Thioxanthenone-based derivatives as multitarget therapeutic leads for Alzheimer's disease.

A set of twenty-five thioxanthene-9-one and xanthene-9-one derivatives, that were previously shown to inhibit cholinesterases (ChEs) and amyloid ß (Aß40) aggregation, were evaluated for the inhibition of tau protein aggregation. All compounds exhibited a good activity, and eight of them (5-8, 10, 14, 15 and 20) shared comparable low micromolar inhibitory potency versus Aß40 aggregation and human acetylcholinesterase (AChE), while inhibiting human butyrylcholinesterase (BChE) even at submicromolar concentration. Compound 20 showed outstanding biological data, inhibiting tau protein and Aß40 aggregation with IC50 = 1.8 and 1.3 µM, respectively. Moreover, at 0.1-10 µM it also exhibited neuroprotective activity against tau toxicity induced by okadoic acid in human neuroblastoma SH-SY5Y cells, that was comparable to that of estradiol and PD38. In preliminary toxicity studies, these interesting results for compound 20 are somewhat conflicting with a narrow safety window. However, compound 10, although endowed with a little lower potency for tau and Aß aggregation inhibition additionally demonstrated good inhibition of ChEs and rather low cytotoxicity. Compound 4 is also worth of note for its high potency as hBChE inhibitor (IC50 = 7 nM) and for the three order of magnitude selectivity versus hAChE. Molecular modelling studies were performed to explain the different behavior of compounds 4 and 20 towards hBChE. The observed balance of the inhibitory potencies versus the relevant targets indicates the thioxanthene-9-one derivatives as potential MTDLs for AD therapy, provided that the safety window will be improved by further structural variations, currently under investigation.

Optimization of the clofazimine structure leads to a highly water-soluble C3-aminopyridinyl riminophenazine endowed with improved anti-Wnt and anti-cancer activity in vitro and in vivo.

Triple-negative breast cancer (TNBC) is a cancer subtype critically dependent upon excessive activation of Wnt pathway. The anti-mycobacterial drug clofazimine is an efficient inhibitor of canonical Wnt signaling in TNBC, reducing tumor cell proliferation in vitro and in animal models. These properties make clofazimine a candidate to become first targeted therapy against TNBC. In this work, we optimized the clofazimine structure to enhance its water solubility and potency as a Wnt inhibitor. After extensive structure-activity relationships investigations, the riminophenazine 5-(4-(chlorophenyl)-3-((2-(piperazin-1-yl)ethyl)imino)-N-(pyridin-3-yl)-3,5-dihydrophenazin-2-amine (MU17) was identified as the new lead compound for the riminophenazine-based targeted therapy against TNBC and Wnt-dependent cancers. Compared to clofazimine, the water-soluble MU17 displayed a 7-fold improved potency against Wnt signaling in TNBC cells resulting in on-target suppression of tumor growth in a patient-derived mouse model of TNBC. Moreover, allowing the administration of reduced yet effective dosages, MU17 displayed no adverse effects, most notably no clofazimine-related skin coloration.

Synthesis and Antiplasmodial Activity of Novel Chloroquine Analogues with Bulky Basic Side Chains.

Chloroquine is commonly used in the treatment and prevention of malaria, but Plasmodium falciparum, the main species responsible for malaria-related deaths, has developed resistance against this drug. Twenty-seven novel chloroquine (CQ) analogues characterized by a side chain terminated with a bulky basic head group, i.e., octahydro-2H-quinolizine and 1,2,3,4,5,6-hexahydro-1,5-methano-8H-pyrido[1,2-a][1,5]diazocin-8-one, were synthesized and tested for activity against D-10 (CQ-susceptible) and W-2 (CQ-resistant) strains of P. falciparum. Most compounds were found to be active against both strains with nanomolar or sub-micromolar IC50 values. Eleven compounds were found to be 2.7- to 13.4-fold more potent than CQ against the W-2 strain; among them, four cytisine derivatives appear to be of particular interest, as they combine high potency with low cytotoxicity against two human cell lines (HMEC-1 and HepG2) along with easier synthetic accessibility. Replacement of the 4-NH group with a sulfur bridge maintained antiplasmodial activity at a lower level, but produced an improvement in the resistance factor. These compounds warrant further investigation as potential drugs for use in the fight against malaria.

