Objective

In the present study, a series of twenty benzotriazolyl-N-phenylalkylamide derivatives (7a-7j) and (8a-8j) were synthesized, characterized by physicochemical and spectral data (IR, ¹H NMR, ¹³C NMR and mass spectroscopy) and evaluated for their anti-HIV activity with the aim to develop novel substituted benzotriazole derivatives with broad-spectrum chemotherapeutic properties. The synthesized compound have been further evaluated as antibacterial and antifungal activity.

Method

A series of twenty benzotriazolyl-N-phenylalkylamide derivatives were synthesized by reacting substituted anilines (1) with 2-chloroacetyl chloride (2) and 3-chloropropionyl chloride (3) to form intermediate (4a-4j) and (5a-5j). Intermediates further reacted with benzotraizole (6) to form benzotriazolyl-N-alkylamide derivatives (7a-7j) and (8a-8j). The synthesized test compounds (7a-7j) and (8a-8j) were assessed by MTT colorimetric assay on C8166 cells and screened for antibacterial activity against Gram-positive bacteria: Staphylococcus aureus (NCIM 2122), Bacillus subtilis (MTCC 121) and Gram-negative bacteria: Escherichia coli (MTCC118), Pseudomonas aeruginosa (MTCC 647), Salmonella typhi (NCIM 2501), Klebsiella pneumonia (MTCC 3384) and in vitro antifungal activity against Candida albicans (MTCC 227) and Aspergillus niger (NCIM 1056) by two fold broth serial dilution method.

Result- Compounds 7h, 7j, 8i and 8j being the most active showed therapeutic index that were >24.4, 31.1, 30.5 and 51.5 compared to Zidovudine (AZT) having therapeutic index (TI) 514342.6. The test compounds 7h, 7i, 7j, 8h, 8i and 8j exhibited very high activity against all the strains of Gram (+) ve and Gram (-) ve bacteria and antifungal strains.

Passion fruit oil has been used on the skin as a moisturizer, antioxidant, and depigmenting, due to its high content of polyphenols. The objective of this work was to evaluate different methods of preparation of microemulsions containing passion fruit seeds oil. For this, we used sonication and high pressure homogenization (HPH) methods under different conditions. In order to characterize the microemulsions, accelerated stability tests, pH, and internal phase droplets size measurements were performed. Then, a double face hydrophilic and lipophilic stick was prepared from the microemulsion chosen and characterized according to their hardness and coloration. The results showed that the microemulsion did not change its internal phase droplets size throughout the study. The stick hydrophilic face had lower hardness and showed less color change than lipophilic face. This study showed that it is possible to develop sticks from microemulsions containing passion fruit seeds oil with good stability characteristics.

Design of novel non-platinum DNA and protein targeting metal-based anticancer agents with potential in vitro toxicity have gained importance in recent years (Bertini, I. et al, Biological Inorganic Chemistry. Structure and Reactivity. University Science Books. Sausalito, CA. 2007. The non-platinum antitumor complexes could be alternatives to platinum-based drugs due to their better characteristics and less negative side effects. (Pessoa, J.C., et al. J. Inorg. Biochem. 2011, 105, 637-644).

The mechanism of substitution from tetrahedral [ZnCl₂(en)] and square-pyramidal [ZnCl₂(terpy)] complexes (where en = 1,2-diaminoethane or ethylenediamine; terpy= 2,2′:6′,2′′-terpyridine) by guanosine-5’-monophospahate (5’-GMP) have been investigated by ¹H NMR spectroscopy. Information about the structures of the final products in solution were obtained from the DFT calculations (B3LYP/6-31G(d)) and experimental ¹H NMR data acquired during the course of the reaction. The cytotoxic activity of zinc(II) complexes was tasted on human breast cancer cell line MDA-MB-231, human colon cancer cell line HCT-116 and normal human lung fibroblast cell line MRC-5. Both complexes reduced cell viabilities, while [ZnCl₂(terpy)] complex was significantly cytotoxic on MDA-MB-231 after 72 h, and HCT-116 after 24 h without dose dependence. The differences in reactivity toward 5’-GMP and cytotoxic activity of Zn(II) complexes may be attributed to the very stable square-pyramidal geometry of [ZnCl₂(terpy)] complex in solution, while weak ligand effect of the en compared to the terpy affected slow interaction of tetrahedral [ZnCl₂(en)] complex with the target bio-molecule (Soldatović, E., et al. J. Coord. Chem. 2019, 72(4), 690-706).

