Catalyst- and excess reagent recycling in aza-Michael additions.

16α-Azolyl-pregnenolone derivatives were prepared via 2-butyl-1,1,3,3-tetramethylguanidine (n-Bu-TMG) catalysed aza-Michael addition of 16-dehydropregnenolone (16-DHP) carried out in [bmim][BF4]. The application of the guanidine base and the imidazolium ionic liquid made it possible to recycle not only the catalyst/solvent mixture but also the excess of the N-heterocyclic reagent. By the introduction of CO2 at the end of the reaction, both the guanidine base and the unreacted (excess) reagent could be converted into ionic species that remained dissolved in the ionic liquid phase, while the steroid components were extracted with an apolar solvent. After the removal of CO2, the experiment could be repeated by the addition of the steroid substrate and only an equimolar amount of the N-heterocycle. The methodology was successfully applied to a number of N-heterocycles, such as imidazole, pyrazole, 1,2,3- and 1,2,4-triazoles, and benzimidazole. Indazole and indole could also be converted into the corresponding products, but a stronger base had to be used to obtain a recyclable system.

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.

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.

Convergent cross-species pro-cognitive effects of RGH-235, a new potent and selective histamine H3 receptor antagonist/inverse agonist.

The histamine H3 receptor is a favourable target for the treatment of cognitive deficits. Here we report the in vitro and in vivo profile of RGH-235, a new potent, selective, and orally active H3 receptor antagonist/inverse agonist developed by Gedeon Richter Plc. Radioligand binding and functional assays were used for in vitro profiling. Procognitive efficacy was investigated in rodent cognitive tests, in models of attention deficit hyperactive disorder (ADHD) and in cognitive tests of high translational value (rat touch screen visual discrimination test, primate fixed-foreperiod visual reaction time task). Results were supported by pharmacokinetic studies, neurotransmitter release, sleep EEG and dipsogenia. RGH-235 displayed high affinity to H3 receptors (Ki = 3.0-9.2 nM, depending on species), without affinity to H1, H2 or H4 receptors and >100 other targets. RGH-235 was an inverse agonist ([35S] GTPγS binding) and antagonist (pERK1/2 ELISA), showing favourable kinetics, inhibition of the imetit-induced dipsogenia and moderate effects on sleep-wake EEG. RGH-235 stimulated neurotransmitter release both in vitro and in vivo. RGH-235 was active in spontaneously hypertensive rats (SHR), generally considered as a model of ADHD, and revealed a robust pro-cognitive profile both in rodent and primate tests (in 0.3-1 mg/kg) and in models of high translational value (e.g. in a rodent touch screen test and in non-human primates). The multiple and convergent procognitive effects of RGH-235 support the view that beneficial cognitive effects can be linked to antagonism/inverse agonism of H3 receptors.

Estrogens regulate neuroinflammatory genes via estrogen receptors α and ß in the frontal cortex of middle-aged female rats.

