Design, synthesis and SAR of a novel series of benzimidazoles as potent NPY Y5 antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.09.064

A novel class of benzimidazole NPY Y5 receptor antagonists was prepared exploiting a privileged spirocarbamate moiety. The structure-activity relationship of this series and efforts to achieve a profile suitable for further development and an appropriate pharmacokinetic profile in rat are described. Optimisation led to the identification of the brain penetrant, orally bioavailable Y5 antagonist 9b which significantly inhibited the food intake induced by a Y5 selective agonist with a minimal effective dose of 30 mg/kg po.

Full text of DOI:10.1016/j.bmcl.2010.09.064

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7120–7123
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and SAR of a novel series of benzimidazoles as potent
NPY Y5 antagonists
⇑
Domenica Antonia Pizzi , Colin Philip Leslie, Angelica Mazzali, Catia Seri, Matteo Biagetti,
Jonathan Bentley , Thorsten Genski à, Romano Di Fabio, Stefania Contini, Fabio Maria Sabbatini,
Laura Zonzini, Laura Caberlotto
GlaxoSmithKline, Neurosciences Centre of Excellence for Drug Discovery, Medicines Research Centre, Via A. Fleming 4, 37135 Verona, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2010
Revised 9 September 2010
Accepted 10 September 2010
Available online 17 September 2010
A novel class of benzimidazole NPY Y5 receptor antagonists was prepared exploiting a privileged
spirocarbamate moiety. The structure–activity relationship of this series and efforts to achieve a profile
suitable for further development and an appropriate pharmacokinetic profile in rat are described.
Optimisation led to the identification of the brain penetrant, orally bioavailable Y5 antagonist 9b which
significantly inhibited the food intake induced by a Y5 selective agonist with a minimal effective dose of
30 mg/kg po.
Keywords:
NPY Y5
Ó 2010 Elsevier Ltd. All rights reserved.
Benzimidazoles
Neuropeptide Y (NPY) is a 36-amino acid peptide originally dis-
covered in extracts of porcine brain.1 The peptide belonging to a
broad family of peptides (which includes peptide YY and pancre-
atic polypeptide PP) has a wide distribution in the central2–4 and
peripheral nervous system.5–7 NPY is expressed in hypothalamic
regions and this suggests that it may be implicated in a variety
of physiological functions, notably the central regulation of feeding
behaviour and energy homeostatis.8,9 The biological effects of NPY
are mediated through its interaction with five G-protein coupled
receptors (Y1, Y2, Y4, Y5 and y6).10 A number of pharmacological
studies conducted with transgenic mice and/or subtype selective
agonists and antagonists have suggested that the Y5 receptors
are involved in the regulation of food intake.11–14 Additionally,
small molecule Y5 antagonists have proven efficacious in animal
models of addiction to drugs of abuse15 and in animal models of
depression.16 Hence, antagonism of the Y5 receptor represents an
interesting pharmacological target with potential therapeutic
applications for the treatment of eating disorders, drug depen-
dency or depression.
benzimidazoles, endowed with potent Y5 antagonist activity and
a profile suitable for further development.
As part of a campaign to identify novel, orally bioavailable and
brain penetrant Y5 antagonists, the urea lead 1, characterised by a
spirocyclic carbamate moiety, was discovered from a number of
prospective diversity arrays (Fig. 1).17
Considering that a spirocyclic lactone group, such as that shown
in structure 2, appears to be a privileged substructure, recurring in
several series of Y5 antagonists reported in literature,18–22 we
hypothesised that the spirocarbamate group may represent an
isosteric privileged substructure that could bestow Y5 affinity
upon other templates. Moreover, if this hypothesis held true, then
judicious selection of the new template may potentially lead to
structures with improved brain penetration with respect to 1.
