Antimycobacterial activity of 1-deaza-7,8-dihydropteridine derivatives against Mycobacterium tuberculosis and Mycobacterium avium complex in vitro

William J. Suling,* and Joseph A. Maddry

Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Twenty-five 1-deaza-7,8-dihydropteridine derivatives were screened for antimycobacterial activity against Mycobacterium tuberculosis strain H37Ra and three Mycobacterium avium clinical isolates (serovar 1, 4 or 6). Antibacterial activity was determined with a colorimetric microdilution broth assay. Seventeen of the compounds inhibited growth in the range >1.28 to <=12.8 mg/L against one or more of the test strains. The presence of an alkoxycarbonyl group on the amino nitrogen at position 2 was not required for activity. Activity was dependent upon the type and location of group substitutions on the 6-phenyl ring and, in some cases, the presence of a 7-alkyl group.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Effective chemotherapy of persons with AIDS who are also infected with Mycobacterium avium or Mycobacterium tuberculosis, especially multidrug-resistant strains, can be difficult.1,2 Although some success has been achieved through the rational use of multiple drug combination therapy, the resistant nature of clinical isolates of M. avium and the appearance of drug-resistant tuberculosis emphasize the need for new drugs to treat these infections. As part of a continuing programme at Southern Research Institute for the development of putative antifolates, a series of 1-deaza-7,8-dihydropteridines was synthesized and tested initially for anticancer activity.3,4 Although some of the derivatives were active as anticancer agents, they were not good inhibitors of their predicted target, dihydrofolate reductase. The inhibitors were found subsequently to cause mitotic arrest in cancer cells5 and further studies established a correlation between cytotoxicity and tubulin binding of the compounds.6

More recently, several of the deazapteridine derivatives were submitted to the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF; NIH, NIAID Contract AI45246) for screening against M. tuberculosis; some of them demonstrated activity against M. tuberculosis strain H37Rv. We later confirmed the antimicrobial activity of this class of compounds and report here on the activity of 1-deaza-7,8-dihydropteridines against both M. tuberculosis and M. avium in vitro. Also, we present data which suggest that these compounds do not inhibit mycobacterial dihydrofolate reductase. This class of compounds does not appear to inhibit the polymerization of mycobacterial FtsZ, the bacterial homologue of tubulin.7


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Synthesis of 1-deaza-7,8-dihydropteridine derivatives

The methods for the synthesis of 1-deaza-7,8-dihydropteridines have been reported elsewhere.3 A list of the compounds and their structures is presented in Tables I and IIGoGo.


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Table I. Effect of substitutions on the 2-N, and 7- and 8-positions of 6-phenyl-2,4-diamino-1-deaza-7,8-dihydropteridine on antimycobacterial activity
 

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Table II. Effect of substitutions on the 7-position of the heterocyclic ring and the 3', 4', 5' and 6' positions of the 6-phenyl group
 
Screening tests for antibacterial activity

The inhibitory activity of the test compounds against M. tuberculosis H37Ra (ATCC 25177; American Type Culture Collection, Rockville, MD, USA) and M. avium clinical isolates (serovar 1, 4 or 6; National Jewish Center for Immunology and Respiratory Diseases, Denver, CO, USA) was determined with a colorimetric microdilution broth assay described elsewhere.8 The compounds were dissolved in dimethylsulphoxide (DMSO), then diluted serially in assay medium with 10-fold dilutions. The greatest concentration of DMSO in the assay medium was 1.3%. This concentration did not affect the growth of the test organisms. The antimycobacterial drug ethambutol (Sigma, St Louis, MO, USA) was used as a positive control.

