Total synthesis of the potent cAMP signaling agonist (-)-Alotaketal A

Article abstract of DOI:10.1021/ja303529z

We have developed a convergent synthetic route to the potent cAMP signaling agonist (-)-alotaketal A that employs two stages of SmI₂-mediated reductive allylation reactions for assembling the polycycle and fragment coupling. Also notable are a Hg(OAc)₂-mediated selective alkene oxidation and the subtlety of the formation of the unprecedented spiroketal ring system. The probes AKAR4 and ICUE3 were used to evaluate the cAMP singaling agonistic activity of (-)-alotaketal A and elucidate its structure-activity relationship.

Full text of DOI:10.1021/ja303529z

Communication
pubs.acs.org/JACS
Total Synthesis of the Potent cAMP Signaling Agonist (−)-Alotaketal
A
Jinhua Huang,† Jessica R. Yang,‡ Jin Zhang,*,‡ and Jiong Yang*,†
†Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States
‡Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland
21205, United States
S
* Supporting Information
ABSTRACT: We have developed a convergent synthetic
route to the potent cAMP signaling agonist (−)-alotaketal
A that employs two stages of SmI2-mediated reductive
allylation reactions for assembling the polycycle and
fragment coupling. Also notable are a Hg(OAc)2-mediated
selective alkene oxidation and the subtlety of the formation
of the unprecedented spiroketal ring system. The probes
AKAR4 and ICUE3 were used to evaluate the cAMP
singaling agonistic activity of (−)-alotaketal A and
elucidate its structure−activity relationship.
Figure 1. Alotaketals and phorbaketals.
ignaling through cyclic adenosine monophosphate
spiroketals. Contemporaneous to the Andersen report, the Rho
S
(cAMP), the paradigm for the second messenger concept,
lab described the isolation of the closely related phorbaketals
A−C (3−5) from the sponge Phorbas sp.7 Their studies
suggested that an unknown endosymbiotic microorganism
might be the true producer of phorbaketals. We initiated our
synthetic study of alotaketals and phorbaketals as part of a
research program aimed at functionally characterizing natural
products with useful biological properties. Herein we report the
results of our efforts, which culminated in the first
enantioselective total synthesis of (−)-alotaketal A and
elucidation of the structure−activity relationship (SAR) of
this potent agonist of cAMP signaling.
Our convergent synthetic design leading to alotaketal A is
depicted in Scheme 1. We planned to construct the tricyclic
molecular skeleton by spiroketalization of the alcohol derived
from silyl deprotection of 6. Unknown at the outset was the
compatibility of the Δ11,23 alkene with the acidic reaction
conditions that would be necessary to elaborate this
unprecedented spiroketal ring system. Specifically, allylic
activation of the C10 methylene by both the Δ11,23 alkene
and the C9 oxocarbenium, to be transiently formed during
spiroketalization, would cause the Δ11,23 alkene to be
susceptible to undesired exo-to-endo isomerization. With the
expectation that conditions to suppress such isomerization
could be identified, we pursued this route because of the
efficiency gained by convergent coupling of bicyclic lactone 7
with allyl iodide 8 to afford the fully functionalized hemiketal 6.
These two fragments would in turn be prepared from 5β-
hydroxycarvone (9) and ethyl acetoacetate (10), respectively.
is fundamental to a diverse range of cellular processes.1 Such
signaling is typically initiated by the binding of hormones to
cell-surface G protein-coupled receptors (GPCRs), which leads
to the recruitment of cellular guanine-nucleotide binding
proteins (G proteins) and activation of adenylyl cyclases
(ACs), the enzymes responsible for converting adenosine
triphosphate (ATP) to cAMP. The elevated level of cAMP in
turn regulates downstream cellular functions through effectors
such as cAMP-dependent protein kinase (PKA) and the
cAMP−GTP exchange factor Epac.2,3 Formation of cAMP by
ACs and degradation by cAMP-specific phosphodiesterases
(PDEs) collectively determine cellular cAMP levels.
Traditional pharmacological regulation of cAMP signaling
has employed GPCR agonists or antagonists and PDE
inhibitors. ACs have also been pharmacologically targeted by
the diterpenoid forskolin, which binds to ACs and activates
their enzymatic activity.4 Development of new modulators of
cAMP signaling has implications for treating heart failure,
cancer, and neurodegenerative diseases.5 Thus, we were
intrigued by a recent report from the Andersen lab describing
the isolation of alotaketals A (1) and B (2) from the marine
sponge Hamigera sp. collected in Papua New Guinea (Figure
1).6 These compounds were found to cause potent activation of
cAMP cell signaling in the absence of hormone binding in a
cell-based pHTS-CRE luciferase reporter gene assay with half-
maximal effective concentration (EC50) values of 18 and 240
nM, respectively. In contrast, forskolin activates cAMP signaling
with an EC50 of 3 μM. Alotaketals possess a sesterterpenoid
carbon skeleton that cyclizes into a unique tricyclic spiroketal.
