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.
We recently reported N
4-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; K
i = 0.673 nM) and 4-iodo (MRS4620 18; K
i = 0.436 nM) substitution of the N
4-benzyloxy group decreased K
i 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 N
4-benzyl groups
in adenine inhibitors. Molecular dynamics identified stabilizing interactions
and predicted conformational diversity. Thus, by N
4-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.
The orthosteric ATP-binding site of the P2X receptors
is poorly
understood. Only a few compounds were well characterized for their
P2X receptor functional activity and subtype selectivity. This study
represents the first fully functional characterization of various
ATP derivatives combined with in silico studies to advance the understanding
of SARs at the orthosteric binding sites of P2X receptors leading
to the identification of 2-chloro-3-trifluoromethylbenzoyl ATP ester
as a novel pan-P2X receptor agonist and several subtype-selective
P2X receptor agonists. Furthermore, esterification of both hydroxyl
functions of ATP using 1-naphthoic acid has led to compound 26 acting as an antagonist at P2X1-4 and P2X2/3 receptors
and an agonist at P2X7 receptors. This particular ATP derivative will
allow interrogating the P2X7 receptor function while antagonizing
all other P2X receptor subtypes and therefore serve as a valuable
pharmacological tool in the future.
The orthosteric ATP-binding site of the P2X receptors is poorly understood. Only a few compounds were well characterized for their P2X receptor functional activity and subtype selectivity. This study represents the first fully functional characterization of various ATP derivatives combined with in silico studies to advance the understanding of SARs at the orthosteric binding sites of P2X receptors leading to the identification of several subtype-selective P2X receptor agonists and compounds with agonistic as well as antagonistic profiles.
Adenosine in the tumor microenvironment acts on A2A and A2B adenosine receptors (ARs) on immune cells (T cells, dendritic cells, NK cells, macrophages and neutrophils) to prevent their activation. Therefore, a means of lowering extracellular adenosine would be beneficial in cancer immunotherapy either as a co‐therapy or monotherapy. Inhibitors of CD73 (ecto‐5’‐nucleotidase, the main extracellular adenosine‐forming enzyme) are already in clinical trials for cancer immunotherapy. Many purine (ADP) analogues have been shown to inhibit this enzyme, but we have explored pyrimidine (UDP/CDP) analogues. We recently reported N4‐substituted 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as potent CD73 inhibitors. We now expand the structure activity relationship (SAR) of pyrimidine nucleotides as human CD73 inhibitors, focusing mainly on cytidine functionalization at the N4 position. 4‐Chloro (MRS4598 16, Ki 0.673 nM) and 4‐iodo (MRS4620 18, Ki 0.436 nM) substitution of the N4‐benzyloxy group increased inhibitory potency Ki by ~20‐fold. Primary alkylamine derivatives coupled through a p‐amido group and varying methylene chain length (24and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. hCD73 X‐ray structures with two of the newly synthesized inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between C‐terminal and N‐terminal domains) as N4‐benzyl groups in potent adenine‐derived inhibitors. Molecular dynamics simulation identified stabilizing interactions and predicted conformational diversity around the N4‐benzyloxy moiety. Thus, by structure‐guided substitution of the N4‐benzyloxy moiety, we have greatly enhanced their inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in human head and neck squamous cell carcinoma (HNSCC) and palatine tonsils in situ.
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