Potent and selective fluoroketone inhibitors of group VIA calcium-independent phospholipase A2

Article abstract of DOI:10.1021/jm901872v

Group VIA calcium-independent phospholipase A₂ (GVIA iPLA ₂) has recently emerged as a novel pharmaceutical target. We have now explored the Structure-activity relationship between fluoroketones and GVIA iPLA₂ inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA₂ (X_I(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA ₂ than for GIVA cPLA₂ and GV sPLA₂, respectively, makes it a valuable tool to explore the role of GVIA iPLA ₂ in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8- (naphthalene-2-yl)octan-4-one inhibited GVIA iPLA₂ with a X _I(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA₂ and GV sPLA₂ at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent inhibitors of GVIA iPLA ₂; however, they are not selective.

Full text of DOI:10.1021/jm901872v

3602 J. Med. Chem. 2010, 53, 3602–3610
DOI: 10.1021/jm901872v
Potent and Selective Fluoroketone Inhibitors of Group VIA Calcium-Independent Phospholipase A2
George Kokotos,*,† Yuan-Hao Hsu,‡ John E. Burke,‡ Constantinos Baskakis,† Christoforos G. Kokotos,†
Victoria Magrioti,† and Edward A. Dennis*,‡
†Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece, and
‡Department of Chemistry and Biochemistry and Department of Pharmacology, School of Medicine, MC 0601, University of California,
San Diego, La Jolla, California 92093-0601
Received December 19, 2009
Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as a novel
pharmaceutical target. We have now explored the structure-activity relationship between fluoroke-
tones and GVIA iPLA2 inhibition. The presence of a naphthyl group proved to be of paramount
importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of
GVIA iPLA2 (XI(50) = 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA2
than for GIVA cPLA2 and GV sPLA2, respectively, makes it a valuable tool to explore the role of GVIA
iPLA2 in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl)octan-4-one inhibited
GVIA iPLA2 with a XI(50) value of 0.001 while inhibiting the other intracellular GIVA cPLA2 and GV
sPLA2 at least 90 times less potently. Hexa- and octafluoro ketones were also found to be potent
inhibitors of GVIA iPLA2; however, they are not selective.
Introduction
from 85 to 88 kDa and expressed as several distinct splice
variants of the same gene.8 GVIA iPLA2 has long been
proposed as a homeostatic enzyme involved in basal metabo-
lism within the cell.9-15 However, a number of studies
suggest that GVIA iPLA2 also plays important roles in
numerous cell types, although they may differ from cell to
cell. Recent review articles discuss the role of GVIA iPLA2 in
signaling and pathological conditions (for example, cancer
and ischemia).16-20
The phospholipase A2 (PLA2) superfamily consists ofmany
different groups of enzymes that catalyze the hydrolysis of the
ester bond at the sn-2 position of various phospholipids.1 The
products of the hydrolysis are a free fatty acid and a lysopho-
spholipid, both of which may generate second messengers that
play important physiological roles. The PLA2 superfamily
currentlycontains 15 separate, identifiablegroupsandvarious
subgroups.2,3 The three predominant types of PLA2 found in
The GVIA iPLA2 enzyme contains a consensus lipase
motif, Gly-Thr-Ser*-Thr-Gly, with the catalytic serine con-
firmed by site-directed mutagenesis.8,21 Both of the intracel-
lular enzymes GIVA cPLA2 and GVIA iPLA2 share the same
catalytic mechanism utilizing a serine residue as the nucleo-
phile. The various inhibitor classes of both enzymes are
summarized in a recent review article.22 Arachidonyl triflu-
oromethyl ketone has been shown to function as a tight bind-
ing, reversible inhibitor of both GIVA and GVIA PLA2,23,24
while methylarachidonyl fluorophosphonate functions as an
irreversible inhibitor of both enzymes.25 Bromoenol lactone
(BEL) (1, Figure 1) has previously been considered to be a
selective and irreversible GVIA iPLA2 inhibitor and has been
widely applied to study potential biological roles for GVIA
iPLA2.11,26 Turk et al. have recently studied the inactivation
mechanism of GVIA iPLA2 by 1 (BEL),27 and they concluded
that it is likely that this inhibitor affects multiple enzymes and
should be used with appropriate caution when studying
potential roles of GVIA iPLA2. Our laboratories have pre-
viously reported on the development of 2-oxoamide inhibitors
targeting GIVA cPLA2.28-32 We have demonstrated that
2-oxoamides containing a free carboxyl group are selective
inhibitors of GIVA cPLA2, and most recently we have deter-
mined the location of such an inhibitor bound in the active site
of GIVA cPLA2 using a combination of deuterium exchange
a
human tissues are the cytosolic (such as the GIVA cPLA2 ),
the secreted (such as the GIIA and GV sPLA2), and the
calcium-independent (such as the GVIA iPLA2) enzymes.
GIVA cPLA2 is generally considered a proinflammatory
enzyme that is the rate-limiting provider of arachidonic acid
and lysophospholipids.4 In many cases, the activity of secreted
PLA2 has been shown to be dependent on or linked to the
activity of GIVA cPLA2.5-7 The calcium-independent group
VIA iPLA2 (GVIA iPLA2), typically referred to in the litera-
ture as iPLA2, is actually a group of cytosolic enzymes ranging
*To whom correspondence should be addressed. For G.K.:
(phone) (30210) 7274462; (fax) (30210) 7274761; (e-mail) gkokotos@
chem.uoa.gr. For E.A.D.: (phone) 858-534-3055; (fax) 858-534-7390;
(e-mail) edennis@ucsd.edu.
a Abbreviations: ATP, adenosine triphosphate; BEL, bromoenol lac-
tone, BSA, bovine serum albumin; DAST, diethylaminosulfur trifluoride;
DMAP, 4-dimethylaminopyridine; DPPC, 1,2-dipalmitoylphosphatidyl-
choline; DTT, dithiothreitol; EtOAc, ethyl acetate; GIVA cPLA2, group
IVA cytosolic phospholipase A2; GV sPLA2, group V secreted phospho-
lipase A2; GVIA iPLA2, group VIA calcium-independent phospholipase
A2; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NMM,
N-methylmorpholine; PAPC, 1-palmitoyl-2-arachidonylphosphatidyl-
choline; PIP2, phosphatidylinositol (4,5)-bisphosphate; TBAF, tetra-n-
butylammonium fluoride; TFA, trifluoroacetic acid; THF, tetrahydrofuran;
TLC, thin-layer chromatography; Tris, tris(hydroxymethyl)aminomethane;
TMS, tetramethylsilane; WSCI HCl, N-(3-dimethylaminopropyl)-N0-ethyl-
carbodiimide hydrochloride.
3
r
pubs.acs.org/jmc
Published on Web 04/06/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3603
Scheme 1a
a Reagents and conditions: (a) (i) (COCl)2, CH2Cl2; (ii) (C3F7CO)2O,
pyridine, CH2Cl2.
