Revisiting 1,3,4-Oxadiazol-2-ones: Utilization in the Development of ABHD6 Inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.08.030

This article describes our systematic approach to exploring the utility of the 1,3,4-oxadiazol-2-one scaffold in the development of ABHD6 inhibitors. Compound 3-(3-aminobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (JZP-169, 52) was identified as a potent i

Full text of DOI:10.1016/j.bmc.2015.08.030

Bioorganic & Medicinal Chemistry 23 (2015) 6335–6345
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Revisiting 1,3,4-Oxadiazol-2-ones: Utilization in the Development
of ABHD6 Inhibitors
a
a
Jayendra Z. Patel a, , John van Bruchem , Tuomo Laitinen , Agnieszka A. Kaczor a,b, Dina Navia-Paldanius c,
⇑
Teija Parkkari a, Juha R. Savinainen c, Jarmo T. Laitinen c, Tapio J. Nevalainen a
a School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland
b Department of Synthesis and Chemical Technology of Pharmaceutical Substances, Faculty of Pharmacy with Division of Medical Analytics, Medical University of Lublin, 4a Chodzki
St., PL-20093 Lublin, Poland
c School of Medicine, Institute of Biomedicine/Physiology, University of Eastern Finland, PO Box 1627, FIN-70211 Kuopio, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
This article describes our systematic approach to exploring the utility of the 1,3,4-oxadiazol-2-one scaf-
fold in the development of ABHD6 inhibitors. Compound 3-(3-aminobenzyl)-5-methoxy-1,3,4-oxadiazol-
2(3H)-one (JZP-169, 52) was identified as a potent inhibitor of hABHD6, with an IC50 value of 216 nM.
Received 17 June 2015
Revised 6 August 2015
Accepted 25 August 2015
Available online 28 August 2015
This compound at 10
FAAH, MAGL and ABHD12, or bind to the cannabinoid receptors (CB1 and CB2). Moreover, in competitive
activity-based protein profiling (ABPP), compound 52 (JZP-169) at 10 M selectively targeted ABHD6 of
lM concentration did not inhibit any other endocannabinoid hydrolases, such as
l
Dedicated to Dr. Preeti Raval, principal
scientist at Zydus Research Centre (ZRC,
India).
the serine hydrolases of mouse brain membrane proteome. Reversibility studies indicated that compound
52 inhibited hABHD6 in an irreversible manner. Finally, homology modelling and molecular docking
studies were used to gain insights into the binding of compound 52 to the active site of hABHD6.
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
a/b-Hydrolase domain-containing 6
2-Arachidonoylglycerol
Monoacylglycerol lipase
Fatty acid amide hydrolase
1,3,4-Oxadiazol-2-ones
1. Introduction
(MAGL) and a/b-hydrolase domain-containing 12 (ABHD12) which
participate in 2-AG degradation, with the former enzyme account-
ing for the majority, 85%, and the latter for 9% of the hydrolytic
activity, with the remainder of the degradation (ꢀ2%) being carried
out by other enzymes, including fatty acid amide hydrolase
(FAAH).1,4 It is believed that ABHD6, MAGL and ABHD12 have dif-
ferent tissue distributions and subcellular localizations, giving rise
to the possibility that all of these hydrolases have their own dis-
tinct roles in controlling the duration of action and fate of 2-AG.1
It would be advantageous to develop selective ABHD6 inhibitors
since these could help in dissecting the roles of these enzymes
and obtaining an in-depth understanding of their physiological
significance.
Several recent studies have identified ABHD6 as an emerging
therapeutic target for the treatment of inflammation, metabolic
disorders (obesity and type II diabetes mellitus) and epilepsy.5–8
Moreover, it has been speculated that ABHD6 inhibitors may offer
certain advantages over inhibitors of MAGL and ABHD12. Firstly,
both a global genetic inactivation of MAGL and a chronic treatment
with irreversibly acting MAGL inhibitors have evoked a substantial
The a/b-hydrolase domain-containing 6 (ABHD6) is an integral
membrane serine hydrolase, accounting for ꢀ4% of bulk 2-arachi-
donoylglycerol (2-AG) degradation in the central nervous system
(CNS).1 2-AG is an endogenous cannabinoid ligand (endocannabi-
noid) which acts as a full agonist at the cannabinoid receptors
CB1 and CB2.2,3 In addition to ABHD6, there are two additional ser-
ine hydrolases in mouse brain tissue, monoacylglycerol lipase
Abbreviations: 2-AG, 2-arachidonoylglycerol; hABHD6, human recombinant
hydrolase domain 6; hMAGL, human recombinant monoacylglycerol lipase; hFAAH,
human recombinant fatty acid amide hydrolase; hABHD12, human recombinant
a/b
a
/
b hydrolase domain 12; WWL70, N-methyl-N-[[3-(4-pyridinyl)phenyl]-methyl]-40-
(aminocarbonyl)[1,10-biphenyl]-4-yl carbamic acid ester; JZP-327A, S-3-(1-(4-
isobutylphenyl)ethyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one; JZP-430, 4-mor-
pholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate; JZP-361, (4-(8-chloro-
5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11(6H)-ylidene)piperidin-1-yl)(1H-1,2,4-
triazol-1-yl)methanone; THL, tetrahydrolipstatin (orlistat); PE, petroleum ether;
EtOAc, ethyl acetate; DCM, dichloromethane.
⇑
Corresponding author. Tel.: +358 40 355 3887.
E-mail addresses: jayendra.patel@uef.fi, jayorgchem137@gmail.com (J.Z. Patel).
http://dx.doi.org/10.1016/j.bmc.2015.08.030
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

6336
J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
increase in brain 2-AG levels, triggering psychotropic side effects
and cannabinoid receptor desensitization.9–11 Secondly, genetically
ABHD12 deficient mice display age-dependent symptoms that
resemble a serious human neurodegenerative disorder, PHARC
(polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and
cataract).12 In contrast, inhibition of ABHD6 causes only a marginal
increase in brain 2-AG levels, evidence that ABHD6 inhibitors are
likely to possess less CNS-related side-effects.4,6,13
Figure 2. Advancement in 1,3,4-oxadiazol-2-ones (I–III).
At present, only a limited number of ABHD6 inhibitors have
been described (Fig. 1). Cravatt’s group prepared the first potent
and selective carbamate-based ABHD6 inhibitor, WWL70 (1) using
activity-based protein profiling (ABPP).14 Marrs and colleagues
synthesized UCM710 (2) and characterized it as a dual inhibitor
of ABHD6 and FAAH.15 Methylarachidonoyl fluorophosphonate
(MAFP), orlistat (tetrahydrolipstatin, THL), RHC-80267, and the
triterpene pristimerin have been identified as non-selective ABHD6
inhibitors.16 Recently, Cravatt and coworkers noted that a carba-
mate-based compound, WWL123 (3), as well as triazole urea ana-
logues such as KT195 (4) and KT182 (5), were potent and selective
ABHD6 inhibitors.17–19 Very recently, Janssen et al. revealed the
glycine sulfonamide-based analogue LEI-106 (6) to be a highly
towards mouse brain membrane serine hydrolases and the inhibi-
tory data have been used to make a preliminary exploration of the
structure–activity relationships (SARs). Finally, the reversibility of
the synthesized compounds has been evaluated by both experi-
mental approaches and molecular modelling.
