The Evolution of Pleconaril: Modified O-Alkyl Linker Analogs Have Biological Activity towards Coxsackievirus B3 Nancy

Coxsackieviruses type B are one of the most common causes of mild upper respiratory and gastrointestinal illnesses. At the time of writing, there are no approved drugs for effective antiviral treatment for Coxsackieviruses type B. We used the core-structure of pleconaril, a well-known antienteroviral drug candidate, for the synthesis of novel compounds with O-propyl linker modifications. Some original compounds with 4 different linker patterns, such as sulfur atom, ester, amide, and piperazine, were synthesized according to five synthetic schemes. The cytotoxicity and bioactivity of 14 target compounds towards Coxsackievirus B3 Nancy were examined. Based on the results, the values of 50% cytotoxic dose (CC50), 50% virus-inhibiting dose (IC50), and selectivity index (SI) were calculated for each compound. Several of the novel synthesized derivatives exhibited a strong anti-CVB3 activity (SI > 20 to > 200). These results open up new possibilities for synthesis of further new selective anticoxsackievirus compounds.


Introduction
Enteroviruses belonging to the Picornaviridae family are a diverse group of small (30-32 nm size) icosahedral non-enveloped viruses with single-stranded non-segmented positive RNA genome with a poly(A) tail. They are able to survive in harsh environments and can cause both self-limiting infections as well as polio, hand-foot-mouth disease, and heart and central nervous system diseases [1]. Currently, the genus Enterovirus encompasses 15 species: Enterovirus A-L and Rhinovirus A-C. Coxsackieviruses type B (CVBs) are members of Enterovirus B species and include six serotypes (CVB1-6). CVBs are lytic viruses but persistent infection responsible for chronic inflammation within target organs can be established. CVB3 often leads to mild upper respiratory and gastrointestinal illnesses, but it can also cause myocarditis [2,3]. Viral myocarditis is usually associated with dyspnea, arrhythmia, and chest pain and can lead to acute heart failure and sudden death. Currently available treatment is supportive and focuses on the symptomatic factors of disease [4,5].
To date, there are no approved antiviral agents for effective therapy of CVB3 infections. Currently the most advanced approaches for anti-CVB drug design are focused on the search for new direct antivirals, the modification of existing antiviral compounds, and drug repurposing screening [6]. 2 of 18 Pleconaril, a well-known antienteroviral drug candidate with the capsid-binding mechanism of action, does not cover all of the Coxsackievirus B serotypes, including the typical representative, Coxsackievirus B3 Nancy, which is explored in this article [7][8][9].
Previously, we have reported that pleconaril resistance was overcome by unsubstituted analogues or by monosubstitution in the central phenyl ring [10]. In our most recent work, we showed the impact of the substitution pattern in the isoxazole and phenyl rings of the pleconaril core structure and their effect on antiviral activity [11]. The most active compound to date contains the 3-N,N-dimethylcarbamoyl group in the isoxazole ring and the 3-methyl group in the phenyl ring ( Figure 1).
It is interesting to investigate how the replacement of the alkyl linker with different substituents affects the antienteroviral activity, because in the original pleconaril research project, G.D. Diana et al. varied the length of the aliphatic chain only [12,13]. Thus, in the present article, we have continued our investigation to study the influence of the pleconaril core structure and various modifications on the observed antiviral activity. We synthesized compounds with the general structure shown in Figure 1, which have the N,N-dimethylcarbamoyl or ethoxycarbonyl or methyl (like pleconaril) groups in the isoxazole ring and the 3-methyl group in phenyl ring, and then examined their inhibition activity against Coxsackievirus B3 Nancy.
Molecules 2020, 25, x FOR PEER REVIEW 2 of 18 [6]. Pleconaril, a well-known antienteroviral drug candidate with the capsid-binding mechanism of action, does not cover all of the Coxsackievirus B serotypes, including the typical representative, Coxsackievirus B3 Nancy, which is explored in this article [7][8][9]. Previously, we have reported that pleconaril resistance was overcome by unsubstituted analogues or by monosubstitution in the central phenyl ring [10]. In our most recent work, we showed the impact of the substitution pattern in the isoxazole and phenyl rings of the pleconaril core structure and their effect on antiviral activity [11]. The most active compound to date contains the 3-N,Ndimethylcarbamoyl group in the isoxazole ring and the 3-methyl group in the phenyl ring ( Figure 1).
