Chiral disubstituted piperidinyl ureas: A class of dual diacylglycerol lipase-α and ABHD6 inhibitors

Article abstract of DOI:10.1039/c7md00029d

Inhibitors of diacylglycerol lipases and α,β-hydrolase domain containing protein 6 (ABHD6) are potential leads for the development of therapeutic agents for metabolic and neurodegenerative disorders. Here, we report the enantioselective synthesis and structure activity relationships of triazole ureas featuring chiral, hydroxylated 2-benzylpiperidines as dual inhibitors of DAGLα and ABHD6. The chirality of the carbon bearing the C2 substituent, as well as the position of the hydroxyl (tolerated at C5, but not at C3) has profound influence on the inhibitory activity of both DAGLα and ABHD6, as established using biochemical assays and competitive activity-based protein profiling on mouse brain extracts.

Full text of DOI:10.1039/c7md00029d

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Chiral Disubstituted Piperidinyl Ureas: a Class of Dual
Diacylglycerol Lipase-α and ABHD6 Inhibitors†
Hui Denga, Tom van der Wela, Richard J. B. H. N. van den Bergb, Adrianus M.C.H. van den
Nieuwendijkb, Freek J. Janssena, Marc P. Baggelaara, Hermen S. Overkleeftb & Mario van der Stelta*
eceived 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Inhibitors of diacylglycerol lipases and α,β-hydrolase domain containing protein 6 (ABHD6) are potential leads for the
development of therapeutic agents for metabolic and neurodegenerative disorders. Here, we report the enantioselective
synthesis and structure activity relationships of triazole ureas featuring chiral, hydroxylated 2-benzylpiperidines as dual
inhibitors of DAGLα and ABHD6. The chirality of the carbon bearing the C2 substituent, as well as the position of the
hydroxyl (tolerated at C5, but not at C3) has profound influence on the inhibitory activity of both DAGLα and ABHD6, as
established using biochemical assays and competitive activity-based protein profiling on mouse brain extracts.
www.rsc.org/
brain, as well as anapyrexia and refeeding in fasted mice15. Of
note, most DAGL inhibitors cross-react with α,β-hydrolase
domain containing protein 6 (ABHD6), which has a minor role
Introduction
in
the
hydrolysis
of
2-AG16,
and acts
degrades
as
Diacylglycerol lipase
DAGL ) are intracellular, multi-domain, transmembrane serine
hydrolases that employ Ser-His-Asp catalytic triad to
α and diacylglycerol lipase β (DAGLα and
bis(monoacylglycero)phosphate17,
a
β
lysophosphatidyl hydrolase18. Inhibition of ABHD6 produces
neuroprotective, anti-obesity and anti-inflammatory effects in
preclinical disease models19,20. Thus, dual inhibition of DAGLs
and ABHD6 may actually be advantageous from a therapeutic
point of view.
a
specifically hydrolyse arachidonate-containing diglycerides to
form the endocannabinoid 2-arachidonoylglycerol (2-AG) in
the brain and peripheral tissues1,2. Endocannabinoid signalling
is involved in various neurophysiological functions, such as
learning, memory, pain sensation, adult neurogenesis and
regulation of the energy balance3-5. 2-AG is hydrolysed by
monoacylglycerol lipase into arachidonic acid, which is a
The design of DH376 and DO34 was inspired by (R)-KT109
21,22
(
2a)
, the first in vivo active DAGLβ inhibitor. Both
compounds are covalent irreversible inhibitors that feature a
2-benzylpiperidine moiety that confers selectivity and activity
towards DAGLs and ABHD6. We have reported previously an
enantioselective synthesis route for DH376 based on our
ample experience with the synthesis of chiral piperidines from
easily available starting materials following a strategy that
encompasses enzyme-catalysed cyanohydrin synthesis
precursor
for
pro-inflammatory
prostaglandins6-8
.
Consequently, the development of DAGL inhibitors that
perturb 2-AG production is an emerging strategy for potential
therapeutic intervention in various human diseases, including
metabolic
neuroinflammation9,10
Previously, we have reported the discovery of
ketoheterocycles11-13, glycinesulfonamides14 and triazole ureas
(e.g. DO34 and DH376 (
))15, as selective DAGL inhibitors (Fig.
syndrome
related
diseases
and
.
followed
by
a
transamination-reduction-ring-closing
α
-
metathesis series of events23-25
.