2. Chemistry
The syntheses of 3-benzyl-1,3,4-oxadiazol-2-ones (38–41, 43–
45 and 49) have been reported in our earlier reports.29,30 By utiliz-
ing the same procedure, several other analogues (42, 48, 50, 60–69
and 71, Scheme 1) were also prepared by condensation of the
appropriate aldehyde (8–21) with a suitable carbazate to produce
a Schiff’s base which was reduced to form the hydrazine carboxy-
late derivative (22, 25–37) and subsequently cyclized with phos-
gene into 3-benzyl-1,3,4-oxadiazol-2-ones (42, 48, 50, 60–69 and
71). Nitro containing 1,3,4-oxadiazol-2-ones (48–50, 69 and 71)
which had been obtained through Scheme 1 were reduced by
10% Pd/C to the corresponding amino derivatives (51–53, 70 and
72, respectively, Scheme 1). Selected amino derivatives (51–53)
were coupled with either acetyl or benzoyl chloride to produce
the desired acetamides (54–56) and benzamides (57–59), respec-
tively (Scheme 1). For the naphthalene analogues (46 and 47),
the appropriate naphthaldehyde was used as the starting material
as shown in Scheme 2. The synthesis of hydrazine carboxylate
derivatives (22–37) was performed as according to published pro-
cedures with minor modifications (see Supporting information).
selective dual inhibitor of sn-1-diacylglycerol lipase
a (DAGL-a)
and ABHD6.20 In addition, we recently identified the 1,2,5-thiadia-
zole carbamate-based compound JZP-430 (7) as a potent and selec-
tive ABHD6 inhibitor and based on ABPP, it displayed good
selectivity over other serine hydrolases in the mouse brain mem-
brane proteome.21
Several research groups, including ours, have now described the
exploitation of the 1,3,4-oxadiazol-2-one scaffold (Fig. 2, general
structure I and II) in the development of serine hydrolases
inhibitors.22–30 For example, our chiral 1,3,4-oxadiazol-2-one
(Fig. 2, general structure II) based compound S-3-(1-(4-isobutylphe-
nyl)ethyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (JZP-327A) was
demonstrated to be a potent and reversible FAAH inhibitor.29 In
the same report, we also demonstrated that removal of the methyl
group from the 3-position of 1,3,4-oxadiazol-2-ones led to the
identification of 3-benzyl-1,3,4-oxadiazol-2-ones (Fig. 2, general
structure III) which were dual FAAH–MAGL inhibitors. From the
1,3,4-oxadiazol-2-ones (II and III), we selected our previously
reported compound 38 (see Table 1) for further optimization since
3. Results and discussion
3.1. Structure–activity relationships (SARs)
it achieved ꢀ40% inhibition of hABHD6 at a concentration of 1
but importantly no inhibition of hFAAH or hMAGL was evident at
10
M.29
Here, building on our prior experience with the 1,3,4-oxadiazol-
lM
The inhibitory activities of the synthesized compounds were
l
initially screened at a concentration of 1
lM against hABHD6 and
hABHD12, and at 10 M against hFAAH and hMAGL. The effects
l
2-ones, we have now conducted a systematic structural modifica-
tion of 3-benzyl-1,3,4-oxadiazol-2-ones and report activity data for
these compounds against the endocannabinoid hydrolases
(ABHD6, FAAH, MAGL and ABHD12). In addition, we evaluated
their selectivity towards the cannabinoid receptors as well as
on hABHD6, hFAAH and hMAGL are presented in Tables 1 and 2,
while those on hABHD12 appear in Table S2 (see Supplementary
information).
As shown in Table 1, the unsubstituted analogue 38 displayed
no inhibitory activity against either FAAH or MAGL. However, at
a concentration of 1
l
M, analogue 38 inhibited ABHD6 by ꢀ40%.
Analogues with bulky substituents on the para position of the ben-
zyl moiety, such as isobutyl (compound 39), Ph (compound 40),
and OPh (compound 43), exhibited dual FAAH–MAGL inhibition.
Switching the substituent from the para (compounds 40 and 43)
to the meta position (compounds 41 and 44) resulted in a reduction
in FAAH inhibitory activity. However, this change resulted in a
complete loss of MAGL inhibition. Similarly, switching of the para
substituents (compounds 40 and 43) to the ortho position (com-
pounds 42 and 45) led to a marginal reduction, or even a complete
loss of FAAH and/or MAGL inhibition. Among the analogues 38–45,
compounds 43–45 with a bulky OPh group only weakly (20–40%)
inhibited ABHD6 at 1 lM concentration. From the bicyclic naph-
thalene analogues, the 1-naphthyl analogue 46 was inactive
towards FAAH while the 2-naphthyl analogue 47 was a weak FAAH
inhibitor (IC50 3.98 lM). However, both analogues were inactive
Figure 1. Representative structures of selective and non-selective ABHD6 inhibi-
tors (1–7).

J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
6337
Scheme 1. Synthesis of 1,3,4-oxadiazol-2-ones 42, 48, 50–72. Reagents and conditions: (a) NH2NHCOOR,2 MeOH, 4 Å molecular sieves, reflux, 2–16 h; (b) NaBH3CN, MeOH,
methanolic HCl, 0–25 °C, 24–72 h; (c) COCl2, CH2Cl2, pyridine, 0–25 °C, 2–16 h (for compounds 60 and 61, the reaction was carried out at ꢁ40 °C, 10 min); (d) 10% Pd/C, H2
(atm), 20–25 °C, 4–8 h, MeOH; (e) MeCOCl, DCM, Et3N, 0–25 °C, 16–24 h; (f) PhCOCl, DCM, Et3N, 0–25 °C, 16–24 h.
and MAGL inhibition for the para-substituted analogue 59 over
its meta (compound 58) and ortho (compound 57) counterparts,
in line with previous findings with the bulky para-substituted
compounds 40 and 43. As expected, analogues 60 and 61, possess-
ing bulky benzyloxy substituents at their meta and para positions
respectively, displayed improved abilities to inhibit both FAAH
and MAGL. Moreover, the meta-benzyloxy analogue (compound
60) inhibited ABHD6 by nearly 50% at a concentration of 1 lM.
Scheme 2. Synthesis of 1,3,4-oxadiazol-2-ones 46 and 47. Reagents and conditions:
(a) NH2NHCOOMe, MeOH, 4 Å molecular sieves, 3–4 drops AcOH, reflux, 2–16 h; (b)
NaBH3CN, MeOH, methanolic HCl, 0–25 °C, 24–72 h; (c) COCl2, CH2Cl2, pyridine,
0–25 °C, 2–16 h.
The majority of the analogues having either smaller EWGs at either
meta (compounds 62, 64, 66 and 67) or para positions (compounds
63 and 65) or smaller ERG at the meta position (compound 68)
were found to be inactive towards FAAH and MAGL although ana-
logue 65 which had a para substituted EWG did exhibit weak FAAH
inhibition. Moreover, among these analogues, only meta-substi-
tuted analogues 62, 67 and 68 appreciably inhibited ABHD6
whereas compounds 64 and 66 inhibited ABHD6 in the sub-micro-
towards MAGL while only the 1-naphthyl analogue weakly inhib-
ited ABHD6 at 1 lM concentration. Since no significant improve-
ments in ABHD6 inhibitory activity or selectivity over FAAH or
MAGL were obtained with the bicyclic analogues 46 and 47, we
shifted our focus to study the electronic effect on unsubstituted
analogue 38.
molar range with IC50 values of 0.45 and 0.87 lM, respectively.
