Beigi Farideh, Bertucci Carlo, Zhu Weizhong, Chakir Khalid, Wainer Irving W, Xiao Rui-Ping, Abernethy Darrell R
Laboratory of Clinical Investigation, National Institute on Aging Intramural Research Program, Baltimore, Maryland, USA.
Chirality. 2006 Nov;18(10):822-7. doi: 10.1002/chir.20317.
rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR.
R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes.
Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect.
Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure.
消旋非诺特罗是一种用于治疗哮喘的β2肾上腺素能受体激动剂（β2-AR）。它有两个手性中心，以R,R'-和S,S'-非诺特罗（R-F和S-F）的外消旋混合物形式上市。在此，我们报告了R-F和S-F对映体的分离及其与β2-AR结合和激活的评估。
通过手性色谱从对映体混合物中分离出R-F和S-F，并通过圆二色性确定其绝对构型。使用含有表达人β2-AR的HEK-293细胞固定化膜的固定相通过前沿亲和色谱评估β2-AR结合，并使用相同的膜进行标准膜结合研究。还使用新鲜分离的成年大鼠心肌细胞研究了R-F和S-F对心肌细胞收缩力的影响。
消旋非诺特罗的手性色谱产生了分离的峰，对映选择性因子为1.21。保留较少的峰被指定为S-F的绝对构型，保留较多的峰为R-F。使用膜结合的β2-AR作为固定相和消旋3H-非诺特罗作为标记配体的前沿色谱表明，向流动相中添加浓度不断增加的R-F会导致消旋3H-非诺特罗保留率呈浓度依赖性降低，而类似地添加S-F则不会改变消旋3H-非诺特罗的保留率。计算得出R-F的解离常数为472 nM，可用结合位点数量为176 pmol/柱，这与膜结合研究的结果一致，即460±55 nM（R-F）和109,000±10,400 nM（S-F）。在心肌细胞中，R-F使最大收缩反应从静息细胞长度的（265±11.6）%增加到（306±11.8）%（P<0.05），并使半数有效浓度（EC50）从-7.0±0.270降低到-7.
