With the continuous development of medicine, people gradually discovered that the occurrence of many diseases was closely related to the activity of proteins in the human body, such as kinases, channel proteins, etc. [1,2]. Upon administering a specific quantity of chemical agents to suppress the activity of these proteins, such as through the application of molecule-targeted drugs, a therapeutic impact on disease conditions was realized [3]. Traditional molecule-targeted drugs mainly bound tightly to the active pocket of the target protein, changing its conformation and inhibiting its activity to achieve the goal of treating diseases [4]. Unfortunately, due to the lack of suitable drug binding active pockets, it was difficult for about 80 % of disease-related proteins to develop corresponding molecule-targeted drugs [5].

In the early 21st century, the famous scientist Craig Crews from Yale University first proposed the concept of Proteolysis Targeting Chimera (PROTAC), which uses a heterobifunctional molecule to specifically bind the two ends of this molecule as ligands to the target protein and E3 ligase, forming a ternary complex. After the ternary complex was ubiquitinated by the E2-E3 complex ligase, the intracellular ubiquitin-proteasome system recognized and degraded the target protein [6]. Although PROTAC still needed to bind to target proteins, it did not necessarily depend on the presence of target protein active pockets. Non-active pockets could also be used in the design of PROTAC, allowing it to be applied to target proteins that were not suitable for traditional molecule-target drugs [7,8]. In addition, since PROTAC did not rely on inhibitory effects on target proteins, it still had strong pharmacological activity against some target proteins with drug-resistant mutations [9]. Based on these characteristics of PROTAC, it has great potential in future disease applications.

PROTAC is mainly composed of three parts: a warhead of the protein of interest (POI), an intermolecular linker, and an E3 ligand. In the early stages, researchers mainly focused on the application of high-affinity and high-selectivity warhead of the POI and the E3 ligand. As the research deepened, more and more reports indicated that the linker had a significant impact on the degradation activity and selectivity of PROTAC [[10], [11], [12], [13]].

In early literature on PROTAC, the design of the linker was mainly based on flexible chains such as alkyl chains, polyethylene glycol (PEG) chains, and polyethylene glycol-like chains [[14], [15], [16], [17]]. Although PROTAC with alkyl flexible chains as the linker could perform well in vitro bioactivity evaluation, the introduction of lipophilic alkyl flexible chains reduced the hydrophilicity of these drug molecules, resulting in generally poor pharmacokinetic characteristics and pharmacological activity in vivo biological experiments [13]. In addition, recent reports have also shown that the introduction of alkyl flexible chains in PROTAC posed some risks to causing hemolysis in vivo [18], which further constrained the application of alkyl chains in clinical PROTAC. The application of PEG chains and PEG-like chains in PROTAC could compensate for the shortcomings of alkyl chains in some kinds, but the use of excessively long PEG chains could cause a large accumulation of oxygen atoms, thereby increasing the polarity surface area of PROTAC and reducing cell permeability. This prevented the PROTAC from being taken up by cells to degrade the target proteins [19]. In addition, studies on drug metabolism have shown that PROTAC containing PEG chains and PEG-like chains were prone to undergo O-dealkylation reactions in organisms, leading to a decrease in the metabolic stability of the drugs [20]. Another issue worth mentioning is that regardless of the use of alkyl chains, PEG chains, and PEG-like chains, when the length was too long, due to its increased flexibility, the overall molecular conformation would gradually become more complex. At the microscopic level, it showed that flexible chains were easily intertwined with proteins, which was not conducive to the stability of the ternary complex. Consequently, flexible chains were only suitable for the early stage of the PROTAC exploration, and continuous optimization of linker was still needed for subsequent development.

In recent years, the utilization of rigid structures, including nitrogen heterocycles, alkynes, and aromatic rings, in the design and chemical synthesis of PROTAC linkers has been extensively documented [21]. The introduction of these rigid structures was proven to effectively enhance the drug activity and the stability of metabolism [[22], [23], [24], [25], [26]]. Furthermore, it was reported that the incorporation of rigid structures into PROTAC linkers significantly advanced the development of PROTAC [27]. Among these rigid structures, nitrogen heterocycles were highly favored by various research institutions and pharmaceutical companies, including triazole, pyridine, and piperazine. Since the first PROTAC molecule ARV-110, developed by Arvinas, progressed into clinical trials, nitrogen heterocycles have been extensively employed as linkers in PROTAC molecules [28]. Compared with flexible chains, nitrogen heterocycles retained a certain degree of flexibility and rigidity as linker, which not only reduced the possibility of linker adhering to the protein surface during the formation of ternary complexes by PROTAC, making the ternary complexes more stable, but also allowed PROTAC to connect with target protein and E3 ligase in an appropriate conformation to further enhance degradation activity. Moreover, the nitrogen atoms within the nitrogenous heterocycles imparted a finely tuned equilibrium between polar surface area and lipophilicity to the entire PROTAC molecule. This delicate balance allowed the PROTAC molecule to be better taken up by cells. The application of ring structure could delay the metabolism of drugs in the organism, thereby prolonging the efficacy time [29]. These reports indicate that the introduction of nitrogen heterocycles in linker could also enhance the selectivity of PROTAC [30,31]. Based on the characteristics of nitrogen heterocycles, it was believed that more and more nitrogen heterocycles would appear in the design of clinical PROTAC linker in the future.

In this work, we reviewed the application progress of nitrogen heterocycles in PROTAC linker design in recent years, and we also outlined the challenges faced in optimizing linkers using nitrogen heterocycles. Finally, we summarized some regular experiences in the optimization process of nitrogen heterocycles applied to linkers to help future researchers optimize linkers.