Further studies on 2-arylacetamide pyridazin-3(2H)-ones: design, synthesis and evaluation of 4,6-disubstituted analogs as formyl peptide receptors (FPRs) agonists.

AbstractFormyl peptide receptors (FPRs) play an essential role in the regulation of endogenous inflammation and immunity. In the present studies, a large series of pyridazin-3(2H)-one derivatives bearing an arylacetamide chain at position 2 was synthesized and tested for FPR agonist activity. The pyridazin-3(2H)-one ring was confirmed to be an appropriate scaffold to support FPR agonist activity, and its modification at the 4 and 6 positions led to the identification of additional active agonists, which induced intracellular Ca2+ flux in HL-60 cells transfected with either FPR1, FPR2, or FPR3. Seven formyl peptide receptor 1 (FPR1)-specific and several mixed FPR1/FPR2 dual agonists were identified with low micromolar EC50 values. Furthermore, these agonists also activated human neutrophils, inducing intracellular Ca2+ flux and chemotaxis. Finally, molecular docking studies indicated that the most potent pyridazin-3(2H)-ones overlapped in their best docking poses with fMLF and WKYMVM peptides in the FPR1 and FPR2 ligand binding sites, respectively. Thus, pyridazinone-based compounds represent potential lead compounds for further development of selective and/or potent FPR agonists

Synthesis, enantioresolution and activity profile of chiral 6-methyl-2,4-disubstituted pyridazin-3(2H)-ones as potent N-formyl peptide receptor agonists.

A series of chiral pyridazin-3(2H)-ones was synthesized, separated as pure enantiomers, and evaluated for N-formyl peptide receptor (FPR) agonist activity. Characterization of the purified enantiomers using combined chiral HPLC and chiroptical studies (circular dichroism, allowed unambiguous assignment of the absolute configuration for each pair of enantiomers). Evaluation of the ability of racemic mixtures and purified enantiomers to stimulate intracellular Ca(2+) flux in FPR-transfected HL-60 cells and human neutrophils and to induce β-arrestin recruitment in FPR-transfected CHO-K1 cells showed that many enantiomers were potent agonists, inducing responses in the sub-micromolar to nanomolar range. Furthermore, FPRs exhibited enantiomer selectivity, generally preferring the R-(−)-forms over the S-(+)-enantiomers. Finally, we found that elongation of the carbon chain in the chiral center of the active compounds generally increased biological activity. Thus, these studies provide important new information regarding molecular features involved in FPR ligand preference and report the identification of a novel series of FPR agonists

Synthesis, HPLC enantioresolution and X-ray analysis of a new series of C5-methyl pyridazines as N-formyl peptide receptor (FPR) agonists

The synthesis of three racemates and the corresponding non chiral analogues of a C5-methyl pyridazine series is described here, as well as the isolation of pure enantiomers and their absolute configuration assignment. In order to obtain optically active compounds, direct chromatographic methods of separation by HPLC-UV were investigated using four chiral stationary phases (CSPs: Lux Amylose-2(®), Lux Cellulose-1(®), Lux Cellulose-2(®) and Lux Cellulose-3(®)). The best resolution was achieved using amylose tris(5-chloro-2-methylphenylcarbamate) (Lux Amylose-2(®)), and single enantiomers were isolated on a semipreparative scale with high enantiomeric excess, suitable for biological assays. The absolute configuration of optically active compounds was unequivocally established by X-ray crystallographic analysis and comparative chiral HPLC-UV profile. All compounds of the series were tested for formyl peptide receptor (FPR) agonist activity, and four were found to be active, with EC(50) values in the micromolar range

Synthesis and pharmacological evaluation of indole derivatives as deaza analogues of potent human neutrophil elastase inhibitors

A number of N-benzoylindoles were designed and synthesized as deaza analogues of our previously reported potent and selective HNE inhibitors with an indazole scaffold. The new compounds containing substituents and functions that were most active in the previous series were active in the micromolar range (the most potent had IC(50)=3.8 µM) or inactive. These results demonstrated the importance of N-2 in the indazole nucleus. Docking studies performed on several compounds containing the same substituents but with an indole or an indazole scaffold, respectively, highlight interesting aspects concerning the molecule orientation and H-bonding interactions, which could help to explain the lower activity of this new series

Экспериментальная модель сахарного диабета 2-го типа у крыс, вызванная диетой с высоким содержанием жиров и стрептозотоцином в низкой дозе

Aim. To develop a pathogenetically reasonable model of type 2 diabetes with marked peripheral insulin resistance and relative insulin deficiency in rats using a high-fat diet and a single injection of streptozotocin in the low dose.Materials and methods. Experiments were conducted on 16 outbred male rats. Type 2 diabetes model in experimental animals was achieved by feeding them with high-fat diet (55% of energy from fat) for 28 days followed by a single injection of streptozotocin (35 mg/kg). The serum glucose and insulin concentrations in rats were measured before streptozotocin administration and at the end of the experiment. To estimate insulin resistance, insulin tolerance test and glucose tolerance test were performed. Total protein, albumin, total and direct bilirubin, urea, uric acid, total cholesterol, high-density lipoproteins and low-density lipoproteins, and activity of alanine aminotransferase and aspartate aminotransferase were measured in the blood serum.Results. A high-fat diet with a single injection of streptozotocin resulted in lipid and protein metabolism disorders and peripheral tissues insulin resistance in experimental animals. Basal insulin levels did not change against the backdrop of high glucose level.Conclusions. These results indicate that feeding rats with a high-fat diet (55% of calories from fats) and a single administration of streptozotocin at a low dose (35 mg/kg) reproduce general pathological processes of type 2 diabetes. This model can be used to study the pathogenesis of type 2 diabetes as well as to investigate the effect of potential hypoglycemic agents.Цель исследования – разработать с помощью диеты с высоким содержанием жиров и однократной инъекции стрептозотоцина в низкой дозе патогенетически обоснованную модель сахарного диабета 2-го типа у крыс с выраженной периферической инсулинорезистентностью и относительным дефицитом инсулина.Материалы и методы. Эксперименты проводили на 16 аутбредных самцах крыс. Сахарный диабет 2-го типа моделировали кормлением экспериментальных животных высокожировой диетой (55% калорий за счет жиров) в течение 28 сут с последующей однократной интраперитонеальной инъекцией стрептозотоцина в дозе 35 мг/кг. Концентрацию глюкозы и инсулина в сыворотке крови крыс измеряли до введения стрептозотоцина и по окончании эксперимента. Для оценки инсулинорезистентности проводили глюкозотолерантный и инсулинотолерантный тесты. В сыворотке крови определяли содержание общего белка, альбуминов, общего и прямого билирубина, мочевины, мочевой кислоты, общего холестерина, холестерина липопротеинов высокой плотности и низкой плотности, активности аланинаминотрансферазы и аспартатаминотрансферазы.Результаты. Диета с высоким содержанием жиров в сочетании с однократной инъекцией стрептозотоцина приводила у экспериментальных животных к нарушению липидного и белкового обменов и развитию инсулинорезистентности. Уровень базального инсулина не изменялся на фоне выраженной гликемии.Заключение. Полученные результаты свидетельствуют о том, что при кормлении крыс диетой с высоким содержанием жиров и однократном введении стрептозотоцина в низкой дозе (35 мг/кг) воспроизводятся патологические процессы, характерные для сахарного диабета 2-го типа. Созданная модель может использоваться для изучения патогенеза сахарного диабета 2-го типа, а также для исследования действия потенциальных гипогликемических средств