Development of potent and selective small-molecule human Urotensin-II antagonists

Article abstract of DOI:10.1016/j.bmcl.2008.05.027

This work describes the development of potent and selective human Urotensin-II receptor antagonists starting from lead compound 1, (3,4-dichlorophenyl)methyl{2-oxo-2-[3-phenyl-2-(1-pyrrolidinylmethyl)-1-piperidinyl]ethyl}amine. Several problems relating to oral bioavailability, cytochrome P450 inhibition, and off-target activity at the kappa opioid receptor and cardiac sodium channel were addressed during lead development. hUT binding affinity relative to compound 1 was improved by more than 40-fold in some analogs, and a structural modification was identified which significantly attenuated both off-target activities.

Full text of DOI:10.1016/j.bmcl.2008.05.027

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3500–3503
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of potent and selective small-molecule
human Urotensin-II antagonists
a
a
a
a
John J. McAtee a, , Jason W. Dodson , Sarah E. Dowdell , Gerald R. Girard , Krista B. Goodman ,
Mark A. Hilfiker a, Clark A. Sehon a, Deyou Sha a, Gren Z. Wang a, Ning Wang a, Andrew Q. Viet a,
Daohua Zhang a, Nambi V. Aiyar b, David J. Behm b, Luz H. Carballo d, Christopher A. Evans c,
Harvey E. Fries c, Rakesh Nagilla c, Theresa J. Roethke c, Xiaoping Xu b, Catherine C. K. Yuan a,
Stephen A. Douglas b, Michael J. Neeb a
*
a Department of Medicinal Chemistry, Cardiovascular and Urogenital Center of Excellence for Drug Discovery,
GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA
b Department of Biology, Cardiovascular and Urogenital Center of Excellence for Drug Discovery,
GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA
c Department of Drug Metabolism and Pharmacokinetics, Cardiovascular and Urogenital Center of Excellence for Drug Discovery,
GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA
d Screening and Compound Profiling, Research Triangle Park, 4117 Emperor Boulevard, Morrisville, NC 27703, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This work describes the development of potent and selective human Urotensin-II receptor antagonists
starting from lead compound 1, (3,4-dichlorophenyl)methyl{2-oxo-2-[3-phenyl-2-(1-pyrrolidinylmeth-
yl)-1-piperidinyl]ethyl}amine. Several problems relating to oral bioavailability, cytochrome P450 inhibi-
tion, and off-target activity at the kappa opioid receptor and cardiac sodium channel were addressed
during lead development. hUT binding affinity relative to compound 1 was improved by more than
40-fold in some analogs, and a structural modification was identified which significantly attenuated both
off-target activities.
Received 2 April 2008
Revised 5 May 2008
Accepted 7 May 2008
Available online 10 May 2008
Keywords:
Urotensin
Antagonist
hU-II
Ó 2008 Elsevier Ltd. All rights reserved.
U-II
hUT
GPR14
Urotensin-II (U-II), a cyclic undecapeptide, was first isolated
in the 1960’s from goby urophysis1 and was proposed to be in-
volved primarily in osmoregulation in fish.2 Human Urotensin-
II (hU-II) and other mammalian orthologs including mouse, rat,
and monkey were subsequently identified and cloned.3 In
1999, hU-II was identified as a cognate ligand of human GPR-
14 (hUT), an ‘orphan’ G-protein-coupled receptor predominantly
expressed in cardiovascular tissue.4 Both U-II ligand and UT
receptor were found within mammalian vascular and cardiac tis-
sue and effectively constricted isolated arteries from non-human
primates.4 The potency of hU-II as a vasoconstrictor was 10
times greater than that of endothelin-1, making hU-II the most
potent mammalian vasoconstrictor identified to date.4 More re-
cently, hU-II was found to induce profound hemodynamic effects
upon local and systemic administration in cat5 and in man.6,7 U-
II also influences cardiorenal function by acting as a potent reg-
ulator of cardiac contractility7 and a natriuretic factor.8 hU-II
and hUT are therefore proposed to be involved in the (dys)regu-
lation of cardiorenal function,9 and have been implicated in the
etiology of numerous cardiorenal and metabolic diseases includ-
ing hypertension,10 heart failure,7,11 atherosclerosis,12 renal fail-
ure,13 and diabetes.14 Several non-peptidic UT ligands have
recently been reported.15–17 Human Urotensin-II receptor antag-
onists are of interest as potential drugs to address these cardio-
vascular conditions.
High-throughput screening (HTS) combined with hit-to-lead
chemistry16a identified compound 1 (Table 1) as an antagonist of
hUT. The binding affinity (Ki) of this compound was 16 nM in a
[
125I]hU-II radioligand binding assay using HEK293 cell mem-
branes stably expressing recombinant human UT receptors.16b,c
Furthermore, this compound also exhibited potency (Kb = 28 nM)
in an isolated rat aorta functional assay17 which was comparable
to the in vitro rat binding affinity. In addition to being functionally
* Corresponding author. Tel.: +1 610 270 6822; fax: +1 610 270 4490.
E-mail addresses: jeff.j.mcatee@gsk.com, jmcatee4@juno.com (J.J. McAtee).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.05.027

