二氢呋喃香豆素对耐药大肠杆菌的多重抗菌作用：抑制生物膜形成、破坏细胞膜、干扰代谢功能以及引发氧化应激损伤

《Journal of Materials Chemistry B》：Multifaceted antibacterial action of dihydrofurocoumarins against drug-resistant Escherichia coli: biofilm inhibition, membrane disruption, metabolic dysfunction, and oxidative stress damage

本研究通过双功能合成策略，设计并合成了两类二氢呋喃香豆素衍生物（硝基苯并呋喃和香豆素类似物），其中最佳活性化合物16b和21e对大肠杆菌的MIC值仅为1.56 μg/mL，显著优于阿莫西林。其通过破坏细菌膜、诱导氧化应激反应、抑制生物膜形成等多靶点机制发挥作用，且与人类血清白蛋白的结合特性差异显著（16b结合Sudlow site I，21e结合heme site），提示靶向递送潜力。量子化学分析表明， narrower HOMO-LUMO gap增强了分子活性。该成果为新型广谱抗菌剂的开发提供了理论依据。（摘要：98字）

　　
The alarming rise in antibiotic resistance has necessitated innovative approaches to developing effective therapeutic agents. This study reports a bifunctional strategy to synthesize dihydrofurocoumarin analogues (DHFCs) incorporating naphthalimide and coumarin moieties, designed to target multiple mechanisms of bacterial resistance.

### Key Findings and Analysis:
1. **Synthetic Approach**:
DHFC analogues were synthesized via a multistep process involving Knoevenagel condensation and Michael-type addition. This strategy enabled structural diversity by introducing electron-withdrawing groups (e.g., fluorine, chlorine) at specific positions, enhancing antibacterial potency. For instance, analogues like **16b** (fluorine-substituted) and **21e** (nitro-substituted) exhibited exceptional activity, with MIC values as low as 1.56 μg/mL against *E. coli*, surpassing commercial antibiotics like amoxicillin.

2. **Mechanisms of Action**:
- **Membrane Disruption**: Both analogues compromised bacterial membrane integrity, leading to cytoplasmic leakage and cell death. This was confirmed through SEM imaging showing ruptured membranes and fluorescence assays indicating DNA intercalation.
- **Oxidative Stress**: The analogues induced reactive oxygen species (ROS) production, depleting glutathione (GSH) levels and disrupting cellular redox balance. This oxidative damage synergized with membrane targeting to enhance antibacterial efficacy.
- **Biofilm Inhibition**: They effectively inhibited biofilm formation in *E. coli*, a critical resistance mechanism. Dose-dependent studies showed inhibition up to 86.5% at higher concentrations.

3. **Human Serum Albumin (HSA) Binding**:
- **16b** preferentially bound to Sudlow site I (critical for drug transport), while **21e** associated with the heme site. This binding influenced pharmacokinetics, with both analogues showing optimal drug-likeness profiles (low MW, favorable logP, and no P-glycoprotein interaction).
- Spectroscopic and docking studies confirmed non-covalent interactions (π–π stacking, hydrogen bonds) and distinct binding sites, ensuring targeted delivery without compromising efficacy.

4. **Resistance Development**:
Unlike traditional antibiotics, **16b** and **21e** showed minimal resistance even after prolonged bacterial exposure. Rapid bactericidal action and disruption of biofilm formation likely contributed to this stability.

5. **Safety and Selectivity**:
Cytotoxicity assays against mammalian HEK293 cells revealed low toxicity (81.7–86.4% cell viability at 100 μM), indicating high specificity for bacterial targets. Quantum chemical studies suggested narrower HOMO-LUMO gaps in **21e**, enhancing molecular reactivity and membrane permeability.

### Implications and Future Directions:
- **Novel Therapeutic Agents**: DHFCs represent a promising class of broad-spectrum antibiotics, effective against multidrug-resistant *E. coli* and other Gram-negative/b正极菌. Their dual targeting (membrane + metabolic) reduces the likelihood of resistance.
- **Drug Delivery Optimization**: HSA binding studies highlight potential for albumin-mediated targeting, improving bioavailability and reducing off-target effects.
- **Clinical Potential**: The analogues’ ability to disrupt biofilms and induce rapid cell death positions them as candidates for treating chronic infections. Further studies on pharmacokinetics, pharmacodynamics, and clinical trials are recommended.

### Conclusion:
This work underscores the value of structural diversity and multifunctional mechanisms in combating antibiotic resistance. The DHFC analogues demonstrate promise as potent, stable, and selective antibacterial agents, offering a new frontier in infection control and drug design.