Conformationally constrained analogues of diacylglycerol (DAG). 31. Modulation of the biological properties of diacylgycerol lactones (DAG-lactones) containing rigid-rod acyl groups separated from the core lactone by spacer units of different lengths

Article abstract of DOI:10.1021/jm900186m

Diacylglycerol lactones built with a rigid 4-[(methylphenyl)ethynyl]phenyl rod that is separated from the exocyclic acylcarbonyl of the DAG-lactone core by a spacer unit of variable length were synthesized and studied. Binding affinities for a panel of cl

Full text of DOI:10.1021/jm900186m

3274
J. Med. Chem. 2009, 52, 3274–3283
Conformationally Constrained Analogues of Diacylglycerol (DAG). 31. Modulation of the
Biological Properties of Diacylgycerol Lactones (DAG-lactones) Containing Rigid-Rod Acyl
Groups Separated from the Core Lactone by Spacer Units of Different Lengths
Maria J. Comin,† Gabriella Czifra,‡ Noemi Kedei,‡ Andrea Telek,‡ Nancy E. Lewin,‡ Sofiya Kolusheva,§ Julia F. Velasquez,‡
Ryan Kobylarz,‡ Raz Jelinek,§ Peter M. Blumberg,‡ and Victor E. Marquez*,†
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer InstitutesFrederick, National Institutes of Health, 376
Boyles Street, Frederick, Maryland 21702, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, and Department of Chemistry and Ilse Katz Institute for Nanotechnology, Ben Gurion
UniVersity, Beer SheVa 84105, Israel
ReceiVed February 13, 2009
Diacylglycerol lactones built with a rigid 4-[(methylphenyl)ethynyl]phenyl rod that is separated from the
exocyclic acylcarbonyl of the DAG-lactone core by a spacer unit of variable length were synthesized and
studied. Binding affinities for a panel of classical and novel PKC isozymes in two different phospholipid
environments, one corresponding to the plasma membrane of cells, were determined. The kinetics and site
of translocation for the PKC isozymes R and δ upon treatment with the compounds were also studied as
well as the early response of ERK phosphorylation and the late response of induction of apoptosis in the
human prostatic carcinoma cell line LNCaP. Finally, the compounds were evaluated in terms of their
interaction with biomimetic lipid/polydiacetylene membranes by the associated chromatic response. The
different spatial disposition of the rigid structural motif on the DAG-lactones contributes to differential
activity.
Introduction
were investigated for their ability to bind and translocate PKC
R and δ isoforms to plasma and internal membranes. Some of
the DAG-lactones were able to induce a substantially prolonged
translocation state relative to the parent DAG-lactone containing
an equivalent flexible acyl chain.6 More recently, we studied
the thermodynamic features of some new DAG-lactones with
specific small groups attached to the terminus of a rigid
4-(phenylethynyl)benzoyl chain which affected both the self-
assembly of the molecules and their interactions with phospho-
lipids.7 The results suggested that these DAG-lactones are
predominantly incorporated within fluid phospholipid phases
rather than in the condensed phases formed by cholesterol and
that the size and charge of the phospholipid headgroups did
not seem to affect the DAG-lactone interactions with lipids.7
Occupancy of a wide range of G-protein-coupled receptors
and receptor tyrosine kinases triggers the hydrolysis of phos-
phatidylinositol 4,5-bisphosphate (PIP2 a), leading to the release
of membrane-bound diacylglycerol (DAG).1 DAG in turn
interacts with the DAG recognition domains, termed C1
domains, on multiple families of signaling proteins, including
isoforms of protein kinase C, the chimerins, RasGRP, PKD,
MRCK, Unc13, and DAG kinase.2 The activation of conven-
tional (cPKCs R, ꢀI, ꢀII, γ) and novel (nPKCs δ, ε, η, θ) protein
kinase C isoforms has been studied in great detail and involves
both recruitment to membranes and allosteric activation by
DAG.3 Specific molecular interactions of DAG with the lipid
environment into which it and the C1 domains insert influence
both processes. The physical properties of the membranes and
the interplay between the membranes and the alkyl chains of
the DAG are thus of great importance. The combination of these
interactions controls the localization of an isozyme to specific
intracellular compartments and correspondingly controls which
substrates are accessible.4
Because different extents of membrane penetration and
preference for membrane subdomains might yield different
patterns of response in the ability of DAG-lactones to interact
or translocate different GFP-tagged isozymes, we designed a
series of DAG-lactones with a rigid 4-[(methylphenyl)ethy-
nyl]phenyl rod (red) that is separated from the acylcarbonyl of
the DAG-lactone core [2-(hydroxymethyl)-4-(methylethylidene)-
The effective use of a DAG-lactone template as a more potent
DAG surrogate has been well documented in our laboratory.5
In a recent study, we extended this concept to include a series
of DAG-lactones containing rigid rods composed of ethynylene-
substituted aromatic spacers [oligo-(p-phenyleneethynylene)] as
acyl groups.6 These compounds were designed to achieve
various levels of membrane organization and penetration and
5-oxo-2-2,3-dihydrofuryl]methyloxocarbonyl] (blue) by
a
-O(CH2)n- spacer of 1, 2, or 3 methylene units (1a-c, Figure
1). We compared these three new targets with compound 2
without a spacer.
We evaluated the compounds in both in vitro and in cellular
systems: (1) We determined their binding affinities for a panel
of classical and novel PKC isozymes, assayed in two different
phospholipid environments, either 100% phosphatidylserine or
a lipid mixture corresponding to that of the plasma membrane
of cells; (2) we compared the kinetics and site of translocation
for the PKC isozymes R and δ upon treatment with the
compounds; (3) we measured the potencies of the compounds
for the early response of ERK phosphorylation and for the late
response of induction of apoptosis in the human prostatic
* To whom correspondence should be addressed. Phone: 301-846-5954.
Fax: 301-846-6033. E-mail: marquezv@dc37a.nci.nih.gov.
†
National Cancer InstitutesFrederick.
National Cancer Institute.
Ben Gurion University.
‡
§
a Abbreviations: PKC, protein kinase C; DAG, diacylglycerol; PKD,
protein kinase D; RasGRP, Ras guanyl releasing protein; MRCK, mytonic
dystrophy kinase-related Cdc42-binding kinase; Unc13, uncoordination gene
13 product; PIP2, phosphatidylinositol 4,5-bisphosphate.
10.1021/jm900186m CCC: $40.75
2009 American Chemical Society
Published on Web 04/20/2009

Properties of Diacylgycerol Lactones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3275
product of phenol to the triple bond, and cross-reaction between
the starting phenol and desired product 4b, successful separation
of 4b was achieved by simple column chromatography. The
synthesis of the 3-methylene unit derivative was first attempted
by reaction between 3 and γ-butyrolactone, but again, this
method failed when applied to our system.12 Starting from
4-bromobutanoic acid, the corresponding tert-butyl 4-bromobu-
tanoate13 was obtained in quantitative yield, and following
nucleophilic displacement with 3 resulted in the desired 3-me-
thylene unit, tert-butyl ester 4c, in 83% yield (Scheme 1).
