Synthesis and evaluation of paracetamol esters as novel fatty acid amide hydrolase inhibitors

Article abstract of DOI:10.1021/jm901891p

Fatty acid amide hydrolase (FAAH) is the key hydrolytic enzyme for the endogenous cannabinoid receptor ligand anandamide. The synthesis and evaluation for their FAAH inhibitory activities of a series of 18 paracetamol esters are described. Structure-activity relationship studies indicated that the ester (33) with a 2-(4-(2-(trifluoromethyl)pyridin-4-ylamino)phenyl)acetic acid substituent was the most potent analogue in this series. The compound inhibited FAAH activity in a competitive manner with a K_i value of 0.16 μM. The compound was also able to inhibit the FAAH activity in rat basophilic leukemia cells as assessed by measuring either the hydrolysis of anandamide, the FAAH-dependent cellular accumulation of anandamide, or the FAAH-dependent recycling of tritium to the cell membranes. The compound also inhibited the activity of monoacylglycerol lipase (MGL), the enzyme responsible for the hydrolysis of the endogenous cannabinoid receptor ligand 2-arachidonoylglycerol, with an IC₅₀ value of 1.9 μM. It is concluded that the compound may be a useful template for the design of potent novel inhibitors of FAAH.

Full text of DOI:10.1021/jm901891p

2286 J. Med. Chem. 2010, 53, 2286–2298
DOI: 10.1021/jm901891p
Synthesis and Evaluation of Paracetamol Esters As Novel Fatty Acid Amide Hydrolase Inhibitors
,†
Valentina Onnis,* Cenzo Congiu, Emmelie Bjorklund, Franziska Hempel, Emma Soderstrom, and Christopher J. Fowler
†
‡
‡
‡
‡
€
€
€
†Department of Toxicology, Unit of Medicinal Chemistry, University of Cagliari, via Ospedale 72, Cagliari I-09124, Italy, and
‡Department of Pharmacology and Clinical Neuroscience, Umea University, SE-901 87 Umea, Sweden
Received December 22, 2009
Fatty acid amide hydrolase (FAAH) is the key hydrolytic enzyme for the endogenous cannabinoid receptor
ligand anandamide. The synthesis and evaluation for their FAAH inhibitory activities of a series of 18
paracetamol esters are described. Structure-activity relationship studies indicated that the ester (33) with a
2-(4-(2-(trifluoromethyl)pyridin-4-ylamino)phenyl)acetic acid substituent was the most potent analogue in
this series. The compound inhibited FAAH activity in a competitive manner with a Ki value of 0.16 μM. The
compound was also able to inhibit the FAAH activity in rat basophilic leukemia cells as assessed by
measuring either the hydrolysis of anandamide, the FAAH-dependent cellular accumulation of ananda-
mide, or the FAAH-dependent recycling of tritium to the cell membranes. The compound also inhibited the
activity of monoacylglycerol lipase (MGL), the enzyme responsible for the hydrolysis of the endogenous
cannabinoid receptor ligand 2-arachidonoylglycerol, with an IC50 value of 1.9 μM. It is concluded that the
compound may be a useful template for the design of potent novel inhibitors of FAAH.
Introduction
can be used as a template for the design of more potent
compounds. In this respect, we have reported an analogue
of ibuprofen, Ibu-am5 (N-(3-methylpyridin-2-yl)-2-(40-iso-
butylphenyl)propionamide), that retaines the cyclooxygenase
(COX) inhibitory properties of ibuprofen but is a more potent
FAAH inhibitor.45
Fatty acid amide hydrolase (FAAHa)1,2 is a membrane-
bound serine hydrolase that catalyzes the deactivating hydro-
lysis of the fatty acid ethanolamide family of signaling lipids,3,4
which includes endogenous ligands for cannabinoid receptors
such as arachidonoylethanolamide (anandamide, AEA)5 and
peroxisome proliferator-activated receptors R (oleoylethanol-
amide6,7 and palmitoylethanolamide8,9).
Endogenous FAAH substrates such as AEA serve key regu-
latory functions in the body and have been implicated in a
variety of pathological conditions including pain,10-12 inflam-
mation,13 sleep disorders,14,15 anxiety, depression, and vascular
hypertension,16-18 and there has been an increasing interest in
the development of inhibitors of this enzyme.19-25 Different
structural classes of FAAH inhibitors have been reported
including R-ketoheterocycles,26-33 (thio)hydantoins,34 piperi-
dine/piperazine ureas,35 and carbamate derivatives.34-42 When
tested, these compounds have been shown to efficacious in
models of inflammatory, visceral, and in some cases neuro-
pathic pain without producing the central effects seen with
directly acting cannabinoid receptor agonists.20,21,36
The contribution of the endocannabinoid system to para-
cetamol (acetaminophen) analgesia has been a subject of parti-
cular speculation. Paracetamol itself does not inhibit FAAH.43
However, Hoegestaett et al.46 recently described that parace-
tamol undergoes a FAAH-dependent two-step metabolic
transformation in the brain, liver, and spinal cord to form the
bioactive N-acylphenolamine AM404 (N-(4-hydroxyphenyl)-
5Z,8Z,11Z,14Z-eicosatetraenamide). This compound inhibits
FAAH by acting as a competing substrate, inhibits COX,
affects the cellular accumulation of AEA, interacts with both
CB1 and TRPV1 (vanilloid) receptors, and has analgesic effects
in vivo.46-51 Three independent studies have shown that block-
ade of CB1 receptors affects the antinociceptive properties of
paracetamol,52-54 suggesting that this metabolic pathway can
at least contribute to the pharmacological properties of para-
cetamol. This may also be true of the paracetamol analogue
N-(4-hydroxybenzyl)acetamide), which shows analgesic pro-
perties in vivo in the formalin test of persistent pain but does not
inhibit FAAH or interact with CB receptors in vitro, while
its equivalent arachidonoyl analogue is more potent than
AM404 at these targets.55 AM404 derivatives in which the
4-aminophenol moiety has been replaced by NSAIDs or in
which mefenamic and flufenamic acid served as arachidonic
acid mimetics were capable of interacting with TRPV1 and
retained the COX inhibition properties of the parent NSAIDs.56
Furthermore, FAAH inhibitors targeting also other proteins
involved in pain tranduction migth be more efficacious than selec-
tive FAAH inibitors, as shown by the case of N-arachidonyl-
serotonin, a dual FAAH/TRPV1 inhibitor, that has been
reported to be efficacious against pain and anxiety.57,58
An intriguing aspect of FAAH inhibition is that some
currently marketed nonsteroidal anti-inflammatory drugs
(NSAIDs) have also been shown to inhibit FAAH. The
NSAID ibuprofen is a weak inhibitor of FAAH,43,44 but
*To whom correspondence should be addressed: Phone:
þ390706758632. Fax: þ390706758612. E-mail: vonnis@unica.it.
