The design, synthesis, and evaluation of C7 diversified bryostatin analogs reveals a hot spot for PKC affinity

Article abstract of DOI:10.1021/ol801235h

(Chemical Equation Presented) The first series of systematically varied C7-functionalized bryostatin analogs (12 members in all) have been synthesized through an efficient and convergent route. A new hotspot for PKC affinity, not present in the natural products, has been discovered, allowing for affinity control and potentially for selective regulation of PKC isozymes. Several analogs exhibit single-digit nanomolar affinity to PKC and display superior activity compared to bryostatin against the leukemia cell line K562.

Full text of DOI:10.1021/ol801235h

ORGANIC
LETTERS
2008
Vol. 10, No. 15
3331-3334
The Design, Synthesis, and Evaluation
of C7 Diversified Bryostatin Analogs
Reveals a Hot Spot for PKC Affinity
Paul A. Wender* and Vishal A. Verma
Department of Chemistry, Department of Chemical and Systems Biology, Stanford
UniVersity, Stanford, California 94305-5080
wenderp@stanford.edu
Received June 1, 2008
ABSTRACT
The first series of systematically varied C7-functionalized bryostatin analogs (12 members in all) have been synthesized through an efficient
and convergent route. A new hotspot for PKC affinity, not present in the natural products, has been discovered, allowing for affinity control
and potentially for selective regulation of PKC isozymes. Several analogs exhibit single-digit nanomolar affinity to PKC and display superior
activity compared to bryostatin against the leukemia cell line K562.
The bryostatins are structurally complex, marine-derived
macrolactones (Figure 1)1 with a unique and expanding
portfolio of biological activities. Bryostatin 1 is currently in
clinical trials for the treatment of cancer. It exhibits
exceptional potency, being dosed in humans at ca. 50 µg/
m2.2 It restores apoptotic function in cancer cells,3 reverses
multidrug resistance,4 and unlike most antineoplastic agents,
stimulates the immune system.5 It also acts synergistically
with several anticancer agents when used in combination
therapy.6 Remarkably, bryostatin also facilitates learning and
extends memory in animal models,7 serving as a significant
lead for treating cognitive dysfunction including Alzheimer’s
disease.8
Figure 1. Bryostatins 1 and 2, superior lead analog 1, and designed
C7 analogs.
There are two major problems that have impeded clinical
advancement of the bryostatin leads. Bryostatins are naturally
scarce and are isolated in low yields (0.00014%).9 Second, these
complex compounds have proven to be difficult to selectively
modify. Thus, relatively few bryostatin derivatives have been
prepared and fewer still have been evaluated preclinically.10
The importance of accessing new functional and tunable analogs
(1) Pettit, G. R.; Herald, C. L.; Doubek, D. L.; Herald, D. L.; Arnold,
E.; Clardy, J. J. Am. Chem. Soc. 1982, 104, 6846–6848.
(2) For current information, see http://clinicaltrials.gov.
(3) Wall, N. R.; Mohammad, R. M.; Al-Katib, A. M. Leuk. Res. 1999,
23, 881–888.
(4) Elgie, A. W.; Sargent, J. M.; Alton, P.; Peters, G. J.; Noordhuis, P.;
Williamson, C. J.; Taylor, C. G. Leuk. Res. 1998, 22, 373–378.
10.1021/ol801235h CCC: $40.75
Published on Web 06/28/2008
2008 American Chemical Society

is great as the natural product itself elicits off target effects that
limit or preclude clinical applications.
