a Dipartimento di Scienze Biomediche (sez. Microbiologia), Università di Trieste, via A. Fleming 22, I-34127 Trieste; b Dipartimento di Scienze Farmaceutiche, Università di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy
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Abstract |
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Introduction |
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In previous papers we described the inhibiting activity of different pyridine carboxamidrazone derivatives on different species of the genus Mycobacterium and against different human clinical strains.37 In order to better define the antimycobacterial properties of compounds characterized by the pyridine-2-carboxamidrazone moiety, we recently synthesized a series of new N1-substituted pyridine-2-carboxamidrazone derivatives and evaluated their antimycobacterial activity.8 The significant activity of some of those compounds against a single M. avium strain prompted us to examine their activity against a number of different M. avium human isolates, because of their great heterogeneity in terms of drug susceptibility.
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Materials and methods |
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All 17 M. avium strains were clinical isolates cultured from specimens submitted over several years to the Modulo di Microbiologia Polmonare, Ospedale di Cattinara, in Trieste, for investigation of mycobacterial disease in patients with or without AIDS. The strains were respiratory or blood isolates from different patients. Isolates were identified by classical culture and biochemical characteristics or DNA probe (Gene-Probe, San Diego, CA, USA).
Antimicrobial agents
We have synthesized a series of 24 molecules, N1-{1-[3-aryl-1-(pyridin-2-, 3- or 4-yl)-3-oxo] propyl}-2-pyridinecarboxamidrazones variously substituted on the phenyl residue, as described in a previous paper;1 the chemical structure and substituted chemical groups in the three different series of molecules, 2-pyridyl, 3-pyridyl and 4-pyridyl derivatives, are shown in the Figure.
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MIC determination
We employed the agar macrodilution method on Middlebrook 7H11 agar (Becton Dickinson Microbiology Systems, Milan, Italy) to evaluate the antimycobacterial activity of the new molecules. Serial two-fold dilutions of each compound were made in quadrant Petri dishes coated with 7H11 agar medium supplemented with 10% albumin dextrose complex (ADC; Becton Dickinson) from stock solutions (final concentration ranging from 0.5 to 64 mg/L). Mycobacteria grown on LowensteinJensen slants (Becton Dickinson) were freshly suspended in 0.02% Tween phosphate-buffered saline to give a concentration of 15 x 104 colony forming units (cfu) (previously stated to correspond to a 10-2 McFarland 0.5 equivalent turbidity standard). Ten microlitres of each mycobacterial suspension were distributed on to each quadrant surface and allowed to incubate for 1520 days; ofloxacin (Sigma Aldrich, Milan, Italy) (concentrations ranging from 1 to 8 mg/L) was evaluated as a control. Colony forming units were counted and the MIC was defined as the lowest concentration of each compound that allowed the growth of <1% of the initial bacterial inoculum compared with the antibiotic-free quadrant. MIC90s and MIC50s have been defined as the MICs for 90% and 50%, respectively, of all strains tested.
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Results and discussion |
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The Figure shows the chemical structure of the derivatives and the different substituted chemical groups in the three different series of molecules, 2-pyridyl (compounds 19), 3-pyridyl (compounds 1017) and 4-pyridyl (compounds 1824) derivatives. The results obtained are reported as MIC90 and MIC50, defined as the concentration inhibiting 99% of growth of 90% or 50% of the strains tested (Table). The range of MICs is also reported for each compound. All strains tested (except one with comparable susceptibility to the new compounds but susceptible to ofloxacin 4 mg/L) were ofloxacin resistant at concentrations >4 mg/L.
Most compounds were active at 1632 mg/L against 50% of M. avium strains, most 4-pyridyl molecules being active at lower inhibitory concentrations. Compounds 6, 20 and 21 (3,4-di-chlorophenyl and 4-chlorophenyl derivatives), had MICs of 32 mg/L for 94% of the strains tested (16 out of 17), with MICs of 4 or 8 mg/L for four organisms, i.e. 23.5% of M. avium strains. The results obtained with the 3-pyridyl derivatives show that these compounds are largely less active, having an MIC of 160320 mg/L for 90% of strains, with the exception of compound 14, a 3,4-di-chlorophenyl derivative, which had an MIC90 of 80 mg/L and an MIC50 of 32 mg/L.
We have recently described the synthesis of a series of new compounds containing the pyridine nucleus together with the 2-pyridinecarboxamidrazone moiety and reported their interesting antimycobacterial activity.8 In this work, we report the inhibitory activity of the newly synthesized compounds against 17 different strains of M. avium isolated from clinical specimens. The promising results obtained will prompt us to examine more strains of M. avium, of M. tuberculosis and other Mycobacterium species to evaluate their susceptibility and to study the pharmacokinetics of the most active compounds, together with the molecular mechanism of action of this new group of molecules.
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Acknowledgements |
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Notes |
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References |
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2 . Nolan, C. M. (1997). Nosocomial multidrug-resistant tuberculosis. Global spread of the third epidemic. Journal of Infectious Diseases 176, 74851.[ISI][Medline]
3 . Banfi, E., Mamolo, M. G., Vio, L. & Fabris, C. (1991). Preliminary evaluation of in-vitro antimycobacterial properties of N1-(aryliden)-2-pyridine-carboxamidrazones. Journal of Chemotherapy 3, 668.[Medline]
4 . Banfi, E., Mamolo, M. G., Vio, L. & Predominato, M. (1993). In-vitro antimycobacterial activity of new synthetic amidrazone derivatives. Journal of Chemotherapy 5, 1647.[Medline]
5 . Mamolo, M. G., Vio, L., Banfi, E. & Predominato, M. (1992). Synthesis and antimycobacterial activity of some 2-pyridinecarboxyamidrazone derivatives. Farmaco 7, 10559.
6 . Mamolo, M. G., Vio, L., Banfi, E. & Predominato, M. (1993). Synthesis and antimycobacterial activity of some 4-pyridinecarboxyamidrazone derivatives. Farmaco 48, 52932.[ISI][Medline]
7 . Mamolo, M. G., Vio, L. & Banfi, E. (1996). Synthesis and antimycobacterial activity of some indole derivatives of pyridine-2-carboxamidrazone and quinoline-2-carboxamidrazone. Farmaco 51, 6571.[ISI][Medline]
8 . Mamolo, M. G., Falagiani, V., Vio, L. & Banfi, E. (1999). Synthesis and antimycobacterial activity of some N1-{1-[3-aryl-1-(pyridin-2-,3- or 4-yl)-3-oxo] propyl}-2-pyridinecarboxamidrazones. Farmaco 54, 7617.[ISI][Medline]
Received 9 April 2001; returned 25 June 2001; revised 19 July 2001; accepted 23 August 2001