BACKGROUND: Estrogens exert anti-inflammatory and neuroprotective effects in the brain mainly via estrogen receptors α (ERα) and ß (ERß). These receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors. This study was aimed at the elucidation of the effects of ERα and ERß agonists on the expression of neuroinflammatory genes in the frontal cortex of aging female rats. METHODS: To identify estrogen-responsive immunity/inflammation genes, we treated middle-aged, ovariectomized rats with 17ß-estradiol (E2), ERα agonist 16α-lactone-estradiol (16α-LE2) and ERß agonist diarylpropionitrile (DPN), or vehicle by Alzet minipump delivery for 29 days. Then we compared the transcriptomes of the frontal cortex of estrogen-deprived versus ER agonist-treated animals using Affymetrix Rat230 2.0 expression arrays and TaqMan-based quantitative real-time PCR. Microarray and PCR data were evaluated by using Bioconductor packages and the RealTime StatMiner software, respectively. RESULTS: Microarray analysis revealed the transcriptional regulation of 21 immunity/inflammation genes by 16α-LE2. The subsequent comparative real-time PCR study analyzed the isotype specific effects of ER agonists on neuroinflammatory genes of primarily glial origin. E2 regulated the expression of sixteen genes, including down-regulation of complement C3 and C4b, Ccl2, Tgfb1, macrophage expressed gene Mpeg1, RT1-Aw2, Cx3cr1, Fcgr2b, Cd11b, Tlr4 and Tlr9, and up-regulation of defensin Np4 and RatNP-3b, IgG-2a, Il6 and ER gene Esr1. Similar to E2, both 16α-LE2 and DPN evoked up-regulation of defensins, IgG-2a and Il6, and down-regulation of C3 and its receptor Cd11b, Ccl2, RT1-Aw2 and Fcgr2b. CONCLUSIONS: These findings provide evidence that E2, 16α-LE2 and DPN modulate the expression of neuroinflammatory genes in the frontal cortex of middle-aged female rats via both ERα and ERß. We propose that ERß is a promising target to suppress regulatory functions of glial cells in the E2-deprived female brain and in various neuroinflammatory diseases.

Ionic liquid-promoted Wagner-Meerwein rearrangement of 16α,17α-epoxyandrostanes and 16α,17α-epoxyestranes.

Ionic liquids 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim](+)[PF(6)](-)) and 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim](+)[BF(4)](-)) were found to promote an unusual Wagner-Meerwein rearrangement of steroidal 16α,17α-epoxides leading to unnatural 13-epi-18-nor-16-one derivatives as the main products. These compounds were isolated in good to excellent yields. 16α-Hydroxy-Δ(13)-18-norsteroids, the results of the usual rearrangement, were obtained as minor components of the reaction mixtures. The ionic liquid [bmim](+)[PF(6)](-) was shown to induce C-ring aromatization of 16α,17α-epoxyestranes due to the formation of HF, the hydrolysis product of [PF(6)](-). Increasing amounts of HF and [PO(2)F(2)](-) were detected by (19)F and (31)P NMR when the ionic liquid was reused. The structures of the steroidal products, 16-oxo-18-nor-13α-steroid derivatives, 16α-hydroxy-Δ(13)-18-norsteroids, and C-aromatic compounds were determined by two-dimensional NMR techniques and high-resolution mass spectrometry (HRMS). The ionic liquids were recirculated efficiently.

A Temperature-Controlled Switch between Fürst-Plattner Rule and Anti-Fürst-Plattner Rule Ring Opening of 2,3-Epoxy-steroids with Various Halide Sources in the Presence of Imidazolium Ionic Liquids.

The ring opening of 2α,3α- and 2ß,3ß-epoxy-5α-androstan-17-one with halide reagents (AlCl3, TMSCl, LiCl, and LiBr) was investigated using imidazolium ionic liquids in the dual role of solvent and catalyst. The application of the ionic liquid was shown to result in an increase in the amount of the unusual diequatorial halohydrins especially at temperatures above 100 °C. With a careful choice of reaction conditions, the latter derivatives could be produced with 43-96% selectivity depending on the nature of the halide ion. Moreover, the usual diaxial products could also be isolated in 70-85% yields by a proper change in the reaction conditions. The reusability of the ionic liquid was demonstrated in both types of reactions. The structures of the products were proved unequivocally by nuclear magnetic resonance (NMR) measurements including two-dimensional (2D) techniques as well as high-resolution mass spectrometry (HRMS). Based on quantum chemical calculations, the effect of the ionic liquid could be explained by the stabilization of the transition state leading to the diequatorial product.

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.

Stereocontrolled synthesis of the four possible 3-methoxy and 3-benzyloxy-16-triazolyl-methyl-estra-17-ol hybrids and their antiproliferative activities.