O
O
O
O
N
N
Herein we report our efforts to elaborate a proprietary series of
NPY Y5 antagonists that led to the discovery of a novel class of
hNPY Y5 fpKi 8.7
hNPY Y1/2 fpKi<4.5
CYP450 >10 uM
hNPY Y5 pIC50 = 8.9
N
N
Clint (h/r) = 0.6/0.5 ml/min/g liver
Clb=9 ml/min/kg
F=49%
B/B<0.1
O
NH
O
NH
S
N
N
⇑
Corresponding author. Tel.: +39 045 8218931; fax: +39 045 8218196.
E-mail addresses: Domenica.a.pizzi@gsk.com, pizzidomenica@hotmail.com (D.A.
Pizzi).
N
F
Present address: Evotec, 114 Milton Park, Abingdon, Oxfordshire OX14 4SA,
United Kingdom.
1
2
à
Present address: AnalytiCon Discovery GmbH, Hermannswerder Haus 17, 14473
Potsdam, Germany.
Figure 1. Structure of lead compound 1 and spirolactone 2.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.064

D. A. Pizzi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7120–7123
7121
R1
O
Consequently, we set about transferring the novel spirocarbamate
building block, present in derivative 1, to a benzimidazole scaffold
to give compound 3a (Fig. 2).
N linker
N
O
Gratifyingly, compound 3a demonstrated good Y5 activity
verifying our hypothesis. In addition, the in vivo pharmacokinetic
profile23 of 3a was characterised by exceptionally low clearance
and high bioavailability; as hoped brain penetration improved
with respect to 1 although it remained moderate in absolute terms
(B/B = 0.2). The in vitro profile was tarred by potent CYP450
inhibition of the 2C9 isoform and limited selectivity with respect
to a1F (fpKi = 7.0). The initial optimisation strategy was directed
at increasing the brain penetration of this class whilst exploring
the structure–activity relationships (SARs) at CYP450 and the
a1F receptor. Three different approaches were followed for the
optimisation: (i) variation of the substituent on the benzimidazole;
(ii) reduction of the polar surface area (PSA) via removal of hetero-
atoms, switching from a N-linker to a C-linker between the spiroc-
arbamate building block and the benzimidazole; (iii) introduction
of an ‘ortho-substituent’ on the N-phenyl ring of the spirocarba-
mate to help mask the polarity of carbamate group.
R1
O
R2
N
a
N
N
O
Cl
+
N
H
N
NH
N
H
4
5
R2
3a-d,f
O
R1
R1
O
N
N
O
O
b
N
N
NH
NH
N
N
The synthetic route used to access the N-linked compounds 3
and the C-linked derivatives 9 is outlined in Scheme 1.
To prepare the N-linked benzimidazoles, amine 424 and
2-chlorobenzimidazoles 5, which were either purchased or pre-
pared according to reported methods,19 were reacted in the
presence of Hunig’s base, in DMSO at 180 °C in a microwave reac-
tor for 40 min, to give the products 3. Additionally, the bromo
derivative 3c was arylated under Suzuki conditions to afford 3e.
For the C-linked benzimidazoles, the trans-carboxylic acids 617
and the substituted phenyl-1,2-diamines 7 were easily converted
into a mixture of regioisomeric amides 8 with EDCÁHCl in pyridine
at room temperature. The amides were subsequently cyclised with
p-toluenesulphonic acid, in a mixture of toluene and dioxane, heat-
ing the mixture to 150 °C in a microwave reactor for 30 min, to af-
ford the final derivatives 9. Initially, a similar synthetic sequence
was followed using the corresponding cis-isomer of 6 (R = Ph) to
give the cis-analogue of 9a, however, this derivative had low target
activity (NPY Y5 fpKi = 5.6 vs 8.8 for 9a) hence SAR studies were
conducted exclusively with the trans-isomer.
