Benzoxazolone Carboxamides as Potent Acid Ceramidase Inhibitors: Synthesis and Structure-Activity Relationship (SAR) Studies

Article abstract of DOI:10.1021/acs.jmedchem.5b01188

Ceramides are lipid-derived intracellular messengers involved in the control of senescence, inflammation, and apoptosis. The cysteine amidase, acid ceramidase (AC), hydrolyzes these substances into sphingosine and fatty acid and, by doing so, regulates their signaling activity. AC inhibitors may be useful in the treatment of pathological conditions, such as cancer, in which ceramide levels are abnormally reduced. Here, we present a systematic SAR investigation of the benzoxazolone carboxamides, a recently described class of AC inhibitors that display high potency and systemic activity in mice. We examined a diverse series of substitutions on both benzoxazolone ring and carboxamide side chain. Several modifications enhanced potency and stability, and one key compound with a balanced activity-stability profile (14) was found to inhibit AC activity in mouse lungs and cerebral cortex after systemic administration. The results expand our arsenal of AC inhibitors, thereby facilitating the use of these compounds as pharmacological tools and their potential development as drug leads.

Full text of DOI:10.1021/acs.jmedchem.5b01188

Subscriber access provided by EPFL | Scientific Information and Libraries
Article
Benzoxazolone Carboxamides as Potent Acid Ceramidase Inhibitors:
Synthesis and Structure-Activity Relationship (SAR) Studies
Anders Bach, Daniela Pizzirani, Natalia Realini , Valentina Vozella, Debora
Russo, Ilaria Penna, Laurin Melzig, Rita Scarpelli, and Daniele Piomelli
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01188 • Publication Date (Web): 11 Nov 2015
Downloaded from http://pubs.acs.org on November 12, 2015
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Benzoxazolone Carboxamides as Potent Acid
Ceramidase Inhibitors: Synthesis and Structureꢀ
Activity Relationship (SAR) Studies
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†
,
,‡
†,‡
†
†
†
⊥
Anders Bach
, Daniela Pizzirani, Natalia Realini, Valentina Vozella, Debora Russo, Ilaria
†
†,ǁ
†
†,§,
Penna, Laurin Melzig, Rita Scarpelli, Daniele Piomelli *
†
Drug Discovery and Development, Fondazione Istituto Italiano di Tecnologia, Via Morego 30,
Iꢀ16163 Genova, Italy
§
Departments of Anatomy and Neurobiology, Biological Chemistry and Pharmacology,
University of California, Irvine, California 92697ꢀ4625, United States
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 70
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
ABSTRACT
Ceramides are lipidꢀderived intracellular messengers involved in the control of senescence,
inflammation, and apoptosis. The cysteine amidase, acid ceramidase (AC), hydrolyses these
substances into sphingosine and fatty acid and, by doing so, regulates their signalling activity.
AC inhibitors may be useful in the treatment of pathological conditions, such as cancer, in which
ceramide levels are abnormally reduced. Here, we present a systematic SAR investigation of the
benzoxazolone carboxamides, a recently described class of AC inhibitors that display high
potency and systemic activity in mice. We examined a diverse series of substitutions on both
benzoxazolone ring and carboxamide side chain. Several modifications enhanced potency and
stability, and one key compound with a balanced activityꢀstability profile (14) was found to
inhibit AC activity in mouse lungs and cerebral cortex after systemic administration. The results
expand our arsenal of AC inhibitors thereby facilitating the use of these compounds as
pharmacological tools and their potential development as drug leads.
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
ACS Paragon Plus Environment

Page 3 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Acid ceramidase (AC) is a cysteine amidase involved in the metabolism of ceramides, bioactive
1
lipid molecules that belong to the class of sphingolipids. Sphingolipids act as cellular
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
messengers throughout the body, and are involved in the survival, growth and differentiation of
2
3
cells, as well as in pathophysiological process such as inflammation and neuropathic pain. AC
4
plays a pivotal role in balancing sphingolipidꢀmediated signalling. The enzyme is mainly, albeit
5
not uniquely located in lysosomes, where it cleaves the amide bond of ceramides producing the
aliphatic amino alcohol sphingosine along with fatty acid (typically 14ꢀ26 carbon atoms in
length) (Figure 1). Sphingosine is a metabolic precursor of sphingosineꢀ1ꢀphosphate, which
6
enhances cell survival and proliferation, while ceramides exert proꢀsenescent and proꢀapoptotic
4
, 6c, 7
effects in both normal and tumor cells.
Interestingly, ceramide concentrations increase in
7f
cells that are stressed with chemotherapeutic agents, DNA damage and ionizing radiation,
either through stimulation of de novo ceramide biosynthesis, hydrolysis of sphingomyelin or the
1
a
salvage pathway; moreover, the cytotoxic effects of certain anticancer drugs partly depend on
8
9
de novo ceramide biosynthesis. AC is overꢀexpressed in several cancer types and in prostate
cancer this upꢀregulation renders cells more resistant to chemotherapy and radiotherapy, while
1
0
genetic or pharmacological AC inhibition restores sensitivity to therapy in vivo. Thus,
modulating the ceramide/sphingosineꢀ1ꢀphosphate axis by inhibiting AC could provide a new
strategy for future therapeutics against cancer and, possibly, other pathologies in which ceramide
metabolism is dysregulated. To test this hypothesis, several AC inhibitors have been developed
1
1
over the past decades.
We have recently disclosed a small set of benzoxazolone carboxamides as the first example of
12
systemically active inhibitors of intracellular AC activity.
We demonstrated that these
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 70
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
molecules covalently inhibit AC through Sꢀacylation of its catalytic nucleophile, Cysꢀ143, with
the benzoxazolone ring acting as leaving group. Preliminary structureꢀactivity relationship
(SAR) studies showed that a secondary carboxamide moiety is mandatory for activity, as in the
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
initial hit compound 1 (hꢀAC IC = 64 nM) (Figure 2), and that introducing a bromine atom at
5
0
position 6 on the heterocyclic scaffold leads to a 2ꢀfold increase in inhibitory potency (2, IC =
5
0
31 nM), diminishing at the same time chemical and metabolic stability in aqueous buffer and
mouse plasma. Also, we observed that replacing bromine with a pꢀfluorophenyl group, as in 6ꢀ
1
2
(4ꢀfluorophenyl)ꢀ2ꢀoxoꢀNꢀ(4ꢀphenylbutyl)ꢀ1,3ꢀbenzoxazoleꢀ3ꢀcarboxamide (3, ARN14974)
allows for balancing potency (IC50 = 79 nM) with enhanced chemical and metabolic stability
Figure 2). We found that compound 3 inhibits AC in intact cells and in vivo, causing a
(
substantial reduction in AC activity in multiple organs and resulting in expected changes of
ceramide and sphingosine levels.
In the present study we expanded our initial SAR study around compound 1 with the objective
of demonstrating whether the benzoxazolone carboxamide scaffold is generally capable of
providing potent AC inhibitors. We synthesized a series of benzoxazolone carboxamides to
explore the roles of both position and stereoelectronic properties of substituents on the
heterocyclic scaffold, as well as the effect of side chain modifications on AC inhibition (Figure
2). Finally, we tested select derivatives for their stability in aqueous buffer and demonstrated in
vivo AC inhibitory activity of the optimised compound, 6ꢀchloroꢀ2ꢀoxoꢀNꢀ(4ꢀphenylbutyl)ꢀ1,3ꢀ
benzoxazoleꢀ3ꢀcarboxamide (14).
CHEMISTRY
A series of benzoxazolone carboxamide derivatives (4ꢀ36) bearing different substituents on
the bicyclic ring system were synthesized, as outlined in Schemes 1ꢀ5. In analogy to hit
ACS Paragon Plus Environment

