Synthesis of polyfluoro ketones for selective inhibition of human phospholipase A2 enzymes

Article abstract of DOI:10.1021/jm800649q

The development of selective inhibitors for individual PLA₂ enzymes is necessary in order to target PLA₂-specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA₂ inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA₂, a very important enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA₂ (X_I(50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the α′ position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA₂ inhibitors.

Full text of DOI:10.1021/jm800649q

J. Med. Chem. 2008, 51, 8027–8037
8027
Synthesis of Polyfluoro Ketones for Selective Inhibition of Human Phospholipase A2 Enzymes
Constantinos Baskakis,† Victoria Magrioti,† Naomi Cotton,‡ Daren Stephens,‡ Violetta Constantinou-Kokotou,§
Edward A. Dennis,*,‡ and George Kokotos*,†
Laboratory of Organic Chemistry, Department of Chemistry, UniVersity of Athens, Panepistimiopolis, Athens 15771, Greece, Department of
Chemistry and Biochemistry and Department of Pharmacology, School of Medicine, MC 0601, UniVersity of California,
San Diego, La Jolla, California 92093-0601, Chemical Laboratories, Agricultural UniVersity of Athens, Athens 11855, Greece
ReceiVed May 29, 2008
The development of selective inhibitors for individual PLA2 enzymes is necessary in order to target PLA2-
specific signaling pathways, but it is challenging due to the observed promiscuity of known PLA2 inhibitors.
In the current work, we present the development and application of a variety of synthetic routes to produce
pentafluoro, tetrafluoro, and trifluoro derivatives of activated carbonyl groups in order to screen for selective
inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate
that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA2, a very important
enzyme for which specific, potent, reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-
phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA2 (XI(50) ) 0.0073). Furthermore, we
conclude that the introduction of an additional fluorine atom at the R′ position of a trifluoromethyl ketone
constitutes an important strategy for the development of new potent GVIA iPLA2 inhibitors.
Introduction
Various classes of synthetic compounds have been studied
as inhibitors of human GIVA cPLA2, GVIA iPLA2, and GV
sPLA2; and the results are summarized in recent review
articles.11,12 One of the most potent inhibitors of GIVA cPLA2
is pyrrophenone (1, Figure 1).13 Other recently reported inhibi-
tors include 2-propanone derivatives combined with the indole
ring (e.g., 2, Figure 1)14-16 and a series of indole derivatives17-19
presented by Wyeth (for example compounds 3a and 3b, Figure
1), of which Efipladib (3b) is currently in phase I clinical trials.19
Our laboratories have reported on the development of 2-oxoa-
mide inhibitors of GIVA cPLA2 (e.g., 4a-d, Figure 1).20-26
Historically, the first potent inhibitor of GIVA cPLA2 was a
trifluoromethyl ketone analogue of arachidonic acid (AACOCF3)
in which the carboxyl group was replaced by COCF3 (5, Figure
2).27 This analogue was shown to be a slow- and tight-binding
inhibitor of GIVA cPLA2, and its mechanism of inhibition has
been characterized via 19F NMR and 13C NMR.28 Trifluoro-
methyl ketone analogues of γ-linolenic and linoleic acid as well
as the analogue of palmitic acid (6, Figure 2) also inhibit GIVA
cPLA2.29,30 Furthermore, a variety of trifluoromethyl ketones
have been analyzed with phospholipid vesicle-, detergent-
phospholipid mixed micelle-, and natural membrane-based
assays.31
Phospholipase A2 (PLA2) enzymes catalyze the hydrolysis
of the sn-2 ester bond of glycerophospholipids, producing free
fatty acids and lysophospholipids.1,2 Both products are precursor
signaling molecules that are involved in a plethora of biological
functions. The PLA2 superfamily currently consists of 15 groups
and many subgroups, of which a number of enzymes differ in
primary sequence, structure and catalytic mechanism.1 Among
the various PLA2 enzymes, Group IVA cPLA2 (GIVA
cPLA2)a is considered the rate-limiting provider of arachidonic
acid and lysophospholipids that can be converted into prostag-
landins, leukotrienes, and PAF, respectively.1-3 Another major
intracellular PLA2, the calcium-independent PLA2 (GVIA
iPLA2) appears to be the primary phospholipase for basal
metabolic functions within the cell.1,2,4,5 Both intracellular
enzymes share the same catalytic mechanism of utilizing a serine
residue as the nucleophile. The PLA2 superfamily also includes
a type of small, secreted phospholipase (sPLA2) that is
characterized by a catalytic His/Asp dyad as well as a catalytic
Ca2+ 1,2,6
A well-studied example of this class is the human
.
Group V secreted phospholipase A2 (GV sPLA2).7 In many
cases, the activity of sPLA2 has been shown to be dependent
on or linked to the activity of GIVA cPLA2.8-10
AACOCF3 has been used as a tool to study the role of GIVA
cPLA2 inhibition in various animal models. Using this inhibitor,
it was demonstrated that GIVA cPLA2 plays an important role
in the pathogenesis of experimental autoimmune encephalomy-
elitis (EAE), the animal model of multiple sclerosis.32 AA-
COCF3 was also used to study possible contributions of central
nervous PLA2 enzymes to the development of allodynia after
facial carrageenan injection in mice.33 Intrathecal administration
of AACOCF3 prevented thermal hyperalgesia induced by
intraplantar carrageenan as well as formalin-induced flinching
in a dose-dependent manner.34 Intrathecal injection of AA-
COCF3, at antihyperalgesic doses, decreased the release of
prostaglandin PGE-2 into spinal dialysate-evoked N-methyl-D-
aspartate (NMDA).34 Similarly, treatment of prion-infected cell
lines indicated a pivotal role for PLA2 enzymes in prion
diseases.35 Even so, the various in vivo activities of AACOCF3
* 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.
†
University of Athens.
University of California, San Diego.
Agricultural University of Athens.
‡
§
a Abbreviations: AACOCF3, arachidonyl trifluoromethyl ketone; ATP,
adenosine triphosphate; BEL, bromoenol lactone, DAST, diethylaminosulfur
trifluoride; DIBALH, diisobutylaluminium hydride; DPPC, 1,2-dipalmi-
toylphosphotidylcholine; DTT, dithiothreitol; EAE, experimental autoim-
mune encephalomyelitis; EtOAc, ethyl acetate; GIVA cPLA2, Group IVA
cytosolic phospholipase A2; GV sPLA2, Group V secreted phospholipase
A2; GVIA iPLA2, Group VIA calcium-independent phospholipase A2;
NMDA, N-methyl-D-aspartate; PAF, platelet activating factor; PAPC,
1-palmitoyl, 2-arachidonoyl phosphatidylcholine; PIP2, phosphatidyl inositol
(4,5)-bisphosphate; TBAF, tetra-n-butylammonium fluoride; TEMPO, 2,2,6,6-
tetramethylpiperidine-1-yloxy free radical; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; TLC, thin-layer chromatography; TMS, tetramethylsilane.
10.1021/jm800649q CCC: $40.75
2008 American Chemical Society
Published on Web 11/24/2008

8028 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Baskakis et al.
Figure 1. Some known inhibitors of GIVA cPLA2.
Figure 3. Polyfluoro ketone functionalities.
routes for the synthesis of polyfluoro ketones and demonstrate
their inhibition of the three major human PLA2 enzymes: GIVA
cPLA2, GVIA iPLA2, and GV sPLA2 but with vastly different
specificities. Of particular note is the development of specific
GVIA iPLA2 inhibitors.
Figure 2. Trifluoromethyl ketone inhibitors of GIVA cPLA2 and GVIA
iPLA2.
