10A Inhibitors Research Articles

Fragment-assisted hit investigation involving integrated HTS and fragment screening: Application to the identification of phosphodiesterase 10A (PDE10A) inhibitors

Fragment-based drug design (FBDD) relies on direct elaboration of fragment hits and typically requires high resolution structural information to guide optimization. In fragment-assisted drug discovery (FADD), fragments provide information to guide selection and design but do not serve as starting points for elaboration. We describe FADD and high-throughput screening (HTS) campaign strategies conducted in parallel against PDE10A where fragment hit co-crystallography was not available. The fragment screen led to prioritized fragment hits (IC50's ∼500μM), which were used to generate a hypothetical core scaffold. Application of this scaffold as a filter to HTS output afforded a 4μM hit, which, after preparation of a small number of analogs, was elaborated into a 16nM lead. This approach highlights the strength of FADD, as fragment methods were applied despite the absence of co-crystallographical information to efficiently identify a lead compound for further optimization.

Discovery of pyrazolopyrimidine phosphodiesterase 10A inhibitors for the treatment of schizophrenia.

Herein, we present the identification of a novel class of pyrazolopyrimidine phosphodiesterase 10A (PDE10A) inhibitors. Beginning with a lead molecule (1) identified through a fragment-based drug discovery (FBDD) effort, lead optimization was enabled by rational design, X-ray crystallography, metabolic and off-target profiling, and fragment scaffold-hopping. We highlight the discovery of PyP-1, a potent, highly selective, and orally bioavailable pyrazolopyrimidine inhibitor of PDE10A. PyP-1 exhibits sub-nanomolar potency (PDE10A Ki=0.23nM), excellent pharmacokinetic (PK) and physicochemical properties, and a clean off-target profile. It displays dose-dependent efficacy in numerous pharmacodynamic (PD) assays that measure potential for anti-psychotic activity and cognitive improvement. PyP-1 also has a clean preclinical profile with respect to cataleptic potential in rats, prolactin secretion, and weight gain, common adverse events associated with currently marketed therapeutics. Further, PyP-1 displays in vivo preclinical target engagement as measured by PET enzyme occupancy in concert with [(11)C]MK-8193, a novel PDE10A PET tracer.

Biochemical and behavioral effects of PDE10A inhibitors: Relationship to target site occupancy

Phosphodiesterase 10A (PDE10A) inhibitors increase the functionality of striatal medium spiny neurons and produce antipsychotic-like effects in rodents by blocking PDE10A mediated hydrolysis of cAMP and/or cGMP. In the current study, we characterized a radiolabeled PDE10A inhibitor, [3H]BMS-843496, and developed an ex vivo PDE10 binding autoradiographic assay to explore the relationship between PDE10 binding site occupancy and the observed biochemical and behavioral effects of PDE10 inhibitors in mice. [3H]BMS-843496 is a potent PDE10A inhibitor with a binding affinity (KD) of 0.15 nM and a functional selectivity of >100-fold over other PDE subtypes tested. Specific [3H]BMS-843496 binding sites were dominant in the basal ganglia, especially the striatum, with low to moderate binding in the cortical and hippocampal areas, of the mouse and monkey brain. Systemic administration of PDE10 inhibitors produced a dose- and plasma/brain concentration-dependent increase in PDE10A occupancy measured in the striatum. PDE10A occupancy was positively correlated with striatal pCREB expression levels. PDE10A occupancy was also correlated with antipsychotic-like effects measured using the conditioned avoidance response model; a minimum of ∼40% occupancy was typically required to achieve efficacy. In contrast, a clear relationship between PDE10A occupancy and catalepsy scores, a potential extrapyramidal symptom readout in rodent, was not evident.

Synthesis of Fluorine-Containing Phosphodiesterase 10A (PDE10A) Inhibitors and the In Vivo Evaluation of F-18 Labeled PDE10A PET Tracers in Rodent and Nonhuman Primate.