kinome, but its functional role could be significant because
many of these kinases are implicated in major diseases
including cancer, cardiovascular, and cognitive indications.15
Ligands for the selective regulation (activation or inhibition)
of C1 domain proteins are not available, adding further
importance to the search for new high-affinity, selective
agents. Related to the search for ligand structural features
that could influence potency, selectivity, and function, it is
noteworthy that no systematic studies on the role of C7
functional variations on biological activity have been reported
(Figure 1). Significantly, docking studies using PKC sub-
domain crystal structures and homology models suggest that
the C7 functionality of bryostatin is proximate to a conserved
tryptophan residue in the novel class of PKC isozymes and
a conserved tyrosine residue in the conventional class of PKC
isoforms (Figure 2).16 The biorelevancy of such models has
Total synthesis offers a means to address this problem,
but current syntheses, while impressive in content, are as
yet too long (>70 steps) to impact immediate supply needs.11
Engineered biosynthesis is another notable and promising
source under investigation.12 In 1986, we initiated a third
approach to addressing this problem based on function-
oriented synthesis.13 In this approach, the structural features
of the complex bryostatin target that putatively influence
function (activity) are recapitulated on a simplified scaffold
to produce a functional analog that is designed for rapid,
step-economical, and practical synthesis.13a Illustrative of this
approach, designed analog 1 (Figure 1) is found to be more
potent than bryostatin and can be readily synthesized in a
highly convergent fashion (<30 steps, 19 LL) that can be
scaled to meet clinical needs.14
The current study is directed at a hitherto unexplored but
fundamentally and clinically significant issue, namely the
role of A-ring C7 functionality in the activities of bryostatin
and analogs related to 1. Bryostatin 1’s activity is mediated
through its potent binding to the C1 domain of various
proteins, including kinases such as protein kinase C (PKC).
This domain is found in only a small subset of the human
(5) Oz, H. S.; Hughes, W. T.; Rehg, J. E.; Thomas, E. K. Microb.
Pathog. 2000, 29, 187–190.
(6) For a recent clinical trial, see: Ajani, J. A.; Jiang, Y.; Faust, J.; Chang,
B. B.; Ho, L.; Yao, J. C.; Rousey, S.; Dakhil, S.; Cherny, R. C.; Craig, C.;
Bleyer, A InVest. New Drugs 2006, 24, 353–357.
Figure 2. Docking of bryostatin 1 to the C1b domain of novel
(7) Hongpaisan, J.; Alkon, D. L Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 19571–19576.
PKCδ. The distance between the C7 acetate carbonyl and tryp-
tophan hydrogen is 1.9 Å, and the bond angle is O-H-N )
152°.
(8) Etcheberrigaray, R.; Tan, M.; Dewachter, I.; Kuiperi, C.; Van der
Auwera, I.; Wera, S.; Qiao, L. X.; Bank, B.; Nelson, T. J.; Kozikowski,
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not been tested, but such differences in binding selectivity
could have profound therapeutic ramifications. It is note-
worthy that these residues of PKC have recently been found
to be critical for selective isozyme activation by the endog-
enous ligand, diacylglycerol.17 We report herein the efficient
and convergent synthesis of 12 members of the first class of
C7-functionalized bryostatin analogs and their initial biologi-
cal evaluation.
(10) (a) Wender, P. A.; DeBrabander, J.; Harran, P. G.; Jimenez, J. M.;
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Our overall strategy employs a Yamaguchi esterification
followed by a macroacetalization to convergently couple the
target recognition (10) and spacer domains generated from
intermediates 8 or 9. The synthesis of C7 diversifiable spacer
domains draws in turn on a stereospecific Prins cyclization18
to convergently generate the syn-pyran A-ring from bis-
nucleophile 4 and 2 equiv of aldehyde 3, a strategy that
exploits the pseudosymmetry of the designed spacer domain.
The synthesis of spacer domain intermediate 8 began with
(13) (a) Wender, P. A.; Verma, V. A.; Paxton, T. J.; Pillow, T. H. Acc.
Chem. Res. 2008, 41, 40–49. (b) Wender, P. A.; Koehler, K. F.; Sharkey,
N. A.; Dellaquila, M. L.; Blumberg, P. M Proc. Natl. Acad. Sci. U.S.A.
1986, 83, 4214–4218.
(14) (a) Wender, P. A.; Baryza, J. L.; Bennett, C. E.; Bi, F. C.; Brenner,
S. E.; Clarke, M. O.; Horan, J. C.; Kan, C.; Lacoˆte, E.; Lippa, B. S.; Nell,
P. G.; Turner, T. M. J. Am. Chem. Soc. 2002, 124, 13648–13649. For other
recent analog work, see: (b) Wender, P. A.; DeChristopher, B. A.; Schrier,
A. J. J. Am. Chem. Soc. 2008, 130, 6658–6659. (c) Keck, G. E.; Kraft,
M. B.; Truong, A. P.; Li, W.; Sanchez, C. C.; Kedei, N.; Lewin, N. E.;
Blumberg, P. M. J. Am. Chem. Soc. 2008, 130, 6660–6661. (d) Wender,
P. A.; Horan, J. C.; Verma, V. A Org. Lett. 2006, 8, 5299–5302, and
references therein. (e) For a review of our bryostatin analog program, 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.