Design and Synthesis of Polyfluoro Ketones. We designed
a variety of polyfluoro ketones, and examples of such activated
carbonyl functionalities are depicted in Figure 3. The rationale
behind our design of polyfluoro ketones was based on: (a)
Increase of the carbonyl reactivity by introduction of additional
fluorine atoms at the ꢀ- or R′- positions. The inductive effect
of additional fluorine atoms may increase carbonyl reactivity
against nucleophiles, such as the active-site serine hydroxyl
group in GIVA cPLA2 and GVIA iPLA2. (b) Increase of the
inhibitor binding affinity to the target enzymes. Additional
fluorine atoms at the ꢀ- or R′- position may contribute to the
development of additional interactions, further stabilizing the
enzyme-inhibitor complex. Recently, it has become clear that
fluorine can enhance binding efficacy and selectivity in phar-
maceuticals due to a variety of multipolar C-F· · ·H-N,
C-F· · ·CdO, and C-F· · ·H-CR interactions between a flu-
orinated ligand and protein binding-site.43,44 Because the natural
substrates of PLA2 enzymes are long-chain phospholipids, we
chose to attach the polyfluoro ketone functionality to a long
aliphatic chain as well as to short or medium chains carrying a
nonsubstituted or para-alkoxy (or aryloxy) substituted ring.
should be viewed with some caution because this inhibitor is
not selective for GIVA cPLA2 and has been reported to cause
cell lysis.36 Additional trifluoromethyl ketone derivatives are
also observed to inhibit GIVA cPLA2.37-40 For example, BMS-
22972441 (7, Figure 2) was reported to be a tight-binding
inhibitor of GIVA cPLA2 possessing anti-inflammatory activity
in skin inflammation models.41
Trifluoromethyl ketone analogues of arachidonic and palmitic
acids also inhibit GVIA iPLA2.42 Both compounds inhibited
macrophage GVIA iPLA2 in a concentration-dependent manner
and, in contrast to GVIA cPLA2, GIVA iPLA2 showed a
preference for the saturated fatty chain.42 Inhibition studies of
a variety of trifluoromethyl ketones as inhibitors of GVIA iPLA2
in mixed-micelle assays found that one trifluoromethyl ketone
(8, Figure 2) is a potent inhibitor of GVIA iPLA2 presenting a
XI(50) value of 0.0043, which is 10-fold more potent than the
corresponding value against GIVA cPLA2.31
Continuing our efforts to synthesize selective inhibitors for
the various PLA2 enzyme types, we designed a variety of
polyfluoro ketone-based derivatives. In this work, we present

Polyfluoro Ketone Inhibitors of Phospholipase A2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8029
Figure 4. Reagents and conditions: (a) (i) (COCl2)2, CH2Cl2; (ii)
(CF3CF2CO)2O, pyridine, CH2Cl2.
Among the existing methods, the synthesis of trifluoromethyl
ketones through conversion of carboxylic acids into chlorides
followed by subsequent treatment with trifluoroacetic anhydride
and pyridine45 has found wide application. We observe that
simple carboxylic acids, amino acids and peptides,46 and even
lipophilic glyceride analogues, as we have demonstrated for the
synthesis of potent gastric lipases inhibitors,47 are able to
produce trifluoromethyl ketones in satisfactory yields. For the
synthesis of pentafluoroethyl ketones, carboxylic acids 9a-c
were converted to chlorides by treatment with oxalyl chloride
and then to the target compounds 10a-c using pentafluoropro-
pionic anhydride and pyridine (Figure 4). For comparison
purposes, we prepared pentafluoroethyl ketone 11 corresponding
to palmitic acid as well as trifluoromethyl ketones 12a,b
corresponding to pentafluoro derivatives 10b,c.
Figure 5. Reagents and conditions: (a) NaOCl, TEMPO, NaBr,
NaHCO3, toluene/EtOAc, H2O; (b) Ph3PdCHCOOCH3, CH2Cl2; (c)
C2H5OOCHdCHCH2P(dO)(OC2H5)2, LiOH, THF; (d) (i) H2, 10% Pd,
(ii) NaOH, CH3OH, (e) (i) (COCl2)2, CH2Cl2, (ii) (CF3CO)2O, pyridine,
CH2Cl2.
The synthesis of various trifluoromethyl and pentafluoroethyl
ketones is depicted in Figure 5. The hydroxymethyl group of
compounds 13a,b was oxidized to an aldehyde by the NaOCl/
TEMPO method.48 Wittig olefination of aldehydes 14a,b and
Wadsworth-Horner-Emmons reaction led to elongation of the
chain by two or four carbon atoms, respectively. After hydro-
genation and saponification, carboxylic acids 17a,b and 18a,b
were converted to fluoroketones 19a,b, 20a,b, and 21 as
described above. The trifluoromethyl ketone 23 was prepared
from the known carboxylic acid 22 (Figure 6).
Figure 6. Reagents and conditions: (a) (i) (COCl2)2, CH2Cl2, (ii)
(CF3CO)2O, pyridine, CH2Cl2.
Tetrafluoro derivative 26 was synthesized as shown in Figure
7. The replacement of the hydroxyl group of methyl 2-hydroxy-
hexadecanoate (24) with fluorine was carried out by treatment
with diethylaminosulfur trifluoride (DAST), a well-known
fluorinating agent.49 Treatment of methyl ester 25 by (trifluo-
romethyl)trimethylsilane in the presence of a catalytic amount
of cesium fluoride, followed by hydrolysis of silyl ether
intermediate,50 led directly to tetrafluoro derivative 26. It should
be noted that a 2-fluorocarboxylic acid cannot transform into a
trifluoromethyl ketone by conversion to chloride and treatment
with anhydride and pyridine, probably because the intermediate
ketene required for such a transformation45 cannot be formed.
To synthesize pentafluoro derivative 30, we explored two
different routes (Figures 8 and 9). Reaction of diethyl oxalate
with Grignard reagent51 27 led to 2-oxoester 28 (Figure 8).
DAST is an efficient reagent for the conversion of 2-oxoesters
to 2,2-difluoroesters;52,53 therefore, 2-oxoester 28 was fluorinated
by treatment with DAST and ethyl ester 29 was converted to
trifluoromethyl ketone 30 as described above. Alternatively,
compound 30 was prepared starting from aldehyde 31 (Figure
Figure 7. Reagents and conditions: (a) DAST, dry CH2Cl2; (b) (i)
(CH3)3SiCF3, CsF, CH3OCH2CH2OCH3, (ii) conc HCl.
9). Formation of cyanohydrin 32 was followed by methanolysis
and finally oxidation to produce 2-oxoester 34. By similar
procedures to those described above, the pentafluoro derivative
30 was prepared.
Electrophilic ketones, like fluoroketones, may exist in equi-
librium with their corresponding hydrates (gem diols) depending
1
on the environment. On the basis of the H NMR data, the
trifluoromethyl ketones and the pentafluoroethyl ketones syn-

8030 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Baskakis et al.
Figure 8. Reagents and conditions: (a) dry Et2O, diethyl oxalate; (b) Et2NSF3; (c) (i) (CH3)3SiCF3, CsF, CH3OCH2CH2OCH3, (ii) conc HCl.
Figure 9. Reagents and conditions: (a) NaHSO3, KCN, CH2Cl2; (b) HCl, MeOH; (c) Dess-Martin periodinane, CH2Cl2; (d) Et2NSF3, CH2Cl2; (e)
(i) (CH3)3SiCF3, CsF, CH3OCH2CH2OCH3, (ii) conc HCl.
thesized in this work were found to exist solely in their ketone
forms in chloroform solution. However, tetrafluoro derivative
26 appears to be a mixture of ketone-hydrate form in a ratio
1:2, whereas pentafluoro derivative 30 is completely hydrated
(see NMR data in Experimental Section). 19F NMR spectro-
scopic data confirm the existence of the hydrated form in the
cases of compounds 26 and 30.
In Vitro Inhibition of GIVA cPLA2, GVIA iPLA2 and
GV sPLA2. All synthesized inhibitors were tested for inhibition
of human GIVA cPLA2, GVIA iPLA2, and GV sPLA2 using
previously described mixed micelle-based assays.20,21,24,25 The
resulting degrees of inhibition are presented in Table 1 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 estimated for
compounds that displayed greater than 90% inhibition. The
XI(50) is the mole fraction of the inhibitor in the total substrate
interface required to inhibit the enzyme by 50%.
In accordance with the literature, the long-chain saturated
palmitoyl trifluoromethyl ketone 6 inhibits both intracellular
enzymes GIVA cPLA2 and GVIA iPLA2 at a similar level. In
this work, we show that compound 6 is also a weak inhibitor
of GV sPLA2 (79% inhibition at 0.091 mol fraction). However,
compound 8 is considered to be a selective inhibitor of GVIA
iPLA2 with an observed XI(50) 0.0096, while high mole fraction
of the inhibitor causes only 38% inhibition of GIVA cPLA2
and does not affect GV sPLA2.
