The Discovery of a Novel Phosphodiesterase (PDE) 4B-Preferring Radioligand for Positron Emission Tomography (PET) Imaging

Article abstract of DOI:10.1021/acs.jmedchem.7b01050

As part of our effort in identifying phosphodiesterase (PDE) 4B-preferring inhibitors for the treatment of central nervous system (CNS) disorders, we sought to identify a positron emission tomography (PET) ligand to enable target occupancy measurement in vivo. Through a systematic and cost-effective PET discovery process, involving expression level (B_max) and biodistribution determination, a PET-specific structure-activity relationship (SAR) effort, and specific binding assessment using a LC-MS/MS "cold tracer" method, we have identified 8 (PF-06445974) as a promising PET lead. Compound 8 has exquisite potency at PDE4B, good selectivity over PDE4D, excellent brain permeability, and a high level of specific binding in the "cold tracer" study. In subsequent non-human primate (NHP) PET imaging studies, [¹⁸F]8 showed rapid brain uptake and high target specificity, indicating that [¹⁸F]8 is a promising PDE4B-preferring radioligand for clinical PET imaging.

Full text of DOI:10.1021/acs.jmedchem.7b01050

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Article
The Discovery of a Novel Phosphodiesterase (PDE) 4B-preferring
Radioligand for Positron Emission Tomography (PET) Imaging
Lei Zhang, Laigao Chen, Elizabeth M. Beck, Thomas A. Chappie, Richard V Coelho, Shawn
D Doran, Kuo-Hsien Fan, John M. Humphrey, Zoe Hughes, Kyle Kuszpit, Erik A Lachapelle,
John T. Lazzaro, Robert J. Mather, Nandini C. Patel, Marc B Skaddan, Simone Sciabola,
Patrick R. Verhoest, Joseph Michael Young, Kenneth Zasadny, and Anabella Villalobos
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01050 • Publication Date (Web): 28 Sep 2017
Downloaded from http://pubs.acs.org on September 29, 2017
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The Discovery of a Novel Phosphodiesterase (PDE) 4Bꢀ
preferring Radioligand for Positron Emission
Tomography (PET) Imaging
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Lei Zhang,1* Laigao Chen,2 Elizabeth M. Beck,1 Thomas A. Chappie,1 Richard V. Coelho,3 Shawn D.
Doran,4 Kuo-Hsien Fan,3 John M. Humphrey,5 Zoe Hughes,6 Kyle Kuszpit,3 Erik A. Lachapelle,5 John
T. Lazzaro,4 Robert J. Mather,6 Nandini C. Patel,1 Marc B. Skaddan,3 Simone Sciabola,1 Patrick R.
Verhoest,1 Joseph M. Young,5 Kenneth Zasadny,3 Anabella Villalobos7
2
1Medicine Design, Medicinal Chemistry, Pfizer Inc., Cambridge, MA 02139, USA; Clinical &
Translational Imaging, Early Clinical Development, Pfizer Inc., Cambridge, MA 02139, USA;
4
3Bioimaging Center, Pfizer Inc., Groton, CT 06340, USA; Medicine Design, Pharmacokinetics,
5
Dynamics and Metabolism, Groton, CT 06340, USA; Medicine Design, Medicinal Chemistry, Pfizer
6
Inc., Groton, CT 06340, USA; Internal Medicine Research Unit, Pfizer Inc., Cambridge, MA 02139,
USA; 7Medicinal Synthesis Technologies, Pfizer Inc., Groton, CT 06340, USA.
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
Keywords: Positron emission tomography, PET, CNS PET MPO, PDE4B-preferring, LC-MS/MS cold
tracer method, PET imaging, target occupancy
ABSTRACT:
As part of our effort in identifying PDE4Bꢀpreferring inhibitors for the treatment of central
nervous system (CNS) disorders, we sought to identify a positron emission tomography (PET) ligand to
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enable target occupancy measurement in vivo. Through a systematic and costꢀeffective PET discovery
process, involving expression level (Bmax) and bioꢀdistribution determination, a PETꢀspecific structureꢀ
activity relationship (SAR) effort, and specific binding assessment using a LCꢀMS/MS “cold tracer”
method, we have identified 8 (PFꢀ06445974) as a promising PET lead. Compound 8 has exquisite
potency at PDE4B, good selectivity over PDE4D, excellent brain permeability, and a high level of
specific binding in the “cold tracer” study. In subsequent nonꢀhuman primate (NHP) PET imaging
studies, [18F]8 showed rapid brain uptake and high target specificity, indicating that [18F]8 is a
promising PDE4Bꢀpreferring radioligand for clinical PET imaging.
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INTRODUCTION
Positron emission tomography (PET) imaging is an important technology used in the development
of drug candidates in preclinical and clinical studies.1 With a suitable radiotracer, PET imaging has
been utilized to measure target occupancy and confirm that drug candidates have access to the
appropriate tissue compartment, which is especially important for central nervous system (CNS)
indications. As such, PET imaging can play a crucial role in setting clinical doses and defining
therapeutic indices. PET imaging has also played a role in disease endophenotyping.2 With PET
ligands available for targets associated with a disease, PET imaging has been helpful in defining
appropriate patient populations, as well as assessing disease stage and progression (e.g. Aβ amyloid and
tau PET ligands).
Cost-effective specific
PET-specific SAR
binding assessment
using LC-MS/MS
method
guided by CNS PET
MPO, Fu_b,
RRCK/MDR
Target-specific
clinical PET
imaging
NHP PET imaging;
dosimetry and GLP
toxicity study
Early determination
of Bmax and bio-
distribution
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Figure 1. A rational and costꢀeffective CNS PET ligand discovery process
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As part of our efforts to identify and advance novel PET ligands for our portfolio of CNS
programs, we have utilized an efficient and costꢀeffective approach to our PET ligand discovery process,
as illustrated in Figure 1.3 As a first step, we seek to measure the expression level (Bmax) and brain bioꢀ
distribution for the biological target of interest. Data for both parameters can be generated in vitro using
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3
brain tissues or slices and a Hꢀradiolabeled ligand. This ligand needs to be potent and selective but not
necessarily brainꢀpenetrant, thus broadening the scope of chemical matter suitable as a tool. Bmax
information is critical in defining the potency range necessary for the novel PET ligand given that most
successful PET ligands have a Bmax/Kd ratio >10.4 Biodistribution data confirms areas of the brain
enriched in the biological target of interest and establishes whether a particular brain area can be used as
a reference region to determine the ratio of specific to nonꢀspecific binding. With this knowledge, we
then proceed to PETꢀspecific structureꢀactivity relationship (SAR) efforts, guided by a set of PET design
and selection criteria previously disclosed by our group.5 Specifically we focus on the following criteria:
high potency (Bmax/Kd > 10) and selectivity (> 30x), favorable physicochemical properties (CNS PET
MPO > 3), high passive transꢀcellular permeability (RRCK Papp AB > 5 x10ꢀ6cm/s), and low human pꢀ
glycoprotein (Pꢀgp) liability (MDR1 BA/AB ≤ 2.5) to optimize brain permeability, and fraction unbound
in brain (Fu_b) > 0.05 for low risk of nonꢀspecific binding. Once PET ligand leads with suitable
properties are identified, we assess their specific binding ex vivo in rodents using a liquid
chromatographyꢀmass spectroscopy/mass spectroscopy (LCꢀMS/MS) method with unlabeled compound
(“cold tracer” method)6 prior to triggering the more costly PET radiochemistry and nonꢀhuman primate
(NHP) imaging studies. Implementation of these steps enables a rational and costꢀeffective approach to
brainꢀpenetrant PET ligand discovery and makes it possible to identify a viable PET ligand through
advancement of only one or two radioligand leads into imaging studies.
