Abstract
Herein, we have synthesized and tested a selected set of N1-(coumarin-7-yl)-4,5-dihydro-1,2,4-triazin-6(1H)-ones (8) incorporating masked a-amino acids within the triazinone skeleton as synthetic fluorogenic substrates for amino peptidases using human nasal epithelium that expressed aminopeptidases. These chiral triazinones, obtained via direct interaction of a-amino esters with N1-(coumarin-7-yl) nitrile imine (generated in situ from its hydrazonoyl chloride by the action of triethylamine), bear close resemblance to 7-amino-4-methylcoumarin (AMC) with labeled a-amino acids (AA-AMC's/7). The aminopeptidase activity of the synthesized compounds (0.125 and 0.5 mM) was compared with that of a model compound L-alanine-4-methylcoumaryl-7-amide (Ala-MCA), following in-house validation of the aminopeptidase activities of the human nasal primary culture. In comparison to the model aminopeptidase substrate (Ala-MCA), the synthesized compounds yielded fluorescent 7-amino-4-methylcoumarin following incubation with nasal epithelial cells. However, the yield was significantly lower than was observed for the control compound.
Highlights
A number of 1,2,4-triazinone derivatives possess significant biological activities, such as antimicrobial, fungicide, pesticide, herbicide and crop protection agents, as well as blood platelet aggregation-inhibition (Neunhoeffer et al, 1978; El Ashry et al, 1994; Abdel-Rahman, 2001), while some others exhibit anti-HIV and antitumoral activity against leukemia, ovarian cancer, small and large lung cancer cells, and breast cancer (Abdel-Rahman et al, 1999; El-Gendy et al, 2001; Krauth et al, 2010)
The intermediate arene diazonium chloride solution was freshly prepared by diazotization of 7-amino-4-methylcoumarin which, in turn, was prepared from m-aminophenol according to literature procedure (Pozdnev, 1990). α-Amino esters (11a-k), acting as nitrogen nucleophiles, are expected to add onto nitrile imine, generated in situ from the corresponding hydrazonoyl chloride (10) by the action of triethyl amine, to give initially the corresponding Z-amidrazone ester as the kinetically controlled product, the latter transient acyclic adducts undergo spontaneous intramolecular condensation, involving the amidrazone nitrogen and the ester carbonyl group, to yield the respective targeted heterocycles (8a-k) (Scheme 2)
We demonstrated that 7-amino-4-methylcoumarin can be formed from specific substrates for APN (L-alanine-4-methylcoumaryl-7-amide, APB (L-arginine-4-methylcoumaryl-7-amide) and DPPIV (Agu et al, 2009)
Summary
A number of 1,2,4-triazinone derivatives possess significant biological activities, such as antimicrobial, fungicide, pesticide, herbicide and crop protection agents, as well as blood platelet aggregation-inhibition (Neunhoeffer et al, 1978; El Ashry et al, 1994; Abdel-Rahman, 2001), while some others exhibit anti-HIV and antitumoral activity against leukemia, ovarian cancer, small and large lung cancer cells, and breast cancer (Abdel-Rahman et al, 1999; El-Gendy et al, 2001; Krauth et al, 2010). As a result, such substrates offer rapid detection, visualization and assay of various aminopeptidase enzymes from different / target microorganisms in samples or plant extracts (Iwaki et al, 1986; Acosta et al, 2008; Budič et al, 2009; Semashko et al, 2008; Chung, 2008; Townsend, 2009) In this context, we sought to prepare a selected set of 1-(chromen-7-yl)-4,5-dihydro-1,2,4-triazin-6(1H)-ones incorporating masked α-amino acid residues within the triazinone ring system, as exemplified by 8 (Fig. 3). These derivatives (8) bear close resemblance to their AA-AMC’s counter parts (7), shown in Fig. 3, and might act as fluorogenic substrates for aminopeptidases enzymes in a similar manner to 7
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