Since the discovery of penicillin in 1928, antibiotics have become the most commonly used drugs for treating a variety of infections, and the widespread use of antibiotics has saved billions of patients' lives and extended human being's life for 15–20 years on average. However, the abuse and misuse of antibiotics have led to a drastic increase in bacterial resistance, posing a serious threat to public health [1]. Bacterial persisters, a small phenotype mutation subgroup, are often in a dormant, or inactive state, and can tolerate lethal concentrations of antibiotics and thus escape antibiotic treatment, leading to enhanced bacterial resistance [2]. Bacterial persisters play a critical role in perpetuating chronic and recurrent infections, which can not be treated with conventional antibiotics [3,4]. Hence, the development of novel antimicrobial agents effective for treating resistant bacterial persisters is urgently needed.

However, few compounds have been reported with activity against persister cells [5]. Therefore, addressing drug resistance by eliminating persisters has become a research hotspot worldwide. Three strategies have been put forward to eliminate persisters [2]. One approach seeks to directly kill metabolically dormant persister cells [6,7]. An alternative approach sensitizes the persister cells for conventional antibiotics through resuscitation, activating the antibiotic targets, or stimulating the antibiotic influx [8,9]. The third approach employs molecules to disrupt or reduce the formation of persister cells. Triphenylphosphonium cation (TPP+), a lipophilic cation, can easily cross lipid bilayers due to its high lipophilicity and stable cationic charge, and the potential gradient drives the accumulation of TPP+-conjugates in the mitochondrial matrix [10]. There have been reports on the conjugation of TPP+ with drugs to improve their anticancer efficacy by increasing their distribution in the mitochondria [11,12]. Since the bacterial cell wall is a phospholipid bilayer, TPP-conjugates could potentially cross the bacterial cell wall via similar interactions, accumulating in the bacterial cytoplasm and leading to an enhanced antibacterial effect [13,14]. For example, the TPP-linked prodrugs ciprofloxacin-ester-PPh3 and ciprofloxacin-amide-PPh3 induced significant morphological changes in MRSA ST5 5016, including irregular deformation and membrane disruption, and some leakage of the cytoplasmic contents of bacteria to the extracellular environment [14]. Based on the membrane permeability of TPP+, we hypothesized that conjugation of TPP+ with natural products could further enhance its antibacterial activities, and have the potential to directly destroy persister cells.

Thymol is a naturally occurring phenolic monoterpene, which is one of the major constituents of the essential oils (10–64%) of thyme (Thymus vulgaris L., Lamiaceae), a medicinal plant with multiple therapeutic properties [15]. Thymol exhibits antimicrobial, antioxidant, anticarcinogenesis, anti-inflammatory, and antispasmodic activities, as well as a potential as a growth enhancer and immunomodulator [16]. However, due to the high concentrations required to reach therapeutic effects, the efficiency of Thymol in clinical applications was limited. Continuing our studies on novel antibiotic discovery [13,[17], [18], [19], [20], [21], [22], [23]], we set out to conduct Structure-Activity Relationship (SAR) studies on Thymol in the hope of improving its antibacterial activity.

We synthesized a series of derivatives by introducing a substituent to the 4-position of Thymol. Antibacterial evaluation of these derivatives against the priority bacteria (Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) as defined by WHO identified derivatives with significantly improved antibacterial activities. We then selected the potent Thymol derivatives to form conjugates with TPP+, and further studied their SAR and antibacterial activities, leading to the discovery of Thymol-TPP conjugates effective against bacterial persisters. We also established their preliminary antibacterial mechanisms. The strategy reported in this study could help develop novel antibiotics to address the drug resistance challenges, from natural products that are often neglected in traditional bioassays due to their weak antibacterial activity.