Basmisanil, a highly selective GABAA-α5 negative allosteric modulator: preclinical pharmacology and demonstration of functional target engagement in man.

GABAA-α5 subunit-containing receptors have been shown to play a key modulatory role in cognition and represent a promising drug target for cognitive dysfunction, as well as other disorders. Here we report on the preclinical and early clinical profile of a novel GABAA-α5 selective negative allosteric modulator (NAM), basmisanil, which progressed into Phase II trials for intellectual disability in Down syndrome and cognitive impairment associated with schizophrenia. Preclinical pharmacology studies showed that basmisanil is the most selective GABAA-α5 receptor NAM described so far. Basmisanil bound to recombinant human GABAA-α5 receptors with 5 nM affinity and more than 90-fold selectivity versus α1, α2, and α3 subunit-containing receptors. Moreover, basmisanil inhibited GABA-induced currents at GABAA-α5 yet had little or no effect at the other receptor subtypes. An in vivo occupancy study in rats showed dose-dependent target engagement and was utilized to establish the plasma exposure to receptor occupancy relationship. At estimated receptor occupancies between 30 and 65% basmisanil attenuated diazepam-induced spatial learning impairment in rats (Morris water maze), improved executive function in non-human primates (object retrieval), without showing anxiogenic or proconvulsant effects in rats. During the Phase I open-label studies, basmisanil showed good safety and tolerability in healthy volunteers at maximum GABAA-α5 receptor occupancy as confirmed by PET analysis with the tracer [11C]-Ro 15-4513. An exploratory EEG study provided evidence for functional activity of basmisanil in human brain. Therefore, these preclinical and early clinical studies show that basmisanil has an ideal profile to investigate potential clinical benefits of GABAA-α5 receptor negative modulation.

Preclinical Evaluation of 18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 as Novel PET Radiotracers for Imaging Tau Aggregates in Alzheimer Disease.

Tau aggregates and amyloid-ß (Aß) plaques are key histopathologic features in Alzheimer disease (AD) and are considered targets for therapeutic intervention as well as biomarkers for diagnostic in vivo imaging agents. This article describes the preclinical in vitro and in vivo characterization of 3 novel compounds-RO6958948, RO6931643, and RO6924963-that bind specifically to tau aggregates and have the potential to become PET tracers for future human use. Methods: RO6958948, RO6931643, and RO6924963 were identified as high-affinity competitors at the 3H-T808 binding site on native tau aggregates in human late-stage AD brain tissue. Binding of tritiated compounds to brain tissue sections of AD patients and healthy controls was analyzed by macro- and microautoradiography and by costaining of tau aggregates and Aß plaques on the same tissue section using specific antibodies. All 3 tracer candidates were radiolabeled with a PET nuclide and tested in vivo in tau-naïve baboons to assess brain uptake, distribution, clearance, and metabolism. Results:3H-RO6958948, 3H-RO6931643, and 3H-RO6924963 bound with high affinity and specificity to tau aggregates, clearly lacking affinity for concomitant Aß plaques in human AD Braak V tissue sections. The specificity of all 3 radioligands for tau aggregates was supported, first, by binding patterns in AD sections comparable to the tau-specific radioligand 3H-T808; second, by very low nonspecific binding in brain tissue devoid of tau pathology, excluding significant radioligand binding to any other central nervous system target; and third, by macroscopic and microscopic colocalization and quantitative correlation of radioligand binding and tau antibody staining on the same tissue section. RO6958948, RO6931643, and RO6924963 were successfully radiolabeled with a PET nuclide at high specific activity, radiochemical purity, and yield. After intravenous administration of 18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 to baboons, PET scans indicated good brain entry, rapid washout, and a favorable metabolism pattern. Conclusion:18F-RO6958948, 11C-RO6931643, and 11C-RO6924963 are promising PET tracers for visualization of tau aggregates in AD. Head-to-head comparison and validation of these tracer candidates in AD patients and healthy controls will be reported in due course.

Identification of Three Novel Radiotracers for Imaging Aggregated Tau in Alzheimer's Disease with Positron Emission Tomography.