Page 5 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compound 1, we initially coupled substituted benzoxazolones with the commercial 4ꢀphenylbutyl
isocyanate, in the presence of 4ꢀdimethylaminopyridine (DMAP), under basic conditions
1
3
(
Method A). First, we prepared a set of derivatives (4ꢀ19) bearing groups with different
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
stereoelectronic properties on the heterocyclic ring (Scheme 1). Coupling reactions proceeded
smoothly to give the final carboxamides 4ꢀ19 in good yield, with the exception of compounds 4
and 7, for which no or low conversion was observed, seemingly due to the substituent at position
4
leading to chemical instability. In most cases, the starting benzoxazolone scaffolds were
commercially available (37b, 37eꢀf, 37hꢀk); alternatively the properly substituted heterocycles
37a, 37cꢀd, 37g, 37lꢀp) were obtained from the corresponding 2ꢀaminophenols by
intramolecular cyclization reaction in the presence of 1,1'ꢀcarbonyldiimidazole (CDI) (Scheme
(
1
4
1).
Secondly, we introduced aromatic substituents on the benzoxazolone scaffold by Suzukiꢀ
Miyaura coupling reaction between the appropriate bromo heterocycles (37aꢀc, 38) and
commercially available boronic acids (Scheme 2). Couplings at position 5 and 6 of the
benzoxazolone system were accomplished in satisfactory yields, while the reactions to get 4ꢀ and
7ꢀphenyl benzoxazolones 39a and 39d led to a complex mixture, and the intermediates were
used as crudes in the following step. Nꢀacylation of 39aꢀg with 4ꢀphenylbutyl isocyanate yielded
the targeted compounds 20ꢀ26.
Next, we synthesized a small set of benzoxazolone carboxamide derivatives (27ꢀ30) with
alkyl/alkenylꢀcyclohexyl/phenyl groups at position 6. The appropriate vinyl boronic acid was
reacted with 6ꢀbromoꢀbenzoxazolone 38 under SuzukiꢀMiyaura reaction conditions to afford the
corresponding alkenylic derivatives (40aꢀb) in good yields. Hydrogenation of the double bond
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 70
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
yielded compounds 41aꢀb quantitatively. Coupling of 40aꢀb and 41aꢀb to 4ꢀphenylbutyl
isocyanate gave the targeted derivatives 27ꢀ28 and 29ꢀ30, respectively, in good yield (Scheme 3).
To further expand the series of 6ꢀsubstituted derivatives, compounds 31ꢀ33 bearing electronꢀ
donating and bulky alkoxy groups were prepared as shown in Scheme 4. The benzoxazolone 42
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
15
was obtained by orthogonal protection of the commercial 6ꢀhydroxy derivative 43 (Scheme 4).
Oꢀsilyl group cleavage by treatment with tetrabutylammonium fluoride (TBAF) gave the Nꢀ
protected 6ꢀhydroxy derivative 44, which was readily alkylated with commercial alcohols under
Mitsunobu reaction conditions, using triphenylphosphine and diꢀtertꢀbutylazodicarboxylate
16
(
DBAD). DBAD was degraded under mild conditions in trifluoroactic acid (TFA), affording Nꢀ
protectedꢀ6ꢀalkoxy benzoxazolones 45aꢀc in good yield (Scheme 4). Cleavage of the
1
5
methoxyethoxymethyl (MEM) group with refluxing TFA led only to partial deprotection (46aꢀ
c); however, subsequent treatment in dimethylformamide (DMF) at 153 °C provided efficient
and clean reaction conditions for obtaining benzoxazolones 47aꢀc. Coupling with 4ꢀphenylbutyl
isocyanate delivered the desired final compounds 31ꢀ33 (Scheme 4).
Scheme 5 shows the syntheses of benzoxazolone carboxamides 34ꢀ36 bearing a benzoyl
group at either position 5 or 6. Acylation of the benzoxazolone ring could be efficiently achieved
by means of FriedelꢀCraft reaction. To get compound 34, the commercial Nꢀacetyl 2ꢀ
aminophenol was treated with a mild electrophile, such as the complex AlCl ·DMF in the
3
presence of benzoyl chloride to regioselectively acylate position 4 (Scheme 5A). Subsequent Nꢀ
acetyl group cleavage and ring closure with CDI afforded the targeted 5ꢀbenzoylꢀ3Hꢀ1,3ꢀ
benzoxazolꢀ2ꢀone 49, which was then coupled to 4ꢀphenylbutyl isocyanate. To obtain
compounds 35ꢀ36, the unsubstituted benzoxazolone 50 was first Nꢀacylated with benzoyl
chloride or 4ꢀchlorobenzoyl chloride to give 51a and 51b, respectively (Scheme 5B). Heating in
ACS Paragon Plus Environment

Page 7 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
the presence of AlCl promoted the migration of acyl group from the nitrogen to the carbon atom
3
17
at position 6 (52aꢀb), according to a “Friesꢀlike” rearrangement mechanism. Final Nꢀacylation
of nitrogen N3 with 4ꢀphenylbutyl isocyanate led to the targeted compounds.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finally, a series of unsubstituted benzoxazolone derivatives with different side chains at the
carboxamide moiety was accessed as depicted in Scheme 6. Compounds 53ꢀ66 were obtained by
activating benzoxazolone 50 with triphosgene, either in pyridine (Method B) (54, 62ꢀ63, 66) or
in DCM in the presence of Et N (Method C) (53, 55ꢀ61, 64ꢀ65), and in situ quenching with the
3
proper amine (67aꢀn) to incorporate the desired side chains (Scheme 6). Nonꢀcommercial amines
(67aꢀj) were synthesized through a twoꢀstep sequence starting from alcohols 68aꢀi and bromide
69 under Mitsunobu and Gabriel reaction conditions, respectively (Scheme 7). Hydrazineꢀ
mediated cleavage of phthalimide derivatives 70aꢀj led to the desired amines as hydrochloride
salts. Nonꢀcommercial alcohols 68aꢀe were readily obtained by reduction of the corresponding
ester (71a) or acids (71bꢀe).
RESULTS AND DISCUSSION
We recently disclosed benzoxazolone carboxamides as the first class of potent and
12
systemically active inhibitors of intracellular AC. We demonstrated that these compounds
covalently inhibit AC, and identified derivative 3 as the first chemical probe that may be used to
study the impact of AC inhibition in vivo (Figure 2). A preliminary SAR study around hit
compound 1 revealed that a secondary carboxamide moiety is mandatory for activity, and
substitution at position 6 of the benzoxazolone ring allows for modulation of the inhibitory
potency against AC. These results prompted us to expand the class of benzoxazolone
carboxamides by systemically investigating the effects of different substituents on the
heterocyclic scaffold as well as of modifications of the side chain. The new compounds were
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 70
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
tested for their ability to inhibit the hydrolysis of Nꢀ[(1S,2R)ꢀ2ꢀhydroxyꢀ1ꢀ(hydroxymethyl)ꢀ4ꢀ(2ꢀ
oxochromenꢀ7ꢀyl)oxyꢀbutyl]dodecanamide by recombinant human AC (hꢀAC) in a fluorescenceꢀ
1
8
based assay.
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
To probe for stereoelectronic effects on the benzoxazolone system, we initially
monosubstituted 1 with bromine or methyl groups at positions 4, 5, 6 or 7 (Table 1). As
previously observed with the 6ꢀbromo derivative 2 (IC = 31 nM), introduction of bromine at
5
0
either position 5 or 7 led to a 2ꢀ to 4ꢀfold increase in potency relative to 1 (5, IC = 22 nM and 6,
5
0
IC50 = 18 nM). Bromine introduction at position 4 yielded the unstable derivative 4, and the
same result was obtained when a methyl group was introduced at the same position (7). As for
the small set of methylꢀsubstituted derivatives, with the exception of compound 10 bearing a
methyl at position 7 and having an IC of 23 nM, the introduction of this group led to a decrease
5
0
in potency when the methyl was placed in position 5 (8, IC50 = 117 nM) or to an equipotent
compound, compared to 1, when the methyl was placed in position 6 (9, IC = 66 nM). Next, we
50
expanded the set of substituted analogs at positions 5 and 6 investigating compounds 11ꢀ19,
which were 2ꢀ to 20ꢀfold more potent than 1 (Table 1). The 5ꢀfluoro derivative 11 (IC = 12
5
0
nM) turned out to be ~3ꢀfold more potent than the corresponding 6ꢀsubstituted analog 12 (IC =
5
0
31 nM). The same trend in potency associated to halide introduction at either position 5 or 6 was
observed for the chloroꢀsubstituted compounds 13 and 14 (and for the bromineꢀsubstituted 5 and
2). Replacement of halides with strong electronꢀwithdrawing groups such as trifluoromethyl and
nitro groups diminished the difference in potency between 5 and 6ꢀsubstituted derivatives (15ꢀ
18). Of particular interest were the results obtained with 5ꢀnitro benzoxazolone carboxamide 17
and the corresponding 6ꢀnitro analog 18, which represent the first singleꢀdigit nanomolar
compounds in this class, both inhibiting AC with an IC value of 3 nM. Finally, we explored
5
0
ACS Paragon Plus Environment

Page 9 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
whether incorporating simultaneously two favorable substitutions would provide additive effects.
We observed that the disubstituted compound 19 was potent (IC = 18 nM), but not significantly
50
more so than the related monosubstituted analogs 13 and 14 (Table 1).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As a continuation of the SAR study around the benzoxazolone system, we prepared a small
series of derivatives bearing a phenyl ring (Table 2). First, we systematically explored the
introduction of this moiety at positions 4, 5, 6 and 7. Interestingly, stability issues did not prevent
isolation and testing of the 4ꢀphenyl derivative 20, which turned out to be 75 folds more potent
than 1 (IC50 = 0.8 nM). Derivatives 21ꢀ23 bearing a phenyl ring at positions 5, 6, and 7,
respectively, were approximately as potent as 1. Next, we focused on positions 5 and 6, and
prepared a small series of arylꢀsubstituted derivatives (24ꢀ36). Overall, decorating the phenyl
ring with fluorine or methoxy group in the para position (3, 24 and 25ꢀ26) did not improve
potency over the unsubstituted benzoxazolone derivative 1. However, replacement of the phenyl
with aryl ketones (34ꢀ36) led to 2ꢀ to 5ꢀfold increases in potency relative to 1, with compound 36
(
IC = 14 nM) being the most potent within this small series. Finally, introduction of an alkylic
50
or alkenylic spacer between the benzoxazolone system and the phenyl ring as well as the
replacement of the phenyl with a cyclohexyl led to a loss in potency, with compounds 27ꢀ30
(Scheme 3) showing no inhibitory activity against AC at the concentrations tested (50 and 500
nM). Similarly, compounds 31ꢀ33 (Scheme 4) bearing various alkoxy groups at position 6 were
inactive.
We next examined the role of the side chain by preparing a series of unsubstituted
benzoxazolone carboxamides. First, we evaluated the length of the carbon linker between the
benzoxazolone and the phenyl ring of 1. Compounds 53ꢀ56, in which the spacer was
progressively increased from two to six methylene units, were tested for their inhibitory activity
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 70
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
(
Table 3). The short linker with two methylene units led to a 2ꢀfold decrease in potency (53, IC50
=
121 nM) compared to 1, while compounds 54ꢀ56 – which contain a 3ꢀ, 5ꢀ or 6ꢀcarbon atom
linker, respectively – turned out to be as potent as 1. Replacement of one methylene unit of the
fourꢀcarbon atom chain of 1 with a heteroatom (62ꢀ64) was detrimental for activity, with the
exception of the thioether derivative 63, which was equipotent to 1. Next, to evaluate the role of
the phenyl ring, we replaced it with a naphtyl (57) or thiophene (65) group, observing an increase
in potency with the latter derivative (65, IC50 = 37 nM). Introduction of substituents with
different electronic properties in the para position of the phenyl ring was generally well
tolerated, as shown by compounds 59ꢀ61, with the exception of derivative 58 (IC = 312 nM),
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
which turned out to be 5ꢀfold less potent than 1. Interestingly, the most potent compound of this
series showed the same side chain length of 1, but with the phenyl ring situated one carbon atom
from the N3ꢀcarboxamide group (66, IC = 19 nM).
5
0
Previously, we reported that introducing bromine in position 6 (2) destabilizes compounds
and leads to lower halfꢀlife in PBS and blood plasma compared to 1, but that having a pꢀ
fluorophenyl in position 6 (3) enhanced chemical and metabolic stability dramatically.
Conversely, stability under standard AC assay conditions (pH 4.5) was not affected by these
12
substitutions. In order to gain a more comprehensive understanding of the structureꢀstability
relationship of benzoxazolone carboxamides, compounds 14, 19ꢀ26 and 35ꢀ36 were tested for
stability in aqueous buffer at physiological pH (Table 4). Representative compounds (14, 17ꢀ18,
2
1, 35ꢀ36) were also evaluated for their stability at pH 4.5 (Table S1, Supporting Information).
At acidic pH, all compounds were stable, as previously observed with derivatives 1ꢀ3. The
analysis at physiological pH showed that, in contrast to bromine, chlorine in position 6 (14) or
positions 5 and 6 (19) did not induce instability relative to the unsubstituted compound 1.
ACS Paragon Plus Environment