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S Science 2002, 298, 1912–1934.
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M. L.; Horan, J. C.; Meyer, T. Curr. Drug DiscoV. Tech. 2004, 1, 1–11.
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3332
Org. Lett., Vol. 10, No. 15, 2008

the selective reduction of commercially available diester 219
and oxidation to the key aldehyde 3 (Scheme 1). Allylation
analogs in only 3-4 steps (Scheme 2). Toward this end, 8,
Scheme 2.
Completion of C7-Functionalized Analogsa
Scheme 1
.
Highly Convergent Synthesis of C7-Functionalized
Intermediates
aSee the Supporting Information for experimental details.
9, or their derivatives were selectively converted to the C1
carboxylic acid through an efficient TBSOTf-mediated
procedure. The resultant spacer domains were then esterified
with recognition domain 1014 using Yamaguchi conditions.
Our remarkably general14,23 and mild tandem global depro-
tection and macroacetalization procedure was then employed
to form the B-ring which also serves to complete the
macrocycle and set the C15 stereocenter under thermody-
namic control, providing the analog in good yield. In total,
12 C7-functionalized analogs were efficiently synthesized
in this manner, with the spacer domains prepared in as few
as seven overall steps.
The newly synthesized analogs were tested for their ability
to bind to PKC, providing a first-ever benchmark comparison
to bryostatin and other analogs as well as to whether C7
modifications influence potency (Figure 3). The C7 acetate
analog 11 bound with a Ki of 13 nM while the benzoate and
pivaloate analogs (12 and 13) exhibited comparable but
higher potency, indicating that steric bulk is tolerated in this
region. The more polar furoyl analog 14 displayed a lower
affinity, comparable to the carbonate or carbamate analogs
15 and 16. Remarkably, the free hydroxyl analog 17 was
found to bind with a Ki ) 1000 nM, a 4 orders of magnitude
loss in binding potency compared to lead analog 1 simply
due to the incorporation of a hydroxyl moiety at C7. This
analog was found to be stable to the assay buffer conditions
and ROESY NMR studies indicated that the analog adopts
a similar conformation to that of other potent bryologs.
Interestingly, inversion of this stereocenter to the C7 R-OH
of this aldehyde with stannane 4,20 while efficient, was not
highly disatereoselective with various catalytic and stoichio-
metric Lewis acid systems (Ti/BINOL or Zr/BINOL)21 as
well as additives (B(OMe)3 or i-PrSBEt2).22 The undesired
R-alcohol 5 was therefore efficiently converted to the desired
ꢀ-alcohol 6 using Mitsunobu conditions. Reduction of ester
6 and protection of the resultant alcohol led to Prins precursor
7. The Prins cyclization of 7 using aldehyde 3 a second time
proceeded smoothly to give pyran 8 in good yield and with
high if not complete diastereoselectivity. The use of the more
robust TBDPS protecting group for the primary alcohol was
critical, as silyl exchange and deprotection was problematic
under TMSOTf-promoted conditions using a primary TBS
group. Ozonolysis and reductive workup then selectively
installed C7 oxygenation as the desired ꢀ-alcohol in only
seven overall steps from commercial material through 6 or
nine steps through 5. Notably, these syntheses of spacer
domains through intermediates 8 and 9 are the shortest of
any spacer domain synthesis reported thus far.
An attractive aspect of this strategy is that alkene 8, alcohol
9, or their derivatives can be converted into bryostatin
(19) Item 49.60 from Ju¨lich Chiral Solutions (www.codexis.com) for
33 euros/g.
(20) Available in one step: (a) Clive, D. L. J.; Paul, C. C.; Wang, Z. J.
Org. Chem. 1997, 62, 7028–7032.
(21) (a) Yu, C. M.; Lee, J. Y.; So, B.; Hong, J. Angew. Chem., Int. Ed
2002, 41, 161–163. (b) Kurosu, M.; Lorca, M. Tetrahedron Lett. 2002, 43,
1765–1769.
