Convergent Synthesis of Two Fluorescent Ebselen-Coumarin Heterodimers

The organo-seleniumdrug ebselen exhibits a wide range of pharmacological effects that are predominantly due to its interference with redox systems catalyzed by seleno enzymes, e.g., glutathione peroxidase and thioredoxin reductase. Moreover, ebselen can covalently interact with thiol groups of several enzymes. According to its pleiotropic mode of action, ebselen has been investigated in clinical trials for the prevention and treatment of different ailments. Fluorescence-labeled probes containing ebselen are expected to be suitable for further biological and medicinal studies. We therefore designed and synthesized two coumarin-tagged activity-based probes bearing the ebselen warhead. The heterodimers differ by the nature of the spacer structure, for which—in the second compound—a PEG/two-amide spacer was introduced. The interaction of this probe and of ebselen with two cysteine proteases was investigated.


Introduction
Ebselen represents a lipid-soluble, selenium-containing, multifunctional drug with a broad range of pharmacological effects including both beneficial and harmful actions. The general mechanism of action is mainly based on reactions with specific thiol groups. This reactivity makes it a potent modulator for proteins that require cysteine for normal function [1,2]. Ebselen has been shown to be an efficient antioxidantin vivo. It was considered to be a relatively nontoxic compound because its selenium is not bioavailable [1][2][3][4]. However, it can act detrimentally through the depletion of glutathione [5,6]. Ebselen targets a wide variety of enzymes and modulates several biological processes. The inhibition of enzymes is based on the high reactivity of ebselen with critical protein thiol groups, leading to the reversible formation of relatively stable seleno-sulfide bonds [1,3]. However, such formation can be reversed by the addition of reducing agents [1], as has been shown, for example, for cerebral Na + , K + -ATPase and indoleamine 2,3-dioxygenase [7,8]. Ebselen exhibits anti-inflammatory effects due to its ability to directly inhibit inflammation-related enzymes [1,3,9].
The drug ebselen interferes with certain selenoenzymes, an important class of antioxidant biocatalysts that include glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Glutathione peroxidase protects biomembranes and other cellular components by using glutathione as the reducing substrate for the detoxification of a variety of hydroperoxides. Thioredoxin reductase catalyzes the reduction of thioredoxin (Trx) with NADPH as the cofactor. These transformations create a basis for a number of processes, such as defense against oxidative stress, the synthesis transformations create a basis for a number of processes, such as defense against oxidative stress, the synthesis of desoxyribonucleotides, redox regulation of gene expression, and signal transduction [5,10,11]. Ebselen, on the one hand, mimics GPx activity by catalyzing the reduction of peroxides with glutathione, and, on the other hand, has been demonstrated to be an excellent substrate for human TrxR [1,9,12]. As an antioxidant compound and a GPx mimic, ebselen appears to be a potential drug for the treatment of several disorders including diabetes-related diseases associated with reduced GPx levels such as atherosclerosis and nephropathy as well as a prospective treatment for cerebral ischemia. Hence, ebselen has been used in clinical trials for the prevention and treatment of different disorders [1,3].
In order to provide tool compounds for the ongoing scientific efforts to further characterize the biological activity of ebselen, we designed two activity-based probes containing the intact ebselen structure and a rigidified 7-amino coumarin (coumarin 343) as the fluorescent tag. Coumarins represent a widely used type of fluorophores and are characterized by their small molecular size, large Stokes shifts as well as chemical and enzymatic stability [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. In the second heterodimer, the coumarin tag should be connected via a PEG/two-amide linker with the ebselen substructure ( Figure  1). It was the aim of this study to synthesize these probes as biochemical tools for future studies. Moreover, we investigated the interaction of both ebselen and the second probe with two model cysteine proteases, i.e. the human cathepsins B and L.

