Design, Synthesis, and Characterization of Novel sn-1 Heterocyclic DAG-Lactones as PKC Activators

Article abstract of DOI:10.1021/acs.jmedchem.1c00739

DAG-lactones represent useful templates for the design of potent and selective C1 domain ligands for PKC isozymes. The ester moiety at the sn-1 position, a common feature in this template, is relevant for C1 domain interactions, but it represents a labile

Full text of DOI:10.1021/acs.jmedchem.1c00739

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Design, Synthesis, and Characterization of Novel sn-1 Heterocyclic
DAG-Lactones as PKC Activators
Eleonora Elhalem, Ana Bellomo, Mariana Cooke, Antonella Scravaglieri, Larry V. Pearce,
Megan L. Peach, Lucía Gandolfi Donadío, Marcelo G. Kazanietz,* and María J. Comin*
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ABSTRACT: DAG-lactones represent useful templates for the
design of potent and selective C1 domain ligands for PKC
isozymes. The ester moiety at the sn-1 position, a common feature
in this template, is relevant for C1 domain interactions, but it
represents a labile group susceptible to endogenous esterases. An
interesting challenge involves replacing the ester group of these
ligands while still maintaining biological activity. Here, we present
the synthesis and functional characterization of novel diacylglycer-
ol-lactones containing heterocyclic ring substituents at the sn-1
position. Our results showed that the new compound 10B12, a
DAG-lactone with an isoxazole ring, binds PKCα and PKCε with nanomolar affinity. Remarkably, 10B12 displays preferential
selectivity for PKCε translocation in cells and induces a PKCε-dependent reorganization of the actin cytoskeleton into peripheral
ruffles in lung cancer cells. We conclude that introducing a stable isoxazole ring as an ester surrogate in DAG-lactones emerges as a
novel structural approach to achieve PKC isozyme selectivity.
RasGRPs, which are guanyl exchange factors for Ras;
chimaerins, which are GTPase activating proteins for Rac;
DAG kinases (DGKs), enzymes responsible for DAG
phosphorylation and the generation of phosphatidic acid;
MRCK (myotonic dystrophy kinase-related cdc42-binding
kinase), protein kinase D isoforms (PKDs), and Munc-13
isoforms.8,9 Despite the similar overall structure of C1 domains
in these proteins, subtle differences may confer differential
ligand binding and membrane association properties to
individual members of the family, thus encouraging the
generation of agents capable of displaying binding selectivity
among the different phorbol ester receptors and specificity for
activation. This would also be useful to dissect PKC isozyme-
specific responses in cellular models.
Among the multiple C1 domain ligands generated as agents
capable of activating PKC isozymes, diacylglycerol lactones
(DAG-lactones) represent a unique class of DAG mimetics in
which the flexibility of the structure has been constrained to
reduce the entropic loss due to binding. Indeed, the natural
second messenger DAG displays a rather weak affinity for
cPKCs and nPKCs, largely reflecting the flexibility of the
INTRODUCTION
■
Protein kinase C (PKC) isozymes comprise a family of signaling
proteins that play essential roles in the control of cellular
functions as diverse as cell cycle progression, differentiation,
apoptosis, proliferation, motility, and gene expression.1 The
central role of PKC in cellular signal transduction has established
it as a validated therapeutic target for various pathologies such as
cancer, diabetes, Alzheimer’s, and cardiovascular diseases.2−5
All members of the PKC family contain in their N-terminal
regulatory domain one or two copies of C1 domains, a zinc-
binding, cysteine-rich motif of 50−51 amino acid residues. In
classical/conventional (cPKCs: α, β1, β2, and γ) and novel
isoforms (nPKCs: δ, ε, η, and θ), the second messenger
diacylglycerol (DAG) binds to the C1a and C1b domain present
in their regulatory region, which promotes their translocation to
the plasma membrane or other intracellular compartments and
subsequent enzyme activation. The C1 domains in cPKCs and
nPKCs are also high-affinity binding sites for the phorbol ester
tumor promoters, natural compounds that bind to the same site
as DAG. Unlike the C1 domains in cPKCs and nPKCs, a single
C1 domain present in atypical isoforms (aPKCs: ζ and ι/λ) is
unable to bind either phorbol esters or DAG.6 Individual
members of the PKC family have been shown to confer
distinctive patterns of cellular responses, including opposite
responses in the context of tumor growth.1Although PKCs are
the best-studied class of signaling proteins with C1 domains,7
other families of proteins containing DAG/phorbol ester-
responsive C1 domains have been later identified. These include
Received: April 23, 2021
https://doi.org/10.1021/acs.jmedchem.1c00739
© XXXX American Chemical Society
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glycerol backbone, with the consequence that the immobiliza-
tion of the glycerol upon binding is entropically unfavorable.
The design of DAG-lactones, pioneered by the Marquez and
Blumberg laboratories, constitutes the greatest synthetic efforts
to date, leading to the development of potent C1 domain ligands
in which the glycerol backbone of DAG has been cyclized,
leading to a rigid structure that eliminates the entropic penalty.
Through appropriate manipulation of the hydrophobic side
chains, it has been possible to obtain DAG-lactone analogues
displaying higher affinity than the natural DAGs and, in many
cases, achieving nanomolar binding affinities similar to those of
phorbol esters or related compounds derived from natural
sources.10−12
libraries of DAG-lactones were produced, leading to compounds
with marked selectivity for the RasGRP family relative to their
affinity for PKCα and other PKC isozymes.11,12,15 A study by
Ann et al.16 designed to generate isozyme-specific PKC ligands
by the incorporation of linoleic acid derivatives as well as
saturated and unsaturated alkyl chains into the side chains of
DAG-lactones led to the identification of DAG-lactones with
prominent isozyme selectivity. One of these compounds, the
DAG-lactone (E)-(2-(hydroxymethyl)-4-(3-isobutyl-5-methyl-
hexylidene)-5-oxotetrahydrofuran-2-yl)methyl pivalate (re-
ferred to as AJH-836), exhibits pronounced selectivity in vitro
for novel PKCε and PKCδ relative to classical PKCα and
PKCβ.17 Remarkably, AJH-836 displayed significant selectivity
for the translocation of PKCε to the plasma membrane (a
readout of activation) relative to PKCα. This selectivity could
also be observed at a functional level in cellular models. For
example, AJH-836 is capable of inducing major changes in the
reorganization of the actin cytoskeleton, specifically inducing
the formation of peripheral membrane ruffles, a response that is
mediated primarily by PKCε.17 On the other hand, AJH-836 has
limited ability to induce transcriptional activation of genes in
lung cancer cells, a response that is mainly mediated by PKCα,18
ultimately reflecting its selectivity for the nPKCs relative to
cPKCs.
Modeling and docking experiments have revealed two
different modes of binding for diacylglycerol itself and the
DAG-lactones, defined by the interaction between the C1
domain and either the sn-1 carbonyl or the sn-2 carbonyl in the
DAG-lactone structure (Figure 1).13 These two comparable
A fundamental feature common to DAG-lactones is the
presence of an ester moiety at the sn-1 position that, as discussed
above, is important in mediating membrane and receptor
interactions; however, this makes the DAG-lactones susceptible
to hydrolysis by endogenous esterases, reducing their stability in
cells.19 Previous attempts to replace this labile functional group
led to a significant loss in biological activity. Interestingly, the
isosteric replacement of the side-chain ester group at the sn-1
position with an amide or an N-hydroxyl amide group,
developed by Marquez and colleagues, revealed a dramatic
drop in binding affinity of more than 3 orders of magnitude,
suggesting that the new functional groups were not able to
adequately recapitulate the polar interactions that the sn-1
carbonyl makes in the C1 domain−membrane−DAG-lactone
ternary complex.19,20 In the present study, we designed and
synthesized heterocycle-containing DAG-lactones in order to
expand the chemical space covered by these ligands, leading to
the development of novel activators of DAG-responsive C1
domain-containing proteins. Using AJH-836 as a lead DAG-
lactone structure, we synthesized six new compounds in which
we varied the nature of the heterocyclic ring at the sn-1 position,
specifically replacing the ester moiety with isoxazole (1−2),
triazole (3−4), and tetrazole (5−6) groups (Figure 2).
Figure 1. Schematic illustration of the C1 domain hydrogen bonding
interactions in the alternative sn-1 (A) and sn-2 (B) binding modes for
DAG-lactones.
binding modes form identical networks of hydrogen bonds with
receptor amino acids Thr 12, Leu21, and Gly23, as is observed in
the crystal structure of PKCδ C1b with phorbol 13-O-acetate.14
For convenience in comparing C1 domains from different
isozymes, we use residue numbering internal to the C1 domain,
with residue 1 corresponding to the first zinc-binding histidine
and residue 50 corresponding to the last zinc-binding cysteine.
The carbonyl that is not directly involved in C1 domain
binding contributes to the binding energy through interactions
with the C1 domain surface and the lipid bilayer in the C1
domain−ligand−membrane ternary complex. Thus, both
carbonyl groups have been shown to be essential for a strong
interaction with PKC. The distinct patterns of acyl (R1) and
alkyl (R2) substitution are key elements that determine
selectivity, providing significant differences in biological out-
come patterns. When this approach was applied, combinatorial
Figure 2. Structure of new heterocyclic DAG-lactones 1−6 and AJH-836.
B
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Evaluation of their binding and functional properties in cellular
models revealed that a stable isoxazole ring can function as an
ester surrogate to allow binding to the C1 domain.
RESULTS AND DISCUSSION
■
Design of the Compounds. Heterocycle rings play a
central role in modern drug design. According to statistics, more
than 85% of all biologically active chemical entities contain a
heterocycle. The presence of heterocycles provides a useful tool
for the modification of solubility, lipophilicity, polarity, and
hydrogen bonding capacity of biologically active agents, which
results in the optimization of the ADME/Tox properties of
drugs or drug candidates.21 With the aim of increasing metabolic
stability, we selected three popular heterocyclic ring moieties
present in numerous biologically active compounds for the
design of the new DAG-lactones derivatives. In this sense,
isoxazole, triazole, and tetrazole heterocycles have provided
improved pharmacological properties that have resulted in the
development of wide synthetic strategies and methods for the
synthesis of these valuable building blocks.22
Isoxazole is an azole with an oxygen atom attached to nitrogen
often found in natural products and in various pharmaceutical
and therapeutic products such as insecticides, antibacterials,
antibiotics, antifungals, among others. It is interesting to obtain
the isoxazolol derivative since this group represents a bioisostere
of the ester group present in our lead structure at the sn-1
position.23,24
Figure 3. Docked structures of compound 2 in sn-1 (A) and sn-2
orientations (B) and compounds 4 (C) and 6 (D) in sn-1 orientation.
Hydrogen bonds are indicated with dashed orange lines. The receptor
crystal structure is the PKCδ C1b domain14 (PDB ID: 1PTR).
The 1,2,3-triazole unit is a fundamental building block found
in different bioactive compounds. One of the most interesting
features of triazoles is their ability to mimic the characteristics of
different functional groups. This aromatic heterocycle is stable
to metabolic and chemical degradation and is capable of acting
as a hydrogen bond donor and acceptor, which can be
convenient in the binding of biomolecular targets. This explains
its wide use as a bioisostere for the synthesis of new active
molecules, mainly amide bonds, although there are also some
examples where it is used as an ester group isostere to reduce the
susceptibility to enzymatic degradation in vivo.25
Tetrazole is a five-member, planar heterocycle with four
nitrogen atoms arranged in a regular fashion rarely present in
nature. It has attracted significant attention, especially in
medicinal chemistry, because it increases drug bioavailability
and is resistant to biological degradation, extending their
action.26,27
Molecular Modeling. After planning the synthesis, a series
of heterocycle-containing DAG-lactones was docked into the
crystal structure of the PKCδ C1b domain bound to phorbol
ester.14 This domain has a 62% sequence identity with the PKCα
and PKCε C1b domains, and the ligand-binding residues are
strictly conserved. These docking experiments allowed us to
ensure that the designed heterocyclic compounds fit into the
binding site, to compare their binding mode to other known and
previously studied DAG-lactones, and to analyze the inter-
actions formed by the new heterocyclic rings and the C1
domain.
