Novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives and their antitrypanosomal activities against T.brucei.

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense and is invariably fatal unless treated. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work, informed by previous findings, presents novel 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidine derivatives with promising antitrypanosomal activity. In particular, 32 exhibits an in vitro EC50 value of 0.5 µM against Trypanosoma brucei rhodesiense, and analogues 29, 30 and 33 show antitrypanosomal activities in the <1 µM range. We have demonstrated that substituted 4-[4-(4-methylpiperazin-1-yl)phenyl]-6-arylpyrimidines present promising antitrypanosomal hit molecules with potential for further preclinical development.

CYP1-Activation and Anticancer Properties of Synthetic Methoxylated Resveratrol Analogues.

Naturally occurring stilbenoids, such as the (E)-stilbenoid resveratrol and the (Z)-stilbenoid combretastatin A4, have been considered as promising lead compounds for the development of anticancer drugs. The antitumour properties of stilbenoids are known to be modulated by cytochrome P450 enzymes CYP1A1 and CYP1B1, which contribute to extrahepatic phase I xenobiotic and drug metabolism. Thirty-four methyl ether analogues of resveratrol were synthesised, and their anticancer properties were assessed, using the MTT cell proliferation assay on a panel of human breast cell lines. Breast tumour cell lines that express CYP1 were significantly more strongly affected by the resveratrol analogues than the cell lines that did not have CYP1 activity. Metabolism studies using isolated CYP1 enzymes provided further evidence that (E)-stilbenoids can be substrates for these enzymes. Structures of metabolic products were confirmed by comparison with synthetic standards and LC-MS co-elution studies. The most promising stilbenoid was (E)-4,3',4',5'-tetramethoxystilbene (DMU212). The compound itself showed low to moderate cytotoxicity, but upon CYP1-catalysed dealkylation, some highly cytotoxic metabolites were formed. Thus, DMU212 selectively affects proliferation of cells that express CYP1 enzymes.

Advance toward isolation, extraction, metabolism and health benefits of kaempferol, a major dietary flavonoid with future perspectives.

As a major ubiquitous secondary metabolite, flavonoids are widely distributed in planta. Among flavonoids, kaempferol is a typical natural flavonol in diets and medicinal plants with myriad bioactivities, such as anti-inflammatory activity, anti-cancer activity, antioxidant activity, and anti-diabetic activity. However, the natural sources, absorption and metabolism as well as the bioactivities of kaempferol have not been reviewed comprehensively and systematically. This review highlights the latest research progress and the effect of kaempferol in the prevention and treatment of various chronic diseases, as well as its protective health effects, and provides a theoretical basis for future research to be used in nutraceuticals. Further, comparison of the different extraction and analytical methods are presented to highlight the most optimum for PG recovery and its detection in plasma and body fluids. Such review aims at improving the value-added applications of this unique dietary bioactive flavonoids at commercial scale and to provide a reference for its needed further development.

Effect of the Citrus Flavone Nobiletin on Circadian Rhythms and Metabolic Syndrome.

The importance of the circadian clock in maintaining human health is now widely acknowledged. Dysregulated and dampened clocks may be a common cause of age-related diseases and metabolic syndrome Thus, circadian clocks should be considered as therapeutic targets to mitigate disease symptoms. This review highlights a number of dietary compounds that positively affect the maintenance of the circadian clock. Notably the polymethoxyflavone nobiletin has shown some encouraging results in pre-clinical experiments. Although many more experiments are needed to fully elucidate its exact mechanism of action, it is a promising candidate with potential as a chronotherapeutic agent.

Artemisinin Cocrystals for Bioavailability Enhancement. Part 1: Formulation Design and Role of the Polymeric Excipient.

