Improved protein kinase C affinity through final step diversification of a simplified salicylate-derived bryostatin analog scaffold

Article abstract of DOI:10.1021/ol502492b

Bryostatin 1, in clinical trials or preclinical development for cancer, Alzheimer's disease, and a first-of-its-kind strategy for HIV/AIDS eradication, is neither readily available nor optimally suited for clinical use. In preceding work, we disclosed a new class of simplified bryostatin analogs designed for ease of access and tunable activity. Here we describe a final step diversification strategy that provides, in only 25 synthetic steps, simplified and tunable analogs with bryostatin-like PKC modulatory activities.

Full text of DOI:10.1021/ol502492b

Letter
pubs.acs.org/OrgLett
Improved Protein Kinase C Affinity through Final Step Diversification
of a Simplified Salicylate-Derived Bryostatin Analog Scaffold
Paul A. Wender* and Daryl Staveness
Departments of Chemistry and Chemical and Systems Biology, Stanford University, Stanford, California 94305, United States
*
S Supporting Information
ABSTRACT: Bryostatin 1, in clinical trials or preclinical development
for cancer, Alzheimer’s disease, and a first-of-its-kind strategy for HIV/
AIDS eradication, is neither readily available nor optimally suited for
clinical use. In preceding work, we disclosed a new class of simplified
bryostatin analogs designed for ease of access and tunable activity. Here
we describe a final step diversification strategy that provides, in only 25
synthetic steps, simplified and tunable analogs with bryostatin-like PKC
modulatory activities.
iscovered over 46 years ago by Pettit and collaborators in a
limited, as found for bryostatin, and they are rarely optimal
D
marine extract, bryostatin 1 (1) is a densely functionalized
candidates for human use (taxol notwithstanding), more often
macrolide that has since attracted much interest due to its novel
serving as the inspiration for more effective derivatives or
1
13
structure and its clinical potential (evaluated in 37 clinical trials
designed clinical agents. More generally, analogs of a natural
2
to date ). In addition to its proposed clinical use for the
treatment of cancer and its largely under-explored potential as a
small molecule immunomodulator, bryostatin has also shown
promise for the treatment of stroke based on animal models of
ischemic damage. It has also been shown to facilitate learning
and extend memory in animal conditioning experiments,
apparently through induction of synaptogenesis, leading to its
recent entry into clinical trials for the treatment of Alzheimer’s
product containing only those substructures critical for activity
could be designed to enable facile synthesis, thereby solving the
issue of supply while generating clinical candidates with
improved function. Toward this end, our group has extensively
investigated the bryostatin scaffold both in silico and syntheti-
3
4
14
cally in an effort to better understand its interaction with
5
protein kinase C, the intracellular target putatively responsible
15
for its desirable effects. There are eight isoforms of PKC
(conventional PKCs: α, βI/βII, γ; novel PKCs: δ, ε, η, θ) that
contain the regulatory C1 domains allowing for allosteric
regulation by bryostatin, phorbol esters, or the endogenous
2
a,6
7
disease.
Bryostatin and other PKC modulators (e.g.,
8
prostratin) also activate transcription of the HIV provirus,
which, when combined with antiretroviral therapy, could
eliminate latent viral reservoirs, thus potentially providing a
first-of-its-kind strategy to reduce disease burden or eradicate
16
ligand, diacylglycerol. Using this FOS approach, our group has
prepared analogs that either mimic or differ from the pan-PKC
selectivity profile of bryostatin, some of which perform as well or
even better than the natural product in various in vitro and ex vivo
9
,10
HIV.
These clinical indications have justifiably driven interest in
bryostatin, but the lack of a reliable natural supply and off target
effects (dose-limiting toxicity of myalgia) have provided barriers
to its development as a therapeutic. Aquaculture, engineered
biosynthesis, and total synthesis have been explored as sources of
17
evaluations.
