Pharmacophore Identification and Scaffold Exploration to Discover Novel, Potent, and Chemically Stable Inhibitors of Acid Ceramidase in Melanoma Cells

Article abstract of DOI:10.1021/acs.jmedchem.7b00472

Acid ceramidase (AC) hydrolyzes ceramides, which are central lipid messengers for metabolism and signaling of sphingolipids. A growing body of evidence links deregulation of sphingolipids to several diseases, including cancer. Indeed, AC expression is abn

Full text of DOI:10.1021/acs.jmedchem.7b00472

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Pharmacophore identification and scaffold exploration to discover novel,
potent, and chemically stable inhibitors of acid ceramidase in melanoma cells
Jose Antonio Ortega, Jose M. Arencibia, Giuseppina La Sala, Marco Borgogno, Inga Bauer, Luca
Bono, Clarissa Braccia, Andrea Armirotti, Stefania Girotto, Anand Ganesan, and Marco De Vivo
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017
Downloaded from http://pubs.acs.org on June 13, 2017
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 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Pharmacophore identification and
scaffold exploration to discover novel,
potent, and chemically stable inhibitors of
acid ceramidase in melanoma cells
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, #
1, #
1
Jose Antonio Ortega, Jose M. Arencibia, Giuseppina La Sala,
1
4
3
3
3
Marco Borgogno, Inga Bauer, Luca Bono, Clarissa Braccia, Andrea Armirotti,
4
5
1, 2, *
Stefania Girotto, Anand Ganesan, Marco De Vivo
1
Laboratory of Molecular Modeling & Drug Discovery,
Istituto Italiano di Tecnologia, Via Morego 30, 16163, Genoa, Italy
2
IASꢀ5 / INMꢀ9 Computational Biomedicine Forschungszentrum Jülich
WilhelmꢀJohnenꢀStraße 52428 Jülich, Germany
3
D3ꢀPharmaChemistry, Istituto Italiano di Tecnologia,
Via Morego 30, 16163 Genoa, Italy
4
CompuNet, Istituto Italiano di Tecnologia,
Via Morego 30, 16163 Genoa, Italy
5
Department of Dermatology and Biological Chemistry,
University of California, 202 Sprague Hall, 92697ꢀ2400, Irvine, United States of America
*
Corresponding author:
Dr. Marco De Vivo
Phone: +39 01071781577
Marco.devivo@iit.it
# Equal contributors
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 56
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
Acid ceramidase (AC) hydrolyses ceramides, which are central lipid messengers
for metabolism and signaling of sphingolipids. A growing body of evidence links
deregulation of sphingolipids to several diseases, including cancer. Indeed, AC
expression is abnormally high in melanoma cells. AC inhibition may thus be key to
treating malignant melanoma. Here, we have used a systematic scaffold exploration to
design a general pharmacophore for AC inhibition. This pharmacophore comprises a
6+5 fused ring heterocycle linked to an aliphatic substituent via a urea moiety. We
have thus identified the novel benzimidazole derivatives 10, 21, 27, and 30, which are
highly potent AC inhibitors. Their chemical and metabolic stability is comparable or
superior to that of previously reported AC inhibitors. Moreover, they are potent
against endogenous AC in intact melanoma cells. These novel inhibitors merit further
characterization and can serve as a promising starting point for the discovery of new
antimelanoma therapeutics.
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
ACS Paragon Plus Environment

Page 3 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
1
ꢀ2
The incidence of cutaneous melanoma continues to rise. At the same time,
treating unresectable or metastatic melanoma with conventional cytotoxic agents
3
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
(such as dacarbazine) has failed to produce the expected survival benefit. The
survival of patients with advanced melanoma has been increased by using BRAF and
MEK inhibitors, and by using antibodies that block immuneꢀcheckpoint molecules
(such as ipilimumab, which targets the cytotoxic T lymphocyte antigen 4 (CTLAꢀ
4ꢀ6
4)).
Nevertheless, progression of melanoma due to intrinsic and/or acquired
7
resistance to these novel treatments remains a major issue. In addition, clinicians are
8
concerned about the toxicity, tolerability, and effectiveness of these new agents.
Combination therapy is an alternative and promising approach, where novel targeted
agents and traditional anticancer modalities (radiation, chemotherapy, or surgery) are
9
used together to enhance treatment efficacy and reduce sideꢀeffects. Combination
therapy, however, has not yet demonstrated a significant decrease in the mortality of
melanoma patients. For all these reasons, treatment of advanced melanoma is still an
6
1
unmet medical need, and novel therapeutic strategies are urgently required.
Recent findings have shown that the acid ceramidase (AC) enzyme may be a
10ꢀ12
novel target for treating several types of cancer, including melanoma.
AC is a
cysteine amidase that hydrolyzes the proapoptotic lipidꢀderived messenger ceramide,
13ꢀ15
a central molecule in sphingolipid metabolism and signaling.
The expression of
AC is abnormally high in several tumors, suggesting that the dysregulation of
1
6ꢀ18
ceramide metabolism could be linked to cancer development and progression.
Indeed, AC overexpression has been detected in various types of human cancer (e.g.
melanoma, head and neck, prostate, and colon cancer). Moreover, autoantibodies
against AC have been found in the serum of melanoma patients, suggesting that AC
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 56
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
9
might also be used as a biomarker for disease prognosis. There is also evidence that
overexpression of AC may be related to higher resistance to pharmacological
20ꢀ21
induction of apoptosis,
while AC inhibition sensitizes cancer cells to the action of
anticancer therapeutics,20, 22 Taken together, there is a growing body of evidence that
indicates AC inhibition as an effective and innovative therapeutic approach to treat
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
10, 23ꢀ25
melanoma, as well as other cancers.
In the last two decades, a few potent AC inhibitors have been discovered.
26ꢀ27
28
29
Initially, inhibitors such as NOE and DꢀeꢀMAPP were designed for their similarity
to ceramide, the endogenous AC ligand (Figure 1). However, these inhibitors have
low potency and selectivity, being also active against Czꢀceramideꢀactivated
phosphatase (CAPP) and protein kinase C (PKC). Optimization of these firstꢀ
generation inhibitors led to slightly better activity in vitro and in vivo (ceranib 1 and 2,
23, 26
see Figure 1).
Recently, however, more potent ceramideꢀunrelated inhibitors of
AC have been identified by synthesizing new derivatives of 5ꢀfluorouracil (5ꢀFU) and
by screening commercial chemical libraries, which returned promising AC blockers
30ꢀ32
such as the benzoxazolone carboxamide compounds.
These inhibitors are fairly
potent, being active in the nanomolar range (see benzoxazolone 1, 2, and 3 in Figure
). This class of inhibitors blocks AC by forming a covalent adduct with the catalytic
1
Cysꢀ143. This covalent ligandꢀtarget interaction is due to the presence of a urea group
in the benzoxazolone carboxamide derivatives. The urea is cleaved to generate the
33
covalent ACꢀinhibitor complex.
Although promising, these benzoxazolone
carboxamides derivatives suffer from limited stability, which may hinder their
development as clinically viable therapeutic agents.
4
ACS Paragon Plus Environment

