Third and fourthgeneration quinolones

These newer compounds are characterized by increasing structural novelty and complexity (Figure 7), which has resulted in new and useful characteristics. Increased activity against Gram-positive cocci (particularly S. pneumoniae) over that of ciprofloxacin is the criterion applied to place agents in the third generation of quinolones, while potent activity against anaerobes was used to separate a subset of these agents into a fourth generation. In some cases, enhanced potency is combined with improved pharmacokinetics, such that the agents can be dosed once daily [22].

In this section, the structural modifications in the newer agents that have resulted in this enhanced antibacterial potency against important pathogens are highlighted. Additional characteristics that are central to the development of improved human therapeutants, such as pharmacokinetics, selectivity, and minimized potential for adverse effects, are also examined for their response to chemical modification. It is worthy of mention that, although certain substituents can impart improvements in a particular biological or chemical property, the overall characteristics of each molecule are derived from the interaction of all the substituents with each other and with the specific nucleus employed. For more detailed discussion of SAR, generally organized by position about the quinolone nucleus, the reader is referred to a number of earlier reviews (e.g., [2-5]).

FIGURE 7 Structural evolution of quinolones.

In vitro Potency Overall Potency

The N-1 cyclopropyl group, which was originally described for ciprofloxacin, remains one of the most effective functionalities for providing broad-spectrum activity against aerobic organisms. Combination of the N-1 cyclopropyl with a fluorine or chlorine substituent at C-8, as in a number of newer quinolones (Figures 5 and 6), further enhances antibacterial activity [23]. Clinafloxacin, sitafloxacin, and BAY y 3118, all of which bear a chlorine atom at C-8, are among the most potent broad-spectrum agents that have been in development, and are the only compounds shown here that exhibit Gram-negative activity superior to that of ciprofloxacin [2,24,25]. Unusual SAR was found in the case of gemiflox-acin (Figure 5), where the naphthyridone nucleus provided MICs improved over those of the corresponding C-8 chloroquinolone [26]. The typical potency enhancement observed on C-8 halogenation was originally suggested to result from improvement in cell penetration [11,23]. More recent work has shown, however, that the presence of a C-8 chlorine improves activity against both DNA gyrase and topoisomerase IV enzymes [27,28]. Excellent correlations were found between quinolone activity against E. coli DNA gyrase and MICs versus E. coli for a number of marketed and developmental agents. Similar correlations exist between antibacterial activity against S. aureus and inhibitory potency against S. aureus topoisomerase IV [27,28].

A C-8 methoxy group, in combination with an N-1 cyclopropane, reliably increases Gram-positive activity compared to the corresponding 8-unsubstituted quinolone, although a halogen atom can be more effective in this regard. Gram-negative potency is increased in some cases, but by less than the improvement offered by fluorine or chlorine [29]. An example is the decreased activity of moxifloxacin (Figure 6) compared to BAY y 3118 [24,30]. The only difference between these compounds is in replacement of the C-8 chlorine of the discontinued BAY y 3118 by a methoxy group in moxifloxacin, which nonetheless exhibits potent broad-spectrum activity, roughly equivalent to that of trovafloxacin [30].

The activity of the N-1 cyclopropyl C-8 substituted agents can depend further on the group at C-7. A pyrrolidine substituent in particular (see clinafloxacin, Figure 5) gives the most dramatic increase in activity on C-8 halogenation [31]. It is important to note, however, that, in addition to their potency benefits, some C-8 substituents can bring in undesirable effects, either on their own (see the section on "Phototoxicity") or in combination with an N-1 cyclopropyl group (see the section on "Selectivity").

Activity against Representative Gram-Positive Pathogens

Staphylococcus aureus

This is an area in which all of the third- and fourth-generation fluoroquinolones show improvement over ciprofloxacin [2,24,33]. As mentioned above, combination of the N-1 cyclopropane with a C-8 substituent can improve both Gram-positive and Gram-negative activity. Increasing the lipophilicity of a quinolone, however, as has been effected in many third- and fourth-generation agents, generally tends to increase potency against Gram-positive organisms while somewhat attenuating Gram-negative potency. Thus, the third- and fourth-generation compounds (Figures 5 and 6) exhibit improved activity over ciprofloxacin against S. aureus but, with the exception of clinafloxacin, sitafloxacin, and BAY y 3118, lesser activity against the Enterobacteriaceae [2,33]. This may arise in part through the opposing effects of hydrophobicity on penetration: increasing log D tends to enhance accumulation in S. aureus while decreasing penetration into Gram-negative organisms [34].

