Design and synthesis of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one as a novel class of GlyT1 inhibitors: achieving selectivity against the mu opioid and nociceptin/orphanin FQ peptide (NOP) receptors

A novel class of 4-substituted-8-(2-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-ones have been discovered and developed as potent and selective GlyT1 inhibitors. The molecules are devoid of activity at the GlyT2 isoform and display excellent selectivities against the mu opioid receptor as well as the nociceptin/orphanin FQ peptide (NOP) receptor. A novel, straightforward and efficient synthetic strategy for the assembly of the target molecules is also presented.     

4-Substituted-8-(1-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-one as a novel class of highly selective GlyT1 inhibitors with superior pharmacological and pharmacokinetic parameters

A novel class of 4-substituted-8-(1-phenyl-cyclohexyl)-2,8-diaza-spiro[4.5]decan-1-ones have been discovered and developed as potent and selective GlyT1 inhibitors. The molecules are devoid of activity at the GlyT2 isoform and display excellent selectivities against the mu opioid receptor as well as the nociceptin/orphanin FQ peptide (NOP) receptor. These molecules also exhibit superior pharmacological and pharmacokinetic parameters, relative to all GlyT1 inhibitors of the spiropiperidine family, culminating in the identification of 16b with an oral bioavailability of approximately 60%. In addition, a straightforward two-step procedure for the assembly of the target molecules is also presented.     

Novel complex crystal structure of prolyl hydroxylase domain-containing protein 2 (PHD2): 2,8-Diazaspiro[4.5]decan-1-ones as potent,orally bioavailable PHD2 inhibitors

We have discovered a novel complex crystal structure of the PHD2 enzyme with its inhibitor, the 2,8-diazaspiro[4.5]decan-1-one analogue 4b. The widely reported salt bridge between Arg383 of the enzyme and its inhibitors in all complex structures published thus far was not observed in our case. In our complex structure compound 4b forms several novel interactions with the enzyme, which include a hydrogen bond with Arg322, a π-cation interaction with Arg322, a π–π stacking with Trp389, and a π–π stacking with His313. Guided by the structural information, SAR studies were performed on the 2,8-diazaspiro[4.5]decan-1-one series leading to the discovery of compound 9p with high potency and good oral pharmacokinetic profile in mice.     

ORL1 receptor ligands: structure-activity relationships of 8-cycloalkyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-ones

We have investigated 8-cycloalkyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-o nes as ligands for the ORL1 receptor. These unsophisticated, achiral compounds show remarkable affinity for the ORL1 receptor. Optimizing for selectivity we show that the maximum of affinity and selectivity versus the other opioid receptors is achieved for 8-cyclodecyl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-o ne 2e and 8-(cis-4-isopropyl-cyclohexyl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one 2q. The identified compounds (2e, 2q) are more or less equipotent to the natural ligand itself, both in the binding assay and in the functional GTPgammaS assay.     

Discovery of N-(2-aryl-cyclohexyl) substituted spiropiperidines as a novel class of GlyT1 inhibitors

Screening of the Roche compound library led to the identification of cis-N-(2-phenyl-cyclohexyl)-spiropiperidine 1 as structurally novel GlyT1 inhibitor. The SAR, which was developed in this series, resulted in the discovery of highly potent compounds displaying excellent selectivity against the GlyT2 isoform.     

Microwave assisted synthesis and anti-influenza virus activity of 1-adamantyl substituted N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide derivatives

A microwave-assisted three-component one-pot cyclocondensation method was applied for the synthesis of novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide compounds carrying an adamantyl moiety. The structures of the compounds were confirmed by spectral and elemental analysis. All compounds were evaluated for antiviral activity against influenza A (H1N1 and H3N2) and influenza B virus in MDCK cell cultures. The compounds displayed a confined structure-activity relationship. The N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)adamantane-1-carboxamide 3b was the most potent inhibitor [antiviral EC₅₀: 1.4 μM against influenza A/H3N2 virus]. Its strong inhibitory effect in a virus hemolysis assay supports that 3b acts as an influenza virus fusion inhibitor by preventing the conformational change of the influenza virus hemagglutinin at low pH.     