The introduction of a pentafluoroethyl ketone functionality
led to adverse effects depending on the nature of the chain.
1,1,1,2,2-Pentafluoro-7-phenyl-heptan-3-one (10a, FKGK11)
presents slightly higher inhibitory activity on GVIA iPLA2
(XI(50) 0.0073) than the corresponding trifluoromethyl derivative
8. The dose-response curve for the inhibition of GVIA iPLA2
by pentafluoroethyl ketone 10a is shown in Figure 10. In
addition, it demonstrates selective inhibition for GVIA iPLA2
because high mole fractions (0.091) do not affect GIVA cPLA2
and caused slight inhibition (28%) of GV sPLA2. Interestingly,
the long-chain saturated pentafluoroethyl ketone 11 abolished
the inhibitory potency and selectivity, demonstrating only 50%
inhibition of GVIA iPLA2 and 43% inhibition of GV sPLA2 at
0.091 mol fraction.
carbonyl group and the aromatic ring resulted in decreased
selectivity for GVIA iPLA2. Derivatives 10b and 10c (five and
six carbon atoms, respectively) inhibit GVIA iPLA2 at a similar
level as inhibitor 10a (XI(50) 0.0065). However, both 10b and
10c are weak inhibitors of GIVA cPLA2 (56% and 65%,
respectively) and GV sPLA2 (46% and 75%, respectively). For
the trifluoromethyl ketone derivatives 12a and 12b, the inhibi-
tory activity increased as the chain length increased between
the carbonyl group and the aromatic ring. Both 12a and 12b
are more potent inhibitors of GVIA iPLA2 (XI(50) 0.0025 and
XI(50) 0.0018, respectively) than compound 8; however, these
compounds also weakly inhibit GIVA cPLA2 (62% and 68%,
respectively) and GV sPLA2 (48% and 53%, respectively) at
0.091 mol fraction. These results demonstrate that an increase
of carbon atoms between the activated carbonyl group and the
aromatic ring leads to a loss in selectivity.
Trifluoromethyl ketones 19a, 19b, 20a, and 20b containing
a medium (hexyloxy) or a long (decyloxy) chain substituent at
the para position of the aromatic ring inhibit both GIVA cPLA2
and GVIA iPLA2. The dose-response curves for the inhibition
of GVIA iPLA2 and GIVA cPLA2 by 1,1,1-trifluoro-6-(4-
hexyloxy-phenyl)-hexan-2-one (20a, FKGK2) are shown in
Figure 11. Comparison of 19a with 20a and 19b with 20b shows
that the increase of the chain length between the carbonyl group,
and the aromatic ring from two to four carbon atoms results in
increased inhibitory potency for both GIVA cPLA2 and GVIA
iPLA2. All of these compounds (19a, 19b, 20a, and 20b) also
inhibit GV sPLA2. Thus, trifluoromethyl ketones containing an
alkoxy group at the para position of the aromatic group can be
considered to be pan inhibitors of the all three enzymes: GIVA
cPLA2, GVIA iPLA2, and GV sPLA2. In particular, compound
20a is an inhibitor suitable for applications involving the
inhibition of both intracellular and extracellular PLA2 enzymes.
The replacement of the hexyloxy by a benzyloxy group led to
derivative 23, which weakly inhibited all the three PLA2
enzymes. Comparison of inhibitors 8, 20a, 20b, and 23
demonstrates that the introduction of an alkoxy or a benzyloxy
group in the aromatic ring destroys the selectivity for GVIA
iPLA2.
Comparison of pentafluoroethyl ketone 21 with the corre-
sponding trifluoromethyl ketone 19a reinforces our observation
that pentafluoroethyl ketone functionality favors the inhibition
of GVIA iPLA2 (XI(50) 0.0075). However, the presence of a
In pentafluoroethyl derivatives, increasing the chain length
(from four to five or six carbon atoms) between the activated

Polyfluoro Ketone Inhibitors of Phospholipase A2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8031
Table 1. Inhibition of PLA2 by Fluoroketonesa
a Average percent inhibition and standard error (n ) 3) reported for each compound at 0.091 mole fraction. XI(50) values determined for inhibitors with
greater than 90% inhibition. N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition).
hexyloxy substituent leads to loss of selectivity for GVIA iPLA2
because compound 21 weakly inhibits GIVA cPLA2 (73%) and
GV sPLA2 (86%) at 0.091 inhibitor mole fraction.
is the most potent inhibitor of GVIA iPLA2 reported, indicating
that introduction of an additional fluorine atom at the R′ position
constitutes an important strategy for the development of new
potent GVIA iPLA2 inhibitors. However, the tetrafluoro deriva-
tive 26 also inhibits GIVA and GV PLA2. Interestingly, the
introduction of two fluorine atoms at the R′ position in an
aromatic ring containing derivative destroyed the inhibitory
potency and the selectivity for GVIA iPLA2. For example, at
0.091 mol fraction, derivative 30 is a weak inhibitor of GVIA
iPLA2 (49%), GV sPLA2 (59%), and presents no significant
inhibition of GIVA cPLA2 (27%).
Comparison of compound 26 with 6 shows that the introduc-
tion of an additional fluorine atom at the R′ position in a long
chain saturated derivative results in a derivative with slightly
better activity for GIVA cPLA2 (XI(50) 0.0167) than the parent
trifluoromethyl ketone 6 (XI(50) 0.0223). More importantly,
tetrafluoro derivative 26 is approximately 20-fold more potent
inhibitor of GVIA iPLA2 (XI(50) 0.0011) than the trifluoro
derivative 6 (XI(50) 0.0195). To our knowledge, compound 26

8032 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Baskakis et al.
However, Turk et al. have recently reported that BEL inactivates
GVIA iPLA2 by generating a diffusible bromomethyl keto acid
that alkylates cysteine thiols, rather than creating an acyl-enzyme
intermediate with the active-site serine.57 Therefore, it is likely
that BEL affects multiple enzymes and should be used with
appropriate caution when studying potential roles of GVIA
iPLA2.57 These observations lead us to design selective inhibitors
of GVIA iPLA2 such as the pentafluoroethyl ketone 10a.
In conclusion, we developed and applied a variety of synthetic
routes to produce various pentafluoro, tetrafluoro, and trifluoro
derivatives containing activated carbonyl groups. We studied
their in vitro activity on the three major human PLA2 enzyme
classes and demonstrated that the pentafluoroethyl ketone
functionality favors GVIA iPLA2 inhibition. Furthermore,
1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (10a) was shown
to be a selective inhibitor of GVIA iPLA2. Additionally,
introduction of an additional fluorine atom at the R′ position of
a trifluoromethyl ketone constitutes an important strategy for
the development of new potent GVIA iPLA2 inhibitors. The
tetrafluoro derivative of palmitic acid 26 is observed to be the
most potent inhibitor of GVIA iPLA2 to date; however, it also
inhibits GIVA cPLA2 and GV sPLA2. Polyfluoro ketones
displaying an array of selectivities for the major PLA2 enzyme
classes will prove to be valuable tools for the in vivo
characterization of the roles of PLA2 enzymes. Furthermore,
we found that these compounds do not show cytotoxicity toward
cells in culture and we are currently utilizing these polyfluoro
ketone derivatives for the comparison of intracellular versus
extracellular PLA2 enzyme roles in animal models of neurologi-
cal disorders such as multiple sclerosis, spinal cord injury, and
peripheral nerve injury.58
Figure 10. Inhibition curve for pentafluoro ketone 10a in a mixed-
micelle assay with human GVIA iPLA2. Nonlinear regression (hyper-
bolic) estimated a XI(50) value of 0.0073 ( 0.0007. Compound 10a
inhibited GIVA cPLA2 less than 25% and GV sPLA2 approximately
28% at 0.091 mol fraction.
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) and are referenced in
ppm relative to TMS for 1NMR and 13C 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 phospho-
molybdic acid, in EtOH stain. Tetrahydrofuran (THF), 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 the products gave
satisfactory elemental analysis results.