Page 11 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Aromatic substitutions in positions 5ꢀ7 enhanced stability in all cases (3, 21ꢀ26), with pꢀ
fluorophenyl in position 6 (3) being the most efficient substituent and pꢀmethoxyphenyl in the
same position (26) the least efficient one. Noticeably, incorporating a phenyl group in position 4
resulted in a very unstable compound (20), which tallies well with the chemical instability
observed with other compounds (4, 7) with substitutions in the same position. Finally, compound
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
35 with the benzoyl group in position 6 demonstrated enhanced stability, and interestingly its
close analog 36 was even more stable with a halfꢀlife 6ꢀfold higher than 1 (Table 4). Overall,
these results demonstrate that stability properties are not simply explained by the
electronegativity of the substituents on the benzoxazolone ring, and that increased activity is not
necessarily linked with decreased stability. Indeed, we see that it is possible to increase potency
without decreasing stability, as exemplified by compounds 14 and 19, and that it is even possible
to enhance both potency and stability relative to 1 as seen for compounds 35 and 36. However,
the most potent compound in this series, compound 20 (IC = 0.8 nM, Table 2) also showed the
50
lowest halfꢀlife, providing a case in which activity and stability are seemingly related.
To test whether the explored structural modifications of the benzoxazolone carboxamide
scaffold could also provide novel systemically active compounds, we selected derivative 14 for
further pharmacological studies in vivo, based on its balanced profile in terms of potency,
stability, solubility and drugꢀlikeness. Indeed, compound 14 shows an improved potency
compared to 3, and an adequate solubility in the vehicle used for systemic administration. In this
regard, other slightly more potent and stable derivatives of this series, such as compounds 35ꢀ36,
were found to be poorly soluble and thus difficult to handle in in vivo studies. As additional
criteria of selection, we considered the drugꢀlikeness of 14 in comparison with other
considerably more potent compounds, such as the singleꢀdigit nanomolar inhibitors 17 and 18.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 70
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 nitroꢀsubstituted benzoxazolone ring makes these derivatives important for SAR purposes
but not promising as candidates for further development, due to the potential in vivo toxicity of
aromatic nitro groups.
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
Compound 14 was injected in mice at the dose of 10 mg kg (i.p.) and AC activity was
evaluated at different time points in lysosomal fractions prepared from lung tissue and cerebral
cortex. We selected the lungs because of the high basal AC activity in this tissue, and the
cerebral cortex to evaluate the ability of 14 to reach the brain. As shown in Figure 3, the
compound significantly inhibited AC activity in both compartments. The effect was long lasting,
as AC inhibition was still observed 24 hours after administration, although a slight recovery in
activity was seen at this time point (Figure 3AꢀC). In the lungs, AC inhibition was accompanied
by an increase in the levels of ceramide and dihydroceramide (namely d18:1/16:0 and
d18:0/16:0) for up to 6 hours; sphingosine and sphingosine 1ꢀphosphate were also decreased at 3
hours (Figure 3B). No statistically significant changes in sphingolipid levels were observed in
the cerebral cortex (Figure 3D), presumably owing to the relative low level of AC inhibition
achieved in that brain region. The results indicate that 14 engages its intended target in two
relevant tissues, with results that are comparable to those previously obtained with compound 3
12
under the same experimental conditions.
CONCLUSIONS
The present study describes a systematic SAR investigation of a recently disclosed class of
12
AC inhibitors that features the benzoxazolone carboxamide scaffold. We show that this
scaffold is able to provide stable systemically active AC inhibitors, and may thus represent a
good starting point for further discovery. We present various synthetic approaches to obtain
diversified analogs in high yields and with great feasibility. We show that electronꢀwithdrawing
ACS Paragon Plus Environment

Page 13 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
groups in positions 5ꢀ7 of the benzoxazolone scaffold (2, 5ꢀ6, 10ꢀ19) lead to enhanced inhibitory
activity toward AC. This is consistent with the mechanism of action of these agents, as electronꢀ
withdrawing groups are likely to make the carboxamide carbonyl more prone to nucleophilic
attack by AC´s active site cysteine. Introducing a phenyl group at position 4 gave a highly potent
but very unstable compound (20), which correlates with the chemical instability seen with other
molecules substituted in the same position (4 and 7). Additionally, we investigated a range of
aromatic substitutions on positions 5ꢀ7 of the benzoxazolone scaffold (21ꢀ26), which all provide
stable compounds but do not improve activity towards AC. Also, we demonstrated that long and
bulky aliphatic groups are not favorable for inhibitory activity, as seen with compounds 27ꢀ30
and 31ꢀ33. Instead, we found that substitutions with benzoyl (34ꢀ35) or 4ꢀchlorobenzoyl (36)
groups combine good potency and high stability: the most favorable of these derivatives, 36,
shows a 5ꢀ6ꢀfold improvement in AC inhibition relative to 1 and 3 and a 6ꢀfold improvement in
stability relative to 1. Finally, we addressed the role of the carboxamide side chain and
demonstrated that the inhibitory potency of 1 could be improved 2ꢀ3 folds by incorporating the
phenyl group within the linker (66) or replacing it with thiophene (65). Based on potency,
stability and adequate solubility, we chose to investigate compound 14 for in vivo activity. We
show that following systemic administration in mice, this compound inhibits AC in peripheral
tissues as well as in the brain, leading to the expected variations in the sphingolipid profile. This
provides essential evidence that AC inhibition can be successfully obtained in live animals using
potent and reasonably stable benzoxazolone carboxamides. Compound 14 might be used as a
tool to unravel the biological role of AC and define the potential value of this enzyme as a
therapeutic target.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 70
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
EXPERIMENTAL SECTION
Chemistry. General. All commercial available reagents and solvents were used as purchased
from vendors without further purification. Dry solvents (pyridine, DCM) were purchased from
SigmaꢀAldrich. Automated column chromatography purifications were done using a Teledyne
ISCO apparatus (CombiFlash® Rf) with preꢀpacked silica gel columns of different sizes (from 4
g up to 40 g). Mixtures of increasing polarity of cyclohexane and ethyl acetate (EtOAc) were
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
®
used as eluents (unless otherwise stated). Microwave heating was performed using Explorer ꢀ48
TM
positions instrument (CEM). Hydrogenation reactions were performed using HꢀCube
continuous hydrogenation equipment (SSꢀreaction line version), employing disposable catalyst
TM
cartridges (CatCart ) preloaded with the required heterogeneous catalyst. NMR experiments
1
13
were run on a Bruker Avance III 400 system (400.13 MHz for H, and 100.62 MHz for C),
equipped with a BBI probe and Zꢀgradients. Spectra were acquired at 300 K, using deuterated
dimethylsulfoxide (DMSOꢀd ) or deuterated chloroform (CDCl ) as solvents. Chemical shifts for
6
3
1
13
H and C spectra were recorded in parts per million using the residual nonꢀdeuterated solvent
1
13
as the internal standard (for CDCl : 7.26 ppm, H and 77.16 ppm, C; for DMSOꢀd : 2.50 ppm,
3
6
1
13
H; 39.52 ppm, C). UPLCꢀMS analyses were run on a Waters ACQUITY UPLCꢀMS system
consisting of a SQD (Single Quadrupole Detector) Mass Spectrometer equipped with an
Electrospray Ionization interface and a Photodiode Array Detector. PDA range was 210ꢀ400 nm.
Analyses were performed on an ACQUITY UPLC HSS T3 C18 column (50 × 2.1 mm ID,
particle size 1.8 µm) with a VanGuard HSS T3 C preꢀcolumn (5 × 2.1 mm ID, particle size 1.8
1
8
µm). Mobile phase was either 10 mM NH OAc in H O at pH 5 adjusted with AcOH (A) and 10
4
2
mM NH OAc in MeCNꢀH O (95:5) at pH 5 (B). Electrospray ionization in positive and negative
4
2
1
13
1
1
mode was applied. All final compounds showed ≥ 95% purity by NMR ( H, C, Hꢀ H COSY,
ACS Paragon Plus Environment

