Design, synthesis, and in vitro evaluation of carbamate derivatives of 2-benzoxazolyl- and 2-benzothiazolyl-(3-hydroxyphenyl)-methanones as novel fatty acid amide hydrolase inhibitors

Article abstract of DOI:10.1021/jm070501w

Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase, which catalyzes the hydrolysis of the endocannabinoid N-arachidonoylethanolamide to arachidonic acid and ethanolamine. FAAH also hydrolyzes another endocannabinoid, 2-arachidonoylglyc

Full text of DOI:10.1021/jm070501w

4236
J. Med. Chem. 2007, 50, 4236-4242
Design, Synthesis, and In Vitro Evaluation of Carbamate Derivatives of 2-Benzoxazolyl- and
2-Benzothiazolyl-(3-hydroxyphenyl)-methanones as Novel Fatty Acid Amide Hydrolase
Inhibitors
Mikko J. Myllyma¨ki,† Susanna M. Saario,‡ Antti O. Kataja,† Joel A. Castillo-Melendez,† Tapio Nevalainen,‡ Risto O. Juvonen,§
Tomi Ja¨rvinen,‡ and Ari M. P. Koskinen*,†
Laboratory of Organic Chemistry, Department of Chemical Technology, Helsinki UniVersity of Technology, Post Office Box 6100,
Kemistintie 1, FIN-02015 TKK, Finland, and Department of Pharmaceutical Chemistry and Department of Pharmacology and Toxicology,
UniVersity of Kuopio, Post Office Box 1627, FIN-70211 Kuopio, Finland
ReceiVed April 30, 2007
Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase, which catalyzes the hydrolysis of
the endocannabinoid N-arachidonoylethanolamide to arachidonic acid and ethanolamine. FAAH also
hydrolyzes another endocannabinoid, 2-arachidonoylglycerol (2-AG). However, 2-AG has been assumed to
be hydrolyzed mainly by monoacylglycerol lipase (MAGL) or a MAGL-like enzyme. Inhibition of FAAH
or MAGL activity might lead to beneficial effects in many physiological disorders such as pain, inflammation,
and anxiety due to increased endocannabinoid-induced activation of cannabinoid receptors CB1 and CB2.
In the present study, a total of 34 novel compounds were designed, synthesized, characterized, and tested
against FAAH and MAGL-like enzyme activity. Altogether, 16 compounds were found to inhibit FAAH
with half-maximal inhibition concentrations (IC50) between 28 and 380 nM. All the active compounds belong
to the structural family of carbamates. Compounds 14 and 18 were found to be the most potent FAAH
inhibitors, which may serve as lead structures for novel FAAH inhibitors.
Introduction
Arachidonoylethanolamide (AEA,a Figure 1) and 2-arachi-
donoylglycerol (2-AG, Figure 1) are considered to be the most
important endogenous agonists for the G protein-coupled
cannabinoid receptors CB1 and CB2.1-3 CB1 receptors are
predominantly located on presynaptic terminals in the central
nervous system (CNS), whereas CB2 receptors are located
mainly in peripheral tissues.4-6 However, it has been reported
very recently that also CB2 receptors are expressed in the CNS.7
The endocannabinoids are inactivated rapidly by cellular re-
uptake followed by intracellular hydrolysis by specific en-
zymes.8,9 AEA is assumed to be transported into the cell by a
specific transporter10-12 and rapidly hydrolyzed by the enzyme
fatty acid amide hydrolase (FAAH).8 Also, like AEA, 2-AG is
Figure 1. Structures of AEA, 2-AG, and some potent inhibitors of
FAAH (1a, 1b, 2, and 3).
thought to be removed from its sites of action by cellular uptake
and then hydrolyzed enzymatically. Although 2-AG can be
hydrolyzed by FAAH,13 the main enzyme responsible for 2-AG
the decrease of inflammation, lowering of blood pressure, and
hydrolysis in vivo is probably monoacylglycerol lipase14
(MAGL; EC 3.1.1.23) or MAGL-like enzyme.15 Due to the rapid
inactivation, the cannabimimetic effects of the endocannabinoids
remain very weak. The increase in the concentration of the
endocannabinoids in the extracellular space can lead to several
beneficial therapeutic effects16 such as treatment of pain17 and
anxiety,18 reduction of intraocular pressure19 as well as increase
of appetite.20 The activation of CB2 receptors is involved in
suppression of peripheral pain.21 Several potent synthetic CB
receptor agonists as well as antagonists have been described
(see for review Lambert and Fowler, 2005).22 In addition,
inhibitors have been developed for the transport protein,12,23,24
although its existence is under debate.12,25,26 The enzyme
inhibition could be a convenient way to elevate the endocan-
nabinoid levels and thus increase the receptor activity.27
Endocannabinoids are biosynthesized upon demand and released
immediately from neurons afterward. By inhibiting FAAH or
MAGL, the effect of endocannabinoids could be enhanced and
more selective therapeutic effects achieved. Thus, the inhibition
of these enzymes is of great interest to medicinal chemists
nowadays.
