New P2X3 receptor antagonists. Part 1: Discovery and optimization of tricyclic compounds.

Purinergic P2X3 receptors are trimeric ligand-gated ion channels whose antagonism is an appealing yet challenging and not fully validated drug development idea. With the aim of identification of an orally active, potent human P2X3 receptor antagonist compound that can penetrate the central nervous system, the compound collection of Gedeon Richter was screened. A hit series of tricyclic compounds was subjected to a rapid, two-step optimization process focusing on increasing potency, improving metabolic stability and CNS penetrability. Attempts resulted in compound 65, a potential tool compound for testing P2X3 inhibitory effects in vivo.

New P2X3 receptor antagonists. Part 2: Identification and SAR of quinazolinones.

Numerous potent P2X3 antagonists have been discovered and the therapeutic potential of P2X3 antagonism already comprises proof-of-concept data obtained in clinical trials with the most advanced compound. We have lately reported the discovery and optimization of thia-triaza-tricycle compounds with potent P2X3 antagonistic properties. This Letter describes the SAR of a back-up series containing a 4-oxo-quinazoline central ring. The discovery of the highly potent compounds 51 is presented.

4-Aryl-3-arylsulfonyl-quinolines as negative allosteric modulators of metabotropic GluR5 receptors: From HTS hit to development candidate.

High throughput screening of our corporate compound library followed by hit-to-lead development resulted in a 4-aryl-3-arylsulfonyl-quinoline derivative lead (2) with mGluR5 negative allosteric modulator activity. During the lead optimization process, our objective was to improve affinity and metabolic stability. Modifications at the three targeted regions of the lead structure resulted in compounds with nanomolar affinity and acceptable metabolic stability. One of the most promising compounds (3), showing excellent in vivo efficacy, was selected for preclinical development and subsequent phase I clinical studies.

The influence of 5-HT(2A) activity on a 5-HT(2C) specific in vivo assay used for early identification of multiple acting SERT and 5-HT(2C) receptor ligands.

As a result of our exploratory programme aimed at elaborating dually acting compounds towards the serotonin (5-HT) transporter (SERT) and the 5-HT2C receptor a novel series of 3-amino-1-phenylpropoxy substituted diphenylureas was identified. From that collection two promising compounds (2 and 3) exhibiting highest 5-HT2C receptor affinity strongly inhibited the 5-HT2C receptor agonist 1-(3-chlorophenyl)piperazine (mCPP) induced hypomotility in mice. In further pursuance of that objective (2-aminoethyl)(benzyl)sulfamoyl diphenylureas and diphenylpiperazines have also been elaborated. Herein we report the synthesis of potent multiple-acting compounds from this new class. However, when two optimized representatives (6 and 14) possessing the desired in vitro profile were tested neither reduced the motor activity of mCPP treated animals. Comparative albeit limited in vitro structure-activity relationship (SAR) analysis and detailed in vivo studies are discussed and explanation for their intricate behaviour is proposed.

Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: Lead optimization.

An HTS campaign of our corporate compound library, and hit-to lead development resulted in thieno[2,3-b]pyridine derivative leads with mGluR5 negative allosteric modulator effects. During the lead optimization process, our objective was to improve affinity and metabolic stability. Modification of the first two targeted regions resulted in compounds with nanomolar affinity, then optimal substitution of the third region improved metabolic stability. One of the most promising compounds showed excellent in vivo efficacy and is a potential development candidate.

Cell-based and virtual fragment screening for adrenergic α2C receptor agonists.

Fragment-based drug discovery has emerged as an alternative to conventional lead identification and optimization strategies generally supported by biophysical detection techniques. Membrane targets like G protein-coupled receptors (GPCRs), however, offer challenges in lack of generic immobilization or stabilization methods for the dynamic, membrane-bound supramolecular complexes. Also modeling of different functional states of GPCRs proved to be a challenging task. Here we report a functional cell-based high concentration screening campaign for the identification of adrenergic α2C receptor agonists compared with the virtual screening of the same ligand set against an active-like homology model of the α2C receptor. The conventional calcium mobilization-based assay identified active fragments with a similar incidence to several other reported fragment screens on GPCRs. 16 out of 3071 screened fragments turned out as specific ligands of α2C, two of which were identified by virtual screening as well and several of the hits possessed surprisingly high affinity and ligand efficiency. Our results indicate that in vitro biological assays can be utilized in the fragment hit identification process for GPCR targets.

