New structure-activity relationship studies in a series of N,N-bis(cyclohexanol)amine aryl esters as potent reversers of P-glycoprotein-mediated multidrug resistance (MDR).

As a continuation of previous research on a new series of potent and efficacious P-gp-dependent multidrug resistant (MDR) reversers with a N,N-bis(cyclohexanol)amine scaffold, we have designed and synthesized several analogs by modulation of the two aromatic moieties linked through ester functions to the N,N-bis(cyclohexanol)amine, aiming to optimize activity and to extend structure-activity relationships (SAR) within the series. This scaffold, when esterified with two different aromatic carboxylic acids, gives origin to four geometric isomers (cis/trans, trans/trans, cis/cis and trans/cis). The new compounds were tested on doxorubicin-resistant erythroleukemia K562 cells (K562/DOX) in the pirarubicin uptake assay. Most of them resulted in being potent modulators of the extrusion pump P-gp, showing potency values ([I](0.5)) in the submicromolar and nanomolar range. Of these, compounds 2b, 2c, 3d, 5a-d and 6d, showed excellent efficacy with a α(max) close to 1. Selected compounds (2d, 3a, 3b, 5a-d) were further studied to evaluate their doxorubicin cytotoxicity potentiation (RF) on doxorubicin-resistant erythroleukemia K562 cells and were found able to enhance significantly doxorubicin cytotoxicity on K562/DOX cells. The results of both pirarubicin uptake and the cytotoxicity assay, indicate that the new compounds of the series are potent P-gp-mediated MDR reversers. They present a structure with a mix of flexible and rigid moieties, a property that seems critical to allow the molecules to choose the most productive of the several binding modes possible in the transporter recognition site. In particular, compounds 5c and 5d, similar to the already reported analogous isomers 1c and 1d,(29) are potent and efficacious modulators of P-gp-dependent MDR and may be promising leads for the development of MDR-reversal drugs.

Inhibition of P-glycoprotein-mediated Multidrug Resistance (MDR) by N,N-bis(cyclohexanol)amine aryl esters: further restriction of molecular flexibility maintains high potency and efficacy.

Conformational modulation of the aryl portion of a set of N,N-bis(cyclohexanol)amine aryl esters (1a-d) that are potent Pgp-dependent MDR inhibitors has been performed. Toward this end the trans-3-(3,4,5-trimethoxyphenyl)acrylic acid present in set 1 was substituted with 3-(3,4,5-trimethoxyphenyl)propanoic and 3-(3,4,5-trimethoxyphenyl)propiolic moieties to give sets 2 and 3, respectively. While the introduction of 3-(3,4,5-trimethoxyphenyl)propanoic moiety resulted in a definite drop in potency and efficacy, esterification with 3-(3,4,5-trimethoxyphenyl)propiolic acid gave four isomers (3a-d) that maintain high potency and possess optimal efficacy. These results are discussed in terms of conformational flexibility of the different sets of compounds.

Design, synthesis and nootropic activity of new analogues of sunifiram and sapunifiram, two potent cognition-enhancers.

A series of amides and sulfonamides, structurally related to DM235 (sunifiram) and MN19 (sapunifiram), derived by ring expansion or contraction, or by inversion of the exocyclic amide function, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, and with a potency similar to the parent compounds.

Docking analyses on human muscarinic receptors: unveiling the subtypes peculiarities in agonists binding.

The study presents a docking analysis for the interaction capabilities of some muscarinic receptors (i.e., M(1), M(2), and M(5)) whose full-length models were previously generated by us. In detail, the docking simulations involved a dataset of 30 agonists, taken from the literature, including a first series of oxathiolane/dioxolane congeners and a second subset of more heterogeneous ligands. The obtained results unveil that it is possible to discriminate among the binding modes of considered muscarinic receptors, developing specific interaction patterns which are significantly different for the arrangement of both polar and hydrophobic interactions. Thus, the M(1) subtype possesses the widest binding site, while the M(2) receptor is characterized by a large but asymmetric region that accommodates the ligand's ammonium head and finally the M(5) binding site is quite similar to that of M(2) subtype being univocally characterized by a second aspartate residue which interacts with the ammonium head. The significant correlations between docking scores and affinity values afford an encouraging validation for the reported binding modes and confirm the key role of molecular flexibility in the ligand recognition of muscarinic receptors.

Design, synthesis and preliminary pharmacological evaluation of new analogues of DM232 (unifiram) and DM235 (sunifiram) as cognition modulators.

