Total synthesis and structure-activity relationship study of the potent cAMP signaling agonist (-)-alotaketal A

Article abstract of DOI:10.1039/c3ob40120k

A detailed account of the first total synthesis of alotaketal A, a tricyclic spiroketal sesterterpenoid that potently activates the cAMP signaling pathway, is provided. The synthesis employs both intra- and intermolecular reductive allylation of esters for assembling one of the fragments and their coupling. A Hg(OAc)₂-mediated allylic mercuration is used to introduce the C22-hydroxyl group. The subtle influence of substituents over the course of the spiroketalization process is revealed. The synthesis confirms the relative and absolute stereochemistry of (-)-alotaketal A and allows verification of alotaketal A's effect over cAMP signaling using reporter-based FRET imaging assays with HEK 293T cells. Our studies also revealed alotaketal A's unique activity in selectively targeting nuclear PKA signaling in living cells. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob40120k

Organic &
Biomolecular Chemistry
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Total synthesis and structure–activity relationship study
of the potent cAMP signaling agonist (−)-alotaketal A†
Jinhua Huang,a Jessica R. Yang,b Jin Zhang*b and Jiong Yang*a
Cite this: Org. Biomol. Chem., 2013, 11,
3212
A detailed account of the first total synthesis of alotaketal A, a tricyclic spiroketal sesterterpenoid that
potently activates the cAMP signaling pathway, is provided. The synthesis employs both intra- and inter-
molecular reductive allylation of esters for assembling one of the fragments and their coupling. A
Hg(OAc)2-mediated allylic mercuration is used to introduce the C22-hydroxyl group. The subtle influence of
substituents over the course of the spiroketalization process is revealed. The synthesis confirms the relat-
ive and absolute stereochemistry of (−)-alotaketal A and allows verification of alotaketal A’s effect over
cAMP signaling using reporter-based FRET imaging assays with HEK 293T cells. Our studies also revealed
alotaketal A’s unique activity in selectively targeting nuclear PKA signaling in living cells.
Received 21st January 2013,
Accepted 22nd March 2013
DOI: 10.1039/c3ob40120k
www.rsc.org/obc
phosphodiesterases (PDEs), which hydrolyze cAMP to give ade-
nosine monophosphate (AMP). Thus, ACs and PDEs collec-
1. Introduction
Nature provides a rich repertoire of small molecules with tively determine cellular cAMP levels. In addition to using
useful biological properties. Many of these small-molecules agonists and antagonists of GPCRs, cAMP signaling also can
target and regulate disease-relevant cellular signaling path- be pharmacologically regulated using modulators of ACs and
ways/processes and find applications in drug development for PDEs. For example, ACs are activated by the diterpenoid
treating human diseases.1 Indeed, half of clinical anti-cancer natural product forskolin (1, Fig. 1), which interacts with ACs
drugs are derived from natural products, i.e. they are either at the hydrophobic site created by the C1 and C2 catalytic sub-
analogs of natural products or natural products themselves.2 units and activates their enzymatic activity for generating
Bioactive natural products that selectively target biological cAMP.6 Inhibition of cAMP-specific PDEs by their small-
pathways and processes are also used as probes to gain molecule inhibitors also leads to upregulation of cellular cAMP
insights into complex biological systems.3 This so-called levels. Since cAMP signaling is relevant to a number of disease
“small-molecule approach” was instrumental in the study of
cellular signaling events, including the cellular cyclic adeno-
sine monophosphate (cAMP) signaling pathway.4 The acti-
vation of this pathway is initiated with hormone binding to
cell-surface G protein-coupled receptors (GPCRs), which leads
to the activation of trimeric guanine-nucleotide binding pro-
teins (G proteins) and subsequent activation of adenylyl
cyclases (ACs), the enzyme responsible for converting adeno-
sine triphosphate (ATP) to cAMP. This “second messenger” in
turn binds to its downstream effectors, such as cAMP depen-
dent protein kinase (PKA) and exchange proteins activated by
cAMP (Epac).5 Production of cAMP by ACs is countered by
aDepartment of Chemistry, Texas A&M University, College Station, TX 77843, USA.
E-mail: yang@mail.chem.tamu.edu
bDepartment of Pharmacology and Molecular Sciences, The Johns and Hopkins
University School of Medicine, Baltimore, Maryland 21205, USA.
E-mail: jzhang32@jhmi.edu
†Electronic supplementary information (ESI) available: Copies of the NMR
spectra of all new compounds. See DOI: 10.1039/c3ob40120k
Fig. 1 Structures of forskolin, alotaketals and phorbaketals.
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states, such as heart failure, cancer, and neurodegenerative
diseases, the development of new modulators of this signaling
pathway is therapeutically relevant.7
Alotaketal A (2) and B (3) belong to a new class of terpe-
noids isolated by Andersen and co-workers from the marine
sponge Hamigera sp. collected in Papua New Guinea (Fig. 1).8
These natural products feature an “alotane” sesterterpenoid
molecular skeleton that cyclizes into a unique tricyclic spiro-
ketal ring system in which the spiroketal center was simul-
taneously substituted with a vinyl group and an allyl group. To
the best of our knowledge, similarly substituted spiroketals are
unprecedented in natural products. Along with their unique
molecular structures, these compounds also possess interest-
ing biological activities. For example, using HEK 293T cells
transformed with pHTS-CRE luciferase reporter genes, alotake-
tal A and B were found to potently activate the cAMP signaling
pathway with EC50 values of 18 nM and 240 nM in the absence
of hormone binding. Forskolin also activated cAMP signaling
in this reporter gene assay with an EC50 value (3 μM) that is
167-fold less potent than that of alotaketal A. On the other
hand, forskolin elicited a stronger response in the reporter
gene assay, suggesting that different mode-of-action might be
involved. Contemporaneous to the report of Andersen and co-
workers, the Rho group reported isolation of phorbaketals A–C
(4–6) from Korean marine sponge Phorbas sp.9 These com-
pounds possess a molecular skeleton that is identical to that
of alotaketals and are moderately cytotoxic toward human col-
orectal, hepatoma, and lung cancer cell lines. In addition, a
recent study by Hong and co-workers demonstrated that phor-
baketal A stimulated murine C3H10T1/2 cells and human
mesenchymal stem cells for osteoblast differentiation through
TAZ mediated Runx 2 activation, possibly by activation of extra-
cellular signal regulated kinase (ERK).10 While cross talk
between the cAMP and ERK signalings has been well esta-
blished,11 the effect of phorbaketal A on the cAMP signaling
remains unknown and the potential correlation between the
bioactivities of alotaketal A and phorbaketal A remains to be
elucidated. Our interests in developing small molecule tools
for biomedical discoveries prompted us to start a research
project to elucidate the mode-of-action and explore potential
biomedical applications of this family of natural products.
Herein we report in detail our study, which culminated in the
first enantioselective total synthesis of (−)-alotaketal A and illu-
minated the preliminary structure–activity relationship (SAR)
of this potent cAMP signaling agonist.12,13 Our study also
Scheme 1 Synthetic design.
