Identification of a novel arylpiperazine scaffold for fatty acid amide hydrolase inhibition with improved drug disposition properties

Article abstract of DOI:10.1016/j.bmcl.2012.11.035

We herein describe the systematic approach used to develop new analogues of compound 2, recently identified as a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Aiming at identifying new scaffolds endowed with improved drug disposition properties with respect to the phenylpyrrole-based lead, we subjected it to two different structural modification strategies. This process allowed the identification of derivatives 4b and 5c as potent, reversible and non-competitive FAAH inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.11.035

Bioorganic & Medicinal Chemistry Letters 23 (2013) 492–495
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a novel arylpiperazine scaffold for fatty acid amide
hydrolase inhibition with improved drug disposition properties
Stefania Butini a, Sandra Gemma a, Margherita Brindisi a, Samuele Maramai a, Patrizia Minetti b,
,
⇑
Diana Celona b, Raffaella Napolitano b, Franco Borsini b, Walter Cabri b, Filomena Fezza c, Lucio Merlini d,
c
Sabrina Dallavalle d, Giuseppe Campiani a, , Mauro Maccarrone
⇑
a European Research Centre for Drug Discovery and Development (NatSynDrugs), Università degli Studi di Siena, Via Aldo Moro, 53100 Siena, Italy
b Sigma-Tau Industrie Farmaceutiche Riunite spa, Via Pontina Km 30,400, 00040 Pomezia, Italy
c Dipartimento di Scienze Biomediche, Università degli Studi di Teramo, Piazza A. Moro 45, 64100 Teramo, Italy
d DeFENS, Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università di Milano, Via Celoria 2, 20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein describe the systematic approach used to develop new analogues of compound 2, recently
identified as a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Aiming at identifying
new scaffolds endowed with improved drug disposition properties with respect to the phenylpyrrole-
based lead, we subjected it to two different structural modification strategies. This process allowed the
identification of derivatives 4b and 5c as potent, reversible and non-competitive FAAH inhibitors.
Ó 2012 Published by Elsevier Ltd.
Received 15 October 2012
Revised 7 November 2012
Accepted 9 November 2012
Available online 22 November 2012
Keywords:
Fatty acid amide hydrolase (FAAH)
Enzyme inhibitors
Solubility
Cannabinoid system
Drug disposition properties
Fatty acid amide hydrolase (FAAH) is a membrane bound en-
zyme which regulates the intracellular level of anandamide (AEA)
and other endocannabinoids (ECs).1 AEA is transported across the
plasma membrane and then by intracellular transporters to gain
facilitated access to its final targets.2–6 Subsequent hydrolysis by
FAAH drives AEA uptake by creating and maintaining a concentra-
tion gradient across the plasma membrane. FAAH utilizes a Ser-
Ser-Lys catalytic triad as confirmed by X-ray analysis.7 Inactivation
of FAAH elevates the endogenous concentrations of its substrates
thus potentiating their beneficial effects on the modulation of pain,
inflammation, and anxiety.8 This modulation of ECs catabolizing
enzymes for controlling the endocannabinoid system is an intrigu-
ing possibility with respect to direct global activation of the recep-
tors.2 Indeed, the therapeutic effects could be elicited by avoiding
cannabinoid agonists side effects (hypomotility, hypothermia,
and catalepsy). The design of selective and druggable small-mole-
cule inhibitors of FAAH remains an essential step in the exploita-
tion of this enzyme as a therapeutic target.9–12 However, for the
identification of potential clinical candidates the use of the sub-
strate as template may lead to inhibitors characterized by a num-
ber of drawbacks such as poor drug disposition properties (high
lipophilicity).
The FAAH inhibitors can be classified as irreversible and revers-
ible inhibitors. The early designed irreversible inhibitors were sub-
strate-inspired compounds (e.g., the one digit nanomolar inhibitor
methoxyarachidonoyl fluorophosphonate). Successively, other
structurally diverse FAAH inhibitors were developed. Among them,
carbamates and ureas act as irreversible FAAH inhibitors such as
URB59713 and JP8314 (1, Fig. 1), while the
a-ketooxazole
OL135,15 and the recently identified enol carbamates,16 and
ST391317,18 (2, Fig. 1), were classified as reversible inhibitors. Fur-
thermore, the piperazine urea JNJ-166101019 (3, Fig. 1) was found
as a covalent but slowly reversible inhibitor.
