In vitro Anti-HMPV activity of new synthetic phenytoin derivatives

New derivatives of synthetic 5,5-diphenylhydantoin (phenytoin) were prepared by N-alkylation with 1,3-dibromopropane. Subsequent treatment with sodium azide led to the respective azide. Reaction of the azide with phenylacetylene and 2-hydroxy-3-butyne and oxidation of the resulting alcohol with MnO2 resulted in three triazolic compounds that were evaluated in vitro for their antiviral activity against human metapneumovirus (HMPV). 5,5-Diphenyl-3-[3-(4-phenyl-1H-1,2,3-triazol-1-yl)propyl]imidazolidine-2,4-dione was the most active of the three compounds tested, with selectivity index of 129.87, even higher than ribavirin, the control substance. The three compounds showed activity in the early stages of viral replication presenting virucidal activity and binding to cellular receptors, preventing the adsorption of viral particles. These compounds showed higher activity in both experiments, inhibiting 98.3% of infection as virucidal and 98.9% when interacting with cellular receptors. Furthermore, they showed 73.8% of activity during the penetration of HMPV particles into cells. The derivative 3-{3-[4-(1-hydroxyethyl)-1H-1,2,3-triazol-1-yl]propyl}-5,5-diphenylimidazolidine-2,4-dione presented a mild anti-HMPV activity, with selectivity index of 2.74. 3-[3-(4-acetyl-1H-1,2,3-triazol-1-yl)propyl]-5,5-diphenylimidazolidine-2,4-dione inhibited less than 50% of HMPV replication

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.</p> <p>Conclusions</p> <p>We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant <it>P. pastoris </it>clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of <it>P. pastoris </it>clones with high expression levels of aquaporins and other classes of membrane proteins.</p

Factors associated with disease evolution in Greek patients with inflammatory bowel disease

BACKGROUND: The majority of Crohn's disease patients with B1 phenotype at diagnosis (i.e. non-stricturing non-penetrating disease) will develop over time a stricturing or a penetrating pattern. Conflicting data exist on the rate of proximal disease extension in ulcerative colitis patients with proctitis or left-sided colitis at diagnosis. We aimed to study disease evolution in Crohn's disease B1 patients and ulcerative colitis patients with proctitis and left-sided colitis at diagnosis. METHODS: 116 Crohn's disease and 256 ulcerative colitis patients were followed-up for at least 5 years after diagnosis. Crohn's disease patients were classified according to the Vienna criteria. Data were analysed actuarially. RESULTS: B1 phenotype accounted for 68.9% of Crohn's disease patients at diagnosis. The cumulative probability of change in disease behaviour in B1 patients was 43.6% at 10 years after diagnosis. Active smoking (Hazard Ratio: 3.01) and non-colonic disease (non-L2) (Hazard Ratio: 3.01) were associated with behavioural change in B1 patients. Proctitis and left-sided colitis accounted for 24.2%, and 48.4% of ulcerative colitis patients at diagnosis. The 10 year cumulative probability of proximal disease extension in patients with proctitis and left-sided colitis was 36.8%, and 17.1%, respectively (p: 0.003). Among proctitis patients, proximal extension was more common in non-smokers (Hazard Ratio: 4.39). CONCLUSION: Classification of Crohn's disease patients in B1 phenotype should be considered as temporary. Smoking and non-colonic disease are risk factors for behavioural change in B1 Crohn's disease patients. Proximal extension is more common in ulcerative colitis patients with proctitis than in those with left-sided colitis. Among proctitis patients, proximal extension is more common in non-smokers

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

A genetic investigation of sex bias in the prevalence of attention-deficit/hyperactivity disorder

