Design, Synthesis, and Antiviral Activities of New Benzotriazole-Based Derivatives

Figures

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  Figure 1. Previously reported benzotriazole-based compounds with biological activities, BTZ1 [41], BTZ2 [42], BTZ3 [43], BTZ4 [44], BTZ5 [46], BTZ6 [47], BTZ7 [48].

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  Figure 2. General structures of our benzotriazole-based antiviral compounds: series (A), active against CVB (EC50 values between 8–10 µM) and RSV (EC50 values 3–7 µM). Series (B), active on HTNV (EC50 < 5 µM), CVB (EC50 values between 4–8 µM) and RSV (EC50 values 2–3 µM) [51,52,53,54].

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  Figure 3. The precursor N1,N4-bis(4-(5,6-dichloro-1H-benzo[d][1,2,3]triazol-1-yl)phenyl)succinamide (A) and its active derivative N1,N4-bis(4-((5,6-dichloro-1H-benzo[d][1,2,3]triazol-1-yl)methyl)phenyl)succinamide (B).

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  Figure 4. Structural modifications on our hit compounds: introduction of a methylene bridge (red) and displacement of the substituent on the benzene ring (blue).

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  Scheme 1. General procedure for intermediates compounds: 3a, b; 4a,b; 5b; 6a,b; 8a–e; 9a–e; 10e, 11a,b,d; 12a–e Reaction conditions: I, from 1a,b, anhydrous N,N-Dimethylformamide (DMF) or N,N-dimethylacetamide (DMA), Cs2CO3 at t = 50/70 °C; ii, from 1a–e, ethanol, hydrated hydrazine in molar ratio 1:10, 10% palladium on activated charcoal at t = 80/90 °C or methylhydrazine in 1:10 molar ratio, at 100 °C in autoclave for 48 h.

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  Scheme 2. I. stirred at r.t.; II. DMA or DMF at 80 °C; III. DMF at 100 °C. General procedure to obtain the final compounds: amides 23b–29b, 75b, and 76b and urea-derivatives 30b–34b were obtained by using benzotriazol-2-ylphenylamino derivatives 5b and 11b as starting compounds.

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  Scheme 3. I. stirred at r.t.; II. DMA or DMF at 80 °C; III. DMF at 100 °C. General procedure to obtain the final compounds: Amides 15e–22e, 35a, 36a, 37b, 38b, 39a–48a, 49b–57b, 77b, 78b, 79c, 80c, 81d, 82d, 83b, 84b, 85c–88c, 89d–94d. Urea-derivatives 58a–62a, 63b–74b, 95a–98a, 99b, 100b, 101c–103c, 104d–107d were gained by using benzotriazol-1ylphenylamino derivatives 6a, 6b, 12a–e as starting compounds.

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  Figure 5. (A–C) SARs analysis from the synthetized compounds.

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  Figure 6. Evaluation of cell monolayer permeabilization as transepithelial electrical resistance (TEER) assay. Caco-2 cell monolayers were incubated with LPS at 1 μg/mL (black squares) as negative control, compound 18e at 20 µM (blue triangles), compound 43a at 20 µM (green triangles), and Control (red circles) as positive control. Statistically significant differences are expressed as follows: * = p < 0.05 LPS/Control; *** = p < 0.001 LPS vs. Control. Each value represents the mean ± SD of independent experiments (n = 3).

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  Figure 7. Derivative 18e protected Vero 76 cells from apoptosis induced by CVB5 infection. The percentage of live, apoptotic, and necrotic cells were measured by flow cytometry using the PI-annexin V assay. The graph shows the percentage of live, apoptotic, and necrotic cells. Each value represents the mean ± SD of independent experiments (n = 3).

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  Figure 8. Virucidal activity (expressed as plaque-forming units (PFU/mL) of benzotriazole 18e (20 µM) against CVB5 infectivity at either 4 °C or 37 °C for 2 h. Blue columns, viral titer for benzotriazole 18e and viral solution; yellow columns indicate the viral titer of untreated solution.

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  Figure 9. Pre-treatment and time course experiment. Vero-76 cells were pre-treated with compound 18e (20 µM) for 2 h. After this period, the monolayers were infected with CVB5 (m.o.i. = 1). After infection period for time of addition condition, the compound 18e (20 µM) was added at the indicated times. Viral yields were then determined by plaque assay. Dark columns, the viral yield for control cells; grey columns; viral yield for cells treated with 18e.