Advanced generation of paeonol-phenylsufonyl derivatives as potential anti-HBV agents

Abstract

Hepatitis B virus (HBV) infection causes serious liver diseases, and the development of effective drugs for chronic hepatitis B treatment remains an important step towards the eradication of HBV worldwide. Recently, our group designed paeonol-phenylsulfonyl derivatives and found that the compound 2-acetyl-5-methoxyphenyl 4-methoxybenzenesulfonate (6f) had the most potent antiviral effect against HBV, with an IC₅₀ value of 0.36 μM and a high selectivity index (SI; TC₅₀/IC₅₀) of 47.75. In this research, we modified compound 6f with a 2-aminothiazole scaffold and generated 13 novel paeonol derivatives by utilizing various benzoyl and acyl chlorides. Among this new generation, compound 2-(2-benzamidothiazol-4-yl)-5-methoxyphenyl 4-methoxybenzenesulfonate (8a) showed the highest SI value of 59.14 which exceeds those of compound 6f and lamivudine (3TC), a commercially available antiviral drug. Hence, we believe that our studies may offer some useful information for the development of antiviral medicines.

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1. Introduction

Hepatitis B virus (HBV), a member of the Hepadnaviridae family, is a causative agent in progressive liver diseases. Nowadays, over two billion people are infected with HBV worldwide and approximately 400 million are chronically infected carriers.^1,2 Moreover, 80% of chronic HBV carriers have varying degrees of liver damage, which could progress to liver cirrhosis and hepatocellular carcinoma.³ At present, the clinically available anti-HBV agents are all nucleoside or nucleotide analogs that target the activity of viral reverse transcriptase (RT), which regulates minus strand DNA synthesis, the first step in viral genome replication from the pregenomic RNA template;⁴ however, these anti-HBV drugs are reported to have viral resistance owing to specific RT mutations. Thus, it is important to develop new therapeutic agents that adopt other approaches to ameliorate the disease.

Many compounds isolated from botanical resources are alleged to have antiviral activity toward HBV.^5–7 In our previous study,⁸ we derivatized paeonol (4; Scheme 1), the main active component of a traditional Chinese herbal medicine Moutan Cortex that has anti-inflammatory,^9,10 analgesic,¹⁰ and antiatherogenic¹¹ activity, with various phenylsulfonyl groups (Scheme 1). The results demonstrated that the compound 2-acetyl-5-methoxyphenyl 4-methoxybenzenesulfonate (6f; Scheme 1) had the most potent inhibitory effect on viral gene expression and viral propagation in a cell culture model, with an IC₅₀ value of 0.36 μM and a high selectivity index (SI; TC₅₀/IC₅₀) of 47.75. We then modified 6f with a 2-aminothiazole scaffold (7; Scheme 2), a moiety contained in a number of commercially available drugs such as abafungin (1; Fig. 1), an antimicrobial agent, meloxicam (2; Fig. 1), a nonsteroidal anti-inflammatory drug used in arthritis, dysmenorrhea and fever, and talipexole (3; Fig. 1), which is used as an anti-Parkinson molecule.¹² Caravatti et al. presented a potent and selective phosphatidylinositol-3 kinase alpha inhibitor which bore this moiety and had therapeutic potential for treating cancers.¹³ Costantino et al. reported N-substituted 2-aminothiazole derivatives with inhibitory activity toward Mycobacterium tuberculosis H₃₇Rv.¹⁴ From the perspective of medicinal chemistry, the 2-aminothiazole moiety may serve as a stable bioisostere of a phenol group.¹⁵ Like the phenol group, such a structure also has antioxidant properties; in addition, compared to the phenol group, the 2-aminothiazole scaffold is more lipophilic and displays improved oral availability. In this study, we developed a 2-aminothiazole core embodying paeonol derivatives with a 4-methoxyphenylsulfonyl side chain on its phenol group, and further substituted the amino group with para-position substituted benzoyl chlorides and acyl chlorides. The anti-HBV effects of these novel compounds in HepG2 2.2.15 cells were evaluated and the results are described in the following text.

Scheme 1 Synthesis of paeonol-phenylsulfonyl derivatives.

Scheme 2 Synthesis of 2-aminothiazole embodying paeonol-4-methoxyphenylsulfonyl derivatives.

Fig. 1 Examples of commercially available drugs containing 2-aminothiazole scaffold.

