The Discovery of a Novel Phosphodiesterase (PDE) 4B-Preferring

Article Cite This: J. Med. Chem. 2017, 60, 8538-8551

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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,§ Christopher J. Helal,⊥ John M. Humphrey,⊥ Zoe Hughes,# Kyle Kuszpit,§ Erik A. Lachapelle,⊥ John T. Lazzaro,∥ Chewah Lee,⊥ Robert J. Mather,# Nandini C. Patel,† Marc B. Skaddan,§ Simone Sciabola,† Patrick R. Verhoest,† Joseph M. Young,⊥ Kenneth Zasadny,§ and Anabella Villalobos∇ †

Medicine Design, Medicinal Chemistry, Pfizer Inc., Cambridge, Massachusetts 02139, United States Clinical & Translational Imaging, Early Clinical Development, Pfizer Inc., Cambridge, Massachusetts 02139, United States § Bioimaging Center, Pfizer Inc., Groton, Connecticut 06340, United States ∥ Medicine Design, Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Groton, Connecticut 06340, United States ⊥ Medicine Design, Medicinal Chemistry, Pfizer Inc., Groton, Connecticut 06340, United States # Internal Medicine Research Unit, Pfizer Inc., Cambridge, Massachusetts 02139, United States ∇ Medicinal Synthesis Technologies, Pfizer Inc., Groton, Connecticut 06340, United States ‡

S Supporting Information *

ABSTRACT: 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 (Bmax) 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.

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INTRODUCTION

efficient and cost-effective 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 biodistribution for the biological target of interest. Data for both parameters can be generated in vitro using brain tissues or slices and a 3H-radiolabeled ligand. This ligand needs to be potent and selective but not necessarily brain-penetrant, 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

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). As part of our efforts to identify and advance novel PET ligands for our portfolio of CNS programs, we have utilized an © 2017 American Chemical Society

Received: July 19, 2017 Published: September 28, 2017 8538

DOI: 10.1021/acs.jmedchem.7b01050 J. Med. Chem. 2017, 60, 8538−8551

Journal of Medicinal Chemistry

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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 PDE4B-preferring PET radioligand, utilizing the PET ligand discovery strategies described above, to provide accurate target occupancy assessment in the clinic.

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Figure 1. A rational and cost-effective CNS PET ligand discovery process.

RESULTS AND DISCUSSION We initiated our efforts on the discovery of PDE4B-preferring PET ligands by surveying existing project chemical matter, with the goal of identifying an early radiotracer lead to allow for Bmax and biodistribution 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) helices 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 potency and selectivity observed for PDE4A−C in humans and old world monkeys. This unique structure-driven selectivity profile prompted us to focus on 1 as a promising lead for Bmax and biodistribution 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 off-target activities up to 10 μM (see Supporting Information, Table 21).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 × 10−6 cm/s), low P-gp liability (MDR1 BA/AB = 1.17), and low risk of nonspecific 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). Bmax and Biodistribution 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 nonselective PDE4 radiotracer [3H]rolipram. In mice, we observed equivalent Bmax for these two tracers (Figure 3A,B). Interestingly, in NHP we observed a significant

nonspecific binding. With this knowledge, we then proceed to PET-specific structure−activity 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 (>30×), favorable physicochemical properties (CNS PET MPO > 3), high passive transcellular permeability (RRCK Papp AB > 5 × 10−6 cm/s), and low human p-glycoprotein (P-gp) liability (MDR1 BA/AB ≤ 2.5) to optimize brain permeability, and fraction unbound in brain (Fu_b) > 0.05 for low risk of nonspecific binding. Once PET ligand leads with suitable properties are identified, we assess their specific binding ex vivo in rodents using a liquid chromatography−mass spectroscopy/ mass spectroscopy (LC-MS/MS) method with unlabeled compound (“cold tracer” method)6 prior to triggering the more costly PET radiochemistry and non-human primate (NHP) imaging studies. Implementation of these steps enables a rational and cost-effective approach to brain-penetrant 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 (PDE4A−D), among which PDE4A, 4B, and 4D are widely expressed in brain.7 PDE4 inhibition has been associated with beneficial pharmacological outcomes in preclinical models of antiinflammatory, procognitive, and antipsychotic effects, and pan-PDE4 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 pan-PDE4 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 brain-penetrant PDE4B-preferring inhibitors for the treatment of CNS disorders, selective versus PDE4D, to potentially

