New potent and selective polyfluoroalkyl ketone inhibitors of GVIA calcium-independent phospholipase A2

Article abstract of DOI:10.1016/j.bmc.2013.07.010

Group VIA calcium-independent phospholipase A₂ (GVIA iPLA ₂) has recently emerged as an important pharmaceutical target. Selective and potent GVIA iPLA₂ inhibitors can be used to study its role in various neurological diso

Full text of DOI:10.1016/j.bmc.2013.07.010

Bioorganic & Medicinal Chemistry 21 (2013) 5823–5829
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New potent and selective polyfluoroalkyl ketone inhibitors of GVIA
calcium-independent phospholipase A2
Victoria Magrioti a, Aikaterini Nikolaou a, Annetta Smyrniotou b, Ishita Shah c,d
,
Violetta Constantinou-Kokotou b, Edward A. Dennis c,d, , George Kokotos a,
⇑
⇑
a Laboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, Athens 15771, Greece
b Chemical Laboratories, Agricultural University of Athens, Athens 11855, Greece
c Department of Chemistry and Biochemistry, School of Medicine, MC 0601, University of California, San Diego, La Jolla, CA 92093-0601, USA
d Department of Pharmacology, School of Medicine, MC 0601, University of California, San Diego, La Jolla, CA 92093-0601, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as an important
pharmaceutical target. Selective and potent GVIA iPLA2 inhibitors can be used to study its role in various
neurological disorders. In the current work, we explore the significance of the introduction of a substitu-
ent in previously reported potent GVIA iPLA2 inhibitors. 1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)hep-
tan-3-one (GK187) is the most potent and selective GVIA iPLA2 inhibitor ever reported with a XI(50) value
of 0.0001, and with no significant inhibition against GIVA cPLA2 or GV sPLA2. We also compare the inhi-
bition of two difluoromethyl ketones on GVIA iPLA2, GIVA cPLA2, and GV sPLA2.
Received 17 May 2013
Accepted 6 July 2013
Available online 16 July 2013
Keywords:
Phospholipase A2
Inhibitor
Ó 2013 Elsevier Ltd. All rights reserved.
GVIA iPLA2
Polyfluoroalkyl ketone
Pentafluoroethyl ketones
1. Introduction
GVIA iPLA2 is a phospholipase A2 that can be characterized by
its calcium-independent activity. It was purified and characterized
Phospholipases A2 are the enzymes that hydrolyse the ester
bond of phospholipids at the sn-2 position releasing free fatty acids
and lysophospholipids.1 Both of the products of this hydrolysis
may generate second messengers that play significant pharmaco-
logical roles, and especially when the released free fatty acid is ara-
chidonic acid. The PLA2 superfamily currently consists of 16
different groups and various subgroups.2 Three of the most impor-
tant types of PLA2s that can be found in human tissues are the se-
creted (such as GIIA and GV sPLA2), the cytosolic GIVA cPLA2 and
the calcium-independent GVIA iPLA2.
GIVA cPLA2 is considered to be a proinflammatory enzyme that
is the rate-limiting provider of arachidonic acid and lysophospho-
lipids.3 GIVA cPLA2 is regulated by intracellular calcium, and cal-
cium binding to the C2 domain of GIVA PLA2 can activate the
enzyme, resulting in the localization of the enzyme to the
phospholipid membrane.4,5 Furthermore, the activity of sPLA2s
has been suggested to be dependent on or linked to the activity
of cPLA2.6–8
from macrophages in 19949 and it functions through a catalytic
serine at the active site in a patatin-like a/b-hydrolase domain. It
is a 752-amino acid protein with a molecular mass of 85 kDa that
contains eight ankyrin repeats and a catalytic domain.10–12 Both
intracellular enzymes GIVA cPLA2 and GVIA iPLA2 share the same
catalytic mechanism utilizing a serine residue as the nucleophile,
while the active site serine of GVIA iPLA2 lies within a lipase con-
sensus sequence (Gly-X-Ser519-X-Gly) on top of the catalytic
domain.2 GVIA iPLA2 is known to be a homeostatic enzyme in-
volved in basal metabolism within the cell.13–19 Several studies
also suggest that GVIA iPLA2 plays significant 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 path-
ological conditions (e.g., diabetes, Barth syndrome, ischemia and
cancer).20–27
Various GVIA iPLA2 inhibitor classes have been discussed in re-
cent review articles.2,28–30 The first reported GVIA iPLA2 inhibitors
were the trifluoromethyl ketones,31 tricarbonyls32 and methyl flu-
orophosphonates33 of fatty acids, such as arachidonic acid. They
were not very potent, nor selective inhibitors, while the methyl flu-
orophosphonates were also irreversible. Most recently, cardiolipin
was found to inhibit iPLA2 and cPLA2 activity towards PC in vitro.34
Bromoenol lactone (BEL, Fig. 1) was considered to be a selective,
irreversible GVIA iPLA2 inhibitor and was used to study potential
⇑
Corresponding authors. Tel.: +1 858 534 3055; fax: +1 858 534 7390 (E.A.D.);
tel.: +30 210 727 4462; fax: +30 210 727 4761 (G.K.).
E-mail addresses: edennis@ucsd.edu (E.A. Dennis), gkokotos@chem.uoa.gr
(G. Kokotos).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.010

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V. Magrioti et al. / Bioorg. Med. Chem. 21 (2013) 5823–5829
Br
O
Furthermore, compounds 12 and 13 were synthesized as the
structurally restricted analogues of the inhibitor FKGK11 to deter-
mine the effect that the second phenyl group would have on the
activity of the inhibitor.
Finally, two difluoromethyl ketones were prepared that resem-
bled the structure of inhibitor FKGK11 in order to identify the ef-
fect that different number of fluorine atoms would have on the
activity of polyfluoroalkyl ketones.
C2F5
O
O
FKGK11
BEL
O
CF3
The inhibition studies showed high activity and selectivity for
compounds 5d and 6d that had a methoxy group at para position.
Thus, we prepared another series of substituted polyfluoroalkyl ke-
tones bearing one or two methoxy groups in different positions of
the phenyl or in the naphthalene group to see the effect on inhibi-
tion and selectivity.
FKGK18
Figure 1. Some known iPLA2 inhibitors.
biological functions of GVIA iPLA2.13,31 The inactivation mechanism
of GVIA iPLA2 by BEL has been studied by Turk and co-workers.35 It
is likely that this inhibitor affects multiple enzymes and should be
used with appropriate caution when studying potential roles of
GVIA iPLA2.
