1,3‐Disubstituted‐4‐Aminopyrazolo [3, 4‐d] Pyrimidines, a New Class of Potent Inhibitors for Phospholipase D

Abstract

Phospholipase D enzymes cleave lipid substrates to produce phosphatidic acid, an important precursor for many essential cellular molecules. Phospholipase D is a target to modulate cancer-cell invasiveness. This study reports synthesis of a new class of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure. These molecules were synthesized and used to perform initial screening for the inhibition of purified bacterial phospholipase D, which is highly homologous to the human PLD1 . Initially tested with the bacterial phospholipase D enzyme, then confirmed with the recombinant human PLD1 and PLD2 enzymes, the molecules presented here exhibited inhibition of phospholipase D activity (IC50 ) in the low-nanomolar to low-micromolar range with both monomeric substrate diC4 PC and phospholipid vesicles and micelles. The data strongly indicate that these inhibitory molecules directly block enzyme/vesicle substrate binding. Preliminary activity studies using recombinant human phospholipase Ds in in vivo cell assays measuring both transphosphatidylation and head-group cleavage indicate inhibition in the mid- to low-nanomolar range for these potent inhibitory novel molecules in a physiological environment.

The phospholipase D superfamily as therapeutic targets

“…When possible, use of multiple inhibitors in experimental strategies should be pursued to reduce the possibility that interpretations will be confounded by off-target effects such as that reported for one of the isoform-selective inhibitors [28]. A new class of PLD inhibitors has been developed starting with an approach based on a pyrimidine core structure [35]. While also quite potent, these inhibitors have not been characterized as well.…”

Phospholipase D in Cell Signaling: From a Myriad of Cell Functions to Cancer Growth and Metastasis

“…Based on these factors, 4-aminopyrazolopyrimidines (used as kinase inhibitors) have been developed, which have IC 50 values of 5 and 15 nM for PLD1 and PLD2, respectively (89). Although targeting PLD isoforms is the main focus for abrogating the effects of PLD on cancer growth, using indirect inhibitors of upstream regulators of PLD is another approach.…”

The Role of Phospholipase D Enzyme(s) in Modulating Cell Signaling: Implications for Cancer Drug Development

“…These novel inhibitors were tested for inhibition in several biochemical assays using various forms of phospholipids from monomer substrate, micelle and SUVs, which are based on 4-aminopyrazolopyrimidine core structure (Scheme 1). 4-aminopyrazolopyrimidines have earlier been used as tyrosine kinase inhibitors [33] and dual inhibitors of tyrosine and phosphoinositide kinases [34] among other applications [35].…”

“…The synthesis was accomplished from economic and commercially available starting materials. A considerable diversity in the basic core structure could be achieved by using various hydrazines and acyl/aroyl chlorides [33].…”