Characterization of tunable piperidine and piperazine carbamates as inhibitors of endocannabinoid hydrolases

Article abstract of DOI:10.1021/jm9016976

Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the serine hydrolase superfamily that degrade the endocannabinoids 2-arachidonoylglycerol and, anandamide, respectively. We have recently discovered that, MAGL and, FAAH are both inhibited by carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous functions of MAGL and FAAH. Herein, we disclose a full account of the syntheses, structure-activity relationships, and, inhibitory activities of piperidine/piperazine carbamates against members of the serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH inhibition by the attachment of an, appropriately substituted bisarylcarbinol or aryloxybenzyl moiety, respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated inhibitory activity, suggesting a strict requirement for a six-membered ring to maintain potency.

Full text of DOI:10.1021/jm9016976

1830 J. Med. Chem. 2010, 53, 1830–1842
DOI: 10.1021/jm9016976
Characterization of Tunable Piperidine and Piperazine Carbamates as Inhibitors of Endocannabinoid
Hydrolases
Jonathan Z. Long, Xin Jin, Alexander Adibekian, Weiwei Li, and Benjamin F. Cravatt*
The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 N. Torrey Pines Road,
La Jolla, California 92037
Received November 16, 2009
Monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) are two enzymes from the
serine hydrolase superfamily that degrade the endocannabinoids 2-arachidonoylglycerol and anand-
amide, respectively. We have recently discovered that MAGL and FAAH are both inhibited by
carbamates bearing an N-piperidine/piperazine group. Piperidine/piperazine carbamates show excellent
in vivo activity, raising brain endocannabinoid levels and producing CB1-dependent behavioral effects
in mice, suggesting that they represent a promising class of inhibitors for studying the endogenous
functions of MAGL and FAAH. Herein, we disclose a full account of the syntheses, structure-activity
relationships, and inhibitory activities of piperidine/piperazine carbamates against members of the
serine hydrolase family. These scaffolds can be tuned for MAGL-selective or dual MAGL-FAAH
inhibition by the attachment of an appropriately substituted bisarylcarbinol or aryloxybenzyl moiety,
respectively, on the piperidine/piperazine ring. Modifications to the piperidine/piperazine ring ablated
inhibitory activity, suggesting a strict requirement for a six-membered ring to maintain potency.
The endogenous cannabinoid system is composed of the
G-protein-coupled receptors CB1 and CB2, their endogenous
ligands anandamide (AEAa) and 2-arachidonoylglycerol
(2-AG) (the “endocannabinoids”), and the enzymes that
biosynthesize and degrade endocannabinoids.1 The magni-
tude and duration of brain endocannabinoid signaling are
tightly regulated by enzymatic hydrolysis, a process that
involves distinct enzymes for each endocannabinoid. Fatty
acid amide hydrolase (FAAH) is the principal hydrolytic
enzyme for AEA.2 Genetic or pharmacological disruption of
FAAH in rodents causes dramatic elevations (>10-fold) in
brain AEA levels and CB1-dependent analgesic responses in
several acute and chronic pain models.3-5 Although several
enzymes can hydrolyze 2-AG in vitro, this activity appears to
be principally mediated by monoacylglycerol lipase (MAGL)
in the rodent brain.6,7 Recently, we described a highly selective
and efficacious MAGL inhibitor 1 (JZL184, Figure 1), a
piperidine carbamate that, upon administration to mice,
reduces brain 2-AG hydrolysis activity, elevates total brain
2-AG levels by >8-fold, and causes CB1-dependent hypo-
motility and analgesia.7,8 We have also described 2 (JZL195,
Figure 1), a dual FAAH-MAGL inhibitor based on a piper-
azine carbamate scaffold, that elevates total brain AEA and
2-AG to levels comparable to selective inhibitors of each
individual enzyme.9 Interestingly, 2 produces strong antino-
ciceptive and cataleptic effects that provide evidence for
crosstalk between AEA and 2-AG signaling pathways in vivo.
Concurrently with these studies, highly selective inhibitors of
FAAH have been introduced that use a piperazine or pipe-
ridine urea to inactivate this enzyme. Examples include 3
(PF-622) and 4 (PF-3845) (Figure 1).5,10 Collectively, these
data indicate that piperazine/piperidine carbamates and ureas
may representprivileged chemical scaffolds11,12 for generating
covalent, selective, and efficacious inhibitors of endocanna-
binoid hydrolases. Herein, we provide a full account of the
discovery, synthesis, structure-activity relationships, and
inhibitory activities of compounds that led to the identifica-
tion of 1 and 2 and we show that by attachment of an
appropriately substituted 4-bisarylcarbinol or 4-aryloxyben-
zyl moiety, the versatile piperidine/piperazine carbamate
scaffold can be tuned to generate MAGL-selective or dual
FAAH-MAGL inhibitors.
Results and Discussion
Lead Endocannabinoid Hydrolase Inhibitors Discovered by
Competitive Activity-Based Protein Profiling (ABPP). Both
FAAH and MAGL are members of the serine hydrolase
superfamily of enzymes that use a conserved serine nucleo-
phile for catalysis.13-15 Our search for selective inhibitors of
MAGL or dual inhibitors for FAAH-MAGL has therefore
benefited from some unusual features of this family. First,
the catalytic serine is susceptible to covalent inactivation by
several electrophilic groups, including fluorophosphonates
and carbamates, that show little cross-reactivity with other
enzyme classes (Figure 2A).16-18 Carbamates are a privi-
leged scaffold in this respect because their selectivity among
members of the serine hydrolases can be tuned in two
ways, either by modulation of the carbonyl electrophilicity
*To whom correspondence should be addressed. Phone: 858-784-
8633. Fax: 858-784-8023. E-mail: cravatt@scripps.edu.
aAbbreviations: CB, cannabinoid receptor; AEA, anandamide;
2-AG, 2-arachidonoylglycerol; FAAH, fatty acid amide hydrolases;
MAGL, monoacylglycerol lipase; ABPP, activity-based protein profil-
ing; ABHD6, R/β hydrolases domain-containing 6; NTE, neuropathy
target esterase; TRP, transient receptor potential.
r
pubs.acs.org/jmc
Published on Web 01/25/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1831
Figure 1. Structures of the MAGL-selective inhibitor 1, the dual
FAAH-MAGL inhibitor 2, and the FAAH-selective inhibitors 3
and 4.
(reactivity) or by modification of substituents distal to the
reactive carbonyl (binding). A second attribute of serine
hydrolases is that their activity state can be concurrently
profiled in complex biological samples using the func-
tional proteomic method activity-based protein profiling
(ABPP).16,19,20 ABPP of serine hydrolases is typically per-
formed using fluorophore- or biotin-conjugated fluoropho-
sphonate probes [e.g., FP-rhodamine (FP-Rh) or FP-biotin]
that covalently label serine hydrolases, which are then re-
solved by SDS-PAGE and detected by in-gel fluorescence
scanning (for FP-Rh) or enriched by avidin chromatography
and identified by liquid chromatography-mass spectrome-
try (for FP-biotin).21,22 When performed in a competitive
mode, where inhibitors are preincubated with cell or tissue
proteomes prior to addition of FP probes, ABPP provides a
highly versatile screen for serine hydrolase inhibitors.7,23-26
Competitive ABPP has the important advantage of testing
inhibitors against numerous serine hydrolases in parallel
(i.e., all of the FP-reactive enzymes that are expressed in a
given cell or tissue) and, therefore, offers a powerful way to
concurrently optimize the potency and selectivity of inhibi-
tors directly in native proteomes (Figure 2B).
In our initial screen of a diverse carbamate library against
the mouse brain membrane proteome, we identified two
compounds (5 and 6) that showed good potency and promis-
ing selectivity for MAGL over the other serine hydrolases
(Figure 2C,D and Table 1). At higher concentrations, both
compounds also inhibited FAAH and a second brain 2-AG
hydrolase, ABHD6.6 Compounds 5 and 6 were structurally
similar, each containing a six-membered ring in the form of
a piperidine or piperazine, an activated N-4-nitrophenoxy
carbamate, and a bisarylcarbinol moiety distal to the elec-
trophilic carbonyl. However, despite their 27- and 68-fold
greater activity for MAGL over FAAH in brain proteomes
(Table 1), compounds 5 and 6 were not sufficiently selective
for in vivo studies, since their administration to mice at doses
that reduced MAGL activity also significantly blocked
FAAH activity (data not shown). We therefore undertook
an effort to improve the selectivity of piperazine/piperidine
carbamates for inhibiting MAGL.
Figure 2. Competitive activity-based protein profiling (ABPP) for
discovering lead carbamates that selectively inhibit MAGL.
(A) Carbamates irreversibly inactivate serine hydrolases (shown
as gray shapes) by carbamoylation of the nucleophilic serine. (B)
Schematic depiction of competitive ABPP. Native tissue proteomes
containing various serine hydrolases (gray shapes) are first incu-
bated with a carbamate inhibitor (box) and then with an ABPP
probe such as fluorphosphonate conjugated rhodamine (FP-Rh,
circles). Separation and visualization of FP-Rh-labeled proteins
by SDS-PAGE and in-gel fluorescence scanning, respectively,
reveal the disappearance of bands that represent the serine
hydrolase targets of the carbamate inhibitor (fluorescent gel
shown in grayscale). (C) Structures of the lead compounds 5 and 6.
(D) Competitive ABPP showing the concentration-dependent
effects of 5 and 6 on serine hydrolases activities in the mouse brain
membrane proteome. 5 and 6 were incubated with proteomes for
30 min at 37 °C before addition of FP-Rh. Note that MAGL
migrates as several bands between 33 and 35 kDa, as reported
previously.45
of the commercially available secondary amines 7a and 7b
using 4-nitrophenylchloroformate (8) afforded 9a and 9b.
Addition of 4-methoxyphenylmagnesium bromide (11) to
methyl 1-benzyl-5-oxopyrrolidine-3-carboxylate (10) in re-
fluxing THF gave the amide 12, which was reduced to 13
using LiAlH4. Benzyl deprotection using standard Pd/C
hydrogenation conditions to afford secondary amine 14
and finally carbamoylation afforded pyrrolidine carbamate
15. When 9a, 9b, and 15 were assessed by competitive ABPP
against the mouse brain membrane proteome, they were
found to be inactive against FAAH, MAGL, and other
serine hydrolases (Table 2). These data therefore suggested
that the six-membered ring in compounds 5 and 6 was
required for MAGL inhibition.
Structure-Activity Relationships of MAGL-Selective Inhi-
bitors. We first asked whether the six-membered piperazine/
piperidine ring was required for activity. Carbamates 9a and
9b, which maintained the N,N-dialkyl 4-nitrophenoxy carba-
mate moiety but had unbranched alkyl linkers to a bisphenyl
substituted terminal carbon, and carbamate 15, in which
the piperidine was contracted into a pyrrolidine ring, were
synthesized as outlined in Schemes 1 and 2. Carbamoylation

1832 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Table 1. In Vitro Activity of Lead Compoundsa
Long et al.
Scheme 3. Synthesis of Carbamates 17, 19, and 20
compd
FAAH
MAGL
ABHD6
selectivity
5
6
5410
2660
200
40
900
2110
27
68
a IC50 values are shown in nM unless otherwise indicated.
Scheme 1. Synthesis of Carbamates 9a and 9b
Scheme 4. Synthesis of Carbamate 23
Scheme 2. Synthesis of Carbamate 15
Scheme 5. Synthesis of Carbamate 29
Table 2. In Vitro Activity of Carbamates Lacking a Six-Membered
Ring
compd
FAAH
MAGL
ABHD6
carbamoylation/deprotection sequence from Boc-pipera-
zine, was used to alkylate 26 and afford 29.
9a
9b
15
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
>50 μM
17, 19, 20, 23, and 29 were evaluated against brain
membrane proteomes using competitive ABPP. Comparison
of 17 and 6 revealed that the 4-methoxy group on the distal
aryl rings conferred both additional potency and selectivity
for MAGL (Table 3). However, rigidification of any portion
of this distal substitutent, either by installation of an olefin
(19) or epoxide (20) linker between the phenyl groups and the
piperidine ring or by covalently tethering the aryl rings with
an oxygen bridge (29), reduced the activity and/or selecti-
vity for MAGL in comparison to lead carbamates 5 and 6
(IC50 = 200 nM to 6 μM for 19, 20, and 29, versus IC50 = 200
and 40 nM for 5 and 6, respectively). 23, in which the
methanol linker of compound 17 was replaced with a
methane, exhibited excellent selectivity for MAGL over
FAAH (>500-fold), despite being of slightly lower potency
compared to lead compound 6 (IC50 = 70 nM versus 40 nM).
