The novel non-hallucinogenic compound DM506 (3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole) induces sedative- and anxiolytic-like activity in mice by a mechanism involving 5-HT2A receptor activation.

The anxiolytic and sedative-like effects of 3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole (DM506), a non-hallucinogenic compound derived from ibogamine, were studied in mice. The behavioral effects were examined using Elevated O-maze and novelty suppressed feeding (NSFT) tests, open field test, and loss of righting reflex (LORR) test. The results showed that 15 mg/kg DM506 induced acute and long-lasting anxiolytic-like activity in naive and stressed/anxious mice, respectively. Repeated administration of 5 mg/kg DM506 did not cause cumulative anxiolytic activity or any side effects. Higher doses of DM506 (40 mg/kg) induced sedative-like activity, which was inhibited by a selective 5-HT2A receptor antagonist, volinanserin. Electroencephalography results showed that 15 mg/kg DM506 fumarate increased the transition from a highly alert state (fast γ wavelength) to a more synchronized deep-sleeping activity (δ wavelength), which is reflected in the sedative/anxiolytic activity in mice but without the head-twitch response observed in hallucinogens. The functional, radioligand binding, and molecular docking results showed that DM506 binds to the agonist sites of human 5-HT2A (Ki = 24 nM) and 5-HT2B (Ki = 16 nM) receptors and activates them with a potency (EC50) of 9 nM and 3 nM, respectively. DM506 was relatively less potent and behaved as a partial agonist (efficacy <80%) for both receptor subtypes compared to the full agonist DOI (2,5-dimethoxy-4-iodoamphetamine). Our study showed for the first time that the non-hallucinogenic compound DM506 induces anxiolytic- and sedative-like activities in naïve and stressed/anxious mice in a dose-, time-, and volinanserin-sensitive manner, likely through mechanisms involving 5-HT2A receptor activation.

Tetrazole and oxadiazole derivatives as bioisosteres of tariquidar and elacridar: New potent P-gp modulators acting as MDR reversers.

New 2,5- and 1,5-disubstituted tetrazoles, and 2,5-disubstituted-1,3,4-oxadiazoles were synthesized as tariquidar and elacridar derivatives and studied as multidrug resistance (MDR) reversers. Their behaviour on the three ABC transporters P-gp, MRP1 and BCRP was investigated. All compounds inhibited the P-gp transport activity in MDCK-MDR1 cells overexpressing P-gp, showing EC50 values even in the low nanomolar range (compounds 15, 22). Oxadiazole derivatives were able to increase the antiproliferative effect of doxorubicin in MDCK-MDR1 and in HT29/DX cells confirming their nature of P-gp modulators, with derivative 15 being the most potent in these assays. Compound 15 also displayed a dual inhibitory effect showing good activities towards both P-gp and BCRP. A computational study suggested a common interaction pattern on P-gp for most of the potent compounds. The bioisosteric substitution of the amide group of lead compounds allowed identifying a new set of potent oxadiazole derivatives that modulate MDR through inhibition of the P-gp efflux activity. If compared to previous amide derivatives, the introduction of the heterocycle rings greatly enhances the activity on P-gp, introduces in two compounds a moderate inhibitory activity on MRP1 and maintains in some cases the effect on BCRP, leading to the unveiling of dual inhibitor 15.

Chemical, Pharmacological, and Structural Characterization of Novel Acrylamide-Derived Modulators of the GABAA Receptor.

Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABAA receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed "DM compounds") derived from the previously characterized GABAA and the nicotinic α7 receptor modulator (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary αßγ GABAA receptor. Using electrophysiology, we show that the DM compounds, and the structurally related (E)-3-furan-2-yl-N-phenylacrylamide (PAM-4), have concurrent potentiating and inhibitory effects that can be isolated and observed under appropriate recording conditions. The potentiating efficacies of the DM compounds are similar to those of neurosteroids and benzodiazepines (ΔG ∼ -1.5 kcal/mol). Molecular docking, functionally confirmed by site-directed mutagenesis experiments, indicate that receptor potentiation is mediated by interactions with the classic anesthetic binding sites located in the transmembrane domain of the intersubunit interfaces. Inhibition by the DM compounds and PAM-4 was abolished in the receptor containing the α1(V256S) mutation, suggestive of similarities in the mechanism of action with that of inhibitory neurosteroids. Functional competition and mutagenesis experiments, however, indicate that the sites mediating inhibition by the DM compounds and PAM-4 differ from those mediating the action of the inhibitory steroid pregnenolone sulfate. SIGNIFICANCE STATEMENT: We have synthesized and characterized the actions of novel acrylamide-derived compounds on the mammalian GABAA receptor. We show that the compounds have concurrent potentiating effects mediated by the classic anesthetic binding sites, and inhibitory actions that bear mechanistic resemblance to but do not share binding sites with, the inhibitory steroid pregnenolone sulfate.