2.2. Synthesis of inhibitors
For the synthesis of trifluomethyl and pentafluoroethyl ketones
5a–j and 6a–j, a Wadsworth–Horner–Emmons olefination reaction
of the corresponding commercially available substituted aromatic
aldehydes 1a–j with triethyl phosphonocrotonate yielded the usat-
urated esters 2a–j (Scheme 1). Hydrogenation with 10% Pd/C gave
esters 3a–j, followed by saponification to afford acids 4a–j.
After treating compounds 4a–j with oxalyl chloride, the corre-
sponding chlorides were treated with trifluoroacetic or pentaflu-
oropropionic anhydride and pyridine to yield trifluoromethyl
ketones 5a–j and pentafluoropropyl ketones 6a–j. In the case of
heptafluorobutyl ketone 7a, the corresponding chloride was trea-
ted with heptafluorobutyric anhydride and pyridine.
The development of selective inhibitors for the three main hu-
man PLA2 enzymes is of paramount importance. Our groups have
previously synthesized and assayed a series of polyfluoroalkyl ke-
tones for their activity on GIVA cPLA2, GVIA iPLA2, and GVs-
PLA2.36,37 When compared to a trifluoromethyl ketone, it was
found that the corresponding pentafluoroethyl ketone favored
selective GVIA iPLA2 inhibition. FKGK11 (Fig. 1) was found to be
a selective GVIA iPLA2 inhibitor, while FKGK18 (Fig. 1) was identi-
fied as the most potent GVIA PLA2 inhibitor yet reported.37 Selec-
tive PLA2 inhibitors may contribute to the clarification of the role
of each PLA2 class in various disorders. Using the selective GVIA
iPLA2 inhibitor FKGK11, a selective GIVA cPLA2 inhibitor, and a
pan-PLA2 inhibitor, the role of the various classes of PLA2 in an ani-
mal model of multiple sclerosis, EAE, was studied.38 According to
the results of that study, GIVA cPLA2 plays a role in the onset of
the disease, while GVIA iPLA2 plays a key role in both the onset
and the progression of the disease. Therefore, it appears that GVIA
iPLA2 is a target enzyme for the development of novel therapies for
multiple sclerosis.38 Furthermore, in a very recent article, the inhi-
bition mechanism of GVIA iPLA2 by a fluoroketone ligand was
examined using a combination of deuterium exchange mass spec-
trometry (DXMS) and molecular dynamics (MD), while models for
iPLA2 were built by homology with the known structure of pata-
tin.39 The discovery of the precise binding mode of fluoroketone li-
gands to iPLA2 should greatly improve our ability to design new
inhibitors with higher potency and selectivity.
O
O
O
a
b
Ar
O
Ar
Ar
O
3a-j
1a-j
2a-j
O
O
Ar
CF3
O
d, e
c
5a-j
Ar
OH
d, f
4a-j
Ar
CF2CF3
d, g
O
6a-j
CF2CF2CF3
Based on previous results, we have explored further this family
of GVIA iPLA2 inhibitors and herein we describe our most recent
results.
7a
Ar
Ar
1-6
a
1-6
f
2. Results and discussion
2.1. Design of inhibitors
O
g
h
i
b
c
d
O
O
The design of the novel polyfluoroalkyl ketones was based on
the optimization of the activity and selectivity of iPLA2 inhibitors
that we have presented in previous work, such as FKGK11 and
FKGK18 (Fig. 1).36,37 Having established that the best linker be-
tween a polyfluoroalkyl ketone and an aromatic ring is a chain of
four methylene groups, we introduced in the aromatic ring differ-
ent substituents and studied the effect of these substituents on the
affinity towards GVIA iPLA2, as well as the selectivity towards GVIA
iPLA2 when compared to GIVA cPLA2 and GV sPLA2 activity. Several
substituents were introduced in para position, such as a fluorine
atom, a methoxy group, a phenyl group, and a trifluoromethyl
group. Also, the isomer of the most potent iPLA2 inhibitor FKGK18
was prepared, where the naphthyl group was attached to the linker
at the 1-position.
F
O
F3C
O
O
O
O
e
j
O
Scheme 1. Reagents and conditions: (a) C2H5OOCCH@CHCH2P(@O)(OC2H5)2, LiOH,
THF, reflux; (b) H2, 10% Pd/C, EtOH; (c) NaOH 1 N, EtOH; (d) (COCl)2, DMF, CH2Cl2;
(e) pyridine, (CF3CO)2O, CH2Cl2, 0 °C to rt; (f) pyridine, (CF3CF2CO)2O, CH2Cl2, 0 °C to
rt; (g) pyridine, (CF3CF2CF2CO)2O, CH2Cl2, 0 °C to rt.

V. Magrioti et al. / Bioorg. Med. Chem. 21 (2013) 5823–5829
5825
b
For the synthesis of trifluomethyl and pentafluoroethyl ketones
a
CHF2
MgBr
, n=1
( )n
Br
( )n
( )n
12 and 13, a Wittig olefination reaction between aldehyde 8 and
methyl (triphenylphosphanylidene)acetate yielded usaturated es-
ter 9 (Scheme 2). Catalytic hydrogenation, followed by saponifica-
tion gave compound 11. Ketones 12 and 13 were prepared
similarly as described above.
The difluoromethyl ketones were prepared from bromides 14a
and 14b, after being treated with magnesium, and the correspond-
ing Grignard reagents were slowly added to ethyl difluoroacetate
at À78 °C to yield ketones 16a and 16b (Scheme 3).
O
16a
14a
15a
15b, n=2
, n=1
, n=1
14b, n=2
16b, n=2
Scheme 3. Reagents and conditions: (a) Mg, dry Et2O; (b) CHF2COOEt, dry Et2O,
À78 °C.
both present XI(50) values of 0.0001 and they are much more selec-
tive to GVIA iPLA2 when compared to GIVA cPLA2 and GV sPLA2.
Taking into consideration these results, we prepared and tested
in vitro a series of other polyfluoroalkyl ketones bearing one or two
methoxy groups in different positions of the phenyl group, in order
to find even more potent and selective GVIA iPLA2 inhibitors. How-
ever, compounds 5g–j and 6g–j lost the potency that inhibitors 5d
and 6d presented. The most potent of this group were compounds
5j and 6j, bearing a dioxolane ring on the phenyl group.
