Discovery libraries targeting the major enzyme classes: The serine hydrolases

Article abstract of DOI:10.1016/j.bmcl.2014.06.063

Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subs

Full text of DOI:10.1016/j.bmcl.2014.06.063

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3807–3813
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery libraries targeting the major enzyme classes: The serine
hydrolases
⇑
Katerina Otrubova, Venkat Srinivasan, Dale L. Boger
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla CA 92037, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyra-
zoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors.
Within each library is a small but powerful subset of compounds that serve as a chemotype fragment
screening library capable of subsequent structural diversification. Elaboration of the pyrazole-based
ureas provided remarkably potent irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent
Ki = 100–200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based
ureas.
Received 7 June 2014
Revised 19 June 2014
Accepted 20 June 2014
Available online 27 June 2014
Keywords:
Serine hydrolase inhibitors
Enzyme inhibitors
Screening library
Ó 2014 Elsevier Ltd. All rights reserved.
Fatty acid amide hydrolase
The serine hydrolases constitute a superfamily of enzymes rep-
resenting more than 1% of the predicted proteins in the human
genome for which at least 40–50% are still uncharacterized, lacking
known roles or identified endogenous substrates.1 Not only are
they one of the largest and most diverse enzyme class in mammals,
but they perform crucial roles in many biological processes.1,2
There are approximately 240 human serine hydrolases composed
of a series of lipases, peptidases, esterases, thioesterases, and
amidases that hydrolyze small molecules, signaling lipids, peptides
or post-translational ester and thioester protein modifications.
They share a conserved mechanism that enlists a key active site
serine nucleophile within a catalytic triad that is activated by a
proton relay often involving an acidic (aspartate/glutamate) and
basic residue (histidine/lysine). More than half of the serine hydro-
lases (>120 enzymes) remain poorly annotated, with no described
physiological function or identified substrates, and an even greater
number of these enzymes (>80%) lack selective inhibitors to aid in
their characterization. In cases where a comprehensive under-
standing has been established, this has not only improved our fun-
damental understanding of biology, but it has also led to invaluable
new therapies to treat disease.1
synthesis of an extensive series of a
-ketoheterocycles5 (ca. 1000
compounds) many of which target fatty acid amide hydrolase
(FAAH),6 the efforts herein provide two libraries of modestly reac-
tive urea derivatives capable of irreversible7 serine hydrolase inhi-
bition by virtue of selective active site carbamoylation of the
catalytic serine.8 Such ureas, typically unreactive unless activated
for nucleophilic acylation within a serine hydrolase active site,
can be profiled against all serine hydrolases in the proteome9 with
confidence that they are not routinely reactive with enzymes or
proteins outside the serine hydrolase family.9,10 Because of their
irreversible mechanism of action, those that are sufficiently selec-
tive may serve as initial in vivo pharmacological probes to define
the function of an uncharacterized serine hydrolase,11 used to con-
fidently validate its potential as a therapeutic target, and optimized
into drug candidates themselves.1,12–15 First identified as a class of
inhibitors effective for selective and potent inhibition of FAAH,12–15
their utility for serine hydrolase inhibition requires a modestly
reactive urea bearing an amine capable of behaving as an effective
leaving group (e.g., electron-deficient aniline,13–15 tetrazole,16 tria-
zole,17 or imidazole17), was found often to be dependent on the
nature, size, and substitution (e.g., tertiary vs secondary) of the sec-
ond attached urea amine,13–15 and can be exquisitely responsive to
the recognition elements used to target the enzyme active site.
Opportunities to create such comprehensive screening libraries
for the serine hydrolases are enhanced with the availability and
continued refinement of activity-based protein profiling (ABPP)
proteomic screening technology9–11 that offer the advantage of
testing enzymes in their native state and eliminate the need for
their recombinant expression, purification, and the development
Herein, we report two distinct efforts designed to assemble a
powerful screening library targeting the serine hydrolases. As a
complement to our early ad hoc synthesis of a trifluoromethyl
ketone library (ca. 300 compounds),3 additional candidate inhibi-
tors bearing activated electrophilic carbonyls,4 and the more recent
⇑
Corresponding author. Tel.: +1 858 784 7522; fax: +1 858 784 7550.
E-mail address: boger@scripps.edu (D.L. Boger).
http://dx.doi.org/10.1016/j.bmcl.2014.06.063
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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K. Otrubova et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3807–3813
Scheme 2.
assess proteome-wide selectivity, provide a powerful paradigm
for discovery of selective chemical probes of new targets, for
optimization of an enzyme inhibitor selectivity concurrent with
target affinity,19 and for the detection, identification, and
characterization of new therapeutic targets.1,2
Figure 1. Aniline-based urea library of candidate serine hydrolase inhibitors.
