1-Indol-1-yl-propan-2-ones and related heterocyclic compounds as dual inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase

Article abstract of DOI:10.1016/j.bmc.2009.11.028

Cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH) are enzymes, which have emerged as attractive targets for the development of analgetic and anti-inflammatory drugs. We recently reported that 1-[3-(4-octylphe

Full text of DOI:10.1016/j.bmc.2009.11.028

Bioorganic & Medicinal Chemistry 18 (2010) 945–952
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Bioorganic & Medicinal Chemistry
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1-Indol-1-yl-propan-2-ones and related heterocyclic compounds as dual
inhibitors of cytosolic phospholipase A2a and fatty acid amide hydrolase
Laura Forster, Joachim Ludwig, Martina Kaptur, Stefanie Bovens, Alwine Schulze Elfringhoff,
*
Angela Holtfrerich, Matthias Lehr
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58-62, D-48149 Münster, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Cytosolic phospholipase A2a (cPLA2a) and fatty acid amide hydrolase (FAAH) are enzymes, which have
emerged as attractive targets for the development of analgetic and anti-inflammatory drugs. We recently
reported that 1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid (10) and related compounds
Received 29 September 2009
Revised 10 November 2009
Accepted 12 November 2009
Available online 17 November 2009
are inhibitors of cPLA2
screened this substance series together with some new derivatives for FAAH inhibition. Some of the
assayed compounds proved to be selective cPLA2 inhibitors, while others showed high FAAH and mod-
erate cPLA2 inhibitory potency. Furthermore, several derivatives were favorably active against both
enzymes and, therefore, could represent agents, which have improved analgetic and anti-inflammatory
qualities in comparison with selective cPLA2 and FAAH inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
a. Since cPLA2a and FAAH possess several common structural features, we now
a
Keywords:
a
Cytosolic phospholipase A2
Fatty acid amide hydrolase
Monoacylglycerol lipase
Inhibitors
a
a
1-Indol-1-yl-propan-2-one
1. Introduction
arachidonic acid and ethanolamine.13–15 Inhibition of FAAH is sup-
posed to enhance the action of anandamide. Therefore, like cPLA2
a
In mammalian organism, derivatives of arachidonic acid play
important roles as algesic and pro-inflammatory as well as analgesic
and anti-inflammatory mediators. On the one hand, oxidation prod-
ucts of arachidonic acid such as prostaglandin E2 and leukotriene B4
formed via the arachidonic acid cascade are involved in the
pathophysiology of pain and inflammation.1 On the other hand,
the arachidonic acid amide anandamide generated via the endocan-
nabinoid pathway has analgetic and anti-inflammatory properties.2
The key enzyme in the formation of oxidized derivatives of ara-
inhibitors, inhibitors of FAAH may represent new agents against
pain and inflammation.16,17 In the past years many potent inhibi-
tors of FAAH have been found such as the carbamate 5 (URB
597) and the
FAAH and cPLA2
a
-ketoheterocycle 6 (PHOP) (Fig. 1).18–29
a
have several common features.30–32 Both en-
zymes are serine hydrolases cleaving arachidonoyl residues from
their appropriate substrates. Following formation of the enzyme–
substrate complexes, the active site serines of the enzymes attack
the arachidonoyl-ester and -amide, respectively, resulting in the
formation of oxyanions. These tetrahedral transition states are sta-
bilized by ‘oxyanion holes’ built by the backbone amide groups of
two glycine residues of each enzyme. Upon collapse of the oxya-
nions, serine-arachidonoyl intermediates are generated, which fi-
nally are hydrolyzed by water. Many inhibitors of FAAH and
chidonic acid is cytosolic phospholipase A2
zyme provides arachidonic acid for prostaglandin and leukotriene
synthesis by cleaving membrane phospholipids at the sn-2
a (cPLA2a
).3,4 This en-
position. Therefore, cPLA2
a is considered as a target for treatment
of pain and inflammatory diseases.5,6 First-generation cPLA2
a
inhibitors were analogues of arachidonic acid with the COOH
cPLA2
the enzymes. So-called serine-trap inhibitors of FAAH are the car-
bamate 5 and the inhibi-
-ketoheterocycle 6.18–22 Known cPLA2
a, respectively, bind covalently to the active site serines of
group replaced by COCF3 (AACOCF3, 1)7,8 (Fig. 1) or CH2PO(OCH3)F
(MAFP, 2).9 Compounds with high in vitro cPLA2
a
inhibitory po-
a
a
tency reported later are benzhydrylindoles from Wyeth,10 thiazo-
lidinedione compounds from Shionogi (3),11 and propan-2-ones,
such as 4 (AR-C70484XX), from AstraZeneca (Fig. 1).12
tors acting in this way are the trifluoromethylketone 1, the
fluorophosphonate 2 and the activated acetone derivative 4.7–9,12
Interestingly, 1 and 2, initially developed as cPLA2
a
inhibitors, also
An important enzyme in the endocannabinoid metabolism is
fatty acid amide hydrolase (FAAH), which rapidly cleaves the
analgetic and anti-inflammatory lipid mediator anandamide into
inhibit FAAH activity.33–36
Recently, we have described a series of cPLA2a inhibitors such
as 5-carboxyindol-1-ylpropan-2-one 10 (Fig. 1) structurally related
to 4.37–39 On account of the described similarities between cPLA2
and FAAH, we have now tested our cPLA2 inhibitors for FAAH
inhibition too. This was of special interest since dual inhibitors of
a
a
* Corresponding author. Tel.: +49 251 83 33331; fax: +49 251 83 32144.