Aggregates of tau and beta amyloid (Aß) plaques constitute the histopathological hallmarks of Alzheimer's disease and are prominent targets for novel therapeutics as well as for biomarkers for diagnostic in vivo imaging. In recent years much attention has been devoted to the discovery and development of new PET tracers to image tau aggregates in the living human brain. Access to a selective PET tracer to image and quantify tau aggregates represents a unique tool to support the development of any novel therapeutic agent targeting pathological forms of tau. The objective of the study described herein was to identify such a novel radiotracer. As a result of this work, we discovered three novel PET tracers (2-(4-[11C]methoxyphenyl)imidazo[1,2-a]pyridin-7-amine 7 ([11C]RO6924963), N-[11C]methyl-2-(3-methylphenyl)imidazo[1,2-a]pyrimidin-7-amine 8 ([11C]RO6931643), and [18F]2-(6-fluoropyridin-3-yl)pyrrolo[2,3-b:4,5-c']dipyridine 9 ([18F]RO6958948)) with high affinity for tau neurofibrillary tangles, excellent selectivity against Aß plaques, and appropriate pharmacokinetic and metabolic properties in mice and non-human primates.

Direct photocatalytic fluorination of benzylic C-H bonds with N-fluorobenzenesulfonimide.

The late-stage fluorination of common synthetic building blocks and drug leads is an appealing reaction for medicinal chemistry. In particular, fluorination of benzylic C-H bonds provides a means to attenuate drug metabolism at this metabolically labile position. Here we report two complimentary strategies for the direct fluorination of benzylic C-H bonds using N-fluorobenzenesulfonimide and either a decatungstate photocatalyst or AIBN-initiation.

Adventures in drug-like chemistry space: from oxetanes to spiroazetidines and beyond!

Recently we have documented research efforts aimed at new classes of oxetanes as well as spiroheteroalicyclic ring systems (which we have termed 'Compact Modules') designed to expand the palette of tailored module scaffolds available to medicinal chemists, which constitute an important role for synthetic chemistry in the drug discovery process. An essential component for this process is to provide access to specific molecular topologies with functional group diversity, essential for generating leads that discriminate among biological targets, therefore promoting selectivity and enhancing the safety profile of the final clinical candidates.

Mapping the binding pocket of a novel, high-affinity, slow dissociating tachykinin NK3 receptor antagonist: biochemical and electrophysiological characterization.

The NK3 receptor is a GPCR that is prominently expressed in limbic areas of the brain, many of which have been implicated in schizophrenia. Phase II clinical trials in schizophrenia with two selective NK3 antagonists (osanetant and talnetant) have demonstrated significant improvement in positive symptoms. The objective of this study was to characterize the properties of a novel dual NK2/NK3 antagonist, RO5328673. [(3)H]RO5328673 bound to a single saturable site on hNK2, hNK3 and gpNK3 with high-affinity. RO5328673 acted as an insurmountable antagonist at both human and guinea-pig NK3 receptors in the [(3)H]IP accumulation assay. In binding kinetic analyses, [(3)H]RO5328673 had fast association and dissociation rates at hNK2 while it had a fast association rate and a remarkably slow dissociation rate at gp and hNK3. In electrophysiological recordings of gp SNpc, RO5328673 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurons with an insurmountable mechanism of action. RO5328673 exhibited in-vivo activity in gerbils, robustly reversing the senktide-induced locomotor activity. The TM2 residue gpNK3-A114(2.58) (threonine in all other species) was identified as the critical residue for the RO5328673's slower dissociation kinetics and stronger insurmountable mode of antagonism in the guinea-pig as compared to hNK3-T139(2.58). Using site-directed mutagenesis, [(3)H]RO5328673 binding and rhodopsin-based modeling, the important molecular determinants of the RO5328673-binding pocket of hNK3 were determined. A comparison of the RO5328673-binding pocket with that of osanetant showed that two antagonists have similar contact sides on hNK3 binding crevice except for three mutations V95L(1.42), Y247W(5.38), V255I(5.46), which behaved differently between interacting modes of two antagonists in hNK3.

Conformationally restricted pyrrolidines by intramolecular [2+2] photocycloaddition reactions.

Intramolecular [2+2] photocycloaddition reactions of diversely substituted N-Boc protected 4-(allylaminomethyl)-2(5H)-furanones resulted in rigid products (53-75%) with three spatially defined positions for further functionalisation.