The first of the screening libraries detailed herein constitutes a
693-membered library of activated ureas that systematically vary
the reactivity of an aniline leaving group (head group) as well as
the structure and flexibility of the additional urea amine (linking
of specific substrate assays. Because the inhibitors are screened
against many enzymes in the proteome in parallel, both their
potency and selectivity can be simultaneously evaluated.18,19 Such
libraries, in combination with use of this technology to rapidly
unit) that bears
a common resident functionalization site
Scheme 1.
Figure 2. The 21 acid chlorides, 40 carboxylic acids, and 16 sulfonyl chlorides used in the library.

K. Otrubova et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3807–3813
3809
lead identification using a solution-phase synthetic protocol for
the parallel synthesis of individual compounds where each inter-
mediate and final product is isolated and purified by a simple
acid/base liquid/liquid extraction,20 the library immediately
informs on the influence of the urea reactivity, the importance of
the linker and its rigidity or orientation, and the nature of the
active site recognition features.
Three leaving group amines were selected for incorporation into
the library: 2-aminopyridine (2-AP), 4-trifluoromethylaniline
(4-TFMA), and 2-fluoroaniline (2-FA). Both the former 2-AP13d
and the latter 2-FA14a,d have been examined in series irreversibly
targeting FAAH, whereas 4-TFMA appears unique to the studies
herein (Fig. 1, head group). A set of three linker domains were
incorporated as mono Boc protected derivatives of three symmet-
rical diamines: a six-membered cyclic diamine (6-CDA, piperazine)
widely used in past efforts,13–15 a seven-membered cyclic diamine
(7-CDA, 1,4-diazepine or homopiperazine), and an acyclic diamine
(ACDA, N,N0-dimethyl-1,2-diaminoethane), each of which bear a
protected amine spatially proximal within the series for
subsequent divergent functionalization (Fig. 1, linker group). The
nine individual combinations of the three head groups and three
linkers were assembled using one of two protocols (Scheme 1).
The first (method A) illustrated with 1, entailed treatment of
4-Boc-piperazine with carbonyldiimidazole (CDI, THF, 25 °C,
14 h) to provide the activated urea (93%) followed by MeI treat-
ment (4 equiv MeI, CH3CN, 25 °C, 24 h) to provide the N-methyl
imidazolium salt (quant.) activated for subsequent displacement.21
Its treatment with the lithium salt of 2-aminopyridine generated
in situ (n-BuLi, THF) provided 1 (55%). Alternatively and in a proce-
dure that proved more general and effective for all nine intermedi-
ates (method B), treatment of 2-AP (58%), 4-TFMA (97%), or 2-FA
(88%) with 2-propenyl chloroformate (N-methylmorpholine,
THF)22 provided the corresponding activated 2-propenyl
carbamates that undergo smooth urea formation upon reaction
with the mono Boc protected diamines (N-methylpyrrolidine,
THF, reflux, 57–70%).
Figure 3. Digested data (summed % inhibition) from the urea compound library
screening (at 5 M) against a panel of eight serine hydrolases in the proteome.
l
Each of the nine core structures (1–9) was Boc deprotected by
treatment with anhydrous HCl in dioxane (25 °C) and each crude
amine hydrochloride salt was subjected to coupling (30 mg/reac-
tion) with the identical series (Fig. 2) of 21 acid chlorides (2 equiv,
amenable to a final divergent introduction of a variable unit
capable of modulating active site recognition (Fig. 1). In addition
to providing a template that may be rapidly expanded following
Figure 4. N-Acyl pyrazole amides, carbamates, and ureas examined against FAAH.

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K. Otrubova et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3807–3813
sublibraries, each composed of 77 compounds (61 amides and 16
sulfonamides) (Scheme 2). Isolation and purification of the final
products was conducted by simple acid/base extraction of the
crude reaction mixture following aqueous workup for removal of
excess reagents, their byproducts, and unreacted starting materi-
als. Assessment of the 693-membered library by LCMS revealed
that nearly all expected products were generated (ca. random
5-10% failure rate in each sublibrary) and a subsequent chromato-
graphic purification of the individual compounds provided the
individual library members in purified amounts ranging from 1
to 33 mg (>95% purity).
In principle, the systematic variations in the candidate inhibi-
tors provide an initial SAR immediately following its screening that
informs the subsequent optimization of screening hits. Illustrating
this feature of the library, a small screening campaign with the
library (screened at 5 lM) was conducted against a modestly sized
serine hydrolase library to establish its utility. In addition to pro-
viding a distinct lead compound for inhibiting the individual serine
hydrolases, the informative and digested screening data (sum %
inhibition) for the library against the eight serine hydrolases is
summarized in Figure 3. Using as an example the serine hydrolase
ABHD6-2 that is upregulated in metastatic tumors, both the leav-
ing group aniline (2-FA > 4-TFMA > 2-AP) and the linker domain
(6-CDA > 7-CDA > ACDA) follow well-defined trends that can be
exploited immediately in ongoing second generation optimization
libraries directly following a screening campaign.