The structurally restricted analogues of inhibitor FKGK11 12
and 13 did not give optimum potency; instead they were weak,
yet selective, iPLA2 inhibitors.
2.3. In vitro inhibition of GIIA sPLA2, GIVA cPLA2 and GVIA iPLA2
All synthesized inhibitors were tested for inhibition of human
GIVA cPLA2, GVIA iPLA2 and GV sPLA2 using previously described
mixed micelle-based assays.40–42 The inhibition results are pre-
sented in Table 1, either as percent inhibition or as XI(50) values.
At first, the percent of inhibition for each PLA2 enzyme at 0.091
mole fraction of each inhibitor was determined; and, then the
XI(50) values were measured for compounds that displayed greater
than 95% inhibition. The XI(50) is the mole fraction of the inhibitor
in the total substrate interface required to inhibit the enzyme by
50%.
The isomer of the most potent iPLA2 inhibitor FKGK18, com-
pound 5a seems to be a 9-times weaker inhibitor towards GVIA
iPLA2, while there is no significant selectivity towards GIVA cPLA2.
Interestingly enough, the pentafluoro and heptafluoro ketone ana-
logues 6a and 7a are even weaker iPLA2 inhibitors. The methoxy
group in position 6 of the naphthalene group also seems to lower
the inhibitory potency of FKGK18 in compounds 5f and 6f.
Compounds 5b–e and 6b–e were prepared as substituted ana-
logues of FKGK11. Most of these compounds presented excellent
iPLA2 inhibition, with the exception of compounds 5e and 6e,
which were 13-fold and ninefold weaker towards iPLA2 than
FKGK11. It was interesting though that trifluoromethyl ketone
5e, 6-(biphenyl-4-yl)-1,1,1-trifluorohexan-2-one (GK174), seemed
to be a more potent inhibitor towards GIVA cPLA2 than for GVIA
iPLA2. Compounds 5b (GK176) and 5c (GK178) proved to be as po-
tent as FKGK18, and compound 5c showed even better selectivity
when compared to GIVA cPLA2 and GV sPLA2. The most potent
inhibitors proved to be compounds 5d (1,1,1-trifluoro-6-(4-
methoxyphenyl)hexan-2-one, GK177) and 6d (1,1,1,2,2-pentaflu-
oro-7-(4-methoxyphenyl)heptan-3-one, GK187) bearing a meth-
oxy group at the para position of the phenyl substituent. They
Finally, the two difluoromethyl ketones 16a and 16b that are
analogues of FKGK11 presented good selectivity, but low activity,
towards GVIA iPLA2 when compared with GIVA cPLA2 and GV
sPLA2.
3. Conclusion
In the present study, we identified six fluoroketones (5b, 5c, 5d,
6b, 6c, and 6d) that are very potent inhibitors of GVIA iPLA2. All of
them are more potent than the previous lead inhibitor FKGK11,
which has been successfully used in animal models of neurological
disorders,38 but compounds 5d and 6d are also more potent than
the most potent iPLA2 inhibitor FKGK18 in the literature. Espe-
cially, compound 6d (GK187) is the most potent, but also the most
selective iPLA2 inhibitor presented, since it shows less that 25%
inhibition against GIVA cPLA2 and 32.8% against GV sPLA2 at
0.091 mol fraction.
In conclusion, a series of potent GVIA iPLA2 inhibitors was
developed. The introduction of a methoxy group at the para posi-
tion of the phenyl group of the lead compound FKGK11 resulted
in the most potent GVIA iPLA2 inhibitor ever reported
(XI(50) = 0.0001). By the use of these inhibitors in studies in animal
models, the role of GVIA iPLA2 in inflammatory conditions or neu-
rological diseases may be further explored. Since GVIA iPLA2 has
emerged as a novel target for drug discovery, the identification of
potent and selective iPLA2 inhibitors is of paramount importance.
4. Experimental section
4.1. General
O
O
O
a
b
O
O
9
10
8
Melting points were determined on a Buchi 530 apparatus and
are uncorrected. Nuclear magnetic resonance spectra were ob-
tained on a Varian Mercury spectrometer (1H NMR recorded at
200 MHz, 13C NMR recorded at 50 MHz, 19F NMR recorded at
188 MHz) and were recorded in chloroform (CDCl3), using CHCl3
residual peak as the 1H internal reference (7.27 ppm); and the cen-
tral peak of CDCl3 at 77.0 ppm for 13C NMR. All 19F NMR chemical
shifts were referenced to CFCl3 (0.0 ppm). Thin layer chromatogra-
phy (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
O
O
c
d, e
OH
CF3
11
13
12
d, f
O
CF2CF3
Scheme 2. Reagents and conditions: (a) CH3OOCCH@PPh3, dry THF; (b) H2, 10% Pd/
C, MeOH; (c) NaOH 1 N, MeOH; (d) (COCl)2, DMF, CH2Cl2; (e) pyridine, (CF3CO)2O,
CH2Cl2, 0 °C to rt; (f) pyridine, (CF3CF2CO)2O, CH2Cl2, 0 °C to rt.

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V. Magrioti et al. / Bioorg. Med. Chem. 21 (2013) 5823–5829
Table 1
Inhibition of PLA2 by fluoroketonesa
Number
GVIA iPLA2
GIVA cPLA2
GV sPLA2
% Inhibition at 0.091
99.4
XI(50)
% Inhibition at 0.091
% Inhibition at 0.091
FKGK1137
FKGK1837
5a
6a
7a
5b
6b
5c
6c
5d
6d
5e
6e
5f
6f
5g
6g
5h
6h
5i
6i
5j
6j
12
13
0.0014 0.0001
0.0002 0.0000
0.0018 0.0002
0.0034 0.0002
N.D.
80.8
77.4
64.0
60.4
41.4
N.D.
83.9
28.0
63.0
31.9
29.4
57.8
N.D.
29.3
30.4
36.9
N.D.
32.8
40.5
38.3
41.7
63.1
N.D.
38.7
N.D.
N.D.
N.D.
39.1
N.D.
29.4
99.9
98.7
96.4
76.7
98.5
98.2
99.9
98.9
99.8
99.8
97.9
94.6
92.6
92.5
89.6
95.6
62.5
78.3
82.8
95.5
98.7
99.5
75.0
90.4
86.5
87.4
0.0002 0.0000
0.0003 0.0001
0.0002 0.0000
0.0003 0.0001
0.0001 0.0000
0.0001 0.0000
0.0189 0.0045
0.0134 0.0017
62.5
54.3
N.D.