E-mail address: lehrm@uni-muenster.de (M. Lehr).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.028

946
L. Forster et al. / Bioorg. Med. Chem. 18 (2010) 945–952
imidazole and benzimidazole, respectively, with 1-bromo-3-(4-
octylphenoxy)propan-2-one (29), obtained by Dess–Martin oxida-
tion of 1-bromo-3-(4-octylphenoxy)propan-2-ol.37
Pyrazole-4-carboxylic acid 38 was synthesized by the route out-
lined in Scheme 2. Ethyl pyrazole-4-carboxylate was coupled with
2-(4-octylphenoxy)methyloxirane37 by heating without solvent.
Oxidation of the resulting alcohol intermediate 36 to the ketone
37 was carried out with Dess–Martin periodinane reagent. Finally,
the ethyl ester group of 37 was hydrolyzed with aqueous KOH in
ethanol to give the target compound 38.
Reaction of methyl pyrazole-3-carboxylate and methyl imidaz-
ole-4-carboxylate with 2-(4-octylphenoxy)methyloxirane led to
the formation of two regioisomers in each case, namely methyl
pyrazole-3- and -5-carboxylate derivatives 33a and 33b and
methyl imidazole-4- and -5-carboxylate derivatives 40a and 40b.
The corresponding isomers were separated by silica gel chroma-
tography and further reacted as described for the synthesis of 38
(Scheme 2) to afford the test compounds 35, 39, 41 and 42.
The preparation of the benzotriazole derivative 47 is shown in
Scheme 3. Reaction of benzotriazole with epichlorohydrin led to
the oxiranylmethyl-substituted compound 45. Opening of the
epoxy ring of 45 with 4-octylphenol was achieved without solvent
in the presence of catalytic amounts of 4-dimethylaminopyridine.
Oxidation of the resulting alcohol intermediate 46 to the target ke-
tone 47 was carried out with Dess–Martin reagent.
3. Biological evaluation
All target compounds were tested for cPLA2
a inhibition apply-
ing cPLA2
a
isolated from human platelets.40 Enzyme activity was
measured in a solution containing covesicles of 1-stearoyl-2-arach-
idonoyl-sn-glycero-3-phosphocholine (SAPC) and 1,2-dioleoyl-
sn-glycerol (DOG). Inhibitory potency of the test compounds was
assessed by comparing the amount of arachidonic acid released
from SAPC in their absence and presence after an incubation time
of 60 min with reversed-phase HPLC and UV-detection.
For evaluation of FAAH inhibition, microsomes from rat brain
served as enzyme source. N-(2-Hydroxyethyl)-4-pyren-1-ylbu-
tanamide was used as substrate in combination with the micelle
forming agent Triton X-100.41 The FAAH activity was determined
by measuring the amount of 4-pyren-1-ylbutanoic acid released
by the enzyme during 45 min with reversed-phase HPLC and fluo-
Figure 1.
cPLA2
a
and FAAH could have additional benefits in comparison
with selective ones, because they would inhibit the formation of
pro-inflammatory and the degradation of anti-inflammatory lipid
mediators simultaneously.
2. Chemistry
The syntheses of most of the compounds investigated have al-
ready been described.37–39 The imidazole derivative 30 (Scheme 1)
and the benzimidazole derivative 44 were prepared by reaction of
rescence-detection.cPLA2a and FAAH are enzymes that work at the
surface of the lipophilic cell membranes. A possible problem of as-
says with such enzymes is that a test compound could inhibit the
enzyme not by binding to its active site but merely by altering the
lipophilic substrate assembly and hence causing the enzyme to
desorb from the lipid–water-interface. To exclude this path of ac-
tion, the mole fraction of inhibitor in the interface should be kept
low.42 Thus, in our enzyme assays the concentration of the
substances forming the lipophilic substrate phase was chosen
Scheme 1.
Scheme 2.

L. Forster et al. / Bioorg. Med. Chem. 18 (2010) 945–952
947
Scheme 3.
Table 2
significantly higher (300
in case of FAAH) than the maximal concentration of inhibitor
(10 M).
lM in case of cPLA2a and about 3000 lM
Inhibition of FAAH and cPLA2
a
O
l
O
N
R
4. Results and discussion
C8H17
The known dual cPLA2
of about 3 M against both enzymes in our assays. The fluor-
ophosphonate 2 blocked cPLA2 (IC50 = 0.64 M) and especially
FAAH (IC50 = 0.023 M) with considerable higher potency (Table
1). The investigation of the regioisomeric 1-(2-oxopropyl)indole-
carboxylic acids 7–11 has revealed, that the derivative with the
a/FAAH inhibitor 1 exhibited IC50 values
Compd
R
Inhibition of
Inhibition of
cPLA2aIC50 (lM)
a
a
FAAH IC50
(l
M)
l
a
l
10
12
13
14
15
16
17
18
19
COOH
H
CH3
Cl
OCH3
CN
COOCH3
CHO
CONH2
2.8
0.035
n.a.b
n.a.b
n.a.b
n.a.b
>10e
>10f
4.3
n.a.b
n.a.b
n.a.b
>10c
>10d
n.a.b
>10g
6.4
l
carboxylic acid group at position 5 (10) is a potent cPLA2
(IC50 = 0.035
M).37 Screening 10 for FAAH inhibition now showed
that it also possesses a substantial activity against this enzyme.
With an IC50 of 2.8 M it is about as active as the reference 1. In
a inhibitor
l
0.12
l
a
contrast, the other isomers are significantly less active or even
inactive.
Values are the means of at least two independent determinations; errors are
within 20%.
b
Not active at 10 lM.
