The identification of a series of novel, soluble non-peptidic neuropeptide y Y2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.10.065

The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with enhanced CNS exposure.

Full text of DOI:10.1016/j.bmcl.2010.10.065

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7341–7344
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The identification of a series of novel, soluble non-peptidic 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 L. Caberlotto c, Amanda Emmons b, Ian P. Holmes a, , Dino Montanari c,
Roz Norris b, Gemma V. Puckey a, Dewi J. Walters a, Steve P. Watson a, John Willis 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:
Received 11 July 2010
Revised 13 October 2010
Accepted 14 October 2010
Available online 10 November 2010
The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is
described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with
enhanced CNS exposure.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
NPY antagonist
NPY Y2 antagonist
NPY
NPY Y2
Neuropeptide Y (NPY) is a member of the pancreatic polypep-
tide family that includes pancreatic polypeptide (PP) and peptide
YY (PYY).1 NPY is thought to be involved in regulating a number
of physiological processes2–4 including: food intake; water
consumption; anxiety; circadian rhythm; memory processing;
endocrine and cardiovascular functions via action at six specific
receptors in mammalian systems (Y1, Y2, Y3, Y4, Y5 and Y6).5,6
We previously reported studies leading to the identification of
pyridine 1 as a potent, selective NPY Y2 receptor antagonist
(Fig. 1, Table 1).7
generally poor physiochemical properties and only one such study
has been described. Caberlotto and co-workers have demonstrated
hNPY Y2 fpKi = 7.6
rNPY Y2 fpKi = 7.3
BTB (rat) = 97.1%
Clint mL/min/g = 0.7 (human), 2.7 (rat)
cLogP = 4.32
Solubility = 56 µg/mL
O
Ph
HN
O
N
N
N
Cl
MeO
.HCl
1
Figure 1. Structure and profile of pyridine 1.
Whilst compound 1 has a promising in vitro pharmacokinetic
(PK) profile, it exhibited disappointing in vivo rat PK with a brain
to blood ratio of 0.6 and low exposure in the brain (6 ng/g) follow-
ing a single subcutaneous (sc) administration of 3 mg/kg (Table 1).
Herein we report further optimisation studies of the diamide
series exemplified by 1 resulting in the identification of amine 2
(Fig. 2) as a selective NPY Y2 antagonist with improved in vivo
CNS exposure.
Table 1
In vivo PK profile of pyridine 1 after sc administration
Dose
(mg/kg)
Br:Bl
Cmax (Br) (ng/g)
Cmax (Bl) (ng/mL)
Sample time (h)
1.0
3.0
0.6
6
13
A number of selective small molecule NPY Y2 antagonists have
now been described.4,7–10 Unfortunately, the utility of these to date
for in vivo target validation studies has been limited by their
HN
N
N
N
O
Cl
⇑
Corresponding author.
E-mail address: gill@lunniss.net (G.E. Lunniss).
F3C
N
2
Present address: Cancer Therapeutics, Monash Institute of Pharmaceutical
Sciences, 381 Royal Parade, Parkville, Vic. 3052, Australia.
Figure 2. Selective, soluble NPY Y2 antagonist 2.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.065

7342
G. E. Lunniss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7341–7344
HN
O
N
N
N
N
R1
HN
N
N
Cl
Cl
O
Cl
13
O
HN
O
R1
hNPY Y2 fpKi = 7.3
rNPY Y2 fpKi = 7.1
BTB (rat) = 99.6%
Clint mL/min/g = <0.53 (human), <0.53 (rat)
MW = 488
Figure 3. SAR for the comparison of amines and amides.
cLogP = 5.88
Solubility = 27 µg/mL
Table 2
Profiling results for the comparison of amine and amide analogues
Figure 4. Profile of piperidine 13.
R1
hNPY Y2 (fpKi)a
Solubilitya
(
lg/mL)
3
4
5
6
H
6.6 (6.3)
6.4 (6.9)
6.7 (6.6)
6.7 (6.3)
38 (40)
25 (53)
4 (52)
2-F
3-F
4-F
comparable potency14 to the corresponding amides but exhibited
lower aqueous solubility.
