Discovery and optimization of novel 4-[(aminocarbonyl)amino]-N-[4-(2-aminoethyl)phenyl]benzenesulfonamide ghrelin receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.08.076

This Letter describes optimization of ghrelin receptor antagonists and inverse agonists starting from a screening hit.

Full text of DOI:10.1016/j.bmcl.2009.08.076

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6237–6240
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of novel 4-[(aminocarbonyl)amino]-N-[4-(2-ami-
noethyl)phenyl]benzenesulfonamide ghrelin receptor antagonists
*
Alexander Pasternak , Stephen D. Goble, Reynalda K. deJesus, Donna L. Hreniuk, Christine C. Chung,
Michael R. Tota, Paul Mazur, Scott D. Feighner, Andrew D. Howard, Sander G. Mills, Lihu Yang
Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
This Letter describes optimization of ghrelin receptor antagonists and inverse agonists starting from a
screening hit.
Received 22 June 2009
Accepted 20 August 2009
Available online 26 August 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Ghrelin
Antagonist
Inverse agonist
Obesity
Ghrelin receptor antagonists have been proposed as potential
therapeutic agents for treatmentof obesity,1 and more recently, type
II diabetes.2 Ghrelin, a 28 amino acid peptide bearing unusual acyl-
ation by octanoic acid at Ser 3, was recently identified as the endog-
enous ligand for the growth hormone secretagogue receptor-1a
(GHS-R1a, ghrelin receptor).3 Activation of the GHS-R1a by ghrelin
leads to release of growth hormone (GH) from pituitary tissue, and
potently stimulates appetite in rodents4 and humans.5 The ghrelin
receptor is a member of the seven-transmembrane G-protein-cou-
pled receptor (GPCR) family, and though widely expressed, is most
abundant in the hypothalamus and pituitary.6 The GHS-R1a is a
Gq-coupled GPCR and is notable for possessing a high level of li-
gand-independent, constitutive receptor activity as measured in
an inositol phosphate (IP) accumulation assay.7 Consequently, the
ghrelin receptor may not be quiescent during inter-prandial periods
even when circulating ghrelin levels are low, and for this reason, it
has been suggested that an inverse agonist may be required to most
effectively block ghrelin receptor mediated orexigenic signaling.7c
Despite the enthusiasm surrounding antagonism of the ghrelin
receptor as a target for obesity, no small molecule ghrelin antagonist
has yet advanced to clinical trials and considerable debate persists
with regard to ghrelin-GHS-R1a function and the prospects for treat-
ment of obesity by targeting this pathway.1b,8 Small molecule ghre-
lin agonists, antagonists, and inverse agonists should serve as useful
tools to address this debate. While GHS-R1a agonists (growth hor-
mone secretagogues) have been widely described in the literature,
relatively fewer antagonists are known, and most of these were dis-
closed recently.1b,9 Several reports claim decreased food intake and
body weight in rodent models following dosing with small molecule
ghrelin antagonists,9,10 although it was not always rigorously estab-
lished that these effects were totally mechanism based (for example,
by showing efficacy in wild type but not in GHS-R1a knockout mice).
As part of our effort to develop ghrelin receptor antagonists for
preclinical proof of concept studies, we identified a number of clo-
sely related compounds typified by compound 1 as ghrelin recep-
tor ligands.
OH
H
N
O
O
O
S
O
N
OH
H
N
N
H
H
1
Compound 1 was highly potent in our GHS-R1a competitive binding
assay employing
35S]-MK-0677 as the radiolabeled probe,11
[
(IC50 = 13 nM) and was determined to be an antagonist in an aequo-
rin bioluminescence functional assay (IC50 = 40 nM; ghrelin at
10 nM) measuring inhibition of ghrelin stimulated intracellular cal-
cium mobilization in HEK-293 cells expressing hGHS-R1a.6,11 Com-
pound 1 and analogs have previously been described as potent b3
* Corresponding author.
