Discovery and evaluation of spirocyclic derivatives as antagonists of the neuropeptide Y5 receptor

Article abstract of DOI:10.1016/j.bmcl.2012.02.098

A novel series of spirocyclic derivatives was synthesized and evaluated as NPY Y5R antagonists for the treatment of obesity. Cis and trans analogs 7a and 8a were equipotent in a Y5R binding assay (K_i's ≤ 1 nM) and displayed good stability in human and rat liver microsome preparations. Compound 7a failed to demonstrate weight loss activity in a diet-induced obese (DIO) rat model at unbound drug levels in the brain that exceeded the Y5R K_i value by 25-fold over a 24-h time-period.

Full text of DOI:10.1016/j.bmcl.2012.02.098

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2738–2743
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and evaluation of spirocyclic derivatives as antagonists
of the neuropeptide Y5 receptor
Michael Fichtner a, Eunsun Lee b, Elizabeth Tomlinson b, Dennis Scott c, Peter Cornelius b,
Terrell A. Patterson b, Philip A. Carpino a,
⇑
a Department of Worldwide Medicinal Chemistry, Pfizer PharmaTherapeutics, Groton, CT 06340, USA
b Department of Cardiovascular, Metabolic and Endocrine Diseases, Pfizer Global Research & Development, Groton, CT 06340, USA
c Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer PharmaTherapeutics, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of spirocyclic derivatives was synthesized and evaluated as NPY Y5R antagonists for the
treatment of obesity. Cis and trans analogs 7a and 8a were equipotent in a Y5R binding assay (Ki’s 6 1 nM)
and displayed good stability in human and rat liver microsome preparations. Compound 7a failed to
demonstrate weight loss activity in a diet-induced obese (DIO) rat model at unbound drug levels in
the brain that exceeded the Y5R Ki value by 25-fold over a 24-h time-period.
Received 19 December 2011
Revised 24 February 2012
Accepted 28 February 2012
Available online 6 March 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Neuropeptide Y
Obesity
NPY Y5R
Drug discovery
MK-0557
Velneperit
Neuropeptide Y (NPY) is an amidated 36 amino acid neurohor-
mone that is part of the pancreatic family of polypeptides.1,2 NPY is
widely distributed throughout the central nervous system (CNS),
including areas such as the hypothalamus, brainstem, and cerebral
cortex that are believed to play a role in the regulation of feeding
behavior, energy metabolism, anxiety, and reproductive health.
The pharmacological actions of NPY are mediated through at least
five G-protein coupled receptor subtypes (Y1R, Y2R, Y4R, Y5R, y6)
that are expressed in both the periphery (Y1R, Y2R) and CNS (Y1R,
Y4R, Y5R).3 Dysregulation of the NPYergic system has been impli-
cated in the pathophysiology of a number of diseases including
obesity, diabetes, and various cancers.4–6
Y5R has attracted considerable interest as a therapeutic target
for the treatment of obesity. Two Y5R antagonists, MK-0557 (1)
and S-2367 (2, velneperit), have been shown to decrease food in-
take and body weight gain in obese animal models and were ad-
vanced into clinical development (Fig. 1).7–9 Compound 2 is
reportedly in phase II clinical trials, however, the development of
1 has been halted because the weight loss observed in phase II
trials was not considered ‘clinically meaningful’.10 A variety of
other Y5R antagonists have also been reported in the literature,
including the diarylimidazole 3 from our laboratories and the urea
derivative 4.11–13 Compound 3 was shown to block Y5R agonist-in-
duced feeding responses in Sprague–Dawley (SD) rats following
oral administration, but exhibited no anorectic effects in either
Wistar or SD models of natural feeding behavior despite high
unbound drug exposure in the CNS, the primary site of action. Sim-
ilar results have been reported with other structurally distinct Y5R
H
F
N
N
N
O
S
H
O O
N
N
N
N
H
O
CF3
O
O
1 MK-0557, Ki = 1.6 nM
2 S-2367, hY5R = 5 nM
N
O
H
CN
N
O
N
N
N
N
H
N
O
O
O
3 hY5R = 1.2 nM
4
⇑
Corresponding author. Tel.: +1 860 441 5694; fax: +1 860 441 8067.