Lipophilicity can be increased by addition of one or more alkyl groups, which can be introduced at C-5 (grepafloxacin, Figure 5) and/or on the C-7 diamine sidechain (e.g., grepafloxacin, sparfloxacin, gatifloxacin, temafloxacin; see Figure 5). Surprisingly, introduction of the 5-amino group in sparfloxacin also serves to increase lipophilicity [35]. The improvement in Gram-positive activity seen for 5-amino introduction in sparfloxacin, however, is dependent on the presence of the N-1 cyclopropyl group. For most N-1 ethyl derivatives, activity is reduced by a 5-amino group [31].

Use of a 3-aminopyrrolidine sidechain at C-7 generally results in increased Gram-positive activity compared to the piperazine derivative [23,31], whereas Gram-negative potency tends to be competitive with the corresponding piperazine analogue. Clearly, many of the newer agents (e.g., clinafloxacin, sitafloxacin, BAY y 3118, moxifloxacin, tosufloxacin) take advantage of this, using pyrrolidine rings appended at C-7. Tosufloxacin (Figure 5) shows substantially more potent activity versus S. aureus than does the related piperazinyl quinolone temafloxacin [33]. In gemifloxacin, the core sidechain is a 3-aminomethylpyrrolidine. In this case, the methyl oxime ether moiety was added to enhance the drug's lipophilicity, improving its Gram-positive activity over that of the des-oxime analogue [26].

Although generally not providing the potency of the N-1 cyclopropyl/C-8 halogenated quinolone motif, use of the 2,4-difluorophenyl N-1 substituent (tosufloxacin, trovafloxacin, temafloxacin; see Figures 5 and 6) also enhances Gram-positive antibacterial activity over the classical ethyl substitution. This substituent, discovered independently by chemists at Abbott and Toyama [3], expanded the established SAR at the time, which held that only small N-1 substituents, similar in size to ethyl and cyclopropyl, could provide good antibacterial activity [7,36]. The 2,4-difluorophenyl and 4-fluorophenyl analogues were among the most potent of a number of substituted aryl groups [36] and have been held relatively constant in N-1 aryl compounds prepared since. Interestingly, combination of the N-1 2,4-difluorophenyl group with a C-8 halogen does not provide the enhanced potency observed for the N-1 cyclopropyl group [37].

Streptococcus pneumoniae

The new compounds, with the exception of pazufloxacin (Figure 5), all show improved activity against S. pneumoniae compared to ciprofloxacin. The most potent of these agents are gemifloxacin and BAY y 3118 [24,38], followed by clinafloxacin, sitafloxacin, moxifloxacin and trovafloxacin [2,39]. Balofloxacin, sparfloxacin, tosufloxacin, grepafloxacin, and gatifloxacin (Figure 5) have slightly less activity [2,40,41]. Structurally, it seems that N-1 cyclopropyl, C-8 substituted quinolones, and N-1 cyclopropyl and aryl naphthyridones can provide potent activity against S. pneumoniae. The relative activities of the third- and fourth-generation quinolones against S. pneumoniae generally mirror their performance against S. aureus, but in a number of cases the typical MIC90 for S. pneumoniae is two to four times higher than that for S. aureus [2].

The smallest improvement over ciprofloxacin is seen for levofloxacin and temafloxacin (Figure 5). The pyridobenzoxazine nucleus found in pazufloxacin and levofloxacin provides Gram-positive potency roughly equivalent to a C-8 unsubstituted N-1 cyclopropyl quinolone, as evidenced by the general similarity in Gram-positive activity of ofloxacin and ciprofloxacin [15]. For these pyri-dobenzoxazines, a C-7 piperazine, as in levofloxacin, is more successful at imparting S. pneumoniae potency than is the carbon-linked cyclopropylamine of pazufloxacin. Levofloxacin, as the more potent enantiomer of ofloxacin (see the section on "Stereochemistry"), demonstrates twice the in vitro activity of the racemic ofloxacin [42]. In combination with its high serum peak level, this moderate improvement in potency makes levofloxacin a clinically useful drug against S. pneumoniae infections.