a Abbreviations: AA-maleimide, N-arachidonoylmaleimide; AEA, ana-
ndamide; AM404, N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraena-
mide; CB, cannabinoid; COX, cyclooxygenase; DMF-DMA, N,N-
dimethylformamide dimethylacetal; EDC, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride; FAAH, fatty acid amide hydrolase;
HOBt, hydroxybenzotriazole; Ibu-am5, (N-(3-methylpyridin-2-yl)-2-(40-
isobutylphenyl)propionamide); MGL monoacylglycerol lipase; NSAIDs,
nonsteroidal anti-inflammatory drugs; OTMK, oleoyl trifluoromethyl
ketone; SAR structure-activity relationship; URB597, 30-carbamoyl-bi-
phenyl-3-yl-cyclohexylcarbamate.
r
pubs.acs.org/jmc
Published on Web 02/09/2010
2010 American Chemical Society

Article
The findings above that paracetamol (indirectly) and
NSAIDs (directly) interact with FAAH raise the possibility
that compounds containing elements of both structures may
be a new source of FAAH inhibitors. In the present study, we
report the synthesis and characterization of a novel series of
paracetamol esters, designed as inhibitors of FAAH by
progressive modification of the structure of 4-acetamidophenyl
2-(2-(trifluoromethyl)pyridin-4-ylamino)benzoate (21).
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2287
hydrolysis of compounds 5a-i produced the required acids
6a-i.
A chemistry similar to that described in the synthesis of
acids 6a-h was used for the preparation of acid 11. As
reported in Scheme 3, ester 7 was sequentially treated with
compound 2, then with DMF-DMA, NH4OAc, and succes-
sively hydrolyzed to 11.
Carboxylic acids 17-20 were obtained by reaction of ethyl
glycinate hydrochloride (12) with the appropriate acid 6e-h
by EDC method and subsequent hydrolysis of esters 13-16
(Scheme 4).
Chemistry
The synthesis of paracetamol esters was performed by
condensation of paracetamol with the appropriate acid, using
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC), in the presence of hydroxybenzotriazole (HOBt) in
acetonitrile (MeCN) solution. (Scheme 1). This method was
found to be clean and high yielding.
The bulk of the syntheses required acids 6a-i that were not
commercially available. These were synthesized using our
previously described procedure that allows the construction
of the trifluoromethylpyridine moiety linked by an amino
nitrogen to an aromatic ring.59 According to Scheme 2, methyl
esters 1a-i were reacted with trifluoroacetylvinyl ether 2, in a
1.5 molar ratio, in boiling MeCN solution affording high yields
of intermediates 3a-i. Treatment of 3a-i with an excess
of N,N-dimethylformamide dimethylacetal (DMF-DMA) in
refluxing toluene provided 1,1,1-trifluorohexadienones 4a-i.
Intermediates 4a-i readily underwent pyridine ring closure
to give compounds 5a-i upon treatment with ammonium
acetate (NH4OAc) in hot DMF. Sodium hydroxide catalyzed
Biology
Compounds 21-38, along with their parent compound
paracetamol, were evaluated for their ability to inhibit [3H]-
AEA hydrolysis in rat brain homogenates. In all cases re-
ported here, concentration-response curves were constructed
from values from at least three experiments using at least five
concentrations over the appropriate range (in half log units, i.
e., 0.3, 1, 3, 10, 30, 100 μM) (for examples, see Figure 1), with
the exception of (R,S)-ibuprofen, where four concentrations
(30, 100, 300, and 500 μM) were used. The effects of para-
cetamol esters21-38uponthe FAAH-catalyzed hydrolysis of
AEA by rat brain membranes at an assay pH of 7.4 are shown
in Tables 1 and 2. Experiments investigating FAAH activityin
intact cells and effects upon MGL and COX were also
performed for selected compounds.
Results and Discussion
Although paracetamol was devoid of FAAH inhibitory
activity (IC50 value >300 μM), a result consistent with the
literature,54,55 its conversion into esters resulted in generation
of FAAH inhibitors. To address this issue, an SAR study on
paracetamol esters, focused on aromatic nucleus variation,
was systematically undertaken. Different groups on the
anthranilic acid scaffold were first investigated (Table 1). In
some cases, a complete inhibition of FAAH was not seen.
This was usually the case for the weaker compounds and
Scheme 1. Synthesis of Target Paracetamol Esters 21-37a
a Reagents and conditions: (i) EDC, HOBt, MeCN, rt, 24 h.
Scheme 2. Synthesis of Intermediates Compounds 3-6a
a Reagents and conditions: (i) MeCN, reflux, 2 h; (ii) DMF-DMA, PhMe, reflux, 1 h; (iii) NH4OAc, DMF, reflux, 1.5 h; (iv) 10% aq NaOH, reflux, 30 min.

2288 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Scheme 3. Synthesis of Intermediates Compounds 8-11a
Onnis et al.
a Reagents and conditions: (i), (ii), (iii), (iv) steps (i), (ii), (iii), (iv) in Scheme 2.