Page 5 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
HN
HO
OH
Ceramide
O
HO
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
H
N
H
N
O
O
OMe
7
7
N-Oleoylethanolamine (NOE)
R
N
H
O
5
Ceranib 1, R= Me
Ceranib 2, R= H
(1S,2R)-N-myristoylamino-
phenylpropanol-1 (D-e-MAPP)
H
N
O
N
O
O
R
Benzoxazolone 1, R= H, IC50 64 nM, t1/2(pH 7.4) 45 min, t1/2(mouse plasma) 60 min
Benzoxazolone 2, R= Br, IC50 32 nM, t1/2(pH 7.4) 24 min, t1/2(mouse plasma) 8 min
Benzoxazolone 3, R= p-F-Ph, , IC50 79 nM, t1/2(pH 7.4) >300 min, t1/2(mouse plasma) >120 min
Figure 1. Ceramide and known acid ceramidase (AC) inhibitors
Here, we report on an extensive exploration of different scaffolds that act by
covalently inhibiting AC, just like their template benzoxazolone carboxamide
derivatives. Our goal was to understand which chemical features are necessary for
potent AC inhibition, while improving chemical stability. Thus, we present a general
pharmacophore for AC inhibition that combines 6+5 fused ring heterocycles with
different aliphatic chains, linked via a urea functional group (Figure 2). We obtained a
set of 30 new compounds, with seven different chemical cores. The four most potent
and stable AC inhibitors 10, 21, 27, and 30 also inhibited AC in intact cells. This
information will be useful for further optimizing these promising compounds.
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 56
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
Results and discussion
Strategy for pharmacophore exploration. Previous studies described a set of potent,
but only fairly stable benzoxazolone inhibitors of the acid ceramidase (AC) enzyme
(Figure 1). These were all characterized by the presence of the urea functional group,
which was demonstrated to be crucial for activity against AC. These potent
compounds are active in the low nanomolar range, acting by forming an adduct with
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
33
the nucleophilic Cysꢀ143 residue of AC. Notably, all these covalent inhibitors are
characterized by an aliphatic substituent, which mimics the alkylic tail of the AC
natural substrate, and which is linked through the urea functional group to the
34
benzoxazolone heterocyclic core.
In an effort to elucidate and improve the pharmacological shortcomings of the
existing covalent inhibitors of AC, we explored the chemistry of a general
pharmacophore for AC inhibition. This was designed by combining 6+5 fused ring
heterocycles and different aliphatic tails (Figure 2). Seven different 6+5 fused ring
heterocycles (benzothiazole, benzoxazole, benzimidazole, indazole, benzotriazole, 4ꢀ
azabenzotriazole, and phathalimide) were combined via a urea functional group to
different aliphatic tails. In this way, these seven different cores were linked to both
hexylꢀ and 4ꢀphenylbutyl fragment tails, which were selected from previous inhibition
31ꢀ34
studies
(Figure 2), obtaining an initial set of 18 compounds. In addition, we
synthesized a set of four 2ꢀbenzimidazole derivatives to study the effect of these
modifications on potency and stability. We also considered a benzylꢀcontaining tail,
generating eight additional compounds, for a total of 30 new derivatives that block the
targeted AC enzyme (Table 1).
6
ACS Paragon Plus Environment

