Lead diversification through a prins-driven macrocyclization strategy: Application to C13-diversified bryostatin analogues

Article abstract of DOI:10.1055/s-0033-1338860

The design, synthesis, and biological evaluation of a novel class of C13-diversified bryostatin analogues are described. An innovative and general strategy based on a Prins macrocyclization-nucleophilic trapping cascade was used to achieve late-stage dive

Full text of DOI:10.1055/s-0033-1338860

PAPER
▌1815
paper
Lead Diversification through a Prins-Driven Macrocyclization Strategy:
Application to C13-Diversified Bryostatin Analogues
C13-Diversified Bryostatin Analogues
Paul A. Wender,*a Kelvin L. Billingsleyb
a
Department of Chemistry, Department of Chemical and Systems Biology, Stanford University, Stanford, CA 94305-5080, USA
Fax +1(650)7250259; E-mail: wenderp@stanford.edu
b
Department of Radiology, Stanford University School of Medicine, Stanford, CA 94305-5105, USA
Received: 19.04.2013; Accepted: 06.05.2013
To a friend and scholar in recognition of his towering contributions to education and to mechanistic and synthetic chemistry
signal transduction cascades, including regulation of cell
growth, modulation of cell membrane structure, and con-
trol of transcription. Dysfunction of certain PKC iso-
zymes is implicated in a variety of pathologies.10
Abstract: The design, synthesis, and biological evaluation of a
novel class of C13-diversified bryostatin analogues are described.
An innovative and general strategy based on a Prins macrocycliza-
tion-nucleophilic trapping cascade was used to achieve late-stage
diversification. In vitro analysis of selected library members re- Although many ligands for PKC serve as kinase inhibitors
vealed that modification at the C13 position of the bryostatin scaf-
fold can be used as a diversification handle to regulate biological
activity.
at the ATP binding site, bryostatin 1 binds to the C1 reg-
ulatory domain, permitting up- or down-regulation of par-
ticular PKC isozymes. This additional activity of the
natural product has many functional therapeutic ramifica-
Key words: macrocycles, cyclizations, drugs, polyketides
tions, including induction of apoptosis in cancer cells, res-
toration of kinase activity in various disorders, and
Bryostatin 1 is a marine-derived macrocyclic polyketide transcriptional regulation. In addition to interacting with
isolated from the bryozoan Bugula neritina (Figure 1).1 It PKC, bryostatin 1 also interacts with other C1-domain-
exhibits clinically relevant, highly potent, and diverse bi- containing proteins. For example, the natural product and
ological activities,2 and is currently undergoing Phase I its analogues have been shown to activate RasGRP1 in
and Phase II clinical trials for treatment of a variety of cultured T cells,11 and a recent study has linked RasGRP
cancers as well as Alzheimer’s disease.3 Bryostatin 1 has activation with apoptotic induction in Toledo non-
also been shown to reverse multidrug resistance,4 to pro- Hodgkin’s lymphoma.12
mote immunostimulant effects,5 to enhance memory and
Despite its therapeutic potential, bryostatin 1, like many
learning in animal models,6 and to induce repair of neuro-
natural products, is isolated in variable low yields (1.4 ×
nal damage resulting from stroke.7 More recently, bryo-
10–4%) yields from its source.1 In addition, although sev-
statin has also been shown to induce clearance of HIV
eral members of the bryostatin family (bryostatins 1,13 2,14
viral reservoirs, an activity that could figure in the as yet
3,15 7,16 9,17 and 1618) have been prepared by total synthe-
unachieved goal of eradicating HIV/AIDS.8
sis during the past two decades, from about 40 to as many
as 89 steps have been required to produce the natural
products. In 1988, anticipating the problems associated
HO
13
7
OAc
MeO2C
with obtaining a practical supply of bryostatin and with
the view that bryostatin was not evolved for clinical use,
we initiated an alternative approach to address problems
associated with both its supply and its potential clinical
performance.19 Through bioassay-informed computer
modeling, our function-oriented synthesis (FOS)
strategy20 sought to identify those structural features of
the natural product that might affect its biological activity.
The resulting hypotheses were then used to design new
structures with potentially superior activities that might be
produced in a practical manner.21 By using this computer-
assisted synthesis-informed design approach, we reported
in 1998 the first designed analogues of bryostatin (bryo-
logs) with potencies that match that of bryostatin.22 We
subsequently reported several analogues (Figure 2) whose
potencies exceed that of the natural product while emulat-
ing its functional activity in various biological assays.
Significantly, these analogues can be prepared in far few-
er steps than required to synthesize the natural product (19
longest linear steps, 29 total steps).23 Importantly, this
B
A
O
O
O
1
HO
OH
O
C
O
19
26
OH
O
C3H7
O
CO2Me
Figure 1 Bryostatin 1 (PKC binding affinity Ki = 1.4 nM)
The unique biological activities of this natural product
might arise in part from its ability to target and modulate
the protein kinase C (PKC) family of serine/threonine ki-
nases.9 PKCs are implicated in a wide range of cellular
SYNTHESIS 2013, 45, 1815–1824
Advanced online publication: 28.05.2013
DOI: 10.1055/s-0033-1338860; Art ID: SS-2013-C0300-OP
© Georg Thieme Verlag Stuttgart · New York
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X

1816
P. A. Wender, K. L. Billingsley
PAPER
FOS strategy provides access to analogues that exhibit Diversification at C13, which is needed to explore the role
PKC isoform selectivities corresponding to that of the nat- of this center in PKC activity, required cleavage of the
ural product, as well as access to other analogues that ex- C13 exo-alkene, ketone reduction, alcohol activation, and
hibit unique selectivities that are not observed with the nucleophilic substitution. We reasoned that a Prins mac-
natural products but which might be exploited as tools or rocyclization–nucleophilic trapping process would, in a
leads in various preclinical studies.24
single operation, form the macrocycle, assemble the B-
ring, establish two new stereocenters, and permit incorpo-
ration of various functional groups at C13 (Scheme 1).27
Although the Prins macrocyclization reaction is emerging
as a versatile synthesis strategy that has been used suc-
cessfully in our work17,25a and in syntheses of kendomy-
cin,28 neopeltolide,29 and polycavernoside A,30 the
reaction has not been used to diversify a lead scaffold, a
strategy that might be exploited in advancing many natu-
ral hydropyranyl lead compounds.31 In the current study,
we sought to explore the applicability of Prins macrocy-
clization–nucleophilic trapping methods in synthesizing
bryostatin scaffolds, with the aims of establishing whether
modifications to the THP-based B-ring confer advanta-
geous biological activities, such as increased potency, ef-
ficacy, or therapeutic index, and of exploring the
generality of this diversification strategy. These studies
were also conducted to set the stage for positron emission
tomography (PET) and NMR labeling studies on the
structures and functions of PKC–ligand complexes. Here,
we report the successful implementation of this strategy
through the efficient and convergent synthesis of a novel
class of C13-diversified bryostatin analogues, together
with their preliminary biological evaluation.
X
13
7
7
Y
B
A
B
A
O
O
O
O
O
O
O
O
O
1
1
HO
HO
OH
OH
O
C
O
O
19
19
C
26
26
O
OH
OH
C7H15
O
CO2Me
C7H15
O
CO2Me
1 Ki = 3.1 nM
2 X = Y = H
Ki = 1.6 nM
Ki = 2.5 nM
Ki = 0.9 nM
3 X = CO2Me, Y = H
4 X = H, Y = CO2Me
Figure 2 PKC binding affinities of synthetic analogues of bryostatin 1
We recently showed that a Prins-driven macrocyclization
reaction, a variant of our initially reported macrotransace-
talization strategy, can be used to prepare a new class of
B-ring tetrahydropyran (THP)-based bryostatin ana-
logues.25 This transformation is presumed to proceed
through formation of an oxocarbenium ion (as inferred in
our earlier work) that is subsequently captured by an allyl-
silane nucleophile to furnish a C13-substituted exocyclic
olefin (Scheme 1).26 Deprotection of this compound with
hydrogen fluoride·pyridine gives analogue 2, which can
be elaborated by selective ozonolysis and Horner–
Wadsworth–Emmons olefination, followed by global de-
protection, to give bryostatin analogues 3 and 4. Impor-
tantly, the THP-based B-ring bryostatin analogues exhibit
significant potencies that exceed the in vitro antiprolifer-
ative activity of 1 by up to two orders of magnitude. In ad-
dition, in terms of its PKC binding affinity, the natural
C13-(Z)-enoate 4 is one of the most active analogues that
has been discovered to date.