Figure 11. Inhibition curves for trifluoromethyl ketone 20a in a mixed-
micelle assay with human GVIA cPLA2 and GIVA iPLA2. Nonlinear
regressions (hyperbolic) estimated XI(50) values of 0.0169 ( 0.0021
and 0.0098 ( 0.0006 for GIVA cPLA2 and GVIA iPLA2, respectively.
Compound 20a inhibited GV sPLA2 approximately 86% at 0.091 mol
fraction.
Figure 12. Structures of difluoro ketone inhibitor 36 of cobra venom
PLA2 and BEL inhibitor 37.
General Procedure for the Synthesis of Pentafluoroethyl
Ketones. Oxalyl chloride (0.38 g, 3 mmol) and N,N-dimethylfor-
mamide (40 µL) were added to a solution of carboxylic acid (1
mmol) in dry dichloromethane (40 mL). After 3 h stirring at room
temperature, the solvent and excess reagent were evaporated under
reduced pressure and the residue was dissolved in dry dichlo-
romethane (10 mL). Pyridine (0.64 mL, 8 mmol) and pentafluo-
ropropionic anhydride (0.85 mL, 6 mmol) were added dropwise to
this solution at 0 °C consecutively. After stirring 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
stirring 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 (Na2-
SO4). The solvent was evaporated under reduced pressure, and the
residual oil was purified by flash column chromatography [EtOAc-
petroleum ether (bp 40-60 °C) 1/9].
Our data indicate the importance of screening selective
inhibitors against multiple enzyme classes within the PLA2
superfamily. As mentioned above, our work shows that the
known inhibitor palmitoyl trifluoromethyl ketone 6, reported
to strictly inhibit intracellular GVIA iPLA2 and GIVA cPLA2,
also weakly inhibits GV sPLA2. Similarly, some of our
synthesized trifluoromethyl, pentafluoroethyl and tetrafluoro
derivatives (for example, compounds 20a, 21, 26) were found
to inhibit GV sPLA2. Futhermore, Gelb et al. demonstrated that
difluoro ketones similar to 36 (Figure 12) inhibit cobra venom
PLA2.54 Therefore activated ketones, such as polyfluoro ketones,
are likely to inhibit serine enzymes, GIVA cPLA2 and GVIA
iPLA2, as well as histidine enzymes like secreted PLA2.
Bromoenol lactone (BEL) 37 (Figure 12) is considered to be
a selective and irreversible GVIA iPLA2 and has been widely
applied to study potential biological roles for GVIA iPLA2.55,56
1,1,1,2,2-Pentafluoro-7-phenyl-heptan-3-one (10a). Yield 53%;
1
yellowish oil. H NMR (CDCl3): δ 7.31-7.17 (5H, m, Ph), 2.80

Polyfluoro Ketone Inhibitors of Phospholipase A2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8033
(2H, t, J ) 6.2 Hz, CH2), 2.66 (2H, t, J ) 6.6 Hz, CH2), 1.73-1.67
(4H, m, 2 × CH2). 13C NMR: δ 194.2 (t, JC-C-F ) 26 Hz, CO),
1,1,1-Trifluoro-4-(4-hexyloxy-phenyl)-butan-2-one (19a). Yield
53%; yellowish oil. 1H NMR (CDCl3): δ 7.10 (2H, d, J ) 8.6 Hz,
Ph), 6.84 (2H, d, J ) 8.6 Hz, Ph), 3.93 (2H, t, J ) 6.2 Hz, CH2O),
3.10-2.92 (4H, m, 2 × CH2), 1.82-1.62 (2H, m, CH2), 1.55-1.22
(6H, m, 3 × CH2), 0.91 (3H, t, J ) 6.6 Hz, CH3). 13C NMR: δ
190.7 (q, JC-C-F ) 35 Hz, COCF3), 157.9 (Ph), 131.0 (Ph), 129.2
(Ph), 115.5 (q, JC-F ) 292 Hz, CF3), 114.7 (Ph), 68.0 (OCH2),
38.3 (CH2), 31.6 (CH2), 29.2 (CH2), 27.5 (CH2), 25.7 (CH2), 22.6
(CH2), 13.9 (CH3). 19F NMR: δ -1.5 (CF3). MS (ESI) m/z (%):
301 (M-, 100). Anal. (C16H21F3O2) C, H.
141.6 (Ph), 128.4 (Ph), 128.3 (Ph), 125.9 (Ph), 117.8 (qt, JC-F3
287 Hz, JC-CF2 ) 34 Hz, CF3), 106.8 (tq, JC-F2 ) 267 Hz, JC-CF3
)
)
38 Hz, CF2), 37.1 (CH2), 35.5 (CH2), 30.3 (CH2), 21.9 (CH2). 19
F
NMR: δ -4.1 (CF3), -45.5 (CF2). MS (ESI) m/z (%): 279 (M-,
100). Anal. (C13H13F5O) C, H.
1,1,1,2,2-Pentafluoro-8-phenyl-octan-3-one (10b). Yield 75%;
1
yellowish oil. H NMR (CDCl3): δ 7.35-7.21 (5H, m, Ph), 2.79
(2H, t, J ) 6.8 Hz, CH2), 2.68 (2H, t, J ) 7.4 Hz, CH2), 1.80-1.68
(4H, m, 2 × CH2), 1.48-1.40 (2H, m, CH2). 13C NMR: δ 194.3
(t, JC-C-F ) 26 Hz, CO), 142.2 (Ph), 128.3 (Ph), 128.2 (Ph), 125.7
(Ph), 117.8 (qt, JC-F3 ) 285 Hz, JC-CF2 ) 34 Hz, CF3), 106.9 (tq,
JC-F2 ) 265 Hz, J ) 37 Hz, CF2), 37.2 (CH2), 35.6 (CH2), 31.0
(CH2), 28.2 (CH2), 22.1 (CH2). 19F NMR: δ -4.2 (CF3), -45.6
(CF2). MS (ESI) m/z (%): 293 (M-, 100). Anal. (C14H15F5O) C,
H.
4-(4-Decyloxy-phenyl)-1,1,1-trifluoro-butan-2-one (19b). Yield
46%; yellowish oil; 1H NMR (CDCl3): δ 7.12 (2H, d, J ) 8.6 Hz,
Ph), 6.85 (2H, d, J ) 8.6 Hz, Ph), 3.95 (2H, t, J ) 6.6 Hz, CH2O),
3.05-2.85 (4H, m, 2 × CH2), 1.81-1.62 (2H, m, CH2), 1.56-1.22
(14H, m, 7 × CH2), 0.92 (3H, t, J ) 6.8 Hz, CH3). 13C NMR: δ
190.5 (q, JC-C-F ) 35 Hz, COCF3), 157.9 (Ph), 131.0 (Ph), 129.2
(Ph), 115.5 (q, JC-F ) 292 Hz, CF3), 114.6 (Ph), 68.0 (CH2O),
38.3 (CH2), 31.8 (CH2), 29.6 (CH2), 29.3 (CH2), 29.2 (CH2), 27.4
(CH2), 26.0 (CH2), 22.7 (CH2), 14.0 (CH3). 19F NMR: δ -1.5 (CF3).
MS (FAB) m/z (%): 358 (M+, 85). Anal. (C20H29F3O2) C, H.
1,1,1-Trifluoro-6-(4-hexyloxy-phenyl)-hexan-2-one (20a).
1,1,1,2,2-Pentafluoro-9-phenyl-nonan-3-one (10c). Yield 60%;
1
yellowish oil. H NMR (CDCl3): δ 7.31-7.18 (5H, m, Ph), 2.76
(2H, t, J ) 6.8 Hz, CH2), 2.64 (2H, t, J ) 8.0 Hz, CH2), 1.72-1.58
(4H, m, 2 × CH2), 1.44-1.34 (4H, m, 2 × CH2). 13C NMR: δ
194.4 (t, JC-C-F ) 26 Hz, CO), 142.5 (Ph), 128.4(Ph), 128.3 (Ph),
125.7 (Ph), 117.8 (qt, JC-F3 ) 285 Hz, JC-CF2 ) 34 Hz, CF3), 106.9
(tq, JC-F2 ) 265 Hz, JC-CF3 ) 37 Hz, CF2), 37.3 (CH2), 35.8 (CH2),
31.1 (CH2), 28.8 (CH2), 28.5 (CH2), 22.2 (CH2). 19F NMR: δ -4.2
(CF3), -45.6 (CF2); MS (ESI) m/z (%): 307 (M-, 100). Anal.