* To whom correspondence should be addressed. Tel.: +358 9 451 2526.
Fax: +358 9 451 2538. E-mail: ari.koskinen@tkk.fi.
† Helsinki University of Technology.
‡ Department of Pharmaceutical Chemistry, University of Kuopio.
§ Department of Pharmacology and Toxicology, University of Kuopio.
a Abbreviations: FAAH, fatty acid amide hydrolase; MAGL, mono-
acylglycerol lipase; AEA, N-arachidonoylethanolamine; 2-AG, 2-arachi-
donoylglycerol; CNS, central nervous system; IBX, o-iodoxybenzoic acid;
DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; MW, microwave irra-
diation; BSA, bovine serum albumin.
We have designed and synthesized a series of potential FAAH
inhibitors and determined their inhibitory activities on FAAH
and MAGL-like enzyme. We compared the structures of
previously reported FAAH inhibitors and found that the most
10.1021/jm070501w CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/01/2007

NoVel Fatty Acid Amide Hydrolase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4237
Scheme 1. Synthesis of Presented Compoundsa
a Reagents and conditions: (a) benzoxazole or benzothiazole, n-BuLi, 1 M ZnCl2/Et2O, CuI, THF, -75 °C f 0 °C, 54-55%; (b) BF3Et2O, Me2S,
CH2Cl2, rt, 84-91% or N-1-BuPyrBr, MW, 30 s, 90%; (c) Et3N or pyridine, RNCO, RCOCl or ROCOCl, toluene or CH2Cl2, rt or heating up to 93 °C,
41-99%.
potent compounds had very different structural moieties. Boger
et al. (2000) have published potent and selective FAAH
inhibitors that comprise of either 2-acyl-oxazolo[4,5-b]pyri-
dines (1a and 1b), 2-acyl-5-(2-pyridyl)-1,3,4-oxadiazoles (2),
or with an alkyl tail (Figure 1).28,29 Additionally, the series
of carbamates, including the highly active compound 3
(URB597),30,31 with promising pharmacological features have
been developed.
reference compounds. These results correlate with those reported
in the literature.28-31 The structures of compounds and their
inhibition potencies for FAAH and MAGL-like enzyme activity
are presented in the Table 1.
The most potent FAAH inhibitor in this series was a
cyclopentyl carbamate 14 (IC50, 28 nM). Compounds 13, 15,
18, 22, 23, and 26 inhibit FAAH with IC50 values (32-56 nM)
almost equal to that obtained for compound 14. The inhibition
of FAAH activity by these compounds was dependent on the
carbamate group, as the compounds lacking this functionality
were unable to inhibit FAAH even at 10 µM. This supports the
mechanism by which carbamates like 3 inhibit FAAH by
carbamylation of the enzyme’s nucleophilic serine.41 Com-
pounds 11, 12, and 13 as well as the corresponding sulfur
analogs (19, 20, and 21) were found to have a trend for
increasing potency for FAAH with increasing length of the alkyl
group. It was noteworthy that compounds 18 and 26, containing
3-methylbenzyl carbamate, had clearly higher inhibition activity
against MAGL-like enzyme activity compared to the other
compounds in this series. Thus, the introduction of the methyl
group in the 3-position of the benzyl ring increased FAAH as
well as MAGL-like enzyme inhibition. However, none of these
compounds could inhibit MAGL-like activity at the nanomolar
concentration range. Furthermore, there were no significant
differences in FAAH or MAGL-like enzyme inhibition between
the benzoxazoles and the benzothiazoles. Compounds 17 and
25 were not stable enough to give reliable inhibition activity.
Apparently the carbamate was hydrolyzed.