Given the excellent activity of a number of the new quinolones against S. pneumoniae, and the fact that they are equally potent against sensitive and penicillin- and macrolide-resistant S. pneumoniae [2], these compounds have also been examined for their activity against other organisms important in respiratory tract infections. Like ciprofloxacin, essentially all the third- and fourth-generation quinolones exhibit excellent activity versus Haemophilus influenzae [2,43] and Legionella pneumophila [2,43-46]. The third- and fourth-generation agents that have been tested against Chlamydia pneumoniae, with the exception of temaflox-acin, exhibit improved activity over second-generation quinolones. Levofloxacin, trovafloxacin, and moxifloxacin are two- to fourfold more potent than earlier agents, while grepafloxacin, gatifloxacin, gemifloxacin, sitafloxacin, and spar-floxacin are the most active, with MIC90s as low as 0.03-0.06 |g/ml [43,47,48]. Overall, a number of these new agents are very promising for the empirical treatment of respiratory tract infections.

Activity against Mycobacterium tuberculosis

Of the second-generation quinolones, ciprofloxacin and ofloxacin have received the most examination for utility against M. tuberculosis. Substantially heightened in vitro activity against this pathogen was obtained for the more recent agents sparfloxacin, BAY y 3118, moxifloxacin, sitafloxacin, and gatifloxacin [49-54]. Each of these compounds bears the 8-substituted N-1 cyclopropyl motif. This is in agreement with SAR studies carried out around mycobacterial activity, which have shown that the N-1 cyclopropane provides better activity than the N-1 2,4-difluorophenyl group [55], and that 8-substitution is beneficial [56]. Indeed, none of the three N-aryl agents shown in Figures 5 and 6 have useful activity against M. tuberculosis [49,57]. Naphthyridones have been identified as a negative factor in a quantitative structure-activity relationship (QSAR) study around mycobacterial activity [58], which may account for the weak activity of gemi-floxacin [59] and further contribute to the poor performance of tosufloxacin and trovafloxacin against M. tuberculosis. Levofloxacin, as might be expected, exhibits twofold better activity against M. tuberculosis than does the corresponding racemate ofloxacin [49,60].

The lesser activity of clinafloxacin, which displays M. tuberculosis activity similar to or just twofold improved over that of ciprofloxacin [49,61], is interesting, considering its generally equivalent activity to the highly potent sitafloxacin and BAY y 3118 against other organisms. All three compounds are built on the same 8-chloroquinolone nucleus, with the only differences residing in the 7-substituent and in the presence of a fluorine on the N-1 cyclopropane of sitafloxacin. The sidechains of sitafloxacin and BAY y 3118 can be looked at as alkylated versions of clinafloxacin, however, consistent with the observation that more highly lipophilic moieties at the 7-position confer improved antimycobac-terial activity [55]. The C-7 piperazines of sparfloxacin and gatifloxacin, which are both methylated, may enhance activity in the same way.

Activity against Mycoplasma pneumoniae

Although the second-generation quinolones exhibited only moderate activity against M. pneumoniae, a number of the newer agents possess activity substantially improved over that of ciprofloxacin and ofloxacin against this pathogen. In particular, clinafloxacin, sitafloxacin, and BAY y 3118 possess very potent activity [2,62]. Sparfloxacin, trovafloxacin, gatifloxacin, moxifloxacin, gre-pafloxacin, and gemifloxacin have also been characterized as having excellent activity against this pathogen [63-67], whereas balofloxacin and temafloxacin are closer in activity to ciprofloxacin and ofloxacin [63,68]. These activities seem to correlate generally with the S. aureus and S. pneumoniae potencies of these agents, except for the lowered activity of balofloxacin, which may be due to its somewhat unusual piperidine sidechain.

Activity against Gram-Negative Pathogens

The activity of the newer quinolones against the Enterobacteriaceae is generally strong. Again, sitafloxacin, clinafloxacin, and BAY y 3118 are the most potent agents, outperforming ciprofloxacin against these pathogens [2,25]. Most of the third- and fourth-generation quinolones are slightly less potent than ciprofloxacin; this subset includes sparfloxacin, trovafloxacin, moxifloxacin, pazufloxacin, tosufloxacin, gatifloxacin, gemifloxacin, and levofloxacin [42,69-72]. Agents with reduced activity against the Enterobacteriaceae include balofloxacin and temafloxacin [40,73].

