Reactivity assessment of chalcones by a kinetic thiol assay

Abstract

The electrophilic nature of chalcones (1,3-diphenylprop-2-en-1-ones) and many other α,β-unsaturated carbonyl compounds is crucial for their biological activity, which is often based on thiolmediated regulation processes. To better predict their biological activity a simple screening assay for the assessment of the secondorder rate constants (k 2 ) in thia-Michael additions was developed.Hence, a clear structure-activity relationship of 16 differentially decorated hydroxy-alkoxychalcones upon addition of cysteamine could be established. Moreover, amongst other naturally occurring α,β-unsaturated carbonyl compounds k 2 values for curcumin and cinnamaldehyde were gained while cinnamic acids or esters gave no or very slow reactions.Chalcones (1,3-diphenylprop-2-en-1-ones) are natural products from the class of plant polyphenols and the biochemical precursors of cyclic flavonoids. They display anti-inflammatory, antioxidative, anti-mitotic, bactericidal, antifungal, antimalarial, antileishmanial to chemoprotective and chemopreventive activity, but also cytotoxic and antiviral properties were found. 1 Their activity is mostly based either on the Michael acceptor activity of the α,β-unsaturated carbonyl system, or their radical scavenging or reductive potential which are often referred to as antioxidative behaviour.2 The Michael acceptor activity is affected both by the decoration of the aromatic rings, and also, even more effectively, by an α-X-substitution of the double bond of the enone system. 3 Despite the fact that Michael acceptors are a neglected class of compounds in drug development their unique capability to address certain cysteine residues qualifies them as valuable tools to modulate biological activity. 1d Nevertheless, there is no simple, efficient quantitative method to assess and compare the reactivity of different chalcones and other α,β-unsaturated carbonyl compounds to be able to identify suitable electrophiles. Therefore, we developed a facile screening assay to determine the second-order rate constants (k 2 ) of chalcones in thia-Michael additions. As a starting point, the reactivity of the α-H-chalcones was assessed since this is the pattern found in almost all natural products of this class -the only exception being the α-hydroxychalcones.

Reactivity of Biliatresone, a Natural Biliary Toxin, with Glutathione, Histamine, and Amino Acids

“…9,10,26 In this study, a 96-well microtiter plate-based screening system was used in a thiol reactivity screen for a simple and quick determination of binding to cysteamine, dithiothreitol, 2-mercaptoethanol, GSH, and cysteine. 27 These reports led us to examine the reactivity of biliatresone toward EVK, one of the more reactive compounds studied.…”

Covalent Modifiers: A Chemical Perspective on the Reactivity of α,β-Unsaturated Carbonyls with Thiols via Hetero-Michael Addition Reactions

“…95 The second order rate constants for the addition of cysteamine, a model protein surface thiol due to its pK a of 8.3, were calculated from the rates observed under pseudo-first order conditions (40 μM chalcone, 480 to 20000 μM cysteamine) by monitoring the decrease in UV absorbance of the enone peak between 350 and 375 nm. 96 These reactions were performed in a solution of Tris-HCl buffer (pH = 7.4) and ethylene glycol (1:4), which contained 2 mM EDTA to prevent thiol oxidation, and a 12-500 fold excess of cysteamine. 96 A chalcone bearing a 2'-hydroxy group ( 244 ) reacted the fastest with cysteamine (k 2 = 5.1 M −1 s −1 ) and a chalcone with no aromatic substituents ( 243 , R = H) reacted the second fastest (k 2 = 3.0 M −1 s −1 ).…”

“…96 A chalcone bearing a 2'-hydroxy group ( 244 ) reacted the fastest with cysteamine (k 2 = 5.1 M −1 s −1 ) and a chalcone with no aromatic substituents ( 243 , R = H) reacted the second fastest (k 2 = 3.0 M −1 s −1 ). 96 All other chalcones with hydroxy and alkoxy substituents at various positions on the aromatic rings reacted with a k 2 between 0.1 and 1 M −1 s −1 , and it was observed that a 2'-hydroxy group generally increased the rate of thiol addition while 2'-alkoxy groups reacted more slowly. 96 When 2'-hydroxy groups were present, cyclization of the pendant amine from cysteamine into the ketone led to formation of the 1,4-tetrahydrothiazepine 245 , 96 which has also been reported to occur with unsaturated aldehydes as discussed later (Scheme 13).…”

DARC: Mapping Surface Topography by Ray-Casting for Effective Virtual Screening at Protein Interaction Sites

“…While the SAR described above is consistent with our model of M0 binding, these results are also notably inconsistent with the behavior expected if these compounds were simply carrying out thia-Michael additions. Kinetic studies using a model thiol (cysteamine) demonstrate that the presence of the 6’-methoxy group decreases reactivity, and the 2’-hydroxy group increases reactivity [84]. Through the comparisons above, we find that the most potent compounds for inhibition of Mcl-1 correspond to those expected to be least reactive, further implying that the inhibition we observe is not due to reactivity of these compounds with Mcl-1’s unpaired cysteine sidechain.…”