At this point, hydrolysis of tert-butyl esters was necessary
to perform the acylation of the DAG-lactone moiety. Probably
because of the presence of the triple bond, acidic hydrolysis of
tert-butyl ester produced complex mixtures, even when trieth-
ylsilane was added as a carbocation scavenger.14 However,
thermolysis in quinoline at 205 °C 15 led cleanly to the desired
free acids in quantitative yield which were isolated as crystalline
solids (5a-c, Scheme 2). The use of bis(2-oxo-3-oxazolidi-
nyl)phosphinic acid chloride (BOPCl) and dimethylaminopy-
ridine (DMAP)16 allowed the one-pot coupling of the acids with
the free hydroxyl group of the monoprotected O-PMP-DAG-
lactone 66 (for n ) 1 and 3) and O-benzyl-DAG-lactone 76 (for
n ) 2) in very good yield. Removal of the p-methoxyphenyl
(PMP) protecting group in the presence of ceric ammonium
nitrate (CAN) to give the target compounds 1a and 1c resulted
in low yields (54% and 50%) due to the presence of the
additional phenoxy ether linkage in 8a and 8c. A better strategy
was the coupling of the O-benzyl protected DAG-lactone 7 that
led to the desired DAG-lactone 1b in a markedly better yield
(80%) after treatment with boron trichloride at low temperature
(Scheme 2).
Figure 1. (A) ω-(4-[2-(Methylphenyl)ethynyl]phenoxy)alkanoyl DAG-
lactones 1a-c. The rigid rod moiety is indicated in red, the DAG-
lactone in blue, and the variable linker in black. (B) [2-(Hydroxymethyl)-
4-(methylethylidene)-5-oxo-2-2,3-dihydrofuryl]methyl, 4-[2-(4-
methylphenyl)ethynyl]benzoate (compound 2).
Scheme 1. Synthesis of tert-Butyl Esters
Biological Activity. Binding Affinity for PKC Isozymes.
Enzyme-ligand interactions were assessed in vitro in terms of
the ability of the ligand to displace bound [20-3H]phorbol 12,13-
dibutyrate (PDBu) from the recombinant human PKC isozymes
in the presence of phosphatidylserine.17 The partition coefficients
(log P) were calculated according to the atom-based program
MOE SlogP18 (Table 1).
Each of the PKC isozymes showed a similar pattern of
dependence on the length of the spacer for compounds 1a-c
(Table 1). In each case, the affinities increased (lower Ki) with
the distance of the rigid rod from the DAG-lactone core, with
compound 1c (n ) 3) being the most potent. In sharp contrast,
however, the most potent of all ligands (2) lacks a spacer
between these two units, suggesting a qualitatively different
interaction between it, the enzymes, and the lipid environment.
In terms of isozyme specificity, PKCε stood out from the other
isoforms in showing a 5- to 4-fold lower affinity for compounds
1a and 1b with the shorter spacers. The other isozymes
responded within similar ranges of concentration for each
individual compound.
The above assays were carried out under standard conditions
in which the PKC isoforms were assayed in the presence of
100 µg/mL phosphatidylserine, which provides a highly anionic
surface. These conditions maximize the interaction of the PKC
isoforms with the phospholipid bilayer. To detect differences
in interactions that might depend on a more physiological lipid
composition, we also assayed the isoforms in the presence of
100 µg/mL of a mixture of phosphatidylcholine/phosphatidyle-
thanolamine/phosphatidylserine/phosphatidylinositol/choles-
terol (12:35:22:9:21), a plasma membrane lipid (PML) mixture
designed to mimic that of the inner leaflet of the plasma
membrane.19 We observed (Table 2, Figure 2) that the rigid
rod compounds were generally several-fold less potent for the
carcinoma cell line LNCaP; and (4) we evaluated the interaction
of the compounds with a biomimetic lipid/polydiacetylene
membrane by the associated chromatic response.
Results
Synthesis. Compound 2 was synthesized according to our
previously published approach.7 The preparation of racemic
ω-(4-[2-(methylphenyl)ethynyl]phenoxy)alkanoyl monoesters of
DAG-lactones (Figure 1) with one to three methylene spacers
between the lactone and the rigid chain was achieved as depicted
in Schemes 1 and 2. The rigid-rod component, 4-[2-(4-
methylphenyl)ethynyl]phenol (3),8 was prepared in excellent
yield by a modified Sonogashira coupling between 4-iodophenol
and 4-ethynyltoluene.9 This phenol derivative was used as a
building block for the preparation of all tert-butyl esters shown
in Scheme 1. Thus, 4a (n ) 1) was obtained straightforwardly
by treatment of 3 and tert-butyl 2-bromoacetate with cesium
carbonate in excellent yield (92%).10 Unfortunately, because
of ꢀ-elimination, this was not the case when tert-butyl 3-bro-
mopropanoate was used. Although the successful alkylation of
phenols with ꢀ-propiolactone as the alkylating agent has been
reported,10 our reaction of 3 with ꢀ-propiolactone in the presence
of t-BuOK or CsCO3 led to mixtures of products. On the other
hand, Michael addition of 3 to tert-butyl acrylate in the presence
of catalytic amounts of sodium and hydroquinone11 gave the
desired tert-butyl ester 4b (n ) 2) in sufficient quantities to
continue the synthesis. Despite the fact that several byproducts
were obtained, including starting material 3, the self-addition

3276 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Scheme 2. Synthesis of DAG-lactones
Comin et al.
Table 1. Binding Affinities (Ki, nM) for PKC Isozymes (Average of Three Determinations ( SEM)
isozyme
1a (n ) 1), log P ) 3.31
1b (n ) 2), log P ) 3.72
1c (n ) 3), log P ) 4.11
2, log P ) 3.56
PKCR
PKCꢀ
PKCγ
PKCδ
PKCε
38 ( 14
76 ( 15
50.8 ( 6.0
46.4 ( 3.6
268 ( 72
20.8 ( 5.2
57 ( 13
35.0 ( 4.1
35.8 ( 6.1
153 ( 12
19.5 ( 0.74
15.2 ( 3.1
12.3 ( 1.2
11.9 ( 0.4
37.9 ( 4.4
6.0 ( 1.5
4.1 ( 1.1
6.2 ( 1.6
5.4 ( 0.8
10.0 ( 1.2
Table 2. Binding Affinities (Ki, nM) for PKC Isozymes (Average of Three Determinations ( SEM) Assayed in the Presence of Plasma Membrane
Lipid Mimetic Mixture
isozyme
1a (n ) 1), log P ) 3.31
1b (n ) 2), log P ) 3.72
1c (n ) 3), log P ) 4.11
2, log P ) 3.56
PKCR
PKCꢀ
PKCγ
PKCδ
PKCε
165.1 ( 2.8
100.3 ( 6.9
139.6 ( 3.7
47.7 ( 6.1
147 ( 16
143 ( 37
66 ( 21
78.0 ( 4.5
31.3 ( 0.82
78.7 ( 9.1
76 ( 15
43.8 ( 4.7
31.7 ( 2.8
11.6 ( 2.3
31.9 ( 1.3
15.8 ( 2.7
12.6 ( 0.72
15.7 ( 2.1
2.74 ( 0.43
8.3 ( 1.5
classic PKC isoforms in the plasma membrane mimetic mem-
branes as compared to the 100 µg/mL phosphatidylserine,
whereas for the novel PKC isoforms they were equal or more
potent.