Scheme 4. Synthesis of Intermediates Compounds 13-20a
a Reagents and conditions: (i) EDC, HOBt, TEA, MeCN, rt, 4 h; (ii) 50% aq NaOH, rt, 24 h.
were kept in para for further SAR studies. Next, connect-
ing patterns of these groups (Table 2) were evaluated. Intro-
duction of an extra methylene linker between phenyl ring and
trifluoromethylpyridine-amino group, such as in compound
32, led to drop in potency (IC50 22 μM). We observed the
opposite effect by introduction of an extra methylene linker
between phenyl ring and ester moiety (ester 31, IC50 0.18 μM)
that caused an increase in activity with respect to 29. Although
less potent when compared to 31, ester 38 bearing an ethylene
linker between phenyl ring and ester moiety (IC50 1.5 μM)
showed increased activity as compared with 29. Branching the
methylene linker with a methyl produced further increase of
activity (ester 33, IC50 0.10 μM). This compound contains
structural elements of ibuprofen and Ibu-am5, and so data
for these compounds are included in Table 2 for comparative
purposes.
Figure 1. Inhibition of FAAH-catalyzed hydrolysis of 0.5 μM
[3H]AEA by rat brain homogenates by compounds 6g, 6h, 31, and
33. Data are means and SEM (when not enclosed by the symbols),
n = 3-15.
On the most active compounds introduction of an extra
three atom linker was evaluated. Even though derivatives 34
and 35 were slightly stronger than the parent 29 and 30, in the
case of 31 and 33, the same modification led to weaker
compounds 36 and 37. The substitution of the phenyl ring
was also probed. Methylated derivatives 30 and 35 have
essentially the same potency as that of 29.
The stability of ester derivatives 31 and 33 was investigated
both toward acid (0.1 M aq trifluoroacetic acid) and base (0.1
M aq NaOH) hydrolysis. After 48 h at room temperature, the
formation of the corresponding acids 6g and 6h was moni-
tored by TLC (ethyl acetate/n-hexane 1:1). In all cases,
examination of TLC plates revealed a single spot with rf
identical to starting compound. Thus, these paracetamol
esters do not appear to be hydrolytically labile. Furthermore,
acids 6g and 6h do not inhibit FAAH (IC50 values >100 μM)
(Figure 1), confirming that it is the paracetamol esters rather
than the theoretical hydrolysis products that inhibit FAAH.
pH Sensitivity of [3H]-AEA Hydrolysis by Compounds 22,
33, and 34. The FAAH inhibitory effect of acidic NSAIDs is
sensitive to the assay pH used. Thus (R,S)-ibuprofen was
more potent at pH 6 than at pH 8.45 Rat brain membranes in
assay buffer of pH 6 or pH 9 were preincubated with 22, 33,
and 34 or (R,S)-ibuprofen prior to addition of [3H]-AEA and
presumably reflects a solubility issue with these lipophilic
compounds (for discussion, see ref 60). However, the IC50
values here are comparable because they reflect the inhibitable
component of the FAAH activity.
It was found that antranoyl ester (21) inhibited FAAH with
an IC50 value of 64 μM. While the introduction of 5-Me (23) or
4-Cl (24) substituent resulted in about 2-fold increased activity,
it was a chorine atom in 5-position (22) that produced a
significant improvement in activity (IC50 4.3 μM). Replace-
ment of trifluoromethylpyridine mojety of 21 with a trifluoro-
methylbenzene to give the flufenamic acid ester (25) also led
to significant enhancement in potency (IC50 8.7 μM). In
contrast, the substitution of benzene ring of 21 with a pyridine
(ester 26, IC50 58 μM) did not change the activity with respect
to ester 21. Replacement of trifluoromethylpyridine mojety of
26 with a trifluoromethylbenzene to give the niflumic acid ester
(27) resulted in a loss of activity. An important finding arising
from the comparison of the IC50 values for esters 21, 28, and 29
was that going from ortho- (IC50 64 μM) to meta- (IC50 47 μM)
to para-substituted (IC50 2.7 μM) analogues resulted in gradual
increase of potencies. This fact indicated potential favorable
interaction of the para-substituted ester 29 with the enzyme.
Therefore, ester and trifluoromethylpyridine-amino groups

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2289
Table 1. IC50 Values for Inhibition of Rat Brain AEA Hydrolysis by Compounds 21-29 and Paracetamol
a pI50 values (i.e., -log10(IC50) values) are indicated because the analysis method returns these ( SE values, which are then antilogged to give the IC50
values for the inhibitable component (max inhibition) of the FAAH activity.

2290 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Onnis et al.
Table 2. IC50 Values for Inhibition of Rat Brain AEA Hydrolysis by Compounds 30-37, Ibuprofen, and Ibu-am5
incubation for 10 min at 37 °C. As expected, ibuprofen was
more potent at pH 6 than at pH 9: at pH 9, the IC50 value was
greater than the highest concentration tested (500 μM),
whereas at pH 6, the lowest concentration tested (100 μM),
produced 71 ( 2% inhibition (Figure 2). At pH 7.4, (R,S)-
ibuprofen inhibited [3H]AEA hydrolysis with an IC50 value
of 240 μM (data not shown). The ionization properties of
esters 22, 33, and 34 are very reduced as compared to acidic
compounds such as ibuprofen. Consistent with this, these
compounds showed little pH dependency, and if anything,
the potencies were lower at pH 6 than pH 9 (see Figure 2 for
data with 22, 33, and 34).

Article
Mode of Inhibition of FAAH by 31 and 33. To determine
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2291
similar to that seen with 30 μM of Ibu-am5 (Figure 4A), which is
consistent with the relative potencies of these compounds in cell
free homogenates (Table 2 and ref 45). However, the blank
values seen in this assay were high, and so two indirect models
were used to corroborate the finding of Figure 4A, namely
FAAH-driven accumulation and membrane processing of tri-
tium in RBL2H3 cells incubated with AEA labeled in the
arachidonate part of the molecule. In RBL2H3 cells, FAAH
regulates the extra-intracellular concentration of AEA by me-
tabolizing the intracellularly accumulated AEA. In conse-
quence, inhibition of FAAH reduces the observed rate of
uptake.61,62 This is shown in Figure 4B, where 1 μM of the
selective FAAH inhibitor 30-carbamoyl-biphenyl-3-yl-cyclo-
hexylcarbamate (URB597) reduces the uptake of AEA by
about 2/3. Compound 33 behaved in a similar manner, consis-
tent with its FAAH inhibitory properties. In RBL2H3 cells,
arachidonic acid produced as a result of the FAAH-catalyzed
hydrolysis of AEA is recycled back to the plasma membrane.63
Measurement of membrane tritium accumulation following
incubation of RBL2H3 cells with [3H]AEA is thus a simple
way of assessing the FAAH inhibitory properties of com-
pounds.64 As expected, URB597 almost completely blocked
the accumulation of membrane tritium, whereas the monoacyl-
glycerol lipase (MGL)-selective 4-nitrophenyl-4-(dibenzo[d]-
[1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate65 39
(JZL184) was without effect (Figure 4C). Compound 33 pro-
duced a concentration-dependent inhibition of the tritium
accumulation (Figure 4C). Thus, in all three models, 33 pro-
duces effects consistent with an inhibition of FAAH in intact
RBL2H3 cells.