Page 7 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
6
+5 fused rings
R= Aliphatic tail
heterocycle
X= CH and/or N
X= S, O, N
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
Figure 2. Proposed pharmacophore for AC covalent inhibitors.
First, we investigated how the presence of either an exocyclic or endocyclic urea
linked to each different compound core affects their inhibitory activity against AC.
We used either 2ꢀaminobenzothiazole or 2ꢀaminobenzoxazole to synthesize an initial
set of four derivatives containing the exocyclic urea (1ꢀ4 in Table 1). Notably, these
compounds only have an exocyclic amino group accessible for chemical synthesis.
Then, four additional derivatives were synthesized from 2ꢀaminobenzimidazole,
where both endocyclic and exocyclic amino groups could be derivatized (5ꢀ8 in Table
1
). In a second step, after identifying endocyclic ureas as the most active ones, we
synthesized a group of 10 compounds bearing different heterocycles and both hexylꢀ
and 4ꢀphenylbutyl tails (9ꢀ18 in Table 1). In addition, we synthesized a third set of
four compounds containing the 4ꢀphenylbutyl fragment tail, which produced the best
inhibitory results against AC, and different 2ꢀsubstituted benzimidazoles. Finally, we
synthesized a group of eight compounds containing a benzyl group with a single
methyl substituent at different positions along the aliphatic fragment, as an alternative
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 56
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
tail. Encouraged by the results obtained with compounds bearing the 4ꢀphenylbutyl
tail, which resulted in more potent inhibitors, we investigated additional tails formed
by combining an aromatic group with an alkyl fragment.
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
Chemistry. The synthesis of all the differently substituted ureas was efficiently
accomplished by a general strategy, which involved reacting different heterocycle
cores (Het, see Table 1) with an appropriate isocyanate in the presence of a catalytic
amount of 4ꢀ(dimethylamino)pyridine (DMAP) (Scheme 1).
i
Het
1-4, 7-30
Scheme 1. General pathway for urea synthesis. Reagents and conditions: (i) DMAP
(0.1 eq), pyridine, N , room temperature, 5 h.
2
All heterocycles were commercially available, with the exception of 2ꢀmethoxyꢀ
1
Hꢀbenzimidazole, which was synthesized following an adapted procedure from a
35
previously described protocol (Scheme 2).
i
3
4
Scheme 2. Synthesis of 2ꢀmethoxyꢀ1Hꢀbenzimidazole. Reagents and conditions: (i)
Tetramethylortho carbonate (1.4 eq), Ar, 90 °C, 24 h.
Compounds 1ꢀ4 and 7ꢀ30 were obtained in moderate to good yields as indicated in
the SI. The reaction with heterocycles 2ꢀaminobenzimidazole (7ꢀ8) and with 1Hꢀ
36ꢀ37
1
,2,3ꢀtriazolo[4,5ꢀb]pyridine (15ꢀ16) was regioselective, as previously described.
Compounds 19 and 20 were obtained by adding one or two equivalents of the
corresponding isocyanate, respectively.
8
ACS Paragon Plus Environment

Page 9 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Compounds 5 and 6 bear a urea in the exocyclic amino group attached to the 2ꢀ
aminobenzimidazole. They were obtained via an alternative strategy (Scheme 3). The
heterocycle was first selectively protected on N1 with a tert-butyl carbamoyl group,
38
adapting a known procedure. Then, intermediate 31 permitted regioselective urea
formation on the 2ꢀamino group with both hexyl and 4ꢀphenylbutyl isocyanates, in the
presence of catalytic DMAP at 80 °C. Finally, elimination of the protecting group in
pyridine was achieved by heating at 80 °C, yielding the 2ꢀamino derivatives 5 and 6.
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
Table 1. Synthesized compounds.
Cpd
1
Heterocycle
R
Yield
64 %
85 %
64 %
57 %
70 %
70 %
64 %
64 %
20 %
43 %
70 %
61 %
Hexyl
2
4ꢀphenylbutyl
Hexyl
3
4
4ꢀphenylbutyl
Hexyl
5
6
4ꢀphenylbutyl
Hexyl
7
8
4ꢀphenylbutyl
Hexyl
9
1
1
1
0
1
2
4ꢀphenylbutyl
Hexyl
4ꢀphenylbutyl
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 56
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
Hexyl
4ꢀphenylbutyl
Hexyl
64 %
64 %
61 %
62 %
38 %
12 %
1
1
4
5
4ꢀphenylbutyl
Hexyl
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
16
1
7
4
ꢀphenylbutyl
18
1
9
4ꢀphenylbutyl
4ꢀphenylbutyl
23 %
56 %
2
0
2
2
1
2
4ꢀphenylbutyl
4ꢀphenylbutyl
45 %
57 %
2
2
2
3
4
5
Benzyl
64 %
64 %
35 %
60 %
56 %
66 %
68 %
66 %
2ꢀMethylbenzyl
3ꢀMethylbenzyl
4ꢀMethylbenzyl
Benzyl
26
2
2
2
7
8
9
2ꢀMethylbenzyl
3ꢀMethylbenzyl
4ꢀMethylbenzyl
30
1
0
ACS Paragon Plus Environment

Page 11 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
O
R
N
N
O
R
N
i
NH2
ii
N
N
N
H
iii
N
N
H
NH2
N
H
N
H
N
H
BOC
N
H
BOC
3
1
32, R=
33, R=
5, R=
6, R=
Ph
Ph
Scheme 3. Synthesis of 2ꢀaminobenzimidazole exocyclic ureas 5 and 6. Reagents and
conditions: (i) Boc O (1 eq), Et N (3 eq), THF (dry), room temperature, 5 h, Yield
5%. (ii) RꢀNCO (1 eq), DMAP (0.1 eq), pyridine, N , 80 °C, 3 h. For R= Hexyl yield
1%. For R= 4ꢀphenylbutyl yield 12%. (iii) Pyridine, 80 °C, 16 h. For R= Hexyl yield
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
3
9
1
2
70%. For R= 4ꢀphenylbutyl yield 70%
Structure-activity relationships. For the first set of compounds, those bearing
endocyclic ureas are more active than exocyclic ones (Table 2). Compounds 1ꢀ6,
which all have the urea fragment linked to the exocyclic 2ꢀamino group, were inactive
(at the highest concentration tested), whereas compounds 7ꢀ8, which present the same
type of substitution in the endocyclic N1, displayed potent AC inhibition (IC50 2.4 nM
and 0.9 nM, respectively).
Table 2. Exocyclic amine ureas versus endocyclic amine ureas
a
Cpd
Het
R
IC50 (nM)
Carmofur
2
0.7±4.1
1
2
3
4
>10000
>10000
>10000
>10000
1
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 56
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
5
6
7
8
>10000
>10000
2.4±1.1
0.9±0.2
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
a
IC50 values are reported as mean values of three determinations
Based on this evidence, we focused on those heterocycles bearing endocyclic ureas.
Four more 6+5 fused ring heterocycles with an endocyclic amino group were used to
synthesize new derivatives with either a hexyl or 4ꢀphenylbutyl group (compounds 9ꢀ
18, Table 3).
Table 3. Exploration of 5+6 fused ring heterocycles.
Stability t1/2 (min)
IC50
Cpd
9
Het
R
Buffer Buffer Mouse
a
(nM)
pH 4.5 pH 7.4 plasma
6.6±1.1
2.5±0.7
9.9±4.5
4.7±1.5
4.1±1.7
2.1±0.7
>120
>120
>120
>120
>120
>120
59
50
81
134
17
0
>120
>120
>120
>120
13
1
1
0
1
12
13
14
0
1
2
ACS Paragon Plus Environment