Design, syntheses, and SAR of 2,8-diazaspiro[4.5]decanones as T-type calcium channel antagonists

It was hypothesized that an appropriately substituted 2,8-diazaspiro[4.5]decan-1-one could effectively approximate a 5-feature T-type pharmacophore model published in the literature. Compounds were designed and synthesized to test our hypothesis and were found to be potent T-type calcium channel inhibitors with modest selectivity over L-type calcium channels. The synthesis and SAR of the series is described.     

Preparation of an affinity chromatography matrix for the selective purification of the dopamine D2 receptor from bovine striatal membranes

A ligand affinity matrix has been developed and utilized to purify the dopamine D2 receptor approx. 2100 fold from bovine striatal membranes. 3-[2-Aminoethyl]-8-[3-(4-fluorobenzoyl)propyl]-4-oxo-1-phenyl-1,3,8- triazaspiro[4.5]decan-4-one (AES) was synthesized and used to prepare the affinity matrix by coupling to epoxy-activated Sepharose 6B (AES-Sepharose). AES (Ki approximately 1.7 nM) is similar in potency to the parent compound, spiperone (Ki approximately 0.8 nM), in competing for [3H]spiperone-binding activity. AES has no significant potency in competing for the dopamine D1 receptor as assessed by competition for [3H]SCH23390 binding (Ki greater than 1 microM). Covalent photoaffinity labeling of the dopamine D2 receptor in bovine striatal membranes with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS) was prevented by AES at nanomolar concentrations. The dopamine D2 receptor was solubilized from bovine striatal membranes using 0.25% cholate in the presence of high ionic strength, followed by precipitation and subsequent treatment with 0.5% digitonin. Nearly 100% of the [3H]spiperone-binding activity in the cholate-digitonin solubilized preparation was absorbed at a receptor-to-resin ratio of 2:1 (v/v). Dopamine D2 receptor was eluted from the affinity resin using a competing dopaminergic antagonist molecule, haloperidol. Recovery of dopamine D2 receptor activity from the affinity matrix was approx. 9% of the activity adsorbed to the resin. The [3H]spiperone-binding activity in AES-Sepharose affinity purified preparations is saturable and of high affinity (0.2 nM). Affinity-purified preparations maintain the ligand-binding characteristics of a dopamine D2 receptor as assessed by agonist and antagonist competition for [3H]spiperone binding.     

Pre-synaptic glycine GlyT1 transporter--NMDA receptor interaction: relevance to NMDA autoreceptor activation in the presence of Mg2+ ions

Rat hippocampal glutamatergic terminals possess NMDA autoreceptors whose activation by low micromolar NMDA elicits glutamate exocytosis in the presence of physiological Mg(2+) (1.2 mM), the release of glutamate being significantly reduced when compared to that in Mg(2+)-free condition. Both glutamate and glycine were required to evoke glutamate exocytosis in 1.2 mM Mg(2+), while dizocilpine, cis-4-[phosphomethyl]-piperidine-2-carboxylic acid and 7-Cl-kynurenic acid prevented it, indicating that occupation of both agonist sites is needed for receptor activation. D-serine mimicked glycine but also inhibited the NMDA/glycine-induced release of [(3H]D-aspartate, thus behaving as a partial agonist. The NMDA/glycine-induced release in 1.2 mM Mg(2+) strictly depended on glycine uptake through the glycine transporter type 1 (GlyT1), because the GlyT1 blocker N-[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl])sarcosine hydrochloride, but not the GlyT2 blocker Org 25534, prevented it. Accordingly, [(3)H]glycine was taken up during superfusion, while lowering the external concentration of Na(+), the monovalent cation co-transported with glycine by GlyT1, abrogated the NMDA-induced effect. Western blot analysis of subsynaptic fractions confirms that GlyT1 and NMDA autoreceptors co-localize at the pre-synaptic level, where GluN3A subunits immunoreactivity was also recovered. It is proposed that GlyT1s coexist with NMDA autoreceptors on rat hippocampal glutamatergic terminals and that glycine taken up by GlyT1 may permit physiological activation of NMDA pre-synaptic autoreceptors.     

Synthesis and structure-activity relationship of 2-(aminoalkyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives: a novel series of 5-HT(2A/2C) receptor antagonists. Part 1.

