Development and characterization of endocannabinoid hydrolases FAAH and MAGL inhibitors bearing a benzotriazol-1-yl carboxamide scaffold

Article abstract of DOI:10.1016/j.bmc.2012.09.011

A series of (1H-benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-1-yl) methanones and of (1H-benzo[d][1,2,3]triazol-1-yl)(4-phenylpiperazin-1-yl) methanones has been prepared and tested on human fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). In the benzylpiperazinyl series, compound 29 (ML30) exhibited an IC₅₀ value of 0.54 nM on MAGL, combined with a 1000-fold selectivity versus FAAH, while compounds 11 and 16 acted as potent dual FAAH-MAGL inhibitors (IC₅₀ <10 nM). In the phenylpiperazinyl series, compounds 37, 38, 42, and 43 displayed IC₅₀ values against MAGL in the nanomolar range, whilst being between one and two orders of magnitude less potent on the FAAH, while compounds 31 and 32 were potent FAAH inhibitors (IC₅₀ <20 nM) and over 12-fold selective versus MAGL. The key structural determinants driving the structure-activity relationships were explored by the minimization of the inhibitors inside the active site of both enzymes.

Full text of DOI:10.1016/j.bmc.2012.09.011

Bioorganic & Medicinal Chemistry 20 (2012) 6260–6275
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development and characterization of endocannabinoid hydrolases FAAH
and MAGL inhibitors bearing a benzotriazol-1-yl carboxamide scaffold
Ludovica Morera a, , Geoffray Labar b, , Giorgio Ortar a, Didier M. Lambert b
⇑
,
⇑
,
a Sapienza Università di Roma, Dipartimento di Chimica e Tecnologie del Farmaco, P.le Aldo Moro 5, Rome 00185, Italy
b Université Catholique de Louvain, LDRI Louvain Drug Research Institute, Medicinal Chemistry Dpt, Avenue E. Mounier 73, Brussels 1200, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 June 2012
Revised 5 September 2012
Accepted 7 September 2012
Available online 15 September 2012
A series of (1H-benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-1-yl)methanones and of (1H-benzo[d]
[1,2,3]triazol-1-yl)(4-phenylpiperazin-1-yl)methanones has been prepared and tested on human fatty
acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). In the benzylpiperazinyl series,
compound 29 (ML30) exhibited an IC50 value of 0.54 nM on MAGL, combined with a 1000-fold selectivity
versus FAAH, while compounds 11 and 16 acted as potent dual FAAH-MAGL inhibitors (IC50 <10 nM). In
the phenylpiperazinyl series, compounds 37, 38, 42, and 43 displayed IC50 values against MAGL in the
nanomolar range, whilst being between one and two orders of magnitude less potent on the FAAH, while
compounds 31 and 32 were potent FAAH inhibitors (IC50 <20 nM) and over 12-fold selective versus MAGL.
The key structural determinants driving the structure–activity relationships were explored by the minimi-
zation of the inhibitors inside the active site of both enzymes.
Keywords:
FAAH
Fatty acid amide hydrolase
MAGL
Monoacylglycerol lipase
FAAH and MAGL inhibitors
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
the second foremost enzyme in the endocannabinoid system. Since
1997 and the cloning of its gene,9 much work has been devoted to
The quest for enzyme inhibitors intended to increase endocan-
nabinoid levels, in particular N-arachidonoylethanolamine
(ananda-mide, AEA) and 2-arachidonoylglycerol (2-AG), is well
underway. Since the early nineties and the identification of these
endogenous lipids able to bind to CB1 and CB2 cannabinoid recep-
tors, thus producing a wide range of physiological effects, the
unraveling of the enzymes controlling this type of signaling as well
as the design of compounds counteracting their action have consti-
tuted—and still do—major challenges.1–7
Fatty acid amide hydrolase (FAAH), which terminates ananda-
mide signaling, is undoubtedly the best known representative of
these enzymes. Its gene was cloned in 1996 and since then, numer-
ous increasingly potent and selective inhibitors have been
reported.1–5 These efforts have culminated with compound
PF04457845 from Pfizer, which completed phase II clinical trials
(NCT00981357) for relief of pain in patients with osteoarthritis of
the knee, after a phase I study demonstrating a good tolerability
as well as a complete FAAH inhibition and an increase in AEA and
three other fatty acid amides levels.8
the characterization of MAGL. In mammals, this enzyme is ubiqui-
tous and appears exclusively dedicated to the hydrolysis of monoa-
cylglycerols. In the last years, several teams have contributed,
using various approaches, to unravel the leading role played by
MAGL in the degradation of 2-AG, the most abundant endocannab-
inoid in the brain,10–13 generating an intense interest for the design
of MAGL inhibitors. The role of MAGL in promoting the malignancy
of several aggressive cancer cell lines has recently further under-
scored the need for such compounds.14–16 Moreover, the possibility
of taking advantage of an inhibition of the enzyme in other patho-
logical conditions, including neuroinflammation, pain and colitis,
has also been demonstrated.11,17,18
However, apart from the O-phenyl carbamate-based compound
JZL184, the N,N-diphenyl tetrazole-carboxamide 1 (Fig. 1)19 and
the O-hexafluoroisopropyl (HFIP) carbamates, very recently re-
ported by Cravatt and co-workers,20 MAGL inhibitors exhibiting
both selectivity and potency have been lacking for a long time,
and we became therefore interested into a project aimed at the
development of such compounds. Thus, besides the determination
of the tridimensional structure of the human MAGL,21 we have re-
ported in the last four years that a number of carbamoyl tetrazoles
(e.g. 2) turned out to constitute potent, although non selective,
MAGL inhibitors22 and that one analogue of the mammalian lipase
inhibitor tetrahydrolipstatin was a potent and relatively selective
MAGL-inhibitor (OMDM169).23 Additional structural classes of
MAGL inhibitors described recently by us and other groups are tri-
azole-carboxamides,24 bis(dialkylaminethiocarbonyl)disulfides,25
Beside FAAH—and other enzymes whose existence was discov-
ered more recently—monoacylglycerol lipase (MAGL) constitutes
⇑
Corresponding authors. Tel.: +39 6 49913893; fax: +39 6 49693268 (L.M.);
tel.: +32 2 7647341; fax: +32 2 7647363 (G.L.).
E-mail addresses: ludovica.morera@yahoo.it (L. Morera), geoffray.labar@
uclouvain.be (G. Labar).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.011

L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
6261
O
O
Cl
N
O2N
O
N
N
N
N
O
N
N
N
N
OH
Cl
N
N
O
O
O
O
JZL184
1
2
N
N
N
N
N
N
O
N
O
O
N
N
HN
N
N
O
O
O
O
S
N
S
N
N
S
N
S
F
F
OMDM169
SAR629
3
4
O
O
H
N
O
N
N
O
N
O
O
N
O
O
N
S
HO
F3C
5
6
Pristimerin
PF3845
Figure 1. Structures of some MAGL inhibitors and of the FAAH inhibitor PF3845.
maleimides,26 isothiazolinones,27 and pentacyclic triterpenoids,28
exemplified by compounds SAR629 and 3, 4, 5, 6 and pristimerin,
respectively, and most of them target MAGL cystein residues.
At the time, during a preliminary screening of our chemical li-
brary, we identified compound 7 (Fig. 2), an urea based on a ben-
zotriazole-1-carboxamide template, as a MAGL inhibitor. This
maltose-binding protein. Compounds 8–11 turned out to consti-
tute potent inhibitors in this assay as well. In particular, compound
11, which exhibited a very high inhibitory activity for both FAAH
and MAGL, has the potential to serve as a useful pharmacological
probe for the evaluation of behavioral consequences of simulta-
neous elevations in AEA and 2-AG.29 Although the issue of selectiv-
ity of inhibition of FAAH versus MAGL (or vice versa) is certainly
the most important one,20,30 the identification of dual inhibitors
with nanomolar potencies on both targets appears to be of interest
in view of their potential therapeutic exploitation. In this respect,
Makriyannis and co-workers reported this year that the dual
FAAH-MAGL inhibitor 2 is more protective against seizure pathol-
ogy that its 1,5-regioisomer which is more selective for FAAH than
MAGL.31 In order to achieve selectivity for MAGL over FAAH, we
next synthesized compounds 12–43, starting from inhibitors 8
and 9, which were chosen as lead compounds, on the basis of their
good affinity for the principal target, the opportunities that they of-
fered for pharmacomodulations and the gain in solubility that
could be achieved compared to compounds bearing the piperidinyl
moiety (compound 11).
compound was of limited value (IC50hMAGL = 10
lM), and we there-
fore envisaged a few simple pharmacomodulations, after which we
could identify compound 8 and the phenylpiperazinyl moiety as
promising candidates for further medicinal chemistry efforts. This
compound showed a more than respectable potency for the pure
MAGL, with an IC50 value of 21 nM (Table 1).
We first explored with compounds 9–11 some linkers joining
the phenyl ring to the piperazinyl moiety and the replacement of
the latter by a piperidinyl ring. Comparison of compounds 8 and
9 revealed that the introduction of a methylene linker conferred
additional potency towards MAGL, while the contemporaneous
replacement of the core piperazine of compound 9 with a piperi-
dine resulted in a seven times increase of the potency (compound
11). Besides this, as one of the aims of this work was the design of
selective MAGL inhibitors, the affinity for the human FAAH
was also measured, using a recombinant enzyme fused to the
2. Chemistry
Compounds 8–43 were synthesized as shown in Scheme 1.
Compounds 8–14, 24, and 29–43 were synthesized by the reaction
of 1H-benzotriazole-1-carbonyl chloride, generated by treatment
of benzotriazole with triphosgene, with the appropriate substi-
tuted piperazine/piperidine. Compounds 15–18 and 25–28 were
prepared by reaction of appropriate benzyl halides with (1H-ben-
zotriazol-1-yl)(piperazin-1-yl)methanone trifluoroacetate (45), in
turn obtained from 1H-benzotriazole-1-carbonyl chloride and
N-Boc-piperazine and deprotection with trifluoroacetic acid in
H
N
O
O
N
N
N
OH
OH
N
N
N
N
N
7
8
Figure 2. Structures of compounds 7 and 8.

6262
L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
Table 1
chloroform of the Boc-derivative 44. Benzamides 19–23 were pre-
pared by condensing the carboxylic group of compound 18 with
appropriate amines, using 1-hydroxybenzotriazole (HOBt)/N-ethyl-
N0-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as
the carboxylate activator.
In vitro activity of benzotriazol-1-yl carboxamides 7–11 and JZL184 on human MAGL
and FAAH recombinant enzymea
O
R
Non commercially available phenylpiperazines 47a–g and
49a–b were prepared by reaction of the appropriate chloro nitro
benzoic acid with thionyl chloride, followed by addition of the
appropriate amine and heating in DMF with an excess of pipera-
zine of the resulting benzamides 46a–g and 48a–b (Scheme 2).
N
N
N
Compds
R=
IC50, nM
Selectivityb
hMAGL
hFAAH
3. Results and discussion
H
N
OH
OH
7
8
9
10471
21
NDc
—
We started our optimization efforts by exploring first the ben-
zylpiperazinyl series. For this series, substitution in the para or
meta position of the phenyl ring of the benzyl moiety with halo-
gens or a nitro group generally maintained the activity towards
both MAGL and FAAH (Table 2, compounds 12–17) to afford some
promising potent dual FAAH-MAGL inhibitors, in particular com-
pound 16, with a slight increase of the selectivity in the case of
compounds 15 and 17. The introduction of a carboxylic group in
the meta position (compound 18) led, on the contrary, to a dra-
matic 80 and 1500 times decrease of the activity for MAGL and
FAAH, respectively, although resulting in a twenty times improve-
ment of the selectivity. Therefore, we synthesized a limited series
of benzylpiperazines bearing various carboxamide groups in the
meta position of the benzyl moiety (compounds 19–23), with the
purpose of recovering the activity for MAGL, whilst maintaining
low FAAH activities. However, this strategy turned out to be ineffi-
cient, as we were not able to dissociate the improvement of the
activity for MAGL from that for FAAH. The addition of a second
phenyl ring to various positions of the benzyl moiety (compounds
24–26) or using a 1- or 2-naphtylmethyl moiety (compounds 27–
28) also led us to the same result.
N
N
N
N
11
0.5
2.4
8
19
28
N
N
N
10
33
0.8
11
3
2
0.7
JZL184
—
37
36308
981
a
Values are the means of three independent experiments performed in duplicate.
Standard errors are not shown for the sake of clarity and were never higher than
10% of the means.
b
c
hMAGL
Selectivity is expressed as IC50hFAAH/IC50
ND, not determined.
.
a
e
8-14, 24, 29-43
18
19-23
H
N
N
N
O
N
N
O
N
NBoc
CF3COOH
d
NH
N
N
c
N
b
15-18, 25-28
N
N
44
45
Scheme 1. Synthesis of compounds 8–43. Reagents and conditions: (a) (Cl3CO)2CO, TEA, CH2Cl2, rt, 1 h, then appropriate piperazine or piperidine, TEA, rt, 16 h; (b)
(Cl3CO)2CO, TEA, CH2Cl2, rt, 1 h, then N-Boc-piperazine, TEA, rt, 16 h; (c) CF3CO2H, CHCl3, rt, 2 h; (d) ArCH2X, K2CO3, DMF, rt, 16 h; (e) EDC, HOBt, DMF, 0 °C, 15 min, rt, 45 min,
then RNH2, rt, 16 h.
Cl
NO2
Cl
NO2
R'
HN
N
NO2
a
a
b
b
R'
COOH
N
N
O
O
R''
R''
46 a-g
47 a-g
O
N
O
Cl
O2N
COOH
Cl
O2N
HN
N
R''
N R''
R'
48 a-b
O2N
49 a-b
R'
a R' = H R'' = CH(CH3)2
b
R', R'' = (CH2CH2)2O c R', R'' = CH3
d
R' = H R'' = C6H5
e
f
g
R' = H R'' = C6H11
R', R'' = (CH2CH2)2CH2
R', R'' = (CH2CH2)2NCH3
Scheme 2. Synthesis of phenylpiperazines 47a–g and 49a–b. Reagents and conditions: (a) SOCl2, 80 °C, 2 h, then R0R00NH, CH2Cl2, rt, 16 h; (b) piperazine, DMF, 130 °C, 1 h.

