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Synlett 2009(16): 2682-2684  
DOI: 10.1055/s-0029-1217971
LETTER
© Georg Thieme Verlag Stuttgart ˙ New York

A New Synthesis of 2-Cyano-6-hydroxybenzothiazole, the Key Intermediate of d-Luciferin, Starting from 1,4-Benzoquinone

Giuseppe Meronia, Paolo Cianab, Adriana Maggib, Enzo Santaniello*a
a Department of Medicine, Surgery and Dentistry, University of Milan, Polo Universitario S. Paolo, Via A. di Rudinì 8, 20142 Milan, Italy
Fax: +39(02)50323275; e-Mail: enzo.santaniello@unimi.it;
b Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
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Publication History

Received 26 June 2009
Publication Date:
09 September 2009 (online)

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Abstract

2-Cyano-6-hydroxybenzothiazole is the key intermediate for the synthesis of d-luciferin, the natural substrate of firefly luciferases. A new synthesis of 2-cyano-6-hydroxybenzothiazole has been realized starting from the reaction of 1,4-benzoquinone with l-cysteine ethyl ester, followed by oxidation-cyclization of the intermediate ethyl (R)-2-amino-3-(2,5-dihydroxyphenylsulfanyl)propan­oate hydrochloride to 2-carbethoxy-6-hydroxybenzothiazole. A suitable protection of this intermediate and a conversion to the corresponding nitrile gave, after deprotection, 2-cyano-6-hydroxybenzothiazole (32% yield from 1,4-benzoquinone). This nitrile reacts with d-cysteine to afford d-luciferin at room temperature in nearly quantitative yield (90-95%).

Key words

d-luciferin - 1,4-benzoquinone - firefly luciferase - 2-cyano-6-hydroxybenzothiazole

    References and Notes

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20

According to the original procedure, extraction with EtOAc removed most of the impurities, and evaporation of the solvents afforded the hygroscopic ester 9. In our hands, samples of different preparations showed variable purity for the final product that contained also variable amount of H2O. Therefore, the recovery (96%) refers to the weight of the final product.

22

The one-pot procedure for the preparation of the benzothiazolyl ester 10 starting from 1,4-benzoquinone 8 proceeds less satisfactorily (35-45%).

23

Among protecting groups, we have tested tert-butyldi-methyl­silyl, tetrahydropyranyl, and 2-methoxy ethoxy-methyl (MEM) ethers that did not survive or were partially hydrolyzed in the conditions required for the conversion of the 2-carboxyamide into the 2-nitrile function. Benzyl was an effective protecting group in the transfor-mation of amide 12 into nitrile 13, but removal of the protecting group by hydrogenolysis of 2-cyano-6-O-benzyl-oxy benzothiazole caused the reduction of the nitrile moiety.

25

The progress of all reactions and column chromatography were monitored by TLC (PE-EtOAc, 8:2) using silica gel 60 F254 precoated plates with a fluorescent indicator (Merck). Purification of products by chromatography was performed using silica gel 60 (230-400 mesh, Merck). All compounds gave satisfactory elemental analysis.

27

Ester 11 To a solution of the ester 10 (1.20 g, 5.38 mmol) in DMF (15 mL), K2CO3 was added (1.11 g, 8.07 mmol) and the mixture stirred at r.t. (30 min). After addition of allyl bromide (0.746 mL, 8.07 mmol) the reaction was refluxed for 1 h. After H2O addition, extraction with EtOAc, washing of the organic solution with aq NaCl, drying, and evaporation of solvent, the product is purified by chromatography (PE-EtOAc, 9:1). The 6-O-allyl ester 11 is obtained as a yellow solid (1.353 g, 96% yield); mp 97-99 ˚C. ¹H NMR (500 MHz, CDCl3): δ = 1.48 (t, 3 H, J = 8.7 Hz), 4.53 (q, 2 H, J = 8.7 Hz), 4.63 (d, 2 H, J = 5.2 Hz), 5.35 (dd, 1 H, J = 1.5, 10.5 Hz), 5.45 (dd, 1 H, J = 1.8, 17.5 Hz), 6.05 (m, 1 H), 7.18 (dd, 1 H, J = 2.4, 9.1 Hz), 7.36 (d, 1 H, J = 2.4 Hz), 8.10 (dd, 1 H, J = 2.4, 9.1 Hz). ¹³C NMR (125 MHz, CDCl3): δ = 14.90, 63.00, 69.50, 104.50, 118.05, 118.50, 126.30, 132.90, 139.00, 148.00, 156.00, 158.90, 161.00.
Amide 12 Compound 11 (1.10 g, 4.198 mmol) was added to a solution containing concentrated aq NH3 (20 mL) in EtOH (50 mL) and the solution refluxed (5 h). After evaporation at reduced pressure, EtOAc was added and the solution passed through a column of Florisil (20 g). Evaporation of the solvent left the pure amide 12 as a yellow solid (0.929 g, 95% yield); mp 173-175 ˚C.

29

2-Cyano-6- O -allyloxybenzothiazole (13) A solution containing compound 12 (0.950 g, 4.077 mmol) and imidazole (0.277 g, 4.077 mmol) in anhyd pyridine (25 mL) was cooled to -10 ˚C under nitrogen. Through a syringe, a solution of phosphorous oxychloride (0.746 mL, 8.154 mmol) in CH2Cl2 (5 mL) was slowly added, and the resulting solution was kept in ice under stirring (1 h). The temperature was slowly raised to ambient and stirred for additional 12 h, then CH2Cl2 and H2O were sequentially added. The organic phase was separated, washed with H2O, dried over Na2SO4 and evaporated. The crude was purified by chromatography (PE-EtOAc, 9:1), obtaining compound 13 (0.713 g, 81%) as a yellow solid; mp 74-75 ˚C. ¹H NMR (500 MHz, CDCl3): δ = 4.62 (d, 2 H, J = 5.2 Hz), 5.35 (dd, 1 H, J = 1.5, 10.5 Hz), 5.45 (dd, 1 H, J = 1.8, 17.5 Hz), 6.07 (m, 1 H), 7.21 (dd, 1 H, J = 2.4 Hz), 7.36 (d, 1 H, J = 2.4, 9.1 Hz), 8.05 (dd, 1 H, J = 2.4, 9.1 Hz). ¹³C NMR (125 MHz, CDCl3): δ = 69.50, 104.10, 113.70, 118.20, 118.90, 126.20, 132.80, 133.50, 137.80, 147.00, 159.70.

31

LiBH4 was also used as a reducing agent and reaction was faster, as described in ref. 31. However, with this stronger reducing reagent a competing reduction of the nitrile was observed.

32

2-Cyano-6-hydroxybenzothiazole (2) To a solution of compound 13 (0.600 g, 2.777 mmol) in anhyd THF under N2, Pd(PPh3)4 (0.02 mmol) and NaBH4 (0.422 g, 11.1 mmol) were added sequentially. After 24 h at r.t., the reaction was filtered and the solvent evaporated to leave a residue that was purified by column chromatography (PE-EtOAc, 8:2). The nitrile 2 was obtained as a pale yellow solid (0.406 g, 83%). Chemico-physical data were in agreement with those reported previously.9,¹0