Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate) dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the P_α,β-bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1’s catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-β-bisphosphonate, 10, was identified as an NPP1 inhibitor (K_i 16.3 μM vs. the artificial substrate p-nitrophenyl thymidine-5′-monophosphate (p-Nph-5′-TMP), and 9.60 μM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (K_i 12.6 μM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100 μM.

中文翻译：

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鉴定腺嘌呤-N9-（甲氧基）乙基-β-双膦酸盐为NPP1抑制剂可减弱人骨关节炎软骨细胞中的NPPase活性。

由于NPP1水解ATP导致的细胞外二磷酸过度生产会导致软骨中病理性二磷酸钙（焦磷酸）二水合物沉积（CPPD），从而导致退行性关节疾病，目前尚无法治愈。在这里，我们的目标是鉴定新型NPP1抑制剂作为CPPD沉积疾病的潜在治疗剂。具体来说，我们合成了AMP（NPP1反应产物）和ADP（NPP1抑制剂）的新型类似物。这些衍生物结合了天然核苷酸的几种化学修饰，包括（1）取代_Pα，β的亚甲基-桥接氧原子以提供代谢抗性;（2）取代磺酸根的磺酸盐基团，以改善与NPP1催化锌离子的结合;（3）无环核苷酸类似物，以允许在NPP1催化位点中灵活结合，以及（ 4）苯并咪唑碱替代腺嘌呤。在研究的化合物中，腺嘌呤-N 9-（甲氧基）乙基-β-双膦酸酯10被鉴定为NPP1抑制剂（K _i 16.3μM，而人工底物对-硝基苯基胸苷5'-单磷酸（p -Nph -5'-TMP）和9.60μM（相对于天然底物ATP）。化合物10对人NPP3，人CD39和组织非特异性碱性磷酸酶（TNAP）具有选择性，但对人CD73的抑制作用（K _i 12.6μM）。因此，10是双重NPP1 / CD73抑制剂，它不仅可用于治疗CPPD沉积疾病和钙化主动脉瓣疾病，而且还可考虑用于癌症的免疫治疗。化合物10被证明是一种有前途的抑制剂，当浓度为100μM时，它几乎完全降低人骨关节炎软骨细胞中的NPPase活性。