Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.0c01561

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.

Full text of DOI:10.1021/acs.jmedchem.0c01561

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Article
Design, Synthesis, and Biological Evaluation of a Series of
Oxazolone Carboxamides as a Novel Class of Acid Ceramidase
Inhibitors
Samantha Caputo,¶ Simona Di Martino,¶ Vincenzo Cilibrasi, Piero Tardia, Marco Mazzonna,
Debora Russo, Ilaria Penna, Maria Summa, Sine Mandrup Bertozzi, Natalia Realini, Natasha Margaroli,
Marco Migliore, Giuliana Ottonello, Min Liu, Peter Lansbury, Andrea Armirotti, Rosalia Bertorelli,
Soumya S. Ray, Renato Skerlj,* and Rita Scarpelli*
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ABSTRACT: Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides,
important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes
ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of
the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized.
Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-
oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-
piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic
properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice,
following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as
useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated
disorders.
produced in response to stressful stimuli via two major
pathways: the de novo pathway from serine and palmitoyl-CoA
in the endoplasmic reticulum and the salvage pathway from the
recycling of sphingosine (So), bypassing the formation of
dihydroceramide. The generated Cer can be transported to
distinct cellular compartments and further modified to more
complex SLs, for example, glycosylated to hexosylceramides
INTRODUCTION
■
Ceramides (Cer) and their metabolites are members of the
sphingolipid (SL) family that play important roles as integral
components of the eukaryotic cell membranes and signaling
molecules in apoptosis, cell growth, differentiation, senescence,
diabetes, insulin resistance, inflammation, neurodegenerative
disorders, and atherosclerosis.1−4 The proper regulation of
ceramide biosynthesis and metabolism is controlled by a
complex, highly compartmentalized, and interconnected net-
work of enzymatic pathways essential to maintaining the cellular
homeostasis and development.1 Although a detailed analysis of
this complex network is beyond the scope of this paper, a
simplified representation of the ceramide metabolism is shown
in Figure 1. Cer are the centerpiece of the SL metabolism,
Received: September 7, 2020
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Figure 1. Overview of the ceramide metabolism and some related enzymes.
(HexCer) and, in turn, to more complex glycosylceramides;
metabolized into sphingomyelin (SM) and ceramide-1-
phosphate (Cer-1P); and catabolized to produce So, which is
further phosphorylated to sphingosine-1-phosphate (So-1P).
Cellular Cer can also be generated through catabolic pathways in
distinct sub-cellular compartments. In the lysosomes, for
example, SM and HexCer (glucosylceramide and galactosylcer-
amide) participate in distinct degradative pathways that
contribute to the formation of lysosomal Cer. In the same
compartment, HexCer can be hydrolyzed through distinct
pathways to generate the corresponding lysosomal glycosyl-
sphingosines.5 Evidence to date suggests that imbalances in this
complex network because of an altered expression and/or
regulation of SL-modifying enzymes can lead to dysregulated
cell signaling responses that contribute to the initiation and
progression of several SL-related disorders.3,4 During the past
years, aided by the impressive advances of the modern biological
and analytical technologies, the scientific community has
focused much attention on improving the understanding of
the functional roles of some basic components of this metabolic
network, under physiological and pathological conditions.6
Ceramidases (CDases) have attracted particular attention as
key SL-metabolizing enzymes that regulate the levels and
functions of different bioactive lipids, especially, Cer and So.7
Thus far, five human CDases (hCDase) have been identified,
which can be characterized by their different optimal pH for
catalytic activity and localization in cells: acid ceramidase (AC),
neutral ceramidase (NC), alkaline ceramidase 1 (ACER1),
alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3
(ACER3).7,8 Because of differences in tissue distribution and
expression level, cellular localization, optimum pH, and
substrate specificity, these CDases appear to play distinct
physiological roles in cellular responses.8 The overexpression of
NC has been implicated in colon carcinogenesis9 and, therefore,
it has emerged as a potential new therapeutic target for cancer
therapy.10 Recent reports have shown the implication of ACER1
in keratinocyte differentiation11,12 and that of ACER2 in
programmed cell death in response to DNA damage.13
ACER3 has been reported to control both cell proliferation
and apoptosis14 and to be involved in motor coordination-
associated Purkinje cell degeneration.15,16 Despite these
fundamental studies on the functional roles of NC and
ACER1-3 in certain biological processes, further investigations
are still on going to better clarify their implications in human
diseases.8 By contrast, a growing body of evidence describes the
important role of AC in the development and progression of
different human pathological conditions, suggesting human AC
(hAC) as a potential target for promising therapeutic
applications. hAC (also known as N-acylsphingosine amidohy-
drolase-1, ASAH-1) is a lysosomal cysteine amidase that, at an
optimal pH of 4.5, hydrolyzes Cer into So and fatty acids (Figure
1).17,18 Because the phosphorylation of So is the only pathway
for the formation of So-1P, cellular So-1P is highly dependent on
the availability of So; hence, hAC is a critical enzyme in
regulating not only the hydrolysis of Cer but also the generation
of both So and So-1P in cells. Cer and So-1P have opposing
effects in the control of cell fate.19 While Cer favor cell-cycle
arrest20 and apoptosis,21,22 So-1P promotes angiogenesis, cell
survival, and proliferation.23−26 Hence, the altered Cer/So
balance determines the shifting of cell fate toward apoptosis and
proliferation, respectively, and contributes to the pathogenesis
of some human diseases. For example, various common diseases,
including inflammation, pain, and several pulmonary disorders,
have been associated with aberrant hAC activities.27 hAC is also
deficient in two rare inherited disorders: spinal muscular atrophy
with myoclonic epilepsy and Farber’s disease.28 By contrast,
collected evidence has shown that hAC is abnormally expressed
in various types of human cancer, for example, prostate,29
melanoma,30 head and neck,31 colon,32 and glioblastoma.33 It
has been observed that the overexpression of hAC renders the
cells more resistant to pharmacological induction of apopto-
sis.29,34 Therefore, the inhibition of hAC has been proposed as a
potential strategy to enhance the therapeutic efficacy of standard
antineoplastic agents and radiation.34,35 Relevant evidence has
B
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Figure 2. Representative known and structurally diversified AC inhibitors.
shown that Alzheimer’s disease (AD) brains exhibit elevated
level and activity of hAC, suggesting a potential role of AC in
controlling neuronal apoptosis and in the molecular mechanism
of AD.36 Notably, recent reports are proposing the role of hAC
inhibition as an emerging strategy for treating some types of rare
inherited metabolic disorders called lysosomal storage diseases
(LSDs),37−39 in particular, some severe neuropathic conditions
related to Gaucher’s40 disease (GD) and Krabbe’s41 disease
(KD). GD and KD are caused by the defective functions of some
specific lysosomal proteins, acid β-glucocerebrosidase (GCase,
β-glucosyl ceramidase) for GD and β-galactocerebrosidase
(GALCase, β-galactosyl ceramidase) for KD. In GD patients,
recent evidence suggests an active role of hAC in the catabolism
of the lysosomal glucosylceramide, which is responsible for the
accumulation of toxic glucosylsphingosine (Figure 1).42 In KD
patients, deficiency of GALCase activity results in the buildup of
the galactosylceramide and the galactosylsphingosine (psycho-
sine) in nervous tissues, especially in the brain. Notably, a recent
report suggests that genetic ablation or pharmacological
inhibition of AC could eliminate the accumulation of the
neurotoxic psychosine and prolong the life span of the KD
mouse model.43 There are no approved treatments for
neuropathic GD and KD; targeting the inhibition of hAC may
provide an innovative approach for treating these severe
diseases.
Although many efforts in the past decade have been made to
identify new classes of hAC inhibitors, to date, these activities
have resulted in limited success and a very limited number of
suitable candidates for in vivo experiments are currently
available. In a recent study, Gebai and co-workers reported
the crystal structure analysis of mammalian AC (PDB code:
5U7Z),44 which may assist future structure-guided drug
discovery programs. First generation hAC inhibitors were
designed on the basis of substrate (Cer)-based structures, for
example, N-oleoylethanolamine (OEA, median inhibitory
concentration (IC50) ∼ 500 μM,45 Figure 2). Despite being
the first Cer-mimicking inhibitor to be described, the ability of
OEA to inhibit hAC was not always reproducible.45−47 Further
representative examples are D-erithro-2-(N-myristoylamino)-1-
phenyl-1-propanol (D-e-MAPP, IC50 > 500 μM in HL-60 cell
lysates45 and IC50 = 500 μM in HaCaT cell lysates,48 Figure 2)
and its more water soluble derivative N-NMAPPD (B13, IC50 ∼
10 μM in HaCaT cell lysates,48 Figure 2). Efforts to ameliorate
these Cer-mimicking molecules led to several structurally varied
analogues of B13, as compounds DP24a (IC50 = 1.287 μM,49
Figure 2) and the potent irreversible AC inhibitor SABRAC
C
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Figure 3. Rational design of the novel class of AC inhibitors and hit identification.
a
Scheme 1. Synthesis of 8a−d
a
Reagents and conditions: (a) KNCO, AcOH, and i-PrOH, 70 °C, 3 h (36−53%) and (b) RNCO and DMAP, CH3CN, rt, 3−16 h (15−82%).
(IC50 = 0.052 μM,50,51 Figure 2). By contrast, the quinolinone-
based compounds, Ceranib-1 and its optimized analogue
Ceranib-2, represent the first class of non-Cer-mimicking
inhibitors of hCDase identified by Draper and co-workers by
screening a chemical library (hCDase IC50 = 55 μM and 28 μM
in SKOV3 cells, respectively,52 Figure 2). In another study by
Yildiz-Oze and co-workers, Ceranib-2 was found to inhibit hAC
activity by 44% at 25 μM in H460 cells.53 More recently, Cho
and co-workers reported the identification of the hit compound
co-workers reported the identification of benzimidazole
derivatives 3a−d (3a, IC50 = 2.5 nM; 3b, IC50 = 13.9 nM; 3c,
IC50 = 22.5 nM; and 3d, IC50 = 14.8 nM,58 Figure 2 and Table
S1) with promising AC inhibitory activity in different melanoma
cell lines.58 A screening campaign of a small compound library
was exploited by Pizzirani and co-workers resulting in the
identification of the benzoxazolone (abbreviated as BOA,
hereafter) carboxamide series, exemplified by the initial hit 4a
(IC50 = 64 nM,59 Figure 2 and Table S1).59 Preliminary studies
led to the more advanced and systematically active analogues 4b
(IC50 = 79 nM,59 Figure 2 and Table S1) and 4c (IC50 = 33
nM,60 Figure 2 and Table S1).59,60 Although these molecules
showed potent inhibitory effects on hAC activity, they generally
suffered from low aqueous solubility and moderate metabolic
stability, which impede their further development as oral drugs.
During recent years, we directed the scope of our research work
to solve these limitations. As part of our continued efforts in the
optimization of the BOA carboxamide series, we recently
reported the discovery of the piperidine 4d (IC50 = 166 nM,61
Figure 2 and Table S1) as a lead compound with good oral
bioavailability, excellent brain penetration, and target engage-
ment in two animal models of neuropathic GD and KD.61 As an
extension of this work while adopting a different strategy, we
started an exploratory drug discovery program directed to the
search for a novel class of hAC inhibitors with optimal drug-like
properties, suitable for investigational studies in cellular and in
vivo model systems. In the present study, we describe our
strategies for the design and synthesis of a novel chemotype of
hAC inhibitors (general structure, compound 5, Figure 3). The
disruption of the molecular planarity of the fused bicyclic BOA
moiety resulted in the identification of two initial hits, 2-oxo-4-
phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-
5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a (Figure
3). Herein, we present the structure−activity relationship (SAR)
exploration of this novel series of substituted oxazol-2-one-3-
carboxamides and the chemical optimization which resulted in
the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)-
phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent
[1,1′-biphenyl]-4-yl-2-(4-guanidinophenyl)acetate (E2, IC50
=
52 μM,54 Figure 2) from 68 guanidine-based derivatives tested
for the discovery of new antiangiogenic inhibitors and
determined the role of hAC as the E2-binding protein. Although
a comparative analysis of the AC inhibitory activities of these
different molecules is limited by the fact that the reported
pharmacological data have been collected using different assay
conditions and protein sources (Table S1),45−50,52−54 overall,
these AC inhibitors are characterized by low inhibitory potency
(as those with IC50 values in the μM range)45−49,52−54 and poor
drug-likeness (as those with, e.g., long lipophilic carbon
chains).45−50
A significant breakthrough was made by Realini and co-
workers with the identification of carmofur [rat AC (rAC) IC50
= 29 nM,55 Figure 2 and Table S1] and some close uracil
analogues, for example, compounds 1a−d, as nanomolar
inhibitors of AC activity (1a, rAC IC50 = 67 nM;55 1b, rAC
IC50 = 12 nM;55 1c, rAC IC50 = 16 nM56 and hAC IC50 = 7.7
nM;30 and 1d, hAC IC50 = 12.8 nM,30 Figure 2 and Table S1).
Despite being potent AC inhibitors with some potential
applications as chemo-sensitizing agents, the uracil derivatives
showed low chemical and metabolic stability. Successively, using
a ligand-based virtual screening approach, with carmofur as the
template, Diamanti and co-workers identified a new class of
potent hAC inhibitors, exemplified by the pyrazole carboxamide
2 (IC50 = 14 nM,57 Figure 2 and Table S1). However, these
molecules exhibited low metabolic stability (2, mouse plasma
half-life, t1/2 = 9 min),57 limiting their therapeutic potential.
Through a systematic computational investigation, Ortega and
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a
Scheme 2. Synthesis of 11a−q
a
Reagents and conditions: (a) TZD, K2CO3, and DMF, rt, 1−2 h. (b) LiOH and THF, rt, 30 min−1 h (6%- quant. over two steps for 11a, 11c−p)
or t-BuOK and THF, rt, 30 min (for 11b and 11q: 10 and 38% over two steps; respectively).
and drug-like hAC inhibitorstructurally differentiated from
commercially available chiral (4S)-14a- and (4R)-14b-phenyl-
previously reported series.
oxazolidin-2-ones by carboxamide formation under standard
conditions (Scheme 4A). A similar synthetic strategy was
adopted for the preparation of analogues 18a−b upon the
formation of the enantiomers (5S)-17a- and (5R)-17b-phenyl-
oxazolidin-2-ones starting from the enantiomerically pure 2-
amino-1-phenylethanols 16a−b via 1,1′-carbonyldiimidazole
(CDI)-mediated intramolecular cyclization (Scheme 4B).
Similar procedures were exploited for the preparation of the
targeted oxazolone carboxamides 25c−f and 32a−c, bearing a 4-
methylpiperidine moiety at the C(3′)- and C(2′)-positions of
the phenyl ring, respectively (Schemes 5 and 6). The methyl
ketones 22a−b and 28 were prepared in two steps, starting from
the corresponding bromophenyls 20a−b and 26, using Pd-
catalyzed cross-coupling reactions, in the presence of the
commercially available boronic pinacol ester 19, followed by
hydrogenation in EtOH at 60 °C in the presence of 10% Pd/C
and cyclohexene (as in the synthesis of 22a−b), or using
Pd(OH)2 and ammonium formate in MeOH at reflux (as in the
synthesis of 28). The resulting methyl ketones 22a−b and 28
were transformed into the corresponding α-bromo ketones
22c−d and 29, through a slightly modified reported
procedure,66 consisting of an in situ addition of N-
bromosuccinimide (NBS) to the corresponding silyl enol ethers
in the presence of Et3N at a controlled low temperature
(Schemes 5 and 6). The α-bromo ketones 22c−d and 29 were
directly reacted with TZD to afford the corresponding
intermediates 23a−b and 30 and then converted, through an
intramolecular cyclization, to the 2-oxazolones 24a−b and 31a,
respectively, as described above. Standard reaction conditions
were exploited to convert the piperidines 24b and 31a to the
corresponding 4-methylpiperidines 24d and 31c, which
involved N-Boc removal and reductive amination in the
presence of NaBH(OAc)3 and 37% aqueous solution of
HCHO. Alternatively, as in the synthesis of 25c, these synthetic
steps were performed on the corresponding carboxamide
CHEMISTRY
■
The synthetic routes for the preparation of all target compounds
are described in Schemes 1−6. We introduced different
substituents at the C(4)- and C(5)-positions of the 2-oxazolone
core scaffold by exploring the synthetic pathways depicted in
Schemes 1 and 2. The substituted 4-phenyl-oxazol-2-one
derivatives 7a−c were obtained starting from the corresponding
α-hydroxy ketones 6a−c, through the condensation reaction
with potassium cyanate and in situ intramolecular cyclization
under acidic conditions (Scheme 1).62 A series of substituted 5-
phenyl- and 5-heteroaryl-oxazol-2-one derivatives 11a−q were
synthesized starting from the commercially available α-bromo
ketones 9a−q by condensation with 2,4-thiazolidinedione
(TZD), followed by intramolecular cyclization of the
intermediates 10a−q under basic conditions (LiOH or t-
BuOK) (Scheme 2).63 We introduced the carboxamide
functionalities using standard conditions, by reacting inter-
mediates 7a−c or 11a−q with the corresponding commercially
available isocyanates, as in the synthesis of 8a−d (Scheme 1) or
12a−b, g−w (Scheme 3). Alternatively, the isocyanates were
generated in situ, through the activation of the corresponding
amines by reaction with Boc2O in the presence of 4-
(dimethylamino)-pyridine (DMAP)64 (12c−d, Scheme 3) or
by reaction with triphosgene in the presence of N,N-
diisopropylethylamine (DIPEA) or Et3N65 (12e−f, Scheme
3). The N-methylated analogue 13a and the carbamate 13b were
prepared upon the activation of 11a with triphosgene in the
presence of DIPEA, followed by the addition of N-methyl-4-
phenylbutylamine and 4-phenyl-1-butanol, respectively
(Scheme 3). On the other hand, 11a was converted to the
corresponding amide 13c by reaction with the corresponding
freshly prepared 6-phenylhexanoic chloride. The oxazolidin-2-
one analogues 15a−b were prepared starting from the
E
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a
Scheme 3. Synthesis of 12a−y and 13a−c
a
Reagents and conditions: (a) RNCO and DMAP (10−83% for 12a−b, h−w) or Et3N (78% for 12g) and CH3CN, rt, 3−16 h or RNH2, Boc2O,
DMAP and CH3CN, rt, 1−3 h (48−80% for 12c−d) or RNH2, triphosgene, and DIPEA (76% for 12e) or Et3N (24% for 12f) and DCM, rt, 3−12
h; (b) 4 M HCl, 1,4-dioxane, rt, 2 h; (c) HCHO, AcOH, NaBH(OAc)3, and CH3CN, rt, 1 h (34% over two steps); (d) for 13a: N-methyl-4-
phenylbutylamine, triphosgene, DIPEA, and DCM, 0 °C to rt, 3 h (98%); for 13b: 4-phenyl-1-butanol, triphosgene, DIPEA, and DCM, 0 °C to rt,
3 h (53%); for 13c: 6-phenylhexanoic acid, SOCl2, and DCM, rt, 6 h; then 11a, Et3N and THF, 0 °C to rt, 16 h (55%).
a
Scheme 4. Synthesis of 15a−b and 18a−b
a
Reagents and conditions: for the synthesis of 15a−b: (a) 4-phenylbutyl isocyanate, DMAP, DMF, 50 °C, 2 h, (47−81%); for the synthesis of
18a−b: (a′) CDI, imidazole, DCM, rt, 16 h (86−92%); (b′) 4-phenylbutyl isocyanate, DMAP, DMF, 50 °C, 4 h (68−79%).
derivative 25a, obtained by reacting 24a with 4-phenylbutyl
isocyanate (Scheme 5). Finally, the carboxamide functionality of
the targeted compounds 25d−f and 32a−c was introduced
using standard reaction conditions, as described above.
F
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a
Scheme 5. Synthesis of 25c−f
a
Reagents and conditions: (a) Pd(PPh3)4, 2 M Na2CO3, and 1,4-dioxane, reflux, 16 h (77−87%); (b) 10% Pd/C, cyclohexene, and EtOH, 60 °C,
24 h (65%); (c) TMSOTf, Et3N, and THF, −78 to −50 °C, 4 h; then, NBS and THF, −40 to −20 °C, 30 min; (d) TZD, K2CO3, and DMF, rt, 1−
2 h (93% over three steps for 23b); (e) t-BuOK and THF, rt, 1 h (29% over 4 steps for 24a) or LiOH and THF, rt, 30 min (72% for 24b); (f) 4-
phenylbutyl isocyanate, DMAP, and pyridine, rt, 16 h (69%); (g) 4 M HCl and 1,4-dioxane, rt, 2 h (46%); (h) HCHO, AcOH, NaBH(OAc)3, and
CH3CN, rt, 1 h (60%) (i) 4 M HCl and 1,4-dioxane, rt, 2 h; (j) HCHO, AcOH, NaBH(OAc)3, and CH3CN, rt, 1 h (71% over two steps); and (k)
RNCO, DMAP, and pyridine, rt, 16 h (25−36% for 25d−e) or i-BuNH2, triphosgene, Et3N, and DCM, rt, 3 h (82% for 25f).
workers, on the crystal structure analysis of carmofur covalently
bound to Cys143 at a 2.7 Å resolution.67 While potent and, in
certain cases, systemically active, for example, analogues 4b−
c,59,60 these potent hAC inhibitors share two characteristics that
hamper their applications as oral drugs. First, the chemical
warheads which, on the one hand, are responsible for the
covalent binding mechanisms of these inhibitors and, on the
other hand, can contribute to the poor chemical and plasma
stability of these molecules (e.g., carmofur, 1a−d and 2);56,57
second, the hydrophobic linear side-chains, although funda-
RESULTS AND DISCUSSION
■
A common characteristic of some classes of known AC
inhibitors is the presence of a cysteine (Cys)-targeting warhead
as the α-bromo acetyl moiety or the urea-like functionality that
can undergo a chemical reaction with the thiol group of the
catalytic Cys143 of hAC to produce a covalent bond,44 as
reported for 2-bromoacetamide SABRAC51 and the carbox-
amides 3a−b58 and 4a59 (Figure 2). This evidence has been
supported by recent studies, reported by Dementiev and co-
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a
Scheme 6. Synthesis of 32a−c
a
Reagents and conditions: (a) Pd(PPh3)4, 2 M Na2CO3, and 1,4-dioxane, reflux, 16 h (85%); (b) HCO2NH4, 20% Pd(OH)2, and MeOH, 60 °C, 4
h (69%); (c) TMSOTf, Et3N, and THF, −78 to −50 °C, 4 h; then, NBS and THF, −40 to −20 °C, 30 min; (d) TZD, K2CO3, and DMF, rt, 2 h
(66% over three steps); (e) t-BuOK and THF, rt, 30 min (67%); (f) 4 M HCl and 1,4-dioxane, rt, 2 h; (g) HCHO, AcOH, NaBH(OAc)3, and
CH3CN, rt, 1 h and (h) RNCO, DMAP, and pyridine, rt, 16 h (62% for 32a; 64% for 32b) or i-BuNH2, triphosgene, Et3N, and DCM, rt, 3 h (20%
for 32c).
mental for target recognition and some degree of specificity,
negatively affect the drug-like properties of these molecules (e.g.,
SABRAC and 4a).51,59 Thus, there is a strong need for novel and
optimized hAC inhibitors. In this respect, our continued efforts
dedicated to the chemical optimization of the BOA carboxamide
series, exemplified by 4a−c,59,60 have recently led to the
identification of the lead 4d as a potent and orally bioavailable
hAC inhibitor with excellent brain penetration in mice and
target engagement in two animal models of LSDs (Figure 2).61
As part of our more exploratory research program, our medicinal
chemistry strategies were also focused on expanding the
chemical diversity of the existing hAC inhibitors for the
identification of new chemotypes with optimal physicochemical
and metabolic properties suitable for cellular and in vivo studies.
In this regard, by the disruption of the molecular planarity of the
fused bicyclic aromatic BOA system, we designed a series of
compounds with the general structure 5 and synthesized a few
initial molecules, for example, the 2-oxo-4-phenyl-N-(4-
phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-
(4-phenylbutyl)oxazole-3-carboxamide 12a (Figure 3). In
particular, we were interested in studying the substituted-
oxazolone ring system as a potential and attractive strategy for
the BOA bioisosteric replacement. In addition, we envisaged
that the insertion of this relatively unexplored heterocycle
system, by reducing the molecular planarity of the core scaffold
and, therefore, varying the nature of the leaving group at the
reactive electrophilic functionality, might be a valuable strategy
for the subsequent optimization of our targeted molecules.68 By
contrast, in order to somehow preserve the hAC recognition, we
initially designed scaffolds that bear a lipophilic group on the
lateral chain of the urea-like functionality, as the butyl phenyl
group of 8a and 12a, already described in other series of known
inhibitors (e.g., 3a−b and 4a−c, Figure 1) to be suitable for
chemical optimization. Compounds 8a and 12a were screened
against hAC using a fluorogenic assay and were able to inhibit
the enzymatic activity with IC50 values equal to 0.007 and 0.090
μM, respectively (Table 1). These initial results encouraged us
to start a preliminary SAR exploration around these new
scaffolds in the three main Regions A, B, and C, as depicted in
Figure 4, with the objective of identifying the pharmacophore
necessary for target inhibition.
Figure 4. Planned SAR exploration.
In this regard, to first validate our initial hits, we prepared a set
of representative analogues around the 4-phenyl-oxazol-2-one
(4-POA) and 5-phenyl-oxazol-2-one (5-POA) carboxamide
compounds 8a and 12a (Table 1). Interestingly, in the 4-POA
carboxamide series, although a slight drop in potency was
detected with the removal of the terminal aromatic ring, as in the
n-pentyl analogue 8b (hAC IC50 = 0.025 μM), the insertion of a
Cl atom at the para phenyl position was tolerated, with
compound 8d (hAC IC50 = 0.005 μM) being equipotent to the
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parent 8a (hAC IC50 = 0.007 μM) (Table 1). In contrast, the
insertion of a methyl group on the C(5)-position of the
oxazolone ring, as in the di-substituted analogue 8c, resulted in a
6-fold loss of potency (hAC IC50 = 0.042 μM). A slightly
different trend was observed in the 5-POA carboxamide series;
the removal of the terminal phenyl ring, as in the n-pentyl
analogue 12b (hAC IC50 = 0.039 μM), resulted in a weak
improvement in potency compared to the parent compound 12a
(hAC IC50 = 0.090 μM). Both the insertion of a Cl atom at the
para phenyl position, as in 12i, and the insertion of a methyl
group at the C(4)-position of the oxazolone ring, as in 12h,
afforded analogues (hAC IC50 = 0.083 and 0.069 μM,
respectively) with similar potency compared to 12a (Table 1).
These encouraging preliminary results confirmed that both
the 4- and 5-(POA) carboxamide series were promising scaffolds
and warranted further exploration. Nevertheless, a head-to-head
comparison of the two hit series directed future investigations
toward the 5-POA carboxamide series. Specifically, although
being very potent hAC inhibitors, the 4-POA carboxamide series
suffered from significantly poorer chemical stability compared to
the 5-POA carboxamide series, as measured by performing the
stability assay in aqueous media [8a, t1/2 = 10 min, in phosphate
buffered saline (PBS), pH 7.4; 12a, t1/2 = >12 h, in PBS, pH 7.4].
First, we demonstrated the importance of the reactive
carboxamide functionality of 12a because the corresponding
unsubstituted analogue 11a was not active against hAC at the
concentrations tested (Scheme 3 and Figure 5A), suggesting
that inhibition by 12a could occur through covalent AC
modification. Preliminary kinetic studies on hAC-enriched
lysates showed that 12a causes a concentration-dependent
reduction in the maximal catalytic velocity of AC (Vmax) without
Table 1. Inhibitory Potencies of Compounds 8a−d and 12a−
n and 12y on the Activity of hAC
Figure 5. (A) Concentration−response curve for the inhibition of hAC
activity by 11a and 12a; (B) Michaelis−Menten analysis of the reaction
a
IC50 values are the mean of at least three independent experiments
b
●
▲
;
of hAC in the presence of vehicle (DMSO 1%, ) or 12a (25 nM,
performed in three technical replicates. IC50 values were not
determined for compounds showing less than 50% inhibition at
concentrations of 10 μM for hAC. Partial degradation was observed
in the 10 mM DMSO solution.
