Discovery of Novel Imidazo[4,5-b]pyridines as Potent and Selective Inhibitors of Phosphodiesterase 10A (PDE10A)

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Inhibition of the striatum-enriched phosphodiesterase PDE10A: a novel approach to the treatment of psychosis

Phosphodiesterase 10A (PDE10A) is a recently identified cyclic nucleotide phosphodiesterase expressed primarily in dopaminoreceptive medium spiny neurons of the striatum. We report that papaverine is a potent, specific inhibitor of PDE10A and use this compound to explore the role of PDE10A in regulating striatal function. Papaverine administration produces an increase in striatal tissue levels of cGMP and an increase in extracellular cAMP measured by microdialysis. These cyclic nucleotide changes are accompanied by increases in the phosphorylation of CREB and ERK, downstream markers of neuronal activation. In rats, papaverine potentiates haloperidol-induced catalepsy, consistent with the hypothesis that inhibition of PDE10A can increase striatal output and prompting a further evaluation of papaverine in models predictive of antipsychotic activity. Papaverine is found to inhibit conditioned avoidance responding in rats and mice and to inhibit PCP- and amphetamine-stimulated locomotor activity in rats. The effects of papaverine on striatal cGMP and CREB and ERK phosphorylation, as well as on conditioned avoidance responding, were absent in PDE10A knockout mice, indicating that the effects of the compound are the result of PDE10A inhibition. These results indicate that PDE10A regulates the activation of striatal medium spiny neurons through effects on cAMP- and cGMP-dependent signaling cascades. Furthermore, the present results demonstrate that papaverine has efficacy in behavioral models predictive of antipsychotic activity. Thus, inhibition of PDE10A may represent a novel approach to the treatment of psychosis.     

Discovery of highly selective and orally available benzimidazole-based phosphodiesterase 10 inhibitors with improved solubility and pharmacokinetic properties for treatment of pulmonary arterial hypertension

Optimization efforts were devoted to discover novel PDE10A inhibitors in order to improve solubility and pharmacokinetics properties for a long-term therapy against pulmonary arterial hypertension (PAH) starting from the previously synthesized inhibitor A. As a result, a potent and highly selective PDE10A inhibitor, 14·3HCl (half maximal inhibitory concentration, IC₅₀ = 2.8 nmol/L and >3500-fold selectivity) exhibiting desirable solubility and metabolic stability with a remarkable bioavailability of 50% was identified with the aid of efficient methods of binding free energy predictions. Animal PAH studies showed that the improvement offered by 14·3HCl [2.5 mg/kg, oral administration (p.o.)] was comparable to tadalafil (5.0 mg/kg, p.o.), verifying the feasibility of PDE10A inhibitors for the anti-PAH treatment. The crystal structure of the PDE10A−14 complex illustrates their binding pattern, which provided a guideline for rational design of highly selective PDE10A inhibitors.     

A concisely automated synthesis of TSPO radiotracer [18F]FDPA based on spirocyclic iodonium ylide method and validation for human use

Fluorine-18 labeled N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ([¹⁸F]FDPA) is a potent and selective radiotracer for positron-emission tomography (PET) imaging of the translocator protein 18 kDa (TSPO). Our previous in vitro and in vivo evaluations have proven that this tracer is promising for further human translation. Our study addresses the need to streamline the automatic synthesis of this radiotracer to make it more accessible for widespread clinical evaluation and application. Here, we successfully demonstrate a one-step radiolabeling of [¹⁸F]FDPA based on a novel spirocyclic iodonium ylide (SCIDY) precursor using tetra-n-butyl ammonium methanesulfonate (TBAOMs), which has demonstrated the highest radiochemical yields and molar activity from readily available [¹⁸F]fluoride ion. The nucleophilic radiofluorination was completed on a GE TRACERlab FX2 N synthesis module, and the formulated [¹⁸F]FDPA was obtained in nondecay corrected (n.d.c) radiochemical yields of 15.6 ± 4.2%, with molar activities of 529.2 ± 22.5 GBq/μmol (14.3 ± 0.6 Ci/μmol) at the end of synthesis (60 minutes, n = 3) and validated for human use. This methodology facilitates efficient synthesis of [¹⁸F]FDPA in a commercially available synthesis module, which would be broadly applicable for routine production and widespread clinical PET imaging studies.     