The four possible isomers of each of 3-methoxy- and 3-benzyloxyestra-1,3,5(10)-trien-17-ols (5-8 and 9-12) were converted through 16-p-tosyloxymethyl- or 16-bromomethyl derivatives into their 3-methoxy- and 3-benzyloxy-16-azidomethylestra(1,3,5(10)-triene derivatives (13-16 and 17-20). The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of these compounds with different terminal alkynes afforded novel 1,4-disubstituted diastereomers (21a-f, 22a-f, 23a-f, 24a-f and 25a-f, 26a-f, 27a-f, 28a-f). The antiproliferative activities of the structurally related triazoles were determined in vitro with the microculture tetrazolium assay on four malignant human cell lines of gynecological origin (Hela, SiHa, MCF-7 and MDA-MB-231).

Facile ring opening of 2,3-epoxy-steroids with aromatic amines in ionic liquids.

Efficient ring opening of steroidal 2,3-epoxides with stoichiometric amount of aromatic amines has been carried out using an ionic liquid ([bmim](+)[BF(4)](-)) both as solvent and catalyst. The reactions were completely regio- and stereoselective in each case. The aminoalcohol products have chair conformations in ring A. The ionic liquid-mediated ring opening can efficiently be carried out with aliphatic amines like morpholine as well.

Facile synthesis of 12-carboxamido-11-spirostenes via palladium-catalyzed carbonylation reactions.

12-Carboxamido- and 12-carboxyl-11-spirostenes were synthesized from the corresponding 12-iodo-11-ene derivative in palladium-catalyzed carbonylation reactions under mild reaction conditions. The synthesis of the iodo-alkene substrate is based on the transformation of the 12-keto derivative (hecogenin) to hydrazone, which was treated with iodine in the presence of a base (1,1,3,3-tetramethyl guanidine). While various 12-carboxamides were synthesized in moderate to high yields by using simple alkyl/arylamines or amino acid methylesters as N-nucleophiles, low yields can be achieved with alcohols as O-nucleophiles. The homogeneous carbonylation reactions tolerate the 3-hydroxy substituent and the spiroacetal moiety.

Synthesis and structure-activity relationships of neuromuscular blocking agents.

The first use of neuromuscular blocking agents (muscle relaxants) in clinical practice (1942) revolutionised the practice of anaesthesia and started the modern era of surgery. Since 1942 introduction of tubocurarine (18) neuromuscular blocking agents have been used routinely to provide skeletal muscle relaxation during surgical procedures allowing access to body cavities without hindrance from voluntary or reflex muscle movement. After the introduction of tubocurarine and the depolarizing suxamethonium chloride (4) (1949) several nondepolarizing steroidal and nonsteroidal neuromuscular blocking agents with different onset time and duration of effect were introduced e.g. gallamine triethiodide (1) (1949), methocurine (2) (1949), alcuronium chloride (3) (1963), pancuronium bromide (9) (1968), vecuronium bromide (11) (1982), pipecuronium bromide (10) (1982), atracurium besylate (5) (1982), doxacurium chloride (6) (1991), mivacurium chloride (8) (1992), rocuronium bromide (12) (1994) cisatracurium besylate (7) (1996), and rapacuronium bromide (13) (2000). SZ 1677 (14) a steroid type nondepolarizing neuromuscular blocking agent under development (preclinical phase). This review article deals with a comprehensive survey of the progress in chemical, pharmacological and, in some respects, of clinical studies of neuromuscular blocking agents used in the clinical practice and under development, including the synthesis, structure elucidation, pharmacological actions, structure activity relationships studies of steroidal and nonsteroidal derivatives.

Detection by HPLC and structural characterization by NMR and MS of a natural deuterium isotopologue of ulipristal acetate.

Herein we discuss the structure elucidation of an unknown peak detected by HPLC in the active pharmaceutical ingredient ulipristal acetate during analytical method development. An extensive chromatographic, MS and NMR spectroscopic study gave the surprising result that the detected component is the natural-abundance mono-deuterium isotopologue of the API. To the best of our knowledge this is the first example where such a mono-deuterium isotopologue could be resolved from its mother component by HPLC and structurally fully characterized by NMR and MS. The reason for this separation could be rationalized in terms of some special structural features of the molecule.

