Comprehensive Analysis of Structure-Activity Relationships of α-Ketoheterocycles as sn-1-Diacylglycerol Lipase α Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01627

Diacylglycerol lipase α (DAGLα) is responsible for the formation of the endocannabinoid 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLα inhibitors are required to study the physiological role of 2-AG. Previously, we identified the α-ketoheterocycles as potent and highly selective DAGLα inhibitors. Here, we present the first comprehensive structure-activity relationship study of α-ketoheterocycles as DAGLα inhibitors. Our findings indicate that the active site of DAGLα is remarkably sensitive to the type of heterocyclic scaffold with oxazolo-4N-pyridines as the most active framework. We uncovered a fundamental substituent effect in which electron-withdrawing meta-oxazole substituents increased inhibitor potency. (C₆-C₉)-acyl chains with a distal phenyl group proved to be the most potent inhibitors. The integrated SAR data was consistent with the proposed binding pose in a DAGLα homology model. Altogether, our results may guide the design of future DAGLα inhibitors as leads for molecular therapies to treat neuroinflammation, obesity, and related metabolic disorders.

Full text of DOI:10.1021/acs.jmedchem.5b01627

Article
pubs.acs.org/jmc
Comprehensive Analysis of Structure−Activity Relationships of
α‑Ketoheterocycles as sn-1-Diacylglycerol Lipase α Inhibitors
Freek J. Janssen,† Marc P. Baggelaar,† Jessica J. A. Hummel,† Herman S. Overkleeft,‡ Benjamin F. Cravatt,∥
Dale L. Boger,§ and Mario van der Stelt*,†
†Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden,
Netherlands
‡Department of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden,
Netherlands
§Department of Chemistry, The Scripps Research Institute, La Jolla, California 92037, United States
∥Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: Diacylglycerol lipase α (DAGLα) is responsible
for the formation of the endocannabinoid 2-arachidonoylgly-
cerol (2-AG) in the central nervous system. DAGLα inhibitors
are required to study the physiological role of 2-AG.
Previously, we identified the α-ketoheterocycles as potent
and highly selective DAGLα inhibitors. Here, we present the
first comprehensive structure−activity relationship study of α-
ketoheterocycles as DAGLα inhibitors. Our findings indicate
that the active site of DAGLα is remarkably sensitive to the
type of heterocyclic scaffold with oxazolo-4N-pyridines as the
most active framework. We uncovered a fundamental substituent effect in which electron-withdrawing meta-oxazole substituents
increased inhibitor potency. (C6−C9)-acyl chains with a distal phenyl group proved to be the most potent inhibitors. The
integrated SAR data was consistent with the proposed binding pose in a DAGLα homology model. Altogether, our results may
guide the design of future DAGLα inhibitors as leads for molecular therapies to treat neuroinflammation, obesity, and related
metabolic disorders.
activity, and hence 2-AG biosynthesis, are of great impor-
tance.11 In addition, these inhibitors may serve as valuable
probes to evaluate DAGLα as a novel target to treat human
conditions like obesity, diabetes, cardiovascular, and neuro-
degenerative diseases.7
INTRODUCTION
■
Sn-1-specific diacylglycerol lipases (DAGLs), of which two
isoforms exist (DAGLα and β), catalyze the formation of the
signaling lipid 2-arachidonoylglycerol (2-AG) from diacylgly-
cerols.1 2-AG is a full cannabinoid CB1 receptor agonist and
modulates synaptic plasticity at GABAergic and glutamatergic
synapses by regulating neurotransmitter release.2,3 In the
periphery, 2-AG is mainly produced by DAGLβ, a key enzyme
involved in the regulation of marcrophage pro-inflammatory
responses.4 2-AG signaling is linked to diet-induced obesity and
related metabolic disorders, as well as to addiction and
(neuro)inflammation.2,3,5
2-AG is hydrolyzed by monoacylglycerol lipase (MAGL), α/
β-hydrolase domain 6 and 12 (ABHD6, ABHD12) to give
arachidonic acid. Both 2-AG and arachidonic acid can be
converted by cyclooxygenase-2 into eicosanoids, including pro-
inflammatory prostaglandins (and their ester derivatives) that
contribute to neuroinflammation.6−9 The many players
involved in 2-AG metabolism signify that 2-AG and its
metabolic products have a wide array of physiological functions,
of which many are still poorly understood.10 For a better
understanding of 2-AG-mediated physiological processes, the
development of inhibitors that selectively perturb DAGL
Recently, we discovered α-ketoheterocycles as a novel and
highly potent class of DAGLα inhibitors.12,13 α-Ketohetero-
cycles have previously been applied to the discovery of potent
inhibitors of diverse serine and cysteine proteases such as fatty
acid amide hydrolase (FAAH),14 elastase,15−17 thrombin,18,19
factor Xa,20 chymase,21 tryptase,22 cathepsin K, and cathepsin
S.23,24 The α-ketoheterocycle scaffold provides an electrophilic
ketone group with tunable reactivity, as well as a structural
template to introduce important interactions with key amino
acids in the binding site to obtain potency and selectivity.24 α-
Ketoheterocycles have been shown to be orally bioavailable and
have entered human clinical trials and thus provide an
interesting scaffold for probe and drug discovery purposes.
We identified 1-(oxazolo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-
one (LEI104, OL-100)25 via pharmocophore-based screening
Received: October 16, 2015
© XXXX American Chemical Society
A
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approach as the first covalent and reversible inhibitor for
DAGLα. Compound 1 (LEI104) had an IC50 of 37 5 nM in a
colorimetric surrogate DAGLα substrate assay and was highly
selective over a panel of serine hydrolases as assessed by gel-
based competitive activity-based protein profiling (ABPP).
FAAH was identified as its only detected off-target, which was
not surprising because 1 was originally designed as a potent
inhibitor of FAAH,14 an enzyme that inactivates the other
endocannabinoid anandamide.
Table 1. Structure−Activity Relationship of Compounds
with a Varying Central Heterocyclic Scaffold (1−42)
Here, we report the first extensive structure−activity
relationship (SAR) study of α-ketoheterocycles as DAGLα
inhibitors. We screened a 1040-member, focused library of
FAAH inhibitors, mainly based on the α-ketoheterocycle
scaffold. We included newly synthesized analogues in our
screens, and by this means systematically investigated the
structural requirements for interaction of α-ketoheterocycles
with DAGLα.
RESULTS AND DISCUSSION
■
To investigate the SAR of 1, we screened a focused library
consisting of 1040 previously published α-ketoheterocycles and
their corresponding precursors14,26−33 using a colorimetric
DAGLα activity assay (Figure 1).12 In total, 64 active
Figure 1. Results of a focused library of serine hydrolase inhibitors.
The 1040 compounds were screened in duplicate (N = 2) at 10 μM
using a colorimetric 96-well assay in which para-nitrophenylbutyrate
was used as a surrogate substrate on membranes of HEK293T cells
overexpressing recombinant human DAGLα.12 A total of 64 actives
(indicated in green) showed >50% inhibitory activity and were
selected for further concentration response analysis.
compounds were identified with more than 50% inhibitory
activity at 10 μM final inhibitor concentration. These active
compounds were further analyzed in concentration response
experiments. To complement the SAR analysis of the focused
library, we synthesized and tested 19 additional α-ketohetero-
cycles (3−16, 106−110).12 The combined structure−activity
relationships are described in a topological fashion below.
Central Heterocyclic Scaffold Modifications. First, we
explored the influence of the central heterocyclic scaffold on
DAGLα activity. A diverse set of α-ketoheterocycles, including
benzoxazole (2), benzimidazole (3), benzothioazole (4), and
their 4-pyridine analogues (1, 5, and 6, respectively), were
analyzed. N4-Oxazolopyridine (1) was identified as the most
potent scaffold with a pIC50 of 7.4 0.05, whereas the other
scaffolds have a pIC50 < 6 (Table 1) or are completely inactive
(17−42). The imidazolopyridine (5) and thiazolopyridine (6)
are predicted to reduce electrophilicity of the C-2 carbonyl
(lower σi),12,13 thereby possibly reducing inhibitor activity.15,34
The basic nitrogen of the oxazolopyridine scaffold is another
important feature of the scaffold because its removal resulted in
a 100-fold drop in potency (compare 1 and 2). This
observation is corroborated by the fact that other regioisomers
of the oxazolopyridine scaffold (7 and 8) are also less potent
(Table 2). Introduction of other electron withdrawing groups
such as fluorine (9−12) and nitro-substituents (13, 14)
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inactive compounds. These results align with the observation,
noted above, that the pyridyl provides important interactions
with amino acids in the binding pocket. Activity of the oxazole
scaffold can be (partly) rescued, however, by introducing small
electron withdrawing groups at the meta-position (X4: 45−55)
but not at the para-position (X5: 62−75) (Table 2). A clear
correlation between the electron withdrawing effect of the
substituents and pIC50 was observed. A plot of the inhibition
(pIC50 values) versus the Hammet σm constants for the
substituents (Figure 2) was found to follow a well-defined
Table 2. Structure−Activity Relationship of Isoxazoles and
Oxadiazoles 43−10526,27,31−33
Figure 2. Effect of meta electron withdrawing substituent on potency
of oxazoles. Electron withdrawing effect of meta (X5) subsituted
oxazoles, Hammet constant σm versus pIC50 (for exact values see
Supporting Information). A linear correlation (R2 = 0.78) with a slope
ρ = 2.95 was found. Potency of inhibitors 46 and 49 (−CC(O)CH3
and −CC(O)tBu) is higher than predicted solely by the electron
withdrawing effect, indicating a potential additional interaction with
the enzyme.
correlation (r2 = 0.78) and the substituent effect was large (ρ =
2.95). This resulted in an almost 1000-fold increase in potency
per unit change in σm, which indicates that the electron
withdrawing effect of the substituent is the dominant factor
contributing to the rescue of inhibitory activity. This may be
explained by the increased electrophilic character of the
reacting C-2 ketone imparted by the electron withdrawing C-
4 substituent that leads to an increased strength of the covalent
transient state in which Ser472 of the enzyme forms a
hemiketal with the inhibitor, thereby increasing its affinity. The
magnitude of the effect is similar as that reported previously for
the activity of the α-ketoheterocycle inhibitors on FAAH, which
indicates that this is a fundamental relationship for α-
ketoheterocycles as serine hydrolase inhibitors.32
The aforementioned hypothesis allowed us to establish that
both the aldehyde (52) and trifluoromethylketone (45) inhibit
DAGLα as carbonyl active species and not as gem-diols, as
previously observed for the FAAH inhibitors,32 because the σm
values for CH(OH)2 and C(OH)2CF3 (0.02 and 0.33,
respectively) do not explain the observed inhibition, while
the σm values for C(O) and C(O)CF3 (0.35 and 0.63,
respectively) do correlate with DAGLα perturbation. The
variation in assay buffer pH (7.4 vs 9.0 for the DAGLα assay
and FAAH assay, respectively) may explain the observed
differences in hydration state of the activated ketones. Of note,
compounds featuring a methyl ketone (46) or a t-butyl ketone
(49) display higher than expected activity based on their σm
values. This would indicate that these inhibitors exhibit
additional H-bond or van der Waals interactions with the
enzyme. Remarkably, all para-substituted compounds (62−75)
that are able to directly conjugate with the electrophilic
carbonyl did not show any substantial inhibitor activity,
resulted in a 5−50-fold increased potency compared to their
benzoxazole analogue (2), but they did not reach the same
extent of inhibition as observed with the oxazolopyridine (1).
This indicates that the nitrogen at the 4-position is not only
required for its electron withdrawing properties but may also
have a specific interaction, likely an H-bond acceptor, with
amino acid residues in the binding site. Interestingly, halogens
at the 7-position in the heterocyclic scaffold (12, 16) are not
allowed.
Deconstruction of the oxazolopyridine scaffold by removal of
the pyridyl moiety is not allowed because using a simple
oxazole (43) or oxadiazole (44, 61) as scaffolds results in
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whereas the meta-substituted derivatives (45−60), which exert
their effects only through inductive electron-withdrawing
properties, do inhibit the enzyme. This might be explained by
steric hindrance of the para-substituents, thereby generating a
steric clash that precludes their interaction with DAGLα
(Figure 3B). The oxazoles could, however, at least theoretically,
Table 3. Structure−Activity Relation of C2-Acyl Derivatives
106−11112,14
entry
n
pIC50 SEM
cLogP
LipE
106
107
108
1
2
3
4
5
6
7
8
4.74 0.15
5.65 0.10
6.69 0.07
7.43 0.05
6.28 0.10
7.33 0.07
8.44 0.04
2.63
3.01
3.54
4.07
4.60
5.13
5.66
2.11
2.64
3.15
3.35
1.68
2.20
2.78
109
110
111
Figure 3. Proposed binding mode of substituted α-keto oxazoles. (A)
Meta (X4) substituted oxazole (exemplified by 45) forms a hydrogen
bond with His650 and positions the substituent away from the highly
sterically restricted pocket at X5. (B) Para (X5) substituted oxazoles
(exemplified by 62) have a potential steric clash with the enzyme,
possibly explaining their low potency on DAGLα. (C) If the oxazole
flips to avoid steric clash (para substituted), the oxazole nitrogen is
excluded from hydrogen bond formation with His650.