In particular, simultaneous substitution of the spiroketal center
by both allyl and vinyl groups is unprecedented in natural
Received: April 12, 2012
Published: May 7, 2012
© 2012 American Chemical Society
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Communication
iodosuccinimide (NIS) in CH2Cl2, while a complex mixture
was obtained when dimethyldioxirane or CF3CO3H was used.
Thus, after extensive experimentation, we were pleased to find
that selective functionalization of the Δ7,22 alkene could be
achieved through reaction of 16 with Hg(OAc)2 to give
allylmercury chloride 17 as the only product. This somewhat
surprising chemoselectivity is likely due to facile rearrangement
of the reversibly formed Δ7,22 mercurinium intermediate upon
enolization of the lactone carbonyl of 16. Since attempts at
direct oxidation (NaBH4, O2) of the C−Hg bond of 17 led only
to proto-demercuration product 18,14,15 the C22 hydroxyl
group was introduced by iodinolysis of 17 followed by
substitution of the resulting allyl iodide 19 with sodium
formate. The initially formed formic ester was hydrolyzed upon
basic workup. Protection of the C22 hydroxyl group as its p-
methoxybenzyl (PMB) ether gave 7.
Scheme 1. Synthetic Design
We developed a reductive allylation approach to bicyclic
lactone 7 (Scheme 2). Regioselective allylic chlorination of 9,
The synthesis of allyl iodide 8 started from the known β-keto
ester 20 (Scheme 3).16 Ester 20 was stereoselectively (>20:1)
a
Scheme 2. Synthesis of Bicyclic Lactone 7
a
Scheme 3. Synthesis of 8
a
Reagents and conditions: (a) (i) Tf2O, LiOH(aq), hexanes, 98%; (ii)
MeMgBr, CuCN, Et2O, 95%. (b) (i) DIBAL-H, THF; (ii) DMP,
CH2Cl2, 93% for two steps. (c) 4-Isopropyl-N-acyl-1,3-thiazolidine-2-
thione, Sn(OTf)2, N-ethylpiperidine, CH2Cl2, −50 °C, 4 h; then 22,
CH2Cl2, −78 °C, 80%. (d) (i) TBSOTf, 2,6-lutidine, CH2Cl2; (ii)
K2CO3, MeOH, 97% for two steps. (e) Me3SiCH2Li, CeCl3, THF,
−78 °C to RT; silica gel. (f) (i) NBS, propylene oxide, THF, RT; (ii)
NaI, acetone, RT, 12 h, 76% for three steps.
a
Reagents and conditions: (a) HClO, CH2Cl2, 64%. (b) HCO2H,
DEAD, PPh3, THF, 70%. (c) (i) NaBH4, CeCl3·7H2O, MeOH; (ii)
TBSCl, imidazole, DMF, 88% for two steps; (iii) NaI, acetone. (d)
SmI2, THF, 73% for two steps. (e) IBX, DMSO, 72%. (f) Hg(OAc)2,
toluene; aq. KCl. (g) I2, CH2Cl2, 81% for two steps. (h) (i) HCO2H,
NaHCO3, DMF; MeOH/H2O, 86%; (ii) PMBOC(NH)CCl3, pTSA,
CH2Cl2, 92%.
converted to 21 by the CuCN-mediated methylation of the
corresponding (Z)-enol triflate,17 which was prepared by
treating 20 with Tf2O/LiOH(aq) under biphasic conditions.18
While similar methylation reactions could be catalyzed by
Fe(acac)3 in high yield,19 significant isomerization of the alkene
was observed under Fe catalysis. Application of a diisobutyla-
luminum hydride (DIBAL-H) reduction/Dess−Martin period-
inane (DMP) oxidation sequence to ester 21 gave aldehyde 22,
which was subjected to the Nagao−Fujita aldol protocol to give
23 with excellent diastereoselectivity (>20:1 based on 1H NMR
analysis).20,21 Alcohol 23 was converted to 24 by silylation and
methanolysis.22 Allylsilane 25 was prepared in good yield by
reacting 24 with Me3SiCH2Li/CeCl3 and then exposing the
crude reaction mixture to silica gel for the Peterson elimination
of the bis(trimethylsilyl)methylcarbinol intermediate.23 Treat-
ment of 25 with freshly recrystallized N-bromosuccinimide
(NBS) at −78 °C in the dark24 followed by Finkelstein reaction
of the allyl bromide intermediate gave 8.