Chemistry
Compound 1a (Figure 1) was prepared as described by Boger
et al.28 The synthesis of the actual library was started from 3-
or 4-hydroxybenzoic acid or 3-aminobenzoic acid. The phenol
functionality was protected as benzyl ether and then the acids
were converted to acid chlorides 4 and 5 (Scheme 1) according
to literature procedures.32-34 Compound 4 was then coupled with
benzoxazole or benzothiazole to prepare compounds 6a, 6b, and
7 following the method described by Harn et al.35 Aniline 8
was prepared from 5 in a similar manner. Deprotection of the
phenolic hydroxyl was first attempted with hydrogenation
catalyzed by Pd/C. Unfortunately, under these conditions the
methanone was reduced to alcohol before the cleavage of benzyl
group. Although the alcohols were easily oxidized back to
ketone with IBX36 or DDQ,37,38 another method was needed to
avoid the extra step. Finally, the removal of the benzyl protecting
group without reducing the ketone was carried out using
BF3Et2O and Me2S.39 Ionic liquid-based microwave-assisted
cleavage of benzyl ether also proved to be as efficient a
method.40 Deprotected phenols 9a, 9b, 10, and aniline 7 were
then coupled with electrophiles (isocyanates, chloroformates,
and acid chlorides; Scheme 1)
To clarify the importance of the carbamate group, we prepared
some other carbonyl derivatives for comparison. Carbonyl com-
pounds 27 (ester), 28 (amide), 29 (urea), 30 (reverse carbamate),
and 31a-d and 32a-d (carbonates) were not effective FAAH
inhibitors. It was surprising that 30 did not inhibit FAAH or
MAGL because its structural difference compared to compound
14 is only in the direction of the carbamate bond. Carbonates
31a-d and 32a-d were not stable enough in the assay
conditions. The decomposition of compounds 17, 25, 31a-d,
and 32a-d was detected with TLC (data not shown). 4-Sub-
stituted compounds 33 and 34 were also synthesized. The
activity of these compounds against FAAH was still in nano-
molar range (288 and 137 nM). It was noteworthy that a 10-
fold difference was found between meta- (14, 28 nM) and para-
substituted (33, 288 nM) cyclopentyl carbamates. In addition,
Results and Discussion
We have designed and prepared the novel series of FAAH
inhibitors by combining the structural features of the known
inhibitors presented in Figure 1. The carbamate and other
carbonyl containing groups were attached to 2-methanone-
benzothiazole and -benzoxazole via a phenylmethanone linker.
The compounds were tested against FAAH and MAGL-like
enzyme. From this series the most promising alkyl moiety was
selected and other carbonyl derivatives were prepared. Ad-
ditionally, two previously reported potent FAAH inhibitors, 1a
and 3 (see Figure 1), were also tested in our FAAH assay as

4238 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Myllyma¨ki et al.
Table 1. IC50 Values for the Inhibition of FAAH and MAGL-Like Enzymes Activity by Compounds Tested
a Values represent the mean of three independent experiments (n ) 3) performed in duplicate (95% confidence intervals (95% CI) are given in parentheses).
b Remaining enzyme activity (%) at 100 µM compound concentration (n ) 2). c Not stable in used assay conditions. d Remaining enzyme activity at 1 mM
was ∼11-24%. e Remaining enzyme activity (%) at 10 µM compound concentration (n ) 2).
compound 33 was found clearly more active against MAGL-
like enzyme activity than 14.
beneficial therapeutic effects. This point of view calls for closer
evaluation because the selectivity against other hydrolase
enzymes might suffer.
Conclusion
Experimental Section
A series of carbonyl compounds as potential FAAH inhibitors
were synthesized and evaluated for their FAAH and MAGL-
like enzyme inhibition activity. Altogether, 16 carbamate
containing compounds were found to inhibit FAAH with IC50
values between 28 and 380 nM. These carbamate compounds
11-16, 18-24, 26, 33, and 34 were selective for FAAH as
they were not able to inhibit MAGL-like enzyme at the high
nanomolar concentration range. The most potent compounds
for FAAH in this series containing a cyclopentyl carbamate (14)
or 3-methyl-benzyl carbamate (18) group may serve as lead
structures for novel FAAH inhibitors. Against MAGL-like
enzyme activity, the best inhibitor was the para-substituted
derivative of cyclopentyl carbamate (33) with 14 µM IC50 value.
These findings will further help designing more potent and
selective inhibitors of FAAH and MAGL-like enzyme activity.
It is also noteworthy that inhibiting both AEA- and 2-AG-
hydrolyzing enzymes simultaneously through a combination of
selective FAAH and MAGL inhibitors could lead to even more
Chemistry. All solvents and reagents were obtained from
commercial suppliers and used without further purification unless
otherwise noted. Tetrahydrofuran (THF) was distilled from Na/
benzophenone. All dry reactions were performed under argon in
flame-dried glassware. Analytical thin-layer chromatography was
carried out on Merck silica gel F254 (60 Å, 40-63 µm, 230-400
mesh) precoated aluminum sheets and detected under UV light.