■
100 nM, ). Rbm14-12: fluorogenic substrate of hAC. The graph is
representative of two independent experiments, each performed in
three technical replicates.
c
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replacement of the N−H of the urea-like functionality of 12a
with a N−Me (13a), with an oxygen (13b) or with a methylene
(13c) were detrimental to activity (Scheme 3), as these
analogues were not active against hAC at the concentrations
tested (1 and 10 μM). Moreover, we demonstrated that the
presence of the oxazol-2-one ring of 12a was essential to
maintaining inhibitory potency, since the corresponding chiral
1,3-oxazolidinone carboxamide analogues 18a−b were not
active against hAC at the concentrations tested (1 and 10
μM) (Scheme 3). A similar outcome was also observed with the
chiral 1,3-oxazolidinone carboxamide analogues of the more
potent 8a, compounds 15a−b (Scheme 3).57
Table 2. Inhibitory Potencies of Compounds 12o−v on the
Activity of hAC
Therefore, based on these results, we continued with a more
focused SAR exploration, by targeting additional analogues
bearing small linear and branched alkyl substituents on the side-
chain at the N-terminal urea moiety of 12a series (Region C,
Figure 4). Replacement of one methylene unit with an oxygen in
the n-pentyl chain of 12b (as ethers 12c−d) significantly
affected the inhibitory potency; specifically, 12c showed an hAC
IC50 = 2.04 μM, while a complete loss in potency was observed
for 12d (Table 1). These results suggested that the lipophilic
side-chains at region C were very likely occupying a hydrophobic
channel of the enzyme. Our SAR exploration continued with the
insertion of branched alkyl groups, the i-butyl 12e inhibited AC
with an IC50 of 1.70 μM, while the s-butyl analogue 12f was not
active up to 10 μM. No improvement was also observed with the
aryl urea 12g.
In parallel, our medicinal chemistry efforts were also focused
on the exploration of region A by introducing different
substituents on the phenyl ring of the 12a series. Besides, the
p-Cl atom of 12i, both electron-withdrawing (F) and electron-
donating (OCH3) groups at different positions of the phenyl
ring were tolerated, resulting in compounds, such as 12j (p-F)
and 12k−m (p-, m- and o-OCH3), with hAC IC50 values in the
submicromolar ranges (Table 1). In addition, the di-substituted
derivative 12n (p-F, m-OCH3) was almost equipotent (hAC
IC50 = 0.080 μM) to 12a.
With these results in hand, we explored the replacement of the
substituted phenyl rings at the C(5)-position of the oxazol-2-
one moiety with a series of heteroaryl groups by preparing
compounds 12o−v (Table 2). Different 5- and 6-membered-, 6
+ 5-, and 6 + 6-fused heteroaryl groups were introduced on
Region A. Interestingly, we identified potent AC inhibitors, for
example, the 2- and 4-pyridyl analogues 12o and 12q, showing
hAC IC50 = 0.025 and 0.018 μM, respectively; whereas, the 3-
pyridyl isomer 12p (hAC IC50 = 0.070 μM) was almost
equipotent to 12a (Table 2). A similar improvement of
inhibitory potency was also observed with other nitrogen
containing heteroaryl derivatives, for example, the pyrazine
(12r) and quinoxaline (12s) analogues (hAC IC50 = 0.032 and
0.037 μM, respectively). On the other hand, while the thiazole
isomers 12u and 12v showed a moderately improved inhibitory
activity (hAC IC50 = 0.059 and 0.044 μM, respectively), the 1-
methylindazole 12t (hAC IC50 = 0.092 μM) was equipotent to
the parent 12a.
A comparison of several of these analogues in terms of
aqueous kinetic solubility (PBS, pH 7.4) and in vitro metabolism
(t1/2 in mouse plasma and mouse liver microsomes) underlined
some important differences (Table 3), which informed the next
steps of the SAR exploration. In general, these compounds
showed poor aqueous solubility, which was not ameliorated by
the removal of the terminal lipophilic phenyl group (e.g., n-
pentyl analogue 12b), or by the introduction of more polar
a
IC50 values are the mean of at least three independent experiments
performed in three technical replicates.
Table 3. Aqueous Kinetic Solubility and In Vitro Metabolism
of Some Selected Compounds
a
b
c
solubility (μM)
m-plasma t1/2 (min) m-LM t1/2 (min)
compound
(PBS, pH 7.4)
[% at 120 min]
[% at 60 min]
12a
12b
12c
12e
12j
<1
<1
20
<1
<1
<1
<1
<1
<1
<1
<1
<5
40
<5
<5
50
60
30
>120 [60%]
80
>120 [70%]
90
>120 [64%]
>120 [64%]
40
60
30
12l
12n
12o
12p
12q
12r
>60 [74%]
10
>60 [55%]
>60 [75%]
40
60
a
b
c
Aqueous kinetic solubility in PBS. Mouse plasma. Mouse liver
microsomes. a,b,cValues are reported as the mean of at least two
independent experiments performed in two technical replicates.
influencing the Michaelis−Menten constant (KM) (Figure 5B
and Table S2) supporting irreversible binding. In addition, the
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microsomal stability was also detected with the i-butyl analogue
12e (m-liver microsomal t1/2 = 50 min). A similar effect of the F-
atom was shown by some nitrogen containing heteroaryl
analogues, bearing an “aza”-group in the phenyl ring, for
example, the 4-pyridyl 12q (m-liver microsomal t1/2 > 60 min,
75% compound remaining at 1 h) and the pyrazine 12r (m-liver
microsomal t1/2 = 60 min) (Table 3).
Table 4. Inhibitory Potencies of Compounds 25c−f and 32a−
c on the Activity of hAC
Based on these results, we continued the SAR exploration by
modifying the scaffold with the insertion of some potential
solubilizing groups. We specifically focused our attention on
regions A and B due to the fact that, as mentioned above, our SAR
study suggested region C to be more involved in lipophilic
interactions with hAC (Table 1). In this respect, our preliminary
exploration of this series showed that the insertion of an
hydrophilic group, such as the N-methyl-piperidine ring at the
C(5)-position of the oxazol-2-one moiety (12y, Table 1),
although detrimental for the inhibitory potency (hAC IC50
=
1.40 μM), was essential for significantly improving the aqueous
solubility (12y, kinetic solubility = 248 μM). As a consequence,
we decided to investigate the effect of inserting the N-methyl-
piperidine group directly at the C(3′)- position of the phenyl
ring of the 5-POA carboxamide series by preparing analogues
25c and 25d (Table 4). Although a similar decrease in potency
(hAC IC50 = 0.153 and 0.341 μM, respectively) was observed
compared to the corresponding parent compounds 12a and 12j,
we were pleased to notice that, overall, these structural
modifications were tolerated. Moreover, a similar trend was
observed when the N-methyl-piperidine ring was moved to the
C(2′)- position of the phenyl ring, with 32a (hAC IC50 = 0.337
μM) being equipotent to 25d. Notably, we observed that these
targeted compounds showed moderately improved solubility
(kinetic solubility > 30 μM, Table 5) compared to the parent
compounds 12a and 12j.
These results prompted us to continue a more focused SAR
study on the lateral chains of 25d and 32a scaffolds, selecting the
optimal moieties previously identified in the exploration of
region C (Tables 1 and 3) and, hence, synthetizing the
corresponding n-pentyl analogues 25e and 32b and the i-butyl
analogues 25f and 32c. Although, a complete loss in potency was
observed with both i-butyl analogues 25f and 32c, the n-pentyl
derivatives 25e and 32b gave unexpected results. While 25e
showed a hAC IC50 of 1.9 μM, surprisingly, compound 32b
inhibited hAC with an IC50 equal to 0.129 μM. In addition,
compound 32b, bearing both the N-methyl piperidine ring and
the small linear alkyl chain, showed a high solubility value
(kinetic solubility > 250 μM) (Table 5). Finally, the most
promising compounds were evaluated for in vitro metabolism
(Table 5). In general, we were pleased to observe that the
selected compounds exhibited good mouse plasma stabilities
with t1/2 values ≥2 h. On the other hand, the poor mouse liver
microsomal stability observed for 25c was ameliorated by the
insertion of a F-atom at the para-position of the phenyl ring
(25d, m-liver microsomal t1/2 > 60 min, 70% compound
remaining at 1 h). A similar trend was observed with the other
fluorinated analogues, compound 32a−b. Furthermore, addi-
tional in vitro metabolism studies were performed on 32b which
showed acceptable h-plasma (t1/2 = 40 min) and good h-liver
microsomal stability (t1/2 > 60 min, 80% compound remaining
at 1 h).
a
IC50 values are the mean of at least three independent experiments
b
performed in three technical replicates. IC50 values were not
determined for compounds showing less than 50% inhibition at
concentrations of 10 μM for hAC.
Table 5. Aqueous Kinetic Solubility and In Vitro Metabolism
of Some Selected Compounds
a
b
c
solubility (μM)
(PBS,pH 7.4)
m-plasma t1/2 (min) m-LM t1/2 (min)
compound
[% at 120 min]
[% at 60 min]
25c
25d
32a
32b
70
30
60
>250
>120 [73%]
>120 [80%]
>120 [54%]
30
>60 [70%]
>60 [51%]
>60 [74%]
110
a
b
c
Aqueous kinetic solubility in PBS. Mouse plasma. Mouse liver
microsomes. a,b,cValues are reported as the mean of at least two
independent experiments performed in two technical replicates.
groups, such as an oxygen atom on the lateral chain (12c) or
heteroaryl rings at the C(5)-position of the oxazol-2-one moiety
(e.g., the pyridines 12o−q and the pyrazine 12r) (Table 3). On
the contrary, more pronounced differences were observed by
comparing the mouse plasma and mouse liver microsomal
stability properties of these analogues. In general, except for the
parent 12a and the heteroaryls 12o and 12q−r, the selected
compounds showed good mouse plasma stability. In contrast,
poor mouse microsomal stabilities were observed within the
phenyl derivatives, except when a F-atom was inserted in the
phenyl ring, for example, p-F analogues 12j (m-liver microsomal
Based on its inhibitory potency and good overall drug-like
properties, compound 32b was selected for further biological
and pharmacological characterizations. As previously antici-
pated for the initial hit 12a and based on our previous work with
t
1/2 = 60 min) and 12n (m-liver microsomal t1/2 > 60 min, 74%
compound remaining at 1 h). An improvement of the mouse
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Figure 6. Concentration dependence of the effects of 32b in SH-SY5Y cells on hAC activity after a 3 h incubation (A) and SL levels (B−F). GraphPad
Prism software (GraphPad Software, Inc., USA) was used for statistical analysis. Data were analyzed using the Student t-test or 1-way ANOVA followed
by the Bonferroni post hoc test for multiple comparisons. Differences between groups were considered statistically significant at values of p < 0.05.
Values are expressed as means SEM of at least six determinations. Experiments were repeated twice with similar results.
Figure 7. Time course of the effects of 32b (10 μM) in SH-SY5Y cells on hAC activity (A) and SL levels (B−F). GraphPad Prism software (GraphPad
Software, Inc., USA) was used for statistical analysis. Data were analyzed using the Student t-test or 1-way ANOVA followed by the Bonferroni post hoc
test for multiple comparisons. Differences between groups were considered statistically significant at values of p < 0.05. Values are expressed as means
SEM of at least six determinations. Experiments were repeated twice with similar results.
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into the structural bases of these biological results, we then
performed molecular modeling and docking studies using the X-
ray crystal structures of hAC (PDB code: 6MHM)67 and
hNAAA (PDB code: 6DXX).69 The protein (hAC)−ligand
(targeted compound) binding site was prepared by adding
hydrogen atoms, optimizing hydrogen bonds, and verifying the
protonation states of His, Gln, and Asn. The energy
minimization was carried out using a default constraint of 0.3
Å rmsd and OPLS 3e force field. The SiteMap tool was used to
identify binding pockets of hAC. The docking calculation on the
̈
SAR series was carried out using Schrodinger covalent docking
program CovDock. The docking procedure used the distance
cutoffs (8 Å Cβ to the ligand, 5 Å to the ligand reactive carbonyl
group) to decide if the covalent reaction can indeed happen or a
molecule pose should be kept for further consideration. Cys143
was used as the reactive nucleophile in the calculations. While
under experimental conditions, the leaving group would in fact
cleave from the molecule and only a part of the molecule would
remain attached to Cys143; the docking calculation actually
captures the binding pose quality of the tetrahedral intermediate
(reaction intermediate). The binding strength and quality of the
pose for this is likely to have an impact on the overall ligand
potency. A series of compounds from the described SAR studies
were analyzed using this docking model. We observed that, while
the docking model could account for the large changes in
potency and the scores were well correlated with the
experimental potency, it was hard to account for the smaller
incremental changes in the SAR data. Nevertheless, these
computational analyses gave useful insights in our experimental
results. In particular, in silico docking studies of 32b to hAC
suggests a binding mode of the corresponding tetrahedral
intermediate (reaction intermediate, docking score = −7.3), as
depicted in Figure 8B. The docking pose suggests the alkyl side-
chain (n-pentyl) of 32b sharing the same binding mode of the n-
hexyl chain of carmofur (Figure 2) within the hAC binding
pocket,67 where lipophilic residues, such as Phe163, Tyr137,
Leu211, and Meth161, are localized. In addition, hydrogen
bonding interaction stabilizes the transition state around the
catalytic Cys143 residue between the side-chain carboxyl group
of Glu225 (2.77 Å) and the hydroxy group (tetrahedral
intermediate) on the alpha carbon of the reaction center. The
ligand pose itself is stabilized by a cation−pi interaction between
the protonated N-methyl amino group of the piperidine ring and
Trp395 and a second edge to face the pi−pi interaction between
the fluorophenyl group of the ligand and Phe165 (4.56 and 3.43
Figure 8. Concentration−response curve for the inhibition of hAC
activity by 31c and 32b (A) and putative docking pose of compound
32b in hAC (PDB code: 6MHM) (B).
4d,61 the mechanism of inhibition of 32b occurs through
covalent hAC modification. This was further supported by the
corresponding analogue 31c, lacking the reactive urea-like
functionality of 32b, which was not active against hAC (Figure
8A). Based on these considerations, we prioritized the selectivity
evaluation of 32b against human N-acylethanolamine acid
amidase (hNAAA), a lysosomal cysteine amidase that shares
33−34% sequence identity and a very similar reactive site with
hAC.69 Notably, 32b showed no effect at up to 125 μM against
hNAAA under our assay conditions. In an effort to gain insights
Figure 9. Putative docking poses of compound 32b in hAC (PDB code: 6MHM) (A) and in hNAAA (PDB code: 6DXX) (B).
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7B,C) and decreased So levels (Figure 7D), which persisted up
to 6 h. No major variations were observed in the levels of SM
(d18:1/16:0) (Figure 7E) and HexCer (d18:1/16:0) (Figure
7F).
Table 6. PK Properties of 32b after Intravenous (3 mg/kg)
and Oral Administration (10 mg/kg) in C57BL/6 Mice
a
parameters
max (min)
(3 mg/kg, i.v.)
(10 mg/kg, p.o.)
t
30
278
Finally, we then took 32b for pharmacokinetic (PK) studies in
C57BL/6 mice, following intravenous (i.v.) and oral admin-
istration (p.o.). The relevant PK parameters are reported in
Table 6. Values of plasma clearance (Clp), volume of
distribution (Vdss), and plasma elimination half-life (t1/2) were
calculated after i.v. administration of 32b at 3 mg/kg. Clp was
relatively low (72 mL/min/kg) with acceptable plasma t1/2 (119
min) and high Vdss (12426 mL/kg), indicating that 32b is well
distributed out of the circulating mouse plasma compartment.
Compound 32b is an orally bioavailable hAC inhibitor at 10 mg/
kg (oral bioavailability, F = 40%) and is rapidly adsorbed in the
plasma compartment (tmax = 30 min), with a maximal plasma
concentration (Cmax) of 278 ng/mL and acceptable plasma t1/2
(147 min). Moreover, 32b shows significant exposures in mouse
plasma, after both i.v. and p.o. doses (AUC = 31978 and 42525
min × ng/mL, respectively).
C
max (ng/mL)
t
1/2 (min)
Cl (mL/min/kg)
dss (mL/kg)
119
72
12 426
31 978
147
155
32 877
42 525
40
V
AUC (min × ng/mL)
F (%)
a
PK parameters were calculated using PK solutions.
Å). It is conceivable that the electron-withdrawing nature of the
fluorophenyl might promote the strength of the pi−pi
interaction.
Analysis of 32b in the anti-target hNAAA suggests that the
binding site of the two enzymes share over 90% similarity in their
amino acid composition. However, when compound 32b is
docked into hNAAA,69 the resultant equivalent pose has a very
low score. Careful examination of the docking poses (modeling
details in Experimental Section) show that there is a critical
difference between hAC and hNAAA around residue 182 in the
binding site. While hAC has Leu at position 182, the equivalent
position is occupied by much larger Trp181 in hNAAA. The n-
pentyl side-chain on the compound 32b easily fits in the wider
pocket of hAC (Figure 9A), but it causes steric clashes, as seen in
the Figure 9B, in the case of hNAAA because of the much bulkier
Trp181 side-chain. We believe this to be one of key reasons for
selectivity observed in 32b that might be further exploited in
future drug design programs.
Because of the overall property profile of 32b, this compound
was selected for additional pharmacological studies with the aim
of testing its ability to inhibit hAC in intact cells. In particular, we
examined the effects of compound 32b treatment using human
neuroblastoma SH-SY5Y cells, which are a well-characterized
and widely used in vitro cell model.70,71 Notably, Kyriakou and
co-workers recently established and characterized a stable AC
knockdown human neuroblastoma SH-SY5Y cell line, as a
human in vitro cell model for studying the effects of AC
deficiency.72 Human neuroblastoma SH-SY5Y cells were
incubated in the presence of 32b at different concentrations
for 3 h (1, 2.5, 5, and 10 μM, Figure 6) and in the presence of
32b (10 μM) at different incubation times (1, 3, and 6 h, Figure
7). hAC activity was measured and SL levels were identified and
quantified with a liquid chromatography/mass spectrometry
(LC/MS)-based activity assay, as previously described.55,56,61
We indeed demonstrated that 32b is effectively able to engage
hAC in the complex cellular environment under our
experimental conditions, causing the expected changes in the
cellular levels of SL. Treatment of SH-SY5Y cell cultures with
32b caused a concentration (Figure 6A) and time-dependent
reduction of hAC activity (Figure 7A). After 3 h incubation, we
observed an intracellular accumulation of various Cer species,
including Cer (d18:0/16:0) and Cer (d18:1/16:0) (Figure
6B,C), and a corresponding decrease in So levels in a
concentration-dependent manner (Figure 6D). Conversely, no
major variations were observed in the levels of SM (d18:1/16:0)
(Figure 6E) and HexCer (d18:1/16:0) (Figure 6F). The effect
of 32b (10 μM) on the inhibition of hAC activity and the
intracellular SL levels is reported in Figure 7A−F. Specifically,
we observed that 32b inhibits hAC in SH-SY5Y cells leading to
an increased Cer (d18:0/16:0) and Cer (d18:1/16:0) (Figure
Taking into account its overall profile, 32b was selected for
further development studies aimed to elucidate the potential
therapeutic applications of AC inhibition in cellular and in vivo
model systems of relevant SL-mediated disorders, which will be
described elsewhere in due course.
CONCLUSIONS
■
Although hAC inhibition has been the focus of intense discovery
in the last decade, only a very limited number of valuable
candidates for in vivo experiments are available. The scope of
this work was directed to solve this limitation. The design and
synthesis of a series of substituted oxazol-2-one-3-carboxamide
derivatives were presented, resulting in the identification of two
initial hits, 8a and 12a as a novel and versatile class of hAC
inhibitors. Preliminary results of the hit expansion around these
new scaffolds in the three main Regions A, B, and C contributed
to the definition of the pharmacophore necessary for target
inhibition and directed the strategies for chemical optimization.
Our medicinal chemistry efforts around the most promising 5-
substituted oxazol-2-one-3-carboxamide series led to the
identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-
2-oxo-N-pentyl-oxazole-3-carboxamide (32b) as an optimized
hAC inhibitor, structurally distinct from previous reported
inhibitors, with good drug-like properties. Furthermore, 32b
showed target engagement in human neuroblastoma SH-SY5Y
cells and desirable PK properties in mice, with good F % and
significant exposures in plasma, after intravenous and oral
administrations. Compound 32b is a valuable lead that increases
the arsenal of suitable hAC-targeting molecules, which can
directly probe the link of hAC function to distinct physiological
processes and investigate how the inhibition of its activity can
provide health benefits under severe pathological conditions.
The identification of novel hAC-modulating compounds,
targeting active Cys143 with optimal drug-like properties,
remains a challenging task and can be only achieved by a
critical and balanced modulation of different parameters, whose
objectives often clash during the chemical optimization process.
Utilizing the recently reported crystal structures, we were able to
dock our covalent inhibitors into the reactive site highlighting
the basis for selectivity observed with 32b for hAC compared to
hNAAA. In addition, the modeling that was undertaken can
guide future optimization of this lead series accelerating the field
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compounds were diluted to 50 μM in CH3CN/H2O and analyzed.
Leucine Enkephalin (2 ng/mL) was used as the lock mass reference
compound for spectra recalibration. All final compounds displayed
≥95% purity as determined by NMR and UPLC/MS analysis.
General Procedure for the Synthesis of 4-Substituted-
oxazol-2-ones (Procedure A). To a mixture of the appropriate α-
hydroxy ketone (1.0 equiv) and KNCO (2.0 equiv) in i-PrOH (0.2 M)
was added dropwise AcOH (2.0 equiv) with stirring. The resulting
suspension was heated at 70 °C for 3 h, then poured into an ice/H2O
bath, and extracted with DCM or EtOAc. The organic phase was dried
over Na2SO4 and concentrated under reduced pressure. The crude was
purified by column chromatography (SiO2), eluting with Cy/EtOAc or
used as crude in the next step without further purification.
of research and contributing to the identification of additional
novel and efficacious hAC inhibitors.
EXPERIMENTAL SECTION
■
Chemicals, Materials, and Methods. Solvents and reagents were
obtained from commercial suppliers and were used without further
purification. Automated column chromatography purifications were
done using a Teledyne ISCO apparatus (CombiFlash Rf) with pre-
packed silica gel or neutral alumina columns of different sizes (from 4 g
until 120 g). Mixtures of increasing polarity of Cy and EtOAc or
dichloromethane (DCM) and MeOH were used as eluents. Thin-layer
chromatography (TLC) analyses were performed using Supelco silica
gel on TLC Al foils 0.2 mm with a fluorescence indicator 254 nm. NMR
experiments of all the intermediates and final compounds were run on a
Bruker AVANCE III 400 system (400.13 MHz for 1H, and 100.62 MHz
for 13C), equipped with a BBI probe and Z-gradient coil. Spectra were
acquired at 300 K using DMSO-d6 or CDCl3 as solvents. Chemical
shifts for 1H and 13C spectra were recorded in parts per million (ppm)
using the residual nondeuterated solvent as the internal standard (for
General Procedure for the Synthesis of 5-Substituted-
oxazol-2-ones (Procedure B). Step 1: to a stirred solution of the
appropriate α-bromoketone (1.0 equiv) and TZD (1.2 equiv) in DMF
(0.1−1 M) was added K2CO3 (1.5 equiv). The resulting mixture was
stirred at rt for 1−2 h and then poured into an ice/H2O bath. In some
cases, the resulting solid was filtered off and washed with H2O. In other
cases, the aq phase was extracted with EtOAc, and the organic layer was
washed with 5% aq LiCl and brine and dried over Na2SO4. After
evaporation of the solvent, the crude was purified by flash
chromatography (SiO2) eluting with Cy/EtOAc or used as a crude in
the next step without further purification, as indicated in each case. Step
2: the corresponding intermediate from step 1 was dissolved in THF
(0.1 M) and treated with LiOH (4.0 equiv) or t-BuOK (2.0−4.0 equiv)
as indicated in each case. The resulting mixture was stirred at rt for 30−
60 min and then poured into a cold aq AcOH solution (5.0−10.0
equiv). In some cases, the resulting solid was filtered off, washed with
H2O, and used in the next step without further purification.
Alternatively, the aq phase was extracted with EtOAc and the organic
phase was dried over Na2SO4. After evaporation of the solvent, the
crude was purified by flash chromatography (SiO2 or Al2O3) eluting
with Cy/EtOAc or DCM/MeOH or DCM/EtOH or used as a crude in
the next step without further purification, as indicated in each case.
General Procedure for the Synthesis of 5-Phenyl-oxazolidin-
2-ones (Procedure C). To a solution of (S) or (R)-2-amino-1-
phenylethanol (1.0 equiv) in DCM (0.1 M) was added imidazole (0.5
equiv) followed by the addition of CDI (1.1 equiv). The reaction
mixture was stirred at rt 16 h, then washed with H2O, and dried over
Na2SO4. After evaporation of the solvent, the crude was purified by flash
chromatography (SiO2) eluting with DCM/MeOH, as indicated in
each case.
General Procedure for the Synthesis of Carboxamides
(Procedure D). Method A: to a stirred solution of the appropriate
oxazol-2-one, or oxazolidin-2-one, (1.0 equiv) in CH3CN, or pyridine,
or DMF (0.2 M) were added DMAP (0.1−1.1 equiv), or Et3N (4.0
equiv), and the appropriate isocyanate (1.1−2.0 equiv). The resulting
solution was stirred at rt (or at 50 °C, as indicated in each case) for 3−
16 h, then diluted with EtOAc, washed with sat. aq NH4Cl solution and
brine, and dried over Na2SO4. After evaporation of the solvent, the
crude was purified by column chromatography (SiO2 or Al2O3), eluting
with Cy/EtOAc or DCM/MeOH, as indicated in each case. Method B:
To a stirred solution of Boc2O (2.0 equiv) in CH3CN (0.4 M) were
added DMAP (2.0 equiv) and the appropriate amine (1.1−2.0 equiv).
The resulting solution was stirred at rt for 10 min then the appropriate
oxazol-2-one derivative (1.0 equiv) was added, and the mixture was
stirred at rt for 1−3 h. After evaporation of the solvent, the crude was
purified by flash chromatography (SiO2) eluting with Cy/EtOAc or
DCM/MeOH, as indicated in each case. Method C: To a stirred
solution of triphosgene (0.33 equiv) in dry DCM (0.2 M), a solution of
the appropriate amine (1.0 equiv) and Et3N, or DIPEA, (2.0 equiv) in
DCM (0.2 M) was added at −15 °C. The resulting mixture was stirred
at rt for 30 min under a N2 atmosphere and then added to a solution of
the appropriate oxazol-2-one (0.33 equiv) and Et3N, or DIPEA, (1.0
equiv) in DCM (0.2 M). The reaction mixture was stirred under a N2
atmosphere at rt for 3−12 h, then diluted with DCM, washed with sat.
aq NH4Cl solution and brine, and dried over Na2SO4. After evaporation
of the solvent, the crude was purified by column chromatography
1
1
DMSO-d6: 2.50 ppm, H; 39.52 ppm, 13C; for CDCl3: 7.26 ppm, H
and 77.16 ppm, 13C). Data are reported as follows: chemical shift
(ppm), multiplicity (indicated as: bs, broad singlet; s, singlet; d,
doublet; t, triplet; q, quartet; p, quintet, sx, sextet; m, multiplet; and
combinations thereof), coupling constants (J) in Hertz (Hz), and
integrated intensity. Quantitative 1H NMR analyses of the freshly
prepared 10 mM DMSO-d6 stock solutions (used for biological
screenings) of the final compounds were performed using the
PULCON method (pulse length-based concentration determination,
Bruker software, topspin 3.0. References: (a) Wider, G.; Reires, L. J. Am.
Chem. Soc. 2006, 128 (8), 2571−2576; (b) Burton, I. W.; Quilliam, M.
A.; Valter, J. A. Anal. Chem. 2005, 77, 3123−3131). UPLC/MS analyses
of all the intermediates and final compounds were performed on a
Waters Acquity UPLC/MS system consisting of single quadrupole
detection (SQD) mass spectrometry (MS) equipped with an
electrospray ionization (ESI) interface and a photodiode array
(PDA) detector. The PDA range was 210−400 nm. Analyses were
performed on an Acquity UPLC BEH C18 column (50 × 2.1 mm ID,
particle size 1.7 μm) with a VanGuard BEH C18 pre-column (5 × 2.1
mm ID, particle size 1.7 μm). The mobile phase was 10 mM NH4OAc
in H2O at pH 5 adjusted with AcOH (A) and 10 mM NH4OAc in
CH3CN/H2O (95:5) at pH 5 (B). ESI in both positive and negative
modes was used in the mass scan range 100−650 Da. Analyses were
performed with method A, B, C, or D. Method A: Gradient: 5−95% B
over 2.5 min. Flow rate 0.5 mL/min. Temperature 40 °C. Method B:
Gradient: 50−100% B over 2.5 min. Flow rate 0.5 mL/min.