2 (36)
a
Amines (3–6) were synthesized using the synthetic route
detailed in Scheme 1. A SNAr reaction of Boc-piperazine and
2-chloro-1-fluoro-4-nitrobenzene (7) gave nitro compound 8. Sub-
sequent reduction using iron/ammonium chloride of the nitro
group gave aniline 9 with retention of the aromatic chloride. Ani-
lide formation using HATU afforded 10. Deprotection of the Boc
group under acidic conditions gave aryl piperazine 11 which was
subsequently reductively alkylated to afford the desired com-
pounds 12.15
Data in parentheses is for the amide analogue.
Boc
N
Boc
N
Boc
N
F
N
N
N
Cl
Cl
Cl
(i)
(ii)
(iii)
Cl
Alternatives to the piperazine ring including 3 and 4-substi-
tuted piperidines, morpholine, homopiperidine and 3-substituted
pyrrolidines were then investigated. Using a synthetic strategy
similar to that described in Scheme 1 a diverse range of amines
were substituted on to 7 and then elaborated to give analogues
of 12. Throughout this study the benzhydryl group was used as
the R2 group of choice. The majority of compounds prepared dis-
played low potency at NPY Y2 with the exception of piperidine
13 (Fig. 4). Compound 13 displayed encouraging potency (hNPY
Y2 pIC50 = 7.3), low intrinsic clearance and promising aqueous sol-
ubility despite its high lipophilicity.
NO2
R2 NH
NO2
NH2
9
7
8
10
O
R3
H
N
N
N
N
(v)
(iv)
Cl
Cl
R2 NH
R2 NH
Initially, SAR explorations around 13 focused on replacing the
benzhydryl group to reduce lipophilicity (Fig. 5, Table 3). As previ-
ously observed for the diamide series, it was found that the benz-
hydryl group could be replaced with substituted phenylacetamides
for example 14–17. Additionally, meta substitution of the phenyl-
acetamide with a lipophilic group, for example methyl 16 or tri-
fluoromethyl 17, gave compounds with enhanced NPY Y2
potency compared to the unsubstituted derivative 14. Increasing
the bulk of the benzylic substituents as in cyclopentyl 15 also re-
sulted in a potency increase. All the phenylacetamide compounds
tested displayed improved aqueous solubility compared to benz-
hydryl 13. Attempts at reducing the lipophilicity by introduction
of a heteroatom into the benzhydryl group (e.g., -18) resulted in
a reduction in the NPY Y2 potency.
Modification of the distal piperidine group was then studied
and a series of mono substituted 4-amino piperdines were pre-
pared (Fig. 6, Table 4). It was shown that the size of the 4-amino
substituent affected NPY Y2 potency and for an alkyl chain potency
increased from ethyl to i-butyl (19–22). Methylene cyclopropyl
substitution was well tolerated, as in 23, and inferred similar po-
tency to an i-butyl chain but with reduced lipophilicity. As previ-
O
11
O
12
Scheme 1. Representative synthesis of amine analogues. Reagents and conditions:
(i) Boc-piperazine, Hünigs base, MeCN, 150 °C, microwave; (ii) iron, ammonium
chloride, water, methanol, 80 °C; (iii) R2CO2H, HATU, Hünigs base, NMP; (iv) HCl in
diethyl ether; (v) R3CHO, AcOH, NMP, NaBH(OAc)3.
that the potent and selective pseudo-peptide BIIE0246 (NPY Y2
IC50 = 3.3 nM), when administered via intracerebroventricular
(icv) injection, induced an anxiolytic effect in rats in the elevated
plus maze model.11 Whilst encouraging this result is open to inter-
pretation as the use of icv administration in an anxiety model is
not ideal.
The aim of this study was to identify selective NPY Y2 antago-
nists suitable for in vivo target validation studies when adminis-
tered either orally or via sc dosing. Additionally, we sought to
investigate whether total or free brain concentration of a selective
NPY Y2 antagonist correlated with in vivo receptor occupancy and
hence pharmacodynamic (PD) effects. It was anticipated that by
synthesizing compounds with a range of brain tissue binding
(BTB)12 that suitable tool molecules could be identified to study
the interplay between potency and brain free fraction in achieving
receptor occupancy for the NPY Y2 receptor.13
Ar
R4
HN
O
N
N
Using our previously reported diamide series7 as a starting
point the effect of modifying the piperazine amide was examined
(Fig. 3, Table 2). It was found that the amines (3–6) were of
R5
Cl
Figure 5. SAR for replacements of the benzhydryl amide.

