Pyrimidine Schiff Bases: Synthesis, Structural Characterization and Recent Studies on Biological Activities.

Recently, 5-[(4-ethoxyphenyl)imino]methyl-N-(4-fluorophenyl)-6-methyl-2-phenylpyrimidin-4-amine has been synthesized, characterized, and evaluated for its antibacterial activity against Enterococcus faecalis in combination with antineoplastic activity against gastric adenocarcinoma. In this study, new 5-iminomethylpyrimidine compounds were synthesized which differ in the substituent(s) of the aromatic ring attached to the imine group. The structures of newly obtained pyrimidine Schiff bases were established by spectroscopy techniques (ESI-MS, FTIR and 1H NMR). To extend the current knowledge about the features responsible for the biological activity of the new 5-iminomethylpyrimidine derivatives, low-temperature single-crystal X-ray analyses were carried out. For all studied crystals, intramolecular N-H∙∙∙N hydrogen bonds and intermolecular C-H∙∙∙F interactions were observed and seemed to play an essential role in the formation of the structures. Simultaneously, their biological properties based on their cytotoxic features were compared with the activities of the Schiff base (III) published previously. Moreover, computational investigations, such as ADME prediction analysis and molecular docking, were also performed on the most active new Schiff base (compound 4b). These results were compared with the highest active compound III.

New Insights on the Feature and Function of Tail Tubular Protein B and Tail Fiber Protein of the Lytic Bacteriophage φYeO3-12 Specific for Yersinia enterocolitica Serotype O:3.

For the first time, we are introducing TTPBgp12 and TFPgp17 as new members of the tail tubular proteins B (TTPB) and tail fiber proteins (TFP) family, respectively. These proteins originate from Yersinia enterocolitica phage φYeO3-12. It was originally thought that these were structural proteins. However, our results show that they also inhibit bacterial growth and biofilm formation. According to the bioinformatic analysis, TTPBgp12 is functionally and structurally similar to the TTP of Enterobacteria phage T7 and adopts a ß-structure. TFPgp17 contains an intramolecular chaperone domain at its C-terminal end. The N-terminus of TFPgp17 is similar to other representatives of the TFP family. Interestingly, the predicted 3D structure of TFPgp17 is similar to other bacterial S-layer proteins. Based on the thermal unfolding experiment, TTPBgp12 seems to be a two-domain protein that aggregates in the presence of sugars such as maltose and N-acetylglucosamine (GlcNAc). These sugars cause two unfolding events to transition into one global event. TFPgp17 is a one-domain protein. Maltose and GlcNAc decrease the aggregation temperature of TFPgp17, while the presence of N-acetylgalactosamine (GalNAc) increases the temperature of its aggregation. The thermal unfolding analysis of the concentration gradient of TTPBgp12 and TFPgp17 indicates that with decreasing concentrations, both proteins increase in stability. However, a decrease in the protein concentration also causes an increase in its aggregation, for both TTPBgp12 and TFPgp17.

Synthesis and Biological Activity of New 7-Amino-oxazolo[5,4-d]Pyrimidine Derivatives.

The synthesis of a series of novel 7-aminooxazolo[5,4-d]pyrimidines 5, transformations during their synthesis and their physicochemical characteristics have been described. Complete detailed spectral analysis of the intermediates 2-4, the N'-cyanooxazolylacetamidine by-products 7 and final compounds 5 has been carried out using MS, IR, 1D and 2D NMR spectroscopy. Theoretical research was carried out to explain the privileged formation of 7-aminooxazolo[5,4-d]pyrimidines in relation to the possibility of their isomer formation and the related thermodynamic aspects. Additionally, the single-crystal X-ray diffraction analysis for 5h was reported. Ten 7-aminooxazolo[5,4-d]pyrimidines 5 (SCM1-10) were biologically tested in vitro to preliminarily evaluate their immunological, antiviral and anticancer activity. Compounds SCM5 and SCM9 showed the best immunoregulatory profile. The compounds displayed low-toxicity and strongly inhibited phytohemagglutinin A-induced proliferation of human peripheral blood lymphocytes and lipopolysaccharide-induced proliferation of mouse splenocytes. Compound SCM9 caused also a moderate suppression of tumor necrosis factor α (TNF-α) production in a human whole blood culture. Of note, the compounds also inhibited the growth of selected tumor cell lines and inhibited replication of human herpes virus type-1 (HHV-1) virus in A-549 cell line. Molecular investigations showed that the compounds exerted differential changes in expression of signaling proteins in Jurkat and WEHI-231 cell lines. The activity of SCM5 is likely associated with elicitation of cell signaling pathways leading to cell apoptosis. The compounds may be of interest in terms of therapeutic utility as inhibitors of autoimmune disorders, virus replication and antitumor agents.

Highly regioselective and diastereoselective synthesis of novel pyrazinoindolones via a base-mediated Ugi-N-alkylation sequence.