L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
6263
Table 2
Table 2 (continued)
In vitro activity of 4-benzyl piperazinylbenzotriazole carboxamides on human MAGL
Compds
R=
IC50, nM
Selectivityb
and FAAH recombinant enzymesa
hMAGL
hFAAH
O
N
N
R
28
3
13
4
N
N
N
Compds
R=
IC50, nM
Selectivityb
29 (ML30)
0.54
562
1040
hMAGL
hFAAH
a
Values are the means of three independent experiments performed in duplicate.
12
13
14
15
10
11
9
15
1.5
1.5
2.5
7.6
F
Standard errors are not shown for the sake of clarity and were never higher than
10% of the means.
17
Cl
Br
Selectivity is expressed as IC50hFAAH/IC50
.
b
hMAGL
22
12
2
89
9
NO2
Long et al. reported compound JZL184 (Fig. 1), the first MAGL
inhibitor displaying high efficacy and selectivity against MAGL
(IC50MAGL = 10 nM; IC50FAAH = 4690 nM,11 although in other studies
JZL184 exhibited lower inhibition activity on MAGL,32,33) with the
selectivity in part due to the inability of the bulky lipophilic bisa-
rylcarbinol moiety to fit into the relatively narrow FAAH acyl chain
binding pocket. On the basis of these results, we introduced a benz-
hydryl group on the 4-piperazine position. As expected, a spectacu-
lar increase in both potency and selectivity for MAGL was obtained
when adding steric hindrance with the bulkier diphenylmethyl
substituent (compound 29, ML30). Compound 29 allowed us to
gain potency for the MAGL by a factor of ten (IC50 = 0.54 nM) whilst
substantially decreasing FAAH inhibitory activity (IC50 = 562 nM),
thus resulting in a three order of magnitude selectivity, and, being
70 times more potent, compares favorably with JZL184 in the same
assay conditions (Table 1).
Other close analogues of ML30 are a couple of triazolo-carbox-
amides described by Sanofi-Aventis, SAR629 and 3 (Fig. 1). The
triazolopyridine carboxamide-based compound 3 has been shown
to inhibit MAGL with an IC50 value of 4 nM,24 but the inhibitory po-
tency against FAAH has not been determined, while no details of
the inhibitory potency against MAGL nor of its capability of inhib-
iting MAGL selectively over FAAH have been provided in the case of
SAR629.34
Based on structure and reactivity similarities, the mechanism of
MAGL inhibition by ML30 and its mode of recognition should be
similar to those of JZL184 and Sanofi-Aventis triazolo-carboxam-
ides (Fig. 3). To investigate the reversibility of the inhibition of
MAGL by ML30, we first used the rapid dilution assay (Fig. 4a).
Concentrations were carefully chosen according to the IC50 curve
to achieve an almost complete inactivation of the enzyme at the
initial concentration, whilst the final concentration—that is, after
a 300-fold dilution—should not lead to inhibition. After dilution
however, the enzyme did not recover activity, indicating the irre-
versible character of the inhibition. We next recorded the kinetics
of MAGL inhibition over time in the presence of ML30. MAGL enzy-
matic velocity decreased over time in the presence of the inhibitor
(Fig. 4b), giving us an additional evidence of the irreversible char-
acter of the inhibition. The kinact and Ki inactivation constants,
which are often used in the case of an irreversible inhibition, were
also calculated using kobs, the first-order constant for enzyme inac-
tivation (Fig. 4c).
16
17
18
4.5
5.4
41
Cl
8
43
NO2
676
27542
COOH
N
19
20
21
102
36
275
58
2.7
1.6
4.3
O
O
O
N
324
1380
N
N
22
23
13
44
35
3.4
4.8
NH
O
O
7
NH
24
25
62
23
18
9
0.3
0.4
26
27
33
9
32
12
1.0
1.3
The tridimensional structure of FAAH has been known since
2002,35 and we as well as other groups have recently reported that
of human MAGL as the apoenzyme or as the complex with several
inhibitors.21,34 Therefore, in order to gain a better understanding of

6264
L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
100
a
80
60
40
20
0
.
.
l
l
l.
i
i
i
di
d
d
d
r
r
e
e
r
e
e
t
r
t
o
f
f
o
f
f
A
A
e
e
B
B
[ ]i = 10-9M
[ ]i = 10-8M
100
80
60
40
20
0
b
100 nM
125 nM
167 nM
250 nM
500 nM
1000 nM
Ctrl
Figure 3. Overlay of the minimized structures of compound 29, JZL184 and SAR629
(carbon atoms in green, blue and magenta, respectively).
the structure–activity relationships obtained within benzylpiper-
azinyl series, we then studied the binding mode of several inhibi-
tors inside the MAGL and FAAH active sites, as covalent adducts
by carbamoylation of a nucleophilic serine of the two enzymes.
We first minimized compound 9 inside the FAAH active site, bound
to the nucleophilic Ser241 (Fig. 5a). To this end, we used the crystal
coordinates of the rat humanized FAAH in complex with the inhib-
itor PF3845 (Fig. 1).36 Noteworthy, we considered for these studies
both the open and closed conformations of the Phe432, which con-
stitutes a lock controlling the access to the active site and the
shape of the FAAH membrane access channel and acyl-binding
pocket.37 As expected, the benzylic ring of compound 9 closely
superimposes with the proximal phenyl ring of PF3845 and makes
0
1000
2000
3000
time (s)
1200
1000
800
600
400
200
0
c
a CH–p interaction with Phe192. This minimization also shows us
that the meta position of the phenyl ring is well oriented to project
toward the distal pocket which, in the experimental structure, is
filled by the 4-trifluoromethyl-2-pyridyl group of the inhibitor
PF3845. The lipophilicity of this distal pocket, lined with Met495,
Val491 and Phe432 side chains, likely accounts for the dramatic
loss of potency observed for compound 18, due to the negative
charge of the carboxylate. Similarly, it explains the progressive
recovery of activity when filling the pocket with N,N-dimethylcarb-
oxamide, piperidin-1-ylmethanone, N-cyclohexylcarboxamide or
N-phenylcarboxamide groups (compounds 19, 20, and 22, 23).
Due to the positively charged N-methylpiperazinyl moiety, com-
pound 21 is also twenty times less potent than 20 on the FAAH.
In the biphenyl series, the meta-linked biphenyl compound 25
was the most potent FAAH inhibitor. This position allows to reach
the distal pocket of the acyl-binding pocket of FAAH, in which com-
pound 25 superimposes nicely with PF3845 (Fig. 5b). In order to fit
into the FAAH active site, the benzhydryl group of 29 adopts an
energetically unfavorable conformation (Fig. 5b). Moreover, the
only possibility for the second phenyl group is to protrude toward
a pocket lined, among others, by the hydroxyl groups of Ser213 and
Thr488 and by the main chain carbonyl of Phe192, Leu401, Cys400,
Pro484, and Gly485. This pocket, which constitutes the bottom of
the channel leading from the membrane access port to the active
site, has thus a less lipophilic character which, together with the
torsion which is imposed to fit into the active site of the FAAH,
likely account for the loss of potency of compound 29 compared
to 9 or 25.
kinact/Ki = 207650
Ki = 7.57x10-8
2.5×106
5.0×106
7.5×106 1.0×107
0
1/[inh] (M-1)
Figure 4. (a) Rapid dilution assay. After a 30 min incubation of MAGL with ML30
(10ꢀ8 or 10ꢀ9 M) followed by a 300-fold dilution, the 2-OG hydrolase activity was
measured to investigate the reversibility of MAGL inhibition. The percentage of
residual activity observed using 10ꢀ8 or 10ꢀ9 M of the inhibitor (without dilution) is
also indicated. The experiment was performed two times in triplicate. (b) MAGL
residual activity in the presence of ML30 was measured over time using
p-nitrophenylpropionate as a substrate. The experiment was done four times in
quadruplicate. (c) kobs (first order constant for enzyme inactivation) values at each
inhibitor concentration were used to calculate kinact and Ki values for MAGL
inhibition by ML30.
MAGL active site. Several tridimensional MAGL structures have
been reported to date.21,34,38 A wide hydrophobic site able to
accommodate the acyl chain of 2- AG is present in each, with the
major difference being observed in the organization of the cap,
which occludes or not part of the active site entry. We used, for
our study, the coordinates of the human enzyme in complex with
the inhibitor SAR629. As expected, the diphenylmethyl group of 29
In order to better understand the high selectivity of compound
29 for the MAGL, we next studied its binding mode inside the

L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
6265
Table 3
In vitro activity of 4-aryl piperazinylbenzotriazole carboxamides on human MAGL
and FAAH recombinant enzymesa
O
N
N R
N
N
N
Compds R=
IC50, nM
hMAGL hFAAH
Selectivityb
30
31
30
71
13
3
0.44
0.04
F
32
224
372
19
0.08
0.43
O2N
O2N
O2N
O
33
34
158
HN
O
N
955
1950
2.0
O
35
36
O
32
20
15
34
0.47
1.7
NO2
NO2
N
37
63
5012
79
O
NO2
N
38
39
40
38
162
141
2291
40
60
O
O
O
O
NO2
N
% inh (10ꢀ5 M)>100
% inh (10ꢀ 5M)>100
O
N
NO2
N
40
Figure 5. (a) Minimization of compounds 9 (magenta) and 23 (green) in the active
site of the rat humanized FAAH. (b) Minimization of compounds 25 (cyan) and 29
(blue) in the active site of the rat humanized FAAH. (c) Minimization of compounds
29 (blue) in the active site of the human MAGL. The FAAH inhibitor PF3845 (Fig. 5a
and b) or the MAGL inhibitor SAR629 (Fig. 5c) are also represented (orange).
NO2
NH
41
42
43
234
41
2089
490
8.9
NO2
closely superimposes with the corresponding substituent of
H
N
12
19
SAR629, making CH–p interactions either with Leu205 and
O
Leu241 alkyl chains or with Leu148 (Fig. 5c). Unlike in the case
of FAAH, compound 29 fits in the MAGL active site in a energeti-
cally favorable conformation and makes Van der Waals interac-
tions with both P1 and P2 pockets.
NO2
H
N
19
363
O
We next explored the phenylpiperazinyl series (Table 3). Substi-
tution in ortho with a fluoro, methyl or nitro group gradually de-
creased the activity for MAGL (compounds 30–32 compared to 8)
while maintaining excellent activity for FAAH. In particular, com-
pounds 31 and 32 were potent FAAH inhibitors (IC50 <20 nM) and
over 12-fold selective versus MAGL. The association of a nitro
a
Values are the means of three independent experiments performed in duplicate.
Standard errors are not shown for the sake of clarity and were never higher than
10% of the means.
b
hMAGL
Selectivity is expressed as IC50hFAAH/IC50
.

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L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
Figure 6. Minimization of compounds 8 (green) and 9 (magenta) in the active site
of the human MAGL.
group in ortho and of a carboxamide in the para position led to a
general loss of potency (compounds 33–34). However, when the
nitro group was placed in para, the potency against MAGL was
slightly increased, while the activity for FAAH was reduced by a
factor of three (compound 36). In the hope to increase the MAGL
selectivity, we planned therefore to synthesize a series of 1-(4-
nitrophenyl)piperazines bearing various carboxamides in the meta
position of the aromatic ring (compounds 37–43). In the case of
tertiary amides, a substantial decrease of the potency at FAAH
was observed, ranging from a ꢁ70 times diminution for compound
38 compared to compound 36 to an almost complete loss of
activity (compounds 39–40). This effect was less spectacular with
secondary amides, although the presence of a meta N-isopropyl-
carboxamide group led to a compound (41) 60 times less potent
than the meta unsubstituted analogue 36 on FAAH.
Although the majority of the inhibitors bearing a carboxamide
group showed also a decreased potency on MAGL, the diminution
was less dramatic than that on FAAH. Indeed, only a slight
decrease was observed for compounds 38 and 42, and compound
43 was equipotent to compound 8. These differences in struc-
ture–activity relationships of the two enzymes resulted in an im-
proved MAGL selectivity. In fact, when compared to the parent
inhibitor 8, compound 43 gained more than one order of magni-
tude in selectivity and compounds 37, 38, and 39, although two
to eight times less potent than compound 8, were 120–200 times
more selective.
Figure 7. (a) Minimization of compound 43 in the active site of the human MAGL.
(b) Minimization of compounds 36 (yellow) in the active site of the FAAH. The FAAH
inhibitor PF3845 is represented in orange. The position of the acyl-binding pocket
(ACB) and the membrane access channel (MAC) is also indicated.
and, in MAGL, this charge is enclosed in an apolar environment, di-
rectly facing the CD1 and CD2 atoms of Leu148 and Leu241 alkyl
chains. It is thus likely that, regarding compound 8, the disappear-
ance of this unfavourable condition counterbalances the lack of
CH–p interaction involving its phenyl ring. Besides this, the ortho
substitution on the phenyl ring leads to less active MAGL inhibitors
(30–32). This can be explained by the very close proximity of the
phenyl ortho position with the Leu205 CD2, Leu213 CD1, and
Leu241 main chain oxygen (Fig. 6). This situation creates, in the
near environment of the phenyl ring, a steric hindrance, leading
to a steric clash when a substituent is introduced in the ortho posi-
tion. On the contrary, compound 36, with a nitro group in the para
position, fits well in the active site, even if the nitro is not optimal
for the hydrophobic cavity, which is lined by Ala151, Leu214,
Leu205 and Leu176 side chains.
As for the benzylpiperazine series, we then tried, based on the
tridimensional structure of both enzymes, to gain further under-
standing of the structure–activity relationships obtained in the
phenylpiperazinyl series. We first minimized 8 in the cavity of
the MAGL, covalently linked to the Ser122 (Fig. 6). Due to the ab-
sence of a methylene bridge between the phenyl ring and the pip-
erazinyl moiety, the aromatic ring fails to project toward the P1 or
P2 pocket. Moreover, despite partly filling the hydrophobic acyl-
binding channel, Leu148 and Val217 are not well oriented or not
A minimization of compound 43 in the MAGL showed that the
inhibitor can project the phenylcarboxamide group toward the
top of the active site, just below the hydrophobic a4 helix that con-
stitutes the lid that controls access to the enzyme (Fig. 7a).
On the other hand, although compound 36 fits in the FAAH cav-
ity, the 4-nitro group comes in close contact with Thr488 and
Phe432 side chains, two residues that form the junction between
the acyl binding pocket and the membrane access channel
(Fig. 7b). Along this line, the geometric constrains imposed by
the combination of nitro and carboxamide groups in the para and
meta positions, respectively, seem to explain the poor inhibitory
potency of compounds 37–43 on FAAH.
close enough to be engaged in stabilizing CH–p interactions with
the inhibitor. Therefore, at first glance, it could be rather surprising
to see that 8 was almost equipotent to its benzylpiperazine ana-
logue, compound 9. However, at the pH of the enzymatic assay, un-
like compound 8, compound 9 bears a positively charged nitrogen,