The second library evolved in a different manner and ultimately
resulted in a library designed to screen for minimal, more promis-
cuous serine hydrolase inhibitors capable of subsequent immedi-
ate lead optimization. In the course of efforts targeting FAAH,23,24
we examined a series of N-acyl pyrazole amides, carbamates and
ureas as candidate inhibitors of the enzyme. Precedent for their
potential utility was based on the reported behavior of related
azoles (tetrazoles,16 triazoles,17 imidazoles17, and indazoles17),
although we were not aware of detailed accounts of N-acyl pyra-
zole ureas or carbamates reacting with serine hydrolases.25 The
candidate acyl pyrazoles display an intrinsic modest reactivity
toward nucleophilic attack and represent an activating group capa-
ble of formation of a metal-chelated stable tetrahedral intermedi-
ate. Additionally, the pyrazoles represent a series in which its
acylating reactivity and leaving group propensity may be tuned
not only through the nature of the second acyl site (amide,
carbamate, urea), but also through symmetrical substitution of
the pyrazole with electron-withdrawing or electron-donating sub-
stituents. The importance and remarkable impact of these features
were examined and defined with a series of 111 acyl pyrazoles as
inhibitors of FAAH (Fig. 4), a collection in itself that represents a
useful serine hydrolase inhibitor screening library.
Figure 5. Recombinant rFAAH inhibition, apparent Ki (3 h preincubation with
enzyme).
Several key structural features emerged from examination of
the small library where the impact on FAAH inhibitory activity dis-
played clear pronounced effects, improving substantially as one
alters both the nature of the reacting acyl group (urea > carba-
mate > amide) and the pyrazole C4 substituent (CN > H > Me). This
is illustrated in Figure 5 with a representative series of increasingly
potent FAAH inhibitors, the most potent of which display apparent
Ki values of 100-200 pM when substituted with acyl chains known
to be especially effective for targeting FAAH.5 Two of these potent
series, the three pyrazole ureas bearing the naphthyl tail as well as
the three containing the benzyloxyphenyl tail, were each con-
firmed to be irreversible FAAH inhibitors (dialysis dilution, not
shown). Interestingly, it was the least reactive chemotype
(urea > carbamate > amide) that proved to provide the more potent
inhibitors, suggesting a unique FAAH active site activation for
carbamoylation.
Figure 6. C4-Substituent impact on pyrazole urea inhibition of FAAH.
10 equiv NaHCO3, 0.2 M in DMF, 0–25 °C, 12 h), 40 carboxylic acids
(1 equiv, 1.2 equiv EDCI, 1 equiv HOAt, 10 equiv NaHCO3, 0.2 M in
DMF, 25 °C, 24 h), and 16 arylsulfonyl chlorides (2 equiv, 10 equiv
NaHCO3, 0.2–0.4 M in DMF, 25 °C, 12 h) to provide nine
Especially interesting in the series was the unusually large and
remarkable impact of the pyrazole C4 substituent. Although this

K. Otrubova et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3807–3813
3811
Figure 7. Key members of a serine hydrolase screening library.
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Me
1.2
1
CN
0.8
0.6
0.4
0.2
0
Figure 8. Identification of serine hydrolase targets for 10 (top) and 12 (bottom) in the HeLa cell membrane protein fraction established by ABPP-SILAC.17a Cells were cultured
with each inhibitor at 20 M (or DMSO controls) for 4 h before isolation of the membrane proteins and analysis by ABPP-SILAC (two independent replicates, FP-biotin
probe9b,17a). Ratio light/heavy = 1, no inhibition; ratio = 0, complete inhibition. Only those serine hydrolases displaying some inhibition are shown.
l
effect could reflect both an electronic and steric impact of the
substituent on the activity, FAAH possess a large cytosolic port
adjacent to the active site capable of accommodating large substi-
tuted heterocycles,26 suggesting the impact is largely or solely due
to the electronic properties of the pyrazole C4 substituent. In order
to establish whether this truly reflects 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 the substituent size or polarity (Fig. 6). Beautifully, the ser-
ies 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 appar-
ent Ki values across the series spans a stunning 103-fold.