E-mail address: philip.a.carpino@pfizer.com (P.A. Carpino).
Figure 1. Structures of Y5R receptor antagonists 1–4.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.098

M. Fichtner et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2738–2743
2739
antagonists in lean animal models at doses that give high Y5R
O
W
O
receptor occupancies in the CNS, suggesting that the lack of activity
observed with the diarylimidazole series was not compound
specific.14
O
O
O
Following the advancement of 1 into human clinical trials, we
decided to re-investigate the role of Y5R in the regulation of
feeding behavior and body weight in obese rodent models. As
toxicological studies had uncovered several issues with diarylimi-
dazoles such as 3, a new series of spirocyclic Y5R antagonists exem-
plified by 5a and 6a was designed by combining structural and
pharmacophoric features from several different Y5R antagonists
including 1 and 4 (Fig. 2a). Minimized conformers of 5a and 6a did
not show high overlap with prototypical Y5R antagonists such as 1
(Fig. 2b), howeverour previous experience in the area suggestedthat
Y5R was tolerant to a wide array of structurally diverse chemotypes.
Herein, we describe the synthesis and pharmacological evaluation of
a novel series of spirocyclic derivatives that led to the identification
of 1-methyl-1-[10-oxospiro[cyclohexane-1,30(10H)-furo[3,4-c]pyri-
din]-4-yl]-3-(5-phenylpyr azin-2-yl) urea 7a as a potent Y5R antag-
onist with good CNS exposure.
Spirocyclic compounds 5a–g, 6a,b, 7a–e, and 8a were synthe-
sized in one step from intermediates 9a–i, prepared by reductive
animation of spirocyclohexanone derivatives 10a–f (Fig. 3) or by
a Curtius rearrangement of 11 (Scheme 1).15 Final compounds
were generally prepared as cis/trans mixtures which were then
separated where possible by silica gel chromatography. Stereo-
chemical assignments of the cis (less polar) diastereomer 7a and
the trans (more polar) diastereomer 8a were made on the basis
of NOE (nuclear overhauser effect) and HMBC (heteronuclear
multiple bond correlation) interactions between axial hydrogen
10a W = N
10b W = C
10c
O
O
W
O
O
O
10d
10e W = N
10f W = C
Figure 3. Structures of spirocyclohexanone derivatives 10a–f.
atoms on the cyclohexyl ring and various protons on the
furo[3,4-c]pyridin-1(3H)-one ring (Fig. 4).16 The cis geometry in
7a was confirmed by condensing phenyl 5-phenylpyrazin-2-ylcar-
bamate 13 with cis-9a, prepared with high diastereoselectivity as
part of the optimization of a bulk synthesis route.17 Stereochemical
assignments of the other final compounds were all inferred, with
the less polar compounds assigned cis geometries. Syntheses of
intermediates 10a,1710b,8 10c,18 and 10d19 have been previously
described in the literature. Intermediates 10e,f were prepared by
treatment of the dianions of 2-methyl-N-phenylbenzamide (14a)
or 3-methyl-N-phenylisonicotinamide (14b) with 1,4-dioxaspi-
ro[4.5]decan-8-one (15) to yield 16a,b, followed by deprotection
of the ketal group under acidic conditions (Scheme 2). Acetamide
7f was generated from 10f in 4 steps: (1) condensation with
methyl triphenylphosphorylideneacetate under microwave condi-
tions; (2) catalytic hydrogenation of the mixture of methyl 2-cyclo-
H
N
N
(a)
N
O
N
N
F
O
hexylideneacetate
and
methyl
2-(cyclohex-1-enyl)acetate
O
derivatives 17 and 18, followed by ester hydrolysis; and finally
(3) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-medi-
ated coupling of the acid 20 with 5-phenylpyrazin-2-amine
(Scheme 3).