Bis(arylvinyl)pyrazines, -pyrimidines, and -pyridazines as imaging agents for tau fibrils and ß-amyloid plaques in Alzheimer's disease models.

The in vivo diagnosis of Alzheimer's disease (AD) is of high socioeconomic interest and remains a demanding field of research. The biopathological hallmarks of the disease are extracellular plaques consisting of aggregated ß-amyloid peptides (Aß) and tau protein derived intracellular tangles. Here we report the synthesis and evaluation of fluorescent pyrazine, pyrimidine,and pyridazine derivatives in vitro and in vivo aiming at a tau-based diagnosis of AD. The probes were pre-evaluated on human brain tissue by fluorescence microscopy and were found to label all known disease-related alterations at high contrast and specificity. To quantify the binding affinity, a new thiazine red displacement assay was developed and selected candidates were toxicologically profiled. The application in transgenic mouse models demonstrated bioavailability and brain permeability for one compound. In the course of histological testing, we discovered an AD-related deposition of tau aggregates in the Bowman's glands of the olfactory epithelium, which holds potential for an endoscopic diagnosis of AD in the olfactory system.

Expanding the azaspiro[3.3]heptane family: synthesis of novel highly functionalized building blocks.

The preparation of versatile azaspiro[3.3]heptanes carrying multiple exit vectors is disclosed. Expedient synthetic routes enable the straightforward access to these novel modules that are expected to have significance in drug discovery and design.

Synthesis of novel angular spirocyclic azetidines.

The syntheses of a variety of novel angular azaspiro[3.3]heptanes are reported. gem-Difluoro and gem-dimethyl variants of the angular 1,6-diazaspiro[3.3]heptane module were prepared in high yields using efficient sequences. Additionally, a practical one-pot synthesis of 5-oxo-2-azaspiro[3.3]heptanes and subsequent conversions into functionalized derivatives are described. The methods reported are amenable to the synthesis of these building blocks for drug discovery as members of a library or individually on a preparative scale.

Rational design of novel pyrrolidine derivatives as orally active neurokinin-3 receptor antagonists.

The rational design of a novel series of pyrrolidine derivatives as neurokinin-3 receptor antagonists is reported starting from a selective neurokinin-1 receptor antagonist. Typical representatives in this series showed in vivo efficacy after oral administration in a NK3 mediated functional assay. This series of NK3 antagonists shows promise to deliver a novel antipsychotic.

Synthesis and structural analysis of a new class of azaspiro[3.3]heptanes as building blocks for medicinal chemistry.

Straightforward access toward previously unreported substituted, heterocyclic spiro[3.3]heptanes is disclosed. These spirocyclic systems may be considered as alternatives to 1,3-heteroatom-substituted cyclohexanes, which are otherwise insufficiently stable to allow their use in drug discovery. Conformational details are discussed on the basis of X-ray crystallographic structures.

Imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepines as potent and highly selective GABAA alpha5 inverse agonists with potential for the treatment of cognitive dysfunction.

In a search for GABAA alpha5 ligands that combine high subtype binding selectivity with a marked inverse agonism imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepines were identified as a promising class. A short tandem reaction allowed rapid access to this chemical series, thereby facilitating rapid SAR generation which guided the optimization process. Two compounds (10e and 11f) were found to be active in an in vivo paradigm for cognitive improvement.

The discovery and unique pharmacological profile of RO4938581 and RO4882224 as potent and selective GABAA alpha5 inverse agonists for the treatment of cognitive dysfunction.

Lead optimisation of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine class led to the identification of two clinical leads [RO4882224 (11) and RO4938581 (44)] functioning as novel potent and selective GABAA alpha5 inverse agonists. The unique pharmacological profiles and optimal pharmacokinetic profiles resulted in in vivo activity in selected cognition models.

Discovery of the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine scaffold as a novel, potent and selective GABA(A) alpha5 inverse agonist series.

Through iterative design cycles we have discovered a number of novel new classes where the imidazo[1,5-a][1,2,4]-triazolo[1,5-d][1,4]benzodiazepine was deemed the most promising GABA(A) alpha5 inverse agonist class with potential for cognitive enhancement. This class combines a modest subtype binding selectivity with inverse agonism and has the most favourable molecular properties for further lead optimisation towards a central nervous system (CNS) acting medicine.