Notable in this work, the pyrazole ureas 10–12 that contain
only a simple piperidine 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 (Fig. 5). Rather, they and a small series of pyrazole ureas
including those containing pendant protected secondary amines
in the acyl chain may serve as effective screening compounds used
to identify chemotypes selective for inhibition of a subset of serine
hydrolases, being more promiscuous across the enzyme class
(10–21, Fig. 7).17a Combined with 1–9, 10–21 represent a small,
but powerful serine hydrolase screening library. Following detec-
tion of a targeted serine hydrolase inhibition, the informative
screening leads, which define trends in acyl urea reactivity (head
group) and the importance of the second attached amine (linker),
may be divergently modified to improve active site recognition
features, simultaneously improving potency and selectivity for a
targeted serine hydrolase.
Representative of such screening results and highlighted in
Fig. 8, the comparison of 10 and 12 screened at 20 lM against all
proteins in the HeLa cell membrane fraction indicate the more
selective nature of the least reactive pyrazole urea 10 and the more
sensitive nature of the most reactive pyrazole urea 12. Combined
their concurrent screening allows the identification of serine
hydrolases sensitive to inhibition by the pyrazole urea chemotype,
provide insights into how reactivity can impact selectivity among
the distinguished candidate serine hydrolases, define candidate
off target enzymes in the class whose activity should be monitored
when addressing optimization against a single subsequently cho-
sen enzyme, and provide superb starting fragments that can be fur-
ther elaborated to tailor the potency and selectivity of the inhibitor
class to a targeted serine hydrolase. Most notably and by using the
nitrile substituted pyrazole urea 12, serine hydrolases sensitive to
pyrazole urea inhibition are detected that would be overlooked in
such fragment screening if only the unsubstituted pyrazole 10 was
employed.
Complementary to our screening libraries comprehensively
targeting protein-protein29,31 or protein-DNA32 interactions, the
urea-based serine hydrolase libraries disclosed herein and the

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Chem. Lett. 2011, 21, 6538; (f) Meyers, J. M.; Long, A. S.; Pelc, J. M.; Wang, L. J.;
Bowen, J. S.; Schweitzer, A. B.; Wilcox, C. M.; McDonald, J.; Smith, E. S.; Foltin,
S.; Rumsey, J.; Yang, S. Y.; Walker, C. M.; Kamtekar, S.; Beidler, F.; Thorarensen,
A. Bioorg. Med. Chem. Lett. 2011, 21, 6545.
related libraries of candidate inhibitors bearing electrophilic car-
bonyls3,4 represent our first of several screening libraries assem-
bled to effectively target the major enzyme classes. The results of
these and related studies, as well as their utility will be reported
in due course.
14. (a) Keith, J. M.; Apocada, R.; Xiao, W.; Seierstad, M.; Pattabiraman, K.; Wu, J.;
Webb, M.; Karbarz, M. J.; Brown, S.; Wilson, S.; Scott, B.; Tham, C.-S.; Luo, L.;
Palmer, J.; Wennerholm, M.; Chaplan, S.; Breitenbucher, J. G. Bioorg. Med. Chem.
Lett. 2008, 18, 4838; (b) Karbarz, M. J.; Luo, L.; Chang, L.; Tham, C.-S.; Palmer, J.
A.; Wilson, S. J.; Wennerholm, M. L.; Brown, S. M.; Scott, B. P.; Apocada, R. L.;
Keith, J. M.; Wu, J.; Breitenbucher, J. G.; Chaplan, S. R.; Webb, M. Anesth. Analg.
2009, 108, 316; (c) Keith, J. M.; Apodaca, R.; Tichenor, M.; Xiao, W.; Jones, W.;
Pierce, J.; Seierstad, M.; Palmer, J.; Webb, M.; Karbarz, M.; Scott, B.; Wilson, S.;
Luo, L.; Wennerholm, M.; Chang, L.; Brown, S.; Rizzolio, M.; Rynberg, R.;
Chaplan, S.; Breitenbucher, J. G. ACS Med. Chem. Lett. 2012, 3, 823; (d) Tichenor,
M. S.; Keith, J. M.; Jones, W. M.; Pierce, J. M.; Merit, J.; Hawryluk, N.; Seierstad,
M.; Palmer, J. A.; Webb, M.; Karbarz, M. J.; Wilson, S. J.; Wennerholm, M. L.;
Woestenborghs, F.; Beerens, D.; Luo, L.; Brown, S. M.; De Boeck, M.; Chaplan, S.
R.; Breitenbucher, J. G. Bioorg. Med. Chem. Lett. 2012, 22, 7357.
15. (a) Kono, M.; Matsumoto, T.; Kawamura, T.; Nishimura, A.; Kiyota, Y.; Oki, H.;
Miyazaki, J.; Igaki, S.; Behnke, C. A.; Shimojo, M.; Kori, M. Bioorg. Med. Chem.