Temperature 40 °C. Method C: Gradient: 0−100% B over 2.5 min.
Flow rate 0.5 mL/min. Temperature 40 °C. Method D: Isocratic 55% B
over 5 min. Flow rate 0.5 mL/min. Temperature 40 °C. UPLC/MS
analyses of freshly prepared 10 mM DMSO-d6 stock solutions (used for
biological screenings) of the final compounds were performed with
method E or method F. DMSO-d6 stock solution (10 μL, 10 mM) was
diluted 20-fold or 100-fold with a 1:1 CH3CN/H2O solution and
directly analyzed. An Acquity UPLC BEH C18 (100 × 2.1 mm ID,
particle size 1.7 μm) with a VanGuard BEH C18 pre-column (5 × 2.1
mm ID, particle size 1.7 μm). The mobile phase was either 10 mM
NH4OAc in H2O at pH 5 adjusted with AcOH (A) and 10 mM
NH4OAc in CH3CN/H2O (95:5) at pH 5 (B). ESI in positive and
negative modes was applied in the mass scan range 100−650 Da.
Method E: Gradient: 10−90% B over 6 min. Flow rate 0.5 mL/min.
Temperature 40 °C. Method F: Gradient: 50−100% B over 6 min. Flow
rate 0.5 mL/min. Temperature 40 °C. The detection wavelength (λ)
was set at 215 nm for relative purity determination. Rt of the final
compounds under method E or F UPLC/MS analytical conditions are
reported in Table S3. Optical rotations were measured on a Rudolf
Research Analytical Autopol II Automatic polarimeter using a sodium
lamp (589 nm) as the light source, concentrations are expressed in g/
100 mL using CHCl3 as a solvent and a 1 dm cell. Accurate mass
measurements were performed on a Synapt G2 Quadrupole-ToF
Instrument (Waters, USA), equipped with an ESI ion source; the
O
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(SiO2), eluting with Cy/EtOAc, or DCM/MeOH, as indicated in each
case.
(m, 1H). UPLC/MS (method A): Rt 1.71 min. MS (ES): C9H6ClNO2
requires, 195; found, 196 [M + H]+, 194 [M − H]−.
Synthesis of 2-Oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-car-
boxamide (8a). Compound 8a was prepared according to general
procedure D (method A) using 7a (0.161 g, 1.00 mmol), DMAP (0.012
g, 0.10 mmol), and 4-phenylbutyl isocyanate (0.350 g, 2.00 mmol) in
CH3CN. The crude was purified by column chromatography (SiO2),
eluting with Cy/EtOAc (9:1), to afford 8a as a white solid (0.243 g,
72%). 1H NMR (400 MHz, CDCl3): δ 8.10 (bs, 1H), 7.56−7.34 (m,
5H), 7.34−7.27 (m, overlapped with CDCl3 signal, 2H), 7.26−7.11 (m,
3H), 6.75 (s, 1H), 3.42−3.26 (m, 2H), 2.63 (t, J = 7.2 Hz, 2H), 1.78−
1.60 (m, 4H). 13C NMR (101 MHz, CDCl3): δ 154.57, 148.99, 142.09,
129.36, 129.00, 128.85, 128.53, 128.50, 128.14, 127.43, 125.99 (2C),
40.28, 35.57, 29.12, 28.68. UPLC/MS (method A): Rt 2.55 min. MS
(ES): C20H20N2O3 requires, 336; found, 337 [M + H]+. HRMS:
C20H20N2O3 [M + H]+ calcd 337.1552; measured, 337.1549, Δppm
−0.9.
General Procedure for Palladium-Catalyzed Cross-Coupling
Reaction (Procedure E). To a solution of the appropriate phenyl
bromide (1.0 equiv) in dry 1,4-dioxane (0.1 M, previously degassed
under a nitrogen atmosphere), 19 (1.1 equiv) was added followed by
the addition of Pd(PPh3)4 (0.05 equiv) and Na2CO3 (2.2 equiv, 2 M aq
solution). The suspension was stirred at reflux on, cooled to rt, and then
diluted with EtOAc and filtered through a pad of celite. The filtrate was
concentrated under reduced pressure, diluted with EtOAc, washed with
sat. aq NH4Cl solution and brine, and dried over Na2SO4. After
evaporation of the solvent, the crude was purified by flash
chromatography (SiO2), eluting with Cy/EtOAc, as indicated in each
case.
General Procedure for Catalytic Hydrogenation Reaction
(Procedure F). Method A: To a suspension of the appropriate
unsaturated intermediate (1.0 equiv) in EtOH (0.1 M) were added 10%
Pd/C (0.1 equiv) and cyclohexene (20.0 equiv,) and the mixture was
stirred at 60 °C until disappearance of the starting material, as indicated
by UPLC/MS analysis. The suspension was filtered through a pad of
celite, and the filtrate was quickly evaporated under reduced pressure.
The crude was purified by flash chromatography (SiO2), eluting with
Cy/EtOAc or used in the next step without further purification, as
indicated in each case. Method B: To a suspension of the appropriate
unsaturated intermediate (1.0 equiv) in MeOH (0.1 M) were added
20% Pd(OH)2 on carbon (10% wt) and HCO2NH4 (7.0 equiv), and
the mixture was stirred at 60 °C for 4 h. The suspension was filtered
through a pad of celite, and the filtrate was quickly evaporated under
reduced pressure. The crude was purified by flash chromatography
(SiO2), eluting with Cy/EtOAc, or used in the next step without further
purification, as indicated in each case.
Synthesis of 2-Oxo-N-pentyl-4-phenyl-oxazole-3-carboxamide
(8b). Compound 8b was prepared according to general procedure D
(method A) using 7a (0.161 g, 1.00 mmol), DMAP (0.012 g, 0.10
mmol), and n-pentyl isocyanate (0.170 g, 1.50 mmol) in CH3CN. The
crude was purified by column chromatography (SiO2), eluting with Cy/
EtOAc (9:1), to afford 8b as yellow oil (0.225 g, 82%). 1H NMR (400
MHz, CDCl3): δ 8.09 (bs, 1H), 7.45−7.32 (m, 5H), 6.74 (s, 1H),
3.34−3.23 (m, 2H), 1.64−1.49 (m, overlapped with a H2O signal, 2H),
1.40−1.23 (m, 4H), 0.91 (t, J = 6.8 Hz, 3H). 13C NMR (101 MHz,
CDCl3): δ 154.57, 148.94, 129.33, 128.97, 128.85, 128.12, 127.45,
125.80, 40.45, 29.16, 29.08, 22.42, 14.09. UPLC/MS (method A): Rt
2.40 min. MS (ES): C15H18N2O3 requires, 274; found, 275 [M + H]+.
HRMS: C15H18N2O3Na [M + Na]+ calcd 297.1215; measured,
297.1210, Δppm −1.7.
Synthesis of 5-Methyl-2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-
3-carboxamide (8c). Compound 8c was prepared according to general
procedure D (method A) using 7b (0.130 g, 0.74 mmol), DMAP (0.099
g, 0.81 mmol), and 4-phenylbutyl isocyanate (0.142 g, 0.81 mmol) in
pyridine. The crude was purified by column chromatography (SiO2),
eluting with Cy/EtOAc (4:1), to afford 8c as a white solid (0.040 g,
15%). 1H NMR (400 MHz, CDCl3): δ 8.09 (t, J = 4.4 Hz, 1H), 7.42−
7.37 (m, 3H), 7.33−7.24 (m, overlapped with CDCl3 signal, 4H),
7.21−7.12 (m, 3H), 3.34−3.23 (m, 2H), 2.61 (t, J = 7.4 Hz, 2H), 2.05
(s, 3H), 1.71−1.56 (m, overlapped with H2O signal, 4H). 13C NMR
(101 MHz, CDCl3): δ 149.26, 142.13, 135.35, 131.21, 129.77, 129.14,
128.87, 128.53, 128.47, 128.11, 126.59, 125.95, 40.16, 35.58, 29.16,
28.69, 10.19. UPLC/MS (method A): Rt 2.71 min. MS (ES):
C21H22N2O3 requires, 350; found, 351 [M + H]+. HRMS:
C21H22N2O3Na [M + Na]+ calcd 373.1528; measured, 373.1523,
Δppm −1.3.
Synthesis of 4-(4-Chlorophenyl)-2-oxo-N-(4-phenylbutyl)-
oxazole-3-carboxamide (8d). Compound 8d was prepared according
to general procedure D (method A) using 7c (0.100 g, 0.51 mmol),
DMAP (0.068 g, 0.56 mmol), and 4-phenylbutyl isocyanate (0.098 g,
0.56 mmol) in pyridine. The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (95:5), to afford 8d
as yellow oil (0.040 g, 21%). 1H NMR (400 MHz, CDCl3): δ 8.10 (t, J =
5.2 Hz, 1H), 7.40−7.34 (m, 2H), 7.32−7.26 (m, overlapped with
CDCl3 signal, 4H), 7.22−7.12 (m, 3H), 6.75 (s, 1H), 3.41−3.23 (m,
2H), 2.63 (t, J = 7.3 Hz, 2H), 1.90−1.49 (m, 4H). 13C NMR (101 MHz,
CDCl3): δ 154.33, 148.93, 142.02, 135.54, 130.32, 128.50 (2C), 128.43
(3C), 126.00, 125.88, 40.29, 35.54, 29.07, 28.65. UPLC/MS (method
A): Rt 2.71 min. MS (ES): C20H19ClN2O3 requires, 370; found, 371 [M
+ H]+. HRMS: C20H19ClN2O3 [M + Na]+ calcd 393.0982; measured,
393.0973, Δppm −2.3.
General Procedure for N-Boc Removal (Procedure G). To a
suspension of the appropriate N-Boc protected intermediate (1.0
equiv) in 1,4-dioxane (0.1 M) was added HCl (30.0 equiv, 4 M in 1,4-
dioxane), and the reaction mixture was stirred at rt for 2 h. After
evaporation of the solvent, the crude was triturated with Et2O, or used
as a crude, in the next step without further purification.
General Procedure for Reductive Amination Reaction
(Procedure H). To a solution of the appropriate substituted piperidine
intermediate (1.0 equiv) in CH3CN (0.1 M) were added 37% aq
solutions of HCHO (2.0−5.0 equiv), AcOH (2.0 equiv), and
NaBH(OAc)3 (3.0 equiv). The mixture was stirred at rt for 1 h.
Then, the reaction mixture was poured into a saturated aqueous
NaHCO3 solution and extracted with EtOAc. The organic phase was
washed with brine and dried over Na2SO4. After evaporation of the
solvent, the crude was purified by flash chromatography (SiO2) eluting
with DCM/MeOH, or used as a crude, in the next step without further
purification, as indicated in each case.
Synthesis of 4-Phenyl-3H-oxazol-2-one (7a). Compound 7a was
prepared according to general procedure A using 6a (1.360 g, 10.00
mmol). The crude was purified by column chromatography (SiO2),
eluting with Cy/EtOAc (4:1) to afford 7a as a yellow solid (0.858 g,
53%). 1H NMR (400 MHz, DMSO-d6): δ 11.34 (bs, 1H), 7.68 (d, J =
1.2 Hz, 1H), 7.62−7.53 (m, 2H), 7.49−7.40 (m, 2H), 7.39−7.29 (m,
1H). UPLC/MS (method A): Rt 1.48 min. MS (ES): C9H7NO2
requires, 161; found, 162 [M + H]+, 160 [M − H]−.
Synthesis of 5-Methyl-4-phenyl-3H-oxazol-2-one (7b). Com-
pound 7b was prepared according to general procedure A using 6b
(0.350 g, 2.33 mmol). The crude was used in the next step without
1
further purification. H NMR (400 MHz, CDCl3): δ 9.39 (bs, 1H),
7.48−7.41 (m, 2H), 7.40−7.32 (m, 3H), 2.32 (s, 3H). UPLC/MS
(method A): Rt 1.84 min. MS (ES): C10H9NO2 requires, 175; found,
176 [M + H]+, 174 [M − H]−.
Synthesis of 4-(4-Chlorophenyl)-3H-oxazol-2-one (7c). Com-
pound 7c was prepared according to general procedure A using 6c
(0.241 g, 1.41 mmol). The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (9:1), to afford 7c
as a yellow solid (0.10 g, 36%). 1H NMR (400 MHz, CDCl3): δ 9.61
(bs, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H), 7.11−7.08
Synthesis of 3-Phenacylthiazolidine-2,4-dione (10a). Compound
10a was prepared according to general procedure B, Step 1, using 9a
(1.990 g, 10.00 mmol). The crude was used in Step 2 without further
purification. UPLC/MS (method A): Rt 1.69 min. MS (ES):
C11H9NO3S requires, 235; found, 234 [M − H]−.
Synthesis of 3-(1-Methyl-2-oxo-2-phenyl-ethyl)thiazolidine-2,4-
dione (10b). Compound 10b was prepared according to general
procedure B, Step 1, using 9b (0.500 g, 2.35 mmol). The crude was
P
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Article
purified by column chromatography (SiO2), eluting with Cy/EtOAc
(4:1), to afford 10b as a white solid (0.550 g, 94%). H NMR (400
(method D): Rt 1.08 min. MS (ES): C13H11N3O3S requires, 289; found,
290 [M + H]+, 288 [M − H]−.
1
MHz, CDCl3): δ 7.75−7.68 (m, 2H), 7.58−7.52 (m, 1H), 7.47−7.41
(m, 2H), 5.55 (q, J = 7.0 Hz, 1H), 3.90 (d, J = 17.5 Hz, 1H), 3.82 (d, J =
17.5 Hz, 1H), 1.65 (d, J = 7.0 Hz, 3H). UPLC/MS (method A): Rt 1.83
min. MS (ES): C12H11NO3S requires, 249; found, 250 [M + H]+.
Synthesis of 3-[2-(4-Chlorophenyl)-2-oxo-ethyl]thiazolidine-2,4-
dione (10c). Compound 10c was prepared according to general
procedure B, Step 1, using 9c (1.170 g, 5.00 mmol). The crude was used
in Step 2 without further purification. UPLC/MS (method A): Rt 2.01
min. MS (ES): C11H8ClNO3S requires, 269; found, 268 [M − H]−.
Synthesis of 3-[2-(4-Fluorophenyl)-2-oxo-ethyl]thiazolidine-2,4-
dione (10d). Compound 10d was prepared according to general
procedure B, Step 1, using 9d (2.170 g, 10.00 mmol). The crude was
used in Step 2 without further purification. UPLC/MS (method A): Rt
1.81 min. MS (ES): C11H8FNO3S requires, 253; found, 254 [M + H]+,
252 [M − H]−.
Synthesis of 3-(2-Oxo-2-thiazol-2-yl-ethyl)thiazolidine-2,4-dione
(10o). Compound 10o was prepared according to general procedure B,
Step 1, using 9o (0.500 g, 2.43 mmol). The crude was used in Step 2
without further purification. UPLC/MS (method A): Rt 1.47 min. MS
(ES): C8H6N2O3S2 requires, 242; found, 243 [M + H]+, 241 [M − H]−.
Synthesis of 3-(2-Oxo-2-thiazol-4-yl-ethyl)thiazolidine-2,4-dione
(10p). Compound 10p was prepared according to general procedure B,
Step 1, using 9p (0.130 g, 0.63 mmol). The crude was used in Step 2
without further purification. UPLC/MS (method A): Rt 1.27 min. MS
(ES): C8H6N2O3S2 requires, 242; found, 243 [M + H]+, 241 [M − H]−.
Synthesis of tert-Butyl 4-[2-(2,4-Dioxothiazolidin-3-yl)acetyl]-
piperidine-1-carboxylate (10q). Compound 10q was prepared
according to general procedure B, Step 1, using 9q (0.310 g, 1.00
mmol). The crude was used in Step 2 without further purification. 1H
NMR (400 MHz, CDCl3): δ 4.46 (s, 2H), 4.17−4.06 (m, 2H), 4.03 (s,
2H), 2.89−2.77 (m, 2H), 2.61 (tt, J = 11.2, 3.8 Hz, 1H), 1.92−1.81 (m,
2H), 1.61 (qd, J = 11.9, 4.3 Hz, 1H), 1.45 (s, 9H). UPLC/MS (method
A): Rt 1.98 min. MS (ES): C15H22N2O5S requires, 342; found, 343 [M
+ H]+.
Synthesis of 3-[2-(4-Methoxyphenyl)-2-oxo-ethyl]thiazolidine-
2,4-dione (10e). Compound 10e was prepared according to general
procedure B, Step 1, using 9e (2.290 g, 10.00 mmol). The crude was
1
used in Step 2 without further purification. H NMR (400 MHz,
DMSO-d6): δ 8.13−7.92 (m, 2H), 7.14−7.05 (m, 2H), 5.06 (s, 2H),
4.39 (s, 2H), 3.87 (s, 3H). UPLC/MS (method A): Rt 1.78 min. MS
(ES): C12H11NO4S requires, 265; found, 266 [M + H]+, 264 [M − H]−.
Synthesis of 3-[2-(3-Methoxyphenyl)-2-oxo-ethyl]thiazolidine-
2,4-dione (10f). Compound 10f was prepared according to general
procedure B, Step 1, using 9f (1.260 g, 5.50 mmol). The crude was used
in Step 2 without further purification. UPLC/MS (method A): Rt 1.83
min. MS (ES): C12H11NO4S requires, 265; found, 266 [M + H]+, 264
[M − H]−.
Synthesis 3-[2-(2-Methoxyphenyl)-2-oxo-ethyl]thiazolidine-2,4-
dione (10g). Compound 10g was prepared according to general
procedure B, Step 1, using 9g (0.500 g, 2.18 mmol). The crude was used
in Step 2 without further purification. UPLC/MS (method A): Rt 1.85
min. MS (ES): C12H11NO4S requires, 265; found, 266 [M + H]+, 264
[M − H]−.
Synthesis of 3-[2-(4-Fluoro-3-methoxy-phenyl)-2-oxo-ethyl]-
thiazolidine-2,4-dione (10h). Compound 10h was prepared according
to general procedure B, Step 1, using 9h (0.250 g, 1.01 mmol). The
crude was used in Step 2 without further purification. UPLC/MS
(method A): Rt 1.85 min. MS (ES): C12H10FNO4S requires, 283; found,
284 [M + H]+, 282 [M − H]−.
Synthesis of 3-[2-Oxo-2-(2-pyridyl)ethyl]thiazolidine-2,4-dione
(10i). Compound 10i was prepared according to general procedure
B, Step 1, using 9i (0.400 g, 2.00 mmol). The crude was used in Step 2
without further purification. UPLC/MS (method A): Rt 1.38 min. MS
(ES): C10H8N2O3S requires, 236; found, 237 [M + H]+.
Synthesis of 3-[2-Oxo-2-(3-pyridyl)ethyl]thiazolidine-2,4-dione
(10j). Compound 10j was prepared according to general procedure
B, Step 1, using 9j (2.000 g, 10.00 mmol). The crude was used in Step 2
without further purification. UPLC/MS (method A): Rt 1.21 min. MS
(ES): C10H8N2O3S requires, 236; found, 237 [M + H]+.
Synthesis of 3-[2-Oxo-2-(4-pyridyl)ethyl]thiazolidine-2,4-dione
(10k). Compound 10k was prepared according to general procedure
B, Step 1, using 9k (1.000 g, 5.00 mmol). The crude was used in Step 2
without further purification. UPLC/MS (method A): Rt 1.15 min. MS
(ES): C10H8N2O3S requires, 236; found, 237 [M + H]+.
Synthesis of 3-(2-Oxo-2-pyrazin-2-yl-ethyl)thiazolidine-2,4-dione
(10l). Compound 10l was prepared according to general procedure B,
Step 1, using 9l (0.600 g, 2.99 mmol). The crude was used in Step 2
without further purification. UPLC/MS (method A): Rt 1.25 min. MS
(ES): C9H7N3O3S requires, 237; found, 238 [M + H]+.
Synthesis of 3-(2-Oxo-2-quinoxalin-2-yl-ethyl)thiazolidine-2,4-
dione (10m). Compound 10m was prepared according to general
procedure B, Step 1, using 9m (0.200 g, 0.80 mmol). The crude was
used in Step 2 without further purification. UPLC/MS (method A): Rt
1.76 min. MS (ES): C13H9N3O3S requires, 287; found, 288 [M + H]+.
Synthesis of 3-[2-(1-Methylindazol-3-yl)-2-oxo-ethyl]-
thiazolidine-2,4-dione (10n). Compound 10n was prepared according
to general procedure B, Step 1, using 9n (0.120 g, 0.47 mmol). The
crude was used in Step 2 without further purification. UPLC/MS
Synthesis of 5-Phenyl-3H-oxazol-2-one (11a). Compound 11a was
prepared using 10a (2.350 g, 10.00 mmol) and LiOH (0.960 g, 40.00
mmol), according to general procedure B, Step 2. After aq. work-up, the
resulting solid was collected by filtration to afford 11a as a white solid
1
(1.20 g, 74%). H NMR (400 MHz, DMSO-d6): δ 10.83 (bs, 1H),
7.53−7.46 (m, 3H), 7.44−7.36 (m, 2H), 7.31−7.23 (m, 1H). 13C NMR
(101 MHz, DMSO-d6): δ 155.16, 138.51, 128.82, 128.80, 127.90,
127.43, 122.30, 122.27, 109.05. UPLC/MS (method A): Rt 1.50 min.
MS (ES): C9H7NO2 requires, 161; found, 162 [M + H]+, 160 [M −
H]−. HRMS: C9H8NO2 [M + H]+ calcd 162.0555; measured,
162.0546, Δppm −5.0.
Synthesis of 4-Methyl-5-phenyl-3H-oxazol-2-one (11b). Com-
pound 11b was prepared using 10b (0.150 g, 0.60 mmol) and t-BuOK
(0.130 g, 1.20 mmol), according to general procedure B, Step 2. The
crude was purified by column chromatography (Al2O3), eluting with
DCM/EtOH (9:1 to 6:4), to afford 11b as a white solid (0.010 g, 10%).
1H NMR (400 MHz, CDCl3): δ 9.30 (bs, 1H), 7.50−7.45 (m, 2H),
7.43−7.37 (m, 2H), 7.32−7.26 (m, overlapped with CDCl3 signal, 1H),
2.31 (s, 3H). UPLC/MS (method A): Rt 1.61 min. MS (ES): C10H9NO2
requires, 175; found, 176 [M + H]+, 174 [M − H]−.
Synthesis of 5-(4-Chlorophenyl)-3H-oxazol-2-one (11c). Com-
pound 11c was prepared using 10c (1.345 g, 5.00 mmol) and LiOH
(0.479 g, 20.00 mmol), according to general procedure B, Step 2. After
aq work-up, the resulting solid was collected by filtration to afford 11c
1
as a pink solid (0.975 g, quant.). H NMR (400 MHz, DMSO-d6): δ
10.82 (bs, 1H), 7.55 (s, 1H), 7.53−7.43 (m, 4H). UPLC/MS (method
A): Rt 1.79 min. MS (ES): C9H6ClNO2 requires, 195; found, 196 [M +
H]+, 194 [M − H]−
Synthesis of 5-(4-Fluorophenyl)-3H-oxazol-2-one (11d). Com-
pound 11d was prepared using 10d (2.530 g, 10.00 mmol) and LiOH
(0.958 g, 40.00 mmol), according to general procedure B, Step 2. After
aq work-up, the resulting solid was collected by filtration to afford 11d
1
as a white solid (0.300 g, 17%). H NMR (400 MHz, DMSO-d6): δ
10.84 (bs, 1H), 7.59−7.49 (m, 2H), 7.46 (s, 1H), 7.30−7.20 (m, 2H).
UPLC/MS (method A): Rt 1.60 min. MS (ES): C9H6FNO2 requires,
179; found, 180 [M + H]+, 178 [M − H]−.
Synthesis of 5-(4-Methoxyphenyl)-3H-oxazol-2-one (11e). Com-
pound 11e was prepared using 10e (2.650 g, 10.00 mmol) and LiOH
(0.958 g, 40.00 mmol), according to general procedure B, Step 2. After
aq work-up, the resulting solid was collected by filtration to afford 11e
1
as a white solid (1.040 g, 54%). H NMR (400 MHz, DMSO-d6): δ
10.62 (bs, 1H), 7.48−7.39 (m, 2H), 7.30 (s, 1H), 7.03−6.91 (m, 2H),
3.77 (s, 3H). UPLC/MS (method A): Rt 1.55 min. MS (ES): C10H9NO3
requires, 191; found, 192 [M + H]+, 190 [M − H]−.
Synthesis of 5-(3-Methoxyphenyl)-3H-oxazol-2-one (11f). Com-
pound 11f was prepared using 10f (1.458 g, 5.50 mmol) and LiOH
(0.527 g, 22.00 mmol), according to general procedure B, Step 2. After
aq work-up, the resulting solid was collected by filtration to afford 11f as
a white solid (0.627 g, 60%). 1H NMR (400 MHz, DMSO-d6): δ 10.85
Q
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(bs, 1H), 7.52 (s, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.19−6.95 (m, 2H),
6.85 (ddd, J = 8.4, 2.6, 0.9 Hz, 1H), 3.78 (s, 3H). UPLC/MS (method
A): Rt 1.59 min. MS (ES): C10H9NO3 requires, 191; found, 192 [M +
H]+, 190 [M − H]−.
Synthesis of 5-(2-Methoxyphenyl)-3H-oxazol-2-one (11g). Com-
pound 11g was prepared using 10g (0.578 g, 2.18 mmol) and LiOH
(0.210 g, 8.72 mmol), according to general procedure B, Step 2. After aq
work-up, the resulting solid was collected by filtration to afford 11g as a
white solid (0.250 g, 60%). 1H NMR (400 MHz, CDCl3): δ 9.56 (bs,
1H), 7.66 (dd, J = 7.7, 1.6 Hz, 1H), 7.31−7.21 (m, overlapped with
CDCl3 signal, 1H), 7.11 (d, J = 1.9 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H),
6.93 (d, J = 8.3 Hz, 1H), 3.93 (s, 3H). UPLC/MS (method A): Rt 1.65
min. MS (ES): C10H9NO3 requires, 191; found, 192 [M + H]+, 190 [M
− H]−.
Synthesis of 5-(4-Fluoro-3-methoxy-phenyl)-3H-oxazol-2-one
(11h). Compound 11h was prepared using 10h (0.286 g, 1.01 mmol)
and LiOH (0.097 g, 4.04 mmol), according to general procedure B,
Step 2. The crude was purified by flash chromatography (SiO2), eluting
with DCM/MeOH (95:5), to afford 11h as a yellow solid (0.055 g,
26%). 1H NMR (400 MHz, CDCl3): δ 8.67 (bs, 1H), 7.13−7.04 (m,
2H), 7.04−6.97 (m, 1H), 6.78 (s, 1H), 3.93 (s, 3H). UPLC/MS
(method A): Rt 1.61 min. MS (ES): C10H8FNO3 requires, 209; found,
210 [M + H]+, 208 [M − H]−.
7.70−7.64 (m, 1H), 7.59 (d, J = 2.2 Hz, 1H), 7.49−7.43 (m, 1H),
7.25−7.19 (m, 1H), 4.06 (s, 3H). UPLC/MS (method D): Rt 0.49 min.
MS (ES): C11H9N3O2 requires, 215; found, 216 [M + H]+, 214 [M −
H]−.
Synthesis of 5-Thiazol-2-yl-3H-oxazol-2-one (11o). Compound
11o was prepared using 10o (0.588 g, 2.43 mmol) and LiOH (0.233 g,
9.72 mmol), according to general procedure B, Step 2. After aq work-
up, the resulting solid was collected by filtration to afford 11o as a
yellow solid (0.200 g, 49%). 1H NMR (400 MHz, DMSO-d6): δ 11.29
(bs, 1H), 7.86 (d, J = 3.2 Hz, 1H), 7.73 (d, J = 3.2 Hz, 1H), 7.72−7.68
(m, 1H). UPLC/MS (method A): Rt 1.09 min. MS (ES): C6H4N2O2S
requires, 168; found, 169 [M + H]+, 167 [M − H]−.
