Discovery of Novel Steroid-Based Histamine H3 Receptor Antagonists/Inverse Agonists.

Steroid-based histamine H3 receptor antagonists (d-homoazasteroids) were designed by combining distinct structural elements of HTS hit molecules. They were characterized, and several of them displayed remarkably high affinity for H3 receptors with antagonist/inverse agonist features. Especially, the 17a-aza-d-homolactam chemotype demonstrated excellent H3R activity together with significant in vivo H3 antagonism. Optimization of the chemotype was initiated with special emphasis on the elimination of the hERG and muscarinic affinity. Additionally, ligand-based SAR considerations and molecular docking studies were performed to predict binding modes of the molecules. The most promising compounds (XXI, XXVIII, and XX) showed practically no muscarinic and hERG affinity. They showed antagonist/inverse agonist property in the in vitro functional tests that was apparent in the rat in vivo dipsogenia test. They were considerably stable in human and rat liver microsomes and provided significant in vivo potency in the place recognition and novel object recognition cognitive paradigms.

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).

Effects of Chemical Structures Interacting with Amine Oxidases on Glucose, Lipid and Hydrogen Peroxide Handling by Human Adipocytes.

Benzylamine is a natural molecule present in food and edible plants, capable of activating hexose uptake and inhibiting lipolysis in human fat cells. These effects are dependent on its oxidation by amine oxidases present in adipocytes, and on the subsequent hydrogen peroxide production, known to exhibit insulin-like actions. Virtually, other substrates interacting with such hydrogen peroxide-releasing enzymes potentially can modulate lipid accumulation in adipose tissue. Inhibition of such enzymes has also been reported to influence lipid deposition. We have therefore studied in human adipocytes the lipolytic and lipogenic activities of pharmacological entities designed to interact with amine oxidases highly expressed in this cell type: the semicarbazide-sensitive amine oxidase (SSAO also known as PrAO or VAP-1) and the monoamine oxidases (MAO). The results showed that SZV-2016 and SZV-2017 behaved as better substrates than benzylamine, releasing hydrogen peroxide once oxidized, and reproduced or even exceeded its insulin-like metabolic effects in fat cells. Additionally, several novel SSAO inhibitors, such as SZV-2007 and SZV-1398, have been evidenced and shown to inhibit benzylamine metabolic actions. Taken as a whole, our findings reinforce the list of molecules that influence the regulation of triacylglycerol assembly/breakdown, at least in vitro in human adipocytes. The novel compounds deserve deeper investigation of their mechanisms of interaction with SSAO or MAO, and constitute potential candidates for therapeutic use in obesity and diabetes.

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.

Discovery of novel steroidal histamine H3 receptor antagonists/inverse agonists. Part 2. Versatile steroidal carboxamide derivatives.

To further proceed with our previous work, novel steroid-based histamine H3 receptor antagonists were identified and characterized. Using an 'amine-to-amide' modification strategy at position 17, in vitro and in vivo potent monoamino steroid derivatives were found during the lead optimization. Usage of the non-basic amide moiety resulted in beneficial effects both in activity and selectivity. The 15α-carboxamido derivative 10 was not only highly active at human and rat H3 receptors, but also showed negligible activity at rat muscarinic receptors. Furthermore, it proved to be considerably stable in human and rat microsomes and showed significant in vivo potency in the pharmacodynamic rat dipsogenia test and in the water-labyrinth cognitive model. Based on all of these considerations, compound 10 was appointed to be a preclinical candidate.

Discovery of novel steroidal histamine H3 receptor antagonists/inverse agonists.

Emerging from an HTS campaign, novel steroid-based histamine H3 receptor antagonists were identified and characterized. Structural moieties of the hit compounds were combined to improve binding affinities which resulted in compound 4 as lead molecule. During the lead optimization due to the versatile modifications of diamino steroid derivatives, several in vitro potent compounds with subnanomolar binding affinities to histamine H3 receptors were found. The unfavorable binding to rat muscarinic receptors was successfully reduced by tuning the basicity. Compound 20 showed significant in vivo activity in the rat dipsogenia model and could serve as a pharmacological tool in the future.

