New hydrophilic riminophenazines as potent antiprotozoal agents

Malaria and leishmaniasis are life-threatening human parasitosis caused by protozoa-infected insect vectors. In most of affected countries, the expansive and hazardous therapies available to fight protozoan infections are generally harmed by the spread of drug resistance phenomena upon prolonged treatments. This scenario highlights the need of novel antiprotozoal agents hopefully able to act trough new mechanism(s) of action. Interestingly, the fat-soluble antimycobacterial drug Clofazimine was reported to exhibit a moderate antiprotozoal action and some interesting antileishmanial in vitro and in vivo effects were reported in few preliminary, yet promising, studies.1,4 Intrigued by these results, we have previously prepared a series of basic Clofazimine analogues which demonstrated the beneficial effects of the introduction of a basic head on the antiprotozoal activity.5 Here, to further investigate the role of balancing between the lipo- and hydrophilicity on the antiparasitic activity of these riminophenazines, we report the synthesis and the in vitro evaluation as leishmanicidal (L. tropica and L. infantum promastigotes) and antiplasmodial (chloroquine sensitive and resistant P. falciparum strains) agents of a family of hydrophilic C-2 aminopyridinyl substituted riminophenazines, bearing in C-3 differently decorated basic side chains. Results showed that most of the new compounds potently inhibited the growth of protozoa with IC50 in the high nanomolar range and underlined the key role of the hydrophilic C-2 aminopyridinyl moiety to improve the leishmanicidal activity. In addition, the length and the nature of the basic side chain differently influenced the antiprotozoal activity and the selectivity index versus mammalian cells, providing useful information for further structural optimizations

Target-Oriented Development Of Novel Antiprotozoal Agents: Celastrol Carboxamides As Inhibitors Of Leishmania Hsp90

The Leishmania isoform of the 90kDa Heat Shock Protein (LsHsp90), a chaperone known to assist the folding of more than 200 client proteins, was reported to be generally involved in parasite differentiation from promastigote to amastigote possessing a pivotal role during heat-induced cellular stress. Moreover, it was demonstrated that an impair of the native functions of LsHsp90 through the action of active-site inhibitors can exert a detrimental effect on the natural parasite life-cycle ultimately leading to its death. Celastrol is natural triterpene exhibiting a plethora of in vitro and in vivo activities. Among them, this pentacyclic compound is reported to possess a promising antiproliferative activity thanks to its ability of interacting with the chaperone cycle of the human isoform of Hsp90 (hHsp90). Moreover, celastrol derivatives (e.g. the methyl ester pristimerin, Figure 1) have also exhibited an interesting antiprotozoal activity. With the aim of building a target-oriented approach to treat Leishmania infections based on the inhibition of LsHsp90, we prepared two basic carboxamides celastrol derivatives (SS-1 and SS-2) to enhance its leishmanicidal activity and selectivity of action by deducting its unspecific cytotoxicity (measured as IC50 on HMEC-1 cell lines). Accordingly, celastrol and the two basic derivatives SS-1 and SS-2 were in vitro tested for their leishmanicidal activity against promastigotes of Leishmania tropica and L. infantum and, in parallel, their mechanism of action was investigated as well via ad hoc in vitro experiments using a recombinant Hsp90 from L. braziliensis (LbHsp90). In virtue of their pH sensitive basic heads, both SS-1 and SS-2 were found to be more potent (IC50 in the nanomolar range) and selective leishmanicidal agents than celastrol itself. Furthermore, we were able to demonstrate that SS-1 and SS-2 successfully (in vitro) inhibited the native kinase activity of LbHsp90 highlighting the key role of the inhibition of this chaperone in their mechanism of action

High antiplasmodial activity of novel plasmepsins I and II inhibitors

The aim of this study was to develop new antiplasmodial compounds acting through distinct mechanisms during both the liver and the blood stages of the parasite life cycle. Compounds were designed on the basis of the "double-drug" approach: primaquine, which has been linked to statine-based inhibitors of plasmepsins (PLMs), the plasmodial aspartic proteases involved in degradation of hemeoglobin. The compounds were tested in vitro for anti-PLM I/PLM II activities and against chloroquine-sensitive (D10) and chloroquine-resistant (W2) strains of P. falciparum. An antiplasmodial activity (IC50) as low as 0.1 M was obtained, an excellent improvement in comparison with inhibitors previously reported (IC50 = 2-20 M). The killing activity was equally directed against both P. falciparum strains and was correlated to lipophilicity (calculated as ALogP), for all compounds but one (9). All compounds inhibited PLM I and PLM II in the nanomolar range (Ki = 1-700 nM). The most promising compounds (2, 6, 10) were not cytotoxic against human fibroblasts at 100 M and were highly selective for PLMs vs human cathepsin

