Discovery of potent, selective, and metabolically stable 4-(pyridin-3-yl)cinnolines as novel phosphodiesterase 10A (PDE10A) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.01.086

We report the discovery of 6,7-dimethoxy-4-(pyridin-3-yl)cinnolines as novel inhibitors of phosphodiesterase 10A (PDE10A). Systematic examination and analyses of structure-activity-relationships resulted in single digit nM potency against PDE10A. X-ray co-crystal structure revealed the mode of binding in the enzyme's catalytic domain and the source of selectivity against other PDEs. High in vivo clearance in rats was addressed with the help of metabolite identification (ID) studies. These findings combined resulted in compound 39, a promising potent inhibitor of PDE10A with good in vivo metabolic stability in rats and efficacy in a rodent behavioral model.

Full text of DOI:10.1016/j.bmcl.2012.01.086

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2262–2265
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent, selective, and metabolically stable
4-(pyridin-3-yl)cinnolines as novel phosphodiesterase 10A (PDE10A) inhibitors
Essa Hu a, , Roxanne K. Kunz a, Shannon Rumfelt a, Ning Chen a, Roland Bürli d, Chun Li f,
⇑
Kristin L. Andrews c, Jiandong Zhang c, Samer Chmait c, Jeffrey Kogan g, Michelle Lindstrom b,
Stephen A. Hitchcock a,e, James Treanor b
a Department of Small Molecule Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA
b Department of Neuroscience, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA
c Department of Molecular Structures, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA
d Currently at BioFocus, Chesterford Research Park, Saffron Walden CB10 1XL, United Kingdom
e Currently at Envoy Therapeutics, 555 Heritage Drive, Jupiter, FL 33458, USA
f Currently at Genomics Institute of the Novartis Research Foundation,10675 John J. Hopkins Drive, San Diego, CA 92121, USA
g Currently at Astellas Research Institute of America, Skokie, IL 60077, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the discovery of 6,7-dimethoxy-4-(pyridin-3-yl)cinnolines as novel inhibitors of phosphodies-
terase 10A (PDE10A). Systematic examination and analyses of structure–activity-relationships resulted in
single digit nM potency against PDE10A. X-ray co-crystal structure revealed the mode of binding in the
enzyme’s catalytic domain and the source of selectivity against other PDEs. High in vivo clearance in rats
was addressed with the help of metabolite identification (ID) studies. These findings combined resulted
in compound 39, a promising potent inhibitor of PDE10A with good in vivo metabolic stability in rats and
efficacy in a rodent behavioral model.
Received 22 November 2011
Revised 19 January 2012
Accepted 23 January 2012
Available online 8 February 2012
Keywords:
PDE10A
Schizophrenia
Cinnolines
Ó 2012 Elsevier Ltd. All rights reserved.
Phosphodiesterase
Metabolism
Phosphodiesterase 10A belongs to a family of phosphodiester-
ase (PDE) enzymes whose role is to hydrolyze the 30–50 phosphodi-
ester bonds of cyclic adenosine monophosphate (cAMP) and cyclic
guanosine monophosphate (cGMP) to produce adenosine mono-
phosphate (AMP) and guanosine monophosphate (GMP), respec-
tively. Consequently, phosphodiesterases effectively regulate cell
signalling by modulating the levels of critical secondary messen-
gers cAMP/cGMP.1 The eleven PDE isozyme families identified to
date vary in their affinities for cAMP and cGMP and differ in their
relative tissue distributions.2 In particular, the predominance of
PDE10A in the medium spiny neurons of the striatum captured
our interest.3 Inhibition of PDE10A enzyme activity in the striatum
could present a new approach to treat neurological disorders
whose pathology has been linked to striatal brain regions. Several
groups have reported findings to suggest inhibition of PDE10A
could have therapeutic benefit in treatment of schizophrenia and
potentially other neurodegenerative disorders.4 PDE10A knock
out (PDE10Aꢀ/ꢀ) mice demonstrated reduced hyperactivity re-
sponse in the locomotor activity model where rodents were chal-
lenged with PCP to trigger schizophrenic like behaviors.5 Of the
PDE10A inhibitors reported to date,6 TP-10 (Fig. 1) was compre-
hensively described to be effective in several rodent behavioral
models of schizophrenia such as the locomotor activity model,
the Conditioned Avoidance Response model (CAR), and the Pre-
pulse Inhibition model (PPI).7 These findings further supported
the hypothesis that the symptoms of schizophrenia could be ame-
liorated by a PDE10A inhibitor. The ultimate therapeutic utility of
the novel biological target PDE10A will be demonstrated from
clinical data. Notably, PF-02545920 (MP-10) is currently being
evaluated in clinical trials for the treatment of schizophrenia.8
Our search for a novel and potent inhibitor of PDE10A enzyme
activity began with compound 1 that exhibited PDE10A potency
of 590 nM (Table 1). Removal of the chlorine atom resulted in sig-
nificant loss of activity (2), indicating the importance of substitu-
tion at the 2-pyridyl position for PDE10A activity. Replacement of
the chlorine atom with a methyl group (3) improved potency.