An efficient base-mediated/metal-free approach has been developed for the synthesis of 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide derivatives via intramolecular indole N-H alkylation of novel bis-amide Ugi-adducts. In this protocol the Ugi reaction of (E)-cinnamaldehyde derivatives, 2-chloroaniline, indole-2-carboxylic acid and different isocyanides was designed for the preparation of bis-amides. The main highlight of this study is the practical and highly regioselective preparation of new polycyclic functionalized pyrazino derivatives. This system is facilitated by Na2CO3 mediation in DMSO and 100 °C conditions.

Synthesis, Structural Characterization and Anticancer Activity of New 5-Trifluoromethyl-2-thioxo-thiazolo[4,5-d]pyrimidine Derivatives.

Thiazolo[4,5-d]pyrimidine derivatives are considered potential therapeutic agents, particularly in the development of anticancer drugs. In this study, new 7-oxo-(2a-e), 7-chloro-(3a-e) and also three 7-amino-(4a-c) 5-trifluoromethyl-2-thioxo-thiazolo[4,5-d]pyrimidine derivatives have been synthesized and evaluated for their potential anticancer activity. These derivatives were characterized by spectroscopic methods and elemental analysis, and the single-crystal X-ray diffraction was further performed to confirm a 3D structure for compounds 2e and 4b. The antiproliferative activity evaluation of twelve new compounds was carried out on a variety of cell lines including four human cancer (A375, C32, DU145, MCF-7/WT) and two normal cell lines (CHO-K1 and HaCaT). Four of them (2b, 3b, 4b and 4c) were selected by the National Cancer Institute and evaluated for their in vitro anticancer activity using the NCI-60 screening program. 7-Chloro-3-phenyl-5-(trifluoromethyl)[1,3]thiazolo[4,5-d]pyrimidine-2(3H)-thione (3b) proved to be the most active among the newly synthesized compounds.

Structure and cytotoxic properties of 1-hydroxy-5-methyl-7-phenylpyrido[3,4-d]pyridazin-4(3H)-one and its mono- and disubstituted ethyl acetates.

Derivatives of pyrido[3,4-d]pyridazine, namely, 1-hydroxy-5-methyl-7-phenylpyrido[3,4-d]pyridazin-4(3H)-one dimethylformamide monosolvate, C14H11N3O2·C3H7NO (2), ethyl [1-(2-ethoxy-2-oxoethoxy)-5-methyl-4-oxo-7-phenyl-3,4-dihydropyrido[3,4-d]pyridazin-3-yl]acetate, C18H17N3O4 (3), and ethyl [(5-methyl-4-oxo-7-phenyl-3,4-dihydropyrido[3,4-d]pyridazin-1-yl)oxy]acetate, C22H23N3O6 (4), were synthesized with the aim of discovering new potential biologically active agents. The properties of all three derivatives were characterized by 1H NMR, 13C NMR and FT-IR spectroscopic analysis. All the crystals were obtained by a solvent diffusion method from dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) and characterized by single-crystal X-ray diffraction. The collected X-ray data revealed that the crystals of 2 and 4 belong to the triclinic space group P-1, whereas the crystal of 3 belongs to the monoclinic space group P21/c. The presented derivatives crystallized with one molecule in the asymmetric unit, but only compound 2 crystallized as a solvate with DMF. Structure analysis showed that the molecule of 2 exists as its amide-imidic acid tautomer and that O-alkylation occurred before N-alkylation during the synthesis of the mono- and disubstituted derivatives, i.e. 3 and 4, respectively. The molecular geometries of the 5-methyl-7-phenylpyrido[3,4-d]pyridazine core within the studied derivatives differ in the mutual orientation of the rings. The interplanar angles between the heterocyclic ring and the bound aromatic ring are 1.71 (7), 18.16 (3) and 3.1 (1)° for 2, 3 and 4, respectively. The potential cytotoxicity of these compounds was evaluated against one normal (HaCat) and four human cancer cell lines (A549, DU145, MDA-MB-231 and SKOV-3).

φYeO3-12 phage tail fiber Gp17 as a promising high specific tool for recognition of Yersinia enterocolitica pathogenic serotype O:3.

Yersiniosis is an infectious zoonotic disease caused by two enteropathogenic species of Gram-negative genus Yersinia: Yersinia enterocolitica and Yersinia pseudotuberculosis. Pigs and other wild and domestic animals are reservoirs for these bacteria. Infection is usually spread to humans by ingestion of contaminated food. Yersiniosis is considered a rare disease, but recent studies indicate that it is overlooked in the diagnostic process therefore the infections with this bacterium are not often identified. Reliable diagnosis of Yersiniosis by culturing is difficult due to the slow growth of the bacteria easily overgrown by other more rapidly growing microbes unless selec-tive growth media is used. Phage adhesins recognizing bacteria in a specific manner can be an excellent diagnostic tool, es-pecially in the diagnosis of pathogens difficult for culturing. In this study, it was shown that Gp17, the tail fiber protein (TFP) of phage φYeO3-12, specifically recognizes only the pathogenic Yersinia enterocolitica serotype O:3 (YeO:3) bacteria. The ELISA test used in this work confirmed the specific interaction of this protein with YeO:3 and demonstrated a promising tool for developing the pathogen recognition method based on phage adhesins.

Synthesis, Crystal Structure, and Biological Evaluation of Novel 5-Hydroxymethylpyrimidines.

