Synthesis and biological evaluation of bryostatin analogues: The role of the A-ring

Article abstract of DOI:10.1016/S0040-4039(99)02195-4

The first biologically active member and a key intermediate of a new family of simplified bryostatin analogues are synthesized through an optimized esterification-macrotransacetalization strategy. This family incorporates both an ester linkage between C5 and C9 in addition to a C9 t- butyl substituent to mimic the bryostatin A-ring. Importantly, a free C7 alcohol is revealed late in the synthesis, allowing access to numerous C7 derivatized analogues. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(99)02195-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 1007–1011
Synthesis and biological evaluation of bryostatin analogues:
the role of the A-ring
Paul A. Wender ∗ and Blaise Lippa
Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA
Received 10 September 1999; revised 7 October 1999; accepted 8 October 1999
Abstract
The first biologically active member and a key intermediate of a new family of simplified bryostatin analogues
are synthesized through an optimized esterification–macrotransacetalization strategy. This family incorporates
both an ester linkage between C5 and C9 in addition to a C9 t-butyl substituent to mimic the bryostatin A-
ring. Importantly, a free C7 alcohol is revealed late in the synthesis, allowing access to numerous C7 derivatized
analogues. © 2000 Elsevier Science Ltd. All rights reserved.
The bryostatins are a unique and promising class of emerging chemotherapeutic candidates first
isolated in trace quantities from the marine invertibrae Bugula neritina.1 Bryostatin 1 has been shown
to exhibit therapeutic efficacy in a number of human clinical trials, including the induction of several
complete responses to advanced, refractory forms of cancer.2 While their molecular mode of action is
not known, the bryostatins potently bind to protein kinase C (PKC) and stimulate enzymatic activity
both in vitro and in vivo.3 Other noteworthy effects induced by bryostatin include stimulation of immune
system responses,4 increased expression of p53 and decreased expression of bcl-2 coupled to induction
of apoptosis in cancer cells,5 and induction of differentiation of chronic lymphocytic leukemia to a less
invasive cell type.6
∗
Corresponding author.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(99)02195-4
tetl 16123

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Efforts to identify the structural basis for these and related activities and to develop more effective
clinical candidates have been hampered by the low supply of the bryostatins and difficulties associated
with their modification.1b,7 As a potential solution to these problems, we have recently reported the first
series of totally synthetic bryostatin analogues based on computer, pharmacophoric, X-ray, and structure
activity analyses.8,1c These simplified analogues have been shown to have similar or better activity than
the bryostatins in terms of PKC binding affinity and in vitro growth inhibition of human cancer cells.
In order to advance this approach to potentially superior clinical candidates and mechanistic probes, it
is necessary to simplify the target structure and to allow expedient access to a large number of analogues,
possibly in a combinatorial fashion. For these purposes, analogue class 1 has been investigated. Incorpo-
rated in the design of 1 is a C7 (bryostatin numbering) alcohol which is designed to be liberated at a late
stage of the synthesis to serve as a branching point for the synthesis of a number of analogues. In an effort
to systematically simplify the analogue framework, 1 also contains a C9 t-butyl substituent to simulate
the bryostatin A-ring in addition to an ester linkage between C5 and C9. The ester is expected to prefer
the s-cis orientation which would, in principle, serve to rigidify this key portion of the target analogue.
The incorporation of this ester linkage also allows for an improvement in the convergency of the C1–C13
fragment synthesis. Described herein is the synthesis of the first member and the key branching point
intermediate of this new class of bryostatin analogues.
The synthesis of the key C7 hydroxy intermediate began with menthone aldehyde 2 (Scheme 1).8a
A convenient prenyl indium addition9 to 2 gave a 3:1 (α:β) diastereomeric mixture of alcohols, which
could be recycled through an oxidation reduction protocol to provide 4. Ozonolysis of 4 followed by a
reductive workup yielded a diol which was immediately esterified with chloroacetic anhydride to give the
selectively protected chloroacetate derivative 5. The secondary alcohol of intermediate 5 and the known
t-butyl alcohol 68c were then taken through a parallel sequence. Thus, esterification10 of alcohols 5 and
6 independently with acid 7,11 gave the ester derivatives 8 and 9 in good yield. Removal of the chiral
auxiliaries then afforded the completed C1–C13 subunits 10 and 11 in only ten and eight linear steps,
respectively.
Independent Yamaguchi esterification10 of both 10 and 11 with the previously synthesized C15–27
enal 128a gave seco aldehydes 13 and 14 (Scheme 2). Utilization of the previously described two step
macrotransacetalization conditions8a on 13 afforded the target macrocycle 15, but in only 35% yield.
Optimization efforts conducted independently on aldehydes 13 and 14 subsequently revealed that in one
step, using excess 70% HF/pyridine, the C3 TBS group and the menthone ketal of 13 and 14 can be
removed, and the 20-membered macrocycles 15 and 16 can be formed with the stereocenter at C15 set
in both to the desired thermodynamically preferred orientation in high yield. Importantly, this procedure
has been found to be superior in yield in all substrates studied to date.8