R2
Br
3c
3e
R1
O
C linker
N
R1
O
O
N
c
R2
NH2
NH2
O
+
O
NH
NH2
HO
O
R2
6
7
8
d
The SAR exploration focused on the introduction of different
substituents on the benzimidazole resulted in a fair number of
potent derivatives (Table 1). It was found that a substituent at
the 5-position was essential for high target activity and that larger,
more lipophilic substituents gave the highest Y5 activities;
compounds 3e and 3f were most active, showing 10-fold higher
potencies than the lead 3a. Most substituents improved the P450
inhibition profile at the 2C9 isoform, with the fluorophenyl deriv-
R1
O
R2
N
N
N
H
O
9
Scheme 1. Reagents and conditions: (a) DIPEA, DMSO, 180 °C, microwave reactor,
40 min; (b) R2B(OH)2, Pd(PPh3)4, Na2CO3, dioxane:water (5:1), 140 °C, microwave
reactor, 10 min; (c) EDCÁHCl, Py, rt, 1.5 h; (d) TsOH, dioxane:toluene (1:1), 150 °C,
30 min.
ative 3e being of particular note (>10 lM at all isoforms). Little or
no change was observed in a1A activity, however, metabolic
stability proved highly susceptible to changes in the substituent
in the 5-position, where only trifluoromethyl (3a) or trifluoro-
methoxy (3f) groups gave acceptable intrinsic clearances (Clint
)
O
values. Compound 3f was progressed to rat PK studies where it
demonstrated a similar profile to 3a but with slightly improved
brain penetration (Clb = 3 ml/min/kg, F = 90%, B/B = 0.3) although
this was at the cost of higher protein binding—the unbound frac-
tion in the brain (Fubr) was determined as this is considered an
influential parameter in driving the in vivo efficacy of a compound.
The strategy to exchange the N-linker of the aminobenzimidaz-
ole 3a for a C-linker led to compound 9n; as well as maintaining
excellent Y5 activity this modification also led to an improved
P450 inhibition profile on the 2C9 isoform and a slight increase
in brain penetration (B/B = 0.3),25 but, more importantly, activity
at the a1A receptor dropped 10-fold to give >1000-fold selectivity
against Y5.
O
N
N
O
hNPY Y5 fpKi 8.8
hNPY Y1/2 fpKi<4.5
P450 2C9=0.3 uM
Clint (h/r) = 0.5/0.5 ml/min/g
Clb=2 ml/min/kg
T1/2=8 h
O
N
N
N
O
NH
N
HN
F=91%
S
B/B=0.2
α1A fpKi= 7.0
N
F3C
1
3a
Figure 2. Structure of novel lead compound 3a.

7122
D. A. Pizzi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7120–7123
Table 1
NPY Y5, a1A and hERG activities, in vitro metabolic stability, c log P, tPSA, CYP450 inhibitions, Fubr and brain penetration of benzimidazole derivatives 3a–f and 9a–s
f
h
Compds
R1
R2
hNPY Y5 fpKia
Clint h/rb
(ml/min/g liver)
c log Pc
PSAd (Å2) a1A fpKie
hERG pIC50
CYP450 inh.
(l
M)g
Fubr
B/Bi
3a
3b
3c
3d
3e
3f
9a
9b
9c
9d
9e
9f
Ph
Ph
Ph
Ph
Ph
Ph
Ph
CF3
H
Br
F
8.8
6.5
8.9
7.7
9.7
9.8
9.5
9.2
9.5
8.3
8.6
8.9
9.4
7.1
8.9
7.6
7.7
9.2
8.3
8.4
9.5
9.2
8.1
0.5/0.5
9.8/1.0
2.5/0.5
7/0.5
3.75
2.73
3.67
2.95
4.76
4.14
5.15
5.29
5.29
5.65
5.86
3.65
4.15
4.15
3.88
3.65
3.88
4.75
3.26
3.48
3.35
3.49
1.85
61
61
61
61
61
71
67
67
67
67
67
80
80
80
80
80
80
58
71
71
82
82
95
7.0
7.0
7.4
7.0
5.7
6.0
na
na
na
>10/0.3/2/5/>10
>10/>10/>10/2/>10
>10/-/2/3/>10
>10/-/7/2/>10
All >10
>10//2/2/1/>10
>10/3/2/4/>10
>10/4/2/>10/>10
>10/4/3/1/>10
>10/7/3/>10/>10
>10/5/2/6/10
>10/3/2/4/>10
>10/2/2/1/>10
All >10
1.0
0.2
4-F-phenyl
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
OCF3
CF3
7/5
na
0.5/0.5
0.5/0.5
0.5/0.5
0.5/0.5
0.5/0.5
0.5/0.5
0.5/0.5
0.6/0.7
0.6/0.7
0.5/0.5
0.5/0.5
na
0.5/0.5
0.5/0.5
na
0.5/0.5
0.5/0.5
15/14
6.7
5.9
6.1
6.1
na
na
5.7
5.8
<5.6
6.3
<5.6
na
5.7
5.9
na
<5.6
<5.6
<5.6
na
0.6
0.3
0.9
0.3
0.6
0.8