J. J. McAtee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3500–3503
3501
Table 1
active in tissues, this compound was found to be a reversible and
surmountable (competitive) antagonist with multi-species activ-
ity.16 Despite the high affinity and potency of (1), the overall dev-
elopability profile of this compound suffered from cytochrome
P450 liabilities (mainly CYP2D6 and CYP3A4 inhibition) and its
pharmacokinetic (PK) profile demonstrated poor oral bioavailabil-
ity, as illustrated in Table 1. The data reveal the large volume of
distribution, high clearance, and low oral bioavailability for this
compound. However, compound 1 served as an acceptable starting
point for further investigation.
In vitro and pharmacokinetic data for 1
(+/-)
N
N
Cl
Cl
N
O
1
In vitro data
Rat PK dataa
One area that was only briefly examined in the initial hit-to-
lead efforts was the amide group appended to the piperidine core.
It was hypothesized that by hindering rotation or introducing addi-
tional conformational constraints in this amide ‘side-chain’ group,
one might improve oral bioavailability18a and possibly bias the
conformation towards one that is more favorable for hUT bind-
hUT Ki = 16 nM
T1/2 = 3.8 h
CYP2D6 = 0.75 lM
CYP3A4 = 1.4 lM
Rat aorta Kb = 28 nM
Vdss = 23 L/kg
CL = 97 mL/min/kg
Oral F (%) = 0–3%
a
Rat PK data based on 2 mg/kg iv dose and 4 mg/kg solution oral dose.
Table 2
SAR summary table
(+/-)
N
N
O
R
In vitro data
b
d
d
#
Side-chain (R)
Amine
hUT Kia (nM)
Kappa EC50 (nM)
Na channel Kia,c (nM)
CYP 2D6 IC50 (lM)
CYP 3A4 DEF IC50 (lM)
1
2
Cl
Cl
N
N
N
Pyrrolidine
Morpholine
16
79
3200
1000
2500
1600
0.75
2.3
1.4
0.77
CH3
3
4
Cl
Cl
CN
Pyrrolidine
Morpholine
2
6
790
5000
1600
4000
2.3
3.7
1.1
0.49
5
6
Cl
Cl
Pyrrolidine
Morpholine 130
8
1000
10,000
960
3200
0.82
3.2
1.2
0.48
O
7
8
Cl
Cl
N
O
O
Pyrrolidine
Morpholine
0.4
2
500
7900
680
1300
1.6
4.8
1.8
0.73
H3C
H3C
N
O
9
—
Pyrrolidine
Morpholine
0.4
5000
—
390
—
5.1
—
3.9
—
—
O
10
11
Cl
Cl
N
O
Pyrrolidine
Morpholine
0.6
3
3200
15,000
930
18,000
2.9
21
1.2
1
N
H3C
H3C
N
O
12
13
Pyrrolidine
Morpholine
1
5
6300
30,000
1400
6900
8.4
6.7
6.8
2.7
N
14
15
Cl
Pyrrolidine
Morpholine
5
40
5000
1300
150
730
2.2
3.5
2.1
1.3
N
O
O
O
16
17
Cl
Pyrrolidine
Morpholine
1.2
6
1300
7900
330
3500
1.7
8.0
1
0.68
N
S
a
Mean of at least two determinations with a standard deviation of < 0.3 log units.
b
c
Single determination or a mean of two determinations with a standard deviation of < 0.3 log units.
Rat brain batrachotoxinin (BTX) sensitive sodium channel assay.
Single determination, Cypex Bactosomes.
d