Phosphodiesterase 4 (PDE4) isozymes carry out selective hydrolytic degradation of adenosine 3’,
5’ꢀcyclic monophosphate (cAMP). PDE4 has four known isoforms (PDE4 A–D), among which
PDE4A, 4B, and 4D are widely expressed in brain.7 PDE4 inhibition has been associated with
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beneficial pharmacological outcomes in preclinical models of antiꢀinflammatory, proꢀcognitive, and
antipsychotic effects, and panꢀPDE4 inhibitors such as roflumilast and apremilast have been approved
for the treatment of chronic obstructive pulmonary disease and psoriatic arthritis, respectively.8
However, a common side effect of these panꢀPDE4 inhibitors has been the induction of gastrointestinal
(GI) symptoms such as nausea and emesis, which have been hypothesized to be at least partially
associated with inhibition of the PDE4D isoform.9 We therefore became interested in developing brainꢀ
penetrant PDE4Bꢀpreferring inhibitors for the treatment of CNS disorders, selective versus PDE4D, to
potentially enhance the therapeutic window between efficacy and GI side effects.10 Clinical PET
imaging would be critical in validation of this hypothesis and determination of whether high PDE4B
target occupancy could indeed be achieved without eliciting GI side effects. Existing PDE4 PET ligands
such as (R)ꢀ[11C]rolipram11 are not suitable due to a lack of isoform selectivity over PDE4D that could
lead to underestimation of target occupancy for selective inhibitors. We were keen to develop a PDE4Bꢀ
preferring PET radioligand, utilizing the PET ligand discovery strategies described above, to provide
accurate target occupancy assessment in the clinic.
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RESULTS AND DISCUSSION
We initiated our efforts on the discovery of PDE4Bꢀpreferring PET ligands by surveying existing
project chemical matter, with the goal of identifying an early radiotracer lead to allow for Bmax and bioꢀ
distribution determination. An imidazopyridazine series,12 represented by compound 1, was particularly
intriguing, as it demonstrated unprecedented selectivity for the long forms of PDE4A–C over PDE4D in
humans and old world monkeys, although not in other species. Structural studies revealed that
compounds from this chemical series form a hydrogen bond between the amide carbonyl oxygen and the
phenol functionality in a tyrosine residue (Y274) in one of the upstream conserved region 2 (UCR2)
helixes in PDE4. This tyrosine residue is present in PDE4A–C in all species and in PDE4D in most
species except in humans and old world monkeys where it is replaced by a phenylalanine residue. The
loss of this critical hydrogen bond between compounds of this series and PDE4D results in the greater
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potency and selectivity observed for PDE4A–C in humans and old world monkeys. This unique
structureꢀdriven selectivity profile prompted us to focus on 1 as a promising lead for Bmax and bioꢀ
distribution assessment. As shown in Figure 2, 1 exhibited good potency for the PDE4A–C isoforms and
excellent selectivity over PDE4D as well as other PDE subtypes. Further testing in a broad spectrum
selectivity screen revealed no significant offꢀtarget activities up to 10 µM (see supporting information,
Table 2ꢀ1).13 Compound 1 met our PET ligand design parameters, with a high CNS PET MPO score
(4.96), high passive permeability (RRCK Papp AB= 32.1 x 10ꢀ6 cm/s), low Pꢀgp liability (MDR1
BA/AB= 1.17), and low risk of nonꢀspecific binding (cFu_b = 0.19). Tritiation at the methoxy moiety
was readily achieved via alkylation of the corresponding phenol precursor 17 with [3H]CH3I (Scheme 1,
Chemistry section).
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Figure 2. Profile of an early tritiated radiotracer lead 1
Bmax and Bio-distribution Determinations
With [3H]1 in hand, we carried out saturation binding studies using frontal cortex brain tissues of
mouse and NHP to determine Bmax and compare with the nonꢀselective PDE4 radiotracer [3H]rolipram.
In mice, we observed equivalent Bmax for these two tracers (Figure 3A, 3B). Interestingly, in NHP we
observed a significant discrepancy in Bmax determined with these two radiotracers: 107.41 fmol/mg
protein using [3H]rolipram (Figure 3C) and a roughly 40% lower level (62.07 fmol/mg) using [3H]1
(Figure 3D). This result confirmed the distinct speciesꢀspecific isoform selectivity profile of 1 (only
selective over PDE4D in humans and old world monkeys). For development of a PET ligand aimed at
clinical applications, the Bmax value determined by [3H]1 (62.07 fmol/mg protein, ~ 3.1 nM based on an
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assumption of 50 mg protein/g wet tissue) in NHP would be the relevant value, and was used for our
subsequent PET ligand design and prioritization. PDE4B isozyme has been reported to be widely
distributed in the brain, as demonstrated by in situ hybridization histochemistry and [3H]rolipram in
vitro autoradiography studies.7 Our in vitro autoradiography study with [3H]1 recapitulated these
findings in mice, showing binding throughout the brain with higher expression in prefrontal cortex,
striatum, and cerebellum.14 No reference region (free of PDE4 expression) was identified in brain.
Therefore, it was necessary to perform both baseline and blocking studies for specific binding
assessment of PET ligand leads.
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Saturation binding of [3H]rolipram and [3H]1 in mouse cortex
A
B
[3H]Rolipram (nM)
[3H]1 (nM)
Bmax = 488 fmol/mg
Kd = 10.6 nM
Bmax = 445 fmol/mg
Kd = 1.8 nM
Saturation binding of [3H]rolipram and [3H]1 in NHP cortex
C
D
[3H]Rolipram (nM)
[3H]1 (nM)
Bmax = 62.07 fmol/mg
Bmax = 107.41 fmol/mg
Kd = 2.6 nM
Kd = 3.6 nM
Figure 3. Saturation binding study in homogenated mouse frontal cortex brain tissue using A) nonꢀ
selective PDE4 radiotracer [3H]rolipram or B) PDE4Bꢀpreferring radiotracer [3H]1; Saturation binding
study in NHP homogenated frontal cortex using C) nonꢀselective PDE4 radiotracer [3H]rolipram or D)
PDE4Bꢀpreferring radiotracer [3H]1.
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PET Ligand Design
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Based on the above Bmax determination, it became clear that additional improvement in potency
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would be necessary for an effective PET ligand (Bmax/Kd ≥ 10). Our initial SAR effort focused on
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improving PDE4B potency while maintaining high selectivity over PDE4D. Matchedꢀpair analysis of
existing analogs in the imidazopyridazine series revealed an encouraging SAR trend, as illustrated by
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compounds 2 and 3 (Figure 4). Compound 2, bearing an
showed moderate PDE4B potency (IC50 of 429 nM). A substantial improvement in PDE4B potency
(35ꢀfold increase) was observed in its corresponding ꢀH amide analog 3. Importantly, this
Nꢀmethyl group at the amide side chain,
N
improvement in potency did not come at the expense of PDE4D selectivity (336x). Molecular dynamics
(MD) simulations of compounds 2 and 3 revealed differences in the conformation of the amide side
chains. As shown in the bar chart in Figure 4, the highest probability density for the
N
ꢀmethyl amide
bond orientation in 2 was at an angle of 70o to the imidazopyridazine core, while the
NꢀH amide in 3
preferred to adopt a coꢀplanar conformation (highest probability density at 0o). Further examination of
detailed atom interactions with the protein residues indicated that the ꢀmethyl analog 2 suffered a
significant drop in hydrogenꢀbond interaction with tyrosine 274, compared with the ꢀH analog 3. This
N
N
analysis suggested that adding a conformational constraint to promote coplanarity could be an effective
design strategy to further improve potency while maintaining the hydrogen bond critical for selectivity
versus PDE4D.
Cl
Cl
R1
N
O
N
N
N
O
N
N
O
N
N
R2
N
N
N
R
conformational
constraint for planarity
4
PDE4B IC50: 429 nM
PDE4D IC50: >30 M
2, R = Me
3, R = H
PDE4B IC50: <1 nM
PDE4D IC50: 59 nM
CNS PET MPO = 3.58
RRCK = 21.5 x 10ꢀ6 cm/s
MDR BA/AB = 1.32
35x
PDE4B IC50: 12 nM
PDE4D IC50: 4028 nM
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Figure 4. Design of highly potent novel tricyclic pyrrolopyridine PDE4Bꢀpreferring inhibitor 4: a) a
schematic depiction of ligand atomꢀprotein residue interactions that occur more than 30% of the MD
simulation time in the selected trajectory; b) probability density bar charts of the amide bond throughout
the course of the MD simulation with energy potentials (in kcal/mol) of the rotatable bond shown on the
yꢀaxis of the chart.