1
O
R
OH
1). DH376 and DO34 are brain active DAGL inhibitors that
reduce 2-AG levels in a time- and dose-dependent manner in
mouse brain. They also reduce lipopolysaccharide-induced
pro-inflammatory prostaglandin and cytokine levels in mouse
O
O
O
OH
OH
O
O
DAG lipases
2-AG
DAG
F
O
O
O
N
N
N
N
OCF3
N
N
N
N
OH
N
N
O
N
N
N
2a
(R)-KT109(
)
DO34
O
O
1
)
DH376 (
F
Fig. 1 Conversion of diacylglycerol (DAG) into 2-arachidonoylglycerol (2-AG) by
DAG lipases and chemical structures of their inhibitors DO34, DH376 and (R)-
KT109.
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Fig. 2 Design of compounds 3, 4a-7a, 4b-7b, 6c, 8 and 9-18 based on lead 2a.
Our strategy, as we demonstrated earlier in the synthesis
of polyhydroxylated piperidines (termed iminosugars), is
especially suited for the construction of chiral, enantiopure 2-
alkylpiperidines bearing one or more hydroxyl substituents.
We realised that, in this way, piperidinylureas bearing multiple
substituents, amongst which solubilizing hydroxyl groups,
would be easy to accomplish. To demonstrate the validity of
this reasoning, and to extend our panel of putative serine
hydrolase inactivators, we set out to make a small library of
chiral, disubstituted piperidinylureas. Here, these synthesis
efforts as well as the inhibitory potential of the resulting
Scheme 1. Reagents and conditions: (a) 10% Pd/C, H2, MeOH, 95%; (b) TBAF,
THF, r.t., 98% (21), 92% (24); (c) BnBr, TBAI, NaH, DMF, 90% (22), 90% (25); (d)
25% TFA, DCM, 84% (26), 85% (28); (e) DIPEA, triphosgene, THF, 0 oC; (f) DIPEA,
DMAP, 1,2,3-triazole, THF, 60 oC, 25% (
3), 30% (5a), 40% (30) over 2 steps; (g)
1,4-dioxane: H2O (2:1), biphenyl boronic acid, PdCl2(dppf), 80 oC, 75%; (h) HF-
pyridine, THF : pyridine = 1:1 (v/v), 20% over 3 steps (based on 28).
compounds 3, 4a-7a, 4b-7b, 6c, 8 and 9-18, in comparison with
lead compounds 2a and 2b against DAGLα and ABHD6 are
Following a related sequence of events, we obtained
compound 5a (Scheme 1). Compound 5b (the enantiomer of
5a) was synthesized in the same fashion as described for 5a
(see supporting information, Scheme. S1). For the synthesis of
compound 6a, we first prepared key intermediate 27 by
employing a previously reported method15,24. Subsequently,
removal of the Boc group using 25% (v/v) TFA in DCM
generated amine 28 that was directly coupled with 4-([1,1'-
reported.
Results and discussion
Chemistry
To systematically investigate the structure-activity relationship
of the covalent irreversible inhibitors, we focused our
attention first on the modification of 2-alkylpiperidine group,
biphenyl]-4-yl)-1H-1,2,3-triazole.
After
silica
gel
chromatography, 1,4-regioisomer 29 was isolated and ensuing
desilylation with HF-pyridine yielded target compound 6a
resulting 1,2,3-triazole ureas
3, 4a-7a, 4b-7b, 6c, 8 (Fig. 2,
Table 1 and 2). Next, we explored the influence of
(Scheme 1). In a similar manner, we prepared compounds 4a
,
electrophilicity of the leaving group (i.e. triazole scaffold) by
4b, 6b, 6c and 8 with different stereochemistry and
synthesizing compounds 9-18. The synthesis started with
substitution pattern on the piperidine ring (see for synthesis
details the supporting information). The synthesis of
compound 7b started with piperidene 33 that was prepared
according to previously reported method27,28. Deprotection of
33 with a catalytic amount of p-TsOH yielded diol intermediate
34 that was then regioselectively benzylated using boronic
amide as catalyst29 (Scheme 2). After O-silylation and N-Boc
deprotection, we obtained free amine 37 that was successfully
coupled with triazole using triphosgene. Finally, desylilation
compound 3, as a close homologue of lead compound 2a with
a methyloxy moiety inserted into the benzylic position. The
synthesis route commenced with O-TBDPS-protected
intermediate 19 that was prepared according to previously
established procedure26. Treatment of 19 with hydrogen and
10% Pd/C in MeOH gave hydrogenated intermediate 20, and
ensuing desilylation and benzylation of the primary alcohol
yielded Boc-protected intermediate 22 (Scheme 1). Removal of
the Boc group using 25% (v/v) TFA in DCM gave amine 23 in
near quantitative yield. Finally, triphosgene-mediated
condensation of 23 with 4-([1,1'-biphenyl]-4-yl)-1H-1,2,3-
triazole and isolation of the 1,4-regioisomer by silica gel
(HF-pyridine) gave target compound 7b. Compound 7a
(diastereoisomer of 7b) was obtained in the same fashion (see
supporting information).