The synthesis of a series of novel 2-(aminoalkyl)-2,3,3a,8-tetrahydrodibenzo[c,f]isoxazolo[2,3-a]azepine derivatives as well as their 5-HT(2A/2C) and H(1) receptor binding affinities are described. The in vivo activity as potential anxiolytics of the synthesised compounds was measured in a mCPP challenge test. One of the compounds, 2a, proved to be a potent 5-HT(2A/2C) receptor antagonist showing as well oral activity and therefore could be considered as a potential anxiolytic/antidepressant agent.     

Novel 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids: discovery and hit-to-lead evolution of a selective CRTh2 receptor antagonist chemotype

Hit-to-lead evolution of 2-(2-((2-(4-chlorophenoxy)ethyl)thio)-1H-benzo[d]imidazol-1-yl)acetic acid (1), discovered in a high-throughput screening campaign as a novel chemotype of CRTh2 receptor antagonist, is presented. SAR development as well as in vitro and in vivo DMPK properties of selected representatives of substituted 2-(2-(benzylthio)-1H-benzo[d]imidazol-1-yl)acetic acids are discussed.     

8-(1-Naphthalen-2-yl-vinyl)-6,7,10-trioxaspiro (4.5) decane, a new 1,2,4-trioxane effective against rodent and simian malaria

A new series of 8-(1-aryl-vinyl)-6,7,10-trioxaspiro [4.5] decanes 7a-e and 3-(1-aryl-vinyl)-l,2,5-trioxaspiro [5.5] undecanes 8a-e have been prepared and screened against multi-drug resistant Plasmodium yoelii in mice. 8-(1-Naphthalen-2-yl-vinyl)-6,7,10-trioxaspiro [4.5] decane 7b, the most active trioxane of the series, has also shown promising activity against Plasmodium cynomolgi in rhesus monkeys.     

Synthesis and biological characterization of novel hybrid 7-[[2-(4-phenyl-piperazin-1-yl)-ethyl]-propyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol and their heterocyclic bioisosteric analogues for dopamine D2 and D3 receptors

In a recent preliminary communication we described the development of a series of hybrid molecules for the dopamine D2 and D3 receptor subtypes. The design of these compounds was based on combining pharmacophoric elements of aminotetralin and piperazine molecular fragments derived from known dopamine receptor agonist and antagonist molecules. Molecules developed from this approach exhibited high affinity and selectivity for the D3 receptor as judged from preliminary [(3)H]spiperone binding data. In this report, we have expanded our previous finding by developing additional novel molecules and additionally evaluated functional activities of these novel molecules in the [(3)H]thymidine incorporation mitogenesis assay. The binding results indicated highest selectivity in the bioisosteric benzothiazole derivative N6-[2-(4-phenyl-piperazin-1-yl)-ethyl]-N6-propyl-4,5,6,7-tetrahydro-benzothiazole-2,6-diamine (14) for the D3 receptor whereas the racemic compound 7-([2-[4-(2,3-dichloro-phenyl)-piperazin-1-yl]-ethyl]-propyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol (10c) showed the strongest potency. Mitogenesis studies to evaluate functional activity demonstrated potent agonist properties in these novel derivatives for both D2 and D3 receptors. In this regard, compound 7-[[4-(4-phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol (7b) exhibited the most potent agonist activity at the D3 receptor, 10 times more potent than quinpirole and was also the most selective compound for the D3 receptor in this series. Racemic compound 10a was resolved; however, little separation of activity was found between the two enantiomers of 10a. The marginally more active enantiomer (-)-10a was examined in vivo using the 6-OH-DA induced unilaterally lesioned rat model to evaluate its activity in producing contralateral rotations. The results demonstrated that in comparison to the reference compound apomorphine, (-)-10a was quite potent in inducing contralateral rotations and exhibited longer duration of action.     

3-(4-Piperidinyl)- and 3-(8-aza-bicyclo[3.2.1]oct-3-yl)-2-phenyl-1H-indoles as bioavailable h5-HT2A antagonists

A series of 3-(4-piperidinyl)- and 3-(8-aza-bicyclo[3.2.l]oct-3-yl)-2-phenyl-1H-indoles have been prepared and evaluated as ligands for the h5-HT_2A receptor. 3-(8-Phenethyl-8-aza-bicyclo[3.2.l]oct-3-yI)-2-phenyl-1H-indole is a high-affinity (1.2 nM), selective (>800 fold over h5-HT_2C and hD₂ receptors) antagonist at the h5-HT_2A receptor with oral bioavailability in rats.     

