Antimicrobial/Antibiofilm Activity and Cytotoxic Studies of ß-Thujaplicin Derivatives.

Natural ß-thujaplicin displays a remarkable array of biological activities for the prevention or treatment of various disorders while its tropolone scaffold inspired the synthesis of new analogs. The main goal of the current study was to evaluate the influence of 4-substituted piperazine moieties at position 7 of the ß-thujaplicin scaffold, on the antimicrobial activity. In order to determine the biological activity of the ß-thujaplicin derivatives, a microdilution method was used against a wide variety of bacteria and fungi. Pseudomonas aeruginosa PAO 1 was used for testing antiquorum and antibiofilm effects. Four human tumor cell lines (MCF-7, NCI-H460, HeLa, and HepG2) and a porcine liver derived cell line (PLP2) were used for testing antitumor and cytotoxic activity. The compounds present better antibacterial and antifungal activity in comparison with approved antimicrobials used as control agents. ß-Thujaplicin showed strong antibacterial and antifungal activities against all tested species. Further studies of their antibacterial activity revealed that all compounds presented good antibiofilm and antiquorum effects. Fungi were more susceptible than bacteria to the tested compounds, with the exception of MK150, which possessed the best antibacterial effect. None of the tested compounds, at the GI50 values obtained for the tumor cell lines, have shown toxicity for non-tumor liver cells (PLP2). The prediction of physicochemical properties of the compounds was performed to further explain the structure-activity relationship. Finally, in order to explore a possible mechanism of action of the synthesized compounds, molecular docking studies were performed on CYP51 (14-a lanosterol demethylase), an important component of the fungal cell membrane.

Vitamin D regulates microbiome-dependent cancer immunity.

A role for vitamin D in immune modulation and in cancer has been suggested. In this work, we report that mice with increased availability of vitamin D display greater immune-dependent resistance to transplantable cancers and augmented responses to checkpoint blockade immunotherapies. Similarly, in humans, vitamin D-induced genes correlate with improved responses to immune checkpoint inhibitor treatment as well as with immunity to cancer and increased overall survival. In mice, resistance is attributable to the activity of vitamin D on intestinal epithelial cells, which alters microbiome composition in favor of Bacteroides fragilis, which positively regulates cancer immunity. Our findings indicate a previously unappreciated connection between vitamin D, microbial commensal communities, and immune responses to cancer. Collectively, they highlight vitamin D levels as a potential determinant of cancer immunity and immunotherapy success.

Discovery of 6-[4-(6-nitroxyhexanoyl)piperazin-1-yl)]-9H-purine, as pharmacological post-conditioning agent.

Novel purine analogues bearing nitrate esters were designed and synthesized in an effort to develop compounds triggering endogenous cardioprotective mechanisms such as ischemic preconditioning (IPC) or postconditioning (PostC). The majority of the compounds reduced infarct size compared to the control group in anesthetized rabbits, whereas administration of the most active analogue 16 at a dose of 3.8 µmol/kg resulted on a significant reduction of infarct size, compared to PostC group (13.4 ± 1.9% vs 26.4 ± 2.3%). These findings introduce a novel class of promising pharmacological compounds that could be used as mimics or enhancers of PostC.

Chemotherapy-induced COX-2 upregulation by cancer cells defines their inflammatory properties and limits the efficacy of chemoimmunotherapy combinations.

Cytotoxic therapies, besides directly inducing cancer cell death, can stimulate immune-dependent tumor growth control or paradoxically accelerate tumor progression. The underlying mechanisms dictating these opposing outcomes are poorly defined. Here, we show that cytotoxic therapy acutely upregulates cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) production in cancer cells with pre-existing COX-2 activity. Screening a compound library of 1280 approved drugs, we find that all classes of chemotherapy drugs enhance COX-2 transcription whilst arresting cancer cell proliferation. Genetic manipulation of COX-2 expression or its gene promoter region uncover how augmented COX-2/PGE2 activity post-treatment profoundly alters the inflammatory properties of chemotherapy-treated cancer cells in vivo. Pharmacological COX-2 inhibition boosts the efficacy of the combination of chemotherapy and PD-1 blockade. Crucially, in a poorly immunogenic breast cancer model, only the triple therapy unleashes tumor growth control and significantly reduces relapse and spontaneous metastatic spread in an adjuvant setting. Our findings suggest COX-2/PGE2 upregulation by dying cancer cells acts as a major barrier to cytotoxic therapy-driven tumor immunity and uncover a strategy to improve the outcomes of immunotherapy and chemotherapy combinations.

