Novel Multi-Target Agents Based on the Privileged Structure of 4-Hydroxy-2-quinolinone.

In this work, the privileged scaffold of 4-hydroxy-2quinolinone is investigated through the synthesis of carboxamides and hybrid derivatives, as well as through their bioactivity evaluation, focusing on the ability of the molecules to inhibit the soybean LOX, as an indication of their anti-inflammatory activity. Twenty-one quinolinone carboxamides, seven novel hybrid compounds consisting of the quinolinone moiety and selected cinnamic or benzoic acid derivatives, as well as three reverse amides are synthesized and classified as multi-target agents according to their LOX inhibitory and antioxidant activity. Among all the synthesized analogues, quinolinone-carboxamide compounds 3h and 3s, which are introduced for the first time in the literature, exhibited the best LOX inhibitory activity (IC50 = 10 µM). Furthermore, carboxamide 3g and quinolinone hybrid with acetylated ferulic acid 11e emerged as multi-target agents, revealing combined antioxidant and LOX inhibitory activity (3g: IC50 = 27.5 µM for LOX inhibition, 100% inhibition of lipid peroxidation, 67.7% ability to scavenge hydroxyl radicals and 72.4% in the ABTS radical cation decolorization assay; 11e: IC50 = 52 µM for LOX inhibition and 97% inhibition of lipid peroxidation). The in silico docking results revealed that the synthetic carboxamide analogues 3h and 3s and NDGA (the reference compound) bind at the same alternative binding site in a similar binding mode.

Investigation of the cytotoxicity of bioinspired coumarin analogues towards human breast cancer cells.

Coumarins possess a wide array of therapeutic capabilities, but often with unclear mechanism of action. We tested a small library of 18 coumarin derivatives against human invasive breast ductal carcinoma cells with the capacity of each compound to inhibit cell proliferation scored, and the most potent coumarin analogues selected for further studies. Interestingly, the presence of two prenyloxy groups (5,7-diprenyloxy-4-methyl-coumarin, 4g) or the presence of octyloxy substituent (coumarin 4d) was found to increase the potency of compounds in breast cancer cells, but not against healthy human fibroblasts. The activity of potent compounds on breast cancer cells cultured more similarly to the conditions of the tumour microenvironment was also investigated, and increased toxicity was observed. Results suggest that tested coumarin derivatives could potentially reduce the growth of tumour mass. Moreover, their use as (combination) therapy in cancer treatment might have the potential of causing limited side effects.

Novel quinolinone-pyrazoline hybrids: synthesis and evaluation of antioxidant and lipoxygenase inhibitory activity.

The present project deals with the investigation of structure-activity relationship of several quinolinone-chalcone and quinolinone-pyrazoline hybrids, in an effort to discover promising antioxidant and anti-inflammatory agents. In order to accomplish this goal, four bioactive hybrid quinolinone-chalcone compounds (8a-8d) were synthesized via an aldol condensation reaction, which were then chemically modified, forming fifteen new pyrazoline analogues (9a-9o). All the synthesized analogues were in vitro evaluated in terms of their antioxidant and soybean lipoxygenase (LOX) inhibitory activity. Among all the pyrazoline derivatives, compounds 9b and 9m were found to possess the best combined activity, whereas 9b analogue exhibited the most potent LOX inhibitory activity, with IC50 value 10 µM. The in silico docking results revealed that the synthetic pyrazoline analogue 9b showed high AutoDock Vina score (- 10.3 kcal/mol), while all the tested derivatives presented allosteric interactions with the enzyme.

Synthesis, Bioactivity, Pharmacokinetic and Biomimetic Properties of Multi-Substituted Coumarin Derivatives.

