Undoubtedly, effective, widely applicable, and affordable antibacterial therapy is one of the most important medical developments of the 20th century. Unfortunately, several decades of antibiotic misuse and abuse in humans, animals, and agricultural practices have created a health emergency. Antibiotics misapplication has spread resistance and ineffectiveness through the community, threatening the enormous gains made by the availability of therapies. Analysis of epidemiological data suggests that the evolution and spread of multidrug-resistant organisms have accelerated dramatically over the past decades. [[1], [2], [3]]

The report of World Health Organization (WHO) –Antimicrobial Resistance (ABR), Global Report on Surveillance– provides an extensive global review of ABR including surveillance and resistance data for bacteria that are classified as “bacteria of international concern”. [4]

The explanation corresponds to poor infection control practices, negligent antibiotic use, misdiagnose, bad dosage, rampant irrational antibiotic use and nonattendance of side effects, among others. [5] This emergence of drug-resistant infections has encouraged research community to develop new mechanisms against bacterial infections, mainly focused on multi-target strategies as opposed to specific targets that characterizes conventional antibiotics.

One strategy that has been extensively explored over the past decades is the use of light-based bacterial inactivation termed “Antibacterial Photodynamic Therapy (aPDT)”. [[6], [7], [8]] In this treatment, a photoactive molecule (commonly referred to as a photosensitizer), is activated by specific wavelength light to generate several reactive oxygen species (ROS) that include singlet oxygen (1O2), superoxide radical anion (O2•-), hydrogen peroxide (H2O2) and/or hydroxyl radical (HO•). Singlet oxygen corresponds to an excited state of oxygen, able to oxidize a wide range of biomolecules including proteins, lipids and nucleic acids, leading to bacterial death. [9] Due to its reactivity towards biomolecules, the use of singlet oxygen as mediator permits a multi-target focus against bacterial infection, with the benefit of avoiding resistance, as reported to date. [6,7] Singlet oxygen is the preferred ROS to be generated in aPDT since there are no specific enzymes or biomolecules in living cells that can counteract its effect. [10] In contrast, superoxide radical anion or hydrogen peroxide can be eliminated by specific antioxidant enzymes that are overexpressed by the organism, to maintain redox balance. [11] However, its short lifetime in aqueous media (3.09 μs) [12] restricts the oxidative damage of singlet oxygen to the vicinity of the photosensitizer, around 150 nm, which limits the damage exerted by a free photosensitizer. [6,7] These considerations make it crucial to increase photosensitizer solubility in aqueous environments but still able to partition. Additionally, for use in antimicrobial photodynamic therapy, the photosensitizer must be: toxic only to microbes and water soluble (as mentioned), in addition to having an efficient production of reactive oxygen species and absorbing preferably visible light and photostability under conditions of use, is not always considered a fundamental requirement. [13]

Phenalenone (perinaphthenone, 1H-phenalen-1-one), an aromatic ketone, which was characterized and introduced by Oliveros et al. in the 90s as an exceptional photosensitizer with a very high quantum yield of singlet oxygen generation and is soluble in a large number of solvents [14] and has been widely used as a singlet oxygen sensitizer in photochemistry and photobiology. [15] Furthermore, phenalenone derivatives, found in plants and fruits, are potent antifungal phytoalexins that protect against pathogens through photosensitization involving singlet oxygen. [[16], [17], [18], [19], [20]] Hydroxylated phenalenone derivatives serve as chromophore models for haemocorin, a colored dihydroxy phenalenone found in Australian plants. [21,22] Electron-acceptor or electron-donor substituents on phenalenone derivatives increase sensitivity to local polarity, making their spectroscopic behavior highly susceptible to environmental properties. [23]. In previous work we have demonstrated that the presence of an alkoxy substituent in the position 6 of phenalenone scaffold (6-alkoxy-PNF), reduces the energy of electronic states, promoting a bathochromic shift of UV–Vis absorption when compared to clean phenalenone (PNF). The 6-alkoxy derivative of PNF presents a maximum absorption wavelength centered at 430–450 nm depending on the solvent (a significant displacement of absorbance compared with PNF), and its generation quantum yield of singlet oxygen remained high, with exception of methanol, where instead an increased fluorescence is observed.

To take advantage of the photo-physical properties of 6-alkoxy-PNF framework, a trimethylammonium group was introduced in the molecule to improve solubility in water (PNF is slightly soluble in water) and depending on the length of methylene chain included, to promote interfacial activity and keep partition between aqueous and non-aqueous media. This substitution through a methylene linker would maintain distance from the 6-alkoxy-PNF, keeping unchanged its visible absorption bands (displaced to the red) and its singlet oxygen generation capacity. Amphiphilic properties are useful in permeating bacterial membranes and ultimately leading to cell death, as reported for cationic antimicrobial peptides. [24] The addition of cationic charges enhances the photodynamic efficacy of photosensitizers against Gram-negative bacteria due to electrostatically attractive interactions that allow better attachment to the bacterial surface. [25]

The synthesis of 6-hydroxyphenalenone (6OH-PNF), from which the corresponding alkoxy derivatives are prepared, was described in a protocol proposed by Cook et Al. [26] and later improved by Otalvaro et Al., [27] but there is still a serious yield-related drawback. To construct the discoid tricyclic motif of phenalenone (avoiding the low yields achieved in the previously mentioned procedures), we employed a synthetic procedure proposed by Matsuhita et al. [28] involving a naphthalene derivative and allenic acid under acidic conditions. This procedure, despite the incorporation of a methyl group in position 3, noticeably improves reaction yields. The compounds here prepared correspond to the series shown in Fig. 1. Their capacity to generate singlet oxygen and hence inactivate bacteria was tested. Our results show that the behavior of this family of compounds is dependent on the length of the alkyl chain, particularly in micro-heterogeneous systems.