Synthesis of 5-Thiazol-4-yl-3H-oxazol-2-one (11p). Compound
11p was prepared using 10p (0.152 g, 0.63 mmol) and LiOH (0.060 g,
2.52 mmol), according to general procedure B, Step 2. After aq work-
up, the resulting solid was collected by filtration to afford 11p as a
brown solid (0.100 g, 94%). 1H NMR (400 MHz, DMSO-d6): δ 10.92
(bs, 1H), 9.16 (d, J = 1.9 Hz, 1H), 7.68 (d, J = 1.9 Hz, 1H), 7.32 (s, 1H).
UPLC/MS (method A): Rt 1.09 min. MS (ES): C6H4N2O2S requires,
168; found, 169 [M + H]+, 167 [M − H]−.
Synthesis of tert-Butyl 4-(2-Oxo-3H-oxazol-5-yl)piperidine-1-
carboxylate (11q). Compound 11q was prepared using 10q (0.300
g, 0.88 mmol) and t-BuOK (0.197 g, 1.75 mmol) in THF, according to
general procedure B, Step 2. The crude was purified by column
chromatography (SiO2), eluting with DCM/EtOAc (3:7 to 0:10), to
afford 11q as a pale yellow solid (0.091 g, 38%). 1H NMR (400 MHz,
DMSO-d6): δ 10.27 (bs, 1H), 6.58 (d, J = 1.4 Hz, 1H), 4.02−3.79 (m,
2H), 2.97−2.68 (m, 2H), 2.63−2.52 (m, overlapped with DMSO
signal, 1H), 1.86−1.73 (m, 2H), 1.39 (s, 9H), 1.28 (qd, J = 12.1, 4.3 Hz,
2H). UPLC/MS (method A): Rt 1.72 min. MS (ES): C13H20N2O4
requires, 268; found, 267 [M − H]−.
Synthesis of 5-(2-Pyridyl)-3H-oxazol-2-one (11i). Compound 11i
was prepared using 10i (0.472 g, 2.00 mmol) and LiOH (0.192 g, 8.00
mmol), according to general procedure B, Step 2. After aq work-up, the
resulting solid was collected by filtration to afford 11i as an orange solid
1
(0.308 g, 93%). H NMR (400 MHz, DMSO-d6): δ 8.56−8.49 (m,
1H), 7.82 (td, J = 7.8, 1.8 Hz, 1H), 7.55 (s, 1H), 7.47−7.42 (m, 1H),
7.34−7.19 (m, 1H). UPLC/MS (method A): Rt 1.02 min. MS (ES):
C8H6N2O2 requires, 162; found, 163 [M + H]+, 161 [M − H]−.
Synthesis of 5-(3-Pyridyl)-3H-oxazol-2-one (11j). Compound 11j
was prepared using 10j (2.360 g, 10.00 mmol) and LiOH (0.958 g,
40.00 mmol), according to general procedure B, Step 2. The crude was
purified by flash chromatography (SiO2), eluting with DCM/MeOH
(95:5), to afford 11j as an orange solid (0.375 g, 23%). 1H NMR (400
MHz, CDCl3): δ 8.78−8.74 (m, 1H), 8.55 (dd, J = 4.9, 1.7 Hz, 1H),
7.79 (dt, J = 8.0, 2.0 Hz, 1H), 7.34 (ddd, J = 8.2, 5.0, 0.8 Hz, 1H), 6.93
(d, J = 2.1 Hz, 1H). UPLC/MS (method A): Rt 1.00 min. MS (ES):
C8H6N2O2 requires, 162; found, 163 [M + H]+, 161 [M − H]−.
Synthesis of 5-(4-Pyridyl)-3H-oxazol-2-one (11k). Compound 11k
was prepared using 10k (1.180 g, 5.00 mmol) and LiOH (0.479 g, 20.00
mmol), according to general procedure B, Step 2. The crude was
purified by flash chromatography (SiO2), eluting with DCM/MeOH
(95:5), to afford 11k as an orange solid (0.122 g, 15%). 1H NMR (400
MHz, DMSO-d6): δ 11.18 (bs, 1H), 8.60−8.52 (m, 2H), 7.85 (s, 1H),
7.49−7.40 (m, 2H). UPLC/MS (method A): Rt 0.83 min. MS (ES):
C8H6N2O2 requires, 162; found, 163 [M + H]+, 161 [M − H]−.
Synthesis of 5-Pyrazin-2-yl-3H-oxazol-2-one (11l). Compound 11l
was prepared using 10l (0.709 g, 2.99 mmol) and LiOH (0.958 g, 40.00
mmol), according to general procedure B, Step 2. The crude was
purified by flash chromatography (SiO2), eluting with DCM/MeOH
(95:5), to afford 11l as a brown solid (0.030 g, 6%). UPLC/MS (method
A): Rt 0.86 min. MS (ES): C7H5N3O2 requires, 163; found, 162 [M −
H]−. 1H NMR (400 MHz, CDCl3): δ 8.94−8.88 (m, 1H), 8.64−8.60
(m, 1H), 8.55−8.52 (m, 1H), 8.27−7.87 (m, 1H).
Synthesis of 2-Oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-car-
boxamide (12a). Compound 12a was prepared according to general
procedure D (method A) using 11a (0.161 g, 1.00 mmol), DMAP
(0.012 g, 0.10 mmol), and 4-phenylbutyl isocyanate (0.350 g, 2.00
mmol) in CH3CN. The crude was purified by column chromatography
(SiO2), eluting with Cy/EtOAc (9:1), to afford 12a as a white solid
1
(0.276 g, 82%). H NMR (400 MHz, CDCl3): δ 7.95 (t, J = 5.7 Hz,
1H), 757−7.49 (m, 2H), 7.46 (s, 1H), 7.45−7.32 (m, 3H), 7.32−7.25
(m, overlapped with CDCl3 signal, 2H), 7.22−7.15 (m, 3H), 3.49−3.35
(m, 2H), 2.67 (t, J = 7.2 Hz, 2H), 1.79−1.60 (m, 4H). 13C NMR (101
MHz, CDCl3): δ 152.83, 148.59, 142.01, 139.77, 129.29, 129.12 (2C),
128.53, 126.41, 126.03, 123.72, 106.08, 40.44, 35.58, 29.21, 28.65.
UPLC/MS (method A): Rt 2.74 min. MS (ES): C20H20N2O3 requires,
336; found, 337 [M + H]+. HRMS: C20H20N2O3Na [M + Na]+ calcd
359.1372; measured, 359.1366, Δppm −0.6.
Synthesis of 2-Oxo-N-pentyl-5-phenyl-oxazole-3-carboxamide
(12b). Compound 12b was prepared according to general procedure
D (method B) using 11a (0.100 g, 0.62 mmol) and n-pentyl amine
(0.059 g, 0.68 mmol). The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (9:1), to afford 12b
as a white solid (0.107 g, 63%). 1H NMR (400 MHz, CDCl3): δ 7.94
(bs, 1H), 7.56−7.49 (m, 2H), 7.47 (s, 1H), 7.45−7.38 (m, 2H), 7.38−
7.32 (m, 1H), 3.45−3.33 (m, 2H), 1.68−1.51 (m, overlapped with H2O
signal, 2H), 1.43−1.29 (m, 4H), 0.99−0.86 (m, 3H). 13C NMR (101
MHz, CDCl3): δ 152.52, 148.24, 139.39, 128.92, 128.77, 126.11,
123.37, 105.79, 40.29, 28.96, 28.73, 22.11, 13.75. UPLC/MS (method
A): Rt 2.62 min. MS (ES): C15H18N2O3 requires, 274; found, 275 [M +
H]+, 160 [M−CONH(CH2)4CH3]−. HRMS: C15H18N2O3Na [M +
Na]+ calcd 297.1215; measured, 297.1207, Δppm −2.7.
Synthesis of 5-Quinoxalin-2-yl-3H-oxazol-2-one (11m). Com-
pound 11m was prepared using 10m (0.230 g, 0.80 mmol) and LiOH
(0.077 g, 3.20 mmol), according to general procedure B, Step 2. After aq
work-up, the resulting solid was collected by filtration to afford 11m as
1
an orange solid (0.158 g, 93%). H NMR (400 MHz, DMSO-d6): δ
Synthesis of N-(2-Ethoxyethyl)-2-oxo-5-phenyl-oxazole-3-car-
boxamide (12c). Compound 12c was prepared according to general
procedure D (method B) using 11a (0.161 g, 1.00 mmol) and 2-
ethoxyethyl amine (0.178 g, 2.00 mmol). The crude was purified by
column chromatography (SiO2), eluting with Cy/EtOAc (8:2), to
11.46 (bs, 1H), 9.17 (s, 1H), 8.18 (d, J = 2.3 Hz, 1H), 8.12−7.99 (m,
2H), 7.90−7.65 (m, 2H). UPLC/MS (method A): Rt 1.29 min. MS
(ES): C11H7N3O2 requires, 213; found, 214 [M + H]+, 212 [M − H]−.
Synthesis of 5-(1-Methylindazol-3-yl)-3H-oxazol-2-one (11n).
Compound 11n was prepared using 10n (0.136 g, 0.47 mmol) and
LiOH (0.045 g, 1.88 mmol), according to general procedure B, Step 2.
The crude was purified by flash chromatography (SiO2), eluting with
DCM/MeOH (8:2), to afford 11n as a yellow solid (0.050 g, 49%). 1H
NMR (400 MHz, DMSO-d6): δ 10.97 (bs, 1H), 7.97−7.92 (m, 1H),
1
afford 12c as a white solid (0.222 g, 80%). H NMR (400 MHz,
CDCl3): δ 8.21 (bs, 1H), 7.56−7.49 (m, 2H), 7.46 (s, 1H), 7.45−7.38
(m, 2H), 7.38−7.32 (m, 1H), 3.62−3.59 (m, 4H), 3.54 (q, J = 7.0 Hz,
2H), 1.23 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 152.66,
148.72, 139.77, 129.27, 129.10, 126.41, 123.71, 106.03, 68.63, 66.78,
R
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40.56, 15.23. UPLC/MS (method A): Rt 2.19 min. MS (ES):
C14H16N2O4 requires, 276; found, 277 [M + H]+, 275 [M − H]−,
160 [M−CONH(CH2)2OCH2CH3]−. HRMS: C14H16N2O4 [M + H]+
calcd 277.1188; measured, 277.1187, Δppm −0.4.
128.51, 127.12, 126.68, 126.00, 119.54, 40.17, 35.61, 29.16, 28.74,
12.16. UPLC/MS (method B): Rt 1.97 min. MS (ES): C21H22N2O3
requires, 350; found, 351 [M + H]+, 174 [M−CONH(CH2)4Ph]−.
HRMS: C21H22N2O3 [M + H]+ calcd 351.1709; measured, 351.1714,
Δppm 1.4.
Synthesis of N-(3-Methoxypropyl)-2-oxo-5-phenyl-oxazole-3-
carboxamide (12d). Compound 12d was prepared according to
general procedure D (method B) using 11a (0.100 g, 0.62 mmol) and 3-
methoxypropyl amine (0.061 g, 0.68 mmol). The crude was purified by
column chromatography (SiO2), eluting with DCM/MeOH (8:2), to
Synthesis of 5-(4-Chlorophenyl)-2-oxo-N-(4-phenylbutyl)-
oxazole-3-carboxamide (12i). Compound 12i was prepared accord-
ing to general procedure D (method A) using 11c (0.100 g, 0.51 mmol),
DMAP (0.006 g, 0.05 mmol), and 4-phenylbutyl isocyanate (0.135 g,
0.77 mmol) in CH3CN. The precipitate was collected by filtration to
1
afford 12d as a white solid (0.082 g, 48%). H NMR (400 MHz,
1
CDCl3): δ 8.24 (bs, 1H), 7.56−7.49 (m, 2H), 7.46 (s, 1H), 7.45−7.38
(m, 2H), 7.38−7.32 (m, 1H), 3.56−3.47 (m, 4H), 3.38 (s, 3H), 1.89 (p,
J = 6.2 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ 152.71, 148.59,
129.24, 129.11, 128.44, 126.48, 123.71, 106.13, 70.86, 58.94, 38.70,
29.29. UPLC/MS (method A): Rt 2.11 min. MS (ES): C14H16N2O4
requires, 276; found, 277 [M + H]+, 160 [M−CONH(CH2)3OCH3]−.
HRMS: C14H16N2O4 [M + H]+ calcd 277.1188; measured, 277.1183,
Δppm −1.8.
afford 12i as a white solid (0.055 g, 29%). H NMR (400 MHz,
CDCl3): δ 7.91 (t, J = 5.9 Hz, 1H), 7.50−7.42 (m, 3H), 7.41−7.36 (m,
2H), 7.33−7.24 (m, overlapped with CDCl3 signal, 2H), 7.22−7.15 (m,
3H), 3.51−3.34 (m, 2H), 2.67 (t, J = 7.2 Hz, 2H), 1.78−1.61 (m, 4H).
13C NMR (101 MHz, CDCl3): δ 152.61, 148.43, 141.98, 138.81,
135.18, 129.45, 128.53 (2C), 126.04, 124.96, 124.91, 106.51, 40.48,
35.57, 29.19, 28.63. UPLC/MS (method B): Rt 2.08 min. MS (ES):
C20H19ClN2O3 requires, 370; found, 369 [M − H]− and 194 [M−
CONH(CH2)4Ph]−. HRMS: C20H19ClN2O3 [M + H]+ calcd
371.1184; measured, 371.1168, Δppm 1.6.
Synthesis of 5-(4-Fluorophenyl)-2-oxo-N-(4-phenylbutyl)-
oxazole-3-carboxamide (12j). Compound 12j was prepared accord-
ing to general procedure D (method A) using 11d (0.150 g, 0.84 mmol),
DMAP (0.112 g, 0.92 mmol), and 4-phenylbutyl isocyanate (0.161 g,
0.92 mmol) in pyridine. The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (8:2), to afford 12j
as a white solid (0.180 g, 59%). 1H NMR (400 MHz, CDCl3): δ 7.92 (t,
J = 5.9 Hz, 1H), 7.54−7.46 (m, 2H), 7.41 (s, 1H), 7.32−7.24 (m,
overlapped with CDCl3, signal 2H), 7.23−7.15 (m, 3H), 7.15−7.08 (m,
2H), 3.46−3.37 (m, 2H), 2.67 (t, J = 7.2 Hz, 2H), 1.78−1.61 (m, 4H).
13C NMR (101 MHz, CDCl3): δ 163.16 (d, JC−F = 250.4 Hz), 152.70,
148.51, 141.99, 138.97, 128.52 (2C), 125.77 (d, JC−F = 8.2 Hz, ×2),
122.69, 116.38 (d, JC−F = 22.1 Hz), 105.78, 40.45, 35.56, 29.19, 28.63.
UPLC/MS (method A): Rt 2.76 min. MS (ES): C20H19FN2O3 requires,
354; found, 353 [M − H]− and 178 [M−CONH(CH2)4Ph]−. HRMS:
C20H19FN2O3 [M + H]+ calcd 355.1458; measured, 355.1451, Δppm
1.1.
Synthesis of N-Isobutyl-2-oxo-5-phenyl-oxazole-3-carboxamide
(12e). Compound 12e was prepared according to general procedure D
(method C) using 11a (0.050 g, 0.31 mmol), 2-methylpropan-1-amine
(0.069 g, 0.94 mmol), and DIPEA (0.243 g, 1.88 mmol). The crude was
purified by column chromatography (SiO2), eluting with Cy/EtOAc
1
(95:5), to afford 12e as a white solid (0.061 g, 76%). H NMR (400
MHz, CDCl3): δ 8.01 (t, J = 6.0 Hz, 1H), 7.55−7.49 (m, 2H), 7.47 (s,
1H), 7.45−7.38 (m, 2H), 7.38−7.32 (m, 1H), 3.23 (t, J = 6.4 Hz, 2H),
1.97−1.82 (m, 1H), 0.98 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz,
CDCl3): δ 152.87, 148.69, 139.71, 129.24, 129.08, 126.43, 123.69,
106.14, 47.87, 28.61, 20.10. UPLC/MS (method A): Rt 2.42 min. MS
(ES): C14H16N2O3 requires, 260; found, 261 [M + H]+, 160 [M−
CONHCH2CH(CH3)2]−. HRMS: C14H16N2O3Na [M + Na]+ calcd
283.1059; measured, 283.1049, Δppm −3.5.
Synthesis of ( ) 2-Oxo-5-phenyl-N-s-butyl-oxazole-3-carboxa-
mide (12f). Compound 12f was prepared according to general
procedure D (method B) using 11a (0.161 g, 1.00 mmol) and butan-
2-amine (0.110 g, 1.50 mmol). The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (95:5), to afford 12f
as a white solid (0.062 g, 24%). 1H NMR (400 MHz, CDCl3): δ 7.78 (d,
J = 7.5 Hz, 1H), 7.58−7.49 (m, 2H), 7.46 (s, 1H), 7.45−7.38 (m, 2H),
7.38−7.32 (m, 1H), 4.02−3.85 (m, 1H), 1.60 (q, J = 7.3 Hz, overlapped
with H2O signal, 2H), 1.25 (d, J = 6.6 Hz, 3H), 0.97 (t, J = 7.4 Hz, 3H).
13C NMR (101 MHz, CDCl3): δ 152.86, 148.00, 139.67, 129.24,
Synthesis of 5-(4-Methoxyphenyl)-2-oxo-N-(4-phenylbutyl)-
oxazole-3-carboxamide (12k). Compound 12k was prepared
according to general procedure D (method A) using 11e (0.150 g,
0.84 mmol), DMAP (0.105 g, 0.86 mmol), and 4-phenylbutyl
isocyanate (0.150 g, 0.86 mmol) in pyridine. The crude was purified
by column chromatography (SiO2), eluting with Cy/EtOAc (8:2), to
129.11, 126.48, 123.71, 106.13, 48.44, 29.63, 20.50, 10.40. UPLC/MS
(method A): Rt 2.44 min. MS (ES): C14H16N2O3 requires, 260; found,
261 [M + H]+. HRMS: C14H16N2O3Na [M + Na]+ calcd 283.1059;
measured, 283.1059, Δppm 0.0.
1
afford 12k as a yellow solid (0.187 g, 65%). H NMR (400 MHz,
CDCl3): δ 7.95 (t, J = 5.9 Hz, 1H), 7.48−7.41 (m, 2H), 7.32 (s, 1H),
7.31−7.24 (m, overlapped with CDCl3 signal, 2H), 7.22−7.14 (m, 3H),
6.98−6.89 (m, 2H), 3.84 (s, 3H), 3.47−3.35 (m, 2H), 2.67 (t, J = 7.3
Hz, 2H), 1.78−1.60 (m, 4H). 13C NMR (101 MHz, CDCl3): δ 160.46,
152.91, 148.71, 142.02, 139.86, 128.53, 128.50, 126.00, 125.34, 119.05,
114.59, 104.39, 55.51, 40.38, 35.56, 29.20, 28.62. UPLC/MS (method
A): Rt 2.72 min. MS (ES): C21H22N2O4 requires, 366; found, 190 [M−
CONH(CH2)4Ph]−. HRMS: C21H22N2O4 [M + H]+ calcd 367.1658;
measured, 367.1646, Δppm −1.2.
Synthesis of 2-Oxo-N,5-diphenyl-oxazole-3-carboxamide (12g).
Compound 12g was prepared according to general procedure D
(method A) using 11a (0.050 g, 0.31 mmol), Et3N (0.125 g, 1.24
mmol), and phenyl isocyanate (0.046 g, 0.34 mmol) in CH3CN. The
crude was purified by column chromatography (SiO2), eluting with Cy/
EtOAc (95:5), to afford 12g as a white solid (0.068 g, 78%). 1H NMR
(400 MHz, CDCl3): δ 9.95 (bs, 1H), 7.63−7.52 (m, 5H), 7.49−7.34
(m, 5H), 7.22−7.15 (m, 1H). 13C NMR (101 MHz, CDCl3): δ 152.87,
145.86, 140.24, 136.43, 129.54, 129.39, 129.18, 126.17, 125.23, 123.85,
120.41, 105.83. UPLC/MS (method B): Rt 2.48 min. MS (ES):
C16H12N2O3 requires, 280; found, 160 [M−CONHPh]−. HRMS:
C16H13N2O2 [M + H]+ calcd 281.0923; measured, 281.0926, Δppm 0.
Synthesis of 4-Methyl-2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-
3-carboxamide (12h). Compound 12h was prepared according to
general procedure D (method A) using 11b (0.010 g, 0.057 mmol),
DMAP (0.008 g, 0.06 mmol), and 4-phenylbutyl isocyanate (0.011 g,
0.06 mmol) in CH3CN. The crude was purified by column
chromatography (Al2O3), eluting with Cy, to afford 12h as a white
solid (0.007 g, 35%). 1H NMR (400 MHz, CDCl3): δ 8.33 (t, J = 5.3 Hz,
1H), 7.52−7.47 (m, 2H), 7.47−7.41 (m, 2H), 7.40−7.34 (m, 1H),
7.32−7.26 (m, 2H, overlapped with CDCl3 signal), 7.22−7.15 (m, 3H),
3.38 (td, J = 6.8, 5.6 Hz, 2H), 2.67 (t, J = 7.3 Hz, 2H), 2.58 (s, 3H),
1.78−1.56 (m, overlapped with H2O signal, 4H). 13C NMR (101 MHz,
CDCl3): δ 154.02, 150.34, 142.12, 135.72, 128.92, 128.86, 128.56,
Synthesis of 5-(3-Methoxyphenyl)-2-oxo-N-(4-phenylbutyl)-
oxazole-3-carboxamide (12l). Compound 12l was prepared according
to general procedure D (method A) using 11f (0.191 g, 1.00 mmol),
DMAP (0.012 g, 0.10 mmol), and 4-phenylbutyl isocyanate (0.350 g,
2.00 mmol) in CH3CN. The precipitate was collected by filtration to
1
afford 12l as a white solid (0.150 g, 41%). H NMR (400 MHz,
CDCl3): δ 7.94 (t, J = 5.8 Hz, 1H), 7.46 (s, 1H), 7.36−7.24 (m,
overlapped with CDCl3 signal, 3H), 7.22−7.15 (m, 3H), 7.11 (dt, J =
7.7, 1.2 Hz, 1H), 7.06−7.02 (m, 1H), 6.92−6.87 (m, 1H), 3.84 (s, 3H),
3.45−3.38 (m, 2H), 2.67 (t, J = 7.3 Hz, 2H), 1.78−1.60 (m, 4H). 13C
NMR (101 MHz, CDCl3): δ 160.15, 152.77, 148.55, 142.01, 139.63,
130.27, 128.54, 127.61, 126.03, 116.12, 115.34, 108.94, 106.35, 104.88,
55.54, 40.45, 35.58, 29.21, 28.65. UPLC/MS (method A): Rt 2.73 min.
MS (ES): C21H22N2O4 requires, 366; found, 367 [M + H]+, 190 [M−
CONH(CH2)4Ph]−. HRMS: C21H22N2O4 [M + H]+ calcd 367.1658;
measured, 367.1654, Δppm −1.1.
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Synthesis of 5-(2-Methoxyphenyl)-2-oxo-N-(4-phenylbutyl)-
oxazole-3-carboxamide (12m). Compound 12m was prepared
according to general procedure D (method A) using 11g (0.120 g,
0.63 mmol), DMAP (0.084 g, 0.69 mmol), and 4-phenylbutyl
isocyanate (0.121 g, 0.69 mmol) in pyridine. The crude was purified
by column chromatography (SiO2), eluting with Cy/EtOAc (8:2), to
according to general procedure D (method A) using 11k (0.120 g,
0.74 mmol), DMAP (0.099 g, 0.81 mmol), and 4-phenylbutyl
isocyanate (0.142 g, 0.81 mmol) in pyridine. The crude was purified
by column chromatography (SiO2), eluting with DCM/MeOH (9:1),
to afford the free base of 12q. The free base of 12q was then dissolved in
1.0 mL of 1,4-dioxane and 4.0 M HCl solution (3.7 mL, 14.8 mmol,
20.0 equiv) was added. The evaporation of solvents afforded 12q as a
white solid (0.120 g, 43%). 1H NMR (400 MHz, DMSO-d6): δ 8.84−
8.78 (m, 2H), 8.72 (s, 1H), 8.08 (t, J = 5.9 Hz, 1H), 8.03−7.97 (m, 2H),
7.32−7.24 (m, 2H), 7.22−7.12 (m, 3H), 3.43−3.24 (m, overlapped
1
afford 12m as a yellow solid (0.060 g, 26%). H NMR (400 MHz,
CDCl3): δ 8.01 (t, J = 5.2 Hz, 1H), 7.65 (s, 1H), 7.63 (dd, J = 7.8, 1.6
Hz, 1H), 7.35−7.25 (m, overlapped with CDCl3 signal, 3H), 7.23−7.12
(m, 3H), 7.03 (td, J = 7.7, 0.8 Hz, 1H), 6.99−6.93 (m, 1H), 3.95 (s,
3H), 3.46−3.37 (m, 2H), 2.67 (t, J = 7.3 Hz, 2H), 1.78−1.60 (m, 4H).
13C NMR (101 MHz, CDCl3): δ 155.98, 152.63, 148.84, 142.05,
136.36, 129.69, 128.55, 128.51, 126.00, 125.53, 120.93, 115.43, 110.73,
110.35, 55.59, 40.38, 35.59, 29.23, 28.66. UPLC/MS (method A): Rt
2.79 min. MS (ES): C21H22N2O4 requires, 366; found, 367 [M + H]+,
190 [M−CONH(CH2)4Ph]−. HRMS: C21H22N2O4 [M + H]+ calcd
367.1658; measured, 367.1655, Δppm −1.0.
with H2O signal, 2H), 2.61 (t, J = 7.2 Hz, 2H), 1.68−1.49 (m, 4H). 13
C
NMR (101 MHz, DMSO): δ 151.14, 147.38, 144.25, 141.99, 134.41,
128.28, 128.22, 125.67, 118.87, 116.84, 40.35 (overlapped with DMSO
signal), 34.70, 28.51, 28.05. UPLC/MS (method A): Rt 2.26 min. MS
(ES): C19H19N3O3 requires, 337; found, 338 [M + H]+, 161 [M−
CONH(CH2)4Ph]−. HRMS: C19H19N3O3 [M + H]+ calcd 338.1505;
measured, 338.1503, Δppm −0.6.
Synthesis of 5-(4-Fluoro-3-methoxy-phenyl)-2-oxo-N-(4-
phenylbutyl)oxazole-3-carboxamide (12n). Compound 12n was
prepared according to general procedure D (method A) using 11h
(0.055 g, 0.26 mmol), DMAP (0.035 g, 0.29 mmol), and 4-phenylbutyl
isocyanate (0.051 g, 0.29 mmol) in pyridine. The crude was purified by
column chromatography (SiO2), eluting with DCM/MeOH (9:1), and
then was triturated with pentane to afford 12n as a white solid (0.045 g,
45%). 1H NMR (400 MHz CDCl3): δ 7.91 (t, J = 5.2 Hz, 1H), 7.41 (s,
1H), 7.32−7.24 (m, overlapped with CDCl3 signal, 2H), 7.23−7.15 (m,
3H), 7.15−7.03 (m, 3H), 3.93 (s, 3H), 3.47−3.37 (m, 2H), 2.67 (t, J =
7.0 Hz, 2H), 1.77−1.60 (m, 4H). 13C NMR (101 MHz, CDCl3): δ
154.13, 152.89 (d, JC−F = 250.1 Hz), 152.65, 148.47, 141.99, 138.98,
128.53 (2C), 126.04, 122.93 (d, JC−F = 3.9 Hz), 116.91 (d, JC−F = 19.5
Hz), 116.51 (d, JC−F = 7.1 Hz), 108.86, 105.92, 56.47, 40.46, 35.56,
29.18, 28.64. UPLC/MS (method A): Rt 2.70 min. MS (ES):
C21H21FN2O4 requires, 384; found, 385 [M + H]+, 208 [M−
CONH(CH2)4Ph]−. HRMS: C21H21FN2O4 [M + H]+ calcd
385.1564; measured, 385.1566, Δppm 0.5.
