Synthesis of 2-substituted-6-hydroxy and 6-methoxy benzothiazoles from 1,4-benzoquinone

A few 2-substituted-6-hydroxy and 6-methoxybenzothiazoles have been prepared from ethyl 6-hydroxybenzothiazole-2-carboxylate, obtained by oxidation of ethyl ( R )-2-amino-3-(2,5-dihydroxyphenylsulfanyl)propanoate hydrochloride, in turn prepared from 1,4-benzoquinone.


Introduction
The luciferase from the North American firefly Photinus pyralis (PpyLuc) catalyzes the conversion of D-luciferin [(S)-2-(6′-hydroxy-2′-benzothiazolyl)-Δ 2 -thiazoline-4-carboxylic acid] to oxyluciferin in the presence of ATP, Mg 2+ , and oxygen with production of a yellow-green light characterized by a broad emission spectrum and a peak at 560 nm (Figure 1).PpyLuc is a well characterized enzyme that finds a large number of biotechnological applications 2 and is, at present, the preferred enzyme for in vivo optical imaging of small animals. 3We have recently undertaken a project aimed to prepare compounds related to Dluciferin labelled with positron emitting fluorine ( 18 F) for the in vivo imaging 4 of a transgenic mouse that expresses a luciferase reporter gene under the control of activated estrogen receptors. 5e have considered the introduction of 18 F into the benzothiazole core of D-luciferin (compounds 1, Figure1) provided that affinity for PpyLuc could be demonstrated.2,6-Disubstituted benzothiazoles (compounds 2a) were required as intermediates for the preparation of compounds 1 or as potential probes for imaging, in view of the fact that compounds such as 2b or 2c are competitive inhibitors of luciferase.Within the above project, we have prepared a few compounds structurally related to compounds 2a (X=H) and evaluated their in vitro affinity for PpyLuc. 82-Substituted-6-hydroxy and 6-methoxybenzothiazoles such as compounds 4a-d (Scheme 1) were prepared from 2-cyano-6-methoxybenzothiazole 3 that is commercially available.It should be mentioned that the nitrile 3 is an important intermediate for the synthesis of Dluciferin 9,10 and that the synthesis of this compound has been the target of several publications. 10- 16We report here the synthesis of a few 2-substituted-6-hydroxy and 6-methoxybenzothiazoles from ethyl 6-hydroxybenzothiazole-2-carboxylate, in turn obtained from 1,4-benzoquinone.

Results and discussion
The reaction of quinones and cysteine has been extensively studied [17][18][19] and from 1,4benzoquinone 5 ethyl (R)-2-amino-3-(2,5-dihydroxyphenylsulfanyl)propanoate hydrochloride 6 has been prepared and characterized. 20The oxidation of the ester 6 has been studied by a few authors 20,21 and the synthetic procedure most reliable from a preparative point of view is that recently described by Löwik et al. 21When the oxidation was carried out with potassium ferricyanide K3Fe(CN)6 in the presence of NaOH and methanol or ethanol, mixtures of the thiazines 7a or 7b and the ethyl ester 8 were obtained (Scheme 2).The thiazines 7a or 7b could be isolated by flash chromatography and converted into the benzothiazole ester 8 by treatment with 1 M HCl in methanol or ethanol.The ester 8 could also be obtained by acidic treatment of the crude mixtures 7a/8 or 7b/8.We have prepared our 2-substituted-6-hydroxy and 6methoxybenzothiazoles from ethyl (R)-2-amino-3-(2,5-dihydroxyphenylsulfanyl) propanoate hydrochloride 6 basically relying upon the original experimental protocol. 20Specifically, we have carried out the reaction of 1,4-benzoquinone 5 with L-cysteine ethyl ester hydrochloride in methanol at room temperature, excluding water from the reaction.The ethyl ester 6 obtained after the work-up (95%) could be stored as a solid product 23 and later oxidized with K3Fe(CN)6 in aqueous NaOH in the presence of an alcohol, as described by Löwik et al. 21We have selected isopropanol 23 and carried out the reaction at room temperature.Purification of the crude mixture of products by silica gel column chromatography afforded the ester 8 essentially pure (68%).Furthermore, in order to avoid the high molar ratio (6:1) of K3Fe(CN)6 reported in the original procedure, 21 we have attempted a few alternative oxidations. 24Finally, we have found that the reaction of cupric salts with the ester 6 (molar ratio, 3:1) in isopropanol/water can constitute an alternative to the procedure involving K3Fe(CN)6. 25he synthesis of 2-substituted 6-hydroxy and 6-methoxybenzothiazoles from the ester 8 is illustrated in Scheme 3. Thus, protection of the ester 8 as the corresponding 6-O-methyl ether 9 (92% yield) and following quantitative reaction with concentrated aqueous ammonia 26 afforded the amide 4a.This amide could also be prepared by methylation of 2-carboxyamide-6hydroxybenzothiazole 4c, in turn obtained from the ester 8 by treatment with concentrated aqueous ammonia (98%).However, methylation of the amide 4c was less straightforward, due to the formation of side products, presumably occurring from the methylation of the 2carboxyamide function.Finally, the transformation of the amide 4a into the nitrile 3 was carried out using POCl3 as dehydrating agent (72% yield). 27RKAT USA, Inc.

Conclusions
We have reported a new approach to the synthesis of a few 2-substituted 6-hydroxy and 6methoxybenzothiazoles starting from 1,4-benzoquinone 5. Ethyl (R)-2-amino-3-(2,5dihydroxyphenylsulfanyl)propanoate hydrochloride 6 has been prepared by an improved procedure and the oxidation of this intermediate to the benzothiazole ester 8 has been ameliorated.According to the experimental protocol outlined in our work, the key intermediate for the synthesis of D-luciferin, 2-cyano-6-methoxybenzothiazole 3, can be prepared in 42 % yield.This is worth of note, since yields are higher than those obtained following the classical approach (9.9%) 10 and comparable with the most convenient synthesis based on a Sandmeyer approach, using poisonous cyanides (48,6%). 13,14Finally, our synthetic approach opens the possibility of preparing 2,6-disubstituted benzothiazoles suitable for 18 F labeling such as compounds 2a starting from properly substituted 1,4-benzoquinones.Alternatively, from the ester 8 also new 18 F-labeled amides 2d may become available.

Experimental Section
General.Melting points were recorded on a Stuart Scientific SMP3 instrument and are uncorrected. 1H-and 13 C-NMR spectra were recorded at 303 K on a Bruker AM-500 spectrometer equipped with an Aspect 3000 computer, a process control and an array processor.
(10 mL)to wash the crude product and then is removed by decantation.The operation is repeated with diethyl ether and dichloromethane and the ester 6 is obtained as a solid.The ester 6 is a hygroscopic compound that can be obtained and stored at room temperature if ambient moisture is properly b.Reaction with Cu(II) salts.