DM506 (3-Methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole fumarate), a Novel Derivative of Ibogamine, Inhibits α7 and α9α10 Nicotinic Acetylcholine Receptors by Different Allosteric Mechanisms.

The main objective of this study was to determine the pharmacological activity and molecular mechanism of action of DM506 (3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole fumarate), a novel ibogamine derivative, at different nicotinic acetylcholine receptor (nAChR) subtypes. The functional results showed that DM506 neither activates nor potentiates but inhibits ACh-evoked currents at each rat nAChR subtype in a non-competitive manner. The receptor selectivity for DM506 inhibition follows the sequence: α9α10 (IC50 = 5.1 ± 0.3 µM) ≅ α7ß2 (5.6 ± 0.2 µM) ∼ α7 (6.4 ± 0.5 µM) > α6/α3ß2ß3 (25 ± 1 µM) > α4ß2 (62 ± 4 µM) ≅ α3ß4 (70 ± 5 µM). No significance differences in DM506 potency were observed between rat and human α7 and α9α10 nAChRs. These results also indicated that the ß2 subunit is not involved or is less relevant in the activity of DM506 at the α7ß2 nAChR. DM506 inhibits the α7 and α9α10 nAChRs in a voltage-dependent and voltage-independent manner, respectively. Molecular docking and molecular dynamics studies showed that DM506 forms stable interactions with a putative site located in the α7 cytoplasmic domain and with two intersubunit sites in the extracellular-transmembrane junction of the α9α10 nAChR, one located in the α10(+)/α10(─) interface and another in the α10(+)/α9(─) interface. This study shows for the first time that DM506 inhibits both α9α10 and α7 nAChR subtypes by novel allosteric mechanisms likely involving modulation of the extracellular-transmembrane domain junction and cytoplasmic domain, respectively, but not by direct competitive antagonism or open channel block.

The Antinociceptive Activity of (E)-3-(thiophen-2-yl)- N -(p-tolyl)acrylamide in Mice Is Reduced by (E)-3-(furan-2-yl)- N -methyl- N -(p-tolyl)acrylamide Through Opposing Modulatory Mechanisms at the α7 Nicotinic Acetylcholine Receptor.

BACKGROUND: The primary objective of this study was to characterize the pharmacological and behavioral activity of 2 novel compounds, DM497 [(E)-3-(thiophen-2-yl)- N -(p-tolyl)acrylamide] and DM490 [(E)-3-(furan-2-yl)- N -methyl- N -(p-tolyl)acrylamide], structural derivatives of PAM-2, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor (nAChR). METHODS: A mouse model of oxaliplatin-induced neuropathic pain (2.4 mg/kg, 10 injections) was used to test the pain-relieving properties of DM497 and DM490. To assess possible mechanisms of action, the activity of these compounds was determined at heterologously expressed α7 and α9α10 nAChRs, and voltage-gated N-type calcium channel (Ca V 2.2) using electrophysiological techniques. RESULTS: Cold plate tests indicated that 10 mg/kg DM497 was able to decrease neuropathic pain in mice induced by the chemotherapeutic agent oxaliplatin. In contrast, DM490 induced neither pro- nor antinociceptive activity but inhibited DM497's effect at equivalent dose (30 mg/kg). These effects are not a product of changes in motor coordination or locomotor activity. At α7 nAChRs, DM497 potentiated whereas DM490 inhibited its activity. In addition, DM490 antagonized the α9α10 nAChR with >8-fold higher potency than that for DM497. In contrast, DM497 and DM490 had minimal inhibitory activity at the Ca V 2.2 channel. Considering that DM497 did not increase the mouse exploratory activity, an indirect anxiolytic mechanism was not responsible for the observed antineuropathic effect. CONCLUSIONS: The antinociceptive activity of DM497 and the concomitant inhibitory effect of DM490 are mediated by opposing modulatory mechanisms on the α7 nAChR, whereas the involvement of other possible nociception targets such as the α9α10 nAChR and Ca V 2.2 channel can be ruled out.