Compound 23 provided the first evidence that it would be
possible to achieve selectivity for MAGL over FAAH on the
order of 300- to 1000-fold with the piperidine carbamate
scaffold. We therefore continued to search for compounds
that displayed this high degree of selectivity while also being
more potent for MAGL than the original compound 6.
Our next series of carbamates was motivated by the
improved potency of 6 compared to 17, which suggested
that further gains in MAGL active site binding interactions
could be achieved by additional substitution and/or increas-
ing the steric bulk of the arene rings. We reasoned that such
modifications might have the further benefit of increasing
We next focused on making structural modifications to the
portion of the molecules distal to the piperazine or piperidine
ring. The synthesis of these compounds is outlined in
Schemes 3-5. Carbamoylation under the usual conditions
of commercially available diphenyl(piperidin-4-yl)methanol
(16) afforded 17 (Scheme 3). Alternatively, the dibenzylic
tertiary hydroxyl of 16 could be readily eliminated using
TFA to afford 18, which was then converted to the corre-
sponding carbamate 19 using chloroformate 8. Treatment
of olefin 19 with mCPBA afforded epoxide 20 (Scheme 3).
Compound 23, containing a fully saturated bisaryl to piperi-
dine linker, was synthesized starting from commercially
available 1-tert-butyl 4-ethyl piperidine-1,4-dicarboxylate
21 (Scheme 4). Addition of Grignard 11 under refluxing
conditions afforded Boc-protected piperidine 22, which was
then subjected to a three-step procedure involving sequential
treatment with TFA/CH2Cl2, which concomitantly removed
the Boc group and eliminated the hydroxyl, Pd/C under the
usual hydrogenation conditions, which saturated the incipi-
ent olefin, and chloroformate 8, to afford the saturated
piperidine carbamate 23. We also rigidified the bisaryl ring
system by installing an ortho-oxygen bridge. This compound
(29) was synthesized starting from xanthen-9-one 24, which
was reduced with NaBH4 and chlorinated with SOCl2 to
afford 26. Piperazine 28, which was made using a two-step

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1833
Table 3. In Vitro Activity of Carbamates Modified with Different
Linkersa
Table 4. In Vitro Activity of Carbamates Modified at the Distal Arene
Groupa
a IC50 values are shown in nM unless otherwise indicated.
Scheme 6. Synthesis of Carbamates 33a-f
a IC50 values are shown in nM unless otherwise indicated.
maintained a comparable MAGL-selectivity window to 23
but gained a 7-fold improvement in potency. An additional
advantage was that, unlike many other compounds in the
piperidine/piperazine carbamate series, 1 exhibited low ac-
tivity for ABHD6, another enzyme that has been shown to
hydrolyze 2-AG in vitro.6 Further efforts to refine the aryl
substitution patterns by expanding the steric bulk, by using
4-N,N-dimethylanilinebenzene (33d) or 2-naphthyl (33f)
groups, or by modifying the aryl group electronics, with
substituents such as 4-chlorobenzene (33e), did not produce
more potent or more selective compounds. Since both larger
(33c, 33d, and 33f) and smaller (17) substituents reduce the
potency of these piperidine carbamates for MAGL, we
attribute, in part, the high potency and selectively of 1 to
be due to its unique substitution pattern and steric proper-
ties, since the methylenedioxy group exploits two points of
substitution along the arene ring while maintaining a rela-
tively small van der Waals volume.
Tuning the Piperazine Carbamate Scaffold To Create Dual
FAAH-MAGL Inhibitors. Our competitive ABPP screens
revealed that many compounds of the piperidine/piperazine
carbamate class with activity for MAGL also tended to
inhibit a select number of additional serine hydrolases in
the mouse brain proteome, principally FAAH and ABHD6.
This suggested that, despite their low sequence homology,
MAGL, FAAH, and ABHD6 share active site similarity, a
hypothesis that is reinforced by the structural relatedness of
some FAAH-selective (3 and 4) and MAGL-selective (1)
inhibitors, which all shared an N-electrophile tethered to a
piperidine or piperazine ring motif. Since 3 and 4 derive their
selectivity for FAAH by containing a relatively unreactive
urea electrophile, and since 1 derives its selectivity for MAGL
from the bulky piperidine bisarylcarbinol scaffold, we rea-
soned that an appropriate combination of 1 and 3 could
potentially provide a dual FAAH-MAGL inhibitor.28,29
the selectivity window for MAGL versus FAAH, since we
hypothesized that these compounds’ selectivity was in part
due to the inability of the sterically encumbering bisaryl
groups to fit into the relatively narrow FAAH acyl-chain
binding pocket.27 Since 6 remained at this point the most
potent compound identified, we chose to maintain the pipe-
ridine carbinol scaffold while varying the aryl groups. Com-
pounds 33a-f and 1 were prepared according to Scheme 6.
Addition of variously substituted aryl groups to the ester of
1-benzyl 4-ethyl piperidine-1,4-dicarboxylate (30) was ac-
complished using aryllithium anions at -78 °C, which were
generated by treating the appropriate aryl bromide with
t-BuLi at the same temperature. The resultant Cbz-protected
4-bisarylcarbinolpiperidine compounds 32a-f were depro-
tected by hydrogenation using Pd/C (32a-d, 32f) or aqueous
KOH (32e) to give the crude amines, which were used directly
in the carbamoylation reaction with 8 to afford the carba-
mates 33a-f.
Evaluation of 33a and 33b by competitive ABPP showed
that a single methoxy substituent at the 3- or 2- position of
the distal aryl ring could be tolerated and that these com-
pounds could still maintain good selectivity for MAGL
(Table 4, ∼10-fold selective). Remarkably, when we incor-
porated two oxygen substituents, in the form of either the
3,4-dimethoxybenzene (33c) or 3,4-methylenedioxybenzene
(1), we achieved excellent selectivity for MAGL (about 40- to
400-fold selective) without inhibiting any of the other serine
hydrolases in mouse brain membranes as judged by our
competitive ABPP gels. Compound 1, which was ultimately
advanced to in vivo pharmacological and behavioral studies,

1834 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Long et al.
Table 5. In Vitro Activity of Initial Dual FAAH-MAGL Inhibitorsa
Figure 3. Concentration-dependent effects of 36a on serine hydro-
lases activities in the mouse brain membrane proteome as deter-
mined by competitive ABPP. 36a was incubated with proteomes at
the indicated concentrations for 30 min at 37 °C before addition of
FP-Rh probe.
Scheme 7. Synthesis of Carbamates 36a-q
In such a compound, the FAAH-selective urea electrophile
would be replaced with a carbamate to enable inactivation of
MAGL (4-nitrophenoxy carbamate), and the MAGL-selec-
tive bulky bisarylcarbinol motif would be substituted with a
distal group that interacted with both the FAAH and
MAGL active sites.
a IC50 values are shown in nM unless otherwise indicated.
Table 6. In Vitro Activity of Dual FAAH-MAGL Inhibitors That
Showed Selectivity away from NTEa
With these general concepts in mind, we targeted the
piperazine p-nitrophenoxy carbamate as a scaffold that
was both likely to yield a dual inhibitor and also amenable
to facile N-modification (36a-q). These compounds were
synthesized by one of two routes shown in Scheme 7. Boc-
piperazine was alkylated by displacement of substituted
benzyl bromides using K2CO3 in refluxing CH2Cl2, and the
resultant products were deprotected and carbamoylated in
the usual way to afford the products. Alternatively, alkyla-
tion could be accomplished by reductive amination of Boc-
piperazine with arylaldehydes using Na(OAc)3BH at room
temperature.
Our initial compound in this series, the 2-napthyl sub-
stituted carbamate 36a, showed good activity for FAAH and
MAGL but also inhibited another serine hydrolase, the
150 kDa neuropathy target esterase (NTE) (Figure 3).30,31
Since the inhibition of NTE is believed to be responsible for
the delayed onset mortality induced by organophosphate
reagents and also lower limb paralysis in humans,32-34 we
sought to reduce the potency of the piperazine carbamates
for this target and pursued derivatives of 36a (Table 5).
While many compounds of this series fully blocked labeling
of NTE at high concentrations and also inhibited NTE when
administered to mice (for example, compounds 36h at 20 mg/
kg, ip; data not shown), a small subset of three compounds
showed only partially blocked NTE activity at high concen-
a IC50 values are shown in nM unless otherwise indicated.
trations (100 μM, Table 6). Of these compounds, 2 was most
promising, since it showed the least inhibition toward NTE
while maintaining excellent activity for MAGL and FAAH.
Additional derivatives that also contained the N-3-aryloxy-
benzyl motif of 2, such as 36m-q, were also unable to fully
block NTE activity at high doses (Table 7), suggesting that
the poor inhibition of NTE exhibited by these compounds is

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1835
Table 7. In Vitro Activity of Dual FAAH-MAGL Inhibitors Modified
at the 3-Aryloxybenzyl Positiona
would likely have gone undetected without the broad and
unbiased screening output provided by competitive ABPP.
From a structure-activity perspective, our data suggest that
the MAGL-selective inhibitors derive much of their selectivity
from the piperidine ring as well as the bulky bisarylcarbinol
motif distal to the electrophilic carbonyl. We have also
demonstrated that by rationally changing the substitution at
the 4-position of the six-membered ring, from bisarylcarbinol
to 3-aryloxybenzyl, we can selectively tune the inhibitory
properties of the piperidine/piperazine carbamate scaffold
from being a MAGL-selective inhibitor to a dual inhibitor
of MAGL and FAAH.
Since their disclosure, 1 and 2 have proven useful for
selectively enhancing 2-AG signaling or concurrently aug-
menting both 2-AG and anandamide signaling, respectively,
in a variety of in vitro and in vivo systems.35-39 Nevertheless,
we anticipate that even within this general chemical class there
remains much potential for improvements in the potency
and selectivity of future MAGL-selective inhibitors or dual
FAAH-MAGL inhibitors. For instance, compound 36e,
while originally designed to be a dual FAAH-MAGL inhibi-
tor, in fact displayed remarkable selectivity for MAGL over
FAAH. We attribute this unique property of 36e to the rigid
and linear biphenyl group and speculate that this compound
might serve as a lead for the development of next generation
MAGL inhibitors with a scaffold distinct from the bisaryl-
carbinol of 1. Moreover, we elected to use a 4-nitrophenoxy
carbamate, since our initial structure-activity relationship
efforts indicated that MAGL inhibition requires an activated
leaving group. However, we hypothesize that, similar to the
urea series of FAAH inhibitors,5 hydroxypyridine leaving
groups and derivatives thereof may also be sufficiently acti-
vated for MAGL inhibition and that incorporation of addi-
tional substitutions on the pyridine ring might offer a new way
to tune the selectivity window of these inhibitors for MAGL
versus FAAH. Recently, two crystal structures of human
MAGL have been solved, one of which contained the piper-
azine inhibitor SAR629 bound to the catalytic serine of
MAGL.40,41 These crystal structures may help in the design
of future irreversible or reversible MAGL inhibitors. Lastly,
piperazine carbamates have been also reported to act as dual
FAAH inhibitors/transient receptor potential V1 (TRPV1)
antagonists.42 That TRP channels can also be activated
by endogenous FAAH substrates such as AEA43 or the
N-acyltaurines44 suggests a high degree of homology between
the binding pockets of these proteins and furthermore indicates
that the piperidine/piperazine scaffold could potentially be
exploited to generate polypharmacological tools that interact
with MAGL, FAAH, and members of the TRP channel family.
a IC50 values are shown in nM unless otherwise indicated.
due to the unique steric and/or electronic properties of the
aryloxybenzyl substitution. That 3,5-dichlorobenzene phenyl
ether 36q was unable to inhibit any of the hydrolases in our
screen indicated that there is an upper limit to the steric bulk
that can be appended to the distal arene ring. Thus, while
the piperidine bisarylcarbinol scaffold results in MAGL-
selective inhibition, a piperazine aryloxybenzyl scaffold can
be used as a scaffold for the selective dual blockade of FAAH
and MAGL.