Identification of N-(2-(azepan-1-yl)-2-phenylethyl)-benzenesulfonamides as novel inhibitors of GlyT1

A novel series of glycine transporter 1 (GlyT1) inhibitors is described. Scoping of the heterocycle moiety of hit 4-chlorobenzenesulfonamide 1 led to replacement of the piperidine with an azepane for a modest increase in potency. Phenyl sulfonamides proved superior to alkyl and non-phenyl aromatic sulfonamides, while subsequent ortho substitution of the 2-(azepan-1-yl)-2-phenylethanamine aromatic ring yielded 39 (IC₅₀ 37 nM, solubility 14 μM), the most potent GlyT1 inhibitor in this series. Favorable brain–plasma ratios were observed for select compounds in pharmacokinetic studies to evaluate CNS penetration.     

Novel GlyT1 inhibitor chemotypes by scaffold hopping. Part 2: Development of a [3.3.0]-based series and other piperidine bioisosteres

This Letter describes the development and SAR of a novel series of GlyT1 inhibitors derived from a scaffold hopping approach, in lieu of an HTS campaign, which provided intellectual property position. Members within this new [3.3.0]-based series displayed excellent GlyT1 potency, selectivity, free fraction, and modest CNS penetration. Moreover, enantioselective GlyT1 inhibition was observed, within this novel series and a number of other piperidine bioisosteric cores.     

Development of 3,4-dihydro-2H-benzo[1,4]oxazine derivatives as dual thromboxane A2 receptor antagonists and prostacyclin receptor agonists

We discovered a novel series of 3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxyacetic acid derivatives as potent dual-acting agents to block the TXA2 receptor and to activate the PGI2 receptor. We report the synthesis, structure-activity relationship, and in vitro, ex vivo, and in vivo pharmacology of this series of compounds. 4-[2-(1,1-Diphenylethylsulfanyl)ethyl]-3,4-dihydro-2H-benzo[1,4]oxazin-8-yloxyacetic acid N-methyl-D-glucamine salt (7) is a promising candidate for a novel treatment in the anti-thrombotic and the cardiovascular fields avoiding hypotensive side effects.     

3-(4-Piperidinyl)- and 3-(8-aza-bicyclo[3.2.1]oct-3-yl)-2-phenyl-1H-indoles as bioavailable h5-HT2A antagonists

A series of 3-(4-piperidinyl)- and 3-(8-aza-bicyclo[3.2.l]oct-3-yl)-2-phenyl-1H-indoles have been prepared and evaluated as ligands for the h5-HT_2A receptor. 3-(8-Phenethyl-8-aza-bicyclo[3.2.l]oct-3-yI)-2-phenyl-1H-indole is a high-affinity (1.2 nM), selective (>800 fold over h5-HT_2C and hD₂ receptors) antagonist at the h5-HT_2A receptor with oral bioavailability in rats.     

Synthesis and screening for acetylcholinesterase inhibitor activity of some novel 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-ones: derivatives of irbesartan key intermediate

The association of bioactive nucleus with other pharmacological agents is hoped to improve the efficacy of the treatment by combining the effects of different pharmacological mechanisms of action. Keeping this in view, a series of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one derivatives have been synthesized by interaction of 2-butyl-1,3-diaza-spiro[4,4]non-1-en-4-one with different bioactive aralkyl halides in presence of powdered potassium carbonate by two different methods viz., conventional and microwave irradiation. The yields under conventional and microwave irradiation methods were in the range of 60-65% and 80-90%, respectively. The structure elucidation of the new compounds has been carried out with the help of elemental analysis and spectral data. All the synthesized compounds have been screened for their efficacy as acetylcholinesterase (AChE) inhibitor. AChE inhibitory activity study was carried out by using Ellman colorimetric assay with neostigmine as a reference standard against targets from different species, such as pure electric eel AChE, human serum AChE, and rat brain AChE. Among the compounds synthesized, compounds 5a, 5b, 5j showed good inhibition against AChE.     

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.