Proinflammatory cytokine interleukin (IL)-1β is a key mediator of the inflammatory response in atherosclerosis. Targeting IL-1β represents a new approach for the anti-inflammatory therapy of cardiovascular diseases. Based on our previous data demonstrating that cardioprotective 6-piperazinyl purines can effectively inhibit IL-1β release in vascular cells, in this study, we present the synthesis of a next generation of purine analogues bearing either a furoxan moiety as a nitric oxide (NO) donor, or a (methylsulfonyl)thio group, a benzothioamide, or a 5-phenyl-3H-1,2-dithiole-3-thione as putative hydrogen sulfide (H2S) donor moieties. NO and H2S are gaseous signaling molecules that can reduce vascular inflammation. The new purine analogues were evaluated for their anti-inflammatory activity by assessing their inhibitory effect on the secretion of lipopolysaccharide (LPS)-induced IL-1β in human aortic smooth muscle cells (HAoSMCs). Our initial findings revealed that compounds bearing a [methylsulfonyl)thio]propanoyl or [methylsulfonyl)thio]hexanoyl group (compounds MK175 and MK169, respectively) could effectively inhibit LPS-induced IL-1β release in HAoSMCs.

Background The nitrate ester-bearing 6-piperazinyl-purine analogue MK128 is a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor with cardioprotective, anti-inflammatory, and antiplatelet properties. However, structure-activity relationships of this compound class have not been systematically explored. This study aimed to elucidate the impact of spacer length on the antiplatelet activity of closely related piperazinyl-purine analogues and to identify the most active candidate for further evaluation. Methods Antiplatelet activity of piperazinyl-purine analogues bearing short (MK128), intermediate (MK176), and long (MK118) spacers was evaluated by lumi-aggregometry and flow cytometry in isolated platelets and whole blood. Platelets were activated by proteinase-activated receptor-1 agonist SFLLRN-OH or collagen. Binding interactions with ROCK1/ROCK2 were examined using SelectScreen™ Kinase Profiling, in silico docking, and molecular dynamics simulations. Results MK128 exhibited the strongest inhibition of platelet aggregation and secretion in isolated platelets and whole blood, and rapidly promoted disaggregation of preformed aggregates. The rank order of potency in whole blood was MK128>MK176>MK118. All analogues inhibited ROCK1 and ROCK2 in cell-free assays, while broader kinase profiling demonstrated high selectivity. Docking studies suggested stable bidentate interactions between the purine core and hinge region, whereas molecular dynamics simulations identified spacer length-dependent differences in nitrate ester tail interactions. Reduced spacer flexibility in MK128 enabled productive interactions with catalytically relevant residues, providing a mechanistic explanation for its superior functional activity. Conclusion Spacer length critically determines the antiplatelet effect of piperazinyl-purine analogues. The one-carbon spacer analogue MK128 demonstrated superior platelet and ROCK inhibition, making it a promising lead candidate for further evaluation in ischemic disease models.

The exponential growth of the aged population worldwide is followed by an increase in the prevalence of age-related disorders. Oxidative stress plays central role in damage accumulation during ageing and cell senescence. Thus, a major target of today’s anti-ageing research has been focused on antioxidants counteracting senescence. In the current work, six novel 5,7,8-trimethyl-1,4-benzoxazine/catechol or resorcinol hybrids were synthesized connected through a methoxymethyl-1,2,3-triazolyl or a 1,2,3-triazoly linker. The compounds were evaluated for their antioxidant capacity in a cell-free system and for their ability to reduce intracellular ROS levels in human skin fibroblasts, both young (early-passage) and senescent. The most efficient compounds were further tested in these cells for their ability to induce the expression of the gene heme oxygenase-1 (ho-1), known to regulate redox homeostasis, and cellular glutathione (GSH) levels. Overall, the two catechol derivatives were found to be more potent than the resorcinol analogues. Furthermore, these two derivatives were shown to act coordinately as radical scavengers, ROS inhibitors, ho-1 gene expression inducers, and GSH enhancers. Interestingly, one of the two catechol derivatives was also found to enhance human skin fibroblast viability. The properties of the synthesized compounds support their potential use in cosmetic applications, especially in products targeting skin ageing.

