Substitution on the A-ring confers to bryopyran analogues the unique biological activity characteristic of bryostatins and distinct from that of the phorbol esters

Article abstract of DOI:10.1021/ol8027253

(Chemical Equation Presented) A close structural analogue of bryostatin 1, which differs from bryostatin 1 only by the absence of the C₃₀ carbomethoxy group (on the C₁₃ enoate of the B-ring), has been prepared by total synthesis. Bio

Full text of DOI:10.1021/ol8027253

ORGANIC
LETTERS
2009
Vol. 11, No. 3
593-596
Substitution on the A-Ring Confers to
Bryopyran Analogues the Unique
Biological Activity Characteristic of
Bryostatins and Distinct From That of
the Phorbol Esters
Gary E. Keck,*,† Yam B. Poudel,† Dennie S. Welch,‡ Matthew B. Kraft,† Anh
P. Truong,§ Jeffrey C. Stephens,† Noemi Kedei,| Nancy E. Lewin,| and Peter
M. Blumberg|
Department of Chemistry, UniVersity of Utah, 315 South 1400 East, RM 2020, Salt
Lake City, Utah 84112 and LCBG, Center for Cancer Research, NCI, National
Institutes of Health, Bethesda, Maryland 20892
keck@chemistry.utah.edu
Received November 26, 2008
ABSTRACT
A close structural analogue of bryostatin 1, which differs from bryostatin 1 only by the absence of the C30 carbomethoxy group (on the C13
enoate of the B-ring), has been prepared by total synthesis. Biological assays reveal a crucial role for substitution in the bryostatin 1 A-ring
in conferring those responses which are characteristic of bryostatin 1 and distinct from those observed with PMA.
The bryostatins are a family of highly complex marine natural
products originally isolated by Pettit using activity against
P-388 leukemia cells to guide the fractionation of the crude
extracts.1 Of the 20 known members, by far the most
extensively studied is bryostatin 1 (1) (Figure 1).2 Bryostatin
1 has been shown to exhibit a remarkable profile of biological
effects, including potent activity against a number of cancers.
Bryostatin 1 has also been shown to stimulate the immune
† University of Utah.
‡ Present address: Abbott Laboratories, North Chicago, IL 60064.
§ Present address: Elan Corporation, South San Francisco, CA 94080.
| National Institutes of Health.
Figure 1. Structure of bryostatin 1.
(1) Pettit, G. R.; Herald, C. L.; Douobek, D. L.; Herald, D. L. J. Am.
Chem. Soc. 1982, 104, 6846–6848.
system,3 which stands in contrast to many established
oncolytic agents and which may play a role in the observed
(2) Hale, K. J.; Hummersome, M. G.; Manaviazar, S.; Frigerio, M. Nat.
Prod. Rep. 2002, 19, 413–453.
10.1021/ol8027253 CCC: $40.75
Published on Web 12/29/2008
2009 American Chemical Society