Role of neuronal nicotinic acetylcholine receptors (nAChRs) on learning and memory in zebrafish.

RATIONALE: Neuronal nicotinic acetylcholine receptors (nAChRs) play a modulatory role in cognition, and zebrafish provide a preclinical model to study learning and memory. OBJECTIVES: We investigated the effect of nicotine (NIC) and some new cytisine-derived partial agonists (CC4 and CC26) on spatial memory in zebrafish using a rapid assay on T-maze task. The role of α4/α6ß2 and the α7 nAChRs in NIC-induced memory enhancement was evaluated using selective nAChR antagonists. RESULTS: Low and high doses of NIC, cytisine (CYT), CC4 and CC26 respectively improved and worsened the mean running time, showing an inverted U dose-response function. The effective dose (ED50) (×10⁻5 mg/kg) was 0.4 for CC4, 4.5 for CYT, 140 for NIC and 200 for CC26. NIC-induced cognitive enhancement was reduced by the selective nAChR subtype antagonists: methyllycaconitine (MLA) for α7, α-conotoxin (MII) for α6ß2, dihydro-ß-erythroidine (DhßE) for α4ß2, the nonselective antagonist mecamylamine (MEC) and the muscarinic antagonist scopolamine (SCOP), with DhßE being more active than MLA or MII. All the partial agonists blocked the cognitive enhancement. The improvement with the maximal active dose of each partial agonist was blocked by low doses of DhßE (0.001 mg/kg) and MII (0.01 mg/kg). MLA reduced the effects of CC26 and CC4 at doses of 0.01 and 1 mg/kg, respectively, but did not antagonize CYT-induced memory improvement at any of the tested dose. No change in swimming activity was observed. CONCLUSIONS: Our findings demonstrate that zebrafish make a useful model for the rapid screening of the effect of new α4ß2 nAChR compounds on spatial memory.

Primary anti-proliferative activity evaluation of 1-(quinolizidin-1'-yl)methyl- and 1-(ω-tert-amino)alkyl-substituted 2-phenyl-, 2-benzyl- and 2-[(benzotriazol-1/2-yl)methyl]benzimidazoles on human cancer cell lines.

Twenty benzimidazole derivatives bearing in position 1 a ([Formula: see text]-tert-amino)alkyl chain (mainly quinolizidin-1-ylmethyl) and in position 2 an aromatic moiety (phenyl, benzyl or benzotriazol-1/2-ylmethyl) were evaluated at the National Cancer Institute (NCI) for anti-proliferative activity against a panel of 60 human cancer cell lines. Four compounds (6, 7, 9 and 10) displayed a large spectrum of activity with [Formula: see text] 10 [Formula: see text] on 24-57 cell lines, while thirteen compounds exhibited sub-micromolar or even nanomolar activity against single cell lines, such as leukemia CCRF-CEM, HL-60 and MOLT-4, CNS cancer SF-268 and, particularly, renal cancer UO-31, sometimes with outstanding selectivity (compounds 5-7, 11, 13 and 18).

(+)-Laburnamine, a natural selective ligand and partial agonist for the α4ß2 nicotinic receptor subtype.

(+)-Laburnamine (1), a rare alkaloid extracted from Laburnum anagyroides seeds (∼4 mg from 1 kg), was shown to bind with high affinity (Ki, 293 nM) to the α4ß2 nicotinic receptor subtype, which is, respectively, 126 and 136 times higher than to the α3ß4 (Ki 37 µM) and α7 subtypes (Ki 40 µM). When its ability to release [(3)H]-dopamine from striatal slices was tested in a functional assay, compound 1 behaved as a partial agonist with an EC50 of 5.8 µM and an Emax that was 43% that of nicotine. When incubated with nicotine in the same assay, 1 prevented a maximal effect from being reached.