Acknowledgements: T. Soldatović and E. Selimović gratefully acknowledge financial support from State University of Novi Pazar, Novi Pazar, Republic of Serbia. R. Puchta would like to thank the Regionales Rechenzentrum Erlangen (RRZE) for a generous allotment of computer time, Prof. Tim Clark for hosting this work at the CCC. B.M. Alzoubi thanks Al-Balqa Applied University for its support. The authors are grateful for the support to the Ministry of Education, Science and Techhnological Development of the Republic of Serbia (Projects No. III41010, OI172016 and OI172036)

It has been reported that the antioxidant activity of the passion fruit seeds oil is comparable to that of ascorbic acid, due to a high content of polyphenols such as stilbenes. The aim of this study was to use the residues from the Madeira Island food industry to produce passion fruit seed extracts by Soxhlet and Ultrasound methods, using acetone and ethanol, in order to determine the presence of stilbenes (piceatannol and resveratrol) by High-performance liquid chromatography (HPLC). The extracts obtained by the ultrasound method showed high piceatannol and resveratrol content, while the extracts produced by Soxhlet and the commercial oil showed none of these stilbenes. These results suggest that passion fruit oil from Madeira Island can be used by the pharmaceutical and cosmetics industry because of its potential to reduce oxidative stress (ROS).

The topic to this study is determination of mass spectrometric diffusion parameters “D_SD” of oligomeric associates of glycylhomopeptides and their Ag^I–complexes according to our “stochastic dynamic” approach and model equations under electrospray ionization condition. The problematic has recently gained attenion thanks to the latter formulas connecting among “D_SD” data; the measurable outcome “intensity” of analyte ions; and the experimental parameter “temperature,” respectively. The equations are empirically testable and verifiable. In advancing this innovative view upon which models we will carry out analysis of ions of oligomeric associates of peptides, we should point out that it is crucial to further test our formulas on a larger set of chemical classes and experimental conditions in order to verify their universal applicability to different mass spectrometric ionization methods. Because of, on the concept sketched above the D_SD parameters correlate excellent linearly with kinetic parameters of fragment reactions and quantum chemical diffusions according to the Arrhenius’s approximation which reflects the 3D molecular structures of analytes. The most important point regarding our concept is that it extends crucially the capability of the mass spectrometry of multidimentional structural analysis when is applied to high accuracy quantum chemical static and molecular dynamic approaches.

Breast-milk α_S1-casein was suggested as an agonist of the Toll-like receptor 4 (TLR4).¹ Pathogen recognition receptor TLR4 responds to lipopolysaccharides and a wide range of molecules, from proteins to metal ions. In consequence, three criteria are required to validate agonists which directly activate TLR4 and exclude TLR4-agonisticity through contaminants.² Recently, we demonstrated that α_S1-casein fulfilled two of these criteria. (i) α_S1-Casein required TLR4/MD2 complex as well as cofactor CD14 to induce IL-8 secretion via TLR4 and (ii) α_S1-casein bound TLR4, MD2 and CD14.³ Aim of this study was to (iii) identify a synthetic amino acid sequence derived from human α_S1-casein responsible for TLR4-agonistic effects.

For this, we analyzed the amino acid sequence (AAS) of α_S1-casein in silico. α_S1‑Casein showed to be α-helical and was likely to be intrinsically disordered in the region corresponding to R¹⁶-K⁹⁹ of α_S1-casein. Six truncated variants of α_S1-casein coding for parts of the AAS were purified from Escherichia coli. These variants were tested for binding to HEK293 cells transfected with TLR4 (TLR4⁺) by flow cytometry and their induction of IL-8 secretion via TLR4. Variants of α_S1-casein truncated at the N-terminus (E³⁵-W¹⁸⁵, R⁵⁷-W¹⁸⁵, V⁷⁷-W¹⁸⁵) bound TLR4⁺ induced lower IL-8 secretion with less AAS (7.5 ng/ml, 4.8 ng/ml, 3.6 ng/ml). Variant corresponding to E⁹³-W¹⁸⁵ of α_S1-casein was neither binding TLR4⁺ nor inducing IL-8 secretion. Therefore, we postulated V⁷⁷-E⁹² derived from α_S1-casein as TLR4-agonist. This was confirmed by a synthetic peptide V⁷⁷-E⁹² derived from α_S1-casein, which induced an IL-8 secretion of 0.95 ng/ml. Hence, the third criteria of TLR4-agonists fulfilled and activation of TLR4 through contamination was excluded.