Though 2 showed weak inhibition of NTE in vitro (∼50%
blockade at 100 μM), no inhibition of NTE was observed
following in vivo administration to mice at doses that
completely blocked FAAH and MAGL (20 mg/kg ip or
100 mg/kg orally), and chronic administration of 2 to mice
(20 mg/kg ip daily for 6 days) did not produce death or overt
signs of discomfort.9 That ABHD6 was also inhibited at
higher concentrations suggests that 2 has the potential to
function as a poly-pharmacological inhibitor for several
endocannabinoid hydrolases (FAAH and two of the major
brain 2-AG hydrolases, MAGL and ABHD6). The FAAH-
selective inhibitor 4, the MAGL-selective inhibitor 1, and the
dual FAAH-MAGL inhibitor 2 thus constitute a versatile
arsenal of efficacious and selective pharmacological probes
that can be used to interrogate the function of endocanna-
binoid-degrading enzymes in vivo.
Experimental Section
Chemistry. General Methods. All reagents were purchased
from Sigma-Aldrich, Acros, Fisher, Fluka, or Maybridge and
used without further purification, except where noted. Dry
solvents were obtained by passing commercially available pre-
dried, oxygen-free formulations through activated alumina
columns. All reactions were carried out under a nitrogen atmo-
sphere using oven-dried glassware unless otherwise noted. Flash
chromatography was performed using 230-400 mesh silica gel.
NMR spectra were recorded in CDCl3 on a Varian Inova-400 or
a Bruker DMX-600 spectrometer and were referenced to tri-
methylsilane (TMS) or the residual solvent peak. Chemical
shifts are reported in ppm relative to TMS, and J values are
reported in Hz. High resolution mass spectrometry (HRMS)
Conclusions
Herein, we have described the discovery, synthesis, struc-
ture-activity relationships, and inhibitory activities of a series
of piperidine/piperazine carbamates that function either as
selective inhibitors of MAGL or as dual inhibitors of FAAH
and MAGL invivo. Thedevelopment of compoundssuch as 1
and 2 underscores the utility of competitive ABPP to concur-
rently optimize inhibitor potency and selectivity directly in
native proteomes. Indeed, considering that NTE shares little
to no sequence homology with either FAAH or MAGL,31 this
common “off-target” of dual FAAH-MAGL inhibitors

1836 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Long et al.
experiments were performed at The Scripps Research Institute
Mass Spectrometry Core on an Agilent mass spectrometer using
electrospray ionization-time-of-flight (ESI-TOF). LC/MS ana-
lysis was performed on an Agilent 1100 LC/MS instrument,
using a ZORBAX SB-C18, 3.5 mm, 4.6 mm ꢀ 50 mm column, a
flow rate of 0.75 mL/min, detection at 220 and 254 nm, a
10-98% acetonitrile/water/0.1% formic acid gradient, and a
50-98% acetonitrile/water/0.1% formic acid gradient. The
purity of compounds (g95%) was confirmed by clean NMR
spectra and elution as a single peak by LC/MS.
mixture. After 4 h, TLC indicated complete consumption of
the starting material. The mixture was diluted with CH2Cl2,
filtered over a pad of Celite, and concentrated in vacuo. The
crude was taken up in CH2Cl2 (10 mL), and triethylamine (1 mL,
7 mmol) and 4-nitrochloroformate (80 mg, 0.4 mmol) were
sequentially added. After 2 h, TLC indicated complete con-
sumption of the starting material. The mixture was diluted with
CH2Cl2 and poured onto saturated aqueous Na2CO3. The
organic layer was washed thrice with 2 N aqueous NaOH, once
with brine, dried over Na2SO4, and concentrated in vacuo.
Purification of the crude oil by flash chromatography (6:1
Hex/EtOAc) gave the 15 (30 mg, 28% yield over two steps):
1H NMR (CDCl3, 400 MHz) δ 8.28-8.17 (m, 2H), 7.40-7.22
(m, 6H), 6.85 (dd, J = 12.5, 5.8 Hz, 4H), 3.78 (dd, J = 3.2, 1.3
Hz, 6H), 3.71-3.30 (m, 4H), 2.34 (d, J = 14.1 Hz, 1H),
2.01-1.89 (m, 2H). HRMS calculated for C26H26N2NaO7
[M þ Na]þ 501.1632, found 501.1681.
Compounds 1-6 have been described previously.5,7,9,10
4-Nitrophenyl 3,3-Diphenylpropyl(methyl)carbamate (9a). Gene-
ral Procedure A. To a stirring solution of N-methyl-3,3-diphenyl-
propan-1-amine (70 mg, 0.27 mmol) in CH2Cl2 (3 mL) was
sequentially added triethylamine (0.1 mL, 0.7 mmol) and 4-nitro-
chloroformate (143 mg, 0.71 mmol). After 2 h, TLC indicated
complete consumption of the starting material. The mixture was
diluted with CH2Cl2 and poured onto saturated aqueous Na2CO3.
The organic layer was washed thrice with 2 N NaOH, once with
brine, dried over Na2SO4, and concentrated in vacuo. Purification of
the crude oil by flash chromatography (1-2% MeOH in CH2Cl2)
gave 9a (52 mg, 49% yield): 1H NMR (CDCl3, 400 MHz) δ 8.09
(d, J = 9.2 Hz, 2H), 7.28-7.16 (m, 10H), 6.90 (d, J = 9.2 Hz, 2H),
3.78 (t, J = 7.8 Hz, 2H), 3.12 (t, J = 7.8 Hz, 2H), 2.81 (s, 3H), 2.31
(m, 2H). MS (ESIþ) m/z 391 [M þ H]þ. HRMS calculated for
C23H23N2O4 [M þ H]þ 391.1652, found 391.1650.
4-Nitrophenyl 4-(Hydroxydiphenylmethyl)piperidine-1-car-
boxylate (17). 17 was prepared according to general procedure
A, using 4-benzhydrylpiperidine (192 mg, 0.72 mmol), 4-nitro-
chloroformate (230 mg, 1.1 mmol), triethylamine (0.2 mL,
1.4 mmol), and CH2Cl2 (10 mL). Purification of the crude oil
by flash chromatography (2-3% MeOH in CH2Cl2) gave 17
(100 mg, 32% yield): 1H NMR (CDCl3, 400 MHz) δ 8.22 (d, J =
9.1 Hz, 2H), 7.47 (d, J = 8.0 Hz, 4H), 7.35-7.22 (m, 8H), 4.30
(bs, 2H), 3.02 (t, J = 12.5 Hz, 1H), 2.89 (t, J = 12.2 Hz, 1H),
2.64 (m, 1H), 1.64-1.60 (m, 2H), 1.47 (m, 2H). HRMS calcu-
lated for C25H24N2NaO5 [M þ Na]þ 455.1577, found 455.1586.
4-(Diphenylmethylene)piperidine (18). To a stirring solution of
4-benzhydrylpiperidine (1.77 g, 6.7 mmol) in CH2Cl2 (15 mL)
was added TFA (5 mL). After 7 h, TLC indicated complete
consumption of the starting material. The mixture was concen-
trated in vacuo and then diluted with CH2Cl2 and saturated
aqueous Na2CO3. The aqueous layer was extracted twice with
CH2Cl2, and the combined organic layers were dried over
Na2SO4 and concentrated in vacuo. The crude was passed
through a pad of silica (5% MeOH in CH2Cl2) to give 18 (1.6
4-Nitrophenyl Benzhydryl(methyl)carbamate (9b). 9b was pre-
pared according to general procedure A, using N-methyl-1,
1-diphenylmethanamine (280 mg, 1.42 mmol), 4-nitrochloro-
formate (313 mg, 1.56 mmol), triethylamine (1 mL, 7 mmol),
and CH2Cl2 (10 mL). Purification of the crude oil by flash
chromatography (12:1 Hex/EtOAc) gave 9b (201 mg, 39%
yield): 1H NMR (CDCl3, 400 MHz) δ 8.24 (d, J = 8.1 Hz,
2H), 7.42-7.25 (m, 12H), 6.73 (s, 1H), 2.93 (s, 3H). HRMS
calculated for C21H19N2O4 [M þ H]þ 363.1339, found 363.1364.
1-Benzyl-4-(hydroxybis(4-methoxyphenyl)methyl)pyrrolidin-
2-one (12). To a stirring solution of methyl 1-benzyl-5-oxopyr-
rolidine-3-carboxylate (494 mg, 2.12 mmol) in dry ether (10 mL)
was added 4-methoxyphenylmagesium bromide (0.5 M in THF,
10 mL, 5 mmol). The mixture was heated to reflux, and TLC
indicated completion consumption of the starting material after
12 h. The mixture was diluted with CH2Cl2 and poured onto
saturated aqueous Na2CO3. The organic layer was washed once
with brine, dried over Na2SO4, and concentrated in vacuo.
Purification of the crude oil by flash chromatography (1:1
1
g, 96% yield): H NMR (CDCl3, 400 MHz) δ 7.34-7.23 (m,
6H), 7.11-7.08 (m, 4H), 3.24 (bs, 4H), 2.64 (m, 4H). MS (ESIþ)
m/z 250 [M þ H]þ.
4-Nitrophenyl 4-(Diphenylmethylene)piperidine-1-carboxylate
(19). 19 was prepared according to general procedure A, using
18 (182 mg, 0.73 mmol), 4-nitrochloroformate (142 mg, 0.71
mmol), triethylamine (0.3 mL, 2.1 mmol), and CH2Cl2 (10 mL).
The crude product was passed through a pad of silica (CH2Cl2)
to afford 19 (281 mg, 93% yield): 1H NMR (CDCl3, 400 MHz) δ
8.23 (d, J = 9.3 Hz, 2H), 7.36-7.09 (m, 12H), 3.69 (m, 2H), 3.61
(m, 2H), 2.47 (m, 4H). HRMS calculated for C25H23N2O4 [M þ
H]þ 415.1652, found 415.1676.
1
Hex/EtOAc) gave 12 (190 mg, 21% yield): H NMR (CDCl3,
400 MHz) δ 7.31-7.19 (m, 7H), 7.15 (d, J = 7.4 Hz, 2H),
6.81-6.64 (m, 4H), 4.43 (d, J = 14.8 Hz, 1H), 4.11 (d, J = 15.3
Hz, 1H), 3.70 (s, 6H), 3.48- 3.33 (m, 1H), 3.23 (dd, J = 10.0, 6.3
Hz, 1H), 3.08 (t, J = 9.5 Hz, 1H), 2.45 (ddd, J = 27.2, 17.5, 8.5
Hz, 2H). MS (ESIþ) m/z 440 (M þ Na)þ.
4-Nitrophenyl 2,2-Diphenyl-1-oxa-6-azaspiro[2.5]octane-6-
carboxylate (20). To a stirring solution of 19 (23 mg, 0.056
mmol) in CH2Cl2 (5 mL) was added mCPBA (<72%, 49 mg,
<0.2 mmol). After 2 h, TLC indicated complete consumption of
the starting material, and a saturated aqueous solution of
Na2S2O3 (1 mL) was added to the reaction. After being stirred
for 30 min, the mixture was diluted with EtOAc and the organic
layer was washed twice with water, once with saturated aqueous
Na2CO3, once with brine, dried over Na2SO4, and concentrated
in vacuo. The crude was passed through a pad of silica to afford
20 (15 mg, 63% yield): 1H NMR (CDCl3, 400 MHz) δ 8.24 (d,
J = 9.2 Hz, 2H), 7.47 (d, J = 7.6 Hz, 2H), 7.39 -7.22 (m, 8H),
4.03 (m, 2H), 3.51 (t, J = 11 Hz, 1H), 3.39 (t, J = 11 Hz, 1H),
1.87 (m, 2H), 1.38 (d, J = 13.8 Hz, 1H). HRMS calculated for
C25H23N2O5 [M þ H]þ 431.1602, found 431.1626.
(1-Benzylpyrrolidin-3-yl)bis(4-methoxyphenyl)methanol (13).