ERK Phosphorylation in LNCaP Cells. Activation of the
ERK pathway, as reflected by ERK phosphorylation, is a
prominent early biological response to PKC stimulation by
DAG-lactones and phorbol ester.6 Because of the rapidity of
the response, phosphorylation of ERK is relatively independent
of issues of compound stability, so it provides a convenient
measure of overall biological potency. All of the compounds
caused phosphorylation of ERK in the concentration range of
1-10 µM (Figure 3). Similar to the potencies in the binding
assays, increasing potency was observed as the length of the
spacer was increased, with compounds 1b and 1c being the most
potent. Likewise, compound 2, which lacked any spacer, was
the most potent of all four rigid rod compounds examined.
Apoptotic Activity. In the human prostatic carcinoma cell
line LNCaP, apoptosis is induced both by phorbol esters and
by DAG-lactones.20 This response, which we measure at 2 days
after treatment, lies downstream of several PKC isoforms, in
particular PKCR and PKCδ, and will be influenced by the
stability of the compounds as well as by their potencies and
selectivities. As seen in Figure 4, all the compounds induced
apoptosis, with an apparent preference for the longer spacers
of 1b and 1c. It is noteworthy that compound 1c induced a
Figure 2. Effect of the lipid environment on the potencies of the rigid
rod compounds for PKC isoforms. Compounds were assayed with the
various PKC isoforms in the presence of either phosphatidylserine (PS)
or a lipid mixture corresponding to the composition of the inner leaflet
of the plasma membrane, as described in the methods section. Values
represent the ratio of the Ki value with the plasma membrane lipid
(PML) mimetic mixture to that with phosphatidylserine, where the Ki
values are the average of three independent experiments. Error bars
represent (SEM.

Properties of Diacylgycerol Lactones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3277
of response, and may uncover differences in the distribution of
an isoform between various membrane compartments.21 We
examined the pattern of response of PKCR (Figure 5) and PKCδ
(Figure 6) as representatives of the classic and novel PKC
isoforms, using green fluorescent protein fusion constructs
heterologously expressed in Chinese hamster ovary cells. Results
are summarized in Table 3.
For all derivatives, PKCR translocated to the plasma mem-
brane, which is the typical response for this isoform. Compound
2 was the fastest acting compound on PKCR. As the length of
the linker increased in the series 1a-c the response became
more rapid, although the response to 1c remained slower than
that to 2. PKCδ shows a more variable pattern of response to
various ligands, with PMA inducing initial translocation to the
plasma membrane followed by redistribution to internal mem-
branes and hydrophilic derivatives inducing the initial translo-
cation to the internal membranes (not shown). Compound 2
translocated PKCδ mainly to the plasma membrane and nuclear
membrane, but it was not PMA-like for PKCδ; there was no
sequential translocation to the plasma membrane followed by
a shift to nuclear membrane. Compounds 1a-c translocated
PKCδ mainly to the internal membranes, but it was noteworthy
that localization was predominantly perinuclear rather than to
the nuclear membrane per se.
Interactions of DAG-lactones with Artificial Mem-
branes. Interactions of DAG-lactones with membranes are
expected to cause perturbations that affect the structural and
dynamic properties of the phospholipid bilayers. Visualization
of these interactions is possible with the use of membranes that
containpolydiacetylene(PDA)patchesthatareeasilyperturbed.22,23
The PDA-based vesicle assay reports on the occurrence of
membrane interactions through color and fluorescence trans-
formations of the vesicle-embedded PDA patches induced
through interactions of membrane-associated molecules with the
vesicle bilayers.24
Figure 7 depicts the dose-dependent fluorescence chromatic
response (% FCR) curves induced by the DAG-lactones fol-
lowing addition to biomimetic dimyristoylphosphatidylcholine
(DMPC)/PDA vesicles. All four compounds gave rise to
increasing fluorescence signals upon higher concentrations,
indicating that the molecules exhibited membrane interactions.
However, differences were apparent among the compounds with
regards to the extent of induced chromatic response (intensity
of fluorescence signal) and particularly the shape of the
dose-response curves. Specifically, Figure 7 shows that 1a, 1b,
and 1c exhibited a relatively steep initial increase in % FCR
with a subsequent moderate rise of the dose-response curve
[or a slight decrease in % FCR in the case of 1a (Figure 7,
curve i), most likely due to severe precipitation]. Compound 2,
on the other hand, induced a very low chromatic response up
to approximately 2 mM, followed by a pronounced increase in
fluorescence emission, which subsequently exceeded the % FCS
values induced by the three other molecules (Figure 7, iv).
Figure 3. ERK phosphorylation induced by compounds 1a-c and 2
in LNCaP cells. Cells were treated with the indicated compounds at
the indicated nanomolar concentrations or with PMA (100 nM) for 30
min, and then ERK phosphorylation was determined by Western
blotting. (A) Representative Western blot showing the phosphorylated
p42/44 bands indicated by arrows (two additional independent experi-
ments gave similar results). Control blots (data not shown) confirmed
that the amounts of ERK did not change as a function of the ligand
concentration. (B) Intensities of the bands of phosphorylated ERK were
determined by densitometry. Bars represent the mean values from the
three independent experiments. Error bars indicate the SEM.
Previous studies have shown that the features of the chromatic
lipid/PDA dose-response curves are intimately affected by the
mechanisms of membrane binding and insertion.25 Accordingly,
the pronounced difference between the fluorescence curve
induced by 2 and the curves of 1a-c indicates that DAG-lactone
2 exhibits a distinct lipid binding profile. In particular, the
apparent parabolic shape of the chromatic response curve of 2
suggests that a self-assembly process precedes membrane
binding and that this self-association might be a precondition
for bilayer interactions.26,27 Compounds 1a-c, on the other
hand, most likely undergo a simple concentration-dependent
Figure 4. Dose-response of compounds 1a-c and 2 for induction of
apoptosis in LNCaP cells relative to 1000 nM PMA. Data are the mean
( SEM of three experiments (2 days of treatment).
higher level of apoptosis than did compound 2, contrasting with
the higher potency of 2 in the in vitro binding assays (Table 1).
Cellular Translocation of GFP-Tagged PKC Isoforms
in CHO Cells. The above biological assays do not distinguish
between PKC isoforms. Translocation of individual PKC
isoforms from cytosol to membrane compartments provides a
measure of their activation, can reveal differences in the kinetics

3278 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Comin et al.
