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 N-terminal 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).

Scheme 1

Scheme 2

Phenylserine (R = H) produces the corresponding benzoxazole 2l as the product, while phenylserine methyl ester (R = CH3) reacts in a different way. The latter produces benzoxazole 2m in a reaction involving C-C bond cleavage, in accordance with our previous results with 2-aminoethanol derivatives carrying benzylic hydroxyls [12] (Scheme 3).

Scheme 3

Dipeptides with N-terminal glycine (Gly-Gly, Gy-Leu, Gly-Tyr) afforded benzoxazole derivatives 2n, 2o, 2p (Scheme 4).

Scheme 4

As expected, dipeptides carrying at the N-terminal an amino acid other than glycine (Ala-Gly, Phe- Phe and Leu-Gly) underwent Correy´s oxidative deamination [13] with formation of ketoacylamino acids 3a, 3b, 3c, (Scheme 5). Two of them were characterized by their 2,4-dinitrophenylhydrazones 4a, 4b (Scheme 6).

Scheme 5

Scheme 6

Biological activity

Antimycobacterial activity was evaluated against a set of four mycobacterium strains: Mycobacterium tuberculosis CNCTC My 331/88, Mycobacterium kansasii CNCTC My 235/80, M. kansasii 6509/96 and Mycobacterium avium CNCTC My 330/88 using the micro method for determination of the minimum inhibitory concentration (MIC), the lowest concentration of a substance, at which the inhibition of growth of mycobacteria occurred. The following concentrations were used: 1000, 500, 250, 125, 62.5, 32, 16, 8 and 4 μmol·L-1. Results of the tests are shown in Table 1. The tested compounds have shown promising activity, ranging from 16 μmol·L-1 to 500 μmol·L-1. The most active was the benzoxazole 4-(5,7-di-tert-butylbenzoxazole-2-yl-methyl)-phenol (2h).

Table 1

In vitro antimycobacterial activity of compounds expressed as MIC (μmol·L-1)

Compound	Strains
	Mycobacterium kansasii 6 509/96 conc. 10-4			Mycobacterium kansasii My 235/80 conc. 10-4			Mycobacterium avium My 330/88 conc. 10-5		Mycobacterium tuberculosis My 331/88 conc. 10-3
Time	7d	14 d	21 d	7d	14 d	21 d	7d	14 d	14 d	21 d
2e	500	1000	1000	500	125	250	1000	>1000	500	500
2h	16	32	62.5	32	62.5	125	250	500	125	125
2i	62.5	125	125	62.5	125	500	250	250	250	250
2j	62.5	62.5	62.5	62.5	125	125	125	250	125	125
2n	250	500	500	250	500	500	1000	1000	500	500
2o	125	500	500	250	500	1000	1000	500	250	250
2p	500	1000	1000	1000	1000	1000	1000	>1000	500	1000
INH	2	2	4	>250	>250	>250	>250	>250	0.5a	1.0a

a conc. 10-4

The in vitro antifungal activity was tested against Candida albicans ATCC 44859 (CA), Candida tropicalis 156 (CT), Candida krusei E28 (CK), Candida glabrata 20/I (CG), Trichosporon beigelii 1188 (TB), Trichophyton mentagrophytes 445 (TM), Aspergillus fumigatus 231 (AF) and Absidia corymbifera 272 (AC) by using the microdilution broth test [14]. All strains, except CA were clinical isolates, identified by conventional morphological and biochemical methods. All the studied compounds were nearly inactive in concentrations of less than 500 μmol·L-1 MIC against all strains, with the exception of compound 2j, which showed 62.5 μmol·L-1 MIC against CA and 125 μmol·L-1 MIC against AC.

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.