Design and Identification of a Novel, Functionally Subtype Selective GABAA Positive Allosteric Modulator (PF-06372865)

Article abstract of DOI:10.1021/acs.jmedchem.9b00322

The design, optimization, and evaluation of a series of novel imidazopyridazine-based subtype-selective positive allosteric modulators (PAMs) for the GABA_A ligand-gated ion channel are described. From a set of initial hits multiple subseries were designed and evaluated based on binding affinity and functional activity. As designing in the desired level of functional selectivity proved difficult, a probability-based assessment was performed to focus the project's efforts on a single subseries that had the greatest odds of delivering the target profile. These efforts ultimately led to the identification of two precandidates from this subseries, which were advanced to preclinical safety studies and subsequently to the identification of the clinical candidate PF-06372865.

Full text of DOI:10.1021/acs.jmedchem.9b00322

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Drug Annotation
Design and identification of a novel, functionally subtype
A
selective GABA positive allosteric modulator (PF-06372865).
Robert M. Owen, David C Blakemore, Lishuang Cao, Neil Flanagan, Rebecca Fish, Karl R Gibson,
Rachel Gurrell, Chan Woo Huh, Juha Kammonen, Elisabeth Mortimer-Cassen, Sarah Nickolls,
Kiyoyuki Omoto, Dafydd R Owen, Andrew Pike, David C. Pryde, David Reynolds, Rosemarie
Roeloffs, Colin R. Rose, Clara Stead, Mifune Takeuchi, Joseph S Warmus, and Christine Watson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00322 • Publication Date (Web): 09 Apr 2019
Downloaded from http://pubs.acs.org on April 10, 2019
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Design and identification of a novel, functionally
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subtype selective GABA positive allosteric
A
modulator (PF-06372865).
Robert M. Owen,b* David Blakemore, Lishuang Cao, Neil Flanagan,
b
c
e
Rebecca Fish, c Karl R. Gibson, f Rachel Gurrell, c Chan Woo Huh,g
Juha Kammonen, c Elisabeth Mortimer-Cassen, Sarah Nickolls,
d
c
b
g
a
b
Kiyoyuki Omoto, Dafydd Owen , Andy Pike, David C. Pryde,
David S. Reynolds, c Rosemarie Roeloffs, h Colin Rose, g Clara
Stead, c Mifune Takeuchi, f Joseph S. Warmus,g Christine Watson f
a
b
Pharmacokinetics, Dynamics and Metabolism, Worldwide Medicinal
c
d
Chemistry, Pfizer Neusentis, Global Safety Group,
ePharmaceutical Sciences, Pfizer Ltd., The Portway, Granta Park,
f
Cambridge, CB21 6GS; Worldwide Medicinal Chemistry Sandwich
Laboratories, Ramsgate Road, Sandwich, Kent CT13 9NJ, United
g
Kingdom; Worldwide Medicinal Chemistry, Pfizer Global Research &
Development, 558 Eastern Point Road, Groton, Connecticut 06340,
h
United States; Pfizer Icagen, Research Triangle Park, North
Carolina 27703, United States
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KEYWORDS: GABA , selective positive allosteric modulator, PAM,
A
ion channel, CNS, clinical
ABSTRACT: The design, optimization and evaluation of a series
of novel imidazopyridazine-based subtype-selective positive
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allosteric modulators (PAMs) for the GABA ligand-gated ion
A
channel are described. From a set of initial hits multiple
subseries were designed and evaluated based on binding affinity
and functional activity. As designing in the desired level of
functional selectivity proved difficult, a probability-based
assessment was performed to focus the project’s efforts on a
single subseries which had the greatest odds of delivering the
target profile. These efforts ultimately led to the
identification of two pre-candidates from this subseries, which
were advanced to pre-clinical safety studies and subsequently to
the identification of the clinical candidate PF-06372865.
