Anti-inflammatory and antioxidant activity of 8-methoxy-1,3-dimethyl-2,6-dioxo-purin-7-yl derivatives with terminal carboxylic, ester or amide moieties in animal models

The previous studies in a series of 8-methoxy-1,3-dimethyl-2,6-dioxo-purin-7-yl derivatives revealed their analgesic properties. We extended the study with these compounds in aim to assess their impact on inflammatory process. For this purpose we used: the zymosan-induced peritonitis and the carrageenan induced edema model. Furthermore, the antioxidant activity of the investigated compounds by the FRAP assay was determined. For the most active derivatives from evaluated series their influence on plasma TNF-α level was also tested in vivo. All investigated purine-2,6-dione derivatives 1-11 decreased neutrophils count and inhibited intensity of early vascular permeability. Furthermore, all evaluated compounds reduced the volume of edema caused by subcutaneous injection of carrageenan. Derivatives 1 (with ester moiety), 3 and 4 (with carboxylic group) showed the highest activity in the zymosan-induced peritonitis. In addition, a significant inhibition of plasma TNF-α level in rats with endotoxemia was observed following intraperitoneal administration of these compounds. In turn, compounds 6 and 8-11 containing amide moiety showed the greatest anti-inflammatory (antiedematous) effect in the carrageenan-induced paw edema model. All compounds did not show significant antioxidant properties. The present studies revealed that the presented purine-2,6-dione derivatives exhibit a significant anti-inflammatory activity and this effect may result from their ability to lower TNF-α level

Pan-Phosphodiesterase Inhibitors Attenuate TGF-β-Induced Pro-Fibrotic Phenotype in Alveolar Epithelial Type II Cells by Downregulating Smad-2 Phosphorylation

Airway remodeling is a pathological process that accompanies many chronic lung diseases. One of the important players in this process are epithelial cells, which under the influence of pro-inflammatory and pro-fibrotic factors present in the airway niche, actively participate in the remodeling process by increasing extracellular matrix secretion, acquiring migration properties, and overproducing pro-fibrotic transducers. Here, we investigated the effect of three new 8-arylalkylamino- and 8-alkoxy-1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl-N-(5-(tert-butyl)-2-hydroxyphenyl)butanamides (1, 2, and 3), representing prominent pan-phosphodiesterase (pan-PDE) inhibitors on transforming growth factor type β (TGF-β)-induced alveolar epithelial type II cells (A549 cell line) of a pro-fibrotic phenotype. Our results demonstrate for the first time the strong activity of pan-PDE inhibitors in the prevention of TGF-β-induced mesenchymal markers’ expression and A549 cells’ migration. We also showed an increased p-CREB and decreased p-Smad-2 phosphorylation in TGF-β-induced A549 cells treated with 1, 2, and 3 derivatives, thereby confirming a pan-PDE inhibitor mesenchymal phenotype reducing effect in alveolar epithelial type II cells via suppression of the canonical Smad signaling pathway. Our observations confirmed that PDE inhibitors, and especially those active against various isoforms involved in the airway remodeling, constitute an interesting group of compounds modulating the pro-fibrotic response of epithelial cells

Synthesis and characterization of two new TiO2-containing benzothiazole-based imine composites for organic device applications

