Lead Optimization of Benzoxazolone Carboxamides as Orally Bioavailable and CNS Penetrant Acid Ceramidase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b02004

Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex network of enzymatic pathways. A disturbance in these pathways leads to lipid accumulation and initiation of several SphL-related disorders. Acid ceramidase is one of the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which are important members of the SphL family. Herein, we describe the lead optimization studies of benzoxazolone carboxamides resulting in piperidine 22m, where we demonstrated target engagement in two animal models of neuropathic lysosomal storage diseases (LSDs), Gaucher's and Krabbe's diseases. After daily intraperitoneal administration at 90 mg kg^-1, 22m significantly reduced the brain levels of the toxic lipids glucosylsphingosine (GluSph) in 4L;C? mice and galactosylsphingosine (GalSph) in Twitcher mice. We believe that 22m is a lead molecule that can be further developed for the correction of severe neurological LSDs where GluSph or GalSph play a significant role in disease pathogenesis.

Full text of DOI:10.1021/acs.jmedchem.9b02004

Subscriber access provided by NATIONAL UNIV OF SINGAPORE
Article
Lead Optimization of Benzoxazolone Carboxamides as Orally
Bioavailable and CNS Penetrant Acid Ceramidase Inhibitors.
Simona Di Martino, Piero Tardia, Vincenzo Cilibrasi, Samantha Caputo, Marco Mazzonna,
Debora Russo, Ilaria Penna, Natalia Realini, NATASHA MARGAROLI, Marco Migliore, Daniela
Pizzirani, Giuliana Ottonello, Sine Mandrup Bertozzi, Andrea Armirotti, Duc Nguyen, Ying
Sun, Ernesto Bongarzone, Peter Lansbury, Min Liu, Renato T. Skerlj, and Rita Scarpelli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02004 • Publication Date (Web): 16 Mar 2020
Downloaded from pubs.acs.org on March 17, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Lead Optimization of Benzoxazolone Carboxamides
as Orally Bioavailable and CNS Penetrant Acid
Ceramidase Inhibitors.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Simona Di Martino,a,¥# Piero Tardia,a,¥# Vincenzo Cilibrasi,a,¥ Samantha Caputo,a,¥ Marco
Mazzonna,a,¥ Debora Russo, Ilaria Penna, Natalia Realini, Natasha Margaroli,a,¥ Marco
a,§
a,§
a,¥
Migliore,a,¥ Daniela Pizzirani,a,¥ Giuliana Ottonello,a,^ Sine Mandrup Bertozzi,a,^ Andrea
Armirotti,a,^ Duc Nguyen, Ying Sun, Ernesto R. Bongarzone, Peter Lansbury, Min Liu,
c
d
c
b
b
Renato Skerljb,†* and Rita Scarpellia,¥*
a
¥
Fondazione Istituto Italiano di Tecnologia, Drug Discovery and Development (D3)-
§
^
Validation, D3-Pharma Chemistry, Analytical Chemistry Lab, Via Morego 30, I-16163
Genova, Italy
bLysosomal Therapeutics Inc., 19 Blackstone Street, Cambridge, Massachusetts 02139, USA
cThe Myelin Regeneration Group at the Dept. Anatomy & Cell Biology, College of Medicine,
University of Illinois, Chicago. Chicago, IL. 60612, USA.
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dThe Division of Human Genetics, Cincinnati Children's Hospital Medical Center,
Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH
45229-3039, USA.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
KEYWORDS. Acid ceramidase inhibitors, lysosomal storage diseases, sphingolipids,
structure-activity relationship (SAR).
#
These authors contributed equally.
*Corresponding authors:
rita.scarpelli@iit.it; phone: +39 010 2896233
renato.skerlj@x4pharma.com; phone: +1 617 610 8624
ACS Paragon Plus Environment
2

Page 3 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
ABSTRACT
Sphingolipids (SphLs) are a diverse class of molecules that are regulated by a complex
network of enzymatic pathways. A disturbance in these pathways leads to lipid
accumulation and initiation of several SphL- related disorders. Acid ceramidase is one of
the key enzymes that regulate the metabolism of ceramides and glycosphingolipids, which
are important members of the SphL family. Herein, we describe the lead optimization
studies of benzoxazolone carboxamides resulting in piperidine 22m, where we
demonstrated target engagement in two animal models of neuropathic lysosomal storage
diseases (LSDs), Gaucher’s and Krabbe’s diseases. After daily intraperitoneal
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
administration at 90 mg Kg , 22m significantly reduced the brain levels of the toxic lipids
glucosylsphingosine (GluSph) in 4L;C* mice and galactosylsphingosine (GalSph) in
Twitcher mice. We believe that 22m is a lead molecule that can be further developed for
the correction of severe neurological LSDs, where GluSph or GalSph play a significant role
in disease pathogenesis.
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
INTRODUCTION
Sphingolipids (SphLs) are a large class of diverse amphipathic molecules found in
abundance in plasma membranes.1-2 Besides being important as structural cellular
components, SphLs play a central role in different biological processes, which are essential
to maintain the homeostasis and the development of eukaryotic cells. These processes
include signaling, angiogenesis, cell growth, proliferation and death, senescence,
inflammation, immune responses, metabolism, autophagy, brain development and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
function. Aided by recent technological advances, much has been accomplished in terms of
the identification of the basic biological components of the complex network in dynamic
and interconnected enzymatic pathways that regulate the biosynthesis of SphLs and the
formation of a variety of bioactive metabolites in distinct cellular compartments.1
In recent years, both academia and industry have shown a growing interest in advancing
our understanding of the multifaceted roles of SphL species under physiological and
2
pathological conditions. Collected evidence suggests that a disturbance between the
synthesis and catabolism of SphLs leads to their accumulation in specific cellular
compartments, such as the lysosomes, and the initiation of several SphL- related disorders.
3
Lysosomes are critical organelles responsible for cellular homeostasis. They contain
different degradative enzymes that can hydrolyze proteins, DNA, RNA, polysaccharides,
and lipids.4
Acid ceramidase (AC, also known as N-acylsphingosine amidohydrolase-1, ASAH-1) is a
lysosomal cysteine amidase that catalyzes the hydrolysis of ceramides (Cer) into fatty acids
and sphingosine, which is then converted into sphingosine 1-phosphate (Sph1P) by
sphingosine kinase.5-7 Cer and Sph1P are important members of the SphLs class and have
ACS Paragon Plus Environment
4

