The target compound was synthesized according to the literature with some modifications (Scheme 1). The omission of an aqueous recrystallization step leads to near-quantitative yields of the hydrolytically unstable chlorokojic acid (1) without sacrificing purity. Furthermore, 4-iodobenzylamine could be replaced with 4-bromobenzylamine (g), which is less costly and not sensitive towards light and air [22]. The HCl/acetic acid protocol as written in the original publication [21] did not provide us with the final inhibitor in good yield (some 39% crude yield). Reductive debenzylation (step i) did prove to be a very reliable method to obtain the deprotected molecule. The complete synthetic protocol is included below for the convenience of the reader.
2.1.1. Chemistry: General Procedures
Proton NMR spectra were recorded using a Bruker Avance 300 (300 MHz) device using tetramethylsilane as internal standard for CDCl3 and DMSO-d6. 13C-NMR spectra were recorded using a Bruker Avance 300 (75 MHz) device, using the deuterated solvent as an internal standard. FT-IR spectra were recorded on a Bruker alpha-P. Reagents were purchased from various companies, most notably TCI, Sigma-Aldrich, and Acros. Reagents were not purified prior to use unless specifically noted otherwise.
(1) Preparation of Compound
1
(Chlorokojic Acid). Kojic acid (TCI, 15 g, 0.105 mol) is placed in a round bottom flask equipped with a magnetic stirring bar. The flask is cooled in an ice bath and thionyl chloride (60 mL) is slowly added after which the ice bath is removed and the mixture is placed under an argon atmosphere. After stirring for 1.5 h a yellow mass is filtered off and washed extensively with petroleum ether. The product was an off-white solid (16.6 g, 98%): 1H-NMR (DMSO-d6) δ 4.6 (s, 2H, Cl-CH2-6), 6.6 (s, 1H, 5-H), and 8.1 (s, 1H, 2-H-) in agreement with the literature [19].
(2) Preparation of Compound
2
(Allomaltol). Chlorokojic acid 1 (2.5 g, 0.0156 mol, 1 eq) is dissolved in water (8 mL) and heated to 50°C under an argon atmosphere in a flask equipped with a magnetic stirring bar. Zinc dust (2.1 g, 0.031 mol, 2 eq) is added. An aqueous solution of HCl (4.7 mL, 0.055 mol, 3.5 eq) is slowly added to the reaction mixture under vigorous stirring, during which the temperature is increased to 70°C. After 4 h the excess zinc dust is filtered off while being hot, and the resultant liquid is extracted with dichloromethane (
3
∗
20
mL). The combined organic phase is dried over magnesium sulfate and the solvent is removed by evaporation under reduced pressure. The crude material is purified via recrystallization from isopropanol to obtain an off-white solid (1.2 g; 52%): 1H-NMR (CDCl3) δ 2.3 (s, 3H), 6.2 (s, 1H), and 7.8 (s, 1H) in agreement with the literature [19].
(3) Preparation of Compound
3
(3-Hydroxy-2-(hydroxymethyl)-6-methyl-4H-pyran-4-one). Sodium hydroxide (0.696 g, 0.017 mol, 1.1 eq) is dissolved in water (16 mL). To this solution 2 (2 g, 0.016 mol, 1 eq) a magnetic stirring bar is added. The solution is stirred for 10 minutes under an argon atmosphere. A 35% aqueous formaldehyde solution (0.36 mL, 0.017 mol, 1.1 eq) is added and the mixture is allowed to stir overnight. After acidification to pH 1 with 35% aqueous hydrochloric acid the mixture is cooled to 2°C and allowed to stand overnight. The crystalline mass is filtered off, washed with 5 mL of water, and dried via lyophilisation. An off-white solid is obtained (1.71 g, 71%): 1H-NMR (DMSO-d6) δ 2.26 (s, 3H), 4.38 (s, 2H), 5.36 (b, s, 1H), 6.21 (s, 1H), and 8.86 (b, s, 1H) in agreement with the literature [19].