Our initial synthetic studies were aimed at a convergent
preparation of the cyclization precursor 11. Allylation of
the previously reported lactone 5,32 followed by reduction
of the resulting hemiacetal gave the allyl derivative 6 in
55% yield (Scheme 2). The terminal olefin group was then
oxidized with potassium osmate and sodium periodate to
give the corresponding aldehyde 7 in 87% yield. Conver-
sion of aldehyde 7 into the corresponding homoallylic al-
cohol was accomplished by an asymmetric Brown
allylation, and the resulting secondary alcohol was pro-
tected by treatment with chloro(triethyl)silane (67% yield,
two steps). To complete the synthesis of the spacer do-
main 9, the C1 benzyl group was removed by treatment
X
13
13
13
Nuc
7
OH
O
O
O
O
O
O
O
O
O
O
H
O
O
O
1
X = CH2TMS
TBDPSO
X = H
RO
OH
RO
OH
O
O
OH
this work
previous
work
O
O
19
OR
O
OR
26
OTBS
C7H15
O
CO2Me
C7H15
O
CO2Me
C7H15
O
CO2Me
direct functionalization of B-ring
(Prins macrocyclization
nucleophilic trapping)
precursor of 2–4
(Prins-driven macrocyclization)
Scheme 1 Synthetic strategies for preparing substituted THP-derived B-ring bryostatin analogues
Synthesis 2013, 45, 1815–1824
© Georg Thieme Verlag Stuttgart · New York

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C13-Diversified Bryostatin Analogues
1817
O
O
conversion was detected when the halo-Prins macrocycli-
zation was conducted with bromo(trimethyl)silane and
iron(III) bromide at –78 °C (Scheme 4), the transforma-
tion was effective when performed at –40 °C and gave the
C13 bromide in 57% yield (82% based on recovered start-
H
O
O
O
c
a, b
TBDPSO
OBn
TBDPSO
OBn
TBDPSO
OBn
ing
material).
Deprotection
with
hydrogen
5
6
7
fluoride·pyridine resulted in clean production of analogue
12.
A key advantage of the Prins macrocyclization–nucleo-
philic trapping process is its ability to facilitate efficient
synthesis of the THP-based B-ring. For example, upon
coupling of fragments 9 and 10 by Yamaguchi esterifica-
tion, the resulting product could be treated with hydrogen
fluoride·pyridine to initiate directly a fluoro-Prins macro-
cyclization. This remarkable cascade assembles the mac-
rocycle, introduces a fluorine atom at the C13 position,
OR
O
OR
O
f, g
d, e
O
TBDPSO
8, R = TES
OBn
TBDPSO
OH
9, R = TES
spacer domain
Scheme 2 Synthesis of spacer domain 9. Reaction conditions: (a)
AllMgBr, THF, –78 °C, 1.5 h; (b) TES, TFA, CH2Cl2, –30 °C, 1.5 h, and achieves global removal all the silyl protecting groups
55%; (c) K2OsO4, NaIO4, 1,4-dioxane–H2O (3:1), 87%; (d) (–)-(+)-
methoxy(diisopinocampheyl)borane, AllMgBr, Et2O, –78 °C, 3 h,
to give the fluoro derivative 13 (Scheme 4). This transfor-
mation is only the second example of a hydrogen fluoride
then 30 % H2O2, 1 M aq NaOH, 1 h, 69%; (e) TESCl, imidazole,
promoted Prins reaction,34 and only the second report of a
CH2Cl2, 1 h, 98%; (f) lithium naphthalenide, THF, –30 °C, 30 min,
–
fluoro-Prins macrocyclization.28 Although not yet opti-
mized in terms reaction time, the reaction provides a one-
step route to fluorine-labeled analogues. Importantly, the
sequence to produce 13 represents the shortest synthesis
(longest linear sequence 19, and 31 total steps) of a potent
THP-derived B-ring bryostatin analogue (see below).
87%; (g) (1) Pr4N+ RuO4 , NMO, CH2Cl2, r.t., 30 min; (2) NaClO2,
NaH2PO4, CH2Cl2, r.t., 30 min, 75% (two steps).
with lithium naphthalenide (87% yield), and the resultant
alcohol was oxidized to give the carboxylic acid in a two-
step sequence (75% yield).
Because of the efficiency of the initial macrocyclization
process, we next investigated the applicability of a range
of nucleophiles, with the aim of accessing novel C13 sub-
stitution patterns. For example, employing a nitrile as the
trapping agent in a tandem Prins–Ritter reaction would be
expected to provide bryostatin analogues substituted with
amide groups at the C13 position.35 Although few exam-
ples of nitrogen-centered nucleophiles have been reported
to participate in the Prins reaction, they have been restrict-
ed to formation of simple THP moieties. The application
of this method to complex polyfunctionalized molecular
scaffolds has yet to be realized.
Spacer domain 9 was then coupled to the recognition do-
main 10 by means of a Yamaguchi esterification (Scheme
3). Upon TES deprotection, the resulting cyclization pre-
cursor 11 was produced in a 58% yield over three steps.
OR
O
R = TES
O
OH
H
O
TBDPSO
+
OH
O
H
O
O
9
TBDPSO
a–c
The tandem Prins–Ritter macrocyclization was initially
examined by treating a solution of enal 11 in acetonitrile
with trimethylsilyl triflate added dropwise at –40 °C
(Scheme 4). Interestingly, this protocol resulted in the de-
sired macrocyclization with a concomitant global deprot-
ection to give analogue 14 directly. Although only a
modest yield (22%) was obtained, the one-step reaction
involves four distinct transformations and many more
mechanistic steps. Further optimization of solvents,
amounts of reagents, and their rate of addition did not re-
sult in any appreciable increase in the yield of the desired
product. However, the order of addition proved to be cru-
cial in improving the efficiency of the reaction. For exam-
ple, when a solution of enal 11 in acetonitrile was added
dropwise to a solution of trimethyl triflate in dichloro-
methane at –40 °C, 14 was isolated in a markedly im-
proved yield of 75%. Furthermore, this reverse addition
protocol could be applied to other nitriles. When benzoni-
trile was used as the trapping agent, a 55% yield of ana-
OH
OH
O
OH
O
O
OTBS
O
O
OTBS
11
C7H15
O
CO2Me
C7H15
O
CO2Me
recognition domain (10)
Scheme 3 Synthesis of cyclization precursor, 11. Reaction condi-
tions: (a) 9, 2,4,6-trichlorobenzoyl chloride, Et3N, toluene, r.t., 3 h;
(b) 10, DMAP, toluene, r.t., 1 h; (c) PPTS, 20% H2O–THF, 20 h, 58%
(three steps).
Next, we turned our attention to the evaluation of the Prins
macrocyclization–nucleophilic trapping transformation
with a halogen nucleophile (Scheme 4).33 Preliminary in-
vestigations indicated that enal 11 was unstable when ex-
posed to various Lewis acids at 0 °C or above;
optimizations of reaction conditions were therefore un-
dertaken at lower temperatures. Although only modest
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 1815–1824

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P. A. Wender, K. L. Billingsley
PAPER
Nuc
Nuc
13
OH
H
O
B
A
O
O
O
O
O
O
O
O
O
O
O
O
O
Prins-driven
O
O
macrocyclization
TBDPSO
RO
OH
HO
OH
O
RO
OH
O
O
O
OH
O
O
O
C
OR
OH
O
OR
O
OTBS
C7H15
O
CO2Me
11
C7H15
CO2Me
C7H15
O
CO2Me
C7H15
O
CO2Me
H
H
Br
F
N
N
O
O
O
O
O
O
12
57% (A)
13
14
15
55% (C)
75% (C)
35% (B)
MeO
OMe
MeO
H
N
O
O
O
O
16
17
18
56% (D)
14% (C)
(2.3:1.0 ratio of 2- and 4-anisole regioisomers)
Scheme 4 Synthesis of bryostatin analogues by Prins macrocyclization (only the B-rings are shown for products 12–18; the macrocyclization
yields are given). Reaction conditions: (A, halide–Prins macrocyclization): (i) TMSBr, FeBr3, CH2Br2, –40 °C, 2 h; (ii) HF·pyridine, –78 °C to
r.t.; (B, fluoro-Prins macrocyclization): HF·pyridine, –78 °C to r.t.; (C, Prins–Ritter macrocyclization): TMSOTf, nitrile, CH2Cl2, –40 °C, 3 h;
H2O, –40 °C to r.t.; (D, Prins–Friedel–Crafts macrocyclization): (i) TMSOTf, arene (solvent), –40 °C, 2 h; (ii) HF·pyridine, –78 °C to r.t. In the
case of B, C11 is the TES-protected alcohol in the starting material.
logue 15 was obtained, whereas only a 20% yield was vious studies) (Figure 3).25a This remarkably high potency
found under the initial standard conditions.
toward the enzyme mixture was comparable to that of the
parent natural product. However, 13 can be prepared in as
few as 31 total transformations, and is therefore a more
easily accessible target for synthesis. Furthermore, 13 rep-
resents one of the most potent bryostatin analogues that
has been reported to date, and is therefore a superb candi-
date for PET and NMR studies.