INTRODUCTION
Neuronal signaling via the γ-amino butyric acid (GABA)-type A
(
GABA ) receptor plays a critical role in a wide range of
A
processes within the central nervous system (CNS). The GABAA
receptor belongs to the Cys-loop family of ligand gated ion
channels and modulates the flow of chloride anions across the
synaptic junction upon binding of its endogenous ligand GABA.1
The receptor is comprised of five subunits, of which sixteen
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different subtypes have been identified (α1–6, β1–3, γ1–3, δ, ε,
θ and π). However, relatively few combinations have been shown
to exist in the CNS with the overwhelming majority of receptors
consisting of two α, two β and one γ subunit, although other
subunit compositions, such as 2α2βδ have also been identified.2-4
GABA is the major inhibitory neurotransmitter in the CNS and
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drugs targeting the GABA receptor show diverse pharmacology,
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including anxiolytic, hypnotic and anti-convulsant effects, as
well as the receptor being involved in the processes of
5-7
cognition and the perception of pain. There are several
different classes of marketed GABAergic drugs, and modulation of
the function of the GABA receptor via small molecules has
A
remained an active area of research since the initial discovery
and clinical success of the 1,4-benzodiazapine class of
therapeutics in the 1960s.8
Classical benzodiazepines act as non-selective positive
allosteric modulators (PAMs) of the action of GABA on α1-, α2-,
α3- and α5-subunit-containing receptors where present with a γ2
subunit. While they are highly effective drugs, with both
anxiolytic and anti-convulsant properties, they also exhibit
undesirable properties including sedation, cognitive deficits,
potential for abuse and dependence, and significant potentiation
of these effects by alcohol.
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However, it has been demonstrated, using a combination of
studies in genetically modified mice and using compounds showing
subtype-selective modulation, that different aspects of
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benzodiazepine pharmacology can be attributed to different 
subunits. For example, signaling via α1-containing receptors
contributes significantly to the sedative effects of 1,4-
9
benzodiazepines. In contrast, the
α2- and
α3-containing
receptors have stronger links to anxiety related activities,10-12
while some of the effects on cognitive function are tied to α5-
13
containing receptors. In addition, α1- and α2-containing
receptors have been linked to the anticonvulsant activity of
benzodiazepines.10,
14
Finally, modulation of α2- and α3-
containing GABA receptors in the spinal cord, results in pain
A
10
relief in preclinical studies. Therefore, subunit selective-
allosteric modulators have the potential to retain the
therapeutic benefits of classical benzodiazepines while
minimizing the associated side effects.8-9, 15
With these ideas in mind, various research groups have explored
the development of α1-sparing, α2/3 PAMs for the treatment of
anxiety and other disorders.8-9, 16 During the course of these
studies it proved difficult to identify compounds that exhibited
the desired selectivity profile by selectively binding to the α2
or 3-subunit-containing receptors over those containing α1
subunits. This is presumably due to conservation of key-binding
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residues in the benzodiazepine binding site which is located at
17-18
the α/γ interface of the GABA receptor complex . Instead, the
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researchers found that selectivity could be more readily
obtained functionally, i.e. a given compound binds to different
receptor subunit combination with comparable affinity but
promotes very different levels of functional activity.
Ultimately this approach proved successful and has led to the
identification of a broad range of both clinical and preclinical
compounds (vide infra). However, to date, no new approved drugs
have resulted from this work.
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Herein we describe our efforts towards the identification of a
novel α1-sparing GABA PAM. From initial file screening leads, we
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successfully optimized the imidazopyridazine series to
ultimately yield two compounds with preclinical profiles that
were suitable for advancement to pre-clinical safety studies.
From these safety studies, compound PF-06372865 was selected for
advancement into clinical studies to explore the benefits of
8-9, 16
subtype selective GABA PAMs in humans.
A
TARGET COMPOUND PROFILE
Beyond the standard desired preclinical profile for a safe and
developable CNS-penetrant agent,19-21 our initial goal was to
establish the required levels of functional selectivity for α2
over α1 as measured in our assays in order to ensure that any
potential candidate would exhibit the desired pharmacology
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profile in the clinic. As mentioned previously, multiple
functionally selective GABA PAMs have been advanced into
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clinical trials. Of these, clinical data has been disclosed for
three key compounds, MRK-409, TPA-023 and TPA-023B (Figure 1)8,
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22-26.
MRK-409, which exhibits selectivity for α2 over α1 in
functional assays but still shows moderate α1 activity, was
markedly sedative in man even though this was not predicted from
27
preclinical species. In contrast, TPA-023 and TPA-023B, which
both show selectivity as well as decreased efficacy at α1
relative to MRK-409, were both well tolerated and demonstrated
reduced sedative effects in human clinical trials.28-30 TPA-023
also showed preliminary evidence of efficacy in both anxiety and
30
schizophrenia in man. Based on this analysis, we set a maximal
α1 enhancement of the GABA response in our in vitro systems of
around 20% and a minimum α2 enhancement of 50% as our target PAM
profile for any potential candidates. We also wanted to avoid
significant negative allosteric modulation of α1-containing
receptors, as this had been demonstrated to cause seizures in
preclinical studies. Subsequent to the initiation of our
internal studies, two additional functionally selective GABAA
31
32
PAMs (AZD7325 and NS11821 ) have been reported in the
literature.
