Chiral analogues of (+)-cyclazosin as potent α1B-adrenoceptor selective antagonist.

(+)-Cyclazosin [(+)-1] is one of most selective antagonists of the α1B-adrenoceptor subtype (selectivity ratios, α1B/α1A = 13, α1B/α1D = 38-39). To improve the selectivity, we synthesized and pharmacologically studied the blocking activity against α1-adrenoceptors of several homochiral analogues of (+)-cyclazosin featuring different substituents on the carbonyl or amine groups, namely (-)-2, (+)-3, (-)-4-(-)-8, (+)-9. Moreover, we studied the activity of some their opposite enantiomers, namely (-)-1, (-)-3, (+)-6, and (-)-9, to evaluate the influence of stereochemistry on selectivity. The benzyloxycarbonyl and methyl (4aS,8aR) analogues (+)-3 and (-)-6 improved in a significant way the α1B selectivity of the progenitor compound: 4 and 14 time vs. the α1D subtype and 35 and 77 times vs. the α1A subtype, respectively. The study confirmed the importance of the hydrophobic cis-octahydroquinoxaline moiety of these molecules for the establishment of interactions with the α1-adrenoceptors as well that of their (4aS,8aR) stereochemistry to grant selectivity for the α1B subtype. Hypotheses on the mode of interaction of these compounds were advanced on the basis of molecular modeling studies performed on compound (+)-3.

Quinazoline based α1-adrenoreceptor antagonists with potent antiproliferative activity in human prostate cancer cell lines.

New α1-adrenoreceptor (α1-AR) antagonists related to prazosin and doxazosin were synthesized by replacing piperazine ring with (S)- or (R)-3-aminopiperidine. Binding studies indicated that the S configuration at the 3-C position of the piperidine ring is crucial for an optimal interaction of the compounds at all three α1-AR subtypes. Quinazolines 9 and 10, bearing a quinone ring on the lateral chain, exhibited also potent antiproliferative activity in LNCaP androgen-sensitive prostate cancer cell lines, higher than that of doxazosin. Compound 10 increased apoptosis, in terms of DNA fragmentation, without triggering cell necrosis. The prooxidant activity found in compound 10 may underlie its ability to inhibit cell proliferation in synergy with the effect mediated by α1-AR antagonism. Due to its better biological profile compared to doxazosin for LNCaP cell line, compound 10 might be a valuable lead compound for the design of new prostate antitumor agents.

The replacement of the 2-methoxy substituent of N-((6,6-diphenyl-1,4-dioxan-2-yl)methyl)-2-(2-methoxyphenoxy)ethan-1-amine improves the selectivity for 5-HT1A receptor over α1-adrenoceptor and D2-like receptor subtypes.

N-((6,6-diphenyl-1,4-dioxan-2-yl)methyl)-2-(2-methoxyphenoxy)ethan-1-amine (3) is a potent 5-HT1A receptor and α1d-adrenoceptor (α1d-AR) ligand. Analogues 5-10 were rationally designed and prepared to evaluate whether electronic and/or lipophilic properties of substituents in the ortho position of its phenoxy moiety exert any favorable effects on the affinity/activity at 5-HT1A receptor and improve selectivity over α1-ARs. To rationalize the experimental observations and derive information about receptor-ligand interactions of the reported ligands, docking studies, using 5-HT1A and α1d-AR models generated by homology techniques, and a retrospective computational study were performed. The results highlighted that proper substituents in position 2 of the phenoxy moiety of 3 selectively address the ligands toward 5-HT1A receptor with respect to α1-ARs and D2-like receptor subtypes. Methoxymethylenoxy derivative 9 showed the best 5-HT1A selectivity profile and the highest potency at 5-HT1A receptor, behaving as a partial agonist. Finally, 9, tested in light/dark exploration test in mice, significantly reduced anxiety-linked behaviors. Therefore, it may be considered a lead for the design of partial agonists potentially useful in the treatment of disorders in which 5-HT1A receptor is involved.