Figure 5. Translocation of GFP-PKCR in CHO cells in response to addition of DAG-lactones (10 µM). Cells expressing the GFP-PKCR were
treated with 10 µM of the compounds. The redistribution of the fluorescent proteins was monitored with a Zeiss MRC 1024 confocal microscope
as a function of time after the addition of the compounds. Images were captured every 30 s. Each panel represents a typical example from four
independent experiments.
bilayer binding, as reflected in the dose-response curves in
Figure 7 (i-iii).
ligands for PKC to induce different biological responses, by
the ability of bryostatin to antagonize many responses induced
by phorbol esters, and by the demonstration that one PKC
isoform may have effects antagonistic of another.28 Opportuni-
ties for antagonism are further enhanced by the additional classes
of C1 domain containing targets. For example, DAG kinases
will terminate DAG signaling, and the chimerins, by inhibiting
Rac, would be expected to be tumor suppressors.2 In contrast
to kinase inhibitors, a great opportunity for design of selectivity
for C1 domain ligands is provided by the nature of the ligand
interaction site, where not only the C1 domain but also the lipid
environment with which it is associated contributes to ligand
recognition.4 Thus, for example, we have described very
different selectivity for PMA relative to bryostatin between the
PKCR and PKCδ isoforms in mouse 3T3 cells and mouse
keratinocytes.33,34 Likewise, using several libraries of DAG-
lactones differing only in their patterns of side chains, modulat-
ing their lipid interactions, we have shown that different
compounds were selective for different biological end points.4
We describe here our initial characterization of a small series
of compounds in which we have manipulated the length and
Discussion
Protein kinase C has attracted great interest as a therapeutic
target for cancer and other conditions in consequence of its
central role in cellular signal transduction, mediating responses
to the second messenger DAG.28,29 Current PKC targeted drugs
in clinical trials reflect two complementary strategies. Enzas-
taurin, an orally active PKC inhibitor that is relatively selective
for PKCꢀ among the PKC isoforms, is directed at the catalytic
site.30 Like most kinase inhibitors, it faces the imposing
challenge of distinguishing among the 500 plus kinases of the
mammalian kinome, and it indeed shows off-target activity on
several other kinases. Bryostatin 131 and PEP005,32 on the other
hand, are natural products, which interact with the DAG-phorbol
ester responsive C1 domains shared by PKC, RasGRP, and the
chimerins.
Although the mechanistic rationale for the therapeutic activity
of C1 directed agents is less clear than for catalytic site directed
agents, interest is driven by the demonstrated ability of different

Properties of Diacylgycerol Lactones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3279
Figure 6. Translocation of GFP-PKCδ in CHO cells upon addition of DAG-lactones (10 µM). Cells expressing GFP-PKCδ were treated with 10
µM of the compounds as indicated. The redistribution of the fluorescent proteins was monitored with a Zeiss MRC 1024 confocal microscope as
a function of time after the addition of the drugs. Images were captured every 30 s. Each panel represents a typical example from three independent
experiments.
Table 3. Pattern of Translocation for PKCR and PKCδ in Response to
DAG-lactonesa
relative distribution
of GFP-PKCδ
kinetics of
translocation of
GFP-PKCR,
plasma
nuclear
internal
compd plasma membrane membrane membrane membranes
1a
1b
1c
2
slowest
++
+
++
+++
++
+
++
+++
+++
+++
++
intermediate
intermediate
fast
+
a Intensities of localization are represented by the number of “+” marks.
Results from three independent experiments are summarized.
Figure 7. Chromatic dose-response curves. Fluorescence chromatic
response (% FCR) induced by the DAG-lactones in DMPC/PDA
vesicles: (i) 1a; (ii) 1b; (iii) 1c; (iv) 2.
nature of the connection of the DAG-lactone template and a
rigid rod terminus on one side chain. We have previously
observed that incorporation of a rigid rod structure into the side
chain caused exclusion of the ligand from cholesterol rich
subdomains, suggesting that such rigid structural motifs might
contribute to differential recognition.7 Lipid rafts rich in
cholesterol are proposed to represent specialized areas for
signaling complexes in the plasma membrane,35 and plasma
membranes differ from those of internal membranes such as
the nuclear membrane in being overall richer in cholesterol.36
Other types of specialized lipid subdomains have also been
described.37 Here, we observed that the rigid rod compounds
were relatively less active on the classic PKC isoforms in
membranes containing cholesterol and relatively deficient in
phosphatidylserine compared to those constituted solely of
phosphatidylserine. We also observed that the potencies of the
compounds in vitro depended not only on the log P but also on
the particular nature of the linker, emphasizing the importance

3280 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Comin et al.
YO-PRO-1 (Invitrogen, Carlsbad, CA) was added to a final
concentration of 1 µM, and the cells were incubated for 20 min at
4 °C in the dark. 7-Aminoactinomycin D (7-AAD) (Invitrogen,
Carlsbad, CA) was then added at a 5 µg/mL final concentration 10
min before analysis by flow cytometry using the FACSCalibur
system (Becton Dickinson, Mountain View, CA). Data were
analyzed with FlowJo 7 (Tree Star, Inc. Ashland, OR). The Yo-
Pro-1 positive cells were considered apoptotic and were expressed
as % of total cells.
Translocation of GFP-Tagged PKC Isoforms in CHO
Cells. CHO cells were purchased from the American Type Culture
Collection (Manassas, VA) and cultured in F12-K medium supple-
mented with 10% fetal bovine serum and antibiotics (penicillin at
50 units/mL and streptomycin at 0.05 mg/mL). CHO cells plated
onto T delta dishes (Bioptechs Inc., Butler, PA) were transfected
with GFP-tagged PKCR or PKCδ using Lipofectamine reagent
(Invitrogen, Carlsbad, CA). Cells were visualized with a Zeiss LSM
510 confocal microscope (Carl Zeiss Inc., Thornwood, NY) with
an Axiovert 100M inverted microscope operating with a 25 mW
argon laser tuned to 488 nm. Cells were imaged with a 63 × 1.4
NA Zeiss Plan Apochromat oil immersion objective and with
varying zooms (1.4-2). Time-lapse images were collected every
30 s using the Zeiss AIM software, in which the green emission
was collected in a PMT with a LP 505 filter.
of the specific interactions with the lipid. At the biological level,
we noted differences in induction of the downstream response
of ERK phosphorylation by the various compounds, as we did
for apoptotic response and PKC isoform translocation.
The biomimetic lipid/PDA vesicle assay showed that DAG-
lactones exhibited significant membrane interactions; however,
there appears to be a pronounced difference between the
mechanism of membrane association between DAG-lactone 2
and the other three compounds with variable tethers. Specifi-
cally, the chromatic dose-response analysis in Figure 6 might
suggest that 2 undergoes a distinct self-assembly process
(possibly forming micelles or similar aggregates) prior to
membrane binding; the consequent aggregates are therefore the
likely entities exhibiting affinity to the membrane bilayer rather
than the individual molecules.
