Molecular modeling, total synthesis, and biological evaluations of c9-deoxy bryostatin 1

Article abstract of DOI:10.1002/anie.201001200

(Chemical Equation Presented) 'OH' no you don't: The title compound 1 has been synthesized and evaluated for biological function. Molecular modeling of bryostatin 1 with the Cl domain of protein kinase C δ indicates that the C9OH of bryostatin 1 makes a hydrogenbonding contact to the protein. Despite the absence of the hydrogen-bonding contact for 1, it displays bryostatin-like biological effects in four assays using either U937 leukemia cells or prostate LNCaP cells.

Full text of DOI:10.1002/anie.201001200

Communications
DOI: 10.1002/anie.201001200
Structure–Activity Relationship
Molecular Modeling, Total Synthesis, and Biological Evaluations of
C9-Deoxy Bryostatin 1**
Gary E. Keck,* Yam B. Poudel, Arnab Rudra, Jeffrey C. Stephens, Noemi Kedei,
Nancy E. Lewin, Megan L. Peach, and Peter M. Blumberg
The bryostatins are a family of natural products of marine
origin that display both intriguing structural complexity and a
fascinating profile of biological activity.[1] These materials
were isolated (from Bugula neritina) and their structures
determined through the pioneering work of Pettit and co-
workers.[2] Subsequently, a monumental large-scale collection
and isolation effort managed to yield some 18 g of bryosta-
tin 1, the most abundant and now most thoroughly inves-
established that bryostatin 1 binds with high affinity to the
regulatory C1 domains of protein kinase C (PKC) isozymes
and thereby activates these enzymes.[6] Likewise, it binds to
the homologous regulatory C1 domains of six other families
of signaling proteins, for example, the chimaerins and
RasGRPs, to modulate their activities.[7] Physiologically, all
of these proteins function, through their C1 domains, as
sensors for the lipophilic second messenger sn-1,2-diacylgly-
cerols. Paradoxically, however, whereas bryostatin 1 binds to
the same binding site as do the diacylglycerols or their high-
affinity analogues, the phorbol esters, bryostatin 1 induces
only a subset of the responses observed with these other
ligands.[8] Moreover, bryostatin 1 blocks those responses that
it does not itself induce and, in particular, is not tumor
promoting, which is in contrast to most phorbol esters (e.g.,
phorbol 12-myristate-13-acetate, PMA). Despite the intense
synthetic attention this family of compounds has attracted,
bryostatin 1 has not as yet been synthesized, although the
structurally similar and high-affinity bryostatins 2, 3, and 7
have been prepared.[9] In addition, bryostatin 16, which has
markedly diminished affinity for PKC (Ki = 118 nm) relative
to bryostatin 1 (Ki = 1.35 nm), has also been prepared.[10]
Several analogues of bryostatin have also been prepared,
primarily by the group of Wender and by our group.[1,11]
Recently, our group has been focused on elucidating the
structural features of bryostatin that are responsible for its
function as a phorbol ester antagonist, as distinct from its
activity simply as a ligand for PKC. We have previously
reported on the synthesis of the bryopyran core structure[12]
and of bryopyran analogues with greatly simplified A and
B rings that function as phorbol ester mimics,[13] and have
shown that functionality on the A ring of bryostatin 1 is
critical in preserving bryostatin-like biological effects.[14]
Herein, we describe the results of studies designed to reveal
the influence of the C9 hydroxy substituent of the A ring on
the biological responses elicited by bryostatin 1.
