Introduction of Linking Groups by Alkylation of Fluorescent Heterocycles

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1 Seite 1 von 5 Sixth International Electronic Conference on Synthetic Organic Chemistry (ECSOC-6), September 1-30, 2002 [A####] Introduction of Linking Groups by Alkylation of Fluorescent Heterocycles Wolfgang Stadlbauer *, Werner De Cecco, Gerhard Hojas, Jenny Kremsner, Marek Pazicky, Anamaria Terec-Suciu, Pedro Traar and Georg Uray Institut für Chemie, Organic Synthesis Group and Analytical Organic Chemistry Group, Karl-Franzens-University of Graz Heinrichstrasse 28, A-8010 Graz (Austria) wolfgang.stadlbauer@uni-graz.at Received: 24. July 2002/ Uploaded: ##. #### 2002 Contents: 1. Introduction 2. Synthesis of 6,7-dimethoxy-2-quinolones 3. N-Alkylation of 6,7-dimethoxy-2-quinolones 4. O-Alkylation of 6,7-dihydroxy-2-quinolones 5. Synthesis of reactive intermediates and reaction with substrates 6. Discussion and conclusion 7. Acknowledgement 8. References 1. Introduction Quinolin-2-ones (carbostyrils) A with electron donating substituents such as amino or methoxy groups in position 6 and 7 and preferably an electron deficient substituent in position 4 are fluorescent dyes absorbing close to the visible [1,2]. Such push-pull substituted carbostyrils have the important advantage that they are highly stable against chemical and photochemical stress, in contrast to a number of similar fluorescent heterocycles such as coumarins. These properties makes them also interesting to be used in sensor devices utilizing the new blue laser diodes. In this contribution investigations are described to introduce in such carbostyrils A a reactive linker group which allows binding without loss of the fluorescence properties to amino groups of natural substrates such as proteins or immobilization at polymers. The linker groups were introduced either by exchange of the methoxy groups in the benzo part against analogues having reactive substituents. Another versatile variation lead to almost selective functionalization of the nitrogen in position 1 by alkylation with suitably substituted alkyl groups. These compounds have been converted subsequently to reactive intermediates B. In turn nucleophiles such as amino acids or proteins were acylated at room temperature to give

2 Seite 2 von 5 fluorescent products showing large Stoke s shifts and high quantum yield. 2. Synthesis of 6,7-dimethoxy-2-quinolones The synthesis of 6,7-methoxyquinolones 4 was achieved by cyclocondensation of 3,4-dimethoxyanilines 1 with acetoacetates 2 using literature procedures [1, 3]. For comparison purposes also N-methyl and N-ethyl derivatives were synthesized using a 2-step procedure via N-alkylformanilides followed by a Chapman rearrangement [4, 5]. The non-fluorescent isomer 4-oxo-quinolones 3 were synthesized following the procedure of reference [6]. 3. N-Alkylation of 6,7-dimethoxy-2-quinolones 6,7-Dimethoxy-2-quinolones 4 have 2 positions which are suitable for introduction of a linker group by alkylation: One is the heterocyclic nitrogen atom at position 1, the other is the methoxy function in the benzo part of the quinoline nucleus. In both cases, by-products are possible deriving from the keto-enole tautomeric forms at the oxo-function of the quinolin-2-one at position 2. When quinolones 4 were reacted with omega-chloro alkanoates, the N- and O-alkyl-quinolines 5 and 6 were formed. Their ratio was dependent mainly on reaction conditions. Methyl 2-chloroethanoate (n=1) reacts with 4- methyl- and 4-trifluoromethylquinolones 4 in DMF/potassium carbonate to a 30:70 ratio of the desired 1-alkyl product 5 and the O-alkyl byproduct 6. Attempts with sodium hydroxide as the base in ethanol gave unsufficient results. Using crown ethers such as 18-crown-6 in dichloromethane with sodium hydroxide as base, the desired 1-alkyl product 5 was formed in only 10:90 ratio; the main product was the O-alkyl product 6. With dibenzo-18-crown-6 as catalyst and 2-bromoethanoate (n=1) as alkylation agent and sodium carbonate as the base in dichloromethane the ratio could be reversed to 90:10 for the N-alkyl product 5. So this method is most suitable for the synthesis of functionalized 1-alkyl-carbostyrils 5, which should be a general improvement over most existing protocols.