Whereas bulky trifluoromethyl group (4) had a modest impact
on activity, a small nitrile substituent (5) increased potency.
Unsubstituted amine (6) was also tolerated. Alkylation of the
amine with an isopropyl group produced a single digit nM potency
inhibitor (7). Nitrogen atom appeared to be the optimal linker as
replacement with carbon (8) or oxygen (9) decreased potency.
⇑
Corresponding author. Tel.: +1 805 313 5300.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.086

E. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2262–2265
2263
Figure 1. TP-10 and MP-10.
Table 1
SAR of 2-pyridine analogs
yield. Bromination with phosphorous oxybromide followed by Su-
zuki coupling with the appropriately substituted (6-fluoropyridin-
e-yl)boronic acid produced the 4-(6-fluoropyridin-3-yl)-6,7-
dimethoxycinnoline scaffold (12). The synthesis of final com-
pounds (13) was accomplished by heating the cinnoline pyridyl
fluoride scaffold in DMSO with the appropriate amine.
R
N
N
Keeping the amine linker fixed, we proceeded to examine the
effects of various substituents (Table 2). Compared to compound
7, the hydrogen bond donor on the amine did not appear to be
essential to the activity (14). Increasing the chain length (15) and
inserting an oxygen atom (16) into the alkyl chain did not result
in significant impact on potency. Cyclopropyl group (17) decreased
its PDE10A activity while cyclobutyl (18) and cyclopentyl (19)
groups produced comparable or slight improvement in potency.
Extending the cyclopropyl substituent by one carbon (20) resulted
in a tenfold improvement in potency versus compound 17. Elec-
tron-withdrawing groups like allyl (21) and trifluoromethyl (22)
did not further increase activity. Cyclic amine substituents were
also examined. Variation of ring size (23, 24), location of oxygen
atom (25), and presence of a hydrogen bond donating group (26)
all afforded less potent compounds.
O
O
N
Compound#
R
PDE10 IC50 (nM)
1
2
3
4
5
6
7
8
9
Cl
H
CH3
CF3
CN
NH2
NHiPr
CH2iPr
OiPr
590
16300
181
320
92.8
124
7.6
40.6
24.4
With single digit nM potency achieved, we began to profile se-
lect compounds in the in vivo rat pharmacokinetic (PK) studies.
Compounds 16 and 24, for example, were initially selected due
The improvement in potency with small modifications of sub-
stituents on the pyridine ring was surprisingly dramatic. These
early findings convinced us of need for a focused SAR effort and
the need for an efficient synthesis to the 6,7-dimethoxy-4-(pyri-
din-3-yl)cinnoline core that would enable rapid analoging.9 The
synthetic route began with commercially available 1-(2-amino-
4,5-dimethoxyphenyl)ethanone (10). Scheme 1 Cyclization with
sodium nitrite afforded 6,7-dimethoxycinnolin-4-ol (11) in 71%
to low in vitro rat liver microsomal clearance of 51 and 76 lL/
min/mg, respectively. However, higher than expected in vivo clear-
ance (1 mg/kg dose, IV), was observed (5.53 L/h/kg for 16, 6.35 L/h/
kg for 24). Metabolite ID studies in rat hepatocytes showed exten-
sive metabolism of the aniline substituents. We reasoned that a
Scheme 1. Reagents and conditions. (a) NaNO2, HCl, water, 75 °C, 71% yield; (b) POBr3, MeCN, 70 °C, 77% yield; (c) Cs2CO3, Pd(PPh3)4, DME, water; 40–95% yield; (d) amine,
K2CO3, DMSO, 90 °C.

