ω-Heteroarylalkylcarbamates as inhibitors of fatty acid amide hydrolase (FAAH)

Article abstract of DOI:10.1039/c4md00181h

Fatty acid amide hydrolase (FAAH) is a serine hydrolase that terminates the analgetic and anti-inflammatory effects of endocannabinoids such as anandamide. Herein we describe structure-activity relationship studies on a new series of ω-heteroarylalkylcarb

Full text of DOI:10.1039/c4md00181h

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u-Heteroarylalkylcarbamates as inhibitors of fatty
acid amide hydrolase (FAAH)†
Cite this: DOI: 10.1039/c4md00181h
*
Tobias Terwege, Helmut Dahlhaus, Walburga Hanekamp and Matthias Lehr
Fatty acid amide hydrolase (FAAH) is a serine hydrolase that terminates the analgetic and anti-inflammatory
effects of endocannabinoids such as anandamide. Herein we describe structure–activity relationship
studies on a new series of u-heteroarylalkylcarbamate inhibitors of FAAH. The most active compounds
exhibit IC50 values in the low nanomolar range. Investigations on selectivity and metabolic stability of
these inhibitors are also presented.
Received 22nd April 2014
Accepted 15th May 2014
DOI: 10.1039/c4md00181h
www.rsc.org/medchemcomm
Anandamide is one principal endocannabinoid in the substituents into the ring at the free carbon site, which could
mammalian organism.1,2 It is formed “on demand” during lead to additional binding interactions with FAAH. Therefore,
several pathological disorders and mediates analgetic and anti- we have synthesized the pyrrole analogue of 2 and used it as
inammatory effects by activation of the cannabinoid receptors starting point for structure–activity relationship studies.
CB1 and CB2. These two receptors are differentially distributed
The pyrrolyl-substituted N-alkylphenylcarbamates investi-
within the various cells.3–5 While the CB1 receptor is preferen- gated were synthesized using one of the three routes outlined
tially expressed in neuronal tissues, the CB2 receptor occurs in Scheme 1. In the rst instance, the appropriate nitrogen
mainly in cells of the immune system such as macrophages.
heterocycle was alkylated with a BOC-protected u-bro-
An important enzyme in the endocannabinoid metabolism moalkylamine followed by cleaving of the BOC group with
is fatty acid amide hydrolase (FAAH), which rapidly cleaves the triuoroacetic acid and reaction of obtained amine with
lipid mediator anandamide into arachidonic acid and etha- phenyl chloroformate. Alternatively, the heterocycle was alky-
nolamine.6–9 Inhibition of FAAH is supposed to potentiate the lated with
a dibromoalkane. The obtained bromoalkyl
action of anandamide. Therefore, inhibitors of FAAH may compound was treated with potassium phthalimide yielding a
represent new agents against pain and inammation. In the phthalimide-protected alkylamine. Removal of the protecting
past years many potent inhibitors of FAAH have been found.10–14 group was accomplished using hydrazine hydrate and the
The majority of these compounds are substances, which form formed amine was directly reacted with phenyl chloroformate
covalent binding interactions with the catalytic serine of the to yield the target compound. Instead, the phthalimide inter-
active site of FAAH such as carbamates like 1 (URB 597)15 and mediate was directly prepared by reaction of the nitrogen
2,16 activated ketone derivatives like the a-ketoheterocycle 3 heterocycle with u-bromoalkyphthalimide as shown for the
(PHOP)17 and the propan-2-one 4,18 (Fig. 1), and urea derivatives synthesis of 27 and 30.
like 5 (PF-04457845).19 The latter compound is being examined
in clinical studies for fear response and cannabis withdrawl.20
Several years ago we have described dual inhibitors of
cyclooxygenase and 5-lipoxygenase, which contain a pyrrole
scaffold with vicinal phenyl substituents (e.g. 6)21 (Fig. 2)
showing structural similarities with the heterocyclic part of the
FAAH inhibitor 2.16 This initiated our interest in the question,
in which way inhibitory potency of 2 is inuenced when the
2-methyl-4,5-diphenylimidazole system of 2 is replaced by a
2,3-diphenyl-5-methylpyrrole substituent. Moreover, this
pyrrole ring system offered the opportunity to introduce further
¨
Institute of Pharmaceutical and Medicinal Chemistry, University of Munster,
¨
Corrensstrasse 48, D-48149 Munster, Germany. E-mail: lehrm@uni-muenster.de;
Fax: +49 251 8332144; Tel: +49 251 8333331
† Electronic supplementary information (ESI) available: Syntheses and analytical
data of the test compounds; procedures for biological evaluation. See DOI:
10.1039/c4md00181h
Fig. 1 Structure of known inhibitors of FAAH.