From this breakdown, some structural themes emerge. The 8-chloro compounds once again exhibit the highest level of activity, but 8-methoxy and 8-fluoro groups also provide potency approaching that of ciprofloxacin. Naphthyridone compounds bearing a pyrrolidine-based sidechain and an N-1 aryl group also yield good Gram-negative activity. The potent activity of the second-generation NM394 indicates that the sulfur-containing four-membered ring containing N-1 and C-2 of the quinolone nucleus can replace the N-1 cyclopropane of ciprofloxacin as far as Gram-negative in vitro activity is concerned [70]. Again, a correlation can be seen between the MIC against E. coli and the inhibition of E. coli gyrase [74], although additional factors such as ease of penetration into the bacterial cell also contribute to in vitro potency.

Versus P. aeruginosa, ciprofloxacin exhibits the most potent activity of the second-generation quinolones. This activity has been difficult to improve upon. Although clinafloxacin and sitafloxacin exhibit lower MICs in some studies, they, along with BAY y 3118 and the second-generation NM394, are often found to be equivalent to ciprofloxacin [2,25,75,76]. Pazufloxacin, trovafloxacin, tosufloxacin, and gemifloxacin are the next most potent agents after ciprofloxacin [57,77,78], with sparfloxacin, grepafloxacin, moxifloxacin, gatifloxacin, and levofloxacin in the following tier [40,42,79-82]. The least potent agents against P. aeruginosa are balofloxacin and temafloxacin [40,73].

It is interesting to note that most of the more potent derivatives against P. aeruginosa bear C-7 sidechains that are based on a pyrrolidine core (clinafloxacin, sitafloxacin, BAY y 3118, gemifloxacin, trovafloxacin, and tosufloxacin). This may be a reflection of the preponderance of alkylated piperazines in this group of compounds; as mentioned previously, although alkylation improves activity against Gram-positive pathogens, it generally reduces Gram-negative potency somewhat. P. aeruginosa clearly highlights the potency-enhancing effect of the C-8 chlorine, as evidenced by the differing activities of BAY y 3118 and moxifloxacin. Of the less commonly used sidechains, it would appear that the aminocyclopropane of pazufloxacin is more favorable for activity against P. aeruginosa than the piperazine found in the otherwise identical compound levofloxacin. Comparison of balofloxacin and gatifloxacin indicates that the piperidine sidechain of balofloxacin provides less effectiveness against Gramnegative organisms than does the piperazine moiety. It has been suggested that this results from lower permeation of balofloxacin into P. aeruginosa, as its IC50 against gyrase from P. aeruginosa is less than threefold higher than that of ciprofloxacin, while its MICs are substantially elevated [83].

Activity against Anaerobes

Activity against anaerobes such as Bacteroides fragilis is less widespread among these agents than is improved activity against Gram-positive pathogens. The 8-chloro quinolones clinafloxacin, sitafloxacin, and BAY y 3118, which exhibit markedly improved overall activity against aerobes, display excellent potency against anaerobes as well [2,84]. The next tier of agents includes trovafloxacin and moxifloxacin, which possess broad-spectrum anaerobe activity [85,86]. The potential for clinical application of these agents to anaerobic infections is the basis for their separation into a fourth generation of quinolones. Sparfloxacin, gatiflox-acin, and grepafloxacin exhibit anaerobe activity improved over ciprofloxacin, but weaker than that of trovafloxacin, moxifloxacin, and the C-8 chloro compounds [87-89]. In a systematic study, introduction of a C-8 halogen, methyl, or methoxy group was shown to enhance activity against B. fragilis for both piperazine and 3-aminopyrrolidine-substituted quinolones [3]; utility of the chloro and methoxy groups is exemplified in the fourth-generation compounds. Trova-floxacin exhibits enhanced activity against anaerobes over the structurally related tosufloxacin [87,90], however, indicating a contribution for the C-7 substituent as well. Gemifloxacin exhibits potent activity against many Gram-positive anaerobes, equivalent to sitafloxacin in some cases. Versus Gram-negative anaerobes, however, its activity is variable [91].

