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Trovafloxacin, a novel fluoronaphthyridone with a unique 6-amino-3-azabicyclo[3.1.0]hexyl side chain (Fig. 1), is active against Toxoplasma gondii both in vitro and in murine models of acute toxoplasmosis either when administered alone (6) or in combination with other drugs (5). We examined 11 structurally related analogs of trovafloxacin for their in vitro activity against T. gondii. In addition, we examined structure-activity relationships in an effort to determine if structural features can be identified that may help in understanding the antiparasitic activity of the fluoroquinolones and possibly lead to development of compounds with greater potency. The structure-activity relationship study was conducted because of previous observations (6) showing that tosufloxacin, a compound structurally very similar to trovafloxacin, was not active against T. gondii either in vitro or in vivo. Unlike trovafloxacin, which has a 3-azabicyclo[3.1.0]hexane substituent at position C-7 on the naphthyridone nucleus, tosufloxacin has a 3-aminopyrrolidine substituent. The loss in activity due to the single differing substituent in tosufloxacin indicated that derivatization at the C-7 position in the naphthyridone ring is critical for activity against T. gondii. Other compounds that had substituents other than a 3-azabicyclo[3.1.0]hexane at the C-7 position, such as a piperazine in ciprofloxacin or a methylpiperazine in fleroxacin, ofloxacin, and temafloxacin, did not have in vitro or in vivo activities against T. gondii in our earlier study (6). Therefore, it was considered of interest to evaluate compounds with substitutions for the 3-azabicyclo[3.1.0]hexane groups at C-7 and additional changes at N-1 and C-5 in either the naphthyridone or quinolone ring for in vitro activity against T. gondii.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Chemical structure of trovafloxacin.

Tachyzoites of the RH strain of T. gondii were obtained from the peritoneal cavities of Swiss Webster mice 2 days postinfection, as previously described (1). Human foreskin fibroblast (HFF) cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, N.Y.) containing 100 U of penicillin, 1 µg of streptomycin/ml, and 10% heat-inactivated bovine serum free of antibodies to T. gondii (HyClone Laboratories, Logan, Utah). Trovafloxacin (CP-99,219-27) and the 11 related analogs, 1 to 11, were obtained from Pfizer Inc., Groton, Conn.

In vitro studies. Trovafloxacin and the analogs were examined at concentrations ranging from 0 to 60 µM. Each drug was dissolved in a small volume of dimethyl sulfoxide and brought to the required volume for testing with Dulbecco's modified Eagle's medium. All solutions were made and stored in the dark. Inhibition of intracellular replication of T. gondii was determined by the [³H]uracil incorporation technique (6). Briefly, confluent monolayers of HFF cells were infected with 6 × 10⁴ tachyzoites. Four hours later, the monolayers were washed to remove extracellular parasites and different concentrations of the drugs were added to triplicate wells. Addition of the drugs marked the starting time point. Incorporation of radioactivity was determined at 48 h by the addition of [³H]uracil (1 µCi/well) 4 h prior to harvesting the cells. The cells were collected with a cell harvester, and radioactivity was counted with a scintillation counter. Infected monolayers treated with medium that contained only 1% dimethyl sulfoxide served as controls. Fifty percent inhibitory concentrations (IC₅₀) were calculated by CalcuSyn, a dose-effect analysis computer program (Biosoft, Ferguson, Mo.).

Results. Each of the 11 analogs of trovafloxacin demonstrated potent in vitro activity against T. gondii. IC₅₀ ranged from 0.53 to 14.09 µM (Table 1). Six of the analogs had IC₅₀ lower than that of trovafloxacin, indicating that their in vitro activities against T. gondii were significantly higher than that of the parent compound. The activity against T. gondii of one representative analog, 1, and trovafloxacin as the control drug is shown in Fig. 2. Both analog 1 and trovafloxacin demonstrated toxicity for HFF cells (34 to 49% inhibition) only at the highest concentration (60 µM) (data not shown). However, analog 1 and trovafloxacin had parasite inhibition values of 98 and 99%, respectively, at a concentration that was fivefold lower than the concentration showing toxicity. Similar results were obtained with other analogs.

View larger version (14K): [in this window] [in a new window]   FIG. 2.   Effect of exposure to trovafloxacin and a representative analog (1) on the growth of intracellular tachyzoites of T. gondii in vitro measured by [3H]uracil incorporation.

View larger version (11K): [in this window] [in a new window]   FIG. 3.   Chemical structures of trovafloxacin and analog 6.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Chemical structures of trovafloxacin and analogs 11, 4, and 9.

View larger version (12K): [in this window] [in a new window]   FIG. 5.   Chemical structures of trovafloxacin and analog 4.

View larger version (10K): [in this window] [in a new window]   FIG. 6.   Chemical structures of analogs 1, 2, and 3.

View larger version (10K): [in this window] [in a new window]   FIG. 7.   Chemical structures of trovafloxacin and analogs 5 and 8.

Discussion. The results described above reveal that each of the 11 novel fluoroquinolone compounds that are structurally related to trovafloxacin has potent in vitro activity against T. gondii. Six of these compounds had greater activity than the parent compound, trovafloxacin. Favorable pharmacokinetic parameters of trovafloxacin in humans, such as long half-life of elimination, rapid oral absorption, a linear increase in serum levels with increasing dose, and achievement of well-tolerated concentrations in serum (8), combined with excellent in vitro and in vivo activities against T. gondii (5, 6) make it an attractive candidate for further development for treatment of human toxoplasmosis. In addition, an understanding of changes in activity with structural modification may help the search for better drug candidates.