2. Experimental section

2.1. Chemistry

All reactions were carried out in oven-dried glassware (120 °C) under an atmosphere of nitrogen. Acetone, dichloromethane, ethyl acetate and hexane from Mallinckrodt Chemical Co. were dried and distilled from CaH₂. Paeonol, 4-methoxybenzenesulfonyl chloride, potassium carbonate, thiourea, iodine, sodium hydroxide, benzoyl chloride, 4-fluorobenzoyl chloride, 4-chlorobenzoyl chloride, 4-bromobenzoyl chloride, 4-toluoylbenzoyl chloride, 4-(trifluoromethyl)benzoyl chloride, 4-methoxybenzoyl chloride, 4-nitrobenzoyl chloride, acetyl chloride, propionyl chloride, and butyryl chloride were purchased from Sigma-Aldrich Chemical Co without further purification. Analytical thin layer chromatography (TLC) was performed on precoated plates (silica gel 60 F-254), purchased from Merck Inc. Purification by column chromatography was carried out with Merck Reagents Silica Gel 60 (particle size 0.063–0.200 mm, 70–230 mesh ASTM).

Proton NMR spectra were obtained on a Bruker Avance 500 (500 MHz) and a Varian Unity-400 (400 MHz) with d-chloroform and d-acetone as solvent. Proton NMR chemical shifts are referenced to the CDCl₃ singlet (7.24 ppm) and the center of (CD₃)₂CO quintet (2.05 ppm). Carbon-13 NMR spectra were obtained on a Varian UNITY INOVA 500 (125 MHz) by use of chloroform-d and d-acetone as solvent. Carbon-13 chemical shifts are referenced to the center of the CDCl₃ triplet (77.0 ppm) and (CD₃)₂CO heptet (29.85 ppm). Multiplicities are recorded by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; J, coupling constant (Hertz). High-resolution mass spectra were obtained with a VARIAN 901-MS (FT-ICR Mass) mass spectrometer. High-performance liquid chromatography (HPLC) analyses were carried out by Agilent 1100 series system with CNW Athena C18 column (120 Å, 4.6 mm × 250 mm, 5 μm) and UV detection at 254 nm.

2.1.1 General synthetic procedures. The method for generating compound 6f can be found in the previous literature.⁸ The 6f (1.00 equiv.) was further reacted with thiourea (3.00 equiv.) and iodine (1.10 equiv.) in 20.0 mL ethanol, and refluxed for 12.0 hours. Then, it was quenched with NaOH_(aq) (2.00 equiv.) and the ethanol was removed under reduced pressure. The residue was extracted with ethyl acetate (3 × 30 mL) and the combined organic layer was washed with brine and dried over MgSO_4(S). After filtering and condensing under reduced pressure, the crude product was purified by column chromatography on silica gel to give compound 7. Finally, compound 7 (1.00 equiv.) was derivatized with various para-substituted benzoyl and acyl chlorides (1.20 equiv.) in 10.0 mL dichloromethane, and stirred overnight at room temperature. Quenched with water, the mixture was extracted with dichloromethane (3 × 20 mL) and the combined organic layer was washed with brine and dried over MgSO_4(S). After filtration and condensation under reduced pressure, purification by column chromatography on silica gel afforded the desired products.

2.1.2 2-(2-Benzamidothiazol-4-yl)-5-methoxyphenyl 4-methoxybenzenesulfonate (8a). Pale orange solid (21.05%; 3 steps); ¹H NMR (500 MHz, d-acetone): δ 8.22 (d, J = 9.5 Hz, 2H), 8.05 (d, J = 7 Hz, 1H), 7.698–7.499 (m, 5H), 7.25 (s, 1H), 6.98 (dd, J_AB = 8.5 Hz, J_CD = 2.5 Hz, 1H), 6.94–6.91 (m, 3H), 3.86 (s, 3H), 3.80 (s, 3H); ¹³C NMR (125 MHz, d-acetone): δ 167.62, 165.77, 165.24, 160.69, 158.11, 148.40, 145.31, 133.78, 133.52, 133.38, 131.88, 131.51, 130.44, 129.61, 129.35, 128.85, 127.26, 122.10, 115.00, 113.90, 112.15, 109.70, 56.25, 56.10; HRMS (ESI) calculated for C₂₄H₂₀N₂O₆S₂, 496.0763, found 496.0761.

