Triazole Ureas Act as Diacylglycerol Lipase Inhibitors and Prevent Fasting-Induced Refeeding

Article abstract of DOI:10.1021/acs.jmedchem.6b01482

Triazole ureas constitute a versatile class of irreversible inhibitors that target serine hydrolases in both cells and animal models. We have previously reported that triazole ureas can act as selective and CNS-active inhibitors for diacylglycerol lipases (DAGLs), enzymes responsible for the biosynthesis of 2-arachidonoylglycerol (2-AG) that activates cannabinoid CB₁ receptor. Here, we report the enantio- and diastereoselective synthesis and structure-activity relationship studies. We found that 2,4-substituted triazole ureas with a biphenylmethanol group provided the most optimal scaffold. Introduction of a chiral ether substituent on the 5-position of the piperidine ring provided ultrapotent inhibitor 38 (DH376) with picomolar activity. Compound 38 temporarily reduces fasting-induced refeeding of mice, thereby emulating the effect of cannabinoid CB₁-receptor inverse agonists. This was mirrored by 39 (DO34) but also by the negative control compound 40 (DO53) (which does not inhibit DAGL), which indicates the triazole ureas may affect the energy balance in mice through multiple molecular targets.

Full text of DOI:10.1021/acs.jmedchem.6b01482

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Article
Triazole Ureas act as Diacylglycerol Lipase
Inhibitors and Prevent Fasting-induced Refeeding
Hui Deng, Sander Kooijman, Adrianus M.C.H. van den Nieuwendijk, Daisuke Ogasawara, Tom van
der Wel, Floris van Dalen, Marc P. Baggelaar, Freek J. Janssen, Richard J.B.H.N van den Berg, Hans
den Dulk, Benjamin F. Cravatt, Herman S. Overkleeft, Patrick C.N. Rensen, and Mario van der Stelt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01482 • Publication Date (Web): 07 Dec 2016
Downloaded from http://pubs.acs.org on December 9, 2016
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Triazole Ureas act as Diacylglycerol Lipase
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Inhibitors and Prevent Fastingꢀinduced Refeeding
Hui Deng †, Sander Kooijman §, Adrianus M.C.H. van den Nieuwendijk ‡, Daisuke Ogasawara ∥,
Tom van der Wel †, Floris van Dalen †, Marc P. Baggelaar †, Freek J. Janssen †, Richard
J.B.H.N. van den Berg ‡, Hans den Dulk †, Benjamin F. Cravatt ∥, Herman S. Overkleeft ‡,
Patrick C.N. Rensen § & Mario van der Stelt †,*
† Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden,
The Netherlands.
‡ Department of Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden,
The Netherlands.
§ Department of Medicine, Division of Endocrinology and Einthoven Laboratory for
Experimental Vascular Medicine, Leiden University Medical Center, Leiden, The Netherlands.
∥ Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California,
United States of America.
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ABSTRACT
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Triazole ureas constitute a versatile class of irreversible inhibitors that target serine hydrolases
both in cells and animal models. We have previously reported that triazole ureas can act as
selective and CNSꢀactive inhibitors for diacylglycerol lipases (DAGLs), enzymes responsible for
the biosynthesis of 2ꢀarachidonoylglycerol (2ꢀAG) that activates cannabinoid CB1 receptor.
Here, we report the enantioꢀ and diastereoselective synthesis and structureꢀactivity relationship
studies. We found 2,4ꢀsubstituted triazole ureas with a biphenylmethanol group provided the
most optimal scaffold. Introduction of a chiral ether substituent on the 5ꢀposition of the
piperidine ring provided ultrapotent inhibitor 38 (DH376) with picomolar activity. Compound 38
temporarily reduces fastingꢀinduced refeeding of mice, thereby emulating the effect of
cannabinoid CB1ꢀreceptor inverse agonists. This was mirrored by 39 (DO34), but also by the
negative control compound 40 (DO53) (that does not inhibit DAGL), which indicates the triazole
ureas may affect the energy balance in mice through multiple molecular targets.
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INTRODUCTION
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Compound libraries that contain a 1,2,3ꢀtriazole urea scaffold have previously been applied to
the discovery of potent inhibitors of diverse serine hydrolases, such as diacylglycerol
lipaseꢀβ (DAGLβ), α,βꢀhydrolase domain (ABHD) 6/11, DDHD2, APEH and PAFAH2.1ꢀ
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1,2,3ꢀTriazole ureas constitute a versatile chemotype for the covalent, irreversible and
selective inhibition of serine hydrolases. They contain an electrophilic carbonyl group
with tunable reactivity as well as a scaffold to introduce functional groups conferring
enzyme potency and/or specificity. 1,2,3ꢀTriazole ureas irreversibly inhibit serine
hydrolases via carbamoylation of the activeꢀsite serine nucleophile. Some reported
triazole urea inhibitors were proven to be potent and selective for specific serine
hydrolases both in cells and mouse models, and are effective chemical probes to study the
biological function of serine hydrolases in diverse biological systems.1, 2 For example,
1
(KT109)4, a selective and in vivoꢀactive DAGLβ inhibitor reduces 2ꢀarachidonoylglycerol
(2ꢀAG), arachidonic acid and eicosanoids levels in peritoneal macrophages of
lipopolysaccharide (LPS)ꢀtreated mice and significantly decreases the proꢀinflammatory
cytokine, tumour necrosis factor α (TNFα) in LPSꢀtreated mice.4
Two isoforms of DAGL exist, and that are expressed in a tissueꢀdependent manner.
Both isoforms, termed DAGLα and DAGLβ employ a SerꢀHisꢀAsp catalytic triad
characteristic for serine hydrolases to hydrolyse an ester bond of diacylglycerol (DAG) in
a
snꢀ1 specific manner. DAGLα and DAGLβ share extensive homology, but differ in size:
DAGLα is about 120 kDa and DAGLβ is around 70 kDa.6, 7 DAGLα is the principal
regulator of 2ꢀAG formation in the nervous system, where it controls the activity of this
endocannabinoid, which activates the cannabinoid CB1 receptor, as a retrograde
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messenger at neuronal synapses. DAGLβ in turn is the dominant enzyme for 2ꢀAG
production in the periphery during inflammation.8, 9
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To study the function of DAGLs in a temporal and dynamic manner, in vivoꢀactive
inhibitors of these enzymes would be of great value. Particularly, a CNSꢀactive chemical
probe is required for DAGLα (mainly expressed in the brain) that can be used to acutely
perturb 2ꢀAG production in the central nervous system. The known DAGL inhibitors can
be classified into six different chemotypes: αꢀketoheterocycles, glycine sulfonamides
(both reversible, competitive DAGL inhibitor classes), bisꢀoximinoꢀcarbamates, βꢀ
lactones, fluorophosphonates and 1,2,3ꢀtriazole ureas (the latter four being mechanismꢀ
based and irreversible).4, 10ꢀ14 These inhibitors have been used to study the function of 2ꢀ
AG in cellular models and brain slice preparations, but they lack selectivity over serine
hydrolases, potency and/or chemical properties required for central activity. Of note, with
the exception of the αꢀketoheterocycles, all DAGL inhibitors reported to date also inhibit
ABHD6. Since the latter enzyme hydrolyses the DAGL product 2ꢀAG, relating the
physiological effect of such dual inhibitors to modulating 2ꢀAG levels through DAGL
inactivation is complicated.
