Application of difluoromethylene phosphonic acids to development of biologically active nucleotides

1)根据9-(5‘，5’-二氟-5‘-膦戊基)鸟嘌呤(9-(5’，5‘-Difluoro-5’-phosphonopentyl)-9-deazaguanine-G)与小牛脾嘌呤核苷磷酸化酶(PNP)的二元络合物的X-射线晶体结构数据，设计了一种多底物类似物抑制剂DFPP-DG。DFPP-DG及其类似物通过9-去氮基-9-碘鸟嘌呤衍生物与ω-炔基二氟亚甲基膦酸酯之间的索诺加希拉偶联反应得到的连接基长度作为关键反应进行调节。DFPP-DG是一种非常有效的PNP抑制剂，在1 mM磷酸盐存在下，对小牛脾和人红细胞PNPs的表观抑制常数分别为4.4 nM和8.1nM。它的类似物之一Homo-DFPP-DG具有更长的链连接磷酸盐和9-去氮鸟嘌呤，比人的酶更有效，在1 mM磷酸盐存在下的表观抑制常数为53 nM。2)天然存在的核苷的二磷酸衍生物，包括腺苷3‘，5’-二磷酸(A3P5P)，被报道作为P2Y1受体的竞争性拮抗剂或部分激动剂。选择性的P2Y1受体拮抗剂被认为具有抗血栓形成的潜力，而选择性受体激动剂可能具有抗高血压或抗糖尿病的潜力。因此，人们合成了多种修饰的核苷酸类似物，以探讨其与P2Y1受体拮抗剂和激动剂的构效关系。其中，MRS2179被报道对P2Y1受体具有选择性拮抗活性。然而，MRS2179对体外血小板聚集的影响较强，但作用时间较短。为了开发一种耐磷酸酶的P2Y1受体拮抗剂，我们探索了P2Y1受体配体的修饰核苷酸类似物的合成路线，其中MRS2216的一个或两个磷酸基团被一个二氟亚甲基膦酰基取代。

1) 9-(5',5'-Difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) was designed as a multi-substrate analogue inhibitor against purine nucleoside phosphorylase (PNP) on the basis of X-ray crystallographic data obtained for a binary complex of 9-(5',5'-difluoro-5'-phosphonopentyl)guanine (DFPP-G) with calf spleen PNP. DFPP-DG and its analogous compounds were adjusted by length of the linker achieved by the Sonogashira coupling reaction between a 9-deaza-9-iodoguanine derivative and ω-alkynyldifluoromethylene phosphonates as a key reaction. DFPP-DG is a very potent PNP inhibitor with apparent inhibition constants (in the presence of 1 mM phosphate) of 4.4 nM and 8.1 nM vs calf spleen and human erythrocyte PNPs, respectively. One of its analogues, homo-DFPP-DG, with longer chain linking phosphonate and 9-deazaguanine is even more potent vs human enzyme, with an apparent inhibition constant of 53 nM (in the presence of 1mM phosphate).2) The bisphosphate derivatives of naturally occurring nucleosides including adenosine 3',5'-bisphosphate (A3P5P) are reported to act as competitive antagonists or partial agonists of P2Y1 receptors. A selective P2Y1 receptor antagonist is believed to have potential as an antithrombotic agent, while a selective receptor agonist may have potential as an antihypertensive or antidiabetic agent. Therefore, a variety of modified nucleotide analogues were synthesized to discuss the structure-activity relationships to P2Y1 receptor antagonists and agonists. Among them, MRS2179 is reported to show selective antagonist activities against P2Y1 receptor. However, the effect of MRS2179 on ex vivo platelet aggregation was strong but of short duration. To develop a phosphatase-resistant P2Y1 receptor antagonist, we examined to synthetic routes to modified nucleotide analogues for P2Y1 receptor ligands, in which one or two of phosphate groups for MRS2216 are replaced by a difluoromethylenephosphonyl group.