Discovery and structure-activity relationship of a novel spirocarbamate series of NPY Y5 antagonists

Article abstract of DOI:10.1016/j.bmcl.2010.08.041

A novel series of trans-8-aminomethyl-1-oxa-3-azaspiro[4.5]decan-2-one derivatives was identified with potent NPY Y5 antagonist activity. Optimization of the original lead furnished compounds 23p and 23u, which combine sub-nanomolar Y5 activity with metabolic stability, oral bioavailability, brain penetration and strong preclinical profile for development. Both compounds significantly inhibited the food intake induced by a Y5 selective agonist with minimal effective doses of 3 mg/kg po.

Full text of DOI:10.1016/j.bmcl.2010.08.041

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6103–6107
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and structure–activity relationship of a novel spirocarbamate
series of NPY Y5 antagonists
Colin P. Leslie , Jonathan Bentley , Matteo Biagetti, Stefania Contini, Romano Di Fabio, Daniele Donati à,
⇑
Thorsten Genski §, Sebastien Guery, Angelica Mazzali, Giancarlo Merlo, Domenica A. Pizzi, Fabiola Sacco,
Catia Seri, Michela Tessari, Laura Zonzini, Laura Caberlotto
Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline SpA, 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:
A novel series of trans-8-aminomethyl-1-oxa-3-azaspiro[4.5]decan-2-one derivatives was identified with
potent NPY Y5 antagonist activity. Optimization of the original lead furnished compounds 23p and 23u,
which combine sub-nanomolar Y5 activity with metabolic stability, oral bioavailability, brain penetration
and strong preclinical profile for development. Both compounds significantly inhibited the food intake
induced by a Y5 selective agonist with minimal effective doses of 3 mg/kg po.
Received 13 July 2010
Revised 6 August 2010
Accepted 7 August 2010
Available online 12 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keyword:
NPY Y5
Neuropeptide Y (NPY) is a 36-amino acid peptide neurotrans-
mitter discovered in 1982.1 A member of the pancreatic polypep-
tide family, it is abundant in both the central and the peripheral
nervous systems2 and it is implicated in a variety of physiological
functions, notably the central regulation of feeding behavior and
energy homeostasis.3
therapeutic applications for the treatment of eating disorders, drug
dependency and depression.
We report here our efforts to identify a proprietary series of Y5
antagonists that led to the discovery of a novel spirocarbamate
scaffold, endowed with potent Y5 antagonist activity and a strong
preclinical profile for development.
The actions of NPY are mediated through five G-protein coupled
receptors (Y1, Y2, Y4, Y5, y6).4 A number of pharmacological stud-
ies conducted with transgenic mice and/or sub-type selective ago-
nists and antagonists have suggested that the Y5 receptors are
involved in the regulation of food intake.5 Additionally, small mol-
ecule Y5 antagonists have proven efficacious in animal models of
addiction to drugs of abuse6 and in animal models of depression.7
To date, clinical studies reported with small molecule Y5 antago-
nists have demonstrated modest, albeit significant, anti-obesity
effects in obese human subjects.8 However, considering the poten-
tial implication of NPY Y5 in regulating addiction and depression
there is ample scope for Y5 antagonists to offer greater efficacy
in psychiatric eating disorders with stress and/or compulsive/
impulsive components. Hence, antagonism of the Y5 receptor rep-
resents an attractive pharmacological approach with potential
A screening campaign of our corporate compound collection
generated a number of promising chemotypes from which the
aminothiazolepyridine 1 was identified as an efficient, orally bio-
available and brain penetrant Y5 antagonist (Fig. 1).
A direct comparison of compound 1 with the structures of
known Y5 antagonists9 revealed some recurrent pharmacophoric
features and suggested that the opportunity to further optimize
this molecule lay with the modification of the N-phenyl side chain.
Accordingly, a number of prospective compound arrays were pre-
pared from commercially available 4-(2-pyridinyl)-1,3-thiazol-2-
amine 2. From amongst the derivatives synthesized, attention
was drawn to the spirocyclic urea derivative 3, which was charac-
terized by potent Y5 antagonist activity and promising in vitro
criteria for development (Fig. 2).
