Computer-guided design, synthesis, and protein kinase C affinity of a new salicylate-based class of Bryostatin analogs

Article abstract of DOI:10.1021/ol502491f

Bryostatin 1 is in clinical trials for the treatment of cancer and Alzheimer's disease and is a candidate for a first-in-class approach to HIV/AIDS eradication. It is neither readily available nor optimally suited for clinical use. Using a function oriented synthesis strategy, a new class of bryostatin-inspired analogs was designed with a simplified salicylate-derived subunit, enabling step-economical synthesis (23 total steps) of agents exhibiting bryostatin-like affinity to protein kinase C (PKC).

Full text of DOI:10.1021/ol502491f

Letter
pubs.acs.org/OrgLett
Computer-Guided Design, Synthesis, and Protein Kinase C Affinity of
a New Salicylate-Based Class of Bryostatin Analogs
Paul A. Wender,* Yu Nakagawa,† Katherine E. Near, and Daryl Staveness
Departments of Chemistry and Chemical and Systems Biology, Stanford University, Stanford, California 94305, United States
S
* Supporting Information
ABSTRACT: Bryostatin 1 is in clinical trials for the treatment of
cancer and Alzheimer’s disease and is a candidate for a first-in-class
approach to HIV/AIDS eradication. It is neither readily available nor
optimally suited for clinical use. Using a function oriented synthesis
strategy, a new class of bryostatin-inspired analogs was designed with
a simplified salicylate-derived subunit, enabling step-economical
synthesis (23 total steps) of agents exhibiting bryostatin-like affinity
to protein kinase C (PKC).
he bryostatins are a family of 20 macrolide natural products
the vast majority of research on these indications has been
limited to the natural product itself. This raises unresolved issues
already encountered in earlier cancer trials regarding bryostatin’s
supply and off-target effects. In fact, patient accrual in a recent
clinical study was stopped due to these concerns.10 Given that
natural products are evolved for purposes other than treating
human disease (humans arrived late in this evolutionary
process), it is expected that analogs inspired by these
information-rich natural prototypes could potentially serve as
more accessible and efficacious clinical candidates.11 The key to
realizing this goal is understanding the structural basis for
bryostatin’s activity and using that information to design simpler
and more effective analogs.
T
isolated from the marine bryozoan Bugula neritina.1
Extracts from these animals displayed marked antineoplastic
activity when first tested against murine leukemia in the late
1960s by Pettit et al.2 The structure of bryostatin 1, the
biologically active constituent, was subsequently reported in
1982 (Figure 1).3 Numerous investigations have since sought to
The activity of bryostatin is thought to be mediated through
binding to the regulatory C1 domains of protein kinase C (PKC)
isoforms,12 a family of serine/threonine kinases integral to
proper cellular signal transduction.13 Two of the three
mammalian PKC classes (conventional PKCs: α, βI/βII, γ;
novel PKCs: δ, ε, η, θ) are endogenously activated by association
of diacylglycerol (DAG) to the C1 domains, with this binding
interaction requiring PKC to transiently associate with a
phospholipid bilayer.14 This regulatory mechanism influences a
multitude of downstream signaling events, though the basis for
its selectivity is still a work in progress.15 DAG and bryostatin are
competitive PKC binders, though the latter is orders of
magnitude more potent. Developing small molecule modulators
of PKC is key to understanding the system biology and chemistry
of this signaling pathway and thus its therapeutic potential.
Early on and now, bryostatin’s limited availability has
hampered research on its mode of action and clinical use.
Isolation yields are prohibitively low (just 18 g of 1 were obtained
from 14 tons of the source organism in the only GMP isolation),
and large scale harvesting in marine ecosystems raises environ-
Figure 1. Comparison of bryostatin 1, first generation designed analogs,
and new designed analogs.
explore the therapeutic potential of this highly potent agent.
