Comparative study of new benzenesulphonamide fluoroquinolones structurally related to ciprofloxacin against selected ciprofloxacin-susceptible and -resistant Gram-positive cocci

F. Aloveroa, A. Barnesa,b, M. Nietoa, M. R. Mazzieria and R. H. Manzoa,*

a Departamento de Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, (5000) Ciudad Universitaria, Córdoba; b Hospital Tránsito Cáceres de Allende, Córdoba, Argentina


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The in vitro activities of benzenesulphonamide fluoroquinolones (BSFQs) I–III, new fluoroquinolones with a p-substituted benzenesulphonyl moiety attached to the C7 piperazinyl ring of ciprofloxacin, were assessed in comparison with those of N-sulfanilylpiperazinyl fluoroquinolone (NSFQ)-105 and ciprofloxacin for 133 Gram-positive clinical isolates. NSFQ-105 and BSFQ-I were the most active drugs. They were 16- to 64-fold more active than ciprofloxacin against Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, including ciprofloxacin-resistant strains, and Streptococcus pneumoniae (eight- to 32-fold). A high degree of correlation was found between the MICs of the new compounds and ciprofloxacin for Staphylococcus spp. and E. faecalis. Staphylococci and enterococci exhibit cross-resistance to BSFQs and ciprofloxacin.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
In the development of quinolones, from nalidixic acid to the latest extended-spectrum fluoroquinolones, structural modifications have allowed significant improvements of their properties. The fluoroquinolones developed after norfloxacin, exemplified by ciprofloxacin, had good activity against Gram-negative species and a number of atypical pathogens, good-to-moderate activity against Grampositive species, and were well absorbed and distributed. The growth in understanding of structure–activity relationships of fluoroquinolones has enabled the development of even better compounds. In recent years, interest has focused on improvements in pharmacokinetic properties, greater activity against Gram-positive cocci and anaerobes, activity against fluoroquinolone-resistant strains and improvements in activity against non-fermentative Gram-negative species. The compounds developed in recent years have fulfilled some, but not all, of these goals.1 On the basis of their in vitro activity, newer fluoroquinolones such as gatifloxacin, clinafloxacin, moxifloxacin and gemifloxacin provide a potentially important breakthrough in chemotherapy against Gram-positive bacteria.1,2 These fluoroquinolones have either been introduced recently into clinical use or reached the stage of clinical evaluation. Nevertheless, some of them are not quite as active as ciprofloxacin against Enterobacteriaceae, and show no substantial improvements in activity against non-fermentative species.1 The N-sulfanilylpiperazinyl fluoroquinolones, NSFQ-104 and NSFQ-105, are a novel class of antibacterial fluoroquinolones, structurally related to norfloxacin and ciprofloxacin, respectively.3 NSFQs have a 4-amino benzenosulphonylamide group attached to the C7 piperazinyl ring of norfloxacin or ciprofloxacin and display signifi-cantly enhanced activity against Staphylococcus aureus.4 Later, different p-substitutions introduced in the benzenesulphonylamide group of NSFQs yielded a series of 16 new benzenesulphonamide fluoroquinolones (BSFQs).5–7

In this study, we compared the in vitro antibacterial activity of several BSFQs with that of NSFQ-105 and ciprofloxacin (Figure 1Go) against medically significant Grampositive strains such as S. aureus, Streptococcus pneumoniae and Enterococcus faecalis because Gram-positive bacteria are rapidly becoming the most important pathogens in hospital infections,1 adding new data to our earlier study.4,6



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Figure 1. Chemical structure of the fluoroquinolones used in this study.

 

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

Clinical isolates were collected and identified by standard techniques at the Hospital Tránsito Cáceres de Allende, Córdoba, Argentina, from 1996 to 2000.

Susceptibility tests

The in vitro activity was assayed by the agar dilution technique as recommended by the NCCLS,8 using Mueller– Hinton agar (Merck Química Argentina SAIC, Buenos Aires, Argentina). NSFQ-105 and BSFQs I–III were synthesized in our laboratory as described previously.3,5,7 Ciprofloxacin was obtained from Amifarma (Madrid, Spain). The effect of pH on activity was determined against S. aureus ATCC 29213 by the macrodilution tube method with Mueller–Hinton broth as reported previously.4 The final inoculum size was 5 x 105 cfu/mL.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The table shows the MIC50s, MIC90s and MIC ranges of the new fluoroquinolones and ciprofloxacin for 133 Gram-positive bacteria. The isolates were pooled according to their ciprofloxacin susceptibility or resistance.9

