Synthesis of derivatives of methoxydibenzo[b, f]oxepine in the presence of sodium azide

Dibenzo[b,f]oxepine is an important scaffold in medicinal chemistry and its derivatives occur in several medicinally important plants. A new approach to methoxydibenzo[b,f]oxepines (15–21) proceeding under mild reaction conditions, has been developed. Notably, the use of sodium azide in the reaction allows access to new substituted dibenzo[b,f]oxepines. In order to study their shape and conformation, the optimum structures of these compounds were calculated using the DFT B3LYP/6-311++G(2d,p) method. A docking simulation was performed to insert compound 20 into the crystal structure of tubulin at the colchicine binding site to determine the probable binding model. The information from this work can be helpful for the investigation of new tubulin polymerization inhibitors exhibiting stronger activity

Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein.

In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), alpha-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3-->Arg(+) revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB(-)) strain in comparison with the AB1157 (alkB(+)) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd(+), and no repair in BS87 mfd(-) cells. Determination of specificity of Arg(+) revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT-->TA transversions by supL suppressor formation (1meA) and GC-->AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB(-)mfd(-) bacteria but not in the alkB(+)mfd(-) counterpart

Unsymmetrically Substituted Dibenzo[b,f][1,5]-diazocine-6,12(5H,11H)dione—A Convenient Scaffold for Bioactive Molecule Design

A novel approach for the synthesis of unsymmetrically substituted dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones has been developed. This facile three-step method uses variously substituted 1H-benzo[d][1,3]oxazine-2,4-diones (isatoic anhydrides) and 2-aminobenzoic acids as a starting materials. The obtained products were further transformed into N-alkyl-, N-acetyl- and dithio analogues. Developed procedures allowed the synthesis of unsymmetrical dibenzo[b,f][1,5]diazocine-6,12(5H,11H)diones and three novel heterocyclic scaffolds: benzo[b]naphtho[2,3-f][1,5]diazocine-6,14(5H,13H)dione, pyrido[3,2-c][1,5]benzodiazocine-5,11(6H,12H)-dione and pyrazino[3,2-c][1,5]benzodiazocine-6,12(5H,11H)dione. For 11 of the compounds crystal structures were obtained. The preliminary cytotoxic effect against two cancer (HeLa, U87) and two normal lines (HEK293, EUFA30) as well as antibacterial activity were determined. The obtained dibenzo[b,f][1,5]diazocine(5H,11H)6,12-dione framework could serve as a privileged structure for the drug design and development

Selected ALKBH dioxygenases are overexpressed in salivary gland tumours.

Salivary gland tumours (SGTs) are a heterogeneous group of benign tumours of various origins and pathologies, showing a number of DNA modifications. Previously, in malignant head and neck cancer (HNSCC), we found overexpression of ALKBH proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate and Fe(II) dependent dioxygenase. Moreover, we proved the connection of some of these dioxygenases with cancer development. Here, we studied the expression of five of these ALKBH dioxygenases: 1, 3, 4, 5, and FTO in benign SGTs. Using Western blot analysis, we found overexpression of three proteins: ALKBH1, 4, and FTO in SGT as compared to the surrounding, unaffected tissue. ALKBH4 was overexpressed in 76% of patient samples, whereas ALKBH1 and FTO in 65% of the samples. These results differ from those obtained in HNSCC, where FTO overexpression has been observed in 90% of patient samples. We also investigated the relationships between ALKBHs’ expression levels in normal and SGT tissues and identified two correlated pairs: ALKBH1-ALKBH3 and ALKBH1-ALKBH5. Additionally, in tumour tissue ALKBHs: ALKBH1, ALKBH3, ALKBH4, and ALKBH5 levels were correlated with each other. Together, these findings show that the ALKBH proteins exhibit pro cancerogenic action in SGT, even though the levels ALKBHs are generally lower in benign SGT than in malignant HNSCC. We suggest that the overexpression of the ALKBHs, especially FTO, may be used as a cancer marker and for its grading

Unsymmetrically-Substituted 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione Scaffold—A Useful Tool for Bioactive Molecules Design

Unsymmetrically N-substituted and N,N’-disubstituted 5,12-dihydrodibenzo [b,f][1,4]diazocine-6,11-diones were synthesized in the new protocol. The desired modifications of the dibenzodiazocine scaffold were introduced at the stages of proper selection of building blocks as well as post-cyclization modifications with alkylation or acylation agents, expanding the structural diversity and possible applications of synthesized molecules. The extension of developed method resulted in the synthesis of novel: tricyclic 5,10-dihydrobenzo[b]thieno[3,4-f][1,4]diazocine-4,11-dione scaffold and fused pentacyclic framework possessing two benzodiazocine rings within its structure. Additionally, the unprecedented rearrangement of 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-diones to 2-(2-aminophenyl)isoindoline-1,3-diones was observed under the basic conditions in the presence of sodium hydride for secondary dilactams. The structures of nine synthesized products have been established by single-crystal X-ray diffraction analysis. Detailed crystallographic analysis of the investigated tri- and pentacyclic systems has shed more light on their structural features. One cell line derived from non-cancerous cells (EUFA30—human fibroblasts) and three tumor cells (U87—human primary glioblastoma, HeLa—cervix adenocarcinoma, BICR18—laryngeal squamous cell carcinoma) were used to determine the cytotoxic effect of the newly synthesized compounds. Although these compounds showed a relatively weak cytotoxic effect, the framework obtained for 5,12-dihydrodibenzo[b,f][1,4]diazocine-6,11-dione could serve as a convenient privilege structure for the design and development of novel bioactive molecules suitable for drug design, development and optimization programs