Docking predicted that the isoxazole-containing compounds,
which retain an ether oxygen in the linkage between the
heterocycle and the lactone ring, are capable of binding in both
sn-1 and sn-2 orientations, like the parent DAG-lactone
structures (Figure 3A,B). The triazole and tetrazole DAG-
lactones, with a single methylene spacer between the two rings,
bind exclusively sn-1 (Figure 3C,D). These compounds cannot
bind sn-2 because the shorter linker does not give enough space
to the heterocyclic ring, and instead of extending out of the back
of the binding slot, it bumps against a side loop without making
any productive interactions.
In the sn-1 orientation, the hydrogen bonding interaction
between the C1 domain backbone at Gly23 and the DAG-
lactone is stronger in the tri- and tetrazole compounds because,
with the shorter linker, the ring can adopt an orientation such
that the hydrogen bond is in the plane of the ring. With the
additional ether oxygen in the linker, in the isoxazole
compounds, the NH donor is not aligned with the ring plane,
and the hydrogen bonding is a weaker, out-of-plane interaction
with an isoxazole oxygen atom.28
Previous studies have shown that DAG-lactones prefer to bind
in the sn-2 orientation, particularly when there is a large,
branched alkyl chain at R2, adjacent to the sn-2 carbonyl (Figure
1) as in compounds 2, 4, and 6. In this binding mode, the
entropic advantage of constraining the DAG backbone into a
lactone ring is realized, with a concomitant increase in binding
affinity relative to an equivalent open-chain DAG.29 This
suggested that the isoxazole compounds would exhibit stronger
PKC C1 domain binding overall than the other compounds,
even though their sn-1 binding is predicted to be weaker.
Chemistry. We started the synthesis of new analogues with
orthogonally protected lactone 7 bearing a benzyl and a silyl
ether group.30 In order to explore the chemical compatibility
between reaction conditions required to build the heterocycles
and for hydroxyl group deprotection, we decided to synthesize
simple derivatives bearing a propan-2-ylidene group at the sn-2
position. This alkyl substituent was introduced through a well-
established procedure using an alkylation−elimination sequence
to give lactone 8 according to published methods (Scheme
1).13,31
Considering that deprotection conditions used for silyl ethers
cleavage were, in principle, compatible with the presence of the
isoxazole ring, we decided first to selectively remove the benzyl
C
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a
Scheme 1. Synthesis of Isoxazole (1) and Triazole (3−4) Derivatives Employing Orthogonally Protected Lactones
a
Reagents and conditions: (a) [1] R1R2CO, LiHMDS, THF, −78 °C, 2−4 h; [2] (i) CH3SO2Cl, Et3N, 0 °C, 5 h; (ii) DBU, CH2Cl2, room temp,
24 h, 62% for 8, 35% for 14 (E/Z regioisomers 69%); (b) BCl3, CH2Cl2, −78 °C, 2 h, 97% for 9, 89% for 15; (c) 5-methy-3-isoxazolol, PPh3,
DIAD, THF, 24 h, 52%; (d) TBAF, THF, 0 °C, 1 h, 80% for 1, 80% for 3, 60% for 4; (e) p-TsCl, pyr, CH2Cl2, 24 h, (40 °C) 88% for 11, (90 °C)
90% for 16 (f) NaN3, DMF, 100 °C, 48 h, 96% for 12, 72% for 17; (g) 4-ethynyltoluene, sodium ascorbate 1 M, CuSO4·5H2O 0.3 M, t-BuOH/
H2O, 48 h, 75% for 13, 77% for 18.
ether group, with BCl3 at low temperatures, to obtain the known
lactone 9, already described in our previous work, in a good
yield.31
75% yield, although a long reaction time (48 h) was required to
achieve full conversion (Scheme 1). Deprotection of the TBDPS
group was accomplished under standard conditions (TBAF) to
obtain triazole 3 in 80% yields.
The introduction of variations in the patterns of the
substituents attached to the sn-1 and sn-2 positions on the
DAG-lactone has resulted, over the years, in the development of
very powerful agonists in the low nM range. One of the most
The heterocyclic ring 5-methy-3-isoxazolol, a commercial
product known as himexazole that is employed as a pesticide,
was previously synthesized according to methods described in
the literature.32 A convergent strategy based on the Mitsunobu
reaction was used to couple this heterocyclic ring to the free
hydroxyl group of lactone 9, using DIAD and PPh3, to produce
compound 10 in a moderate yield (52%). Selective desilylation
of the tert-butyldiphenylsilyl ether (TBDPS) group under
standard conditions (TBAF) was carried out in an excellent
yield, obtaining the first heterocyclic-containing DAG-lactone 1
(Scheme 1).
In the past decade, the use of triazoles in medicinal chemistry
has received marked attention, leading to the development of
regioselective methodologies based on Huisgen 1,3-dipolar
cycloaddition. We envisioned the synthesis of triazole analogues
in the archetypical “click” reaction: the Huisgen [3+2]
cycloaddition between alkynes and azides catalyzed by copper-
(I) salts (Cu-AAC) that conduce to the regioselective formation
of 1,4-regioisomer.33 With that idea in mind, the hydroxyl group
in compound 9 was transformed to the corresponding tosylate
with tosyl chloride in pyridine, which in turn was treated with
sodium azide in DMF at elevated temperatures, yielding azide
12 in 85% yield, for both steps. Preparation of triazole 13 was
carried out using the click reaction as a key step, between azide
12 and a commercial alkyne, 4-ethynyl toluene, in the presence
of CuSO4 and sodium ascorbate, a well-known catalytic
system.34,35 The reaction proceeded regioselectively at room
temperature to give 13, as the 1,4-regioisomer exclusively, in
́
potent analogues prepared to date, obtained by Marquez’s
group, was the DAG-lactone AJH-836. Encouraged by this, we
decided to synthesize heterocyclic DAG-lactones maintaining
the same alkyl substituent present in AJH-836 at the sn-2
position. In this sense, aldehyde 5-methyl-3-(2-methylpropyl)-
hexan-1-one was prepared according to published methods13,19
and immediately employed in an aldol condensation with
lactone 7, followed by olefination through the alkylation−
elimination sequence already mentioned, yielding 69% of the
mixture of E/Z isomers (Scheme 1). Separation of E/Z
geometric isomers was achieved at this stage by column
chromatography. The vinyl proton corresponding to the E-
isomer (14) appears in its 1H NMR spectrum as a characteristic
multiplet that is further downfield compared to that of the
corresponding Z-isomer. Following the same synthetic sequence
described for the preparation of triazole 3, we obtained DAG-
lactone 4 with similar yields for almost every step, except for
TBDPS remotion with TBAF that renders a lower yield.
Attempts to obtain the branched isoxazole 2, starting with
compound 15 and following the strategy based on Mitsunobu
coupling employed to synthesize DAG-lactone 1, were
unsuccessful. Consequently, we decided to change the starting
material and prepared the known lactone 1936 protected with
D
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a
Scheme 2. Different Strategies to the Synthesis of Isoxazole 2
a
Reagents and conditions: (a) [1] 3-isobutyl-5-methylhexanal, LiHMDS, THF, −78 °C, 2−4 h; [2] (i) CH3SO2Cl, Et3N, 0 °C, 5 h; (ii) DBU,
CH2Cl2, room temp, 24 h, 35% for 20 (69% for E/Z mixture); (b) CAN, H2O/CH3CN, 75%; (c) 5-methy-3-isoxazolol, PPh3, DIAD, THF, 24 h,
37% ; (d) BCl3, CH2Cl2, −78 °C, 0.5 h, 67%; (e) I2, PPh3, imidazole, toluene, 90 °C, 1 h, 89%; (f) K2CO3, DMF, sealed tube, 140 °C, 4 h, 62%.
a
Scheme 3. Synthesis of Tetrazole Analogues DAG-Lactone 5 and DAG-Lactone 6
a
Reagents and conditions: (a) [1] acetone, LiHMDS, THF, −78 °C, 2−4 h; [2](i) CH3SO2Cl, Et3N, 0 °C, 5 h; (ii) DBU, CH2Cl2, room temp, 24
h, 90% for 24; (b) CAN, H2O/CH3CN, 92%; (c) I2, PPh3, imidazole, toluene, 90 °C, 1 h, 95%; (d) 5-phenyl-1H-tetrazole, K2CO3, DMF, sealed
tube, 120 °C, 20 h, 74%; (e) BCl3, CH2Cl2, −78 °C, 0.5 h, 90% for 5, 78% for 6; (f) 5-phenyl-1H-tetrazole, PPh3, DIAD, THF, 24 h, 53%.
benzyl and p-methoxyphenyl (PMP) groups. The sn-2 alkyl
substituent was introduced through condensation with the
branched aldehyde mentioned before,37 and after elimination of
the p-methoxyphenyl (PMP)-protecting group, Mitsunobu
coupling with isoxazolol was performed to yield compound 23
in 37% yield. Finally, deprotection of the benzyl group with BCl3
afforded DAG-lactone 2 in a good yield (Scheme 2).
In view of the low yields obtained, it was decided to test an
alternative synthetic route. The conversion of the hydroxyl
group in compound 21 to an iodide group was carried out with
iodine in the presence of PPh3 and imidazole in excellent yield.38
To favor the substitution reaction, compound 22 was treated
with isoxazolol and a base (K2CO3) at an elevated temperature,
in a sealed tube, obtaining the same intermediate 23 in higher
yield (55% in two steps) than the Mitsunobu approach.39
Both synthetic strategies used in the preparation of isoxazole
derivatives could, in principle, be applied in obtaining tetrazole
analogues. We decided to prepare derivatives from 5-phenyl-1H-
tetrazole, a commercially available compound that is easily
prepared according to literature procedures by a cycloaddition
reaction between benzonitrile and sodium azide catalyzed by
copper.40 Considering that the Mitsunobu route was straightfor-
ward, we tried the coupling between tetrazole 28 and the known
lactone 2541 (Scheme 3). Unfortunately, conversion was low
even when adding a large excess of PPh3 and DIAD. The
reaction rendered a complex mixture, and several attempts to
purify the desired compound were unsuccessful. Compound 27
was always obtained with variable amounts of byproducts.
Deprotection under classical conditions for this type of lactones
(BCl3) allowed us to obtain 5 in 17% total yield from compound
25. Hence, the alternative strategy based on the nucleophilic
substitution of a good leaving group, such as iodine, by the
phenyltetrazole anion generated in situ in the presence of base
was attempted. Indeed, compound 27 was obtained in 70% yield
from 25 (two steps). Based on two-dimensional NMR
experiments, it was concluded that the 2,5-disubstituted
regioisomer was obtained as the only product. Finally, benzyl
deprotection led to derivative 5. Unlike the previous case, when
we tested the Mitsunobu reaction employing the branched
lactone 21 and compound 28, we could introduce the tetrazole
ring into compound 29, although in a moderate yield. The
complete conversion was observed, and the byproducts were
removed efficiently by column chromatography. Benzyl
deprotection under standard conditions gave the tetrazolic
DAG-lactone 6 (Scheme 3).
