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Abstract

Background:

In continuation of our search for new anti-HIV and anti-HCV agents, the suggestion, synthesis and structure elucidation of a new series of 2,6-diamino-4-alkylthio- or (2-benzylhydrazinyl)-5-p-chlorophenylazopyrimidines), 2,6-diamino-4-(2-benzylhydrazinyl)-5-(aryl-[1,1′-biphenyl]-4-yl)pyrimidines, 2,6-diamino-4-(aryl)-5-(aryl[1,1′-biphenyl]-4-yl) pyrimidines), 6-(aryl)-1,3-dimethyl-5-nitro pyrimidine-2,4-dione and 6-amino-4-methoxy-N,N-dimethyl-6-arylpyrimidines were described.

Methods:

The anti-HIV-1 (strain III_B) and HIV-2 (strain ROD) activity of the newly synthesized pyrimidine analogues was evaluated in vitro in human MT-4 cells using the MT-4/MTT assay. Similarly, the same compounds were evaluated in vitro for their selective antiviral activity against HCV in the Huh 5–2 replicon system (type 1b, Con1 strain).

Results:

None of the tested compounds exhibited inhibition of HIV-1 and HIV-2 replication in cell culture. Even though many compounds yielded a 50% effective concentration in the HCV replicon system with selectivity indexes up to 6.9, none of the compounds matched the selection criteria of a selective inhibitor of virus replication in this assay (that is, >70% inhibition at concentrations that do not elicit an anti-metabolic effect on the host cells).

Conclusions:

Structural modification of these compounds might optimize their anti-HCV activity by introducing diverse and potent functional groups at the pyrimidine backbone, like nitrile residue. Because of the nature of the molecules, these new derivatives will also be evaluated for their potential anti-HIV activity.

Introduction

Pyrimidines are compounds that in vitro possess biological activity against a wide spectrum of unrelated viruses, such as poliovirus [1], herpes virus [2] and HIV [3 –5]. For the latter, two diarylpyrimidines, rilpivirine [6] (1; Figure 1) and etravirine [7,8] have been clinically approved for the treatment of people infected by HIV. These drugs have been classified as non-nucleoside reverse transcriptase inhibitors. Furthermore, pyrimidine-based compounds were shown to have cardiovascular effects [9,10], as well as diuretic [11,12], anti-bacterial and anti-tumour properties [13 –17]. Bacimethrin (=4-amino-5-(hydroxymethyl)-2-methoxypyrimidine) [18] (2; Figure 1), a naturally-occurring thiamine anti-metabolite that was isolated from Bacillus megatherium is active against several yeasts and bacteria in vitro. Anti-bacterial activity of Bacimethrin against Staphyloccocal infection has been reported in vivo [18]. Since 1980, methoprim, 5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine (3) [19,20] (Figure 1) is used, in combination with sulfamethoxazole, as a bacteriostatic antibiotic (co-trimoxazole) and mainly prescribed for the treatment of urinary tract infections and Pneumocystis jirovecii pneumonia [21].

Figure 1.

Compounds 1, 2 and 3

Hydrazine-pyrimidine-5-carbonitrile derivatives inhibit the growth of a wide range of cancer cell lines at concentrations as low as 10⁻⁷ M [22], whereas, 2,4-diamino-N⁴-6-diarylpyrimidines were identified to block the proliferation of tumour cell lines in vivo, especially duodenal cancer (DU145; 50% inhibitory concentration =0.23 μM) [15]. The dynamic behaviour of this compound scaffold in multiple biological systems encouraged us to further investigate their properties within the anti-infectives field [23]. In particular, the synthesis of 4-aryl-2,6-diaminopyrimidines is still largely unexplored, despite their potentially interesting agrochemical and medicinal properties [24].

Recently, we have synthesized a series of 5-nitro-and 5-amino-6-arylsulfanyl-1-propyl-pyrimidine-2-4-dione, their 6-arylsulfanyl-1,3-dimethyl and the 2-amino analogues [25], as well as a range of substituted 1,2,4-triazolo thymidines [26], with evaluation of their anti-HIV activity.

We next aimed to explore the anti-HCV activity of the compounds. HCV belongs to the family of Flaviviridae and is an important aetiological agent causing chronic hepatitis that can progress further to hepatocellular carcinoma [27]. The current therapeutic protocol for HCV infection used to consist of interferon in combination with ribavirin that is usually accompanied by strong side effects and a moderately successful rate [28,29].

Recently, two protease inhibitors have been approved for the treatment of HCV infection and several more selective antivirals are in advanced clinical development. However, there is still a need for compounds with an alternative mechanism of action and compounds are an important tool to further unravel the exact mechanism by which the virus replicates.

In the present work, we report the synthesis of new mono- and diaryl pyrimidines via the Suzuki cross-coupling procedure [30 –32] and their in vitro activity on the replication of HIV-1, HIV-2 and HVC-1b.