2013, 21, 28; (b) Kono, M.; Matsumoto, T.; Imaeda, T.; Kawamura, T.; Fujimoto,
S.; Kosugi, Y.; Odani, O.; Shimizu, Y.; Matsui, H.; Shimojo, M.; Kori, M. Bioorg.
Med. Chem. 2014, 22, 1468.
16. Moore, S. A.; Nomikos, G. G.; Dickason-Chesterfield, A. K.; Sohober, D. A.;
Schaus, J. M.; Ying, B. P.; Xu, Y. C.; Phebus, L.; Simmons, R. M.; Li, D.; Iyengar, S.;
Felder, C. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17852.
Acknowledgement
This work was supported by the National Institutes of Health
(DA015648). We thank Landon Whitby for digestion of the screen-
ing results obtained with the aniline urea library and William Par-
sons and Professor B. F. Cravatt for assistance with the ABPP-SILAC.
Supplementary data
Supplementary data (full details of the synthesis, characteriza-
tion and purity of the screening library) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2014.06.063.
17. (a) Adibekian, A.; Martin, B. R.; Wang, C.; Hsu, K. L.; Bachovchin, D. A.; Niessen,
S.; Hoover, H.; Cravatt, B. F. Nat. Chem. Biol. 2011, 7, 469; (b) Morera, L.; Labar,
G.; Ortar, G.; Lambert, D. M. Bioorg. Med. Chem. 2012, 20, 6260; (c) Aaltonen, N.;
Savinainen, J. R.; Ribas, C. R.; Ronkko, J.; Kuusisto, A.; Korhonen, J.; Navia-
Paldanius, D.; Hayrinen, J.; Takabe, P.; Kasnanen, H.; Pantsar, T.; Laitinen, T.;
Lehtonen, M.; Pasonen-Seppanen, S.; Poso, A.; Nevalainen, T.; Laitinen, J. T.
Chem. Biol. 2013, 20, 379; (d) Ebdrup, S.; Sorensen, L. G.; Olsen, O. H.; Jacobsen,
P. J. Med. Chem. 2004, 47, 400; (e) Crocetti, L.; Schepetkin, I. A.; Cilibrizzi, A.;
Graziano, A.; Vergelli, C.; Giomi, D.; Khlebnikov, A. I.; Quinn, M. T.; Giovannoni,
M. P. J. Med. Chem. 2013, 56, 6259.
18. Leung, D.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Nat. Biotechnol. 2003, 21, 687.
19. Leung, D.; Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.; Cravatt, B. F.; Boger, D. L.
Bioorg. Med. Chem. Lett. 2005, 15, 1423.
20. (a) Cheng, S.; Comer, D. D.; Williams, J. P.; Boger, D. L. J. Am. Chem. Soc. 1996,
118, 2567; (b) Boger, D. L.; Tarby, C. M.; Caporale, L. H. J. Am. Chem. Soc. 1996,
118, 2109; (c) Cheng, S.; Tarby, C. M.; Comer, D. D.; Williams, J. P.; Caporale, L.
H.; Boger, D. L. Bioorg. Med. Chem. 1996, 4, 727.
21. Saha, A. K.; Schultz, P.; Rapoport, H. J. Am. Chem. Soc. 1989, 111, 4856.
22. Jouin, P.; Castro, B.; Zeggaf, A.; Pantaloni, A.; Senet, J. P.; Lecolier, S.; Sennyey, G.
Tetrahedron Lett. 1987, 28, 1661.
23. (a) Cravatt, B. F.; Lichtman, A. H. Curr. Opin. Chem. Biol. 2003, 7, 469; (b) Ahn, K.;
Johnson, D. S.; Cravatt, B. F. Exp. Opin. Drug Discov. 2009, 4, 763.
24. (a) Cravatt, B. F.; Giang, D. K.; Mayfield, S. P.; Boger, D. L.; Lerner, R. A.; Gilula, N.
B. Nature 1996, 384, 83; (b) Giang, D. K.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A.
1997, 94, 2238; (c) Cravatt, B. F.; Prospero-Garcia, O.; Suizdak, G.; Gilula, N. B.;
Henriksen, S. J.; Boger, D. L.; Lerner, R. A. Science 1995, 268, 1506.
25. (a) Lone, A. M.; Bachovchin, D. A.; Westwood, D. B.; Speers, A. E.; Spicer, T. P.;
Fernandez-Vega, V.; Chase, P.; Hodder, P. S.; Rosen, H.; Cravatt, B. F.;
Saghatelian, A. J. Am. Chem. Soc. 2011, 133, 11665; (b) Chang, J. W.; Cognetta,
A. B., III; Niphakis, M. J.; Cravatt, B. F. ACS Chem. Biol. 2013, 8, 1590; (c)
Blankman, J. L.; Cravatt, B. F. Pharmacol. Rev. 2013, 65, 849; For N-acyl pyrazole
amides, see: (d) Schepetkin, I. A.; Khlebnikov, A. I.; Quinn, M. T. J. Med. Chem.