E
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Biological Results. In Vitro Binding of Novel Heterocyclic
DAG-Lactones to PKC Isozymes. In order to determine the
affinity of the newly synthesized DAG-lactones, we used an
established in vitro competition binding assay, with [3H]phorbol
12,13-dibutyrate ([3H]PDBu) as a radioligand. The assay was
carried out in the presence of 100 μg/mL phosphatidylserine, as
previously described (for more details, see the General
Procedures section).42 We first examined the affinity of the
compounds for PKCα. Results revealed that DAG-lactone 2,
from now on 10B12, could compete [3H]PDBu, displaying a Ki
= 79.3 nM. The other DAG-lactones only marginally displaced
the radioligand at a concentration of 30 μM. For comparison,
the DAG-lactone AJH-836 bound to PKCα, with a Ki = 23.6
nM.17 We next determined the ability of 10B12 to bind to
PKCε. Competition assays revealed that the Ki for PKCε was
2.4-fold lower than that for PKCα (33.6 nM), suggesting a
marginal, yet significant preference for the nPKC, at least in vitro.
For comparison, AJH-836 displayed a Ki = 1.89 nM for PKCε,
therefore showing 12.4-fold lower Ki than that for PKCα.17 Two
of the DAG-lactones with limited binding for PKCα (4 and 6)
were also examined for their ability to bind PKCε, and they were
found to display limited binding for this nPKC using a fixed
concentration of 30 μM (Table 1).
lactones to translocate PKC isozymes to membranes, a well-
established hallmark of enzyme activation in cells. As an
experimental strategy, we expressed Green Fluorescence Protein
(GFP)-fused PKCα or PKCε in HeLa cervical adenocarcinoma
cells and examined the ability of DAG lactones to induce kinase
relocalization to the plasma membrane using fluorescence
microscopy. Coupling this imaging analysis to the determination
of membrane to cytosol fluorescent ratio allows accurate
quantification of enzyme translocation and the precise
determination of potencies in cellular models. Using this
approach, we determined that AJH-836 displays 43-fold higher
potency for translocation of PKCε relative to PKCα.17 As shown
in the images from Figure 4A and subsequent quantification of
Table 1. DAG-Lactones Binding Affinities for PKCα and
a
PKCε In Vitro
KI (nM) (or % inhibition, if partial)
compound
ClogP
PKCα
PKCε
AJH-836
1
2 (10B12)
3
4
5
6
5.52
0.78
4.75
2.61
6.58
2.08
6.05
23.6 2.017
31.3% at 30 μM
79.3 20.7
11.7% at 30 μM
21.3% at 30 μM
23.5% at 30 μM
37.5% at 30 μM
1.89 0.2017
ND
33.6 6.8
ND
17% at 10 μM
ND
69.2% at 30 μM
a
Values represent the mean SEM of three independent experiments
and were calculated from the ID50 values determined from the
competition curves. ND: nondetermined. ClogP calculated in
ChemDraw.
Figure 4. Translocation of PKC isozymes by DAG-lactones. HeLa cells
expressing GFP (control), GFP-PKCα, or GFP-PKCε were stimulated
for 30 min with the indicated compounds, fixed, counterstained with
DAPI, and visualized by fluorescent microscopy. (A) Representative
micrographs are shown. (B) Representative graphs obtained using
Image G that depicts the quantification of membrane/cytosol ratio. (C)
Membrane/cytosol ratio (n = 10−15 cells/group). Results are
Previous studies with chemical series of DAG-lactones have
shown that there is a parabolic relationship between C1 domain
binding affinity and logP, with a peak at a logP of approximately
5. A logP close to this value gives compounds adequate
lipophilicity required for partitioning into membranes with the
lowest possible amount of nonspecific membrane affinity.10 Of
the heterocyclic DAG-lactones, compounds 10B12 and 6 are
closest in logP value to the model compound AJH-836 and
closest to this log P “sweet spot”, suggesting that these
compounds have the ideal balance between the membrane
and C1 domain binding ability.
Differential Translocation of PKCα and PKCε by Hetero-
cyclic DAG-Lactones in Cells. While the [3H]PDBu binding
assay using reconstituted 100% phosphatidylserine vesicles is a
well-accepted approach to determine the in vitro binding
affinities of C1 domain ligands to PKC isozymes, assessing
their effects in cellular models provides more reliable
information about their ability to activate these kinases in
their physiological environment. Indeed, small differences in Ki’s
for individual PKCs, as determined in vitro, may translate into
significant differences in isozyme specificity in cellular-based
assays. We, therefore, examined the ability of heterocyclic DAG
expressed as mean
SD *, p < 0.05. ****, p < 0.001 vs control.
Similar results were observed in two additional experiments.
the membrane/cytosol fluorescence ratio in Figure 4B, 10B12
(2) induced an evident redistribution of fluorescence in GFP-
PKCε expressing HeLa cells. On the other hand, compounds 1,
3, 4, and 5 had essentially no effect on GFP-PKCε
redistribution, and fluorescence remained cytoplasmic after
treatment. Despite its weak in vitro binding affinity, compound 6
also displays some PKCε translocation effect, although smaller
than that of AJH-836, possibly reflecting other mechanisms
contributing to its efficacy in cells such as preferential partition
into membranes or unique lipid associations. As controls, we
found that the phorbol ester PMA (100 nM), which displays
similar activity on cPKCs and nPKCs, fully translocated GFP-
PKCα and GFP-PKCε to the plasma membrane, whereas the
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PKCε-selective DAG lactone AJH-836 (1 μM) only trans-
located GFP-PKCε, as we established in a previous study.17
10B12 Induces Major Changes in Cytoskeletal Morphol-
ogy. In order to pursue a deeper biological characterization of
the heterocyclic DAG-lactones, we next examine a cellular
function known to be specifically mediated by PKCε.
Specifically, PKCε, but not PKCα, is the PKC implicated in
the formation of membrane ruffles, actin-rich protrusions
involved in cancer cell motility. Indeed, both PMA- and AJH-
836-induced ruffle formation in A549 lung cancer cells was
severely affected upon silencing PKCε by means of RNA
interference (RNAi) or pretreatment with a selective PKCε
pharmacological inhibitor.17 To address the effect of the newly
synthesized DAG-lactones on ruffle formation, we treated A549
cells with the different compounds (1 μM, 30 min) and then
subjected cells to polymerized actin staining using phalloidin.
Upon microscopy examination, we observed a prominent
stimulation of ruffle formation in response to 10B12 treatment.
No effects were observed for the other synthesized DAG-
lactones, other than a small response by DAG-lactone 6 (Figure
5A). Quantification of ruffle formation using a densitometric
Figure 6. PKCε mediates the ruffle formation response of compound
10B12. A549 cells were subjected to PKCε RNAi by delivery of specific
siRNA duplexes (PKCε1 and PKCε2). As a control, cells were
transfected with a nontarget control (NTC) siRNA duplex. Experi-
ments were carried out 48 h later. (A) PKCε mRNA levels relative to
NTC (dotted line). (B) PKCε protein levels. Upper panel,
representative Western blot. Lower panel, densitometric analysis of
PKCε protein levels, expressed as arbitrary units relative to β-actin. (C)
A549 cells were stimulated for 30 min with 10B12 (1 μM), fixed, and
stained with phalloidin-rhodamine. Upper panel. Representative
micrographs. (D) Quantification of ruffle area/cell using ImageJ.
****, p < 0.001 vs 10B12-treated cells, NTC.
depletion (Figure 6C,D), as was previously observed with AJH-
836 using a similar approach.17 Together with the membrane
translocation studies, these actin reorganization experiments
establish 10B12 as a bona fide PKCε-activating DAG-lactone.
CONCLUSION
■
Over the past decades, a large number of DAG-lactones
analogues with broad structural variation have been designed
and synthesized, generating diversity at the sn-1 and sn-2
positions and covering an extensive chemical space. Most of
these compounds have an ester moiety at the sn-1 position that is
important for receptor interaction, and only a few attempts have
been made to replace it. In this study, different types of
heterocyclic groups were introduced as substituents for the
DAG-lactone structure at the sn-1 position. Synthetic strategies
were developed for the introduction of isoxazolol, triazole, and
tetrazole rings as ester moiety replacements. The isoxazolol ring
was introduced in DAG-lactone 1 by a Mitsunobu approach
starting from lactone 7. A change in the protective groups of the
starting material, following the Mitsunobu route, led the
analogues with a saturated-branched alkyl chain at the sn-2
position (DAG-lactone 2, 10B12) in low yields. An improve-
ment in the 2 yield was achieved by employing a synthetic
alternative based on a substitution reaction. Triazole analogues 3
and 4 were obtained in good yields by methodologies based on
the copper(I)-catalyzed variant of the Huisgen 1,3-dipolar
cycloaddition between alkynes and azides catalyzed. The first
attempts to introduce a tetrazole ring in DAG-lactones 5 and 6
were carried out in Mitsunobu conditions. Compound 6 was
straightforwardly obtained by this route, but the strategy did not
proceed well for compound 5, which was obtained using the
alternative based on the nucleophilic substitution of a good
leaving group by the phenyltetrazole anion.
Figure 5. Stimulation of ruffle formation by DAG-lactones. A549 cells
were stimulated for 30 min with DAG-lactones (1 μM) or PMA (0.1
μM). Cells were fixed and stained with phalloidin-rhodamine. (A)
Representative micrographs. (B) Quantification of ruffle area/cell using
ImageJ. ****, p < 0.001 vs vehicle.
approach17 revealed that the effect of 10B12 was near the
maximum response caused by 1 μM AJH-836 and 100 nM PMA
(Figure 5B). We also tested a higher dose of the DAG lactones
(10 μM), but at this concentration, those compounds that
induce ruffles at 1 μM showed significant toxicity, as evidenced
by cell detachment and death (data not shown). The rank in
potency observed in these experiments essentially matched that
observed for GFP-PKCε peripheral translocation.
Finally, in order to confirm the involvement of PKCε in the
formation of ruffles induced by 10B12, we used PKCε RNAi to
silence the expression of this kinase in A549 cells. In order to
minimize misinterpretations due to the “off-target” effects of
RNAi duplexes, we used two different RNAi duplexes (ε1 and
ε2). We observed an 80−90% reduction in PKCε mRNA levels
(as determined by Q-PCR) (Figure 6A) and ∼70% reduction in
PKCε protein expression (as determined by Western blot) 48 h
after transfection of either RNAi duplex (Figure 6B). Notably,
the formation of ruffles by 10B12 was markedly impaired in
PKCε-depleted cells in a magnitude that was consistent with its
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Modeling predicted that compound 10B12, with its higher
logP and its ability to adopt the favored sn-2 binding mode for
DAG-lactones, would have a stronger C1 domain binding
ability, and this was in agreement with the in vitro binding assay
results. However, as has been shown previously with PKCα-
binding and RasGRP-binding DAG-lactones,43 the binding
affinity to C1 domains is only one aspect of the biological effects
of a DAG-lactone, and compound 6, with relatively weak C1
domain binding, was also able to induce GFP-PKCε trans-
location and a small effect on ruffle formation. Agents that
belong to the different families of C1 domain ligands, such as
phorbol esters, deoxyphorbol esters, and bryostatins, display
similar affinities in vitro for individual PKC isoforms, yet these
ligands have widely diverse biological functions.44,45 This is
epitomized by bryostatins and deoxyphorbol esters, compounds
that display antitumor-promoting activities in mouse models
rather than the typical tumor promoting effects caused by
phorbol esters. Although not fully understood, it may be possible
that additional mechanisms, namely, the interactions of ligands
with lipid microdomains, the time-dependent concentration of
ligand in different subcellular membranes, and/or the formation
of specific ligand−protein−membrane ternary complexes, may
greatly influence the biological responses of C1 domain-binding
compounds. While these mechanisms still remain subject to
experimental examination, it is clear that such rich diversity in
the effects of C1 domain ligands underscores opportunities for
the generation of PKC isozyme-specific ligands. In this regard,
our data is in line with previous studies demonstrating that it is
possible to achieve remarkable selectivity upon introduction of
side chains in cyclized DAG structures, as has been previously
demonstrated by means of combinatorial libraries for DAG-
lactones with different alkyl-chain and acyl-chain substitu-
tions.12
The present results highlight the feasibility of generating C1
domain ligands with preferential selectivity toward specific
members of the PKC family, a highly complex task due to the
remarkable similarity among C1 domains. In previous studies,
we demonstrated significant selectivity toward PKCε for the
DAG-lactone AJH-836. This derivative represents, to date, the
best-characterized example for a cyclized DAG with preferential
selectivity for an nPKC relative to cPKCs.16−18 Similar to AJH-
836, the DAG-lactone 10B12 synthesized in the present study,
in which the ester moiety at the sn-1 position has been replaced
by an isoxazole ring, displays a significant degree of specificity for
PKCε relative to PKCα, particularly in a cellular context. Indeed,
although the [3H]PDBu competition binding assays revealed a
lower PKCε/PKCα selectivity index for 10B12 compared to
AJH-836, it shows a preferred ability to translocate PKCε
relative to PKCα. This selectivity is reflected in its ability to
induce the reorganization of the cellular actin cytoskeleton into
peripheral ruffles, a well-described PKCε-mediated effect. We
carefully validated this conclusion by means of PKCε RNAi
silencing, which was able to abrogate the activity of the new sn-1
derivative to form ruffles in lung cancer cells. Thus, a stable
isoxazole ring can function as an ester surrogate to allow binding
to C1 domain and dictate specificity toward PKCε as a receptor
in a cellular environment. Since PKCε is very important for
tumor growth and metastasis and has been implicated in cancer
cell motility and invasiveness,46,47 novel sn-1 derivatives may
have significant utility for the study of this kinase in cancer
progression. Considering the limited availability of isozyme-
specific PKC activators, this class of C1 domain ligands may
represent a novel tool that promotes PKC isozyme biological
response patterns.