Table 4. Structure−Activity Relation of of C2-Acyl
derivatives 112−12314
simply flip their orientation in the active site, reversing the
location of the nitrogen and oxygen atom of the heterocycle in
a manner that places the substituent in a comparable location as
the meta-substituents. Apparently, this does not happen, which
indicates that the electron-withdrawing effect is required but
not sufficient to inhibit DAGLα with meta- or para-substituted
compounds. It also suggests that an additional interaction is
required with the meta-substituted compounds to perturb
DAGLα activity (Figure 3A), which cannot be formed by their
para-substituted analogues (Figure 3C). Indeed, the nitrogen of
the oxazole-scaffold has previously been implicated in H-
bonding with the histidine residue of the catalytic triad of
elastase.17 It is, therefore, reasonable to suggest that in our α-
ketoheterocycles the oxazole nitrogen is also required for its
interaction with the catalytic His650 from DAGLα. Finally,
substituted oxazoles with more sterically demanding side
groups (e.g., phenyl/pyridine, entries 77−87, such as potent
FAAH inhibitor OL-135)26 are not active on DAGLα, which
suggest that the meta-substituents are located in a pocket that is
restricted in size.
Length of C2-Acyl Substituents. To investigate the
influence of the C-2 acylphenyl spacer length, we measured the
activity of analogues (1, 106−111) in which the number of
methylene groups was increased from 2 to 8 (Table 3). The
inhibitory activity was higher with increasing number of
methylene groups and was highest at n = 8 (compound 111),
thereby making it the most potent inhibitor identified in this
compound 122 with an arachidonoyl substituent (C20:4)
displayed almost 100-fold less activity compared to its oleoyl
analogue. This might indicate that the C-2 acyl chain is located
in the hydrophobic channel that harbors the sn-1 acyl chain of
the natural substrate of DAGLα.
FAAH Off-Target Activity. The compounds of the focused
library were originally developed as FAAH inhibitors; therefore,
we have plotted the DAGLα pIC50 data of the hits against
previously reported FAAH pKi values (Figure 4). Most of the
hits are dual FAAH/DAGLα inhibitors and display high FAAH
activity (pKi > 8). Oleoyl-based benzimidazole (123, Table 4)
and compound 45 were the only two inhibitors selective over
FAAH, but they were only moderately potent in our DAGLα
assay (pIC50 < 7).
study with a pIC50 of 8.44
0.04. Compound 109 (n = 6)
exhibits a >10-fold drop in activity compared to compound 1 (n
= 5). The reason for this reduced activity is not easily explained.
When taking lipophilicity into account (i.e., lipophilic
efficiency: LipE = pIC50 − cLogP), the most efficient linker
length was n = 5 (compound 1). A similar trend was also
observed for α-ketoheterocycles in which the C-2 acyl chain
consisted of either saturated or monounsaturated fatty acids
(112−123, Table 4) Compounds bearing C2−C6 chains were
inactive, but DAGLα inhibition increased upon further
elongation of the acyl chain and was found to be optimal
with an oleoyl chain (C18:1) in compound 121. Interestingly,
Binding Mode. Previously, we reported the development of
a homology model for DAGLα and performed a molecular
dynamics simulation with 1 to understand its interaction with
hDAGLα at a molecular level (Figure 5).12 The model
represented the typical α,β-hydrolase fold and the catalytic
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withdrawing character of the functional group on the meta-
position of substituted oxazoles, but not on the para-position,
increased to large extent inhibitor potency. As previously
observed, the C-2 carbonyl (i.e., site of reversible covalent
attachment) is key to inhibitor activity and its reduction to an
hydroxyl group abolished DAGLα inhibition.12,13 Increasing C-
2 acyl chain length enhanced inhibitor activity and was optimal
for an oleoyl (C18:1) group, while an arachidonoyl (C20:4) chain
was less preferred. C6−C9 acyl chains with a distal phenyl group
yielded the most potent inhibitors. These detailed SAR results
provided valuable insight in the structural requirements for
DAGLα inhibition by α-ketoheterocycles and was fully
consistent with the proposed binding pose of 1 in our
homology model. We have successfully applied this homology
model to guide the design of new DAGLα inhibitors, which led
to the identification of 6-phenyl-1-(6-(p-tolyl)oxazolo[4,5-
b]pyridin-2-yl)hexan-1-one (LEI105) as a highly selective,
reversible, and dual DAGLα/DAGL-β inhibitor that was active
in cells and reduced cannabinoid CB1-receptor-dependent
synaptic plasticity.13 Our current efforts are directed toward
optimizing the physicochemical properties of the α-ketohetero-
cycles to improve their pharmacokinetic properties. Of note,
the reversible character of the α-ketoheterocycle inhibitors may
have less probability to induce idiosyncratic toxic side effects,
which may be associated with covalent irreversible inhibitors.
Consequently, α-ketoheterocycles may provide potential leads
for small molecule therapies to treat human conditions like
obesity, diabetes, cardiovascular, and neurodegenerative dis-
eases where high 2-AG signaling and/or 2-AG metabolite levels
play a crucial role.
Figure 4. Graphical representation of DAGLα inhibition (pIC50)
versus FAAH activity (pKi). FAAH activity is that reported in the
literature.14,26−33 For a list with exact values, see Supporting
Information.
Figure 5. Proposed binding mode of 1 in DAGLα.
EXPERIMENTAL SECTION
■
Experimental Procedures Computational Chemistry. The
homology model was constructed as previously reported based on the
S146A Mutant Of Thermomyces (Humicola) Lanuginosa Lipase in
Complex With Oleic Acid (PDB code 1GT6) as template.12
Experimental Procedures Biochemistry. Cloning Procedures.
Cloning procedures were performed as previously reported.12 In brief,
full-length human hDAGL-α cDNA was purchased from Biosource
and cloned into mammalian expression vector pcDNA3.1, containing
genes for ampicillin and neomycin resistance. The empty vector was
used as a negative control (mock). All plasmids were grown in XL-10
Z-competent cells and prepped (Maxi Prep, Qiagen). The sequences
were confirmed by sequence analysis at the Leiden Genome
Technology Centre.
Cell Culture and Membrane Preparation. Cell culture and
membrane preperations were performed as previously reported.12 In
brief, HEK293T cells were grown in DMEM with stable glutamine and
phenol red (PAA) with 10% new born calf serum, penicillin, and
streptomycin. Cells were passaged every 2−3 days by resuspension in
medium and seeding to the appropriate confluence. Membranes were
prepared from transiently transfected HEK293T cells. Twenty-four
hours 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
polyethylenimine (60 μg) and plasmid DNA (20 μg) in 2 mL of
serum free medium. The medium was refreshed after 24 h, and after
72 h the cells were harvested by suspending them in 20 mL of
medium. The supernatant was removed by centrifuge for 10 min at
1000 rpm. The cell pellet was quickly frozen in liquid nitrogen and
stored at −80 °C until use. Cell pellets were thawed on ice and
suspended in lysis buffer A (20 mM Hepes, pH 7.2, 2 mM DTT, 0.25
M sucrose, 1 mM MgCl2, 1× cocktail (Roche cOmplete EDTA free),
25 U/mL benzonase). The suspension was homogenized by polytrone
(3 × 7 s) and incubated for 30 min on ice. The membrane fraction was
separated by ultracentrifuge (100000g, 30 min, 4 °C, Beckman
Coulter, type Ti70 rotor), and the pellet was resuspended in lysis
triad, represented by Ser472, His650, and Asp524, was
appropriately aligned in the binding cavity. The tetrahedral
transition state of 1, which is formed through the nucleophilic
attack of Ser472 on the α-carbonyl, was minimized and
subjected to a short molecular dynamics refinement. The
oxyanion intermediate was stabilized by the backbone N−H of
Leu473 as well as by the backbone N−H and side chain O−H
of Thr400. The oxazole nitrogen of 1 formed hydrogen-bond
interactions with His650 and the pyridine nitrogen showed
hydrogen-bond interactions with His471 and His650, both of
which could further stabilize the tetrahedral intermediate.
These proposed hydrogen bonds are in line with the 4N-
oxazolopyridine being the optimal heterocycle. In addition,
aliphatic amino acids like Leu427 and Val419 line the
hydrophobic pocket, accommodating the flexible acyl chain of
1. This study indicates that this large pocket might normally
accommodate sn-1 acyl chain of the natural substrate. The
proposed binding mode is thus consistent with the observed
structure−activity relationships reported in this study.
CONCLUSIONS
■
Screening of an extensive focused library of α-ketoheterocyclic
FAAH inhibitors, combined with the synthesis and analysis of
novel α-ketoheterocycles, resulted in the rapid generation of a
comprehensive and detailed set of structure−activity relation-
ships for DAGLα inhibitors. We showed that the binding site of
DAGLα is remarkably sensitive to the type of α-ketohetero-
cycle with oxazolo-4N-pyridine as the optimal scaffold. The
potency of the α-ketoheterocycle is also strongly influenced by
a fundamental substituent effect in which the electron-
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buffer B (20 mM Hepes, pH 7.2, 2 mM DTT, 1× cocktail (Roche
cOmplete EDTA free)). The protein concentration was determined
with Qubit protein assay (Invitrogen). The total protein concentration
was diluted to 1 mg/mL, and the samples were quickly frozen in liquid
nitrogen and stored in small aliquots at −80 °C until use.
Biochemical hDAGL-α Activity Assay. The biochemical hDAGL-α
activity assay was performed as previously reported.12 In brief, the
biochemical hDAGL-α activity assay is based on the hydrolysis of para-
nitrophenylbutyrate (PNP-butyrate) by membrane preparations from
HEK293T cells transiently transfected with hDAGL-α. Reactions (200
μL) were performed in a flat bottom Greiner 96-well plates, 50 mM
HEPES pH 7.2 buffer with 0.05 μg/μL (final protein concentration)
hDAGL-α transfected membrane fractions.
chromatography to yield 1-(oxazolo[4,5-b]pyridin-2-yl)-6-phenylhex-
an-1-ol (34 mg, 0.58 mmol, 20%). Spectroscopic data are in agreement
with those previously reported.12
1-(Oxazolo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-one (1). The title
compound was synthesized from 1-(oxazolo[4,5-b]pyridin-2-yl)-6-
phenylhexan-1-ol (125) as previously reported.12
1-(Benzo[d]oxazol-2-yl)-6-phenylhexan-1-one (2). The title com-
pound was synthesized from 1-(benzo[d]oxazol-2-yl)-6-phenylhexan-
1-ol as previously reported.12
1-(1H-Benzo[d]imidazol-2-yl)-6-phenylhexan-1-ol (126). The title
compound was synthesized from 2-hydroxy-7-phenylheptanenitrile
(124, 100 mg, 0.49 mmol) and commercially available benzene-1,2-
diamine (55 mg, 0.51 mmol) according to the procedures described
for compound 125. This yielded 1-(1H-benzo[d]imidazol-2-yl)-6-
The focused library hit identification was performed using the 96-
well plate protocol. Compound plates (13 plates, N = 2) were
screened over a total of four days. A total of 68 actives were identified
(<50% activity at 10 μM inhibitor concentration, 6.54%).
1
phenylhexan-1-ol (136 mg, 0.46 mmol, 94%). H NMR (CDCl3, 400
MHz): δ 7.47 (dd, J = 7.0, 3.4 Hz, 2H), 7.25−7.11 (m, 3H), 7.06 (d, J
= 7.4 Hz, 2H), 6.77−6.65 (m, 2H), 5.29 (bs, 2H), 4.92 (d, J = 6.7 Hz,
2H), 2.43 (t, J = 7.7 Hz, 2H), 1.94−1.56 (m, 4H), 1.55−1.04 (m, 4H).
13C APT NMR (CDCl3, 101 MHz): δ 157.55, 142.67, 137.57, 134.67,
128.44 (2C), 128.31 (2C), 125.69, 122.81, 120.49, 116.92, 115.00,
68.43, 36.90, 35.86, 31.31, 29.04, 25.23.
Focused Library Dose Response Analysis. Dose response analysis
was performed on the 64 hits of the hit identification screen. The hits
were analyzed (10-fold serial dilution) using the above protocol12 with
minor adjustments for high throughput: 384-well plate, 50 μL total
volume, OD405 was measured after 60 min incubation with PNP-
butyrate (final concentration 0.3 mM) on an Envision plate reader.
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, and
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 coevaporation with toluene. 1H and 13C NMR spectra were
recorded on a Bruker AV 400 MHz spectrometer at 400.2 (1H) and
100.6 (13C) MHz using the reported deuterated solvent. Chemical
shift values are reported in ppm with tetramethylsilane or solvent
1-(1H-Benzo[d]imidazol-2-yl)-6-phenylhexan-1-one (3). The title
compound was synthesized from 1-(1H-benzo[d]imidazol-2-yl)-6-
phenylhexan-1-ol (126, 65 mg, 0.22 mmol) according to the
procedures described for compound 1. This yielded 1-(1H-benzo[d]-
imidazol-2-yl)-6-phenylhexan-1-one (22 mg, 0.075 mmol, 34%).
