Vol. 34, No. 3
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1990, p. 496497 0066-4804/901030496-02$02.00/0 Copyright 0 1990, American Society for Microbiology
Influence of N Substitution on Antimycobacterial Activity of Ciprofloxacin ACHIEL HAEMERS,l* DIRK C. LEYSEN,1 WILLY BOLLAERT,' MINGQIANG ZHANG,' AND STEFAAN R. PATTYN2 Department of Pharmaceutical Sciences' and Department of Microbiology,2 University of Antwerp, B-2610 Antwerp, Belgium Received 20 September 1989/Accepted 5 December 1989
Ciprofloxacin analogs with various substitutions on the piperazine nitrogen were tested aginst several mycobacteria. In contrast to what has been found with other gram-positive and gram-negative bacteria, alkyl analogs such as N-isopropylclproloxacin were shown to be snicandy more active than ciprofloxacin. MICs of 0.125 ,g/ml against Mycobacterium tuberculosis were found. These compounds were tested against a number of mycobacterial strains from our own collection (4) by the agar dilution method. The inoculum was a 1-,ul suspension of about 104 CFU grown in Dubos broth without Tween. Middlebrook 7H10 agar with 10%o oleic acid-albumin-dextrose solution was used. The incubation time was 5 days to 2 weeks, depending on the growth rate of the strains. Compounds were dissolved in 0.1 N potassium hydroxide solution. The results are summarized in Tables 1, 2, and 3. They show an important difference between mycobacteria and other bacteria. Indeed, N-alkylated ciprofloxacin, with alkyl chains as ethyl or propyl, were mostly two to four times more active against Mycobacterium tuberculosis than was ciprofloxacin. N-Isopropylciprofloxacin had an MIC of 0.125 igtml against each strain of M. tuberculosis tested. Such low MICs have never been reported for a fluoroquinolone. Against Mycobacterium avium these alkylated ciprofloxacin derivatives were at least twice as active, and against several strains they were even 10 to 20 times more active. Some strains, however, remained unsusceptible. Only a few strains of other mycobacteria were tested. In most cases N alkylation also increased the activity. N phenylation or N benzylation did not change (M. avium)
The fluoroquinolones show pronounced activity against mycobacteria. Among the most interesting compounds in clinical use currently are ciprofloxacin and ofloxacin. They are possible candidates to be used in the treatment of mycobacteriosis (6). Most fluoroquinolones possess a nitrogen-containing cyclic substituent on the quinolone nucleus. Although several ring structures are possible, the 1-piperazinyl substituent and the 4-methyl-1-piperazinyl substituent are the most common ones. Higher alkyl chains yield compounds that are less active against gram-positive and gram-negative bacteria (1, 2, 5). Our aim was to investigate whether both substituents were also the best when tested against mycobacteria. Hence we tested several N-substituted ciprofloxacin analogs against a series of mycobacteria. N-Methyl- and N-ethylciprofloxacin (enrofloxacin) were prepared from ciprofloxacin, respectively, by an Eschweiler-Clark methylation and with an ethyl iodide alkylation as described in the literature (3, 5) (K. Grohe, H.-J. Zeiler, K. Metzger, and H. Heitzer; German patent DE 3142854; Bayer AG, October 1981). N-Isopropyl-, N-propyl-, N-phenyl-, and N-benzylciprofloxacin were kindly provided by Bayer AG, Leverkusen, Federal Republic of Germany.
Species
M. tuberculosis M. avium
TABLE 1. MICs of ciprofloxacin and N-substituted analogs against M. tuberculosis and M. avium MIC (Lg/mw N-Ethylciprofloxacin N-Methylciprofloxacin Ciprofloxacin 50%o 50%O 90%o Range Range 50%o 90%o Range 0.5 1 0.125-0.5 (74) 0.125 0.5 0.125-0.250 (74) 0.125 0.125-1 (74)b 0.25->16 (32) 2 16 0.125->16 (32) 1 8 >16 0.25->16 (32)
a50% and 90%, MIC for 50 and 90% of strains tested, respectively.
b
*
Numbers within parentheses are numbers of strains tested. Corresponding author.