Recent Advances in the Discovery of Nicotinic Acetylcholine Receptor Allosteric Modulators.

Positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), silent agonists, allosteric activating PAMs and neutral or silent allosteric modulators are compounds capable of modulating the nicotinic receptor by interacting at allosteric modulatory sites distinct from the orthosteric sites. This survey is focused on the compounds that have been shown or have been designed to interact with nicotinic receptors as allosteric modulators of different subtypes, mainly α7 and α4ß2. Minimal chemical changes can cause a different pharmacological profile, which can then lead to the design of selective modulators. Experimental evidence supports the use of allosteric modulators as therapeutic tools for neurological and non-neurological conditions.

Synthetic Approaches to Piperazine-Containing Drugs Approved by FDA in the Period of 2011-2023.

The piperazine moiety is often found in drugs or in bioactive molecules. This widespread presence is due to different possible roles depending on the position in the molecule and on the therapeutic class, but it also depends on the chemical reactivity of piperazine-based synthons, which facilitate its insertion into the molecule. In this paper, we take into consideration the piperazine-containing drugs approved by the Food and Drug Administration between January 2011 and June 2023, and the synthetic methodologies used to prepare the compounds in the discovery and process chemistry are reviewed.

New Dual P-Glycoprotein (P-gp) and Human Carbonic Anhydrase XII (hCA XII) Inhibitors as Multidrug Resistance (MDR) Reversers in Cancer Cells.

In a continuing search of dual P-gp and hCA XII inhibitors, we synthesized and studied new N,N-bis(alkanol)amine aryl diester derivatives characterized by the presence of a coumarin group. These hybrids contain both P-gp and hCA XII binding groups to synergistically overcome the P-gp-mediated multidrug resistance (MDR) in cancer cells expressing both P-gp and hCA XII. Indeed, hCA XII modulates the efflux activity of P-gp and the inhibition of hCA XII reduces the intracellular pH, thereby decreasing the ATPase activity of P-gp. All compounds showed inhibitory activities on P-gp and hCA XII proteins taken individually, and many of them displayed a synergistic effect in HT29/DOX and A549/DOX cells that overexpress both P-gp and hCA XII, being more potent than in K562/DOX cells overexpressing only P-gp. Compounds 5 and 14 were identified as promising chemosensitizer agents for selective inhibition in MDR cancer cells overexpressing both P-gp and hCA XII.

The Tetrahydroisoquinoline Scaffold in ABC Transporter Inhibitors that Act as Multidrug Resistance (MDR) Reversers.

BACKGROUND: The failure of anticancer chemotherapy is often due to the development of resistance to a variety of anticancer drugs. This phenomenon is called multidrug resistance (MDR) and is related to the overexpression of ABC transporters, such as P-glycoprotein, multidrug resistance- associated protein 1 and breast cancer resistance protein. Over the past few decades, several ABC protein modulators have been discovered and studied as a possible approach to evade MDR and increase the success of anticancer chemotherapy. Nevertheless, the co-administration of pump inhibitors with cytotoxic drugs, which are substrates of the transporters, does not appear to be associated with an improvement in the therapeutic efficacy of antitumor agents. However, more recently discovered MDR reversing agents, such as the two tetrahydroisoquinoline derivatives tariquidar and elacridar, are characterized by high affinity towards the ABC proteins and by reduced negative properties. Consequently, many analogs of these two derivatives have been synthesized, with the aim of optimizing their MDR reversal properties. OBJECTIVE: This review aims to describe the MDR modulators carrying the tetraidroisoquinoline scaffold reported in the literature in the period 2009-2021, highlighting the structural characteristics that confer potency and/or selectivity towards the three ABC transport proteins. RESULTS AND CONCLUSION: Many compounds have been synthesized in the last twelve years showing interesting properties, both in terms of potency and selectivity. Although clear structure-activity relationships can be drawn only by considering strictly related compounds, some of the compounds reviewed could be promising starting points for the design of new ABC protein inhibitors.

The piperazine scaffold for novel drug discovery efforts: the evidence to date.