HRMS (ESI+) m/z: calculated for C19H21N2O ([M + H]),
1
293.1648; found, 293.1648. H NMR (CDCl3, 400 MHz): δ 10.75
(bs, 1H), 7.91 (bs, 1H), 7.55 (bs, 1H), 7.40 (bs, 2H), 7.29−7.23 (m,
2H), 7.21−7.13 (m, 3H), 3.31 (t, J = 7.5 Hz, 2H), 2.62 (t, J = 7.7 Hz,
2H), 1.85 (p, J = 7.5 Hz, 2H), 1.70 (p, J = 7.7 Hz, 2H), 1.55−1.41 (m,
2H). 13C BBDEC NMR (CDCl3, 101 MHz): δ 194.82, 147.60, 142.86
(bs), 142.52, 134.18 (bs), 128.40 (2C), 128.27 (2C), 125.66, 124.83
(bs, 2C), 121.42 (bs), 112.62 (bs), 38.36, 35.77, 31.30, 28.84, 23.82.
Purity of >95% as determined by LC/MS.
1
resonance as the internal standard (CDCl3: δ 7.26 for H, δ 77.16 for
13C; CD3OD: δ 3.31 for 1H, δ 49.00 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, quintet =
quint, b = broad, m = multiplet), coupling constants J (Hz), and
integration. High resolution mass spectra were recorded on a Thermo
Scientific LTQ Orbitrap XL. Compound purity (>95% unless stated
otherwise) was measured by liquid chromatography on a Finnigan
Surveyor LC/MS system equipped with a C18 column. Flash
chromatography was performed using SiliCycle silica gel type
SiliaFlash 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 phosphomo-
lybdic 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),
and H2O (600 mL)).
1-(Benzo[d]thiazol-2-yl)-6-phenylhexan-1-ol (127). The title
compound was synthesized from 2-hydroxy-7-phenylheptanenitrile
(124, 50 mg, 0.25 mmol) and commercially available 2-amino-
benzenethiol (0.03 mL, 0.28 mmol) according to the procedures
described for compound 125. This yielded 1-(benzo[d]thiazol-2-yl)-6-
1
phenylhexan-1-ol (63 mg, 0.20 mmol, 83%). H NMR (CDCl3, 400
MHz): δ 7.97 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 8.2, 1H), 7.51−7.41 (m,
1H), 7.41−7.32 (m, 1H), 7.31−7.21 (m, 2H), 7.21−7.10 (m, 4H),
5.08 (t, J = 7.9, 1H), 2.59 (t, J = 7.8, 2H), 2.11−1.26 (m, 8H). 13C
APT NMR (CDCl3, 101 MHz): δ 152.76, 142.61, 134.81, 130.91,
128.41 (2C), 128.26 (2C), 126.11, 125.64, 125.04, 122.86, 121.86,
72.29, 38.05, 35.83, 31.30, 29.01, 24.98.
1-(Benzo[d]thiazol-2-yl)-6-phenylhexan-1-one (4). The title com-
pound was synthesized from 1-(benzo[d]thiazol-2-yl)-6-phenylhexan-
1-ol (127, 50 mg, 0.16 mmol) according to the procedures described
for compound 1. This yielded 1-(1H-imidazo[4,5-b]pyridin-2-yl)-6-
phenylhexan-1-one (16 mg, 0.05 mmol, 32%). HRMS (ESI+) m/z:
calculated for C19H20NOS ([M + H]), 310.1260; found, 310.1260. 1H
NMR (CDCl3, 400 MHz): δ 8.23−8.13 (m, 1H), 8.01−7.93 (m, 1H),
7.61−7.48 (m, 2H), 7.31−7.23 (m, 2H), 7.21−7.13 (m, 3H), 3.27 (t, J
= 7.4 Hz, 2H), 2.63 (t, J = 7.8 Hz, 2H), 1.84 (p, J = 7.8 Hz, 2H), 1.70
(p, J = 7.8 Hz, 2H), 1.54−1.42 (m, 2H). 13C APT NMR (CDCl3, 101
MHz): δ 195.65, 166.67, 153.69, 142.65, 137.38, 128.53 (2C), 128.39
(2C), 127.73, 127.07, 125.78, 125.51, 122.57, 38.65, 35.88, 31.37,
28.96, 23.94. Purity of >95% as determined by LC/MS.
1-(1H-Imidazo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-ol (128). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 254 mg, 1.25 mmol) and commercially available pyridine-
2,3-diamine (54 mg, 0.40 mmol) according to the procedures
described for compound 125. This yielded 1-(1H-imidazo[4,5-
b]pyridin-2-yl)-6-phenylhexan-1-ol (24 mg, 0.08 mmol, 16%). 1H
NMR (MeOD, 400 MHz): δ 8.33 (d, J = 4.7 Hz, 1H), 7.95 (dd, J =
8.1, 1.5 Hz, 1H), 7.28 (dd, J = 8.0, 4.9 Hz, 1H), 7.23−7.17 (m, 2H),
7.16−7.05 (m, 3H), 4.98−4.91 (m, 1H), 2.57 (t, J = 8.0 Hz, 2H),
2.06−1.82 (m, 2H), 1.61 (p, J = 7.4 Hz, 2H), 1.51−1.42 (m, 2H),
2-Hydroxy-7-phenylheptanenitrile (124). The title compound was
synthesized from commercially available 6-phenylhexan-1-ol (1.70 g,
9.51 mmol) to yield 2-hydroxy-7-phenylheptanenitrile (1.67 g, 8.22
mmol, 86% over two steps) using previously reported procedures.
Spectroscopic data are in agreement with those previously reported.12
1-(Oxazolo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-ol (125). To a
dry round-bottom flask containing a solution of dry EtOH (1.4 mL,
17 mmol) and dry CHCl3 (1.4 mL) was added dropwise AcCl (1.4
mL, 19 mmol) at 0 °C under argon. The reaction mixture was stirred
for 30 min, after which a solution of 2-hydroxy-7-phenylheptanenitrile
(124, 117 mg, 0.58 mmol) in dried CHCl3 (1.0 mL) was added
dropwise at 0 °C under argon. The reaction mixture was stirred for 2
h, slowly warmed up to rt, and concentrated at 25 °C in vacuo. The
crude mixture was coevaporated with toluene (3 × 5 mL) until the
white solid immidate was obtained. The solid was dissolved in dry
EtOH (1.0 mL) and was added under argon to a sealed and dried
microwave tube containing a prestirred solution (80 °C for 30 min,
then to rt) of commercially available 2-amino-3-hydroxypyridine (68.8
mg, 0.63 mmol) with pyridine (50 μL, 0.63 mmol) in dry EtOH (4.0
mL). The reaction mixture was heated to reflux (80 °C) for 8 h. The
reaction mixture was concentrated in vacuo and purified by flash
F
DOI: 10.1021/acs.jmedchem.5b01627
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1.41−1.31 (m, 2H). 13C APT NMR (MeOD, 101 MHz): δ 162.22,
153.23 (bs), 144.53, 143.79, 131.27 (bs), 129.36 (2C), 129.22 (2C),
126.60, 124.00 (bs), 119.27, 69.40, 37.70, 36.76, 32.58, 29.99, 25.97.
1-(1H-Imidazo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-one (5). The
title compound was synthesized from 1-(1H-imidazo[4,5-b]pyridin-2-
yl)-6-phenylhexan-1-ol (128, 23.7 mg, 0.08 mmol) according to the
procedures described for compound 1. This yielded 1-(1H-imidazo-
[4,5-b]pyridin-2-yl)-6-phenylhexan-1-one (10 mg, 0.034 mmol, 43%).
HRMS (ESI+) m/z: calculated for C18H20N3O ([M + H]), 294.1601;
found, 294.1600. 1H NMR (CDCl3, 400 MHz): δ 14.92 (bs, 1H), 8.91
(d, J = 4.3 Hz, 1H), 8.30 (d, J = 8.1 Hz, 1H), 7.43 (dd, J = 8.2, 4.7 Hz,
1H), 7.29−7.24 (m, 2H), 7.21−7.14 (m, 3H), 3.32 (t, J = 8.0, 7.0 Hz,
2H), 2.65 (t, J = 7.6 Hz, 2H), 1.89 (p, J = 7.5 Hz, 2H), 1.78−1.65 (m,
2H), 1.57−1.45 (m, 2H). 13C APT NMR (CDCl3, 101 MHz): δ
194.41, 149.12, 147.18, 142.52, 136.08, 136.10, 130.78, 128.42 (2C),
128.27 (2C), 125.67, 119.55, 38.17, 35.78, 31.28, 28.87, 23.75. Purity
of >95% as determined by LC/MS.
2-Aminopyridine-3-thiol (129). Commercially available 3-(tert-
butylthio)pyridin-2-amine (235.4 mg, 1.291 mmol) was refluxed in
37% aq HCl (5 mL, 60.0 mmol) for 14 h until completion. The
mixture was concentrated in vacuo and coevaporated with toluene (3
× 20 mL). The resulting solid was taken up in satd NaHCO3 (40 mL),
extracted with EtOAc (3 × 20 mL), washed with brine, dried, and
concentrated in vacuo to obtain 2-aminopyridine-3-thiol (155 mg,
1.228 mmol, 95% yield) without further purification. 1H NMR
(MeOD, 400 MHz): δ 7.95 (dd, J = 5.0, 1.8 Hz, 1H), 7.32 (dd, J = 7.4,
1.8 Hz, 1H), 6.51 (dd, J = 7.5, 5.0 Hz, 1H). 13C BBDEC NMR
(MeOD, 101 MHz): 161.06, 150.74, 146.34, 114.42, 114.36.
1-(Thiazolo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-ol (130). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 52.3 mg 0.257 mmol) and 2-aminopyridine-3-thiol (129,
41.9 mg, 0.332 mmol) according to the procedures described for
compound 125. This yielded 1-(thiazolo[4,5-b]pyridin-2-yl)-6-phenyl-
hexan-1-ol (15.4 mg, 0.049 mmol, 19%). 1H NMR (CDCl3, 400
MHz): δ 8.68 (dd, J = 4.7, 1.6 Hz, 1H), 8.22 (dd, J = 8.0, 1.6 Hz, 1H),
7.30 (dd, J = 8.0, 4.7 Hz, 1H), 7.28−7.22 (m, 2H), 7.19−7.13 (m,
3H), 5.19 (dd, J = 8.0, 4.4 Hz, 1H), 3.97 (bs, 1H), 2.58 (t, J = 7.6 Hz,
2H), 2.12−1.86 (m, 2H), 1.69−1.48 (m, 4H), 1.46−1.35 (m, 2H). 13C
APT NMR (CDCl3, 101 MHz): δ 181.08, 163.86, 148.00, 142.72,
131.12, 128.65, 128.51 (2C), 128.36 (2C), 125.74, 119.85, 72.49,
37.88, 35.95, 31.40, 29.12, 24.99.
1-(Thiazolo[4,5-b]pyridin-2-yl)-6-phenylhexan-1-one (6). The
title compound was synthesized from 1-(thiazolo[4,5-b]pyridin-2-yl)-
6-phenylhexan-1-ol (130, 15.4 mg, 0.049 mmol) according to the
procedures described for compound 1. This yielded 1-(thiazolo[4,5-
b]pyridin-2-yl)-6-phenylhexan-1-one (10.9 mg, 0.035 mmol, 71%).
HRMS (ESI+) m/z: calculated for C18H19N2OS ([M + H]), 311.1213;
found, 311.1213. 1H NMR (CDCl3, 400 MHz): δ 8.89 (dd, J = 4.5, 1.7
Hz, 1H), 8.38 (dd, J = 8.2, 1.7 Hz, 1H), 7.47 (dd, J = 8.2, 4.6 Hz, 1H),
7.31−7.23 (m, 2H), 7.18 (d, J = 7.3 Hz, 3H), 3.35 (t, J = 7.4 Hz, 2H),
2.63 (t, J = 7.8 Hz, 2H), 1.86 (p, J = 7.5 Hz, 2H), 1.75−1.64 (m, 2H),
1.54−1.43 (m, 2H). 13C BBDEC NMR (CDCl3, 101 MHz): δ 195.55,
169.14, 163.78, 149.94, 142.60, 131.94, 131.48, 128.53 (2C), 128.40
(2C), 125.79, 122.10, 38.89, 35.87, 31.32, 28.96, 23.96. Purity of >95%
as determined by LC/MS.
h, slowly warmed up to rt, and concentrated at 25 °C in vacuo. The
immidate was dissolved in dry EtOH (1.0 mL) and was added under
argon to a sealed microwave tube containing 3-amino-4-hydroxypyr-
idine (131, 121 mg, 1.1 mmol) in dry EtOH (4.0 mL). The reaction
mixture was heated to reflux (80 °C) for 8 h using microwave
irridiation. The reaction mixture was concentrated in vacuo and
purified by flash chromatography to yield 1-(oxazolo[4,5-c]pyridin-2-
1
yl)-6-phenylhexan-1-ol (16 mg, 0.054 mmol, 5%). H NMR (CDCl3,
400 MHz): δ 9.03 (s, 1H), 8.56 (dd, J = 5.6, 1.4 Hz, 1H), 7.50 (dt, J =
5.6, 1.0 Hz, 1H), 7.28−7.24 (m, 2H), 7.21−7.10 (m, 3H), 5.00 (dd, J
= 7.6, 5.3 Hz, 1H), 2.60 (t, J = 7.2 Hz, 2H), 2.13−1.93 (m, 2H), 1.64
(p, 2H), 1.58−1.47 (m, 2H), 1.46−1.39 (m, 2H).13C APT NMR
(CDCl3, 101 MHz): δ 168.81, 156.07, 145.56, 142.85, 142.59, 138.46,
128.49 (2C), 128.38 (2C), 125.80, 106.91, 68.04, 35.91, 35.52, 31.33,
28.97, 24.92.