A series of fluorine-containing PDE10A inhibitors were designed and synthesized to improve the metabolic stability of [(11)C]MP-10. Twenty of the 22 new analogues had high potency and selectivity for PDE10A: 18a-j, 19d-j, 20a-b, and 21b had IC50 values <5 nM for PDE10A. Seven F-18 labeled compounds [(18)F]18a-e, [(18)F]18g, and [(18)F]20a were radiosynthesized by (18)F-introduction onto the quinoline rather than the pyrazole moiety of the MP-10 pharmacophore and performed in vivo evaluation. Biodistribution studies in rats showed ~2-fold higher activity in the PDE10A-enriched striatum than nontarget brain regions; this ratio increased from 5 to 30 min postinjection, particularly for [(18)F]18a-d and [(18)F]20a. MicroPET studies of [(18)F]18d and [(18)F]20a in nonhuman primates provided clear visualization of striatum with suitable equilibrium kinetics and favorable metabolic stability. These results suggest this strategy may identify a (18)F-labeled PET tracer for quantifying the levels of PDE10A in patients with CNS disorders including Huntington's disease and schizophrenia.

Design and synthesis of a novel 2-oxindole scaffold as a highly potent and brain-penetrant phosphodiesterase 10A inhibitor

Highly potent and brain-penetrant phosphodiesterase 10A (PDE10A) inhibitors based on the 2-oxindole scaffold were designed and synthesized. (2-Oxo-1,3-oxazolidin-3-yl)phenyl derivative 1 showed the high P-glycoprotein (P-gp) efflux (efflux ratio (ER)=6.2) despite the potent PDE10A inhibitory activity (IC50=0.94nM). We performed an optimization study to improve both the P-gp efflux ratio and PDE10A inhibitory activity by utilizing structure-based drug design (SBDD) techniques based on the X-ray crystal structure with PDE10A. Finally, 1-(cyclopropylmethyl)-4-fluoro-5-[5-methoxy-4-oxo-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-1(4H)-yl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-one (19e) was identified with improved P-gp efflux (ER=1.4) and an excellent PDE10A inhibitory activity (IC50=0.080nM). Compound 19e also exhibited satisfactory brain penetration, and suppressed PCP-induced hyperlocomotion with a minimum effective dose of 0.3mg/kg by oral administration in mice.

Discovery and Optimization of a Series of Pyrimidine-Based Phosphodiesterase 10A (PDE10A) Inhibitors through Fragment Screening, Structure-Based Design, and Parallel Synthesis.

Screening of a fragment library for PDE10A inhibitors identified a low molecular weight pyrimidine hit with PDE10A Ki of 8700 nM and LE of 0.59. Initial optimization by catalog followed by iterative parallel synthesis guided by X-ray cocrystal structures resulted in rapid potency improvements with minimal loss of ligand efficiency. Compound 15 h, with PDE10A Ki of 8.2 pM, LE of 0.49, and >5000-fold selectivity over other PDEs, fully attenuates MK-801-induced hyperlocomotor activity after ip dosing.

Phosphodiesterase 10A inhibitor, MP-10 (PF-2545920), produces greater induction of c-Fos in dopamine D2 neurons than in D1 neurons in the neostriatum

Studies described here tested the hypothesis that phosphodiesterase 10A inhibition by a selective antagonist, MP-10, activates the dopamine D2 receptor expressing medium spiny neurons to a greater extent than the D1 receptor expressing neurons. We used regional pattern of c-Fos induction in the neostriatal subregions of rodents and direct assessment of D1-positive and -negative neurons in the DRd1a-tdTomato mice for the purpose. MP-10 (1, 3, 10 or 30 mg/kg, PO) dose-dependently increased c-Fos immunopositive nuclei in all regions of neostriatum. However, the effect was statistically greater in the dorsolateral striatum, a region known to be activated preferentially by the D2 antagonism, than the D1-activated dorsomedial striatum. The D2 antagonist, haloperidol (0.3, 1, or 3 mg/kg, PO) produced an identical, regional pattern of c-Fos induction favoring the dorsolateral striatum of the rat. In contrast, the D1 agonist, SKF82958 (0.5, 1, or 2 mg/kg, PO), induced greater expression of c-Fos in the dorsomedial striatum. The C57Bl/6 mouse also showed regionally preferential c-Fos activation by haloperidol (2 mg/kg, IP) and SKF82858 (3 mg/kg, IP). In the Drd1a-tdTomato mice, MP-10 (3 or 10 mg/kg, IP) increased c-Fos immunoreactivity in both types of neurons, the induction was greater in the D1-negative neurons. Taken together, both the regional pattern of c-Fos induction in the striatal sub-regions and the greater induction of c-Fos in the D1-negative neurons indicate that PDE10A inhibition produces a small but significantly greater activation of the D2-containing striatopallidal pathway.