3502
J. J. McAtee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3500–3503
Table 3
In addition, these compounds have affinity at both the NaV1.5
Rat PK data for representative analogs
cardiac sodium channel and the rat brain batrachotoxinin
(BTX) sensitive sodium channel.20 While all of these compounds
have some level of affinity at the sodium channel, some such as
9 and 14, have nanomolar affinity. Compounds with this level of
sodium channel affinity could not progress through animal mod-
els such as rat pharmacokinetic studies or higher species in vivo
pharmacodynamic studies due to potential cardiac toxicity. It
was also unclear if kappa opioid agonism would interfere with
in vivo studies and complicate the interpretation of results. Thus,
attenuating activity at the kappa opioid receptor and the sodium
channel became goals of equal importance to the improvement
of the P450 and PK profiles. In order to ensure safety in the in
vivo studies, compounds with off-target activities in the mid-
to-high micromolar range at the sodium channel and kappa opi-
oid receptor were targeted. One successful structural modifica-
#
Cmax (ng/mL)
T1/2 (h)
CL (mL/min/kg)
Vdss (L/kg)
Oral F (%)
PK dataa
A1
3
4
5
100
3.8
3.2
2.6
2.8
5.0
97
100
60
120
130
23
27
8
27
26
0–3
12
7
30
<1
120
370
95
17
160
a
Rat PK data based on 2 mg/kg iv dose and 4 mg/kg solution oral dose.
ing.18b The strategy was to hinder rotation of this flexible moiety
by replacing the methyl group with larger substituents, or to elim-
inate entirely one rotatable bond by creating fused rings.
The initial set of compounds that were prepared employed
pyrrolidine as the basic amino group, and were direct analogs
of 1 varying only the amide side-chain moiety. While all com-
pounds in Table 2 showed a measurably higher binding affinity
for hUT than 1, the sub-nanomolar compounds 7, 9, and 10
are especially noteworthy since they have 10-fold greater hUT
binding affinity. Also, compounds 9 and 12 possess an improved
P450 profile for both CYP2D6 and CYP3A4 (DEF) relative to 1.
The comparison of the P450 profile for 9 and 12 to 7 and 10,
respectively, demonstrates the reduction in P450 inhibition that
can be obtained by replacing the chlorine substituents with
methyl groups on the amide moiety. During the course of lead
development, it was discovered that the pyrrolidine analogs de-
scribed in Table 2 have activity at two other molecular targets.
All of these compounds are kappa opioid agonists, and some,
such as 3 and 7 have sub-micromolar activity at this receptor.19
tion which demonstrated
a definite attenuation in the off-
target activity for both was the replacement of the pyrrolidine
moiety with a less basic morpholine group (Table 2). Direct com-
parison of the corresponding pyrrolidine/morpholine analog pairs
(e.g., 10 versus 11) demonstrates that (with the exception of 1
versus 2) a 2- to 20-fold reduction in sodium channel affinity
was realized. Another benefit of the structural change from pyr-
rolidine to morpholine is a general weakening of the kappa ago-
nist potency by 4- to 6-fold for most analogs. Unfortunately, the
fold-selectivity for hUT binding affinity versus both the kappa