α1 PAM ±
SEM (%)
α2 PAM ±
SEM (%)
Structure
Name
α2 Ki ±
a
b
SEM
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(
nM)
48.3 ±
Cl
Diazepa
m
216 ± 3 293 ± 4
(n=422) (n=410)
2
7.4
N
O
(n=8)
N
1
1
1
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1
1
2
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0
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0
F
N N
N
N
0.81 ±
MRK-409 0.4
F
5
(
6 ± 5
n=26)
80 ± 3
(n=31)
O
N
(n=10)
N
N
F
N N
N
N
0.54 ±
2
(
1 ± 1
n=56)
41 ± 2
(n=59)
TPA-023 0.2
(n=31)
O
N
N
N
N
N
2.14 ±
TPA-
0.7
19 ± 1
81 ± 1
N
OH
N
N
023B
(n=273) (n=315)
(n=37)
F
F
Table 1. GABA PAMs for which clinical data has been reported. α2
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Ki and functional activity values were measured as described
here.33-35 Percentage values refer to the level of enhancement
measured after activation of the receptor with GABA as described
here.33-35 a,b GABA functional activity were measured at 100-times
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the compound’s Ki.
IDENTIFICATION AND INITIAL OPTIMIZATION OF IMIDAZOPYRIDAZINE
SERIES
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Based on historical literature precedent and our own internal
experience, although SAR for functional selectivity exists
within a given chemotype, generally it is quite “spiky” with
minor changes in structure leading to very different functional
profiles. As a result, early in the lifetime of the program we
adopted a strategy of profiling multiple series in parallel to
maximize our chances of identifying a potent, functionally
selective molecule that could be developed as a once daily
agent.
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As a starting point for our design efforts, the project team
applied a range of ligand-based techniques based on structures
from the literature that functioned as GABA PAMs to find
starting points from within our internal compound collection.
These methods included similarity-based searches as well as
molecular overlays to determine key pharmacophoric elements
which, allowed us to ultimately arrive at the following three
series (Figure 1). As a general design strategy, we explored a
range of different heterocyclic cores decorated with
substituents as indicated including directly linked aromatic or
heterocyclic groups, alkyl groups and heteroatom (e.g. O, N)
linked substituents. Each of these series was progressed in
parallel and evaluated on their ability to deliver affinity,
functional activity and an appropriate in vitro ADME profile.
For the purpose of this discussion we will focus on the
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optimization of one of these series, the imidazopyridazines,
series 2, but will revisit the overall merits of each series in
terms of providing the required affinity, ADME and functional
profiles.
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X X
R1
R1
N
Y
N
R1
X
X
X
N
X
X
X
O
NH2
R3
X
X = C,N
R2
R2
X = C,N
Series 2
Y = OR , NR R
2
3
2
Series 1
Series 3
Figure 1. Markush structures for the series 1-3.
Table 2 outlines some of the initial hits within this series,
which were identified via a mixture of ligand-based methods.
Compound 1 and 2 suggested that reasonable affinity for the GABAA
receptor could be obtained via an imidazopyridazine core
substituted by a simple aryl group and that alkyl ether groups
were tolerated on this aryl ring system. A para-fluoro group
could provide a modest improvement in affinity over the
equivalent des-fluoro scaffold (Compound 2 vs 3). Optimization
of these initial leads rapidly demonstrated that the overall
affinity of this scaffold could be improved via introduction of
either a second direct-linked or ether-linked aromatic group
(Compound 4-6). Although neither compound 4 or 5 exhibited a
promising functional profile, compound 6 exhibited the desired
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level of functional selectivity as well as an in vitro profile
consistent with good CNS-penetration, which suggested that
further work within this scaffold was warranted.