L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
6267
C17H17N5O: C, 66.43; H, 5.58; N, 22.79. Found: C, 66.58; H, 5.57;
4. Conclusion
N, 22.75.
A series of (1H-benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-
1-yl)methanones (compounds 9, 12–29) and of (1H-benzo[d] [1,
2,3]triazol-1-yl)(4-phenylpiperazin-1-yl)methanones (compounds
8, 30–43) has been synthesized and its inhibitory activity mea-
sured against human FAAH and MAGL recombinant enzymes.
Starting from compounds 8 and 9, we were able to identify some
MAGL- and FAAH-selective or dual FAAH-MAGL inhibitors. In par-
ticular, compound 29 (ML30) exhibited an IC50 value of 0.54 nM on
MAGL, combined with a 1000-fold selectivity versus FAAH, while
compounds 11 and 16 acted as potent dual FAAH-MAGL inhibitors
(IC50 <10 nM). Compounds 37, 38, 42, and 43 displayed IC50 values
against MAGL in the nanomolar range, whilst being between one
and two orders of magnitude less potent on the FAAH, while com-
pounds 31 and 32 were potent FAAH inhibitors (IC50 <20 nM) and
over 12-fold selective versus MAGL. The structural features driving
the recognition profile on FAAH versus MAGL and vice versa were
also studied by a comparison of the binding mode of the inhibitors
inside the enzyme active sites.
5.1.3. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-1-yl)
methanone (9)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-benzylpiperazine instead of
1-phenylpiperazine. Chromatography on silica gel (8:2 CH2Cl2/
AcOEt as eluent) afforded pure 9 as solid. Yield = 29%. Mp = 91–
92 °C; IR 2815, 2771, 1712, 1450, 1246, 1011, 749; 1H NMR d
2.64 (4H, m), 3.60 (2H, s), 3.93 (4H, m), 7.27–7.35 (5H, m), 7.45
(1H, t, J = 7.6 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.98 (1H, d, J = 8.2 Hz),
8.09 (1H, d, J = 8.2 Hz); 13C NMR d 52.82, 62.82, 113.57, 119.86,
125.20, 127.38, 128.40, 129.15, 129.34, 133.21, 137.45, 145.34,
149.36. Anal. Calcd for C18H19N5O: C, 67.27; H, 5.96; N, 21.79.
Found: C, 67.48; H, 5.97; N, 21.73.
5.1.4. (E)-(1H-Benzo[d][1,2,3]triazol-1-yl)(4-cinnamylpiperazin-
1-yl)methanone (10)
The title compound was prepared following the same proce-
dure used for the synthesis of 8, using 1-cinnamylpiperazine in-
stead of 1-phenylpiperazine. Chromatography on silica gel (5:5
n-hexane/AcOEt as eluent) afforded pure 10 as solid. Yield = 46%.
Mp = 111 °C; IR 2936, 2813, 1711, 1417, 1229, 999, 747; 1H NMR
d 2.70 (4H, t, J = 5.1 Hz), 3.25 (2H, d, J = 6.7 Hz), 3.96 (4H, m),
6.28 (1H, dt, J = 15.8, 6.7 Hz), 6.57 (1H, d, J = 15.8 Hz), 7.21–
7.28 (1H, m), 7.28–7.36 (2H, m), 7.40 (2H, d, J = 7.5 Hz), 7.45
(1H, t, J = 7.6 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.99 (1H, d,
J = 8.3 Hz), 8.10 (1H, d, J = 8.3 Hz); 13C NMR d 52.92, 53.42,
60.84, 113.57, 119.87, 125.23, 125.66, 126.36, 127.71, 128.61,
129.38, 133.19, 133.73, 136.62, 145.34, 149.36. Anal. Calcd for
Taken together, these data suggest that these inhibitors could
serve as promising leads for the development of therapeutics based
on the inhibition of AEA and/or 2-AG degradation.
5. Experimental section
5.1. Chemistry
5.1.1. General
All chemical reagents were commercially available unless
otherwise indicated and were used without further purification.
Solvents were of analytical grade. Column chromatographies were
carried out using Merck silica gel 60 (230–400 mesh). Melting
points were determined on a Kofler hot-stage apparatus or in open
capillaries using the Electro thermal 9100 apparatus and are re-
ported uncorrected. IR spectra were recorded on a Perkin-Elmer
1000 FT-IR spectrophotometer as CHCl3 solutions unless otherwise
indicated. For compounds 20, 21 and 23, IR spectra were run on a
SpectrumOne FT-ATR spectrophotometer (Perkin Elmer). Band po-
C20H21N5O: C, 69.14; H, 6.09; N, 20.16. Found: C, 68.98; H,
6.10; N, 20.17.
5.1.5. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperidin-1-yl)
methanone (11)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 4-benzylpiperidine instead of
1-phenylpiperazine. Chromatography on silica gel (8:2 n-hexane/
AcOEt as eluent) afforded pure 11 as solid. Yield = 95%. Mp = 80–
81 °C; IR 2948, 2929, 1705, 1430, 1250, 1051, 749; 1H NMR d
1.49 (2H, m), 1.74–1.98 (3H, m), 2.63 (2H, d, J = 6.9 Hz), 3.00–
3.13 (2H, m), 4.51 (2H, d, J = 12.6 Hz), 7.14–7.35 (5H, m), 7.44
(1H, t, J = 7.6 Hz), 7.59 (1H, t, J = 7.6 Hz), 7.96 (1H, d, J = 8.4 Hz),
8.09 (1H, d, J = 8.4 Hz); 13C NMR d 32.10, 38.19, 42.89, 53.45,
113.47, 119.82, 125.11, 126.17, 128.38, 129.12, 129.24, 133.20,
139.78, 145.37, 149.39. Anal. Calcd for C19H20N4O: C, 71.23; H,
6.29; N, 17.49. Found: C, 71.38; H, 6.28; N, 17.47.
sition and absorption ranges are given in cmꢀ1 1H and 13C NMR
.
spectra were obtained on a Bruker Avance 400 spectrometer using
CDCl3 as solvent unless otherwise indicated and TMS as internal
standard. Satisfactory elemental analyses were obtained for the
newly synthesized compounds (C, H, N 0.4%).
5.1.2. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-phenylpiperazin-1-yl)
methanone (8)
Bis(trichloromethyl) carbonate (1.000 g, 3.42 mmol) was added
to a solution of benzotriazole (1.230 g, 10.3 mmol) and triethyl-
amine (TEA) (1.5 mL) in CH2Cl2 (15 mL) at 0 °C, and the reaction
mixture was stirred at 0 °C for 1 h. Then, a solution of 1-phenylpip-
erazine (1.330 g, 8.2 mmol) and TEA (1.0 mL) in CH2Cl2 (15 mL)
was added dropwise, and the reaction mixture was stirred at room
temperature overnight. The reaction was quenched by adding a
saturated NaHCO3 solution and extracted with CH2Cl2. The organic
phase was dried over Na2SO4, and evaporated in vacuo to give a
residue of 770 mg, which was chromatographed on silica gel
(98:2 CH2Cl2/n-hexane as eluent) to give 600 mg (57%) of pure 8
as solid. Mp = 152–153 °C; IR (KBr) 2829, 1695, 1441, 1233, 1011,
749; 1H NMR d 3.38 (4H, m), 4.10 (4H, m), 6.92–6.99 (3H, m),
7.31 (2H, t, J = 7.8 Hz), 7.47 (1H, t, J = 7.6 Hz), 7.62 (1H, t,
J = 7.6 Hz), 8.02 (1H, d, J = 8.4 Hz), 8.11 (1H, d, J = 8.4 Hz); 13C
NMR d 49.60, 113.65, 116.77, 119.92, 120.80, 125.34, 129.32,
129.50, 133.22, 145.38, 149.42, 150.75. Anal. Calcd for
5.1.6. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(4-fluorobenzyl) piper-
azin-1-yl)methanone (12)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-(4-fluorobenzyl)piperazine in-
stead of 1-phenylpiperazine. Chromatography on silica gel (99:1
CH2Cl2/MeOH as eluent) afforded pure 12 as white solid.
Yield = 62%. Mp = 113 °C; IR (KBr) 3417, 2811, 1716, 1510, 1428,
1209, 1024, 750; 1H NMR d 2.63 (4H, m), 3.56 (2H, s), 3.93 (4H,
m), 7.03 (2H, m), 7.32 (2H, m), 7.46 (1H, t, J = 7.6 Hz), 7.60 (1H, t,
J = 7.6 Hz), 7.99 (1H, d, J = 8.4 Hz), 8.09 (1H, d, J = 8.4 Hz); 13C
NMR d 44.96, 52.76, 61.99, 113.59, 115.26 (d, JC–F = 21.1 Hz),
119.88, 125.23, 129.37, 130.0 (d, JC–F = 7.9 Hz), 130.64, 133.19,
133.22, 145.36, 149.37, 162.17 (d, JC–F = 244.9 Hz). Anal. Calcd for
C18H18FN5O: C, 63.70; H, 5.35; N, 20.64. Found: C, 63.87; H, 5.36;
N, 20.60.

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5.1.7. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(4-chlorobenzyl) piperazin-
1-yl)methanone (13)
5.1.11. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(3-nitrobenzyl) piperazin-
1-yl)methanone (17)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-(4-chlorobenzyl)piperazine in-
stead of 1-phenylpiperazine. Chromatography on silica gel (8:2
CH2Cl2/AcOEt as eluent) afforded pure 13 as white solid.
Yield = 70%. Mp = 121–122 °C; IR (KBr) 3417, 2812, 1703, 1433,
1229, 990, 760; 1H NMR d 2.62 (4H, m), 3.56 (2H, s), 3.92 (4H,
m), 7.30 (4H, m), 7.46 (1H, t, J = 7.6 Hz), 7.60 (1H, t, J = 7.6 Hz),
7.99 (1H, d, J = 8.4 Hz), 8.09 (1H, d, J = 8.4 Hz); 13C NMR d 52.79,
62.01, 63.82, 113.56, 119.87, 125.25, 128.56, 129.39, 130.37,
133.11, 133.18, 136.03, 145.33, 149.35. Anal. Calcd for
The title compound was prepared from 1-(chloromethyl)-3-
nitrobenzene following the same procedure used for the synthesis
of 15. Chromatography on silica gel (9:1 CH2Cl2/AcOEt as eluent)
afforded pure 17 as yellow solid. Yield = 66%. Mp = 164–165 °C;
IR (KBr) 2799, 2780, 1698, 1533, 1438, 1342, 1018, 735; 1H NMR
d 2.68 (4H, m), 3.69 (2H, s), 3.96 (4H, m), 7.45 (1H, t, J = 7.6 Hz),
7.52 (1H, t, J = 8.0 Hz), 7.60 (1H, t, J = 7.6 Hz), 7.71 (1H, d,
J = 7.6 Hz), 7.99 (1H, d, J = 8.4 Hz) 8.09 (1H, d, J = 8.4 Hz), 8.14
(1H, d, J = 8.0 Hz), 8.26 (1H, s); 13C NMR d 46.83, 52.75, 61.66,
113.49, 119.76, 122.41, 123.61, 125.17, 129.27, 129.32, 133.10,
134.84, 139.95, 145.24, 148.41, 149.25. Anal. Calcd for
C18H18ClN5O: C, 60.76; H, 5.10; N, 19.68. Found: C, 60.93; H,
5.10; N, 19.63.
C18H18N6O3: C, 59.01; H, 4.95; N, 22.94. Found: C, 59.12; H, 4.95;
N, 22.99.
5.1.8. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(4-bromobenzyl)
piperazin-1-yl)methanone (14)
5.1.12. 3-((4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)methyl)benzoic acid (18)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-(4-bromobenzyl)piperazine in-
stead of 1-phenylpiperazine. Chromatography on silica gel (98:2
CH2Cl2/MeOH as eluent) afforded pure 14 as white solid.
Yield = 66%. Mp = 131 °C; IR (KBr) 3418, 2807, 1692, 1433, 1228,
991, 760; 1H NMR d 2.62 (4H, t, J = 4.4 Hz), 3.54 (2H, s), 3.92 (4H,
m), 7.23 (2H, d, J = 8.0 Hz), 7.46 (3H, m), 7.60 (1H, t, J = 7.6 Hz),
7.98 (1H, d, J = 8.0 Hz), 8.09 (1H, d, J = 8.0 Hz); 13C NMR d 47.51,
52.77, 62.05, 113.56, 119.87, 121.21, 125.25, 129.40, 130.74,
131.53, 133.19, 136.57, 145.33, 149.36. Anal. Calcd for
A solution of 3-(chloromethyl)benzoic acid (512 mg, 3.0 mmol)
in DMF (9 mL) was added dropwise to a stirring solution of 7
(1.04 g, 3.0 mmol) and DIPEA (2.5 mL, 15.0 mmol) in DMF (6 mL),
and the mixture was stirred at room temperature overnight. The
reaction was quenched by adding a 2 N HCl solution and extracted
with Et2O; the aqueous layer was basified with saturated NaHCO3
and extracted with Et2O. The aqueous phase was neutralized (pH
ꢁ6.5) and extracted with AcOEt. The organic phase was dried over
Na2SO4, and evaporated in vacuo to give a residue of 603 mg,
which was chromatographed on silica gel (9:1 AcOEt/MeOH as elu-
ent) to give 476 mg (43%) of pure 18 as white solid. Mp = 197–
199 °C; IR (KBr) 3649, 3447, 1708, 1434, 1285, 1235, 981, 740;
1H NMR (DMSO-d6) d 2.57 (4H, m), 3.64 (2H, s), 3.78 (4H, t,
J = 4.6 Hz), 7.48 (1H, t, J = 7.6 Hz), 7.54 (1H, t, J = 7.6 Hz), 7.59
(1H, d, J = 7.6 Hz), 7.70 (1H, t, J = 7.6 Hz), 7.86 (1H, d, J = 7.6 Hz),
7.93 (2H, m), 8.18 (1H, d, J = 8.3 Hz); 13C NMR d 52.15, 61.14,
113.10, 119.50, 125.29, 128.04, 128.42, 129.37, 129.67, 130.89,
132.43, 133.27, 138.19, 144.59, 148.61, 167.27. Anal. Calcd for
C18H18BrN5O: C, 54.01; H, 4.53; N, 17.50. Found: C, 54.17; H,
4.54; N, 17.49.
5.1.9. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(4-nitrobenzyl) piperazin-
1-yl)methanone (15)
A solution of 4-nitrobenzylbromide (65 mg, 0.3 mmol), 45
(104 mg, 0.3 mmol), and potassium carbonate (250 mg, 1.8 mmol)
in DMF (1 mL) was stirred overnight at room temperature. The
reaction was quenched by adding a saturated NaHCO3 solution
and extracted with AcOEt. The organic phase was dried over
Na2SO4 and evaporated in vacuo to give a residue of 109 mg, which
was chromatographed on silica gel (55:45 n-hexane/AcOEt as elu-
ent) to give 74 mg (67%) of pure 15 as yellow solid. Mp = 170–
172 °C; IR (KBr) 2813, 1715, 1605, 1513, 1429, 1345, 1231, 1025,
1001, 754; 1H NMR d 2.67 (4H, m), 3.70 (2H, s), 3.96 (4H, m),
7.46 (1H, t, J = 7.7 Hz), 7.56 (2H, d, J = 8.6 Hz), 7.61 (1H, t,
J = 7.7 Hz), 7.99 (1H, d, J = 8.3 Hz), 8.09 (1H, d, J = 8.3 Hz), 8.21
(2H, d, J = 8.6 Hz); 13C NMR d 46.52, 52.93, 61.86, 113.58, 119.89,
123.70, 125.31, 129.46, 129.54, 133.20, 145.35, 147.42, 149.37.
Anal. Calcd for C18H18N6O3: C, 59.01; H, 4.95; N, 22.94. Found: C,
59.07; H, 4.95; N, 22.99.
C19H19N5O3: C, 62.46; H, 5.24; N, 19.17. Found: C, 62.61; H, 5.22;
N, 19.23.
5.1.13. 3-((4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)methyl)-N,N-dimethylbenzamide (19)
To a stirring solution of 18 (82 mg, 0.22 mmol) in DMF (2 mL) 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(EDC) (45 mg, 0.23 mmol) and hydroxybenzotriazole (HOBt)
(40 mg, 0.23 mmol) were added at 0 °C and the mixture was stirred
at 0 °C for 15 min, then at room temperature for 45 min. Dimethyl-
amine (33% in ethanol) (30 ll, 0.22 mmol) was added dropwise and
the reaction mixture was stirred at room temperature overnight.
The reaction was quenched by adding a saturated NaHCO3 solution
and extracted with AcOEt. The organic phase was dried over
Na2SO4, and evaporated in vacuo to give a residue of 87 mg, which
was chromatographed on silica gel (8:2 AcOEt/n-hexane as eluent)
to afford 65 mg (75%) of pure 19 as oil. IR 3011, 2932, 1701, 1626,
1449, 1433, 999, 787, 697; 1H NMR d 2.67 (4H, m), 2.99 (3H, s),
3.12 (3H, s), 3.63 (2H, s), 3.94 (4H, m), 7.32–7.47 (5H, m), 7.60
(1H, t, J = 7.7 Hz), 7.98 (1H, d, J = 8.3 Hz), 8.09 (1H, d, J = 8.3 Hz);
13C NMR d 35.40, 39.62, 42.52, 52.78, 62.34, 113.57, 119.85,
125.25, 126.17, 127.71, 128.48, 129.39, 130.25, 133.19, 136.49,
137.62, 145.33, 149.33, 171.55. Anal. Calcd for C21H24N6O2: C,
64.27; H, 6.16; N, 21.41. Found: C, 64.36; H, 6.17; N, 21.37.
5.1.10. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(3-chlorobenzyl)
piperazin-1-yl)methanone (16)
The title compound was prepared from 1-(bromomethyl)-3-
chlorobenzene following the same procedure used for the synthe-
sis of 15. Chromatography on silica gel (6:4 n-hexane/AcOEt as elu-
ent) afforded pure 16 as white solid. Yield = 91%. Mp = 69–70 °C; IR
(KBr) 2925, 2800, 1698, 1439, 1235, 1020, 752; 1H NMR d 2.62 (4H,
m), 3.55 (2H, s), 3.93 (4H, m), 7.21–7.28 (3H, m), 7.36 (1H, br s),
7.44 (1H, t, J = 7.7 Hz), 7.59 (1H, t, J = 7.7 Hz), 7.98 (1H, d,
J = 8.3 Hz) 8.08 (1H, d, J = 8.3 Hz); 13C NMR d 45.20, 47.64, 52.75,
62.07, 113.55, 119.80, 125.18, 127.10, 127.51, 128.96, 129.32,
129.62, 133.15, 134.28, 139.74, 145.28, 149.28. Anal. Calcd for
5.1.14. (3-((4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)methyl)phenyl)(piperidin-1-yl)methanone (20)
The title compound was prepared following the same procedure
used for the synthesis of 19, using piperidine as amine. Chromatography
C18H18ClN5O: C, 60.76; H, 5.10; N, 19.68. Found: C, 60.83; H,
5.11; N, 19.65.

