Dicyclic and tricyclic diaminopyrimidine derivatives as potent inhibitors of Cryptosporidium parvum dihydrofolate reductase: Structure-activity and structure-selectivity correlations

A structurally diverse library of 93 lipophilic di- and tricyclic diaminopy rimidine derivatives was tested for the ability to inhibit recombinant dihy drofolate reductase (DHFR) cloned from human and bovine isolates of Cryptos poridium parvum (J. R. Vasquez et al., Mol. Biochem. Parasitol. 79:153-165, 1996). In parallel, the library was also tested against human DHFR and, fo r comparison, the enzyme from Escherichia coli. Fifty percent inhibitory co ncentrations (IC(50)s) were determined by means of a standard spectrophotom etric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrat es. Of the compounds tested, 25 had IC(50)s in the 1 to 10 muM range agains t one or both C. parvum enzymes and thus were not substantially different f rom trimethoprim (IC(50)s, ca. 4 muM). Another 25 compounds had IC(50)s of < 1.0 muM, and 9 of these had IC(50)s of < 0.1 muM and thus were at least 4 0 times more potent than trimethoprim. The remaining 42 compounds were weak inhibitors (IC(50)s, > 10 muM) and thus were not considered to be of inter est as drugs useful against this organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition a ssays and those obtained recently in a yeast complementation assay (V. H. B rophy et al., Antimicrob. Agents Chemother. 44:1019-1028, 2000; H. Lau et a l., Antimicrob. Agents Chemother. 45:187-195, 2001). Although many of the c ompounds in the library were more potent than trimethoprim, none had the de gree of selectivity of trimethoprim for C. parvum versus human DHFR. Collec tively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The com pounds in the library were also tested as inhibitors of the proliferation o f intracellular C. parvum oocysts in canine kidney epithelial cells culture d in folate-free medium containing thymidine (10 muM) and hypoxanthine (100 muM). After 72 h of drug exposure, the number of parasites inside the cell s was quantitated by indirect immunofluorescence microscopy. Sixteen compou nds had IC(50)s of < 3 muM, and five of these had IC(50)s of < 0.3 muM and thus were comparable in potency to trimetrexate. The finding that submicrom olar concentrations of several of the compounds in the library could inhibi t in vitro growth of C. parvum in host cells in the presence of thymidine ( dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in co mbination with leucovorin, may find use in the treatment of intractable C. parvum infections.