Artemisinin (ART) is a most promising antimalarial agent, which is both effective and well tolerated in patients, though it has therapeutic limitations due to its low solubility, bioavailability, and short half-life. The objective of this work was to explore the possibility of formulating ART cocrystals, i.e., artemisinin-orcinol (ART-ORC) and artemisinin-resorcinol (ART2-RES), as oral dosage forms to deliver ART molecules for bioavailability enhancement. This is the first part of the study, aiming to develop a simple and effective formulation, which can then be tested on an appropriate animal model (i.e., mouse selected for in vivo study) to evaluate their preclinical pharmacokinetics for further development. In the current work, the physicochemical properties (i.e., solubility and dissolution rate) of ART cocrystals were measured to collect information necessary for the formulation development strategy. It was found that the ART solubility can be increased significantly by its cocrystals, i.e., 26-fold by ART-ORC and 21-fold by ART2-RES, respectively. Screening a set of polymers widely used in pharmaceutical products, including poly(vinylpyrrolidone), hydroxypropyl methylcellulose, and hydroxypropyl methylcellulose acetate succinate, based on the powder dissolution performance parameter analysis, revealed that poly(vinylpyrrolidone)/vinyl acetate (PVP-VA) was the most effective crystallization inhibitor. The optimal concentration of PVP-VA at 0.05 mg/mL for the formulation was then determined by a dissolution/permeability method, which represented a simplified permeation model to simultaneously evaluate the effects of a crystallization inhibitor on the dissolution and permeation performance of ART cocrystals. Furthermore, experiments, including surface dissolution of single ART cocrystals monitored by Raman spectroscopy, scanning electron microscopy and diffusion properties of ART in solution measured by 1H and diffusion-ordered spectroscopy nuclear magnetic resonance spectroscopy, provided insights into how the excipient affects the ART cocrystal dissolution performance and bioavailability.

Artemisinin Cocrystals for Bioavailability Enhancement. Part 2: In Vivo Bioavailability and Physiologically Based Pharmacokinetic Modeling.

We report the evaluation and prediction of the pharmacokinetic (PK) performance of artemisinin (ART) cocrystal formulations, that is, 1:1 artemisinin/orcinol (ART-ORC) and 2:1 artemisinin/resorcinol (ART2-RES), using in vivo murine animal and physiologically based pharmacokinetic (PBPK) models. The efficacy of the ART cocrystal formulations along with the parent drug ART was tested in mice infected with Plasmodium berghei. When given at the same dose, the ART cocrystal formulation showed a significant reduction in parasitaemia at day 4 after infection compared to ART alone. PK parameters including Cmax (maximum plasma concentration), Tmax (time to Cmax), and AUC (area under the curve) were obtained by determining drug concentrations in the plasma using liquid chromatography-high-resolution mass spectrometry (LC-HRMS), showing enhanced ART levels after dosage with the cocrystal formulations. The dose-response tests revealed that a significantly lower dose of the ART cocrystals in the formulation was required to achieve a similar therapeutic effect as ART alone. A PBPK model was developed using a PBPK mouse simulator to accurately predict the in vivo behavior of the cocrystal formulations by combining in vitro dissolution profiles with the properties of the parent drug ART. The study illustrated that information from classical in vitro and in vivo experimental investigations of the parent drug of ART formulations can be coupled with PBPK modeling to predict the PK parameters of an ART cocrystal formulation in an efficient manner. Therefore, the proposed modeling strategy could be used to establish in vitro and in vivo correlations for different cocrystals intended to improve dissolution properties and to support clinical candidate selection, contributing to the assessment of cocrystal developability and formulation development.

Analysis of plant secondary metabolism using stable isotope-labelled precursors.