A series of amides, structurally related to DM232 (unifiram) and DM235 (sunifiram), characterized by a 1,2,3,4-tetrahydropyrazino[2,1-a]isoindol-6(2H)-one, 1,4-diamino-cyclohexane or 1,4-diaminobenzene ring, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, while some cyclohexane derivatives are endowed with amnesia inducing properties.

Muscarinic antagonists with multiple stereocenters: Synthesis, affinity profile and functional activity of isomeric 1-methyl-2-(2,2-alkylaryl-1,3-oxathiolan-5-yl)pyrrolidine sulfoxide derivatives.

Completing a long-lasting research on 1,3-oxathiolane muscarinic ligands, we have synthesized a set of isomeric 1-methyl-2-(2,2-alkylaryl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide derivatives, containing three or four stereogenic centers. In general the compounds are very potent antagonists even if, unlike the corresponding agonists, they show modest subtype selectivity.

Design, synthesis and preliminary pharmacological evaluation of new piperidine and piperazine derivatives as cognition-enhancers.

A series of 2-oxopiperazine, 4-aminomethyl-, 3-amino- and 3-aminomethylpiperidine analogues of DM235 (sunifiram) and MN19 (sapunifiram), two previously reported potent cognition-enhancers, have been synthesized and tested in the mouse passive-avoidance test. The compounds display minimal effective doses in the range 0.3-10mg/kg. Although the new substances do not show improved activity when compared to the parent compounds, some useful information has been obtained to understand structure-activity relationships. In addition, the 3-aminopiperidine moiety appears to be a promising scaffold to synthesize new drugs endowed with cognition-enhancing activity.

Synthesis, affinity profile, and functional activity of muscarinic antagonists with a 1-methyl-2-(2,2-alkylaryl-1,3-oxathiolan-5-yl)pyrrolidine structure.

Starting from a previously studied muscarinic ligand, characterized by a 1,3-oxathiolane nucleus, a new series of muscarinic antagonists were designed by increasing the stereochemical complexity of the molecules. A small library of enantiomeric and diastereomeric 2,2-diphenyl- and 2-cyclohexyl-2-phenyl substituted compounds was thus obtained. All the tested compounds show a high affinity toward cloned human muscarinic hm1-hm5 receptors expressed in CHO cells and a good antagonistic activity on functional assays, with a modest selectivity on rabbit vas deferens.

Isomeric N,N-bis(cyclohexanol)amine aryl esters: the discovery of a new class of highly potent P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors.

A new series of P-glycoprotein (Pgp)-dependent multidrug resistance (MDR) inhibitors having a N,N-bis(cyclohexanol)amine scaffold have been designed, following the frozen analog approach. With respect to the parent flexible molecules, the new compounds show improved potency and efficacy. Among them, compound 1d, on anthracycline-resistant erythroleukemia K562 cells, is able to completely reverse Pgp-dependent MDR at low nanomolar concentration.

Design, synthesis, and preliminary pharmacological evaluation of new quinoline derivatives as nicotinic ligands.

A series of nicotinic ligands, carrying a quinoline nucleus, and characterized by a pharmacophoric distance between the quinoline nitrogen (H-bond acceptor) and the cationic nitrogen atoms higher than that proposed in the classical pharmacophoric models, have been synthesized and tested for their affinity for the central nicotinic receptor. The enantiomers of the nicotine analogue 1-methyl-2-pyrrolidinyl-6-quinoline and of its methiodide display enantioselectivity in binding studies, but not when tested in vivo; on alpha7* nicotinic receptor enantioselectivity is inverted with respect to the alpha4beta2* subtype. N,N,N-Trimethyl-4-(quinolin-6-yl)but-3-yn-1-ammonium iodide (3c) and trans-N,N,N-trimethyl-4-(quinolin-6-yl)but-3-en-1-ammonium iodide (4c), showing pharmacophoric distances in the range 8.5-10.4 A, interact with the alpha4beta2* nicotinic receptor with Ki in the microM range; compound 3c shows preference for the alpha7* subtype.

Highly chiral muscarinic ligands: the discovery of (2S,2'R,3'S,5'R)-1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide methyl iodide, a potent, functionally selective, M2 partial agonist.