The carboxylic acid group of 10 is important for both cPLA2
a
c
d
e
f
Indicates 22% of enzyme activity at 10
Indicates 42% of enzyme activity at 10
Indicates 25% of enzyme activity at 10
Indicates 36% of enzyme activity at 10
Indicates 45% of enzyme activity at 10
lM.
lM.
lM.
lM.
lM.
and FAAH inhibition. This can be seen by the fact that the unsub-
stituted indole derivative 12 and the compounds with a methyl,
chloro, methoxy, cyano, methoxycarbonyl and formyl group in
position 5 (13–18) are no or only poor inhibitors of the enzymes
(Table 2). Best tolerated is the replacement of the 5-carboxylic acid
by a carboxamide-moiety. In this case inhibitory potency against
g
cPLA2
Introduction of substituents at position 3 of the indole-5-car-
boxylic acid 10 created striking different effects on cPLA2 and
FAAH inhibitory potency. While this structural variation led to sev-
eral compounds with significant higher cPLA2 inhibitory potency
a
and FAAH is reduced only two to threefold.
22–25, it concurrently resulted in a loss of FAAH inhibition in all
cases (Table 3).
Like the indole 12, the analogous pyrrole 27 did not inhibit
either of the enzymes. However, introduction of a second nitrogen
in the pyrrole heterocycles led to compounds, which affected
a
a
such as the cyano, formyl, acetyl and methoxycarbonyl derivatives
cPLA2a as well as FAAH activity. While the IC50 values of the pyr-
azole 28 lay in the micromolar range, the imidazole derivative 30
Table 1
Table 3
Inhibition of FAAH and cPLA2
a
Inhibition of FAAH and cPLA2a
O
R
O
O
N
COOH
O
N
C8H17
COOH
Inhibition of cPLA2a
C8H17
Compd
Position of the carboxylic
acid moiety at the
indole ring
Inhibition
of FAAH IC50
M)
Inhibition
of cPLA2
(lM)
a
a
a
IC50
Compd
R
Inhibition of FAAH
a
a
(
l
IC50
(lM)
IC50 (lM)
7
8
9
10
2
3
4
5
6
n.a.b
n.a.b
>10c
2.8
n.a.b
>10c
>10c
0.035
1.1
10
20
21
22
23
24
25
26
H
2.8
0.035
0.34
0.11
0.015
0.016
0.012
0.005
0.89
C(CH3)3
Cl
CN
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
n.a.b
11
n.a.b
3.7
CHO
1 (AACOCF3)
2 (MAFP)
2.3
0.64
COCH3
COOCH3
COOH
0.023
a
Values are the means of at least two independent determinations; errors are
within 20%.
a
Values are the means of at least two independent determinations; errors are
within 20%.
b
Not active at 10
Indicates about 40% inhibition of enzyme activity at 10
l
M.
c
b
lM.
Not active at 10 lM.

948
L. Forster et al. / Bioorg. Med. Chem. 18 (2010) 945–952
Table 6
Inhibition of FAAH and cPLA2
exhibited even submicromolar inhibition values (IC50 = 0.76
and 0.50 M, respectively) (Table 4).
lM
a
l
O
O
N
N
O
O
Introduction of carboxylic acid groups in the pyrrole, pyrazole
and imidazole ring, respectively, of 27, 28 and 30 produced similar
O
O
N
effects on cPLA2
group in -position to the N1-nitrogen led to compounds, which
were inactive or only weakly active (IC50 >10 M) (31, 39, 42).
Shifting the acid group into -position, however, increased inhibi-
tory activity in all cases. The pyrazole-4-carboxylic acid 38 was the
most active cPLA2 inhibitor (IC50 = 1.8 M) and FAAH inhibitor
(IC50 = 0.11 M) of this series (Table 5).
a and FAAH inhibitory potency (Table 5). A carboxy
12
43
47
C8H17
C8H17
a
N
N
O
O
N
N
l
N
a
44
C8H17
C8H17
Inhibition of FAAH
a
l
l
Compd
Inhibition of cPLA2a
a
a
Replacement of the indole heterocycle of 12 by indazole (43),
benzimidazole (44) and benzotriazole (47) successively increased
inhibitory potency against FAAH (Table 6). While indole 12 was
IC50
(l
M)
IC50 (lM)
12
43
44
47
n.a.b
5.0
1.0
n.a.b
n.a.b
>10c
2.2
even inactive at 10
potent showing an IC50 of 0.047
27, introduction of nitrogens into the five-membered ring resulted
in more active FAAH inhibitors. Inhibition of cPLA2 was not ef-
lM, the benzotriazole derivative 47 was highly
0.047
l
M. Thus, as in case of the pyrrole
a
Values are the means of at least two independent determinations; errors are
within 20%.
a
b
Not active at 10
lM.
fected as dramatically as FAAH inhibition by this structural modi-
c
Indicates 36% inhibition of enzyme activity at 10
lM.
Table 4
Inhibition of FAAH and cPLA2
fication. Only the benzotriazole 47 possessed a moderate activity
against cPLA2 (IC50 = 2.2 M).
Substitution of indole 12, indazole 43, benzimidazole 44 and
benzotriazole 47 with a carboxy group in position 5 or 6 of the het-
a
a
l
O
N
N
O
O
O
N
erocyclic ring system elevated cPLA2a inhibitory potency in each
28
27
C8H17
C8H17
case significantly (Table 7). Contrary, the effect of this structural
variation on FAAH inhibition was inconsistent. A carboxy group
in position 6 of the indole, indazole and benzimidazole ring,
respectively, reduced activity of the compounds (11, 49, 51), while
such a substitution in 5-position increased it (10, 48, 50). In case of
benzotriazole 47, however, a three to fourfold reduction of potency
against FAAH occurs with the introduction of a carboxy group in
position 5 or 6 (52,53).
N
O
O
N
30
C8H17
Compd
Inhibition of FAAH
Inhibition of cPLA2a
IC50 (lM)
a
a
IC50
(
lM)
27
28
30
n.a.
2.0
0.76
n.a.
Finally, we also tested some other inhibitors of cPLA2
FAAH, respectively, for inhibition of these enzymes (Table 8). The
known potent FAAH inhibitors 5 and 6 did not inhibit cPLA2 at
the highest concentration tested (10 M). Vice versa, the thiazolid-
inhibitor 3 was inactive against FAAH. However,
a and
>10b
0.50
a
a
Values are the means of at least two independent determinations, errors are
within 20%.
l
b
inedione cPLA2
a
Indicates about 43% inhibition of enzyme activity at 10 lM.
