Synthesis of Novel Aminothiazole Derivatives as Promising Antiviral, Antioxidant and Antibacterial Candidates

It is well-known that thiazole derivatives are usually found in lead structures, which demonstrate a wide range of pharmacological effects. The aim of this research was to explore the antiviral, antioxidant, and antibacterial activities of novel, substituted thiazole compounds and to find potential agents that could have biological activities in one single biomolecule. A series of novel aminothiazoles were synthesized, and their biological activity was characterized. The obtained results were compared with those of the standard antiviral, antioxidant, antibacterial and anticancer agents. The compound bearing 4-cianophenyl substituent in the thiazole ring demonstrated the highest cytotoxic properties by decreasing the A549 viability to 87.2%. The compound bearing 4-trifluoromethylphenyl substituent in the thiazole ring showed significant antiviral activity against the PR8 influenza A strain, which was comparable to the oseltamivir and amantadine. Novel compounds with 4-chlorophenyl, 4-trifluoromethylphenyl, phenyl, 4-fluorophenyl, and 4-cianophenyl substituents in the thiazole ring demonstrated antioxidant activity by DPPH, reducing power, FRAP methods, and antibacterial activity against Escherichia coli and Bacillus subtilis bacteria. These data demonstrate that substituted aminothiazole derivatives are promising scaffolds for further optimization and development of new compounds with potential influenza A-targeted antiviral activity. Study results could demonstrate that structure optimization of novel aminothiazole compounds may be useful in the prevention of reactive oxygen species and developing new specifically targeted antioxidant and antibacterial agents.