Novel purine analogues regulate IL-1ß release via inhibition of JAK activity in human aortic smooth muscle cells.

Purine analogues bearing a nitrate ester motif were previously discovered as cardioprotective and anti-inflammatory agents, but the anti-inflammatory mechanism remains to be established. We therefore investigated the anti-inflammatory effect of two purine analogues, MK118 bearing a nitrate ester moiety and the methyl-substituted analogue MK196 in Aortic Smooth Muscle Cells (AoSMCs), with emphasis on IL-1ß release. The AoSMCs were stimulated with LPS with or without purine analogue, followed by ELISA, Olink proteomics, Western blot and real time PCR of NLRP3 inflammasome components. Both purine analogues inhibited the release of proteins involved in inflammation, such as TRAIL, CCL4, CSF1 and IL-1ß in AoSMCs, as well as intracellular gene and protein expression of IL-1ß and NLRP3 inflammasome components. MK196, but not MK118, also inhibited the LPS-induced release of IL-7, CXCL10, PD-L1, FLT3L and CCL20. We also showed that MK118 and possibly MK196 act via inhibition of JAKs. In silico studies showed that the purine moiety is a competent hinge binding motif and that the purine-piperazine scaffold is well accommodated in the lipophilic groove of JAK1-3. Both compounds establish interactions with catalytic amino acids in the active site of JAK1-3 and the terminal nitrate ester of MK118 was revealed as a promising pharmacophore. Our data suggest that MK118 and MK196 inhibit the release of proinflammatory proteins in AoSMCs, and targets JAK1-3 activation. Purine analogues also inhibit the expression of NLRP3 inflammasome genes and proteins and may in the future be evaluated for anti-inflammatory aspects on inflammatory diseases.

Isoxazole substituted chromans against oxidative stress-induced neuronal damage.

We have previously reported that catechol-bearing regioisomers of 5-isoxazolyl-6-hydroxy-chroman display higher in vitro neuroprotective activity, compared to hybrids with other nitrogen heterocycles, but their activity is hampered by cytotoxicity at higher concentrations. In an effort to discover non-cytotoxic isoxazole substituted chromans of high neuroprotective activity, 20 new 3- and 5-substituted (chroman-5-yl)-isoxazoles and (chroman-2-yl)-isoxazoles were synthesized using the copper(I)-catalysed cycloaddition reaction between in situ generated nitrile oxides and terminal acetylenes. An additional aim was to further explore the effect of the isoxazole ring substituents on the neuroprotective activity. The activity of these compounds against oxidative stress-induced death (oxytosis) of neuronal HT22 cells was evaluated and interesting SARs for this group of analogues were derived. The vast majority of new chroman analogues displayed high in vitro neuroprotective activity displaying EC(50) values below 1 µM and lacked cytotoxicity. The position of substituents on the isoxazole ring influences the activity of the regioisomers, with the 3-aryl-5-(chroman-5-yl)-isoxazoles, 17 and 18 and bis-chroman 20 displaying higher neuroprotective activity (EC(50)∼0.3 µM) compared to other (chroman-5-yl) and (chroman-2-yl)-isoxazoles.

The Adhesome Network: Key Components Shaping the Tumour Stroma.

Beyond the conventional perception of solid tumours as mere masses of cancer cells, advanced cancer research focuses on the complex contributions of tumour-associated host cells that are known as "tumour microenvironment" (TME). It has been long appreciated that the tumour stroma, composed mainly of blood vessels, cancer-associated fibroblasts and immune cells, together with the extracellular matrix (ECM), define the tumour architecture and influence cancer cell properties. Besides soluble cues, that mediate the crosstalk between tumour and stroma cells, cell adhesion to ECM arises as a crucial determinant in cancer progression. In this review, we discuss how adhesome, the intracellular protein network formed at cell adhesions, regulate the TME and control malignancy. The role of adhesome extends beyond the physical attachment of cells to ECM and the regulation of cytoskeletal remodelling and acts as a signalling and mechanosensing hub, orchestrating cellular responses that shape the tumour milieu.