In the preceding manuscript, we reported the development of
a 16-membered macrolactone analog (2, Figure 1; contracted
from the natural 20-membered ring) that substituted the densely
functionalized A- and B-ring system of bryostatin with a
2c
11
bryostatin with varying degrees of success, but a practical
supply of bryostatin has not yet been realized.
1
8
salicylate-derived linker. Since the southern half of the
molecule contains the functionality implicated in binding PKC,
this drastic simplification of the northern fragment did not have a
This situation has no doubt contributed to the relative lack of
advanced studies and absence of clinical studies on analogs.
Unlike most other natural product leads, the clinical case for
bryostatin has thus far been exclusively based on one compound,
bryostatin 1 itself. Significantly, the supply of the natural product
and issues related to off-target effects of bryostatin 1 could be
ameliorated or eliminated with simplified designed analogs that
retain efficacy using a function-oriented synthesis (FOS)
major deleterious effect on PKC affinity (K = 18 nM vs 1.1 nM
i
for bryostatin 1), a promising first data point for this new scaffold.
Described herein is a final step diversification strategy of this
salicylate-derived bryostatin analog scaffold based on a C7′-
bromide handle and requiring only one step from the point of
diversification to reach library members. The diversifiable C7′-Br
1
2
strategy. While natural products represent a vast and
exceptional library of leads, encoding 3.8 billion years of
chemical experimentation, their availability is often variable and
Received: August 22, 2014
Published: September 19, 2014
©
2014 American Chemical Society
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Scheme 2. Fragment Coupling and Preparation of C7′-Br
Analog 3
Figure 1. Simplification through function-oriented, synthesis-informed
19
design: new diversifiable analog 3.
analog 3 is reached in just 24 total steps. The reduced step count
and ability to tune PK properties provide a practical foundation
for the synthesis and study of agents with improved accessibility
and clinical potential.
The synthesis of analog 3, incorporating a diversifiable
bromide group, was achieved in a similar manner to that for
18
the original salicylate-derived analog (2), although a different
northern fragment was used and improvements were made in the
late-stage macrocyclization step.
Northern fragment 4 was prepared in 5 steps (Scheme 1) from
commercially available 5-bromosalicylic acid (5). Fischer
Scheme 1. Synthesis of the Northern Fragment 4
a
See Figure 2 for structures of R and yields.
Zn, AcOH in DMF) cleanly afforded the desired seco acid and set
the stage for the necessary macrolactonization.
While macrocyclizations driven by acid activation did not
compete with a Mitsunobu-based lactonization in the synthesis
of the parent salicylate scaffold, it was speculated that simply
reducing the basicity of these methods could improve the yield.
a
Tce = 2,2,2-trichloroethyl.
̈
Gratifyingly, substituting pyridine for Hunig’s base during the
anhydride formation in a traditional Yamaguchi cyclization led to
a reproducible 38% yield over the final three steps (C1
deprotection, macrocyclization, C26 deprotection). Notably,
the best yields were achieved on ∼350 mg scale. Acidic hydrolysis
of the silyl ether furnished the free C26 alcohol, affording C7′-Br
analog 3 in 24 steps overall (19 longest linear sequence) from
commercial materials. Two sets of Suzuki conditions were
esterification afforded the desired methyl salicylate (6), allowing
for clean alkylation of the C4′ phenol with 3-bromo-1-propanol
to generate phenyl ether 7. Oxidation of the resultant C1 alcohol
and esterification with 2,2,2-trichloroethanol provided bis-ester
8
. This was then demethylated without loss of the C1 protecting
group with in situ generated TMSI in refluxing DCE in 84% yield,
thus affording northern fragment 4 in 55% yield over five steps.