Results and Discussion
The two convergent synthetic routes start with the procedure for synthesizing ebselen (2-phenyl-1,2-benzoselenazol-3-one) [28] (Scheme 1). Anthranilic acid (1) as the starting material was converted into a diazonium salt 2, which was treated with a disodium diselenide containing solution to obtain 2,2'-diselenobisbenzoate (3). The diselenide required for this step was obtained through a hydrazine-promoted reduction of selenium. After reacting 3 with thionyl chloride and a catalytic amount of DMF, Boc-semiprotected 4-aminomethylaniline was added. The resulting intermediate 5 was deprotected with trifluoroacetic acid to give the ebselen building block 6. The fluorescent coumarin 343 (8) was synthesized by submitting 8-hydroxyjulolidine-9-carboxaldehyde to a Knoevenagel condensation [20]. This was further coupled with building block 6 by using HATU in DMF to yield the first heterodimeric ebselen-coumarin probe 9.
To incorporate a PEG linker between the ebselen and coumarin substructures, we successfully employed a synthetic strategy in which the linker was first connected with the coumarin and the product was subsequently finalized by introducing the ebselen building block (Scheme 2). To generate the linker structure 13 [20], the amino group of 2-(2-aminoethoxy)ethanol (10) was Cbz-protected with benzyl chloroformate, then alkylated at the hydroxy group with tert-butyl bromoacetate using potassium tert-butoxide as a base to give compound 12 [29]. Afterwards, the linker structure 13 was achieved by a Cbz deprotection with hydrogen and palladium/carbon [20]. The fluorophore 8 was then reacted with 13 by using HATU in dichloromethane. The resulting compound 14 was deprotected with trifluoroacetic acid and finally coupled to the ebselen building block 6 by means of HATU in DMF to assemble the second probe 15.

Results and Discussion
The two convergent synthetic routes start with the procedure for synthesizing ebselen (2-phenyl-1,2-benzoselenazol-3-one) [28] (Scheme 1). Anthranilic acid (1) as the starting material was converted into a diazonium salt 2, which was treated with a disodium diselenide containing solution to obtain 2,2'-diselenobisbenzoate (3). The diselenide required for this step was obtained through a hydrazine-promoted reduction of selenium. After reacting 3 with thionyl chloride and a catalytic amount of DMF, Boc-semiprotected 4-aminomethylaniline was added. The resulting intermediate 5 was deprotected with trifluoroacetic acid to give the ebselen building block 6. The fluorescent coumarin 343 (8) was synthesized by submitting 8-hydroxyjulolidine-9-carboxaldehyde to a Knoevenagel condensation [20]. This was further coupled with building block 6 by using HATU in DMF to yield the first heterodimeric ebselen-coumarin probe 9.
To incorporate a PEG linker between the ebselen and coumarin substructures, we successfully employed a synthetic strategy in which the linker was first connected with the coumarin and the product was subsequently finalized by introducing the ebselen building block (Scheme 2). To generate the linker structure 13 [20], the amino group of 2-(2-aminoethoxy)ethanol (10) was Cbz-protected with benzyl chloroformate, then alkylated at the hydroxy group with tert-butyl bromoacetate using potassium tert-butoxide as a base to give compound 12 [29]. Afterwards, the linker structure 13 was achieved by a Cbz deprotection with hydrogen and palladium/carbon [20]. The fluorophore 8 was then reacted with 13 by using HATU in dichloromethane. The resulting compound 14 was deprotected with trifluoroacetic acid and finally coupled to the ebselen building block 6 by means of HATU in DMF to assemble the second probe 15. The structures of the new compounds were confirmed by analytical data, including high-resolution mass spectra (HRMS) which gave two main signals according to the two predominant isotopes of selenium. We have recorded UV/Vis and fluorescence spectra of 9 and 15 in three different solvents. Coumarins exhibit advantageous features, i.e. a high fluorescent intensity and a large Stokes shift. Such properties were also obtained for our heterodimers, as shown in Figure  2 and Table 1.  The structures of the new compounds were confirmed by analytical data, including high-resolution mass spectra (HRMS) which gave two main signals according to the two predominant isotopes of selenium. We have recorded UV/Vis and fluorescence spectra of 9 and 15 in three different solvents. Coumarins exhibit advantageous features, i.e. a high fluorescent intensity and a large Stokes shift. Such properties were also obtained for our heterodimers, as shown in Figure  2 and Table 1. The structures of the new compounds were confirmed by analytical data, including high-resolution mass spectra (HRMS) which gave two main signals according to the two predominant isotopes of selenium. We have recorded UV/Vis and fluorescence spectra of 9 and 15 in three different solvents. Coumarins exhibit advantageous features, i.e. a high fluorescent intensity and a large Stokes shift. Such properties were also obtained for our heterodimers, as shown in Figure 2 and Table 1. Wavelengths of absorption were used for excitation. The heterodimer 9 was measured similarly (see Table 1). All fluorescent measurements were carried out with a PMT value of 200 V. Ebselen was reported to react with its targets through the formation of covalent seleno-sulfide bonds. This covalent mode of interaction provides an impetus for the development of activity-based probes. Recently, an ebselen-cyanine probe was reported to be utilized for the real-time imaging of the cellular redox status changes [30]. In this study, we have coupled the ebselen warhead with coumarin 343. The fluorophor coumarin 343 is valued for its bathochromic shift of absorption and emission and its high fluorescence quantum yield, even in aqueous medium [31]. These desirable properties arise from its rigidified tetracyclic structure. Compared to less rigid 7-donor substituted coumarins, the rotation of the amino group in coumarin 343 is constrained and the nitrogen lone pair can maximally interact with the aromatic system [32,33]. Accordingly, coumarin 343 was incorporated as a fluorescence label in various activity-based probes [13,18,20].