The Prins macrocyclization–nucleophilic trapping pro-
cess was further developed through the investigation of a
tandem Prins–Friedel–Crafts macrocyclization.36 This
method proved to be successful for the production of C13-
aryl bryostatin analogues (Scheme 4). In this reaction, 11
was dissolved in the appropriate arene and then treated
with trimethylsilyl triflate. Lower reaction temperatures The development of the Prins macrocyclization–nucleo-
were again necessary to prevent decomposition of the philic trapping method provided a novel class of C13-
starting material. When anisole was used as the trapping substituted THP-based B-ring bryostatin analogues for bi-
agent, the corresponding C13-aryl products were isolated ological evaluation. These compounds were further inves-
in 56% yield as a 2.3:1.0 ratio of the ortho- and para-re-
gioisomers. The corresponding analogues 17 and 18 could
F
then be obtained by hydrogen fluoride·pyridine mediated
O
O
silyl deprotection. This process proved to be limited in
scope to electron-rich arenes, as electron-deficient arenes
gave little or no product. The reverse addition protocol did
not improve the yield of the desired macrocyclization
product.
O
Ki = 1.6 nM
HO
OH
O
potency comparable to natural product .
in only 31 total synthetic steps
O
O
(longest linear sequence = 19)
O
OH
To determine whether a representative example of this
new C13-functionalized class of bryostatin analogues
would effectively target PKC, we conducted a preliminary
competitive binding assay with analogue 13. Importantly,
this compound displayed a 1.6 nM affinity to rodent-
brain-derived PKC (used to permit comparisons with pre-
C7H15
CO2Me
13
Figure 3 Binding affinity of a new C13-substituted bryostatin ana-
logue for rat-brain PKC
Synthesis 2013, 45, 1815–1824
© Georg Thieme Verlag Stuttgart · New York

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C13-Diversified Bryostatin Analogues
1819
in a Hickman apparatus immediately before use. Powdered 4-Å MS
(<5 μm) were purchased from Aldrich and stored in a vacuum oven
(140 °C, 90 mmHg). All other reagents were purchased from
Aldrich and used as received without additional purification.
(3H)phorbol dibutyrate [(3H)PDBu] was obtained from American
Radiolabeled Chemicals, Inc. (St. Louis, MO) as a soln in acetone.
Samples prepared for biological evaluation were purified by prepar-
ative HPLC with a H2O–MeCN gradient using a Varian Pro-Star
(model 320) system equipped with an AllTech Alltima C18 column
(10 μm, 10 × 250 mm).
NMR spectra were recorded on Varian INOVA 500 (1H at 500
MHz, 13C at 125 MHz), Varian 400 (1H at 400 MHz, 13C at 100
MHz), or Varian INOVA 600 MHz (1H at 500 MHz, 13C at 150
MHz) spectrometers, as noted. 1H chemical shifts are reported rela-
tive to the residual solvent peak (CHCl3; δ = 7.26 ppm, benzene;
δ = 7.15 ppm). 13C chemical shifts are reported relative to the 13C
signals for the deuterated solvent (CDCl3; δ = 77 ppm). IR spectra
were recorded on a PerkinElmer 1600 Series Fourier-transform
spectrometer. Optical rotation data were obtained by using a
JASCO DIP. High-resolution mass spectra were recorded at the
Stanford University Mass Spectrometry facility.
tigated for their growth inhibitory effects against the K562
human erythroleukemia cell line (Table 1). In our initial
analysis, analogue 12 showed a promising level of activity
(EC50 = 471 nM). Interestingly, in the examination of the
corresponding C13-fluoride bryostatin analogue 13, we
found a remarkable increase in potency (EC50 = 14.2 nM).
A double-digit nanomolar in vitro activity was also ob-
served for each member of the C13-amide-derived bryo-
statin analogue series 14–16. Furthermore, the C13-aryl-
based bryostatin analogues 17 and 18 showed activity
with potencies that were in the single-digit to low-double-
digit nanomolar range. These results collectively show
that, although the B-ring is positioned outside the pro-
posed binding pocket of the enzyme, modifications to the
spacer domain can result in significant changes in the bi-
ological profile of the corresponding bryostatin analogue,
as would be expected from its potential role in transloca-
tion and membrane association. Thus, the C13 position
appears to be a tunable element that can be utilized to reg-
ulate biological activity and pharmacological perfor-
mance.
Protein Kinase C Isozyme Mixture Binding Assay
Preparation of assay materials. In a polypropylene vial, a buffer
soln was prepared by diluting 1 M aq Tris-HCl (pH 7.4, 1 mL), 1 M
aq KCl (2 mL), 0.1 M aq CaCl2 (30 μL), and bovine serum albumin
(40 mg) with deionized H2O (20 mL). The buffer was stored on ice
until required. Phosphatidyl serine (Avanti Polar Lipids, 10 mg/mL
in CHCl3, 350 μL) was isolated by removing the CHCl3 under a
stream of N2 and then suspended as vesicles by adding the prepared
buffer soln (3.5 mL), followed by sonication (Branson Sonifier 250;
power = 6; 40% duty cycle) for four 30-s periods with a 30 s rest be-
tween sonications. The resulting suspension was stored on ice until
required. Assay Protein Kinase C (PKC) was prepared by dissolv-
ing an aliquot of a PKC rat-brain isozyme mixture (600 μL) in buf-
fer soln (14 mL). This mixture was stored on ice for immediate use.
Table 1 Effective Concentrations of C13-Functionalized Analogues
against Human Leukemia Cell Line K562 after 48 Hours of Incuba-
tion: Average of Two Experiments.
Analogue
12
13
14
15
16
17
18
EC50 (nM) 471
14.2
55.0 30.0
45.2 5.8
23.3
In summary, we have described a novel Prins macrocycli-
zation–nucleophilic trapping diversification strategy for
accessing C13 halogen-, carbon-, and nitrogen-substituted
THP-derived bryostatin analogues. This transformation
was applicable to a range of nucleophiles, including ha-
lides, nitriles, and arenes, and it served as an efficient
method for preparing a range of bryostatin analogues for
further in vitro analysis. Notably, modifications at the C13
position of the bryostatin scaffold were found to affect the
binding and potency of the analogues. Further biological
evaluation of these analogues, including isozyme-selec-
tive activity, is currently underway and will be reported in
due course.
Preparation of dilutions of (3H)PDBu and test compounds. A com-
mercial 1 mCi/mL soln of (3H)PDBu in acetone (American Radio-
labeled Chemicals, Inc.) was diluted 10-fold in EtOH. This EtOH
soln was further diluted by a factor of 16.7 (96 μL into 1504 μL) in
DMSO for use in assays. The assay concentration of (3H)PDBu for
any specific batch of dilutions was measured by competitive bind-
ing of (1H)PDBu [IC50 = assay (3H)PDBu concentration]. For the
data presented above (Figure 3), the assay concentration of
(3H)PDBu was 9.4 nM.
Dilutions of the test compounds were prepared in DMSO, with se-
rially dilution from a high concentration of 2.0 μM (assay concen-
tration 130 nM) by factors of three to a low concentration of 2.7 nM
(assay concentration: 180 pM).
Assay protocol. Polypropylene assay vials (in triplicate) were
charged with phosphatidyl serine vesicle suspension (60 μL), PKC
soln (200 μL), and the diluted test compound (20 μL). Nonspecific
(3H)PDBu binding was analyzed in triplicate by using analogue 4
(75 μM, 20 μL, assay concentration: 5 μM) instead of the test com-
pound. Maximum (3H)PDBu binding was analyzed in triplicate by
substitution of DMSO (20 μL) for the test compound. Finally,
(3H)PDBu [diluted in DMSO (20 μL), assay concentration: 9.4 nM,
determined by titration with (1H)PDBu] was added to all the vials.