tigated member of this family, from some 13000 kg of the
source organism.[3] This worldꢀs supply of material has
supported numerous biological investigations and roughly
80 clinical trials against various cancers.[4] Recently, a clinical
trial against Alzheimerꢀs disease has also commenced.[5]
Despite this intense interest in the bryostatins as potential
therapeutics, the mechanisms by which bryostatin 1 elicits its
biological responses are only partially understood. It has been
[*] Prof. G. E. Keck, Y. B. Poudel, A. Rudra, J. C. Stephens
Department of Chemistry, University of Utah
315 South 1400 East, Rm 2020, Salt Lake City, UT 84112 (USA)
Fax: (+1)801-585-0024
E-mail: keck@chem.utah.edu
Among the models that have been put forward previously
for the binding of bryostatin 1 to the C1 domain of PKC, the
computationally derived model of Itai and co-workers
proposed an explicit hydrogen-bonding interaction (one of
N. Kedei, N. E. Lewin, Dr. P. M. Blumberg
Laboratory for Cancer Biology and Genetics, CCR, NCI, NIH
Bethesda, MD 20892 (USA)
M. L. Peach
À
four) between the C9 OH and the C1 domain of the
protein.[15] We began by independently examining the docking
of bryostatin 1 and its C9-deoxy analogue to the C1 domain.
Before beginning the docking we first performed a conforma-
tional search of bryostatin 1 in implicit water and octanol
solvents. The global energy-minimum conformation found in
both solvents was essentially identical to the crystal[2] and
NMR[16] conformations, and is characterized by an intra-
molecular hydrogen-bonding network in which the proton of
Basic Research Program SAIC-Frederick, Inc., NCI-Frederick
Frederick, MD 21702 (USA)
[**] Financial support for this work was provided by the National
Institutes of Health through grant GM28961, in part by the
intramural research program of the NIH, NCI, CCR, and in part with
federal funds from the National Cancer Institute, National Institutes
of Health, under contract HHSN261200800001E.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201001200.
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the OH group on C3 makes a bifurcated hydrogen bond
between the pyran oxygen atoms of the A and B rings, and a
second hydrogen bond is established between the proton of
the OH group on C19 and the oxygen atom of the OH group
on C3 (Figure 1b). The four lowest-energy conformations
from the search were saved, and corresponding structures of
C9-deoxy bryostatin 1 for each of these conformations were
built by simply replacing the C9 hydroxy group with a
hydrogen atom.
Merle 28.[14] The synthesis of the new C9-deoxy analogue
Merle 30 is outlined in Scheme 1.
The regio- and stereoselective deoxygenation of the C9
methyl ketal in the presence of the C19 methyl ketal was
carried out by treating the bisketal compound 1 with TMSOTf
and triethylsilane which afforded the reduced product 2 in
excellent yield. To install the enoate on the B ring, a cross-
metathesis with methyl acrylate was first attempted.[19]
However, this reaction provided an inseparable mixture of
E/Z-isomeric products in low yield. Alternatively, installation
of this enoate could be approached by an asymmetric
Horner–Wadsworth–Emmons (HWE) reaction with the cor-
responding ketone.[20] The oxidative cleavage of the C13–C33
olefin in the presence of the C16–C17 olefin proved to be
unselective utilizing OsO4/NaIO4; however, when the bisole-
fin 2 was subjected to stoichiometric ozonolysis conditions
using an ozone solution, the reaction was selective for the
exocyclic olefin and after reductive workup provided the
bisketone 3.
When the asymmetric Horner–Wadsworth–Emmons
reaction was conducted on bisketone 3 using the Fuji
phosphonate reagent 8 derived from (R)-binol, once again,
an inseparable 1:1.4 mixture of olefin isomers resulted. This
result prompted us to attempt the HWE reaction later on a
macrocyclic compound.
Figure 1. Bryostatin 1 (a) and C9-deoxy bryostatin 1 (b) docked into
the C1 domain of PKCd.
To prepare the seco acid 4 required for macrolactoniza-
tion, the PMB group was removed under standard oxidative
conditions and the thiolester was hydrolyzed using mCPBA/
H2O (1:1) in THF.[21] Yamaguchi macrolactonization of the
seco acid then afforded the macrolactone in good yield.[22]
With the macrolactone in hand, an asymmetric Horner–
Wadsworth–Emmons reaction was carried out using the
reagent 8[20] to give a 4:1 mixture of Z/E-olefin isomers,
which were separated by preparative thin layer chromatog-
raphy.