Scheme 2a
Figure 1. Some known inhibitors of GVIA iPLA2.
mass spectrometry and molecular dynamics.33 2-Oxoamides
based on amino acid esters show cross-reactivity for both
GIVA cPLA2 and GVIA iPLA2,30,32 while most recently we
identified a 2-oxoamide based on a pseudodipeptide that
preferentially inhibits GVIA iPLA2.34
The development of selective inhibitors for the three main
human PLA2 enzymes is an important goal, and we have syn-
thesized and assayed a variety of polyfluoroketones for their
activity on GIVA cPLA2, GVIA iPLA2, and GV sPLA2. We
previously found that 1,1,1,2,2-pentafluoro-7-phenylheptan-
3-one (FKGK11)35 (2, Figure 1) is a selective inhibitor of
GVIA iPLA2.36 Trifluoromethyl ketone 3 (FKGK2, Figure 1)
can be considered to be a pan inhibitor of all three enzymes:
GIVA cPLA2, GVIA iPLA2, and GV sPLA2. The tetrafluoro
derivative 4 was found to be the most potent GVIA iPLA2
inhibitor, although it is not selective.36 The selective GVIA
iPLA2 inhibitor 2 was successfully used tostudythe role of this
enzyme in neurological disorders such as peripheral nerve
injury and multiple sclerosis.37,38 We successfully demon-
strated that inhibitor 2 causes a beneficial therapeutic effect
in experimental autoimmune encephalomyelitis,38 the animal
model of multiple sclerosis. This indicates that GVIA iPLA2 is
a novel target for the development of new therapies for
multiple sclerosis. The recently emerged important pharma-
ceutical significance of GVIA iPLA2 and the lack of potent
and selective GVIA iPLA2 inhibitors prompted us to extend
our studies toward the discovery of such inhibitors. In this
work, we report the synthesis of a variety of new fluoroke-
tones and the study of their selectivity on the three main
human phospholipase A2 compounds.
a Reagents and conditions: (a) C2H5OOCCHdCHCH2P(dO)-
(OC2H5)2, LiOH, THF; (b) NaOH, 1,4-dioxane; (c) DMAP, NMM,
WSCI HCl, CH3ONHCH3 HCl, CH2Cl2; (d) CF3CF2I, CH3Li LiBr,
3
3
3
Et2O.
(7) with triethyl phosphonocrotonate,39 followed by saponi-
fication, led to unsaturated acid 8 (Scheme 2). We previously
showed that R,β-unsaturated acids may be converted into
pentafluoroethyl ketones by treatment of the corresponding
Weinreb amide with (pentafluoroethyl)lithium.40 Following
this procedure, the Weinreb amide 9 was converted to the
unsaturated pentafluoroethyl ketone 10.
Various trifluoromethyl, pentafluoroethyl, and heptafluoro-
propyl ketones 12a-i were synthesized as depicted in
Scheme 3. Reaction of furfural (13) with triethyl phosphono-
crotonate, followed by hydrogenation and saponification,
produced acid 11b. However, treatment of this acid with
oxalyl chloride, followed by (C2F5CO)2O and pyridine, led
to the formylated derivative 12b. Under these conditions we
were unable to prepare the nonformylated derivative.
The synthesis of tetrafluoro derivatives 18a,b was accom-
plished by procedures developed earlier36 (Scheme 4). On the
basis of 1H and 19F NMR data, tetrafluoro derivatives 18a,b
appear to be a mixture of ketone-hydrate form.
Design and Synthesis of Polyfluoroketones
Fluoroketones 19-25 (for structures, see Table 1), which
were used in the in vitro assays, were prepared as described
previously.40
The rationale behind our design of polyfluoroketones was
based on the hypothesis that the introduction of more than
three fluorine atoms adjacent to a carbonyl group may
increase either the carbonyl reactivity or the inhibitor binding
affinity to the target enzyme.36 This hypothesis was con-
firmed, and in fact, such a design led to the selective GVIA
iPLA2 inhibitor 2 (pentafluoroethyl ketone) and the tetra-
fluoro derivative 4, which is a potent GVIA iPLA2 inhibitor,
although it is not a selective inhibitor. In the present work, our
aim was to extend the structure-activity relationship studies
on the potency and the selectivity of heptafluoropropyl
ketones and analogues of the lead GVIA iPLA2 inhibitors 2,
3, and 4.
In Vitro Inhibition of GVIA iPLA2, GIVA cPLA2, and GV
sPLA2
All synthesized inhibitors were tested for inhibition of
human GVIA iPLA2 based on a modification of the pre-
viously described mixed micelle-based assay.30 The mixed
micelle assay employed herein used 1-palmitoyl-2-arachido-
nylphosphatidylcholine (PAPC) as substrate, and the specific
conditions employed herein were somewhat different from
those employed in the previous mixed micelle assay which
employed 1,2-dipalmitoylphosphatidylcholine (DPPC) as
substrate. This change was made in order to use the same
substrate for iPLA2 as for cPLA2, to better compare the
specificities of both iPLA2 and cPLA2 toward the same
substrate. This also improved the consistency of the standard
For the synthesis of heptafluoropropyl ketones, carboxylic
acids 5a,b were converted to chlorides by treatment with
oxalyl chloride and then to the target compounds 6a,b using
heptafluorobutanoic anhydride and pyridine (Scheme 1).
Wadsworth-Horner-Emmons reaction of benzaldehyde

3604 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Kokotos et al.
Scheme 3a
and GV sPLA2, the previously reported mixed micelle-based
assays were used.28,29,31 The resulting values of GVIA iPLA2
inhibition arepresented in Figure 2 as either percent inhibition
or XI(50) values. Initially, the percent of inhibition for each
PLA2 enzyme at 0.091 mol fraction of each inhibitor was
determined, and XI(50) values were determined for all com-
pounds toward GVIA iPLA2 and for the other two enzymes
for all inhibitors that displayed greater than 90% inhibition.
However, for two additional iPLA2 inhibitor examples, we
also determined their XI(50) toward cPLA2 in order to
calculate their relative specificities. The XI(50) is the mole
fraction of the inhibitor in the total substrate interface re-
quired toinhibit the enzyme by 50%. Theinhibitionresults for
all three enzymes are summarized in Table 1.
The replacement of the pentafluoroethyl group of inhibitor
2 (XI(50) = 0.0014) by the heptafluoropropyl group led to
inhibitor 6a (XI(50) = 0.0022), which resulted in a slightly
decreased potency of the GVIA iPLA2 inhibition. Extension
of the carbon chain by one carbon atom produced inhi-
bitor 6b, which also resulted in a slightly decreased potency
(XI(50) = 0.0030). Compounds 19 and 20, which carry a
hydroxyl group instead of the carbonyl group of inhibitors 2
and 6a, were surprisingly found to be inhibitors of GVIA
iPLA2, although they were not potent. They were also weak
inhibitors of the other two PLA2 compounds.
We observed that the insertion of two unsaturated bonds,
while keeping the distance between the phenyl and the acti-
vated carbonyl group constant, significantly reduced the
inhibitory activity of 10 by 22 times. When the carbon atom
next to the phenyl group of 2 was replaced by oxygen,
compound 12a was 2.5 times less potent than inhibitor 2. At
the same time, we found that inhibitor 12a is selective for
GVIA iPLA2, since a high mole fraction of the inhibitor
(0.091) does not inhibit either GIVA cPLA2 or GV sPLA2 at
all. The furan-based inhibitor 12b was not a potent inhibitor.