HLMe
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α1
α2 PAM
Clint
α2 Ki ± Lip PAM ±
LogD
MDR
er
Structure
ID
± SEM
a
d
f
SEM (nM)
E
SEM
(L/m
in/mg
)
c
(%)
b
(%)
F
274.6 ±
1
57.4
4.3 ND
ND
ND
ND
2.3 105
2.5 ND
2.5 ND
2.3 47
ND
ND
ND
ND
N
(n=2)
N
N
N
O
367.3 ±
114.4 4.0 ND
(n=2)
2
3
4
N
N
N
N
F
O
119.5 ±
20.3
(n=2) 4.5 ND
N
N
N
N
N
F
N
-55 ±
10 ± 7.4
n=2)
-4 ± 3
(n=6)
5.8 2
(
N
N
(n=4)
N
N
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F
N
2
8.9 ±
6.6
n=5)
-48 ±
4.9 10
(n=2)
10 ± 1
n=4)
5
6
2.6 78
1.0
2.7
(
N
N
(
N
O
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
F
33.3 ±
N
-
22 ± 4 54 ± 5
n=4) (n=7)
15.1 4.8
(n=3)
2.6 65
(
N
N
N
N
Table 2. Initial hits within the imidazopyridazine series. α2 Ki
and α1/α2 PAM values were measured as described here.33-35
aLogD
are measured values or if calculated (internal logD model) are
b,c
in italics. GABA functional activity were measured at 100-
A
d
times the compound’s K . LipE values are calculated (pK – logD)
i
i
from measured logD values when possible or from calculated logD.
eHLM Clint (L/min/mg), in vitro human microsomal stability
f
measurement. MDR er: Ratio from the MS-based quantification of
apical/basal and basal/apical transfer rates of test compound at
2
μM across confluent monolayers of MDR1-transfected MDCK cells.
Based on the results obtained for Compound 6, we sought to
further optimize this aryl-ether linked subseries towards a
potential candidate. In order to synthesize a broad range of
compounds within this subseries, we initially examined whether
the more synthetically-accessible pyridyl linked ethers would
provide the desired activity profile. Compound 7, while weaker
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Journal of Medicinal Chemistry
Page 12 of 81
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
than our other starting points, was still quite efficient and
changing from a methoxy to phenyl ether provided a significant
boost in affinity with equal efficiency (LipE) but without the
desired functional profile. Unfortunately, attempts to further
optimize the profile of any pyridyl-based ethers met with
limited success and when compared with the fluoro-phenyl group
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Compound 9 vs 10) the pyridyl moiety did not bring any
advantages and was not explored any further. Additional linkers
attached to the ether moiety were also explored (Compound 11 and
12) and provided good affinity and efficiency but did not yield
the required functional profile. Examining the profile of
compound 10, we then explored other polar aromatic groups with
the hope of improving efficiency, the functional selectivity
profile as well as overall metabolic stability. Changing from
the phenyl sulfone (10) to a pyrimidyl nitrile (Compound 13)
provided a small improvement in efficiency and stability and
more importantly a marked improvement in the functional
selectivity profile. Further optimization yielded Compound 14
which again had the desired functional profile but lacked the
desired level of metabolic stability for further progression.
Based on metabolite identification work for related compounds,
we hypothesized that compound 14 was being N-dealkylated at the
imidazo ring system which may contribute to its high turnover in
human microsomes. Conversion of the ethyl group to either a
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Page 13 of 81
Journal of Medicinal Chemistry
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4
5
6
7
8
9
cyclopropyl (15) or isopropyl (16) led to compounds with an
improvement in overall stability while maintaining the
functional selectivity. However, in both cases, the compounds
lost affinity and efficiency and could not be progressed
further.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
HLMe
α1
α2 PAM
Clint
α2 Ki ± Lip PAM ±
LogD
MDR
er
Structure
ID
± SEM
a
d
f
SEM (nM)
E
SEM
(%)
(L/m
in/mg
)
c
(%)
b
O
N
N
961.6 ±
7
23.4 4.6 ND
ND
1.5 ND
ND
N
N
(n=2)
N
O
N
-
24 ±
4.6 1
n=5)
6
1.2 ±
13 ± 3
(n=5)
8
9
2.6 51
1.2
2.0
N
N
11 (n=4)
(
N
N
N
O
1
141.9 ±
160.8 4.2 ND
n=3)
ND
1.6 <8
S
O
N
N
O
(
N
N
F
O
1
4 ±
3
77.3 ±
10 19.1 4.8
3
7 ± 4
