Discovery and characterization of AZD9272 and AZD6538 - Two novel mGluR5 negative allosteric modulators selected for clinical development

Article abstract of DOI:10.1016/j.bmcl.2012.08.100

AZD9272 and AZD6538 are two novel mGluR5 negative allosteric modulators selected for further clinical development. An initial high-throughput screening revealed leads with promising profiles, which were further optimized by minor, yet indispensable, structural modifications to bring forth these drug candidates. Advantageously, both compounds may be synthesized in as little as one step. Both are highly potent and selective for the human as well as the rat mGluR5 where they interact at the same binding site than MPEP. They are orally available, allow for long interval administration due to a high metabolic stability and long half-lives in rats and permeate the blood brain barrier to a high extent. AZD9272 has progressed into phase I clinical studies.

Full text of DOI:10.1016/j.bmcl.2012.08.100

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6974–6979
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and characterization of AZD9272 and AZD6538—Two novel
mGluR5 negative allosteric modulators selected for clinical development
Patrick Raboisson a, , Anna Breitholtz-Emanuelsson a, Henrik Dahllöf a, Louise Edwards b,
⇑
William L. Heaton c, Methvin Isaac b, Keith Jarvie b, Annika Kers a, Alexander B. E. Minidis a,
Anna Nordmark a, Susan M. Sheehan c, Abdelmalik Slassi b, Peter Ström a, Ylva Terelius a,
David Wensbo a, Julie M. Wilson b, Tao Xin b, Donald A. McLeod c
a AstraZeneca R&D, Södertälje S-15185, Sweden
b NPS Pharmaceuticals, 6850 Goreway Dr., Mississauga, ON L4V 1V7, Canada
c NPS Pharmaceuticals 420 Chipeta Way, Salt Lake City, UT 84108, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2012
Revised 26 August 2012
Accepted 27 August 2012
Available online 21 September 2012
AZD9272 and AZD6538 are two novel mGluR5 negative allosteric modulators selected for further clinical
development. An initial high-throughput screening revealed leads with promising profiles, which were
further optimized by minor, yet indispensable, structural modifications to bring forth these drug candi-
dates. Advantageously, both compounds may be synthesized in as little as one step. Both are highly
potent and selective for the human as well as the rat mGluR5 where they interact at the same binding
site than MPEP. They are orally available, allow for long interval administration due to a high metabolic
stability and long half-lives in rats and permeate the blood brain barrier to a high extent. AZD9272 has
progressed into phase I clinical studies.
Keywords:
mGluR5
Negative allosteric modulator
GPCR
Ó 2012 Elsevier Ltd. All rights reserved.
In vitro pharmacology
Pain
Clinical candidate
The metabotropic glutamate receptor subtype 5 (mGluR5) is a
family C G protein-coupled receptor, linked via Gq to the activation
of the phospholipase C cascade and formation of protein kinase C.1
It has a wide distribution in the central nervous system and is pre-
dominantly expressed post-synaptically, where it is believed to en-
hance neuronal signaling. Given the wide distribution and
modulatory function of mGluR5 in the CNS, pharmacological
manipulation of mGluR5 signaling will influence multiple systems
in the brain and spinal cord. This is reflected by the interest in
mGluR5 modulators as potential therapeutics in various neurolog-
ical, psychiatric and pain disorders.2–11 In addition, mGluR5 was
have been identified. Focused drug discovery efforts by industry
have generated compounds with appropriate properties, of which
some have reached clinical stages and provided the first evidence
in patients for the therapeutic utility of mGluR5 NAMs in GERD16
,
levodopa-induced dyskinesia in Parkinson’s disease6 and Fragile X
syndrome.17
We describe herein the discovery and pharmacological charac-
terization of AZD9272 (2) and AZD6538 (3), two novel mGluR5
NAMs selected for clinical development for the treatment of neuro-
pathic pain (Fig. 1).
The pyridyl and phenyl substituted oxadiazole motif of 2 and 3
was identified by a high-throughput screening of NPS Pharmaceuti-
suggested to play
a role in gastroesophageal reflux disease
(GERD).12
Over the years following the discovery of the first subtype selec-
tive mGlu5 receptor antagonists SIB-1757 (6-methyl-2-(pheny
lazo)-3-pyridinol) and SIB-1893 (2-methyl-6-((E)-styryl)-pyri-
dine),13 followed by MPEP (2-methyl-6-(phenyethynyl) pyridine
(1) and the structurally related MTEP (3-[(2-methyl-1,3-thiazol-4-
yl]-pyridine),14,15 various new classes of negative allosteric modu-
lators (NAM), also called non-competitive mGluR5 antagonists,
R1
F
N
N
N
N
O
CN
2 (R1 = F , AZD9272)
3 (R1 = CN, AZD6538)
1, MPEP
⇑
Corresponding author. Tel.: +46 73 354 2055.