Based on this analysis, we designed a novel tricyclic pyrrolopyridine series, exemplified by
compound 4, wherein the amide side chain is fused to the central core through an ethylene linker (Figure
4). This structural modification indeed led to a significant increase in PDE4B potency into the subꢀnM
range. Furthermore, favorable selectivity versus PDE4D (>59ꢀfold) was maintained. The predicted
binding mode of 4 in the active site of the longꢀform PDE4B structure (PDB code: 4X0F) is illustrated
in Figure 5. The 4ꢀchlorophenyl group is nicely buried in the hydrophobic pocket found at the interface
between the catalytic domain and one of the UCR2 helixes (in yellow). Key hydrogen bonds between
the carbonyl oxygen and tyrosine 274 as well as between the pyridine nitrogen and glutamine 615 are
optimized by the coplanarity of the tricyclic core. These interactions were maintained 97% and 98% of
the time, respectively, during our 50 ns molecular dynamics simulation session. As mentioned above,
the hydrogen bond with tyrosine 274 is responsible for maintaining the desired selectivity over PDE4D.
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Figure 5. Predicted binding mode of tricyclic inhibitor 4 in the active site of the longꢀform PDE4B
structure (PDB code: 4X0F)
Encouraged by the favorable potency and selectivity profile of 4, we next carried out PETꢀspecific
SAR efforts varying the pendant aryl group, focusing on rings with substituents appropriate for either
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11C or F incorporation while maintaining favorable PET properties. As shown in Table 1, a total of 8
analogs were targeted and all had excellent CNS PET MPO scores (> 3) and calculated fraction unbound
in brain within the preferred PET property space (cFu_b > 0.05). In addition, all analogs displayed good
passive permeability (RRCK
our criteria. In terms of potency, when R is phenyl, electronꢀwithdrawing substituents appeared to be
preferred. For example, replacement of ꢀCl (4) with a ꢀmethoxy (5) led to a moderate potency loss (<1
nM vs. 6 nM). The potency could be maintained or incrementally increased by adding one or two ꢀF
substituents (IC50 = 6 nM for 7 and IC50 = 3 nM for 6). The best potency was achieved with the ꢀCN,
F phenyl derivative (compound 8, PF-06445974) which exhibited an IC50 < 1 nM that could not be
accurately determined due to the lower limit of our enzymatic assay. Its closeꢀin analog (9) bearing a
F, ꢀCN phenyl also demonstrated good PDE4B potency (IC50 = 2 nM) but with a slight deterioration in
Papp AB > 5) and low Pꢀgp liability (MDR1 BA/AB < 2.5) consistent with
p
p
m
p
mꢀ
p
ꢀ
m
PDE4D selectivity. Limited SAR investigation of heteroaryl substituents indicated that substituted 3ꢀ
pyridyls were tolerated, as exemplified by compounds 10 (IC50 = 6 nM) and 11 (IC50 = 6 nM), while
incorporation of a methylpyrazole ring (12) led to almost complete loss of PDE4B potency.
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Table 1. PETꢀspecific SAR with variations at the pendant R group
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PDE4D IC50
RRCK Papp
AB
PDE4B
CNS PET
MPO c
MDR1
Compound
5
R
(nM)
cFu_b f
IC50 (nM)a
BA/ABe
(selectivity)b
(10-6 cm/s)d
6
3
212 (35x)
125 (42x)
4.31
3.98
24.2
26.3
1.10
1.28
0.11
F
O
F
0.09
0.09
6
7
F
O
6
281 (47x)
3.97
31.2
1.24
< 1
2
36 ( > 36x)
44 (22x)
4.00
4.00
5.01
20.6
20.9
26.3
1.16
1.08
1.07
0.13
0.12
0.23
8
9
6
480 (80x)
10
6
159 (26x)
4.00
26.1
1.34
0.08
11
2006
3540 (1.8x)
5.41
33.6
1.20
0.39
12
aIC50 values obtained from a human recombinant fullꢀlength PDE4B1 scintillation proximity assay;
values represent the geometric mean of at least three experiments; bIC50 values obtained from a human
recombinant fullꢀlength PDE4D3 scintillation proximity assay; values represent the geometric mean of
at least three experiments; cCNS PET MPO represents the summation of scores of six commonly used
individual physicochemical properties using the transformed functions described in reference 5;d RRCK
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cells with low transporter activity were isolated from MardinꢀDarby canine kidney cells and were used
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2
3
4
e
to estimate passive permeability; Ratio from the MSꢀbased quantification of Papp,A→B and Papp,B→A of
f
the test compound (1 µM) across contiguous monolayers from MDR1ꢀtransfected MDCK cells; In
silico calculated fraction unbound in brain.
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7
8
From this PETꢀspecific SAR effort, 8 emerged as the best PET ligand lead for further profiling
(Figure 6). Structurally, 8 is amenable to 18Fꢀlabeling at the pendant phenyl moiety, due to activation by
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the pꢀCN group. In terms of pharmacology, 8 exhibited subꢀnM PDE4B IC50 and favorable selectivity
over PDE4D (> 36x) with moderate selectivity for PDE4C (> 17x) and PDE4A (> 5x). Importantly, 8
demonstrated minimal offꢀtarget activities in broadꢀspectrum selectivity panels, with only weak µM
activities at PDE10 (IC50 = 2290 nM), PDE5A (IC50 = 4640 nM) and GABAA (
K
i = 3850 nM). In terms
app AB =
of properties, 8 exhibited high CNS PET MPO score (4.0), high passive permeability (RRCK
P
20.6 x10ꢀ6 cm/s), low Pꢀgp liability (MDR1 BA/AB= 1.16), and good fraction unbound in brain (Fu_b =
0.11) similar to the predicted value (0.13). Compound 8 therefore met most of our PET ligand design
and selection parameters. Neuropharmacokinetic study in rats (0.1 mg/kg, IV) confirmed high brain
permeability with a total brain to plasma ratio (B/P_total) of 0.76, corresponding to a free brain to
plasma ratio (B/P_free) of 0.70. This overall favorable potency, selectivity, and PK profile further
solidified 8 as a promising radiotracer lead for specific binding assessment.
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Figure 6. Selectivity and PK properties of compound 8
Chemistry
Our synthesis of radiotracer lead 1, illustrated in Scheme 1, began with the
condensation/cyclization of pyridazinꢀ3ꢀamine (13) with ethyl 3ꢀbromoꢀ2ꢀoxopropanoate to afford
intermediate 14. Direct arylation catalyzed by palladium acetate, followed by ester saponification
provided acid 16, and subsequent HATUꢀmediated formation of the azetidine amide afforded 1.
Incorporation of the CT3 label was accomplished by deꢀmethylation with BBr3 followed by reꢀalkylation
with CT3I to afford the labelled target [3H]1.
Scheme 1a
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aReagents and conditions: a) ethyl 3ꢀbromoꢀ2ꢀoxopropanoate, EtOH, 78 C, 20%; b) 5ꢀbromoꢀ1,3ꢀ
o
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difluoroꢀ2ꢀmethoxybenzene, Pd(OAc)2, P(PPh3)4, K2CO3, DMF, 110 C, 58%; c) LiOH, EtOH/H2O, rt;
d) azetidine hydrochloride, HATU, DIPEA, DMF, rt, 32% for two steps; e) BBr3, DCM, 0 oC to rt, 27%;
f) CT3I, K2CO3, DMF, rt, 20 mCi, 73.6 Ci/mmol specific activity.
The synthesis of compounds 2 and 3 followed a similar sequence of reactions from common
intermediate 14, with the primary difference being replacement of direct arylation by a
bromination/Suzuki sequence as illustrated in Scheme 2. Thus, bromination of 14 afforded intermediate
18. Suzuki coupling with (4ꢀchlorophenyl)boronic acid provided 19, which upon ester saponification
gave carboxylic acid 20. Conversion into the final products 2 and 3 was accomplished via amide
coupling using
N
ꢀmethylcyclopropylamine and cyclopropylamine, respectively.