Figure 2. Profile of an early tritiated radiotracer lead 1. 8539



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Scheme 1a

aimed at clinical applications, the Bmax value determined by [3H]1 (62.07 fmol/mg protein, ∼3.1 nM based on an 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. PET Ligand Design. Based on the above Bmax determination, it became clear that additional improvement in potency would be necessary for an effective PET ligand (Bmax/Kd ≥ 10). Our initial SAR effort focused on improving PDE4B potency while maintaining high selectivity over PDE4D. Matched-pair analysis of existing analogs in the imidazopyridazine series revealed an encouraging SAR trend, as illustrated by compounds 2 and 3 (Figure 4). Compound 2, bearing an Nmethyl group at the amide side chain, showed moderate PDE4B potency (IC50 of 429 nM). A substantial improvement in PDE4B potency (35-fold increase) was observed in its corresponding N-H amide analog 3. Importantly, this improvement in potency did not come at the expense of PDE4D selectivity (336×). 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 70° to the imidazopyridazine core, while the N-H amide in 3 preferred to adopt a coplanar conformation (highest probability density at 0°). Further examination of detailed atom interactions with the protein residues indicated that the N-methyl analog 2 suffered a significant drop in hydrogen-bond interaction with tyrosine 274, compared with the N-H analog 3. This 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. 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 subnanomolar range. Furthermore, favorable selectivity versus PDE4D (>59×) was maintained. The predicted binding mode of 4 in the active site of the long-form PDE4B structure (PDB code: 4X0F) is illustrated in Figure 5. The 4-chlorophenyl 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. Encouraged by the favorable potency and selectivity profile of 4, we next carried out PET-specific SAR efforts varying the

a

Reagents and conditions: (a) ethyl 3-bromo-2-oxopropanoate, EtOH, 78 °C, 20%; (b) 5-bromo-1,3-difluoro-2-methoxybenzene, 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 °C to rt, 27%; (f) CT3I, K2CO3, DMF, rt, 20 mCi, 73.6 Ci/mmol specific activity.

Figure 3. Saturation binding study in homogenated mouse frontal cortex brain tissue using (A) nonselective PDE4 radiotracer [3H]rolipram or (B) PDE4B-preferring radiotracer [3H]1; Saturation binding study in NHP homogenated frontal cortex using (C) nonselective PDE4 radiotracer [3H]rolipram or (D) PDE4B-preferring radiotracer [3H]1.

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 species-specific isoform selectivity profile of 1 (only selective over PDE4D in humans and old world monkeys). For development of a PET ligand 8540



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Figure 4. Design of highly potent novel tricyclic pyrrolopyridine PDE4B-preferring inhibitor 4: (a) a schematic depiction of ligand atom−protein 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 y-axis of the chart.

within the preferred PET property space (cFu_b > 0.05). In addition, all analogs displayed good passive permeability (RRCK Papp AB > 5) and low P-gp liability (MDR1 BA/AB < 2.5) consistent with our criteria. In terms of potency, when R is phenyl, electron-withdrawing substituents appeared to be preferred. For example, replacement of p-Cl (4) with a pmethoxy (5) led to a moderate potency loss (3) and calculated fraction unbound in brain 8541



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Table 1. PET-Specific SAR with Variations at the Pendant R Group

a

IC50 values obtained from a human recombinant full-length PDE4B1 scintillation proximity assay; values represent the geometric mean of at least three experiments. bIC50 values obtained from a human recombinant full-length 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 ref 5. dRRCK cells with low transporter activity were isolated from Mardin−Darby canine kidney cells and were used to estimate passive permeability. eRatio from the MS-based quantification of Papp,A→B and Papp,B→A of the test compound (1 μM) across contiguous monolayers from MDR1-transfected MDCK cells. fIn silico calculated fraction unbound in brain.