More extensive exploration of the influence of the positioning
of rigid rod substituents on the recognition of DAG lactones
and clarification of the linkage between mechanistic differences
and biological consequences should be of considerable interest.
Experimental Section
Analysis of Inhibition of [3H]PDBu Binding by Nonra-
dioactive Ligands. Enzyme-ligand interactions were analyzed by
competition with [3H]PDBu binding to a single isozyme essentially
as described previously.17 As indicated in the text, the PKC
isozymes were assayed in the presence of 100 µg/mL phosphati-
dylserine or else 100 µg/mL total lipid, where the lipid composition
was chosen to mimic that of the inner leaflet of the plasma
membrane. This latter composition was phosphatidylcholine/
phosphatidylethanolamine/phosphatidylserine/phosphatidylinositol/
cholesterol (12:35:22:9:21). The ID50 values were determined by
least-squares fitting of the theoretical sigmoidal competition curve
to the binding data. The Ki values were calculated from the ID50
values according to the relationship Ki ) ID50/(1 + L/Kd), where L
is the concentration of free [3H]PDBu at the ID50 and Kd is the
dissociation constant for [3H]PDBu under the assay conditions.
Values represent the mean ( standard error of the mean for three
independent experiments. The octanol/water partition coefficients
(log P) were calculated according to the atom-based program MOE
SlogP.18
Vesicle Preparation. Vesicle emulsions containing DMPC and
TRCDA (2:3 molar ratio) were prepared as previously described.25
Briefly, the lipid constituents were dried together in vacuo followed
by addition of deionized water and sonication at 70 °C. The vesicle
emulsion was then cooled to room temperature and kept at 4 °C
overnight; it was then polymerized by irradiation at 254 nm for
30 s. The resulting emulsion exhibits a blue appearance. Vesicle
samples for experiments were prepared at concentrations of 0.5
mM (total lipids) in 50 mM Tris-Cl, pH 8. The various compounds
at different concentrations were mixed with the vesicles prior to
addition of the Tris buffer. All compounds were dissolved in DMSO
(10 mg/mL).
Multiwell Fluorescence Spectroscopy. Samples were prepared
by adding different volumes of the compounds in DMSO solutions
to 30 µL of the dimyristoylphosphatidylcholine (DMPC) and
diacetylene 10,12-tricosadiynoic acid (TRCDA) vesicles, followed
by addition of 30 µL of 50 mM Tris base buffer (pH 8). Samples
were placed in Greiner 96-microwell plates. Fluorescence intensity
was measured using a multiwell fluorescence plate reader (Fluo-
roscan Ascent; Thermo, Finland) with a 485/555 nm excitation/
emission long path filter set. The fluorescence percentage was
calculated as percentage intensity compared to the fully transformed
red vesicles. All measurements were carried out at 27 °C.
Synthetic Methods. General. All chemical reagents were
commercially available. Melting points were taken on a Fisher-
Jones apparatus and are uncorrected. Combi-flash column chro-
matography was performed on silica gel 60 (230-240 mesh)
employing a Teledyne Isco instrument, and analytical TLC was
performed on Analtech Uniplates silica gel GF. Unless otherwise
indicated, NMR spectra were determined in CDCl3 (99.8%) with
residual CHCl3 as the reference peak (7.26 and 77.0 ppm) and were
recorded on a Varian 400 MHz spectrophotometer. The coupling
constants are reported in hertz, and the peak shifts are reported in
the δ (ppm) scale; abbreviations used are s (singlet), d (doublet),
dd (doublet-of-doublets), ddd (doublet-of-doublet-of-doublets), t
(triplet), q (quartet), and m (multiplet). Positive-ion fast-bombard-
ment mass spectra (FABMS) were obtained on a VG 7070E mass
spectrometer at an accelerating voltage of 6 kV and a resolution of
2000. Glycerol was used as the sample matrix, and ionization was
effected by a beam of xenon atoms. Combustion analyses were
performed for all intermediates and final products to confirm that
their level of purity was g95%; they were performed by Atlantic
Microlab. Inc., Norcross, GA. Infrared spectroscopy data were
obtained neat with a Jasco FT-IR/615 spectrometer. Specific optical
rotations were measured in a Perkin-Elmer model 241 polarimeter.
All reaction glassware was oven-dried and cooled to room tem-
perature in an argon atmosphere prior to use.
[3H]PDBu was from PerkinElmer (Waltham, MA). 1-Palmitoyl-
2-oleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidyle-
thanolamine, 1-palmitoyl-2-oleoylphosphatidylserine, and bovine
liver phosphatidylinositol were from Avanti Polar Lipids (Alabaster,
AL). Cholesterol was from Sigma (St. Louis, MO). The human
PKCR, PKCꢀ, PKCγ, PKC δ, and PKCε were from Invitrogen
(Carlsbad, CA).
ERK Phosphorylation in LNCaP Cells. Androgen-sensitive
LNCaP prostate cancer cells were purchased from the American
Type Culture Collection (Manassas, VA) and cultured in RPMI-
1640 medium supplemented with 10% fetal bovine serum and
antibiotics (penicillin at 50 units/mL and streptomycin at 0.05 mg/
mL). Cells were plated in 6 cm dishes at about 60% confluency
and 24 h later were treated with different concentrations of the test
compound and 100 nM PMA for 30 min. After treatment, the cells
were washed with phosphate buffered saline, total cell lysates were
prepared, and the lysates were analyzed by 10% SDS-PAGE
followed by electrotransfer and immunostaining. The primary
antibodies used for immunostaining against phospho-ERK (9101)
and total ERK (9102) were from Cell Signaling (Danvers, MA).
The immunostaining was visualized by ECL (Amersham Bio-
sciences). Films were scanned, and densitometry was performed
using Image J (developed at National Institute of Mental Health,
NIH).
Apoptosis in LNCaP Cells. LNCaP cells plated in 10 cm dishes
at about 60% confluency were treated with different concentrations
of the test compound and of 1 µM PMA for 48 h. After treatment,
the cells were washed and resuspended in phosphate buffered saline.

Properties of Diacylgycerol Lactones
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10 3281
4-[2-(4-Methylphenyl)ethynyl]phenol (3). 4-Ethynyltoluene (5.0
g, 43.04 mmol) was added to a solution of 4-iodophenol (9.2 g,
41.79 mmol), Pd(PPh3)2Cl2 (590 mg, 0.84 mmol), CuI (320 mg,
1.67 mmol), and Et3N (8.5 mL, 60.9 mmol) in THF (50 mL) at
room temperature under an argon atmosphere. The reaction mixture
was stirred overnight. The solids were separated by filtration, and
the volatiles were evaporated under reduced pressure. The residue
was purified by combi-flash column chromatography (silica gel;
hexanes/ethyl acetate, 4:1) to give 8.3 g (95% yield) of pure 3 as
a yellowish solid: mp 124 °C (lit.7 103-104 °C); FTIR (neat) 3307
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.42 (m, 4 H, Ph), 7.14 (dm,
J ) 7.8 Hz, 2 H, Ph), 6.80 (dm, J ) 8.8 Hz, 2 H, Ph), 4.80 (bs, 1
H, -OH), 2.36 (s, 3 H, -CH3); 13C NMR (100 MHz, CDCl3) δ
155.45, 138.07, 133.20, 133.14, 131.35, 131.29, 129.08, 120.37,
115.45, 88.49, 88.19, 21.43. FABMS m/z (relative intensity): 209
(MH+, 36), 208 (M+•, 100). Anal. Calcd for C14H10O·0.2H2O: C,
85.06; H, 5.30. Found: C, 84.98; H, 5.59.