Introduction
Progress in organic and medicinal chemistry allows for the design, synthesis and optimization of the structures of novel thiazole compounds. Thiazole compounds are widely used in the pharmaceutical industry for the design of therapeutics and novel biosensors for their antioxidant [1][2][3], antibacterial [4][5][6], anti-proliferative [7], antiparasitic [8], anti-inflammatory [9], analgesic [10], neuroprotective [11], antiviral (including SARS-CoV-2) [12], and anti-HIV activities [13]. Studies of thiazole-structure activities promote the improvement of their chemical/biochemical and therapeutic properties and anti-TB activity, mainly against resistant Mycobacterium tuberculosis (Mtb) strains [14]. The data demonstrate that a reaction in THF allowed the preparation of compound 2, providing the highest yield of 70%. Triplets at 2.47 (CH 2 CO) and 3.20 (NHCH 2 ), the singlet at 3.77 (NHCH 2 ), and a broad singlet at 11.81 (COOH) in 1 H NMR for the compound confirmed the formation of the β-alanine fragment in the structure.
The cyclization of β-alanine moiety to 2-thioxotetrahydropyrimidinedione 3 was performed by refluxing carboxylic acid 2 with potassium thiocyanate in acetic acid. Compound 3 was separated by dilution of the reaction mixture with water. The obtained product was applied for the preparation of thioureido acid 4 (Scheme 1). For this purpose, compound 3 was dissolved in hot, aqueous 5% sodium hydroxide, and the obtained solution of the carboxylic acid sodium salt was filtered off and transferred to an acidic form by acidifying the filtrate with acetic acid to pH 6. report a 77.4% yield, the reaction under the specified conditions yielded only 7% of the product. This led to the study of the above-mentioned reaction using different solvents Reactions were carried out in toluene, 1,4-dioxane, 2-propanol, and tetrahydrofuran (THF) at reflux for 24 h. The results are shown in Table 1. The data demonstrate that a reaction in THF allowed the preparation of compound 2, providing the highest yield of 70%. Triplets at 2.47 (CH2CO) and 3.20 (NHCH2), the singlet at 3.77 (NHCH2), and a broad singlet at 11.81 (COOH) in 1 H NMR for the com pound confirmed the formation of the β-alanine fragment in the structure.
The cyclization of β-alanine moiety to 2-thioxotetrahydropyrimidinedione 3 was per formed by refluxing carboxylic acid 2 with potassium thiocyanate in acetic acid. Com pound 3 was separated by dilution of the reaction mixture with water. The obtained prod uct was applied for the preparation of thioureido acid 4 (Scheme 1). For this purpose compound 3 was dissolved in hot, aqueous 5% sodium hydroxide, and the obtained solu tion of the carboxylic acid sodium salt was filtered off and transferred to an acidic form by acidifying the filtrate with acetic acid to pH 6. The prepared thioureido acid 4 was applied for the synthesis of thiazoles 5a-e. The products 5a-e have been achieved by a reaction of thioureido acid 4 and the correspond ing bromoacetophenone in water, with the presence of sodium carbonate in the reaction mixture. The 1 H NMR spectra of these compound singlets, in the range of 7.07-7.42 ppm ( 13 C, 102.09-106.41 ppm), and the additional peaks in the aromatic region, confirm the presence of a 4-arylthiazole moiety.
The next goal of this study was the transformation of the acetamide fragment to an amine group. Reactions occurred easily and rapidly and were completed after refluxing in aqueous 5% hydrochloric acid for 1 h. Products were isolated by neutralizing the reac tion mixtures with sodium acetate to pH 6. By careful assignment of the peaks in the 1 H and 13 C NMR spectra, the structures 6 were elucidated. Comparison of the spectra of com pounds 5 and 6 showed obvious differences, i.e., the spectra of the latter compounds do not contain singlets of the methyl group of the acetamide moiety, but broad singlets o amino group are visible at approximately 5.35 ppm. In addition, the additional proof o the new structure is the absence of the resonance of the carbonyl carbon of the CH3CO Scheme 1. Synthesis of compounds 2-6.
The prepared thioureido acid 4 was applied for the synthesis of thiazoles 5a-e. The products 5a-e have been achieved by a reaction of thioureido acid 4 and the corresponding bromoacetophenone in water, with the presence of sodium carbonate in the reaction mixture. The 1 H NMR spectra of these compound singlets, in the range of 7.07-7.42 ppm ( 13 C, 102.09-106.41 ppm), and the additional peaks in the aromatic region, confirm the presence of a 4-arylthiazole moiety.
The next goal of this study was the transformation of the acetamide fragment to an amine group. Reactions occurred easily and rapidly and were completed after refluxing in aqueous 5% hydrochloric acid for 1 h. Products were isolated by neutralizing the reaction mixtures with sodium acetate to pH 6. By careful assignment of the peaks in the 1 H and 13 C NMR spectra, the structures 6 were elucidated. Comparison of the spectra of compounds 5 and 6 showed obvious differences, i.e., the spectra of the latter compounds do not contain singlets of the methyl group of the acetamide moiety, but broad singlets of amino group are visible at approximately 5.35 ppm. In addition, the additional proof of the new structure is the absence of the resonance of the carbonyl carbon of the CH 3 CO fragment in the 13 C NMR spectra of compounds 6, which are clearly visible in the analogous spectra of derivatives 5 at approximately 168.45 ppm.
To obtain thiazolone derivative 7, a ring-closure reaction was carried out where thioureido acid 4 was reacted with monochloroacetic acid in an aqueous sodium carbonate solution, where acidification to pH 6 after completion of the reaction produced the target compound 7 (Scheme 2). The structure of 7 was approved based on the data of elemental analysis and NMR as well as IR spectroscopy. fragment in the 13 C NMR spectra of compounds 6, which are clearly visible in the analo gous spectra of derivatives 5 at approximately 168.45 ppm.
To obtain thiazolone derivative 7, a ring-closure reaction was carried out where thioureido acid 4 was reacted with monochloroacetic acid in an aqueous sodium carbonate solution, where acidification to pH 6 after completion of the reaction produced the targe compound 7 (Scheme 2). The structure of 7 was approved based on the data of elementa analysis and NMR as well as IR spectroscopy. Scheme 2. Synthesis of thiazolone derivatives 7-9.