(C15H17F5O) C, H.
1
Yield 45%; yellowish oil. H NMR (CDCl3): δ 7.09 (2H, d, J )
8.0 Hz, Ph), 6.85 (2H, d, J ) 8.0 Hz, Ph), 3.95 (2H, t, J ) 6.6 Hz,
CH2O), 2.74 (2H, t, J ) 6.6 Hz, CH2), 2.60 (2H, t, J ) 6.2 Hz,
CH2), 1.82-1.62 (6H, m, 3 × CH2), 1.46-1.25 (6H, m, 3 × CH2),
0.94 (3H, t, J ) 6.8 Hz, CH3). 13C NMR: δ 191.4 (q, JC-C-F ) 34
Hz, COCF3), 157.9 (Ph), 133.4 (Ph), 129.1 (Ph), 115.4 (q, JC-F
)
290 Hz, CF3), 114.4 (Ph), 67.9 (CH2O), 36.1 (CH2), 34.5 (CH2),
31.6 (CH2), 30.6 (CH2), 29.3 (CH2), 25.7 (CH2), 22.6 (CH2), 21.8
(CH2), 13.9 (CH3). 19F NMR: δ -1.6 (CF3). MS (FAB) m/z (%):
330 (M+, 23). Anal. (C18H25F3O2) C, H.
1,1,1,2,2-Pentafluoro-octadecan-3-one (11). Yield 24%; color-
1
less oil. H NMR (CDCl3): δ 2.75 (2H, t, J ) 7.4 Hz, CH2), 1.67
(2H, t, J ) 7.0 Hz, CH2), 1.38-1.20 (24H, m, 12 × CH2), 0.88
(3H, t, J ) 7.0 Hz, CH3). 13C NMR: δ 194.5 (t, JC-CF2 ) 26 Hz,
CO), 117.8 ppm (qt, JC-F3 ) 285 Hz, JC-CF2 ) 34 Hz, CF3), 106.9
ppm (tq, JC-F2 ) 265 Hz, JC-CF3 ) 38 Hz, CF2), 37.4 (CH2), 31.9
(CH2), 30.3 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2),
29.2 (CH2), 28.7 (CH2), 22.7 (CH2), 22.3 (CH2), 14.1 (CH3). 19F
NMR: δ -4.2 (CF3), -45.6 (CF2). MS (ESI) m/z (%): 357 (M-,
93). Anal. (C18H31F5O) C, H.
6-(4-Decyloxy-phenyl)-1,1,1-trifluoro-hexan-2-one (20b). Yield
46%; yellowish oil. 1H NMR (CDCl3): δ 7.08 (2H, d, J ) 8.6 Hz,
Ph), 6.84 (2H, d, J ) 8.6 Hz, Ph), 3.94 (2H, t, J ) 6.6 Hz, CH2O),
2.73 (2H, t, J ) 6.6 Hz, CH2), 2.59 (2H, t, J ) 7.0 Hz, CH2),
1.82-1.62 (6H, m, 3 × CH2), 1.45-1.22 (14H, m, 7 × CH2), 0.90
(3H, t, J ) 6.8 Hz, CH3). 13C NMR: δ 191.6 (q, JC-C-F ) 35 Hz,
COCF3), 157.7 (Ph), 133.7 (Ph), 129.4 (Ph), 115.8 (q, JC-F ) 292
Hz, CF3), 114.6 (Ph), 68.2 (CH2O), 36.4 (CH2), 34.8 (CH2), 32.1
(CH2), 30.9 (CH2), 29.8 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2),
1,1,1,2,2-Pentafluoro-5-(4-hexyloxy-phenyl)-pentan-3-one (21).
1
Yield 76%; yellowish oil. H NMR (CDCl3): δ 7.10 (2H, d, J )
26.6 (CH2), 26.3 (CH2), 22.9 (CH2), 22.1 (CH2), 14.32 (CH3). 19
F
8.6 Hz, Ph), 6.85 (2H, d, J ) 8.6 Hz, Ph), 3.94 (2H, t, J ) 6.6 Hz,
CH2O), 3.02 (2H, t, J ) 7.0 Hz, CH2), 2.96 (2H, t, J ) 7.0 Hz,
CH2), 1.86-1.73 (2H, m, CH2), 1.60-1.25 (6H, m, 3 × CH2), 0.93
NMR: δ -1.5 (CF3). MS (FAB) m/z (%): 386 (M+, 100). Anal.
(C22H33F3O2) C, H.
(3H, t, J ) 6.4 Hz, CH3). 13C NMR (CDCl3): δ 193.5 (t, JC-C-F
26 Hz, CO), 157.9 (Ph), 130.9 (Ph), 129.2 (Ph), 117.8 (qt, JC-F3
)
)
4-(4-Benzyloxy-phenyl)-1,1,1-trifluoro-butan-2-one (23). Yield
43%; yellowish solid; mp 71-72 °C. 1H NMR (CDCl3): δ
7.46-7.35 (5H, m, Ph), 7.15 (2H, d, J ) 8.4 Hz, Ph), 6.95 (2H, d,
J ) 8.4 Hz, Ph), 5.07 (2H, s, PhCH2), 3.12-2.85 (4H, m, 2 ×
CH2). 13C NMR: δ 190.7 (q, JC-C-F ) 35 Hz, COCF3), 157.4 (Ph),
136.9 (Ph), 131.5 (Ph), 129.2 (Ph), 128.5 (Ph), 127.9 (Ph), 127.4
(Ph), 115.4 (q, JC-F ) 290 Hz, CF3), 114.9 (Ph), 70.0 (CH2O),
38.3 (CH2), 27.4 (CH2). 19F NMR: δ -1.4 (CF3). MS (ESI) m/z
(%): 307 (M-, 100). Anal. (C17H15F3O2) C, H.
286 Hz, JC-CF2 ) 34 Hz, CF3), 114.7 (Ph), 106.8 (tq, JC-F2 ) 265
Hz, JC-CF3 ) 38 Hz, CF2), 68.0 (CH2O), 39.4 (CH2), 31.6 (CH2),
29.3 (CH2), 27.5 (CH2), 25.7 (CH2), 22.6 (CH2), 13.9 (CH3). 19F
NMR: δ -4.2 (CF3), -45.6 (CF2). MS (ESI) m/z (%): 351 (M-,
100). Anal. (C17H21F5O2) C, H.
Synthesis of Trifluoromethyl Ketones. The synthesis of triflu-
oromethyl ketones was carried out following the procedure
described above for pentafluoromethyl ketones, except that trifluo-
roacetic anhydride was used instead of pentafluoropropionic
anhydride. The products were purified by flash column chroma-
tography [EtOAc-petroleum ether (bp 40-60 °C) 3/7].
Intermediate compounds 14a,b and 22 were prepared by known
methods, and their spectroscopic data were in accordance with those
in the literature.60,61
Horner-Wadsworth-Emmons Olefination. A suspension of
aldehyde 14a or 14b (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 tetrahydro-
furan (10 mL) was refluxed under argon for 24 h. The reaction
mixture was then cooled to room temperature, filtered through a
thin pad of celite and the solvent evaporated under reduced pressure.
The residual oil was purified by chromatography on silica gel eluting
with ether-petroleum ether (bp 40-60 °C) 1/9.
Ethyl (2E,4E)-5-(4-Hexyloxy-phenyl)-penta-2,4-dienoate (16a).