With respect to the analogues with a smaller electron with-
drawing group (EWG) nitro (compounds 48–50), and for those ana-
logues with an electron releasing group (ERG) amino (compounds
51–53), it was found that substituents at different positions on the
benzyl moiety present at the 3-position of 1,3,4-oxadiazol-2-ones
were inactive towards FAAH and MAGL. However, ortho- and
meta-NO2 substituted analogues (compounds 48 and 49) showed
As shown in Table 2, replacement of the methoxy group
present at the 5-position of 1,3,4-oxadiazol-2-ones in compounds
49 and 52 with an ethoxy group (compounds 69 and 70) resulted
in the loss of inhibitory activities towards all studied enzymes,
with the sole exception being compound 69 which at 1 lM con-
centration weakly inhibited ABHD6. However, when a phenoxy
group was incorporated (compounds 71 and 72), the compounds
inhibited FAAH weakly and MAGL moderately whereas they
weak ABHD6 inhibition at a concentration of 1 lM while the
meta-amino analogue 52 showed significant ABHD6 inhibition
with an IC50 value of 216 nM. Protection of the amino group in
compounds 51–53 with an acetyl group (compounds 54–56)
achieved no improvement of FAAH and MAGL inhibition. The loss
of ABHD6 inhibition seen with the meta-substituted compound
55 revealed the importance of the free amino group of compound
52. In contrast, protection with the bulky benzoyl group of com-
pounds 51–53 (compounds 57–59) resulted in enhanced FAAH
potently inhibited ABHD6 (IC50 values of 0.13 and 0.073 lM,
respectively). Overall, analogues with a bulky or smaller meta
substituent displayed weak to considerable ABHD6 inhibition;
among these, amino analogues 52 and 72 in particular showed
much improved ABHD6 inhibition (see Tables 1 and 2). Finally,
we further tested compound 52 at
(20 M) to obtain an indication of selectivity towards putative
significant off-targets, FAAH and MAGL. Compound 52 still
a higher concentration
l

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J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
Table 1
Inhibitory activities of 1,3,4-oxadiazol-2-ones 38–68⁄ towards FAAH, MAGL and ABHD6
Compd
R1
pIC50 SEM [IC50
,
l
M]a
% inhibition at 10
l
Mb
% remaining enzymatic ABHD6 activity at 1
hABHD6
l
Mc
hFAAH
hMAGL
d
38
39
40
H
NI
NI
61%
NI
NI
p-Isobutyl
p-Ph
7.05 0.09 [0.091]
6.79 0.10 [0.16]
5.87 0.13 [1.3]
5.79 0.15 [1.6]
41
m-Ph
46%
32%
NI
42
o-Ph
23%
8%
NI
43
44
45
46
p-OPh
m-OPh
o-OPh
—
7.0 0.06 [0.10]
6.01 0.05 [0.98]
5.20 0.13 [6.3]
35%
6.40 0.23 [0.40]
15%
NI
11%
NI
77%
70%
60%
64%
NI
47
—
5.40 0.21 [3.98]
48
49
50
51
o-NO2
m-NO2
p-NO2
o-NH2
m-NH2
p-NH2
o-NHCOMe
m- NHCOMe
p-NHCOMe
o-NHCOPh
m-NHCOPh
p-NHCOPh
m-OBn
p-OBn
m-CN
36%
7%
18%
8%
43%
32%
36%
4%
21% (33% at 20
23%
3%
12%
16%
NI
57%
65%
NI
NI
e
52 (JZP-169)
53
54
55
56
57
58
59
18% (41% at 20
lM)
lM)
6.66 0.04 [0.216]
NI
NI
NI
NI
NI
79%
88%
34%
11%
NI
37%
NI
NI
48%
6.21 0.12 [0.62]
5.82 0.06 [1.51]
6.39 0.08 [0.41]
NI
6.25 0.08 [0.56]
5.49 0.04 [3.23]
6.28 0.07 [0.52]
23%
60
61
62
53%
NI
63%
63
p-CN
NI
25%
NI
64
65
66
67
m-COOMe
p-COOMe
m-CF3
m-Cl
20%
7%
15%
48%
43%
6.35 0.05 [0.45]
NI
6.06 0.05 [0.87]
52%
5.17 0.06 [6.76]
12%
37%
68
m-OMe
—
—
NI
6%
16%
NI
70%
NI
JZP-327Ae
JZP-430 (7)f
JZP-361g
7.94 0.05 [0.011]
18%
5.14 0.08 [7.24]
7.36 0.05 [0.044]
5.75 0.04 [1.78]
—
7.34 0.12 [0.046]
*
Compounds 38–41,29 43–4529 and 4930 had been reported earlier but are included here to provide better insights into SARs.
pIC50 values (ꢁlog10 [IC50]) represent the mean SEM from three independent experiments performed in duplicates for the most potent compounds while for the less
a
active ones, values from two independent experiments were used.
b
The percentage (%) of inhibition is represented as the mean from two independent experiments performed in duplicate.
c
Remaining activity (%) at 1
NI, no inhibition.
lM is given when it is <90%.
d
e
f
S-3-(1-(4-Isobutylphenyl)ethyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (JZP-327A)29 was used as a reference FAAH inhibitor.
4-Morpholino-1,2,5-thiadiazol-3-yl cyclooctyl(methyl)carbamate (JZP-430)21 was used as a reference ABHD6 inhibitor.
g
(4-(8-Chloro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11(6H)-ylidene)piperidin-1-yl)(1H-1,2,4-triazol-1-yl)methanone (JZP-361)31 was used as a reference MAGL inhibitor.
Table 2
Inhibitory activities of 1,3,4-oxadiazol-2-ones 69–72 towards FAAH, MAGL and ABHD6
Compd
R1
R2
pIC50 SEM [IC50, l
M]a
% inhibition at 10
l
Mb
% remaining enzymatic ABHD6 activity at 1
hABHD6
l
Mc
hFAAH
hMAGL
69
70
71
m-NO2
m-NH2
m-NO2
m-NH2
Et
Et
Ph
Ph
NId
10%
38%
25%
30%
15%
63%
NI
5.49 0.06 [3.31]
5.1 0.08 [7.94]
6.88 0.05 [0.13]
7.13 0.05 [0.073]
72 (JZP-JVB-053)
a–d
See footnotes for Table 1.

J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
6339
Table 4
Inhibition of ABHD6 by JZP-169 (52) and JZP-430 (7) indicates the irreversible mode
of inhibition after a rapid 40-fold dilution of inhibitor-treated hABHD6 preparation
Incubation time (min)
pIC50 SEM
JZP-169 (52)
JZP-430 (7)
THL
10
30
60
90
5.39 0.08
5.42 0.07
5.44 0.06
5.48 0.05
7.04 0.03
7.06 0.03
7.06 0.03
7.03 0.03
7.88 0.05⁄⁄⁄
7.85 0.04⁄⁄⁄
7.70 0.06⁄⁄
7.43 0.06
In contrast to compounds (52) and (7), potency of THL significantly declines during
the 90 min incubation (an asterisk denotes a statistically significant difference
compared to the pIC50 value at 90 min time-point: one-way Anova, followed by
Tukey’s multiple comparison test (p <0.01⁄⁄, p <0.001⁄⁄⁄)). Data are means SEM
from three to seven independent experiments. Note also that due to methodological
limitations, the IC50 values obtained by the dilution method are not directly com-
parable to those obtained using the routine assay protocol.34,38
3.2. Selectivity among serine hydrolases and cannabinoid
receptors
(I) Activity based protein profiling (ABPP).
Next, we explored in more detail the selectivity of the 1,3,4-
oxadiazol-2-one analogues JZP-169 (52) and JZP-JVB-053 (72)
using competitive ABPP of the mouse brain membrane proteome.
We used earlier reported inhibitor WWL7014 (1) at the indicated
concentration to locate the band of ABHD6. As shown in Figure 3,
JZP-169 (52) at 10 lM concentration efficiently competed with
probe binding to ABHD6 but no visible effect on other serine
hydrolases could be detected by this approach. Only at a very high
concentration of 32.5
hydrolases (indicated by black arrows). JZP-JVB-053 (72) achieved
almost complete inhibition of the ABHD6 band at 1 M (Fig. S1, see
lM did JZP-169 (52) inhibit the other serine
l
Supporting information) although at a 10-fold concentration it
targeted also other serine hydrolases. In brief, when tested at
10 lM, JZP-169 (52) appeared to be selective for ABHD6 over the
other recognized brain serine hydrolases, including FAAH, MAGL
and ABHD12.
Figure 3. Competitive ABPP reveals the selectivity of compound 52 (JZP-169)
among the serine hydrolases of mouse whole brain membrane proteome. Molecular
weight markers are indicated on the left. The reference inhibitor WWL70 (1) was
used at the indicated concentration to indicate the ABHD6 protein band.14 At a
(II) Selectivity over the cannabinoid receptors.
concentration of 10
probe labelling to other visible serine hydrolases was not affected. However, at very
high concentration (32.5 M) FAAH and additional serine hydrolase (indicated by
black arrows) appear as off-target of this compound. The gel is representative from
two ABPP experiments with similar outcomes.
lM, compound 52 (JZP-169) selectively targeted ABHD6 as
To rule out cross-activity with additional targets of the endo-
cannabinoid system, compound JZP-169 (52), which appeared to
be the best ABHD6 inhibitor in this series in terms of potency
and selectivity, was evaluated for its possible agonist or antago-
nist activity at the cannabinoid CB1 and CB2 receptors, using a
l
previously validated [35S]GTP S binding assay.32,33 In this sys-
c
tem, compound 52 did not display any significant agonist or
caused less than 50% inhibition towards both of these enzymes
(see Table 1) thereby achieving nearly >90-fold selectivity
towards ABHD6 over FAAH and MAGL.
antagonist activities when tested at 10
(Table 3).
lM
concentration
Table 3
Activity of compound JZP-169 (52) at CB1 and CB2 receptors
Compd
Agonist activity
CB1R antagonist activitya
CB1Ra
CB2Rb
[ cS binding % HU210-evoked
35S]GTP
response (mean (range), n = 2)
[
35S]GTP
c
S binding % basal
(mean (range), n = 2)
JZP-169 (52)
96 (92–100)
107 (102–111)
104 (101–106)
HU210 (1
HU210 (10 nM)
AM251 (1 M)
SR144528 (1 M)
l
M)
306 (302–311)
—
—
—
—
—
—
167 (165–169)
—
56 (54–59)
l
38 (36–40)
—
l
a
Rat cerebellar membranes.
hCB2R-CHO cell membranes.
b

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J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
oxyanion hole along with good shape complementarity between
the enzyme and ligand. JZP-169 achieved good docking convergence
by making hydrogen bond interactions with the oxyanion hole
(main chain amide of Phe80) when the putative nucleophile
Ser148 was in close vicinity to the carbonyl carbon ready for nucle-
ophilic attack. Furthermore, our homology model and docking stud-
ies suggested that the amino group present at the meta-position in
this 3-benzyl-1,3,4-oxadiazol-2-one was able to make hydrogen
bond contact with the side chains of Glu190 and Glu253, which
may have enhanced its inhibitory activity by stabilizing the covalent
reaction intermediate or slowing the rate of the product release.