INTRODUCTION: Analysis of biochemical pathways typically involves feeding a labelled precursor to an organism, and then monitoring the metabolic fate of the label. Initial studies used radioisotopes as a label and then monitored radioactivity in the metabolic products. As analytical equipment improved and became more widely available, preference shifted the use stable 'heavy' isotopes like deuterium (2 H)-, carbon-13 (13 C)- and nitrogen-15 (15 N)-atoms as labels. Incorporation of the labels could be monitored by mass spectrometry (MS), as part of a hyphenated tool kits, e.g. Liquid chromatography (LC)-MS, gas chromatography (GC)-MS, LC-MS/MS. MS offers great sensitivity but the exact location of an isotope label in a given metabolite cannot always be unambiguously established. Nuclear magnetic resonance (NMR) can also be used to pick up signals of stable isotopes, and can give information on the precise location of incorporated label in the metabolites. However, the detection limit for NMR is quite a bit higher than that for MS. OBJECTIVES: A number of experiments involving feeding stable isotope-labelled precursors followed by NMR analysis of the metabolites is presented. The aim is to highlight the use of NMR analysis in identifying the precise fate of isotope labels after precursor feeding experiments. As more powerful NMR equipment becomes available, applications as described in this review may become more commonplace in pathway analysis. CONCLUSION AND PROSPECTS: NMR is a widely accepted tool for chemical structure elucidation and is now increasingly used in metabolomic studies. In addition, NMR, combined with stable isotope feeding, should be considered as a tool for metabolic flux analyses.

A Brief Overview of Potential Treatments for Viral Diseases Using Natural Plant Compounds: The Case of SARS-Cov.

The COVID-19 pandemic, as well as the more general global increase in viral diseases, has led researchers to look to the plant kingdom as a potential source for antiviral compounds. Since ancient times, herbal medicines have been extensively applied in the treatment and prevention of various infectious diseases in different traditional systems. The purpose of this review is to highlight the potential antiviral activity of plant compounds as effective and reliable agents against viral infections, especially by viruses from the coronavirus group. Various antiviral mechanisms shown by crude plant extracts and plant-derived bioactive compounds are discussed. The understanding of the action mechanisms of complex plant extract and isolated plant-derived compounds will help pave the way towards the combat of this life-threatening disease. Further, molecular docking studies, in silico analyses of extracted compounds, and future prospects are included. The in vitro production of antiviral chemical compounds from plants using molecular pharming is also considered. Notably, hairy root cultures represent a promising and sustainable way to obtain a range of biologically active compounds that may be applied in the development of novel antiviral agents.

Flavones as tyrosinase inhibitors: kinetic studies in vitro and in silico.

INTRODUCTION: Tyrosinase is a multifunctional copper-containing oxidase enzyme that catalyses the first steps in the formation of melanin pigments. Identification of tyrosinase inhibitors is of value for applications in cosmetics, medicine and agriculture. OBJECTIVE: To develop an analytical method that allows identification of drug-like natural products that can be further developed as tyrosinase inhibitors. Results of in vitro and in silico studies will be compared in order to gain a deeper insight into the mechanism of action of enzyme inhibition. METHOD: Using an in vitro assay we tested tyrosinase inhibitor effects of five structurally related flavones, i.e. luteolin (1), eupafolin (2), genkwanin (3), nobiletin (4), and chrysosplenetin (5). The strongest inhibitors were further investigated in silico, using enzyme docking simulations. RESULTS: All compounds tested showed modest tyrosinase inhibitory effect compared to the positive control, kojic acid. The polymethoxy flavones 4 and 5 exhibited the strongest tyrosinase inhibitory effect with the half maximal inhibitory concentration (IC50 ) values of 131.92 ± 1.75 µM and 99.87 ± 2.38 µM respectively. According to kinetic analysis 2, 4 and 5 were competitive inhibitors, whereas 1 and 3 were non-competitive inhibitors of tyrosinase. Docking studies indicated that methoxy groups on 4 and 5 caused steric hindrance which prevented alternative binding modes in the tyrosinase; the methoxy groups on the B-ring of these flavones faced the catalytic site in the enzyme. CONCLUSIONS: The docking simulations nicely complemented the in vitro kinetic studies, opening the way for the development of predictive models for use in drug design.

The synthesis of 4,6-diaryl-2-pyridones and their bioactivation in CYP1 expressing breast cancer cells.

As part of a programme to develop anticancer prodrugs which are activated by cytochrome P450 (CYP)1B1, a library of 4,6-diaryl-2-pyridones was synthesised in yields of 6-60% from the corresponding chalcones. A number of these derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing little toxicity towards a non-tumour breast cell line with no CYP expression. Metabolism studies provided evidence supporting the involvement of CYP1 enzymes in the bioactivation of these compounds.