CC4, a dimer of cytisine, is a selective partial agonist at α4ß2/α6ß2 nAChR with improved selectivity for tobacco smoking cessation.

BACKGROUND AND PURPOSE: Many of the addictive and rewarding effects of nicotine are due to its actions on the neuronal nicotinic ACh receptor (nAChR) subtypes expressed in dopaminergic mesocorticolimbic cells. The partial agonists, cytisine and varenicline, are helpful smoking cessation aids. These drugs have a number of side effects that limit their usefulness. The aim of this study was to investigate the preclinical pharmacology of the cytisine dimer1,2-bisN-cytisinylethane (CC4). EXPERIMENTAL APPROACH: The effects of CC4 on nAChRs were investigated using in vitro assays and animal behaviours. KEY RESULTS: When electrophysiologically tested using heterologously expressed human subtypes, CC4 was less efficacious than cytisine on neuronal α4ß2, α3ß4, α7 and muscle-type receptors, and had no effect on 5-hydroxytryptamine3 receptors. Acting through α4ß2 and α6ß2 nAChRs, CC4 is a partial agonist of nAChR-mediated striatal dopamine release and, when co-incubated with nicotine, prevented nicotine's maximal effect on this response. In addition, it had low affinity for, and was less efficacious than nicotine and cytisine on the α3ß4 and α7-nAChR subtypes. Like cytisine and nicotine, CC4-induced conditioned place preference (CPP), and its self-administration shows an inverted-U dose-response curve. Pretreatment with non-reinforcing doses of CC4 significantly reduced nicotine-induced self-administration and CPP without affecting motor functions. CONCLUSION AND IMPLICATIONS: Our in vitro and in vivo findings reveal that CC4 selectively reduces behaviours associated with nicotine addiction consistent with the partial agonist selectivity of CC4 for ß2-nAChRs. The results support the possible development of CC4 or its derivatives as a promising drug for tobacco smoking cessation.

Efficacy of novel acridine derivatives in the inhibition of hPrP90-231 prion protein fragment toxicity.

Quinacrine is one of the few molecules tested to treat patients affected by prion diseases, although the clinical outcome is largely unsatisfactory. To identify novel derivatives with higher neuroprotective activity, we evaluated the effects of a small library of acridine derivatives. The 6-chloro-2-methoxyacridine derivatives bearing on position 9 a quinolizidin-1-ylamino (Q1, Q2) or a quinolizidin-1-ylalkylamino residue (Q3, Q4, Q6, Q7), the thio-bioisoster of Q3 (Q5), the 9-(N-lupinylthiopropyl)amino derivative (Q8) and simple acridines (Q9 and Q10) were considered. We compared the effects of quinacrine and these novel analogues in the inhibition of the cytotoxic activity and protease K (PK) resistance of the human prion protein fragment 90-231 (hPrP90-231). We demonstrate that quinacrine caused a significant reduction of hPrP90-231 toxicity due to its binding to the fragment and the prevention of its conversion in a toxic isoform. All acridine derivatives analyzed showed high affinity binding for hPrP90-231, but only Q3 and Q10, caused a significant reduction of hPrP90-231 cytotoxicity, with higher efficacy than quinacrine. We attempted to correlate the cytoprotective effects of the new compounds with some biochemical parameters (binding affinity to hPrP90-231, intrinsic fluorescence quenching, hydrophobic amino acid exposure), but a direct relationship occurred only with the reduction of PK resistance, likely due to the prevention of the acquisition of the ß-sheet-rich toxic conformation. These data represent interesting leads for further modifications of the basic side chain and the substituent pattern of the acridine nucleus to develop novel compounds with improved antiprion activity to be tested in in vivo experimental setting.

Antiviral and cytotoxic activities of aminoarylazo compounds and aryltriazene derivatives.