(process I) while the other involved a similar process through
lactol 8 (process II). The former lactol (7) could be assembled
by intermolecular reductive allylation of bicyclic lactone 10
with allyl halide 11 while the latter (8) could be assembled by
intramolecular reductive allylation of ester 9, which in turn
would be synthesized by coupling of 10 and 12. These building
blocks (i.e. 10, 11 and 12) would be prepared from 5β-hydroxy
carvone (13) and ethyl acetoacetate. Unknown at the outset of
our study was the stability of the exocyclic Δ11,23 alkene under
the acidic reaction conditions that are necessary for the for-
mation of the spiroketal. It was expected that allylic activation
of the C10 methylene by both the Δ11,23 alkene and the oxocar-
benium intermediates formed during spiroketalization would
significantly increase the chance of exo-to-endo migration of
the alkene through a process of deprotonation and re-protona-
tion. Despite such a concern, building upon the assumption
that the alkene isomerization could be suppressed by fine-
tuning the reaction conditions and the substrates themselves,
we initiated our synthetic studies.
2.2 Efforts toward a ring-closing metathesis approach to 10
revealed alotaketal A’s unique activity in selectively targeting Our initial efforts focused on developing a ring-closing meta-
PKA signaling in the nucleus of living cells.
thesis approach to bicyclic lactone 15 en route to hydrobenzo-
pyranone 10 (Scheme 2). The metathesis substrates 14a/14b were
synthesized by Mitsunobu reaction of acrylic/trans-2-pentenoic
acid and 5β-hydroxycarvone 13,14 readily prepared from R-(−)-
carvone by vinylogous oxidation of the corresponding silyl
dienol ether under the conditions we recently developed.15
2. Results and discussion
2.1 Synthetic design
Our convergent synthetic design of alotaketal A relied on a late Unfortunately, only the starting materials were recovered when
stage spiroketalization to assemble its tricyclic spiroketal ring 14a or 14b was subjected to common ring-closing metathesis
system. Two such cyclization processes could be envisioned conditions.16 We then turned to the “relay metathesis”
(Scheme 1): one involved spiroketalization through lactol 7 approach, which was expected to facilitate cyclization by
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Scheme 2 Studies of a ring-closing metathesis approach. (a) RCO2H, DEAD,
PPh3, THF, RT, 14a 47%, 14b 83%, 14c 56%. (b) NaBH4, CeCl3·7H2O, MeOH,
−78 °C, 92%. (c) TBSOTf, 2,6-lutidine, CH2Cl2, 97%. DEAD
= diethyl
azodicarboxylate.
assisted formation of the initiating ruthenium alkylidene
species.17 To this end, ester 14c was similarly prepared and
subjected to reaction with the second-generation Grubbs cata-
lyst.18 To our surprise, ester 14a was isolated as the sole
product. The formation of 14a suggested that the β-carbonyl-
carbene species [Ru]=CH(CO)R was indeed generated from 14c
and was competent for intermolecular cross metathesis with
styrene, but not for the intramolecular ring-closing meta-
thesis.19 Despite their electronically and sterically deactivated
nature, acrylates and 1,1-disubstituted alkenes have been
shown to be effective for ring-closing metathesis to give related
benzohydropyranones.20 Thus, the difficulty of cyclization of
14 likely was due to unfavourable conformational effects, such
as developing 1,2-interactions during the formation of the
metallocyclobutane intermediate (e.g. A). Esters 16a/b, pre-
pared by diastereoselective Luche reduction of 14b and TBS-
protection of the allyl hydroxyl group thus formed, also
remained intact under ring-closing metathesis reaction
conditions.
Scheme 3 Synthesis of bicyclic lactone 10. (a) Ca(ClO)2, CO2, CH2Cl2–H2O,
64%; (b) HCO2H, DEAD, PPh3, THF, 70%; (c) NaBH4, CeCl3·7H2O, MeOH, 92%;
(d) TBSCl, imidazole, CH2Cl2, 73% for 2 steps; (e) MOMCl, iPr2NEt, CH2Cl2, 95%;
(f) NaI, acetone, RT; (g) SmI2, THF, 73% for 22b over 2 steps, 76% for 22c over 2
steps; (h) IBX, DMSO, RT, 72% for 23b, 75% for 23c; (i) DBU, CH2Cl2, RT, 95%; ( j)
Hg(OAc)2, toluene; aq. KCl; (k) I2, CH2Cl2, 81% for 2 steps; (l) HCO2H, NaHCO3,
DMF; MeOH–H2O, 86%; (m) PMBOC(NH)CCl3, pTSA, CH2Cl2, 92%.
allylation to give epimers of bicyclic lactol 22a under the con-
ditions developed by Keck and co-workers.24 However, despite
our efforts, further functionalization of 22a was found to be
problematic. Thus, allyl alcohol 20a was protected as its tert-
butyldimethylsilyl ether 20b, and converted to allyl iodide 21b
by reaction with NaI. Again, SmI2-mediated reductive allylation
of 21b gave bicyclic lactol 22b. Subsequent oxidation of 22b
with IBX gave β,γ-unsaturated lactone 23b. A MOM-protected
variant 23c was similarly prepared from 20c, obtained by
MOM-protection of 20a. However, it was not further pursued
because of the relatively harsh conditions that would be
necessary for removal of the MOM-protecting group.
Further functionalization of 23b required oxidative
migration of the Δ7,22 alkene to introduce the C22-allyl
hydroxyl group, which proved to be surprisingly difficult. For
2.3 An intramolecular reductive allylation approach to
bicyclic lactone 10
The difficulty encountered in ring-closing metathesis of 14 example, β,γ-unsaturated lactone 23b was recovered when it
and 16 prompted us to develop a second-generation approach was subjected to epoxidation with mCPBA at RT to 40 °C while
to bicyclic lactone 10 based on intramolecular reductive allyla- complex mixtures were obtained when it was treated with
tion of esters (Scheme 3). It commenced with regioselective DMDO or CF3CO3H. Attempts at allylic halogenation of 23
allylic chlorination of 13 with hypochloric acid, generated in with NIS, I2, or Br2 led to complex reaction mixtures or recov-
situ from calcium hypochlorite and CO2, to give allyl chloride ery of 23b. Efforts to prepare the silyl dienol ether of 23 for
18.21 Formate 19 was obtained when 18 was subjected to Mit- vinylogous oxidation were also unsuccessful.15 Only α,β-un-
sunobu reaction with formic acid, DEAD, and PPh3. The allylic saturated bicyclic lactone 24 was isolated upon reaction of 23b
chloride of 18 remained intact in the presence of nucleophilic with TBSOTf-Et3N or under other silylation conditions. This
PPh3. To prepare for intramolecular reductive allylation, cyclo- bicyclic lactone 24 could also be obtained in 95% yield upon
hexenone 19 was subjected to diastereoselective Luche treating 23b with DBU. A solution was eventually identified
reduction to give 20a,22 which was converted to allyl iodide 21a after extensive experimentation. It started from migratory mer-
by reaction with NaI under Finkelstein conditions.23 Despite curation of 23 with Hg(OAc)2 to give allylmercury intermediate
some initial concerns, the free hydroxyl group of 21a proved to 25, apparently through facile rearrangement of the reversibly
be compatible with SmI2-mediated intramolecular reductive formed Δ7,22 mercuronium intermediate under the influence
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of the ester group. Iodinolysis of 25 with I2 gave allyl iodide 26,
which was subjected to nucleophilic substitution with sodium
formate to give allyl alcohol 27 after basic workup. The
hydroxyl group of 27 was protected as its PMB ether to give
bicyclic lactone 10. We also tested the possibility of displacing
allyl iodide 26 with p-methoxybenzyl alcohol, which was
expected to provide access of 10 directly. However, common
substitution reaction conditions, such as with NaH, KHMDS,
Cs2CO3, K2CO3, or Ag2O, all failed to deliver 10. Interestingly, a
mixture of 23b and 24 was obtained under Pd(0)-catalyzed sub-
stitution conditions.25 Attempts at direct oxidation of allyl-
mercury chloride 23b to allyl alcohol 27 gave reductive-
demercuration product 24 only.26
Scheme 5 Preparation of allyl iodide 11. (a) DIBAL-H, THF; DMP, CH2Cl2, 93%
for 2 steps; (b) 4-isopropyl-N-acyl-1,3-thiazolidine-2-thione, Sn(OTf)2, N-ethyl-
piperidine, CH2Cl2, −50 °C, 4 h, then 35, CH2Cl2, −78 °C, 80%; (c) TBSOTf, 2,6-luti-
dine, CH2Cl2; (d) K2CO3, MeOH, 97% for 2 steps; (e) TMSCH2Li, CeCl3, THF,
−78 °C to RT; silica gel; (f) NBS, propylene oxide, THF, RT; NaI, acetone, RT, 12 h,
76% for 3 steps; (g) KHMDS, PhNTf2, THF, 98%; (h) TMSCH2MgBr, LiCl, Pd-
(PPh3)4, Et2O, 85%.