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. Chemistry. Our synthesis of radiotracer lead 1, illustrated in Scheme 1, began with the condensation/cyclization of pyridazin-3-amine (13) with ethyl 3-bromo-2-oxopropanoate

over PDE4D (>36×) with moderate selectivity for PDE4C (>17×) and PDE4A (>5×). Importantly, 8 demonstrated minimal off-target activities in broad-spectrum selectivity panels, with only weak micromolar activities at PDE10 (IC50 = 2290 nM), PDE5A (IC50 = 4640 nM), and GABAA (Ki = 3850 nM). In terms of properties, 8 exhibited high CNS PET MPO score (4.0), high passive permeability (RRCK Papp AB = 20.6 × 10−6 cm/s), low P-gp 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 8542



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using N-methylcyclopropylamine and cyclopropylamine, respectively. 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 4−12. LC-MS/MS “Cold Tracer” Study in Rodents. Prior to the NHP PET imaging study, we explored a LC-MS/MS based “cold tracer” method to assess the specific binding of 8 in vivo. Considering the unique species-dependent PDE4B selectivity profile of our chemical series (selective over PDE4D in higher species but not in rodents), we chose 129/B6 PDE4D knockout (KO) mouse as a higher-species 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 (99%, with chemical purities >93%. Oxygen-18 enriched (97%) water was purchased from Huayi Isotopes (cat. no. WT-98-5), Taiyo Nippon Sanso Corp. (cat. no. F030027) or Sigma-Aldrich (cat. no. 329878). Radiochemistry was performed on a GE TracerLab FXFN synthesis module. [18F]8 was purified by high-performance liquid chromatography (HPLC) available on the GE TracerLab FXFN module, consisting of a Sykam S-1021 pump, a Knauer K-2001 UV detector (λ = 254 nm) in series with a Berthold β+-flow detector, on a Phenomenex Luna

Phenyl Hexyl column (cat. no. 00G-4257-N0, 10 mm × 250 mm, 4 μm) at 6 mL/min with 30% v/v acetonitrile/0.05 M aqueous ammonium acetate as the mobile phase. Quality control analysis of [18F]8 was performed on an Agilent 1100 using a Phenomenex PolarRP column (4.6 mm × 150 mm, 4 μm) and 45% acetonitrile/water (1.0 mL/min) as the mobile phase for radiochemical and chemical purity. The identity of the labeled compound was confirmed by coinjection of authentic unlabeled 8 on HPLC. The specific activity was determined by injection of an aliquot of the final solution with known radioactivity on the analytical HPLC system described above. The area of the UV peak measured at 254 nm corresponding to the carrier product was measured and compared to a standard curve relating mass to UV absorbance. Radioactivity was measured with a Capintec CRC15 PET dose calibrator. NHP PET Imaging. All procedures performed on animals in this study were in accordance with established guidelines and regulations and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee. Pfizer animal care facilities that supported this work are fully accredited by AAALAC International. Two male cynomolgus monkeys (8.2 and 8.0 kg) from the animal housing facility located at Pfizer Groton laboratories were evaluated using PET imaging with [18F]8. Each monkey was scanned twice, once at baseline and once at blocking study with a selective PDE4B-preferring inhibitor.15 For the blocking study, the selective PDE4B-preferring inhibitor was dosed subcutaneously at 0.5 mg/kg at 4 h prior to the PET scan. Both monkeys were surgically outfitted with arterial ports (femoral artery access to descending aorta) for blood sampling and fasted overnight prior to PET imaging. In preparation for PET scanning, monkeys were anesthetized with ketamine (10 mg/kg) and glycopyrrolate (0.01 mg/kg), intubated, and maintained under isoflurane anesthesia (1.5−2.0% in oxygen) for the duration of the preparation and scan procedure. An intravenous catheter was placed into the saphenous vein for tracer injection. The head of the monkey was fixed in the center field of view in a Siemens Focus 220 microPET scanner (Siemens Medical). Before radiotracer injection, an 8 min transmission scan was acquired for attenuation and scatter correction using a 57Co point source. PET scans were acquired for 180 min following a 3 min infusion of [18F]8 by syringe pump (1 mL/min), followed by flush at the same rate for 3 min. Arterial blood was collected at 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 60, 90, 120, 150, and 180 min post-tracer-infusion after accounting for IV dead volume, for measurement of the whole blood and plasma radioactivity. Samples from 5, 15, 30, 60, 90, 120, 150, and 180 min were analyzed by radio HPLC for parent and metabolite fractions. PET images were reconstructed using OSEM3D-MAP (fastMAP, Siemens Medical) with decay, randoms, attenuation, and scatter correction. PET images were co-registered to the baseline PET scan of the same monkey subject in which the brain regions of interest (ROIs) were previously defined. ROIs were defined for frontal, temporal, parietal, occipital and anterior cingulate cortex, putamen, caudate, thallamus, cerebellum, and hippocampus. Time−activity curves were generated for the 120 or 180 min PET scans. Tracer equilibrium volume of distribution, VT, was calculated with the Ichise MA1 multilinear method19 using the PMOD software (PMOD 2.85, PMOD Technologies). Target occupancy was estimated in blocking studies using the Lassen plot method.20 On a Lassen plot, the difference between VT values of baseline and drug pretreatment conditions for each brain region were plotted against their respective baseline VT values. A linear fit was then calculated based on all points on the Lassen plot, with each point presenting one brain region. The slope of the fitted line corresponded to the target occupancy, and the x-intercept was the nondisplaceable volume of distribution.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01050. 8549