A comparative study of binding affinities for 6,7-dimethoxy-4-pyrrolidylquinazolines as phosphodiesterase 10A inhibitors using the linear interaction energy method

The linear interaction energy (LIE) method was used to estimate the free energies of binding for a set of 27 pyrrolidylquinazoline derivatives as phosphodiesterase 10A inhibitors. Twenty-six X-ray crystal structures of phosphodiesterase 10A and two sampling methods, minimization and Hybrid Monte Carlo, were used to assess the affinity models based on the linear interaction energies. The best model was obtained based on the parameters α=0.16 and β=0.04, which represent non-polar and polar interactions, respectively, with a root mean square error (RMSE) of 0.42kcal/mol (R2=0.71) and 0.52kcal/mol (R2=0.86) for the training and test sets, respectively. In addition, the applicability domain of the model was investigated. After validation of the models, the best model was subsequently used in a virtual screening process, which resulted in a set of optimized compounds. The models developed in this study could be useful as filter for virtual screening and lead optimization processes for phosphodiesterase 10A drug developments.

BN/CC isosterism in borazaronaphthalenes towards phosphodiesterase 10A (PDE10A) inhibitors

The application of BN/CC isosterism is explored as a method of expanding the scope of core scaffolds in biologically active compounds. The viability of potential drug candidates incorporating BN-heteroaromatic moieties was investigated through the synthesis of BN-substituted analogs to known phosphodiesterase (PDE10A) inhibitors, namely MP10 and a selection of N-methylanilide analogs. These in some cases revealed unexpectedly potent and relatively stable derivatives, providing further support for the potential of BN-incorporation in medicinal chemistry.

Discovery of [¹¹C]MK-8193 as a PET tracer to measure target engagement of phosphodiesterase 10A (PDE10A) inhibitors.

Phosphodiesterase 10A (PDE10A) inhibition has recently been identified as a potential mechanism to treat multiple symptoms that manifest in schizophrenia. In order to facilitate preclinical development and support key proof-of-concept clinical trials of novel PDE10A inhibitors, it is critical to discover positron emission tomography (PET) tracers that enable plasma concentration/PDE10A occupancy relationships to be established across species with structurally diverse PDE10A inhibitors. In this Letter, we describe how a high-throughput screening hit was optimized to provide [(11)C]MK-8193 (8j), a PET tracer that supports the determination of plasma concentration/PDE10A occupancy relationships for structurally diverse series of PDE10A inhibitors in both rat and rhesus monkey.

Pharmacological evaluation of a novel phosphodiesterase 10A inhibitor in models of antipsychotic activity and cognition

In this study, we report the pharmacological effects of a novel PDE10A inhibitor, SEP-39. SEP-39 is a potent (1.0nM) inhibitor of human PDE10A in vitro, with good selectivity (>16000-fold) against other PDEs. In an in vivo occupancy study, the RO50 value was determined to be 0.7mg/kg (p.o.), corresponding to plasma and brain exposures of 28ng/mL and 43ng/g, respectively. Using microdialysis, we show that 3mg/kg (p.o.) SEP-39 significantly increased rat striatal cGMP concentrations. Furthermore, SEP-39 inhibits PCP-induced hyperlocomotion at doses of 1 and 3mg/kg (p.o.) corresponding to 59–86% occupancy. At similar doses in a catalepsy study, the time on the bar was increased but the maximal effect was less than that seen with haloperidol. In an EEG study, 3 and 10mg/kg (p.o.) SEP-39 suppressed REM intensity and increased the latency to REM sleep. We also demonstrate the procognitive effects of SEP-39 in the rat novel object recognition assay. These effects appear to require less PDE10A inhibition than the reversal of PCP-induced hyperlocomotion or EEG effects, as improvements in recognition index were seen at doses of 0.3mg/kg and above. Our data demonstrate that SEP-39 is a potent, orally active PDE10A inhibitor with therapeutic potential in a number of psychiatric indications.

Phosphodiesterase 10A inhibitors: analysis of US/EP patents granted since 2012.