To a -78 °C stirring solution of 12 (185 mg, 0.44 mmol) in
dry ether/CH2Cl2 (4:1 v/v, 25 mL total) was added LiAlH4 (4 M
in ether, 0.5 mL, 2 mmol). The dry ice bath was removed, and the
mixture was heated to reflux. After 2 h, TLC indicated complete
consumption of the starting material. The mixture was diluted
with CH2Cl2 and poured onto water. The organic layer was
washed once with brine, dried over Na2SO4, and concentrated in
vacuo. The crude oil was passed through a pad of silica to afford
13 (160 mg, 90% yield): 1H NMR (CDCl3, 400 MHz) δ
7.30-7.15 (m, 9H), 6.78-6.52 (m, 4H), 3.65 (d, J = 5.2 Hz,
6H), 3.52 (d, J = 12.8 Hz, 1H), 3.41 (d, J = 12.9 Hz, 1H),
3.16-3.08 (m, 1H), 2.95-2.87 (m, 1H), 2.77 (d, J = 9.5 Hz, 1H),
2.16 (dd, J = 9.5, 6.5 Hz, 1H), 2.03 (q, J = 8.9 Hz, 1H),
1.91-1.74 (m, 2H). MS (ESIþ) m/z 404 [M þ H]þ.
tert-Butyl 4-(Hydroxybis(4-methoxyphenyl)methyl)piperidine-
1-carboxylate (22). To a 0 °C stirring solution of 1-tert-butyl
4-ethyl piperidine-1,4-dicarboxylate (1.16 g, 4.5 mmol) in dry
THF (10 mL) was added 4-methoxyphenylmagnesium bromide
(26 mL, 13 mmol) dropwise. After the reagent addition was
complete, the ice bath was removed and the mixture was heated
4-Nitrophenyl 3-(Hydroxybis(4-methoxyphenyl)methyl)pyr-
rolidine-1-carboxylate (15). General Procedure B. To a stirring
solution of the 13 (90 mg, 0.22 mmol) in EtOH (5 mL) was added
10% Pd/C (20 mg), and H2 gas was bubbled through the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1837
to 60 °C. TLC indicated complete consumption of the starting
material after 9 h. The mixture was diluted with EtOAc and
poured onto water. The organic layer was washed once with
water and once with brine, dried over Na2SO4, and concentrated
in vacuo. Purification of the crude oil by flash chromatography
5.1 mmol) dropwise. After 30 min, a solution of 30 (300 mg, 1.03
mmol) in dry THF (3 mL) was added to the reaction. After the
mixture was stirred for 4 h at the same temperature, TLC
indicated complete consumption of the starting material. The
mixture was diluted with CH2Cl2 and poured onto water. The
organic layer was washed twice with water, once with brine,
dried over Na2SO4, and concentrated in vacuo. Purification of
the crude oil by flash chromatography (10:1 and then 4:1 Hex/
1
(12:1 and then 3:1 Hex/EtOAc) gave 22 (1.6 g, 82% yield): H
NMR (CDCl3, 400 MHz) δ 6.82 (d, J = 8.8 Hz, 2H), 7.34 (d, J =
8.8 Hz, 2H), 4.11 (s, 2H), 3.75 (s, 6H), 2.68 (t, J = 11.7 Hz, 2H),
2.44 (dd, J = 13.2, 10.5 Hz, 1H), 2.16 (s, 1H), 1.60-1.45 (m, 2H),
1.42 (s, 9H), 1.33-1.19 (m, 2H). MS (ESIþ) m/z 410 (M - H2O þ
H)þ.
1
EtOAc) gave 32a (290 mg, 61% yield): H NMR (CDCl3, 400
MHz) δ 7.37-7.26 (m, 5H), 7.20 (t, J = 8.0 Hz, 2H), 7.09-6.99
(m, 4H), 6.71 (ddd, J = 8.2, 2.5, 0.7 Hz, 2H), 5.06 (s, 2H), 4.20
(bs, 2H), 3.75 (s, 6H), 2.77 (bs, 2H), 2.61-2.43 (m, 1H), 2.31
(s, 1H), 1.51 (s, 1H), 1.34 (qd, J = 12.6, 4.4 Hz, 2H). MS (ESIþ)
m/z 444 (M - H2O þ H)þ.
4-Nitrophenyl 4-(Hydroxybis(4-methoxyphenyl)methyl)pipe-
ridine-1-carboxylate (23). To a stirring solution of 22 (90 mg,
0.21 mmol) in CH2Cl2 (2 mL) was added TFA (2 mL). The
mixture turned red, and vigorous bubbling ensued. After 1 h,
TLC indicated complete consumption of the starting material.
Saturated aqueous Na2CO3 was added until the reaction was
completely neutralized, and the aqueous layer was extracted
thrice with CH2Cl2. The combined organic layers were washed
once with brine, dried over Na2SO4, and concentrated in vacuo.
The crude was taken up in EtOH (3 mL), and 10% Pd/C
(100 mg) was added. After 12 h, the mixture was diluted with
CH2Cl2, filtered over a pad of Celite, and concentrated in vacuo.
The crude was taken up in CH2Cl2 (5 mL), and triethylamine
(0.15 mL, 1.1 mmol) and 4-nitrochloroformate (66 mg, 0.33
mmol) were sequentially added. After 2 h, TLC indicated
complete consumption of the starting material. The mixture
was diluted with CH2Cl2 and poured onto saturated aqueous
Na2CO3. The organic layer was washed once with brine, dried
over Na2SO4, and concentrated in vacuo. Purification of the
crude oil by flash chromatography (12:1 and then 3:1 Hex/
EtOAc) gave 23 (16 mg, 15% yield over three steps): 1H NMR
(CDCl3, 400 MHz) δ 7.30 (d, J = 9.2 Hz, 2H), 8.25 (d, J = 9.2
Hz, 2H), 7.09-6.99 (m, 4H), 6.90-6.78 (m, 4H), 3.80 (s, 6H),
3.68 (m, 2H), 3.61 (m, 2H), 2.52-2.43 (m, 4H). HRMS calcu-
lated for C27H29N2O6 [M þ H]þ 477.2020, found 477.2054.
4-Nitrophenyl 4-(9H-Xanthen-9-yl)piperazine-1-carboxylate
(29). To a stirring solution of N-Boc piperazine (100 mg, 0.54
mmol) in CH2Cl2 (5 mL) was sequentially added 4-nitrophenyl-
chloroformate (109 mg, 0.54 mmol) and Et3N (75 µL, 0.54 mmol)
at room temperature. After 3 h, the mixture was concentrated in
vacuo. Purification of the crude mixture via flash chromatography
(3:1 Hex/EtOAc) gave 27 (160 mg, 83% yield): 1H NMR (CDCl3,
400 MHz) δ 8.24 (d, J = 9 Hz, 2H), 7.31 (d, J = 9 Hz, 2H), 3.67
(bs, 2H), 3.54 (bs, 6H), 1.50 (s, 9H). To the intermediate (100 mg,
0.28 mmol) was added 1:1 v/v TFA/CH2Cl2 (2 mL) at room
temperature. After 2 h, the mixture was concentrated in vacuo to
afford crude 28, which was used without further purification. To a
stirring solution of 9H-xanthene-9-one (98 mg, 0.5 mmol) in EtOH
(5 mL) was added NaBH4 (38 mg, 1 mmol) at room temperature.
After 4 h, the mixture was poured onto water (10 ml), stirred for
1 h, and then the product was filtered off, dried, and used directly
in the next step. To the crude 25 in CH2Cl2 (5 mL) was added
sulfurous dichloride (52 µL, 0.6 mmol) and Et3N (84 µL, 0.6 mmol)
dropwise at room temperature. After 2 h, the mixture was con-
centrated in vacuo to afford crude 26. Crude 26 was redissolved in
CH3CN (10 mL), and to it was sequentially added crude 28 and
Et3N (70 µL, 0.5 mmol). After the reagent addition was complete,
the mixture was heated to reflux overnight. The next morning,
the mixture was concentrated in vacuo. Purification of the crude
mixture via flash chromatography (1:1 Hex/EtOAc) gave 29 (201 mg,
84% yield over three steps): 1H NMR (CDCl3, 600 MHz) δ 8.20
(d, J = 9 Hz, 2H), 7.35 (m, 4H), 7.26 (m, 4H), 6.84 (d, J = 9 Hz,
2H), 4.90 (s, 1H), 3.57 (d, J = 27 Hz, 4H), 2.44 (d, J = 27 Hz, 4H).
HRMS calculated for C24H21N3O5 [M þ H]þ 432.1554, found
432.1559.
Benzyl 4-(Hydroxybis(2-methoxyphenyl)methyl)piperidine-1-
carboxylate (32b). 32b was prepared according to general
procedure C, using 1-bromo-2-methoxybenzene (980 mg, 5.2
mmol), 1-benzyl 4-ethyl piperidine-1,4-dicarboxylate (280 mg,
0.96 mmol), t-BuLi (1.7 M in pentane, 3 mL, 5.1 mmol), and dry
THF (20 mL). Purification of the crude oil by flash chromato-
graphy (10:1 and then 4:1 Hex/EtOAc) gave 32b (210 mg, 49%
yield): 1H NMR (CDCl3, 400 MHz) δ 7.66 (d, J = 7.1 Hz, 2H),
7.41-7.26 (m, 5H), 7.20 (ddd, J = 8.1, 7.4, 1.7 Hz, 2H), 7.02 (td,
J = 7.7, 1.1 Hz, 2H), 6.78 (dd, J = 8.2, 1.0 Hz, 2H), 5.14 (s, 2H),
4.24 (bs, 2H), 3.51 (s, 6H), 3.03 (t, J = 10.8 Hz, 1H), 2.85 (bs,
2H), 1.74-1.40 (m, 4H). MS (ESIþ) m/z 444 (M - H2O þ H)þ.
Benzyl 4-(Bis(3,4-dimethoxyphenyl)(hydroxy)methyl)piperidine-
1-carboxylate (32c). 32c was prepared according to general proce-
dure C, using 4-bromo-1,2-dimethoxybenzene (485 mg, 2.2 mmol),
1-benzyl 4-ethyl piperidine-1,4-dicarboxylate (215 mg, 0.74 mmol),
t-BuLi (1.7 M in pentane, 1.2 mL, 2.0 mmol), and dry THF
(10 mL). Purification of the crude oil by flash chromatography
(6:1 Hex/EtOAc) gave 32c (50 mg, 13% yield): 1H NMR (CDCl3,
400 MHz) δ 7.40-7.28 (m, 5H), 7.00 (s, 2H), 6.94 (d, J = 8.4 Hz,
2H), 6.80 (d, J = 8.4 Hz, 2H), 5.10 (s, 2H), 4.23 (bs, 2H), 3.85
(s, 6H), 3.84 (s, 6H), 2.78 (bs, 2H), 2.45 (t, J = 11.9 Hz, 1H),
1.70-1.47 (m, 2H), 1.38-1.15 (m, 2H). MS (ESIþ) m/z 444
(M þ Na)þ.
Benzyl
4-(Bis(4-(dimethylamino)phenyl)(hydroxy)methyl)-
piperidine-1-carboxylate (32d). 32d was prepared according
to general procedure C, using 4-bromo-N,N-dimethylaniline
(890 mg, 4.5 mmol), 1-benzyl 4-ethyl piperidine-1,4-dicarboxy-
late (220 mg, 0.75 mmol), t-BuLi (1.7 M in pentane, 2.5 mL, 4.3
mmol), and dry THF (20 mL). Purification of the crude oil by
flash chromatography (4:1 Hex/EtOAc) gave 32d (210 mg, 57%
yield): 1H NMR (CDCl3, 400 MHz) δ 7.39-7.19 (m, 9H), 6.65
(d, J = 8.8 Hz, 4H), 5.08 (s, 2H), 4.20 (bs, 2H), 2.90 (s, 12H),
2.81-2.71 (m, 2H), 2.45 (dd, J = 13.3, 10.4 Hz, 1H), 1.63 (bs,
2H), 1.34-1.18 (m, 2H). MS (ESIþ) m/z 470 (M - H2O þ H)þ.
Benzyl 4-(Bis(4-chlorophenyl)(hydroxy)methyl)piperidine-1-
carboxylate (32e). 32e was prepared according to general pro-
cedure C, using 1-bromo-4-chlorobenzene (920 mg, 4.8 mmol),
1-benzyl 4-ethyl piperidine-1,4-dicarboxylate (220 mg, 0.75
mmol), t-BuLi (1.7 M in pentane, 2.7 mL, 4.6 mmol), and dry
ether (15 mL). Purification of the crude oil by flash chromato-
graphy (6:1 Hex/EtOAc) gave 32e (220 mg, 63% yield): 1H
NMR (CDCl3, 400 MHz) δ 7.46-7.18 (m, 13H), 5.01 (d, J = 9.2
Hz, 2H), 4.19 (bs, 2H), 2.80-2.64 (m, 2H), 2.55-2.38 (m, 1H),
1.54-1.20 (m, 4H). MS (ESIþ) m/z 452 (M - H2O þ H)þ.
