Design and synthesis of a novel and potent series of inhibitors of cytosolic phospholipase A2 based on a 1,3-disubstituted propan-2-one skeleton

Article abstract of DOI:10.1021/jm011050x

Using knowledge of the substrate specificity of cPLA₂ (phospholipases A₂), a novel series of inhibitors of this enzyme were designed based upon a three point model of inhibitor binding to the enzyme active site comprising a lipophilic anchor, an electrophilic serine "trap", and an acidic binding moiety. The resulting 1,3-diheteroatom-substituted propan-2-ones were evaluated as inhibitors of cPLA₂ in both aggregated bilayer and soluble substrate assays. Systematic variation of the lipophilic, electrophilic, and acidic groups revealed a well-defined structure-activity relationship against the enzyme. Optimization of each group led to compound 22 (AR-C70484XX), which contains a decyloxy lipophilic side chain, a 1,3-diaryloxypropan-2-one moiety as a unique serine trap, and a benzoic acid as the acidic binding group. AR-C70484XX was found to be among the most potent in vitro inhibitors of cPLA₂ described to date being more than 20-fold more active against the isolated enzyme (IC₅₀ = 0.03 μM) than the standard cPLA₂ inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃), and also greater than 10-fold more active than AACOCF₃ against the cellular production of arachidonic acid by HL60 cells (IC₅₀ = 2.8 μM).

Full text of DOI:10.1021/jm011050x

1348
J . Med. Chem. 2002, 45, 1348-1362
Design a n d Syn th esis of a Novel a n d P oten t Ser ies of In h ibitor s of Cytosolic
P h osp h olip a se A2 Ba sed on a 1,3-Disu bstitu ted P r op a n -2-on e Sk eleton
Stephen Connolly,*,† Colin Bennion,† Sarah Botterell,‡ Pamela J . Croshaw,§ Catherine Hallam,§ Kim Hardy,†
Paul Hartopp,† Clive G. J ackson,§ Sarah J . King,† Louise Lawrence,† Antonio Mete,† David Murray,§
David H. Robinson,† Gillian M. Smith,‡ Linda Stein,† Iain Walters,† Edward Wells,§ and W. J ohn Withnall†
Departments of Medicinal Chemistry, Molecular Biology, and Discovery BioScience, AstraZeneca R&D Charnwood,
Bakewell Road, Loughborough, Leicestershire LE11 5RH, United Kingdom
Received October 8, 2001
Using knowledge of the substrate specificity of cPLA2 (phospholipases A2), a novel series of
inhibitors of this enzyme were designed based upon a three point model of inhibitor binding to
the enzyme active site comprising a lipophilic anchor, an electrophilic serine “trap”, and an
acidic binding moiety. The resulting 1,3-diheteroatom-substituted propan-2-ones were evaluated
as inhibitors of cPLA2 in both aggregated bilayer and soluble substrate assays. Systematic
variation of the lipophilic, electrophilic, and acidic groups revealed a well-defined structure-
activity relationship against the enzyme. Optimization of each group led to compound 22 (AR-
C70484XX), which contains a decyloxy lipophilic side chain, a 1,3-diaryloxypropan-2-one moiety
as a unique serine trap, and a benzoic acid as the acidic binding group. AR-C70484XX was
found to be among the most potent in vitro inhibitors of cPLA2 described to date being more
than 20-fold more active against the isolated enzyme (IC50 ) 0.03 µM) than the standard cPLA2
inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), and also greater than 10-fold more
active than AACOCF3 against the cellular production of arachidonic acid by HL60 cells (IC50
) 2.8 µM).
In tr od u ction
First, for a long time, the only PLA2s that had been
described were all low molecular mass (∼14 kD) en-
zymes that have been secreted from cells and hence
have subsequently been termed sPLA2s. These sPLA2s
are characterized by 7 (or 8) disulfide bonds that endow
the enzyme with extracellular stability. In addition, the
enzymes demonstrate a lack of specificity for the fatty
acid cleaved from the sn-2 position of phospholipids
(although in contrast a reasonable specificity toward a
phosphoethanolamine group in the sn-3 position) and
require a high concentration (>µM) of Ca2+ for activa-
tion.6 While none of these attributes would be expected
of an intracellular PLA2, it was nonetheless believed for
some time that the cellular PLA2 would be structurally
and mechanistically similar; hence, much effort has
been expended7 on the search for inhibitors of the
sPLA2s as possible antiinflammatory agents. The com-
bined Lilly and Shionogi groups have advanced an
sPLA2 inhibitor to clinical trials for the treatment of
septic shock; however, it is now unclear whether the
mechanism of action of this class of compound is through
enzyme inhibition or antagonism of an sPLA2 receptor.8
Phospholipases A2 (PLA2s) are a class of esterases
that cleave the sn-2 ester bond of membrane phospho-
lipids in both a regiospecific and a stereospecific man-
ner.1 Because the vast majority of cellular arachidonic
acid is found esterified at this position of glycerophos-
pholipids and free arachidonate is only produced by cells
when appropriately activated, a PLA2 is generally
accepted as being the enzyme that is responsible for the
cellular production of this important intermediate.2
Arachidonic acid is further converted by cells into a
number of proinflammatory metabolites, including pros-
taglandins and leukotrienes, molecules that have been
the subject of many medicinal chemistry efforts,3 either
to block their biosynthesis or to antagonize the endpoint
action of these mediators at specific receptors. In
addition, the byproduct of arachidonic acid production
by PLA2 is a lysophospholipid, which on further enzy-
matic acetylation can lead to the formation of platelet-
activating factor, yet another proinflammatory molecule
of medicinal interest.4 Thus, inhibition of the action of
PLA2 has the clear potential to block the cellular
production of a wide range of proinflammatory lipid
mediators and has therefore been the subject of intense
study by the pharmaceutical industry.5 It may therefore
seem surprising that no inhibitor of PLA2 has emerged
as an antiinflammatory drug to date. However, there
are good reasons for the lack of progress toward this
aim.
However, in the early 1990s, an intracellular PLA2
was described,9 the properties of which were much more
consistent with those anticipated for the enzyme re-
sponsible for intracellular arachidonic acid production.
This enzyme had a far higher molecular mass (ap-
proximately 85 kD) but contained no disulfide bridges.
The enzyme showed a distinct preference for the hy-
drolysis of an arachidonoyl ester at the sn-2 position of
phospholipids, both against purified substrates and in
a natural phospholipid environment.10 Furthermore, it
was activated by much lower (<µM) levels of Ca2+ than
sPLA2, consistent with the levels required for the
* To whom correspondence should be addressed. Tel.: +44 01509
644160. Fax: +44 01509 5571. E-mail: steve.connolly@astrazeneca.com.
† Department of Medicinal Chemistry.
‡ Department of Molecular Biology.
§ Department of Discovery BioScience.
10.1021/jm011050x CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/08/2002

1,3-Disubstituted Propan-2-one Skeleton
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1349
production of arachidonic acid (and its metabolites)
within cells.11 Notably, this intracellular PLA2 was
found within the cytosol and was named cPLA2. Two
further members of the cPLA2 class, cPLA2â and
cPLA2γ, have been described recently.12 Later, it was
found13 that when cells are activated cPLA2 translocates
largely to the nuclear membrane where the arachidonic
acid can be produced in close proximity to the down-
stream enzymes 5-lipoxygenase (5-LO) and cyclooxyge-
nase (CO). This provides compelling evidence that
cPLA2 production of arachidonic acid is concerted with
its further biochemical transformation to proinflamma-
tory eicosanoids14 and lends support to the hypothesis
that the inhibition of cPLA2 would provide a valuable
target in the search for a novel antiinflammatory
agent.15 This hypothesis became further strengthened
with the publication from two separate laboratories that
cPLA2 knockout mice have a much reduced output of
inflammatory eicosanoids and associated resistance to
downstream effects such as anaphylactic response,
bronchial reactivity, and postischemic brain injury.16
F igu r e 1. Simple model for substrate binding to cytosolic
PLA2.
F igu r e 2. Simple model for inhibitor binding to cytosolic
PLA2.
A second reason for the apparent lack of progress in
finding inhibitors of PLA2s that might lead to clinical
candidates is the complex nature of the in vitro screen-
ing of these enzymes. PLA2s as their name suggests are
lipases and as such have evolved to work optimally at
a lipid-water interface. A large variety of screens have
been described that involve the action of PLA2 at an
aggregated assembly of substrate such as that formed
in bilayers, vesicles, and mixed micelles.17 The great
problem with all such screens is that inhibitors may
work by interfering with the structure of the interface
and inhibit the enzyme action not by binding to its
catalytic site but merely by causing the enzyme to
desorb from the lipid-water interface. This may be
described as “nonspecific inhibition”. Many different
assay strategies have been employed in attempts to
overcome this difficulty. For instance, Gelb and J ain
have described18 a “scooting assay” in which the enzyme
is so strongly bound to (modified) substrate vesicles that
nonspecific inhibition cannot occur. On the other hand,
Lehr19 has dispensed with isolated enzyme screening
altogether, preferring to screen only in a cellular situ-
ation. We have previously described20 a “dual-screening”
strategy for sPLA2, in which inhibitors are tested
against the enzyme in the presence of a natural sub-
strate bilayer and also against the enzyme working
against a soluble substrate where the complexities
associated with lipid aggregation are minimized. In this
paper, we describe the use of both bilayer and soluble
substrate screens for the discovery of novel and potent
inhibitors of the cytosolic enzyme cPLA2.
natural substrate. A simple model of how one might
expect substrate to bind to cPLA2 is shown in Figure 1.
Features that intrigued us were cPLA2s selectivity for
cleaving arachidonic acid from the sn-2 position of
substrate, which suggested that there might be a
specific binding site in the enzyme for the arachidonoyl
group. In fact, Gelb has previously demonstrated18b that
the double bonds of the arachidonate moiety, and
particularly the first (∆5,6) double bond, are highly
beneficial for selectivity. In contrast, there was little
evidence that the sn-2 ester was recognized at all.
Turning to the phosphocholine group, it appeared that
an anionic phosphate was required for recognition by
the enzyme,11 but there was no evidence that the
choline, or any other quaternary ammonium group, was
necessary suggesting that this part of the substrate
plays little role in binding to enzyme. The model is
completed by a proposed22 catalytic diad with an acti-
vated serine OH group acting as the nucleophile23 to
react with the sn-2 ester group, thereby producing an
intermediate oxyanion. This intermediate is likely to be
stabilized by an “oxyanion hole” within the enzyme
active site.
This knowledge led to the design of novel inhibitors
of cPLA2 based on a model for inhibitor binding, which
is shown in Figure 2. The model consists of three parts:
first, a lipophilic substituent that incorporates aromatic
rings to mimic (at least part of) the arachidonic acid-
skipped tetraene unit; second, an electrophilic ketone
as a serine “trap”; and third, an anionic group at a few
atoms distance from the serine trap to mimic the
phosphate group of the substrate. Our experience in the
sPLA2 area with the design of AR-C67047 showed that
a carboxylic acid can be an excellent bioisostere for the
monoanionic phosphodiester of substrate.21 Therefore,
in this model for a cPLA2 inhibitor, we also replaced
the phosphodiester with a carboxylic acid, conveniently
positioned on an alkyl chain attached to the chosen
serine trap. Subsequent publication24 of the X-ray
crystal structure of cPLA2 showed this model to be
largely correct; however, confirmation of the binding
In h ibitor Design
At the outset of this work, we tested our entire
collection of sPLA2 inhibitors against cPLA2, including
the extremely potent sPLA2 inhibitor AR-C67047, which
we have previously reported.21 None of the compounds
showed significant inhibition of cPLA2, as might be
expected since data that later emerged showed the two
classes of enzyme to have completely different catalytic
mechanisms and quite distinct selectivities toward
phospholipid substrate. We therefore turned our atten-
tion to the a priori design of inhibitors based on the