2.4 Synthesis of the allyl iodide fragment
Our efforts toward synthesis of fragments 11 and 12 were built
upon the ease of access of β-keto ester 28 through γ-alkylation
of the dienolate of ethyl acetoacetate with 4-bromo-2-methyl-
but-1-ene (Scheme 4).27 We envisioned employing transition
metal-catalyzed methylation to convert 28 to E-enoate 30
through the intermediacy of Z-enol triflate 29, which could be
prepared from 28 under biphasic reaction conditions with aq.
LiOH-Tf2O.28 Whereas Fe-catalyzed methylation of enol tri-
flates often proceeds with excellent yield and stereoselecti-
vity,29 we were surprised that the reaction of 29 under such
conditions led to extensive isomerization of the enoate alkene
of 30 (E : Z ∼ 3 : 1 to 1 : 1). To our relief, such an isomerization
was suppressed when CuCN-mediated methylation with
MeMgBr was used and enoate 30 was obtained in 93% yield as
a single isomer.30 Reaction of 30 with N,O-dimethylhydroxyl-
amine by the Merck procedure gave Weinreb amide 31.31 It was
converted to ketone 33 upon alkylation with allylmagnesium
chloride 32. No isomerization of the β,γ-enone moiety of 33
was observed. Preparation of allyl alcohol 12 required enantio-
selective reduction of 33. However, this β,β-disubstituted
enone was found to be unreactive under Noyori transfer hydro-
genation and LiAlH4/R-(+)-BINOL reduction conditions.32,33
While allyl alcohol 12 could be obtained by CBS reduction of
33,34 no enantioselectivity was observed during this process.
We then turned to an aldol approach to introduce the
C13 hydroxyl group. For this purpose, reaction of ester 30 with
DIBAL-H followed by oxidation of the resulting allyl alcohol
with Dess–Martin periodinane gave aldehyde 34, which was
subjected to Nagao–Fujita aldol reaction with 35 to give 36 in
80% yield and with excellent diastereoselectivity (Scheme 5).35
Protection of 36 as its tert-butyldimethylsilyl ether and metha-
nolysis of the thione chiral auxiliary gave ester 37, which was
subjected to a reaction with TMSCH2Li in the presence of
anhydrous CeCl3.36 The initially formed bis-trimethylsilyl-
methyl carbinol was stirred with silica gel at room temperature
to afford allyl silane 38 in 86% yield. Consistent with previous
reports, successful preparation of the allylsilane required that
the trimethylsilylmethylcerium reagent be meticulously pre-
pared. Under less stringent conditions, methyl ketone 39 was
formed as the product (>95% yield).37 However, it could be
converted to 38 via a procedure that consisted of transforming
39 into its enol triflate followed by Pd-catalyzed Kumada coup-
ling with trimethylsilylmethylmagnesium bromide. Various
protocols for iodinolysis of allylsilane 38 were tested. The
optimal results were obtained when 38 was treated with freshly
recrystallized NBS followed by Finkelstein reaction with NaI to
give allyl iodide 11 in 88% yield. It was crucial to maintain the
reaction in the dark to suppress radical bromination of the
remote terminal 1,1-disubstituted alkene.
2.5 Synthesis of 22-deoxyalotaketal A
To shed light on potential obstacles that we might encounter
along the rest of the synthetic route, a pilot study of fragment
coupling and subsequent transformations was carried out
using 11 and 24 as model substrates. This study not only
would illuminate nuances in assembling the unique spiroketal
ring system of alotaketal A, but also would provide access to
22-deoxy and other analogues of alotaketal A for structure–
activity relationship (SAR) studies. Pathway I of our synthetic
design (Scheme 1) was employed because of the perceived
Scheme 4 Preparation of fragment 12. (a) 2.2 equiv. LDA, THF, −78 °C, 82%;
(b) Tf2O, aq. LiOH, hexanes, 98%; MeMgBr, CuCN, Et2O, 95%; (d) MeNH-
(OMe)·HCl, iPrMgBr, THF, 92%; (e) 32, THF, 98%; (f) (R)-(+)-methyl-CBS-oxaza-
borolidine, BH3·Me2S, THF, 85%, ee <5%.
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Scheme 7 Synthesis of (−)-alotaketal A (2). (a) SmI2, THF, 0 °C; (b) TBAF, THF,
RT; (c) PPTS, CH2Cl2, RT, 40% for 3 steps; (d)–(e) IBX, DMSO, 89%.
mediated reductive allylation under Barbier conditions to give
46. This was followed by global desilylation with TBAF and
spiroketalization of the reaction crude (47) with PPTS. We were
pleased to find that only the desired spiroketal 48 was formed
and the exo-to-endo isomerization of Δ7,22 alkene was not
observed. Since 41 and 47 differ by a p-methoxybenzyloxyl sub-
stituent only, we speculate that the inductive electron-with-
drawing effect of the alkoxyl functionality or the cation-π
stabilization of the oxocarbenium by the 4-methoxyphenyl
group contributed to the different reactivities of 41 and 47.