antitumor effects. Recently, bryostatin 1 has been shown to
exhibit profound effects on memory in animal models4 and
to have significant activity against Alzheimer’s disease in
transgenic mouse models.5 An even more recent exciting
finding on the neurological effects of this agent is its ability
to reverse damage from stroke and to effect neural growth
and repair.6
Bryostatin 1 has also shown remarkable synergistic effects
with a number of established oncolytic agents, including
vincristine, paclitaxel, gemcitibine, and flavopyridol.7 These
intriguing synergies have been shown to be quite complex
in that the results depend heavily upon dosing schedules
(including which agent is administered first) and methods
of administration. In some cases, studies focusing on the
underlying mechanisms for the synergies have been per-
formed.8
Figure 2. Structures of previous bryopyran analogues.
bryostatin 1 is not tumor promoting and functionally
antagonizes many of the responses induced by the phorbol
esters.
Although the precise mechanisms by which bryostatin 1
leads to these observed biological results have not been
rigorously established, it is thought that they are a conse-
quence of binding to the C1 domains of PKC isozymes9 and
to other C1 domain containing proteins such as the RasGRPs,
the chimerins, and the Munc13 proteins.10 Thus bryostatin
1 is well-known to have very high binding affinity for the
PKCs (Ki ) 1.35 nM with PKCR), and these proteins play
critical roles in cell signaling processes relevant to cellular
events including proliferation, differentiation, motility, adhe-
sion, and apoptosis.11 In addition to the natural activators of
PKC (diacylglycerols, DAGs), numerous other activators
with high affinity for PKC are known; the most thoroughly
studied of these are the phorbol esters. However, whereas
bryostatin 1 binds PKC (Ki with PKCR ) 1.35 nM) with
similar affinity to that of phorbol-12-myristate-13-acetate
(PMA, Ki ) 1.17 nM), the events induced subsequent to
binding are quite different for the two agents.12 In particular,
In an effort to identify the mechanisms responsible for
the unique activity of bryostatin 1, we are attempting to
determine, through chemical synthesis, the structural features
of bryostatin that are responsible for its unique biological
profile. Toward this end, we have developed and reported
on powerful enabling methodology for the construction of
pyran rings bearing flexible and malleable substitution
precisely where it is needed for the preparation of a variety
of bryostatin analogues13 and applied this to the synthesis
of the bryopyran core structure.14
In an earlier report, we described the synthesis of three
agents (2-4, Figure 2) based on the bryostatin trispyran core
structure.15 These close mimics of the bryostatin 1 structure
were found to have very high affinity for PKC (Ki with
PKCR ) 0.70-1.05 nM) but to be similar to PMA in terms
of the results of both proliferation and attachment assays with
U937 leukemia cells, a system where exposure to bryostatin
1 and PMA has been established to give very different
biological end points and where bryostatin 1 antagonizes the
PMA response.16 Characterization in multiple additional
assays where bryostatin 1 is distinguished from phorbol esters
in terms of biological response further supports the initial
finding that these bryostatin analogues largely function as
phorbol ester mimics (unpublished observations). No de-
pendence on the nature of the C20 substituent was seen. Thus,
although these compounds are close structural analogues of
bryostatin 1, they are close functional analogues of PMA.
In this paper, we report studies which reveal that the A-ring
functionality of bryostatin 1 is critical in conferring bryosta-
tin-like biological responses as opposed to those characteristic
(3) Koutcher, J. A.; Motwani, M.; Zakian, K. L.; Li, X.-K.; Matei, C.;
Dyke, J. P.; Ballon, D.; Yoo, H.-H.; Schwartz, G. K Clin. Cancer Res.
2000, 6, 1498–1507.
(4) (a) Alkon, D. L.; Epstein, H.; Kuzirian, A.; Bennett, M. C.; Nelson,
T. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16432–16437. (b) Sun, M.-K.;
Alkon, D. L. Eur. J. Pharmacol. 2005, 512, 43–51.
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der Auwera, I.; Wera, S.; Qiao, L.; 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. (b) Alkon, D. L.; Sun, M.-K.; Nelson, T. J. Trends
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Acad. Sci. U.S.A. 2008, 105, 13620–13625.
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Clin. Cancer Res. 2000, 6, 4950–4956. (b) Koutcher, J. A.; Motwani, M.;
Zakian, K. L.; Li, X.-K.; Matei, C.; Dyke, J. P.; Ballon, D.; Yoo, H.-H.;
Schwartz, G. K. Clin. Cancer Res. 2000, 6, 1498–1507. (c) El-Rayes, B. F.;
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Munksgaard: Copenhagen, 1992; pp 273-285.
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Org. Lett., Vol. 11, No. 3, 2009

Scheme 1. Synthesis of the “Almost Bryostatin” Bryopyran 12
of the tumor-promoting phorbol esters. This was accom-
plished through total synthesis and biological evaluation of
the “almost bryostatin” analogue 12, which lacks only the
C30 carbomethoxy group of bryostatin 1 itself.
reduction and acylation of the resulting alcohol provided 10
as essentially a single diastereomer. The correct C20 and C34
stereochemistry was confirmed by the observation of a strong
NOE between the respective protons at these positions.
This intermediate was readied for macrolactonization by
a sequence designed to avoid conducting this reaction on a
dihydroxy acid, which has proven problematic. Removal of
the BPS group at C3 proved necessary to allow for hydrolysis
of the thiolester using LiOH and H2O2. Attempted hydrolysis
with the BPS protected thiolester was slow and resulted in
the hydrolysis of the C7 acetate as well. The resulting
hydroxy acid was reprotected at C3 using TESCl. Following
removal of the PMB group at C25, macrolactonization using
the Yamaguchi20 conditions afforded the desired macrolac-
tone in 62% yield. Global deprotection (hydrolysis of two
mixed ketals, C3TES, and BOM ether) using the Lipshutz21
method (LiBF4 in aqueous acetonitrile) then gave the desired
“almost bryostatin” analogue 12.
The synthetic plan uses our pyran annulation methodol-
ogy13 to join preformed A-ring and C-ring subunits with
concomitant formation of the B-ring, according to an overall
strategy which has been suggested previously.14,17 Although
superficially similar to the route used previously to prepare
2-4, this synthesis reverses the roles of the coupling partners
for the critical pyran annulation. The resulting differences
then track back to a synthetic route for the A-ring component
totally different from that utilized previously, as well as
significant differences in the functionality present after pyran
annulation.
As shown in Scheme 1, reaction of the A-ring aldehyde 5
with hydroxy allylsilane 6,17 using TMSOTf in ether, gave
the desired tricyclic adduct 7.18 Oxidative functionalization
of the C-ring glycal afforded ketone 8, which was converted
to enoate 9 by an aldol-elimination sequence.19 Luche
Initial assay of this analogue for PKC binding showed, as
expected, high affinity for PKCR like that shown by 1 (Ki
) 0.52 ( 0.06 nM, average of three experiments). Initial
assays for function used the U937 leukemia proliferation and
attachment assays previously mentioned in connection with
(17) (a) Keck, G. E.; Welch, D. S.; Vivian, P. K. Org. Lett. 2006, 8,
3667–3670. (b) Keck, G. E.; Welch, D. S.; Poudel, Y. B. Tetrahedron Lett.
2006, 47, 8267–8270
.
(18) Unreacted aldehyde 5 and the O-trimethylsilyl derivative of 6 were
isolated in 35% and 27% yields, respectively, and subsequently recyled.
(19) Use of 1.1 equiv of LDA gave incomplete conversion of ketone 8
to the desired aldol adduct. Attempts to increase conversion resulted in
reaction also occurring at the C7 acetate.
(20) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. Jpn. 1979, 52, 1989–1993.
(21) Lipschutz, B. H.; Harvey, D. F. Synth. Commun. 1982, 14, 267–
277.
Org. Lett., Vol. 11, No. 3, 2009
595