The current study reports the synthesis and the in vitro biolog-
ical investigation of a series of novel FAAH inhibitors (4a–e and 5a–
f, Fig. 2) the development of which was based on the model of our
previously identified potent and selective FAAH reversible inhibi-
tor 2. The new scaffolds were conceived following the lines of ap-
proach described in Figure 2, by taking into account two key issues
for the development of a good (pre)clinical candidate, namely: (i)
the drug disposition profile and (ii) the possibility of a rapid ana-
loging. Since a successful drug candidate should possess a balance
of potency and drug-like properties (e.g., water solubility), our lead
⇑
Corresponding authors. Tel.: +39 06 91393906; fax: +39 06 91393638 (P.M.);
tel:+39 0577 234172; fax: +39 0577 234333 (G.C.).
E-mail addresses: patrizia.minetti@sigma-tau.it (P. Minetti), campiani@unisi.it
(G. Campiani).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.11.035

S. Butini et al. / Bioorg. Med. Chem. Lett. 23 (2013) 492–495
493
NH
MeO
O
H
N
H
N
Br
N
O
O
O
( )6
OMe
( )6
N
S
b
c,d
O
O
R
N
N
N
N
N
6a, R = H
6b, R = CHO
6c, R = CN
a
NH2
R
R
NH2
2, ST3913
7a, R = H
8a, R = H
O
7b, R = CN
8b, R = CN
O
N
H
1, JP83
e
O
3, JNJ1661010
N
N
N
H
N
6
6
Figure 1. Reference FAAH inhibitors.
N
optimization strategies include solubility enhancement, performed
through introduction of ionizable groups and of H-bond donors/
acceptors. The insertion of a piperazine moiety represents a com-
mon approach for providing reduced lipophilicity while increasing
solubility. We therefore rationally modified the core structure of
our lead 2 by appending a piperazine ring on the 1-phenylpyrrole
system. This represented a drug disposition properties optimiza-
tion, leading to scaffold A analogues (Fig. 2). An additional cycle
of optimization involved the replacement of the pyrrole system
by a piperazine ring (scaffold B analogues, Fig. 2). Accordingly,
from the structural point of view, all the newly developed
piperazine-containing compounds can be clustered in two main
subgroups (i) the bis-aryl derivatives 4a–e, and (ii) the arylpipera-
zines 5a–f.
Schemes 1 and 2 describe the synthesis of compounds 4a–e
belonging to the first template. Compounds 4a–d (Scheme 1) were
synthesized starting from the appropriate dimethoxytetrahydrofu-
ran derivatives (6a,c) which were subjected to a Clauson–Kaas
reaction to provide the corresponding 1-phenylpyrrole derivatives
(7a,b). A palladium catalyzed amination of Boc-piperazine20,21 fol-
lowed by Boc-deprotection22 afforded the key intermediates 8a,b.
Reaction of the piperazine-derivatives with phenylhexyl bromide
provided the alkylated piperazine 4a, and after partial hydrolysis
of the nitrile functionality the amido-derivative 4b. Reaction of
8a,b with phenylhexylisocyanate provided urea derivatives 4c
and 4d. For the synthesis of compound 4e (Scheme 2), the key
intermediate 10 was reacted with Boc-piperazine by a highly yield-
ing coupling procedure performed in the presence of N-bromosuc-
cinimide and triphenylphosphine.23 Boc-deprotection followed by
piperazine N4-alkylation and partial nitrile hydrolysis led to 4e.
g and
f, for 4d
N
N
4a, R = CN
4b R = CONH2
R
4c, R = H
4d, R = CONH2
f
R
Scheme 1. Reagents and conditions: (a) I2, NH4OH, THF, rt, 3 h, 46%; (b) 3-
bromoaniline, HCl 5 N, dioxane, reflux, 30 min, 92%; (c) Boc-piperazine, Pd2(dba)3,
( )-BINAP, NaO-t-Bu, dry toluene, 70 °C, 10 h, 70%; (d) AcCl, MeOH, rt, 30 min, 99%;
(e) phenylhexyl bromide, MeCN, TEA, reflux, 15 h, 82%; (f) 6 N NaOH, 35% wt H2O2,
EtOH, reflux, 12 h, 55–63%; (g) phenylhexyl isocyanate, TEA, dry THF, reflux, 8 h,
52%.