Materials and methods

Chemistry

Melting points are uncorrected and were measured on a Büchi melting point apparatus B-545 (Büchi Labortechnik AG, Flawil, Switzerland). NMR data were obtained on 400 and 600 MHz (¹H) and 150.91 MHz (¹³C) spectrometers (Avance III; Bruker, Reinstetten, Germany) with TMS as internal standard and on the δ scale in ppm. Heteronuclear assignments were verified by heteronuclear single quantum coherence spectroscopy experiments. Microanalytical data were obtained with a Vario Elemental analyzer (Shimadzu; Kyoto, Japan). Analytical silica gel TLC plates 60 F254 were purchased from Merck (Darmstadt, Germany). All reagents were obtained from commercial suppliers and were used without further purification.

2,6-Diamino-4-phenylthio-5-p-chlorophenylazopyrimidine

A solution of 2,6-diamino-4-chloro-5-p-chlorophenylazopyrimidine (5; 250 mg, 0.89 mmol) in benzene (20 ml) containing NaSPh (110 mg, 0.89 mmol) was heated under reflux. After 8 h, the colour of the solution was changed to yellow, where the completion of the reaction was monitored by TLC. After cooling, the solution was concentrated and left overnight at a low temperature. The yellow crystalline material was collected and was recrystallized from ethanol to give 2,6-diamino-4-phenylthio-5-p-chlorophenylazopyrimidine (6; 281 mg, 89%), melting point 237–238°C. ¹H NMR (DMSO-d₆): δ = 9.25 (br s., 2H, C₆-NH₂); 7.90–7.30 (m, 9H, H_arom), 6.88 (br s., 2H, C₂-NH₂). ¹³C NMR (DMSO-d₆): δ = 182.1 (C-4); 164.6 (C-2); 155.4 (C-6); 139.8 (C_arom-Cl); 133.4 (C^1″ _arom-S); 129.4, 129.3, 128.7, 128.3, 127.5, 125.4, 124.1, 122.9 (C_arom); 118.5 (C-5). Anal. Calcd. for C₁₆H₁₃ClN₆S (356.83): C, 53.85; H, 3.67; N, 23.55. Found; C, 53.53; H, 3.54; N, 23.72.

2,6-Diamino-4-ethylthio-5-p-chlorophenylazopyrimidine

The method was analogous to the previous procedure, using 5 (250 mg, 0.87 mmol) and NaSEt (74 mg, 0.887 mmol) as starting materials. Yield: 235 mg 2,6-diamino-4-ethylthio-5-p-chlorophenylazopyrimidine (7; 90%), melting point 267–268°C. ¹H NMR (DMSO-d₆): δ = 9.25 (d, 2H, J = 5.0 Hz, C₆-NH₂); 8.10 (d, 2H, J = 5.1 Hz, C₂-NH₂); 7.78 (d, 2H, J = 7.0 Hz, H_arom-3 + H_arom-5); 7.54 (d, 2H, J = 7.0 Hz, H_arom-2 + H_arom-6). ¹³C NMR (DMSO-d₆): δ = 164.7 (C-4); 161.2 (C-2); 155.9 (C-6); 133.5 (C_arom-Cl); 129.3, 123.3 (C_arom); 118.7 (C-5). Anal. Calcd. for C₁₂H₁₃ClN₆S (308.79): C, 46.68; H, 4.24; N, 27.22. Found; C, 46.38; H, 4.18; N, 27.39.

2,6-Diamino-4-(2-benzylhydrazinyl)-5-p-chlorophenylazopyrimidine

To a solution of 5 (1.0 g, 3.54 mmol) in EtOH (30 ml) was added benzylhydrazine hydrochloride (0.45 g, 2.84 mmol) and the mixture was heated under reflux for 2 h. After cooling, the orange solution was concentrated and left overnight at 0°C. The orange crystals were filtered, and recrystallized from EtOH to give 2,6-diamino-4-(2-benzylhydrazinyl)-5-p-chlorophenylazopyrimidine (8; 1.14 g, 88%), melting point 211–215°C. ¹H NMR (DMSO-d₆): δ = 9.31 (br s., 2H, C₆-NH₂), 9.00–8.96 (m, 2H, 2xNH); 8.26 (br s., 2H, C₄-NH₂); 7.99–7.35 (m, 9H, H_arom); 4.06 (s, 2H, CH₂). ¹³C NMR (DMSO-d₆): δ = 164.7 (C-4); 160.5 (C-2); 155.4 (C-6); 139.8 (C¹_{phenylhydraz.}); 133.4 (C_arom-Cl); 129.3, 129.2, 128.4, 128.1 (C_arom); 103.8 (C-5); 53.6 (CH₂). Anal. Calcd. for C₁₇H₁₇ClN₈. (368.82): C, 55.36; H, 4.65; N, 30.38. Found: C, 55.36; H, 4.50; N, 30.21.

General procedure of Suzuki reaction for preparation of 9–14

A mixture of halopyrimidine and arylboronic acid in n-propanol (15 ml) was stirred for 15 min. To this mixture was added Pd(OAc)₄ (650 mg, 0.19 mmol), triphenylphosphine (498 mg, 0.19 mmol) and 2M aqueous solution of Na₂CO₃ (3.5 ml). The reaction mixture was refluxed under nitrogen for 4–6 h and the completion of the reaction was monitored by TLC. After cooling, water was added (7 ml), followed by stirring for 5 min. The mixture was partitioned with ethyl acetate (3×10 ml) and the combined organic layers were washed subsequently with 5% Na₂CO₃ solution (2×10 ml), brine solution (2×10 ml) and finally with water (10 ml). The organic phase was decolourized with charcoal, filtered and the filtrate was dried (Na₂SO₄), filtered through Celite and evaporated to dryness to give the desired product after purification.