L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
6269
on silica gel (8:2 AcOEt/n-hexane as eluent) afforded pure 20 as oil.
Yield = 81%. IR (neat) 2934, 1698, 1626, 1424, 1281, 1228, 996, 747;
1H NMR d 1.53 (2H, br s), 1.69 (4H, m), 2.65 (4H, m), 3.35 (2H, br s),
3.62 (2H, s), 3.72 (2H, br s), 3.93 (4H, m), 7.30 (1H, m), 7.39 (3H, m),
7.45 (1H, t, J = 7.7 Hz), 7.60 (1H, t, J = 7.7 Hz), 7.99 (1H, d, J = 8.3 Hz),
8.09 (1H, d, J = 8.3 Hz); 13C NMR d 24.61, 25.61, 26.54, 43.17, 48.86,
52.83, 62.41, 113.59, 119.87, 125.24, 125.88, 127.39, 128.53, 129.38,
130.01, 133.22, 136.75, 137.86, 145.36, 149.37, 170.18. Anal. Calcd for
(99:1 CH2Cl2/MeOH as eluent) afforded pure 24 as white solid.
Yield = 72%. Mp = 100–101 °C; IR (KBr) 3427, 2818, 1707, 1430,
1230, 998, 759; 1H NMR d 2.68 (4H, m), 3.64 (2H, s), 3.95 (4H,
m), 7.34 (1H, t, J = 7.2 Hz), 7.43 (5H, m), 7.59 (5H, m), 7.99 (1H,
d, J = 8.4 Hz), 8.09 (1H, d, J = 8.4 Hz); 13C NMR d 52.84, 53.42,
62.49, 113.57, 119.86, 125.22, 127.06, 127.14, 127.27, 128.77,
129.36, 129.57, 133.20, 136.51, 140.35, 140.82, 145.34, 149.36.3.
Anal. Calcd for C24H23N5O: C, 72.52; H, 5.83; N, 17.62. Found: C,
72.67; H, 5.84; N, 17.63.
C24H28N6O2: C, 66.65; H, 6.53; N, 19.43. Found: C, 66.83; H, 6.54; N,
19.48.
5.1.19. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(biphenyl-3-ylmethyl)
piperazin-1-yl)methanone (25)
5.1.15. (3-((4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)methyl)phenyl)(4-methylpiperazin-1-yl)methanone (21)
The title compound was prepared following the same proce-
dure used for the synthesis of 19, using 1-methylpiperazine as
amine. Chromatography on silica gel (95:5 CH2Cl2/MeOH as elu-
ent) afforded pure 21 as oil. Yield = 77%. IR (neat) 2924, 2794,
1698, 1629, 1424, 1288, 997, 747; 1H NMR d 2.34 (3H, s), 2.38
(2H, br s), 2.51 (2H, br s), 2.64 (4H, m), 3.47 (2H, br s), 3.62 (2H,
s), 3.82 (2H, br s), 3.93 (4H, m), 7.31 (1H, m), 7.42 (4H, m), 7.60
(1H, t, J = 7.7 Hz), 7.98 (1H, d, J = 8.3 Hz), 8.08 (1H, d, J = 8.3 Hz);
13C NMR d 41.97, 45.94, 47.55, 52.80, 55.14, 62.34, 113.55,
119.82, 125.21, 126.01, 127.57, 128.54, 129.35, 130.27, 133.17,
135.95, 138.18, 145.30, 149.31, 170.19. Anal. Calcd for
The title compound was prepared from 3-(bromomethyl)biphe-
nyl following the same procedure used for the synthesis of 15.
Chromatography on silica gel (8:2 CH2Cl2/AcOEt as eluent) affor-
ded pure 25 as oil. Yield = 81%. IR 3009, 2812, 1700, 1449, 1433,
1018, 999, 675; 1H NMR d 2.63 (4H, m), 3.61 (2H, s), 3.91 (4H,
m), 7.29–7.43 (6H, m), 7.48–7.60 (5H, m), 7.97 (1H, d, J = 8.3 Hz),
8.05 (1H, d, J = 8.3 Hz); 13C NMR d 45.48, 47.55, 52.73, 62.71,
113.52, 119.75, 125.11, 126.11, 127.08, 127.29, 127.79, 127.99,
128.71, 128.76, 129.23, 133.14, 137.95, 140.88, 141.25, 145.24,
149.23. Anal. Calcd for C24H23N5O: C, 72.52; H, 5.83; N, 17.62.
Found: C, 72.77; H, 5.84; N, 17.58.
C
24H29N7O2: C, 64.41; H, 6.53; N, 21.91. Found: C, 64.58; H, 6.51;
5.1.20. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(biphenyl-2-ylmethyl)
piperazin-1-yl)methanone (26)
N, 21.87.
The title compound was prepared from 2-(bromomethyl)biphe-
nyl following the same procedure used for the synthesis of 15.
Chromatography on silica gel (75:25 n-hexane/AcOEt as eluent)
afforded pure 26 as oil. Yield = 84%. IR 3025, 3011, 2816, 1699,
1449, 1433, 1214, 775, 717; 1H NMR d 2.51 (4H, m), 3.50 (2H, s),
4.10 (4H, m), 7.25–7.42 (9H, m), 7.54 (2H, m), 7.95 (1H, d,
J = 8.3 Hz), 8.05 (1H, d, J = 8.3 Hz); 13C NMR d 47.93, 52.39, 59.52,
113.49, 119.75, 125.11, 126.91, 127.09, 127.20, 127.88, 129.23,
129.32, 129.87, 130.17, 133.12, 134.93, 141.29, 142.79, 145.24,
149.21. Anal. Calcd for C24H23N5O: C, 72.52; H, 5.83; N, 17.62.
Found: C, 72.35; H, 5.82; N, 17.63.
5.1.16. 3-((4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)methyl)-N-cyclohexylbenzamide (22)
The title compound was prepared following the same procedure
used for the synthesis of 19, using cyclohexylamine as amine.
Chromatography on silica gel (7:3 AcOEt/n-hexane as eluent) affor-
ded pure 22 as white solid. Yield = 92%. Mp = 158–159 °C; IR (KBr)
3321, 2936, 1722, 1631, 1525, 1435, 1281, 754; 1H NMR d 1.13–
1.27 (4H, m), 1.37 (2H, m), 1.62 (1H, m), 1.72 (2H, m), 2.00 (2H,
m), 2.59 (4H, m), 3.57 (2H, s), 3.88 (4H, m), 6.22 (1H, d,
J = 7.7 Hz), 7.34 (1H, t, J = 7.6 Hz), 7.43 (2H, m), 7.56 (1H, t,
J = 7.6 Hz), 7.63 (1H, d, J = 7.7 Hz), 7.74 (1H, br s), 7.94 (1H, d,
J = 8.3 Hz), 8.04 (1H, d, J = 8.3 Hz); 13C NMR d 24.99, 25.57, 33.19,
48.80, 52.80, 62.40, 113.56, 119.82, 125.23, 125.86, 127.75,
128.54, 129.37, 131.88, 133.18, 135.37, 138.00, 145.31, 149.31,
166.53. Anal. Calcd for C25H30N6O2: C, 67.24; H, 6.77; N, 18.82.
Found: C, 67.33; H, 6.75; N, 18.77.
5.1.21. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(naphthalen-2-ylmethyl)
piperazin-1-yl)methanone (27)
The title compound was prepared from 2-(bromomethyl)naph-
thalene following the same procedure used for the synthesis of 15.
Chromatography on silica gel (8:2 CH2Cl2/AcOEt as eluent) affor-
ded pure 27 as oil. Yield = 70%. IR 3009, 2817, 1699, 1449, 1433,
1018, 999, 677; 1H NMR d 2.65 (4H, m), 3.70 (2H, s), 3.92 (4H,
m), 7.41–7.51 (4H, m), 7.56 (1H, t, J = 7.7 Hz), 7.73 (1H, s), 7.81
(3H, m), 7.99 (1H, d, J = 8.4 Hz), 8.07 (1H, d, J = 8.4 Hz); 13C NMR
d 45.76, 52.84, 62.84, 113.53, 119.78, 125.13, 125.75, 126.06,
127.17, 127.63, 127.66, 127.73, 128.06, 129.27, 132.83, 133.16,
133.25, 134.99, 145.28, 149.27. Anal. Calcd for C22H21N5O: C,
71.14; H, 5.70; N, 18.85. Found: C, 71.36; H, 5.69; N, 18.82.
5.1.17. 3-((4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)methyl)-N-phenylbenzamide (23)
The title compound was prepared following the same procedure
used for the synthesis of 19, using aniline as amine. Chromatogra-
phy on silica gel (6:4 AcOEt/n-hexane as eluent) afforded pure 23
as white solid. Yield = 93%. Mp = 65–68 °C; IR (neat) 3308, 2919,
2814, 1696, 1529, 1429, 1320, 1228, 997, 749; 1H NMR d 2.66
(4H, m), 3.65 (2H, s), 3.93 (4H, m), 7.15 (1H, t, J = 7.4 Hz), 7.36
(2H, t, J = 8.0 Hz), 7.44 (2H, m), 7.52 (1H, d, J = 7.6 Hz), 7.59 (1H,
t, J = 7.7 Hz), 7.68 (2H, d, J = 7.8 Hz), 7.79 (1H, d, J = 7.7 Hz), 7.92
(1H, br s), 7.98 (1H, d, J = 8.3 Hz), 8.07 (1H, d, J = 8.3 Hz), 8.12
(1H, br s); 13C NMR d 52.82, 60.49, 62.38, 113.59, 119.87, 120.29,
124.60, 125.30, 126.21, 127.93, 128.88, 129.08, 129.44, 132.48,
133.21, 135.36, 138.02, 145.35, 149.34, 165.67. Anal. Calcd for
5.1.22. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(naphthalen-1-ylmethyl)
piperazin-1-yl)methanone (28)
The title compound was prepared from 1-(chloromethyl)naph-
thalene following the same procedure used for the synthesis of
15. Chromatography on silica gel (95:5 CH2Cl2/AcOEt as eluent)
afforded pure 28 as oil. Yield = 27%. IR 3009, 2817, 1700, 1448,
1433, 1231, 997, 677; 1H NMR d 2.71 (4H, m), 3.90 (4H, m), 4.00
(2H, s), 7.40–7.61 (6H, m), 7.81 (1H, d, J = 8.1 Hz), 7.87 (1H, d,
J = 8.3 Hz), 7.98 (1H, d, J = 8.3 Hz), 8.08 (1H, d, J = 8.3 Hz), 8.31
(1H, d, J = 8.1 Hz); 13C NMR d 42.73, 53.02, 61.01, 113.58, 119.86,
124.66, 125.13, 125.20, 125.77, 125.92, 127.73, 128.39, 128.51,
129.34, 132.50, 133.23, 133.94, 145.36, 149.36. Anal. Calcd for
C25H24N6O2: C, 68.17; H, 5.49; N, 19.08. Found: C, 68.25; H, 5.48;
N, 19.13.
5.1.18. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(biphenyl-4-ylmethyl)
piperazin-1-yl)methanone (24)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-(biphenyl-4-ylmethyl)pipera-
zine instead of 1-phenylpiperazine. Chromatography on silica gel
C22H21N5O: C, 71.14; H, 5.70; N, 18.85. Found: C, 70.95; H, 5.70;
N, 18.89.