2.1.3 2-(2-(4-Fluorobenzamido)thiazol-4-yl)-5-methoxyphenyl 4-methoxybenzenesulfonate (8b). Pale orange solid (25.62%; 3 steps); ¹H NMR (500 MHz, d-acetone): δ 8.31–8.28 (m, 2H), 7.74 (d, J = 9 Hz, 1H), 7.57–7.55 (m, 1H), 7.39–7.35 (m, 2H), 7.30–7.25 (m, 2H), 6.97 (dd, J_AB = 8.5 Hz, J_CD = 2.5 Hz, 1H), 6.94–6.92 (m, 3H), 3.86 (s, 3H), 3.81 (s, 3H); ¹³C NMR (125 MHz, d-acetone): δ 167.26, 166.64, 165.23, 164.77, 160.71, 158.08, 148.40, 145.30, 133.29, 133.21, 131.86, 131.74, 131.67, 131.51, 116.65, 116.47, 116.40, 116.23, 115.00, 113.91, 112.20, 109.72, 56.26, 56.107; HRMS (ESI) calculated for C₂₄H₁₉FN₂O₆S₂, 514.0669, found 514.0670.

2.1.4 2-(2-(4-Chlorobenzamido)thiazol-4-yl)-5-methoxyphenyl 4-methoxybenzenesulfonate (8c). Pale orange solid (17.62%; 3 steps); ¹H NMR (500 MHz, d-acetone): δ 8.28 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 9 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 9 Hz, 2H), 7.26 (s, 1H), 6.97 (dd, J_AB = 9 Hz, J_CD = 2.5 Hz, 1H), 6.94–6.91 (m, 3H), 3.86 (s, 3H), 3.81 (s, 3H); ¹³C NMR (125 MHz, d-acetone): δ 165.23, 164.84, 160.72, 157.97, 148.40, 145.37, 139.17, 132.16, 131.86, 131.50, 130.70, 129.79, 127.26, 122.05, 115.00, 113.91, 112.28, 109.72, 56.27, 56.11; HRMS (ESI) calculated for C₂₄H₁₉ClN₂O₆S₂, 530.0373, found 530.0369.

2.1.5 2-(2-(4-Bromobenzamido)thiazol-4-yl)-5-methoxyphenyl 4-methoxybenzenesulfonate (8d). Orange solid (20.63%; 3 steps); ¹H NMR (500 MHz, d-acetone) δ 8.15 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 9 Hz, 2H), 7.26 (s, 1H), 6.97 (dd, J_AB = 8.5 Hz, J_CD = 2.5 Hz, 1H), 6.94–6.92 (m, 3H), 3.86 (s, 3H), 3.81 (s, 3H); ¹³C NMR (125 MHz, d-acetone): δ 165.23, 160.73, 148.40, 132.81, 131.92, 131.86, 131.50, 130.83, 127.76, 127.25, 122.00, 115.69, 115.00, 113.92, 113.73, 112.29, 109.74, 109.47, 56.27, 56.11; HRMS (ESI) calculated for C₂₄H₁₉BrN₂O₆S₂, 573.9868, found 573.9869.

2.1.6 5-Methoxy-2-(2-(4-methylbenzamido)thiazol-4-yl)phenyl 4-methoxybenzenesulfonate (8e). White solid (14.68%; 3 steps); ¹H NMR (400 MHz, d-acetone): δ 8.12 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 9.2 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.23 (s, 1H), 6.99–6.91 (m, 4H), 3.85 (s, 3H), 3.81 (s, 3H); ¹³C NMR (125 MHz, d-acetone): δ 167.62, 165.63, 165.23, 160.67, 158.18, 148.39, 144.20, 131.87, 131.51, 130.52, 130.22, 129.95, 128.90, 122.14, 115.00, 113.89, 112.06, 109.69, 56.25, 56.10; HRMS (ESI) calculated for C₂₅H₂₂BrN₂O₆S₂, 510.0919, found 510.0922.

2.1.7 5-Methoxy-2-(2-(4-(trifluoromethyl)benzamido)thiazol-4-yl)phenyl 4-methoxybenzenesulfonate (8f). Pale orange solid (25.39%; 3 steps); ¹H NMR (500 MHz, CDCl₃): δ 8.05 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 1H), 7.51 (d, J = 9 Hz, 2H), 7.12 (s, 1H), 6.89 (d, J = 2.5 Hz, 1H), 6.82 (dd, J_AB = 9 Hz, J_CD = 2.5 Hz, 1H), 6.75 (d, J = 9 Hz, 2H), 3.81 (s, 3H), 3.73 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 163.96, 163.31, 160.01, 157.14, 147.35, 144.52, 134.99, 134.61, 134.34, 130.55, 127.94, 126.54, 126.06, 126.03, 124.51, 122.34, 120.46, 113.95, 113.50, 111.81, 109.09, 55.64; HRMS (ESI) calculated for C₂₅H₁₉F₃N₂O₆S₂, 564.0637, found 564.0637.

