Discovery of a Potent, Long-Acting, and CNS-Active Inhibitor (BIA 10-2474) of Fatty Acid Amide Hydrolase

Article abstract of DOI:10.1002/cmdc.201800393

Fatty acid amide hydrolase (FAAH) can be targeted for the treatment of pain associated with various medical conditions. Herein we report the design and synthesis of a novel series of heterocyclic-N-carboxamide FAAH inhibitors that have a good alignment of potency, metabolic stability and selectivity for FAAH over monoacylglycerol lipase (MAGL) and carboxylesterases (CEs). Lead optimization efforts carried out with benzotriazolyl- and imidazolyl-N-carboxamide series led to the discovery of clinical candidate 8 l (3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine 1-oxide; BIA 10-2474) as a potent and long-acting inhibitor of FAAH. However, during a Phase I clinical trial with compound 8 l, unexpected and unpredictable serious neurological adverse events occurred, affecting five healthy volunteers, including the death of one subject.

Full text of DOI:10.1002/cmdc.201800393

Accepted Article
Title: Discovery of a Potent, Long Acting and CNS Active Inhibitor (BIA
10-2474) of Fatty Acid Amide Hydrolase
Authors: Laszlo Kiss, Alexandre Beliaev, Humberto S. Ferreira, Carla
P. Rosa, Maria Joao Bonifacio, Ana I. Lourerio, Nuno M.
Pires, Nuno P. Palma, and Patricio Soares-da-Silva
This manuscript has been accepted after peer review and appears as an
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800393
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800393
A Journal of
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Discovery of a Potent, Long Acting and CNS Active Inhibitor (BIA
10-2474) of Fatty Acid Amide Hydrolase
László E. Kiss*[a], Alexandre Beliaev[a], Humberto S. Ferreira[a], Carla P. Rosa[a], Maria João
Bonifácio[b], Ana I. Loureiro[b], Nuno M. Pires[b], P. Nuno Palma[b] and Patrício Soares-da-Silva[b,c]
[a]
Dr. L. E. Kiss, Dr. A. Beliaev, Dr. H. S. Ferreira, Dr. C. P. Rosa
Laboratory of Chemistry, Department of Research & Development
BIAL – Portela & Cª., S.A.
À Avenida da Siderurgia Nacional, 4745-457 Coronado (S. Romão and S. Mamede), Portugal
E-mail: laszlo.kiss@bial.com
[b]
[c]
Dr. M. J. Bonifácio, Dr. A. I. Loureiro, Dr. N. M. Pires, Dr. P. N. Palma, Dr. (M.D., Ph.D.) P. Soares-da-Silva
Laboratory of Pharmacology, Department of Research & Development
BIAL – Portela & Cª., S.A.
À Avenida da Siderurgia Nacional, 4745-457 Coronado (S. Romão and S. Mamede), Portugal
Dr. (M.D., Ph.D.) P. Soares-da-Silva
MedInUp - Center for Drug Discovery and Innovative Medicines
University of Porto
Praça Gomes Teixeira, 4099-002 Porto, Portugal
Supporting information for this article is given via a link at the end of the document.
Abstract: Fatty acid amide hydrolase (FAAH) enzyme can be
targeted for the treatment of pain associated with various medical
conditions. Herein, we report the design and synthesis of a novel
series of heterocyclic-N-carboxamide FAAH inhibitors that achieve
good alignment of potency, metabolic stability and selectivity over
monoacylglycerol lipase (MAGL) and carboxylesterases (CEs)
enzymes. Lead optimization efforts carried out with benzotriazolyl-
and imidazolyl-N-carboxamide series led to the discovery of clinical
candidate 8l (3-(1-(cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-
yl)pyridine 1-oxide; BIA 10-2474) as a potent and long acting inhibitor
of FAAH. However, during a Phase I clinical trial with compound 8l,
unexpected and unpredictable serious neurological adverse events
occurred affecting five healthy volunteers, including the death of one
subject.
The X-ray crystal structure of the rat catalytically active
transmembrane domain-deleted FAAH was first unveiled in 2002,
in complex with an irreversible inhibitor (methoxy arachidonyl
fluorophosphonate (MAFP)).11 Later on, an engineered
‘‘humanized’’ rat (h/r) FAAH was produced by interconverting the
active site residues of rat and human FAAH, using site-directed
mutagenesis.12 This h/rFAAH combined the efficient rFAAH
recombinant expression with the inhibitor sensitivity profile of
hFAAH, thus greatly facilitating the design of new inhibitors. Since
then, a number of inhibitors have been co-crystallized with this
form of the enzyme.12-19
The most important classes of compounds with different
mechanism of action are the reversible inhibitor heterocyclic
ketones20 exemplified by 1 (OL-135), and the irreversible,
covalently binding inhibitors such as carbamates21 (e.g. 2
(URB597)) and ureas13 (e.g. 3 (PF3845)) (Figure 1). It is
interesting to note, that that despite the quite high chemical
Introduction
Fatty acid amide hydrolase (FAAH, EC 3.5.1.99), an integral
membrane bound enzyme, is a member of the extensive family of
serine hydrolases referred to as amidase signature (AS) family.1
It catalyses degradation of lipid signalling fatty acid amides
including the sleep-inducing oleamide and endogenous
cannabinoid anandamide (AEA).2 AEA binds and activates both
central (CB1) and peripheral (CB2) cannabinoid receptors of the
endocannabinoid system (ECS).3 The modulation of levels of AEA
via inhibition of FAAH has potentially clinical relevance in a wide
range of diseases and pathological conditions. Therefore, both
the ECS and FAAH have become recognized as promising
therapeutic targets for the treatment of a range of central and
peripheral disorders.4-5 Importantly, phenotypes observed in
FAAH knockout mice allowed to expect the efficiency of FAAH
inhibitors not being compromised by typical side effects of direct
CB1 agonists.6 Due to increasing interest over the last two
decades, numerous small molecule inhibitors of FAAH belonging
to various chemical classes have been reported (for recent
reviews see5-10 and references cited therein).
Figure 1. Chemical structure of OL-135 1, URB597 2, PF3845 3, JNJ-42165279
4, PF-04457845 5 and MK-4409 6.
1
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stability of the urea functional group in 3, FAAH is yet inhibited in
an irreversible manner by covalently modifying the enzyme's
active site.22 Of the urea class22-24 the most interesting
compounds which have been advanced into clinical trial are 4
(JNJ-42165279)25 developed by Johnson & Johnson and 5 (PF-
04457845)26 by Pfizer (Figure 1). Unfortunately, the clinical
development of 4 and 5 have been suspended as a precautionary
measure following serious safety issues with 8l (BIA 10-2474) and
due to lack of therapeutic effect,27 respectively. Recent efforts
from Merck led to the discovery of oxazole 6 (MK-4409)28 which
has also advanced into clinical development. Compound 6 is
described as a reversible FAAH inhibitor but no clinical results
published up to now.
in refluxing tetrahydrofuran (THF) containing base (N,N-
diisopropyl ethyl amine (DIPEA) or pyridine) to provide the target
compounds 8a-b, 8d and 8f-k in moderate yield. The 3-pyridyl
imidazole analogue 8k was further oxidized to pyridine N-oxide 8l
(BIA 10-2474) on reaction with meta-chloroperbenzoic acid
(MCPBA) in dichloromethane (DCM) at room temperature. Due to
reduced reactivity of 7e and 9a-b different reaction conditions
were applied for preparing compounds 8c, 10a, 10g and 10o-p,
wherein 7e and 9a-b were deprotonated with sodium hydride in
THF at 0 °C followed by reaction with the corresponding
dialkylcarbamoyl chlorides to give phenyl imidazole 8c and
benzotriazoles 10a, 10g and 10o-p in fairly good yields.