Coronavirus disease 2019 (COVID-19) is caused by a new, highly pathogenic severe-acute-respiratory syndrome coronavirus 2 (SARS-CoV-2) that infects human cells through its transmembrane spike (S) glycoprotein. The receptor-binding domain (RBD) of the S protein interacts with the angiotensin-converting enzyme II (ACE2) receptor of the host cells. Therefore, pharmacological targeting of this interaction might prevent infection or spread of the virus. Here, we performed a virtual screening to identify small molecules that block S-ACE2 interaction. Large compound libraries were filtered for drug-like properties, promiscuity and protein-protein interaction-targeting ability based on their ADME-Tox descriptors and also to exclude pan-assay interfering compounds. A properly designed AI-based virtual screening pipeline was applied to the remaining compounds, comprising approximately 10% of the starting data sets, searching for molecules that could bind to the RBD of the S protein. All molecules were sorted according to their screening score, grouped based on their structure and postfiltered for possible interaction patterns with the ACE2 receptor, yielding 31 hits. These hit molecules were further tested for their inhibitory effect on Spike RBD/ACE2 (19-615) interaction. Six compounds inhibited the S-ACE2 interaction in a dose-dependent manner while two of them also prevented infection of human cells from a pseudotyped virus whose entry is mediated by the S protein of SARS-CoV-2. Of the two compounds, the benzimidazole derivative CKP-22 protected Vero E6 cells from infection with SARS-CoV-2, as well. Subsequent, hit-to-lead optimization of CKP-22 was effected through the synthesis of 29 new derivatives of which compound CKP-25 suppressed the Spike RBD/ACE2 (19-615) interaction, reduced the cytopathic effect of SARS-CoV-2 in Vero E6 cells (IC50 = 3.5 mu M) and reduced the viral load in cell culture supernatants. Early in vitro ADME-Tox studies showed that CKP-25 does not possess biodegradation or liver metabolism issues, while isozyme-specific CYP450 experiments revealed that CKP-25 was a weak inhibitor of the CYP450 system. Moreover, CKP-25 does not elicit mutagenic effect on Escherichia coli WP2 uvrA strain. Thus, CKP-25 is considered a lead compound against COVID-19 infection.

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-1β 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-1β in AoSMCs, as well as intracellular gene and protein expression of IL-1β 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.

In this study, the first mechanism-based monoclonal antibodies have been produced that recognize and differentiate diethoxy- and monoethoxyphosphorylated serine residues. Haptens were synthesized as the stable phosphonate form of phosphoserine esters to improve the immunoresponse. Following condensation with a glutaric anhydride to link the phosphoserine moieties to carrier protein, the hapten densities attached to bovine serum albumin and keyhole limpet henocyanin were determined by partial trypsin digestion and MALDI mass spectrometry, and confirmed using a fluorescent assay (FITC) to quantify unmodified lysine residues. The conjugation reactions were pH optimized to improve hapten density. Screening of subclones led to the identification of two monoclonal antibodies: (a) N257/25.11 that specifically recognizes (EtO)2P(O)-Ser as the phosphylated or inhibited form, and (b) N262/16 that recognizes (EtO)(HO)P(O)-Ser as the 'aged' form. Analysis of blood samples treated with paraoxon (EtO)2P(O)-OPhNO2 showed a concentration dependent recognition of the phosphylated form.

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.

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.

Modem 2D-NMR spectroscopy and Molecular Modeling are valuable tools in 3D-QSAR applications of flexible molecules. CoMFA and CoMSIA have been applied to a novel synthetic class of flexible ring substituted ether phospholipids. A correlation was observed, relating the steric, electrostatic, and hydrophobic properties of the new compounds with the in vitro antileishmanial activity against two promastigote Leishmania strains, L. infantum and L. donovani. The obtained 3D-maps indicate the necessary structural features for biological activity and the pharmacophores. These results will assist in the design of new analogs possessing improved biological profile.

Four heteroaromatic compounds bearing nitrate esters were selected using a virtual-screening procedure as putative sterol 14-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, insilico 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)-1-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.

A convenient method is described for the rapid synthesis of 5-(4-methoxyphenyl)-3H-1,2-dithiole-3thione (ADT-OMe) from anethole and elemental sulfur using microwave irradiation. Various reaction conditions were applied to reduce the reaction time from several hours to 10 min, resulting in an improvement in yield and overcoming the undesired by-product formation associated with conventional methods. 5-(4-Hydroxypheny1)-3H-1,2-dithio1-3-thione (ADT-OH) was obtained by the deprotection of ADT-OMe using pyridine hydrochloride under microwave irradiation. (C) 2017 Elsevier Ltd. All rights reserved.

Natural β‐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 β‐thujaplicin scaffold, on the antimicrobial activity. In order to determine the biological activity of the β‐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. β‐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 GI 50 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.

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.

AbstractA series of 3,5‐disubstituted isoxazole derivatives is synthesized by microwave‐assisted 1,3‐dipolar cycloaddition of alkynes bearing protected antioxidant substituents with in situ generated nitrile oxides.