(Indolylalkyl)piperidine carbamates as inhibitors of fatty acid amide hydrolase (FAAH)

Article abstract of DOI:10.1039/c6md00683c

A series of phenyl 4-[(indol-1-yl)alkyl]piperidine carbamates was synthesized and tested for inhibition of the endocannabinoid degrading enzyme fatty acid amide hydrolase (FAAH) and for metabolic stability in rat liver S9 fractions and porcine blood plasma. Structure-activity relationship studies revealed that variation of the length of the alkyl spacer connecting the indole and the piperidine heterocycle, introduction of substituents into the indole ring, replacement of the piperidine by a piperazine scaffold as well as opening of the piperidine ring system affect activity significantly. The metabolic stability of this compound class proved to be significantly higher than that of corresponding phenyl N-(indol-1-ylalkyl)carbamates.

Full text of DOI:10.1039/c6md00683c

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(Indolylalkyl)piperidine carbamates as inhibitors of fatty
acid amide hydrolase (FAAH)†
Cite this: DOI: 10.1039/x0xx00000x
Helmut Dahlhaus, Walburga Hanekamp and Matthias Lehr*
†The authors declare no competing interests.
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of phenyl 4ꢀ[(indolꢀ1ꢀyl)alkyl]piperidine carbamates was synthesized and tested for inhibition of
the endocannabinoid degrading enzyme fatty acid amide hydrolase (FAAH) and for metabolic stability in
rat liver S9 fractions and porcine blood plasma. Structureꢀactivity relationship studies revealed that
variation of the length of the alkyl spacer connecting the indole and the piperidine heterocycle,
introduction of substituents into the indole ring, replacement of the piperidine by a piperazine scaffold
as well as opening of the piperidine ring system affect activity significantly. The metabolic stability of
this compound class proved to be significantly higher than that of corresponding phenyl Nꢀ(indolꢀ1ꢀ
ylalkyl)carbamates.
The endocannabinoid system is involved in numerous physiological
and pathophysiological processes. Central elements of this system
are the cannabinoid receptors CB1 and CB2, endogenous ligands
such as anandamide and 2ꢀarachidonoyl glycerol, enzymes that
synthesize and degrade those ligands, as well as proteins for their
transportation.1ꢀ3An important player in the endocannabinoid metabꢀ
olism is fatty acid amide hydrolase (FAAH).4 This enzyme rapidly
cleaves anandamide into arachidonic acid and ethanolamine and, by
this way, it terminates its beneficial effects, like analgesia and reꢀ
duction of inflammation. Since inhibition of FAAH is supposed to
prolong and enhance the action of anandamide, inhibitors of FAAH
may represent new agents against pain and inflammation.5 In the
past years many potent inhibitors of FAAH have been found6 like
ꢀ
ketoheterocycles,7 heteroarylpropanꢀ2ꢀones,8 urea compounds9 and
carbamate derivatives.10 Despite some drawbacks during the evaluaꢀ
tion of such agents in man,3,11,12 FAAH still can be considered as an
interesting target for new therapeutic approaches.3,13,14
Fig. 1 Inhibitors of FAAH with carbamate structure
The carbamate inhibitors of FAAH can be divided in compounds, in
which the carbamate nitrogen is part of a cyclic structure such as
piperidine or piperazine (e.g. compounds 1 and 2),15ꢀ17 and in derivꢀ
atives, in which this nitrogen is substituted with one alkyl or cycloꢀ
alkyl residue (e.g. compounds 3 and 4) (Figure 1).18ꢀ20
We have developed FAAH inhibitors of the latter class with Nꢀalkyl
substituents bearing heteroarylꢀresidues at the terminal alkylcarbon
like the indolylalkylcarbamates 5ꢀ9 (Figure 1).21 In the present inꢀ
vestigation we wanted to evaluate the effect of an incorporation of
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DOI: 10.1039/C6MD00683C
the carbamate nitrogen and the adjoining alkyl carbons of these peridine 24. Carbamoylation of the piperidine nitrogen of 24 with
alkylcarbamates into a piperidine ring system on their FAAH inhibiꢀ phenyl chloroformate afforded the target carbamate 25.
tory potency and in vitro metabolic stability. Furthermore, more
detailed structureꢀactivity relationship studies with this type of comꢀ
pounds should be performed.