2,6-Diamino-4-(2-benzylhydrazinyl)-5-(4′-fluoro-[1,1′-biphenyl]-4-yl)pyrimidine

From 8 (70 mg, 0.19 mmol) and p-fluorophenylboronic acid (27 mg, 0.19 mmol). Yield: 63 mg 2,6-diamino-4-(2-benzylhydrazinyl)-5-(4′-fluoro-[1,1′-biphenyl]-4-yl)pyrimidine (9; 78%), as brown crystals, melting point 180–182°C (dec), R_f = 0.67 (eluent: ethyl acetate/hexane 3:2). ¹H NMR (DMSO-d₆): δ = 8.03 (br s., 2H, C₆-NH₂). 7.82–7.67 (m, 4H, C₂-NH₂+2xNH); 7.64–7.31 (m, 13H, H_arom); 4.31 (d, 2H, J = 5.5 Hz, CH₂). ¹³C NMR (DMSO-d₆): δ = 166.9 (C-4); 162.1 (C-2); 161.1 (d, J_C4″,F = 250 Hz, C_4″-F); 152.2 (C-6); 139.5 (C-4′ + C¹_{phenylhydraz.}); 139.2 (d, J_C-1″,F = 2.4 Hz, C-1″); 133.9, 133.1, 131.9, 131.4, 129.2, 128.7, 128.6, 127.2, 119.2 (C_arom); 104.7 (C-5); 55.4 (CH₂). Anal. Calcd. for C₂₃H₂₁FN₈ (428.46): C, 64.47; H, 4.94; N, 26.15. Found: C, 64.24; H, 4.90; N, 25.94.

4,6-Diamino-4-(2-benzylhdrazinyl)-5-(3′,4′-dimethoxy[1,1′-biphenyl]-4-yl)pyrimidine

From 8 (100 mg, 0.27 mmol) and 3,4-dimethoxyphenylboronic acid (50 mg, 0.27 mmol). Yield: 110 mg 4,6-diamino-4-(2-benzylhdrazinyl)-5-(3′,4′-dimethoxy[1,1′-biphenyl]-4-yl)pyrimidine (10; 87%), as brown crystals, melting point 173–176°C, R_f = 0.66 (eluent: ethyl acetate/hexane 3:2). ¹H NMR (DMSO-d₆): δ = 9.41 (br s., 2H, C₆-NH₂); 7.68–7.46 (m, 12H, H_arom); 7.00–6.51 (m, C₂-NH₂+2xNH); 4.32 (d, 2H, J = 5.2 Hz, CH₂); 3.31 (s, 6H, OMe). ¹³C NMR (DMSO-d₆): δ = 164.5 (C-4); 161.0 (C-2); 155.8 (C-6); 149.6 (C_3″-OMe + C_4″-OMe); 141.6 (C-4′ + C¹_{phenylhydraz.}); 131.9; 131.4, 131.3, 129.2, 128.7, 128.6, 122.9 (C_arom); 113.5 (C-2″ + C-5″); 105.5 (C-5); 56.2 (OMe); 55.4 (CH₂). Anal. Calcd. for C₂₅H₂₆N₈O₂ (470.53): C, 63.82; H, 5.57; N, 23.81. Found: C, 62.95; H, 5.39; N, 23.92.

2,6-Diamino-4-(3,4-dimethoxyphenyl)-5-(3′,4′-dimethoxy[1,1′-biphenyl]-4-yl)pyrimidine

From 5 (122 mg, 0.40 mmol) and 3,4-dimethoxyphenylboronic acid (155 mg, 0.85 mmol). Yield: 148 mg 2,6-diamino-4-(3,4-dimethoxyphenyl)-5-(3′,4′-dimethoxy[1,1′-biphenyl]-4-yl)pyrimidine (11; 92%), as red crystals, melting point 165–166°C, R_f = 0.54 (eluent: ethyl acetate/hexane 3:2). ¹H NMR (DMSO-d₆): δ = 9.00 (br s, 2H, C₆-NH₂); 7.73–7.65 (m, 10H, H_arom); 6.71 (br s, 2H, C₂-NH₂); 3.83, 3.76, 3.74 (m, 12H, 4xOMe). ¹³C NMR (DMSO-d₆): δ = 163.7 (C-2); 161.7 (C-4), 160.9 (C-6), 151.8, 147.0 (4xC-OMe); 141.0 (C-1′); 131.9, 131.4, 131.3, 129.2, 128.7, 128.5 (C_arom); 122.9 (C-5); 113.5, 112.1, 110.3 (C_arom); 55.1 (4xOMe). Anal. Calcd. for C₂₆H₂₆N₆O₄ (486.52): C, 64.19; H, 5.39; N, 17.27. Found: C, 64.56; H, 5.31; N, 17.20.