1-(Oxazolo[4,5-c]pyridin-2-yl)-6-phenylhexan-1-one (7). To a
solution of 1-(oxazolo[4,5-c]pyridin-2-yl)-6-phenylhexan-1-ol (132,
15 mg, 0.050 mmol) in dry CH2Cl2 (3 mL) was added Dess−Martin
periodinane (43 mg, 0.1 mmol). The reaction mixture was stirred for
10 h and quenched with 5 mL of satd NaHCO3 (aq) upon completion.
The organic layer was washed with satd NaHCO3 (aq), brine, dried on
MgSO4, filtered, concentrated in vacuo, and purified by flash
chromatography to obtain 1-(oxazolo[4,5-c]pyridin-2-yl)-6-phenyl-
hexan-1-one (9.7 mg, 0.033 mmol, 66%). HRMS (ESI+) m/z:
1
calculated for C18H19N2O2 (M + H+) 295.1441; found 295.1440. H
NMR (CDCl3, 400 MHz): δ 9.26 (s, 1H), 8.77−8.70 (d, J = 3.6 Hz,
1H), 7.64 (d, J = 5.6 Hz, 1H), 7.29−7.25 (m, 2H), 7.19−7.15 (m,
3H), 3.23 (t, J = 7.4 Hz, 2H), 2.64 (t, J = 7.7 Hz, 2H), 1.87 (p, J = 7.5
Hz, 2H), 1.69 (p, J = 7.7 Hz, 2H), 1.53−1.42 (m, 2H). 13C APT NMR
(CDCl3, 101 MHz): δ 189.78, 157.42, 155.66, 148.03, 145.64, 142.46,
128.51 (2C), 128.42 (2C), 125.85, 107.73, 39.77, 35.82, 31.26, 28.78,
23.71. Purity of 90% as determined by LC/MS.
1-(Oxazolo[5,4-b]pyridin-2-yl)-6-phenylhexan-1-ol (133). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 59 mg, 0.29 mmol) and commercially available 3-amino-2-
hydroxypyridine (35 mg, 0.31 mmol) according to the procedures
described for compound 132. This yielded 1-(oxazolo[5,4-b]pyridin-2-
1
yl)-6-phenylhexan-1-ol (5 mg, 0.018 mmol, 6%). H NMR (CDCl3,
400 MHz): δ 8.36 (dd, J = 5.0, 1.6 Hz, 1H), 8.03 (dd, J = 7.9, 1.6 Hz,
1H), 7.35 (dd, J = 7.8, 5.0 Hz, 1H), 7.30−7.27 (m, 2H), 7.20−7.14
(m, 3H), 5.01−4.95 (m, 1H), 2.60 (t, J = 7.6 Hz, 2H), 2.14−1.90 (m,
2H), 1.82−1.73 (m, 2H), 1.60−1.50 (m, 4H). 13C APT NMR (CDCl3,
101 MHz): δ 168.19, 159.96, 144.98, 142.65, 132.46, 128.82 (2C),
128.51 (2C), 128.39, 125.78, 121.16, 68.30, 35.93, 35.45, 31.37, 29.02,
24.85.
1-(Oxazolo[5,4-b]pyridin-2-yl)-6-phenylhexan-1-one (8). The title
compound was synthesized from 1-(oxazolo[5,4-b]pyridin-2-yl)-6-
phenylhexan-1-ol (133, 5 mg, 0.018 mmol) according to the
procedures described for compound 7. This yielded 1-(oxazolo[5,4-
b]pyridin-2-yl)-6-phenylhexan-1-one (3 mg, 0.010 mmol, 57%).
HRMS (ESI+) m/z: calculated for C18H19N2O2 (M + H+),
295.1441; found, 295.1441. 1H NMR (CDCl3, 400 MHz): δ 8.58
(dd, J = 4.9, 1.6 Hz, 1H), 8.24 (dd, J = 8.0, 1.6 Hz, 1H), 7.48 (dd, J =
8.0, 4.9 Hz, 1H), 7.30−7.24 (m, 2H), 7.19−7.16 (m, 3H), 3.21 (t, J =
7.4 Hz, 2H), 2.64 (t, J = 7.6 Hz, 2H), 1.85 (p, J = 7.5 Hz, 2H), 1.75−
1.66 (m, 2H), 1.50−1.44 (m, 2H). 13C APT NMR (CDCl3, 101
MHz): δ 189.73, 156.64, 148.66, 142.39, 132.41, 131.24, 128.40 (2C),
128.30 (2C), 125.72, 122.15, 39.40, 35.71, 31.15, 28.68, 23.56. Purity
of >95% as determined by LC/MS.
1-(4-Fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol (134). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 100 mg, 0.49 mmol) and commercially available 2-amino-3-
fluorophenol (62 mg, 0.49 mmol) according to the procedures
described for compound 125. This yielded 1-(4-fluorobenzo[d]oxazol-
2-yl)-6-phenylhexan-1-ol (99 mg, 0.32 mmol, 65%). 1H NMR (CDCl3,
400 MHz): δ 7.34−7.29 (m, 1H), 7.29−7.23 (m, 3H), 7.19−7.13 (m,
3H), 7.05 (ddd, J = 9.3, 7.8, 1.3 Hz, 1H), 4.97 (dd, J = 7.7, 5.2 Hz,
1H), 2.60 (t, J = 7.6 Hz, 2H), 2.09−1.91 (m, 2H), 1.68−1.60 (m, 2H),
1.56−1.46 (m, 2H), 1.44−1.37 (m, 2H). 13C APT NMR (CDCl3, 101
MHz): δ 168.28, 153.47 (d, J = 256.54 Hz), 153.03 (d, J = 7.1 Hz),
3-Amino-4-hydroxypyridine (131). To a solution of commercially
available 4-hydroxy-3-nitropyridine (500 mg, 3.58 mmol) in methanol
(25 mL) was added 100 mg of 10% Pd/C. The reaction mixture was
stirred under hydrogen atmosphere for 10 h. Upon completion, the
solution was filtered and concentrated in vacuo to obtain 3-amino-4-
1
hydroxypyridine (350 mg, 3.18 mmol, 89%). H NMR (DMSO-d6,
400 MHz): δ 7.34 (dd, J = 6.7, 1.6 Hz, 1H), 7.12 (s, 1H), 5.99 (d, J =
6.6 Hz, 1H), 4.52 (s, 2H).
1-(Oxazolo[4,5-c]pyridin-2-yl)-6-phenylhexan-1-ol (132). To a
dry round-bottom flask containing a solution of dry EtOH (1.0 mL,
17 mmol) and dry CHCl3 (1.0 mL) was added dropwise AcCl (1.0
mL, 14 mmol) at 0 °C under argon. The reaction mixture was stirred
for 30 min, after which a solution of 2-hydroxy-7-phenylheptanenitrile
(124, 213 mg, 1.05 mmol) in dry CHCl3 (1.0 mL) was added
dropwise at 0 °C under argon. The reaction mixture was stirred for 2
G
DOI: 10.1021/acs.jmedchem.5b01627
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Journal of Medicinal Chemistry
Article
142.67, 129.23 (d, J = 16.2 Hz), 128.49 (2C), 128.36 (2C), 125.76 (d,
J = 7.1 Hz), 125.74, 110.96 (d, J = 17.6 Hz), 107.07 (d, J = 4.5 Hz),
68.09, 35.91, 35.55, 31.36, 28.99, 24.90.
(2C), 125.78, 120.41 (d, J = 10.1 Hz), 112.69 (d, J = 24.2 Hz), 99.01
(d, J = 28.3 Hz), 68.11, 35.93, 35.52, 31.38, 29.01, 24.92.
1-(6-Fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-one (11). The
title compound was synthesized from 1-(6-fluorobenzo[d]oxazol-2-yl)-
6-phenylhexan-1-ol (137, 44 mg, 0.14 mmol) according to the
procedures described for compound 7. This yielded 1-(6-fluorobenzo-
[d]oxazol-2-yl)-6-phenylhexan-1-one (37 mg, 0.12 mmol, 85%).
HRMS (ESI+) m/z: calculated for C19H19FNO2 ([M + H]),
1-(4-Fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-one (9). The
title compound was synthesized from 1-(4-fluorobenzo[d]oxazol-2-
yl)-6-phenylhexan-1-ol (134, 63 mg, 0.20 mmol) according to the
procedures described for compound 7. This yielded 1-(4-fluorobenzo-
[d]oxazol-2-yl)-6-phenylhexan-1-one (41 mg, 0.13 mmol, 66%).
HRMS (ESI+) m/z: calculated for C19H19FNO2 ([M + H]),
1
312.1394; found, 312.1393. H NMR (CDCl3, 400 MHz): δ 8.23−
1
8.13 (m, 1H), 8.01−7.93 (m, 1H), 7.61−7.48 (m, 2H), 7.31−7.23 (m,
2H), 7.21−7.13 (m, 3H), 3.27 (t, J = 7.4 Hz, 2H), 2.63 (t, J = 7.8 Hz,
2H), 1.84 (p, J = 7.8 Hz, 2H), 1.70 (p, J = 7.8 Hz, 2H), 1.54−1.42 (m,
2H). 13C APT NMR (CDCl3, 101 MHz): δ 189.79, 162.71 (d, J =
250.5 Hz), 158.04 (d, J = 4.0 Hz), 151.03 (d, J = 15.2 Hz), 142.50,
136.98 (d, J = 1.0 Hz), 128.49 (2C), 128.38 (2C), 125.80, 123.11 (d, J
= 10.1 Hz), 114.66 (d, J = 25.3 Hz), 99.73 (d, J = 27.3 Hz), 39.48,
35.82, 31.25, 28.81, 23.79. Purity of >95% as determined by LC/MS.
1-(7-Fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol (138). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 124 mg, 0.61 mmol) and commercially available 2-amino-6-
fluorophenol (75 mg, 0.59 mmol) according to the procedures
described for compound 125. This yielded 1-(7-fluorobenzo[d]oxazol-
2-yl)-6-phenylhexan-1-ol (157 mg, 0.50 mmol, 85%). 1H NMR
(CDCl3, 400 MHz): δ 7.50 (dd, J = 8.0, 0.9 Hz, 1H), 7.33−7.22 (m,
3H), 7.20−7.13 (m, 3H), 7.13−7.06 (m, 1H), 4.97 (dd, J = 7.7, 5.1
Hz, 1H), 3.06 (bs, 1H), 2.60 (t, J = 7.9 Hz, 2H), 2.19−1.88 (m, 2H),
1.73−1.58 (m, 2H), 1.58−1.46 (m, 2H), 1.46−1.33 (m, 2H). 13C
BBDEC NMR (CDCl3, 101 MHz): δ 168.34, 147.16 (d, J = 253.5
Hz), 143.82 (d, J = 2.0 Hz), 142.66, 138.05 (d, J = 11.1 Hz), 128.51
(2C), 128.38 (2C), 125.78, 125.15 (d, J = 5.0 Hz), 115.93 (d, J = 4.0
Hz), 112.17 (d, J = 16.2 Hz), 68.13, 35.93, 35.60, 31.38, 29.00, 24.90.
1-(7-Fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-one (12). The
title compound was synthesized from 1-(7-fluorobenzo[d]oxazol-2-yl)-
6-phenylhexan-1-ol (138, 46 mg, 0.15 mmol) according to the
procedures described for compound 1. This yielded 1-(7-fluorobenzo-
[d]oxazol-2-yl)-6-phenylhexan-1-one (39 mg, 0.13 mmol, 86%).