Evaluation of the Escherichia coli HK82 and BS87 strains as tools for AlkB studies

Within a decade the family of AlkB dioxygenases has been extensively studied as a one-protein DNA/RNArepair system in Escherichia coli but also as a group of proteins of much wider functions in eukaryotes.Two strains, HK82 and BS87, are the most commonly used E. coli strains for the alkB gene mutations. Theaim of this study was to assess the usefulness of these alkB mutants in different aspects of research onAlkB dioxygenases that function not only in alkylated DNA repair but also in other metabolic processes incells. Using of HK82 and BS87 strains, we found the following differences among these alkB−derivatives:(i) HK82 has shown more than 10-fold higher MMS-induced mutagenesis in comparison to BS87; (ii)different specificity of Arg+revertants; (iii) increased induction of SOS and Ada responses in HK82; (iv)the genome of HK82, in comparison to AB1157 and BS87, contains additional mutations: nalA, sbcC, andnuoC. We hypothesize that in HK82 these mutations, together with the non-functional AlkB protein, mayresult in much higher contents of ssDNA, thus higher in comparison to BS87 MMS-induced mutagenesis.In the light of our findings, we strongly recommend using BS87 strain in AlkB research as HK82, bearingseveral additional mutations in its genome, is not an exact derivative of the AB1157 strain, and showsadditional features that may disturb proper interpretation of obtained results

Hypoxic 3D in vitro culture models reveal distinct resistance processes to TKIs in renal cancer cells

The aim of this study is to determine the effect of hypoxia on axitinib and sorafenib-treated renal cell carcinoma (RCC) cells. Hypoxia is a crucial factor influencing transcription process via protein modulation, which was shown i.e. in pancreatic cancer. Until now, hypoxia has been defined as associated with poorer outcome and inducing chemotherapy resistance in solid tumors. The unique phenomenon of pseudo-hypoxia connected with vhl mutation was observed in clear-cell, but not in papillary RCC, and the treatment of this subtype of cancer is still challenging. Despite the introduction of new antiangiogenic targeted therapies (inter alia tyrosine kinase inhibitors, TKIs), patients still develop both primary and acquired resistance. Overcoming resistance to TKIs, also in papillary RCC, may be possible by finding significantly modified protein expression. To do this, hypoxic 3D in vitro models must be developed to mimic both molecular pathways typical for low oxygen tension and cell–cell dynamics in tumor-like spatial structures

Hypoxic 3D in vitro culture models reveal distinct resistance processes to TKIs in renal cancer cells

The aim of this study is to determine the effect of hypoxia on axitinib and sorafenib-treated renal cell carcinoma (RCC) cells. Hypoxia is a crucial factor influencing transcription process via protein modulation, which was shown i.e. in pancreatic cancer. Until now, hypoxia has been defined as associated with poorer outcome and inducing chemotherapy resistance in solid tumors. The unique phenomenon of pseudo-hypoxia connected with vhl mutation was observed in clear-cell, but not in papillary RCC, and the treatment of this subtype of cancer is still challenging. Despite the introduction of new antiangiogenic targeted therapies (inter alia tyrosine kinase inhibitors, TKIs), patients still develop both primary and acquired resistance. Overcoming resistance to TKIs, also in papillary RCC, may be possible by finding significantly modified protein expression. To do this, hypoxic 3D in vitro models must be developed to mimic both molecular pathways typical for low oxygen tension and cell–cell dynamics in tumor-like spatial structures

Structure and Function of Enterocyte in Intrauterine Growth Retarded Pig Neonates

The intestine of intrauterine growth retarded (IUGR) neonates showed different morphology compared to neonates born with normal body weight (NBW). The aim of the present study was to investigate the ultrastructure and proteomic profile of the gut epithelium in IUGR pig neonates with special attention to the digestive and absorptive function. Intestine tissue samples were investigated in 7-day-old IUGR and NBW littermate piglets using histometry, immunofluorescence, scanning electron microscopy (SEM), and mass spectrometry analysis. IUGR piglets have shown reduced mucosa and muscularis thickness and an enhanced number of foetal type enterocytes (FTE). SEM studies have shown the lack of the characteristic large-size vacuole in IUGR’s enterocytes. Delayed removal of FTE in IUGR neonates was probably due to the inhibited apoptosis in the apical part of villi and increased apoptosis and reduced mitosis in the crypt region. In the expression of proteins in the intestinal mucosa such as hexokinase I, histones, and prelamin A/C, carbamoyl phosphate was reduced in IUGR neonates. Finally, IUGR intestines showed higher expression of HSPA9 and HSPA5 as apoptosis markers. The data indicate modifications of gut mucosa in IUGRs that may result in slower gut mucosa maturation and reduced utilisation of nutrient as compared to NBW pig neonates