Identification and Characterization of Rotigotine Degradation Products by HPLC Coupled DAD and CAD Detectors and HRMS Through Q-Orbitrap and Electrospray Ionization.

Rotigotine (RTG) is a dopamine agonist used in the treatment of Parkinson's disease. As it is susceptible to oxidation, stability studies must be carefully designed for the identification and characterization of all possible degradation products. Here, RTG degradation was evaluated according to the International Conference on Harmonization guidelines under various stress conditions, including acidic and basic hydrolysis, oxidative, metallic, photolytic, and thermal conditions. Additionally, more severe stress conditions were applied to induce RTG degradation. Significant degradation was only observed under oxidative and photolytic conditions. The samples were analyzed by high performance liquid chromatography coupled to photodiode array detectors, charged aerosol, and high-resolution mass spectrometry. Chromatographic analyses revealed the presence of eight substances related to RTG, four of which were already described and were qualified impurities (impurities B, C, K and E) and four new degradation products (DP-1 - DP-4), whose structures were characterized by high-resolution mass spectrometry through Q-Orbitrap and electrospray ionization. In the stress testing of the active pharmaceutical ingredient in solid form, significant RTG degradation was observed in the presence of the oxidative matrix. The results corroborate the literature that confirm the high susceptibility of RTG to oxidation and the importance of using different detectors to detect degradation products in forced degradation studies.

N, N'-disubstituted Ureas as Novel Antiplatelet Agents: Synthesis, Pharmacological Evaluation and In Silico Studies.

INTRODUCTION: Thrombotic disorders are among the leading causes of morbidity and mortality worldwide. Drugs used in the prevention and treatment of atherothrombosis have pharmacokinetic limitations and adverse effects such as hemorrhagic conditions, highlighting the importance of developing more effective antiplatelet agents. ethod: In this work, we synthesized N,N'-disubstituted ureas 3a-3j and evaluated their antiplatelet profiles through in vitro, ex vivo, and in silico studies. The synthesized derivatives exhibited a selective inhibitory profile against platelet aggregation induced by arachidonic acid (AA) in vitro, without significantly affecting other aspects of primary hemostasis and blood coagulation. The compounds that showed inhibition greater than 85% were submitted to the analysis of their potency by calculating the concentration required to inhibit 50% of platelet aggregation induced by AA (IC50). Urea derivative 3a was the most potent with IC50 of 1.45 µM. Interestingly, this derivative inhibited more than 90% of platelet aggregation induced by AA ex vivo, with a similar effect to acetylsalicylic acid. In the hemolysis assay, most of the urea derivatives presented values below 10% suggesting good hemocompatibility. Additionally, the compounds tested at 100 µM also showed no cytotoxic effects in HepG2 and Vero cells. RESULT: The in silico results suggested that compound 3a may bind to the key residue of COX-1 similar to AA and known COX-1 inhibitors, and the results are also in agreement with our SAR, which suggests that the inhibition of this enzyme is the most likely mechanism of antiplatelet activity. CONCLUSION: Therefore, these results demonstrated that N,N'-disubstituted ureas are promising candidates for the development of novel antiplatelet agents.

Antimycobacterial and anti-inflammatory activities of thiourea derivatives focusing on treatment approaches for severe pulmonary tuberculosis.

Tuberculosis (TB) remains a serious public health problem and one of the main concern is the emergence of multidrug-resistant and extensively resistant TB. Hyper-reactive patients develop inflammatory necrotic lung lesions that aggravate the pathology and facilitate transmission of mycobacteria. Treatment of severe TB is a major clinical challenge that has few effective solutions and patients face a poor prognosis, years of treatment and different adverse drug reactions. In this work, fifteen novel and thirty-one unusual thiourea derivatives were synthesized and evaluated in vitro for their antimycobacterial and anti-inflammatory potential and, in silico for ADMET parameters and for structure-activity relationship (SAR). Thioureas derivatives 10, 15, 16, 28 and 29 that had shown low cytotoxicity and high activities were selected for further investigation, after SAR study. These five thioureas derivatives inhibited Mtb H37Rv growth in bacterial culture and in infected macrophages, highlighting thiourea derivative 28 (MIC50 2.0 ± 1.1 and 2.3 ± 1.1 µM, respectively). Moreover, these compounds were active against the hypervirulent clinical Mtb strain M299, in bacterial culture, especially 16, 28 and 29, and in extracellular clumps, highlighting 29, with MIC50 5.6 ± 1.2 µM. Regarding inflammation, they inhibited NO through the suppression of iNOS expression, and also inhibited the production of TNF-α and IL-1ß. In silico studies were carried out suggesting that these five compounds could be administered by oral route and have low toxicological effects when compared to rifampicin. In conclusion, our data show that, at least, thiourea derivatives 16, 28 and 29 are promising antimycobacterial and anti-inflammatory agents, and candidates for further prospective studies aiming new anti-TB drugs, that can be used on a dual approach for the treatment of severe TB cases associated with exacerbated inflammation.