Background Attention-deficit/hyperactivity disorder (ADHD) shows substantial heritability and is 2-7 times more common in males than females. We examined two putative genetic mechanisms underlying this sex bias: sex-specific heterogeneity and higher burden of risk in female cases. Methods We analyzed genome-wide autosomal common variants from the Psychiatric Genomics Consortium and iPSYCH Project (20,183 cases, 35,191 controls) and Swedish populationregister data (N=77,905 cases, N=1,874,637 population controls). Results Genetic correlation analyses using two methods suggested near complete sharing of common variant effects across sexes, with rg estimates close to 1. Analyses of population data, however, indicated that females with ADHD may be at especially high risk of certain comorbid developmental conditions (i.e. autism spectrum disorder and congenital malformations), potentially indicating some clinical and etiological heterogeneity. Polygenic risk score (PRS) analysis did not support a higher burden of ADHD common risk variants in female cases (OR=1.02 [0.98-1.06], p=0.28). In contrast, epidemiological sibling analyses revealed that the siblings of females with ADHD are at higher familial risk of ADHD than siblings of affected males (OR=1.14, [95% CI: 1.11-1.18], p=1.5E-15). Conclusions Overall, this study supports a greater familial burden of risk in females with ADHD and some clinical and etiological heterogeneity, based on epidemiological analyses. However, molecular genetic analyses suggest that autosomal common variants largely do not explain the sex bias in ADHD prevalence

In vitro Anti-HMPV Activity of New Synthetic Phenytoin Derivatives

New derivatives of synthetic 5,5-diphenylhydantoin (phenytoin) were prepared by N-alkylation with 1,3-dibromopropane. Subsequent treatment with sodium azide led to the respective azide. Reaction of the azide with phenylacetylene and 2-hydroxy-3-butyne and oxidation of the resulting alcohol with MnO 2 resulted in three triazolic compounds that were evaluated in vitro for their antiviral activity against human metapneumovirus (HMPV). 5,5-Diphenyl-3-[3-(4-phenyl-1H-1,2,3-triazol-1-yl)propyl]imidazolidine-2,4-dione was the most active of the three compounds tested, with selectivity index of 129.87, even higher than ribavirin, the control substance. The three compounds showed activity in the early stages of viral replication presenting virucidal activity and binding to cellular receptors, preventing the adsorption of viral particles. These compounds showed higher activity in both experiments, inhibiting 98.3% of infection as virucidal and 98.9% when interacting with cellular receptors. Furthermore, they showed 73.8% of activity during the penetration of HMPV particles into cells. The derivative 3-{3-[4-(1-hydroxyethyl)-1H-1,2,3-triazol-1-yl]propyl}-5,5-diphenylimidazolidine-2,4-dione presented a mild anti-HMPV activity, with selectivity index of 2.74. 3-[3-(4-acetyl-1H-1,2,3-triazol-1-yl)propyl]-5,5-diphenylimidazolidine-2,4-dione inhibited less than 50% of HMPV replication. Keywords: phenytoin, 1,2,3-triazoles, metapneumovirus, antiviral assay, real-time RT-PCR Introduction Human metapneumovirus (HMPV) is a negative-sense, single-stranded RNA virus first isolated in 2001 by van den Hoogen and colleagues 1,2 in previously virus-negative nasopharyngeal aspirates from children with respiratory tract infections. It has been recognized as a common cause of respiratory infections in children, immunocompromised and elderly populations. 12 Many hydantoin (imidazolidine-2,4-dione) derivatives have pharmacological activities (anticonvulsant, antifungal, antibacterial and antiparasitic), Phenytoin (5,5-diphenyl-2,4-imidazolidindione or 5,5-diphenylhydantoin) belongs to a group of antiepileptic drugs with clinical use in cases of partial and generalized seizures. It displays various effects such as inhibition of sodium, potassium and calcium channels in the neurons membrane, changes in local concentrations of neurotransmitters such as GABA, acetylcholine and noradrenaline and decreased neuronal excitation in general. Benzil (4) 19 A mixture of 2-hydroxy-1,2-diphenylethanone (3) (27.5 g; 0.13 mol) and concentrated nitric acid (140 mL) was refluxed for 2 h, until nitrogen oxide gas evolution stopped. The reaction mixture was poured into cold distilled water (400 mL), stirred and allowed to freeze for 24 h. After vacuum filtration, the crystals were washed with cold distilled water to completely remove the nitric acid. The product recrystallized from hot ethanol (66 mL) forming crystals as pale yellow needles (26.16 g; 0.12 mol; 96%); mp 94-97 °C; IR (KBr) ν / cm -1 3551, 3477, 3416, 1676, 1657, 1211, 998, 875, 794, 718, 642 Potassium hydroxide 70% (8.1 mL), urea (2.30 g, 0.04 mol) and benzil 4 (4.20 g, 0.02 mol) were added to ethanol (45 mL) in a round-bottom flask. The dough formed was refluxed for 3 h and became clear by heating. Then cold distilled water was added (100 mL) and the diphenylacetylenediurein precipitate was discarded. A solution of 50% sulfuric acid was added to the filtrate, with stirring, until acidic pH. The precipitate so formed was filtered under vacuum and washed with cold distilled water. The crystals were recrystallized from a solution of hot distilled water (4.5 mL) and sodium hydroxide (1.88 g, 0.05 mol) and treated with activated charcoal (0.3 g). After filtration on Celite ® , a solution of 50% sulfuric acid was added to the filtrate until precipitation of the product as a beige colored solid, which was filtered and dried in oven at 100 ºC, yielding a beige powder (3.28 g, 0.013 mol, 65%); mp 297-300 ºC; IR (KBr) ν / cm -1 3273, 3208, 1773, 1741, 1718, 1494, 1449, 1401, 1235, 1015, 787, 641; 1 H NMR (300 MHz, DMSO-d 6 ) d 11.13 (s, 1H), 9.33 (s, 1H), 7.31-7.44 (m, 10H); 13 C NMR (75.46 MHz, DMSO-d 6 ) d 174. 9, 156.1, 139.9, 128.6, 128.1, 126.7, 70.3. 3-(3-Bromopropyl)-5,5-diphenylimidazolidine-2,4-dione In vitro Anti-HMPV Activity of New Synthetic Phenytoin Derivatives J. Braz. Chem. Soc. 4 brine (20 mL), dried over Na 2 SO 4 and concentrated under vacuum. The crude residue was purified by recrystallization (EtOH) yielding a white solid (6.50 g; 17.4 mmol; 69%); mp 140-144 °C; IR (KBr) ν / cm -1 3454, 3406, 3220, 3099, 1772, 1706, 1494, 1417, 1255, 789, 700, 693, 518; 1 H NMR (300 MHz, CDCl 3 ) d 7.36 (s, 10H), 7.14 (s, 1H), 3.72 (t, 2H, J 6.9), 3.34 (t, 2H, J 6.7), 2.16-2.25 (m, 2H); 13 C NMR (75.46 MHz, CDCl 3 ) d 173. 2, 156.7, 138.9, 128.7, 128.5, 126.6, 70.0, 37.6, 31.0, 29.5 Sodium azide (5.10 g; 78.6 mmol) was added to a mixture of 3-(3-bromopropyl)-5,5-diphenylimidazolidine-2,4-dione 5 (9.80 g; 26.0 mmol) in ethanol (80 mL) and distilled water (16 mL). The white mixture was stirred under reflux for 35 h. Ethanol was evaporated and the aqueous phase extracted with CH 2 Cl 2 (80 mL). The organic layer was washed with distilled water (2 × 40 mL), brine (40 mL), dried over Na 2 SO 4 , and concentrated under vacuum. The yellowish oil was dissolved in ethyl acetate, treated with charcoal and filtered through Celite ® . After solvent evaporation and purification by recrystallization (ethanol) a pale yellow solid was obtained (8.80 g; 24.3 mmol; 93%); mp 118-120 °C; IR (KBr) ν / cm -1 3448, 3413, 3222, 3171, 3094, 2099, 2079, 2053, 1770, 1708, 1495, 1245, 759, 156.9, 138.9, 128.6, 128.4, 126.6, 69.9, 48.7, 36.4, 27 2-Hydroxy-3-butyne (1.60 mL; 20.0 mmol) was added to a mixture of CuSO 4 .5H 2 O (3.70 g; 15.0 mmol), sodium ascorbate (4.50 g; 22.5 mmol) and 3-(3-azidopropyl)-5,5-diphenylimidazolidine-2,4-dione 7 (5.00 g; 15.0 mmol) in a mixture of distilled water (50 mL) and CH 2 Cl 2 (25 mL). The orange mixture was stirred under reflux for 24 h. Then, CH 2 Cl 2 (100 mL) and distilled water (100 mL) were added to the reaction mixture. The organic layer was washed with distilled water (50 mL), brine (50 mL), dried over Na 2 SO 4 and concentrated under vacuum. The brown oil obtained was dissolved in ethyl acetate, treated with charcoal and filtered through Celite ® . After solvent evaporation and purification by recrystallization (CH 2 Cl 2 /hexane), a white solid was recovered (4.00 g; 9.92 mmol; 66%); mp 78-81 °C; IR (KBr) ν / cm -1 3548-3238, 3464, 3418, 3171, 2974, 2958, 2926, 1766, 1788, 1634, 1617, 1491, 1447, 1308, 1150, 1077, 759, 529; 1 H NMR (300 MHz; CDCl 3 ) d 7.59 (s, 1H), 7.35-7.38 (m, 10H), 6.69 (s, 1H), 5.05 (q, 1H, J 6.7), 4.32 (t, 2H, J 6.9), 3.63 (t, 2H, J 6.6), 2.24-2.33 (m, 2H), 1.67 (s, 1H), 1.57 (d, 3H, J 6.7); 13 C NMR (75.46 MHz; DMSO-d 6 ) d 173. 