E-mail address: alexander_pasternak@merck.com (A. Pasternak).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.076

6238
A. Pasternak et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6237–6240
adrenergic receptor agonists,12 however they suffer from relatively
high molecular weights and poor pharmacokinetic properties.13 In
an effort to eliminate the b3 adrenergic receptor agonist activity,
as well as decrease molecular weight, we undertook the systematic
truncation of lead 1. We began by attempting to remove the 1-ami-
no-3-(aryloxy)propan-2-ol moiety, known to be a critical pharma-
cophore required for b3 adrenergic receptor activity.12a Analogs
were prepared as described previously for the structurally related
b3 adrenergic receptor agonists.12 The analogs were evaluated for
their binding affinities to the cloned hGHS-R1a and for aequorin
functional agonist activities at the hGHS-R1a,6,11 in our attempt to
minimize agonist liability. Table 1 shows that the entire 1-amino-
3-(aryloxy)propan-2-ol moiety is not required for ghrelin receptor
binding potency, however the basic amine appears to contribute
(4). The gem-dimethyl substitution (5) improves binding potency
compared to the unsubstituted aminoethyl (4) by about sixfold.
The N-ethyl analog of 5 had similar potency (6) suggesting no sig-
nificant contribution of the added ethyl to binding affinity. None
of the analogs were agonists based upon our aequorin functional as-
say where all analogs tested showed little or no ghrelin receptor
activation. Importantly, compound 5 showed minimal binding
Table 2
Binding affinities for SAR focused on the aminoethyl subunit
O
O
R
S
O
N
H
N
H
N
H
Compd Position
R
hGHS-R1a
binding
IC50 (nM)
Max
activationb
%
a
5
7
8
p
p
p
p
p
p
m
m
p
–CH2C(CH3)2NH2
–CH2C(CH3)2OH
–CH2NH2
–CH2CH2CH2NH2
–CH2CH2C(CH3)2NH2
–CH2C(CH3)2CH2NH2
–CH2CH2NH2
46
4
—
5
1
3
48%@ 6.25
1715
108
310
444
l
M
9
10
11
12
13
14
2
625
429
13
51
1
–CH2CH2CH2NH2
–CH2C(CH3)2NHCH2CF3 682
a
Values are means of two or three experiments, standard deviations were gen-
erally less than 20% of the mean values.
b
Maximum % activation (agonist activity) relative to ghrelin, hGHS-R1a aequorin
assay, maximum compound concentration = 10 lM.
affinity12 to the human b3 adrenergic receptor (IC50 = 5.2
lM),
and no functional activation12 (hb3 EC50 >10
lM), indicating that
truncation of lead 1 serves to abolish its original b3 adrenergic ago-
nist activity.
NHBoc
d, e
a, b, c
NH2
We further evaluated the importance and position of the amino
group and several relevant analogs are presented in Table 2.
Replacing the amino with a hydroxyl (7) and substitution with
2,2,2-trifluoroethyl (14) led to dramatic potency loss, supporting
that a basic group is required and that reducing basicity is detri-
mental. Shortening the aryl to amine chain length (8) results in po-
tency loss, while increasing the chain length (9) is tolerated.
Installing the gem-dimethyl substitutions into the 3-carbon chain
length analog (10 and 11) did not improve potency as it had in
the original two-carbon chain length series (4 to 5). m-Substituted
aminoethyl (12) and aminopropyl (13) analogs were less potent
than the original p-substituted analogs, and showed increased
functional agonism at the ghrelin receptor as measured in the
aequorin bioluminescence assay.
H2N
NHBoc
f, g
O
S
N
H
O
H2N
NH2
O
O
S
O
N
H
R
N
N
H
H
Scheme 1. Reagents and conditions: (a) KNO3, H2SO4, 95%; (b) Boc2O; (c) H2, Pd/C,
MeOH, 87%, two steps; (d) 4-nitro-benzenesulfonyl chloride, saturated NaHCO3
solution, DCM, vigorous stirring, 89%; (e) H2, (balloon) Pd/C, MeOH, 100%; (f) alkyl
and aryl isocyanates, toluene or dioxane, 60 °C; (g) TFA.