Table 5
Inhibition of FAAH and cPLA2
Table 7
Inhibition of FAAH and cPLA2
a
a
O
N
N
O
O
O
O
O
N
N
O
N
N
O
O
R
R
35,38,39
31,32
C8H17
C8H17
R
C8H17
10,11
48,49
R
R
C8H17
N
O
N
O
N
O
N
N
O
N
R
O
N
C8H17
41,42
R
C8H17
50,51
52,53
C8H17
Compd
R
Inhibition of FAAH
Inhibition of cPLA2a
IC50 (lM)
n.a.b
5.0
8.2
a
a
Compd
R
Inhibition of FAAH
Inhibition of cPLA2a
IC50
(
lM)
a
a
IC50
(l
M)
IC50 (lM)
31
32
35
38
39
41
42
2-COOH
3-COOH
3-COOH
4-COOH
5-COOH
4-COOH
5-COOH
n.a.b
3.6
10
11
48
49
50
51
52
53
5-COOH
6-COOH
5-COOH
6-COOH
5-COOH
6-COOH
5-COOH
6-COOH
2.6
0.035
1.1
0.005
0.026
0.15
0.085
0.026
0.016
n.a.b
0.16
>10c
0.61
3.1
>10c
0.11
n.a.b
1.9
1.8
>10d
5.3
n.a.b
>10e
0.14
0.20
a
Values are the means of at least two independent determinations; errors are
within 20%.
a
b
Values are the means of at least two independent determinations; errors are
within 20%.
Not active at 10 lM.
c
d
e
Indicates 39% inhibition of enzyme activity at 10
Indicates 21% inhibition of enzyme activity at 10
Indicates 18% inhibition of enzyme activity at 10
lM.
lM.
lM.
b
Not active at 10
lM.
c
Indicates 23% inhibition of enzyme activity at 10
lM.

L. Forster et al. / Bioorg. Med. Chem. 18 (2010) 945–952
949
Table 8
Inhibition of FAAH and cPLA2
pounds was determined by HPLC with UV-detection at 254 nm. A
cyano phase (LiChrospher 100 CN, 5
a
by reference inhibitors
Inhibition of FAAH Inhibition of cPLA2
l
m, 3.0 mm (ID) Â 250 mm,
Merck, Darmstadt, Germany) was applied eluting with an isohex-
ane/THF gradient containing 0.1% trifluoroacetic acid (35, 38, 39,
47), an isohexane/THF gradient containing 0.1% ethyldiisopropyl-
amine (30, 44) or acetonitrile/water/H3PO4 (85%) (70:30:0.1, v/v/
v) (41, 42). The flow rate was 0.5 mL/min. All target compounds
showed purities P97%.
Compd
a
a
a
IC50
(lM)
IC50
(lM)
1 (AACOCF3)
2 (MAFP)
3.7
2.3
0.023
0.64
3 (Thiazolidinedione derivative) n.a.b
0.031
0.011
n.a.b
4 (AR-C70484XX)
5 (URB597)
6 (PHOP)
0.059
0.24
0.009
n.a.b
5.1.2. 1-Bromo-3-(4-octylphenoxy)propan-2-one (29)
a
Values are the means of at least two independent determinations; errors are
within 20%.
A
solution of 1-bromo-3-(4-octylphenoxy)propan-2-ol37
b
(110 mg, 0.32 mmol) in dry CH2Cl2 (3 mL) was treated with
Dess–Martin periodinane reagent (204 mg, 0.48 mmol) and stirred
under a nitrogen atmosphere at room temperature for 4 h. Then a
solution of sodium thiosulfate (0.4 g) in saturated sodium bicar-
bonate solution (15 mL) was added. After stirring for 5 min, the
reaction mixture was extracted exhaustively with CH2Cl2. The
combined organic layers were dried (Na2SO4) and the solvent
was evaporated. The residue was purified by silica gel chromatog-
raphy (hexane/ethyl acetate, 8:2) to yield 29 as a solid (90 mg,
82%); mp 50 °C. 1H NMR (CDCl3) d 0.88 (t, J = 7.1 Hz, 3H), 1.26–
1.30 (m, 10H), 1.53–1.67 (m, 2H), 2.55 (t, J = 7.8 Hz, 2H), 4.17 (s,
2H), 4.76 (s, 2H), 6.62 (d, J = 8.6 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H).
MS (EI): m/z (%) 342 (23), 340 (24) [M+], 241 (100).
Not active at 10 lM.
the potent cPLA2
blocked FAAH activity with high potency (IC50 = 0.059
Besides anandamide, 2-arachidonoyl glycerol (2-AG) is the sec-
ond important endocannabinoid. FAAH is also able to hydrolyze
this compound.43 However, the main enzyme for inactivation of
2-AG has been suggested to be monoacylglycerol lipase (MAGL).
a
inhibitor 4 of AstraZeneca (IC50 = 0.011
lM) also
lM).
Like cPLA2
a and FAAH, MAGL is a serine hydrolase, which is
strongly inhibited by methylarachidonoyl fluorophosphonate
(2).43 The propane-2-ones presented in Tables1–7, therefore, were
evaluated for inhibition of MAGL additionally. A test system anal-
ogous to that for measuring FAAH inhibition was applied, utilizing
a synthetic pyrene labelled fluorescence substrate (here: 1,3-
dihydroxypropan-2-yl-4-pyren-1-ylbutanoate) as well.44 We
found that none of the compounds was active at the highest test
concentration of 10
not show MAGL inhibitory potency. In contrast, the dual cPLA2
and FAAH 2 exhibited an IC50 of 0.062 M in our assay.