the likely mechanism of the inhibition, a time dependent
inhibition study was performed using as reference com-
pounds 31 and 33. In theory, if a compound inhibits the
enzyme via an irreversible mechanism, upon prolonged
preincubation the potency should become greater; a constant
IC50, conversely, supports a reversible mechanism. Com-
pounds 31 and 33 showed no time-dependent improvement
of potency, arguing against an irreversible mechanism of
action (Figure 3). The kinetics of inhibition were also
investigated. The inhibition was essentially competitive in
nature, with Ki values calculated from slope replots of
0.19 μM and 0.16 μM for 31 and 33, respectively (Figure 3).
FAAH Inhibition in Intact Cells. The effects of 33 were
investigated in RBL2H3 rat basophilic leukemia cells
(Figure 4). Initially, cells were incubated with 100 nM
[3H]AEA (labeled in the ethanolamine part of the molecule)
and the tritium recovered in the aqueous phase after metha-
nol and chloroform extraction (corresponding to [3H]etha-
nolamine) was determined. A significant reduction of tritium
recovery was found at 1 and 3 μM of 33, consistent with
inhibition of FAAH. The inhibition seen with 3 μM of 33 was
Inhibition of MGL. MGL is the primary enzyme respon-
sible for the hydrolysis of the endocannabinoid 2-arachido-
noylglycerol in the brain,66,67 and its selective inhibition by
39 produces not only analgesic effects but effects upon body
temperature and locomotion.65 OMDM169, a potent inhi-
bitor of 2-AG hydrolysis, has been reported to enhance 2-
AG levels and to exert analgesic activity via indirect activa-
tion of cannabinoid receptors.68
Figure 2. Inhibition of FAAH-catalyzed hydrolysis of 0.5 μM
[3H]AEA by rat brain homogenates by (R,S)-ibuprofen and com-
pounds 22, 33, and 34. Data are means, n = 3 for the assay pH
values shown.
Figure 3. Mode of inhibition of rat brain FAAH by 31 (A) and 33 (B). Top left (for each panel): homogenates were preincubated with the
compounds for the times shown prior to addition of 0.5 μM [3H]AEA and assay for FAAH activity (means and SEM, n = 3-4). Bottom (for
app
each panel): AEA hydrolysis at the substrate and inhibitor concentrations shown (means and SEM, n = 3). Secondary slope (Kmapp/Vmax
and intercept (1/Vmaxapp) replots are shown for the mean data at the top right (for each panel).
)

2292 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Onnis et al.
Figure 5. Inhibition of recombinant human MGL by: (A) 31, 33,
37, and as positive controls AA-maleimide and OTMK; (B) para-
cetamol. The compounds were incubated together with enzyme and
substrate (0.25 mM 4-nitrophenyl acetate) for 20 min. Unless
otherwise indicated, a preincbuation phase between inhibitor and
enzyme prior to addition of substrate was not used. Shown are
means and SEM, n = 3-4.
Figure 4. Inhibtion of [3H]AEA (100 nM) hydrolysis in intact
RBL2H3 cells by 33. In (A) (hydrolysis), cells were preincubated
with 33 or Ibu-am5 for 10 min prior to addition of [3H-
ethanolamine]-labeled AEA and incubation for a further 20 min.
In (B) (uptake), cells or wells alone (shaded columns) were pre-
incubated with 33 or Ibu-am5 for 10 min prior to addition of [3H-
arachidonoyl]-labeled AEA and incubation for a further 4 min. In
(C) (membrane recycling) cells were preincubated with 33, URB597,
or 39 for 10 min prior to addition of [3H-arachidonoyl]-labeled AEA
and incubation for a further 5 min. Shown are means and SEM, n =
3-7. *Significantly different (p < 0.05 Dunnett’s multiple compar-
ison test) from the corresponding control (vehicle) value following
significant one-way factorial ANOVA. #p < 0.05 vs the correspond-
ing vehicle value, two-tailed t test.
paracetamol toward recombinant human MGL is rather
weak (20 and 34% inhibition at 300 and 1000 μM, respec-
tively, following 60 min of preincubation, Figure 5B), and it
is in any case unwise to overinterpret in vitro data in terms of
the situation in vivo.
Although the IC50 values for 33 and 31 are about an order
of magnitude higher than seen for inhibition of FAAH, the
MGL and FAAH assays were undertaken in rather different
conditions, so it is unwise to claim a FAAH selectivity for the
compounds. However, the relative potencies of compounds
within a given assay can be compared, and it is noted that the
IC50 values for 33 and 37 relative to 31 are rather similar for
FAAH (1.8 and 7.2, respectively) as for MGL (1.2 and 9.3,
respectively, calculated from the mean pI50 values). Thus, in
this limited series, the structural elements conferring an
increased potency toward FAAH also increase the potency
toward MGL.
