The bryostatin 1 A-ring acetate is not the critical determinant for antagonism of phorbol ester-induced biological responses

Article abstract of DOI:10.1021/ol900585t

The contribution of the A-ring C₇ acetate to the function of bryostatin 1 has been investigated through synthesis and biological evaluation of an analogue incorporating this feature into the bryopyran core structure. No enhanced binding affinit

Full text of DOI:10.1021/ol900585t

ORGANIC
LETTERS
2009
Vol. 11, No. 11
2277-2280
The Bryostatin 1 A-Ring Acetate is Not
the Critical Determinant for Antagonism
of Phorbol Ester-Induced Biological
Responses
Gary E. Keck,* Wei Li, Matthew B. Kraft, Noemi Kedei,† Nancy E. Lewin,† and
Peter M. Blumberg†
UniVersity of Utah, Department of Chemistry, Salt Lake City, Utah 84112
keck@chemistry.utah.edu
Received March 19, 2009
ABSTRACT
The contribution of the A-ring C7 acetate to the function of bryostatin 1 has been investigated through synthesis and biological evaluation of
an analogue incorporating this feature into the bryopyran core structure. No enhanced binding affinity for protein kinase C (PKC) was observed,
relative to previously characterized analogues lacking the C7 acetate. Functional assays showed biological responses characteristic of those
induced by the phorbol ester PMA and distinctly different from those observed with bryostatin 1.
The protein kinase C (PKC) family of kinases plays a central
role in the regulation of crucial cellular events such as
proliferation, differentiation, and apoptosis. Furthermore,
alterations in the expression or activity of PKC isoforms have
been linked to various human diseases such as cancer,1
diabetes,2 and Alzheimer’s disease.3 PKC has thus emerged
as an attractive target for drug development. Attempts at
targeting the catalytic domain of PKC have been hindered
by issues of selectivity since the human genome encodes
approximately 500 protein kinases, each containing the highly
conserved ATP binding pocket.4 The novel and classical
PKC isozymes possess cysteine-rich C1 domains which
recognize endogenous 1,2-diacyglycerols (DAGs) ultimately
leading to the activation of the enzyme. Targeting the C1
domain rather than the catalytic domain provides a potential
strategic advantage in drug selectivity since only a small
number of other enzymes are known to contain a C1 domain,
e.g., the chimaerins, RasGRPs, PKDs, Unc-13s, and the
diacylglycerol kinases.5
The first known exogenous ligands for the PKC C1 domain
were the tumor promoting phorbol esters such as phorbol
12-myristate-13-acetate (PMA, 2) (Figure 1), leading to the
hypothesis that their tumor promoting activity was in part
† Laboratory of Cancer Biology and Genetics, Center for Cancer
Research, NCI, NIH, Bethesda, MD 20892.
(1) Griner, E. M.; Kazanietz, M. G. Nat. ReV. Cancer 2007, 7, 281–
294.
(2) Way, K. J.; Katai, N.; King, G. L. Diabetic Med. 2001, 18, 945–
959.
(5) (a) Blumberg, P. M.; Kedei, N.; Lewin, N. E.; Yang, D.; Czifra, G.;
Pu, Y.; Peach, M. L.; Marquez, V. E. Curr. Drug Targets 2008, 9, 641–
652. (b) Teicher, B. A. Clin. Cancer Res. 2006, 12, 5336–5345. (c)
Kazanietz, M. Biochim. Biophys. Acta 2005, 1754, 296–304. (d) Gould,
C. M.; Newton, A. C. Curr. Drug Targets 2008, 9, 614–625.
(3) Alkon, D. L.; Sun, M.-K.; Nelson, T. J. Trends Pharmacol. Sci. 2007,
28, 51–60.
(4) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam,
S. Science 2002, 298, 1912–2934.
10.1021/ol900585t CCC: $40.75
Published on Web 05/06/2009
 2009 American Chemical Society