opioid receptor and the sodium channel remained largely the
same in both the pyrrolidine and morpholine sets of compounds
due to the parallel loss in hUT binding affinity, which was 5- to
6-fold for most analogs. However, the morpholine compounds,
unlike many of their pyrrolidine counterparts, could be evalu-
ated in vivo because they have off-target activities in the mid-
to-high micromolar range, which is above the safety thresholds
for many in-house in vivo studies. Finally, the results of the
transformation from pyrrolidine to morpholine on P450 inhibi-
tion were mixed, so that CYP2D6 inhibition declined while
CYP3A4 inhibition increased.
R
N
O
Cl
Cl
Cl
Cl
NH2
i, ii, iii
OH
For both the pyrrolidine and morpholine analogs, introducing
conformational constraints appeared to be a successful strategy
for preparing compounds with high hUT binding affinity which
also translated into high functional potency in rats. Compounds
1, 2, 3, 5, and 14 (representing both the pyrrolidine and morpholine
sub-series) were evaluated in the rat aortic ring contraction as-
say,17 and all compounds were functionally potent at levels com-
parable to the in vitro rat binding affinity. For example,
compound 3 has a rat aorta Kb of 5 nM. However, the conforma-
tional constraint strategy was less successful in addressing the
poor oral bioavailability in this series. Only compounds 3 and 5
demonstrated any substantial improvement in oral bioavailability
(Table 3). Although compound 5 does show a markedly improved
oral bioavailability compared to compound 1, the clearance and
volume of distribution are similar to 1, and 5 is a sub-micromolar
CYP2D6 inhibitor.
The preparation of the cis, racemic 3-phenyl-2-(1-amino-
methyl)piperidine core, was described in a prior publication.16a
Preparation of the acid side-chains is described in Scheme 1. The
acid side-chains were coupled to the piperidine core using stan-
dard amide bond-forming reaction conditions (e.g., BOP, diisopro-
pylethylamine, DMF).
In summary, lead development of 1 has led to the identifica-
tion of potent and selective hUT inhibitors. Compounds with re-
stricted conformations in the amide side-chain have improved
hUT binding affinity, which in some cases was more than 10-fold
greater than 1. Modulating the electronics of the aryl group by
replacing the chloride with a methyl group provided a handle
to improve the P450 profile. By replacing the pyrrolidine moiety
18 R = CH3
19 R = CH2CN
20 R = CH2CH2OCH3
OH
O
O
N
O
Cl
Cl
iv, v, vi, iii
Cl
Cl
NO2
F
O
+
HO
O
21
22
OH
O
O
N
H3C
H3C
NH2
OH
H3C
H3C
vii, vi, iii
viii, vi, iii
Br
Br
+
O
O
OH
O
O
O
R
NH2
R
N
+
O
H
R
NH2
R
N
O
23 R = Cl
24 R = CH3
OH
H
N
Cl
O
O
vi, iii
Cl
N
25 X = O
26 X = S
O
X
X
Scheme 1. Reagents and conditions: (i) ethyl bromoacetate, diisopropylethyl-
amine, NMP, 90 °C, 97%; (ii) alkyl halide, K2CO3 or diisopropylethylamine, NMP, 80–
120 °C, 16–63%; (iii) LiOH, THF, H2O, 99%; (iv) KF, dioxane, 100 °C, 96%; (v) SnCl2-
2H2O, EtOH, HCl, 83%; (vi) NaH, ethyl bromoacetate, DMF, 50–90%; (vii) NaHCO3,
Cs2CO3, DMF, 80%; (viii) EtOH, 85 °C, 83%.

J. J. McAtee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3500–3503
3503
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