5a
H
N
N
N
[6,5]- and [6,6]-Spirocyclic derivatives were evaluated in
125I-PYY binding displacement assays using Sf9 membranes over-
O
N
N
F
O
expressing
a recombinant human chimeric Y5/Y1 receptor
(Y5R).20 Metabolic stability of the compounds was measured in hu-
man and rat microsomal preparations.21 Cis and trans spirocyclic
analogs 5b and 6b displayed high Y5 receptor binding affinities,
validating the design strategy (Table 1). Cis diastereomer 5b (less
polar isomer, vide supra) was more potent than trans diastereomer
6b, a trend observed with many of the other pairs of diastereomers
(data not reported). Ligand efficiency and lipophilic efficiency
(LipE) for the more potent 5b were high (0.39 and 4.99, respec-
tively).22 Compound 5b showed moderate-to-high turnover in
human and rat liver microsome preparations, presumably because
of its lipophilicity. Replacement of the isobenzofuran-1(3H)-one
moiety in 5b with a more polar furo[3,4-c]pyridin-1(3H)-one group
as in 5a did not result in any loss of Y5R binding affinity, but LipE
improved significantly (6.3). Compound 5a displayed good stability
in both human and rat liver microsomes. Trans diastereomer 6a
could not be isolated in diastereomerically pure form and was
not tested. Removal of the N-methyl group on the urea moiety in
5a led to a significant drop in affinity (5c). Compounds 5d,e which
lack carbonyl groups on the spirocyclohexane rings were 11- and
14-fold less potent than 5b, but still showed nanomolar affinity
for Y5R. The carbonyl group appears important for reducing the
global lipophilicity of the molecules and thus improving metabolic
stability. [6,6]-Spiro isochroman-1-one analog 5f showed good
O
6a
(b)
Figure 2. (a) Structures of 5a and 6a; (b) Overlay of minimized conformer of 5a and
6a with minimized conformer of 1.

2740
M. Fichtner et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2738–2743
O
X
Y
W
N
Z
H
a or b,c (for 10a only)
N
R1
X
10a-f
W
Z
d,e
Y
O
9a W = N, X = absent, Y = CO, Z = O, R1 = Me
OH
9b W = N, X = absent, Y = CO, Z = O, R1 = H
9c W = C, X = absent, Y = CO, Z = O, R1 = Me
9d W = C, X = absent, Y = CH2, Z = O, R1 = Me
9e W = C, X = absent, Y = O, Z = CH2, R1 = Me
9f W = C, X = CH2, Y = CO, Z = CH2, R1 = Me
9g W = C, X = CH2, Y = CO, Z = O, R1 = Et
9h W = C, X = CH2, Y = CO, Z = O, R1 = CH2CF3
9i W = N, X = CH2, Y = CO, Z = O, R1 = Me
O
O
11
R1
H
N
N
X
Y
W
O
N
N
F
Z
f
5a-g (cis isomers), 6a,b (trans)
(see Table 1 for W, X, Y, Z, and R1 groups)
g
R1
N
H
N
N
X
Y
W
O
N
Z
7a-e (cis isomers), 8a (trans)
(see Table 1 for W, X, Y, Z, and R1 groups)
Scheme 1. Reagents and conditions: (a) R1NH2ꢀHCl, NEt3, NaBH(OAc)3; 1,2-dichloroethane, 23 °C; (b) R1NH2ꢀHCl, NEt3, Ti(O-i-Pr)4, EtOH, 23 °C; (c) NaBH4; (d) (PhO)2PON3,
NEt3, t-BuOH, 85 °C; (e) concd HCl, EtOH, 23 °C; (f) phenyl 1-(2-fluorophenyl)-1H-pyrazol-3-ylcarbamate (12), DMSO, 23 °C; (g) phenyl 5-phenylpyrazin-2-ylcarbamate (13);
DMSO, 23 °C.
