Discovery of aryl ureas and aryl amides as potent and selective histamine H3 receptor antagonists for the treatment of obesity (Part I)

Article abstract of DOI:10.1016/j.bmcl.2013.03.080

A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective histamine H₃ receptor (H₃R) antagonists. The SAR was explored and the data obtained set up the starting point and foundation for further optimization. The most potent tool compounds, as exemplified by compounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-H₃R FLIPR assays and rhesus monkey H₃R binding assays.

Full text of DOI:10.1016/j.bmcl.2013.03.080

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3416–3420
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of aryl ureas and aryl amides as potent and selective
histamine H3 receptor antagonists for the treatment of obesity
(Part I)
b,
Zhongli Gao a, , William J. Hurst , Etienne Guillot c, Werngard Czechtizky d, Ulrike Lukasczyk d,
⇑
Raisa Nagorny b, Marie-Pierre Pruniaux c, Lothar Schwink d, Juan Antonio Sanchez c, Siegfried Stengelin d,
Lei Tang b, Irvin Winkler d, James A. Hendrix b, Pascal G. George c
a LGCR SMRPD Chemical Research, Sanofi US, 153-1-122, 153 2nd Ave, Waltham, MA 02451, United States
b Sanofi US, 55C-420A, 55 Corporate Drive, Bridgewater, NJ 08807, United States
c Sanofi R&D, Chilly-Mazarin, France
d Sanofi R&D, Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, 65926 Frankfurt, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 29 March 2013
A series of structurally novel aryl ureas was derived from optimization of the HTS lead as selective his-
tamine H3 receptor (H3R) antagonists. The SAR was explored and the data obtained set up the starting
point and foundation for further optimization. The most potent tool compounds, as exemplified by com-
pounds 2l, 5b, 5d, and 5e, displayed antagonism potencies in the subnanomolar range in in vitro human-
H3R FLIPR assays and rhesus monkey H3R binding assays.
Keywords:
Histamine H3 receptor
Antagonist/inverse agonist
Obesity
Ó 2013 Elsevier Ltd. All rights reserved.
Aryl urea
Obesity1 is characterized by accumulation of excess body fat
and can be conceptualized as the physical manifestation of chronic
energy excess. Thus, the fundamental cause of obesity and over-
weight is an energy imbalance between calories consumed and cal-
ories expended. Obesity is rising globally and severe obesity (body
mass index BMI of 40 kg/m2 or 35 kg/m2 with co-morbidity)2 is
growing at an even faster rate. Worldwide obesity has more than
doubled since 1980. Today, one American in three is obese and this
epidemic threatens many other countries, too. Obesity is an impor-
tant risk factor for both cardiovascular disease and diabetes.
Eating behavior is regulated by a complex interplay of central
neurotransmitter systems, peripheral endocrine stimuli, the circa-
dian rhythm, and environmental cues, all factors that change the
behavioral state and alter homeostatic aspects of appetite and en-
ergy expenditure. Brain histamine has long been considered a sati-
ety signal in the central nervous system.3 Histaminergic neural
circuits arise in the tuberomammillary nucleus, and project into
the satiety centers of the hypothalamus and participate in regula-
tion of energy homeostasis.4 The intrasynaptic level of histamine is
primarily controlled by feedback signals from pre-synaptic hista-
dine to histamine and the release of histamine into the synaptic
cleft. Despite conflicting preclinical data, insights are emerging
into the potential role of H3R as a target of anti-obesity therapeu-
tics.5 This has attracted many pharmaceutical companies to set up
programs in discovery of H3R antagonists for the treatment of
obesity. The efforts in the field have resulted in many H3R antago-
nists in diverse structural types.6 When we started our H3R pro-
gram, we aimed to identify H3R antagonists that inhibit food
intake in the standard animal models, possess a predictable phar-
macokinetic (PK) profile consistent with once-a-day dosing in hu-
mans, and demonstrate an acceptable safety profile.
High-throughput screening (HTS) of the in-house collections
against a HEK293 cell-line stably expressing human H3 receptors
(h-H3R) using imetit as an agonist under the standard calcium
mobility based assay conditions (FLIPR)7 in 384 well-format iden-
tified among other hits, a set of biaryl urea derivatives exemplified
by compound 1.
This set of the compounds is unique in that the molecules are
symmetrical, with phenethylamines connected by a urea core.