Page 13 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
5
6
7
8
2.2±0.5
1.3±0.3
7.0±2.8
2.4±0.4
>120
2
2
9
6
<5
<5
29
23
>120
>120
>120
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
Interestingly, all the considered heterocycles generated potent AC inhibitors. All
the compounds bearing the same aliphatic tail can be considered equipotent,
suggesting that a 6+5 fused ring heterocycle anchored by an endocyclic amino group
is sufficient for AC inhibition. Larger differences were observed when comparing
both aliphatic tails. In all cases, compounds bearing the 4ꢀphenylbutyl tail returned an
IC50 value that was on average twice as potent as those bearing the hexyl tail. Notably,
the presence of a phenyl group in the aliphatic tail of the molecules provided an
advantage in terms of AC inhibition. This slight improvement in potency may be due
3
9
to the preference that the enzyme has for substrates containing more double bonds.
Potency, chemical and metabolic stability. The previously reported covalent
33ꢀ34
inhibitors of AC have limited stability.
In this respect, we selected our most active
compounds 9ꢀ18 to further characterize their stability (Table 3). We tested their
chemical stability in neutral buffered solution in PBS at pH 7.4 and acetate at pH 4.5,
and used mouse plasma to test their metabolic stability. Notably, depending on the
heterocycle, the halfꢀlife of each compound ranged from a few minutes (13ꢀ18) to one
or two hours (9ꢀ12) in neutral buffered solution. This difference in stability was even
more pronounced in plasma where, remarkably, 9ꢀ12 had a halfꢀlife of over 2 hours.
1
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 56
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
To further investigate whether additional modifications of the heterocycle core
could improve the potency and stability of our best AC inhibitors, we focused on the
benzimidazole heterocycle core of 9 and 10, which presented the best compromise
between potency and stability (Table 3). Since a phenyl group is preferred in the
aliphatic tail, we studied a new set of compounds with modifications at the
benzimidazole 2 position, while maintaining the 4ꢀphenylbutyl tail linked to the urea
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 4). The presence of an amino group at postion 2 (8) returned an IC50=0.9 nM,
twice as potent as the nonsubstituted benzimidazole (10). Interestingly, the presence
of a methyl group at the same position (22) also produce a compound that was twice
as potent as the unsubstituted benzimidazole 10. In contrast, introducing a methoxide
substituent (21) resulted in a 4ꢀfold decrease in potency, with an IC50=13.9 nM.
Finally, we considered the 3ꢀdihydrobenzimidazolꢀ2ꢀone heterocycle, which has two
nitrogen atoms that can be derivatized. Thus, we obtained 19 with just one urea,
which was surprisingly inactive, while 20 with two substituents was almost 10ꢀfold
less potent than the unsubstituted benzimidazole 10. The activity with two
substituents as in 20, compared to the inactive derivative 19, is also confirmed by
analogs 35 and 36 (see supporting information), and remains a puzzling result that
merits further investigation. In terms of stability, substituents introduced at the
benzimidazole 2ꢀposition, as in 8 and 22, resulted in less chemically stable
compounds (t1/2 14 and 7 min, respectively). This suggests that modifications
introduced to make the urea group more sensitive to the Cysꢀ143 nucleophilic attack
may potentially render these compounds more easily hydrolysable in solution.
1
4
ACS Paragon Plus Environment

