N-Acyl pyrazoles: Effective and tunable inhibitors of serine hydrolases

Article abstract of DOI:10.1016/j.bmc.2019.03.020

A series of N-acyl pyrazoles was examined as candidate serine hydrolase inhibitors in which the active site acylating reactivity and the leaving group ability of the pyrazole could be tuned not only through the nature of the acyl group (reactivity: amide

Full text of DOI:10.1016/j.bmc.2019.03.020

Accepted Manuscript
N-Acyl pyrazoles: effective and tunable inhibitors of serine hydrolases
Katerina Otrubova, Shreyosree Chatterjee, Srijana Ghimire, Benjamin F.
Cravatt, Dale L. Boger
PII:
S0968-0896(19)30292-5
https://doi.org/10.1016/j.bmc.2019.03.020
BMC 14812
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 February 2019
8 March 2019
8 March 2019
Please cite this article as: Otrubova, K., Chatterjee, S., Ghimire, S., Cravatt, B.F., Boger, D.L., N-Acyl pyrazoles:
effective and tunable inhibitors of serine hydrolases, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/
10.1016/j.bmc.2019.03.020
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N-Acyl pyrazoles: effective and tunable inhibitors of serine hydrolases
Katerina Otrubova, Shreyosree Chatterjee, Srijana Ghimire, Benjamin F. Cravatt, and Dale L. Boger*
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research
Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
Abstract. A series of N-acyl pyrazoles was examined as candidate serine hydrolase inhibitors in which
the active site acylating reactivity and the leaving group ability of the pyrazole could be tuned not only
through the nature of the acyl group (reactivity: amide > carbamate > urea), but also through pyrazole
C4 substitution with electron-withdrawing or electron-donating substituents. Their impact on enzyme
inhibitory activity displayed pronounced effects with the activity improving substantially as one alters
both the nature of the reacting carbonyl group (urea > carbamate > amide) and the pyrazole C4
substituent (CN > H > Me). It was further demonstrated that the acyl chain of the N-acyl pyrazole ureas
can be used to tailor the potency and selectivity of the inhibitor class to a targeted serine hydrolase.
Thus, elaboration of the acyl chain of pyrazole-based ureas provided remarkably potent, irreversible
inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki = 100–200 pM), dual inhibitors of FAAH and
monoacylglycerol hydrolase (MGLL), or selective inhibitors of MGLL (IC50 = 10–20 nM) while
simultaneously minimizing off-target activity (e.g., ABHD6 and KIAA1363).
Key words: serine hydrolase inhibitor, N-acyl pyrazole, fatty acid amide hydrolase, monoacylglycerol
hydrolase, activity-based protein profiling (ABPP)
*Corresponding author. e-mail address: dale.boger@outlook.com (D L. Boger)
1. Introduction
The endocannabinoids anandamide1,2 and 2-arachidonylglycerol (2-AG)3,4 are endogenous lipid
signaling molecules5 that activate cannabinoid (CB1 and CB2) receptors (Figure 1).2,6 The
characterization and pharmacologic inactivation of the major enzymes that terminate the signaling of
anandamide and 2-AG, fatty acid amide hydrolase (FAAH)7 and monoacylglycerol lipase (MGLL),
respectively,8-11 have shown that each elevate endocannabinoid signaling and that each affect many

of the same pharmacological processes, including pain and inflammation.12-18 Less has been described
about simultaneous inhibition of both enzymes, but indications are that it results in more pronounced
pharmacological activity.19 Inhibitors of each enzyme individually or combined could provide
therapeutics that replace opioid analgesics for either severe (surgical) or chronic neuropathic pain,
avoiding the opioid side effects of dependence, desensitization with chronic dosing, dose-limiting
respiratory depression, and constipation that extends hospital stays. Similarly, it is expected such
individual or dual enzyme inhibitors could serve as alternatives to medical marijuana or its active
constituents in clinical indications where it is presently used, including oncology pain and nausea.
Importantly and because they only act on activated signaling pathways, increasing the endogenous
concentration of the released signaling molecules only at a site of stimulation, they provide a spatial
and temporal pharmacological control not available to direct acting opioid or CB receptor agonists.
Herein, we report full details of a study that identified N-acyl pyrazole ureas as candidate serine
hydrolase inhibitors and their elaboration to provide selective FAAH inhibitors, dual FAAH/MGLL
inhibitors, or selective MGLL inhibitors through modification of distinguishable recognition elements
used to target the individual enzyme active sites.
Figure 1. Structures of anandamide and 2-AG.
2. Results and Discussion
In the course of efforts targeting serine hydrolases,12,13 we prepared a series of N-acyl pyrazole
amides, carbamates and ureas and examined them as candidate inhibitors of the FAAH in order to
define their potential utility (Figure 2).20 The compounds were first evaluated in a substrate (oleamide)
hydrolysis assay with the pure recombinant rat enzyme (rFAAH) and the results are reported as an
o
apparent Ki that was measured after 3 h preincubation at 25 C with the enzyme (typically 1 nM, but
0.3 nM for measurement of app Ki < 2 nM).21 A subsequent gel based activity-based protein profiling