Besides the direct connection of the ebselen structure with that of coumarin 343, realized in compound 9, we have designed the second probe 15 comprising a PEG/two-amide spacer. The incorporation of this flexible spacer is thought to facilitate the interaction of the ebselen part with putative targets by preventing a possible steric hindrance caused by the coumarin part of the heterodimer. Moreover, 15 was unsurprisingly found to be more soluble than 9 in different organic solvents. Compounds 9 and 15 expand the portfolio of ebselen homo-and heterodimers [2,4,12,30,34,35]. Our ebselen containing fluorescence-labeled probes are expected to be suitable pharmacological tools to continuously elucidate the biological activity of ebselen.
The known reactivity of ebselen with cysteine residues of several proteins [1,7,8,[38][39][40] prompted us to investigate its interaction with two model cysteine proteases, the human cathepsins B and L. An inactivation of these enzymes through a seleno-sulfide bond formation would provide a possible starting point for the development of probes for cysteine proteases. Ebselen and probe 15 were added at a final concentration of 10 μM to the cathepsin activity assays [41,42]. Duplicate experiments revealed no inhibition of the proteolytic activity by using peptidic chromogenic substrates. However, the covalent interaction of ebselen and assumedly of 15 with critical protein thiol groups can be reversed by the addition of reducing compounds, such as dithiothreitol (DTT) [1,8,38,39]. In our usual cathepsin measurements, DTT is applied for the activation of the cathepsins, were used for excitation. The heterodimer 9 was measured similarly (see Table 1). All fluorescent measurements were carried out with a PMT value of 200 V. Ebselen was reported to react with its targets through the formation of covalent seleno-sulfide bonds. This covalent mode of interaction provides an impetus for the development of activity-based probes. Recently, an ebselen-cyanine probe was reported to be utilized for the real-time imaging of the cellular redox status changes [30]. In this study, we have coupled the ebselen warhead with coumarin 343. The fluorophor coumarin 343 is valued for its bathochromic shift of absorption and emission and its high fluorescence quantum yield, even in aqueous medium [31]. These desirable properties arise from its rigidified tetracyclic structure. Compared to less rigid 7-donor substituted coumarins, the rotation of the amino group in coumarin 343 is constrained and the nitrogen lone pair can maximally interact with the aromatic system [32,33]. Accordingly, coumarin 343 was incorporated as a fluorescence label in various activity-based probes [13,18,20].
Besides the direct connection of the ebselen structure with that of coumarin 343, realized in compound 9, we have designed the second probe 15 comprising a PEG/two-amide spacer. The incorporation of this flexible spacer is thought to facilitate the interaction of the ebselen part with putative targets by preventing a possible steric hindrance caused by the coumarin part of the heterodimer. Moreover, 15 was unsurprisingly found to be more soluble than 9 in different organic solvents. Compounds 9 and 15 expand the portfolio of ebselen homo-and heterodimers [2,4,12,30,34,35]. Our ebselen containing fluorescence-labeled probes are expected to be suitable pharmacological tools to continuously elucidate the biological activity of ebselen.