Nanoparticles Loaded with a New Thiourea Derivative: Development and In vitro Evaluation Against Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a neglected tropical disease caused by protozoa of the genus Leishmania. Current treatments are restricted to a small number of drugs that display both severe side effects and a potential for parasites to develop resistance. A new N-(3,4-methylenedioxyphenyl)-N'- (2-phenethyl) thiourea compound (thiourea 1) has shown promising in vitro activity against Leishmania amazonensis with an IC50 of 54.14 µM for promastigotes and an IC50 of 70 µM for amastigotes. OBJECTIVE: To develop a formulation of thiourea 1 as an oral treatment for leishmaniasis, it was incorporated into Nanoparticles (NPs), a proven approach to provide long-acting drug delivery systems. METHODS: Poly (D,L-Lactic-co-Glycolic Acid) (PLGA) polymeric NPs containing thiourea 1 were obtained through a nanoprecipitation methodology associated with solvent evaporation. The NPs containing thiourea 1 were characterized for Encapsulation Efficiency (EE%), reaction yield (% w/w), surface charge, particle size and morphology by Transmission Electron Microscopy (TEM). RESULTS: NPs with thiourea 1 showed an improved in vitro leishmanicidal activity with a reduction in its cytotoxicity against macrophages (CC50>100 µg/mL) while preserving its IC50 against intracellular amastigotes (1.46 ± 0.09 µg/mL). This represents a parasite Selectivity Index (SI) of 68.49, which is a marked advancement from the reference drug pentamidine (SI = 30.14). CONCLUSION: The results suggest that the incorporation into NPs potentiated the therapeutic effect of thiourea 1, most likely by improving the selective delivery of the drug to the phagocytic cells that are targeted for infection by L. amazonensis. This work reinforces the importance of nanotechnology in the acquisition of new therapeutic alternatives for oral treatments.

Forced degradation studies of norepinephrine and epinephrine from dental anesthetics: Development of stability-indicating HPLC method and in silico toxicity evaluation.

Injectable solutions containing epinephrine (EPI) and norepinephrine (NE) are not stable, and their degradation is favored mainly by the oxidation of catechol moiety. As studies of these drugs under forced degradation conditions are scarce, herein, we report the identification of their degradation products (DP) in anesthetic formulations by the development of stability-indicating HPLC method. Finally, the risk assessment of the major degradation products was evaluated using in silico toxicity approach. HPLC method was developed to obtain a higher selectivity allowing adequate elution for both drugs and their DPs. The optimized conditions were developed using a C18 HPLC column, sodium 1-octanesulfonate, and methanol (80:20, v/v) as mobile phase, with a flow rate of 1.5 mL/min, UV detection at 199 nm. The analysis of standard solutions with these modifications resulted in greater retention time for EPI and NE, which allow the separation of these drugs from their respective DPs. Then, five DPs were identified and analyzed by in silico studies. Most of the DPs showed important alerts as hepatotoxicity and mutagenicity. To the best of our acknowledgment, this is the first report of a stability-indicating HPLC method that can be used with formulations containing catecholamines.

Self-cured resin modified by quaternary ammonium methacrylates and chlorhexidine: Cytotoxicity, antimicrobial, physical, and mechanical properties.

OBJECTIVE: To evaluate the addition of dimethylaminohexadecyl methacrylate (DMAHDM) and chlorhexidine diacetate on cytotoxicity, antimicrobial activity, physical, and mechanical properties of a self-cured resin. METHODS: 132 disk-shaped and 48 rectangular specimens were divided into four experimental groups as described: Control Group (CG - no addition), dCHX (1%), DMAHDM (5%), and DMAHDM+dCHX (5%+1%). The biofilm viability, flexural strength (FS - ISO 20795-1:2013), surface roughness (SR), and color stability (ΔE) were analyzed after being stored for 4 weeks in distilled water and immersed for 72h in coffee. Cytotoxicity was measured after 24h, 3, and 7 days of elution using an MTT test on L929 cells (ISO 10993-5:2009). SR and ΔE were measured by a contact profilometer and a spectrophotometer using the CIELab parameter. Data were submitted to ANOVA and Bonferroni's/Tukey's tests (p≤0.05). RESULTS: Significant antimicrobial activity against Streptococcus mutans and Candida albicans was detected in all groups when compared to the CG (p<0.05). Only the dCHX group, in 24h of elution, demonstrated no cytotoxicity effects. There was a statistical difference for FS on the tested groups (p<0.05). No differences were detected in the initial roughness' measurements among the groups (p>0.05). However, after storage and immersion in coffee, the groups containing DMAHDM presented with rougher surfaces and significantly lower color stability compared to the control (p<0.05). SIGNIFICANCE: The addition of dCHX and DMAHDM in self-cured resin presented antimicrobial properties; however, cytotoxicity, physical, and mechanical properties were compromised.

