Deconstruction of sulfonamide inhibitors of the urotensin receptor (UT) and design and synthesis of benzylamine and benzylsulfone antagonists

Article abstract of DOI:10.1016/j.bmcl.2013.01.105

Potent small molecule antagonists of the urotensin receptor are described. These inhibitors were derived via systematically deconstructing a literature inhibitor to understand the basic pharmacophore and key molecular features required to inhibit the prot

Full text of DOI:10.1016/j.bmcl.2013.01.105

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2177–2180
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Deconstruction of sulfonamide inhibitors of the urotensin receptor
(UT) and design and synthesis of benzylamine and benzylsulfone
antagonists
a
a
b
b
b
Steven J. Taylor a, , Fariba Soleymanzadeh , Ingo Muegge , Isamu Akiba , Naoyuki Taki , Saisoku Ueda ,
⇑
Elizabeth Mainolfi c, Anne B. Eldrup d
a Department of Medicinal Chemistry, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877-036, USA
b Department of Molecular and Cellular Biology, Kobe Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd, 6-7-5, Minatojima-Minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047, Japan
c Department of Inflammation and Immunology, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., PO Box 368, Ridgefield, CT 06877-036, USA
d R&D Project Management, Boehringer Ingelheim Pharmaceuticals, Inc., H81/01.006.01 Birkendorfer Str. 6, Biberach, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Potent small molecule antagonists of the urotensin receptor are described. These inhibitors were derived
via systematically deconstructing a literature inhibitor to understand the basic pharmacophore and key
molecular features required to inhibit the protein receptor. The series of benzylamine and benzylsulfone
antagonists herein reported display a combination of nanomolar molecular and cellular potency as well
as acceptable in vitro permeability and metabolic stability.
Received 20 November 2012
Revised 16 January 2013
Accepted 22 January 2013
Available online 4 February 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Urotensin receptor
Urotensin II
Cardiovascular disease
Benzylamines
Benzylsulfones
Urotensin II (U-II) is a small cyclic peptide originally isolated in
1967 from the fish species Gillichtys mirabilis.1 Subsequent to its
discovery, U-II was identified as an extremely powerful vasocon-
strictor, many times more potent than endothelin.2 In 1999, hu-
man U-II was cloned and identified as the natural ligand of the
G-protein coupled receptor GPR14 (urotensin receptor, UT).3 Upon
binding of U-II to UT, a variety of intracellular signaling molecules
are released in the cell that effect a wide range of physiological
conditions, primarily related to cardiovascular function.4 The phys-
iology of UT antagonism is complex; in some tissues binding of U-II
to the UT results in vasoconstriction induced by phospholipase C
and RhoA, whereas in others U-II binding and activation of UT leads
to vasodilatation, hypothesized to be mediated by nitrous oxide
and prostacyclin.5
teams have also disclosed non-peptidic small molecule inhibitors
of the UT,7b however to our knowledge none have been advanced
to clinical evaluation.
To determine the effect of UT antagonism in vivo it is critical to
develop chemical tools that can provide the appropriate receptor
coverage. Towards that goal we examined a number of literature-
based compounds to better understand the pharmacophoric
elements critical for non-peptidic, small molecule receptor antago-
nists of the UT. Initially we chose to deconstruct the known sulfon-
amide inhibitors, 2 and 3 (Fig. 2), to determine the importance of
the various regions of the molecule, and to aid in the construction
of novel chemical matter.8 This series of inhibitors was chosen
Based upon the reported variable pharmacological effects of
U-II, a number of research groups have pursued small molecule
inhibitors of UT, primarily for cardiovascular indications.6 The
most advanced antagonist, Palosuran (Fig. 1, 1), reached Phase II
clinical trials for diabetic neuropathy in 2004.7 Other research
OH
O
N
N
N
H
N
H
1
UT
IC50 (nM)
HLM
%Qh
UT Cell
IC50 (nM)
⇑
Corresponding author.
E-mail addresses: steven.taylor@boehringer-ingelheim.com, steventaylor76@
<5
39
53
gmail.com (S.J. Taylor).
Figure 1. Structure of the urotensin receptor antagonist Palosuran.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.105