A series of novel multi-substituted coumarin derivatives were synthesized, spectroscopically characterized, and evaluated for their antioxidant activity, soybean lipoxygenase (LOX) inhibitory ability, their influence on cell viability in immortalized human keratinocytes (HaCaT), and cytotoxicity in adenocarcinomic human alveolar basal epithelial cells (A549) and human melanoma (A375) cells, in vitro. Coumarin analogues 4a-4f, bearing a hydroxyl group at position 5 of the coumarin scaffold and halogen substituents at the 3-phenyl ring, were the most promising ABTS•+ scavengers. 6,8-Dibromo-3-(4-hydroxyphenyl)-4-methyl-chromen-2-one (4k) and 6-bromo-3-(4,5-diacetyloxyphenyl)-4-methyl-chromen-2-one (3m) exhibited significant lipid peroxidation inhibitory activity (IC50 36.9 and 37.1 µM). In the DCF-DA assay, the 4'-fluoro-substituted compound 3f (100%), and the 6-bromo substituted compounds 3i (80.9%) and 4i (100%) presented the highest activity. The 3'-fluoro-substituted coumarins 3e and 4e, along with 3-(4-acetyloxyphenyl)-6,8-dibromo-4-methyl-chromen-2-one (3k), were the most potent lipoxygenase (LOX) inhibitors (IC50 11.4, 4.1, and 8.7 µM, respectively) while displaying remarkable hydroxyl radical scavenging ability, 85.2%, 100%, and 92.9%, respectively. In silico docking studies of compounds 4e and 3k, revealed that they present allosteric interactions with the enzyme. The majority of the analogues (100 µΜ) did not affect the cell viability of HaCaT cells, though several compounds presented over 60% cytotoxicity in A549 or A375 cells. Finally, the human oral absorption (%HOA) and plasma protein binding (%PPB) properties of the synthesized coumarins were also estimated using biomimetic chromatography, and all compounds presented high %HOA (>99%) and %PPB (60-97%) values.

Exploring the 2'-Hydroxy-Chalcone Framework for the Development of Dual Antioxidant and Soybean Lipoxygenase Inhibitory Agents.

2'-hydroxy-chalcones are naturally occurring compounds with a wide array of bioactivity. In an effort to delineate the structural features that favor antioxidant and lipoxygenase (LOX) inhibitory activity, the design, synthesis, and bioactivity profile of a series of 2'-hydroxy-chalcones bearing diverse substituents on rings A and B, are presented. Among all the synthesized derivatives, chalcone 4b, bearing two hydroxyl substituents on ring B, was found to possess the best combined activity (82.4% DPPH radical scavenging ability, 82.3% inhibition of lipid peroxidation, and satisfactory LOX inhibition value (IC50 = 70 µM). Chalcone 3c, possessing a methoxymethylene substituent on ring A, and three methoxy groups on ring B, exhibited the most promising LOX inhibitory activity (IC50 = 45 µM). A combination of in silico techniques were utilized in an effort to explore the crucial binding characteristics of the most active compound 3c and its analogue 3b, to LOX. A common H-bond interaction pattern, orienting the hydroxyl and carbonyl groups of the aromatic ring A towards Asp768 and Asn128, respectively, was observed. Regarding the analogue 3c, the bulky (-OMOM) group does not seem to participate in a direct binding, but it induces an orientation capable to form H-bonds between the methoxy groups of the aromatic ring B with Trp130 and Gly247.

Novel 3-aryl-5-substituted-coumarin analogues: Synthesis and bioactivity profile.

Eighteen 3-aryl-5-substituted-coumarins-six 5-acetyloxy-derivatives, six 5-hydroxy-derivatives, and six 5-geranyloxy-derivatives-were synthesized, structurally characterized and their antioxidant activity, lipoxygenase inhibitory ability, as well as their cytotoxic activity against human neuroblastoma SK-N-SH and HeLa adenocarcinoma cell lines were evaluated. The 5-acetyloxy-compounds 3a-3f were found to be the best cytotoxic agents among all the compounds studied. The bromo-substituted coumarins 3a and 3b were remarkably active against HeLa cell line showing IC50 1.8 and 6.1 µM, respectively. Coumarin 5e possessing a geranyloxy-chain on position 5 of the coumarin scaffold presented dual bioactivity, while 5-geranyloxy-coumarin 5f was the most competent soybean lipoxygenase inhibitor of this series (IC50 10 µM). As shown by in silico docking studies, the studied molecules present allosteric interactions with soybean lipoxygenases.