E-mail address: patrick.raboisson.1@gmail.com (P. Raboisson).
Figure 1. Molecular structure of MPEP, AZD9272 and AZD6538.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.100

P. Raboisson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6974–6979
R1
6975
R1
R1
i
ii
R2
NH2
R2
R3
HOOC
N
N
CN
N
N
N
N
R3
O
4a (R1 = H)
4b (R1 = F)
4c (R1 = CN)
HO
5a-c
7a-s (R1= H)
6a-s
2
3
(R1= F, R2=CN, R3=F)
(R1= CN, R2=CN, R3=F)
Scheme 1. (i) NH2OH⁄HCl, NaOH, EtOH, 60%–80% yield. (ii) Oxalyl chloride, cat. dimethylformamide, dichloromethane, 6a–s. then 5a–c, 24–38% yield.
Table 1
SAR of compounds 7a–s (R1 = H)
Table 2
SAR of left and centre heterocycles
R2
R3
IC50 (lM)
a
F
Compound
Q
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
3-Cl
H
H
5-Cl
3-Cl
2-Cl
4-Cl
3-Cl
4-Cl
4-Cl
3-Me
3-OMe
3-OCF3
3-Br
3-CN
3-F
3-NO2
2-CONH2
5-F
5-CF3
5-CN
5-F
0.142
0.116
0.436
>10
0.274
1.7
W
CN
H
2-Cl
2-Cl
3-Cl
H
H
H
H
H
H
H
a
Compound
Ring Q
Ring W
IC50 (lM)
>10
N
0.443
0.54
8a
0.022
0.023
0.061
1.1
N
N
O
0.7
N
0.118
0.043
0.845
0.236
3.3
0.154
>10
0.176
0.025
8b
8c
9a
O
N
O
H
3-F
3-CF3
3-CN
3-CN
N
N
N
N
N
N
N
O
a
FLIPR data. See text for more details.
N
9b
4.7
O
N
cal’s compound collection. The chemistry used for most of the
structure–activity relationships (SAR) exploration was based on
the methodology developed by Chiou and Shine,18 as outlined in
Scheme 1.
a
FLIPR data. See text for more details.
The central oxadiazole is thus formed by a simple one-pot, two-
step coupling and cyclization reaction with optional isolation of
the intermediate acylated hydroxyamidine (Scheme 1). Depending
on commercial availability, precursors may be made in two to
three steps, as described in the literature.19 By employment of
the unsubstituted pyridyhydroxyl amidine 5a as a common start-
ing material, a series of homologous derivatives 7a–s with varia-
tion of substituents R2 and R3 was initially synthesized (Table 1).
With regards to the phenyl moiety, electron-withdrawing groups,
especially di-substitution in the 3,5-position, appeared as a pre-
ferred substitution pattern. Cyano, as one of the substituents, is
especially well tolerated, whereas bulkier electron withdrawing
groups, such as OCF3, are not equally well tolerated.20 Homologue
7s, carrying fluoro and cyano in meta-position relative the oxadiaz-
ole ring, turned out to be one of the most potent compounds in this
series. This substitution pattern was therefore chosen for further
exploration of the SAR of other parts of the motif.
We next turned our attention to the central heterocycle and the
pyridyl substituent. With regard to in vitro potency, it was found
that the former allowed much greater variation in comparison to
the latter (Table 2). While deviations from the 2-pyridyl substitu-
ent, as illustrated by 9a and 9b, furnished analogs which were sig-
nificantly less potent, replacement of the central oxadiazole with
the corresponding oxazole, regioisomeric variant, or furan as in
8a–c, respectively, resulted in only minor variations of potency.