Synthesis and structure-activity relationships of novel arylpiperazines as potent antagonists of α1-adrenoceptor.

Arylpiperazines 2-11 were synthesized, and their biological profiles at α1-adrenergic receptors (α1-ARs) assessed by binding assays in CHO cells expressing human cloned subtypes and by functional experiments in isolated rat vas deferens (α1A), spleen (α1B), and aorta (α1D). Modifications at the 1,3-benzodioxole and phenyl phamacophoric units resulted in the identification of a number of potent compounds (moderately selective with respect to the α1b-AR), in binding experiments. Notably, compound 7 (LDT451) showed a subnanomolar pKi of 9.41 towards α1a-AR. An encouragingly lower α1B-potency was a general trend for all the series of compounds, which showed α1A/D over α1B selectivity in functional assays. If adequately optimized, such peculiar selectivity could have relevance for a potential LUTS/BPH therapeutic application.

Insights into the interaction of negative allosteric modulators with the metabotropic glutamate receptor 5: discovery and computational modeling of a new series of ligands with nanomolar affinity.

Metabotropic glutamate receptor 5 (mGlu5) is a biological target implicated in major neurological and psychiatric disorders. In the present study, we have investigated structural determinants of the interaction of negative allosteric modulators (NAMs) with the seven-transmembrane (7TM) domain of mGlu5. A homology model of the 7TM receptor domain built on the crystal structure of the mGlu1 template was obtained, and the binding modes of known NAMs, namely MPEP and fenobam, were investigated by docking and molecular dynamics simulations. The results were validated by comparison with mutagenesis data available in the literature for these two ligands, and subsequently corroborated by the recently described mGlu5 crystal structure. Moreover, a new series of NAMs was synthesized and tested, providing compounds with nanomolar affinity. Several structural modifications were sequentially introduced with the aim of identifying structural features important for receptor binding. The synthesized NAMs were docked in the validated homology model and binding modes were used to interpret and discuss structure-activity relationships within this new series of compounds. Finally, the models of the interaction of NAMs with mGlu5 were extended to include important non-aryl alkyne mGlu5 NAMs taken from the literature. Overall, the results provide useful insights into the molecular interaction of negative allosteric modulators with mGlu5 and may facilitate the design of new modulators for this class of receptors.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 11. (1) reversed enantioselectivity of 1,4-dioxane derivatives in α1-adrenergic and 5-HT1A receptor binding sites recognition.

5-HT(1A) receptor and α(1)-adrenoreceptor (α(1)-AR) binding sites recognized by the 1,4-dioxanes 2-4 display reversed stereochemical requirements. (S)-2 proved to be a potent 5-HT(1A) receptor agonist highly selective over α(1)-AR subtypes. Chirality influenced the anticancer activity of 3 and 4 in human prostate cancer cells (PC-3): (R)-4, eutomer at the α(1d)-AR subtype, was the most potent. The decreased effect of 4 and (R)-4 in α(1d)-AR silenced PC-3 cells confirmed that their anticancer activity was α(1d)-AR-dependent.

Exploring multitarget interactions to reduce opiate withdrawal syndrome and psychiatric comorbidity.

Opioid addiction is often characterized as a chronic relapsing condition due to the severe somatic and behavioral signs, associated with depressive disorders, triggered by opiate withdrawal. Since prolonged abstinence remains a major challenge, our interest has been addressed to such objective. Exploring multitarget interactions, the present investigation suggests that 3 or its (S)-enantiomer and 4, endowed with effective α2C-AR agonism/α2A-AR antagonism/5-HT1A-R agonism, or 7 and 9-11 producing efficacious α2C-AR agonism/α2A-AR antagonism/I2-IBS interaction might represent novel multifunctional tools potentially useful for reducing withdrawal syndrome and associated depression. Such agents, lacking in sedative side effects due to their α2A-AR antagonism, might afford an improvement over current therapies with clonidine-like drugs.