2.3 127
2.8
S
O
(n=5)
N
N
O
(n=2)
(n=9)
N
N
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Journal of Medicinal Chemistry
Page 14 of 81
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
N
F
O
2
2.8 ±
2.6
n=2)
7
± 4 10 ± 2
1
1
2
5.2
2.5 41
4.8
3.4
(n=8) (n=19)
N
N
(
N
N
F
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
33.3 ±
-6 ±
5.8 4
6
± 5
O
1
3.9
1.6 <8
N
N
(n=6)
(n=2)
(n=6)
N
N
F
O
N
8
1.4 ±
13 97.3 5.1 2
n=4) (n=6)
13 ±
87 ± 5
N
N
N
N
2.0 49
0.8
1.4
(n=9)
N
N
(
N
N
N
N
F
O
-
3 ±
30.6 ±
N
3
96 ± 5
14
5.7
5.2
2.3 65
(n=15 (n=19)
N
N
(n=2)
)
N
F
O
N
219.1 ±
10 ± 78 ±
14
(n=4) (n=6)
1
5 28.1 4.3 2
n=2)
2.3 17
1.1
1.1
N
N
(
N
F
O
N
358.3 ±
225
(n=2)
89 ±
10
3
± 4
N
16
3.7
2.7 38
N
N
(n=3)
(n=4)
N
N
Table 3. Ether-based imidazopyridazine subseries. α2 Ki and
α1/α2 PAM values were measured as described here.33-35
aLogD are
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Page 15 of 81
Journal of Medicinal Chemistry
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2
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4
5
6
7
8
9
measured values or if calculated (internal logD model) are in
b,c
italics. GABA functional activity were measured at 100-times
A
d
the compound’s K . LipE values are calculated (pK – logD) from
i
i
e
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
measured logD values when possible or from calculated logD. HLM
Clint (L/min/mg), in vitro human microsomal stability
f
measurement. MDR er: Efflux ratio from the MS-based
quantification of apical/basal and basal/apical transfer rates
of test compound at 2 μM across confuent monolayers from MDR1-
transfected MDCK cells.
In parallel to the optimization efforts described above, we
also sought to optimize the profile of compound 5, looking for
modifications that could improve the overall functional profile
of the compounds. Changing the pyridine isomer led to compound
17 which immediately showed an improvement in efficiency and met
our target profile for functional selectivity. As before in the
ethers, this compound was too unstable to progress and attempts
to mitigate that problem through altering the imidazole
substituent were unsuccessful (Compound
18 and
19).
Interestingly, the three different substituents did not have a
profound effect on the affinity of the compounds unlike the
ether-linked compounds. However, the isopropyl substituent did
lead to an increase in α1 functional activity relative to the α2
activity. As a result of these two observations, the N-ethyl
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Journal of Medicinal Chemistry
Page 16 of 81
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
imidazole became the preferred substituent in subsequent rounds
of optimization. Moving away from the pyridyl ring demonstrated
that phenyl rings containing polar groups could provide good
affinity along with excellent functional selectivity (Compound
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
20). However, while a subtle change in the arrangement of those
polar groups maintained affinity (Compound 21), they also led to
a large decrease in functional activity. Other polar groups
were well tolerated in terms of the affinity of these directly
linked aryl groups (Compounds 22 – 27) and also led to compounds
that were both stable and CNS penetrant. However, as before,
small changes in structure could lead to moderate to large
changes in function. For example, presentation of either a
sulfone or amide moiety in the para position of the aryl ring
(Compound 22 and Compound 26) was the most optimal in terms of
binding affinity. In the case of compound 22, while this
compound showed the desired functional selectivity, truncation
of the ethyl sulfone to the methyl sulfone decreased both α1 and
α2 activity below our desired threshold. In addition, the methyl
amide (26) appeared to be a good bioisostere for the ethyl
sulfone in terms of binding affinity, but this compound lost its
functional selectivity. One final interesting SAR observation is
the effect of ortho substituents on binding affinity with the
ortho-fluoro group (27) providing an improvement in affinity and
efficiency.