Our group set out to design a fast onset, long acting and CNS
active FAAH inhibitor, which may be useful for the treatment of
pain associated with various medical conditions. Preliminary
screening of selected compounds sourced from both commercial
vendors and those cherry-picked from our in-house collections for
FAAH inhibition led to the identification of the imidazole 8a (Figure
2,
N-methyl-N-phenyl-1H-imidazole-1-carboxamide),
which
showed moderate in vitro activity (88
%
inhibition at
a
concentration of 10 M in rat brain homogenates).
Figure 2. Chemical structure of screening hit 8a.
At that time, compound 8a represented a novel scaffold29 in the
field of FAAH inhibitors, although during our research programme
a patent application filed by an academic group has independently
disclosed similar heterocyclic-N-carboxamide scaffolds claiming
them as dual inhibitors of MAGL and FAAH enzymes.30 The
structure of 8a was deemed to be readily amenable to exploration
of structure-activity relationships (SAR) and it was therefore
selected as the starting point for
programme.
a medicinal chemistry
Herein, we describe a novel series of potent in vivo CNS active
benzotriazolyl- and imidazolyl-N-carboxamides that have been
designed and synthesized in our laboratory, including 8l (BIA 10-
2474), which in January 2016 faced a severe incident in a Phase
I clinical trial, where one person died and others suffered adverse
neurological effects.
Scheme 1. Preparation of imidazole and benzotriazole analogues
The general synthetic routes to support SAR exploration within
the series of N-benzotriazolyl carboxamide derivatives are
depicted in Scheme 2. Benzotriazoles 9a and 9c-g were acylated
with 20% toluenic solution of phosgene in THF. Benzotriazole-N-
carbonyl chloride intermediates thus obtained were treated with
secondary amines in THF using pyridine as base to provide the
desired N,N-disubstituted benzotriazole carboxamides (10b-c,
10f, 10h-n, 11a-c, 11e-g) in moderate yield. The carboxamide
derivative 11h was prepared from carboxylic acid intermediate
10p. Activation of 10p with thionyl chloride (SOCl2) afforded the
corresponding acyl chloride intermediate, which was
subsequently reacted with ethanolic ammonia solution to provide
11h. Mono-substituted benzotriazole N-carboxamides 10d-e
were obtained by treating benzotriazole 9a with isocyanates in
DCM at room temperature. Construction of the biphenyl ring in
11d was accomplished via Suzuki coupling reaction. 6-Bromo
Results and Discussion
Chemistry
The synthetic routes employed to obtain the initial heterocyclic-N-
carboxamide inhibitors are outlined in Scheme 1. The necessary
dialkylcarbamoyl chloride intermediates were obtained either from
commercial sources or prepared by a literature method.31 The
target heterocyclic-N-carboxamides were synthesised under two
different conditions. More reactive heterocycles such as 7a-d and
7f were smoothly carbamoylated with dialkylcarbamoyl chlorides
2
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benzimidazole 9b was smoothly converted to 11d on the reaction
with phenyl boronic acid in a mixture of 1-propanol – water using
palladium catalyst (Pd(PPh3)4) and sodium carbonate as base.
8e
00
00
615
10
10a
[a] % of Control, results are means ± SD (n=4), determined in rat brain
homogenates after 15 min of preincubation time.
In the first phase of our hit-to-lead optimization programme,
modification by substitution or replacement of the imidazole ring
in 8a with other common heterocycles were investigated. To
facilitate the interpretation of the SAR, all prepared analogues
shared the same N-methylaniline moiety on the right-hand side of
the molecule and were evaluated at two different concentrations.
The results for a representative selection of compounds are
provided in Table 1 and reported in residual enzymatic activity.
Whilst the initial screening hit 8a exhibited fairly promising activity
at a concentration of 10 µM, it was found to be completely inactive
at a hundred-fold lower concentration. The 4-phenyl derivative 8b
displayed slightly increased inhibition over the parent 8a, whereas
shifting the phenyl ring to C2 position of the imidazole ring (8c)
led to drastic reduction in FAAH inhibition. Within the series of
heterocycles with three heteroatoms, the unsubstituted 1,2,4-
triazole 8d was found to possess enhanced inhibition over the
screening hit 8a. Inclusion of p-chloro-phenyl group at 3-position
of the imidazole ring (8e) made little difference in potency
compared to imidazole analogue 8b. However, it was then
discovered that in vitro inhibition could markedly be enhanced by
condensing the triazole core in 8d with phenyl ring (10a).
Benzotriazole 10a was clearly the most potent inhibitor with an
Scheme 2. Preparation of benzotriazole analogues
Pharmacology
In vitro screening of all new compounds was carried out in rat
brain membranes. FAAH activity was determined by measuring
IC50 of
7
nM, and therefore, it was further studied
3
the formation of H-ethanolamine using tritium (3H) labelled AEA
pharmacologically. Target selectivity of 10a for FAAH relative to
CEs and MAGL enzymes was evaluated in vitro in rat liver
microsomes and in rat cerebellum cytosol, respectively. Inhibitor
10a showed significant selectivity for FAAH (IC50 = 7 nM) over CE
(IC50 > 1 M) and MAGL (IC50 > 100 M) enzymes, although it
inhibited CEs to a greater extent than reference compounds 2 and
3 (Table 2).
as has been described previously.32
Table 1. In vitro FAAH inhibition (expressed as percentage of control values) by
selected heterocyclic N-carboxamide analogues in rat brain homogenates.
In another experiment 2, 3 and 10a were assessed for in vivo
FAAH inhibition. Compounds were orally administered to
overnight fasted mice at a dose of 30 mg/kg, and thereafter at 1h
post-administration the FAAH activity was determined. As seen in
Table 2, whilst 2 and 3 exhibited over 90 % inhibition in both the
liver and brain, compound 10a only moderately inhibited FAAH
both centrally and peripherally. At this point compound 10a was
selected as an early lead for further optimization. The SAR around
our lead structure 10a was investigated by varying the amino side-
chain of the carboxamide core structure. Accordingly, we
proceeded to synthesize a restricted series of benzotriazoles
(10b-d) aimed at enhancing in vitro efficacy whilst maintaining
good target selectivity over CEs and MAGL. Introduction of
electron withdrawing groups (EWGs) such as fluorine in the
aromatic ring (10b) had no beneficial effect on in vitro inhibitory
potency. Increasing the electron density on the aromatic ring in
10a by appropriate substitution at the meta positions led to a
weakly active compound (10c). Removal of the N-methyl group in
10b completely abolished FAAH inhibition (10d). Moreover,
compounds 10b-d inhibited CE and MAGL to an appreciable
extent. The next round of SAR concentrated on analogues which
No.
HetAr
10 M[a]
123
0.1 M[a]
11220
8a
8b
00
847
8c
8d
936
00
11537
172
3
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10.1002/cmdc.201800393
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incorporate benzylic, cycloaliphatic and saturated heterocyclic
sidechains (10e-k). Compounds 10e-k were evaluated both in
vitro (FAAH, CEs and MAGL) and in vivo (30 mg/kg, p.o., mice 1
h post-dose). Despite the promising in vitro FAAH activity of the
N-benzyl derivative 10e, only residual in vivo inhibition was seen
both centrally and peripherally. In contrast, the in vitro also highly
potent N-methyl-N-benzyl 10f derivative showed evidence of in
vivo efficacy, but it was uninteresting in terms of selectivity.