Pyrimidine displays a wide array of bioactivities, and thence, it is still considered a potent unit of new drug research. Its derivative, 5-hydroxymethylpyrimidine, can be found as a scaffold of nontypical nitrogen bases in DNA and as a core of some natural bioactive compounds. In this study, we obtained a series of 5-hydroxymethylpyrimidines that vary in the 4-position by the reduction of proper esters. All compounds were characterized by spectroscopic analysis, and single-crystal X-ray diffraction was performed for some of them. Biological investigations estimated cytotoxic properties against normal (RPTEC) and cancer (HeLa, HepaRG, Caco-2, AGS, A172) cell lines. It was found that the derivatives with an aliphatic amino group at the 4-position are generally less toxic to normal cells than those with a benzylsulfanyl group. Moreover, compounds with bulky constituents exhibit better anticancer properties, though at a moderate level. The specific compounds were chosen due to their most promising IC50 concentration for in silico study. Furthermore, antimicrobial activity tests were performed against six strains of bacteria and one fungus. They demonstrated that only derivatives with at least three carbon chain amino groups at the 4-position have weak antibacterial properties, and only the derivative with 4-benzylsulfanyl constituent exhibits any antifungal action.

Biochemical features of the novel Tail Tubular Protein A of Yersinia phage phiYeO3-12.

Tail Tubular Protein A (TTPA) was long thought to be strictly a structural protein of environmental bacteriophages. However, our recent work has suggested that some TTPAs have additional functional features and thus are dual-function proteins. This study introduces a new TTPA family member, TTPAgp11, which belongs to Yersinia phage phiYeO3-12. We cloned the gene, expressed it and then purified the phage protein. The protein, including its hydrolytic activity, was characterized. Our enzymatic activity tests showed that TTPAgp11 displayed hydrolytic activity towards Red-starch, suggesting that this enzyme could be classified as part as the α - 1, 4-glucosidase family. Protein folding and aggregation tests indicated that TTPAgp11 is a single-domain protein whose aggregation can be induced by maltose or N-acetylglucosamine. The spatial structure of TTPAgp11 seemed to resemble that of the first reported dual-function TTPA, TTPAgp31, which was isolated from Klebsiella pneumoniae phage 32.

Tail tubular protein A: a dual-function tail protein of Klebsiella pneumoniae bacteriophage KP32.

Tail tubular protein A (TTPA) is a structural tail protein of Klebsiella pneumoniae bacteriophage KP32, and is responsible for adhering the bacteriophage to host cells. For the first time, we found that TTPA also exhibits lytic activity towards capsular exopolysaccharide (EPS) of the multiresistant clinical strain of Klebsiella pneumoniae, PCM2713, and thus should be regarded as a dual-function macromolecule that exhibits both structural and enzymatic actions. Here, we present our crystallographic and enzymatic studies of TTPA. TTPA was crystallized and X-ray diffraction data were collected to a resolution of 1.9 Å. In the crystal, TTPA molecules were found to adopt a tetrameric structure with α-helical domains on one side and ß-strands and loops on the other. The novel crystal structure of TTPA resembles those of the bacteriophage T7 tail protein gp11 and gp4 of bacteriophage P22, but TTPA contains an additional antiparallel ß-sheet carrying a lectin-like domain that could be responsible for EPS binding. The enzymatic activity of TTPA may reflect the presence of a peptidoglycan hydrolase domain in the α-helical region (amino acid residues 126 to 173). These novel results provide new insights into the enzymatic mechanism through which TTPA acts on polysaccharides.

Hydrolytic activity determination of Tail Tubular Protein A of Klebsiella pneumoniae bacteriophages towards saccharide substrates.

In this paper, the enzymatic activity, substrate specificity and antibiofilm feature of bacteriophage dual-function tail proteins are presented. So far, tail tubular proteins A-TTPAgp31 and TTPAgp44-have been considered as structural proteins of Klebsiella pneumoniae bacteriophages KP32 and KP34, respectively. Our results show that TTPAgp31 is able to hydrolyze maltose as well as Red-starch. The activity of 1 µM of the protein was calculated as 47.6 milli-Units/assay relating to the α-amylase activity. It degrades capsular polysaccharides (cPS), slime polysaccharides (sPS) and lipopolysaccharide (LPS) of K. pneumoniae PCM 2713 and shows antibiofilm reactivity towards S. aureus PCM 519 and E. faecalis PCM 2673. TTPAgp44 hydrolyses trehalose and cPS of E. faecium PCM 1859. TTPAgp44's activity was also observed in the antibiofilm test against P. aeruginosa PCM 2710 and B. subtilis PCM 2021. TTPAgp31 has been identified as α-1,4-glucosidase whereas, TTPAgp44 exhibits trehalase-like activity. Both proteins contain aspartate and glutamate residues in the ß-stranded region which are essential for catalytic activity of glycoside hydrolases. The significant novelty of our results is that for the first time the bacteriophage tubular proteins are described as the unique enzymes displaying no similarity to any known phage hydrolases. They can be used as antibacterial agents directed against bacterial strains producing exopolysaccharides and forming a biofilm.