The condensation of the obtained thiazolone 7 with various aromatic aldehydes was performed under analogous conditions, such as synthesis, instead of the monochloroace tic acid using the corresponding aromatic aldehydes. The reactions afforded 5-[(substi tuted phenyl)methylidene] thiazolones 8a-d. The spectra of compounds 8 do not contain the proton singlet of the SCH2, which arises at 3.91 ( 1 H) and 40.59 ( 13 C) ppm in the NMR spectra for compound 7. In the structure of compounds 8, a = CHAr moiety is attached to the 5-position of the thiazole ring. The NMR spectra of formed molecules 8 show obvious differences in comparison with the NMR spectra of 7. The increase in spectral lines in the aromatic region of the 1 H (in the interval of 7.59-7.61 ppm for CH and in the range of 7.21-7.63 ppm for HAr) and 13 C (observed in the interval of 129−134 ppm) NMR spectra corre spond to the number of hydrogen and carbon atoms of the new fragment. Then, 3-((4 aminophenyl)(4-(4-chlorobenzylidene)-5-oxo-4,5-dihydrothiazol-2-yl)amino)propanoic acid (9) was obtained by the deacetylation of compound 8c in the aqueous 5% hydrochlo ric-acid solution. The formed amino group was confirmed by a broad singlet, which was visible at 5.61 ppm.
Next, 2-thioxotetrahydropyrimidinedione 3 was used to prepare derivative 10 with an amino group in its structure. For this reason, compound 3 was refluxed in an aqueous 5% HCl solution. Product 10 was isolated by neutralizing the reaction mixture with sodium acetate to pH 6.
The obtained product 10 was used for the preparation of thioureido acid 11 (Scheme 3). For this purpose, compound 10 was dissolved in a hot, aqueous 5% sodium hydroxide solution, then filtered off and acidified with acetic acid to pH 6, to transfer sodium salt to the acidic form. Comparison of the spectra of compounds 3 and 10 showed that the single of the methyl group of the acetamide moiety is absent, but a broad singlet of an amino group is visible at 5.20 ppm. The condensation of the obtained thiazolone 7 with various aromatic aldehydes was performed under analogous conditions, such as synthesis, instead of the monochloroacetic acid using the corresponding aromatic aldehydes. The reactions afforded 5-[(substituted phenyl)methylidene] thiazolones 8a-d. The spectra of compounds 8 do not contain the proton singlet of the SCH 2 , which arises at 3.91 ( 1 H) and 40.59 ( 13 C) ppm in the NMR spectra for compound 7. In the structure of compounds 8, a = CHAr moiety is attached to the 5-position of the thiazole ring. The NMR spectra of formed molecules 8 show obvious differences in comparison with the NMR spectra of 7. The increase in spectral lines in the aromatic region of the 1 H (in the interval of 7.59-7.61 ppm for CH and in the range of 7.21-7.63 ppm for H Ar ) and 13 C (observed in the interval of 129-134 ppm) NMR spectra correspond to the number of hydrogen and carbon atoms of the new fragment. Then, 3-((4-aminophenyl)(4-(4chlorobenzylidene)-5-oxo-4,5-dihydrothiazol-2-yl)amino)propanoic acid (9) was obtained by the deacetylation of compound 8c in the aqueous 5% hydrochloric-acid solution. The formed amino group was confirmed by a broad singlet, which was visible at 5.61 ppm.
Next, 2-thioxotetrahydropyrimidinedione 3 was used to prepare derivative 10 with an amino group in its structure. For this reason, compound 3 was refluxed in an aqueous 5% HCl solution. Product 10 was isolated by neutralizing the reaction mixture with sodium acetate to pH 6.
The obtained product 10 was used for the preparation of thioureido acid 11 (Scheme 3). For this purpose, compound 10 was dissolved in a hot, aqueous 5% sodium hydroxide solution, then filtered off and acidified with acetic acid to pH 6, to transfer sodium salt to the acidic form. Comparison of the spectra of compounds 3 and 10 showed that the singlet of the methyl group of the acetamide moiety is absent, but a broad singlet of an amino group is visible at 5.20 ppm. solution, where acidification to pH 6 after completion of the reaction produced the target compound 7 (Scheme 2). The structure of 7 was approved based on the data of elemental analysis and NMR as well as IR spectroscopy. The condensation of the obtained thiazolone 7 with various aromatic aldehydes was performed under analogous conditions, such as synthesis, instead of the monochloroacetic acid using the corresponding aromatic aldehydes. The reactions afforded 5-[(substituted phenyl)methylidene] thiazolones 8a-d. The spectra of compounds 8 do not contain the proton singlet of the SCH2, which arises at 3.91 ( 1 H) and 40.59 ( 13 C) ppm in the NMR spectra for compound 7. In the structure of compounds 8, a = CHAr moiety is attached to the 5-position of the thiazole ring. The NMR spectra of formed molecules 8 show obvious differences in comparison with the NMR spectra of 7. The increase in spectral lines in the aromatic region of the 1 H (in the interval of 7.59-7.61 ppm for CH and in the range of 7.21-7.63 ppm for HAr) and 13 C (observed in the interval of 129−134 ppm) NMR spectra correspond to the number of hydrogen and carbon atoms of the new fragment. Then, 3-((4aminophenyl)(4-(4-chlorobenzylidene)-5-oxo-4,5-dihydrothiazol-2-yl)amino)propanoic acid (9) was obtained by the deacetylation of compound 8c in the aqueous 5% hydrochloric-acid solution. The formed amino group was confirmed by a broad singlet, which was visible at 5.61 ppm.
Next, 2-thioxotetrahydropyrimidinedione 3 was used to prepare derivative 10 with an amino group in its structure. For this reason, compound 3 was refluxed in an aqueous 5% HCl solution. Product 10 was isolated by neutralizing the reaction mixture with sodium acetate to pH 6.
The obtained product 10 was used for the preparation of thioureido acid 11 (Scheme 3). For this purpose, compound 10 was dissolved in a hot, aqueous 5% sodium hydroxide solution, then filtered off and acidified with acetic acid to pH 6, to transfer sodium salt to the acidic form. Comparison of the spectra of compounds 3 and 10 showed that the singlet of the methyl group of the acetamide moiety is absent, but a broad singlet of an amino group is visible at 5.20 ppm.

