HETEROCYCLES

Short Paper | Regular issue | Vol. 87, No. 6, 2013, pp. 1349-1358
Received, 9th April, 2013, Accepted, 7th May, 2013, Published online, 13th May, 2013.
DOI: 10.3987/COM-13-12726

■ A FACILE SYNTHESIS OF (5-HYDROXY-4-OXO-4H-PYRAN-2-YL)METHYL CARBOXYLATES AND THEIR ANTIVIRAL ACTIVITY AGAINST HEPATITIS C VIRUS

Tetsuro Shimo,* Yuki Taketsugu, Takuya Goto, Masaaki Toyama, Kohji Yoshimura, and Masanori Baba

Department of Chemistry, Faculty of Engineering, Kagoshima University, Korimoto, Kagoshima 890-0065, Japan

Abstract

5-Benzyloxy-2-hydroxymethyl-4H-pyran-4-one (2) was synthesized from kojic acid (1) and subsequently reacted with carboxylic anhydride (3a) and a series of carboxylic acid chlorides (3b–l) to give the corresponding (5-benzyloxy-4-oxo-4H-pyran-2-yl)methyl carboxylates (4a–l). These compounds were then reductively debenzylated to afford the (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl carboxylates (5a-l), which were tested for their inhibitory activities against the hepatitis C virus.

Hepatitis C virus (HCV) infection is a worldwide problem. In general, HCV infection proceeds to chronic infection,1 which often induces cirrhosis of the liver and hepatocellular carcinoma.2 Liver transplantation is currently the only treatment available to patients with the severe end-stage liver disorders caused by HCV infection.3 To date, no protective vaccines have been developed for HCV, and pegylated interferon (PEG-IFN) and the nucleoside analogue ribavirin are the standard treatments for HCV infection.4-6 Unfortunately, however, many patients cannot tolerate the serious side effects associated with the use of PEG-IFN and ribavirin. It is well known that kojic acid, which is otherwise known as 5-hyroxy-2-hydroxymethyl-4H-pyran-4-one and isolated as a fermentation product of the Aspergillus species, inhibits tyrosinase activity through the chelation of copper,7 which is essential for tyrosinase activity.8 Kojic acid and its derivatives have also been reported to prevent photodamage9 by inhibiting nitric oxide (NO) production,10 express depigment activity,11 and act as Histamine H3 receptor ligands.12 To the best of our knowledge, however, kojic acid and its derivatives have not been examined for their anti-HCV activity. Since kojic acid possesses a chelating moiety that enables it to form bidentate complexes with a variety of different metals, we describe herein a facile synthesis of (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl carboxylates (5) possessing two such functional groups for chelating, and provide an evaluation of their anti-HCV activity.

(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl carboxylates (5a-l) were synthesized in three steps from kojic acid (1), as shown in Scheme 1. Thus, kojic acid (1) was reacted with benzyl chloride under basic conditions to give 5-benzyloxy-2-hydroxymethyl-4H-pyran-4-one (2). The 2-hydroxymethyl group of 2 was then esterified with carboxylic anhydride (3a) and a series of carboxylic acid chlorides (3b–l) to give the corresponding (5-benzyloxy-4-oxo-4H-pyran-2-yl)methyl carboxylates (4a–l), which were reductively debenzylated to afford the (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl carboxylates (5a-l).　 Compound 2 was prepared from 1 in 88% yield according to the method previously described in the literature.13a The reaction of the compound 2 with propionic anhydride (3a) in pyridine gave (5-benzyloxy-4-oxo-4H-pyran-2-yl)methyl propionate 4a in 75% yield, and the material was subsequently reductively debenzylated with hydrogen in the presence of Pd/C to give (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl propionate 5a in 72% yield. Similarly, the reactions of 2 with a variety of acyl chlorides (3b-c) and aroyl chlorides (3d-l) afforded the corresponding products (4b-l), which were also reductively debenzylated to give the corresponding products (5b-l). The results of these reactions are summarized in Table 1.