(ABPP) screen was used to assess activity and selectivity,22 which offers the advantage of testing
enzymes in their native state and eliminates the need for their recombinant expression, purification,
and the development of specific substrate assays. Because the inhibitors are screened against many
enzymes in the complex proteome in parallel, their potency and selectivity as well as their performance
in cell lysates can be simultaneously evaluated.23
Figure 2. Design and synthesis of candidate N-acyl pyrazole inhibitors.
Precedent for the potential utility of N-acyl pyrazoles was based on the reported behaviour of
related azoles (tetrazoles,24 triazoles,25 imidazoles,25 and indazoles25). At the time of our initial
communication,20 there were no accounts of detailed or systematic examinations of N-acyl pyrazole
ureas or carbamates reacting with or inhibiting serine hydrolases although isolated cases have been
disclosed.26 Acyl pyrazoles display an intrinsic modest reactivity toward nucleophilc attack, being less
reactive than tetrazoles, triazoles or imidazoles, though they conceivably could be further activated by
(enzymatic) protonation or metal chelation. They also represent compounds that contain an activating
group potentially capable of formation of a stable (chelated) tetrahedral intermediate.27 The lone pair
on the pyrazole nitrogen bound to the carbonyl is not intimately conjugated with the acyl group, as this
would diminish the pyrazole aromaticity, making it a potentially good leaving group. Additionally, they
are expected to adopt a preferential well-defined acyl orientation with the carbonyl oxygen eclipsed with
the in plane pyrazole C5-H and opposite the pyrazole N2 in order to avoid a destabilizing lone pair/lone
pair electrostatic interaction (Figure 2). Finally, N-acyl pyrazoles represent a chemotype series in which
its acylating reactivity and the leaving group propensity of the pyrazole could be tuned not only by the

nature of the acyl group (reactivity: amide > carbamate > urea), but also through simple symmetrical
C4 substitution of the pyrazole with electron-withdrawing or electron-donating substituents (vs
unsymmetrical substitution for all other azoles). Notably, electron-withdrawing substituents would be
expected to increase the intrinsic leaving group ability of the pyrazole, but would diminish their
capabilities for activation by protonation or metal chelation. The importance and impact of these
features were first examined and defined with a series N-acyl pyrazoles as inhibitors of FAAH, some of
which bear acyl chains known to promote FAAH active site binding (1–9 and 17–23) (Figure 3). Each
pyrazole inhibitor subset was prepared as an amide, carbamate, or urea and each contained a series
of pyrazole substituents (R = Me, H, CN; a–c) designed to establish their electronic impact on inhibitor
activity (reactivity) and its magnitude. This collection of 69 compounds in itself represents a useful
serine hydrolase inhibitor screening sublibrary.20 Several key structural features emerged from their
examination. The impact on FAAH inhibitory activity displayed clear and pronounced effects with the
activity improving substantially as one alters both the nature of the reacting carbonyl group (urea >
carbamate > amide) and the pyrazole C4 substituent (CN > H > Me). Interestingly, the N-acyl pyrazole
ureas provided the most potent inhibitors even though they are intrinsically less reactive than the
corresponding amides and carbamates, suggesting a unique active site activation for carbamoylation.
In contrast, it was the N-acyl pyrazole ureas with the intrinsically more reactive pyrazole leaving group,
those bearing the electron-withdrawing C4 substituent (CN), that provided the most potent inhibitors.
Additionally, the trends in the impact of the acyl chain were found to follow those expected from
observations made with -ketoheterocycle12,13,23 or carbamate/urea12 inhibitors of FAAH. Thus, the
phenalkyl chain inhibitors that contain 5 or 6 atom spacers between the reacting carbonyl and phenyl
group12d (4 and 7 vs 1) displayed good activity as did those that contain the now characteristic extended
and rigidified aryl acyl chains12d (20–23), the most potent of which displayed apparent Ki’s of 10–20 nM.
Expectedly, the candidate inhibitors with an even shorter phenylalkyl chain were inactive against FAAH
(not shown) as were the shorter benzyl (15–16), naphthyl (10–11), naphthylmethyl (12–14) (Figure 3)
and related compounds (Figure 4).

Figure 3. Recombinant rFAAH inhibition, apparent Ki (25 oC, 3 h preincubation with enzyme, 1 nM).