Synthesis of 2-Oxo-N-(4-phenylbutyl)-5-(2-pyridyl)oxazole-3-car-
boxamide (12o). Compound 12o was prepared according to general
procedure D (method A) using 11i (0.280 g, 1.73 mmol), DMAP (0.232
g, 1.90 mmol), and 4-phenylbutyl isocyanate (0.333 g, 1.90 mmol) in
pyridine. The crude was purified by column chromatography (SiO2),
eluting with DCM/MeOH (9:1), to afford 12o as a white solid (0.058
g, 10%). 1H NMR (400 MHz, CDCl3): δ 8.60 (ddd, J = 4.8, 1.7, 0.9 Hz,
1H), 7.92 (t, J = 4.8 Hz, 1H), 7.86 (s, 1H), 7.76 (td, J = 7.8, 1.7 Hz, 1H),
7.55−7.48 (m, 1H), 7.31−7.22 (m, overlapped with CDCl3 signal, 3H),
7.21−7.15 (m, 3H), 3.48−3.36 (m, 2H), 2.67 (t, J = 7.2 Hz, 2H), 1.79−
1.60 (m, 4H). 13C NMR (101 MHz, CDCl3): δ 152.66, 150.17, 148.31,
145.92, 142.01, 139.13, 137.10, 128.54 (2C), 126.03, 123.56, 118.81,
109.78, 40.50, 35.57, 29.18, 28.64. UPLC/MS (method A): Rt 2.38 min.
MS (ES): C19H19N3O3 requires, 337; found, 338 [M + H]+, 161 [M−
CONH(CH2)4Ph]−. HRMS: C19H19N3O3 [M + H]+ calcd 338.1505;
measured, 338.1497, Δppm −2.4.
Synthesis of 2-Oxo-N-(4-phenylbutyl)-5-pyrazin-2-yl-oxazole-3-
carboxamide (12r). Compound 12r was prepared according to general
procedure D (method A) using 11l (0.027 g, 0.16 mmol), DMAP (0.022
g, 0.18 mmol), and 4-phenylbutyl isocyanate (0.033 g, 0.18 mmol) in
pyridine. The crude was purified by column chromatography (SiO2),
eluting with DCM/MeOH (9:1), to afford 12r as a white solid (0.045 g,
83%). 1H NMR (400 MHz, CDCl3): δ 8.83−8.77 (m, 1H), 8.59−8.50
(m, 2H), 7.92 (s, 1H), 7.89 (bs, 1H), 7.33−7.24 (m, overlapped with
CDCl3 signal, 2H), 7.22−7.15 (m, 3H), 3.49−3.38 (m, 2H), 2.67 (t, J =
7.2 Hz, 2H), 1.80−1.58 (m, 4H). 13C NMR (101 MHz, CDCl3): δ
152.28, 147.97, 144.62, 144.40, 141.91, 140.07, 136.89, 128.50 (2C),
126.02, 111.67, 40.55, 35.52, 29.11, 28.58. UPLC/MS (method A): Rt
2.28 min. MS (ES): C18H18N4O3 requires, 338; found, 339 [M + H]+,
162 [M−CONH(CH2)4Ph]−. HRMS: C18H18N4O3 [M + H]+ calcd
339.1457; measured, 339.1455, Δppm −0.6.
2-Oxo-N-(4-phenylbutyl)-5-quinoxalin-2-yl-oxazole-3-carboxa-
mide (12s). Compound 12s was prepared according to general
procedure D (method A) using 11m (0.081 g, 0.38 mmol), DMAP
(0.051 g, 0.42 mmol), and 4-phenylbutyl isocyanate (0.074 g, 0.42
mmol) in pyridine. The crude was purified by column chromatography
(SiO2), eluting with DCM/MeOH (9:1), to afford 12s as a white solid
(0.063 g, 43%). 1H NMR (400 MHz, CDCl3): δ 9.08 (s, 1H), 8.16−
8.06 (m, 3H), 7.93 (t, J = 5.3 Hz, 1H), 7.86−7.74 (m, 2H), 7.32−7.25
(m, overlapped with CDCl3 signal, 2H), 7.23−7.15 (m, 3H), 3.45−3.40
(m, 2H), 2.68 (t, J = 7.2 Hz, 2H), 1.80−1.61 (m, 4H). 13C NMR (101
MHz, CDCl3): δ 152.37, 148.02, 142.23, 142.10, 141.96, 140.84,
140.49, 137.53, 131.23, 130.65, 129.61, 129.48, 128.56 (2C), 126.08,
112.35, 40.64, 35.57, 29.15, 28.64. UPLC/MS (method A): Rt 2.49 min.
MS (ES): C22H20N4O3 requires, 388; found, 389 [M + H]+, 212 [M−
CONH(CH2)4Ph]−. HRMS: C22H20N4O3 [M + H]+ calcd 389.1614;
measured, 389.1611, Δppm −0.8.
Synthesis of 5-(1-Methylindazol-3-yl)-2-oxo-N-(4-phenylbutyl)-
oxazole-3-carboxamide (12t). Compound 12t was prepared accord-
ing to general procedure D (method A) using 11n (0.041 g, 0.19 mmol),
DMAP (0.026 g, 0.21 mmol), and 4-phenylbutyl isocyanate (0.037 g,
0.21 mmol) in pyridine. The crude was purified by column
chromatography (SiO2), eluting with DCM/MeOH (9:1), and, then,
triturated with pentane to afford 12t as a white solid (0.040 g, 54%). 1H
NMR (400 MHz, CDCl3): δ 7.99 (t, J = 5.2 Hz, 1H), 7.91 (d, J = 8.2 Hz,
1H), 7.65 (s, 1H), 7.50−7.40 (m, 2H), 7.34−7.23 (m, overlapped with
CDCl3 signal, 3H), 7.23−7.14 (m, 3H), 4.12 (s, 3H), 3.50−3.38 (m,
2H), 2.67 (t, J = 7.2 Hz, 2H), 1.80−1.59 (m, 4H). 13C NMR (101 MHz,
CDCl3): δ 152.64, 148.56, 142.02, 140.96, 135.22, 131.42, 128.55,
128.52, 127.27, 126.02, 122.10, 120.94, 120.82, 109.62, 107.28, 40.47,
36.07, 35.58, 29.20, 28.66. UPLC/MS (method A): Rt 2.63 min. MS
(ES): C22H22N4O3 requires, 390; found, 391 [M + H]+, 214 [M−
CONH(CH2)4Ph]−.
Synthesis of 2-Oxo-N-(4-phenylbutyl)-5-(3-pyridyl)oxazole-3-car-
boxamide (12p). Compound 12p was prepared according to general
procedure D (method A) using 11j (0.200 g, 1.23 mmol), DMAP (0.165
g, 1.35 mmol), and 4-phenylbutyl isocyanate (0.236 g, 1.35 mmol) in
pyridine. The crude was purified by column chromatography (SiO2),
eluting with DCM/MeOH (9:1), to afford 12p as a white solid (0.187
g, 45%). 1H NMR (400 MHz, CDCl3): δ 8.85−8.79 (m, 1H), 8.60 (dd,
J = 4.9, 1.5 Hz, 1H), 7.89 (t, J = 5.0 Hz, 1H), 7.83 (dt, J = 8.0, 1.8 Hz,
1H), 7.59 (s, 1H), 7.39 (ddd, J = 8.0, 4.9, 0.7 Hz, 1H), 7.32−7.24 (m,
overlapped with CDCl3 signal, 2H), 7.22−7.15 (m, 3H), 3.51−3.35 (m,
2H), 2.67 (t, J = 7.2 Hz, 2H), 1.92−1.60 (m, overlapped with H2O
signal, 4H). 13C NMR (101 MHz, CDCl3): δ 152.52, 149.90, 148.26,
144.99, 141.97, 137.06, 131.08, 128.55 (2C), 126.07, 123.89, 122.98,
107.73, 40.54, 35.57, 29.17, 28.62. UPLC/MS (method A): Rt 2.34 min.
MS (ES): C19H19N3O3 requires, 337; found, 338 [M + H]+, 161 [M−
CONH(CH2)4Ph]−. HRMS: C19H19N3O3 [M + H]+ calcd 338.1505;
measured, 338.1497, Δppm −2.4.
Synthesis of 2-Oxo-N-(4-phenylbutyl)-5-thiazol-2-yl-oxazole-3-
carboxamide (12u). Compound 12u was prepared according to
general procedure D (method A) using 11o (0.100 g, 0.59 mmol),
DMAP (0.079 g, 0.65 mmol), and 4-phenylbutyl isocyanate (0.114 g,
0.65 mmol) in pyridine. The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (8:2), to afford 12u
Synthesis of 2-Oxo-N-(4-phenylbutyl)-5-(4-pyridyl)oxazole-3-car-
boxamide Hydrochloride (12q). Compound 12q was prepared
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as a white solid (0.150 g, 73%). 1H NMR (400 MHz CDCl3): δ 7.89 (d,
J = 3.2 Hz, 1H), 7.86 (t, J = 5.4 Hz, 1H), 7.80 (s, 1H), 7.42 (d, J = 3.2
Hz, 1H), 7.34−7.24 (m, overlapped with CDCl3 signal, 2H), 7.23−7.12
(m, 3H), 3.47−3.35 (m, 2H), 2.66 (t, J = 7.2 Hz, 2H), 1.77−1.60 (m,
overlapped with H2O signal, 4H). 13C NMR (101 MHz, CDCl3): δ
151.81, 147.95, 144.46, 144.42, 141.94, 140.61, 128.53 (2C), 126.05,
119.74, 109.26, 40.58, 35.54, 29.11, 28.61. UPLC/MS (method A): Rt
2.41 min. MS (ES): C17H17N3O3S requires, 343; found, 344 [M + H]+,
167 [M−CONH(CH2)4Ph]−. HRMS: C17H17N3O3S [M + H]+ calcd
344.1069; measured, 344.1054, Δppm −4.4.
Synthesis of 2-Oxo-N-(4-phenylbutyl)-5-thiazol-4-yl-oxazole-3-
carboxamide (12v). Compound 12v was prepared according to
general procedure D (method A) using 11p (0.120 g, 0.71 mmol),
DMAP (0.100 g, 0.78 mmol). and 4-phenylbutyl isocyanate (0.140 g,
0.78 mmol) in pyridine. The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (8:2), to afford 12v
as a white solid (0.122 g, 50%). 1H NMR (400 MHz, CDCl3): δ 7.90 (d,
J = 3.2 Hz, 1H), 7.86 (t, J = 4.8 Hz, 1H), 7.83 (s, 1H), 7.42 (d, J = 3.2
Hz, 1H), 7.33−7.25 (m, overlapped with CDCl3 signal, 2H), 7.22−7.15
(m, 3H), 3.47−3.36 (m, 2H), 2.66 (t, J = 7.2 Hz, 2H), 1.76−1.59 (m,
4H). 13C NMR (101 MHz, CDCl3): δ 153.82, 151.80, 147.15, 144.36,
141.94, 138.47, 128.57 (2C), 126.05, 119.76, 109.41, 40.59, 35.55,
29.12, 28.62. UPLC/MS (method A): Rt 2.41 min. MS (ES):
C17H17N3O3S requires, 343; found, 344 [M + H]+, 167 [M−
CONH(CH2)4Ph]−. HRMS: C17H17N3O3S [M + H]+ calcd
344.1069; measured, 344.1061, Δppm −2.3.
mmol, 4.0 equiv). The crude was purified by column chromatography
(SiO2), eluting with Cy/EtOAc (91:9), to afford 13a as yellow oil
(0.109 g, 98%). 1H NMR (400 MHz, DMSO-d6): δ 7.82 (s, 1H), 7.62−
7.56 (m, 2H), 7.50−7.42 (m, 2H), 7.40−7.32 (m, 1H), 7.30−7.23 (m,
2H), 7.22−7.14 (m, 3H), 3.46−3.37 (m, 2H), 3.00 (s, 3H), 2.65−2.56
(m, 2H), 1.65−1.51 (m, 4H). 13C NMR (101 MHz, DMSO-d6): δ
150.11, 149.58, 141.88, 139.14, 128.93, 128.52, 128.23 (2C), 126.66,
125.67, 122.99, 110.18, 49.52, 36.47, 34.69, 27.69 (×2). UPLC/MS
(method B): Rt 1.56 min. MS (ES): C21H22N2O3 requires, 350; found,
351 [M + H]+. HRMS: C21H22N2O3 [M + H]+ calcd 351.1704;
measured, 351.1709, Δppm −1.4.
Synthesis of 4-Phenylbutyl-2-oxo-5-phenyl-oxazole-3-carboxy-
late (13b). Compound 13b was prepared according to general
procedure D (method C) using 11a (0.160 g, 1.00 mmol), 4-phenyl-
1-butanol (0.050 g, 0.051 mL, 0.33 mmol, 0.33 equiv), and DIPEA
(0.260 g, 0.350 mL, 2.01 mmol). The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (94:6), to afford 13b
as a white solid (0.059 g, 53%). 1H NMR (400 MHz, CDCl3): δ 7.58−
7.50 (m, 2H), 7.49−7.37 (m, 3H), 7.33−7.24 (m, overlapped with
CDCl3 signal, 2H), 7.24−7.14 (m, 4H), 4.42 (t, J = 6.1 Hz, 2H), 2.70 (t,
J = 7.1 Hz, 2H), 1.90−1.71 (m, 4H). 13C NMR (101 MHz, CDCl3): δ
149.39, 148.73, 141.80, 140.13, 129.56, 129.12, 128.57 (2C), 126.26,
126.12, 123.91, 105.95, 68.52, 35.43, 28.16, 27.45. UPLC/MS (method
B): Rt 2.48 min. MS (ES): C20H19NO4 requires, 337; found, 338 [M +
H]+. HRMS: C20H19NO4Na [M + Na]+ calcd 360.1212; measured,
360.1207, Δppm −1.4.
Synthesis of 5-Phenyl-3-(6-phenylhexanoyl)oxazol-2-one (13c).
To a solution of 6-phenylhexanoic acid (0.086 g, 0.45 mmol, 1.2 equiv)
in dry DCM (7.5 mL, 0.06 M) was added slowly SOCl2 (0.169 g, 0.104
mL, 0.53 mmol, 1.5 equiv) at 0 °C. The mixture was stirred at rt for 6 h
and, then, the solvent was evaporated. The residue was taken up in dry
THF (1.0 mL) and added dropwise over 10 min to a solution of 11a
(0.060 g, 0.37 mmol, 1.0 equiv) and Et3N (0.163 g, 0.225 mL, 1.61
mmol, 4.3 equiv) in dry THF (0.37 M) at 0 °C. The reaction mixture
was stirred, and then, sat. aq NH4Cl was added and extracted with
DCM. The combined organic layers were dried over Na2SO4. After the
evaporation of the solvent, the crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (97:3), to afford
13c as a white solid (0.069 g, 55%). 1H NMR (400 MHz, DMSO-d6): δ
8.06 (s, 1H), 7.71−7.63 (m, 2H), 7.50−7.42 (m, 2H), 7.42−7.35 (m,
1H), 7.30−7.23 (m, 2H), 7.22−7.13 (m, 3H), 2.94 (t, J = 7.3 Hz, 2H),
2.58 (t, J = 7.7 Hz, 2H), 1.71−1.52 (m, 4H), 1.44−1.30 (m, 2H). 13C
NMR (101 MHz, DMSO-d6): δ 170.12, 150.51, 142.16, 139.35, 128.98,
128.96, 128.25, 128.19, 126.25, 125.59, 123.49, 106.70, 34.97, 34.65,
30.70, 27.97, 23.06. UPLC/MS (method B): Rt 2.16 min. MS (ES):
C21H21NO3 requires, 335; found, 336 [M + H]+, 334 [M − H]−.
HRMS: C21H21NO3Na [M + Na]+ calcd 358.1419; measured,
358.1416, Δppm −0.8.
Synthesis of tert-Butyl 4-[2-Oxo-3-(4-phenylbutylcarbamoyl)-
oxazol-5-yl]piperidine-1-carboxylate (12w). Compound 12w was
prepared according to general procedure D (method A) using 11q
(0.090 g, 0.34 mmol), DMAP (0.045 g, 0.37 mmol). and 4-phenylbutyl
isocyanate (0.065 g, 0.37 mmol) in CH3CN. The crude was purified by
column chromatography (SiO2), eluting with Cy/EtOAc (65:35), to
1
afford 12w as a white solid (0.025 g, 17%). H NMR (400 MHz,
CDCl3): δ 7.88 (t, J = 5.7 Hz, 1H), 7.32−7.26 (m, overlapped with
CDCl3 signal, 2H), 7.21−7.12 (m, 3H), 6.87 (d, J = 1.4 Hz, 1H), 4.23−
4.05 (m, 2H), 3.37 (q, J = 6.5 Hz, 2H), 2.90−2.75 (m, 2H), 2.65 (t, J =
7.3 Hz, 2H), 2.62−2.53 (m, 1H), 1.95−1.85 (m, 2H), 1.77−1.49 (m,
6H), 1.46 (s, 9H). UPLC/MS (method B): Rt 1.91 min. MS (ES):
C24H33N3O5 requires, 443; found, 444 [M + H]+.
Synthesis of 2-Oxo-N-(4-phenylbutyl)-5-(4-piperidyl)oxazole-3-
carboxamide Hydrochloride (12x). Compound 12x was prepared
according to general procedure G using 12w (0.025 g, 0.06 mmol). The
crude was used in the next step without further purification. 1H NMR
(400 MHz, DMSO-d6): δ 8.79 (bs, 2H), 7.98 (t, J = 5.9 Hz, 1H), 7.30−
7.23 (m, 2H), 7.22−7.12 (m, 4H), 3.33−3.17 (m, overlapped with H2O
signal, 4H), 2.94 (td, J = 12.2, 3.1 Hz, 2H), 2.88−2.77 (m, 1H), 2.59 (t,
J = 7.3 Hz, 2H), 2.04−1.94 (m, 2H), 1.78−1.64 (m, 2H), 1.62−1.44
(m, 4H). UPLC/MS (method A): Rt 1.89 min. MS (ES): C19H25N3O3
requires, 343; found, 344 [M + H]+.
Synthesis of 5-(1-Methyl-4-piperidyl)-2-oxo-N-(4-phenylbutyl)-
oxazole-3-carboxamide (12y). Compound 12y was prepared
according to general procedure H using 12x (0.022 g, 0.06 mmol)
and 37% aq solution of HCHO (0.023 g, 0.022 mL, 0.29 mmol). The
crude was purified by column chromatography (SiO2), eluting with
DCM/MeOH (88:12), to afford 12y as a white solid (0.007 g, 34%). 1H
NMR (400 MHz, DMSO-d6): δ 7.97 (t, J = 5.9 Hz, 1H), 7.30−7.23 (m,
2H), 7.21−7.13 (m, 3H), 7.05 (d, J = 1.5 Hz, 1H), 3.26 (q, J = 6.4 Hz,
2H), 2.74 (dt, J = 11.7, 3.6 Hz, 2H), 2.58 (t, J = 7.3 Hz, 2H), 2.44−2.33
(m, 1H), 2.15 (s, 3H), 1.91 (td, J = 11.6, 2.5 Hz, 2H), 1.84−1.74 (m,
2H), 1.63−1.42 (m, 6H). 13C NMR (101 MHz, DMSO-d6): δ 152.30,
148.08, 144.06, 142.00, 128.27, 128.21, 125.65, 106.25, 54.32 (2C),
46.06, 39.77 (overlapped with DMSO signal), 34.69, 31.87, 28.58,
28.09. UPLC/MS (method A): Rt 1.90 min. MS (ES): C20H27N3O3
requires, 357; found, 358 [M + H]+. HRMS: C20H27N3O3 [M + H]+
calcd 358.2131; measured, 358.2121, Δppm −2.8.
Synthesis of (4S)-(+)-2-Oxo-4-phenyl-N-(4-phenylbutyl)-
oxazolidine-3-carboxamide (15a). Compound 15a was prepared
according to general procedure D (method A) at 50 °C using 14a (0.250
g, 1.53 mmol), DMAP (0.206 g, 0.17 mmol), and 4-phenylbutyl
isocyanate (0.279 g, 1.70 mmol) in DMF. The crude was purified by
column chromatography (SiO2), eluting with Cy/EtOAc (85:15), to
1
afford 15a as a white solid (0.420 g, 81%). H NMR (400 MHz,
CDCl3): δ 7.81 (bs, 1H), 7.42−7.23 (m, overlapped with CDCl3 signal,
7H), 7.21−7.10 (m, 3H), 5.43 (dd, J = 8.9, 3.8 Hz, 1H), 4.71 (t, J = 8.9
Hz, 1H), 4.28 (dd, J = 8.8, 3.8 Hz, 1H), 3.37−3.12 (m, 2H), 2.61 (t, J =
7.5 Hz, 2H), 1.70−1.44 (m, overlapped with H2O signal, 4H). 13C
NMR (101 MHz, CDCl3): δ 156.01, 151.05, 142.17, 139.70, 129.32,
128.80, 128.50, 128.45, 125.95, 125.91, 70.47, 57.72, 40.05, 35.56,
29.31, 28.65. [α]3D0 +71.8 (c 0.1, CHCl3). UPLC/MS (method A): Rt
2.48 min. MS (ES): C20H22N2O3 requires, 338; found, 339 [M + H]+.
HRMS: C20H22N2O3 [M + H]+ calcd 339.1709; measured, 339.1697,
Δppm −1.2.
Synthesis of N-Methyl-2-oxo-5-phenyl-N-(4-phenylbutyl)-
oxazole-3-carboxamide (13a). Compound 13a was prepared
according to general procedure D (method C) using 11a (0.155 g,
0.96 mmol, 1.0 equiv), N-methyl-4-phenylbutylamine hydrochloride
(0.061 g, 0.32 mmol, 0.33 equiv), and DIPEA (0.496 g, 0.668 mL, 3.84
Synthesis of (4R)-(−)-2-Oxo-4-phenyl-N-(4-phenylbutyl)-
oxazolidine-3-carboxamide (15b). Compound 15b was prepared
according to general procedure D (method A) using 14b (0.326 g, 2.00
mmol), DMAP (0.268 g, 2.20 mmol), and 4-phenylbutyl isocyanate
(0.385 g, 2.20 mmol) in pyridine. The crude was purified by column
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chromatography (SiO2), eluting with Cy/EtOAc (85:15), to afford 15b
as a white solid (0.320 g, 47%). 1H NMR (400 MHz, CDCl3) 7.81 (bs,
1H), 7.42−7.23 (m, overlapped with CDCl3 signal, 7H), 7.21−7.10 (m,
3H), 5.43 (dd, J = 8.9, 3.8 Hz, 1H), 4.71 (t, J = 8.9 Hz, 1H), 4.28 (dd, J =
8.8, 3.8 Hz, 1H), 3.37−3.12 (m, 2H), 2.61 (t, J = 7.5 Hz, 2H), 1.70−
1.44 (m, overlapped with H2O signal, 4H). 13C NMR (101 MHz,
CDCl3): δ 155.92, 150.96, 142.08, 139.65, 129.20, 128.67, 128.41,
128.35, 125.85, 125.82, 70.36, 57.59, 39.92, 35.45, 29.21, 28.55. [α]D30
−39.8 (c 0.1, CHCl3). UPLC/MS (method A): Rt 2.48 min. MS (ES):
C20H22N2O3 requires, 338; found, 339 [M + H]+. HRMS: C20H22N2O3
[M + H]+ calcd 339.1709; measured, 339.1714, Δppm 0.5.
Synthesis of (5S)-(+)-5-Phenyloxazolidin-2-one (17a). Compound
17a was prepared according to general procedure C using 16a (0.100 g,
0.73 mmol). The crude was purified by column chromatography
(SiO2), eluting with DCM/MeOH (8:2), to afford 17a as a white solid
(0.103 g, 86%). 1H NMR (400 MHz, CDCl3): δ 7.49−7.31 (m, 5H),
5.72−5.49 (m, 2H), 3.98 (t, J = 8.6 Hz, 1H), 3.58−3.51 (m, 1H). [α]D30
+28.3 (c 0.6, EtOH), consistent with data reported in the literature
(lit:73 [α]D30 +26.1 (c 0.88, EtOH)). UPLC/MS (method A): Rt 1.41
min. MS (ES): C9H9NO2 requires, 163; found, 164 [M + H]+.
Synthesis of (5R)-(−)-5-Phenyloxazolidin-2-one (17b). Compound
17b was prepared according to general procedure C using 16b (0.100 g,
0.73 mmol). The crude was purified by column chromatography
(SiO2), eluting with DCM/MeOH (9:1), to afford 17b as a whitish
solid (0.109 g, 92%). 1H NMR (400 MHz, CDCl3): δ 7.49−7.31 (m,
5H), 5.63 (t, J = 8.1 Hz, 1H), 5.35 (bs, 1H), 4.02−3.95 (m, 1H), 3.58−
3.51 (m, 1H). [α]3D0 −26.0 (c 0.5, EtOH), consistent with data reported
in the literature (lit:74 [α]2D2 −24.6° (c 0.045, EtOH)). UPLC/MS
(method A): Rt 1.41 min. MS (ES): C9H9NO2 requires, 163; found, 164
[M + H]+.
7.87−7.81 (m, 1H), 7.60−7.54 (m, 1H), 7.43 (t, J = 7.7 Hz, 1H), 6.15−
6.08 (m, 1H), 4.12−4.07 (m, 2H), 3.66 (t, J = 5.7 Hz, 2H), 2.62 (s, 3H),
2.59−2.52 (m, 2H), 1.50 (s, 9H). UPLC/MS (method A): Rt 2.42 min.
MS (ES): C18H23NO3 requires, 301; found, 302 [M + H]+.
Synthesis of tert-Butyl 4-(5-Acetyl-2-fluoro-phenyl)-3,6-dihydro-
2H-pyridine-1-carboxylate (21b). Compound 21b was prepared
according to general procedure E using 20b (1.000 g, 4.61 mmol)
and 19 (1.568 g, 5.07 mmol). The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (9:1), to afford 20b as
colorless oil (1.280 g, 87%). 1H NMR (400 MHz, CDCl3): δ 7.91−7.81
(m, 2H), 7.11 (dd, J = 10.4, 8.5 Hz, 1H), 6.04−5.94 (m, 1H), 4.11−
4.06 (m, 2H), 3.63 (t, J = 5.7 Hz, 2H), 2.59 (s, 3H), 2.54−2.49 (m, 2H),
1.50 (s, 9H). UPLC/MS (method A): Rt 2.56 min. MS (ES):
C18H22FNO3 requires, 319; found, 320 [M + H]+.
Synthesis of tert-Butyl 4-(3-Acetylphenyl)piperidine-1-carboxy-
late (22a). Compound 22a was prepared according to general
procedure F (method A) using 21a (0.510 g, 1.69 mmol). The crude
was used in the next step without further purification. 1H NMR (400
MHz, CDCl3): δ 7.88−7.76 (m, 2H), 7.44−7.38 (m, 2H), 4.32−4.21
(m, 2H), 2.81 (td, J = 13.0, 2.7 Hz, 2H), 2.72 (tt, J = 12.1, 3.7 Hz, 1H),
2.60 (s, 3H), 1.89−1.78 (m, 2H), 1.65 (qd, J = 12.7, 4.5 Hz, 2H), 1.49
(s, 9H). UPLC/MS (method A): Rt 2.43 min. MS (ES): C18H25NO3
requires, 303; found, 304 [M + H]+.
Synthesis of tert-Butyl 4-(5-Acetyl-2-fluoro-phenyl)piperidine-1-
carboxylate (22b). Compound 22b was prepared according to general
procedure F (method A) using 21b (1.000 g, 3.13 mmol). The crude
was purified by column chromatography (SiO2), eluting with Cy/
1
EtOAc (9:1), to afford 21b as colorless oil (0.650 g, 65%). H NMR
(400 MHz, CDCl3): δ 7.87−7.84 (m, 1H), 7.84−7.78 (m, 1H), 7.09
(dd, J = 9.9, 8.5 Hz, 1H), 4.34−4.22 (m, 2H), 3.02 (tt, J = 12.1, 3.6 Hz,
1H), 2.83 (td, J = 13.2, 2.7 Hz, 2H), 2.58 (s, 3H), 1.86−1.78 (m, 2H),
1.68 (qd, J = 12.6, 4.1 Hz, 2H), 1.48 (s, 9H). UPLC/MS (method A): Rt
2.54 min. MS (ES): C18H24FNO3 requires, 321; found, 322 [M + H]+.
Synthesis of tert-Butyl 4-[3-(2-Bromoacetyl)phenyl]piperidine-1-
carboxylate (22c). To a solution of 22a (0.370 g, 1.22 mmol, 1.0 equiv)
in dry THF (12 mL, 0.1 M), Et3N (0.494 g, 0.68 mL, 4.88 mmol, 4.0
equiv) was added at 0 °C under a N2 atmosphere. After 30 min,
TMSOTf (0.425 g, 0.35 mL, 1.92 mmol, 1.5 equiv) was added dropwise
at −78 °C. The mixture was stirred at −50 °C for 4 h and then
quenched with cold 5% aq NaHCO3 solution and extracted with cold
EtOAc (3 times). The combined organic layer was dried over Na2SO4.