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889–890. (b) Yu, C. M.; Choi, H. S.; Jung, W. H.; Kim, H. J.; Shin, J.
Chem. Commun. 1997, 761–762.
(23) (a) Wender, P. A.; Verma, V. A Org. Lett. 2006, 8, 1893–1896.
For a related application, see: (b) Smith, A. B.; Razler, T. M.; Meis, R. M.;
Pettit, G. R. Org. Lett. 2006, 8, 797–799
.
Org. Lett., Vol. 10, No. 15, 2008
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human leukemia cell line K562 (Figure 4). Analog 1 is
Figure 3. PKC binding affinities of C7 functionalized analogs (nM).
Only A-rings are shown.
Figure 4. Effective concentrations of analogs 11 and 17 against
human leukemia cell line K562 after 48 h incubation.
effective against this line (EC50 ) 32 nM), while bryostatin
1 is significantly less active (EC50 ) 4.0 µM).14 In line with
their binding potencies, analog 11 was more effective than
analog 17 against this cell line. Importantly, the synthesis
of the first C7 acetate bryolog allows for retention of activity
when compared with lead analog 1 and superior activity
when compared to bryostatin 1.
The first C7 A-ring functionalized bryologs have been
synthesized in a scalable and step-economical fashion, with
spacer domains available in 7-10 steps (overall 26-29
steps). These 12 analogs represent the first in a series of C7
modified bryologs, some of which retain potent, single-digit
nanomolar affinity to PKC. A critical hotspot for PKC affinity
has been discovered at the C7 position of the bryostatin
analogs. The discovery of this hotspot adds weight to prior
data17 implicating this region of PKC as critical for potency
and potentially for controlling selectivity and optimizing
pharmacological function. Indeed, the C7 acetate analog 11
exhibits superior in vitro antileukemic activity when com-
pared to bryostatin 1. Further biological evaluation of these
analogs, including isozyme selective activity, is currently
underway and will be reported in due course.
analog 19 results in a 10-fold increase in binding potency
(Ki ) 100 nM) compared to 17, while the homologated
hydroxyl analog 20 increases relative affinity by only 3-fold
(Ki ) 330 nM). It appears that analogs retaining a free
hydroxyl in this region of the bryologs lose a significant
degree of potency compared to other C7 functionalized
analogs, raising the hypothesis that C7 groups are at the
protein complex-membrane interface or other hydrophobic
region. This hypothesis is further supported by the better
binding of methoxy analog 18 relative to 17. Even more
dramatically, exomethylene analog 22, nearly isosteric with
ketone analog 21, exhibits single digit nanomolar binding.
Interestingly, bryostatin 2 (Figure 1), containing a C7
hydroxyl group as in analog 17, is only about 4-fold less
potent than bryostatin 1 with a C7 acetate. The effect of the
neighboring gem-dimethyl groups in the natural series could
enhance membrane association by offsetting or shielding the
C7 differences (Figure 2). The absence of a crystal structure
of full length PKC and more significantly, of a solution
structure involving membrane association, makes this ligand-
based probe of the complex structure a singularly critical
means of detailing molecular level interactions. It is clear
from the binding data that PKC and the lipid cofactors
required for proper protein folding24 represent a more
complex system than the hypothetical binding model of the
analogs docked to the C1 domain would indicate. These
studies do show that, in contrast to the natural bryostatins,
the C7 region of the bryologs can play a significant role in
binding affinity and could be potentially exploited for
improved pharmacological function such as PKC selectivity.
To further compare the cellular effects of these analogs,
11 and 17 were tested for their effectiveness against the
Acknowledgment. Support of this work through a grant
(CA31845) provided by the NIH is gratefully acknowledged.
Fellowship support from Amgen (V.A.V.) is also recognized.
We thank the Daria Mochly-Rosen group (Department of
Chemical and Systems Biology) for support with biological
studies.
Supporting Information Available: Available experi-
mental conditions and spectral data for compounds reported
in this paper. This material is available free of charge via
the Internet at http://pubs.acs.org.
(24) Wender, P. A.; Irie, K.; Miller, B. L Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 239–243.
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