The in situ click chemistry approach to lead discovery employs the biological target itself for assembling inhibitors from complementary building block reagents via irreversible connection chemistry. The present publication discusses the optimization of this target-guided strategy using acetylcholinesterase (AChE) as a test system. The application of liquid chromatography with mass spectroscopic detection in the selected ion mode for product identification greatly enhanced the sensitivity and reliability of this method. It enabled the testing of multicomponent mixtures, which may dramatically increase the in situ screening throughput. In addition to the previously reported in situ product syn-TZ2PA6, we discovered three new inhibitors, syn-TZ2PA5, syn-TA2PZ6, and syn-TA2PZ5, derived from tacrine and phenylphenanthridinium azides and acetylenes, in the reactions with Electrophorus electricus and mouse AChE. All in situ-generated compounds were extremely potent AChE inhibitors, because of the presence of multiple sites of interaction, which include the newly formed triazole nexus as a significant pharmacophore.

The target-guided, in situ click chemistry approach to lead discovery has been successfully employed for discovering acetylcholinesterase (AChE) inhibitors by incubating a selected enzyme/tacrine azide combination with a variety of acetylene reagents that were not previously known to interact with the enzyme's peripheral binding site. The triazole products, formed by the enzyme, were identified by HPLC-mass spectrometry analysis of the crude reaction mixtures. The target-guided lead discovery search was also successful when performed with reagent mixtures containing up to 10 components. From 23 acetylene reagents, the enzyme selected two phenyltetrahydroisoquinoline (PIQ) building blocks that combined with the tacrine azide within the active center gorge to form multivalent inhibitors that simultaneously associate with the active and peripheral binding sites. These new inhibitors are up to 3 times as potent as our previous phenylphenanthridinium-derived compounds, and with dissociation constants as low as 33 femtomolar, they are the most potent noncovalent AChE inhibitors known. In addition, the new compounds lack a permanent positive charge and aniline groups and possess fewer fused aromatic rings. Remarkably, despite the high binding affinity, the enzyme displayed a surprisingly low preference for one PIQ enantiomer over the other.

An efficient room temperature method for the synthesis of 1-sulfonyl-1,2,3-triazoles from in situ generated copper(I) acetylides and sulfonyl azides is described. Copper(I) thiophene-2-carboxylate (CuTC) catalyst produces the title compounds under both non-basic anhydrous and aqueous conditions in good yields.As latent diazo compounds, 1-sulfonyl-1,2,3-triazoles serve as convenient progenitors of reactive azavinyl carbenes. 1 These electron-deficient heterocycles stand out as an important exception in the family of generally stable and unreactive 1,2,3-triazoles: the weakened N1-N2 bond in 1-sulfonyl derivatives facilitates their ring-chain isomerism, which leads to the formation of diazoimines, and subsequent decomposition to the transition metal stabilized carbenes.Copper(I) catalyzed azide-alkyne cycloaddition (CuAAC) 2 could well be the most direct and practical route to 1-sulfonyl-1,2,3-triazoles. High fidelity, efficiency, and compatibility with a broad range of functional groups and conditions have made it a widely utilized method for the synthesis of small molecules, bioconjugates, and complex molecular architectures. 3

Copper(i) efficiently catalyzes the "fusion" of organic azides with terminal alkynes to produce regioselectively 1,4-disubstituted-1,2,3-triazoles.[1] The very broad scope, simple experimental conditions, and exceptional reliability of this transformation have placed it among the most reliable "stitching" reactions [2] available today. It has resulted in applications in organic synthesis, molecular biology, macromolecular chemistry, and materials science. A stepwise mechanism involving the coordination of the azide to the copper(i) acetylide through the proximal nitrogen atom, followed by formation of a (1,2,3-triazol-5-yl)copper species has been proposed.[1] This mechanism is supported by computational studies, which indicate that the activation barrier for this catalytic version of the Huisgen cycloaddition is reduced by as much as 11 kcal mol À1 , which corresponds to the experimentally observed rate acceleration of seven to eight orders of magnitude.