Table 2. In vitro FAAH, CE, MAGL and in vivo FAAH inhibition (expressed as % of control values) by selected benzotriazole N-carboxamides.
No.
HetAr
100 nM[a,b]
00
10 nM[a,b]
153
Liver[a,c]
61
Brain[a,c]
92
CEs[a,d]
725
MAGL[a,e]
874
2
3
74
10131
22
10
9517
8712
10a
10
21
4212
353
345
349
312
401
734
706
10b
10c
ND
ND
760
ND
ND
ND
ND
ND
10d
1005
21
ND
ND
ND
ND
ND
10e
10f
131
846
877
674
41
00
30
00
62
10
241
332
22
10g
10h
3910
415
472
102
00
00
00
00
61
81
242
271
75
74
10i
154
205
10j
00
12
10
10
305
10
887
00
282
301
124
733
10k
10l
9412
151
ND
ND
ND
ND
ND
577
302
8018
586
10m
626
4
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10n
717
ND
ND
ND
281
355
10o
11a
173
21
712
ND
ND
607
816
8425
905
7825
21
00
[a] % of Control, results are means ± SD (n=4). [b] Determined in rat brain homogenates after 15 min of preincubation time. [c] 30 mg/kg, po, in mice, FAAH activity
was determined 1 h after administration. [d] Cinhib. = 10 M. in rat liver microsomes. [e] Cinhib. = 100 M in rat cerebellum cytosol.
Morpholine 10g exhibited weaker inhibitory activity both in vitro
and in vivo than 10f without improving target selectivity. The
piperidine 10h and piperazine analogues 10i-j exhibited higher in
vitro inhibition either than 2 or 3 at a concentration of 10 nM
irrespectively of type of substitution. Unfortunately, both CEs and
MAGL were inhibited by 10h-j to high extent. In vivo efficacy of
10h-j both centrally and peripherally was comparable to that of 2
and 3 with the exception of 10j, which showed a clear preference
for peripheral FAAH. Another effort within the benzotriazole N-
carboxamide series focused on further functionalization of the
amino moiety. We envisioned that compounds endowed with
higher steric hindrance in the neighbourhood of the carbonyl
group might improve the poor target selectivity of earlier N,N-
dialkyl analogues (10f-j) without compromising the excellent in
vitro and good in vivo FAAH inhibitory ability. Indeed, this
hypothesis was borne out, the in vitro highly potent N-methyl-N-
cyclohexyl derivative 10k exhibited greater in vivo efficacy over
10f-j with considerably improved selectivity over MAGL. Thus,
compound 10k exhibited the greatest promise at this point and
subsequent efforts focused on this particular N-alkyl, N-cycloalkyl
series, leading to the elaboration of derivatives 10l-n shown in the
lower half of Table 2. Elongation of the N-methyl group of 10k with
improved target selectivity over regioisomer 10o, but more
importantly, demonstrated potent oral activity. On the basis of
these findings, 11a was selected for further pharmacological
studies. The IC50 of 11a was calculated to be 19 nM. The dose-
dependent central- and peripheral inhibitory ability of 11a was
assessed in the mouse (1 h post-dose) given increasing doses of
compound 11a (0.001-3.0 mg/kg, p.o.).
Table 3. In vitro CYP450 metabolic stability in different species by 11a, 11h, 8h
and 8l.
No.
11a
11h
8h
mouse[a]
50
rat[a]
dog[a]
10
primates[a]
00
human[a]
529
260
870
886
911
879
620
992
1016
540
942
573
968
413
754
8l
881
951
[a] Cinhib. = 5 M in liver microsomes after 1h of incubation, data are given in %
of remaining, results are means ± SD (n=4).
Compound 11a was found to inhibit both peripheral and cerebral
FAAH with ED50’s of 7 µg/kg and 61 µg/kg, respectively.
Reference compound 3 was a slightly weaker inhibitor of FAAH in
the liver than 11a in the same conditions but in the brain found to
be equipotent (ED50(liver) = 23 µg/kg; ED50(brain) = 63 µg/kg). In
another experiment, inhibitor 11a was incubated with liver
microsomes (at a concentration of 5 µM) prepared from different
species to evaluate the potential likelihood of metabolic instability.
Moderate to high metabolism was observed in different species
with the highest degradation seen in mouse, primates and dog
(Table 3).
a
methylene spacer resulted in inactive derivative 10l.
Incorporation of eight-membered (10m) macrocyclic amines was
less tolerated in vitro without providing improvement in selectivity
over both MAGL and CEs. A structural isomer of 10m, which
includes 2 methyl groups at 2,6-positions of the piperidine ring
(10n), was weakly potent in vitro. However, the 5-membred 2,2-
dimethyloxazolidine analogue 10o, which displayed moderate in
vitro FAAH inhibition, was endowed with better target selectivity
than any other previously synthesized analogue. Moving the two
methyl groups to 4-position of the oxazolidine ring to change the
steric crowding around the carbonyl group gave 11a. This
compound had slightly higher in vitro FAAH inhibition and
Table 4. In vivo FAAH and in vitro CE and MAGL inhibition data (expressed as % of control values) of selected benzotriazole N-carboxamides.
No.
2
R1
R2
R3
X
Liver[a,b]
7420
74
Brain[a,b]
917
51
CEs(10 M)[a,c]
725
MAGL(100 M)[a,d]
CYPs(50 M) [e]
874
8712
905
ND
3
9517
ND
11a
H
H
H
CH
104
247
816
252
5
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11b
11c
11d
11e
11f
H
H
Br
CH
CH
CH
N
166
199
546
147
397
84
7729
7110
7618
124
739
825
922
6111
7913
725
817
6712
683
774f
725
961f
71
82
F
H
F
81
H
H
H
H
H
H
Ph
H
732
ND
H
H
OCH3
OCH3
H
CH
CH
CH
12538
93
00
11g
11h
OCH3
CONH2
550
672
61
61
962
[a] % of Control, results are means ± SD n=4. [b] 0.1 mg/kg, po, in mice, FAAH activity was determined 8 h after administration. [c] In rat liver microsomes. [d] In
rat cerebellum cytosol. [e] eCinhib. = 50 M in mouse liver microsomes after 1 h of incubation, data are given in % of remaining.
Despite the outstanding in vivo efficacy, benzotriazole derivative
11a was not considered for further development, but it was
selected for additional optimization to improve its pharmacological
profile. Blocking the 4,4-dimethyl oxazolidinyl moiety in structure
of 11a, further investigation into the effect of the aromatic
substitution on CYP450 stability (50 M, 1 h of incubation) and in
vivo efficacy (0.1 mg/kg, p.o., 8h post-dose) were carried out.
Table 4 details results for new compounds and includes
comparative data for 2 and 3. Despite the high in vitro potency of
2 (IC50 = 5 nM)33, only residual in vivo FAAH inhibition was
observed in both liver and brain (0.1 mg/kg, p.o., 8h post-dose).
In contrast, compound 11a displayed comparable in vivo potency
to that of 3 in both liver and brain. Inclusion of bromine (11b) atom
at C-6 position of the benzotriazole ring provided a metabolically
unstable compound and made little difference in in vivo inhibitory
profile. The 4,6-difluoro derivative 11c did not perform better in
vivo than the mono bromo derivative 11b and failed to improve
metabolic stability. Incorporation of a bulky phenyl group as in 11d
was found to be detrimental for in vivo potency.
Table 5. In vivo FAAH and in vitro CE and MAGL inhibition data (expressed as % of control values) of selected imidazole N-carboxamides.
No.
