation of aldehyde
6 followed by TBS protection to give silyl ether 11.11 Cross
metathesis with p-bromo styrene12 and subsequent reduction
of the olefin using Rh on alumina13 gave silyl ether 12.
lation generated inseparable mixtures of the product alcohols
and the pinanol byproduct from the allylation reagent.
Subsequent TBS protection allowed for isolation of the
diastereomerically pure silyl ethers 19 and 22. Silyl ether
20 (R ) Ph) was isolated as an inseparable mixture of
diastereomers that could be separated after removal of the
silyl group. Reprotection provided silyl ether 20 in diaste-
reomerically pure form. Oxidative cleavage of the terminal
olefins using KMnO4 and NaIO4 gave the completed spacer
domains 23, 24, and 25.
Each of the spacer domains was coupled individually to
the existing recognition domain 267 using the PyBroP
coupling reagent (Scheme 3).15 The macrocycles were closed
Scheme 3. Completion of Analogues 3, 4, and 5
After desilylation of 12, each of the secondary alcohols
was carried independently through a parallel synthetic
sequence to complete the individual spacer domains (Scheme
2). Allylation of the individual alcohols with allyl bromide
Scheme 2. Completion of Spacer Domains 23, 24, and 25
and the silyl protecting groups removed in a remarkably
general one-step, mild, and diastereoselective macrotrans-
acetalization, providing the completed analogues 3, 4, and
5. The newly formed stereocenter in each is set under
thermodynamic control affording only the cis-diequatorial
dioxolane B-ring.
These new analogues exhibited single-digit nanomolar
binding affinities for rat brain PKC when tested in a
competition binding assay against the known PKC ligand
phorbol 12,13-dibutyrate (3, Ki ) 6.5 nM; 4, Ki ) 2.3 nM;
5, Ki ) 1.9 nM).16 Significantly, these analogues exhibit
binding potencies superior to analogue 2 and on par with
bryostatin 1. These data demonstrate that extensive modifica-
tions can be made to the A-ring region without affecting
binding affinity, indicating that the C9 region could be
modified as needed to tune ADME and pharmacokinetic
characteristics. To ascertain whether these new analogues
will elicit biological responses similar to bryostatin 1, studies
exploring the response of individual PKC isozymes to these
new ligands, as well as the in vitro functional differences
among them, are currently underway.
gave terminal olefins 13, 14, and 15. Hydroboration of these
olefins followed by Dess-Martin periodinane oxidation gave
aldehydes 16, 17, and 18, respectively.14 Asymmetric ally-
(10) (a) Wender, P. A.; DeBrabander, J.; Harran, P. G.; Hinkle, K. W.;
Lippa, B.; Pettit, G. R. Tetrahedron Lett. 1998, 39, 8625-8628. (b) Wender,
P. A.; Lippa, B. Tetrahedron Lett. 2000, 41, 1007-1011.
(11) Brown, H. C.; Jadhav, P. K. J. Am. Chem. Soc. 1983, 105, 2092-
2093.
(12) Morgan, J. P.; Grubbs, R. H. Org. Lett. 2000, 2, 3153-3155.
(13) Danheiser, R. L.; Helgason, A. L. J. Am. Chem. Soc. 1994, 116,
9471-9479.
(14) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277-
7287.
(15) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. Jpn. 1979, 52, 1989-1993.
(16) All PKC binding experiments were performed against a rat brain
isozyme mixture as described in refs 6 and 7.
Org. Lett., Vol. 7, No. 10, 2005
1997

Acknowledgment. Support of this work through a grant
(CA31845) provided by the NIH is gratefully acknowledged.
We would like to thank the Daria Mochly-Rosen group for
assistance with the PKC assay. HRMS analyses for some
compounds were performed at UCSF.
Supporting Information Available: Experimental condi-
tions and spectral data for compounds reported in this paper.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL0504650
1998
Org. Lett., Vol. 7, No. 10, 2005