Oxidative removal of the PMB protection of 48 by DDQ gave
diol 49, an alotaketal A analogue with the enone moiety
reduced to an allyl alcohol. Oxidation of 48 with IBX followed
by removal of the PMB protecting group with DDQ gave syn-
Scheme 6 Synthesis of 22-deoxyalotaketal A (44). (a) SmI2, THF, 0 °C; (b) TBAF,
THF, RT; (c) pTSA, CH2Cl2, 29% for 3 steps, 42 : 43 = 1 : 3 to 6; with PPTS, 31%
for 3 steps, 42 : 43 = 1 : 1; (d) IBX, DMSO, 87% for 44, 89% for 45.
simplicity of its fragment coupling event. Whereas initial
attempts at fragment coupling through the intermediacy of
allyllithium or allylmagnesium reagents of 11 were hampered
by competing dimerization of the allyl iodide, the two frag-
ments were smoothly coupled under Barbier conditions with
SmI2 through an intermolecular reductive allylation process to
give lactol 40 (Scheme 6). No over-reduction was observed even
though excess of SmI2 was used. Our attempts at global desily-
lation and spiroketalization of 40 under acidic conditions
(HF-Py or pTSA) in one-pot were unsuccessful. However, the
desired transformations could be effected in a stepwise
manner. Thus, global desilylation of crude 40 with TBAF gave
lactol 41, which was subjected to pTSA-mediated spiroketaliza-
tion without purification. This led to the formation of tricyclic
spiroketal 42 and its Δ10,11 isomer 43 due to migration of
Δ11,23 alkene under the acidic reaction conditions, with the
latter isolated as the major product (1 : 3 to 1 : 6). Interestingly,
spiroketalization of 41 by PPTS led to the formation of spiro-
ketals 42 and 43 in equal amounts. Since the same oxocar-
benium intermediate is involved in the spiroketalization of 41
with either pTSA or PPTS, it is unlikely that the different
product distribution reflects the kinetics of the spiroketaliza-
tion (to give 42) versus the alkene isomerization (to give 43
after cyclization). Instead, it is more likely that isomerization
of the initially formed products contributed to the observed
product distribution. Indeed, treatment of 42 with pTSA in
CH2Cl2 led to the formation of 43. Under otherwise identical
conditions, compound 42 remained intact when treated with
the less acidic PPTS. Both 42 and 43 were oxidized with IBX
to give 22-deoxyalotaketal A (44) and its Δ10,11 isomer (45),
respectively.
1
thetic alotaketal A (2). Its H and 13C NMR spectra were con-
sistent with those reported for the natural product, as was its
optical rotation. In addition, synthetic (−)-alotaketal A was
found to be identical to an authentic sample of the natural
product by TLC and HPLC.
2.7. Investigating the bioactivity of alotaketal A and analogs
The goal of the next series of experiments was to examine the
effects of alotaketal A and analogs on the cAMP/PKA signaling
pathway. These effects were studied in HEK 293T cells by per-
forming fluorescence imaging using an optimized genetically
encoded A-kinase activity reporter (AKAR4).38 As the name
suggests, AKAR4 is able to directly report PKA activity by its
change in fluorescence. The change in fluorescence is due to
the phosphorylation-dependent conformational change of a
tandem fusion domain, composed of a substrate peptide for
PKA and a phosphorylation recognition domain (Fig. 2a). The
conformational change alters the distance or orientation
between the two fluorescent proteins at the N and C termini of
the reporter, generating a change in Förster resonance energy
transfer (FRET) between them. In the context of AKAR4, the
phosphorylation of the substrate domain by activated PKA
causes a conformational change that brings the fluorescent
2.6 Synthesis of (−)-alotaketal A
Building upon our preliminary studies, successful synthesis of proteins in close proximity to produce an increase in FRET
alotaketal A was carried out as depicted in Scheme 7. The which is detected as an increase in the ratio of yellow to cyan
coupling of fragments 10 and 11 went smoothly by SmI2- emission.
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Fig. 2 Effects of different alotaketal A analogs on cAMP/PKA signaling. (A)
Schematic diagram of the AKAR4 FRET-based biosensor used in this series of
experiments. (B) Bar graph showing AKAR4 responses in HEK 293T cells follow-
ing treatment with 42 (1 μM; n = 11), 43 (1 μM; n = 8), 44 (1 μM; n = 5), 45
(1 μM; n = 8), 49 (1 μM; n = 24), and 2 (1 μM; n = 13). (C) Representative time
course graphs depicting AKAR4 responses to the inactive compound 42 (left)
and active compound 49 (right).
First, the HEK 293T cells were transfected with AKAR4 for
24 hours and treated with 1 μM of alotaketal A (2) or one of the
analogs (42–45 and 49). Of the six compounds, 42–45 were
unable to induce a significant change in the ratio of yellow to
cyan emission from the reporter, increasing the emission ratio
by 1.07 0.3% (n = 11; n = number of cells), 1.55 0.8% (n =
8), 1.28 0.6% (n = 5), 1.22 0.4% (n = 8), respectively (Fig. 2b
and c). The relatively small responses from analogs 42–45 were
confirmed to be due to the inactivity of the analogs and not
from poorly functioning AKAR4 because the probes were able
Fig. 3 Alotaketal A and 49 preferentially activate nuclear cAMP/PKA signaling.
(A) Representative YFP and ratiometric images of AKAR4-expressing HEK 293T
to respond maximally upon the addition of a cAMP-elevating cells treated with 49. (B) Time course graph depicting AKAR4 responses in the
nucleus and cytoplasm of HEK 293T cells after the addition of 49 (top panel)
cocktail of the AC activator forskolin (Fsk) and the general PDE
and 2 (bottom panel). (C) YFP and ratiometric images of AKAR4 response after
inhibitor 3-isobutyl-1-methylxanthine (IBMX).39 In contrast to
Fsk (50 μM) and IBMX (100 μM) treatment (top panel). Corresponding time
course graph depicting AKAR4 responses in the nucleus and cytoplasm after the
analogs 42–45, 49 and alotaketal A (2) elicited responses of 6.7
2.2% (n = 24) and 5.3 2.5% (n = 13) from AKAR4, respect-
ively (Fig. 2b and c). Thus, these results show that 49 and 2 are
active and involved in PKA activation. Together with the data
treatment (n = 8) (bottom panel).
showing that 49 and 2 induced responses from a FRET-based while 2 also had a similar response of 6.3 2.5% (n = 6) in the
cAMP indicator,12 these results suggest that these compounds nucleus and 2.5 0.21% (n = 6) in the cytosol (Fig. 3b). These
activate PKA by inducing cAMP production.
results are quite different from what has been seen when naïve
The mechanisms by which these compounds induce cAMP HEK 293T cells expressing AKAR4 are treated with forskolin
production and PKA activation are not clear. But an interesting and IBMX. As previously observed,40 the magnitude of the
observation from these imaging experiments suggested that AKAR4 responses is smaller and kinetically slower in the
they exhibit subcellular specificity when activating this nucleus than in the cytosol (Fig. 3c), 16.1 0.11% (n = 8) and
pathway in living cells. As illustrated in the ratiometric images, 70.8 0.09% (n = 8) respectively, due to the activation of cyto-
the nuclear region showed a more drastic color change com- solic PKA and subsequent translocation of the catalytic
pared to the cytosol upon the addition of 49 or 2 (Fig. 3a). We domain of cytosolic PKA to the nucleus. These results show
then quantitated the responses in the two cellular compart- that alotaketals are possibly targeting a different pool of
ments. Compound 49 elicited a response of 7.2 0.71% (n = 5) cAMP/PKA signaling components, more specifically those
in the nucleus and a mere 2.5 0.72% (n = 5) in the cytosol, found in the nucleus.
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2.50 (dd, J = 16.3, 3.9 Hz, 1H), 2.38 (dd, J = 16.4, 13.8 Hz, 1H),
1.79–1.78 (m, 3H), 1.76–1.75 (m, 3H); 13C NMR (125 MHz,
3. Conclusions
We described in detail the first total synthesis of the tricyclic CDCl3) δ 198.5, 147.4, 143.0, 135.1, 114.8, 68.4, 52.7, 40.8, 19.0,
sesterterpenoid (−)-alotaketal A, a potent cAMP signaling 15.4.
agonist that features an unprecedented spiroketal ring system.