INTRODUCTION: Piperazine is a structural element present in drugs belonging to various chemical classes and used for numerous different therapeutic applications; it has been considered a privileged scaffold for drug design. AREAS COVERED: The authors have searched examples of piperazine-containing compounds among drugs recently approved by the FDA and in some research fields (nicotinic receptor modulators, compounds acting against cancer, and bacterial multidrug resistance), looking in particular to the design behind the insertion of this moiety. EXPERT OPINION: Piperazine is widely used due to its peculiar characteristics, such as solubility, basicity, chemical reactivity, and conformational properties. This moiety has represented an important tool to modulate pharmacokinetic and pharmacodynamic properties of drugs.

Overcoming Multidrug Resistance (MDR): Design, Biological Evaluation and Molecular Modelling Studies of 2,4-Substituted Quinazoline Derivatives.

Some 2,4-disubstituted quinazolines were synthesized and studied as multidrug resistance (MDR) reversers. The new derivatives carried the quinazoline-4-amine scaffold found in modulators of the ABC transporters involved in MDR, as the TKIs gefitinib and erlotinib. Their behaviour on the three ABC transporters, P-gp, MRP1 and BCRP, was investigated. Almost all compounds inhibited the P-gp activity in MDCK-MDR1 cells overexpressing P-gp, showing EC50 values in the nanomolar range (1 d, 1 e, 2 a, 2 c, 2 e). Some compounds were active also towards MRP1 and/or BCRP. Docking results obtained by in silico studies on the P-gp crystal structure highlighted common features for the most potent compounds. The P-gp selective compound 1 e was able to increase the doxorubicin uptake in HT29/DX cells and to restore its antineoplastic activity in resistant cancer cells in the same extent of sensitive cells. Compound 2 a displayed a dual inhibitory effect showing good activities towards both P-gp and BCRP.

Type I and type II positive allosteric modulators of α7 nicotinic acetylcholine receptors induce antidepressant-like activity in mice by a mechanism involving receptor potentiation but not neurotransmitter reuptake inhibition. Correlation with mTOR intracellular pathway activation.

The aim of this study is to determine whether type I and type II positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (nAChRs) induce antidepressant-like activity in mice after acute, subchronic, and chronic treatments, and to assess whether α7-PAMs inhibit neurotransmitter transporters and activate mTOR (mammalian target of rapamycin) and/or ERK (extracellular signal-regulated protein kinases) signaling. The forced swim (FST) and tail suspension (TST) test results indicated that NS-1738 (type I PAM), PNU-120596 and PAM-2 (type II PAMs) induce antidepressant-like activity after subchronic treatment, whereas PAM-2 was also active after chronic treatment. Methyllycaconitine (α7-antagonist) inhibited the observed effects, highlighting the involvement of α7 nAChRs in this process. Drug interaction studies showed synergism between PAM-2 and bupropion (antidepressant), but not between PAM-2 and DMXBA (α7-agonist). The studied PAMs showed no high affinity (< 1 µM) for the human dopamine, serotonin, and noradrenaline transporters, suggesting that transporter inhibition is not the underlying mechanism for the observed activity. To assess whether mTOR and ERK signaling pathways are involved in the activity of α7-PAMs, the phosphorylation status of key signaling nodes was determined in prefrontal cortex and hippocampus from mice chronically treated with PAM-2. In conclusion, the antidepressant-like activity of type I and type II PAMs is mediated by a mechanism involving α7 potentiation but not α7 desensitization or neurotransmitter transporter blockade, and is correlated with activation of both mTOR and ERK signaling pathways. These results support the view that α7-PAMs might be clinically used to ameliorate depression disorders .

(E)-3-Furan-2-yl-N-p-tolyl-acrylamide and its Derivative DM489 Decrease Neuropathic Pain in Mice Predominantly by α7 Nicotinic Acetylcholine Receptor Potentiation.