The solns were mixed with a vortexer, incubated at 37 °C for 90
min, and placed on ice for 15 min. Filters (Whatman GF/B 21 mm)
were treated by soaking them for 90 min in 10 vol% aq poly(ethyl-
eneamine) (6 mL) diluted in H2O (200 mL). A rinse buffer of 20
mM aq Tris (500 mL, pH 7.4) was prepared and cooled on ice for
the duration of the incubation period and for the remainder of the as-
say. The contents of the assay vials were vacuum filtered through
the poly(ethyleneamine)-soaked filter papers, washing the residual
contents of the vial with additional rinse buffer (0.5 mL). The filters
were then washed by dropwise addition of buffer (4.5 mL) and
Unless otherwise noted, all reactions were performed under N2 in
flame- or oven-dried glassware. Mixtures were stirred by using Tef-
lon-coated magnetic stirrer bars. Reactions were monitored by TLC
on 0.25-mm silica gel 60F plates with a fluorescent indicator
(Merck). Plates were visualized by treatment with an acidic p-an-
isaldehyde or KMnO4 stain and gentle heating. Products were puri-
fied by column chromatography on silica gel 60 (230–400 mesh)
(EM) using the solvent systems indicated.
When necessary, solvents and reagents were purified before use.
THF was distilled from sodium benzophenone ketyl. Et2O, CH2Cl2,
and toluene were passed through a drying column of alumina (Solv-
Tek Inc.) under N2 pressure. Anhyd DMF and DMSO were obtained
from Acros Organics. EtOAc, PE, pentane, and MeOH were ob-
tained from Fisher Scientific. DMSO used to prepare biological
samples was obtained from Fisher BioReagents (Class III). DMSO
used in the PKC and cellular assay protocols was purchased from
Aldrich. TMSOTf was obtained from Aldrich and distilled under N2
© Georg Thieme Verlag Stuttgart · New York
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P. A. Wender, K. L. Billingsley
PAPER
placed into scintillation vials. These vials were filled with Bio-Safe
scintillation fluid (5 mL) and their radioactivity was immediately
measured for 1 min each in a scintillation counter (Beckman LS
6000SC). Counts per minute (cpm) were averaged for each of the
three triplicate dilutions. The data were then plotted [cpm versus
log(concentration)] by using Prism software (GraphPad), and the
value of the IC50 was calculated by using one-site-competition least-
squares regression analysis. Ki values were then calculated by
means of the equation: Ki = IC50/(1 + [(3H)PDBu])/[Kd of
(3H)PDBu]. The Kd of (3H)PDBu was measured under these assay
conditions to be 1.55 nM.
13C NMR (125 MHz, CDCl3): δ = 201.9, 138.5, 135.9, 134.4, 134.3,
129.5 (×2), 128.2, 127.5, 127.4 (×2), 127.3, 74.5, 72.7, 72.5, 68.2,
66.8, 49.9, 44.4, 37.7, 31.4, 31.3, 30.9, 27.0, 23.3, 19.4.
HRMS (ES+): m/z calcd for C34H44NaO4Si: 567.2901; found:
567.2906.
(2S)-1-[(2R,6S)-6-((2R)-4-(Benzyloxy)-2-{[tert-butyl(diphe-
nyl)silyl]oxy}butyl)tetrahydro-2H-pyran-2-yl]pent-4-en-2-ol
(8; R = H)
(–)-(+)-Methoxy(diisopinocampheyl)borane (460 mg, 1.45 mmol)
was weighed into a flask in a dry box. Et2O (15.0 mL) was added
and the soln was cooled to –78 °C. A 1.0 M soln of
CH2=CHCH2MgBr in Et2O (1.21 mL, 1.21 mmol) was added and
the mixture stirred for 5 min at –78 °C then for 1 h at r.t. The mixture
was cooled to –78 °C, a soln of aldehyde 7 (440 mg, 0.808 mmol)
in Et2O (5 mL) was added, and the mixture was stirred for 2.5 h. The
reaction was quenched with 30% H2O2 (1 mL) and 1 M aq NaOH
(400 μL), and the mixture was stirred at 30 °C for 30 min. The soln
was then extracted with Et2O (3 × 30 mL) and the organic layers
were combined, dried (MgSO4), filtered, and concentrated in vacuo.
The crude product was dissolved in MeOH (3 × 10 mL) and con-
centrated in vacuo to remove the remaining borane. The resulting
product was purified by flash chromatography (10% EtOAc–PE) to
give an oil; yield: 55 mg (69%); [α]D24.2 –2.4 (c 4.0, CH2Cl2);
Rf = 0.40 (10% EtOAc–PE) [stained with anisaldehyde (red), UV
active].
K562 Cellular Assay: MTT Cellular Viability Readout
K562 cells were cultured in RPMI medium 1640 (Gibco) supple-
mented with fetal bovine serum( FBS) (10% v/v), penicillin (100
units/mL), and streptomycin (50 units/mL) at 37 °C, under 5% CO2
in air in a humidified incubator. Cells were subcultured every 72–
96 h once the cellular concentration had reached > 1,000,000
cells/mL, to a starting concentration of 100,000 cells per mL. The
cell concentration was determined with a hemocytometer. To assay
the compounds, cells were plated in plasma-treated 96-well polysty-
rene plates at a density of 10,000–14,000 cells/well in 50 μL of cul-
ture medium, and incubated for 1 h while compound dilutions were
prepared. Compounds were diluted from a 4 mM DMSO stock soln
with culture medium to a concentration of 20 μM. Triplicate serial
dilutions were then prepared in culture medium from 20 μM to 80
pM across 10 wells of a 96-well plate (diluted by factors of 4).
Plates containing cells were dosed with the compound (50 μL/well)
to afford final assay concentrations of 10 μM to 40 pM. The maxi-
mum DMSO concentration was 0.25%. Cells were then incubated
at 37 °C under 5% CO2–air in a humidified incubator for 48 h. A 5
mg/mL soln of thiazolyl blue tetrazolium bromide (MTT; Aldrich)
in cell culture medium (10 μl) was then added to each well. The
cells were allowed to incubate for an additional 2.5 h then lysed
with 100 μL of a detergent solvent [Triton-X 100 (Aldrich) (20 mL)
dissolved in 0.1 M HCl in i-PrOH (180 mL)]. After thorough mix-
ing, the plates were analyzed with a VERSAmax tunable microplate
reader (Molecular Devices) using SOFTmax Pro software (version
3.1.1), reading at 570 nm and subtracting at 690 nm. The sigmoidal
dose–response curves generated by plotting the corrected signal
versus the log(drug concentration) were analyzed by least-squares
regression using Prism software (GraphPad) to generate the EC50
values.
IR (thin film): 3497, 3070, 2932, 2857, 1640, 1589, 1427, 1106, 702
cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.68 (d, 8 Hz, 4 H), 7.24–7.42 (m,
11 H), 5.85 (m, 1 H), 5.07–5.12 (m, 2 H), 4.36 (s, 2 H), 4.08 (dt,
J = 2, 6 Hz, 1 H), 3.68 (m, 1 H), 3.64 (s, 1 H), 3.47 (dt, J = 3, 7 Hz,
2 H), 3.34 (m, 1 H), 3.27 (m, 1 H), 2.10–2.26 (m, 2 H), 1.78 (q,
J = 7 Hz, 2 H), 1.62–1.70 (m, 2 H), 1.55–1.60 (m, 1 H), 1.39–1.49
(m, 3 H), 1.24–1.32 (m, 2 H), 1.10–1.20 (m, 1 H), 1.04 (s, 9 H).
13C NMR (125 MHz, CDCl3): δ = 138.5, 135.9, 135.8, 135.2, 134.3,
134.2, 129.5 (×2), 128.2, 127.6, 127.5 (×2), 127.3, 116.8, 78.6,
74.8, 72.7, 70.9, 68.4, 66.7, 44.5, 42.2, 41.9, 37.5, 31.8, 31.3, 26.9,
23.2, 19.4.
[((1S)-1-{[(2R,6S)-6-((2R)-4-(Benzyloxy)-2-{[tert-butyl(diphe-
nyl)silyl]oxy}butyl)tetrahydro-2H-pyran-2-yl]methyl}but-3-
en-1-yl)oxy](triethyl)silane (8, R = TES)
Imidazole (22.5 mg, 0.331 mmol) was added to a soln of alcohol 8
(R = H; 55 mg, 0.094 mmol) in CH2Cl2 (1.0 mL) under ambient air.