96.5 XI(50): 0.0074 0.0003
72.6
85.5
54.7
64.3
35.6
28.8
N.D.
58.9
30.5
41.2
38.3
N.D.
N.D.
N.D.
N.D.
0.0553 0.0056
0.0263 0.0047
0.0025 0.0002
0.0019 0.0003
16a
16b
N.D.
N.D.
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 95% inhibition. N.D. signifies compounds with less than 25% inhibition (or no detectable inhibition).
reagent grade and used without further purification. All the prod-
ucts gave satisfactory elemental analysis results.
7.17–6.93 (m, 4H, arom), 2.74 (t, 2H, CH2, J = 6.6 Hz), 2.63 (t, 2H,
CH2, J = 7.2 Hz), 1.80–1.56 (m, 4H, CH2CH2); 13C NMR (50 MHz,
CDCl3): d 191.3 (q, CO, J = 45 Hz), 161.3 (d, C-F, J = 242 Hz), 137.2,
129.6 (d, J = 8 Hz), 115.5 (q, CF3, J = 291 Hz), 115.1 (d, J = 21 Hz),
36.1, 34.6, 30.5, 21.8; 19F NMR (188 MHz, CDCl3): d À79.8 (CF3),
À118.0 (F); MS (ESI) m/z (%): 247.2 ([MÀH]À, 85); Anal. Calcd for
4.2. Chemistry
4.2.1. Synthesis of trifluoromethyl ketones
Oxalyl chloride (1.5 mL, 3 mmol) and N,N-dimethylformamide
C12H12F4O: C, 58.07; H, 4.87. Found: C, 58.16; H, 4.85.
(40 lL) were added to a solution of carboxylic acid 4a–j or 11
(1 mmol) in dry dichloromethane (40 mL). After 2 h stirring at
room temperature, the solvent and excess reagent were evapo-
rated under reduced pressure and the residue was dissolved in
dry dichloromethane (10 mL). Pyridine (0.64 mL, 8 mmol) and tri-
fluoroacetic 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 tem-
perature, 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 chromatography
[EtOAc–petroleum ether (bp 40–60 °C) 5/95 to 1/9].
4.2.1.3. 1,1,1-Trifluoro-6-(4-(trifluoromethyl)phenyl)hexan-2-
one (5c).
Yield 16%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
7.53 (d, 2H, arom, J = 8.0 Hz), 7.27 (d, 2H, arom, J = 8.0 Hz), 2.71
(t, 4H, CH
J = 7.0 Hz), 1.78–1.60 (m, 4H, CH2); 13C NMR
,
2
(50 MHz, CDCl3): d 191.4 (t, CO, J = 35 Hz), 145.9, 132.6, 128.2,
125.6, 124.5 (q, CF3, J = 270 Hz), d 115.8 (q, CF3, J = 290 Hz), 36.2,
35.5, 30.3, 22.1; 19F NMR (188 MHz, CDCl3): d À62.8 (CF3), À79.8
(CF3); MS (ESI) m/z (%): 297.1 ([MÀH]À, 100); Anal. Calcd for
C13H12F6O: C, 52.36; H, 4.06. Found: C, 52.48; H, 4.01.
4.2.1.4.
1,1,1-Trifluoro-6-(4-methoxyphenyl)hexan-2-one
(5d)43,44
.
Yield 40%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
7.09 (d, 2H, arom, J = 8.6 Hz), 6.83 (d, 2H, arom, J = 8.6 Hz), 3.79
(s, 3H, OCH3), 2.74 (t, 2H, CH2, J = 6.6 Hz), 2.68 (t, 2H, CH2,
J = 6.8 Hz), 1.80–1.60 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3): d
191.4 (q, CO, J = 34 Hz), 157.8, 133.6, 129.9, 115.5 (q, CF3,
J = 290 Hz), 113.7, 55.2, 36.2, 34.5, 30.6, 21.9; 19F NMR (188 MHz,
CDCl3): d À79.8 (CF3). MS (ESI) m/z (%): 259.2 ([MÀH]À, 100); Anal.
Calcd for C13H15F3O2: C, 60.00; H, 5.81. Found: C, 60.11; H, 5.76.
4.2.1.1.
(5a).
1,1,1-Trifluoro-6-(naphthalen-1-yl)hexan-2-one
Yield 26%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
8.10–7.30 (m, 7H, arom), 3.13 (t, 2H, CH2, J = 5.8 Hz), 2.77 (t, 2H,
CH2, J = 5.8 Hz), 1.86–1.79 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3):
d 191.39 (q, CO, J = 35 Hz), 137.62, 133.87, 131.66, 128.82, 126.78,
125.98, 125.83, 125.49, 123.55, 115.51 (q, CF3, J = 290 Hz), 36.20,
32.67, 29.71, 22.36; 19F NMR (188 MHz, CDCl3): d À79.7 (CF3);
MS (ESI) m/z (%): 279.2 ([MÀH]À, 100); Anal. Calcd for
4.2.1.5.
(5e).
6-(Biphenyl-4-yl)-1,1,1-trifluorohexan-2-one
Yield 39%; Yellowish oil; 1H NMR (200 MHz, CDCl3): d
7.70–7.20 (m, 9H, arom), 2.80–2.60 (m, 4H, CH2), 1.90–1.60 (m,
4H, CH2); 13C NMR (50 MHz, CDCl3): d 191.4 (q, CO, J = 35 Hz),
140.9, 140.7, 138.9, 128.9, 128.8, 128.7, 127.2, 127.1, 127.0,
126.9, 115.5 (q, CF3, J = 290 Hz), 36.2, 35.1, 30.4, 22.0; 19F NMR
(188 MHz, CDCl3): d À79.7 (CF3); MS (ESI) m/z (%): 305.2 ([MÀH]À,
C16H15F3O: C, 68.56; H, 5.39. Found: C, 68.47; H, 5.42.
4.2.1.2.
(5b).
1,1,1-Trifluoro-6-(4-fluorophenyl)hexan-2-one
Yield 38%; Colorless oil; 1H NMR (200 MHz, CDCl3): d

V. Magrioti et al. / Bioorg. Med. Chem. 21 (2013) 5823–5829
5827
100); Anal. Calcd for C18H17F3O: C, 70.58; H, 5.59. Found: C, 70.69;
H, 5.54.
