Over the past decades, transition metal complexes have attracted considerable attention in medicinal inorganic chemistry, especially as synthetic metallonucleases and metal-based anticancer drugs that are able to bind to DNA under physiological conditions (Pessoa, J.C., et al. J. Inorg. Biochem. 2011, 105, 637-644). Copper(II) complexes offer various potential advantages as antimicrobial, antiviral, anti-inflammatory, antitumor agents, enzyme inhibitors, chemical nucleases, and they are also beneficial against several diseases like copper rheumatoid and gastric ulcers (Fricker, S.P., Dalton Trans. 2007, 43, 4903-4917).

Substitution reactions of square-planar [CuCl₂(en)] and square-pyramidal [CuCl₂(terpy)] complexes (where en= 1,2-diaminoethane and terpy= 2,2’:6’,2’’- terpyridine) with bio-relevant nucleophiles have been investigated at pH 7.4 in the presence of 0.010 M NaCl. Mechanism of substitution was probed via mole-ratio, kinetic, mass spectroscopy and EPR studies. In the presence of an excess of chloride, the octahedral complex anion [CuCl₄(en)]^2- forms rapidly while equilibrium reaction was observed for [CuCl₂(terpy)]. Different order of reactivity of selected bio-molecules toward Cu(II) complexes was observed. The nature of the buffer just affects the Cu(II) complexes conformational dynamics. According to EPR data L-Methionine forms a most stable complex with [CuCl₂(en)] among the bio-ligands considered while [CuCl₂(terpy)] complex is very stable and there are no significant changes in its square-pyramidal geometry in the presence of buffers or bio-ligands. The obtained results represent progress in investigation of the mechanism of substitution reactions between Cu(II) complexes and biological relevant nuclepohiles. Also, they provide very useful information for the future design of potential copper-based anticancer drugs (Selimović, E., et al. J. Coord. Chem. 2018, 71(7), 1003-1019).

The tyrosine-protein kinase Src, also known as proto-oncogene c-Src or simply c-Src, is a non-receptor tyrosine kinase protein that has been shown to be involved in the regulation of important cellular processes including migration, survival and proliferation. In fact, Src activation has been associated with multiple cancers, such as colon, breast, pancreas, lung, or brain (Roskoski, R. Jr. Pharmacol. Res. 2015, 94, 9-25; Creedon, H., et al., Crit. Rev. Oncog. 2012, 17, 145-159). There are only few Src inhibitors in clinical development, therefore, there is an urgent need to identify new low molecular weight therapeutics able to inhibit Src and, thus, to modulate aberrant pathways leading to malignant transformation of cells (Lu, X.L., et al., Curr. Med. Chem. 2012, 19, 1821-1829). Heterocyclic compounds attracted a lot of attention because of their wide spread biological activities. Thus, we have previously reported the synthesis of biological active heterocyclic derivatives based on the reactivity of the amidine moiety of 2-amino-2-oxazolines 2 with bis-electrophiles (Massip, S., et al., Bioorg. Med. Chem. 2006, 14, 2697-2719).

https://ibb.co/hzkMnJ

In a preliminary screening testing our heterocycles library, we have identified a “hit” (compound 1d) derived from various substituted 6-formyl-oxazolo[3,2-a]pyrimidines as a new Src kinase inhibitor (IC₅₀ = 4 µM). These original oxazolo[3,2-a]pyrimidine derivatives 1a-k were synthesized through a Diels-Alder cycloaddition of alkylidene derivatives of 2-amino-2-oxazoline (compounds 3a-k) with acrolein, as an electron-poor dienophile, a reaction previously described by our group (Guillon, J., et al., Synlett 2002, 8, 1249-1252). Versatility given by this reaction allowed us to access a promising family of diversely substituted 6-formyl-oxazolo[3,2-a]pyrimidines with inhibitory effect on Src kinase.

Acknowledgments: This work was supported by a grant from Ligue Contre le Cancer (Gironde, Bordeaux, France).

Ocmium teniflorum (Local name: Tulsi, Family: Lamiaceae) is a plant well known for its medicinal uses in unani and ayuveda medicine. It is called the ‘queen of herbs’ for its antimicrobial, antiinflammatory, hypotensive, hypoglycemic antipyretic and analgesic activities. Azadirachta indica (Local name: Neem, Family: Meliaceae) is a medicinal plant best known for its antimicrobial, antiinflammatory, anticancer and antiviral activities. It is a fact that the individual exudates of each plant attract a specific species of microbes and induce them to produce possible novel compounds under the influence of the plant environment. With this idea in mind, we investigated the antimicrobial activity of endophytic actinomycetes inhabiting tulsi and neem plants. The preliminary screening was done using agar plug method and it displayed 12 isolates with prominent bioactivity. Further biological screening of their active metabolites showed that the compounds were most active against Salmonella enteritidis, Campylobacter jejuni and Proteus mirabilis. The thin layer chromatography (TLC) and high performance liquid chromatography-UV (HPLC-UV) displayed UV absorbing polar and non polar compounds. Our study reveals tulsi and neem plant microenvironment as an unexplored niche harboring endophytes that are prominently bioactive against multidrug resistant (MDR) poultry and human pathogens.