5.1.3. 1-Imidazol-1-yl-3-(4-octylphenoxy)propan-2-one (30)
Under a nitrogen atmosphere a solution of imidazole (17 mg,
0.25 mmol) in dry DMF (3 mL) was treated with K2CO3 (35 mg,
0.25 mmol). At 50 °C a solution of 29 (77 mg, 0.23 mmol) in dry
DMF (2 mL) was added dropwise and heating was continued at this
temperature for 1 h. After addition of half-saturated brine (50 mL),
the reaction mixture was extracted exhaustively with ethyl ace-
tate. The combined organic layers were washed with half-satu-
rated brine, dried (Na2SO4) and the solvent was evaporated. After
chromatography on silica gel (CH2Cl2/methanol, 95:5), the ob-
tained product was dissolved in a small amount of acetonitrile
and precipitated by addition of water. The solvents were removed
by freeze drying to afford 30 as a solid (29 mg, 35 %); mp 92 °C. 1H
NMR (CDCl3) d 0.88 (t, J = 7.0 Hz, 3H), 1.26–1.27 (m, 10H), 1.51–
1.61 (m, 2H), 2.57 (t, J = 7.4 Hz, 2H), 4.66 (s, 2H), 5.11 (s, 2H),
6.84 (d, J = 8.6 Hz, 2H), 6.87–6.89 (m, 1H), 7.12–7.13 (m, 1H),
7.15 (d, J = 8.7 Hz, 2H), 7.45 (s, 1H). MS (EI): m/z (%) 329 (1) [M+],
82 (100).
lM. In the same way, propane-2-one 4 did
a
l
In conclusion, we have found that several of the indol-1-ylpro-
pan-2-ones and structurally related heterocyclic compounds re-
cently published as cPLA2
FAAH. Inhibitory potency against the two enzymes can be con-
trolled by the substitution pattern of the heterocycles. For high
a
inhibitors37–39 are also inhibitors of
inhibition of cPLA2a, a carboxylic acid group at the heterocyclic
part of the molecules with a certain distance to the activated ke-
tone is necessary. On the contrary, such a carboxylate moiety is
not required unconditionally for a strong interaction with FAAH.
Introduction of substituents like chloro, acetyl, cyano and
methoxycarbonyl in position 3 of the indole-5-carboxylic acid 10
leads to a selective cPLA2
acid 38 and the benzotriazole derivative 47 are potent FAAH inhib-
itors exhibiting only moderate activity against cPLA2 . The inda-
a inhibition. The pyrazole-4-carboxylic
a
zole-5-carboxylic acid 48 and the benzotriazole-5- and 6-
carboxylic acids 52 and 53 can be considered as favorably active
5.1.4. Methyl 1-[2-hydroxy-3-(4-octylphenoxy)propyl]pyrazole-
3-carboxylate (33a) and -5-carboxylate (33b)
dual cPLA2
cPLA2 inhibition lie in the low nanomolar range and their IC50
against FAAH is lower or equal than 0.20 M. The best dual
cPLA2 /FAAH inhibitor tested so far, however, was reference com-
pound 4, published by Connolly and co-workers from AstraZeneca
(IC50 = 0.011 M and 0.059 M, respectively (Table 8).
a/FAAH inhibitors. The IC50s of these compounds for
A
mixture of methyl pyrazole-3-carboxylate (283 mg,
a
s
2.2 mmol) and 2-(4-octylphenoxymethyl)oxirane (577 mg,
2.2 mmol) was heated at 100 °C for 6 h. The obtained melt was dis-
solved in toluene and chromatographed on silica gel (CH2Cl2/meth-
anol, 98:2) to yield 33a (188 mg, 22%) and 33b (29 mg, 3 %) as
solids.
l
a
l
l
Compound 33a: Mp 92 °C. 1H NMR (CDCl3): d 0.87 (t, J = 7.0 Hz,
3H), 1.26–1.29 (m, 10H), 1.55–1.59 (m, 2H), 2.53 (t, J = 7.4 Hz, 2H),
3.03 (d, J = 5.1 Hz, 1H), 3.92 (s, 3H), 3.89–3.92 (m, 2H), 4.36–4.46
(m, 2H), 4.50 (dd, J = 12.7 Hz and 2.5 Hz, 1H), 6.80 (dd, J = 8.6 Hz,
J = 2.0 Hz, 2H), 6.82 (d, J = 2.4 Hz, 1H), 7.08 (d, J = 8.6 Hz, 2H),
7.52 (d, J = 2.4 Hz, 1H). MS (EI): m/z (%) 388 (1) [M+], 183 (100).
Compound 33b: Mp 42 °C. 1H NMR (CDCl3): d 0.88 (t, J = 7.0 Hz,
3H), 1.26–1.30 (m, 10H), 1.54–1.58 (m, 2H), 2.53 (t, J = 7.4 Hz, 2H),
3.58 (d, J = 5.5 Hz, 1H), 3.86 (s, 3H), 3.91 (dd, J = 9.6 Hz and 5.4 Hz,
1H), 4.01 (dd, J = 9.6 Hz and 5.0 Hz, 1H), 4.36–4.43 (m, 1H), 4.86–
4.89 (m, 2H), 6.82 (dd, J = 9.0 and 2.4 Hz, 2H), 6.87 (d, J = 2.0 Hz,
1H), 7.08 (dd, J = 9.0 Hz and 2.4 Hz, 2H), 7.55 (d, J = 2.0 Hz, 1H).
MS (EI): m/z (%) 388 (2) [M+], 183 (100).
