Currently, oncology treatment widely involves surgery, chemotherapy, and radiation therapy. However, all these approaches have significant drawbacks, including systemic toxicity for the organism [[1], [2], [3]]. The low selectivity of chemotherapeutic agents can lead to life-threatening conditions [4], which has prompted the development of novel anticancer therapies, including photodynamic therapy which acts locally on the tumor cells with minimal side effects. The raised selectivity of photodynamic therapy (PDT) toward tumor tissues is associated with the specific properties of the tetrapyrrolic photosensitizers used in the method, including porphyrins, phthalocyanines, chlorins, and bacteriochlorins [5]. These compounds, which are relatively hydrophobic, readily penetrate into tumor cells through the cell membrane and remain therein for longer periods than in healthy tissues. This effect is attributed to several factors, including the presence of nucleophilic nitrogen atoms in the macroheterocycles, the structural features of malignant tumors, the acidic microenvironment of tumor tissues, etc. [5]. The therapeutic effect of photosensitizers is based on their ability, upon excitation by light of a specific wavelength, to generate reactive oxygen species leading to the death of tumor cells [6]. In the absence of irradiation, cellular toxicity remains at a minimum level. Thus, the application of PDT for the treatment of solid tumors enables complete eradication of tumor cells in the irradiated area, without affecting the surrounding healthy tissue. However, despite the significant advantages of this method, there are several limitations. Firstly, the side photosensitizers accumulation in healthy tissues exposed to sunlight, forces patients to endure the continuous dark isolation during the treatment. Secondly, the low reach of light for photosensitizers accumulated in the deep layers of the tumor is another challenge. Several methods have been developed to address these issues. The development of specialized delivery systems for photosensitizers allows for increased selectivity of their accumulation in tumors, while chemical modification of the macrocycle shifts the absorption maximum to a longer wavelength, enabling interaction with light that penetrates deeper into tissues [6].

The application of PDT for the treatment of oncological and precancerous conditions is often associated with pain [[7], [8], [9]]. Pain arises from tissue damage during the PDT procedure, particularly in areas with dense innervation [10]. In addition, thermal injury caused by laser irradiation used in PDT can also induce a pain syndrome [11,12]. Currently, a range of strategies is employed to alleviate pain during PDT, including intravenous administration of analgesics, application of topical anesthetics or cold air to the treatment area, and modification of the light source or radiation dose [13]. However, despite the fact that anesthetics are relatively efficient in relieving pain, their action is often non-selective.

Apart from the antitumor effects, the reactive oxygen species generated by photoexcited photosensitizers exhibit a pronounced antibacterial activity, including effects against microorganisms that have developed resistance to pharmaceutical agents that have been in use for a long time. For example, the application of a chlorin e6-based photosensitizer reduces the viability of bacterial strains Moraxella catarrhalis, Streptococcus pneumoniae, and non-typeable Haemophilus influenzae by 99.9 % [14]. Furthermore, the administration of a compound based on the same photosensitizer for the treatment of patients with necrotic pulp containing microflora resistant to prior antibacterial therapy results in 100 % eradication of microorganisms that remained viable after preliminary antibiotic treatment [15]. Importantly, the development of microbial resistance to the destructive effects of reactive oxygen species generated during PDT has proven to be impossible, even in targeted attempts [16]. Despite their highly efficient bactericidal activity, photosensitizers are unable to alleviate the pronounced pain syndrome associated with inflammatory pulp diseases and other endodontic conditions. Currently, pain management relies on systemically acting analgesics and anti-inflammatory drugs that may cause side effects.

One strategy to enhance the selectivity of local anesthetics involves the use of compounds capable of modulating their activity in response to external stimuli, such as light irradiation. Among such compounds, a prominent position belongs to photoresponsive local anesthetics based on aromatic azo derivatives [[17], [18], [19]]. These agents remain inactive in the absence of stimulation and do not exert analgesic effects; however, upon exposure to light with a specific wavelength, they undergo structural transformation to give an isomer capable of providing analgesia [20]. By incorporating both a photosensitizer and a photo-controlled anesthetic moiety within a single molecule, it becomes possible to address pain management during antimicrobial and antitumor PDT, while simultaneously enhancing the selectivity of local anesthetics. However, it is worth noting that this strategy may also have some drawbacks. For example, the selectivity of the resulting conjugate accumulation in tumor tissues may differ from the selectivity of the non-modified chlorin accumulation. Additionally, when developing such compounds as pharmaceutical prototypes, it is crucial to consider the concentrations at which each part of the conjugate is active and create conjugates with an appropriate ratio of active fragments. In this work, a conjugate with a chlorine-to-azobenzene ratio of 1:1 was studied, as it was necessary to focus on the photophysical aspects of the system, which was poorly investigated before. Therefore, it was decided to create and investigate a simple model.

A linker is introduced to minimize steric interactions between the moiety with potential local anesthetic activity and the photosensitizer within a conjugate structure. This linker is designed to undergo cleavage either in response to external stimuli or by metabolism.

To date, there are relatively few studies in the literature dealing with the interactions between tetrapyrrolic macroheterocycles and azobenzenes. For example, Durandin et al. conducted a comprehensive study on this topic [21], in which they examined the photophysical properties, including energy transfer processes, in a system composed of non-bound azobenzene and porphyrin. In studies reporting conjugates in which azobenzenes are covalently bound to porphyrins to form a common conjugated system [[21], [22], [23], [24], [25]], it was reported that azobenzenes lost their ability to undergo photoisomerization. In the work by Peters et al. [22], the azobenzene moiety was shown to play a role in quenching porphyrin fluorescence due to the presence of Förster resonance energy transfer (FRET) between two photoactive centers along with photoinduced electron transfer (PET).

The system composed of three photoactive components — a natural chlorin, azobenzene, and a photocleavable linker — was investigated. Moreover, the photophysical processes occurring upon irradiation of this system were comprehensively examined and quantitatively described for the first time. A comparative analysis of the photophysical properties of compounds with covalently bound and non-bound chlorin and azobenzene was conducted. This article presents the synthesis, stability assessment, and physicochemical characterization of a conjugate of a natural chlorin with azobenzene. Investigated conjugate could be considered as a model intended to serve as the basis for developing a series of similar conjugates for studying the effect of structure on biological activity. Studies on the selectivity of the conjugate accumulation in tumors are also of great interest, and they will be conducted in the future as part of our ongoing research.