Compounds 9-17 were prepared by triphosgene-mediated
condensation of free amine 26 with the appropriate
heterocycle.
chromatography provided compound
determined by chiral HPLC.
3 in >95% ee as
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Table 1. pIC50 ± SEM values of triazole ureas 3, 4a-7a, 4b-7b, 6c, and 8. Inhibition of recombinant human DAGLα or ABHD6 was measured by indicated
assays. Data represent average values ± SEM; n = 4 group.
pIC50 ± SEM
(DAGLα)
pIC50 ± SEM
(ABHD6)
pIC50 ± SEM
(DAGLα)
pIC50 ± SEM
(ABHD6)
Entry
2a
R
Entry
2b
R
9.1±0.1
7.1±0.1
9.1±0.1
8.6±0.1
8.5±0.1
8.6±0.1
7.4±0.1
7.8±0.1
7.1±0.1
6.2±0.1
8.5±0.2
7.6±0.1
3
8
4a
4b
5a
6a
7.6±0.1
7.6±0.1
7.9±0.1
8.3±0.1
5b
6b
5.9±0.2
7.0±0.1
6.5±0.1
<5
6c
7a
7.5±0.2
<5
8.0±0.1
6.1±0.1
7b
<5
6.6±0.1
As an example, heterocycle 17 (Scheme 1) was synthesized by
coupling of 26 with 3-bromo-1H-1,2,4-triazole followed by
Suzuki coupling with (1,1'-biphenyl)-4-ylboronic acid (Scheme
Biological evaluation
The potency of of
3, 4a-7a, 4b-7b, 6c, 8 and 9-18, as DAGLα
1). Finally, the para-nitrophenyl carbamate derivative 18 was inhibitors was established in a colorimetric assay using para-
prepared following a strategy as followed for heterocycle 5a
with 4-nitrophenol instead of 4-([1,1'-biphenyl]-4-yl)-1H-1,2,3-
triazole (see supporting information).
nitrophenylbutyrate as a surrogate substrate and membrane
fractions from HEK293T cells overexpressing recombinant
human DAGLα. As a reference, we report the biochemical data
of (R)-KT109 (2a)30, which we established to be more potent
than its enantiomer, (S)-KT109
(
2b)30. The same
stereochemistry at the C-2 position was preferred for
compounds tested (e.g. compare compounds 4a-6a vs 4b-6b).
A
30-100-fold drop in potency of benzyloxy-containing
compounds (3 and 5a) was found. This may suggest that a
lipophilic pocket in DAGLα, which accommodates the 2-
benzylpiperidine moiety, is restricted in size or, alternatively,
that a polar, flexible linker is less preferred. Introduction of
polar hydroxyl groups at other positions in the unsaturated
Scheme 2. Reagents and conditions: (a) cat. p-TsOH, MeOH, 86%; (b) BnBr,
K2CO3, KI, MeCN, 60 oC, 89%; (c) TBS-Cl, imidazole, DMF, 95%; (d) 10% TFA, DCM,
0 oC, 69%. (e) DIPEA, triphosgene, THF, 0 oC; (f) DIPEA, DMAP, 1,2,3-triazole, THF,
60 oC; (g) HF-pyridine, THF : pyridine = 1:1 (v/v), 15% over 3 steps.
piperidines (e.g. 4a vs 8) also reduced the activity over 20-fold.
