HKSA secara In-Silico Senyawa 1-Benzil-3- Benzoilurea dan Analognya sebagai Penghambat Reseptor Bruton Tyrosine Kinase (BTK)

Abstract—In this study, a new anticancer drug design for non-Hodgkin’s lymphoma was carried out, with a molecular docking approach from the compound 1-benzyl-3-benzoylurea parent and its analog as an anticancer compound. The purpose of the study was to obtain the best quantitative structure-activity relationship (QSAR). The in-silico activity test was carried out on the new 1-benzyl-3-benzoilurea and its analog compound against the Bruton Tyrosine Kinase receptor (BTK) PDB code (5FBN) by using the Molegro Virtual Docker 5.5 program and producing a RS (Rerank Score) value for the test compound and Acalabrutinib was used as a comparison. This study also conducted bioavailability by predicting the value of F (intestinal human absorption) in the pkCSM program and toxicity studies by predicting LD50 values using the Protox II program. Correlation and regression were performed using the RS, F, and LD50 values that we obtained on the physicochemical properties of the test compound using the IBM SPSS version 24 program. The best equation is obtained as follows: (1) F = 0.851 Es Taft - 6.116 σ - 1.969 π² + 3.620 π + 90.809; (2) RS = 4.376 Es Taft - 88.802; (3) LD50 = 672.518 CMR - 669.385 ClogP - 813.806. From the results of the best equation is obtained that the activity is influenced by the parameters of steric physicochemical properties (Es Taft). Keywords: 1-benzyl-3-benzoylurea, code pdb:5fbn, in-silico, non-hodgkin lymphoma Abstrak—Pada penelitian ini dilakukan rancangan obat baru antikanker Limfoma non-Hodgkin, dengan pendekatan penambatan molekul dari senyawa induk 1-benzil-3-benzoilurea dan analognya sebagai senyawa antikanker.Tujuan penelitian ini untuk mendapatkan persamaan hubungan struktur aktivitas (HKSA) terbaik. Uji aktivitas in-silico dilakukan terhadap senyawa baru 1-benzil-3-benzoilurea dan analognya terhadap reseptor Bruton Tyrosine Kinase (BTK) kode PDB 5FBN dengan menggunakkan program Molegro Virtual Docker 5.5 dan menghasilkan nilai RS (Rerank Score) untuk senyawa uji dan Acalabrutinib digunakan sebagai pembanding. Penelitian ini juga dilakukan studi bioavaibilitas dengan memprediksi nilai F (intestinal human absorbtion) pada program pkCSM dan studi toksisitas dengan memprediksi nilai LD50 menggunakan program Protox II. Korelasi dan regresi dilakukan menggunakan nilai RS, F dan LD50 yang telah diperoleh terhadap parameter sifat fisikokimia senyawa uji menggunakan program IBM SPSS versi 24. Persamaan terbaik yang diperoleh sebagai berikut: (1) F = - 1.969 π² + 0.851 Es Taft - 6.116 σ + 3.620 π + 90.809 (2) RS = 4.376 Es Taft - 88.802 (3) LD50 = 672.518 CMR - 669.385 ClogP - 813.806. Dari hasil persamaan terbaik tersebut diperoleh bahwa aktivitas dipengaruhi oleh parameter sifat fisikokimia sterik (Es Taft). Kata kunci: 1-benzil-3-benzoilurea, in-silico, kode pdb: 5fbn, limfoma non-hodgkin

Antimicrobial and Antibiofilm Activities of 4,5-Dihydro-1H-pyrazole-1-carboximidamide Hydrochloride against Salmonella spp.

In the present study, the antimicrobial and antibiofilm activities of two 4,5-dihydro-1H-pyrazole-1-carboximidamide hydrochloride, (trifluoromethyl) phenyl-substituted (compound 1) and bromophenyl-substituted (compound 2), were evaluated against four Salmonella spp. serotypes through broth microdilution and biofilm-forming activity. Further, the cytotoxicity of the compounds was evaluated by cell viability assays using cultures of HeLa and Vero cell lines, and the mutagenic potential was assessed by the Ames test. In the broth microdilution test, compound 1 inhibited 90% of the strains tested at the minimum inhibitory concentration of 62.5 μg mL−1. Furthermore, both compounds prevented biofilm formation, with a reduction of up to 5.2 log10. HeLa and Vero cells exhibited 100% viability in the presence of compound 1. In contrast, low cell viability was observed in the presence of 15 µg mL−1 of compound 2. Furthermore, no mutagenic potential was detected at any of the tested concentrations of compound 1.