Pentafluoroethyl derivative 21 based on the oleyl chain
inhibited GVIA iPLA2 better than the corresponding palmi-
toyl derivative.36 Heptafluoropropyl derivative 22 was a
weaker inhibitor of GVIA iPLA2 than derivative 21. Both
21 and 22 were able to inhibit weakly the other two enzymes.
These data indicate that for the inhibition of GVIA iPLA2 a
chain bearing an aromatic ring rather than a long aliphatic
saturated or unsaturated chain has to be attached to the
activated carbonyl. Reducing the distance between the phenyl
and the carbonyl group and inserting a trans double bond led
to compound 23, which inhibits GVIA iPLA2 with XI(50) =
0.0066. This derivative does not inhibit at all the other
intracellular enzyme GIVA cPLA2 and inhibits weakly GV
sPLA2 (77%) at 0.091 mol fraction.
a Reagents and conditions:(a) (i) (COCl)2, CH2Cl2; (ii) (CF3CO)2O or
(C2F5CO)2O or (C3F7CO)2O, pyridine, CH2Cl2; (b) C2H5OOCC-
HdCHCH2P(dO)(OC2H5)2, LiOH, THF; (c) H2, 10% Pd/C; (d)
NaOH, CH3OH; (e) Br(CH2)3COOEt, K2CO3, acetone.
Scheme 4a
Both the p-hexyloxy substituted pentafluoroethyl and hep-
tafluoropropyl derivatives12c and 12d inhibited GVIA iPLA2
with XI(50) of 0.0084 and 0.0136, respectively. Comparing 12c
and 12d with 2 and 6a, it seems that the p-hexyloxy substitu-
tion had a negative effect on the GVIA iPLA2 inhibition.
Interestingly, when the oxygen atom was moved between the
aromatic group and the carbonyl next to the phenyl group,
both the trifluoromethyl and the pentafluoroethyl derivatives
12e and 12f strongly inhibited GVIA iPLA2 (XI(50) of 0.0029
and 0.0024, respectively). However, all the derivatives bearing
a substituent at the para position failed to be selective for any
PLA2 enzyme.
a Reagents and conditions: (a) DAST, CH2Cl2 or (CH3OCH2CH2)2-
NSF3, CH2Cl2; (b) (i) (CH3)3SiCF3, CsF, CH3OCH2CH2OCH3 or
(CH3)3SiCF3, TBAF, toluene; (ii) conc HCl or TBAF, CH3COOH,
THF.
error in the assay. By use of this more refined assay, a
XI(50) = 0.0014 was determined for the lead inhibitor 2,
lower than that determined previously (0.0073).36 To test the
selectivity of the synthesized inhibitors toward GIVA cPLA2
The replacement of the phenyl group of inhibitor 2 by a
naphthyl group led to excellent results. 1,1,1-Trifluoro-
6-(naphthalen-2-yl)hexan-2-one (FKGK18, 12g)35 proved to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3605
Table 1. Inhibition of PLA2 by Fluoroketonesa
a Average percent inhibition and standard error (n = 3) are reported for each compound at 0.091 mol fraction. XI(50) values were determined for
inhibitors with greater than 90% inhibition. N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition).
be a very potent inhibitor of GVIA iPLA2 (XI(50) = 0.0002),
exhibiting 7 times higher inhibition than inhibitor 2. Com-
pound 12g also inhibited GIVA cPLA2 but at a significant
lower level, so we determined its XI(50) which was 0.039 (
0.001. It is 195 times more potent on GVIA iPLA2 than on
GIVA cPLA2. It was also a very weak inhibitor of GV sPLA2
(37% at 0.091 mol fraction), which implies that it is >455
times selective for iPLA2. Both pentafluoroethyl and hepta-
fluoropropyl derivatives 12h and 12i were potent inhibitors of
GVIA iPLA2 (XI(50) of 0.0006 and 0.0010, respectively).
However, both 12h and 12i inhibited weakly GIVA
cPLA2 and GV sPLA2. The dose response curves for the
inhibition of GVIA iPLA2 by 12g and 12i are presented in
Figure 3.
Both the tetrafluoro derivatives 18a and 18b were potent
inhibitors of GVIA iPLA2 (XI(50) of 0.0025 and 0.0013,
respectively). 1,1,1,2,2,4-Hexafluoro-7-phenylheptan-3-one 24
(FKGK21)35 and 1,1,1,2,2,3,3,5-octafluoro-7-phenyloctan-
4-one 25 (FKGK22)35 were even more potent inhibitors of
GVIA iPLA2 (XI(50) = 0.0005 for both). Comparing 24 and 25
with 2 and 6a, we observe that the insertion of an additional
fluorine atom at the R0 position of either the pentafluoroethyl or
the heptafluoropropyl ketone results in improved inhibitory
potency. However, none of the tetra-, hexa-, and octafluoro
derivatives proved selective.
The chemical mapping of the GVIA iPLA2 active site
through structure-activity studies, carried out in the present
and the previous work,36 allowed us to understand some of its
features, although no crystal structure has been reported. The
enzyme-inhibitor complex is likely to be stabilized by an
“oxyanion hole” and other features shown in Figure 4.
Although long saturated or unsaturated chains may be bound
by the enzyme, the presence of an aromatic ring facilitates
inhibitor-enzyme binding. The aromatic system, preferably
an extended one such as the naphthyl moiety, may be accom-
modated in an enzyme binding pocket able to create aromatic-
aromatic interactions. The aromatic system should have a
distance from the carbonyl group corresponding to a four-
carbon chain. We propose that the fluorine atoms, apart from
their role in increasing the carbonyl reactivity, may contribute
to additional interactions of the inhibitor with a “fluorophi-
lic” region of the enzyme. The perfluoroalkyl chain may
interact with a “fluorophilic” binding site through a variety
of bonds involving fluorine. Recently, it has become clear that

3606 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Kokotos et al.
Figure 2. GVIA iPLA2 % inhibition at 0.091 mol fraction of inhibitor in mixed micelles (top) and XI(50) values (bottom). Standard error
(n = 3) for average % inhibition and for XI(50) values for all synthesized compounds is indicated.
Figure 3. Dose-response curves for GVIA iPLA2 inhibition by inhibitors 12g and 12i. Inhibition of the activity of human GVIA iPLA2 was
tested on mixed micelles containing 100 μM PAPC and 400 μM Triton X-100. Inhibition curves were generated using Graphpad Prism with a
nonlinear regression targeted at symmetrical sigmoidal curves based on plots of % inhibition versus log(inhibitor concentration). The reported
XI(50) values were calculated from the resultant plots.
should be large enough to accommodate even a heptafluor-
opropyl group.