Twelve aminoarylazocompounds (A-C) and 46 aryltriazene 7 derivatives (D-G) have been synthesized and evaluated in cell-based assays for cytotoxicity and antiviral activity against a panel of 10 RNA and DNA viruses. Eight aminoazocompounds and 27 aryltriazene derivatives exhibited antiviral activity, sometimes of high level, against one or more viruses. A marked activity against BVDV and YFV was prevailing among the former compounds, while the latter type of compounds affected mainly CVB-2 and RSV. None of the active compounds inhibited the multiplication of HIV-1, VSV and VV. Arranged in order of decreasing potency and selectivity versus the host cell lines, the best compounds are the following; BVDV: 1>7>8>4; YFV: 7>5; CVB-2: 25>56>18; RSV: 14>20>55>38>18>19; HSV-1: 2. For these compounds the EC(50) ranged from 1.6 microM (1) to 12 microM (18), and the S. I. from 19.4 (1) to 4.2 (2). Thus the aminoarylazo and aryltriazene substructures appear as interesting molecular component for developing antiviral agents against ss RNA viruses, particularly against RSV and BVDV, which are important human and veterinary pathogens. Finally, molecular modeling investigations indicated that compounds of structure A-C, active against BVDV, could work targeting the viral RNA-dependent RNA-polymerase (RdRp), having been observed a good agreement between the trends of the estimated IC(50) and the experimental EC(50) values.

Organometallic compounds in oncology: implications of novel organotins as antitumor agents.

Since the introduction of cisplatin in cancer therapy, metal complexes and organometallic compounds have been gaining growing importance in oncology. The impressive clinical effectiveness of cisplatin is limited by significant side effects and the emergence of drug resistance. Thus, novel classic and unconventional Pt(II) and Pt(IV) complexes have been introduced in therapy or are presently in advanced clinical trials. Moreover, innovative non-platinum metal-based antitumor agents, whose activity does not rely on direct DNA damage and may involve proteins and enzymes, have been developed. Gold and tin derivatives are enjoying an increasing interest and appear very promising as potential drug candidates.

In vitro cytotoxic activity of tri-n-butyltin(IV)lupinylsulfide hydrogen fumarate (IST-FS 35) and preliminary antitumor activity in vivo.

The cytotoxicity in vitro and antitumor activity in vivo of the organotin compound tri-n-butyltin(IV)lupinylsulfide hydrogen fumarate (IST-FS 35) have been investigated. The IC50 values obtained in a panel of tumor cell lines were compared to those of the parental compound IST-FS 29 in the same cells. IST-FS 35 resulted significantly more active than IST-FS 29 with IC50 values in the range 0.16-1.8 microM. Toxicity studies in vivo, after intravenous administration of escalating concentrations of IST-FS 35, provided the identification of the maximal tolerated dose (3.5 mg/kg) which was employed as therapeutic dose in the antitumor activity experiments. Preliminary results, in transplanted murine tumor models, revealed that both the P388 myelomonocytic leukaemia and the B16-F10 melanoma, implanted subcutaneously in BDF1 mice, were inhibited about 96% in their tumor volume at day 11, following a single intravenous injection of the compound. Additional studies are mandatory to unravel the mechanism of action for the development of IST-FS 35 as potential antitumor drug.

Long-term exposure to the new nicotinic antagonist 1,2-bisN-cytisinylethane upregulates nicotinic receptor subtypes of SH-SY5Y human neuroblastoma cells.