HRMS (ESI+) m/z: calculated for C19H19FNO2 ([M + H]),
312.1394; found, 312.1395. 1H NMR (CDCl3, 400 MHz): δ 7.68
(dd, J = 8.1, 1.0 Hz, 1H), 7.41 (td, J = 8.2, 4.5 Hz, 1H), 7.31−7.24 (m,
3H), 7.21−7.14 (m, 3H), 3.22 (t, J = 7.4 Hz, 2H), 2.64 (t, J = 7.9 Hz,
2H), 1.85 (p, J = 7.5 Hz, 2H), 1.74−1.65 (m, 2H), 1.52−1.41 (m,
2H). 13C BBDEC NMR (CDCl3, 101 MHz): δ 189.70, 157.49, 147.54
(d, J = 255.5 Hz), 143.65 (d, J = 1.0 Hz), 142.53, 138.34 (d, J = 11.1
Hz) 128.53 (2C), 128.42 (2C), 126.26 (d, J = 6.1 Hz), 125.84, 118.10
(d, J = 4.04 Hz), 114.89 (d, J = 16.2 Hz), 39.84, 35.85, 31.31, 28.81,
23.72. Purity of >95% as determined by LC/MS.
1-(5-Nitrobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol (139). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 131 mg, 0.64 mmol) and commercially available 2-amino-4-
nitrophenol (99 mg, 0.64 mmol) according to the procedures
described for compound 125. This yielded 1-(5-nitrobenzo[d]-
oxazol-2-yl)-6-phenylhexan-1-ol (120 mg, 0.35 mmol, 55%). 1H
NMR (CDCl3, 400 MHz): δ 8.58 (d, J = 2.2 Hz, 1H), 8.30 (dd, J =
8.9, 2.2 Hz, 1H), 7.62 (d, J = 8.9 Hz, 1H), 7.30−7.23 (m, 2H), 7.18−
7.13 (m, 3H), 5.01 (dd, J = 7.7, 5.2 Hz, 1H), 2.60 (t, J = 7.2 Hz, 2H),
2.11−1.93 (m, 2H), 1.64 (p, J = 7.8 Hz, 2H), 1.57−1.50 (m, 2H),
1.45−1.37 (m, 2H). 13C APT NMR (CDCl3, 101 MHz): δ 171.10,
154.28, 145.41, 142.49, 140.96, 128.42 (2C), 128.33 (2C), 125.75,
121.43, 116.49, 111.18, 68.06, 35.84, 35.41, 31.27, 28.90, 24.87.
1-(5-Nitrobenzo[d]oxazol-2-yl)-6-phenylhexan-1-one (13). The
title compound was synthesized from 1-(5-nitrobenzo[d]oxazol-2-
yl)-6-phenylhexan-1-ol (139, 74 mg, 0.22 mmol) according to the
procedures described for compound 7. This yielded 1-(5-nitrobenzo-
[d]oxazol-2-yl)-6-phenylhexan-1-one (55 mg, 0.16 mmol, 74%).
HRMS (ESI+) m/z: calculated for C38H37N4O8 ([2 M + H]),
677.2606; found, 677.2605. 1H NMR (CDCl3, 400 MHz): δ 8.79 (d, J
= 2.2 Hz, 1H), 8.47 (dd, J = 9.0, 2.3 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H),
7.29−7.24 (m, 2H), 7.21−7.15 (m, 3H), 3.24 (t, J = 7.4 Hz, 2H), 2.64
(t, J = 7.2 Hz, 2H), 1.86 (p, J = 7.5 Hz, 2H), 1.76−1.66 (m, 2H),
1.53−1.43 (m, 2H) 13C APT NMR (CDCl3, 101 MHz): δ 189.60,
312.1394; found, 312.1393. H NMR (CDCl3, 400 MHz): δ 8.23−
8.13 (m, 1H), 8.01−7.93 (m, 1H), 7.61−7.48 (m, 2H), 7.31−7.23 (m,
2H), 7.21−7.13 (m, 3H), 3.27 (t, J = 7.4 Hz, 2H), 2.63 (t, J = 7.8 Hz,
2H), 1.84 (p, J = 7.8 Hz, 2H), 1.70 (p, J = 7.8 Hz, 2H), 1.54−1.42 (m,
2H). 13C APT NMR (CDCl3, 101 MHz): δ 190.01, 157.23, 154.85 (d,
J = 260.6 Hz), 152.67 (d, J = 6.1 Hz), 142.52, 129.79 (d, J = 20.2 Hz),
129.16 (d, J = 7.1 Hz), 128.51 (2C), 128.40 (2C), 125.81, 111.73 (d, J
= 17.2 Hz), 108.16 (d, J = 5.1 Hz), 39.56, 35.83, 31.29, 28.78, 23.72.
Purity of >95% as determined by LC/MS.
1-(5-Fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol (135). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 124 mg, 0.61 mmol) and commercially available 2-amino-4-
fluorophenol (72 mg, 0.57 mmol) according to the procedures
described for compound 132. This yielded 1-(5-fluorobenzo[d]oxazol-
2-yl)-6-phenylhexan-1-ol (133 mg, 0.42 mmol, 75%). 1H NMR
(CDCl3, 400 MHz): δ 7.45 (dd, J = 8.9, 4.2 Hz, 1H), 7.39 (dd, J = 8.3,
2.6 Hz, 1H), 7.30−7.22 (m, 2H), 7.20−7.12 (m, 3H), 7.08 (td, J = 9.1,
2.6 Hz, 1H), 4.94 (dd, J = 7.6, 5.1 Hz, 1H), 2.70 (bs, 1H), 2.60 (t, J =
7.8 Hz, 2H), 2.12−1.87 (m, 2H), 1.70−1.57 (m, 2H), 1.56−1.45 (m,
2H), 1.45−1.33 (m, 2H). 13C BBDEC NMR (CDCl3, 101 MHz): δ
169.65, 160.15 (d, J = 241.4 Hz), 147.24 (d, J = 1.0 Hz), 142.65,
141.35 (d, J = 13.1 Hz), 128.51 (2C), 128.39 (2C), 125.78, 113.05 (d,
J = 26.3 Hz), 111.26 (d, J = 10.1 Hz), 106.66 (d, J = 25.3 Hz), 68.21,
35.93, 35.58, 31.39, 29.01, 24.89.
1-(5-Fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-one (10). The
title compound was synthesized from 1-(5-fluorobenzo[d]oxazol-2-yl)-
6-phenylhexan-1-ol (135, 44 mg, 0.14 mmol) according to the
procedures described for compound 7. This yielded 1-(5-fluorobenzo-
[d]oxazol-2-yl)-6-phenylhexan-1-one (33 mg, 0.10 mmol, 74%).
HRMS (ESI+) m/z: calculated for C19H19FNO2 ([M + H]),
312.1394; found, 312.1395. 1H NMR (CDCl3, 400 MHz): δ 7.61
(dd, J = 9.0, 4.2 Hz, 1H), 7.56 (dd, J = 8.0, 2.5 Hz, 1H), 7.32−7.24 (m,
2H), 7.21−7.13 (m, 3H), 3.20 (t, J = 7.4 Hz, 2H), 2.64 (t, J = 7.5 Hz,
2H), 1.84 (p, J = 7.5 Hz, 2H), 1.75−1.63 (m, 2H), 1.52−1.41 (m,
2H). 13C BBDEC NMR (CDCl3, 101 MHz): δ 190.10, 160.66 (d, J =
244.4 Hz), 158.69, 147.20 (d, J = 1.0 Hz), 142.53, 141.35 (d, J = 14.1
Hz), 128.53 (2C), 128.42 (2C), 125.84, 116.97 (d, J = 27.3 Hz),
112.71 (d, J = 10.1 Hz), 108.28 (d, J = 25.3 Hz), 39.63, 35.85, 31.31,
28.82, 23.75. Purity of >95% as determined by LC/MS.
2-Amino-5-fluorophenol (136). The title compound was synthe-
sized from commercially available 5-fluoro-2-nitrophenol (500 mg,
3.18 mmol) according to the procedures described for compound 131.
1
This yielded 2-amino-5-fluorophenol (388 mg, 3.05 mmol, 96%). H
NMR (DMSO-d6, 400 MHz): δ 6.53 (dd, J = 8.3, 6.3 Hz, 1H), 6.46
(dd, J = 10.3, 2.6 Hz, 1H), 6.35 (td, J = 8.7, 2.7 Hz, 1H). 13C APT
NMR (DMSO-d6, 101 MHz): δ 154.63 (d, J = 230.8 Hz), 144.99,
133.47, 114.32 (d, J = 9.3 Hz), 105.16 (d, J = 21.3 Hz), 102.28 (d, J =
24.9 Hz).
1-(6-Fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol (137). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 110 mg, 0.54 mmol) and 2-amino-5-fluorophenol (136, 72
mg, 0.57 mmol) according to the procedures described for compound
132. This yielded 1-(6-fluorobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol
(66 mg, 0.21 mmol, 39%). 1H NMR (CDCl3, 400 MHz): δ 7.62 (dd, J
= 8.8, 4.8 Hz, 1H), 7.29−7.25 (m, 2H), 7.24−7.21 (m, 1H), 7.19−7.13
(m, 3H), 7.08 (ddd, J = 9.5, 8.7, 2.4 Hz, 1H), 4.93 (dd, J = 7.6, 5.2 Hz,
1H), 2.59 (t, J = 6.4 Hz, 2H), 2.08−1.89 (m, 2H), 1.63 (p, J = 7.8 Hz,
2H), 1.55−1.46 (m, 2H), 1.44−1.36 (m, 2H). 13C APT NMR (CDCl3,
101 MHz): δ 168.45 (d, J = 4.0 Hz), 160.72 (d, J = 244.4 Hz), 150.84
(d, J = 15.2 Hz), 142.65, 136.83 (d, J = 1.0 Hz), 128.50 (2C), 128.38
H
DOI: 10.1021/acs.jmedchem.5b01627
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Journal of Medicinal Chemistry
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159.38, 153.98, 146.13, 142.38, 140.83, 128.45 (2C), 128.36 (2C),
125.80, 124.05, 118.84, 112.55, 39.74, 35.76, 31.19, 28.71, 23.61.
Purity of >95% as determined by LC/MS.
(dd, J = 7.9, 1.4 Hz, 1H), 6.67 (t, J = 8.0 Hz, 1H). 13C APT NMR
(MeOD, 101 MHz): δ 145.56, 132.00, 127.50, 122.52, 119.45, 111.94.
1-(7-Bromobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol (143). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 98 mg, 0.49 mmol) and 2-amino-6-bromophenol (142, 92
mg, 0.49 mmol) according to the procedures described for compound
125. This yielded 1-(7-bromobenzo[d]oxazol-2-yl)-6-phenylhexan-1-
1-(6-Nitrobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol (140). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 100 mg, 0.49 mmol) and commercially available 2-amino-5-
nitrophenol (76 mg, 0.49 mmol) according to the procedures
described for compound 125. This yielded 1-(6-nitrobenzo[d]-
1
ol (132 mg, 0.36 mmol, 73%). H NMR (CDCl3, 400 MHz): δ 7.61
1
(dd, J = 7.9, 1.0 Hz, 1H), 7.46 (dd, J = 8.0, 1.0 Hz, 1H), 7.29−7.22 (m,
2H), 7.22−7.11 (m, 4H), 4.97 (dd, J = 7.8, 5.1 Hz, 1H), 2.58 (t, J = 7.6
Hz, 2H), 2.11−1.91 (m, 2H), 1.67−1.58 (m, 2H), 1.56−1.47 (m, 2H),
1.43−1.37 (m, 2H). 13C APT NMR (CDCl3, 101 MHz): δ 168.30,
148.97, 142.60, 141.12, 128.44 (2C), 128.31 (2C), 125.80, 119.09,
102.74, 67.99, 35.86, 35.49, 31.31, 28.93, 24.95.
oxazol-2-yl)-6-phenylhexan-1-ol (59 mg, 0.17 mmol, 35%). H NMR
(CDCl3, 400 MHz): δ 8.42 (d, J = 2.1 Hz, 1H), 8.30 (dd, J = 8.8, 2.1
Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.28−7.22 (m, 2H), 7.18−7.12 (m,
3H), 5.01 (dd, J = 7.7, 5.1 Hz, 1H), 2.60 (t, J = 7.6 Hz, 2H), 2.12−1.93
(m, 2H), 1.64 (p, J = 7.8 Hz, 2H), 1.57−1.49 (m, 2H), 1.45−1.38 (m,
2H). 13C APT NMR (CDCl3, 101 MHz): δ 172.53, 150.10, 145.85,
145.50, 142.54, 128.48 (2C), 128.40 (2C), 125.82, 120.85, 120.21,
107.65, 68.25, 35.90, 35.55, 31.32, 28.94, 24.87.
1-(7-Bromobenzo[d]oxazol-2-yl)-6-phenylhexan-1-one (16). The
title compound was synthesized from 1-(7-bromobenzo[d]oxazol-2-
yl)-6-phenylhexan-1-ol (143, 37 mg, 0.10 mmol) according to the
procedures described for compound 1. This yielded 1-(7-bromobenzo-
[d]oxazol-2-yl)-6-phenylhexan-1-one (41 mg, 0.13 mmol, 66%).