Previous patent applications21,22 reveal that oxadiazoles, together
with oxazoles, are among the more potent heterocycles in compar-
ison to homologous furans, thiazoles and imidazoles for this type
of compounds. Merck subsequently disclosed tetrazole analogs
with potency in the same range as the corresponding
oxadiazoles.23,24
Although there appears to be a similarity of MPEP (1) and e.g. 7s
due to a common 2-pyridyl-substituent, methyl-substitution in
position 6 (corresponding to position 2 of MPEP) on this part
showed significantly reduced activity in comparison to 6-H pyri-
dines. Further exploration of allowed substitution on the pyridine
moiety by small group probes revealed that the 5-position toler-
ated well smaller groups without loss of potency. In addition, the
metabolic stability of these substituted analogs was greatly en-
hanced by the blocking of this position (Table 3, entries 2 and 3).
Members of the group I mGluR family (mGluR1 and mGluR5)
are 7-TM receptors which couple through G
a
q, leading to the
mobilization of intracellular Ca2+ 25
.
Intracellular Ca2+ levels were
measured using a fluorescence imaging plate reader (FLIPRÒ,
Molecular Devices, Silicon Valley, CA, USA) to determine potency
and efficacy of AZD9272 and AZD6538 at inhibiting recombinant
human or rat mGluR5 expressed in HEK293 cells constitutively
expressing the glutamate/aspartate transporter (mGluR5-GHEK).
AZD9272 and AZD6538 caused a concentration dependent de-
crease in the magnitude of the intracellular Ca2+ response to
1.5 lM of the mGluR group I selective agonist (S)-3,5-dihydroxy-
phenylglycine (DHPG) in both the human and the rat mGluR5
expressing cell lines. The maximal inhibition was 100%. The mean
IC50 ( SD) values at the human mGluR5 were 7.6 1.1 nM (n = 13)
and 13.4 1.3 nM (n = 27) for AZD9272 and AZD6538, respectively.
The mean IC50 values at the rat mGluR5 were 2.6 0.3 nM (n = 3)

6976
P. Raboisson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6974–6979
Table 3
(3 nM) were incubated (in the presence or absence of 10 lM MPEP
Comparison of compounds 7s, 2 and 3
to define non-specific binding) for varying periods of time
(1–30 min). In a parallel series of reactions, [3H]AZD9272 was incu-
Entry
Parameter
Compound
bated in the absence of 10
lM MPEP for a period of 30 min. Subse-
7s (R1 = H)
2 (R1 = F)
3 R1 = CN
quently MPEP (10 M) was added to initiate dissociation and the
l
1
2
3
4
5
6
FLIPRa
(
l
M)
0.025
29
92
78.1
>33
2.07
0.007
10
13.5
4.2
>33
2.25
0.013
6
3
16.1
6.35
1.65
reaction was allowed to proceed for varying amounts of time
(0–30 min). Reactions were terminated by rapid filtration.
[3H]AZD9272 binding increased monotonically, achieving apparent
equilibrium at approximately 20 min. Association kinetics (Kon) aver-
aged 3.95 Â 107 MÀ1 minÀ1, corresponding to a t1/2 value of 3.45 min
(n = 2). Dissociation was monotonic and consistent with reversible
binding of [3H]AZD9272 to mGluR5. Dissociation kinetics (Koff) aver-
aged 8.9 Â 10À2 minÀ1, corresponding to a t1/2 value of 7.85 min,
respectively (n = 2). Kinetic estimates of the Kd (Koff/Kon) yielded affin-
ity estimates of 2.25 nM. In a parallel set of reactions binding was as-
Rat CLINTb (mic)
Hu CLINTb (mic)
Solubilityc
(
l
M)
hERGd
cLogP
(
l
M)
a
b
c
FLIPR data. See text for more details.
Intrinsic Clearance CLINT by human/rat microsomes (l
l/min mgÀ1).
Based on a ‘Dried-DMSO rapid throughput solubility assay’.37
IonWorks based hERG assay.38
d
sessed over this 30-min period in the absence of MPEP (10 lM).
AZD9272
AZD6538
Binding was stable and linear over this time period, indicating stable
retention over the range of 30–60 min total incubation time.
In order to allow for a direct comparison of the maximal binding
densities (Bmax) of [3H]MPEP and [3H]AZD9272, parallel assess-
ments were completed. The density of saturable binding sites for
[3H]AZD9272 and [3H]MPEP on human mGluR5-GHEK membranes
was comparable (Table 4), providing support for the contention
that both radioligands interact with a common binding site.
Equilibrium binding studies were conducted to determine the Kd
of [3H]AZD9272. Additionally, incubations were done in the absence,
or presence of moderate concentrations of MPEP (10 and 20 nM).