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We selected compound
1 as a suitable starting point for the rational design of new,
potent and selective inhibitors for DAGLα, because it inhibits DAGLα with an IC50 of 2.3
µM in a competitive activityꢀbased protein profiling (ABPP) assay.4 In a first round of
optimization, we converted compound
1 into 38 (DH376), a highly potent, in vivo active
compound that inhibits DAGLα in a timeꢀ and dose dependent manner in mouse brain.15
Using compound 38, as well as the structurally distinct compound 39 (DO34)15 (see
Supporting Information, Figure S6), we performed functional studies on DAGLα in
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nervous system.15 We found that DAGLs coordinate crosstalk between several brain lipid
signalling networks and modulate neuro(immuno)logical functions in central nervous
system.
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Here, we provide a full account of the discovery, development and in vivo efficacy
data of compound 38 as an inhibitor of DAGLα.15 We systematically investigated the
influence of regioselectivity of the 1,4ꢀ and 2,4ꢀtriazole moiety, the nature of the
substituents on the triazole core, the chirality of the benzylpiperidine and the substituent
pattern of the piperidine ring on DAGLα and ABHD6 activity. To this end, we developed
an enantioselective synthesis route to obtain both enantiomers of the benzylpiperidines
and their derivatives. Finally, we determined the selectivity profiles of the 1,2,3ꢀtriazole
ureas in mouse brain membrane proteome and assessed whether compound 38 is able to
inhibit fastingꢀinduced refeeding in mice, a typical cannabinoid CB1 receptor mediated
response.
RESULTS AND DISCUSSION
Discovery of 2,4ꢀsubstituted 1,2,3ꢀtriazole urea as new chemotype of DAGLα inhibitor.
In our search for CNSꢀactive DAGLα inhibitors, we employed a rational design drug
discovery approach in which compound 1 and a closely related analogue,
2
(ML226)1,
served as starting points (structures are shown in Figure 1). Compound 1 is a peripherally
restricted DAGLβ inhibitor with 60ꢀfold selectivity over DAGLα. Compound 2 in turn, is
a potent, cellular and in vivo active ABHD11 inhibitor with excellent physicochemical
properties.1, 4 First, we tested the activity of compounds 1 and
2 on HEK293T membranes
overexpressing human DAGLα and mouse DAGLβ in a colorimetric assay using para
ꢀ
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nitrophenylbutyrate (PNP) as a surrogate substrate (Figure 1b, c; Table S.1 in supporting
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information).10, 11 We found that compound
1 inhibited mouse DAGLβ with a pIC50 of 7.1
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±0.2, which is consistent with previously reported in a gelꢀbased ABPP assay using HTꢀ
01 as a chemical probe (pIC50 = 7.4)4. However, compound 1 (pIC50 8.9±0.1) was much
more potent on human DAGLα in our assay, than previously reported in a gelꢀbased
ABPP assay using HTꢀ01 as a chemical probe (pIC50 = 5.6)4. The difference might be due
to the weak labeling efficiency of HTꢀ01 for DAGLα. In contrast, compound 2
demonstrated weak DAGLα activity with a pIC50 7.2±0.1 and poor DAGLβ activity in the
PNPꢀassay. At first sight this is in line with the previously reported preference of DAGLβ
for 1,4ꢀregioisomers of the triazole ureas over the corresponding 2,4ꢀregioisomers.16
Compound 2 lacks, however, the 2ꢀbenzyl substituent on the piperidine moiety for an
appropriate comparison between the two inhibitors. Therefore, we synthesized
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compounds
this end, the triazole building blocks and the final compounds were synthesized as
previously reported (see supporting information). Interestingly, compound , a 2ꢀ
benzylpiperidine urea of a 2,4ꢀtriazole with a 1,1ꢀdiphenylmethanol substituent at the 4ꢀ
position (as in compound ), showed the highest DAGLα and DAGLβ inhibitory activity
with pIC50 of 8.6±0.1 and 7.9±0.1, respectively. Its 1,4ꢀregioisomer (compound ) is 10ꢀ
3ꢀ6 as hybrid structures harbouring elements of both compounds 1 and 2. To
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fold less potent. This indicates that 2,4ꢀtriazole is the preferred regioisomer for DAGLα
and DAGLβ inhibition (Figure 1d, e; Table S.1 in supporting information). Hybrid
compounds (
5
and
6
) with an ethyl substituent at the 2ꢀposition of the piperidine ring
, which suggests that the benzyl substituent is
appeared less potent than compound
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required to address an additional lipophilic pocket near the active site in the enzymes
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(Figure 1d, e). We next employed competitive ABPP assays to confirm the inhibitory
activities of compounds 1ꢀ6 against recombinant human DAGLα and human DAGLβ. A
previously reported DAGLꢀtailored activityꢀbased probeꢀDH379 was used for these
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studies. The results of gelꢀbased ABPP assay were in line with the above PNPꢀassay
that compound
1 potently inhibited DAGLα and DAGLβ labeling by DH379, and
compound showed the highest potency against human DAGLα and DAGLβ among the
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hybrid compounds (Figure 1 f and g; Figure S.1 in supporting information).
Figure 1. (a) The structures of 1,2,3ꢀtriazole ureas 1ꢀ6. (b, d) Concentrationꢀdependent inhibition
of recombinant human DAGLα by compounds 1ꢀ6 as measured with a colorimetric assay based
on the hydrolysis of PNP butyrate in DAGLꢀtransfected HEK293T cells. (c, e) Concentrationꢀ
dependent inhibition of recombinant mouse DAGLβ by compounds 1ꢀ6 as measured with PNP
butyrate substrate assay. Data represent average values ± SEM; n = 4 per group. (f, g)
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Representative fluorescent gelꢀbased competitive ABPP with compound 1ꢀ6 against recombinant
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human DAGLα and DAGLβ by tailored activityꢀbased probe DH379 (1 µM, 30 min).