NSFQ-105 and BSFQ-I were the most active compounds tested. The MIC90s of both NSFQ-105 and BSFQ-I for ciprofloxacin-susceptible or intermediate S. aureus, S. epidermidis and E. faecalis were 0.015, 0.015 and 0.125 mg/L, respectively. These compounds were 16- to 64-fold more active than ciprofloxacin. Among the 46 S. aureus tested, 14 were methicillin resistant (MRSA). However, for both NSFQ-105 and BSFQ-I no change in MIC profiles was noticeable between the two populations.

MICs of all newly tested compounds were increased for ciprofloxacin-resistant staphylococci and enterococci. However, NSFQ-105 and BSFQ-I yielded MICs of <=1 mg/L and <=2 mg/L, against ciprofloxacin-resistant MRSA or methicillin-resistant S. epidermidis (MRSE) and E. faecalis, respectively.

The Table also includes 11 S. pneumoniae isolates; some of them exhibited intermediate susceptibility or low-level ciprofloxacin resistance (MIC range 2–4 mg/L). Nevertheless, no changes were observed in the MICs of NSFQ-105 and BSFQ-I, which remained as low as 0.03 and 0.125 mg/L, respectively. This behaviour is in agreement with published results regarding the activity of these derivatives against a panel of S. pneumoniae strains with defined resistance mutations.10

Although BSFQ-II was more active than NSFQ-105 and BSFQ-I against a few ciprofloxacin-resistant coagulase-negative staphylococci, in general its MICs were increased two- to 16-fold in comparison with those of NSFQ-105 and BSFQ-I. The MIC range of BSFQ-II for ciprofloxacin-resistant Gram-positive isolates was 0.125–16 mg/L. However, BSFQ-II is more active than ciprofloxacin against Gram-positive bacteria.

BSFQ-III was less active than the other BSFQs against Staphylococcus spp. Nevertheless, its MICs were <=0.25 mg/L for all quinolone-susceptible staphylococci, which means that BSFQ-III was two- to eight-fold more active than ciprofloxacin. However, BSFQ-III MICs rose to values similar to those of ciprofloxacin against quinolone-resistant isolates. Furthermore, BSFQ-III did not show good anti-pneumococcal activity.

The antibacterial activities of NSFQ-105 and other BSFQs against staphylococci were affected similarly by changes in the pH of Mueller–Hinton broth between 8.5 and 5.5. All BSFQs were four- to eight-fold more active at pH 5.5 than at pH 7.4. In addition, the activities were reduced by shifting the pH from 7.4 to 8.5 (data not shown). Previously, we had related such behaviour to the high proportion of the uncharged form of the NSFQs at pH 5.5.4,6 It is also documented that the activity of zwitterionic fluoroquinolones drops in acidic media.11 Hence, NSFQ-105 and BSFQ-I were 128-fold more active than ciprofloxacin at pH 5.5. This observation may have clinical relevance for the use of the new derivatives at sites of infection such as macrophages, abscesses or the urinary tract.

In order to compare the activity of the test compounds, log MICs of each were correlated with those of ciprofloxacin. High degrees of correlation between quinolone log MICs have been reported previously for other fluoroquinolones, with slopes of regression close to unity and regression coefficients usually >0.85.12Figure 2Go shows a plot of BSFQ-I log MIC versus ciprofloxacin log MIC for 91 Staphylococcus spp., including MSSA, MRSA and coagulase-negative staphylococci. Such a plot reveals a bimodal distribution of the strains assayed; a linear regression analysis shows a slope of 1.05 with r2 = 0.89. Similar distributions and linear correlations were also observed for NSFQ-105, BSFQ-II and BSFQ-III. The same behaviour was observed for E. faecalis but not for pneumococci. These results indicate cross-resistance between new BSFQs and ciprofloxacin in S. aureus, S. epidermidis and E. faecalis, but not in pneumococci. Previously, we have shown that the primary target of NSFQ-105 and other BSFQs in pneumococci is DNA gyrase rather than topoisomerase IV (the primary target for ciprofloxacin in pneumococci).10 This observation, together with the present results, could indicate a different target affinity in pneumococci compared with the other bacteria.



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Figure 2. Relationship between BSFQ-I log MICs and ciprofloxacin log MICs for staphylococci (n = 91).

