The synthesis of phosphodiesterase 10 inhibitors in the group of indazol-pyrolidine derivatives with potential receptor activity

We wstępie przedstawiono rolę receptora w sygnalizacji komórkowej na przykładzie modelu „zamka” i „klucza”. Następnie opisano na przykładzie receptorów związanych z białkiem G syntezę wtórnych przekaźników. Przedstawiono charakterystykę wybranych wtórnych przekaźników: cyklicznego 3',5'-adenozynomonofosforanu (cyclic adenosine monophosphate, cAMP) oraz cyklicznego 3',5'-guanozyno monofosforanu (cyclic guanosine monophosphate, cGMP). Dokonano charakterystyki fosfodiesteraz (phosphodiesterases, PDEs) ze szczególnym uwzględnieniem PDE10, będącej potencjalnym celem molekularnym w terapii schorzeń ośrodkowego układu nerwowego. W dalszej części pracy przedstawiono poszukiwania inhibitorów PDE10, uwzględniając w nich takie związki jak papaweryna, Pfizer 489, PF-2545920 oraz związki o budowie hybrydowej. Na końcu uwagę poświęcono znakowaniu radioaktywnemu związku PF-2545920, co zostało wykorzystano w celu przeprowadzenia badań z wykorzystaniem pozytonowej tomografii emisyjnej (Positron emission tomography, PET).W części doświadczalnej opracowano metodę syntezy dwóch serii pochodnych indazolu. Reakcje prowadzono etapami. Pierwszy etap stanowiła synteza 1-arylo-4-(pirolidyn-3-ylo-metylo)piperazyn. W drugim etapie otrzymywano związki końcowe w reakcji N-alkilacji pomiędzy produktami z etapu pierwszego i 7-chloroindazolem. Przeprowadzono również jednoetapowe reakcje alkilacji pomiędzy 7-(piperazyn-1-yl)-1H-indazololem i 8-chlorochinazoliną, 2-chlorometylooksazolo[2,3-f]teofiliną, 2-chlorometylobenzimidazolem i 2-chlorometylochinoliną. Opracowano również metodę syntezy związków pochodnych piperazynowych izosterycznego w stosunku do indazolu układu 1,3-dihydro-2H-benzimidazol-2-onu. Do kontroli czystości opracowano odpowiednie warunki rozdziału chromatograficznego. Strukturę otrzymanych związków potwierdzono wynikami analizy spektroskopowej1H i 13C NMR oraz MS.In the introduction, the role of receptor in cell signaling was explained using the model of "lock and key". Afterwards, synthesis of secondary messengers was presented, connected with G-protein transmission. Several secondary messengers were characterized, including: cyclic 3',5'-adenosine monophosphate (cAMP) and cyclic 3',5'-guanosine monophosphate (cGMP). There was also portrayed a characteristics of enzymes - phosphodiesterases (PDE) with particular emphasis on PDE10, which may be a potential biological target in therapy of central nervous system diseases. In the following part, discovered PDE10 inhibitors were presented, such as papaverine, Pfizer 489, PF-2545920 and compounds with a hybrid structure. Then, a radiolabeling of compund PF-2545920 was described, which was used for testing using positron emission tomography (PET).In the experimental part, a method of synthesis of two series of indazole derivatives was elaborated. The reaction was carried out in stages. First stage consisted of synthesis of 1-aryl-4-(pyrrolidin-3-yl-methyl)piperazines. In the second stage, final compounds were obtained in the reaction of N-alkylation between products from first step and 7-chloroindazole. There were also made an one-step reactions of alkylation between 7-(piperazine-1-yl)-1H-indazole and 8-chloroquinazoline, 2-chloromethoxyoxazole[2,3-f]theophylline, 2-chloromethylbenzoimidazole and 2-chloromethylquinoline. There was evaluated a method of synthesis of piperazine derivatives with structure of 1,3-dihydro-2H-benzimidazole-2-one, isosteric against indazole. Obtained compounds were purificated using different conditions of chromatographic separation. Structures of final products were determined by results of 1H NMR, 13C NMR and mass spectre analysis

Molecular Dynamics Scoring of Protein–Peptide Models Derived from Coarse-Grained Docking