R1
R2
100 nM[a,b]
Liver[a,c]
Brain[a,c]
CEs(10 M)[a,d]
1055
MAGL(100 M)[a,e]
8b
-Ph
847
115f
1417f
ND
8f
-Ph
-Ph
9410
10
536
927
956
522
923
616
8g
ND
ND
8h
8i
-Ph
-Ph
417
102
124
114
978
967
928
961
10418
3818
8j
-Ph
373
757
332
134
62
865
7516
82
9011
973
913
944
964
8k
8l
3-Py
3-Py-N-oxide
8511
1025
[a] % of Control, results are means ± SD (n=4). [b] Determined in rat brain homogenates after 15 min of preincubation time. [c] 0.1 mg/kg, po, in mice, FAAH activity
was determined at 8 h after administration. [d] In rat liver microsomes. [e] In rat cerebellum cytosol. [f] 30 mg/kg, po, in mice, FAAH activity was determined at 1 h
after administration.
6
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10.1002/cmdc.201800393
ChemMedChem
FULL PAPER
Replacement of the phenyl ring in 11a with a pyridyl ring gave
11e, which demonstrated over 85% peripheral and cerebral
inhibition with slight increase in inhibition of CEs. As expected,
methoxy derivative 11f was poorly active in vivo most probably
due to extensive CYP450 mediated metabolism. Surprisingly, the
5,6-dimethoxy derivative 11g was endowed with reasonable good
metabolic stability and displayed impressive FAAH inhibition,
although it is a good inhibitor of MAGL. Finally, the optimal
125
100
75
50
25
0
3
8h
8l
11h
compound from this series was achieved by
a simple
incorporation of a carboxyamide group at C-5 position of 11a to
produce a compound 11h with high CNS efficacy and good
CYP450 stability in vitro over 11a in various species (Table 3).
For completeness of the SAR, we considered the replacement of
benzotriazole ring in 11h with other heterocycles such as
imidazole and triazole. We were interested to see what sort of
pharmacological effect in terms of in vivo efficacy and target
selectivity could be expected by this modification. From the early
imidazole and triazole inhibitors (Table 1) the latter class was
ruled out due to poor selectivity over CEs. Imidazole 8b was
devoid of CEs inhibition and despite the lower in vitro potency, it
was endowed with more enhanced in vivo inhibition (30 mg/kg,
po, and 1h post-dose) both centrally and peripherally over our
initial benzotriazole derivative 10a. (Table 5). Therefore, we were
encouraged to synthesize further imidazole analogues (8f-g) for
in vitro and in vivo comparison to their benzotriazole counterparts
(10i, 10j and 11a). Compounds that exhibited over 90 % of control
in the MAGL and CEs assays were evaluated in vivo in mice (0.1
mg/kg, p.o., 8h post-dose). The results are shown in Table 5.
Majority of the newly synthesized compounds were much less
efficacious in vitro than their benzotriazole analogues. Compound
8f, the analogue of 11a, demonstrated some degree of peripheral
inhibition, but centrally was ineffective. Although piperazine
analogue 8g fully abolished FAAH activity at a concentration of
100 nM, somewhat it inhibited both CEs and MAGL. Increasing
the steric hindrance near the carbonyl group led to a more
selective compound 8h, which exhibited over 90 % in vivo FAAH
inhibition in the brain and liver. Replacement of the N-benzyl
group (8h) with an oxygen atom (8i) or methylene group (8j) led
to a reduction in central inhibition. The more polar and basic
pyridyl analogue 8k failed to improve in vivo efficacy in the brain
over the parent 8j. However, the even more polar pyridine N-oxide
derivative 8l displayed impressive inhibition in both brain and liver
with no measurable effect in vitro in CEs and MAGL assays.
Furthermore, compound 8l displayed favourable in vitro metabolic
stability in various species (Table 3). Generally, we can say that
within the imidazole scaffold few correlations could be established
between results of in vitro and in vivo FAAH activity. Possible
explanations include in vivo accumulation of the inhibitors, which
has not been seen (manuscript in preparation) or their interaction
with the enzyme might be affected by the enzyme environment in
vitro (brain membranes) or in vivo; further studies will be required
to understand this effect. Having identified from these series 11h,
8h and 8l possessing excellent in vivo FAAH inhibitory activity (>
90 % of control in brain and liver at a dose of 0.1 mg/kg, p.o.) and
good selectivity over CEs and MAGL (> 80 % of control),
subsequent experiments with 3, 8h, 8l and 11h were designed.
Figure 3 highlights their FAAH inhibition profile in mouse liver and
brain homogenates at a dose of 0.1 mg/kg, p.o. Compounds 3,
8h, 8l and 11h were found to achieve their maximum inhibitory
effect at 1 h post-dose in the liver.
0
8
16
24
32
40
48
T im e / h
Figure 3. Liver FAAH activity after oral administration of 0.1 mg/kg of
compounds 3 (■), 8h (●), 8l (○) and 11h (●). Results are means ± SD (n = 4).
Thereafter, the constant peripheral inhibition of FAAH (> 90 %)
was sustained over the next 7 h, followed by gradual return to
lower levels at later time points.
The profile of central inhibition by 3, 8h and 8l was found to be
different to that of their liver inhibition. Inhibitors 3, 8h and 8l
produced their maximal inhibitory effect (> 90 %) in the brain only
at 8 h post dose, and significant central inhibition by 8h and 8l (>
50 %) was maintained up to 48 h post-administration (Figure 4).
In contrast, compound 11h displayed similar inhibitory profile
(Tmax = 1 h) both centrally and peripherally and found to achieve
long lasting effect.
125
3
100
75
50
25
0
8h
8l
11h
0
8
16
24
32
40
48
T im e / h
Figure 4. Brain FAAH activity after oral administration of 0.1 mg/kg of
compounds 3 (■), 8h (●), 8l (○) and 11h (●). Results are means ± SD (n = 4).
150
8h
8l
100
50
0
11h
-4
-3
-2
-1
0
1
Log [com pound] / m g/kg
Figure 5. Effect of increasing doses of compounds 8h (●), 8l (○) and 11h (●) on
mice liver FAAH activity 8 h after oral administration of each compound. Results
are means ± SD (n=4).
Finally, we examined the dose dependent FAAH inhibitory profile
by 8h, 8l and 11h. Mice were given increasing doses of
7
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10.1002/cmdc.201800393
ChemMedChem
FULL PAPER
compounds 8h, 8l and 11h ranging from 0.001 to 3 mg/kg p.o.,
thereafter, at 8 h post-administration the FAAH activity was
assayed in liver and brain. The results demonstrate (Figure 5 and
6) that all three compounds were very efficacious in these assays.
In the liver compounds 8l and 11h are essentially equipotent with
ED50’s of 7 µg/kg, whereas inhibitor 8h was slightly less potent
with an ED50 value of 28 µg/kg. Again, 11h was the most potent
compound out of 8h, 8l and 11h at inhibiting cerebral FAAH with
an ED50 value of 14 µg/kg. Imidazoles 8h and 8l displayed less
marked inhibition with ED50’s of approximately 79 and 27 µg/kg,
respectively.
devoid of off-target effects (CEs and MAGL). Further optimization
resulted in other selective (CEs and MAGL) in vivo potent
imidazolyl N-carboxiamides such as 8h and 8l. On the basis of
overall pharmacological profile, inhibitor 8l was selected for
clinical development. Since the accident in the clinical trial
concerning 8l in which one person died and four were hospitalised
with neurological symptoms35 there has been much speculation
as to its cause. The investigations conducted by the French
authorities concluded that it was an unexpected effect of the test
item, having ruled out other extraneous causes. Their conclusion
was that the accident was likely to have been caused by an
unknown off-target effect of 8l.36 However, the nature of this off-
target effect remains obscure, although much speculation has
centred around the idea that 8l is not particularly selective for the
FAAH enzyme. In fact, a recent activity-based profiling of serine
hydrolases revealed that 8l inhibited not only FAAH, but ABHD6
and some other serine hydrolases namely FAAH2, CES1, CES2,
CES3, ABHD11, LIPE, and PNPLA6 albeit at concentrations
several folds higher than those needed for FAAH inhibition.37
Although the published data provide information about the
selectivity of 8l, it does not allow to conclude which of the
identified off-targets could be responsible for the toxicity found.