Novel Hit Compounds as Putative Antifungals: The Case of Aspergillus fumigatus.

The prevalence of invasive fungal infections has been dramatically increased as the size of the immunocompromised population worldwide has grown. Aspergillus fumigatus is characterized as one of the most widespread and ubiquitous fungal pathogens. Among antifungal drugs, azoles have been the most widely used category for the treatment of fungal infections. However, increasingly, azole-resistant strains constitute a major problem to be faced. Towards this direction, our study focused on the identification of compounds bearing novel structural motifs which may evolve as a new class of antifungals. To fulfil this scope, a combination of in silico techniques and in vitro assays were implemented. Specifically, a ligand-based pharmacophore model was created and served as a 3D search query to screen the ZINC chemical database. Additionally, molecular docking and molecular dynamics simulations were used to improve the reliability and accuracy of virtual screening results. In total, eight compounds, bearing completely different chemical scaffolds from the commercially available azoles, were proposed and their antifungal activity was evaluated using in vitro assays. Results indicated that all tested compounds exhibit antifungal activity, especially compounds 1, 2, and 4, which presented the most promising minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values and, therefore, could be subjected to further hit to lead optimization.

Exploring new scaffolds for angiotensin II receptor antagonism.

Nowadays, AT1 receptor (AT1R) antagonists (ARBs) constitute the one of the most prevalent classes of antihypertensive drugs that modulate the renin-angiotensin system (RAS). Their main uses include also treatment of diabetic nephropathy (kidney damage due to diabetes) and congestive heart failure. Towards this direction, our study has been focused on the discovery of novel agents bearing different scaffolds which may evolve as a new class of AT1 receptor antagonists. To fulfill this aim, a combination of computational approaches and biological assays were implemented. Particularly, a pharmacophore model was established and served as a 3D search query to screen the ChEMBL15 database. The reliability and accuracy of virtual screening results were improved by using molecular docking studies. In total, 4 compounds with completely diverse chemical scaffolds from potential ARBs, were picked and tested for their binding affinity to AT1 receptor. Results revealed high nanomolar to micromolar affinity (IC50) for all the compounds. Especially, compound 4 exhibited a binding affinity of 199nM. Molecular dynamics simulations were utilized in an effort to provide a molecular basis of their binding to AT1R in accordance to their biological activities.

Aurones as histone deacetylase inhibitors: identification of key features.

In this study, a total of 22 flavonoids were tested for their HDAC inhibitory activity using fluorimetric and BRET-based assays. Four aurones were found to be active in both assays and showed IC50 values below 20 µM in the enzymatic assay. Molecular modelling revealed that the presence of hydroxyl groups was responsible for good compound orientation within the isoenzyme catalytic site and zinc chelation.

Structural modifications of coumarin derivatives: Determination of antioxidant and lipoxygenase (LOX) inhibitory activity.

In the present project, a series of coumarin analogues, were synthesised and evaluated for their antioxidant and soybean lipoxygenase inhibitory activity. A variety of structural modifications on the coumarin scaffold revealed interesting structure­activity relationships concerning the different biological assays. Prenyloxy-coumarins 9 and 10 displayed the best combined inhibition of lipid peroxidation and soybean lipoxygenase. Thiocoumarins 11 and 14 were identified as potent lipoxygenase inhibitors whereas hydrazone analogues 15 and 16 were efficient DPPH radical scavengers.

Evaluation of anti-oxidant and acetylcholinesterase activity and identification of polyphenolics of the invasive weed Dittrichia viscosa.