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The indole derivatives with 3-pyridyl carbamate residues
(52 and 54) were synthesized with the latter method utilizing
3-pyridyl chloroformate, synthesized from diphosgene and
pyridin-3-ol, instead of phenyl chloroformate in the last step
(Scheme 2).
Inhibition of FAAH hydrolase by the test compounds was
determined with an assay using microsomes from rat brain as
enzyme source and N-(2-hydroxyethyl)-4-pyren-1-ylbutanamide
as uorogenic substrate.23 Inhibitory potencies of the tested
compounds were assessed by comparing the amount of 4-pyren-
1-ylbutanoic acid released from the substrate in their absence
and presence aer an incubation time of 60 min by reversed-
phase HPLC with uorescence detection (see ESI†). With this
assay for the diphenylimidazole 2 an IC50 of 0.35 mM was
measured (Table 1). Replacement of the imidazole scaffold of 2
by a pyrrole (9) led to an about sevenfold loss of activity.
Shortening of the alkyl chain connecting the carbamate group
and the pyrrole heterocycle from six to four and two carbon
atoms further decreased inhibitory potency. With an IC50 of 6.2
mM the butyl derivative 13 was only about half as active as the
hexyl compound 9. The derivative with an ethyl linker (15) even
was inactive at the highest test concentration of 10 mM. Because
removing of the methyl group in position 2 of the imidazole ring
of 2 did not change the activity of the compound,24 the impact of
the corresponding methyl group of the pyrrole 9 on inhibitory
potency was not examined.
Fig.
inhibitor 6.
2 Structure of the dual cyclooxygenase–5-lipoxygenase
Introduction of polar ethoxycarbonyl, acetyl and cyano
substituents at the free position of the pyrrole ring of 9 was also
detrimental. The inhibition values of obtained compounds 18,
21 and 24 were in the magnitude of 10 mM (Table 2). Interest-
ingly, moving the phenyl group from position 3 to position 4 of
the pyrrole ring, to give compound 27, as well as completely
removing the phenyl ring, to give compound 30, signicantly
increased FAAH inhibition. The activity of these two inhibitors
was comparable to that of the diphenylimidazole 2.
Similar results were obtained with analogous indole deriva-
tives (Table 3). With IC50 values of about 5 mM the 2,3-dipheny-
lindole 32 and the 3-phenylindole 34 possessed the lowest potency
of the series. The 2-phenylindole 36 was about tenfold more active
than the 3-phenylderivative 34. Thus, a phenyl substituent is
much better tolerated in position 2 than in position 3 of the indole
scaffold. The indole derivative 38, which has no phenyl-substitu-
ents in 2- and 3-position, possessed the highest activity (IC50
0.25 mM) showing that phenyl substituents are even unfavourable.
Scheme 1 Reagents and conditions: (a) tert-butyl-N-(6-bromohexyl)-
carbamate–tert-butyl-N-(2-bromoethyl)carbamate, K-tert-butylate,
DMSO, 60/80 ꢀC; (b) (1) trifluoroacetic acid, CH2Cl2, 0 ꢀC; (2) phenyl
chloroformate, triethylamine, CH2Cl2, room temp.; (c) 1,4-dibromo-
butane, tetrabutylammonium bromide, toluene, 50% aqueous NaOH,
45 ꢀC; (d) potassium phthalimide, DMF, 130 ꢀC; (e) (1) hydrazine-
hydrate, ethanol, reflux; (2) phenyl chloroformate, triethylamine,
CH2Cl2, room temp.; (f) N-(6-bromohexyl)phthalimide, K-tert-buty-
late, DMSO, 50/70 ꢀC; (g) (1) hydrazine-hydrate, ethanol, reflux; (2)
phenyl chloroformate, triethylamine, CH2Cl2, 0 ꢀC to room temp.