(4) Preparation of Compound
4
(3-(Benzyloxy)-2-(hydroxymethyl)-6-methyl-4H-pyran-4-one). Sodium hydroxide (0.56 g, 8.93 mmol, 1.1 eq) is dissolved in water (2.4 mL) and added to a solution of 3 (2 g, 8.12 mmol, 1 eq) in methanol (1.2 mL) in a two-necked flask under argon equipped with a reflux condenser, a septum, and a magnetic stirring bar. The mixture is heated to reflux. Benzyl bromide (1.7 mL, 8.93 mmol, 1.1 eq) is slowly added and the mixture is allowed to stir overnight. The mixture is concentrated in vacuo and taken up in dichloromethane (approximately 20 mL). Inorganic salts are filtered off. The mixture is then extracted with 5% aqueous sodium hydroxide (
2
∗
20
mL) and brine (
1
∗
20
mL). The organic phase is dried with anhydrous sodium sulfate after which the solvent is removed under reduced pressure. The solid is dissolved in as little dichloromethane as possible after which heptane (or petroleum ether) is added until no more precipitation occurs. The precipitate is filtered off and dried to yield a white to off-white powdery solid (2.3 g, 73%): 1H-NMR (CDCl3) δ 2.25 (s, 3), 2.28–4.30 (d, 2H, 6.8 Hz), 5.18 (s, 2H), 6.19 (s, 1H), and 7.37 (s, 5H) in agreement with the literature [19].
(5) Preparation of Compound
5
(3-(Benzyloxy)-6-methyl-4-oxo-4H-pyran-2-carbaldehyde). Compound 4 (2 g, 1 eq, 8.18 mmol) is dissolved in 37.8 mL chloroform and 10.2 mL DMSO. To this mixture triethylamine (7 mL, 0.050 mol, 6 eq) and pyridine (0.1 mL) are added. The mixture is stirred in an ice bath under argon using a magnetic stirring bar until a temperature of 3–5°C is reached. Sulfur trioxide-pyridine complex (6.48 g, 0.040 mol, 5 eq) is subsequently added and the mixture is allowed to stir overnight. It is then extracted (
2
∗
20
mL water,
1
∗
20
mL brine) and dried over anhydrous sodium sulfate. The solvent is removed under reduced pressure using a rotavapor equipped with a cold trap (dry ice/isopropanol) to obtain the crude product as orange oil. This is diluted with 10 mL diethyl ether and transferred onto a flash column packed with silica/diethyl ether. After eluting with diethyl ether the solvent is removed under reduced pressure using a rotavapor equipped with a cold trap. Remaining volatiles are removed by a stream of nitrogen. A white to yellow crystalline solid is obtained (1.38 g, 69%): 1H-NMR (CDCl3) δ 2.32 (s, 3H), 5.49 (s, 2H), 6.31 (s, 1H), 7.35 (s, 5H), and 9.85 (s, 1H) in agreement with the literature [19]; FT-IR 1690.33 cm−1 (C=O aldehyde), 1641.24 cm−1 (C=O pyrone), 1613.12 cm−1, and 1583.88 cm−1 (C=C pyrone).
(6) Preparation of Compound
6
(3-(Benzyloxy)-6-methyl-4-oxo-4H-pyran-2-carboxylic acid). Compound 5 (1.57 g, 6.4 mmol, 1 eq) is dissolved in acetone/water (20 mL/20 mL) with a magnetic stirring bar. Sulfamic acid (0.86 g, 8.9 mmol, 1.4 eq) and sodium chlorite (80%, 0.76 g, 6.7 mmol, 1.05 eq) are added and the mixture is allowed to stir for 1 h in an open vessel. Some precipitation may occur. The mixture is subsequently evaporated under reduced pressure to remove the acetone after which the crude product is filtered off and washed with a small amount of ethanol. The product is then dried in vacuo to obtain a stark white powder (1.3 g, 78%): 1H-NMR (DMSO-d6) δ 2.29 (s, 3H), 5.10 (s, 2H), 6.40 (s, 1H), and 7.32–7.45 (m, 5H) in agreement with the literature [19]; FT-IR 1720.37 cm−1 (C=O acid), 1625.50 cm−1 (C=O pyrone), 1554.48 cm−1 and 1496.36 cm−1 (C=C pyrone), and MP (precipitated from water/acetone) 180°C.