Figure 4. Additional compounds found to be inactive against rFAAH.
The representative members 22a–c of this series were examined by gel based ABPP in mouse
brain proteome as a prelude to the examination of additional inhibitors disclosed later herein (Figure
5). The mouse brain proteome is known to contain FAAH as well as many important off target enzymes
observed with earlier classes of FAAH inhibitors, including MGLL and KIAA1363 (also known as
arylacetamide deacetylase-like 1 (AADACL1) and neutral cholesterol ester hydrolase 1 (NCEH)).
Inhibitors 22a–c proved to be remarkably selective given their structural simplicity, notably displaying
no activity against KIAA1363. By contrast, MGLL was established to be competitively inhibited, and
both FAAH and MGLL were inhibited by 22 with near equivalent potencies. Moreover, this near
equivalent activity against FAAH and MGLL was observed regardless of the pyrazole substituent,
indicating that their impact on intrinsic reactivity and inhibitor potency do not serve to discriminate
between the two enzymes. Thus, the same relative potencies of 22a–c against FAAH in the pure
enzyme assay were observed in the gel based assay and their activity against MGLL paralleled these
same tends (potency: 22c > 22b > 22a, R = CN > H > Me), providing dual balanced inhibitors of the
two enzymes.
Figure 5. ABPP assays performed after preincubation (1 h, 25 oC) of mouse brain proteome (1 mg/mL) with 22
over a range of inhibitor concentrations. FP-Rhodamine probe (100 nM) was then added and incubated for an
additional 20 min.

In many regards, the observation of effective FAAH inhibition with the pyrazole ureas (1, 4, 7,
20 and 22) is surprising given the incorporation of monosubstituted carbamides. Typically, FAAH
inhibition with urea or carbamate inhibitors is observed with disubstituted carbamide-based ureas or
carbamates and seen best with incorporation of rigid, cyclic disubstituted carbamides. With ureas,
catalysis of the acylation reaction has been suggested to be derived from an enzyme active site binding-
induced conformational change in the urea that diminishes the stabilizing conjugation of the nitrogen
lone pair with the carbonyl, activating the urea for nucleophile attack (Figure 6).28 The potential that this
binding-induced activation of a urea is FAAH specific has been used to rationalize both the potency
and selectivity of other urea-based inhibitors of FAAH.29 As a result, we examined an additional series
of 57 N-acyl pyrazole ureas that contain acyclic and cyclic disubstituted carbamides with expectations
they may prove to be even more effective inhibitors of FAAH (Figure 6). Each candidate inhibitor set
was composed of one of three linkers and one of a series of acyl chains in which each set contained
the same three pyrazole substituents (R = Me, H, CN; a–c) designed to further establish a leaving
group substituent electronic impact on inhibitor activity (reactivity) and its magnitude.
Figure 6. Cyclic and acyclic N-acyl pyrazole ureas containing disubstituted carbamides.
The results of their assessment are summarized in Figure 7 and provided a series of potent
FAAH inhibitors. The most potent of these display apparent Ki values of 100–200 pM when substituted
with acyl chains we have shown to be especially effective for targeting the FAAH active site.30 Several
conclusions are apparent from the results. Even the simple pyrazole ureas 24–27 now display effective
inhibition of FAAH, highlighting the productive impact of a fully substituted urea. Moreover, even the
more flexible acyclic acyl group found in 27 provided remarkably good inhibition of FAAH. The impact
of the C4 pyrazole substituent on FAAH inhibitory activity displayed clear and pronounced effects with
the activity improving substantially through each set (R = CN > H > Me).

Figure 7. Recombinant rFAAH inhibition, apparent Ki (25 oC, 3 h preincubation with enzyme at 1 nM for Ki’s > 2
nM and at 0.3 nM for Ki’s < 2 nM). Inhibitors 29–34c with apparent Ki’s = 0.1–0.2 nM represent stoichiometric
irreversible enzyme inhibition under these conditions.

Not only did the electron-withdrawing nitrile group substantially improve activity (CN > H, avg =
25-fold), but even the introduction of a modest electron-donating group diminished activity (H > Me,
avg = 8-fold). These distinctions were most pronounced with the rigid, cyclic ureas 28–34 and 35–38,
but still substantial even with the simple (24–27) or more flexible acyclic ureas (39–42). Although this
effect could reflect both an electronic and steric impact of the substituent on the activity, we have shown
that FAAH possess a large cytosolic port adjacent to the active site capable of accommodating large
substituted heterocycles.31 This suggests that the impact is largely or solely due to the electronic
properties of the pyrazole C4 substituent. In order to establish whether this truly reflects only the
electronic impact of the substituent, an expanded series of substituted pyrazoles was examined in
which the electron-withdrawing properties of the C4 substituent was systematically varied without
significantly altering its size or polarity (Figure 8). This expanded series exhibited pronounced and
incremental increases in potency as the electron-withdrawing properties of the C4 substituent increases
(CN > CO2Me > Br > H > Me), where the change in apparent Ki values across the series spans a
stunning 103-fold.
Figure 8. Impact of pyrazole C4-substituent on inhibition of FAAH.
The time-dependent inhibition of rFAAH was examined with 33a–c (Figure 9). These three
inhibitors are nearly identical in structure, containing the same acyl chain and differing only in the
pyrazole C4 substitution. When used in near stoichiometric amounts, all three inhibitors exhibited time-
dependent inhibition of rFAAH consistent with irreversible versus reversible competitive inhibition of the
enzyme. The intrinsically most reactive of the three inhibitors (33c) not only produced the greatest
enzyme inhibition at each time point, but also required the shortest time period to reach complete