Silica gel (230-400 mesh) for column chromatography was
purchased from Merck. Melting points (mp) were determined in
open capillaries using a Gallenkamp melting point apparatus and
are uncorrected. Nuclear magnetic resonance (1H NMR and 13C
NMR) spectra were recorded on a Bruker Avance DPX 400
spectrometer operating at 400 MHz for H and 100 MHz for 13C.
1
Chemical shifts are reported in ppm on the δ scale from an internal
standard of residual solvent (CDCl3 7.26 and 77.0 ppm; DMSO-d6
2.50 and 39.52 ppm). Coupling constants (J) are reported in Hz.
Infrared (IR) spectra were recorded using a Perkin-Elmer Spectrum
One FT-IR spectrometer, and values are reported as frequency (ν)

NoVel Fatty Acid Amide Hydrolase Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4239
and expressed in cm-1. Elemental analyses were recorded on a
Perkin-Elmer 2400 CHN.
lization from EtOAc/hexane yielded 11 (121 mg, 93%) as a white
cotton-like solid: mp 141-142 °C; Rf (5% Et2O in CH2Cl2) 0.70;
1H NMR (CDCl3) δ 8.46 (d, 1H, J ) 7.7 Hz), 8.32 (s, 1H), 7.94
(d, 1H, J ) 8.1 Hz), 7.70 (d, 1H, J ) 8.2 Hz), 7.57-7.45 (m, 4H),
5.19 (br s, 1H), 3.33 (qui, 2H, J ) 6.8 Hz), 1.23 (t, 3H, J ) 7.2
Hz); 13C NMR (CDCl3) δ 179.4, 156.9, 154.0, 151.2, 150.4, 140.7,
136.0, 129.4, 128.5, 127.9 , 127.8, 125.7, 123.9, 122.4, 111.8, 36.2,
15.0; IR (KBr) 3345, 2976, 1712, 1662, 1527; Anal. (C17H14N2O4)
C, H, N.
3-(Benzo[d]oxazole-2-carbonyl)phenyl cyclopentanecarboxy-
late (27). A mixture of 6a (130 mg, 0.54 mmol, 100 mol %),
cyclopentanoyl chloride (86 mg, 0.65 mmol, 120 mol %), and TEA
(76 µL, 0.54 mmol, 100 mol %) was stirred in dry toluene (6 mL)
for 20 min at room temperature, then quenched with ice (5 mL)
and satd NaHCO3 (5 mL), and extracted with EtOAc (2 × 25 mL).
The combined organic phase was washed with brine (20 mL), dried
(Na2SO4), filtered, and evaporated. Recrystallization from EtOAc/
hexane gave 27 (169 mg, 93%) as white crystals: mp 147-148
°C; Rf (5% MeOH in CH2Cl2) 0.90; 1H NMR (CDCl3) δ 8.53 (ddd,
1H, J ) 7.8, 1.5, 1.1 Hz), 8.27-8.25 (m, 1H), 7.96 (td, 1H, J )
8.0, 0.7 Hz), 7.74-7.70 (m, 1H), 7.61-7.54 (m, 2H), 7.49 (ddd,
1H, J ) 8.4, 7.4, 1.1 Hz), 7.43 (ddd, 1H, J ) 8.1, 2.4, 1.0 Hz),
3.00-3.09 (m, 1 H), 2.11-1.95 (m, 4H), 1.85-1.75 (m, 2H), 1.73-
1.64 (m, 2H); 13C NMR (CDCl3) δ 179.4, 175.0, 156.8, 151.0,
150.4, 140.7, 136.2, 129.6, 128.6, 128.5, 127.8, 125.8, 123.9, 122.5,
111.9, 43.8, 30.1, 25.9; IR (KBr) 2971, 2872, 1750, 1661, 1531;
Anal. (C20H17NO4) C, H, N.