With our compounds in hand, we proceeded to investigate the cytotoxicity and anti-HCV activity of kojic acid (1) and its derivatives (5a-l) in subgenomic HCV replicon cells (LucNeo#2). The results of these experiments are shown in Table 2. Compounds 5e and 5l, which contained a p-methoxy phenyl or 2-naphthyl group as their carboxylates, respectively, showed higher anti-HCV activity than the other compounds, but were less active than the HCV NS3 protease inhibitor telaprevir. Since products 5e and 5l, which have hydrophobic or bulky substituent, showed higher anti-HCV activity, the structure of the acceptor may have hydrophilic substituent and relatively wide cavity.
In summary, a series of (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl carboxylates (5a-l) were easily synthesized from kojic acid in relatively good yields over three steps. Given that compounds 5e and 5l showed the highest levels of anti-HCV activity of this particular compound series, our laboratory is currently involved in the synthesis of further kojic acid derivatives.

EXPERIMENTAL
All melting points were measured on Yanagimoto Melt-temp apparatus and uncorrected. NMR spectra were measured at 400 MHz on the JNM GSX-400 (TMS as an internal standard). IR spectra were recorded with a JASCO IR Report-100 spectrometer. Mass spectra were recorded with a JEOL JMS-HX110A (FABMS) using m-nitrobenzyl alcohol as matrix. Elemental analysis was made using a Yanaco MT-5. PLC Silicagel 60 F254 (2 mm) was used for preparative TLC and Wakogel 200 was used for preparative column chromatography.

5-Benzyloxy-2-hydroxymethyl-4H-pyran-4-one (2) ….. A solution of kojic acid (1) (3.00 g, 21.1 mmol) benzyl chloride (3.85 g, 22.5 mmol) and sodium hydroxide (0.870 g, 21.8 mmol) in MeOH (40 mL) was refluxed for 5 h. After removing the solvent in vacuo, to the reaction mixture was added cold water (40 mL). The resulting solid was filtered and recrystallized from EtOH to give 2 (4.30 g, 88% yield).
2: mp 131 – 133 °C (lit., mp 132 °C,13a 134 –136 °C13b). 1H NMR (CDCl3) δ 2.12 (1H, s), 4.45 (2H, s), 5.08 (2H, s), 6.51 (1H, s), 7.36 (5H, m), 7.52 (1H, s). LR MS m/z 233(MH+). HR MS (MH+) calcd for C13H13O3 233.0814. Found: 233.0808.

(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl propionate (5a)….. Propionic anhydride (3a) (252 mg, 1.94 mmol) was added to a solution of 2 (300 mg, 1.29 mmol) in pyridine (12 mL) and the solution was heated at 60 °C for 20 h. After the solution was evaporated in vacuo, a mixture of 1M NaHCO3 aqueous solution (50 mL) and CHCl3 (50 mL) was added to the residue. The separated organic layer was dried by MgSO4 and the filtrate was evaporated in vacuo to give 4a (300 mg, 75% yield) which was used to the next reaction without further purification.
4a: 1H NMR (CDCl3) δ 1.20 (3H, t, J = 7.6 Hz), 2.45 (q, J = 7.6 Hz), 4.83 (2H, s), 5.05 (2H, s), 6.42 (1H, s), 7.33 (5H, m), 7.54 (1H, s).
A solution of 4a (100 mg, 0.35 mmol) in MeOH (5 mL) containing 5% Pd-C (50 mg) was vigorously stirred for 1 h at room temperature under a hydrogen atmosphere. After the catalyst was removed by filtration, the filtrate was evaporated to dryness in vacuo. The residue was purified by the use of preparative TLC (eluent: EtOAc:hexane = 1:1) to give 5a (50 mg, 72% yield).
5a: mp 77 – 79 °C (from EtOAc:hexane = 1:1, v/v). 1H NMR (CDCl3) δ 1.19 (3H, q, J = 7.6 Hz), 2.43 (2H, t, J = 7.6 Hz), 4.93 (2H, s), 6.48 (1H, s), 7.84 (1H, s). IR (KBr) 1730, 1679 cm-1. LR MS m/z 199 (MH+). HR MS calcd for C9H11O5 199.0603. Found: 199.0621.