However, researchers from Johnson and Johnson8 outlined issues
observed with diamine compounds such as 1a (JNJ-5207852)
showing excessive brain residence time. Coincidentally, Zulli
et al.6n also disclosed a lead compound (1b) which displayed an ex-
tremely high brain to plasma ratio (B/P = 50). It was hypothesized
that the exceptionally long brain residence of the diamines might
mine H3 receptors (H3R) that inhibit both the conversion of L-histi-
⇑
Corresponding authors. Tel.: +1 781 434 3635.
E-mail addresses: zhongli.gao@sanofi.com (Z. Gao), william.hurst@sanofi.com
(W.J. Hurst).
Tel.: +1 908 981 3723.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.080

Z. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3416–3420
3417
be either associated with their high basicity or due to the di-basic
property of the molecule. We hence aimed at replacing one basic
side chain with a non-basic functional group to reduce the overall
basicity and, at the same time, eliminate one of the two basic cen-
ters of the molecule. In further modification of the lead compound,
we adopted phenoxy-propylamine, a privileged fragment in the
H3R antagonist research field, to replace the other side chain
(Fig. 1, 1 and 2). Herein we describe the discovery of a novel series
of selective H3R antagonists through series of structural modifica-
tions of the lead structure 1 and SAR studies and identification of
the new lead series (2) as tool compounds for further optimization
in our H3R program.
methylhistamine in membranes isolated from a CHO cell line sta-
bly transfected with the cloned human H3 receptors (h-H3R), the
rhesus monkey H3 receptors (rh-H3R) or the rat H3 receptors (r-
H3R) (Table 2).
As shown in Table 2, the assessed compounds (2m, 2o, and 2p)
displayed reasonably low species variations even though the num-
bers of available data points were limited. The project team took
advantage of this by using rhesus monkey H3R binding data as
the first tier screening for all the synthesized compounds. The spe-
cies variability was checked from time to time for selected com-
pounds to ensure that the most interested compounds and each
of the new series displayed good human and rat affinity. The choice
of rhesus monkey H3R binding data as the first tier screening was
driven by the rationale that the data could be more useful for
in vivo pharmacological screening of a few more structurally di-
verse novel compounds in rhesus monkey acute models in the fu-
ture when the program advanced to that stage. The selected
compounds could be profiled further and the development candi-
date could be selected.
Analogs 2a–2p were synthesized by coupling of commercially
available substituted phenylamines 3 with substituted 4-amino-
phenyether derivatives 4 using carbonyl di-imidazole (CDI) as cou-
pling agent at elevated temperature in DMF (Scheme 1).
Analogs 2a–2p were tested in h-H3R FLIPR assays (using the
same protocol as for HTS) and their antagonism was confirmed
by GTPc
S assays.9,10 The data are collected in Table 1. In general,
lipophilic substituents with variations of substitution positions in
the left-hand side aromatic ring were tolerated. The length of the
side chain was found to be critical (2a vs 2b; 2f vs 2g; 2j vs 2k;
and 2o vs 2n) with n = 3 being optimal. Introduction of a piperidine
as NR1R2-group improved potency (2o vs 2g; and 2n vs 2e).
The species variability of the H3R affinity of this series across
human, rhesus monkey, and rat cell lines were then evaluated. Se-
lected compounds (2m, 2o and 2p) were assessed in in vitro radi-
Due to the less than optimal permeability and metabolic stabil-
ity of analogs 2a–2p (e.g., 2m displayed solubility of 0.006 mg/mL,
and metabolic liability of 10%, 34%, and 36% in human, mouse, and
rat liver microsomes, respectively), we decided to optimize the
side chain further. In this regard, the rigid bipyrrolidine side chain
was introduced to replace the flexible side chain in 2 to give ana-
logs 5 (Fig. 2).
Analogs 5a–5u were synthesized effectively by condensing
commercially available BOC-protected 3-pyrrolidinone (6) with
2-methyl-pyrrolidine (7) under reductive amination conditions to
obtain intermediate 8 (Scheme 2). After de-protection, the desired
intermediate 9 ([1,30]-pyrrolidinyl-pyrrolidine) was realized. Con-
densation of 9 with para-fluoro-nitrobenzenes afforded intermedi-
ates 10. Hydrogenation of 10 yielded anilines 11, which were then
coupled with various amines by using CDI in DMF to obtain arylu-
rea analogs 5a–5u.