Interaction of Compounds 22, 25, 32, and 33 with COX. The
effects of using as reference compounds 22, 25, 32, and 33
upon ovine COX-1 and human COX-2 were assessed using
an oxygen electrode.73 The assay was undertaken in two
ways, either preincubating the enzyme and test compound
for 5 min before starting the reaction with 10 μM arachidonic
acid as substrate (“preincubation method”) or mixing the
test compound and the substrate and starting the assay by
addition of the COX (“non-preincubation method”). Parts
A and B of Figure 6 show the effects of (R)- and (S)-
ibuprofen and 33 upon the activity of COX-1 using the
preincubation method. As expected,73,74 (S)-ibuprofen was
more potent than the (R)-enantiomer (Figure 6A), and a
concentration of 50 μM of (S)-ibuprofen produced a large
The inhibition of MGL by compounds 31, 33, and 37 was
investigated using recombinant human MGL and 4-nitro-
phenyl acetate as substrate.69 (Figure 5A) As positive con-
trols, oleoyltrifluoromethyl ketone (OTMK) and N-
arachidonoylmaleimide (AA-maleimide) were used because
these compounds are known to inhibit MGL.70,71 In a
subsequent experiment, the selective MGL inhibitor 39 was
also tested and found potently to inhibit the hydrolysis of
4-nitrophenylacetate in a time-dependent manner.72 Com-
pounds 31, 33, and 37 all inhibited MGL activity. The IC50
values (with pI50 values and max inhibition attained given in
brackets) were: 31, IC50 2.3 μM (pI50 5.64 ( 0.11, max
inhibition 78 ( 5%); 33, IC50 1.9 μM (pI50 5.73 ( 0.09,
max inhibition 76 ( 4%); 37, IC50 18 μM (pI50 4.74 ( 0.16,
max inhibition 100%). In contrast, indomethacin, Ibu-am5,
and (R,S)-ibuprofen, at concentrations of 100 μM, were
without effect upon the MGL activity (data not shown).
Paracetamol itself produced no inhibition up to a concentra-
tion of 1 mM. However, when the compound was preincu-
bated with MGL for 60 min prior to assay, some inhibition
was seen (Figure 5B). In theory, inhibition of MGL could
explain the CB receptor-mediated analgesic properties of
paracetamol52-54 because the effects of 39 are also blocked
by a CB1 receptor antagonist.65 However, the potency of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2293
Figure 6. The effects of (R)- and (S)-ibuprofen (A) and 33 (B) upon the oxygenation of 10 μM arachidonic acid by ovine COX-1 using the
preincubation assay. In (C), the effect of 33 upon the activity of COX-1 was measured using the nonpreincubation assay. In (D), the effects of
33, (R)- and (S)-ibuprofen, indomethacin, and valdecoxib upon the activity of human recombinant COX-2 using the preincubation assay. Note
that one of the three valdecoxib experiments was conduced using a different control to those shown in the graph. Shown are means and SEM
(when not enclosed by the symbols), n = 3-4.
inhibition of oxygen consumption. In contrast, 33 at a
concentration of 100 and 200 μM (i.e., 2-3 orders of
magnitude higher than required for inhibition of FAAH)
had very little effect upon COX-1 activity. The highest
concentration tested (500 μM) produced a slight lag prior
to the start of oxygen consumption by the enzyme
(Figure 6B). With the nonpreincubation method, similar
results were found for the ibuprofen enantiomers (data not
shown), whereas the slight delay produced by the 500 μM
concentration of 33 before the enzyme started to metabolize
arachidonic acid was more pronounced (Figure 6C). Curve
fitting of the data indicated that the plateau length was 7, 17,
23, and 44 s after addition of enzyme to the assay mixture to
start the reaction at concentrations of 33 of 0, 100, 200, and
500 μM, respectively. This delay was still seen if the non-
preincubation assays were undertaken in the presence of
detergent (0.3 mM Triton X-100) if the COX-1 was premixed
with hematin rather than being added to the hematin solu-
tion or if the assays were undertaken at pH 8.0 rather than at
pH 7.4 (data not shown). Compounds 22, 25, and 32 were
without effect upon COX-1 activity at the concentrations
tested (100, 100, and 500 μM, respectively, nonpreincubation
method, data not shown). Human COX-2 is more expensive
than ovine COX-1, and so the number of experiments under-
taken was limited and the preincubation assay alone was
used. However, under these conditions, the COX-2-selective
inhibitor valdecoxib75 (1 μM) produced a complete blockade
of activity and the ibuprofen enantiomers produced a partial
inhibition at 500 μM. In contrast, 33 was without effect upon
the COX-2 activity at 500 μM (Figure 6D). These data
indicate that 33 has at best minor effects upon COX enzymes
that are only seen at very high concentrations relative to
those needed for FAAH inhibition, which is perhaps not
surprising given the weak potency of paracetamol toward
COX-1 and COX-2.76
Conclusions
In the present study, a series of paracetamol esters with
FAAH inhibitory activity have been described. The most
potent compound, 33, inhibits rat brain FAAH in homo-
genates in an essentially competitive manner with submicro-
molar potency and can inhibit the hydrolysis of AEA by intact
RBL2H3 rat basophilic leukemia cells. The compound, how-
ꢀ
ever, can also inhibit MGL activity, and so selectivity vis a vis
this enzymeisless thanideal. Nevertheless, 33may be useful as
a template for the design of novel reversible FAAH inhibitors
derived from a much used, but incompletely understood,
analgesic agent.
Experimental Section
Chemistry. Unless otherwise noted, all solvents, including
anhydrous solvents and chemicals, were purchased from
Aldrich Co. and/or AlfaAesar, and used without further puri-
fication. Melting points were recorded on a Stuart Scientific

2294 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5
Onnis et al.
melting point SMP1 apparatus and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded at
300 MHz (Varian Unity 300) using DMSO-d6 as solvent and TMS
as the internal standard. Splitting patterns are designated as singlet
(s), doublet (d), triplet (t), quartet (q), and multiplet (m). Infrared
spectra were obtained with a Bruker Vector 22 spectrophotometer.
Elemental analyses were carried out with a Carlo Erba model 1106
elemental analyzer, and all values were within 0.4% of the
calculated values, which indicates >95% purity of the tested
compounds. Analytical thin layer chromatography (TLC) was
carried out on E. Merck TLC plates coated with silica gel 60 F254
(0.25 mm layer thickness). TLC visualization was carried out using
either a UV lamp. N-(2-(Trifluoromethyl) pyridin-4-yl)anthranilic
acid,59 2-(2-(trifluoromethyl)pyridin-4-ylamino)nicotinic acid,59
1a,77 1f,78 1h,79 1i,80 2,81 and 782 were obtained with previously
described procedures.