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Journal of Medicinal Chemistry
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7
8
9
HLMe
α1
α2 PAM
Clint
α2 Ki ± Lip PAM ±
LogD
MDR
er
Structure
ID
± SEM
a
d
f
SEM (nM)
E
SEM
(L/m
in/mg
)
c
(%)
b
(%)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
F
13.4 ±
5.8
-5 ±
5.0 8
(n=3)
7
1 ± 3
n=11)
17
2.9 157
1.1
1.2
(
(n=2)
N
N
N
N
F
N
20.3 ±
4.8
-6 ±
4.8 4
(n=8)
6
(
8 ± 5
n=10)
18
2.9 42
N
N
(n=25)
N
N
F
N
5
7 ± 103 ±
4.4 10 12
n=2) (n=3)
34.0 ±
19
20
21
3.1 102
3.1 66
3.2 32
ND
ND
16 (n=2)
N
(
N
N
N
O
F
23.6 ±
-4 ± 181 ±
4.6 6
N
0.8
9
(n=2)
(n=8) (n=7)
N
N
N
N
F
N
O
8.8 ±
-40 ±
4.9 2
(n=6)
31 ± 2
(n=6)
2.2
1.4
(n=2)
N
N
N
N
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Journal of Medicinal Chemistry
Page 18 of 81
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
S
F
-
13 ±
1
21.7 ±
2.4
85 ± 3
22
4.8
2.9 <13
1.9
(n=32 (n=36)
)
(n=31)
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
N
F
373.1 ±
3 21.3 4.0 ND
n=2)
O S
O
N
2
ND
2.4
9
ND
(
N
F
N
N
O
S
O
17.7 ±
5.9
-15 ±
5.2 7
33 ± 5
24
25
2.5 10
2.2
2.4
(n=8)
(n=3)
(n=3)
N
N
N
N
F
O
NH
23.2 ±
-10 ±
4.8 7
30 ± 4
9.5
(n=2)
2.8 40
(n=9)
N
N
(n=3)
N
N
HN
O
F
6.7 ±
3.7
-41 ±
5.3 4
(n=4)
21 ± 2
26
2.9 16
3.4
(n=28)
(n=3)
N
N
N
N
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Page 19 of 81
Journal of Medicinal Chemistry
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4
5
6
7
8
9
F
F
H
N
6
.8 ±
3.7
(n=4)
-11 ±
5.1 4
(n=9)
O
17 ± 5
(n=12)
2
7
3.0 34
5.2
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Direct linked aryl-based imidazopyridazine subseries.
α2 Ki and α1/α2 PAM values were measured as described here.33-35
aLogD are measured values or if calculated (internal logD model)
b,c
are in italics. GABA functional activity were measured at 100-
A
d
times the compound’s K . LipE values are calculated (pK – logD)
i
i
from measured logD values when possible or from calculated logD.
eHLM Clint (L/min/mg), in vitro human microsomal stability
f
measurement. MDR er: Ratio from the MS-based quantification of
apical/basal and basal/apical transfer rates of test compound at
2
μM across contiguous monolayers from MDR1-transfected MDCK
cells.
PROBABILITY ASSESSMENT OF ACTIVE SERIES
Although good progress had been made with the various rounds of
optimization described above for the imidazopyridazine series,
at this stage in the project we did not feel we had identified a
compound that was of sufficient quality to nominate as a
development candidate. In addition to the work undertaken on the
imidazopyridazine series, the project team also worked to
optimize two orthogonal series to provide alternative chemotypes
with similar selectivity and affinity profiles. Given the spiky
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Journal of Medicinal Chemistry
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
SAR observed across all three series for functional activity, in
order to focus our efforts on the series with the best chance or
delivering a candidate, we analyzed the probability that each
series could deliver in terms of affinity, HLM and CNS-
penetration (as measured by MDR efflux ratios) and functional
selectivity (Figure 2, Imidazopyridazine series = Series 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Page 21 of 81
Journal of Medicinal Chemistry
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5
6
7
8
9
Figure 2. Probability analysis of the project’s three main
series (Series 2 is the imidazopyradine series) on the basis of:
Affinity: α2 K < 10 nM Green, > 40 nM Red. ADME: HLM < 20 AND
i
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MDR er < 2.5 Green, HLM > 40 AND MDR er > 4.0 Red. Functional:
α1 PCT < 20 and α2 PCT > 50 Green, α1 PCT > 50 AND α2 < 40 Red.
The total number of compounds contained in each pie chart is
shown at the chart’s center.