1009
Scheme 1. Syntheses of two C1–C13 bryostatin analogue fragments. (a) PrenylBr, in powder, DMF, 84% (3:1, 3:4); (b)
Dess–Martin Periodinane, CH2Cl2; (c) NaBH4, CeCl3·7H2O, MeOH, 0°C, 71%, (3:1, 4:3), two steps; (d) 4, O3, CH2Cl2,
MeOH, −78°C, then NaBH4, 0°C; (e) (ClCH2CO)2O, Pyr, CH2Cl2, 0°C, 63% (based on recovered diol), two steps; (f) 7,
2,4,6-trichlorobenzoyl chloride, DMAP, Et3N, Tol., 83% for 8 from 5; 79% for 9 from 6; (g) H2, Pd(OH)2, EtOAc, 95% for 10
from 8; 80% for 11 from 9
Scheme 2. Convergent esterification–macrotransacetalization approach to bryostatin analogue synthesis. (a) 2,4,6-trichloroben-
zoyl chloride Et3N, DMAP, Tol., then 12, 75% for 13 from 10; 70% for 14 from 11; (b) 1:1 HF:Pyr, THF; (c) Amberlyst-15, 4 Å
MS, CH2Cl2, 35% for 15 from 13; (d) excess HF/pyridine, THF, 62% for 15 from 13; 79% for 16 from 14; (e) H2, Pd(OH)2/C,
EtOAc, 92%
In order to determine the biological activity of this new analogue framework and to allow a direct
comparison to previously synthesized analogues, the C26 benzyl ether of 16 was cleaved to reveal
analogue 17. Binding assays to PKC12 revealed that 17 binds with high affinity (8.42 ± 0.05 nM), similar
to that of the bryostatins. Importantly, 17 is also an effective agent for growth inhibition of the P388
murine lymphocytic leukemia cell line (ED50=113 nM).
Initial efforts to remove the C7 chloroacetate protecting group of 15 with ammonia unfortunately
led to low yields and a significant amount of the 1,3-acyl migrated product 19 (Scheme 3).13 While
19 represents a novel ring expanded bryostatin analogue precursor, our current efforts were focused on
the optimization of the yield of 18. These efforts revealed that the use of excess thiourea in THF gave
excellent yields of 18 without any detectable acyl migration despite the complexity of the molecular

1010
framework and the presence of four other ester functional groups. Intermediate 18 is a key precursor for
the investigation of the role of the C7 group in binding and functional performance.
Scheme 3. Deprotection of the C7 chloroacetate group. (a) NH3, MeOH, THF, 1 h, 26%, 1:1, 18:19; (b) excess thiourea, THF,
4 days, 92%, >11.5:1, 18:19
In conclusion, the successful syntheses of macrocycles 15 and 16 utilizing improved macrotransace-
talization conditions has been achieved. The synthesis of simplified t-butyl analogue 17 has revealed
that high affinity binding is maintained even in analogues lacking the A-ring of bryostatin. Selective
deprotection of the C7 chloroacetate protecting group of 15 has afforded a pivotal intermediate (18) in
excellent yield, allowing the study of the role of the C7 substituent in binding and function. Studies on
these novel analogues will be reported shortly.
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 Professor Daria Mochly-Rosen
(Stanford University). HRMS analyses were performed at the UC Riverside mass spectrometry facility.
Fellowship support from Eli Lilly and Hoffmann-La Roche is also gratefully recognized.
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