General Procedure for the Preparation of Paracetamol Esters
21-38. A mixture of the appropriate acid (1 mmol), EDC (0.19
g, 1.1 mmol), and HOBt (0.13 g, 1 mmol) in dry MeCN (10 mL)
was stirred at room temperature for 30 min and then treated
with paracetamol (0.15 g, 1 mmol). The mixture was stirred at
room temperature for an additional 24 h. Then the solution was
evaporated to dryness in vacuo. The residue was dissolved in
ethyl acetate (20 mL) and washed with brine (2ꢀ 5 mL), 5%
aqueous sodium hydroxide (2ꢀ 5 mL), and water (2ꢀ 5 mL).
The organic layer was dried over anhydrous magnesium sulfate.
Concentration of the dried extract yielded a residue, which was
triturated with isopropyl ether. The formed precipitate was
filtered off and purified by crystallization from the adequate
solvent to give the ester derivatives 21-38.
Example: 4-Acetamidophenyl 2-(2-(trifluoromethyl)pyridin-4-
ylamino)benzoate (21). Following the general procedure, the title
compound was obtained from N-(2-(trifluoromethyl) pyridin-4-
yl)anthranilic acid;59 yield 87%; mp 149-150 °C (isopropyl
ether). 1H NMR (DMSO-d6) δ 2.16 (s, 3H), 7.23 (m, 3H), 7.29
(m, 1H), 7.46 (m, 2H), 7.73 (m, 3H), 7.84 (m, 1H), 8.27 (d, J =
5.8, 1H), 8.48 (d, J = 4.2, 1H), 9.55 (s, 1H), 10.15 (s, 1H). IR
(Nujol) 3314, 1692, 1666 cm-1. Anal. (C21H16F3N3O3) C, H, N.
General Procedure for the Synthesis of 3a-i, 8. A mixture of
methyl esters 1a-i, 7 (10 mmol), and enol ether 2 (2.52 g, 15
mmol) in anhydrous MeCN (10 mL) was refluxed for 2 h. After
cooling, the formed precipitate was collected by filtration,
washed with isopropyl ether, dried, and used without further
purification.
1H NMR (DMSO-d6) δ 2.40 (s, 3H), 3.96 (s, 3H), 7.14 (m, 1H),
7.35 (s, 1H), 7.55 (m, 1H), 7.93 (m, 1H), 8.02 (m, 1H), 8.45 (d,
J = 5.8, 1H), 9.04 (s, 1H). IR (nujol) 3328, 1698 cm-1. Anal.
(C15H13F3N2O2) C, H, N.
General Procedure for Synthesis of Acids 6a-i, 11. A mixture
of 5a-i, 10 (1 mmol) in 10% aqueous sodium hydroxide (15 mL)
was refluxed for 30 min, during which a homogeneous solution
was formed. After cooling, the solution was acidified with 20%
aqueous hydrochloric acid to pH 3-4. The formed solid was
filtered off, washed with water, air-dried, and crystallized from
ethanol to give 6a-i, 11.
Example: 5-Methyl-2-(2-(trifluoromethyl)pyridin-4-ylamino)-
benzoic Acid (6a). Yield 94%; mp 248-250 °C. 1H NMR
(DMSO-d6) δ 2.33 (s, 3H), 6.55 (m, 1H), 7.12 (m, 1H), 7.74
(m, 2H), 8.60 (d, J = 5.8 Hz, 1H), 9.83 (s, 1H, NH). IR (nujol)
3239, 2499, 1863, 1681 cm-1. Anal. (C14H11F3N2O2) C, H, N.
General Procedure for the Preparation of Compounds 13-16.
A mixture of the acid 6e-h (2 mmol), EDCI (0.39 g, 2.2 mmol),
and HOBt (0.27 g, 2 mmol) in dry MeCN (10 mL) was stirred at
room temperature. After 30 min, TEA (0.4 mL, 4 mmol) and 12
(0.56 g, 4 mmol) were added. The mixture was stirred at room
temperature for an additional 4 h. Then the solution was
evaporated to dryness in vacuo. The residue was dissolved in
ethyl acetate (20 mL) and washed with brine (2ꢀ 5 mL), 10%
aqueous hydrochloric acid (2ꢀ 5 mL), 5% aqueous sodium
hydroxide (2ꢀ 5 mL), and water (2ꢀ 5 mL). The organic layer
was dried over anhydrous magnesium sulfate. Concentration of
the dried extract yielded a residue, which was triturated with
isopropyl ether. The formed precipitate was filtered off and
purified by crystallization from the adequate solvent to give
derivatives 13-16.
Example: Ethyl 2-(4-(2-(Trifluoromethyl)pyridin-4-ylamino)-
benzamido)acetate (13). Yield 75%; mp 170-172 °C (2-PrOH).
1H NMR (DMSO-d6) δ 1.30 (t, J = 7.8, 3H), 4.12 (s, 2H), 4.16
(q, J = 7.8, 2H), 7.33 (m, 4H), 7.99 (d, J = 8.4 Hz, 2H), 8.44 (d,
J = 5.8 Hz, 1H), 9.05 (s, 1H, NH). IR (nujol) 3360, 3303, 1729,
1636 cm-1. Anal. (C17H16F3N3O3) C, H, N.
General Procedure for the Synthesis of Acids 17-20. To a
solution of 13-16 (1 mmol) in ethanol (10 mL), 50% aqueous
sodium hydroxide (2 mL) and water (2 mL) were added. The
mixture was stirred at room temperature for 24 h. The mixture
was concentrated in vacuo, and then ice was added. Then the
solution was acidified with 20% aqueous hydrochloric acid to
pH 3-4. The formed solid was filtered off, washed with water,
air-dried, and crystallized from ethanol to give 17-20.