These conformations of bryostatin 1 and C9-deoxy bryo-
statin were then docked into the crystal structure of the C1b
domain of PKCd.[17] We included a similarity constraint to the
crystallized phorbol-13-acetate ligand to bias the optimization
toward solutions where the acceptor atoms in bryostatin 1 and
phorbol are close in space. The highest-scoring pose for both
compounds was the global energy-minimum conformation in
solution, suggesting that bryostatin 1 does not undergo a
conformational change upon binding to the C1 domain. The
docked structures are shown in Figure 1.
The overall binding mode does not change with the
presence or absence of the C9 hydroxy group, and is very
similar to the model proposed by Itai et al.[15] The A and
B rings lie above the binding site in the plane of the bilayer. In
both compounds, the C26 hydroxy group is buried in the
binding site and forms hydrogen bonds with the backbone
carbonyl group of Leu 251 and the backbone NH of Thr 242.
The ester oxygen atom adjacent to C1 appears to make a
weak hydrogen bond to the NH of Gln 257. The methoxy-
carbonyl group at C34 extends over the edge of the binding
site and hydrogen bonds to the backbone NH of Gly 253.
Bryostatin 1 also forms an additional hydrogen bond between
the C9 hydroxy group and the backbone carbonyl group of
Met 239. This hydrogen bond is not formed by the phorbol
ester compounds, lending support to the hypothesis that the
C9 hydroxy group might be an important structural determi-
nant for bryostatinꢀs biological activity.
To install the enoate functionality on the C ring, an aldol
reaction was attempted between the C20 ketone and methyl
glyoxylate using 1.1 equivalents of LDA. Surprisingly, and in
contrast to previous experience with similar compounds, the
aldol reaction took place exclusively on the C7 acetate of the
C9-deoxy compound 5. Gratifyingly, when the ketone 5 was
subjected to reaction with the methyl acetal of methyl
glyoxylate,[23] using K2CO3/MeOH,
a mixture of aldol
adduct and the condensed product were obtained in which
the C7 acetate had been hydrolyzed in both cases. A simple
acetylation of the crude product both completed conversion
of the aldol product into the desired enoate and reinstalled
the C7 acetate, yielding the desired enoate 6 as a single
isomer. Finally, Luche reduction of the C20 ketone and
subsequent esterification with octadienoic anhydride pro-
vided the protected form of C9-deoxy bryostatin 1. The
removal of the BPS (tert-butyldiphenylsilyl) group using
HF·Py and then global deprotection using LiBF4 in aqueous
CH3CN completed the synthesis of C9-deoxy bryostatin 1
(Merle 30; Scheme 1).[24]
À
To evaluate experimentally the contribution of the C9
OH to the observed biological profile associated with
bryostatin 1, we targeted C9-deoxy bryostatin 1 (Merle 30)
for synthesis.[18] Here we were able to utilize the advanced
intermediate 1 that we had prepared previously in our
laboratories enroute to another “almost bryostatin” analogue,
Biological evaluation of this new analogue began with an
assay of the binding affinity with PKCa, which gave Ki =
0.38 nm. Thus, this analogue binds comparably to bryostatin 1,
despite the absence of the proposed interaction between the
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PMA-like
character
(larger decrease in cell
proliferation and greater
attachment). Both ana-
logues
Merle 28
and
Merle 30, which corre-
spond in structure to dele-
tion of just one polar
group from the
A or
B ring of bryostatin 1, are
only slightly less effective
than bryostatin 1 itself in
blocking
the
effects
induced by PMA. Presum-
ably, deletion of both
polar groups simultane-
ously would provide an
analogue that is much less
bryostatin-like. Although
that precise experiment
has not yet been accom-
plished, it is of interest to
note that deletion of both
of these polar groups as
well as deletion of the gem
dimethyl group provides
analogue
Merle 27
(Figure 2), which we have
previously characterized