Scheme 2a
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o
aReagents and conditions: a) NBS, DCM, 0 C to rt, 90%; b) (4ꢀchlorophenyl)boronic acid, CsF,
o
o
Pd(amphos)Cl2, dioxane/H2O, 100 C, 83%; c) NaOH, MeOH/H2O, 60 C, 97%; d) HATU, DIPEA,
DMF, ꢀmethylcyclopropylamine (for compound 2, 61%) or HBTU, DIPEA, THF, cyclopropylamine
N
(for compound 3, 81%), rt.
Synthesis of tricyclic analogs 4˗12 is shown in Scheme 3. Mitsunobu alkylation of 21 with
bromoethanol yielded intermediate 22 which upon treatment with cyclopropylamine afforded 23.
Subsequent lactamization mediated by magnesium methoxide then afforded the tricycle 24 in good
yield. Bromination with NBS followed by Suzuki coupling with a variety of aryl boronates afforded the
requisite targets 4ꢀ12.
Scheme 3a
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aReagents and conditions: a) 2ꢀbromoethanol, PPh3, DIAD, THF, rt, 70%; b) cyclopropylamine,
K2CO3, CH3CN, 60 oC; c) Mg(OMe)2, MeOH, 80 oC, 97% over two steps; d) NBS, DCM, 25 oC, 72%;
e) RꢀB(OH)2, [Pd] catalyst, Na2CO3 or K2CO3, H2O, dioxane, 90 oC.
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LC-MS/MS “Cold Tracer” Study in Rodents
8
9
Prior to the NHP PET imaging study, we explored a LCꢀMS/MS based “cold tracer” method to
assess the specific binding of 8 in vivo. Considering the unique speciesꢀdependent PDE4B selectivity
profile of our chemical series (selective over PDE4D in higher species, but not in rodents), we chose
129/B6 PDE4D knockꢀout (KO) mouse as a higherꢀspecies surrogate for in vivo binding assessment of
8. This experiment allowed us to gain an early read on whether sufficient specific binding could be
achieved for compound 8, given that its PDE4B potency (< 1 nM) could not be accurately determined
due to the lower limit of our enzymatic assay. Toward this end, a tracer amount of unlabeled “cold”
compound 8 (10 µg/kg) was dosed intravenously in mice, and brain distribution was quantified in
regions of interest using high sensitivity LCꢀMS/MS. The level of specific binding was then determined
by comparing a baseline group (pretreated with vehicle) with a blocking group pretreated with PDE4
inhibitor rolipram. As shown in Figure 7A, 8 showed excellent brain uptake and reached peak
concentrations at around 20 minutes. The concentration of 8 was evaluated in five different brain
regions, with the highest concentration observed in frontal cortex, followed by striatum and cerebellum,
and the lowest concentration observed in hippocampus and brain stem. After pretreatment with a high
dose of rolipram (3 mg/kg, IV), the concentration of 8 in all brain regions was significantly reduced
(Figure 7B), suggesting that the binding of 8 in these brain regions was targetꢀspecific.
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1
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Brain Stem
Cerebellum
Striatum
Frontal Cortex
Hippocampus
0
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Survival Interval (min)
B
Vehicle
Rolipram
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Figure 7. LCꢀMS/MS cold tracer evaluation of 8 in PDE4D KO mice: A) %ID/g of 8 (10 µg/kg, IV, 5ꢀ
75 min survival interval) in five brain regions: brain stem, cerebellum, frontal cortex, hippocampus, and
striatum; B) Comparison %ID/g of 8 (10 µg/kg, IV, 45 min survival interval) between a vehicle group
and a blocking group (rolipram, 3 mg/kg, IV, 15 min preꢀtreatment) in brain stem, cerebellum, frontal
cortex, hippocampus and striatum.
PET Ligand Synthesis
The favorable binding profile of 8 in the cold tracer LCꢀMS/MS experiment prompted us to further
investigate radiofluorination of 8 and subsequent PET imaging in NHPs. As shown in Scheme 4,
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pinacolborane 27 was prepared in one step from commercially available 4ꢀbromoꢀ2ꢀnitrobenzonitrile
(26). Suzuki coupling of 27 with tricyclic template 25 yielded nitro precursor 28. 18FꢀLabeling of 8
proceeded by nucleophilic aromatic substitution (SNAr) displacement of the nitro group using 18F
Kryptofix 222/K2CO3 in DMF at 130 °C. As expected, the strong electronꢀwithdrawing influence of the
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9
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ꢀnitrile led to high conversion to the desired product. Purification by semiꢀpreparative HPLC led to
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[18F]8 in good radiochemical yields and excellent specific radioactivities.
Scheme 4a
CN
CN
CN
18F
NO2
NO2
c
b
N
N
O
O
X
N
N
N
26, X= Br
N
a
O
B
27, X=
O
[
18F]8
28
aReagents and conditions: a) bis(pinacolato)diborane, Pd(dppf)Cl2, KOAc, dioxane, 80oC; b)
o
Pd(amphos)2Cl2, 25, CsF, 2ꢀMeTHF/H2O, 80 C, 45% over two steps; c) [18F]fluoride/Kryptofix 222,
o
K2CO3, DMF, 130 C, 10 min, 36 ± 8% radiochemical yield, 688.79 ± 214.71 GBq/µmol specific
radioactivity.
NHP PET Imaging.
Due to the lack of reference region within the brain, the specific binding of [18F]8 was evaluated in
cynomolgus monkeys under both baseline and blocking conditions. We were gratified to see that, similar
to the LCꢀMS/MS study, [18F]8 showed excellent brain penetration in NHPs, with the highest
accumulation in thalamus, followed by putamen and caudate, various cortical regions, and lower levels
in cerebellum and hippocampus. The baseline timeꢀactivity curves (TACs) (Figure 8A) demonstrated
that [18F]8 reached peak concentration (standardized uptake value, or SUV ~3) in less than 30 minutes
post tracer injection in all brain regions. The MA1 model fitted the measured time activity data well and
generated robust estimates of the total volume of distribution (VT). The PET images, TACs, and
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measured VT all indicated significant blockade of the brain uptake of [18F]8 upon pretreatment with a
structurally distinct PDE4Bꢀpreferring inhibitor15 (0.5 mg/kg, sc) four hours prior to the tracer injection.
The target occupancy in the blocking study was estimated to be ~92% (Figure 8B).
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Time (min)
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Frontal Cortex
Cerebellum
Putamen
Hippocampus
Caudate
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Thalamus
SUV 0
4
Figure 8. Timeꢀactivity curves and transverse and coronal SUV images of [18F]8 in NHPs under A)
baseline, and B) blocking (a PDE4B inhibitor, 0.5 mg/kg, sc, 4 h pretreatment) conditions.
CONCLUSIONS
Phosphodiesterase 4 (PDE4) inhibitors have shown promising efficacy in a number of preclinical
models and in the clinic, with doseꢀlimiting GI side effects (nausea and emesis) thought to be mediated
by the PDE4D isoform. To facilitate a drug discovery effort with brainꢀpenetrant PDE4Bꢀpreferring
inhibitors, we aimed to develop a PET ligand with a similar selectivity profile to provide target
occupancy measurements associated with efficacy and GI side effects. Following an efficient and costꢀ
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effective CNS PET ligand discovery process, we quickly identified a viable PDE4Bꢀpreferring PET
ligand, [18F]8, via rational design and a PETꢀfocused SAR effort. Our work started by identifying an
earlier radiotracer [3H]1 with the requisite high selectivity over the PDE4D isoform for Bmax
determinations. In vitro autoradiography study confirmed literatureꢀreported widespread expression of
PDE4 throughout the brain with no reference region. In NHP cortex, Bmax was determined to be
approximately 3.1 nM, suggesting that subꢀnM binding affinity would likely be necessary to achieve
specific in vivo target engagement with PDE4Bꢀpreferring PET ligands.