Two Barbier-type SmI2-mediated reductive coupling of allyl
iodides and esters were employed in this convergent synthetic
(1R,6R)-3-Methyl-4-oxo-6-(prop-1-en-2-yl)cyclohex-2-en-1-yl
acrylate (14a)
route: an intramolecular reductive coupling was crucial for the To a solution of 5β-hydroxycarvone 13 (83 mg, 0.5 mmol),
preparation of the key bicyclic lactone fragment whereas an acrylic acid (103 μL, 1.5 mmol) and triphenylphosphine
intermolecular reductive coupling was employed to join the (394 mg, 1.5 mmol) in THF (2.5 mL) was added diethyl azodi-
fragments and complete the sesterterpenoid molecular skele- carboxylate (0.68 mL, 40% in toluene, 1.5 mmol) dropwise at
ton. Our study revealed the subtle interplay of the structure 0 °C under nitrogen. The resulting mixture was stirred at room
and functional group stability/reactivity during spiroketaliza- temperature for 3 h, diluted with EtOAc (30 mL), and washed
tion to form the unique spiroketal ring system. Also high- with H2O and brine. The organic layer was dried over Na2SO4,
lighted is the Hg(OAc)2-mediated allylic mercuration of concentrated and purified by flash column chromatography
β,γ-unsaturated lactone to introduce the C22-hydroxyl group. (silica gel, ethyl acetate–petroleum ether, 1/5) to provide 14a
Using FRET imaging assays with genetically encoded AKAR4 (52 mg, 47%) as a light yellow oil. [α]2D0 −98.3 (c 1.96, CHCl3);
reporter genes, we verified that the cAMP signaling pathway IR (film, cm−1) 2976, 2919, 1729, 1685, 1407, 1262, 1182, 1042,
was activated by alotaketal A. Our preliminary SAR studies 900, 809; 1H NMR (500 MHz, CDCl3) δ 6.79 (dd, J = 5.6, 1.5 Hz,
revealed the C22-hydroxyl group to be important to alotaketal 1H), 6.38 (dd, J = 17.3, 1.4 Hz, 1H), 6.07 (dd, J = 17.3, 10.4 Hz,
A’s bioactivity. On the other hand, reduction of the enone 1H), 5.84 (dd, J = 10.4, 1.4 Hz, 1H), 5.63–5.61 (m, 1H), 4.94 (s,
moiety to an allyl alcohol was tolerated, suggesting the enone 1H), 4.75 (s, 1H), 2.86 (d, J = 12.6 Hz, 2H), 2.55 (dd, J = 12.5,
moiety to be nonessential. Our FRET imaging studies revealed 0.9 Hz, 1H), 1.83 (dd, J = 1.4, 0.8 Hz, 3H), 1.78 (s, 3H); 13C
that alotaketal A and analog 49 activated the cAMP/PKA NMR (125 MHz, CDCl3) 199.2, 165.4, 142.8, 138.9, 138.6, 131.4,
pathway in a subcellular specific manner. Recent studies have 128.0, 113.0, 66.4, 44.3, 37.9, 21.9, 15.6; HRMS (ESI): calcu-
suggested that cAMP elicits highly specific cellular response to lated for C13H16O3 [M + Li+] 227.1259, found 227.1249.
external stimuli via highly compartmentalized cAMP signal-
(E)-(1R,6R)-3-Methyl-4-oxo-6-(prop-1-en-2-yl)cyclohex-2-en-1-yl
pent-2-enoate (14b)
ing.41 Evidence of subcellular cAMP signaling domains in
specific regions such as the nucleus has been presented
through the identification of a functional pool of nuclear PKA To a solution of 5β-hydroxycarvone 13 (50 mg, 0.3 mmol),
holoenzyme, and its regulators, the phosphodiesterase, PDE4, 2-pentenoic acid (0.09 mL, 0.9 mmol) and triphenylphosphine
and scaffolding proteins such as A-kinase anchoring proteins (237 mg, 0.9 mmol) in THF (2 mL) was added diethyl azodicar-
(AKAPs).42 Therefore, future experiments will focus on the boxylate (0.42 mL, 40% in toluene, 0.9 mmol) dropwise at 0 °C
identification of cellular targets of alotaketals and elucidation under nitrogen. The resulting mixture was stirred at room
of their functions within the nucleus. Furthermore, these alota- temperature for 3 h, diluted with EtOAc (30 mL) and washed
ketals may also serve as molecular tools to provide a better with H2O and brine. The organic layer was dried over Na2SO4,
understanding of the function of nuclear PKA as well as the concentrated and purified by flash column chromatography
details of the mechanism that underlies compartmentalized (silica gel, ethyl acetate–petroleum ether, 1/5) to provide
cAMP signaling.
product 14b (61.8 mg, 83%) as a light yellow oil. [α]2D1 −287.77
(c 2.14, CHCl3); IR (film, cm−1) 2970, 2919, 1720, 1679, 1649,
1244, 1176, 1116, 1025, 971; 1H NMR (300 MHz, CDCl3) δ
7.05–6.96 (m, 1H), 6.79 (dd, J = 5.6, 1.5 Hz, 1H), 5.76 (dt, J =
15.7, 1.7 Hz, 1H), 5.61–5.58 (m, 1H), 4.94 (s, 1H), 4.75 (s, 1H),
2.88–2.80 (m, 2H), 2.60–2.47 (m, 1H), 2.27–2.17 (m, 2H), 1.83
(dd, J = 1.5, 0.8 Hz, 3H), 1.78 (s, 3H), 1.06 (t, J = 7.4 Hz, 3H);
4. Experimental section
(4S,5R)-4-Hydroxy-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-enone
(13)
To a solution of nitrosobenzene (10.80 g, 100.7 mmol) in 13C NMR (75 MHz, CDCl3) δ 199.3, 166.0, 151.8, 142.9, 139.0,
CH2Cl2 (210 mL) were added acetic acid (5.76 mL, 100 mmol) 138.7, 119.7, 112.9, 66.1, 44.3, 37.9, 25.4, 21.9, 15.6, 12.0;
and (5-isopropenyl-2-methyl-cyclohexa-1,3-dienyloxy)-trimethyl- HRMS (ESI): molecular ion was not observed.
silane43 (9.33 g, 42.0 mmol) at −78 °C. The solution was
(E)-4,4-Diethyl 1-((1R,6R)-3-methyl-4-oxo-6-(prop-1-en-2-yl)-
cyclohex-2-en-1-yl) hepta-1,6-diene-1,4,4-tricarboxylate (14c)
stirred at this temperature for 12 h before being warmed to
room temperature and further stirred for 4 h. The solvent was
removed under reduced pressure and the residue was purified To a solution of triethyl pent-4-ene-1,2,2-tricarboxylate44 (5 g,
by flash column chromatography (silica gel, ethyl acetate– 16 mmol) in EtOH (20 mL) was added 1 N NaOH (19.2 mL,
petroleum ether, 1/2) to provide 13 (4.12 g, 59%) as a yellow oil. 19.2 mmol) at room temperature. The resulting mixture was
1H NMR (500 MHz, CDCl3) δ 6.71–6.70 (m, 1H), 4.98–4.97 (m, stirred for 4 h before it was acidified with 2 N HCl. The
1H), 4.95–4.94 (m, 1H), 4.46–4.42 (m, 1H), 2.72–2.67 (m, 1H), aqueous layer was extracted with EtOAc. The combined
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organic layer was dried over Na2SO4, concentrated and purified (E)-(1R,4R,6R)-4-((tert-Butyldimethylsilyl)oxy)-3-methyl-6-
by flash column chromatography (silica gel, ethyl acetate– (prop-1-en-2-yl)cyclohex-2-en-1-yl pent-2-enoate (16b)
petroleum ether, 1/2) to provide 3,3-bis(ethoxycarbonyl)hex-5-
To a solution of allyl alcohol 16a (12 mg, 0.048 mmol) in
CH2Cl2 (1 mL) were added 2,6-lutidine (14 μL, 0.12 mmol) and
TBSOTf (22 μL, 0.096 mmol) at 0 °C. After 15 min, the reaction
was quenched with a sodium potassium phosphate buffer
solution (pH = 7, 1 mL) and extracted with CH2Cl2 (3 × 10 mL).