In summary, our study provides a proof of principle for the
generation of DAG-lactones with replacements in the labile ester
group at the sn-1 position having significant potency for PKC
activation. DAG-lactones with a stable isoxazole ring that can
function as an ester surrogate represent a novel class of PKC
activators, thus expanding the diversity of C1 domain ligands
with PKC isozyme selectivity. Given the simplicity of the DAG-
lactone structure for chemical modifications, the search for
additional substitutions could help in the design of additional
potent nPKC selective agents for experimentally assessing the
functional involvement of these kinases in human diseases.
EXPERIMENTAL SECTION
■
General Procedures. All chemical compounds were purchased
from commercial sources or synthesized. Reaction monitoring was
performed on Merck silica gel 254F TLC plates. Column chromatog-
raphy was performed on a Teledyne Isco CombiFlash Rf+ instrument
under gradient elution conditions with RediSep disposable flash
columns. All melting points were determined on an Electrothermal
IA9000 series digital melting point apparatus and are uncorrected. 1H
NMR and 13C NMR spectra were carried out on a Bruker Fourier 300,
300 MHz or Bruker Avance DPX 400, 400 MHz spectrometer using
CDCl3, MeOD, or DMSO-d6 as the solvent and TMS as the internal
standard. HRMS was performed at CIBION (Bioanalytical Mass
Spectrometry Group). High-resolution positive ion electrospray
ionization MS was conducted on a Xevo G2S Q-TOF spectrometer
(Waters Corporation, Manchester, UK) operated at both positive and
negative modes. Data processing was carried out using MassLynx v4.1
software (Waters Corp.). Data was corrected during acquisition using a
reference compound (LockSpray). The purity was determined by high-
performance liquid chromatography (HPLC). The purity of all final
compounds was 95% or higher. HPLC analysis was performed on a
Shimadzu SPD-M20A UV−vis photo diode array (PDA) detector,
HPLC system (Shimadzu, Tokyo, Japan). The column used was a
Zorbax Eclipse XDB C8 (5 μm, 150 mm × 4.60 mm) at a temperature
of 40 °C, using a linear gradient of 0−100% H2O (0.1% HAcO)/
acetonitrile at a flow rate of 1.0 mL/min over 20 min. All title
compounds were examined for PAINS and passed.48
Docking Studies. Compounds were docked into the crystal
structure of the C1B domain of PKC δ14 (PDB ID: 1PTR) using the
program GOLD, version 5.2.2.49,50 We used standard default genetic
algorithm (GA) settings, with the binding site defined by atoms within a
10.0 Å radius of the Nε atom of residue Q257. The scoring function was
GoldScore, and we added a template similarity constraint to the
hydrogen bond acceptor atoms in bound phorbol-13-O-acetate from
the crystal structure. Twenty GA runs were performed for each
compound. After docking, the docked poses were refined with 500 steps
of conjugate gradient energy minimization using the Embrace facility in
MacroModel.51 The ligand and residues 240−242, 250−253, and 257
in the C1 domain were free to move, residues 237−239, 243, and 254−
256 were restrained with a force constant of 100 kJ/mol Å2, and all
other protein residues were fixed in place.
Chemistry. General Procedure A: Aldol Condensation Followed
by Olefination. Standard Alkylation Procedure. A solution of lactone
7 or 19 (1 equiv) in THF (5 mL/mmol) at −78 °C was treated
dropwise with LiHMDS (1.5−2.0 equiv, 1 M in THF). After the
mixture was stirred at −78 °C for 1 h, a solution of R2CHO (1.5 equiv)
or acetone (5 equiv) in THF (1 mL/mmol) was added, and the stirring
continued for 2−3 h at −78 °C. The reaction was quenched by the slow
addition of a saturated aqueous solution of NH4Cl and allowed to warm
to room temperature. The layers were separated, and the aqueous layer
was extracted with Et2O (3×). The combined organic phases were
washed with H2O (1×) and brine (1×), dried over MgSO4, and
concentrated in vacuo. Purification by silica gel flash column
chromatography (gradient 0−20% EtOAc/hexanes) gave a mixture of
alcohol diastereomers, which were used directly in the next step.
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Standard Mesylation−Olefination Procedure. A solution of the
alkylation product in dichloromethane (10 mL/mmol) at 0 °C was
treated with methanesulfonyl chloride (2 equiv) and triethylamine (4
equiv) and then stirred at room temperature for 2−5 h. DBU (3−5
equiv) was added at 0 °C, and the resulting solution was stirred
overnight at room temperature. The reaction mixture was treated with a
saturated solution of NH4Cl (10 mL/mmol) and extracted with
CH2Cl2 (3×). The combined organics were washed with H2O (2×) and
brine (1×), dried (Na2SO4), and concentrated in vacuo. Purification by
silica gel flash column chromatography (gradient 0−20% EtOAc/
hexanes) gave 14, 20, and 24.
General Procedure B: BnO Deprotection. A solution of 14, 22, 27,
or 29 (1 equiv) in anhydrous dichloromethane (20 mL/mmol) at −78
°C was treated dropwise with boron trichloride (1 M in CH2Cl2, 2
equiv). The reaction was monitored by TLC and quenched upon
completion (0.5−1 h) by the slow addition of NaHCO3, and the
aqueous phase was extracted with dichloromethane (2 × 15 mL). The
organic phase was dried (Na2SO4) and concentrated in vacuo.
Purification by silica gel flash column chromatography (gradient 0−
20% EtOAc/hexanes) gave 15, 2, 5, and 6.
General Procedure C: PMP Deprotection. Ceric ammonium nitrate
(3 equiv) was added to a solution of 20 or 24 (1 equiv) in 80%
CH3CN/H2O (v/v, 10 mL/mmol) at 0 °C. The reaction mixture was
stirred for 15 min. A solution of 5% Na2S2O3 was added at room
temperature, and the aqueous phase was extracted with CH2Cl2 (3×).
The organic phase was dried (Na2SO4) and concentrated in vacuo.
Purification by silica gel flash column chromatography (gradient 0−
30% EtOAc/hexanes) gave 21 and 25.
5-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-(((5-methylisoxazol-3-
yl)oxy)methyl)-3-(propan-2-ylidene)dihydrofuran-2(3H)-one (10). A
solution of triphenylphosphine (557 mg, 2.0 mmol), lactone 931 (638
mg, 1.5 mmol), and 5-methylisoxazol-3-ol32 (119 mg, 1.2 mmol) in
anhydrous tetrahydrofuran (6 mL) was treated with diisopropyl
azodicarboxylate (410 μL, 2.0 mmol) and stirred at room temperature
overnight. The reaction mixture was evaporated, and the residue was
purified by flash chromatography using hexane/ethyl acetate (8:2) as an
eluent to afford 10 as a white solid (314 mg, 52%). Mp: 93−94 °C. 1H
NMR (400 MHz, CDCl3): δ 7.67−7.65 (m, 4H), 7.42−7.40 (m, 6H),
5.62 (s, 1H), 4.38−4.32 (m, 2H), 3.78 (s, 2H), 2.90 (AB d, J = 16.4 Hz,
1H), 2.78 (AB d, J = 16.4 Hz, 1H), 2.34 (s, 3H), 2.28 (s, 3H), 1.88 (s,
3H), 1.05 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 171.6, 170.6, 169.1,
150.2, 135.6, 135.6, 132.7, 132.5, 129.9, 127.8, 127.8, 119.5, 92.9, 81.2,
70.5, 66.0, 35.3, 26.6, 24.5, 19.8, 19.1, 12.9. HRMS (ESI): m/z calcd for
C29H35NO5Si [M + H]+, 506.2363; found, 506.2364.
5-(Hydroxymethyl)-5-(((5-methylisoxazol-3-yl)oxy)methyl)-3-
(propan-2-ylidene)dihydrofuran-2(3H)-one (1). To a stirred solution
of 10 (160 mg, 0.32 mmol) in anhydrous THF (3 mL) was added
tetrabutylammonium fluoride (1.0 M in THF, 320 μL, 0.32 mmol) at 0
°C. After the mixture was stirred for 1 h, the reaction was evaporated.
The residue was purified by column chromatography over silica gel with
hexane/ethyl acetate (6:4) as an eluent to afford 1 (68 mg, 80%) as a
white solid. Mp: 139−140 °C. 1H NMR (400 MHz, CDCl3): δ 5.63 (s,
1H), 4.33−4.27 (AB q, J = 11.1 Hz, 2H), 3.69 (m, 2H), 3.15 (t, J = 6.6
Hz, 1H), 2.85 (AB d, J = 16.5 Hz, 1H), 2.73 (AB d, J = 16.5 Hz, 1H),
2.30 (s, 3H), 2.23 (s, 3H), 1.86 (s, 3H). 13C NMR (101 MHz, CDCl3):
δ 171.8, 170.9, 169.1, 151.9, 118.7, 92.9, 81.3, 70.5, 64.3, 31.9, 24.5,
19.9, 12.8. HRMS (ESI): m/z calcd for C13H18NO5 [M + H]+,
268.1185; found, 268.1190.
(2-(((tert-Butyldiphenylsilyl)oxy)methyl)-5-oxo-4-(propan-2-
ylidene)tetrahydrofuran-2-yl)methyl 4-methylbenzenesulfonate
(11). To a solution of compound 9 (127 mg, 0.30 mmol) in CH2Cl2
(2 mL) in a sealed vial were added triethylamine (606 mg, 6 mmol) and
p-toluenesulfonyl chloride (114 mg, 0.6 mmol). The reaction mixture
was heated at 40 °C overnight and treated with 1 N HCl (5 mL). The
aqueous layer was extracted with CH2Cl2 (3 × 5 mL). The combined
organic layers were washed with NaHCO3 (sat. solution, 5 mL) and
NaCl (sat. solution, 5 mL), dried over Na2SO4, and concentrated under
a vacuum. The residue was purified by silica gel flash column
chromatography hexane/ethyl acetate (9:1) to afford 11 as a white
solid (148 mg, 88%). Mp: 44−46 °C. 1H NMR (400 MHz, CDCl3): δ
7.75 (d, J = 8.3 Hz, 2H), 7.63−7.56 (m, 4 H), 7.47−7.35 (m, 6H), 7.31
(d, J = 8.0 Hz, 1H), 4.12 (d, J = 10.1 Hz, 1H), 4.05 (d, J = 10.1 Hz, 1H),
3.60 (s, 2H), 2.79−2.65 (m, 2H), 2.43 (s, 3H), 2.21 (t, J = 2.0 Hz, 3H),
1.82 (s, 3H), 0.98 (s, 9H). 13C NMR (101 MHz, CDCl3): δ 168.5,
151.1, 145.2, 135.5, 132.4, 132.3, 132.1, 129.9, 128.0, 127.8, 127.8,
118.7, 80.3, 70.2, 66.0, 32.0, 26.5, 24.5, 21.6, 19.8, 19.1. HRMS (ESI):
m/z calcd for C32H39O6SSi [M + H]+, 579.2237; found, 579.2231.