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We next investigated the contribution of the phenyl groups of the 1,1ꢀ
diphenylmethanol substituent in compound
4
to DAGLα inhibition (Table 1). To this end,
); removed one ( ) or both (10) phenyl
we replaced the phenyl rings by cyclohexyls (
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groups; replaced them by a pyridyl (11) or introduced fluorine atoms (12). The
biochemical assay revealed that paraꢀfluoro substituted inhibitor (12) was the most potent
agent in this series against hDAGLα with a pIC50 of 9.0, which suggested that its
increased lipophilicity and/or electron withdrawing effect is beneficial. The ~100ꢀfold
drop in potency of the more polar pyridylꢀcontaining compound (11), suggested that
lipophilicity is more important than electron withdrawing properties. Indeed, the
lipophilic interactions of the phenyl groups are essential features of the DAGLα inhibitor,
because their removal led to a 160ꢀ2000 fold decrease in potency (
8ꢀ10), whereas
retaining two bulky cyclohexyl groups ( ) resulted in only a 40ꢀfold drop in potency. A
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role for piꢀsigma/cation interactions can, however, also not be excluded. A 10ꢀfold
decrease in potency was observed when the tertiary alcohol group was methylated, as in
compound (13), suggesting that a hydrogen bond donor is important (or alternatively, the
grafted methyl group has a steric clash with the enzyme). To reduce the lipophilicity we
substituted the 2ꢀbenzyl substituent of the piperidine ring for a phenoxymethylꢀ (14
, 15)
or (4ꢀfluoro)phenoxymethyl group (16 17) and introduced polar methoxy substituents on
,
the phenyl ring (18
,
19). The substitutions were tolerated (Table 1), but led to a fiveꢀfold
reduced activity.
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Table 1. Structureꢀactivity relationship of triazole ureas with N2ꢀisomers as leaving
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groups
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Regioꢀ
isomer
pIC50±SEM
(DAGLα)
pIC50±SEM
(ABHD6)
Entry
3
R1
R2
1,4ꢀ
7.6±0.1
7.4±0.1
2,4ꢀ
8.6±0.1
7.7±0.1
4
2,4ꢀ
2,4ꢀ
7.0±0.1
5.8±0.1
5.1±0.2
5.5±0.1
7
8
2,4ꢀ
6.4±0.1
5.5±0.1
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2,4ꢀ
2,4ꢀ
5.3±0.2
6.8±0.1
<5
10
11
5.5±0.2
2,4ꢀ
2,4ꢀ
2,4ꢀ
2,4ꢀ
2,4ꢀ
2,4ꢀ
2,4ꢀ
9.0±0.1
7.9±0.2
8.1±0.1
8.3±0.1
8.2±0.1
8.3±0.1
8.5±0.5
7.6±0.1
<5
12
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18
6.8±0.1
6.7±0.1
6.6±0.1
6.7±0.1
6.8±0.2
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6.6±0.1
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29a is the most active DAGL inhibitor.
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Previously, the eutomer of compound
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was found to be 100ꢀfold more potent than the
distomer against DAGLβ.16 The absolute configuration of the enantiomers was, however,
not assigned. To determine whether 29a or 29b is the most active enantiomer, we
developed a novel enantioselective synthesis route (Scheme 1 and Supporting
Information). Our synthesis of the separate enantiomers of compound
1 began with the
preparation of chiral amine 23a in four steps from commercially available Bocꢀprotected
Lꢀphenyl alanine 20a.17 We reacted amine 23a with 3ꢀpentene nitrile 24 to give secondary
amine 25a via a oneꢀpot DIBALꢀH reductionꢀtransiminationꢀNaBH4 reduction
sequence.18 Subsequent Bocꢀprotection of the amine, ringꢀclosing metathesis,
hydrogenation and Bocꢀdeprotection led to the construction of the key chiral 2ꢀ
benzylpiperidine building block 28a in a 44% overall yield. Direct coupling of the chiral
piperidine with 4ꢀ([1,1'ꢀbiphenyl]ꢀ4ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazole using triphosgene, provided
final compound 29a in >95% e.e. as determined by chiral HPLC. The synthesis of the
other enantiomer 29b proceeded in a similar fashion using chiral amine 23b (see
Supporting Information, Scheme S.3).
To correlate the activity of the compounds with their stereochemistry, we tested both
compounds and a 1:1 mixture in the colorimetric surrogate PNPꢀsubstrate assay using
HEK293T membranes expressing recombinant human DAGLα. Compound 29a proved
to be the eutomer with a pIC50 of 9.1±0.1, while compound 29b showed ~100ꢀfold less
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activity (pIC50 of 7.4±0.1) (Figure 2a) in the PNPꢀassay. The 1:1 racemic mixture
demonstrated a pIC50 of 8.2±0.1. We also employed a realꢀtime, fluorescenceꢀbased assay
to test the activity of the inhibitors on DAGLαꢀmediated hydrolysis of its natural substrate
1ꢀstearoylꢀ2ꢀarachidonolyꢀsnꢀglycerol.19 Again, compound 29a was the most active
DAGLα inhibitor with a pIC50 of 7.6±0.1 (Figure 2b). Since ABHD6 was previously
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reported as an offꢀtarget of compound
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(
29a and 29b) against human ABHD6.11, 20 Compound 29a (pIC50 8.6±0.1) was ~100ꢀfold
more potent than 29b (pIC50 6.2±0.1) (Figure 2c), which indicates that the inhibitory
activity for both DAGLα and ABHD6 resides in the ( )ꢀenantiomer. To assess the
R
activity and selectivity of compounds 29a and 29b on endogenously expressed DAGLα in
mouse brain membrane proteome, we used the previously reported ABPP method with
MB064, a tailored activityꢀbased probe.10 Consistent with the biochemical assays, we
found that compounds 29a and 29b blocked DAGLα labeling by MB064 with pIC50 of
8.1±0.1 and 6.2±0.1, respectively (Figure 2d and Figure S.2).