Among the chemical world of flavonoids, prenylated derivatives have been attracting the attention because of the myriad of their biological activities, with chalcones being widely reported for their antitumor activity against a variety of tumor cell lines [1]. In fact, it has been demonstrated that isoprenylation of flavonoids significantly increased their growth inhibitory effect on human tumor cell lines [2]. A series of prenylchalcones was synthesized and evaluated for the ability to inhibit the MDM2-p53 interaction using a yeast-based assay [3]. The capacity of all synthesized prenylchalcones and their non-prenylated precursors to inhibit the growth of human colon tumor HCT116 cells was evaluated and compared [3]. The overall results led to the identification of a hit compound, which behaved as potential inhibitor of the MDM2-p53 interaction in yeast, and showed improved cytotoxicity against human tumor cells expressing wild-type p53. In HCT116 cancer cells, it was also shown that the growth inhibitory effect of this prenylchalcone was associated with the induction of cell cycle arrest, and apoptosis.

1. S. Venturelli et al. Nutrition, 2016, 32(11-12), 1171-1178.
2. M.P. Neves et al. Chem. Biodivers., 2012, 9, 1133-1143.
3. P. Brandão et al. Eur J Med Chem, 2018, 156, 711-721.

This research was partially supported by the Strategic Funding UID/Multi/04423/2013 and UID/MULTI/04378/2013 through national funds provided by FCT and ERDF, in the framework of the programme PT2020, the projects POCI-01-0145-FEDER-028736, PTDC/MAR-BIO/4694/2014 (reference POCI-01-0145-FEDER-016790; Project 3599–PPCDT), PTDC/AAGTEC/0739/2014 (reference POCI-01-0145-FEDER-016793; Project 9471–PPCDT), and PTDC/DTPFTO/1981/2014 (reference POCl-01-0145-FEDER-016581), as well as by the project INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, within Research Line NOVELMAR), supported by North Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF).

Oxime ethers have attracted much interest as important precursors and intermediates for the preparation of a wide variety of drugs and natural products. They can be easily converted into important functional groups such as amino alcohols and hydroxy ketones. Therefore, the development of methodologies for the preparation of oxime ethers is of considerable interest. Various researchers have studied the interesting biological properties of oxime ether derivatives such as anticonvulsant, anti-inflammatory, antineoplastic, anti-enteroviral, antimicrobial, antitumor, and anti-Helicobacter pylori activities [1].

Since clinical use of almost all anticancer drugs has been limited by the toxicity to normal tissues, important goal of cancer chemotherapy is to amplify the selective inhibition of tumor cells while decreasing toxicity to normal tissues.

In order to develop more efficient and selective antitumor agents, here we report the efficient synthesis of 17-substituted O-alkylated androstane derivatives, and investigate their antiproliferative activity (IC₅₀ after 72 h, MTT test) against three tumor cell lines (MDA-MB-231, HeLa and HT-29) and one healthy cell line (MRC-5).

In continuation of our work on nitrogen containing androstane derivatives [2-4], synthesis of respective novel compounds and their evaluation in a human carcinoma cell lines will be presented and discussed.

Acknowledgements:

This work was financialy supported by Ministry of Education, Science and Technological development of the Republic of Serbia (Project No. 172021).

[1] K. Sharma, S. B. Mishra, A. K. Mishra, Helv. Chim. Acta 94 (2011), 2256.

[2] J. Ajduković, E. Djurendić, E. Petri, O. Klisurić, A. Ćelić, M. Sakač, D. Jakimov, K. Penov Gaši, Bioorg. Med. Chem. 21 (2013), 7257.

[3] E. Djurendić, J. Ajduković, M. Sakač, J. Csanádi, V. Kojić, G. Bogdanović, K. Penov Gaši, Eur. J. Med. Chem. 54 (2012), 784.

[4] J. J. Ajduković, K. M. Penov Gaši, D. S. Jakimov, O. R. Klisuric, S. S. Jovanovic-Šanta, M. N. Sakac, L. D. Aleksic, E. A. Djurendic, Bioorg. Med. Chem. 23 (2015), 1557.