5-(Azidomethyl)-5-((tert-butyldiphenylsilyloxy)methyl)-3-(prop-
an-2-ylidene)dihydrofuran-2(3H)-one (12). To a solution of tosylate
11 (140 mg, 0.245 mmol) in DMF (3 mL) in a sealed vial was added
sodium azide (319 mg, 4.9 mmol). The reaction mixture was heated at
120 °C for 4 h, then cooled to room temperature, and evaporated. The
residue was dissolved in CH2Cl2 and washed with H2O and brine, dried
over anhydrous Na2SO4, and concentrated under a vacuum. The
residue was purified by silica gel flash column chromatography hexane/
ethyl acetate (95:5) to afford 12 (105 mg, 96%) as a colorless oil. 1H
NMR (400 MHz, CDCl3): δ 7.67−7.62 (m, 4H), 7.47−7.36 (m, 6H),
3.65 (AB m, 2H), 3.52 (d, J = 12.8 Hz, 1H), 3.46 (d, J = 12.8 Hz, 1H),
2.73 (AB m, 2H), 2.25 (s, 3H), 1.85 (s, 3H), 1.04 (s, 9H). 13C NMR
(101 MHz, CDCl3): δ 168.7, 150.7, 135.6, 135.6, 132.6, 132.5, 129.9,
127.8, 119.3, 81.9, 66.8, 55.1, 32.8, 26.6, 24.5, 19.9, 19.2. HRMS (ESI):
m/z calcd for C25H32N3O3Si [M + H]+, 450.2213; found, 450.2218.
5-((tert-Butyldiphenylsilyloxy)methyl)-3-(propan-2-ylidene)-5-
((4-p-tolyl-1H-1,2,3-triazol-1-yl)methyl)dihydrofuran-2(3H)-one
(13). 4-Ethynyltoluene (25 mg, 26 μL, 0.21 mmol) and compound 12
(95 mg, 0.21 mmol) were dissolved in 5 mL of a 1:1 tert-BuOH/H2O
mixture. While the mixture was being stirred, sodium ascorbate (1 M in
H2O, 189 μL, 0.189 mmol) was added, followed by copper(II) sulfate
pentahydrate (0.3 M, 280 μL, 0.084 mmol). The resulting mixture was
stirred at room temperature for 18 h before dilution with ethyl acetate
(10 mL) and saturation with EDTA solution (0.4 mL) and water (10
mL). The aqueous layer was extracted with ethyl acetate (10 mL × 3).
The combined organic layers were dried over Na2SO4, filtered through
a short silica gel column, and concentrated in vacuo. The residue was
purified by silica gel flash column chromatography hexane/ethyl acetate
(9:1) to afford 13 (89 mg, 75%) as a white solid. Mp: 59−62 °C. 1H
NMR (400 MHz, CDCl3): δ 7.88 (s, 1H), 7.68 (d, J = 8.3 Hz, 2H),
7.66−7.63 (m, 4H), 7.46−7.36 (m, 6H), 7.23 (d, J = 8.0 Hz, 2H), 4.72
(AB d, J = 14.5 Hz, 1H), 4.61 (AB d, J = 14.5 Hz, 1H), 3.72 (AB d, J =
10.9 Hz, 1H), 3.65 (AB d, J = 10.8 Hz, 1H), 2.75 (AB q, J = 16.6 Hz,
2H), 2.38 (s, 3H), 2.06 (s, 3H), 1.74 (s, 3H), 1.08 (s, 9H). 13C NMR
(101 MHz, CDCl3): δ 168.5, 151.8, 148.3, 138.1, 135.6, 135.5, 132.3,
132.2, 130.1, 129.5, 127.9, 127.4, 125.7, 121.0, 118.3, 81.5, 67.2, 54.0,
32.3, 26.7, 24.5, 21.3, 19.9, 19.2. HRMS (ESI): m/z calcd for
C34H40N3O3Si [M + H]+, 566.2839; found, 566.2847.
5-(Hydroxymethyl)-3-(propan-2-ylidene)-5-((4-p-tolyl-1H-1,2,3-
triazol-1-yl)methyl) dihydrofuran-2(3H)-one (3). To a stirred solution
of 13 (60 mg, 0.106 mmol) in anhydrous THF (3 mL) was added
tetrabutylammonium fluoride (TBAF, 1.0 M solution in THF, 106 μL,
0.106 mmol) at 0 °C. After the mixture was stirred for 1 h, the reaction
was evaporated. The residue was purified by column chromatography
over silica gel with hexane/ethyl acetate (2:8) as an eluent to give the
alcohol 3 (28 mg, 80%) as a white solid. Mp: 167−168 °C. 1H NMR
(400 MHz, CDCl3): δ 7.88 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.23 (d, J =
8.0 Hz, 2H), 4.69 (AB q, J = 14.6 Hz, 2H), 3.61 (dq, J = 12.2 Hz, 6.5 Hz,
2H), 3.02 (t, J = 6.6 Hz, 1H), 2.84 (AB q, J = 16.5 Hz, 2H), 2.37 (s, 3H),
2.10 (s, 3H), 1.80 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 168.5,
153.5, 148.3, 138.3, 129.5, 127.3, 125.7, 121.1, 117.6, 81.4, 65.3, 53.9,
32.5, 24.6, 21.3, 20.0. HRMS (ESI): m/z calcd for C18H22N3O3 [M +
H]+, 328.1661; found, 328.1665.
(E)-5-((Benzyloxy)methyl)-5-(((tert-butyldiphenylsilyl)oxy)-
methyl)-3-(3-isobutyl-5-methylhexylidene)dihydrofuran-2(3H)-one
(14). Starting from lactone 730 (1.654 g, 3.46 mmol) and 3-isobutyl-5-
methylhexanal13 (940 mg, 5.45 mmol), and following general
procedure A, the reaction product was isolated as a 1:1 mixture of E-
and Z-isomers. Two fractions were isolated after purification by silica
gel flash column chromatography with hexane/ethyl acetate (98:2) as
an eluent. The first fraction corresponded to the Z-isomer (724 mg,
34%), and the second fraction was the E-isomer 14 (763 mg, 35%). 1H
NMR (400 MHz, CDCl3): δ 7.64 (d, J = 6.9 Hz, 4H), 7.46−7.22 (m,
I
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11H), 6.74 (m, 1H), 4.53 (s, 2H), 3.73 (AB q, J = 10.7 Hz, 2H), 3.59 (d,
J = 10.1 Hz, 1H), 3.53 (d, J = 10.1 Hz, 1H), 2.82−2.69 (AB m, 2H),
2.10 (t, J = 6.2 Hz, 2H), 1.64−1.60 (m, 3H), 1.14−1.06 (m, 4H), 1.02
(s, 9H), 0.85 (d, J = 6.5 Hz, 12H). 13C NMR (101 MHz, CDCl3): δ
170.3, 139.2, 137.7, 135.6, 135.6, 132.9, 132.6, 129.8, 129.8, 128.4,
127.9, 127.8, 127.7, 127.6, 84.4, 73.7, 71.9, 66.2, 43.9, 43.8, 34.7, 32.8,
30.3, 26.7, 25.2, 23.0, 22.9, 22.7, 22.6, 19.2. HRMS (ESI): m/z calcd for
C40H55O4Si [M + H]+, 627.3870; found, 627.3879.
(E)-5-((tert-Butyldiphenylsilyloxy)methyl)-5-(hydroxymethyl)-3-
(3-isobutyl-5-methylhexylidene)dihydrofuran-2(3H)-one (15). Start-
ing from 14 (644 mg, 1.03 mmol) and following general procedure B,
15 (490 mg, 89%) was obtained as a colorless oil after purification by
silica gel flash column chromatography with hexane/ethyl acetate (9:1).
1H NMR (400 MHz, CDCl3): δ 7.64−7.62 (m, 4H), 7.48−7.35 (m,
6H), 6.76 (m, 1H), 3.77−3.62 (m, 4H), 2.87−2.65 (AB m, 2H), 2.12 (t,
J = 6.7 Hz, 2H), 1.93 (t, J = 6.8 Hz, 1H), 1.75−1.56 (m, 3H), 1.14−1.07
(m, 4H), 1.03 (s, 9H), 0.86 (d, J = 6.5 Hz, 12H). 13C NMR (101 MHz,
CDCl3): δ 170.2, 140.0, 135.6, 135.6, 132.7, 132.5, 130.0, 129.9, 127.8,
127.7, 85.1, 66.0, 65.3, 43.9, 43.8, 34.8, 32.8, 29.7, 26.7, 25.2, 23.0, 22.9,
22.7, 22.6, 19.2. HRMS (ESI): m/z calcd for C33H48O4SiNa [M + Na]+,
559.3220; found, 559.3224.
(E)-(2-((tert-Butyldiphenylsilyloxy)methyl)-4-(3-isobutyl-5-meth-
ylhexylidene)-5-oxotetra hydro furan-2-yl)methyl 4-methylbenze-
nesulfonate (16). To a solution of 15 (474 mg, 0.88 mmol) in pyridine
(4 mL) was added p-toluenesulfonyl chloride (421 mg, 2.2 mmol). The
reaction mixture was heated at 90 °C overnight and treated with 1 N
HCl (8 mL). The aqueous layer was extracted with CH2Cl2 (3 × 10
mL). The combined organic layers were washed with NaHCO3 (sat.
solution, 10 mL) and NaCl (sat. solution, 10 mL), dried over Na2SO4,
and concentrated under a vacuum. The residue was purified by silica gel
flash column chromatography with hexane/ethyl acetate (98:2) to
afford 16 (423 mg, 90%) as a white solid. Mp: 135−136 °C. 1H NMR
(400 MHz, CDCl3): δ 7.74 (d, J = 8.1 Hz, 2H), 7.63−7.55 (m, 4 H),
7.48−7.34 (m, 6H), 7.31 (d, J = 8.1 Hz, 1H), 6.74 (m, 1H), 4.14 (d, J =
10.2 Hz, 1H), 4.06 (d, J = 10.2 Hz, 1H), 3.62 (s, 2H), 2.79−2.65 (m,
2H), 2.43 (s, 3H), 2.08 (t, J = 6.4 Hz, 2H), 1.73−1.55 (m, 3H), 1.15−
1.02 (m, 4H), 0.98 (s, 9H), 0.86 (m, 12H). 13C NMR (101 MHz,
CDCl3): δ 169.2, 145.2, 140.9, 135.5, 132.3, 132.2, 130.0, 130.0, 128.0,
127.9, 126.3, 82.1, 70.1, 65.9, 43.9, 43.8, 34.9, 32.8, 30.0, 26.6, 25.2,
22.9, 22.9, 22.6, 22.6, 21.6, 19.2. HRMS (ESI): m/z calcd for
C40H55O6SSi [M + H]+, 691.3489; found, 691.3475.