Yield 71%; white solid; mp 68-69 °C. 1H NMR (CDCl3): δ
7.48-7.20 (3H, m, CH, Ph), 6.90-6.75 (3H, m, CH, Ph), 6.71
(1H, d, J ) 15.4 Hz, CH), 5.94 (1H, d, J ) 15.4 Hz, CHCOO),
4.23 (2H, q, J ) 7.4 Hz, OCH2CH3), 3.97 (2H, t, J ) 6.2 Hz,
CH2O), 1.85-1.62 (2H, m, CH2CH2O), 1.45-1.02 (9H, m, 3 ×
CH2, CH3), 0.92 (3H, t, J ) 6.8 Hz, CH3). 13C NMR: δ 167.2
1,1,1-Trifluoro-7-phenylheptan-2-one (12a).59 Yield 45%; yel-
lowish oil. 1H NMR (CDCl3): δ 7.34-7.19 (5H, m, Ph), 2.76-2.62
(4H, m, 2 × CH2), 1.77-1.66 (4H, m, 2 × CH2), 1.46-1.39 (2H,
m, CH2). 13C NMR: δ 191.8 (q, JC-C-F ) 35 Hz, COCF3), 142.2
(Ph), 128.3 (Ph), 128.2 (Ph), 125.7 (Ph), 115.5 (q, JC-F ) 290 Hz,
CF3), 36.2 (CH2), 35.7 (CH2), 30.9 (CH2), 28.2 (CH2), 22.2 (CH2).
19F NMR: δ -1.5 (CF3). MS (ESI) m/z (%): 243 (M-, 100).
1,1,1-Trifluoro-8-phenyloctan-2-one (12b).59 Yield 42%; yel-
lowish oil. 1H NMR (CDCl3): δ 7.28-7.17 (5H, m, Ph), 2.72-2.60
(4H, m, 2 × CH2), 1.70-1.61 (4H, m, 2 × CH2), 1.42-1.24 (4H,
m, 2 × CH2). 13C NMR: δ 191.4 (q, JC-C-F ) 35 Hz, COCF3),
142.5 (Ph), 128.3 (Ph), 128.2 (Ph), 125.7 (Ph), 115.6 (q, JC-F
)
291 Hz, CF3), 36.3 (CH2), 35.8 (CH2), 31.1 (CH2), 28.7 (CH2),
28.6 (CH2), 22.3 (CH2). 19F NMR: δ -1.5 (CF3). MS (ESI) m/z
(%): 257 (M-, 100).

8034 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Baskakis et al.
(COO), 160.0 (Ph), 145.0 (CH), 140.2 (CH), 131.9 (Ph), 128.6 (Ph),
123.9 (CH), 119.9 (CH), 114.7 (Ph), 68.0 (CH2O), 60.2 (OCH2CH3),
31.6 (CH2), 29.1 (CH2), 25.7 (CH2), 22.6 (CH2), 14.3 (CH3), 14.0
(CH3). Anal. (C19H26O3) C, H.
(CH2O), 35.9 (CH2), 31.9 (CH2), 29.7 (CH2), 29.5 (CH2), 29.3
(CH2), 26.0 (CH2), 22.6 (CH2), 14.1 (CH3). Anal. (C19H30O3) C,
H.
5-(4-Hexyloxy-phenyl)-pentanoic acid (18a). Yield 96%; white
1
solid; mp 90-91 °C. H NMR (CDCl3): δ 7.03 (2H, d, J ) 8.4
Ethyl (2E,4E)-5-(4-Decyloxy-phenyl)-penta-2,4-dienoate (16b).
Yield 65%; white solid; mp 80-81 °C. 1H NMR (CDCl3): δ
7.45-7.38 (3H, m, CH, Ph), 6.88-6.80 (3H, m, CH, Ph), 6.78
(1H, d, J ) 12 Hz, CH), 5.94 (1H, d, J ) 15.4 Hz, CHCOO), 4.23
(2H, q, J ) 7.4 Hz, OCH2CH3), 3.97 (2H, t, J ) 6.6 Hz, CH2O),
1.81-1.75 (2H, m, CH2CH2O), 1.50-1.14 (17H, m, 7 × CH2,
CH3), 0.89 (3H, t, J ) 6.8 Hz, CH3). 13C NMR: δ 167.3 (COO),
160.0 (Ph), 145.1 (CH), 140.2 (CH), 131.9 (Ph), 128.6 (Ph), 124.0
(CH), 119.9 (CH), 114.8 (Ph), 68.1 (CH2O), 60.2 (OCH2CH3) 31.9
(CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 26.0 (CH2),
22.7 (CH2), 14.3 (CH3), 14.1 (CH3). Anal. (C23H34O3) C, H.
Wittig Olefination. A solution of aldehyde 14a or 14b (1 mmol)
and methyl (triphenylphosphanylidene)acetate (0.334 g, 1 mmol)
in dry dichloromethane (3 mL) was refluxed under argon for 24 h.
The reaction mixture was then cooled to room temperature and the
solvent evaporated under reduced pressure. The residual oil was
purified by flash column chromatography on silica gel eluting with
EtOAc-petroleum ether (bp 40-60 °C) 1/9.
Methyl (E)-3-(4-Hexyloxy-phenyl)-acrylate (15a). Yield 93%;
white solid; mp 84-85 °C. H NMR (CDCl3): δ 7.63 (1H, d, J )
15.8 Hz, CH ) CHCO), 7.43 (2H, d, J ) 8.8 Hz, Ph), 6.87 (2H,
d, J ) 8.8 Hz, Ph), 6.28 (1H, d, J ) 15.8 Hz, CHCOO), 3.95 (2H,
t, J ) 6.4 Hz, CH2O), 3.77 (3H, s, OCH3), 1.76 (2H, m, CH2CH2O),
1.46-1.21 (6H, m, 3 × CH2), 0.89 (3H, t, J ) 6.8 Hz, CH3). 13C
NMR: δ 167.7 (COO), 161.0 (Ph), 144.6 (CH), 129.6 (Ph), 126.8
(Ph), 115.0 (CH), 114.7 (Ph), 68.1 (CH2O), 51.5 (OCH3), 31.5
(CH2), 29.0 (CH2), 25.6 (CH2), 22.5 (CH2), 13.9 (CH3). Anal.
(C16H22O3) C, H.
Methyl (E)-3-(4-Decyloxy-phenyl)-acrylate (15b). Yield 92%;
white solid; mp 75-76 °C. 1H NMR (CDCl3): δ 7.63 (1H, d, J )
15.8 Hz, CH ) CHCOO), 7.37 (2H, d, J ) 8.8 Hz, Ph), 6.85 (2H,
d, J ) 8.8 Hz, Ph), 6.23 (1H, d, J ) 15.8 Hz, CHCOO), 3.87 (2H,
t, J ) 6.6 Hz, CH2O), 3.71 (3H, s, OCH3), 1.78-1.62 (2H, m,
CH2CH2O), 1.40-1.22 (14H, m, 7 × CH2), 0.84 (3H, t, J ) 7 Hz,
CH3). 13C NMR: δ 167.4 (COO), 160.8 (Ph), 144.3 (CH), 129.4
(Ph), 126.6 (Ph), 114.8 (CH), 114.5 (Ph), 67.8 (CH2O), 51.2
(OCH3), 31.7 (CH2), 29.4 (CH2), 29.2 (CH2), 29.1 (CH2), 29.0
(CH2), 25.8 (CH2), 22.5 (CH2), 13.9 (CH3). Anal. (C20H30O3) C,
H.
Hydrogenation and Saponification of Unsaturated Esters. A
mixture of the unsaturated ester (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 compound.
Hz, Ph), 6.77 (2H, d, J ) 8.4 Hz, Ph), 3.88 (2H, t, J ) 6.2 Hz,
CH2O), 2.52 (2H, t, J ) 6.8 Hz, CH2), 2.32 (2H, t, J ) 6.7 Hz,
CH2COO), 1.80-1.60 (6H, m, 3 × CH2), 1.60-1.21 (6H, m, 3 ×
CH2), 0.89 (3H, t, J ) 6.7 Hz, CH3). 13C NMR: δ 180.1 (COO),
157.3 (Ph), 133.9 (Ph), 129.2 (Ph), 114.4 (Ph), 68.0 (CH2O), 34.6
(CH2), 33.9 (CH2), 31.6 (CH2), 31.0 (CH2), 29.3 (CH2), 25.7 (CH2),
24.2 (CH2), 22.6 (CH2), 14.0 (CH3). Anal. (C17H26O3) C, H.