oligand binding assays11 by displacement of [3H]N-
a
-
H3C
CH3
N
H
N
N
H3C
N
H
CH3
O
The analogs 5a–5u were tested in human H3R FLIPR and GTPcS
functional assays, and rhesus monkey H3R radioligand binding as-
1
says. The data are collected in Table 3.
h-H3R FLIPR IC50 = 6.5 nM
h-H3R GTPγS % inhibition @ 10 μM: 94.7%
As shown in Table 3, introduction of the more rigid bi-pyrroli-
dine side chain did not cause a significant drop of the potency
(5a vs 2l). On the contrary, the potency was substantially enhanced
compared with the open chain analogs 2a–2p for most of the com-
pounds. The substituents at the central aryl residue were explored
first. Compounds with R0 = H or 2-methyl were usually approxi-
mately equipotent (5a–5b and 5d–5e). These compounds were
more potent than compounds with R0 = 3-methyl, except 5g. When
analyzing the influence of substituents at the left-hand side aro-
matic ring towards rh-H3R Ki, clear trends were observed: (1) lipo-
philic substituents were preferred over polar functional groups (5d
vs 5g and 5e vs 5h); (2) the orientation of the hydrogen-bond
forming atoms (oxygen and nitrogen) appeared not critical (Ki of
5j was comparable with that of 5n), suggesting that hydrogen
bonding interactions might play a less important role in this por-
tion of the molecules; (3) the size of the substituents was impor-
tant. For example, affinity of 5t was twofold lower than that of
5a and 5u was 3-fold lower in affinity than 5c. It is worth noting
R1
R
O
N
O
n
R2
N
H
N
H
2
O
O
N
O
N
N
O
N
1a
1b
Figure 1. Structures of H3R antagonists.
R1
N
R1
R
O
R2
R
n
+
O
N
a
O
n
R2
NH2
H2N
N
H
N
H
3
4
2
Scheme 1. Syntheses of analogs 2a–2p. Reagents and conditions: (a) CDI, DMF, 80 °C, 2–14 h.

3418
Z. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3416–3420
Table 1
H3R antagonist activity of compounds 2a–2p
R1
R
O
N
X
n
R2
N
H
N
H
2
R1R2N–
h-H3R FLIPR IC50 (nM)
h-H3R GTPcS I% @ 10 l
Mb (%)
a
Compd No.
R
n
X
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2,3-Di-Me
2,3-Di-Me
4-Cl
2,4-Di-Cl
2,4-Di-F
2,4-Di-F
2,4-Di-F
2-EtO, 5-Me
2,6-Di-Cl
4-Cl
2
3
2
2
2
2
3
2
2
2
3
3
3
2
3
2
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
Me2N–
Me2N–
Me2N–
Et2N–
Me2N–
Et2N–
Et2N–
Et2N–
73.1
14.7
16.4
39.1
148.6
111.4
7.9
9.1
1.0
27.5
0.7
0.5
94
125
99
118
102
117
124
107
116
105
119
125
133
115
128
129
Et2N–
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Et2N–
4-Cl
3,5-Di-Cl
4-Me, 3,5-di-Cl
2,4-Di-F
2,4-Di-F
4-iBuO–
0.2
40.5
0.5
0.6
a
h-H3R FLIPR IC50 were determined in HTS with a 384-well format, using Imetit agonist.
b
h-H3R GTP
c
S assays were performed in triplicates at 10
lM of the compounds tested; the data were expressed as percent of inhibitions (I%) of basal GTP
c
S binding.
Table 2
Assessment of species variation of H3R binding (Ki) across human, rhesus monkey, and rat
Compd No.
h-H3R FLIPR IC50 (nM)
h-H3R binding Kia (nM)
rh-H3R binding Kia (nM)
r-H3R binding Kia (nM)
h-H3R GTP
c
S I% @ 10 l
Mb (%)
2m
2o
2p
0.2
0.56
0.63
112.3
52.0
16.8
18.2
3.4
15.7
135.0
33
39.9
133
128
129
a
Ki values were averages of three or more determinations.
h-H3R GTPcS assays were performed in triplicates at 10 lM of the compounds tested; the data were expressed as percent of inhibitions (I%) of basal GTPcS binding.
b
R1
In spite of these positive results, this series was not optimal for
being an oral drug candidate due to multiple drawbacks (poor sol-
ubility, hERG and Cyp inhibition; e.g., 5b displayed solubility of
R
O
N
O
n
R2
0.0105 mg/mL, cyp 3A4 inhibition with IC50 of 13
lM, and hERG
N
H
N
H
2
percent inhibition of 74% at 10 M). However, the series merited
l
further optimization due to its potency at H3R, low interspecies
variability, and high selectivity. The continued work will be de-
scribed in a subsequent letter (Part II) dealing with the amide
replacement of the urea moiety.