Example: 2-(4-(2-(Trifluoromethyl)pyridin-4-ylamino)benz-
amido)acetic Acid (17). Yield 90%; mp 210-212 °C. 1H NMR
(DMSO-d6) δ 4.14 (s, 2H), 6.66 (m, 2H), 7.68 (m, 2H), 7.70 (m,
2H), 8.42 (d, J = 5.4 Hz, 1H), 9.42 (s, 1H), 10.03 (s, 1H). IR
(nujol) 3411, 3278, 3183, 2518, 1895, 1713, 1638 cm-1. Anal.
(C15H12F3N3O3) C, H, N.
Example: (E)-Methyl 5-Methyl-2-(5,5,5-trifluoro-4-oxopent-
2-en-2-ylamino)benzoate (3a). Yield 90%; mp 114-116 °C (n-
hexane). 1H NMR (DMSO-d6) δ 2.28 (s, 3H), 2.40 (s, 3H), 3.97
(s, 3H), 5.80 (s, 1H), 7.52 (d, J = 8.1, 1H), 7.77 (s, 1H), 8.01 (d,
J = 8.6, 1H), 12.78 (s, 1H, NH). IR (nujol) 3142, 1702 cm-1
.
Anal. (C14H14F3N3O3) C, H, N.
General Procedure for the Synthesis of 4a-i, 9. A mixture of
3a-i, 8 (5 mmol), and DMF-DMA (1.79 g, 15 mmol) in
anhydrous toluene (20 mL) was refluxed for 1 h, then allowed
to reach the room temperature and stirred for additional 24 h.
The mixture was carefully concentrated in vacuo to give 4a-i, 9.
Example: Methyl 2-((1E,3E)-1-(Dimethylamino)-6,6,6-tri-
fluoro-5-oxohexa-1,3-dien-3-ylamino)-5-methylbenzoate (4a).
Assay of FAAH Activity. Membrane preparations were ob-
tained from brains (without cerebellum) from adult Wistar and
Sprague-Dawley rats. The frozen brains were thawed on ice
and homogenized with a glass homogenizator in 20 mM
HEPES, 1 mM MgCl2, pH 7.0. The homogenates were centri-
fuged at 17000 rpm for 20 min (4 °C), resuspended in 20 mL
buffer, and recentrifuged at 17000 rpm for 20 min (4 °C). The
pellets were resuspended in 10 mL of buffer and incubated at
37 °C for 15 min in order to remove any endogenous FAAH
substrates which otherwise might interfere with the assay. After
this, they were recentrifuged at 17000 rpm for 20 min (4 °C) and
the pellets were resuspended in Tris-HCl buffer, pH 7.4, contain-
ing 1 mM EDTA and 3 mM MgCl2. The FAAH assay was
carried out essentially as described previously.83 Membrane
homogenates were diluted with buffer (10 mM Tris-HCl,
1 mM EDTA pH 7.4) to the appropriate protein concentrations
so that initial velocities were measured. Test compounds (10 μL,
in ethanol except for URB 597 (in dimethyl sulphoxide
(DMSO)), 165 μL of homogenates and 25 μL of [3H]AEA
1
Yield 66%; mp 124-125 °C (2-PrOH). H NMR (DMSO-d6) δ
2.34 (s, 3H), 2.87 (s, 3H), 3.30 (s, 3H), 3.91 (s, 3H), 5.00 (d, J=11.5,
1H), 5.84 (s, 1H), 7.46 (m, 1H), 7.68 (s, 1H), 8.02 (m, 2H), 12.86 (s,
1H). IR (nujol) 1719 cm-1. Anal. (C17H19F3N2O3) C, H, N.
General Procedure for the Synthesis of 5a-i, 10. To a solution
of 4a-i, 9 (2 mmol) in dry DMF (5 mL), ammonium acetate
(0.308 g, 4 mmol) was added and the mixture was gently refluxed
for 1.5 h. The mixture was carefully concentrated in vacuo, and
then ice-water (15 mL) was added. The formed solid was
filtered off, washed with water, air-dried, and then crystallized
from the appropriate solvent to give 5a-i, 10.
Example: Methyl 5-Methyl-2-(2-(trifluoromethyl)pyridin-4-
ylamino)benzoate (5a). Yield 85%; mp 90-92 °C (n-hexane).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 5 2295
labeled in the ethanolamine part of the molecule (American
Radiolabeled Chemicals Inc., St. Louis, MO, in 10 mM Tris-
HCl, 1 mM EDTA, pH 7.4, containing 1% w/v fatty acid-free
bovine serum albumin, final substrate concentration of 0.5 μM)
were added to the tubes. The tubes were incubated for 10 min at
37 °C. The reaction was stopped by placing the tubes on ice and
adding 400 μL of activated charcoal mixture (80 μL of activated
charcoal, 320 μL 0.5 M HCl). The samples were mixed and left
at room temperature for about 30 min and then centrifuged at
2500 rpm for 10 min. Aliquots (200 μL) of the supernatants were
removed from each tube and analyzed for titrium content by
liquid scintillation spectroscopy with quench correction. Blanks
contained buffer instead of the membrane homogenates.
Hydrolysis of AEA by Intact RBL2H3 Cells. Rat basophilic
leukemia (RBL2H3) cells were obtained from American Type
Culture Collection (Manassas, VA) and cultured in MEM with
Earl’s salts, 2 mM L-glutamine, 15% fetal bovine serum, and 1%
100 U mL-1 penicilin þ 100 μg mL-1 streptomycin. Hydrolysis
of [3H-ethanolamine]AEA was measured in 24-well culture
plates.84 Cells were plated at a density of 2 ꢀ 105 cells per well
and incubated overnight at 37 °C in an atmosphere of 5% CO2.