Synthesis and α1-adrenoceptor antagonist activity of tamsulosin analogues.

Tamsulosin (-)-1 is the most utilized α(1)-adrenoceptor antagonist in the benign prostatic hyperplasia therapy owing to its uroselective antagonism and capability in relieving both obstructive and irritative lower urinary tract symptoms. Here we report the synthesis and pharmacological study of the homochiral (-)-1 analogues (-)-2-(-)-5, bearing definite modifications in the 2-substituted phenoxyethylamino group in order to evaluate their influence on the affinity profile for α(1)-adrenoceptor subtypes. The benzyl analogue (-)-3, displaying a preferential antagonist profile for α1A-than α1D-and α1B-adrenoceptors, and a 12-fold higher potency at α1A-adrenoceptors with respect to the α1B subtype, may have improved uroselectivity compared to (-)-1.

Discovery of a new series of 5-HT1A receptor agonists.

Starting from compounds previously identified as alpha(1)-adrenoceptor antagonists that were also found to bind to the 5-HT(1A) receptor, in an attempt to separate the two activities, a new series of 5-HT(1A) receptor agonists was identified and shown to have high potency and/or high selectivity. Of these, compound 13, which combines high selectivity (5-HT(1A)/alpha(1)=151) and good agonist potency (pD(2)=7.82; E(max)=76), was found to be the most interesting.

Doxazosin-related alpha1-adrenoceptor antagonists with prostate antitumor activity.

Doxazosin analogues 1-3 and 1a were synthesized and investigated at alpha1-adrenoceptors and PC-3, DU-145, and LNCaP human prostate cancer cells. Compound 1 (cyclodoxazosin) was a potent alpha(1B)-adrenoceptor antagonist displaying antiproliferative activity higher than that of doxazosin in cancer cells in vitro and in vivo, respectively. Because of its antitumor efficacy at low concentrations, lower apoptotic activity in NHDF vs tumor cells, and antiangiogenetic effect, 1 showed a better therapeutic profile relative to doxazosin.

1,3-Dioxolane-based ligands as rigid analogues of naftopidil: structure-affinity/activity relationships at alpha1 and 5-HT1A receptors.

Conformational restriction of naftopidil proved to be compatible with binding at alpha(1) adrenoceptor subtypes and 5-HT receptor 1A (5-HT(1A)), and led to the discovery of a new class of ligands with a 1,3-dioxolane (1,3-oxathiolane, 1,3-dithiolane) structure. Compound 7 shows the highest affinity toward alpha(1a) and alpha(1d) adrenoceptor subtypes (pK(i) alpha(1a) = 9.58, pK(i) alpha(1d) = 9.09) and selectivity over 5-HT(1A) receptors (alpha(1a)/5-HT(1A) = 100, alpha(1d)/5-HT(1A) = 26). In functional experiments it behaves as a potent competitive alpha(1a) and alpha(1d) adrenoceptor antagonist (pK(b) alpha(1A) = 8.24, pK(b) alpha(1D) = 8.14), whereas at 5-HT(1A) receptors it is a potent partial agonist (pD(2) = 8.30). Compounds 8 and 10 display high affinity (pK(i) = 8.29 and 8.26, respectively) and selectivity for 5-HT(1A) (5-HT(1A)/alpha(1) = 18 and 10). In functional experiments at the 5-HT(1A) receptor, compound 8 appears to be neutral antagonist (pK(b) = 7.29), whereas compound 10 is a partial agonist (pD(2) = 6.27). Therefore, 1,3-dioxolane-based ligands are a versatile class of compounds useful for the development of more selective ligands for one (alpha(1)) or the other (5-HT(1A)) receptor system.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 9. From 1,4-benzodioxane to 1,4-dioxane ring as a promising template of novel alpha1D-adrenoreceptor antagonists, 5-HT1A full agonists, and cytotoxic agents.