Page 15 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
13
Hꢀ C HSQC) and UPLCꢀMS (UV). DMSO stock solutions of final compounds (10 mM) used
for biological tests were evaluated prior to tests (NMR, LCꢀMS), and concentration was assessed
1
by quantitative HꢀNMR.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General Procedure for the Synthesis of Final Benzoxazolone N-Carboxamides 4ꢀ36 via
Method A (Scheme 1ꢀ5, Table 1ꢀ2). The properly substituted 3Hꢀ1,3ꢀbenzoxazolꢀ2ꢀone (1.0
equiv) was dissolved in dry pyridine (6 mL per mmol benzoxazolꢀ2ꢀone). DMAP (1.1 equiv) was
added and the reaction mixture stirred under nitrogen atmosphere at rt for 30 min. 4ꢀPhenylbutyl
isocyanate (1.1 equiv) was added and the resulting mixture was stirred for 15 h. The solvent was
removed under reduced pressure, and the compound was purified by silica gel column
chromatography using the Teledyne ISCO apparatus (cyclohexane/EtOAc).
5-Bromo-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (5). The reaction was
carried out following method A, using 37b (100 mg, 0.467 mmol) and 4ꢀphenylbutyl isocyanate
1
(
90 mg, 0.088 mL, 0.514 mmol). White solid (88 mg, 48%). H NMR (400 MHz, CDCl ) δ 1.64
3
–
1.77 (m, 4H), 2.67 (t, J = 7.2 Hz, 2H), 3.44 (td, J = 6.8, 5.6 Hz, 2H), 7.11 (d, J = 8.6 Hz, 1H),
.16 – 7.21 (m, 3H), 7.26 – 7.31 (m, 2H), 7.37 (dd, J = 8.6, 2.1 Hz, 1H), 7.97 (t, J = 4.6 Hz, 1H),
7
1
3
8.27 (d, J = 2.0 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.7, 29.1, 35.6, 40.4, 111.3, 117.9,
3
119.0, 126.0, 127.6, 128.5 (4C), 129.2, 140.8, 142.0, 149.5, 152.9. MS (ESI) m/z: 212 and 214
˗
[
M ˗ CONH(CH ) Ph] .
2
4
7-Bromo-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (6). The reaction was
carried out following method A, using 37c (140 mg, 0.654 mmol) and 4ꢀphenylbutyl isocyanate
1
(
126 mg, 0.123 mL, 0.720 mmol). White solid (143 mg, 56%). H NMR (400 MHz, CDCl ) δ
3
1
.61 – 1.78 (m, 4H), 2.67 (t, J = 7.1 Hz, 2H), 3.44 (q, J = 6.4 Hz, 2H), 7.13 – 7.21 (m, 4H), 7.26
7.31 (m, 2H), 7.38 (dd, J = 8.3, 1.1 Hz, 1H), 7.99 (t, J = 6.0 Hz, 1H), 8.02 (dd, J = 8.2, 1.0 Hz,
–
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 70
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
13
1
H). C NMR (101 MHz, CDCl ) δ 28.7, 29.1, 35.6, 40.4, 102.4, 114.7, 126.0, 126.2, 128.0,
3
1
28.5 (4C), 129.0, 140.0, 142.0, 149.6, 152.4. MS (ESI) m/z: nonꢀionizable compound under
routine conditions used.
-Methyl-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (7). The reaction was
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
carried out following method A, using 37d (94 mg, 0.630 mmol) and 4ꢀphenylbutyl isocyanate
1
(
121 mg, 0.119 mL, 0.693 mmol). Clear colorless oil (49 mg, 24%). H NMR (400 MHz, CDCl )
3
δ 1.64 – 1.79 (m, 4H), 2.53 (s, 3H), 2.68 (t, J = 7.2 Hz, 2H), 3.45 (q, J = 6.6 Hz, 2H), 7.04 – 7.09
13
(m, 2H), 7.11 – 7.21 (m, 4H), 7.26 – 7.31 (m, 2H), 7.60 (t, J = 4.5 Hz, 1H). C NMR (101 MHz,
CDCl ) δ 21.5, 28.6, 29.1, 35.6, 40.9, 107.6, 124.8, 126.0, 126.4, 126.6, 128.4, 128.5 (2C), 128.5
3
+
+
(
2C), 142.1, 143.0, 149.3, 154.0. MS (ESI) m/z: 325 [M + H] , 342 [M + NH ] .
4
5-Methyl-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (8). The reaction was
carried out following method A, using 37e (50 mg, 0.334 mmol) and 4ꢀphenylbutyl isocyanate
1
(
64 mg, 0.063 mL, 0.367 mmol). White solid (99 mg, 91 %). H NMR (400 MHz, CDCl ) δ 1.64
3
–
1.77 (m, 4H), 2.41 (s, 3H), 2.67 (t, J = 7.2 Hz, 2H), 3.44 (td, J = 6.7, 5.5 Hz, 2H), 7.00 – 7.04
(
m, 1H), 7.08 – 7.11 (m, 1H), 7.16 – 7.21 (m, 3H), 7.26 – 7.30 (m, 2H), 7.90 (br s, 1H), 8.08 (t, J
1
3
= 4.9 Hz, 1H). C NMR (101 MHz, CDCl ) δ 21.7, 28.7, 29.2, 35.6, 40.2, 109.5, 116.1, 125.0,
3
1
26.0, 128.0, 128.5 (2C), 128.5 (2C), 135.2, 139.9, 142.0, 150.1, 153.6. MS (ESI) m/z: 325 [M +
+
+
H] , 342 [M + NH ] .
4
6-Methyl-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (9). The reaction was
carried out following method A, using 37f (50 mg, 0.334 mmol) and 4ꢀphenylbutyl isocyanate
1
(
64 mg, 0.063 mL, 0.367 mmol). White solid (73 mg, 68%). H NMR (400 MHz, CDCl ) δ 1.63
3
–
1.78 (m, 4H), 2.41 (s, 3H), 2.67 (t, J = 7.2 Hz, 2H), 3.44 (td, J = 6.7, 5.5 Hz, 2H), 7.03 – 7.08
(
m, 2H), 7.15 – 7.21 (m, 3H), 7.26 – 7.30 (m, 2H), 7.89 – 7.92 (m, 1H), 8.04 (t, J = 5.4 Hz, 1H).
ACS Paragon Plus Environment

Page 17 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
C NMR (101 MHz, CDCl ) δ 21.6, 28.7, 29.2, 35.6, 40.2, 110.5, 115.2, 125.6, 125.7, 126.0,
3
+
128.5 (2C), 128.5 (2C), 135.0, 142.0, 142.1, 150.0, 153.5. MS (ESI) m/z: 325 [M + H] , 342 [M
+
+
NH ] .
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
-Methyl-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (10). The reaction was
carried out following method A, using 37g (83 mg, 0.556 mmol) and 4ꢀphenylbutyl isocyanate
1
(
107 mg, 0.105 mL, 0.612 mmol). White solid (128 mg, 71%). H NMR (400 MHz, CDCl ) δ
3
1
.63 – 1.78 (m, 4H), 2.39 (s, 3H), 2.67 (t, J = 7.1 Hz, 2H), 3.44 (q, J = 6.6 Hz, 2H), 7.04 (d, J =
7.8 Hz, 1H), 7.12 – 7.20 (m, 4H), 7.26 – 7.31 (m, 2H), 7.88 (dd, J = 8.0, 1.2 Hz, 1H), 8.09 (t, J =
13
5
.5 Hz, 1H). C NMR (101 MHz, CDCl ) δ 14.5, 28.7, 29.2, 35.6, 40.2, 113.11, 120.5, 124.9,
3
1
26.0, 126.1, 127.8, 128.5 (2C), 128.5 (2C), 140.5, 142.0, 150.1, 153.5. MS (ESI) m/z: 325 [M +
+
+
H] , 342 [M + NH ] .
4
5
-Fluoro-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (11). The reaction was
carried out following method A, using 37h (61 mg, 0.398 mmol) and 4ꢀphenylbutyl isocyanate
1
(
76.7 mg, 0.075 mL, 0.438 mmol). White solid (111 mg, 85%). H NMR (400 MHz, CDCl ) δ
3
1
.64ꢀ1.78 (m, 4H), 2.67 (t, J = 7.1 Hz, 2H), 3.44 (q, J = 6.6 Hz, 2H), 6.94 (td, J = 9.0, 2.7 Hz,
1H), 7.15 – 7.21 (m, 4H), 7.26 – 7.31 (m, 2H), 7.85 (dd, J = 8.5, 2.7 Hz, 1H), 8.01 (t, J = 6.3 Hz,
1
3
1
H). C NMR (101 MHz, CDCl ) δ 28.6, 29.1, 35.5, 40.3, 104.3 (d, J = 31.3 Hz), 110.5 (d, J =
3
9
.5 Hz), 111.2 (d, J = 25.1 Hz), 126.0, 128.5 (4C), 128.7 (d, J = 14.0 Hz), 137.8 (d, J = 2.2 Hz),
˗
142.0, 149.5, 153.4, 159.8 (d, J = 242.4 Hz). MS (ESI) m/z: 152 [M ˗ CONH(CH ) Ph] .
2
4
6
-Fluoro-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (12). The reaction was
carried out following method A, using 37i (51 mg, 0.334 mmol) and 4ꢀphenylbutyl isocyanate
1
(
64 mg, 0.063 mL, 0.367 mmol). White solid (87 mg, 79%). H NMR (400 MHz, CDCl ) δ 1.63ꢀ
3
1
.78 (m, 4H), 2.67 (t, J = 7.2 Hz, 2H), 3.44 (td, J = 6.7, 5.6 Hz, 2H), 6.96 – 7.04 (m, 2H), 7.16 –
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 70
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
13
7
.21 (m, 3H), 7.25 – 7.31 (m, 2H), 7.96 (t, J = 5.7 Hz, 1H), 8.00 – 8.05 (m, 1H). C NMR (101
MHz, CDCl ) δ 28.7, 29.1, 35.6, 40.3, 99.1 (d, J = 28.8 Hz), 111.8 (d, J = 23.4 Hz), 116.3 (d, J =
3
9
.0 Hz), 124.3 (d, J = 2.6 Hz), 126.0, 128.5 (4C), 141.8, 142.0 (d, J = 2.3 Hz), 149.6, 153.2,
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
+
+
159.7 (d, J = 245.2 Hz). MS (ESI) m/z: 329 [M + H] , 346 [M + NH ] .
4
5-Chloro-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (13). The reaction was
carried out following method A, using 37j (68 mg, 0.401 mmol) and 4ꢀphenylbutyl isocyanate
1
(
76.7 mg, 0.075 mL, 0.438 mmol). Clear colorless oil (127 mg, 92%). H NMR (400 MHz,
CDCl ) δ 1.64 – 1.79 (m, 4H), 2.67 (t, J = 7.1 Hz, 2H), 3.44 (td, J = 6.7, 5.5 Hz, 2H), 7.13 – 7.24
3
13
(
m, 5H), 7.26 – 7.31 (m, 2H), 7.98 (t, J = 5.2 Hz, 1H), 8.12 (d, J = 2.1 Hz, 1H). C NMR (101
MHz, CDCl ) δ 28.7, 29.1, 35.6, 40.4, 110.8, 116.3, 124.7, 126.0, 128.5 (4C), 128.9, 130.8,
3
+
140.3, 142.0, 149.5, 153.0. MS (ESI) m/z: 345 and 347 [M + H] .
6
-Chloro-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (14). The reaction was
carried out following method A, using 37k (56 mg, 0.334 mmol) and 4ꢀphenylbutyl isocyanate
1
(
64 mg, 0.063 mL, 0.367 mmol). White solid (77 mg, 67%). H NMR (400 MHz, CDCl ) δ 1.64
3
–
1.78 (m, 4H), 2.67 (t, J = 7.2 Hz, 2H), 3.44 (td, J = 6.7, 5.6 Hz, 2H), 7.16 – 7.20 (m, 3H), 7.24
1
3
– 7.31 (m, 4H), 7.93 – 8.02 (m, 2H). C NMR (101 MHz, CDCl ) δ 28.7, 29.1, 35.6, 40.3, 110.9,
3
1
16.4, 125.4, 126.1, 126.8, 128.5 (4C), 130.2, 142.0, 142.1, 149.6, 152.9. MS (ESI) m/z: 168 and
˗
170 [M ˗ CONH(CH ) Ph] .
2
4
2-Oxo-N-(4-phenylbutyl)-5-(trifluoromethyl)-1,3-benzoxazole-3-carboxamide (15). The
reaction was carried out following method A, using 37l (128 mg, 0.630 mmol) and 4ꢀphenylbutyl
1
isocyanate (121 mg, 0.119 mL, 0.693 mmol). White solid (166 mg, 70%). H NMR (400 MHz,
CDCl ) δ 1.65 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 3.46 (q, J = 6.5 Hz, 2H), 7.16 – 7.21 (m, 3H),
3
7
.26 – 7.31 (m, 2H), 7.34 (d, J = 8.5 Hz, 1H), 7.53 – 7.57 (m, 1H), 7.96 (t, J = 5.7 Hz, 1H), 8.40
ACS Paragon Plus Environment