4, 155.3, 152.7, 139.6, 129.8, 127.8, 125.7, 120.2, 69.2, 62.6, 46.6, 40.3, 24.7, 6.8 MnO 2 (0.87 g; 10.0 mmol) was added to a mixture of 3-{3-[4-(1-hydroxyethyl)-1H-1,2,3-triazol-1-yl]propyl}-5,5-diphenylimidazolidine-2,4-dione 8 (0.40 g; 1.00 mmol) in CH 2 Cl 2 (20 mL). The reaction mixture was stirred at room temperature for 24 h. Then, it was filtered through Celite ® and concentrated under vacuum. The pale yellow semi-solid was purified by recrystallization (CH 2 Cl 2 ) yielding a white solid (0.37 g; 0.92 mmol, 92%); mp 151-152 °C; IR (KBr) ν / cm -1 3456, 3351, 3149, 1780, 1673 1713, 1493, 1528, 1449, 1236, 1207, 1035, 768 7, 173.3, 155.2, 146.9, 139.6, 128.6, 128.2, 127.4, 126.7, 69.2, 47.5, 35.5, 28.3, 27 Mendes et al. 5 Vol. 27, No. 1, 2016 In vitro antiviral assays against HMPV The cell culture used was LLC-MK2 (Macaca mulatta, monkey, rhesus) grown in Dulbecco&apos;s modified Eagle&apos;s medium (DMEM), supplemented with L-glutamine (3 mmol L -1 ), garamicin (50 mg mL -1 ), fungizon (2.5 mg mL -1 ), sodium bicarbonate at 0.25%, and 10% of heat-inactivated fetal bovine serum (FBS) and maintained at 37 °C in an atmosphere of 5% CO 2 . All experiments were carried out using the same amount of cells (5 × 10 5 cells mL -1 ). A sample of HMPV NL/1/00 was kindly provided by ViroNovative BV, Erasmus University Rotterdam. Because the in vitro viral replication is dependent on trypsin, this enzyme was added to the culture medium for a final concentration of 1 μg mL -1 in all antiviral and cytotoxicity experiments. The cytotoxicity assay was performed by incubating LLC-MK2 cell monolayers cultivated in 96-well microplates with two-fold serial dilutions of compounds 7, 8 and 9 in triplicate, for seven days at 34 °C. The cellular viability was further evaluated by the neutral red dye-uptake method. 20 In this experiment, the cytotoxic concentration for 20% of cell culture (CC 20 ), used in the antiviral assays, and the cytotoxic concentration for 50% of cell culture (CC 50 ) were determined for subsequent determination of the selectivity index (SI). In the antiviral assay, LLC-MK2 cell monolayers cultivated in 48-well microplates were treated with compounds 7, 8 and 9 at the concentration chosen according to the cytotoxicity results. The wells reserved for cell and virus controls were not treated with the compounds. Afterwards, 100 µL of an HMPV suspension diluted at 10 -1 corresponding to 1.12 × 10 7 copies mL -1 were added to treated and untreated cell cultures and incubated in a 5% CO 2 atmosphere at 34 °C for seven days. All experiments were carried out in triplicate. After incubation, the supernatant of the cell monolayers was removed and then lysated using guanidine thiocyanate buffer. The viral RNA was extracted and a real time RT-PCR for detection of HMPV genome was performed using primers that amplify a 151 bp fragment of the HMPV N gene, as previously described by Brittain-Long et al. The dose-response curve was established starting from the non-cytotoxic concentrations, and the 50% effective dose (ED 50 ) was defined as the concentration of the compounds that inhibits 50% of viral replication. The selectivity index (SI) was determined as the ratio of CC 50 to EC 50 . Viral RNA was extracted from the cells supernatant lysate using the commercial kit Totally RNA™ (Applied Biosystems/Ambion, USA), according to the manufactory&apos;s instructions. Reverse transcription (RT-PCR) was performed in a 10 μL reaction mixture containing 5 μL of extracted viral RNA and 360 nmol L -1 of the antisense primer, using ImProm-II Reverse Transcriptase (Promega, Madison, WI, USA). Real-time