prepared via pyran annulation as previously described.11 The
exomethylene was used to introduce the requisite C7
oxygenation by ozonolysis followed by NaBH4 reduction
(94%) to give the C7 alcohol 4 as a single diastereomer.
Temporary protection of the alcohol as its TMS derivative14
was followed by Bunnelle reaction15 to give the desired
hydroxy allylsilane 5 in 71% yield.
Pyran annulation between 5 and C-ring enal 6 using our
standard conditions of TMSOTf in ether gave the desired
tricyclic adduct, as a single diastereomer, in 92% isolated
yield. Protection of the C7 OH using TBSOTf and 2,6-
lutidine gave 7 in 98% yield.
Figure 1. Structures of bryostatin 1 and PMA.
Aldol condensation with methyl glyoxylate gave a dia-
stereomeric mixture of aldol products (92%) which under-
went elimination (93%) by treatment with Ac2O and DMAP
in pyridine at 60 °C. The resulting enoate 8 was then
subjected to Luche reduction16 followed by acylation with
octanoic anhydride to give 9. Selective removal of the BPS
(tert-butyldiphenylsilyl) group at C1 using TBAF and HOAc
in DMF (89%) was followed by oxidation to the acid 10
(quantitative yield over 2 steps) by sequential Parikh-
Doering17 and Pinnick18 oxidations. Removal of both TBS
groups (at C7 and C25) using HF·Py then gave the corre-
sponding dihydroxy acid.
linked to PKC activation.6 Bryostatin 1 (1), isolated by Pettit
from the marine bryozoan Bugula neritina, is also a potent
activator of PKC.7 However, unlike most phorbol esters,
bryostatin 1 is not tumor promoting and antagonizes many
of the responses induced by the phorbol esters. Bryostatin 1
has potent activity toward a number of cancer cell lines,
displays immunostimulant effects,8 and shows promise in
the treatment of Alzheimer’s disease.9,3 Recently, bryostatin
1 has also been shown to induce repair of neuronal damage
resulting from stroke.10
Attempted macrolactonization of this dihydroxy acid using
the Yamaguchi protocol19 afforded a macrolactone in which
the C7 alcohol had also been acylated by 2,4,6-trichloroben-
zoyl chloride, in excellent (79%) yield. To preclude the
undesired acylation of C7, this hydroxyl was selectively
protected by reaction with TESCl and DMAP to give 11;
the Yamaguchi reaction then afforded the desired macro-
lactone 12. Removal of the TES group followed by acety-
lation gave the desired C7 acetate derivative 13 (95% over 2
steps). Finally, protecting group removal (DDQ then LiBF4
in aqueous CH3CN) gave analogue Merle 27 (87% over 2
steps). The C7 trichlorobenzoate derivative obtained inadvert-
ently was also carried through a parallel sequence to give
analogue 14.
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. Recently, we have prepared and evaluated analogues
(Merle 21-2311 and Merle 28,12 vide infra) which now limit
the functionality largely responsible for the unique biological
profile exhibited by bryostatin 1 to that present in the C7-C9
region of the A-ring.13 In this paper, we report studies which
rule out the A-ring C7 acetate as being the critical determinant
of the unique biological responses characteristic of bryostatin
1, through synthesis and biological evaluation of analogue
Merle 27. The preparation of this new bryopyran is outlined
in Scheme 1.
Analogue Merle 27 was found to bind to PKC with slightly
lower affinity than that of Merle 21-23 (Ki ) 3.0 ( 0.6
nM with PKCR). Assays for differential response were
conducted using leukemia U937 cells.20 In this assay, PMA
inhibits cell proliferation and induces attachment. Bryostatin
1 has much less effect on either response and blocks the
effect of the phorbol ester when both agents are administered
together. The results are shown in Figures 2 and 3. Results
Analogue Merle 27 was prepared using a convergent pyran
annulation strategy to unite preformed A- and C-rings with
concomitant formation of the B-ring, as shown in Scheme
1. Preparation of the A-ring pyran began with pyran 3,
(6) Castagna, M.; Takai, Y.; Kaibuchi, K.; Sano, K.; Kikkawa, U.;
Nishizuka, Y. J. Biol. Chem. 1982, 257, 7847–7851.
(7) Pettit, G. R.; Herald, C. L.; Douobek, D. L.; Herald, D. L. J. Am.
Chem. Soc. 1982, 104, 6846–6848.
(8) 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.
(14) Stereochemistry at C7 was confirmed at this point by observation
of NOEs between the axial proton at C7 and those at C5 and C9.
(15) Bunnelle, W. H.; Narayanan, B. A. Organic Syntheses; Wiley &
Sons: New York, 1993; Collect. Vol. 8, pp 602-605.
(16) (a) Luche, J. L. J. Am. Chem. Soc. 1978, 100, 2226–2667. (b)
Wender, P. A.; Baryza, J. L.; Bennet, C. E.; Bi, F. C.; Brenner, S. E.; Clarke,
M. O.; Horan, J. C.; Kan, C.; Lacote, E.; Lippa, B.; Nell, P. G.; Turner,
T. M. J. Am. Chem. Soc. 2002, 124, 13648–13649.
(9) (a) Etcheberrigaray, R.; Tan, M.; Dewachter, I.; Kuiperi, C.; Van
der Au; wera, 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
.
(10) Sun, M.-K.; Hongpaisan, J.; Nelson, T. J.; Alkon, D. L. Proc. Natl.
Acad. Sci., U.S.A. 2008, 105, 13620–13625.
(11) Keck, G. E.; Kraft, M. B.; Truong, A. P.; Li, W.; Sanchez, C. C.;
Kedei, N.; Lewin, N.; Blumberg, P. M. J. Am. Chem. Soc. 2008, 130, 6660–
6661.
(17) Parikh, J. R.; Doering, W. v. E. J. Am. Chem. Soc. 1967, 89, 5505–
5507.
(18) Bal, B. S.; Childers, W. E., Jr.; Pinnick, H. W. Tetrahedron 1981,
37, 2091–2096.
(12) Keck, G. E.; Poudel, Y. B.; Welch, D. S.; Kraft, M. B.; Truong,
A. P.; Stephens, J. C.; Kedei, N.; Lewin, N. E.; Blumberg, P. M. Org. Lett.
2009, 11, 593–596.
(19) Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull.
Chem. Soc. Jpn. 1979, 52, 1989–1993.
(13) Here we adopt permanent identifiers (“Merle numbers”) for these
analogues which will remain invariant from publication to publication.
(20) Vrana, J. A.; Saunders, A. M.; Srikumar, P. C.; Grant, S.
Differentiation 1998, 63, 33–42.
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Org. Lett., Vol. 11, No. 11, 2009