The increasing use of healthy unsaturated fats, more prone to oxidation, the understanding of the pathology of oxidative stress in the organisms, and the negative side-effects of some synthetic antioxidant compounds point to the research of natural antioxidants of plant origin which could be used in pharma or food industry.

In this study, the polyphenolic composition and in vitro antioxidant activity of the acetone extracts of hazelnut involucre (HI) were assayed. Optimum extraction conditions, based on an experimental design, were developed to obtain HI extracts rich in bioactive compounds. The best responses per gram of dry weight (dw) HI were in the range of 332 – 377 mg GAE (gallic acid equivalents) for total phenolic content (TPC); 37 – 43 mg QE (quercetin equivalents) for total flavonoid content (TFC); 24 – 28 mg CE (catechin equivalents) for condensed tannin content (CTC). The highest values for total antioxidant activity (TAA), determined by TEAC method, were in the range of 1,296 to 1,811 mg TE (Trolox equivalents) per gram of dw HI. A good relationship was observed between TPC, TFC, CTC, and TAA, the best results were found for the equal mixture (1:1) of acetone and water, as the extraction solvent. Further, for the richest polyphenolic HI extract, the phytochemical profile was examined by HPLC-MS/MS and several phenolics (epicatechin, catechin, syringic acid, gallic acid, protocatechuic acid, vanillic acid, hyperoside, quercitrin, isoquercitrin) and phytosterols (stigmasterol, campesterol, beta-sitosterol) were quantified.

To the best of our knowledge, this is the first study to assay the detailed composition in both hydrophilic and lipophilic bioactive compounds in hazelnut involucre. The phytochemical profile and the biological activity of the analyzed extract prove that this plant matrix can be a valuable source of bioactive compounds for food and/or pharmaceutical industry.

Intervention trials attempt to clarify the possible effects of certain challenge tests on study subjects (e.g. drugs effectiveness, environmental exposure experiments), while observational studies employ free-living populations to analyze the relationship between a particular effect and possible triggering factors. Based on the hypothesis under investigation, the researcher will choose the appropriate study design. Nevertheless, here we report the utility of combining observational and interventional studies to discover confident biomarkers in the clinical field (1). We contrasted metabolomic profiles related with diabetes and the oral glucose tolerance test (OGTT), a clinical test used to simulate the hyperinsulinemia observed in diabetes. We found that the main metabolic changes occur in the same metabolite classes, including energy-related metabolites, amino acids (especially brain chain amino acids, BCAA) and multiple lipids, such as free fatty acids, acyl-carnitines, triglycerides and phospholipids, among them. Hence, challenge tests such as the OGTT guarantee to be a great strategy to investigate pathological signatures associated with the development of diseases as a previous step before performing validation works in observational studies.

(1) Á. González-Domínguez, A.M. Lechuga-Sancho, R. González-Domínguez. Intervention and Observational Trials are Complementary in Metabolomics: Diabetes and the Oral Glucose Tolerance Test. Curr. Top. Med. Chem. 18 (2018) 896-900

The main subjects of this keynote are protein kinase CK2 and human leukocyte elastase (HLE), two biomedically important enzymes and pharmacologically attractive targets.

CK2 - more precisely its catalytic subunit CK2alpha - is a member of the superfamily of eukaryotic protein kinases. Its antiapoptotic activity is exploited by tumour cells in order to escape cell death. The indeno[1,2-b]indole scaffold, a flat annulated 4-ring system, is a relatively novel lead structure for the development of ATP-competitive CK2 inhibitors. Complex structures of CK2alpha and a number of indeno[1,2-b]indole-type compounds had been predicted previously. In such an in silico model the inhibitor sticks in the ATP cavity in an apparently plausible way, namely such that its hydrophobic side is directed inwards while its hydrophilic side has access to the solvent. However, when we determined the first co-crystal of CK2alpha with an indeno[1,2-b]indole-type inhibitor, we realized to our surprise that the orientation of the inhibitor was reversed: the "hydrophobic-out/oxygen-out" binding mode that we discovered is determined by hydrogen bonds of the inhibitor to a hidden and conserved water molecule. This molecular arrangement requires an inhibitor orientation in which hydrophobic substitutents are at the outer surface which opens the possibility for further modifications.