These concentrations were chosen to approximate one and twofolds
the Kd for the binding of MPEP to the human mGluR5. Summary re-
sults for these studies are provided in Table 5. [3H]AZD9272 bound
with high affinity (Kd = 3.8 0.7 nM; SD, n = 3) to the human
mGluR5. Specific binding was greater than 95% of total binding at
concentrations approximating the Kd. MPEP caused a concentration
dependent reduction in the apparent affinity of AZD9272 binding to
mGluR5 without significant change in the Bmax. These data further
support the idea that [3H]AZD9272 and MPEP interact with the same
binding site on the human mGluR5 receptor.
0 nM
10
30
100
300
100
50
0
-8
-7
-6
-5
-4
-8
-7
-6
-5
-4
[DHPG] M
[DHPG] M
Figure 2. AZD9272 and AZD6538 inhibition of DHPG-stimulated intracellular Ca2+
release (FLIPR) in HEK cells expressing human mGluR5. Data are presented as the
mean S.E.M. of 3 separate experiments.
and 3.2 0.1 nM (n = 3) for AZD9272 and AZD6538, respectively. In
contrast, 10
the response to 10
l
M of either AZD9272 or AZD6538 did not diminish
l
M ATP in the background GHEK cells. Increas-
ing concentrations of AZD9272, AZD6538 and MPEP caused a de-
crease in the potency and the maximal response of DHPG. The
decreased maximal response reached a plateau, such that increas-
ing the concentration of DHPG was not able to overcome the inhi-
bition (Fig. 2). This result is consistent with AZD9272 and AZD6538
acting through a non-competitive interaction with DHPG.
AZD9272 and AZD6538 in vitro activity was further assessed
using a phosphatidyl inositol hydrolysis assay. Accumulation of
inositol phosphates (IP) in the cytoplasm provides a functional
index of mGluR5 activation where antagonists will reverse ago-
nist-induced IP accumulation. AZD9272 and AZD6538 completely
Displacement by AZD6538 of either [3H]MPEP or [3H]AZD9272
binding to human mGluR5-GHEK membranes was monotonic
and consistent with a competitive interaction at a unitary binding
site (see Fig. 3). Dissociation constants for AZD6538 displacement
were comparable (Ki values of 28.0 2.9 nM ([3H]MPEP, n = 4)
and 17.9 9.5 nM ([3H]AZD9272, n = 3)).
In vitro autoradiographic methods27 on fresh frozen tissue slices
were used to describe the distribution and basic pharmacological
properties of AZD9272 in the rat CNS. [3H]AZD9272 binding
(Fig. 4A) was region-dependent and in agreement with the immu-
nohistological localization of mGluR528 and with previous ligand
binding studies using other mGluR5 NAMs in rodents.29,30 In gen-
eral, binding was higher in forebrain than hindbrain and very low
in cerebellum. The highest binding levels were observed in the stri-
atum, lateral septum and the CA1 region of the hippocampus. Mod-
erate binding was seen in the dorsal cortex, dentate gyrus,
amygdala, the dorsal cortex of the inferior colliculus, ventral layers
of the cortex and the superficial layers of the spinal cord. Lower
binding densities were observed in the lateral globus pallidus, peri-
aqueductal gray, nucleus of the solitary tract, inferior olivary nuclei
and hypothalamus. Binding in the spinal cord was dense in the dor-
sal superficial layers and moderate to low in the remaining gray
matter regions, corresponding to the known distribution of
mGluR5 mRNA.31 [3H]AZD9272 binding in rat striatum was of high
affinity and finite capacity (Kd = 9.4 1.9 nM; Bmax = 1553 474
fmol/mg T.E.; SD, n = 5) and could be inhibited by MPEP, MTEP
and AZD6538 (IC50 AZD6538, 14.5 4.7 nM; MTEP, 24 6.1;
n = 2; Fig. 5), confirming that these compounds bind to a common
site also at the native mGluR5 in rat brain sections.
reversed the glutamate-stimulated (EC80, 80 lM) phosphatidyl
inositol hydrolysis in human mGluR5-GHEK cells in a concentra-
tion-dependent manner, with IC50 of 26 3 nM (n = 21) and
51 3 nM (n = 97), respectively.
