A facile synthesis of (5-hydroxy-4-oxo-4H-pyran-2-YL)methyl carboxylates and their antiviral activity against hepatitis C virus

Article abstract of DOI:10.3987/COM-13-12726

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.

Full text of DOI:10.3987/COM-13-12726

HETEROCYCLES, Vol. 87, No. 6, 2013
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HETEROCYCLES, Vol. 87, No. 6, 2013, pp. 1349 - 1358. © 2013 The Japan Institute of Heterocyclic Chemistry
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,a,* Yuki Taketsugu,a Takuya Goto,a Masaaki Toyama,b
Kohji Yoshimura,b and Masanori Babab
aDepartment of Chemistry, Biotechnology and Chemical Engineering, Graduate
School of Science and Engineering, Kagoshima University, 1-21-40, Korimoto,
Kagoshima 890-0065, Japan
bDivision of Antiviral Chemotherapy, Center for Chromic Viral Diseases,
Graduate School of Medicinal and Dental Sciences, Kagoshima University,
8-35-1, Sakuragaoka, Kagoshima, 890-8544, 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,

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HETEROCYCLES, Vol. 87, No. 6, 2013
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.

HETEROCYCLES, Vol. 87, No. 6, 2013
1351
OH
OH
OCOR
OCOR
(RCO)2O 3a or
2
O
O
O
BnCl
3b l
O
RCOCl
-
Pd/C, H2
NaOH
pyridine
5
O
O
OBn
OH
O
OBn
O
OH
2
1
4
5
3a: R = Et, 3b:R = Me(CH2)16,
3c: R = C6H5, 3d:R=C6H4CH3(p),
3e: R = C6H4OMe(p),
4a, 5a: R = Et
4b, 5b: R = Me(CH2)16
4c, 5c: R = C6H5
4d 5d
, : R = C6H4Me(p)
4e, 5e: R = C6H4OMe(p)
4f, 5f: R = C6H4OEt(p)
3f
: R = C6H4OEt(p),
3g: R = C6H4F(p), 3h:R=C6H4Cl(p),
3i: R = C6H4CF3(p),
3j: R = C6H3Me2(3,5-),
4g 5g
,
: R = C6H4F(p)
4h 5h
, : R = C6H4Cl(p)
3k
3l
: R = 1-naphthyl, :R=2-naphthyl
4i, 5i: R = C6H4CF3(p)
4j, 5j: R = C6H3Me2(3,5-)
4k 5k
,
: R = 1-naphthyl
4l, 5l: R = 2-naphthyl
Scheme 1
Table 1. Reaction of compound 2 with propionic anhydride (3a) and the acid
chlorides (3b-l)
Product (yield, %)
Entry
3
4
5
1
2
4a (75)
5a (72)
3a
3b
3c
3d
3e
3f
4b (69)
4c (88)
4d (86)
4e (63)
4f (33)
4g (80)
4h (85)
4i (63)
4j (80)
4k (84)
4l (37)
5b (72)
5c (88)
5d (80)
5e (79)
5f (45)
5g (81)
5h (79)
5i (48)
5j (80)
5k (81)
5l (88)
3
4
5
6
7
3g
3h
3i
8
9
10
11
12
3j
3k
3l

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HETEROCYCLES, Vol. 87, No. 6, 2013
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.
Table 2. Anti-HCV activity of compounds 1 and 5a-l in LucNeo#2 cells
Inhibitory activity M)
Compound
R
EC50
CC50
>100
1(Kojic acid)
>100
Et
>100
63
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5a
5b
5c
5d
5e
5f
Me(CH2)16
C6H5
23.49
C6H4Me(p)
C6H4OMe(p)
C6H4OEt(p)
C6H4F(p)
5.6
2.55±0.61
5.13±3.44
7.3
5g
5h
5i
C6H4Cl(p)
C6H4CF3(p)
C6H3Me2(3,5-)
1-naphthyl
2-naphthyl
4.15
7.91±2.24
31
5j
10.54
5k
5l
2.74±0.22

HETEROCYCLES, Vol. 87, No. 6, 2013
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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)  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.
1
4a: H 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),

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HETEROCYCLES, Vol. 87, No. 6, 2013
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 C 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)  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.
1
4c: H NMR (CDCl3)  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 C).14 1H NMR (CDCl3)  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).

HETEROCYCLES, Vol. 87, No. 6, 2013
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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
(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl
p-fluorobenzoate
p-chlorobenzoate (5h),
(5g),
(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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