5.6
5.2
6.1
na
2-F-phenyl
4-F-phenyl
2-Me-phenyl
2-Cl-phenyl
2-Py
6-Me-2-Py
2-Me-3-Py
6-F-2-Py
3-Py
2-F-3-Py
Ph
2-Py
6-F-2-Py
Ph
4-F-phenyl
2- Py
na
5.6
5.7
4.3
5.4
5.1
4.8
5.4
5.4
na
1.0
0.5
9g
9h
9i
>10/5/2/3/-
9l
>10/8/5/2/>10
>10/10/5/>10/>10
>10/4/2/3/>10
>10/2/2/8/>10
na
>10/>10/7/>10/>10
>10/10/5/7/>10
All >10
9m
9n
9o
9p
9q
9r
9s
0.7
3.4
0.3
CF3
CF3
CN
CN
5.0
5.8
4.9
<0.03
CN
a
The functional activity (fpKi) at the human NPY Y5 receptor stably expressed in HEK293 cells was assessed using FLIPR/Ca2+ methodology in a 384-well format. Each
determination lies within 0.3 log units of the mean with a minimum of two replicates.
b
Intrinsic clearance values (Clint) were determined in human (h) and rat (r) liver microsomes and are expressed as ml/min/g liver.
ACD log P version 11.
c
d
Topological polar surface area (PSA) calculated using freeware by Peter Ertl.
e
The functional activity (fpKi) at the human a
1A receptor stably expressed in rat 1 fibroblast cells was assessed using FLIPR/Ca2+ methodology in a 384-well format. Each
determination lies within 0.3 log units of the mean with a minimum of two replicates.
f
The binding affinity (pIC50) for the human ether-a-go-go related gene (hERG) ion channel stably expressed in CHO cells was assessed using [3H]-dofetilide in a SPA
displacement assay in a 384-well format. Each determination lies within 0.3 log units of the mean with a minimum of two replicates.
g
CYP450 inhibition potential at 1A2/2C9/2C19/2D6/3A4 isoforms, respectively, determined in Cypex bactosomes.
Fraction unbound determined in an equilibrium dialysis assay using homogenised rat brain.
Brain/blood partition determined 1 h after iv administration of 0.5 mg/kg test compound to male rat; na = not available.
h
i
The combination of OCF3 on the benzimidazole and the C-linker
afforded compound 9a, with high target activity and high selectiv-
ity. The PK profile of this compound in rat confirmed the high met-
abolic stability and good oral absorption typically seen in this
series (Clb = 7 ml/min/kg, F = 69%) and brain penetration was sig-
nificantly higher than previous analogues (B/B = 0.6) although,
again, this was compensated by a lower free fraction in the brain
(Fubr = 0.3%). In an effort to reduce brain tissue binding, while
maintaining the desirable characteristics of 9a, an attempt was
made to reduce the lipophilicity by exchanging the 5-trifluoro-
methoxy group on the benzimidazole for a 5-cyano group. This
gave compound 9q which was endowed with the best in vitro
profile observed in the series offering high Y5 activity (fpKi = 9.5),
high metabolic stability (Clint h/r = 0.5/0.5 ml/min/kg), high selec-
tivity and greatly improved P450 inhibition profile (all isoforms
functional assays). In addition an ortho-fluoro or methyl group had
a beneficial effect on CYP 2D6 inhibition. Compound 9b was fur-
ther characterised in rat in vivo PK studies, where it exhibited
the best overall profile seen within the series displaying good brain
penetration (Clb = 8 ml/min/kg, F = 75%, B/B = 0.8) without erosion
of the free fraction in the brain (Fubr = 0.90%). Considering its
whole profile, compound 9b was selected for further characterisa-
tion in a pharmacodynamic model to verify its in vivo efficacy as a
NPY Y5 antagonist. Compound 9b dose dependently reversed the
food intake induced by icv administration of 0.6 nM27 of the NPY
Y5 selective agonist, [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]hPP,
with a minimal effective dose of 30 mg/kg po.