By further steric complication of previously studied highly chiral muscarinic agonists, we have obtained a small chiral library of enantiomeric and diasteromeric 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxides. Binding studies on cloned human muscarinic receptors expressed in CHO cells show that the introduction of a fourth stereogenic center gives undetectable affinity for hm1, hm3, hm4 and hm5 subtypes while leaving a quite modest affinity only for hm2 subtypes. However, functional studies on model M1-M4 muscarinic tissues have shown that three compounds of the series [(-)-5, (-)-7, (+)-8] are endowed with functional activity and behave as M2 selective partial agonists. Among them, compound (2S,2'R,3'S,5'R)-1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine 3-sulfoxide methyl iodide [(+)-8] is particularly interesting, as it is a potent partial agonist on guinea pig atrium (force) (M2; pD2=7.65, alpha=0.41) while being a poor antagonist on M1, M3, and M4 model tissues (pKb<5).

Exploratory chemistry toward the identification of a new class of multidrug resistance reverters inspired by pervilleine and verapamil models.

On the basis of the present knowledge of the substrate recognition site of ABC transporter proteins and inspired by the structures of verapamil and pervilleine A, a new class of Pgp-mediated multidrug resistance (MDR) reverters has been designed and synthesized. The new compounds are flexible molecules carrying one or two basic nitrogen atoms flanked, at properly modulated distance, by two aromatic moieties. Most of the molecules studied possess MDR inhibitory activity on anthracycline-resistant erythroleukemia K 562 cells, showing a potency that is higher than that of the reference compound verapamil and, in a few cases (7, 12, 13,17, 20, 22, 28), is in the high nanomolar range. These compounds may be useful leads to develop new MDR reverting agents. In fact, the chemical structure of the class is fairly simple and can be implemented in a variety of ways that will allow the synthesis of new compounds that might be useful leads for the development of drugs to control Pgp-dependent MDR.

Design, synthesis, and preliminary pharmacological evaluation of a set of small molecules that directly activate gi proteins.

Heterotrimeric G proteins play a pivotal role in the communication of cells with the environment. G proteins are stimulated by cell surface receptors (GPCR) that catalyze the exchange of GDP, bound to Galpha subunit, with GTP and can per se be the target of drugs. Based on the structure of two nonpeptidic modulators of Gi proteins, a series of new molecules characterized by a long hydrophobic chain and at least two nitrogen atoms protonated at physiological pH was designed. The compounds were tested for their ability to stimulate binding of GTPgammaS to recombinant Gi proteins. Gi activation properties were also evaluated by inhibition of adenylyl cyclase activity in intact lymphocytes. Most compounds were able to stimulate GTPgammaS binding and to inhibit cAMP production at micromolar doses. Among the active compounds, 34 showed good efficacy and was the most potent compound studied, particularly on alpha(o) subtype; its regioisomer, 36, was the most efficacious one. Compound 7 showed also an interesting profile as it showed selectivity toward the alpha(o) subtype, in both efficacy and potency. Some of the compounds synthesized and found to be active may be useful leads to develop more potent and selective Gi protein modulators.

Muscarinic subtype affinity and functional activity profile of 1-methyl-2-(2-methyl-1,3-dioxolan-4-yl)pyrrolidine and 1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine derivatives.

Starting from two previously studied muscarinic full agonists, characterized by a 1,3-dioxolane ((+)-1) and a 1,3-oxathiolane ((+)-2) cycle, two new series of muscarinic ligands were designed, obtained by the steric complication of the parent compounds produced by freezing the aminoalkyl chain into a pyrrolidine ring. Both tertiary amines and the corresponding iodomethyl derivatives were synthesised and studied, and several compounds of the series which behaved as muscarinic agonists have been selected, on the basis of preliminary binding experiments on rat cortex homogenates, for the present work. Results are presented obtained from testing the affinity of the selected compounds against cloned human muscarinic receptors expressed in CHO cells, in order to evaluate subtype selectivity. Their functional activity on classical models of M1-M4 receptors, in guinea pig and rabbit tissues is also reported. With respect to parent compounds, the new molecules present some selectivity toward hm2 receptors; fair M2 selectivity is also evident in functional studies, where these compounds behave as partial agonists. Among the other compounds of the series (2S, 4'R, 2'S)-1,1-dimethyl-2-(2-methyl-1,3-dioxolan-4-yl)pyrrolidinium iodide (-)-3 and (2R, 5'S, 2'S)-1-methyl-2-(2-methyl-1,3-oxathiolan-5-yl)pyrrolidine (+)-5 present a promising pharmacological profile. Compound (-)-3 shows modest hm2 selectivity in binding experiments but a clearcut M2 selectivity in functional tests, where it behaves as a weak antagonist on M1 and M4 subtypes, as a weak full agonist on the M3 subtype and as a potent partial agonist on M2 subtype. Tertiary amine (+)-5 presents a quite similar profile but appears more interesting since, lacking a permanent charge on the nitrogen atom, it may represent an interesting tool to study CNS muscarinic receptors. Our results confirm that sterical complication of parent compounds (+)-1 and (+)-2 produces more selective muscarinic agonists.