Scheme 1. Enantioselective synthesis of 29a
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4
5
6
7
8
c, d, e
a, b
N
HO
O
NHBoc
NHBoc
20a
NHBoc
22a
f, g
yield based
on 20a 86%
O
O
21a
9
89%
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h, i, j, k
44%
l
NH
NBoc
92%
NH2
23a
CN
26a
25a
24
m
68%
O
n, f
70%
o, p
NH
N
N
N
NBoc
30%
N
28a
29a
27a
HN
N
N
Reagents and conditions: (a) Me(OMe)NH·HCl; (b) EDCI, NMM; (c) LiAlH4; (d) H3O+;
o
(e) (Ph)3P=CH2; (f) MeOH, HCl; (g) NaOH; (h) 24, diethyl ether, DIBALꢀH, ꢀ80 C to 0
oC; (i) MeOH, ꢀ90 oC; (j) corresponding amine 23a (3 equiv), r.t., 20h; (k) NaBH4, 0 oC to
o
r.t., 5h; (l) Boc2O, Et3N, THF, 50 C, 20h; (m) Grubbs I cat. 4 mol%, DCM, reflux, 48h;
o
(n) H2, Pd/C, MeOH; (o) DIPEA, triphosgene, THF, 0 C; (p) DIPEA, DMAP, triazole ,
THF, 60 oC.
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Figure 2. Characterization of both enantiomers of compound
1
as DAGLα inhibitors: (a)
29b and racemic
(1:1) as measured with a colorimetric assay based on the hydrolysis of PNP
butyrate. (b) Concentrationꢀdependent inhibition of recombinant hDAGLα by 29a 29b
and racemic compound (1:1) as measured with a SAG substrate assay in DAGLαꢀ
transfected HEK293T cells. (c) Concentrationꢀdependent inhibition of hABHD6 by 29a
29b and racemic compound (1:1) as measured with a 2ꢀAG substrate assay. Data
Concentrationꢀdependent inhibition of recombinant hDAGLα by 29a
compound
,
1
,
1
,
1
represent average values ± SEM; n = 4 per group. (d, e) Representative fluorescent gelꢀ
based competitive ABPP with 29a and 29b in mouse brain proteome by tailored activityꢀ
based probe MB064 (0.25 µM, 30 min).
Discovery of highly potent DAGL inhibitors.
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Having discovered that the (
R)ꢀenantiomer is the most active compound in the 1,4ꢀtriazole
5
6
7
8
series, we transferred this knowledge to the 2,4ꢀtriazole series. To this end we coupled the
chiral amine building block 23a to the triazole scaffold to provide compound )ꢀ12. We
found that )ꢀ12 had a pIC50 of 9.1±0.1, which was slightly higher than the racemic
(
R
9
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(
R
mixture (Figure 3a). To improve solubility and to mimic the natural substrate
diacylglycerol, we designed several new analogues with a chiral hydroxyl group at the Cꢀ
5 position (Scheme 2 and S.4; 30ꢀ33). The chiral, diastereomers were synthesized
according to Scheme 2. In short, the cyanohydrin 40 was enzymatically produced by the
)ꢀhydroxynitrile lyase using crotonic aldehyde 39 as a substrate.21 After silyl
almond (
R
protection of the alcohol, we generated key intermediate 44 by the same strategy as
described for the synthesis of 29a. After ꢀBoc deprotection, and optional hydrogenation,
compounds 45 and 48, were coupled to the 1,2,3ꢀtriazole building block, yielding ꢀsilyl
protected intermediates 46 and 49. Deprotection gave (final) compounds 30 and 31
Further alkylation of intermediate 50 ꢀBoc deprotection and coupling with 1,2,3ꢀ
triazole building block afforded compounds 34 and 35. Compounds 32 33 36 and 37
N
O
.
,
N
,
,
were synthesized in the same fashion as described for the corresponding diastereoisomers
(Scheme S.4).
We tested compounds 30
ꢀ38 in the PNPꢀassay and found that the free alcohol
derivatives 30 33 are less potent than compound 12 (Table 1 and 2). Capping the
ꢀ
secondary hydroxyl group with an alkyl moiety yielded (ultra) potent inhibitors. For
example, compounds 34 and 35 demonstrated picomolar activity with pIC50 values of
9.1±0.1 and 9.2±0.1, respectively (Table 2). Comparison of the diastereoisomers (34 vs
36
;
35 vs 37) revealed that the back isomer at Cꢀ5 is the active diastereomer (34
,
35
)
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(with ~10ꢀfold higher potency). To visualize target engagement, we surmised that we
could introduce a propargyl at Cꢀ5, which serves as a ligation handle to introduce reporter
groups by copperꢀcatalyzed azideꢀalkyne cycloaddition (or “click”ꢀchemistry). This
yielded inhibitor 38 with a pIC50 = 8.9±0.1.
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Scheme 2. Enantioselective synthesis of 1,2,3 triazole ureas 30, 31, 34 and 35.
Reagents and conditions: (a) HCN, EtOAc, 0.1 M aq. citrate buffer, pH 5.4,
Hydroxynitrile lyases; (b) TBDPSꢀCl, imidazole, DMF, 0 oC; (c) diethyl ether, DIBALꢀH,
ꢀ80 oC to 0 oC; (d) MeOH, ꢀ90 oC; (e) (
S
)ꢀamine (23a) (3 equiv), r.t., 20h; (f) NaBH4; (g)
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o
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4
Boc2O, Et3N, THF, 50 C, 20h; (h) Grubbs G1 cat. 4 mol%, DCM, reflux, 48h; (i)
5
6
o
o
Hydrazine, CuSO4, EtOH, 0 C to 70 C; (j) 25% TFA, DCM, r.t.; (k) DIPEA,
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8
9
triphosgene, THF, 0 oC; (l) DIPEA, DMAP, triazole, THF, 60 oC; (m) HFꢀPyridine, THF :
pyridine = 1:1 (v/v); (n) TBAF, THF, r.t.; (o) NaH, corresponding bromide.
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Table 2. Structureꢀactivity relationship of N2ꢀtriazole urea isomers with functionalized
chiral pure (2ꢀbenzyl)ꢀpiperidine staying groups
pIC50± SEM
(DAGLα)
pIC50± SEM
(ABHD6)
pIC50± SEM
(DAGLα)
pIC50± SEM
(ABHD6)
R
R
Entry
30
Entry
35
7.9±0.1
7.7±0.1
7.7±0.1
7.4±0.1
6.8±0.1
6.6±0.1
9.2±0.1
8.1±0.1
7.7±0.1
7.4±0.1
6.9±0.2
5.6±0.1
31
32
36
37
5.2±0.1
9.1±0.1
6.7±0.2
7.3±0.1
8.9±0.1
8.6±0.2
33
34
38
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Activity and selectivity on endogenous DAGLα and ABHD6 in brain membrane proteome.