HRMS (ESI+) m/z: calculated for C19H19BrNO2 ([M + H]),
372.0594 and 374.0573; found, 372.0596 and 374.0574. 1H NMR
(CDCl3, 400 MHz): δ 7.83 (dd, J = 8.1, 1.0 Hz, 1H), 7.68 (dd, J = 8.0,
1.0 Hz, 1H), 7.35 (t, J = 8.0 Hz, 1H), 7.30−7.25 (m, 2H), 7.20−7.16
(m, 3H), 3.20 (t, J = 7.4 Hz, 2H), 2.63 (t, J = 7.6 Hz, 2H), 1.85 (p, J =
7.5 Hz, 2H), 1.74−1.65 (m, 2H), 1.52−1.43 (m, 2H). 13C APT NMR
(CDCl3, 101 MHz): δ 189.67, 157.18, 149.16, 142.54, 141.24, 131.57,
128.53 (2C), 128.42 (2C), 126.96, 125.84, 121.37, 103.90, 39.77,
35.85, 31.30, 28.82, 23.80. Purity of 95% as determined by LC/MS.
2-Hydroxy-4-phenylbutanenitrile (144). The title compound was
synthesized from commercially available 3-phenylpropanal (1.00 g 7.45
mmol) according to the previously reported procedure.12 This yielded
2-hydroxy-4-phenylbutanenitrile (810 mg, 5.02 mmol, 68%). 1H NMR
(CDCl3, 400 MHz): δ 7.38−7.33 (m, 2H), 7.30−7.23 (m, 3H), 4.40
(t, J = 6.8 Hz, 1H), 4.19 (bs, 1H), 2.83 (t, J = 8.0 Hz, 2H), 2.18−2.07
(m, 2H). 13C APT NMR (CDCl3, 101 MHz): δ 139.56, 128.54 (2C),
128.40 (2C), 126.35, 119.96, 60.02, 36.28, 30.46.
1-(Oxazolo[4,5-b]pyridin-2-yl)-3-phenylpropan-1-ol (145). The
title compound was synthesized from 2-hydroxy-4-phenylbutanenitrile
(144, 190 mg, 1.18 mmol) and commercially available 2-amino-3-
hydroxypyridine (142 mg, 1.29 mmol) according to the procedures
described for compound 125. This yielded 1-(oxazolo[4,5-b]pyridin-2-
yl)-3-phenylpropan-1-ol (44 mg, 0.17 mmol, 15%). 1H NMR (CDCl3,
400 MHz): δ 8.52 (dd, J = 4.9, 1.4 Hz, 1H), 7.78 (dd, J = 8.2, 1.4 Hz,
1H), 7.30−7.25 (m, 2H), 7.24−7.19 (m, 3H), 7.19−7.13 (m, 1H),
5.06 (dd, J = 7.8, 5.0 Hz, 1H), 4.31 (bs, 1H), 2.92−2.79 (m, 2H),
2.47−2.26 (m, 2H). 13C BBDEC NMR (CDCl3, 101 MHz): δ 171.12,
154.90, 146.51, 143.15, 140.90, 128.68 (2C), 128.53 (2C), 126.16,
120.40, 118.83, 67.33, 36.91, 31.17.
1-(6-Nitrobenzo[d]oxazol-2-yl)-6-phenylhexan-1-one (14). The
title compound was synthesized from 1-(6-nitrobenzo[d]oxazol-2-
yl)-6-phenylhexan-1-ol (140, 40 mg, 0.12 mmol) according to the
procedures described for compound 7. This yielded 1-(6-nitrobenzo-
[d]oxazol-2-yl)-6-phenylhexan-1-one (35 mg, 0.10 mmol, 86%).
HRMS (ESI+) m/z: calculated for C38H37N4O8 ([2M + H]),
1
677.2606; found, 677.2605. H NMR (CDCl3, 400 MHz): δ 8.55 (s,
1H), 8.39 (d, J = 8.9 Hz, 1H), 8.02 (d, J = 8.9 Hz, 1H), 7.30−7.25 (m,
2H), 7.21−7.15 (m, 3H), 3.23 (t, J = 7.3 Hz, 2H), 2.64 (t, J = 7.7 Hz,
2H), 1.86 (p, J = 7.5 Hz, 2H), 1.71 (p, J = 7.6 Hz, 2H), 1.53−1.43 (m,
2H). 13C APT NMR (CDCl3, 101 MHz): δ 189.50, 160.31, 149.92,
147.49, 145.34, 142.38, 128.45 (2C), 128.37 (2C), 125.82, 122.69,
121.43, 108.76, 39.83, 35.76, 31.17, 28.72, 23.59. Purity of >95% as
determined by LC/MS.
1-(6-Bromobenzo[d]oxazol-2-yl)-6-phenylhexan-1-ol (141). The
title compound was synthesized from 2-hydroxy-7-phenylheptaneni-
trile (124, 124 mg, 0.61 mmol) and commercially available 2-amino-5-
bromophenol (93 mg, 0.50 mmol) according to the procedures
described for compound 132. This yielded 1-(6-bromobenzo[d]-
oxazol-2-yl)-6-phenylhexan-1-ol (116 mg, 0.31 mmol, 62%). 1H NMR
(CDCl3, 400 MHz): δ 7.84 (d, J = 1.9 Hz, 1H), 7.46 (dd, J = 8.6, 1.9
Hz, 1H), 7.40 (d, J = 8.6 Hz, 1H), 7.30−7.22 (m, 2H), 7.21−7.11 (m,
3H), 4.94 (dd, J = 7.6, 5.1 Hz, 1H), 2.60 (t, J = 7.8 Hz, 2H), 2.60 (bs,
1H), 2.16−1.85 (m, 2H), 1.63 (p, J = 7.6 Hz, 2H), 1.57−1.45 (m,
2H), 1.45−1.34 (m, 2H). 13C BBDEC NMR (CDCl3, 101 MHz): δ
169.03, 149.94, 142.64, 142.18, 128.51 (2C), 128.41, 128.39 (2C),
125.79, 123.21, 117.44, 112.18, 68.16, 35.93, 35.57, 31.38, 29.00,
24.88.
1-(6-Bromobenzo[d]oxazol-2-yl)-6-phenylhexan-1-one (15). The
title compound was synthesized from 1-(6-bromobenzo[d]oxazol-2-
yl)-6-phenylhexan-1-ol (141, 43 mg, 0.11 mmol) according to the
procedures described for compound 7. This yielded 1-(6-bromobenzo-
[d]oxazol-2-yl)-6-phenylhexan-1-one (38 mg, 0.10 mmol, 89%).
HRMS (ESI+) m/z: calculated for C19H19BrNO2 ([M + H]),
372.0594 and 374.0573; found, 372.0596 and 374.0575. 1H NMR
(CDCl3, 400 MHz): δ 8.03 (d, J = 1.9 Hz, 1H), 7.64 (dd, J = 8.8, 1.8
Hz, 1H), 7.54 (d, J = 8.7 Hz, 1H), 7.31−7.23 (m, 2H), 7.21−7.14 (m,
3H), 3.20 (t, J = 7.4 Hz, 2H), 2.64 (t, J = 7.7 Hz, 2H), 1.84 (p, J = 7.5
Hz, 2H), 1.75−1.63 (m, 2H), 1.52−1.41 (m, 2H). 13C APT NMR
(CDCl3, 101 MHz): δ 190.09, 158.00, 149.77, 142.52, 142.13, 131.77,
128.53 (2C), 128.42 (2C), 125.85, 125.23, 118.67, 113.38, 39.68,
35.85, 31.30, 28.81, 23.74. Purity of >95% as determined by LC/MS.
2-Amino-6-bromophenol (142). To a solution of commercially
available 2-bromo-6-nitrophenol (200 mg, 0.92 mmol) in EtOH (3
mL) under argon atmosphere was added SnCl2 (870 mg, 4.59 mmol),
and the reaction mixture was heated to 70 °C. After full conversion (5
min), the reaction mixture was cooled to rt and poured into ice water
(20 mL). The pH was set to 10 (3 M NaOH), and the mixture was
stirred for 30 min. The water layer was extracted with 3× 30 mL
EtOAc. The organic layer was washed with 50 mL of brine, treated
with charcoal and filtered, dried (MgSO4), filtered, and concentrated
in vacuo to yield 2-amino-6-bromophenol (42 mg, 0.22 mmol, 25%).
1H NMR (MeOD, 400 MHz): δ 7.07 (dd, J = 8.1, 1.5 Hz, 1H), 6.93
1-(Oxazolo[4,5-b]pyridin-2-yl)-3-phenylpropan-1-one (106). The
title compound was synthesized from 1-(oxazolo[4,5-b]pyridin-2-yl)-
3-phenylpropan-1-ol (145, 43.8 mg, 0.17 mmol) according to the
procedures described for compound 1. This yielded 1-(oxazolo[4,5-
b]pyridin-2-yl)-3-phenylpropan-1-one (40 mg, 0.16 mmol, 93%).
HRMS (ESI+) m/z: calculated for C15H13N2O2 ([M + H]),
253.0972; found, 253.0970. 1H NMR (CDCl3, 400 MHz): δ 8.76
(dd, J = 4.8, 1.5 Hz, 1H), 8.00 (dd, J = 8.3, 1.5 Hz, 1H), 7.50 (dd, J =
8.3, 4.7 Hz, 1H), 7.34−7.27 (m, 4H), 7.25−7.16 (m, 1H), 3.63 (t, J =
7.7 Hz, 2H), 3.16 (t, J = 7.6 Hz, 2H). 13C BBDEC NMR (CDCl3, 101
MHz): δ 189.38, 158.53, 154.20, 148.90, 143.77, 140.10, 128.72 (2C),
128.58 (2C), 126.53, 123.36, 120.44, 41.43, 29.70. Purity of >95% as
determined by LC/MS.
2-Hydroxy-5-phenylpentanenitrile (146). The title compound was
synthesized commercially available 4-phenylbutan-1-ol (500 mg, 3.33
mmol) according to the previously reported two-step procedure.12
This yielded 2-hydroxy-5-phenylpentanenitrile (439 mg, 2.51 mmol,
1
75% over two steps). H NMR (CDCl3, 400 MHz): δ 7.32−7.25 (m,
2H), 7.22−7.14 (m, 3H), 4.46−4.38 (m, 1H), 3.10 (bs, 1H), 2.69−
2.65 (m, 2H), 1.90−1.74 (m, 4H). 13C APT NMR (CDCl3, 101
MHz): δ 141.24, 128.59 (2C), 128.49 (2C), 126.23, 120.04, 61.16,
35.10, 34.61, 26.27.
I
DOI: 10.1021/acs.jmedchem.5b01627
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1-(Oxazolo[4,5-b]pyridin-2-yl)-4-phenylbutan-1-ol (147). The
title compound was synthesized from 2-hydroxy-5-phenylpentaneni-
trile (146, 206 mg, 1.18 mmol) and commercially available 2-amino-3-
hydroxypyridine (129 mg, 1.17 mmol) according to the procedures
described for compound 125. This yielded 1-(oxazolo[4,5-b]pyridin-2-
BBDEC NMR (CDCl3, 101 MHz): δ 142.51, 128.31 (2C), 128.20
(2C), 125.58, 120.14, 60.97, 35.76, 34.85, 31.20, 28.88, 28.68, 24.40.
1-(Oxazolo[4,5-b]pyridin-2-yl)-7-phenylheptan-1-ol (151). The
title compound was synthesized from 2-hydroxy-8-phenyloctanenitrile
(150, 231 mg, 1.06 mmol) and commercially available 2-amino-3-
hydroxypyridine (101 mg, 0.92 mmol) according to the procedures
described for compound 125. This yielded 1-(oxazolo[4,5-b]pyridin-2-
yl)-7-phenylheptan-1-ol (38 mg, 0.12 mmol, 13%). 1H NMR (CDCl3,
400 MHz): 8.53 (dd, J = 4.9, 1.4 Hz, 1H), 7.81 (dd, J = 8.1, 1.4 Hz,
1H), 7.30−7.23 (m, 3H), 7.19−7.13 (m, 3H), 5.03 (dd, J = 7.6, 5.3
Hz, 1H), 3.91 (bs, 1H), 2.58 (t, J = 7.8 Hz, 2H), 2.10−1.91 (m, 2H),
1.65−1.54 (m, 2H), 1.54−1.44 (m, 2H), 1.41−1.29 (m, 4H). δ 13C
APT NMR (CDCl3, 101 MHz): δ 171.29, 154.97, 146.54, 143.15,
142.82, 128.48 (2C), 128.34 (2C), 125.70, 120.37, 118.82, 68.21,
36.01, 35.54, 31.48, 29.27, 29.20, 24.96. Spectroscopic data are in
agreement with reported literature.14
1-(Oxazolo[4,5-b]pyridin-2-yl)-7-phenylheptan-1-one (109). The
title compound was synthesized from 1-(oxazolo[4,5-b]pyridin-2-yl)-
7-phenylheptan-1-ol (151, 38 mg, 0.12 mmol) according to the
procedures described for compound 1. This yielded 1-(oxazolo[4,5-
b]pyridin-2-yl)-7-phenylheptan-1-one (23 mg, 0.07 mmol, 61%).