Study of Cytotoxic Activity of Compounds 3-11
To explore the in vitro cytotoxicity of compounds 3-11, we used A549 human pulmonary endothelial cells and a MTT viability assay. We exposed the cells to the fixed concentration of 100 µM of each compound for 48 h and subsequently measured the viability. Compounds 3-11 demonstrated overall favorable properties with low cytotoxicity. Among all tested compounds, 6d, bearing 4-cianophenyl substituent in the thiazole ring, demonstrated the highest cytotoxic properties by decreasing the A549 viability to 87.2%. All compounds failed to reduce the A549 viability by 50%, suggesting good in vitro safety profiles ( Figure 1). Scheme 3. Synthesis of compounds 10 and 11.

Study of Cytotoxic Activity of Compounds 3-11
To explore the in vitro cytotoxicity of compounds 3-11, we used A549 hum monary endothelial cells and a MTT viability assay. We exposed the cells to the fix centration of 100 µ M of each compound for 48 h and subsequently measured the v Compounds 3-11 demonstrated overall favorable properties with low cytot Among all tested compounds, 6d, bearing 4-cianophenyl substituent in the thiazo demonstrated the highest cytotoxic properties by decreasing the A549 viability to All compounds failed to reduce the A549 viability by 50%, suggesting good in vitr profiles ( Figure 1). Figure 1. The in vitro cytotoxicity of compounds 3-11 on A549 human pulmonary cells. T cells were treated with 100 µ M of each compound or cisplatin (CP) that served as control and the post-treatment viability was measured by using MTT assay. The viability of untrea trol (UC) was used for the post-treatment-viability normalization. Data are shown as me from three experimental replicates.