It is worthy to note that a number of highly potent NPY Y5
antagonists containing other spiropiperidine substructures have
⇑
Corresponding author. Tel.: +39 045 8218546; fax: +39 045 8218196.
N
hNPY Y5 fpKi 7.8
hNPY Y1/2 fpKi<4.5
CYP450 1A2 <0.1 uM
Clb 27 ml/min/kg
F= 30%
B/B 4.2
CF3
S
E-mail address: colin.p.leslie@gsk.com (C.P. Leslie).
Present address: Evotec, 114 Milton Park, Abingdon, Oxfordshire OX14 4SA,
United Kingdom.
N
N
H
à
Present address: Nerviano Medical Sciences Srl, Via Pasteur 10, 20014 Nerviano,
1
Milan, Italy.
§
Present address: AnalytiCon Discovery GmbH, Hermannswerder Haus 17, 14473
Potsdam, Germany.
Figure 1. Structure of hit compound 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.041

6104
C. P. Leslie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6103–6107
Commercially available ethyl 4-oxocyclohexanecarboxylate (8)
O
O
was reacted with a trimethylsulfoxonium iodide derived ylide to
give an inseparable diastereoisomeric mixture of epoxides 9 that
favoured the cis diastereoisomer (cis/trans ratio ꢀ85:15). The mix-
ture of epoxides was opened with an excess of aniline to give a
similar diastereomeric mixture of the b-hydroxyanilines 10, that
was subsequently cyclized with triphosgene at low temperature
to give, after chromatographic separation on silica gel, the trans
and cis spirocarbamates 11 and 12. Ester saponification and amide
coupling gave the trans and cis amide derivatives 4 and 5,
respectively.
N
NH2
S
hNPY Y5 fpKi 8.7
hNPY Y1/2 fpKi<6.2
CYP450 >10 uM
N
N
N
O
NH
Clint (h/r) = 0.6/0.5 ml/min/g
N
S
2
N
Alternatively, ester reduction with lithium aluminium hydride
followed by oxidation with Dess–Martin periodinane gave the
trans and cis aldehydes 17 and 18 which underwent reductive ami-
nation to give the N-alkylated derivatives 6 and 7, respectively. The
amide derivatives 4 and 5 were found to be poorly active at Y5
(fpKi = 6.8 and <5.3, respectively) but the N-alkylated derivates 6
and 7 showed excellent target activity (fpKi = 10.0 and 8.8, respec-
tively). In both pairs of products a notable preference was evident
for the trans diastereoisomer.
Bolstered by this result, the potent trans N-alkyl derivative 6
was progressed into rat pharmacokinetic studies, where it was
found to have moderate blood clearance (Clb = 47 ml/min/kg) and
moderate bioavailability (F = 19%) but more importantly a greatly
improved brain penetration (B/B = 1.2) compared to 3. Before
embarking on a full blown lead optimization campaign around lead
6 it was necessary to develop an alternative synthetic sequence
giving more efficient access to the more potent trans diastereoiso-
mer (Scheme 2). Improved diastereoselectivity was achieved by
exploiting the known propensity of trimethylsulfonium derived
ylides to attack cyclohexanones with axial methylene transfer,12
leading to a complementary stereoselectivity with respect to the
trimethylsulfoxonium derived ylide used in our initial synthesis.
In this specific case, the choice of base employed to generate the
ylide proved fundamental. Several bases promoted the reaction,
all giving epoxide 9 with ꢀ60:40 trans/cis diastereoselectivity,
but high yields were only observed with a Verkade nonionic super-
3
Figure 2. Structure of lead compound 3.
been disclosed in the literature10 suggesting that such a spirocyclic
system is particularly efficacious in presenting the relevant phar-
macophoric features of the molecules in a suitable orientation for
interaction with the receptor.