5
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8
9
To determine the activity and selectivity of our inhibitors in native proteomes, we incubated the
most potent chiral inhibitors 34ꢀ38 for 30 min with mouse brain membrane homogenates and
performed a gelꢀbased ABPPꢀassay using ABPs MB064 and FPꢀTAMRA. All compounds block
DAGLα labeling in a concentration dependent manner. Complete blockade of DAGLα was
already observed at 10 nM for compounds 34, 35 and 38, whereas the diastereoisomers 36 and
37 were less active (Figure 3b and Figure S.3). Compound 35 inhibited labeling of DAGLα and
ABHD6 with pIC50 of 8.7±0.1 and 6.5±0.1, respectively. This indicated that 35 is ~160ꢀfold
selective over ABHD6 (Figure 3c). Of note, compound 38 showed ~126 fold selectivity over
ABHD6. No additional offꢀtargets were identified using FPꢀTAMRA as a probe (Figure S.4).
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Figure 3. (a) Concentrationꢀdependent inhibition of hDAGLα by 12 and (R)ꢀ12 as
measured with a colorimetric assay based on the hydrolysis of PNP butyrate; Data
represent average values ± SEM; n = 4 per group. (b) Representative fluorescent gelꢀ
based competitive ABPP with 34ꢀ38 in mouse brain proteome by activityꢀbased probe
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MB064 (0.25 µM, 30 min). (c) pIC50 ± SEM and selectivity of compounds 34ꢀ38 against
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DAGLα and ABHD6 as determined by competitive ABPP (n=3 per group).
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In vivo efficacy of compound 38.
Activation of the cannabinoid CB1 receptor is a clinically proven signalling pathway controlling
the energy balance in humans.22 Rimonabant, a cannabinoid CB1 receptor inverse agonist
reduced body weight and waist circumference in obese patients and improved cardiovascular risk
factors, possibly via reducing food intake and increasing energy expenditure through activation
of thermogenic brown adipose tissue.23 DAGLα knockout mice have shown hypophagia and
leanness similar to that of cannabinoid CB1 receptor knockout mice, while mice in which the
gene for DAGLβ is disrupted do not share this phenotype.24 Fastingꢀinduced refeeding of mice is
a typical CB1ꢀreceptor mediated behaviour that is accompanied by increased 2ꢀAG levels in the
hypothalamus and can be prevented by preꢀtreatment with rimonabant.25 Since we have
previously shown that compound 38 is able to reduce brain levels of 2ꢀAG in a doseꢀ and time
15
dependent manner , we set out to test whether compound 38 could also inhibit fastingꢀinduced
refeeding. Based on our previous in vivo results with compound 38, we have chosen a dose of 50
mg/kg, because this leads to complete DAGL inhibition for more than 4h.15 Mice were fasted for
18h and received a single intraperitoneal injection of vehicle (saline/ethanol/PEG40 (18:1:1;
v/v/v)) or compound 38 (50 mg/kg) 30 min before refeeding, and cumulative food intake was
measured up to 16h. Within 2h, compound 38ꢀtreated mice only consumed a third of the food
compared to the vehicleꢀtreated mice, and the effect of reduction in foodꢀtake maintained up to
4h (Figure 4a). After 16h, however, food intake in compound 38ꢀtreated mice was back to the
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same levels as in vehicleꢀtreated mice (Figure S.5), which paralleled the recovery of brain 2ꢀAG
levels. Consistent with the delay in refeeding of compound 38ꢀtreated mice, indirect calorimetric
measurements indicated that carbohydrate and fat oxidation remained respectively low and high
for a prolonged period (Figure 4bꢀc). Importantly, compound 38ꢀtreated mice showed no effect
on locomotor activity (Figure 4d, over 4h; Figure S.5, over 24h) as measured by infrared beam
breaks in Y and Z planes. Altogether, these results support the notion that DAGLα regulates food
intake.
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Figure. 4 (aꢀd) In vivo effects of DAGL inhibitors 38, 39 and control compound 40 on
5
6
7
8
food intake (a), fat/carbohydrate oxidation (b, c) and locomotor activity (d, 0ꢀ4h) in mice.
Cumulative food intake was measured in 18h fasted mice. The animals received a single
intraperitoneal injection of vehicle (black), or 38 (red), 39 (blue), and 40 (green) (50
mg/kg) 30 min before the start of refeeding and the testing period. The fat and
carbohydrate oxidation of the animals were calculated from the V·O2 and V·CO2 in
metabolic cages (in 20 min bins), and the locomotor activity over 4h of the animals was
measured by infrared beam breaks in Y and Z axis. Data represent average values ± SEM;
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n = 8 mice per group. **
group.
P < 0.01; ***P < 0.001 for all groups vs. the vehicleꢀtreated
Previously, we have reported another centrally active DAGL inhibitor 39 and a
structurally related control probe 40 (DO53)15 (structures are shown in Supporting
Information, Figure S.6). Compound 39 reduced brain 2ꢀAG levels in doseꢀ and time
dependent manner, whereas the control probe 40 did not affect brain 2ꢀAG levels.
Therefore, we examined the effects of 39 and 40 on fastingꢀinduced refeeding, indirect
calorimetry and locomotor activity and observed that food intake was completely blocked
in 39ꢀtreated mice, which was again also reflected by the calorimetric measurements.
Surprisingly, control probe 40ꢀtreated mice also showed significant reductions in fastingꢀ
induced refeeding, and both 39ꢀ and 40ꢀtreated mice were hypolocomotive (Figure 4).
These data suggest that there are one or more offꢀtargets shared by the DAGL inhibitors
(
38 and 39) and control probe 40 that may also be involved in controlling food intake in
fastingꢀinduced mice. For example, carboxylesterase was identified by ex vivo
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competitive ABPP in mouse liver as a major offꢀtarget shared by all three compounds
(Figure S.7).
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CONCLUSION
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Here, we described the enantioselective synthesis and structureꢀactivityꢀrelationship
(SAR) studies of 2,4ꢀregioisomer of 1,2,3ꢀtriazole ureas as a novel chemotype of DAGLα
inhibitors. We found that (R)ꢀbenzylpiperidine substituted triazole ureas constitute the
active enantiomer for DAGLα inhibition as measured in biochemical assays and activityꢀ
based protein profiling. We showed that 29a is a potent DAGLα inhibitor. Our
investigations culminated in the discovery of compound 38, which is a CNSꢀactive
DAGL inhibitor. We found that DAGL inhibitors 38 and 39 prevented fastingꢀinduced
refeeding of mice, which is typical cannabinoid CB1ꢀreceptor mediated behaviour.
Importantly, compound 38 did not affect locomotion. These effects are in line with the
phenotypes observed with DAGLα and CB1 receptor knockout mice. Our results also
stress the importance of using negative control compounds, because compound 40 (that is
a triazole urea which does not inhibit DAGL) also reduced food intake and did affect
locomotion activity, which indicated that the triazole ureas may affect the energy balance
and locomotion in mice through multiple, yet to be discovered, molecular targets.