Page 15 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 4. Additional 2ꢀbenzimidazole derivatives exploration.
Stability t1/2 (min)
IC50
Cpd
Structure
Buffer Buffer
Mouse
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
a
(nM)
pH 4.5 pH 7.4
plasma
8
0.9±0.2
2.5±0.7
>10000
43
14
50
9
10
19
>120
>120
>120
>120
>120
2
0
21.0±6.9 >120
13.9±3.9 >120
>120
>120
21
>120
7
>120
11
2
2
1.0±0.4
8
a
IC50 values are reported as mean values of three determinations
Encouraged by the fact that a phenyl group in the aliphatic tail consistently
increased the potency of our inhibitors, we further explored the presence of a benzyl
group in this part of the molecule. All the resulting compounds had a benzyl group
directly attached to the urea. We expected this to affect the electrophilic properties of
the carbonyl group. For these compounds, we used benzimidazole and 2ꢀ
aminobenzimidazole cores, which offer the best balance between potency and
stability. This generated eight new derivatives (Table 5, 23ꢀ26 and 27ꢀ30, i.e. four
new derivatives per core).
1
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 56
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
Substituting the 4ꢀphenylbutyl tail for a benzyl tail in the 2ꢀaminobenzimidazole
and benzimidazole heterocycles produced 23 and 27, which both had diminished
potency (35ꢀfold and 7ꢀfold decrease, respectively). Moreover, the position of a
methyl group in the benzyl tail affected AC activity in different ways. For example,
ortho-substitution in 24 and 28 resulted in a ~2ꢀfold decrease in potency compared
with nonsubstituted benzyl derivatives 23 and 27. This is likely due to the bigger
steric hindrance of the ortho-substituted compounds, which occlude the urea
electrophilic center. In fact, meta- and para-substitutions, 25-26 and 29ꢀ30 have a
better inhibitory potency, even compared to the benzyl derivatives 23 and 27. Again,
this is probably due to an increased lipophilicity of these meta- and para-substituted
compounds, compared to nonsubstituted benzyl urea. This is in accordance with the
hydrophobic nature of the natural AC substrate.
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. Exploration of benzyl ureas.
Stability t1/2 (min)
IC50
Cpd
8
R
X
Buffer
pH 4.5
43
Buffer
pH 7.4
14
Mouse
plasma
9
a
(nM)
0.9±0.2
2
2
3
4
29.3±6.2
42
27
27
13
10
11
13
<5
<5
NH
2
68.4±3.3
8.5±3.3
25
1
6
ACS Paragon Plus Environment

Page 17 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
6
0
7.0±0.3
34
23
<5
1
2.5±0.7
>120
94
50
>120
108
27
22.5±4.6
>120
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
2
3
8
9
0
55.9±10.2
8.3±1.5
82
58
56
44
71
94
H
>120
>120
14.8±2.8
>120
a
IC50 values are reported as mean values of three determinations
Related to the stability of these benzyl derivatives, a significant effect was again
observed depending on the heterocycles. In agreement with 8 containing 4ꢀ
phenylbutyl urea (Table 5), derivatives based on the 2ꢀaminobenzimidazole
heterocycle were less stable (chemically and metabolically) than those based on
benzimidazole heterocycle, resulting in halfꢀlife values of less than 30 min. However,
for benzimidazole derivatives, both benzyl and 4ꢀmethylbenzyl substituents returned
more stable compounds, reaching >120 min as halfꢀtime, with potency in the low nM
range (see 27 and 30 in Table 5). These results support this para-substituted benzyl
fragment as a valid alternative to our pharmacophore aliphatic tails.
Study of the mechanism of inhibition. A covalent mechanism of action has been
33ꢀ34
proved for previously reported benzoxazolone AC inhibitors.
Specifically, it was
confirmed that Cysꢀ143 attacks to urea carbonyl group of the benzoxazolone, with the
butyl 4ꢀphenylbutyl remained attached to the enzyme AC (Figure 3). As for the
3
3ꢀ34
benzoxazolone carboxamides,
here we used liquid chromatography mass
spectrometry (LCꢀMS) to characterize the mode of action of compounds 10 and 21.
1
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 56
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
As expected, results demonstrate that these compounds act through the same
mechanism of action observed for benzoxazolone AC inhibitors (Adduct A, Figure
3
A). Incubation with purified recombinant human AC, followed by trypsin digestion
and peptide analysis by LCꢀMS, showed the formation of an adduct with the Nꢀ
terminal peptide of AC (CTSIVAEDK, Figure 3B, red trace), for both inhibitors. In
contrast, only native unmodified peptide was observed after control incubation of AC
with DMSO. For both compounds 10 and 21, tandem MS analysis showed the
formation of the same adduct (Figure 3C). That is, it was observed a pick of the 4ꢀ
phenylbutyl carboxamide fragment (Adduct A) present in all three compounds (see
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
3ꢀ34
supporting information).
No evidence supporting alternative mode of action and
no adducts with other AC tryptic peptides were observed, which indicate that our new
compounds covalently bind to Cysꢀ143.
1
8
ACS Paragon Plus Environment

Page 19 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A
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
Benzoxazolone
Adduct A
Adduct B
R= H, Compound 10
R= OMe, Compound 21
Adduct A
B
C
Figure 3. A. Adduct formed by covalent modification of catalytic Cysꢀ143 by
33ꢀ34
reported benzoxazolone AC inhibitor
and possible adducts formed upon
nucleophilic attack of the catalytic Cysꢀ143 on compounds 10 and 21. B.
Chromatograms of peptide analysis after AC trypsin digestion incubated with
compound 10 or 21 (red trace) and DMSO (blue trace). C. Tandem mass spectrum
1
9
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 56
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
confirming CTSIVAEDK peptide sequence and mass increased of covalently
modified Cysꢀ143 (+ 175.10 Da), in concordance with adduct A formation (+
11
C H13NO). See also supporting information.
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
Computational analysis. On the most potent and promising AC inhibitors
(compounds 8, 10, 19ꢀ22), we performed a coordinate scan analysis as a function of
the dihedral angle ψ centered along the N1ꢀC1’ axis of the C2ꢀN1ꢀC1’ꢀO1’ atoms of
40ꢀ41
the urea moiety.
In this way, we could identify the most probable (i.e. lowest
energy) conformations, which are likely adopted by the urea during nucleophilic
attack by the Cysꢀ143 residue in AC, generating the covalent enzymeꢀligand
42ꢀ45
adduct.
Figure 4. Coordinate scan of the urea torsional angles of compounds 8, 10, 19-22.
The pink sphere represents the global minimum conformation, which is shown as a
2D structure. The exo conformation is preferred for 8, 10, and 22 and the endo
2
0
ACS Paragon Plus Environment