Page 5 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
opposing actions in the control of the cellular fate:8-10 while Cer mediate cellular
1
1
12-13
Sph1P promotes cell survival and proliferation.14-17 Recent
senescence and apoptosis,
studies have shown that AC is abnormally expressed in various types of human cancer (for
example, prostate, head and neck, colon and glioblastoma) and serum AC levels are
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
elevated in patients with melanoma relative to control subjects. Therefore, inhibition of
AC has been envisaged as a potential cancer drug target (Figure 1). Aberrant AC activity
has also been described in several other common diseases, including, inflammation, pain
and various pulmonary disorders.19-20
Over the recent years, the multifaceted catabolic role of AC has attracted much attention
for its potential therapeutic applications in many other altered conditions. Important
genetic studies have identified specific mutations in several genes that encode defective
expressions of some lysosomal enzymes as the causes of the onset and progression of
severe pathological conditions, called lysosomal storage diseases (LSDs).21-24 For example,
Gaucher’s disease (GD) is caused by a defective function of acid β-glucocerebrosidase
(GCase), a lysosomal membrane-associated protein responsible for the hydrolysis of
glucosylceramide (GluCer) to glucose and ceramides.25-27 Krabbe’s disease (KD) is
associated with defective -galactosyl-ceramidase (GALC) activity, a lysosomal enzyme
responsible for the hydrolysis of galactosylceramide (GalCer).28
As a result of either enzyme absences or deficiencies, these metabolic lysosomal
disorders are characterized by an abnormal storage of substrates or metabolites to
concentration levels that are toxic or otherwise detrimental to the cells in various
compartments, including the skeleton, skin, liver, spleen, lung, heart, and central nervous
system (CNS). The substrate or metabolite accumulations are believed to be responsible for
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
24
the disease progression. In GD patients, for example, the accumulation of GluCer (x 3 fold)
and/or glucosylsphingosine (GluSph) (x 200 fold) has been related to the brain
pathogenesis of neuronopathic GD patients, due to neuronal death, which is propagated by
the toxic effects of GluCer and /or GluSph.29-30 Recent evidence demonstrates an active role
of AC in an alternative catabolic pathway, which causes GluSph accumulation through the
deacylation of the lysosomal GluCer.31-32 In KD patients, deficiency of GALC activity leads to
accumulation of neurotoxic galactosylsphingosine (GalSph or psychosine) in tissues,
especially in the brain. It is possible that accumulation of GalSph mediates pathology of KD.
A very recent report suggests that genetic ablation of AC or pharmacological inhibition of
AC could eliminate psychosine accumulation and prolong the life span of Twitcher mice, a
model of KD.33-34 No approved therapeutic approaches are available to treat neuropathic
GD and KD; inhibiting AC may provide an efficacious strategy for treating these two
devastating diseases.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Efforts over the last decade to develop potent AC inhibitors, has resulted in limited
success. The first structural analysis of mammalian AC was recently solved by Gebai and
3
5
co-workers, which may aid future medicinal chemistry programs. In 2013, Realini et al.
reported the discovery of carmofur 1 and some close uracil analogs, as the first class of
single-digit nanomolar inhibitors of intracellular AC activity and studied their potential use
as chemosensitizing agents (Figure 2).36-37 Despite being potent AC inhibitors, the uracil
derivatives suffered from low chemical and metabolic stability. Subsequently, Diamanti et
al. selected compound 1 as a ligand template for a computational-assisted virtual screening
approach, leading to the identification of a new class of potent AC inhibitors, the pyrazole
carboxamides.38 However, although very potent against AC activity, as exemplified by
ACS Paragon Plus Environment
6

Page 7 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
pyrazole 3, this class of molecules suffered from low metabolic stability, limiting their
therapeutic potential (Figure 2).
An alternative approach, consisting of a screening campaign of a small compound library,
led to the identification of a novel and very promising class of covalent AC inhibitors, the
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
9
benzoxazolone carboxamides (BOAs), exemplified by the hit 2a (Figure 2). Preliminary
chemical exploration of this series led to the identification of 2b39 and 2c40 as more
advanced and systematically active analogs. More recently, Ortega et al. reported a
systematic computational investigation of the general pharmacophore model for AC
inhibition, comprising a 6+5 fused ring heterocycle linked to an aliphatic substituent via a
urea moiety. These studies resulted in the identification of the novel class of benzimidazole
derivatives 4a-d with promising activity in different melanoma cell lines (Figure 2).41
Although some of the molecules discussed above exhibited potent inhibitory effect
toward AC, they generally suffer from low aqueous solubility and moderate chemical or
metabolic stability, which hamper their further development. As part of our continued
efforts to optimize the class of BOAs, we further extended the preliminary studies around
2
b39 (and 2c)40 and performed a focused structure-activity relationship (SAR) study
around this scaffold (compound 5, Figure 2), with the aim of identifying an optimal
compound with improved physicochemical and pharmacokinetic profiles favoring oral
administration. The subject of this manuscript describes the lead optimization and
medicinal chemistry strategies that led to the discovery of 22m as a lead candidate with
improved oral bioavailability and excellent distribution to the CNS.
CHEMISTRY
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
All target compounds were prepared by synthetic routes outlined in Schemes 1-11.
Compounds 8a-d were synthetized under standard conditions by reacting 7a-d with 4-
phenylbutyl isocyanate (Scheme 1). The novel core scaffold of 12a was prepared in three
steps from the -bromo ketone 9 (Scheme 2A). Reaction with TZD gave compound 10,
which was then converted in moderate yield to the fused bicyclic derivative 11 via an
intramolecular cyclization under basic condition in anhydrous THF. Subsequent coupling of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1 to 4-phenylbutyl isocyanate gave 12a which upon removal of the N-Boc protecting
group, gave the key intermediate 12b which was subsequently transformed to 12c-e, via
standard reductive amination and acetylation reactions. Alternatively, the isomeric key
intermediate 17b was prepared in four steps from the commercially available epoxide 13
4
2
(Scheme 2B). Ring opening and subsequent oxidation of the corresponding alcohol 14a,
followed by intramolecular cyclization of 15 afforded compound 16. Finally, as discussed
for the synthesis of 12b, standard reactions transformed 16 to 17b.
We introduced cyclic and heterocyclic groups at C(5)-, C(6)- and C(7)- positions of the
benzoxazolone cores by exploring different synthetic pathways (Schemes 3-11). The
exploration at the C(4)- position of the benzoxazolone scaffold was abandoned, because, in
accordance with previously reported results on the 4-Me and 4-Ph derivatives of 2a (Figure
2
),40 we experienced a pronounced chemical instability of our targeted C(4)-derivatives.
The C(6)- substituted benzoxazolones 21a-d were prepared in three steps starting from
boronic esters 18a-c, using Pd-catalyzed cross coupling reactions with the corresponding
bromo-nitrophenols, followed by hydrogenation and intramolecular cyclization in the
presence of CDI (Scheme 3). An additional step consisting of the in situ formation of
boronic ester 29b, from ketone 28 via enol triflate 29a, was necessary for the preparation
ACS Paragon Plus Environment
8