The southern fragment of analog 3 was derived from C17
employed to modify C7′, using Pd(OAc) in both cases: S-Phos
2
20
and CsF or PPh with K CO . The monodentate ligand strategy
3
2
3
22
(drawing from previous diversification efforts in our group )
2
1
alcohol 9, a versatile intermediate available in 12 steps and used
en route to numerous other bryostatin analogs including parent
salicylate analog 2 (Scheme 2). The northern and southern
fragments were coupled through simple DCC-mediated
esterification, cleanly bringing together the two pieces in 96%
yield when run on gram scale. Sharpless asymmetric dihydrox-
ylation of the C25−C26 olefin proceeded with a modest ∼2:1 dr
and was followed by deprotection of the C19 hemiketal and
selective TBS protection of the primary C26 alcohol to generate
C25 β-alcohol 11. Notably, 10% THF was required for sufficient
solvation of olefin 10 during dihydroxylation. Deprotection of
the 2,2,2-trichloroethyl ester with nucleophilic (NaSeH or
generally outperformed the more basic PPh /K CO conditions,
3
2
3
though the latter were necessary for certain boronic acids (e.g.,
3,5-dimethylisoxazole-4-boronic acid). Of note, only boronic
acids proved to be useful for this scaffold. Thermal instability at
60 °C under the reaction conditions limited the time course of
the reaction to 2−3 h. Boronic esters or trifluoroborate salts
resulted in slow reactions and were thus not further used.
The 13 C7′-substituted analogs were tested for their affinity to
24
two full-length PKC isoforms, βI and δ, in a heterogeneous
3
25
competitive binding assay with [ H]-phorbol dibutyrate. These
isoforms are representative members of the conventional and
novel PKC subfamilies. The 4-substituted electron-rich arenes of
14 and 15 showed enhanced potency for PKCδ, while PKCβI
affinity was more or less unchanged from the parent scaffold
(Figure 2). Moving the methoxy or isopropoxy groups to the 2
NaTeH) or reductive methods (e.g., SmI ) was either inefficient
2
or led to decomposition. However, a slight modification of
standard Troc removal conditions (Zn, PPTS in THF rather than
5
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hydrogen-bond acceptors but not donors are tolerated. This is
intriguing given that these ligands are generally expected to
27
provide a hydrophobic cap for the PKC C1 domain, but again,
more structural detail is needed on the spatial orientation of the
ligand−substrate complex within a membrane before these new
results can be rationalized. Interestingly, these analogs displayed
the largest selectivity difference between PKCδ and PKCβI at
nearly a full order of magnitude, perhaps facilitated by an S−π
interaction with M239 of PKCδ (M9 of C1b domain). While this
difference is still modest, it differs from the pan-selectivity of
bryostatin 1.
The consequences of these various selectivities with respect to
functional activity remains to be established, especially given that
it is not known whether pan-PKC selectivity is required for
activity or contributes to side effects, but even slight
perturbations in selectivity could lead to unique phenotypic
results given the time- and dose-dependent nature of PKC-driven
2
8
signal transduction. The incorporation of such polar
functionality is particularly intriguing, as it implies that late-
stage control over PK properties is possible without carrying
major concerns over the abrogation of activity.
a
Figure 2. Diversified library of salicylate-derived analogs. R represents
functional group cross-coupled onto diversifiable scaffold 3 (for analogs
b
This 5-bromosalicylate-derived analog 3 has thus proven to be
an effective scaffold for versatile, final step diversification of a
potent yet highly simplified variation on bryostatin 1.
Transitioning to less basic Yamaguchi conditions improved the
macrocyclization relative to the closure for parent salicylate
analog 2, and importantly, it was most efficient when run on scale
(∼350 mg). The library resulting from standard Suzuki coupling
conditions with C7′-Br analog 3 demonstrated that a variety of
functionalities are tolerated by this approach while still producing
highly potent (<10 nM) analogs. Electron-rich or -poor arenes as
well as lipophilic or hydrophobic moieties (at least when
avoiding hydrogen bond donors) were all tolerated, a valuable
trait when tuning ADME properties for clinical needs.