General Procedure for the Preparation of 6a, 6b, 7, and 8
from Acid Chlorides 4 and 5; Benzo[d]oxazol-2-yl(3-(benzyloxy)-
phenyl)methanone (6a). To a solution of benzoxazole (727 mg,
6.1 mmol, 100 mol %) in THF (35 mL) was added dropwise n-BuLi
(1.8 M in hexane, 3.7 mL, 6.7 mmol, 110 mol %) at -75 °C over
a period of 10 min. After 30 min, ZnCl2 (1.66 g, 12.2 mmol, 200
mol %) in Et2O (20 mL) was added. The mixture was warmed to
0 °C, and after 45 min, CuI (1.16 mg, 6.1 mmol, 100 mol %) was
added. After another 10 min, acid chloride 4 (1.5 g, 6.1 mmol, 100
mol %) in THF (10 mL) was added. The mixture was stirred at 0
°C for another 45 min, diluted with EtOAc (400 mL), and washed
successively with 1:1 H2O/25% aq ammonia (100 mL), H2O (100
mL), and brine (100 mL). The organic phase was dried over
anhydrous Na2SO4, filtered, and evaporated to yield the crude
product as a tan solid, which was purified with flash chromatog-
raphy (5% EtOAc in hexane) and recrystallized (EtOAc/hexane)
to give 6a (1.08 g, 54%) as a yellow solid: mp 95-97 °C; Rf (20%
1
EtOAc in hexane) 0.50; H NMR (CDCl3) δ 8.22 (d, 1H, J ) 7.7
Hz), 8.13 (dd, 1H, J ) 2.4, 1.6 Hz), 7.93 (dd, 1H, J ) 7.9 Hz),
7.70 (d, 1H, J ) 8.2 Hz), 7.54 (td, 1H, J ) 7.8, 1.0 Hz), 7.49-
7.44 (m, 4H), 7.41-7.28 (m, 4H), 5.17 (s, 2H); 13C NMR (CDCl3)
δ 180.1, 158.8, 157.1, 150.4, 140.7, 136.4, 136.2, 129.7, 128.6,
128.4, 128.1, 127.6, 125.7 , 124.1, 122.4, 121.7, 116.0, 111.8, 70.3;
IR (KBr) 3030, 2861, 1648, 1604; Anal. (C21H15NO3) C, H, N.
General Procedure for the Deprotection of 6a, 6b, and 7 to
Prepare 9a, 9b, and 10. Benzo[d]oxazol-2-yl(3-hydroxyphenyl)-
methanone (9a). Compound 6a (5.0 g, 15.2 mmol, 100 mol %)
was stirred in a solution of boron trifluoride diethyl etherate (6.9
mL, 55 mmol, 360 mol %) and dimethylsulfide (10 mL, 136 mmol,
900 mol %) in dry CH2Cl2 (100 mL) at room temperature for 72 h.
The mixture was then quenched with H2O (120 mL) and diluted
with CH2Cl2 (350 mL). The organic phase was washed with brine
(2 × 100 mL), dried over anhydrous Na2SO4, filtered, and
evaporated to yield a crude product as a red solid, which was
recrystallized (EtOAc/hexane) to yield 9a (3.06 g, 84%) as light
yellow crystals: mp 125-128 °C; Rf (50% EtOAc/hexane) 0.61;
1H NMR (CDCl3) δ 8.17 (ddd, 1H, J ) 7.8, 1.6, 1.0 Hz), 8.04 (dd,
1H, J ) 2.4, 1.6 Hz), 7.94 (ddd, 1H, J ) 8.0, 1.3, 0.7 Hz), 7.69
(dt, 1H, J ) 8.2, 0.9 Hz), 7.57-7.52 (m, 1H), 7.48-7.41 (m, 2H),
7.19 (ddd, 1H, J ) 8.1, 2.7, 0.9 Hz), 5.74 (s, 1H); 13C NMR (CDCl3)
δ 180.2, 157.1, 156.0, 150.4, 140.6, 136.2, 130.0, 128.5, 125.8,
123.7, 122.3, 121.9, 117.2, 111.9; IR (KBr) 3463, 1651, 1593, 1525;
Anal. (C14H9NO3) C, H, N.
N-(3-(Benzo[d]oxazole-2-carbonyl)phenyl)cyclopentanecar-
boxamide (28). To a solution of 8 (71 mg, 0.30 mmol, 100 mol
%) in dry CH2Cl2 (3.2 mL) was added cyclopentanoyl chloride (54
mg, 0.41 mmol, 136 mol %). An immediate reaction formed a thick
suspension that was quenched with ice and partitioned between
CH2Cl2 (20 mL) and water (10 mL). The organic layer was washed
with satd NaHCO3 (2 × 10 mL) and brine (10 mL), dried over
Na2SO4, and evaporated. Recrystallization from 30% EtOAc in
hexane yielded 28 (61 mg, 61%) as white crystals: mp 180-182
°C; Rf (50% EtOAc in hexane) 0.51; 1H NMR (DMSO-d6) δ 10.20
(s, 1H), 8.56 (t, 1H, J ) 1.8 Hz), 8.22-8.18 (m, 1H), 8.07-8.02
(m, 2H), 7.97-7.94 (m, 1H), 7.69-7.64 (m, 1H), 7.59-7.54 (m,
2H), 2.83 (m, 1H), 1.88 (m, 2H), 1.73 (m, 4H), 1.57 (m, 2H); 13
C
NMR (DMSO-d6) δ 179.9, 174.8. 156.9, 149.8, 140.2, 139.8, 135.2,
129.0, 128.7, 126.0, 125.7, 124.7, 122.1, 120.4, 112.0, 45.3, 30.1,
25.7; IR (KBr) 3289, 2960, 1656, 1603, 1536; Anal. (C20H18N2O3)
C, H, N.