Molecular modelling was used to confirm the irreversibility of
JZP-169 (52) along with other established irreversible ABHD6 inhi-
bitors (WWL70 (1), KT182 (5) and JZP-430 (7)) and a reversible inhi-
bitor, THL. First, HOMO and LUMO energy and the HOMO–LUMO gap
for the studied inhibitors were calculated with Gaussian09 (Table 5).
It was found that the irreversible inhibitors, JZP-169, JZP-430, KT182
and WWL70 could be characterized with a lower HOMO–LUMO gap
than could be obtained with THL, that is, they were more reactive. In
order to investigate in detail the reversibility of the investigated
inhibitors, the QM/MM approach of Discovery Studio 3.1 was
applied; this is a well-established technique for this purpose.39 This
method was used to minimize hypothetical covalent complexes and
respective transition state complexes on the QM/MM level and to
calculate their final energy. For the transition state systems, it was
assumed that the carbonyl group of the ligands reacted with the side
chain of Ser148 as earlier reported.19,40 The data are presented in
Table 5. It can be seen that THL (orlistat) displayed an energy of acti-
vation of complex dissociation below 20 kcal/mol, which is the
energy barrier for reactions at room temperature.41 In contrast, the
activation energies for dissociation of JZP-169, JZP-430, KT182 and
WWL70were over 20 kcal/mol, meaning that these inhibitors would
be irreversible at room temperature. This may be caused by car-
bamoylation of Ser148 by the irreversible inhibitors which leads to
trapping of this residue within an acyl-enzyme like intermediate
(carbamoyl serine structure) as earlier reported.19,40
Figure 4. Most favourable Glide docking pose of compound 52 (JZP-169) into the
ABHD6 active site in a homology model. Catalytic residues are coloured using
yellow carbons and the surface of the active site is presented.
3.3. Reversibility of ABHD6 inhibition
To explore in more detail the ABHD6-binding mode of JZP-169
(52), we tested its ability to inhibit hABHD6 using a 96-well format
dilution method suitable for testing compound reversibility.34 In
this assay, our previously reported irreversible ABHD6 inhibitor
JZP-430 (7)21 and compound JZP-169 (52) fully retained their
potencies during the 90 min incubation period after a rapid 40-fold
dilution of the enzyme–inhibitor complex (Table 4) suggesting that
in these time-frame studies, JZP-169 inactivated hABHD6 in an
irreversible manner. In contrast, the inhibitory properties of orlis-
tat (tetrahydrolipstatin, THL) were significantly reduced during the
90 min incubation, indicating that this compound functioned as a
reversible ABHD6 inhibitor. The inactivation of ABHD6 by 1,3,4-
oxadiazol-2-one based compound JZP-169, and THL are quite
remarkable. This is because our previously reported 1,3,4-oxadia-
zol-2-one based compound JZP-327A inhibited FAAH in a reversi-
ble manner29 whereas here the 1,3,4-oxadiazol-2-one based
compound JZP-169 inhibited ABHD6 irreversibly. It is also note-
worthy that THL is generally believed to irreversibly inhibit serine
hydrolases such as pancreatic lipase35,36 and ABHD1237 while the
present studies show that it inhibited ABHD6 in a reversible
manner.
4. Conclusions
To summarize, a convenient synthesis of 3-benzyl-1,3,4-oxadi-
azol-2-ones was developed and out of the series of compounds
synthesized, JZP-169 (52) was identified as a potent hABHD6 inhi-
bitor (IC50 = 214 nM), having >90-fold selectivity over other princi-
pal off-targets such as FAAH and MAGL. Competitive ABPP
indicated that compound 52 selectively targeted ABHD6 in prefer-
ence over other serine hydrolases in the mouse brain membrane
proteome. Moreover, the detailed inhibition mechanism studies
conducted with both experimental assays and molecular mod-
elling indicated that compound 52 acted as an irreversible inhibi-
tor of hABHD6. Molecular modelling studies further
demonstrated that compound 52 underwent favourable interac-
tions within the active site of hABHD6, including important hydro-
gen-bonding of the carbonyl oxygen to the oxyanion hole.
Moreover, within the still restricted classes of known ABHD6 inhi-
bitors, the 1,3,4-oxadiazol-2-one scaffold represents a novel class
of compounds. Finally, 1,3,4-oxadiazol-2-one represents a versatile
scaffold for the further development of inhibitors with diverse
mechanisms targeting other serine hydrolases.
3.4. Molecular modelling
Subsequently, we conducted a molecular docking study of our
compound 52 (JZP-169) (see Fig. 4) with a previously generated
homology model.21 The catalytic machinery of several serine hydro-
lases consists of a catalytic triad supplemented by an oxyanion hole
to allow the activation of the carbonyl carbon. Previous docking
studies have indicated that inhibitory activity can be partly
explained by means of easy access to the residues making up the
Table 5
Selected parameters for the compounds involved in reversibility studies
Compd
HOMO
(kcal/mol)
LUMO
(kcal/mol)
HOMO–LUMO
gap (kcal/mol)
Energy of
activation
of complex
dissociation
(kcal/mol)
5. Experimental
JZP-169 (52)
JZP-430 (7)
KT182 (5)
WWL70 (1)
THL
ꢁ8.408
ꢁ8.758
ꢁ8.810
ꢁ9.133
ꢁ10.143
0.180
ꢁ0.533
ꢁ0.371
ꢁ0.491
0.658
8.587
8.224
8.438
8.642
10.801
36.181
42.530
37.454
34.226
17.541
5.1. Materials and methods
All reagents and solvents were purchased from commercial
suppliers and were used without further purification. Reactions

J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
6341
were monitored by thin-layer chromatography (TLC) using alu-
minium sheets coated with silica gel F245 (60 Å, 40–63 m, 230–
5.1.3. General procedures for preparation of novel 3-benzyl-
1,3,4-oxadiazol-2(3H)-ones (54–59)
l
400 mesh) with suitable UV visualization. Purification was carried
out by flash chromatography (FC) on J.T. Baker’s silica gel for chro-
matography (pore size 60 Å, particle size 50 nm) or by RediSepÒ
normal-phase silica flash columns using CombiFlash Companion
(Teledyne Isco, USA). Petroleum ether (PE) of fraction 40–60 °C
was used for chromatography. 1H NMR and 13C NMR were
recorded on a Bruker Avance AV 500 (Bruker Biospin, Switzerland)
spectrometer operating on 500.1 and 125.8 MHz, respectively.
Tetramethylsilane (TMS) was used as an internal standard for 1H
NMR. Chemical shifts are reported in ppm on the d scale from an
internal standard of solvent (CDCl3 7.26 and 77.0 ppm, DMSO
2.50). All spectra were processed from the recorded FID files with
TOPSPIN 2.1 software or ACD/NMR Processor Academic Edition.
The following abbreviations are used: s, singlet; br s, broad singlet;
d, doublet; t, triplet; q, quartet; m, multiplet. The coupling con-
stants are reported in Hz. ESI-MS spectra were acquired using an
LCQ quadrupole ion trap mass spectrometer equipped with an
electrospray ionization source (Thermo LTQ, San Jose, CA, USA).
Elemental analyses were performed on a ThermoQuest CE instru-
ment (EA 1110 CHNS-O) or a Perkin–Elmer PE 2400 Series II
CHNS-O Analyzer. The purity of the synthesized 1,3,4-oxadiazol-
2-ones (42, 46–48, 50–72) are P95% (see Table S1 of Supplemen-
tary information).