Page 9 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
of the benzoxazolone 21l (Scheme 5). A Pd-catalyzed cross coupling procedure was also
used for the synthesis of the C(5)- substituted benzoxazolone 33 (Scheme 6) and other
C(6)- substituted benzoxazolones, such as 37a and 41a (Schemes 7-8). In contrast, to
overcome some synthetic problems in the Pd-catalyzed cross coupling reaction, we
performed an alternative synthetic approach for the preparation of the benzoxazolone 21m
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
3
(Scheme 9A). Lithium-halogen exchange of 6-bromo-3H- 1,3-benzoxazol-2-one, followed
by the addition of the ketone 42a afforded the alcohol 43, which upon dehydration and
hydrogenation, led to the key intermediate 21m. A similar synthetic procedure was applied
to insert the functionalization at the C(7)- position of the benzoxazolone, as in 47 (Scheme
9
B).
Other C(6)-substituted benzoxazolones, as examples 23a-d and 50j (Schemes 10 and 11),
were prepared in three steps and in satisfactory yield using a nucleophilic aromatic
4
4
substitution (SNAr) reaction of activated fluoro-phenyls with a set of heterocyclic amines,
followed by hydrogenation and intramolecular cyclization reaction with CDI. An alternative
approach was used for the synthesis of 50k (Scheme 11). In this case, a Cu-catalyzed cross
coupling N -arylation of O-Bn protected bromo-nitrophenol 51b with 4-methylpiperazin-2-
one afforded 52b in acceptable yield,45 which upon standard reactions, led to the
benzoxazolone 50k.
Finally, the carboxamide functionalities were introduced by standard conditions, which
involved the reaction of the benzoxazolone intermediates with the corresponding
commercially available isocyanates, as, for example, in the preparation of 22a-c (Schemes
3
) and 23a-d (Scheme 10). Alternatively, the isocyanates were prepared in situ, upon
activation of the corresponding amines by reaction with Boc O in the presence of DMAP in
2
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
46
MeCN, as in the synthesis of 22j, l and 22n-o (Scheme 4) or by reaction with triphosgene
4
7
in the presence of Et N in DCM, as in the synthesis of 22m (Scheme 4).
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
RESULTS AND DISCUSSION
The four most potent classes of AC inhibitors described to date are illustrated in Figure 2.
Each class is defined by the presence of a common chemical warhead – the urea-like
functionality – that can covalently react with the catalytic cysteine (Cys-143) of AC to form
a thioester bond.35 It has been reported that carboxamides 2a39 and 4a-b41 form, upon
incubation experiments with the protein, the corresponding cysteine adducts. This was
recently confirmed by Dementiev and coworkers who described the crystal structural
analysis of the uracil 1 covalently bound to Cys-143 at 2.7 Å resolution.48
While potent and, in some cases, systemically active,39-40 these molecules share two
features that limit their use as oral drugs. First, the presence of a reactive warhead on the
molecular scaffolds described to date contributes to their chemical and metabolic
37
instability (e.g. uracil 1) and, secondly, the hydrophobic linear side chain, that ensures
target recognition and some degree of specificity, negatively affects their drug-likeness (e.g.
benzoxazolone 2a).39 Thus, the need for optimised AC inhibitors remains an important
issue to be addressed.49
As previously reported, preliminary structural modifications of 2a by variation of the
lateral-side chain of the urea functionality (Region A) and substitution of the
benzoxazolone moiety (Region B) led to the identification of 2b39 and 2c40 (Figure 3).
3
7
Despite good potency and enhanced drug-likeness compared to the previous uracil series,
compounds 2b-c suffer from low solubility in aqueous media, and moderate chemical and
ACS Paragon Plus Environment
1
0

Page 11 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
metabolic stability that limit their utility as oral drugs.39-40 To address these issues, our lead
optimization strategy focused on designing additional structural modifications on Region A
and B (compound 5, Figure 2) with the aim of improving the physicochemical and
metabolic properties, while maintaining inhibitory potency.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
We initially investigated modifications of the lateral side chain (Region A) of 2c
confirming that, as previously reported with 2a analogs,40 this region is involved in
lipophilic interactions important for target recognition (Figure 3). In fact, different
attempts to improve solubility and metabolic stability by reducing lipophilicity of the side
chain was detrimental regarding potency (Figure 3). Although, the removal of the phenyl
ring was tolerated, as for the n-pentyl analog 2e (hAC IC = 60 nM), no enhancement of
5
0
solubility was observed (< 1 M, PBS, pH 7.4). Replacement of one methylene unit with an
oxygen (e.g. ethers 2d and 2f-g) to increase the hydrophilicity significantly reduced the
inhibitory potency to the M range. A similar trend was observed for the corresponding
analogs in the 2b series (data not shown), indicating that the lipophilic side chain of the
urea was very likely occupying a hydrophobic pocket.
We then shifted our attention to the left-hand side (Region B) of the scaffold by
evaluating the replacement of the benzoxazolone moiety with some bioisosteric 6+5 fused
ring heterocyclic systems (Figure 3), alternative to those already reported by Ortega et al.41
However, both the isatin analog 8c and the oxindole analog 8d were inactive at
concentrations up to 10 M. We then investigated the bioisosteric insertion of an aza-group
in the phenyl ring of the benzoxazolone moiety and this change resulted in very potent
compounds. For example, compounds 8a and 8b gave hAC IC ’s of 6 and 3 nM,
5
0
39
respectively, compared to the earlier compound 2a, which has an hAC IC of 64 nM. We
5
0
ACS Paragon Plus Environment
1
1

Journal of Medicinal Chemistry
Page 12 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
envisaged that the insertion of a polar group on the left-hand side of the scaffold (Region B)
could have an impact on the solubility of this series in aqueous buffer (PBS, pH 7.4), but,
unfortunately, both 8a and 8b had very poor chemical stability in these conditions (t1/2 <15
min).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
These findings prompted us to evaluate the inhibitory potency of the fused bicyclic
derivatives 12b and 17b (Figure 3). We speculated that changing the left hand side leaving
group at the urea functionality could have an effect on the chemical stability of the scaffold.
Although, we generally observed a loss in potency to the sub-M range, regardless of the
substituent (12b-e) or the position of the nitrogen atom (17b), we were pleased to notice
that, as for 12b and 17b, this novel class of hAC inhibitors showed improved chemical
stability in PBS at pH 7.4 (t1/2 > 8 h) and improved aqueous solubility (82 M and 230 M,
respectively). Despite the novel chemotype of these AC inhibitors with promising
physicochemical properties, our attempts to improve the potency of this series were
unsuccessful (data not shown). In addition, although these compounds exhibited high
mouse plasma stability (e.g. t1/2 > 2 h, for 12b and 17b), this class of molecules also
suffered from poor mouse liver microsomal stability (t1/2 < 15 min).
Overall, these results confirmed the benzoxazolone moiety as a ‘privileged scaffold’ thus
focusing our SAR strategy on region B with the intention of reducing the lipophilicity by
replacing the phenyl ring of 2c at the C(6)-position with aliphatic heterocyclic rings (5,
2
Figure 2). We envisaged that the reduction of the number of sp -hybridized carbon atoms
and the insertion of heteroatoms in this region could improve the overall physicochemical
and metabolic stability of this class of inhibitors.50-51
ACS Paragon Plus Environment
1
2

Page 13 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
We were pleased to observe that both the cyclohexyl analog 22a and the
tetrahydropyrane analog 22b resulted in equipotent inhibition (hAC IC = 0.089 and 0.068
50

