CD73 converts AMP to immunosuppressive adenosine, and its inhibition was proposed as a new strategy for cancer treatment. We synthesized 5′-O-[(phosphonomethyl)phosphonic acid] derivatives of purine and pyrimidine nucleosides, which represent nucleoside diphosphate analogs, and compared their CD73 inhibitory potencies. In the adenine series, most ribose modifications and 1-deaza and 3-deaza were detrimental, but 7-deaza was tolerated. Uracil substitution with N3methyl, but not larger groups, or 2-thio, was tolerated. 1,2-Diphosphono-ethyl modifications were not tolerated. N 4 -(Aryl)alkyloxy-cytosine derivatives, especially with bulky benzyloxy substituents, showed increased potency. Among the most potent inhibitors were the 5′-O-[(phosphonomethyl)phosphonic acid] derivatives of 5-fluorouridine (4l), N 4 -benzoyl-cytidine (7f), N 4 -[O-(4-benzyloxy)]-cytidine (9h), and N 4 -[O-(4-naphth-2-ylmethyloxy)]-cytidine (9e) (K i values 5-10 nM at human CD73). Selected compounds tested at the two UDP-activated P2Y receptor subtypes showed high CD73-selectivity, especially those with large nucleobase substituents. These nucleotide analogs are among the most potent CD73 inhibitors reported and may be considered for development as parenteral drugs.

CD73 inhibitors are promising drugs for the (immuno)therapy of cancer. Here, we present the synthesis, structure− activity relationships, and cocrystal structures of novel derivatives of the competitive CD73 inhibitor α,β-methylene-ADP (AOPCP) substituted in the 2-position. Small polar or lipophilic residues increased potency, 2-iodo-and 2-chloro-adenosine-5′-O-[(phosphonomethyl)phosphonic acid] (15, 16) being the most potent inhibitors with K i values toward human CD73 of 3−6 nM. Subject to the size and nature of the 2-substituent, variable binding modes were observed by X-ray crystallography. Depending on the binding mode, large species differences were found, e.g., 2-piperazinyl-AOPCP (21) was >12-fold less potent against rat CD73 compared to human CD73. This study shows that high CD73 inhibitory potency can be achieved by simply introducing a small substituent into the 2-position of AOPCP without the necessity of additional bulky N 6 -substituents. Moreover, it provides valuable insights into the binding modes of competitive CD73 inhibitors, representing an excellent basis for drug development.

Imatinib is the first protein kinase inhibitor approved for clinical use and is a seminal drug for the concept of targeted therapy. Herein we report on the design, synthesis, photokinetic properties, and in vitro enzymatic evaluation of a photoactivatable caged prodrug of imatinib. This approach allows spatial and temporal control over the activation of imatinib triggered by ultraviolet light. The successful application of the photoactivation concept to this significant kinase inhibitor provides further evidence for the caging technique as a feasible approach in the kinase field. The presented photoactivatable imatinib prodrug will be highly useful as a pharmacological tool to study the impact of imatinib toward biological systems in greater detail.

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Ecto-5′-nucleotidase (CD73) catalyzes the hydrolysis of AMP to anti-inflammatory, immunosuppressive adenosine. It is expressed on vascular endothelial, epithelial, and also numerous cancer cells where it strongly contributes to an immunosuppressive microenvironment. In the present study we designed and synthesized fluorescent-labeled CD73 inhibitors with low nanomolar affinity and high selectivity based on N 6 -benzyl-α,βmethylene-ADP (PSB-12379) as a lead structure. Fluorescein was attached to the benzyl residue via different linkers resulting in PSB-19416 (14b, K i 12.6 nM) and PSB-18332 (14a, K i 2.98 nM) as fluorescent high-affinity probes for CD73. These compounds are anticipated to become useful tools for biological studies, drug screening, and diagnostic applications.

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

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. Keywords Ecto-nucleotide pyrophosphatase/phosphodiesterase1 (NPP1). AMP analogs. ADP analogs. Acyclic nucleotide Human chondrocytes. Calcium pyrophosphate dihydrate (CPPD) Abbreviations eNPP Ecto-nucleotide pyrophosphatase/phosphodiesterase NTP Nucleoside-5′-triphosphate p-Nph-5′-TMP p-nitrophenyl ester thymidine 5′-monophosphate SD Standard deviation CPPD Calcium pyrophosphate dihydrate PP i Pyrophosphate/diphosphate TEAB Triethylammonium bicarbonate DCM Dichloromethane

Nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) inhibitors have been suggested as a potential treatment for calcium pyrophosphate dihydrate (CPPD) deposition disease.