After evaporation of the solvent, the crude was dissolved in dry THF
(12 mL, 0.1 M) and NBS (0.230 g, 1.28 mmol, 1.0 equiv) was added at
−40 °C, and the mixture was stirred at −20 °C for additional 30 min.
The reaction was diluted with H2O and extracted with EtOAc. The
combined organic layer was washed with brine and dried over Na2SO4.
After evaporation of the solvent, 22c was used in the next step without
further purification. UPLC/MS (method A): Rt 2.66 min. MS (ES):
C18H24BrNO3 requires, 381; found, 382 [M + H]+.
Synthesis of tert-Butyl 4-[5-(2-Bromoacetyl)-2-fluoro-phenyl]-
piperidine-1-carboxylate (22d). To a solution of 22b (0.450 g, 1.40
mmol, 1.0 equiv) in dry THF (14 mL, 0.1 M), Et3N (0.570 g, 0.780 mL,
5.60 mmol, 4.0 equiv) was added at 0 °C under a N2 atmosphere. After
30 min, TMSOTf (0.470 g, 0.380 mL, 2.10 mmol, 1.5 equiv) was added
dropwise at −78 °C. The mixture was stirred at −50 °C for 4 h. Then,
the mixture was quenched with cold 5% aq NaHCO3 solution and
extracted with cold EtOAc (3 times). The combined organic layer was
dried over Na2SO4 and the solvent was removed under vacuum. The
crude was dissolved in dry THF (14 mL, 0.1 M) and NBS (0.230 g, 1.28
mmol, 1.0 equiv) was added at −40 °C, and the mixture was stirred at
−20 °C for an additional 30 min. The reaction was diluted with H2O
and extracted with EtOAc. The combined organic layer was washed
with brine and dried over Na2SO4. After evaporation of the solvent, 22d
was used as the crude in the next step without further purification.
UPLC/MS (method B): Rt 1.68 min. MS (ES): C18H23BrFNO3
requires, 399; found, 400 [M + H]+.
Synthesis of (5S)-(−)-2-Oxo-5-phenyl-N-(4-phenylbutyl)-
oxazolidine-3-carboxamide (18a). Compound 18a was prepared
according to general procedure D (method A) at 50 °C using compound
17a (0.103 g, 0.63 mmol), DMAP (0.084 g, 0.69 mmol), and 4-
phenylbutyl isocyanate (0.122 g, 0.69 mmol) in DMF. The crude was
purified by column chromatography (SiO2), eluting with DCM/MeOH
1
(97:3), to afford 18a as colorless oil (0.146 g, 68%). H NMR (400
MHz, CDCl3): δ 7.80 (bs, 1H), 7.47−7.39 (m, 3H), 7.39−7.34 (m,
2H), 7.31−7.24 (m, overlapped with CDCl3 signal, 2H), 7.22−7.14 (m,
3H), 5.58 (t, J = 8.3 Hz, 1H), 4.43 (dd, J = 10.5, 8.9 Hz, 1H), 3.96 (dd, J
= 10.5, 7.7 Hz, 1H), 3.40−3.26 (m, 2H), 2.65 (t, J = 7.4 Hz, 2H), 1.77−
1.57 (m, 4H). 13C NMR (101 MHz, CDCl3): δ 155.35, 151.61, 142.17,
137.15, 129.54, 129.24, 128.55, 128.49, 125.96, 125.89, 75.45, 49.86,
40.18, 35.62, 29.38, 28.71. [α]3D0 −8.70 (c 0.1, CHCl3). UPLC/MS
(method B): Rt 1.50 min. MS (ES): C20H22N2O3 requires, 338; found,
339 [M + H]+. HRMS: C20H22N2O3 [M + H]+ calcd 339.1709;
measured, 339.1703, Δppm −1.8.
Synthesis of (5R)-(+)-2-Oxo-5-phenyl-N-(4-phenylbutyl)-
oxazolidine-3-carboxamide (18b). Compound 18b was prepared
according to general procedure D (method A) using 17b (0.109 g, 0.67
mmol), DMAP (0.084 g, 0.74 mmol), and 4-phenylbutyl isocyanate
(0.130 g, 0.74 mmol) in pyridine. The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (9:1), to afford 18b as
colorless oil (0.180 g, 79%). 1H NMR (400 MHz, CDCl3): δ 7.80 (bs,
1H), 7.47−7.39 (m, 3H), 7.39−7.34 (m, 2H), 7.31−7.24 (m,
overlapped with CDCl3 signal, 2H), 7.22−7.14 (m, 3H), 5.58 (t, J =
8.3 Hz, 1H), 4.43 (dd, J = 10.5, 8.9 Hz, 1H), 3.96 (dd, J = 10.5, 7.7 Hz,
1H), 3.40−3.26 (m, 2H), 2.65 (t, J = 7.4 Hz, 2H), 1.77−1.57 (m, 4H).
13C NMR (101 MHz, CDCl3): δ 155.35, 151.61, 142.17, 137.15,
129.54, 129.24, 128.55, 128.49, 125.96, 125.89, 75.45, 49.86, 40.18,
35.62, 29.38, 28.71. [α]3D0 +18.9 (c 0.1, CHCl3). UPLC/MS (method
B): Rt 1.50 min. MS (ES): C20H22N2O3 requires, 338; found, 339 [M +
H]+. HRMS: C20H22N2O3 [M + H]+ calcd 339.1709; measured,
339.1704, Δppm −1.5.
Synthesis of tert-Butyl 4-(3-Acetylphenyl)-3,6-dihydro-2H-pyri-
dine-1-carboxylate (21a). Compound 21a was prepared according to
general procedure E using 20a (0.500 g, 2.50 mmol) and 19 (0.850 g,
2.75 mmol). The crude was purified by column chromatography
(SiO2), eluting with Cy/EtOAc (8:2), to afford 21a as yellowish oil
(0.580 g, 77%). 1H NMR (400 MHz, CDCl3): δ 7.99−7.94 (m, 1H),
Synthesis of tert-Butyl 4-[3-[2-(2,4-Dioxothiazolidin-3-yl)acetyl]-
phenyl]piperidine-1-carboxylate (23a). Compound 23a was prepared
according to general procedure B, Step 1, using 22c (0.466 g, 1.22
mmol). The crude was used in Step 2 without further purification.
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UPLC/MS (method A): Rt 2.46 min. MS (ES): C21H26N2O5S requires,
418; found, 419 [M + H]+, 417 [M − H]−.
(m, 4H). UPLC/MS (method B): Rt 1.72 min. MS (ES): C25H29N3O3
requires, 419; found, 420 [M + H]+, 418 [M − H]−.
Synthesis of tert-Butyl 4-[5-[2-(2,4-Dioxothiazolidin-3-yl)acetyl]-
2-fluoro-phenyl]piperidine-1-carboxylate (23b). Compound 23b was
prepared according to general procedure B, Step 1, using 22d (0.560 g,
1.40 mmol, 1.0 equiv). The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (9:1), to afford 23b
as a white solid (0.570 g, 93%). 1H NMR (600 MHz, CDCl3): δ 7.87−
7.80 (m, 2H), 7.15 (dd, J = 9.8, 8.4 Hz, 1H), 5.00 (s, 2H), 4.32−4.24
(m, 2H), 4.10 (s, 2H), 3.04 (tt, J = 12.2, 3.5 Hz, 1H), 2.83 (td, J = 13.1,
2.6 Hz, 2H), 1.86−1.78 (m, 2H), 1.67 (qd, J = 12.8, 4.4 Hz, 2H), 1.48
(s, 9H). UPLC/MS (method A): Rt 2.50 min. MS (ES): C21H25FN2O5S
requires, 436; found, 437 [M + H]+.
Synthesis of 5-[3-(1-Methyl-4-piperidyl)phenyl]-2-oxo-N-(4-
phenylbutyl)oxazole-3-carboxamide (25c). Compound 25c was
prepared according to general procedure H using 25b (0.046 g, 0.01
mmol) and 37% aq solution of HCHO (0.016 g, 0.015 mL, 0.19 mmol).
The crude was purified by trituration with Et2O to afford 25c as a white
solid (0.025 g, 60%). 1H NMR (400 MHz, CDCl3): δ 7.99 (t, J = 5.9 Hz,
1H), 7.48−7.37 (m, 3H), 7.34−7.22 (m, overlapped with CDCl3 signal,
4H), 7.25−7.16 (m, 3H), 3.48−3.39 (m, 2H), 3.11−2.98 (m, 2H), 2.80
(tt, J = 11.9, 3.8 Hz, 1H), 2.67 (t, J = 7.2 Hz, 2H), 2.38 (s, 3H), 2.23−
2.09 (m, 2H), 2.03−1.87 (m, 2H), 1.86−1.78 (m, 2H), 1.78−1.60 (m,
overlapped with H2O signal, 4H). 13C NMR (101 MHz, CDCl3): δ
152.89, 148.67, 144.41, 141.99, 139.54, 130.49, 129.13, 128.53 (2C),
127.01, 126.68, 126.04, 125.01, 108.93, 56.16, 46.11, 40.44, 38.30,
35.57, 32.89, 29.22, 28.65. UPLC/MS (method B): Rt 1.10 min. MS
(ES): C26H31N3O3 requires, 433; found, 434 [M + H]+, 257 [M−
CONH(CH2)4Ph]−. HRMS: C26H31N3O3 [M + H]+ calcd 434.2444;
measured, 434.2450, Δppm 1.4.
Synthesis of tert-Butyl 4-[3-(2-Oxo-3H-oxazol-5-yl)phenyl]-
piperidine-1-carboxylate (24a). Compound 24a was prepared using
23a (0.509 g, 1.22 mmol) and LiOH (0.117 g, 4.88 mmol), according to
general procedure B, Step 2. The crude was purified by flash
chromatography (SiO2), eluting with Cy/EtOAc (9:1), to afford 24a
as colorless oil (0.120 g, 29%). 1H NMR (400 MHz, CDCl3): δ 7.43−
7.39 (m, 2H), 7.34−7.31 (m, 1H), 7.29−7.24 (m, overlapped with
CDCl3 signal, 1H), 6.95 (s, 1H), 4.29−4.19 (m, 2H), 2.94 (tt, J = 11.8,
3.3 Hz, 1H), 2.84−2.73 (m, 2H), 1.83−1.75 (m, 2H), 1.70−1.60 (m,
overlapped with H2O signal, 2H), 1.48 (s, 9H). UPLC/MS (method A):
Rt 2.83 min. MS (ES): C19H24N2O4 requires, 344; found, 345 [M + H]+.
Synthesis of tert-Butyl 4-[2-Fluoro-5-(2-oxo-3H-oxazol-5-yl)-
phenyl]piperidine-1-carboxylate (24b). Compound 24b was pre-
pared using 23b (0.560 g, 1.30 mmol) and t-BuOK (0.586 g, 5.22
mmol, 4.0 equiv), according to general procedure B, Step 2. The crude
was purified by flash chromatography (SiO2), eluting with Cy/EtOAc
(6:4), to afford 24b as an orange solid (0.340 g, 72%). 1H NMR (600
MHz, CDCl3): δ 9.22−9.12 (m, 1H), 7.36−7.29 (m, 2H), 7.04 (dd, J =
9.9, 8.5 Hz, 1H), 6.79−6.76 (m, 1H), 4.35−4.20 (m, 2H), 3.01 (tt, J =
12.2, 3.5 Hz, 1H), 2.87−2.76 (m, 2H), 1.84−1.75 (m, 2H), 1.66 (qd, J
= 12.7, 4.4 Hz, 2H), 1.48 (s, 9H). UPLC/MS (method A): Rt 2.30 min.
MS (ES): C19H23FN2O4 requires, 362; found, 363 [M + H]+, 361 [M −
H]−.
Synthesis of 5-[4-Fluoro-3-(4-piperidyl)phenyl]-3H-oxazol-2-one
Hydrochloride (24c). Compound 24c was prepared according to
general procedure G using 24b (0.150 g, 0.41 mmol). The crude was
used in the next step without further purification. UPLC/MS (method
A): Rt 1.22 min. MS (ES): C14H15FN2O2 requires, 262; found, 263 [M
+ H]+,261 [M − H]−.
Synthesis of 5-[4-Fluoro-3-(1-methyl-4-piperidyl)phenyl]-3H-ox-
azol-2-one (24d). Compound 24d was prepared according to general
procedure H using 24c (0.123 g, 0.41 mmol) and 37% aq. solution of
HCHO (0.066 g, 0.061 mL, 0.82 mmol). The crude was purified by
trituration with Et2O to afford 24d as a white solid (0.080 g, 71%). 1H
NMR (400 MHz, CDCl3): δ 7.48−7.41 (m, 1H), 7.38−7.31 (m, 1H),
7.08−6.99 (m, 1H), 6.91 (s, 1H), 3.48−3.36 (m, 2H), 3.10−2.98 (m,
1H), 2.67 (s, 3H), 2.66−2.53 (m, 2H), 2.37−2.20 (m, 2H), 2.02−1.91
(m, 2H). UPLC/MS (method A): Rt 1.23 min. MS (ES): C15H17FN2O2
requires, 276; found, 277 [M + H]+, 275 [M − H]−.
Synthesis of tert-Butyl 4-[3-[2-Oxo-3-(4-phenylbutylcarbamoyl)-
oxazol-5-yl]phenyl]piperidine-1-carboxylate (25a). Compound 25a
was prepared according to general procedure D (method A) using 24a
(0.120 g, 0.35 mmol), DMAP (0.047 g, 0.39 mmol) and 4-phenylbutyl
isocyanate (0.068 g, 0.067 mL, 0.39 mmol) in pyridine. The crude was
purified by column chromatography (SiO2), eluting with Cy/EtOAc
(9:1), to afford 25a as yellowish oil (0.125 g, 69%). UPLC/MS (method
B): Rt 2.46 min. MS (ES): C30H37N3O5 requires, 519; found, 520 [M +
H]+.
Synthesis of 2-Oxo-N-(4-phenylbutyl)-5-[3-(4-piperidyl)phenyl]-
oxazole-3-carboxamide Hydrochloride (25b). Compound 25b was
prepared according to general procedure G using 25a (0.125 g, 0.24
mmol). The crude was purified by trituration with Et2O to afford 25b as
a white solid (0.046 g, 46%). 1H NMR (400 MHz, DMSO-d6): δ 8.66
(bs, 2H), 8.12 (t, J = 5.9 Hz, 1H), 7.57 (s, 1H), 7.52−7.45 (m, 2H),
7.39−7.32 (m, 2H), 7.31−7.24 (m, 2H), 7.24−7.11 (m, 3H), 3.43−
3.26 (m, overlapped with H2O signal, 4H), 3.24−3.13 (m, 1H), 3.09−
2.90 (m, 1H), 2.61 (t, J = 7.2 Hz, 2H), 1.95−1.77 (m, 4H), 1.68−1.48
Synthesis of 5-[4-Fluoro-3-(1-methyl-4-piperidyl)phenyl]-2-oxo-
N-(4-phenylbutyl)oxazole-3-carboxamide (25d). Compound 25d
was prepared according to general procedure D (method A) using
24d (0.075 g, 0.27 mmol), DMAP (0.037 g, 0.30 mmol), and 4-
phenylbutyl isocyanate (0.095 g, 0.092 mL, 0.54 mmol) in pyridine at
50 °C. The crude was purified by column chromatography (SiO2),
eluting with DCM/MeOH (92:8), to afford 25d as a white solid (0.030
g, 25%). 1H NMR (400 MHz, CDCl3): δ 7.92 (t, J = 5.8 Hz, 1H), 7.41
(dd, J = 6.9, 2.3 Hz, 1H), 7.38 (s, 1H), 7.34 (ddd, J = 8.5, 4.8, 2.3 Hz,
1H), 7.31−7.26 (m, overlapped with CDCl3 signal, 2H), 7.22−7.13 (m,
3H), 7.06 (dd, J = 9.9, 8.5 Hz, 1H), 3.45−3.36 (m, 2H), 3.14−3.00 (m,
2H), 2.94−2.81 (m, 1H), 2.66 (t, J = 7.2 Hz, 2H), 2.40 (s, 3H), 2.26−
2.13 (m, 2H), 2.01−1.80 (m, 4H), 1.77−1.60 (m, 4H). 13C NMR (101
MHz, CDCl3): δ 152.70, 148.52, 142.02, 139.03, 128.56, 128.53,
126.03, 123.38, 122.98, 116.42 (d, JC−F = 24.2 Hz), 105.90, 55.80,
45.60, 40.46, 35.57, 34.37, 30.85, 29.20, 28.64. UPLC/MS (method A):
Rt 2.30 min. MS (ES): C26H30FN3O3 requires, 451; found, 452 [M +
H]+, 275 [M−CONH(CH2)4Ph]−. HRMS: C26H30FN3O3 [M + H]+
calcd 452.2349; measured, 452.2340, Δppm −2.0.
Synthesis of 5-[4-Fluoro-3-(1-methyl-4-piperidyl)phenyl]-2-oxo-
N-pentyl-oxazole-3-carboxamide (25e). Compound 25e was pre-
pared according to general procedure D (method A), using 24d (0.070
g, 0.25 mmol), Et3N (0.050 g, 0.070 mL, 0.05 mmol) and pentyl
isocyanate (0.034 g, 0.038 mL, 0.30 mmol) in CH3CN at 50 °C. The
crude was purified by column chromatography (SiO2), eluting with
DCM/MeOH (9:1), to afford 25e as a white solid (0.035 g, 36%). 1H
NMR (400 MHz, DMSO-d6): δ 8.09 (s, 1H), 8.01 (t, J = 5.8 Hz, 1H),
7.68−7.62 (m, 1H), 7.55−7.49 (m, 1H), 7.23 (dd, J = 10.3, 8.7 Hz,
1H), 3.33−3.22 (m, overlapped with H2O signal, 2H), 3.02−2.89 (m,
2H), 2.79 (tt, J = 11.7, 3.5 Hz, 1H), 2.28 (s, 3H), 2.20−2.02 (m, 2H),
1.82 (qd, J = 12.4, 3.3 Hz, 2H), 1.75−1.66 (m, 2H), 1.53 (p, J = 7.2 Hz,
2H), 1.37−1.21 (m, 4H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6): δ 159.96 (d, JC−F = 246.6 Hz), 151.80, 147.92, 142.44,
137.69, 132.80, 123.23, 123.15 (d, JC−F = 3.1 Hz), 116.08 (d, J = 24.1
Hz), 107.61, 54.89, 44.95, 40.11 (overlapped with DMSO signal),
33.82, 30.38, 28.61, 28.34, 21.76, 13.85. UPLC/MS (method A): Rt 2.12
min. MS (ES): C21H28FN3O3 requires, 389; found, 390 [M + H]+, 275
[M−CONH(CH2)4CH3]−. HRMS: C21H28FN3O3 [M + H]+ calcd
390.2193; measured, 390.2195, Δppm 0.5.
Synthesis of 5-[4-Fluoro-3-(1-methyl-4-piperidyl)phenyl]-N-iso-
butyl-2-oxo-oxazole-3-carboxamide (25f). Compound 25f was
prepared according to general procedure D (method C) using 24d
(0.035 g, 0.13 mmol), 2-methylpropan-1-amine (0.028 g, 0.030 mL,
0.39 mmol), and Et3N (0.079 g, 0.109 mL, 0.78 mmol). The crude was
purified by column chromatography (SiO2), eluting with DCM/MeOH
(9:1), to afford 25f as a white solid (0.040 g, 82%). 1H NMR (400 MHz,
CDCl3): δ 7.98 (t, J = 4.9 Hz, 1H), 7.45−7.41 (m, 1H), 7.41 (s, 1H),
7.35 (ddd, J = 8.6, 4.8, 2.2 Hz, 1H), 7.07 (dd, J = 10.0, 8.5 Hz, 1H), 3.23
(t, J = 6.4 Hz, 2H), 3.20−3.11 (m, 2H), 2.92 (tt, J = 11.5, 2.9 Hz, 1H),
2.47 (s, 3H), 2.40−2.25 (m, 2H), 2.11−1.96 (m, 2H), 1.95−1.82 (m,
3H), 0.98 (d, J = 6.7 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 161.07
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(d, JC−F = 249.2 Hz), 152.75, 148.63, 139.04, 133.22 (d, J = 15.4 Hz),
123.45, 123.40, 122.93 (d, JC−F = 3.5 Hz), 116.38 (d, JC−F = 24.2 Hz),
105.86 (d, JC−F = 2.1 Hz), 55.93, 47.89, 45.88, 34.56, 31.19, 28.61,
20.11. UPLC/MS (method A): Rt 1.96 min. MS (ES): C20H26FN3O3
requires, 375; found, 376 [M + H]+, 275 [M−CONHCH2CH-
(CH3)2]−. HRMS: C20H26FN3O3 [M + H]+ calcd 376.2036 measured
376.2028, Δppm −2.1.
Synthesis of tert-Butyl 4-(2-Acetyl-5-fluoro-phenyl)-3,6-dihydro-
2H-pyridine-1-carboxylate (27). Compound 27 was prepared
according to general procedure E using 26 (2.000 g, 9.22 mmol) and
19 (3.990 g, 12.90 mmol). The crude was purified by column
chromatography (SiO2), eluting with Cy/EtOAc (9:1), to afford 27 as
yellow oil (2.530 g, 85%). 1H NMR (400 MHz, CDCl3): δ 7.60 (dd, J =
8.6, 5.7 Hz, 1H), 7.05−6.99 (m, 1H), 6.92 (dd, J = 9.5, 2.6 Hz, 1H),
5.60−5.51 (m, 1H), 4.02 (q, J = 2.8 Hz, 2H), 3.65 (t, J = 5.6 Hz, 2H),
2.46 (s, 3H), 2.40−2.32 (m, 2H), 1.49 (s, 9H). UPLC/MS (method B):
Rt 1.40 min. MS (ES): C18H22FNO3 requires, 319; found, 320 [M +
H]+.
Synthesis of tert-Butyl 4-(2-Acetyl-5-fluoro-phenyl)piperidine-1-
carboxylate (28). Compound 28 was prepared according to general
procedure F (method B) using 27 (1.500 g, 4.70 mmol). The crude was
purified by column chromatography (SiO2), eluting with Cy/EtOAc
(9:1), to afford 28 as colorless oil (1.040 g, 69%). 1H NMR (400 MHz,
CDCl3): δ 7.65 (dd, J = 8.6, 5.9 Hz, 1H), 7.05 (dd, J = 10.6, 2.6 Hz, 1H),
6.98−6.92 (m, 1H), 4.31−4.16 (m, 2H), 3.43 (dddd, J = 10.0, 8.6, 3.7,
1.6 Hz, 1H), 2.81 (t, J = 12.8 Hz, 2H), 2.58 (s, 3H), 1.85−1.75 (m, 2H),
1.61−1.52 (m, overlapped with H2O signal, 2H), 1.48 (s, 9H). UPLC/
MS (method B): Rt 1.51 min. MS (ES): C18H24FNO3 requires, 321;
found, 322 [M + H]+.
Synthesis of tert-Butyl 4-[2-(2-Bromoacetyl)-5-fluoro-phenyl]-
piperidine-1-carboxylate (29). To a solution of 28 (0.410 g, 1.28
mmol, 1.0 equiv) in dry THF (13 mL, 0.1 M), and Et3N (0.516 g, 0.710
mL, 5.10 mmol, 4.0 equiv) was added at 0 °C under a N2 atmosphere.
After 30 min, TMSOTf (0.425 g, 0.346 mL, 1.92 mmol, 1.5 equiv) was
added dropwise at −78 °C. The mixture was stirred at −50 °C for 4 h.
Then, the mixture was quenched with cold 5% aq NaHCO3 solution
and extracted with cold EtOAc (3 times). The combined organic layer
was washed with brine and dried over Na2SO4. After evaporation of the
solvent, the crude was dissolved in dry THF (13 mL, 0.1 M), NBS
(0.227 g, 1.28 mmol, 1.0 equiv) was added at −40 °C, and the mixture
was stirred at −20 °C for additional 30 min. The reaction was diluted
with H2O and extracted with EtOAc. The combined organic layer was
washed with brine and dried over Na2SO4. After evaporation of the
solvent, the crude of 29 was used in the next step without further
purification. UPLC/MS (method B): Rt 1.70 min. MS (ES):
C18H23BrFNO3 requires, 399; found, 400 [M + H]+, 398 [M − H]−.
Synthesis of tert-Butyl 4-[2-[2-(2,4-Dioxothiazolidin-3-yl)acetyl]-
5-fluoro-phenyl]piperidine-1-carboxylate (30). Compound 30 was
prepared according to general procedure B, Step 1, using 29 (0.512 g,
1.28 mmol). The crude was purified by column chromatography
(SiO2), eluting with Cy/EtOAc (8:2), to afford 30 as colorless oil
(0.366 g, 66%). 1H NMR (400 MHz, CDCl3): δ 7.67 (dd, J = 8.7, 5.6
Hz, 1H), 7.10 (dd, J = 10.5, 2.6 Hz, 1H), 7.05−6.98 (m, 1H), 4.85 (s,
2H), 4.27−4.16 (m, 2H), 4.10 (s, 2H), 3.36−3.25 (m, 1H), 2.81 (td, J =
13.4, 13.0, 2.5 Hz, 2H), 1.84−1.74 (m, 2H), 1.58−1.51 (m, overlapped
with H2O signal, 2H), 1.47 (s, 9H). UPLC/MS (method B): Rt 1.42
min. MS (ES): C21H25FN2O5S requires, 436; found, 437 [M + H]+, 435
[M − H]−.
Synthesis of tert-Butyl 4-[5-Fluoro-2-(2-oxo-3H-oxazol-5-yl)-
phenyl]piperidine-1-carboxylate (31a). Compound 31a was prepared
using 30 (0.366 g, 0.84 mmol) and t-BuOK (0.376 g, 3.35 mmol),
according to general procedure B, Step 2. The crude was purified by
flash chromatography (SiO2), eluting with DCM/MeOH (95:5), to
afford 31a as yellow foam (0.204 g, 67%). 1H NMR (400 MHz,
CDCl3): δ 9.14 (bs, 1H), 7.39 (dd, J = 8.6, 5.8 Hz, 1H), 7.02 (dd, J =
10.3, 2.6 Hz, 1H), 6.99−6.92 (m, 1H), 6.59 (d, J = 1.9 Hz, 1H), 4.41−
4.14 (m, 2H), 3.05−2.90 (m, 1H), 2.89−2.70 (m, 2H), 1.84−1.75 (m,
2H), 1.71−1.53 (m, overlapped with H2O signal, 2H), 1.48 (s, 9H).
UPLC/MS (method A): Rt 2.25 min. MS (ES): C19H23FN2O4 requires,
362; found, 363 [M + H]+, 361 [M − H]−.
Synthesis of 5-[4-Fluoro-2-(4-piperidyl)phenyl]-3H-oxazol-2-one
Hydrochloride (31b). Compound 31b was prepared according to
general procedure G using 31a (0.150 g, 0.41 mmol). The crude was
used in the next step without further purification. UPLC/MS (method
A): Rt 1.27 min. MS (ES): C14H15FN2O2 requires, 262; found, 263 [M
+ H]+, 261 [M − H]−.
Synthesis of 5-[4-Fluoro-2-(1-methyl-4-piperidyl)phenyl]-3H-ox-
azol-2-one (31c). Compound 31c was prepared according to general
procedure H using 31b (0.123 g, 0.41 mmol) and 37% aq solution of
HCHO (0.165 g, 0.150 mL, 2.05 mmol). The crude was used in the
next step without further purification. 1H NMR (400 MHz, DMSO-d6):
δ 10.85 (bs, 1H), 7.43 (dd, J = 8.7, 6.1 Hz, 1H), 7.21 (dd, J = 10.8, 2.7
Hz, 1H), 7.11 (td, J = 8.4, 2.7 Hz, 1H), 7.06 (s, 1H), 2.94−2.84 (m,
2H), 2.83−2.73 (m, 1H), 2.23 (s, 3H), 2.10−1.96 (m, 2H), 1.78−1.62
(m, 4H). 13C NMR (101 MHz, DMSO-d6): δ 163.57, 161.12, 155.27,
146.81, 137.06, 130.53, 123.11, 113.52, 113.38, 113.30, 113.17, 55.42,
45.73, 37.72, 32.11. UPLC/MS (method A): Rt 1.27 min. MS (ES):
C15H17FN2O2 requires, 276; found, 277 [M + H]+, 275 [M − H]−.