which was readily prepared from (R)-(−)-carvone (11) in two
steps using the vinylogous O-nitroso Mukaiyama aldol
approach we recently developed,8 with HClO gave allylic
chloride 12.9 Mitsunobu reaction of 12 with formic acid went
smoothly to give 13 in 70% yield in the presence of the
electrophilic allyl chloride moiety.10 Diastereoselective Luche
reduction of the enone of 13,11 protection of the hydroxyl
group with TBSCl, and Finkelstein reaction gave iodide 14 as a
single diastereomer.12 As expected, the powerful yet underex-
plored reductive allylation approach reported by Keck,13
achieved by treatment of 14 with excess SmI2, led to smooth
cyclization to give lactol 15 as an inconsequential mixture of
epimers through intramolecular Barbier-type allylation of the
formate. Even though excess SmI2 was employed, further
reduction of 15 was not observed. Oxidation of 15 with 2-
iodoxybenzoic acid (IBX) furnished hydrobenzopyranone 16.
Further functionalization of lactone 16 was complicated by
its unexpected low reactivity toward common electrophilic
reagents required for selective functionalization of the
disubstituted Δ7,22 alkene in the presence of the trisubstituted
Δ2,3 alkene. For example, no reaction occurred when 16 was
treated with m-chloroperoxybenzoic acid (mCPBA) or N-
We anticipated that the fragments of alotaketal A could be
joined through allylation of bicyclic lactone 7 with allyl iodide
8. To investigate the nuances of this transformation and also to
provide the 22-deoxy analogue of alotaketal A for SAR studies,
we explored the coupling of 8 (or the corresponding bromide)
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Scheme 5. Isomerization of the Δ11,23 Alkene
and bicyclic lactone 18 under a variety of conditions (Scheme
4). Whereas all attempts at coupling through the intermediacy
Scheme 4. Synthesis of 22-Deoxyalotaketal A (33) and
a
Alotaketal A (1)
However, no isomerization of 29 was observed when it was
treated with PPTS. These results suggested that part of the exo-
to-endo isomerization of the Δ11,23 alkene occurred through the
intermediacy of oxocarbenium A prior to spiroketalization.
However, the significant isomerization of the alkene in the
pTSA-promoted spiroketalization of 27 was to a large extent
due to the unchecked equilibration of 29 to its thermodynami-
cally more favorable Δ10,11 isomer 30.
On the basis of this model study, the completion of the
alotaketal A synthesis involved the coupling of 7 and 8 with
SmI2 under Barbier conditions to give hemiketal 6 (Scheme 4).
Desilylation of 6 with TBAF to give 28 was again followed by
spiroketalization with PPTS. Interestingly, the spiroketalization
proceeded smoothly to give 31 as a single diastereomeric
product without Δ11,23 alkene isomerization. Since 27 and 28
differ only by the C22 p-methoxybenzyloxy group, we speculate
that the electron-withdrawing inductive effect of the alkoxy
group might be responsible for their differential reactivity
profiles for spiroketalization. Alotaketal A was obtained by IBX
oxidation of 31 and removal of the PMB protecting group with
2,3-dichloro-5,6-dicyanobenzoquinone (DDQ). The 1H and
13C NMR spectra of the synthetic alotaketal A (1) were
consistent with those of the natural product, as was its specific
optical rotation ([α]2D5 = −40.2 (c 0.15, MeOH) for synthetic 1,
[α]2D5 = −38.9 (c 0.01, MeOH) reported for the natural
product). Synthetic 1 was also identical to an authentic sample
on the basis of TLC and HPLC.25
a
Reagents and conditions: (a) SmI2, THF. (b) TBAF, THF. (c) pTSA,
CH2Cl2, 29% for three steps, 29:30 = 1:3−6; with PPTS, 31% for
three steps, 29:30 = 1:1; 40% for 31 over three steps with PPTS. (d)
DDQ, CH2Cl2/H2O (10:1), 92% for 32, 92% for 1. (e) pTSA,
CH2Cl2, RT. (f) IBX, DMSO, 87% for 33, 89% for 34, 89% for 35.
