Xu Bin, Stephens Andrew, Kirschenheuter Gary, Greslin Arthur F, Cheng Xiaoquin, Sennelo Joe, Cattaneo Marco, Zighetti Maddalena L, Chen Aishe, Kim Soon-Ai, Kim Hak Sung, Bischofberger Norbert, Cook Gary, Jacobson Kenneth A
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, USA.
J Med Chem. 2002 Dec 19;45(26):5694-709. doi: 10.1021/jm020173u.
Activation by ADP of both P2Y(1) and P2Y(12) receptors in platelets contributes to platelet aggregation, and antagonists at these receptor subtypes have antithrombotic properties. In an earlier publication, we have characterized the SAR as P2Y(1) receptor antagonists of acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine. In this study, we have focused on antiaggregatory effects of P2Y antagonists related to a 2-chloro-N(6)-methyladenine-9-(2-methylpropyl) scaffold, containing uncharged substitutions of the phosphate groups. For the known nucleotide (cyclic and acyclic) bisphosphate antagonists of P2Y(1) receptors, there was a significant correlation between inhibition of aggregation induced by 3.3 microM ADP in rat platelets and inhibition of P2Y(1) receptor-induced phospholipase C (PLC) activity previously determined in turkey erythrocytes. Substitution of the phosphate groups with nonhydrolyzable phosphonate groups preserved platelet antiaggregatory activity. Substitution of one of the phosphate groups with O-acyl greatly reduced the inhibitory potency, which tended to increase upon replacement of both phosphate moieties of the acyclic derivatives with uncharged (e.g., ester) groups. In the series of nonsymmetrically substituted monophosphates, the optimal antagonist potency occurred with the phenylcarbamate group. Among symmetrical diester derivatives, the optimal antagonist potency occurred with the di(phenylacetyl) group. A dipivaloyl derivative, a representative uncharged diester, inhibited ADP-induced aggregation in both rat (K(I) 3.6 microM) and human platelets. It antagonized the ADP-induced inhibition of the cyclic AMP pathway in rat platelets (IC(50) 7 microM) but did not affect hP2Y(1) receptor-induced PLC activity measured in transfected astrocytoma cells. We propose that the uncharged derivatives are acting as antagonists of a parallel pro-aggregatory receptor present on platelets, that is, the P2Y(12) receptor. Thus, different substitution of the same nucleoside scaffold can target either of two P2Y receptors in platelets.
血小板中P2Y(1)和P2Y(12)受体被ADP激活均有助于血小板聚集，而这些受体亚型的拮抗剂具有抗血栓形成特性。在早期的一篇论文中，我们已对腺嘌呤核苷酸的无环类似物作为P2Y(1)受体拮抗剂的构效关系进行了表征，这些类似物在对称支链脂肪族链上含有两个磷酸基团，连接在腺嘌呤的9位上。在本研究中，我们重点关注了与2-氯-N(6)-甲基腺嘌呤-9-(2-甲基丙基)骨架相关的P2Y拮抗剂的抗聚集作用，该骨架含有不带电荷的磷酸基团取代基。对于已知的P2Y(1)受体核苷酸（环状和无环）双磷酸拮抗剂，在大鼠血小板中3.3 microM ADP诱导的聚集抑制与先前在火鸡红细胞中测定的P2Y(1)受体诱导的磷脂酶C（PLC）活性抑制之间存在显著相关性。用不可水解的膦酸基团取代磷酸基团可保留血小板抗聚集活性。用O-酰基取代其中一个磷酸基团会大大降低抑制效力，在用不带电荷的（如酯）基团取代无环衍生物的两个磷酸部分后，抑制效力往往会增加。在一系列非对称取代的单磷酸酯中，苯氨基甲酸酯基团的拮抗剂效力最佳。在对称二酯衍生物中，二（苯乙酰）基团的拮抗剂效力最佳。二新戊酰基衍生物是一种代表性的不带电荷的二酯，可抑制大鼠（K(I) 3.6 microM）和人血小板中ADP诱导的聚集。它拮抗大鼠血小板中ADP诱导的环磷酸腺苷途径抑制（IC(50) 7 microM），但不影响在转染的星形细胞瘤细胞中测定的hP2Y(1)受体诱导的PLC活性。我们提出，不带电荷的衍生物作为血小板上存在的一种平行促聚集受体即P2Y(12)受体的拮抗剂发挥作用。因此，相同核苷骨架的不同取代可靶向血小板中的两种P2Y受体中的任何一种。