1-(3-(Benzo[d]oxazole-2-carbonyl)phenyl)-3-cyclopentyl-
urea (29). To a solution of 8 (71 mg, 0.30 mmol, 100 mol %) in
dry CH2Cl2 (3.2 mL) was added cyclopentyl isocyanate (88 µL,
0.76 mmol, 250 mol %). After stirring at room temperature for 16
h, another portion of cyclopentyl isocyanate (88 µL, 0.76 mmol,
250 mol %) was added, and the mixture was refluxed for another
24 h. The mixture was diluted with EtOAc (10 mL), washed with
H2O (5 mL) and brine (5 mL), dried over Na2SO4, filtered, and
evaporated. Purification with flash chromatography (2% MeOH in
CH2Cl2) and recrystallization from MeOH (5 mL) yielded 29 (58
mg, 55%) as off-white crystals: mp 216-217 °C; Rf (5% MeOH
in CH2Cl2) 0.35; 1H NMR (DMSO-d6) δ 8.62 (s, 1H), 8.34 (t, 1H,
J ) 1.9 Hz), 8.06-8.00 (m, 2H), 7.95 (app d, 1H, J ) 8.2 Hz),
7.84-7.80 (m, 1H), 7.69-7.64 (m, 1H), 7.49 (t, 1H, J ) 7.9 Hz),
6.25 (d, 1H, J ) 7.2 Hz), 3.96 (sext, 1H, J ) 6.7 Hz), 1.90-1.81
(m, 2H), 1.70-1.49 (m, 4H), 1.43-1.34 (m, 2H); 13C NMR
(DMSO-d6) δ 180.1, 157.0, 154.7, 149.8, 140.9, 140.1, 135.2, 129.0,
128.6, 125.9, 123.6, 123.3, 122.1, 118.9, 112.0, 50.9, 32.8, 23.1;
IR (KBr) 3335, 2956, 2925, 2857, 1666, 1646, 1626, 1564; Anal.
(C20H19N3O3) C, H, N.
Benzo[d]oxazol-2-yl(4-hydroxyphenyl)methanone (9b). Com-
pound 6b (510 mg, 1.55 mmol, 100 mol %) and N-butylpyridinium
bromide (820 mg, 3.8 mmol, 245 mol %) were added to a 10 mL
CEM reaction tube, closed with a septum, and irradiated with CEM
microwave apparatus for 30 s (power 125 W, Tmax ) 125 °C). The
reaction mixture was dissolved in EtOAc (70 mL), washed with
water (50 mL) and brine (50 mL), dried over Na2SO4, filtered, and
evaporated. The yellow solid was then purified with flash chro-
matography (50% EtOAc in hexane) and recrystallization (EtOAc/
hexane) giving 9b (310 mg, 86%) as a yellow solid: mp 188-189
°C; Rf (50% EtOAc in hexane) 0.60; 1H NMR (DMSO-d6) δ 10.80
(s, 1H), 8.45-8.42 (m, 2H), 8.04 (d, 1H, J ) 7.9 Hz), 7.93 (d, 1H,
J ) 8.2 Hz), 7.66-7.62 (m, 1H), 7.57-7.53 (m, 1H), 7.02-6.99
(m, 2H); 13C NMR (DMSO-d6) δ 177.9, 163.6, 157.2, 149.7, 140.1,
133.6, 128.3, 126.1, 125.8, 121.9, 115.5, 111.9; IR (KBr) 3254,
3050, 1599, 1579; Anal. (C14H9NO3) C, H, N.
General Procedure for the Preparation of Phenolic Carbam-
ates 11-26, 33, and 34 from 9a, 9b, and 10. 3-(Benzo[d]oxazole-
2-carbonyl)phenyl ethylcarbamate (11). To a solution of benzo-
[d]oxazol-2-yl(3-hydroxyphenyl)methanone (100 mg, 0.42 mmol,
100 mol %) in dry toluene (4 mL) were added triethylamine (60
µL, 0.42 mmol, 100 mol %) and ethyl isocyanate (166 µL, 2.1
mmol, 500 mol %). After stirring at room temperature for 12 h,
the reaction was complete, as judged by TLC (5% Et2O in
CH2Cl2), and the mixture was diluted with EtOAc (8 mL), filtered
through a pad of silica gel, and evaporated to dryness. Recrystal-
Cyclopentyl 3-(Benzo[d]oxazole-2-carbonyl)phenylcarbamate
(30). A solution of triphosgene (353 mg, 1.19 mmol, 435 mol %),
cyclopentanol (330 µL, 3.60 mmol, 1300 mol %), and pyridine
(250 µL, 3.09 mmol, 1100 mol %) in dry CH2Cl2 (1.2 mL) was
stirred in an ice bath for 2 h. Compound 8 (65 mg, 0.273 mmol,
100 mol %) dissolved in dry CH2Cl2 (4 mL) was added to the
mixture. The mixture was stirred for an hour and quenched with

4240 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Myllyma¨ki et al.