Study of Antiviral Activity of Compounds 3-11
To explore the potential antiviral ability of synthesized compounds, we u MDCK influenza in vitro infection model [39,40]. Prior to infection, we pretrea MDCK cells with 100 µ M of each compound, or oseltamivir and amantadine that as antiviral controls. Compounds 3-11 showed structure-dependent antiviral against influenza A/Puerto Rico/8/34 H1N1 strain and were able to significantly (p restore MDCK viability in comparison to the untreated control (UC) (Figure 2 pounds 8d, 5e, 5d, and 6e showed the highest antiviral activity (p < 0.001) in com to UC. The antiviral activity of compounds 8d, 5e, 5d, and 6e at 100 µ M was sim greater than oseltamivir and amantadine. Furthermore, compound 5e, bearing 4 romethylphenyl substituent in the thiazole ring, showed significantly higher antiv tivity (p < 0.0162) than oseltamivir ( Figure 2). The in vitro cytotoxicity of compounds 3-11 on A549 human pulmonary cells. The A549 cells were treated with 100 µM of each compound or cisplatin (CP) that served as control for 48 h, and the post-treatment viability was measured by using MTT assay. The viability of untreated control (UC) was used for the post-treatment-viability normalization. Data are shown as mean ± SD from three experimental replicates.

Study of Antiviral Activity of Compounds 3-11
To explore the potential antiviral ability of synthesized compounds, we used an MDCK influenza in vitro infection model [39,40]. Prior to infection, we pretreated the MDCK cells with 100 µM of each compound, or oseltamivir and amantadine that served as antiviral controls. Compounds 3-11 showed structure-dependent antiviral activity against influenza A/Puerto Rico/8/34 H1N1 strain and were able to significantly (p < 0.05) restore MDCK viability in comparison to the untreated control (UC) (Figure 2). Compounds 8d, 5e, 5d, and 6e showed the highest antiviral activity (p < 0.001) in comparison to UC. The antiviral activity of compounds 8d, 5e, 5d, and 6e at 100 µM was similar or greater than oseltamivir and amantadine. Furthermore, compound 5e, bearing 4-trifluoromethylphenyl substituent in the thiazole ring, showed significantly higher antiviral activity (p < 0.0162) than oseltamivir ( Figure 2).
These data demonstrate that substituted aminothiazole derivatives are promising scaffolds for further optimization and the development of new compounds with potential influenza A-targeted antiviral activity.  After 24 h, the viability was measured using MTT assay. Uninfected control (UIC) cells were used as a comparison demonstrating the fully viable cells. * shows significant comparisons between test compounds and untreated control (UC), # shows significant comparisons between test compounds and oseltamivir, and Ψ shows significant comparisons between test compounds and amantadine. Statistical significance was tested with one-way ANOVA, and error bars show mean ± SD from three experiments. * p < 0.05, ** p < 0.0021, , **** p < 0.0001, ΨΨΨ p < 0.0001.
These data demonstrate that substituted aminothiazole derivatives are promising scaffolds for further optimization and the development of new compounds with potential influenza A-targeted antiviral activity.
Measuring the increasing absorption at 593 nm using a spectrophotometer monitors this reduction, and results are expressed as a Fe 2+ µmol/L concentration. [44].
Measuring the increasing absorption at 593 nm using a spectrophotometer monitors this reduction, and results are expressed as a Fe 2+ µmol/L concentration. [44].

Evaluation of Antibacterial Activity
The novel derivatives were screened for their in vitro antibacterial activity against Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive) bacteria strains. Antimicrobial tests were conducted using the agar well-diffusion method. Ciprofloxacin was used as the reference antibiotic for the in vitro antibacterial activity.
The results of the antibacterial study against E. coli ( Figure 6) illustrated that the com- Data are shown as mean ± SD from three experimental replicates.

Evaluation of Antibacterial Activity
The novel derivatives were screened for their in vitro antibacterial activity against Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive) bacteria strains. Antimicrobial tests were conducted using the agar well-diffusion method. Ciprofloxacin was used as the reference antibiotic for the in vitro antibacterial activity.

Evaluation of Antibacterial Activity
The novel derivatives were screened for their in vitro antibacterial activity against Escherichia coli (Gram-negative) and Bacillus subtilis (Gram-positive) bacteria strains. Antimicrobial tests were conducted using the agar well-diffusion method. Ciprofloxacin was used as the reference antibiotic for the in vitro antibacterial activity.