Scheme 1. Synthesis of C7-Functionalized Bryopyrans
for the previously reported bryostatin-like analogue Merle
28 are also included for comparison.
tion on, at most, two carbons.21 Efforts to determine, through
synthesis, the role of substituents at each of these carbons
are in progress.
Merle 27 can be seen to induce biological responses
characteristic of those shown by PMA rather than bryostatin
1. Thus, the C7 acetate cannot be singly responsible for the
switch in activity observed between the PMA-like analogues
Merle 21-23 (and now Merle 27) and the bryo-like analogue
Merle 28. Instead, it appears that the critical residues must
be the C9 OH and/or the C8 gem-dimethyl. With these results,
the puzzling aspects of bryostatin 1 biology have been
pinpointed as originating primarily from effects of substitu-
It should be noted that Wender and Verma have recently
reported the synthesis of the C7 acetate 16 and a number of
other C7 derivatives, including the C7-exomethylene com-
pound 15, using the B-ring acetal platform developed
(21) Since modifications of the bryostatin 1 structure by definition give
different compounds, these will of course interact somewhat differently with
PKC or other C1 domain-containing proteins. Here we are concerned with
substantial changes in the biological profiles exhibited by these analogues
and not the minor differences anticipated for different structures, such as
relatively minor changes in binding affinity or potency in other assays.
Org. Lett., Vol. 11, No. 11, 2009
2279

Figure 2. Effect of Merle 27 on U937 cell attachment.
Figure 4. Structures of other recent analogues.
directly by the pyran annulation reaction. All other modifica-
tions at C7 examined gave materials with decreased affinity
for PKCs. The same is true of the C7 acetate Merle 27 and
the C7 trichlorobenzoate 14 (Ki ) 16.8 ( 4.2 nM with PKCR)
prepared by us using the bryopyran platform, which are less
potent than the exomethylene compounds Merle 21-23.
Thus, on both platforms, the exomethylene compounds are
approximately 2.5-3-fold more potent in terms of binding
than the compounds incorporating the natural C7 acetate
functionality.
Acknowledgment. Financial support was provided by the
National Institutes of Health through NIH grant GM28961
and by the Intramural Research Program, Center for Cancer
Research, National Cancer Institute, National Institutes of
Health.
Figure 3. Effect of Merle 27 on U937 cell proliferation.
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.
previously by that group.22 These authors also utilized our
pyran annulation approach for construction of the A-ring
bearing the versatile C7 exomethylene group. Using this
platform, the acetate 16 was found to have Ki ) 13 ( 3.8
nM against a mixture of PKC isozymes isolated from rat
brain, while the exomethylene compound 15 had Ki ) 5.3
nM.23 (Note Figure 4.)
Interestingly, of the 12 C7-functionalized derivatives
reported by Wender and Verma, the highest affinity for PKCs
was shown by the C7-exomethylene derivative 15, i.e., the
analogue incorporating the C7 functionality introduced
OL900585T
(22) (a) Wender, P. A.; Verma, V. A. Org. Lett. 2008, 10, 3331–3334.
(b) For an overview of bryostatin analogue synthesis by the Wender group,
see: Wender, P. A.; Baryza, J. L.; Hilinski, M. K.; Horan, J. C.; Kan, C.;
Verma, V. A. Beyond Natural Products: Synthetic Analogues of Bryostatin
1. In Drug DiscoVery Research: New Frontiers in the Post-Genomic Era;
Huang, Z., Ed.; Wiley-VCH: Hoboken, NJ, 2007; pp 127-162.
(23) These authors suggest that the carbonyl oxygen of this acetate
substituent may be involved in hydrogen bonding to a residue in the C1
domain.
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