Thieno[2,3-b]pyridines as negative allosteric modulators of metabotropic GluR5 receptors: hit-to-lead optimization.

An HTS campaign of our corporate compound library resulted in thieno[2,3-b]pyridines derivative hits with mGluR5 negative allosteric modulator effects. During the hit-to-lead development our objective was to improve affinity, and to keep the ligand efficiency values at an acceptable level. After different modifications of the linker resulted in a 2-sulfonyl-thieno[2,3-b]pyridines derivative, which fulfilled the lead criteria.

Optimization of Novel α7 Nicotinic Acetylcholine Receptor Positive Allosteric Modulators and the Discovery of a Preclinical Development Candidate Molecule (RGH-560).

During optimization of a previously identified lead compound, attempts were made to optimize the reactive indole structural element, the suboptimal metabolic stability, as well as the low kinetic solubility. It was concluded that the indole was important for in vitro activity. With the aim of further improvements, more thorough modifications were also carried out. As a result, a new chemotype (the azetidinespirochromone family) was identified, which proved to be 1 order of magnitude less lipophilic retaining the same high level of in vitro potency as the lead series itself, however, with improved metabolic stability and kinetic solubility. Compound 53 showed the most balanced physicochemical and pharmacological profile with significant in vivo efficacy in the scopolamine-induced amnesia test. Based on these promising results, cognitive enhancement through the positive modulation of α7 nAChRs appears to be a viable approach. Compound 53 was selected to be a preclinical development candidate (as RGH-560).

Quinolinyl- and phenantridinyl-acetamides as bradykinin B1 receptor antagonists.

A new series of quinolinyl- and phenantridinyl-acetamides were synthesizer and evaluated against bradykinin B1 receptor. In vitro metabolic stability data were reported for the key compounds.The analgesic effect of compound 20 from the phenantridine series was proved in-vivo.

Fragment-Based Optimization of Dihydropyrazino-Benzimidazolones as Metabotropic Glutamate Receptor-2 Positive Allosteric Modulators against Migraine.

Our previous scaffold-hopping attempts resulted in dihydropyrazino-benzimidazoles as metabotropic glutamate receptor-2 (mGluR2) positive allosteric modulators (PAMs) with suboptimal drug-like profiles. Here, we report an alternative fragment-based optimization strategy applied on the new dihydropyrazino-benzimidazolone scaffold. Analyzing published high-affinity mGluR2 PAMs, we used a pharmacophore-guided approach to identify suitable growing vectors and optimize the scaffold in these directions. This strategy resulted in a new fragment like lead (34) with improved druglike properties that were translated to sufficient pharmacokinetics and validated proof-of-concept studies in migraine. Gratifyingly, compound 34 showed reasonable activity in the partial infraorbital nerve ligation, a migraine disease model that might open this indication for mGluR2 PAMs.

HTS-based discovery and optimization of novel positive allosteric modulators of the α7 nicotinic acetylcholine receptor.

HTS campaign of the corporate compound collection resulted in a novel, oxalic acid diamide scaffold of α7 nACh receptor positive allosteric modulators. During the hit expansion, several derivatives, such as 4, 11, 17 demonstrated not only high in vitro potency, but also in vivo efficacy in the mouse place recognition test. The advanced hit molecule 11 was further optimized by the elimination of the putatively mutagenic aromatic-amine building block that resulted in a novel, aminomethylindole compound family. The most balanced physico-chemical and pharmacological profile was found in case of compound 55. Docking study revealed an intersubunit binding site to be the most probable for our compounds. 55 demonstrated favorable cognitive enhancing profile not only in scopolamine-induced amnesia (place recognition test in mice) but also in natural forgetting (novel object recognition test in rats). Compound 55 was, furthermore, active in a cognitive paradigm of high translational value, namely in the rat touch screen visual discrimination test. Therefore, 55 was selected as a lead compound for further optimization. Based on the obtained favorable results, the invented aminomethylindole cluster may provide a viable approach for cognitive enhancement through positive allosteric modulation of α7 nAChRs.