New Hydrogels Enriched with Antioxidants from Saffron Crocus Can Find Applications in Wound Treatment and/or Beautification.

Saffron extracts have a long history of application as skin protectant, possibly due to their ability to scavenge free radicals. In this work, the performance of a hydrogel enriched with antioxidant compounds isolated from saffron crocus (Crocus sativus L.) petals was tested. These hydrogels could be considered as new drug delivery system. Hydrogels are crosslinked polymer networks that absorb large quantities of water but retain the properties of a solid, thus making ideal dressings for sensitive skin. We tested antioxidant-enriched hydrogels on primary mouse fibroblasts. Hydrogels enriched with kaempferol and crocin extracted from saffron petals showed good biocompatibility with in vitro cultured fibroblasts. These new types of hydrogels may find applications in wound treatment and/or beautification.

Investigation of Linum flavum (L.) Hairy Root Cultures for the Production of Anticancer Aryltetralin Lignans.

Linum flavum hairy root lines were established from hypocotyl pieces using Agrobacterium rhizogenes strains LBA 9402 and ATCC 15834. Both strains were effective for transformation but induction of hairy root phenotype was more stable with strain ATCC 15834. Whereas similar accumulation patterns were observed in podophyllotoxin-related compounds (6-methoxy-podophyllotoxin, podophyllotoxin and deoxypodophyllotoxin), significant quantitative variations were noted between root lines. The influence of culture medium and various treatments (hormone, elicitation and precursor feeding) were evaluated. The highest accumulation was obtained in Gamborg B5 medium. Treatment with methyl jasmonate, and feeding using ferulic acid increased the accumulation of aryltetralin lignans. These results point to the use of hairy root culture lines of Linum flavum as potential sources for these valuable metabolites as an alternative, or as a complement to Podophyllum collected from wild stands.

The discovery of novel antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines.

Human African trypanosomiasis, or sleeping sickness, is a neglected tropical disease caused by Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense which seriously affects human health in Africa. Current therapies present limitations in their application, parasite resistance, or require further clinical investigation for wider use. Our work herein describes the design and syntheses of novel antitrypanosomal 4-phenyl-6-(pyridin-3-yl)pyrimidines, with compound 13, the 4-(2-methoxyphenyl)-6-(pyridine-3-yl)pyrimidin-2-amine demonstrating an IC50 value of 0.38 µM and a promising off-target ADME-Tox profile in vitro. In silico molecular target investigations showed rhodesain to be a putative candidate, supported by STD and WaterLOGSY NMR experiments, however, in vitro evaluation of compound 13 against rhodesain exhibited low experimental inhibition. Therefore, our reported library of drug-like pyrimidines present promising scaffolds for further antikinetoplastid drug development for both phenotypic and target-based drug discovery.

Activity of Antioxidants from Crocus sativus L. Petals: Potential Preventive Effects towards Cardiovascular System.

The petals of the saffron crocus (Crocus sativus L.) are considered a waste material in saffron production, but may be a sustainable source of natural biologically active substances of nutraceutical interest. The aim of this work was to study the cardiovascular effects of kaempferol and crocin extracted from saffron petals. The antiarrhythmic, inotropic, and chronotropic effects of saffron petal extract (SPE), kaempferol, and crocin were evaluated through in vitro biological assays. The antioxidant activity of kaempferol and crocin was investigated through the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay using rat cardiomyoblast cell line H9c2. The MTT assay was applied to assess the effects of kaempferol and crocin on cell viability. SPE showed weak negative inotropic and chronotropic intrinsic activities but a significant intrinsic activity on smooth muscle with a potency on the ileum greater than on the aorta: EC50 = 0.66 mg/mL versus EC50 = 1.45 mg/mL. Kaempferol and crocin showed a selective negative inotropic activity. In addition, kaempferol decreased the contraction induced by KCl (80 mM) in guinea pig aortic and ileal strips, while crocin had no effect. Furthermore, following oxidative stress, both crocin and kaempferol decreased intracellular ROS formation and increased cell viability in a concentration-dependent manner. The results indicate that SPE, a by-product of saffron cultivation, may represent a good source of phytochemicals with a potential application in the prevention of cardiovascular diseases.