Experimental composites containing quaternary ammonium methacrylates reduce demineralization at enamel-restoration margins after cariogenic challenge.

OBJECTIVE: This study evaluated the influence of experimental composites containing quaternary ammonium monomers (QAM) at different concentrations and alkyl chains on demineralization at enamel-composite margins after cariogenic challenge. METHODS: Standardized 4×4mm cavities were cut into 35 bovine enamel blocks, which were randomly divided into seven groups (n=5) and restored with the following experimental composites and commercial materials: (G12.5) - 5% dimethylaminododecyl methacrylate (DMADDM) with a 12-carbon alkyl chain (G12.10) - 10% DMADDM, (G16.5) - 5% dimethylaminohexadecyl methacrylate (DMAHDM) with a 16-carbon alkyl chain (G16.10) - 10% DMAHDM, (CG) - control group (without QAM), (GZ250) - commercial composite (Filtek Z250®), and (GIC) - glass ionomer cement (Maxxion R®). After restorative procedures, initial microhardness was measured and experimental composites were subjected to Streptococcus mutans biofilm formation for 48h. After cariogenic challenge, the samples were washed and microhardness was reassessed. A 3D non-contact profilometer was used to determine surface roughness and enamel demineralization was assessed by micro-CT. Microhardness results were analyzed by the Kruskal-Wallis and Mann-Whitney tests and micro-CT results were analyzed by Tukey's HSD test (95% confidence interval). RESULTS: None of the materials could prevent mineral loss at the enamel-restoration margins. The addition of 10% DMAHDM yielded the lowest, albeit statistically significant, mineral loss (p<0.05). 3D non-contact profilometry showed enamel surface roughness modification after biofilm exposure. The CG had the highest roughness values. Micro-CT analysis revealed mineral loss, except for GIC. SIGNIFICANCE: The addition of 10% QAM with a 16-carbon chain in experimental composites reduced mineral loss at the enamel-restoration margins after cariogenic challenge.

Physical and chemical properties of model composites containing quaternary ammonium methacrylates.

OBJECTIVE: Investigate physical and chemical properties of model composites formulated with quaternary ammonium salt monomers (QAS) at different concentrations and alkyl chains lengths METHODS: QAS with 12 dimethylaminododecyl methacrylate (DMADDM) and 16 dimethylaminohexadecyl methacrylate (DMAHDM) chains lengths were synthesized and incorporated at 5 and 10% in model composites, resulting in four groups: G12.5 (DMADDM 5%), G12.10 (DMADDM 10%), G16.5 (DMAHDM 5%), G16.10 (DMAHDM 10%). One group was used as control group (CG 0%). Degree of conversion (DC); water sorption (WS) and solubility (SL); hygroscopic expansion (HE); degradation temperature (DT); glass transition temperature (Tg) and polymerization shrinkage (PS) were determined. Knoop hardness (KNH), flexural strength (FS) and elastic modulus (EM) were measured before and after storage Data were submitted to ANOVA and Tukey's test (p≤0.05). RESULTS: DC ranged between 76.1 (G12.10) and 70.7 (G16.5) %; CG had the lowest WS, SL and HE. There was no statistical difference for PS and FS. KHN values ranged between 30.2 (GC) and 25 (G16.10) and after storage the performance was depended on QAS concentration and chain length. For EM, CG had the highest values before and after storage and no difference was observed in the QAS groups before storage. After storage, the results were dependent on QAS concentration (3.5-4.3GPa). SIGNIFICANCE: In general, the addition of QAS increased composite's degradation compared with the CG. In the tested QAS, the addition of DMADDM at 5% concentration resulted in a less degradable material.

Antileishmanial Thioureas: Synthesis, Biological Activity and in Silico Evaluations of New Promising Derivatives.