It is interesting to investigate how the replacement of the alkyl linker with different substituents affects the antienteroviral activity, because in the original pleconaril research project, G.D. Diana et al. varied the length of the aliphatic chain only [12,13]. Thus, in the present article, we have continued our investigation to study the influence of the pleconaril core structure and various modifications on the observed antiviral activity. We synthesized compounds with the general structure shown in Figure 1, which have the N,N-dimethylcarbamoyl or ethoxycarbonyl or methyl (like pleconaril) groups in the isoxazole ring and the 3-methyl group in phenyl ring, and then examined their inhibition activity against Coxsackievirus B3 Nancy.

Results and Discussion
To explore the impact of the O-alkyl linker modification we, firstly, exchanged an oxygen atom for a sulfur atom with conservation of the 3-carbon chain; secondly, we introduced an ester or amide group into the linker; finally, we completely replaced the alkyl linker with piperazine. Syntheses of the compounds are presented in Schemes 1-4.
In the Scheme 1, the initial benzonitrile 1 was reacted with 5-chloro-1-pentyne in the presence of potassium carbonate and potassium iodide in NMP to produce pentynylthiobenzonitrile 2. The reaction of 2 with excess of hydroxylamine hydrochloride and potassium carbonate in absolute refluxing ethanol provided amidoxime 3 with a yield of 92%. Cyclization into 1,2,4-oxadiazole 4 was carried out using treatment of 3 with trifluoroacetic anhydride in pyridine. The cycloaddition of the S-pentyn-1 linker in 4 and 2-chloro-2-(hydroxyimino)acetic acid ethyl ester 5 or commercial acetaldoxime resulted in the target compounds 6a,b with yields of 40% and 32%, respectively. Finally, (carbethoxy-isoxazolyl)propyl)thio)phenyl)oxadiazole 6a reacted with dimethylamine solution in order to synthesize 7 with a yield of 57%.

Results and Discussion
To explore the impact of the O-alkyl linker modification we, firstly, exchanged an oxygen atom for a sulfur atom with conservation of the 3-carbon chain; secondly, we introduced an ester or amide group into the linker; finally, we completely replaced the alkyl linker with piperazine. Syntheses of the compounds are presented in Schemes 1-4.
In the Scheme 1, the initial benzonitrile 1 was reacted with 5-chloro-1-pentyne in the presence of potassium carbonate and potassium iodide in NMP to produce pentynylthiobenzonitrile 2. The reaction of 2 with excess of hydroxylamine hydrochloride and potassium carbonate in absolute refluxing ethanol provided amidoxime 3 with a yield of 92%. Cyclization into 1,2,4-oxadiazole 4 was carried out using treatment of 3 with trifluoroacetic anhydride in pyridine. The cycloaddition of the S-pentyn-1 linker in 4 and 2-chloro-2-(hydroxyimino)acetic acid ethyl ester 5 or commercial acetaldoxime resulted in the target compounds 6a,b with yields of 40% and 32%, respectively. Finally, (carbethoxy-isoxazolyl)propyl)thio)phenyl)oxadiazole 6a reacted with dimethylamine solution in order to synthesize 7 with a yield of 57%. 2-Methyl-4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)phenyl ester of 3-(3-substituted isoxazol-5yl)propionic acids 12a,b were synthesized according to Scheme 2. Because of the ester hydrolytic instability, synthesis of these compounds was started with formation of 1,2,4-oxadiazole cycle. Phenyloxadiazole 10 was successfully obtained via standard procedure (Scheme 1) from amidoxime 9 with a yield of 43%. The Steglich esterification of phenol 10 with 4-pentynoic acid provided 11 in 72%. In the final stage, treatment of pentynoate 11 by 2-chloro-2-(hydroxyimino)acetic acid ethyl ester 5 or acetaldoxime led to target compounds 12a,b with low yields. Unfortunately, the reaction of 12a with non-water dimethylamine solution did not provide the corresponding