2.1.8 5-Methoxy-2-(2-(4-methoxybenzamido)thiazol-4-yl)phenyl 4-methoxybenzenesulfonate (8g). Pale yellow solid (15.48%; 3 steps); ¹H NMR (400 MHz, d-acetone): δ 8.20 (d, J = 9.2 Hz, 2H), 7.74 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.21 (s, 1H), 7.12 (d, J = 9.2 Hz, 2H), 6.97 (dd, J_AB = 8.4 Hz, J_CD = 2.4 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 3.92 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H); ¹³C NMR (125 MHz, d-acetone): δ 165.22, 165.16, 164.27, 160.64, 158.32, 148.38, 145.19, 131.86, 131.50, 130.87, 127.25, 125.41, 122.17, 114.98, 114.84, 113.87, 111.95, 109.68, 56.25, 56.10, 56.00; HRMS (ESI) calculated for C₂₅H₂₂N₂O₇S₂, 526.0868, found 526.0871.

2.1.9 5-Methoxy-2-(2-(4-nitrobenzamido)thiazol-4-yl)phenyl 4-methoxybenzenesulfonate (8h). Yellow solid (20.95%; 3 steps); ¹H NMR (500 MHz, CDCl₃): δ 8.33 (d, J = 8.5 Hz, 2H), 8.10 (d, J = 8.5 Hz, 2H), 7.55–7.53 (m, 3H), 7.13 (s, 1H), 6.83–6.77 (m, 4H), 3.79 (s, 3H), 3.75 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 164.00, 162.83, 160.06, 157.32, 150.29, 147.33, 144.61, 137.25, 130.59, 130.54, 128.71, 126.56, 124.10, 120.39, 114.02, 113.41, 111.90, 108.96, 55.68, 55.66; HRMS (ESI) calculated for C₂₄H₁₉N₃O₈S₂, 541.5530, found 541.0615.

2.1.10 5-Methoxy-2-(2-propionamidothiazol-4-yl)phenyl 4-methoxybenzenesulfonate (9a). White solid (18.84%; 3 steps); ¹H NMR (400 MHz, CDCl₃): δ 7.53 (d, J = 8.8 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.02 (s, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.83 (dd, J_AB = 8.8 Hz, J_CD = 2.4 Hz, 1H), 6.73 (d, J = 9.2 Hz, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 2.43 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 171.37, 163.90, 159.90, 157.07, 147.39, 143.80, 130.57, 130.54, 126.47, 120.59, 113.85, 113.53, 111.27, 109.05, 55.68, 55.62, 29.44, 9.01; HRMS (ESI) calculated for C₂₀H₂₀N₂O₆S₂, 448.0763, found 448.0763.

2.1.11 2-(2-Butyramidothiazol-4-yl)-5-methoxyphenyl 4-methoxybenzenesulfonate (9b). Pale yellow solid (15.63%; 3 steps); ¹H NMR (400 MHz, CDCl₃): δ 9.46 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 9.2 Hz, 2H), 7.03 (s, 1H), 6.94 (d, J = 2.8 Hz, 1H), 6.83 (dd, J_AB = 8.4 Hz, J_CD = 2.8 Hz, 1H), 6.74 (d, J = 8.8 Hz, 2H), 3.81–3.79 (m, 6H), 2.37 (t, J = 7.6 Hz, 2H), 1.78–1.72 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 170.56, 163.92, 159.86, 156.79, 147.38, 143.92, 130.57, 130.56, 126.51, 120.68, 113.85, 113.51, 111.28, 108.99, 55.67, 55.63, 38.14, 18.47, 13.64; HRMS (ESI) calculated for C₂₁H₂₂N₂O₆S₂, 462.0919, found 462.0919.

2.1.12 2-(2-(N-Acetylacetamido)thiazol-4-yl)-5-methoxyphenyl 4-methoxybenzenesulfonate (9c). Orange oil (16.80%; 3 steps); ¹H NMR (500 MHz, CDCl₃): δ 7.72 (d, J = 8.5 Hz, 1H), 7.68 (s, 1H), 7.54 (d, J = 9 Hz, 2H), 6.89 (d, J = 2.5 Hz, 1H), 6.82 (dd, J_AB = 8.5 Hz, J_CD = 2.5 Hz, 1H), 6.79 (d, J = 9 Hz, 2H), 3.79 (s, 6H), 2.34 (s, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 176.39, 168.11, 163.84, 160.11, 158.59, 147.36, 143.17, 130.49, 130.44, 126.17, 120.11, 113.76, 113.65, 111.20, 109.42, 55.68, 55.52, 22.92, 21.04; HRMS (ESI) calculated for C₂₁H₂₀N₂O₇S₂, 476.0712, found 476.0712.