Even in the case of PNPLA6, which has previously been linked to
organophosphate-based neurotoxicity in humans, it was shown
that carbamoylation of the enzyme (expected effect of 8l) was not
neuropathic.38 Detailed pharmacological characterization of the
clinical candidate 8l will be addressed in a separate publication
including a discussion on off target effects, in addition to the
preliminary data already disclosed.39-41 Preliminary information
concerning the safety profile of 8l during clinical trial BIA-102474-
101 have been provided, the conclusions drawn already revealed
that 1) following multiple-ascending-doses up to 20 mg, Inhibitor
8l was found to be safe and well tolerated and systemic exposure
to anandamide increased in a dose dependent manner42 and 2)
five out of six subjects exposed to 8l at 50 mg/day reported
unexpected CNS related serious adverse events, for which
causality cannot be excluded; the available data does not allow
discerning about specific reasons that could have caused the
SAEs, which were considered unexpected and unpredictable.43
150
8h
8l
100
50
0
11h
-4
-3
-2
-1
0
1
Log [com pound] / m g/kg
Figure 6. Effect of increasing doses of compounds 8h (●), 8l (○) and 11h (●) on
mice brain FAAH activity 8 h after oral administration of each compound.
Results are means ± SD (n=4).
The overall pharmacological profiles of 8h, 8l and 11h did not
demonstrate marked differences and all three inhibitors were
efficacious in the low µg/kg range. Therefore, other factors such
as ease of large scale manufacturing and cost of synthesis played
a role in preclinical candidate selection.
Our synthetic feasibility analysis revealed difficulties in synthesis
of 11h. Any kind of 1H-benzotriazole compound substituted at 5-
position exist in three tautomeric forms, which may result in
different products in the carbamoylation step. Indeed, 11h was
always contaminated with the unwanted regioisomer (substituted
at 6-position of the benzotriazole ring) and isolation of a pure
sample of 11h was not really feasible by a simple recrystallization.
In contrast, the most efficacious imidazole compound 8l could
easily be prepared from cheap raw materials in a simple few steps
process.34 Furthermore, inhibitor 8l was screened against a panel
of 97 off-targets (Caliper Life Sciences), including
neurotransmitter related targets, ion channels, prostaglandins,
growth factors/hormones, peptides and enzymes and at a
concentration of 10 M none of those off-targets was found to be
inhibited/blocked by 8l (see supporting information). Therefore,
compound 8l was selected as preclinical candidate and later on
advanced into Phase I clinical studies.
Experimental Section
Chemistry
NMR spectra were recorded on
a Bruker Avance III (600 MHz)
Spectrometer with solvent used as internal standard, and data are reported
in the order: chemical shift (ppm), number of protons, multiplicity (s, singlet;
d, doublet; dd, doublet of doublet; t, triplet; q, quartet; m, multiplet; br,
broad), approximate coupling constant (J) in Hertz and assignment of a
signal. Elemental analyses were performed on a CE Instruments, EA 1110
CHNS analyser. The purity of the compounds in all cases was higher than
95%. Analytical TLC was performed on precoated silica gel plates (Merck
60 Kieselgel F 254) and visualized with UV light. Solvents and reagents
were purchased from Aldrich, Merck and Fluka and were used without
further purification.
Conclusions
A novel series of potent benzotriazolyl- and imidazolyl-N-
carboxamides have been developed from an in vitro imidazolyl-N-
carboxamide hit 8a. The 4,4-dimethyloxazolidinyl derivative 11a
was the most potent compound from this series with limited in vitro
metabolic stability in different animal species. Consequently, the
carboxamide substituted derivative 11h was prepared and found
to be highly efficacious in the CNS, metabolically stable and
N-Methyl-N-phenyl-1H-imidazole-1-carboxamide (8a). To
a stirred
solution of imidazole 7a (681 mg, 10 mmol) in THF (15 mL) at 0 ºC was
added a solution of methyl(phenyl)carbamic chloride (848 mg, 5 mmol) in
toluene (6 mL). After being stirred for 2 h at room temperature, the reaction
was filtered and the filtrate was evaporated. Chromatography (in DCM)
followed by trituration with diethyl ether afforded 8a as a white powder (446
8
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10.1002/cmdc.201800393
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FULL PAPER
1
mg, 44%), mp.: 58 °C. H NMR (CDCl3):  7.57 (1H, s), 7.39 (2H, t, J =
1H-imidazole-1-carboxamide 8k (90 mg, 0.317 mmol) was added MCPBA
(149 mg, 0.475 mmol) at 20-25 ºC in one portion. The reaction was allowed
to stir at 20-25 ºC for 20 h. The mixture was evaporated to dryness, the
residue was then triturated in diethyl ether. The resulting white crystals
were collected, dried on air and recrystallized from isopropanol to afford 8l
(46 mg, 48% yield). mp.: 227.5 °C. 1H NMR (DMSOd6):  8.70 (1H, t, J =
1.5 Hz), 8.28 (1H, s), 8.20 (1H, s), 8.11 (1H, d, J = 6.5 Hz), 7.78 (1H, d, J
= 8.1 Hz), 7.45 (1H, d d, J = 6.5, 8.1 Hz), 3.80 (1H, m), 2.92 (3H, s), 1.77
(4H, m), 1.56 (3H, m), 1.30 (2H, m), 1.11 (1H, m). 13C NMR (DMSOd6): 
DMSO: 150.4, 138.3, 137.1, 135.7, 134.7, 132.8, 126.7, 121.2, 117, 56.8,
31.4, 29, 25.1, 24.8. Elemental analysis: (C16H20N4O2): Calc: C, 63.98; H,
6.71; N, 18.65. Found: C, 63.76; H, 6.94; N, 18.74.
7.7 Hz), 7.32 (1H, t, J = 7.3 Hz), 7.13 (2H, d, J = 7.7 Hz), 6.85 (1H, s), 6.81
(1H, s), 3.50 (3H, s). 13C NMR (CDCl3):  150.2, 142.9, 137.7, 130.3, 129,
128, 125.9, 118.4, 40.1. Elemental analysis: (C11H11N3O): Calc: C, 65.66;
H, 5.51; N, 20.88. Found: C, 65.68; H, 5.11; N, 21.03.
N-Methyl-N,4-diphenyl-1H-imidazole-1-carboxamide (8b). To a stirred
solution of 4-phenyl-1H-imidazole 7b (433 mg, 3.0 mmol) in THF (10 mL)
was added DIPEA (0.78 mL, 4.5 mmol) followed by a solution of
methyl(phenyl)carbamic chloride (534 mg, 3.15 mmol) in toluene (4 mL).
The reaction mixture was stirred at room temperature for 1 h, then was
heated at 80 °C for 1 h. After being cooled to room temperature the
reaction was evaporated and partitioned between DCM and water. The
organic layer was separated, dried over MgSO , filtered and evaporated.