The combined organic layers were washed with sat. aq.
enoic acid (3.51 g, 77%) as a colorless oil. IR (film, cm−1) 2985,
1735, 1700, 1652, 1282, 1208, 1096, 927, 856; 1H NMR
(500 MHz, CDCl3) δ 6.92 (dt, J = 15.5, 7.7 Hz, 1H), 5.87 (dd, J =
15.5, 0.8 Hz, 1H), 5.66–5.58 (m, 1H), 5.15 (dd, J = 2.8, 0.9 Hz,
1H), 5.12 (d, J = 0.9 Hz, 1H), 4.20 (q, J = 7.1 Hz, 4H), 2.78 (d, J =
7.7 Hz, 2H), 2.65 (d, J = 7.4 Hz, 2H), 1.25 (t, J = 7.1 Hz, 6H); 13
C
NaHCO3 and dried over Na2SO4. After concentration in vacuo,
the residue was purified by flash column chromatography
(silica gel, ethyl acetate–petroleum ether, 1/15) to provide 16b
(16.7 mg, 97%) as a yellow oil. [α]1D9 −188.8 (c 1.58, CHCl3); IR
(film, cm−1) 2958, 2928, 2854, 1717, 1646, 1253, 1179, 1105,
891, 832; 1H NMR (300 MHz, CDCl3) δ 6.96 (dt, J = 15.7, 6.3 Hz,
1H), 5.74 (dt, J = 15.6, 1.6 Hz, 1H), 5.65–5.63 (m, 1H),
5.36–5.33 (m, 1H), 4.85 (s, 1H), 4.73 (s, 1H), 4.14 (t, J = 8.0 Hz,
1H), 2.34–2.23 (m, 1H), 2.22–2.14 (m, 2H), 1.94–1.82 (m, 2H),
1.75 (s, 6H), 1.04 (t, J = 7.4 Hz, 3H), 0.92 (s, 9H), 0.11 (s, 3H),
0.11 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 166.4, 150.5, 144.9,
144.8, 121.0, 120.5, 111.5, 71.4, 66.8, 43.3, 32.5, 25.9, 25.6,
25.3, 22.1, 19.6, 12.1, −4.1, −4.9; HRMS (ESI): calculated for
C21H36O3Si [M + Li+] 371.2594, found 371.2606.
NMR (125 MHz, CDCl3) δ 171.1, 170.1, 145.8, 131.7, 124.2,
119.8, 61.6, 56.9, 37.4, 35.3, 14.1; HRMS (ESI): calculated for
C14H20O6 [M + Li+] 291.1420, found 291.1433.
To a solution of 5β-hydroxycarvone 13 (83 mg, 0.5 mmol),
3,3-bis(ethoxycarbonyl)hex-5-enoic acid (426 mg, 1.5 mmol)
and triphenylphosphine (394 mg, 1.5 mmol) in THF (2.5 mL)
was added diethyl azodicarboxylate (0.68 mL, 40% in toluene,
1.5 mmol) dropwise at 0 °C under nitrogen. The resulting
mixture was stirred at room temperature for 3 h, diluted with
EtOAc (30 mL), and washed with H2O and brine. The organic
layer was dried over Na2SO4, concentrated and purified by
flash column chromatography (silica gel, ethyl acetate–pet-
roleum ether, 1/5) to provide 14c (121 mg, 56%) as a light
yellow oil. [α]1D9 −174.17 (c 1.81, CHCl3); IR (film, cm−1) 2985,
1726, 1685, 1649, 1448, 1173, 1045, 915; 1H NMR (500 MHz,
CDCl3) δ 6.82–6.76 (m, 2H), 5.81 (d, J = 15.6 Hz, 1H), 5.65–5.56
(m, 2H), 5.13 (d, J = 1.2, 1H), 5.10 (dd, J = 9.3, 1.9 Hz, 1H), 4.92
(s, 1H), 4.73 (s, 1H), 4.19–4.16 (m, 4H), 2.88–2.77 (m, 2H), 2.74
(dd, J = 7.7, 1.3 Hz, 2H), 2.62 (d, J = 7.5 Hz, 2H), 2.53 (dd, J =
15.2, 2.6 Hz, 1H), 1.83 (s, 3H), 1.76 (s, 3H), 1.29–1.18 (m, 6H);
13C NMR (125 MHz, CDCl3) δ 199.2, 170.1, 170.1, 165.0, 143.9,
142.9, 138.8, 138.7, 131.7, 124.3, 119.7, 112.9, 66.3, 61.6, 56.9,
44.3, 37.9, 37.3, 35.3, 21.9, 15.6, 14.1; HRMS (ESI): calculated
for C24H32O7 [M + Li+] 439.2308, found 439.2288.
(1R,4R,6R)-6-(3-Chloroprop-1-en-2-yl)-4-(methoxymethoxy)-3-
methylcyclohex-2-en-1-yl formate (20c)
To a solution of allyl alcohol 20a (400 mg, 1.73 mmol) in
dichloromethane (11 mL) was added N,N-diisopropylethyl-
amine (0.58 mL, 3.46 mmol). The reaction mixture was cooled
to 0 °C before methyl chloromethyl ether (2.1 mL, 2.1 M in
toluene, 4.35 mmol) was introduced. The solution was stirred
at room temperature for 12 h before it was poured into water
(20 mL) and extracted with ethyl acetate. The combined
organic layers were dried over Na2SO4. After concentration
(E)-(1R,4R,6R)-4-Hydroxy-3-methyl-6-(prop-1-en-2-yl)cyclohex-2- in vacuo, the residue was purified by flash column chromato-
en-1-yl pent-2-enoate (16a)
graphy (silica gel, ethyl acetate–petroleum ether, 1/5) to
provide 20c (450 mg, 95%) as a yellow oil. [α]2D1 −209.4 (c 0.71,
CHCl3); IR (film, cm−1) 2949, 2931, 2890, 1720, 1173, 1143,
1101, 1034, 927; 1H NMR (500 MHz, CDCl3) δ 7.97 (s, 1H), 5.70
(d, J = 5.5 Hz, 1H), 5.43 (brs, 1H), 5.30 (s, 1H), 5.06 (s, 1H),
4.82 (d, J = 6.9 Hz, 1H), 4.70 (d, J = 6.9 Hz, 1H), 4.16 (d, J =
11.9, 1H), 4.14–4.10 (m, 1H), 4.04 (d, J = 11.9, 1H), 3.44 (s, 3H),
2.73 (d, J = 13.5 Hz, 1H), 2.17–2.12 (m, 1H), 1.96–1.88 (m, 1H),
1.83 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 160.5, 144.2, 143.8,
121.2, 117.1, 95.8, 75.5, 65.9, 55.7, 47.5, 38.4, 28.9, 19.3; HRMS
(ESI): molecular ion not observed.