An effective QWBA/UHPLC-MS/tissue punch approach: solving a pharmacokinetic issue via quantitative Met-ID

Background: Methods to provide absolute quantitation of the administered drug and corresponding metabolites in tissue in a spatially resolved manner is a challenging but much needed necessity in pharmaceutical research. Quantitative whole-body autoradiography (QWBA) after a single-dose intravenous (3 mg/kg) and extravascular (30 mg/kg) administrations of an in vitro metabolically stable test compound (structure not reported here) indicated quick tissue distribution and excretion. Objective: Good bioavailability and short in vivo half-lives were determined formerly for the same test compound. For closing gaps in the understanding of pharmacokinetic data and in vitro results, radioactive hot spots on whole-body tissue sections had been profiled. Method: Punches from selected tissue regions containing high radioactivity in the tissue sections previously analyzed by QWBA were extracted by a highly organic solvent and analyzed without any consecutive sample preparation step, applying ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) and off-line radioanalysis to maximize signal levels for metabolite identification and profiling. Results: The analysis revealed that the test compound was metabolized intensively by phase I reactions in vivo and the metabolites formed were excreted in bile and in urine. The predominant metabolites showed abundant signal intensities both by MS and by radioanalysis but the MS signal intensities generally underestimated the real abundances of metabolites relative to the unchanged drug. Conclusions: This work illustrates that maximizing the sensitivity of tissue punch radioanalysis and the combination with UHPLC-MS leads to a better insight of pharmacokinetic processes by providing quantitative data with high molecular selectivity.

Physcion Induces Potential Anticancer Effects in Cervical Cancer Cells

Background: The extent of morphological and ultrastructural changes in HeLa cells was assessed by optical, fluorescence and electron microscopy after exposure to various concentrations of physcion, taking into account the biological properties of the test compound. Methods: Cell viability was assessed by MTT assay, while the cell cycle, LC3 expression, apoptosis, change of mitochondrial potential, Bcl-2 protein expression level and the level of reactive oxygen species were analyzed by flow cytometry. Results: As a result of physcion encumbrance, concentration-dependent inhibition of HeLa cell viability and the G0/G1 phase of the cell cycle was observed. Activation of the lysosomal system was also revealed, which was expressed by an increased number of lysosomes, autophage vacuoles and increased expression of the LC3 protein, a marker of the autophagy process. Transmission electron microscopy and fluorescence microscopy showed that physcion induced clear changes in cervical cancer cells, especially in the structure of the nucleus and mitochondria, which correlated with the production of reactive oxygen species by the test compound and indicated the induction of the oxidative process. At the same time, the pro-apoptotic effect of physcion was demonstrated, and this mechanism was dependent on the activation of caspases 3/7 and the reduction in Bcl-2 protein expression. Conclusion: The obtained results indicate an antitumor mechanism of action of physcion, based on the induction of oxidative stress, autophagy and apoptosis.

