The pyrimidine core has attracted much attention from researchers. For chemists, pyrimidine is of interest due to the wide possibilities of structural variability [1–4], and for biologists, pyrimidine is a molecule with a wide spectrum of proven biological activity [5–8], which plays a unique role in the human body.

Among antiviral drugs, there are a number of compounds that include a pyrimidine (raltegravir, sofosbuvir, cidofovir), thiazole (nitazoxanide, tizoxanide, ritonavir) or phosphonate (foscavir) fragments (Scheme 1). At the same time, sofosbivur and cidofovir simultaneously combine pyrimidine and phosphorus-containing fragments in the molecule.

Scheme
scheme 1

1.

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The starting 6-aryl-5-cyano-2-thiouracils were obtained by the typical method based on a three-component reaction between thiourea, ethyl cyanoacetate, and aromatic aldehyde in methanol in the presence of sodium methoxide [9]. The yields of the obtained 6-aryl-5-cyano-2-thiouracils were 62–90% (Scheme 2).

Scheme
scheme 2

2.

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Previously, we have already carried out phosphonylation reactions of a number of 2-thiouracils. It was shown that the main route of the reaction with chloroethynylphosphonates is intramolecular cyclization leading to the formation of thiazolo[3,2-a]pyrimidines [10]. We found that, depending on the structure of the 2-thiouracils used, the formation of various cyclization products both with the participation of the N3 and N1 nitrogen atoms is possible. The introduction of a strong electron acceptor (CF3 group) into position 6 of the 2-thiouracil molecule directs the initial attack of chloroethynylphosphonate to the N3 nitrogen atom. Thus, the reactivity of 2-thiouracils is diverse, its study is topical and can lead to new unexpected results.

In the present work, we studied the features of the reaction of chloroethinylphosphonate with a number of 6-aryl-5-cyano-2-thiouracils, as well as studied the cytotoxicity and antiviral activity of the starting thiouracils and their phosphonylated derivatives. It was found that regardless of the aryl substituent nature the reaction of 6-aryl-5-cyano-2-thiouracils with diethylchloroethynylphosphonates proceeds with high chemo- and regioselectivity, leading to the formation of diethyl (6-cyano-5-oxo-7-aryl-5H-thiazolo[3,2-a]pyrimidin-3-yl)phosphonates 2а–2d in 62–90% yield (Scheme 3). Structure of the obtained compounds was confirmed by 1Н, 13С, and 31Р NMR spectroscopy data.

Scheme
scheme 3

3.

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Data on the antiviral activity of the obtained compounds against the influenza A virus, as well as their cytotoxicity against the MDCK cell culture are presented in Table. 1. It was found that the starting 6-aryl-5-cyano-2-thiouracils 1a–1d have low cytotoxicity. The introduction of a phosphonate group leads to some increase in cytotoxicity, which is especially evident when comparing the values for pairs of compounds 1a–2a and 1d–2d. In both cases, the cytotoxicity of the tested compounds increased. From the data presented in Table 1, it can be seen that the studied compounds do not have antiviral activity against the influenza virus under study. Compound 2b, containing a chlorine atom in the position 4 of the benzene ring, exhibited high activity. In previous studies [11], the most active compounds contained two chlorine atoms in the benzene ring, one of which was in position 4. These results allow us to make an assumption about the effect of this atom on the appearance of antiviral activity. Research on other viruses is ongoing.

Table 1. Antiviral activity of compounds 1a–1d and 2a–2d against influenza A virus and cytotoxicity against MDCK cell culture
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In conclusion, a series of new 6-aryl-5-cyano-2-thiouracils and their phosphonylated derivatives was synthesized. It was found that the reaction proceeds with high regio- and chemoselectivity, leading to the formation of a cyclization product with the participation of the N3 nitrogen atom. One of the obtained (5-oxo-7-aryl-6-cyano-5H-thiazolo[3,2-a]pyrimidin-3-yl)phosphonates showed high antiviral activity against influenza A (H1N1).

EXPERIMENTAL

1Н, 13С, and 31Р NMR spectra were taken on a Bruker Avance III HD 400 NanoBay spectrometer at frequencies of 400.17 (1H), 100.62 (13C), 161.98 MHz (31P). Mass spectra were registered on a Bruker micrOTOF instrument. Melting points were measured on a Kofler table (VEB Wägetechnik Rapido, PHMK 81/2969).