Synthesis of a second generation chroman/catechol hybrids and evaluation of their activity in protecting neuronal cells from oxidative stress-induced cell death.

A new generation of chroman/catechol hybrids bearing heterocyclic five-membered rings, such as 1,2,4-oxadiazole 1,3,4-oxadiazole, 1,2,3-triazole, tetrazole and isoxazole, were designed and synthesized. The activity of the new derivatives against oxidative stress induced neuronal damage, was evaluated using glutamate-challenged hippocampal HT22 cells. Compound 3 in which a 3,4-dimethoxyphenyl moiety, is directly attached to the 1,2,4-oxadiazole ring was the most active among the 2-substituted chroman analogues, with EC(50)=254+/-65nM. Concerning the 5-subtituted chroman analogues, isoxazole derivative 29 exhibited the strongest activity (EC(50)=245+/-38nM). However, 29 was cytotoxic at concentrations higher than 1microM, while the triazole analogue 24 (EC(50)=801+/-229nM), was non-toxic at all concentrations tested.

Design, Synthesis, and in†vitro Evaluation of P2X7 Antagonists.

The P2X7 receptor is a promising target for the treatment of various diseases due to its significant role in inflammation and immune cell signaling. This work describes the design, synthesis, and in vitro evaluation of a series of novel derivatives bearing diverse scaffolds as potent P2X7 antagonists. Our approach was based on structural modifications of reported (adamantan-1-yl)methylbenzamides able to inhibit the receptor activation. The adamantane moieties and the amide bond were replaced, and the replacements were evaluated by a ligand-based pharmacophore model. The antagonistic potency of the synthesized analogues was assessed by two-electrode voltage clamp experiments, using Xenopus laevis oocytes that express the human P2X7 receptor. SAR studies suggested that the replacement of the adamantane ring by an aryl-cyclohexyl moiety afforded the most potent antagonists against the activation of the P2X7 cation channel, with analogue 2-chloro-N-[1-(3-(nitrooxymethyl)phenyl)cyclohexyl)methyl]benzamide (56) exhibiting the best potency with an IC50 value of 0.39 µM.

Design and synthesis of novel neuroprotective 1,2-dithiolane/chroman hybrids.

Novel 1,2-dithiolane/chroman hybrids bearing heterocyclic rings such as 1,2,4- and 1,3,4-oxadiazole, 1,2,3-triazole and tetrazole were designed and synthesized. The neuroprotective activity of the new analogues was tested against oxidative stress-induced cell death of glutamate-challenged HT22 hippocampal neurons. Our results show that bioisosteric replacement of amide group in 2-position of the chroman moiety, by 1,3,4-oxadiazole did not affect activity. However, analogue 5 bearing the 1,2,4-oxadiazole moiety showed improved neuroprotective activity. The presence of nitrogen heterocycles strongly influences the neuroprotective activity of 5-substituted chroman derivatives, depending on the nature of heterocycle. Replacement of the amide group of the first generation analogues by 1,2,4-oxadiazole or 1,2,3-triazole resulted in significant improvement of the activity against glutamate induced oxidative stress.

A novel purine analogue bearing nitrate ester prevents platelet activation by ROCK activity inhibition.

Natural purines like ATP, ADP and adenosine have crucial roles in platelet physiology. This knowledge has been significant in drug development and today ADP receptor antagonists are widely used for prevention of thrombotic events following myocardial infarction and ischaemic stroke. Recent studies have shown that a purine analogue bearing nitrate ester group (denoted MK128) has anti-inflammatory effects probably due to its ability to donate nitric oxide (NO). However, other pharmacological mechanisms may contribute to the observed effect. The aim of the present study was to establish the anti-platelet activity and elucidate the underlying molecular mechanism(s) of the purine analogue MK128. We found that MK128 reduced aggregation and secretion induced by the thrombin receptor agonist SFLLRN and nearly abolished aggregation and secretion induced by thromboxane A2 (TxA2) and collagen receptor agonists. The inhibition took place despite blockage of the NO/cGMP signalling system. Furthermore, interaction between MK128 and platelet purinergic receptors did not explain the observed inhibition. Instead, we found that MK128 concentration-dependently inhibited Rho-associated kinase (ROCK), which led to decreased ROCK-dependent myosin phosphatase target subunit (MYPT)-1 phosphorylation and suppression of platelet functional responses.