1350 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Connolly et al.
mode of inhibitors will need to wait until the structure
of cPLA2 complexed to an inhibitor is reported.
in comparison to the trifluoromethyl ketones and may
be further substituted on the CF2 group. For instance,
some very elegant work describing substituted difluo-
romethyl ketone inhibitors of interleukin-1â converting
enzyme has been reported.29 However, while potent
inhibitors of this type were found, these compounds
remain highly electrophilic, and it is still difficult to
control the electrophilicity of the pharmacophoric ketone
group. Furthermore, it is likely that such highly elec-
trophilic ketones will be rapidly metabolized. Indeed,
the facile cellular reduction of compound 1 has been
disclosed.27a
The choice of electrophilic ketone to act as a serine
trap deserves some discussion. Described in the litera-
ture, there are a number of different types of electro-
philic ketones that may serve to interact with nucleo-
philic serine residues25 (mainly within the context of
serine protease inhibitors). For example, trifluoromethyl
ketones are known to interact well with serine hydroxyl
groups at the active site of serine proteases.25 Indeed,
while this work was in progress, the Merck-Frosst group
described26 arachidonyl trifluoromethyl ketone 1 (AA-
COCF3) as a potent inhibitor of cPLA2.
In our work, we felt it was essential to incorporate a
ketone in which the electrophilicity of the carbonyl
group could be altered over a wide range by structural
modification. We chose to investigate the little-known
1,3-diheteroatom-substituted propan-2-one30,31 group,
where the flanking heteroatoms were either O or S
atoms, as the interactive serine trap. On one side of the
propan-2-one group, we introduced an aryloxy substitu-
ent to both activate the ketone group to nucleophilic
attack and also to incorporate a planar all sp2 moiety
with high π-electron density to mimic the ∆5,6 double
bond of arachidonic acid. We also appended a long alkyl
chain on the distal side of the aromatic ring, to mimic
the remainder of the lipid side chain of substrate. This
alkyl chain was conveniently attached as an alkyloxy
group. With the further incorporation of an alkyl-linked
carboxylic acid group on the other side of the central
ketone, we arrived at the prototype cPLA2 inhibitor
shown in Figure 2.
Later, it was shown27 that this compound (1) func-
tionally inhibits the products of cPLA2 action in whole
cell systems, although the mechanism of this cellular
inhibition has been questioned.28 However, while useful
as a probe, this inhibitor suffers from some serious
drawbacks as a starting point in the search for a clinical
candidate. First, the skipped diene units of the arachi-
donyl side chain are metabolically labile, especially to
oxidation. Second, because of the very strong electron-
withdrawing effect of the trifluoromethyl group, tri-
fluoromethyl ketones are exceptionally electrophilic and
exist largely as hydrates. It is not possible to modify
the electrophilicity of the trifluoromethyl ketone group
in a controlled way, and because it is only the free
ketone that can interact with the serine hydroxyl group,
hydrated trifluoromethyl ketones may not appear to be
as potent inhibitors of serine enzymes as might be
expected. Additionally, the nature of the trifluoromethyl
group makes it difficult to substitute on the CF3 side of
the ketone without changing the electron-withdrawing
nature of the activating group substantially. Obviously,
difluoromethyl analogues have reduced electrophilicity
Ch em istr y
Scheme 1 outlines the chemical approach used to
synthesize compounds 2-12 and 14-18, as demon-
strated by the preparation of compound 4. 4-Hydroxy-
phenol was monoalkylated using decyl bromide under
basic conditions to afford the 4-alkoxyphenol 4a in
moderate yield. This phenol was further reacted with
epichlorohydrin to give the epoxy intermediate 4b in
Sch em e 1a
a
t
(a) (1) NaH, DMF; (2) C10H21Br. (b) Epichlorohydrin, Cs2CO3, MeCN, reflux. (c) DABCO, HS(CH2)2CO2 Bu, DMF, 100 °C. (d) Dess-
Martin reagent, DCM. (e) TFA, DCM. (f) MCPBA, DCM.

1,3-Disubstituted Propan-2-one Skeleton
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1351
Sch em e 2a
were not characterized but were oxidized directly to the
keto-acid products using PCC, albeit in moderate overall
yields.
Compounds 22-41, 46, and 49 were prepared by the
route outlined for compound 22 in Scheme 3. In this
series of compounds, the epoxide intermediate 4b was
ring-opened by a substituted phenol to afford the
hydroxy intermediate 22a . Subsequent oxidation of this
alcohol with Dess-Martin reagent gave keto-ester 22b,
which upon deprotection gave the desired acid 22 in
good yields.
Scheme 4 outlines the synthesis of the two des-oxy
analogues 13 and 42. The phenol 4a was cleanly
alkylated with 1,3-dibromopropane to afford an inter-
mediate alkyl bromide in excellent yield. Reaction of this
bromo compound with methyl 4-hydroxybenzoate gave
the ester 42a , deprotection of which gave the acid 42.
Similarly, reaction of the same bromo intermediate
derived from 4a with tert-butyl 3-mercaptopropanoate
followed by acidic deprotection gave 13.
For the synthesis of analogues with only one hetero-
atom flanking the central ketone group, quite different
chemistry was required. Scheme 5 outlines the synthesis
of compound 44, which contains a 2 carbon link to the
benzoic acid moiety. 4-(5-Phenylpentylthio)phenol (pre-
pared from 4-mercaptophenol in analogous fashion to
4a ) was alkylated very efficiently by 2-chloroacetonitrile
to give 44a . This compound was reduced with Dibal-H
in toluene, and vinylmagnesium bromide was added
cleanly to the resulting aldehyde (44b) to afford the
allylic alcohol 44c. This alcohol underwent the Heck
reaction with methyl 4-iodobenzoate to give 44d in
moderate yield, which on deprotection gave 44.
a
(a) (1) NaH, DMF; (2) HO(CH2)nOH. (b) PCC, DMF.
excellent yield. Ring opening of the 4b with tert-butyl
3-mercaptopropanoate was best achieved in the pres-
ence of a catalytic amount of DABCO to give 4c.
Oxidation to give the intermediate ketone 4d was
carried out with mild oxidants such as the Dess-Martin
reagent or the acetic anhydride-DMSO (dimethyl sul-
foxide) system. In general, yields for this transformation
were much lower when harsher oxidants, such as
pyridinium dichromate (PDC) or PCC, were employed.
Deprotection of the tert-butyl ester under standard
conditions gave compound 4. Alternatively, oxidation of
4d with 1 or 2 equiv of MCPBA, followed by deprotection
of the ester, afforded the sulfoxide 17 and sulfone 18,
respectively.
Oxygen-linked compounds 19-21 were prepared as
shown in Scheme 2. Thus, the epoxy intermediate 4b
was reacted with the appropriate diol under strongly
basic conditions. The resulting dihydroxy intermediates
Sch em e 3a
a
(a) (1) HOC6H4CO2allyl, DABCO, DMF, 100 °C. (b) Dess-Martin reagent, DCM. (c) Pd(PPh3)4AcOH, THF.
Sch em e 4a
a
t
(a) (1) Cs2CO3, DMF; (2) 1,3-dibromopropane. (b) 4-HOC6H4CO2Me, Cs2CO3, MeCN, reflux. (c) LiOH, THF/H2O. (d) HS(CH2)2CO2 Bu,
DABCO, DMF, 100 °C. (e) TFA, DCM.

1352 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Connolly et al.
Sch em e 5a
exists. Testing inhibitors in both assays (a dual screen-
ing strategy as described above) gave information on
compounds that behaved differently in the two screens
and that thus could be inhibiting in a nonspecific
fashion. Finally, inhibitors were also tested in a cellular
screen based on the stimulated production of arachi-
donic acid by HL60 cells.
Str u ctu r e-Activity Rela tion sh ip s a n d
Discu ssion
On the basis of our design principle as outlined above,
we initially targeted propan-2-ones substituted on one
side of the carbonyl group by a propanoic acid (linked
by an S atom) to mimic the phosphate group and on the
other side by aryloxy groups (to mimic part of the
skipped tetraene of arachidonic acid). Thus, the 2-naph-
thoxy compound (2) and the 4-phenoxyphenoxy com-
pound (3) were prepared but were shown to have no
activity in the bilayer assay (see Table 1). Incorporation
of a decyloxyphenoxy substituent, as in compound 4,
gave the first sub-micromolar inhibitor of cPLA2 with
an IC50 of 0.27 µM in the bilayer assay. This latter
compound has a much higher lipophilicity than the
former compounds, and at that point, it was not clear if
the increased potency was due to the decyloxy group
interacting with the enzyme or merely anchoring the
inhibitor in the bilayer. However, the requirement for
high lipophilicity is perhaps not surprising given the
physical properties of the natural phospholipid sub-
strate.
To investigate this lipophilic requirement, we syn-
thesized analogues of compound 4 with the decyl chain
truncated to 2 and 6 carbon atoms (compounds 5 and 6
in Table 1). These compounds had much reduced activ-
ity, with the least lipophilic (compound 5) being totally
inactive, again emphasizing the need for a lipophilic
substituent at this position in the molecule. Surpris-
ingly, increasing the lipophilicity by 2 orders of magni-
tude over compound 4 by extending the lipophilic alkyl
group to 14 carbon atoms (as in compound 7) gave no
further increase in activity. Replacement of the decyloxy
group of 4 with a 2-naphthylmethoxy group (common
in inhibitors of 5-LO)33 gave a compound (8) with
somewhat reduced potency, but the residual activity did
show that bulky aromatic groups can indeed be tolerated
in this region of the inhibitor and that a linear alkyl
group is not a necessity. Moreover, when the lipophi-
a
(a) Cs2CO3, DMF, ClCH2CN. (b) Dibal-H, toluene, -40 °C. (c)
VinylMgBr, THF, -10 °C. (d) Pd(OAc)2, DMF, TEA, 4-IC6H4CO2Me.
(e) LiOH, THF/H2O.
Compound 45, which contains a 2 carbon link to the
decyloxyphenyl substituent, was prepared by the route
shown in Scheme 6. Thus, 4-(4-hydroxyphenyl)-2-oxo-
butane was alkylated with decyl bromide to afford 45a .
This methyl ketone was brominated using tetrabutyl-
ammonium tribromide to afford the corresponding bro-
momethyl ketone 45b in good yield. Reaction of this
compound with allyl 3-hydroxybenzoate proceeded ef-
ficiently with KF as the base to give 45c, which was
deprotected under standard conditions to give the target
acid 45.
Eva lu a tion of In h ibitor s
Initially, inhibitors were tested against the action of
cPLA2 in a bilayer-based assay in which the substrate
is comprised of an aggregated form of phospholipid to
which the enzyme binds reversibly. Subsequently, when
it became available,32 inhibitors were also tested in an
assay where cPLA2 is acting upon a simple, synthetic,
and soluble substrate, wherein no aggregated assembly
Sch em e 6a
a
(a) Cs2CO3, C10H21Br, DMF. (b) BuN4‚Br3, DCM/MeOH. (c) KF, 3-HOC6H4CO2allyl, DMF, 100 °C. (d) Pd(PPh3)4, THF/AcOH.