In the present study, we identified five fluoroketones (12g,
12h, 12i, 24, and 25) that are more potent inhibitors of GVIA
iPLA2 than the lead inhibitor 2, which has been successfully
used in animal models of neurological disorders.37,38 The
introduction of one fluorine atom at the R0 position of a
pentafluoroethyl or a heptafluoropropyl ketone (compounds
24 and 25) significantly increased the inhibitory potency for
GVIA iPLA2. We therefore determined the XI(50) for cPLA2
Figure 4. Model for the binding mode of fluoroketone inhibitors in
which was 0.038 ( 0.002. Inhibitor 25 is 76 times and >180
the active-site crevice of GVIA iPLA2.
times more potent for GVIA iPLA2 than for GIVA cPLA2
and GV sPLA2, respectively. Inhibitor 12a is also of interest
fluorine may enhance binding efficacy and selectivity in
pharmaceuticals because of variety of multipolar
C-F H-N, C-F CdO, and C-F H-CR interac-
tions between a fluorinated ligand and protein binding
sites.41,42 The “fluorophilic” binding site of GIVA iPLA2
because it inhibits GVIA iPLA2 (XI(50) = 0.0036) without
affecting at all GIVA cPLA2 and GV sPLA2. The presence of
a naphthyl group proved to be of paramount importance.
Trifluoromethyl ketone 12g is the most potent inhibitor of
GVIA iPLA2 (XI(50) = 0.0002) ever reported. Being 195
a
3 3 3
3 3 3
3 3 3

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3607
and >455 times more potent for GVIA iPLA2 than for GIVA
cPLA2 and sPLA2, respectively, makes it a valuable tool for
ex vivo and in vivo studies.
(CF2). MS (ESI) m/z (%): 343 [(M - H)-, 100]. Anal.
(C15H15F7O) C, H.
1,1,1,2,2,3,3-Heptafluoro-8-(4-hexyloxyphenyl)octan-4-one (12d).
Yield 62%; yellowish oil. 1H NMR (CDCl3): δ 7.05 (2H, d, J = 8.2
Hz, Ph), 6.87 (2H, d, J = 8.2 Hz, Ph), 3.91 (2H, t, J = 6.6 Hz,
OCH2), 2.74 (2H, t, J = 7.7 Hz, CH2), 2.56 (2H, t, J = 7.7 Hz,
CH2), 1.78-1.22 (12H, m, 6 ꢀ CH2), 0.88 (3H, t, J = 6.2 Hz, CH3).
13C NMR: δ 194.2 (t, JC-C-F = 26.0 Hz, CO), 157.6 (Ph), 132.0
(Ph), 130.2-103.5 (m, 2 ꢀ CF2, CF3), 129.1 (Ph), 114.5 (Ph), 68.0
(CH2O), 38.0 (CH2), 34.8 (CH2), 31.8 (CH2), 30.8 (CH2), 29.5
(CH2), 25.9 (CH2),22.8(CH2),22.1(CH2), 14.3 (CH3).19FNMR:δ
-9.4 (CF3), -49.9 (CF2), -55.4 (CF2). Anal. (C20H25F7O2) C, H.
1,1,1,2,2,3,3-Heptafluoro-8-(naphthalen-2-yl)octan-4-one (12i).
In conclusion, we developed new, very potent inhibitors of
the calcium-independent GVIA iPLA2. Some of them present
interesting selectivity over the intracellular GIVA cPLA2 and
the secreted GV sPLA2. By application of these inhibitors as
tools for studies in animal models, the role of GVIA iPLA2 in
various inflammatory diseases may be explored. Since it has
become clear that GVIA iPLA2 is a novel target for the
development of novel therapies, fluoroketone inhibitors may
become leads for the development of novel medicines, in
particular for complex neurological disorders such as multiple
sclerosis.
1
Yield 45%; yellowish oil. H NMR (CDCl3): δ 7.90-7.20 (7H,
m, Ph), 2.85-2.70 (4H, m, 2 ꢀ CH2), 1.85-1.70 (4H, m, 2 ꢀ CH2).
13C NMR: δ 194.2 (t, JC-CF2 = 26 Hz, CO), 139.3 (Ph), 133.8
(Ph), 132.3 (Ph), 128.4 (Ph), 127.9 (Ph), 127.7 (Ph), 127.4 (Ph),
126.7 (Ph), 126.2(Ph), 125.9 (Ph), 125.0-102.0 (m, CF3, 2 ꢀ CF2),
37.7 (CH2), 35.6 (CH2), 30.2(CH2), 22.0(CH2). 19F NMR:δ -8.8
(CF3), -50.0 (CF2), -55.5 (CF2). MS (ESI) m/z (%): 379 [(M -
H)-, 100]. Anal. (C18H15F7O) C, H.
Experimental Section
Synthesis of Fluoroketone Inhibitors. Melting points were
determined on a Buchi 530 apparatus and are uncorrected.
Nuclear magnetic resonance spectra were obtained on a Varian
Mercury spectrometer (1H NMR recorded at 200 MHz, 13C
NMR recorded at 50 MHz, 19F NMR recorded at 188 MHz)
(2E,4E)-N-Methoxy-N-methyl-5-phenylpenta-2,4-dienamide (9).45
To a stirred solution of carboxylic acid (175 mg, 1 mmol) in
CH2Cl2 (7 mL) were added DMAP (122 mg, 1 mmol), N,
O-dimethylhydroxyamine hydrochloride (98 mg, 1 mmol),
and are referenced in ppm relative to TMS for 1H NMR and 13
C
NMR and relative to TFA as an internal standard for 19F NMR.
Thin layer chromatography (TLC) plates (silica gel 60 F254) and
silica gel 60 (230-400 mesh) for flash column chromatography
were purchased from Merck. Visualization of spots was effected
with UV light and/or phosphomolybdic acid, in EtOH stain.
Tetrahydrofuran, toluene, and Et2O were dried by standard
procedures and stored over molecular sieves or Na. All other
solvents and chemicals were reagent grade and used without
further purification. All tested compounds possessed g95%
purity as determined by combustion analysis. Intermediates 11a
and 11e were prepared by known methods,43,44 and its spectro-
scopic data were in accordance with those in the literature.
General Procedure for the Synthesis of Heptafluoropropyl
Ketones. Oxalyl chloride (0.38 g, 3 mmol) and N,N-dimethyl-
formamide (40 μL) were added to a solution of carboxylic acid
(1 mmol) in dry dichloromethane (40 mL). After the mixture was
stirred for 3 h at room temperature, the solvent and excess
reagent were evaporated under reduced pressure and the residue
was dissolved in dry dichloromethane (10 mL). Pyridine (0.64
mL, 8 mmol) and heptafluorobutanoic anhydride (1.5 mL,
6 mmol) were added dropwise to this solution at 0 °C consecu-
tively. After being stirred at 0 °C for 30 min and at room
temperature for 1.5 h, the reaction mixture was cooled again
at 0 °C and water (2 mL) was added dropwise. After being stirred
for 30 min at 0 °C and another 30 min at room temperature, the
reaction mixture was diluted with dichloromethane (10 mL).
The organic phase was then washed with brine and dried
(Na2SO4). The solvent was evaporated under reduced pressure,
and the residual oil was purified by flash column chromatogra-
phy [EtOAc-petroleum ether (bp 40-60 °C), 5/95].