To a solution of enone 14b (19 mg, 0.076 mmol) in MeOH
(1.5 mL) was added CeCl3·7H2O (43 mg, 0.12 mmol) at room
temperature under nitrogen. The resulting suspension was
stirred for 10 min, and cooled to −78 °C; then NaBH4 (3.5 mg,
0.092 mmol) was added in one portion. Once the reaction was
complete (typically <10 min), it was immediately quenched
with sat. aq. NaHCO3. The aqueous phase was extracted with
EtOAc. The combined organic layers were dried over Na2SO4,
concentrated and purified by flash column chromatography
(silica gel, ethyl acetate–petroleum ether, 1/4) to provide 16a
(17.5 mg, 92%) as a yellow oil. [α]1D9 −261.6 (c 2.02, CHCl3); IR
(film) 2967, 2875, 2854, 1714, 1694, 1653, 1448, 1285, 1178,
(1R,4R,6R)-6-(3-Iodoprop-1-en-2-yl)-4-(methoxymethoxy)-3-
methylcyclohex-2-en-1-yl formate (21c)
1
1119, 1093, 1045, 974, 918, 885; H NMR (300 MHz, CDCl3) δ
6.95 (dt, J = 15.6, 6.3 Hz, 1H), 5.80–5.65 (m, 2H), 5.44–5.32 (m, To a solution of allyl chloride 20c (450 mg, 1.64 mmol) in
1H), 4.85 (s, 1H), 4.74 (s, 1H), 4.14 (brs, 1H), 2.31 (d, J = acetone (16 mL) was added anhydrous NaI (2.46 g, 16.4 mmol)
13.1 Hz, 1H), 2.25–2.03 (m, 3H), 1.95–1.84 (m, 1H), 1.83 (s, 3H), at room temperature. The resulting mixture was stirred over-
1.75 (s, 3H), 1.04 (t, J = 7.4 Hz, 3H); 13C NMR (75 MHz, CDCl3) night before it was concentrated. The mixture was suspended
δ 166.3, 150.7, 144.6, 143.5, 121.9, 120.4, 111.6, 70.8, 66.8, in ether (20 mL) and filtered through a short pad of silica gel.
43.3, 32.2, 25.3, 22.0, 18.8, 12.1; HRMS (ESI): calculated for After concentration in vacuo, the crude allyl iodide 21c was
C15H22O3 [M + Li+] 257.1729, found 257.1724.
used without further purification.
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(4aR,6R,8aR)-6-(Methoxymethoxy)-7-methyl-4-methylene-
3,4,4a,5,6,8a-hexahydro-2H-chromen-2-one (23c)
(E)-2-(((4-Methoxybenzyl)oxy)methyl)-6,10-dimethylundeca-
1,5,10-trien-4-one (33)
To an ice-cold solution of SmI2 (93 mL, 0.1 M in THF, To a well-stirred suspension of magnesium turnings (1.38 g,
9.28 mmol) was added a solution of allyl iodide 21c (485 mg, 57.0 mmol) in anhydrous THF (12 mL) was added 1,2-dibro-
1.32 mmol) in dry THF (5 mL) via cannula under nitrogen. moethane (0.17 mL, 1.97 mmol) under nitrogen. The reaction
The deep blue mixture was stirred at 0 °C for 10 min before it mixture was stirred at room temperature for 30 min and a solu-
was quenched by a mixture of sat. aq. NaHCO3 (30 mL) and a tion of the corresponding allyl chloride (1.77 g, 7.86 mmol) in
sodium potassium phosphate buffer solution (pH = 7, 30 mL). anhydrous THF (4 mL) was introduced dropwise in 30 min.
After 15 min, the reaction mixture was extracted with EtOAc. After an additional 1.5 h, the freshly prepared Grignard
The combined organic layers were washed with water, satu- reagent was transferred to Weinreb amide 31 (565 mg,
rated aqueous NaHCO3, brine, and dried over Na2SO4. After 2.68 mmol) in THF (10 mL) at −78 °C via cannula. The reac-
concentration in vacuo, the residue was purified by flash tion mixture was stirred at −78 °C for 30 min and quenched
column chromatography (silica gel, eluted with ethyl acetate– with aq. NH4Cl. After warming to room temperature, the
petroleum ether = 1/2) to provide lactol 22c (1 : 1 mixture of aqueous layer was extracted with ethyl acetate. The combined
epimers, 258 mg, 76%) as a colorless oil.
organic layers were washed with H2O, brine and dried over
To a solution of lactol 22c (113 mg, 0.47 mmol) in DMSO Na2SO4. After concentration in vacuo, the residue was purified
(4 mL) was added 2-iodoxybenzoic acid (266 mg, 0.95 mmol) at by flash column chromatography (silica gel, ethyl acetate–
room temperature. The resulting mixture was stirred at 45 °C. petroleum ether, 1/10) to give 33 (910 mg, 98% yield) as a col-
The reaction was quenched with H2O (3 mL) when it was com- orless oil. IR (film, cm−1) 2937, 2854, 1679, 1611, 1513, 1244,
1
plete. The mixture was filtered and the aqueous phase was 1173, 1099, 1028, 883, 814; H NMR (300 MHz, CDCl3) δ 7.28
extracted with EtOAc. The combined organic layers were (d, J = 8.1 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 6.14 (s, 1H), 5.27 (s,
washed with brine and dried over Na2SO4. After concentration 1H), 5.07 (s, 1H), 4.76 (s, 1H), 4.71 (s, 1H), 4.44 (s, 2H), 4.01 (s,
in vacuo, the residue was purified by column chromatography 2H), 3.83 (s, 3H), 3.24 (s, 2H), 2.16–2.11 (m, 5H), 2.03 (t, J = 7.6
(silica gel, eluted with ethyl acetate–petroleum ether = 1/2) to Hz, 2H), 1.74 (s, 3H), 1.68–1.58 (m, 2H); 13C NMR (75 MHz,
provide 23c (84 mg, 75%) as a light yellow oil. [α]2D1 −27.12 (c CDCl3) 198.2, 159.6, 159.1, 145.2, 140.4, 130.3, 129.3, 122.7,
2.12, CHCl3); IR (film, cm−1) 2949, 2925, 2889, 1741, 1448, 115.9, 113.7, 110.4, 72.7, 71.8, 55.3, 48.8, 40.7, 37.2, 25.3, 22.3,
1
1241, 1152, 1102, 1028, 968, 906; H NMR (500 MHz, CDCl3) δ 18.4; HRMS (ESI): calculated for C22H30O3 [M + H+] 343.2273,
5.69–5.66 (m, 1H), 5.08–5.06 (m, 2H), 4.79 (d, J = 7.0 Hz, 1H), found 343.2278.