1,3-Disubstituted Propan-2-one Skeleton
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1353
Ta ble 1. Inhibition of Cytosolic PLA2: Variation of the
Ta ble 2. Inhibition of Cytosolic PLA2: Changes to the Ketone
Lipophilic Aryl Substituent
and Acid
bilayer assay
compd
X, Y
dO
dO
H, OH
H, H
R
IC50 (µM)a
cLog Pb
4
11
12
13
H
Me
H
0.27
>10c
3.6
6.3
6.9
5.9
6.9
H
1.9
a
Inhibition of cPLA2 measured against an aggregated phos-
pholipid substrate. IC50 values are the means of at least two
b
independent determinations. Errors are within (20%. Lipophi-
licity as calculated by Daylight.35 c No inhibition at this concen-
tration.
Ta ble 3. Inhibition of Cytosolic PLA2: Variation of the Acidic
Chain
bilayer assay
compd
Y
n
IC50 (µM)a
cLog Pb
4
14
15
16
17
18
19
20
21
S
S
S
S
SO
SO2
O
2
1
3
4
2
2
2
3
4
0.27
1.4
0.15
0.07
1.0
0.50
1.2
0.24
0.03
6.3
6.0
6.3
6.9
5.4
5.3
5.9
5.6
6.0
O
O
a
Inhibition of cPLA2 measured against an aggregated phos-
a
Inhibition of cPLA2 measured against an aggregated phos-
pholipid substrate. IC50 values are the means of at least two
pholipid substrate. IC50 values are the means of at least two
b
independent determinations. Errors are within (20%. Lipophi-
b
licity as calculated by Daylight.35 c No inhibition at this concen-
tration.
independent determinations. Errors are within (20%. Lipophi-
licity as calculated by Daylight.35
licity and activity of compound 8 are compared with that
of compound 6, it is clear that the sole reason for the
reduced activity of 8 over 4 is its reduced lipophilicity.
This could be taken as evidence that the role of the alkyl
group may be only to partition the molecule into the
bilayer. However, shape was also shown to be of some
importance since the isomeric meta- and ortho-decyloxy
compounds 9 and 10 (Table 1) are reduced in activity
as compared to the prototype para-substituted com-
pound 4.
That an anionic group is a requirement for activity
is suggested by the inactivity of the methyl ester 11
(Table 2). Although reduction of the ketone to the
corresponding alcohol 12 or even to the methylene
compound 13 gave weaker inhibition (3.6 and 1.9 µM,
respectively) as compared to ketone 4, these reduced
analogues appear to have a surprising level of inhibitory
activity. However, the similar degree of potency ob-
served for both 12 and 13 strongly suggests that this
level of activity (a few micromolars) might possibly be
due to nonspecific inhibition in the aggregated bilayer
assay. Indeed, a large number of lipophilic acids of
varied structure produced apparent activity at this level
in the bilayer assay and served to highlight the difficul-
ties of screening solely with such aggregated assay
systems (see following). At this stage, compound 4 was
unique in our hands in that it inhibited cPLA2 at sub-
micromolar concentrations, and it gave us the confi-
dence to explore this series further.
We next explored the nature of the chain linking the
acid group to the electrophilic ketone (see Table 3).
Shortening the carbon chain between the S atom and
the acid to one methylene, as in compound 14, gave a
reduction in activity to 1.4 µM (possibly at the nonspe-
cific level) whereas extending the chain to 3 or 4 carbon
atoms gave compounds 15 and 16 with improved activ-
ity. At 70 nM, compound 16 was a particularly potent
inhibitor of cPLA2, and this low concentration of inhibi-
tor dispelled any doubts about nonspecific inhibition by
this compound, even in the bilayer assay. Attempts to
increase the potency of compound 4 by making the
ketone more electrophilic met with mixed success. For
instance, oxidizing the linking sulfur atom to sulfoxide
17 or sulfone 18 gave weaker inhibitors, but this was
probably due to the concomitant reduction in lipophi-
licity achieved by this transformation. Exchanging the
linking sulfur atom for oxygen gave inhibitors 19-21
with broadly similar activity to the S series 4, 15, and
16. In the oxygen series, the inhibitors have probably
gained potency by having a more electrophilic ketone
to interact with the serine hydroxyl, but they have also
lost activity by having a shorter chain to the acidic group
as well as being slightly less lipophilic. This balance of
effects is likely to account for the similar overall

1354 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Connolly et al.
Ta ble 4. Inhibition of Cytosolic PLA2: Introduction of an
Aromatic Group
nated AR-C70484XX) is a potent inhibitor of cPLA2 both
in the presence and in the absence of a lipid-water
interface.
The increased activity of compound 22 over compound
4 is probably due to both the conformational restriction
of the acidic linking group and the increased electron-
withdrawing effect of a second aryloxy group on the
electrophilic ketone serine trap. In addition, incorpora-
tion of the benzoic acid moiety leads to an increase in
lipophilicity. However, because the increased potency
is seen in both the bilayer assay and the soluble
substrate assay, this effect cannot be attributed solely
to increased membrane anchoring (since no membrane
exists in the latter assay).
The isomeric meta-substituted benzoic acid 23 is still
very potent but less so in comparison with 22, and the
phenyl acetic acids 24 and 25 show a similar trend with
the para-substituted compound (24) being the more
active with an IC50 of 17 nM. Extending the acidic chain
by a further oxygen atom, as in compound 26, gave no
change in potency as compared to the analogous meta-
substituted compound 25. To examine electronic effects
further, we prepared compound 27 containing an extra
methoxy group on the aryl ring and observed a drop in
potency to 74 nM. This is possibly due to the electron-
releasing effect of the ortho-methoxy group being re-
layed to the central carbonyl group via the aryl ring and
the linking oxygen atom. Conversely, inclusion of an
extra electron-withdrawing nitro group on the benzoic
acid aryl ring, as in compound 28, did give a potent
inhibitor of cPLA2 with an IC50 of 20 nM, although still
2- to 4-fold less active than the parent 22. Steric effects
accompanying the inclusion of these ortho substituents
cannot be ruled out. Compounds 29 and 30, in which a
methylene has been inserted between the oxygen atom
and the benzoic acid, are not as active as compound 22,
although in this case the reduced activity may be as a
result of the poorer electron-withdrawing effect of
benzyloxy as compared to aryloxy.
With compound 22 in hand, we reinvestigated the
effect of the lipophilic substituent in this exceptionally
potent benzoic acid series. From Table 5, it can be seen
that systematically reducing the chain length of the
decyloxy chain from 10 carbons to 8, 6, 4, and 2
(compounds 31-34, respectively) produces a near linear
reduction in potency. This effect is observed in both the
bilayer and the soluble substrate assay, and because (as
mentioned previously) there is no lipid interface present
in the latter screen, the fall in activity cannot be due to
membrane partitioning. A more likely explanation for
this activity-lipophilicity correlation is that the decyl-
oxy group is actually binding to a lipophilic pocket in
the enzyme. Again, this would not be surprising given
the lipophilic nature of the natural substrate. Similar
to the result seen in Table 1, extending the chain beyond
10 carbon atoms to the dodecyloxy analogue (35) does
not result in any further increase in potency in the
a
Inhibition of cPLA2 measured against an aggregated phos-
pholipid substrate. IC50 values are the means of at least two
b
independent determinations. Errors are within (20%. Inhibition
of cPLA2 measured against a soluble substrate. c Lipophilicity as
calculated by Daylight.35
Not tested. e Flat dose response.
d
Compound was poorly soluble.
inhibition observed. Nonetheless, compound 21 was the
most potent inhibitor of this alkanoic acid series of
cPLA2 inhibitors having a measured IC50 of 30 nM in
the bilayer assay.
Both the sulfur and the oxygen-linked series of
inhibitors described in Table 3 have optimal activity
with pentanoic acid substituents, and we were con-
cerned that these compounds were extremely linear and
flexible molecules. Therefore, we next turned our at-
tention to restricting the conformation of the alkyl chain
linking the ketone group to the acid function. After some
investigation, compound 22 (Table 4), containing a para
benzoic acid group linked by an oxygen atom to the
propan-2-one serine trap, was discovered. This com-
pound was extremely potent in the bilayer assay with
an IC50 of 8 nM. At this time in our program, we also
began testing our inhibitors against cPLA2 in an assay
that used a simplified and soluble substrate.32 Com-
pound 22 is also highly potent in this soluble substrate
assay with an IC50 of 30 nM, notably inhibiting the
action of the enzyme in a situation where there is no
aggregated lipid present. Thus, compound 22 (desig-

1,3-Disubstituted Propan-2-one Skeleton
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1355
Ta ble 5. Inhibition of Cytosolic PLA2: Variation of the
Ta ble 6. Inhibition of Cytosolic PLA2: Changes to the Ketone
Lipophilic Aryl Substituent in the Benzoic Acid Series
and Acid in the Benzoic Acid Series
bilayer assay soluble assay
bilayer assay soluble assay
compd
X, Y
dO
dO
H, OH
H, H
R
IC50 (µM)a
IC50 (µM)b
cLog Pc
compd
RX
C10H21
IC50 (µM)a
IC50 (µM)b
cLog Pc
22
40
41
42
H
allyl
H
0.008
>10d
1.3
0.030
NTe
53
7.8
8.7
7.7
8.8
22
31
32
33
34
35
36
37
38
39
O
0.008
0.046
0.28
4.3
0.030
0.41
4.6
7.8
6.8
5.7
4.7
3.6
8.9
6.6
4.9
4.8
4.6
C8H17
C6H13
O
O
H
>30d
26
C4H9O
C2H5O
28
28
67
a
Inhibition of cPLA2 measured against an aggregated phos-
pholipid substrate. IC50 values are the means of at least two
C
12H25
O
NTd
0.048
2.2
0.026
0.46
16
20
5.3
Ph(CH2)5O
PhCH2O
4-MeOPhCH2O
4-NO2PhCH2O
b
independent determinations. Errors are within (20%. Inhibition
of cPLA2 measured against a soluble substrate. c Lipophilicity as
>3e
d
calculated by Daylight.35 No inhibition at this concentration.
>1e
e Not tested.
a
Inhibition of cPLA2 measured against an aggregated phos-
pholipid substrate. IC50 values are the means of at least two
On the other hand, convincing evidence for the specific
interaction of the active ketones of this series of inhibi-
tors of cPLA2 is demonstrated by simple changes to the
activation of the electrophilic ketone moiety, as shown
in Table 7. Compound 43 differs from 22 only by having
a phenylpentylthio substituent on the aryl ring instead
of the decyloxy group of the latter compound and shows
similar high levels of potency against cPLA2 in both
aggregated and soluble substrate assays. Simple re-
placement of one of the linking oxygen atoms, which also
serve to activate the ketone serine trap, by a methylene
gives a compound (44) with very similar physical
properties (compare cLogPs) but drastically reduced
activity.
Similarly, in the meta-benzoic acid series, replace-
ment of one of the activating oxygen atoms of compound
23 (Table 7) with methylene, as in compound 45, leads
to a substantial reduction in potency. Surprisingly,
retaining the oxygen atom but inserting a methylene
group between the oxygen and the decyloxyphenyl
group, to give compound 46, also reduces activity to a
similar extent. While this insertion may reduce the
electron withdrawing, and hence carbonyl-activating
effect, of this group to a small degree, this is unlikely
to be sufficient to account for the observed fall in
potency. Indeed, an analogous insertion of a methylene
group on the benzoic acid side, as in compound 30,
produced no fall in potency. Taken together, these
results suggest that this series of inhibitors may be more
tightly bound to enzyme around the alkoxy-substituted
aryl ring than around the benzoic acid region. Because
this alkoxy-substituted aryl ring was designed (see
above) to mimic the ∆5,6 double bond of arachidonic
acid, it may be hypothesized that insertion of an extra
methylene group in this region of the inhibitor (as in
compound 46) dislocates the aryl ring such that it is no
longer recognized by the enzyme as a bioisostere for the
∆5,6 double bond of the natural substrate.
b
independent determinations. Errors are within (20%. Inhibition
of cPLA2 measured against a soluble substrate. c Lipophilicity as
d
calculated by Daylight.35
concentration.
Not tested. e No inhibition at this
soluble substrate assay, suggesting that if the decyloxy
chain binds to cPLA2 then the lipophilic pocket is
optimally filled with this substituent. This implies that
the decyloxyphenoxymethyl carbonyl moiety of com-
pound 22, which was designed to mimic the arachidonyl
group in substrate, is an excellent fit for the enzyme
catalytic site.
Replacement of the decyloxy group of 22 with phen-
ylpentyloxy gives a reasonably potent inhibitor of cPLA2
(compound 36, Table 5). However, comparison of this
compound with the octyloxy-substituted analogue (31)
shows that it is almost identical in terms of lipophilicity
and potency. If the substituent at this position of the
inhibitor is indeed binding within the enzyme, then a
phenyl group at the end of the chain is clearly quite
acceptable. Likewise, truncating the phenylpentyl group
to a simple benzyl substituent produces a less potent
inhibitor (37), but the similarity of the lipophilicity and
activity of this compound with the butyloxy compound
(33) further demonstrates that it is probably only the
lipophilicity of each alkyl chain that contributes to
activity. The corresponding para-methoxybenzyl com-
pound 38 and the para-nitrobenzyl compound 39 show
very similar potencies to the parent 37.
Simple changes to the highly potent keto-acid 22, such
as esterification (compound 40) or reduction (41 and 42),
again largely destroy activity (see Table 6). While these
effects were previously demonstrated for the earlier less
potent inhibitor 4 (see Table 2), a comparison between
the two series is quite illuminating. For instance, it is
notable that the reduced hydroxy compounds of each
series (12 and 41, respectively) give similar IC50 values
in the bilayer assay, despite the parent ketones 4 and
22 having quite different potencies. This comparison
lends support to the hypothesis that the level of activity
seen with the alcohols in the bilayer assay is likely to
be due to nonspecific inhibition. Furthermore, when
alcohol 41 was tested in the soluble substrate assay,
where no inhibition by interference with an aggregated
substrate is possible per se, it was essentially inactive
(see Table 6).
In summary, we have described the design and
synthesis of a completely novel series of inhibitors of
cPLA2 based on a 1,3-diheteroatom-substituted propan-
2-one skeleton. These inhibitors show well-defined
structure-activity relationships that point to the re-
quirement of both lipophilic and electrophilic substitu-
ents for optimal potency. Most notably, the inhibitors
show high levels of inhibitory potency in both ag-
gregated bilayer-based and soluble substrate-based as-