2007, 50, 4928.
26. (a) Bracey, M. H.; Hanson, M. A.; Masuda, K. R.; Stevens, R. C.; Cravatt, B. F. Science
2002, 298, 1793; (b) Mileni, M.; Johnson, D. S.; Wang, Z.; Everdeen, D. S.; Liimatta,
M.; Pabst, B.; Bhattacharya, K.; Nugent, R. A.; Kamtekar, S.; Cravatt, B. F.; Ahn, K.;
Stevens, R. C. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 12820; (c) Mileni, M.;
Garfunkle, J.; DeMartino, J. K.; Cravatt, B. F.; Boger, D. L.; Stevens, R. C. J. Am. Chem.
Soc. 2009, 131, 10497; (d) Mileni, M.; Garfunkle, J.; Kimball, F. S.; Cravatt, B. F.;
Stevens, R. C.; Boger, D. L. J. Med. Chem. 2010, 53, 230; (e) Mileni, M.; Garfunkle, J.;
Ezzili, C.; Cravatt, B. F.; Stevens, R. C.; Boger, D. L. J. Am. Chem. Soc. 2011, 133, 4092.
27. (a) Boger, D. L.; Henriksen, S. J.; Cravatt, B. F. Curr. Pharm. Des. 1998, 4, 303; (b)
Cravatt, B. F.; Lerner, R. A.; Boger, D. L. J. Am. Chem. Soc. 1996, 118, 580; (c)
Lerner, R. A.; Siuzdak, G.; Prospero-Garcia, O.; Henriksen, S. J.; Boger, D. L.;
Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 9505; (d) Devane, W. A.;
Hanus, L.; Breuer, A.; Pertwee, R. G.; Stevenson, L. A.; Griffin, G.; Gibson, D.;
Mandelbaum, A.; Etinger, A.; Mechoulam, R. Science 1992, 258, 1946.
28. (a) Boger, D. L.; Fecik, R. A.; Patterson, J. E.; Miyauchi, H.; Patricelli, M. P.;
Cravatt, B. F. Bioorg. Med. Chem. Lett. 2000, 10, 2613; (b) Ezzili, C.; Otrubova, K.;
Boger, D. L. Bioorg. Med. Chem. Lett. 2010, 20, 5959.
29. (a) Boger, D. L.; Desharnais, J.; Capps, K. Angew. Chem., Int. Ed. 2003, 42, 4138;
(b) Whitby, L. R.; Boger, D. L. Acc. Chem. Res. 2012, 45, 1698.
30. (a) Whitby, L. R.; Ando, Y.; Setola, V.; Vogt, P. K.; Roth, B. L.; Boger, D. L. J. Am.
Chem. Soc. 2011, 133, 10184; (b) Shaginian, A.; Whitby, L. R.; Hong, S.; Hwang,
I.; Farooqi, B.; Searcey, M.; Chen, J. C.; Vogt, P. K.; Boger, D. L. J. Am. Chem. Soc.
2009, 131, 5564.
31. (a) Whitby, L. R.; Boyle, K. E.; Cai, L.; Yu, X.; Gochin, M.; Boger, D. L. Bioorg. Med.
Chem. Lett. 2012, 22, 2861; (b) Ambrus, G.; Whitby, L. R.; Singer, E. L.; Trott, O.;
Choi, E.; Olson, A. J.; Boger, D. L.; Gerace, L. Bioorg. Med. Chem. 2010, 18, 7611;
(c) Chang, M. W.; Giffin, M. J.; Muller, R.; Savage, J.; Lin, Y. C.; Hong, S.; Jin, W.;
References and notes
1. Bachovchin, D. A.; Cravatt, B. F. Nat. Drug Disc. Rev. 2012, 11, 52.
2. Long, J. Z.; Cravatt, B. F. Chem. Rev. 2011, 111, 6022.
3. Boger, D. L.; Sato, H.; Lerner, A. E.; Austin, B. J.; Patterson, J. E.; Patricelli, M. P.;
Cravatt, B. F. Bioorg. Med. Chem. Lett. 1999, 9, 265.
4. (a) Patterson, J. E.; Ollmann, I. R.; Cravatt, B. F.; Boger, D. L.; Wong, C.-H.; Lerner,
R. A. J. Am. Chem. Soc. 1996, 118, 5938; (b) Patricelli, M. P.; Patterson, J. P.;
Boger, D. L.; Cravatt, B. F. Bioorg. Med. Chem. Lett. 1998, 8, 613.