4.2.2. Synthesis of pentafluoroethyl ketones
The synthesis of pentafluoroethyl ketones was carried out fol-
lowing the procedure described above for trifluoromethyl ketones,
except that pentafluoropropionic anhydride was used instead of
trifluoroacetic anhydride. The products were purified by flash col-
umn chromatography [EtOAc–petroleum ether (bp 40–60 °C) 5/95
to 1/9].
4.2.1.6. 1,1,1-Trifluoro-6-(6-methoxynaphthalen-2-yl)hexan-2-
one (5f).
Yield 53%; Yellow solid; mp 51–53 °C; 1H NMR
(200 MHz, CDCl3): d 7.70 (d, 2H, arom, J = 8.2 Hz), 7.55 (s, 1H,
arom), 7.30 (d, 1H, arom, J = 8.0 Hz), 7.16 (d, 1H, arom, J = 8.2 Hz),
7.14 (s, 1H, arom), 3.92 (s, 3H, OCH3), 2.90–2.55 (m, 4H, CH2),
1.90–1.50 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3): d 191.4 (q,
CO, J = 35 Hz), 157.2, 136.7, 133.0, 129.0, 128.8, 127.6, 126.8,
126.2, 118.7, 115.5 (q, CF3, J = 290 Hz), 105.5, 55.2, 36.1, 35.3,
30.3, 21.9; 19F NMR (188 MHz, CDCl3): d À79.7 (CF3); MS (ESI) m/
z (%): 309.3 ([MÀH]À, 100); Anal. Calcd for C17H17F3O2: C, 65.80;
H, 5.52. Found: C, 65.88; H, 5.48.
4.2.2.1. 1,1,1,2,2-Pentafluoro-7-(naphthalen-1-yl)heptan-3-one
(6a).
Yield 65%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
8.06–7.32 (m, 7H, arom), 3.13 (t, 2H, CH2, J = 7.2 Hz), 2.81 (t, 2H,
CH2, J = 7.0 Hz), 1.86–1.80 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3):
d 194.2 (t, CO, J = 27 Hz), 137.6, 133.9, 131.7, 128.8, 126.8, 126.0,
125.8, 125.5, 123.5, 117.8 (qt, CF3, J1 = 285 Hz, J2 = 34 Hz), 109.5
(tq, CF2, J1 = 265 Hz, J2 = 38 Hz), 37.2, 32.7, 29.7, 22.3; 19F NMR
(188 MHz, CDCl3): d À82.3 (CF3), À123.7 (CF2); MS (ESI) m/z (%):
329.2 ([MÀH]À, 100); Anal. Calcd for C17H15F5O: C, 61.82; H,
4.58. Found: C, 61.87; H, 4.55.
4.2.1.7.
(5g).
6-(2,4-Dimethoxyphenyl)-1,1,1-trifluorohexan-2-one
Yield 16%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
7.02 (d, 1H, arom, J = 7.2 Hz), 6.45 (s, 1H, arom), 6.42 (d, 1H, arom,
J = 7.2 Hz), 3.80 (s, 6H, OCH3), 2.75 (t, 2H, CH2, J = 6.6 Hz), 2.58 (t,
2H, CH2, J = 6.8 Hz), 1.80–1.50 (m, 4H, CH2); 13C NMR (50 MHz,
CDCl3): d 191.6 (q, CO, J = 35 Hz), 159.2, 158.2, 129.9, 122.4,
115.5 (q, CF3, J = 291 Hz), 103.7, 98.4, 55.3, 55.2, 36.2, 29.1, 29.0,
21.9; 19F NMR (188 MHz, CDCl3): d À79.7 (CF3); MS (ESI) m/z (%):
289.3 ([MÀH]À, 100); Anal. Calcd for C14H17F3O3: C, 57.93; H,
5.90. Found: C, 57.87; H, 5.92.
4.2.2.2. 1,1,1,2,2-Pentafluoro-7-(4-fluorophenyl)heptan-3-one
(6b).
Yield 53%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
7.20–7.00 (m, 2H, arom), 6.98–6.80 (m, 2H, arom), 2.78 (t, 2H,
CH2, J = 6.8 Hz), 2.62 (t, 2H, CH2, J = 7.0 Hz), 1.82–1.60 (m, 4H,
CH2); 13C NMR (50 MHz, CDCl3): d 194.2 (t, CO, J = 24 Hz), 161.3
(d, C-F, J = 242 Hz), 137.2, 129.6 (d, J = 8 Hz), 115.1 (d, J = 21 Hz),
122.0–100.0 (m, CF2, CF3), 37.1, 34.7, 30.5, 21.8; 19F NMR
(188 MHz, CDCl3): d À82.3 (CF3), À118.0 (F), À123.8 (CF2); MS
(ESI) m/z (%): 297.1 ([MÀH]À, 100); Anal. Calcd for C13H12F6O: C,
52.36; H, 4.06. Found: C, 52.42; H, 4.03.
4.2.1.8.
(5h).
6-(3,4-Dimethoxyphenyl)-1,1,1-trifluorohexan-2-one
Yield 26%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
6.90–6.50 (m, 3H, arom), 3.83 (s, 3H, OCH3), 3.81 (s, 3H, OCH3),
2.70 (t, 2H, CH2, J = 5.8 Hz), 2.55 (t, 2H, CH2, J = 7.0 Hz), 1.85–1.40
(m, 4H, CH2); 13C NMR (50 MHz, CDCl3):
d 191.3 (q, CO,
4.2.2.3. 1,1,1,2,2-Pentafluoro-7-(4-(trifluoromethyl)phenyl)hep-
tan-3-one (6c).
CDCl3): 7.57 (d, 2H, arom, J = 5.2 Hz), 7.31 (d, 2H, arom,
Yield 65%; Yellow oil; 1H NMR (200 MHz,
J = 35 Hz), 148.7, 147.1, 134.1, 120.0, 115.4 (q, CF3, J = 291 Hz),
111.4, 111.0, 55.7, 55.6, 36.1, 35.0, 30.5, 21.8; 19F NMR (188 MHz,
CDCl3): d À79.8 (CF3); MS (ESI) m/z (%): 289.1 ([MÀH]À, 100);
Anal. Calcd for C14H17F3O3: C, 57.93; H, 5.90. Found: C, 57.82; H,
5.94.
d
J = 5.2 Hz), 2.81 (t, 2H, CH2, J = 4.4 Hz), 2.74 (t, 2H, CH2,
J = 4.4 Hz), 1.80–1.66 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3): d
194.1 (t, CO, J = 27 Hz), 145.7, 132.4, 129.3, 128.6, 128.0, 125.3 (q,
C-CF3, J = 4 Hz), 121.6, 117.8 (qt, CF3, J1 = 285 Hz, J2 = 34 Hz),
109.5 (tq, CF2, J1 = 265 Hz, J2 = 38 Hz), 37.1, 35.4, 30.0, 21.8; 19F
NMR (188 MHz, CDCl3): d À62.8 (CF3), À82.4 (CF3), À123.8 (CF2);
MS (ESI) m/z (%): 347.1 ([MÀH]À, 95); Anal. Calcd for C14H12F8O:
C, 48.29; H, 3.47. Found: C, 48.38; H, 3.43.