1356 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Connolly et al.
Ta ble 7. Inhibition of Cytosolic PLA2: Effect of the Flanking Heteroatoms on Potency
bilayer assay
soluble assay
compd
RZ
X
Y
acid
IC50 (µM)a
IC50 (µM)b
cLog Pc
43
44
23
45
46
30
Ph(CH2)5S
Ph(CH2)5S
O
O
O
CH2
CH2O
O
O
CH2
O
O
O
para
para
meta
meta
meta
meta
0.004
1.2
0.050
0.690
0.660
0.035
0.08
6.5
7.2
7.3
7.8
8.0
7.8
7.7
C
C
C
C
10H21
10H21
10H21
10H21
O
O
O
O
NTd
2.0
3.3
0.15
OCH2
a
Inhibition of cPLA2 measured against an aggregated phospholipid substrate. IC50 values are the means of at least two independent
determinations. Errors are within (20%. Inhibition of cPLA2 measured against a soluble substrate. c Lipophilicity as calculated by
b
Daylight.35 Not tested.
d
Ta ble 8. Inhibition of Cytosolic PLA2: Comparison of
Arachidonoyl Trifluoromethyl Ketone 1 and AR-C70484 22
mmol) in dry dimethylformamide (DMF, 700 mL) under
nitrogen. After a further 20 min, a solution of decyl bromide
(59.7 g, 270 mmol) in dry DMF (50 mL) was added over 5 min.
The brown solution was stirred for 3 h, poured into water, and
acidified with dilute hydrochloric acid. The reaction product
was extracted with ethyl acetate, and the organic extract was
washed with water, dried (MgSO4), and evaporated under
reduced pressure. The residue was purified by flash chroma-
tography (hexane/ethyl acetate, 19:1 to 9:1) to give a white
solid 4a (24.23 g, 36%); mp 69-70 °C. 1H nuclear magnetic
resonance (NMR) (CDCl3): δ 0.88 (t, 3H), 1.25-1.30 (m, 12H),
1.42 (m, 2H), 1.71-1.78 (m, 2H), 3.89 (t, 2H), 6.73-6.82 (m,
4H). Mass spectroscopy (MS) electron impact (EI): m/z 250
(M)+.
bilayer soluble
assay
IC50
assay
IC50
HL60 cell
assay
compd
name
(µM)a
(µM)b IC50 (µM)c cLog Pd
1
22
AACOCF3
AR-C70484XX 0.008
NTe
0.800
0.030
29
2.8
7.9
7.8
a
Inhibition of cPLA2 measured against an aggregated phos-
pholipid substrate. IC50 values are the means of at least two
b
independent determinations. Errors are within (20%. Inhibition
of cPLA2 measured against a soluble substrate. c Inhibition of 3H-
d
arachidonic acid production by stimulated HL60 cells. Lipophi-
licity as calculated by Daylight.35 e Not tested.
2-[(4-(Decyloxy)p h en oxy)m eth yl]oxir a n e (4b). A mix-
ture of 4a (23.0 g, 92 mmol), epichlorohydrin (42.6 g, 460
mmol), and cesium carbonate (30 g, 92 mmol) in acetonitrile
(200 mL) was heated under reflux for 3 h. The mixture was
cooled, poured into water, and extracted with ethyl acetate.
The separated organic solution was washed with saturated
brine solution and dried (MgSO4), and the solvent was
evaporated under reduced pressure. The residue was purified
by flash chromatography (hexane/diethyl ether, 4:1) to give a
white solid 4b (25.56 g, 91%); mp 57-58 °C. 1H NMR
(CDCl3): δ 0.88 (t, 3H), 1.27-1.30 (m, 12H), 1.44 (m, 2H),
1.71-1.77 (m, 2H), 2.74-2.91 (m, 2H), 3.34 (m, 1H), 3.88 (t,
2H), 3.92-4.18 (m, 2H), 6.80-6.87 (m, 4H). MS (EI): m/z 306
(M)+. Anal. (C19H30O3) C, H.
says and are among the most potent and druglike
inhibitors of cPLA2 described to date.34 For comparison,
compound 22 (AR-C70484XX) is more than 20-fold more
potent than arachidonyl trifluoromethyl ketone (1) in
the soluble substrate assay and also displays higher
potency against arachidonic acid production in a cellular
(HL60 cells) assay (see Table 8). Most importantly,
compound 22, by virtue of its design, is a “modular”
molecule that, unlike arachidonyl trifluoromethyl ke-
tone, lends itself to simple synthetic manipulation and
therefore provides an excellent and potent starting point
with which to further explore structure-activity rela-
tionships with cPLA2. Studies on the reduction of both
the high lipophilicity and the electrophilicity of this
initial series of cPLA2 inhibitors, while retaining potency
against the enzyme, will be the subject of future papers.
1,1-Dim et h ylet h yl 3-[(3-(4-(Decyloxy)p h en oxy)-2-h y-
d r oxyp r op yl)th io]p r op a n oa te (4c). A solution of 4b (2 g,
6.5 mmol) in acetonitrile (200 mL) was treated with 1,1-
dimethylethyl 3-thiopropanoate (1.1 g, 9.8 mmol) and a
catalytic amount of DABCO. The mixture was heated under
reflux under nitrogen for 3 h, cooled, and then poured into
water (400 mL). The precipitate was collected, dissolved in
ethyl acetate, washed with brine, and dried (MgSO4). The
solvent was removed under reduced pressure, and the oily
residue was purified by flash chromatography (hexane/ethyl
Exp er im en ta l Section
Gen er a l. Proton magnetic resonance spectra were obtained
on a Bruker AM360 (360 MHz) or Bruker AMX500 (500 MHz)
instrument. Chemical shifts are reported as δ values (parts
per million) downfield relative to Me4Si as an internal stan-
dard. Mass spectra were obtained with Micromass Platform,
Micromass LCT, and VG Sector spectrometers; ESI indicates
electrospray ionization, APCI/NH3 indicates atmospheric pres-
sure chemical ionization in the presence of ammonia, FAB-
(Xe) indicates fast atom bombardment ionization using xenon.
The molecular ions were detected in either negative (-ve) or
positive (+ve) mode. Combustion analyses were performed
with a Carlo Erba EA1108 instrument. Melting points were
determined on a Buchi 510 melting point apparatus with a
silicone oil bath and are uncorrected. Chromatographies were
carried out in flash mode using silica gel 60 (230-400 mesh)
from E. Merck.
1
acetate, 4:1) to give a colorless oil 4c (1.76 g, 57%). H NMR
(CDCl3): δ 0.88 (t, 3H), 1.24-1.27 (m, 14H), 1.45 (s, 9H), 1.71-
1.77 (m, 2H), 2.52-2.56 (t, 2H), 2.71-2.90 (m, 4H), 3.88-3.91
(t, 2H), 3.97-3.99 (d, 2H), 4.07-4.15 (m, 1H), 6.83-6.86 (m,
4H). MS (EI): m/z 468 (M)+.
1,1-Dim eth yleth yl 3-[(3-(4-(Decyloxy)p h en oxy)-2-oxo-
p r op yl)th io]p r op a n oa te (4d ). A solution of 4c (1.79 g, 3.8
mmol) in dry DCM (5 mL) was treated with the Dess-Martin
periodinane reagent (2.43 g, 5.7 mmol) and stirred under
nitrogen at room temperature for 2 h. Ether (75 mL) and a
solution of sodium thiosulfate (9.15 g, 37 mmol) in saturated
sodium bicarbonate solution (75 mL) were added to the
reaction mixture. After the mixture was stirred for 5 min, the
organic layer was separated, washed with brine, and dried
(MgSO4). The solvent was evaporated under reduced pressure,
and the residue was purified by flash chromatography (hexane/
4-(Decyloxy)p h en ol (4a ). Sodium hydride (10.92 g, 270
mmol, 60% dispersion in oil) was added batchwise over half
an hour to a stirred solution of 1,4-hydroquinone (30 g, 270
1
ethyl acetate, 9:1) to give a colorless oil 4d (1.14 g, 64%). H