2264
E. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2262–2265
Table 2
Table 3
Investigation of aniline substitution
Examination of disubstitution on pyridine ring
Compound#
NR1R2
PDE10 IC50 (nM)
–N(iPr)H
–N(iPr)Me
–N(iBu)H
7.6
5.8
7.5
Compound#
R3
R4
PDE10 IC50 (nM)
7
14
15
7
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Cl
7.6
125
2.0
4.0
1.1
1.5
2.4
9.7
17.3
7.0
27
28
29
30
31
32
33
34
35
36
37
38
NH
CH2CH3
16
10.1
cPr
CHF2
F
CN
Vinyl
C(O)NH2
C(CH3)2OH
CH2OH
O
17
18
19
20
21
22
–N(cPr)H
36.9
10.9
4.4
3.0
16.4
12.2
–N(cBu)H
–N(cPentyl)H
–N(CH2cPr)H
–N(Allyl)H
–N(CH2CF3)H
16.3
2.6
1.6
N
23
21.4
O
Table 4
SAR of piperidine ring
N
24
18.6
O
N
25
26
13
O
N
OH
13.2
Compound# R4
R5
R6
PDE10 IC50
(nM)
CL (L/hr/
kg)
N
26
39
40
H
OH
OH
OH
2-
Pyridyl
2-
Pyridyl
13.2
0.15
0.18
0.38
Me
Cl
2.6
5.8
polar substituent on the aniline should reduce the metabolic liabil-
ity in that region. Gratifyingly, compound 26 emerged with good
in vitro and in vivo data. The cyclic amine alcohol exhibited rat li-
2-
Pyridyl
cPr
Me
H
H
H
41
42
43
44
45
46
Me
Me
Me
OH
OH
C(CH3)2OH
1.8
3.2
4.9
1.2
6.2
1.3
0.86
2.15
1.29
0.80
1.84
0.94
ver microsomal clearance of 28
ance of 0.146 L/h/kg.
lL/min/mg and rat in vivo clear-
CHF2 C(CH3)2OH
Since cyclic amines generally afforded less potent compounds
than acyclic amines (Table 2), but provided viable PK profiles, we
next turned to examine substitution on other positions of the pyr-
idine ring in hopes of regaining potency in the single digit nM
range (Table 3). Introduction of a methyl group ortho to the pyri-
dine nitrogen (27) resulted in significant loss of potency. Adding
the same functional group ortho to the aniline amine (28), how-
ever, led to an improvement in potency. Replacement with a chlo-
rine atom was not as favorable (29). Bulkier substituents such as
ethyl (30), cyclopropyl (31), and difluoroethyl (32) demonstrated
similar potency improvements. Electron-withdrawing groups like
fluorine (33), nitrile (34), and vinyl (35) were less tolerated. Unlike
primary amide (36) which afforded decreased PDE10A activity, ter-
tiary (37) and primary alcohols (38) all generated similar improve-
ments in potency as compared to methyl analog 28.
Cl
C(CH3)2OH
C(CH3)2OH
cPr
H
group at the R4 position such as methyl (39) and chloro (40) in-
creased PDE10A potency to single digit nM compared to compound
26. Compounds 39 and 40 also maintained the low rat in vivo
clearance of compound 26. Replacement of the bulky 2-pyridyl
groups on the piperidine with smaller cyclopropyl (41) and methyl
(42) groups, and extending the alcohol with a gem-dimethyl meth-
ylene group (43–46) all maintained the single digit nM potency. In
vivo rat clearance of compounds 41, 43–44, and 46 remained
acceptable.