2,6-Diamino-4-(4-fluorophenyl)-5-(4′-fluoro[1,1′-biphenyl]-4-yl)pyrimidine

From 5 (86 mg, 0.30 mmol) and 4-nitrophenylboronic acid (140 mg, 0.60 mmol). Yield: 93 mg 2,6-diamino-4-(4-fluorophenyl)-5-(4′-fluoro[1,1′-biphenyl]-4-yl) pyrimidine (12; 99%), as red crystals, melting point 179–180°C, R_f = 0.70 (eluent: etheyl acetate/hexane 2:1). ¹H NMR (DMSO-d₆): δ = 9.43 (br s, 2H, C₆-NH₂) 8.05–7.31 (m, 12H, H_arom); 7.06 (br s, 2H, C₂-NH₂). ¹³C NMR (DMSO-d₆): δ = 165.4 (C-2); 161.0 (m, C-4 + 2xC_4″-F); 155.9 (C-6); 139.7 (C-1′); 133.9, 133.1, 132.1, 131.45, 131.36, 130.67, 129.9, 127.2 (C_arom); 122.3 (C-5); 120.8, 116.2, 115.3 (C_arom-c+ C_arom-e+C-3″+C5″). Anal. Calcd. for C₂₂H₁₆F₂N₆ (402.40): C, 65.66; H, 4.01; N, 20.88. Found: C, 65.42: H, 3.96; N, 20.65.

2,6-Diamino-4-(2-fluorophenyl)-5-(2′-fluoro[1,1′-biphenyl]-4-yl)pyrimidine

From 5 (213 mg, 0.75 mmol) and 2-fluorophenylboronic acid (210 mg, 1.50 mmol). Yield: 220 mg 2,6-diamino-4-(2-fluorophenyl)-5-(2′-fluoro[1,1′-biphenyl]-4-yl) pyrimidine (13; 95%), as a red coloured material, melting point 176–178°C, R_f = 0.71 (eluent: ethyl acetate/hexane 2:1). ¹H NMR (DMSO-d₆): δ = 8.22 (s, 2H, C₆-NH₂); 7.65–7.23 (m, 12H, H_arom); 7.18 (br s., 2H, C₂-NH₂). ¹³C NMR (DMSO-d₆): δ = 168.1 (C-2); 162.2 (C-4); 158.5 (d, J_C,F = 249 Hz, 2xC_2″-F); 151.1 (C-6); 140.2 (C-1′); 131.3, 129.96, 129.9, 129.4, 128.9, 126.8 (C_arom); 119.9 (C-5); 117.1, 116.5 (C_arom-c+C-3″). Anal. Calcd. for C₂₂H₁₆F₂N₆ (402.40): C, 65.66; H, 4.01; N, 20.88. Found: C, 65.31; H, 3.91; N, 20.61.

2,6-Diamino-4-(4-nitrophenyl)-5-(4′-nitro[1,1′-biphenyl]-4-yl)pyrimidine

From 5 (213 mg, 0.75 mmol) and 4-nitrophenylboronic acid (250 mg, 1.50 mmol). Yield: 250 mg 2,6-diamino-4-(4-nitrophenyl)-5-(4′-nitro[1,1′-biphenyl]-4-yl) pyrimidine (14; 94%), as red crystals, melting point 185–187°C, R_f = 0.70 (eluent: ethyl acetate/hexane 2:1). ¹H NMR (DMSO-d₆): δ = 9.24 (s, 2H, C₆-NH₂); 8.16–6.96 (m, 12H, H_arom); 6.94 (s, 2H, C₂-NH₂). ¹³C NMR (DMSO-d₆): δ = 164.5 (C-2); 161.0 (C-4); 155.7 (C-6); 150.9 (C-1″ + 2xC4″-NO₂); 133.2, 131.8, 131.2, 129.1, 126.0, 122.6, 121.9 (C_arom); 118.5 (C-5). Anal. Calcd. for C₂₂H₁₆N₈O₄ (456.41): C, 57.89; H, 3.53; N, 24.55. Found: C, 57.76; H, 3.48; N, 24.71.

2,6-Diamino-5-(4-chlorophenylazo)-3H-pyrimidin-4-one

A solution of 5 (100 mg, 0.35 mmol) in DMF (5 ml) containing 2M aq. NaOH (1 ml) was heated at 60°C for 30 min. The solution was neutralized with HOAc to pH 5. After cooling, the precipitate was filtered, washed with water, dried and recrystallized from EtOH to give 2,6-diamino-5-(4-chlorophenylazo)-3H-pyrimidin-4-one (15; 54 mg, 75%), melting point 183–186°C. ¹H NMR (DMSO-d₆): δ = 9.22 (br s., 2H, C₆-NH₂); 8.10 (br s., 2H, C₂-NH₂); 7.78 (d, 2H, J = 7.1 Hz, H_3′,5′); 7.54 (d, 2H, J = 7.1 Hz, H_2′,6′). ¹³C NMR (DMSO-d₆): δ = 164.7 (C-6); 161.2 (C-4); 155.9 (C-2); 151.0 (C-1′); 133.5, 129.3, 123.0 (C_arom); 118.7 (C-5). Anal. Calcd. for C₁₀H₉ClN₆O (264.05): C, 45.38; H, 3.43; N, 31.75. Found: C, 45.11; H, 3.75; N, 31.81.