product 13. In this case, the C-O bond of the ester breaks rapidly, forming 10, for which the structure was confirmed by MS and NMR data. 2-Methyl-4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)phenyl ester of 3-(3-substituted isoxazol-5-yl)propionic acids 12a,b were synthesized according to Scheme 2. Because of the ester hydrolytic instability, synthesis of these compounds was started with formation of 1,2,4-oxadiazole cycle. Phenyloxadiazole 10 was successfully obtained via standard procedure (Scheme 1) from amidoxime 9 with a yield of 43%. The Steglich esterification of phenol 10 with 4-pentynoic acid provided 11 in 72%. In the final stage, treatment of pentynoate 11 by 2-chloro-2-(hydroxyimino)acetic acid ethyl ester 5 or acetaldoxime led to target compounds 12a,b with low yields. Unfortunately, the reaction of 12a with non-water dimethylamine solution did not provide the corresponding product 13. In this case, the C-O bond of the ester breaks rapidly, forming 10, for which the structure was confirmed by MS and NMR data. 2-Methyl-4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)phenyl ester of 3-(3-substituted isoxazol-5yl)propionic acids 12a,b were synthesized according to Scheme 2. Because of the ester hydrolytic instability, synthesis of these compounds was started with formation of 1,2,4-oxadiazole cycle. Phenyloxadiazole 10 was successfully obtained via standard procedure (Scheme 1) from amidoxime 9 with a yield of 43%. The Steglich esterification of phenol 10 with 4-pentynoic acid provided 11 in 72%. In the final stage, treatment of pentynoate 11 by 2-chloro-2-(hydroxyimino)acetic acid ethyl ester 5 or acetaldoxime led to target compounds 12a,b with low yields. Unfortunately, the reaction of 12a with non-water dimethylamine solution did not provide the corresponding product 13. In this case, the C-O bond of the ester breaks rapidly, forming 10, for which the structure was confirmed by MS and NMR data.  At first, we thought of using the same sequence of steps described above (Schemes 1 and 2) for the synthesis of 2-methyl-4-(5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl)phenylamide of 3-(3-substituted isoxazol-5-yl)propionic acids 20a,b and 21, but this was not possible for two reasons. The first reason was asphaltization of the product as a result of the amidoxime formation reaction. Yet another reason was the reaction of trifluoroacetic anhydride with the free amino group of 4-amino-N'-hydroxy-3-methylbenzimidamide at the second 1,2,4-oxadiazole cyclization stage. Therefore, another approach was designed.
N-Boc-protected aminobenzonitrile 15 was obtained from reaction of 4-amino-3-methylbenzonitrile 14 with Boc 2 O in non-solvent conditions with a yield of 93%. The consistent treatment of 15 with hydroxylamine hydrochloride and trifluoroacetic anhydride provided oxadiazole 17. For selective cleavage of the N-Boc group, compound 17 was treated by trifluoroacetic acid to give aniline 18 with a yield of 99%. The reaction of 18 with 4-pentynoic acid in the presence of EDCl as dehydrating agent, DMAP as catalyst in the medium of DCM provided pentynamide 19. Final compounds 20a,b were synthesized as described above (Schemes 1 and 2). Unlike the ester (Scheme 2), the amide group is more hydrolytically stable. Treatment of 20a with dimethylamine solution prepared the corresponding product 21 with a yield of 63%.
Two different ways for the synthesis of piperazine derivatives were developed as indicated in Schemes 4 and 5. The initial Scheme 4 was designed based on intermediate 22, which was coupled with two isoxazoles, and in the final stages, 1,2,4-oxadiazole formation was provided. Scheme 5 was developed in order to improve Scheme 4, and in this case, the synthesis of target piperazine derivatives was dependent on the key intermediate, compound 34, which was especially prepared for these goals.