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S. J. Taylor et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2177–2180
Cl
O
CF3
CF3
NH
O
O
N
N
N
O
H
CF3
NH2
P.S. Na(CN)BH3
DCM 12 h
Cl
Br
O
O
O
O
NH
HN
S
N
+
S
R
O
O
F3C
Cl
R
O
3
2
Scheme 1. Reagents and conditions: polymer supported Na(CN)BH3, dichloro-
methane, rt, 12 h.
Figure 2. Sulfonamide antagonists of the urotensin receptor.
based upon their conformational rigidity as well as reported po-
tency and in vivo efficacy.9 These compounds are characterized
by two substituted lipophilic aromatic groups linked together by
a sulfonamide moiety. One of the aryl rings is consistently tethered
to an ionizable amine, often restrained into an aliphatic ring sys-
tem (2). We were further interested in the linear amine 3 as this
compound (with an obvious linage to 2), was reported to display
efficacy in a disease-relevant model of heart failure in rodents (cor-
onary arterial ligation).10
Our initial structure–activity relationship (SAR) campaign fo-
cused on determining the role of the sulfonamide linker, as this
was hypothesized to serve as a spacer, primarily positioning the
flanking aryl groups and basic amine in the proper orientation
for antagonist activity. As such, we chose to systematically
examine a variety of two-atom replacements for this moiety.
Compounds were profiled for their ability to displace a radiola-
beled ligand, or inhibit calcium flux in a cell line overexpressing
the UT receptor. Replacement of the nitrogen atom of the sulfon-
amide linker with a carbon resulted in a compound that was
almost equipotent to the analogous literature compound 4 (Table 1,
entries 1 and 2), implying that the amine hydrogen is not involved
in a direct hydrogen bond with the receptor. Reduction of the sul-
fone to either the sulfoxide 7 or the thio ether 8 resulted in a 10-
fold loss in potency (Table 1, entries 4 and 5). The simple benzyl
amine 9 was an order of magnitude less potent than baseline
sulfonamide 4. Furthermore, translocation of the amine to the
right-hand side aromatic group resulted in an additional 10-fold
loss in potency (Table 1, entry 9), suggesting a role for a hydrogen
bond acceptor at this site. This hypothesis was confirmed by profil-
ing of the ether analog 11 that partially recovered the potency lost
with analog 12. A strong enantiomeric preference was observed
favoring the (R) enantiomer of the pyrrolidine ether side chain in
both the benzylamine and benzylsulfone analogs (Table 1, entries
2, 3, 6 and 7). The initial sets of analogs were also profiled in hu-
man liver microsomes as a predictor of in vivo clearance. Of the
analogs tested, benzylamine 9 showed a preferred in vitro meta-
bolic profile and was therefore used as a probe substrate for further
exploration of the SAR on the right hand side of the molecule.
A high-throughput method was developed to evaluate the effect
of substitution of the benzyl aromatic ring of the baseline molecule
9 (Scheme 1). Combining the aniline and a variety of aldehydes at
room temperature and employing polystyrene-supported sodium
cyanoborohydride amines were cleanly obtained by simple filtra-
tion followed by silica gel purification. Using this method, more
than 30 analogs were rapidly synthesized, representatives of which
are shown in Table 2.
Removal of all substitution of the benzyl ring resulted in com-
plete loss of activity against the enzyme (Table 2, entry 3).
Mono-substitution at the 2-, 3- or 4- position (compounds 17–
20) of the aryl ring regained some of the activity that was lost with
compound 15. Cyclization of the two pendant methoxy groups in
13 resulted in a deterioration in activity against the UT receptor
(Table 2, entries 9 and 10). Additionally, isosteric replacement of
the phenyl ring with a thiophene was not tolerated either (24),
however activity could be restored by the addition of a halogen
to the thiophene (Table 2, entry 13). Substituted phenyl analogs
were consistently more active when coupled with the pyrroli-
dine-CF3 aniline B, compared with the straight chain amine chlo-
ride core A (Table 2, entries 1 vs 2 and 4 vs 5).
Table 1
Data for linker replacements/sulfonamide replacementsa,b
N
O
Br
Cl
R
OMe
OMe
Entry Compd Linker (R)
UT IC50 (nM) UT cell IC50 (nM) HLM %Qh
H
N
1
4
23
43
65
S
S
S
O
O
O
O
O
O
2
3
5
34
60
85
83
6*
1280
742
S
4
7
245
nt
72
O
5
6
8
9
440
237
nt
76
28
S
H
N
1328
H
N
7
8
9
10*
11
1350
669
1450
nt
36
53
11
O
The difference in potency between compound pairs 13–14, and
16–17 suggested that the basic amine could be sensitive to simple
modifications and should be explored further. When the sulfon-
amide linker was employed in place of the amine linker, the SAR
of the basic amine tracked with the previous rank order observa-
tions (Table 3 compd 26–27, and 13–14). Conformational restric-
tion of the amine by cyclication lead to compounds that were
five times more potent in both the UT binding and functional as-
says (Table 3, entries 1 and 2). Ring expansion from the pyrrolidine
N
H
12
3060
nt
*
(S) enantiomer was tested.
a
Values are means of a minimum of two experiments (nt = not tested).
b
(S) Enantiomer tested. UT IC50 assay determines the ability of a ligand to inhibit
binding of 125I-U-II to the receptor. UT Cell determines the ability to inhibit U-II
stimulated Ca2+ mobilization in a CHO cell line expressing the human U-II receptor.
HLM in vitro metabolism of compounds in the presence of human liver microsomes,
represented a percentage of total liver blood flow. For a complete description of the
assays see Ref. 11.