ice and extracted with CH2Cl2 (10 mL). The organic phase was
washed with water (5 mL) and brine (5 mL), dried over Na2SO4,
filtered, and evaporated. Recrystallization with EtOAc/hexane
yielded 30 (50 mg, 52%) as off-white solid: mp 140-141 °C; Rf
at 37 °C (60 µL). At the 10 min time point, arachidonoylethano-
lamide was added so that its final concentration was 2 µM
(containing 50 × 10-3 µCi of 60 Ci/mmol [3H]AEA) and the final
incubation volume was 100 µL. The incubations proceeded for 10
min at 37 °C. Ethyl acetate (400 µL) was added at the 20 min time
point to stop the enzymatic reaction. Additionally, 100 µL of
unlabeled ethanolamine (1 mM) was added as a “carrier” for
radioactive ethanolamine. Samples were centrifuged at 16 000 g
for 4 min at room temperature, and aliquots (100 µL) from aqueous
phase containing [ethanolamine 1-3H] were measured for radio-
activity by liquid scintillation counting (Wallac 1450 MicroBeta;
Wallac Oy, Finland).
1
(35% EtOAc in hexane) 0.49; H NMR (CDCl3) δ 8.39 (t, 1H, J
) 1.9 Hz), 8.28-8.24 (m, 1H), 7.98-7.89 (m, 2H), 7.72 (d, 1H, J
) 8.2 Hz), 7.59-7.45 (m, 3H), 6.76 (br s, 1H), 5.27-5.21 (m,
1H), 1.96-1.86 (m, 2H), 1.84-1.70 (m, 4H), 1.68-1.58 (m, 2H);
13C NMR (CDCl3) δ 180.0, 157.0, 153.3, 150.4, 140.7, 138.6, 135.6,
129.5, 128.5, 125.8, 125.7, 124.3, 122.4, 120.4, 111.9, 78.5, 32.8,
23.6; IR (KBr) 3303, 2961, 1701, 1663, 1591, 1528; Anal.
(C20H18N2O4) C, H, N.
General Procedure for the Preparation of Phenolic Carbon-
ates from 9 and 10. 3-(Benzo[d]oxazole-2-carbonyl)phenyl Ethyl
Carbonate (31a). To a solution of compound 9 (100 mg, 0.42
mmol, 100 mol %) in dry toluene (4 mL) was added triethylamine
(70 µL, 0.50 mmol, 120 mol %) and ethyl chloroformate (48 µL,
0.50 mmol, 120 mol %). The yellow color of the mixture
disappeared immediately after the addition of chloroformate. After
stirring at room temperature for 5 min, the reaction was complete
(TLC, 5% Et2O in CH2Cl2), and the mixture was evaporated to
dryness. Recrystallization from EtOAc/hexane yielded 31a (113
mg, 86%) as a white solid: mp 116-117 °C; Rf (5% Et2O in
CH2Cl2) 0.50; 1H NMR (CDCl3) δ 8.51 (dt, 1H, J ) 7.9, 1.4 Hz),
8.44 (t, 1H, J ) 1.8 Hz), 7.96 (d, 1H, J ) 8.1 Hz), 7.72 (d, 1H, J
) 8.2 Hz), 7.62-7.46 (m, 4H), 4.46 (q, 2H, J ) 7.1 Hz), 1.42
(t, 3H, J ) 7.1 Hz); 13C NMR (CDCl3) δ 179.1, 156.8, 153.3,
151.2, 150.5, 140.7, 136.3, 129.7, 128.6, 128.6, 127.1, 125.8, 123.6,
122.5, 111.9, 65.2, 14.2; IR (KBr) 1762, 1667, 1582, 1526; Anal.
(C17H13NO5) C, H, N.