In conclusion, α_S1-casein was proofed as an agonist directly activating TLR4. This supported our postulate that α_S1-casein has at least two functions, a nutritional and an immune active one.

1. Vordenbaumen, S. et al. Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling. Mol Nutr Food Res 60, 1079-89 (2016).
2. Mancek-Keber, M. & Jerala, R. Postulates for validating TLR4 agonists. Eur J Immunol 45, 356-70 (2015).
3. Saenger, T. et al. Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex. Biochim Biophys Acta Gen Subj 1863, 632-643 (2019).

Breast-milk α_S1-casein is a Toll-like receptor (TLR4) agonist which induced proinflammatory cytokine secretion. Phosphorylated α_S1-casein (P- α_S1-casein) is non-agonistic.^1,2 The objective of this study was to analyze structural characteristics underlying these observations.

Recombinant α_S1-casein was shown to exist in two conformations, an α-helical TLR4-agonistic conformation and a non-agonistic conformation with lower α‑helical and higher random coil content. TLR4-agonstic α_S1-casein conformation was found at a pH-range between 7.4 and 2. α_S1-Casein bound itself (K_D-value: 2 µM) formed large aggregates (between Ø 73 nm [pH7] and Ø 826.2 nm [pH2]). Using Thioflavin T assay and atomic force microscopy showed that α_S1-casein adopted fibril-like structure. P-α_S1-casein was observed in a less α‑helical conformation, not inducing IL-8 secretion. P-α_S1-casein bound itself stronger (K_D-value: 0.5 µM) than α_S1-casein and did not form fibrils.

In conclusion, TLR4-agonistic and non-agonistic conformations of α_S1-casein could be differentiated. It was demonstrated that human caseins are able to adopt fibril structure. These kind of structures are often disease related. We postulate, that phosphorylation could be a switch of two conformations regulating immunomodulatory effects of human α_S1-casein especially in immune system development.

1. Vordenbaumen, S. et al. Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling. Mol Nutr Food Res 60, 1079-89 (2016).
2. Saenger, T. et al. Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex. Biochim Biophys Acta Gen Subj 1863, 632-643 (2019).

Currently drug resistance is rising to dangerously high levels worldwide and threatening our ability to treat even common infectious diseases. Secondary metabolites, especially alkaloids containing an indole group and structurally related to fumiquinazolines, are of crucial importance in the area of drug discovery, having representatives such as fiscalin B that was reported as substance P antagonist and neofiscalin A, a potent antibacterial agent active in both reference and multidrug-resistant isolates. [1] Herein, the synthesis of quinazolinone alkaloid derivatives containing an indole moiety is reported, using two different methodologies – a highly efficient three-component one-pot microwave-assisted and a multi-step Mazurkiewicz-Ganesan approach. With this approach, 38 derivatives were obtained in low to moderate yields and were further tested for their antitumor [2,3], neuroprotection [2], antibacterial, and antifungal activities. While 16 compounds exhibited weak to moderate tumor cell growth inhibitory activity, other four compounds showed potential for in vitro neuroprotection in Parkinson disease. It was also observed for some derivatives a good antibacterial activity against clinical Staphylococcus aureus resistant to methicillin (MRSA). Structure-activity relationship was established and four hit compounds containing the quinazolinone scaffold emerged as potential drug candidates.

Acknowledgements: We thank the UID/Multi/04423/2019 through national funds provided by FCT-Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the program PT2020. This research was developed under Project No. POCI-01-0145-FEDER-028736, co-financed by COMPETE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT through national funds, and Solida Long thanks Erasmus-Lotus+ program (LOTUS+, LP15DF0205).