5. Experimental
5.1. Chemistry
5.1.1. General
Column chromatography was performed on Merck Silica Gel 60,
0.040–0.063 mm (230–400 mesh). Melting points were deter-
mined on a Büchi B-540 apparatus and are uncorrected. 1H NMR
spectra were recorded on a Varian Mercury Plus 400 spectrometer
(400 MHz). Mass spectra were obtained on Finnigan GCQ and LCQ
apparatuses applying electron beam ionization (EI) and electro-
spray ionization (ESI), respectively. Purity of the new target com-

950
L. Forster et al. / Bioorg. Med. Chem. 18 (2010) 945–952
5.1.5. Methyl 1-[3-(4-octylphenoxy)-2-oxopropyl]pyrazole-3-
carboxylate (34)
2.55 (t, J = 7.4 Hz, 2H), 4.68 (s, 2H), 5.33 (s, 2H), 6.83 (d,
J = 8.6 Hz, 2H), 7.11 (d, J = 8.6 Hz, 2H), 7.98 (s, 1H), 8.01 (s, 1H).
MS (EI): m/z (%) 373 (2) [M+], 167 (100).
Compound 33a (95 mg, 0.24 mmol) was oxidized with Dess–
Martin periodinane (156 mg, 0.37 mmol) as described above for
the synthesis of 29. The crude product was purified by silica gel
chromatography (CH2Cl2/methanol, 98:2) to yield 34 as a solid
(91 mg, 96%); mp 105 °C. 1H NMR (CDCl3): d 0.87 (t, J = 7.0 Hz,
3H), 1.26–1.29 (m, 10H), 1.55–1.59 (m, 2H), 2.55 (t, J = 7.4 Hz,
2H), 3.92 (s, 3H), 4.65 (s, 2H), 5.39 (s, 2H), 6.82 (dd, J = 8.6 Hz
and 2.3 Hz, 2H), 6.90 (d, J = 2.4 Hz, 1H), 7.13 (dd, J = 9.0 Hz and
5.1.10. 1-[3-(4-Octylphenoxy)-2-oxopropyl]pyrazole-5-
carboxylic acid (39)
Compound 39 was prepared from 33b by the procedures de-
scribed for the preparation of 35. Chromatography on silica gel
(CH2Cl2/methanol/formic acid, 98:2:0.5) yielded 39 as a solid; mp
97 °C. 1H NMR (DMSO-d6): d 0.83 (t, J = 7.0 Hz, 3H), 1.22–1.24 (m,
10H), 1.49–1.52 (m, 2H), 2.46–2.5 (m, 2H), 4.94 (s, 2H), 5.55 (s,
2H), 6.84 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 2.0 Hz, 1H), 7.07 (d,
J = 8.6 Hz, 2H), 7.57 (d, J = 2.0 Hz, 1H). MS (ESI-): m/z 371 [MÀH]À.
1
1
2.4 Hz, 2H), 7.46 (d, J = 2.3 Hz, 1H). NOE (CDCl3): d Hirr/d Hres
7.46 (pyrazol-5-CH)/6.90 (pyrazol-4-CH), 5.39 (propyl-1-CH2);
6.90 (pyrazol-4-CH)/7.46 (pyrazol-5-CH); 5.39 (propyl-1-CH2)/
7.46 (pyrazol-5-CH), 6.82 (phenyl-2-CH, phenyl-6-CH), 4.65 (pro-
pyl-3-CH2); 4.65 (propyl-3-CH2)/6.82 (phenyl-2-CH, phenyl-6-
CH), 5.39 (propyl-1-CH2). MS (EI): m/z (%) 386 (5) [M+], 181 (100).
5.1.11. Methyl 1-[2-hydroxy-3-(4-
octylphenoxy)propyl]imidazole-4-carboxylate (40a) and -5-
carboxylate (40b)
5.1.6. 1-[3-(4-Octylphenoxy)-2-oxopropyl]pyrazole-3-
carboxylic acid (35)
A
mixture of methyl imidazole-4-carboxylate (96 mg,
0.76 mmol) and 2-(4-octylphenoxymethyl)oxirane (200 mg,
0.76 mmol) was heated at 80 °C for 5 h. The obtained melt was
chromatographed on silica gel (CH2Cl2/methanol, 99.5:0.5) to yield
40a (60 mg, 20%) and 40b (15 mg, 5 %) as solids.
A solution of 34 (80 mg, 0.21 mmol) in ethanol (9 mL) was trea-
ted with a solution of 10% aqueous KOH (3 mL). The mixture was
stirred at room temperature for 2 h. After acidification with 3 M
HCl, the reaction mixture was extracted exhaustively with
CH2Cl2/ethyl acetate (3:1). The combined organic layers were
washed with water, dried (Na2SO4) and the solvent was distilled
off. Chromatography on silica gel (CH2Cl2/methanol/formic acid,
98:2:0.5) yielded 35 as a solid (5 mg, 6%). 1H NMR (DMSO-d6): d
0.84 (t, J = 6.8 Hz, 3H), 1.19–1.29 (m, 10H), 1.47–1.53 (m, 2H),
2.45–2.51 (m, 2H), 4.91 (s, 2H), 5.40 (s, 2H), 6.71–6.73 (m, 1H),
6.85 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 7.75–7.77 (m, 1H).
MS (ESI-): m/z 371 [MÀH]À.
Compound 40a: mp 111 °C. 1H NMR (CDCl3): d 0.88 (t, J = 7.1 Hz,
3H), 1.26–1.29 (m, 10H), 1.55–1.59 (m, 2H), 2.55 (t, J = 7.4 Hz, 2H),
3.86 (s, 3H), 3.92–4.01 (m, 2H), 4.11 (dd, J = 14.0 Hz and 7.6 Hz,
1H), 4.27 (dd, J = 14.0 Hz and 3.4 Hz, 1H), 4.31–4.37 (m, 1H), 6.83
(dd, J = 8.6 Hz and 2.0 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 7.50 (s,
1H), 7.64 (d, J = 1.2 Hz, 1H). MS (EI): m/z (%) 389(18) [M+], 303
(100).