496
90%o 0.250 8
VOL. 34, 1990
NOTES
497
TABLE 2. MICs of ciprofloxacin and N-substituted analogs against Mycobacterium chelonae, M. fortuitum, and M. malmoense Species (n)
Ciprofloxacin Range 50%
M. chelonae (10) M. fortuitum (16) M. malmoense (17)
0.125->16
0.016-0.5 0.25-4
4 0.125 1
MIC (pLg/ml)a N-Methylciprofloxacin Range 50%o 90%o
90%0 16 0.25 2
0.125->16 0.016-0.5 0.125-1
4 0.125 0.125
Range
>16 0.25 0.5
N-Ethylciprofloxacin 50%
0.125->16 16 0.125 0.25
a See footnote a of Table 1.
TABLE 3. MICs of ciprofloxacin and N-substituted analogs against a series of mycobacteria MIC (,ug/ml) Species (n) CiproN-MethylN-Ethylciprofloxciprofloxfloxacin acin acin M. duvalii (1) 0.125 0.125 0.125 M. flavescens (1) 0.5 0.5 0.5 M. aurum (1) 0.125 0.5 0.25 M. gastri (2) 162a 162 82 M. gordonae (1) 2 2 1 M. terrae (1) 64 32 16 M. nonchromogenicum (3) 82, 4 43 23 M. simiae (3) 162 8, 42 4, 22 M. scrofulaceum (4) >162, 16, 8 >16, 2, 12 16, 12, 0.5 M. phlei (4) 0.1254 0.1254 0.1254 M. xenopi (1) 0.125 0.125 0.125 M. thermoresistibile (3) 0.1253 0.1253 0.0633 a The subscript indicates the number of isolates with the MIC indicated.
quinolones against mycobacteria. They deserve attention as potential antimycobacterial agents. It is also clear that when newly developed series of quinolones are tested against mycobacteria, derivatives with higher alkyl substitutions should be investigated in detail. Additional studies need to be conducted to determine whether these compounds could be active against Mycobacterium leprae.
1. 2.
3. 4.
decreased (M. tuberculosis) the activity of ciprofloxacin. It is not clear why higher alkyl substitutions increased the activity of ciprofloxacin against mycobacteria. The higher lipophilicity may play an important role. We can conclude from these results that N-ethyl- and N-isopropylciprofloxacin, although less active against grampositive or gram-negative bacteria (2, 5), are the most active or
5.
6.
LITERATURE CITED Chu, D. T. W., and P. B. Fernandes. 1989. Structure-activity relationships of the fluoroquinolones. Antimicrob. Agents Chemother. 33:131-135. Domagala, J. M., L. D. Hanna, C. L. Heifetz, M. P. Hutt, T. F. Mich, J. P. Sanchez, and M. Solomon. 1986. New structureactivity relationships of quinolone antibacterials using the target enzyme. The development and application of a DNA-gyrase assay. J. Med. Chem. 29:384404. Grohe, K., and H. Heitzer. 1987. Cycloaracylierung von Enaminen. I. Synthese von 4-Chinolon-3-carbonsauren. Liebigs Ann. Chem., p. 29-37. Jenkins, P. A., S. R. Pattyn, and F. Portaels. 1982. Diagnostic bacteriology, p. 441-470. In C. Ratledge and J. Stanford (ed.), The biology of the mycobacteria, vol. 1. Academic Press, Inc. (London), Ltd., London. Koga, H., A. Itoh, S. Murayama, S. Suzue, and T. Izikura. 1980. Structure-activity relationship of antibacterial 6,7- and 7,8-disubstituted 1-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxylic acids. J. Med. Chem. 23:1358-1363. Leysen, D. C., A. Haemers, and S. R. Pattyn. 1989. Mycobacteria and the new quinolones. Antimicrob. Agents Chemother. 33:1-5.