as exhibiting PMA-like
biology in the U937 cell
system.[26]
Scheme 1. Synthesis of C9-deoxy bryostatin 1. a) TMSOTf, Et3SiH, CH2Cl2, À788C, 82%; b) O3, CH2Cl2, À788C,
then DMS, 89%; c) DDQ, pH 7 buffer, CH2Cl2, 08C, 91%; d) mCPBA, THF/H2O (1:1), 83%; e) 2,4,6-Cl3PhCOCl,
Et3N, THF, then DMAP, tol, 408C, 79%; f) 8, NaHMDS, THF, À788C, then diketone, 08C, Z/E=4:1, 85%;
g) K2CO3, MeOH, MeO(OH)CCO2Me; h) Ac2O, DMAP, Py, 60% 2 steps; i) NaBH4, CeCl3, MeOH, À408C;
j) (C8H11O)2O, DMAP, Py, d.r.=5:1, 75% 2 steps; k) HF·Py, THF/MeOH/Py (1:1:1), l) LiBF4, MeCN/H2O (20:1),
808C , 68% 2 steps. BOM=benzyloxymethyl, DDQ= 2,3-dichloro-5,6-dicyano-p-benzoquinone, DMAP=4-
dimethylaminopyridine, DMS=dimethyl sulfide, HMDS=hexamethyldisilazide, mCPBA=meta-chloroperben-
zoic acid, PMB=para-methoxybenzyl, Py=pyridine, Tf=trifluoromethanesulfonyl, THF=tetrahydrofuran,
TMS=trimethylsilyl.
The biological activity
of Merle 30 was also
examined
in
another
system, the androgen-de-
À
C9 OH and the Met 239 carbonyl group. This result is not
À
unexpected as other bryopyrans lacking the C9 OH have
shown high affinity for PKCs.[11c,13]
Functional activity of Merle 30 in living cells was assessed
using the U937 leukemia cell system that we have employed
previously. These cells give differential responses to PMA and
bryostatin 1.[25] PMA is strongly anti-proliferative whereas
bryostatin 1 causes only a minor, biphasic decrease in cell
proliferation. When used in combination, bryostatin 1 is
observed to block the anti-proliferative effect of PMA in a
dose dependent manner. In addition, PMA induces attach-
ment of the U937 cells whereas bryostatin 1 does not; again,
bryostatin 1 antagonizes the PMA effect. When the results for
these experiments are displayed as a function of dose, a
Figure 2. Structures of Merle 27, Merle 28, and PMA.
characteristic visual “fingerprint” for each agent results,
which can then be easily compared to those for PMA and
for bryostatin 1. The results with Merle 30 in these assays, as
well as the results for the previous bryostatin-like analogue
Merle 28 (Figure 2), are shown in Figure 3.
pendent human prostate cancer cell line LNCaP, for two
different endpoints: proliferation and secretion of tumor
necrosis factor a (TNFa), a key mediator of inflammation. In
this system PMA inhibits cell proliferation and induces
apoptosis whereas bryostatin 1 does not.[27] For proliferation,
Notably, analogue Merle 30 is largely bryostatin-like in its
biological response in these assays, although with a little more
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itself. Both specific substituents on the bryopyran as well as its
overall physico-chemical properties might be expected to
make contributions. The diversity of these interactions could
well underlie the complex structure–activity relations of the
bryostatins and their bryopyran analogues.
Received: February 27, 2010
Published online: May 20, 2010
Keywords: biological activity · cancer · natural products ·
.
structure–activity relationships · total synthesis
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Figure 3. Results of proliferation (a) and attachment (b) assays in
U937 leukemia cells treated for 72 h with C9-deoxy bryostatin 1
(Merle 30) and Merle 28.
Merle 30 behaves almost identically to bryostatin 1; it does
not inhibit LNCaP proliferation but does antagonize the
inhibition by PMA. For induction of TNFa secretion, the
three agents behave differently: PMA induces a potent
response, bryostatin 1 induces no response, and Merle 30
induces a weak biphasic response. (see the Supporting
Information.) Once again, those compounds which them-
selves do not induce secretion block the response to PMA. We
conclude that the C9 hydroxy group of bryostatin 1, at first
hypothesized to be a key substituent, makes only a minor
specific contribution to both the binding affinity for bryosta-
tin 1 and the unique patterns of biological response to
bryostatin 1.
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