Considering its attractive in vitro profile, compound 3 was fur-
ther characterized for its pharmacokinetic profile in rat.11 In agree-
ment with its low in vitro intrinsic clearance (Clint = 0.5 ml/min/g
liver) compound 3 showed low blood clearance (Clb = 9 ml/min/
kg) and high bioavailability (F = 49%) but unfortunately it did not
exhibit any appreciable brain penetration (B/B <0.1). Hypothesiz-
ing that the low brain penetration may have been a consequence
of the high polar surface area (PSA) of 3 (PSA = 88), the initial opti-
mization strategy focused on lowering PSA via removal of hetero-
atoms. Initial attempts to replace the oxazolidinone ring with less
polar five membered heterocycles led to a dramatic loss of Y5
activity (data not shown) suggesting that the carbamate enters into
crucial pharmacophoric interactions with the Y5 receptor. Atten-
tion was therefore focused on the urea linker, which proved more
amenable to modification. The chemistry used to access the de-
sired analogs will be described next. The initial synthetic route
used to access the amide and amine analogues 4–7 is outlined in
Scheme 1.
O
O
O
O
O
O
O
O
HN
N
N
O
N
N
O
HO
c
b
a
d
+
+
O
O
O
O
O
O
O
O
O
O
O
OH
O
OH
8
9
10
11
12
13
14
f
e
O
O
O
O
O
O
O
O
O
O
O
O
O
N
N
N
N
N
N
N
N
O
O
O
h
g
+
+
OH
OH
NH
NH
NH
NH
O
O
O
O
N
N
S
N
S
N
S
N
S
+
17
18
15
16
N
N
N
6
7
4
5
Scheme 1. Reagents and conditions: (a) Me3SOI, tBuOK, DMSO, rt; (b) PhNH2, tBuOH, 150 °C, microwave irradiation; (c) triphosgene, Et3N, CH2Cl2, ꢁ50 °C; (d) LiOH, MeOH, rt;
(e) amine 2, HOBt, EDC, MeCN, rt; (f) LiAlH4, THF, ꢁ20 °C; (g) Dess–Martin periodinane, CH2Cl2, rt; (h) amine 2, TiCl(OiPr)3, CH2Cl2, 0 °C to rt, then NaBH(OAc)3, AcOH.

C. P. Leslie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6103–6107
6105
R1
R1
R1
O
O
O
O
O
O
O
O
O
O
H
H
N
O
N
N
N
N
O
c
f
d,e
a
b
+
O
O
O
O
O
O
O
O
O
O
O
NH
R2
20
19
21
22
23
8
9
Scheme 2. Reagents and conditions: (a) Me3SI, 2,8,9-triisobutyl-2,5,8,9-tetraaza-1-phosphabicyclo[3.3.3]undecane, MeCN, rt; (b) ethyl carbamate, tBuOK, DMPU or DMF,
130 °C; (c) R1Br or R1I, CuI, trans-1,2-diaminocyclohexane, Cs2CO4 or K3PO4, toluene or 1,4-dioxane, 80–120 °C; (d) LiAlH4, THF, ꢁ20 °C; (e) Dess–Martin periodinane, CH2Cl2,
rt; (h) amine R2NH2, Ti(OiPr)4, CH2Cl2, rt, then NaBH4.
base whereas inorganic bases such as NaH and tBuOK gave low
Table 1
yields. A convenient one step formation of the oxazolidinone ring
using ethyl carbamate and tBuOK was preferred as it afforded a
more convergent sequence and hence facilitated SAR exploration.
After chromatographic separation from the cis diastereoisomer
20, the desired spirocarbamate intermediate 19 was arylated via
an Ullmann–Goldberg type coupling to give 21. The synthetic route
was completed with similar functional group modification and
reductive amination as before. When opportune the order of the
steps could be modified, postponing the Ullmann–Goldberg cou-
pling to the end of the sequence. Furthermore, in some instances
the amine RNH2 utilized for the reductive amination step was a
halogenated arylamine, which was subjected to an additional Su-
zuki coupling with an appropriate boronic acid to prepare the final
compounds.