Dihydrofolate reductase inhibition

The inhibition of M. avium dihydrofolate reductase was determinined using purified recombinant enzyme as described elsewhere.8 Enzyme activity was measured by following the decrease in absorbance at 340 nm. Dihydrofolate (0.1 mM) was used to initiate the reaction after a 3 min preincubation of a mixture containing 10 mM 2- mercaptoethanol, 0.1 mM NADPH, 1 mM EDTA, 55 mM potassium phosphate (pH 7), enzyme and inhibitor as appropriate. The total assay volume was 1 mL and the temperature 30°C.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Structure–activity relationships

Overall, 17 of the 25 compounds tested were active (MIC <= 12.8 mg/L) against one or more of the bacterial strains. Growth inhibition by the positive control agent, ethambutol, for M. tuberculosis H37Ra was in the range >0.128 to <=12.8 mg/L and that for the M. avium strains was in the range >1.28 to <=12.8 mg/L (data not shown). Although most of the compounds tested contained an alkoxycarbonyl group (R1, Table IGo) attached to the 2-amino nitrogen, this substitution was not required for activity. This is apparent by comparing compound 6 with compounds 3 and 4. Also, compounds with an acetoxycarbonyl substitution on the 2-N position and a phenyl substitution at position 6 (Table IGo, compounds 1, 2, 3, 4, 5 and 11) were active against one or more of the strains only when a methyl or ethyl group was present on the 7 or 8 position (Table IGo, R3 and R2, respectively). Interestingly, only the S-(–) enantiomer of the methyl substitution at position 6 was active (compare compound 4 with compound 5). In contrast to the observation that no substitutions on the 6-phenyl ring were required for activity as long as the heterocyclic ring contained an alkyl substitution at the 7- or 8-position, methyl-, methoxy-, chloro- and trifluoromethyl-6-phenyl substituted derivatives were active against one or more strains without an alkyl group at these positions (Table IIGo, compounds 12, 13, 18, 19, 20, 21 and 23). An amino- or fluoro-6-phenyl substitution, however, was inactive (compounds 22 and 24)

Inhibition of dihydrofolate reductase

We assayed compounds 2 and 6 for their ability to inhibit recombinant M. avium dihydrofolate reductase activity. No inhibition was seen with compound 2 or 6 at 32.5 and 40.4 µM, the highest concentrations assayed, respectively. Active pteridine derivatives, which have been assayed by us under the same conditions, inhibit enzyme activity by 50% at nanomolar concentrations.8


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although synthesized originally as putative antifolates with potential anticancer activity, several 1-deaza-7,8-dihydropteridines were found in the present study to possess antimycobacterial activity. Compounds of this type are poor inhibitors of dihydrofolate reductase from Streptococcus faecium, presumably because of the importance of the nitrogen at the 1-position to tight binding to the enzyme.9 Our examination of compounds 2 and 6 in micromolar concentrations showed a similar lack of activity for recombinant M. avium dihydrofolate reductase.

Certain 1-deaza-7,8-dihydropteridines cause mitotic arrest in cancer cells in vitro.5 Also, two of the compounds used for the present study, compounds 1 and 3, inhibit competitively the binding of colchicine to tubulin and the polymerization of tubulin.6 Recently FtsZ, a bacterial protein essential for cell division, has been proposed to be the tubulin homologue in eubacteria, and the solution of the crystal structure of recombinant FtsZ from Methanococcus jannaschii has revealed its strong similarity to that of tubulin.7 Since 1-deazadihydropteridines interact with tubulin, their mechanism of action in mycobacteria may involve a similar interaction with FtsZ. Recombinant FtsZ from M. tuberculosis was expressed recently in Escherichia coli and purified.10 Several 1-deaza-7,8-dihydropteridines that were active against M. tuberculosis H37Rv (TAACF, NIH, NIAID contract) did not inhibit M. tuberculosis FtsZ polymerization (E. Lucile White, personal communication). These results support the conclusion that these compounds did not interfere with the function of the FtsZ protein in mycobacteria.

In summary, our results demonstrate that 1-deaza-7,8-dihydropteridine derivatives inhibit the growth of M. tuberculosis and M. avium, and therefore could be useful as antimycobacterial drugs. Studies of the mechanism of action of these compounds indicate that they do not inhibit mycobacterial dihydrofolate reductase and do not interfere with the essential function of FtsZ in cell division. Further investigations are needed to determine how these compounds inhibit the growth of mycobacteria.