Phosphodiesterases are enzymes that metabolically inactivate the intracellular second messengers 3',5'-cyclic adenosine and guanosine monophosphate contributing to the control of multiple biological processes. Among them, PDE10A has the most restricted distribution with high expression in striatal medium spiny neurons. Dysfunction of this key brain circuit has been associated with different psychiatric and neurodegenerative disorders. The unique role of PDE10A, together with its increased pharmacological characterization, have prompted enormous interest in investigating the potential of inhibitors of this enzyme as potential novel therapeutic agents This article reviews PDE10A related patents issued in the period 2012-2014 in the USA and Europe offering also a perspective on potential avenues for the future clinical development of phosphodiesterase 10A inhibitors.

Addressing phototoxicity observed in a novel series of biaryl derivatives: Discovery of potent, selective and orally active phosphodiesterase 10A inhibitor ASP9436

We synthesized several biaryl derivatives as PDE10A inhibitors to prevent phototoxicity of 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1) and found that the energy difference between the energy-minimized conformation and the coplanar conformation of the biaryl moiety helped facilitate prediction of the phototoxic potential of biaryl compounds. Replacement of the quinoline ring of 1 with N-methyl benzimidazole increased this energy difference and prevented phototoxicity in the 3T3 NRU test. Further optimization identified 1-methyl-5-(1-methyl-3-{[4-(1-methyl-1H-benzimidazol-4-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (38b). Compound 38b exhibited good selectivity against other PDEs, and oral administration of 38b improved visual-recognition memory deficit in mice at doses of 0.001 and 0.003mg/kg in the novel object recognition test. ASP9436 (sesquiphosphate of 38b) may therefore be used for the treatment of schizophrenia with a low risk of phototoxicity.

Characterization of binding and inhibitory properties of TAK-063, a novel phosphodiesterase 10A inhibitor.

Phosphodiesterase 10A (PDE10A) inhibition is a novel and promising approach for the treatment of central nervous system disorders such as schizophrenia and Huntington’s disease. A novel PDE10A inhibitor, TAK-063 [1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)-pyridazin-4(1H)-one] has shown high inhibitory activity and selectivity for human recombinant PDE10A2 in vitro; the half-maximal inhibitory concentration was 0.30 nM, and selectivity over other phosphodiesterases (PDEs) was more than 15000-fold. TAK-063 at 10 µM did not show more than 50% inhibition or stimulation of 91 enzymes or receptors except for PDEs. In vitro autoradiography (ARG) studies using rat brain sections revealed that [3H]TAK-063 selectively accumulated in the caudate putamen (CPu), nucleus accumbens (NAc), globus pallidus, substantia nigra, and striatonigral projection, where PDE10A is highly expressed. This [3H]TAK-063 accumulation was almost entirely blocked by an excess amount of MP-10, a PDE10A selective inhibitor, and the accumulation was not observed in brain slices of Pde10a-knockout mice. In rat brain sections, [3H]TAK-063 bound to a single high-affinity site with mean ± SEM dissociation constants of 7.2 ± 1.2 and 2.6 ± 0.5 nM for the CPu and NAc shell, respectively. Orally administered [14C]TAK-063 selectively accumulated in PDE10A expressing brain regions in an in vivo ARG study in rats. Striatal PDE10A occupancy by TAK-063 in vivo was measured using T-773 as a tracer and a dose of 0.88 mg/kg (p.o.) was calculated to produce 50% occupancy in rats. Translational studies with TAK-063 and other PDE10A inhibitors such as those presented here will help us better understand the pharmacological profile of this class of potential central nervous system drugs.

Identification of a novel orally bioavailable phosphodiesterase 10A (PDE10A) inhibitor with efficacy in animal models of schizophrenia.

We report the continuation of a focused medicinal chemistry program aimed to further optimize a series of imidazo[1,2-a]pyrazines as a novel class of potent and selective phosphodiesterase 10A (PDE10A) inhibitors. In vitro and in vivo pharmacokinetic and pharmacodynamic evaluation allowed the selection of compound 25a for its assessment in preclinical models of psychosis. The evolution of our medicinal chemistry program, structure-activity relationship (SAR) analysis, as well as a detailed pharmacological profile for optimized lead 25a are described.

Synthesis and in vitro characterization of cinnoline and benzimidazole analogues as phosphodiesterase 10A inhibitors

Fifteen cinnoline analogues and six benzimidazole phosphodiesterase 10A (PDE10A) inhibitors were synthesized as potential PET radiopharmaceuticals and their in vitro activity as PDE10A inhibitors was determined. Nine out of twenty-one compounds were potent inhibitors of PDE10A with IC50 values ranging from 1.5 to 18.6nM. Notably, the IC50 values of compounds 26a, 26b, and 33c were 1.52±0.18, 2.86±0.10, and 3.73±0.60nM, respectively; these three compounds also showed high in vitro selectivity (>1000-fold) for PDE10A over PDE 3A/3B, PDE4A/4B. The high potency and selectivity of these three compounds suggests that they could be radiolabeled with PET radionuclides for further evaluation of their in vivo pharmacological behavior and ability to quantify PDE10A in the brain.

