2,6-Disubstituted benzothiazoles, analogues of the aromatic core of D-luciferin: synthesis and evaluation of the affinity for Photinus pyralis luciferase

A few 2,6-disubstituted benzothiazoles have been prepared as reference compounds or starting material for the preparation of derivatives containing positron emitting fluorine in the aromatic ring. Their affinity for Photinus pyralis luciferase has been evaluated and values of IC 50 (8.8-45.2 µM) suggest that they are competitive inhibitors of the enzyme


Introduction
The luciferase from the North American firefly Photinus pyralis (PpyLuc) catalyzes the conversion of D-luciferin [(S)-2-(6′-hydroxy-2′-benzothiazolyl)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 has been used, for example, as an indicator of cell proliferation, gene delivery or gene expression in cell culture and in living animals as a transgenic marker. 3PpyLuc is at present the preferred enzyme for in vivo optical imaging of small animals, 4 a useful modality of molecular imaging that presents the limit of light absorption and scattering by organs or the high dose of the reporter probe required for a sufficient emission. 5Furthermore, it is not certain to what degree the high contrast images obtained from the luciferin/luciferase bioluminescence are the result of selective substrate conversion or might be mainly dependent on substrate distribution.This could be better approached by other imaging modalities, such as single photon emission computed tomography (SPECT) and positron emission tomography (PET) that use radionuclides for imaging of reporter genes. 6In this respect, only two examples of radioactive isotopomers of D-luciferin (1a) are available, i. e. 6′-[ 11 C-methyl]-D-luciferin 1b 7 and 7′-[ 123 I]iodo-D-luciferin 1c. 8 However, 1b and 1c were unable to locate the tumor and showed poor cell uptake. 7,9e have recently undertaken a project aimed to prepare compounds related to D-luciferin labeled with positron emitting fluorine ( 18 F) for the in vivo imaging of a transgenic mouse that expresses a luciferase reporter gene under the control of activated estrogen receptors. 10Initially, we evaluated the possibility of introducing 18 F into the benzothiazole moiety of D-luciferin (1a) or 6′-O-methyl luciferin 1d that is a well known inhibitor of PpyLuc (IC 50 1µM). 11,12However, due to the instability of the thiazoline moiety of 1a or 1d, chemical manipulations of these compounds were excluded. 13We next considered to introduce 18 F into benzothiazoles corresponding to the aromatic core of compounds 1a or 1d and 2,6-disubstituted benzothiazoles 2a were selected as starting material for the preparation of compounds 1e.Furthermore, compounds such as 2a could be used themselves as tracers, provided that they could show affinity for Ppyluc.We relied on early kinetic studies showing that 2,6-disubstituted benzothiazoles 2b are competitive inhibitors of luciferase at pH 7.7, K i ranging from 25 to 58 µM. 11Moreover, in a recent paper other benzothiazoles 2c have been examined as possible inhibitors of luciferase within a high-throughput screening (HTS) of a library of 70,000 small molecules.Among these compounds, a series of benzothiazole analogues showed an activity approaching an IC 50 0.1 µM. 14Based on this encouraging premise, we have prepared a few model compounds structurally related to compounds 2a (X=H) and have evaluated their in vitro affinity for PpyLuc.