(7) Preparation of Compound
7
(3-(Benzyloxy)-N-(4-bromobenzyl)-6-methyl-4-oxo-4H-pyran-2-carboxamide). Compound 6 (0.1 g, 0.38 mmol, 1 eq) is dissolved in dry tetrahydrofuran (4.16 mL) in a flame-dried round bottom flask equipped with a magnetic stirring bar under an argon atmosphere. N-Hydroxysuccinimide (0.045 g, 0.39 mmol, 1.02 eq) is added and the mixture stirred for 30 min. Dicyclohexylcarbodiimide (0.079 g, 0.38 mmol, 1 eq) is added as a solid and the mixture is stirred for an additional 3 h at RT under an argon atmosphere. Dicyclohexylurea is filtered off and the precipitate is washed with tetrahydrofuran (2 mL). 4-Bromobenzylamine (0.05 mL, 0.385 mmol, 1.01 eq) is added to the filtrate in a dried round bottom flask equipped with a magnetic stirring bar and a reflux condenser under an argon atmosphere. The mixture is heated to 60°C and allowed to stir overnight. The solvent is removed under reduced pressure and the residue taken up in chloroform. This solution is transferred to a chromatography column (silica, eluent chloroform/methanol 1%). After chromatography, the product is obtained as an off-white solid (0.15 g, 91%): 1H-NMR (CDCl3) δ 2.37 (s, 3H), 4.3–4.38 (d, 2H,
J
=
5.6
), 5.33 (s, 2H), 6.28 (s, 1H), 6.9–7.5 (m, 9H), and 8.06 (b, s, 1H); FT-IR 3363.82 cm−1 (ν(NH)), 1677.31 cm−1 (ν(C=O) pyrone), 1643.58 cm−1 (amide I band), 1621.41 cm−1 (ν(C=C) pyrone), 1584.93 cm−1 (ν(C=C) pyrone), 1536.21 cm−1 (amide II band), and MP (recrystallized from methanol) 118°C.
(8) Preparation of Compound
8
(N-([1,1′:4′,1′′-Terphenyl]-4-ylmethyl)-3-(benzyloxy)-6-methyl-4-oxo-4H-pyran-2-carboxamide). Compound 7 (90 mg, 0.21 mmol, 1 eq) is dissolved in toluene in a round bottom flask equipped with a reflux condenser, magnetic stirring bar, and an argon atmosphere. To this mixture are added aqueous potassium carbonate (2 M solution, 10 mL), triphenylphosphine (5.5 mg, 0.021 mmol, 0.1 eq), [1,1′-biphenyl]-4-ylboronic acid (41.5 mg, 0.21 mmol, 1 eq), and palladium acetate (4.6 mg, 0.02 mmol, 0.1 eq) after which the mixture is heated to 135°C with vigorous stirring. The mixture is allowed to stir for 24 h before being extracted with toluene (
3
∗
20
mL) and dried over sodium sulfate and the solvent is removed under reduced pressure. The crude product is purified via column chromatography (silica, chloroform/1-2% methanol): 1H-NMR (CDCl3) δ 2.37 (s, 3H), 4.44–4.5 (d, 2H,
J
=
5.7
Hz), 5.34 (s, 2H), 6.29 (s, 1H), 7.10–7.75 (m, 18H), and 8.12 (b, 1H).
(9) Preparation of Compound
9
(N-([1,1′:4′,1′′-Terphenyl]-4-ylmethyl)-3-hydroxy-6-methyl-4-oxo-4H-pyran-2-carboxamide). Compound 8 (186.5 mg) is dissolved in dichloromethane/methanol (70/30, 35 mL) in a thick-walled glass vessel. After flushing with argon, the hydrogenation catalyst is added (10% palladium on carbon). The vessel is placed in a shaker-type hydrogenation apparatus and placed under 40 psi hydrogen gas for 20 h after flushing with hydrogen gas to remove traces of oxygen. The catalyst is filtered off and the filtrate washed with 10 mL dichloromethane/methanol (50/50). The combined organic phase is evaporated in vacuo to yield an orange solid (138.5 mg, 90%): 1H-NMR (CDCl3) 2.24 (s, 3H), 4.6 (s, 2H), 6.16 (s, 1H), and 7–7.8 (m, 13H); FT-IR 2339.5 cm−1 (OH), 1633.1 cm−1 (amide I), 1591.8 cm−1 (C=O pyrone), 1537.5 cm−1 (amide II), 1483.9 cm−1 (C=C pyrone), and MP (recrystallized from toluene) 253–270°C.