Novel 1,4-dioxane compounds structurally related to WB 4101 (1) were prepared in order to investigate the possibility that the quite planar 1,4-benzodioxane template of 1 might be replaced by the less conformationally constrained 1,4-dioxane ring. The biological profiles of the new compounds were assessed using binding assays at human cloned alpha 1-adrenoreceptor (alpha 1-AR) subtypes and 5-HT 1A receptors, expressed in Chinese hamster ovary and HeLa cell membranes, respectively, and by functional experiments in isolated rat vas deferens (alpha 1A), spleen (alpha 1B), and aorta (alpha 1D). Moreover, the cytotoxic effects of the novel compounds were determined in PC-3 prostate cancer cells. The results showed that the properly substituted 1,4-dioxane nucleus proved to be a suitable scaffold for selective alpha 1D-AR antagonists (compound 14), potential anticancer agents (compound 13), and full 5-HT 1A receptor agonists (compound 15). In particular, compound 15 may represent a novel lead in the development of highly potent 5-HT 1A receptor full agonists useful as antidepressant and neuroprotective agents.

Effect of selective antagonists of group I metabotropic glutamate receptors on the micturition reflex in rats.

OBJECTIVE: To investigate the role of Group I metabotropic glutamate (mGlu) receptor subtypes on reflex-induced micturition in anaesthetized and conscious rats using selective mGlu1 (NPS 2407 and R214127) and mGlu5 (MPEP, MTEP, and SIB1893) allosteric antagonists. MATERIALS AND METHODS: The affinity of the compounds at mGlu1 and mGlu5 receptor subtypes was evaluated by displacement of tritiated R214127 and MPEP, respectively, from rat brain tissue. Effects of intravenous (i.v.) administration of the compounds on isovolumic bladder contractions were evaluated in anaesthetized rats. Effects of MPEP and NPS 2407 on bladder filling and voiding were evaluated by cystometry using saline or diluted (0.2%) acetic acid (MPEP only) infusion of bladders in conscious rats. RESULTS: Binding studies confirmed the selectivity of the mGlu1 (NPS 2407 and R214127) and mGlu5 (MPEP, MTEP, and SIB1893) compounds. Isovolumic bladder contractions were blocked after i.v. administration of all compounds. However, the mGlu5 antagonists were generally more potent than mGlu1 antagonists. In conscious rats with bladders infused with saline, MPEP dose-dependently and significantly increased bladder capacity starting from oral administration of 10 mg/kg. Oral administration of NPS 2407 (up to 30 mg/kg) did not induce consistent changes in bladder capacity or micturition pressure. MPEP (10 mg/kg, orally) was also evaluated in conscious rats with bladders infused with diluted acetic acid. In this model, MPEP reduced bladder instability counteracting the decrease of bladder volume capacity induced by acetic acid. There were no consistent effects on bladder contractility. CONCLUSIONS: The results indicate that i.v. and oral administration of selective mGlu5 antagonists, but not those selective for the mGlu1 subtype, have a marked inhibitory effect on reflex micturition pathways in the rat.

Synthesis and alpha(1)-adrenoceptor antagonist activity of derivatives and isosters of the furan portion of (+)-cyclazosin.

alpha(1)-Adrenoceptor selective antagonists are crucial in investigating the role and biological functions of alpha(1)-adrenoceptor subtypes. We synthesized and studied the alpha(1)-adrenoceptor blocking properties of new molecules structurally related to the alpha(1B)-adrenoceptor selective antagonist (+)-cyclazosin, in an attempt to improve its receptor selectivity. In particular, we investigated the importance of substituents introduced at position 5 of the 2-furan moiety of (+)-cyclazosin and its replacement with classical isosteric rings. The 5-methylfuryl derivative (+)-3, [(+)-metcyclazosin], improved the pharmacological properties of the progenitor, displaying a competitive antagonism and an 11 fold increased selectivity for alpha(1B) over alpha(1A), while maintaining a similar selectivity for the alpha(1B)-adrenoceptor relative to the alpha(1D)-adrenoceptor. Compound (+)-3 may represent a useful tool for alpha(1B)-adrenoceptor characterization in functional studies.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 8.(1) {2-[2-(4-chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-dimethoxyphenoxy)ethyl]amine (clopenphendioxan) as a tool to highlight the involvement of alpha1D- and alpha1B-adrenoreceptor subtypes in the regulation of human PC-3 prostate cancer cell apoptosis and proliferation.