2.1.13 5-Methoxy-2-(2-(N-propionylpropionamido)thiazol-4-yl)phenyl 4-methoxybenzenesulfonate (9d). Pale yellow solid (30.45%; 3 steps); ¹H NMR (400 MHz, CDCl₃): δ 7.72 (d, J = 8.8 Hz, 1H), 7.66 (s, 1H), 7.53 (d, J = 9.2 Hz, 2H), 6.92 (d, J = 2.8 Hz, 1H), 6.83–6.79 (m, 3H), 3.80–3.79 (m, 6H), 2.65 (t, J = 7.2 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 175.69, 164.16, 160.04, 157.56, 147.53, 147.09, 131.08, 130.65, 126.05, 120.07, 118.63, 114.09, 113.34, 108.49, 55.68, 55.62, 31.67, 8.74; HRMS (ESI) calculated for C₂₃H₂₄N₂O₇S₂, 504.1025, found 504.1027.

2.1.14 2-(2-(N-Butyrylbutyramido)thiazol-4-yl)-5-methoxyphenyl 4-methoxybenzenesulfonate (9e). Orange oil (20.63%; 3 steps); ¹H NMR (400 MHz, CDCl₃): δ 7.46–7.42 (m, 3H), 6.98 (s, 1H), 6.97 (d, J = 2.4 Hz, 2H), 6.84 (dd, J_AB = 8.8 Hz, J_CD = 2.4 Hz, 1H), 6.70 (d, J = 8.8 Hz, 2H), 3.82 (s, 3H), 3.76 (s, 3H), 2.43–2.30 (m, 4H), 1.80–1.65 (m, 6H), 1.02 (t, J = 7.2, 4H); ¹³C NMR (125 MHz, CDCl₃): δ 170.85, 163.87, 159.97, 157.61, 147.37, 143.53, 130.53, 130.50, 126.31, 120.41, 113.78, 113.59, 111.23, 109.21, 55.68, 55.57, 38.09, 18.48, 13.66; HRMS (ESI) calculated for C₂₅H₂₈N₂O₇S₂, 532.1338, found 532.1339.

2.2. Biology

2.2.1 Cell culture and reagents. HepG2 2.2.15 cells, derived from HepG2 human hepatocellular carcinoma cells, were stably transfected with a head-to-tail HBV DNA dimer¹⁶ and were maintained in MEM with heat-inactivated 10% fetal bovine serum (FBS) and 1% antibiotics and grown at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Lamivudine (2′,3′-dideoxy-3′-thiacytidine, commonly known as 3TC) was purchased from Sigma (St. Louis, MO, USA). Power SYBR Green PCR master mix was purchased from Applied Biosystems (Foster City, CA, USA). MTS reagent was purchased from Promega (Madison, WI, USA).

2.2.2 Cell viability assay. The cytotoxic effects of compounds were determined using a CellTiter 96® AQueous one solution cell proliferation assay kit (MTS) (Promega, Madison, WI, USA). In order to pinpoint the toxicity limits in HepG2 2.2.15, they were plated into 96-well plates at a density of 4 × 10⁴ cells per mL for 24 h. Cells were then treated with serial dilutions of compounds, ranging 1–1000 μM, and the mixture was incubated for 3 days. Cell toxicity was measured according to the manufacturer's protocol. All measurements were performed in four replicates, and results are presented as relative percentages over that of the control group.

2.2.3 Determination of HBsAg and HBeAg expression levels. After treating HepG2 2.2.15 cells, the levels of the HBsAg and HBeAg proteins were measured in culture media using an EIA kit (Johnson and Johnson, Skillman, NJ, USA) according to the manufacturer's instructions.

2.2.4 Determination of HBV replicated DNA levels. To determine the viral DNA level from cell cultures, virion HBV DNA from conditioned media that had been either untreated or treated with compounds or lamivudine (3TC) was isolated using NucleoSpin Blood mini-kit (Macherey-Nagel) and subjected to real-time PCR analysis. A real-time PCR analysis of HBV DNA used 5′-AGGAGGCTGTAGGCATAAATTGG-3′ as the forward primer and 5′-CAGCTTGGAGGCTTGAACAGT-3′ as the reverse primer.¹⁷ The PCR was performed using SYBR Green PCR master mix, and the reaction protocol was as follows: initial denaturation at 50 °C for 2 min and 95 °C for 10 min, followed by 45 cycles of amplification at 95 °C for 15 s and annealing/extending at 58 °C for 1 min.¹⁸

2.2.5 Statistical analysis and quantification of data. The data were expressed as the mean and the standard deviation (SD) of the mean from three independent experiments and the TC₅₀ or IC₅₀ value was determined according to non-linear regression fitting analysis procedure by using GraphPad Prism (GraphPad Software Inc.). Variance analysis and the Student's t-test were used for data analysis. Differences were considered significant when P < 0.05.