    Acknowledgments
 
The compounds used in this study were synthesized originally by Dr Carroll Temple Jr, Southern Research Institute. The M. avium strains were kindly provided by Dr Leonid Heifets, National Jewish Center for Immunology and Respiratory Diseases, Denver, CO, USA. We thank Sabrina Zywno-van Ginkel, Molecular Genetics Laboratory, Southern Research Institute, for providing the recombinant dihydrofolate reductase and Louise Westbrook for performing the dihydrofolate reductase inhibition assays. We also thank E. Lucile White, Biochemistry and Molecular Biology Department, Southern Research Institute, for sharing with us the results of her M. tuberculosis FtsZ polymerization inhibition studies. Part of this work was supported by National Institutes of Health grant AI38667 (J. A. Maddry, Principal Investigator) and by a Southern Research Institute research and development grant. These results were presented in part at the Ninety-Eighth General Meeting of the American Society for Microbiology, March 17–21, 1998, Atlanta, GA, USA.


    Notes
 
* Corresponding author. Tel: +1-205-581-2558; Fax: +1-205-581-2877; E-mail: suling{at}sri.org Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Ellner, J. J., Goldberger, M. J. & Parenti, D. M. (1991). Mycobacterium avium infection and AIDS: a therapeutic dilemma in rapid evolution. Journal of Infectious Diseases 163, 1326–35.[ISI][Medline]

2 . Snider, D. E. & La Montagne, J. R. (1994). The neglected global tuberculosis problem: a report of the 1992 World Congress on Tuberculosis. Journal of Infectious Diseases 169, 1189–96.[ISI][Medline]

3 . Temple, C., Wheeler, G. P., Elliott, R. D., Rose, J. D., Comber, R. N. & Montgomery, J. A. (1983). 1,2-Dihydropyrido[3,4-b]pyrazines: structure–activity relationships. Journal of Medicinal Chemistry 26, 91–5.[ISI][Medline]

4 . Wheeler, G. P., Bowdon, B. J., Werline, J. A. & Temple, C. (1981). 1-Deaza-7,8-dihydropteridines, a new class of mitotic inhibitors with anticancer activity. Biochemical Pharmacology 30, 2381–4.[ISI][Medline]

5 . Wheeler, G. P., Bowdon, B. J., Werline, J. A., Adamson, D. J. & Temple, C. G. (1982). Inhibition of mitosis and anticancer activity against experimental neoplasms by ethyl 5-amino-1,2-dihydro- 3-[(N-methylanilino)methyl]-pyrido[3,4-b]pyrazin-7-yl carbamate (NSC 181928). Cancer Research 42, 791–8.[Abstract]

6 . Bowdon, B. J., Waud, W. R., Wheeler, G. P., Hain, R., Dansby, L. & Temple, C. (1987). Comparison of 1,2-dihydropyrido[3,4-b]pyrazines (1-deaza-7,8-dihydropteridines) with several other inhibitors of mitosis. Cancer Research 47, 1621–6.[Abstract]

7 . Lowe, J. & Amos, L. A. (1998). Crystal structure of the bacterial cell-division protein FtsZ. Nature 391, 203–6.[ISI][Medline]

8 . Suling, W. J., Seitz, L. E., Pathak, V., Westbrook, L., Barrow, E. W., Zywno-Van Ginkel, S. et al. (2000). Antimycobacterial activities of 2.4-diamino-5-deazapteridine derivatives and effects on mycobacterial dihydrofolate reductase. Antimicrobial Agents and Chemotherapy 44, 2784–93.[Abstract/Free Full Text]

9 . Williams, J. W., Duggleby, R. G., Cutler, R. & Morrison, J. F. (1980). The inhibition of dihydrofolate reductase by folate analogues: structural requirements for slow- and tight-binding inhibition. Biochemical Pharmacology 29, 589–95.[ISI][Medline]

10 . White, E. L., Ross, L. J., Reynolds, R. C., Seitz, L. E., Moore,G. D. & Borhani, D. W. (2000). Slow polymerization of Mycobacterium tuberculosis FtsZ. Journal of Bacteriology 182, 4028–34.[Abstract/Free Full Text]

Received 1 August 2000; returned 18 October 2000; revised 22 November 2000; accepted 1 December 2000