Fischer B, Shahar L, Chulkin A, Boyer J L, Harden K T, Gendron F P, Beaudoin A R, Chapal J, Hillaire-Buys D, Petit P
Department of Chemistry, Gonda-Goldschmied Medical Research Center, Bar-Ilan University, Ramat Gan, Israel.
Isr Med Assoc J. 2000 Jul;2 Suppl:92-8.
P2-receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were identified also on pancreatic B cells and are involved in insulin secretion. The aim of our study was to synthesize and evaluate pharmacologically the novel P2Y-R ligands, 2-thioether-5'-O-phosphorothioate adenosine derivatives, as potential insulin secretagogues. An efficient synthesis of these nucleosides and a facile method for separation of the chiral products is described. The enzymatic stability of the compounds towards pig-pancreas NTPDase was evaluated. The rate of hydrolysis of 2-hexylthio-5'-O-(1-thiotriphosphate)-adenosine (2-hexylthio-ATP-alpha-S) isomers by NTPDase was 28% that of ATP. The apparent affinity of the compounds to P2Y1-R was determined by measurement of P2Y-receptor-promoted phospholipase C activity in turkey erythrocyte membranes. 2-RS-ATP-alpha-S derivatives were agonists, stimulating the production of inositol phosphates with K0.5 values in the nM range. 2-RS-AMP-S derivatives were full agonists although 2 orders of magnitude less potent. All the compounds were more potent than ATP. The effect on insulin secretion and pancreatic flow rate was evaluated on isolated and perfused rat pancreas. A high increase, up to 500%, in glucose-induced insulin secretion was due to addition of 2-hexylthio-ATP-alpha-S in the nM concentration range, which represents 100-fold enhancement of activity relative to ATP. 2-Hexylthio-AMP-S was 2.5 orders of magnitude less effective. A high chemical hydrolytic stability was observed for 2-hexylthio-ATP-alpha-S. Hydrolysis of the phosphoester bond, which was the only detectable degrading reaction under the investigation conditions (pH 7.4, 37 degrees C), was slow, with a half-life of 264 hours. Moreover, even at gastric juice conditions (pH 1.4, 37 degrees C), hydrolysis of the terminal phosphate was the only detectable reaction, with a half-life of 17.5 hours. 2-Hexylthio-ATP-alpha-S isomers are enzymatically and chemically stable. These isomers are highly potent and effective insulin secretagogues, increasing, however, pancreatic vascular resistance.
P2受体（P2-Rs）是新型药物研发的重要靶点。在胰腺β细胞上也发现了P2-Rs，其与胰岛素分泌有关。我们研究的目的是合成并从药理学角度评估新型P2Y-R配体，即2-硫醚-5'-O-硫代磷酸腺苷衍生物，作为潜在的胰岛素促分泌剂。本文描述了这些核苷的高效合成方法以及手性产物的简便分离方法。评估了这些化合物对猪胰腺NTPDase的酶稳定性。NTPDase对2-己硫基-5'-O-(1-硫代三磷酸)-腺苷（2-己硫基-ATP-α-S）异构体的水解速率为ATP的28%。通过测量火鸡红细胞膜中P2Y受体促进的磷脂酶C活性，确定了这些化合物对P2Y1-R的表观亲和力。2-RS-ATP-α-S衍生物是激动剂，能刺激肌醇磷酸的产生，其K0.5值在纳摩尔范围内。2-RS-AMP-S衍生物是完全激动剂，但其效力低两个数量级。所有化合物的效力均高于ATP。在离体灌注的大鼠胰腺上评估了其对胰岛素分泌和胰腺流速的影响。在纳摩尔浓度范围内添加2-己硫基-ATP-α-S可使葡萄糖诱导的胰岛素分泌大幅增加，最高可达500%，相对于ATP，其活性增强了100倍。2-己硫基-AMP-S的效力低2.5个数量级。观察到2-己硫基-ATP-α-S具有较高的化学水解稳定性。在研究条件（pH 7.4，37℃）下，磷酸酯键的水解是唯一可检测到的降解反应，水解缓慢，半衰期为264小时。此外，即使在胃液条件（pH 1.4，37℃）下，末端磷酸的水解也是唯一可检测到的反应，半衰期为17.5小时。2-己硫基-ATP-α-S异构体在酶和化学方面都很稳定。这些异构体是高效且有效的胰岛素促分泌剂，但会增加胰腺血管阻力。