General procedure for the preparation of compounds 1a–1d. To a solution of 0.02 mol of sodium methoxide in 20 mL of anhydrous methanol was added 0.02 mol of thiourea, 0.02 mol of the corresponding aromatic aldehyde, and 0.02 mol of ethyl ester of cyanoacetic acid. The mixture was boiled for 12–18 h with stirring. After cooling, the mixture was poured into ice water and acidified with concentrated hydrochloric acid. The precipitate was filtered off, washed with cold ethanol, and recrystallized from ethanol.

4-Oxo-2-thio-6-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1a). Yield 88%, white crystals. 1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 7.38 d (2H, HAr, 3JHH 8.0), 7.57 d (2H, HAr, 3JHH 8.0), 13.14 s (1H, NH), 13.26 s (1H, NH). 13C NMR spectrum (DMSO-d6), δС, ppm: 30.73 (С14), 90.39 (С5), 114.89 (C7), 126.40 (CAr), 128.78 (CAr), 129.00 (CAr), 142.51 (CAr), 158.59 (C6), 160.93 (C4), 176.22 (C2).

6-(4-Chlorophenyl)-4-oxo-2-thio-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1b). Yield 83%, white crystals. 1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 7.68 m (4H, HAr), 13.19 s (1H, NH). 13C NMR spectrum (DMSO-d6), δС, ppm: 91.08 (С5), 114.56 (C7), 128.16 (CAr), 128.61 (CAr), 130.77 (CAr), 136.94 (CAr), 158.38 (C6), 159.91 (C4), 176.16 (C2).

6-(4-Bromophenyl)-4-oxo-2-thio-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1c). Yield 81%, white crystals. 1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 7.62 d (2H, HAr, 3JHH 8.5), 7.80 d (2H, HAr, 3JHH 8.5), 13.21 s (1H, NH), 13.37 s (1H, NH). 13C NMR spectrum (DMSO-d6), δС, ppm: 90.97 (С5), 114.64 (C7), 125.86 (CAr), 130.87 (CAr), 131.55 (CAr), 132.50 (CAr), 158.44 (C6), 160.07 (C4), 176.25 (C2).

6-(4-Carbomethoxy)-4-oxo-2-thio-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (1d). Yield 91%, white crystals. 1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 7.82 d (2H, HAr, 3JHH 8.0), 8.11 d (2H, HAr, 3JHH 8.0), 13.22 s (1H, NH), 13.45 s (1H, NH). 13C NMR spectrum (DMSO-d6), δС, ppm: 52.58 (С14), 91.28 (С5), 114.46 (C7), 129.08 (CAr), 129.33 (CAr), 132.48 (CAr), 158.36 (CAr), 160.09 (C6), 166.48 (C4), 176.22 (C2).

General procedure for the preparation of compounds 2a–2d. A mixture of 0.001 mol of diethylchloroethynylphosphonate, 0.001 mol of the corresponding 2-thiouracil 1a–1d, and 0.0012 mol of potassium carbonate in 10 mL of anhydrous acetonitrile was vigorously stirred at room temperature for 2–3 h. The reaction progress was monitored by 31P NMR spectroscopy. After the reaction completed, the mixture was filtered. The filtrate was evaporated in vacuum, the residue was recrystallized from ethyl acetate.

Diethyl {5-oxo-7-(p-tolyl)-6-cyano-5H-thiazolo[3,2-a]pyrimidin-3-yl}phosphonate (2a). Yield 79%, white crystals, mp 183°C. 1H NMR spectrum (DMSO-d6), δ, ppm (J, Hz): 1.31 t (6H, H18,20, 3JHH 7.1), 2.41 s (3H, H21), 4.21–4.23 m (4H, H17,19), 7.40 d (2H, HAr, 3JHH 8.1), 7.87 d (2H, HAr, 3JHH 8.1), 8.28 d (1H, H2, 3JHP 7.3). 13C NMR spectrum (DMSO-d6), δС, ppm (J, Hz): 16.21 d (C18,20, 3JСР 6.4), 21.10 (C21), 63.61 d (C17,19, 2JСР 6.0), 89.06 (C6), 116.14 (C10), 127.19 d (C3, 1JСР 216.6), 128.82 (CAr), 129.23 (CAr), 129.90 d (C2, 2JСР 13.9), 132.16 (CAr), 142.09 (CAr), 157.06 (C7), 165.89 (C5), 166.87 d (C9, 3JСР 9.6). 31P NMR spectrum (DMSO-d6): δР 0.48 ppm. Mass spectrum (HRMS-ESI), m/z: 404.0828 [M + H]+ (calcd for C18H18N3O4PS: 403.0832).