CHARACTERISATION AND STUDY OF 1- [2- (2-BENZOYLPHENOXY) ETHYL] -6-METHYLURACIL MECHANISM OF ACTION

The aim of the study is to identify 1-[2-(2-benzoylphenoxy) ethyl]-6-methyluracil using various methods of analysis, as well as to study its action mechanism against wild-type and mutant forms of HIV-1 reverse transcriptase (RT).Materials and methods. To characterize the structure of the test substance, a few kinds of analysis (X-ray diffraction, elemental, thermal) as well as a few kinds of spectroscopy (UV, IR, and NMR) have been used. The study of the action mechanism of the compound as a potential drug was carried out by evaluating the inhibitory activity against HIV-1 RT wild-type and its mutant forms corresponding to drug-resistant viral strains.Results. The studies have been carried out to confirm the structure of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil. The UV spectrum has a pronounced absorption maximum when measuring a solution of the substance in tetrahydrofuran at the concentration of 0.10 mg / ml. In the IR spectrum, there are specific bands in the range of 4000-370 cm–1. These factors make it possible to use UV and IR spectra to identify the test compound in the substance. It has also been established that the number and mutual arrangement of functional groups, the integrated intensity of signals in the 1H-NMR spectrum, as well as the structure of the carbon skeleton, correspond to the structure of 1-[2-(2-benzoylphenoxy) ethyl]-6-methyluracil. The results of studying the action mechanism showed that the test compound is an effective inhibitor of wild-type HIV-1 RT with an inhibition constant of 0.2 µM, as well as an enzyme inhibitor (mutation G190A) with an inhibition constant of 8 µM; enzyme (mutation Y181C) with an inhibition constant of 10 µM, as well as a reverse transcriptase (RT) inhibitor (mutation L100I, K103N, V106A) and a double mutant K103N / Y181C with an inhibition constant of more than 20 µM.Conclusion. As a result of the performed X-ray structural, elemental, 1H-NMR and 13C-NMR analyzes, the structure of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil has been confirmed. The possibility of using UV, IR and NMR spectroscopy, as well as thermal analyzes to confirm the authenticity during the verification of 1-[2-(2-benzoylphenoxy)ethyl]-6-methyluracil, has been shown. The developed methods can be used in the quality control and included in the draft of practice guidelines for the investigated substance. The studies of the action mechanism of the compound of HIV-1 RT reverse transcriptase have shown that this compound belongs to the group of non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1.

Evaluation of Analgesic and Anti-inflammatory Effects of 5-amino-3-phenyl-1,3,5-thiadiazine-2-thione in Mice

Purpose: The purpose of the study is to evaluate the analgesic and anti-inflammatory activity of new synthetic compound: 5-amino-3-phenyl-1,3,5-thiadiazine-2-thione and to compare its effects with the available standard drug, diclofenac sodium in mice. Methods: This test compound was evaluated for its anti-nociceptive activity using hot plate and writhing models, while for antiinflammatory activity evaluation, carrageenan and histamine-induced paw edema models were used. Results: In hotplate test, the latency time (in seconds), at 90 minutes of the administered doses (60 and 120 mg/kg) of 5-amino-3- phenyl -1.3.5-thiadiazine-2-thione were 19.5 seconds (p < 0.05) (Mean latency time) and 21.5 seconds (p < 0.01) (Mean latency time) respectively. In writhing test maximum inhibitory effect was demonstrated at a dose of 120 mg/kg with an average of 1.4 (p < 0.001) wriths (this inhibition was more significant) followed by 60 mg/kg (Mean number of wriths = 1.6) (p < 0.001) . The anti-inflammatory effect of the compound for 60 and 120 mg/kg doses was found to be significant and was more significant for 120 mg/kg in both histamine and carrageenan-induced paw edema models. Conclusion: It has been concluded that 5-amino-3-phenyl-1,3,5-thiadiazine-2-thione represents, dose-dependent antinociceptive and anti-inflammatory properties, which are statistically significant. The test compound showed a significant in-vivo peripheral analgesic effect in the writhing test, in comparison to the diclofenac sodium.

A study of acute toxicity of newly synthesized compound on adult hydrobiont Danio rerio