R'
In summary, we have described the SAR around an initial urea
series of H3R ligands. The HTS derived hit compound 1 was modi-
fied through a series of medicinal chemistry approaches, such as
re-scaffolding to remove the di-basic property of 1, and side chain
optimization including installation of rigidity. These efforts yielded
a series of H3R antagonists with subnanomolar affinity in a rh-H3R
binding assay as exemplified by compounds 2l, 5b, 5d, and 5e. This
series provided a set of tool compounds for further optimization to
derive a drug candidate for preclinical and clinical evaluations. Fu-
ture efforts around this chemotype will be focused on maintaining
the excellent potency and selectivity while improving the pharma-
cokinetic profile.
O
R
N
N
H
N
H
N
5
Figure 2. Structures of H3R antagonists.
that urea analogs of secondary amines (5i–5n) were also active,
suggesting that the left-hand side NH of the urea moiety was not
essential for binding. This piece of the data provided the founda-
tion for our amide bioisosteric replacement.
In the next step, the selectivity of the compounds toward H1R,
H2R, and MCH1R12 was assessed. The compounds in Table 3 were
tested for human H1R and H2R binding as well as in human MCH1R
FLIPR assays. They all showed weak to no activities toward these
Acknowledgments
The authors are thankful to Sanofi R&D management for the
strong support, to all the H3R project team members for their con-
targets (percent inhibition <40% @ 10 lM).

Z. Gao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3416–3420
3419
b
HN
a
N
+
N
BOC
N
BOC
HN
O
6
7
8
R1
O
O
c
N+
d
N
N
N
10
9
R1
R1
O
R
e
N
H
H2N
N
N
N
H
N
N
5
11
Scheme 2. Syntheses of analogs 5a–5u. Reagents and conditions: (a) NaBH(OAc)3, DCE, rt overnight, 84% yield; (b) 4 M HCl in dioxane, rt, 1 h, 100% yield; (c) (optionally
further substituted) para-fluoro-nitro-benzene, K2CO3, DMSO, 80 °C, 2 h; 80% yield; (d) H2, 10% Pd–C, MeOH, rt 2 h, 100% yield; (e) amine, CDI, DMF, 80 °C, 2–14 h, 80–84%
yield.
Table 3
SAR of arylurea analogs 5a–5u
R'
2
3
H
N
R
N
N
O
5
Compd No.
R
R0
h-H3R FLIPR IC50 (nM)
h-H3R GTP
c
S I% @ 10
l
Ma (%)
rh-H3R binding Kib (nM)
2l
0.5
1.2
0.9
3.1
0.1
0.2
0.2
0.2
0.8
1.1
0.5
2.3
0.4
0.3
125
103
126
103
122
110
130
132
120
118
127
115
122
108
3.4
19.1
22.2
43.0
4.1
5a
5b
5c
5d
5e
5f
5g
5h
5i
3,5-Di-Cl-phenyl-NH–
3,5-Di-Cl-phenyl-NH–
3,5-Di-Cl-phenyl-NH–
Cyclohexyl-NH–
Cyclohexyl-NH–
Cyclohexyl-NH–
1-Methyl-piperazinyl
1-Methyl-piperazinyl
1-Methyl-piperazinyl
1-Ac-piperazinyl
1-Ac-piperazinyl
1-Phenyl-piperazinyl
Piperidinyl
H
2-Me
3-Me
H
2-Me
3-Me
H
2-Me
3-Me
H
3-Me
H
4.6
11.2
25.7
10.5
12.7
34.1
23.0
10.6
6.0
5j
5k
5l
5m
H
5n
5o
H
3-Me
1.1
1.1
109
118
35.5
89.7
O
N
N
H3C
CH3
NH
5p
5q
H
3-Me
0.1
0.2
129
125
1.9
8.4
S
5r
5s
5t
5u
3,5-Di-Cl-benzyl-NH–
3,5-Di-Cl-benzyl-NH–
4-PhO-Ph-NH–
H
3-Me
H
0.2
1.1
1.4
0.6
144
130
113
126
7.1
28.8
46.8
4-PhO-Ph-NH–
3-Me
115.2
a
h-H3R GTPcS assays were performed in triplicates at 10 lM of the compounds tested; the data were expressed as percent of inhibitions (I%) of basal GTPcS binding.
