3'-[4-Aryl-(1,2,3-triazol-1-yl)]-3'-deoxythymidine Analogues as Potent and Selective Inhibitors of Human Mitochondrial Thymidine Kinase
TL;DR: The synthesis of new thymidine analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3'-position of the 2'-deoxyribofuranosyl ring is described.
Abstract: In an effort to increase the potency and selectivity of earlier identified substrate-based inhibitors of mitochondrial thymidine kinase 2 (TK-2), we now describe the synthesis of new thymidine analogues containing a 4- or 5-substituted 1,2,3-triazol-1-yl substituent at the 3′-position of the 2′-deoxyribofuranosyl ring. These analogues were prepared by Cu- and Ru-catalyzed cycloadditions of 3′-azido-3′-deoxythymidine and the appropriate alkynes, which produced the 1,4- and 1,5-triazoles, respectively. Selected analogues showed nanomolar inhibitory activity for TK-2, while virtually not affecting the TK-1 counterpart. Enzyme kinetics indicated a competitive and uncompetitive inhibition profile against thymidine and the cosubstrate ATP, respectively. This behavior is rationalized by suggesting that the inhibitors occupy the substrate-binding site in a TK-2−ATP complex that maintains the enzyme’s active site in a closed conformation through the stabilization of a small lid domain.
Summary (1 min read)
Introduction of a -CH
- Of an electron-withdrawing Cl in the para position of the phenyl (8f) significantly improved the inhibitory activity.
- A similar effect had previously been observed in the thiourea series.
- This revealed that 14b inhibited the enzyme in a purely competitive fashion and had Ki values as low as 0.012 µM.
- Its Ki/Km ratios were markedly lower than 1 (0.011), pointing to an affinity for the enzyme that largely exceeds the affinity of the natural substrate.
- Combination of the 3'-modification and the 5'-O-trityl substituent led, as expected, to an analogue that was completely devoid of affinity for TK-2.
Molecular modeling and str uctur e-activity r elationship
- To gain insight into the mode of binding of this new class of inhibitors, docking experiments were undertaken using the previously reported homology-based model of TK-2 as the target.
- This "lid loop" appears as a disordered region in many dNK crystal structures apparently due to its high mobility, and side-chain orientation varies depending on the nature of the molecules binding in the long active-site cleft and on the presence of an additional negative charge (e.g. sulfate ions) on the enzyme surface.
- -the 3'-OH of the thymidine substrate can be held in place with the aid of two direct hydrogen bonds, one with the carboxylate of Glu201 and another one with the phenol of Tyr99.
- O -rotation of the triazole ring relative to the sugar would lead to steric clash of the attached phenyl ring with the lid loop, and also that this model does not favor substitution at position 5 of the triazole ring, in good agreement with the much lower inhibitory activity measured for the 1,5-substituted derivatives 8i and 8j.
- To reveal whether the TK-2 inhibitors are able to be taken up by the cells, the most active compound 14b was chosen for further studies.
- Cycloaddition of organic azides and alkynes is the most direct route to 1,2,3-triazoles.
- The authors used two different catalysts to achieve this reaction: the Cu(I) catalyst, which provided the 1,4- ] catalyst, which has recently been described for regioselective synthesis of 1,5-disubstituted 1,2,3-triazole systems.
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