Chloro-substituted pyridine squaramates as new DNase I inhibitors: Synthesis, structural characterization, in vitro evaluation and molecular docking studies.

Having continued our recent study on the synthesis and DNase I inhibition of several monosquaramides, two new chloro-substituted pyridine squaramates were synthesized and their structure was identified by X-ray. Their inhibitory properties towards deoxyribonuclease I (DNase I) and xanthine oxidase (XO) were evaluated in vitro. 3-(((6-Chloropyridin-3-yl)methyl)amino)-4-ethoxycyclobut-3-ene-1,2-dione (compound 3a) inhibited DNase I with an IC50 value of 43.82 ± 6.51 µM, thus standing out as one of the most potent small organic DNase I inhibitors tested to date. No cytotoxicity to human tumor cell lines (HL-60, MDA-MB-231 and MCF-7) was observed for the tested compounds. In order to investigate the drug-likeness of the squaramates, the ADME profile and pharmacokinetic properties were evaluated. Molecular docking was performed to reveal the binding mode of the studied compounds on DNase I.

Synthesis and DNase I Inhibitory Properties of New Squaramides.

Three new monosquaramides (3a-c) were synthesized, characterized by IR, NMR and X-ray, and evaluated for inhibitory activity against deoxyribonuclease I (DNase I) and xanthine oxidase (XO) in vitro. The target compounds inhibited DNase I with IC50 values below 100 µM, being at the same time more potent DNase I inhibitors than crystal violet, used as a positive control. 3-Ethoxy-4-((1-(pyridin-3-yl)propan-2-yl)amino)cyclobut-3-ene-1,2-dione (3c) stood out as the most potent compound, exhibiting a slightly better IC50 value (48.04 ± 7.98 µM) compared to the other two compounds. In order to analyze potential binding sites for the studied compounds with DNase I, a molecular docking study was performed. Compounds 3a-c are among the most potent small organic DNase I inhibitors tested to date.

Interaction of thrombophilic SNPs in patients with unexplained infertility-multifactor dimensionality reduction (MDR) model analysis.

PURPOSE: Our aim was to evaluate the frequency and SNP-SNP interactions between factor V Leiden (FVL) G1691A, prothrombin G20210A mutation, and C677T MTHFR and PAI-1 4G/5G gene polymorphisms in female IVF patients with unexplained infertility (UI) by using a multifactor dimensionality reduction (MDR) model analysis. METHODS: A total of 225 subjects were enrolled in the study. There were 105 females in UI group and 120 healthy controls. Designated SNPs were determined by using allele-specific PCR methods. The difference in thrombophilia prevalence was assessed by a chi-square test and logistic regression analysis. Four-locus SNP interaction model was tested using the MDR approach. A ten-fold cross-validation consistency (CVC) and permutation testing were performed. RESULTS: There was a significant difference of MTHFR C677T polymorphism frequency between the groups. Significantly less UI patients had MTHFR CC genotype (p = 0.005), while the risk allele T was more frequent (OR = 1.83, p = 0.0018). Logistic regression determined a significant association only for MTHFR C677T in our patients (TT genotype OR = 2.99). The MDR analysis confirmed the significance of a single-locus model for MTHFR C677T polymorphism (p = 0.015; OR = 2.93). However, the best, significant predictive model was the two-locus model comprising MTHFR C677T and FVL (CVC = 10/10, testing accuracy = 60.95%, p = 0.013; OR = 3.02). CONCLUSION: The MTHFR C677T polymorphism was significantly associated with UI, with minor allele T being more frequent. Additionally, there was a significantly increased presence of MTHFR C677T with FVL mutation in these patients. Therefore, MTHFR and its interaction with FVL should be recognized as contributing factors in the pathogenesis of infertility.

3'-Methyl-4-thio-1H-tetrahydropyranspiro-5'-hydantoin platinum complex as a novel potent anticancer agent and xanthine oxidase inhibitor.