Compound 40b: mp 83 °C. 1H NMR (CDCl3): d 0.87 (t, J = 7.0 Hz,
3H), 1.26–1.3 (m, 10H), 1.55–1.58 (m, 2H), 2.54 (t, J = 7.4 Hz, 2H),
3.45 (d, J = 5.1 Hz, 1H), 3.84 (s, 3H), 3.95 (dd, J = 9.6 Hz and
5.1 Hz, 1H), 4.0 (dd, J = 9.6 Hz and 4.8 Hz, 1H), 4.25–4.32 (m, 1H),
4.41 (dd, J = 14.1 Hz and 7.5 Hz, 1H), 4.69 (dd, J = 14.1 Hz and
3.3 Hz, 1H), 6.82 (dd, J = 8.6 Hz and 2.0 Hz, 2H), 7.09 (dd,
J = 8.6 Hz and 2.0 Hz, 2H), 7.67 (d, J = 0.8 Hz, 1H), 7.72 (d,
J = 1.2 Hz, 1H). MS (EI): m/z (%) 389 (6) [M+], 107 (100).
5.1.7. Ethyl 1-[2-hydroxy-3-(4-octylphenoxy)propyl]pyrazole-
4-carboxylate (36)
A mixture of ethyl pyrazole-4-carboxylate (200 mg, 1.4 mmol)
and 2-(4-octylphenoxymethyl)oxirane (380 mg, 1.4 mmol) was
heated at 80 °C for 4 h. The obtained melt was dissolved in toluene
and chromatographed on silica gel (CH2Cl2/methanol, 99:1) to
yield 36 (373 mg, 65 %) as solid; mp 84 °C. 1H NMR (CDCl3): d
0.87 (t, J = 7.0 Hz, 3H), 1.26–1.29 (m, 10H), 1.33 (t, J = 7.2 Hz, 3H),
1.53–1.60 (m, 2H), 2.54 (t, J = 7.2 Hz, 2H), 3.50 (s, br, 1H), 3.86
(dd, J = 9.7 Hz and 5.6 Hz, 1H), 3.95 (dd, J = 9.7 Hz and 4.8 Hz,
1H), 4.28 (q, J = 7.2 Hz, 2H), 4.31–4.34 (m, 1H), 4.36–4.41 (m,
1H), 4.44 (dd, J = 12.9 Hz and 2.8 Hz, 1H), 6.80 (dd, J = 8.6 Hz and
2.0 Hz, 2H), 7.09 (dd, J = 8.6 Hz and 2.4 Hz, 2H), 7.94 (s, 1H), 7.97
(s, 1H). MS (EI): m/z (%) 402 (4) [M+], 197 (100).
5.1.12. 1-[3-(4-Octylphenoxy)-2-oxopropyl]imidazole-4-
carboxylic acid (41)
Compound 41 was prepared from 40a by the procedures de-
scribed for the preparation of 35; mp 168 °C. 1H NMR (DMSO-
d6): d 0.83 (t, J = 6.7 Hz, 3H), 1.22–1.29 (m, 10H), 1.45–1.54 (m,
2H), 2.44–2.52 (m, 2H), 4.88 (s, 2H), 5.25 (s, 2H), 6.87 (d,
J = 6.9 Hz, 2H), 7.10 (d, J = 8.2 Hz, 2H), 7.60 (s, 1H), 7.70 (s, br,
1H). MS (ESI-): m/z 371 [MÀH]À.
5.1.8. Ethyl 1-[3-(4-octylphenoxy)-2-oxopropyl]pyrazole-4-
carboxylate (37)
5.1.13. 1-[3-(4-Octylphenoxy)-2-oxopropyl]imidazole-5-
carboxylic acid (42)
Compound 36 (167 mg, 0.41 mmol) was oxidized with Dess–
Martin periodinane (264 mg, 0.62 mmol) as described above for
the synthesis of 29. The crude product was purified by silica gel
chromatography (CH2Cl2/methanol, 99:1) to yield 37 as a solid
(121 mg, 73%); mp 88 °C. 1H NMR (CDCl3): d 0.88 (t, J = 7.1 Hz,
3H), 1.27–1.30 (m, 10H), 1.34 (t, J = 7.0 Hz, 3H), 1.54–1.61 (m,
2H), 2.56 (t, J = 7.8 Hz, 2H), 4.30 (q, J = 7.2 Hz, 2H), 4.67 (s, 2H),
5.30 (s, 2H), 6.83 (d, J = 8.6 Hz, 2H), 7.13 (d, J = 8.6 Hz, 2H), 7.93
(s, 1H), 7.96 (s, 1H). MS (EI): m/z (%) 400 (16) [M+], 167 (100).
Compound 42 was prepared from 40b by the procedures de-
scribed for the preparation of 35; mp 154 °C. 1H NMR (DMSO-
d6): d 0.84 (t, J = 6.7 Hz, 3H), 1.22–1.24 (m, 10H), 1.48–1.52 (m,
2H), 2.46–2.50 (m, 2H), 4.91 (s, 2H), 5.33 (s, 2H), 6.86 (d,
J = 8.6 Hz, 2H), 7.07 (d, J = 8.6 Hz, 2H), 7.54 (s, br, 1H), 7.79 (s, br,
1H). MS (ESI-): m/z 371 [MÀH]À.
5.1.14. 1-Benzimidazol-1-yl-3-(4-octylphenoxy)propan-2-one
(44)
Compound 44 was synthesized from benzimidazole (54 mg,
0.46 mmol) analogously to the corresponding imidazole derivative
30. Chromatography on silica gel (CH2Cl2/methanol, 98:2) yielded
41 as a solid (20 mg, 12%); mp 101 °C. 1H NMR (CDCl3): d 0.84 (t,
J = 6.8 Hz, 3H), 1.18–1.29 (m, 10H), 1.46–1.54 (m, 2H), 2.45–2.51
(m, 2H), 5.01 (s, 2H), 5.48 (s, 2H), 6.89 (d, J = 8.6 Hz, 2H), 7.11 (d,
J = 8.5 Hz, 2H), 7.17–7.23 (m, 2H), 7.46–7.50 (m, 1H), 7.63–7.67
(m, 1H), 8.10 (s, 1H). MS (EI): m/z (%) 378 (3) [M+].