2.2.6 Giemsa staining and flow cytometry. The assessments of cell morphology and the cell cycle of HepG2 2.2.15 cells after treatment with compound 8a were determined by Giemsa staining and propidium iodide (PI) staining. HepG2 2.2.15 cells were seeded into 6 cm dishes at a density of 2 × 10⁵ cells per dish for 24 h. Cells were then treated with serial dilution of compound 8a for 72 h. After 72 h, cells were washed with ice-cold phosphate-buffered saline (PBS) for drying, then fixed with methanol and stained with Giemsa stain solution (Gibco, Grand Island, NY, USA) to observe the nuclei and morphology. For flow cytometry, cells were harvested and fixed with ice cold 70% ethanol at 4 °C for 24 h. After the washes, cells were stained PI staining solution (20 mg mL⁻¹ PI in 0.3 mL of PBS containing 200–400 unit of RNase A), followed by incubation at 37 °C for 30 min in the dark. The cells were analyzed by flow cytometry (Accuri C6, Becton Dickenson) to determine the proportion of cells within cycle.

3. Results and discussion

3.1. Synthesis

To obtain various 2-aminothiazole core-modified paeonol derivatives with 4-methoxyphynylsulfonyl side chains on the phenol group, we first treated paeonol with 4-methoxyphenylsulfonyl chloride to yield 6f (Scheme 1). Then, 6f was reacted with thiourea and iodine in ethanol under reflux conditions, which afforded compound 7 (Scheme 2). Amino groups on thiazole rings were then processed through nucleophilic substitutions with 4-position substituted benzoyl chlorides and aliphatic acyl chlorides to generate the desired compounds (Scheme 2). All the products were purified to more than 95% by column chromatography for bioassays.

3.2. Cytotoxic effect of the compounds on HepG2 2.2.15 hepatoma cells

To understand the structure–activity relationship (SAR) and determine the cytotoxic effect of compounds 8a–9e on HepG2 2.2.15 cells, the cells were subjected to treatment with each compound which was diluted in a two-fold serial order, for 72 h, and the viability of the cells was measured according to the manufacturer's protocol. All measurements were performed four times and results were presented as a relative percentage of those of the control group. In Table 1, according to TC₅₀ values, compound 8a, 8e, 8f, 8g and 8h are less cytotoxic when compared to 5-FU, and substituting H atom at para positions on benzoyl rings with halogen atoms F, Cl and Br, which correspond to compounds 8b, 8c and 8d, increases the cytotoxicity. To our surprise, replacing the aromatic ring with more flexible aliphatic alkyl chains, which corresponds to compounds 9a–9e, significantly enhances the cytotoxic effect. Additionally, the activity of compound 8b bearing F, the bioisostere of H, is similar to that of compound 8a. Another two bioisostere sets, Cl and Br on compounds 8c and 8d and CH₃ and CF₃ on compounds 8e and 8f, show the same phenomenon as those of 8a and 8b. Compared with compounds 8a–8d, activities of compounds 8f–8h are much lower, and this may result from relatively larger steric hindrances caused by the 4-position substituting groups on benzene rings of 8f–8h. These results suggest that the 4-position on the benzoyl ring may play a crucial role in the anti-HBV actions of these compounds. In addition, displacing a relatively rigid aromatic moiety with a more flexible aliphatic alkyl chain on the side chain of amide group may, however, endow the compound a much higher cytotoxicity thus lowering its SI value drastically.

Table 1 Cytotoxic effect of compounds 8a–9e on HepG2 2.2.15 cells, inhibition potential of HBV viral antigen and DNA replication for 72 h treatment^a