Enhancement of artemisinin concentration and yield in response to optimization of nitrogen and potassium supply to Artemisia annua.

BACKGROUND AND AIMS: The resurgence of malaria, particularly in the developing world, is considerable and exacerbated by the development of single-gene multi-drug resistances to chemicals such as chloroquinone. Drug therapies, as recommended by the World Health Organization, now include the use of antimalarial compounds derived from Artemisia annua--in particular, the use of artemisinin-based ingredients. Despite our limited knowledge of its mode of action or biosynthesis there is a need to secure a supply and enhance yields of artemisinin. The present study aims to determine how plant biomass can be enhanced while maximizing artemisinin concentration by understanding the plant's nutritional requirements for nitrogen and potassium. METHODS: Experiments were carried out, the first with differing concentrations of nitrogen, at 6, 31, 56, 106, 206 or 306 mg L(-1) being applied, while the other differing in potassium concentration (51, 153 or 301 mg L(-1)). Nutrients were supplied in irrigation water to plants in pots and after a growth period biomass production and leaf artemisinin concentration were measured. These data were used to determine optimal nutrient requirements for artemisinin yield. KEY RESULTS: Nitrogen nutrition enhanced plant nitrogen concentration and biomass production successively up to 106 mg N L(-1) for biomass and 206 mg N L(-1) for leaf nitrogen; further increases in nitrogen had no influence. Artemisinin concentration in dried leaf material, measured by HPLC mass spectroscopy, was maximal at a nitrogen application of 106 mg L(-1), but declined at higher concentrations. Increasing potassium application from 51 to 153 mg L(-1) increased total plant biomass, but not at higher applications. Potassium application enhanced leaf potassium concentration, but there was no effect on leaf artemisinin concentration or leaf artemisinin yield. CONCLUSIONS: Artemisinin concentration declined beyond an optimal point with increasing plant nitrogen concentration. Maximization of artemisinin yield (amount per plant) requires optimization of plant biomass via control of nitrogen nutrition.

Anticancer effects of the flavonoid diosmetin on cell cycle progression and proliferation of MDA-MB 468 breast cancer cells due to CYP1 activation.

Flavonoids constitute a large class of polyphenolic compounds with cancer preventative properties. We have examined the ability of the natural flavone diosmetin to inhibit proliferation of breast adenocarcinoma MDA-MB 468 and normal breast MCF-10A cells and found that this compound is selective for the cancer cells with slight toxicity in the normal breast cells. Diosmetin was metabolised to the structurally similar flavone luteolin in MDA-MB 468 cells, whereas no metabolism was seen in MCF-10A cells. Diosmetin caused G1 arrest at 10 microM in MDA-MB 468 cells after 48-h treatment whereas this effect was not observed in MCF-10A cells. We suggest that diosmetin exerts cytostatic effects in MDA-MB 468 cells, due to CYP1A1 and CYP1B1 catalyzed conversion to the flavone luteolin.

The methoxylated flavones eupatorin and cirsiliol induce CYP1 enzyme expression in MCF7 cells.

Flavonoids have often been associated with cancer prevention and activity of the human cytochrome P450 enzymes CYP1A1 and CYP1B1 with the occurrence of cancer. The flavones eupatorin (1) and cirsiliol (2) enhanced CYP1 enzyme activity in a concentration-dependent manner in MCF7 human breast adenocarcinoma cells. In the range of 0-2.5 microM, 2 caused a dose-dependent increase in CYP1B1 mRNA levels and an increase in CYP1A1 mRNA. Compound 1 caused an increase in CYP1A1 and CYP1B1 mRNA at higher doses (approximately 5 microM). Both CYP1B1 and CYP1A1 catalyzed the conversion of 2 into an as yet unidentified compound. Application of the CYP1 family inhibitor, acacetin, significantly increased the IC(50) value of 2 in MCF7 cells, but did not significantly affect the action of 1. The data suggest that 2 induces CYP1 enzyme expression in cancer cells and is subsequently converted by CYP1B1 or CYP1A1 into an antiproliferative agent.

