Anti-melanogenesis and anti-tyrosinase properties of aryl-substituted acetamides of phenoxy methyl triazole conjugated with thiosemicarbazide: Design, synthesis and biological evaluations.

A series of aryl phenoxy methyl triazole conjugated with thiosemicarbazides were designed, synthesized, and evaluated for their tyrosinase inhibitory activities in the presence of l-dopa and l-tyrosine as substrates. All the compounds showed tyrosinase inhibition in the sub-micromolar concentration. Among the derivatives, compound 9j bearing benzyl displayed exceptionally high potency against tyrosinase with IC50 value of 0.11 µM and 0.17 µM in the presence of l-tyrosine and l-dopa as substrates which is significantly lower than that of kojic acid as the positive control with an IC50 value of 9.28 µM for l-tyrosine and 9.30 µM for l-dopa. According to Lineweaver-Burk plot, 9j demonstrated an uncompetitive type of inhibition in the kinetic assay. Also, in vitro antioxidant activities determined by DPPH assay recorded an IC50 value of 68.43 µM for 9i. The melanin content of 9j was determined on B16F10 melanoma human cells which demonstrated a significant reduction of the melanin content. Moreover, the binding energies corresponding to the same ligand as well as computer-aided drug-likeness and pharmacokinetic studies were also carried out. Compound 9j also possessed metal chelation potential correlated to its high anti-TYR activity.

A rapid and sensitive LC-MS/MS method for evaluation of the absolute oral bioavailability of a novel c-Met tyrosine kinase inhibitor QBH-196 in rats.

A sensitive, selective and high-throughput UPLC-MS/MS method was developed and validated for the determination of a novel c-Met tyrosine kinase inhibitor, QBH-196, in rat plasma. QBH-196 and its analog BH357 (IS) were extracted from rat plasma using a mixture of dichloromethane and N-hexane (2:3, v/v). The chromatographic separation was carried out on Phenomenex C18 column (50 × 2.1 mm, 2.6 µm particle size) with a gradient mobile phase of methanol (A) and water containing 0.05% formic acid (B) at a flow rate of 0.2 mL/min. The assay was performed by positive electrospray ionization in multiple reaction monitoring mode using transitions of m/z 622.68 → 140.41 for QBH-196 and m/z 591.19 →126.21 for the IS, respectively. Good linearity was obtained over the concentration range of 8.0-4000 ng/mL (r(2) > 0.99) for QBH-196 and the lower limit of quantification was 8.0 ng/mL in rat plasma. Validations of the method, including its sensitivity, extraction recovery, matrix effect, intra- and inter-day precision, accuracy and stability, were all within acceptable limits. The established method was successfully applied to determine absolute oral bioavailability of QBH-196 in rats for the first time. The mean oral absolute bioavailability of QBH-196 was found to be about 40.8% and the elimination half-life was 40.0 ± 13.1 h. This result suggested that QBH-196 exhibits good oral absorption in vivo, which is very important for the further development of QBH-196 as a new oral anticancer drug.

Antinociceptive effect of [Met5]enkephalin semicarbazide is not affected by dipeptidyl carboxypeptidase-I.

Dipeptidyl carboxypeptidase-I is an enzyme involved in the biological degradation of enkephalins. It has been suggested that C-terminal amidation of enkephalins enhances their resistance to dipeptidyl carboxypeptidase-I-mediated biodegradation. In this study, a novel [Met5]enkephalin amide (MEA) analogue [Met5]enkephalin (ME)-semicarbazide synthesized by another laboratory in our group was assessed for its antinociceptive effects compared with ME-ethylamide, MEA and ME, using tail flick test. To protect the administered drugs from biodegradation, rats were pretreated with peptidase inhibitors including amastatin, phosphoramidon and captopril. Then captopril (dipeptidyl carboxypeptidase-I inhibitor) was deleted from the peptidase inhibitors' combination for evaluating in vivo resistance of the synthetic drugs to dipeptidyl carboxypeptidase-I. According to the results, ME-semicarbazide and MEA were resistant enough to dipeptidyl carboxypeptidase-I to exert their strong antinociception following intrathecal administration even in the absence of captopril, whereas the antinociceptive effects produced by ME-ethylamide (10 nmol) were abolished in rats not pretreated with captopril, indicating that significant amounts of the ME-ethylamide were degraded by dipeptidyl carboxypeptidase-I. Replacement of the amide moiety of MEA with semicarbazide provides a new ME derivative, with high analgesic effects as well as more resistance to dipeptidyl carboxypeptidase-I-mediated biodegradation.