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H and 13C NMR spectra for compound 8 and the nitro precursor 28, with peak assignments, selectivity profile of compounds 1 and 8 in CEREP and PDE selectivity panels, neuroPK of compound 8 in male Wistar-Han rats, protocols for the preparation of recombinant full-length human PDE4A3, 4B1, 4C1 and 4D3, in vitro autoradiography of [3H]1 in mice brain, and PDE selectivity profile of the PDE 4B-preferring inhibitor used as the blocking compound in in vitro autoradiography and NHP PET imaging experiments (PDF) Molecular formula strings (CSV)

D.; Liebsack, C.; Skovronsky, D. M.; Sabbagh, M. N. PET imaging of amyloid with Florbetapir F-18 and PET imaging of dopamine degeneration with 18F-AV-133 (florbenazine) in patients with Alzheimer’s disease and Lewy body disorders. BMC Neurol. 2014, 14, 79. (3) Zhang, L.; Villalobos, A. Strategies to facilitate the discovery of novel CNS PET ligands. EJNMMI Radiopharm. Chem. 2017, 1, 13. (4) Patel, S.; Gibson, R. In vivo site-directed radiotracers: a minireview. Nucl. Med. Biol. 2008, 35, 805−815. (5) Zhang, L.; Villalobos, A.; Beck, E. M.; Bocan, T.; Chappie, T. A.; Chen, L.; Grimwood, S.; Heck, S. D.; Helal, C. J.; Hou, X.; Humphrey, J. M.; Lu, J.; Skaddan, M. B.; McCarthy, T. J.; Verhoest, P. R.; Wager, T. T.; Zasadny, K. Design and selection parameters to accelerate the discovery of novel central nervous system positron emission tomography (PET) ligands and their application in the development of a novel phosphodiesterase 2A PET ligand. J. Med. Chem. 2013, 56, 4568−4579. (6) (a) Chernet, E.; Martin, L. J.; Li, D.; Need, A. B.; Barth, V. N.; Rash, K. S.; Phebus, L. A. Use of LC/MS to assess brain tracer distribution in preclinical, in vivo receptor occupancy studies: Dopamine D2, serotonin 2A and NK-1 receptors as examples. Life Sci. 2005, 78, 340−346. For additional applications, please see: (b) Pike, V. W.; Rash, K. S.; Chen, Z.; Pedregal, C.; Statnick, M. A.; Kimura, Y.; Hong, J.; Zoghbi, S. S.; Fujita, M.; Toledo, M. A.; Diaz, N.; Gackenheimer, S. L.; Tauscher, J. T.; Barth, V. N.; Innis, R. B. Synthesis and evaluation of radioligands for imaging brain nociceptin/ orphanin FQ peptide (NOP) receptors with positron emission tomography. J. Med. Chem. 2011, 54, 2687−2700. (c) Mitch, C. H.; Quimby, S. J.; Diaz, N.; Pedregal, C.; de la Torre, M. G.; Jimenez, A.; Shi, Q.; Canada, E. J.; Kahl, S. D.; Statnick, M. A.; McKinzie, D. L.; Benesh, D. R.; Rash, K. S.; Barth, V. N. Discovery of aminobenzyloxyarylamides as κ opioid receptor selective antagonists: Application to preclinical development of a κ opioid receptor antagonist receptor occupancy tracer. J. Med. Chem. 2011, 54, 8000−8012. (7) Pérez-Torres, S.; Miró, X.; Palacios, J. M.; Cortés, R.; Puigdoménech, P.; Mengod, G. Phosphodiesterase type 4 isozymes expression in human brain examined by in situ hybridization histochemistry and [3H]rolipam binding autoradiography: Comparison with monkey and rat brain. J. Chem. Neuroanat. 