The identification and optimisation of novel and selective diamide neuropeptide Y Y2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2009.06.035

A novel small molecule NPY Y2 antagonist (3) identified from high throughput screening is described. A subsequent SAR study and optimisation programme based around this molecule is also described, leading to the identification of potent and soluble pyridyl analogue 36.

Full text of DOI:10.1016/j.bmcl.2009.06.035

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4022–4025
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The identification and optimisation of novel and selective diamide
neuropeptide Y Y2 receptor antagonists
Gillian E. Lunniss a, , Ashley A. Barnes b, Nick Barton b, Matteo Biagetti c, Federica Bianchi c,
Stephen M. Blowers a, Laura Caberlotto c, Amanda Emmons b, Ian P. Holmes a, , Dino Montanari c,
Ros Norris b, Dewi J. Walters a, Steve P. Watson a,
*
a GlaxoSmithKline, Neurosciences Centre of Excellence for Drug Discovery, New Frontiers Science Park, Harlow, UK
b Molecular Discovery Research, New Frontiers Science Park, Harlow, UK
c Neurosciences Centre of Excellence for Drug Discovery, Medicine Research Centre, Verona, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel small molecule NPY Y2 antagonist (3) identified from high throughput screening is described. A
subsequent SAR study and optimisation programme based around this molecule is also described, leading
to the identification of potent and soluble pyridyl analogue 36.
Received 28 April 2009
Revised 5 June 2009
Accepted 8 June 2009
Available online 13 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
NPY antagonist
NPY Y2 antagonist
NPY
NPY Y2
BIIE0246
Neuropeptide Y (NPY) is a member of the pancreatic polypep-
tide family that also includes pancreatic polypeptide (PP) and pep-
tide YY (PYY)1 and is highly expressed in both the periphery and in
a number of specific brain regions.2,3 Its localisation implies that it
may be involved in a variety of physiological responses (such as
food intake, water consumption, anxiety, circadian rhythms, mem-
ory processing, endocrine and cardiovascular functions).2 Six NPY
receptors (Y1, Y2, Y3, Y4, Y5 and Y6), five of which have been
cloned,4 have been identified from pharmacological and molecular
biology studies on NPY.2,5
Whilst there are extensive literature reports of Y1 and Y5 antag-
onists,1,6,7 and compounds have been progressed into the clinic
(e.g., MK-05578), small molecule Y2 antagonists have until very re-
cently proved elusive, and hence the therapeutic potential of this
receptor has remained relatively unexplored.9 Doods et al.10 re-
ported the pseudo-peptide BIIE0246 as a potent and selective
NPY Y2 antagonist (IC50 = 3.3 nM), and Caberlotto and co-workers
subsequently administered BIIE0246 via intracerebroventricular
injection to successfully demonstrate its anxiolytic-effect in rats
in the elevated plus maze model.11
The first small molecule NPY Y2 receptor antagonists were dis-
closed by Grant-Young and co-workers,12 who identified a series of
amines (Fig. 1) from a high throughput screen (HTS) as exemplified
by 1 (IC50 = 450 nM) although no comment was made on func-
tional activity. Jablonowski et al.13 disclosed a series of indolylpi-
peridines, again identified from a HTS, and optimised to obtain
JNJ-5207787 (2), a compound with good affinity for the NPY Y2
receptor (IC50 = 100 nM in a binding assay) and demonstrated to
be
35S]GTP
Herein we report the discovery and optimisation of a series of
a
functional antagonist via inhibition of PYY-stimulated
[
cS binding (pIC50 = 7.2) (Fig. 1).
small molecule NPY Y2 functional antagonists based on diamide
3 (Fig. 2).
O
N
N
N
N
S
O
O
S
NMe2
Ph
CN
* Corresponding author. Tel.: +44 1279 875004.
1
E-mail address: steve.p.watson@gsk.com (S.P. Watson).
Present address: Cancer Therapeutics CRC, Monash Institute of Pharmaceutical
Sciences, 381 Royal Parade, Parkville, VIC 3052, Australia.
JNJ-5207787 (2)
Figure 1. Structures of known selective NPY Y2 antagonists.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.035

G. E. Lunniss et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4022–4025
4023
hNPY Y2 fpKi = 6.3
rNPY Y2 fpKi = 6.4
NPY Y1 fpKi <5.2
NPY Y5 fpKi <5.3
Clint mL/min/g = 0.5 (human)
1.6 (rat)
O
H
N
N
N
O
H
N
N
N
O
Cl
R3
Ph
O
Cl
Figure 3. SAR for replacements of phenyl amide.
MW = 399
cLogP = 4.04
3
Solubility = 46 µg/mL
Table 1
Profiling results for phenyl amide SAR
Figure 2. Initial HTS hit diamide 3.