HRMS: C15H18FN2O2 [M + H]+ calcd 277.1352; measured, 277.1354,
Δppm 0.7.
Synthesis of 5-[4-Fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-
N-(4-phenylbutyl)oxazole-3-carboxamide (32a). Compound 32a
was prepared according to general procedure D (method A) using
31c (0.048 g, 0.174 mmol), DMAP (0.023 g, 0.19 mmol), and 4-
phenylbutyl isocyanate (0.033 g, 0.033 mL, 0.19 mmol) in DMF at 50
°C. The crude was purified by column chromatography (SiO2), eluting
with DCM/MeOH (95:5), to afford 32a as a white solid (0.050 g,
62%). 1H NMR (400 MHz, DMSO-d6): δ 8.06 (t, J = 5.9 Hz, 1H), 7.51
(dd, J = 8.6, 6.0 Hz, 1H), 7.45 (s, 1H), 7.31−7.24 (m, 3H), 7.23−7.11
(m, 4H), 3.36−3.22 (m, overlapped with H2O signal, 2H), 2.92−2.82
(m, 2H), 2.82−2.71 (m, 1H), 2.61 (t, J = 7.2 Hz, 2H), 2.19 (s, 3H), 1.96
(td, J = 11.1, 3.9 Hz, 2H), 1.79−1.51 (m, 8H). 13C NMR (101 MHz,
DMSO-d6): δ 163.03 (d, JC−F = 247.8 Hz), 151.80, 148.00, 147.96,
141.98, 137.25, 131.49 (d, JC−F = 8.6 Hz), 128.27, 128.21, 125.65,
121.58 (d, JC−F = 3.2 Hz) 113.76, 113.42 (d, JC−F = 22.3 Hz), 109.67,
55.52 (2C), 46.02, 40.01 (overlapped with DMSO signal), 38.03, 34.71,
32.42 (2C), 28.61, 28.12. UPLC/MS (method A): Rt 2.27 min. MS
(ES): C26H30FN3O3 requires, 451; found, 452 [M + H]+, 275 [M−
CONH(CH2)4Ph]−. HRMS: C26H30FN3O3 [M + H]+ calcd 452.2349;
measured, 452.2358, Δppm 2.0.
Synthesis of 5-[4-Fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-
N-pentyl-oxazole-3-carboxamide (32b). Compound 32b was pre-
pared according to general procedure D (method A) using 31c (0.040 g,
0.14 mmol), DMAP (0.020 g, 0.17 mmol), and n-pentyl isocyanate
(0.019 g, 0.022 mL, 0.17 mmol) in CH3CN at 50 °C. The crude was
purified by column chromatography (SiO2), eluting with DCM/MeOH
1
(9:1), to afford 32b as a white solid (0.035 g, 64%). H NMR (400
MHz, DMSO-d6): δ 8.04 (t, J = 5.8 Hz, 1H), 7.51 (dd, J = 8.7, 6.0 Hz,
1H), 7.45 (s, 1H), 7.26 (dd, J = 10.7, 2.7 Hz, 1H), 7.15 (td, J = 8.4, 2.7
Hz, 1H), 3.31−3.23 (m, overlapped with H2O signal, 2H), 2.90−2.82
(m, 2H), 2.81−2.70 (m, 1H), 2.18 (s, 3H), 1.94 (td, J = 11.0, 3.8 Hz,
2H), 1.78−1.62 (m, 4H), 1.54−1.48 (m, 2H), 1.36−1.25 (m, 4H), 0.88
(t, J = 6.8 Hz, 3H). 13C NMR (101 MHz, DMSO-d6): δ 151.85, 148.13,
147.95, 137.28, 131.50 (d, JC−F = 9.0 Hz), 121.60, 113.78, 113.43 (d,
J
C−F = 23.0 Hz), 109.67, 55.56, 46.08, 40.15 (overlapped with DMSO
signal), 38.09, 32.47, 28.61, 28.35, 21.76, 13.85. UPLC/MS (method
A): Rt 2.12 min. MS (ES): C21H28FN3O3 requires, 389; found, 390 [M
+ H]+, 275 [M−CONH(CH2)4CH3]−. HRMS: C21H28FN3O3 [M +
H]+ calcd 390.2193; measured, 390.2204, Δppm 2.8.
Synthesis of 5-[4-Fluoro-2-(1-methyl-4-piperidyl)phenyl]-N-iso-
butyl-2-oxo-oxazole-3-carboxamide Hydrochloride (32c). Com-
pound 32c was prepared according to general procedure D (method
C) using 31c (0.034 g, 0.12 mmol), 2-methylpropan-1-amine (0.027 g,
0.029 mL, 0.37 mmol), and Et3N (0.075 g, 0.104 mL, 0.74 mmol). The
crude was purified by column chromatography (SiO2), eluting with
DCM/MeOH (95:5), to afford the free base of 32c (44%, 0.020 g,
0.053 mmol). The free base of 32c (0.020 g, 0.05 mmol) was then
dissolved in 0.5 mL of 1,4-dioxane and 4.0 M HCl solution (0.27 mL,
1.06 mmol, 20.0 equiv) was added. After evaporation of the solvent, the
residue was triturated with Et2O to afford 32c as a white solid (0.009 g,
X
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20%). 1H NMR (400 MHz, CDCl3): δ 12.88 (bs, 1H), 7.96 (t, J = 5.9
Hz, 1H), 7.39 (dd, J = 8.6, 5.6 Hz, 1H), 7.29−7.21 (m, overlapped with
CDCl3 signal, 2H), 7.05−6.97 (m, 1H), 3.67−3.55 (m, 2H), 3.24 (dd, J
= 6.8, 5.9 Hz, 2H), 3.05 (tt, J = 12.0, 2.7 Hz, 2H), 2.91−2.77 (m, 5H),
2.69−2.52 (m, 2H), 2.07−1.97 (m, 2H), 1.96−1.84 (m, 1H), 0.99 (d, J
= 6.7 Hz, 6H). 13C NMR (101 MHz, CDCl3): δ 165.58, 152.78, 149.42,
purchased from Open Biosystems (clone ID 3923451) and
subcloned in the mammalian expression vector pCDNA3.1,
containing the neomycin resistance gene. HEK293 cells were
transfected with the hAC-pCDNA3.1 construct using a JetPEI
reagent (Polyplus Transfection, Illkirch-Graffenstaden, France)
and following the manufacturer’s instructions. A stable cell line
was generated by selection with G418 (1 mg/mL), and cell
clones were derived by limited dilution plating. HEK293-hAC
were grown in Dulbecco’s modified Eagle medium (DMEM)
containing 10% fetal bovine serum (FBS), 1% glutamine, 1 mM
sodium pyruvate, and 500 μg/mL G418. Cells were harvested
and pellets were stored at −80 °C until lysosomal-enriched
lysate preparation. Cells were suspended in 20 mM Tris HCl
(pH 7.5) with 0.32 M sucrose, sonicated, and centrifuged at 800
× g for 30 min at 4 °C. Supernatants were then centrifuged at
12 000 × g for 30 min at 4 °C. Pellets were re-suspended in PBS
(pH 7.4) and subjected to three freeze−thaw cycles at −80 °C.
The suspension was finally centrifuged at 105 000 × g for 1 h at 4
°C, and protein concentration was measured in the supernatant
with a bicinchoninic acid based protein assay. This hAC-
enriched preparation allowed us to further optimize the
enzymatic assay and to use small amounts of lysate (2 μg/
well) at the 5 μM substrate (Rbm14-12) around its KM (KM =
5.0 μM).
Fluorogenic hAC Assay. The assay was performed in
Optiplate 96-wells black plates, with each reaction well
containing a mixture of 25 mM NaOAc buffer (pH 4.5) and a
fixed amount of protein (2 μg) in a volume of 85 μL. After 10
min of pre-incubation with the test compounds (diluted 20×
from DMSO stock solutions at different concentrations), the
fluorogenic probe was added (diluted 40× from EtOH stock
solution, final concentration 5 μM). After 3 h incubation at 37
°C, reactions were stopped with 50 μL of MeOH and 100 μL of a
2.5 mg/mL NaIO4 fresh solution in 100 mM glycine/NaOH
buffer (pH 10.6). The plates were further incubated for 2 h at 37
°C in the dark and fluorescence intensities were measured at
excitation/emission wavelengths of 355/460 nm. Negative
control samples consisted of the same incubation mixture in
the absence of protein-enriched extracts. Data were plotted as a
function of compound concentrations. IC50 values were
calculated by nonlinear regression analysis using GraphPad
Prism 5 (GraphPad Software Inc., CA, USA) applying a standard
slope curve fitting. The reported IC50 values are the mean of at
least three independent experiments performed in three
technical replicates.
148.51, 144.71, 138.82, 131.80 (d, JC−F = 8.7 Hz), 115.01 (d, JC−F
=
22.0 Hz), 114.91, 109.29, 55.10, 47.96, 44.00, 36.67, 29.99, 28.64,
20.11. UPLC/MS (method A): Rt 1.93 min. MS (ES): C20H26FN3O3
requires, 375; found, 376 [M + H]+, 275 [M−CONHCH2(CH3)2]−.
HRMS: C20H26FN3O3 [M + H]+ calcd 376.2031; measured, 376.2036,
Δppm −1.3.
MOLECULAR MODELING AND DOCKING STUDIES
■
Covalent Docking. To carry out the covalent docking, we
used docking modules available in a Schrodinger platform:
CovDoc in Schrodinger 2018-4 version. These covalent docking
tools require that the ligand set must be a series of compounds all
of which should react with the receptor at the same site and by
the same mechanism. The first step is the regular noncovalent
docking of the ligands having the potential to form a covalent
bond with the reactive residue of the receptor. Then, the
program allows for the different conformations of the side-chain
of the reactive residue, which is temporarily mutated to alanine
at this stage. The reason for this temporary mutation is only to
avoid the bias toward the particular ligand conformation if the
side-chain of the reactive residue is present. After the
noncovalent docking, the original side-chain of the reactive
residue is replaced with the cysteine and the covalent bond is
formed. The ligands where the covalent bond lengths between
the reactive center of the ligand and the reactive residue of the
receptor are longer compared to the standard chemical bonds
are discarded. After the bond formation between the potential
covalent ligand and the receptor, the complex is minimized using
the Prime module of Schrodinger suite. Finally, the docked
poses of the covalently linked ligands are to be visualized and
rank ordered by energy and the docked score. The reaction site
of ligands was determined using a SMART2 search pattern
implemented in the covalent docking program. After determin-
ing the reactive sites from both the ligands and the receptor, a 12
A3 grid box was generated making sure that Cys143 would be in
that grid box. The docked poses and the energetics were
analyzed and 43 covalent inhibitors were showing good docking
poses with lower complex energies and having docking scores
≥6.0. Some of the ligands lacked the proper SMARTS for the
reaction and were rejected from the calculations. Docking
calculations for hNAAA were carried out under identical
conditions using the X-ray structure (PDB code: 6DXX).
Compound 32b did not natively dock into this receptor using
the standard CovDock protocol. To understand the binding
mode in greater details, each step of the docking run was
analyzed and the raw unrefined poses were examined. It was
clear that Trp181 was occluding binding of the n-pentyl side-
chain of compound 32b leading to a docking score >10,000
(indicate no binding affinity to NAAA). The compound was
thus modeled using a flexible ligand overlay using the AC
docking pose for the purpose of generating a figure.
KINETIC STUDIES
■
Michaelis−Menten Analysis. Assay conditions for the
kinetic studies were the same as those described for the
fluorogenic hAC assay. The enzyme-enriched lysate (2 μg) was
incubated with the following concentrations of substrate
Rbm14-12: 0.25, 0.5, 1, 2.5, 5, 10, 12.5, 15 μM. Compound
12a was used at a final concentration of 25 and 100 nM. Initial
velocities (V0) were determined and automatically fitted to the
Michaelis−Menten equation to obtain kinetic parameters (KM
and Vmax). The graph is representative of two independent
experiments, each performed in three technical replicates.
Graphs and data analysis were performed using GraphPad
Prism 5 software (GraphPad Software Inc., CA, USA).
IN VITRO PHARMACOLOGICAL ASSAY
■
In Vitro hAC Fluorescent Assay. Cell Culture Conditions
and Preparation of the hAC-Enriched Lysate. HEK293 cells
stably expressing hAC (HEK293-hAC) were generated in our
laboratory using a protocol, as previously described.30 Briefly,
hAC (variant 1 coding sequence, NM_177924) cDNA was
In Vitro hNAAA Fluorescent Assay. Cell Culture and
Preparation of the hNAAA-Enriched Lysate. HEK-293 cells
stably transfected with the hNAAA coding sequence cloned
from a human spleen cDNA library (catalog no. 639124,
Y
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Clontech, Mountain View, CA, USA) were used as the enzyme
source. Cells were grown in DMEM containing 10% FBS, 1%
glutamine, 1 mM sodium pyruvate, and 500 μg/mL G418. Cells
were harvested and pellets were stored at −80 °C until
lysosomal-enriched lysate preparation. Cells were suspended
in 20 mM Tris HCl (pH 7.4) with 0.32 M sucrose, sonicated,
and centrifuged at 800 × g for 30 min at 4 °C. Supernatants were
then ultracentrifuged at 12 000 × g for 30 min at 4 °C. Pellets
were re-suspended in PBS buffer (pH 7.4) and subjected to
three freeze−thaw cycles at −80 °C. The suspension was finally
ultracentrifuged at 105 000 × g for 1 h at 4 °C, supernatants were
collected, protein concentration was measured, and samples
aliquoted and stored at −80 °C until use.
Fluorogenic hNAAA Assay. The assay was run in 96-well
microplates (Black OptiPlate-96 F; PerkinElmer, Massachu-
setts, USA) in a total reaction volume of 200 μL. hNAAA protein
preparation (4.0 μg) was pre-incubated for 30 min with various
concentrations of test compounds or vehicle control (DMSO
5%) in 100 mM citrate/phosphate buffer (pH 4.5) containing
3.0 mM dithiothreitol (DTT), 0.1% NP40 0.1%, 0.05% bovine
serum albumin, and 150 mM NaCl. N-(4-Methyl-2-oxo-
chromen-7-yl)-hexadecanamide (PAMCA) was used as a
substrate (2.0 μM) and the reaction carried out for 50 min at
37 °C. Fluorescence was measured with an EnVision 2014
Multilabel Reader (PerkinElmer, Massachusetts, USA) using an
excitation wavelength of 355 nm and emission 460 nm. IC50
values were calculated by nonlinear regression analysis of
log[concentration]/inhibition curves using GraphPad Prism 5
(GraphPad Software Inc., CA, USA) applying a standard slope
curve fitting. The values are the mean of at least three
independent experiments performed in three technical repli-
cates.
Lipid Extraction and Ceramide Analysis. Lipid extraction
and SL measurements were performed as previously de-
scribed.59,61,75 Lipids were extracted with a CHCl3/MeOH
mixture (2:1, 3 mL) spike with internal standards. The organic
phase was collected, dried under N2, and dissolved in CHCl3/
MeOH (1:3) for LC/MS analyses. Ceramides and sphingosine
were analyzed by LC/MS/MS, using a Waters Acquity UPLC
coupled to a Waters Xevo triple quadrupole mass spectrometer
(TQMS) and interfaced with an ESI ion source. Separation was
performed on a Waters Acquity BEH C18 1.7 μm column (2.1 ×
50 mm) at 60 °C. A linear gradient of 0.1% HCOOH in
CH3CN/i-PrOH (20:80) as solvent B in 0.1% HCOOH in
CH3CN/H2O (20:80) as solvent A and was applied at a flow
rate of 0.4 mL/min. The detection of SLs was performed in
positive ion mode. Capillary voltage was 3.5 kV and cone voltage
was 25 V. The source temperature and desolvation temperatures
were set at 120 and 600 °C, respectively. Desolvation gas and
cone gas (N2) flows were 800 and 20 L/h, respectively.
Ceramides were identified by comparison of their LC retention
times and MS/MS fragmentation patterns with those of
authentic standards (Avanti Polar Lipids). Multiple reaction
monitoring (MRM) ion chromatograms were used to quantify
dihydroceramide (Cer(d18:0/16:0), Rt = 4.6 min, m/z: 540.5 >
522.5), palmitoyl ceramide (Cer(d18:1/16:0), Rt = 4.5 min, m/
z: 520.3 > 264.2) sphingosine (So(d18:1), Rt = 2.6 min, m/z:
300.2 > 282.2), sphingomyelin (SM(d18:1/16:0), Rt = 4.2 min,
m/z: 703.2 > 184.1) and hexosylceramide (HexCer (d18:1/
16:0), 4.3 min, m/z: 682.2 > 264.2). Detection and analysis were
controlled by Waters MassLynx software version 4.1. Calibra-
tion curves were prepared for every experiment.
Statistics. GraphPad Prism software (GraphPad Software,
Inc., USA) was used for statistical analysis. Data were analyzed
using the Student t-test or 1-way ANOVA followed by
Bonferroni post hoc test for multiple comparisons. Differences
between groups were considered statistically significant at values
of p < 0.05. Results are expressed as mean SEM.
CELL CULTURE AND TREATMENTS
■
SH-SY5Y cells were purchased from Sigma Aldrich (Italy) and
cultured in DMEM containing 10% FBS at 37 °C and 5% CO2.
Drugs were dissolved in DMSO (10 mM) and diluted in cell
culture medium with reduced serum (1%) for cell treatments.
hAC LC/MS-Based Activity Assay. hAC activity measure-
ment was performed as previously described.59,61 Total lysates
from cells were diluted in assay buffer (100 mM sodium
phosphate, 0.1% Nonidet P-40, 150 mM NaCl, 3 mM DTT, 100
mM sodium citrate, pH 4.5). Reactions were started by the
addition of 50 μM N-lauroyl ceramide (Nu-Chek Prep, Elysian,
MN) and carried out for 1 h at 37 °C. Reactions were stopped by
the addition of a mixture of CHCl3/MeOH (2:1) spike with 11-
lauroleic acid (1.0 nmol, NuChek Prep). The organic phases
were collected, dried under N2, and analyzed by UPLC/MS
(Acquity, Waters) in the negative-ion mode monitoring the
reaction product (lauric acid, m/z: 199) using 11-lauroleic acid
as the internal standard. Lipids were eluted on an Acquity UPLC
BEH C18 column (50 mm length, 2.1 mm ID, 1.7 μm pore size,
Waters) column at 0.5 mL/min for 1.5 min with a gradient of
CH3CN and H2O, both containing 0.25% AcOH and 5 mM
NH4OAc (70−100% CH3CN in 0.5 min, 100% CH3CN for 0.5
min, 70% CH3CN for 0.4 min). The column temperature was 40
°C. ESI was in the negative mode, capillary voltage was 1 kV, and
cone voltage was 50 V. N2 was used as a drying gas at a flow rate
of 500 L/h and at a temperature of 400 °C. The [M − H]− ion
was monitored in the selected-ion monitoring mode (m/z
values: lauric acid 199, 11-lauroleic acid 197.35). Calibration
curves were generated with authentic lauric acid (Nu Check
Prep).
IN VITRO PHYSICOCHEMICAL AND METABOLIC
STABILITY ASSAY
■
Aqueous Kinetic Solubility Assay. The aqueous kinetic
solubility was determined from a 10 mM CH3CN stock solution
of the test compound in PBS at pH 7.4. The study was
performed by the incubation of an aliquot of 10 mM CH3CN
stock solution in PBS (pH 7.4) at a target concentration of 250
μM. The incubation was carried out under shaking at 25 °C for 1
h, followed by centrifugation at 21 100 × g for 30 min. The
supernatant was analyzed by UPLC/MS for the quantification of
the dissolved compound (in μM) by UV at a specific wavelength
(215 nm). The aqueous kinetic solubility (in μM) was calculated
by dividing the peak area of the dissolved test compound
(supernatant) by the peak area of the test compound in the
reference (250 μM in CH3CN) and further multiplied by the
target concentration and dilution factor. The UPLC/MS
analyses were performed on a Waters Acquity UPLC/MS
system consisting of SQDMS equipped with an ESI interface
and a PDA detector. The PDA range was 210−400 nm. ESI in
positive mode was used in the mass scan range 100−650 Da.
The analyses were run on an Acquity UPLC BEH C18 column
(50 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH
C18 pre-column (5 × 2.1 mm ID, particle size 1.7 μm), using 10
mM NH4OAc in H2O at pH 5 adjusted with AcOH (A) and 10
mM NH4OAc in CH3CN/H2O (95:5) at pH 5 (B) as the
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mobile phase. The values are the mean of at least two
independent experiments performed in two technical replicates.
Chemical Stability Assay. Chemical stability of the
selected compounds was evaluated under physiological pH
conditions (0.01 M PBS, pH 7.4) for up to 8 h. The buffer was
added with 10% of CH3CN. Stock solutions of each compound
(10 mM) were freshly prepared in CH3CN. Each compound
was incubated at a final concentration of 1 μM in pre-heated
buffer (37 °C). The sample solutions were divided into aliquots
in glass vials (preheated at 37 °C) for each time point. The
samples were maintained at 37 °C in an UPLC/MS autosampler
during the study (no shaking). A reference solution of each
compound (final concentration: 1 μM) in preheated CH3CN
was prepared from the stock solutions and maintained at 37 °C
in the UPLC/MS autosampler during the study. For each time
point, the samples were analyzed directly by LC/MS without
any further sample preparation. The samples were analyzed by
integrating the corresponding MRM peak areas. The relative
compound concentration was calculated by dividing the peak
area at each time point by the peak area at t = 0 min. The
reference solution was analyzed at the beginning (t = 0 min) and
at the end of the study (t = 8 h). The apparent half-life (t1/2) of
the disappearance of the compound was calculated using the
best fitting equation by GraphPad Prism (GraphPad Software,
Inc., USA). The analyses were performed on a Waters Acquity
UPLC/MS triple quadrupole detection (TQD) system
consisting of TQD MS equipped with an ESI interface and a
PDA detector. The analyses were run on an Acquity UPLC BEH
C18 1.7 μm 2.1 × 50 mm column with a VanGuard BEH C18
1.7 μm pre-column at 40 °C. For each compound, the
appropriate mobile phase was chosen. ESI was applied in
positive mode. The values are the mean of at least two
independent experiments performed in two technical replicates.
In Vitro Plasma Stability Study. Freshly prepared 10 mM
CH3CN stock solution of the test compound was diluted 50-fold
with DMSO/H2O (1:1) and incubated at 37 °C for 2 h with
mouse/human plasma added 5% DMSO (preheated at 37 °C for
10 min). The final concentration was 2 μM. At each time point
(0, 5, 15, 30, 60, and 120 min), 50 μL of the incubation mixture
was diluted with 200 μL of cold CH3CN spiked with 200 nM of
the internal standard, followed by centrifugation at 3300 × g for
20 min. The supernatant was further diluted with H2O (1:1) for
analysis. The concentration of the test compound was quantified
by LC/MS−MS on a Waters Acquity UPLC/MS TQD system
consisting of TQD MS equipped with an ESI interface. The
analyses were run on an Acquity UPLC BEH C18 (50 × 2.1 mm
ID, particle size 1.7 μm) with a VanGuard BEH C18 pre-column
(5 × 2.1 mm ID, particle size 1.7 μm) at 40 °C. For each
compound, the appropriate mobile phase was chosen. ESI was
applied in positive mode. The response factors, calculated on the
basis of the internal standard peak area, were plotted over time.
When possible, response versus time profiles were fitted with
Prism (GraphPad Software, Inc., USA) to estimate compounds
t1/2 in the plasma. The values are the mean of at least two
independent experiments performed in two technical replicates.
In Vitro Microsomal Stability Study. Freshly prepared 10
mM CH3CN stock solution of the test compound was pre-
incubated at 37 °C for 15 min with mouse/human liver
microsomes added 0.1 M Tris-HCl buffer (pH 7.4). The final
concentration was 4.6 μM. After pre-incubation, the cofactors
(NADPH, G6P, G6PDH, and MgCl2 pre-dissolved in 0.1 M
Tris-HCl) were added to the incubation mixture and the
incubation was continued at 37 °C for 1 h. At each time point (0,
5, 15, 30, and 60 min), 30 μL of the incubation mixture was
diluted with 200 μL of cold CH3CN spiked with 200 nM of the
internal standard, followed by centrifugation at 3300g for 15
min. The supernatant was further diluted with H2O (1:1) for
analysis. The concentration of the test compound was quantified
by LC/MS−MS on a Waters Acquity UPLC/MS TQD system
consisting of TQD MS equipped with an ESI interface. The
analyses were run on an Acquity UPLC BEH C18 (50 × 2.1 mm
ID, particle size 1.7 μm) with a VanGuard BEH C18 pre-column
(5 × 2.1 mm ID, particle size 1.7 μm) at 40 °C. For each
compound, the appropriate mobile phase was chosen. ESI was
applied in positive mode. The percentage of the test compound
remaining at each time point relative to t = 0 was calculated. The
t1/2 were determined by a one-phase decay equation using a
nonlinear regression of the compound concentration versus
time. The values are the mean of at least two independent
experiments performed in two technical replicates.
ANIMAL MODELS
■
In Vivo PK Study. C57 B6/J male mice, 8 weeks old (22−24
g), were used (Charles River, Calco). All procedures were
performed in accordance with the Ethical Guidelines of
European Communities Council (Directive 2010/63/EU of
22 September 2010) and accepted by the Italian Ministry of
Health. All efforts were made to minimize animal suffering and
to use the minimal number of animals required to produce
reliable results, according to the “3Rs rules”. Animals were
group-housed in ventilated cages and had free access to food and
water. They were maintained under a 12 h light/dark cycle
(lights on at 8:00 am) at a controlled temperature (21 1 °C)
and relative humidity (55
10%). 32b was administrated
intravenously (i.v.) at 3 mg/kg/5 mL, vehicle: PEG400/Tween
80/saline solution (10/10/80% in volume, respectively) via tail
vein injection and via oral administration (p.o.) at 10 mg/kg/10
mL, vehicle: PEG400/Tween 80/saline solution (10/10/80% in
volume, respectively) by an oral gavage. Sample collection.
Samples were collected at 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h
(i.v. administration) and at 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h
(p.o. administration). N = 3 animals per dose time point were
treated. Plasma samples were centrifuged at 21,100g for 15 min
at 4 °C. An aliquot of each sample was extracted (1:3) with cold
CH3CN containing 200 nM of an appropriate internal standard
being a close analogue of compound 32b. A calibration curve
was prepared in blank mouse plasma over a 1 nM−10 μM range.
Three quality controls were prepared by spiking compound 32b
in the blank mouse plasma to 20, 200, and 2000 nM as final
concentrations. The calibrators and quality controls were
extracted (1:3) with the same extraction solution as the plasma
samples. The plasma samples, the calibrators, and quality
controls were centrifuged at 3270 × g for 15 min at 4 °C. The
supernatants were further diluted (1:1) with H2O and analyzed
by LC/MS−MS on a Waters Acquity UPLC/MS TQD system
consisting of TQD) MS equipped with an ESI interface and a
PDA detector. ESI was applied in positive mode. Compound-
dependent parameters such as MRM transitions and collision
energy were developed for compound 32b and the internal
standard. The analyses were run on an Acquity UPLC BEH C18
(50 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH
C18 pre-column (5 × 2.1 mm ID, particle size 1.7 μm) at 40 °C.
For plasma samples, the mobile phase was H2O + 0.1%
HCOOH (A) and CH3CN + 0.1% HCOOH (B) at a flow rate =
0.5 mL/min. A linear gradient was applied starting at 20% B with
an initial hold for 0.2 min, then 20−100% B in 2 min. All samples
AA
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(plasma samples, calibrators, and quality controls) were
quantified by a MRM peak area response factor in order to
determine the levels of compound 32b in the plasma. The
concentrations versus time were plotted and the profiles were
fitted using PK Solutions Excel Application (Summit Research
Service, USA) in order to determine the PK parameters.