Upon dissolution of the solids, TMSCl (28 μL, 0.166 mmol) was
added in one portion to give a white suspension. The mixture was
stirred for 15 min and then diluted with sat. aq NH4Cl (5 mL) and
Et2O (5 mL). The organic and aqueous layers were separated and
the aqueous layer was extracted with Et2O (3 × 5 mL). The organic
phases were combined, dried (MgSO4), filtered, and concentrated.
The crude product was purified chromatography (silica gel, 5%
EtOAc–PE) to give a clear colorless oil; yield: 65 mg (98%);
[α]D24.2 +15.2 (c 3.35, CH2Cl2); Rf = 0.50 (5% EtOAc–pentane)
[stained with anisaldehyde (green), UV active].
[(2R,6S)-6-((2R)-4-(Benzyloxy)-2-{[tert-butyl(diphenyl)si-
lyl]oxy}butyl)tetrahydro-2H-pyran-2-yl]acetaldehyde (7)
An oven-dried round-bottomed flask equipped with stirrer bar was
charged with terminal olefin 637 (468 mg, 0.862 mmol) and the flask
was evacuated, backfilled with N2. A 3:1 mixture of 1,4-dioxane
and H2O (16 mL) followed by 2,6-lutidine (0.200 mL, 0.276 mmol)
were added at r.t by syringe through a septum. K2OsO4·2H2O (1.5
mg, 1.72 mmol) and NaIO4 (736 mg, 3.44 mmol) were added direct-
ly to the flask under N2, and the mixture was stirred at r.t. for 15 h.
The reaction was quenched with sat. aq NaHCO3 (25.0 mL) and di-
luted with Et2O (30 mL). The mixture was then extracted with Et2O
(3 × 30 mL) and the organic layers were combined, dried (MgSO4),
filtered, and concentrated in vacuo. The resulting crude product was
purified by flash chromatography (10% EtOAc–PE) to give an oil;
yield: 410 mg (87%); [α]D24.2 +13.7 (c 1.65, CH2Cl2); Rf = 0.50
(15% EtOAc–PE) (stained with KMnO4).
IR (thin film): 3071, 2932, 2875, 2857, 1641, 1589, 1455, 1427,
1106, 1005, 736, 707 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.71 (m, 4 H), 7.24–7.42 (m, 11
H), 5.84 (m, 1 H), 5.06 (s, 1 H), 5.03 (d, J = 4 Hz, 1 H), 4.37 (s, 2
H), 4.13 (dt, J = 2, 5 Hz, 1 H), 3.86 (p, J = 6 Hz, 1 H), 3.48 (dt,
J = 3, 7 Hz, 2 H), 3.29 (m, 2 H), 2.17–2.29 (m, 2 H), 1.79 (q,
J = 7 Hz, 2 H), 1.45–1.75 (m, 6 H), 1.28–1.38 (m, 2 H), 1.07 (s, 9
H), 1.02 (m, 2 H), 0.97 (t, J = 8 Hz, 9 H), 0.61 (q, J = 8 Hz, 6 H).
13C NMR (125 MHz, CDCl3): δ = 138.6, 135.8, 135.0, 134.4 (×2),
129.4 (×2), 128.2, 127.5 (×2), 127.4, 127.3, 116.9, 74.4, 74.3, 72.7,
68.7, 68.5, 66.9, 44.8, 43.5, 42.0, 37.7, 31.9, 31.3, 27.0, 23.6, 19.4,
6.9, 5.0.
IR (thin film): 3069, 2931, 2856, 1726, 1427, 1110, 821, 737, 702
cm–1.
1H NMR (500 MHz, CDCl3): δ = 9.48 (t, 3 Hz, 1 H), 7.66 (d, 8 Hz,
4 H), 7.24–7.42 (m, 11 H), 4.35 (s, 2 H), 4.12 (pent, J = 7 Hz, 1 H),
3.47 (t, J = 7 Hz, 2 H), 3.42 (m, 1 H), 3.32 (m, 1 H), 2.23–2.36 (m,
2 H), 1.78 (m, 2 H), 1.70 (m, 1 H), 1.56 (m, 2 H), 1.48 (m, 1 H),
1.25–1.34 (m, 3 H), 1.09 (m, 1 H), 1.03 (s, 9 H).
Synthesis 2013, 45, 1815–1824
© Georg Thieme Verlag Stuttgart · New York

PAPER
C13-Diversified Bryostatin Analogues
1821
HRMS (ES+): m/z calcd for C43H64NaO4Si2: 723.4235; found:
723.4246.
as a viscous colorless oil; yield: 23 mg (75%, two steps);
[α]D24.2 +9.0 (c 0.95, CH2Cl2); Rf = 0.20 (20% EtOAc–PE) [stained
with anisaldehyde (green), UV active].
(2S)-1-[(2R,6S)-6-((2R)-2-{[tert-Butyl(diphenyl)silyl]oxy}-4-hy-
droxybutyl)tetrahydro-2H-pyran-2-yl]pent-4-en-2-ol
IR (thin film): 3072, 2931, 1712, 1641, 1590, 1428, 1111, 1084,
1006, 702 cm–1.
A ~1 M soln of lithium naphthalenide in THF was prepared by dis-
solving Li metal (high sodium content; 347 mg, 50 mmol) and
naphthalene (7.69 g, 60 mmol) in THF (50 mL) with sonication un-
der N2 at r.t. The resulting deep green soln was stored under N2 in a
Schlenk flask at –20 °C until required. Solns of this reagent could
be stored for several weeks with only slight loss of activity.
1H NMR (500 MHz, CDCl3): δ = 7.68 (m, 4 H), 7.36–7.45 (m, 6 H),
5.77 (m, 1 H), 5.02 (s, 1 H), 4.98 (dd, J = 2, 7 Hz, 1 H), 4.25 (p,
J = 5 Hz, 1 H), 3.78 (p, J = 6 Hz, 1 H), 3.24 (m, 1 H), 3.12 (m, 1 H),
2.55 (m, 2 H), 2.19 (m, 2 H), 1.50–1.74 (m, 5 H), 1.45 (m, 1 H),
1.18–1.32 (m, 3 H), 1.04 (s, 9 H), 1.00 (m, 1 H), 0.93 (t, J = 8 Hz, 9
H), 0.57 (q, J = 8 Hz, 6 H); the CO2H proton was not observed.
A soln of 8 (R = TES; 307 mg, 0.438 mmol) in THF (7.0 mL) was
cooled in a CO2/MeCN bath (external temperature –25 °C) and then
the ~1.0 M soln of lithium naphthalenide in THF (5.39 mL, 5.39
mmol) was added dropwise in 200-μL portions at 1 min intervals.
When addition of the lithium naphthalenide reagent was complete,
the resulting opaque green mixture was stirred for 10 min until the
starting material was completely consumed (TLC). The reaction
was then quenched with sat. aq NH4Cl (5 mL), which caused the
color to disappear immediately. The biphasic mixture was diluted
with H2O (10 mL) and Et2O (20 mL). The organic and aqueous lay-
ers were separated and the aqueous layer was extracted with Et2O
(2 × 20 mL). The organic layers were combined, dried (MgSO4),
filtered, and concentrated. The residue was purified by chromatog-
raphy (silica gel, 10 to 20% EtOAc–PE) to give a clear, colorless
oil; yield: 232 mg (87%); [α]D24.2 +10.7 (c 1.1, CH2Cl2); Rf = 0.20
(10% EtOAc–pentane) [stained with anisaldehyde (green), UV ac-
tive].
13C NMR (125 MHz, CDCl3): δ = 174.6, 135.9, 135.8, 134.7, 133.8,
133.3, 129.8, 129.7, 127.7, 127.6, 117.1, 74.7 (×2), 68.7, 68.5, 43.9,
43.3, 42.6, 42.0, 31.8, 31.1, 26.9, 23.4, 19.3, 6.9, 5.0.
HRMS (ES+): m/z calcd for C36H56NaO5Si2: 647.3558; found:
647.3569.