Ki values were averages of three or more determinations.
b
2. Kellett, S.; Poirier, P.; Dewailly, E.; Sampasa, H.; Chateau-Degat, M. L. Am. J.
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9. The rhesus H3 GTP
modified standard protocol [Br. J. Pharmacol., 2002, 135, 383]. Assay mixture
contained 50 l of rhesus H3 receptor membranes (prepared as described in the
literature; 20–30 g protein/well), diluted in assay buffer (20 mM HEPES–
NaOH, pH 7.4, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 3 M GDT) along with
50 S (Amersham), 50 l of WGA SPA beads (0.1 g)
l of 1 nM [35S] GTP
(Amersham) and 50 l of compound in a 96-well opti-plate (Packard). Total
assay volume 200 l. Assay plates were sealed with TopSeal (Perkin Elmer),
cS radioligand binding assay was performed utilizing a
l
l
l
l
c
l
l
l
l
incubated (25 °C, 90 min) and centrifuged (1000 rpm, 5 min). Assay plates were
read on TopCount scintillation counter (Packard). Non-specific binding was
determined by running the assay in the presence of 20
Inverse agonism was measured by percent inhibition of basal GTP
This assay was also evaluated using parental cell membranes (Flp-In T-REx 293
lM GTP
c
c
S (Sigma).
S binding.
Cell Line, Invitrogen) to confirm that the measured GTPcS binding was rhesus
H3-mediated and not due to endogenous receptors.
10. Greasley, P. J.; Clapham, J. C. Eur. J. Pharmacol. 2006, 553, 1.
11. H3 radioligand binding assay was performed using H3 receptor membranes
prepared from the Flp-In T-REx 293 Cell Line (Invitrogen) stably transfected
with pcDNA5/FRT/TO (Invitrogen) containing the human or rhesus monkey
(Macacca Mulatta) or rat 445 amino acid H3 receptor. [3H]-methylhistamine
(Perkin Elmer) and WGA SPA beads (wheat germ agglutinin scintillation
proximity assay) beads (Amersham). The assay was performed in 96-well
opti-plates (Packard). Each reaction contained 50
30 g total protein), 50 l WGA SPA beads (0.1 g) and 50
[3H]-methylhistamine (final concentration 2 nM) and 50
l
l
H3 membranes (20–
l of 83 Ci/mmol
of tested
l
l
l
l
ll
compound. The exemplified compounds and/or vehicle were diluted with
binding buffer from 10 mM DMSO stocks. Assay plates were sealed with
TopSeal (Perkin Elmer) and mixed on shaker (25 °C, 1 h). Assay plates were
read on TopCount scintillation counter (Packard). Results were analyzed by
Hill transformation and Ki values were determined by Cheng–Prusoff
equation.
7. Hurst, D. P.; Lynch, D. L.; Barnett-Norris, J.; Hyatt, S. M.; Seltzman, H. H.; Zhong,
M.; Song, Z. H.; Nie, J.; Lewis, D.; Reggio, P. H. Mol. Pharmacol. 2002, 62, 1274.
8. (a) Barbier, A. J.; Berridge, C.; Dugovic, C.; Laposky, A. D.; Wilson, S. J.; Boggs, J.;
Aluisio, L.; Lord, B.; Mazur, C.; Pudiak, C. M.; Langlois, X.; Xiao, W.; Apodaca, R.;
Carruthers, N. I.; Lovenberg, T. W. Br. J. Pharmacol. 2004, 143, 649; (b) Ly, K. S.;
Letavic, M. A.; Keith, J. M.; Miller, J. M.; Stocking, E. M.; Barbier, A. J.;
Bonaventure, P.; Lord, B.; Jiang, X.; Boggs, J. D.; Dvorak, L.; Miller, K. L.;
12. (a) Schwink, L.; Stengelin, S.; Gossel, M.; Boehme, T.; Hessler, G.; Rosse, G.;
Walser, A. WO2004011438A1, 2004, 113.; (b) Schwink, L.; Stengelin, S.; Gossel,
M.; Boehme, T.; Hessler, G.; Stahl, P.; Gretzke, D. WO2004072025A2, 2004, p.
390.