Activity against Quinolone-Resistant Organisms

Clinafloxacin, sitafloxacin, and BAY y 3118, all of which bear a C-8 chlorine atom, exhibit potent activity against quinolone-resistant strains, particularly quinolone-resistant S. aureus [2,75]. In one study [92], the MICs of clinafloxacin against staphylococci, Enterobacteriaceae, and P. aeruginosa strains were less affected by increasing resistance to ciprofloxacin than were those of sparfloxacin and ofloxacin. Sitafloxacin, although exhibiting MIC and gyrase activity similar to those of tosufloxacin and ciprofloxacin against a quinolone-sensitive P. aeruginosa strain, proved much more potent than the comparative agents against a set of quinolone-resistant P. aeruginosa strains. Examination of sitafloxacin analogues in which either the N-1 fluorine, C-8 chlorine, or C-7 spirocyclo-propane had been removed demonstrated that the C-8 chlorine, although having little effect on activity against a sensitive P. aeruginosa strain, imparted improved activity against quinolone-resistant strains. Measurement of the inhibitory effects of these analogues against gyrase isolated from each of these strains revealed that the heightened activity provided by the 8-chloro substituent was mediated at the level of the gyrase enzyme. For the most resistant P. aeruginosa strain, the gyrase supercoiling IC50 for sitafloxacin was 19-fold that of the quinolone-sensitive strain, whereas the corresponding increase for the des-chloro derivative was 196-fold [93].

The 8-methoxy substituent also enhances activity, although to a lesser extent, against quinolone-resistant S. aureus, as exemplified by gatifloxacin, moxiflox-acin, and balofloxacin [94-96]. A specific role for the 8-methoxy group was originally suggested by a study [94] investigating the bactericidal activity of balofloxacin against quinolone-resistant S. aureus. More extensive work has been carried out examining the effects of 8-substitution on bactericidal potency as well as resistance emergence. Addition of an 8-methoxy group to ciprofloxacin increased lethal activity against wild-type S. aureus by a factor of 4. Against a parC mutant of S. aureus, however, the cidal activity was enhanced over ciprofloxacin by 30-fold. Interestingly, an 8-ethoxy analogue gave results similar to ciprofloxacin itself. Evidence was also obtained for an 8-methoxy group enhancing the ability to kill nongrowing cells [97]. In E. coli, ciprofloxacin and the same 8-methoxy analogue were shown to exhibit very different abilities to select resistant mutants: at 10 x MIC, numerous colonies were obtained with ciprofloxacin from wild-type E. coli, and none with the 8-methoxy analogue. Using an E. coli strain with a preexisting parC mutation, however, resulted in selection of equal numbers of colonies with the two agents, indicating that topoisomerase IV in E. coli is involved in the ability of the C-8 methoxy group to reduce initial selection of resistant mutants [98]. Further studies examining gatifloxacin and its des-methoxy analogue against E. coli mutants yielded suggestions for the manner in which gatifloxacin interacts with gyrase [99] (see section on "Models of Fluoroquinolone Interactions with the Topoisomerases").

Fluoroquinolone resistance in S. pneumoniae remains low but is increasing in some geographical areas. Strains with decreased susceptibility to quinolones generally carry parC and gyrA mutations. Gemifloxacin, sitafloxacin, and cli-nafloxacin retain the highest level of potency against these strains, with MIC90s 4- to 16-fold lower than those of moxifloxacin and trovafloxacin [100,101].

Quinolone resistance can also occur through efflux of drug from the bacterial cell. The most common mechanism for this in S. aureus is through elevated expression of the efflux system NorA. It has been noted repeatedly that the more recently developed quinolones are less affected by efflux-mediated resistance, which is frequently attributed to their lower hydrophilicity compared to older quinolones such as norfloxacin and ciprofloxacin [102]. Additional structural factors have been found to decrease interaction with NorA, including the bulkiness of the C-7 sidechain, and the hydrophobicity of the C-8 substituent [103].

As is evident from this discussion, discovery and exploitation of SAR in the quinolone area have resulted in the synthesis of increasingly potent and broad-spectrum agents. In vitro activity, however, is just one component of the profile necessary for successful advancement of a compound through clinical study for human use. In the following sections, the effects of structural modification on additional characteristics such as pharmacokinetics, adverse effects, and physico-chemical properties are summarized.

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