enzyme inhibition (1 h). Both 33a and 33b required a longer and near identical time period to reach
maximal enzyme inhibition (ca. 3 h) after which little further decrease in enzyme activity was observed,
presumably due to near stoichiometric use of inhibitor and its competitive consumption under the
strongly basic conditions of the assay (pH 9), and the intrinsically least reactive of the inhibitors 33a
displayed the smallest degree of enzyme inhibition at each time point. Not only did this confirm time-
dependent irreversible enzyme inhibition, but the qualitative results mirror those of the quantitatively
measured enzyme inhibition that is reported as apparent Ki. It also established the inhibitor–enzyme
preincubation time period (3 h) that we utilized in examination of the inhibitors in the FAAH assay.
o
Figure 9. Time-dependent rFAAH inhibition by 33. rFAAH (2 nM) preincubation (0–9 h, 25 C, pH 9) with
inhibitors 33a–c (5 nM) followed by measurement of residual enzyme activity.
Two of the potent sets of inhibitors, the three pyrazole ureas bearing the naphthyl tail (29a–c)
as well as the three containing the 4-benzyloxyphenyl tail (33a–c), were each confirmed to be
irreversible FAAH inhibitors. Thus, rFAAH was treated with each inhibitor (3 h, 25 oC) at concentrations
that resulted in >70% enzyme inhibition. The inhibitor–enzyme mixtures were then subjected to dialysis
dilution (4 oC, 18 h) and the residual enzyme activities were measured both before and following dialysis
dilution (Figure 10). No recovery of enzyme activity was observed for all six inhibitors after 18 h
indicative of irreversible enzyme inhibition by active site carbamoylation of a catalytic serine.

Figure 10. Irreversible rFAAH inhibition by 29 and 33. rFAAH (2 nM) preincubation (3 h, 25 oC) with inhibitors (a
at 100 nM, b at 50 nM, c at 10 nM) and measurement of residual enzyme activity pre and post dialysis dilution
(18 h, 4 oC).
Six representative members of this series were examined by gel based ABPP in mouse brain
proteome, defining both their potency and intrinsic selectivity in complex proteomes. As highlighted
earlier, the mouse brain proteome contains FAAH as well as many of the important off target enzymes
observed with earlier classes of FAAH inhibitors, including KIAA1363, MGLL, and ABHD6
(hydrolase containing domain 6). Like inhibitor 22, none of the compounds examined displayed
activity against KIAA1363. Whereas 24, lacking an active site targeting acyl tail, inhibited FAAH, MGLL
and ABHD6 with near equivalent activities, the inhibitors 29, 33, and 37 displayed a divergence in
selectivity (Figure 11). Each of these four inhibitors effectively targeted ABHD6, often more effectively
than either FAAH or MGLL. MGLL was established to be competitively but less effectively inhibited by
29 and 33, whereas the further small change in the closely related inhibitor 37 resulted in near complete
disruption of MGLL inhibition. This latter compound 37 proved to be quite selective versus MGLL, and
displayed dual, often balanced activity against both FAAH and ABHD6. These trends in activity towards
the enzymes were observed regardless of the pyrazole substituent, indicating that their impact on
intrinsic reactivity and inhibitor potency do not serve to discriminate between these enzymes. Finally,
the same relative potencies of a–c against rFAAH in the pure enzyme assay were observed in the gel
based assay and their relative activity against the competitive enzymes (MGLL and ABHD6) typically
also paralleled these same tends (potency: c > b > a, R = CN > H > Me).

