JAC
Journal of Antimicrobial Chemotherapy (1998) 42, 811–815
Susceptibilities of Mycobacterium tuberculosis and Mycobacterium avium complex to lipophilic deazapteridine derivatives, inhibitors of dihydrofolate reductase W. J. Suling*, R. C. Reynolds, E. W. Barrow, L. N. Wilson, J. R. Piper and W. W. Barrow Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA Twelve lipophilic 2,4-diamino-5-methyl-5-deazapteridine derivatives and trimethoprim were evaluated for activity against Mycobacterium tuberculosis and Mycobacterium avium in vitro. Six of the compounds had MICs of 12.8 mg/L and 1.28 mg/L against M. tuberculosis and M. avium, respectively; trimethoprim MICs were >128 mg/L and >12.8 but 128 mg/L, respectively. Two compounds, with either a 2-methyl-5-methoxy phenyl or 2-methoxy-5trifluoromethyl phenyl linked at the 6-position of the deazapteridine moiety by a CH2NH bridge, had MICs of 0.13 mg/L against M. avium; the two compounds also had apparent I50 values for dihydrofolate reductase of 2 and 8 nM, respectively, compared with an I50 of 400 nM with trimethoprim. Four of the compounds were selectively toxic to mycobacteria as compared with Vero cells. These results demonstrated that lipophilic antifolates can be synthesized which are more active against mycobacteria than trimethoprim and which possess selective toxicity.
Introduction Among the opportunistic infections associated with AIDS, the Mycobacterium avium complex is a major cause of systemic disease. 1 Also of concern is the serious problem in people co-infected with Mycobacterium tuberculosis and HIV2 and the global problem tuberculosis presents for others. The above, together with the resistant nature of M. avium to antitubercular drugs1 and the appearance of multidrug-resistant clinical isolates of M. tuberculosis,2 emphasize the need for new agents to treat these infections. The enzyme dihydrofolate reductase (DHFR, EC 1.5.1.3) is an important target for medicinal chemistry.3 Inhibitors of this enzyme have been used to treat cancer as well as bacterial and protozoal diseases. DHFR catalyses the reduction of dihydrofolate to tetrahydrofolate, the reduced form of folate that is involved in a variety of biochemical functions involving single-carbon transfers at various oxidation states. Inhibition of DHFR results in the depletion of intracellular reduced folate cofactors necessary for the biosynthesis of thymidylate, purine nucleotides, methionine, serine and glycine required for DNA, RNA and protein synthesis. Bacterial DHFRs differ sufficiently from mammalian DHFR to allow the development of selective inhibitors such as trimethoprim
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which is used to treat a variety of bacterial infections. However, this drug is reported to have poor activity against mycobacteria. 4 The objective of the present study was to evaluate the in-vitro activity of a series of lipophilic deazapteridine derivatives against M. tuberculosis and M. avium and to determine the activity of selected analogues against M. avium DHFR.
Materials and methods Compounds The structures of the compounds used in this study are presented in Table I. The syntheses of these specific deazapteridines have been reported.5
MICs MICs for M. tuberculosis strain H37Ra (ATCC 25177, American Type Culture Collection, Rockville, MD, USA) and the M. avium clinical isolates (National Jewish Center for Immunology and Respiratory Diseases, Denver, CO, USA) were determined using the colorimetric microdilution broth assay described below, which uses the dye Alamar blue.6,7,8 A frozen culture in Middlebrook 7H9 broth (Difco, Detroit, MI, USA) supplemented with 10%
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811 © 1998 The British Society for Antimicrobial Chemotherapy
W. J. Suling et al. Table I. Activity of 2,4-diamino-5-methyl-5-deazapteridine derivatives and trimethoprim against M. tuberculosis H37Rv, M. tuberculosis H37Ra and Vero cells MIC (mg/L) Compound
Structure
SRI number
H37Rv
H37Ra
IC50 Vero cells (mg/L)
1
8709
12.5
1.28,
12.8
2
8117
12.5
1.28,
12.8
717
57
3
7714
12.5
12.8,
128
ND
ND
4
8922
12.5
ND
ND
5
8710
12.8
222
71
6
8686
12.5
64
ND
ND
7
8687
12.5
ND
ND
8
8202
6.25
1.28,
12.8
461
74
9
8229
6.25
1.28,
12.8
471
75
10
8911
ND
ND
3.13
12.5
812
ND
1.28,
32,
ND
ND
40.6
SI 3.2
Mycobacteria and dihydrofolate reductase inhibitors Table I. Continued MIC (mg/L) Compound
Structure
SRI number
H37Rv
H37Ra
1.56
0.128,
11
8228
12
8691
12.5
13
8708
12.5
Trimethoprim
none
ND
IC50 Vero cells (mg/L) 1.28
64
32,
64
128
1.03
SI 0.66
322
26
ND
ND
ND
ND
ND, not determined.