Discussion
It is important to develop an understanding of these novel aminothiazole compounds by integrating their synthesis and biological activities (antiviral, antioxidant, antibacterial). The present study results demonstrated that the compound 3-{(4-aminophenyl)[4-

Discussion
It is important to develop an understanding of these novel aminothiazole compounds by integrating their synthesis and biological activities (antiviral, antioxidant, antibacterial). The present study results demonstrated that the compound 3-{(4-aminophenyl)[4-(4-cianophenyl)-1,3-thiazol-2-yl]amino}propanoic acid (6d) showed the highest cytotoxic properties. Screening of the tested compounds for antiviral activity has revealed that the compounds 3-((4-aminophenyl){4-[4-(trifluoromethyl)phenyl]thiazol-2-yl}amino)propanoic acid (6e) and 3-{(4-acetamidophenyl)[4-(4-trifluoromethylphenyl)-1,3-thiazol-2-yl]amino} propanoic acid (5e) exhibited significant influenza A-targeted antiviral activity, in comparison with oseltamivir and amantadine. The principle of antioxidant activity is focused on the availability of electrons to neutralize any free radicals. Moreover, antioxidant activity is related to the nature of the hydroxylation pattern on the aromatic ring. Various antioxidant methods have been described to evaluate antioxidant properties of pharmaceuticals, antioxidants, and other bioactive samples [43]. The antioxidant bioassays should be based on the elucidation of the structure-antioxidant-activity relationship of the bioactive molecules. Screening of the reduction power of the bioactive compounds gives information not only about their reducing ability but also reveals their thermodynamic parameters. It was determined that thiazole compounds bearing 4-cyanophenyl 6d, 4-chlorophenyl 6c, and 4-trifluoromethylphenyl 6e substituents in the thiazole ring showed the highest 6d > 6c > 6e > BHT > 11 > 6a > 6b > 5c antioxidant-reducing activity. The compounds 8a, 8d, 8c, 8b, and 7 demonstrated the lowest antioxidant-reducing power. It could be concluded that aminothiazole compounds bearing 4-chlorophenyl 6c, 4-trifluoromethylphenyl 6e, phenyl 6a, 4-fluorophenyl 6b, and 4-cianophenyl 6d substituents in the thiazole ring exhibited the highest 6c > 6a > 6e > 6b > 6d > 11 > 5a > 5d > 5b > BHT reduced FRAP power. The model of scavenging the stable DPPH radical is a widely used method to evaluate the free-radical-scavenging ability of various samples. It is a stable nitrogen-centered free radical, the color of which changes from violet to yellow upon reduction by either the process of hydrogen or electron donation. It was determined that the thiazole compounds 6b > 11 > 6a > 4 > BHT showed the highest antioxidant activity by DPPH assay. The acquired results of the antibacterial activity showed that compounds bearing 4-cianophenyl 6d, 4-fluorophenyl 6b, 4-chlorophenyl 8c, 4-trifluoromethylphenyl 5e, and phenyl 6a substituents in the thiazole ring have been indicated as the most active antibacterial agents against E. coli. The compounds 6d = 6a = 6b = 8c = 5a = 5b = 5e > 5d = 5c = 8d showed the highest antibacterial activity against B. subtilis. The antibacterial activity of the novel compounds could be related with the existence of electron-withdrawing groups in the thiazole ring.

Synthesis of Novel Compounds
Melting points were determined on a MEL-TEMP (Electrothermal, A Bibby Scientific Company, Burlington, NJ, USA) melting point apparatus and are uncorrected. FT-IR spectra (ν, cm −1 ) were recorded on a Perkin-Elmer Spectrum BX FT-IR spectrometer (Perkin-Elmer Inc., Waltham, MA, USA) using KBr pellets. 1 H and 13 C-NMR spectra were recorded in DMSO-d 6 on a Bruker Avance III (400 MHz, 101 MHz and 700 MHz, 176 MHz) spectrometer. Chemical shifts (δ) are reported in parts per million (ppm) calibrated from TMS (0 ppm) as an internal standard for 1 H-NMR and DMSO-d 6 (39.43 ppm) for 13 C-NMR. The reaction course and purity of the synthesized compounds was monitored by TLC using aluminum plates coated with silica gel 60 F254 (MerckKGaA, Darmstadt, Germany). Reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Cytotoxicity Assay
The cytotoxicity of compounds 3-11 on A549 human lung cells were evaluated by using a commercial MTT assay (CyQUANT MTT Cell Viability Assay, Thermo Fisher Scientific, Eugene, Oregon, USA). A549 was plated to flat-bottomed 96-well plates (1 × 10 4 cells/well) and incubated overnight to facilitate the attachment. The compounds at fixed 100 µM concentration were added, and plates were further incubated for 48 h at 37 • C, 5% CO 2 . After incubation, the commercial MTT reagent was added, and the % of viability was determined, in accordance with the description of the manufacturer, using untreated cells as a control. All experiments were performed in triplicate.

