Click Chemistry for the development of enzyme inhibitors (I)

Click chemistry, first proposed by the American chemist Sharpless in 2001, is a combinatorial chemistry method based on carbon-heteroatom bond synthesis. This method has the advantages of simple reaction conditions, easy availability of raw materials and reagents, no solvent or easy removal with solvent, and easy separation of products.

Click chemistry mainly includes four major types of reactions, namely: cycloaddition reaction, nucleophilic ring-opening reaction, non-aldol carbonyl reaction and carbon-carbon multi-bond addition reaction. These reactions have the following points in common:

1. Click components are mostly alkene or alkyne derivatives, and the carbon-carbon multiple bonds provide energy and mechanical pathways for the fragment structure constructed by the reaction.


2. Most click chemistry reactions involve the formation of carbon-heteroatoms (mainly N, O, S) bonds.


3. Because the chemical energy of the raw materials is higher or the product is more stable, the click chemistry reaction is often accompanied by the release of a large amount of energy.


4. The click chemistry reaction is usually a cyclization process or a condensation process, with relatively high by-products, which is very economical.

Azide-alkyne cycloaddition (AAC) is one of the most widely used types of cycloaddition reactions. It is often used for the rapid construction of 1,2,3-triazole. According to the reaction principle, it is classified as a 1,3-dipolar cycloaddition reaction. However, due to the lack of regioselectivity, higher reaction temperature and longer reaction time, this type of addition reaction did not get the attention of scientists in the next hundred years. It was not until 2001 that Sharpless et al. discovered the Cu (I) -catalyzed alkyne-azide cycloaddition (CuAAC) that can occur at room temperature. The azide-alkyne cycloaddition reaction once again attracted the attention of the scientific community. Although the introduction of monovalent copper greatly improves the efficiency of the cycloaddition reaction, it also limits the direct application of CuAAC in biological systems. In 2004, Agard's research group proposed a strain-promoted azide-alkyne cycloadditon (SPAAC) promoted by ring tension, which solved this problem. Compared with the terminal alkynes, the activation energy of cyclooctyne is very low, and the cycloaddition reaction can be completed without a catalyst, making the application of this type of reaction in biological systems possible. Through more than ten years of development, CuAAC and SPAAC have become advantageous tools for the discovery of lead compounds due to their high reliability, complete specificity, and biocompatibility of reactants, and they are widely used in anti-HIV, anti-virus and anti-bacterial applications.

The 1,2,3-triazole structure constructed by the azide-alkyne cycloaddition reaction is not only a stable linking fragment, but also a safe amide bio-isostere. Compared with amide, triazole structure has stronger target protein interaction ability, stronger biological environment stability and better cytochrome P450 metabolic enzyme inertness. It is a more favorable biological experiment fragment than amide group.

Target-guided synthesis (TGS) is a synthetic method that uses the biological target itself to directly assemble selective ligands from a pool of fragments containing complementary reactive functional groups. It is mainly divided into two categories: kinetic TGS (KTGS) for the formation of irreversible reactions and thermodynamic control TGS for the formation of reversible reactions, also known as dynamic combinatorial chemistry (DCC). The application of the azide-alkyne cycloaddition reaction in TGS is called "in situ click chemistry" (in situ click chemistry), using AAC technology to complete the in vivo assembly of selective ligands.

Enzymes play an important role in the organism and are closely related to the occurrence of many major diseases such as cancer, Alzheimer's disease, diabetes, and tuberculosis. Regulating the activity of related enzymes is an important means of treating diseases. In-situ click chemistry accelerates the research and development of enzyme inhibitors in a faster and more effective manner, and provides a feasible method for constructing new enzyme inhibitor compound libraries. The following are the applications of in-situ click chemistry in the development of enzyme inhibitors.