(4,4-Dimethyloxazolidin-3-yl)(4-phenyl-1H-imidazol-1-yl)methanone
(8f). To a stirred solution of 4-phenyl-1H-imidazole 9b (433 mg, 3.00 mmol)
in THF (10 mL) at 0 ºC was added dropwise a solution of 4,4-
dimethyloxazolidine-3-carbonyl chloride (515 mg, 3.15 mmol) in THF (10
mL) followed by pyridine (0.48 mL, 5.93 mmol). The reaction was allowed
to stir at room temperature for 1 h, then heated at reflux for 4 h. The mixture
was cooled to room temperature and evaporated. The residue was
partitioned between DCM and water. The organic phase was separated,
washed with 2N HCl, water and brine, then dried over MgSO4, filtered and
evaporated in vacuum. Column chromatography (petroleum ether–ethyl
acetate, 2:1) followed by recrystallization from isopropanol afforded the
title compound as a white powder (120 mg, 14 %). mp.: 89 °C. 1H NMR
(CDCl3):  7.96 (1H, d, J = 1.3 Hz), 7.79 (2H, m, J = 8.3 Hz), 7.52 (1H, d,
J = 1.3 Hz), 7.41 (2H, m, J = 7.9 Hz), 7.30 (1H, m, J = 7.3 Hz), 5.13 (2H,
s), 3.88 (2H, s), 1.62 (6H, s). 13C NMR (CDCl3):  146.5, 142.6, 136.2,
132.7, 128.7, 127.7, 125.2, 112, 81.1, 80.3, 61.8, 22.8. Elemental analysis:
(C15H17N3O2): Calc: C, 66.40; H, 6.32; N, 15.49. Found: C, 65.59; H, 6.35;
N, 15.32.
4
Trituration in a mixture of petroleum ether–ethyl acetate (3:1) followed by
filtration and drying gave 8b as a beige powder (465 mg, 56%), mp.: 104-
1
105 °C. H NMR (CDCl3):  7.60 (2H, d, J = 7.7 Hz), 7.53 (1H, s), 7.40
(2H, t, J = 7.7 Hz), 7.34 (1H, m), 7.32 (2H, t, J = 7.6 Hz), 7.25 (1H, m), 7.19
(2H, m), 7.18 (1H, s), 3.52 (3H, s). 13C NMR (CDCl3):  150.1, 142.9, 141.4,
137.7, 132.8, 130.4, 128.5, 128.2, 127.4, 126, 125, 113.5, 40.2. Elemental
analysis: (C17H15N3O): Calc: C, 73.63; H, 5.45; N, 15.15. Found: C, 73.25;
H, 5.29; N, 15.05.
N-Methyl-N,2-diphenyl-1H-imidazole-1-carboxamide (8c). To a stirred
suspension of sodium hydride (166 mg, 4.15 mmol, 60% dispersion in
mineral oil) in THF (10 mL) at 0 ºC was added a solution of 2-phenyl-1H-
imidazole 7e (500 mg, 3.47 mmol) in tTHF (5 mL). The mixture was stirred
in the cold for 30 min then was allowed to stir at room temperature for 2 h.
The mixture was cooled again to
0
ºC and
a
solution of
methyl(phenyl)carbamic chloride (616 mg, 3.63 mmol) in THF (5 mL) was
added dropwise. The reaction was stirred in the cold for 0.5 h, then was
allowed to stir at room temperature for 2 days. Thereupon, water was
added at 0 ºC and the solvent was evaporated and then the oily residue
was partitioned between DCM and water. The organic phase was
separated, washed with water and brine, then dried over MgSO4, filtered
and evaporated to leave an oily solid. Chromatography (petroleum ether–
ethyl acetate, 2:1) allowed the separation of the product as a pale yellow
solid (166 mg, 17%), mp.: 123-125 °C. 1H NMR (CDCl3):  7.43-7.24 (6H,
m), 7.14-6.93 (4H, m), 6.33 (2H, br), 3.37 (3H, s br). 13C NMR (CDCl3): 
151.9, 146.5, 141.4, 130.3, 129.3, 129, 128.9, 128.3, 127.3, 126.9, 124.1,
120, 38.9. Elemental analysis: (C17H15N3O): Calc: C, 73.63; H, 5.45; N,
15.15. Found: C, 73.83; H, 5.74; N, 14.82.
N-(2,4-Difluorophenyl)-N-methyl-1H-benzo[d][1,2,3]triazole-1-
carboxamide (10b). To a stirred solution of phosgene (15 mL, 28.5 mmol,
20 % solution in toluene) at 0 ºC was added a solution of 1H-
benzo[d][1,2,3]triazole 9a (1 g, 8.39 mmol) in THF (20 mL) dropwise. The
resulting mixture was stirred in the cold for 0.5 h, then allowed to stir at
room temperature overnight. A strong stream of nitrogen was bubbled
through the mixture for 0.5 h, then the solvent was removed by evaporation
under
reduced
pressure
to
give
the
intermediate
1H-
benzo[d][1,2,3]triazole-1-carbonyl chloride as a clear oil that solidified on
standing (762 mg, 4.2 mmol). The above intermediate was suspended in
THF (20 mL), cooled to 0 ºC and treated with pyridine (0.36 mL, 4.41 mmol)
followed by dropwise addition of 2,4-difluoro-N-methylaniline (601 mg,
4.20 mmol). The reaction mixture was allowed to stir at room temperature
overnight, then cooled to 0 ºC, diluted with water and ethyl acetate. The
organic layer was separated, washed with 1N HCl and brine, then dried
over MgSO4 filtered and evaporated. Recrystallization from isopropanol
gave 10b as a white solid (378 mg, 31%), mp.: 91-92 °C. 1H NMR (CDCl3):
 8.10 (1H, d, J = 8.4 Hz), 8.01 (1H, d, J = 8.1 Hz), 7.63 (1H, m, J = 7.8
Hz), 7.45 (1H, t, J = 7.7 Hz), 7.30 (1H, m), 6.88 (1H, m), 6.84 (1H, m), 3.60
(3H, s). 13C NMR (CDCl3):  161.8 (d d, J = 11.5, 251.0 Hz), 157.7 (d d, J
= 12.5, 252.0 Hz), 150.1, 144.9, 132.7, 129.6, 129.3 (d d, J = 1.5, 10.0 Hz),
127.7 (d d, J = 5.0, 12.0 Hz), 125.4, 119.9, 113.5, 112.1 (d d, J = 4.0, 23.0
N-Cyclohexyl-N-methyl-4-(pyridin-3-yl)-1H-imidazole-1-carboxamide
(8k). To
a
stirred suspension of 3-(1H-imidazol-4-yl)pyridine
dihydrochloride 7f (0.654 g, 3 mmol) in THF (10 mL) was added potassium
tert-butoxide (0.673 g, 6.00 mmol) and the mixture was refluxed for 1 h.
Then, N,N-dimethyl formamide (1 mL) was added and the heating was
continued for additional 30 min The resultant brown suspension was
cooled to room temperature and treated with pyridine (0.37 mL, 4.50 mmol)
and N,N-dimethylpyridin-4-amine (0.037 g, 0.300 mmol) followed by
addition of cyclohexyl(methyl)carbamic chloride (0.553 g, 3.15 mmol). The
reaction was heated to 90 °C overnight. After cooling the mixture was
diluted with water and extracted with ethyl acetate. The organic phase
was dried over MgSO4 and filtered. After evaporation, the crude product
was recrystallized from 2-propanol, crystals were collected and dried
under vacuum to afford 8k (62 mg, 6.9 % yield). mp.: 161-163 °C. 1H NMR
(CDCl3):  9.01 (1H, d d, J = 0.8, 2.3 Hz), 8.53 (1H, d d, J = 1.7, 4.8 Hz),
8.12 (1H, d d d, J = 1.8, 2.2, 8.0 Hz), 7.94 (1H, d, J = 1.3 Hz), 7.58 (1H, d,
J = 1.3 Hz), 7.34 (1H, d d d, J = 0.8, 4.9, 8.0 Hz), 3.95 (1H, m), 3.01 (3H,
s), 1.87 (4H, m), 1.71 (1H, d br, J = 13.5 Hz), 1.59 (2H, d q, J = 3.5, 12.5
Hz), 1.38 (2H, t q, J = 3.5, 13.0 Hz), 1.13 (1H, t q, J = 3.5, 13.5 Hz). 13C
NMR (CDCl3):  151, 148.5, 146.7, 139.2, 137.3, 132.4, 129, 123.6, 114,
57.6, 31.4, 30, 25.4, 25.2. Elemental analysis: (C16H20N4O): Calc: C,
67.58; H, 7.09; N, 19.7. Found: C, 66.77; H, 7.36; N, 19.95.
