Application of Suzuki-Miyaura and Buchwald-Hartwig Cross-coupling Reactions to the Preparation of Substituted 1,2,4-Benzotriazine 1-Oxides Related to the Antitumor Agent Tirapazamine.

Many 1,2,4-benzotriazine 1,4-dioxides display the ability to selectively kill the oxygen-poor cells found in solid tumors. As a result, there is a desire for synthetic routes that afford access to substituted 1,2,4-benzotriazine 1-oxides that can be used as direct precursors in the synthesis of 1,2,4-benzotriazine 1,4-dioxides. Here we describe the use of Suzuki-Miyaura and Buchwald-Hartwig cross-coupling reactions for the construction of various 1,2,4-benzotriazine 1-oxide analogs bearing substituents at the 3-, 6-, and 7-positions.

Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor-Electrophile Conjugate.

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 102 M-1 min-1. Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Redox regulation of protein tyrosine phosphatases: structural and chemical aspects.

Protein tyrosine phosphatases (PTPs) are important targets of the H(2)O(2) that is produced during mammalian signal transduction. H(2)O(2)-mediated inactivation of PTPs also may be important in various pathophysiological conditions involving oxidative stress. Here we review the chemical and structural biology of redox-regulated PTPs. Reactions of H(2)O(2) with PTPs convert the catalytic cysteine thiol to a sulfenic acid. In PTPs, the initially generated sulfenic acid residues have the potential to undergo secondary reactions with a neighboring amide nitrogen or cysteine thiol residue to yield a sulfenyl amide or disulfide, respectively. The chemical mechanisms by which formation of sulfenyl amide and disulfide linkages can protect the catalytic cysteine residue against irreversible overoxidation to sulfinic and sulfonic oxidation states are described. Due to the propensity for back-door and distal cysteine residues to engage with the active-site cysteine after oxidative inactivation, differences in the structures of the oxidatively inactivated PTPs may stem, to a large degree, from differences in the number and location of cysteine residues surrounding the active site of the enzymes. PTPs with key cysteine residues in structurally similar locations may be expected to share similar mechanisms of oxidative inactivation.

Protection of a single-cysteine redox switch from oxidative destruction: On the functional role of sulfenyl amide formation in the redox-regulated enzyme PTP1B.

Model reactions offer a chemical mechanism by which formation of a sulfenyl amide residue at the active site of the redox-regulated protein tyrosine phosphatase PTP1B protects the cysteine redox switch in this enzyme against irreversible oxidative destruction. The results suggest that 'overoxidation' of the sulfenyl amide redox switch to the sulfinyl amide in proteins is a chemically reversible event, because the sulfinyl amide can be easily returned to the native cysteine thiol residue via reactions with cellular thiols.