The known reactivity of ebselen with cysteine residues of several proteins [1,7,8,[36][37][38] prompted us to investigate its interaction with two model cysteine proteases, the human cathepsins B and L. An inactivation of these enzymes through a seleno-sulfide bond formation would provide a possible starting point for the development of probes for cysteine proteases. Ebselen and probe 15 were added at a final concentration of 10 µM to the cathepsin activity assays [39,40]. Duplicate experiments revealed no inhibition of the proteolytic activity by using peptidic chromogenic substrates. However, the covalent interaction of ebselen and assumedly of 15 with critical protein thiol groups can be reversed by the addition of reducing compounds, such as dithiothreitol (DTT) [1,8,36,37]. In our usual cathepsin measurements, DTT is applied for the activation of the cathepsins, leading to rather high final DTT concentrations of 100 µM (cathepsin B) and 200 µM (cathepsin L) in the assays. Hence, the strong excess of DTT might prevent enzyme inhibition by the two compounds. We have modified the assays as follows: (i) the final DTT concentration was reduced to 3.5 µM; (ii) the final DMSO concentration was changed from 2% to 5%; (iii) the enzymes were preincubated for 10 min with the substrate prior to the addition of the test compounds; and (iv) the final enzyme concentration was increased 3.5-fold (cathepsin B) and 1.75-fold (cathepsin L). Under such conditions, the enzymes could be assayed appropriately with the decreased amount of DTT. Ebselen and probe 15 were investigated at final concentrations of 1 µM and 0.2 µM in duplicate measurements. Unexpectedly, the proteolytic activity was stimulated, in particular that of cathepsin B, when each of the two compounds was added. Obviously, the test compounds were able to interfere with the redox equilibrium between the protein and DTT. It could therefore be concluded that probe 15 is not suitable for labeling cysteine cathepsins. Nevertheless, the fluorescent ebselen derivatives might be helpful to identify proteins which are targeted by this drug and might therefore be valuable in future biological studies.

General Methods and Materials
Thin-layer chromatography was carried out on Merck aluminum sheets, silica gel 60 F254. Detection was performed with UV light at 254 nm. Preparative column chromatography was performed on Merck silica gel 60 (70-230 mesh). Melting points were determined on a Büchi 510 oil bath apparatus (Büchi, Essen, Germany). Mass spectra were recorded on an API 2000 mass spectrometer (electron spray ion source, Applied Biosystems, Darmstadt, Germany) coupled with an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) using a Phenomenex Luna HPLC C 18 column (50ˆ2.00 mm, particle size 3 µm).

Syntheses
2,2'-Diselenobisbenzoic acid (3). A disodium diselenide solution was prepared by the reaction of selenium powder (5.9 g, 75 mmol), 100% hydrazine hydrate (0.98 g, 0.95 mL, 20 mmol) and sodium hydroxide (9 g, 22.5 mmol) in MeOH (150 mL) carried out at rt for 24 h. Meanwhile, to a stirred solution of anthranilic acid (1, 10.3 g, 75 mmol) in 3N HCl (60 mL) cooled with an ice/salt bath, a solution of sodium nitrite (5.7 g, 83 mmol) in water (15 mL) was added dropwise, and the temperature was maintained below 5˝C. After stirring for additional 15 min, this solution was added dropwise to the stirred solution containing disodium diselenide cooled in an ice/salt bath below´5˝C. The temperature was kept for 2.5 h and then increased to rt for another 20 h. The reaction mixture was filtrated and the filtrate was acidified to pH 3 by adding 3N HCl (300 mL). After 30 min, the obtained precipitate was filtered off, washed with hot water (2 L), dried at 90˝C for 4 h, then on air for another 24 h, and recrystallized from 1,4-dioxane resulting in the pure product 3 (2.7 g, 18%); m.p. > 250˝C, lit. [41]  tert-Butyl 4-(3-oxobenzo[d] [1,2]selenazol-2(3H)-yl)benzylcarbamate (5) [34]. Compound 