Page 21 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
conformation for 19-21. The dark gray spheres in position 1 represent the phenylbutyl
moiety.
As expected, in all the compounds we detected two energetic minima for
was located at = 180°, where the oxygen atom of the urea group at position 1 is
oriented toward the ‘outer’ side of the molecule (hereafter referred to as the exo
conformation, Figure 4). One was located at = 360°, where the oxygen atom points
ψ. One
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
ψ
ψ
toward the ‘inner’ side of the molecule (hereafter referred to as the endo conformation,
Figure 4). Interestingly, the most active compounds 8, 10, and 22 (IC of 0.9 ± 0.2
50
nM, 2.5 ± 0.1 nM, and 1.0 ± 0.4 nM, respectively) prefer the exo conformation, while
the others prefer endo conformations. Compound 8 is highly stabilized in exo thanks
to the formation of an intramolecular Hꢀbond between the amino group in position 2
and the oxygen atom of the urea. However, exo conformations are also preferred in 10
and 22 in the absence of this Hꢀbond, indicating that this conformation is stabilized by
the nature of the central core. In fact, the less active compounds 19ꢀ21, (IC50 of
>
10000 nM, 21.0 ± 6.9 nM, and 13.9 ± 3.9 nM, respectively) prefer the endo
conformation in which they form an intramolecular Hꢀbond (Figure 4). These results
suggest that the nucleophilic attack performed by Cysꢀ143 is favored when the urea
assumes the exo conformation, which therefore identifies more potent compounds. In
other words, based on our computational analysis, we hypothesize that the AC active
site is more selective toward the exo conformation of the amide group of the inhibitor.
Then, when bound, the amideꢀbond might undergo to a slight distortion, which has
46ꢀ47
been suggested to facilitate the nucleophilic attack for amide hydrolysis.
In conclusion, the minimal energy conformation assumed by and, in particular,
the stabilization of the exo conformation seems to be an important prerequisite for the
ψ
48
rational design of a potent compound against AC.
2
1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 56
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
Targeting AC in melanoma cells.
Based on in vitro activity results on AC inhibition, as well as on the stability
studies, benzimidazole derivatives 10, 21, 27, and 30 were also tested for their ability
to inhibit endogenous AC in intact cells. The assay is based on the intracellular
enzymoꢀchemical transformation of the AC substrate Rbmꢀ14ꢀ12 into fluorescent
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
umbelliferone. To this end, we used two different melanoma cell lines, A375 and Gꢀ
3
61, which are representative of the invasive and proliferative phenotype of the
4
9
disease, respectively. After 2 h of treatment, all four compounds inhibited
endogenous AC activity with variable potencies in both cell lines (Table 6). The A375
cell line was more sensitive to AC inhibition than Gꢀ361. This difference is likely due
to higher levels of expression of catalytically competent AC in proliferative Gꢀ361
49
melanoma cells than in invasive A375 cells. The inhibitory activity of these
compounds against AC in intact cells correlated well with that obtained in vitro, with
1
0 being the most potent and promising compound. In addition, the benzimidazole
derivatives had improved stability and comparable potency to carmofur, which has
49
been used to generate similarly active derivatives to block AC. The effect observed
for these compounds in melanoma cells can be attributed to their ability to block AC
activity. In fact, under these experimental conditions, we did not observe any
cytotoxic effect of the compounds, alone. Moreover, cytotoxicity assays treating the
cells for 72 hours demonstrated the lack of cytotoxicity (see supporting information).
2
2
ACS Paragon Plus Environment

Page 23 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
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
Table 6. Effect of AC inhibitors in melanoma cells
IC50 (nM)
IC50 (nM)
IC50 (nM)
Compound
Structure
a
a
a
Enzyme
A375 cells
G-361 cells
Carmofur
20.7±4.1
190.8±2.5
758.5±434.9
10
2.5±0.7
13.9±3.9
22.5±4.6
75.6±3.3
229.7±69.6
2
2
3
1
7
0
966.9±80.9
2784.1±781.6
1061.5±72.8 2799.0±883.9
14.8±2.8
505.3±29.6
928.3±54.8
a
IC50 values are reported as mean values of three determinations
2
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 56
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
Conclusions
In the present work, we have identified a pharmacophore model that captures
essential features for covalent inhibition of the AC enzyme. Our results show that a
chemical structure formed by a 6+5 fused ring heterocycles, functionalized at the
endocyclic amino group, is critical for AC inhibition. Although all the fused ring
heterocycles considered were quite potent, each returned different results in terms of
chemical stability. In this regard, benzimidazole derivatives showed the best balance
between potency and stability. Exploration of 2ꢀbenzimidazole substituents yielded
slightly more potent compounds when an electrodonating group was inserted, as in 8
and 22. However, electrowithdrawing substituents helped both chemical and
metabolic stability, with a compound halfꢀlife of more than two hours. Our findings
also show that compounds with an aliphatic chain that contains an aromatic group are
more potent in blocking AC. We have demonstrated this for tails based on a 4ꢀ
phenylbutyl fragment as in 10, 12, 14, 16, and 18, compared to those with the hexyl
group as in 9, 11, 13, 15, and 17. Along these lines, a benzyl fragment in the tail
showed different effects depending on methyl substituent position. While a naked
benzyl fragment resulted in less potent compounds, as in 23 and 27, the presence of a
methyl group showed different effects depending on its position. Orthoꢀ substitution,
as in 24 and 28, had diminished potency, probably due to a larger steric hindrance.
We also observed a slight decrease in potency for metaꢀ derivatives, as in 25 and 29,
and paraꢀ, as in 26 and 30, especially for benzimidazole heterocycle. Benzyl
substituents also affected chemical and metabolic stability. For example, 2ꢀ
aminobenzimidazole derivatives are unstable compounds. However, when the
benzimidazole core is linked to either a benzyl or a paraꢀmethylbenzyl group,
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
4
ACS Paragon Plus Environment