The derivative of compound 5, in which the first three carbon atoms
and the nitrogen of the Nꢀbutyl carbamate chain were integrated into
a piperidine ring was synthesized as outlined in Scheme 1. Piperidinꢀ
4ꢀylmethanol was reacted with phenyl chloroformate to give the
phenyl carbamate 11. The alcohol group of this compound was
substituted with bromine in an Appel reaction using tetrabromoꢀ
methane and triphenylphosphine. Treatment of obtained intermediate
Scheme 3. Reagents and conditions: (a) Methanesulfonyl chloride,
12 with indole, deprotonated by NaH in DMF, afforded the target
compound 13.
triethylamine, CH2Cl2, room temperature, 5 h; (b) indole, NaH,
o
DMF, 60 C, 5 h followed by conc. HCl, methanol, room temperaꢀ
ture, 2 h; (c) phenyl chloroformate, NꢀethylꢀN,Nꢀdiisopropylamine,
CH2Cl2, room temperature, 5 h.
The derivatives of 17 with varying substituents at the phenyl ring of
the indole scaffold (26ꢀ34) and the azaindole compounds 39ꢀ42 were
synthesized by reaction of 16 with the appropriate indole employing
conditions similar to those described for the preparation of 17.
The piperazinecarbamate 38 (Scheme 4) was prepared from 35 using
a reaction sequence analogous to that outlined for the synthesis of 25
(Scheme 3).
Scheme 1 Reagents and conditions: (a) Phenyl chloroformate, Nꢀ
ethylꢀN,Nꢀdiisopropylamine, THF, room temperature, 12 h; (b)
tetrabromomethane, triphenylphosphine, CH2Cl2, room temperature,
4 d; (c) indole, NaH, DMF, room temperature, 20 h.
The homologous derivatives of 13, in which the methylene group
linking the indole with the piperidine ring was replaced by ethylene
and propylene bridges, respectively, were prepared starting from the
piperidinyl alcohols 14 and 18 (Scheme 2). Treatment with phenyl
chloroformate afforded the carbamates 15 and 19. The primary
Scheme 4 Reagents and conditions: (a) Methanesulfonyl chloride,
alcohol groups of these compounds were transformed into mesyl
triethylamine, CH2Cl2, room temperature, 3 h; (b) 6ꢀfluoroindole,
esters utilizing methanesulfonyl chloride and triethylamine. Finally,
o
NaH, DMSO, 60 C, 6 h; (c) conc. HCl, methanol, room temperaꢀ
the mesylates 16 and 20 were reacted with the sodium salt of indole
ture, 24 h followed by phenyl chloroformate, NꢀethylꢀN,Nꢀdiisoꢀ
to yield the desired indolylalkylꢀsubstituted piperidine carbamates 17
propylamine, THF, room temperature, 5 h.
and 21.
Scheme 2 Reagents and conditions: (a) 15: Phenyl chloroformate,
triethylamine, CH2Cl2, room temperature, overnight; 19: phenyl
chloroformate, NꢀethylꢀN,Nꢀdiisopropylamine, THF, room temperaꢀ
ture, 12 h; (b) methanesulfonyl chloride, triethylamine, THF, room
temperature, 2 h; (c) indole, NaH, KI, DMF, 60 oC, 2 h.