From ancient times the people used different poisons as a treatment for diseases of different genesis. To date, almost nothing has changed. One of the main conditions for the development of a new drug is its low toxicity. Literature analysis has shown that 1,2,4-triazole-3-thione derivatives are low-toxic compounds and exhibit a wide range of pharmacological activities. The aim of this work was the investigate acute toxicity LC50 in vivo of sodium 2-((4-amino-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-3-yl)thio)acetate on the Zebrafish (Danio rerio) aquatic model according to OECD instruction № 203. Materials and methods. Determination of acute toxicity LC50 of the test compound was performed in vivo on a model of the aquatic organism Zebrafish (Danio rerio) according to the instruction OECD № 203 (Fish, Acute Toxicity Test) for testing chemical compounds (acute toxicity test on fish from 10.12.2009). It was used fish 2 months of age, 11.8 ± 0.1 mm long, and weighing 2.6 ± 0.2 g in the experiment. The concentration of the test compound ranged from 5.0 mg/l to 100.0 mg/l. Test water-soluble compounds were dissolved in distilled water. Each mini-aquarium with a certain dose of the compound contained at least 7 individuals of Danio rerio. During the experiments, the fish were kept on a diet for a test 96 hours and their mortality was checked every 24, 48, 72 and 96 hours. Statistical analysis of the results was performed using the program Statistica 6. Results. According to the obtained data, graphs of the dependence of the concentration on the fish mortality percentage were constructed. Then the corresponding values of LC50 sodium 2-((4-amino-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-3-yl)thio)acetate were calculated. According to the acute toxicity of LC50 (96 hours) of sodium 2-((4-amino-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-3-yl)thio)acetate, which according to the classification of D. R. Passino and co-authors allowed it to be classified as a moderately toxic compound. Conclusions. The acute toxicity LC50 in vivo of sodium 2-((4-amino-5-(thiophen-2-ylmethyl)-4H-1,2,4-triazole-3-yl)thio)acetate on the Zebrafish (Danio rerio) aquatic model was investigated. The research was conducted in accordance with the national “General Ethical Principles of Animal Experimentation” approved by the First National Congress on Bioethics and the “Bioethical Expertise of Preclinical and Other Animal Research”. The highest fish mortality occurs on the last day of observation (96 hours). The highest number of Zebrafish deaths is at a minimum concentration of the substance. The acute toxicity LC50 (96 hours) of the test substance was 4.5364 mg/l.

Stimulus Control of Odorant Concentration: Pilot Study of Generalization and Discrimination of Odor Concentration in Canines

Despite dogs’ widespread use as detection systems, little is known about how dogs generalize to variations of an odorant’s concentration. Further, it is unclear whether dogs can be trained to discriminate between similar concentration variations of an odorant. Four dogs were trained to an odorant (0.01 air dilution of isoamyl acetate) in an air-dilution olfactometer, and we assessed spontaneous generalization to a range of concentrations lower than the training stimulus (Generalization Test 1). Dogs generalized to odors within a 10-fold range of the training odorant. Next, we conducted discrimination training to suppress responses to concentrations lower than a concentration dogs showed initial responding towards in Generalization Test 1 (0.0025 air dilution). Dogs successfully discriminated between 0.0025 and 0.01, exceeding 90% accuracy. However, when a second generalization test was conducted (Generalization Test 2), responding at the 0.0025 concentration immediately recovered and was no different than in Generalization Test 1. Dogs were then tested in another generalization test (Compound Discrimination and Generalization) in which generalization probes were embedded within discrimination trials, and dogs showed suppression of responding to the 0.0025 concentration and lower concentrations in this preparation. These data suggest dogs show limited spontaneous generalization across odor concentration and that dogs can be trained to discriminate between similar concentrations of the same odorant. Stimulus control, however, may depend on the negative stimulus, suggesting olfactory concentration generalization may depend on relative stimulus control. These results highlight the importance of considering odor concentration as a dimension for generalization in canine olfactory research.

ANTIRADIKALSKI KAPACITET (E)-N’-1-(2,4-DIOKSO-2H-HROMEN-3(4H)-ILIDENE)ETIL)-4-HIDROKSI-3-METOKSIBENZOHIDRAZIDA

Coumarins are a widespread group of compounds. They have a wide range of physiological effects, from antifungal, through anticoagulant to anticancer, and therefore they have found wide application in the treatment of various diseases. In this paper, the ability of 4-hydroxy-3-methoxy-N'-((E)-1-(2,4-dioxo-2H-chromen-3(4H)-ylidene)ethyl)benzohydrazide to inactivate ROS was investigated. The analysis of the obtained results showed that the SET-PT (Single Electron Transfer Followed By Proton Transfer) mechanism is non-operative. On the other hand, the thermodynamic parameters describing the HAT *Hydrogen Atom Transfer) and the first step of the SPLET (Sequential Proton Loss-Electron Transfer) mechanism indicate that both mechanisms will be operative. The antiradical capacity of the test compound with the radicals decreases in the following order: •OH> •OCH3> •OC(CH3)3> •OOH> •OOCH3> •OOCH2CH3.