20 mL). The organic phase was washed with brine (10 mL), dried
(MgSO4), and concentrated. The residue was purified by combi-
flash column chromatography (silica gel; hexanes/EtOAc, 1:1) to
give 35 mg (54% yield) of pure 1a as a white solid: mp 147 °C.
FTIR (neat) 3447, 1746 cm-1
;
1H NMR (400 MHz,
CDCl3-CD3OD) δ 7.30 (dm, J ) 8.9 Hz, 2 H, Ph), 7.24 (dm, J )
8.2 Hz, 2 H, Ph), 7.00 (dm, J ) 8.2 Hz, 2 H, Ph), 6.71 (dm, J )
8.9 Hz, 2 H, Ph), 4.55 (s, 2 H, H-2), 4.18 (ABq, J ) 11.7 Hz, 2 H,
-CH2OCOCH2OAr), 3.44 (mAB, 2 H, -CH2OH), 2.59 (dm, J )
16.5 Hz, 1 H, H-3a), 2.42 (dm, J ) 16.5 Hz,1 H, H-3b), 2.21 (s,
3 H, -CH3), 2.09 (br s, 3 H, -CH3), 1.70 (s, 3 H, -CH3); 13C
NMR (100 MHz, CDCl3) δ 169.54, 168.47, 157.23, 152.19, 138.02,
132.77, 131.05, 128.84, 119.97, 118.50, 116.65, 114.29, 88.40,
87.93, 81.14, 65.88, 64.68, 64.11, 31.56, 24.23, 21.08, 19.63.
FABMS m/z (relative intensity): 435 (MH+, 99), 434 (M+•, 100).
Anal. Calcd for C26H26O6 ·0.3H2O: C, 71.04; H, 6.10. Found: C,
71.06; H, 6.07.
4-Methyl-1-[(phenyl)ethyny]benzene, tert-Butyl 2-hydroxy-
acetate (4a). tert-Butyl bromoacetate (0.85 mL, 5.58 mmol) was
added to a suspension of 3 (1.02 g, 4.90 mmol) and CsCO3 (2.55
g, 7.76 mmol) in anhydrous DMF (12 mL) at room temperature
under argon. The reaction mixture was stirred for 5 h. Et2O (50
mL) was added, and the solution was washed with NaHCO3 (2 ×
15 mL) and water (1 × 15 mL) before it was dried (MgSO4) and
concentrated. The residue was purified by combi-flash column
chromatography (silica gel; hexanes/EtOAc, 9:1) to give 1.43 g
(92% yield) of pure 4a as a yellowish solid: mp 85 °C, FTIR (neat)
1751 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.45 (dm, J ) 8.9 Hz,
2 H, Ph), 7.40 (dm, J ) 8.1 Hz, 2 H, Ph), 7.14 (dm, J ) 8.1 Hz,
2 H, Ph), 6.86 (dm, J ) 8.9 Hz, 2 H, Ph), 4.52 (s, 2 H, H-2), 2.36
(s, 3 H, -CH3), 1.48 (s, 9 H, -C(CH3)3); 13C NMR (100 MHz,
CDCl3) δ 167.69, 157.74, 138.07, 132.95, 131.33, 129.05, 120.37,
116.52, 114.60, 88.44, 88.40, 82.52, 65.65, 28.01, 21.47. FABMS
m/z (relative intensity): 323 (MH+, 44), 322 (M+•, 100). Anal. Calcd
for C21H22O3: C, 78.23; H, 6.88. Found: C, 78.29; H, 6.91.
(2-[(4-Methoxyphenoxy)methyl]-4-(methylethylidene)-5-oxo-
2-2,3-dihydrofuryl)methyl 2-(4-[2-(4-Methylphenyl)ethynyl]phe-
noxy)acetate (8a). A solution of tert-butyl ester 4a (1.34 g, 0.42
mmol) in quinoline (30 mL) was heated to 205 °C for 3 h. The
mixture was cooled to room temperature and was poured into 1.2
M HCl (30 mL) at 0 °C. The solid free acid was filtered, washed
with 1.2 M HCl and water, and dried under vacuum overnight at
100 °C. A solution of 6 (151 mg, 0.52 mmol) in CH2Cl2 (5 mL)
was added to a suspension of free acid 5a (151 mg, 0.57 mml) in
CH2Cl2 (10 mL). DMAP (158 mg, 1.29 mmol) and BOPCl (263
mg, 1.38 mmol) were successively added, and the reaction mixture
was stirred for 1 h at room temperature under argon. NH4Cl (ss,
10 mL) was added, and the mixture was extracted with CH2Cl2 (3
× 20 mL). The combined organic phases were washed with brine
(2 × 10 mL), dried (MgSO4), and concentrated. The residue was
purified by combi-flash column chromatography (hexanes/EtOAc,
4:1) to give a 210 mg (75%) of 8a as a foam: FTIR (neat) 1748
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.41 (m, 4 H, Ph), 7.15 (dm,
J ) 7.9 Hz, 2 H, Ph), 6.76-6.84 (m, 6 H, Ph), 4.68 (s, 2 H, H-2),
4.68 (s, 2 H, -CH2OCOCH2OAr), 3.95 (d, J ) 9.5 Hz, 1 H,
-CHaHOPMP), 3.84 (d, J ) 9.5 Hz, 1 H, -CHHbOPMP), 3.74
(s, 3 H, -OCH3), 2.89 (dm, J ) 16.7 Hz,1 H, H-3a), 2.64 (dm,
J ) 16.7 Hz,1 H, H-3b), 2.36 (s, 3 H, -CH3), 2.27 (br s, 3 H,
-CH3), 1.86 (s, 3 H, -CH3); 13C NMR (100 MHz, CDCl3) δ
168.39, 168.27, 157.40, 154.46, 152.34, 152.13, 138.16, 132.96,
133.04, 131.21, 129.07, 120.27, 118.02, 116.87, 115.57, 114.68,
114.46, 88.67, 88.24, 79.10, 69.99, 66.23, 64.91, 55.65, 32.67,
24.60, 21.45, 19.99. FABMS m/z (relative intensity): 541 (MH+,
78), 540 (M+•, 100). Anal. Calcd for C33H32O7 ·0.8H2O: C, 71.46;
H, 6.11. Found: C, 71.33; H, 5.86.
tert-Butyl 3-(4-[2-(4-Methylphenyl)ethynyl]phenoxy)propanoate
(4b). A solution of 3 (1.05 g, 5.04 mmol) in tert-butyl acrylate (5
mL) was treated with sodium (3.5 mg, cat.) and hydroquinone (1
mg, cat.). The mixture was stirred at room temperature for 2 h.