5-(4-Decyloxy-phenyl)-pentanoic acid (18b). Yield 94%; white
solid; mp 101-102 °C. 1H NMR (CDCl3): δ 7.08 (2H, d, J ) 8.4
Hz, Ph), 6.82 (2H, d, J ) 8.4 Hz, Ph), 3.93 (2H, t, J ) 6.2 Hz,
CH2O), 2.57 (2H, t, J ) 6.8 Hz, PhCH2), 2.37 (2H, t, J ) 7 Hz,
CH2COOH), 1.80-1.60 (6H, m, 3 × CH2), 1.51-1.22 (14H, m, 7
× CH2), 0.89 (3H, t, J ) 6.6 Hz, CH3). 13C NMR: δ 179.5 (COO),
157.2 (Ph), 133.8 (Ph), 129.1 (Ph), 114.3 (Ph), 67.9 (CH2O), 34.5
(CH2), 33.8 (CH2), 31.8 (CH2), 30.9 (CH2), 29.5 (CH2), 29.4 (CH2),
29.3 (CH2), 29.2 (CH2), 26.0 (CH2), 24.2 (CH2), 22.6 (CH2), 14.0
(CH3). Anal. (C21H34O3) C, H.
Methyl 2-Fluoro-hexadecanoate (25). Compound 24 (1 mmol)
was added to a solution of DAST (0.14 mL, 1 mmol) in dry
dichloromethane (0.2 mL) at -78 °C. After stirring for 2 h at -78
°C and another 3 h at room temperature, the reaction mixture
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. Yield 64%; yellowish
oil. 1H NMR (CDCl3): δ 4.86 (1H, dt, JH-F ) 49.2 Hz, JH-H ) 6.6
Hz, CH), 3.74 (3H, s, OCH3), 2.00-1.72 (2H, m, CH2), 1.45-1.10
(24H, br, 12 × CH2), 0.83 (3H, t, J ) 6.2 Hz, CH3). 13C NMR: δ
170.4 (d, JC-C-F ) 24 Hz, COO), 88.9 (d, JC-F ) 183 Hz, CF),
52.0 (OCH3), 32.3 (d, JC-C-F ) 21, CH2), 31.9 (CH2), 29.6 (CH2),
29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 29.0 (CH2), 24.6
(CH2), 24.4 (CH2), 24.3 (CH2), 22.7 (CH2), 14.0 (CH3). 19F NMR:
δ -120.8 (m, CF). Anal. (C17H33FO2) C, H.
1,1,1,3-Tetrafluoro-heptadecan-2-one (in Equilibrium with
1,1,1,3-Tetrafluoro-heptadecane-2,2-diol) (26). A solution of
compound 25 (173 mg, 0.6 mmol) and trifluoromethyltrimethyl-
silane (170 µL, 1.15 mmol) in ethylene glycol dimethyl ether (0.55
mL) at 0 °C was treated with cesium fluoride (3 mg). After stirring
for 30 min at 0 °C and another 18 h at 25 °C the reaction mixture
was treated with concentrated HCl (1 mL). After stirring 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.
Yield 58%; white solid; mp 34-35 °C. 1H NMR (CDCl3): δ 5.23
(1/3H, dt, JH-F ) 48.2 Hz, JH-H ) 6.2 Hz, CH), 4.65 (2/3H, dt,
JH-F ) 49.4 Hz, JH-H ) 6.6 Hz, CH), 3.74 (2/3H, s, OH), 3.49
(2/3H, s, OH), 2.08-1.27 (26H, m, 13 × CH2), 0.89 (3H, t, J ) 7
Hz, CH3). 13C NMR: δ 122.6 (q, JC-F3 ) 286 Hz, CF3), 115.4 (q,
JC-F3 ) 290 Hz, CF3), 92.9 [C(OH)2], 92.4 (d, JC-F ) 186 Hz,
CF), 32.1 (CH2), 31.6 (d, JC-C-F ) 20 Hz, CH2), 29.9 (CH2), 29.8
(CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.1 (CH2),
28.5 (CH2), 28.1 (CH2), 22.7 (CH2), 22.3 (CH2), 14.3 (CH3). 19F
NMR: δ 1.6 (CF3), -5.3 (CF3), -121.7 (CF). MS (ESI) m/z (%):
343 (M-, 100).
Ethyl 2,2-Difluoro-5-phenyl-pentanoate (29). To a stirring
mixture of magnesium (350 mg, 14.6 mmol) and iodine in dry THF
(10 mL), (3-bromo-propyl)-benzene (2.87 g, 14.4 mmol) was added
dropwise under N2 atmosphere. Once the Grignard reagent was
formed, the resulting mixture was added dropwise to a cooled (-78
°C) solution of diethyl oxalate (1.6 mL, 11.8 mmol) in dry ether
(17.3 mL). The reaction mixture was stirred at -78 °C for 45 min
and then was quenched with 1N HCl. The aqueous layer was
extracted with ether (3 × 25 mL) and the combined organic layers
The solution of the saturated ester in methanol (1.4 mL) was
treated with sodium hydroxide 1N (1 mL, 1 mmol). The mixture
was stirred at room temperature for 12 h, acidified with 1N HCl
and extracted with EtOAc (3 × 10 mL). The solvent was removed
in vacum to afford the saturated acid as a white solid.
3-(4-Hexyloxy-phenyl)-propanoic acid (17a). Yield 90%; white
1
solid; mp 70-72 °C. H NMR (CDCl3): δ 7.14 (2H, d, J ) 8.2
Hz, Ph), 6.86 (2H, d, J ) 8.2 Hz, Ph), 3.96 (2H, t, J ) 6.6 Hz,
CH2O), 2.93 (2H, t, J ) 7.6 Hz, CH2), 2.67 (2H, t, J ) 7.6 Hz,
CH2), 1.76-1.60 (2H, m, CH2), 1.41-1.30 (6H, m, 3 × CH2), 0.92
(3H, t, J ) 6.7 Hz, CH3). 13C NMR: δ 179.0 (COO), 157.6 (Ph),
132.0 (Ph), 129.1 (Ph), 114.5(Ph), 67.9 (CH2O), 35.9 (CH2), 31.5
(CH2), 29.7 (CH2), 29.2 (CH2), 25.7 (CH2), 22.5 (CH2), 14.0 (CH3).
Anal. (C15H22O3) C, H.
3-(4-Decyloxy-phenyl)-propanoic acid (17b). Yield 96%; white
1
solid; mp 74-76 °C. H NMR (CDCl3): δ 7.14 (2H, d, J ) 8.2
Hz, Ph), 6.86 (2H, d, J ) 8.2 Hz, Ph), 3.95 (2H, t, J ) 6.5 Hz,
CH2O), 2.93 (2H, t, J ) 7.7 Hz, CH2CH2COO), 2.67 (2H, t, J )
7.7 Hz, CH2COO), 1.85-1.68 (2H, m, CH2CH2O), 1.50-1.21
(14H, br s, 7 × CH2), 0.92 (3H, t, J ) 6.2 Hz, CH3). 13C NMR: δ
179.3 (COO), 157.6 (Ph), 132.0 (Ph), 129.1 (Ph), 114.5 (Ph), 67.9

Polyfluoro Ketone Inhibitors of Phospholipase A2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8035
were washed with brine, dried (Na2SO4) and the solvent was
evaporated in vacuo. After flash column chromatography, a mixture
of methyl 2-oxo-5-phenyl-pentanoate (28) with diethyl oxalate was
obtained and treated with DAST (1 equib) at room temperature.
After stirring for 4 h at 45 °C, the reaction mixture was quenched
with ice-water. The reaction mixture was diluted with dichlolom-
ethane, and 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) 1/9.
(CDCl3): δ 7.31-7.15 (5H, m, Ph), 3.85 (3H, s, OCH3), 2.86 (2H,
t, J ) 6.6 Hz, CH2), 2.63 (t, J ) 6.6 Hz, 2H, CH2), 1.71-1.62
(2H, m, CH2). 13C NMR: 194.0 (CO), 161.2 (COO), 141.8 (Ph),
128.3 (Ph), 128.0 (Ph), 125.8 (Ph), 52.8 (OCH3), 35.5 (CH2), 30.5
(CH2), 22.5 (CH2). Anal. (C12H14O3) C, H.