The main objective of this study was to determine whether (E)-3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) and its structural derivative DM489 produce anti-neuropathic pain activity using the streptozotocin (STZ)- and oxaliplatin-induced neuropathic pain animal models. To assess possible mechanisms of action, the pharmacological activity of these compounds was determined at α7 and α9α10 nicotinic acetylcholine receptors (nAChRs) and CaV2.2 channels expressed alone or coexpressed with G protein-coupled GABAB receptors. The animal results indicated that a single dose of 3 mg/kg PAM-2 or DM489 decreases STZ-induced neuropathic pain in mice, and chemotherapy-induced neuropathic pain is decreased by PAM-2 (3 mg/kg) and DM489 (10 mg/kg). The observed anti-neuropathic pain activity was inhibited by the α7-selective antagonist methyllycaconitine. The coadministration of oxaliplatin with an inactive dose (1 mg/kg) of PAM-2 decreased the development of neuropathic pain after 14, but not 7, days of cotreatment. The electrophysiological results indicated that PAM-2 potentiates human (h) and rat (r) α7 nAChRs with 2-7 times higher potency than that for hCaV2.2 channel inhibition and an even greater difference compared to that for rα9α10 nAChR inhibition. These results support the notion that α7 nAChR potentiation is likely the predominant molecular mechanism underlying the observed anti-nociceptive pain activity of these compounds.

Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Dual P-Glycoprotein and CA XII Inhibitors: A New Strategy to Reverse the P-gp Mediated Multidrug Resistance (MDR) in Cancer Cells.

A new series of N,N-bis(alkanol)amine aryl diesters was synthesized and studied as dual P-glycoprotein (P-gp) and carbonic anhydrase XII inhibitors (CA XII). These hybrids should be able to synergistically overcome P-gp mediated multidrug resistance (MDR) in cancer cells. It was reported that the efflux activity of P-gp could be modulated by CA XII, as the pH reduction caused by CA XII inhibition produces a significant decrease in P-gp ATPase activity. The new compounds reported here feature both P-gp and CA XII binding moieties. These hybrids contain a N,N-bis(alkanol)amine diester scaffold found in P-glycoprotein ligands and a coumarin or benzene sulfonamide moiety to target CA XII. Many compounds displayed a dual activity against P-gp and CA XII being active in the Rhd 123 uptake test on K562/DOX cells and in the hCA XII inhibition test. On LoVo/DOX cells, that overexpress both P-gp and CA XII, some coumarin derivatives showed a high MDR reversal effect in Rhd 123 uptake and doxorubicin cytotoxicity enhancement tests. In particular, compounds 7 and 8 showed higher activity than verapamil and were more potent on LoVo/DOX than on K562/DOX cells overexpressing only P-gp. They can be considered as valuable candidates for selective P-gp/CA XII inhibition in MDR cancer cells.

6,7-Dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline amides and corresponding ester isosteres as multidrug resistance reversers.

Aiming to deepen the structure-activity relationships of the two P-glycoprotein (P-gp) modulators elacridar and tariquidar, a new series of amide and ester derivatives carrying a 6,7-dimethoxy-2-phenethyl-1,2,3,4-tetrahydroisoquinoline scaffold linked to different methoxy-substituted aryl moieties were synthesised. The obtained compounds were evaluated for their P-gp interaction profile and selectivity towards the two other ABC transporters, multidrug-resistance-associated protein-1 and breast cancer resistance protein, showing to be very active and selective versus P-gp. Two amide derivatives, displaying the best P-gp activity, were tested in co-administration with the antineoplastic drug doxorubicin in different cancer cell lines, showing a significant sensitising activity towards doxorubicin. The investigation on the chemical stability of the derivatives towards spontaneous or enzymatic hydrolysis, showed that amides are stable in both models while some ester compounds were hydrolysed in human plasma. This study allowed us to identify two chemosensitizers that behave as non-transported substrates and are characterised by different selectivity profiles.

Design, synthesis and biological evaluation of stereo- and regioisomers of amino aryl esters as multidrug resistance (MDR) reversers.

Stereo- and regioisomers of a series of N,N-bis(alkanol)amine aryl ester derivatives have been prepared and studied as multidrug resistance (MDR) modulators. The new compounds contain a 2-(methyl)propyl chain combined with a 3-, 5- or 7-methylenes long chain and carry different aromatic ester portions. Thus, these compounds have a methyl group on the 3-methylenes chain and represent branched homologues of previously studied derivatives. The introduction of the methyl group gives origin to a stereogenic center and consequently to (R) and (S) enantiomers. In the pirarubicin uptake assay on K562/DOX cell line these compounds showed good activity and efficacy and in many cases enantioselectivity was observed. Docking studies confirmed the influence of the stereocenter on the interaction in the P-gp pocket. The P-gp interaction mechanism and selectivity towards MRP1 and BCRP were also evaluated on MDCK transfected cells overexpressing the three transporters. Almost all these compounds inhibited both P-gp and BCRP, but only derivatives with specific structural characteristics showed MRP1 activity. Moreover, two compounds, (S)-3 and (R)-7, showed the ability to induce collateral sensitivity (CS) against MDR cells. Therefore, these two CS-promoting agents could be considered interesting leads for the development of selective cytotoxic agents for drug-resistant cells.