NMM (0.11 mL, 1 mmol), and WSCI HCl (192 mg, 1 mmol)
3
consecutively at room temperature. The reaction mixture was left
stirring for 18 h. It was then washed with an aqueous solution of 1
N HCl (3 ꢀ 10 mL), brine (1 ꢀ 10 mL), an aqueous solution of 5%
NaHCO3 (3 ꢀ 10 mL), and brine (1 ꢀ 10 mL). The organic layer
was dried (Na2SO4) and concentrated under reduced pressure.
The amide was purified by flash chromatography, eluting with the
appropriate mixture of EtOAc-petroleum ether (40-60 °C), 1/9,
to afford the desired product. Yield 67%; white solid. 1H NMR
(CDCl3): δ 7.55-7.20 (6H, m, Ph, CH), 6.90-6.80 (2H, m, 2 ꢀ
CH), 6.57 (1H, d, J = 15 Hz, CH), 3.70 (3H, s, CH3O), 3.25 (3H, s,
CH3). 13C NMR: δ 167.0 (CO), 143.2 (CH), 139.6 (CH), 136.2
(Ph), 129.8 (Ph), 128.7 (Ph), 126.9 (Ph), 126.8 (CH), 119.0 (CH),
61.7 (CH3O), 32.3 (CH3). MS (ESI) m/z (%): 218 (Mþ, 100).
(4E,6E)-1,1,1,2,2-Pentafluoro-7-phenylhepta-4,6-dien-3-one (10).
To a stirring solution of the Weinreb amide 9 (78 mg, 0.36 mmol) in
Et2O (5 mL) at -78 °C was added pentafluoroiodoethane (0.7 mL,
1.80 mmol) followed by dropwise addition of a MeLi LiBr solution
3
1.6 M in ether (1.2 mL, 1.80 mmol). The reaction mixture was
stirred at -78 °C for 3 h. Once the reaction was finished, the
reaction mixture was poured into H2O and acidified with a 10%
solution of KHSO4. The layers were separated, and the aqueous
layer was extracted with Et2O (3 ꢀ 15 mL). The combined organic
layers were washed with a 5% solution of NaHCO3 (40 mL) and
dried over MgSO4. The organic solvent was evaporated in vacuo
and the residue was purified by column chromatography, eluting
with EtOAc-petroleum ether (40-60 °C), 2/98. Yield 90%; yellow
oil. 1H NMR (CDCl3): δ 7.74 (1H, dd, J1 = 15.0 Hz, J2 = 10.6 Hz,
CH), 7.56-7.44 (2H, m, Ph), 7.42-7.32 (3H, m, Ph), 7.25-6.88
(2H, m, CH), 6.65 (1H, d, J = 15.4 Hz, CH). 13C NMR: δ 182.1
(t, JC-C-F = 25.4 Hz, CO), 149.8 (CH), 146.7 (CH), 135.3 (Ph),
130.4 (Ph), 129.0 (Ph), 127.9 (Ph), 125.9 (CH), 119.9
(CH), 130.0-107.0 (m, CF2, CF3). 19F NMR: δ -4.3 (CF3),
-46.0 (CF2). MS (ESI) m/z (%): 276 (M-, 100). Anal.
(C13H9F5O) C, H.
1,1,1,2,2,3,3-Heptafluoro-8-phenyloctan-4-one (6a).40 Yield
1
59%; yellowish oil. H NMR (CDCl3): δ 7.32-7.15 (5H, m,
Ph), 2.77 (2H, t, J = 6.2 Hz, CH2), 2.65 (2H, t, J = 6.6 Hz, CH2),
1.71-1.59 (4H, m, 2 ꢀ CH2). 13C NMR: δ 194.0 (t, JC-CF2 = 26
Hz, CO), 141.6 (Ph), 130.0-103.5 (m, 2 ꢀ CF2, CF3), 128.4 (Ph),
128.3 (Ph), 125.9 (Ph), 37.7 (CH2), 35.4 (CH2), 30.3 (CH2), 21.9
(CH2). 19F NMR: δ -9.4 (CF3), -49.9 (CF2), -55.4 (CF2). MS
(ESI) m/z (%): 329 [(M - H)-, 100].
Synthesis of Pentafluoroethyl Ketones. The synthesis of
pentafluoroethyl ketones was carried out following the proce-
dure described above for heptafluoropropyl ketones except that
pentafluoropropionic anhydride was used instead of hepta-
fluorobutanoic anhydride. The products were purified by
flash column chromatography [EtOAc-petroleum ether (bp
40-60 °C), 1/9].
1,1,1,2,2,3,3-Heptafluoro-9-phenylnonan-4-one (6b). Yield
1
76%; yellowish oil. H NMR (CDCl3): δ 7.38-7.15 (5H, m,
Ph), 2.74 (2H, t, J = 6.2 Hz, CH2), 2.63 (2H, t, J = 6.6 Hz, CH2),
1.78-1.60 (4H, m, 2 ꢀ CH2), 1.42-1.35 (2H, m, CH2). 13C
NMR: δ 194.4 (t, JC-CF2 = 26 Hz, CO), 142.4 (Ph),
130.2-103.5 (m, 2 ꢀ CF2, CF3), 128.6 (Ph), 128.5 (Ph), 126.4
(Ph), 38.1 (CH2), 35.9 (CH2), 31.3 (CH2), 29.9 (CH2),
22.5 (CH2). 19F NMR: δ -9.4 (CF3), -49.9 (CF2), -55.4
1,1,1,2,2-Pentafluoro-6-phenoxyhexan-3-one (12a). Yield
1
60%; yellowish oil. H NMR (CDCl3): δ 7.40-7.20 (2H, m,
Ph), 7.00-6.83 (3H, m, Ph), 4.02 (2H, t, J = 7 Hz, OCH2), 3.02

3608 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9
Kokotos et al.
(2H, t, J = 6.6 Hz, CH2CO), 2.30-2.10 (2H, m, CH2). 13C
NMR: δ 194.0 (t, JC-C-F = 26.4 Hz, CO), 158.5 (Ph), 133.4
(Ph), 129.5 (Ph), 121.0 (Ph), 125.0-110.0 (m, CF3), 114.4 (Ph),
106.8 (tq, JC-F2 = 265 Hz, JC-CF3 = 38 Hz, CF2), 68.0 (CH2O),
34.1 (CH2), 22.3 (CH2). 19F NMR: δ -4.1 (CF3), -45.6 (CF2).
MS (ESI) m/z (%): 281 [(M - H)-, 100]. Anal. (C12H11F5O2)
C, H.
5-(6,6,7,7,7-Pentafluoro-5-oxoheptyl)furan-2-carboxaldeyde
(12b). Yield 34%; yellowish oil. 1H NMR (CDCl3): δ 9.49 (1H, s,
CHO), 7.16 (1H, d, J = 3.8 Hz, arom), 6.26 (1H, d, J = 3.6 Hz,
arom), 2.78-2.74 (4H, m, 2 ꢀ CH2), 1.76-169 (4H, m, 2 ꢀ
CH2). 13C NMR: δ 193.9 (t, JC-C-F = 26.4 Hz, CO), 177.0
(CHO), 162.7 (arom), 151.9 (arom), 123.7 (arom), 117.6 (qt,
(CH3). 19F NMR: δ -1.5 (s, CF3). MS (ESI) m/z (%): 343 [(M -
H)-, 100]. Anal. (C19H27F3O2) C, H.