To a solution of an appropriate amino containing 3-benzyl-
1,3,4-oxadiazol-2-one (51–53) (1.0 equiv) in dichloromethane
(0.23 M), triethylamine (Et3N) (2 equiv) was added followed by
either acetyl chloride or benzoyl chloride (1 equiv) at 0–5 °C. The
reaction mixture was allowed to warm to 22–25 °C and stirred at
the same temperature for 16–24 h. The progress of the reaction
was monitored by TLC using 20% EtOAc in PE as the mobile phase.
Solvents were concentrated under vacuum and the residue
obtained was dissolved in EtOAc and washed with H2O and brine.
The organic layer was dried over sodium sulfate, filtered and con-
centrated under vacuum to afford crude 3-benzyl-1,3,4-oxadiazol-
2-one which were purified by flash column chromatography using
PE/EtOAc (9:1) as an eluent. The desired fractions were collected
and solvents were evaporated on a rotatory evaporator to afford
pure acetamides (54–56) and benzamides (57–59).
5.1.4. 3-([1,10-Biphenyl]-2-ylmethyl)-5-methoxy-1,3,4-
oxadiazol-2(3H)-one (42)
Light orange oil (290 mg, 20%); 1H NMR (CDCl3): d 7.44–7.41 (m,
2H), 7.37–7.34 (m, 6H), 7.28–7.25 (m, 1H), 4.78 (s, 2H), 3.92 (s,
3H); 13C NMR (CDCl3): d 155.2, 151.5, 141.8, 140.2, 132.3, 130.4,
129.2, 128.4 (2C), 128.3, 128.0, 127.8, 127.6, 127.4, 57.6, 47.1; Anal.
Calcd for C16H14N2O3: C, 68.08; H, 5.00; N, 9.92; Found: C, 68.12; H,
4.98; N, 9.89; ESI-MS: 283.03 [M+H]+.
5.1.1. General procedures for preparation of novel 1,3,4-
oxadiazol-2(3H)-ones (42, 46–48, 50, 60–69 and 71)
5.1.5. 5-Methoxy-3-(naphthalen-1-ylmethyl)-1,3,4-oxadiazol-2-
(3H)-one (46)
The 1,3,4-oxadiazol-2-one (42, 46–48, 50, 60–69 and 71)
were prepared according to our earlier reported method with
minor modifications.29 To a solution of appropriate hydrazine
carboxylate (22–37) (1.0 equiv) in dichloromethane (0.2 M) pyri-
dine (3.0 equiv) was added at 0 °C followed by a drop wise addi-
tion of phosgene solution (20% in toluene, 5.1 equiv) under an
inert atmosphere. The reaction mixture was allowed to warm
and stirred at 20–25 °C for another 2–16 h (Note: for the synthe-
sis of compounds 60 and 61, the reaction was stirred at ꢁ40 °C
for 10 min.). The progress of the reaction was monitored by TLC
using 20% EtOAc in PE as the mobile phase. Solvents were con-
centrated under vacuum and the residue obtained was dissolved
in EtOAc. It was washed with H2O and brine. The organic layer
was dried over sodium sulfate, filtered and concentrated under
vacuum to afford crude 1,3,4-oxadiazol-2-one which were puri-
fied by flash column chromatography using PE/EtOAc (9:1) as
an eluent. The desired fractions were collected and solvents were
evaporated on a rotatory evaporator to afford pure 1,3,4-oxadia-
zol-2-ones.
White solid (100 mg, 13%); 1H NMR (CDCl3, 500 MHz): d 8.19 (d,
J = 8.5 Hz, 1H), 7.89–7.85 (m, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.52 (t,
J = 7.2 Hz, 2H), 7.46 (t, J = 7.7 Hz, 1H), 5.22 (s, 2H), 3.89 (s, 3H);
13C NMR (CDCl3): d 155.6, 151.2, 133.8, 131.2, 130.4, 129.3,
128.8, 128.0, 126.9, 126.0, 125.2, 123.4, 57.3, 47.4; Anal. Calcd for
C
14H12N2O3: C, 65.62; H, 4.72; N, 10.93; Found: C, 65.66; H, 4.71;
N, 10.95; ESI-MS: 257.06 [M+H]+.
5.1.6. 5-Methoxy-3-(naphthalen-2-ylmethyl)-1,3,4-oxadiazol-2-
(3H)-one (47)
White solid (135 mg, 17%); 1H NMR (CDCl3, 500 MHz): d 7.85–
7.83 (m, 3H), 7.80 (s, 1H), 7.50–7.45 (m, 3H), 4.94 (s, 2H), 3.93 (s,
3H); 13C NMR (CDCl3): d 155.6, 151.5, 133.2, 133.1, 132.4, 128.7,
127.9, 127.7, 127.4, 126.4, 126.3, 125.8, 57.4, 49.7; Anal. Calcd
for C14H12N2O3: C, 65.62; H, 4.72; N, 10.93; Found: C, 65.59; H,
4.75; N, 10.91; ESI-MS: 257.07 [M+H]+.
5.1.7. 5-Methoxy-3-(2-nitrobenzyl)-1,3,4-oxadiazol-2(3H)-one
(48)
White solid (500 mg, 32%); 1H NMR (CDCl3, 500 MHz): d 8.11 (d,
J = 8.0 Hz, 1H), 7.65 (t, J = 7.25 Hz, 1H), 7.51 (t, J = 7.75 Hz, 1H), 7.39
(d, J = 8.0 Hz, 1H), 5.27 (s, 2H), 3.97 (s, 3H); 13C NMR (CDCl3): d
155.97, 151.53, 148.06, 133.89, 130.62, 129.26, 129.02, 125.35,
57.55, 46.67; Anal. Calcd for C10H9N3O5: C, 47.81; H, 3.61; N,
16.73; Found: C, 47.83; H, 3.58; N, 16.71; ESI-MS: 252.08 [M+H]+.
5.1.2. General procedures for preparation of novel 3-benzyl-
1,3,4-oxadiazol-2(3H)-ones (51–53, 70 and 72)
To a solution of an appropriate nitro containing 3-benzyl-1,3,4-
oxadiazol-2-one (48–50, 69 and 71) (1.0 equiv) in methanol
(0.28 M), 10% Pd/C (catalyst loading 10% w/w) was added and the
mixture was stirred under an H2 atmosphere at 20–25 °C for 4–
8 h. The progress of the reaction was monitored by TLC using
40% EtOAc in PE as the mobile phase. Subsequently, the reaction
mixture was filtered through Celite. The solvents were concen-
trated under vacuum and the residue obtained was dissolved in
EtOAc and washed with H2O and brine. The organic layer was dried
over sodium sulfate, filtered and concentrated under vacuum to
obtain a crude amino analogue which was purified by flash column
chromatography using PE/EtOAc (8:2) as an eluent. The desired
fractions were collected and solvents were evaporated on a rota-
tory evaporator to produce the pure desired amino containing 3-
benzyl-1,3,4-oxadiazol-2-ones (51–53, 70 and 72).
5.1.8. 5-Methoxy-3-(4-nitrobenzyl)-1,3,4-oxadiazol-2(3H)-one
(50)
White solid (387 mg, 91%); 1H NMR (CDCl3, 500 MHz): d 8.24 (d,
J = 8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 4.88 (s, 2H), 3.97 (s, 3H); 13
C
NMR (CDCl3): d 155.9, 151.3, 147.9, 141.9, 129.0 (2C), 124.0 (2C),
57.5, 48.6; Anal. Calcd for C10H9N3O5: C, 47.81; H, 3.61; N, 16.73;
Found: C, 47.80; H, 3.63; N, 16.75; ESI-MS: 252.05 [M+H]+.
5.1.9. 3-(2-Aminobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one
(51)
Light yellow oil (295 mg, 89%); 1H NMR (CDCl3, 500 MHz): d
7.22 (d, J = 7.5 Hz, 1H), 7.15 (t, J = 8.2 Hz, 1H), 6.74 (t, J = 7.2 Hz,

6342
J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
1H), 6.70 (d, J = 8.0 Hz, 1H), 4.70 (s, 2H), 4.24–4.22 (br s, 2H), 3.95
(s, 3H); 13C NMR (CDCl3): d 155.6, 151.6, 145.6, 131.7, 130.1, 119.2,
118.3, 116.3, 57.4, 46.7; Anal. Calcd for C10H11N3O3: C, 54.30; H,
5.01; N, 19.00; Found: C, 54.28; H, 5.03; N, 19.02; ESI-MS: 222.20
[M+H]+.