Development of a series of novel carbon‐11 labeled PDE10A inhibitors

Phosphodiesterase 10A (PDE10A) is a member of the PDE family of enzymes that degrades cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Our aim was to label a series of structurally related PDE10A inhibitors with carbon‐11 and evaluate them as potential positron emission tomography (PET) radioligands for PDE10A using nonhuman primates. The series consisted of seven compounds based on the 3‐(1H‐pyrazol‐5‐yl)pyridazin‐4(1H)‐one backbone. These compounds were selected from the initial larger library based on a number of parameters such as affinity, selectivity for hPDE10A in in vitro tests, lipophilicity, and on the results of multidrug resistance protein 1 (MDR1)‐LLCPK1 and the parallel artificial membrane permeability assays. Seven radioligands (KIT‐1, 3, 5, 6, 7, 9, and 12) were radiolabeled with carbon‐11 employing O‐methylation on the hydroxyl moiety using [¹¹C]methyl triflate. In vivo examination of each radioligand was performed using PET in rhesus monkeys; analysis of radiometabolites in plasma also was conducted using HPLC. All seven radioligands were labeled with high (>90%) incorporation of [¹¹C]methyl triflate into their appropriate precursors and with high specific radioactivity. Carbon‐11 labeled KIT‐5 and KIT‐6 showed high accumulation in the striatum, consistent with the known anatomical distribution of PDE10A in brain, accompanied by fast washout and high specific binding ratio. In particular [¹¹C]KIT‐6, named [¹¹C]T‐773, is a promising PET tool for further examination of PDE10A in human brain.     

Direct radiofluorination of [18F]MH.MZ for 5‐HT2A receptor molecular imaging with PET

Imaging the serotonin 2A neuroreceptor with positron emission tomography has been carried out with [¹¹C]MDL 100907 and [¹⁸F]altanserin for years. Recently, the MDL 100907 analogue [¹⁸F]MH.MZ was developed by combining the increased selectivity profile of MDL 100907 and the favourable radiophysical properties of fluorine‐18. Here, we want to report the synthesis of [¹⁸F]MH.MZ via direct radiofluorination. Unfortunately, the direct radiofluorination did not have any significant benefits over the indirect labelling method. This is mainly because the precursor for the direct labelling approach is not completely stable and slowly decomposes. However, only one HPLC separation is necessary for the direct ¹⁸F‐nucleophilic labelling procedure, and accordingly, automation is easier. Copyright © 2012 John Wiley & Sons, Ltd.     

Use of LC–MS–MS for the rapid,specific and sensitive quality control measurement of carrier in a PET radioligand: [18F]FECNT

In the production of radioligands for imaging low concentrations of target proteins (e.g. receptors or transporters) in human subjects with positron emission tomography, control of specific radioactivity is necessary for efficacy and safety. Such quality control requires a fast method to be available for measuring carrier (non‐radioactive ligand) in each batch of radioligand, preceding its release for administration. Measurement is usually achieved with HPLC equipped with an ultraviolet (UV) absorbance detector. However, this method is not easily applicable to radioligands that have low UV extinction coefficients and are produced at high specific radioactivity, such as [¹⁸F] (2β‐carbomethoxy‐3β‐(4‐chlorophenyl)‐8‐(2‐fluoroethyl)nortropane; [¹⁸F]FECNT). Here we describe a fast, specific and sensitive LC–MS–MS method for measuring carrier in [¹⁸F]FECNT preparations. Small samples of formulated [¹⁸F]FECNT plus an added internal standard (2β‐carbomethoxy‐3β‐(4‐chlorophenyl)‐8‐(n‐propyl)nortropane; INTSTD) are rapidly eluted from a short reverse phase HPLC column into an MS probe. Following electrospray ionization, the molecular ions ([MH]⁺) of FECNT (m/z=326) and INTSTD (m/z=322) are isolated and energized for collision‐induced dissociation. The product ions from FECNT (m/z=294) and INTSTD (m/z=290) are monitored selectively. The calibration curve for MS response is linear for FECNT concentrations in the range 2–20 pg/µl and suitable for reproducibly (RSD 5%) and rapidly (<3 min) measuring low concentrations of carrier in [¹⁸F]FECNT preparations. Copyright © 2005 John Wiley & Sons, Ltd.     