One of the major challenges in the computational prediction of protein–peptide complexes is the scoring of predicted models. Usually, it is very difficult to find the most accurate solutions out of the vast number of sometimes very different and potentially plausible predictions. In this work, we tested the protocol for Molecular Dynamics (MD)-based scoring of protein–peptide complex models obtained from coarse-grained (CG) docking simulations. In the first step of the scoring procedure, all models generated by CABS-dock were reconstructed starting from their original C-alpha trace representations to all-atom (AA) structures. The second step included geometry optimization of the reconstructed complexes followed by model scoring based on receptor–ligand interaction energy estimated from short MD simulations in explicit water. We used two well-known AA MD force fields, CHARMM and AMBER, and a CG MARTINI force field. Scoring results for 66 different protein–peptide complexes show that the proposed MD-based scoring approach can be used to identify protein–peptide models of high accuracy. The results also indicate that the scoring accuracy may be significantly affected by the quality of the reconstructed protein receptor structures

Effect of a Diet Supplemented with Nettle (<i>Urtica dioica</i> L.) or Fenugreek (<i>Trigonella Foenum-Graecum</i> L.) on the Content of Selected Heavy Metals in Liver and Rabbit Meat

The literature on herbal additives for rabbit feed offers little information on the use of nettle and fenugreek. Both of these herbs are valuable sources of vitamins and minerals. These herbs affect the growth, health, and meat quality of rabbits. They regulate the digestive system, stimulate the appetite, have a positive effect on the functioning of the immune system, and exhibit antibacterial activity. The purpose of the present study was to determine the effect of nettle (Urtica dioica L.) leaves or fenugreek (Trigonella foenum-graecum L.) seeds in the feed on the content of selected heavy metals in the liver and meat of the rabbit. The rabbits were divided into three groups: group C (n = 20; 10♂ and 10♀) was fed ad libitum with a complete feed, N group (n = 20; 10♂ and 10♀) was fed a complete mixture with 1% added nettle, and group F (n = 20; 10♂ and 10♀) was fed with a complete mixture with 1% added fenugreek. The experiment lasted 7 weeks (from the 35th to the 84th day of the rabbits’ lives). All the rabbits were slaughtered on the 84th day of age, with a body weight of about 2.6 kg. The concentration of heavy metals (Zn, Cu, Ni, Mn, Fe, Pb, Cd) was determined by the atomic absorption spectrometry (AAS). The additives to the feed significantly affected the content of elements in both the liver and the meat of rabbits (p p p p < 0.05). It is important to study the content of heavy metals in the used animal herbal feed additives and their interaction with each other, as they affect the distribution of elements in tissues and organs

Non-Invasive Measurement of Thyroid Hormones in Domestic Rabbits

Thyroid hormones are essential for metabolism, energy homeostasis and reproduction. Hormones can be measured in various biological source materials: blood, feces, urine, saliva and others. The aim of our study was to verify usefulness of thyroid hormone analysis in the urine and feces of the domestic rabbit (Oryctolagus cuniculus f. domesticus), comparing them with the serum analyses. Samples were collected from 27 does in the age of 12–14 weeks. Total thyroxine (tT4), total triiodothyronine (tT3), free thyroxine (fT4) and free triiodothyronine (fT3) were tested using the radioimmunological method in serum, feces and urine. The highest concentration of tT4 was found in feces (104.72 ± 59.52 nmol/mg) and the lowest in urine (3.03 ± 3.11 nmol/mL). The highest tT3 concentration was found in blood serum (3.19 ± 0.64 nmol/L) and the lowest in urine (0.31 ± 0.43 nmol/L). The highest concentration of fT4 was observed in feces (43.71 ± 4.79 pmol/mg) and the lowest in blood serum (14.97 ± 3.42 pmol/L). The statistically highest concentration of fT3 (28.56 ± 20.79 pmol/L) was found in urine, whereas the lowest concentration of this hormone was found in feces (3.27 ± 1.33 pmol/mg). There was a positive and statistically significant correlation between serum and urine fT3 (r = 0.76) and a high positive correlation between serum and feces fT3 concentration (r = 0.62). Correlations between concentrations of other thyroid hormones between serum, urine and feces were found to be insignificant. The results suggest that fT3 can be accurately and reliably measured in the feces and urine of the domestic rabbit

Novel Tetrazole-Based Antimicrobial Agents Targeting Clinical Bacteria Strains: Exploring the Inhibition of <i>Staphylococcus aureus</i> DNA Topoisomerase IV and Gyrase