Page 19 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
13
(
d, J = 1.9 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.7, 29.1, 35.5, 40.4, 110.2, 113.5 (q, J =
3
4
.0 Hz), 122.2 (q, J = 3.9 Hz), 123.8 (q, J = 272.0 Hz), 126.0, 127.8 (q, J = 33.3 Hz), 128.5,
+
+
1
28.5 (4C), 142.0, 143.8, 149.4, 152.8. MS (ESI) m/z: 379 [M + H] , 396 [M + NH ] .
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
-Oxo-N-(4-phenylbutyl)-6-(trifluoromethyl)-1,3-benzoxazole-3-carboxamide (16). The
reaction was carried out following method A, using 37m (81 mg, 0.399 mmol) and 4ꢀ
1
phenylbutyl isocyanate (76.7 mg, 0.075 mL, 0.438 mmol). White solid (91 mg, 60%). H NMR
(
400 MHz, CDCl ) δ 1.64 – 1.79 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 3.46 (q, J = 6.6 Hz, 2H), 7.15
3
– 7.21 (m, 3H), 7.26 – 7.31 (m, 2H), 7.51 (d, J = 1.7 Hz, 1H), 7.55 – 7.59 (m, 1H), 7.98 (t, J =
13
5
.9 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.6, 29.1, 35.6, 40.4,
3
1
07.6 (q, J = 4.0 Hz), 115.9, 122.6 (q, J = 3.9 Hz), 123.7 (q, J = 272.1 Hz), 126.0, 127.2 (q, J =
33.3 Hz), 128.5 (4C), 130.9, 141.6, 141.9, 149.3, 152.8. MS (ESI) m/z: 202 [M ˗
˗
CONH(CH ) Ph] .
2
4
5
-Nitro-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (17). The reaction was
carried out following method A, using 37n (124 mg, 0.688 mmol) and 4ꢀphenylbutyl isocyanate
1
(
133 mg, 0.130 mL, 0.757 mmol). White solid (150 mg, 61%). H NMR (400 MHz, CDCl ) δ
3
1.65 – 1.79 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 3.47 (td, J = 6.7, 5.6 Hz, 2H), 7.16 – 7.21 (m, 3H),
7
.26 – 7.31 (m, 2H), 7.37 (d, J = 8.9 Hz, 1H), 7.88 (t, J = 5.3 Hz, 1H), 8.24 (dd, J = 8.9, 2.4 Hz,
1
3
1
H), 8.98 (d, J = 2.4 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.6, 29.1, 35.5, 40.5, 110.2,
3
112.0, 121.1, 126.1, 128.5 (2C), 128.5 (2C), 128.6, 141.9, 145.3, 145.6, 148.9, 152.6. MS (ESI)
+
m/z: 354 [M + H] .
6
-Nitro-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (18). The reaction was
carried out following method A, using 37o (72 mg, 0.400 mmol) and 4ꢀphenylbutyl isocyanate
1
(
76.7 mg, 0.075 mL, 0.438 mmol). White solid (86 mg, 60%). H NMR (400 MHz, CDCl ) δ
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 70
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
.64 – 1.79 (m, 4H), 2.68 (t, J = 7.0 Hz, 2H), 3.47 (q, J = 6.4 Hz, 2H), 7.15 – 7.21 (m, 3H), 7.26
1
3
–
7.31 (m, 2H), 7.94 (t, J = 5.8 Hz, 1H), 8.15 (t, J = 1.2 Hz, 1H), 8.23 – 8.28 (m, 2H). C NMR
(101 MHz, CDCl ) δ 28.6, 29.0, 35.5, 40.5, 106.3, 115.6, 121.5, 126.1, 128.5 (2C), 128.6 (2C),
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
133.3, 141.3, 141.9, 144.7, 149.0, 152.7. MS (ESI) m/z: nonꢀionizable compound under routine
conditions used.
5
,6-Dichloro-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (19). The reaction
was carried out following method A, using 37p (121 mg, 0.593 mmol) and 4ꢀphenylbutyl
1
isocyanate (114 mg, 0.112 mL, 0.652 mmol). White solid (203 mg, 90%). H NMR (400 MHz,
CDCl ) δ 1.63 – 1.78 (m, 4H), 2.67 (t, J = 7.1 Hz, 2H), 3.44 (td, J = 6.7, 5.5 Hz, 2H), 7.16 – 7.21
3
13
(m, 3H), 7.26 – 7.31 (m, 2H), 7.37 (s, 1H), 7.84 (t, J = 5.8 Hz, 1H), 8.22 (s, 1H). C NMR (101
MHz, CDCl ) δ 28.6, 29.1, 35.6, 40.4, 112.0, 117.3, 126.1, 127.5, 128.5 (4C), 128.6, 129.3,
3
140.4, 141.9, 149.2, 152.6. MS (ESI) m/z: nonꢀionizable compound under routine conditions
used.
2
-Oxo-4-phenyl-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (20). The reaction was
carried out following method A, using crude 39a (140 mg) and 4ꢀphenylbutyl isocyanate (127
mg, 0.124 mL, 0.724 mmol). In total, four portions of 4ꢀphenylbutyl isocyanate were added with
1
about 3 h in between. Waxy white solid (18 mg, 6% over steps from 37a). H NMR (400 MHz,
CDCl ) δ 1.47 – 1.58 (m, 2H), 1.59 – 1.68 (m, 2H), 2.61 (t, J = 7.5 Hz, 2H), 3.20 (td, J = 6.8, 5.7
3
1
3
Hz, 2H), 7.09 (t, J = 5.1 Hz, 1H), 7.15 – 7.25 (m, 4H), 7.26 – 7.38 (m, 9H). C NMR (101 MHz,
CDCl ) δ 28.5, 29.1, 35.6, 40.8, 108.8, 124.9, 125.0, 126.0, 126.4, 127.3, 127.3 (2C), 127.6,
3
+
128.4 (2C), 128.5 (2C), 128.5 (3C), 139.4, 142.1, 143.6, 147.9. MS (ESI) m/z: 387 [M + H] , 404
+
[
M + NH ] .
4
ACS Paragon Plus Environment