Synthesis of novel WAY 100635 derivatives containing a norbornene group and radiofluorination of [18F]AH1.MZ as a serotonin 5‐HT1A receptor antagonist for molecular imaging

5‐HT_1A receptors are involved in a variety of psychiatric disorders and in vivo molecular imaging of the 5‐HT_1A status represents an important approach to analyze and treat these disorders. We report herein the synthesis of three new fluoroethylated 5‐HT_1A ligands (AH1.MZ, AH2.MZ and AH3.MZ) as arylpiperazine derivatives containing a norbornene group. AH1.MZ (K_i= 4.2 nM) and AH2.MZ (K_i=30 nM) showed reasonable in vitro affinities to the 5‐HT_1A receptor, whereas AH3.MZ appeared to be non‐affine toward the 5‐HT_1A receptor. The receptor profile of AH1.MZ and AH2.MZ showed selectivity within the 5‐HT system. ¹⁸F‐labelling via [¹⁸F]FETos to [¹⁸F]AH1.MZ was carried out in radiochemical yields of >70%. The final formulation of injectable solutions including [¹⁸F]FETos synthon synthesis, radiosynthesis and semi‐preparative high‐performance liquid chromatography (HPLC) separation took no longer than 130 min and provided [¹⁸F]AH1.MZ with a purity of >98% as indicated by analytical HPLC analyses. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis,radiofluorination and first evaluation of (±)‐[18F]MDL 100907 as serotonin 5‐HT2A receptor antagonist for PET

In some psychiatric disorders 5‐HT_2A receptors play an important role. In order to investigate those in vivo there is an increasing interest in obtaining a metabolically stable, subtype selective and high affinity radioligand for receptor binding studies using positron emission tomography (PET). Combining the excellent in vivo properties of [¹¹C]MDL 100907 for PET imaging of 5‐HT_2A receptors and the more suitable half‐life of fluorine‐18, MDL 100907 was radiofluorinated in four steps using 1‐(2‐bromoethyl)‐4‐[¹⁸F]fluorobenzene as a secondary labelling precursor. The complex reaction required an overall reaction time of 140 min and (±)‐[¹⁸F]MDL 100907 was obtained with a specific activity of at least 30 GBq/µmol (EOS) and an overall radiochemical yield of 1–2%. In order to verify its binding to 5‐HT_2A receptors, in vitro rat brain autoradiography was conducted showing the typical distribution of 5‐HT_2A receptors and a very low non‐specific binding of about 6% in frontal cortex, using ketanserin or spiperone for blocking. Thus, [¹⁸F]MDL 100907 appears to be a promising new 5‐HT_2A PET ligand. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis,labelling and first evaluation of [18F]R91150 as a serotonin 5‐HT2A receptor antagonist for PET

In psychiatric disorders such as anxiety, depression and schizophrenia, 5‐HT_2A receptors play an important role. In order to investigate them in vivo there is an increasing interest in selective and high‐affinity radioligands for receptor binding studies using positron emission tomography (PET). Since available radioligands have disadvantages, R91150, which is a selective and high‐affinity ligand for 5‐HT_2A receptors, was labelled with fluorine‐18. This was accomplished in six steps via 4‐[¹⁸F]fluorophenol and 1‐(3‐bromopropoxy)‐4‐[¹⁸F]fluorobenzene within 190 min starting from no‐carrier‐added [¹⁸F]fluoride. The overall radiochemical yield was 3.8±2% and the specific activity was at least 335 GBq/µmol at the end of the synthesis. First ex vivo studies in mice proved the uptake of [¹⁸F]R91150 in the brain. Radiometabolite studies revealed no radiometabolites in the brain, whereas in the plasma at least two could be detected 30 min p.i. Further preclinical studies are encouraged to evaluate the potential of this new 5‐HT_2A ligand as a radiotracer for PET. Copyright © 2008 John Wiley & Sons, Ltd.