Schemes 3 and 4 describe the synthesis of compounds 5a–f. The
phenylpiperazines 12a,b,d were reacted with functionalized aryl or
aralkyl isocyanates, and after appropriate deprotection of the
piperazine functionality, compounds 5a–d were obtained. The
preparation of compound 5e was accomplished by an alkylation
reaction of the Cbz-protected 3-hydroxyphenylpiperazine 12d
with 4-fluorophenoxybutyl bromide, followed by Cbz removal.
Oxime carbamate derivative 5f (Scheme 4) was prepared by reac-
tion of the ketone 13 with hydroxylamine in ethanol, followed by
treatment of the oxime 14 with 1-piperidine carbonylchloride.
For the development of novel FAAH inhibitors endowed with an
optimized solubility profile, we modified our potent and selective
lead candidate 2 (Ki = 0.16 nM, on mFAAH)17 while preserving
nanomolar inhibitory activity. Accordingly, starting from the core
scaffold of 2, we adopted a strategy involving the two main struc-
tural tunings outlined in Figure 2 (see calculated LogS in Table 1).
The first subgroup of compounds (scaffold A analogues, 4a–e)
Figure 2. Strategies for optimizing the drug disposition properties of 2.

494
S. Butini et al. / Bioorg. Med. Chem. Lett. 23 (2013) 492–495
Table 1
O
Inhibition of mouse brain FAAH activity (Ki, nM) by compounds 4a–e and 5a–f
O
OH
MeO
O
HO
a
X
Y
OMe
N
CN
6c
NH2
N
N
CN
b,c
9
10
N
R
R1
Scaffold A
Scaffold B
O
O
4a-e
5a-f
N
N
Compd
R
X
Kia (nM)
LogSb
À6.18
N
NH
2HCl
6
d,e
N
N
N
4a
CN
270
N
( )6
( )6
N
N
4e
CONH2
11
CN
4b
4c
CONH2
H
10
À5.39
À6.91
N
N
Scheme 2. Reagents and conditions: (a) 5 N HCl, dioxane, reflux, 30 min, 45%; (b)
Boc-piperazine, N-bromosuccinimide, Ph3P, pyridine, dry DCM, rt, 5 h, 87%; (c) AcCl,
MeOH, rt, 1 h, 99%; (d) phenylhexylbromide, MeCN, TEA, reflux, 15 h, 77%; (e) 6 N
NaOH, 35% wt H2O2, EtOH, rt, 12 h, 36%.
H
N
263
( )6
O
N
O
H
N
N
4d
4e
CONH2
CONH2
>500
>500
À6.49
À5.10
( )6
O
N
OH
b, then
H
N
O
R2
N
O
O
( )6
N
c for 5a
N
d for 5c,d
Compd
R1
H
Y
Kia (nM)
LogSb
À1.93
N
N
O-nBu
R1
R1
5a-d
R2
5a
>500
12a, R1 = Boc
12b, R1 = Me
12c, R1 = H
O
O
N
H
R1
Cpd
5a
a
H
N
nBu
12d, R = Cbz
H
5b
5c
Me
H
240
13
À3.39
À2.38
o
( )6
O
( )6
( )6
Me
5b
e,d
H
N
5c
H
H
O
( )6
O
O
( )4
nHep
5d
O
O
o
O-nHep
F
5d
5e
H
H
>500
>500
À2.89
À1.69
N
O
O
N
H
O
N
H
( )4
5e
F
Scheme 3. Reagents and conditions: (a) KHCO3, benzyl chloroformate, H2O/
acetone, 0 °C, 1 h, 89%; (b) alkyl or aralkyl isocyanate, TEA, dry THF, rt, 8 h, 46–
67%; (c) AcCl, MeOH, rt, 1 h, 99%; (d) H2, Pd/C, AcOH, rt, 12 h, 59%; (e) K2CO3, 4-
fluorophenoxybutyl bromide, acetone, 60 °C, 10 h, 70%.