The effect of the presence of titanium dioxide in two new imines, (E,E)-(butane-1,4-diyl)bis(oxybutane-4,1-diyl) bis(4- {[(benzo[d][1,3]thiazol-2-yl)methylidene]amino}benzoate) (SP1) and (E)-N-[(benzo[d][1,3]thiazol-2-yl)methylidene]-4-dodecylaniline (SP2), on the properties and stability of imine:TiO2 composites for organic device applications were examined. The investigated titanium dioxide (in anatase form, obtained via the sol–gel method) exhibited a surface area of 59.5 m2 /g according to Brunauer–Emmett–Teller theory, and its structure is a combination of both meso- and microporous. The average pore diameter calculated by the Barrett–Joyner–Halenda method was 6.2 nm and the cumulative volume of pores was 0.117 m3 /g. The imine SP1 exhibited columnar organization (Col), while SP2 revealed a hexagonal columnar crystalline phase (Colhk). The imine:TiO2 mixtures in various weight ratio (3:0, 3:1, 3:2, 3:3) showed a lower energy gap and HOMO–LUMO energy levels compared to pure TiO2 . This implies that TiO2 provides not only a larger surface area for sensitizer adsorption and good electron collection, but also causes a shift of the imine energy levels resulting from intermolecular interaction. Also the temperature of the phase transition was slightly affected with the increase of TiO2 concentration in imine-based composites. The changes observed in the Fourier transform middle-infrared absorption (FT-MIR) spectra confirmed the significant influence of TiO2 on structural properties of both investigated imines. Similar interactions of oxygen vacancies existing on the TiO2 surface with SP1 and SP2 were observed. The imine:TiO2 mixtures showed good air stability and reusability, which demonstrates its potential for organic device applications

Synthesis and characterization of two new TiO2-containing benzothiazole-based imine composites for organic device applications

The effect of the presence of titanium dioxide in two new imines, (E,E)-(butane-1,4-diyl)bis(oxybutane-4,1-diyl) bis(4- {[(benzo[d][1,3]thiazol-2-yl)methylidene]amino}benzoate) (SP1) and (E)-N-[(benzo[d][1,3]thiazol-2-yl)methylidene]-4-dodecylaniline (SP2), on the properties and stability of imine:TiO2 composites for organic device applications were examined. The investigated titanium dioxide (in anatase form, obtained via the sol–gel method) exhibited a surface area of 59.5 m2/g according to Brunauer–Emmett–Teller theory, and its structure is a combination of both meso- and microporous. The average pore diameter calculated by the Barrett–Joyner–Halenda method was 6.2 nm and the cumulative volume of pores was 0.117 m3/g. The imine SP1 exhibited columnar organization (Col), while SP2 revealed a hexagonal columnar crystalline phase (Colhk). The imine:TiO2 mixtures in various weight ratio (3:0, 3:1, 3:2, 3:3) showed a lower energy gap and HOMO–LUMO energy levels compared to pure TiO2. This implies that TiO2 provides not only a larger surface area for sensitizer adsorption and good electron collection, but also causes a shift of the imine energy levels resulting from intermolecular interaction. Also the temperature of the phase transition was slightly affected with the increase of TiO2 concentration in imine-based composites. The changes observed in the Fourier transform middle-infrared absorption (FT-MIR) spectra confirmed the significant influence of TiO2 on structural properties of both investigated imines. Similar interactions of oxygen vacancies existing on the TiO2 surface with SP1 and SP2 were observed. The imine:TiO2 mixtures showed good air stability and reusability, which demonstrates its potential for organic device applications

A novel, pan-PDE inhibitor exerts anti-fibrotic effects in human lung fibroblasts via inhibition of TGF-β\beta signaling and activation of cAMP/PKA signaling

Phosphodiesterase (PDE) inhibitors are currently a widespread and extensively studied group of anti-inflammatory and anti-fibrotic compounds which may find use in the treatment of numerous lung diseases, including asthma and chronic obstructive pulmonary disease. Several PDE inhibitors are currently in clinical development, and some of them, e.g., roflumilast, are already recommended for clinical use. Due to numerous reports indicating that elevated intracellular cAMP levels may contribute to the alleviation of inflammation and airway fibrosis, new and effective PDE inhibitors are constantly being sought. Recently, a group of 7,8-disubstituted purine-2,6-dione derivatives, representing a novel and prominent pan-PDE inhibitors has been synthesized. Some of them were reported to modulate transient receptor potential ankyrin 1 (TRPA1) ion channels as well. In this study, we investigated the effect of selected derivatives (832-a pan-PDE inhibitor, 869-a TRPA1 modulator, and 145-a pan-PDE inhibitor and a weak TRPA1 modulator) on cellular responses related to airway remodeling using MRC-5 human lung fibroblasts. Compound 145 exerted the most considerable effect in limiting fibroblast to myofibroblasts transition (FMT) as well as proliferation, migration, and contraction. The effect of this compound appeared to depend mainly on its strong PDE inhibitory properties, and not on its effects on TRPA1 modulation. The strong anti-remodeling effects of 145 required activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway leading to inhibition of transforming growth factor type β1 (TGF-β1) and Smad-dependent signaling in MRC-5 cells. These data suggest that the TGF-β pathway is a major target for PDE inhibitors leading to inhibitory effects on cell responses involved in airway remodeling. These potent, pan-PDE inhibitors from the group of 7,8-disubstituted purine-2,6-dione derivatives, thus represent promising anti-remodeling drug candidates for further research