A series of new alpha1-adrenoreceptor antagonists (5-18) was prepared by introducing various substituents (Topliss approach) into the ortho, meta, and para positions of the benzyloxy function of the phendioxan open analogue 4 ("openphendioxan"). All the compounds synthesized were potent antagonists and generally displayed, similarly to 4, the highest affinity values at alpha1D- with respect to alpha1A- and alpha1B-AR subtypes and 5-HT1A subtype. By sulforhodamine B (SRB) assay on human PC-3 prostate cancer cells, the new compounds showed antitumor activity (estimated on the basis of three parameters GI50, TGI, LC50), at low micromolar concentration, with 7 ("clopenphendioxan") exhibiting the highest efficacy. Moreover, this study highlighted for the first time alpha1D- and alpha1B-AR expression in PC3 cells and also demonstrated the involvement of these subtypes in the modulation of apoptosis and cell proliferation. A significant reduction of alpha1D- and alpha1B-AR expression in PC3 cells was associated with the apoptosis induced by 7. This depletion was completely reversed by norepinephrine.

Non-competitive inhibitors of metabotropic glutamate receptor 5 (mGluR5).

Based on a pharmacophore alignment on known non-competitive mGluR5 inhibitors applying 4SCan technology, a new lead series was identified and further structurally investigated. K(i)'s as low as around 100 nM were achieved.

1,2,4-Benzothiadiazine derivatives as alpha1 and 5-HT1A receptor ligands.

A series of new 1,2,4-benzothiadiazine derivatives with an arylpiperazine mojety linked at position 3 of the heterocyclic ring were synthesized and assessed for their pharmacological profiles at alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) by functional experiments and by in vitro binding studies at human cloned 5-HT(1A) receptor. Compound 1 was identified as a novel alpha(1D) antagonist (pK(b)alpha(1D)=7.59; alpha(1D)/alpha(1A)>389; alpha(1D)/alpha(1B)=135) with high selectivity over 5-HT(1A) receptor (5-HT(1A)/alpha(1D)<0.01), while compound 6, a 3,4-dihydro-derivative, was characterized as a novel 5-HT(1A) receptor ligand, highly selective over alpha(1D)-adrenoceptor subtype (pK(i)5-HT(1A)=8.04; 5-HT(1A)/alpha(1D)=1096). Further pharmacological studies demonstrated that 6 is a partial agonist at 5-HT(1A) receptor (E(max)=23, pD(2)=6.92).

Synthesis and structure-activity relationships of 1-aralkyl-4-benzylpiperidine and 1-aralkyl-4-benzylpiperazine derivatives as potent sigma ligands.

In the attempt to define more accurately structure-affinity relationships for sigma(1) and sigma(2) ligands, we synthesized and tested on sigma subtype receptors a series of aralkyl derivatives of 4-benzylpiperidine, in which the effect of modifications on the aralkyl moiety was studied in a systematic way. The affinity of the compounds here described varied to a great extent, with a sigma(2)/sigma(1) selectivity ranging from 0.1 to 9. Thus, to confirm the ability of the piperazine derivative to bind to sigma(1) receptors in a different way than piperidines, we synthesized and tested a series of piperazine compounds; the comparison of their affinity with that of the corresponding piperidines strongly supports the possibility of a different binding mode. While the compounds here described are on the whole selective for sigma vs serotonin 5-HT(1A) and dopamine D(2) receptors, 9aa, 9ba and 9ab possess a remarkable affinity for both sigma and 5-HT(1A) receptors, with K(i) in the nanomolar range, and are selective with respect to D(2) receptors. They displayed also a partial agonist profile in a human 5-HT(1A) [(35)S]GTP gamma S binding assay, suggesting their potential use as atypical antipsychotic agents.