4.2.1.9.
(5i).
1,1,1-Trifluoro-6-(2-methoxyphenyl)hexan-2-one
Yield 29%; Yellowish oil; 1H NMR (200 MHz, CDCl3): d
7.30–7.05 (m, 2H, arom), 6.95–6.75 (m, 2H, arom), 3.82 (s, 3H,
OCH3), 2.82–2.54 (m, 4H, CH2), 1.83–1.50 (m, 4H, CH2); 13C NMR
(50 MHz, CDCl3): d 191.6 (q, CO, J = 35 Hz), 157.3, 130.0, 129.8,
127.1, 120.3, 115.5 (q, CF3, J = 291 Hz), 110.1, 55.1, 36.1, 29.6,
28.8, 22.0; 19F NMR (188 MHz, CDCl3): d À79.9 (CF3). MS (ESI) m/
z (%): 259.2 ([MÀH]À, 100); Anal. Calcd for C13H15F3O2: C, 60.00;
H, 5.81. Found: C, 59.87; H, 5.87.
4.2.2.4.
one (6d).
1,1,1,2,2-Pentafluoro-7-(4-methoxyphenyl)heptan-3-
Yield 31%; Colorless oil; 1H NMR (200 MHz, CDCl3):
d 7.12 (d, 2H, arom, J = 5.6 Hz), 6.87 (d, 2H, arom, J = 5.8 Hz), 3.83 (s,
3H, OCH3), 2.79 (t, 2H, CH2, J = 6.6 Hz), 2.62 (t, 2H, CH2, J = 6.8 Hz),
1.79–1.61 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3): d 194.3 (t, CO,
J = 27 Hz), 157.8, 133.7, 129.2, 113.8, 122.0–100.0 (m, CF2, CF3),
55.2, 37.2, 34.6, 30.6; 19F NMR (188 MHz, CDCl3): d À82.3 (CF3),
À123.8 (CF2); MS (ESI) m/z (%): 309.2 ([MÀH]À, 72); Anal. Calcd
for C14H15F5O2: C, 54.20; H, 4.87. Found: C, 54.32; H, 4.84.
4.2.1.10.
one (5j).
6-(Benzo[d][1,3]dioxol-5-yl)-1,1,1-trifluorohexan-2-
Yield 70%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
6.82–6.42 (m, 3H, arom), 5.92 (s, 2H, OCH2O), 2.73 (t, 2H, CH2,
J = 6.6 Hz), 2.57 (t, 2H, CH2, J = 6.6 Hz), 1.90–1.50 (m, 4H, CH2);
13C NMR (50 MHz, CDCl3): d 191.4 (q, CO, J = 35 Hz), 147.6, 145.7,
135.4, 121.0, 115.5 (q, CF3, J = 291 Hz), 108.7, 108.1, 100.7, 36.1,
35.1, 30.6, 21.8; 19F NMR (188 MHz, CDCl3): d À79.8 (CF3); MS
(ESI) m/z (%): 273.4 ([M+H]+, 100); Anal. Calcd for C14H15F3O2: C,
61.76; H, 5.55. Found: C, 61.87; H, 5.49.
4.2.2.5.
(6e).
7-(Biphenyl-4-yl)-1,1,1,2,2-pentafluoroheptan-3-one
Yield 63%; Yellow low mp solid; mp 32–34 °C; 1H NMR
(200 MHz, CDCl3): d 7.80–7.20 (m, 9H, arom), 2.83 (t, 2H, CH2,
J = 6.8 Hz), 2.73 (t, 2H, CH2, J = 7.0 Hz), 1.95–1.60 (m, 4H, CH2);
13C NMR (50 MHz, CDCl3): d 194.2 (t, CO, J = 27 Hz), 141.0, 140.7,
138.9, 128.8, 128.7, 127.1, 127.0, 126.9, 126.4, 125.7, 117.8 (qt,
CF3, J1 = 285 Hz, J2 = 34 Hz), 106.9 (tq, CF2, J1 = 265 Hz, J2 = 38 Hz),
37.1, 35.1, 30.3, 21.9; 19F NMR (188 MHz, CDCl3): d À82.3 (CF3),
À123.7 (CF2); MS (ESI) m/z (%): 355.2 ([MÀH]À, 100); Anal. Calcd
for C19H17F5O: C, 64.04; H, 4.81. Found: C, 64.16; H, 4.78.
4.2.1.11.
(12).
4-(Biphenyl-2-yl)-1,1,1-trifluorobutan-2-one
Yield 67%; Yellow oil; 1H NMR (200 MHz, CDCl3): d
7.60–7.10 (m, 9H, arom), 3.04 (t, 2H, CH2, J = 7.0 Hz), 2.83 (t, 2H,
CH2, J = 7.0 Hz); 13C NMR (50 MHz, CDCl3):
d
190.5 (q, CO,
J = 35 Hz), 142.0, 141.1, 136.6, 130.4, 128.4, 127.7, 127.2, 126.7,
115.4 (q, CF3, J = 291 Hz), 37.3, 26.0; 19F NMR (188 MHz, CDCl3):
d À79.7 (CF3). MS (ESI) m/z (%): 277.2 ([MÀH]À, 100); Anal. Calcd
for C16H13F3O: C, 69.06; H, 4.71. Found: C, 69.15; H, 4.68.
4.2.2.6.
yl)heptan-3-one (6f).