albumin–dextrose–catalase (ADC, Difco) and 0.2% glycerol (Difco) was thawed and diluted in broth to 2 105 and 1 105 cfu/mL for M. avium and M. tuberculosis, respectively, and used as the inoculum. The assay used a 96-well ( -shaped) microtitre plate and a format designed to accommodate seven compounds in 4-log10 dilutions. The assay plates also contained colour, medium and viability controls. Each test compound was dissolved in dimethylsulphoxide (DMSO) then diluted in broth at twice the desired concentration; 0.05 mL was added to duplicate assay wells. The final concentration of DMSO in the assay medium was 1.3%. Each plate was then inoculated with 0.05 mL of standardized culture and the plates incubated at 37°C for 6 or 13 days, depending upon the assay strain. The oxidation–reduction indicator Alamar blue (Acumed International, Inc., Westlake, OH, USA) was then added to each well (0.035 mL) as a mixture with Tween 80 (Difco) (one part dye to 2.5 parts 5% Tween 80) and the plates were incubated for an additional 18–22 h. The plates were read in an optical microtitre plate reader programmed to subtract the absorbance at 600 nm from that at 570 nm to blank out turbidity and absorbance due to oxidized dye. The MIC was reported as the lowest concentration of drug yielding a differential absorbance of zero or less. Growth and viability were indicated visually by the metabolic reduction of the dye from blue to red.
MICs of the test compounds were also obtained for M. tuberculosis strain H37Rv through the Tuberculosis Antimicrobial Coordinating Facility (TAACF) (NIH, NIAID Contract No. NO1-AI45246). These data, along with the Vero cell cytotoxicity determinations described below, were obtained through the above contract to assess the selective toxicity of the compounds. The MIC of each drug was determined with the radiometric BACTEC assay and was defined as the lowest concentration of drug which inhibits 99% of the bacterial population present at the beginning of the assay.8
Vero cell cytotoxicity The cytotoxicity of each drug for Vero cells was determined using the Promega CellTiter 96 Non-radioactive Cell Proliferation Assay (Promega Corp., Madison, WI, USA) through the TAACF and reported as the IC50, or the concentration of drug which inhibited growth by 50% of the control. The selectivity index (SI) was determined as the ratio of the IC50 to the MIC (BACTEC assay).
Dihydrofolate reductase assay A purified cell-free preparation of enzyme was obtained from a culture of M. avium serovar 4. Washed cells
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W. J. Suling et al. were suspended in 10 mM Tris–HCl (pH 7.4) containing 150 mM NaCl, 10 mM EDTA and 0.1% Tween 80 to which was added leupeptin (0.5 mg/L), pepstatin (0.67 mg/L), 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 0.6% DNase and 0.6% RNase (Sigma, St Louis, MO, USA) and broken in a Bead Beater (Biospec Products, Bartlesville, MD, USA). Following centrifugation, the supernatant was filtered sequentially through 0.45 and 0.2 m Supor Acrodisc filters (Belman Sciences, Ann Arbor, MI, USA) and the proteins then precipitated in ice with ammonium sulphate to 80% saturation and stored at 20°C. Portions were removed and centrifuged in the cold, and the precipitate was dissolved in standard assay buffer (50 mM potassium phosphate–1 mM EDTA, pH 7). DHFR activity was measured as described elsewhere9 as the decrease in absorbance at 340 nm. The reaction was initiated by the addition of dihydrofolate (Sigma) after preincubation of the other components (NADPH, enzyme, buffer, 2-mercaptoethanol) for 3 min. Activity was corrected for NADPH oxidase activity. For inhibition assays, varying amounts of inhibitor were added to the mixture before the 3 min preincubation period. The I50 was determined as the amount of inhibitor required to inhibit the reaction rate by 50%.