Epimerization of Lupinine to Epilupinine and viceversa. Reexamination of the Structures of Lupinal and Epilupinal

Although the epimerization of lupinine (I) has been largely investigated, a previously not obsd. compd. of formula C10H17NO was now isolated from the mixt. of alkaloids that remains after the sepn. of epilupinine (II). It is insol. in dry Et2O but sol. in EtOH, from which it is recovered as an Et2O-sol. oil that slowly returns to the Et2O-insol. solid form. For these characteristics and based on GC/MS, 1H-NMR, and IR data, it is considered as the inner salt III of the common enolic form IV of lupinal and epilupinal, with which it is in equil. when standing in soln. The oily form, but not the solid one, is able to improve the conversion of I to II, establishing the role of the aldehydes in the epimerization process. It was obsd. that also II can be converted to I. Finally, the solid lupinal described by Zaboev should be considered as being identical to the now isolated inner salt III, while the oily epilupinal of Wicky and Schumann is, indeed, a mixt. of epilupinal with a minor amt. of lupinal, which, on standing, is converted to the inner salt III of the common enolic form IV

Modulation of thiol homeostasis induced by a novel H2S-releasing compound

Recently, the physiological function of hydrogen sulfide (H2S) has been discovered and a potential therapeutic use of this gas for the treatment of diseases characterized by its altered concentrations has been suggested. A possible approach for a therapeutic administration of H2S is represented by molecules able to release H2S in a controlled manner, mimicking what happens physiologically. Dithiolethiones have been found to behave as H2S donors in physiological conditions. N-Acetylcysteine (NAC) is under investigation as potential therapeutic agent against several different pathologies characterized by the occurrence of oxidative stress and a decrease in GSH although results deriving from large, multi-center, prospective clinical trials are on most case contradictory and inconclusive. It is possible that the scarce efficacy of NAC is due to its low oral bioavailability (about 8%). We have recently observed that both dithiolethione containing molecules and the derivative of NAC, N-acetylcysteine ethylester (NACET) are able to significantly reduce circulating and tissue levels of hyperomocysteinemia (hCys), probably via an increase of the thiol to disulfide ratio in extracellular fluids. Mild hCys is considered an independent risk factor for cardiovascular and cerebrovascular disease. Starting from these observations, we synthesized new dithiolethione\u2013cysteine hybrids (ACS94, ACS96, ACS97) with the assumption that they could have synergic effect in reducing plasma hCys, as well (by tissue glutathione increase) correcting the redox imbalance process present in several diseases. The effects on thiols pool in different organs and in plasma, after iv or oral administration of NAC (10mg/kg) or equimolar ACS94 to healthy rats and after ip administration of paracetamol (as a model of hepatic toxicity), have been investigated. The results clearly indicate that ACS94 protects from paracetamol induced hepatic toxicity better than NAC and that ACS94 prevents paracetamol induced thiol depletion in kidney and liver. In addition a more significant decrease of hCys compared to NAC, was observed in some rat target organs and in plasma

Novel Hydrophilic Riminophenazines as Potent Antiprotozoal Agents

SAR studies on a set of novel hydrophilic C-2 aminopyridinyl riminophenazines bearing variously functionalized basic side chains at C-3 were conducted. The novel compounds were evaluated for in vitro activity against two different species of Leishmania promastigotes, intramacrophage Leishmania amastigotes, chloroquine-sensitive and chloroquine-resistant strains of P. falciparum, and also against mature-stage P. falciparum gametocytes. Their cytotoxicity was evaluated as well on BMDM cell lines. Most of the new compounds potently inhibited the growth of both genera of protozoa with IC50 values in the high nanomolar range and good selectivities versus mammalian cells. Besides their potent activity against asexual intraerythrocytic stages of P. falciparum, three compounds showed potential as transmission-blocking agents. The key role of the hydrophilic C-2 aminopyridinyl substituent to improve the leishmanicidal activity and the influence of the length and the nature of the basic side chain on the antiprotozoal activity and cytotoxicity were underlined

H2S releasing aspirin protects amyloid beta induced cell toxicity in BV-2 microglial cells