1,3-Disubstituted Propan-2-one Skeleton
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1357
NMR (CDCl3): δ 0.88 (t, 3H), 1.24-1.28 (m, 14H), 1.45 (s, 9H),
1.71-1.77 (m, 2H), 2.53 (t, 2H), 2.77 (t, 2H), 3.42 (s, 2H), 3.89
(t, 2H), 4.71 (s, 2H), 6.84 (s, 4H). MS (EI): m/z 466 (M)+.
3-[(3-(4-(Decyloxy)ph en oxy)-2-oxopr opyl)th io]pr opan o-
ic Acid (4). A solution of 4d (1.6 g, 3.3 mmol) in dry DCM (50
mL) was treated with trifluoroacetyl (TFA, 10 mL) and stirred
at room temperature under nitrogen for 2 h. Toluene (30 mL)
was added, and the solvents were evaporated under reduced
pressure. The residue was recrystallized from ethyl acetate/
3.65 (s, 2H), 3.90 (t, 2H), 4.67 (s, 2H), 6.83 (m, 4H). MS (EI):
m/z 396 (M)+. Anal. calcd for (C21H32O5S) C, H, S, 8.09; found,
7.38.
4-[(3-(4-(Decyloxy)p h en oxy)-2-oxop r op yl)th io]bu ta n o-
1
ic Acid (15). mp 90-91 °C. H NMR (CDCl3): δ 0.88 (t, 3H),
1.27 (m, 12H), 1.44 (m, 2H), 1.71-1.77 (m, 2H), 1.89-1.96 (m,
2H), 2.48 (t, 2H), 2.59 (t, 2H), 3.39 (s, 2H), 3.89 (t, 2H), 4.71
(s, 2H), 6.84 (m, 4H). MS (EI): m/z 424 (M)+. Anal. (C23H36O5S)
C, H, S.
1
hexane to give a white solid 4 (0.92 g, 80%); mp 95-96 °C. H
5-[(3-(4-(Decyloxy)ph en oxy)-2-oxopr opyl)th io]pen tan o-
NMR (CDCl3): δ 0.88 (t, 3H), 1.27 (m, 12H), 1.44 (m, 2H),
1.71-1.77 (m, 2H), 2.68 (t, 2H), 2.81 (t, 2H), 3.44 (s, 2H), 3.89
(t, 2H), 4.70 (s, 2H), 6.84 (s, 4H). MS (EI): m/z 410 (M)+. Anal.
(C22H34O5S) C, H, S.
1
ic Acid (16). mp 88-89 °C. H NMR (CDCl3): δ 0.88 (t, 3H),
1.27 (m, 12H), 1.43 (m, 2H), 1.64-1.77 (m, 6H), 2.37 (t, 2H),
2.54 (t, 2H), 3.38 (s, 2H), 3.89 (t, 2H), 4.72 (s, 2H), 6.84 (m,
4H). MS (ESI -ve): m/z 437 (M - H)-. Anal. (C24H38O5S) C,
H, S.
The synthetic route described for the preparation of 4 was
used to prepare compounds 2, 3, 5-12, and 14-16, starting
with the appropriate protected acids and alkoxyphenols.
3-[(3-(2-Naph th yloxy)-2-oxopr opyl)th io]pr opan oic Acid
(2). mp 90-2 °C. 1H NMR (CDCl3): δ 2.68 (t, 2H), 2.82 (t, 2H),
3.48 (s, 2H), 4.88 (s, 2H), 7.01 (d, 1H), 7.23 (dd, 1H), 7.39 (t,
1H), 7.46 (t, 1H), 7.72 (d, 2H), 7.79 (d, 2H). MS (EI): m/z 304
(M+). Anal. (C16H16O4S) C, H, S.
3-[(3-(4-(Decyloxy)p h en oxy)-2-oxop r op yl)su lfin yl]p r o-
p a n oic Acid (17). A solution of 4d (0.5 g, 1.1 mmol) in DCM
(20 mL) was treated with m-chloroperbenzoic acid (0.27 g, 1.1
mmol). The reaction mixture was stirred at room temperature
for 1 h and poured into a saturated aqueous sodium meta-
bisulfite solution (20 mL). The organic layer was separated,
washed with water and brine, and dried (MgSO4). The solvent
was removed under reduced pressure, and the residue was
purified by flash column chromatography (hexane/ethyl ace-
tate, 2:1) to give a solid (0.44 g). This solid was treated with a
4:1 mixture of TFA:DCM (25 mL) for 2 h at ambient temper-
ature. The solvent was removed under reduced pressure, and
the residue was purified by flash column chromatography
(hexane/ethyl acetate, 2:1 + 1% acetic acid) to give a white
3-[(3-(4-P h en oxyp h en oxy)-2-oxop r op yl)th io]p r op a n o-
1
ic Acid (3). mp 94-96 °C. H NMR (CDCl3): δ 2.69 (t, 2H),
2.82 (t, 2H), 3.45 (s, 2H), 4.75 (s, 2H), 6.88-7.06 (m, 6H), 7.26
(s, 1H), 7.31 (t, 2H). MS (EI): m/z 346 (M+). Anal. (C18H18O5S)
C, H, S.
3-[(3-(4-Eth oxyp h en oxy)-2-oxop r op yl)th io]p r op a n oic
Acid (5). mp 78-79 °C. 1H NMR (CDCl3): δ 1.39 (t, 3H), 2.67
(t, 2H), 2.80 (t, 2H), 3.44 (s, 2H), 3.95-4.00 (q, 2H), 4.70 (s,
2H), 6.83 (s, 4H). MS (EI): m/z 370 (M + tetramethylsilane
(TMS))+. Anal. (C14H18NO5S) C, H, S.
1
solid 17 (0.24 g, 62%); mp 116-117 °C. H NMR (DMSO-d6):
δ 0.85 (t, 3H), 1.25 (m, 12H), 1.38 (bs, 2H), 1.64-1.68 (m, 2H),
2.66 (t, 2H), 2.94-3.16 (m, 2H), 3.87 (t, 2H), 3.99-4.22 (dd,
2H), 4.83 (s, 2H), 6.82 (m, 4H), 12.55 (bs, 1H). MS (FAB-Xe):
m/z 427 (M + H)+. Anal. (C22H34O6S) C, H, S.
3-[(3-(4-Hexyloxyp h en oxy)-2-oxop r op yl)th io]p r op a n o-
1
ic Acid (6). mp 88-89 °C. H NMR (CDCl3): δ 0.89 (t, 3H),
1.3-1.5 (m, 6H), 1.73-1.77 (m, 2H), 2.67 (t, 2H), 2.81 (t, 2H),
3.44 (s, 2H), 3.89 (t, 2H), 4.70 (s, 2H), 6.83 (s, 4H). MS (EI):
m/z 354 (M)+. Anal. (C18H26O5S) C, H, S.
3-[(3-(4-(Decyloxy)p h en oxy)-2-oxop r op yl)su lfon yl]p r o-
p a n oic Acid (18). A solution of 4d (1.76 g, 3.8 mmol) in dry
DCM (50 mL) was treated with m-chloroperbenzoic acid (2.04
g, 8.3 mmol) and stirred at room temperature for 2 h. Workup
and deprotection were carried out as for 17 to give a white
3-[(3-(4-(Tetr adecyloxy)ph en oxy)-2-oxopr opyl)th io]pr o-
1
p a n oic Acid (7). mp 91-93 °C. H NMR (CDCl3): δ 0.87 (t,
3H), 1.2-1.4 (m, 20H), 1.45 (m, 2H), 1.72-1.76 (m, 2H), 2.67
(t, 2H), 2.81 (t, 2H), 3.44 (s, 2H), 3.89 (t, 2H), 4.70 (s, 2H),
6.83 (m, 4H). MS (EI): m/z 466 (M)+. Anal. calcd for (C26H42O5S)
C, H, S, 6.87; found, 6.28.
1
solid 18 (1.05 g, 63%); mp 138-140 °C. H NMR (DMSO-d6):
δ 0.85 (t, 3H), 1.25 (m, 12H), 1.38 (m, 2H), 1.63-1.68 (m, 2H),
2.71 (t, 2H), 3.48 (t, 2H), 3.88 (t, 2H), 4.62 (s, 2H), 4.89 (s,
2H), 6.83 (m, 4H), 12.64 (bs, 1H). MS (ESI -ve): m/z 441 (M
- H)-. Anal. (C22H34O7S‚0.5H2O) C, H, S.
3-[(3-(4-((2-Na p h th yl)m eth oxy)p h en oxy)-2-oxop r op yl)-
th io]p r op a n oic Acid (8). mp 137-138 °C. 1H NMR
(CDCl3): δ 2.66 (t, 2H), 2.79 (t, 2H), 3.43 (s, 2H), 4.69 (s, 2H),
5.18 (s, 2H), 6.85 (d, 2H), 6.95 (d, 2H), 7.47-7.53 (m, 3H),
7.82-7.87 (m, 4H). MS (EI): m/z 410 (M)+. Anal. (C23H22O5S‚
0.5H2O) C, H, S.
3-[3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy]p r op a n oic
Acid (19). 1,3-Propanediol (1.1 mL, 15 mmol) was added to a
stirred suspension of sodium hydride (0.36 g, 15 mmol) in
anhydrous DMF (25 mL). After 15 min, 4b (2 g, 6.5 mmol)
was added, and the mixture was heated at 65 °C for 3.5 h.
The reaction mixture was cooled and poured into dilute
hydrochloric acid, and the product was extracted with ethyl
acetate, which was washed with water and dried (MgSO4). The
solvent was evaporated under reduced pressure, and the
residue was purified by flash chromatography (hexane/ethyl
acetate, 1:2) to give 3-[3-(4-(decyloxy)phenoxy)-2-hydroxypro-
poxy]propan-1-ol as a white solid (1.3 g). This solid (0.5 g, 1.3
mmol) and PDC (3.4 g, 9 mmol) in anhydrous DMF (25 mL)
were stirred at room temperature for 18 h. The reaction
mixture was poured into aqueous sodium metabisulfite solu-
tion and extracted with ethyl acetate, which was washed with
brine and dried (MgSO4). The solvent was evaporated under
reduced pressure, and the residue was purified by flash
chromatography (hexane/ethyl acetate, 2:1 + 0.5% acetic acid)
3-[(3-(3-(Decyloxy)ph en oxy)-2-oxopr opyl)th io]pr opan o-
1
ic Acid (9). mp 88 °C. H NMR (CDCl3): δ 0.88 (t, 3H), 1.27
(m, 12H), 1.43 (m, 2H),1.76 (m, 2H), 2.65 (t, 2H), 2.81 (t, 2H),
3.44 (s, 2H), 3.92 (t, 2H), 4.74 (s, 2H), 6.46-6.56 (m, 3H), 7.18
(t, 1H). MS (EI): m/z 410 (M)+. Anal. (C22H34O5S) C, H, S.
3-[(3-(2-(Decyloxy)ph en oxy)-2-oxopr opyl)th io]pr opan o-
1
ic Acid (10). mp 77 °C. H NMR (CDCl3): δ 0.88 (t, 3H), 1.27
(m, 12H), 1.45 (m, 2H), 1.81 (m, 2H), 2.67 (t, 2H), 2.80 (t, 2H),
3.54 (s, 2H), 3.99 (t, 2H), 4.74 (s, 2H), 6.87-7.00 (m, 4H). MS
(EI): m/z 410 (M)+. Anal. (C22H34O5S) C, H, S.
Meth yl 3-[(3-(4-(Decyloxy)p h en oxy)-2-oxop r op yl)th io]-
1
p r op a n oa te (11). mp 36-40 °C. H NMR (CDCl3): δ 0.88 (t,
3H), 1.27-1.30 (m, 12H), 1.44 (m, 2H), 1.77 (m, 2H), 2.63 (t,
2H), 2.81 (t, 2H), 3.43 (s, 2H), 3.70 (s, 3H), 3.90 (t, 2H), 4.71
(s, 2H), 6.84 (bs, 4H). MS (EI): m/z 424 (M)+. Anal. (C23H36O5S‚
0.25H2O) C, H, S.
1
to give a white solid 19 (0.19 g, 37%); mp 69-70 °C. H NMR
(CDCl3): δ 0.88 (t, 3H), 1.27 (m, 12H), 1.43 (m, 2H), 1.73-
1.77 (m, 2H), 2.72 (t, 2H), 3.82 (t, 2H), 3.89 (t, 2H), 4.42 (s,
2H), 6.82 (m, 4H). MS (EI): m/z 394 (M)+. Anal. (C22 H34 O6‚
0.25H2O) C, H.
3-[(3-(4-(Decyloxy)ph en oxy)-2-h ydr oxypr opyl)th io]pr o-
p a n oic Acid (12). mp 102-104 °C. H NMR (CDCl3): δ 0.88
(t, 3H), 1.27-1.30 (m, 12H), 1.44 (m, 2H), 1.71-1.77 (m, 2H),
2.67 (t, 2H), 2.80-2.90 (m, 4H), 3.90 (t, 2H), 3.99 (d, 2H), 4.11
(m, 1H), 6.80-6.87 (m, 4H). MS (EI): m/z 412 (M)+. Anal.
(C22H36O5S) C, H, S.
3-[(3-(4-(Decyloxy)p h en oxy)-2-oxop r op yl)t h io]a cet ic
Acid (14). mp 76-77 °C. 1H NMR (CDCl3): δ 0.88 (t, 3H),
1.27 (m, 12H), 1.44 (m, 2H), 1.71-1.77 (m, 2H), 3.34 (s, 2H),
1
4-[3-(4-(Decyloxy)ph en oxy)-2-oxopr opoxy]bu tan oic Acid
(20). The procedure described for the preparation of 19 was
applied to 1,4-butanediol to give 20 as a white solid; mp 70-
72 °C. 1H NMR (CDCl3): δ 0.88 (t, 3H), 1.27 (m, 12H), 1.44
(m, 2H), 1.73-1.77 (m, 2H), 1.97 (m, 2H), 2.51 (t, 2H), 3.58 (t,