Interestingly, several of these agents displayed modest selectivity
profiles between PKCδ and PKCβI which could reveal distinct
trends when moving to more advanced biological evaluations of
these compounds. Efforts to assess the therapeutic potential of
this library toward Alzheimer’s disease, HIV eradication, and
other high priority indications are underway and will be reported
in due course. It is clear from these studies that readily tunable
analogs comparable in potency to the natural product can be
accessed in significantly fewer steps than that required to access
the natural product. This FOS strategy thus provides an
alternative to the “all or nothing” efforts to achieve clinical
relevancy based on the use of the natural product alone.
1
2−23); see Scheme 2 for full structure. CC yield = yield of Pd-based
cross-coupling; analogs 12 and 14−21 were prepared with Method A
whereas analogs 13, 22, and 23 were prepared with Method B (see
Scheme 2 for methods). c K values (nM) from a heterogeneous
i
3
d
competitive binding as against [ H]-phorbol dibutyrate. Two-step
yield resulting from use of Method A with trans-octenyl boronic acid
followed by hydrogenation: Pd/C, THF, H2 (1 atm), rt. 2,6-
Bis(isopropoxy)phenyl boronic acid was prepared in two steps from 2-
e
23
bromoresorcinol.
position generated the first compounds (16 and 17) with single-
digit nanomolar affinity for both isoforms, each favoring PKCδ
by only about 2-fold (as opposed to 5+-fold for 14 and 15) and
reaching affinities comparable to bryostatin itself. While further
research is needed, this modest difference in selectivity might be
explained by a hydrogen bonding interaction between the −OMe
or −OiPr of analogs 16 and 17 with serine 110 of PKCβI (9th
C1b residue; methionine in PKCδ ), an interaction that would
likely not be accessible to the 4-substituted variants, as their
substituents point away from the binding pocket. This
interaction would also explain the additional gain in potency
when moving to the 2,6-bis-substituted analogs 18 and 19, as the
ligand would no longer need to preorganize itself to one
atropisomer over the other for favorable ligand−protein
interactions. Given the lack of information on the interaction
between bryostatin and PKC, a more detailed understanding of
2
6
26
ASSOCIATED CONTENT
the basis for these effects is not possible. However, we are
attempting to rectify this problem through molecular dynamics
and REDOR NMR studies currently in progress, as the structure
of PKC in a membrane environment is currently not known.
At this time, the electron-rich nature of the arene substituents
and overall lipophilicity of certain analogs (e.g., C7′-octyl analog
■
*
S
Supporting Information
Expanded description of PKC C1 domain structure and
implications for targeting isoform selectivity. Synthetic proce-
dures for all transformations shown above. Characterization data
1
13
for all novel compounds with H and C NMR spectra included.
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
2) raised concerns about potential metabolic toxicities and
pharmacokinetic properties, respectively. The isopropyl ben-
zoate analog 20 was prepared as a comparator to the 4-OiPr-Ph
analog 15 in terms of lipophilicity but with inverted electronic
properties. Since the two analogs displayed comparable function,
the electron-rich nature of the arene is, at least in this first
generation study, not significant. While the introduction of an
indole moiety greatly reduced PKC binding affinity, both a
sulfonamide (22) and isoxazole (23) were successfully
incorporated and afforded potent ligands, suggesting that
AUTHOR INFORMATION
■
Corresponding Author
*
E-mail: wenderp@stanford.edu.
Notes
The authors declare no competing financial interest.
5
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Letter
(
20) Prior to the development of this method, an alternative route
ACKNOWLEDGMENTS
■
(
51% over six steps) was used to provide the necessary material for the
This research was supported by the National Institutes of Health
CA031845 and CA031841). Additional funding was provided
by the Amgen Graduate Fellowship.
1
00+ mg preparation of C7′-Br analog 3. See Supporting Information
(
for details.
(21) Wender, P. A.; Baryza, J. L.; Bennett, C. E.; Bi, 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.
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(
19) For ease of comparison with prior work, the bryostatin 1 carbon
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