O
O
NHPh
O
W
O
a,b
O
NHPh
OH
H
N
W
N
N
O
N
H
O
N
14a W = N
14b W = C
16a W = N
16b W = C
NOE interaction
7a
O
c (W = C)
d (W = N)
W
O
N
O
N
H
HMBC
N
N
O
H
O
H
10e W = N
10f W = C
O
N
NOE interaction
Scheme 2. Reagents and conditions: (a) 2 equiv n-BuLi, THF, ꢁ78 °C; (b) 1,4-
dioxaspiro[4.5]decan-8-one (15), ꢁ78 °C to 23 °C; (c) H2O, HOAc, H2SO4, 115 °C, 5 h;
(d) H2O, HOAc, H2SO4, 65 °C, 18 h.
8a
Figure 4. NOE (nuclear overhauser effect) and HMBC (heteronuclear multiple bond
correlation) interactions in cis and trans diastereomers 7a and 8a.
no change in binding affinity was observed with a similar atom
exchange (e.g., 5a,b).
The difficulty of separating compound 5a from 6a via column
chromatography prompted an investigation into replacements for
binding affinity, but interestingly, analog 5g with an extra nitrogen
atom in the aromatic ring in the spiro moiety was less active in the
binding assay, in contrast to the [6,5]-spiro series of analogs where

M. Fichtner et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2738–2743
2741
O
CO2Me
CO2Me
a
+
O
O
O
O
O
O
10f
17
18
H
N
CO2R2
N
e
b,c
O
N
O
O
O
O
19 R2 = Me
20 R2 = H
7f (~1:1 mixture of cis/trans isomers)
d
Scheme 3. Reagents and conditions: (a) Ph3P = CHCO2Me, CH3CN, microwave irradiation, 190 °C, 0.5 h; (b) H2, PtO2, EtOAc, 23 °C; (c) aq. LiOH, MeOH, 23 °C; (d) concd HCl; (d)
5-phenylpyrazin-2-amine, EDCꢀHCl, pyridine, 23 °C.
Table 1
Y5R binding affinities, calculated lipophilicities, and intrinsic clearances of compounds 1, 5a–g, 6b, 7a–f, and 8a
H
H
A
N
A
N
X
Y
N
W
O
N
N
X
Y
W
O
N
F
Z
Z
5a-g (less polar cis compound)
7a-f (less polar cis compound)
H
H
N
N
N
N
N
N
O
N
N
O
N
F
O
O
O
O
6b (more polar trans urea)
8a (more polar trans urea)
X
Y
cLogPa
Y5R Ki (nM)b,c
Clint, human/rat (ml/mg/kg)d
nd
W
N
Compd
W
X
Y
Z
A
Z
1
2.0
1.6
5a
N
Absent
CO
O
NMe
1.9
0.8
<7/28
O
O
O
O
5b
6b
C
C
Absent
Absent
CO
CO
O
O
NMe
NMe
3.2
3.2
0.7
22
24/165
nd
‘‘
N
5ce
N
Absent
CO
O
NH
2.0
849
nd
O
O
O
5d
5e
C
C
Absent
Absent
CH2
O
O
NMe
NMe
4.0
4.6
10
8
67/>1120
68/>1120
CH2
O
5f
C
CH2
CO
O
NMe
4.7
2.4
28/290
O
O
(continued on next page)

2742
M. Fichtner et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2738–2743
Table 1 (continued)
X
Y
W
Compd
W
N
X
Y
Z
A
cLogPa
Y5R Ki (nM)b,c
Clint, human/rat (ml/mg/kg)d
Z
N
N
5g
CH2
CO
O
NMe
3.3
188
nd
O
O
O
7a
8a
N
N
Absent
Absent
CO
CO
O
O
NMe
NMe
2.1
2.1
1
<6/<25
<7/12
O
‘‘
0.9
N
7b
N
CH2
CO
O
NMe
NMe
3.5
82
<26/86
59/333
O
O
7c
C
CH2
CO
O
4.9
4.6
O
O
7d
7e
7fe
‘‘
‘‘
‘‘
C
C
C
CH2
CH2
CH2
CO
CO
CO
O
O
O
NEt
NCH2CF3
CH2
5.3
5.6
5.5
11
2330
301
nd
<7/nd
nd
nd: not determined.
a
Calculated using ACD/logP predictor.
b
c
Human Y5R radioligand binding assay.20
Data represent single determinations obtained in triplicate.