1358 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Connolly et al.
2H), 3.89 (t, 2H), 4.36 (s, 2H), 4.67 (s, 2H), 6.83 (m, 4H). MS
(s and d, 3H), 6.82-6.88 (m, 4H), 7.18 (t, 1H), 13.05 (bs, 1H).
MS (FAB-Xe): m/z 473 (M + H)+. Anal. (C27H36O7) C, H.
4-[3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy]-3-m eth oxy-
ben zoic Acid (27). mp 157-158 °C. 1H NMR (CDCl3): δ 0.88
(t, 3H), 1.27 (m, 12H), 1.44 (m, 2H), 1.74-1.78 (m, 2H), 3.90
(t, 2H), 3.95 (s, 3H), 4.82 (s, 2H), 5.06 (s, 2H), 6.79 (d, 1H),
6.85 (m, 4H), 7.63-7.72 (m, 3H). MS (EI): m/z 472 (M)+. Anal.
(C27H36O7) C, H.
(ESI -ve): m/z 407 (M - H)-. Anal. (C23H36O6) C, H.
5-[3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy]p en t a n oic
Acid (21). The procedure described for the preparation of 19
was applied to 1,5-pentanediol to give 21 as a white solid; mp
1
69-71 °C. H NMR (DMSO-d6): δ 0.85 (t, 3H), 1.25 (m, 12H),
1.40 (m, 2H), 1.54 (m, 2H), 1.64 (m, 2H), 2.24 (m, 2H), 2.50 (t,
2H), 3.44 (t, 2H), 3.87 (t, 2H), 4.27 (s, 2H), 4.79 (s, 2H), 6.82
(bs, 4H), 12.04 (bs, 1H). MS (ESI -ve): m/z 421 (M - H)-.
Anal. (C24H38O6) C, H.
4-[3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy]-3-n itr oben -
1
zoic Acid (28). mp 165-167 °C. H NMR (DMSO-d6): δ 0.86
(t, 3H), 1.25 (m, 12H), 1.39 (m, 2H), 1.67 (m, 2H), 3.88 (t, 2H),
4.95 (s, 2H), 5.35 (s, 2H), 6.84-6.90 (m, 4H), 7.39 (d, 1H), 8.12
(d, 1H), 8.37 (s, 1H), 13.40 (bs, 1H). MS (EI): m/z 487 (M)+.
Anal. (C26H33NO8‚0.25H2O) C, H, N.
3-P r op en yl 4-[3-(4-(Decyloxy)p h en oxy)-2-h yd r oxyp r o-
p oxy]ben zoa te (22a ). 3-Propenyl 4-hydroxy benzoate (3.24
g, 18 mmol), prepared from 4-hydroxybenzoic acid, TMS-
chloride, and 3-hydroxypropene, and 4b (5.57 g, 18 mmol) in
dry DMF (90 mL) were treated with a catalytic amount of
DABCO (0.2 g) under nitrogen, and the mixture was heated
under reflux for 3 h. The cooled solution was then poured into
2 N HCl (300 mL), extracted with ethyl acetate (3 × 75 mL),
washed with brine, and dried (MgSO4). The solvent was
removed under reduced pressure, and the residue was purified
by flash chromatography (hexane/ethyl acetate, 4:1) to give a
4-[(3-(4-(Decyloxy)ph en oxy)-2-oxopr opoxy)m eth yl]ben -
1
zoic Acid (29). mp 125-126 °C. H NMR (DMSO-d6): δ 0.86
(t, 3H), 1.25 (m, 12H), 1.38 (m, 2H), 1.63-1.68 (m, 2H), 3.87
(t, 2H), 4.42 (s, 2H), 4.63 (s, 2H), 4.83 (s, 2H), 6.83 (m, 4H),
7.48 (d, 2H), 7.94 (d, 2H), 12.96 (bs, 1H). MS (ESI -ve): m/z
455 (M - 1)-. Anal. (C27H36O6‚0.5H2O) C, H.
3-[(3-(4-(Decyloxy)ph en oxy)-2-oxopr opoxy)m eth yl]ben -
zoic Acid (30). mp 95-97 °C. 1H NMR (CDCl3): δ 0.88 (t,
3H), 1.30 (m, 12H), 1.43 (m, 2H), 1.72-1.76 (m, 2H), 3.89 (t,
2H), 4.43 (s, 2H), 4.68 (s, 2H), 4.71 (s, 2H), 6.82 (m, 4H), 7.50
(t, 1H), 7.63 (d, 1H), 8.01 (m, 2H). MS (EI): m/z 456 (M)+. Anal.
(C27H36O6‚0.5H2O) C, H.
1
white powder 22a (5.61 g, 64%). H NMR (CDCl3): δ 0.88 (t,
3H), 1.26 (m, 12H), 1.42 (m, 2H), 1.76 (m, 2H), 2.59 (d, 1H),
3.90 (t, 2H), 4.11 (m, 2H), 4.19 (m, 2H), 4.39 (m, 1H), 4.80 (d,
2H), 5.43-5.27 (dd, 2H), 6.06 (m, 1H), 6.95 (m, 4H), 6.97 (d,
2H), 8.04 (d, 2H). MS (FAB-Xe): m/z 485 (M + H)+.
4-[3-(4-(Octyloxy)ph en oxy)-2-oxopr opoxy]ben zoic Acid
(31). mp 162-163 °C. 1H NMR (DMSO-d6): δ 0.85 (t, 3H), 1.2-
1.45 (m, 10H), 1.63-1.68 (m, 2H), 3.88 (t, 2H), 4.93 (s, 2H),
5.12 (s, 2H), 6.83-6.89 (m, 4H), 7.01 (m, 2H), 7.87 (m, 2H),
12.67 (bs, 1H). MS (ESI -ve): m/z 413 (M - H)-. Anal.
(C24H30O6) C, H.
3-P r op en yl 4-[3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy]-
ben zoa te (22b). Compound 22a (4.85 g, 10 mmol) was
oxidized by the procedure described for 4d to afford a white
solid 22b (4.26 g, 88%); mp 68-69 °C. 1H NMR (CDCl3): δ
0.88 (t, 3H), 1.27 (m, 12H), 1.43 (m, 2H), 1.76 (m, 2H), 3.91 (t,
2H), 4.79 (s, 2H), 4.84 (d, 2H), 4.99 (s, 2H), 5.29-5.39 (dd, 2H),
6.04 (m, 1H), 6.86 (m, 4H), 6.93 (d, 2H), 8.04 (d, 2H). MS (EI):
m/z 482 (M)+. Anal. (C29H38O6) C, H.
4-[3-(4-(Decyloxy)ph en oxy)-2-oxopr opoxy]ben zoic Acid
(22). A solution of 22b (4.25 g, 8.8 mmol) in tetrahydrofuran
(THF, 70 mL) was treated with Pd(PPh3)4 (1.15 g, 1 mmol),
and nitrogen gas was bubbled through the solution for 20 min.
Acetic acid (10 mL) was then added, and the reaction was
stirred at room temperature under a nitrogen atmosphere for
4 h. A precipitate formed, which was collected and recrystal-
lized from ethyl acetate to give a white solid 22 (3.02 g, 77%);
mp 165-166 °C. 1H NMR (CDCl3): δ 0.88 (t, 3H), 1.27 (m,
12H), 1.44 (m, 2H), 1.72-1.76 (m, 2H), 3.90 (t, 2H), 4.85 (s,
2H), 5.03 (s, 2H), 6.82 (d, 2H), 6.87 (d, 2H), 6.94 (d, 2H), 7.97
(d, 2H), 12.24 (bs, 1H). MS (ESI): m/z 442 (M + H)+. Anal.
(C26H34O6, 0.25H2O) C, H.
The synthetic route described for the preparation of 22 was
adapted to prepare compounds 23-41, 43, and 46 starting
with the appropriate protected acids and alkoxyphenols, or in
the case of 46, alkoxybenzyl alcohol.
3-[3-(4-(Decyloxy)ph en oxy)-2-oxopr opoxy]ben zoic Acid
(23). mp 133-134 °C. 1H NMR (DMSO-d6): δ 0.86 (t, 3H), 1.25
(m, 12H), 1.38 (m, 2H), 1.66 (m, 2H), 3.88 (t, 2H), 4.95 (s, 2H),
5.10 (s, 2H), 6.85 (m, 4H), 7.21 (d, 2H), 7.41 (t, 1H), 7.44 (s,
1H), 7.55 (d, 1H), 13.09 (bs, 1H). MS (EI): m/z 442 (M)+. Anal.
(C26H34O6) C, H.
4-[3-(4-(Hexyloxy)ph en oxy)-2-oxopr opoxy]ben zoic Acid
1
(32). mp 162-163 °C. H NMR (CDCl3): δ 0.90 (t, 3H), 1.3-
1.4 (m, 4H), 1.73-1.77 (m, 2H), 3.90 (t, 2H), 4.81 (s, 2H), 4.98
(s, 2H), 6.85 (m, 4H), 6.91 (d, 2H), 8.00 (d, 2H). MS (EI): m/z
386 (M)+. Anal. (C22H26O6) C, H.
4-[3-(4-(Bu toxy)p h en oxy)-2-oxop r op oxy]ben zoic Acid
1
(33). mp 156-157 °C. H NMR (CDCl3): δ 0.92 (t, 3H), 1.36-
1.47 (m, 2H), 1.62-1.70 (m, 2H), 3.89 (t, 2H), 4.94 (s, 2H), 5.13
(s, 2H), 6.83-6.90 (m, 4H), 7.02 (d, 2H), 7.88 (d, 2H), 12.69
(bs, 1H). MS (ESI +ve): m/z 359 (M + H)+. Anal. (C20H22O6)
C, H.
4-[3-(4-(Eth oxy)p h en oxy)-2-oxop r op oxy]ben zoic Acid
(34). mp 172-173 °C. 1H NMR (DMSO-d6): δ 1.29 (t, 3H),
3.92-3.98 (q, 2H), 4.94 (s, 2H), 5.13 (s, 2H), 6.83-6.90 (m, 4H),
7.02 (d, 2H), 7.87 (d, 2H), 12.68 (bs, 1H). MS (ESI -ve): m/z
329 (M - H)-. Anal. (C18H18O6) C, H.
4-[3-(4-(Dod ecyloxy)p h en oxy)-2-oxop r op oxy]b en zoic
1
Acid (35). mp 169-170 °C. H NMR (CDCl3): δ 0.87 (t, 3H),
1.2-1.3 (m, 16H), 1.43 (m, 2H), 1.71 (m, 2H), 3.88 (t, 2H), 4.89
(s, 2H), 5.07 (s, 2H), 6.82-6.86 (m, 4H), 6.96 (d, 2H), 7.91 (d,
2H). MS (FAB-Xe): m/z 471 (M + H)+. Anal. (C28H38O6) C, H.
4-[3-(4-(5-P h en ylp en t yloxy)p h en oxy)-2-oxop r op oxy]-
ben zoic Acid (36). mp 163-164 °C. 1H NMR (CDCl3):
δ
1.45-1.55 (m, 2H), 1.67-1.82 (m, 4H), 2.65 (t, 2H), 3.91 (t,
2H), 4.95 (s, 2H), 5.01 (s, 2H), 6.86 (m, 4H), 6.94 (d, 2H), 7.18
(m, 3H), 7.28 (m, 2H), 8.05-8.08 (m, 2H). MS (EI): m/z 448
(M)+. Anal. (C27H28O6, H2O) C, H.
4-[3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy]p h en yla ce-
tic Acid (24). mp 119-120 °C. 1H NMR (CDCl3): δ 0.88 (t,
3H), 1.27 (m, 12H), 1.44 (m, 2H), 1.73-1.75 (m, 2H), 3.61 (s,
2H), 3.90 (t, 2H), 4.81 (s, 2H), 4.87 (s, 2H), 6.85 (m, 4H), 6.88
(d, 2H), 7.30 (d, 2H). MS (EI): m/z 456 (M)+. Anal. (C27H36O6)
C, H.
4-[3-(4-(P h en ylm eth oxy)p h en oxy)-2-oxop r op oxy]ben -
1
zoic Acid (37). mp 173-175 °C. H NMR (DMSO-d6): δ 3.77
(s, 2H), 3.89 (s, 2H), 4.01 (s, 2H), 6.32-6.49 (dd, 4H), 6.52 (d,
2H), 6.86-7.06 (m, 5H), 7.64 (d, 2H). MS (ESI -ve): m/z 391
(M - H)-. Anal. (C23H20O6, 0.25H2O) C, H.
3-[3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy]p h en yla ce-
tic Acid (25). mp 107-108 °C. 1H NMR (CDCl3): δ 0.88 (t,
3H), 1.27 (m, 12H), 1.44 (m, 2H), 1.73-1.77 (m, 2H), 3.63 (s,
2H), 3.90 (t, 2H), 4.82 (s, 2H), 4.88 (s, 2H), 6.85 (m, 6H), 6.94
(d, 1H), 7.26 (d, 1H). MS (EI): m/z 456 (M)+. Anal. (C27H36O6)
C, H.
[3-(3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy)p h en oxy]-
a cetic Acid (26). mp 111-112 °C. 1H NMR (DMSO-d6): δ
0.86 (t, 3H), 1.25 (m, 12H), 1.39 (m, 2H), 1.63-1.69 (m, 2H),
3.88 (t, 2H), 4.65 (s, 2H), 4.92 (s, 2H), 4.99 (s, 2H), 6.50-6.54
4-[3-(4-((4-Met h oxyp h en yl)m et h oxy)p h en oxy)-2-oxo-
p r op oxy]ben zoic Acid (38). mp 196-198 °C. 1H NMR
(CDCl3): 3.75 (s, 3H), 4.94 (bs, 4H), 5.14 (s, 2H), 6.91 (m, 6H),
7.00 (d, 2H), 7.35 (d, 2H), 7.87 (d, 2H). MS (FAB-Xe): m/z 422
(M)+. Anal. (C24H22O7) C, H.
4-[3-(4-((4-Nit r op h en yl)m et h oxy)p h en oxy)-2-oxop r o-
1
p oxy]ben zoic Acid (39). mp 207-209 °C. H NMR (CDCl3):
4.87 (s, 2H), 5.03 (s, 2H), 5.18 (s, 2H), 6.92 (m, 6H), 7.67 (d,
2H), 7.97 (d, 2H), 8.23 (d, 2H). MS (TOF ES -ve): m/z
436.1027 (M - H)-. (C23H19NO8) requires 436.1033.