of the allyllithium or allyl Grignard reagents prepared in situ
were unsuccessful, the desired transformation occurred under
Barbier conditions in which the allylsamarium reagent,
generated in situ from 8 by treatment with SmI2, combined
with 18 to give 26 smoothly. Again, despite the presence of
excess SmI2, no over-reduction was observed. Since hemiacetal
26 was relatively unstable, it was subjected to desilylation with
tetrabutylammonium fluoride (TBAF) to give 27 followed by
spiroketalization with p-toluenesulfonic acid (pTSA) without
purification of any intermediates. The desired spiroketalization
to give 29 did occur, but it was accompanied by significant
isomerization of the Δ11,23 alkene to afford the Δ10,11 isomer 30
as the major product (1:3−6). 29 and 30 were oxidized with
IBX to give 22-deoxyalotaketal A (33) and its isomer 34,
respectively. The stereochemistry of the spiroketal centers was
assigned by analogy to that of the natural product.
We examined the effects of 1 and its analogues 29, 30, and
32−34 on cAMP/PKA signaling using a genetically encoded A
kinase activity reporter (AKAR4).26 AKAR4 serves as a
surrogate substrate for PKA and reports endogenous PKA
activity via a change in Forster resonance energy transfer
̈
(FRET). First, we tested each of these compounds in HEK
293T cells transfected with the AKAR4 biosensor. 1 and 32
produced significant increases in the emission ratio of yellow
over cyan [6.7
2.2% (n = 24) and 5.3
2.5% (n = 13),
respectively; Figure 2], whereas no response was observed with
the addition of 29, 30, 33, or 34. We further evaluated the
specificity of the alotaketal-induced AKAR4 responses by
utilizing an AKAR4 T/A mutant probe containing a mutated
PKA phosphorylation site within the PKA substrate domain.
This mutation abolishes PKA phosphorylation and the PKA-
activity-induced FRET changes. No response was detected
when cells expressing the AKAR4 T/A mutant were treated
with 1 and 32, confirming that these compounds induce PKA
activity via the cAMP/PKA signaling pathway (Figure S1 in the
Supporting Information).
The formation of the Δ10,11 isomer 30 was mechanistically
interesting because it could arise either from isomerization of
oxocarbenium intermediate A by deprotonation/reprotonation
to form D followed by cyclization to give 30 or from
isomerization of 29 through the intermediacy of oxocarbenium
A and/or B under the acidic conditions (Scheme 5). To
illuminate the mechanistic subtleties of this process, the
spiroketalization of 27 was tested using less acidic pyridinium
p-toluenesulfonate (PPTS). Isomerization of the Δ11,23 alkene
was again observed, but the two isomers 29 and 30 were
formed in a ratio of ∼1:1. Further experiments showed that 29
could be readily isomerized to 30 upon treatment with pTSA.
To examine further the effects of 1 and 32 on cAMP
accumulation, we used ICUE3, a FRET-based reporter for
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Communication
discussions. Financial support was provided by the Welch
Foundation (A-1700) and Texas A&M University to J.Y. and by
NIH (R01 DK073368) to J.Z.
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(−)-alotaketal A and confirmed its assigned absolute
configuration. The synthesis features two Barbier-type intra-
and intermolecular SmI2-mediated reductive allylations for the
efficient formation of two key C−C bonds. These reactions will
likely find further applications in complex natural product
synthesis. Also notable are the Hg(OAc)2-mediated selective
functionalization of the Δ7,22 alkene and the subtlety of the
spiroketalization/isomerization of the unprecedented spiroketal
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of alotaketal A using the FRET-based AKAR4 and ICUE3
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and spectroscopic data. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
jzhang32@jhmi.edu; yang@mail.chem.tamu.edu
■
Notes
(25) The retention time of 1 was identical to that of the coinjected
authentic alotaketal A using an Agilent ZORBAX Eclipse Plus C18 3.5
μM HPLC column eluted with 90:10 acetonitrile/water.
(26) Depry, C.; Allen, M. D.; Zhang, J. Mol. BioSyst. 2011, 7, 52.
(27) DiPilato, L. M.; Zhang, J. Mol. BioSyst. 2009, 5, 832.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Prof. Raymond Andersen of the University of British
Columbia for an authentic sample of alotaketal A. Profs.
Weiping Tang of University of Wisconsin-Madison, Tarek
Sammakia of University of Colorado Boulder, and Daniel
Romo of Texas A&M University are acknowledged for helpful
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