4-Arylthiosemicarbazide derivatives - Pharmacokinetics, toxicity and anti-Toxoplasma gondii activity in vivo.

The increasing resistance of Toxoplasma gondii to drugs and side effects of therapy indicate that specific treatment for these parasites is still needed. The 4-arylthiosemicarbazide derivatives seem to be a solution to this challenge because they have low cytotoxicity against host cells and high anti-T. gondii activity. The molecular mechanism for these compounds is related to the inhibition of tyrosine amino acids involved in the proliferation and parasitophorous vacuole formation. The pharmacokinetic analysis shows that 1-(4-Methylimidazol-5-oyl)-4-(4-nitrophenyl)thiosemicarbazide and 4-(3-Iodophenyl)-1-(4-methylimidazol-5-oyl)thiosemicarbazide administered intragastrically pass into the bloodstream and cross the blood-brain barrier, and the absorption of both compounds is first-order absorption. Toxicity analysis shows that our derivatives possess lower toxicity than the routinely used drugs trimethoprim, sulfadiazine and pyrimethamine, as was observed in the level of liver enzymes and creatinine. Both derivatives are highly potent antiparasitic agents against T. gondii, prolonged survival and cure parasite-infected mice. Additionally, significant reductions in cyst formation in the brain and heart were observed, but the highest decreases were noted in muscle and the level of bradyzoites was similar to these observed in mice treated with commercially used drugs. Collectively, the obtained results support the conclusion that both compounds are highly efficacious in a mouse model of acute and chronic toxoplasmosis.

Synthesis, characterization, DFT calculation, antioxidant activity, ADMET and molecular docking of thiosemicarbazide derivatives and their Cu (II) complexes.

In this work, new thiosemicarbazides (ECA-1, ECA-2) and their Cu (II) complexes (ECA-1-Cu, ECA-2-Cu) were synthesized and their structures were characterized by 1H NMR, 13C NMR, FT-IR, LC-MS, UV-Vis, and thermogravimetric analysis methods. Also, the surface morphology of the all compounds were examined by SEM (Scanning Electron Microscope). In the second stage, in vitro antioxidant capacity of the obtained compounds was investigated. The evaluation of the antioxidant properties of both synthesized ligands and complexes in this study was carried out by DPPH and FRAP methods. According to the results, both complexes exhibited more antioxidant capacity than the corresponding ligands. When antioxidant effects are compared for DPPH (SC50 = 5.27 ± 0.05 µM) and for FRAP (7845.69 ± 16.75 mmolTE/g), compound ECA-2-Cu appears to have the best inhibition effect. The complexes were found non-electrolytic in nature with melting point of above 250 °C, and electronic spectra and magnetic behavior demonstrated that the complexes were found to be tetrahedral geometry. Further, in silico the ADMET properties which studies are a significant role in improving and predicting drug compounds were calculated using web-based platforms. The theoretical calculations were made using the method of Density Functional Theory (Frontier molecular orbital analyze and Nonlinear optical properties). Also, molecular docking studies were performed to evaluate the binding interactions between the ligand and complex compounds and Human Peroxiredoxin 2. Both in vitro and in silico results indicated that synthesized compounds could act as potent antioxidant agents.

A novel series of IKKß inhibitors part I: Initial SAR studies of a HTS hit.

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKß. In this Letter we document our early efforts at optimization of the quinoline core, the imidazole and the semithiocarbazone moiety. Most potency gains came from substitution around the 6- and 7-positions of the quinoline ring. Replacement of the semithiocarbazone with a semicarbazone decreased potency but led to some measurable exposure.

A novel series of IKKß inhibitors part II: description of a potent and pharmacologically active series of analogs.