Enantioselective synthesis and preliminary pharmacological evaluation of the enantiomers of unifiram (DM232), a potent cognition-enhancing agent.

The enantiomers of the potent cognition-enhancer DM232 ((1), unifiram) and of its isopropylsulfonyl analog (2), which is endowed with amnesic properties, have been synthesized using (S)- and (R)-5-(hydroxymethyl)-2-pyrrolidinone as chiral precursors. The enantiomeric excess was determined by means of capillary electrophoresis, and found higher than 99.9 %. DM232 enantiomers were tested as cognition-enhancers in the passive-avoidance and social learning tests, and their ability to induce ACh release from rat cerebral cortex was also determined; in all the performed essays, (R)-(+)-(1) displayed higher potency than its (S)-(-) enantiomer, being able to elicit comparable effects at 3-fold to 10-fold lower doses. On the contrary, (R)-(+) and (S)-(-)-(2) showed the same amnesic potency when tested in the passive-avoidance test. These findings may be useful to clarify the mechanism of action of these substances.

Design, synthesis, and preliminary pharmacological evaluation of 4-aminopiperidine derivatives as N-type calcium channel blockers active on pain and neuropathic pain.

Several compounds with a 4-aminopiperidine scaffold decorated on both nitrogen atoms by alkyl or acyl moieties containing the structural motifs of verapamil and of flunarizine, as well as those that are more frequent in known N-type calcium channel antagonists, have been synthesized. Antinociceptive activity on the mouse hot-plate test was used to select molecules to be submitted to further studies. Active compounds were tested in vitro on a PC12 rat pheochromocytoma clonal cell line, to evaluate their action on N-type calcium channels, and on a rat model of neuropathic pain. Two compounds that show N-type calcium channel antagonism and are endowed with potent action on pain and neuropathic pain (3 and 18) have been selected for further studies.

2-pyrrolidinone moiety is not critical for the cognition-enhancing activity of piracetam-like drugs.

Following the indications of previous work, 2-pyrrolidinone moiety of piracetam and piracetam-like compounds has been opened to the corresponding amide derivatives. As found previously in the case of 1,4-diazabicyclo[4.3.0]nonan-9-one compounds, the cognition-enhancing activity of 2-pyrrolidinone compounds is maintained in most cases, suggesting that this moiety is not crucial for activity.

Synthesis and pharmacological evaluation of some (pyridyl)cyclopropylmethyl amines and their methiodides as nicotinic receptor ligands.

A series of 3- and (4-pyridyl)cyclopropylmethyl amines and their quaternary ammonium derivatives have been synthesized; they can be considered as rigid analogues of nicotine. The compounds have been tested on rat cerebral cortex to measure the affinity for the central nicotinic receptor. Only the methiodides show affinity in the micromolar range. The results obtained can provide useful information on the topography of the nicotinic receptor-binding site.

Molecular modulation of muscarinic antagonists. Synthesis and affinity profile of 2,2-diphenyl-2-ethylthio-acetic acid esters designed to probe the binding site cavity.

The synthesis and preliminary pharmacological profile of a new series of muscarinic antagonists, derived from previously studied 2,2-diphenyl-2-ethylthio-acetic acid esters, are reported. The parent molecules were decorated with linkers of different length, carrying an amino group to catch a putative anionic function outside the recognition site of the receptor. It was hoped that the interception of this function would give molecules with higher potency and selectivity. The attempt has not been successful, but a new series of compounds with a peculiar pharmacological profile has been identified.

Synthesis and biological evaluation of 3,7-diazabicyclo[4.3.0]nonan-8-ones as potential nootropic and analgesic drugs.

A series of cis and trans 3,7-diazabicyclo[4.3.0]nonan-8-ones has been synthesized and tested for their ability to revert scopolamine-induced amnesia in the mouse passive-avoidance test. The racemates of the most potent compounds 4 and 7 were separated and tested, but no enantioselectivity was found for the nootropic activity. Compounds 4 and 7 and their enantiomers displayed interesting antihyperalgesic activity in two models of neuropathic pain (streptozotocin-induced and oxalilplatin-induced neuropathy) in comparison with pregabalin.