Antiproliferative and cytostatic effects of the natural product eupatorin on MDA-MB-468 human breast cancer cells due to CYP1-mediated metabolism.

INTRODUCTION: The natural product eupatorin has been reported to have antiproliferative activity in tumour cell lines, but the exact mechanism is unclear. The cytochromes P450 CYP1B1, CYP1A1, and CYP1A2 have been shown to participate in the activation of various xenobiotics, compounds derived from the diet as well as chemotherapeutic drugs. CYP1B1 and CYP1A1 have also been proposed as targets for cancer chemotherapy for their differential and selective overexpression in tumour cells. In this study, we aimed to identify a possible mechanism of action for the antiproliferative effect of eupatorin, which can be attributed to CYP1 family-mediated metabolism. METHODS: The study focuses on the antiproliferative action of eupatorin on the human breast carcinoma cell line MDA-MB-468 and on a cell line derived from normal mammary tissue, MCF-10A. The cytotoxicity of the flavone, its effect on the cell cycle of the abovementioned cell lines, and its metabolism by CYP1 family enzymes were examined. RESULTS: Eupatorin showed a dose-dependent inhibitory effect of cell growth on MDA-MB-468 cells with a submicromolar median inhibition concentration (IC50) whereas the IC50 of this compound in MCF-10A cells was considerably higher. The antiproliferative effect, as measured by EROD (ethoxyresorufin-O-deethylase) assay and Western immunoblotting, was attributed mainly to CYP1A1 expression in MDA-MB-468 cells but not in MCF-10A cells. Moreover, CYP1 family enzymes were shown to metabolise eupatorin in vitro to the flavone cirsiliol and two other unidentified metabolites. Metabolism of eupatorin was also detected in MDA-MB-468 cell cultures, whereas metabolism by MCF-10A cells was negligible. Eupatorin was further shown to arrest the cell cycle of the CYP1-expressing cell line MDA-MB-468 in G2/M phase, whereas no effect was observed in MCF-10A cells, which do not express CYP1 enzymes. The effect of eupatorin on the MDA-MB-468 cell cycle could be reversed by co-application of the CYP1 inhibitor acacetin. CONCLUSION: The flavone eupatorin is selectively activated in breast cancer cells, but not in normal breast cells, due to CYP1 family metabolism. This provides a basis for selectivity which is desired against breast tumour cells. In this sense, eupatorin is shown by this study to be a very promising chemopreventative candidate that should be examined further in an in vivo study.

The Synthesis of Chalcones as Anticancer Prodrugs and their Bioactivation in CYP1 Expressing Breast Cancer Cells.

BACKGROUND: Although the expression levels of many P450s differ between tumour and corresponding normal tissue, CYP1B1 is one of the few CYP subfamilies which is significantly and consistently overexpressed in tumours. CYP1B1 has been shown to be active within tumours and is capable of metabolising a structurally diverse range of anticancer drugs. Because of this, and its role in the activation of procarcinogens, CYP1B1 is seen as an important target for anticancer drug development. OBJECTIVE: To synthesise a series of chalcone derivatives based on the chemopreventative agent DMU-135 and investigate their antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1. METHOD: A series of chalcones were synthesised in yields of 43-94% using the Claisen-Schmidt condensation reaction. These were screened using a MTT assay against a panel of breast cancer cell lines which have been characterised for CYP1 expression. RESULT: A number of derivatives showed promising antiproliferative activities in human breast cancer cell lines which express CYP1B1 and CYP1A1, while showing significantly lower toxicity towards a non-tumour breast cell line with no CYP expression. Experiments using the CYP1 inhibitors acacetin and α-naphthoflavone provided supporting evidence for the involvement of CYP1 enzymes in the bioactivation of these compounds. CONCLUSION: Chalcones show promise as anticancer agents with evidence suggesting that CYP1 activation of these compounds may be involved.