o
Figure 11. ABPP assays performed after preincubation (1 h, 25 C) of mouse brain proteome (1 mg/mL) with
the each inhibitor over a range of inhibitor concentrations. FP-Rhodamine probe (100 nM) was then added and
incubated for an additional 20 min.
By contrast, the pyrazole ureas 41 and 42, which contained a noncyclic disubstituted carbamide
with the same attached groups found in 33 and 37, were less active in the brain proteome and displayed
essentially equal activity against FAAH, MGLL, and ABHD6, displaying little selectivity between the
three enzymes (Figure 12). In addition, the same relative potencies of a–c against rFAAH in the pure
enzyme assay were observed in the gel based assay and their relative activity against the competitive

enzymes (MGLL and ABHD6) smoothly paralleled these same tends (potency: c > b > a, R = CN > H
> Me). Cumulatively, these studies demonstrate that N-acyl pyrazole ureas may be used as potent
serine hydrolase inhibitors whose (re)activity can be predictably tuned by introduction of pyrazole
substituents and whose acyl chain can be tailored to selectively target individual enzymes or more
broadly target several closely related enzymes.
o
Figure 12. ABPP assays performed after preincubation (1 h, 25 C) of mouse brain proteome (1 mg/mL) with
the each inhibitor over a range of inhibitor concentrations. FP-Rhodamine probe (100 nM) was then added and
incubated for an additional 20 min.
In the preceding studies, the candidate inhibitors were outfitted with acyl chains likely to promote
binding to MGLL as well as FAAH, and dual balanced FAAH/MGLL inhibitors were discovered (e.g., 41
and 42) as well as several that displayed greater activity against FAAH (FAAH > MGLL; e.g., 29 and
33). The series also provided very selective FAAH inhibitors (FAAH >> MGLL, 37) that can serve as
key closely related comparison compounds in the same inhibitor class. However, the series was not
designed for and did not define a set of N-acyl pyrazole ureas that act as MGLL selective inhibitors
(FAAH < MGLL). Advances in the discovery of selective MGLL inhibitors15 has been more recent
compared to FAAH and even fewer reports have detailed inhibitors with dual FAAH/MGLL
inhibition.19,32,33 Early on, the irreversible MGLL-selective carbamate-based inhibitors JZL18416 and

KLM2917 and the carbamate-based dual FAAH/MGLL inhibitor JZL195 were reported (Figure 13).33
Although useful, both JZL184 and JZL195 contain a p-nitrophenol leaving group that limited in vivo use.
To better define the impact of selective MGLL inhibition, continued efforts provided high quality,
selective, and in vivo active inhibitors of the enzyme that bear alternative activated carbamates,
including the N-hydroxysuccinimide (NHS; e.g. MJN11034) and hexafluoroisopropanol (HFIP; e.g.,
KLM2917) derived carbamates. Related inhibitors have since been disclosed.35,36 Based on these
studies, we examined a series of additional N-acyl pyrazole ureas to illustrate that the acyl chain can
be tuned to more selectively target other serine hydrolases, including MGLL.
Figure 13. Representative MGLL inhibitors and distinctions with a comparable dual FAAH/MGLL inhibitor.
FAAH and MGLL bind substrates that contain fatty acid chains and often display similar, but not
identical acyl chain preferences.37 The distinguishing feature is that MGLL can bind bulkier hydrophobic
groups than FAAH (e.g., JZL184 vs JZL195, Figure 13). Since the disclosure of JZ184, a tactic now
widely employed relies on the addition of a second, identical and sterically large aryl group to the tail of
the acyl chain, disrupting binding to FAAH and maintaining or enhancing binding to MGLL. An additional
attractive site to vary is the linker, where a large impact on the FAAH versus MGLL inhibitor activity has
also been observed. Consequently, we chose four of the sets of the three related inhibitors (22, 41, 29,

and 33) with which to probe these effects (43 vs 22, 44 vs 41, 45 vs 29, and 46 vs 33). Two of these
with the most flexible linker regions composed of either a secondary urea (43) or the flexible tertiary
urea (44) not only abolished nearly all FAAH activity as expected (5,000-fold and 50,000-fold reduction
in apparent Ki for 43c vs 22c and 44a vs 41a, respectively), but also resulted in disrupted activity against
all enzymes for which 22 and 41 were effective, including MGLL (e,g., IC50 reduced 100-fold for 44c vs
41c) (Figure 14).
Figure 14. Additional N-acyl pyrazole ureas. ABPP assays performed after preincubation (1 h, 25 oC) of mouse
brain proteome (1 mg/mL) with the each inhibitor over a range of inhibitor concentrations. FP-Rhodamine probe
(100 nM) was then added and incubated for an additional 20 min.
In contrast, 45 and 46, which contain the six-membered cyclic tertiary urea, displayed the
expected complete disruption of FAAH inhibition and a maintained or enhanced MGLL inhibition relative
to 29 and 33, respectively (Figure 15). Both 45c and 46c displayed excellent potency against MGLL
(IC50 = 8 and 20 nM, respectively). In addition, the competitive or even preferential inhibition of ABHD6
observed with 29 and 33 was also dramatically reduced such that little residual activity was observed
with 45 and 46. For the most potent of the compounds, 45c and 46c displayed a 70-fold and >1,000-