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L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
5.1.23. (4-Benzhydrylpiperazin-1-yl)(1H-benzo[d][1,2,3]triazol-
1-yl)methanone (29)
azine. Chromatography on silica gel (8:2 AcOEt/n-hexane as elu-
ent) afforded pure 33 as yellow solid. Yield = 46%. Mp = 209–
210 °C; IR (KBr) 3417, 3299, 2969, 1721, 1698, 1626, 1521, 1434,
1220, 1009, 749; 1H NMR d 1.28 (6H, d, J = 6.4 Hz), 3.36 (4H, t,
J = 5.2 Hz), 4.12 (4H, m), 4.30 (1H, m), 5.99 (1H, d, J = 7.6 Hz),
7.20 (1H, d, J = 8.8 Hz), 7.48 (1H, t, J = 7.6 Hz), 7.63 (1H, t,
J = 7.6 Hz), 7.97 - 8.03 (2H, m), 8.11 (1H, d, J = 8.4 Hz), 8.23 (1H,
d, J = 2.0 Hz); 13C NMR d 22.82, 42.29, 51.21, 53.44, 113.61,
119.97, 120.71, 124.99, 125.48, 128.38, 129.66, 132.63, 133.15,
141.59, 145.36, 147.42, 149.51, 163.95. Anal. Calcd for
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-benzhydrylpiperazine instead
of 1-phenylpiperazine. Chromatography on silica gel (9:1 CH2Cl2/
AcOEt as eluent) afforded pure 29 as white solid. Yield = 65%.
Mp = 164–165 °C; IR (KBr) 3418, 2814, 1692, 1426, 1231, 992,
754; 1H NMR d 2.59 (4H, m), 3.93 (4H, m), 4.33 (1H, s), 7.21 (2H,
m), 7.30 (4H, m), 7.43 (5H, m), 7.59 (1H, t, J = 7.6 Hz), 7.97 (1H,
d, J = 8.4 Hz), 8.07 (1H, d, J = 8.4 Hz); 13C NMR d 51.72, 58.93,
75.92, 113.55, 119.85, 125.17, 127.27, 127.87, 128.68, 129.31,
133.17, 141.93, 145.30, 149.30. Anal. Calcd for C24H23N5O: C,
72.52; H, 5.83; N, 17.62. Found: C, 72.38; H, 5.83; N, 17.66.
C21H23N7O4: C, 57.66; H, 5.30; N, 22.41. Found: C, 57.76; H, 5.31;
N, 22.47.
5.1.28. (4-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-3-nitrophenyl)(morpholino) methanone (34)
5.1.24. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(2-fluorophenyl) piper-
azin-1-yl)methanone (30)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 49b instead of 1-phenylpiper-
azine. Chromatography on silica gel (9:1 AcOEt/n-hexane as
eluent) afforded pure 34 as yellow solid. Yield = 86%. Mp = 167–
168 °C; IR (KBr) 3416, 2853, 1698, 1631, 1426, 1249, 1113, 1007,
751; 1H NMR d 3.25–3.44 (6H, m), 3.73 (6H, m), 4.12 (4H, m),
7.23 (1H, d, J = 8.2 Hz), 7.49 (1H, t, J = 7.6 Hz), 7.64 (2H, m), 7.96
(1H, s), 8.02 (1H, d, J = 8.3 Hz), 8.12 (1H, d, J = 8.3 Hz); 13C NMR d
30.95, 51.38, 53.46, 66.78, 113.62, 119.97, 121.14, 125.48, 125.87,
128.96, 129.66, 132.95, 133.16, 142.17, 145.37, 146.52, 149.50,
167.77. Anal. Calcd for C22H23N7O5: C, 56.77; H, 4.98; N, 21.06.
Found: C, 56.88; H, 4.96; N, 21.03.
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-(2-fluorophenyl)piperazine in-
stead of 1-phenylpiperazine. Chromatography on silica gel (98:2
CH2Cl2/AcOEt as eluent) afforded pure 30 as solid. Yield = 91%.
Mp = 115–116 °C; IR (KBr) 2841, 1698, 1500, 1426, 1239, 1012,
749; 1H NMR d 3.29 (4H, t, J = 5.0 Hz), 4.11 (4H, m), 6.98–7.12
(4H, m), 7.47 (1H, t, J = 7.6 Hz), 7.62 (1H, t, J = 7.6 Hz), 8.02 (1H,
d, J = 8.2 Hz), 8.11 (1H, d, J = 8.2 Hz); 13C NMR d 50.68, 53.45,
113.63, 116.35 (d, JC–F = 20.5 Hz), 119.37 (d, JC–F = 2.6 Hz), 119.93,
123.42 (d, JC–F = 7.9 Hz), 124.64 (d, JC–F = 3.6 Hz), 125.35, 129.49,
133.24, 139.35 (d, JC–F = 8.6 Hz), 145.40, 149.45, 155.83 (d, JC–F
=
244.6 Hz). Anal. Calcd for C17H16FN5O: C, 62.76; H, 4.96; N, 21.53.
Found: C, 62.83; H, 4.95; N, 21.47.
5.1.29. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(4-methoxyphenyl)
piperazin-1-yl)methanone (35)
5.1.25. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(o-tolyl)piperazin-1-
yl)methanone (31)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-(4-methoxyphenyl)piperazine
instead of 1-phenylpiperazine. Chromatography on silica gel (7:3
n-hexane/AcOEt as eluent) afforded pure 35 as white solid.
Yield = 71%. Mp = 131 °C; IR (KBr) 3414, 2928, 2829, 1715, 1513,
1437, 1232, 1013, 755; 1H NMR d 3.26 (4H, m), 3.78 (3H, s), 4.09
(4H, m), 6.87 (2H, d, J = 9.2 Hz), 6.96 (2H, d, J = 9.2 Hz), 7.47 (1H,
t, J = 7.6 Hz), 7.62 (1H, t, J = 7.6 Hz), 8.02 (1H, d, J = 8.4 Hz), 8.11
(1H, d, J = 8.4 Hz); 13C NMR d 46.39, 51.11, 55.54, 113.62, 114.56,
119.08, 119.91, 125.31, 129.47, 133.21, 145.04, 145.37, 149.39,
154.56. Anal. Calcd for C18H19N5O2: C, 64.08; H, 5.68; N, 20.76.
Found: C, 64.28; H, 5.67; N, 20.82.
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-o-tolylpiperazine dihydrochlo-
ride instead of 1-phenylpiperazine and TEA (3.6 equiv). Chroma-
tography on silica gel (98:2 CH2Cl2/AcOEt as eluent) afforded
pure 31 as solid. Yield = 61%. Mp = 136 °C; IR (KBr) 2823, 1695,
1490, 1414, 1225, 1009, 758; 1H NMR d 2.38 (3H, s), 3.10 (4H, t,
J = 4.8 Hz), 4.08 (4H, m), 7.04 (2H, m), 7.21 (3H, m), 7.47 (1H, t,
J = 7.4 Hz), 7.62 (1H, t, J = 7.4 Hz), 8.02 (1H, d, J = 8.4 Hz), 8.11
(1H, d, J = 8.4 Hz); 13C NMR d 17.84, 47.97, 51.85, 113.60, 119.39,
119.91, 124.02, 125.28, 126.78, 129.43, 131.25, 132.80, 133.26,
145.39, 149.55, 150.60. Anal. Calcd for C18H19N5O: C, 67.27; H,
5.96; N, 21.79. Found: C, 67.32; H, 5.96; N, 21.77.
5.1.30. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(4-nitrophenyl)piperazin-
1-yl)methanone (36)
5.1.26. (1H-Benzo[d][1,2,3]triazol-1-yl)(4-(2-nitrophenyl) piperazin-
1-yl)methanone (32)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-(4-nitrophenyl)piperazine in-
stead of 1-phenylpiperazine. Chromatography on silica gel (98:2
CH2Cl2/AcOEt as eluent) afforded pure 36 as yellow solid.
Yield = 57%. Mp = 220 °C dec; IR (KBr) 2865, 1682, 1596, 1473,
1301, 1148, 1004, 753; 1H NMR d 3.67 (4H, t, J = 4.8 Hz), 4.15
(4H, m), 6.89 (2H, d, J = 9.4 Hz), 7.50 (1H, t, J = 7.6 Hz), 7.65 (1H,
t, J = 7.6 Hz), 8.04 (1H, d, J = 8.2 Hz), 8.13 (1H, d, J = 8.2 Hz), 8.18
(2H, d, J = 9.4 Hz); 13C NMR d 46.93, 66.42, 113.13, 113.69,
120.01, 125.57, 125.99, 129.76, 133.13, 139.34, 145.36, 149.49,
154.31. Anal. Calcd for C17H16N6O3: C, 57.95; H, 4.58; N, 23.85.
Found: C, 58.09; H, 4.59; N, 23.80.
The title compound was prepared following the same procedure
used for the synthesis of 8, using 1-(2-nitrophenyl)piperazine39 in-
stead of 1-phenylpiperazine. Chromatography on silica gel (75:25
n-hexane/AcOEt as eluent) afforded pure 32 as yellow solid.
Yield = 87%. Mp = 136–138 °C; IR (KBr) 3037, 2852, 2833, 1706,
1526, 1446, 1430, 1364, 1348, 1283, 1227, 1011, 783, 748; 1H
NMR
d 3.27 (4H, t, J = 4.7 Hz), 4.08 (4H, m), 7.15 (1H, t,
J = 7.9 Hz), 7.23 (1H, d, J = 7.9 Hz), 7.45 (1H, t, J = 7.9 Hz), 7.53
(1H, t, J = 7.9 Hz), 7.61 (1H, t, J = 7.9 Hz), 7.81 (1H, d, J = 7.9 Hz),
8.00 (1H, d, J = 8.3 Hz), 8.09 (1H, d, J = 8.3 Hz); 13C NMR d 46.20,
51.98, 113.59, 119.86, 121.81, 123.31, 125.32, 125.80, 129.47,
133.18, 133.66, 144.33, 145.33, 145.40, 149.46. Anal. Calcd for
5.1.31. 5-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-N,N-dimethyl-2-nitrobenzamide (37)
C17H16N6O3: C, 57.95; H, 4.58; N, 23.85. Found: C, 57.75; H, 4.60;
N, 23.77.
The title compound was prepared following the same procedure
used for the synthesis of 8, using 47c instead of 1-phenylpiper-
azine. Chromatography on silica gel (1:1 CH2Cl2/AcOEt as eluent)
afforded pure 37 as yellow solid. Yield = 62%. Mp = 208–210 °C;
IR (KBr) 3020, 2932, 1704, 1651, 1574, 1433, 1318, 1241, 1005,
767; 1H NMR d 2.83 (3H, s), 3.16 (3H, s), 3.69 (4H, t, J = 5.0 Hz),
5.1.27. 4-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-N-isopropyl-3-nitrobenzamide (33)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 49a instead of 1-phenylpiper-