EXPERIMENTAL SECTION
Experimental Procedures: Biochemistry.
Cloning Procedures. For the preparation of the different constructs, full length human
cDNA was purchased from Source Bioscience and cloned into mammalian expression
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vector pcDNA3.1, containing genes for ampicillin and neomycin resistance. DAGLα and
ABHD6 constructs were obtained as reported previously.10, 15 Plasmids were isolated
from transformed XLꢀ10 Zꢀcompetent cells (Maxi Prep, Qiagen) and verified by Sanger
sequencing (BaseClear). The sequences were confirmed by sequence analysis at the
Leiden Genome Technology Centre.
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Cell culture and membrane preparation. Cell culture was performed as previously
15
reported. In brief, HEK293T cells were grown in DMEM with stable glutamine and
phenolred (PAA or Sigma) with 10% New Born Calf serum, penicillin and streptomycin.
Cells were passaged every 2ꢀ3 days by resuspending in medium and seeding them to
appropriate confluence. Membranes were prepared from transiently transfected HEK293T
cells. One day prior to transfection 107 cells were seeded in a 15 cm petri dish. Cells were
transfected by the addition of a 3:1 mixture of polyethyleneimine (60 µg) and plasmid
DNA (20 µg) in 2 mL serum free medium. The medium was refreshed after 24h, and after
72h the cells were harvested by suspending them in 20 mL medium. The suspension was
centrifuged for 10 min at 1000 rpm, and the supernatant was removed. The cell pellet was
stored at ꢀ80 oC until use.
Cell pellets were thawed on ice and suspended in lysis buffer A (20 mM HEPES, 2
mM DTT, 0.25 M sucrose, 1 mM MgCl2, 1x Cocktail (Roche cOmplete EDTA free), 25
U/mL Benzonase). The suspension was homogenized by polytrone (3 × 7 sec) and
incubated for 30 min on ice. The suspension was subjected to ultracentrifugation (93,000
× g, 30 min, 4 oC, Beckman Coulter, Type Ti70 rotor) to yield the cytosolic fraction in the
supernatant and the membrane fraction as a pellet. The pellet was resuspended in lysis
buffer B (20 mM HEPES, 2 mM DTT, 1x Cocktail (Roche cOmplete EDTA free)). The
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protein concentration was determined with Quick Start Bradford reagent (BioRad) or
QubitTM fluorometric quantitation (Life Technologies). The protein fractions were diluted
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o
to a total protein concentration of 1 mg/mL and stored in small aliquots at ꢀ80 C until
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use.
Biochemical DAGL activity assay. The biochemical hDAGLα assay was performed as
10
reported previously. In brief, the biochemical hDAGLα activity assay is based on the
hydrolysis of paraꢀnitrophenylbutyrate (PNPꢀbutyrate) by membrane preparations from
HEK293T cells transiently transfected with hDAGLα. Reactions were performed in 50
mM pH 7.2 HEPES buffer with 0.05 ꢁg/ꢁL final protein concentration hDAGLα
transfected protein.
Natural substrate based fluorescence assay (DAGLα and ABHD6). The natural
substrate assay was performed as reported previously 11, 19. Standard assay conditions: 0.2
U/mL glycerol kinase (GK), glycerolꢀ3ꢀphosphate oxidase (GPO) and horseradish
peroxidase (HRP), 0.125 mM ATP, 10 µM Amplifu™Red, 5% DMSO in a total volume
of 200 µL. For ABHD6, the assay additionally contained 25 µM 2ꢀAG and 0.5%
acetonitrile, with a final protein concentration of 40 µg/mL. For DAGLα, the assay
additionally contained 5 µg/mL MAGLꢀoverexpressing membranes, 100 µM SAG and
0.0075% (w/v) Triton Xꢀ100, with a final protein concentration of 50 µg/mL.
Preparation of mouse brain membrane proteome. Mouse brain membrane proteome
preparation was performed as previously reported. 15 In brief, mouse brains were isolated
according to guidelines approved by the ethical committee of Leiden University
(DEC#10095). Mouse brains were Dounce homogenized in pH 7.2 lysis buffer A (20 mM
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HEPES pH 7.2, 2 mM DTT, 1 mM MgCl2, 25 U/mL Benzonase) and incubated for 5 min
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o
on ice, followed by low speed spin (2,500 × g, 3 min, 4 C) to remove debris. The
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supernatant was subjected to ultracentrifugation (100,000 × g, 45 min, 4 C, Beckman
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Coulter, Type Ti70 rotor) to yield the cytosolic fraction in the supernatant and the
membrane fraction as a pellet. The pellet was resuspended in storage buffer B (20 mM
HEPES pH 7.2, 2 mM DTT). The total protein concentration was determined with Quick
Start Bradford reagent (BioꢀRad) or QubitTM fluorometric quantitation (Life
Technologies). Membranes and supernatant were flash frozen in liquid nitrogen and
stored in aliquots at ꢀ80 oC until use.
Activity based protein profiling in mouse brain. Mouse brain proteome (2 mg/mL, 19.5
µL) was incubated with DMSO or inhibitor in 0.5 µL DMSO for 30 min at r.t. and
subsequently incubated with 250 nM (final concentration) ABP MB064, or 500 nM (final
concentration) ABP TAMRAꢀFP for 20 min at r.t. before the reaction was quenched with
standard 3x Laemmli sample buffer. The gels were scanned using a ChemiDoc MP
system and analyzed using Image Lab 4.1.
ABPP inhibitor activity measurements. The percentage of activity remaining was
determined by measuring the integrated optical intensity of the fluorescent protein bands
using Image Lab 4.1. The relative intensity was compared to the vehicleꢀtreated proteins,
which were set to 100%. IC50 values were determined by plotting a log (inhibitor) vs.
normalized response (Variable slope) doseꢀresponse curve generated using Prism
software (Graphpad Prism 5.0).
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In vivo studies with 38, 39 and 40. All animal experiments were approved by the Ethics
Committee on Animal Care and Experimentation of the Leiden University Medical
Center. 12ꢀweekꢀold male C57Bl/6J mice (Charles River, SaintꢀGermainꢀNuelles, France)
were single housed in metabolic cages (LabMaster System, TSE Systems, Bad Homburg,
Germany) with a regular 12:12h light/dark cycle (6 a.m.ꢀ 6 p.m.) and free access to food
and water unless noted otherwise. After 3 days of acclimatization, mice were fasted for
18h starting at midnight followed by an intraperitoneal injection with 38 (50 mg/kg), 39
(50 mg/kg), 40 (50 mg/kg) or vehicle 30 min prior to refeeding. Solutions were prepared
in mixture of saline/ethanol/PEG40 (18:1:1; v/v/v). Cumulative food intake, oxygen
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uptake (V
were monitored. Carbohydrate oxidation was calculated using the formula ((4.585*
CO2)ꢀ(3.226* V O2))*4, in which the 4 represents the conversion from mass per time
unit to kcal per time unit. Similarly, fat oxidation was calculated using the formula
((1.695* V O2)ꢀ(1.701* V CO2))*9.