Page 21 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
-Oxo-5-phenyl-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (21). The reaction was
carried out following method A, using 39b (14.9 mg, 0.0705 mmol) and 4ꢀphenylbutyl
1
isocyanate (33 mg, 0.032 mL, 0.187 mmol). Clear colourless sticky oil (17 mg, 63%). H NMR
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
400 MHz, CDCl ) δ 1.65 – 1.80 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 3.46 (q, J = 6.4 Hz, 2H), 7.15
3
– 7.21 (m, 3H), 7.25 – 7.31 (m, 3H), 7.33 – 7.38 (m, 1H), 7.41 – 7.47 (m, 3H), 7.57 – 7.61 (m,
1
3
2
H), 8.08 (t, J = 5.3 Hz, 1H), 8.34 (d, J = 1.9 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.7, 29.2,
3
3
5.6, 40.3, 110.1, 114.2, 123.6, 126.0, 127.5 (2C), 127.7, 128.5 (2C), 128.5 (2C), 128.6, 129.0
˗
(2C), 139.0, 140.4, 141.3, 142.0, 150.0, 153.5. MS (ESI) m/z: 210 [M ˗ CONH(CH ) Ph] .
2
4
2
-Oxo-6-phenyl-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (22). The reaction was
carried out following method A, using 39c (29 mg, 0.138 mmol) and 4ꢀphenylbutyl isocyanate
1
(29 mg, 0.028 mL, 0.165 mmol). White solid (33 mg, 62%). H NMR (400 MHz, CDCl ) δ 1.65
3
–
–
1.79 (m, 4H), 2.68 (t, J = 7.2 Hz, 2H), 3.47 (td, J = 6.7, 5.6 Hz, 2H), 7.16 – 7.21 (m, 3H), 7.25
7.31 (m, 2H), 7.34 – 7.40 (m, 1H), 7.43 – 7.50 (m, 4H), 7.55 – 7.59 (m, 2H), 8.06 (t, J = 5.3
1
3
Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.7, 29.2, 35.6, 40.3, 108.7,
3
1
15.8, 124.1, 126.0, 127.3 (2C), 127.3, 127.9, 128.5 (2C), 128.6 (2C), 129.1 (2C), 138.6, 140.05,
+
+
142.0, 142.4, 149.9, 153.4. MS (ESI) m/z: 387 [M + H] , 404 [M + NH ] .
4
2
-Oxo-7-phenyl-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (23). The reaction was
carried out following method A, using crude 39d (399 mg) and 4ꢀphenylbutyl isocyanate (145
mg, 0.141 mL, 0.825 mmol). The reaction was stirred for 68 h. Yellow solid (52 mg, 18% over 2
1
steps from 37c). H NMR (400 MHz, CDCl ) δ 1.65 – 1.80 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H),
3
3
.47 (q, J = 6.6 Hz, 2H), 7.16 – 7.21 (m, 3H), 7.25 – 7.32 (m, 2H), 7.33 – 7.36 (m, 1H), 7.38 –
7
.44 (m, 2H), 7.46 – 7.52 (m, 2H), 7.69 – 7.73 (m, 2H), 8.07 (dd, J = 7.9, 1.3 Hz, 1H), 8.11 (t, J
13
=
5.6 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.7, 29.2, 35.6, 40.3, 114.6, 124.4, 124.5, 125.4,
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 70
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
26.0, 128.5 (4C), 128.5 (3C), 128.7, 129.0 (2C), 134.4, 139.0, 142.0, 150.0, 153.3. MS (ESI)
+
+
m/z: 387 [M + H] , 404 [M + NH ] .
4
5
-(4-Fluorophenyl)-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (24). The
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
reaction was carried out following method A, using 39e (26 mg, 0.113 mmol) and 4ꢀphenylbutyl
1
isocyanate (33 mg, 0.032 mL, 0.187 mmol). White solid (26 mg, 58%). H NMR (400 MHz,
CDCl ) δ 1.64 – 1.78 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 3.46 (q, J = 6.5 Hz, 2H), 7.09 – 7.16 (m,
3
2H), 7.17 – 7.21 (m, 3H), 7.26 – 7.31 (m, 3H), 7.39 (dd, J = 8.4, 1.9 Hz, 1H), 7.51 – 7.57 (m,
1
3
2H), 8.07 ( (t, J = 5.5 Hz, 1H), 8.28 (d, J = 1.9 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.7,
3
29.2, 35.6, 40.3, 110.2, 114.4, 115.9 (d, J = 21.5 Hz, 2C), 123.5, 126.0, 128.5 (2C), 128.5 (2C),
128.7, 129.1 (d, J = 7.7 Hz, 2C), 136.5, 136.6, 141.3, 142.0, 150.0, 153.4, 162.8 (d, J = 246.9
˗
Hz). MS (ESI) m/z: 228 [M ˗ CONH(CH ) Ph] .
2
4
5
-(4-Methoxyphenyl)-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (25). The
reaction was carried out following method A, using 39f (41 mg, 0.170 mmol) and 4ꢀphenylbutyl
1
isocyanate (33 mg, 0.032 mL, 0.187 mmol). White solid (33 mg, 47%). H NMR (400 MHz,
CDCl ) δ 1.65 – 1.78 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 3.46 (q, J = 6.6 Hz, 2H), 3.86 (s, 3H),
3
6.94 – 6.99 (m, 2H), 7.15 – 7.21 (m, 3H), 7.24 – 7.30 (m, 3H), 7.40 (dd, J = 8.4, 1.9 Hz, 1H),
1
3
7.50 – 7.54 (m, 2H), 8.09 (t, J = 5.3 Hz, 1H), 8.29 (d, J = 1.8 Hz, 1H). C NMR (101 MHz,
CDCl ) δ 28.7, 29.2, 35.6, 40.3, 55.5, 110.0, 114.1, 114.4 (2C), 123.1, 126.0, 128.5 (2C), 128.5
3
(2C), 128.6 (2C), 128.6, 132.9, 138.6, 140.9, 142.0, 150.1, 153.5, 159.5. MS (ESI) m/z: 417 [M +
+
+
H] , 416 [M + NH ] .
4
6
-(4-Methoxyphenyl)-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (26). The
reaction was carried out following method A, using 39g (36 mg, 0.149 mmol) and 4ꢀphenylbutyl
1
isocyanate (29 mg, 0.028 mL, 0.165 mmol). White solid (52 mg, 84%). H NMR (400 MHz,
ACS Paragon Plus Environment

Page 23 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
CDCl ) δ 1.65 – 1.78 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 3.46 (td, J = 6.8, 5.6 Hz, 2H), 3.86 (s,
3
3H), 6.96 – 7.01 (m, 2H), 7.15 – 7.21 (m, 3H), 7.26 – 7.31 (m, 2H), 7.41 (d, J = 1.5 Hz, 1H),
1
3
7
.44 (dd, J = 8.4, 1.7 Hz, 1H), 7.48 – 7.52 (m, 2H), 8.03 – 8.09 (m, 2H). C NMR (101 MHz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CDCl ) δ 28.7, 29.2, 35.6, 40.3, 55.5, 108.2, 114.6 (2C), 115.7, 123.6, 126.0, 126.8, 128.3 (2C),
3
128.5 (2C), 128.6 (2C), 132.6, 138.2, 142.0, 142.5, 149.9, 153.4, 159.7. MS (ESI) m/z: nonꢀ
ionizable compound under routine conditions used.
2
-Oxo-N-(4-phenylbutyl)-6-[(E)-styryl]-1,3-benzoxazole-3-carboxamide (27). The reaction
was carried out following method A, using 40a (80 mg, 0.337 mmol) and 4ꢀphenylbutyl
1
isocyanate (65 mg, 0.064 mL, 0.371 mmol). White solid (72 mg, 52%). H NMR (400 MHz,
CDCl ) δ 1.64 – 1.79 (m, 4H), 2.68 (t, J = 7.1 Hz, 2H), 3.45 (q, J = 6.6 Hz, 2H), 7.09 (br s, 2H),
3
7.16 – 7.21 (m, 3H), 7.24 – 7.31 (m, 3H), 7.35 – 7.42 (m, 4H), 7.49 – 7.53 (m, 2H), 8.02 (d, J =
13
8
1
1
.2 Hz, 1H), 8.02 – 8.06 (m, 1H). C NMR (101 MHz, CDCl ) δ 28.7, 29.2, 35.6, 40.3, 107.2,
3
15.7, 124.0, 126.0, 126.7 (2C), 127.3, 127.5, 128.1, 128.5 (2C), 128.6 (2C), 128.9 (2C), 129.6,
34.8, 136.9, 142.0, 142.5, 149.8, 153.4. MS (ESI) m/z: nonꢀionizable compound under routine
conditions used.
6-[(E)-2-Cyclohexylvinyl]-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (28).
The reaction was carried out following method A, using 40b (58 mg, 0.238 mmol) and 4ꢀ
1
phenylbutyl isocyanate (46 mg, 0.045 mL, 0.262 mmol). White solid (81 mg, 82%). H NMR
(400 MHz, CDCl ) δ 1.13 – 1.39 (m, 5H), 1.63 – 1.86 (m, 9H), 2.08 – 2.18 (m, 1H), 2.67 (t, J =
3
7
.1 Hz, 2H), 3.44 (q, J = 6.4 Hz, 2H), 6.16 (dd, J = 15.9, 6.8 Hz, 1H), 6.33 (dd, J = 15.9, 1.2 Hz,
H), 7.15 – 7.27 (m, 5H), 7.26 – 7.30 (m, 2H), 7.93 (d, J = 8.3 Hz, 1H), 8.04 (t, J = 5.6 Hz, 1H).
1
1
3
C NMR (101 MHz, CDCl ) δ 26.1 (2C), 26.3, 28.7, 29.2, 33.0 (2C), 35.6, 40.26, 41.3, 106.8,
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 70
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
15.4, 123.3, 126.0, 126.3, 126.6, 128.5 (2C), 128.5 (2C), 135.6, 137.9, 142.0, 142.3, 149.9,
+
+
153.5. MS (ESI) m/z: 419 [M + H] , 436 [M + NH ] .
4
2
-Oxo-6-phenethyl-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (29). The reaction
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
was carried out following method A, using 41a (48 mg, 0.201 mmol) and 4ꢀphenylbutyl
1
isocyanate (46 mg, 0.045 mL, 0.262 mmol). White solid (65 mg, 78%). H NMR (400 MHz,
CDCl ) δ 1.65 – 1.80 (m, 4H), 2.70 (t, J = 7.2 Hz, 2H), 2.91 – 3.03 (m, 4H), 3.46 (q, J = 6.4 Hz,
3
2H), 7.03 (d, J = 1.6 Hz, 1H), 7.07 (dd, J = 8.2, 1.6 Hz, 1H), 7.14 – 7.17 (m, 2H), 7.18 – 7.24 (m,
13
4H), 7.27 – 7.33 (m, 4H), 7.95 (d, J = 8.2 Hz, 1H), 8.07 (t, J = 5.7 Hz, 1H). C NMR (101 MHz,
CDCl ) δ 28.7, 29.2, 35.6, 37.9, 38.0, 40.2, 110.0, 115.4, 125.2, 126.0, 126.1, 126.3, 128.5 (2C),
3
1
28.6 (4C), 128.6 (2C), 138.1, 141.1, 141.9, 142.1, 150.0, 153.5. MS (ESI) m/z: nonꢀionizable
compound under routine conditions used.
ꢀ(2-Cyclohexylethyl)-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (30). The
6
reaction was carried out following method A, using 41b (42 mg, 0.173 mmol) and 4ꢀphenylbutyl
1
isocyanate (40 mg, 0.033 mL, 0.194 mmol). White solid (55 mg, 75%). H NMR (400 MHz,
CDCl ) δ 0.87 – 0.98 (m, 2H), 1.10 – 1.29 (m, 4H), 1.45 – 1.56 (m, 2H), 1.62 – 1.78 (m, 9H),
3
2.62 – 2.69 (m, 4H), 3.44 (q, J = 6.6 Hz, 2H), 7.05 (s, 1H), 7.07 (d, J = 1.2 Hz, 1H), 7.15 – 7.20
1
3
(m, 3H), 7.26 – 7.31 (m, 2H), 7.92 (d, J = 8.2 Hz, 1H), 8.05 (t, J = 5.8 Hz, 1H). C NMR (101
MHz, CDCl ) δ 26.4 (2C), 26.8, 28.7, 29.2, 33.3, 33.4 (2C), 35.6, 37.3, 39.5, 40.2, 109.8, 115.3,
3
125.0, 125.8, 126.0, 128.5 (2C), 128.5 (2C), 140.3, 142.0, 142.1, 150.1, 153.5. MS (ESI) m/z:
nonꢀionizable compound under routine conditions used.
6
-Butoxy-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (31). The reaction was
carried out following method A, using 47a (49 mg, 0.238 mmol) and 4ꢀphenylbutyl isocyanate
1
(46 mg, 0.045 mL, 0.262 mmol). White solid (67 mg, 74%). H NMR (400 MHz, CDCl ) δ 0.98
3
ACS Paragon Plus Environment