L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
6271
4.13 (4H, m), 6.70 (1H, d, J = 2.7 Hz), 6.87 (1H, dd, J = 2.7, 9.4 Hz),
7.49 (1H, t, J = 7.7 Hz), 7.64 (1H, t, J = 7.7 Hz), 8.04 (1H, d,
J = 8.3 Hz), 8.12 (1H, d, J = 8.3 Hz), 8.17 (1H, d, J = 9.4 Hz); 13C
(1H, m), 5.64 (1H, d, J = 8.0 Hz), 6.79 (1H, d, J = 2.4 Hz), 6.85 (1H,
dd, J = 2.4, 9.2 Hz), 7.50 (1H, t, J = 7.6 Hz), 7.65 (1H, t, J = 7.6 Hz),
8.02–8.13 (3H, m); 13C NMR d 22.40, 42.31, 46.68, 60.40, 112.70,
113.27, 113.70, 120.01, 125.60, 127.29, 129.79, 133.11, 135.77,
136.09, 145.35, 149.47, 153.51, 166.65. Anal. Calcd for
NMR
d 34.87, 38.05, 46.60, 111.33, 113.03, 113.73, 120.02,
125.61, 127.34, 129.79, 133.14, 135.34, 135.93, 145.39, 149.50,
153.95, 168.58. Anal. Calcd for C20H21N7O4: C, 56.73; H, 5.00; N,
23.16. Found: C, 56.83; H, 5.01; N, 23.14.
C21H23N7O4: C, 57.66; H, 5.30; N, 22.41. Found: C, 57.75; H, 5.31;
N, 22.39.
5.1.32. (5-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-2-nitrophenyl)(piperidin-1-yl)methanone (38)
5.1.36. 5-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-N-cyclohexyl-2-nitrobenzamide (42)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 47f instead of 1-phenylpiper-
azine. Chromatography on silica gel (7:3 AcOEt/n-hexane as
eluent) afforded pure 38 as yellow solid. Yield = 73%. Mp = 174–
177 °C; IR (KBr) 2917, 2850, 1701, 1637, 1447, 1323, 1245, 1232,
1007, 759; 1H NMR d 1.45 (1H, m), 1.52 (1H, m), 1.66 (3H, m),
1.78 (1H, m), 3.16 (2H, t, J = 5.5 Hz), 3.67 (4H, m), 3.75 (2H, m),
4.13 (4H, m), 6.67 (1H, d, J = 2.7 Hz), 6.86 (1H, dd, J = 2.7, 9.4 Hz),
7.49 (1H, t, J = 7.5 Hz), 7.64 (1H, t, J = 7.5 Hz), 8.03 (1H, d,
J = 8.3 Hz), 8.11 (1H, d, J = 8.3 Hz), 8.15 (1H, d, J = 9.4 Hz); 13C
NMR d 24.50, 25.05, 25.82, 42.68, 46.65, 47.75, 111.25, 113.00,
113.73, 120.01, 125.59, 127.33, 129.77, 133.17, 135.45, 136.02,
145.40, 149.51, 153.92, 166.88. Anal. Calcd for C23H25N7O4: C,
59.60; H, 5.44; N, 21.15. Found: C, 59.73; H, 5.43; N, 21.19.
The title compound was prepared following the same procedure
used for the synthesis of 8, using 47e instead of 1-phenylpiper-
azine. Chromatography on silica gel (8:2 CH2Cl2/AcOEt as eluent)
afforded pure 42 as yellow solid. Yield = 43%. Mp = 218–219 °C;
IR (KBr) 3269, 2930, 2853, 1702, 1644, 1600, 1579, 1504, 1428,
1323, 1241, 1229, 1005, 752; 1H NMR (DMSO-d6) d 0.64 (5H, m),
0.95 (1H, m), 1.09 (2H, m), 1.26 (2H, m), 2.56 (4H, m), 3.08 (4H,
m), 3.38 (1H, m), 6.22 (1H, d, J = 2.7 Hz), 6.40 (1H, dd, J = 2.7,
9.4 Hz), 6.91 (1H, t, J = 7.7 Hz), 7.08 (1H, t, J = 7.7 Hz), 7.35 (1H, d,
J = 8.3 Hz), 7.39 (1H, d, J = 9.4 Hz), 7.55 (1H, d, J = 8.3 Hz), 7.61
(1H, d, J = 7.9 Hz); 13C NMR d 24.38, 25.05, 31.75, 45.62, 48.26,
78.27, 78.60, 78.93, 112.00, 112.33, 113.23, 119.33, 125.22,
126.42, 129.26, 132.38, 134.82, 136.37, 144.54, 148.78, 153.03,
165.71. Anal. Calcd for C24H27N7O4: C, 60.37; H, 5.70; N, 20.53.
Found: C, 60.58; H, 5.71; N, 20.59.
5.1.33. (5-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-2-nitrophenyl)(morpholino) methanone (39)
5.1.37. 5-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-2-nitro-N-phenylbenzamide (43)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 47b instead of 1-phenylpiper-
azine. Chromatography on silica gel (9:1 AcOEt/n-hexane as
eluent) afforded pure 39 as yellow solid. Yield = 70%. Mp = 213–
215 °C; IR (KBr) 2922, 2858, 1703, 1641, 1427, 1259, 1005, 755;
1H NMR d 3.23 (2H, br s), 3.57–3.83 (8H, m), 3.86–3.97 (2H, m),
4.09–4.15 (4H, m), 6.70 (1H, d, J = 2.7 Hz), 6.89 (1H, dd, J = 2.7,
9.4 Hz), 7.50 (1H, t, J = 7.7 Hz), 7.65 (1H, t, J = 7.7 Hz), 8.04 (1H, d,
J = 8.3 Hz), 8.13 (1H, d, J = 8.3 Hz), 8.19 (1H, d, J = 9.4 Hz); 13C
NMR d 42.20, 46.57, 47.00, 66.39, 111.21, 113.18, 113.71, 120.04,
125.65, 127.51, 129.85, 133.13, 134.98, 135.36, 145.43, 149.45,
153.96, 167.29. Anal. Calcd for C22H23N7O5: C, 56.77; H, 4.98; N,
21.06. Found: C, 56.66; H, 4.99; N, 21.11.
The title compound was prepared following the same procedure
used for the synthesis of 8, using 47d instead of 1-phenylpiper-
azine. Chromatography on silica gel (7:3 CHCl3/AcOEt as eluent)
afforded pure 43 as yellow solid. Yield = 56%. Mp = 136–138 °C;
IR (KBr) 3278, 2917, 1681, 1598, 1578, 1443, 1318, 1222, 1004,
752; 1H NMR d 3.48 (4H, m), 3.96 (4H, m), 6.51 (1H, dd, J = 1.9,
9.3 Hz), 6.62 (1H, d, J = 1.9 Hz), 7.04 (1H, t, J = 7.5 Hz), 7.21 (2H, t,
J = 7.8 Hz), 7.40 (1H, t, J = 7.5 Hz), 7.54 (3H, m), 7.67 (1H, d,
J = 9.3 Hz), 7.91 (1H, d, J = 8.3 Hz), 7.99 (1H, d, J = 8.3 Hz), 9.10
(1H, br s); 13C NMR d 45.97, 60.40, 111.79, 112.49, 113.62,
119.72, 120.29, 124.58, 125.54, 127.02, 128.92, 129.67, 132.96,
134.50, 135.19, 138.00, 145.11, 149.35, 153.07, 165.94. Anal. Calcd
for C24H21N7O4: C, 61.14; H, 4.49; N, 20.80. Found: C, 61.33; H,
4.50; N, 20.83.
5.1.34. (5-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-2-nitrophenyl)(4-methylpiperazin-1-yl)methanone (40)
The title compound was prepared following the same procedure
used for the synthesis of 8, using 47g instead of 1-phenylpiper-
azine. Chromatography on silica gel (95:5 CH2Cl2/MeOH as eluent)
afforded pure 40 as yellow solid. Yield = 40%. Mp = 99–103 °C; IR
(KBr) 3482, 2937, 2797, 1702, 1639, 1598, 1578, 1446, 1319,
1228, 1002, 754; 1H NMR d 2.31 (5H, m), 2.48 (1H, m), 2.58 (1H,
m), 3.23 (2H, br s), 3.70 (5H, m), 3.91 (1H, m), 4.13 (4H, m), 6.67
(1H, d, J = 2.7 Hz), 6.86 (1H, dd, J = 2.7, 9.4 Hz), 7.48 (1H, t,
J = 7.7 Hz), 7.63 (1H, t, J = 7.7 Hz), 8.02 (1H, d, J = 8.3 Hz), 8.10
(2H, m); 13C NMR d 41.64, 45.98, 46.46, 46.58, 54.08, 54.49,
111.10, 113.02, 113.70, 119.91, 125.54, 127.32, 129.70, 133.12,
135.23, 135.39, 145.32, 149.44, 153.88, 166.95. Anal. Calcd for
5.1.38. tert-Butyl 4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)
piperazine-1-carboxylate (44)
The title compound was prepared following the same procedure
used for the synthesis of 8, using tert-butylpiperazine-1-carboxyl-
ate instead of 1-phenylpiperazine. Chromatography on silica gel
(8:2 n-hexane/AcOEt as eluent) afforded pure 44 as white solid.
Yield = 91%. Mp = 105–106 °C; IR (KBr) 2971, 1722, 1697, 1420,
1247, 1169, 998, 748; 1H NMR d 1.50 (9H, s), 3.65 (4H, t,
J = 5.2 Hz), 3.90 (4H, m), 7.47 (1H, t, J = 7.7 Hz), 7.62 (1H, t,
J = 7.7 Hz), 8.00 (1H, d, J = 8.4 Hz), 8.10 (1H, d, J = 8.4 Hz); 13C
NMR d 28.40, 80.57, 113.63, 119.96, 125.39, 129.56, 133.20,
145.40, 149.58, 154.54. Anal. Calcd for C16H21N5O3: C, 57.99; H,
6.39; N, 21.13. Found: C, 57.10; H, 6.39; N, 21.17.
C23H26N8O4: C, 57.73; H, 5.48; N, 23.42. Found: C, 57.87; H, 5.48;
N, 23.47.
5.1.39. (1H-Benzo[d][1,2,3]triazol-1-yl)(piperazin-1-yl)methanone
2,2,2-trifluoroacetate (45)
5.1.35. 5-(4-(1H-Benzo[d][1,2,3]triazole-1-carbonyl)piperazin-
1-yl)-N-isopropyl-2-nitrobenzamide (41)
To a solution of 44 (4.50 g, 13.6 mmol) in CHCl3 (5 mL) a solu-
tion of 2,2,2-trifluoroacetic acid (10 mL) in CHCl3 (10 mL) was
added, and the mixture was stirred at room temperature for 2 h.
The reaction mixture was then evaporated in vacuo and the residue
was triturated with Et2O and filtered to give 4.59 g of pure 45 (98%)
as white solid. Mp = 205–207 °C; IR (KBr) 2983, 2480, 1723, 1675,
1440, 1202, 1122, 1002; 1H NMR (CD3OD) d 3.47 (4H, t, J = 5.3 Hz),
The title compound was prepared following the same procedure
used for the synthesis of 8, using 47a instead of 1-phenylpiper-
azine. Chromatography on silica gel (7:3 AcOEt/n-hexane as elu-
ent) afforded pure 41 as yellow solid. Yield = 94%. Mp = 129 °C; IR
(KBr) 3333, 2972, 1687, 1651, 1578, 1317, 1271, 1006, 755; 1H
NMR d 1.28 (6H, d, J = 6.4 Hz), 3.67 (4H, m), 4.13 (4H, m), 4.30

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L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
4.17 (4H, m), 7.55 (1H, t, J = 7.4 Hz), 7.70 (1H, t, J = 7.4 Hz), 8.02
(1H, d, J = 8.4 Hz), 8.11 (1H, d, J = 8.4 Hz); 13C NMR d 41.70, 44.47,
114.89, 120.66, 127.00, 131.03, 134.40, 146.59, 150.70. Anal. Calcd
for C13H14F3N5O3: C, 45.22; H, 4.09; N, 20.28. Found: C, 45.16; H,
4.10; N, 20.25.
Calcd for C13H15ClN2O3: C, 55.23; H, 5.35; N, 9.91. Found: C, 55.15;
H, 5.36; N, 9.89.
5.1.39.6.
(5-Chloro-2-nitrophenyl)(piperidin-1-yl)methanone
(46f). The title compound was prepared following the same proce-
dure used for the synthesis of 46a, using piperidine as amine. Chro-
matography on silica gel (7:3 n-hexane/AcOEt as eluent) afforded
pure 46f as white solid. Yield = 71%. Mp = 109–111 °C; IR (KBr)
2935, 2864, 1638, 1527, 1443, 1342, 1273, 1005, 830; 1H NMR d
1.53 (2H, m), 1.69 (4H, m), 3.20 (2H, t, J = 5.6 Hz), 3.74 (2H, m),
7.38 (1H, d, J = 2.4 Hz), 7.54 (1H, dd, J = 2.4, 8.8 Hz), 8.15 (1H, d,
J = 8.8 Hz); 13C NMR d 24.31, 25.00, 25.70, 42.71, 47.90, 126.26,
127.92, 129.69, 134.90, 140.99, 143.44, 164.62. Anal. Calcd for
5.1.39.1. 5-Chloro-N-isopropyl-2-nitrobenzamide (46a). A solu-
tion of 5-chloro-2-nitrobenzoic acid (1.209 g, 6 mmol) in thionyl
chloride (3.9 mL, 54 mmol) was stirred at 80 °C for 2 h. The reac-
tion mixture was then evaporated in vacuo and to the residue, dis-
solved in CH2Cl2 (5 mL), isopropylamine (567 ll, 13.2 mmol) was
added dropwise. The reaction mixture was stirred at room temper-
ature overnight, and then quenched by adding a 1 N NaOH solution
and extracted with AcOEt. The organic phase was washed succes-
sively with brine, 2 N HCl, and brine until neutral, dried over
Na2SO4, and evaporated in vacuo to give a residue of 1.3 g (89%)
of 46a as white solid. Mp = 172 °C; IR (KBr) 3270, 3098, 2978,
1644, 1554, 1526, 1364, 1150, 906, 835; 1H NMR d 1.26 (6H, d,
J = 6.4 Hz), 4.23 (1H, m), 7.44 (1H, d, J = 2.0 Hz), 7.50 (1H, dd,
J = 2.0, 8.8 Hz), 8.01 (1H, d, J = 8.8 Hz); 13C NMR d 22.28, 42.51,
125.99, 128.94, 130.19, 134.76, 140.35, 144.35, 164.29. Anal. Calcd
for C10H11ClN2O3: C, 49.50; H, 4.57; N, 11.54. Found: C, 49.63; H,
4.57; N, 11.52.
C12H13ClN2O3: C, 53.64; H, 4.88; N, 10.43. Found: C, 53.66; H,
4.88; N, 10.40.
5.1.39.7.
(5-Chloro-2-nitrophenyl)(4-methylpiperazin-1-yl)
methanone (46g). The title compound was prepared following
the same procedure used for the synthesis of 46a, using 1-methyl-
piperazine as amine. Chromatography on silica gel (7:3 AcOEt/
CH2Cl2 as eluent) afforded pure 46g as white solid. Yield = 87%.
Mp = 146–151 °C; IR (KBr) 2980, 2806, 1636, 1528, 1346, 1294,
839, 762; 1H NMR d 2.32 (3H, s), 2.36 (2H, m), 2.54 (2H, m), 3.26
(2H, m), 3.81 (2H, m), 7.39 (1H, d, J = 2.0 Hz), 7.55 (1H, dd, J = 2.0,
8.8 Hz), 8.15 (1H, d, J = 8.8 Hz); 13C NMR d 41.77, 45.92, 46.72,
54.03, 54.39, 126.28, 128.05, 129.91, 134.35, 141.05, 143.60,
164.73. Anal. Calcd for C12H14ClN3O3: C, 50.80; H, 4.97; N, 14.81.
Found: C, 50.98; H, 4.95; N, 14.77.
5.1.39.2.
(5-Chloro-2-nitrophenyl)(morpholino)methanone
(46b). The title compound was prepared following the same proce-
dure used for the synthesis of 46a, using morpholine as amine. The
extraction afforded 46b as white solid. Yield = 92%. Mp = 156–
157 °C; IR (KBr) 2860, 1644, 1528, 1342, 1115, 1023, 844; 1H
NMR d 3.24 (2H, m), 3.64 (2H, m), 3.84 (4H, m), 7.39 (1H, d,
J = 2.2 Hz), 7.55 (1H, dd, J = 2.2, 8.8 Hz), 8.17 (1H, d, J = 8.8 Hz);
13C NMR d 42.33, 47.19, 66.23, 66.36, 126.37, 128.15, 130.12,
134.01, 141.40, 143.62, 165.07. Anal. Calcd for C11H11ClN2O4: C,
48.81; H, 4.10; N, 10.35. Found: C, 48.87; H, 4.11; N, 10.36.
5.1.40. N-Isopropyl-2-nitro-5-(piperazin-1-yl)benzamide (47a)
A solution of 46a (500 mg, 2 mmol) and piperazine (4.437 g,
51.5 mmol) in DMF (7 mL) was stirred at 130 °C for 1 h. After cool-
ing to room temperature, the reaction was quenched by adding a
1 N NaOH solution and extracted with CH2Cl2. The organic phase
was dried over Na2SO4, and evaporated in vacuo. The residue
was chromatographed on silica gel (9:1 CH2Cl2/MeOH as eluent)
to give 600 mg (99%) of pure 47a as yellow solid. Mp = 186–
189 °C; IR (KBr) 3253, 2967, 1652, 1579, 1327, 1246, 1080, 854;
1H NMR d 1.26 (6H, d, J = 6.4 Hz), 3.01 (4H, t, J = 5.3 Hz), 3.40 (4H,
t, J = 5.3 Hz), 4.28 (1H, m), 6.74 (1H, d, J = 2.8 Hz), 6.79 (1H, dd,
J = 2.8, 9.2 Hz), 8.05 (1H, d, J = 9.2 Hz); 13C NMR d 22.40, 42.24,
45.64, 47.89, 112.23, 112.85, 127.28, 134.76, 136.08, 154.34,
167.06. Anal. Calcd for C14H20N4O3: C, 57.52; H, 6.90; N, 19.17.
Found: C, 57.42; H, 6.92; N, 19.22.
5.1.39.3. 5-Chloro-N,N-dimethyl-2-nitrobenzamide (46c)40. The
title compound was prepared following the same procedure used
for the synthesis of 46a, using dimethylamine (33% in ethanol) as
amine. Chromatography on silica gel (6:4 n-hexane/AcOEt as eluent)
afforded pure 46c as white solid. Yield = 80%. Mp = 96–102 °C; IR
(KBr) 3093, 2928, 1640, 1567, 1529, 1402, 1343, 1099, 859; 1H
NMR d 2.86 (3H, s), 3.16 (3H, s), 7.39 (1H, d, J = 2.2 Hz), 7.52 (1H,
dd, J = 2.2, 8.8 Hz), 8.16 (1H, d, J = 8.8 Hz); 13C NMR d 34.94, 38.21,
126.22, 128.27, 129.80, 134.94, 141.27, 143.43, 166.40.
5.1.39.4. 5-Chloro-2-nitro-N-phenylbenzamide (46d). The title
compound was prepared following the same procedure used for
the synthesis of 46a, using aniline as amine. The extraction affor-
ded 46d as white solid. Yield = 91%. Mp = 166–168 °C; IR (KBr)
3259, 3058, 1658, 1531, 1443, 1354, 1321, 750. 1H NMR 7.20
(1H, t, J = 7.6 Hz), 7.38 (2H, t, J = 7.6 Hz), 7.55–7.61 (4H, m), 8.09
(1H, d, J = 8.7 Hz); 13C NMR d 120.57, 125.49, 126.26, 128.87,
129.26, 130.76, 134.44, 137.02, 140.64, 144.56, 166.98. Anal. Calcd
for C13H9ClN2O3: C, 56.43; H, 3.28; N, 10.13. Found: C, 56.33; H,
3.28; N, 10.15.
5.1.41. Morpholino(2-nitro-5-(piperazin-1-yl)phenyl)methanone
(47b)
The title compound was prepared from 46b following the same
procedure used for the synthesis of 47a. Chromatography on silica
gel (9:1 CH2Cl2/MeOH as eluent) afforded pure 47b as yellow solid.
Yield = 74%. Mp = 160–162 °C; IR (KBr) 3208, 2850, 1634, 1495,
1327, 1248, 1110, 1024; 1H NMR d 3.00 (4H, m), 3.22 (2H, m),
3.40 (4H, m), 3.60 (2H, m), 3.76 (2H, m), 3.87 (2H, m), 6.62 (1H,
d, J = 2.2 Hz), 6.83 (1H, dd, J = 2.2, 9.6 Hz), 8.10 (1H, d, J = 9.6 Hz);
13C NMR d 42.09, 45.52, 46.94, 47.71, 50.21, 53.57, 66.22, 66.26,
110.53, 112.69, 127.37, 134.08, 134.90, 154.70, 167.66. Anal. Calcd
for C15H20N4O4: C, 56.24; H, 6.29; N, 17.49. Found: C, 56.35; H,
6.31; N, 17.47.
5.1.39.5. 5-Chloro-N-cyclohexyl-2-nitrobenzamide (46e). The ti-
tle compound was prepared following the same procedure used for
the synthesis of 46a, using cyclohexylamine as amine. Chromatog-
raphy on silica gel (AcOEt as eluent) afforded pure 46e as white so-
lid. Yield = 65%. Mp = 198–200 °C; IR (KBr) 3266, 2937, 2852, 1638,
1562, 1524, 1346, 1085, 839, 749; 1H NMR (DMSO-d6) d 1.10–1.35
(5H, m), 1.57 (1H, m), 1.71 (2H, m), 1.85 (2H, m), 3.68 (1H, m), 7.66
(1H, d, J = 2.2 Hz), 7.76 (1H, dd, J = 2.2, 8.7 Hz), 8.08 (1H, d,
J = 8.7 Hz), 8.56 (1H, d, J = 7.9 Hz); 13C NMR d 24.33, 25.07, 31.76,
48.15, 125.98, 128.81, 130.13, 134.58, 138.09, 145.29, 162.99. Anal.
5.1.42. N,N-Dimethyl-2-nitro-5-(piperazin-1-yl)benzamide (47c)
The title compound was prepared from 46c following the same
procedure used for the synthesis of 47a. Chromatography on silica
gel (9:1 CH2Cl2/MeOH as eluent) afforded pure 47c as yellow solid.
Yield = 96%. Mp = 63–70 °C; IR (KBr) 3405, 2924, 1653, 1612, 1599,
1492, 1329, 1257, 1116; 1H NMR d 2.82 (3H, s), 3.01 (4H, t,
J = 5.3 Hz), 3.15 (3H, s), 3.42 (4H, t, J = 5.3 Hz), 6.64 (1H, d,

