Myrtus communis Liquor Byproduct as a Source of Bioactive Compounds.

The fatty acid (FA), polyphenol content and evaluation of the antioxidant capacity of exhausted Myrtus communis berries (EMB) resulting from the production of myrtle liqueur were assessed. All parts of the exhausted berries exhibited high concentrations of carbohydrates, proteins, lipids and phenolic compounds. The lipid fraction contained a high amount of poly unsaturated fatty acids (PUFA), mainly represented by linoleic acid (>70%). Of the phenolic acids evaluated by liquid chromatography/mass spectrometry, ellagic acid was the most predominant (>50%), followed by gallic and quinic acids. Quercetin and quercetin3-O-rhamnoside were the most abundant flavonoids. The seed extracts showed a higher antioxidant potential than the pericarp extracts; the same trend was observed for total phenolic compounds evaluated by spectrophotometric assay. The overall high content of bioactive compounds and the high antioxidant potential of this byproduct sustain its suitability for a number of industrial applications, such as a food ingredient in novel foods, an additive in cosmetic formulations or a component of animal feed formulations.

Inhibitory Effect of 2,3,5,6-Tetrafluoro-4-[4-(aryl)-1H-1,2,3-triazol-1-yl]benzenesulfonamide Derivatives on HIV Reverse Transcriptase Associated RNase H Activities.

The HIV-1 ribonuclease H (RNase H) function of the reverse transcriptase (RT) enzyme catalyzes the selective hydrolysis of the RNA strand of the RNA:DNA heteroduplex replication intermediate, and represents a suitable target for drug development. A particularly attractive approach is constituted by the interference with the RNase H metal-dependent catalytic activity, which resides in the active site located at the C-terminus p66 subunit of RT. Herein, we report results of an in-house screening campaign that allowed us to identify 4-[4-(aryl)-1H-1,2,3-triazol-1-yl]benzenesulfonamides, prepared by the "click chemistry" approach, as novel potential HIV-1 RNase H inhibitors. Three compounds (9d, 10c, and 10d) demonstrated a selective inhibitory activity against the HIV-1 RNase H enzyme at micromolar concentrations. Drug-likeness, predicted by the calculation of a panel of physicochemical and ADME properties, putative binding modes for the active compounds, assessed by computational molecular docking, as well as a mechanistic hypothesis for this novel chemotype are reported.

Identification and quantitation of 4-bromo-2,5-dimethoxyamphetamine in seized blotters.

Blotters are usually impregnated with hallucinogens such as lysergic acid diethylamide (LSD); only rarely other psychoactive substances are detected. In this work we identified 4-bromo-2,5-dimethoxyamphetamine (DOB) and 2,5-dimethoxyamphetamine (DMA) in illicit blotters seized in Italy. This report describes a rapid method for the simultaneous identification and quantitation of DOB and its precursor (DMA) by liquid chromatography tandem mass spectrometry (LC-MS-MS), using 2,3-dimethoxyphenethylamine-d3 as internal standard. Regression equations were linear over the tested concentration range with good correlation coefficients. The achieved levels of sensitivity may be suitable to confirm the possible presence of DOB and DMA also in low concentration or in traces in seized material for forensic analysis. The developed method showed good reproducibility and sensitivity, and could be used for similar routine analysis. To our knowledge, this is the first report describing the detection of DOB and DMA from blotters.

Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer.

BACKGROUND: Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling. METHODS: Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots. RESULTS: Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity. CONCLUSION: In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies. GENERAL SIGNIFICANCE: Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.

Synthesis and anti-mycobacterial activities of triazoloquinolones.

A number of quinolone derivatives have been reported to possess anti-mycobacterial activity. Generally. Mycobacterium tuberculosis isolates expressing resistance to both isoniazid and rifampin are susceptible to fluoroquinolones. Benzotriazole is a hetero-bicyclic aromatic ring endowed with interesting chemical and biological properties and pharmacological activities. In a preliminary study we have recently reported the activity of triazolo[4,5-h]quinolone-carboxylic acids, a new class of benzotriazole derivatives active against multi-drug resistant M. tuberculosis (MDR-Mtb). In this study we confirm that this novel class of quinolones is endowed with a selective anti-mycobacterial activity, coupled with absence of cytotoxicity. The SAR analysis of the new derivatives in comparison with the previous series shows that the methyl group is the most effective substituent in both N-3 and N-9 positions of the ring system.