Benzyl 4-(Hydroxydinaphthalen-2-ylmethyl)piperidine-1-car-
boxylate (32f). 32f was prepared according to general procedure
C, using 2-bromonaphthalene (300 mg, 1.45 mmol), 1-benzyl
4-ethyl piperidine-1,4-dicarboxylate (106 mg, 0.36 mmol),
t-BuLi (1.7 M in pentane, 0.6 mL, 1.1 mmol), and dry THF
(6 mL). Purification of the crude oil by flash chromatography
(4:1 Hex/EtOAc) gave 32f (79 mg, 44% yield): 1H NMR
(CDCl3, 400 MHz) δ 8.04 (m, 2H), 7.74-7.84 (m, 6H),
7.56-7.43 (m, 6H), 7.31-7.25 (m, 5H), 5.08 (s, 2H), 4.24 (bs,
2H), 2.98-2.81 (m, 3H), 1.61-1.39 (m, 2H), 1.34-1.20 (m, 2H).
MS (ESIþ) m/z 524 (M þ Na)þ.
Benzyl 4-(Hydroxybis(3-methoxyphenyl)methyl)piperidine-1-
carboxylate (32a). General Procedure C. To a -78 °C stirring
solution of 1-bromo-3-methoxybenzene (980 mg, 5.2 mmol) in
dry THF (10 mL) was added t-BuLi (1.7 M in pentane, 3 mL,

1838 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Long et al.
4-Nitrophenyl 4-(Hydroxybis(3-methoxyphenyl)methyl)pipe-
ridine-1-carboxylate (33a). 33a was prepared according to gen-
eral procedure B, using 32a (290 mg, 0.63 mmol), 10% Pd/C
(70 mg), EtOH (4 mL) in the first step and CH2Cl2 (10 mL),
triethylamine (1 mL, 7 mmol), and 4-nitrochloroformate (150
mg, 0.75 mmol) in the second step. Purification of the crude oil by
flash chromatography (5:1 and then 2:1 Hex/EtOAc) gave the
product (220 mg, 73% yield over two steps): 1H NMR (CDCl3,
400 MHz) δ 8.22 (d, J = 9.1 Hz, 2H), 7.33-7.18 (m, 4H),
7.11-7.00 (m, 4H), 6.75 (dd, J = 8.2, 2.4 Hz, 2H), 4.29 (m,
2H), 3.79 (s, 6H), 3.01 (t, J = 12.0 Hz, 1H), 2.88 (t, J = 12.2 Hz,
1H), 2.59 (tt, J = 11.9, 3.3 Hz, 1H), 2.22-2.12 (m, 1H),
1.69-1.55 (m, 2H), 1.53-1.40 (m, 2H). HRMS calculated for
C27H28N2NaO7 [M þ Na]þ 515.1789, found 515.1829.
material and the mixture was diluted with CH2Cl2 and poured
onto water. The organic layer was washed thrice with 2 N
NaOH, once with brine, dried over Na2SO4, and concentrated
in vacuo. Purification of the crude oil by flash chromatography
(2:1 Hex/EtOAc) gave 33e (50 mg, 54% yield over two steps): 1H
NMR (CDCl3, 400 MHz) δ 8.23 (d, J = 8.2 Hz, 2H), 7.41-7.23
(m, 10H), 4.31 (bs, 2H), 3.02 (m, 1H), 2.88 (m, 1H), 2.57 (t, J =
2.9 Hz, 1H), 1.66-1.38 (m, 4H). HRMS calculated for
C25H22Cl2N2NaO5 [M þ Na]þ 523.0798, found 523.0823.
4-Nitrophenyl 4-(Hydroxydinaphthalen-2-ylmethyl)piperi-
dine-1-carboxylate (33f). 33f was prepared according to general
procedure B, using 32f (50 mg, 0.1 mmol), 10% Pd/C (50 mg),
EtOH/CH2Cl2 (4:1 v/v, 10 mL total) in the first step and CH2Cl2
(10 mL), triethylamine (1 mL, 7 mmol), and 4-nitrochlorofor-
mate (61 mg, 0.3 mmol) in the second step. Purification of the
crude oil by flash chromatography (3:1 Hex/EtOAc) gave 33f
(10 mg, 19% yield over two steps): 1H NMR (CDCl3, 400 MHz)
δ 8.22 (d, J = 9.2 Hz, 2H), 8.06 (s, 2H), 7.85 (d, J = 7.4 Hz, 2H),
7.79 (d, J = 7.6 Hz, 4H), 7.57 (d, J = 8.7 Hz, 2H), 7.53-7.42 (m,
4H), 7.26 (dd, J = 4.9, 4.3 Hz, 2H), 4.32 (d, J = 13.5 Hz, 2H),
3.09 (t, J = 12.9 Hz, 1H), 3.03-2.85 (m, 2H), 1.75 (dd, J = 26.3,
13.0 Hz, 2H), 1.65-1.47 (m, 2H). HRMS calculated for
C33H28N2NaO5 [M þ Na]þ 555.1890, found 555.1916.
4-Nitrophenyl 4-(Hydroxybis(2-methoxyphenyl)methyl)pipe-
ridine-1-carboxylate (33b). 33b was prepared according to gen-
eral procedure B, using 32b (210 mg, 0.47 mmol), 10% Pd/C
(50 mg), EtOH (5 mL) in the first step and CH2Cl2 (10 mL) and
triethylamine (1 mL, 7 mmol) and 4-nitrochloroformate (170
mg, 0.85 mmol) in the second step. Purification of the crude oil
by flash chromatography (8:1 and then 2:1 Hex/EtOAc) gave
the product (150 mg, 66% yield over two steps): 1H NMR
(CDCl3, 400 MHz) δ 8.24 (d, J = 9.3 Hz, 2H), 7.65 (d, J = 6.0
Hz, 2H), 7.29 (d, J = 9.3 Hz, 2H), 7.21 (ddd, J = 8.1, 7.4, 1.7 Hz,
2H), 7.08- 6.95 (m, 2H), 6.80 (dd, J = 8.2, 0.9 Hz, 2H), 4.28 (t,
J = 10.8 Hz, 2H), 3.54 (s, 6H), 3.13- 2.98 (m, 2H), 2.98- 2.85
(m, 1H), 1.82-1.52 (m, 4H). HRMS calculated for C27H28N2-
NaO7 [M þ Na]þ 515.1789, found 515.1837.
tert-Butyl 4-(Naphthalen-2-ylmethyl)piperazine-1-carboxy-
late (35a). General Procedure D. To a stirring solution of
N-Boc-piperazine (370 mg, 2.0 mmol) in CH2Cl2 (20 mL) was
sequentially added 2-(bromomethyl)naphthalene (350 mg, 1.6
mmol) and K2CO3 (1 g, 7.2 mmol). The mixture was heated to
reflux. After 10 h, TLC indicated complete consumption of the
starting material, so the mixture was diluted with EtOAc and
poured onto water. The organic layer was washed once with
brine, dried over Na2SO4, and concentrated in vacuo. Purifica-
tion of the crude oil by flash chromatography (5:1 to 3:1 Hex/
4-Nitrophenyl 4-(Bis(3,4-dimethoxyphenyl)(hydroxy)methyl)-
piperidine-1-carboxylate (33c). 33c was prepared according to
general procedure B, using 32c (50 mg, 0.1 mmol), 10% Pd/C (50
mg), EtOH/CH2Cl2 (4:1 v/v, 10 mL total) in the first step and
CH2Cl2 (10 mL), triethylamine (1 mL, 7 mmol), and 4-nitro-
chloroformate (51 mg, 0.25 mmol) in the second step. Purifica-
tion of the crude oil by flash chromatography (3:1 Hex/EtOAc)
gave 33c (20 mg, 38% yield over two steps): 1H NMR (CDCl3,
400 MHz) δ 8.24 (d, J = 9.2 Hz, 2H), 7.35-7.19 (m, 2H), 7.02 (d,
J = 1.8 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz,
2H), 4.31 (bs, 1H), 3.87 (s, 6H), 3.86 (s, 6H), 3.02 (t, J = 12.4 Hz,
1H), 2.89 (t, J = 13.0 Hz, 1H), 2.54 (t, J = 11.7 Hz, 1H),
1.79-1.60 (m, 2H), 1.46-1.41 (m, J = 11.9 Hz, 2H). HRMS
calculated for C29H32N2NaO9 [M þ Na]þ 575.2000, found
575.2015.
4-Nitrophenyl 4-(Bis(4-(dimethylamino)phenyl)(hydroxy)-
methyl)piperidine-1-carboxylate (33d). 33d was prepared accord-
ing to general procedure B, using 32d (40 mg, 0.08 mmol), 10%
Pd/C (50 mg), EtOH/CH2Cl2 (4:1 v/v, 5 mL total) in the first
step and CH2Cl2 (8 mL), triethylamine (1 mL, 7 mmol), and
4-nitrochloroformate (40 mg, 0.2 mmol) in the second step.
Purification of the crude oil by flash chromatography (6:1 Hex/
EtOAc) gave 33d (14 mg, 35% yield over two steps): 1H NMR
(CDCl3, 400 MHz) δ 8.22 (d, J = 9.2 Hz, 2H), 7.27 (m, 6H), 6.68
(d, J = 8.9 Hz, 4H), 4.27 (bs, 2H), 3.13-2.71 (m, 14H), 2.53 (t,
J = 11.9 Hz, 1H), 1.72 (d, J = 11.9 Hz, 2H), 1.39 (q, J = 12.2
Hz, 2H). HRMS calculated for C29H33N4O4 [M - H2O þ H]þ
501.2496, found 501.2543.
4-Nitrophenyl 4-(Bis(4-chlorophenyl)(hydroxy)methyl)pipe-
ridine-1-carboxylate (33e). To a stirring solution of 32e (90
mg, 0.19 mmol) in water/EtOH (1:3 v/v, 20 mL total) was added
KOH (1.0 g, 18 mmol), and the mixture was heated to reflux.
After the mixture was stirred overnight, TLC the next morning
indicated complete consumption of the starting material. The
mixture was diluted with CH2Cl2 and poured onto water. The
organic layer was partitioned, and the aqueous layer was
extracted thrice with CH2Cl2. The combined organic layers were
washed once with brine, dried over Na2SO4, and concentrated in
vacuo. The crude product was taken up in CH2Cl2 (10 mL), and
to the solution was sequentially added triethylamine (1 mL,
7 mmol) and 4-nitrochloroformate (90 mg, 0.45 mmol). After
1 h, TLC indicated complete consumption of the starting
1
EtOAc) gave 35a (420 mg, 76% yield): H NMR (CDCl3, 400
MHz) δ 7.82 (t, J = 7.6 Hz, 3H), 7.73 (s, 1H), 7.52-7.41
(m, 3H), 3.67 (s, 2H), 3.44 (bs, 4H), 2.43 (bs, 4H), 1.45 (s, 9H).
MS (ESIþ) m/z 327 [M þ H]þ.
tert-Butyl 4-Benzylpiperazine-1-carboxylate (35c). 35c was
prepared according to general procedure D, using N-Boc-pipe-
razine (116 mg, 0.62 mmol), CH2Cl2 (6 mL), benzyl bromide
(0.1 mL, 0.94 mmol), and K2CO3 (0.5 g, 3.6 mmol). Purifica-
tion of the crude oil by flash chromatography (5:1 Hex/EtOAc)
gave the 35c (120 mg, 69% yield): 1H NMR (CDCl3, 400 MHz) δ
7.35-7.20 (m, 5H), 3.49 (s, 2H), 3.46-3.37 (m, 4H), 2.43-2.29
(m, 4H), 1.45 (s, 9H). MS (ESIþ) m/z 277 [M þ H]þ.
tert-Butyl 4-(4-Methoxybenzyl)piperazine-1-carboxylate (35d).