The optimization of lead 6 was initially divided into two parallel
explorations (i) substitution on the pyridinethiazole biaryl portion
or replacement with alternative biaryl groups (ii) substitution on
the N-phenyl ring or replacement of the phenyl with isosteric het-
erocycles. In actual fact, the biaryl exploration was performed with
a 2-pyridyl on the carbamate as the analogue 23a showed identical
activity at Y5 and similar pharmacokinetics to 6 but presents a
much preferable lipophilicity (c log P = 2.9 vs 4.4).
NPY Y5 activities,a CYP450 inhibition,b c log Pc and Fubrd of N-pyridyl derivatives 23a–
j
N
H
R2
N
N
O
O
Compds R2
NPY
Y5
CYP450 Inh.
M)b
c log Pc Fubr
d
(
l
(%)
fpKia
N
S
23a
23b
10.1
10.5
>10/3/6/>10/3
2.9
3.4
1.0
—
N
N
N
>10/4/2/>10/
>10
F
F
F
F
F
N
N
23c
23d
23e
23f
9.5
10.6
10.2
8.3
6/4/1/>10/5
3.5
3.7
3.6
4.3
<1.0
0.2
1.6
—
N
>10/2/0.3/>10/
>10
N
N
N
A representative selection of compounds prepared during the
explorations is shown in Tables 1 and 2.
5/2/1/2/>10
—/9/1/2/9
Examples of simple substitution, such as methylation or fluori-
nation, of the pyridinethiazole are not shown as they did not lead
to any appreciable improvement in the CYP450 inhibition profile or
the protein binding—the unbound fraction in the brain (Fubr) was
deemed an important parameter to increase during the optimiza-
tion process as experience gained previously across multiple leads
series for numerous CNS targets has taught that pharmacodynamic
efficacy most closely correlates to the free concentration of test
compound in the brain. A selection of alternative biaryl groups
was identified that maintain high target potency; of these 23b,
23e and 23i offered somewhat improved CYP450 profiles and/or
N
N
F
>10/5/2/>10/
>10
F
23g
23h
9.0
9.8
3.2
3.4
0.5
—
F
N
>10/7/2/2/3
O
N
N
>10/10/5/>10/
>10
23i
23j
9.8
9.9
1.9
2.7
3.5
—
N
higher Fubr
.
N
S
The exploration of the carbamate N-aryl group was undertaken
with the phenylpyrazole biaryl group from 23b. Again, in this case
simple substitution was unproductive (data not shown) and again
a wide variety of heterocycles were found to be tolerated by the
target receptor (Table 2). Modification of this part of the molecule
proved especially effective in improving the CYP450 profile and the
Fubr. In particular, derivative 23p presented an in vitro profile suit-
able for progression into in vivo studies.
>10/3/0.3/1/7
N
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
CYP450 inhibition potential at 1A2/2C19/2C9/2D6/3A4 isoforms, respectively,
determined in Cypex bactosomes.
c
Calculated octanol/water partition coefficient using DaylightÒ 4.81 software.13
Fraction unbound determined in an equilibrium dialysis assay using homoge-
d
Building on the learning’s from the initial exploration, further
iterative rounds of optimization were conducted.
nized rat brain expressed as a percentage.
Indeed, the propensity of the 3-pyridazinyl substituent to
simultaneously improve CYP450 and Fubr was confirmed with
other biaryl groups (Table 3) and a number of further derivatives
suitable for progression into in vivo studies (23t–x) were
identified.