In conclusion, rational design exploiting a privileged spirocar-
bamate building block led to the discovery of a novel class of benz-
imidazole NPY Y5 receptor antagonists. Optimisation of the lead
structure for a1A selectivity, CYP450 inhibition and brain penetra-
tion led to the identification of compound 9b which combines
potent NPY Y5 antagonist activity with high selectivity, good
developability, high metabolic stability, high bioavailability and
brain penetration.
>7 lM) and protein binding (Fubr = 3.4%). As usual the rat PK profile
showed low clearance and good bioavailability (Clb = 7 ml/min/kg,
F = 66%), but brain penetration was exceedingly low (B/B <0.03%)
most likely due to active efflux.26 Attempts to reduce lipophilicity
via replacement of the phenyl group in 9a,n,q with a pyridyl group
in 9f,o,s were accompanied by a modest reduction in Y5 potency,
and in the case of 9s a dramatic increase in intrinsic clearance (Clint
h/r = 15/14 ml/min/g liver).
Acknowledgements
Finally, a number of ortho-substituents were introduced on the
N-phenyl ring of the spirocarbamate with the intent to mask the
polarity of the carbamate and increase the brain penetration. Y5
activities tended to decrease (9d,e) but compound 9b, with a small
ortho-fluoro substituent, maintained acceptably high NPY Y5 activ-
ity (fpKi = 9.2) and good selectivity including NPY Y1 and Y2 recep-
The authors thank Dr. Carla Marchioro and members of the
Analytical Chemistry department, Verona for support in the analyt-
ical characterisation of the compounds described. The authors also
thank Francesca Graziani and Alberto Buson of Molecular Discov-
ery Research, Verona for support in the in vitro characterisation
of the compounds described.
tors (all compounds 3 and 9 showed fpKi <5.3 in Y1 and Y2 GTPcS

D. A. Pizzi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7120–7123
7123
19. Ogino, Y.; Ohtake, N.; Nagae, Y.; Matsuda, K.; Moriya, M.; Suga, T.; Ishikawa,
M.; Kanesaka, M.; Mitobe, Y.; Ito, J.; Kanno, T.; Ishihara, A.; Iwaasa, H.; Ohe, T.;
Kanatani, A.; Fukami, T. Bioorg. Med. Chem. Lett. 2008, 18, 5010.
20. Bakthavatchalam, R.; Blum, C. A.; Brielmann, H. L.; Darrow, J. W.; De Lombaert,
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200,20,48,152 A2, 2002; PCT Int. Appl., 2002.
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23. All the work involving animals was carried out in accordance with European
directive 86/609/EEC governing animal welfare and protection, which is
acknowledged by Italian Legislative Decree No. 116, 27 January 1992, and
according to internal review performed by the GlaxoSmithKline Committee on
Animal Research & Ethics (CARE) and to the company policy on the care and
use of laboratory animals.
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25. It is noteworthy that various factors may be influencing the modest brain/
blood partitions observed with some of the derivatives 3 and 9. For instance,
compound 3f has an observed B/B ratio (B/B = 0.3) close to its Kbb (Kbb = 0.4)
(Kbb = fraction unbound in blood/fraction unbound in brain) and it tested
negative in an in vitro p-glycoprotein assay, suggesting its observed B/B ratio is
merely a consequence of its natural disposition at equilibrium to distribute
into these two compartments. In contrast compound 9n has an observed B/B
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27. The dose of 0.6 nM of [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]hPP used in the
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curve which indicated this dose induced a near-maximal response.