The couplings of piperazinylbenzonitrile 22 and isoxazoles 23 or 24 in the presence of potassium carbonate in refluxing acetonitrile give the corresponding compounds 25a,b with yields of 40% and 56%, respectively. In the case of derivative 25a, having the carbethoxy group in the isoxazole, it was not possible to carry out the reaction of amidoxime formation directly, in our opinion, due to hydrolysis of the ester. Therefore, the ester group was replaced with the amide group using dimethylamine solution to provide 26. In the case of 25b, direct reaction with hydroxylamine hydrochloride successfully provided amidoxime 27b. Finally, oxadiazole cyclization with the formation of piperazine derivatives 28a,b took place under typical conditions (Schemes 1-3).  In another approach, 1-(4-bromo-2-methylphenyl)piperazine 29 was N-protected with acetic anhydride to yield 30. The bromine atom in 30 was exchanged to a cyano group by copper(I) cyanide in NMP at high temperature in accordance with the procedure [14]. Benzonitrile 31 was treated in two steps under typical conditions for 1,2,4-oxadiazole cyclization (Schemes 1-4) to provide compound 33 with a yield of 62%. For selective cleavage of the acetyl group, 33 was worked up with  In another approach, 1-(4-bromo-2-methylphenyl)piperazine 29 was N-protected with acetic anhydride to yield 30. The bromine atom in 30 was exchanged to a cyano group by copper(I) cyanide in NMP at high temperature in accordance with the procedure [14]. Benzonitrile 31 was treated in two steps under typical conditions for 1,2,4-oxadiazole cyclization (Schemes 1-4) to provide compound 33 with a yield of 62%. For selective cleavage of the acetyl group, 33 was worked up with In another approach, 1-(4-bromo-2-methylphenyl)piperazine 29 was N-protected with acetic anhydride to yield 30. The bromine atom in 30 was exchanged to a cyano group by copper(I) cyanide in NMP at high temperature in accordance with the procedure [14]. Benzonitrile 31 was treated in two steps under typical conditions for 1,2,4-oxadiazole cyclization (Schemes 1-4) to provide compound 33 with a yield of 62%. For selective cleavage of the acetyl group, 33 was worked up with hydrochloric acid in ethanol to give ((piperazinyl)phenyl)oxadiazole 34 in 65%. While working on piperazine analogue synthesis, we came across an article in which compounds with similar structural fragments like ours (the phenyl ring is bound to piperazine, which through a carbonyl group is bound to isoxazole) were studied against influenza virus A [15]. It was interesting to investigate this structural fragment in the skeleton of our compound. For this purpose, compound 34 was coupled with commercially available 3-phenylisoxazole-5-carboxylic acid or 5-methyl-3-phenylisoxazole-4-carboxylic acid in the presence of EDCl, DMAP, and DCM to obtain the products 35a,b with yields of 44% and 38%, respectively. Moreover, 34 was reacted with isoxazole 23 to provided 28c in 80%. Finally, 34 was successively treated with propargyl bromide and 2-chloro-2-(hydroxyimino)acetic acid methyl ester 36 to give 28d. The structures of the all derivatives were characterized by 1 H-NMR spectroscopy and mass spectrometry.
Molecules 2020, 25, x FOR PEER REVIEW 6 of 18 hydrochloric acid in ethanol to give ((piperazinyl)phenyl)oxadiazole 34 in 65%. While working on piperazine analogue synthesis, we came across an article in which compounds with similar structural fragments like ours (the phenyl ring is bound to piperazine, which through a carbonyl group is bound to isoxazole) were studied against influenza virus A [15]. It was interesting to investigate this structural fragment in the skeleton of our compound. For this purpose, compound 34 was coupled with commercially available 3-phenylisoxazole-5-carboxylic acid or 5-methyl-3-phenylisoxazole-4carboxylic acid in the presence of EDCl, DMAP, and DCM to obtain the products 35a,b with yields of 44% and 38%, respectively. Moreover, 34 was reacted with isoxazole 23 to provided 28c in 80%. Finally, 34 was successively treated with propargyl bromide and 2-chloro-2-(hydroxyimino)acetic acid methyl ester 36 to give 28d. The structures of the all derivatives were characterized by 1 H-NMR spectroscopy and mass spectrometry. The final compounds 6a,b, 7, 12a,b, 20a,b, 21, 28a,b, 35a,b, and 28c,d were tested for their activity against Coxsackievirus B3 Nancy in the viral yield reduction assay. Derivatives 35a,b were also tested for anti-influenza A activity. Based on the results obtained, 50% cytotoxic concentration (CC50) and 50% inhibiting concentration (IC50) were calculated for each compound. The selectivity index was calculated for each compound as a ratio of CC50 to IC50. The biological results are summarized in Table 1 and Table 2 below. Pleconaril was used for comparison. Table 1. Antiviral activity of synthesized compounds towards coxsackievirus B3 strain Nancy.  6a,b, 7, 12a,b, 20a,b, 21, 28a,b, 35a,b, and 28c,d were tested for their activity against Coxsackievirus B3 Nancy in the viral yield reduction assay. Derivatives 35a,b were also tested for anti-influenza A activity. Based on the results obtained, 50% cytotoxic concentration (CC 50 ) and 50% inhibiting concentration (IC 50 ) were calculated for each compound. The selectivity index was calculated for each compound as a ratio of CC 50 to IC 50 . The biological results are summarized in Tables 1 and 2 below. Pleconaril was used for comparison. against Coxsackievirus B3 Nancy in the viral yield reduction assay. Derivatives 35a,b were also tested for anti-influenza A activity. Based on the results obtained, 50% cytotoxic concentration (CC50) and 50% inhibiting concentration (IC50) were calculated for each compound. The selectivity index was calculated for each compound as a ratio of CC50 to IC50. The biological results are summarized in Table 1 and Table 2 below. Pleconaril was used for comparison.  When the carbamoyl group was introduced instead of the O-linker (20a,b), the antiviral activity was reduced again. However, for compound 21, this change was reflected positively: this derivative has a good IC 50 (6.8 µM) and, subsequently, the highest selectivity index (248) in this series.