4.69–4.67 (m, 1H), 4.67 (d, J = 7.0 Hz, 1H), 4.12–4.09 (m, 1H),
3.41 (s, 3H), 3.31 (d, J = 17.6 Hz, 1H), 3.25 (d, J = 17.6 Hz, 1H),
2.72 (d, J = 13.2 Hz, 1H), 2.23–2.09 (m, 1H), 1.85 (s, 3H),
(E)-2-(((4-Methoxybenzyl)oxy)methyl)-6,10-dimethylundeca-
1,5,10-trien-4-ol (12)
1.80–1.73 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 170.2, 144.5,
139.8, 120.2, 113.1, 95.8, 74.6, 73.0, 55.7, 37.9, 36.9, 31.2, 19.4;
A solution of (R)-(+)-2-methyl-CBS-oxazaborolidine (60 μL,
0.06 mmol) in THF (0.3 mL) was treated with BH3·DMS (30 μL,
2 M in THF, 0.06 mmol) at room temperature. After stirring for
15 min, the mixture was cooled to −78 °C and a solution of
enone 33 (17 mg, 0.05 mmol) in THF (0.3 mL) was added
through cannula. The reaction mixture was stirred at −78 °C
HRMS (ESI): calculated for C13H18O4 [M + H+] 239.1284, found
239.1288.
(E)-N-Methoxy-N,3,7-trimethylocta-2,7-dienamide (31)
To a solution of E-alkenoic ester 30 (904 mg, 4.61 mmol) for 3 h before it was slowly warmed to room temperature. The
and N,O-dimethylhydroxyamine hydrochloride (899 mg, reaction was quenched by the addition of MeOH followed by
9.22 mmol) in THF (12 mL) was added i-PrMgCl (10.4 mL, 2 M H2O. The aqueous phase was extracted with ethyl acetate. The
in THF, 20.75 mmol) dropwise at −5 °C. The mixture was combined organic layers were dried over Na2SO4. After concen-
stirred for 30 min and quenched with sat. aq. NH4Cl (30 mL). tration in vacuo, the residue was purified by flash column
The mixture was extracted with ethyl acetate. The combined chromatography (silica gel, ethyl acetate–petroleum ether, 1/2)
organic layers were washed with brine and dried over Na2SO4. to give 12 (14.6 mg, 85% yield) as a colorless oil. IR (film,
After concentration in vacuo, the residue was purified by flash cm−1) 2935, 2854, 1649, 1617, 1513, 1442, 1247, 1170, 1037,
1
column chromatography (silica gel, ethyl acetate–petroleum 891, 820; H NMR (500 MHz, CDCl3) δ 7.27 (d, J = 8.6 Hz, 2H),
ether = 1/5) to give Weinreb amide 31 (896 mg, 92% yield) as a 6.88 (d, J = 8.7 Hz, 2H), 5.20 (d, J = 8.4 Hz, 1H), 5.16 (s, 1H),
colorless oil. IR (film, cm−1) 2970, 2940, 1658, 1635, 1436, 5.05 (s, 1H), 4.70 (s, 1H), 4.67 (s, 1H), 4.52–4.48 (m, 1H), 4.47
1
1362, 1173, 1099, 980, 883; H NMR (300 MHz, CDCl3) δ 6.10 (d, J = 11.7 Hz, 1H), 4.45 (d, J = 11.5 Hz, 1H), 3.99 (d, J = 11.3
(s, 1H), 4.71 (s, 1H), 4.67 (s, 1H), 3.65 (s, 3H), 3.20 (s, 3H), Hz, 1H), 3.95 (d, J = 11.9 Hz, 1H), 3.80 (s, 3H), 2.35–2.26 (m,
2.15–2.10 (m, 5H), 2.01 (t, J = 7.5, 2H), 1.70 (s, 3H), 1.66–1.56 2H), 1.98 (t, J = 7.7 Hz, 4H), 1.71 (s, 3H), 1.66 (d, J = 1.3 Hz,
(m, 2H); 13C NMR (75 MHz, CDCl3) 145.3, 114.0, 110.2, 61.3, 3H), 1.58–1.51 (m, 2H); 13C NMR (125 MHz, CDCl3) 159.2,
40.6, 37.2, 25.4, 22.3, 18.6; HRMS (ESI): calculated for 145.8, 142.8, 138.2, 129.9, 129.5, 127.3, 116.3, 113.8, 109.9,
C12H21NO2 [M + H+] 212.1651, found 212.1653.
73.2, 72.0, 67.2, 55.3, 42.7, 39.0, 37.3, 25.6, 22.4, 16.6; HRMS
3220 | Org. Biomol. Chem., 2013, 11, 3212–3222
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(ESI): calculated for C22H32O3 [M + Li+] 351.2511, found with Na2SO4 and concentrated in vacuo. Purification of the
351.2503.
residue by flash column chromatography (silica gel, ethyl
acetate–petroleum ether, 1/20) gave 38 (1.51 g, 85%) as a clear
oil.
(S,E)-4-((tert-Butyldimethylsilyl)oxy)-6,10-dimethylundeca-5,10-
dien-2-one (39)
This was formed as the major product during synthesis of 38
when a less stringently prepared TMSCH2Li-CeCl3 reagent was
used.45 It was isolated by flash column chromatography (silica
Acknowledgements
gel, ethyl acetate–petroleum ether = 1/20) as a light yellow oil. We thank Prof. Raymond Andersen (University of British
[α]2D1 −24.28 (c 2.72, CHCl3); IR (film) 2955, 2931, 2860, 1362, Columbia) for an authentic sample of alotaketal A. Profs.
1
1256, 1075, 838; H NMR (300 MHz, CDCl3) δ 5.13 (dd, J = 8.8, Weiping Tang (University of Wisconsin), Tarek Sammakia (Uni-
1.2 Hz, 1H), 4.87–4.80 (m, 1H), 4.70 (s, 1H), 4.66 (s, 1H), 2.69 versity of Colorado Boulder), and Daniel Romo (Texas A&M
(dd, J = 14.3, 8.4 Hz, 1H), 2.34 (dd, J = 14.3, 4.6 Hz, 1H), 2.15 University) are acknowledged for helpful discussions. Finan-
(s, 3H), 1.96 (dd, J = 15.1, 7.4 Hz, 4H), 1.70 (s, 3H), 1.64 (d, J = cial support was provided by the Welch Foundation (A-1700)
1.3 Hz, 3H), 1.57–1.47 (m, 2H), 0.84 (s, 9H), 0.01 (s, 3H), 0.00 and Texas A&M University to J.Y. and by NIH (R01 DK073368)
(s, 3H); 13C NMR (75 MHz, CDCl3) δ 207.7, 145.7, 135.7, 127.9, to J.Z.
109.9, 67.0, 51.8, 38.9, 37.3, 31.9, 25.8, 25.5, 22.4, 18.0, 16.5,
−4.3, −5.0; HRMS (ESI): calculated for C19H36O2Si [M + Li+]
331.2645, found 331.2637.
Notes and references
(S,E)-tert-Butyl((6,10-dimethyl-2-((trimethylsilyl)methyl)-
undeca-1,5,10-trien-4-yl)oxy)dimethylsilane (38)
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A solution of methyl ketone 39 (1.50 g, 4.62 mmol) in THF
(10 mL) was cooled to −78 °C and treated with a solution of
potassium hexamethyldisilizide (5.55 mL, 1.0 M in THF,
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5.14–5.11 (m, 2H), 4.97 (d, J = 3.2 Hz, 1H), 4.71 (s, 1H), 4.67 (s,
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