(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl stearate (5b)….. Stearoyl chloride (3b) (312 mg. 1.03 mmol) was added to a solution of 2 (200 mg, 0.86 mmol) in pyridine (12 mL) and the solution was heated at 60 ºC for 24 h. After the solution was evaporated in vacuo, a mixture of 1M NaHCO3 aqueous solution (50 mL) and CHCl3 (50 mL) was added to the residue. The separated organic layer was dried by MgSO4 and the filtrate was evaporated in vacuo and the resulting oily residue was chromatographed by silica gel (eluent: EtOAc/hexane = 1:1, v/v) to afford 4b (189 mg, 69% yield).
4b: δ 0.85 (3H, t, J = 7.6 Hz), 1.55 (28H, m), 1.66 (2H, m), 2.40 (t, J = 7.6 Hz), 4.84 (2H, s), 5.03 (2H, s), 6.42 (1H, s), 7.35 (5H, m), 7.54 (1H, s).
A solution of 4b (100 mg, 0.20 mmol) in a 1:1 mixture of MeOH and CHCl3 (6 mL) containing 5% Pd-C (50 mg) was vigorously stirred for 24 h at room temperature under a hydrogen atmosphere. After the catalyst was removed by filtration, the filtrate was evaporated to dryness in vacuo. The residue was purified by the use of preparative TLC (eluent: EtOAc:hexane = 1:1, v/v) to give 5b (50 mg, 72% yield).
5b: mp 90 – 93 °C (from EtOAc:hexane = 1:1, v/v). 1H NMR (CDCl3) δ 0.88 (3H, t, J = 7.6 Hz), 1.52 (28H, m), 1.64 (2H, m), 2.38 (2H, t, J = 7.6 Hz), 4.92 (2H, s), 6.48 (1H, s), 7.84 (1H, s). IR (KBr) 1732, 1655 cm-1. LR MS m/z 409 (MH+). Anal. Calcd for C24H41O5: C, 70.55, H, 9.87. Found: C, 70.11, H, 9.90.

(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl benzoate (5c)….. The reaction of 2 (300 mg, 1.29 mmol) with benzoyl chloride (3c) (270 mg, 2.0 mmol) in pyridine (10 mL) was carried out, according to the similar reaction of 2 with 3b, to give 4c (380 mg, 88% yield) which was used to the next reaction without further purification.
4c: 1H NMR (CDCl3) δ 5.02 (2H, s), 5.18 (2H, s), 6.54 (1H, s), 7.40 (5H, m), 7.50 (2H, t, J = 8.5 Hz), 7.54 (1H, s), 7.62 (1H, t, J = 8.5 Hz), 8.05 (2H, d, J = 8.5 Hz).
The reduction of 4c (100 mg, 0.30 mmol) in MeOH (5 mL) with Pd-C (50 mg) under a hydrogen atmosphere was carried out, according to the same treatment of 4b, to afford 5c (65 mg, 88%).
5c: mp 180 – 182 °C (from EtOAc:hexane = 1:1, v/v) (180-181 ºC).14 1H NMR (CDCl3) δ 5.18 (2H, s), 6.60 (1H, s), 7.48 (2H, t, J = 8.5 Hz), 7.62 (1H, t, J = 8.5 Hz), 7.84 (1H, s), 8.07 (2H, d, J = 8.5 Hz). IR (KBr) 1742, 1685 cm-1. LR MS m/z 247 (MH+). HR MS (MH+) calcd for C13H11O5.247.0603. Found: 247,0615.

The results of similar reactions of 2 with 3d-l and the reduction of 4d-l to afford (5-hydroxy- 4-oxo-4H-pyran-2-yl)methyl p-methylbenzoate (5d), (5-Hydroxy-4-oxo-4H–pyran-2-yl)methyl p-methoxybenzoate (5e), (5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl p- ethoxybenzoate (5f), (5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl p-fluorobenzoate (5g), (5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl p-chlorobenzoate (5h), (5-Hydroxy-4-oxo-4H- pyran-2-yl)methyl p-trifluoromethylbenzoate (5i), (5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl 3,5-methylbenzoate (5j), (5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl 1-naphthylbenzoate (5k), (5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl 2-naphthylbenzoate (5l) are summarized in Table 3.
The anti-HCV activity of the test compounds was determined in LucNeo#2 cells by the previously described method with some modifications.15 Briefly, the cells (5 × 103 cells/well) were cultured in a 96-well plate in the absence of G418 and in the presence of various concentrations of the compounds. After incubation at 37 °C for 3 days, the culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Lysis buffer was added to each well, and the lysate was transferred to the corresponding well of a non-transparent 96-well plate. The luciferase activity was measured by addition of the luciferase reagent in a luciferase assay system kit (Promega) using a luminometer with automatic injectors (Berthold Technologies).

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