Page 25 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compounds become more stable, reaching an optimal halfꢀtime that exceed two hours,
as for 27 and 30.
In summary, our scaffold exploration and derivatization suggest a general
pharmacophore for AC inhibition formed by a 6+5 fused ring heterocycle bearing a
urea at an endocyclic nitrogen group. This nitrogen should be derivatized with an
alkyl chain presenting a phenyl substituent. In our study, this general scaffold resulted
in benzimidazole derivatives 10, 21, 27, and 30, which are potent and fairly stable AC
inhibitors, with promising activity against AC in different melanoma cell lines. These
novel AC inhibitors merit further characterization, and can serve as a promising
starting point for the discovery of new therapeutics for melanoma.
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
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 56
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
All the commercial available reagents and solvents were used as purchased from
vendors without further purification. Dry solvent pyridine was purchased from Sigmaꢀ
Aldrich. Automated column chromatography purifications were done using a
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
f
Teledyne ISCO apparatus (CombiFlash R ) with prepacked silica gel columns of
different sizes (from 4 g up to 24 g) and mixtures of increasing polarity of
cyclohexane and ethyl acetate (EtOAc) or cyclohexane and tertꢀbutyl methyl ether
(TBME). NMR experiments were run on a Bruker Avance III 400 system (400.13
1
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 (DMSO–d ) or
deuterated chloroform (CDCl ) as solvents. UPLC/MS analyses were run on a Waters
6
3
ACQUITY UPLC/MS system consisting of an SQD (single quadrupole detector)
mass spectrometer equipped with an electrospray ionization interface, and a
photodiode array detector. The PDA range was 210–400 nm. Analyses were
performed on an ACQUITY UPLC BEH C18 column (100x2.1mmID, particle size 1.7
µm) with a VanGuard BEH C18 precolumn (5x2.1 mmID, particle size 1.7 µm).
Mobile phase was 10 mM NH
4 2 3
OAc in H O at pH 5 adjusted with CH COOH (A) and
10 mM NH OAc in CH CN–H O (95:5) at pH 5.0 (B). Depending on the analysis
4
3
2
method used, a different gradient increasing the proportion of mobile phase B was
applied. For analysis method 1, mobileꢀphase B proportion increased from 5% to 95%
in 3 minutes. For analysis method 2, mobileꢀphase B proportion increased from 50%
to 100% in 3 min. Electrospray ionization in positive and negative mode was applied.
All tested compounds showed ≥ 95% purity by NMR and UPLC/MS analysis.
2
6
ACS Paragon Plus Environment

Page 27 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
General procedure for urea synthesis
Under N atmosphere, to a room temperature solution of corresponding
heterocycle (1.0 mmol) and 4ꢀ(Dimethylamino)pyridine (0.1 mmol) in dry pyridine
2.7 ml) was dropwise added corresponding isocyanate (1.0 mmol). Then, the reaction
crude was stirred at room temperature for 5 h, portioned between AcOEt (20 ml) and
NaHCO saturated solution (20 ml), the organic layer extracted with brine (20 ml),
dried over Na SO , and concentrated to dryness at low pressure. The pure compounds
2
(
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
3
2
4
were afforded by purification by typical silica gel flash chromatography, using
mixtures of increasing polarity of cyclohexane and ethyl acetate (EtOAc) or tertꢀbutyl
methyl ether (TBME), followed by trituration in diethyl ether.
Synthesis of 1-(1,3-benzothiazol-2-yl)-3-hexyl-urea (1) – in Table 1
Compound 1 was synthesized following the general procedure previously
described using 2ꢀaminobenzothiazole (100 mg, 0.65mmol) and hexyl isocyanate
(0.10 ml, 0.65 mmol). Purification by typical silica gel flash chromatography
(cyclohexane/TBME from 95:5 to 70:30) and final trituration in diethyl ether twice (2
x 1 ml) afforded 1 (111 mg, 64%) as a white solid. Rt = 2.70 min (analysis method 1);
+
1
MS (ESI) m/z: 278.1 [MꢀH] , calculated 278.1. H NMR (400 MHz, DMSOꢀd
0.58 (s, 1H), 7.85 (d, J = 7.1 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.40 – 7.27 (m, 1H),
7.19 (m, 1H), 6.74 (t, J = 5.6 Hz, 1H), 3.12 – 3.16 (m, 2H), 1.44 (q, J = 7.0 Hz, 2H),
6
) δ
1
13
1
.35 – 1.23 (m, 6H), 0.92 – 0.79 (m, 3H). C NMR (101 MHz, DMSOꢀd ) δ 125.71
6
2 2
(CH), 122.54 (CH), 121.28 (CH), 119.82 (CH), 39.08 (CH ), 30.93 (CH ), 29.36
(
CH ), 25.96 (CH ), 22.06 (CH ), 13.91 (CH ). qNMR: 97.3%.
2
2
2
3
Synthesis of 1-(1,3-benzothiazol-2-yl)-3-(4-phenylbutyl)urea (2)
2
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 56
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
Compound 2 was synthesized following the general procedure previously
described using 2ꢀaminobenzothiazole (50 mg, 0.32mmol) and 4ꢀphenylbutyl
isocyanate (0.06 ml, 0.32 mmol). Purification by typical silica gel flash
chromatography (cyclohexane/TBME from 95:5 to 60:40) and final trituration in
diethyl ether twice (2 x 1 ml) afforded 2 (89 mg, 85%) as a white solid. Rt = 2.73 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
+
1
(
analysis method 1); MS (ESI) m/z: 326.1 [MꢀH] , calculated 326.1. H NMR (400
MHz, DMSOꢀd ) δ 10.59 (s, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H),
.35 (td, J = 8.1, 7.7, 1.2 Hz, 1H), 7.27 (t, J = 7.5 Hz, 2H), 7.23 – 7.19 (m, 3H), 7.21
7.12 (m, 1H), 6.77 (t, 1H), 3.19 (q, J = 6.6 Hz, 2H), 2.60 (t, J = 7.5 Hz, 2H), 1.68 –
6
7
–
13
1.54 (m, 2H), 1.54 – 1.41 (m, 2H). C NMR (101 MHz, DMSOꢀd ) δ 142.09 (CH),
6
1
3
28.29 (CH), 128.24 (CH), 125.72 (CH), 125.67 (CH), 122.56 (CH), 121.29 (CH),
8.98 (CH ), 34.76 (CH ), 29.09 (CH ), 28.33 (CH ). qNMR: 97.5%.
2
2
2
2
Synthesis of 1-(1,3-benzoxazol-2-yl)-3-hexyl-urea (3)
Compound 3 was synthesized following the general procedure previously
described using 2ꢀAminobenzoxazole (100 mg, 0.72 mmol) and Hexyl isocyanate
(
0.11 ml, 0.72 mmol). Purification by typical silica gel flash chromatography
cyclohexane/TBME from 95:5 to 70:30) and final trituration in diethyl ether twice (2
(
x 1 ml) afforded 3 (48 mg, 64%) as a white solid. Rt = 2.81 min (analysis method 1);
+
1
MS (ESI) m/z: 262.1 [MꢀH] , calculated 262.2. H NMR (400 MHz, DMSOꢀd ) δ
6
1
1
1
1.03 (s, 1H), 8.39 (t, J = 5.7 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.50 (d, J = 7.5 Hz,
H), 7.28 (td, J = 7.7, 1.2 Hz, 1H), 7.21 (td, J = 7.7, 1.2 Hz, 1H), 3.30 – 3.20 (m, 2H),
13
.52 (q, J = 7.3 Hz, 2H), 1.31 (m, 6H), 0.98 – 0.77 (m, 3H). C NMR (101 MHz,
) δ 157.18 (CO), 151.95 (C), 146.95 (C), 140.31(C), 124.53 (CH), 122.89
DMSOꢀd
6
(CH), 117.14 (CH), 109.77 (CH), 39.37 (CH ), 30.89 (CH ), 29.29 (CH ), 25.98
2
2
2
(
CH ), 22.04 (CH ), 13.88 (CH ). qNMR: 97.1%.
2
2
3
2
8
ACS Paragon Plus Environment