Based on this analysis, despite reasonable ADME properties
Series 3 was clearly compromised by the low likelihood of
achieving adequate affinity. Based on affinity and ADME the
choice between Series 1 and 2 was less clear, however, Series 2
clearly had the best chance of obtaining the desired level of
functional selectivity. This analysis was extended to the two
main subseries within Series 2 (Figure 3), the direct linked and
ether linked scaffolds discussed above and clearly showed that
the direct linked subseries was far more likely to yield the
desired functional profile. For both analyses, none of the
probabilities quoted for the three parameters indicate that any
series was any more likely than the others for finding a single
molecule that combined all three of these features. However, we
believed that problems like affinity and ADME would be much more
soluble and less “spiky” in terms of SAR than maintaining
functional selectivity within a series and therefore shifted all
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Journal of Medicinal Chemistry
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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
our design efforts to identifying a suitable candidate from the
direct-linked imidazopyridazine series (Series 2).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Probability analysis of two main subseries within
Series 2 on the basis of: Affinity: α2 K < 10 nM Green, >40 nM
i
Red. ADME: HLM < 20 AND MDR er < 2.5 Green, HLM > 40 AND MDR er
>
4.0 Red. Functional: α1 PCT < 20 and α2 PCT > 50 Green, α1 PCT
> 50 AND α2 < 40 Red
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Page 23 of 81
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
FINAL OPTIMIZATION AND IDENTIFICATION AND CHARATERIZATION OF
PRECLINICAL LEADS
With our design strategy focused on the imidazopyridazine
series, the project team next explored a few different core
modifications in order to determine if there were any advantages
to alternative heterocyclic cores (Table 5). Modification of
compound 22 to a pyrazolotriazine (28) provided a boost in
affinity and efficiency while maintaining the desired functional
selectivity. This core was particularly interesting as it
provided access to different substituents in the lower right
quadrant that were not accessible for the imidazopyridazine
scaffold, for example compound 29. Unfortunately, this series
was generally less stable than the imidazopyridazine series and
a majority of the compounds were highly coloured, typically
bright yellow or orange, leading to concerns of photostability
and was therefore not explored further. Compounds 30 and 31
demonstrated that both nitrogens of the pyridazine ring system
were required for good binding affinity. Finally, the
triazolopyridazone core was tolerated but as it was less
efficient than the imidazopyridazine, was also not explored
further. Although ultimately unproductive, this work allowed the
project team to confidently focus on the imidazopyridazine core
in our final rounds of optimization.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Journal of Medicinal Chemistry
Page 24 of 81
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HLMe
α1
α2 Ki ±
SEM
α2 PAM
Clint
Lip PAM ±
LogD
MDR
er
Structure
ID
± SEM
a
d
f
E
SEM
(L/m
in/mg
)
c
(nM)
(%)
b
(%)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
S
F
2.8 ±
9
(
± 5 71 ± 4
n=2) (n=2)
2
8 1.8 5.1
3.5 33
1.1
1.8
N
(n=3)
N
N
N
O
O
S
F
-
13 ±
3 77 ± 4
(n=11 (n=13)
)
29.7 ±
5 (n=4)
2
9
0
4.7
2.8 <8
N
N
N
N
O
O
S
F
O
655.9
3
2.9 ND
ND
ND
3.3 ND
ND
(n=1)
N
N
N
O O
S
F
944.3 ±
3
1 162.2 2.1 ND
n=2)
3.9 30
2.1
(
N
N
N
ACS Paragon Plus Environment
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Page 25 of 81
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
O
O
S
F
81.3 ±
2 17.1 3.8 1
-1 ± 107 ±
3
18
3.3 25
1.4
N
N
(n=3)
(n=5) (n=7)
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
Table 5. The effect of modification to the imidazopyridazine
a
core. LogD are measured values or if calculated (internal logD
b,c
model) are in italics. GABA functional activity were measured
A
d
at 100-times the compound’s IC . LipE values are calculated
50
(
pIC – logD) from measured logD values when possible or from
50
e
calculated logD. HLM Clint (L/min/mg), in vitro human
f
microsomal stability measurement. MDR er: Ratio from the MS-
based quantification of apical/basal and basal/apical transfer
rates of test compound at 2 μM across confluent monolayers from
MDR1-transfected MDCK cells.
Two compounds stood out from the optimization work outlined in
Table 4, the sulfone containing compound 22 and the amide
containing compound 26. Compound 22 exemplified the desired
functional and ADME profile but ideally could have higher
affinity. In contrast, compound 26 had the desired level of
affinity, but was not ideal in terms of functional selectivity
and CNS penetration based on its MDR ratio. Focusing first on
optimizing affinity for compound 22, combining SAR from the
above studies, namely the affinity improvement observed by
ACS Paragon Plus Environment
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Journal of Medicinal Chemistry
Page 26 of 81
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
introducing an ortho-fluoro group (22 to 23), compound 33 was
synthesized. Unfortunately, this modification did not yield an
improvement in affinity. However, increasing the size of the
fluoro group to a methoxy group (34) did provide a large
improvement in both affinity and efficiency. This compound also
maintained an excellent level of functional selectivity and a
desirable ADME profile. Small changes to the structure of
compound 34 suggested that the ethyl sulfone and methoxy groups
were optimal. Attempts to increase the size of the methoxy group
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(35), change the electronics of the ring system (37) or decrease
(36) or increase (38) the size of the sulfone were all
detrimental to the functional profile. The best alternative to
compound 34 in terms of functional selectivity was the
cyclopropyl containing compound 39, but this modification led to
a drop in affinity. The fluorophenyl group central ring system
also appeared to be optimal with replacement of the fluorine
with either a chlorine or a nitrile leading to a reduction in
affinity and some erosion of the functional selectivity. This
work suggested that within the sulfone series, compound 34 was
the optimal compound to profile further.