Compound	TC50 (μM)	HBsAg IC50 (μM)	HBeAg IC50 (μM)	Inhibition of HBV DNA replication IC50 (μM)	HBV DNA replication SI value (TC50/IC50)
a TC50: the concentration of the compound at which cell viability was reduced to 50%. IC50: the concentration of the compound at which anti-HBV effect was reached to 50%. *5-FU: fluorouracil, the positive control for cytotoxic analysis; the structure is shown in Fig. S2 of the ESI. *Lamivudine (3TC), the positive control for anti-HBV analysis; the structure is shown in Fig. S2 of the ESI. SI: selectivity index; TC50/IC50.
8a	373.99 ± 5.52	3.30 ± 0.32	4.27 ± 0.32	6.32 ± 0.31	59.14 ± 3.51
8b	145.47 ± 3.45	3.96 ± 0.12	8.34 ± 1.34	12.67 ± 1.21	11.48 ± 1.32
8c	144.80 ± 4.21	5.52 ± 0.6	5.48 ± 0.45	7.25 ± 1.41	19.97 ± 4.72
8d	204.80 ± 3.36	8.13 ± 0.24	19.88 ± 2.12	17.78 ± 2.87	11.52 ± 2.22
8e	508.32 ± 3.54	107.09 ± 2.25	173.41 ± 3.42	153.26 ± 3.65	3.32 ± 0.1
8f	416.93 ± 4.32	86.21 ± 1.64	116.87 ± 4.5	99.81 ± 1.78	4.18 ± 0.1
8g	479.78 ± 4.65	85.99 ± 2.57	173.76 ± 3.74	161.65 ± 4.43	2.97 ± 0.1
8h	581.53 ± 4.45	83.76 ± 3.68	96.52 ± 3.21	84.24 ± 3.12	6.90 ± 0.3
9a	54.29 ± 2.33	6.22 ± 0.12	7.63 ± 1.36	5.89 ± 0.21	9.22 ± 0.59
9b	12.89 ± 2.24	2.85 ± 0.25	4.02 ± 0.64	4.00 ± 0.87	3.22 ± 1.1
9c	47.20 ± 2.67	3.67 ± 0.64	4.13 ± 1.41	3.44 ± 0.11	13.71 ± 1.03
9d	13.02 ± 1.21	N/A	N/A	N/A	N/A
9e	64.98 ± 3.47	5.73 ± 0.22	11.36 ± 2.21	13.99 ± 0.84	4.65 ± 0.43
5-FU*	216.79 ± 5.74	—	—	—	—
Lamivudine (3TC)*	352.03 ± 4.38	—	—	7.63 ± 0.41	46.13 ± 2.95

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3.3. Antiviral effect of compounds on HBV viral antigen expression in HepG2 2.2.15 cell culture medium

Fig. 2 demonstrates the inhibition effect of compounds 8a–9e on HBV viral antigen expression and secretion in HepG2 2.2.15 cell culture media. All compounds could effectively reduce secretion of HBsAg and HBeAg at low doses, and compound 8a had the most potent inhibitory effect. As concentrations of compounds 8a–d and 9a–e increased, the expression of HBsAg and HBeAg decreased; on the other hand, the results did not show pronounced changes when cells were treated with compounds 8e–8h. Such phenomena may be attributed to the relatively large steric hindrance that the 4-position substituting groups cast on the benzene rings of compounds 8e–8h.

Fig. 2 Effects of compounds 8a–9e on HBV viral (A) HBsAg and (B) HBeAg secretion. HepG2 2.2.15 cells were treated with three concentrations (0.75, 1.5 and 3.0 μM) of compounds 8a–9e or treated with DMSO 0.1% (v/v) (control) for 72 h. The cultural media of each treatment were collected for viral HBsAg and HBeAg EIA analysis. The data are expressed as the mean and the standard deviation of the mean (n = 3, * P < 0.05 vs. untreated cells).

To further confirm the effect of 8a on HepG2 2.2.15 proliferation, we assessed the morphology of growth response of cells employing the Giemsa staining method. The general cellular morphology after 8a treatment is shown in Fig. 3A. There was no significant difference in the morphology of HepG2 2.2.15 cells in the groups treated with compound 8a and the control group (Fig. 3A). To examine the role of compound 8a in HepG2 2.2.15 cell cycle progression, we performed flow cytometry analysis. DNA of the HepG2 2.2.15 cells were stained with PI to analyze the population of DNA content as index of cell cycle after treatment with 8a (Fig. 3B). The ratio of cells in each phase of the cell cycle did not decline as the dose of 8a rose. These results suggest that 8a did not affect the HepG2 2.2.15 cell growth or cell cycle in treated dose range that have inhibitory effect on HBV.

Fig. 3 Effect of 8a on cellular morphology and DNA content analysis of HepG2 2.2.15 cells. HepG2 2.2.15 cells were exposed to three non-cytotoxic concentration (0.75, 1.5 and 3.0 μM) of 8a. After 72 h of culture, cells were fixed and stained with Giemsa's solution to identify the morphology of HepG2 2.2.15 cells. (A) Based on cellular morphology, inhibition of HepG2 2.2.15 cell growth was not observed in cells treated with 8a. (B) Cell cycle distribution analysis of HepG2 2.2.15 treated with 8a was performed by flow cytometry.