Page 25 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
t, J = 7.4 Hz, 3H), 1.44 – 1.55 (m, 2H), 1.63 – 1.81 (m, 6H), 2.67 (t, J = 7.2 Hz, 2H), 3.44 (q, J
=
6.4 Hz, 2H), 3.95 (t, J = 6.5 Hz, 2H), 6.78 (d, J = 2.3 Hz, 1H), 6.79 – 6.82 (m, 1H), 7.14 – 7.21
1
3
(m, 3H), 7.26 – 7.31 (m, 2H), 7.91 (d, J = 8.7 Hz, 1H), 8.00 (t, J = 5.2 Hz, 1H). C NMR (101
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MHz, CDCl ) δ 13.14.0, 19.3, 28.7, 29.2, 31.3, 35.6, 40.2, 68.7, 97.6, 111.1, 116.0, 121.4, 126.0,
3
+
128.5 (2C), 128.5 (2C), 142.0, 142.6, 150.0, 153.5, 156.9. MS (ESI) m/z: 383 [M + H] , 400 [M
+
+
NH ] .
4
6
-(2-Cyclohexylethoxy)-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide
(32).
The reaction was carried out following method A, using 47b (50 mg, 0.190 mmol) and 4ꢀ
1
phenylbutyl isocyanate (37 mg, 0.036 mL, 0.209 mmol). White solid (45 mg, 55%). H NMR
(
400 MHz, CDCl ) δ 0.91 – 1.04 (m, 2H), 1.10 – 1.33 (m, 3H), 1.43 – 1.54 (m, 1H), 1.62 – 1.80
3
(m, 11H), 2.67 (t, J = 7.2 Hz, 2H), 3.43 (q, J = 6.4 Hz, 2H), 3.98 (t, J = 6.7 Hz, 2H), 6.77 (d, J =
2.3 Hz, 1H), 6.79 – 6.82 (m, 1H), 7.15 – 7.20 (m, 3H), 7.26 – 7.30 (m, 2H), 7.91 (d, J = 8.7 Hz,
13
1
H), 8.00 (t, J = 5.7 Hz, 1H). C NMR (101 MHz, CDCl ) δ 26.4 (2C), 26.7, 28.7, 29.2, 33.4
3
(2C), 34.7, 35.6, 36.6, 40.2, 67.0, 97.6, 111.1, 116.0, 121.4, 126.0, 128.5 (2C), 128.5 (2C), 142.1,
˗
1
42.6, 150.01, 153.5, 156.9. MS (ESI) m/z: 260 [M ˗ CONH(CH ) Ph] .
2
4
2-Oxo-6-phenethyloxy-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide
(33).
The
reaction was carried out following method A, using 47c (65 mg, 0.254 mmol) and 4ꢀphenylbutyl
1
isocyanate (49 mg, 0.048 mL, 0.279 mmol). White solid (71 mg, 65%). H NMR (400 MHz,
CDCl ) δ 1.61 – 1.77 (m, 4H), 2.67 (t, J = 7.2 Hz, 2H), 3.10 (t, J = 7.0 Hz, 2H), 3.43 (q, J = 6.4
3
Hz, 2H), 4.17 (t, J = 7.0 Hz, 2H), 6.78 (d, J = 2.2 Hz, 1H), 6.79 – 6.82 (m, 1H), 7.15 – 7.21 (m,
13
4
H), 7.22 – 7.36 (m, 6H), 7.91 (d, J = 8.5 Hz, 1H), 7.99 (t, J = 5.6 Hz, 1H). C NMR (101 MHz,
CDCl ) δ 28.7, 29.2, 35.6, 35.8, 40.2, 69.7, 97.8, 111.2, 116.0, 121.7, 126.0, 126.8, 128.5 (2C),
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 70
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
28.5 (2C), 128.7 (2C), 129.1 (2C), 130.4, 138.0, 142.1, 142.6, 150.0, 156.6. MS (ESI) m/z: 431
+
[
M + H] .
5
-Benzoyl-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (34). The reaction
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
was carried out following method A, using 49 (100 mg, 0.418 mmol) and 4ꢀphenylbutyl
1
isocyanate (81 mg, 0.079 mL, 0.460 mmol). White solid (127 mg, 73%). H NMR (400 MHz,
CDCl ) δ 1.62 – 1.79 (m, 4H), 2.67 (t, J = 7.2 Hz, 2H), 3.44 (td, J = 6.7 Hz, 2H), 7.14 – 7.23 (m,
3
3H), 7.24 – 7.32 (m, 2H), 7.35 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.56 – 7.65 (m, 1H),
13
7.79 (dd, J = 1.5, 8.3 Hz, 3H), 7.98 (t, J = 5.4 Hz, 1H), 8.51 (d, J = 1.7 Hz, 1H). C NMR (101
MHz, CDCl ) δ 28.7, 29.4, 35.6, 40.4, 110.0, 117.7, 126.0, 127.4, 128.0, 128.5 (4C), 128.6 (2C),
3
+
1
30.1 (2C), 132.8, 135.1, 137.4 142.0, 144.5, 149.5, 153.1, 195.2. MS (ESI) m/z: 415 [M + H] ,
+
432 [M + NH ] .
4
6
-Benzoyl-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (35). The reaction
was carried out following method A, using 52a (100 mg, 0.418 mmol) and 4ꢀphenylbutyl
1
isocyanate (81 mg, 0.079 mL, 0.461 mmol). White solid (142 mg, 82%). H NMR (400 MHz,
CDCl ) δ 1.61 – 1.79 (m, 4H), 2.68 (t, J = 6.6 Hz, 2H), 3.47 (q, J = 6.3 Hz, 2H), 7.15 – 7.21 (m,
3
3H), 7.26 – 7.32 (m, 2H), 7.46 – 7.54 (m, 2H), 7.58 – 7.65 (m, 1H), 7.69 – 7.80 (m, 4H), 8.03 (t,
1
3
J = 5.3 Hz, 1H), 8.16 (d, J = 8.6 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.7, 29.1, 35.6, 40.4,
3
111.6, 115.2, 126.1, 128.1, 128.5 (4C), 128.6 (2C), 130.0 (2C), 131.6, 132.8, 134.4, 137.4, 141.8,
˗
142.0, 149.5, 153.2, 194.9. MS (ESI) m/z: 238 [M ˗ CONH(CH ) Ph] .
2
4
6
-(4-Chlorobenzoyl)-2-oxo-N-(4-phenylbutyl)-1,3-benzoxazole-3-carboxamide (36). The
reaction was carried out following method A, using 52b (169 mg, 0.616 mmol) and 4ꢀ
1
phenylbutyl isocyanate (119 mg, 0.116 mL, 0.678 mmol). White solid (66 mg, 26%). H NMR
(400 MHz, CDCl ) δ 1.65 – 1.79 (m, 4H), 2.68 (t, J = 7.0 Hz, 2H), 3.47 (q, J = 6.3 Hz, 2H), 7.16
3
ACS Paragon Plus Environment