Viral Infection Assay
To determine the potential antiviral activity of compounds 3-11 on the virus replication in MDCK cells, we used virus-induced cell death as an experimental output. Briefly, MDCK cells were plated to flat-bottomed 96-well plates (1 × 10 4 cells/well) and incubated overnight to facilitate the attachment. After incubation, the media was removed, cells were gently washed twice with DPBS, and the compounds (100 µM) were dissolved in DMEM supplemented with 5% bovine serum albumin (BSA), 2 µg/mL TPCK-treated trypsin (Thermo Fisher Scientific, Rockford, Illinois, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin. The oseltamivir and amantadine (100 µM) were used as a comparator. Cells were incubated with compounds for 1 h at 37 • C, 5% CO 2 , and were then infected with influenza A/Puerto Rico/8/34 H1N1 strain at MOI 1:5. The infected cells were then further incubated for 24 h to facilitate the infection, and the remaining post-infection viability was measured by using MTT assay.  25 mL, 1%). The mixture was incubated at 50 • C for 20 min. Aliquots (1.25 mL) of trichloroacetic acid (10%) were added to the mixture, which was then centrifuged for 10 min at 9000 rpm. The upper layer of solution (1.25 mL) was mixed with distilled water (1.25 mL) and FeCl 3 (0.25 mL, 0.1%), and the absorbance was measured at 700 nm in a spectrophotometer [47,48]. Butylhydroxytoluene (BHT) was used as a positive control.

Ferric Reducing Antioxidant Power Assay (FRAP)
Reducing properties were investigated using the FRAP method, which is based on the reduction of a ferric-tripyridyl triazine complex to its ferrous-colored form in the presence of antioxidants [49]. The FRAP reagent contained 2.5 mL of 10 mM TPTZ (2,4,6-tripyridyls-triazine) solution in 40 mM HCl as well as 2.5 mL of FeCl 3 (20 mM) and 25 mL of acetate buffer (0.3 M, pH = 3.6). Then, 100 µL of analyzed compounds (20 mM) were mixed with 3 mL of the FRAP reagent. The absorbance of the reaction mixture was measured spectrophotometrically at 593 nm. For comprising the calibration curve, five concentrations of FeSO 4 · 7H 2 O (5, 10, 15, 20, 25 µM) were used, and the absorbance was measured as a sample solution. Each experiment was repeated three times.

1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Assay
The free-radical-scavenging activity of 3-11 compounds was measured by the DPPH method [48,50]. Firstly, a solution (20 mM) of 3-11 compounds was prepared in DMSO. Then, a 1 mM solution of DPPH in ethanol was prepared, and 1 mL of this solution was added to the solutions of the analyzed compounds. The mixture was vigorously stirred and allowed to stand at room temperature. After 20 min, the absorbance of the reaction mixture was measured at 517 nm with a UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan). Each experiment was repeated three time.

Evaluation of Antibacterial Activity
Antibacterial activity of the compounds was screened by using the disk-diffusion method [51]. In this study, inhibition of bacterial growth was investigated against Grampositive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli. The solution (20 mM) of the compounds was prepared in DMSO. Bacterial cultures were cultivated in Petri dishes at 37 • C for 24 h on the Luria-Bertani (LB) agar medium. Then, 50 µL inoculum containing bacterial cells were spread across the LB agar medium. Sterile filter-paper disks were soaked in 25 µL of each compound solution, and then the disks were put on the LB agar medium. Ciprofloxacin (20 mM) was used as positive control, and DMSO was used as the negative control. Petri dishes were incubated aerobically at 37 • C and examined for zones of inhibition after 24 h. The inhibition zones (cm) were measured.