We next turned to optimization of the alkyl urea moiety. A library
of more than one hundred analogs was prepared. A wide variety of
commercially available alkyl and aryl isocyanates were coupled to
1,1-dimethylethyl[2-(4-{[(4-aminophenyl)sulfonyl]amino}
phe-
group, and reduction of the nitro gave the corresponding aniline.
Coupling of the aniline with 4-nitrophenylsulfonyl chloride, fol-
lowed by reduction of the nitro group gave the advanced aniline
intermediate. After coupling to the isocyanate collection, removal
of the Boc-protecting group with TFA afforded the target analogs
Binding potencies for selected urea analogs are presented in Table
3. Modifications to the hexyl group (chain length, branching, exam-
ples 15–20) led to diminished potencies. Phenyl (21), benzyl (22),
and phenethyl (23) ureas had modest potencies; methyl substitu-
tion of phenyl urea indicated a slight potency enhancement com-
pared to the unsubstituted phenyl, with para (26) and meta (25)
isomers appearing most improved. Increasing the substituent size
further improved potency in the p-substituted series only (27–30),
with the optimal substituent being iso-propyl (28). 1-Naphthyl-
methyl urea analog (31) had improved potency relative to the parent
benzyl urea (22). Installation of a benzylic methyl to generate race-
mic 1-naphthylethyl urea 32 led to a sevenfold boost in potency. The
individual enantiomers 33 (R), and 34 (S) were equipotent. Increas-
ing the benzylic substitution from methyl to ethyl (36), or making
the gem-dimethyl analog 35 gave no further advantage relative to
33 and 34. Compound 33 exhibited potent binding to the human
ghrelin receptor (IC50 = 2 nM), was shown to be a hGHS-R1a antago-
nist in the aequorin assay (IC50 = 1 nM), and was further shown to in-
nyl)-1,1-dimethylethyl]carbamate (Scheme 1). This intermediate
was prepared starting from phentermine. Nitration gave the p-nitro
product exclusively. Protection of the amine functionality with a Boc
Table 1
Binding affinities of truncated analogs of 1
R
O
O
S
O
N
H
N
H
N
H
Compd
R
hGHS-R1a binding
IC50 (nM)
Max activationb
%
a
2
3
4
5
6
–H
–Et
4,300
47%@ 6.25 lM
272
46
—
—
5
4
1
–CH2CH2NH2
–CH2C(CH3)2NH2
–CH2C(CH3)2NHEt
30
a
Values are means of two or three experiments, standard deviations were gen-
erally less than 20% of the mean values.
b
Maximum % activation (agonist activity) relative to ghrelin, hGHS-R1a aequorin
assay, maximum compound concentration = 10 lM.

A. Pasternak et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6237–6240
6239
Table 3
as aequorin or FLIPR, and inverse agonism of 33 was only observed
using the IP-SPA assay which measures receptor dependent, phos-
pholipase C mediated accumulation of IP over a 1 h interval.
Compounds 33 and 34 had low binding affinity for the b3
Binding affinities of selected ureas
NH2
O
O
S
O
R
N
adrenergic receptor (b3 IC50 33 >20
had no issues with regard to hERG channel binding (33 hERG
IC50 = 1.9 M). Unfortunately, compounds 33 and 34 demonstrated
lM, IC50 34 = 5.8 lM), and
H
N
N
H
H
l
poor oral bioavailability in rat and mouse, precluding their applica-
tion in oral proof of concept studies. Although compound 33 was
not orally bioavailable in rat (F ꢀ 0%, t1/2 = 2.7 h, Clb = 26 mL/min/
kg, Vdss = 4.6 L/kg), we decided to explore intravenous (IV) dosing.
Dosed at 1 mpk IV in rats the brain/plasma ratio was 0.15 with a
23 nM brain concentration of 33 measured at 4 h post dose. On
dosing 33 in lean SD male rats (3, and 10 mg/kg, IV) no effects were
observed on overnight food intake compared to control animals.