In Vitro Assay for MGL Activity. The assay for MGL has
been described previously.46 Briefly, experiments were carried out
with preincubations (80 µL, 30 min at 25 °C) containing 10 µg
membrane protein, 44 mM Tris-HCl (pH 7.4), 0.9 mM EDTA, 0.5%
(wt/vol) BSA, and 1.25% (vol/vol) DMSO as a solvent for
inhibitors. The preincubated membranes were kept at 0 °C just prior
to the experiments. The incubations (90 min at 25 °C) were initiated
by adding 40 µL of preincubated membrane cocktail, in a final
volume of 400 µL. The final volume contained 5 µg membrane
protein, 54 mM Tris-HCl (pH 7.4), 1.1 mM EDTA, 100 mM NaCl,
5 mM MgCl2, 0.5% (wt/vol) BSA, and 50 µM of 1. At time points
of 0 and 90 min, 100 µL samples were removed from the
incubation, acetonitrile (200 µL) was added to stop the enzymatic
reaction, and the pH of the samples was simultaneously decreased
to 3.0 with phosphoric acid (added to acetonitrile) to stabilize
compound 1 against acyl migration to 1(3)-AG. Samples were
centrifuged at 23 700 g for 4 min at room temperature prior to
HPLC analysis of the supernatant.
Animals and Preparation of Rat Brain Homogenate for
FAAH Assay. Eight-week-old male Wistar rats were used in these
studies. All animal experiments were approved by the local ethics
committee. The animals lived in a 12 h light/12 h dark cycle (lights
on at 0700 h), with water and food available ad libitum.
The rats were decapitated, and whole brains minus cerebellum
were dissected and homogenized in one volume (v/w) of ice-cold
0.1 M potassium phosphate buffer (pH 7.4) with a Potter-Elvehjem
homogenizer (Heidolph). The homogenate was centrifuged at
10 000 g for 20 min at 4 °C, and the resulting supernatant was
used as a source of FAAH activity. The protein concentration of
the supernatant (7.2 mg/mL) was determined by the method of
Bradford with BSA as a standard.42 Aliquots of the supernatant
were stored at -80 °C until use.
Animals and Preparation of Rat Cerebellar Membranes for
MGL Assay. Four-week-old male Wistar rats were used in these
studies. All animal experiments were approved by the local ethics
committee. The animals lived in a 12 h light/12 h dark cycle (lights
on at 0700 h), with water and food available ad libitum. The rats
were decapitated, 8 h after lights on (1500 h), whole brains were
removed, dipped in isopentane on dry ice, and stored at -80 °C.
Membranes were prepared as previously described.43-45
HPLC Method. The analytical HPLC was performed as previ-
ously described.15 Briefly, the analytical HPLC system consisted
of a Merck Hitachi (Hitachi Ltd., Tokyo, Japan) L-7100 pump,
D-7000 interface module, L-7455 diode-array UV detector (190-
800 nm, set at 211 nm), and L-7250 programmable autosampler.
The separations were accomplished on a Zorbax SB-C18 endcapped
reversed-phase precolumn (4.6 × 12.5 mm, 5 µm) and column (4.6
× 150 mm, 5 µm; Agilent). The injection volume was 50 µL. A
mobile phase mixture of 28% phosphate buffer (30 mM, pH 3.0)
in acetonitrile was used at a flow rate of 2.0 mL min-1. Retention
times were 5.8 min for 1, 6.3 min for 1(3)-AG, and 10.2 min for
arachidonic acid. The relative concentrations of 1, 1(3)-AG, and
arachidonic acid were determined by the corresponding peak areas.
This was justified by the equivalence of response factors for the
studied compounds and was supported by the observation that the
sum of the peak areas was constant throughout the experiments.
Data Analyses. The results from the enzyme inhibition experi-
ments are presented as mean ( 95% confidence intervals of at least
three independent experiments performed in duplicate. Data analyses
for the dose-response curves were calculated as nonlinear regres-
sions using GraphPad Prism 4.0 for Windows.
Briefly, cerebella (minus brain stem) from eight animals were
weighed and homogenized in nine volumes of ice-cold 0.32 M
sucrose with a glass Teflon homogenizer. The crude homogenate
was centrifuged at low speed (1000 × g for 10 min at 4 °C) and
the pellet was discharged. The supernatant was centrifuged at high
speed (100 000 × g for 10 min at 4 °C). The pellet was resuspended
in ice-cold deionized water and washed twice, repeating the high-
speed centrifugation. Finally, membranes were resuspended in 50
mM Tris-HCl, pH 7.4, with 1 mM EDTA and aliquoted for storage
at -80 °C. The protein concentration of the final preparation,
Acknowledgment. The study was supported by a grant from
the National Technology Agency of Finland and Academy of
Finland (Grant No. 107300).
Supporting Information Available: Spectroscopic character-
ization and elemental analysis data for all novel compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
measured by the Bradford method,42 was 11 mg mL-1
.
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