Eleven novel imide-tetrazoles were synthesized. In the initial stage of research, in silico structure-based pharmacological prediction was conducted. All compounds were screened for antimicrobial activity using standard and clinical strains. Within the studied group, compounds 1–3 were recognized as leading structures with the most promising results in antimicrobial studies. Minimal inhibitory concentration values for compounds 1, 2, 3 were within the range of 0.8–3.2 μg/mL for standard and clinical Gram-positive and Gram-negative bacterial strains, showing in some cases higher activity than the reference Ciprofloxacin. Additionally, all three inhibited the growth of all clinical Staphylococci panels: Staphylococcus aureus (T5592; T5591) and Staphylococcus epidermidis (5253; 4243) with MIC values of 0.8 μg/mL. Selected compounds were examined in topoisomerase IV decatenation assay and DNA gyrase supercoiling assay, followed by suitable molecular docking studies to explore the possible binding modes. In summary, the presented transition from substrate imide-thioureas to imide-tetrazole derivatives resulted in significant increase of antimicrobial properties. The compounds 1–3 proposed here provide a promising basis for further exploration towards novel antimicrobial drug candidates

Menthol- and thymol-based ciprofloxacin derivatives against Mycobacterium tuberculosis: in vitro activity, lipophilicity, and computational studies

Abstract In this work, we investigated the antitubercular properties of Ciprofloxacin derivatives conjugated with menthol and thymol moieties. For the sixteen derivatives, we established minimal inhibitory concentrations (MIC) using isolates of Mycobacterium tuberculosis that were resistant or susceptible to other antibiotics. For the most potent compound 1‐cyclopropyl‐6‐fluoro‐7‐{4‐[6‐((1R,2S,5R)‐2‐isopropyl‐5‐methylcyclohexyloxy)‐6‐oxohexyl]piperazin‐1‐yl}‐4‐oxo‐1,4‐dihydroquinoline‐3‐carboxylic acid (6), we determined fractional inhibitory concentration index (FICI) values to confirm antibacterial susceptibility and synergistic effects with other reference drugs. In addition, chromatographic studies of all the derivatives demonstrated a significant three to four-fold increase in lipophilicity and affinity to phospholipids compared to Ciprofloxacin. Finally, we conducted structure-based studies of the investigated compounds using molecular docking and taking into account protein target mutations associated with fluoroquinolone resistance. In summary, our findings indicate that the investigated compounds possess tuberculostatic properties, with some showing similar or even better activity against resistant strains compared to reference drugs. Increased lipophilicity and affinity to phospholipids of the new derivatives can offer several advantages for new drug candidates, beyond just improved cell membrane penetration. However, further studies are needed to fully understand their safety, efficacy, and mechanism of action

Polymorphism within <i>IGFBP</i> Genes Affects the Acidity, Colour, and Shear Force of Rabbit Meat

Rabbits are important livestock animals, popular for their nutritional value. Nowadays, the molecular background of traits influencing the quality of meat and meat products is in high demand. Therefore, in the current study, we analyse the sequences of IGFBP1, IGFBP2, IGFBP4, IGFBP5, and IGFBP6 for possible polymorphisms. Based on a bioinformatics analysis in an association study on 466 animals of different breeds (New Zealand White × Flemish Giant crossbreed (9NZWxFG), Termond White (TW), Popielno White (PW), and Flemish Giant (FG)), we analyse the influence of five polymorphisms within the IGFBP genes. Statistically significant differences were found among the carcass and meat quality traits but not for all of the analysed rabbit breeds. The most promising polymorphism was g.158093018A>T within the IGFBP5 gene. The values of pH24 of m.longissimus lumborum (m.l.l.) and biceps femoris muscles (m.b.f.) were higher for the AT genotypes compared to the AA genotypes for the TW and NZWxFG crossbreeds. Also, for pH24, we found differences in ing.41594308T>C for NZWxFG, where the TT genotype values were higher than the TC values. We found differences in L*24 on m.l.l. for g.41592248A>C for NZWxFG. For m.b.f., significant differences were found in b*45 for g.3431insAC in the FG population and a*45 for g.41592248A>C and g.158093018A>T in the TW population. The shear force statistically differed for g.158093018A>T in TW rabbits and g.41592248A>C for NZWxFG. We conclude that this polymorphism may be promising for better quality rabbit meat and may be implemented in selection processes