Results MICs (Tables I and II) Several of the derivatives were more active than trimethoprim. Among the derivatives containing the phenyl group, activity was dependent upon the group substitution and whether or not the bridging amino group was methylated. Substitution of the phenyl ring with methoxy groups resulted in poor activity with a tri-methoxy substitution (3) and greater activity with di-methoxy substitutions (1 and 2). Also, methylation of the bridging amino group resulted in overall greater activity of 5 compared with 1, 2 and 3 but the presence of ethoxy substitutions on the phenyl group caused a loss of activity (6 and 7). Two compounds, a
2-methyl-5-methoxy phenyl derivative (8) and a 2methoxy-5-trifluoromethyl phenyl derivative (11), were very active ( 0.13 mg/L) against M. avium.
Vero cell toxicity (Table I) The types of substitutions on the phenyl group affected the toxicity of the compounds for Vero cells. For example, although 11 was very active against M. tuberculosis and M. avium, its IC50 resulted in a poor selectivity index. Also, the 3,5-methoxy phenyl derivative (1) was more toxic than the 3,6-methoxy phenyl derivative (2). The best selectivities were observed with 5, 8 and 9.
Inhibition of DHFR Two of the 5-deazapteridine derivatives (8 and 11) were compared with trimethoprim for inhibition of DHFR activity. Compounds 8 (I50 2 nM) and 11 (I50 8 nM) were 200- and 50-fold more active, respectively, against DHFR than trimethoprim (I50 400 nM).
Discussion DHFR has been considered as a target for chemotherapy for nearly 50 years. 3 The potential for the development of inhibitors of DHFR with selective toxicity is perhaps best represented by trimethoprim. Although not very active against mycobacteria, this drug has good selectivity for bacterial DHFRs over the mammalian enzyme.3 Two groups in particular have reported on the synthesis of lipophilic inhibitors of mycobacterial DHFR. Colwell et al.10 reported on the syntheses and activities of 2,4diamino-6-substituted pteridines, 8-deazapteridines, 5deazapteridines and quinazolines against the DHFR and whole cells of Mycobacterium sp. 607. Their data support the activity of representatives of each compound class against mycobacteria. The 5-deazapteridines had aryl groups linked either directly or through a methyl bridge to
Table II. Activity of 2,3-diamino-5-methyl-5-deazapteridine derivatives and trimethoprim against M. avium clinical isolates MIC (mg/L) Compound 1 2 5 8 9 11 Trimethoprim
NJ-168 serovar 1
NJ-3404 serovar 4
0.128, 1.28 0.128, 1.28 0.128, 1.28 0.128 0.128, 1.28 0.128 12.8, 128
0.128, 1.28 0.128, 1.28 0.128 0.128 0.128, 1.28 0.128 12.8, 128
814
NJ-211 serovar 4/6 0.128, 1.28 0.128 0.128 0.128 ND 0.128 12.8, 128
Mycobacteria and dihydrofolate reductase inhibitors the pteridine ring at position 6. Others4 have focused on the synthesis of 2,4-diamino-5-benzyl pyrimidines (trimethoprim derivatives) with improved activity against mycobacteria. The data from the present study showed that 2,4-diamino-5-methyl-5-deazapteridines with aryl group substitutions at the 6-position linked through a CH2NH bridge were active against both M. tuberculosis and M. avium. Also, both antimycobacterial activity and Vero cell toxicity could vary depending on the type and position of the phenyl group substitutions and whether or not the bridge was methylated. The limited I50 data for inhibition of DHFR by two of the compounds and trimethoprim suggested that the active derivatives (8 and 11) evaluated in the present study are strong inhibitors of the mycobacterial enzyme whereas trimethoprim is a weaker inhibitor. Also, the inhibition (I50) of rat liver DHFR by 8 and 11 was reported previously.5 We compared the reported I50 values for inhibition of rat liver DHFR with those reported in the present study for bacterial DHFR; the results correlated with the differential toxicity observed between mycobacteria and Vero cells, i.e. 8 was 75-fold more active against M. avium DHFR than against rat liver DHFR, while 11 had little or no selectivity for the two enzymes. These data suggest that it will be possible to synthesize potent inhibitors of mycobacterial DHFR with good selective toxicity.
Acknowledgements This research was funded in part by National Institutes of Health Grant AI41348. MICs for M. tuberculosis strain H37Rv and Vero Cell IC50s were obtained through the Tuberculosis Antimicrobial Coordinating Facility, National Institutes of Health, NIAID Contract No. NO1AI45246. The M. avium strains were kindly provided by Dr Leonid Heifets, National Jewish Center for Immunology and Respiratory Diseases, Denver, CO, USA. Presented in part at the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, September 28–October 1, 1997, Toronto, Ontario, Canada.
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