Pharmacological pre- and post- conditioning agents: reperfusion-injury of the heart revisited.

Ischemic preconditioning (PC) and postconditioning (PostC) are endogenous mechanisms of protection of the ischemic heart. In brief, short cycles of sublethal ischemia separated by brief periods of reperfusion render the heart resistant to infarction from a subsequent lethal episode of prolonged ischemia. Although PC is a powerful form of protection, its clinical application is limited because of ethical and practical reasons. It is of interest that multiple very short periods of ischemia and reperfusion applied at the onset of reperfusion are also capable in limiting the infarct size. In fact, the short ischemic insults in PC have to be applied before the onset of sustained period of ischemia which cannot be precisely anticipated. On the contrary, the very brief insults in postconditioning (PostC) have to be applied immediately after the end of the long ischemia thus making the intervention more easily applicable. Both mechanisms reduce the infarct size by limiting the reperfusion injury. Pharmacological PC and PostC represent ideal alternatives that may substitute the short ischemic insults for pharmaceuticals means. The components of PC share two pathways, one that involves the mitochondrial K(ATP) channels- free radicals and PKC and another one that involves adenosine and PKC. Reperfusion injury salvage kinases (RISK) prevent the mitochondrial permeability transition pores (mPTP) which destroy the mitochondria and cause cell death. PC via PKC and PostC via gradual restoration of pH at reperfusion up-regulate RISK and preserve viable part of the ischemic region of the heart. In order to confer pharmacological protection, novel therapeutic strategies, based on the knowledge of the ligands, of the receptors and of the intracellular signaling pathways have emerged. Adenosine, nicorandil and other agents have been already used as pharmacological mimetics of ischemic PC in multicenter trials. Furthermore, agents that increase RISK or directly prevent mPTP are also under investigation as PostC analogues. We summarize recent studies focused on the pharmacological interventions and on the discovery of novel agents that may reduce the infarct size.

Design and synthesis of nitrate esters of aromatic heterocyclic compounds as pharmacological preconditioning agents.

Ischemic preconditioning (IPC) constitutes an endogenous protective mechanism in which one or more brief periods of myocardial ischemia and reperfusion render the myocardium resistant to a subsequent more-sustained ischemic insult. Pharmacological preconditioning represents an ideal alternative of IPC. We now describe the design and synthesis of indole, quinoline, and purine systems with an attached pharmacophoric nitrate ester group. The indole and quinoline derivatives 4 and 5 possess structural features of the nitrate containing K(ATP) channel openers. Purine analogues 11 and 12, substituted at the position 6 by a piperidine moiety and at position 9 by an alkyl nitrate, could combine the effects of the nitrate containing K(ATP) channel openers and those of adenosine. Compound 13 bears the nicotinamide moiety of nicorandil instead of nitrate ester. Compounds 4, 5, and 11 reduced infarction and the levels of malondialdehyde (MDA) at reperfusion in anesthetized rabbits. Compounds 12 and 13 did not significantly reduce the infarct size. Analogues 4 and 5 increased cGMP and MDA during ischemia, while combined analogue 4 and mitoK(ATP) blocker 5-hydroxydecanoic acid (5-HD) abrogated this benefit suggesting an action through mitoK(ATP) channel opening. Treatment with derivative 11 combined with 5-HD as well as treatment with 11 and adenosine receptor blocker 8-(p-sulfophenyl)theophylline (SPT) did not abrogate cardioprotection. Compound 11 is a lead molecule for the synthesis of novel analogues possessing a dual mode of action through cGMP-mitoK(ATP) channel opening-free radicals and through adenosine receptors.

Nitrate Esters of Heteroaromatic Compounds as Candida albicans CYP51 Enzyme Inhibitors.