Page 27 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
–
7.22 (m, 3H), 7.26 – 7.31 (m, 2H), 7.47 – 7.51 (m, 2H), 7.69 – 7.75 (m, 4H), 8.02 (t, J = 5.4
13
Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H). C NMR (101 MHz, CDCl ) δ 28.7, 29.1, 35.6, 40.4, 111.5,
3
115.2, 126.1, 128.0, 128.5 (4C), 129.0 (2C), 131.4 (2C), 131.7, 133.9, 135.6, 139.4, 141.8, 141.9,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
˗
1
49.4, 153.1 193.7. MS (ESI) m/z: 272 and 274 [M ˗ CONH(CH ) Ph] .
2
4
General Procedure for the Synthesis of Benzoxazolone N-Carboxamides 54, 62-63, and
6 via Method B (Scheme 6, Table 3). Triphosgene (1.0 equiv) was placed in an ovenꢀdried
6
flask, cooled to 0 °C, and dissolved in dry pyridine (5 mL per mmol triphosgene). 3Hꢀ1,3ꢀ
benzoxazolꢀ2ꢀone (50, 1.0 equiv) dissolved in dry pyridine (1.9 mL per mmol 50) was added,
and the reaction mixture was stirred under nitrogen for 30 min at rt. The solution was cooled to 0
°
C and the proper amine (1.5 equiv), dissolved in dry pyridine (3.5 mL per mmol amine), was
added. The reaction was allowed to warm to rt and stirred for 3 h. The solvent was removed
under reduced pressure, and the compound was purified by column chromatography using the
Teledyne ISCO apparatus (cyclohexane/EtOAc).
2
-Oxo-N-(3-phenylpropyl)-1,3-benzoxazole-3-carboxamide (54). The reaction was carried
out following method B, using 50 (70 mg, 0.520 mmol) and 3ꢀphenylpropanꢀ1ꢀamine 67l (106
1
mg, 0.111 mL, 0.784 mmol). White solid (65 mg, 42%). H NMR (400 MHz, CDCl ) δ 1.99 (p, J
3
=
7.4 Hz, 2H), 2.73 (t, J = 7.6 Hz, 2H), 3.46 (q, J = 6.6 Hz, 2H), 7.16 – 7.32 (m, 8H), 8.07 (d, J =
1
3
7
.5 Hz, 1H), 8.07 – 8.14 (m, 1H). C NMR (101 MHz, CDCl ) δ 31.0, 33.2, 39.9, 110.0, 115.7,
3
124.7, 125.1, 126.2, 128.1, 128.5 (2C), 128.6 (2C), 141.1, 141.9, 150.0, 153.3. MS (ESI) m/z:
+
+
2
97 [M + H] , 314 [M + NH ] .
4
2
-Oxo-N-[2-(benzyloxy)ethyl]-1,3-benzoxazole-3-carboxamide (62). The reaction was
carried out following method B, using 50 (70 mg, 0.520 mmol) and 2ꢀ(benzyloxy)ethanꢀ1ꢀamine
hydrochloride 67m (584 mg, 3.11 mmol, 6.0 equiv). The reaction was stirred for 22 h. White
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 70
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
solid (89 mg, 55%). H NMR (400 MHz, CDCl ) δ 3.62 – 3.69 (m, 4H), 4.58 (s, 2H), 7.22 – 7.29
3
1
3
(
m, 4H), 7.31 – 7.39 (m, 4H), 8.03 – 8.08 (m, 1H), 8.36 (br s, 1H). C NMR (101 MHz, CDCl )
3
δ 40.4, 68.3, 73.4, 110.0, 115.7, 124.6, 125.1, 127.9 (3C), 128.1, 128.6 (2C), 137.9, 141.9, 150.1,
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
53.1. MS (ESI) m/z: 313 [M + H] , 330 [M + NH ] .
4
2-Oxo-N-[2-(benzylthio)ethyl]-1,3-benzoxazole-3-carboxamide (63). The reaction was
carried out following method B, using 50 (70 mg, 0.520 mmol) and 2ꢀ(benzylthio)ethanꢀ1ꢀamine
1
hydrochloride 67n (159 mg, 0.780 mmol). White powder (58 mg, 34%). H NMR (400 MHz,
CDCl ) δ 2.69 (t, J = 6.6 Hz, 2H), 3.55 (q, J = 6.5 Hz, 2H), 3.77 (s, 2H), 7.17ꢀ7.37 (m, 8H), 8.03ꢀ
3
13
8
.07 (m, 1H), 8.29 (t, J = 6.2 Hz, 1H). C NMR (101 MHz, CDCl ) δ 31.0, 36.3, 39.4, 110.1,
3
1
15.7, 124.7, 125.1, 127.3, 128.1, 128.7 (2C), 129.0 (2C), 138.1, 141.9, 149.9, 153.1. MS (ESI)
+
+
m/z: 329 [M + H] , 346 [M + NH ] .
4
2
-Oxo-N-[(4-propylphenyl)methyl]-1,3-benzoxazole-3-carboxamide (66). The reaction
was carried out following method B, using 50 (60 mg, 0.444 mmol) and amine 67e (125 mg,
1
0
.673 mmol). White powder (40 mg, 29%). H NMR (400 MHz, CDCl ) δ 0.94 (t, J = 7.3 Hz,
3
3
H), 1.58 – 1.71 (m, 2H), 2.58 (dd, J = 8.4, 6.8 Hz, 2H), 4.58 (d, J = 5.7 Hz, 2H), 7.17 (d, J = 8.1
13
Hz, 2H), 7.21 – 7.32 (m, 5H), 8.07 – 8.14 (m, 1H), 8.35 – 8.46 (m, 1H). C NMR (101 MHz,
DMSO) δ 13.9, 24.7, 37.8, 44.1, 110.0, 115.8, 124.7, 125.1, 128.1, 129.0, 134.6, 141.9, 142.5,
+
+
1
50.0, 153.2. MS (ESI) m/z: 311 [M + H] , 328 [M + NH ] .
4
General Procedure for the Synthesis of Benzoxazolone N-Carboxamides 53, 55-61 and
4-65 via Method C (Scheme 6, Table 3). In an ovenꢀdried flask, triphosgene (1.0 equiv) was
6
dissolved in dry DCM (5 mL per mmol triphosgene). A solution of 3Hꢀ1,3ꢀbenzoxazolꢀ2ꢀone
50, 1.0 equiv) and Et N (4.0 equiv) in dry DCM (5.6 mL per mmol 50) was added at 0 °C, and
(
3
the reaction was stirred for 1 h at rt under nitrogen. Then a solution of the appropriate amine (1.5
ACS Paragon Plus Environment

Page 29 of 70
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
equiv) and Et N (1.5 equiv) in dry DCM (6.5 mL per mmol amine) was added at 0 °C, and the
3
reaction was stirred at rt for 3 h. The reaction mixture was diluted with DCM (20 mL per mmol
5
0) and quenched with sat aq NH Cl (30 mL per mmol 50). The two phases were separated and
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
the aq layer was extracted with DCM (3 × 20 mL per mmol 50). The combined organic phases
were dried over Na SO , evaporated on celite or silica, and the compound was purified by silica
2
4
gel column chromatography using the Teledyne ISCO apparatus (cyclohexane/EtOAc).
-Oxo-N-(phenethyl)-1,3-benzoxazole-3-carboxamide (53). The reaction was carried out
2
following method C, using 50 (60 mg, 0.444 mmol) and 2ꢀphenylethanꢀ1ꢀamine 67k (80 mg,
1
0
.084 mL, 0.660 mmol). White solid (79 mg, 64%). H NMR (400 MHz, CDCl ) δ 2.96 (t, J =
3
7.2 Hz, 2H), 3.69 (td, J = 5.8, 7.2 Hz, 2H), 7.19 – 7.29 (m, 6H), 7.34 (dd, J = 5.8, 8.9 Hz, 2H),
1
3
8.05 – 8.09 (m, 1H), 8.09 – 8.17 (m, 1H). C NMR (101 MHz, CDCl ) δ35.9, 41.8, 110.0, 115.7,
3
124.7, 125.1, 126.9, 128.1, 128.9, 128.9, 138.4, 141.9, 149.9, 153.2. MS (ESI) m/z: 283 [M +
+
+
˗
H] , 300 [M + NH ] . MS (ESI) m/z: 281[M ˗ H] .
4
2
-Oxo-N-(5-phenylpentyl)-1,3-benzoxazole-3-carboxamide (55). The reaction was carried
out following method C, using 50 (68 mg, 0.503 mmol) and amine 67j (100 mg, 0.501 mmol).
1
White solid (83 mg, 51%). H NMR (400 MHz, CDCl ) δ 1.42 – 1.51 (m, 2H), 1.65 – 1.76 (m,
3
4H), 2.66 (t, J = 7.6 Hz, 2H), 3.45 (td, J = 7.1, 5.9 Hz, 2H), 7.16 – 7.22 (m, 3H), 7.24 – 7.32 (m,
1
3
5
H), 8.06 – 8.13 (m, 2H). C NMR (101 MHz, CDCl ) δ 26.4, 29.3, 31.0, 35.7, 40.2, 109.9,
3
115.6, 124.5, 125.0, 125.7, 128.0, 128.3, 128.4, 141.7, 142.3, 149.8, 153.2. MS (ESI) m/z: 325
+
[
M + H] .
2
-Oxo-N-(6-phenylhexyl)-1,3-benzoxazole-3-carboxamide (56). The reaction was carried
out following method C, using 50 (40 mg, 0.300 mmol) and amine 67f (95 mg, 0.440 mmol).
1
White solid (32 mg, 31%). H NMR (400 MHz, CDCl ) δ 1.32 – 1.52 (m, 4H), 1.59 – 1.71 (m,
3
ACS Paragon Plus Environment