Et2O (20 mL) and a drop of acetic acid were added, and the solution
was washed with water (2 × 10 mL), dried (MgSO4), and
concentrated. The residue was purified by combi-flash column
chromatography (silica gel; hexanes/EtOAc, 9:1) to give 550 mg
(33% yield) of pure 4b as a yellowish solid: mp 74 °C. FTIR (neat)
1731 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.42 (m, 4 H, Ph), 7.14
(dm, J ) 7.8 Hz, 2 H, Ph), 6.87 (dm, J ) 8.9 Hz, 2 H, Ph), 4.23
(t, J ) 6.5 Hz, 2 H, H-3), 2.71 (t, J ) 6.5 Hz, 2 H, H-2), 2.36 (s,
3 H, -CH3), 1.47 (s, 9 H, -C(CH3)3); 13C NMR (100 MHz, CDCl3)
δ 170.06, 158.51, 137.91, 132.87, 131.26, 129.00, 120.42, 115.75,
114.55, 88.59, 88.19, 80.92, 63.72, 35.62, 28.02, 21.40. FABMS
m/z (relative intensity): 337 (MH+, 41), 336 (M+•, 100). Anal. Calcd
for C22H24O3: C, 78.24; H, 7.19. Found: C, 78.48; H, 7.21.
(4-(Methylethylidene)-5-oxo-2-[(phenylmethoxy)methyl]-2-
2,3-dihydrofuryl)methyl 3-(4-[2-(4-Methylphenyl)ethynyl]phe-
noxy)propanoate (8b). tert-Butyl ester 4b was treated in the same
way as that described for 4a and the corresponding free acid was
reacted with 7 as described for the synthesis of 8a to give 180 mg
1
(70% yield) of 8b as a colorless oil. FTIR (neat) 1744 cm-1; H
NMR (400 MHz, CDCl3) δ 7.42-7.25 (m, 9 H, Ph), 7.11 (dm, J
) 7.9 Hz, 2 H, Ph), 6.80 (dm, J ) 8.9 Hz, 2 H, Ph), 4.50 (s, 2 H,
-OCH2Ph), 4.25 (s, 2 H, -CH2OCOCH2CH2OAr), 4.18 (t, J )
6.3 Hz, 2 H, H-3), 3.49 (ABq, J ) 9.9 Hz, 2 H, -CH2OBn), 2.77
(t, J ) 6.3 Hz, 2 H, H-2), 2.78 (dm, J ) 16.4 Hz, 1 H, H-3a), 2.62
(dm, J ) 16.4 Hz,1 H, H-3b), 2.33 (s, 3 H, -CH3), 2.20 (t, J ) 2.0
Hz, 3 H, -CH3), 1.76 (s, 3 H, -CH3); 13C NMR (100 MHz, CDCl3)
δ 170.26, 168.83, 158.25, 151.28, 138.05, 137.44, 132.96, 131.31,
129.05, 128.43, 127.86, 127.64, 120.39, 118.74, 116.07, 114.51,
88.48, 79.94, 73.65, 71.72, 66.19, 63.20, 34.40, 32.72, 24.47, 21.47,
19.89. FABMS m/z (relative intensity): 539 (MH+, 31), 538 (M+•,
35). Anal. Calcd for C34H34O6 ·0.6H2O: C, 74.38; H, 6.46. Found:
C, 74.05; H, 6.16.
[2-(Hydroxymethyl)-4-(methylethylidene)-5-oxo-2-2,3-dihy-
drofuryl]methyl 3-(4-[2-(4-Methylphenyl)ethynyl]phenoxy)pro-
panoate (1b). A solution of 8b (141 mg, 0.26 mmol) in anhydrous
CH2Cl2 (5 mL) was treated with 1.0 M BCl3 solution in CH2Cl2 (1
mL, 1.05 mmol) at -78 °C for 15 min. NaHCO3 (ss, 5 mL) was
added, and the mixture was allowed to reach room temperature.
The aqueous layer was extracted with CH2Cl2 (3 × 20 mL),
and the combined organic layers were dried (MgSO4) and concen-
trated. The residue was purified by combi-flash column chroma-
tography (silica gel; hexanes/EtOAc, 1:1) to give 93 mg (80% yield)
of pure 1b as a white solid: mp 125 °C. FTIR (neat) 3472, 1731
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.45-7.38 (m, 4 H, Ph), 7.14
(dm, J ) 8.0 Hz, 2 H, Ph), 6.84 (dm, J ) 8.9 Hz, 2 H, Ph), 4.28
(ABq, J ) 11.8 Hz, 2 H, -CH2OCOCH2CH2OAr), 4.23 (t, J )
6.3 Hz, 2 H, H-3), 3.66 (ABq, J ) 12.1 Hz, 2 H, -CH2OH), 2.83
(t, J ) 6.3 Hz, 2 H, H-2), 2.78 (dm, J ) 16.5 Hz, 1 H, H-3a), 2.64
(dm, J ) 16.4 Hz,1 H, H-3b), 2.35 (s, 3 H, -CH3), 2.23 (t, J ) 2.0
Hz, 3 H, -CH3), 1.80 (s, 3 H, -CH3); 13C NMR (100 MHz, CDCl3)
[2-(Hydroxymethyl)-4-(methylethylidene)-5-oxo-2-2,3-dihy-
drofuryl]methyl 2-(4-[2-(4-Methylphenyl)ethynyl]phenoxy)ac-
etate (1a). Cerium ammonium nitrate (CAN, 247 mg, 0.45 mmol)
was added to a solution of 8a (81 mg, 0.15 mmol) in acetonitrile/
water (4:1, 5 mL) at 0 °C. After 10 min, 5% Na2S2O3 solution (10
mL) was added and the mixture was extracted with EtOAc (3 ×

3282 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 10
Comin et al.
δ 170.66, 168.91, 158.18, 152.14, 138.06, 132.98, 131.30, 129.05,
120.34, 118.59, 116.14, 114.49, 88.43, 88.39, 80393, 65.66, 64.82,
63.22, 34.42, 32.01, 24.53, 21.45, 19.96; FABMS m/z (relative
intensity): 449 (MH+, 85), 448 (M+•, 100). Anal. Calcd for
C27H28O6 ·0.3H2O: C, 71.49; H, 6.35. Found: C, 71.30; H, 6.18.
tert-Butyl 4-(4-[2-(4-Methylphenyl)ethynyl]phenoxy)butanoate
(4c). This compound was prepared similarly as described for 4a
from tert-butyl 4-bromobutanoate (302 mg, 2.43 mmol) and phenol
3 (253 mg, 2.21 mmol). After combi-flash column chromatography
(silica gel; hexanes/ethyl acetate, 9:1) 351 mg (83% yield) of pure
4c was obtained as a yellowish solid: mp 68 °C. FTIR (neat) 1723
Acknowledgment. This research was supported in part by
the Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research. We also thank Drs. James
A. Kelley and Chris Lai for HRMS measurements and Dr. Dina
M. Sigano for help with the calculations of log P values and
assembling the manuscript.