Diethyl {5-oxo-7-(4-chlorophenyl)-6-cyano-5H-thiazolo[3,2-a]pyrimidin-3-yl}phosphonate (2b). Yield 90%, white crystals, mp 202°C. 1H NMR spectrum (CDCl3), δ, ppm (J, Hz): 1.31 t (6H, H18,20, 3JHH 7.1), 4.21–4.23 m (4H, H17,19), 7.68 d (2H, HAr, 3JHH 8.6), 7.96 d (2H, HAr, 3JHH 8.6), 8.31 d (1H, H2, 3JHP 7.4). 13C NMR spectrum (CDCl3), δС, ppm (J, Hz): 16.20 d (C18,20, 3JСР 6.3), 63.64 d (C17,19, 2JСР 6.0), 89.69 (C6), 115.83 (C10), 127.26 (C3, 1JСР 216.4), 128.84 (CAr), 130.18 d (C2, 2JСР 14.4), 130.63 (CAr), 133.82 (CAr), 136.61 (CAr), 156.89 (C7), 164.83 (C5), 167.10 d (C9, 3JСР 9.5). 31P NMR spectrum (CDCl3): δР 0.41 ppm. Mass spectrum (HRMS-ESI), m/z: 446.0072 [M + Na]+ (calcd for C17H15ClN3O4PS: 423.01133).

Diethyl {7-(4-bromophenyl)-5-oxo-6-cyano-5H-thiazolo[3,2-a]pyrimidin-3-yl}phosphonate (2c). Yield 62%, white crystals, mp 225°C. 1H NMR spectrum (CDCl3), δ, ppm (J, Hz): 1.31 t (6H, H18,20, 3JHH 7.0), 4.21–4.23 m (4H, H17,19), 7.82 d (2H, HAr, 3JHH 8.6), 7.88 d (2H, HAr, 3JHH 8.6), 8.31 d (1H, H2, 3JHP 7.3). 13C NMR spectrum (CDCl3), δС, ppm (J, Hz): 16.22 d (C18,20, 3JСР 6.3), 63.66 d (C17,19, 2JСР 6.0), 89.68 (C6), 115.85 (C10), 125.58 (CAr), 127.26 d (C3, 1JСР 216.5), 130.20 d (C2, 2JСР 13.9), 130.79 (CAr), 131.80 (CAr), 134.20 (CAr), 156.90 (C7), 164.96 (C5), 167.14 d (C9, 3JСР 9.5). 31P NMR spectrum (CDCl3): δР 0.42 ppm. Mass spectrum (HRMS-ESI), m/z: 491.9376 [M + Na]+ (calcd for C17H15BrN3O4PS: 468.9575).

Diethyl {7-(4-carbomethoxyphenyl)-5-oxo-6-cyano-5H-thiazolo[3,2-a]pyrimidin-3-yl}phosphonate (2d). Yield 83%, white crystals, mp 207°C. 1H NMR spectrum (CDCl3), δ, ppm (J, Hz): 1.39 t (6H, H18,20, 3JHH 7.1), 3.93 s (3H, H21), 4.37–4.39 m (4H, H17,19), 7.97 d (1H, H2, 3JHP 7.4), 8.06 d (2H, HAr, 3JHH 8.2), 8.15 d (2H, HAr, 3JHH 8.2). 13C NMR spectrum (CDCl3), δС, ppm (J, Hz): 16.53 d (C18,20, 3JСР 6.4), 52.55 (C21), 64.70 d (C17,19, 2JСР 6.3), 91.65 (C6), 114.80 (C10), 127.64 d (C2, 2JСР 13.1), 129.18 (CAr), 129.61 d (C3, 1JСР 215.5), 129.89 (CAr), 133.06 (CAr), 138.67 (CAr), 156.72 (C7), 165.82 (C5), 166.02 d (C9, 3JСР 9.6). 31P NMR spectrum (CDCl3): δР –0.39 ppm. Mass spectrum (HRMS-ESI), m/z: 470.0360 [M + Na]+ (calcd for C18H18N3O5PS: 447.0552).