6-(2-Fluorophenyl)-1,3-dimethyl-5-nitropyrimidine-2,4-dione

From 6-chloro-1,3-dimethyl-5-nitropyrimidine-2,4-dione (16; 165 mg, 0.75 mmol) and 2-fluorophenylboronic acid (105 mg, 0.75 mmol). Yield: 142 mg 6-(2-fluorophenyl)-1,3-dimethyl-5-nitropyrimidine-2,4-dione (17; 68%), as a green powder, melting point 121–123°C, R_f = 0.88 (eluent: ethyl acetate/hexane 1:1). ¹H NMR (DMSO-d₆): δ = 7.64–7.54 (m, 4H, H_arom); 3.32 (s, 3H, N¹-Me); 3.07 (s, 3H, N³-Me). ¹³C NMR (DMSO-d₆): δ = 168.5 (C-4); 159.0 (d, J_C,F = 248 Hz, C_2′-F); 154.9 (C-6); 151.4 (C-2); 131.9, 128.7, 128.5, 116.2 (C_arom); 108.6 (C₅-NO₂); 32.2 (N¹-Me); 27.0 (N³-Me). Anal. Calcd. for C₁₂H₁₀FN₃O₄ (279.22): C, 51.62; H, 3.61; N, 15.05; Found: C, 51.79; H; 3.55; N, 14.87.

1,3-Dimethyl-5-nitro-6-(4-nitrophenyl)pyrimidine-2,4-dione

From 16 (165 mg, 0.75 mmol) and 4-nitrophenylboronic acid (125 mg, 0.45 mmol). Yield: 145 mg 1,3-dimethyl-5-nitro-6-(4-nitrophenyl)pyrimidine-2,4-dione (18; 63%), as a green powder, melting point 139–141°C, R_f = 0.50 (eluent: ethyl acetate/hexane 1:1). ¹H NMR (DMSO-d₆): δ = 8.36 (d, 2H, J = 6.8 Hz, H_arom); 8.10 (d, 2H, J = 6.8 Hz, H_arom); 3.62 (s, 3H, N¹-Me); 3.01 (s, 3H, N³-Me). ¹³C NMR (DMSO-d₆): δ = 162.8 (C-4); 157.2 (C-6); 150.9 (C-2); 147.5 (C_4′-NO₂); 144.0 (C-1′); 128.6, 128.7 124.1 (C_arom); 108.3 (C-5); 32.9 (N¹-Me); 27.0 (N³-Me). Anal. Calcd. for C₁₂H₁₀N₄O₆.H₂O (324.25): C, 44.45; H, 3.73; N, 17.28. Found: C, 44.22; H, 3.59; N, 17.59.

1,3-Dimethyl-6-(2-methylthiophenyl)-5-nitropyrimidine-2,4-dione

From 16 (200 mg, 0.91 mmol) and 2-thiomethylphenylboronic acid (153 mg, 0.91 mmol). Yield: 134 mg 1,3-dimethyl-6-(2-methylthiophenyl)-5-nitropyrimidine-2,4-dione (19; 64%), as a brown powder, melting point 252°C (dec.), R_f = 0.61 (eluent: ethyl acetate/hexane 1:1). ¹H NMR (DMSO-d₆): δ = 7 72–6.93 (m, 4H, H_arom); 3.46 (s, 3H, N¹-Me), 3.18 (s, 3H, N³-Me); 2.50 (s, 3H, SMe). ¹³C NMR (DMSO-d₆): δ = 162.9 (C-4); 157.3 (C-6); 150.9 (C-2); 141.1 (C_2′-SMe); 132.6 (C_arom-1′); 129.1, 126.7, 124.0 (C_arom); 90.3 (C-5); 34.3 (N¹-Me); 29.7 (N³-Me); 16.1 (SMe). Anal. Calcd. for C₁₃H₁₃N₃O₄S (307.33): C, 50.81; H, 4.26; N, 13.67. Found: C, 51.02; H, 4.18; N, 13.44.

Virology

In vitro anti-HIV assay

Evaluation of the antiviral activity of the compounds 6–15 and 16–19 against HIV-1 strain III_B and HIV-2 strain (ROD) in MT-4 cells was performed using the MTT assay as previously described [33,34]. Briefly, stock solutions (10x final concentration) of test compounds were added in 25 μl volumes to two series of triplicate wells so as to allow simultaneous evaluation of their effects on mock- and HIV-infected cells at the beginning of each experiment. Serial fivefold dilutions of test compounds were made directly in flat-bottomed 96-well microtitre trays using a Biomek 3000 robot (Beckman Coulter, Fullerton, CA, USA). Untreated control HIV- and mock-infected cell samples were included for each sample. HIV-1 (III_B) [35] or HIV-2 (ROD) [36] stock (50 μl) at 100–300 50% cell culture infectious dose or culture medium was added to either the infected or mock-infected wells of the microtitre tray. Mock-infected cells were used to evaluate the effect of test compound on uninfected cells in order to assess the cytotoxicity of the test compound. Exponentially growing MT-4 cells [37] were centrifuged for 5 min at 1,000 rpm (220 g) and the supernatant was discarded. The MT-4 cells were resuspended at 6×10⁵ cells/ml and 50 μl volumes were transferred to the microtitre tray wells. Five days after infection, the viability of mock- and HIV-infected cells was examined spectrophotometrically by the MTT assay.