In this study, a Pt(IV) complex with 3'-methyl-4-thio-1H-tetrahydropyranspiro-5'-hydantoin (complex 1) was synthesized. The structure was determined via elemental analyses, infrared, 1 H, and 13 C nuclear magnetic resonance techniques. Density functional theory calculations were applied to optimize the molecular geometry and to calculate structural parameters and vibrational frequencies. The cytotoxicity of the newly synthesized complex 1 was assessed against K-562 and REH cells and compared with the cytotoxic effects of the ligand (L) and its Pd(IV) complex (complex 2). Complex 1 exhibited a better cytotoxic activity (IC50 = 76.9 µM against K-562 and 15.6 µM against REH cells) than L and complex 2, which was closer to the cytotoxic effect of cisplatin (IC50 = 36.9 µM and 1.07 µM against K-562 and REH cells, respectively), as compared with the ligand and complex 2. L and its complexes 1 and 2 were evaluated for inhibitory activity against xanthine oxidase (XO) in vitro, as compared with allopurinol (IC50 = 1.70 µM). Complex 1 was shown as a potent XO inhibitor, with an IC50 value of 19.33 µM, and the binding mode with the enzyme was predicted by molecular docking. Its inhibitory activity against XO is a potential advantage that might result in improved profile and anticancer activity.

Benzimidazole-based dual dipeptidyl peptidase-4 and xanthine oxidase inhibitors.

Multiple-targeting compounds might reduce complex polypharmacy of multifactorial diseases, such as diabetes, and contribute to the greater therapeutic success. Targeting reactive oxygen species-producing enzymes, as xanthine oxidase (XO), might suppress progression of diabetes-associated vascular complications. In this study a small series of benzimidazole derivatives (1-9) was evaluated for inhibitory activity against dipeptidyl peptidase-4 (DPP-4) and XO. One 1,3-disubstituted-benzimidazole-2-imine (5) and 1,3-thiazolo[3,2-a]benzimidazolone derivative (8) were shown as effective dual DPP-4 and XO inhibitors, with IC50 values lower than 200 µM, and predicted binding modes with both target enzymes. Both selected dual inhibitors (compounds 5 and 8) did not show cytotoxicity to a greater extent on Caco-2 cells even at concentration of 250 µM. These structures represent new non-purine scaffolds bearing two therapeutic functionalities, being DPP-4 and XO inhibitors, more favorable in comparison to DPP-4 inhibitors with DPP-4 as a single target due to pleiotropic effects of XO inhibition.

Xanthine oxidase inhibitors beyond allopurinol and febuxostat; an overview and selection of potential leads based on in silico calculated physico-chemical properties, predicted pharmacokinetics and toxicity.

Xanthine oxidase (XO), a versatile metalloflavoprotein enzyme, catalyzes the oxidative hydroxylation of hypoxanthine and xanthine to uric acid in purine catabolism while simultaneously producing reactive oxygen species. Both lead to the gout-causing hyperuricemia and oxidative damage of the tissues where overactivity of XO is present. Over the past years, significant progress and efforts towards the discovery and development of new XO inhibitors have been made and we believe that not only experts in the field, but also general readership would benefit from a review that addresses this topic. Accordingly, the aim of this article was to overview and select the most potent recently reported XO inhibitors and to compare their structures, mechanisms of action, potency and effectiveness of their inhibitory activity, in silico calculated physico-chemical properties as well as predicted pharmacokinetics and toxicity. Derivatives of imidazole, 1,3-thiazole and pyrimidine proved to be more potent than febuxostat while also displaying/possessing favorable predicted physico-chemical, pharmacokinetic and toxicological properties. Although being structurally similar to febuxostat, these optimized inhibitors bear some structural freshness and could be adopted as hits for hit-to-lead development and further evaluation by in vivo studies towards novel drug candidates, and represent valuable model structures for design of novel XO inhibitors.

The importance of Myd88 L265P mutation, clinical and immunohistochemical prognostic factors for the survival of patients with diffuse large B-cell non-Hodgkin lymphoma treated by immunochemotherapy in southeast Serbia.