M, respectively) compared to the corresponding phenyl derivative 2c39 (Table 1 and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2). With these results in hand, we were then interested in evaluating the effect of
increasing the hydrophilicity by the addition of a polar basic amine, as in the piperidine
analogs 22d and 22e. With this modification, we observed that both compounds showed
only a slight loss of potency compared to the aliphatic analog 22a (hAC IC = 0.134 and
5
0
0.129 M, respectively). Encouraged by these results, we explored the effect of other N-
containing heterocyclic systems, such as the piperidine 23a, the morpholine 23b, the 1,1-
dioxothiomorpholine 23c and the piperazines 23e-f. Overall, this set of compounds showed
similar potency compared to the initial cyclohexyl analog 22a. Notably, the piperidine 23a
and the piperazine 23f were the most potent compounds, showing IC values of 0.080 M
5
0
and 0.116 M, respectively (Table 1). Based on these promising results, we selected the
piperidine and piperazine series, exemplified by 22d and 23e, respectively, as novel
scaffolds for further studies, exploiting the presence of a distal nitrogen atom as an anchor
point for additional structural modifications (Table 2). First, we evaluated the SAR
exploration around the piperidine series, by introducing both linear and branched alkyl
chains on the nitrogen atom, such as the ethyl 22f, isopropyl 22g and isobutyl 22h analogs
(Table 2). In general, no significant differences were observed on the inhibitory potency of
these derivatives, 22f-h almost being equipotent to the unsubstituted 22d. Moreover, the
removal of the basic center by the introduction of either an exocyclic (22i) or endocyclic N-
acyl group (22q) was tolerated, showing IC50 values of 0.064 M and 0.105 M,
ACS Paragon Plus Environment
1
3

Journal of Medicinal Chemistry
Page 14 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
respectively. These results further confirmed that different polar groups were tolerated in
this region of the scaffold.
The same strategy was applied to the piperazine series (Table 2). Specifically, both the
N–alkyl derivatives 23g-i and the piperazinones 23j-k resulted in more potent AC inhibition
compared to the parent 23e. For examples, the N-ethyl piperazine 23g and the
piperazinone 23k were almost 7-fold more potent than 23e (hAC IC50 = 0.363 M),
showing IC values of 0.056 and 0.052 M, respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
However, a comparison of the piperazine and piperidine series in terms of aqueous
kinetic solubility (PBS, pH 7.4), in vitro metabolism highlighted some significant
differences (Table 3). Interestingly, the piperidine analogs, bearing small linear alkyl
groups (22d-f), were highly soluble (kinetic solubility > 100 M), and, in some cases (22d),
had acceptable stability profiles both in mouse plasma and in liver microsomes. On the
other hand, the piperidine derivatives, bearing more sterically hindered lipophilic alkyl
groups, such as the isopropyl 22g and isobutyl 22h, or the acyls 22i and 22q suffered from
low solubility and, with the exception of 22h, poor stability in mouse plasma (Table 3).
Conversely, all the piperazine derivatives generally suffered from poor aqueous solubility,
poor microsomal and plasma stability (Table 3). As illustrative examples, the piperazines
2
3f-g showed poor solubility in water (< 1 M), rapid metabolism in liver microsomes and
poor plasma stability (m-plasma and m-liver microsomes, t1/2 = < 5 min). Some
improvement in microsomal stability was observed with the des-methylated 23e and with
2
3k, which bears a heterocyclic ring at a higher oxidative state compared to 23f.
With these results in hand, we focused our efforts on exploring regions A and B of the N-
methyl piperidine 22e, as reported in Table 4. In order to reduce the lipophilicity and
ACS Paragon Plus Environment
1
4

Page 15 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
improve metabolic stability of this scaffold, we followed different strategies: a) insertion of
a heteroatom, removal of the phenyl ring and reduction of the side chain length (Region A);
and b) removal of potential metabolic soft spots (Region A and B).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
An immediate loss in potency was observed with the removal of the lipophilic phenyl ring
(22k), or the insertion of an oxygen on the lateral chain (22j and 22l); whilst the
bioisosteric replacement of a fluorine on the distal phenyl ring resulted in 22o, being
almost equipotent to 22e (Table 4). Nonetheless, exploration of region A continued with
the insertion of branched alkyl groups. We were pleased that the isobutyl analog 22m (hAC
IC = 0.166 M) was equipotent to the corresponding butyl phenyl 22e, demonstrating
5
0
that it was possible to remove the phenyl group and reduce the overall lipophilicity without
compromise potency. On the other hand, a methyl group adjacent to the urea functionality,
such as the sec-butyl analog 22n, was detrimental for potency, with an IC50 of 2.1 M.
Moving the SAR exploration back to region B, insertion of a fluorine on the benzoxazolone
ring 22p boosted the inhibitory potency (IC = 0.024 M), while the difluoro ethyl analog
5
0
2
2r showed similar potency (IC50 = 0.095 M) compared to 22e. The kinetic aqueous
solubility and in vitro metabolic stability of a selection of compounds in the piperidine
series are summarized in Table 4. Notably, while the insertion of an oxygen did not affect
either the solubility or metabolic stability in microsomes of 22j compared to 22e, reducing
lipophilicity with small aliphatic groups (22k and 22m) was particularly beneficial. For
example, 22k and 22m, showed high aqueous solubility (240 M) and improved plasma
and liver microsomal stabilities (t1/2 > 60 min). On the other hand, attempts to improve the
liver microsomal stability of 22e by inserting a fluorine atom at different potential
metabolic soft spots of both Region A and B (compounds 22o-p, 22r) was not successful.
ACS Paragon Plus Environment
1
5

Journal of Medicinal Chemistry
Page 16 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Not surprisingly, these bioisosteric replacements negatively affected the aqueous solubility
of 22o-p, 22r, without a substantial improvement of the metabolic stability in microsomes.
With these results in hand, SAR studies continued on the scaffold of compound 22m
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Table 5). Specifically, we evaluated the effect of the location of both - the N-methyl
piperidine ring -at C(5) and C(7) positions of the benzoxazolone moiety (compounds 24c
and 25) and - the N-methylated nitrogen atom- within the piperidine nucleus (compounds
2
6 and 27). Overall, we generally observed a loss in the inhibitory potency of these
targeted analogs compared to 22m, which was even more pronounced with compounds 26
and 27, showing IC values in the M range. Finally, the evaluation of the kinetic aqueous
5
0
solubility and in vitro metabolism of 22m and close analogs was completed (Table 5). In
general, all the targeted compounds showed high solubility values in aqueous media,
except for 24c, which bears the piperidine ring at the C(5) position of the benzoxazolone
system. On the other hand, major differences were observed comparing their metabolic
stability properties. In particular, we observed that substitution at the C(6) position was
critical to maintaining acceptable mouse plasma and liver microsomal stabilities
(compound 22m, m-plasma t1/2 = 80 min and m-liver microsomes t1/2 > 60 min (76%
remaining at 1 h)). On the other hand, both derivatives with the piperidine ring at the C(5)
and C(7) positions, 24c and 25 showed reduced mouse plasma and liver microsomal
stability. A similar trend was observed by moving the nitrogen atom to a different position
on the piperidine ring, except for 27, which showed a similar liver microsomal stability
compared to 22m. Due to its inhibitory potency and improved overall drug-likeness profile,
the piperidine 22m was selected for further biological and pharmacological investigations.
ACS Paragon Plus Environment
1
6