Of note, introduction of a chiral hydroxyl group at the C-3
position of an unsaturated piperidine ring (compounds 7a and
7b) abolished the activity against DAGLα (pIC50 < 5), whereas a
hydroxyl at the C-5 position (compounds 6a and 6c) was
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Table 2. pIC50 ± SEM values of compounds 9-18. Inhibition of recombinant
human DAGLα or ABHD6 was measured by indicated assays. Data represent
average values ± SEM; n = 4 group.
O
O
N
R
pIC50 ± SEM
(DAGLα)
pIC50 ± SEM
(ABHD6)
Entry
9
R
6.8±0.1
7.8±0.1
6.8±0.1
7.5±0.1
10
11
Fig. 3 Graphical representation of DAGLα versus ABHD6 inhibition
(pIC50) compounds 3, 4a-7a, 4b-7b, 6c, 8 and 9-18.
7.8±0.1
7.8±0.1
12
13
7.6±0.1
<5
8.2±0.1
<5
compounds, we plotted their pIC50 values against both targets
(Fig. 3). Most of the compounds were dual DAGLα/ABHD6
inhibitors and a linear relationship (r2 = 0.85) for the potency
was observed. Compounds 6b, 7a and 7b were inactive against
14
15
<5
<5
<5
<5
DAGLα, but still showed inhibition against ABHD6 (pIC50 > 6).
Therefore, these compounds could be interesting starting
points for the discovery of selective ABHD6 inhibitors.
Finally, to evaluate the selectivity of compounds
3, 4a-7a,
4b
-
7b, 6c, 8 and 9-18 across a broad panel of serine hydrolases,
we applied activity-based protein profiling (ABPP) using mouse
brain membrane proteome. Fluorophosphonate (FP)-based
probes are routinely used in competitive ABPP experiments to
16
<5
<5
17
18
<5
<5
<5
<5
determine the selectivity of serine hydrolase inhibitors31,32,34
.
However, FP-based probes do not label DAGLα. MB064, a
Bodipy-tagged tetrahydrolipstatin based β-lactone probe, was
therefore previously developed by our group, to detect
endogenous DAGLα in brain proteomes31. Thus, we applied
both TAMRA-FP and MB064 to assess the activity and
selectivity of our dual DAGLα and ABHD6 inhibitors. In brief,
allowed. This suggests that the position of the chiral hydroxyl
group plays an important role in the binding site of DAGLα.
However, a change in conformation of the piperidine ring
induced by the double bond can also not be excluded to be
responsible for the decrease in potency. Of note, the
stereochemistry of the chiral hydroxyl at the C-5 position (6a
vs 6c) is not important for DAGL activity, which may suggest
that this functional group does not make any significant
interaction in the binding pocket and may protrude into a
we incubated inhibitors 3, 4a-7a, 4b-7b, 6c, 8 and 9-18 at 10
µM for 30 min with mouse brain membrane homogenates and
performed a gel-based ABPP assay using MB064 (0.25 µM, 20
min) or TAMRA-FP (0.5 µM, 20 min). Almost complete
blockade of DAGLα and ABHD6 was observed by compounds
3,
4a-6a, 4b, 6c and 8-12, which is consistent with the results of
the biochemical assay (Fig. 4a and Table 3). Most compounds
showed excellent selectivity over the other serine hydrolases
solvent exposed region. Compounds 10
-12 were equally
potent as compound 5a, but 9 showed ~10-fold less activity.
(Fig. 4). Compounds
3,
5a,
6c and
9-12 did, however, reduce
The pyrazoles (13 and 15), imidazoles (14 and 16), 1,2,4-
triazole (17) and carbamate (18) were inactive. This is in line
with a reduced electrophilicity of their warhead imparted by
the heterocycle15.
the labeling of DDHD2 (Fig. 4a), while compounds 6c
,
9
and 10
were non-selective and prevented the labelling of several
unknown off-targets (Fig. 4a and 4b).
To screen derivatives
3,
4a-
7a,
4b
-7b
,
6c,
8
and
9-18 for
ABHD6 inhibitory activity, we employed
a
real-time,
Conclusions
fluorescence-based natural substrate assay with membranes
from HEK293T cells expressing recombinant human ABHD6. In
general, the inhibitory potency of the compounds followed the
same trend as observed for DAGLα inhibition (Table 1 and 2).
To compare the DAGLα and ABHD6 activities of the
We reported the enantioselective synthesis and structure–
activity relationship studies of chiral, disubstituted
piperidinylureas as dual inhibitors of DAGLα and ABHD6. The
SAR studies revealed the stereochemistry of the C-2
4 | J. Name., 2012, 00, 1-3
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substitution on the piperidine ring plays an important role.