5.1.9. 1-[3-(4-Octylphenoxy)-2-oxopropyl]pyrazole-4-
carboxylic acid (38)
Compound 37 (91 mg, 0.23 mmol) was hydrolyzed according to
the procedure described above for the preparation of 35. Chroma-
tography on silica gel (CH2Cl2/methanol/formic acid, 99:1:0.5)
yielded 38 as a solid (38 mg, 45%); mp 121 °C. 1H NMR (CDCl3): d
0.87 (t, J = 7.0 Hz, 3H), 1.26–1.33 (m, 10H), 1.55–1.59 (m, 2H),

L. Forster et al. / Bioorg. Med. Chem. 18 (2010) 945–952
951
5.1.15. 1-Oxiranylmethylbenzotriazole (45)
pound with reversed-phase HPLC and fluorescence-detection
(excitation: 340 nm; emission: 380 nm). The only deviation from
the published procedure was that the incubation time with the en-
zyme was 45 min instead of 60 min. This lead to a slight change of
the IC50 values of the reference compounds 5 and 6.
A mixture of benzotriazole (0.50 g, 4.2 mmol), powdered KOH
(88%, 0.48 g, 7.5 mmol), tetrabutylammonium bromide (135 mg,
0.42 mmol) and epichlorohydrin (4 mL) was stirred at room tem-
perature for 4 h. The reaction mixture was purified by silica gel
chromatography (hexane/ethyl acetate, 9:1 followed by 8:2) to
give 45 as an oil (0.44 g, 60%). 1H NMR (CDCl3): d 2.62–2.66 (m,
1H), 2.90–2.94 (m, 1H), 3.42–3.46 (m, 1H), 4.59 (dd, J = 15,1 Hz
and 6.0 Hz, 1H), 5.10 (dd, J = 15.1 Hz and 3.0 Hz, 1H), 7.37–7.42
(m, 1H), 7.49–7.55 (m, 1H), 7.66–7.70 (m, 1H), 8.05–8.09 (m,
1H). MS (EI): m/z (%) 175 (91) [M+], 146 (100).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.11.028.
References and notes
5.1.16. 1-Benzotriazol-1-yl-3-(4-octylphenoxy)propan-2-ol (46)
Compound 45 (0.43 g, 2.5 mmol), 4-octylphenol (0.51 g,
2.5 mmol) and 4-dimethylaminopyridine (61 mg) were mixed
thoroughly and heated under stirring at 120 °C for 45 min. The
warm reaction mixture was dissolved in toluene and subjected to
chromatography on silica gel (hexane/ethyl acetate, 8:2) to give
46 as an oil (0.62 g, 66%). 1H NMR (CDCl3): d (ppm) = 0.87 (t,
J = 6.7 Hz, 3H), 1.20–1.35 (m, 10H), 1.51–1.62 (m, 2H), 2.49–2.58
(m, 2H), 3.02 (s, br, 1H), 3.99 (dd, J = 9.6 Hz and 5.6 Hz, 1H). 4.04
(dd, J = 9.6 Hz and 5.2 Hz, 1H), 4.56–4.65 (m, 1H), 4.84 (dd,
J = 14.3 Hz and 6.5 Hz, 1H), 4.94 (dd, J = 14.4 Hz and 4.2 Hz, 1H),
6.81 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 8.5 Hz, 2H), 7.38 (t, 1H,
J = 7.6 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H),
8.03 (d, J = 8.4 Hz, 1H). MS (EI): m/z (%) 381 (8) [M+], 176 (100).
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5.1.17. 1-Benzotriazol-1-yl-3-(4-octylphenoxy)propan-2-one
(47)
Compound 46 (0.31 g, 0.81 mmol) was oxidized with Dess–
Martin periodinane (0.51 g, 1.2 mmol) analogously to the synthesis
of 29. The crude product was purified by silica gel chromatography
(hexane/ethyl acetate, 8:2) to yield 47 as a solid (0.28 g, 91 %); mp
102–103 °C. 1H NMR (CDCl3): 0.88 (t, J = 6.9 Hz, 3H), 1.23–1.36 (m,
10H), 1.55–1.64 (m, 2H), 2.55–2.61 (m, 2H), 4.75 (s, 2H), 5.80 (s,
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1H), 7.37–7.43 (m, 1H), 7.47–7.52 (m, 1H), 8.08–8.12 (m, 1H). MS
(EI): m/z (%) 379 (28) [M+], 175 (100).
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5.2. Biological evaluation of the target compounds
5.2.1. cPLA2a inhibition assay
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The ability of test compounds to inhibit cPLA2
a isolated from
human platelets was performed as described earlier.40 Briefly, son-
icated covesicles consisting of 1-stearoyl-2-arachidonoyl-sn-glyce-
ro-3-phosphocholine (200
lM) and 1,2-dioleoyl-sn-glycerol
(100 M) were used as substrate. Enzyme reaction was terminated
l
after 60 min by addition of a mixture of acetonitrile, methanol and
0.1 M aqueous EDTA-Na2 solution, which contained 4-undecyloxy-
benzoic acid as internal standard and nordihydroguaiaretic acid
(NDGA) as oxygen scavenger. cPLA2
a activity was determined by
measuring the arachidonic acid released by the enzyme in absence
and presence of a test compound with reversed-phase HPLC and
UV-detection at 200 nm after sample clean-up by solid phase
extraction.
26. Hart, T.; Macias, A. T.; Benwell, K.; Brooks, T.; D’Alessandro, J.; Dokurno, P.;
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