1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (12g). Yield
1
39%; yellowish oil. H NMR (CDCl3): δ 7.81-7.29 (7H, m,
Ph), 2.81-2.73 (4H, m, 2 ꢀ CH2), 1.79-1.73 (4H, m, 2 ꢀ CH2).
13C NMR: δ 194.4 (t, JC-C-F = 26 Hz, CO), 139.4 (Ph), 133.9
(Ph), 132.3 (Ph), 128.4 (Ph), 127.9 (Ph), 127.7 (Ph) 127.4 (Ph),
126.7 (Ph), 126.2 (Ph), 125.9 (Ph), 115.8 (q, JC-F = 292 Hz,
CF3), 36.2 (CH2), 35.6 (CH2), 30.3 (CH2), 22.0 (CH2). 19F
NMR: δ -1.5 (s, CF3). MS (ESI) m/z (%): 279 [(M - H)-,
100]. Anal. (C16H15F3O) C, H.
5-(Furan-2-yl)pentanoic Acid (11b).46 A suspension of alde-
hyde 13 (0.096 g, 1 mmol), triethyl 4-phosphonocrotonate
(0.37 g, 1.5 mmol), lithium hydroxide (0.036 g, 1.5 mmol), and
molecular sieves (beads, 4-8 mesh, 1.5 g/mmol aldehyde) in dry
tetrahydrofuran (10 mL) was refluxed under argon for 24 h. The
reaction mixture was then cooled to room temperature and
filtered through a thin pad of Celite and the solvent evaporated
under reduced pressure. The residual oil was purified by chro-
matography on silica gel, eluting with ether-petroleum ether
(bp 40-60 °C), 1/9. A mixture of the unsaturated ester (135 mg,
0.7 mmol) in dry 1,4-dioxane (7 mL) and 10% palladium on
activated carbon (0.07 g) was hydrogenated for 12 h under
atmospheric conditions. After filtration through a pad of Celite,
the solvent was removed in vacuo to give the saturated com-
pound. The solution of the saturated ester in methanol (1.4 mL)
was treated with 1 N sodium hydroxide (1 mL, 1 mmol). The
mixture was stirred at room temperature for 12 h, acidified with
1 N HCl, and extracted with EtOAc (3 ꢀ 10 mL). The solvent
was removed in vacum to afford the saturated acid. Yield 66%;
white solid; mp 40-41 °C. 1H NMR (CDCl3): δ 10.00 (1H, br,
COOH), 7.29-7.27 (1H, m, arom), 6.27-6.25 (1H, m, arom),
6.00-5.97 (1H, m, arom), 2.64 (2H, t, J = 6.4 Hz, CH2), 2.36
(2H, t, J = 6.0 Hz, CH2), 1.73-1.63 (4H, m, 2 ꢀ CH2). 13C
NMR: δ 178.4 (CO), 155.5 (arom), 140.9 (arom), 110.0 (arom),
104.9 (arom), 33.8 (CH2), 27.6 (CH2), 27.4 (CH2), 24.1 (CH2).
(2E,4E )-5-Phenylpenta-2,4-dienoic Acid (8).47 Benzaldehyde
was treated with triethyl 4-phosphonocrotonate, and the result-
ing ester was saponified as described above. Yield 76%; white
solid; mp 165-166 °C. 1H NMR (CDCl3): δ 7.58-7.22 (6H, m,
Ph, CH), 7.05-6.90 (2H, m, CH), 6.00 (1H, d, J = 15 Hz, CH).
13C NMR: δ 169.3 (CO), 145.5 (CH), 140.6 (CH), 136.4 (Ph),
128.9 (Ph), 128.4 (Ph), 127.1 (Ph), 126.2 (CH), 121.1 (CH).
4-(4-Octylphenoxy)butyric Acid Ethyl Ester (15). A mixture of
p-octylphenol (206 mg, 1 mmol), K2CO3 (415 mg, 3 mmol), and
ethyl 4-bromobutyrate (215 mg, 1.1 mmol) in acetone (7.6 mL)
was refluxed overnight. The reaction mixture was then cooled to
room temperature and the soslvent evaporated under reduced
pressure. The residual oil was purified by flash column chroma-
tography on silica gel, eluting with EtOAc-petroleum ether (bp
40-60 °C), 1/9. Yield 71%; colorless oil. 1H NMR (CDCl3): δ
7.07 (2H, d, J = 8.8 Hz, Ph), 6.81 (2H, d, J = 8.8 Hz, Ph), 4.17
(2H, q, J = 7 Hz, OCH2CH3), 3.92 (2H, t, J = 6.6 Hz, OCH2),
2.60-2.45 (4H, m, 2 ꢀ CH2), 2.18-2.05 (2H, m, CH2CH2COO),
1.65-1.42 (2H, m, CH2), 1.38-1.21 (13H, br, 5 ꢀ CH2, CH3),
0.90 (3H, t, J = 6.8 Hz, CH3). 13C NMR: δ 173.4 (CO), 157.1
(Ph), 135.3 (Ph), 129.4 (Ph), 114.4 (Ph), 66.9 (CH2O), 60.5
(OCH2CH3), 35.3 (CH2), 32.1 (CH2), 32.0 (CH2), 31.5 (CH2),
31.0 (CH2), 29.7 (CH2), 29.5 (CH2), 24.9 (CH2), 22.9 (CH2), 14.5
(CH3), 14.3 (CH3). Anal. (C20H32O3) C, H.
J
C-F3 = 286 Hz, JC-CF2 = 34 Hz, CF3), 109.2 (arom), 106.8
(tq, JC-F2 = 265 Hz, JC-CF3 = 38 Hz, CF2), 36.9 (CH2), 28.1
(CH2), 26.4 (CH2), 21.7 (CH2). 19F NMR: δ -4.0 (CF3), -45.5
(CF2). MS (ESI) m/z (%): 299 [(M þ H)þ, 100]. Anal.
(C12H11F5O3) C, H.
1,1,1,2,2-Pentafluoro-7-(4-hexyloxyphenyl)heptan-3-one (12c).
1
Yield 61%; yellowish oil. H NMR (CDCl3): δ 7.06 (2H, d,
J = 8.4 Hz, Ph), 6.82 (2H, d, J = 8.4 Hz, Ph), 3.93 (2H, t, J = 6.6
Hz, OCH2), 2.75 (2H, t, J = 6.6 Hz, CH2), 2.57 (2H, t, J = 6.2
Hz, CH2), 1.77-1.62 (6H, m, 3 ꢀ CH2), 1.44-1.27 (6H, m,
3 ꢀ CH2), 0.90 (3H, t, J = 6.8 Hz, CH3). 13C NMR: δ 194.2 (t,
JC-C-F = 26.4 Hz, CO), 157.5 (Ph), 133.4 (Ph), 129.2 (Ph),
117.6 (qt, JC-F3 = 286 Hz, JC-CF2 = 34 Hz, CF3), 114.4 (Ph),
106.8 (tq, JC-F2 = 265 Hz, JC-CF3 = 38 Hz, CF2), 68.0 (CH2O),
37.2 (CH2), 34.5 (CH2), 31.8 (CH2), 31.6 (CH2), 29.3 (CH2), 27.5
(CH2), 25.7 (CH2), 22.6 (CH2), 14.0 (CH3). 19F NMR: δ -4.1
(CF3), -45.6 (CF2). MS (ESI) m/z (%): 379 [(M - H)-, 100].