In vivo pharmacological characterization of TAK-063, a potent and selective phosphodiesterase 10A inhibitor with antipsychotic-like activity in rodents.

Phosphodiesterase 10A (PDE10A) is a cAMP/cGMP phosphodiesterase highly expressed in medium spiny neurons (MSNs) in the striatum. We evaluated the in vivo pharmacological profile of a potent and selective PDE10A inhibitor, TAK-063 (1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)-pyridazin-4(1H)-one). TAK-063 at 0.3 and 1 mg/kg p.o., increased cAMP and cGMP levels in the rodent striatum and upregulated phosphorylation levels of key substrates of cAMP- and cGMP-dependent protein kinases. TAK-063 at 0.3 and 1 mg/kg p.o., strongly suppressed MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]-induced hyperlocomotion, which is often used as a predictive model for antipsychotic-like activity in rodents. Upregulation of striatal cAMP/cGMP levels and the antipsychotic-like effect of TAK-063 were not attenuated after 15 days of pretreatment with TAK-063 in mice. The potential side effect profile of TAK-063 was assessed in rats using the clinical antipsychotics haloperidol, olanzapine, and aripiprazole as controls. TAK-063 did not affect plasma prolactin or glucose levels at doses up to 3 mg/kg p.o. At 3 mg/kg p.o., TAK-063 elicited a weak cataleptic response compared with haloperidol and olanzapine. Evaluation of pathway-specific markers (substance P mRNA for the direct pathway and enkephalin mRNA for the indirect pathway) revealed that TAK-063 activated both the direct and indirect pathways of MSNs. These findings suggest that TAK-063 represents a promising drug for the treatment of schizophrenia with potential for superior safety and tolerability profiles.

AMG 580: a novel small molecule phosphodiesterase 10A (PDE10A) positron emission tomography tracer.

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington's disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography-tandem mass spectrometry technology. [(3)H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [(18)F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

Synthesis, SAR study, and biological evaluation of novel quinoline derivatives as phosphodiesterase 10A inhibitors with reduced CYP3A4 inhibition

A novel class of phosphodiesterase 10A inhibitors with potent PDE10A inhibitory activity and reduced CYP3A4 inhibition was designed and synthesized starting from 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1). Replacement of pyridine ring of 1 with N-methyl pyridone ring drastically improved CYP3A4 inhibition, and further optimization of these quinoline analogues identified 1-methyl-5-(1-methyl-3-{[4-(quinolin-2-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (42b), which showed potent PDE10A inhibitory activity and a good CYP3A4 inhibition profile. A PET study with 11C-labeled 42b indicated that 42b exhibited good brain penetration and specifically accumulated in the rodent striatum. Further, oral administration of 42b dose-dependently attenuated phencyclidine-induced hyperlocomotion in mice with an ED50 value of 2.0mg/kg and improved visual-recognition memory impairment at 0.1 and 0.3mg/kg in mice novel object recognition test.

Discovery of 1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (TAK-063), a highly potent, selective, and orally active phosphodiesterase 10A (PDE10A) inhibitor.

A novel series of pyridazinone-based phosphodiesterase 10A (PDE10A) inhibitors were synthesized. Our optimization efforts using structure-based drug design (SBDD) techniques on the basis of the X-ray crystal structure of PDE10A in complex with hit compound 1 (IC50 = 23 nM; 110-fold selectivity over other PDEs) led to the identification of 1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one (27h). Compound 27h has potent inhibitory activity (IC50 = 0.30 nM), excellent selectivity (>15000-fold selectivity over other PDEs), and favorable pharmacokinetics, including high brain penetration, in mice. Oral administration of compound 27h to mice elevated striatal 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) levels at 0.3 mg/kg and showed potent suppression of phencyclidine (PCP)-induced hyperlocomotion at a minimum effective dose (MED) of 0.3 mg/kg. Compound 27h (TAK-063) is currently being evaluated in clinical trials for the treatment of schizophrenia.