R3
hNPY Y2 fpKi
3
10
11
12
13
14
H
6.3
6.7
6.5
7.1
6.9
6.8
Diamide 3 was one of several chemotypes identified from a
functional HTS of the GSK compound collection. The molecule is
selective for NPY Y214,15 over both NPY Y1 and Y5 receptors,16,17
exhibits moderate aqueous solubility,18 and low intrinsic clear-
ance/good in vitro metabolic stability (Clint), and hence repre-
2-OMe
3-OMe
2-CF3
3-CF3
4-CF3
sented
a promising start point for a medicinal chemistry
programme. The initial aim of this programme was to identify sol-
uble, but more potent, NPY Y2 antagonists for use in Target Valida-
tion experiments in animal models of anxiety.
R4
O
N
N
N
R1
Compounds were readily synthesized in the general manner
shown in Scheme 1. SNAr displacement of 2-chloro-1-fluoro-4-
nitrobenzene (4) with N-Boc-piperazine gave nitroarylpiperazine
5, subsequent iron/ammonium chloride reduction of the nitro
group afforded aniline 6. Anilide formation under standard condi-
tions afforded anilides 7. Boc deprotection under acidic conditions
gave aryl piperazines 8 which underwent subsequent amide for-
mation to afford diamides 9.19
Ph
O
Cl
Figure 4. SAR for aliphatic replacements of the t-butyl amide.
Table 2
Profiling results for aliphatic amide SAR
R1
R4
hNPY Y2 fpKi
Solubility (lg/mL)
Initially, the effect on potency of substituents on the piperazine
amide was investigated. Electron donating and withdrawing sub-
stituents were well tolerated 10–14 (Fig. 3, Table 1). An ortho tri-
fluoromethyl group as exemplified by 12 gave a modest increase
in potency compared to diamide 3.
The effect of replacing the t-butyl anilide with alternative ali-
phatic amides was also investigated (Fig. 4, Table 2). It was found
that methylation of the amide nitrogen 15 resulted in a loss in po-
tency although solubility was increased. As the steric bulk of the
amide was increased, for example, by replacement of a methyl
group of the t-butyl amide with an ethyl group to give amide 16,
or introduction of ring constrained analogues such as cyclohexane
15
16
17
18
-19
20
t-Bu
C(CH3)2C2H5
Cyclohexane
1-Methyl cyclohex-1-yl
2-Tetrahydropyran
4-Tetrahydropyran
Me
H
H
H
H
5.6
7.3
6.8
7.7
6.5
<5.2
113
22
4
4
NT
NT
H
17, resulted in an increase in NPY Y2 potency. Replacement of the
cyclohexane ring with 1-methylcyclohexane 18 resulted in nearly
a 10 fold increase in the NPY Y2 potency compared to unsubstitut-
ed derivative 17. Incorporation of more polar groups such as a tet-
rahydropyran moiety ( -19 and 20) did not benefit. In the case of
the 2-tetrahydropyran derivative -19 the potency was only
slightly reduced compared to the cyclohexyl amide 17. However,
in the case of the 4-tetrahydropyran amide 20, the compound
was inactive.
A range of substituted phenylacetamide replacements for the t-
butyl anilide were also investigated (Fig. 5, Table 3). Phenylaceta-
mides with a benzylic substituent, such as 22 or 23, exhibited an
increase in potency compared to the unsubstituted derivative 21.
Incorporation of a small lipophilic meta substituent such as methyl
24 resulted in a further small increase in potency. However, all of
these compounds possessed poor solubility. The potency of 2-pyr-
idyl derivatives 25 and -26 was slightly reduced compared to the
parent phenyl analogues. Pyridyl 25 possessed improved aqueous
solubility compared to the analogous phenyl derivative. Disap-
pointingly, pyridine -26 was no more soluble than corresponding
benzhydryl analogue 22.
Boc
Boc
N
N
F
N
N
Cl
Cl
(i)
(iii)
R2
Cl
(ii)
NO2
NO2
NH2
6
4
5
O
Boc
N
H
N
N
N
N
N
(iv)
(v)
Cl
Cl
Cl
R1
NH
R1
NH
R1
NH
O
O
O
9
O
Ph
7
8
H
Ar
R5
N
N
N
Scheme 1. Representative synthesis of analogues. Reagents and conditions: (i) Boc-
piperazine, Hünig’s base, MeCN, 150 °C, microwave; (ii) iron, ammonium chloride,
water, methanol, 80 °C; (iii) R1CO2H, HATU, Hünigs base, NMP; (iv) HCl in diethyl
ether; (v) R2CO2H, HATU, Hünigs base, NMP.
R6
O
Cl
Figure 5. SAR for aromatic replacements of the t-butyl amide.