PCR assays were performed in a step-one real time PCR System (Applied Biosystems, Carlsbad, CA, USA) and consisted of 10 min activation at 95 °C, followed by 45 cycles of 15 s at 95 °C, and 60 s at 60 °C. Amplification was carried out in 24 μL reaction volumes, including 5 μL of cDNA, 900 nmol L -1 of sense primer and 300 nmol L -1 of antisense primer, and 12 μL of Maxima qPCR SYBR Green (Thermo Scientific/Fermentas, Canada). The quantification of HMPV RNA was performed using a standard curve generated by the Ct (threshold cycle) values obtained from serial 10-fold dilutions of in vitro transcripts containing dilutions varying from 10 0 to 10 7 of the original virus stock. Each dilution was quantified using the Quant-IT™ DNA assay kit (Invitrogen, Carlsbad), which allows us to correlate the amount of DNA, i.e., the number of genome copies, with the Ct of each dilution. The results were analyzed in triplicate, and the average number of copies of the viral DNA was calculated. Once the ability of compounds 7, 8 and 9 to inhibit the replication of HMPV was established, several experiments were carried out to elucidate the mechanism involved in their antiviral activity. The experiments were designed in order to demonstrate whether the activity was on the viral particle (virucidal), on the virus-cell interaction (receptors and cell entry) or in a late stage of virus replication (intracellular activity). Virucidal assay 100 µL of an HMPV suspension diluted at 10 -1 were added to either 900 μL of compounds 7, 8 and 9 or to DMEM-Eagle without serum (control), according to Chen et al. The preincubation of the cells with the compounds for 1 h at 4 °C is to allow the interaction of the substances with the protein in the cell surface and this temperature is important so the cell surface will not be in fluid state. This avoids the substances to penetrate the cells and allows their interaction only with the cell surface proteins. After that period, the cells were washed to remove anything that does not have a strong binding to the cell surface proteins, so if the substance has affinity for any protein in the cell surface, it will remain bound. And only after that the virus is added. So, if the receptor for the virus, which is a cell surface protein, is somehow blocked by the substances, the virus will not infect those cells, resulting in a reduction of the titer, once again measured by RT-real time PCR, comparing to the virus control. This is also a well established methodology. 23-26 Cell entry assay LLC-MK2 cell monolayers were inoculated with 100 μL of an HMPV suspension diluted at 10 -1 and incubated for 1 h at 4 °C. After absorption, the monolayers were washed, treated with compounds 7, 8 and 9, followed by incubation for 1 h at 37 °C. Afterwards, the monolayers were washed, DMEM-Eagle was added and the cells were incubated at 34 °C for seven days in an atmosphere of 5% CO 2 . After incubation, the supernatant of the cell monolayers was removed and lysated using guanidine thiocyanate buffer. The viral RNA was extracted and real time PCR for HMPV was performed as already described. Intracellular assay LLC-MK2 cell monolayers were inoculated with 100 μL of an HMPV suspension diluted at 10 -1 and incubated at 37 °C for 2 h, which is enough time for the viral particles to penetrate the cells, but not enough for the virus to start the translation and transduction stages. After incubation, the cell monolayers were washed and compounds 7, 8 and 9 added. Then, the cells were incubated at 37 °C for 10 h. It is well known that the viral cycle of the HMPV takes approximately 12 hours, which means that after this period of infection the first viral particles will start to emerge from the infected cells. In order to evaluate any activity inside the cells, the experiment has to be stopped before 12 hours of incubation and then a comparison of the amount of RNA produced in this period of time is carried out in the presence and in the absence of the