35d was prepared according to general procedure D, using
N-Boc-piperazine (116 mg, 0.62 mmol), CH2Cl2 (6 mL), 1-(bro-
momethyl)-4-methoxybenzene (0.2 mL, 1.4 mmol), and K2CO3
(0.5 g, 3.6 mmol). Purification of the crude oil by flash chroma-
1
tography (5:1 Hex/EtOAc) gave 35d (134 mg, 71% yield): H
NMR (CDCl3, 400 MHz) δ 7.22 (d, J = 8.6 Hz, 2H), 6.85 (d, J =
8.6 Hz, 2H), 3.79 (s, 3H), 3.45-3.40 (m, 6H), 2.43-2.31 (m, 4H),
1.45 (s, 9H). MS (ESIþ) m/z 307 [M þ H]þ.
4-Nitrophenyl 4-(Naphthalen-2-ylmethyl)piperazine-1-carbo-
xylate (36a). General Procedure E. Compound 35a (420 mg,
1.1 mmol) was taken up in CH2Cl2/TFA (1:1 v/v, 6 mL total).
After the mixture was stirred for 2 h, TLC indicated complete
consumption of the starting material. The crude was concen-
trated in vacuo and used without purification. The intermediate
was then taken up in CH2Cl2 (5 mL) and to the solution was
added triethylamine (2 mL, 14 mmol) and 4-nitrochloroformate
(300 mg, 1.5 mmol). After 3 h, TLC indicated complete con-
sumption of the starting material. Purification of the crude oil by
flash chromatography (5:1 Hex/EtOAc) gave 36a (102 mg, 20%
yield over two steps): 1H NMR (CDCl3, 400 MHz) δ 8.27 (d, J =
9.3 Hz, 2H), 7.88-7.82 (m, 3H), 7.78 (s, 1H), 7.62-7.43 (m, 3H),
7.36-7.30 (m, 2H), 3.76 (s, 2H), 3.72 (bs, 2H), 3.64 (bs, 2H),
2.60 (bs, 2H). HRMS calculated for C22H22N3O4 [M þ H]þ
392.1605, found 392.1638.

Article
4-Nitrophenyl 4-(Quinolin-2-ylmethyl)piperazine-1-carboxy-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1839
TFA (10 mL) in the second step, and CH2Cl2 (5 mL), triethyl-
amine (1 mL, 7 mmol), and 4-nitrochloroformate (220 mg, 1.1
mmol) in the third step. Purification of the crude oil by flash
chromatography (5:1 Hex/EtOAc) gave 36f (90 mg, 20% yield
over three steps): 1H NMR (CDCl3, 400 MHz) δ 8.16 (d, J = 9.2
Hz, 2H), 8.08 (d, J = 9.1 Hz, 2H), 7.39-7.16 (m, 7H), 6.79 (d,
J = 9.2 Hz, 2H), 3.54 (bs, 2H), 3.44 (bs, 4H), 2.34 (bs, 4H).
HRMS calculated for C24H24N3O4 [M þ H]þ 418.1761, found
418.1800.
late (36b). General Procedure F. To a stirring solution of
N-Boc-piperazine (268 mg, 1.44 mmol) in dry MeOH (6 mL)
was sequentially added quinoline-2-carbaldehyde (175 mg, 1.1
mmol), AcOH (60 μL), and Na(OAc)3BH (475 mg, 2.2 mmol).
After 15 h, TLC indicated complete consumption of the starting
material. The mixture was diluted with EtOAc and poured onto
saturated aqueous NaHCO3. The organic layer was washed
once with brine, dried over Na2SO4, and concentrated in vacuo.
The crude oil was passed through a plug of silica, and the
intermediate was taken up in CH2Cl2/TFA (1:1 v/v, 10 mL
total). After the mixture was stirred for 2 h, TLC indicated
complete consumption of the starting material. The crude was
concentrated in vacuo and used without purification. The
intermediate was then taken up in CH2Cl2 (5 mL), and to the
solution was added triethylamine (1 mL, 7 mmol) and 4-nitro-
chloroformate (220 mg, 1.1 mmol). After 3 h, TLC indicated
complete consumption of the starting material. The mixture was
diluted with CH2Cl2 and poured onto saturated aqueous
Na2CO3. The organic layer was washed thrice with 2 N NaOH,
once with brine, dried over Na2SO4, and concentrated in vacuo.
Purification of the crude oil by flash chromatography (3:1 Hex/
EtOAc) gave 36b (87 mg, 20% yield over three steps): 1H NMR
(CDCl3, 400 MHz) δ 8.28-8.21 (m, 2H), 8.16 (d, J = 8.5 Hz,
1H), 8.09 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.72
(ddd, J = 8.4, 7.0, 1.3 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H),
7.57-7.51 (m, 1H), 7.33-7.27 (m, 2H), 3.91 (s, 2H), 3.72 (bs,
2H), 3.64 (bs, 2H), 2.76-2.52 (m, 4H). HRMS calculated for
C21H21N4O4 [M þ H]þ 393.1557, found 393.1590.
4-Nitrophenyl 4-(4-(Benzyloxy)-2-methoxybenzyl)piperazine-
1-carboxylate (36g). 36g was prepared according to general
procedure F, using N-Boc-piperazine (345 mg, 1.9 mmol), dry
MeOH (6 mL), 4-(benzyloxy)-3-methoxybenzaldehyde (364 mg,
1.5 mmol), AcOH (60 μL), and Na(OAc)3BH (575 mg, 2.7
mmol) in the first step, CH2Cl2/TFA (10 mL) in the second
step, and CH2Cl2 (5 mL), triethylamine (1 mL, 7 mmol), and
4-nitrochloroformate (300 mg, 1.5 mmol) in the third step.
Purification of the crude oil by flash chromatography (4:1
1
Hex/EtOAc) gave 36g (46 mg, 6% yield over three steps): H
NMR (CDCl3, 400 MHz) δ 8.24 (d, J = 9.1 Hz, 2H), 7.44 (d,
J = 7.3 Hz, 2H), 7.37 (dd, J = 11.2, 4.2 Hz, 2H), 7.33-7.23 (m,
3H), 6.91 (d, J = 1.4 Hz, 1H), 6.83 (d, J = 8.1 Hz, 1H), 6.77 (d,
J = 8.1 Hz, 1H), 5.15 (s, 2H), 3.90 (s, 3H), 3.67 (bs, 2H), 3.59 (bs,
2H), 3.48 (s, 2H), 2.49 (bs, 4H). HRMS calculated for
C26H28N3O6 [M þ H]þ 478.1973, found 478.2014.
4-Nitrophenyl 4-((5-Phenylthiophen-2-yl)methyl)piperazine-1-
carboxylate (36h). 36h was prepared according to general pro-
cedure F, using N-Boc-piperazine (373 mg, 2.0 mmol), dry THF
(20 mL), 5-phenylthiophene-2-carbaldehyde (372 mg, 2.0
mmol), AcOH (0.2 mL), and Na(OAc)3BH (796 mg, 3.8 mmol)
in the first step, CH2Cl2/TFA (10 mL) in the second step, and
CH2Cl2 (15 mL), triethylamine (2 mL, 14 mmol), and 4-nitro-
chloroformate (400 mg, 2.0 mmol) in the third step. Purification
of the crude oil by flash chromatography (6:1 Hex/EtOAc) gave
36h (250 mg, 30% yield over three steps): 1H NMR (CDCl3, 400
MHz) δ 8.23 (d, J = 9.1 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.36
(t, J = 7.6 Hz, 2H), 7.32-7.22 (m, 3H), 7.16 (d, J = 3.6 Hz, 1H),
6.89 (d, J = 3.5 Hz, 1H), 3.77 (s, 2H), 3.71 (bs, 2H), 3.62 (bs,
2H), 2.65-2.55 (m, 4H). HRMS calculated for C22H22N3O4S
[M þ H]þ 424.1325, found 424.1347.
4-Nitrophenyl 4-Benzylpiperazine-1-carboxylate (36c). 36c
was prepared according to general procedure E, using 35c (113
mg, 0.5 mmol) and CH2Cl2/TFA (6 mL total) in the first step
and CH2Cl2 (5 mL), triethylamine (2 mL, 14 mmol), and
4-nitrochloroformate (160 mg, 0.8 mmol) in the second step.
Purification of the crude oil by flash chromatography (4:1 Hex/
1
EtOAc) gave 36c (61 mg, 69% yield): H NMR (CDCl3, 400
MHz) δ 8.24 (d, J = 9.3 Hz, 2H), 7.36-7.32 (m, 4H), 7.29 (d,
J = 9.3 Hz, 3H), 3.73-3.65 (m, 2H), 3.62-3.56 (m, 4H),
2.57-2.47 (m, 4H). HRMS calculated for C18H20N3O4 [M þ
H]þ 342.1448, found 342.1470.
4-Nitrophenyl 4-((4-Phenylthiophen-2-yl)methyl)piperazine-1-
carboxylate (36i). 36i was prepared according to general proce-
dure F, using N-Boc-piperazine (343 mg, 1.9 mmol), dry MeOH
(10 mL), 4-phenylthiophene-2-carbaldehyde (260 mg, 1.4
mmol), AcOH (0.1 mL), and Na(OAc)3BH (738 mg, 3.48 mmol)
in the first step; CH2Cl2/TFA (10 mL) in the second step, and
CH2Cl2 (5 mL), triethylamine (1 mL, 7 mmol), and 4-nitro-
chloroformate (294 mg, 1.5 mmol) in the third step. Purifica-
tion of the crude oil by flash chromatography (3:1 Hex/EtOAc)
gave 36i (132 mg, 28% yield over three steps): 1H NMR
(CDCl3, 400 MHz) δ 8.28-8.21 (m, 2H), 7.70-7.52 (m, 2H),
7.47-7.34 (m, 3H), 7.33-7.19 (m, 4H), 3.81 (d, J = 0.5 Hz,
2H), 3.72 (bs, 2H), 3.63 (bs, 2H), 2.75-2.52 (m, 4H). HRMS
calculated for C22H22N3O4S [M þ H]þ 424.1325, found
424.1345.
4-Nitrophenyl 4-(4-Methoxybenzyl)piperazine-1-carboxylate
(36d). 36d was prepared according to general procedure E, using
35d (110 mg, 0.36 mmol) and CH2Cl2/TFA (6 mL) in the first
step and CH2Cl2 (5 mL), triethylamine (2 mL, 14 mmol), and
4-nitrochloroformate (160 mg, 0.8 mmol) in the second step.
Purification of the crude oil by flash chromatography (4:1 Hex/
1
EtOAc) gave 36d (43 mg, 32% yield): H NMR (CDCl3, 400
MHz) δ 8.24 (d, J = 9.3 Hz, 2H), 7.29 (d, J = 9.3 Hz, 2H), 7.24
(d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 3.81 (s, 3H),
3.70-3.64 (m, 2H), 3.62-3.55 (m, 2H), 3.51 (s, 2H), 2.58-2.41
(m, 4H). HRMS calculated for C19H22N3O5 [M þ H]þ 372.1554,
found 372.1581.
4-Nitrophenyl 4-(Biphenyl-4-ylmethyl)piperazine-1-carboxy-
late (36e). 36e was prepared according to general procedure F,
using N-Boc-piperazine (302 mg, 1.6 mmol), dry CH2Cl2
(10 mL), biphenyl-4-carbaldehyde (260 mg, 1.4 mmol), AcOH
(0.1 mL), and Na(OAc)3BH (593 mg, 2.8 mmol) in the first step,
CH2Cl2/TFA (10 mL) in the second step, and CH2Cl2 (5 mL),
triethylamine (1 mL, 7 mmol), and 4-nitrochloroformate (280
mg, 1.4 mmol) in the third step. Purification of the crude oil by
flash chromatography (5:1 Hex/EtOAc) gave 36e (300 mg, 51%
yield over three steps): 1H NMR (CDCl3, 400 MHz) δ 8.25 (d,
J = 9.2 Hz, 2H), 7.59 (dd, J = 13.0, 4.7 Hz, 4H), 7.51-7.25 (m,
7H), 3.70 (bs, 2H), 3.58 (bs, 4H), 2.65-2.36 (m, 4H). HRMS
calculated for C24H24N3O4 [M þ H]þ 418.1761, found 418.1802.