4024
G. E. Lunniss et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4022–4025
Table 3
Table 5
Profiling results for replacement of the t-butyl amide
Profiling results of combining potency enhancing modifications
Ar
R5
R6
hNPY Y2 fpKi
Solubility (
lg/mL)
X
R9
hNPY Y2 fpKi
clog P
Solubility (lg/mL)
21
22
23
24
25
Ph
Ph
Ph
m-MePh
2-Py
2-Py
H
H
H
Me
Me
H
7.1
8.2
8.0
8.3
6.5
7.9
3
7
0
0
70
8
33
34
35
36*
C
C
N
N
CF3
OMe
CF3
8.4
8.6
7.9
7.6
6.07
5.27
4.90
4.32
1
2
11
56
Ph
Me
Me
H
OMe
*
Tested as the HCl salt.
-26
Ph
H
Table 6
In vitro profiling results of selected compounds
O
H
N
N
N
Ph
hNPY Y2
fpKi
clog P
Cli rat
(mL/min/g)
Cli human
(mL/min/g)
BTB (rat)%
Ph
R8
R7
O
21
23
-26
7.1
8.0
7.9
7.6
4.34
5.05
4.19
4.32
13.8
7.4
15.3
0.7
0.5
0.6
<0.5
2.7
99.0
99.5
99.7
97.1
Figure 6. SAR for alternate aryl substitution.
36
Table 4
Profiling results for aryl substitution SAR
R7
R8
hNPY Y2 fpKi
Table 7
23
27
28
29
30
31
32
H
H
H
H
H
H
Cl
Cl
8.0
6.2
6.5
7.2
5.6
5.8
5.5
Selected in vitro selectivity data for pyridyl 36
CF3
Me
F
OMe
H
hERG pIC50
4.3
rNPY Y2 fpKi
7.3
NPY Y1 fpKi
NPY Y5 fpKi
<5.0
36
<5.2
H
O
H
N
Ph
N
N
.HCl
N
The effect of altering the substitution pattern in the central dia-
O
Cl
MeO
mino aryl ring was also investigated (Fig. 6, Table 4). It was found
that small lipophilic substituents such as trifluoromethyl 27,
methyl 28 and fluorine 29 were tolerated ortho to the piperazine
but with reduced potency compared to the chloride 23. More polar
substituents such as in methoxy compound (30), no substituent
(31), and substitution meta to the piperazine (e.g., 32) were detri-
mental to potency. The above results suggest that there may be a
small lipophilic pocket in the receptor that the chlorine atom, or
to a lesser extent fluorine may access, and which may also require
a ‘twist’ out of the planar geometry between the piperazine and
aryl moiety.
The more potent anilides and aryl-ortho substituents described
above were combined in a series of compounds (33–36) which dis-
played good potency (Fig. 7, Table 5). However, these potency
enhancing combinations typically afforded highly lipophilic com-
pounds with low solubility (33 and 34).
Figure 8. Compound 36 selected for selectivity profiling.
Pyridine 36 possessed the most promising profile of the com-
pounds screened viz. moderate intrinsic clearance (Clint), good hNPY
Y2potency,moderatesolubilityandmoderatebrainfreefraction.This
compound also displays excellent selectivity against the other NPY
receptors, good potency in the orthologue rat NPY Y2 assay and min-
imal activity at hERG (Table 7, Fig. 8). The compound had an excellent
CYPEX bactosome P450 profile showing inhibition potencies greater
than 10 lM against all isoforms tested and possessed no significant
off-target activity when cross screened against an extensive panel of
aminergic receptors (including dopamine, serotonin, adrenergic and
histamine receptors) and liability targets (data not shown).
In summary, extensive SAR explorations were performed
around highly promising HTS hit (3) and a number of potency
enhancing modifications identified. A highly potent, soluble and
selective NPY Y2 antagonist 36 suitable for in vitro profiling studies
was identified. In vivo studies on this molecule will be published in
due course.
The incorporation of a pyridyl group allowed the lipophilicity to
be reduced, for example, 35 and 36 and some aqueous solubility to
be introduced.
On completion of the SAR study, a number of the more promising
compounds with good NPY Y2 potency were progressed into
in vitro DMPK screens (Table 6). A number of the compounds (21,
23 and -26) proved to be metabolically unstable in the rat intrinsic
clearance assay (values >5 mL/min/g). Disappointingly, with the
exception of pyridine amide 36, these compounds also displayed
high non-specific rat brain tissue binding19 (BTB) which would pre-
dict very low free fraction in the brain in vivo.
References and notes
1. Hammond, M. IDrugs 2001, 4, 920.
2. Adrian, T. E.; Allen, J. M.; Bloom, S. R.; Ghatei, M. A.; Rossor, M. N.; Roberts, G.
W.; Crow, T. J.; Tatemoto, K.; Polak, J. M. Nature 1983, 306, 584.