3.4. Antiviral effect of compounds on HBV DNA replication in HepG2 2.2.15 cells

To investigate the antiviral activity of compounds in HBV DNA replication, HepG2 2.2.15 cells were treated with three non-cytotoxic concentrations of each compound for 48 h. The cultural media were harvested and the viral genomic DNA of the secreted virion particles was isolated using real-time PCR analysis. The results, shown in Fig. 4 reveal that compounds 8a–9e could reduce the replication amounts of viral DNA at low doses, and in comparison with lamivudine (3TC), compounds 8a had apparent inhibitory effects on viral DNA replication whereas 8e–8h did not. Fig. 5 illustrates the selectivity index (SI; TC₅₀/IC₅₀) values of each compound on viral DNA replication, and indicates that the SI value of compound 8a is the highest of all the novel compounds and even better than those of the commercially available antiviral drug 3TC and our previously synthesized compound 6f.

Fig. 4 Effects of compounds 8a–9e on HBV DNA replication in HepG2 2.2.15 cells. Cells were treated with three concentrations (0.75, 1.5 and 3.0 μM) of each compound or treated with DMSO 0.1% for 72 h and culture media of each treatment were collected for viral DNA extraction. The detection of viral DNA was conducted by real-time PCR analysis. The data are expressed as the mean and the standard deviation of the mean (n = 3) (* P < 0.05 vs. untreated cells).

Fig. 5 The selectivity index SI (TC₅₀/IC₅₀) of compound 8a–9e on HBV DNA level.

3.5. Effect of compound 8a on HBV viral gene expression

We further examined the antiviral effect of 8a, which showed potent inhibition of HBV viral gene expression. HepG2 2.2.15 cells were treated with three non-cytotoxic concentrations of compound 8a for 48 h, and the total cellular RNA was extracted and subjected to northern blot analysis of HBV viral RNA levels. As shown in Fig. 6, treatment of 8a significantly lowered the levels of 3.5 kb precore/pregenomic and 2.4-/2.1 kb surface antigen RNA in a dose-dependent manner.

Fig. 6 Effects of 8a on HBV RNA expression in HepG2 2.2.15 cells. (A) HepG2 2.2.15 cells were treated with three non-cytotoxic concentrations (0.75, 1.5 and 3.0 μM) of 8a for 72 h and total cellular RNA was extracted and subjected to northern blot analysis. (B) The intensity of each RNA band was quantitated with a densitometer and the relative amount was normalized with GAPDH loading control. The data shown are representative of three replicate experiments (* P < 0.05 vs. untreated cells).

4. Conclusion

Based on our previous study, we further optimized the 4-methoxyphenylsulfonyl modified paeonol with a 2-aminothiazole core and generated 13 novel paeonol derivatives by utilizing various benzoyl and acyl chlorides. All the compounds were characterized and their anti-HBV activities were assessed. We conducted cell viability assessment, cell cycle analysis, and dose-dependent inhibition experiments on viral DNA replication and HBV gene and viral antigen expression to address the SAR and mechanism issues. The results showed that compound 8a had potent activity against HBV in HepG2 2.2.15 cells with IC₅₀ value of 6.32 μM and a remarkable selectivity index (TC₅₀/IC₅₀) of 59.14 that surpassed those of our previously synthesized compound 6f and 3TC, a commercially available antiviral nucleoside medicine. The compound may be a promising lead for anti-HBV therapy.

Acknowledgements

For financial support, we thank the Ministry of Science and Technology of the Republic of China, National Tsing Hua University, China Medical University Hospital, and Show Chwan Memorial Hospital. This work was supported by the Ministry of Science and Technology of Taiwan (Grant 104-2119-M-007-017-) (Grant 105-2623-E-007-009-), China Medical University Hospital (DMR-103-042) (DMR-103-071) (DMR-105-044), and Show Chwan Memorial Hospital Research Foundation (RD105020). The authors express heartfelt thanks to Dr Pei-Jer Chen for HepG2 2.2.15 cells as a model for antiviral research, the Laboratory Animal Core Facility which is funded by the Agricultural Biotechnology Research Center (ABRC) at Academia Sinica for technical support in cell morphology and flow cytometry experiments, and Ms Miranda Jane Loney (ABRC, Academia Sinica, Taiwan) for her critical editing of this manuscript.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra06119b

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