⋅O2), carbon dioxide production (V⋅CO2) and physical activity (beam breaks)
V⋅
⋅
⋅
⋅
Experimental Procedures: Chemistry.
General Remarks. All reactions were performed using oven or flame‐dried glassware and dry
solvents. Reagents were purchased from Sigma Aldrich, Acros or Merck and used without
further purification unless noted otherwise. All moisture sensitive reactions were performed
under an argon atmosphere. Traces of water were removed from starting compounds by co‐
evaporation with toluene.
1H‐ and 13C‐NMR spectra were recorded on a Bruker AV 400 MHz spectrometer at 400 (1H)
and 101 (13C) MHz, or on a Bruker DMXꢀ600 spectrometer 600 (1H) and 150 (13C) MHz using
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CDCl3, CD3OD or (CD3)2SO as solvent, unless stated otherwise. Chemical shift values are
reported in ppm with tetramethylsilane or solvent resonance as the internal standard (CDCl3, δ
7
8
1
13
1
13
1
7.26 for H, δ 77.16 for C; CD3OD, δ 3.31 for H, δ 49.00 for C; (CD3)2SO, δ 2.50 for H, δ
39.52 for 13C). Data are reported as follows: chemical shifts (δ), multiplicity (s = singlet, d =
doublet, dd = double doublet, td = triple doublet, t = triplet, q = quartet, m = multiplet, br =
broad), coupling constants J (Hz), and integration. HPLC purification was performed on a
preparative LCꢀMS system (Agilent 1200 series) with an Agilent 6130 Quadruple MS detector.
High‐resolution mass spectra (HRMS) were recorded on a Thermo Scientific LTQ Orbitrap XL.
IR spectra were recorded on a Shimadzu FTIR‐8300 and are reported in cmꢀ1. Optical rotations
were measured on a Propol automatic polarimeter (Sodium D‐line, λ = 589 nm). Flash
chromatography was performed using SiliCycle silica gel type SilicaFlash P60 (230 – 400 mesh).
TLC analysis was performed on Merck silica gel 60/Kieselguhr F254, 0.25 mm. Compounds
were visualized using either Seebach’s reagent (a mixture of phosphomolybdic acid (25 g),
cerium (IV) sulfate (7.5 g), H2O (500 mL) and H2SO4 (25 mL)) or a KMnO4 stain (K2CO3 (40
g), KMnO4 (6 g), H2O (600 mL) and 10% NaOH (5 mL)).
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Analysis of Compound Purity by LC/MS. Compound purity was determined by an LCQ
Adventage Max (Thermo Finnigan) ionꢀtrap spectrometer (ESI+) coupled to a Surveyor HPLC
system (Thermo Finnigan) equipped with a C18 (Gemini, 4.6 mm x 50 mm, 3 µm particle size,
Phenomenex) equipped with buffer A: H2O, B: acetonitrile (MeCN) and C: 1% aqueous TFA.
All final compounds were determined to be above 95% pure by this method.
(2ꢀBenzylpiperidinꢀ1ꢀyl)(4ꢀ(hydroxydiphenylmethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)methanone
(3). A solution of 2ꢀbenzylpiperidine (50.0 mg, 0.285 mmol) in THF (4 mL) was treated with
DIPEA (0.249 mL, 1.426 mmol) and bis(trichloromethyl) carbonate (42.3 mg, 0.143 mmol) and
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the reaction mixture was stirred for 30 min at 0 °C. The mixture was poured into water and
extracted with ethyl acetate (3 x 20 mL). The organic layer was washed with water, brine, dried
over MgSO4, filtered and concentrated under reduced pressure. The intermediate was dissolved
in a mixture of THF (8 mL) and DIPEA (0.249 mL, 1.426 mmol), DMAP (34.9 mg, 0.285 mmol)
and diphenyl(1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methanol (71.7 mg, 0.285 mmol) were added to the solution.
The mixture was stirred for two hours at 60 °C and poured into a saturated aqueous NH4Cl
solution. The mixture was extracted with ethyl acetate (3 x 20 mL), washed with water, brine,
dried over MgSO4 and filtered. The solvents were removed under reduced pressure to yield the
crude triazole urea as a mixture of N1ꢀ and N2ꢀcarbamoylated regioisomers. The N1ꢀcarbamoyl
triazole was isolated by silica gel chromatography (pentane/EtOAc 100:1 → 5:1) to afford 1,4ꢀ
triazole urea 3 (29.7 mg, 0.066 mmol, 23% yield). HRMS [ESI+] m/z: calculated for C28H28N4O2
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1
[M+H]+ 453.2285, found: 453.2283. H NMR (400 MHz, CDCl3) δ 7.39 – 7.27 (m, 10H), 7.04 –
6.82 (m, 5H), 4.76 (s, 1H), 4.28 (br s, 1H), 3.71 (br s, 1H), 3.33 – 3.21 (m, 2H), 2.63 (br s, 1H),
2.00 – 1.66 (m, 6H). 13C NMR (101 MHz, CDCl3) δ 145.28, 137.90, 129.03, 128.78, 128.18,
127.76, 127.29, 127.23, 127.22, 126.80, 123.76, 69.63, 57.47, 40.46, 36.50, 29.18, 25.32, 18.75.
(2ꢀBenzylpiperidinꢀ1ꢀyl)(4ꢀ(hydroxydiphenylmethyl)ꢀ2Hꢀ1,2,3ꢀtriazolꢀ2ꢀyl)methanone
(4). The title compound was isolated from the mixture of compound 3. The N2ꢀcarbamoyl
triazole regioisomer was purified by silica gel chromatography (pentane/EtOAc 100:1 → 5:1) to
afford 2,4ꢀtriazole urea 4 (39.0 mg, 0.086 mmol, 30% yield). HRMS [ESI+] m/z: calculated for
1
C28H28N4O2 [M+H]+ 453.2285, found: 453.2284. H NMR (400 MHz, CDCl3) δ 7.52 (s, 1H),
7.35 – 7.27 (m, 10H), 7.22 – 6.97 (m, 5H), 4.75 – 4.17 (m, 1H), 3.68 (br s, 1H), 3.26 – 3.19 (m,
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1H), 3.10 – 3.15 (m, 1H), 2.93 (br s, 1H), 1.79 – 1.60 (m, 6H). C NMR (101 MHz, CDCl3) δ
155.48, 149.55, 145.04, 137.91, 135.14, 129.07, 128.64, 128.21, 127.84, 127.22, 126.64, 69.61,
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53.28, 42.44, 36.31, 26.05, 25.24, 18.71.