INTRODUCTION: The bioactive metabolites derived from weeds have attracted the interest of the food and pharmaceutical industries due to their health benefits. OBJECTIVE: To evaluate the anti-oxidant and acetylcholinesterase activity of Dittrichia viscosa extracts and characterise the polyphenolic metabolites using the LC coupled with diode-array detection (DAD) and positive mode electrospray ionisation (ESI) MS method with a view to evaluating the exploitation potential of this invasive weed. MATERIALS AND METHODS: Roots and aerial parts of D. viscosa were extracted with solvents of increasing polarity and their major polyphenolic metabolites were identified by LC - DAD/ESI(+)/MS. The total phenolic content of the extracts was determined using the Folin-Ciocalteu method, while their anti-oxidant activity was evaluated on the basis of their ability to scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydrogen peroxide. Thin-layer chromatography was used to screen for acetylcholinesterase inhibitors. RESULTS: Stem extracts gave the highest phenolic content, whereas the roots showed the lowest content. Twenty-five polyphenolic constituents of the extracts were tentatively characterised according to their MS and UV spectroscopic data. Among the extracts studied, roots-ethyl acetate and flowers-diethyl ether revealed the highest activity according to the DPPH and chemiluminescence assays respectively. CONCLUSION: The metabolic profile of D. viscosa was studied and the structures of the major polyphenolic metabolites were tentatively assigned based on their MS and UV-vis spectra. The extracts exhibited high levels of anti-oxidant and acetylcholinesterase inhibitory activity and the inhibitors are probably localised mainly in flowers and roots.

Synthesis and anti-parasitic activity of a novel quinolinone-chalcone series.

A series of novel quinolinone-chalcone hybrids and analogues were designed, synthesized and their biological activity against the mammalian stages of Trypanosoma brucei and Leishmania infantum evaluated. Promising molecular scaffolds with significant microbicidal activity and low cytotoxicity were identified. Quinolinone-chalcone 10 exhibited anti-parasitic properties against both organisms, being the most potent anti-L. infantum agent of the entire series (IC50 value of 1.3±0.1 µM). Compounds 4 and 11 showed potency toward the intracellular, amastigote stage of L. infantum (IC50 values of 2.1±0.6 and 3.1±1.05 µM, respectively). Promising trypanocidal compounds include 5 and 10 (IC50 values of 2.6±0.1 and 3.3±0.1 µM, respectively) as well as 6 and 9 (both having IC50 values of <5 µM). Chemical modifications on the quinolinone-chalcone scaffold were performed on selected compounds in order to investigate the influence of these structural features on antiparasitic activity.

Preparation and characterization of solid lipid nanoparticles incorporating bioactive coumarin analogues as photosensitizing agents.

Daphnetin (7,8-dihydroxy-coumarin, DAPH) is a naturally occurring coumarin presenting a wide array of biological activities. In the present study, daphnetin and its novel synthetic analogue 7,8-dihydroxy-4-methyl-3-(4-hydroxyphenyl)-coumarin (DHC) were encapsulated in solid lipid nanoparticles (SLNs) with Encapsulation Efficiency values of 80% and 40%, respectively. Nanoparticles of an average hydrodynamic diameter of approximately 250 nm were formed, showing a good stability in aqueous dispersion (polydispersity index 0.3-0.4), as determined by Dynamic Light Scattering (DLS). The SLNs were also characterized using Fourier Transform-Infrared (FT-IR) spectroscopy and Thermogravimetric Analysis (TGA). TEM images of the blank-SLNs indicated a spherical morphology and a size of 20-50 nm. The release studies of the coumarin analogues indicated a non-Fickian diffusion mechanism, while the release profiles better fitted on the Higuchi kinetic model. Moreover, the coumarin analogues and their SLNs were examined for their antioxidant activity using DPPH and anti-lipid peroxidation assays, exhibiting stronger antioxidant activity when encapsulated than in their free form. The coumarin derivatives and their SLNs were examined for their photodynamic therapy (PDT) efficacy against the human squamous carcinoma A431 cell line, with DHC coumarin both in its free and encapsulated form exhibiting significant PDT activity, reducing the cell viability to 11% after irradiation with a fluence rate of 2.16 J/cm2. Finally, the intracellular localization studies indicated the enhanced cellular uptake of the coumarin analogues when loaded in the SLNs.