1H NMR (200 MHz, CDCl3): d 7.69 (d, 2H, arom, J = 8.8 Hz), 7.54
1,1,1,2,2-Pentafluoro-7-(6-methoxynaphthalen-2-
Yield 60%; Yellow solid; mp 42–44 °C;

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V. Magrioti et al. / Bioorg. Med. Chem. 21 (2013) 5823–5829
(s, 1H, arom), 7.28 (d, 1H, arom, J = 9.4 Hz), 7.15 (d, 1H, arom,
J = 8.2 Hz), 7.13 (s, 1H, arom), 3.91 (s, 3H, OCH3), 2.95–2.60 (m,
4H, CH2), 1.90–1.60 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3): d
194.2 (t, CO, J = 27 Hz), 157.2, 136.7, 133.0, 129.0, 128.9, 127.6,
126.8, 126.3, 118.8, 117.8 (qt, CF3, J1 = 285 Hz, J2 = 34 Hz), 106.9
(tq, CF2, J1 = 265 Hz, J2 = 38 Hz), 105.6, 55.2, 37.2, 35.4, 30.3, 21.9;
19F NMR (188 MHz, CDCl3): d À82.3 (CF3), À123.8 (CF2); MS (ESI)
m/z (%): 359.3 ([MÀH]À, 100); Anal. Calcd for C18H17F5O2: C,
60.00; H, 4.76. Found: C, 60.17; H, 4.71.
100); Anal. Calcd for C17H13F5O: C, 62.20; H, 3.99. Found: C,
62.29; H, 3.95.
4.2.3. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalen-1-yl)octan-4-one
(7a)
The synthesis of heptafluoropropyl ketone 7a was carried out
following the procedure described above for trifluoromethyl ke-
tones, except that heptafluorobutanoic anhydride was used instead
of trifluoroacetic anhydride. The product was purified by flash col-
umn chromatography [EtOAc–petroleum ether (bp 40–60 °C) 5/
95]. Yield 54%; Yellow low mp solid; mp 31–32 °C; 1H NMR
(200 MHz, CDCl3): d 8.06–7.33 (m, 7H, arom), 3.14 (t, 2H, CH2,
J = 7.2 Hz), 2.82 (t, 2H, CH2, J = 7.0 Hz), 1.86–1.80 (m, 4H, CH2);
13C NMR (50 MHz, CDCl3): d 193.9 (t, CO, J = 26 Hz), 137.6, 133.9,
130.0–102.5 (m, CF2, CF3), 128.8, 126.8, 126.0, 125.8, 125.5,
123.5, 37.8, 32.7, 29.6, 22.4; 19F NMR (188 MHz, CDCl3): d À81.05
(CF3), À121.56 (CF2), À127.08 (CF2); MS (ESI) m/z (%): 379.1
([MÀH]À, 100); Anal. Calcd for C18H15F7O: C, 56.85; H, 3.98. Found:
C, 56.72; H, 4.03.
4.2.2.7. 7-(2,4-Dimethoxyphenyl)-1,1,1,2,2-pentafluoroheptan-
3-one (6g).
Yield 52%; Yellow oil; 1H NMR (200 MHz, CDCl3):
d 6.99 (d, 1H, arom, J = 7.8 Hz), 6.42 (s, 1H, arom), 6.40 (d, 1H, arom,
J = 7.8 Hz), 3.77 (s, 6H, OCH3), 2.76 (t, 2H, CH2, J = 6.4 Hz), 2.55 (t,
2H, CH2, J = 7.0 Hz), 1.78–1.50 (m, 4H, CH2); 13C NMR (50 MHz,
CDCl3): d 194.4 (t, CO, J = 26 Hz), 159.2, 158.2, 129.9, 122.4, 117.8
(qt, CF3, J1 = 285 Hz, J2 = 34 Hz), 106.9 (tq, CF2, J1 = 266 Hz,
J2 = 38 Hz), 103.7, 98.4, 55.3, 55.1, 37.2, 29.0, 22.6, 21.9; 19F NMR
(188 MHz, CDCl3): d À82.3 (CF3), À123.8 (CF2); MS (ESI) m/z (%):
339.3 ([MÀH]À, 100); Anal. Calcd for C15H17F5O3: C, 52.94; H,
5.04. Found: C, 52.87; H, 5.07.
4.2.4. Synthesis of difluoromethyl ketones
To a stirring mixture of magnesium (24 mg, 1 mmol) and iodine
in dry Et2O (1 mL), a solution of bromide 14a or 14b (1 mmol) in
dry Et2O (9 mL) was added dropwise under N2 atmosphere. Once
the Grignard reagent was formed, it was added dropwise to a
cooled (À78 °C) solution of ethyl difluoroacetate (62 mg, 0.5 mmol)
in dry ether (0.5 mL). The reaction mixture was stirred at À78 °C
for 45 min and then was quenched with 1 N HCl. The aqueous layer
was extracted with ether (3 Â 25 mL) and the combined organic
layers were washed with brine, dried (Na2SO4) and the solvent
was evaporated in vacuo. The product was purified by flash column
chromatography [EtOAc–petroleum ether (bp 40–60 °C) 5/95].
4.2.2.8. 7-(3,4-Dimethoxyphenyl)-1,1,1,2,2-pentafluoroheptan-
3-one (6h).
Yield 48%; Yellowish oil; 1H NMR (200 MHz,
CDCl3): d 6.90–6.60 (m, 3H, arom), 3.88 (s, 3H, OCH3), 3.86 (s, 3H,
OCH3), 2.78 (t, 2H, CH2, J = 5.4 Hz), 2.59 (t, 2H, CH2, J = 6.4 Hz),
1.90–1.42 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3): d 194.2 (t, CO,
J = 26 Hz), 148.8, 147.2, 134.2, 120.1, 117.8 (qt, CF3, J1 = 285 Hz,
J2 = 34 Hz), 111.5, 111.1, 106.8 (tq, CF2, J1 = 265 Hz, J2 = 38 Hz),
55.8, 55.7, 37.1, 35.0, 30.5, 21.8; 19F NMR (188 MHz, CDCl3): d
À82.4 (CF3), À123.8 (CF2); MS (ESI) m/z (%): 339.3 ([MÀH]À,
100); Anal. Calcd for C15H17F5O3: C, 52.94; H, 5.04. Found: C,
52.87; H, 5.09.
4.2.4.1. 1,1-Difluoro-5-phenylpentan-2-one (16a).
Yield
4.2.2.9.
one (6i).
1,1,1,2,2-Pentafluoro-7-(2-methoxyphenyl)heptan-3-
56%; Colorless oil; 1H NMR (200 MHz, CDCl3): d 7.40–7.10 (m,
5H, CH), 5.66 (t, 1H, CHF2, J = 54.0 Hz), 2.77–2.55 (m, 4H, CH2),
1.99 (quintet, 2H, CH2, J = 8.0 Hz); 13C NMR (50 MHz, CDCl3): d
199.6 (t, CO, J = 26.0 Hz), 140.9, 128.5, 128.4, 126.1, 109.8 (d,
CHF2, J = 250 Hz), 35.2, 34.7, 23.8; 19F NMR (188 MHz, CDCl3): d
À127.4 (d, CHF2, J = 54.5 Hz); MS (ESI) m/z (%): 197.1 ([MÀH]À,
100); Anal. Calcd for C11H12F2O: C, 66.66; H, 6.10. Found: C,
66.78; H, 6.06.