Similarly, no effect on overnight food intake was observed in
diet-induced obese mice at 3 and 10 mpk, IV. In a separate study,
compound 33 at 10 mg/kg IV was shown to inhibit fasting induced
refeeding in lean rats with a 52% reduction in food intake at 1 h
post dose compared to control animals. Unfortunately, at higher
doses (20 mg/kg, IV) adverse effects were noted that suggest that
the inhibition of refeeding may not have been mechanism-based.
The lack of efficacy seen in these models may be the result of the
poor brain/plasma ratio observed for 33. Optimization of the phar-
macokinetic and brain penetration characteristics within this class
was briefly explored, however initial studies which modestly im-
proved oral bioavailability (e.g., by replacing the urea moiety with
heterocycles) led to compounds showing undesired agonist
activity.
In summary, we have described the discovery and optimization
of potent GHS-R1a ligands based on a screening hit. The ligands are
antagonists or inverse agonists, but depending on substitution pat-
terns, can also generate partial agonists. Truncation of the original
screening hit abolished its potent b3 adrenergic activity, while
retaining (and improving) the GHS-R1a binding potency. Milestone
analog 33 was a potent inverse agonist of GHS-R1a. It, and most clo-
sely related compounds, had little oral bioavailability, limiting
in vivo evaluation to the IV route. Compound 33 did not reduce food
intake or body weight in several IV dosed animal models, possibly
owing to its low brain/plasma ratio; higher doses resulted in the
observation of adverse affects. While modifications to improve oral
bioavailability were briefly explored, and further work along these
lines can be envisioned, we suspended work in this area to follow
alternative lead series with better prospects for oral delivery.
Compds
R
hGHS-R1a binding Max activationb
IC50 (nM)
%
a
5
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
n-Hexyl
Ethyl
46
4
4
1
1
1
6
3
1
4
1
1
3
3
1
0
5615
1649
846
474
241
97
263
128
138
161
69
n-Propyl
i-Propyl
n-Butyl
n-Pentyl
n-Heptyl
Ph
Bn
PhCH2CH2
2-Tolyl
3-Tolyl
4-Tolyl
4-Ethylphenyl
77
14
8
4-i-Propylphenyl
4-n-Butylphenyl
4-t-Butylphenyl
1-Napthylmethyl
(R/S)-1-Napthylethyl
(R)-1-Napthylethyl
(S)-1-Napthylethyl
1-Methyl-1-
28
16
21
3
2
2
2
1
1
—
À1
1
1
—
napthylethyl
(R/S)-1-Napthylpropyl
(R/S)-2-Napthylethyl
36
37
4
18
—
—
a
Values are means of two or three experiments, standard deviations were gen-
erally less than 20% of the mean values.
Maximum % activation (agonist activity) relative to ghrelin, hGHS-R1a aequorin
assay, maximum compound concentration = 10 lM.
b
hibit ghrelin stimulated GH release in rat pituitary cells.14 Inclusion
of a 20 nM concentration of 33 led to a 98-fold right shift in the ghre-
lin EC50 for GH release (ghrelin EC50 = 2 nM; EC50 = 196 nM with
20 nM of 33). Compound 33 at 10 lM did not itself increase GH re-
lease over basal levels. Compound 33 was further evaluated for in-
verse agonist activity using an inositol phosphate accumulation-
scintillation proximity assay (IP-SPA)15 in HEK293 cells expressing
rat GHS-R1a, which exhibited robust constitutive activity. As shown
in Figure 1, compound 33 was observed to be both an inverse agonist
(EC50 = 1 nM) and a functional ghrelin antagonist (IC50 = 11 nM;
ghrelin at 1 nM) at the rat ghrelin receptor (rat GHS-R1a binding
IC50 = 6 nM for 33). As noted by Holst, et al.,7a it is difficult to detect
constitutive GPCR signaling using calcium mobilization assays such
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