"Novel chitosan/alginate hydrogels as carriers of phenolic-enriched extracts from saffron floral by-products using natural deep eutectic solvents as green extraction media".

The current saffron production system is generating several hundreds of tons of tepal waste, because only stigmas are used for food. Consequently, the valorization of saffron floral by-products by developing stable functional ingredients could lead to the environmental impact minimization. Thus, the main aim of this study was to develop innovative green extraction processes from saffron floral by-products by using Natural Deep Eutectic Solvents (NaDES) and ultrasound-assisted extraction (UAE) as ecological extraction method. Response surface methodology was used to optimize process parameters. To improve the stability of the optimal extracts, they were incorporated into chitosan/alginate hydrogels, studying their water-uptake and water retention capacity and the total phenolic content (TPC) during the in vitro digestion. The results indicated that the optimal extraction, regarding total phenolic and flavonoid content, was achieved in 20 min, using 180 W ultrasound power and 90% of NaDES. The results of the DPPH assay revealed the potent antioxidant activity of saffron floral by-products. The chitosan/alginate hydrogels incorporating the as-obtained NaDES extracts showed favorable properties whereas the TPC remained stable under intestinal conditions. Therefore, NaDES combined with UAE was an efficient technique to isolate high added-value compounds from saffron flowers, succeeding also the valorization of discarded waste by using green and low-cost strategies. Furthermore, these novel hydrogels could be used as promising candidates for food or cosmetic applications.

Ιnclusion Complexes of Magnesium Phthalocyanine with Cyclodextrins as Potential Photosensitizing Agents.

In this work, the preparation of inclusion complexes, (ICs) using magnesium phthalocyanine (MgPc) and various cyclodextrins (ß-CD, γ-CD, HP-ß-CD, Me-ß-CD), using the kneading method is presented. Dynamic light scattering (DLS) indicated that the particles in dispersion possessed mean size values between 564 to 748 nm. The structural characterization of the ICs by infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy provides evidence of the formation of the ICs. The release study of the MgPc from the different complexes was conducted at pH 7.4 and 37 °C, and indicated that a rapid release ("burst effect") of ~70% of the phthalocyanine occurred in the first 20 min. The kinetic model that best describes the release profile is the Korsmeyer-Peppas. The photodynamic therapy studies against the squamous carcinoma A431 cell line indicated a potent photosensitizing activity of MgPc (33% cell viability after irradiation for 3 min with 18 mW/cm2), while the ICs also presented significant activity. Among the different ICs, the γ-CD-MgPc IC exhibited the highest photokilling capacity under the same conditions (cell viability 26%). Finally, intracellular localization studies indicated the enhanced cellular uptake of MgPc after incubation of the cells with the γ-CD-MgPc complex for 4 h compared to MgPc in its free form.

Eco-friendly synthesis of new olanzapine derivatives and evaluation of their anticancer potential.

New derivatives of the known antipsychotic drug olanzapine have been obtained as potential compounds with anticancer activity in two metabolically different breast cancer cell lines: MCF-7 and triple negative MDA-MB-231. The compounds were obtained under phase transfer catalysis (PTC) in the presence of microwave irradiation (MW) or ultrasound (")))"), evaluating the effect of solvents such as dimethylformamide, water, or choline chloride/urea (natural deep eutectic solvent, NaDES). In the best option, the compounds were obtained within 2 minutes with a yield of 57-86% in MW. Two of the obtained compounds which have a naphthalimide moiety and a pentyl (7) or hexyl chain (8) show pronounced cytotoxicity. Interestingly, neither olanzapine nor desmethylolanzapine (DOLA), which was one of the substrates for the synthesis reaction, showed any significant activity in the study.