AZD9272 and AZD6538 showed a high degree of selectivity for
mGluR5. They did not elicit any agonist, antagonist or positive allo-
steric modulator responses up to a concentration of 30 lM on any
of the other seven mGluR subtypes as measured by changes in
intracellular Ca2+ using FLIPR-based assays and did not interact sig-
nificantly (650% inhibition or stimulation of binding) with 134
receptors, ion channels, nuclear hormone receptors, transporters
and enzymes tested at a concentration of 10
binding assays (MDS Pharma Services).
lM using radioligand
To gain a better understanding of AZD9272 binding to mGluR5,
the binding characteristics of [3H]AZD927226 were assessed in hu-
man mGluR5-GHEK cell membranes and compared to [3H]MPEP.
The time required to achieve equilibrium and the reversibility of
the binding of [3H]AZD9272 were assessed by kinetic experiments.
For association studies, single concentrations of [3H]AZD9272

P. Raboisson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6974–6979
6977
Table 4
[3H]MPEP and [3H]AZD9272 Bmax values (Parallel Assessment)
Assay
1
Radioligand
Kd (nM)
Bmax (pmol/mg protein)
Relative Bmax (% [3H]MPEP)
[3H]MPEP
8.6
2.8
10.6
3.9
4.4
4.4
5.3
5.8
—
99.8%
—
[3H]AZD9272
[3H]MPEP
2
[3H]AZD9272
109%
Table 5
Summary data for the equilibrium binding of [3H]AZD9272 to mGluR5
Assay
Condition
Kd (nM)
Mean Kd (nM)
Std Dev
Bmax (pmol/mg protein)
12.8
7.2
5.3
13.4
7.5
5.1
12.1
7.3
5.0
Relative %
1
2
3
1
2
3
1
2
3
0 MPEP
0 MPEP
0 MPEP
10 nM MPEP
10 nM MPEP
10 nM MPEP
20 nM MPEP
20 nM MPEP
20 nM MPEP
4.5
3.6
3.2
6.8
5.1
4.6
8.6
6.6
6.3
3.8
0.7
1.2
1.2
—
—
—
104.7
103.9
95.8
94.0
101.4
94.1
5.5
7.2
Solubility was in general in the low micro-molar range for the
majority of compounds tested, yet this was sufficiently high for
further in vitro and in vivo testing. Albeit only with a small differ-
ence, the lower solubility of 2 versus 3 was noticeable enough to
have initially biased selection for in vivo testing towards the latter.
However, as described below, the lower solubility of 2 had eventu-
ally little impact on its oral bioavailability. Activity at the hERG
channel, a major cardiac liability target, was in general low within
the series but with a significant difference between 2 and 3 in favor
of the former (Table 3). The lipophilicity of the majority of com-
pounds was around or well below 3 and no obvious correlations
between e.g. potency and many typical drug discovery parameters,
such as clogP, Mrel, number of donors/acceptors, and others were
observed.
100
50
0
100
50
0
-11 -10 -9 -8 -7 -6 -5 -4
AZD6538 [logM]
-11 -10 -9 -8 -7 -6 -5 -4
AZD6538 [logM]
Figure 3. AZD6538 displacement of [3H]MPEP and [3H]AZD9272 binding (repre-
sentative examples).
A
NSB
B
10 nmol/kg
30 nmol/kg
Figure 4. Autoradiograms showing the distribution of [3H]AZD9272 binding in sagittal and coronal sections of the rat CNS. A, in vitro. Sections were incubated with 3 nM
[3H]AZD9272. Nonspecific binding (NSB) was determined in sections co-incubated with 10
and 30 nmol/kg).27
l
M MPEP.; B, ex vivo. 5 min following intravenous infusion of [3H]AZD9272 (10

6978
P. Raboisson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6974–6979
100
80
60
40
20
0
To further examine AZD9272 uptake and distribution in vivo in
the CNS, rats were infused intravenously with 10 and 30 nmol/kg
(196 and 588
Ci/kg) [3H]AZD9272. Five minutes after, brains were
l
removed, sectioned in the sagittal plane and analyzed by conven-
tional autoradiographic methods. [3H]AZD9272 entered the brain
rapidly and demonstrated a distribution pattern (Fig. 4B) similar
to the one observed on the in vitro autoradiograms (Fig. 4A). Re-
cently, a method to label AZD9272 with 11C as a radioligand for
PET has been developed and AZD9272 ability to enter the brain
of cynomolgus monkeys and humans to a high extent has been
established.32–34 AZD9272 and AZD6538 efficacy in animal models
of pain and anxiety will be described elsewhere.35
AZD9272 and AZD6538 are two highly potent, selective and
brain penetrant mGluR5 NAMs with rather long terminal half-lives.