Hz), 105.1 (d d,
J = 24.0, 26.5 Hz), 39.7. Elemental analysis:
(C14H10F2N4O) Calc: C, 58.33; H, 3.5; N, 19.44. Found: C, 58.66; H, 3.52;
N, 19.75.
N-(2,4-Difluorophenyl)-1H-benzo[d][1,2,3]triazole-1-carboxamide
(10d). To an ice-cooled stirred solution of benzotriazole 9a (300 mg, 2.52
mmol) in DCM (18 mL) was added 2,4-difluoro-1-isocyanatobenzene (410
mg, 2.64 mmol) dropwise. After being stirred for 6 h at room temperature
the solvent was evaporated. Recrystallization from
a mixture of
isopropanol-DCM afforded 10d as a white solid (209 mg, 30%), mp.:148-
150 °C. 1H NMR (CDCl3):  9.28 (1H, s), 8.32 (1H, m, J = 8.3 Hz), 8.27
(1H, m, J = 6.0, 8.9 Hz), 8.17 (1H, m, J = 8.3 Hz), 7.70 (1H, m, J = 8.2 Hz),
7.53 (1H, m, J = 8.1 Hz), 7.0 (2H, m). 13C NMR (CDCl3):  160.6 (d d, J =
11.5, 248.0 Hz), 153.4 (d d, J = 12.0, 249.0 Hz), 146.5, 146.4, 131.5, 130.5,
3-(1-(Cyclohexyl(methyl)carbamoyl)-1H-imidazol-4-yl)pyridine
1-
oxide (8l). To a stirred solution of N-cyclohexyl-N-methyl-4-(pyridin-3-yl)-
9
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10.1002/cmdc.201800393
ChemMedChem
FULL PAPER
125.9, 122.7 (d d, J = 2.0, 9.3 Hz), 121.0 (d d, J = 3.7, 11.0 Hz), 120.4,
113.8, 111.6 (d d, J = 4.0, 22.5 Hz), 104.2 (d d, J = 22.5, 27.0 Hz).
Elemental analysis: (C13H8F2N4O) Calc: C, 56.94; H, 2.94; N, 20.43.
Found: C, 56.86; H, 3.09; N, 20.73.
followed by dropwise addition to a 1.75M ethanolic solution of ammonia
(15 mL, 26.3 mmol) at 0 ºC. The reaction mixture was stirred at room
temperature for 0.5 h. Thereupon, water was added and the ethanol was
evaporated under reduced pressure. The resulting aqueous phase was
extracted with DCM. The organic layer was separated, washed with 1N
HCl, then dried over MgSO4 filtered and evaporated. Recrystallization from
a mixture of DCM – ethanol afforded 11h as a beige solid (151 mg, 20%),
mp.: 216-218 °C. 1H NMR (DMSOd6):  8.71 (1H, d d, J = 0.8, 1.3 Hz),
8.26 (1H, s br), 8.21 (1H, d, J = 1.3, 8.6 Hz), 8.16 (1H, d d, J = 0.8, 8.6 Hz),
7.63 (1H, s br), 5.43 (2H, s), 3.90 (2H, s), 1.58 (6H, s). 13C NMR (DMSOd6):
 166.9, 145.4, 144.6, 133.7, 131.9, 129.1, 119, 113.9, 81.7, 78.7, 61.7,
22.5. Elemental analysis: (C13H15N5O3) Calc: C, 53.97; H, 5.23; N, 24.21.
Found: C, 53.65; H, 5.51; N, 24.54.
1-(4,4-Dimethyloxazolidine-3-carbonyl)-1H-benzo[d][1,2,3]triazole-5-
carboxylic acid (10p). To an ice-cooled stirred suspension of sodium
hydride (7.66 g, 192 mmol) in THF (100 mL) was added a solution of 1H-
benzo[d][1,2,3]triazole-5-carboxylic acid 9b (12.5 g, 77 mmol) in a mixture
of THF (280 mL) and DMF (160 mL) dropwise. The suspension was
allowed to stir at room temperature for 0.5 h followed by dropwise addition
of 4,4-dimethyloxazolidine-3-carbonyl chloride (13.16 g, 80 mmol) in THF
(50 mL) at 0 ºC. The reaction mixture was allowed to stir at room
temperature for 5 h. Thereupon, water was carefully added at 0 ºC, the
THF was evaporated, then 2N HCl was added until pH 2 was reached. The
resulting acidic aqueous phase was extracted three times with a mixture
of DCM-isopropanol (7:3). The combined organic layers were dried over
MgSO4 and evaporated. The obtained mobile liquid was azeotroped with
toluene to leave a brown oil. Purification by column chromatography in a
mixture of DCM-methanol (95:5) afforded 10p as a beige solid (1.8 g, 34%).
1H NMR (DMSOd6):  8.71 (1H, d d, J = 0.8, 1.3 Hz), 8.26 (1H, s br), 8.21
(1H, d, J = 1.3, 8.6 Hz), 8.16 (1H, d d, J = 0.8, 8.6 Hz), 7.63 (1H, s br), 5.43
(2H, s), 3.90 (2H, s), 1.58 (6H, s). 13C NMR (DMSOd6):  166.9, 145.4,
144.6, 133.7, 131.9, 129.1, 119, 113.9, 81.7, 78.7, 61.7, 22.5.
Pharmacology
Animal treatment: All animal procedures conformed to the guidelines
from Directive 2010/63/EU of the European Parliament on the protection
of animals used for scientific purposes and the Portuguese law on animal
welfare (Decreto-Lei 113/2013). Adult male NMRI mice were obtained
from Harlan (Spain). Animals were maintained in macrolon cages
(Tecniplast, Italy) with free access to food (2014 Teklad Global 14%
Protein Rodent Maintenance, Envigo) and tap water under controlled
environmental conditions in a colony room (12 hours light/dark cycle, room
temperature: 22±2 ºC and relative humidity: 60±10%). Experiments were
carried out during daylight hours. Animals were then given just water.
Overnight fasted animals were administered compounds via oral route
(gavage) at 8 mL/kg using animal feeding stainless steel needles
(Perfectum, USA). For the 24 h time point the food was removed in the
morning of the day of administration and compounds administered at the
end of the afternoon of the same day. For ED50 determination doses
administered were 0.001, 0.003, 0.01, 0.03 and 0.1 mg/kg in 10% (v/v)
DMSO. The vehicle for other compound administrations was 0.5% (w/v)
carboxymethylcellulose except for compound 2 that was 5% N,N-
dimethylacetamide and 95% 2-hydroxypropyl-β-cyclodextrin (40% w/v in
water). Fifteen minutes before sacrifice animals were anesthetized with 60
mg/kg pentobarbital sodium administered intraperitoneally. A fragment of
liver, and brain without cerebellum were removed and put in plastic vials
containing membrane buffer (3 mM MgCl2, 1 mM EDTA, 50 mM Tris HCl
pH 7.4). Tissues were stored at -30ºC until processed for analysis where
tissues were thawed on ice and were homogenized in 10 volumes of
membrane buffer with Heidolph Diax (position 5, 30 sec for livers and 20
sec for brains). FAAH activity was evaluated in these preparations.