Scheme
5 Reagents and conditions: (a) NaH, 5ꢀbromopenꢀ
o
o
tylphthalimide, DMSO, 60 C, 6 h and 90 C, 3 h; (b) hydrazine
hydrate, (24%), ethanol, reflux, 3 h; (c) paraformaldehyde,
NaOCH3, methanol, room temperature, 5 h, NaBH4, room temperaꢀ
ture, 30 min followed by phenyl chloroformate, NꢀethylꢀN,Nꢀdiisoꢀ
propylamine, THF, room temperature, 3 h.
For the synthesis of the homologous derivative with butyl spacer
between indole and piperidine, the BOCꢀprotected piperidinylbutaꢀ
nol 2222,23 was reacted with methanesulfonyl chloride and triethylꢀ
amine (Scheme 3). Substitution of the mesylate group of obtained
intermediate 23 by indole followed by cleavage of the BOCꢀgroup
with conc. HCl in methanol led to the indolylbutylꢀsubstituted piꢀ
The synthesis of the Nꢀmethylated pentylcarbamte 46 started with
the alkylation of 6ꢀfluoroindole (43) with Nꢀ(6ꢀbromohexyl)phthalꢀ
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imide under basic conditions (Scheme 5). Cleavage of the phthalꢀ 0.1 µM each, the compounds with pentyl and heptyl residues (6 and
imide protecting group of obtained intermediate 44 with hydrazine 8) possessed the highest inhibitory activities in this series of comꢀ
provided the amine 45, which was methylated with paraformaldeꢀ pounds (Table 1). Elongation or shortening of the alkyl spacer of 6
hyde/NaBH424 and finally reacted with phenyl chloroformate to yield and 8, respectively, by one carbon reduced FAAH inhibition by
the target carbamate 46.
approximately threeꢀ to tenfold as reflected by the IC50 values of the
carbamates 5, 7 and 9. Similar structureꢀactivity relationships were
The target compounds were evaluated for FAAH inhibition utilizing found for the analogous piperidine carbamates 13, 17, 21 and 25.
microsomes from rat brain as enzyme source25 and the fluorogenic Stepwise extension of the length of the alkyl linker between the
probe Nꢀ(2ꢀhydroxyethyl)ꢀ4ꢀpyrenꢀ1ꢀylbutanamide26 as substrate. To indole and the piperidine ring from one to four carbons was also
the incubation buffer the detergent Triton Xꢀ100 was added for accompanied with a fluctuation in activity. The highest potencies
solubilization of the substrate and for stabilization of the membrane were observed for the derivatives with ethyl and butyl chains 17 and
bound enzyme.27ꢀ29 Inhibitory potency of the test substances was 25. Notably, in these two (indolylalkyl)piperidine carbamates the
assessed by comparing the amount of 4ꢀpyrenꢀ1ꢀylbutanoic acid number of carbons connecting the indole and the carbamate nitroꢀ
released from the substrate in their absence and presence after an gens is identical with that in the most active indolylalkylcarbamates
incubation time of 60 min by reversedꢀphase HPLC with 6 and 8. Furthermore, it can be seen that incorporation of the carbaꢀ
fluorescence detection applying 6ꢀpyrenꢀ1ꢀylhexanoic acid as
internal standard.25 The metabolic stability in rat liver S9 fractions
and in porcine blood plasma was evaluated with reversed phase
HPLC and MSꢀdetection.30
mate nitrogen and the adjoining αꢀ, βꢀ and γꢀalkyl carbons of the
butyl, hexyl and heptyl chain of 5, 7 and 8 respectively, into a piꢀ
peridine ring led to a twoꢀ to tenfold decrease of activity, while in
case of the derivate with a pentyl spacer (6) this structural modificaꢀ
tion did not have a significant impact on inhibitory potency as shown
by the similar IC50 values of 6 and 17.