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Guided by the CNS PET ligand design and selection criteria previously reported by our group, we
carried out PETꢀspecific SAR efforts to further optimize PDE4B potency, selectivity versus PDE4D,
and favorable PET ligand properties. From this effort, a potent tricyclic series was designed and 4ꢀ(8ꢀ
cyclopropylꢀ9ꢀoxoꢀ6,7,8,9ꢀtetrahydropyrido[2',3':4,5]pyrrolo[1,2ꢀa]pyrazinꢀ10ꢀyl)ꢀ2ꢀfluorobenzonitrile
(8) emerged as the most promising lead. Compound 8 had exquisite potency at the PDE4B isoform and
good selectivity over the PDE4D isoform and other PDEs. Furthermore, 8 showed excellent in vivo
brain permeability (B/P_free = 0.70), consistent with the prediction by favorable in vitro passive
permeability (RRCK) and MDR efflux measurements. Low risk of nonꢀspecific binding was predicted
based on a favorable Fu_b value (0.11). Compound 8 was assessed in a LCꢀMS/MS based “cold tracer”
method in PDE4D KO mice. Brain concentrations in five different brain regions were measured and
found to be consistent with the in vitro PDE4 brain bioꢀdistribution. Importantly, 8 demonstrated a high
level of specific binding across all five brain regions.
These favorable results prompted further evaluation of 8 in PET imaging. Toward this end, [18F]8
was successfully synthesized via a [18F] SNAr displacement of nitro precursor 28. In subsequent NHP
PET imaging studies, [18F]8 showed rapid brain uptake and a brain distribution similar to those observed
in previous autoradiography and LCꢀMS/MS “cold tracer” studies. Importantly, upon blocking with a
PDE4Bꢀpreferring inhibitor, brain radioactivity was significantly reduced, thereby indicating a high level
of specific binding. Overall, these PET imaging data indicate that [18F]8 is a promising PDE4Bꢀ
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preferring PET ligand for target occupancy measurement in vivo, reinforcing the effectiveness of our
PET ligand design and selection approach to identify successful ligands from a small number of leads in
a rational and efficient way. Additional studies including target occupancy of a PDE4Bꢀpreferring
inhibitor, radiation dosimetry in NHPs, and radioactive metabolite analysis will be provided in future
publications.
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EXPERIMENTALS
Biology
Human recombinant full length PDE4A, 4B, 4C and 4D scintillation proximity assay (SPA)
enzymatic assays:
The PDE4A3, PDE4B1, PDE4C1 and PDE4D3 enzymatic assays used the SPA technology to
measure the inhibition of human recombinant PDE4A3, PDE4B1, PDE4C1, and PDE4D3 enzyme
activity by compounds in vitro. Recombinant full length PDE4A3, PDE4B1, PDE4C1, and PDE4D3
were generated and purified following known procedures and assays were run in parallel using identical
parameters, except for the concentration of enzyme (80 pM PDE4A3, 40 pM PDE4B1, 40 pM PDE4C1
and 10 pM PDE4D3). The assays were performed in a 384ꢀwell format with 50 µL per well total volume
of assay buffer (50 mM TRIS pH7.5; 1.3 mM MgCl2; 0.01% Brij) containing sufficient PDE4A3,
PDE4B1, PDE4C1, and PDE4D3 to convert ~20% of substrate (1 µM cAMP consisting of 20 nM
[3H]cAMP + 980 µM cAMP) and a range of inhibitors. Positive control wells containing 10
ꢀM
rolipram and negative control wells containing an equivalent concentration of DMSO were included on
each plate. Reactions were incubated for 30 min at rt and were stopped by the addition of 20 µL of 8
mg/mL yttrium silicate SPA beads (Perkin Elmer). The plates were then sealed (TopSeal, Perkin Elmer)
and the beads were allowed to settle for 8 h, after which they were read on a Trilux Microbeta 1450
counter (Perkin Elmer). Raw data were normalized using the positive and negative controls in each
plate, and the concentration response curves were fitted using a 4ꢀparameter logistic fit model. IC50
results from different experiments were summarized by geometric mean. In cases where IC50 values
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were less than the lower limit of our enzymatic assay, the summarized geometric mean is reported with a
“<” along with the geometric mean.
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Saturation binding in mouse and cynomolgus monkey cortical tissues:
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Saturation binding studies were performed in cortical tissues from mouse or cynomolgus monkey
cortex using in vitro filtration techniques. Frozen mouse brains (PelFreez) were thawed, and frontal
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o
cortex was dissected. Mouse cortical tissue could be used fresh or frozen at −80 C for future use.
o
Frontal cortex was dissected from cynomolgus brain and stored at −80 C. The specific amount of
cynomolgus frontal cortical tissue needed for each experiment could be broken off without thawing.
After thawing, mouse or cynomolgus tissue was homogenized in tissue buffer using a polytron and
centrifuged at 18500 rpm for 10 min. The pellet was then reꢀsuspended in assay buffer and stored on
ice. Tissue buffer contained 50 mM Tris, pH 7.4 with 2 mM MgCl2. Assay buffer contained 50 mM
Tris, pH 8.0 with 2 mM MgCl2. Concentration responses of [3H]1 and [3H]rolipram were carried out
with and without 10
of radioligand in assay buffer, 25
L of prepared tissue were added to each well. 9 mg/mL final concentration of mouse cortical tissue
ꢀM rolipram to determine specific binding. 25
ꢀL of 10 times final concentration
ꢀ
L of assay buffer or 10 times final concentration of rolipram, and 200
ꢀ
and 12 mg/mL final concentration of cynomolgus cortical tissue were used. Assay plates were incubated
for 30 min at rt while shaking, then filtered through PEIꢀsoaked GF/B filterplates using a plate harvester
(Packard) and 50 mM Tris (pH 7.4, 4
°
C). After filter plates had been dried overnight at room
L per well) was added. Plates were sealed and incubated
temperature, Ecolume scintillation fluid (50
ꢀ
for at least 1 h at room temperature, then counted using a Microbeta Trilux 1450 counter. Protein
concentrations were determined using a BCA protein assay kit. Specific binding for each well was
determined by subtracting values from the rolipram containing control wells. Kd and Bmax values were
determined using the specific binding values and a one site – specific binding calculation in Graphpad
Prism.
Chemistry General Information
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All solvents and reagents were of reagent grade and were used as supplied by the manufacturer.
All reactions were run under a N2 atmosphere. Organic extracts were routinely dried over anhydrous
Na2SO4 or MgSO4. Concentration in vacuo refers to rotary evaporation under reduced pressure.
Chromatography refers to flash chromatography using disposable RediSepRf 4 to 120 g silica columns
or Biotage disposable columns on a CombiFlash Companion or Biotage Horizon automatic purification
system. Purification by massꢀtriggered HPLC was carried out using Waters XTerra PrepMS C18
columns, 5 µm, 30 mm x100 mm. Compounds were presalted as TFA salts and diluted with 1 mL of
DMSO. Samples were purified by massꢀtriggered collection using a mobile phase of 0.1% TFA in water
and acetonitrile, with a gradient of 100% aqueous to 100% acetonitrile over 10 min at a flow rate of 20
mL/min. All target compounds were analyzed using ultra high performance liquid
chromatography/ultraviolet/evaporative light scattering detection coupled to time of flight mass
spectrometry (UHPLC/UV/ELSD/TOFMS). LCꢀMS analyses were performed on a Waters Acquity
UPLCꢀMS system with a Waters Aquity HSS T3, 1.7 µm C18 column (50 mm x 2.1 mm). UPLC
conditions: mobile phase A = 0.1% formic acid in water (v/v), mobile phase B = 0.1% formic acid in
acetonitrile; flow rate = 1.25 mL/min; compounds were eluted with a gradient of 5% B/A to 95% B/A
for 1.1 min. All tested compounds were found to be > 95% pure by this method.
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Experimental Procedures
Azetidin-1-yl[3-(3,5-difluoro-4-methoxyphenyl)imidazo[1,2-
b
]pyridazin-2-yl]methanone (1):
ꢀ(7ꢀ
A mixture of 16 (100 mg, 0.328 mmol), azetidine hydrochloride (45 mg, 0.48 mmol),
O
azabenzotriazolꢀ1ꢀyl)ꢀN,N,N’,N’ꢀtetramethyluronium hexafluorophosphate (HATU, 186 mg, 0.489
mmol), DIPEA (126 mg, 0.975 mmol) and DMF (10 mL) was stirred at rt overnight. The reaction
mixture was then diluted with water and extracted with EtOAc (3 x 10 mL). The combined organic
layers were concentrated in vacuo and purified by reversedꢀphase HPLC (Column: Phenomenex Synergi
C18, 4 ꢁm; Mobile phase A: 0.225% formic acid in water; Mobile phase B: acetonitrile; Gradient: 33%
to 53% B) to provide the product as a yellow solid. Yield: 35.7 mg, 32%. LCꢀMS m/z 345.2 [M+H]+. 1H
NMR (400 MHz, DMSOꢀd6
)
δ
8.63 (dd, J = 4.4, 1.6 Hz, 1H), 8.24 (dd, J = 9.3, 1.6 Hz, 1H), 7.56 (br d,
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J = 9.9 Hz, 2H), 7.39 (dd, J = 9.4, 4.4 Hz, 1H), 4.46–4.53 (m, 2H), 4.01–4.07 (m, 2H), 4.01 (br s, 3H),
1
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2.22–2.32 (m, 2H).
