Structure-Activity Relationship of 2,3-Benzodiazepin-4-ones as Noncompetitive AMPA Receptor Antagonists.

摘要

2,3-Benzodiazepin-4-one (BDZ-11) derivatives are a special group of 2,3-benzodiazepine compounds. These compounds have been previously synthesized as AMPA receptor inhibitors, and they are also potential drugs for the treatment of various neurological diseases involving excessive AMPA receptor activity. AMPA receptors are a subtype of glutamate ion channel proteins and are responsible for the majority of excitatory neurotransmission in the mammalian central nervous system. Specifically, they are critically involved in neuronal development and brain activities, including learning and memory. Overstimulation of AMPA receptors is associated with some neurological diseases such as ALS, stroke and Alzheimer's disease. Despite the fact that hundreds of 2,3-benzodiazepine compounds have been synthesized to date, the mechanism of action of these compounds on AMPA receptors at the molecular level is not well understood, and a quantitative structure-activity relationship has not yet been established. Furthermore, there has not been any systematic characterization of the potency and selectivity of these compounds with each of the AMPA receptor subunits. One main problem is that the rate of AMPA receptor channel opening in response to the binding of glutamate occurs in the microsecond time scale, yet the conventional kinetic techniques do not have a sufficient time resolution for a kinetic study of the effect of an inhibitor on the rate of channel opening.;Using a laser-pulse photolysis technique with "caged glutamate", which provides a ∼60 µs time resolution and a rapid solution flow technique, I investigated the effect of the 2,3-BDZ-11 series on the channel-opening process of the homomeric GluA2Qflip and GluA1flip receptors, the two subunits of AMPA receptors. My results show that these compounds bind to a single, noncompetitive site, most likely due to that these compounds share a 7,8-etheylenedioxy ring in the 2,3-benzodiazepine structure. In contrast, GYKI 52466, the prototypic compound in this family, has a 7,8-methylenedioxy feature. My results also show that 2,3-BDZ-11 compounds prefer to inhibit GluA2 and GluA1 over either GluA3 or GluA4, the two remaining subunits of AMPA receptors. Furthermore, chemical modifications of the aminophenyl ring of the BDZ-11 series, such as addition of either a methyl group or a chlorine atom, can yield compounds that are more potent. I further studied the inhibitory property of these compounds with GluA1/GluA2R or GluA2Q/GluA2R AMPA receptor complex channels. As noted, both channel types contained the GluA2R, the edited isoform of GluA2, and GluA2 is a critical AMPA receptor subunit. My results show that a 2,3-benzodiazepin-4-one exhibits the same potency and subunit selectivity on both GluA1/2R and GluA2Q/2R receptor complexes as compared to the GluA1 and GluA2Q, respectively, suggesting that the binding site for the 2,3-BDZ-11 compounds is most likely located in the "Q" isoform (i.e., either GluA1 or GluA2Q). The results from my thesis work are significant in that a detailed mechanistic study and a structure-activity relationship, defined at the functional level and at the single receptor subunit level, provide a better understanding of the receptor structure-function relationship and the site of regulation for these compounds. My results also provide mechanistic clues for design and synthesis of 2,3-benzodiazepin-4-one compounds with higher potency and selectivity.;My thesis work is described in five chapters. In Chapter 1, I provide introduction about AMPA receptors, and the methods as well as techniques I used. In Chapter 2, I introduce the selectivity assay for these compounds against AMPA, kainate and NMDA receptors. In Chapter 3, I describe a detailed mechanistic study of the effect of these compounds on the channel-opening rate process of GluA2 as well as the site characterization for these compounds. In Chapter 4, I present the same type of the study but with the GluA1 receptor channels. In Chapter 5, I describe the work I have done with a set of selected inhibitors with GluA1/2R and GluA2Q/2R AMPA receptor channel complexes.