[1,2,3]Triazolo[4,5-h]quinolones. A new class of potent antitubercular agents against multidrug resistant Mycobacterium tuberculosis strains.

In this preliminary study we report the activity of 3-methyl-9-substituted-6-oxo-6,9-dihydro-3H-[1,2,3]-triazolo[4,5-h]quinolone-carboxylic acids and their esters as a new class of antiinfective agents against MDR Mycobacterium tuberculosis. In antitubercular screening against H37Rv and 11 clinically isolated strains of M. tuberculosis several derivatives (1o,3a,c,i,j,p) showed MIC(90) in the range 0.5-3.2 microg/mL. 3c showed no cytotoxicity and proved to be the most potent derivative exhibiting MIC(90)=0.5 microg/mL against all M. tuberculosis strains and infected human macrophages (J774-A1) tested.

Design of novel bioisosteres of beta-diketo acid inhibitors of HIV-1 integrase.

HIV-1 integrase (IN) is an attractive and validated target for the development of novel therapeutics against AIDS. Significant efforts have been devoted to the identification of IN inhibitors using various methods. In this context, through virtual screening of the NCI database and structure-based drug design strategies, we identified several pharmacophoric fragments and incorporated them on various aromatic or heteroaromatic rings. In addition, we designed and synthesized a series of 5-aryl(heteroaryl)-isoxazole-3-carboxylic acids as biological isosteric analogues of beta-diketo acid containing inhibitors of HIV-1 IN and their derivatives. Further computational docking studies were performed to investigate the mode of interactions of the most active ligands with the IN active site. Results suggested that some of the tested compounds could be considered as lead compounds and suitable for further optimization.

Detection of transcripts of the aflatoxin genes aflD, aflO, and aflP by reverse transcription-polymerase chain reaction allows differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and Aspergillus parasiticus.

The aim of this study was to test the suitability of the RT-PCR (reverse transcription-polymerase chain reaction) technique to differentiate aflatoxin-producing from aflatoxin-non-producing strains of Aspergillus flavus and Aspergillus parasiticus. Total RNAs of 13 strains grown under inducing yeast extract-sucrose (YES) and non-inducing yeast extract-peptone (YEP) media, respectively, were analyzed by using specific primers based on the conserved regions of nine structural genes (aflD, aflG, aflH, aflI, aflK, aflM, aflO, aflP, and aflQ) and two regulatory genes aflS and aflR of the aflatoxin B1 biosynthetic pathway. Transcription was confirmed by the expression of the beta-tubulin gene. The expression of the majority aflatoxin biosynthetic genes including aflR and aflS of all strains varied with regard to the aflatoxin-producing ability and the growth conditions. Nonetheless, we found that the expression profile of the three genes aflD, aflO, and aflP was consistently correlated with a strain's ability to produce aflatoxins or not in YES as well as the inability to produce aflatoxins in YEP. The devised RT-PCR profiling method reflects aflatoxin concentrations ranging from 0.1 to 60 microg/ml of the culture filtrates as determined by fluorescence HPLC. The results are discussed in relation to the suitability of RT-PCR as well as cDNA-based array techniques in diagnostic laboratory settings where individual isolates are being tested for potential toxin production to identify toxigenic isolates of Aspergillus species.

Determination of amphetamine-derived designer drugs in human urine by SPE extraction and capillary electrophoresis with mass spectrometry detection.

In recent years, a number of newer designer drugs have entered the illicit drug market. The methylenedioxy-derivates of amphetamine represent the largest group of designer drugs. This paper describes a method for screening for and quantification of ten 2,5-methylenedioxy-derivates of amphetamine and phenylethylamine in human urine, using capillary electrophoresis coupled to electrospray ionisation-mass spectrometry (CE-ESI-MS). Prior to CE-MS analysis, a simple solid-phase extraction (SPE) was used for sample cleanup. The method was validates according to international guidelines.