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3-(4-Chlorophenyl)-N-cyclopropyl-N-methyl-imidazo[1,2-b]pyridazine-2-carboxamide (2). A
8
9
mixture of compound 20 (60 mg, 0.219 mmol),
N
ꢀmethylcyclopropylamine (31.2 mg, 0.439 mmol),
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HATU (124.8 mg, 0.328 mmol), DIPEA (84.75 mg, 0.657 mmol), and DMF (2 mL) was stirred at rt
overnight. The reaction was diluted with water, and extracted with EtOAc (3 x 10 mL). The combined
organic phases were concentrated and purified by reversedꢀphase HPLC (Column: Boston Symmetrix
ODSꢀH, 5 µm; Mobile phase A: 0.225% formic acid in water (v/v); Mobile phase B: 0.225% formic
acid in acetonitrile (v/v); Gradient: 33% to 53% B) to provide the product as a pale yellow solid. Yield:
36.3 mg, 61%. LCꢀMS m/z 326.9 [M+H]+. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.60 (dd, J = 4.5, 1.5 Hz,
1H), 8.18 (dd, J = 9.0, 1.5 Hz, 1H), 7.79 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 7.35 (dd, J = 9.0,
4.5 Hz, 1H), 2.93 (br s, 3H), 2.64 (br s, 1H), 0.47 (br s, 4H).
3-(4-Chlorophenyl)-
N
-cyclopropyl-imidazo[1,2-
b
]pyridazine-2-carboxamide (3).
TBTU
(97%, 3.21 g, 8.21 mmol) was added to a mixture of 20 (1.50 g, 5.48 mmol) and DIPEA (2.86 mL, 16.4
mmol) in THF (100 mL), and the reaction mixture was stirred at rt for 2 min. Cyclopropylamine (0.77
mL, 11 mmol) was introduced, and stirring was continued at rt overnight. The reaction mixture was
concentrated in vacuo and the residue was subjected to silica gel chromatography (Gradient: 0% to
100% EtOAc in heptane). The resulting solid was triturated with a 10:1 mixture of Et2O and DCM, and
subsequently purified once more via chromatography on silica gel (Gradient: 0% to 100% EtOAc in
heptane) to afford the product as a solid. Yield: 1.39 g, 81%. LCꢀMS m/z 313.1 [M+H]+. 1H NMR (400
MHz, CD3OD)
δ 8.46 (dd, J = 4.3, 1.6 Hz, 1H), 8.06 (dd, J = 9.3, 1.7 Hz, 1H), 7.71 (br d, J = 8.7 Hz,
2H), 7.48 (br d, J = 8.6 Hz, 2H), 7.31 (dd, J = 9.3, 4.4 Hz, 1H), 2.79–2.86 (m, 1H), 0.78–0.84 (m, 2H),
0.62–0.68 (m, 2H).
10-(4-Chlorophenyl)-8-cyclopropyl-7,8-dihydropyrido[2',3':4,5]pyrrolo[1,2-a]pyrazin-9(6H)-
one (4). Bis(triꢀtertꢀbutylphosphine)palladium(0) (83 mg, 0.16 mmol) was added to a mixture of 25
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(500 mg, 1.63 mmol), (4ꢀchlorophenyl)boronic acid (509 mg, 3.26 mmol), dioxane (15 mL), and
Na2CO3 (864 mg, 8.15 mmol, as a 3 M solution in water). The reaction mixture was stirred at 90 °C for
16 h, and then diluted with water (50 mL) and EtOAc (50 mL). After extraction of the aqueous layer
with EtOAc (3 x 30 mL), the combined organic layers were washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. Purification of the combined crude products via silica gel
chromatography (Gradient: 0% to 100% EtOAc in petroleum ether) provided the product as a yellow
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solid. Yield: 870 mg, 39%. LCꢀMS m/z 337.8 [M+H]+. 1H NMR (400 MHz, CDCl3)
δ 8.60 (dd, J = 4.5,
1.4 Hz, 1H), 7.76 (br d, J = 8.5 Hz, 2H), 7.67 (dd, J = 8.4, 1.4 Hz, 1H), 7.43 (br d, J = 8.4 Hz, 2H), 7.29
(dd, J = 8.4, 4.5 Hz, 1H), 4.27–4.32 (m, 2H), 3.86–3.92 (m, 2H), 2.81–2.87 (m, 1H), 0.92–0.99 (m, 2H),
0.73–0.80 (m, 2H).
8-Cyclopropyl-10-(4-methoxyphenyl)-7,8-dihydropyrido[2',3':4,5]pyrrolo[1,2-
a]pyrazin-
9(6H)-one (5). The title compound was prepared from 25 according to the general procedure for the
synthesis of 4. K2CO3 was used as base. Purification was carried out via reversedꢀphase HPLC
(Column: Waters XBridge C18, 5 ꢁm; Mobile phase A: 0.03% NH4OH in water (v/v); Mobile phase B:
0.03% NH4OH in acetonitrile (v/v); Gradient: 10% to 100% B). Yield: 23 mg, 35%. LCꢀMS m/z 334.2
1
[M+H]+. H NMR (600 MHz, DMSOꢀ
d
6) δ 8.51 (dd, J = 4.4, 1.3 Hz, 1H), 8.08 (dd, J = 8.6, 1.1 Hz,
1H), 7.40 (dd, J = 8.4, 4.4 Hz, 1H), 7.33–7.36 (m, 3H), 6.91–6.98 (m, 1H), 4.42 (t, J = 5.7 Hz, 2H),
3.85 (t, J = 5.7 Hz, 2H), 3.83 (s, 3H), 2.88–2.94 (m, 1H), 0.82–0.89 (m, 2H), 0.74–0.82 (m, 2H).
8-Cyclopropyl-10-(3,5-difluoro-4-methoxyphenyl)-7,8-dihydropyrido[2',3':4,5]pyrrolo[1,2-
a
]pyrazin-9(6
H
)-one (6). The title compound was prepared from 25 according to the general procedure
1
for the synthesis of 4. K2CO3 was used as base. Yield: 42 mg, 70%. LCꢀMS m/z 370.2 [M+H]+. H
NMR (400 MHz, DMSOꢀd6) δ 8.59 (d, J = 4.5 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.38–7.46 (m, 2H),
7.29 (dd, J = 8.4, 4.5 Hz, 1H), 4.25–4.30 (m, 2H), 4.02 (s, 3H), 3.85–3.90 (m, 2H), 2.82–2.89 (m, 1H),
0.93–1.00 (m, 2H), 0.73–0.80 (m, 2H).
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Journal of Medicinal Chemistry
8-Cyclopropyl-10-(3-fluoro-4-methoxyphenyl)-7,8-dihydropyrido[2',3':4,5]pyrrolo[1,2-
1
2
3
a
]pyrazin-9(6H)-one (7). The title compound was prepared from 25 according to the general procedure
4
5
6
for the synthesis of 4. K2CO3 was used as base. Purification was carried out via reversedꢀphase HPLC
7
8
(Column: Waters XBridge C18, 5 ꢁm; Mobile phase A: 0.03% NH4OH in water (v/v); Mobile phase B:
9
0.03% NH4OH in acetonitrile (v/v); Gradient: 10% to 100% B). Yield: 23 mg, 33%. LCꢀMS m/z 352.2
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1
[M+H]+. H NMR (400 MHz, DMSOꢀ
d
6) δ 8.47 (dd, J = 4.5, 1.4 Hz, 1H), 8.03 (dd, J = 8.4, 1.4 Hz,
1H), 7.50–7.68 (m, 2H), 7.35 (dd, J = 8.4, 4.5 Hz, 1H), 7.18 (t, J = 9.0 Hz, 1H), 4.30–4.42 (m, 2H),
3.89 (s, 3H), 3.70–3.84 (m, 2H), 2.79–2.93 (m, 1H), 0.68–0.85 (m, 4H).