Page 17 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
We firstly envisaged that the inhibition of 22m, belonging to the same class of the
3
9
benzoxazolone carboxamide 2c, should occur through the same covalent AC modification.
According to our hypothesis, the corresponding benzoxazolone 21g (Scheme 4), tested at 1
and 10 M, was not able to inhibit hAC, due to the lack of the reactive urea -like
functionality. Moreover, kinetic studies on hAC –enriched lysates showed that 22m causes
a concentration-dependent reduction in the maximal catalytic velocity of AC (Vmax) without
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
influencing the Michaelis-Menten constant (K ) (Figure 4B and Table S1) and time
M
-1
-1
dependent inhibition at different 22m concentrations with a k /K = 0.02 M min and k
i
I
i
-1
=
0.15 min (Figure 4C and Figure 4D), suggesting a very fast covalent bond formation to
the enzyme.52-53
The selectivity of 22m was evaluated against a set of related lysosomal enzymes. The
compound showed only a weak inhibitory effect (IC50 = 8.0 M) on human N-
acylethanolamine acid amidase (hNAAA), a lysosomal cysteine amidase that shares 33–
54
3
4% sequence identity and a very similar reactive site with AC. 22m had no effect at the
concentrations tested (1 and 10 µM) on the activity of either acid sphingomyelinase (ASM)
and GCase. We next assessed the selectivity of 22m against two of the most representative
members of serine hydrolases, human fatty acid amide hydrolyase (FAAH)55 and
5
6
monoacylglycerol lipase (MAGL) : 22m showed inhibitory activity on FAAH with an IC of
5
0
0.070 M and no effect on monoacylglycerol lipase (MAGL) at the concentrations tested (1
and 10 µM). Although off-target activity of 22m against FAAH is observed, to our
knowledge, no evidence for biological cross-talk between the sphingolipid-signaling
2
55, 57
pathways and the FAAH-signaling pathway
has been reported that could preclude
further development of 22m.
ACS Paragon Plus Environment
1
7

Journal of Medicinal Chemistry
Page 18 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The favorable overall profile of 22m prompted us to test its ability to inhibit AC in intact
cells. Human neuroblastoma SH-SY5Y cells were incubated in the presence of 22m at
different doses (1, 2.5, 5, 10 M). AC activity was measured with a liquid
chromatography/mass spectrometry (LCMS)-based activity assay after different
incubation times (30 min, 1, 3, 6 h) and SphL levels were identified and quantified by
LC/MS, showing that 22m effectively engages AC in these cells leading to the expected
variations in the SphL levels, as reported in Figure 5 and 6. Treatment of cultures of human
neuroblastoma SH-SY5Y cells with 22m caused a concentration (Figure 5A) and time
dependent reduction of AC activity (Figure 6A). After 3 h incubation, this effect resulted in
an intracellular accumulation of various ceramide species, including Cer (d18:0/16:0) and
Cer (d18:1/16:0) (Figure 5B-C) and a corresponding decrease in the levels of sphingosine
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Figure 5D) in a concentration dependent manner. The effect of 22m (10 M) on AC
activity inhibition and SphL persisted for up to 6 h under our experimental conditions
(Figure 6B-D). The results indicated that 22m inhibits AC in the complex cellular
environment leading to an increased Cer (d18:0/16:0) and Cer (d18:1/16:0) (Figure 6B-C)
and decreased sphingosine levels with a partial recovery of sphingosine levels after 3-6 h
(Figure 6D). Conversely, as expected, no major variations were observed in the levels of
sphingomyelin (SM) (d18:1/16:0) (Figure 5E and 6E) and hexosylceramide (HexCer)
(d18:1/16:0) (Figure 5F and Figure 6F).
Pharmacokinetic studies of 22m were determined in CD1 mice and relevant
pharmacokinetic parameters are reported in Table 6. Values of plasma clearance (Clp),
volume of distribution (Vd ) and plasma half-life (t ) were calculated after intravenous
ss
1/2
-1
administration of 22m at 3 mg Kg . Clearance was moderately high (14.1 L/h/Kg), with a
ACS Paragon Plus Environment
1
8

Page 19 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
relative short plasma half-life (1 h) and high Vdss (12.5 L/Kg) indicating 22m well
distributed out of the circulating plasma compartment. Good oral bioavailability was
-1
observed dosing 22m at 10 mg Kg (F = 58%), with significant exposures in plasma, brain
and cerebrospinal fluid (CSF) (AUC values = 412, 14648 and 119 (h x ng/mL),
respectively). A maximum tolerated dose (MTD) study in mice was also conducted in the
same background as the pharmacodynamic model using C57BL/6 mice at intraperitoneal
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
dose escalation of 20, 40, 80, and 120 mg Kg during a time range of 4 days and no clinical
abnormalities were observed in any animals within the doses and time range used.
Based on these results, we decided to study the effect of dosing 22m in 4L;C* mice, a
validated genetic mutated animal model for neuropathic GD.58 4L;C* mice have marked
increase (20- to 30-fold) of GluSph and moderate elevation (1.5- to 3-fold) of GluCer in the
brain, therefore they are a unique model suitable for testing GluSph reduction therapy.
-1
2
2m was administered at the selected doses of 30 and 90 mg Kg by intraperitoneal
injection (i.p.), once a day for 14 days starting at postnatal day 5. Preliminary results
showed that compound 22m significantly reduces GluSph (d18:1) in the brain of 4L;C*
mice in a dose dependent manner (Figure 7). Target engagement was demonstrated at the
-1
high dose of 90 mg Kg with 54% reduction of the GluSph levels relative to control.
Next, we evaluated 22m in Twitcher mouse, an animal model of Krabbe’s disease. The
Twitcher mice naturally carry a GALC mutation that contains a premature stop codon in
GALC and leads to a complete loss of GALC activity. As a result, dramatic increase of the
extremely toxic lipid GalSph is observed in Twitcher mouse brains. After i.p. administration
-1
at 30 and 90 mg Kg once daily for a treatment period of 20 days starting at postnatal day
ACS Paragon Plus Environment
1
9

Journal of Medicinal Chemistry
Page 20 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
0, 22m showed dose dependent reduction of the toxic lipid GalSph (d18:1) levels in the
brain of Twitcher mice by 72% and 41% at the high and low doses, respectively (Figure 8).
-1
In the group of 4L;C* mice at 90 mg Kg doses, the unbounded drug levels in the brain 1 h
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
post last dose (day 14) are 2.6 M (6.4- fold higher than the EC value) (Table 7); whilst at
5
0
-1
the lower dose of 30 mg Kg the unbounded drug levels are 0.77 M (1.9- fold higher than
-1
the EC value). In the group of Twitcher mice at 90 mg Kg doses, the unbounded drug
5
0
levels in the brain 1 h post last dose (day 20) are 2.15 M (5.2- fold higher than the EC50
-1
value); whilst at the lower dose of 30 mg Kg the unbounded drug levels are 0.80 M (2.0-
fold higher than the EC value). Overall, these data support the observed dose responses in
5
0
the two animal models.
To our knowledge, this is the first report showing the efficacy of inhibiting AC on
reducing the neurotoxic lipids GluSph in the brains of 4L;C* mice. Our result that inhibiting
AC reduces neurotoxic lipid GalSph level in the brains of Twitcher mice is consistent with
the recent report.34
Further pharmacological studies of 22m will be reported in due course.
CONCLUSIONS
The present work outlines the lead optimization studies of a class of benzoxazolone
carboxamides as AC inhibitors. We further extended the preliminary studies around 2b
(
and 2c)39-40 and performed a focused structure-activity relationship (SAR) study on
Region A and B of this scaffold with the aim of improving the physicochemical and
metabolic properties of the series, while maintaining the inhibitory potency. Introduction
of different heterocyclic groups on the benzoxazolone moiety were tolerated regarding
ACS Paragon Plus Environment
2
0