Intrinsic clearance values (Clint) were determined in human and rat liver microsome (HLM, RLM) preparations and refer to total intrinsic clearance obtained from scaling
d
in vitro HLM or RLM half-lives.21
e
Mixture of cis/trans diastereomers.
the 1-(2-fluorophenyl)-1H-pyrazol-3-amine group. Analogs of
ureas 5a, 6a with different biaryl amines were examined, but only
compounds with a 5-phenylpyrazin-2-amine maintained good
potency and high LipE values (>6). Cis pyrazinyl analog 7a and
the easily separable trans diastereomer 8a were equipotent to 5a
and were shown to be stable in human and rat liver microsome
preparations. 3,4-Dihydropyrano[4,3-c]pyridin-1-one 7b showed
a significant drop in binding affinity compared to 7a. Removing
the weakly basic nitrogen atom in 7b to provide 7c resulted in a
significant increase in binding affinity, a trend that was also ob-
served in the [6,6]-spirocyclohexane rings in the pyrazolyl series
(e.g., 5g, 5f). In the isochroman-1-one series, binding affinity de-
creased as the length and size of alkyl group on the urea moiety in-
creased from one carbon atom (methyl analog 7c) to two carbon
atoms (ethyl analog 7d, trifluoroethyl analog 7e) or as the N-
methyl group in the urea moiety was replaced by a methylene
group to give spirocyclic derivative 7f.
From PK modeling studies, it was determined that a 5 mg/kg bid
dose could provide 24 h coverage of 20-fold the Ki value in the
CNS and maintain high receptor occupancy over the period (not
actually measured).
The anti-obesity effects of 7a (5 mg/kg bid) and 1 (1.5 mg/kg
bid) were evaluated in a 4-d study in a DIO rat model.25 Compound
7a induced a small decrease in food intake (<10%) which resulted
in a minimal effect on body weight loss. Unbound drug levels in
the brain exceeded the Y5R Ki value by 25-fold. Compound 1
showed similar anorectic effects at unbound brain concentrations
that exceeded the Y5R Ki value by 20-fold, but induced a greater
decrease in body weight. The changes in body weight observed
with 1 were greater than the changes in body weight seen in a
pair-fed control group. A possible explanation for this effect is that
1 may have decreased increased energy expenditure (EE) in the
DIO rats. Effects of Y5R antagonists on EE have been previously re-
ported.26 The lack of significant body weight changes observed
with compound 7a correlated with the minimal effects on food in-
take, suggesting that additional weight loss mechanisms, such as
changes in EE, were not observed with this compound. The anorec-
tic effects of both 7a and 1 were modest in DIO rats, suggesting Y5R
may not play a major role in regulating food intake in this animal
model or that other feeding mechanisms may compensate when
the Y5R pathway is blocked. Further characterization of 7a in other
models of obesity was not investigated.
Compounds 7a and 8a emerged as promising candidates for
in vivo studies exploring the role of Y5R in obese animal models.
Compound 7a proved easier to isolate on scale-up using silica-gel
column chromatography and so was selected over 8a. Compound
7a showed good Y5R functional activity in a GTP-
say and excellent selectivity against the human Y1R (Ki >10
c
-
35S binding as-
l
M).23
It did not display any significant off-target activity across a panel of
124 receptors, ion channels, transporters, and enzymes (<50% inhi-
bition at 10
l
M).24 Pharmacokinetic evaluation in the rat revealed
In conclusion, we have identified a novel series of spirocyclic Y5R
antagonists. Replacement of the 1-(2-fluorophenyl)-1H-pyrazol-3-
ylamine in 5a with a 5-phenylpyrazin-2-ylamine led to the discovery
of 7a which possessed high Y5R binding affinity, good physicochem-
ical and pharmacokinetic properties, and good CNS exposure. Evalua-
tion of 7a in our DIO rat model failed to demonstrate significant
anti-obesity effects associated with Y5R antagonism.
