In Vitro and In Vivo Properties of Dihydrophthalazine Antifolates, a Novel Family of Antibacterial Drugs

Abstract

Racemic 2,4-diaminopyrimidine dihydrophthalazine derivatives BAL0030543, BAL0030544, and BAL0030545 exhibited low in vitro MICs toward small, selected panels of Enterococcus faecalis, Enterococcus faecium, Streptococcus pneumoniae, Moraxella catarrhalis, and Mycobacterium avium, though the compounds were less active against Haemophilus influenzae. The constellation of dihydrofolate reductases (DHFRs) present in 20 enterococci and 40 staphylococci was analyzed and correlated with the antibacterial activities of the dihydrophthalazines and trimethoprim. DHFRs encoded by dfrB, dfrA (S1 isozyme), dfrE, and folA were susceptible to the dihydrophthalazines, whereas DHFRs encoded by dfrG (S3 isozyme) and dfrF were not. Studies with the separated enantiomers of BAL0030543, BAL0030544, and BAL0030545 revealed preferential inhibition of susceptible DHFRs by the (R)-enantiomers. BAL0030543, BAL0030544, and BAL0030545 were well tolerated by mice during 5-and 10-day oral toxicity studies at doses of up to 400 mg/kg of body weight. Using a nonoptimized formulation, the dihydrophthalazines displayed acceptable oral bioavailabilities in mice, and efficacy studies with a septicemia model of mice infected with trimethoprim-resistant, methicillin-resistant Staphylococcus aureus gave 50% effective dose values in the range of 1.6 to 6.25 mg/kg.Despite sustained efforts to identify new drugs against antibiotic-resistant bacteria, treatment of infections caused by vancomycin-resistant enterococci and methicillin-resistant staphylococci continues to challenge clinicians (5). Enterococci are often associated with urinary tract infections, intra-abdominal abscesses, endocarditis, and bacteremia, whereas staphylococci are common causes of skin and skin structure infections, endocarditis, osteomyelitis, prosthetic joint infections, catheterrelated bacteremia, and nosocomial pneumonia. Methicillinresistant Staphylococcus aureus (MRSA) accounts for approximately 50% of all S. aureus infections in the United States (24). Once confined to healthcare settings, MRSA has emerged as a serious cause of community-acquired skin and skin structure infections (32), and an increased frequency of skin cultures positive for S. aureus is associated with nasal and intestinal colonization (2).Dihydrofolate reductase (DHFR; EC 1.5.1.3) is an essential enzyme in most pathogenic bacteria, and the clinical success of trimethoprim (28,33,41,43) confirms DHFR as an important chemotherapeutic target. A phenylalanine3tyrosine substitution at position 98 in the wild-type, nontransferable, chromosomally encoded DHFR renders S. aureus resistant to trimethoprim (15); a corresponding tyrosyl residue occurs in transferable plasmid-encoded wild-type staphylococcal DHFR isozymes S1, S2, and S3 (13,14,40) and in the enterococcal DHFRs encoded by dfrE and dfrF. The gene encoding DHFR S1 may also be associated with staphylococcal chromosomal cassette mec N1 (SCCmec-N1) (37).BAL0030543, BAL0030544, and BAL0030545 are novel dihydrophthalazine antifolates with potent activities a...

Inhibition of Antibiotic-Resistant Staphylococcus aureus by the Broad-Spectrum Dihydrofolate Reductase Inhibitor RAB1

“…The impact of the F98Y mutation on (R)-RAB1 is consistent with recent observations with related phthalazine-based DHFR inhibitors, such as BAL0030543 (Fig. 2B) (7,9). While the three-dimensional structures with the BAL compounds are not known, they may use a similar binding mode such that the phthalazine group follows the path of RAB1 form 1, while the pyrimidine moiety extending from the phthalazine would occupy the same position as RAB1 form 2.…”

“…Concomitant with the current work, the broad-spectrum activities of related RAB1-like molecules have been demonstrated by Basilea Pharmaceutica International AG. These studies also highlighted the difficulty in generating spontaneous resistant mu-tations of chromosomal DHFR to inhibitors in the dihydrophthalazine series (7,9).…”

Antistaphylococcal Activity of Dihydrophthalazine Antifolates, a Family of Novel Antibacterial Drugs

“…Additionally, an Leu24Ile mutation was detected in the chromosomal DHFR of a clone of S. epidermidis strain CN051 for which BAL0030543 had an MIC of 64 g/ml (Table 6). Isoleucyl, isoleucyl, and asparaginyl residues occur at positions 20, 24, and 46, respectively, in the plasmid-encoded [dfr(G)] DHFR isozyme S3 (35), which is not inhibited by the dihydrophthalazines (11).…”

“…These novel compounds inhibited not only wild-type and mutant chromosomally encoded DHFRs in S. aureus [dfr(B)] and S. epidermidis [dfr(C)] but also the plasmid-borne DHFR isozyme S1 encoded by dfr(A), which is highly resistant to trimethoprim. Unlike trimethoprim, the dihydrophthalazines were active against staphylococci harboring a chromosomal DHFR Phe98Tyr mutation (11). With their good oral bioavailabilities and favorable pharmacological/toxicological properties (20), dihydrophthalazine antifolates BAL0030543, BAL0030544, and BAL0030545 are potentially attractive therapies for multidrug-resistant staphylococoal infections, including those for which long-term (Ͼ2-week) therapy is warranted.…”

Antimicrobial Susceptibility Testing, Drug Resistance Mechanisms, and Therapy of Infections with Nontuberculous Mycobacteria

“…Depending on the species, either the folP1 gene or the sulI genes encode the mycobacterial type 1 DHPS. Although most of the data on the interaction of trimethoprim and sulfonamides come from nonmycobacterial species, several studies demonstrated that trimethoprim and sulfonamides bind to and inhibit the DHFR and DHPS of NTM (7,17,25,38,65,67,72,116,272,326,352).…”