L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
6273
J = 2.8 Hz), 6.81 (1H, dd, J = 2.8, 9.5 Hz), 8.13 (1H, d, J = 9.5 Hz); 13
NMR d 34.86, 38.03, 45.64, 47.88, 110.92, 112.64, 127.28, 135.86,
154.78, 160.33, 168.99. Anal. Calcd for C13H18N4O3: C, 56.10; H,
6.52; N, 20.13. Found: C, 56.27; H, 6.54; N, 20.17.
C
1340, 1051, 851; 1H NMR d 1.28 (6H, d, J = 6.8 Hz), 4.25 (1H, m),
7.59 (1H, d, J = 8.0 Hz), 7.96 (1H, d, J = 8.0 Hz), 8.28 (1H, br s); 13C
NMR d 22.52, 42.58, 124.15, 129.75, 131.72, 132.11, 134.70,
147.49, 163.51. Anal. Calcd for C10H11ClN2O3: C, 49.50; H, 4.57;
N, 11.54. Found: C, 49.67; H, 4.58; N, 11.56.
5.1.43. 2-Nitro-N-phenyl-5-(piperazin-1-yl)benzamide (47d)
The title compound was prepared from 46d following the same
procedure used for the synthesis of 47a. Chromatography on silica
gel (9:1 acetone/MeOH as eluent) afforded pure 47d as yellow so-
lid. Yield = 94%. Mp = 120–123 °C; IR (KBr) 3272, 2952, 2849, 1660,
1597, 1444, 1317, 1251, 1036, 752; 1H NMR (DMSO-d6) d 2.80 (4H,
t, J = 4.9 Hz), 3.42 (4H, t, J = 4.9 Hz), 4.56 (1H, br s), 7.00–7.11 (3H,
m), 7.34 (2H, m), 7.67 (2H, d, J = 7.7 Hz), 8.05 (1H, d, J = 9.4 Hz),
10.42 (1H, s); 13C NMR d 55.73, 68.41, 111.72, 112.41, 119.40,
123.42, 126.81, 128.60, 133.49, 136.13, 139.14, 154.04, 165.12.
Anal. Calcd for C17H18N4O3: C, 62.57; H, 5.56; N, 17.17. Found: C,
62.45; H, 5.56; N, 17.22.
5.1.48. (4-Chloro-3-nitrophenyl)(morpholino)methanone (48b)
The title compound was prepared following the same procedure
used for the synthesis of 46a, using 4-chloro-3-nitrobenzoic acid as
starting material and morpholine as amine. The extraction afforded
48b as white solid. Yield = 55%. Mp = 145–146 °C; IR (KBr) 3071,
2863, 1634, 1524, 1436, 1258, 1108, 1023, 835; 1H NMR d 3.47
(2H, br s), 3.75 (6H, m), 7.58 (1H, dd, J = 2.0, 8.4 Hz), 7.64 (1H, d,
J = 8.4 Hz), 7.95 (1H, d, J = 2.0 Hz); 13C NMR d 42.86, 66.69,
124.63, 128.75, 131.78, 132.35, 135.01, 147.83, 166.73. Anal. Calcd
for C11H11ClN2O4: C, 48.81; H, 4.10; N, 10.35. Found: C, 48.69; H,
4.11; N, 10.33.
5.1.44. N-Cyclohexyl-2-nitro-5-(piperazin-1-yl)benzamide (47e)
The title compound was prepared from 46e following the same
procedure used for the synthesis of 47a. Chromatography on silica
gel (9:1 CH2Cl2/MeOH as eluent) afforded pure 47e as yellow solid.
Yield = 99%. Mp = 170–173 °C; IR (KBr) 3271, 2934, 2853, 1635,
1578, 1501, 1327, 1243, 1038, 828; 1H NMR d 1.18 (3H, m), 1.39
(2H, m), 1.63 (1H, m), 1.73 (2H, m),1.98 (1H, s), 2.08 (2H, m),
3.00 (4H, t, J = 5.2 Hz), 3.39 (4H, t, J = 5.2 Hz), 3.95 (1H, m), 5.76
(1H, d, J = 8.4 Hz), 6.72 (1H, d, J = 2.8 Hz), 6.78 (1H, dd, J = 2.8,
9.2 Hz), 8.02 (1H, d, J = 9.2 Hz); 13C NMR d 24.86, 25.54, 32.69,
45.59, 47.85, 48.92, 112.27, 112.78, 127.22, 134.78, 136.14,
154.34, 167.04. Anal. Calcd for C17H24N4O3: C, 61.43; H, 7.28; N,
16.86. Found: C, 61.55; H, 7.27; N, 16.88.
5.1.49. N-Isopropyl-3-nitro-4-(piperazin-1-yl)benzamide (49a)
The title compound was prepared from 48a following the same
procedure used for the synthesis of 47a. Chromatography on silica
gel (9:1 CH2Cl2/MeOH as eluent) afforded pure 49a as yellow solid.
Yield = 96%. Mp = 60 °C; IR (KBr) 3287, 2973, 1615, 1522, 1235,
1128, 823; 1H NMR d 1.27 (6H, d, J = 6.8 Hz), 3.02 (4H, m), 3.12
(4H, m), 4.27 (1H, m), 7.11 (1H, d, J = 8.8 Hz), 7.92 (1H, dd, J = 2.0,
8.8 Hz), 8.16 (1H, d, J = 2.0 Hz); 13C NMR d 22.82, 42.14, 45.73,
52.19, 120.05, 125.02, 126.52, 132.46, 140.52, 148.13, 164.22. Anal.
Calcd for C14H20N4O3: C, 57.52; H, 6.90; N, 19.17. Found: C, 57.37;
H, 6.92; N, 19.12.
5.1.50. Morpholino(3-nitro-4-(piperazin-1-yl)phenyl)methanone
(49b)
5.1.45. N-(2-Nitro-5-(piperazin-1-yl)benzoyl)piperidine (47f)
Thetitlecompoundwaspreparedfrom46f followingthesamepro-
cedure used for the synthesisof 47a. Chromatographyonsilicagel(9:1
CHCl3/MeOH as eluent) afforded pure 47f as yellow solid. Yield = 92%.
Mp = 67–69 °C; IR (KBr) 3446, 2937, 2852, 1634, 1597, 1577, 1492,
1446, 1322, 1244, 1040, 780; 1H NMR d 1.48 (2H, m), 1.65 (3H, m),
1.75 (1H, m), 2.18 (1H, br s), 2.98 (4H, t, J = 4.9 Hz), 3.16 (2H, m),
3.40 (4H, t, J = 4.9 Hz), 3.74 (2H, m), 6.61 (1H, d, J = 2.6 Hz), 6.84 (1H,
dd, J = 2.6, 9.5 Hz), 8.09 (1H, d, J = 9.5 Hz); 13C NMR d 24.41, 24.99,
25.65, 42.44, 45.50, 47.65, 47.72, 110.37, 112.44, 127.20, 133.89,
135.85, 154.64, 167.17. Anal. Calcd for C16H22N4O3: C, 60.36; H,
6.97; N, 17.60. Found: C, 60.57; H, 6.95; N, 17.58.
The title compound was prepared from 48b following the same
procedure used for the synthesis of 47a. Chromatography on silica
gel (9:1 CH2Cl2/MeOH as eluent) afforded pure 49b as yellow solid.
Yield = 73%. Mp = 196-198 °C; IR (KBr) 3337, 2838, 1619, 1528,
1437, 1330, 1113, 1026, 831; 1H NMR d 3.09 (4H, m), 3.16 (4H,
m), 3.65 (4H, m), 3.71 (4H, m), 7.15 (1H, d, J = 8.8 Hz), 7.57 (1H,
dd, J = 1.6, 8.8 Hz), 7.89 (1H, d, J = 1.6 Hz); 13C NMR d 45.44,
51.79, 66.80, 120.64, 125.96, 127.58, 132.82, 141.39, 147.10,
168.02. Anal. Calcd for C15H20N4O4: C, 56.24; H, 6.29; N, 17.49.
Found: C, 56.38; H, 6.31; N, 17.48.
5.2. Biological evaluation
5.1.46. 1-(2-Nitro-5-(piperazin-1-yl)benzoyl)-4-methylpiperazine
(47g)
5.2.1. MAGL esterase activity assay
MAGL activity was measured by following [3H]-2-oleoyl glyc-
erol ([3H]-2-OG) hydrolysis, as previously described.41 Briefly,
6 ng of pure human MAGL42 was preincubated during 30 min at
22 °C in the presence of the inhibitor at various concentrations
(Tris buffer 50 mM, BSA 0.15% w/v, pH 8.0, inhibitor dissolved
The title compound was prepared from 46g following the same
procedure used for the synthesis of 47a. Chromatography on silica
gel (9:1 CH2Cl2/MeOH as eluent) afforded pure 47g as oil.
Yield = 82%. IR 3690, 3011, 2947, 1634, 1581, 1445, 1328, 1246,
1237, 772; 1H NMR d 2.32 (5H, m), 2.50 (1H, m), 2.58 (1H, m),
3.01 (4H, t, J = 5.1 Hz), 3.23 (2H, t, J = 5.1 Hz), 3.40 (4H, t,
J = 5.1 Hz), 3.70 (1H, m), 3.97 (1H, m), 6.61 (1H, d, J = 2.8 Hz),
6.81 (1H, dd, J = 2.8, 9.4 Hz), 8.12 (1H, d, J = 9.4 Hz); 13C NMR d
41.67, 45.63, 46.02, 46.62, 47.91, 54.13, 54.54, 110.89, 112.72,
127.34, 134.51, 135.38, 154.74, 167.36. Anal. Calcd for
in 10
50,000 dpm, American Radiolabeled Chemicals; 200
ume assay) was then added and the tubes were placed at 37 °C
for 10 min. The reaction was stopped by adding 400 l of a cold
ll DMSO). 2-OG (10
l
M final concentration; [3H]-2-OG
l total vol-
l
l
methanol-chloroform (1-1) mixture and, after 5 min centrifuga-
tion at 700 g, the radioactivity was measured in the upper aque-
ous phase by liquid scintillation. The dpm value obtained for a
blank (containing no enzyme) was systematically subtracted. Re-
sults were then expressed as percent of control activity (without
inhibitor), after which Graph Pad prism software was used to
treat the data and to analyze the dose-response curves. Inhibitors
potency was expressed as IC50 values. JZL184 was purchased from
Cayman Chemical.
C16H23N5O3: C, 57.64; H, 6.95; N, 21.01. Found: C, 57.83; H, 6.96;
N, 20.99.
5.1.47. 4-Chloro-N-isopropyl-3-nitrobenzamide (48a)
The title compound was prepared following the same procedure
used for the synthesis of 46a, using 4-chloro-3-nitrobenzoic acid as
starting material. The extraction afforded 48a as white solid.
Yield = 89%. Mp = 115–116 °C; IR (KBr) 3286, 2978, 1638, 1531,