They seem to interact at the same binding site on mGluR5 as MPEP
and MTEP. Both were selected as clinical candidates, and AZD9272
has been progressed to human clinical trials.
MTEP
AZD6538
-10
-9
-8
log [Ligand] (M)
-7
-6
Figure 5. Displacement of [3H]AZD9272 binding in the rat dorsal striatum by MTEP
and AZD6538 using in vitro autoradiographic techniques. Sequential striatal
sections corresponding to plates # 13–1636 from 2 rats were incubated with 3
nM [3H]AZD9272 together with increasing concentrations of MTEP and AZD6538
(mean SD, n = 2). Nonspecific binding was defined in sections co-incubated with
Acknowledgments
The authors are grateful to Pam Jacobson and the core tissue
culture group at NPS Salt Lake City for their contribution to devel-
oping the cell lines, to Julie Grabell and Josee Normand (NPS Toron-
to) for their technical assistance in developing and implementing
binding studies and IP assay, to Brad VanWagenen (NPS SLC) for
his contribution to chemistry and to the late Allan Johnson (Astra-
Zeneca) who designed and performed the autoradiographic
studies.
10 lM MPEP.
Table 6
Pharmacokinetic parameters of AZD9272 and AZD6538 in male rats (n = 3) following
single administration at 3
lmol/kg
Intravenous administration
We also want to thank Edwin Johnson (AstraZeneca) and Tom
Stormann (NPS) for their support and advice during the course of
the project and all AstraZeneca and NPS scientists involved for
their assistance.
Test comp.
Vss (L/kg)
CL (L/h/kg)
t1/2kz (h)
AZD9272a
AZD6538b
1.6
2.0
0.31
0.24
2.5
5.6
Oral administration
Test comp.
Cmax
(l
mol/L)
tmax (h)
AUC(
lmol h/L)
Fpo (%)
Supplementary data
AZD9272a
AZD6538b
0.46
0.5
4
3-8
5.4
56
46
3.6c
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.100.
a
vehicle: 40% w/w dimethylacetamide (DMA)/40% w/w polyethylene glycol
(PEG) 400/20% w/w water.
b
vehicle: 1.7 mg/ml Polyvidon K30; 0.34 mg/ml sodiumlaurylsulphate; 25.0 mg/
ml glycerin and water.
c
AUC(0–8).
References and notes
The determination of AZD9272 and AZD6538 concentrations in
plasma was performed by liquid–liquid extraction followed by
reversed-phase liquid chromatography and atmospheric pressure
chemical ionization tandem mass spectrometry and electrospray
ionization tandem mass spectrometry, respectively. Stable isotopi-
cally labeled AZD9272/AZD6538 or close analogs were used as
internal standards. The lower limit of quantification of both
AZD9272 and AZD6538 in plasma was 0.01 lmol/L. The in vivo
rat pharmacokinetic parameters for AZD9272 and AZD6538 are
summarized in Table 6.
1. Crawford, J. H.; Wainwright, A.; Heavens, R.; Pollock, J.; Martin, D. J.; Scott, R.
H.; Seabrook, G. R. Neuropharmacology 2000, 39, 621.
2. Varney, M. A.; Gereau, R. W., 4th Curr. Drug Targets CNS Neurol. Disord. 2002, 1,
283.
3. Brodkin, J.; Busse, C.; Sukoff, S. J.; Varney, M. A. Pharmacol. Biochem. Behav.
2002, 73, 359.
4. Chiamulera, C.; Epping-Jordan, M. P.; Zocchi, A.; Marcon, C.; Cottiny, C.;
Tacconi, S.; Corsi, M.; Orzi, F.; Conquet, F. Nat. Neurosci. 2001, 4, 873.
5. Bear, M. F.; Huber, K. M.; Warren, S. T. Trends Neurosci. 2004, 27, 370.
6. Berg, D.; Godau, J.; Trenkwalder, C.; Eggert, K.; Csoti, I.; Storch, A.; Huber, H.;
Morelli-Canelo, M.; Stamelou, M.; Ries, V.; Wolz, M.; Schneider, C.; Di Paolo, T.;
Gasparini, F.; Hariry, S.; Vandemeulebroecke, M.; Abi-Saab, W.; Cooke, K.;
Johns, D.; Gomez-Mancilla, B. Mov. Disord. 2011, 26, 1243.