Finally, we examined the impact of the propyl linker substitution to piperazine on viral inhibition. Derivatives without 1-carbon chain between piperazine and isoxazole cycles, i.e., piperazine linked to isoxazole directly, having the dimethylcarbamoyl group 28a, are more active (SI = 205) than the same derivative with the carbethoxy group 28c (SI = 1). On the other hand, antiviral testing of derivatives with 1-carbon chain 28b and 28d exhibited inconclusive results: 28b has a poor IC 50 and good CC 50 values, whereas 28d showed the highest value of IC 50 in the series, but unfortunately 28d was cytotoxic (CC 50~2 03.8 µM). Derivative 35b with R 1 = 5-methyl-3-phenylisoxazole-4-yl has a selectivity index like the reference compound (SI = 125 for 35b and 130 for pleconaril) and derivative 35a with R 1 = 3-phenylisoxazole-5-yl was inactive towards CVB3. Both of the compounds were inactive against the influenza virus A (see Table 2). The curves demonstrating the cytotoxic and virus-inhibiting properties of the most potent compounds in the series-6b, 21, 28a-are shown in Figure 2.
Molecules 2020, 25, x FOR PEER REVIEW 8 of 18 The curves demonstrating the cytotoxic and virus-inhibiting properties of the most potent compounds in the series-6b, 21, 28a-are shown in Figure 2.

General Information
All reagents and solvents were purchased from commercial suppliers and used without further purification. 1 H and 13 C spectra were measured on a Bruker AC-300 (300 MHz, 1 H) or Bruker AC-200 (50 MHz, 13 C). Chemical shifts were measured in DMSO-d 6 or CDCl 3 , using tetramethylsilane as an internal standard. The following abbreviations are used to indicate the multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet; dd, doublet of doublets; td, triplet of doublets; dt, doublet of triplets; ddd, doublet of doublet of doublets; bs, broad signal. Mass spectra were obtained on a Finnigan SSQ-700 with direct injection. A Waters Micromass ZQ detector was used in EI MS for identification of various products. Melting points were determined on Electrothermal 9001 (10 • C per min) and are uncorrected. Merck silica gel 60 F254 plates were used for analytical TLC; column chromatography was performed on Merck silica gel 60 (70-230 mesh).