The second target enzyme, human leukocyte elastase (HLE), is a chymotrypsin-type serine protease which is produced by neutrophilic granulocytes, the most abundant cells of the innate immune system [therefore the synonym "human neutrophil elastase" (HNE)]. The activity of HLE must be strictly controlled to avoid proteolytic damage of the connective tissue which is a particular problem in chronic obstructive pulmonary disease (COPD) and other inflammatory diseases. Naturally, HLE is downregulated by alpha₁-antitrypsin, a serpin-type protease inhibitor, which is likewise produced by neutrophils. Synthetic HLE inhibitors are useful in cases of inbalance of the natural HLE control system. Typically, HLE inhibibitors block the S1 pocket of the enzyme, the most critical of several substrate binding cavities. The S1 pocket recognizes the side chain of the substrate directly N-terminal of the peptide bond to be hydrolyzed. In our study we co-crystallized HLE with a 1,3-thiazolidine-2,4-dione derivative with antibacterial activity that had been observed to inhibit HLE as well. In the complex structure the inhibitor is bound to the S2' site, i.e. at a region responsible for harbouring residues at the C-terminal side of the scissile peptide bond. In addition, the inhibitor seems to induce a dimerization of the enzyme by which the access to the active site region is prohibited.

Novel betulinic, ursolic, and oleanolic acid derivatives, containing a guanidine moiety have been designed and synthesized in an attempt to develop potent antitumor, antibacterial and antifungal agents. Triterpenoic acids were converted into C-28-aminotriterpenoids in which polyamine moieties were bound with C-28 carboxylic group through an amide or ester bonds. These compounds served as precursors for the synthesis of novel guanidine-functionalized triterpenoic acids derivatives. The cytotoxicity was tested on five human tumor cell lines (Jurkat, K562, U937, HEK, and Hela) and compared with the tests on normal human fibroblasts. The antitumor activities of the most tested guanidine derivatives was lower than that of corresponding amines, but triterpenoids with the guanidine group were less toxic to human fibroblasts. The identified lead molecules with the highest antitumor characteristics were selected for extensive biological testing according to flow cytometry data, which showed that the antitumor activity of these compounds is caused by apoptotic processes and induction of cell cycle arrest in the S-phase. Most of the tested guanidine derivatives showed a good antibacterial effect against Gram-positive bacteria Staphylococcus aureus (MICs values 0.5-4.0 mg/mL) and expressed significant antifungal activity against Candida albicans (4.0 mg/mL) and Cryptococcus neoformans (0.25-4.0 mg/mL), higher than the standard fluconazole (8.0 mg/mL).

Several biological activities from nearly 150 marine-derived sulfated steroids have been reported with both pharmacological (antimicrobial, antitumor, cardiovascular and/ or anti-inflammatory activities) and environmental (antifouling activity) applications [1]. Sulfation is used in Nature to avoid toxicity and therefore marine-inspired sulfated steroids could be an interesting strategy for drug discovery. The sulfated aminosterol squalamine, isolated from the internal organs of the dogfish shark, is in phase III of clinical trials as anti-angiogenic drug [2], which evidences the potential of sulfated steroids.

Sulfation of small molecules using sulfur trioxide-amine complexes entails several advantages, such as persulfation, low degradation, and feasibility in the work-up [3]. Moreover, these complexes appear to be suitable for sulfation of alcohol groups present in steroids [4]. In this direction, sulfation of four sterols was achieved using triethylamine-sulfur trioxide adduct in dimethylacetamide under heating, with yields ranging from 3% to 93%. Purification involved insolubilization with diethyl ether followed by several methods to obtain the sulfated derivatives free of inorganic impurities, including dialysis and/ or chromatographic processes. Structure elucidation of these new compounds was established by infrared (IR), nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). Biological activities will be further studied.

Acknowledgements:

This work was supported through national funds provided by FCT/MCTES - Foundation for Science and Technology from the Ministry of Science, Technology and Higher Education (PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE Programa Operacional Factores de Competitividade (POFC) programme, under the projects PTDC/MAR-BIO/4694/2014 (reference POCI-01-0145-FEDER-016790; Project 3599–PPCDT), PTDC/AAGTEC/0739/2014 (reference POCI-01-0145-FEDER-016793; Project 9471-RIDTI) and POCI-01-0145-FEDER-028736 in the framework of the programme PT2020. Carvalhal F also acknowledges FCT for the grant PTDC/AAG- TEC/0739/2014-018.

References:

[1] Carvalhal, F., M. Correia-da-Silva, M.E. Sousa, M. Pinto, and A. Kijjoa, Journal of Molecular Endocrinology, 2018, 61(2) 211-231.

[2] NCT02727881 (https://clinicaltrials.gov/ct2/show/NCT02727881, October 15, 2018)

[3] Correia-da-Silva, M., E. Sousa, and M.M. Pinto, Medicinal Research Reviews, 2014, 34(2) 223-79.

[4] Al-Horani, R.A., and U.R. Desai, Chemical Sulfation of Small Molecules - Advances and Challenges.Tetrahedron, 2010, 66(16), 2907-2918