Yield 32%; Yellowish oil; 1H NMR (200 MHz, CDCl3):
d 7.35–7.05 (m, 2H, arom), 6.95–6.75 (m, 2H, arom), 3.81 (s, 3H,
OCH3), 2.78 (t, 2H, CH2, J = 6.6 Hz), 2.63 (t, 2H, CH2, J = 6.6 Hz),
1.90–1.50 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3): d 194.4 (t, CO,
J = 26 Hz), 157.3, 130.0, 129.8, 127.2, 120.4, 117.8 (qt, CF3,
J1 = 285 Hz, J2 = 34 Hz), 110.2, 109.5 (tq, CF2, J1 = 265 Hz,
J2 = 38 Hz), 55.1, 37.2, 29.6, 28.8, 21.9; 19F NMR (188 MHz, CDCl3):
d À82.4 (CF3), À123.8 (CF2); MS (ESI) m/z (%): 309.1 ([MÀH]À, 85);
Anal. Calcd for C14H15F5O2: C, 54.20; H, 4.87. Found: C, 54.29; H,
4.84.
4.2.5. 1,1-Difluoro-6-phenylhexan-2-one (16b)45
Yield 45%; Colorless oil; 1H NMR (200 MHz, CDCl3): d 7.37–7.07
(m, 5H, CH), 5.66 (t, 1H, CHF2, J = 54 Hz), 2.78–2.52 (m, 4H, CH2),
1.81–1.58 (m, 4H, CH2); 13C NMR (50 MHz, CDCl3): d 199.7 (t, CO,
J = 26 Hz), 140.2, 128.3, 125.8, 109.8 (d, CHF2, J = 251 Hz), 35.8,
35.5, 30.6, 21.9; 19F NMR (188 MHz, CDCl3): d À127.4 (d, CHF2,
J = 54.5 Hz); MS (ESI) m/z (%): 211.2 ([MÀH]À, 100); Anal. Calcd
for C12H14F2O: C, 67.91; H, 6.65. Found: C, 67.82; H, 6.69.
4.2.2.10. 7-(Benzo[d][1,3]dioxol-5-yl)-1,1,1,2,2-pentafluorohep-
tan-3-one (6j).
Yield 67%; Yellow oil; 1H NMR (200 MHz,
CDCl3): d 6.90–6.50 (m, 3H, arom), 5.92 (s, 2H, OCH2O), 2.78 (t,
2H, CH2, J = 5.8 Hz), 2.57 (t, 2H, CH2, J = 7.0 Hz), 1.85–1.50 (m, 4H,
CH2); 13C NMR (50 MHz, CDCl3): d 194.2 (t, CO, J = 26 Hz), 147.6,
145.7, 135.4, 121.0, 117.8 (qt, CF3, J1 = 285 Hz, J2 = 34 Hz), 108.7,
106.9 (tq, CF2, J1 = 265 Hz, J2 = 38 Hz), 100.8, 37.1, 35.2, 30.6,
21.7; 19F NMR (188 MHz, CDCl3): d À82.4 (CF3), À123.8 (CF2); MS
(ESI) m/z (%): 323.2 ([M+H]+, 100); Anal. Calcd for C15H15F5O2: C,
55.90; H, 4.69. Found: C, 55.98; H, 4.65.
4.3. In vitro PLA2 assays.
The activity of cPLA2, iPLA2 and
sPLA2 were determined using modified Dole Assay.40–42 The buffer
and substrate conditions were optimized for each enzyme assay as
follows: (i) GIVA cPLA2 substrate mixed-micelles were composed
of 400
and 3
2 mM DTT, and 0.1 mg/ml BSA; (ii) GVIA iPLA2 substrate mixed-mi-
celles were composed of 400 M Triton X-100, 98.3 M PAPC, and
1.7
14C-labeled PAPC in buffer containing 100 mM HEPES, pH
7.5, 2 mM ATP, and 4 mM DTT; (iii) GV sPLA2 substrate mixed-mi-
celles were composed of 400 M Triton X-100, 98.3 M PAPC, and
1.7
14C-labeled PAPC in buffer containing 50 mMTris, pH 8.0,
and 5 mM CaCl2.
lM Triton X-100, 97
l
M PAPC, 1.8
l
M
14C-labeled PAPC,
M CaCl2,
4.2.2.11. 5-(Biphenyl-2-yl)-1,1,1,2,2-pentafluoropentan-3-one
lM PIP2 in 100 mM HEPES buffer, pH 7.5, with 90
l
(13).
7.70–7.10 (m, 9H, arom), 3.03 (t, 2H, CH2, J = 7.0 Hz), 2.85 (t, 2H,
CH2, J = 7.0 Hz); 13C NMR (50 MHz, CDCl3):
193.4 (t, CO,
J = 26 Hz), 142.0, 141.1, 136.6, 130.4, 129.1, 128.9, 128.4, 126.7,
117.7 (qt, CF3, J1 = 285 Hz, J2 = 34 Hz), 106.8 (tq, CF2, J1 = 265 Hz,
J2 = 38 Hz), 38.4 (CH2), 26.0 (CH2); 19F NMR (188 MHz, CDCl3): d
À82.4 (CF3), À123.9 (CF2); MS (ESI) m/z (%): 327.2 ([MÀH]À,
Yield 48%; Yellowish oil; 1H NMR (200 MHz, CDCl3): d
l
l
d
lM
l
l
l
M

V. Magrioti et al. / Bioorg. Med. Chem. 21 (2013) 5823–5829
5829
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Initial screening of compounds at 0.091 mole fraction inhibitor
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affect (designated N.D.). We report average percent inhibition for
compounds displaying less than 95% enzyme inhibition. If the per-
cent inhibition was greater than 95%, we determined its XI(50) by
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Acknowledgments
This work was supported by the European Social Fund and Na-
tional Resources Herakleitos II (A.S.) and by NIH Grant GM20501
(E.A.D.).
Supplementary data
Supplementary data (the synthesis and characterization data
of all the intermediates) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmc.2013.
07.010.
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