Cyclization Precursor 11
Carboxylic acid 9 (126 mg, 0.0202 mmol) was dissolved in toluene
(6.00 mL) under N2, and the soln was treated sequentially with Et3N
(163 μL, 1.17 mmol) and 2,4,6-trichlorobenzoyl chloride (60 μL,
0.383 mmol). The resulting soln was stirred for 3 h at r.t., by which
time the formation of salts was observed. A soln of recognition do-
main 10 (80 mg, 0.133 mmol) in toluene (500 μL) was added
through a cannula with the aid of 2 × 150 μL washes. A soln of
DMAP (85 mg, 0.695 mmol) in toluene (200 μL) was then immedi-
ately added and the resulting mixture was stirred for 1 h then loaded
directly onto a silica gel column and eluted with 20% EtOAc–pen-
tane. Fractions containing the product were collected and concen-
trated in vacuo to afford a residue that was used directly in the next
step.
IR (thin film): 3443, 2932, 2876, 2857, 1642, 1427, 1110, 1068,
1046, 702 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.69 (m, 4 H), 7.36–7.45 (m, 6 H),
5.79 (m, 1 H), 5.02 (d, J = 2 Hz, 1 H), 4.98 (dd, J = 2, 7 Hz, 1 H),
4.08 (p, J = 5 Hz, 1 H), 3.75–3.81 (m, 2 H), 3.69 (m, 1 H), 3.19 (m,
1 H), 2.99 (m, 2 H), 2.31 (br s, 1 H), 2.09–2.24 (m, 2 H), 1.81–1.89
(m, 1 H), 1.59–1.76 (m, 4 H), 1.39–1.56 (m, 3 H), 1.16–1.29 (m, 2
H), 1.07 (s, 9 H), 1.02 (m, 1 H), 0.94 (t, J = 8 Hz, 9 H), 0.57 (q,
J = 8 Hz, 6 H).
13C NMR (125 MHz, CDCl3): δ = 135.9, 135.8, 134.9, 134.0, 133.8,
129.7, 129.6, 127.7, 127.6, 116.9, 75.2, 74.6, 71.0, 68.5, 59.6, 43.7,
43.4, 41.8, 39.1, 31.9, 31.3, 27.0, 23.4, 19.3, 6.9, 5.0.
The residue was dissolved in a soln of TsOH (10.6 mg, 0.042 mmol)
in 20% H2O–THF (25 mL) and the soln was stirred at r.t. under air
for 21 h, then diluted with Et2O (50 mL) and sat. aq NaHCO3 (50
mL). The organic and aqueous layers were separated, and the aque-
ous layer was extracted with Et2O (3 × 50 mL). The organic layers
were combined, dried (MgSO4), filtered, and concentrated, and the
residue was purified by chromatography (silica gel, 20% EtOAc–
PE) to afford 11 as a colorless oil; yield: 84.7 mg (58%, two steps);
[α]D24.2 –39.1 (c 1.24, CH2Cl2); Rf = 0.45 (15% EtOAc–PE) [stained
with anisaldehyde (black spot), UV active].
HRMS (ES+): m/z calcd for C36H58NaO4Si2: 633.3766; found:
633.3775.
IR (thin film): 3497, 3072, 3049, 2931, 2857, 2721, 1723, 1689,
1471, 1429, 1388, 1257, 1229, 1156, 1106, 837, 704 cm–1.
(3S)-3-{[tert-Butyl(diphenyl)silyl]oxy}-4-((2S,6R)-6-{(2S)-2-
[(triethylsilyl)oxy]pent-4-en-1-yl}tetrahydro-2H-pyran-2-
yl)butanoic acid (9; Spacer Domain)
1H NMR (500 MHz, CDCl3): δ = 9.56 (d, J = 8 Hz, 1 H), 7.66 (m,
4 H), 7.34–7.43 (m, 7 H), 5.97 (s, 1 H), 5.93 (dd, J = 8, 9 Hz, 1 H),
5.78 (m, 1 H), 5.21 (m, 1 H), 5.09 (s, 1 H), 5.07 (d, J = 4 Hz, 1 H),
5.03 (s, 1 H), 4.32 (m, 1 H), 3.77 (t, J = 9 Hz, 1 H), 3.64 (s, 3 H),
3.52–3.63 (m, 5 H), 3.21 (t, J = 9 Hz, 1 H), 3.10 (m, 1 H), 2.54 (dd,
J = 5, 15 Hz, 1 H), 2.44 (dd, J = 5, 15 Hz, 1 H), 2.20 (m, 1 H), 1.96–
2.10 (m, 6 H), 1.88 (t, J = 11 Hz, 1 H), 1.54–1.75 (m, 6 H), 1.34–
1.52 (m, 9 H), 1.17–1.28 (m, 14 H), 1.19 (s, 3 H), 1.14 (s, 3 H), 1.01
(s, 9 H), 0.89 (s, 3 H), 0.86 (t, J = 7 Hz, 1 H), 0.05 (s, 3 H), 0.04 (s,
3 H).
13C NMR (125 MHz, CDCl3): δ = 194.8, 171.8, 171.5, 167.0, 166.5,
135.8, 135.1, 133.8, 133.7, 129.7 (×2), 127.6, 127.2, 120.1, 116.9,
99.4, 78.5, 74.3, 72.8, 71.0, 70.9, 67.6, 66.3, 64.8, 51.1, 45.7, 44.7,
43.0, 41.7, 37.9, 34.5, 31.7, 31.6, 31.2, 31.1, 28.9 (×2), 26.9, 25.8,
24.4, 23.1, 23.0, 22.5, 20.0, 19.4, 18.2, 14.1, –5.3 (×2).
An oven-dried vial was charged with the alcohol prepared as de-
scribed above (30 mg, 0.049 mmol) and powdered 4-Å MS (250
mg). The vessel was purged twice with N2 and then a soln of NMO
(22 mg, 0.188 mmol) in CH2Cl2 (0.50 mL) was added. A soln of
–
Pr4N+ RuO4 (2.2 mg, 0.00625 mmol) in CH2Cl2 (0.50 mL) was
added and the resulting green mixture was stirred at r.t. for 10 min
until the starting material was completely consumed (TLC). After
15 min, the mixture was loaded directly onto a short pad of silica gel
and eluted with 20% EtOAc–pentane. The filtrate was concentrated
to give a colorless oil that was used directly in the next step.
The oil was dissolved in t-BuOH (1.20 mL) under air, and H2O (600
μL) and 2-methylbut-2-ene (450 μL) were added successively. The
resulting cloudy mixture was stirred vigorously and cooled in an ice
water bath. The suspension was treated with sequentially with
NaH2PO4 (75 mg, 0.625 mmol) and NaClO2 (35 mg, 0.384 mmol),
then stirred for 25 min. The reaction was then quenched with a 1:1
mixture of sat. aq NaCl and sat. aq Na2S2O3 (3 mL). The resulting
mixture was diluted with H2O (10 mL) and Et2O (10 mL), and the
layers were separated. The aqueous layer was extracted with Et2O
(3 × 10 mL) and the organic phases were combined, dried (MgSO4),
filtered, and concentrated. The resulting oil was purified by chroma-
tography (silica gel, 30% EtOAc–PE) to give the carboxylic acid 9
HRMS (ES+): m/z calcd for C61H94O13Si2Na: 1113.6125; found:
1113.6128.
Bryostatin Analogue 12
Cyclization precursor 11 (7.6 mg, 0.0070 mmol) was dissolved in
CH2Br2 (0.250 mL) under N2 and the resulting soln was cooled in a
CO2/MeCN bath. After 10 min, 50 μL of a stock soln of FeBr3 (1.1
mg, 0.00367 mmol) and TMSBr (4.8 μL, 0.0367 mmol) in CH2Br2
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 1815–1824

1822
P. A. Wender, K. L. Billingsley
PAPER
(0.20 mL) was then added dropwise and the resulting mixture was
stirred for 3 h. The reaction was quenched with H2O (0.50 mL) and
the mixture was diluted with Et2O (1.0 mL) and H2O (1 mL). The
organic and aqueous layers were separated and the aqueous layer
was extracted with Et2O (3 × 1 mL). The organic layers were com-
bined, dried (MgSO4), filtered, and concentrated to give a yellow
oil. This oil was purified by chromatography (silica gel, 25%
EtOAc–PE) to give the cyclized, C26 TBS-deprotected product as
a clear colorless viscous residue; yield: 4.1 mg (57% yield);
Rf = 0.40 (25% EtOAc–PE).