Design and synthesis of novel indole beta-diketo acid derivatives as HIV-1 integrase inhibitors.

Diketo acids such as S-1360 (1A) and L-731,988 (2) are potent and selective inhibitors of HIV-1 integrase (IN). A plethora of diketo acid-containing compounds have been claimed in patent literature without disclosing much biological activities and synthetic details (reviewed in Neamati, N. Exp. Opin. Ther. Pat. 2002, 12, 709-724). To establish a coherent structure-activity relationship among the substituted indole nucleus bearing a beta-diketo acid moiety, a series of substituted indole-beta-diketo acids (4a-f and 5a-e) were synthesized. All compounds tested showed anti-IN activity at low micromolar concentrations with varied selectivity against the strand transfer process. Three compounds, the indole-3-beta-diketo acids 5a and 5c, and the parent ester 9c, have shown an antiviral activity in cell-based assays. We further confirmed a keto-enolic structure in the 2,3-position of the diketo acid moiety of a representative compound (4c) using NMR and X-ray crystallographic analysis. Using this structure as a lead for all of our computational studies, we found that the title compounds extensively interact with the essential amino acids on the active site of IN.

Synthesis and antiproliferative activity of 3-aryl-2-[1H(2H)-benzotriazol-1(2)-yl]acrylonitriles variously substituted: Part 4.

A new series of variously substituted 3-aryl-2-[1H(2H)-benzotriazol-1(2)-yl]acrylonitriles was synthesized and tested for antiproliferative and antitubercular activity as part of our continuing research program in the antimicrobial and antitumor fields. The most cytotoxic derivatives (5a,g,i,j,l and 7b) (CC50 < 3.0 microM against MT-4 cells) were evaluated against a panel of human cell lines derived from hematological and solid tumors, using 6-mercaptopurine (6-MP) and etoposide as reference drugs. In particular, E-2-(5,6-dimethyl-1H-benzotriazol-1-yl)-3-(3-nitrophenyl)acrylonitrile (5g) resulted more potent than 6-MP on all cell lines, even if 2-14-fold less potent than etoposide. In the antitubercular screening, the derivatives 5i,j and 7e showed moderate activity against some resistant strains of Mycobacterium tested.

Synthesis and antiproliferative activity of 3-aryl-2-(1H-benzotriazol-1-yl)acrylonitriles. Part III.

A new series of 30 3-aryl-2-(1H-benzotriazol-1-yl)acrylonitriles were synthesized and tested for biological activity as part of our research in the antimicrobial and antitumor fields. In particular, title compounds were evaluated in vitro against representative strains of Gram-positive and Gram-negative bacteria (S. aureus, Salmonella spp), mycobacteria (M. fortuitum, M. smegmatis ATCC 19420 and M. tuberculosis ATCC 27294), yeast and mould (C. albicans ATCC 10231 and A. fumigatus). Furthermore, their antiretroviral activity against HIV-1 was determined in MT-4 cells together with cytotoxicity. In these assays title compounds and 47 additional derivatives described previously (P. Sanna, A. Carta, M.E. Rahbar Nikookar, Eur. J. Med. Chem. 35 (2000) 535-543; P. Sanna, A. Carta, L. Gherardini, M.E. Rahbar Nikookar, Farmaco 57 (2002) 79-87) were tested for their capability to prevent MT-4 cell growth. All compounds resulted devoid of antibacterial, antifungal and anti-HIV-1 activity. In anti-mycobacterial assays several compounds resulted active (MIC(50)=6.0-70 microM) against M. tuberculosis. However, since they showed cytotoxicity against MT-4 cells at lower concentrations (CC(50)=0.05-25 microM), their anti-mycobacterial activity was not selective. For this reason, the most cytotoxic compounds were also evaluated for antiproliferative activity against a panel of human cell lines derived from both hematological and solid tumors. Compound 34 resulted the most potent compound against the above human tumor-derived cell lines.