O
N
N
5f
2c
Me
—
>500
0.16
À2.69
À5.69
O
Me
—
a
Enzyme inhibition tests were performed as in Ref. 17, each value is the mean of
at least three experiments (all SD are within 10% of the mean).
b
Calculated LogS in water at pH 7, ACD/Labs V12.0, Toronto, Canada.
Data from Ref. 17.
c
O
N
OH
N
O
N
springs from the goal of optimizing our lead 2 while maintaining
the 1-phenylpyrrole template. Accordingly, we substituted the car-
bamoyl moiety of 2 by: (i) a piperazine (4a,b); (ii) a piperazine urea
(4c,d); (iii) a piperazine amide (4e). Moreover, these modifications
were combined with different functionalizations at position 3 of
the pyrrole ring of 2 (such as removal of the amide moiety (4c)
or insertion of a nitrile group (4a)). The second subgroup of com-
pounds (scaffold B, analogues 5a–f) stems from a further simplifi-
cation streamline of the structure of 2. Accordingly, we replaced
the pyrrole ring with a piperazine ring thus transforming the origi-
nal 1-phenylpyrrole core in the arylpiperazine core system, charac-
O
Me
b
Me
a
Me
N
N
N
N
Me
14
N
Me
13
N
Me
5f
Scheme 4. Reagents and conditions (a) NH2OHÁHCl, NaOH, EtOH, reflux, 8 h, 62%;
(b) 1-piperidinecarbonyl chloride, NaH, THF, reflux, 30 min, 50%.

S. Butini et al. / Bioorg. Med. Chem. Lett. 23 (2013) 492–495
495
Table 2
In summary, we performed a drug disposition profile optimiza-
tion of compound 2 with the aim of balancing biological activity
and chemico-physical properties. While 4b shows properties sim-
ilar to our lead compound 2, exclusively inhibiting mFAAH, the
arylpiperazine 5c may be considered prototypic of a new class of
potent inhibitors of human and murine FAAH with improved cal-
culated solubility profile. Furthermore, dialysis studies showed
that 5c behaves as a reversible and non-competitive inhibitor of
the mouse enzyme.
Further characterization of mouse and human FAAH inhibition, for compounds 4b and
5c
Type of inhibitiona
IC50 (nM)
Kib (nM)
b
Compd
4b
5c
Reversible non-competitive
Reversible non-competitive
>1000
44
N.D.c
26
a
b
c
Mouse FAAH.
Human FAAH.
N.D. not determined.
Acknowledgments
terized by a protonatable function (5b,c). The arylpiperazine repre-
sents a new scaffold for the development of FAAH inhibitors. Based
on this versatile system, we synthesized analogues decorated by
specific groups that characterize the structure of URB597, 1, 2
and the oximecarbamate analogues.24 As a further modification
aiming at improving the solubility profile of phenylpiperazines,
we explored the outcome of derivatives bearing an ethereal chain
in place of the carbamate moiety (5e, Table 1 for LogS) or an oxi-
mecarbamate function (5f). The potency of inhibition of FAAH for
the newly developed compounds (Table 1) was assessed through
inhibition studies performed on the mouse brain enzyme.