PURPOSE: Immunochemotherapy used in the treatment of non-Hodgkin diffuse large B-cell lymphoma (DLBCL) modifies the course of disease and has a positive effect on overall survival (OS). The purpose of this study was to verify the existence of the important Myd 88 mutation and other immunohistochemical factors on disease prognosis in patients with DLBCL in southeast Serbia. METHODS: Immunohistochemical expression of CD10, Bcl- 2, Bcl-6, Ki-67 and MUM 1 was performed using paraffin blocks of DLBCL. Molecular-genetic study of MyD88 L265P gene polymorphism was done by isolation of genomic DNA from paraffin embedded tissue by means of polymerase chain reaction (PCR). RESULTS: Immunochemotherapy (rituximab+CHOP/R-CHOP) significantly improved the overall survival (OS) of patients with DLBCL compared with patients treated with CHOP alone (p<0.0001). OS in the R-CHOP group was longest in patients with International Prognostic Index (IPI) 2 score (p=0.012) and IPI 4 score (p=0.024). Patients with Bcl-2 +, and MUM 1+ benefited from R-CHOP and their expression had no effect on OS. Analysis of restriction fragment length on the genomic DNA showed a homozygous normal TT genotype. CONCLUSION: Addition of rituximab to CHOP standard protocol improved the OS rate in patients with DLBCL and altered the character and significance of previously recognized prognostic factors. IPI score in the immunochemotherapy era could not reveal possible predictive factors of poor prognosis which would help identify a high-risk subgroup of newly diagnosed DLBCL. In the patient population from Southeast Serbia pathological signaling pathway achieved by Myd 88 L265 mutation was not responsible for the development of DLBCL.

Xanthine oxidase inhibitory properties and anti-inflammatory activity of 2-amino-5-alkylidene-thiazol-4-ones.

Thirty 2-amino-5-alkylidene-thiazol-4-ones were assayed for inhibitory activity against commercial enzyme xanthine oxidase (XO) in vitro and XO in rat liver homogenate as well as for anti-inflammatory response on human peripheral blood mononuclear cells (PBMCs). 4-((2-Benzylamino-4-oxothiazol-5(4H)-ylidene)-methyl)benzonitrile showed the most potent inhibitory effect against commercial XO (IC50 = 17.16 µg/mL) as well as against rat liver XO (IC50 = 24.50 µg/mL). All compounds containing the 4-cyanobenzylidene group or (indol-3-yl)methylene group at the position 5 of thiazol-4-one moiety were moderately potent inhibitors of commercial XO. The assayed compounds were docked into the crystal structures of XO enzyme complexes with three diverse inhibitors (PDB codes: 1FIQ, 1VDV, and 1V97) using OEDocking software. Our results strongly point to a correlation between the data on inhibitory activity against commercial XO and data on antioxidant activity of studied compounds, screened using a lipid peroxidation (LP) method. 2-(Benzylamino)-5-((thiophen-2-yl)methylene)thiazol-4(5H)-one showed the highest anti-inflammatory response on PBMCs, exerted most probably through the NF-κB inhibition. Studied 2-amino-5-alkylidene-thiazol-4-ones obey the "Rule of five" and meet all criteria for good solubility and permeability.

Two 6-(propan-2-yl)-4-methyl-morpholine-2,5-diones as new non-purine xanthine oxidase inhibitors and anti-inflammatory agents.

Two cyclodidepsipeptides, 3-(2-methylpropyl)-6-(propan-2-yl)-4-methyl-morpholine-2,5-dione (1) and 3,6-di(propan-2-yl)-4-methyl-morpholine-2,5-dione (2), were evaluated for inhibitory activity against commercial enzyme xanthine oxidase (XO) in vitro and XO in rat liver homogenate as well as for anti-inflammatory response on human peripheral blood mononuclear cells (PBMCs). Both of cyclodidepsipeptides were excellent inhibitors of XO and significantly suppressed the nuclear factor of κB (NF-κB) activation. Allopurinol, a widely used XO inhibitor and drug to treat gout, relevated stronger inhibitory effect on rat liver XO activity than those of compounds 1 and 2. Molecular docking studies were performed to gain an insight into their binding modes with XO. The studied morpholine-diones derivatives exerting XO inhibition and anti-inflammatory effect may give a promise to be used in the treatment of gout and other excessive uric acid production or inflammatory conditions.