TABLE 1-Continued
N-Isopropylciprofloxacin Range 50%o 90%o
0.125-0.125 (22) 0.125->16 (12)
0.125 1
0.125 8
MIC (p.g/ml)' N-Propylciprofloxacin 50%o Range 90%o
0.125-0.250 (22) 0.25->16 (12)
0.125 1
0.250 8
N-Benzylciprofloxacin 50%o 90%o Range 0.5-2 (22) 0.25->16 (12)
1 4
2 >16
N-Phenylciprofloxacin 50%o 90%o Range 1-4 (22) 0.25->16 (12)
2 4
4 >16
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 1990, p. 496497 0066-4804/901030496-02$02.00/0 Copyright 0 1990, American Society for Microbiology
Influence of N Substitution on Antimycobacterial Activity of Ciprofloxacin ACHIEL HAEMERS,l* DIRK C. LEYSEN,1 WILLY BOLLAERT,' MINGQIANG ZHANG,' AND STEFAAN R. PATTYN2 Department of Pharmaceutical Sciences' and Department of Microbiology,2 University of Antwerp, B-2610 Antwerp, Belgium Received 20 September 1989/Accepted 5 December 1989
Ciprofloxacin analogs with various substitutions on the piperazine nitrogen were tested aginst several mycobacteria. In contrast to what has been found with other gram-positive and gram-negative bacteria, alkyl analogs such as N-isopropylclproloxacin were shown to be snicandy more active than ciprofloxacin. MICs of 0.125 ,g/ml against Mycobacterium tuberculosis were found. These compounds were tested against a number of mycobacterial strains from our own collection (4) by the agar dilution method. The inoculum was a 1-,ul suspension of about 104 CFU grown in Dubos broth without Tween. Middlebrook 7H10 agar with 10%o oleic acid-albumin-dextrose solution was used. The incubation time was 5 days to 2 weeks, depending on the growth rate of the strains. Compounds were dissolved in 0.1 N potassium hydroxide solution. The results are summarized in Tables 1, 2, and 3. They show an important difference between mycobacteria and other bacteria. Indeed, N-alkylated ciprofloxacin, with alkyl chains as ethyl or propyl, were mostly two to four times more active against Mycobacterium tuberculosis than was ciprofloxacin. N-Isopropylciprofloxacin had an MIC of 0.125 igtml against each strain of M. tuberculosis tested. Such low MICs have never been reported for a fluoroquinolone. Against Mycobacterium avium these alkylated ciprofloxacin derivatives were at least twice as active, and against several strains they were even 10 to 20 times more active. Some strains, however, remained unsusceptible. Only a few strains of other mycobacteria were tested. In most cases N alkylation also increased the activity. N phenylation or N benzylation did not change (M. avium)
The fluoroquinolones show pronounced activity against mycobacteria. Among the most interesting compounds in clinical use currently are ciprofloxacin and ofloxacin. They are possible candidates to be used in the treatment of mycobacteriosis (6). Most fluoroquinolones possess a nitrogen-containing cyclic substituent on the quinolone nucleus. Although several ring structures are possible, the 1-piperazinyl substituent and the 4-methyl-1-piperazinyl substituent are the most common ones. Higher alkyl chains yield compounds that are less active against gram-positive and gram-negative bacteria (1, 2, 5). Our aim was to investigate whether both substituents were also the best when tested against mycobacteria. Hence we tested several N-substituted ciprofloxacin analogs against a series of mycobacteria. N-Methyl- and N-ethylciprofloxacin (enrofloxacin) were prepared from ciprofloxacin, respectively, by an Eschweiler-Clark methylation and with an ethyl iodide alkylation as described in the literature (3, 5) (K. Grohe, H.-J. Zeiler, K. Metzger, and H. Heitzer; German patent DE 3142854; Bayer AG, October 1981). N-Isopropyl-, N-propyl-, N-phenyl-, and N-benzylciprofloxacin were kindly provided by Bayer AG, Leverkusen, Federal Republic of Germany.
Species
M. tuberculosis M. avium
TABLE 1. MICs of ciprofloxacin and N-substituted analogs against M. tuberculosis and M. avium MIC (Lg/mw N-Ethylciprofloxacin N-Methylciprofloxacin Ciprofloxacin 50%o 50%O 90%o Range Range 50%o 90%o Range 0.5 1 0.125-0.5 (74) 0.125 0.5 0.125-0.250 (74) 0.125 0.125-1 (74)b 0.25->16 (32) 2 16 0.125->16 (32) 1 8 >16 0.25->16 (32)
a50% and 90%, MIC for 50 and 90% of strains tested, respectively.
b
*
Numbers within parentheses are numbers of strains tested. Corresponding author.