For proof of concept, compound 39 was tested in the Condi-
tioned Avoidance Response (CAR) behavioral model10 to assess
the efficacy of our compounds in a rodent model of schizophrenia.
Sprague-Dawley rats were tested 1 h post dosing at 3, 5.6, and
10 mg/kg given by oral gavage (Fig. 2). Compound 39 was able to
suppress the avoidance response in rats with a MED of 5.6 mg/kg.
Finally, we combined the findings above to determine if they
would result in a potent and metabolically stable PDE10A inhibitor
suitable for testing in behavioral models (Table 4). To improve po-
tency, we were gratified to find that addition of a small functional

E. Hu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2262–2265
2265
500ꢁ fold improvement in potency, increasing the activity of our
initial lead from IC50 of 590 nM (1) to 1 nM (44, 46). The issue of
high rat in vivo clearance was addressed with installation of alco-
hol substituted cyclic amines (26, 39–41, 43–44, 46). Our selective
inhibitor of PDE10A (39) was demonstrated to be efficacious in a
rodent behavioral model of schizophrenia.
References and notes
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Figure 2. Conditioned Avoidance Response study with compound 39.
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L.; Malamas, M. S.; Brandon, N. J.; Kronbach, T. J. Med. Chem. 2010, 53, 4399; (f)
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Figure 3. X-ray co-crystal structure of compound 43 in the catalytic domain of
PDE10A enzyme.
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5188; (b) Clinicaltrials.gov identifier NCT01175135.
9. (a) For more details on synthetic preparations and PDE10A assay conditions,
CAR model, see: Hu, E.; Kunz, R.; Nixey, T.; Hitchock, S. Preparation of quinoline
derivatives and their aza analogs as phosphodiesterase 10 inhibitors.
WO2009025839.; (b) Hu, E.; Kunz, R.; Chen, N.; Nixey, T.; Hitchcock, S.
Preparation of quinoline derivatives and their aza analogs as
phosphodiesterase 10 inhibitors. WO 2009025823.; (c) Arrington, M. P.; Liu,
R.; Hopper, A. T.; Conticello, R. D.; Nguyen, T. M.; Gauss, C. M.; Burli, R.;
Hitchcock, S. A.; Hu, E.; Kunz, R. Cinnoline derivatives as phosphodiesterase 10
inhibitors, their preparation, pharmaceutical compositions, and use in therapy.
WO 2007098169.
Counterscreen assays showed compounds 39–46 exhibited
350ꢁ–5000ꢁ fold selectivity against PDE3 and several were greater
than 2000ꢁ fold selective against other PDE isomers (1–9, 11) as
well. A co-crystal structure of representative compound 43 in the
human PDE10A catalytic domain elucidated the key bonding inter-
actions (Fig. 3).11 The molecule appeared to be anchored by a bifur-
cating hydrogen bonding interaction between both methoxy groups
on the cinnoline core and the conserved Gln716. The substituents on
the aniline extends to a shelf-like area in the enzyme consisting of
four amino acids: Phe686, Ile701, Met703, and Met704. Comparison
against other phosphodiesterase isozymes revealed that amino acid
residues in this region of the catalytic binding domain differed
amongst the PDEs. Thus, this interaction of the piperidine alcohol
with the PDE10A enzyme in that region was likely the source of its
high selectivity.
10. Wadenberg, M.-L. G.; Hicks, P. B. Neuosci. Biobehav. Rev. 1999, 23, 851.
11. (a) Wang, H.; Liu, Y.; Hou, J.; Zheng, M.; Robinson, H.; Ke, H. Proc. Natl. Acad. Sci.
2007, 104, 5782; (b) Coordinates of co-crystal structure of compound 43 in
PDE10 have been deposited in the Protein Data Bank. PDB ID code is 4DDL.
In conclusion, we have described the identification of a novel,
potent, efficacious and selective inhibitor of PDE10A. Systematic
investigation of structure–activity-relationships resulted in over