HRMS (ESI+) m/z: calculated for C19H21N2O2 ([M + H]),
309.1598; found, 309.1596. 1H NMR (CDCl3, 400 MHz): δ 8.77
(dd, J = 4.8, 1.5 Hz, 1H), 8.00 (dd, J = 8.3, 1.5 Hz, 1H), 7.50 (dd, J =
8.3, 4.8 Hz, 1H), 7.30−7.23 (m, 2H), 7.20−7.13 (m, 3H), 3.27 (t, J =
7.4 Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H), 1.83 (p, J = 7.4 Hz, 2H), 1.70−
1.59 (m, 2H), 1.51−1.35 (m, 4H). 13C BBDEC NMR (CDCl3, 101
MHz): δ 190.48, 158.68, 154.25, 148.81, 143.77, 142.75, 128.51 (2C),
128.37 (2C), 125.74, 123.30, 120.47, 39.91, 35.98, 31.38, 29.08, 29.05,
23.86. Purity of 94% as determined by LC/MS. Spectroscopic data are
in agreement with reported literature.14
1
yl)-4-phenylbutan-1-ol (56 mg, 0.21 mmol, 18%). H NMR (CDCl3,
400 MHz): δ 8.48 (dd, J = 4.9, 1.5 Hz, 1H), 7.77 (dd, J = 8.2, 1.5 Hz,
1H), 7.27−7.21 (m, 3H), 7.19−7.12 (m, 3H), 5.08 (dd, J = 7.5, 5.5
Hz, 1H), 4.62 (bs, 1H), 2.67 (t, J = 7.6 Hz, 2H), 2.12−1.98 (m, 2H),
1.94−1.78 (m, 2H). 13C APT NMR (CDCl3, 101 MHz): δ 171.31,
154.87, 146.35, 143.05, 141.86, 128.48 (2C), 128.39 (2C), 125.90,
120.31, 118.84, 67.91, 35.51, 34.96, 26.80.
1-(Oxazolo[4,5-b]pyridin-2-yl)-4-phenylbutan-1-one (107). The
title compound was synthesized from 1-(oxazolo[4,5-b]pyridin-2-yl)-
4-phenylbutan-1-ol (147, 52.4 mg, 0.20 mmol) according to the
procedures described for compound 1. This yielded 1-(oxazolo[4,5-
b]pyridin-2-yl)-4-phenylbutan-1-one (22 mg, 0.08 mmol, 42%).
HRMS (ESI+) m/z: calculated for C16H15N2O2 ([M + H]),
267.1128; found, 267.1126. 1H NMR (CDCl3, 400 MHz): δ 8.76
(dd, J = 4.8, 1.5 Hz, 1H), 7.99 (dd, J = 8.3, 1.5 Hz, 1H), 7.49 (dd, J =
8.3, 4.8 Hz, 1H), 7.31−7.25 (m, 2H), 7.24−7.14 (m, 3H), 3.31 (t, J =
7.3 Hz, 2H), 2.76 (t, J = 7.6 Hz, 2H), 2.22−2.11 (m, 2H). 13C BBDEC
NMR (CDCl3, 101 MHz): δ 190.12, 158.58, 154.22, 148.85, 143.72,
141.19, 128.64 (2C), 128.58 (2C), 123.29, 120.40, 39.25, 35.12, 25.41.
Purity of 94% as determined by LC/MS. Spectroscopic data are in
agreement with reported literature.14
2-Hydroxy-6-phenylhexanenitrile (148). The title compound was
synthesized from commercially available 5-phenylpentanol (513 mg
3.12 mmol) according to the previously reported two-step
procedure.12 This yielded 2-hydroxy-6-phenylhexanenitrile (318 mg,
1
1.68 mmol, 54% over two steps). H NMR (CDCl3, 400 MHz): δ
7.33−7.19 (m, 5H), 4.43 (bs, 1H), 4.18−4.12 (m, 1H), 2.66 (t, J = 7.6
Hz, 2H), 1.86 (q, J = 7.2 Hz, 2H), 1.70 (p, J = 7.6 Hz, 2H), 1.55 (p, J
= 7.6 Hz, 2H). 13C APT NMR (CDCl3, 101 MHz): δ 141.96, 128.81
(2C), 128.32 (2C), 125.79, 120.20, 60.92, 35.56, 34.90, 30.72, 24.21.
1-(Oxazolo[4,5-b]pyridin-2-yl)-5-phenylpentan-1-ol (149). The
title compound was synthesized from 2-hydroxy-6-phenylhexanenitrile
(148, 174 mg, 0.92 mmol) and commercially available 2-amino-3-
hydroxypyridine (102 mg, 0.93 mmol) according to the procedures
described for compound 125. This yielded 1-(oxazolo[4,5-b]pyridin-2-
yl)-5-phenylpentan-1-ol (51 mg, 0.18 mmol, 20%). 1H NMR (CDCl3,
400 MHz): δ 8.49 (dd, J = 5.0, 1.4 Hz, 1H), 7.79 (dd, J = 8.2, 1.4 Hz,
1H), 7.29−7.20 (m, 4H), 7.18−7.10 (m, 3H), 5.05 (dd, J = 7.4, 5.6
Hz, 1H), 2.60 (t, J = 7.6 Hz, 2H), 2.12−1.98 (m, 2H), 1.72−1.62 (m,
2H), 1.60−1.49 (m, 2H). 13C APT NMR (CDCl3, 101 MHz): δ
170.88, 154.90, 146.59, 143.08, 142.26, 128.38 (2C), 128.30 (2C),
125.74, 120.31, 118.68, 68.14, 35.71, 35.34, 31.12, 24.57.
1-(Oxazolo[4,5-b]pyridin-2-yl)-5-phenylpentan-1-one (108). The
title compound was synthesized from 1-(oxazolo[4,5-b]pyridin-2-yl)-
5-phenylpentan-1-ol (149, 40 mg, 0.14 mmol) according to the
procedures described for compound 1. This yielded 1-(oxazolo[4,5-
b]pyridin-2-yl)-5-phenylpentan-1-one (35 mg, 0.10 mmol, 86%).
HRMS (ESI+) m/z: calculated for C17H17N2O2 ([M + H]),
281.1285; found, 281.1282. 1H NMR (CDCl3, 400 MHz): δ 8.75
(dd, J = 4.8, 1.5 Hz, 1H), 8.00 (dd, J = 8.3, 1.4 Hz, 1H), 7.49 (dd, J =
8.3, 4.8 Hz, 1H), 7.30−7.26 (m, 2H), 7.23−7.16 (m, 3H), 3.31 (t, J =
7.2 Hz, 2H), 2.69 (t, J = 7.5 Hz, 2H), 1.93−1.83 (m, 2H), 1.81−1.72
(m, 2H). 13C APT NMR (CDCl3, 101 MHz): δ 190.23, 158.61,
154.26, 148.85, 143.73, 142.01, 128.66 (2C), 128.44 (2C), 125.92,
123.26, 120.35, 39.72, 35.69, 30.87, 23.53. Purity of >95% as
determined by LC/MS. Spectroscopic data are in agreement with
reported literature.14
2-Hydroxy-9-phenylnonanenitrile (152). The title compound was
synthesized from commercially available 8-phenyloctan-1-ol (1.00 g
4.85 mmol) according to the previously reported two-step procedure.
This yielded 2-hydroxy-9-phenylnonanenitrile (850 mg, 4.11 mmol,
1
85% over two steps). H NMR (CDCl3, 400 MHz): δ 7.32−7.23 (m,
2H), 7.22−7.13 (m, 3H), 4.46 (t, J = 6.7 Hz, 1H), 2.60 (t, J = 7.9 Hz,
2H), 2.44 (bs, 1H), 1.90−1.77 (m, 2H), 1.67−1.55 (m, 2H), 1.54−
1.43 (m, 2H), 1.39−1.28 (m, 6H). 13C APT NMR (CDCl3, 101
MHz): δ 142.86, 128.52 (2C), 128.38 (2C), 125.74, 120.03, 61.50,
36.03, 35.32, 31.53, 29.31, 29.19, 28.95, 24.60.
1-(Oxazolo[4,5-b]pyridin-2-yl)-8-phenyloctan-1-ol (153). The title
compound was synthesized from 2-hydroxy-7-phenylheptanenitrile
(152, 337 mg, 1.46 mmol) and commercially available 2-amino-3-
hydroxypyridine (159 mg, 1.44 mmol) according to the procedures
described for compound 125. This yielded 1-(oxazolo[4,5-b]pyridin-2-
yl)-8-phenyloctan-1-ol (104 mg, 0.32 mmol, 22%). 1H NMR (CDCl3,
400 MHz): δ 8.49 (dd, J = 4.9, 1.4 Hz, 1H), 7.79 (dd, J = 8.1, 1.4 Hz,
1H), 7.28−7.23 (m, 3H), 7.18−7.13 (m, 3H), 5.05 (dd, J = 7.4, 5.6
Hz, 1H), 4.55 (bs, 1H), 2.57 (t, J = 8.0 Hz, 2H), 2.12−1.90 (m, 2H),
1.63−1.53 (m, 2H), 1.53−1.40 (m, 2H), 1.39−1.20 (m, 6H). 13C APT
NMR (CDCl3, 101 MHz): δ 171.46, 154.92, 146.34, 143.05, 142.87,
128.44 (2C), 128.28 (2C), 125.62, 120.26, 118.79, 68.06, 35.99, 35.47,
31.53, 29.37, 29.30, 29.26, 25.05. Spectroscopic data are in agreement
with reported literature.14
1-(Oxazolo[4,5-b]pyridin-2-yl)-8-phenyloctan-1-one (110). The
title compound was synthesized from 1-(oxazolo[4,5-b]pyridin-2-yl)-
8-phenyloctan-1-ol (153, 50 mg, 0.15 mmol) according to the
procedures described for compound 1. This yielded 1-(oxazolo[4,5-
b]pyridin-2-yl)-8-phenyloctan-1-one (42 mg, 0.13 mmol, 84%).
HRMS (ESI+) m/z: calculated for C20H23N2O2 ([M + H]),
323.1754; found, 323.1755. 1H NMR (CDCl3, 400 MHz): δ 8.76
(dd, J = 4.8, 1.4 Hz, 1H), 8.00 (dd, J = 8.3, 1.5 Hz, 1H), 7.50 (dd, J =
8.3, 4.8 Hz, 1H), 7.30−7.24 (m, 2H), 7.20−7.13 (m, 3H), 3.27 (t, J =
7.4 Hz, 2H), 2.60 (t, J = 7.8 Hz, 2H), 1.82 (p, J = 7.4 Hz, 2H), 1.62 (p,
J = 7.6 Hz, 2H), 1.48−1.30 (m, 6H). 13C APT NMR (CDCl3, 101
MHz): δ 190.58, 158.65, 154.30, 148.89, 143.74, 142.91, 128.53 (2C),
128.37 (2C), 125.71, 123.30, 120.41, 39.95, 36.06, 31.56, 29.33, 29.22,
29.14, 23.95. Purity of >95% as determined by LC/MS. Spectroscopic
data are in agreement with reported literature.14
2-Hydroxy-8-phenyloctanenitrile (150). The title compound was
synthesized from commercially available 7-phenylheptan-1-ol (500 mg,
2.60 mmol) according to the previously reported two-step
procedure.12 This yielded 2-hydroxy-8-phenyloctanenitrile (430 mg,
1
1.98 mmol, 76% over two steps). H NMR (CDCl3, 400 MHz): δ
7.29−7.20 (m, 2H), 7.18−7.10 (m, 3H), 4.34 (q, J = 6.1 Hz, 1H),
3.86−3.78 (bs, 1H), 2.57 (t, J = 7.7 Hz, 2H), 1.75 (q, J = 7.2 Hz, 2H),
1.59 (t, J = 7.5 Hz, 2H), 1.49−1.38 (m, 2H), 1.37−1.27 (m, 4H). 13C
J
DOI: 10.1021/acs.jmedchem.5b01627
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
A. H.; Conti, B.; Cravatt, B. F. Endocannabinoid hydrolysis generates
brain prostaglandins that promote neuroinflammation. Science 2011,
334, 809−813.
(10) Di Marzo, V. Endocannabinoid signaling in the brain:
biosynthetic mechanisms in the limelight. Nat. Neurosci. 2011, 14,
9−15.
(11) Murataeva, N.; Straiker, A.; Mackie, K. Parsing the players: 2-
arachidonoylglycerol synthesis and degradation in the CNS. Br. J.
Pharmacol. 2014, 171, 1379−1391.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b01627.
■
S
Exact σm values, previously reported FAAH activity, and
LC traces of synthesized final compounds (PDF)
Molecular formula strings (CSV)
(12) Baggelaar, M. P.; Janssen, F. J.; van Esbroeck, A. C.; den Dulk,
H.; Allara, M.; Hoogendoorn, S.; McGuire, R.; Florea, B. I.;
Meeuwenoord, N.; van den Elst, H.; van der Marel, G. A.; Brouwer,
J.; Di Marzo, V.; Overkleeft, H. S.; van der Stelt, M. Development of
an activity-based probe and in silico design reveal highly selective
inhibitors for diacylglycerol lipase-alpha in brain. Angew. Chem., Int. Ed.