RO4938581, a novel cognitive enhancer acting at GABAA alpha5 subunit-containing receptors.

RATIONALE: GABAA alpha5 subunit-containing receptors are primarily expressed in the hippocampus and their role in learning and memory has been demonstrated recently by both genetic and pharmacological approaches. OBJECTIVES: The objective of the study is to evaluate the cognitive effects of a novel GABAA alpha5 receptor inverse agonist, RO4938581 in rats and monkeys. MATERIALS AND METHODS: The in vitro profile was determined using radioligand binding and electrophysiological assays for the GABAA alpha1, alpha2, alpha3, and alpha5 receptors. Long-term potentiation (LTP) was performed in mouse hippocampal slices. Cognitive effects were assessed in rats in the delayed match to position (DMTP) task and the Morris water maze. In monkeys, the object retrieval task was used. Pro-convulsant and anxiogenic potentials were evaluated in mice and rats. In vivo receptor occupancy was determined using [3H]-RO0154513. RESULTS: RO4938581 is a potent inverse agonist at the GABAA alpha5 receptor, with both binding and functional selectivity, enhancing hippocampal LTP. RO4938581 reversed scopolamine-induced working memory impairment in the DMTP task (0.3-1 mg/kg p.o.) and diazepam-induced spatial learning impairment (1-10 mg/kg p.o.). RO4938581 improved executive function in monkeys (3-10 mg/kg p.o.). Importantly, RO4938581 showed no anxiogenic and pro-convulsive potential. RO4938581 dose-dependently bound to GABAA alpha5 receptors and approximately 30% receptor occupancy was sufficient to produce enhanced cognition in the rat. CONCLUSIONS: The data further support the potential of GABAA alpha5 receptors as a target for cognition-enhancing drugs. The dual binding and functional selectivity offers an ideal profile for cognition-enhancing effects without the unwanted side effects associated with activity at other GABAA receptor subtypes.

Total synthesis of (-)-reidispongiolide A, an actin-targeting macrolide isolated from the marine sponge Reidispongia coerulea.

A stereocontrolled total synthesis of the microfilament-destabilizing cytotoxic macrolide (-)-reidispongiolide A, isolated from the New Caledonian marine sponge Reidispongia coerulea, is described. This synthesis utilizes a convergent aldol-based strategy to construct the 26-membered macrolactone, followed by the late-stage coupling of a derived aldehyde with an N-vinylformamide-containing ketone subunit to install the full side chain. Two alternative routes were examined for the introduction of the 2E,4E-dienoate region, and a complex Mukaiyama aldol coupling was used to connect the northern and southern hemispheres to install the C13 stereocenter. This constitutes the first chemical synthesis of any member of the reidispongiolide/sphinxolide family of marine macrolides and unequivocally establishes the relative and absolute configuration.

Synthesis of antimicrofilament marine macrolides: synthesis and configurational assignment of a C5-C16 degradation fragment of reidispongiolide A.

Reidispongiolide A is a representative member of the sphinxolide/reidispongiolide group of cytotoxic 26-membered macrolides of marine origin. By interacting with actin in the cell cytoskeleton, the reidispongiolides and sphinxolides are potent microfilament destabilizing agents that represent a promising mechanism of action for developing novel anticancer drugs. An aldol-based synthesis of a library of diastereomers of C(8)-C(16) and C(5)-C(16) fragments and detailed NMR comparison with a reported degradation fragment enabled a configurational assignment for a major part of the reidispongiolide macrocyclic core, thus setting a solid foundation for ongoing synthetic efforts.

Toward the synthesis of reidispongiolide a: stereocontrolled synthesis of the C17-C22 and C23-C35 degradation fragments.

[structure: see text] By relying on the asymmetric aldol reactions of chiral ketones, a highly stereocontrolled synthesis of each of the C(17)-C(22) and C(23)-C(35) degradation fragments of reidispongiolide A has been achieved. This permits a configurational assignment of the complete C(17)-C(36) region of this antimitotic macrolide, along with providing advanced intermediates for a projected total synthesis.