(E)-5-(Azidomethyl)-5-((tert-butyldiphenylsilyloxy)methyl)-3-(3-
isobutyl-5-methylhexylidene)dihydrofuran-2(3H)-one (17). To a
solution of tosylate 16 (400 mg, 0.58 mmol) in DMF (3 mL) was
added sodium azide (754 mg, 11.6 mmol). The reaction mixture was
heated at 90 °C for 72 h, then cooled to room temperature, and
evaporated. The residue was dissolved in CH2Cl2, washed with H2O
and brine, dried over anhydrous Na2SO4, and concentrated under a
vacuum. The residue was purified by silica gel flash column
chromatography hexane/ethyl acetate (99:1) to afford 17 (235 mg,
72%) as a colorless syrup. 1H NMR (300 MHz, CDCl3): δ 7.66−7.61
(m, 4H), 7.54−7.40 (m, 6H), 6.78 (m, 1H), 3.75−3.67 (mAB, 2H),
3.54 (AB d, J = 12.8 Hz, 1H), 3.47 (AB d, J = 12.8 Hz, 1H), 2.70 (AB m,
2H), 2.11 (t, J = 6.7 Hz, 2H), 1.75−1.56 (m, 3H), 1.17−1.04 (m, 4H),
1.04 (s, 9H),0.86 (m, 12H). 13C NMR (75 MHz, CDCl3): δ 169.5,
140.5, 135.6, 135.6, 132.5, 132.3, 130.0, 127.9, 126.9, 83.7, 66.5, 55.1,
43.9, 43.8, 34.8, 32.8, 30.7, 26.7, 25.2, 23.0, 22.9, 22.7, 22.6, 19.2.
HRMS (ESI): m/z calcd for C33H47N3O3SiNa [M + Na]+, 584.3284;
found, 584.3290.
Na2SO4, filtered through a short silica gel column, and concentrated in
vacuo. The residue was purified by silica gel flash column
chromatography hexane/ethyl acetate (93:7) to afford 18 (95 mg,
77%) as a colorless syrup. 1H NMR (300 MHz, CDCl3): δ 7.85 (s, 1H),
7.67 (d, J = 8.1 Hz, 2H), 7.66−7.61 (m, 4H), 7.49−7.34 (m, 6H), 7.22
(d, J = 8.0 Hz, 2H), 6.62 (m, 1H), 4.74 (AB d, J = 14.5 Hz, 1H), 4.64
(AB d, J = 14.5 Hz, 1H), 3.73 (AB d, J = 11 Hz, 1H), 3.65 (AB q, J = 11
Hz, 1H), 2.82 (AB q, J = 17.6 Hz, 1H), 2.71 (AB q, J = 17.6 Hz, 1H),
2.38 (s, 3H), 1.99 (t, J = 6.6 Hz, 2H), 1.67−1.46 (m, 3H), 1.13−0.93
(m, 4H), 1.08 (s, 9H),0.81 (d, J = 6.5 Hz, 12H). 13C NMR (75 MHz,
CDCl3): δ 169.2, 148.4, 141.5, 138.1, 135.6, 135.5, 132.2, 132.1, 130.1,
129.5, 127.9, 127.4, 125.8, 125.7, 120.8, 83.1, 66.9, 53.9, 43.8, 34.8,
32.7, 30.2, 29.7, 26.7, 25.1, 22.9, 22.6, 22.6, 21.3, 19.2. HRMS (ESI): m/
z calcd for C42H56N3O3Si [M + H]+, 678.4091; found, 678.4121.
(E)-5-(Hydroxymethyl)-3-(3-isobutyl-5-methylhexylidene)-5-((4-
p-tolyl-1H-1,2,3-triazol-1-yl)methyl)dihydrofuran-2(3H)-one (4). To
a stirred solution of 18 (79 mg, 0.12 mmol) in anhydrous THF (3 mL)
was added tetrabutylammonium fluoride (1.0 M solution in THF, 120
μL, 0.12 mmol) at 0 °C. After the mixture was stirred for 1 h, the
reaction was evaporated. The residue was purified by column
chromatography over silica gel with hexane/ethyl acetate (75:25) as
an eluent to afford 4 (31 mg, 60%) as a white solid. Mp: 155−156 °C.
1H NMR (400 MHz, CDCl3): δ 7.88 (s, 1H), 7.69 (d, J = 7.7 Hz, 2H),
7.23 (d, J = 7.7 Hz, 2H), 6.72 (m, 1H), 4.79 (AB d, J = 14.6 Hz, 1H),
4.63 (AB d, J = 14.6 Hz, 1H), 3.70−3.45 (m, 2H), 2.93 (t, J = 6.3 Hz,
1H), 2.83 (s, 2H), 2.37 (s, 3H), 2.07 (m, 2H), 1.71−1.49 (m, 3H),
1.15−0.93 (m, 4H), 0.83 (d, J = 5.9 Hz, 12H). 13C NMR (101 MHz,
CDCl3): δ 169.1, 148.4, 142.7, 138.3, 129.5, 127.2, 125.7, 125.3, 121.0,
83.10, 65.0, 53.7, 43.9, 34.9, 32.8, 30.6, 25.2, 22.9, 22.9, 22.6, 22.5, 21.3.
HRMS (ESI): m/z calcd for C26H38N3O3 [M + H]+, 440.2913; found,
440.2926.
(E)-5-((Benzyloxy)methyl)-5-(hydroxymethyl)-3-(3-isobutyl-5-
methylhexylidene)dihydrofuran-2(3H)-one (21). Compound 21 was
synthesized following a procedure described elsewhere.37
Starting from lactone 1936 (2.52 g, 7.4 mmol) and 3-isobutyl-5-
methylhexanal13 (1.9 g, 11.41 mmol) and following general procedure
A, the reaction product was isolated as a 1:1 mixture of E- and Z-
isomers. Two fractions were isolated after purification by silica gel flash
column chromatography with hexane/ethyl acetate (98:2) as an eluent.
The first fraction corresponded to the Z-isomer (1.23 g, 34%) and the
second fraction corresponded to the E-isomer 20 (1.27 g, 35%).
1
Recorded spectra perfectly matched the published results. H NMR
(400 MHz, CDCl3): δ 7.36−7.26 (m, 5 H), 6.81 (s, 4 H), 6.82−6.76
(m, 1 H), 4.58 (AB q, J = 12.3 Hz, 2H), 4.03 (AB q, J = 9.8, 26.6 Hz,
2H), 3.76 (s, 3H), 3.66 (AB q, J = 10.3, 23.7 Hz, 2H), 2.92−2.77 (AB m,
2H), 2.13 (t, J = 11.6 Hz, 2H), 1.76−1.58 (m, 3H), 1.14−1.06 (m, 4H),
0.85 (m, 12H).
Starting from E-isomer 20 (1.26 g, 2.55 mmol) and following general
procedure C, 21 was obtained as a colorless oil (746 mg, 75% yield). Its
spectrum data perfectly matched with published results. 1H NMR (400
MHz, CDCl3): δ 7.40−7.26 (m, 5H), 6.82−6.73 (m, 1H), 4.56 (m, 2
H), 3.84−3.64 (m, 2H), 3.57 (AB q, J = 9.9, 28.0 Hz, 2H), 2.83−2.65
(m, 2H), 2.19−2.07 (m, 3H), 1.77−1.55 (m, 3H), 1.15−1.05 (m, 4H),
0.84−0.86 (m, 12H).
(E)-5-((Benzyloxy)methyl)-5-(iodomethyl)-3-(3-isobutyl-5-
methylhexylidene)dihydrofuran-2(3H)-one (22). A solution of 2137
(128 mg, 0.33 mmol) in toluene (6 mL) was treated with
triphenylphosphine (173 mg, 0.66 mmol), imidazole (67 mg, 0.99
mmol), and iodine (126 mg, 0.5 mmol). The mixture was heated at 90
°C for 1 h and, after reaching room temperature, was poured into a
saturated solution of NaHCO3. Excess of triphenylphosphine was
destroyed by the addition of iodine until the iodine coloration persisted
in the organic layer. The organic layer was washed with Na2S2O3 (10%,
5 mL) and brine, dried over (MgSO4), filtered, and concentrated in
vacuo. The residue was purified on silica gel (hexanes:ethyl acetate,
(E)-5-((tert-Butyldiphenylsilyloxy)methyl)-3-(3-isobutyl-5-meth-
ylhexylidene)-5-((4-p-tolyl-1H-1,2,3-triazol-1-yl)methyl)-
dihydrofuran-2(3H)-one (18). 4-Ethynyltoluene (23 mg, 25 μL, 0.20
mmol) and 17 (102 mg, 0.18 mmol) were dissolved in 4 mL of a 1:1
tert-BuOH/H2O mixture. While the mixture was being stirred, sodium
ascorbate (0.3 M in H2O, 480 μL, 0.144 mmol) was added, followed by
copper(II) sulfate pentahydrate (0.3 M, 240 μL, 0.072 mmol). The
resulting mixture was stirred at room temperature for 2 h before
dilution with ethyl acetate (10 mL) and saturation with EDTA solution
(0.4 mL) and water (10 mL). The aqueous layer was extracted with
ethyl acetate (3 × 10 mL). The combined organic layers were dried over
1
98:2) to give 22 (146 mg, 89%) as a colorless syrup. H NMR (400
MHz, CDCl3): δ 7.38−7.27(m, 5H), 6.82−6.75 (m, 1H), 4.58 (s, 2H),
3.68 (AB d, J = 10.0 Hz, 1H), 3.63 (AB d, J = 10.0 Hz, 1H), 3.49 (AB d, J
= 10.8 Hz, 1H), 3.43 (AB d, J = 10.8 Hz, 1H), 2.92−2.72 (AB m, 2H),
2.15−2.08 (m, 2H), 1.76−1.67 (m, 1H), 1.66−1.58 (m, 2H), 1.17−
J
https://doi.org/10.1021/acs.jmedchem.1c00739
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
1.02 (m, 4H), 0.85 (m, 12H). 13C NMR (101 MHz, CDCl3): δ 169.3,
140.8, 137.3, 128.5, 127.9, 127.7, 127.1, 81.5, 73.8, 73.1, 43.9, 43.9,
34.8, 34.1, 32.8, 25.2, 23.0, 22.9, 22.6, 11.0. HRMS (ESI): m/z calcd for
C24H36IO3 [M + H]+, 499.1709; found, 499.1703.
2H), 3.68−3.60 (AB m, 2H), 3.49 (AB d, J = 14.2 Hz, 1H), 3.40 (AB d, J
= 14.2 Hz, 1H), 2.91−2.73 (AB m, 2H), 2.26 (t, J = 2.8 Hz, 3H), 1.86 (s,
3H). 13C NMR (101 MHz, CDCl3): δ 168.6, 151.6, 137.4, 128.4, 127.8,
127.7, 119.3, 79.7, 73.7, 73.2, 35.9, 24.6, 20.0, 11.4. HRMS (ESI): m/z
calcd for C16H20IO3 [M + H]+, 387.0457; found, 387.0468.
(E)-5-((Benzyloxy)methyl)-3-(3-isobutyl-5-methylhexylidene)-5-
(((5-methylisoxazol-3-yl)oxy)methyl)dihydrofuran-2(3H)-one (23).
From 21, a solution of triphenylphosphine (325 mg, 1.24 mmol), 21
(160 mg, 0.41 mmol), and 5-methylisoxazol-3-ol (62 mg, 0.62 mmol) in
anhydrous tetrahydrofuran (5 mL) was treated with diisopropyl
azodicarboxylate (243 μL, 1.24 mmol) and stirred at room temperature
overnight. The reaction mixture was evaporated, and the residue was
purified by flash chromatography using hexane/ethyl acetate (95:5) as
an eluent to afford 23 (70 mg, 37%) as a colorless syrup. 1H NMR (400
MHz, CDCl3): δ 7.37−7.25 (m, 5H), 6.83−6.73 (m, 1H), 5.59 (s, 1H),
4.57 (s, 2H), 4.41−4.32 (AB q, 2H), 3.65 (AB d, J = 13.4 Hz, 1H), 3.57
(AB d, J = 13.4 Hz, 1H), 2.92−2.70 (AB m, 2H), 2.32 (s, 3H), 2.11 (t, J
= 8.0 Hz, 2H), 1.74−1.54 (m, 3H), 1.15−1.02 (m, 4H), 0.85 (d, J = 6.5
Hz, 12H). 13C NMR (101 MHz, CDCl3): δ 171.6, 170.7, 169.7, 140.3,
137.4, 128.4, 127.8, 127.6, 126.8, 92.9, 82.2, 73.7, 71.5, 70.8, 43.7, 34.7,
32.7, 30.5, 25.2, 22.9, 22.6, 12.9. HRMS (ESI): m/z calcd for
C28H39NO5Na [M + Na]+, 492.2726; found, 492.2742.