3 Seite 3 von 5 Even with longer-chained alkylhalides preferred ratios of N- vs O-alkylation products were obtained. Therefore linkers could be attached with a larger distance between the fluorescent heterocycle and the labelled target molecule. 3-Bromopropanoate gave with the dibenzo-18-crown-6 method again a 9:1 ratio of N-alkyl and O- alkyl products 5 and 6, however in lower overall yield. 4-Bromobutanoate did not react sufficiently fast. The alternative DMF/potassium carbonate method gave a 1:3 ratio of N-alkyl product 5 and O-alkyl product O-Alkylation of 6,7-dihydroxy-2-quinolones Introduction of functional groups in position 6 and 7 of carbostyrils 4 was started by acid catalyzed cleavage of the methyl groups, which yielded the dihydroxy-carbostyrils 7. The reaction of 7 with halo-ethanoates in DMF/potassium carbonate gave not only ether bonds at the phenolic hydroxy groups, but also O-alkylation of the tautomeric hydroxy group at position 2. From the reaction mixture the tri-alkylated product 8 was isolated as main product in low yield. It has no fluorescent properties. When the alkylation was performed in aqueous sodium hydroxide solution, as main product the dialkyl product 9 was obtained, which revealed unchanged fluorescent properties. Carbostyril 7 could also be successfully converted into a reactive phenyl ester by reaction with acetic anhydride. Diacetate 10 was obtained in good yield and the fluorescence spectrum showed the expected redshift of the fluorescence maximum. Therefore such derivatives could be successfully used as fluorescent indicator for hydrolyzing agents, such as hydrolases.

4 Seite 4 von 5 5. Synthesis of reactive intermediates and reaction with substrates In order to link the fluorescent carbostyril to proteins, compound 5, namely methyl [6,7-dimethoxy-2-oxo-4- (trifluoromethyl)quinolin-1(2h)-yl]acetate, was hydrolyzed and then reacted with N-hydroxy-succinimide in the presence of diisopropyl-carbodimide [7]. As main product the succinimidoyl ester 13 was obtained, together with small amounts of the O-acylurea 14. Both products react readily at room temperature with amines. Also the corresponding acylated amino acids alanin, leucin or asparagin 12 were obtained as fluorescence labelled products. In a similar manner, fluorescence labelled bovine serum albumin was easily obtained and characterized. 6. Discussion and conclusion 6,7-Donor - and 4-acceptor substituted fluorescent carbostyril dyes have well defined ph independent properties, large Stokes shifts and sufficient quantum yields. It has been shown that 7 can be readily functionalized at nitrogen 1 as hydroxysuccinimide acetate. The activated ester 13 reacted at room temperature with amino acids. Fluorescence properties and absorption wavelengths close to the visible remained almost unchanged. The products were highly stable against photolysis, oxygen, acids and bases. This makes such compounds an alternative to less stable coumarins. Cleavage and refunctionalization of both ether groups at position 6 and 7 was shown to be an attractive route to more complex functionalized

5 Seite 5 von 5 carbostyrils such as crown ethers and phenyl esters. 7. Acknowledgement This work was supported by scholarships within the CEEPUS network. 6. References [1] Uray, G., Niederreiter, K. S., Belaj, F., Fabian, W. M. F., Helv. Chim. Acta 82 (1999) [2] Fabian, W. M. F., Niederreiter, K. S., Uray, G., Stadlbauer, W., J. Mol. Structure, 477 (1999) [3] Limpach, L., Ber. Dtsch. Chem. Ges., 64, 970 (1931). [4] Tietze, L.F., Eicher, T., "Reaktionen und Synthesen im organisch-chemischen Praktikum", G. Thieme Verlag Stuttgart New York, 1991; Organic Synthesis, Coll. Vol., 4 (1963) 420. [5] Utimoto, K, Kitai, M, Nozaki, H, Tetrahedron Lett., (1975) 2825; Büchi G., Wüest H, Tetrahedron Lett. (1977) [6] Werner W., Tetrahedron, 25 (1969) [7] Bodanszky, M., Bodanszky, A., The practice of peptide synthesis, Springer Verlag, Berlin, Heidelberg, New York 1994, p. 125; Balcolm B. J., Petersen N. O., J. Org. Chem., 54 (1989)