fold selectivity for MGLL versus ABHD6. Notably, these distinctions were not as obvious with the less
reactive and less active members of the inhibitor sets (R = Me, H), but only apparent with the more
reactive members (R = CN).
Figure 15. MGLL selective N-acyl pyrazole urea inhibitors. ABPP assays performed after preincubation (1 h, 25
oC) of mouse brain proteome (1 mg/mL) with the each inhibitor over a range of inhibitor concentrations. FP-
Rhodamine probe (100 nM) was then added and incubated for an additional 20 min.
Since the pharmacological effects observed with FAAH inhibition require >90% enzyme
inhibition while those derived from MGLL inhibition display smoothly increasing effects with an
increasing extent of inhibition, the most attractive inhibitors likely will be more potent against FAAH than
MGLL. This approach has been validated19a,b using a selective FAAH inhibitor co-administered with a
selective MGLL inhibitor. Thus, a high dose of the irreversible FAAH inhibitor PF-384538 given in
combination with a low dose of the irreversible MGLL inhibitor JZL18416 produced profound increases
in brain anandamide levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels.19 This
combination produced significantly greater antinociceptive effects than single enzyme inhibition and did

not elicit common cannabimimetic side effects, including catalepsy, hypomotility, hypothermia, and
substitution for 9-THC in a drug-discrimination assay. Moreover, repeated administration of this
combination did not lead to tolerance of its antiallodynic actions in the carrageenan assay or to CB1
receptor desensitization. Thus, full FAAH inhibition combined with partial MGLL inhibition reduced
neuropathic and inflammatory pain with minimal cannabimimetic effects.19 Continued exploration of the
N-acyl pyrazole ureas could provide an attractive series of closely related inhibitors with which the
systematic examination of the effects of the full range of dual inhibition embodied in a single compound
could be defined (FAAH > MGLL, balanced FAAH = MGLL, and FAAH < MGLL inhibition potency).
3. Conclusions
A series of N-acyl pyrazoles were examined as candidate serine hydrolase inhibitors and it was
shown that active site acylating reactivity and the leaving group ability of the pyrazole could be tuned
not only through the nature of the acyl group (reactivity: amide > carbamate > urea), but also through
pyrazole C4 substitution with electron-withdrawing or electron-donating substituents. Their impact on
FAAH inhibitory activity displayed clear and pronounced effects with the activity improving as one alters
both the nature of the reacting carbonyl (urea > carbamate > amide) and the pyrazole C4 substituent
(CN > H > Me).42 Interestingly, the N-acyl pyrazole ureas provided the most potent inhibitors even
though they are intrinsically less reactive than the corresponding amides and carbamates, suggesting
a unique active site activation for carbamoylation. In contrast, it was the N-acyl pyrazole ureas with the
intrinsically more reactive pyrazole leaving group that provided the most potent inhibitors. It was further
shown that the acyl chain of the N-acyl pyrazole ureas can be used to tailor the potency and selectivity
of the inhibitor class to a targeted serine hydrolase. Thus, representative modifications of the acyl chain
of pyrazole-based ureas provided selective and remarkably potent, irreversible inhibitors of fatty acid
amide hydrolase (FAAH, apparent Ki = 100–200 pM), dual inhibitors of FAAH and monoacylglycerol
hydrolase (MGLL), or selective potent inhibitors of MGLL (IC50 = 10–20 nM).
Finally, and notable in this work, the pyrazole ureas 24a–c that contain only a simple piperidine
carboxamide displayed robust inhibitory activity against FAAH even though they do not contain a

recognition group mimicking the substrate27,28 fatty acid amide long chain acyl groups (Figure 7).
Rather, they and a small series of pyrazole ureas, including those containing pendant protected
secondary amines in the acyl chain, may serve as useful screening compounds used to identify
chemotypes selective for inhibition of a subset of serine hydrolases, being more promiscuous across
the enzyme class (25–27).17a The combined set of 24–27 represent a small, but powerful serine
hydrolase screening library (Figure 16).39,40 Following detection of a targeted serine hydrolase
inhibition, the informative screening leads, which define trends in acyl urea reactivity and the importance
of the second attached carbamide (linker), may be divergently modified to improve active site
recognition features, simultaneously improving potency and selectivity for a targeted serine hydrolase
as demonstrated herein. Thus, their concurrent screening allows the identification of serine hydrolases
sensitive to inhibition by the pyrazole ureas, provide initial insights into how reactivity can impact
selectivity among the candidate serine hydrolases, define candidate off target enzymes in the class
whose activity should be monitored when addressing optimization against a chosen enzyme, and
provide superb starting fragments that can be further elaborated to tailor the potency and selectivity of
the inhibitor class to a targeted serine hydrolase.43
Figure 16. Key members of a serine hydrolase screening library.
4. Experimental
4.1. Chemistry General Methods. All commercial reagents were used without further purification unless
otherwise noted. THF was distilled prior to use. All reactions were performed in oven-dried (200 C)
glassware and under an inert atmosphere of anhydrous argon unless otherwise noted. Column
chromatography was performed with silica gel 60. TLC was performed on Whatman silica gel (250 m)
F254 glass plates and spots were visualized by UV. PTLC was performed on Whatman silica gel (250
1
and 500 m) F254 glass plates. H NMR spectra was recorded on a Bruker 500 or 400 MHz