4-Nitrophenyl 4-(Biphenyl-2-ylmethyl)piperazine-1-carboxy-
late (36f). 36f was prepared according to general procedure F,
using N-Boc-piperazine (242 mg, 1.3 mmol), dry MeOH (6 mL),
biphenyl-2-carbaldehyde (208 mg, 1.1 mmol), AcOH (60 μL),
and Na(OAc)3BH (467 mg, 2.2 mmol) in the first step, CH2Cl2/
4-Nitrophenyl
4-((5-Phenylfuran-2-yl)methyl)piperazine-1-
carboxylate (36j). 36j was prepared according to general proce-
dure F, using N-Boc-piperazine (348 mg, 1.9 mmol), dry CH2Cl2
(10 mL), 5-phenylfuran-2-carbaldehyde (285 mg, 1.66 mmol),
AcOH (1 mL), and Na(OAc)3BH (807 mg, 3.8 mmol) in the first
step, CH2Cl2/TFA (10 mL) in the second step, and CH2Cl2
(5 mL), triethylamine (1 mL, 7 mmol), and 4-nitrochlorofor-
mate (372 mg, 1.85 mmol) in the third step. Purification of the
crude oil by flash chromatography (3:1 Hex/EtOAc) gave 36j
1
(196 mg, 29% yield over three steps): H NMR (CDCl3, 400
MHz) δ 8.33-8.11 (m, 2H), 7.67 (dd, J = 7.4, 0.9 Hz, 2H),
7.46-7.32 (m, 2H), 7.27 (ddd, J = 5.9, 3.5, 0.9 Hz, 3H), 6.61
(d, J = 3.2 Hz, 1H), 6.32 (d, J = 3.3 Hz, 1H), 3.70 (s, 4H), 3.63
(s, 2H), 2.61 (s, 4H). HRMS calculated for C22H21N3NaO5
[M þ Na]þ 430.1373, found 430.1383.

1840 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Long et al.
4-Nitrophenyl 4-(5-Bromo-2-hydroxybenzyl)piperazine-1-car-
boxylate (36k). 36k was prepared according to general proce-
dure F, using N-Boc-piperazine (367 mg, 2.0 mmol), dry THF
(20 mL), 5-bromo-2-hydroxybenzaldehyde (366 mg, 1.8 mmol),
AcOH (0.2 mL), and Na(OAc)3BH (799 mg, 3.8 mmol) in the
first step, CH2Cl2/TFA (6 mL) in the second step, and CH2Cl2
(5 mL), triethylamine (2 mL, 14 mmol), and 4-nitrochlorofor-
mate (400 mg, 2.0 mmol) in the third step. Purification of the
crude oil by flash chromatography (3:1 Hex/EtOAc) gave 36k
(m, 7H), 3.80 (s, 3H), 3.67 (m, 2H), 3.58 (m, 2H), 3.52 (s,
2H), 2.49 (m, 4H). C25H26N3O6 [M þ H]þ 464.1816, found
464.1882.
4-Nitrophenyl 4-(3-(3-(Trifluoromethyl)phenoxy)benzyl)pipe-
razine-1-carboxylate (36p). 36p was prepared according to gen-
eral procedure G, using N-Boc-piperazine (559 mg, 3.0 mmol),
dry THF (20 mL), 3-(3-trifluoromethylphenoxy)benzaldehyde
(532 mg, 2.00 mmol), AcOH (0.2 mL), and Na(OAc)3BH (848
mg, 4.0 mmol) in the first step, CH2Cl2/TFA (15 mL) in the
second step, and CH2Cl2 (6 mL), triethylamine (3 mL, 21 mmol),
and 4-nitrochloroformate (648 mg, 3.22 mmol) in the third step.
Purification of the crude oil by flash chromatography (2:1
Hex/EtOAc) gave 36p (321 mg, 32% yield over three steps):
1H NMR (CDCl3, 300 MHz) δ 8.26-8.21 (m, 2H), 7.50-6.90
(m, 10H), 3.68 (m, 2H), 3.59 (m, 2H), 3.56 (s, 2H), 2.51 (m, 4H).
C25H23F3N3O5 [M þ H]þ 502.1584, found 502.1639.
1
(140 mg, 16% yield over three steps): H NMR (CDCl3, 400
MHz) δ 8.27 (d, J = 9.3 Hz, 2H), 7.42-7.24 (m, 3H), 7.15 (d,
J = 2.4 Hz, 1H), 6.76 (d, J = 8.6 Hz, 1H), 3.82-3.51 (m, 6H),
2.67 (s, 4H). HRMS calculated for C18H19BrN3O5 [M þ H]þ
436.0503, found 436.0528.
4-Nitrophenyl
4-((5-(2-(Trifluoromethoxy)phenyl)furan-2-
yl)methyl)piperazine-1-carboxylate (36l). 36l was prepared
according to general procedure F, using N-Boc-piperazine
(427 mg, 2.3 mmol), dry MeOH (10 mL), 5-(2-(trifluoro-
methoxy)phenyl)furan-2-carbaldehyde (244 mg, 0.95 mmol),
AcOH (0.1 mL), and Na(OAc)3BH (410 mg, 1.9 mmol) in the
first step;, CH2Cl2/TFA (10 mL) in the second step, and CH2Cl2
(5 mL), triethylamine (1 mL, 7 mmol), and 4-nitrochlorofor-
mate (200 mg, 1.0 mmol) in the third step. Purification of the
crude oil by flash chromatography (4:1 Hex/EtOAc) gave 36l
4-Nitrophenyl 4-(3-(3,5-Dichlorophenoxy)benzyl)piperazine-
1-carboxylate (36q). 36q was prepared according to general
procedure G, using N-Boc-piperazine (373 mg, 2.0 mmol), dry
THF (20 mL), 3-(3,5-dichlorophenoxy)benzaldehyde (534 mg,
2.00 mmol), AcOH (0.2 mL), and Na(OAc)3BH (848 mg, 4.0
mmol) in the first step, CH2Cl2/TFA (15 mL) in the second step,
and CH2Cl2 (6 mL), triethylamine (3 mL, 21 mmol), and
4-nitrochloroformate (798 mg, 3.96 mmol) in the third step.
Purification of the crude oil by flash chromatography (3:1
Hex/EtOAc) gave 36q (422 mg, 42% yield over three steps):
1H NMR (CDCl3, 300 MHz) δ 8.27-8.22 (m, 2H), 7.38-6.93
(m, 7H), 6.86 (m, 2H), 3.70 (m, 2H), 3.61 (m, 2H), 3.54 (s, 2H),
2.51 (m, 4H). C24H22Cl2N3O5 [M þ H]þ 502.0931, found
502.1004.
1
(110 mg, 24% yield over three steps): H NMR (CDCl3, 400
MHz) δ 8.24 (d, J = 9.3 Hz, 2H), 7.91 (ddd, J = 7.6, 1.7, 0.6 Hz,
1H), 7.52-7.15 (m, 5H), 6.84 (d, J = 3.3 Hz, 1H), 6.38 (d, J =
3.4 Hz, 1H), 3.76 (bs, 4H), 3.64 (bs, 2H), 2.63 (bs, 4H). HRMS
calculated for C23H21F3N3O6 [M þ H]þ 492.1377, found
492.1417.
Preparations of Mouse Brain Membrane Proteomes. Tissues
were Dounce-homogenized in PBS, pH 7.5, followed by a low-
speed spin (1400g, 5 min) to remove debris. The supernatant was
then subjected to centrifugation (64000g, 45 min) to provide the
cytosolic fraction in the supernatant and the membrane fraction
as a pellet. The pellet was washed and resuspended in PBS buffer
by sonication. Total protein concentration in each fraction was
determined using a protein assay kit (Bio-Rad). Samples were
stored at -80 °C until use.
Competitive ABPP Experiments. Brain membrane proteomes
were diluted to 1 mg/mL in PBS in a 50 μL total reaction volume.
Inhibitors were added at the indicated concentration, and the
mixtures were incubated for 30 min at 37 °C. FP-rhodamine
was then added at a final concentration of 1 μM. After an
additional 30 min at 25 °C, the reactions were quenched with 4ꢀ
SDS-PAGE loading buffer, boiled for 5 min at 90 °C, subjected
to SDS-PAGE, and visualized in-gel using a flatbed fluore-
scence scanner (Hitachi). Concentration-dependent inhibition
curves were obtained from integrated gel band intensities
(ImageJ) and were fit using Prism software (GraphPad) to
obtain effector concentration for half-maximal response values
(IC50). The IC50 values shown in Tables 1-6 are the mean values
of at least triplicate measurements, and the standard error of the
mean (SEM) is <25% of the indicated value in all cases.
4-Nitrophenyl 4-(3-(p-Tolyloxy)benzyl)piperazine-1-carboxy-
late (36m). 36m was prepared according to general procedure F,
using N-Boc-piperazine (373 mg, 2.0 mmol), dry THF (20 mL),
3-(4-methylphenoxy)benzaldehyde (425 mg, 2.00 mmol), AcOH
(0.2 mL), and Na(OAc)3BH (848 mg, 4.0 mmol) in the first step,
CH2Cl2/TFA (15 mL) in the second step, and CH2Cl2 (6 mL),
triethylamine (3 mL, 21 mmol), and 4-nitrochloroformate (588
mg, 2.92 mmol) in the third step. Purification of the crude oil by
flash chromatography (2:1 Hex/EtOAc) gave 36m (456 mg, 51%
yield over three steps): 1H NMR (CDCl3, 300 MHz) δ 8.26-8.21
(m, 2H), 7.32-6.84 (m, 10H), 3.67 (m, 2H), 3.58 (m, 2H), 3.53
(s, 2H), 2.50 (m, 4H), 2.34 (s, 3H). C25H26N3O5 [M þ H]þ
448.1867, found 448.1911.
4-Nitrophenyl 4-(3-(Pyridin-2-yloxy)benzyl)piperazine-1-car-
boxylate (36n). 36n was prepared according to general procedure
F, using N-Boc-piperazine (305 mg, 1.64 mmol), dry CH2Cl2
(10 mL), 3-(pyridin-2-yloxy)benzaldehyde (104 mg, 0.52 mmol),
AcOH (0.1 mL), and Na(OAc)3BH (233 mg, 1.1 mmol) in the
first step, CH2Cl2/TFA (5 mL) in the second step, and CH2Cl2
(5 mL), triethylamine (1 mL, 7 mmol), and 4-nitrochlorofor-
mate (100 mg, 0.50 mmol) in the third step. Purification of the
crude oil by flash chromatography (2:1 Hex/EtOAc) gave the
product (85 mg, 38% yield over three steps): 1H NMR (CDCl3,
400 MHz) δ 8.31-8.16 (m, 3H), 7.70 (ddd, J = 8.3, 7.2, 1.9 Hz,
1H), 7.37 (t, J = 7.7 Hz, 1H), 7.33-7.24 (m, 2H), 7.17 (d, J = 8.7
Hz, 2H), 7.09-7.03 (m, 1H), 7.01 (ddd, J = 7.2, 5.1, 0.8 Hz, 1H),
6.93 (dd, J = 8.3, 0.8 Hz, 1H), 3.68 (bs, 2H), 3.59 (bs, 4H), 2.53
(bs, 4H). HRMS calculated for C23H23N4O5 [M þ H]þ 435.1663,
found 435.1699.
Acknowledgment. We thank the Cravatt lab for helpful
discussions and critical reading of the manuscript. This work
was supported by the U.S. National Institutes of Health
(Grants DA017259, DA009789, DA025285), the Helen L.
Dorris Institute Child and Adolescent Neuro-Psychiatric
Disorder Institute, and the Skaggs Institute for Chemical
Biology.
4-Nitrophenyl 4-(3-(4-Methoxyphenoxy)benzyl)piperazine-1-
carboxylate (36o). 36o was prepared according to general pro-
cedure F, using N-Boc-piperazine (559 mg, 3.0 mmol), dry THF
(20 mL), 3-(4-methoxyphenoxy)benzaldehyde (457 mg, 2.00
mmol), AcOH (0.2 mL), and Na(OAc)3BH (848 mg, 4.0 mmol)
in the first step, CH2Cl2/TFA (15 mL) in the second step, and
CH2Cl2 (6 mL), triethylamine (3 mL, 21 mmol), and 4-nitro-
chloroformate (653 mg, 3.24 mmol) in the third step. Purifica-
tion of the crude oil by flash chromatography (2:1 Hex/EtOAc)
gave 36o (343 mg, 37% yield over three steps): 1H NMR (CDCl3,
300 MHz) δ 8.26-8.21 (m, 2H), 7.32-7.20 (m, 3H), 7.05-6.80
References
(1) Ahn, K.; McKinney, M. K.; Cravatt, B. F. Enzymatic pathways
that regulate endocannabinoid signaling in the nervous system.