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro.

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence. Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy -performed in a large and diverse chemolibrary- complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening. Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 µM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12-20 µM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7-45 µM). Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known "garbage in, garbage out" machine learning principle.

Evaluation of anti-oxidant activity and identification of major polyphenolics of the invasive weed Oxalis pes-caprae.

INTRODUCTION: Phytochemical analyses of weeds, many of which have been used in traditional medicine worldwide, could lead to the identification of secondary metabolites with significant biological activity. OBJECTIVE: To perform an assessment of the chemical composition and exploitation potential of the invasive weed Oxalis pes-caprae. To evaluate the anti-oxidant activity of its extracts and isolate and characterise polyphenolic metabolites using LC-DAD-MS (ESI+) and NMR methods. METHODOLOGY: Aerial parts of the invasive weed O. pes-caprae were extracted with solvents of increasing polarity and their major polyphenolic metabolites were identified by LC-DAD-MS (ESI+). The total phenolic content of the extracts was determined using the Folin-Ciocalteu method, while their anti-oxidant activity was evaluated on the basis of their ability to scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl and hydrogen peroxide. RESULTS: The major polyphenolic constituents of the extracts were tentatively characterised as chlorogenic acid, quinic ferulate, luteolin glucoside and cernuoside according to their MS and UV spectroscopic data. Cernuoside, an aureusidin glucoside, was isolated from the methanolic extract of the weed's flowers and its structure was unambiguously identified by 1D- and 2D-NMR spectroscopy. The butanol extract of O. pes-caprae displayed the highest anti-oxidant activity. CONCLUSION: The metabolic profile of O. pes-caprae was studied and the structures of the major polyphenolic metabolites based on their MS and UV-vis spectra were tentatively assigned. The aureusidin glucoside cernuoside was isolated and characterised for the first time from O. pes-caprae. The extracts exhibited high levels of anti-oxidant activity.

Synthesis, Conformational Analysis and ctDNA Binding Studies of Flavonoid Analogues Possessing the 3,5-di-tert-butyl-4-hydroxyphenyl Moiety.

Flavanones and their biochemical precursors, chalcones, are naturally occurring compounds and consist of privileged scaffolds used in drug discovery due to their wide range of biological activities. In this work, two novel flavanones (3 and 4), the arylidene flavanone 5, and the chalcone 6, displaying structural analogies with butylated hydroxytoluene (BHT), were synthesized via an aldol reaction. According to the antioxidant activity studies of the synthesized flavanones, the arylidene flavanone 5 was the most potent antioxidant (70.8% interaction with DPPH radical and 77.4% inhibition of lipid peroxidation). In addition, the ability of the synthesized compounds to bind with ctDNA was measured via UV-spectroscopy, revealing that chalcone 6 has the strongest interaction with DNA (Kb = 5.0 × 10-3 M-1), while molecular docking was exploited to simulate the compound-DNA complexes. In an effort to explore the conformational features of the novel synthetic flavanones (3 and 4), arylidene flavanone 5, and chalcone 6, theoretical calculations were applied and the calculation of their physicochemical properties was also performed.

A novel synthesis of 3-aryl coumarins and evaluation of their antioxidant and lipoxygenase inhibitory activity.

A series of coumarin analogues bearing a substituted phenyl ring on position 3 were synthesized via a novel methodology, through an intermolecular condensation reaction of 2-hydroxyacetophenones and 2-hydroxybenzaldehyde, with imidazolyl phenylacetic acid active intermediates. The in vitro antioxidant activity of the synthesized compounds was evaluated using two different antioxidant assays (radical scavenging ability of DPPH stable free radical and inhibition of lipid peroxidation induced by the thermal free radical AAPH). Moreover, the ability of the compounds to inhibit soybean lipoxygenase was determined as an indication of potential anti-inflammatory activity.