Convergent synthesis of a linker-connected fluorescent ebselen-coumarin heterodimer Jim Küppers 1 , Jerzy Palus 2 , Mirosław Giurg 2 , Jacek Skarżewski 2 and Michael Gütschow 1, * 1 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany; 2 Department of Organic Chemistry, Wrocław University of Technology (A2), Wyspiańskiego 27, 50- 370 Wrocław, Poland. 1 * guetschow@uni-bonn.de
Convergent synthesis of a linker-connected fluorescent ebselen-coumarin heterodimer O N Se ebselen O O HO N NH 3 Se O O N 1 st component 2 nd component O N Se O H N O O N H O O O N probe 2
Abstract: Ebselen is a multifunctional drug with a wide range of pharmacological effects that are predominantly due to its interaction with selenoenzymes, e.g. glutathione peroxidase, thioredoxin reductase. Fluorescence-labeled probes containing ebselen can be suitable for further biological and medicinal studies, to profile enzyme activities, identify target enzymes and characterize their functions. The synthetic route starts with the conversion of anthranilic acid into a diazonium salt, treatment with disodium diselenide to a 2,2´-diselenobisbenzoate and activation with thionyl chloride. A reaction with an appropriate para -substituted aniline derivative and deprotection gave the 1 st component. A PEG linker building block was coupled with the 2 nd component, the fluorescent coumarin 343. This was synthesized from 8-hydroxyjulolidine-9-carboxaldehyde. After deprotection, the desired probe was assembled from the ebselen and the PEG derivatives. The new probe will be provided to biochemical and pharmacological studies. Keywords: ebselen; selenoenzymes; activity-based probe 3
Introduction Ebselen is a multifunctional drug with a wide range of pharmacological effects that are predominantly due to its interaction with selenoenzymes, e.g. glutathione peroxidase, thioredoxin reductase [1,2]. Such enzymes play an important role in protecting biomembranes and other cellular components from oxidative stress by catalyzing the reduction of a variety of hydroperoxides [2]. O N Se According to its pleiotropic mode of action which is based on its versatile chemical structure, ebselen is currently under clinical trials for the prevention and treatment of different ailments such as cardiovascular diseases, arthritis, stroke, atherosclerosis, and cancer [2]. Fluorescence-labeled probes containing ebselen can be suitable for further biological and medicinal studies, to profile enzyme activities, identify target enzymes and characterize their functions [3]. [1] Stoyanovsky, D. A.; Jiang, J.; Murphy, M. P.; Epperly, M.; Zhang, X.; Li, S.; Greenberger, J.; Kagan, V.; Bayır , H. ACS Med. Chem. Lett. 2014 , 5 , 1304-1307. [2] Azad, G. K.; Tomar, R. S. Mol. Biol. Rep. 2014 , 41 , 4865-79. [3] Xu, K.; Qiang, M.; Gao, W.; Su, R.; Li, N., Gao, Y.; Xie, Y.; Kong, F.; Tang, B. Chem. Sci. 2013 , 4 , 1079-1086. 4
Synthesis O O O O NaNO 2 Na 2 Se 2 SOCl 2, DMF (cat.) OH OH OH Cl Cl H 2 O, < 5 °C H 2 O/MeOH, 85 °C N N NH 3 Se Se Se Cl Cl < 5 °C 1 2 3 4 HO O O H 2 N O O F 3 CCO 2 N O TFA O H N N N O NH 3 dry CH 2 Cl 2 , rt dry CH 2 Cl 2 , Et 3 N, Se Se H 0 °C to rt 6 5 The synthesis starts with the procedure to prepare ebselen [4]. Anthranilic acid hydrochloride ( 1 ) was converted into a diazonium salt ( 2 ), which was treated with disodium diselenide to obtain the 2,2’ -diselenobisbenzoate ( 3 ). After treating 3 with thionylchloride, an appropriate para -substituted aniline derivative was introduced. Deprotection of 5 led to 6 , the first component for the assembly of the final product 14 . [4] Palus, J.; Młochowski , J.; Juchniewicz, L. Polish J. Chem. 1998 , 72 , 1931-1936. 5
Synthesis O O O Cl O Br O O O H 2 N OH O N OH dry CH 2 Cl 2 , Et 3 N, H Potassium tert - butoxide, 0 °C to rt 7 8 dry THF, 0 °C to rt O H 2, Pd/C O O O O O N O H 2 N O dry MeOH, rt H O O 9 10 To obtain the PEG linker for the connection of the two components, the amino group of 2-(2-aminoethoxy)ethanol was first Cbz-protected, then alkylated at the hydroxyl group with tert -butyl bromoacetate followed by a Cbz deprotection to give compound 10 . 6
Synthesis O O O O O O 1. O Compound 10 , HATU, N H 2 H O OH dry CH 2 Cl 2 , DIPEA, rt N O O N OH abs. EtOH, reflux 2. Trifluoroacetic acid, dry CH 2 Cl 2 , rt 11 12 O O Compound 6 , O O N O OH O HATU N O H H N O N O N O Se N O O dry CH 2 Cl 2 , H O DIPEA, rt 13 14 The fluorescent coumarin 343 12 was synthesized by submitting 8-hydroxyjulolidine- 9-carboxaldehyde to a Knoevenagel condensation. This was further coupled with 10 and deprotected to give the PEG-coumarin 343 building block ( 13 ) which after reaction with the ebselen derivative 6 resulted in the desired probe 14 . 7
Outlook O O O N O H N O N N O Se H O 14 The new probe was designed to contain the intact ebselen structure, a PEG/two-amide spacer to improve solubility and a rigidified 7-amino coumarin, a type of fluorophore valued for its red shift of absorption and emission, even in aqueous medium [5, 6, 7]. Compound 14 will be provided to biochemical and pharmacological studies. [5] Murase, T.; Yoshihara, T.; Yamada, K.; Tobita, S. Bull. Chem. Soc. Jpn. 2013 , 86 , 510 [6] Mertens, M. D.; Schmitz, J.; Horn, M.; Furtmann, N.; Bajorath, J.; Mareš , M.; Gütschow, M. ChemBioChem 2014 , 15 , 955. [7] Kohl, F.; Schmitz, J.; Furtmann, N.; Schulz-Fincke, A.-C.; Mertens, M. D.; Küppers, J.; Benkhoff, M.; Tobiasch, E.; Bartz, U.; Bajorath, J.; Stirnberg, M.; Gütschow, M. Org. Biomol. Chem. 2015 , 13 , 10310-10323. 8
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