5. (a) Boger, D. L.; Sato, H.; Lerner, A. E.; Hedrick, M. P.; Fecik, R. A.; Miyauchi, H.;
Wilkie, G. D.; Austin, B. J.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 5044; (b) Boger, D. L.; Miyauchi, H.; Hedrick, M. P. Bioorg. Med.
Chem. Lett. 2001, 11, 1517; (c) Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.;
Fecik, R. A.; Cheng, H.; Hwang, I.; Hedrick, M. P.; Leung, D.; Acevedo, O.;
Guimarães, C. R. W.; Jorgensen, W. L.; Cravatt, B. F. J. Med. Chem. 2005, 48, 1849;
(d) Romero, F. A.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2006, 68, 14004; (e)
Boger, D. L.; Miyauchi, H.; Hedrick, M. P.; Romero, F. A.; Du, W.; Hwang, I.; Rayl,
T. J.; Kimball, F. S.; Leung, D.; Hoover, H. S.; Apodaca, R. L.; Breitenbucher, J. G.;
Cravatt, B. F.; Boger, D. L. J. Med. Chem. 2007, 50, 1058; (f) Hardouin, C.; Kelso,
M. J.; Romero, F. A.; Rayl, T. J.; Leung, D.; Hwang, I.; Cravatt, B. F.; Boger, D. L. J.
Med. Chem. 2007, 50, 3359; (g) Kimball, F. S.; Romero, F. A.; Ezzili, C.; Garfunkle,
J.; Rayl, T. J.; Hochstatter, D. G.; Hwang, I.; Boger, D. L. J. Med. Chem. 2008, 51,
937; (h) Garfunkle, J.; Ezzili, C.; Rayl, T. J.; Hochstatter, D. G.; Hwang, I.; Boger,
D. L. J. Med. Chem. 2008, 51, 4392; (i) DeMartino, J. K.; Garfunkle, J.; Hochstatter,
D. G.; Cravatt, B. F.; Boger, D. L. Bioorg. Med. Chem. Lett. 2008, 18, 5842; (j) Ezzili,
C.; Mileni, M.; McGlinckey, N.; Long, J. Z.; Kinsey, S. G.; Hochstatter, D. G.;
Stevens, R. C.; Lichtman, A. H.; Cravatt, B. F.; Bilsky, E. J.; Boger, D. L. J. Med.
Chem. 2011, 54, 2805; (k) Otrubova, K.; Brown, M.; McCormick, M. S.; Han, G.
W.; O’Neal, S. T.; Cravatt, B. F.; Stevens, R. C.; Lichtman, A. H.; Boger, D. L. J. Am.
Chem. Soc. 2013, 135, 6289; (l) Otrubova, K.; Cravatt, B. F.; Boger, D. L. J. Med.
Chem. 2014, 57, 1079; (m) Duncan, K. K.; Otrubova, K. Bioorg. Med. Chem. 2014,
22, 2763.
6. Otrubova, K.; Boger, D. L. ACS Chem. Neurosci. 2012, 3, 340.
7. Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Chem. Rev. 2002, 102, 4639.
8. (a) Alexander, J. P.; Cravatt, B. F. J. Am. Chem. Soc. 2006, 128, 9699; (b)
Alexander, J.; Cravatt, B. F. Chem. Biol. 2005, 12, 1179.
9. (a) Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
14694; (b) Patricelli, M. P.; Cravatt, B. F. Biochemistry 1999, 38, 14125.
10. (a) Barglow, K. T.; Cravatt, B. F. Nat. Methods 2007, 4, 822; (b) Evans, M. J.;
Cravatt, B. F. Chem. Rev. 2006, 106, 3279.
11. (a) Li, W.; Blankman, J. L.; Cravatt, B. F. J. Am. Chem. Soc. 2007, 129, 9594; (b)
Chiang, K. P.; Niessen, S.; Saghatelian, A.; Cravatt, B. F. Chem. Biol. 2006, 13,
1041.
12. Otrubova, K.; Ezzili, C.; Boger, D. L. Bioorg. Med. Chem. Lett. 2011, 21, 4674.
13. (a) Ahn, K.; Johnson, D. S.; Fitzgerald, L. R.; Liimatta, M.; Arendse, A.; Stevenson,
T.; Lund, E. T.; Nugent, R. A.; Normanbhoy, T.; Alexander, J. P.; Cravatt, B. F.
Biochemistry 2007, 46, 13019; (b) Johnson, D. S.; Ahn, K.; Kesten, S.; Lazerwith,
S. E.; Song, Y.; Morris, M.; Fay, L.; Gregory, T.; Stiff, C.; Dunbar, J. B., Jr.; Liimatta,
M.; Beidler, D.; Smtih, S.; Nomanbhoy, T. K.; Cravatt, B. F. Bioorg. Med. Chem.