Chem. Rev. 2008, 108, 1687–1707.
(2) Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L.; Lerner,
R. A.; Gilula, N. B. Molecular characterization of an enzyme that

Article
degrades neuromodulatory fatty-acid amides. Nature 1996, 384,
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1841
using fluorescent active site-directed probes. Proteomics 2001, 1,
1067–1071.
83–87.
(3) Cravatt, B. F.; Demarest, K.; Patricelli, M. P.; Bracey, M. H.;
Giang, D. K.; Martin, B. R.; Lichtman, A. H. Supersensitivity to
anandamide and enhanced endogenous cannabinoid signaling
in mice lacking fatty acid amide hydrolase. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 9371–9376.
(4) Kathuria, S.; Gaetani, S.; Fegley, D.; Valino, F.; Duranti, A.;
Tontini, A.; Mor, M.; Tarzia, G.; La Rana, G.; Calignano, A.;
Giustino, A.; Tattoli, M.; Palmery, M.; Cuomo, V.; Piomelli, D.
Modulation of anxiety through blockade of anandamide hydro-
lysis. Nat. Med. 2003, 9, 76–81.
(5) 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 Becelaere, K.; Stevens, R. C.;
Cravatt, B. F. Discovery and characterization of a highly selective
FAAH inhibitor that reduces inflammatory pain. Chem. Biol. 2009,
16, 411–420.
(6) Blankman, J. L.; Simon, G. M.; Cravatt, B. F. A comprehensive
profile of brain enzymes that hydrolyze the endocannabinoid
2-arachidonoylglycerol. Chem. Biol. 2007, 14, 1347–1356.
(7) 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. Selective blockade
of 2-arachidonoylglycerol hydrolysis produces cannabinoid beha-
vioral effects. Nat. Chem. Biol. 2009, 5, 37–44.
(8) Long, J. Z.; Nomura, D. K.; Cravatt, B. F. Characterization of
monoacylglycerol lipase inhibition reveals differences in central
and peripheral endocannabinoid metabolism. Chem. Biol. 2009, 16,
744–753.
(9) 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. Dual blockade of FAAH and
MAGL identifies behavioral processes regulated by endocannabi-
noid crosstalk in vivo. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
20270–20275.
(10) Ahn, K.; Johnson, D. S.; Fitzgerald, L. R.; Liimatta, M.; Arendse,
A.; Stevenson, T.; Lund, E. T.; Nugent, R. A.; Nomanbhoy, T. K.;
Alexander, J. P.; Cravatt, B. F. Novel mechanistic class of fatty
acid amide hydrolase inhibitors with remarkable selectivity. Bio-
chemistry 2007, 46, 13019–13030.
(22) Jessani, N.; Niessen, S.; Wei, B. Q.; Nicolau, M.; Humphrey, M.; Ji,
Y.; Han, W.; Noh, D. Y.; Yates, J. R., 3rd; Jeffrey, S. S.; Cravatt, B.
F. A streamlined platform for high-content functional proteomics
of primary human specimens. Nat. Methods 2005, 2, 691–697.
(23) Li, W.; Blankman, J. L.; Cravatt, B. F. A functional proteomic
strategy to discover inhibitors for uncharacterized hydrolases.
J. Am. Chem. Soc. 2007, 129, 9594–9595.
(24) Hoover, H. S.; Blankman, J. L.; Niessen, S.; Cravatt, B. F.
Selectivity of inhibitors of endocannabinoid biosynthesis evaluated
by activity-based protein profiling. Bioorg. Med. Chem. Lett. 2008,
18, 5838–5841.
(25) Leung, D.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Discovering
potent and selective reversible inhibitors of enzymes in complex
proteomes. Nat. Biotechnol. 2003, 21, 687–691.
(26) Bachovchin, D. A.; Brown, S. J.; Rosen, H.; Cravatt, B. F.
Identification of selective inhibitors of uncharacterized enzymes
by high-throughput screening with fluorescent activity-based
probes. Nat. Biotechnol. 2009, 27, 387–394.
(27) Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.;
Cravatt, B. F. Structural adaptations in a membrane enzyme
that terminates endocannabinoid signaling. Science 2002, 298,
1793–1796.
(28) Morphy, R.; Rankovic, Z. Designed multiple ligands. An emerging
drug discovery paradigm. J. Med. Chem. 2005, 48, 6523–6543.
(29) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti,
V.; Recanatini, M.; Melchiorre, C. Multi-target-directed ligands
to combat neurodegenerative diseases. J. Med. Chem. 2008, 51,
347–372.
(30) Glynn, P.; Read, D. J.; Guo, R.; Wylie, S.; Johnson, M. K.
Synthesis and characterization of a biotinylated organopho-
sphorus ester for detection and affinity purification of a brain
serine esterase: neuropathy target esterase. Biochem. J. 1994, 301
(Part 2), 551–556.
(31) Lush, M. J.; Li, Y.; Read, D. J.; Willis, A. C.; Glynn, P. Neuro-
pathy target esterase and a homologous Drosophila neurodegen-
eration-associated mutant protein contain a novel domain
conserved from bacteria to man. Biochem. J. 1998, 332 (Part 1),
1–4.
(32) Glynn, P. A mechanism for organophosphate-induced delayed
neuropathy. Toxicol. Lett. 2006, 162, 94–97.
(11) DeSimone, R. W.; Currie, K. S.; Mitchell, S. A.; Darrow, J. W.;
Pippin, D. A. Privileged structures: applications in drug discovery.
Comb. Chem. High Throughput Screening 2004, 7, 473–494.
(12) Evans, B. E.; Rittle, K. E.; Bock, M. G.; DiPardo, R. M.;
Freidinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.;
Anderson, P. S.; Chang, R. S.; et al. Methods for drug discovery:
development of potent, selective, orally effective cholecystokinin
antagonists. J. Med. Chem. 1988, 31, 2235–2246.
(13) Patricelli, M. P.; Lovato, M. A.; Cravatt, B. F. Chemical and
mutagenic investigations of fatty acid amide hydrolase: evidence
for a family of serine hydrolases with distinct catalytic properties.
Biochemistry 1999, 38, 9804–9812.
(14) Karlsson, M.; Contreras, J. A.; Hellman, U.; Tornqvist, H.; Holm,
C. cDNA cloning, tissue distribution, and identification of the
catalytic triad of monoglyceride lipase. Evolutionary relationship
to esterases, lysophospholipases, and haloperoxidases. J. Biol.
Chem. 1997, 272, 27218–27223.
(15) Dinh, T. P.; Carpenter, D.; Leslie, F. M.; Freund, T. F.; Katona, I.;
Sensi, S. L.; Kathuria, S.; Piomelli, D. Brain monoglyceride lipase
participating in endocannabinoid inactivation. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 10819–10824.
(33) Casida, J. E.; Nomura, D. K.; Vose, S. C.; Fujioka, K. Organopho-
sphate-sensitive lipases modulate brain lysophospholipids, ether
lipids and endocannabinoids. Chem.-Biol. Interact. 2008, 175,
355–364.
(34) Johnson, M. K.; Glynn, P. Neuropathy target esterase (NTE) and
organophosphorus-induced delayed polyneuropathy (OPIDP):
recent advances. Toxicol. Lett. 1995, 82-83, 459–463.
(35) Pan, B.; Wang, W.; Long, J. Z.; Sun, D.; Hillard, C. J.; Cravatt, B.
F.; Liu, Q. S. Blockade of 2-arachidonoylglycerol hydrolysis
by selective monoacylglycerol lipase inhibitor 4-nitrophenyl
4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxy-
late (JZL184) enhances retrograde endocannabinoid signaling.
J. Pharmacol. Exp. Ther. 2009, 331, 591–597.
(36) Schlosburg, J. E.; Carlson, B. L.; Ramesh, D.; Abdullah, R. A.;
Long, J. Z.; Cravatt, B. F.; Lichtman, A. H. Inhibitors of endo-
cannabinoid-metabolizing enzymes reduce precipitated withdra-
wal responses in THC-dependent mice. AAPS J 2009, 11, 342–352.
(37) Kinsey, S. G.; Long, J. Z.; O’Neal, S. T.; Abdullah, R. A.; Poklis,
J. L.; Boger, D. L.; Cravatt, B. F.; Lichtman, A. H. Blockade of
endocannabinoid-degrading enzymes attenuates neuropathic pain.
J. Pharmacol. Exp. Ther. 2009, 330, 902–910.
(16) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Activity-based protein
profiling: the serine hydrolases. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 14694–14699.
(17) Alexander, J. P.; Cravatt, B. F. Mechanism of carbamate inactiva-
tion of FAAH: implications for the design of covalent inhibitors
and in vivo functional probes for enzymes. Chem. Biol. 2005, 12,
1179–1187.
(18) Tarzia, G.; Duranti, A.; Tontini, A.; Piersanti, G.; Mor, M.;
Rivara, S.; Plazzi, P. V.; Park, C.; Kathuria, S.; Piomelli, D.
Design, synthesis, and structure-activity relationships of alkylcar-
bamic acid aryl esters, a new class of fatty acid amide hydrolase
inhibitors. J. Med. Chem. 2003, 46, 2352–2360.
(38) Straiker, A.; Hu, S. S.; Long, J. Z.; Arnold, A.; Wager-Miller, J.;
Cravatt, B. F.; Mackie, K. Monoacylglycerol lipase limits the
duration of endocannabinoid-mediated depolarization-induced
suppression of excitation in autaptic hippocampal neurons. Mol.
Pharmacol. 2009, 76, 1220–1227.
(39) Nomura, D. K.; Long, J. Z.; Niessen, S.; Hoover, H. S.; Ng, S.-W.;
Cravatt, B. F. Monoacylglycerol lipase regulates a fatty acid net-
work that promotes cancer pathogenesis. Cell 2010, 140, 49–61.
(40) Labar, G.; Bauvois, C.; Borel, F.; Ferrer, J. L.; Wouters, J.;
Lambert, D. M. Crystal structure of the human monoacylglycerol
lipase, a key actor in endocannabinoid signaling. ChemBioChem
2009, DOI: 10.1002/cbic.200900621.
(19) Cravatt, B. F.; Wright, A. T.; Kozarich, J. W. Activity-based
protein profiling: from enzyme chemistry to proteomic chemistry.
Annu. Rev. Biochem. 2008, 77, 383–414.
(41) Bertrand, T.; Auge, F.; Houtmann, J.; Rak, A.; Vallee, F.; Mikol,
V.; Berne, P. F.; Michot, N.; Cheuret, D.; Hoornaert, C.; Mathieu,
M. Structural basis for human mono-glyceride lipase inhibition.
J. Mol. Biol. 2009, DOI: 10.1016/j.jmb.2009.11.060.
(20) Evans, M. J.; Cravatt, B. F. Mechanism-based profiling of enzyme
families. Chem. Rev. 2006, 106, 3279–3301.
(21) Patricelli, M. P.; Giang, D. K.; Stamp, L. M.; Burbaum, J. J. Direct
visualization of serine hydrolase activities in complex proteomes
(42) Morera, E.; De Petrocellis, L.; Morera, L.; Moriello, A. S.; Ligresti,
A.; Nalli, M.; Woodward, D. F.; Di Marzo, V.; Ortar, G. Synthesis
and biological evaluation of piperazinyl carbamates and ureas as

1842 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Long et al.
fatty acid amide hydrolase (FAAH) and transient receptor poten-
tial (TRP) channel dual ligands. Bioorg. Med. Chem. Lett. 2009, 19,
6806–6809.
(44) Saghatelian, A.; McKinney, M. K.; Bandell, M.; Patapoutian, A.;
Cravatt, B. F. A FAAH-regulated class of N-acyl taurines that
activates TRP ion channels. Biochemistry 2006, 45, 9007–9015.
(45) Karlsson, M.; Reue, K.; Xia, Y. R.; Lusis, A. J.; Langin, D.;
Tornqvist, H.; Holm, C. Exon-intron organization and chromo-
somal localization of the mouse monoglyceride lipase gene. Gene
2001, 272, 11–18.
(43) Zygmunt, P. M.; Petersson, J.; Andersson, D. A.; Chuang, H.;
Sorgard, M.; Di Marzo, V.; Julius, D.; Hogestatt, E. D. Vanilloid
receptors on sensory nerves mediate the vasodilator action of
anandamide. Nature 1999, 400, 452–457.