The MTT assay is based on the reduction of yellow coloured 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase of metabolically active cells to a blue-purple formazan that can be measured spectrophotometrically. The absorbances were read in an eight-channel computer-controlled photometer (Safire; Tecan, Männedorf, Switzerland), at two wavelengths (540 and 690 nm). All data were calculated using the median optical density (OD) value of tree wells. The 50% cytotoxic concentration was defined as the concentration of the test compound that reduced the absorbance (OD540) of the mock-infected control sample by 50%. The concentration achieving 50% protection from the cytopathic effect of the virus in infected cells was defined as the 50% effective concentration (EC₅₀).

The results are summarized in Table 1. Nevirapine [38] and azidothymidine [39] were included as reference compounds. All newly synthesized compounds had a selectivity index <1.

Table 1.

In vitro anti-HIV-1^a and HIV-2^b activity and cytotoxicity of some new pyrimidines

Compound	HIV-1 (III_B) IC_50′ μg/ml^c	HIV-2 (ROD) IC_50′ μg/ml_c	CC_50′ μg/ml^d	SI (III_B)	SI (ROD)

6	>11.25	>11.25	11.25	<1	<1
7	>2.05	>2.05	2.05	<1	<1
8	>9.93	>9.93	9.93	<1	<1
9	>45.60	>45.60	45.60	<1	<1
10	>103	>103	103	<1	<1
11	>0.49	>0.49	0.49	<1	<1
12	>12.15	>12.15	12.15	<1	<1
13	>12.05	>12.05	12.05	<1	<1
14	>37.80	>37.80	37.80	<1	<1
15	>57.45	>57.45	57.45	<1	<1
17	>1.92	>1.92	1.92	<1	<1
18	>71.60	>71.60	71.60	<1	<1
19	>51.39	>51.39	51.39	<1	<1
Nevirapine	0.050	>4.00	>4.00	>80	<1
AZT	0.0022	0.00094	>25	>11,363	>26,596

Anti-HIV-1 activity measured with strain III_B.

Anti-HIV-2 activity measured with strain ROD.

Compound concentration required to achieve 50% protection of MT-4 cells from the HIV-1 and HIV-2-induced cytopathic effect (IC₅₀).

Compound concentration that reduces the viability of mock-infected MT-4 cells by 50% (CC₅₀). SI, selectivity index (CC₅₀/IC₅₀).

In vitro anti-HCV assay

Compounds 6, 7, 9–15 and 17–19 were evaluated for their in vitro selective antiviral activity against HCV in the Huh 5–2 replicon system (type 1b, Con1 strain). The results are shown in Table 2.

Table 2.

In vitro anti-HCV-1b activity, cytotoxicity and inhibition percentage of some pyrimidines

Compound	CC_50′ μg/ml	EC_50′ μg/ml	SI	Inhibition percentage	Concentration

6	2.29	2.29	2.36	61.4	5.20
7	12.3	1.78	6.89	67.7	5.78
9	>50	>50	ND	5.15	0.40
10	>50	>50	ND	42.8	50
11	1.0	0.729	1.38	54.5	1.16
12	4.31	1.88	2.30	61.1	3.86
13	7.94	3.40	2.34	60.9	6.52
14	3.64	1.60	2.28	59.4	2.73
15	1.41	0.579	2.44	62.6	1.22
17	>50	>50	ND	35.4	50
18	11.8	ND	ND	50	11.8
19	>50	>50	ND	47.1	50

CC_50′ 50% cytostatic/cytotoxic concentration (concentration at which 50% adverse effect is observed on the host cell); EC_50′ 50% effective concentration (concentration at which 50% inhibition of virus replication is observed); ND, not determined (value could not be derived from the dose–response curve); SI, selectivity index (CC₅₀/EC₅₀).

Cells and HCV

The Huh-5-2 and Huh 9–13 HCV subgenomic replicon-containing cells were provided by R Bartenschlager (University of Heidelberg, Heidelberg, Germany).

Antiviral assays

Huh 5.2 cells, containing the HCV genotype 1b I389lucubi-neo/NS3-3′/5.1 replicon [40] were sub-cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 1% non-essential amino acids, 1% penicillin/streptomycin and 2% Geneticin at a ratio of 1:3 to 1:4 and grown for 3–4 days in 75 cm² tissue culture flasks. One day before addition of the compound, cells were harvested and seeded in assay medium (Dulbecco's modified Eagle's medium, 10% FCS, 1% non-essential amino acids, 1% penicillin/streptomycin) at a density of 6,500 cells/well (100 μl/well) in 96-well tissue culture microtitre plates for evaluation of anti-metabolic effect and CulturPlate (Perkin Elmer, Waltham, MA, USA) for evaluation of the antiviral effect. The microtitre plates were incubated overnight (37°C, 5% CO₂, 95–99% relative humidity), yielding a non-confluent cell monolayer.