Four heteroaromatic compounds bearing nitrate esters were selected using a virtual-screening procedure as putative sterol 14α-demethylase (CYP51) Candida albicans inhibitors. Compounds were examined for their inhibition on C. albicans growth and biofilm formation as well as for their toxicity. NMR spectroscopy studies, in silico docking, and molecular dynamics simulations were used to investigate further the selectivity of these compounds to fungal CYP51. All compounds exhibited good antimicrobial properties, indicated with low minimal inhibitory concentrations and ability to inhibit formation of fungal biofilm. Moreover, all of the compounds had the ability to inhibit growth of C. albicans cells. N-(2-Nitrooxyethyl)-1Η-indole-2-carboxamide was the only compound with selectivity on C. albicans CYP51 that did not exhibit cytotoxic effect on cells isolated from liver and should be further investigated for selective application in new leads for the treatment of candidiasis.

Design and synthesis of novel quinolinone-3-aminoamides and their alpha-lipoic acid adducts as antioxidant and anti-inflammatory agents.

A series of N-substituted-quinolinone-3-aminoamides and their hybrids containing the alpha-lipoic acid functionality were designed and synthesized as potential bifunctional agents combining antioxidant and anti-inflammatory activity. The new compounds were evaluated for their antioxidant activity and for their ability to inhibit in vitro lipoxygenase as well as for their anti-inflammatory activity in vivo. In general, the derivatives were found to be potent antioxidant or anti-inflammatory agents. The results are discussed in terms of structure-activity relationships and an attempt is made to define the structural features required for activity.

Chroman/catechol hybrids: synthesis and evaluation of their activity against oxidative stress induced cellular damage.

Three series of chromans substituted at positions 2 or 5 by catechol derivatives were synthesized, and their activity against oxidative stress induced cellular damage was studied. Specifically, the ability of the new molecules to protect cultured cells from H(2)O(2)-induced DNA damage was evaluated using single cell gel electrophoresis (comet assay), while the neuroprotective activity of the new compounds against oxidative stress induced programmed cell death was studied using glutamate-challanged hippocampal HT22 cells. The majority of the new compounds are stronger neuroprotectants than quercetin. 5-Substituted chroman analogues such as the caffeic acid amides 12 and 16 and the dihydrostilbene analogue 24 were the most potent against both H(2)O(2)- and glutamate-induced damage in Jurkat T cells and HT22 cells, respectively.

Vitamin E derivatives: a patent review (2010 - 2015).

INTRODUCTION: The vitamin E family consists of four tocopherols and four tocotrienols. α-Tocopherol is the most studied member of this family for its antioxidant and non-antioxidant properties, while tocotrienols have attracted recent research interest. The structural motifs of the vitamin E family and specifically the chroman moiety, are amenable to various modifications in order to improve their bioactivities towards numerous therapeutic targets. AREAS COVERED: This review includes the patent literature from 2010 - 2015 related to vitamin E derivatives and it is focused on 2-, 5- or 6-substituted chroman analogues. The patent search was performed using Reaxys® and esp@cenet. EXPERT OPINION: The chroman moiety of vitamin E is a privileged structure and an essential pharmacophore which inspired organic chemists to synthesize new analogues with improved bioactivities. Modifications at the 2- and 5- positions of the chroman ring resulted in very interesting active compounds in cellular and animal models of diseases related to oxidative stress. More recent publications and patents reported 6-substituted chromans as anticancer agents in vitro and in vivo. Additionally, an emerging interest is observed towards the use of vitamin E analogues incorporated in drug delivery systems and for medical imaging as contrast agents or fluorescent probes.

Structure-activity relationships of antineoplastic ring-substituted ether phospholipid derivatives.