spectrometer. Chemical shift are measured and reported relative to an internal standard of residual
1
CHCl3 ( 7.26 for H). H1 NMR data are reported as follows: chemical shift (), multiplicity (ovlp =
operlapping, br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant,
and integration. High resolution mass spectra was obtained on an Agilent ESI-TOF/MS using Agilent
ESI-L low concentration tuning mix as internal high resolution calibration standards. The purity and ESI-
LRMS of each tested compound (95%) were determined on an Agilent 1100 LC/MS instrument using
a ZORBAX SB-C18 column (3.5 mm, 4.6 mm  50 mm, with a flow rate of 0.75 mL/min and detection
at 220 and 253 nm) with a 10–98% acetonitrile/water/0.1% formic acid gradient (two different gradients).
Purity analysis and MS characterization may be found in Supporting Information Table S1.
4.1.1. General Procedure for Pyrazole Urea or Carbamate Synthesis. The appropriate pyrazole (1.0
equiv) was treated with phosgene (10 equiv, 20% in toluene) at 0 C. The reaction mixture was stirred
at room temperature for 1 h. The solvent was removed under reduced pressure and the crude
carbamoyl chloride was dissolved in anhydrous CH2Cl2 (0.5 M). The appropriate amine (1.0 equiv) or
alcohol (1 equiv) and Et3N (1.2 equiv) were dissolved in CH2Cl2 (0.5 M) and cooled to 0 C. The crude
carbamoyl chloride was added dropwise and the reaction mixture was stirred at room temperature for
16 h. The mixture was diluted with EtOAc, washed with saturated aqueous NaCl, and dried over
Na2SO4. Evaporation under reduced pressure yielded the crude coupling product that was purified by
flash chromatography (SiO2).
4.1.2. General Procedure for Pyrazole Amide Synthesis. The appropriate carboxylic acid (1.0 equiv)
was treated with oxalyl chloride (1.1 equiv) in CH2Cl2 (0.5 M) at 0 C. The reaction mixture was stirred
at room temperature for 1 h. The solvent was removed under N2 and the acid chloride (1 equiv) was
dissolved in anhydrous CH2Cl2 (0.5 M). The appropriate pyrazole (1.0 equiv) was dissolved in CH2Cl2
(0.5 M) and cooled to 0 C. The acid chloride (1 equiv) in CH2Cl2 (0.5 M) was added dropwise and the
reaction mixture was stirred at room temperature for 3 h. The mixture was diluted with EtOAc, washed
with saturated aqueous NaCl, and dried over Na2SO4. Evaporation under reduced pressure yielded the
crude product that was purified by flash chromatography (SiO2).