References:

[1] Resende, D. I. S. P.; Boonpothong, P.; Sousa, E.; Kijjoa, A.; Pinto, M. M. M., Chemistry of the fumiquinazolines and structurally related alkaloids. Natural Product Reports 2019, 36, 7-34.

[2] Long, S.; Resende, D. I. S. P.; Kijjoa, A.; Silva, A. M. S.; Fernandes, R.; Xavier, C. P. R.; Vasconcelos, M. H.; Sousa, E.; Pinto, M. M. M., Synthesis of New Proteomimetic Quinazolinone Alkaloids and Evaluation of Their Neuroprotective and Antitumor Effects. Molecules 2019, 24 (3), 534.

[3] Long, S.; Resende, D.; Kijjoa, A.; Silva, A.; Pina, A.; Fernández-Marcelo, T.; Vasconcelos, M.; Sousa, E.; Pinto, M., Antitumor Activity of Quinazolinone Alkaloids Inspired by Marine Natural Products. Mar. Drugs 2018, 16 (8), 261.

Cyclams are macrocyclic polyamines which medical interest was fueled by the therapeutic potential of a bicyclam derivative in HIV infection, inflammatory diseases, cancer and stem-cell mobilization.[1] Taking advantage of the biocompatibility, the high metal chelation stability constants and the possibility of N-functionalization of the cyclam backbone, a variety of compounds have been explored in a wide range of medicinal applications.[2] The use of cyclams and cyclam-based complexes as antimicrobial and antitumoral agents has been described in recent years. In particular, trans-disubstituted cyclam salts revealed to be active antibacterial agents against both Gram-positive and Gram-negative bacteria.[3,4]

In the field of anticancer applications, several attempts are being made, mostly with Cu^II complexes, envisaging their use as ^64/67Cu radionuclides.[5] Recently, we found that trans-disubstituted cyclam derivatives and their Cu^II and Fe^III complexes display relevant antitumoral activity against HeLa cancer cell lines.[6] To the best of our knowledge, this is the first report on an iron-cyclam compound tested as anticancer agent.

[1] De Clercq, E., Nat. Rev. Drug Discov. 2003, 2, 581–587

[2] Liang, X.; Sadler, P. J., Chem. Soc. Rev. 2004, 33, 246-266

[3] Yu, M.; Nagalingam, G.; Ellis, S.; Martinez, E.; Sintchenko, V.; Spain, M.; Rutledge, P. J.; Todd, M. H.; Triccas, J. A., J. Med. Chem. 2016, 59, 5917–5921

[4] Alves, L. G.; Pinheiro, P. F.; Feliciano, J. R.; Dâmaso, D. P.; Leitão, J. H.; Martins, A. M., Int. J. Antimicrob. Agents 2017, 49, 646-649

[5] Cai, Z.; Anderson, C. J., J. Label. Compd. Radiopharm. 2014, 57, 224–230

[6] Pilon, A.; Lorenzo, J.; Rodriguez-Calado, S.; Adão, P.; Martins, A. M.; Valente, A.; Alves, L. G., ChemMedChem, 2019, 14, 770-778

Tuberculosis is still one of the most prevalent diseases worldwide caused by Mycobacterium
tuberculosis (Mtb), bearing a long-term treatment that is not always effective. Admitting this
context, multiple studies have been trying to develop novel substances against Mtb, specially using in silico techniques to predict its effects on a known target. Using a systematic approach, we were able to retrieve and evaluate 46 manuscripts from three different databases that firstly applied an in silico technique to explore new antimycobacterial molecules and secondly attempted to prove its predictive potential by an in vitro or in vivo assay. We found that although all manuscripts followed a similar screening procedure (ligand and/or structure-based screening), they explored a large number of ligands on 29 distinct bacterial enzymes. The following in vitro/vivo analysis showed that the virtual screening was able to decrease the number of tested molecules, saving time and funding, but could only provide a modest correlation to the effectiveness of those molecules in vitro. In short, we found that the preliminary in silico approach is recommended specially on the early steps in developing
a new drug, but call for more studies to evaluate its clinical predictive possibilities.