A SAR analysis of the data reported in Table 1 evidences the best
substitution for the 1-phenylpyrrole series of analogues. Starting
from our lead 2, the replacement of the carbamoyl functionality
by a phenylhexylpiperazine while maintaining the amide function
at the pyrrole ring, led to a new inhibitor (4b) characterized by a
nanomolar potency at mFAAH. Conversely, the introduction of a ni-
trile group at 3 position of the pyrrole system brought to a decrease
of activity (4a) of one order of magnitude. Similarly to 3, an iso-
thiazole inhibitor lacking the amide functionality, introduction of
the piperazine urea moiety, combined or not with the presence
of the amide at the pyrrole ring (4c,d) led to submicromolar FAAH
inhibitors. Spacing the piperazine ring from the 1-phenylpyrrole
scaffold by a carbonyl unit (piperazine amide 4e) was not tolerated
and the compound was found not effective in inhibiting the en-
zyme at the tested concentrations. Taking into account the series
of compounds 4 the modifications performed on lead 2 did not pro-
vide a significant improvement of predicted LogS. In fact com-
pound 2 and its analogue 4b show a comparable calculated LogS
value at pH 7 (Table 1).
Starting from the core structure of 2 we decided also to explore
novel arylpiperazines for FAAH inhibition. Compounds 5a–d based
on scaffold B (Fig. 2) were characterized by the presence of the ure-
thane function and the lack of the pyrrole-3-amide group. Com-
pounds 5c was found as the most potent of this sub-series, being
identified as a nanomolar inhibitor of mFAAH. Furthermore, as
shown in Table 1, its calculated LogS indicate a much higher solu-
bility with respect to compounds 2 and 4b. Replacement of the
urethane function by a polyethereal arylalkyl chain (5e) and the
introduction of an oximecarbamate function led to poorly active
analogues.
In line with the pharmacological in vitro characterization of our
lead 2, dialysis studies performed on compounds 4b and 5c re-
vealed a reversible non-competitive profile for these analogues to-
wards mFAAH (Table 2). Furthermore for compound 5c but not for
4b, the efficacy profile was maintained when tested against human
FAAH.
The authors thank the European Research Centre for Drug Dis-
covery and Development (NatSynDrugs), and MIUR Prin for finan-
cial support; COST Action CM1103 is also acknowledged.
A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
11.035.
References and notes
1. Fezza, F.; De Simone, C.; Amadio, D.; Maccarrone, M. Subcell. Biochem. 2008, 49,
101.
2. Maccarrone, M.; Dainese, E.; Oddi, S. Trends Biochem. Sci. 2010, 35, 601.
3. Fowler, C. J. Trends Pharmacol. Sci. 2012, 33, 181.
4. Fu, J.; Bottegoni, G.; Sasso, O.; Bertorelli, R.; Rocchia, W.; Masetti, M.; Guijarro,
A.; Lodola, A.; Armirotti, A.; Garau, G.; Bandiera, T.; Reggiani, A.; Mor, M.;
Cavalli, A.; Piomelli, D. Nat. Neurosci. 2012, 15, 64.
5. Ligresti, A.; De Petrocellis, L.; Hernan Perez de la Ossa, D.; Aberturas, R.;
Cristino, L.; Moriello, A. S.; Finizio, A.; Gil, M. E.; Torres, A. I.; Molpeceres, J.; Di
Marzo, V. PLoS One 2010, 5, e10239.
6. Kaczocha, M.; Vivieca, S.; Sun, J.; Glaser, S. T.; Deutsch, D. G. J. Biol. Chem. 2012,
287, 3415.
7. Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.; Cravatt, B. F. Science
2002, 298, 1793.
8. Caprioli, A.; Coccurello, R.; Rapino, C.; Di Serio, S.; Di Tommaso, M.; Vertechy,
M.; Vacca, V.; Battista, N.; Pavone, F.; Maccarrone, M.; Borsini, F. J. Pharmacol.
Exp. Ther. 2012, 342, 188.
9. Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem. Lett. 2011, 21, 4674.
10. Petrosino, S.; Di Marzo, V. Curr. Opin. Investig. Drugs 2010, 11, 51.
11. Minkkila, A.; Saario, S.; Nevalainen, T. Curr. Top. Med. Chem. 2010, 10, 828.
12. Seierstad, M.; Breitenbucher, J. G. J. Med. Chem. 2008, 51, 7327.
13. Basso, E.; Duranti, A.; Mor, M.; Piomelli, D.; Tontini, A.; Tarzia, G.; Traldi, P. J.
Mass. Spectrom. 2004, 39, 1450.