\u3b2-Amyloid (A\u3b2) plaques are characteristic hallmarks of Alzheimer's disease. In the present study, we examined the neuroprotective effects of S-aspirin, a hydrogen sulfide (H(2)S)-releasing aspirin, on A\u3b2-induced cell toxicity. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that S-aspirin, but not aspirin, significantly increased cell viability in BV-2 microglial cells, indicating that S-aspirin may protect cells against injury via releasing H(2)S. S-aspirin at 2.5-10 \u3bcM significantly increased cell viability and decreased lactate dehydrogenase release in A\u3b2-treated BV-2 microglial cells. Western blotting analysis showed that S-aspirin suppressed the protein expression levels of cyclooxygenase-2 and growth arrest DNA damage (GADD). These data suggest that S-aspirin may protect microglial cells by inhibition of A\u3b2-induced inflammation and cell cycle re-entry. To study whether S-aspirin can protect mitochondria function, mitochondria membrane potential was measured with molecular probe JC-1. It was found that S-aspirin protected mitochondria from A\u3b2-induced loss of mitochondrial member potential. (\u394\u3a8m). In addition, S-aspirin also prevented A\u3b2-induced activation of p38-mitogen activated protein kinase (MAPK). In conclusion, our results suggest that S-aspirin may protect microglial injury via inhibition of inflammation, prevention of mitochondria function, and stimulation of cell growth via stimulating p38-MAPK pathway. Our study may suggest that S-aspirin may have potential therapeutic value for the treatment of Alzheimer's disease

Quinolizidinyl derivatives as potent inhibitors of acetyl- and butyryl-cholinesterase (ACHE and BCHE) of potential interest for the treatment of Alzheimer's disease

Alzheimer\u2019s disease (AD) is a progressive, neurodegenerative disorder characterized by a gradual decline of cognitive processes. The multifactorial pathogenesis of AD includes accumulation of aggregates of -amyloid and loss of cholinergic neurons, with consequent deficit of the neurotransmitter acetylcholine (ACh). The inhibition of AChE, that is responsible for the breakdown of ACh, has proven useful to relieve some cognitive and behavioral symptoms of AD. In advancing AD, AChE levels in the brain are declining, but a progressive increase (up to 90%)of BChE is observed , which too is able, even if at lower rate, to hydrolize ACh. Selective BChE inhibitors have already been reported to increase ACh levels in the brain, and,interestingly, to also reduce the formation of abnormal amyloid. Potent and selective BChE inhibitors are represented by some physostigmine derivatives as phenethylcymserine and MF-8622 and by some phenothiazine derivatives, as ethopropazine and Astra 1397, whose high activity, with respect to other phenothiazine-derived drugs, is related to their particular basic chains. In order to achieve novel cholinesterase inhibitors, either dual or, even better, selective for BChE, we have prepared and tested a number of derivatives of phenothiazine and related tricyclic systems, bearing a peculiar basic moiety, as the bulky quinolizidine ring, linked through different kinds of spacer. A few quinolizidinyl derivatives of 6-hydroxycoumarin were also included in the set of test compounds, as well as some well known tricyclic drugs(triflupromazine, methyxene, ethopropazine, Astra 1397 and quinacrine) serving as reference inhibitors. Compounds were evaluated for their inhibitory activities of AChE and BChE from bovine erythrocytes and equine serum, respectively. The tested compounds exhibited activity against both enzymes, but the inhibition of BChE was generally stronger than that of AChE, often with submicromolar IC50 values (9-lupinylthioxanthene: IC50, for AChE = 7.0 M; for BChE = 0.15 M). An inverted selectivity was observed for acridine and coumarin derivatives (6-chloro-9-[3-(lupinylthio)propylamino]-2-methoxyacridine: IC50, for AChE = 0.22 M; for BChE = 0.69 M); 6-[(3-lupinylthio)propoxy]coumarin: IC50, for AChE = 0.35 M; for BChE = 5.4 M). Molecular modeling studies provided valuable insights to interpret structure-affinity and structure-selectivity relationships and suggest appropriate structural modifications to improve the binding affinities at either one of the two enzymes

Clofazimine analogs as antimalarial drugs

Clofazimine is a fat-soluble riminophenazine dye used in combination with rifampicin and dapsone as multidrug therapy (MDT) for the treatment of leprosy. Tetramethylpiperidine \u2013substituted phenazines, structurally related to clofazimine, have been described to be endowed with activity against multidrug resistant strains of Plasmodium falciparum.1 With the aim to study more thorougly the antimalarial potentialities of this kind of structures, we synthesized a set of novel aminophenazines bearing a monocyclic or bicyclic basic head linked through a short alkylic chain to the imino nitrogen in position 2 on the phenazine nucleus. The new compounds inhibited the growth of cloroquine sensitive (CQ-S) and chloroquine resistant (CQ-R) strains of P. falciparum with IC50 in low micromolar or submicromolar range. Among the compounds so far synthesized, the quinolizidinyl derivatives were the most active. In this series, activity and resistance index improved with the elongation of the alkyl spacer . Compound VB 667 showing IC50=123 nM against D-10 (CQ-S) strain and IC50=62.6 nM against W-2 (CQ-R) strain, represents our new lead compound worthy of further studies focused on its optimisation