A structure activity relationship study of 3,4'-dimethoxyflavone for ArlRS inhibition in Staphylococcus aureus

Abstract

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are difficult to treat due to their resistance to many β-lactam antibiotics, and their highly coordinated excretion of virulence factors. One way in which MRSA accomplishes this is by responding to environmental stimuli using two-component systems (TCS). The ArlRS TCS has been identified as having a key role in regulating virulence in both systemic and local infections caused by S. aureus. We recently disclosed 3,4'-dimethoxyflavone as a selective ArlRS inhibitor. In this study we explore the structure-activity relationship (SAR) of the flavone scaffold for ArlRS inhibition and identify several compounds with increased activity compared to the parent. Additionally, we identify a compound that suppresses oxacillin resistance in MRSA, and begin to probe the mechanism of action behind this activity.

Figure 1.

Schematic representation of the ArlRS regulatory cascade.

Figure 2.

Parent compound 3,4’-dimethoxyflavone 1

Figure 3.

Inhibition of fluorescence normalized to growth when treated with 50 μM of compounds with different C-3 substitutions than parent compound A: mgrA P2-GFP reporter, B: spx P2-GFP reporter. Student’s t-test, * P < 0.05 from untreated sample, # P < 0.05 from parent compound. C: Inhibition of bacterial growth by compounds 3a, 3b, and 3c

Figure 4.

Inhibition of fluorescence normalized to growth when treated with 50 μM of compounds with phenyl substituted, flavone substituted, and heteroatom substituted derivatives. A: mgrA P2-GFP reporter, B: spx P2-GFP reporter. ☆ P < 0.05 from parent compound. P < 0.05 from untreated sample, # P < 0.05 from parent compound.

Figure 5.

Hybrid derivatives

Figure 6.

Inhibition of fluorescence normalized to growth when treated with 50 μM of hybrid compounds. A: mgrA P2-GFP reporter, B: spx P2-GFP reporter. * P < 0.05 from untreated sample, ☆ P < 0.05 from parent compound. P < 0.05 from untreated sample, # P < 0.05 from parent compound.

Figure 7.

Effects on fluorescence normalized to growth when treated with 50 μM of lead compounds. A: agr P3-YFP reporter, B: P_hla-GFP reporter. P < 0.05 from untreated sample, ☆ P < 0.05 from parent compound.

Scheme 1.

General synthesis of flavone derivatives (1): (a) NaOH, H₂O/EtOH, RT; (b) HCl, H₂O, pH 7; (c) KOH, H₂O₂, H₂O/MeOH; 0 °C (d) HCl, H₂O, pH 2, 0 °C; (e) alkyl halide, K₂CO₃, acetonitrile, 40 °C.

Scheme 2.

Synthesis of final products with hydroxyl groups: (a) chloro(methoxy)methane, DIEA, DCM, RT; (b) NaOH, H₂O/EtOH, RT; (c) HCl, 0 °C, H₂O, pH 7; (d) KOH, H₂O₂, H₂O/MeOH 0 °C; (e) HCl, H₂O, pH 2, 0 °C; (f) alkyl halide, K₂CO₃, acetonitrile, 40 °C (g) TFA, DCM

Scheme 3.

A) Synthesis of 3-methoxy-2-(4-methoxyphenyl)-4H-thiochromen-4-one (10): (a) NaOH, H₂O/EtOH, RT; (b) HCl, H₂O, pH 7, RT; (c) potassium O-ethyl carbonodithioate, Cu(OAc)₂, DMSO, 70 °C; (d) SeO₂, H₂O/dioxane; (e) MeI, K₂CO₃, acetone 40 °C. B) Synthesis of 3-methoxy-2-(4-methoxyphenyl)quinolin-4(1H)-one (15): (f) NaN₃, DMF, 60 °C, 24 h; (g) 1-ethynyl-4-methoxybenzene, n-BuLi, THF, −78 °C; (h) MnO₂, DCM, 0 °C (i) AuCl₃, MeOH