Cytotoxicity assay. The preparations were diluted in a maintenance medium for MDCK cells. Maintenance medium composition: 1 mL of antibiotic solution (ciprofloxacin, Sintez, Kurgan) and 0.1 mL of TPCK-trypsin solution (final concentration in the medium was 2 μg/mL, catalog no. T1426, Sigma, Germany). A series of twofold serial dilutions of preparations was prepared (1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.8 μg/mL, respectively).

One-day culture of MDCK cells grown on 96-well plates (Nunc, Denmark), with cell concentration of 104/well plate was checked visually in an inverted microscope for the monolayer integrity. Plates were selected for work, where the cell density was 95% or more. The plates were washed twice with warm serum-free alpha-MEM medium, after which dilutions of the tested samples at the appropriate concentration in a volume of 100 μL per well were applied to the cells of the monolayer in the plate in 3 repetitions for each tested concentration. The plates were incubated for 24 h at 37°C in the presence of 5% CO2. At the final term, the result was evaluated visually in an inverted microscope, assessing the state of the monolayer in the presence of different concentrations of the tested compound compared to cells in control wells.

Cell viability was assessed using the microtetrazolium test (MTT). The MTT solution was prepared in physiological saline at a concentration of 0.5 mg/mL. Before adding the MTT solution, the cells were washed with 0.1 mL of physiological saline. Next, 0.1 mL of MTT solution was added to each well. After 1.5 h of contact with MTT at 37°C at a CO2 concentration of 5%, the wells were washed and filled with 0.1 mL of ethyl alcohol (96%), after which the optical density in the wells was measured on a Victor 2 1440 reader (PerkinElmer, USA) at 535 nm. Based on the obtained data, CC50 was calculated, i.e., the dose of the drug in the well, at which 50% of cells die, using the method of non-linear regression.

Antiviral activity assay. MDCK cells (c 3×105 cells/mL) were seeded at 100 μl/well into 96LP flat bottom plates and incubated for 24 h at 37°C (5% CO2). After 24 h, upon reaching 100% confluence of the monolayer, the cells were washed once with maintenance medium at c 100 μL/well. For the analyzed substances, a series of threefold dilutions was prepared so that the maximum concentration corresponded to CC50, after which a dilution of the influenza virus strain A/PR/8/34 (H1N1) corresponding to the multiplicity of infection (m.o.i.) of 1 was prepared. Next, 200 µL of the appropriate dilution of the drug at a double concentration was mixed with 200 µL of the virus dilution; for virus control, 200 µL of the maintenance medium was used instead of diluting the drug. This mixture was added in 100 µL to 96LP wells in 4 replications for each dilution of the drug and incubated for 1 h at 37°C (5% CO2). After an hour, the cells were washed once with maintenance medium at 100 μL/well, and then 100 μL of the drug at the appropriate concentration was added to the wells, except for the wells with the virus control. Next, the cells were incubated for 24 h at 37°C (5% CO2). After 24 h, culture fluid was withdrawn from each well and one total run was made for each drug concentration and also virus control. Next, a series of 10-fold dilutions of each stock from 10–1 to 10–7 was prepared, 100 µL was added to the wells of 96LP containing daily culture of MDCK cells, 2 replications for each dilution, and incubated for 72 h at 37°C (5% CO2). The virus titer was determined using the hemagglutination test (HHA). After 72 h, 90 µL of the culture medium was taken and added to a 96LP round bottom plate for immunological reactions (Medpolymer, RF). Then, 90 µL of a 1% suspension of chicken erythrocytes in physiological saline was added to the same round-bottom plate and left at room temperature for 40 min. After this time, the results were recorded. Based on the data obtained, the IC50 was calculated (the dose of the drug at which the virus titer is halved).

Based on the results of experiments to determine the cytotoxicity and antiviral activity of the compounds, such an indicator as SI (chemotherapeutic index) was calculated, equal to the ratio of CC50 to IC50. Drugs with an SI of more than 10 are considered promising.