3 (8.0 g, 20 mmol) was added to thionyl chloride (40 mL) and DMF (1 mL), and the reaction mixture was refluxed at 85˝C for 3 h. The solvent was then evaporated, and the obtained product was recrystallized from n-hexane resulting in pale yellow prisms of 2-(chloroseleno)benzoyl chloride 4 (4.65 g, 46%); m.p. 64-66˝C, lit. [4] mp 64-66˝C, which was used without further characterization. In the next step, a solution of compound 4 (2.54 g, 10 mmol) in dry CH 2 Cl 2 (16 mL) was added dropwise to a stirred solution   Ebselen-coumarin Heterodimer (15). Compound 14 (292 mg, 0.6 mmol) was dissolved in dry CH 2 Cl 2 (30 5 mL) and trifluoroacetic acid (6 mL) was added. The resulting reaction mixture was stirred at rt for 2 h. The solvent was then evaporated, and the residue was diluted with CH 2 Cl 2 (4ˆ10 mL) and evaporated to remove the excess of trifluoroacetic acid. The crude product was dissolved in dry DMF (10 mL), and HATU (255 mg, 0.67 mmol) and DIPEA (0.70 mL, 517 mg, 4 mmol) were added. After stirring at room temperature for 30 min, compound 6 (334 mg, 0.80 mmol) was added and the reaction mixture was stirred at rt for 48 h. The solvent was then evaporated and the resulting residue was redissolved in DMF (3 mL). To this solution silica gel was added and the solvent was again evaporated. The crude product attached to silica gel was added to a column and purified using CH 2 Cl 2 /MeOH (9:1) as eluent. The product-containing fractions were combined and evaporated to dryness. MeOH (

Modified Enzymatic Assays
Cathepsin B. Human isolated cathepsin B (Calbiochem, Darmstadt, Germany) was assayed spectrophotometrically (Cary 50 Bio, Varian, Agilent Technologies, Santa Clara, CA, USA) at 405 nm and at 37˝C. Assay buffer was 100 mM sodium phosphate buffer pH 6.0, 100 mM NaCl, 5 mM EDTA, 0.01% Brij 35. To 2 µL an enzyme stock solution of 1.81 mg/mL in 20 mM sodium acetate buffer pH 5.0 and 1 mM EDTA, a volume of 9.98 µL assay buffer containing 5 mM DTT was added. Then, 988.02 µL of assay buffer was added. This enzyme solution was incubated for 30 min at 37˝C. Stock solutions (10 mM) of ebselen and 15 were prepared in DMSO. A 100 mM stock solution of the chromogenic substrate Z-Arg-Arg-pNA was prepared with DMSO. The final concentration of DMSO was 5%, the final concentration of the substrate was 500 µM, and the final DTT concentration was 3.5 µM. Assays were performed with a final concentration of 253 ng/mL of cathepsin B. Into a cuvette containing 176 µL assay buffer, 7 µL DMSO, 1 µL of the substrate solution and 14 µL of the enzyme solution were added, thoroughly mixed, and incubated for 10 min at 37˝C. The reaction was initiated by adding 2 µL of DMSO or inhibitor solution and followed over 30 min.
Cathepsin L. Human isolated cathepsin L (Enzo Life Sciences, Lörrach, Germany) was assayed spectrophotometrically (Cary 50 Bio, Varian) at 405 nm and at 37˝C. Assay buffer was 100 mM sodium phosphate buffer pH 6.0, 100 mM NaCl, 5 mM EDTA, and 0.01% Brij 35. To 10 µL of an enzyme stock solution of 135 µg/mL in 20 mM malonate buffer pH 5.5, 400 mM NaCl, and 1 mM EDTA, a volume of 10 µL assay buffer containing 5 mM DTT was added. Then, 980 µL of assay buffer was added. This enzyme solution was incubated for 30 min at 37˝C. Stock solutions (10 mM) of ebselen and 15 were prepared in DMSO. A 10 mM stock solution of the chromogenic substrate Z-Phe-Arg-pNA was prepared with DMSO. The final concentration of DMSO was 5%, the final concentration of the substrate was 100 µM, and the final DTT concentration was 3.5 µM. Assays were performed with a final concentration of 94.5 ng/mL of cathepsin L. Into a cuvette containing 176 µL assay buffer, 6 µL DMSO, 2 µL of the substrate solution and 14 µL of the enzyme solution were added, thoroughly mixed, and incubated for 10 min at 37˝C. The reaction was initiated by adding 2 µL of DMSO or inhibitor solution and followed over 30 min.

Conflicts of Interest:
The authors declare no conflict of interest.