Incorporation of a hydroxyl group at the C-5 position on
piperidine ring maintained the activity against DAGLα and
ABHD6, whereas a hydroxyl at the C-3 position completely
abolished all DAGLα activity. Competitive activity-based
protein profiling confirmed the activity of the inhibitors against
endogenous DAGLα and ABHD6 and revealed differences in
the selectivity profile against other serine hydrolases.
Experimental section
Synthesis of compound
The synthesis and characterization of all final compounds
3,
4a-7a, 4b-7b, 6c, 8, 9-18 and intermediates are described in
the Electronic Supplementary Information (ESI).
Biological assays
Cloning Procedures
DAGLα and ABHD6 constructs were obtained as reported
previously31. Plasmids were isolated from transformed XL-10 Z-
competent cells (Maxi Prep, Qiagen) and verified by Sanger
sequencing (BaseClear). The sequences were confirmed by
sequence analysis at the Leiden Genome Technology Centre.
Fig. 4 (a-b) Selectivity profile of compounds 2a, 3, 4a-7a, 4b-7b, 6c, 8 and
18 (10 µM, 30 min) across mouse brain membrane serine hydrolases
as determined by competitive ABPP using two broad-spectrum probes
MB064 (0.25 µM, 20 min) (a) and FP-TAMRA (0.5 µM, 20 min) (b).
Coomassie staining gel were used as a loading control.
9-
Cell culture and membrane preparation
Cell culture was performed as previously reported31. 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 2-3
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
Table 3 . Inhibitory values for compounds 2a, 3, 4a-7a, 4b-7b, 6c, 8 and 9-18
(10 µM, 30 min) against native DAGLα and ABHD6 using competitive
activity–based protein profiling (ABPP) with probe MB064 (0.25 µM, 20
min). Data represent Means±SEM, n=3. Values are corrected for protein
loading per lane as determined by coomassie staining.
were transfected by the addition of
a 3:1 mixture of
Inhibition (%)
DAGLα
Inhibition (%)
polyethyleneimine (60µg) and plasmid DNA (20µg) in 2 mL
serum free medium. The medium was refreshed after 24
hours, and after 72 h 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.
Entry
Entry
ABHD6
95±1
96±0
96±1
96±0
95±2
18±9
96±2
90±3
84±5
50±7
95±2
DAGLα
ABHD6
83±2
2a
3
99±0
96±1
99±0
94±2
87±5
6±13
92±2
83±5
30±12
5±14
85±2
7b
9
18±11
89±5
89±2
4a
5a
6a
7a
8
10
11
12
13
14
15
16
17
18
95±2
93±1
93±3
94±1
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, 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). The protein concentration was
determined with Quick Start Bradford reagent (BioRad) or
QubitTM fluorometric quantitation (Life Technologies). The
protein fractions were diluted to a total protein concentration
of 1 mg/mL and stored in small aliquots at -80 oC until use.
93±3
86±2
ꢀ3±5
ꢀ3±4
ꢀ9±15
ꢀ14±18
ꢀ13±14
0±12
ꢀ15±15
ꢀ3±19
ꢀ28±15
ꢀ16±9
ꢀ12±9
4b
5b
6b
6c
ꢀ11±12
The biochemical hDAGLα assay was performed as reported
previously31. In brief, the biochemical hDAGLα activity assay is
based on the hydrolysis of para-nitrophenylbutyrate (PNP-
butyrate) by membrane preparations from HEK293T cells
transiently transfected with hDAGLα. Reactions were
Biochemical DAGL activity assay
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Brouwer, V. Di Marzo, H. S. Overkleeft and M. van der Stelt,
Angew. Chem. Int. Edit., 2013, 52, 12081-12085.
12. M. P. Baggelaar, P. J. P. Chameau, V. Kantae, J. Hummel, K. L.
Hsu, F. Janssen, T. van der Wel, M. Soethoudt, H. Deng, H. den
Dulk, M. Allara, B. I. Florea, V. Di Marzo, W. J. Wadman, C. G.
Kruse, H. S. Overkleeft, T. Hankemeier, T. R. Werkman, B. F.
Natural substrate based fluorescence assay (ABHD6).