The evaluation of the anti-metabolic as well as the antiviral effect of each compound was performed in parallel. Four-step, 1-to-5 compound dilution series were prepared for the first screen, to collect data for a more detailed dose-response curve, an eight-step, 1-to-2 dilution series was used. Following assay setup, the microtitre plates were incubated for 72 h (37°C, 5% CO₂, 95–99% relative humidity). For the evaluation of anti-metabolic effects, the assay medium was aspirated, replaced with 75 μl of a 5% MTS solution in phenol red-free medium and incubated for 1.5 h (37°C, 5% CO₂, 95–99% relative humidity). Absorbance was measured at a wavelength of 498 nm (Safire²; Tecan), and optical densities (OD values) were converted to percentage of untreated controls. For the evaluation of antiviral effects, assay medium was aspirated and the cell monolayers were washed with PBS. The wash buffer was aspirated and 25 μl of Glo Lysis Buffer (Promega) was added allowing for cell lysis to proceed for 5 min at room temperature. Subsequently, 50 μl of Luciferase Assay System (Promega, Madison, WI, USA) was added, and the luciferase luminescence signal was quantified immediately (1,000 ms integration time/well; Safire²; Tecan). Relative luminescence units were converted into percentage of untreated controls.

The EC₅₀ and EC₉₀ (values calculated from the dose–response curve) represent the concentrations at which 50% and 90% inhibition, respectively, of viral replication, is achieved. The 50% cytotoxic concentration (value calculated from the dose–response curve) represents the concentration at which the metabolic activity of the cells is reduced by 50% as compared to untreated cells.

A concentration of compound is considered to elicit a genuine antiviral effect in the HCV replicon system when the anti-replicon effect is well above the 70% threshold at concentrations where no significant anti-metabolic activity is observed [40].

Results

The azo-pyrimidine derivative 5 has been prepared previously in our laboratory [41] from the commercially available 2,6-diamino-4-chloropyrimidine 4, and was selected here as a starting material for the synthesis of various pyrimidine analogues. Treatment of 5 with NaSPh or NaSEt in DMF yielded, via nucleophilic displacements of the chlorine group, 6 and 7, in 89 and 90% yield, respectively. Similarly, the reaction of 5 with benzylhydrazine afforded 8 (88%), meanwhile, the biaryl-pyrimidines 9 and 10 were obtained (78 and 87%, respectively) via Suzuki cross-coupling reactions of 8 with p-fluorophenyl- and 3,4-dimethoxyphenylboronic acids, respectively. Further, treatment of 5 with 3,4-dimethoxyphenyl-, m- and, o-fluorophenyl-, and p-nitrophenylboronic acids by applying Suzuki coupling gave the triaryl-pyrimidines 11–14 (92–99%). Basic hydrolysis of chloropyrimidine 5 led to the formation of 15 (75%; Figure 2).

Figure 2.

Synthesis of mono- and diarylpyrimidines and analogues

The structures of the newly synthesized compounds 6–15 were determined from the ¹H and ¹³C NMR spectra. The ¹H NMR spectra showed rather similar patterns for the phenyl and ethyl protons, while the doublets at δ = 4.31 and 4.32 ppm were assigned to methylene of the benzylhydrazine group of 9 and 10 (J = 5.5 and 5.1 Hz), respectively. In the ¹³C NMR spectra of 6 and 7, C-4 of the pyrimidine ring resonated at δ = 182.1 and 164.7 ppm, respectively, whereas, C-2, C-5 and C-6 resonated at the regions (δ = 164.6, 160.5 ppm), (δ = 118.5, 118.7 ppm) and (δ = 155.4, 155.9 ppm), respectively. The resonances at the regions δ = 166.9–161.0 ppm were attributed to C-4 and C-2 of 8 and 9, while, the resonances at δ = 104.7 and 105.5 ppm were assigned to C-5. The assignment of protons and carbons of 11–15 was deduced from comparison.

Next, our efforts focused on the synthesis of new 6-aryl-pyrimidine-2,4-dione derivatives to study their effect on HIV and HCV replication in cell culture. Thus, treatment of 16 with aryl boronic acids using Suzuki methodology [32] afforded 17–19 in 68, 63 and 64% yield, respectively (Figure 3).

Figure 3.

Synthesis of 6-aryl-1,3-dimethyl-5-nitropyrimidine 2,4-diones

The assignment of protons and carbons of the pyrimidine and aromatic rings were deduced in comparison to our previously reported data on 6-arylsulfanyl pyrimidines [25]. In the ¹H NMR spectra of 17–19, the N-1 and N-3 methyl groups were resonated at the region (δ = 3.62–3.32 ppm) and (δ = 3.18–2.50 ppm), respectively. The ¹³C NMR spectra of 17–19 showed signals at higher fields (δ = 168.8–162.8 ppm), attributed to the carbonyl group whereas the resonances at (δ = 157.2–154.9 ppm) and (δ = 151.4–150.9 ppm) were assigned to C-6 and C-2, respectively. Resonances at δ = 108.6, 108.3 and 90.3 ppm were assigned to C-5 pyrimidine carbons. From the ¹³C NMR spectra, it is clearly shown a shift in ppm values (approximately 10 ppm) between C₆-C_arom and C₆-Cl, indicative for substitution of a chloride group by aryl moiety.