G. E. Lunniss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7341–7344
7343
Table 3
Profiling results for replacements of the benzhydryl amide
Ar
R4
R5
hNPY Y2 (fpKi)
c log P
Solubility (lg/mL)
14
15
16
17
Ph
Ph
m-MePh
m-CF3Ph
2-Pyridyl
Me
–(CH2)3–
Me
Me
Ph
Me
6.5
7.1
7.2
7.8
6.5
5.24
5.99
5.73
6.12
4.38
92
114
131
96
Me
Me
H
-18
NT
ously observed addition of a meta substituent resulted in increased
potency, for example 24 and 25. Pleasingly, addition of a meta sub-
stituent also resulted in greatly enhanced aqueous solubility.
Physiochemical profiling of project compounds established a
linear relationship between c log P (calculated using ACD software)
and measured log D at pH 7.4 for both the diamide and amine
series. Both parameters were also shown to be predictive of mea-
sured BTB with high c log P correlating with high BTB. We were
therefore able to specifically target compounds for synthesis with
low c log P that were predicted to have a high fraction unbound
in brain tissue.
As pyrimidine 2 demonstrated a balanced profile of NPY Y2
potency, solubility, Clint and BTB ( Tables and 8) it was
7
selected for further profiling. In vitro cross screening showed 2
exhibited comparable potency at both human and rat16 NPY Y2
receptors and selectivity against human NPY Y117 and Y518
receptors (Table 9, Fig. 10). Additionally, 2 had an excellent CYP-
EX bactosome p450 inhibition profile showing inhibition of
>10
lM against the 1A2, 2C9, 2C19, 2D6 and 3A4 (7BQ and
DEF) isoforms.
Consequently, heterocyclic acetamides including the pyridine
26 and pyrimidines 27, 28 and -29 were synthesized and tested
(Fig. 7, Table 5). Unfortunately, these compounds displayed signif-
icantly reduced NPY Y2 potency compared to the corresponding
phenylacetamide derivatives.
R10
X
HN
O
N
NH
R9
N
Cl
R8
In an attempt to increase potency, to allow the incorporation of
heteroatoms, optimisation of the position of the basic nitrogen was
reinvestigated and a number of extended and spirofused amines
synthesized. The majority of compounds prepared were poorly
active (data not shown) with the exception of spiroamine 30
(Fig. 8, Table 6).
Figure 7. SAR for heterocyclic amide replacements.
Table 5
Profiling results of heterocyclic amide replacements
Further gains in potency were achieved when spiroamine 30
was reductively alkylated to afford amine 31 which demonstrated
excellent potency at the NPY Y2 receptor but unsurprisingly with
reduced aqueous solubility (Fig. 9, Table 7). To reduce the lipophil-
icity of 31, the corresponding pyrimidine analogue was prepared.
Pleasingly pyrimidine 2 still maintained significant potency (hNPY
Y2 pIC50 = 6.8) coupled with considerable aqueous solubility.
Profiling of exemplar compounds to determine in vitro intrinsic
clearance (Clint) revealed promising metabolically stability in both
rat and human liver microsomes (Table 8). Notably, the introduc-
tion of a trifluoromethyl group in 25 increased metabolically stabil-
ity and enhanced potency at the NPY Y2 receptor. As predicted, BTB
R8
X
R9
R10 hNPY Y2 (fpKi) c log P Solubility (
lg/mL)
26
27
28
H
C
Me Me 5.9
Me Me 6.4
Me Me 6.1
2.90
2.90
2.44
2.08
NT
CF3
Me
H
N
N
N
112
248
177
-29
Et
H
6.2
NH
HN
O
N
Cl
30
values increased with lipophilicity and incorporation of
a
F3C
pyrimidine acetamide led to a significant reduction in BTB as exem-
plified by 28 and -29.
Figure 8. Spiroamine replacement.
HN
O
N
NH
R7
Table 6
Profiling results for spiroamine 30
Cl
R6
hNPY Y2 (fpKi)
c log P
Solubility (
96
l
g/mL)
g/mL)
30
7.8
5.16
Figure 6. SAR for piperidine replacements.