2013, 52, 12081−12085.
(13) Baggelaar, M. P.; Chameau, P. J.; Kantae, V.; Hummel, J.; Hsu,
K. L.; Janssen, F.; van der Wel, T.; Soethoudt, M.; Deng, H.; den Dulk,
H.; Allara, M.; Florea, B. I.; Di Marzo, V.; Wadman, W. J.; Kruse, C.
G.; Overkleeft, H. S.; Hankemeier, T.; Werkman, T. R.; Cravatt, B. F.;
van der Stelt, M. Highly selective, reversible inhibitor identified by
comparative chemoproteomics modulates diacylglycerol lipase activity
in neurons. J. Am. Chem. Soc. 2015, 137, 8851−8857.
(14) Boger, D. L.; Sato, H.; Lerner, A. E.; Hedrick, M. P.; Fecik, R.
A.; Miyauchi, H.; Wilkie, G. D.; Austin, B. J.; Patricelli, M. P.; Cravatt,
B. F. Exceptionally potent inhibitors of fatty acid amide hydrolase: the
enzyme responsible for degradation of endogenous oleamide and
anandamide. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 5044−5049.
(15) Edwards, P. D.; Wolanin, D. J.; Andisik, D. W.; Davis, M. W.
Peptidyl alpha-ketoheterocyclic inhibitors of human neutrophil
elastase. 2. Effect of varying the heterocyclic ring on in vitro potency.
J. Med. Chem. 1995, 38, 76−85.
(16) Edwards, P. D.; Zottola, M. A.; Davis, M.; Williams, J.; Tuthill,
P. A. Peptidyl alpha-ketoheterocyclic inhibitors of human neutrophil
elastase. 3. In vitro and in vivo potency of a series of peptidyl alpha-
ketobenzoxazoles. J. Med. Chem. 1995, 38, 3972−3982.
(17) Edwards, P. D.; Meyer, E. F.; Vijayalakshmi, J.; Tuthill, P. A.;
Andisik, D. A.; Gomes, B.; Strimpler, A. Design, synthesis, and kinetic
evaluation of a unique class of elastase inhibitors, the peptidyl a-
ketobenzoxazoles, and the x-ray crystal structure of the covalent
complex between porcine pancreatic elastase and Ac-Ala-Pro-Val-2-
benzoxazole. J. Am. Chem. Soc. 1992, 114, 1854−1863.
(18) Costanzo, M. J.; Maryanoff, B. E.; Hecker, L. R.; Schott, M. R.;
Yabut, S. C.; Zhang, H. C.; Andrade-Gordon, P.; Kauffman, J. A.;
Lewis, J. M.; Krishnan, R.; Tulinsky, A. Potent thrombin inhibitors that
probe the S1 subsite: tripeptide transition state analogues based on a
heterocycle-activated carbonyl group. J. Med. Chem. 1996, 39, 3039−
3043.
(19) Costanzo, M. J.; Almond, H. R., Jr.; Hecker, L. R.; Schott, M. R.;
Yabut, S. C.; Zhang, H. C.; Andrade-Gordon, P.; Corcoran, T. W.;
Giardino, E. C.; Kauffman, J. A.; Lewis, J. M.; de Garavilla, L.;
Haertlein, B. J.; Maryanoff, B. E. In-depth study of tripeptide-based
alpha-ketoheterocycles as inhibitors of thrombin. Effective utilization
of the S1′ subsite and its implications to structure-based drug design. J.
Med. Chem. 2005, 48, 1984−2008.
(20) Lin, J.; Deng, H.; Jin, L.; Pandey, P.; Quinn, J.; Cantin, S.;
Rynkiewicz, M. J.; Gorga, J. C.; Bibbins, F.; Celatka, C. A.; Nagafuji, P.;
Bannister, T. D.; Meyers, H. V.; Babine, R. E.; Hayward, N. J.; Weaver,
D.; Benjamin, H.; Stassen, F.; Abdel-Meguid, S. S.; Strickler, J. E.
Design, synthesis, and biological evaluation of peptidomimetic
inhibitors of factor XIa as novel anticoagulants. J. Med. Chem. 2006,
49, 7781−7791.
AUTHOR INFORMATION
Corresponding Author
*Phone: +31 (0)71 527 4768. E-mail: m.van.der.stelt@chem.
leidenuniv.nl.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are thankful for the financial support of the National
Institutes of Health (DA015648, D.L.B.; DA033760, B.F.C.)
and Dutch Research Council-Chemical Sciences (ECHO-
Grant: 711.014.009, M.v.d.S.).
ABBREVIATIONS USED
■
2-AG, 2-arachidonoylglycerol; ABHD6, α/β-hydrolase domain
6; ABHD12, α/β-hydrolase domain 12; DAGLα, diacylglycerol
lipase α; DAGLβ, diacylglycerol lipase β; DAGLs, sn-1-specific
diacylglycerol lipases; FAAH, fatty acid amide hydrolase;
MAGL, monoacylglycerol lipase
REFERENCES
■
(1) Katona, I.; Freund, T. F. Multiple functions of endocannabinoid
signaling in the brain. Annu. Rev. Neurosci. 2012, 35, 529−558.
(2) Tanimura, A.; Yamazaki, M.; Hashimotodani, Y.; Uchigashima,
M.; Kawata, S.; Abe, M.; Kita, Y.; Hashimoto, K.; Shimizu, T.;
Watanabe, M.; Sakimura, K.; Kano, M. The endocannabinoid 2-
arachidonoylglycerol produced by diacylglycerol lipase alpha mediates
retrograde suppression of synaptic transmission. Neuron 2010, 65,
320−327.
(3) Gao, Y.; Vasilyev, D. V.; Goncalves, M. B.; Howell, F. V.; Hobbs,
C.; Reisenberg, M.; Shen, R.; Zhang, M. Y.; Strassle, B. W.; Lu, P.;
Mark, L.; Piesla, M. J.; Deng, K.; Kouranova, E. V.; Ring, R. H.;
Whiteside, G. T.; Bates, B.; Walsh, F. S.; Williams, G.; Pangalos, M. N.;
Samad, T. A.; Doherty, P. Loss of retrograde endocannabinoid
signaling and reduced adult neurogenesis in diacylglycerol lipase
knock-out mice. J. Neurosci. 2010, 30, 2017−2024.
(4) Hsu, K. L.; Tsuboi, K.; Adibekian, A.; Pugh, H.; Masuda, K.;
Cravatt, B. F. DAGLbeta inhibition perturbs a lipid network involved
in macrophage inflammatory responses. Nat. Chem. Biol. 2012, 8,
999−1007.
(5) Di Marzo, V. Targeting the endocannabinoid system: to enhance
or reduce? Nat. Rev. Drug Discovery 2008, 7, 438−455.
(6) Rouzer, C. A.; Marnett, L. J. Endocannabinoid oxygenation by
cyclooxygenases, lipoxygenases, and cytochromes P450: cross-talk
between the eicosanoid and endocannabinoid signaling pathways.
Chem. Rev. 2011, 111, 5899−5921.
(7) Kohnz, R. A.; Nomura, D. K. Chemical approaches to
therapeutically target the metabolism and signaling of the
endocannabinoid 2-AG and eicosanoids. Chem. Soc. Rev. 2014, 43,
6859−6869.
(8) Alhouayek, M.; Muccioli, G. G. COX-2-derived endocannabinoid
metabolites as novel inflammatory mediators. Trends Pharmacol. Sci.
2014, 35, 284−292.
(9) Nomura, D. K.; Morrison, B. E.; Blankman, J. L.; Long, J. Z.;
Kinsey, S. G.; Marcondes, M. C.; Ward, A. M.; Hahn, Y. K.; Lichtman,
(21) Akahoshi, F.; Ashimori, A.; Sakashita, H.; Yoshimura, T.; Imada,
T.; Nakajima, M.; Mitsutomi, N.; Kuwahara, S.; Ohtsuka, T.; Fukaya,
C.; Miyazaki, M.; Nakamura, N. Synthesis, structure-activity relation-
ships, and pharmacokinetic profiles of nonpeptidic alpha-keto
heterocycles as novel inhibitors of human chymase. J. Med. Chem.
2001, 44, 1286−1296.
K
DOI: 10.1021/acs.jmedchem.5b01627
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(22) Costanzo, M. J.; Yabut, S. C.; Almond, H. R., Jr.; Andrade-
Gordon, P.; Corcoran, T. W.; De Garavilla, L.; Kauffman, J. A.;
Abraham, W. M.; Recacha, R.; Chattopadhyay, D.; Maryanoff, B. E.
Potent, small-molecule inhibitors of human mast cell tryptase.
Antiasthmatic action of a dipeptide-based transition-state analogue
containing a benzothiazole ketone. J. Med. Chem. 2003, 46, 3865−
3876.
(23) Tavares, F. X.; Deaton, D. N.; Miller, A. B.; Miller, L. R.; Wright,
L. L. Ketoheterocycle-based inhibitors of cathepsin K: a novel entry
into the synthesis of peptidic ketoheterocycles. Bioorg. Med. Chem. Lett.
2005, 15, 3891−3895.
(24) Maryanoff, B. E.; Costanzo, M. J. Inhibitors of proteases and
amide hydrolases that employ an alpha-ketoheterocycle as a key
enabling functionality. Bioorg. Med. Chem. 2008, 16, 1562−1595.
(25) Commercially available from Aldrich as ALD00032 and also
known as OL-100 first prepared and examined as disclosed in ref 14.
(26) Boger, D. L.; Miyauchi, H.; Du, W.; Hardouin, C.; Fecik, R. A.;
Cheng, H.; Hwang, I.; Hedrick, M. P.; Leung, D.; Acevedo, O.;
Guimaraes, C. R.; Jorgensen, W. L.; Cravatt, B. F. Discovery of a
potent, selective, and efficacious class of reversible alpha-ketohetero-
cycle inhibitors of fatty acid amide hydrolase effective as analgesics. J.
Med. Chem. 2005, 48, 1849−1856.
(27) DeMartino, J. K.; Garfunkle, J.; Hochstatter, D. G.; Cravatt, B.
F.; Boger, D. L. Exploration of a fundamental substituent effect of
alpha-ketoheterocycle enzyme inhibitors: Potent and selective
inhibitors of fatty acid amide hydrolase. Bioorg. Med. Chem. Lett.
2008, 18, 5842−5846.
(28) Ezzili, C.; Mileni, M.; McGlinchey, N.; Long, J. Z.; Kinsey, S. G.;
Hochstatter, D. G.; Stevens, R. C.; Lichtman, A. H.; Cravatt, B. F.;
Bilsky, E. J.; Boger, D. L. Reversible competitive alpha-ketoheterocycle
inhibitors of fatty acid amide hydrolase containing additional
conformational constraints in the acyl side chain: orally active, long-
acting analgesics. J. Med. Chem. 2011, 54, 2805−2822.
(29) Hardouin, C.; Kelso, M. J.; Romero, F. A.; Rayl, T. J.; Leung, D.;
Hwang, I.; Cravatt, B. F.; Boger, D. L. Structure-activity relationships
of alpha-ketooxazole inhibitors of fatty acid amide hydrolase. J. Med.
Chem. 2007, 50, 3359−3368.
(30) Kimball, F. S.; Romero, F. A.; Ezzili, C.; Garfunkle, J.; Rayl, T. J.;
Hochstatter, D. G.; Hwang, I.; Boger, D. L. Optimization of alpha-
ketooxazole inhibitors of fatty acid amide hydrolase. J. Med. Chem.
2008, 51, 937−947.
(31) Leung, D.; Du, W.; Hardouin, C.; Cheng, H.; Hwang, I.;
Cravatt, B. F.; Boger, D. L. Discovery of an exceptionally potent and
selective class of fatty acid amide hydrolase inhibitors enlisting
proteome-wide selectivity screening: concurrent optimization of
enzyme inhibitor potency and selectivity. Bioorg. Med. Chem. Lett.
2005, 15, 1423−1428.
(32) Romero, F. A.; Hwang, I.; Boger, D. L. Delineation of a
fundamental alpha-ketoheterocycle substituent effect for use in the
design of enzyme inhibitors. J. Am. Chem. Soc. 2006, 128, 14004−
14005.
(33) Garfunkle, J.; Ezzili, C.; Rayl, T. J.; Hochstatter, D. G.; Hwang,
I.; Boger, D. L. Optimization of the central heterocycle of alpha-
ketoheterocycle inhibitors of fatty acid amide hydrolase. J. Med. Chem.
2008, 51, 4392−4403.
(34) Taylor, P. J.; Wait, A. R. σi Values for heterocycles. J. Chem. Soc.,
Perkin Trans. 2 1986, 1765−1770.
L
DOI: 10.1021/acs.jmedchem.5b01627
J. Med. Chem. XXXX, XXX, XXX−XXX