Anal. (C19H25F5O2) C, H.
1,1,1,2,2-Pentafluoro-6-(4-octylphenoxy)hexan-3-one (12f ).
1
Yield 70%; yellowish oil. H NMR (CDCl3): δ 7.10 (2H, d,
J = 8 Hz, Ph), 6.81 (2H, d, J = 8 Hz, Ph), 3.99 (2H, t, J = 6.6
Hz, CH2), 3.00 (2H, t, J = 6.6 Hz, CH2), 2.57 (2H, t, J = 6.2 Hz,
CH2), 2.41-2.14 (2H, m, CH2), 1.64-1.58 (2H, m, CH2),
1.38-1.21 (10H, m, 5 ꢀ CH2), 0.91 (3H, t, J = 6.8 Hz, CH3).
13C NMR: δ 194.0 (t, JC-CF2 = 26 Hz, CO), 156.6 (Ph), 135.5
(Ph), 129.1 (Ph), 117.8 (qt, JC-F3 = 287 Hz, JC-CF2 = 34 Hz,
CF3), 114.4 (Ph), 106.8 (tq, JC-F2 = 267 Hz, JC-CF3 = 38 Hz,
CF2), 65.8 (CH2O), 35.4 (CH2), 34.5 (CH2), 31.5 (CH2), 30.6
(CH2), 29.3 (CH2), 25.7 (CH2), 22.6 (CH2), 21.9 (CH2), 14.2
(CH3). 19F NMR: δ -4.2 (CF3), -45.6 (CF2). MS (ESI) m/z
(%): 393 [(M - H)-, 100]. Anal. (C20H27F5O2) C, H.
1,1,1,2,2-Pentafluoro-7-(naphthalen-2-yl)heptan-3-one (12h).
Yield 38%; yellowish oil. 1H NMR (CDCl3): δ 7.88-7.28 (7H,
m, Ph), 2.83-2.78 (4H, m, 2 ꢀ CH2), 1.80-1.74 (4H, m, 2 ꢀ
CH2). 13C NMR: δ 194.4 (t, JC-CF2 = 26 Hz, CO), 139.4 (Ph),
133.9 (Ph), 132.4 (Ph), 128.4 (Ph), 127.9 (Ph), 127.6 (Ph),
127.4 (Ph), 126.7 (Ph), 126.2 (Ph), 125.9 (Ph), 118.1 (qt, JC-F3
=
287 Hz, JC-CF2 = 35 Hz, CF3), 107.2 (tq, JC-F2 = 265 Hz, JC-CF3
= 38 Hz, CF2), 37.4 (CH2), 35.9 (CH2), 30.5 (CH2), 22.2 (CH2).
19F NMR: δ -4.1 (CF3), -45.5 (CF2). MS (ESI) m/z (%): 329
[(M - H)-, 100]. Anal. (C17H15F5O) C, H.
Synthesis of Trifluoromethyl Ketones. The synthesis of triflu-
oromethyl ketones was carried out following the procedure
described above for heptafluoropropyl ketones except that
trifluoroacetic anhydride was used instead of heptafluorobuta-
noic anhydride. The products were purified by flash column
chromatography [EtOAc-petroleum ether (bp 40-60 °C), 3/7].
1,1,1-Trifluoro-5-(4-octylphenoxy)pentan-2-one (12e). Yield
r-Fluorination of r-Hydroxy Methyl Esters. Compound 16a
or 16b (1 mmol) was added to a solution of DAST (0.14 mL,
1 mmol) in dry dichloromethane (0.2 mL) at -78 °C. After being
stirred for 2 h at -78 °C and another 3 h at room temperature,
the reaction mixture was quenched with saturated aqueous
NaHCO3 (2.5 mL). The organic phase was then washed with
brine and dried (Na2SO4). The solvent was evaporated under
reduced pressure, and the residual oil was purified by flash
column chromatography on silica gel, eluting with EtOAc-
petroleum ether (bp 40-60 °C), 3/7.
1
32%; yellowish oil. H NMR (CDCl3): δ 7.10 (2H, d, J = 8
Hz, Ph), 6.80 (2H, d, J = 8 Hz, Ph), 3.99 (2H, t, J = 6.6 Hz,
OCH2), 2.95 (2H, t, J = 6.6 Hz, CH2), 2.54 (2H, t, J = 6.2 Hz,
CH2), 2.20-2.10 (2H, m, CH2), 1.61-1.51 (2H, m, CH2),
1.28-1.21 (10H, m, 5 ꢀ CH2), 0.88 (3H, t, J = 6.8 Hz, CH3).
13C NMR: δ 193.9 (t, JC-CF2 = 26 Hz, CO), 156.5 (Ph), 135.5
(Ph), 129.3 (Ph), 115.8 (q, JC-F = 292 Hz, CF3), 114.2 (Ph), 65.8
(CH2O), 35.0 (CH2), 33.1 (CH2), 31.9 (CH2), 31.7 (CH2), 31.6
(CH2), 31.5 (CH2), 29.5 (CH2), 22.6 (CH2), 22.4 (CH2), 14.0

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 9 3609
Methyl 5-Phenyl-2-fluoropentanoate (17a). Yield 60%; yel-
lowish oil. H NMR (CDCl3): δ 7.35-7.02 (5H, m, Ph), 4.98
as described previously.29,31,34 The specific assay conditions
employed for the studies reported in this manuscript for each
enzyme are as follows: (i) GIVA cPLA2 substrate mixed-micelles
were composed of 400 μM Triton X-100, 97 μM PAPC, 1.8 μM
14C-labeled PAPC, and 3 μM PIP2 in buffer containing 100 mM
HEPES, pH 7.5, 90 μM CaCl2, 2 mM DTT, and 0.1 mg/mL
BSA; (ii) GVI iPLA2 substrate mixed-micelles were composed of
400 μM Triton X-100, 98.3 μM PAPC, and 1.7 μM 14C-labeled
PAPC in buffer containing 100 mM HEPES, pH 7.5, 2 mM
ATP, and 4 mM DTT; (iii) GV sPLA2 substrate mixed-micelles
were composed of 400 μM Triton X-100, 99 μM DPPC, and
1.5 μM 14C-labeled DPPC in buffer containing 50 mM Tris, pH
8.0, and 5 mM CaCl2.