HLMe
α1
α2 PAM
Clint
α2 Ki ± Lip PAM ±
LogD
MDR
er
Structure
ID
± SEM
a
d
f
SEM (nM)
E
SEM
(%)
(L/m
in/mg
)
c
(%)
b
ACS Paragon Plus Environment
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Page 27 of 81
Journal of Medicinal Chemistry
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9
O
O
S
F
18.2 ±
5.4
(n=4)
-31 ±
5.2 2
(n=9)
F
O
O
43 ± 2
n=15)
3
3
2.6 <8
1.6
1.8
(
N
N
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
F
O
O
S
21 ±
2.9 ±
3.7
(n=8)
134 ±
3
(n=34)
2
n=21
)
34
5.9
4.4
2.7 <9
(
N
N
N
F
O
S
O
6
7 ±
8
(n=11
)
18.4 ±
129 ±
17
(n=13)
35
36
37
2.4
3.3 12
1.7
3.3
1.8
(n=4)
N
N
N
O
S
O
F
6
± 2
7.5 ± 1.8
45 ± 4
(n=15)
O
5.6 (n=17
)
2.5 <8
(n=6)
N
N
N
N
O
S
O
F
7.3 ±
1.6
-7 ±
5.0 3
(n=8)
1
(
4 ± 2
n=12)
Cl
3.2
9
(n=4)
N
N
N
N
ACS Paragon Plus Environment
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Journal of Medicinal Chemistry
Page 28 of 81
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
S
O
F
4
0 ±
6
11.6 ±
1.1
111 ±
5
(n=19)
O
3
3
4
8
9
0
5.0
3.0 84
1.5
2.6
(n=15
(n=6)
N
N
)
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
O
O
S
F
12.7 ±
27 ±
5.1 6
(n=4)
115 ± 5
2.2
(n=4)
2.7 <12
O
(n=4)
N
N
N
N
O
S
O
F
O
5
8 ± 171 ±
6.0 6 10
n=3) (n=5)
6.6
n=1)
O
2.2 67
3.1 <8
<8
7.0
2.9
4.0
(
(
N
N
N
N
O
S
O
Cl
22 ±
29.7 ±
3
77 ± 7
O
41
5.3
(n=4)
4.4
(n=12 (n=20)
)
N
N
N
N
N
O
S
O
21 ±
84 ±
12
40.6 ± 3
2
O
42
(n=4)
(n=13
)
(n=13)
N
N
N
N
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Page 29 of 81
Journal of Medicinal Chemistry
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3
4
5
6
7
8
9
O
N
F
7
1.6 ±
43 12.3 4.7 5
(n=2) (n=7)
-14 ±
1
(
3 ± 3
n=8)
2.5 13
2.6 77
2.7 34
1.8
1.7
1.5
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
F
N
-29 ±
2
21.7 ±
O
43 ± 3
4
4
5
6.2
5.1
(n=10 (n=12)
N
N
(n=3)
)
N
N
F
O
N
12.8 ±
-30 ±
5.2 3
(n=8)
47 ± 2
n=9)
4
5.3
n=4)
(
(
N
N
N
N
F
O
O
N
-27 ±
2
6
.6 ±
1.4
96
46
5.6
2.6 14
3.1
(n=25 (n=33)
(n=10)
N
N
)
N
N
Table
6. Optimization of direct linked aryl-based
imidazopyridazine subseries. α2 Ki and α1/α2 PAM values were
measured as described here.33-35 aLogD are measured values or if
calculated (internal logD model) are in italics. b,cGABAA
functional activity were measured at 100-times the compound’s
d
IC . LipE values are calculated (pIC – logD) from measured
5
0
50
e
logD values when possible or from calculated logD. HLM Clint
(
L/min/mg), in vitro human microsomal stability measurement.
ACS Paragon Plus Environment
29