Nicotinic drug treatment can affect the expression of neuronal nicotinic acetylcholine receptors (nAChR) both in vivo and in vitro through molecular mechanisms not fully understood. The present study investigated the effect of the novel cytisine dimer 1,2-bisN-cytisinylethane (CC4) on nAChR natively expressed by SH-SY5Y neuroblastoma cells in culture. CC4 lacked the agonist properties of cytisine and was a potent antagonist (IC50=220 nM) on nAChRs. Chronic treatment of SH-SY5Y cells with 1 mM CC4 for 48 h increased the expression of 3H-epibatidine (3H-Epi; 3-4-fold) or 125I-alpha-bungarotoxin (125I-alphaBgtx; 1.2-fold) sensitive receptors present on the cell membrane and in the intracellular pool. Comparable data were obtained with nicotine or cytisine, but not with carbamylcholine, d-tubocurarine, di-hydro-beta-erythroidine or hexametonium. Immunoprecipitation and immunopurification studies showed that the increase in 3H-Epi-binding receptors was due to the enhanced expression of alpha3beta2 and alpha3beta2beta4 subtypes without changes in subunit mRNA transcription or receptor half-life. The upregulation was not dependent on agonist/antagonist properties of the drugs, and did not concern muscarinic or serotonin receptors. Whole-cell patch clamp analysis of CC4-treated cells demonstrated larger nicotine-evoked inward currents with augmented sensitivity to the blockers alpha-conotoxin MII or methyllycaconitine. In conclusion, chronic treatment with CC4 increased the number of nAChRs containing beta2 and alpha7 subunits on the plasma membrane, where they were functionally active. In the case of beta2-containing receptors, we propose that CC4, by binding to intracellular receptors, triggered a conformational reorganisation of intracellular subunits that stimulated preferential assembly and membrane-directed trafficking of beta2-containing receptor subtypes..

2-Phenyl-3-(quinolizidin-1-yl)-5-substituted indoles as platelet antiaggregating agents.

A set of ten 2-phenyl-3-(quinolizidin-1-yl)-5-substituted indoles was prepared through the Fischer cyclization of lupinyl- and epi-lupinylphenylketone 4-substituted phenylhydrazones. Compounds were tested for antiaggregating activity on human platelets activated by adenosine diphosphate (ADP), collagen and adrenaline. At 2.5 x 10(-4) M concentration most compounds strongly inhibited the aggregation induced by all the agonists considered and many of them still displayed good activity at 0.625 x 10(-4) M concentration. The least active (1c) and one of the most active (1d) compounds were also tested for antiaggregating activity on rabbit platelets activated by ADP, PAF and sodium arachidonate. Both the compounds were active against ADP and PAF, but only 1d inhibited the arachidonate-induced aggregation (100% at 8 x 10(-6) M concentration) and increased the bleeding time in mice. The same compounds were subjected to a general pharmacological screening and found to display several activities; of particular interest was the dose dependent reduction of serum cholesterol and heparin precipitating betalipoproteins in hypercholesterolemic mice exerted by 1c, which was still significant at the oral dose of 10 mg/kg.

Quinolizidinyl derivatives of iminodibenzyl and phenothiazine as multidrug resistance modulators in ovarian cancer cells.

The development of multidrug-resistance (MDR) in neoplastic cells is often responsible for the therapy failure and poor outcome of a number of human cancers. MDR may be associated with the expression of the multidrug transporter glycoprotein p170, encoded by the MDR1 gene, which acts as an ATP-dependent efflux pump by reducing the intracellular accumulation of some cytotoxic agents. A variety of iminodibenzyl and phenothiazine derivatives, characterized by the presence of a bicyclic, strongly basic, and highly lipophilic quinolizidine nucleus, were synthesized to investigate their ability to modulate the MDR phenotype. A set of 10 of them (named 1-10), bearing quinolizidine moiety linked through different connecting chains, were tested as chemoresistance-reversing agents on doxorubicin-resistant ovarian cancer cells (A2780-DX3). A 51-fold resistance to doxorubicin was reported in the A2780-DX3 compared to the parental sensitive A2780 WT with mean IC(50) values of 0.02 and 1.02 muM, respectively. Moreover, overexpression of the glycoprotein p170 in the resistant cell line was detected by Western blot analysis. By cytotoxicity assays and time-course experiments, different treatment schedules with resistance modulators (including clomipramine as reference drug) and doxorubicin were taken into account. The 16 h exposure of cells to 1 muM of modulator before doxorubicin demonstrated to be superior in sensitizing the resistant cell line. In particular, compounds 8, 7, 10, and 4 increasingly potentiated doxorubicin cytotoxicity, up to 5.6-fold in A2780-DX3 cells. The present results suggest promising indications for further development of these compounds as chemosensitizing drugs.

Novel sigma binding site ligands as inhibitors of cell proliferation in breast cancer.