The natural substrate assay was performed as reported
previously14,33. Standard assay conditions: 25 µM 2-AG, 0.2
U/mL glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO)
and horseradish peroxidase (HRP), 0.125 mM ATP, 10 µM
Amplifu™Red, 5% DMSO and 0.5% acetonitrile in a total
volume of 200 µL. The final protein (ABHD6) concentration is
40 µg/mL.
Preparation of mouse brain membrane proteome
Mouse brain membrane proteome preparation was performed
as previously reported15,31. 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
HEPES pH 7.2, 2 mM DTT, 1 mM MgCl2, 25 U/mL Benzonase)
and incubated for 5 min on ice, followed by low speed spin
(2,500 × g, 3 min, 4 oC) to remove debris. The supernatant was
subjected to ultracentrifugation (100,000 × g, 45 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 storage buffer B (20 mM
HEPES pH 7.2, 2 mM DTT). The total protein concentration was
determined with Quick Start Bradford reagent (Bio-Rad) or
QubitTM fluorometric quantitation (Life Technologies).
Membranes and supernatant were flash frozen in liquid
nitrogen and stored in aliquots at -80 oC until use.
Cravatt and M. van der Stelt, J. Am. Chem. Soc., 2015, 137
,
8851-8857.
Activity based protein profiling in mouse brain
13. F. J. Janssen, M. P. Baggelaar, J. J. A. Hummel, H. S. Overkleeft,
B. F. Cravatt, D. L. Boger and M. van der Stelt, J. Med. Chem.,
2015, 58, 9742-9753.
14. F. J. Janssen, H. Deng, M. P. Baggelaar, M. Allara, T. van der
Wel, H. den Dulk, A. Ligresti, A. C. van Esbroeck, R. McGuire, V.
Di Marzo, H. S. Overkleeft and M. van der Stelt, J. Med. Chem.,
2014, 57, 6610-6622.
15. D. Ogasawara, H. Deng, A. Viader, M. P. Baggelaar, A. Breman,
H. den Dulk, A. M. van den Nieuwendijk, M. Soethoudt, T. van
der Wel, J. Zhou, H. S. Overkleeft, M. Sanchez-Alavez, S. Mo,
W. Nguyen, B. Conti, X. Liu, Y. Chen, Q. S. Liu, B. F. Cravatt and
M. van der Stelt, Proc. Natl. Acad. Sci. USA, 2016, 113, 26-33.
16. W. R. Marrs, J. L. Blankman, E. A. Horne, A. Thomazeau, Y. H.
Lin, J. Coy, A. L. Bodor, G. G. Muccioli, S. S. J. Hu, G. Woodruff,
S. Fung, M. Lafourcade, J. P. Alexander, J. Z. Long, W. W. Li, C.
Xu, T. Moller, K. Mackie, O. J. Manzoni, B. F. Cravatt and N.
Stella, Nat. Neurosci., 2010, 13, 951-967.
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 500 nM (final concentration) ABP
TAMRA-FP 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.
Acknowledgements
This work was supported by grants from the Chinese
Scholarship Council (to H.D.); the Netherlands Research
Council– Chemical Sciences ECHO grant (to M.v.d.S.); and an
ECHO-STIP Grant (M.v.d.S.).
17. M. A. Pribasnig, I. Mrak, G. F. Grabner, U. Taschler, O.
Knittelfelder, B. Scherz, T. O. Eichmann, C. Heier, L. Grumet, J.
Kowaliuk, M. Romauch, S. Holler, F. Anderl, H. Wolinski, A.
Lass, R. Breinbauer, G. Marsche, J. M. Brown and R.
Zimmermann, J. Bio. Chem., 2015, 290, 29869-29881.
18. G. Thomas, J. L. Betters, C. C. Lord, A. L. Brown, S. Marshall, D.
Ferguson, J. Sawyer, M. A. Davis, J. T. Melchior, L. C. Blume, A.
C. Howlett, P. T. Ivanova, S. B. Milne, D. S. Myers, I. Mrak, V.
Leber, C. Heier, U. Taschler, J. L. Blankman, B. F. Cravatt, R. G.
Lee, R. M. Crooke, M. J. Graham, R. Zimmermann, H. A. Brown
Notes
The authors declare no competing interest.
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The enantioselective synthesis and structure-activity relationships of deoxy-iminosugar-based triazole
ureas as dual inhibitors of DAGLα and ABHD6 were reported.