14. Alexander, J. P.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179.
15. Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.; Fecik, R. A.; Cheng, H.; Hwang,
I.; Hedrick, M. P.; Leung, D.; Acevedo, O.; Guimaraes, C. R.; Jorgensen, W. L.;
Cravatt, B. F. J. Med. Chem. 2005, 48, 1849.
16. Gattinoni, S.; De Simone, C.; Dallavalle, S.; Fezza, F.; Nannei, R.; Amadio, D.;
Minetti, P.; Quattrociocchi, G.; Caprioli, A.; Borsini, F.; Cabri, W.; Penco, S.;
Merlini, L.; Maccarrone, M. ChemMedChem 2010, 5, 357.
17. Butini, S.; Brindisi, M.; Gemma, S.; Minetti, P.; Cabri, W.; Gallo, G.; Vincenti, S.;
Talamonti, E.; Borsini, F.; Caprioli, A.; Stasi, M. A.; Di Serio, S.; Ros, S.; Borrelli, G.;
Maramai, S.; Fezza, F.; Campiani, G.; Maccarrone, M. J. Med. Chem. 2012, 55, 6898.
18. Cabri, W.; Minetti, P.; Campiani, G.; Butini, S. New carbamate derivatives as
modulators of fatty acid amide hydrolase (FAAH inhibitors), EP09155528.
19. Keith, J. M.; Apodaca, R.; Xiao, W.; Seierstad, M.; Pattabiraman, K.; Wu, J.;
Webb, M.; Karbarz, M. J.; Brown, S.; Wilson, S.; Scott, B.; Tham, C. S.; Luo, L.;
Palmer, J.; Wennerholm, M.; Chaplan, S.; Breitenbucher, J. G. Bioorg. Med. Chem.
Lett. 2008, 18, 4838.
20. Butini, S.; Campiani, G.; Franceschini, S.; Trotta, F.; Kumar, V.; Guarino, E.;
Borrelli, G.; Fiorini, I.; Novellino, E.; Fattorusso, C.; Persico, M.; Orteca, N.;
Sandager-Nielsen, K.; Jacobsen, T. A.; Madsen, K.; Scheel-Kruger, J.; Gemma, S. J.
Med. Chem. 2010, 53, 4803.
21. Fattorusso, C.; Campiani, G.; Kukreja, G.; Persico, M.; Butini, S.; Romano, M. P.;
Altarelli, M.; Ros, S.; Brindisi, M.; Savini, L.; Novellino, E.; Nacci, V.; Fattorusso,
E.; Parapini, S.; Basilico, N.; Taramelli, D.; Yardley, V.; Croft, S.; Borriello, M.;
Gemma, S. J. Med. Chem. 2008, 51, 1333.
22. Butini, S.; Gabellieri, E.; Huleatt, P. B.; Campiani, G.; Franceschini, S.; Brindisi,
M.; Ros, S.; Coccone, S. S.; Fiorini, I.; Novellino, E.; Giorgi, G.; Gemma, S. J. Org.
Chem. 2008, 73, 8458.
23. Bandgar, B. P.; Bettigeri, S. V. Synth. Commun. 2004, 34, 2917.
24. Gattinoni, S.; Simone, C. D.; Dallavalle, S.; Fezza, F.; Nannei, R.; Battista, N.;
Minetti, P.; Quattrociocchi, G.; Caprioli, A.; Borsini, F.; Cabri, W.; Penco, S.;
Merlini, L.; Maccarrone, M. Bioorg. Med. Chem. Lett. 2010, 20, 4406.
Selectivity profile was assessed by Cerep, testing 5c towards 54
different receptors, ion channels and transporters. The compound
was tested at 1 lM concentration and the % of inhibition is shown
in Table 1 of the Supplementary data. At this concentration, 5c
showed low affinity for the majority of receptors tested, except
for serotonin 5-HT1, 5-HT2 receptors, dopamine D1 receptor, opioid
l
receptor, and dopamine transporter. Low affinity for AEA-binding
cannabinoid receptors was also demonstrated.