Synthesis and in vitro evaluation of anti-inflammatory, antioxidant, and anti-fibrotic effects of new 8-aminopurine-2,6-dione-based phosphodiesterase inhibitors as promising anti-asthmatic agents

Phosphodiesterase (PDE) inhibitors are currently an extensively studied group of compounds that can bring many benefits in the treatment of various inflammatory and fibrotic diseases, including asthma. Herein, we describe a series of novel N’-phenyl- or N’-benzylbutanamide and N’-arylidenebutanehydrazide derivatives of 8-aminopurine-2,6-dione (27–43) and characterized them as prominent pan-PDE inhibitors. Most of the compounds exhibited antioxidant and anti-inflammatory activity in lipopolysaccharide (LPS)-induced murine macrophages RAW264.7. The most active compounds (32–35 and 38) were evaluated in human bronchial epithelial cells (HBECs) derived from asthmatics. To better map the bronchial microenvironment in asthma, HBECs after exposure to selected 8-aminopurine-2,6-dione derivatives were incubated in the presence of two proinflammatory and/or profibrotic factors: transforming growth factor type β (TGF-β) and interleukin 13 (IL-13). Compounds 32–35 and 38 significantly reduced both IL-13- and TGF-β-induced expression of proinflammatory and profibrotic mediators, respectively. Detailed analysis of their inhibition preferences for selected PDEs showed high affinity for isoenzymes important in the pathogenesis of asthma, including PDE1, PDE3, PDE4, PDE7, and PDE8. The presented data confirm that structural modifications within the 7 and 8 positions of the purine-2,6-dione core result in obtaining preferable pan-PDE inhibitors which in turn exert an excellent anti-inflammatory and anti-fibrotic effect in the bronchial epithelial cells derived from asthmatic patients. This dual-acting pan-PDE inhibitors constitute interesting and promising lead structures for further anti-asthmatic agent discovery

KM-408, a novel phenoxyalkyl derivative as a potential anticonvulsant and analgesic compound for the treatment of neuropathic pain

BACKGROUND: Epilepsy frequently coexists with neuropathic pain. Our approach is based on the search for active compounds with multitarget profiles beneficial in terms of potential side effects and on the implementation of screening for potential multidirectional central activity. METHODS: Compounds were synthesized by means of chemical synthesis. After antiseizure and neurotoxicity screening in vivo, KM-408 and its enantiomers were chosen for analgesic activity evaluations. Further safety studies included acute toxicity in mice, the effect on normal electrocardiogram and on blood pressure in rats, whole body plethysmography in rats, and in vitro and biochemical assays. Pharmacokinetics has been studied in rats after iv and po administration. Metabolism has been studied in vivo in rat serum and urine. Radioligand binding studies were performed as part of the mechanism of action investigation. RESULTS: Selected results for KM-408: K(i) sigma = 7.2*10(–8); K(i) 5-HT(1A) = 8.0*10(–7); ED(50) MES (mice, ip) = 13.3 mg/kg; formalin test (I phase, mice, ip)—active at 30 mg/kg; SNL (rats, ip)—active at 6 mg/kg; STZ-induced pain (mice, ip)—active at 1 mg/kg (von Frey) and 10 mg/kg (hot plate); hot plate test (mice, ip)—active at 30 mg/kg; ED(50) capsaicin test (mice, ip) = 18.99 mg/kg; tail immersion test (mice)—active at 0.5%; corneal anesthesia (guinea pigs)—active at 0.125%; infiltration anesthesia (guinea pigs)—active at 0.125%. CONCLUSIONS: Within the presented study a novel compound, R,S-2-((2-(2-chloro-6-methylphenoxy)ethyl)amino)butan-1-ol hydrochloride (KM-408) with dual antiseizure and analgesic activity has been developed for potential use in neuropathic pain treatment. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s43440-022-00431-7