1. Cyclooxygenase-2 inhibitor

Cyclooxygenase (COX) can catalyze the metabolism of arachidonic acid into compounds such as prostaglandin, prostacyclin and thromboxane, which is closely related to various physiological and pathological conditions of the human body. There are 3 subtypes in the human body: COX-1, COX-2 and COX-3. Among them, COX-2 is highly expressed in a variety of pathological processes such as inflammation, cancer, neurodegenerative diseases and multidrug resistance symptoms, and is considered to be a key target of related drugs. However, because COX-1 and COX-2 have similar subcellular localization, molecular weight, amino acid composition, and protein homology of more than 60%, the development of selective COX-2 inhibitors faces a huge challenge. The key difference between the active sites of COX-1 and COX-2 is that COX-2 has obvious secondary binding pockets and hydrophobic pockets. Studies have shown that the ideal high-efficiency and highly selective COX-2 inhibitors need to be selectively bound in this secondary binding pocket, there is also enough space volume to block the hydrophobic channel of COX-2.

In view of this, the researchers used different 5-azidopyrazoles and aryl acetylenes as click chemistry building blocks to incubate in pairs with COX-2, using the COX-2 binding site as a reaction vessel to generate a new type of highly efficient and selective COX- 2 Inhibitor, and the progress of the reaction was detected by LC/MS. Compared with clinically used anti-inflammatory drugs, the compounds 1 and 2 obtained by this method show better anti-inflammatory activity and selectivity in vivo, and are highly effective COX-2 inhibitors. The successful development of compounds 1 and 2 demonstrated the high efficiency of in-situ click chemistry in the study of COX-2 inhibitors. When extending this method to the development of COX-1 inhibitors, the researchers found that, unlike COX-2, the fragments extend into the secondary pocket and close to the complementary chemical building blocks to produce an in-situ click reaction. The smaller COX-1 binding site cannot bring the two closers together, so the in-situ click reaction cannot occur. Due to the structural differences between the two enzymes, COX-2 is more suitable than COX-1 to bind two more reactive and bulkier ligands. Such research results also opened up new ideas for the research of selective COX inhibitors.

1. D-amino acid oxidase inhibitor

D-amino acid oxidase (DAO) is the first flavozyme discovered by Krebs in 1935. Its physiological function is to catalyze the oxidative deamination of D-amino acids. DAO (hDAO) in the human brain mainly converts D-serine into hydroxypyruvate to regulate the concentration of D-serine. Abnormal D-serine content can cause a variety of neurological diseases. The overexpression of hDAO in the brain can lead to low D-Serine content, causing mental disorders. Therefore, hDAO has received a lot of attention as a target for the treatment of mental disorders, and related inhibitor research has been widely carried out worldwide.

Through in-situ click chemistry, the complementary azide (3) and alkyne (4) undergo 1,3-dipolar cycloaddition reaction at the hDAO binding site to obtain a series of compound structures, from which compound 5 that can effectively inhibit hDAO was screened out. The formation of the stable triazole structure in this series of compounds makes the process irreversible and provides reliable and effective information for the affinity sites of enzyme ligands. The preparation of compound 5 takes hDAO as the reaction vessel, and uses the in-situ click reaction to produce its own enhanced inhibitor, which proves the effectiveness of the target enzyme as a "casting mold".

1. Aspartic protease inhibitor

Aspartic proteinase is a type of proteolytic enzyme that exhibits activity in an acidic environment, including pepsin, reverse transcriptase, aspartic acid intramembrane cleavage protease, etc. They are widely distributed in eukaryotes and microorganisms, and their main function is to degrade proteins and antigens and promote the activation of other enzymes. These enzymes are related to food digestion, blood pressure regulation, and tumor development. In the research of aspartic protease inhibitors, the discovery of human immunodeficiency virus-1 (HIV-1) protease inhibitors was completed by clicking on chemistry. The synthetic azide of the aspartic protease hydroxy transition state analogue was click-reacted with the alkyne library, and then screened in situ in the form of a microplate, and finally, two effective HIV-1 protease inhibitors (compound 6 and compound 7) were obtained. In addition to helping the rapid discovery of HIV-1 protease inhibitors, click chemistry has also played an important role in the research process of other aspartic protease inhibitors. For example, a series of triazole inhibitor compounds 8-10 can be synthesized by in-situ click chemistry based on the characteristics of the interaction with the target protein. This series of inhibitors occupy the active pocket of cleavage protease in the aspartic acid membrane under physiological conditions. Aspartic proteases showed obvious inhibitory effects.

To be continued in Part II…