2000, 20, 349− 374. (8) Spina, D. PDE4 inhibitors: current status. Br. J. Pharmacol. 2008, 155, 308−315. (9) Robichaud, A.; Stamatiou, P. B.; Jin, S.-L. C.; Lachance, N.; MacDonald, D.; Laliberté, F.; Liu, S.; Huang, Z.; Conti, M.; Chan, C.C. Deletion of phosphodiesterase 4D in mice shortens α2adrenoceptor-mediated anesthesia, a behavioral correlate of emesis. J. Clin. Invest. 2002, 110, 1045−1052. (10) Darout, E.; Menhaji-Klotz, E.; Chappie, T. A. PDE4: Recent Medicinal Chemistry Strategies to Mitigate Adverse Effects. In Phosphodiesterases and Their Inhibitors; Liras, S.; Bell, A. S., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2014; pp 45−64. (11) DaSilva, J. N.; Lourenco, C. M.; Meyer, J. H.; Hussey, D.; Potter, W. Z.; Houle, S. Imaging cAMP-specific phosphodiesterase-4 in human brain with R-[11C]rolipram and positron emission tomography. Eur. J. Nucl. Med. Mol. Imaging 2002, 29, 1680−1683. (12) Chappie, T. A.; Verhoest, P. R.; Patel, N. C.; Hayward, M. M.; Helal, C. J.; Sciabola, S.; Lachapelle, E. A.; Young, J. M. Preparation of Imidazopyridazine Compounds as Inhibitors of PDE4 Isozymes. WO 2016020786, Feb 11, 2016. (13) The detailed selectivity profile of compound 1 in the CEREP selectivity panel and in a PDE selectivity panel can be found in Supporting Information. (14) For in vitro autoradiography study results, please see Supporting Information. (15) Chappie, T. A.; Verhoest, P. R.; Patel, N. C.; Hayward, M. M.; Helal, C. H.; Sciabola, S.; Wager, T. T.; Lachapelle, E. A.; Young, J. M. Pyrazolopyrimidine Compounds. WO 2016012896 A1, Jan 28, 2016.

AUTHOR INFORMATION

Corresponding Author

*Phone: +1 617 395 0640. E-mail: Lei.Zhang3@pfizer.com. ORCID

Lei Zhang: 0000-0001-8118-1402 John M. Humphrey: 0000-0001-8753-9479 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Pfizer safety pharmacology group for providing the PDE selectivity data, the Pfizer ADME technology group for generating the in vitro PK data and developing the in silico RRCK, MDR1 BA/AB, and brain fractions unbound models, BioDuro Inc. for performing the neuroPK study, Covance Inc. for performing the LC-MS/MS cold tracer study, and Dr. Katherine E. Brighty for proofreading. The authors also thank Dr. Timothy McCarthy and Dr. Rikki Waterhouse for helpful discussions and suggestions.