3. Kaga, T.; Fujimiya, M.; Inui, A. Peptides 2001, 22, 501.
4. Antal Zimanyi, I.; Fathi, A.; Poindexter, G. S. Curr. Pharm. Des. 1998, 4, 229.
5. Michel, M. C.; Beck-Sickinger, A.; Cox, H.; Doods, H. N.; Herzog, H.; Larhammar,
D.; Quirion, R.; Schwartz, T.; Westfall, T. Pharmacol. Rev. 1998, 50, 143.
6. Parker, E.; Van Heck, M.; Stamford, A. Eur. J. Pharmacol. 2002, 440, 173.
7. MacNeil, D. J. Curr. Top. Med. Chem. 2007, 7, 1721.
8. Erondu, N.; Gantz, I.; Musser, B.; Suryawanshi, S.; Mallick, M.; Addy, C.; Cote, J.;
Bray, G.; Fujioka, K.; Bays, H.; Hollander, P.; Sanabria-Bohorquez, S. M.; Eng, W.
I.; Langstrom, B.; Hargreaves, R. J.; Burns, H. D.; Kanatani, A.; Fukami, T.;
MacNeil, D. J.; Gottesdiener, Ke. M.; Amatruda, J. M.; Kaufman, K. D.;
Heymsfield, S. B. Cell. Metab. 2006, 4, 275.
O
H
N
R9
Ph
N
N
O
Cl
X
Figure 7. SAR for replacements of phenyl amide.

G. E. Lunniss et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4022–4025
4025
9. During the course of this work, the following patents in which a series of
piperazine and piperidine NPY Y2 antagonists were published: Bonaventure, P.;
Carruthers, N. I.; Chai, W.; Dvorak, C. A.; Jablonowski, J. A.; Rudolph, D. A.;
Seierstad, M.; Shah, C. R.; Swanson, D. M.; Wong, V. D. US 2007100141 A1, 2007
and Dvorak, C. A.; Swanson, D. M.; Wong, V. D. WO 2009/006185 A1.
10. Doods, H.; Gaida, W.; Wieland, H. A.; Dollinger, H.; Schnorrenberg, G.; Esser, F.;
Engel, W.; Eberlein, W.; Rudolf, K. Eur. J. Pharmacol. 1999, 384, R3.
11. Bacchi, F.; Mathé, A. A.; Jiménez, P.; Stasi, L.; Arban, R.; Gerrard, P.; Caberlotto,
L. Peptides 2006, 27, 3202.
15. The functional activity (fpki) against rat NPY Y2 was determined in a human
embryonic kidney (HEK) cell line transiently expressing the receptor, using a
384 well GTPcS35 assay.
16. The functional activity (fpki) against human NPY Y1 was determined in a
Chinese hamster ovarian (CHO) cell line stably expressing the receptor, using a
384 well GTP
17. The functional activity (fpki) at the human NPY Y5 receptor stably expressed in
c
35S assay.
HEK293 cells was assessed using FLIPR/Ca2+ methodology in
format.
a 384 well
12. Andres, C. J.; Antal Zimanyi, I.; Deshpande, M. S.; Iben, L. G.; Grant-Young, K.;
Mattson, G. K.; Zhai, W. Bioorg. Med. Chem. Lett. 2003, 13, 2883.
13. Jablonowski, J. A.; Chai, W.; Li, X.; Rudolph, D. A.; Murray, W. V.; Youngman, M.
A.; Dax, S. L.; Nepomuceno, D.; Bonaventure, P.; Lovenberg, T. W.; Carruthers,
N. I. Bioorg. Med. Chem. Lett. 2004, 14, 1239.
18. Aqueous solubility was measured via Chemiluminescent Nitrogen Detection
(CLND).
19. Compounds were characterised by 1H NMR spectroscopy and hplc coupled-
mass spectrometry. Compound 3: 1H NMR (400 MHz, CDCl3) d: 1.3 (s, 9H), 2.8–
3.2 (br m, 4H), 3.6 (br s, 2H), 4.0 (br s, 2H), 6.9 (d, 1H), 7.4 (d, 1H), 7.8 (s, 1H).
Compound 36: 1H NMR (400 MHz, CDCl3) d: 1.6 (s, 6H), 2.9 (m,2H), 3.1 (m, 2H),
3.3 (m, 2H), 3.9 (s, 3H), 3.9 (m, 2H), 7.1 (d, 1H), 7.2 (m, 1H), 7.3–7.4 (m, 5H), 7.5
(m,1H), 7.6 (s, 1H), 7.6 (m,1H), 8.2 (d, 1H).
14. The functional activity (fpki) against human NPY Y2 was determined in a
human neuroblastoma cell line (KAN-TS) which natively expresses NPY Y2,
using a 384 well GTPcS35 assay, values quoted were a mean of 4 or more expts.