Investigations on the change of inhibitory potency of the phenyl Nꢀ
indolylalkylcarbamates 5ꢀ9 in dependence of the length of the alkyl
chain exhibited two maxima of activity.21 With IC50 values of about
Table 1 Inhibitory potency against FAAH and stability in biological environments
____________________________________________________________________________
Compound
n
Inhibition of FAAH
IC50 (µM)a
Stability in liver
S9 fractions (%)b
Stability in blood
plasma (%)c
____________________________________________________________________________
5
6
7
8
9
1
2
3
4
5
0.96
0.094
0.25
0.090
0.24
53 ± 5
52 ± 2
50 ± 2
59 ± 3
49 ± 1
82 ± 9
91 ± 7
68 ± 8
39 ± 7
64 ± 4
13
17
21
25
1
2
3
4
9.8
63 ± 5
87 ± 4
80 ± 1
78 ± 6
93 ± 4
94 ± 4
93 ± 3
87 ± 8
0.11
0.57
0.18
3 (URB597)
0.060
82 ± 4
77 ± 6
____________________________________________________________________________
a
Values are the means of at least two independent determinations, errors are within ± 20%; the deviations of the IC50 value of URB597
from published data8,25,26,30,34 are due to variations in the assay procedure (for details see supplementary information of ref. 20);
b
Percent of parent remaining after incubation with rat liver S9 fractions for 30 min in presence of the coꢀfactor NADPH; values are means
± standard deviations of independent determinations (n = 3); the lower stability value published for URB597 recently21 was an artefact.
c
Percent of parent remaining after incubation with porcine blood plasma for 30 min; values are means ± standard deviations of
independent determinations (n = 3);
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Recently, we have found that the stability of the alkylcarbamates 5ꢀ9 Table 2 Inhibitory potency against FAAH
against metabolic phase I reactions in rat liver S9 fractions was quite
similar. About 50ꢀ60% of these compounds remained unmetabolized
after incubation in liver homogenate in presence of NADPH. The
corresponding piperidine carbamates 13, 17, 21 and 25 exhibited a
higher stability under the same conditions. Here, 60ꢀ90% of the
parent compounds could be recovered at the end of the metabolic
reactions (Table 1). In porcine plasma the alkycarbamates 5ꢀ9
showed varying stabilities. While the concentration of the butyl and
pentylcarbamates 5 and 6 decreased only slightly, the heptylꢀ
carbamate 8 was metabolized by about 60%. This significant inꢀ
stability of 8 was due to hydrolysis by plasma paraoxonase and
bis(pꢀnitrophenyl)phosphateꢀsensitive carboxylesterases as proven
with inhibitors of these enzymes.30 In contrast, all new piperidine
carbamates displayed a pronounced stability in porcine blood
_____________________________________
plasma.
Compound
R
Inhibition of FAAH
IC50 (µM)a
The piperidine carbamate with the highest FAAH inhibitory activity
was the derivative with ethyl spacer between piperidine and indole
residue. Since this compound (17) possessed a high metabolic
stability in liver homogenate as well as in blood plasma, it was
chosen for further inhibitor development. First, different residues
were introduced at indole 5ꢀposition. While chloro, methyl, methoxy
and cyano substituents reduced inhibition of FAAH by fourꢀ to
tenfold, a fluoro atom did not affect activity significantly (Table 2).
Next, a fluoro atom was introduced in the other three free positions
of the phenyl part of the indole. In position 4, this atom also did not
change FAAH inhibitory potency. In contrast, fluoro atoms in
position 6 (32) and 7 (33) enhanced activity about twofold. As
shown by the IC50 values of 34 and 38, exchange of the indole ring
of 17 by a carbazole heterocycle, and replacement of the piperidine
ring of 32 by a piperazine system resulted in an about fourfold drop
of potency. The four azaindoleꢀsubstituted derivatives 39ꢀ42 (Table
3) were also less active against FAAH than the corresponding indole
17 (Table 2), with the compounds bearing nitrogen atoms in indole 6
and 7 position (41 and 42) possessing a higher activity than the 5ꢀ
and 6ꢀsubstituted analogues 39 and 40.