Supporting Information Available: 1H and 13C spectra for
1a-c. This material is available free of charge via the Internet at
http://pubs.acs.org.
1
cm-1; H NMR (400 MHz, CDCl3) δ 7.45 (dm, J ) 8.3 Hz, 2 H,
References
Ph), 7.40 (dm, J ) 7.8 Hz, 2 H, Ph), 7.14 (dm, J ) 7.8 Hz, 2 H,
Ph), 6.85 (dm, J ) 8.3 Hz, 2 H, Ph), 4.01 (t, J ) 6.2 Hz, 1 H,
H-4), 2.43 (t, J ) 7.3 Hz, 1 H, H-2), 2.36 (s, 3 H, -CH3), 2.07 (m,
2 H, H-3), 1.45 (s, 9 H, -C(CH3)3); 13C NMR (100 MHz, CDCl3)
δ 172.39, 158.78, 137.92, 132.90, 131.27, 129.02, 120.47, 115.52,
114.45, 88.65, 88.14, 80.34, 31.91, 28.07, 24.66, 21.43; FABMS
m/z (relative intensity): 351 (MH+, 16), 350 (M+•, 41). Anal. Calcd
for C23H26O3 ·0.1H2O: C, 78.48; H, 7.50. Found: C, 78.32; H, 7.47.
(2-[(4-Methoxyphenoxy)methyl]-4-(methylethylidene)-5-oxo-
2-2,3-dihydrofuryl)methyl 4-(4-[2-(4-Methylphenyl)ethynyl]phe-
noxy)butanoate (8c). tert-Butyl ester 4c (342 mg, 0.98 mmol) was
treated in the same manner as described for 4a, and the corre-
sponding free acid was reacted with 6 (202 mg, 0.69 mmol) as
described for the synthesis of 8a to give 224 mg (57% yield) of 8c
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1
as a white solid: mp 117 °C; FTIR (neat) 1744 cm-1; H NMR
(400 MHz, CDCl3) δ 7.44-7.39 (m, 4 H, Ph), 7.14 (dm, J ) 7.9
Hz, 2 H, Ph), 6.82 (m, 6 H, Ph), 4.34 (ABq, J ) 11.8 Hz, 2 H,
-CH2OCOCH2CH2CH2OAr), 3.99 (t, J ) 6.0 Hz, 2 H, H-4), 3.98
(ABq, J ) 9.5 Hz, 2 H, -CH2OPMP), 3.76 (s, 3 H, -OCH3), 2.96
(dm, J ) 16.4 Hz, 1 H, H-3a), 2.75 (dm, J ) 16.4 Hz, 1 H, H-3b),
2.55 (t, J ) 7.3 Hz, 2 H, H-2), 2.36 (s, 3 H, -CH3), 2.28 (br s, 3
H, -CH3), 2.09 (m, 2 H, H-3), 1.87 (s, 3 H, -CH3); 13C NMR
(100 MHz, CDCl3) δ 172.55, 168.63, 158.61, 154.43, 152.28,
151.82, 138.00, 132.96, 131.30, 129.05, 120.45, 118.44, 115.61,
114.68, 114.44, 88.60, 88.23, 79.41, 70.22, 66.52, 65.88, 55.69,
32.88, 30.53, 24.60, 24.45, 21.47, 19.97. FABMS m/z (relative
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C35H36O7 ·0.4H2O: C, 73.05; H, 6.45. Found: C, 72.85; H, 6.38.
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1714 cm-1; H NMR (400 MHz, CDCl3) δ 7.44-7.38 (m, 4 H,
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Ph), 7.13 (dm, J ) 7.8 Hz, 2 H, Ph), 6.84 (dm, J ) 8.9 Hz, 2 H,
Ph), 4.23 (ABq, J ) 11.8 Hz, 2 H, -CH2OCOCH2CH2CH2OAr),
4.00 (t, J ) 6.0 Hz, 2 H, H-4), 3.65 (m, 2 H, -CH2OH), 2.80 (dm,
J ) 16.5 Hz, 1 H, H-3a), 2.62 (dm, J ) 16.5 Hz, 1 H, H-3b), 2.56
(t, J ) 7.3 Hz, 2 H, H-2), 2.35 (s, 3 H, -CH3), 2.25 (br s, 3 H,
-CH3), 2.10 (m, 2 H, H-3), 1.85 (s, 3 H, -CH3); 13C NMR (100
MHz, CDCl3) δ 172.91, 168.90, 158.57, 151.94, 138.01, 132.95,
131.29, 129.04, 120.41, 118.70, 115.75, 114.43, 88.55, 88.25, 80.97,
66.54, 65.52, 64.80, 32.09, 30.55, 24.57, 24.43, 21.45, 19.96;
FABMS m/z (relative intensity): 463 (MH+, 100), 462 (M+•, 84).
Anal. Calcd for C28H30O6 ·0.4H2O: C, 71.64; H, 6.61. Found: C,
71.58; H, 6.41.
[2-(Hydroxymethyl)-4-(methylethylidene)-5-oxo-2-2,3-dihy-
drofuryl]methyl 4-[2-(4-Methylphenyl)ethynyl]benzoate (2). This
compound was prepared by the same methodology as described in
ref 7. Pure 2 (260 mg, 76%) was obtained as a yellowish solid: mp
1
167-168 °C; FTIR (neat) 3469, 2214, 1723 cm-1; H NMR (400
MHz, CDCl3) δ 7.96 (m, 2 H, Ph), 7.57 (m, 2 H, Ph), 7.44 (m, 2
H, Ph), 7.18 (m, 2 H, Ph), 4.58 (mAB, 2 H, -Ph-CO2CH2-), 3.76
(ABq, J ) 12.1 Hz, 2 H, HOCH2-), 2.90-2.70 (mAB, 2 H, H-4a,b),
2.38 (s, 3 H, -CH3), 2.25 (t, J ) 2.0 Hz, 3 H, -CH3), 1.86 (s, 3
H, -CH3); 13C NMR (100 MHz, CDCl3) δ 169.10, 165.78, 151.67,
139.09, 131.62, 131.46, 129.61, 129.18, 128.82, 128.23, 119.00,
93.12, 87.88, 81.45, 66.32, 64.78, 32.25, 24.55, 21.53, 19.91.
FABMS m/z (relative intensity): 405 (MH+, 47). Anal. Calcd for
C25H24O5 ·0.3H2O: C, 73.26; H, 6.05. Found: C, 73.21; H, 5.99.
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