1,3-Disubstituted Propan-2-one Skeleton
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6 1359
3-P r op en yl 4-[3-(4-(Decyloxy)p h en oxy)-2-oxop r op oxy]-
ben zoa te (40). mp 68-69 °C. 1H NMR (CDCl3): δ 0.88 (t, 3H),
1.27 (m, 12H), 1.43 (m, 2H), 1.76 (m, 2H), 3.91 (t, 2H), 4.79 (s,
2H), 4.84 (d, 2H), 4.99 (s, 2H), 5.29-5.39 (dd, 2H), 6.04 (m,
1H), 6.86 (m, 4H), 6.93 (d, 2H), 8.04 (d, 2H). MS (EI): m/z 482
(M)+. Anal. (C29H38O6) C, H.
4-[3-(4-(Decyloxy)p h en oxy)-2-h yd r oxyp r op oxy]ben zo-
ic Acid (41). mp 136-137 °C. 1H NMR (DMSO-d6): δ 0.85 (t,
3H), 1.25 (m, 12H), 1.38 (m, 2H), 1.66 (m, 2H), 3.87 (t, 2H),
3.97 (m, 2H), 4.07-4.15 (m, 3H), 5.41 (s, 1H), 6.82-6.90 (m,
4H), 7.04 (d, 2H), 7.88 (d, 2H), 12.64 (bs, 1H). MS (ESI -ve):
m/z 443 (M - H)-. Anal. (C26H36O6‚0.25H2O) C, H.
g, 77%). 1H NMR (CDCl3): δ 1.40-1.48 (m, 2H), 1.56-1.66
(m, 4H), 2.59 (t, 2H), 2.84 (t, 2H), 4.73 (s, 2H), 6.90 (d, 2H),
7.15-7.19 (m, 3H), 7.27-7.32 (m, 2H), 7.35 (d, 2H). MS (EI):
m/z 311 (M)+.
[4-(5-P h en ylp en tylth io)p h en oxy]a ceta ld eh yd e (44b).
Dibal-H (1 M in toluene, 9.5 mL, 9.5 mmol) was added to a
solution of 44a (2 g, 6.5 mmol) in toluene (10 mL) at -40 °C,
under a nitrogen atmosphere. The solution was allowed to
warm to room temperature and stirred at this temperature
for 1 h. Saturated ammonium chloride solution (20 mL) and
ether (20 mL) were added followed by a solution of concen-
trated H2SO4 (8 mL) in water (32 mL). The mixture was stirred
vigorously for 18 h and then extracted with ethyl acetate (250
mL). The organic layer was washed with water and brine,
dried (MgSO4), and evaporated to dryness to give a yellow oil
44b (1.66 g, 82%). 1H NMR (CDCl3): δ 1.43-1.47 (m, 2H),
1.59-1.65 (m, 4H), 2.58 (t, 2H), 2.81 (t, 2H), 4.54 (s, 2H), 6.82
(d, 2H), 7.13-7.19 (m, 3H), 7.24-7.39 (m, 4H), 9.90 (s, 1H).
MS (EI): m/z 314 (M)+.
4-[3-(4-(5-P h en ylp en tylth io)p h en oxy)-2-oxop r op oxy]-
1
ben zoic Acid (43). mp 131-132 °C. H NMR (DMSO-d6): δ
1.34-1.41 (m, 2H), 1.49-1.59 (m, 4H), 2.53 (t, 2H), 2.85 (t,
2H), 5.03 (s, 2H), 5.14 (s, 2H), 6.93 (d, 2H), 7.15-7.17 (s and
d, 3H), 7.24-7.31 (2d, 4H), 7.90 (d, 2H), 12.72 (bs, 1H). MS
(EI): m/z 464 (M)+. Anal. (C27H28O5S) C, H, S.
3-[3-((4-(Decyloxy)p h en yl)m eth yl)-2-oxop r op oxy]ben -
1
4-[4-(5-P h en ylp en t ylt h io)p h en oxy]-3-h yd r oxyb u t -1-
en e (44c). Vinylmagnesium bromide (1 M in THF, 7.5 mL,
7.5 mmol) was added to the crude aldehyde 44b (∼1.7 g, 5.5
mmol) in THF (35 mL) at -10 °C, under a nitrogen atmo-
sphere. It was stirred at -10 °C for 1 h and then allowed to
warm to room temperature. A saturated solution of ammonium
chloride (100 mL) was added, and the product was extracted
into ethyl acetate (3 × 50 mL). The solvent was evaporated to
dryness, and the residue was passed down a silica gel column
(hexane/ethyl acetate, 4:1) to afford a clear oil 44c (0.62 g,
zoic Acid (46). mp 104-106 °C. H NMR (CDCl3): δ 0.88 (t,
3H), 1.2-1.3 (m, 16H), 1.45 (m, 2H), 1.76 (m, 2H), 3.95 (t, 2H),
4.30 (s, 2H), 4.57 (s, 2H), 4.85 (s, 2H), 6.89 (d, 2H), 7.14 (d,
1H), 7.27 (d, 2H), 7.41 (t, 1H), 7.57 (s, 1H), 7.76 (d, 1H). MS
(ESI -ve): m/z 455 (M - H)-. Anal. (C27H36O6) C, H.
Meth yl 4-[3-(4-(Decyloxy)p h en oxy)p r op oxy]ben zoa te
(42a ). A solution of 1,3-dibromopropane (10 g, 50 mmol), 4a
(1.26 g, 5 mmol), and Cs2CO3 (1.8 g, 5.5 mmol) in acetonitrile
was heated under reflux for 6 h. The cold mixture was filtered,
and the solvent was evaporated under reduced pressure. The
residue was taken up in ethyl acetate (250 mL), washed with
water (3 × 75 mL) and brine (100 mL), dried (MgSO4), and
evaporated to dryness. The solid residue was triturated with
ice-cold acetonitrile to afford 3-(4-(decyloxy)phenoxy)propyl
bromide as a white solid (1.23 g, 66%). MS (EI): 370/372 (M)+.
This solid (0.56 g, 1.5 mmol), methyl 4-hydroxybenzoate (0.25
g, 1.6 mmol), and Cs2CO3 (0.6 g, 1.8 mmol) in acetonitrile (25
mL) were heated under reflux for 6 h. The cold mixture was
poured into water (250 mL) and extracted with DCM (250 mL).
The extract was washed with water and brine, dried (MgSO4),
and evaporated under reduced pressure. The residue was
purified by silica gel chromatography (hexane/ethyl acetate,
1
33%). H NMR (CDCl3): δ 1.43-1.48 (m, 2H), 1.57-1.65 (m,
4H), 2.35 (d, 1H), 2.59 (t, 2H), 2.81 (t, 2H), 3.85-4.02 (m, 2H),
4.55 (m, 1H), 5.27-5.47 (m, 2H), 5.89-5.99 (m, 1H), 6.85 (d,
2H), 7.14-7.29 (m, 5H), 7.31 (2d, 2H).
4-[4-(4-(5-P h en ylp en tylth io)p h en oxy)-3-oxobu tyl]ben -
zoic Acid (44). 4-Iodobenzoic acid (0.45 g, 1.8 mmol), 44c (0.60
g, 1.75 mmol), and a catalytic amount of palladium(II) acetate
(0.06 g) in acetonitrile (30 mL) were treated with triethylamine
(10 mL) and heated under reflux for 18 h. The cold reaction
mixture was filtered through Celite, poured into 2 N HCl (100
mL), and extracted with ethyl acetate (3 × 50 mL). The extract
was washed with saturated sodium bicarbonate solution (2 ×
50 mL), water, and brine, dried (MgSO4), and evaporated to
dryness. The residue was passed down a silica gel column
(DCM + 3% MeOH) to give a white solid, which crystallized
from ethyl acetate 44 (0.19 g, 23%); mp 141-142 °C. 1H NMR
(CDCl3): δ 1.43-1.48 (m, 2H), 1.57-1.65 (m, 4H), 2.58 (t, 2H),
2.82 (t, 2H), 2.97-3.03 (m, 4H), 4.51 (s, 2H), 6.76 (d, 2H), 7.14-
7.31 (m and d, 9H), 8.01 (d, 2H). MS (ESI -ve): m/z 461 (M -
H)-. Anal. (C28H30O4S) C, H, S.
4-(4-Decyloxyp h en yl)-2-oxobu ta n e (45a ). 4-(4-Hydroxy-
phenyl)-2-oxobutane (5.0 g, 30 mmol), decyl bromide (6.8 g,
30.5 mmol), and Cs2CO3 (10 g, 30.5 mmol) in DMF (50 mL)
were stirred at room temperature for 18 h. The mixture was
poured into 2 N HCl (500 mL) and extracted with ethyl acetate
(2 × 250 mL). The extract was washed with water and then
brine, dried (MgSO4), and evaporated under reduced pressure
to give a white solid 45a (8.68 g, 94%). 1H NMR (CDCl3): δ
0.88 (t, 3H), 1.24-1.30 (m, 12H), 1.43 (m, 2H), 1.71-1.78 (m,
2H), 2.13 (s, 3H), 2.69-2.74 (m, 2H), 2.80-2.84 (m, 2H), 3.92
(t, 2H), 6.82 (d, 2H), 7.05 (d, 2H).
1
10:1) to give a white solid 42a (0.5 g, 75%). H NMR (CDCl3):
δ 0.88 (t, 3H), 1.26-1.30 (m, 12H), 1.43 (m, 2H), 1.70-1.78
(m, 2H), 2.22-2.28 (m, 2H), 3.88 (s, 3H), 3.90 (t, 2H), 4.11 (t,
2H), 4.21 (t, 2H), 6.82 (bs, 4H), 6.91 (d, 2H), 7.97 (d, 2H). MS
(EI): m/z 422 (M)+.
4-[3-(4-(Decyloxy)p h en oxy)p r op oxy]ben zoic Acid (42).
LiOH‚H2O (0.2 g, 4.8 mmol) and 42a (0.44 g, 1 mmol) in THF/
H2O (3:1, 20 mL) were stirred at room temperature for 18 h.
The mixture was poured into 2 N HCl (100 mL) and extracted
with ethyl acetate (2 × 100 mL). The extract was washed with
water and brine, dried (MgSO4), and evaporated under reduced
pressure to give a white solid that was crystallized from ethyl
acetate 42 (0.42 g, 98%); mp 149-150 °C. 1H NMR (CDCl3): δ
0.88 (t, 3H), 1.26-1.30 (m, 12H), 1.42 (m, 2H), 1.69-1.76 (m,
2H), 2.21-2.28 (m, 2H), 3.91 (t, 2H), 4.10 (t, 2H), 4.21 (t, 2H),
6.78-6.84 (m, 4H), 6.92 (d, 2H), 7.95 (d, 2H). MS (EI): m/z
428 (M)+. Anal. (C26H36O5‚0.25H2O) C, H.
3-[(3-(4-(Decyloxy)ph en oxy)pr opyl)th io]pr opan oic Acid
(13). The synthetic sequence used to prepare 42a and 42 was
1
4-(4-Decyloxyp h en yl)-2-oxobu tyl Br om id e (45b). A so-
lution of 45a (3.05 g, 10 mmol) in DCM (100 mL) and methanol
(50 mL) was treated with tetrabutylammonium tribromide (5.3
g, 11 mmol) and stirred at room temperature for 18 h. The
solvent was removed under reduced pressure, and the residue
was passed down a silica gel column (hexane/ethyl acetate,
20:1) to afford the product 45b as a colorless oil (2.03 g, 53%).
1H NMR (CDCl3): δ 0.88 (t, 3H), 1.27 (m, 12H), 1.44 (m, 2H),
1.72-1.78 (m, 2H), 2.87-2.96 (m, 4H), 3.84 (s, 2H), 3.92 (t,
2H), 6.81 (d, 2H), 7.08 (d, 2H). MS (EI): m/z 382/384 (M)+.
applied to give 13; mp 92-93 °C. H NMR (CDCl3): δ 0.88 (t,
3H), 1.27 (m, 12H), 1.41 (m, 2H), 1.75 (m, 2H), 2.03 (m, 2H),
2.66 (t, 2H), 2.73 (t, 2H), 2.82 (t, 2H), 3.89 (t, 2H), 4.00 (t, 2H),
6.82 (m, 4H). MS (EI): m/z 396 (M)+. Anal. (C22H36O4S) C, H,
S.
[4-(5-P h en ylpen tylth io)ph en oxy]aceton itr ile (44a). 4-((5-
Phenylpentyl)thio)phenol (2.73 g, 10 mmol), bromoacetonitrile
(1.44 g, 12 mmol), and cesium carbonate (4.8 g, 14 mmol) in
acetonitrile (50 mL) were heated under reflux for 4 h. The cold
mixture was poured into 2 N HCl (200 mL) and extracted with
DCM (3 × 100 mL). The organic extract was washed with
water and brine, dried (MgSO4), and evaporated under reduced
pressure to give a solid. This solid was passed down a silica
gel column (hexane/DCM, 1:1) to give a colorless oil 44a (2.39
3-P r op en yl 3-[4-(4-Decyloxyp h en yl)-2-oxobu toxy]ben -
zoa te (45c). 3-Propenyl 3-hydroxybenzoate (0.32 g, 1.8 mmol)
and 45b (0.60 g, 1.5 mmol) in DMF (10 mL) were treated with
KF (0.175 g, 3 mmol) and stirred at room temperature for 2 h