In Vitro PLA2 Inhibition Studies. Initial screening of com-
pounds at 0.091 mol fraction inhibitor in mixed micelles was
carried out. Compounds displaying 25% or less inhibition of the
assays were considered to have no inhibitory affect (designated
N.D.). We report average percent inhibition (and standard
error, n = 3) for compounds displaying less than 90% enzyme
inhibition. If the percent inhibition was greater than 90%, we
determined its XI(50) by plotting percent inhibition vs inhibitor
mole fraction (typically seven concentrations between 0.000 91
and 0.091 mol fraction). Inhibition curves were modeled in
Graphpad Prism 5.0 using nonlinear regression targeted at
symmetrical sigmoidal curves based on plots of % inhibition
versus log(inhibitor concentration) to calculate the reported
XI(50) and associated error values.
1
(1H, dt, JH-F = 48.2 Hz, JH-H = 6.2 Hz, CHF), 3.78 (3H, s,
CH3O), 2.67 (2H, t, J = 6.6 Hz, PhCH2), 2.04-1.78 (4H, m, 2 ꢀ
CH2). 13C NMR: δ 170.2 (d, JC-CF = 23.5 Hz, CO), 141.3 (Ph),
128.4 (Ph), 128.3 (Ph), 125.9 (Ph), 88.8 (d, JC-F = 183.3 Hz,
CHF), 52.1 (CH3), 35.1 (CH2), 31.8 (d, JC-CF = 20.8 Hz,
CH2CHF), 25.9 (CH2). 19F NMR: δ -114.1 (CF). MS (ESI)
m/z (%): 212 [(M þ H)þ,100]. Anal. (C12H15FO2) C, H.
Methyl 6-Phenyl-2-fluorohexanoate (17b). Yield 52%; color-
less oil. 1H NMR (CDCl3): δ 7.35-7.16 (5H, m, Ph), 4.86 (1H,
dt, JH-F = 48.2 Hz, JH-H = 6.2 Hz, CHF), 3.78 (3H, s, CH3O),
2.64 (2H, t, J = 6.6 Hz, PhCH2), 2.10-1.46 (6H, m, 3 ꢀ CH2).
13C NMR: δ 170.2 (d, JC-CF = 23.5 Hz, CO), 141.9 (Ph), 128.2
(Ph), 128.1 (Ph), 125.6 (Ph), 88.8 (d, JC-F = 183.3 Hz, CHF),
52.0 (CH3), 35.4 (CH2), 32.0 (d, JC-CF = 20.8 Hz, CH2CHF),
30.7 (CH2), 23.8 (d, JC-C-CF = 3.0 Hz CH2). 19F NMR: δ
-114.1 (CF). Anal. (C13H17FO2) C, H.
Synthesis of 1,1,1,3-Tetrafluoro Ketones. Method A. A solu-
tion of compound 17a or 17b (1 mmol) and trifluoromethyltri-
methylsilane (283 μL, 1.92 mmol) in ethylene glycol dimethyl
ether (0.92 mL) at 0 °C was treated with cesium fluoride (4 mg).
After being stirred for 30 min at 0 °C and another 18 h at 25 °C,
the reaction mixture was treated with concentrated HCl (1 mL).
After being stirred for another 18 h at 25 °C, the reaction
mixture was diluted with EtOAc (10 mL). The organic phase
was then washed with brine and dried (Na2SO4). The solvent
was evaporated under reduced pressure, and the residual oil was
purified by flash column chromatography on silica gel, eluting
with EtOAc-petroleum ether (bp 40-60 °C), 3/7.
Method B. A solution of compound 17a or 17b (1 mmol) and
trifluoromethyltrimethylsilane (1 mL, 6.9 mmol) in toluene (9
mL) at -78 °C was treated with 1.0 M TBAF (45 μL) in THF.
After the mixture was stirred for 2 h at 25 °C the intermediate
silyl ether was formed and then it was treated with 1.0 M TBAF
(1.2 mmol) in THF and with glacial acetic acid (3 drops). The
reaction mixture was stirred for 30 min at 25 °C and diluted with
EtOAc (10 mL). The organic phase was washed first with
saturated solution of K2CO3 and then with brine and dried
(Na2SO4). The solvent was evaporated under reduced pressure,
and the residual oil was purified by flash column chromatogra-
phy on silica gel, eluting with EtOAc-petroleum ether (bp
40-60 °C), 3/7.
Acknowledgment. This work was supported by the Euro-
peanSocial Fund and NationalResources (G.K.) and by NIH
Grant GM 20,501 (E.A.D.).
References
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(2) Schaloske, R. H.; Dennis, E. A. The phospholipase A(2) super-
family and its group numbering system. Biochim. Biophys. Acta
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(3) Burke, J. E.; Dennis, E. A. Phospholipase A2 structure/function,
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1,1,1,3-Tetrafluoro-6-phenylhexan-2-one (in Equilibrium with
1,1,1,3-Tetrafluoro-6-phenyl-2,2-gem-hexanodiol) (18a). Yield
47% (method A), 93% (method B); yellowish oil. 1H NMR
(CDCl3): δ 7.34-7.15 (5H, m, Ph), 5.23 (1/4H, dm, JH-F = 48.2
Hz, CH), 4.65 (3/4H, dm, JH-F = 48.2 Hz, CH), 3.74 (3/4H, s,
OH), 3.49 (3/4H, s, OH), 2.68 (2H, t, J = 6.2 Hz, CH2),
1.90-1.10 (4H, m, 2 ꢀ CH2). 13C NMR: δ 141.6 (Ph), 128.4
(Ph), 126.1 (Ph), 125.9 (Ph), 122.6 (q, JC-F3 = 286 Hz, CF3),
92.4 (d, JC-F = 175 Hz, CF), 92.2 [m, C(OH)2], 35.4 (CH2), 31.8
(d, JC-F = 20 Hz, CH2), 27.6 (d, JC-F = 20 Hz, CH2). 19F
NMR: δ 1.6 (CF3), -5.3 (CF3), -120.9 (CHF). MS (ESI) m/z
(%): 247 [(M - H)-, 100].
1,1,1,3-Tetrafluoro-7-phenylheptan-2-one (in Equilibrium with
1,1,1,3-Tetrafluoro-7-phenyl-2,2-gem-heptanodiol) (18b). Yield
45% (method A), 94% (method B); yellowish oil. 1H NMR
(CDCl3): δ 7.32-7.15 (5H, m, Ph), 5.20 (1/6H, dm, JH-F = 48.2
Hz, CH), 4.63 (5/6H, dm, JH-F = 48.2 Hz, CH), 2.64 (2H, t, J =
7.4 Hz, CH2), 1.84-1.80 (2H, m, CH2), 1.74-1.42 (4H, m, 2 ꢀ
CH2). 13C NMR: δ 142.1 (Ph), 128.3 (Ph), 125.8 (Ph), 125.3 (Ph),
122.6 (q, JC-F3 = 286 Hz, CF3), 92.3 (d, JC-F = 175 Hz, CF),
92.2 [m, C(OH)2], 35.6 (CH2), 31.0 (CH2), 27.8 (d, JC-F = 20
Hz, CH2), 24.6 (d, JC-C-F = 2.6 Hz, CH2). 19F NMR: δ 1.6
(CF3), -5.3 (CF3), -120.8 (CHF). MS (ESI) m/z (%): 261 [(M -
H)-, 100].
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