_____________________________________
17
26
27
28
29
30
31
32
33
34
38
H
5ꢀF
0.11
0.13
0.41
0.70
0.36
0.97
0.11
0.046
0.052
0.41
0.18
5ꢀCl
5ꢀCH3
5ꢀOCH3
5ꢀCN
4ꢀF
6ꢀF
7ꢀF
_____________________________________
a
Values are the means of at least two independent determinations,
errors are within ± 20%;
Table 3 Inhibitory potency of azaindoleꢀsubstituted 4ꢀethylpiperiꢀ
dine carbamates against FAAH
Finally, we were interested in the question, whether the intact piperiꢀ
dine ring of 32 is necessary for its pronounced activity. Therefore,
we synthesized compound 46, in which carbon 3 of the piperidine
ring is missing. This structural variation led to an about 35ꢀfold
decrease of activity (Table 4). Thus, the rigid carbamate structure is
necessary for good enzyme inhibition. Viewed from the side of the
pentyl carbamate 6 it can be said that methylation of the nitrogen of
this aliphatic carbamate is not well tolerated by the enzyme.
_____________________________________
Compound
Position of the
nitrogen in the
indole ring
Inhibition of FAAH
IC50 (µM)a
FAAH belongs to the large group of serine hydrolase enzymes,
which comprises of about 200 members.31 Phenyl carbamate inhibiꢀ
tors of FAAH have been shown to inactivate the enzyme by covalent
carbamoylation of the enzyme´s catalytic nucleophilic serine.17,32
Since other members of the serine hydrolase family can also react
with carbamates in the same way in principle, we tested the most
active piperidine carbamate 32 for inhibition of the serine hydrolases
monoacylglycerol lipase (MAGL) and cytosolic phospholipase A2α
(cPLA2α). MAGL is also involved in the process of endocannaꢀ
binoid inactivation,1,2 cPLA2α is the key enzyme of the arachidonic
acid cascade.33 Like FAAH, both enzymes hydrolyze very lipophilic
_____________________________________
39
40
41
42
4
5
6
7
1.7
0.79
0.31
0.20
_____________________________________
a
Values are the means of at least two independent determinations,
errors are within ± 20%;
substrates. At the highest test concentration of 10 µM, MAGL34 and
5
cPLA2α
were not affected by compound 32. These data provide
evidence that the compound class investigated exhibits some speciꢀ
ficity for FAAH.
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Table 4 Inhibitory potency against FAAH and stability in biological environments
____________________________________________________________________________
Compound
Inhibition of FAAH
IC50 (µM)a
Stability in liver
S9 fractions (%)b
Stability in blood
plasma (%)c
____________________________________________________________________________
32
46
0.046
1.6
82 ± 1
65 ± 5
93 ± 7
93 ± 3
____________________________________________________________________________
a
Values are the means of at least two independent determinations, errors are within ± 20%;
Percent of parent remaining after incubation with rat liver S9 fractions for 30 min in presence of the coꢀfactor NADPH; values are means
b
± standard deviations of independent determinations (n = 3);
c
Percent of parent remaining after incubation with porcine blood plasma for 30 min; values are means ± standard deviations of
independent determinations (n = 3).
11 J. P. Huggins, T. S. Smart, S. Langman, L. Taylor and T. Young,
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In conclusion, integration of the amino group and the ꢀ, ꢀ and
ꢀ
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porcine plasma can be enhanced by this structural variation. With the
piperidine carbamate 32 a FAAH inhibitor was obtained, which
possesses similar activity and metabolic stability as the known
FAAH standard inhibitor URB597.
16
A. Makriyannis, V. G. Shukla and S. O. Alapafuja,
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Institute of Pharmaceutical and Medicinal Chemistry, University of Münster,
Corrensstrasse 48, Dꢀ48149 Münster, Germany, Eꢀmail: lehrm@uniꢀ
muenster.de; Fax: +49 (251) 8332144; Tel: +49(251)8333331
Electronic Supplementary Information (ESI) available: Syntheses and
analytical data of the test compounds. See DOI:
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