General Procedure for the Synthesis of Compounds 6a, 12a, 20a, 28d
To a solution of 5 or 36 (3 mmol) in DMF, a solution of corresponding compounds 4, or 11, or 19, or 37 in DMF for 20-30 min was added and stirred at rt for 40-50 min. To the reaction solution, Et 3 N (3 mmol) in DMF was added at 80-90 • C for 2 h. The mixture was stirred at 80-90 • C for 1-2 h and at rt for 12 h. The reaction mixture was diluted with water and extracted with ethyl acetate (3-times). The combined organic phases were washed with water (3-times), dried over anhydrous Na 2 SO 4 , and concentrated in vacuo. The residue was treated by water and stored in the refrigerator for 2-4 h. Crystals were collected and recrystallized from the corresponding solvent (in parentheses following mp data).    49.87, 52.15, 52.81, 52.81, 53.11, 104.05, 115.34 (q, J = 273.2), 118.14, 120.03, 127.67, 128.57, 129.85, 150.23,  159.12, 160.31, 166.05, 166.14, 167.32 (q, J = 43.3) ppm. 6b, 12b, 20b To a solution of NCS (2.5 mmol) and 1-2 drops of pyridine in DMF, a solution of acetaldoxime (2.5 mmol) in DMF was added for 30 min and stirred at rt for 1 h; then, to the solution, a solution of 4, or 11, or 19 (1 mmol) in DMF was added for 20 min. To the resulted mixture, Et 3 N (2.5 mmol) in DMF was added at 80-90 • C for 1 h and stirred at 80-90 • C for 3-4 h. The reaction mixture was diluted with water and extracted with ethyl acetate (3 times). The combined organic phases were washed with water (3 times), dried over anhydrous Na 2 SO 4 , and concentrated in vacuo. The residue was recrystallized from the corresponding solvent (in parentheses following mp data).

General Procedure for the Synthesis of Compounds 7, 21, 26
A mixture of 6a, or 20a, or 25a and dimethylamine solution 17 wt.% in dioxane was heated at 50-60 • C for 1-12 h. The cooled reaction mixture was concentrated in vacuo. The residue was treated by water and stored in the refrigerator for 12 h. Crystals were collected and recrystallized from the corresponding solvent (in parentheses following mp data). To a solution of 18 (1 mol) and DMAP (1.3 mol) in DCM, 4-pentynoic acid (1 mol) was added in one portion, followed by the addition of EDCI (1.3 mol) in one portion at rt. The reaction mixture was stirred at rt overnight, after which it was washed successively with 3% aq. HCl and water (3 times

General Procedure for the Synthesis of Compounds 25a,b, 28c
A mixture of the corresponding phenyl-piperazine (1 mol), the corresponding isoxazole (1.4 mol), and finely divided K 2 CO 3 (3 mol) in acetonitrile was refluxed for 24-48 h. The hot mixture was filtered, and the remaining solids were washed with acetonitrile. The combined filtrates were concentrated in vacuo. The residue was recrystallized from ethanol.   To a solution of 33 (1 mmol) in EtOH, concentrated HCl (3 mL) was added. The reaction mixture was refluxed for 4 h. The mixture was dissolved in water, neutralized with saturated aq. NaHCO 3 , and extracted with EtOAc (3 times). The combined organics were dried (Na 2 SO 4 ) and concentrated in vacuo. The residue was purified by column chromatography (eluent CHCl 3 :MeOH To a solution of 34 (1 mol) and DMAP (1.3 mol) in DCM, the corresponding acid (1 mol) was added in one portion, followed by the addition of EDCI (1.3 mol) in one portion at rt. The reaction mixture was stirred at rt overnight, after which it was washed successively with 3% aq. HCl and water (3 times). The organic layer was dried and concentrated in vacuo. The residue was triturated with water to provide the product as solid and recrystallized from ethanol. well). The plates were incubated at 37 • C in 5% CO 2 and observed daily for cytopathic effect (CPE). After 72 h, the viral titer was calculated in TCID 50 using the method of Reed and Muench.

Cytotoxicity Assay
The microtetrazolium test (MTT) was used to study the cytotoxicity of the compounds [24]. The experiment was repeated three times. Vero cells were seeded in 96-well plates in Eagles minimal essential medium (MEM) supplemented with 10% FBS. After 24 h, the media was removed, and the wells were washed with saline. Compounds were dissolved in DMSO, and a series of three-fold dilutions of each compound and pleconaril (1000-4 µg/mL) in MEM without FBS were prepared and added to the cells in quadriplicates (200 µL per well). The maximal concentration of DMSO was 0.5% MEM with 0.5% DMSO and was added to cell control wells. Cells were incubated for 24 h at 37 • C in 5% CO 2 in the presence of the dissolved compounds. The cells were then washed with saline, and a solution of 3-(4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide (ICN Biochemicals Inc., Aurora, OH, USA) (0.5 µg/mL) in MEM was added to the wells (100 µL per well). After 2 h of incubation at 37 • C in 5% CO 2 , the supernatant from wells was discarded, and the formazan residue was dissolved in DMSO (100 µL per well). The optical density of cells was then measured on a Victor 21,440 multifunctional reader (Perkin Elmer, Turku, Finland) at a wavelength of 535 nm and plotted against the concentration of the compounds to generate the dose-response curve. The 50% cytotoxic dose (CC 50 ) of each compound (i.e., the compound concentration that causes the death of 50% of cells in a culture, or decreases the optical density twice as compared to the control wells) was calculated using four-parameter logistic nonlinear regression model. For some compounds (35a and 35b) cytotoxicity towards MDCK cell line was determined using the procedure above, but the cells were exposed to compounds for 72 h.