whereas the phorbol ester PMA is. Moreover, bryostatin 1
is able to block the effect of PMA in a dose-dependent
manner. Both of these aspects of bryostatin 1 induced
biological response are captured by 12.
Taken together with the previous results for analogue 4,
these results clearly show that: (1) the C30 carbomethoxy
group is not essential to obtain bryostatin-like biological
responses with these analogues, and (2) the structure in the
C7-C9 region of the A-ring is critical in conferring bryosta-
tin-like biological responses as opposed to those characteristic
of the tumor-promoting phorbol esters. Practical implications
are that bryostatin analogues simplified in the B-ring may
provide effective biological surrogates of bryostatin 1 for
therapy, whereas bryologues lacking appropriate substitution
in the A-ring instead fall within the broad family of potent
PKC activators but do not capture the special behavior of
bryostatin 1.
Figure 3. Effect of analogue 12 on U937 cell attachment.
analogues 2-4.16 The results for these assays are shown in
Figures 3 and 4.
For a long time, most other work on assessment of
synthetic bryostatin analogues has focused on binding and
simple measures of biological potency, largely skipping over
the nature of the biological responses induced by the
analogues.22 This has led to the view that the A- and B-rings
of the structure serve merely as “spacer domains” which
simply hold other groups in the molecule in the proper
positions.23 Clearly this is not the case when function is
considered. Our results show that analogues such as 2-4,
although based on the bryostatin platform, appear to living
cells to be much like phorbol esters and that bryostatin-like
function can be restored upon inclusion of appropriate A-ring
functionality.
In the attachment assay (Figure 3), PMA induces attach-
ment while bryostatin 1 shows a much diminished effect.
Moreover, when both agents are administered together,
bryostatin 1 blocks the effect of the phorbol ester in a dose-
dependent manner. In marked contrast to analogue 4, the
response of agent 12 in the attachment assay approaches that
of bryostatin 1 itself. Analogue 12 can be seen even to
display the dose-dependent biphasic response characteristic
of exposure to bryostatin 1. In addition, analogue 12, like
bryostatin 1, is a functional antagonist of PMA and blocks
the effect of the phorbol ester when the two agents are
administered together.
The critical structural features of bryostatin 1 primarily
responsible for its unique behavior are now reduced to
substitution at just three carbons. Efforts to elucidate the role
of substituents at these positions on function are in progress.
Acknowledgment. Financial support was provided by the
NIH through grant GM28961 and through the Intramural
Research Program, CCR, NCI, NIH.
Supporting Information Available: Experimental pro-
cedures, assay results, and spectral data. This material is
available free of charge via the Internet at http://pubs.acs.org.
OL8027253
(22) (a) Wender, P. A.; Verma, V. A. Org. Lett. 2008, 10, 3331–3334.
(b) Wender, P. A.; DeChristopher, B. A.; Schrier, A. J. J. Am. Chem. Soc.
2008, 130, 6658–6659. (c) Wender, P. A.; Debrabander, J.; Harran, P. G.;
Jimenez, J. M.; Koehler, M. F. T.; Lippa, B.; Park, C. M.; Shiozaki, M
J. Am. Chem. Soc. 1998, 120, 4534–4535. Note also references therein.
(23) Wender, P. A.; Baryza, J. L.; Hilinski, M. K.; Horan, J. C.; Kan,
C.; Verma, V. A. In Drug DiscoVery Research: New Frontiers in the Post-
Genomic Era; Huang, Z., Ed.; Wiley-VCH: Hoboken, NJ, 2007; pp
127-162. Note also the references cited in ref 22 and references therein.
Figure 4. Inhibition of proliferation assay for analogue 12.
The results with analogue 12 in the proliferation assay
(Figure 4) are likewise very similar to those for bryostatin
1. In this assay, bryostatin 1 is not strongly antiproliferative,
596
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