Compounds 6–15 and 17–19 were evaluated in vitro for their selective antiviral activity against anti-HIV-1 (strain III_B) and anti-HIV-2 (strain ROD) activity in human T-lymphocyte (MT-4) cells using the MT-4/MTT assay [33,34]. None of the compounds inhibit HIV-1 and HIV-2 replication in cell culture.

Discussion

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are notorious for rapidly leading to virus drug resistance development, primarily based on the emergence of the K103N and Y181C mutations in the HIV-1 reverse transcriptase. Newer NNRTIs, such as capravirine, dapivirine (TMC 125) and DPC 083, are resilient to these ‘NNRTI’ mutations, and, therefore, offer considerable promise as future anti-HIV-1 drugs. NNRTIs are targeted at a specific ‘pocket’ binding site within the HIV-1 RT that is distinct from, but both spatially and functionally related to, the catalytic site where the nucleoside reverse transcriptase inhibitors and nucleotide reverse transcriptase inhibitors interact [42]. However, capravirine, an imidazole derivative, is active against HIV variants with the single mutations K103N, V106A or L100I, which confer resistance to NNRTIs and form an extensive hydrogen-bond network with the RT main chain, a network that is unlikely to be disrupted by simple side-chain mutations [43]. Unfortunately, none of our pyrimidine derivatives produced any protective effect against the HIV-induced cytopathogenic effect in MT-4 cells.

The establishment of stable HCV subgenomic replicon systems [44,45] in the human hepatoblastoma cell line Huh7 has provided a useful system for the development of new antiviral approaches against HCV [46 –49]. The overriding aims of the new therapeutic strategies are higher efficacy associated with shortened duration of treatment, favourable mode of administration, and thus improved tolerability and adherence.

However, we have evaluated in vitro our new pyrimidine derivatives 6, 7, 9–15 and 17–19 for selective antiviral activity against HCV in the Huh 5–2 replicon system (type 1b, Con1 strain). Even though for many compounds an EC₅₀ was obtained with a selectivity index of up to 6.89 (for example, compound 7; Table 2), none of the compounds matched the selection criteria of a selective inhibitor of virus replication in this assay (that is, >70% inhibition at concentrations that do not elicit an anti-metabolic effect on the host cells).

Therefore, structural modification of these compounds might optimize their anti-HCV activity by introducing diverse and potent functional groups at the pyrimidine backbone, like nitrile residue. In conclusion, we explored the anti-HIV and anti-HCV activity of a novel series of pyrimidine derivatives in an attempt to identify selective inhibitors of viral replication.

Footnotes

Acknowledgements

We thank U Haunz and A Friemel of the Chemistry Department, University of Konstanz, Germany, for the NMR experiments. We also would like to acknowledge Stijn Delmotte, Tom Bellon, Annelies De Ceulaer and Caroline Collard for their excellent technical assistance in the acquisition of the anti-HCV data.

The authors declare no competing interests.

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Exploration of the in vitro Antiviral Activity of a Series of New Pyrimidine Analogues on the Replication of HIV and HCV

Abstract

Background:

Methods:

Results:

Conclusions:

Introduction

Materials and methods

Chemistry

2,6-Diamino-4-phenylthio-5-p-chlorophenylazopyrimidine

2,6-Diamino-4-ethylthio-5-p-chlorophenylazopyrimidine

2,6-Diamino-4-(2-benzylhydrazinyl)-5-p-chlorophenylazopyrimidine

General procedure of Suzuki reaction for preparation of 9–14

2,6-Diamino-4-(2-benzylhydrazinyl)-5-(4′-fluoro-[1,1′-biphenyl]-4-yl)pyrimidine

4,6-Diamino-4-(2-benzylhdrazinyl)-5-(3′,4′-dimethoxy[1,1′-biphenyl]-4-yl)pyrimidine

2,6-Diamino-4-(3,4-dimethoxyphenyl)-5-(3′,4′-dimethoxy[1,1′-biphenyl]-4-yl)pyrimidine

2,6-Diamino-4-(4-fluorophenyl)-5-(4′-fluoro[1,1′-biphenyl]-4-yl)pyrimidine

2,6-Diamino-4-(2-fluorophenyl)-5-(2′-fluoro[1,1′-biphenyl]-4-yl)pyrimidine

2,6-Diamino-4-(4-nitrophenyl)-5-(4′-nitro[1,1′-biphenyl]-4-yl)pyrimidine

2,6-Diamino-5-(4-chlorophenylazo)-3H-pyrimidin-4-one

6-(2-Fluorophenyl)-1,3-dimethyl-5-nitropyrimidine-2,4-dione

1,3-Dimethyl-5-nitro-6-(4-nitrophenyl)pyrimidine-2,4-dione

1,3-Dimethyl-6-(2-methylthiophenyl)-5-nitropyrimidine-2,4-dione

Virology

In vitro anti-HIV assay

In vitro anti-HCV assay

Cells and HCV

Antiviral assays

Results

Discussion

Footnotes

Acknowledgements

References