Sigma receptors, namely sigma1 and sigma2, have been shown to be expressed in a variety of human cell lines playing a role in cell growth. In the human breast, they are absent in normal mammary tissue but expressed in tumors, particularly in the proliferating stage. The study presented here concerns nine newly synthesized ligands for sigma receptors. The compounds are of general structure consisting of: five (1alpha/1beta-arylalkyl)quinolizidines including two thioisosteres and four spiro-[3,4-dihydro-1,2,4-benzotriazino-3,4'-(1'-substituted) piperidines]. These compounds exhibited varying degrees of affinity for sigma receptors and were able to inhibit the growth of MCF-7 and MDA-MB 231 human breast cancer cell lines, in vitro. Good to moderate binding to at receptors occurred with all tested ligands. However, affinity for sigma2 appeared more evident with compounds FN/C-2 and FN/C-4 (spiro-[3,4-dihydro-1,2,4-benzotriazino-3,4'-(1'-substituted) piperidines] derivatives). In addition, higher cytotoxic activity of FN/C-2 and FN/C-4 with IC50 values below 100 microM in MCF-7 and lower than 40 microM in MDA-MB 231 was revealed. The data from the current study show that these novel sigma receptor ligands exhibit interesting cytotoxic activity and suggest their potential for development as antitumor agents.

Antitumor activity of a new orally active organotin compound: a preliminary study in murine tumor models.

The toxicity and antitumor activity of the novel organotin compound triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29), administered by the oral route, have been evaluated against three transplantable murine tumor models: P388 lymphocytic leukemia, B16F10 melanoma and 3LL Lewis lung carcinoma. Mild and reversible signs of acute toxicity such as behavioral symptoms, weight loss and histological alterations were mainly reported at the highest single dose of 28 mg/kg. Conversely, lower concentrations of compound ranging from 7 to 21 mg/kg did not result in major toxic effects, even after repeated dosing. The antitumor activity studies showed that fractionation dosing, rather than single bolus administration, over 1 week, might prove more active and better tolerated by allowing the achievement of the highest therapeutic total dose of IST-FS 29 (42 mg/kg). Indeed, repeated administrations of IST-FS 29 resulted in marked significant improvement of antitumor activity against B16F10 (50% of tumor volume inhibition, p = 0.0003) and, to a greater extent, 3LL (90% of tumor volume inhibition, p = 0.0001) tumors. These results indicate that IST-FS 29 might be a suitable candidate as an orally administrable anticancer drug and support its further development in human tumor xenografts.

Chemosensitivity of glioblastoma cells during treatment with the organo-tin compound triethyltin(IV)lupinylsulfide hydrochloride.

Malignant gliomas are the most common primary brain tumors in humans. However, poor response to conventional therapeutic approaches, including chemotherapy, leads invariably to disease recurrence and progression. The organo-tin derivative triethyltin(IV)lupinylsulfide hydrochloride (IST-FS 29) was identified and developed as potential antiproliferative agent in human cancer cell lines. However, for its peculiar chemical structure and good lipophilicity, this compound also appeared an eligible candidate for the treatment of gliobastoma cells. The present experiments were designed to explore the in vitro effects of IST-FS 29 on four human glioblastoma cell lines: A-172, DBTRG.05MG, U-87MG and CAS-1. The average IC50 values were obtained by MTT assay and ranged between 3 and 10 microM. Time-course assays with cell recovery after drug withdrawal, demonstrated marked cytotoxicity following exposure to IST-FS 29 for 8, 24 and 72 h. Cultures treated for 8 h were able to partially re-grow by 144 h; on the contrary, longer times of exposure did not allow surviving cells to recover from the damage and actively proliferate. Cell morphology of cultures exposed to IST-FS 29 was assessed by inverted light microscopy after 24 and 72 h and was more consistent with cell death by necrosis which included cell size reduction, vacuolation of cytoplasm, round dying cells. The present results and our previous data, in vitro and in vivo, indicate the relevant cytotoxic activity of this organo-tin compound and suggest that IST-FS 29 might be a promising novel agent to be developed for the treatment of malignant brain neoplasms.