Following oral administration of AZD9272 and AZD6538 to
male rats the maximum plasma concentrations were reached 3
to 8 h after dosing. The clearance of both compounds was low fol-
7. Johnson, K. A.; Conn, P. J.; Niswender, C. M. CNS Neurol. Disord. Drug Targets
2009, 8, 475.
8. Walker, K.; Bowes, M.; Panesar, M.; Davis, A.; Gentry, C.; Kesingland, A.;
Gasparini, F.; Spooren, W.; Stoehr, N.; Pagano, A.; Flor, P. J.; Vranesic, I.;
Lingenhoehl, K.; Johnson, E. C.; Varney, M.; Urban, L.; Kuhn, R.
Neuropharmacology 2001, 40, 1.
9. Walker, K.; Reeve, A.; Bowes, M.; Winter, J.; Wotherspoon, G.; Davis, A.;
Schmid, P.; Gasparini, F.; Kuhn, R.; Urban, L. Neuropharmacology 2001, 40, 10.
10. Tatarczynska, E.; Klodzinska, A.; Chojnacka-Wojcik, E.; Palucha, A.; Gasparini,
F.; Kuhn, R.; Pilc, A. Br. J. Pharmacol. 2001, 132, 1423.
11. Spooren, W. P.; Vassout, A.; Neijt, H. C.; Kuhn, R.; Gasparini, F.; Roux, S.; Porsolt,
R. D.; Gentsch, C. J. Pharmacol. Exp. Ther. 2000, 295, 1267.
12. Jensen, J.; Lehmann, A.; Uvebrant, A.; Carlsson, A.; Jerndal, G.; Nilsson, K.;
Frisby, C.; Blackshaw, L. A.; Mattsson, J. P. Eur. J. Pharmacol. 2005, 519, 154.
13. Varney, M. A.; Cosford, N. D.; Jachec, C.; Rao, S. P.; Sacaan, A.; Lin, F. F.; Bleicher,
L.; Santori, E. M.; Flor, P. J.; Allgeier, H.; Gasparini, F.; Kuhn, R.; Hess, S. D.;
Velicelebi, G.; Johnson, E. C. J. Pharmacol. Exp. Ther. 1999, 290, 170.
lowing a single intravenous dose at 3 lmol/kg and both were elim-
inated from plasma with terminal half-lives between 2 and 6 h.
The terminal half-lives following oral dosing were similar to the
half-lives following intravenous dosing. The volume of distribution
at steady state was intermediate for both AZD9272 and AZD6538.
In vitro protein binding in rats for AZD9272 and AZD6538 was 80%
and 77%, respectively. In vivo, total brain exposure in rats followed
systemic exposure (as measured in plasma) and brain-to-plasma
ratios were approximately 1 for both compounds after oral doses
of 3 lmol/kg.

P. Raboisson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6974–6979
6979
14. Cosford, N. D.; Tehrani, L.; Roppe, J.; Schweiger, E.; Smith, N. D.; Anderson, J.;
25. Conn, P. J.; Pin, J. P. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 205.
26. [3H]AZD9272 synthesis is reported in Supplementary Material.
27. Methods is described in Supplementary Material.
Bristow, L.; Brodkin, J.; Jiang, X.; McDonald, I.; Rao, S.; Washburn, M.; Varney,
M. A. J. Med. Chem. 2003, 46, 204.
15. Gasparini, F.; Lingenhohl, K.; Stoehr, N.; Flor, P. J.; Heinrich, M.; Vranesic, I.;
Biollaz, M.; Allgeier, H.; Heckendorn, R.; Urwyler, S.; Varney, M. A.; Johnson, E.
C.; Hess, S. D.; Rao, S. P.; Sacaan, A. I.; Santori, E. M.; Velicelebi, G.; Kuhn, R.
Neuropharmacology 1999, 38, 1493.
16. Zerbib, F.; Bruley des Varannes, S.; Roman, S.; Tutuian, R.; Galmiche, J. P.; Mion,
F.; Tack, J.; Malfertheiner, P.; Keywood, C. Aliment. Pharmacol. Ther. 2011, 33, 911.
17. Tabolacci, E.; Pirozzi, F.; Gomez-Mancilla, B.; Gasparini, F.; Neri, G. BMC Med.
Genet. 2012, 13, 13.
28. Romano, C.; Sesma, M. A.; McDonald, C. T.; O’Malley, K.; Van den Pol, A. N.;
Olney, J. W. J. Comp. Neurol. 1995, 355, 455.