Bryostatin Analogue 14
TMSOTf (1.0 μL) was dissolved in CH2Cl2 (0.100 mL) under N2 in
an oven-dried vial and the soln was cooled in a CO2/MeCN bath for
about 10 min. A soln of 11 (3.0 mg, 0.0028 mmol) in MeCN (0.100
mL) was then added dropwise over 5 min and the resulting mixture
was stirred for 2 h. The reaction was quenched with H2O (0.300 mL)
and the mixture was allowed to warm to r.t. over 30 min then diluted
with Et2O (0.50 mL). The aqueous layer was extracted with Et2O
(3 × 0.50 mL) and the organic layers were combined, dried
(MgSO4), filtered, and concentrated. The resulting oil was purified
by chromatography (silica gel, EtOAc to 5% MeOH–EtOAc) to
give a colorless oil; yield: 1.6 mg (75%); Rf = 0.40 (5% MeOH–
EtOAc) [stained with anisaldehyde (red)].
The crude oily product was dissolved in THF (5.0 mL) under N2 in
a polypropylene vial and the resulting soln was cooled in an ice wa-
ter bath. HF·pyridine (70% HF; 1.50 mL) was added dropwise over
2 min and the mixture was stirred for 10 min. The cold bath was
then removed and the soln was stirred for an additional 24 h at r.t.
The reaction was then quenched with sat. aq NaHCO3 (2 mL) and
the mixture was diluted with Et2O (5 mL). The organic and aqueous
layers were separated and the aqueous layer was extracted with
Et2O (3 × 5 mL). The organic phases were combined, dried
(MgSO4), filtered, and concentrated. The crude residue was purified
by chromatography (silica gel, 60% EtOAc–PE) to give 12; yield:
2.1 mg (37%, two steps). This product was further purified by C18
reverse-phase HPLC (gradient 60% MeCN–H2O to 100% MeCN)
to give an amorphous white solid; Rf = 0.30 (60% EtOAc–PE)
[stained with anisaldehyde (red)].
IR (thin film): 3442, 3356, 2922, 2852, 1721, 1658, 1463, 1435,
1377, 1259, 1167, 1086, 1031 cm–1.
1H NMR (500 MHz, CDCl3): δ = 5.99 (s, 1 H), 5.74 (d, J = 16 Hz,
1 H), 5.34 (m, 1 H), 5.25 (dd, J = 8, 16 Hz, 1 H), 5.17 (s, 1 H), 5.13
(s, 1 H), 4.47 (d, J = 12 Hz, 1 H), 4.04–4.18 (m, 3 H), 3.85 (m, 1 H),
3.67 (s, 3 H), 3.59–3.75 (m, 4 H), 3.52–3.63 (m, 5 H), 3.53 (t,
J = 12 Hz, 1 H), 3.41 (t, J = 12 Hz, 1 H), 2.49–2.63 (m, 2 H), 2.29
(q, J = 7 Hz, 1 H), 2.18 (s, 1 H), 1.88–2.19 (m, 5 H), 1.76–1.80 (m,
3 H), 1.39–1.60 (m, 7 H), 1.18–1.33 (m, 12 H), 1.10 (s, 3 H), 0.97
(s, 3 H), 0.88 (t, J = 7 Hz, 3 H).
HRMS (ES+): m/z calcd for C41H65NNaO13: 802.4348; found:
802.4354.
IR (thin film): 3395, 2955, 2919, 2850, 2360, 1720, 1656, 1570,
1540, 1464, 1413, 1377, 1261, 1113, 1023 cm–1.
Bryostatin Analogue 15
TMSOTf (1.3 μL) was dissolved in CH2Cl2 (0.100 mL) under N2 in
an oven-dried vial and the soln was cooled in a CO2/MeCN bath for
about 10 min. A soln of 11 (4.0 mg, 0.0037 mmol) in PhCN (0.100
mL) was added dropwise over 5 min, and the resulting mixture was
stirred for 2 h. The reaction was then quenched with H2O (0.300
mL) and the mixture was allowed to warm to r.t. over 30 min, then
diluted with Et2O (0.50 mL). The aqueous layer was extracted with
Et2O (3 × 0.50 mL) and the organic layers were combined, dried
(MgSO4), filtered, and concentrated. The resulting oil was purified
by chromatography (silica gel, EtOAc to 5% MeOH–EtOAc) to
give a colorless oil; yield: 1.7 mg (55%); Rf = 0.50 (5% MeOH–
EtOAc) [stained with anisaldehyde (red)].
1H NMR (500 MHz, CDCl3): δ = 5.99 (s, 1 H), 5.74 (d, J = 16 Hz,
1 H), 5.32–5.37 (m, 2 H), 5.26 (dd, J = 8, 15 Hz, 1 H), 5.13 (s, 1 H),
5.13 (s, 1 H), 4.44 (d, J = 11 Hz, 1 H), 4.16–4.24 (m, 2 H), 4.06 (t,
J = 11 Hz, 1 H), 3.97 (t, J = 9 Hz, 1 H), 3.85 (dd, J = 2, 11 Hz, 1 H),
3.64–3.71 (m, 4 H), 3.55 (t, J = 10 Hz, 1 H), 3.52 (t, J = 11 Hz, 1 H),
3.40 (t, J = 11 Hz, 1 H), 2.22–2.38 (m, 3 H), 1.70–2.13 (m, 3 H),
2.26–2.30 (m, 2 H), 2.00–2.12 (m, 5 H), 1.77–1.82 (m, 3 H), 1.40–
1.65 (m, 7 H), 1.18–1.33 (m, 9 H), 1.09 (s, 3 H), 0.98 (s, 3 H), 0.86
(t, J = 7 Hz, 3 H).
HRMS (ES+): m/z calcd for C39H61BrNaO12: 823.3239; found:
823.3231.
IR (thin film): 3351, 2923, 2852, 1723, 1691, 1664, 1642, 1586,
1462, 1328, 1260, 1158, 1084 cm–1.
Bryostatin Analogue 13
The C11-TES protected cyclization precursor 11 (10.6 mg, 0.0089
mmol) was dissolved in THF (7.0 mL) under N2 in a polypropylene
vial and the soln was cooled to –78 °C. HF·pyridine (70% HF; 2.00
mL) was added dropwise over 1 min and the mixture was stirred at
–78 °C for 3 h and then allowed to warm gradually to r.t. over 24 h
without removal of the cooling bath. The reaction was quenched
with sat. aq NaHCO3 (10 mL) and the mixture was diluted with
Et2O (10 mL). The organic and aqueous layers were separated and
the aqueous layer was extracted with EtOAc (3 × 10 mL). The or-
ganic phases were combined, dried (MgSO4), filtered, and concen-
trated. The crude residue was purified by chromatography (silica
gel, 60% EtOAc–PE) to give a colorless oil; yield: 2.3 mg (35%);
Rf = 0.40 (60% EtOAc–PE) [stained with anisaldehyde (red)].
1H NMR (500 MHz, CDCl3): δ = 7.73 (t, J = 7 Hz, 2 H), 7.49 (t,
J = 7 Hz, 1 H), 7.42 (t, J = 7 Hz, 2 H), 5.98 (s, 1 H), 5.83 (m, 1 H),
5.78 (d, J = 15 Hz, 1 H), 5.27–5.34 (m, 2 H), 5.19 (s, 1 H), 5.14 (s,
1 H), 4.50 (d, J = 11 Hz, 1 H), 4.33 (br s, 1 H), 4.15 (m, 1 H), 4.10
(m, 1 H), 3.86 (m, 1 H), 3.68 (s, 3 H), 3.64–3.74 (m, 3 H), 3.52–3.63
(m, 5 H), 3.55 (t, J = 12 Hz, 1 H), 3.45 (t, J = 12 Hz, 1 H), 2.52–2.60
(m, 2 H), 2.21–2.35 (m, 2 H), 1.91–2.10 (m, 5 H), 1.77–1.84 (m, 3
H), 1.40–1.65 (m, 7 H), 1.18–1.33 (m, 9 H), 1.11 (s, 3 H), 0.99 (s, 3
H), 0.87 (t, J = 7 Hz, 3 H).
HRMS (ES+): m/z calcd for C46H67NNaO13: 864.4505; found:
864.4487.
IR (thin film): 3426, 2919, 2850, 1663, 1449, 1422, 1377, 1263,
1167, 1086, 1031, 704 cm–1.
Bryostatin Analogue 16
Cyclization precursor 11 (3.0 mg, 0.0028 mmol) and 4-methoxy-
benzonitrile (9.2 mg, 0.069 mmol) were dissolved in CH2Cl2 (0.200
mL) under N2, and the resulting soln was cooled in a CO2/MeCN for
about 10 min. TMSOTf (1.0 μL) was then added in one portion and
the mixture was stirred for 4 h. The reaction was then quenched with
H2O (0.300 mL) and the mixture was allowed to warm to r.t. over
30 min then diluted with Et2O (0.50 mL). The separated aqueous
layer was extracted with Et2O (3 × 0.50 mL) and the organic layers
were combined, dried (MgSO4), filtered, and concentrated. The re-
sulting oil was purified by chromatography (silica gel, EtOAc) to
give a colorless oil; yield: 0.3 mg (14%); Rf = 0.250 (EtOAc)
[stained with anisaldehyde (red)].