Discovery of novel positive allosteric modulators of the α7 nicotinic acetylcholine receptor: Scaffold hopping approach.

The paper focuses on the scaffold hopping-based discovery and characterization of novel nicotinic alpha 7 receptor positive modulator (α7 nAChR PAM) ligands around the reference molecule (A-867744). First, substantial efforts were carried out to assess the importance of the various pharmacophoric elements on the in vitro potency (SAR evaluation) by chemical modifications. Subsequently, several new derivatives with versatile, heteroaromatic central cores were synthesized and characterized. A promising, pyrazole-containing new chemotype with good physicochemical and in vitro parameters was identified. Retrospective analysis based on homology modeling was also carried out. Besides its favorable in vitro characteristics, the most advanced derivative 69 also showed in vivo efficacy in a rodent model of cognition (scopolamine-induced amnesia in the mouse place recognition test) and acceptable pharmacokinetic properties. Based on the in vivo data, the resulting molecule with advanced drug-like characteristics has the possibility to improve cognitive performance in a biologically relevant dose range, further strengthening the view of the supportive role of α7 nACh receptors in the cognitive processes.

Carbamoyloximes as novel non-competitive mGlu5 receptor antagonists.

Hit-to-lead optimization of a HTS hit led to new carbamoyloxime derivatives. After identification of an advanced hit (8d) the CYP enzyme inhibitory activity of this class of compounds was successfully eliminated. Systematic exploration of different parts of the advanced hit led us to some promising lead compounds with mGluR5 affinities comparable to that of MPEP.

Hit-to-lead optimization of disubstituted oxadiazoles and tetrazoles as mGluR5 NAMs.

Here we report the discovery and early SAR of a series of mGluR5 negative allosteric modulators (NAMs). Starting from a moderately active HTS hit we synthesized 3,5-disubstituted-oxadiazoles and tetrazoles as mGluR5 NAMs. Based on the analysis of ligand efficiency and lipophilic efficiency metrics we identified a promising lead candidate as a starting point for further optimization.

Discovery of dihydropyrazino-benzimidazole derivatives as metabotropic glutamate receptor-2 (mGluR2) positive allosteric modulators (PAMs).

A scaffold hopping strategy converted the known 1-[(1-methyl-1H-imidazol-2-yl)methyl]-4-phenylpiperidine core (1 and 2) by cyclization to a fused [6 + 5+6] membered heterocyclic mGluR2 PAM scaffold. Pharmacophore guided structure-activity relationship (SAR) studies resulted in a series of potent and metabolically stable mGluR2 PAMs. A representative optimized compound (95) having the most balanced profile, demonstrated efficacy in the PCP-induced hyper-locomotion model in mice that revealed the new chemotype being a promising PAM lead targeting mGluR2 receptors and providing support for further translational studies.

Fragment Based Optimization of Metabotropic Glutamate Receptor 2 (mGluR2) Positive Allosteric Modulators in the Absence of Structural Information.

Metabotropic glutamate receptor 2 (mGluR2) positive allosteric modulators (PAMs) have been implicated as potential pharmacotherapy for psychiatric conditions. Screening our corporate compound deck, we identified a benzotriazole fragment (4) that was rapidly optimized to a potent and metabolically stable early lead (16). The highly lipophilic character of 16, together with its limited solubility, permeability, and high protein binding, however, did not allow reaching of the proof of concept in vivo. Since further attempts on the optimization of druglike properties were unsuccessful, the original hit 4 has been revisited and was optimized following the principles of fragment based drug discovery (FBDD). Lacking structural information on the receptor-ligand complex, we implemented a group efficiency (GE) based strategy and identified a new fragment like lead (60) with more balanced profile. Significant improvement achieved on the druglike properties nominated the compound for in vivo proof of concept studies that revealed the chemotype being a promising PAM lead targeting mGluR2 receptors.

Selective NR1/2B N-methyl-D-aspartate receptor antagonists among indole-2-carboxamides and benzimidazole-2-carboxamides.

