Brehm Lotte, Greenwood Jeremy R, Hansen Kasper B, Nielsen Birgitte, Egebjerg Jan, Stensbøl Tine B, Bräuner-Osborne Hans, Sløk Frank A, Kronborg Tine T A, Krogsgaard-Larsen Povl
Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
J Med Chem. 2003 Apr 10;46(8):1350-8. doi: 10.1021/jm0204441.
We have previously described (RS)-2-amino-3-(3-hydroxy-7,8-dihydro-6H-cyclohepta[d]isoxazol-4-yl)propionic acid (4-AHCP) as a highly effective agonist at non-N-methyl-d-aspartate (non-NMDA) glutamate (Glu) receptors in vivo, which is more potent than (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) but inactive at NMDA receptors. However, 4-AHCP was found to be much weaker than AMPA as an inhibitor of [(3)H]AMPA binding and to have limited effect in a [(3)H]kainic acid binding assay using rat cortical membranes. To shed light on the mechanism(s) underlying this quite enigmatic pharmacological profile of 4-AHCP, we have now developed a synthesis of (S)-4-AHCP (6) and (R)-4-AHCP (7). At cloned metabotropic Glu receptors mGluR1alpha (group I), mGluR2 (group II), and mGluR4a (group III), neither 6 nor 7 showed significant agonist or antagonist effects. The stereoisomer 6, but not 7, activated cloned AMPA receptor subunits GluR1o, GluR3o, and GluR4o with EC(50) values in the range 4.5-15 microM and the coexpressed kainate-preferring subunits GluR6 + KA2 (EC(50) = 6.4 microM). Compound 6, but not 7, proved to be a very potent agonist (EC(50) = 0.13 microM) at the kainate-preferring GluR5 subunit, equipotent with (S)-2-amino-3-(5-tert-butyl-3-hydroxyisothiazol-4-yl)propionic acid [(S)-Thio-ATPA, 4] and almost 4 times more potent than (S)-2-amino-3-(5-tert-butyl-3-hydroxyisoxazol-4-yl)propionic acid [(S)-ATPA, 3]. Compound 6 thus represents a new structural class of GluR5 agonists. Molecular modeling and docking to a crystal structure of the extracellular binding domain of the AMPA subunit GluR2 has enabled identification of the probable active conformation and binding mode of 6. We are able to rationalize the observed selectivities by comparing the docking of 4 and 6 to subtype constructs, i.e., a crystal structure of the extracellular binding domain of GluR2 and a homology model of GluR5.
我们之前曾描述过（RS）-2-氨基-3-（3-羟基-7,8-二氢-6H-环庚[d]异恶唑-4-基）丙酸（4-AHCP）是一种在体内对非N-甲基-D-天冬氨酸（非NMDA）谷氨酸（Glu）受体非常有效的激动剂，它比（RS）-2-氨基-3-（3-羟基-5-甲基异恶唑-4-基）丙酸（AMPA）更有效，但对NMDA受体无活性。然而，发现4-AHCP作为[(3)H]AMPA结合的抑制剂比AMPA弱得多，并且在使用大鼠皮质膜的[(3)H]海藻酸结合试验中作用有限。为了阐明4-AHCP这种相当神秘的药理学特征背后的机制，我们现在已经合成了（S）-4-AHCP（6）和（R）-4-AHCP（7）。在克隆的代谢型Glu受体mGluR1α（I组）、mGluR2（II组）和mGluR4a（III组）中，6和7均未显示出明显的激动剂或拮抗剂作用。立体异构体6而非7激活了克隆的AMPA受体亚基GluR1o、GluR3o和GluR4o，其EC(50)值在4.5 - 15 microM范围内，并且激活了共表达的对海藻酸盐优先的亚基GluR6 + KA2（EC(50) = 6.4 microM）。化合物6而非7被证明是对海藻酸盐优先的GluR5亚基的非常有效的激动剂（EC(50) = 0.13 microM），与（S）-2-氨基-3-（5-叔丁基-3-羟基异噻唑-4-基）丙酸[（S）-硫代-ATPA，4]等效，并且比（S）-2-氨基-3-（5-叔丁基-3-羟基异恶唑-4-基）丙酸[（S）-ATPA，3]效力几乎高4倍。因此，化合物6代表了一种新的GluR5激动剂结构类别。对AMPA亚基GluR2细胞外结合域晶体结构进行分子建模和对接，使得能够确定6的可能活性构象和结合模式。通过比较4和6与亚型构建体（即GluR2细胞外结合域的晶体结构和GluR5的同源模型）的对接，我们能够解释观察到的选择性。