a short half-life (t1/2 = 2 h) and high bioavailability (F = 80%). Stud-
ies measuring unbound fraction of 7a in plasma and brain from
rats showed the drug was modestly brain impaired (fu, plasma/
fu, brain = 3.3). Brain impairment was confirmed in diet-induced
obese (DIO) rats by measuring unbound concentrations of 7a in
plasma (67 nM) and brain (27 nM) following a 5 mg/kg bid dose.

M. Fichtner et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2738–2743
2743
15. All compounds described in this manuscript gave satisfactory spectral data (1H
NMR, MS).
Acknowledgments
16. 1H NMR spectroscopy: compounds 7a and 8a were dissolved in 0.5 mL of
deuterated dimethyl sulfoxide solution with 0.05% (v/v) tetramethylsilane
(TMS) as NMR reference. The 1D proton spectrum was referenced using TMS,
set to 0.00 ppm. The 1D carbon spectrum was referenced to the solvent at
39.5 ppm. All 1D and 2D data were collected at 298 K using a Bruker-Biospin
We thank Fangming Kong and Bao Nguyen for the 1H NMR anal-
yses of compounds 7a and 8a.
References and notes
5 mm TCI cryoprobe on
a Bruker-Biospin AVANCE NMR spectrometer
operating at 700 MHz. Compound 7a: 1H NMR (700 MHz, DMSO-d6, d ppm):
9.43(s, 1H, NH), 9.17 (s, 1H), 9.17 (s, 1H), 8.93 (d, 1H, J = 1.5 Hz), 8.88 (d, 1H,
J = 5.1 Hz), 8.09 (d, 2H, J = 7.3 Hz), 7.85 (d, 1H, J = 5.1 Hz), 7.51 (dd, 2H,
J = 8.0 Hz, 8.0 Hz), 7.44 (t, 1H, J = 7.3 Hz), 4.52 (tt, 1H, J = 12.4 Hz, 3.6 Hz), 2.97
(s, 3H), 2.40 (td, 2H, J = 13.8 Hz, 3.6 Hz), 1.95 (qd, 2H, J = 12.6, 3.6 Hz), 1.85 (d,
2H, J = 13.1 Hz), 1.75 (d, 2H, J = 11.6 Hz); 13C NMR (DMSO-d6, 175 MHz, d ppm):
167.8, 154.6, 150.2, 149.4, 147.3, 144.8, 144.5, 138.7, 136.1, 135.8, 132.5, 128.9,
128.8, 125.8, 118.5, 85.9, 51.7, 34.6, 28.7, 25.6; compound 8a: 1H NMR
(700 MHz, DMSO-d6, d ppm): 9.47 (s, 1H, NH), 9.45 (s, 1H), 9.16 (d, 1H,
J = 1.5 Hz), 8.95 (d, 1H, J = 5.1 Hz), 8.93 (d, 1H, J = 1.5 Hz), 8.09 (d, 2H,
J = 7.3 Hz), 8.07–7.91–7.89 (m, 1H), 7.53–7.49 (m, 2H), 7.45–7.42 (m, 1H),
4.25 (tt, 1H, J = 11.7 Hz, 3.6 Hz), 3.11 (s, 3H), 2.32–2.26 (m, 4H), 1.91 (d, 2H,
J = 11.6), 1.86 (d, 2H, J = 10.2 Hz); 13C NMR (DMSO-d6, 175 MHz, d ppm): 167.5,
154.6, 150.4, 149.4, 146.4, 144.8, 144.6, 138.7, 136.1, 135.9, 133.4, 128.9, 128.8,
125.8, 119.1, 86.9, 53.5, 34.3, 30.4, 25.1.
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