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ABBREVIATIONS USED B/P, brain to plasma ratio; Bmax, receptor expression; cAMP, adenosine 3′,5′-cyclic monophosphate; fs, femtosecond; Fu_b, fraction unbound in brain; GI, gastrointestinal; IFD, Induced Fit Docking; KO, knockout; MD, molecular dynamic; MPO, multiparameter optimization; NHP, non-human primate; Papp, apparent permeability coefficient; PDE, phosphodiesterase; PET, positron emission tomography; P-gp, p-glycoprotein; ps, picosecond; ROI, regions of interest; RRCK, Ralph-Russ canine kidney; SNAr, nucleophilic aromatic substitution; SPA, scintillation proximity assay; TAC, time−activity curve; UCR, upstream conserved region; VT, volume of distribution

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REFERENCES

(1) (a) Brooks, D. J. Positron emission tomography and singlephoton emission computed tomography in central nervous system drug development. NeuroRx 2005, 2, 226−236. (b) Wong, D. F.; Tauscher, J.; Gründer, G. The role of imaging in proof of concept for CNS drug discovery and development. Neuropsychopharmacology 2009, 34, 187−203. (c) Zhang, L.; Villalobos, A. Imaging Techniques for Central Nervous System (CNS) Drug Discovery. In Blood-Brain Barrier in Drug Discovery; Li, D., Kerns, E. H., Eds.; John Wiley & Sons: Hoboken, NJ, 2015; pp 365−384. (2) For recent examples of PET imaging application in patient endophenotyping, please see (a) Villemagne, V. L.; Okamura, N.; Pejoska, S.; Drago, J.; Mulligan, R. S.; Chételat, G.; O’Keefe, G.; Jones, G.; Kung, H. F.; Pontecorvo, M.; Masters, C. L.; Skovronsky, D. M.; Rowe, C. C. Differential diagnosis in Alzheimer’s disease and dementia with Lewy bodies via VMAT2 and amyloid imaging. Neurodegener. Dis. 2012, 10, 161−165. (b) Siderowf, A.; Pontecorvo, M. J.; Shill, H. A.; Mintun, M. A.; Arora, A.; Joshi, A. D.; Lu, M.; Adler, C. H.; Galasko, 8550



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The PDE selectivity profile of the blocking compound can be found in Supporting Information. (16) Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J. Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. W.; Cerutti, D.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. A. OPLS3: a force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 2016, 12, 281−296. (17) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A smooth particle mesh Ewald method. J. Chem. Phys. 1995, 103, 8577−8593. (18) Humphreys, D. D.; Friesner, R. A.; Berne, B. J. A multiple-timestep molecular dynamics algorithm for macromolecules. J. Phys. Chem. 1994, 98, 6885−6892. (19) Ichise, M.; Toyama, H.; Innis, R. B.; Carson, R. E. Strategies to improve neuroreceptor parameter estimation by linear regression analysis. J. Cereb. Blood Flow Metab. 2002, 22, 1271−1281. (20) Lassen, N. A.; Bartenstein, P. A.; Lammertsma, A. A.; Prevett, M. C.; Turton, D. R.; Luthra, S. K.; Osman, S.; Bloomfield, P. M.; Jones, T.; Patsalos, P. N.; O’Connell, M. T.; Duncan, J. S.; Andersen, J. V. Benzodiazepine receptor quantification in vivo in humans using [11C]flumazenil and PET: application of the steady-state principle. J. Cereb. Blood Flow Metab. 1995, 15, 152−165.

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NOTE ADDED AFTER ASAP PUBLICATION After this paper was published ASAP October 9, 2017, authors Christopher J. Helal and Chewah Lee were added to the author list. The corrected version was reposted October 13, 2017.

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The Discovery of a Novel Phosphodiesterase (PDE) 4B-Preferring

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