Results and Discussion
For our initial studies, we have selected 2-substituted-6-hydroxy and 6-methoxybenzothiazoles (compounds 3a-h, Figure 3) that could be prepared from nitriles 3a and 3e.The synthesis of 2cyano-6-methoxybenzothiazole 3e has been fully described, [15][16][17][18] but we started from the commercially available compound.The nitrile 3e can be transformed into the 6-hydroxy analogue 3a carrying out the demethylation by fusion with pyridinium hydrochloride (Py.HCl) at 200 °C. 15This method is still the most efficient for the preparation of 2-cyano-6-hydroxybenzothiazole 3a, because the nitrile function remains intact under these conditions.A few attempts to prepare 3a from 3e with other reagents were much less efficient than Py.HCl. 19The overall preparation of compounds 3a-h is described in Scheme 1. Basic hydrolysis of the nitrile 3a to the amide 3b could not be controlled and this product was obtained in mixture with variable amounts of the acid 3c.However, the 6-hydroxy acid 3c is stable only for a few days as powder and in solution decarboxylates to 6-hydroxybenzothiazole 4 with a reported half-life of 31 hours. 20We have verified the instability of the acid 3c in aqueous solutions at room temperature and consequently the values of IC 50 were not easily reproducible.The amide 3b could also be prepared by a biocatalytic approach, using a specific enzyme that could catalyze the hydrolysis of a nitrile.The superfamily of nitrilases includes nitrilases and nitrile hydrolases and offers this opportunity. 21he properly named nitrilase (EC 3.5.5.1) catalyzes the hydrolysis of a nitrile to the corresponding carboxylic acid and ammonia, whereas nitrile hydrolase (EC 4.2.1.84)transforms a nitrile into an amide. 22Only nitrilases are commercially available, but occasional formation of amide in nitrilase-catalyzed hydolysis of nitriles has been observed. 23This partial hydrolysis may depend on the structure of the nitrile, as in the case reported for a nitrilasecatalyzed hydrolysis of a β-hydroxynitrile. 24We have used the commercially available nitrilase from Arabidopsis thaliana and carried out the reaction in a 10% DMSO aqueous solution of the nitrile 3a.In our case, a quantitative hydrolysis to the required amide 3b was achieved with no formation of the corresponding acid 3c or product of its decarboxylation (compound 4).The same enzymatic reaction could not be repeated on the nitrile 3e, only for the high insolubility of the substrate in the reaction media.For the preparation of the 6-methoxy derivatives, the basic hydrolysis of the nitrile 3e proceeded as described for nitrile 3a.In fact, the amide 3f was prepared in mixture with variable amounts of the acid 3g, that was, in turn, quantitatively obtained by a complete basic hydrolysis.We have also prepared the 6-methoxy imidate 3h from the nitrile 3e, by a modification of a published procedure. 25Under the same conditions, the imidate 3d could not be prepared, confirming the result obtained by Amess et al. 25 The bioluminescent assay was carried out using a recombinant PpyLuc and results are expressed as IC 50 values (Table 1).Comparing the IC 50 values of 6-hydroxybenzothiazole derivatives 3a and 3b, it can be observed that substitution of the 2-cyano with an amide group leads to a slight decrease of affinity (IC 50 from 8.8 to 45.2 µM).In the 6-methoxybenzothiazole series, the presence of the methoxy group at the position 6 does not significantly affect the affinity of the 2-cyano derivatives 3a and 3e (IC 50 8.8 and 14.7 µM for nitrile 3a and 3e, respectively).Furthermore, the 6-methoxy amide 3f shows an IC 50 value nearly identical to that of the corresponding 6methoxynitrile 3e, in contrast with the difference observed between 3a and 3b.The 6-methoxy imidate 3h is characterized by an IC 50 value similar to 3e and 3f.The acid 3g was prepared with a view to improving the limited solubility that all the benzothiazole derivatives prepared by us exhibited in the in vitro assay.However, this acid has revealed to be a poor substrate (IC 50 308.0µM) for PpyLuc.A possible explanation of this result involves the recognition of the substrate by the enzyme that can be disfavored by the presence of a negatively charged group at position 2.This can interfere with some amino acid residue present in the part of the active site where the compound 3g should be located.

Conclusions
Results from our study show that benzothiazoles 3a, 3e, 3f, and 3h present an interesting affinity for PpyLuc (IC50 values 8.8-16.4µM), comparable to that of other benzothiazoles previously reported. 11,14It is reasonable to assume that these small-sized compounds interact in a nonspecific manner with different binding sites of the large enzymatic pocket of PpyLuc.7][28][29] In any event, we have described preliminary observations about the affinity of PpyLuc for 2,6-disubstituted benzothiazole derivatives that can be considered as reference compounds or starting material for the preparation of derivatives containing positron emitting fluorine in the aromatic ring.We are currently investigating the introduction of fluorine or fluorine-containing groups in the benzothiazole nucleus of compounds 3a-h in order to obtain benzothiazoles 2a or 2d, as potential tracers of luciferase reporter gene.