(4-Benzylpiperidine-1-yl)-(6-hydroxy-1H-indole-2-yl)-methanone (6a) derived from (E)-1-(4-benzylpiperidin-1-yl)-3-(4-hydroxy-phenyl)-propenone (5) was identified as a potent NR2B subunit-selective antagonist of the NMDA receptor. To establish the structure-activity relationship (SAR) and to attempt the improvement of the ADME properties of the lead, a series of compounds were prepared and tested. Several derivatives showed low nanomolar activity both in the binding and in the functional assay. In a formalin-induced hyperalgesia model in mice, 6a and (4-benzylpiperidine-1-yl)-[5(6)-hydroxy-1H-benzimidazol-2-yl]-methanone (60a) were as active as besonprodil (2) after oral administration. A CoMSIA model was developed based on binding data of a series of indole- and benzimidazole-2-carboxamides.

Discovery and Preclinical Characterization of 3-((4-(4-Chlorophenyl)-7-fluoroquinoline-3-yl)sulfonyl)benzonitrile, a Novel Non-acetylenic Metabotropic Glutamate Receptor 5 (mGluR5) Negative Allosteric Modulator for Psychiatric Indications.

Negative allosteric modulators (NAM) of metabotropic glutamate receptor 5 (mGluR5) have been implicated as a potential pharmacotherapy for a number of psychiatric diseases, including anxiety and depression. Most of the mGluR5 NAM clinical candidates can be characterized by the central acetylenic moiety that connects the terminal pharmacophores. Identification of a sulfoquinoline hit via high throughput screening (HTS) followed by optimization provided a 4-phenyl-3-aryl-sulfoquinoline lead compound with the minimal pharmacophore. Optimization of the core and aryl appendages was performed by scanning and matrix libraries synthesized by the multiple parallel synthesis approach. Biological evaluation of matrix libraries provided a number of potent, metabolically stable, and in vivo active compounds. One of these compounds, 25 showed high efficacy and safety in preclinical in vivo models; this allowed its nomination as a novel, nonacetylenic mGluR5 NAM clinical candidate. Compound 25 was advanced to first-in-man trials for the treatment of psychiatric conditions.

Functional characterization of sodium channel blockers by membrane potential measurements in cerebellar neurons: prediction of compound preference for the open/inactivated state.

Voltage-gated sodium channel (VGSC) blockers are widely used in the therapy, but most currently available blockers have suboptimal profile. However, discovery of new drug candidates has been hampered by the lack of appropriate in vitro assays. We established a fluorometric, plate reader-based membrane potential assay for testing the inhibitory potency of various VGSC blocking drugs, using primary cultures of cerebellar neurons, and veratridine, as activator of VGSCs. Since inhibition was strongly dependent on the depolarizing effect of veratridine, the EC(80) value of veratridine was determined on each experimental day, and this concentration was used for drug testing. This strict control on agonist effect seems to improve the reliability of the dose-inhibition measurements with antagonists. Veratridine responses could be completely inhibited by tetrodotoxin (TTX, IC(50)=17 nM), consistent with the exclusive expression of TTX-sensitive VGSCs. A variety of compounds known to block sodium channels inhibited veratridine-induced membrane depolarization concentration-dependently. Furthermore, inhibitory potencies of drugs strongly depended on whether their administration preceded or followed veratridine application. Potency of lamotrigine, carbamazepine, phenytoin and lidocaine was approximately 10-fold higher when applied after a steady-state depolarization had been achieved by a supramaximal veratridine dose, compared with those from a different protocol, where cells were preincubated with the antagonists prior to veratridine application. On the contrary, there was only relatively small difference between the IC(50) values of GBR 12909 obtained from the two different protocols (0.51 microM versus 1.23 microM). In contrast with most sodium channel blockers, this compound lacks binding preference to inactivated channels. We suggest that comparison of the results obtained with a particular blocker in the pretreatment and post-treatment schedules may be suitable for drawing conclusions regarding the state-dependency of its action. Thus, relevant information can be obtained about the potential therapeutic utility of different drugs by applying non-electrophysiological methods.

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors.

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.