After incubation, cells were washed with Krebs-Ringer HEPES
(KRH) buffer (120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 10
mM 4-(2-hydroxyethyl)-1-piperazineethyl-sulfonic acid, 0.12
mM KH2PO4, 0.12 mM MgSO4 in Milli-Q deionized water,
pH 7.4) containing 1% of BSA and once with KRH buffer
alone. Cells were preincubated with KRH buffer containing
0.1% of fatty acid-free BSA and the test compounds for 10 min
at 37 °C. After preincubation, 50 μL of [3H]AEA, containing 1%
fatty acid-free BSA (assay substrate concentration 100 nM),
were added and incubated for 20 min. To stop the reaction, 400
μL of ice-cold MeOH was added and the wells were carefully
scraped and then left on ice. Aliquots (400 μL) of the suspension
were then transferred in to glass tubes and 200 μL of chloroform
was added. After two vortex steps, the samples were left at room
temperature for approximately 30 min with subsequent centri-
fugation at 2500 rpm for 5 min. Aliquots (200 μL) of super-
natant were analyzed for titrium content by liquid scintillation
spectroscopy with quench correction. Blanks were defined as the
tritium recovered in samples without cells added.
Uptake of AEA by RBL2H3 Cells. The assays were under-
taken as using a standard method85 modified as described
previously,86 using 24-well culture plates, a preincubation time
of 10 min, and an incubation time of 4 min with substrate (100
nM [3H]AEA, labeled in the arachidonoyl part of the molecule,
American Radiolabeled Chemicals). Parallel experiments were
undertaken using wells alone to determine whether the test
compounds affected the ability of AEA to be retained by inert
surfaces.87
Assay of URB597-Sensitive Accumulation of Tritium in
RBL2H3 Cell Membranes Following Incubation of Cells in
Suspension with [3H]AEA. A recently published method64 was
used. RBL2H3 cells were resuspended in medium in Eppendorf
tubes to a concentration of 2 ꢀ 105 cells per tube (5 ꢀ 105 cells/
ml) and preincubated with 33, URB597, or 39 (both Cayman
Chemical Co.) for 10 min at 37 °C prior to addition of [3H]AEA
(labeled in the arachidonoyl part of the molecule, assay con-
centration 100 nM) in medium and incubation for 5 min (final
assay volume 225 μL). After incubation, the cells were sedimen-
ted using a microcentrifguge (1 min, 1000g) and washed with ice
cold medium, and aliquots (200 μL) of the suspensions were
placed in 96-well plates. The radioactivity retained by the cells
was separated from that in the medium by filtration through
polyethylenimine-coated FilterMAT filters (Skatron Instru-
ments Inc., Sterling, VA) using a Micro cell harvester
(Skatron Instruments Inc.) and a 30 s period of washing with
deionized water to rupture the cells. The filter papers were
analyzed for titrium content by liquid scintillation spectroscopy
with quench correction. Blanks were defined as the tritium
recovered in samples without cells added.
Assay of MGL Activity. Assays were carried out in a 96-well
microtiter plate (100 μL total volume) using a slight modification of
a recently reported method.69 Human recombinant MGL
(Cayman Chemical Co., Ann Arbor, MI) in 10 mM Tris-HCl,
1 mM EDTA, and test compounds were added to each well. To
start the hydrolysis, 20 μL of 4-nitrophenylacetate (Sigma Chemical
Co., St. Louis, MO), final concentration of 0.25 mM, were added.
Blanks contained buffer alone. The concentration of MGL
was chosen to ensure that initial velocities were measured. The
samples were incubated at room temperature. The absorbance was
measured at 405 nm after 0 min (to rule out effects of the
compounds per se upon the absorbance), 20 and 40 min using a
Thermomax microplate reader (ThermoMax Kinetic Microplate
Reader, Molecular Devices, Sunnyvale, CA). The 20 min readings
were used for the analyses.
Assay of COX Activity. An assay based on an oxygen
electrode method73 was used. For the preincubation assays, a
buffer containing 1 μM hematin, 2 mM phenol, 5 mM EDTA,
either ovine COX-1 (cat. no. 60100, Cayman Chemical Co., Ann
Arbor, MI) or human recombinant COX-2 (cat. no. 60122,
Cayman Chemical Co.) (200 U per assay) and 0.1 M tris-HCl,
pH 7.4 (final assay volume 2 mL) at room temperature was added
to an oxygen electrode chamber with an integral stirring unit
(Oxygraph System, Hansatech Instruments, King’s Lynn, U.K.)
that had been calibrated with respect to air pressure and ambient
temperature. After addition of test compound dissolved in DMSO
(20 μL), a baseline was established, usually over a period of 5 min.
Reactions were started by addition of arachidonic acid (10 μM,
unless otherwise stated), and the oxygen consumption was fol-
lowed for the next 5 min. For the nonpreincubation assays, the
assay buffer contained the arachidonic acid, and the reactions were
started by addition of the COX. Data is presented as the Δ[O2]
(μM) from the point of addition of the arachidonic acid
(preincubation assay) or COX-1 (nonpreincubation assay).
Data Analyses. Inhibition curves were expressed as % of
control and pI50, and hence IC50 values were determined using
the built-in equation “sigmoidal dose-response (variable
slope)” in the GraphPad Prism computer program (GraphPad
Software Inc., San Diego, CA). Top (uninhibited) values were
set to 100, and bottom (residual activity) values were either set to
0 (eq 1) or not preset (eq 2). The equation best fitting the data
was then chosen by use of Akaike’s information criteria. In the
case of eq 2, the max inhibition was defined as 100;the residual
app
app
and Vmax
activity. Km
(i.e., the observed Km and Vmax
values in the presence of a given concentration of test
compound) were calculated using the built-in equation “one-
site binding (hyperbola)” in the GraphPad Prism computer
program. For the measurements of oxygen consumption shown
in Figure 6, the data over the first 120 s after addition of COX
was fitted to the built-in equation “plateau followed by one-
phase decay” in the GraphPad Prism computer program.
Acknowledgment. Thestudy was supported bygrantsfrom
ꢁ
the Ministero dell’Universita e della Ricerca (PRIN 2007, Prot.
no. 2007SRY3KR_002), the Swedish Research Council (grant
no. 12158, medicine) and the Research Funds of the Medical
Faculty, Umea University to C.J.F. We are indebted to Britt
Jacobsson and Eva Hallin for expert technical assistance.
Supporting Information Available: Physical and spectral data
for compounds 3b-3i, 4b-4i, 5b-5i, 6b-6i, 8-11, 14-16, and
18-20. This material is available free of charge via the Internet
at http://pubs.acs.org.
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