Methyl 2,2-Difluoro-5-phenyl-pentanoate (35). A solution of
compound 34 (0.404 g, 1.67 mmol) in CH2Cl2 (3.3 mL) was treated
dropwise with DAST (0.489 mL, 3.6 mmol) at room temperature.
After heating at 55 °C for 5 h, it was poured into H2O, cautiously
neutralized by the addition of solid K2CO3, and extracted with
CHCl3 (2 × 15 mL). The organic solvent was dried over Na2SO4,
filtered, and evaporated and the crude product purified by flash
column chromatography on silica gel eluting with EtOAc-petroleum
ether (bp 40-60 °C) 1/9 to give 0.202 g (50%) of the title
compound as an yellowish oil. 1H NMR (CDCl3): δ 7.32-7.12
(5H, m, Ph), 4.10 (3H, s, OCH3), 2.69 (2H, t, J ) 7.4 Hz, PhCH2),
2.21-1.90 (2H, m, CH2CF2), 1.80-1.72 (2H, m, CH2). 13C NMR:
δ 164.2 (t, JC-C-F ) 33 Hz, COO), 140.9 (Ph), 128.4 (Ph), 128.3
(Ph), 126.1 (Ph), 116.2 (t, JC-F ) 248 Hz, CF2), 52.7 (OCH3), 34.9
(CH2), 33.8 (t, JC-C-F ) 23 Hz, CH2CF2), 23.0 (t, JC-C-C-F ) 2.4
Hz, CH2CH2CF2). 19F NMR: δ -28.0 (2F, t, J ) 17 Hz, CF2). MS
(ESI) m/z (%): 229 (M+ + 1, 100). Anal. (C12H14F2O2) C, H.
In Vitro PLA2 Assays. Phospholipase A2 activity was deter-
mined using the previously described modified Dole assay20 with
buffer and substrate conditions optimized for each enzyme as
described previously:20,21,24,25 (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) GVIA iPLA2 substrate mixed-micelles were composed
of 400 µM Triton X-100, 99 µM DPPC, and 1.5 µM 14C-labeled
DPPC in buffer containing 200 mM HEPES pH 7.0, 1 mM ATP,
2 mM DTT, and 0.1 mg/ml BSA, and (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.
1
Yield 69%; yellowish oil. H NMR (CDCl3): δ 7.38-7.12 (5H,
m, Ph), 4.30 (2H, q, J ) 6.8 Hz, OCH2), 2.68 (2H, t, J ) 7.4 Hz,
PhCH2), 2.21-1.93 (2H, m, CH2CF2), 1.90-1.75 (2H, m, CH2),
1.34 (3H, t, J )6.8 Hz, CH3). 13C NMR: δ 164.2 (t, JC-C-F ) 24
Hz, COO), 140.9 (Ph), 128.4 (Ph), 128.3 (Ph), 126.1 (Ph), 116.2
(t, JC-F ) 248 Hz, CF2), 62.7 (OCH2), 34.9 (CH2), 33.8 (t, JC-C-F
) 23 Hz, CH2CF2), 23.0 (t, JC-C-C-F ) 4 Hz, CH2CH2CF2), 13.8
(CH3). 19F NMR: δ 28.0 (t, J ) 17 Hz, CF2). Anal. (C13H16F2O2)
C, H.
1,1,1,3,3-Pentafluoro-6-phenyl-hexane-2,2-diol (30). It was
prepared following the method used for the synthesis of compound
26. Yield 35%; yellowish oil. 1H NMR (CDCl3): δ 7.41-7.18 (5H,
m, Ph), 3.93 (2H, br, 2 × OH), 2.69 (2H, t, J ) 7.6 Hz, PhCH2),
2.22-1.88 (4H, m, 2 × CH2). 13C NMR: δ 141.3 (Ph), 128.4 (Ph),
126.3 (Ph), 126.1 (Ph), 121.5 (q, J ) 286 Hz, CF3), 120.7 (t, J )
249 Hz, CF2), 92.3 [C(OH)2], 35.2 (CH2), 30.8 (t, JC-C-F2 ) 23
Hz, CH2CF2), 22.5 (t, JC-C-C-F2 ) 2.4 Hz, CH2CH2CF2). 19F NMR:
δ -3.2 (CF3), -36.4 (CF2). MS (ESI) m/z (%): 283 (M-, 65), 213
(100). Anal. (C12H13F5O2) C, H.
2-Hydroxy-5-phenyl-pentanenitrile (32).62 A solution of 4-phe-
nylbutanal 31 (0.56 g, 3.78 mmol) and NaHSO3 (0.59 g in 1 mL
H2O) in dichloromethane was stirred for 30 min at room temper-
ature. After the formation of the white salt, the organic solvent
was evaporated and water (3.8 mL) was added. The mixture cooled
to 0 °C and an aqueous solution of KCN (0.368 g, 567 mmol in 1
mL H2O) was added dropwise. The reaction mixture was stirred
for another 18 h at room temperature and then CH2Cl2 (10 mL)
and water (10 mL) were added. The organic phase was 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) 2/8 to give 0.653 g (99%) of the title compound as
a clear oil. 1H NMR (CDCl3): δ 7.32-7.15 (5H, m, Ph), 4.40 (1H,
t, J ) 8.8 Hz, CH), 2.63 (2H, t, J ) 6.6 Hz, CH2), 1.90-1.70 (4H,
m, 2 × CH2). 13C NMR: 141.5 (Ph), 128.7 (Ph), 128.6 (Ph), 126.3
(Ph), 120.4 (CN), 61.2 (CH), 35.3 (CH2), 34.7 (CH2), 26.4 (CH2).
Anal. (C11H13NO) C, H.
In Vitro PLA2 Inhibition Studies. Initial screening of com-
pounds at 0.091 mol fraction inhibitor in mixed-micelles was carried
out. We considered compounds displaying 25% or less inhibition
to have no inhibitory affect (designated N.D.). We report average
percent inhibition (and standard error, n ) 3) for compounds
displaying more than 25% and less than 90% enzyme inhibition.
If percent inhibition was greater than 90%, we determined its XI(50)
by plotting percent inhibition vs inhibitor molar fraction (7 points;
typically 0.005-0.091 mol fraction). Inhibition curves were mod-
eled in Graphpad Prism using either a linear (x, y intercept ) 0) or
nonlinear regression (one-site binding model, hyperbola, BMAX
) 100) to calculate the reported XI(50) and associated error values.
Methyl 2-Hydroxy-5-phenyl-pentanoate (33).63 Compound 32
(0.63 g, 3.59 mmol) was treated with HCl (0.6 mL ·6N) in MeOH
for 18 h at room temperature. The organic solvent was evaporated
and an aqueous solution of K2CO3 was added to neutralize the pH
of the mixture. After extraction with EtOAc (3 × 15 mL), the
combined organic phases were 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
to give 0.56 g (79%) of the title compound as a clear oil. 1H NMR
(CDCl3): δ 7.30-7.12 (5H, m, Ph), 4.22 (1H, t, J ) 4.0 Hz, CH),
3.74 (3H, s, OCH3), 3.18 (1H, s, OH), 2.66 (2H, t, J ) 6.6 Hz,
CH2), 1.85-1.62 (4H, m, 2 × CH2). 13C NMR: 175.4 (COO), 141.6
(Ph), 128.1 (Ph), 128.0 (Ph), 125.5 (Ph), 70.1 (CHOH), 52.1
(OCH3), 35.2 (CH2), 33.6 (CH2), 26.3 (CH2). Anal. (C12H16O3) C,
H.
Methyl 2-oxo-5-Phenyl-pentanoate (34). Compound 33 (0.20
g, 0.96 mmol) was dissolved in CH2Cl2 (20 mL) and treated with
Dess-Martin periodinane (0. 43 g) under stirring for 40 min. The
organic phase was 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 to give
0.195 g (99%) of the title compound as an yellowish oil. 1H NMR
Acknowledgment. This work was supported by NIH GM
20,501 and GM64611 (E.A.D.). The project is cofunded by the
European Social Fund and National Resources (EPEAEK II)
(G.K.).
Supporting Information Available: Elemental analysis results
for the compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
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Polyfluoro Ketone Inhibitors of Phospholipase A2
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8037
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