Synthesis and biological evaluation of a new class of bryostatin analogues: The role of the C20 substituent in protein kinase C binding

Article abstract of DOI:10.1016/S0040-4039(00)01100-X

The synthesis and biological assay of a new series of designed bryostatin analogues modified at the C20 position are described. These compounds are found to bind to protein kinase C with affinities of up to 1.5 nM, demonstrating that variations at C20 can indeed be used to tune affinity and potentially function. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01100-X

Tetrahedron Letters 41 (2000) 6725±6729
Synthesis and biological evaluation of a new class of
bryostatin analogues: the role of the C20 substituent in
protein kinase C binding
Paul A. Wender* and Kevin W. Hinkle
Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA
Received 4 June 1999; revised 23 June 2000; accepted 26 June 2000
Abstract
The synthesis and biological assay of a new series of designed bryostatin analogues modi®ed at the C20
position are described. These compounds are found to bind to protein kinase C with anities of up to 1.5
nM, demonstrating that variations at C20 can indeed be used to tune anity and potentially function.
# 2000 Elsevier Science Ltd. All rights reserved.
The bryostatins are a family of emerging cancer chemotherapeutic candidates isolated from
marine bryozoa.1 These macrocyclic lactones have been shown to exhibit unique biological
activities and are currently in expanded phase II human clinical trials for the treatment of various
types of cancer.2 While the molecular mode of action of bryostatin 1 is not known, it is well
established that it binds potently to and activates protein kinase C (PKC) in a fashion that is
competitive with the phorbol esters but di€erent in physiological consequence.3,4 E€orts to
identify the structural basis for this novel PKC binding and its biochemical rami®cations have
been hampered thus far by the limited current supply of bryostatin 1 which is obtained with
diculty only from ecologically sensitive marine sources. Two total syntheses of a bryostatin
have been completed to date.5,6 However, both of these impressive e€orts required greater than
60 steps. To circumvent this supply problem and more signi®cantly to develop superior clinical
candidates, our laboratory has been involved in the design of a series of bryostatin analogues that
would exhibit enhanced therapeutic performance and would be readily accessible through
synthesis. One such compound, 1, designed on the basis of computer, X-ray, crystallographic and
solution structure analyses, has indeed been shown to exhibit activity similar to bryostatin 1 in
several human cancer cell growth inhibitory assays.7 11
The similar solution structures and activities of 1 and bryostatin suggest that the more readily
available analogue could be used to address questions that cannot be investigated directly with
* Corresponding author.
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01100-X

6726
Figure 1. The bryostatins
bryostatin because of its insucient supply and diculties associated with its modi®cation.12 For
example, relatively little is known about the role of the C20 ester in binding and activity (Fig. 1),
although this site serves as a potential position for tuning pharmacological performance and
could be used mechanistically for anity labeling. Much of the current information on C20
modi®cations is based on the limited number of naturally derived esters. This information is non-
systematic and does not reveal any trends. A few modi®cations of C20 have been achieved
synthetically but this approach is also limited by the complexity of the synthetic intermediates.5,6
For example, initial e€orts in our work to replace directly the C20 ester in the macrocycle 1
proved unsuccessful, presumably due to steric e€ects and instability problems. As a result, we
sought to make this modi®cation on a precursor of 1, generically represented by 3, and to carry
the latter forward to 1 through an esteri®cation±macrotransacetalization sequence that allows
conjunction of the spacer and recognition domains (Fig. 2). We report herein the successful
implementation of this strategy and the synthesis and preliminary binding assays of the ®rst
members of a new class of C20 analogues.
Figure 2. Retrosynthetic analysis
Initial attempts to remove the C20 ester of enal 4 under basic conditions resulted in complete
decomposition of the material. In contrast, protection of the base labile aldehyde subunit of 4
provided a more readily modi®ed compound. Thus, 4 was ®rst converted to its dimethyl acetal
using trimethyl orthorformate and PPTS. The octanoate ester at C20 was then directly cleaved

