Antimycobacterial activity of N1-{1-[3-aryl-1-(pyridin-2-, 3- or 4-yl)-3-oxo] propyl}-2-pyridinecarboxamidrazones

E. Banfia,*, M. G. Mamolob, D. Zampierib, L. Viob and C. Monti Bragadina

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Infections caused by non-tuberculous mycobacteria and multidrug-resistant Mycobacterium tuberculosis are difficult to treat, and so new compounds potentially active against these bacteria are being sought. A series of 2-pyridinecarboxamidrazone derivatives, recently synthesized, have been evaluated for their inhibitory activity against 17 Mycobacterium avium isolates; the agar dilution method showed different degrees of susceptibility to the new molecules. Four molecules, three of which are chlorine derivatives, inhibited 94% of the strains tested with an MIC of 32 mg/L. These data indicate that these new pyridine-2-carboxamidrazones merit further study as antimycobacterial agents.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Mycobacteria are known to be intrinsically resistant to most chemicals and disinfectants and to a number of antibiotics and chemotherapeutic substances. There is an increase in reported cases of morbidity and mortality from Mycobacterium spp. and pulmonary tuberculosis in many institutions worldwide,1 and an increasing incidence of multidrug-resistant Mycobacterium tuberculosis strains.2 Outbreaks are more common among immune-suppressed patients, especially human immunodeficiency virus (HIV)-positive patients, and are characterized by a high fatality rate. The emergence of infections associated with mycobacteria other than M. tuberculosis has focused attention on Mycobacterium avium complex disease, which remains a common cause of morbidity and frequently produces severe disseminated infections in HIV-infected individuals. Antimycobacterial therapy with a combination of drugs is required, and new active molecules to extend the options for first-line therapy are needed.

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.3–7 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.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Bacterial isolates

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 FigureGo.



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Figure. Chemical structure of 2-pyridinecarboxamidrazone derivatives.

 
Stock solutions of each agent were prepared in dimethyl sulphoxide at a concentration of 6.4 g/L.

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 Lowenstein–Jensen slants (Becton Dickinson) were freshly suspended in 0.02% Tween phosphate-buffered saline to give a concentration of 1–5 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 15–20 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.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Activity against M. avium

The FigureGo shows the chemical structure of the derivatives and the different substituted chemical groups in the three different series of molecules, 2-pyridyl (compounds 1–9), 3-pyridyl (compounds 10–17) and 4-pyridyl (compounds 18–24) 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 16–32 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 160–320 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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Table. Antimycobacterial activity of 2-pyridine carboxamidrazone derivatives against 17 strains of M. avium
 

    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
This work had financial support from MURST and University of Trieste, fondi 60%, 1999.


    Notes
 
* Corresponding author. Tel: +39-40-6767187; Fax: +39-40-351668; E-mail: banfi{at}dsbmail.units.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Snider, D. E. J., Raviglione, M. & Kochi, A. (1994). Global burden of tuberculosis. In Tuberculosis: Pathogenesis, Protection and Control, (Bloom, B. R., Ed.), pp. 3–11. American Society for Microbiology, Washington, DC.

2 . Nolan, C. M. (1997). Nosocomial multidrug-resistant tuberculosis. Global spread of the third epidemic. Journal of Infectious Diseases 176, 748–51.[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, 66–8.[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, 164–7.[Medline]

5 . Mamolo, M. G., Vio, L., Banfi, E. & Predominato, M. (1992). Synthesis and antimycobacterial activity of some 2-pyridinecarboxyamidrazone derivatives. Farmaco 7, 1055–9.

6 . Mamolo, M. G., Vio, L., Banfi, E. & Predominato, M. (1993). Synthesis and antimycobacterial activity of some 4-pyridinecarboxyamidrazone derivatives. Farmaco 48, 529–32.[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, 65–71.[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, 761–7.[ISI][Medline]

Received 9 April 2001; returned 25 June 2001; revised 19 July 2001; accepted 23 August 2001





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