7.49 (t, J = 7.2 Hz, 2H), 7.37 (t, J = 7.7 Hz, 1H), 7.13 (d, J = 7.5 Hz,
1H), 4.78 (s, 2H), 3.95 (s, 3H); 13C NMR (CDCl3): d 165.7, 155.6,
151.5, 138.4, 136.0, 134.8, 131.9, 129.6, 129.3, 128.8, 128.5,
127.0, 124.2, 120.1, 119.7, 57.4, 49.3; Anal. Calcd for C17H15N3O4:
C, 62.76; H, 4.65; N, 12.92; Found: C, 62.77; H, 4.68; N, 12.89;
ESI-MS: 326.13 [M+H]+.
5.1.10. 3-(3-Aminobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-
one (52)
5.1.17. N-(4-((5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-
methyl)phenyl)benzamide (59)
Light yellow oil (385 mg, 50%); 1H NMR (CDCl3, 500 MHz): d
7.18 (t, J = 7.75 Hz, 1H), 6.76 (d, J = 7.5 Hz, 1H), 6.68 (s, 1H), 6.66
(d, J = 8.0 Hz, 1H), 4.70 (s, 2H), 3.98 (s, 3H), 3.75–3.72 (br s, 2H);
13C NMR (CDCl3): d 155.5, 151.5, 146.8, 136.2, 129.7, 118.3,
114.9, 114.6, 57.3, 49.4; Anal. Calcd for C10H11N3O3: C, 54.30; H,
5.01; N, 19.00; Found: C, 54.31; H, 4.97; N, 18.97; ESI-MS:
222.11 [M+H]+.
White solid (125 mg, 68%); 1H NMR (DMSO, 500 MHz): d 10.30–
10.27 (br s, 1H), 7.95 (d, J = 7.5 Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.59
(d, J = 6.5 Hz, 1H), 7.53 (t, J = 7.2 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H),
4.76 (s, 2H), 3.93 (s, 3H); 13C NMR (DMSO): d 165.5, 155.0, 150.9,
138.8, 134.8, 131.6, 130.6, 128.3 (2C), 128.0 (2C), 127.6 (2C),
120.5 (2C), 57.7, 48.2; Anal. Calcd for C17H15N3O4: C, 62.76; H,
4.65; N, 12.92; Found: C, 62.79; H, 4.65; N, 12.94; ESI-MS:
326.07 [M+H]+.
5.1.11. 3-(4-Aminobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-
one (53)
Light cream-coloured solid (440 mg, 53%); 1H NMR (CDCl3,
500 MHz): d 7.15 (d, J = 8.0 Hz, 2H), 6.65 (d, J = 8.0 Hz, 2H), 4.65
(s, 2H), 3.93 (s, 3H), 3.72–3.69 (br s, 2H); 13C NMR (CDCl3): d
155.4, 151.3, 146.5, 129.7 (2C), 124.8, 115.1 (2C), 57.2, 49.1; Anal.
Calcd for C10H11N3O3: C, 54.30; H, 5.01; N, 19.00; Found: C, 54.34;
H, 4.98; N, 19.03; ESI-MS: 222.02 [M+H]+.
5.1.18. 3-(3-(Benzyloxy)benzyl)-5-methoxy-1,3,4-oxadiazol-2-
(3H)-one (60)
Dark brown oil (396 mg, 56%) 1H NMR (CDCl3): d 7.43–7.41 (m,
2H), 7.39–7.35 (m, 2H), 7.33–7.30 (m, 1H), 7.28–7.24 (m, 1H),
6.95–6.92 (m, 3H), 5.05 (s, 2H), 4.74 (s, 2H), 3.93 (s, 3H), 13C
NMR (CDCl3): d 159.1, 155.6, 151.4, 136.8, 136.5, 129.9, 128.6
(2C), 128.0, 127.5 (2C), 120.7, 114.7 (2C), 70.0, 57.4, 49.3; Anal.
Calcd for C17H16N2O4: C, 65.38; H, 5.16; N, 8.97; Found: C, 65.41;
H, 5.19; N, 9.01; ESI-MS: 313.14 [M+H]+.
5.1.12. N-(2-((5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-
methyl)phenyl)acetamide (54)
White solid (105 mg, 71%); 1H NMR (CDCl3, 500 MHz): d 8.67–
8.65 (br s, 1H), 7.97 (d, J = 6.5 Hz, 1H), 7.39–7.37 (br s, 2H), 7.14
(s, 1H), 4.76 (s, 2H), 3.98 (s, 3H), 2.29 (s, 3H); 13C NMR (CDCl3): d
169.0, 155.7, 151.8, 136.6, 131.2, 129.9, 125.4, 125.0, 124.4, 57.6,
46.5, 24.3; Anal. Calcd for C12H13N3O4: C, 54.75; H, 4.98; N,
15.96; Found: C, 54.77; H, 4.97; N, 15.99; ESI-MS: 264.09 [M+H]+.
5.1.19. 3-(4-(Benzyloxy)benzyl)-5-methoxy-1,3,4-oxadiazol-2-
(3H)-one (61)
Colourless oil (1.36 g, 55%); 1H NMR (CDCl3): d 7.49–7.45 (m,
2H), 7.44–7.41 (m, 2H), 7.39–7.37 (m, 1H), 7.36–7.34 (m, 1H),
7.33–7.31 (m, 1H), 7.02–6.99 (m, 2H), 5.11 (s, 2H), 4.76 (s, 2H),
3.99 (s, 3H); 13C NMR (CDCl3): d 158.8, 155.5, 151.3, 136.8, 129.8
(2C), 128.6 (2C), 128.0 (2C), 127.5 (2C), 115.1 (2C), 70.1, 57.3,
49.0; Anal. Calcd for C17H16N2O4: C, 65.38; H, 5.16; N, 8.97; Found:
C, 65.39; H, 5.14; N, 8.99; ESI-MS: 312.89 [M+H]+.
5.1.13. N-(3-((5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-
methyl)phenyl)acetamide (55)
Colourless oil (372 mg, 70%); 1H NMR (CDCl3, 500 MHz): d 7.55
(d, J = 7.5 Hz, 1H), 7.44 (s, 1H), 7.31 (t, J = 7.7 Hz, 1H), 7.08 (d,
J = 7.5 Hz, 1H), 4.75 (s, 2H), 3.95 (s, 3H), 2.17 (s, 3H), one –NH
group; 13C NMR (CDCl3): d 168.3, 155.6, 151.5, 138.4, 136.0,
129.5, 124.0, 119.7, 119.3, 57.4, 49.3, 24.6; Anal. Calcd for
5.1.20. 3-((5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)methyl)-
benzonitrile (62)
White solid (137 mg, 18%); 1H NMR (CDCl3): d 7.65–7.59 (m,
3H), 7.51–7.48 (m, 1H), 4.81 (s, 2H), 3.98 (s, 3H); 13C NMR (CDCl3):
d 155.8, 151.3, 136.5, 132.7, 132.1, 131.8, 129.8, 118.3, 113.1, 57.4,
48.6; Anal. Calcd for C11H9N3O3: C, 57.14; H, 3.92; N, 18.17; Found:
C, 57.17; H, 3.88; N, 18.14; ESI-MS: 232.02 [M+H]+.
C
12H13N3O4: C, 54.75; H, 4.98; N, 15.96; Found: C, 54.72; H, 5.02;
N, 15.95; ESI-MS: 264.08 [M+H]+.
5.1.14. N-(4-((5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-
methyl)phenyl)acetamide (56)
5.1.21. 4-((5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)methyl)-
benzonitrile (63)
Off-white solid (75 mg, 50%); 1H NMR (CDCl3, 500 MHz): d 7.49
(d, J = 7.5 Hz, 2H), 7.30 (d, J = 7.5 Hz, 2H), 4.73 (s, 2H), 3.94 (s, 3H),
2.17 (s, 3H), one –NH gr.; 13C NMR (CDCl3): d 168.3, 155.5, 151.4,
138.0, 130.8, 129.1 (2C), 120.0 (2C), 57.4, 49.0, 24.6; Anal. Calcd
for C12H13N3O4: C, 54.75; H, 4.98; N, 15.96; Found: C, 54.74; H,
4.96; N, 15.94; ESI-MS: 264.05 [M+H]+.