substances. After incubation, the supernatant of the cell monolayers was removed and these were washed with culture medium and lysated using guanidine thiocyanate buffer. The viral RNA was extracted and real time PCR for HMPV was performed as already described. By conducting the cytotoxicity tests based on the incorporation of the vital dye neutral red, we could choose the concentrations of each compound to be used in antiviral tests. The chosen concentrations were the ones in which at least 80% of the cells in culture remained viable (CC 20 ), which varied between 500 μg mL -1 (compounds 7 and 9) and 125 μg mL -1 (compound 8). It was not possible to determine the CC 50 (cytotoxic concentration for 50% of cells), for compounds 7 and 9 since the percentage of viable cells at the highest concentration tested (500 μg mL -1 ) was higher than 50%. The CC 50 value for compound 8 was 236.05 μg mL -1 Results and Discussion Phenytoin was synthesized using the method described by Vogel 19 (Scheme 1) in which benzaldehyde undergoes self-condensation followed by oxidation to benzil (4). Benzil condensation with urea then produces phenytoin (1) in good overall yield. Structural modification of pharmacologically active compounds is of interest since the introduction of other pharmacophoric groups in the right position of these compounds can generate a new class of molecules with potential action on living organisms. In this sense, we envisaged the association of the phenytoin nucleus with triazolic rings containing different substituents, since some triazolic compounds have shown a broad spectrum of activities such as antibacterial, anticonvulsant, antiinflammatory, antiviral, anticancer and antitumor, among others. 27 These triazolic rings can be easily obtained by cycloaddition reaction between acetylenes and azides Mendes et al. 7 Vol. 27, No. 1, 2016 catalyzed by copper(I) salts, a process known as Huisgen cycloaddition. 29 This reaction was studied by Huisgen in the 1960&apos;s and, due to its versatility, the alkyne and azide groups can be incorporated into various compounds through many methodologies. 30 In this work, several 3-substituted imidazolic-like compounds were prepared from synthetic phenytoin, aiming at potential new drugs presenting inhibitory activity against human metapneumovirus (HMPV). The synthetic methodology is straightforward and is summarized in Scheme 2. The title compounds (7-9) were obtained in good overall yields from phenytoin and the formation of the triazolic rings was confirmed by NMR analysis in which the CH protons appear between 7.5-9.0 ppm and the carbons between 130-150 ppm. Regarding antiviral activity, compounds 8 and 9 showed a mild potential against HMPV, inhibiting 67.5% and 42.9% of viral replication, respectively, while compound 7 inhibited 99.9%, the same as ribavirin A dose-response curve was performed in order to determine the ED 50 values for each triazolic compound Four different mechanisms of action were evaluated When we studied the mechanisms of action, both compounds (7 and 8) showed activity in the early stages of HMPV replication, mainly virucidal (98.3% and 63.9%, respectively), interacting with the cellular receptors, thus preventing HMPV adsorption to cell surface (98.9% and 67.4%, respectively). Compound 7 also showed activity during the penetration of HMPV particles into cells, inhibiting 73.8% of viral replication. In this stage, compound 9 had no activity and compound 8 a mild one (19.3%). None of the three compounds was capable of inhibiting intracellular events of HMPV replication, unlike ribavirin, probably due to the low solubility of these compounds. Conclusions According to the results shown here, we conclude that compounds 7, 8 and 9 may be used as a prophylactic treatment for infections caused by HMPV because they act by preventing adsorption and penetration of viral particles in the host cell. Probably, its use in combination with ribavirin may be an alternative to minimize the action of the virus