Over 37 clinical trials have been conducted to date, principally
focused on the utility of bryostatin 1 for the treatment of cancer,
both as a single agent and in combination with other oncolytics.4
New clinical indications have also emerged. The ability of
bryostatin to speed recovery after ischemic damage5 and induce
synaptogenesis6 is particularly noteworthy and could figure in its
use to reduce the debilitating effects of stroke relative to current
therapies or to treat neurodegenerative diseases. Indeed,
bryostatin 1 is the focus of a recently opened clinical trial for
Alzheimer’s disease.4a,7 Bryostatin also activates cells latently
infected with the HIV provirus,8 potentially allowing for their
destruction by the immune system or cytopathic effects of viral
production. Since these proviral reservoirs are thought to
resupply active virus, their elimination in conjunction with
antiretroviral therapy could lead to reduction of the provirus
burden if not eradication of the disease.9 Of singular significance,
Received: August 22, 2014
Published: September 19, 2014
© 2014 American Chemical Society
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Organic Letters
Letter
mental issues.16 Aquaculture has been tested and abandoned.17
Supply methods based on de novo synthesis (the shortest total
syntheses of potent bryostatins are around 40 steps)18 and
engineered biosynthesis are still works in progress.19
function of the natural C3 alcohol by engaging the C19 hemiketal
in a hydrogen bond while the aryl group would be positioned for
membrane association. This maintains the critical hydrogen
bond to the C19 hemiketal, while replacing the bifurcated
hydrogen bond network of bryostatin (extending from the C3
alcohol to the A- and B-ring tetrahydropyrans) with covalent
linkages.
To evaluate the conformational correspondence between the
designed analog and bryostatin 1, the conformations of the
analog were determined and compared with the known crystal
structure of bryostatin. Analog 3 was subjected to an extensive
search of its conformational space within the multiconformer
mode of the MMFFs force field provided with the Schrodinger
Suite25 (Macromodel v9.5/Maestro GUI v8.0). The lowest
energy structures from this analysis fell within a single
conformational class, which was subsequently examined for
similarity to the known crystal structure of bryostatin 1. Based on
the lowest energy conformers of both compounds, the hydrogen
bond from the C19 hemiketal to the C3 oxygenation appeared
intact (Figure 2), suggesting that the salicylic acid spacer domain
Recognizing these issues, our group started in the 1980s to
explore a strategy focused on achieving bryostatin-like function
through synthesis- and activity-informed design.20 The premise
behind this function-oriented synthesis (FOS) approach,
influenced by our studies on DAG, bryostatin, and phorbol
estersthree structurally distinct agents with the ability to bind
PKC, albeit with differential selectivitiesis that function is not
uniquely associated with any one structure. A second premise is
that many natural products are encoded with excess structural
information peculiar to the needs of their source organism and
thus possess structural complexity irrelevant to their intended
use in human therapy. It follows that if one could define the
minimum set of features needed for a desired target activity, then
through design one could incorporate that required functionality
into more accessible and effective agents.
Toward this end, our group performed extensive computer-
guided comparisons of the spatial orientation of heteroatoms
(hydrogen bond donors and acceptors) of then known PKC
ligands, resulting in a proposed pharmacophore and the first
designed PKC modulators.21 For bryostatin, this analysis
suggested that the key structural elements that govern binding
were present in its southern half, termed the “recognition
domain,” that is proposed to contact and thus be “recognized” by
PKC. The role of the northern portion of the molecule, the
“spacer domain” (see Figure 1), was proposed to control the
conformation of the recognition domain and potentially
influence PKC translocation and membrane association. It
follows that compounds containing simpler spacer domains that
preserve the conformation of the recognition domain should
elicit PKC affinities comparable to bryostatin.
In 1998, we reported the first designed bryostatin analog with
such a simplified spacer domain.22 An even more potent analog
followed (B-ring dioxane analog 2), exhibiting PKC affinity
comparable to that of bryostatin and requiring ∼40 less steps to
synthesize than the then contemporaneous syntheses of natural
bryostatins.23 Most designed bryostatin analogs have since been
based on this analysis, retaining the recognition domain while
employing simplified spacer domains to improve step econo-
my.24
Reported herein is the application of the FOS approach to the
design of a new and uniquely simplified class of potent functional
bryostatin analogs, in which a salicylate subunit was selected as a
highly simplified mimic of the A- and B-rings of bryostatin. This
drastic reduction in complexity has resulted in the shortest
overall step count to a potent bryostatin analog, a goal of
significant research and clinical importance. Given the potency of
analog 3 (ca. 18 nM) and the ease with which the salicylate
subunit could be modified, this scaffold provides the basis for
iterative design−synthesis−evaluate approaches to targeting
isoform selective modulation of PKC. PKC pathway pharmacol-
ogy is complex, exhibiting activation or formal inhibition as a
function of dose and time.13b Step-economical access to tunable
leads is thus a key to identifying safe and effective clinical
candidates.
