Structure–Activity Relationship of 3‑Methylcytidine-5′-α,β-methylenediphosphates as CD73 Inhibitors

We recently reported N4-substituted
3-methylcytidine-5′-α,β-methylenediphosphates as
CD73 inhibitors, potentially useful in cancer immunotherapy. We now
expand the structure–activity relationship of pyrimidine nucleotides
as human CD73 inhibitors. 4-Chloro (MRS4598 16; Ki = 0.673 nM) and 4-iodo (MRS4620 18; Ki = 0.436 nM) substitution of the N4-benzyloxy group decreased Ki by ∼20-fold. Primary alkylamine derivatives coupled
through a p-amido group with a varying methylene
chain length (24 and 25) were functionalized
congeners, for subsequent conjugation to carrier or reporter moieties.
X-ray structures of hCD73 with two inhibitors indicated a ribose ring
conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal
and N-terminal domains) as N4-benzyl groups
in adenine inhibitors. Molecular dynamics identified stabilizing interactions
and predicted conformational diversity. Thus, by N4-benzyloxy substitution, we have greatly enhanced the
inhibitory potency and added functionality enabling molecular probes.
Their potential as anticancer drugs was confirmed by blocking CD73
activity in tumor tissues in situ.