4.1.3. 1-(4-(4-(Benzyloxy)benzyl)piperazine-1-carbonyl)-1H-pyrazole-4-carbonitrile (33c). The title
compound was prepared from 1H-pyrazole-4-carbonitrile (8 mg, 0.086 mmol) and 4-(4-
benzyloxy)benzylpiperazine (24 mg, 0.086 mmol) by using the general procedure. Flash
1
chromatography (SiO2, 30% EtOAc–hexanes) afforded 33c (13 mg, 40%) as a white solid. H NMR
(CDCl3, 400 MHz) 8.80 (s, 1H), 8.24 (s, 1H), 7.47 (m, 2H), 7.42 (m, 3H), 7.25 (m, 2H), 7.18 (m, 2H),
5.11 (s, 2H), 3.65 (s, 2H), 3.22 (t, 4H, J = 6.7 Hz), 2.54 (t, 4H, J = 6.4 Hz). HRMS-ESI-TOF m/z 402.1921
([M+H]+, C23H24N5O2 requires 402.1930).
4.1.4. 1-(4-(4-(Benzyloxy)benzyl)-1,4-diazepane-1-carbonyl)-1H-pyrazole-4-carbonitrile (37c). The title
compound was prepared from 1H-pyrazole-4-carbonitrile (10 mg, 0.11 mmol) and 4-(4-
(benzyloxy)benzyl)-1,4-diazepane (32 mg, 0.11 mmol) by using the general procedure. Flash
1
chromatography (SiO2, 40% EtOAc–hexanes) afforded 37c (13 mg, 28%) as a white solid. H NMR
(CDCl3, 500 MHz)  8.80 (s, 1H), 8.28 (s, 1H), 7.47–7.45 (m, 2H), 7.36–7.34 (m, 3H), 7.20–7.18 (m,
2H), 6.85–6.84 (s, 2H), 5.12 (s, 2H), 3.68 (s, 2H), 3.64–3.44 (m, 8H), 1.80 (m, 2H). HRMS-ESI-TOF
m/z 416.2083 ([M+H]+, C24H26N5O2 requires 416.2087).
4.2. FAAH Inhibition. 14C-labeled oleamide was prepared from 14C-labeled oleic acid. Truncated rat
FAAH (rFAAH) was expressed in E. coli and purified as described,41 and the purified recombinant
rFAAH was used in the inhibition and reversibility assays. The purity of each tested compound (>95%)
was determined on an Agilent 1100 LC/MS instrument using a ZORBAX SB-C18 column (3.5 mm, 4.6
mm × 50 mm, with a flow rate of 0.75 mL/min and detection at 220 and 253 nm) with a 10–98%
acetonitrile/water/0.1% formic acid gradient (two different gradients, Supporting Information Table S1).
The inhibition assays were performed as described.13,21 The assay was initiated by mixing 1 nM
rFAAH (300 pM rFAAH for inhibitors with Ki ≤ 1–2 nM) in 500 μL of reaction buffer (125 mM TrisCl, 1
mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9.0) at room temperature in the
presence of three different concentrations of inhibitor. After 3 h preincubation at room temperature, The
enzyme reaction as initiated by addition of 20 μM of 14C-labeled oleamide and was terminated by
transferring 20 μL of the reaction mixture to 500 μL of 0.1 N HCl at three different time points. The 14C-

labeled oleamide (substrate) and oleic acid (product) were extracted with EtOAc and analyzed by TLC
as detailed.13,21 The apparent Ki of the inhibitor was calculated using a Dixon plot.
4.3 Competitive ABPP of inhibitors with FP-rhodamine. Mouse tissues were Dounce-homogenized in
PBS buffer (pH 8.0) and membrane proteomes isolated by centrifugation at 4 C (100,000g, 45 min),
washed, resuspended in PBS buffer, and adjusted to a protein concentration of 1 mg/mL. Proteomes
were preincubated with inhibitors (10–100,000 nM; DMSO stocks) for 1 h and then treated with FP-
rhodamine (100 nM, DMSO stock) at room temperature for 20 min. Reactions were quenched with
SDS-PAGE loading buffer, subjected to SDS-PAGE, and visualized in-gel using flatbed fluorescence
scanner (MiraBio). Labeled proteins were quantified by measuring integrated band intensities
(normalized for volume); control samples (DMSO alone) were considered 100% activity. IC50 values (n
= 2–4) were determined from dose-response curves using Prism software.
4.4. Reversibility of FAAH Inhibition (Dialysis Dilution). The reversibility of FAAH inhibition by 29a–c
and 33a–c was assessed by dialysis dilution using purified recombinant rat FAAH (rFAAH). The
enzyme (2 nM) was placed in 15 mL FAAH assay buffer (125 mM Tris, 1 mM EDTA, 0.2% glycerol,
0.02% Triton X-100, 0.4 mM Hepes, pH 9.0). A 3 mL aliquot of membrane homogenate was used for
each sample dialyzed. The dialysis experiment was performed in the pre-dialysis mix at or near the
apparent IC80. The final assay inhibitor concentrations used were: a at 100 nM, b at 50 nM, and c at
10 nM. Samples were pre-incubated with the enzyme (2 nM) for 3 h at room temperature (25 C) before
300 L was removed and assayed in triplicate in a FAAH activity assay. The remaining sample (2.7
mL) was injected into a dialysis cassette employing a 10,000 MW cutoff membrane. The mixture was
dialyzed against 1 L PBS at 4 C on a stir plate for 18 h. The post dialysis FAAH activity was assessed
by assaying 300 L samples taken from the dialysis cassettes in triplicate. FAAH activity is expressed
as a percentage of vehicle treated FAAH (DMSO alone).
Acknowledgements

This work was supported by the National Institutes of Health (DA015648, DK114785, DLB; DA037760,
BFC). We thank Aleksandar Radakovic and Jelena Momirov for the rFAAH Ki determinations reported
in Figures 14 and 15.
Supporting Information
Supplemental data [experimental procedures and characterization data for all tested compounds and
table summary of purity analysis of all tested compounds (Table S1)] to this article can be found online
at xxxxxx.
Competing interests
D.L.B. and B.F.C. have financial interests in Abide Therapeutics, which has interests in the
development of therapeutics derived from inhibition of serine hydrolases.
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