Lett. 2009, 19, 2865; (c) 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. Chem. Biol. 2009, 16, 411; (d)
Johnson, D. S.; Stiff, C.; Lazerwith, S. E.; Kesten, S. R.; Fay, L. K.; Morris, M.;
Beidler, D.; Liimatta, M. B.; Smith, S. E.; Dudley, D. T.; Sadagopan, N.;
Bhattachar, S. N.; Kesten, S. J.; Nomanbhoy, T. K.; Cravatt, B. F.; Ahn, K. ACS
Med. Chem. Lett. 2011, 2, 91; (e) Meyers, J. M.; Long, A. S.; Pelc, J. M.; Wang, L. J.;
Bowen, J. S.; Walker, C. M.; Schweitzer, A. B.; Madsen, M. H.; Tenbrink, E. R.;
McDonald, J.; Smith, E. S.; Foltin, S.; Beidler, F.; Thorarensen, A. Bioorg. Med.

K. Otrubova et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3807–3813
3813
Whitby, L. R.; Elder, J. H.; Boger, D. L.; Torbett, B. E. Biochem. J. 2010, 429, 527;
(d) Lee, A. M.; Rojek, J. M.; Spiropoulou, C. F.; Gundersen, A. T.; Jin, W.;
Shaginian, A.; York, J.; Nunberg, J. H.; Boger, D. L.; Oldstone, M. B. A.; Kunz, S. J.
Biol. Chem. 2008, 283, 18734; (e) Whitby, L. R.; Lee, A. M.; Kunz, S.; Oldstone, M.
B. A.; Boger, D. L. Bioorg. Med. Chem. Lett. 2009, 19, 3771; (f) Thomas, C. J.;
Casquilho, H. E.; York, J.; DeCamp, D. L.; Dai, D.; Petrilli, E. B.; Boger, D. L.;
Slayden, R. A.; Amberg, S. M.; Sprang, S. R.; Nunberg, J. H. J. Biol. Chem. 2011,
286, 6192; (g) Eubanks, L. M.; Hixon, M. S.; Jin, W.; Hong, S.; Clancy, C. M.;
Tepp, W. H.; Malizio, C. J.; Goodnough, M. C.; Barbieri, J. T.; Johnson, E. A.;
Boger, D. L.; Dickerson, T. J.; Janda, K. D. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
2602; (h) Capps, K. J.; Humiston, J.; Dominique, R.; Hwang, I.; Boger, D. L.
Bioorg. Med. Chem. Lett. 2005, 15, 2840; (i) Ambroise, Y.; Yuspan, B.; Ginsberg,
M. H.; Boger, D. L. Chem. Biol. 2002, 9, 1219; (j) Goldberg, J.; Jin, Q.; Ambroise,
Y.; Satoh, S.; Desharnais, J.; Capps, K.; Boger, D. L. J. Am. Chem. Soc. 2002, 124,
544; (h) Berg, T.; Cohen, S. B.; Desharnais, J.; Sonderegger, C.; Maslyar, D. J.;
Goldberg, J.; Boger, D. L.; Vogt, P. K. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 3830;
(k) Shi, J.; Stover, J. S.; Whitby, L. R.; Vogt, P. K.; Boger, D. L. Bioorg. Med. Chem.
Lett. 2009, 19, 6038; (l) Silletti, S.; Kessler, T.; Goldberg, J.; Boger, D. L.; Cheresh,
D. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 119; (m) Boger, D. L.; Goldberg, J.;
Silletti, S.; Kessler, T.; Cheresh, D. A. J. Am. Chem. Soc. 2001, 123, 1280; (n)
Boger, D. L.; Goldberg, J.; Satoh, S.; Ambroise, Y.; Cohen, S. B.; Vogt, P. K. Helv.
Chim. Acta 2000, 83, 1825.
32. (a) Stover, J. S.; Shi, J.; Jin, W.; Vogt, P. K.; Boger, D. L. J. Am. Chem. Soc. 2009, 131,
3342; (b) Boger, D. L.; Fink, B. E.; Hedrick, M. P. J. Am. Chem. Soc. 2000, 122,
6382; (c) Boger, D. L.; Fink, B. F.; Brunette, S. R.; Tse, W.; Hedrick, M. P. J. Am.
Chem. Soc. 2001, 123, 5878; (d) Tse, W.; Boger, D. L. Acc. Chem. Res. 2004, 37, 61;
(e) Hauschild, K. E.; Stover, J. S.; Boger, D. L.; Ansari, A. Z. Bioorg. Med. Chem. Lett.
2009, 19, 3779.