Exploration of the Structural Space in 4(3 H)-Quinazolinone Antibacterials

Abstract

We report herein the syntheses of 79 derivatives of the 4(3H)-quinazolinones and their structure-activity relationship (SAR) against methicillin-resistant Staphylococcus aureus (MRSA). Twenty one analogs were further evaluated in in vitro assays. Subsequent investigation of the pharmacokinetic properties singled out compound 73 ((E)-3-(5-carboxy-2-fluorophenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one) for further study. The compound synergized with piperacillin-tazobactam (TZP) both in vitro and in vivo in a clinically relevant mouse model of MRSA infection. The TZP combination lacks activity against MRSA, yet it synergized with compound 73 to kill MRSA in a bactericidal manner. The synergy is rationalized by the ability of the quinazolinones to bind to the allosteric site of penicillin-binding protein (PBP)2a, resulting in opening of the active site, whereby the β-lactam antibiotic now is enabled to bind to the active site in its mechanism of action. The combination effectively treats MRSA infection, for which many antibiotics (including TZP) have faced clinical obsolescence.

Figure 1.

Antibacterial activities of the 4(3H)-quinazolinones derivatives. The MICs (μg/mL) were determined with S. aureus ATCC29213. The MICs for inactive (MIC ≥ 8 μg/mL) and active compounds (MIC ≤ 4 μg/mL) are in red and blue, respectively. The four previously reported compounds, given here for comparison, are designated in underlined numbers. The MICs for the positive controls vancomycin and linezolid are 1 and 2 μg/mL, respectively.

Figure 2.

Pharmacokinetics of compound 73 after a single 10 mg/kg dose administration to mice (n = 3 per time point per route of administration, total n = 63 mice).

Figure 3.

(a) Time-kill assays for compounds 1 and 73 alone and in combination with TZP with MRSA strain NRS70. Compounds 1, 73, and TZP were used at 0.5 × MIC, with TZP in the 8:1 clinical ratio. (b) Neutropenic thigh-infection study in mice. Compound 73 in combination with TZP shows a significant reduction in colony counts. Mice (n = 8 per group, except untreated mice, where n = 5) were infected in the right thigh with NRS70. Treatment was started subcutaneously 1 h after infection; doses were given every 8 h (3 doses). The TZP dose was at 12 mg/kg of piperacillin and 1.5 mg/kg of tazobactam. Bacteria were counted in the infected thigh homogenates at 24 h after infection. Compound 73 was evaluated at 20 and 40 mg/kg. Data are mean ± standard error of the mean (SEM); statistical significance was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison post hoc test. *p < 0.05, **p < 0.01.

Scheme 1.. General Synthetic Route for the Quinazolinones^a

^aReagents and Conditions: (a) triethyl orthoacetate, reflux, 4 h; (b) R¹NH₂, AcOH or pyridine, reflux or sealed tube at 130 °C, 8 h; or R¹NH₂, toluene, reflux, Dean–Stark, 8 h; (c) R²CHO, AcOH, AcONa, reflux, 8 h; or R²CHO, pyridine, reflux, 8 h; or R²CHO, anhydrous ZnCl₂, neat, sealed tube at 130 °C, 1 h; (d) H₂, Pd/C, room-temperature (rt), 12 h; (e) R¹NCS, AcOH, reflux, 1 h; (f) R²CH₂Br, NaH, anhydrous dimethylformamide (DMF), ice-water to rt, 1 h; (g) PCl₅, POCl₃, reflux, 8 h; (h) R²NH₂, triethylamine, tetrahydrofuran (THF), sealed tube at 100 °C, 8 h.