A novel series of (E)-1-((2-(1-methyl-1H-imidazol-5-yl) quinolin-4-yl) methylene) thiosemicarbazides was discovered as potent inhibitors of IKKß. In this Letter we document our efforts at further optimization of this series, culminating in 2 with submicromolar potency in a HWB assay and efficacy in a CIA mouse model.

Preclinical pharmacokinetics, tissue distribution and excretion studies of a novel anti-candidal agent-thiosemicarbazide derivative of isoniazid (TSC-INH) by validated UPLC-MS/MS assay.

A simple and sensitive UPLC-MS/MS assay was developed and validated for rapid determination of thiosemicarbazide derivative of isoniazid (TSC-INH), a potent anti-candidal agent in rat plasma, tissues, urine and feces. All biological samples were prepared by protein precipitation method using celecoxib as an internal standard (IS). Chromatographic separation was achieved on Acquity BEH™ C18 (50×2.1 mm, 1.7 µm) column using gradient mobile phase of acetonitrile and water (containing 0.1% formic acid) at flow rate of 0.3 mL/min. The MRM transitions were monitored at m/z 305.00→135.89 for TSC-INH and m/z 380.08→316.03 for IS in ESI negative mode. All validation parameter results were within the acceptable range described in guideline for bioanalytical method validation. The pharmacokinetic study showed that the compound TSC-INH was orally active with 66% absolute bioavailability in rats. It was rapidly absorbed with peak plasma concentration of 1985.92 ng/mL achieved within 1 h after single oral dose (10 mg/kg) administration. TSC-INH exhibited rapid distribution across the body with highest levels in liver and lungs. Penetration in brain tissues suggests that TSC-INH crossed the blood brain barrier. Only 5.23% of the orally administered drug was excreted as unconverted form in urine and feces implying that TSC-INH was metabolized extensively before excretion. With the preliminary knowledge of in vivo pharmacokinetics and disposition properties, this study will be beneficial for further development of compound TSC-INH in future studies.

Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity.

Sialic acid-binding immunoglobulin-like lectin-9 (Siglec-9) on leukocyte surface is a counter-receptor for endothelial cell surface adhesin, human primary amine oxidase (hAOC3), a target protein for anti-inflammatory agents. This interaction can be used to detect inflammation and cancer in vivo, since the labeled peptides derived from the second C2 domain (C22) of Siglec-9 specifically bind to the inflammation-inducible hAOC3. As limited knowledge on the interaction between Siglec-9 and hAOC3 has hampered both hAOC3-targeted drug design and in vivo imaging applications, we have now produced and purified the extracellular region of Siglec-9 (Siglec-9-EC) consisting of the V, C21 and C22 domains, modeled its 3D structure and characterized the hAOC3-Siglec-9 interactions using biophysical methods and activity/inhibition assays. Our results assign individual, previously unknown roles for the V and C22 domains. The V domain is responsible for the unusually tight Siglec-9-hAOC3 interactions whereas the intact C22 domain of Siglec-9 is required for modulating the enzymatic activity of hAOC3, crucial for the hAOC3-mediated leukocyte trafficking. By characterizing the Siglec-9-EC mutants, we could conclude that R120 in the V domain likely interacts with the terminal sialic acids of hAOC3 attached glycans whereas residues R284 and R290 in C22 are involved in the interactions with the active site channel of hAOC3. Furthermore, the C22 domain binding enhances the enzymatic activity of hAOC3 although the sialic acid-binding capacity of the V domain of Siglec-9 is abolished by the R120S mutation. To conclude, our results prove that the V and C22 domains of Siglec-9-EC interact with hAOC3 in a multifaceted and unique way, forming both glycan-mediated and direct protein-protein interactions, respectively. The reported results on the mechanism of the Siglec-9-hAOC3 interaction are valuable for the development of hAOC3-targeted therapeutics and diagnostic tools.

Novel Thiosemicarbazide Hybrids with Amino Acids and Peptides Against Hepatocellular Carcinoma: A Molecular Designing Approach Towards Multikinase Inhibitor.