Page 29 of 56
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis of 1-(1,3-benzoxazol-2-yl)-3-(4-phenylbutyl)urea (4)
Compound 4 was synthesized following the general procedure previously
described using 2ꢀAminobenzoxazole (50 mg, 0.36 mmol) and 4ꢀphenylbutyl
isocyanate (0.06 ml, 0.36 mmol). Purification by typical silica gel flash
chromatography (cyclohexane/TBME from 100:0 to 80:20) and final trituration in
diethyl ether twice (2 x 1 ml) afforded 4 (65 mg, 57%) as a white solid. Rt = 2.80 min
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
(
analysis method 1); MS (ESI) m/z: 310.1 [MꢀH] , calculated 310.2. H NMR (400
MHz, DMSOꢀd ) δ 11.03 (s, 1H), 8.39 (t, J = 5.7 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H),
.49 (d, J = 7.7 Hz, 1H), 7.32 – 7.23 (m, 3H), 7.23 – 7.11 (m, 4H), 3.28 (q, J = 6.6 Hz,
6
7
1
3
2H), 2.62 (t, J = 7.5 Hz, 2H), 1.67 – 1.61 (m, 2H), 1.59 – 1.49 (m, 2H). C NMR
(
(
(
101 MHz, DMSOꢀd
C), 128.29 (CH), 128.24 (CH), 125.67 (CH), 124.54 (CH), 122.91 (CH), 117.15
CH), 109.78 (CH), 38.76 (CH ), 34.73 (CH ), 29.02 (CH ), 28.34 (CH ). qNMR:
6
) δ 157.17 (CO), 151.99 (C), 146.97 (C), 142.09 (C), 140.31
2
2
2
2
97.4%.
Synthesis of 1-(1H-benzimidazol-2-yl)-3-hexyl-urea (5)
Step 1. Synthesis of tert-butyl 2-aminobenzimidazole-1-carboxylate (31
)
A room temperature solution of 2ꢀaminobenzimidazole (200.0 mg, 1.46 mmol),
Diꢀtertꢀbutyl dicarbonate (327.8 mg, 1.46 mmol) and triethylamine (0.6 ml, 4.37
mmol) in dry THF (2.4 ml) was stirred at room temperature for 5 h. Then the reaction
crude was portioned between AcOEt (20 ml) and NaHCO saturated solution (20 ml),
3
2 4
the organic layer extracted with brine (20 ml), dried over Na SO and concentrated to
dryness at low pressure. Purification by typical silica gel flash chromatography
(cyclohexane/AcOEt from 75:25 to 65:45) afforded 31 (323 mg, 95%) as a white
+
solid. Rt = 2.03 min (analysis method 1); MS (ESI) m/z: 234.2 [MꢀH] , calculated
1
2
34.1. H NMR (400 MHz, CDCl ) δ 7.61 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 7.5 Hz,
3
2
9
ACS Paragon Plus Environment