1360 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 6
Connolly et al.
and then at 100 °C for 3 h. The mixture was partitioned
between 2 N HCl (100 mL) and ethyl acetate (250 mL). The
organic phase was washed with ammonium chloride water and
brine, dried (MgSO4), and evaporated to dryness. The residue
was passed down a silica gel column (hexane/ethyl acetate,
7:1) to afford a colorless oil 45c (0.59 g, 80%). 1H NMR
(CDCl3): δ 0.88 (t, 3H), 1.27 (m, 12H), 1.44 (m, 2H), 1.72-
1.78 (m, 2H), 2.90 (bs, 4H), 3.92 (t, 2H), 4.56 (s, 2H), 4.82 (d,
2H), 5.28-5.43 (dd, 2H), 5.98-6.07 (m, 1H), 6.81 (d, 2H), 7.04-
7.11 (m, 3H), 7.35 (t, 1H), 7.53 (bs, 1H), 7.71 (d, 1H).
3-[4-(4-Decyloxyph en yl)-2-oxobu toxy]ben zoic Acid (45).
Deprotection of 45c as described for 22 gave a white solid 45;
mp 109-110 °C. 1H NMR (DMSO-d6): δ 0.94 (t, 3H), 1.3-
1.47 (m, 12H), 1.56 (m, 2H), 1.76 (m, 2H), 2.8-2.9 (m, 4H),
3.98 (t, 2H), 4.97 (s, 2H), 6.90 (d, 2H), 7.16-7.20 (m, 3H), 7.46
(t, 2H), 7.61 (d, 1H). MS (EI): m/z 440 (M)+. Anal. (C27H36O5)
C, H.
Assa y of cP LA2 Usin g a Bila yer Su bstr a te. (a ) P r ep a -
r a tion of Su bstr a te. The substrate was prepared by mixing
1-stearoyl-2-[14C]arachidonylphosphatidylcholine (36.8 µL of
a 0.18 MBq/mL solution in ethanol, specific activity 2.02 GBq/
mmol), 1-stearoyl-2-arachidonylphosphatidylcholine (PC) (19.1
µL of a 10 mg/mL solution in ethanol/CHCl3 (4:1)), phospha-
tidylinositol 4,5-bisphosphate (PIP2) (18.1 µL of a 0.5 mg/mL
solution in methanol/CHCl3/H2O/1 M aqueous HCl (20:9:1:
0.01)), and diacylglycerol (DAG) (36.8 µL of a 1 mg/mL solution
in methanol) in a polypropylene test tube (50 mL), and the
mixture was dried thoroughly under a stream of nitrogen. Tris
HCl, pH 7.4, buffer (17 mL of 50 mM solution) was added,
and the mixture was vortex mixed for 60 s and then sonicated
at room temperature for 2 min. The tube was placed in dry
ice until frozen (approximately 20 min). The frozen substrate
was sonicated at room temperature until thawed (approxi-
mately 15 min) and then finally vortex mixed for 60 s.
(b) Assa y of cP LA2 Activity. CaCl2 (50 µL of a 100 µM
aqueous solution) and KCl (50 µL of a 1 M aqueous solution)
were added to Tris HCl, pH 7.4, buffer (135 µL of a 50 mM
solution) in each tube of a 96 well plate, followed by substrate
(250 µL) prepared as above. A solution of inhibitor in DMSO
(5 µL) was then added. cPLA2 enzyme was diluted ap-
propriately in assay buffer to give approximately 10% conver-
sion when 10 µL was used. The final assay composition was
7.4 µM 1-stearoyl-2-arachidonylphosphatidylcholine (contain-
ing 30 000 dpm 14C substrate), 0.24 µM PIP2, 1.7 µM DAG, 10
µM CaCl2, and 100 mM KCl in assay buffer. The final assay
volume was 500 µL. The reaction was started with enzyme
and performed at 37 °C for 10 min and then stopped with a
25 mM aqueous solution of ethylenediaminetetraacetic acid
containing 2.5% Triton X-100 (500 µL). The released arachi-
donate was extracted by adding sufficient anhydrous sodium
sulfate to saturate each tube, followed by acetic acid in hexane
(5 mL, 0.4% (v/v)). The organic layer was transferred to a
scintillation vial and evaporated under a stream of air, and
the released radioactivity was determined by liquid scintilla-
tion counting. Inhibition of enzyme activity was determined
by comparing to control reaction with no inhibitor added, after
correcting for radioactivity released in the absence of enzyme.
inhibitor added, after correcting for background hydrolysis in
the absence of enzyme.
Ion op h or e-In d u ced Relea se of Ar a ch id on ic Acid fr om
DMSO-Differ en tia ted HL60 Cells. The ability of compounds
to inhibit cPLA2 activity in intact cells was evaluated using
HL60s differentiated with DMSO to resemble neutrophils.
HL60 cells were differentiated by resuspending at 106 cells/
mL in 1.3% DMSO in RPMI 1640 medium supplemented with
10% fetal calf serum, 2 mM glutamine, 100 iu/mL penicillin,
100 iu/mL streptomycin, and 2.5 µg/mL amphotericin B.
Cultures were used for compound evaluation on days 4 and 5
at which point they reached their maximum ability to mobilize
incorporated arachidonic acid in response to ionophore. Cells
were incubated with 3H-arachidonic acid for a long enough
time period for labeling of choline, ethanolamine, serine, and
inositol-containing phospholipids to approach equilibrium. 3H-
arachidonic acid (7 GBq/mmol) was added to differentiating
cells on day 3 or 4 giving 5500 Bq/mL of culture. Cells were
spun down and washed twice with 20% of the starting volume
of Tyrodes buffer (137 mM NaCl, 5.7 mM glucose, 10 mM
HEPES buffer, 27 mM KCl, 0.4 mM NaH2PO4, 1.8 mM CaCl2,
and 1.0 mM MgCl2, pH 7.4) and finally resuspended in Tyrodes
buffer. The assay was performed in 96 well polypropylene
plates. Compounds were initially dissolved in DMSO and then
diluted in Tyrodes buffer to give a constant 3.75% DMSO. A
solution of inhibitor (40 µL) was preincubated with cells (90
µL, 4 × 105) for 10 min at 37 °C before initiating release of
arachidonic acid by the addition of calcium ionophore A23187
(20 µL of a 15 µM solution in Tyrodes buffer containing bovine
serum albumin (BSA) (7.5 mg/mL)). Addition of BSA alone to
control wells was used to estimate background release. After
3 min, ice-cold methanol (150 µL) was added to stop the
reaction, and the mixture was left at 0 °C for 20 min. A portion
(200 µL) was removed from each well for filtration through a
96 well filter plate, and the filtrate (100 µL) was dried onto a
Luma-Plate for counting. Recovery of released arachidonic acid
was 80% by this process. Background release in the absence
of ionophore (less than 30% of the release in the presence of
ionophore) was subtracted before calculating the percentage
inhibition.
Refer en ces
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solution of substrate was prepared by adding 7-(2-Oxo-6-
heptenoxy)-2H-1-benzopyran-2-one in DMSO (20 mM) to a
N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES),
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(3.15 mM), to give a final substrate concentration of 31 µM. A
solution of inhibitor in DMSO (5 µL) was added to each well
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cPLA2 enzyme diluted in the same buffer as the substrate (100
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excitation, 460 nm emission filter settings. The plates were
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fluorescence was measured again in the fMax plate reader.
Inhibition of enzyme activity was determined by comparing
the increase in fluorescence with control reaction with no

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