Antiviral Activity Determination
Antiviral activity of the compound against CVB3 was evaluated using viral yield reduction assay. The experiment was repeated three times. Vero cells were seeded in MEM supplemented with 5% FBS in 24-well plates. When the cells confluence reached 100%, giving an approximate cell density of 0.2 × 10 6 per well, the compounds were dissolved in DMSO, and a series of three-fold non-toxic dilutions of each compound (600-6 µg/mL) and pleconaril (600-0.6 µg/mL) in MEM without FBS was prepared, added to the cells (500 µL per well), and incubated at 37 • C in 5% CO 2 . After 1 h, viral suspension in MEM without FBS was added to the all wells at MOI 0.01 (500 µL per well) except cell control, and the plates were incubated at 4 • C for 1 h. Thereafter, the cell supernatant was removed, and MEM without FBS was added to all wells (1 mL). After 24 h of incubation at 37 • C in 5% CO 2 , the viral progeny infectious titers (in TCID 50 ) for each compound concentration, cell control, and virus control wells were determined in Vero cells by endpoint dilution assay. The supernatants from corresponding wells of 24-plates were serially diluted in titer tubes and added to 96-well plates in duplicates (200 µL per well). The plates were incubated at 37 • C in 5% CO 2 and observed daily for cytopathic effect. After 72 h, the viral titer in each compound concentration, cell control, and virus control wells was calculated in TCID 50 using the method of Reed and Muench. The infectious titer of virus progeny was plotted against the concentration of the compounds to generate the dose-response curve. The 50% inhibition concentration (IC 50 ) of each compound tested (i.e., the compound concentration that decreases the infectious viral progeny titer twice as compared to the control wells) was calculated using four-parameter logistic nonlinear regression model. Selectivity index (SI) was calculated for each compound tested as a ratio of CC 50 to IC 50 values.
Antiviral activity of the compound against IAV was evaluated using hemagglutination test. The experiment was repeated three times. MDCK cells were seeded in MEM supplemented with 5% FBS in 96-well plates. When the cells confluence reached 100%, the plate was washed with saline, the tested compounds were dissolved in DMSO, and a series of three-fold non-toxic dilutions of each compound (600-6 µg/mL) in MEM without FBS was prepared and added to the cells (100 µL per well).
Tenfold dilutions of viral suspension in MEM without FBS were added to all wells (100 µL per well) except cell control, and the plates were incubated at 37 • C for 72 h. Thereafter, the cell supernatant (100 µL) was transferred to a "V bottom" 96-well microtiter plate and mixed with 100 µL of 1% chicken erythrocytes (RBC). After 1 h, cell control was checked for complete settling of RBCs, and the viral titer was determined. The 50% inhibitory concentration (IC 50 ) of each compound tested (i.e., the compound concentration that decreases the viral titer twice as compared to the control wells) was calculated using four-parameter logistic nonlinear regression model. Selectivity index (SI) was calculated for each compound tested as a ratio of CC 50 to IC 50 values.

Conclusions
We have synthesized a series of novel pleconaril-based compounds with modified O-alkyl linker. All the derivatives were characterized by their MS and NMR data. Synthesized target compounds were evaluated for their in vitro antiviral activity against coxsackievirus B3 strain Nancy. Among these compounds, 21 with an IC 50 value of 6.8 µM and SI of 248, is the most active anticoxsackievirus agent compared to other studied compounds (including pleconaril) with low cytotoxicity. The results of this study demonstrate the possibility to further improve pleconaril and evolve it to develop novel potent selective anti-coxsackievirus inhibitors that have activity against the B3 Nancy strain.