A systematic approach to the synthesis of androstane-based 3,17-dicarboxamides (homo- and mixed dicarboxamides) via palladium-catalyzed aminocarbonylation.

3,17-Dicarboxamido-androst-3,5,16-triene derivatives possessing various amine moieties were synthesized under mild conditions using palladium-catalyzed homogeneous aminocarbonylation as key reaction. Compounds containing the corresponding iodoalkene functionalities, i.e., 17-iodo-16-ene and 3-iodo-3,5-diene structural motifs, were used in the aminocarbonylation and the N-nucleophiles were varied systematically. Three amines, such as tert-butylamine, piperidine and methyl alaninate were used as N-nucleophiles in the aminocarbonylation. All variations of 3,17-dicarboxamides were synthesized using this methodology. Androst-4-ene-3,17-dione was used as starting material. The synthetic strategy of the multistep synthesis was based on the systematic variation and consecutive use of three different reactions: (i) the protection/deprotection of one of the keto functionalities (3-one or 17-one) as ethylene ketals, (ii) the transformation of the other keto group to iodoalkene functionality via its hydrazone, and (iii) palladium-catalyzed aminocarbonylation of the iodoalkene functionality.

New oxidative decomposition mechanism of estradiol through the structural characterization of a minute impurity and its degradants.

Herein we discuss the structure elucidation of a labile estradiol-related degradant, X1. X1 was detected at Gedeon Richter as an unknown trace impurity in a pharmaceutical formulation containing estradiol (1a) and norethisterone acetate (NA) as active ingredients. The structural identification of X1 proved to be an unusually complex task involving an initial structural hypothesis based on some limited analytical data (UV) obtained from the formulation, synthetic work targeting the proposed structure, chromatographic enrichment from the synthetic reaction mixture, (HPLC)-MS and MS-MS studies of the formulation and of samples from the synthesis using almost all available ionization modes, preparative LC enrichment, and the complementary use of off-line and on-line NMR techniques. Based on these results, X1 was finally characterized as a new oxidative product of estradiol, containing an epoxy function over the C9-C10 bond. During the structure determination of X1 its secondary and tertiary decomposition products were also identified as a new secoepoxy (6) and a known seco derivative (5a) of estradiol, respectively. On this basis a new oxidative decomposition mechanism of estradiol and its analogues could be proposed. A generalization of the mechanism of this pathway can more readily explain the formation of some oxidative secosteroid degradants than the mechanism proposed earlier in the literature.

Systematic investigation on the synthesis of androstane-based 3-, 11- and 17-carboxamides via palladium-catalyzed aminocarbonylation.

3,17-Dicarboxamido-androst-3,5,16-triene, 3-carboxamido-androst-3,5-dien-17-one, 17-carboxamido-androst-4,16-dien-3-one and 11-carboxamido-androst-5,9(11)-dien-3,17-dione derivatives were synthesized in homogeneous carbonylation reactions from the corresponding 3,17-diiodo-androst-3,5,16-triene, 3-iodo-androst-3,5-diene-17-ethylene ketal, 17-iodo-androst-5,16-dien-3-ethylene ketal, 11-iodo-androst-5,9(11)-diene-3,17-bis(ethylene ketal) derivatives, respectively. A highly chemoselective palladium-catalyzed aminocarbonylation of the corresponding iodo-alkene, carried out under mild reaction conditions, can be considered as the key-step for the introduction of the carboxamide functionalities. The synthesis of the iodo-alkene substrate is based on the transformation of the corresponding keto derivative to hydrazone, which was treated with iodine in the presence of a base (1,1,3,3-tetramethyl guanidine). The aminocarbonylation reaction is highly tolerant towards the N-nucleophiles, i.e. various primary and secondary amines including amino acid methyl esters can also be used.