(6-Bromo-1H-benzo[d][1,2,3]triazol-1-yl)(4,4-dimethyloxazolidin-3-
yl)methanone (11b). Compound 11b was prepared by reaction of 6-
bromo-1H-benzo[d][1,2,3]triazole-1-carbonyl chloride (2.63 g, 10.10
mmol) with 4,4-dimethyloxazolidine (1.5 mL, 10.60 mmol, 75% solution in
water) as described for 10b. Recrystallization from ethanol afforded the
title product as a beige solid (253 mg, 7.7%), mp.: 119-121 °C. 1H NMR
(CDCl3):  8.48 (1H, s br), 7.96 (1H, d, J = 8.7 Hz), 7.59 (1H, d d, J = 1.5,
8.7 Hz), 5.53 (2H, s), 3.90 (2H, s), 1.68 (6H, s). 13C NMR (CDCl3):  145.7,
144.1, 133.7, 129.3, 124.2, 120.8, 117.5, 82.4, 79.5, 62.4, 22.9. Elemental
analysis: (C12H13BrN4O2) Calc: C, 44.33; H, 4.03; N, 17.23. Found: C,
44.08; H, 4.05; N, 17.21.
(4,4-Dimethyloxazolidin-3-yl)(6-phenyl-1H-benzo[d][1,2,3]triazol-1-
yl)methanone (11d). To
benzo[d][1,2,3]triazol-1-yl)(4,4-dimethyloxazolidin-3-yl)methanone
a
stirred suspension of 6-bromo-1H-
11b
(0.300 g, 0.92 mmol), phenyl boronic acid (0.12 g, 0.97 mmol) and sodium
carbonate (0.55 mL, 1.11 mmol) in a mixture of 1-propanol (3.4 mL) and
water
(0.7
mL)
at
room
temperature
was
added
Tetrakis(triphenylphosphine) palladium(0) (53 mg, 0.046 mmol). The
resulting yellow mixture was stirred at 90 ºC for 2 h, then cooled to room
Reagent and solutions: Anandamide [ethanolamine -1-3H-] (3H-AEA,
40–60Ci/mmol) and 2-monoleoyl glycerol (2-OG, 40-60 Ci/mmol) were
obtained from American Radiochemicals (USA). 1-oleoyl-rac-glycerol
(OG), arachidonylethanolamide (AEA), fatty acid free bovine serum
albumin, and all other reagents were obtained from Merck (Germany).
Optiphase Supermix was obtained from Perkin Elmer (USA).
temperature and
a
second crop of tetrakis(triphenylphosphine)
palladium(0) (20 mg, 0.16 mmol) was added. The reaction mixture was
allowed to stir for further 2h at 90 °C. After cooling to room temperature,
the solvent was removed under reduced pressure. The residue was
dissolved in ethyl acetate and the organic phase was washed with water.
The organic phase was dried over MgSO4, filtered and evaporated to leave
a yellow oil. Purification by column chromatography in a mixture of
(petroleum ether–ethyl acetate, 20:1) followed by recrystallization from
isopropanol afforded the title compound 11d as a white solid (72 mg, 24%),
mp.: 146-147 °C. 1H NMR (CDCl3):  8.44 (1H, d d, J = 1.7, 1.6 Hz), 8.14
(1H, d d, J = 0.7, 8.7 Hz), 7.74 (1H, d d, J = 1.6, 8.7 Hz), 7.71 (2H, m, J =
8.2 Hz), 7.50 (2H, m, J = 7.9 Hz), 7.42 (1H, m, J = 7.5 Hz), 5.57 (2H, s),
3.92 (2H, s), 1.70 (6H, s). 13C NMR (CDCl3):  146.2, 144.7, 143.1, 140.2,
133.5, 128.9, 128.1, 127.9, 125.7, 119.9, 112.4, 82.5, 79.6, 62.2, 23.
Elemental analysis: (C18H18N4O2) Calc: C, 67.07; H, 5.63; N, 17.38. Found:
C, 66.92; H, 5.62; N, 17.67.
Enzymatic assays: FAAH activity in vitro and in tissues preparations from
treated animals was evaluated essentially as described in Kiss et al.44 In
brief, for compound evaluation in vitro reaction mix (total volume of 200
mL) contained: 2 µM AEA (2 µM AEA + 5 nM 3H-AEA), 0.1% fatty acid free
BSA (bovine serum albumin), 5 µg rat brain membrane preparation and
the compound to evaluate in 1 mM EDTA, 10 mM Tris pH 7.6. After a 15
min pre-incubation period at 37 ºC, reaction was started by the addition of
the substrate solution (cold AEA + radiolabelled AEA + BSA). Reaction
was carried out for 10 min before termination. For tissues from treated
animals the reaction contained 15 µg (brain) or 5 µg (liver) protein, no
compound, and after a preincubation period of 10 min reaction was carried
out for 10 min (brain) or 7 min (liver) before termination. Reactions were
terminated by the addition of 400 mL activated charcoal suspension (8 g
charcoal in 32 mL 0.5 M HCl in continuous agitation). After a 30 min
incubation period at room temperature with agitation, charcoal was
sedimented by centrifugation in microfuge (10 min at 13000 rpm). 200 µL
of the supernatant were added to 800 µL Optiphase Supermix scintillation
1-(4,4-Dimethyloxazolidine-3-carbonyl)-1H-benzo[d][1,2,3]triazole-5-
carboxamide (11h). To a stirred solution of 1-(4,4-dimethyloxazolidine-3-
carbonyl)-1H-benzo[d][1,2,3]triazole-6-carboxylic acid 10p (0.76 g, 2.63
mmol) and pyridine (0.70 mL, 8.67 mmol) in DCM (17 mL) was added
thionyl chloride (0.63 mL, 8.67 mmol) dropwise at room temperature. The
obtained yellow solution was allowed to stir at room temperature for 15 min
10
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10.1002/cmdc.201800393
ChemMedChem
FULL PAPER
cocktail previously distributed in 24-well plates. Counts per minute (cpm)
were determined in a MicrobetaTriLux scintillation counter (Perkin Elmer).
In each assay blanks (without protein) were prepared. The percentage of
remaining enzymatic activity was calculated with respect to controls (no
compound) and after blank subtraction. In some assays reaction was
terminated by the addition of 400 µL chloroform – methanol (1:1 v/v).
Reactions were vortex twice, left on ice for 5 min and were then centrifuged
in microfuge (7 min at 7000 rpm). Counts per minute were determined in
200 µL of the supernatant as described above.
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Keywords: BIA 10-2474 • Fatty acid amide hydrolase (FAAH) •
Anandamide (AEA) • Pain • Target selectivity
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10.1002/cmdc.201800393
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Entry for the Table of Contents
Herein we describe the discovery of BIA 10-2474, a novel inhibitor of Fatty Acid Amide Hydrolase (FAAH). BIA 10-2474 was
developed from a weak in vitro hit. The paper includes a short SAR of the series and then presents the excellent pharmacodynamic
effect of BIA 10-2474 such as ED50 values in the low g/kg range (mouse, p.o.) and significant inhibition up to 48 h post-dose
(mouse, p.o., 0.1 mg/kg).
13
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