The related derivatives with indole, indazole, imidazole and
benzotriazole heterocycle were prepared similarly to Method C
of Scheme 1 using K-tert-butylate–DMSO, NaH–DMSO, NaH–
DMF, K2CO3–DMSO and K2CO3–acetonitrile, respectively, for
the N-alkylation of the appropriate heterocycle with N-(u-bro-
moalkyl)phthalimide.22
Scheme 2 Reagents and conditions: (a) N-(u-bromoalkyl)phthali-
mide, NaH, DMSO, 100 ꢀC; (b) (1) hydrazine-hydrate, ethanol, reflux, 2
h; (2) trichloromethyl chloroformate, pyridin-3-ol, ethyl(diisopropyl)-
amine, CH2Cl2, THF, – 30 ꢀC to room temp.
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Table 1 Inhibition of FAAH
Table 3 Inhibition of FAAH
Compound
R1
R2
Inhibition of FAAH IC50a (mM)
32
34
36
38
Phenyl
H
Phenyl
H
Phenyl
Phenyl
H
4.7
4.9
0.52
0.25
Compound
n
Inhibition of FAAH IC50a (mM)
H
9
13
15
6
4
2
2.6
6.2
a
Values are the means of at least two independent determinations,
n.a.b
0.060
0.35
0.0029
errors are within ꢁ 20%.
1 (URB 597)
2
3 (PHOP)
were about 0.09 mM (Table 5). A more pronounced variation of
the chain length to four (45) or eight (51) carbons resulted in a
reduction of FAAH inhibition.
a
Values are the means of at least two independent determinations,
errors are within ꢁ 20%. b n.a.: not active at 10 mM.
In literature a series of highly potent 3-pyridyl carbamate
inhibitors of FAAH is described.25 Therefore, we nally wanted
to investigate the effect of a replacement of the phenyl group of
the carbamates 47 and 38 by a 3-pyridyl residue. This structural
variation led to a drastic increase of activity (Table 5). With IC50
values of 0.0036 mM and 0.0052 mM obtained compounds 52
and 54 were about as active as the a-ketoheterocycle 3 (PHOP),
which counts to the most active inhibitors of FAAH known
today.
Table 2 Inhibition of FAAH
Besides FAAH, monoacylglycerol lipase (MAGL) is another
important enzyme during the process of endocannabinoid
inactivation. Just like FAAH, MAGL contains a catalytic serine in
the active site. The main substrate of MAGL is 2-arachidonoyl
glycerol. To get some information about the specicity of the
developed substance class, some of the new FAAH inhibitors
were also tested for MAGL inhibition.26 As shown in Table 5, the
evaluated phenyl u-(indol-1-yl)alkylcarbamates did not inhibit
MAGL at the highest test concentration of 10 mM. Contrary, with
Compound
R1
R2
Inhibition of FAAH IC50a (mM)
9
Phenyl
Phenyl
Phenyl
Phenyl
H
H
2.6
18
21
24
27
30
COOCH3
COCH3
CN
Phenyl
H
>10b
>10c
8.7
0.46
0.31
H
a
Values are the means of at least two independent determinations,
b
c
errors are within ꢁ 20%. 21% inhibition at 10 mM. 33% inhibition
at 10 mM.
Table 4 Inhibition of FAAH
Next the effect of an introduction of nitrogen atoms in
position 2 and 3 of the indole ring of 38 was studied. The
indazole and benzotriazole derivatives 40 and 42 bearing a
nitrogen in position 2 of the heterocycle were about three- to
fourfold less active than the indole 38 (Table 4). In contrast, the
imidazole derivative 44 possessing an additional nitrogen only
in position 3 was equipotent to the indole 38.
Taken together, replacement of the diphenylpyrrole hetero-
cycle from the starting compound 9 by an unsubstituted indole
(38) led to an about tenfold increase of FAAH inhibitory potency.