4-(8-Cyclopropyl-9-oxo-6,7,8,9-tetrahydropyrido[2',3':4,5]pyrrolo[1,2-a]pyrazin-10-yl)-2-
fluorobenzonitrile (8): Pd(dppf)Cl2 (242 mg, 0.331 mmol) was added to a mixture of Na2CO3 (1.04 g,
9.81 mmol, as a 3 M solution in water), 25 (1.0 g, 3.3 mmol), and (4ꢀcyanoꢀ3ꢀfluorophenyl)boronic acid
(592 mg, 3.59 mmol) in dioxane (30 mL). The reaction mixture was stirred at 90 °C for 16 h, whereupon
it was diluted with water (30 mL) and EtOAc (30 mL). The aqueous layer was extracted with EtOAc (3
x 30 mL), and the combined organic layers were washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. Chromatographic purification on silica gel (Gradient: 0% to 75% EtOAc in
petroleum ether, followed by a second column using 80% EtOAc in heptane as eluent) afforded a solid,
which was slurried in Et2O, stirred for 30 min, and collected via filtration to afford the product as a
white solid. Yield: 590 mg, 52%. LCꢀMS m/z 347.1 [M+H]+; 1H NMR (400 MHz, CDCl3)
δ 8.62 (br d,
J = 3.5 Hz, 1H), 7.80 (s, 1H), 7.77 (d, J = 2.3 Hz, 1H), 7.73 (br d, J = 7.8 Hz, 1H), 7.67 (dd, J = 7.9,
7.0 Hz, 1H), 7.34 (dd, J = 8.6, 4.7 Hz, 1H), 4.30–4.36 (m, 2H), 3.90–3.95 (m, 2H), 2.87 (m, 1H), 0.96–
13
1.02 (m, 2H), 0.75–0.81 (m, 2H); C NMR (400 MHz, CDCl3)
δ 160.96, 164.34, 146.10, 146.20 (d),
140.02, 132.52, 128.01, 120.37, 119.45, 119.30, 117.89, 115.01, 99.65, 46.87, 41.29, 30.30, 7.94.
5-(8-Cyclopropyl-9-oxo-6,7,8,9-tetrahydropyrido[2',3':4,5]pyrrolo[1,2-a]pyrazin-10-yl)-2-
fluorobenzonitrile (9): The title compound was prepared from 25 according to the general procedure
for the synthesis of 4. K2CO3 was used as base. Purification was carried out via reversedꢀphase HPLC
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(Column: Waters Atlantis dC18 4.6 x 50 mm, 5 µm, Mobile phase A: 0.05% TFA in water (v/v);
1
2
3
Mobile phase B: 0.05% TFA in acetonitrile (v/v); Gradient: 5% to 95% B). Yield: 12.5 mg, 18%. LCꢀ
4
5
6
7
8
9
MS m/z 347.0 [M+H]+. 1H NMR (600 MHz, DMSOꢀ
6.2, 2.2 Hz, 1H), 8.22–8.27 (m, 1H), 8.14 (dd, J = 8.4, 1.3 Hz, 1H), 7.62 (t, J = 9.0 Hz, 1H), 7.45 (dd,
8.4, 4.4 Hz, 1H), 4.43–4.47 (m, 2H), 3.84–3.90 (m, 2H), 2.90–2.96 (m, 1H), 0.84–0.90 (m, 2H), 0.78–
0.83 (m, 2H).
d6) δ 8.57 (dd, J = 4.4, 1.3 Hz, 1H), 8.31 (dd, J =
J
=
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8-Cyclopropyl-10-(6-fluoropyridin-3-yl)-7,8-dihydropyrido[2',3':4,5]pyrrolo[1,2-
a]pyrazin-
9(6H)-one (10). The title compound was prepared from 25 according to the general procedure for the
synthesis of 4. K2CO3 was used as base. Purification was carried out via reversedꢀphase HPLC
(Column: Waters XBridge C18, 5 ꢁm; Mobile phase A: 0.03% NH4OH in water (v/v); Mobile phase B:
0.03% NH4OH in acetonitrile (v/v); Gradient: 10% to 100% B). Yield: 13.6 mg, 43%. LCꢀMS m/z
323.2 [M+H]+. 1H NMR (600 MHz, DMSOꢀ
d6) δ 8.61 (s, 1H), 8.54 (dd, J = 4.2, 1.1 Hz, 1H), 8.36 (td, J
=
8.4, 2.2 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.43 (dd, J = 8.4, 4.4 Hz, 1H), 7.27 (dd, J = 8.6, 2.9 Hz,
1H), 4.44 (t, J = 5.7 Hz, 2H), 3.85 (t, J = 5.7 Hz, 2H), 2.87–2.93 (m, 1H), 0.81–0.87 (m, 2H), 0.76–0.81
(m, 2H).
10-(5-Chloro-6-methoxypyridin-3-yl)-8-cyclopropyl-7,8-dihydropyrido[2',3':4,5]pyrrolo[1,2-
a
]pyrazin-9(6
H
)-one (11). The title compound was prepared from 25 according to the general
1
procedure for the synthesis of 4. K2CO3 was used as base. Yield: 35 mg, 58%. H NMR (400 MHz,
DMSOꢀd6) δ 8.52 (d, J = 2.0 Hz, 1H), 8.51 (dd, J = 4.5, 1.4 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.08 (dd,
J = 8.4, 1.4 Hz, 1H), 7.39 (dd, J = 8.4, 4.5 Hz, 1H), 4.32–4.48 (m, 2H), 4.00 (s, 3H), 3.71–3.91 (m, 2H),
2.78–2.97 (m, 1H), 0.69–0.87 (m, 4H).
8-Cyclopropyl-10-(1-methyl-1H-pyrazol-4-yl)-7,8-dihydropyrido[2',3':4,5]pyrrolo[1,2-
a
]pyrazin-9(6 )-one (12). The title compound was prepared from 25 according to the general
H
procedure for the synthesis of 4. K2CO3 was used as base. Purification was carried out via reversedꢀ
phase HPLC (Column: Waters XBridge C18, 5 ꢁm; Mobile phase A: 0.03% NH4OH in water (v/v);
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Journal of Medicinal Chemistry
Mobile phase B: 0.03% NH4OH in acetonitrile (v/v); Gradient: 10% to 100% B). Yield: 19.9 mg, 66%.
1
2
3
1
LCꢀMS m/z 308.2 [M+H]+. H NMR (600 MHz, DMSOꢀ
d
6) δ 8.80 (s, 1H), 8.67 (s, 1H), 8.57 (dd, J =
4
5
6
7
8
4.4, 1.3 Hz, 1H), 8.05 (dd, J = 8.6, 1.1 Hz, 1H), 7.41 (dd, J = 8.4, 4.4 Hz, 1H), 4.38 (t, J = 5.7 Hz, 2H),
3.97 (s, 3H), 3.81 (t, J = 5.7 Hz, 2H), 2.87–3.03 (m, 1H), 0.86–0.96 (m, 2H), 0.77–0.86 (m, 2H).
9
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Ethyl imidazo[1,2-b]pyridazine-2-carboxylate (14): A mixture of pyridazinꢀ3ꢀamine (20 g, 210
mmol) and ethyl 3ꢀbromoꢀ2ꢀoxopropanoate (82 g, 420 mmol) in EtOH (300 mL) was heated at reflux
for 16 h. After removal of solvent via distillation, the residue was taken up in 2 M aqueous HCl (100
mL) and washed with EtOAc. The aqueous layer was basified to a pH of approximately 8 via addition of
aqueous NaHCO3 solution and then extracted with CHCl3; this organic layer was dried over Na2SO4,
filtered, and concentrated in vacuo. Silica gel chromatography (Eluent: 20% EtOAc in petroleum ether)
1
afforded the product as a brown solid. Yield: 8.0 g, 20%. LCꢀMS m/z 192.1 [M+H]+. H NMR (400
MHz, CDCl3)
δ 8.53 (s, 1H), 8.39 (dd, J = 4.4, 1.6 Hz, 1H), 8.01–8.04 (m, 1H), 7.12 (dd, J = 9.3, 4.4
Hz, 1H), 4.48 (q, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H).