29. Anderson, J. J.; Rao, S. P.; Rowe, B.; Giracello, D. R.; Holtz, G.; Chapman, D. F.;
Tehrani, L.; Bradbury, M. J.; Cosford, N. D.; Varney, M. A. J. Pharmacol. Exp. Ther.
2002, 1044, 303.
30. Anderson, J. J.; Bradbury, M. J.; Giracello, D. R.; Chapman, D. F.; Holtz, G.; Roppe,
J.; King, C.; Cosford, N. D.; Varney, M. A. Eur. J. Pharmacol. 2003, 473, 35.
31. Berthele, A.; Boxall, S. J.; Urban, A.; Anneser, J. M.; Zieglgansberger, W.; Urban,
L.; Tolle, T. R. Brain Res. Dev. Brain Res. 1999, 112, 39.
18. Chiou, S.; Shine, H. J. J. Heterocycl. Chem. 1989, 26, 125.
19. Wang, T.; Lamb, M. L.; Scott, D. A.; Wang, H.; Block, M. H.; Lyne, P. D.; Lee, J. W.;
Davies, A. M.; Zhang, H.; Zhu, Y.; Gu, F.; Han, Y.; Wang, B.; Mohr, P. J.; Kaus, R. J.;
Josey, J. A.; Hoffmann, E.; Thress, K.; MacIntyre, T.; Wang, H.; Omer, C. A.; Yu, D.
J. Med. Chem. 2008, 51, 4672.
20. Rodriguez, A. L.; Grier, M. D.; Jones, C. K.; Herman, E. J.; Kane, A. S.; Smith, R. L.;
Williams, R.; Zhou, Y.; Marlo, J. E.; Days, E. L.; Blatt, T. N.; Jadhav, S.; Menon, U.
N.; Vinson, P. N.; Rook, J. M.; Stauffer, S. R.; Niswender, C. M.; Lindsley, C. W.;
Weaver, C. D.; Conn, P. J. Mol. Pharmacol. 2010, 78, 1105.
32. Andersson, J. D. PhD, Karolinska Institutet, 2010.
33. Andersson, J. D.; Seneca, N.; Truong, P.; Wensbo, D.; Raboisson, P.; Farde, L.;
Halldin, C. Nucl. Med. Biol. submitted for publication
34. Kagedal, M.; Cselenyi, Z.; Nyberg, S.; Jonsson, S.; Raboisson, P.; Stenkrona, P.;
Hooker, A. C.; Karlsson, M. O. Neuroimage 2012, 61, 849.
35. Dahllöf et al, manuscript in preparation.
36. Paxinos, G.; Watson, C. The Rat Brain in Stereotaxic Coordinates; Academic Press:
New York, 1998.
21. VanWagenen, B. C.; Stormann, T. M.; Moe, S. T.; Sheehan, S. M.; Mcleod, D. A.;
Smith, D. L.; Isaac, M. B.; Slassi, A. 2001, WO/2001/012627.
37. Alelyunas, Y. W.; Liu, R.; Pelosi-Kilby, L.; Shen, C. Eur. J. Pharm. Sci. 2009, 37,
172.
22. Slassi, A.; Van Wagenen, B.; Stormann, T. M.; Moe, S. T.; Sheehan, S. M.; Mcleod,
D. A.; Smith, D. L.; Isaac, M. B. 2002, WO/2002/068417.
38. Bridgland-Taylor, M. H.; Hargreaves, A. C.; Easter, A.; Orme, A.; Henthorn, D. C.;
Ding, M.; Davis, A. M.; Small, B. G.; Heapy, C. G.; Abi-Gerges, N.; Persson, F.;
Jacobson, I.; Sullivan, M.; Albertson, N.; Hammond, T. G.; Sullivan, E.; Valentin,
J.-P.; Pollard, C. E. J. Pharmacol Toxicol. Methods 2006, 54, 189.
23. Poon, S. F.; Eastman, B. W.; Chapman, D. F.; Chung, J.; Cramer, M.; Holtz, G.;
Cosford, N. D. P.; Smith, N. D. Bioorg. Med. Chem. Lett. 2004, 14, 5477.
24. Huang, D.; Poon, S. F.; Chapman, D. F.; Chung, J.; Cramer, M.; Reger, T. S.; Roppe,
J. R.; Tehrani, L.; Cosford, N. D. P.; Smith, N. D. Bioorg. Med. Chem. Lett. 2004, 14,
5473.