 
In conclusion, the BSFQs, a group of new fluoroquinolones that exhibit in vitro activity against Grampositive bacteria that is comparable to or better than other quinolones under investigation or recently introduced into clinical use, have been developed. Our results show that of the new BSFQs, NSFQ-105 and BSFQ-I exhibited the lowest MICs for most of the organisms tested, including ciprofloxacin-resistant strains. Although BSFQ-II also displays potent antibacterial activity, it is less active than NSFQ-105 or BSFQ-I against ciprofloxacin-resistant clinical isolates. On the other hand, a structure–activity study with the BSFQs showed BSFQ-II as an outlier.7


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Table. Comparative in vitro activities of new BSFQs and ciprofloxacin against clinical isolates of Gram-positive cocci
 

    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We thank L. Carbajal and C. Culasso from the Hospital de Niños de Córdoba for their kind supply of S. pneumoniae strains. This work was supported by grants from CONICOR and CONICET.


    Notes
 
* Corresponding author. Tel/Fax: +54-351-4334127; E-mail: rubmanzo{at}dqo.fcq.unc.edu.ar Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
1 . Appelbaum, P. C. & Hunter, P. A. (2000). The fluoroquinolone antibacterials: past, present and future perspectives. International Journal of Antimicrobial Agents 16, 5–15.[ISI][Medline]

2 . Blondeau, J. M. (1999). A review of the comparative in-vitro activities of12 antimicrobial agents, with a focus on five new ‘respiratory quinolones’. Journal of Antimicrobial Chemotherapy 43, Suppl. B, 1–11.[Free Full Text]

3 . Manzo, R. H., Allemandi, D. A. & Perez, J. D. (a) 7 March 1995. 7-(4-(4-aminophenyl)sulphonyl)-1-piperazinyl fluoroquinolonic derivatives and synthesis. US Patent 5,395,936. (b) 28 June 1999. Procedimiento para preparar agentes antimicrobianos 7-(4-[4- aminofenil) sulfonil]-1-piperazinil fluoroquinolónicos. Argentinean Patent 253422 (Instituto Nacional de Propiedad Industrial).

4 . Allemandi, D. A., Alovero, F. L. & Manzo, R. H. (1994). In vitro activity of new sulphanilyl fluoroquinolones against Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 34, 261–4.[Abstract]

5 . Manzo, R. H., Mazzieri, M. R., Nieto, M. J. & Alovero, F. L. 21 February 1997. Preparación de nuevos antimicrobianos bencenosulfonamido fluoroquinolónicos (BSFQs). Argentinean Patent Application P970106669.

6 . Alovero, F. L., Nieto, M., Mazzieri, M. R., Then, R. & Manzo, R. H. (1998). Mode of action of sulphanilyl fluoroquinolones (NSFQs). Antimicrobial Agents and Chemotherapy 42, 1495–8.[Abstract/Free Full Text]

7 . Nieto, M. J., Alovero, F. L., Manzo, R. H. & Mazzieri, M. R. (1999). A new class of fluoroquinolones: Benzenesulfonamidefluoroquinolones (BSFQs). Antibacterial activity and SAR studies. European Journal of Medicinal Chemistry 34, 209–14.[ISI]

8 . National Committee for Clinical Laboratory Standards. (1995). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Third Edition: Approved Standard M7-A3. NCCLS, Villanova, PA.

9 . National Committee for Clinical Laboratory Standards. (2000). MIC Testing M7-A5. NCCLS, Villanova, PA.

10 . Alovero, F. L., Pan, X-S., Morris, J., Manzo, R. H. & Fisher, L. M. (2000). Engineering the specificity of antibacterial fluoroquinolones: Benzenesulfonamide modifications at C-7 of ciprofloxacin change its primary target in Streptococcus pneumoniae from topoisomerase IV to gyrase. Antimicrobial Agents and Chemotherapy 44, 320–5.[Abstract/Free Full Text]

11 . Smith, J. T. & Ratcliffe, N. T. (1986). Effect of pH and magnesium on the in vitro activity of ciprofloxacin. Excerpta Medica Current Clinical Practice Service 34, 12–6.

12 . Fass, R. J. (1997). In vitro activity of Bay12-8039, a new 8-methoxyquinolone. Antimicrobial Agents and Chemotherapy 41, 1818–24.[Abstract]

Received 16 January 2001; returned 24 April 2001; revised 25 June 2001; accepted 13 August 2001





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