Leishmaniasis is a neglected tropical disease caused by protozoan parasites belonging to the genus Leishmania. Currently, the drugs available for treatment of this disease present high toxicity, along with development of parasite resistance. In order to overcome these problems, efforts have been made to search for new and more effective leishmanicidal drugs. The aim of this study was to synthesize and investigate the leishmanicidal effect of N,N'-disubstituted thioureas against Leishmania amazonensis, with evaluation of their in silico pharmacokinetics and toxicity profiles. Our results showed that different thioureas could be obtained in high to moderate yields using simple reaction conditions. Nine thiourea derivatives (3e, 3i, 3k, 3l, 3p, 3q, 3v, 3x and 3z) were active against parasite promastigotes (IC50 21.48-189.10 µM), with low cytotoxicity on mice peritoneal macrophages (CC50>200 µM), except for thiourea 3e (CC50=49.22 µM). After that, the most promising thioureas (3k, 3l, 3p, 3q and 3v) showed IC50 ranging from 70 to 150 µM against L. amazonensis amastigotes in infected macrophages. Except for thiourea 3p, the leishmanicidal activity of the derivatives were independent of nitric oxide (NO) production. Thioureas 3q and 3v affected promastigotes cell cycle without disturbing the mitochondrial membrane potential. Furthermore, our derivatives showed satisfactory theoretical absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. These data indicate that thiourea derivatives are good candidates as leading compounds for the development of new leishmanicidal drugs.

Antibiofilm properties of model composites containing quaternary ammonium methacrylates after surface texture modification.

OBJECTIVE: Investigate antimicrobial properties and surface texture of model composites with different concentration and alkyl chain length of quaternary ammonium monomers (QAS). METHODS: Monomers derived from QAS salts with alkyl chain lengths of 12 carbons ((dimethylaminododecyl methacrylate) DMADDM) and 16 carbons (dimethylaminohexadecyl methacrylate-DMAHDM) were obtained from the reactions of their respective organo-halides with the tertiary amine 2-(dimethylamino)ethyl methacrylate (DMAEMA). DMADDM and DMAHDM were incorporated into model composite in concentrations of 5 or 10%, resulting the following groups: G12.5 (DMADDM 5%), G12.10 (DMADDM 10%), G16.5 (DMAHDM 5%), G16.10 (DMAHDM 10%) and GC (control). Biofilm viability, lactic acid production and surface roughness were analysed 24h after samples preparation (initial), repeated after toothbrush abrasion and after polishing simulation. Data were submitted to ANOVA and Tukey's test (p≤0.05). RESULTS: The longer the molecular chain size of QAS and the higher its concentration (G16.10), the lower was the viability and the production of lactic acid by the biofilm. No differences were detected in initial roughness' measurements among groups. However, after abrasion, there was an increase of biofilm viability and lactic acid production. Composites containing QAS presented rougher surfaces compared to the CG. After polishing, biofilm viability and surface roughness were statistically similar for all groups. Nevertheless, DMAHDM at 10% showed reduction in lactic acid production. SIGNIFICANCE: Chain length and concentration of QAS influenced biofilm development and production of lactic acid. Longer chains and higher concentrations of QAS promoted better antimicrobial properties. Changes in surface texture caused by abrasion, decreased antibiofilm properties.

Design, Synthesis and Evaluation of New Fluoroamodiaquine Analogues.

Malaria is one of the most important tropical diseases; the use of amodiaquine as a current chemotherapy in the treatment of malaria has shown some problems such as hepatotoxicity and agranulocytosis. In this work we present the rational design, synthesis, and biological evaluation (antimalarial activity, cytotoxicity and genotoxicity) of four new fluoroamodiaquine analogues. The results showed significant correlation between MolDock score and IC50 values. The molecules 7b and c were the most active of the planned compounds, with lower IC50 against Plasmodium falciparum W2 strain (0.9 and 0.8 µM, respectively) and an excellent cytotoxicity profile. The present study revealed no mutagenicity or genotoxicity for the analogues. Confirming our docking results, the molecular dynamics showed that compound 7b remains stably bound to the heme group by means of π-stacking interactions between quinoline and the porphyrin ring. Based on these findings, this study may prove to be an efficient approach for the rational design of hemozoin inhibiting compounds to treat malaria.

Synthesis and antiplatelet activity of antithrombotic thiourea compounds: biological and structure-activity relationship studies.

The incidence of hematological disorders has increased steadily in Western countries despite the advances in drug development. The high expression of the multi-resistance protein 4 in patients with transitory aspirin resistance, points to the importance of finding new molecules, including those that are not affected by these proteins. In this work, we describe the synthesis and biological evaluation of a series of N,N'-disubstituted thioureas derivatives using in vitro and in silico approaches. New designed compounds inhibit the arachidonic acid pathway in human platelets. The most active thioureas (compounds 3d, 3i, 3m and 3p) displayed IC50 values ranging from 29 to 84 µM with direct influence over in vitro PGE2 and TXA2 formation. In silico evaluation of these compounds suggests that direct blockage of the tyrosyl-radical at the COX-1 active site is achieved by strong hydrophobic contacts as well as electrostatic interactions. A low toxicity profile of this series was observed through hemolytic, genotoxic and mutagenic assays. The most active thioureas were able to reduce both PGE2 and TXB2 production in human platelets, suggesting a direct inhibition of COX-1. These results reinforce their promising profile as lead antiplatelet agents for further in vivo experimental investigations.