Fatty acids profile in compound lipids of eucaryotic cells’ membranes

The aim of the present study was to determine the fatty acid contents in the membrane phospholipids and cholesterol esters. The fatty acid contents were measured in membranes of CD34+ cells, CHO cells and fibroblasts. Membrane lipids were extracted according to Folch et al. They were subsequently fractionated by thin layer chromatography on silica gel. Next, dissolved in methanol residues, they were submitted to methylation according to Morrison and Smith. Fatty acids methylesters were analyzed by capillary gas chromatography with flame ionization detector. According to many studies, diet and metabolic diseases influence fatty acid content in membranes of lymphocytes and erythrocytes. It was proven that a diet rich in ω-3 polyunsaturated fatty acids decreases oxidative stress markers levels, activation of lymphocytes and also modifies plasticity of erythrocytes, and cell membrane permeability. The present study demonstrates that CD34+ cell showed the highest level of ω-3 polyunsaturated fatty acids both in phospholipid and cholesterol ester fractions. Arachidonic acid was the main fatty acid constituent in phospholipid fractions of all the analyzed cells. The disadvantageous values of ω-6/ω-3 index were observed in CHO cells and fibroblasts. In order to determine optimal fatty acid content in culture media for different cell types further studies need to be conducted.Celem niniejszej pracy było określenie profilu kwasów tłuszczowych we frakcjach fosfolipidów i estrów cholesterolu w błonach komórek CD34+, CHO oraz fibroblastów. Służyła temu analiza estrów metylowych kwasów tłuszczowych metodą GC.Do badań użyto komórek CD34+ (pozyskanych z krwi pępowinowej i hodowanych in vitro), fibroblastów oraz komórek linii CHO. Ekstrakcję lipidów przeprowadzono według zmodyfikowanej metody Folcha. Estry metylowe kwasów tłuszczowych analizowano za pomocą kapilarnej chromatografii gazowej z detektorem płomieniowo-jonizacyjnym.Dane zgromadzone na podstawie licznych publikacji ukazują wpływ diety i chorób metabolicznych na zawartość kwasów tłuszczowych w błonach erytrocytów i limfocytów. Przeprowadzono również wiele badań in vitro, których celem była ocena profilu kwasów tłuszczowych w błonach komórkowych i jego wpływu na funkcje i metabolizm komórki. Wykazano, iż wzrost zawartości wielonienasyconych kwasów tłuszczowych serii ω-3 w błonach komórkowych wpływa na redukcję markerów stresu oksydacyjnego, aktywację limfocytów, plastyczność błon erytrocytarnych oraz przepuszczalność błony komórkowej dla jonów.W niniejszej pracy magisterskiej wykazano, że największą zawartością kwasów tłuszczowych serii ω-3, zarówno we frakcji fosfolipidowej, jak i we frakcji estrów cholesterolowych, cechowały się komórki CD34+. Ponadto stwierdzono, iż komórki te mają najkorzystniejszy indeks ω-6/ω-3 w obu frakcjach lipidów. Niekorzystne wartości indeksów ω-6/ω-3 zaobserwowano w lipidach złożonych błon komórek CHO i fibroblastów. Wydaje się zatem celowe prowadzenie dalszych prac badawczych dotyczących optymalnego profilu kwasów tłuszczowych w mediach hodowlanych, dla różnych typów komórek eukariotycznych cechujących się odmiennym metabolizmem