Hepatocellular Carcinoma is the most common primary malignant tumor of the liver. Development of multidrug resistance is the main obstacle to the success of anticancer drugs. In this study, designing and docking study of thiosemicarbazide hybrids with amino acids or peptides against hepatocellular carcinoma was performed since hybrids of biologically active compounds with amino acids or peptides may show target specificity and lower toxicity. All the structures were drawn in 2D platform and converted to the 3D platform using ChemDraw 10.0. Evaluations of ADME properties were done by using QikProp 3.0 to check for the possibility of oral delivery. In silico prediction of LD50 values were performed using Pro-Tox webserver. Interestingly, it was found that conjugation with amino acids decreases toxicity and increases the therapeutic index of thiosemicarbazide. Finally, all the compounds were docked to the crystal structure of the Vascular Endothelial Growth Factor Receptor-2 and Checkpoint kinase-1 utilizing Glide 5.0, Schrödinger 8.5, to understand the interaction of ligands with the receptor. A significant number of derivatives have been found active in both the receptors and also displayed multikinase inhibitory activity similar to Sorafenib, against hepatocellular carcinoma. Further, wet lab synthesis, in vitro ADMET and biological screening studies need to be performed to prove that designed compounds are effective against hepatocellular carcinoma as predicted by molecular modeling. However, as predicted by molecular modeling, the efficacy of designed compounds against hepatocellular carcinoma, needs to be confirmed by wet lab synthesis, in vitro ADMET and biological screening studies.

Synthesis of 1-(6-chloro-1,1-dioxo-1,4,2-benzodithiazin-3-yl)semi-carbazides and their transformation into 4-chloro-2-mercapto-N-(4,5-dihydro-5-oxo-4-phenyl-1H-1,2,4-triazol-3-yl)benzenesulfonamides as potential anticancer and anti-HIV agents.

Synthesis of a series of 1-(6-chloro-1,1-dioxo-1,4,2-benzodithiazin-3-yl)semicarbazides (6-16) and 4-chloro-2-mercapto-N-(4,5-dihydro-5-oxo-4-phenyl-1H-1,2,4-triazol-3-yl)benzenesulfonamides (17-22) were reported. Compounds 7-9, 17, 19-22 were tested at the US National Cancer Institute for their in vitro anticancer and anti-HIV activities. Results of anticancer screening showed moderate activity of 21 and 22, while 19 was found to have encouraging anti-HIV activity at EC(50) = 28.8 microM.

Kinetics of semicarbazide and nitrofurazone in chicken eggs and egg powders.

The accumulation, depletion and partitioning of semicarbazide (SEM) and its parent compound nitrofurazone (NFZ) in eggs were studied using hens fed NFZ at therapeutic and sub-therapeutic levels. Dietary NFZ correlated strongly with NFZ and total SEM in eggs, while 28% of observed SEM was present in the form of parent NFZ. Depletion half-life in eggs was 2.4 days for SEM and 1.1 days for NFZ. NFZ accumulated preferentially in yolk (57-63%) as opposed to albumen, while 71-80% of SEM was found in yolk. In whole egg, 29% of SEM was present as tissue-bound residues compared with 80% in breast muscle. Whilst NFZ and SEM were partly degraded by pasteurization and spray drying, sufficient NFZ remained to suggest it might be detectable in egg powders when SEM is observed at low microg kg(-1) concentrations. NFZ was detectable in whole eggs during ingestion of only 0.1% of the therapeutic NFZ dose, making detection of intact NFZ in eggs a feasible means to prove conclusively the administration of this banned compound.

Benzylamine antihyperglycemic effect is abolished by AOC3 gene invalidation in mice but not rescued by semicarbazide-sensitive amine oxidase expression under the control of aP2 promoter