6727
Scheme 1. (a) CH(OMe)3, PPTS, MeOH, rt; (b) K2CO3, MeOH, rt; (c) Ac2O, DMAP, CH2Cl2, rt, 82% for three steps;
(d) DDQ, CH2Cl2/H2O, rt, 91%; (e) aq. HF, CH3CN, rt, 89%
using K2CO3 in methanol to a€ord the labile C20 alcohol which was immediately acylated using
acetic anhydride and DMAP to give acetate 513 (Scheme 1). Intermediate 5 was prepared for
coupling with the spacer domain 2 by cleavage of the p-methoxybenzyl ether using DDQ to
a€ord the free hydroxyl at C25. Conveniently, the dimethyl acetal is removed under these
conditions. The hemiacetal was revealed with 48% aqueous HF to give enal 6. Completion of the
analogue was then accomplished in a four-step procedure which started with esteri®cation of enal
6 under Yamaguchi's conditions with spacer domain 2 to produce ester 714 (Scheme 2). Cleavage
.
of the C3 silyl ether was accomplished using 1:1 HF pyridine yielding the seco-aldehyde. In a key
step, remarkable for it eciency and tolerance of diverse functionality, macrotransacetalization
of the seco-aldehyde was accomplished with Amberlyst-15 at room temperature to produce the
corresponding benzyl protected analogue.8 The benzyl ether was removed by hydrogenolysis
using Pd(OH)2 as catalyst to a€ord analogue 8.
.
Scheme 2. (a) 2,4,6-Trichlorobenzoylchloride, Et3N, DMAP then 6, CH2Cl2, rt, 63%; (b) HF pyridine, CH3CN, rt,
82%; (c) amberlyst-15 resin, CH2Cl2, rt, 83%; (d) Pd(OH)2, H2, EtOAc, 1 atm, 93%
Syntheses of analogues 13 and 14 were accomplished in good yields using a similar sequence to
that used for analogue 8. Initial esteri®cation of 2 was conducted independently with recognition
domains 9 and 10 following Yamaguchi's protocol to a€ord esters 11 and 1214 (Scheme 3). These
.
esters were then individually treated with HF pyridine in THF at room temperature to a€ord the
macrocyclic products in 83 and 80% yield (for R=C13H27 and R=Ph, respectively).15
Hydrogenolysis of the C26 benzyl ethers was accomplished separately using Pd(OH)2 to provide
analogues 13 and 14.

6728
Scheme 3. (a) 2,4,6-Trichlorobenzoylchloride, Et3N, DMAP then 9 or 10, CH2Cl2, rt, 90% for R=C13H27, 89% for
.
R=Ph; (b) HF pyridine, THF, rt, 83% for R=C13H27, 80% for R=Ph; (c) Pd(OH)2, H2, EtOAc, 1 atm, 97% for 13,
63% for 14
The analogues were tested for binding anity to a rat brain PKC isozyme mixture, a standard
reference system. In view of the limited information on bryostatin and the fact that many
bryostatins have a C20 acetate, it is noteworthy that the C20 acetate analogue 8 showed a
signi®cant reduction in binding anity relative to the octanoate. Furthermore, increasing the
lipophilicity of the analogues (e.g. 13 and 14) caused a signi®cant increase in the binding anity.
Compound 14, selected to test preliminarily whether aromatic groups for anity labeling studies
could be introduced at C20, showed a similar binding anity to the lead analogue. Overall, there
was a correlation between the number of carbons on the ester sidechain (R) and the binding
anity (number of carbons=13>7>6>1), which suggested a previously unrecognized role for
this subunit in binding, membrane association, and potential function (Table 1).
Table 1
Binding anity for rat brain protein kinase C
In summary, a novel series of bryostatin analogues has been assembled which reveals, for the
®rst time, a clear role for the C20 group in PKC binding. These analogues exhibit nanomolar
anities to PKC whose relative order correlates with carbon content. This suggests that one
might use C20 variations to tune the pharmacological performance of these analogues as well as
bryostatin itself. In addition, our ability to introduce aryl groups at C20 without signi®cant loss
of anity bodes well for the use of these compounds in mode of action studies utilizing
photoanity labels. E€orts to further simplify these new medicinal agents, to elucidate the

6729
molecular basis of bryostatin's unique activity, and to develop improved clinical candidates of the
bryostatins are in progress and will be reported in due course.
Acknowledgements
Support of this work through a grant provided by the National Institutes of Health (CA31845)
is gratefully acknowledged. Facilities for PKC assays were supplied by Prof. Daria Mochly-
Rosen (Stanford University). HRMS analyses were performed at UC San Francisco and UC
Riverside Mass Spectrometry Facilities. Fellowship support from the NIH (K.W.H.) is also
gratefully acknowledged.
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