Page 21 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
inhibitory potency, as for the tetrahydropyrane 22b, the piperidines 22d, 23a and the
piperazines 23e, 23f. A more focused exploration around 22d and 23e by changing the
nature of substitution on the distal nitrogen atom led to the identification of novel potent
analogs with improved solubility, as, for example, the piperidines 22e and 22f. Targeted
modifications on different positions of Region A and B of the N-methylated piperazine
series led to compound 22m as a potent and oral bioavailable AC inhibitor with excellent
brain penetration in mice. Preliminary results demonstrated target engagement of 22m
both in the 4L;C* and Twitcher mouse models, where dose dependent reductions in GluSph
and GalSph were observed, supporting further optimized AC inhibitors may be used in the
correction of severe pathological neurological states of LSD, where these toxic lipids may
play a significant role in the pathology, such as GD and KD.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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 SiO columns of different sizes (from 4 g until 40 g). Mixtures of increasing polarity
2
of Cy and EtOAc or DCM and MeOH were used as eluents. TLC analyses were performed
using Supelco on TLC Al foils 0.2 mm with fluorescence indicator 254 nm. Purifications of
basic compounds were done using IST ISOLUTE® SCX packed into SPE cartridges (SCX).
Hydrogenation reaction were performed using H-Cube continuous hydrogenation
equipment (SS-reaction line version), employing disposable catalyst cartridges (CatCart)
preloaded with the required heterogeneous catalyst. Microwave heating was performed
ACS Paragon Plus Environment
2
1

Journal of Medicinal Chemistry
Page 22 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
using Explorer®-48 positions instrument (CEM). NMR experiments of all the intermediates
1
and final compounds were run on a Bruker Avance III 400 system (400.13 MHz for H, and
1
3
100.62 MHz for C), equipped with a BBI probe and Z-gradients. Spectra were acquired at
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
00 K, using deuterated dimethylsulfoxide (DMSO-d ) or deuterated chloroform (CDCl ) as
6
3
1
13
solvents. Chemical shifts for H and C spectra were recorded in parts per million using the
1
residual non-deuterated solvent as the internal standard (for DMSO-d : 2.50 ppm, H; 39.52
6
ppm, 13C; for CDCl : 7.26 ppm, H and 77.16 ppm, 13C). Data are reported as follows:
1
3
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
1
constants (J) in Hertz (Hz) and integrated intensity. Quantitative H-NMR analyses of the
freshly prepared 10 mM DMSO-d stock solutions (used for biological screenings) of the
6
final compounds were performed using 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 a Single
Quadrupole Detector (SQD) Mass Spectrometer (MS) equipped with an Electrospray
Ionization (ESI) interface and a Photodiode Array Detector (PDA). PDA range was 210-400
nm. Analyses were performed on an ACQUITY UPLC BEH C18 column (50 x 2.1 mm ID,
particle size 1.7 µm) with a VanGuard BEH C18 pre-column (5x 2.1 mmID, particle size 1.7
µm). Mobile phase was 10 mM NH OAc in H O at pH 5 adjusted with AcOH (A) and 10 mM
4
2
NH OAc in MeCN-H O (95: 5) at pH 5 (B). ESI in both positive and negative modes was
4
2
used in the mass scan range 100-650Da. Analyses were performed with method A, B, C or
ACS Paragon Plus Environment
2
2

Page 23 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
D. Method A: Gradient 5 to 95% B over 2.5 min. Flow rate 0.5 mL/min. Temperature 40 °C.
Method B: Gradient 50 to 100% B over 2.5 min. Flow rate 0.5 mL/min. Temperature 40 °C.
Method C: Gradient 0 to 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 the final compounds were performed with method E or F using
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
freshly prepared 10 mM DMSO-d stock solutions (used for biological screenings), diluted
6
2
0- fold or 100 fold in MeCN/H O (1:1) and directly analyzed. An ACQUITY UPLC BEH C18
2
(100x 2.1 mmID, particle size 1.7µm) with a VanGuard BEH C18 pre-column (5x 2.1 mm ID,
particle size 1.7 µm). Mobile phase was 10 mM NH OAc in H O at pH 5 adjusted with AcOH
4
2
(
A) and 10 mM NH OAc in MeCN-H O (95: 5) at pH 5 (B). ESI in both positive and negative
4 2
modes was used in the mass scan range 100-650Da. Method E: Gradient: 10 to 90% B over
min. Flow rate 0.5 mL/min. Temperature 40 °C. Method F: Gradient: 50 to 100% B over 6
6
min. Flow rate 0.5 mL/min. Temperature 40 °C. The detection wavelength ( was set at
215 nm for relative purity determination. Rt of the final compounds are reported in Table
S2. Accurate mass measurements were performed on a Synapt G2 Quadrupole-ToF
Instrument (Waters, USA), equipped with an ESI ion source; compounds were diluted to 50
µM in H O/ MeCN and analyzed. Leucine Enkephalin (2 ng/mL) was used as lock mass
2
reference compound for spectra recalibration. All final compounds displayed ≥ 95% purity
as determined by NMR and UPLC/MS analysis.
General procedure for palladium-catalyzed cross coupling reaction (Procedure A).
To a solution of the appropriate phenyl bromide (1.0 eq.) in dry 1,4-dioxane (0.5 M,
previously degassed under nitrogen atmosphere) was added the appropriate boronic acid
or its corresponding boronic ester (1.1 eq.), followed by the addition of Pd(PPh ) or
3
4
ACS Paragon Plus Environment
2
3

Journal of Medicinal Chemistry
Page 24 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Pd(dppf)Cl (0.05- 0.2 eq.) and 2M Na CO (2.5 eq.). The dark reaction mixture was stirred
2
2
3
at reflux for 15 h, then diluted with EtOAc and filtered through a pad of Celite. The filtrate
was concentrated under reduced pressure and the crude was purified by column
chromatography, eluting with Cy/ EtOAc as indicated in each case.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General procedure for catalytic hydrogenation reaction (Procedure B).
Method A: To a suspension of the appropriate 2-nitrophenol (1.0 eq.) in MeOH, or EtOH,
or EtOAc (0.4 M) were added 10% Pd/C (0.25 eq.) and cyclohexene (30 eq.) and the
reaction mixture was stirred at reflux 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 used in the next
step without further purification.
Method B: A suspension of the appropriate 2-nitrophenol (1.0 eq.) in MeOH (0.4 M) was
hydrogenated with the H-Cube apparatus using 10% Pd/C at 60 °C and full H mode. After
2
complete conversion (UPLC/MS analysis monitoring), the solvent was evaporated under
reduced pressure. The crude was used in the next step without further purification.
General procedure for intramolecular cyclization using CDI (Procedure C).
To a solution of the appropriate 2-amino phenol (1.0 eq.) in MeCN (0.1M) was added CDI
(1.0 – 1.5 eq.). The reaction mixture was stirred at rt for 2 h. Then, the solvent was
evaporated under reduced pressure and the crude was re-dissolved in EtOAc, washed with
H O, brine and dried over Na SO . After evaporation of the solvent, the crude was purified
2
2
4
by column chromatography, eluting with Cy/ EtOAc, or DCM/ MeOH, or used in the next
step without further purification, as indicated in each case.
General Procedure for carboxamide synthesis (Procedure D).
ACS Paragon Plus Environment
2
4