Synthesis, screening, and molecular modeling of new potent and selective antagonists at the alpha 1d adrenergic receptor.

In the present study, more than 75 compounds structurally related to BMY 7378 have been designed and synthesized. Structural variations of each part of the reference molecule have been introduced, obtaining highly selective ligands for the alpha(1d) adrenergic receptor. The molecular determinants for selectivity at this receptor are essentially held by the phenyl substituent in the phenylpiperazine moiety. The integration of an extensive SAR analysis with docking simulations using the rhodopsin-based models of the three alpha(1)-AR subtypes and of the 5-HT(1A) receptor provides significant insights into the characterization of the receptor binding sites as well as into the molecular determinants of ligand selectivity at the alpha(1d)-AR and the 5-HT(1A) receptors. The results of multiple copies simultaneous search (MCSS) on the substituted phenylpiperazines together with those of manual docking of compounds BMY 7378 and 69 into the putative binding sites of the alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR, and the 5-HT(1A) receptors suggest that the phenylpiperazine moiety would dock into a site formed by amino acids in helices 3, 4, 5, 6 and extracellular loop 2 (E2), whereas the spirocyclic ring of the ligand docks into a site formed by amino acids of helices 1, 2, 3, and 7. This docking mode is consistent with the SAR data produced in this work. Furthermore, the binding site of the imide moiety does not allow for the simultaneous involvement of the two carbonyl oxygen atoms in H-bonding interactions, consistent with the SAR data, in particular with the results obtained with the lactam derivative 128. The results of docking simulations also suggest that the second and third extracellular loops may act as selectivity filters for the substituted phenylpiperazines. The most potent and selective compounds for alpha(1d) adrenergic receptor, i.e., 69 (Rec 26D/038) and 128 (Rec 26D/073), are characterized by the presence of the 2,5-dichlorophenylpiperazine moiety.

Prazosin-related compounds. Effect of transforming the piperazinylquinazoline moiety into an aminomethyltetrahydroacridine system on the affinity for alpha1-adrenoreceptors.

In a search for structurally new alpha(1)-adrenoreceptor (alpha(1)-AR) antagonists, prazosin (1)-related compounds 2-11 were synthesized and their affinity profiles were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)) and by binding assays in CHO cells expressing human cloned alpha(1)-AR subtypes. Transformation of the piperazinylquinazoline moiety of 1 into an aminomethyltetrahydroacridine system afforded compound 2, endowed with reduced affinity, in particular for the alpha(1A)-AR subtype. Then, to investigate the optimal features of the tricyclic moiety, the aliphatic ring of 2 was modified by synthesizing the lower and higher homologues 3 and 4. An analysis of the pharmacological profile, together with a molecular modeling study, indicated the tetrahydroacridine moiety as the most promising skeleton for alpha(1)-antagonism. Compounds 5-8, where the replacement of the furoyl group of 2 with a benzoyl moiety afforded the possibility to evaluate the effect of the substituent trifluoromethyl on receptor binding, resulted, except for 7, in a rather surprising selectivity toward alpha(1B)-AR, in particular vs the alpha(1A) subtype. Also the insertion of the 2,6-dimethoxyphenoxyethyl function of WB 4101 on the tetrahydroacridine skeleton of 2, and/or the replacement of the aromatic amino function with a hydroxy group, affording derivatives 9-11, resulted in alpha(1B)-AR selectivity also vs the alpha(1D) subtype. On the basis of these results, the tetrahydroacridine moiety emerged as a promising tool for the characterization of the alpha(1)-AR, owing to the receptor subtype selectivity achieved by an appropriate modification of the lateral substituents.