6274
L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
5.2.2. MAGL rapid dilution assay
The reversibility of MAGL inhibition by compound 29 was
investigated using the rapid dilution assay. The enzyme (800 ng
originally bound inhibitor. Following the manual positioning of
the piperazinyl moiety in order to match the conformation of
PF3845 (for the FAAH) or SAR629 (for the MAGL), the inhibitors
as well as key surrounding residues were minimized using the
MM+ force field. For the study of FAAH-inhibitors complexes, both
the open and closed rotamers that were described37 for the Phe432
were considered. Figures were prepared using Pymol.
in 38 ll of a buffer containing Tris 50 mM, BSA 0.15%, pH 8.0)
was incubated during 30 min at room temperature with 2
lL of
compound 29 (concentration of 10ꢀ8M and 10ꢀ9M in the mixture)
dissolved in DMSO. The enzyme-inhibitor mixture was then di-
luted 300-fold with the buffer. After 15 min of incubation, the en-
zyme activity was measured on a 150
ll aliquot (corresponding to
Acknowledgments
10 ng of enzyme) according to the above-described standard
procedure.
G. Labar is postdoctoral Research Fellow at the ‘Fonds National
de la Recherche Scientifique (FNRS)’ (Belgium). This work was sup-
ported by the ‘Fonds National de la Recherche Scientifique’ (FRSM
3.4552.11) and by a FSR Grant from Université catholique de
Louvain.
5.2.3. Measurement of kinact and Ki values
Inactivation constants for MAGL inactivation by compound 29
(1) were measured using p-nitrophenylpropionate as substrate.
In a 96-well plate, MAGL (3 ng in 180 ll of a buffer containing Tris
50 mM, LDAO 0.1%, pH 8.0) was added to a mixture of p-nitrophe-
References and notes
nylpropionate (1.25 mM) and various concentrations of the
inhibitor, both dissolved in 10 ll DMSO, after which the production
1. Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem. Lett. 2011, 21, 4674.
2. Minkkila, A.; Saario, S.; Nevalainen, T. Curr. Top. Med. Chem. 2010, 10, 828.
3. Petrosino, S.; Di Marzo, V. Curr. Opin. Investig. Drugs 2010, 11, 51.
4. Seierstad, M.; Breitenbucher, J. G. J. Med. Chem. 2008, 51, 7327.
5. Vandevoorde, S. Curr. Top. Med. Chem. 2008, 8, 247.
6. Viso, A.; Cisneros, J. A.; Ortega-Gutierrez, S. Curr. Top. Med. Chem. 2008, 8, 231.
7. Maccarrone, M. Curr. Pharm. Des. 2006, 12, 759.
8. Li, G. L.; Winter, H.; Arends, R.; Jay, G. W.; Le, V.; Young, T.; Huggins, J. P. Br. J.
Clin. Pharmacol. 2012, 73, 706.
9. Karlsson, M.; Contreras, J. A.; Hellman, U.; Tornqvist, H.; Holm, C. J. Biol. Chem.
1997, 272, 27218.
10. Dinh, T. P.; Kathuria, S.; Piomelli, D. Mol. Pharmacol. 2004, 66, 1260.
11. Long, J. Z.; Li, W.; Booker, L.; Burston, J. J.; Kinsey, S. G.; Schlosburg, J. E.; Pavon,
F. J.; Serrano, A. M.; Selley, D. E.; Parsons, L. H.; Lichtman, A. H.; Cravatt, B. F.
Nat. Chem. Biol. 2009, 5, 37.
12. Blankman, J. L.; Simon, G. M.; Cravatt, B. F. Chem. Biol. 2007, 14, 1347.
13. Schlosburg, J. E.; Blankman, J. L.; Long, J. Z.; Nomura, D. K.; Pan, B.; Kinsey, S. G.;
Nguyen, P. T.; Ramesh, D.; Booker, L.; Burston, J. J.; Thomas, E. A.; Selley, D. E.;
Sim-Selley, L. J.; Liu, Q. S.; Lichtman, A. H.; Cravatt, B. F. Nat. Neurosci. 2010, 13,
1113.
of p-nitrophenol was followed by measuring the absorbance at
405 nM on a SpectraMax IIe spectrophotometer. Blank values
(without enzyme) were subtracted to the optical density and the
time course of MAGL velocity was calculated using the first deriv-
ative of the optical density vs time curve in the GraphPad Prism
software. Then, the residual enzyme activity (expressed as percent
versus a control without inhibitor) as a function of time was fitted
to a first order decay equation to obtain the rate of inhibition (kobs
,
which is the first-order constant for enzyme inactivation) at each
inhibitor concentration. The inactivation rate (kinact) and inhibitor
dissociation constant (Ki) were then obtained by linear regression
using 1/kobs and 1/[I] values according to equations (2) and (3).
Ki
E þ I $ EI k$inact EIꢂ
ð1Þ
ð2Þ
ð3Þ
14. Nomura, D. K.; Long, J. Z.; Niessen, S.; Hoover, H. S.; Ng, S. W.; Cravatt, B. F. Cell
2010, 140, 49.
15. Nomura, D. K.; Lombardi, D. P.; Chang, J. W.; Niessen, S.; Ward, A. M.; Long, J. Z.;
Hoover, H. H.; Cravatt, B. F. Chem. Biol. 2011, 18, 846.
16. Ye, L.; Zhang, B.; Seviour, E. G.; Tao, K. X.; Liu, X. H.; Ling, Y.; Chen, J. Y.; Wang,
G. B. Cancer Lett. 2011, 307, 6.
1=kobs ¼ Ki=kꢂ ð1 þ ½Sꢃ=KMÞꢂ1=½lꢃ þ 1=kinact
inact
k
inact=Ki ¼ ð1 þ ½Sꢃ=KMÞ=slope
17. Nomura, D. K.; Morrison, B. E.; Blankman, J. L.; Long, J. Z.; Kinsey, S. G.;
Marcondes, M. C.; Ward, A. M.; Hahn, Y. K.; Lichtman, A. H.; Conti, B.; Cravatt, B.
F. Science 2011, 334, 809.
18. Alhouayek, M.; Lambert, D. M.; Delzenne, N. M.; Cani, P. D.; Muccioli, G. G.
FASEB J. 2011, 25, 2711.
5.2.4. FAAH anandamidase activity assay
FAAH activity was measured by following [3H]-anandamide
hydrolysis ([3H]-AEA), as previously described.43 Briefly, 4
g of a
l
19. Makriyannis, A.; Pandarinathan, L.; Zvonok, N.; Parkarri, T.; Chapman, L. Patent
PCT Int. Appl. 2009117444, 2009.
20. Chang, J. W.; Niphakis, W. M.; Lum, K. M.; Cognetta, A. B., III; Wang, C.;
Matthews, M. L.; Niessen, S.; Buczynski, M. W.; Parsons, L. H.; Cravatt, B. F.
Chem. Biol. 2012, 19, 579.
21. Labar, G.; Bauvois, C.; Borel, F.; Ferrer, J. L.; Wouters, J.; Lambert, D. M.
Chembiochem 2010, 11, 218.
22. Ortar, G.; Cascio, M. G.; Moriello, A. S.; Camalli, M.; Morera, E.; Nalli, M.; Di
Marzo, V. Eur. J. Med. Chem. 2008, 43, 62.
preparation of human FAAH fused to the maltose-binding protein
was preincubated during 30 min at 22 °C in the presence of the
inhibitor at various concentrations (Tris buffer 10 mM, EDTA
1 mM, pH 8.0, inhibitor dissolved in 10
(AEA, 2
M final concentration; [3H]-AEA 50,000 dpm, American
radiolabeled Chemicals; 200 l total volume assay) was then
added and the tubes were placed at 37 °C for 10 min. The reaction
was then stopped by adding 400 l of a cold methanol/chloroform
ll DMSO). The substrate
l
l
23. Ortar, G.; Bisogno, T.; Ligresti, A.; Morera, E.; Nalli, M.; Di Marzo, V. J. Med.
Chem. 2008, 51, 6970.
l
(1:1) mixture and, after 5 min centrifugation at 700 g, the radioac-
tivity was measured in the upper aqueous phase by liquid scintil-
lation. The dpm value obtained for a blank (containing no enzyme)
was systematically subtracted. Results were then expressed as per-
cent of control activity (without inhibitor), after which Graph Pad
prism software was used to treat the data and to analyze the dose-
response curves. Inhibitors potency was expressed as IC50 values.
JZL184 was purchased from Cayman Chemical.
24. Hoornaert, C. Patent PCT Int. Appl. 2008145842, 2008.
25. Kapanda, C. N.; Muccioli, G. G.; Labar, G.; Poupaert, J. H.; Lambert, D. M. J. Med.
Chem. 2009, 52, 7310.
26. Matuszak, N.; Muccioli, G. G.; Labar, G.; Lambert, D. M. J. Med. Chem. 2009, 52,
7410.
27. King, A. R.; Lodola, A.; Carmi, C.; Fu, J.; Mor, M.; Piomelli, D. Br. J. Pharmacol.
2009, 157, 974.
28. King, A. R.; Dotsey, E. Y.; Lodola, A.; Jung, K. M.; Ghomian, A.; Qiu, Y.; Fu, J.; Mor,
M.; Piomelli, D. Chem. Biol. 2009, 16, 1045.
29. Long, J. Z.; Nomura, D. K.; Vann, R. E.; Walentiny, D. M.; Booker, L.; Jin, X.;
Burston, J. J.; Sim-Selley, L. J.; Lichtman, A. H.; Wiley, J. L.; Cravatt, B. F. Proc.
Natl. Acad. Sci. U.S.A. 2009, 106, 20270.
5.3. Molecular modeling
30. Fowler, C. J. Br. J. Pharmacol. 2012, 166, 1568.
31. Naidoo, V.; Karanian, D. A.; Vadivel, S. K.; Locklear, J. R.; Wood, J. T.; Nasr, M.;
Quizon, P. M. P.; Graves, E. E.; Shukla, V.; Makriyannis, A.; Bahr, B. A.
Neurotherapeutics. Published [online], 2012.
32. Savinainen, J. R.; Yoshino, M.; Minkkila, A.; Nevalainen, T.; Laitinen, J. T. Anal.
Biochem. 2010, 399, 132.
33. Holtfrerich, A.; Makharadze, T.; Lehr, M. Anal. Biochem. 2010, 399, 218.
34. Bertrand, T.; Auge, F.; Houtmann, J.; Rak, A.; Vallee, F.; Mikol, V.; Berne, P. F.;
Michot, N.; Cheuret, D.; Hoornaert, C.; Mathieu, M. J. Mol. Biol. 2010, 396, 663.
The binding mode of the inhibitors was investigated using
Hyperchem. The compounds were covalently linked, as the result
of a carbamoylation reaction, to the nucleophilic serine of rat
humanized FAAH37 (Ser241, pdb entry 3LJ6) and human MAGL34
(Ser122, pdb entry 3JWE), after removing the coordinates of the

L. Morera et al. / Bioorg. Med. Chem. 20 (2012) 6260–6275
6275
35. Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.; Cravatt, B. F. Science
2002, 298, 1793.
36. Ahn, K.; Johnson, D. S.; Mileni, M.; Beidler, D.; Long, J. Z.; McKinney, M. K.;
Weerapana, E.; Sadagopan, N.; Liimatta, M.; Smith, S. E.; Lazerwith, S.; Stiff, C.;
Kamtekar, S.; Bhattacharya, K.; Zhang, Y.; Swaney, S.; Van, B. K.; Stevens, R. C.;
Cravatt, B. F. Chem. Biol. 2009, 16, 411.
37. Mileni, M.; Johnson, D. S.; Wang, Z.; Everdeen, D. S.; Liimatta, M.; Pabst, B.;
Bhattacharya, K.; Nugent, R. A.; Kamtekar, S.; Cravatt, B. F.; Ahn, K.; Stevens, R.
C. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 12820.
38. Schalk-Hihi, C.; Schubert, C.; Alexander, R.; Bayoumy, S.; Clemente, J. C.;
Deckman, I.; DesJarlais, R. L.; Dzordzorme, K. C.; Flores, C. M.; Grasberger, B.;
Kranz, J. K.; Lewandowski, F.; Liu, L.; Ma, H.; Maguire, D.; Macielag, M. J.;
McDonnell, M. E.; Mezzasalma, H. T.; Miller, R.; Milligan, C.; Reynolds, C.; Kuo,
L. C. Protein Sci. 2011, 20, 670.
39. Lagu, B.; Tian, D.; Nagarathnam, D.; Marzabadi, M. R.; Wong, W. C.; Miao, S. W.;
Zhang, F.; Sun, W.; Chiu, G.; Fang, J.; Forray, C.; Chang, R. S.; Ransom, R. W.;
Chen, T. B.; O’Malley, S.; Zhang, K.; Vyas, K. P.; Gluchowski, C. J. Med. Chem.
1999, 42, 4794.
40. Ohta, T.; Komoriya, S.; Yoshino, T.; Uoto, K.; Nakamoto, Y.; Naito, H.;
Mochizuki, A.; Nagata, T.; Kanno, H.; Haginoya, N.; Yoshikawa, K.;
Nagamochi, M.; Kobayashi, S.; Ono, M. Patent EP1405852 (A1), 2004.
41. Dinh, T. P.; Carpenter, D.; Leslie, F. M.; Freund, T. F.; Katona, I.; Sensi, S. L.;
Kathuria, S.; Piomelli, D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10819.
42. Labar, G.; Bauvois, C.; Muccioli, G. G.; Wouters, J.; Lambert, D. M. Chembiochem
2007, 8, 1293.
43. Labar, G.; Vliet, F. V.; Wouters, J.; Lambert, D. M. Amino Acids 2008, 34, 127.