Page 25 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Method A: To a stirred solution of the appropriate oxazolone (1.0 eq.) and DMAP (1.1 eq.)
in dry MeCN was added the appropriate isocyanate (1.1- 3.0 eq.). The reaction mixture was
stirred at rt for 30 min under nitrogen atmosphere. After evaporation of the solvent, the
crude was purified by column chromatography, eluting with Cy/ EtOAc or DCM/ MeOH as
indicated in each case.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Method B: To a stirred solution of triphosgene (0.33 eq.) in dry DCM (0.2 M) were added
the appropriate amine (1.5- 3.0 eq.) and dry Et N (3.0 eq.) at -15 °C. The resulting mixture
3
was stirred at rt for 30 min under nitrogen atmosphere and then added to a solution of the
appropriate oxazolone (1.0 eq.) and Et N (1.0 eq.) in dry DCM. The reaction mixture was
3
stirred at rt for 30 min under nitrogen and then diluted with DCM. The organic phase was
washed with saturated aqueous NH Cl solution, brine and dried over Na SO . After
4
2
4
evaporation of the solvent, the crude was purified by column chromatography, eluting with
Cy/ EtOAc, or DCM/ MeOH, as indicated in each case.
Method C: To a stirred solution of Boc O (2.0 eq.) in MeCN (0.4 M) were added DMAP (2.0
2
eq.) and the appropriate amine (2.0 eq.). The resulting solution was stirred at rt for 10 min
then the appropriate oxazolone derivative (1.0 eq.) was added and the mixture was stirred
at rt for 1 h. After evaporation of the solvent, the crude was purified by flash column
chromatography, eluting with Cy/ EtOAc, or DCM/ MeOH, as indicated in each case.
General procedure for N-Boc removal (Procedure E).
To a suspension of the appropriate N-Boc protected derivative (1.0 eq.) in 1,4-dioxane or
DCM (0.1 M) HCl (30 eq., 4M in 1,4-dioxane) was added and the reaction mixture was
stirred at rt for 2 h. After evaporation of the solvent, the crude was triturated with Et O or
2
used in the next step without further purification, as indicated in each case.
ACS Paragon Plus Environment
2
5

Journal of Medicinal Chemistry
Page 26 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
General procedure for reductive amination reaction (Procedure F).
To a solution of the appropriate secondary amine (1.0 eq.) in MeCN, or THF (0.1 M) were
added the appropriate aldehyde or ketone (1.6– 5.0 eq.), AcOH (1.6 -5.0 eq.) and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NaBH(OAc) (1.6- 3.0 eq.). The mixture was stirred at rt for 2-16 h under nitrogen
3
atmosphere. Then, the reaction mixture was poured into saturated aqueous NaHCO3
solution and extracted with EtOAc. The organic phase was washed with brine and dried
over Na SO . After evaporation of the solvent, the crude was purified by SCX.
2
4
General procedure for nucleophilic aromatic substitution reaction (SNAr) (Procedure G).
To a solution of the appropriate 4-fluoro nitrobenzene (1.0 eq.) in MeCN were added the
appropriate amine (2.0 eq.) and DIPEA (2.0 eq.). The reaction mixture was refluxed (or
stirred under MW irradiation, 90 °C, power 200W) until disappearance of the starting
material, as indicated by UPLC/MS analysis. After evaporation of the solvent, the crude was
purified by flash column chromatography, eluting with Cy/ EtOAc, or DCM/ MeOH, as
indicated in each case.
General procedure for intramolecular cyclization under basic condition (Procedure H)
To a solution of the appropriate thiazolidinedione derivative (1.0 eq.) in dry THF (0.1 M)
was added tBuOK (2.0- 4.0 eq.) at rt under nitrogen atmosphere. After 30 min, the reaction
mixture was diluted with EtOAc, washed with saturated aqueous NH Cl solution, brine and
4
dried over Na SO . After evaporation of the solvent, the crude was used in the next step
2
4
without further purification.
General procedure for lithium/halogen exchange - addition reaction (Procedure I).
To a solution of the appropriate bromobenzoxazolone (1.0 eq.) in dry THF (0.1M) was
added MeMgBr (1.5 eq., 3.0 M in Et O) at -78 °C under nitrogen atmosphere for 30 min,
2
ACS Paragon Plus Environment
2
6

Page 27 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
followed by the addition of n-BuLi (1.2 eq., 2.5 M in hexanes). After 30 min, a solution of the
appropriate piperidone (1.7 eq.) in dry THF (0.7 M) was added dropwise at -78 °C under
nitrogen atmosphere and, then, the reaction mixture was allowed to warm to rt. After 30
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
min, the reaction was quenched by addition of saturated aqueous NH Cl solution, diluted
4
with EtOAc, washed with brine and dried over Na SO . After evaporation of the solvent, the
2
4
crude was purified by column chromatography, eluting with Cy/ EtOAc, or DCM/ MeOH, as
indicated in each case.
General procedure for dehydration reaction of tertiary alcohols (Procedure L).
To a suspension of the appropriate tertiary alcohol (1.0 eq.) in dry toluene (0.1 M) was
added p-TsOH (3.0 eq.) and the reaction mixture was stirred at reflux for 2 h. After
evaporation of the solvent, the crude was purified by SCX, or used in the next step without
further purification, as indicated in each case.
Synthesis
of
N-(2-benzyloxyethyl)-6-(4-fluorophenyl)-2-oxo-1,3-benzoxazole-3-
carboxamide (2d). Compound 2d was prepared according to the general procedure D
39
(
method B), using 6-(4-fluorophenyl)-3H-1,3-benzoxazol-2-one (0.060 g, 0.26 mmol), 2-
(
benzyloxy)-1-ethanamine hydrochloride (0.073 g, 0.39 mmol) and Et N (0.11 mL, 0.079 g,
3
0.78 mmol), in dry DCM (3 mL). The crude was purified by column chromatography (Cy/
1
EtOAc, 80: 20) to afford 2d as white solid (0.06 g, 57%). H NMR (600 MHz, CDCl ) δ 8.35
3
(bs, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.52 (dd, J = 8.8, 5.2 Hz, 2H), 7.43 (dd, J = 8.3, 1.7 Hz, 1H),
7.40 (d, J = 1.7 Hz, 2H), 7.39 – 7.33 (m, 4H), 7.28 (tt, J = 7.0, 1.6 Hz, 1H), 7.14 (t, J = 8.6 Hz,
13
2
H), 4.59 (s, 2H), 3.74 - 3.65 (m, 4H). C NMR (151 MHz, CDCl ) δ 162.82 (d, J = 247.5
3
C-F
Hz), 153.12, 149.94, 142.43, 137.91, 137.51, 136.18, 136.16, 128.86 (d, JC-F = 8.2 Hz),
128.62, 127.96, 127.95, 127.28, 123.87, 116.03 (d, JC-F = 21.4 Hz), 115.78, 108.57, 73.40,
ACS Paragon Plus Environment
2
7