496
90%o 0.250 8
VOL. 34, 1990
NOTES
497
TABLE 2. MICs of ciprofloxacin and N-substituted analogs against Mycobacterium chelonae, M. fortuitum, and M. malmoense Species (n)
Ciprofloxacin Range 50%
M. chelonae (10) M. fortuitum (16) M. malmoense (17)
0.125->16
0.016-0.5 0.25-4
4 0.125 1
MIC (pLg/ml)a N-Methylciprofloxacin Range 50%o 90%o
90%0 16 0.25 2
0.125->16 0.016-0.5 0.125-1
4 0.125 0.125
Range
>16 0.25 0.5
N-Ethylciprofloxacin 50%
0.125->16 16 0.125 0.25
a See footnote a of Table 1.
TABLE 3. MICs of ciprofloxacin and N-substituted analogs against a series of mycobacteria MIC (,ug/ml) Species (n) CiproN-MethylN-Ethylciprofloxciprofloxfloxacin acin acin M. duvalii (1) 0.125 0.125 0.125 M. flavescens (1) 0.5 0.5 0.5 M. aurum (1) 0.125 0.5 0.25 M. gastri (2) 162a 162 82 M. gordonae (1) 2 2 1 M. terrae (1) 64 32 16 M. nonchromogenicum (3) 82, 4 43 23 M. simiae (3) 162 8, 42 4, 22 M. scrofulaceum (4) >162, 16, 8 >16, 2, 12 16, 12, 0.5 M. phlei (4) 0.1254 0.1254 0.1254 M. xenopi (1) 0.125 0.125 0.125 M. thermoresistibile (3) 0.1253 0.1253 0.0633 a The subscript indicates the number of isolates with the MIC indicated.
quinolones against mycobacteria. They deserve attention as potential antimycobacterial agents. It is also clear that when newly developed series of quinolones are tested against mycobacteria, derivatives with higher alkyl substitutions should be investigated in detail. Additional studies need to be conducted to determine whether these compounds could be active against Mycobacterium leprae.
1. 2.
3. 4.
decreased (M. tuberculosis) the activity of ciprofloxacin. It is not clear why higher alkyl substitutions increased the activity of ciprofloxacin against mycobacteria. The higher lipophilicity may play an important role. We can conclude from these results that N-ethyl- and N-isopropylciprofloxacin, although less active against grampositive or gram-negative bacteria (2, 5), are the most active or
5.
6.
LITERATURE CITED Chu, D. T. W., and P. B. Fernandes. 1989. Structure-activity relationships of the fluoroquinolones. Antimicrob. Agents Chemother. 33:131-135. Domagala, J. M., L. D. Hanna, C. L. Heifetz, M. P. Hutt, T. F. Mich, J. P. Sanchez, and M. Solomon. 1986. New structureactivity relationships of quinolone antibacterials using the target enzyme. The development and application of a DNA-gyrase assay. J. Med. Chem. 29:384404. Grohe, K., and H. Heitzer. 1987. Cycloaracylierung von Enaminen. I. Synthese von 4-Chinolon-3-carbonsauren. Liebigs Ann. Chem., p. 29-37. Jenkins, P. A., S. R. Pattyn, and F. Portaels. 1982. Diagnostic bacteriology, p. 441-470. In C. Ratledge and J. Stanford (ed.), The biology of the mycobacteria, vol. 1. Academic Press, Inc. (London), Ltd., London. Koga, H., A. Itoh, S. Murayama, S. Suzue, and T. Izikura. 1980. Structure-activity relationship of antibacterial 6,7- and 7,8-disubstituted 1-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxylic acids. J. Med. Chem. 23:1358-1363. Leysen, D. C., A. Haemers, and S. R. Pattyn. 1989. Mycobacteria and the new quinolones. Antimicrob. Agents Chemother. 33:1-5.
TABLE 1-Continued
N-Isopropylciprofloxacin Range 50%o 90%o
0.125-0.125 (22) 0.125->16 (12)
0.125 1
0.125 8
MIC (p.g/ml)' N-Propylciprofloxacin 50%o Range 90%o
0.125-0.250 (22) 0.25->16 (12)
0.125 1
0.250 8
N-Benzylciprofloxacin 50%o 90%o Range 0.5-2 (22) 0.25->16 (12)
1 4
2 >16
N-Phenylciprofloxacin 50%o 90%o Range 1-4 (22) 0.25->16 (12)
2 4
4 >16