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(4ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)(2ꢀethylpiperidinꢀ1ꢀyl)methanone (5). The
title compound was synthesized from 2ꢀethylpiperidine (0.059 mL, 0.442 mmol) and 4ꢀ([1,1'ꢀ
biphenyl]ꢀ4ꢀyl)ꢀ1Hꢀ1,2,3ꢀtriazole (98.0 mg, 0.442 mmol) according to the procedures described
for compound 3. The N1ꢀcarbamoyl triazole was isolated by silica gel chromatography
(pentane/EtOAc 100:1 → 5:1) to afford 1,4ꢀtriazole urea 5 (48.0 mg, 0.133 mmol, 30% yield).
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HRMS [ESI+] m/z: calculated for C22H24N4O [M+H]+ 361.2023, found: 361.2022. H NMR
(400 MHz, CDCl3) δ 8.35 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 7.66 – 7.61
(m, 2H), 7.47 – 7.44 (m, 2H), 7.38 ꢀ7.34 (m, 1H), 4.55 (q, J = 6.6 Hz, 1H), 4.29 (br d, J = 13.4
Hz, 1H), 3.20 (br s, 1H), 1.87 – 1.66 (m, 8H), 0.94 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
146.54, 141.42, 140.52, 128.93, 128.71, 127.70, 127.63, 127.08, 126.35, 120.95, 55.26, 53.87,
42.83, 28.15, 25.74, 22.60, 18.86, 10.70.
(4ꢀ([1,1'ꢀBiphenyl]ꢀ4ꢀyl)ꢀ2Hꢀ1,2,3ꢀtriazolꢀ2ꢀyl)(2ꢀethylpiperidinꢀ1ꢀyl)methanone (6). The
title compound was isolated from the mixture of compound 5. The N2ꢀcarbamoyl triazole
regioisomer was isolated by silica gel chromatography (pentane/EtOAc 100:1 → 5:1) to afford
2,4ꢀtriazole urea 6 (36.0 mg, 0.100 mmol, 23% yield). HRMS [ESI+] m/z: calculated for
1
C22H24N4O [M+H]+ 361.2023, found: 361.2024. H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H),
7.94 (d, J = 8.3 Hz, 2H), 7.69 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 7.7 Hz, 2H), 7.46 (t, J = 7.6 Hz,
2H), 7.38 (t, J = 7.5 Hz, 1H), 4.26 (br s, 2H), 3.16 (br s, 1H), 1.88 – 1.63 (m, 8H), 0.96 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 148.64, 142.12, 140.36, 132.95, 128.96, 128.25, 127.77, 127.70,
127.10, 126.90, 53.85, 43.21, 42.24, 28.01, 25.94, 22.78, 18.93, 10.76.
(2ꢀBenzylpiperidinꢀ1ꢀyl)(4ꢀ(dicyclohexyl(hydroxy)methyl)ꢀ2Hꢀ1,2,3ꢀtriazolꢀ2ꢀ
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yl)methanone (7). The title compound was synthesized from 2ꢀbenzylpiperidine (73.6 mg, 0.42
mmol) and dicyclohexyl(2Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl) methanol (122 mg, 0.462 mmol). According to
the procedures described for compound 3. The N2ꢀcarbamoyl triazole was isolated by silica gel
chromatography (pentane/EtOAc 100:1 → 5:1) to afford 2,4ꢀtriazole urea 7 (31.0 mg, 0.067
mmol, 16% yield). HRMS [ESI+] m/z: calculated for C28H40N4O2 [M+H+] 465.3224, found:
465.3221. 1H NMR (400 MHz, CDCl3) δ 7.59 (s, 1H), 7.37 – 7.04 (m, 5H), 4.59 (br s, 1H), 3.81
– 3.68 (m, 1H), 3.28 (td, J = 13.3, 2.9 Hz, 1H), 3.10 (dd, J = 13.3, 6.4 Hz, 1H), 2.99 (t, J = 11.6
Hz, 1H), 1.93 – 1.59 (m, 16H), 1.45 – 1.34 (m, 2H), 1.27 – 1.15 (m, 4H), 1.10 – 0.98 (m, 4H),
0.84 – 0.72 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 153.64, 149.83, 137.99, 134.24, 129.10,
128.60, 126.64, 78.22, 67.99, 43.88, 42.45, 36.10, 29.71, 27.22, 26.52, 26.42, 26.21, 25.46,
18.78.
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(2ꢀBenzylpiperidinꢀ1ꢀyl)(4ꢀ(1ꢀhydroxycyclohexyl)ꢀ2Hꢀ1,2,3ꢀtriazolꢀ2ꢀyl)methanone (8).
The title compound was synthesized from 2ꢀbenzylpiperidine (88.0 mg, 0.500 mmol) and 1ꢀ(2Hꢀ
1,2,3ꢀtriazolꢀ4ꢀyl)cyclohexanꢀ1ꢀol (92 mg, 0.550 mmol). According to the procedures described
for compound 3. The N2ꢀcarbamoyl triazole was isolated by silica gel chromatography
(pentane/EtOAc 100:1 → 5:1) to afford 2,4ꢀtriazole urea 8 (61.0 mg, 0.17 mmol, 34% yield).
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HRMS [ESI+] m/z: calculated for C21H28N4O2 [M+H+] 369.2285, found: 369.2288. H NMR
(400 MHz, CDCl3) δ 7.76 (s, 1H), 7.36 – 7.09 (m, 5H), 4.63 (br s, 1H), 4.14 (br s, 1H), 3.29 (td,
J = 13.2, 2.9 Hz, 1H), 3.16 (dd, J = 13.3, 6.1 Hz, 1H), 3.06 – 2.98 (m, 1H), 2.29 (br s, 1H), 2.02
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– 1.55 (m, 16H), 1.45 – 1.33 (m, 1H). C NMR (101 MHz, CDCl3) δ 157.07, 149.78, 138.07,
132.99, 129.11, 128.59, 126.61, 69.77, 53.47, 42.23, 38.01, 36.04, 26.62, 25.47, 25.25, 21.79,
18.78.
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