Table 4
Profiling results for piperidine replacements
R6
R7
hNPY Y2 (fpKi)
c log P
Solubility (
l
19
20
21
22
23
24
25
H
H
H
H
H
Me
CF3
Et
i-Pr
n-Pr
i-Bu
5.7
6.0
6.6
7.1
6.9
7.6
7.7
3.96
4.26
4.48
4.88
4.40
4.90
5.28
NT
NT
NT
NT
121
268
219
Methylene cyclopropyl
Methylene cyclopropyl
Methylene cyclopropyl

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G. E. Lunniss et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7341–7344
In vivo pharmacokinetic profiling in the rat showed 2 displayed
HN
O
N
N
a brain to blood ratio of 2.3 with 73 ng/g in brain and 32 ng/mL in
blood after a single sc administration at 2 mg/kg (Table 10).
In summary, we have identified a novel series of soluble, selec-
tive small molecule NPY Y2 antagonists exemplified by amine 2.
Compound 2 has a significantly improved in vivo PK profile com-
pared to our previously described diamide series, with comparable
potency at the rat NPY Y2 receptor in vitro, making it a suitable
candidate for in vivo target validation studies.
X
Cl
F3C
X
31 X=C
X=N
2
Figure 9. SAR of substituted spiroamines.
Table 7
Profiling results of substituted spiro amines
hNPY Y2 (fpKi)
c log P
Solubility (lg/mL)
References and notes
31
2
8.7
6.8
6.41
4.03
53
225
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Table 8
Sample compounds selected for in vitro pharmacokinetic profiling
hNPY Y2
(fpKi)
c log P Clint rat (mL/ Clint human
BTB
(rat) %
min/g)
(mL/min/g)
23
24
25
28
-29
2
6.9
7.6
7.7
6.1
6.2
6.8
4.40
4.90
5.28
2.44
2.08
4.03
5.7
16.0
1.4
1.9
0.8
0.8
4.8
<0.5
<0.5
0.7
97.6
99.1
99.6
88.2
75.9
97.2
1.8
0.8
11. Bacchi, F.; Mathé, A. A.; Jiménez, P.; Stasi, L.; Arban, R.; Gerrard, P.; Caberlotto,
L. Peptides 2006, 27, 3202.
12. Brain fractions unbound were determined using equilibrium dialysis in a
96-well format as described in Ferrari, L.; Crestan, V.; Sabattini, G.; Vinco, F.;
Fontana, S.; Gozzi, A. J. Neurosci. Methods 2010, 186, 143.
HN
O
N
N
N
Cl
F3C
N
2
13. Watson, J.; Wright, S.; Lucas, A.; Clarke, K. L.; Viggers, J.; Cheetham, S.; Jeffrey,
P.; Porter, R.; Read, K. D. Drug Metab. Dispos. 2009, 37, 753.
14. The functional activity (fpKi) against human NPY Y2 was determined in a
human neuroblastoma cell line (KAN-TS) which natively expresses NPY Y2,
Figure 10. Pyrimidine 2.
using a 384 well GTPcS35 assay, values quoted were a mean of four or more
experiments.
15. Compounds were characterised by 1H NMR spectroscopy and LC–MS.
16. The functional activity (fpKi) against rat NPY Y2 was determined in a human
embryonic kidney (HEK) cell line transiently expressing the receptor, using a
Table 9
Selected in vitro selectivity data for pyrimidine 2
384 well GTPc35S assay.
hNPY Y2 (fpKi)
rNPY Y2 (fpKi)
NPY Y1 (fpKi)
NPY Y5 (fpKi)
17. The functional activity (fpKi) against human NPY Y1 was determined in a
2
6.8
7.2
<5.2
<5.4
Chinese hamster ovarian (CHO) cell line stably expressing the receptor, using a
384 well GTP
c35S assay.
18. The functional activity (fpKi) at the human NPY Y5 receptor stably expressed in
HEK293 cells was assessed using FLIPR/Ca2+ methodology in
format.
a 384-well
Table 10
In vivo DMPK profile of 2 after sc administration
Dose (mg/kg)
2.0
Br:Bl
2.3
Cmax (Br) (ng/g)
Cmax (Bl) (ng/mL)
Sample time
1.0
73
32