Structure–Activity Relationship of Purine and Pyrimidine Nucleotides as Ecto-5′-Nucleotidase (CD73) Inhibitors

Abstract

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.

Ectonucleoside triphosphate diphosphohydrolases and ecto-5′-nucleotidase in purinergic signaling: how the field developed and where we are now

“…Recently, small molecule inhibitors with subnanomolar K i values at human and rat eN could be developed, which are derivatives of purine and pyrimidine nucleotides. Moreover, high-resolution co-crystal structures revealed insight into the binding mode and represent an excellent basis for drug development [57,[94][95][96]. Similarly, monoclonal antibodies are applied as inhibitors of eN and may be employed as therapeutic agents [97][98][99].…”

Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

“…NM_006258, NM_005021, NM_021572, NM_ 001775, and NM_002526, respectively) were obtained from Origene (Rockville, USA). Soluble enzymes were produced as previously reported with some modifications ( Lee et al, 2015 ; Junker et al, 2019 ). Briefly, the catalytic domains of the enzymes were amplified and sub-cloned into the expression vector p ACGP67 A/B modified with the addition of 9 x histidine tag (His-tag) at the C-terminus (except for NPP1).…”

“…The assay was performed as previously described ( Freundlieb et al, 2014 ). Briefly, it contained with 0.09 µg/ml of soluble human CD73 recombinantly expressed in Sf9 insect cells as described ( Junker et al, 2019 ), the respective test compound, and 5.0 µM [2,8- 3 H]AMP (specific activity 7.4 x 108 Bq/mmol, 20 mCi/mmol) as radioactive substrate in assay buffer consisting of 25 mM Tris buffer, 140 mM NaCl, 25 mM NaH 2 PO 4 pH 7.4. The enzymatic reaction was performed for 25 min at 37°C in a shaking water bath.…”

Purinergic Signaling: Impact of GPCR Structures on Rational Drug Design

“…Dihydropyridine MRS1191 8 and pyridine MRS1523 9 , both selective A 3 AR antagonists, are chemically optimized versions of screening hits identified through binding performed in the Jacobson lab from a manually collected chemical library prior to the commercial availability of libraries. We are also working on inhibitors of ADK (to increase intracellular and, indirectly, extracellular adenosine levels) and CD73 (to prevent the formation of adenosine from nucleotides, useful as co‐therapy in cancer immunotherapy) [28,29] …”