White solid (104 mg, 62% mg); 1H NMR (CDCl3): d 7.68–7.62 (m,
2H), 7.47–7.45 (m, 2H), 4.84 (s, 2H), 3.97 (s, 3H); 13C NMR (CDCl3):
d 155.8, 151.3, 140.1, 132.7, 132.6, 128.9 (2C), 118.4, 112.4, 57.5,
48.9; Anal. Calcd for C11H9N3O3: C, 57.14; H, 3.92; N, 18.17; Found:
C, 57.13; H, 3.90; N, 18.21; ESI-MS: 232.03 [M+H]+.
5.1.15. N-(2-((5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-
methyl)phenyl)benzamide (57)
5.1.22. Methyl 3-((5-methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-
methyl)benzoate (64)
White solid (139 mg, 76%); 1H NMR (CDCl3, 500 MHz): d 9.36–
9.34 (br s, 1H), 8.0–7.90 (m, 3H), 7.47–7.14 (br s, 6H), 4.75 (s, 2H),
3.87 (s, 3H); 13C NMR (CDCl3): d 166.1, 155.7, 151.3, 136.5, 134.4,
132.0, 131.2, 129.8, 128.6 (2C), 127.5 (2C), 126.8, 125.9, 125.7,
57.6, 46.3; Anal. Calcd for C17H15N3O4: C, 62.76; H, 4.65; N, 12.92;
Found: C, 62.74; H, 4.63; N, 12.95; ESI-MS: 326.13 [M+H]+.
White solid (1.8 g, 52%); 1H NMR (CDCl3): d 8.07–8.01 (m, 2H),
7.55 (d, J = 7.6 Hz, 1H), 7.45 (t, J = 7.7 Hz, 1H), 4.83 (s, 2H), 3.95 (s,
3H), 3.92 (s, 3H); 13C NMR (CDCl3): d 166.6, 155.7, 151.4, 135.4,
132.7, 130.8, 129.5, 129.3, 129.0, 57.4, 52.2, 49.0; Anal. Calcd for
C
12H12N2O5: C, 54.55; H, 4.58; N, 10.60; Found: C, 54.58; H, 4.61;
N, 10.57; ESI-MS: 265.01 [M+H]+.
5.1.23. Methyl 4-((5-methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-
methyl)benzoate (65)
5.1.16. N-(3-((5-Methoxy-2-oxo-1,3,4-oxadiazol-3(2H)-yl)-
methyl)phenyl)benzamide (58)
White solid (687 mg, 52%); 1H NMR (CDCl3): d 8.03 (d, J = 8.2 Hz,
2H), 7.41 (d, J = 8.1 Hz, 2H), 4.83 (s, 2H), 3.95 (s, 3H), 3.91 (s, 3H);
Light yellow oil (156 mg, 82%); 1H NMR (CDCl3, 500 MHz): d
7.89–7.86 (m, 3H), 7.70 (d, J = 8.0 Hz, 1H), 7.59–7.54 (m, 2H),

J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
6343
13C NMR (CDCl3): d 166.6, 155.7, 151.4, 139.8, 130.1, 130.1 (2C),
128.1 (2C), 57.4, 52.2, 49.1; Anal. Calcd for C12H12N2O5: C, 54.55;
H, 4.58; N, 10.60; Found: C, 54.56; H, 4.59; N, 10.64; ESI-MS:
265.0 [M+H]+.
J = 7.3 Hz, 1H), 6.63 (t, J = 7.9 Hz, 2H), 4.69 (s, 2H), 3.71–3.69 (br
s, 2H); 13C NMR (CDCl3): d 153.4, 151.9, 151.1, 146.9, 136.0,
129.9 (2C), 129.8, 126.5 (2C), 118.3 (2C), 115.0, 114.6, 49.6; Anal.
Calcd for C15H13N3O3: C, 63.60; H, 4.63; N, 14.83; Found: C,
63.58; H, 4.67; N, 14.79; ESI-MS: 284.11 [M+H]+.
5.1.24. 5-Methoxy-3-(3-(trifluoromethyl)benzyl)-1,3,4-
oxadiazol-2(3H)-one (66)
5.2. Biological evaluation
Colourless oil (455 mg, 55%); 1H NMR (CDCl3): d 7.61–7.50 (m,
4H), 4.84 (s, 2H), 3.97 (s, 3H), 13C NMR (CDCl3): d 155.8, 151.4,
135.9, 131.7, 131.2 (q, JCF = 31.8 Hz), 129.5, 125.3, 125.2, 123.9 (q,
JCF = 272 Hz), 57.5, 48.9; Anal. Calcd for C11H9F3N2O3: C, 48.18; H,
3.31; N, 10.22; Found: C, 48.21; H, 3.35; N, 10.25; ESI-MS:
293.11 [M+NH4]+.
5.2.1. Determination of FAAH activity using anandamide as a
substrate
The inhibitory activities of the synthesized compounds were
determined using membranes of hFAAH-overexpressing COS-7
cells and [3H]-AEA as the substrate, essentially as previously
described.42 Briefly, the assay buffer consisted of 50 mM Tris–HCl
(pH 7.4) and 1 mM EDTA. The solvent for the test compounds
was DMSO (the final concentration was 5% v/v max.). The incuba-
tions were performed in the presence of 0.5% (w/v) BSA (essentially
5.1.25. 3-(3-Chlorobenzyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-
one (67)
Yellow oil (271 mg, 10%); 1H NMR (CDCl3): d 7.36–7.32 (m, 1H),
7.31–7.28 (m, 2H), 7.24–7.22 (m, 1H), 4.75 (s, 2H), 3.96 (s, 3H); 13
C
fatty acid free). The inhibitors (5
with hFAAH-COS-7 cell membranes (55
10 min at 37 °C (60 L). After 10 min, 20 M AEA was added (final
concentration 2
M containing 10 nM of [3H]-AEA; specific activity
60 Ci/mmol and 1 mCi/mL concentration). The final incubation vol-
ume was 100 L. The incubations were carried out for 10 min at
37 °C. Ethyl acetate (400 L) was added at the 20 min time point
to stop the enzymatic reaction. Additionally, 100 l of 50 mM
Tris–HCl, pH 7.4; 1 mM EDTA was added. Then, samples were cen-
l
L) or DMSO were preincubated
NMR (CDCl3): d 155.7, 151.4, 136.9, 134.7, 130.1, 128.5, 128.3,
126.4, 57.5, 48.8; Anal. Calcd for C10H9ClN2O3: C, 49.91; H, 3.77;
N, 11.64; Found: C, 49.88; H, 3.75; N, 11.68; ESI-MS: 240.97 [M
+H]+.
l
L, 1 g protein) for
l
l
l
l
l
5.1.26. 5-Methoxy-3-(3-methoxybenzyl)-1,3,4-oxadiazol-2(3H)-
one (68)
l
l
Colourless oil (104 mg, 12%); 1H NMR (CDCl3): d 7.29–7.25 (m,
1H), 6.92 (d, J = 7.6 Hz, 1H), 6.88–6.85 (m, 2H), 4.75 (s, 2H), 3.95
(s, 3H), 3.81 (s, 3H); 13C NMR (CDCl3): d 159.9, 155.6, 151.4,
136.5, 129.9 (2C), 120.4, 113.7, 57.4, 55.3, 49.4; Anal. Calcd for
trifuged for 4 min at RT 13,000 rpm, and aliquots (100 lL) from the
aqueous phase containing [ethanolamine 1-3H] were measured for
radioactivity by liquid scintillation counting (Wallac 1450
MicroBeta; Wallac Oy, Finland).
C
11H12N2O4: C, 55.93; H, 5.12; N, 11.86; Found: C, 55.96; H, 5.09;
N, 11.82; ESI-MS: 237.05 [M+H]+.