Semicarbazide-sensitive amine oxidase (SSAO) is a transmembrane enzyme that metabolizes primary amines from endogenous or dietary origin. SSAO is highly expressed in adipose, smooth muscle and endothelial cells. In each of these cell types, SSAO is implicated in different biological functions, such as glucose transport activation, extracellular matrix maturation and leucocyte extravasation, respectively. However, the physiological functions of SSAO and their involvement in pathogenesis remain uncompletely characterized. To better understand the role of adipose tissue SSAO, we investigated whether it was necessary and/or sufficient to produce the antihyperglycemic effect of the SSAO-substrate benzylamine, already reported in mice. Therefore, we crossed SSAO-deficient mice invalidated for AOC3 gene and transgenic mice expected to express human SSAO in an adipocyte-specific manner, under the control of aP2 promoter. The aP2-human AOC3 construct (aP2-hAOC3) was equally expressed in the adipose tissue of mice expressing or not the native murine form and almost absent in other tissues. However, the corresponding SSAO activity found in adipose tissue represented only 20 % that of control mice. As a consequence, the benzylamine antihyperglycemic effect observed during glucose tolerance test in control was abolished in AOC3-KO mice but not rescued in mice expressing aP2-hAOC3. The capacity of benzylamine or methylamine to activate glucose uptake in adipocytes exhibited parallel variations in the corresponding genotypes. Although the aP2-hAOC3 construct did not allow a total rescue of SSAO activity in adipose tissue, it could be assessed from our observations that adipocyte SSAO plays a pivotal role in the increased glucose tolerance promoted by pharmacological doses of benzylamine (AU)

Increased monoamine oxidase and semicarbazide-sensitive amine oxidase activities in white adipose tissue of obese dogs fed a high-fat diet

Adipocytes express two types of amine oxidases: the cell surface semicarbazidesensitive amine oxidase (SSAO) and the mitochondrial monoamine oxidase (MAO). In human abdominal subcutaneous adipose tissue, it has been reported that SSAO substrates stimulate glucose transport and inhibit lipolysis while MAO activity is decreased in obese patients when compared to age-matched controls. However, no information has been reported on visceral WAT. To further investigate the obesity-induced regulations of MAO and SSAO in white adipose tissue (WAT) from different anatomical locations, enzyme activities and mRNA abundance have been determined on tissue biopsies from control and high-fat fed dogs, an obesity model already described to be associated with arterial hypertension and hyperinsulinemia. MAO activity was increased in the enlarged omental WAT of diet-induced obese dogs, but not in their mesenteric WAT, another intra-abdominal fat depot. Subcutaneous WAT did not exhibit any change in MAO activity, as did the richest MAO-containing tissue: liver. Similarly, SSAO was increased in omental WAT of diet-induced obese dogs, but was not modified in other WAT and in aorta. The increase in SSAO activity observed in omental WAT likely results from an increased expression of the AOC3 gene since mRNA abundance and maximal benzylamine oxidation velocity were increased. Finally, plasma SSAO was decreased in obese dogs. Although the observed regulations differ from those found in subcutaneous WAT of obese patients, this canine model shows a tissue- and site-specific regulation of peripheral MAO and SSAO in obesity (AU)

Los adipocitos expresan dos tipos de amino-oxidasa: la amino oxidasa sensible a semicarbazida de la superficie celular (SSAO) y la monoamino oxidasa mitocondrial (MAO). En el tejido adiposo subcutáneo abdominal de humanos se ha descrito que los sustratos de la SSAO estimulan el transporte de glucosa e inhiben la lipólisis, mientras que la actividad MAO disminuye en pacientes obesos cuando se compara con controles de su propia edad. Sin embargo, no existe información sobre lo que ocurre en el tejido adiposo visceral. Se investiga, por tanto, sobre la influencia de la obesidad en la regulación de la MAO y SSAO en el tejido adiposo blanco (WAT) de diferentes localizaciones anatomicas, su actividad enzimatica y la riqueza de RNAm en biopsias tisulares procedentes de perros control y tratados con dieta rica en grasa. Este modelo de obesidad ya había sido previamente descrito asociado a hipertensión arterial e hiperinsulinemia. La actividad MAO se incrementó en WAT omental hipertrofiado de perros tratados con dieta rica en grasa, pero este efecto no se apreciaba en su correspondiente tejido adiposo mesentérico, otro depósito graso intra-abdominal. En el tejido adiposo subcutáneo no se pusieron de manifiesto cambios en la actividad MAO, ni tampoco en un tejido como el hígado, muy rico en MAO.De forma similar, la actividad SSAO se incrementó en el WAT omental de perros con obesidad inducida por la dieta, pero no se modificaba en otros WAT y en la aorta. El incremento encontrado en la actividad de la SSAO en el WAT (..) (AU)