Sarcosine-Based Glycine Transporter Type-1 (GlyT-1) Inhibitors Containing Pyridazine Moiety: A Further Search for Drugs with Potential to Influence Schizophrenia Negative Symptoms.

We have synthesized a novel series of N-substituted sarcosines, analogues of NFPS (N-[3-(biphenyl-4- yloxy)-3-(4-fluorophenyl)propyl]-N-methylglycine), as type-1 glycine transporter (GlyT-1) inhibitors. Several compounds incorporated a diazine ring inhibited recombinant hGlyT-1b expressed permanently in CHO cells and GlyT-1 in rat brain synaptosomal preparations. A structure-activity relationship for the newly synthesized compounds was obtained and discussed on the ground of their GlyT-1 inhibitory potencies. Replacement of the biphenyl-4-yloxy moiety in NFPS with a 5-pyridazinylphenoxy moiety (compounds 3, 4, 5, and 6) or a 2-phenyl-5- pyridazinyloxy moiety (compounds 10, 11, and 12) afforded compounds exhibiting potent inhibition on GlyT-1 activity. The GlyT-1 inhibitory properties of NFPS analogues, in which sarcosine was closed into a ring forming (methylamino)pyridazine-3-(2H)-one, were markedly reduced (compounds 13 and 14). The pyridazine-containing GlyT-1 inhibitors with in vitro GlyT-1 inhibitory potency also enhanced extracellular glycine concentrations in conscious rat striatum as was measured by microdialysis technique. In contrast to NFPS, sarcosine-based pyridazine containing GlyT-1 inhibitors failed to evoke compulsive running behavior whereas they inhibited phencyclidine- induced hypermotility in mice. It is believed that increase of extracellular concentrations of glycine by inhibition of its reuptake may probably influence positively glutamate N-methyl-D-aspartate (NMDA)-type ionotropic receptors in the central nervous system. This may have importance in the treatment of neuropsychiatric disorders associated with hypofunctional NMDA receptor-mediated glutamatergic neurochemical transmission. Thus, impaired NMDA receptor functions have been shown to be involved in the development of the negative symptoms and the cognitive deficit of schizophrenia and the treatment of these symptoms is the possible clinical indication of GlyT-1 inhibitors including those containing pyridazine moiety.

Structure-activity relationship of antiparasitic and cytotoxic indoloquinoline alkaloids, and their tricyclic and bicyclic analogues.

Based on the indoloquinoline alkaloids cryptolepine (1), neocryptolepine (2), isocryptolepine (3) and isoneocryptolepine (4), used as lead compounds for new antimalarial agents, a series of tricyclic and bicyclic analogues, including carbolines, azaindoles, pyrroloquinolines and pyrroloisoquinolines was synthesized and biologically evaluated. None of the bicyclic compounds was significantly active against the chloroquine-resistant strain Plasmodium falciparum K1, in contrast to the tricyclic derivatives. The tricyclic compound 2-methyl-2H-pyrido[3,4-b]indole (9), or 2-methyl-beta-carboline, showed the best in vitro activity, with an IC(50) value of 0.45 microM against P. falciparum K1, without apparent cytotoxicity against L6 cells (SI>1000). However, this compound was not active in the Plasmodium berghei mouse model. Structure-activity relationships are discussed and compared with related naturally occurring compounds.

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers.

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.

Synthesis and receptor-binding examination of 16-hydroxymethyl-3,17-estradiol stereoisomers.

The four 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers were synthesized and tested in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities are lower than 2.0% relative to that of the reference molecule estra-1,3,5(10)-triene-3,17beta-diol. The affinities of the tested compounds for the androgen and progesterone receptors are very low (K(i)> 100 microm and 1 microM, respectively). The prepared 16-hydroxymethylestra-1,3,5(10)-triene-3,17-diol isomers are therefore estrogen receptor-selective molecules.