5.2.2. Determination of MAGL activity using 2-AG as a substrate
The inhibitory activity of the synthesized compounds was
determined using lysates of hMAGL overexpressing HEK293 cells
and 2-AG as the substrate, essentially as previously described.43
Briefly, the assay buffer consisted of 50 mM Tris–HCl (pH 7.4)
and 1 mM EDTA. The solvent for the test compounds was DMSO
(the final concentration was max 5% v/v). The incubations were
performed in the presence of 0.5% (w/v) BSA (essentially fatty acid
5.1.27. 5-Ethoxy-3-(3-nitrobenzyl)-1,3,4-oxadiazol-2(3H)-one
(69)
Light cream-coloured solid (965 mg, 40%); 1H NMR (CDCl3): d
8.22–8.19 (m, 2H), 7.70 (d, J = 7.2 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1H),
4.88 (s, 2H), 4.34–4.30 (q, J = 6.7 Hz, 2H), 1.41 (t, J = 6.6 Hz, 3H);
13C NMR (CDCl3): d 155.2, 151.3, 148.5, 131.0, 134.2, 129.9,
123.3, 123.1, 67.6, 48.5, 14.0; Anal. Calcd for C11H11N3O5: C,
49.81; H, 4.18; N, 15.84; Found: C, 49.84; H, 4.21; N, 15.87; ESI-
MS: 266.0 [M+H]+.
free). The inhibitors (5
lysates of hMAGL overexpressing HEK293 cells (2.5
10 min at 37 °C (60 L). At the 10 min time point, 2-AG (125
was added to achieve the final concentration of 50 M with the
final incubation volume of 100 L. Incubation was continued for
10 min at 37 °C. To stop the enzymatic reaction, 400 L of cold
l
L) or DMSO were preincubated with
g) for
M)
l
l
l
5.1.28. 3-(3-Aminobenzyl)-5-ethoxy-1,3,4-oxadiazol-2(3H)-one
(70)
l
l
Light yellow oil (200 mg, 27%); 1H NMR (CDCl3): d 7.13 (t,
J = 7.7 Hz, 1H), 6.72 (d, J = 7.5 Hz, 1H), 6.65–6.62 (m, 2H), 4.66 (s,
2H), 4.32–4.28 (q, J = 7.1 Hz, 2H), 3.71–3.67 (br s, 2H), 1.39 (t,
J = 6.6 Hz, 3H); 13C NMR (CDCl3): d 154.8, 151.5, 146.8, 136.3,
129.7, 118.3, 114.9, 114.6, 67.2, 49.4, 14.1; Anal. Calcd for
l
11 mM H3PO4 in ACN (the pH of the samples was simultaneously
decreased to 3.0) was added to stabilize 2-AG against acyl migra-
tion to 1(3)-AG. All samples were centrifuged for 4 min at RT
14,000 rpm. The formation of arachidonic acid and depletion of
2-AG (and 1(3)-AG) were measured by HPLC.43
C
11H13N3O3: C, 56.16; H, 5.57; N, 17.86; Found: C, 56.19; H, 5.53;
N, 17.88; ESI-MS: 236.0 [M+H]+.
5.2.3. Determination of ABHD6 and ABHD12 activities
5.1.29. 3-(3-Nitrobenzyl)-5-phenoxy-1,3,4-oxadiazol-2(3H)-one
(71)
Glycerol production from the 1-AG hydrolysis was determined
with
a sensitive fluorescent glycerol assay using lysates of
Light yellow solid (725 mg, 34%); 1H NMR (CDCl3): d 8.21–8.19
(m, 2H), 7.68 (d, J = 7.5 Hz, 1H), 7.55 (t, J = 7.7 Hz, 1H), 7.43–7.40
(m, 2H), 7.30–7.26 (m, 3H), 4.90 (s, 2H); 13C NMR (CDCl3): d
154.0, 151.6, 151.0, 148.5, 136.7, 134.2, 130.0 (2C), 129.7, 126.8,
123.5, 120.8 (2C), 119.4, 48.7; Anal. Calcd for C15H11N3O5: C,
57.51; H, 3.54; N, 13.41; Found: C, 57.53; H, 3.51; N, 13.37; ESI-
MS: 314.06 [M+H]+.
HEK293 cells transiently overexpressing hABHD6 or hABHD12 as
previously described.16,34 In this assay, glycerol production is cou-
pled via a three-step enzymatic cascade to hydrogen peroxide-de-
pendent generation of resorufin whose fluorescence (kex 530; kem
590 nm) is kinetically monitored using a Tecan Infinite M200 plate
reader (Tecan Group Ltd, Männedorf, Switzerland). Briefly,
hABHD6- or hABHD12-HEK293 lysates (99
were pretreated for 30 min with the solvent (DMSO) or the inhibi-
tor with desired concentrations (1 L), after which 1-AG (100 L,
12.5 M final concentration) was added. The reaction was then
lL, 0.3 lg protein/well)
5.1.30. 3-(3-Aminobenzyl)-5-phenoxy-1,3,4-oxadiazol-2(3H)-
one (72)
l
l
l
Light yellow oil (118 mg, 13%); 1H NMR (CDCl3): d 7.39 (t,
J = 7.8 Hz, 2H), 7.29–7.24 (m, 3H), 7.11 (t, J = 7.7 Hz, 1H), 6.71 (d,
kinetically monitored for 90 min. The assays contain routinely
0.5% (w/v) BSA (essentially fatty acid free) as a carrier. 1-AG was

6344
J. Z. Patel et al. / Bioorg. Med. Chem. 23 (2015) 6335–6345
used instead of 2-AG, as this is the preferred endocannabinoid iso-
mer for hABHD6 and hABHD12.16 The IC50-values at the 90 min
time-point were calculated after nonlinear fitting of the inhibitors’
dose–response curves. Assay blanks without hABHD6 (or
hABHD12) lysates were included in each experiment and fluores-
cence of the assay blank was subtracted before calculation of the
final results. The reversibility of compounds to inhibit hABHD6
was tested in a 96-well plate format using the 40-fold-dilution
method previously described for testing reversibility of MAGL
inhibitors.34
energies were used to calculate energy of dissociation of covalent
complexes.
Acknowledgments
We thank Ms. Tiina Koivunen, Ms. Minna Glad, Ms. Taija Hukka-
nen, Ms. Satu Marttila and Ms. Miriam Lopez Navarro for their
skillful technical assistance. CSC—Scientific Computing, Ltd is
gratefully acknowledged for software licenses and computational
resources. The Centre of International Mobility (CIMO, Grant TM-
09-6221 for J.Z.P.), Graduate School of Drug Design, UEF (for J.Z.
P.), The Academy of Finland (Grants 139140 for T.J.N., 127653 for
T.P., 139620 for J.T.L.) and Biocenter Finland/DDCB have provided
financial support for this study. Part of the research was performed
under the Marie Curie IEF fellowship for A.A.K. The calculations
with Gaussian09 were performed under a computational Grant
from the Interdisciplinary Center for Mathematical and Computa-
tional Modeling (ICM), Warsaw, Poland, Grant number G30-18.
Dr. Ewen Macdonald and Dr. Gerald G. Netto are acknowledged
for revising the language of this manuscript. J.Z.P. has provided a
personal fund for language editing.
5.2.4. Activity-based protein profiling (ABPP) of serine
hydrolases
Competitive ABPP using mouse whole brain membranes was
conducted to visualize the selectivity of inhibitors towards ABHD6
against serine hydrolases in brain membrane proteome. We used
the active site serine-targeting fluorescent fluorophosphonate
probe TAMRA-FP as previously described.16,34 Briefly, brain mem-
branes (100
inhibitors, after which TAMRA-FP labelling was conducted for 1 h
at RT (final probe concentration 2 M). The reaction was quenched
g protein per
lg) were treated for 1 h with DMSO or the selected
l
by addition of 2ꢂ gel loading buffer, after which 10
l
lane was added into the gel and the proteins were resolved in 10%
SDS–PAGE together with molecular weight standards. Finally,
TAMRA-FP labelling was visualized (kex 552; kem 575 nm) using a
fluorescent scanner (FLA-3000 laser fluorescence scanner, Fujifilm,
Tokyo, Japan).
Supplementary data
Supplementary data (synthesis and spectroscopic characteriza-
tion of all intermediates (22–37); inhibitory activities of novel oxa-
diazolones 42, 46–72 towards ABHD12; selectivity of compound
72 among the serine hydrolases of the mouse brain membrane
proteome using competitive ABPP; inhibitory activity of selected
compounds 52 and 72 towards ABHD6 and related references,
NMR-spectra of selected compounds 52 and 72) associated with
this article can be found, in the online version, at http://dx.doi.
org/10.1016/j.bmc.2015.08.030.
5.2.5. Data analyses
Data analyses for the concentration–response curves were cal-
culated from non-linear regressions using Graph Pad Prism 5.0
for Windows. For comparison of potency values in reversibility
assays, statistical analysis was performed by using one-way Anova,
followed by Tukey’s multiple comparison test.
References and notes
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