S. J. Taylor et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2177–2180
2179
Table 2
Table 3
Data for benzyl substituents on the aniline coresa
Data for basic amine modifications of sulfonamide inhibitorsa
H
N
N
O
N
S
O
O
O
O
O
F3C
Br
Cl
NH
F3C
NH
Entry
1
Compd
LHS linker
UT IC50 (nM)
6
UT cell IC50 (nM)
B
A
O
26
14
Entry Compd LHS Aromatic
UT IC50
(nM)
UT cell IC50
(nM)
N
O
2
3
27
28
134
160
100
1
2
13
14
A
B
1200
217
1300
191
Br
N
OMe
OMe
120
O
O
N
3
15
A
>10,000
nt
4
29
>5000
5500
N
4
5
16
17
A
B
3500
821
6000
1870
O
5
6
30
31
6200
840
nt
nt
O
N
Br
O
N
N
6
7
8
18
19
20
A
B
B
520
714
691
765
7
8
32
800
2100
350
nt
F3C
33*
N
O
OMe
3100
1130
F
9
34
>10,000
nt
*
(S) enantiomer was tested.
a
OMe
Values are means of a minimum of two experiments (nt = not tested). UT IC50
assay determines the ability of a ligand to inhibit binding of 125I-U-II to the receptor.
UT Cell determines the ability to inhibit U-II stimulated Ca2+ mobilization in a CHO
cell line expressing the human U-II receptor. For a complete description of the
assays see Ref. 11.
Br
9
10
11
21
22
23
B
B
B
620
450
520
1400
950
O
O
Br
Table 4
Advanced profiling of benziylamines and benzylsulfone analogsa
O
Cl
CF3
O
O
O
MeO
N
N
OMe
550
O
S
HN
13
5
O
OMe
Br
Br
S
12
13
24
25
B
B
2700
180
2000
600
O
O
Cl
O
O
S
Assay
IC50 UT binding (
13
5
a
Values are means of a minimum of two experiments (nt = not tested). UT IC50
l
M)
M)
M)
0.22
0.19
6.4
3.9
43
<6
>52
>52
2.9
0.22
0.03
0.06
4.7
assay determines the ability of a ligand to inhibit binding of 125I-U-II to the receptor.
UT Cell determines the ability to inhibit U-II stimulated Ca2+ mobilization in a CHO
cell line expressing the human U-II receptor. For a complete description of the
assays see Ref. 11.
UT cell human (
l
UT cell murine (
l
UT cell rat (
HLM (%Qh)
RLM (%Qh)
lM)
2.0
85
66
>87
>87
7.8
32
Solubility pH 4.5 (
Solubility pH 7.4 (
CAC0-2 PEAB (cm/s)
CACO-2 PEBA (cm/s)
l
l
g/mL)
g/mL)
to the piperidine moiety however was not tolerated (28). As previ-
ously observed with the benzylamine and sulfone analogs, there
was a strong enantiomeric preference for the (R) stereoisomer
(Table 3, entries 3 and 4). Reducing the pKa of the amine also
was detrimental to activity (Table 3, entries 5 vs 6). Maintaining
the same distance between the amine and the phenoxy group
but using an alternative pyrrolidine led to a compound (32) that
was significantly less active with the sulfonamide (26) implying
subtle steric constraints at this site. Not unexpectedly, complete
removal of the basic amine resulted in a complete loss in activity
(Table 3 and 34) as has been observed with other UT inhibitors.
Two of the top analogs generated by examining linker replace-
ments were further profiled in more advanced in vitro assays to
a
Values are means of a minimum of two experiments (nt = not tested). UT IC50
assay determines the ability of a ligand to inhibit binding of 125I-U-II to the receptor.
UT Cell determines the ability to inhibit U-II stimulated Ca2+ mobilization in a CHO
cell line expressing the human U-II receptor. HLM in vitro metabolism of com-
pounds in the presence of human liver microsomes, represented a percentage of
total liver blood flow (%Qh). For a complete description of the assays see Ref. 11.
determine other potential liabilities with these classes of com-
pounds (Table 4). The benzylsulfone 5 was a more potent antago-
nist of UT in all species tested, including human, rat, and mouse

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S. J. Taylor et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2177–2180
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than the corresponding benzylamine analog 13. Compounds 13
and 5 both displayed a significant potency shift between the ro-
dent and human receptors. Permeability for the sulfone analog
was acceptable as assessed by CACO2 permeability, whereas the
amine displayed a less attractive profile. The amine however,
was more stable to in vitro metabolism. Solubility for both 5 and
13 were excellent at pH 4.5 and 7.4 (Table 4).
In summary, a potent inhibitor of UT was systematically decon-
structed to determine the generic pharmacophore features neces-
sary for activity. As with many GPCR’s, the basic amine as well as
a properly substituted aromatic right hand side proved critical
for activity. Modification of the central linking atoms was tolerated
although only specific locations of donor and acceptor functional-
ities were tolerated. The linker atoms also played an important
divergent role in in vitro metabolism and permeability assays.
We have applied this information towards designing novel chem-
otypes capable of inhibiting the UT receptor to better understand
the in vivo pharmacological relevance of long term inhibition.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
01.105.
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