Ethyl 3-(3,5-difluoro-4-methoxyphenyl)imidazo[1,2-
b
]pyridazine-2-carboxylate (15):
A
mixture of 14 (500 mg, 2.6 mmol), 5ꢀbromoꢀ1,3ꢀdifluoroꢀ2ꢀmethoxybenzene (864 mg, 3.87 mmol), and
K2CO3 (866 mg, 6.27 mmol) in DMF (10 mL) was degassed with nitrogen several times. Pd(OAc)2 (50
mg, 0.22 mmol) and Pd(PPh3)4 (30 mg, 26 ꢁmol) were added, and the reaction mixture was stirred at
110 °C overnight. After addition of water (50 mL), the mixture was extracted with EtOAc (3 x 30 mL);
the combined organic layers were concentrated in vacuo and purified by chromatography on silica gel to
provide the product as a yellow solid. Yield: 500 mg, 58%. LCꢀMS m/z 334.0 [M+H]+.
3-(3,5-Difluoro-4-methoxyphenyl)imidazo[1,2-b]pyridazine-2-carboxylic acid (16): To a
solution of 15 (500 mg, 1.5 mmol) in EtOH (30 mL) was added a solution of LiOH (75 mg, 3 mmol) in
water (10 mL), and the reaction mixture was stirred at rt for 4 h, whereupon it was concentrated in
vacuo. The residue was diluted with water and acidified to a pH of 4 with hydrochloric acid. After
extraction of the mixture with DCM (3 x 30 mL), the combined organic layers were concentrated under
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reduced pressure to afford the product as a yellow solid, which was used in the next step without
additional purification.
1
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3
4
5
6
7
Azetidin-1-yl[3-(3,5-difluoro-4-hydroxyphenyl)imidazo[1,2-b]pyridazin-2-yl]methanone (17):
8
9
To a solution of 1 (400 mg, 1.16 mmol) in dry DCM (20 mL) was added BBr3 at 0 °C. The resulting
mixture was stirred at rt for 16 h. The reaction was then quenched with water (50 mL) and extracted
with DCM (3 x 30 mL). The organic phase was concentrated and purified via reversedꢀphase HPLC
(Column: Phenomenex Gemini C18, 10 ꢁm; Mobile phase A: 0.03% NH4OH in water (v/v); Mobile
phase B: 0.03% NH4OH in acetonitrile (v/v); Gradient: 0% to 10% B) to afford the product as a yellow
10
11
12
13
14
15
16
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18
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solid (103.8 mg, 27%). LCꢀMS m/z 330.9 [M+H]+. 1H NMR (400 MHz, DMSOꢀ
d6) δ 8.56 (dd, J = 4.0,
1.5 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.24 (dd, J = 8.8, 4.8 Hz, 1H), 7.15 (br s, 2H), 4.19 (br s, 2H),
4.02 (t, J = 7.8 Hz, 2H), 2.13–2.27 (m, 2H).
Ethyl 3-bromoimidazo[1,2-b]pyridazine-2-carboxylate (18): NBS (25.6 g, 144 mmol) was
added to a 0 °C solution of 14 (25.0 g, 131 mmol) in DCM (250 mL). The reaction mixture was allowed
to gradually warm to rt and stir overnight, whereupon the reaction was quenched with 10% aqueous
NaHSO3 solution. The resulting mixture was diluted with additional DCM and washed with saturated
aqueous NaHCO3 solution and with water. The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo. Trituration with tertꢀbutyl methyl ether afforded the product as a mauve solid
(25.4 g). Concentration of the filtrate under reduced pressure was followed by trituration with tertꢀbutyl
methyl ether and hexanes to give a second batch of product (6.46 g). Combined yield: 31.9 g, 90%. LCꢀ
MS m/z 270.0, 272.0 [M+H]+. 1H NMR (400 MHz, CDCl3)
9.3, 1.6 Hz, 1H), 7.22 (dd, J = 9.3, 4.4 Hz, 1H), 4.53 (q, J = 7.1 Hz, 2H), 1.49 (t, J = 7.1 Hz, 3H).
δ 8.55 (dd, J = 4.4, 1.6 Hz, 1H), 8.05 (dd, J
=
Ethyl 3-(4-chlorophenyl)imidazo[1,2-b]pyridazine-2-carboxylate (19): A mixture of 18 (1.08 g,
4.0 mmol), (4ꢀchlorophenyl)boronic acid (686 mg, 4.4 mmol), and CsF (1.824 g, 12 mmol) in
dioxane/H2O (20 mL/2.5 mL) was sparged with N2 for 2 min, then Pd(amphos)Cl2 (142 mg, 0.2 mmol)
o
was added and the reaction was heated at 100 C overnight. The reaction was then filtered through
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Journal of Medicinal Chemistry
Celite and concentrated. The residue was diluted with water (30 mL) and extracted with EtOAc (3 x
130 mL). The organic phases were combined, washed with brine (30 mL) and concentrated in vacuo.
The residue was filtered and further concentrated to remove dioxane. Water (30 mL) was added, and the
residue was extracted with EtOAc (3 x 130 mL). The combined organic phases were washed with brine
(30 mL) and then concentrated. The residue was purified by silica gel chromatography (Gradient: 10%
to 90% EtOAc in petroleum ether) to afford the product as a white solid. Yield: 1.0 g, 83%. LCꢀMS m/z
302.0 [M+H]+. 1H NMR (400 MHz, CDCl3) δ 8.40 (dd, J = 4.3, 1.8 Hz, 1H), 8.10 (dd, J = 9.3, 1.8 Hz,
1H), 7.64 (d, J = 8.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.18 (dd, J = 9.5, 4.5 Hz, 1H), 4.42 (q, J = 7.0
Hz, 2H), 1.37 (t, J = 7.3 Hz, 3H).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
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3-(4-Chlorophenyl)imidazo[1,2-b]pyridazine-2-carboxylic acid (20): To a solution of 19 (660
mg, 2.19 mmol) in MeOH (10 mL) was added a solution of NaOH (175 mg, 4.38 mmol) in water (5 mL)
at rt. The resulting mixture was stirred at 60 °C for 3 h. The reaction was concentrated and the residue
was diluted with water, then acidified to pH = 4 with aqueous HCl. The aqueous layer was extracted
with DCM (3 x 30 mL), and the combined organic layers were concentrated to give the product as a
yellow solid. Yield: 580 mg, 97%. LCꢀMS m/z 274.1 [M+H]+. 1H NMR (400 MHz, CDCl3) 7.22 (dd,
9.3, 4.4 Hz, 1H) 7.48–7.54 (m, 2H) 7.71–7.77 (m, 2H) 8.13 (d, J = 9.2 Hz, 1H) 8.43 (dd, J = 4.3, 1.6
Hz, 1H).
J
=
Ethyl 1-(2-bromoethyl)-1H-pyrrolo[3,2-b]pyridine-2-carboxylate (22). A solution of ethyl 1Hꢀ
pyrrolo[3,2ꢀ ]pyridineꢀ2ꢀcarboxylate (21) (23 g, 0.12 mol), 2ꢀbromoethanol (37.9 g, 0.303 mol), and
b
PPh3 (79.4 g, 0.303 mol) in THF was cooled to 0 °C. Diisopropyl azodicarboxylate (61.2 g, 0.303 mol)
was added dropꢀwise over 20 min and the resulting mixture was warmed to 25 °C and stirred for 18 h.
After the solvent had been removed under reduced pressure, the residue was diluted with EtOAc (300
mL) and extracted with aqueous HCl (1 M, 3 x 100 mL). The combined aqueous extracts were basified
to pH 8–9 using saturated aqueous Na2CO3 solution, and the resulting mixture was extracted with
EtOAc (3 x 100 mL). The combined organic layers were concentrated in vacuo; silica gel
chromatography (Eluent: 5:1 petroleum ether / EtOAc) provided the product as a white solid. Yield: 25
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