Highly Lipophilic Benzoxazoles with Potential Antibacterial Activity

A series of lipophilic 2-substituted 5,7-di-tert-butylbenzoxazoles was prepared in average yields by the reaction of 3,5-di-tert-butyl-1,2-benzoquinone with amino acids and dipeptides bearing N-terminal glycine. Dipeptides having other N-terminal amino acids undergo oxidative deamination. 5,7-Di-tert-butylbenzoxazoles have shown activity against Mycobacterium tuberculosis and some nontuberculous strains where isoniazid has been inactive. Antifungal activity was mediocre.


Introduction
The objectives of this study were the preparation and biological testing of some highly lipophilic 2-substituted-5,7-di-tert-butyl-benzoxazoles. Such lipophilicity may permit their easier penetration through the lipophilic mycobacterial cell walls. Benzoxazoles have been extensively studied for their antibacterial and antifungal activity [1,2], anticancer activity [3], and also as new non-nucleoside topoisomerase I poisons [4] and HIV-1 reverse transcriptase inhibitors [5,6]. Benzoxazoles are also interesting fluorescent probes which show high Stokes shift and present thermal and photophysical stability due to an excited state intramolecular proton transfer mechanism [7]. They interfere with the biosynthesis of coloured carotenoids by inhibiting the enzyme phytoene desaturase so they are studied as potential bleaching herbicides [8]. Benzoxazoles can be considered as structural isosteres of the naturally occurring nucleic bases adenine and guanine, which allow them to interact easily with polymers of living systems. They have shown low toxicity in warm-blooded animals [9].
For preparation of 2-substituted-5,7-di-tert-butylbenzoxazoles the method of choice involves reactions of 3,5-di-tert-butyl-1,2-benzoquinone (DTBBQ) with primary alkyl primary amines, amino acids and some of their derivatives [10]. From di-and oligopeptides those with glycine at the Nterminal also form the desired products.

Results and Discussion
Amino acids (Gly, Ala, Phe, Pgl, Val, Leu, Met, Tyr, Trp) and some of their derivatives such as glycinamide and tryptamine produced with DTBBQ the benzoxazole derivatives 2a-j in average yields. Lysine reacted with both amino groups to form the bis(benzoxazole) derivative 2k. The reaction occurred under mild conditions in ethanol (60-96 %) at a temperature of 50 °C. TLC on silica gel showed a complex mixture of products that were separated by preparative TLC on silica gel plates or by flash column chromatography. All products were characterized by NMR, UV-Vis, IR spectra, elemental analyses and calculated LogP. UV-Vis absorption bands (typical values for 2b: 208, 236, 274 nm) are useful for benzoxazole identification.
The formation of the target compounds from amino acids and DTBBQ is a multistep process that involves a sequence of consecutive reactions in which the intermediates cannot be detected to confirm the proposed reaction scheme (Scheme 1). In the first step the amino group is added to the less hindered carbonyl group in the position 1 of DTBBQ, followed by dehydration and formation of both E/Z isomeric quinone imines. The unstable quinone imines rearrange spontaneously into a mixture of two E/Z isomeric Schiff bases that cyclize into a mixture of two 2,3-dihydrobenzoxazole stereoisomers. The latter is dehydrogenated by a second molecule of DTTBQ into a benzoxazole with loss of carbon dioxide (for a review see [10]). The needed amount of the DTBBQ was produced by air oxidation from the 3,5-di-tert-butylbenzene-1,2-diol produced from DTBBQ during the dehydrogenation process. The main reaction sequence is accompanied by the formation of highly coloured by-products, especially 2,4,6,8-tetra-tert-butylphenoxazine-1-one [11], which complicate isolation of pure benzoxazoles. Formation of these pigments originates in the reaction of the Schiff base or its hydrolysis product, 2-amino-3,5-di-tert-butylphenol, with DTBBQ (Scheme 2).

Conclusions
Preliminary biological evaluation has shown that a number of our newly synthesised highly lipophilic benzoxazole derivatives possess antimycobacterial activity. This included activity against nontuberculous mycobacteria such as Mycobacterium kansasii isolated from a clinical isolate and Mycobacterium avium, where isoniazid is inactive. The possible improvement of the antituberculotic properties of these structures, through the modulation of the benzoxazole substitution and/or further functionalisation warrants further investigation. Antifungal testing against selected strains has not shown any significant activity.

Acknowledgements
This work was supported by the Grant IGA MZ 1A/8238-3 and MSM 002120822.

General
Chemicals were purchased from Aldrich. Melting points (uncorrected) were determined on a Kofler block. Elemental analyses were performed on CHNS-O CE instrument (FISONS EA 1110) and were within ±0.4 % of calculated values. UV spectra were measured on Polarimeter ADP 220 (BS Bellingham Stanley Ltd.). IR spectra were recorded on Nicolet Impact 400 spectrometer in KBr pellets, Nujol mulls or CHCl 3 solutions. NMR spectra were measured in CDCl 3 or DMSO-d 6 solutions at ambient temperature in a Varian Mercury-VxBB 300 spectrometer operating at 300 MHz. The chemical shifts δ are given in ppm related to tetramethylsilane (TMS) as internal standard. The coupling constants (J) are reported in Hz. Log P was calculated by using ChemDraw Ultra version 6.0.1. The reactions were monitored and the purity of the products was checked by TLC (Silufol UV 254, Kavalier Votice, Czech Republic and Merck TLC plates silica gel 60 F 254 , aluminium back) usually with ethyl acetate -light petroleum ether (1:9) or ethyl acetate-toluene (1:4) as eluents. The plates were visualized using UV light, iodine fumes, or carbonyl group detection by reaction with 2,4-dinitrophenylhydrazine. Preparative TLC was carried out on silica gel 60 F 254 (0.015 -0.040 mm, Merck). Silica gel 60 (0.015-0.040 mm, Merck) was used for column chromatography.

Antimycobacterial testing
All strains were obtained from the Czech National Collection of Type Cultures (CNCTC) with exception of M. kansasii 6509/96, which was a clinical isolate. The antimycobacterial activities of the compounds were determined in the Šula semisyntetic medium (SEVAC, Prague). The compounds