At this point we wanted to evaluate the role of the alkyl linker of
38 on activity in more detail. Both shortening and elongation of
the hexyl chain by one carbon increased activity about twofold:
the IC50 values of the pentyl and heptyl derivatives 47 and 49
Compound
X
Y
Inhibition of FAAH IC50a (mM)
38
40
42
44
CH
N
N
CH
CH
N
0.25
0.92
0.81
0.29
CH
N
a
Values are the means of at least two independent determinations,
errors are within ꢁ 20%.
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Table 5 Inhibitory potency against FAAH and MAGL and stability in biological environments of several u-indolylalkylcarbamates
Inhibition of
Inhibition of MAGL
Stability in liver
S9 fractionsb (%)
Stability in blood
plasmac (%)
Compound
n
R
FAAH IC50a (mM)
IC50a (mM)
45
47
38
49
51
52
54
4
5
6
7
8
5
6
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Pyridin-3-yl
Pyridin-3-yl
0.96
0.094
0.25
0.090
0.24
0.0036
0.0052
0.060
n.a.d
n.a.d
n.a.d
n.a.d
n.a.d
0.22
0.46
n.t.e
53 ꢁ 4.7
52 ꢁ 2.3
50 ꢁ 1.5
59 ꢁ 3.1
49 ꢁ 0.3
43 ꢁ 7.0
47 ꢁ 13
13 ꢁ 4.6
82 ꢁ 9.0
91 ꢁ 6.5
68 ꢁ 7.8
39 ꢁ 7.4
64 ꢁ 4.2
0
0
1 (URB 597)
76 ꢁ 12
a
Values are the means of at least two independent determinations, errors are within ꢁ 20%. b Percent of parent remaining aer incubation with rat
liver S9 fractions for 30 min, in presence of the co-factor NADPH; values are means ꢁ standard deviations of independent determinations (n ¼ 3).
c
Percent of parent remaining aer incubation with porcine blood plasma for 30 min; values are means ꢁ standard deviations of independent
determinations (n ¼ 3). d n.a.: not active at 10 mM. e n.t.: not tested.
IC50 values in the submicromolar range, the corresponding
pyridyl carbamates 52 and 54 inhibited MAGL signicantly.
Conclusions
However, the inhibitory potency of these substances against
FAAH is still 60- to 90-fold higher than against MAGL.
In conclusion, starting from the lead compounds 2 and 9,
respectively, a pronounced increase of FAAH inhibitory potency
could be attained by only a few structural variations. The most
active compounds synthesized (52 and 54) exhibited IC50 values
in the low nanomolar range.
Another enzyme of the serine hydrolase family is cytosolic
phospholipase A2a (cPLA2a), which catalyzes the rst step of the
so called arachidonic acid cascade by cleaving membrane
phospholipids in arachidonic acid and lysophospholipids. At
the highest test concentration of 10 mM cPLA2a was not
inhibited27 by the indole compounds listed in Table 5. These
results indicate that the inhibitors investigated display a certain
specicity with regard to their ability to inhibit serine hydrolase
enzymes.
Since carbamates are known to be susceptible to hydrolysis in
biological environments28–30 leading to the inactivation of the
compounds, we also measured the stability of some of the new
substances in rat liver S9 fractions in presence of the co-factor
NADPH31 and in porcine blood plasma (Table 5). Aer incubation
in the liver S9 fractions for 30 min, still about 50% of the selected
indole compounds were present in the incubation mixture. For
comparison, the metabolism rate of the known FAAH inhibitor
URB 597, which also exhibits a carbamate structure, was signif-
icantly higher. Here, only about 13% of the parent compound
could be detected aer the metabolic reactions. Incubation in
blood plasma for 30 min revealed striking different results. The
pyridin-3-yloxy carbamates 52 and 54 had totally disappeared at
the end of the trial most likely due to an extensive hydrolysis or
transcarbamoylation. The stability of the phenoxycarbamates
depended on the chain length of the alkyl spacer. The pentyl-
substituted compound 47 exhibited high stability, with 91%
parent remaining aer incubation. In contrast, only 39% of the
heptyl derivative 49 could be detected under the same conditions.
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