Experimental Section
General Procedures.Melting points were recorded on a Stuart Scientific SMP3 instrument and are uncorrected.IR spectra were recorded as KBr pellets on a Jasco FT-IR 300E spectrophotometer (Jasco Ltd., Tokyo, Japan). 1 H NMR spectra were recorded in DMSO-d 6 solutions at 303 K on a Bruker AM-500 spectrometer equipped with an Aspect 3000 computer, a process control and an array processor.The 1 H-chemical shifts are reported in parts per million, using as reference the signal for residual solvent protons.Mass spectra were recorded on a Finnigan LCQ-Deca (Termoquest) in ESI positive-ion mode (KV 5.00, 225 °C, 15 V).The progress of all reactions and column chromatography were monitored by TLC using Silica Gel 60 F 254 precoated plates with a fluorescent indicator (Merck).Purification of products by chromatography was performed using silica gel 60 (230-400 mesh, Merck).All reagents were obtained from commercial sources and used without further purification.Nitrilase from Arabidopsis thaliana (0.26 U/mg) was purchased from Fluka.The nitrile 3a was prepared from nitrile 3e in 70% yield according to the literature method. 15All products were crystallized from methanol/water.Enzymatic assay.The luciferase assay was carried out in 96 multiwell plates using 100 pg recombinant luciferase (Quantilum, Promega, Madison W.I., U.S.A.).The enzyme was dissolved in a luciferine buffer [(MgCO 3 ) 4 Mg(OH) 2 *5H 2 O, 1mM, Tricine (20 mM), EDTA (0,1 mM), MgSO 4 *7H 2 O (2,5 mM), DTT (33 mM), ATP (530 mM), pH 7.8] D-luciferin potassium salt (Promega, Madison, WI U.S.A.) was added at the final concentration of 30µM in the presence of increasing concentrations of the synthetic compounds to be tested (10 nM, 30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 10 µM, 30 µM, 100 µM, 300 µM, 1mM, 10 mM).The compounds were solubilized in 0,1 M phosphate buffer pH 7.2 with 1% v/v DMSO.The reaction occurred at 37°C for 5 min, then the quantitative analysis of the luminescence was carried out for 10 sec using the Veritas Microplate Luminometer (Turner Biosystems, Sunnyvale, CA U.S.A.).The curve of luciferin activity was generated using 1:3 dilutions of luciferin (at concentrations from 10nM to 1mM).All reactions were carried out in quadruplicate and each experiment was repeated in triplicate.All reagents were from Sigma-Aldrich (Steinheim, Germany).Half maximal inhibitory concentration (IC 50 ) was calculated by means of the Program PRISM5 (GraphPad Software Inc., CA U.S.A.) using Sigmoidal dose-response (variable slope) equation.

General procedure of the hydrolysis of nitriles 3a and 3e.
To a stirred solution of 3a or 3e (500 mg) in 15 mL of tetrahydrofurane, 3 mL of NaOH 1N were added.The reaction was stirred at room temperature for 1 h to afford a mixture of the corresponding amide (3b or 3f) and acid (3c or 3g).The complete hydrolysis of nitriles to the acids was achieved stirring for 3 h at room temperature.At the end of the reaction, a solution of 1N HCl was added and the mixture extracted two times with ethyl acetate.The isolation of amides 3b and 3f required an additional step, consisting in the treatment of the ethyl acetate solution of the amide/acid mixture with a 10% NaHCO 3 solution to remove the sodium salt of the acid 3c or 3g.The organic phase was dried with sodium sulfate and evaporated.

Nitrilase-catalyzed hydrolysis of 2-cyano-6-hydroxybenzothizole (3a): enzymatic preparation of 6-hydroxybenzothiazole-2-carboxyamide (3b)
To a stirred solution of nitrile 3a (120 mg, 0.68 mmol) in 5 mL of a water/dimethyl sulfoxyde solution (9/1) at room temperature, nitrilase from Arabidopsis Thaliana (10 mg) was added and the reaction was stirred for 2 h and only the amide 3b was revealed by TLC (dichloromethane/methanol 95:5).The mixture was filtered, diluted with water (3 mL) and extracted with ethyl acetate.The organic phase was dried over sodium sulfate and the product was recovered in 98% yield as a yellow-brown solid.All chemico-physical characteristics were in agreement with the structure of the amide 3b.

Methyl 6-methoxybenzothiazole-2-carboxyimidate (3h)
To a stirred solution of the nitrile 3e (500 mg, 2.62 mmol) in methanol (15 mL), 1N NaOH (3 mL) was added and the reaction was stirred at room temperature (3 h).The reaction was diluted with water (10 mL) and extracted with ethyl acetate.The organic phase was dried (sodium sulfate) and the product recovered as a white solid (570 mg, 98%).All chemico-physical characteristics were as described for 3h. 25