1H NMR (500 MHz, CDCl3): δ = 5.99 (s, 1 H), 5.75 (d, J = 16 Hz,
1 H), 5.29–5.34 (m, 2 H), 5.17 (s, 1 H), 5.14 (s, 1 H), 4.72 (m, 1 H),
4.47 (d, J = 10 Hz, 1 H), 4.18 (m, 1 H), 4.07 (t, J = 12 Hz, 1 H), 3.95
(t, J = 12 Hz, 1 H), 3.84 (m, 1 H), 3.68 (s, 3 H), 3.64–3.71 (m, 4 H),
3.50–3.57 (m, 2 H), 3.43 (t, J = 12 Hz, 1 H), 2.44–2.51 (m, 2 H),
2.22–2.31 (m, 2 H), 1.94–2.05 (m, 5 H), 1.76–1.80 (m, 3 H), 1.39–
1.60 (m, 8 H), 1.18–1.33 (m, 10 H), 1.12 (s, 3 H), 1.00 (s, 3 H), 0.87
(t, J = 7 Hz, 3 H).
HRMS (ES+): m/z calcd for C39H61FNaO12: 763.4039; found:
763.4045.
Synthesis 2013, 45, 1815–1824
© Georg Thieme Verlag Stuttgart · New York

PAPER
C13-Diversified Bryostatin Analogues
1823
IR (thin film): 3425, 2950, 2919, 2850, 1641, 1547, 1463, 1379,
1261, 1033 cm–1.
2.26–2.30 (m, 2 H), 2.00–2.12 (m, 5 H), 1.77–1.82 (m, 3 H), 1.40–
1.65 (m, 7 H), 1.18–1.33 (m, 9 H), 1.09 (s, 3 H), 0.98 (s, 3 H), 0.86
(t, J = 7 Hz, 3 H).
1H NMR (500 MHz, CDCl3): δ = 7.78 (d, J = 8 Hz, 2 H), 6.94 (d,
J = 8 Hz, 1 H), 5.98 (s, 1 H), 5.83 (m, 1 H), 5.72–5.78 (m, 2 H),
5.31–5.38 (m, 2 H), 5.28 (d, J = 7, 15 Hz, 1 H), 5.18 (s, 1 H), 5.13
(s, 1 H), 4.51 (d, J = 13 Hz, 1 H), 4.27 (m, 1 H), 4.18 (m, 1 H), 4.12
(t, J = 10 Hz, 1 H), 4.07 (t, J = 8 Hz, 1 H), 3.86 (s, 3 H), 3.66 (s, 3
H), 3.61–3.78 (m, 3 H), 3.54 (t, J = 12 Hz, 1 H), 3.44 (t, J = 12 Hz,
1 H), 2.52–2.60 (m, 2 H), 2.17–2.36 (m, 3 H), 1.97–2.06 (m, 4 H),
1.77–1.84 (m, 3 H), 1.40–1.65 (m, 6 H), 1.18–1.33 (m, 14 H), 1.11
(s, 3 H), 0.98 (s, 3 H), 0.86 (t, J = 7 Hz, 3 H).
HRMS (ES+): m/z calcd for C46H68NaO13: 851.4572; found:
851.4575.
Acknowledgment
Support of this work through a grant (CA31845) provided by the
NIH to P.A.W. is gratefully acknowledged.
HRMS (ES+): m/z calcd for C47H69NO14Na: 894.4610; found:
894.4613
References
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Bryostatin Analogues 17 and 18
Cyclization precursor 12 (9.0 mg, 0.0083 mmol) was dissolved in
PhOMe (0.225 mL) under N2 and the resulting soln was cooled in a
CO2/MeCN bath for about 10 min. TMSOTf (1.8 μL, 0.0099 mmol)
was then added in one portion, and the resulting mixture was stirred
for 1.5 h. The reaction was then quenched with NaHCO3 (0.50 mL)
and the mixture was diluted with Et2O (1.5 mL) and H2O (1 mL).
The organic and aqueous layers were separated and the aqueous lay-
er was extracted with Et2O (3 × 2 mL). The organic phases were
combined and concentrated to give a yellow oil that was purified via
chromatography (silica gel, 25% EtOAc–PE) to give the cyclized
C26-TBS-deprotected product as a clear colorless viscous residue;
yield: 4.9 mg (56%); Rf = 0.35 (25% EtOAc–PE)
This product was dissolved in THF (7.0 mL) under N2 in a polypro-
pylene vial and the soln was cooled in an ice water bath.
HF·pyridine (70% HF; 1.82 mL) was added dropwise over 1 min,
and the mixture was stirred for 10 min. The cold bath was then re-
moved and the soln was stirred for an additional 25 h at r.t. The re-
action was quenched with sat. aq NaHCO3 (2 mL) and the mixture
was diluted with EtOAc (5 mL). The organic and aqueous layers
were separated and the aqueous layer was extracted with EtOAc
(3 × 5 mL). The organic phases were combined, dried (Na2SO4), fil-
tered, and concentrated. The crude residue was purified by chroma-
tography (silica gel, 60% EtOAc–PE) to give a 7:3 mixture of the
C13-(2-methoxyphenyl) analogue 17 and the C13-(4-methoxyphe-
nyl) analogue 18 as a white solid; yield: 1.9 mg (27%); Rf = 0.40
(60% EtOAc–PE) [stained with anisaldehyde (red)]. The pure ana-
logues were obtained by C18 reverse-phase HPLC (60% MeCN–
H2O to 100% MeCN) as amorphous white solids.
17
IR (thin film): 3426, 2922, 2852, 1722, 1663, 1463, 1429, 1376,
1260, 1232, 1158, 1109, 704 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.13–7.18 (m, 2 H), 6.91 (dt, J = 2,
7 Hz, 1 H), 6.84 (dd, J = 7, 8 Hz, 1 H), 5.99 (s, 1 H), 5.73 (d,
J = 15 Hz, 1 H), 5.32–5.37 (m, 2 H), 5.27 (s, 1 H), 5.14 (s, 1 H), 4.64
(d, J = 11 Hz, 1 H), 4.21 (m, 1 H), 4.10 (m, 1 H), 3.88 (m, 1 H), 3.82
(s, 3 H), 3.68 (s, 3 H), 3.66–3.74 (m, 3 H), 3.52–3.63 (m, 5 H), 3.54
(m, 1 H), 3.45 (t, J = 12 Hz, 1 H), 3.38 (t, J = 12 Hz, 1 H), 2.52–2.64
(m, 2 H), 2.26–2.30 (m, 2 H), 2.00–2.12 (m, 5 H), 1.77–1.82 (m, 3
H), 1.40–1.65 (m, 7 H), 1.18–1.33 (m, 9 H), 1.09 (s, 3 H), 0.98 (s, 3
H), 0.86 (t, J = 7 Hz, 3 H).
HRMS (ES+): m/z calcd for C46H68NaO13: 851.4572; found:
851.4552.
18
IR (thin film): 3426, 2922, 2852, 1722, 1663, 1463, 1429, 1376,
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1260, 1232, 1158, 1109, 704 cm–1.
1H NMR (500 MHz, CDCl3): δ = 7.08 (d, J = 8 Hz, 1 H), 6.83 (dt,
J = 7, 8 Hz, 2 H), 5.99 (s, 1 H), 5.74 (d, J = 15 Hz, 1 H), 5.32–5.37
(m, 2 H), 5.22 (s, 1 H), 5.14 (s, 1 H), 4.60 (d, J = 11 Hz, 1 H), 4.21
(m, 1 H), 4.10 (m, 2 H), 3.88 (m, 1 H), 3.78 (s, 3 H), 3.68 (s, 3 H),
3.66–3.74 (m, 3 H), 3.52–3.63 (m, 5 H), 3.54 (t, J = 12 Hz, 1 H),
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© Georg Thieme Verlag Stuttgart · New York
Synthesis 2013, 45, 1815–1824

1824
P. A. Wender, K. L. Billingsley
PAPER
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Synthesis 2013, 45, 1815–1824
© Georg Thieme Verlag Stuttgart · New York