From 22, a solution of iodine 22 (50 mg, 0.10 mmol), K2CO3 (140
mg, 1 mmol), and 5-methylisoxazol-3-ol (99 mg, 1 mmol) in DMF (0.5
mL) was stirred at 140 °C for 4 h in a sealed vial. Afterward, the reaction
was cooled to room temperature, diluted with CH2Cl2 (3 mL), and
washed with NaOH (5%) and brine. The organic phase was dried over
anhydrous Na2SO4, filtered, and concentrated under a vacuum. The
residue was purified by silica gel flash column chromatography hexane/
ethyl acetate (95:5) to afford 23 (29 mg, 62%).
(E)-5-(Hydroxymethyl)-3-(3-isobutyl-5-methylhexylidene)-5-(((5-
methylisoxazol-3-yl)oxy)methyl)dihydrofuran-2(3H)-one (2,
10B12). Starting from 22 (26 mg, 0.055 mmol) and following general
procedure B, compound 2 (10B12) was obtained as a white solid (14
mg, 67%). Mp: 81−82 °C. 1H NMR (400 MHz, CDCl3): δ 6.80 (m,
1H), 5.63 (s, 1H), 4.40 (AB d, J = 11.3 Hz, 1H), 4.32 (AB d, J = 11.3 Hz,
1H), 3.78−3.67 (m, 2H), 2.85 (AB d, J = 17.1 Hz, 1H), 2.73 (AB d, J =
17.1 Hz, 1H), 2.71 (t, J = 6.9 Hz, 1H), 2.32 (s, 3H), 2.13 (t, J = 6.5 Hz,
2H), 1.76−1.55 (m, 3H), 1.15−1.03 (m, 4H), 0.85 (d, J = 6.5 Hz,
12H). 13C NMR (101 MHz, CDCl3): δ 171.8, 171.0, 169.7, 141.2,
126.5, 92.9, 83.1, 70.5, 64.4, 43.8, 34.8, 32.8, 30.0, 25.2, 25.2, 22.9, 22.6,
12.9. HRMS (ESI): m/z calcd for C21H34NO5 [M + H]+, 380.2437;
found, 380.2451.
5-((Benzyloxy)methyl)-5-((5-phenyl-2H-tetrazol-2-yl)methyl)-3-
(propan-2-ylidene)dihydrofuran-2(3H)-one (27). A solution of 26
(111 mg, 0.28 mmol), K2CO3 (386 mg, 2.8 mmol), and 5-phenyl-1H-
tetrazole40 (420 mg, 2.8 mmol) in DMF (1 mL) was stirred at 120 °C
overnight in a sealed vial, then cooled to room temperature, diluted
with CH2Cl2 (3 mL), and filtered. The organic phase was washed with
brine, dried over anhydrous Na2SO4, filtered, and concentrated under a
vacuum. The residue was purified by silica gel flash column
chromatography hexane/ethyl acetate (92:8) to afford 27 (84 mg,
74%) as a white solid. Mp: 87−88 °C. 1H NMR (400 MHz, CDCl3): δ
8.15−8.08 (m, 2H), 7.52−7.45 (m, 3H), 7.38−7.27 (m, 5H), 5.00 (AB
d, J = 14.0 Hz, 1H), 4.92 (AB d, J = 14.0 Hz, 1H), 4.67 (AB d, J = 11.9
Hz, 2H), 4.61 (AB d, J = 11.9 Hz, 1H), 3.68 (AB d, J = 10.1 Hz, 1H),
3.60 (AB d, J = 10.1 Hz, 1H), 2.95 (d, J = 16.6 Hz, 1H), 2.75 (d, J = 16.6
Hz, 1H), 2.04 (s, 3H), 1.72 (s, 3H). 13C NMR (101 MHz, CDCl3): δ
168.0, 165.4, 152.0, 137.2, 130.5, 128.9, 128.5, 128.0, 127.8, 127.0,
126.9, 117.6, 79.9, 73.8, 72.7, 56.9, 33.4, 24.4, 19.8.HRMS (ESI): m/z
calcd for C23H25N4O3 [M + H]+, 405.1927; found, 405.1936.
5-(Hydroxymethyl)-5-((5-phenyl-2H-tetrazol-2-yl)methyl)-3-
(propan-2-ylidene)dihydrofuran-2(3H)-one (5). Starting from 27 (78
mg, 0.194 mmol) and following general procedure B, 5 was obtained as
1
a white solid (55 mg, 90%). Mp: 154−155 °C. H NMR (400 MHz,
CDCl3): δ 8.15−8.08 (m, 2H), 7.52−7.45 (m, 3H), 4.96 (s, 2H), 3.79
(d, J = 6.4 Hz, 2H), 2.95 (d, J = 16.4 Hz, 1H), 2.73 (d, J = 16.4 Hz, 1H),
2.74−2.68 (m, 1H), 2.07 (s, 3H), 1.76 (s, 3H). 13C NMR (101 MHz,
CDCl3): δ 168.1, 165.5, 152.9, 130.6, 128.9, 126.9, 126.8, 117.5, 81.0,
65.8, 56.3, 32.4, 24.5, 19.9. HRMS (ESI): m/z calcd for C16H19N4O3
[M + H]+, 315.1457; found, 315.1467.
(E)-5-((Benzyloxy)methyl)-3-(3-isobutyl-5-methylhexylidene)-5-
((5-phenyl-2H-tetrazol-2-yl)methyl)dihydrofuran-2(3H)-one (29). A
solution of triphenylphosphine (226 mg, 0.85 mmol), lactone 21 (110
mg, 0.29 mmol), and 5-phenyl-1H-tetrazole (124 mg, 0.85 mmol) in
anhydrous THF (6 mL) was treated with DIAD (168 μL, 0.85 mmol)
and stirred at room temperature overnight. The reaction mixture was
evaporated, and the residue was purified by flash chromatography using
hexane/ethyl acetate (92:8) as an eluent to afford 29 as a white solid
(78 mg, 53%). Mp: 85−86 °C.1H NMR (400 MHz, CDCl3): δ 8.14−
8.09 (m, 2H), 7.50−7.45 (m, 3H), 7.38−7.28 (m, 5H), 6.68−6.61 (m,
1H), 5.03 (d, J = 14.1 Hz, 1H), 4.93 (d, J = 14.1 Hz, 1H), 4.66 (d, J =
11.9 Hz, 1H), 4.60 (d, J = 11.9 Hz, 1H), 3.68 (d, J = 10.1 Hz, 1H), 3.59
(d, J = 10.1 Hz, 1H), 2.93 (d, J = 17.0 Hz, 1H), 2.76 (d, J = 17.0 Hz,
1H),1.99 (t, J = 6.2 Hz, 2H), 1.66−1.58 (m, 1H), 1.58−1.47 (m, 2H),
1.08−0.91 (m, 4H), 0.82−0.78 (m, 12H). 13C NMR (101 MHz,
CDCl3): δ 168.8, 165.4, 141.4, 137.0, 130.5, 128.9, 128.5, 128.0, 127.8,
126.9, 125.3, 81.5, 73.8, 72.4, 56.6, 43.7, 43.7, 34.7, 32.6, 31.2, 25.1,
22.9, 22.8, 22.6, 22.5. HRMS (ESI): m/z calcd for C31H41N4O3 [M +
H]+, 517.3179; found, 517.3182.
5-((Benzyloxy)methyl)-5-(hydroxymethyl)-3-(propan-2-ylidene)-
dihydrofuran-2(3H)-one (25). Compound 25 was synthesized
following a procedure described elsehwere.41
Starting from lactone 19 (3.51 g, 10.24 mmol) and acetone (1.78 g,
30.72 mmol) and following general procedure A, 24 (3.5 g, 90%) was
obtained as a white solid. Mp: 77−79 °C. Its spectrum data perfectly
matched with published results. 1H NMR (300 MHz, CDCl3): δ 7.37−
7.26 (m, 5H), 6.87 (s, 4H), 4.65 (AB q, J = 12.3 Hz, 2H), 4.11 (AB q, J =
9.6, 2H), 3.82 (s, 3H),3.71 (AB q, J = 10.2, 2H), 2.97−2.84 (AB m,
2H), 2.33 (t, J = 2.1 Hz, 2H), 1.92 (s, 3H).
Starting from 24 (890 mg, 2.32 mmol) and following general
procedure C, 25 was obtained as a colorless oil (586 mg, 92%). Its
spectrum data perfectly matched with published results.1H NMR (400
MHz, CDCl3): δ 7.40−7.26 (m, 5H), 4.56 (AB q, J = 12.1, 2H), 3.70
(AB dq, J = 6.6, 12.1, 34.5 Hz, 2H), 3.55 (AB q, J = 9.9, 27.6 Hz, 2H),
2.82−2.67 (m, 2H), 2.25 (m, 4H), 1.86 (s, 3H).
5-((Benzyloxy)methyl)-5-(iodomethyl)-3-(propan-2-ylidene)-
dihydrofuran-2(3H)-one (26). A solution of 25 (200 mg, 0.73 mmol)
in toluene (13 mL) was treated with triphenylphosphine (380 mg, 1.45
mmol), imidazole (148 mg, 2.18 mmol), and iodine (277 mg, 1.1
mmol). The mixture was heated at 90 °C for 2 h and, after reaching
room temperature, was poured into a saturated solution of NaHCO3.
Excess triphenylphosphine was destroyed by the addition of iodine until
the iodine coloration persisted in the organic layer. The organic layer
was washed with Na2S2O3 (10%, 5 mL) and brine, dried over (MgSO4),
filtered, and concentrated in vacuo. The residue was purified on silica gel
(hexanes/ethyl acetate, 9:1) to give 26 (266 mg, 95%) as a colorless oil.
1H NMR (400 MHz, CDCl3): δ 7.38−7.27 (m, 5H), 4.64−4.53 (AB m,
(E)-5-(Hydroxymethyl)-3-(3-isobutyl-5-methylhexylidene)-5-((5-
phenyl-2H-tetrazol-2-yl)methyl)dihydrofuran-2(3H)-one (6). Start-
ing from 29 (50 mg, 0.097 mmol) and following general procedure B, 6
was obtained as a white solid (32 mg, 78%). Mp: 110−112 °C.1H NMR
(400 MHz, CDCl3): δ 8.20−8.15 (m, 2H), 7.51−7.49 (m, 3H), 6.70
(m, 1H), 5.10−4.94 (AB m, 2H), 3.79−3.76 (m, 2H), 2.94 (d, J = 17.2
Hz, 1H), 2.73 (d, J = 17.2 Hz, 1H), 2.57 (t, J = 6.4 Hz, 1H), 2.03 (t, J =
6.3 Hz, 2H), 1.67−1.60 (m, 1H), 1.60−1.48 (m, 2H), 1.11−0.93 (m,
4H), 0.82 (d, J = 6.5 Hz, 12H). 13C NMR (101 MHz, CDCl3): δ 168.8,
165.6, 142.2, 130.6, 128.9, 126.9, 126.8, 125.3, 82.5, 65.7, 56.0, 43.8,
43.8, 34.8, 32.7, 30.4, 25.1, 22.9, 22.8, 22.6, 22.5. HRMS (ESI): m/z
calcd for C24H35N4O3[M + H]+, 427.2709; found, 427.2733.