Intrinsic clearance (Clint) values for rat and human, and pharma-
cokinetic parameters from in vivo cassette studies in rat for com-
pounds 23a,p,t,u,x are illustrated in Table 4. Blood clearance was

6106
C. P. Leslie et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6103–6107
Table 2
NPY Y5 activities,a CYP450 inhibition,b c log Pc and Fubr of aminopyrazole derivatives 23k–r
d
R1
N
H
N
N
O
N
O
F
R1
NPY Y5
fpKia
CYP450
Inh. ( M)
c log Pc
Fubr (%)
d
Compds
b
l
23k
23l
3-Pyridinyl
2-Methyl-3-pyridinyl
5-Pyrimidinyl
2-Fluoro-3-pyridinyl
2-Pyrazinyl
3-Pyridazinyl
10.0
9.2
>10/8/7/>10/1
All >10
>10/6/>10/>10/2
>10/8/6/>10/5
>10/2/3/2/0.6
All >10
3.3
3.8
2.4
3.6
2.4
2.1
2.1
2.6
2.5
—
23m
23n
23o
23p
23q
23r
10.4
10.0
10.2
10.0
9.7
4.4
1.9
1.7
3.1
4.4
2.6
4-Pyridazinyl
5-Methyl-1,3,4-thiadiazol-2-yl
>10/>10/>10/>10/6
>10/>10/>10/>10/6
9.7
a,b,c,d
See Table 1.
Table 3
Table 4
Selected DMPK parametersa for compounds 23a,p,t,u,x
NPY Y5 activities,a CYP450 inhibition,b c log Pc and Fubr of N-pyridazinyl derivatives
d
23s–x
Compds
Clint r/hb
(ml/min/g liver)
Clb
F%
Rat
B/B
rat
PD model MEDc
(mg/kg po)
(ml/min/kg)
N
23a
23p
23t
23u
23x
3.1/4.0
30
13
18
41
70
35
52
90
75
50
0.7
0.9
0.25
1.6
0.4
10
3
—
3
N
H
<0.5/<0.5
<0.5/<0.5
<0.5/<0.5
ꢁ/ꢁ
R2
N
N
O
O
—
Compds R2
NPY
Y5
CYP450
Inh. ( M)
c log Pc Fubr
d
a
In vivo data determined by 0.5 mg/kg iv and 1 mg/kg po administration in rat.
b
l
(%)
Brain/Blood ratio (B:B) determined from AUC0ꢁ1 following po dosing.
fpKia
b
Intrinsic clearance values (Clint) expressed as ml/min/g liver were determined in
human (h) and rat (r) liver microsomes.
N
N
N
N
c
Minimal effective dose in NPY Y5 agonist driven pharmacodynamic model in
23s
23t
8.8
9.8
All >10
All >10
0.7
2.3
—
rat.
N
4.6
of at least 1000-fold against all of the ꢀ200 targets tested. Addi-
tionally, both derivatives demonstrated potent in vivo antagonist
activity in a pharmacodynamic model, each reversing the food in-
take induced by icv administration of 0.6 nmol of the NPY Y5 selec-
tive agonist, [cPP1-7,NPY19-23,Ala31,Aib32,Gln34]hPP, to male
rat; 23p and 23u caused similar dose-dependent reductions of food
intake with minimal effective doses of 3 mg/kg po.
In conclusion, a novel series of highly potent, functional NPY Y5
antagonists has been prepared based around a spirocarbamate
scaffold. An extensive SAR exploration allowed the shortcomings
of the original lead to be addressed and led to the identification
of compounds 23p and 23u which have been progressed into pre-
clinical disease models and toxicological studies which will be the
subject of a separate publication.
F
F
N N
N N
>10/10/8/>10/
>10
23u
10.0
2.5
1.6
F
23v
10.2
9.6
All >10
All >10
2.4
2.1
5.1
5.6
N
N
N
N
23w
23x
9.5
All >10
1.8
5.2
F
a,b,c,d
See Table 1.
Acknowledgements
The authors thank Dr. Carla Marchioro and members of the
Analytical Chemistry department, Verona for support in the analyt-
ical characterization 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 characterization
of the compounds described.
moderate except for the aminopyridine derivatives 23u,x, which
showed higher clearance. Oral bioavailability was good to excellent
in all cases however brain penetration varied widely. The low
brain/blood ratio observed with compound 23t was verified to be
a consequence of P-glycoprotein (P-gp) efflux (B/B increased five-
fold when a P-gp inhibitor was co-administered), but this issue
could be mitigated through minor structural modifications giving
compound 23u which has a good brain/blood ratio.
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11. 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 and Ethics (CARE) and to the company Policy on the Care and
Use of Laboratory Animals.
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