Figure 2. Left: crystal structure of 1 (red). Right: lowest energy
conformer of 3 (blue). Center: overlay of both structures. The C20 side
chain is depicted as the acetate for clarity.
could be an excellent mimic of that of bryostatin 1. An overlay
between this lowest energy conformer of analog 3 and the crystal
structure of bryostatin 1 showed remarkable similarity in the
spatial location of recognition domain atoms (RMS deviation of
0.05 Å) and prompted our commitment to the synthesis of this
new class.
The synthesis of the benzyl ester of spacer domain 4 began
with readily available methyl salicylate 5 (Scheme 1). Phenolic
Scheme 1. Synthesis of the Spacer Domain 4
alkylation with 3-bromo-propanol gave the phenyl ether 6.
Saponification of the methyl ester followed by Jones oxidation
afforded the desired dicarboxylic acid. Selective benzylation26 of
the aliphatic carboxyl group was then performed utilizing
NaHSO4·SiO2 as a weak acid catalyst in benzyl alcohol to
provide the desired spacer domain 4 with differentiated
functionality (acid and benzylic ester) for coupling to the
recognition domain.
The design of analog 3 started with in silico studies centered on
identifying simple surrogates for the bryostatin spacer domain. In
this case, we focused on salicylates to emulate spacer domain
function which, being commercially available, would thus reduce
step count. It was proposed that an ether at C3 could mimic the
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Letter
Spacer domain 4 was then attached to known truncated
recognition domain fragment 723 via a DCC-mediated
esterification (Scheme 2). Sharpless asymmetric dihydroxylation
aimed at this particular scaffold and the FOS approach in general.
These efforts make possible step-economical access to even more
potent agents (following study) as is critically needed for various
preclinical and intended clinical trials.
By moving from the complex A- and B-ring system of
bryostatin 1 to the salicylate-derived spacer domain of analog 3,
six stereocenters, one ring, a di- and a trisubstituted olefin, and
two quaternary centers (one of which is an all-carbon quaternary
center) were removed from the desired synthetic target, enabling
a decreased step count with the only cost being a modest
decrease in binding affinity. Further exploration of this scaffold
(following manuscript) recovers loss in function through tuning
of this accessible structural class. Ultimately, this new, designed
scaffold becomes a promising lead for future development of
PKC modulators aimed at the treatment of high impact diseases
such as cancer, Alzheimer’s, and HIV. While almost all research
toward these indications has been based on the natural products,
function- and synthesis-informed design offers a unique
opportunity to supply new leads with improved clinical potential.
Scheme 2. Completion of Salicylate-Derived Analog 3
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures for the above transformations. Character-
1
ization data for all novel compounds with H and 13C NMR
spectra included. This material is available free of charge via the
Internet at http://pubs.acs.org.
of the C25−C26 olefin in 8 was followed by deprotection of the
C19 ketal with p-tosic acid. Selective protection of the primary
C26 alcohol with TBSCl afforded macrocyclization precursor 9
in 45% yield over three steps along with 31% of the C25 β-
epimer. Hydrogenolysis of the C1 benzyl ester provided the
requisite seco acid. Reaction of this C25 α-hydroxy seco acid
under Mitsunobu conditions followed by acidic hydrolysis of the
C26 silyl ether successfully generated designed analog 3 in a
three-step yield of 28%, sufficient to reach the critical initial goal
of this study, determining whether our analysis would yield new
active analogs. That noted, the average yield for these steps
(∼65%) is not unreasonable given the challenge of closing a 16-
membered ring with five contiguous sp2 centers, two ester
linkages with a transoid preference, and an out−out bridgehead
across C19/C23. Fortunately, this method did provide the
necessary material for initial biological evaluation. For reference,
the C25 β-alcohol could also be cyclized using acid activation
mechanisms (Yamaguchi macrocyclizations; carbodiimide-medi-
ated esterifications)27 though that approach provided lower
yields, never eclipsing the 20% yield over the final three steps.28
Attempting the macrolactonization with the C19 ketal in place
resulted in little to no product, likely due to the methyl group
providing an additional steric barrier to cyclization. In these
cases, the primary product was the free phenol resulting from
deprotonation at C2 (presumably of the activated form of the C1
acid) and elimination of the phenolate. This elimination
byproduct was only isolated in trace amounts with the C19
hemiketal cyclizations, though these substrates likely suffered
more from C-ring opening pathways.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: wenderp@stanford.edu.
Present Address
†Department of Applied Molecular Biosciences, Nagoya
University, Nagoya, Japan.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of Health
(CA031845 and CA031841). Additional funding was provided
by the National Science Foundation (K.N.) and the Amgen
Graduate Fellowship (D.S.).
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