Natasha Margaroli − Fondazione Istituto Italiano di
Tecnologia, I-16163 Genova, Italy; Drug Discovery and
Development (D3)-Validation, I-16163 Genova, Italy
Marco Migliore − Fondazione Istituto Italiano di Tecnologia, I-
16163 Genova, Italy; Drug Discovery and Development (D3)-
Validation, I-16163 Genova, Italy
Giuliana Ottonello − Fondazione Istituto Italiano di
Tecnologia, I-16163 Genova, Italy; Analytical Chemistry and
Translational Pharmacology, I-16163 Genova, Italy
Min Liu − Lysosomal Therapeutics Inc., Cambridge,
Massachusetts 02139, United States
Peter Lansbury − Lysosomal Therapeutics Inc., Cambridge,
Massachusetts 02139, United States
Andrea Armirotti − Fondazione Istituto Italiano di Tecnologia,
I-16163 Genova, Italy; Analytical Chemistry and
Translational Pharmacology, I-16163 Genova, Italy;
orcid.org/0000-0002-3766-8755
Rosalia Bertorelli − Fondazione Istituto Italiano di Tecnologia,
I-16163 Genova, Italy; Analytical Chemistry and
Translational Pharmacology, I-16163 Genova, Italy
Soumya S. Ray − Lysosomal Therapeutics Inc., Cambridge,
Massachusetts 02139, United States
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01561.
■
sı
AC inhibitory activity data and assay methods for selected
1
literature compounds; Vmax and KM determinations; H
NMR and 13C NMR spectra of the final compounds;
retention times and UPLC analytical methods of the final
compounds; and UPLC traces of the final compounds
(PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Authors
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01561
Renato Skerlj − Lysosomal Therapeutics Inc., Cambridge,
Massachusetts 02139, United States; Phone: +1 617 610
8624; Email: renato.skerlj@x4pharma.com
Rita Scarpelli − Fondazione Istituto Italiano di Tecnologia, I-
16163 Genova, Italy; Drug Discovery and Development (D3)-
Validation, I-16163 Genova, Italy; orcid.org/0000-0002-
2623-306X; Phone: +39 010 2896233;
Author Contributions
¶S.C. and S.D.M. contributed equally. All the authors have given
approval to the final version of the manuscript.
Notes
Email: rita.scarpelli@iit.it
The authors declare the following competing financial
interest(s): All the work in the manuscript was funded by
Lysosomal Therapeutics Inc. A Sponsored Research Agreement
was signed between Fondazione Istituto Italiano di Tecnologia,
Drug Discovery and Development (D3)-Validation and
Lysosomal Therapeutics Inc.
Authors
Samantha Caputo − Fondazione Istituto Italiano di Tecnologia,
I-16163 Genova, Italy; Drug Discovery and Development
(D3)-Validation, I-16163 Genova, Italy
Simona Di Martino − Fondazione Istituto Italiano di
Tecnologia, I-16163 Genova, Italy; Drug Discovery and
Development (D3)-Validation, I-16163 Genova, Italy;
orcid.org/0000-0001-5817-751X
Vincenzo Cilibrasi − Fondazione Istituto Italiano di
Tecnologia, I-16163 Genova, Italy; Drug Discovery and
Development (D3)-Validation, I-16163 Genova, Italy
Piero Tardia − Fondazione Istituto Italiano di Tecnologia, I-
16163 Genova, Italy; Drug Discovery and Development (D3)-
Validation, I-16163 Genova, Italy
Marco Mazzonna − Fondazione Istituto Italiano di Tecnologia,
I-16163 Genova, Italy; Drug Discovery and Development
(D3)-Validation, I-16163 Genova, Italy
Debora Russo − Fondazione Istituto Italiano di Tecnologia, I-
16163 Genova, Italy; D3-Pharma Chemistry, I-16163
Genova, Italy
Ilaria Penna − Fondazione Istituto Italiano di Tecnologia, I-
16163 Genova, Italy; D3-Pharma Chemistry, I-16163
Genova, Italy
Maria Summa − Fondazione Istituto Italiano di Tecnologia, I-
16163 Genova, Italy; Analytical Chemistry and Translational
Pharmacology, I-16163 Genova, Italy
Sine Mandrup Bertozzi − Fondazione Istituto Italiano di
Tecnologia, I-16163 Genova, Italy; Analytical Chemistry and
Translational Pharmacology, I-16163 Genova, Italy
Natalia Realini − Fondazione Istituto Italiano di Tecnologia, I-
16163 Genova, Italy; Drug Discovery and Development (D3)-
Validation, I-16163 Genova, Italy
ACKNOWLEDGMENTS
The authors thank Silvia Venzano, Luca Goldoni and Marina
Veronesi for their technical support.
■
ABBREVIATIONS
■
AC, acid ceramidase; ASM, acid sphingomyelinase; Cer,
ceramide; dh-CerDES, dihydro-ceramide desaturase; CerS,
ceramide synthase; CerK, ceramide kinase; CerPPase, ceramide
phosphate phosphatase; diGalCer, di-galactosylceramide; GAL-
Case, β-galactosyl ceramidase; GD, Gaucher’s disease; GCase,
β-glucosyl ceramidase; GalCer, galactosylceramide; GluCer,
glucosylceramide; CGALT, ceramide galactosyl transferase;
CGT, ceramide glucosyl transferase; LacCer, lactosylceramide;
LSD, lysosomal storage disease; KD, Krabbe’s disease; SL,
sphingolipid; So, sphingosine; So-1P, sphingosine 1-phosphate;
SK, sphingosine kinase; SMase, sphingomyelinase; SPPase,
sphingosine phosphate phosphatase; SMS, sphingomyelin
synthase; AcOH, acetic acid; Al2O3, alumina; CH3CN,
acetonitrile; NH4OAc, ammonium acetate; NH4Cl, ammonium
chloride; HCO2NH4, ammonium formate; aq, aqueous; NBS,
N-bromosuccinimide; CDI, 1,1′-carbonyldiimidazole; CHCl3,
chloroform; Cy, cyclohexane; celite, diatomaceous earth; DCM,
dichloromethane; Et2O, diethyl ether; DIPEA, N,N-diisopropy-
lethylamine; DMAP, 4-(dimethylamino)-pyridine; DMF, N,N-
dimethylformamide; DMSO, dimethylsulfoxide; Boc2O, di-tert-
butyl dicarbonate; equiv, equivalent; EtOH, ethanol; EtOAc,
AB
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(15) Wang, K.; Xu, R.; Schrandt, J.; Shah, P.; Gong, Y. Z.; Preston, C.;
Wang, L.; Yi, J. K.; Lin, C.-L.; Sun, W.; Spyropoulos, D. D.; Rhee, S.; Li,
M.; Zhou, J.; Ge, S.; Zhang, G.; Snider, A. J.; Hannun, Y. A.; Obeid, L.
M.; Mao, C. Alkaline ceramidase 3 deficiency results in purkinje cell
degeneration and cerebellar ataxia due to dyshomeostasis of
sphingolipids in the brain. PLoS Genet. 2015, 11, e1005591−e1005622.
(16) Edvardson, S.; Yi, J. K.; Jalas, C.; Xu, R.; Webb, B. D.; Snider, J.;
Fedick, A.; Kleinman, E.; Treff, N. R.; Mao, C.; Elpeleg, O. Deficiency
of the alkaline ceramidase ACER3 manifests in early childhood by
progressive leukodystrophy. J. Med. Genet. 2016, 53, 389−396.
(17) Bernardo, K.; Hurwitz, R.; Zenk, T.; Desnick, R.; Ferlinz, K.;
Schuchman, E.; Sandhoff, K. Purification, characterization, and
biosynthesis of human acid ceramidase. J. Biol. Chem. 1995, 270,
11098−11102.
ethyl acetate; HCHO, formaldehyde; HCOOH, formic acid;
HCl, hydrochloric acid; LiCl, lithium chloride; LiOH, lithium
hydroxide; MeOH, methanol; N2, nitrogen; on, overnight;
Pd(OH)2, palladium hydroxide; Pd/C, palladium on carbon;
K2CO3, potassium carbonate; KNCO, potassium cyanate; t-
BuOK, potassium tert-butoxide; i-PrOH, 2-propanol; Rt,
retention time; rt, room temperature; sat., saturated; SiO2, silica
gel; NaOAc, sodium acetate; NaHCO3, sodium bicarbonate;
Na2CO3, sodium carbonate; NaCl, sodium chloride; Na2SO4,
sodium sulfate; NaBH(OAc)3, sodium triacetoxyborohydride;
T H F , t e t r a h y d r o f u r a n ; P d ( P P h 3 ) 4 , t e t r a k i s -
(triphenylphosphine) palladium(0); TZD, 2,4-thiazolidine-
dione; SOCl2, thionyl chloride; Et3N, trimethylamine;
TMSOTf, trimethylsilyl trifluoromethanesulfonate; H2O,
water; wt, weight
̈
(18) Ferlinz, K.; Kopal, G.; Bernardo, K.; Linke, T.; Bar, J.; Breiden, B.;
Neumann, U.; Lang, F.; Schuchman, E. H.; Sandhoff, K. Human acid
ceramidase. Processing, glycosylation, and lysosomal targeting. J. Biol.
Chem. 2001, 276, 35352−35360.
REFERENCES
(19) Newton, J.; Lima, S.; Maceyka, M.; Spiegel, S. Revisiting the
sphingolipid rheostat: Evolving concepts in cancer therapy. Exp. Cell
Res. 2015, 333, 195−200.
■
(1) Hannun, Y. A.; Obeid, L. M. Many ceramides. J. Biol. Chem. 2011,
286, 27855−27862.
(20) Huang, X.; Withers, B. R.; Dickson, R. C. Sphingolipids and
lifespan regulation. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 2014,
1841, 657−664.
(2) Gomez-Mun
Trueba, M.; Ordon
̃
oz, A.; Presa, N.; Gomez-Larrauri, A.; Rivera, I.-G.;
̃
ez, M. Control of inflammatory responses by
ceramide, sphingosine 1-phosphate and ceramide 1-phosphate. Prog.
Lipid Res. 2016, 61, 51−62.
(21) Pettus, B. J.; Chalfant, C. E.; Hannun, Y. A. Ceramide in
apoptosis: an overview and current perspectives. Biochim. Biophys. Acta,
Mol. Cell Biol. Lipids 2002, 1585, 114−125.
(3) Hannun, Y. A.; Obeid, L. M. Sphingolipids and their metabolism
in physiology and disease. Nat. Rev. Mol. Cell Biol. 2018, 19, 175−191.
(4) Kurz, J.; Parnham, M. J.; Geisslinger, G.; Schiffmann, S. Ceramides
as novel disease biomarkers. Trends Mol. Med. 2019, 25, 20−32.
(5) van Eijk, M.; Ferraz, M. J.; Boot, R. G.; Aerts, J. M. F. G. Lyso-
glycosphingolipids: presence and consequences. Essays Biochem. 2020,
64, 565.
(22) Morales, A.; Lee, H.; Goni, F. M.; Kolesnick, R.; Fernandez-
̃
Checa, J. C. Sphingolipids and cell death. Apoptosis 2007, 12, 923−939.
(23) Zhang, H.; Desai, N. N.; Olivera, A.; Seki, T.; Brooker, G.;
Spiegel, S. Sphingosine-1-phosphate, a novel lipid, involved in cellular
proliferation. J. Cell Biol. 1991, 114, 155−167.
(24) Payne, S. G.; Milstien, S.; Spiegel, S. Sphingosine-1-phosphate:
dual messenger functions. FEBS Lett. 2002, 531, 54−57.
(25) Spiegel, S.; Milstien, S. Sphingosine-1-phosphate: an enigmatic
signalling lipid. Nat. Rev. Mol. Cell Biol. 2003, 4, 397−407.
(26) Takabe, K.; Spiegel, S. Export of sphingosine-1-phosphate and
cancer progression. J. Lipid Res. 2014, 55, 1839−1846.
(27) Schuchman, E. H. Acid ceramidase and the treatment of
ceramide diseases: the expanding role of enzyme replacement therapy.
Biochim. Biophys. Acta, Mol. Basis Dis. 2016, 1862, 1459−1471.
(28) Yu, F. P. S.; Medin, J. A.; Amintas, S.; Levade, T.; Levade, T.;
Medin, J. A. Acid ceramidase deficiency: Farber disease and SMA-PME.
Orphanet J. Rare Dis. 2018, 13, 121−140.
(6) Sahu, S. K.; Hannun, Y. A.; Yao, N. Emergence of membrane
sphingolipids as a potential therapeutic target. Biochimie 2019, 158,
257−264.
(7) Coant, N.; Sakamoto, W.; Mao, C.; Hannun, Y. A. Ceramidases,
roles in sphingolipid metabolism and in health and disease. Adv. Biol.
Regul. 2017, 63, 122−131.
(8) Parveen, F.; Bender, D.; Law, S.-H.; Mishra, V. K.; Chen, C.-C.;
Ke, L.-Y. Role of ceramidases in sphingolipid metabolism and human
diseases. Cells 2019, 8, 1573−1592.
(9) García-Barros, M.; Coant, N.; Kawamori, T.; Wada, M.; Snider, A.
J.; Truman, J. P.; Wu, B. X.; Furuya, H.; Clarke, C. J.; Bialkowska, A. B.;
Ghaleb, A.; Yang, V. W.; Obeid, L. M.; Hannun, Y. A. Role of neutral
ceramidase in colon cancer. FASEB J. 2016, 30, 4159−4171.
(10) Coant, N.; Hannun, Y. A. Neutral ceramidase: advances in
mechanisms, cell regulation, and roles in cancer. Adv. Biol. Regul. 2019,
71, 141−146.
(29) Seelan, R. S.; Qian, C.; Yokomizo, A.; Bostwick, D. G.; Smith, D.
I.; Liu, W. Human acid ceramidase is overexpressed but not mutated in
prostate cancer. Genes, Chromosomes Cancer 2000, 29, 137−146.
(30) Realini, N.; Palese, F.; Pizzirani, D.; Pontis, S.; Basit, A.; Bach, A.;
Ganesan, A.; Piomelli, D. Acid ceramidase in melanoma: expression,
localization, and effects of pharmacological inhibition. J. Biol. Chem.
2016, 291, 2422−2434.
(11) Liakath-Ali, K.; Vancollie, V. E.; Lelliott, C. J.; Speak, A. O.;
Lafont, D.; Protheroe, H. J.; Ingvorsen, C.; Galli, A.; Green, A.; Gleeson,
D.; Ryder, E.; Glover, L.; Vizcay-Barrena, G.; Karp, N. A.; Arends, M. J.;
Brenn, T.; Spiegel, S.; Adams, D. J.; Watt, F. M.; van der Weyden, L.
Alkaline ceramidase 1 is essential for mammalian skin homeostasis and
regulating whole-body energy expenditure. J. Pathol. 2016, 239, 374−
383.
(12) Sun, W.; Xu, R.; Hu, W.; Jin, J.; Crellin, H. A.; Bielawski, J.; Szulc,
Z. M.; Thiers, B. H.; Obeid, L. M.; Mao, C. Upregulation of the human
alkaline ceramidase 1 and acid ceramidase mediates calcium-induced
differentiation of epidermal keratinocytes. J. Invest. Dermatol. 2008,
128, 389−397.
(13) Xu, R.; Wang, K.; Mileva, I.; Hannun, Y. A.; Obeid, L. M.; Mao,
C. Alkaline ceramidase 2 and its bioactive product sphingosine are
novel regulators of the DNA damage response. Oncotarget 2016, 7,
18440−18457.
(14) Hu, W.; Xu, R.; Sun, W.; Szulc, Z. M.; Bielawski, J.; Obeid, L. M.;
Mao, C. Alkaline ceramidase 3 (ACER3) hydrolyzes unsaturated long-
chain ceramides, and its down-regulation inhibits both cell proliferation
and apoptosis. J. Biol. Chem. 2010, 285, 7964−7976.
(31) Roh, J.-L.; Park, J. Y.; Kim, E. H.; Jang, H. J. Targeting acid
ceramidase sensitises head and neck cancer to cisplatin. Eur. J. Cancer
2016, 52, 163−172.
̌
̌
́
́
́
(32) Klobucar, M.; Grbcic, P.; Pavelic, S. K.; Jonjic, N.; Visentin, S.;
́
Sedic, M. Acid ceramidase inhibition sensitizes human colon cancer
cells to oxaliplatin through downregulation of transglutaminase 2 and
beta1 integrin/FAK-mediated signalling. Biochem. Biophys. Res.
Commun. 2018, 503, 843−848.
(33) Doan, N. B.; Alhajala, H.; Al-Gizawiy, M. M.; Mueller, W. M.;
Rand, S. D.; Connelly, J. M.; Cochran, E. J.; Chitambar, C. R.; Clark, P.;
Kuo, J.; Schmainda, K. M.; Mirza, S. P. Acid ceramidase and its
inhibitors: a de novo drug target and a new class of drugs for killing
glioblastoma cancer stem cells with high efficiency. Oncotarget 2017, 8,
112662−112674.
(34) Mahdy, A. E.; Cheng, J. C.; Li, J.; Elojeimy, S.; Meacham, W. D.;
Turner, L. S.; Bai, A.; Gault, C. R.; McPherson, A. S.; Garcia, N.;
Beckham, T. H.; Saad, A.; Bielawska, A.; Bielawski, J.; Hannun, Y. A.;
Keane, T. E.; Taha, M. I.; Hammouda, H. M.; Norris, J. S.; Liu, X. Acid
AC
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
ceramidase upregulation in prostate cancer cells confers resistance to
radiation: AC inhibition, a potential radiosensitizer. Mol. Ther. 2009,
17, 430−438.
(35) Saad, A. F.; Meacham, W. D.; Bai, A.; Anelli, V.; Elojeimy, S.;
Mahdy, A. E.; Turner, L. S.; Cheng, J.; Bielawska, A.; Bielawski, J.;
Keane, T. E.; Obeid, L. M.; Hannun, Y. A.; Norris, J. S.; Liu, X. The
functional effects of acid ceramidase overexpression in prostate cancer
progression and resistance to chemotherapy. Cancer Biol. Ther. 2007, 6,
1455−1460.
(52) Draper, J. M.; Xia, Z.; Smith, R. A.; Zhuang, Y.; Wang, W.; Smith,
C. D. Discovery and evaluation of inhibitors of human ceramidase. Mol.
Cancer Ther. 2011, 10, 2052−2061.
(53) Yildiz-Ozer, M.; Oztopcu-Vatan, P.; Kus, G. The investigation of
ceranib-2 on apoptosis and drug interaction with carboplatin in human
non small cell lung cancer cells in vitro. Cytotechnology 2018, 70, 387−
396.
(54) Cho, S. M.; Lee, H. K.; Liu, Q.; Wang, M.-W.; Kwon, H. J. A
guanidine-based synthetic compound suppresses angiogenesis via
inhibition of acid ceramidase. ACS Chem. Biol. 2019, 14, 11−19.
(55) Realini, N.; Solorzano, C.; Pagliuca, C.; Pizzirani, D.; Armirotti,
A.; Luciani, R.; Costi, M. P.; Bandiera, T.; Piomelli, D. Discovery of
highly potent acid ceramidase inhibitors with in vitro tumor
chemosensitizing activity. Sci. Rep. 2013, 3, 1035.
(56) Pizzirani, D.; Pagliuca, C.; Realini, N.; Branduardi, D.; Bottegoni,
G.; Mor, M.; Bertozzi, F.; Scarpelli, R.; Piomelli, D.; Bandiera, T.
Discovery of a new class of highly potent inhibitors of acid ceramidase:
synthesis and structure−activity relationship (SAR). J. Med. Chem.
2013, 56, 3518−3530.
(36) Huang, Y.; Tanimukai, H.; Liu, F.; Iqbal, K.; Grundke-Iqbal, I.;
Gong, C.-X. Elevation of the level and activity of acid ceramidase in
Alzheimer’s disease brain. Eur. J. Neurosci. 2004, 20, 3489−3497.
(37) Gieselmann, V. Lysosomal storage diseases. Biochim. Biophys.
Acta, Mol. Basis Dis. 1995, 1270, 103−136.
(38) Ballabio, A.; Gieselmann, V. Lysosomal disorders: From storage
to cellular damage. Biochim. Biophys. Acta, Mol. Cell Res. 2009, 1793,
684−696.
́
́
(39) Cox, T. M.; Cachon-Gonzalez, M. B. The cellular pathology of
lysosomal diseases. J. Pathol. 2012, 226, 241−254.
(40) Stirnemann, J.; Belmatoug, N.; Camou, F.; Serratrice, C.;
Froissart, R.; Caillaud, C.; Levade, T.; Astudillo, L.; Serratrice, J.;
Brassier, A.; Rose, C.; Billette de Villemeur, T.; Berger, M. A review of
gaucher disease pathophysiology, clinical presentation and treatments.
Int. J. Mol. Sci. 2017, 18, 441−471.
(57) Diamanti, E.; Bottegoni, G.; Goldoni, L.; Realini, N.; Pagliuca,
C.; Bertozzi, F.; Piomelli, D.; Pizzirani, D. Pyrazole-based acid
ceramidase inhibitors: design, synthesis, and structure−activity
relationships. Synthesis 2016, 48, 2739−2756.
(58) Ortega, J. A.; Arencibia, J. M.; La Sala, G.; Borgogno, M.; Bauer,
I.; Bono, L.; Braccia, C.; Armirotti, A.; Girotto, S.; Ganesan, A.; De
Vivo, M. Pharmacophore identification and scaffold exploration to
discover novel, potent, and chemically stable inhibitors of acid
ceramidase in melanoma cells. J. Med. Chem. 2017, 60, 5800−5815.
(59) Pizzirani, D.; Bach, A.; Realini, N.; Armirotti, A.; Mengatto, L.;
Bauer, I.; Girotto, S.; Pagliuca, C.; De Vivo, M.; Summa, M.; Ribeiro, A.;
Piomelli, D. Benzoxazolone carboxamides: potent and systemically
active inhibitors of intracellular acid ceramidase. Angew. Chem., Int. Ed.
2015, 127, 495−499.
(60) Bach, A.; Pizzirani, D.; Realini, N.; Vozella, V.; Russo, D.; Penna,
I.; Melzig, L.; Scarpelli, R.; Piomelli, D. Benzoxazolone carboxamides as
potent acid ceramidase inhibitors: synthesis and structure−activity
relationship (SAR) Studies. J. Med. Chem. 2015, 58, 9258−9272.
(61) Di Martino, S.; Tardia, P.; Cilibrasi, V.; Caputo, S.; Mazzonna,
M.; Russo, D.; Penna, I.; Realini, N.; Margaroli, N.; Migliore, M.;
Pizzirani, D.; Ottonello, G.; Bertozzi, S. M.; Armirotti, A.; Nguyen, D.;
Sun, Y.; Bongarzone, E. R.; Lansbury, P.; Liu, M.; Skerlj, R.; Scarpelli, R.
Lead optimization of benzoxazolone carboxamides as orally bioavail-
able and CNS penetrant acid ceramidase inhibitors. J. Med. Chem. 2020,
63, 3634−3664.
(41) Won, J.-S.; Singh, A. K.; Singh, I. Biochemical, cell biological,
pathological, and therapeutic aspects of Krabbe’s disease. J. Neurosci.
Res. 2016, 94, 990−1006.
(42) Ferraz, M. J.; Marques, M. D.; Verhoek, M.; Strijland, A.;
Mirzaian, M.; Scheij, S.; Ouairy, C. M.; Lahav, D.; Wisse, P.; Overkleeft,
H. S.; Boot, R. G.; Aerts, J. M. Lysosomal glycosphingolipid catabolism
by acid ceramidase: formation of glycosphingoid bases during
deficiency of glycosidases. FEBS Lett. 2016, 590, 716−725.
(43) Li, Y.; Xu, Y.; Benitez, B. A.; Nagree, M. S.; Dearborn, J. T.; Jiang,
X.; Guzman, M. A.; Woloszynek, J. C.; Giaramita, A.; Yip, B. K.;
Elsbernd, J.; Babcock, M. C.; Lo, M.; Fowler, S. C.; Wozniak, D. F.;
Vogler, C. A.; Medin, J. A.; Crawford, B. E.; Sands, M. S. Genetic
ablation of acid ceramidase in Krabbe disease confirms the psychosine
hypothesis and identifies a new therapeutic target. Proc. Natl. Acad. Sci.
U.S.A. 2019, 116, 20097−20103.
(44) Gebai, A.; Gorelik, A.; Li, Z.; Illes, K.; Nagar, B. Structural basis
for the activation of acid ceramidase. Nat. Commun. 2018, 9, 1621.
(45) Bielawska, A.; Greenberg, M. S.; Perry, D.; Jayadev, S.; Shayman,
J. A.; McKay, C.; Hannun, Y. A. (1S,2R)-D-erythro-2-(N-myristoyla-
mino)-1-phenyl-1-propanol as an inhibitor of ceramidase. J. Biol. Chem.
1996, 271, 12646−12654.
(62) Wang, Q.; Tan, X.; Zhu, Z.; Dong, X.-Q.; Zhang, X. New
synthetic strategy for chiral 2-oxazolidinones derivatives via rhodium-
catalyzed asymmetric hydrogenation. Tetrahedron Lett. 2016, 57, 658−
662.
(63) Brown, M. L.; Cheung, M.; Dickerson, S. H.; Gauthier, C.;
Harris, P. A.; Hunter, R. N.; Pacofsky, G.; Peel, M. R.; Stafford, J. A.
Chemical compounds. WO 2004032882 A2, 2004.
(46) Sugita, M.; Williams, J. T.; Moser, H. W. Ceramidase and
ceramide synthesis in human kidney and cerebellum: description of a
new alkaline ceramidase. Biochim. Biophys. Acta, Lipids Lipid Metab.
1975, 398, 125−131.
́
(47) Grijalvo, S.; Bedia, C.; Triola, G.; Casas, J.; Llebaria, A.; Teixido,
̀
J.; Rabal, O.; Levade, T.; Delgado, A.; Fabrias, G. Design, synthesis and
activity as acid ceramidase inhibitors of 2-oxooctanoyl and N-
oleoylethanolamine analogues. Chem. Phys. Lipids 2006, 144, 69−84.
(48) Raisova, M.; Goltz, G.; Bektas, M.; Bielawska, A.; Riebeling, C.;
Hossini, A. M.; Eberle, J.; Hannun, Y. A.; Orfanos, C. E.; Geilen, C. C.
Bcl-2 overexpression prevents apoptosis induced by ceramidase
inhibitors in malignant melanoma and HaCaT keratinocytes. FEBS
Lett. 2002, 516, 47−52.
̈
(64) Knolker, H.-J.; Braxmeier, T.; Schlechtingen, G. A Novel method
for the synthesis of isocyanates under mild conditions. Angew. Chem.,
Int. Ed. 1995, 34, 2497−2500.
(65) Eckert, H.; Forster, B. Triphosgene, a crystalline phosgene
substitute. Angew. Chem., Int. Ed. 1987, 26, 894−895.
(66) Guha, S. K.; Wu, B.; Kim, B. S.; Baik, W.; Koo, S. TMS·OTf-
catalyzed α-bromination of carbonyl compounds by N-bromosuccini-
mide. Tetrahedron Lett. 2006, 47, 291−293.
(49) Proksch, D.; Klein, J. J.; Arenz, C. Potent inhibition of acid
ceramidase by novel B-13 analogues. J. Lipids 2011, 2011, 971618.
(67) Dementiev, A.; Joachimiak, A.; Nguyen, H.; Gorelik, A.; Illes, K.;
Shabani, S.; Gelsomino, M.; Ahn, E.-Y. E.; Nagar, B.; Doan, N.
Molecular mechanism of inhibition of acid ceramidase by carmofur. J.
Med. Chem. 2019, 62, 987−992.
́
́
(50) Camacho, L.; Meca-Cortes, O.; Abad, J. L.; García, S.; Rubio, N.;
̀
́
Díaz, A.; Celia-Terrassa, T.; Cingolani, F.; Bermudo, R.; Fernandez, P.
L.; Blanco, J.; Delgado, A.; Casas, J.; Fabrias, G.; Thomson, T. M. Acid
ceramidase as a therapeutic target in metastatic prostate cancer. J. Lipid
̀
(68) Ishikawa, M.; Hashimoto, Y. Improvement in aqueous solubility
in small molecule drug discovery programs by disruption of molecular
planarity and symmetry. J. Med. Chem. 2011, 54, 1539−1554.
(69) Gorelik, A.; Gebai, A.; Illes, K.; Piomelli, D.; Nagar, B. Molecular
mechanism of activation of the immunoregulatory amidase NAAA.
Proc. Natl. Acad. Sci. U.S.A. 2018, 115, E10032−E10040.
Res. 2013, 54, 1207−1220.
́
̃
(51) Ordonez, Y. F.; Abad, J. L.; Aseeri, M.; Casas, J.; Garcia, V.;
Casasampere, M.; Schuchman, E. H.; Levade, T.; Delgado, A.; Triola,
G.; Fabrias, G. Activity-based imaging of acid ceramidase in living cells.
J. Am. Chem. Soc. 2019, 141, 7736−7742.
AD
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