Modulation of the spacer in N,N-bis(alkanol)amine aryl ester heterodimers led to the discovery of a series of highly potent P-glycoprotein-based multidrug resistance (MDR) modulators.

In this study, a new series of N,N-bis(alkanol)amine aryl ester heterodimers was synthesized and studied. The new compounds were designed based on the structures of our previous arylamine ester derivatives endowed with high P-gp-dependent multidrug resistance reversing activity on a multidrug-resistant leukemia cell line. All new compounds were active in the pirarubicin uptake assay on the doxorubicin-resistant erythroleukemia K562 cells (K562/DOX). Compounds bearing a linker made up of 10 methylenes showed unprecedented high reversal activities regardless of the combination of aromatic moieties. Docking results obtained by an in silico study supported the data obtained by the biological tests and a study devoted to establish the chemical stability in phosphate buffer solution (PBS) and human plasma showed that only a few compounds exhibited a significant degradation in the human plasma matrix. Ten selected non-hydrolysable derivatives were able to inhibit the P-gp-mediated rhodamine-123 efflux on K562/DOX cells, and the evaluation of their apparent permeability and ATP consumption on other cell lines suggested that the compounds can behave as unambiguous or not transported substrates. The activity of these the compounds on the transport proteins breast cancer resistance protein (BCRP) and multidrug resistance associated protein 1 (MRP1) was also analyzed. All tested derivatives displayed a moderate potency on the BCRP overexpressing cells; while only four molecules showed to be effective on MRP1 overexpressing cells, highlighting a clear structural requirement for selectivity. In conclusion, we have identified a new very powerful series of compounds which represent interesting leads for the development of new potent and efficacious P-gp-dependent MDR modulators.

EC18 as a Tool To Understand the Role of HCN4 Channels in Mediating Hyperpolarization-Activated Current in Tissues.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are membrane proteins encoded by four genes (HCN1-4) and widely distributed in the central and peripheral nervous system and in the heart. HCN channels are involved in several physiological functions, including the generation of rhythmic activity, and are considered important drug targets if compounds with isoform selectivity are developed. At present, however, few compounds are known, which are able to discriminate among HCN channel isoforms. The inclusion of the three-methylene chain of zatebradine into a cyclohexane ring gave a compound (3a) showing a 5-fold preference for HCN4 channels, and ability to selectively modulate Ih in different tissues. Compound 3a has been tested for its ability to reduce Ih and to interact with other ion channels in the heart and the central nervous system. Its preference for HCN4 channels makes this compound useful to elucidate the contribution of this isoform in the physiological and pathological processes involving hyperpolarization-activated current.

New Rigid Nicotine Analogues, Carrying a Norbornane Moiety, Are Potent Agonists of α7 and α3* Nicotinic Receptors.

A three-dimensional database search has been applied to design a series of endo- and exo-3-(pyridin-3-yl)bicyclo[2.2.1]heptan-2-amines as nicotinic receptor ligands. The synthesized compounds were tested in radioligand binding assay on rat cortex against [3H]-cytisine and [3H]-methyllycaconitine to measure their affinity for α4ß2* and α7* nicotinic receptors. The new derivatives showed some preference for the α4ß2* over the α7* subtype, with their affinity being dependent on the endo/exo isomerism and on the methylation degree of the basic nitrogen. The endo primary amines displayed the lowest Ki values on both receptor subtypes. Selected compounds (1a, 2a, 3a, and 6a) were tested on heterologously expressed α4ß2, α7, and α3ß2 receptors and on SHSY-5Y cells. Compounds 1a and 2a showed α4ß2 antagonistic properties while behaved as full agonists on recombinant α7 and on SHSY5Y cells. On the α3ß2 subtype, only the chloro derivative 2a showed full agonist activity and submicromolar potency (EC50 = 0.43 µM). The primary amines described here represent new chemotypes for the α7 and α3* receptor subtypes.