Determination of In Vitro Binding Affinities for PKC
Isozymes. Recombinant human PKCα and PKCε isozymes were
obtained from ThermoFisher Scientific (Waltham, MA). Compounds
were assayed in vitro by competition for the binding of [3H]PDBu (9
Ci/mmol; prepared as a custom synthesis by Quotient Bioresearch,
Cardiff, United Kingdom). Porcine brain L-α-phosphatidylserine (PS)
K
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was from Avanti Polar Lipids (Alabaster, AL). Immunoglobulin G was
purchased from Sigma-Aldrich (St. Louis, MO). Nonradioactive
phorbol 12,13-dibutyrate (PDBu) was purchased from LC Labo-
ratories (Woburn, MA). Polyethylene glycol (PEG) 6000 was obtained
from EMD Millipore Corporation (Billerica, MA). Briefly, the assay
mixture had a final volume of 250 μL and contained the following
components: 50 mM Tris-HCl, pH 7.4; 100 μg/mL L-α-phosphati-
dylserine; 0.1 mM Ca2+ for PKCα assay and 1 mM EGTA for PKCε
assay; 5 mg/mL bovine immunoglobulin G and 0.003% Tx-100, 10 μM
of nonradioactive phorbol 12,13-dibutyrate (PDBu), 2 nM of
[3H]PDBu and increasing concentrations of the competing ligand.
The assay tubes were incubated at 37 °C for 5 min and then chilled on
ice for 10 min, after which 200 μL of 35% polyethylene glycol (PEG)
was added. The tubes were vortexed and chilled for an additional 10
min. The tubes were then centrifuged at 12,200 rpm for 15 min at 4 °C.
A 200 μL aliquot of each supernatant was transferred to a scintillation
vial for the determination of the free concentration of [3H]PDBu. The
assay pellets were dried, cut off, and placed in a separate scintillation vial
for the determination of bound [3H]PDBu. The supernatant and pellet
vials were immersed in 3 mL of scintillation cocktail (CytoScintEco-
Safe, MP Biomedicals, Santa Ana, CA). In each experiment, triplicate
measurements at each concentration of ligand were performed. MS
Excel was used to process data, and graphing was done using GraphPad
Prism software (GraphPad Software, Inc., San Diego, CA). Final
concentrations for both PKC isozymes assays (fixed): 10 μM cold
PDBu (nonspecific), 2 nM [3H]PDBu, 20 ng/tube enzyme, 100 μg/mL
L-α-phosphatidylserine (porcine brain). In each experiment, at least
triplicate measurements at each concentration of ligand were
performed. Ki values were calculated from ID50 values determined
from the competition curves. MS Excel was used to process data, and
graphing was done using GraphPad Prism software (GraphPad
Software, Inc., San Diego, CA).
Translocation of GFP-Fused PKCs. Experiments were carried out
essentially as previously described.17 Briefly, HeLa cells (5 × 104) were
transfected with 1 μg of either pEGFP-N1-PKCα or pEGFP-N1-PKCε
using Lipofectamine 3000 and plated on cover slides in 24-well plates.
After 24 h, cells were serum-starved for 24 h and then stimulated with
the different DAG-lactones or PMA for 30 min. Cells were then fixed in
methanol, mounted on a glass slide, and visualized with a Nikon
TE2000-U fluorescence microscope. Cells were stained with DAPI for
nucleus visualization. Translocation was quantitated using ImageJ. A
line was traced across the cytoplasm of individual cells (∼10−15 cells/
group), and the signal intensities and profiles were obtained using the
Plot profile tool of the program. The operator was blinded to treatment
assignment.
Western Blotting. A549 cells were harvested in lysis buffer
containing 50 mM Tris-HCl, pH 6.8, 10% glycerol, and 2% β-
mercaptoethanol, and lysates were subjected to SDS-PAGE. After
transferring to polyvinylidene difluoride membranes (Millipore Corp.,
Burlington, MA) and blockade for 1 h with 5% milk in TBS and 0.1%
Tween 20, membranes were incubated overnight with either anti-PKCε
(1:1000 dilution, Cell Signaling Technology, Danvers, MA) or β-actin
(1:500 000, Sigma-Aldrich, St. Louis, MO) antibodies. After extensive
washing, membranes were incubated for 1 h with either antimouse
(1:1000 dilution) or antirabbit (1:3000 dilution) secondary antibodies
conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA). Bands
were visualized and subjected to densitometric analysis using an
Odyssey Fc system (LI-COR Biosciences, Lincoln, NE).
Quantitative PCR (Q-PCR). Total RNA from cultured cells was
extracted using the RNeasy kit as directed by the manufacturer (Qiagen,
Valencia, CA). One μg of mRNA template was added to the reverse-
transcription master mix (Taq-Man Reverse Transcription kit, Applied
Biosystems). The cDNA samples were then diluted with 90 μL of
RNase-free water and stored at −20 °C. Q-PCR primers for PKCε and
UBC (housekeeping gene for normalization) were purchased from
Applied Biosystems. PCR amplifications were performed using an ABI
PRISM 7300 Detection System in a total volume of 20 μL containing
Taqman Universal PCR Master Mix (Applied Biosystems). PCR
product formation was continuously monitored using the Sequence
Detection System software version 1.7 (Applied Biosystems).
Phalloidin Staining for Analysis of Cell Ruffle Formation.
A549 lung cancer cells growing on glass coverslides at low confluence
were serum-starved for 24 h and stimulated with the different
compounds at the concentrations indicated. Following fixation with
4% formaldehyde, F-actin was stained with phalloidin-rhodamine, and
nuclei were counterstained with DAPI. Slides were visualized by
fluorescence microscopy, and five random fields were scored for ruffle
formation. Ruffle area was measured by thresholding for signal intensity
using ImageJ.
RNAi Silencing. A549 cells were transfected with validated siRNA
duplexes for PKCε or a nontarget control (NTC) siRNA duplex
(Dharmacon) using Lipofectamine RNAiMAX (Invitrogen), as
previously described.17 siRNAi’s for PKCε were as follows: J-
004653−06 (ε1) and J-004653−08 (ε2). Experiments were carried
out 48 h after transfection.
Statistical Analysis. We used GraphPad Prism software built-in
analysis tools for ANOVA. A p value <0.05 was considered statistically
significant.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00739.
■
sı
Data for 1H, 13C NMR spectra and HPLC traces for the
title compounds (PDF)
Molecular formula strings (CSV)
Structure representation of isoxazole_sn1 (PDB)
Structure representation of isoxazole_sn2 (PDB)
Structure representation of tetrazole_sn1 (PDB)
Structure representation of triazole_sn1 (PDB)
AUTHOR INFORMATION
Corresponding Authors
■
Marcelo G. Kazanietz − Department of Systems Pharmacology
and Translational Therapeutics, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104,
United States; Phone: +1 215-898-0253; Email: marcelog@
pennmedicine.upenn.edu
María J. Comin − Departamento de Ingredientes Activos y
Biorrefinerías, Instituto Nacional de Tecnología Industrial, San
Martín, Buenos Aires B1650WAB, Argentina; Consejo
Nacional de Investigaciones Científicas y Técnicas
(CONICET), San Martín, Buenos Aires B1650WAB,
Argentina; orcid.org/0000-0003-2187-1932;
Phone: +54-911-44307859; Email: jcomin@inti.gob.ar
Authors
Eleonora Elhalem − Departamento de Ingredientes Activos y
Biorrefinerías, Instituto Nacional de Tecnología Industrial, San
Martín, Buenos Aires B1650WAB, Argentina; Consejo
Nacional de Investigaciones Científicas y Técnicas
(CONICET), San Martín, Buenos Aires B1650WAB,
Argentina
Ana Bellomo − Departamento de Ingredientes Activos y
Biorrefinerías, Instituto Nacional de Tecnología Industrial, San
Martín, Buenos Aires B1650WAB, Argentina; Consejo
Nacional de Investigaciones Científicas y Técnicas
(CONICET), San Martín, Buenos Aires B1650WAB,
Argentina
Mariana Cooke − Department of Systems Pharmacology and
Translational Therapeutics, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, Pennsylvania 19104,
United States; Department of Medicine, Einstein Medical
L
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Article
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Center Philadelphia, Philadelphia, Pennsylvania 19141,
United States
Antonella Scravaglieri − Departamento de Ingredientes Activos
y Biorrefinerías, Instituto Nacional de Tecnología Industrial,
San Martín, Buenos Aires B1650WAB, Argentina
Larry V. Pearce − Laboratory of Cancer Biology and Genetics,
Center for Cancer Research, National Cancer Institute, NIH,
Bethesda, Maryland 20892-4255, United States
Megan L. Peach − Basic Science Program, Chemical Biology
Laboratory, Frederick National Laboratory for Cancer
Research, National Institutes of Health, Frederick, Maryland
21702, United States
Lucía Gandolfi Donadío − Departamento de Ingredientes
Activos y Biorrefinerías, Instituto Nacional de Tecnología
Industrial, San Martín, Buenos Aires B1650WAB, Argentina;
Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), San Martín, Buenos Aires B1650WAB,
Argentina
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Complete contact information is available at:
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(DAG) and DAG-Lactones as Activators of Protein Kinase C (PK-C).
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R. J.; Blumberg, P. M.; Marquez, V. E. Conformationally Constrained
Analogues of Diacylglycerol. 29. Cells Sort Diacylglycerol-Lactone
Chemical Zip Codes to Produce Diverse and Selective Biological
Activities. J. Med. Chem. 2008, 51 (17), 5198−5220.
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S.; Lewin, N. E.; Bhattacharyya, D. K.; Blumberg, P. M.; Marquez, V. E.
Conformationally Constrained Analogues of Diacylglycerol (DAG).
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was partly supported by FONCyT-ANPCyT (PICT-
2015-0362 to M.J.C.), CONICET (PIP2014-2016 GI to
M.J.C.), the National Institute of Industrial Technology
(INTI) (to E.E. and M.J.C.), as well as by National Institutes
of Health grants ES026023, CA189765, and CA196232 (to
M.G.K.). This work was also supported, in part, with Federal
funds from the Frederick National Laboratory for Cancer
Research, National Institutes of Health, under contract no.
HHSN261200800001E. The content of this publication does
not necessarily reflect the views or policies of the Department of
Health and Human Services, nor does mention of trade names,
commercial products, or organizations imply endorsement by
the US Government. The authors are especially grateful to Dr.
Victor E. Marquez and Dr. Peter M. Blumberg for fruitful
discussions and support throw-out the project.
(14) Zhang, G.; Kazanietz, M. G.; Blumberg, P. M.; Hurley, J. H.
Crystal Structure of the Cys2 Activator-Binding Domain of Protein
Kinase C Delta in Complex with Phorbol Ester. Cell 1995, 81 (6), 917−
924.
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Marquez, V. E. A Novel Diacylglycerol-Lactone Shows Marked
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ABBREVIATIONS USED
■
CAN, ceric ammonium nitrate; DAG, diacylglycerol; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DIAD, diisopropyl azodicar-
boxylate; DMAP, 4-dimethylaminopyridine; DMF, dimethyl-
formamide; GEFs, guanine nucleotide exchange factors;
LiHMDS, lithium hexamethyldisilazide; mp, melting point;
MRCKs, myotonic dystrophy kinase-related Cdc42-binding
kinases (MRCKs); PDBu, [20−3H]phorbol 12,13-dibutyrate;
PKC, protein kinase C; PKD, protein kinase D; PMP, p-
methoxyphenyl; pyr, pyridine; RasGRP, Ras guanine nucleo-
tide-releasing protein; TBAF, tetra-n-butylammonium fluoride;
TBDPS, tert-butyldiphenylsilyl; t-BuOH, tert-butylalcohol;
THF, tetrahydrofuran
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