PURPOSE: Previous studies have shown that alkylphosphocholines (APCs) exhibit strong antineoplastic activity against various tumour cell lines in vitro and in several animal models. The current study was designed to investigate the influence of cycloalkane rings on the antiproliferative activity of APCs against a panel of eight human and animal cell lines (PC3, MCF7, A431, Hela, PC12, U937, K562, CHO). Specifically, we explored the effect of the presence of 4-alkylidenecyclohexyl and cycloalkylidene groups in alkoxyethyl and alkoxyphosphodiester ether lipids, respectively. In addition, the haemolytic activity of the new ring-substituted ether phospholipids (EP) was evaluated. METHODS: Cells were exposed to various concentrations of the compounds for 72 h. The cytotoxicity was determined with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye reduction assay. Similarly, red blood cells were distributed in 96-well microplates and treated with the test compounds at concentrations ranging from 100 to 6.25 microM for 1 h. After centrifugation, the absorbance of the supernatants was measured at 550 nm. RESULTS: The majority of the compounds tested exhibited significant cytotoxic activity which depended on both the ring size and position with respect to the phosphate moiety, as well as the head group. Among the cycloalkylidene series the 11-adamantylideneundecyl-substituted N-methylmorpholino EP 13 was the most potent and exhibited broad-spectrum anticancer activity comparable to or superior to that of hexadecylphosphocholine (HePC). All the adamantylidene-substituted EPs were nonhaemolytic (concentration that exhibits 50% haemolytic activity, HC(50), >100 microM). Furthermore, the cyclohexylidene-substituted analogues were more potent against the cell lines tested, with the exception of U937 and K562, than the cyclodecapentylidene-substituted compounds. Hydrogenation of the double bond in the cycloalkylidene-substituted EPs (compounds 14 and 15) resulted in improvement of anticancer activity. Among the 2-(4-alkylidenecyclohexyloxy)ethyl EPs, 2-(4-hexadylidenecyclohexyloxy)ethyl phosphocholine (22) possessed the highest broad-spectrum cytotoxic activity than all the other analogues of this series and was nonhaemolytic (HC(50) >100 microM). In general, the 2-(4-alkylidenecyclohexyloxy)ethyl-substituted EPs were more active against the more resistant cell lines U937, K562 and CHO than HePC. CONCLUSIONS: The presence of cycloalkane rings in the lipid portion of APCs reduces haemolytic effects compared to HePC and in several analogues results in improved antineoplastic activity.

Strategies in the design of prodrugs of anti-HIV agents.

Control of AIDS requires development of special therapeutic strategies in order to reduce the level of monocyte/macrophage HIV infection, to prevent spread of HIV within the monocyte/macrophage reservoir, to maintain a therapeutically effective drug concentration in sanctuaries such as the brain and to overcome the problem of cellular resistance mechanisms. A popular approach towards this end has been the development of prodrugs of anti-HIV drugs. This review covers the different strategies devised for the design of prodrugs of anti-HIV agents with emphasis on the recent findings in this field of research. Thus, prodrugs of nucleoside reverse transcriptase inhibitors (NRTIs) including, 5'-O carboxylic ester derivatives, 5'-O- monophosphate analogues, macromolecular derivatives, prodrugs of purine nucleosides, prodrugs of acyclic nucleosides, homo and hetero dinucleotides, prodrugs of non-classical nucleoside analogues, boranophosphate triesters of NRTIs, and prodrugs of protease inhibitors including acyl-substituted prodrugs, prodrugs with increased water solubility, monophosphate prodrugs, and conjugates of HIV protease inhibitors with a reverse transcriptase inhibitor through spontaneously cleavable linkers, constitute the subject of this review.

Antileishmanial ring-substituted ether phospholipids.

Three series of ring-substituted ether phospholipids were synthesized carrying N,N,N-trimethylammonium, N-methylpiperidino, or N-methylmorpholino headgroups. The first series is substituted by 2-cyclohexyloxyethyl or 2-(4-alkylidenecyclohexyloxy)ethyl groups, the second series by cyclohexylidenealkyl or adamantylidenealkyl moieties, and the third series by 2-aryloxyethyl or 6-aryloxyhexyl groups in the alkyl portion of the molecule. The antileishmanial activity of the new compounds was evaluated in vitro against the promastigote forms of L. donovani and L. infantum using an MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide)-based microassay as a marker of cell viability. Analogues 12, 15, 24, 30, 32, 41, 43, and 45 were more potent than the control compound miltefosine (hexadecylphosphocholine) against both L. donovani and L. infantum while, derivatives 13 and 42 were equipotent to miltefosine. Analogues 16, 17, 19, 20 were more potent than miltefosine against L. infantumand compounds 27, 31, 44 were more active than miltefosine against L. donovani. Differential scanning calorimetry (DSC) was used to probe the role of individual ether phospholipids on the physicochemical properties of model membranes. The DSC scans showed that the active compounds have a more profound effect on the thermotropic properties of model membrane bilayers than the less active ones.