Journal of Medicinal Chemistry
Page 28 of 126
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
6
4
4
8.26, 40.37. UPLC/MS (method A): Rt 2.78 min. MS (ES) C H FN O requires 406, found
2
3
19
2 4
+
+
07 [M+H] . HRMS C H FN O [M+H] : calculated 407.1407, measured: 407.1424, ppm
23 20
2 4
.2.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Synthesis of 6-(4-fluorophenyl)-2-oxo-N-pentyl-1,3-benzoxazole-3-carboxamide (2e).
Compound 2e was prepared according to the general procedure D (method A), using 6-(4-
fluorophenyl)-3H-1,3-benzoxazol-2-one (0.080 g, 0.35 mmol), 1-pentyl isocyanate (0.05
mL, 0.040 g, 0.39 mmol) in dry MeCN (3 mL). The crude was purified by column
1
chromatography (Cy/ EtOAc, 80: 20) to afford 2e as white solid (0.100 g, 82%). H NMR
(400 MHz, CDCl ) δ. 8.10 (d, J = 8.3 Hz, 1H), 8.04 (bs, 1H), 7.52 (dd, J = 8.6, 5.3 Hz, 2H),
3
7.47 – 7.42 (m, 1H), 7.41-7.38 (m, 1H), 7.14 (t, J = 8.6 Hz, 2H), 3.44 (q, J = 6.9 Hz, 2H), 1.66
13
(p, J = 7.2 Hz, 2H), 1.45-1.32 (m, 4H), 0.93 (t, J = 6.9 Hz, 3H). C NMR (101 MHz, CDCl ) δ
3
1
62.70 (d, J = 247.6 Hz), 153.23, 149.71, 142.28, 137.38, 136.06, 128.73 (d, J = 8.8 Hz),
C-F C-F
123.78, 116.00 (d, JC-F = 25.3 Hz), 115.75, 108.42, 99.96, 40.34, 29.12, 28.96, 22.30, 13.94.
+
UPLC/MS (method A): Rt 2.43 min. MS (ES) C H FN O requires 342, found 343 [M+H] .
1
9
19
2 3
+
HRMS C H FN O [M+H] : calculated 343.1458, measured: 343.1449, ppm -2.6.
1
9
20
2 3
Synthesis of N-(2-ethoxyethyl)-6-(4-fluorophenyl)-2-oxo-1,3-benzoxazole-3-carboxamide
2f). Compound 2f was prepared according to the general procedure D (method B), using
-(4-fluorophenyl)-3H-1,3-benzoxazol-2-one (0.130 g, 0.57 mmol), 2-ethoxyethylamine
0.09 mL, 0.080 g, 0.85 mmol) and Et N (0.20 mL, 0.140 g, 1.42 mmol) in dry DCM (15 mL).
(
6
(
3
The crude was purified by column chromatography (Cy/ EtOAc, 80: 20) to afford 2f as
1
white solid (0.03 g, 13%). H NMR (400 MHz, CDCl ) δ 8.32 (bs, 1H), 8.10 (d, J = 8.3 Hz,
3
1
H), 7.57-7.49 (m, 2H), 7.46 – 7.38 (m, 2H), 7.18 – 7.10 (m, 2H), 3.68 - 3.61 (m, 4H), 3.56
ACS Paragon Plus Environment
2
8

Page 29 of 126
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
(
q, J = 7.0 Hz, 2H), 1.24 (t, J = 7.0 Hz, 3H). C NMR (101 MHz, CDCl ) δ 162.69 (d, J
=
C-F
3
2
47.7 Hz), 153.16, 149.98, 142.47, 137.54, 136.20, 128.88 (d, JC-F = 8.2 Hz), 127.34, 123.89,
116.04 (d, JC-F = 21.7 Hz), 115.81, 108.60, 68.63, 66.81, 40.43, 15.25. UPLC/MS (method A):
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
Rt 2.56 min. MS (ES) C H FN O requires 344, found 345 [M+H] . HRMS C H FN O
1
8
17
2
4
18 18
2
4
+
[
M+H] : calculated 345.1251, measured: 345.1258, ppm 2.
Synthesis
of
6-(4-fluorophenyl)-N-(3-methoxypropyl)-2-oxo-1,3-benzoxazole-3-
carboxamide (2g). Compound 2g was prepared according to the general procedure D
(method B), using 6-(4-fluorophenyl)-3H-1,3-benzoxazol-2-one (0.08 g, 0.35 mmol), 3-
methoxypropylamine (0.06 mL, 0.050 g, 0.52 mmol) and Et N (0.12 mL, 0.09 g, 0.88 mmol)
3
in dry DCM (15 mL). The crude was purified by column chromatography (Cy/ EtOAc, 80:
1
20) to afford 2g as white solid (0.020 g, 18%). H NMR (400 MHz, CDCl ) δ 8.35 (bs, 1H),
3
8
7
.10 (d, J = 8.3 Hz, 1H), 7.52 (dd, J = 8.6, 5.3 Hz, 2H), 7.43 (dd, J = 8.4, 1.4 Hz, 1H), 7.41-
.39 (m, 1H), 7.14 (t, J = 8.6 Hz, 2H), 3.55 (dt, J = 16.4, 6.0 Hz, 4H), 3.39 (s, 3H), 1.92 (p, J =
6.1 Hz, 2H). 13C NMR (101 MHz, CDCl ) δ 162.67 (d, J = 247.4 Hz), 153.16, 142.84,
3
C-F
1
42.43, 137.44, 136.23, 128.70 (d, JC-F = 8.1 Hz, 2C), 127.40, 123.84, 116.12, 115.88 (d, JC-F
11.3 Hz, 2C), 108.53, 70.95, 58.95, 38.60, 29.28. UPLC/MS (method A): Rt 2.49 min. MS
=
+
+
(ES) C H FN O requires 344, found 345 [M+H] . HRMS C H FN O [M+H] : calculated
1
8
17
2
4
18 18
2 4
3
45.1251, measured: 345.1258, ppm 2.
Synthesis of 3H-oxazolo[4,5-c]pyridin-2-one (7a). Compound 7a was prepared according to
the general procedure C using 6a (0.10 g, 0.91 mmol) and CDI (0.290 g, 1.82 mmol, 2.0 eq.)
in a mixture of MeCN/ DMF (9 mL, 2: 1). The crude was triturated with DCM to afford 7a as
1
a whitish solid (0.100 g, 80%). H NMR (400 MHz, DMSO-d ) δ 8.34 (s, 1H), 8.32 (d, J = 5.3
6
ACS Paragon Plus Environment
2
9