Tumor Targeting with Novel 6-Substituted Pyrrolo [2,3-d] Pyrimidine Antifolates with Heteroatom Bridge Substitutions via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

Abstract

Targeted antifolates with heteroatom replacements of the carbon vicinal to the phenyl ring in 1 by N (4), O (8), or S (9), or with N-substituted formyl (5), acetyl (6), or trifluoroacetyl (7) moieties, were synthesized and tested for selective cellular uptake by folate receptor (FR) α and β or the proton-coupled folate transporter. Results show increased in vitro anti-proliferative activity toward engineered Chinese hamster ovary cells expressing FRs by 4–9 over the CH2 analog 1. Compounds 4–9 inhibited de novo purine biosynthesis and glycinamide ribonucleotide formyltransferase (GARFTase). X-ray crystal structures for 4 with FRα and GARFTase showed that the bound conformations of 4 required flexibility for attachment to both FRα and GARFTase. In mice bearing IGROV1 ovarian tumor xenografts, 4 was highly efficacious. Our results establish that heteroatom substitutions in the 3-atom bridge region of 6-substituted pyrrolo[2,3-d]pyrimidines related to 1 provide targeted antifolates that warrant further evaluation as anticancer agents.

Fluorine-Substituted Pyrrolo[2,3-d]Pyrimidine Analogues with Tumor Targeting via Cellular Uptake by Folate Receptor α and the Proton-Coupled Folate Transporter and Inhibition of de Novo Purine Nucleotide Biosynthesis

“…The compounds display similar interactions as the native crystal structure ligands 56 (not shown here for clarity), by maintaining key interactions involving the bicyclic scaffolds and the benzoyl L-glutamate tail.…”

“…The pyrrolo[2,3- d ]pyrimidine scaffold is stacked amid the hydrophobic aromatic side chains of Tyr82 (60), Tyr107 (85) and Trp193 (171), similar to that seen with the bicyclic ring of the crystallized ligand (for van der Waals and hydrophobic interactions). 56 The L-glutamate moiety of 5 occupies a similar binding space as the corresponding L-glutamate of the native ligand. 56 The amide NH of 5 forms a hydrogen bond with the backbone carbonyl of His157 (135).…”

“…56 The L-glutamate moiety of 5 occupies a similar binding space as the corresponding L-glutamate of the native ligand. 56 The amide NH of 5 forms a hydrogen bond with the backbone carbonyl of His157 (135). The α -carboxylate of 5 forms a network of hydrogen bonds involving the backbone NH of Gly159 (137) and Trp160 (138), and the side-chain NH of Trp162 (140), while the γ -carboxylic acid of 5 forms ionic interactions with the side-chain protonated amine of Lys158 (136) and the side-chain NH of Gln122 (100) and Trp124 (102).…”

“…The pyrrolo[2,3- d ]pyrimidine scaffold of 5 binds in the same region as that occupied by the bicyclic scaffold of the native ligand in the GARFTase crystal structure (PDB 5J9F, native ligand not shown for clarity). 56 The scaffold is stabilized in the binding site by multiple interactions, a hydrogen bond between the N1 nitrogen of 5 and the backbone of Leu899, 2-NH 2 of 5 and the carbonyl of Leu899, N3 of 5 and the backbone carbonyl of Ala947, and the 4-oxo of 5 and the amide of Asp951. Additionally, the 4-oxo of 5 forms a water-mediated hydrogen bonding network via a conserved water molecule with Ala947, Ala952, and Asp949.…”

“…The X-ray crystal structures of human FR α 56 (PDB 5IZQ, 3.60 Å), FR β 57 (PDB 4KN2, 2.6 Å), and GARFTase 56 (PDB 5J9F, 2.1 Å) were obtained from the protein database. LeadIT 2.1.6 70 was used for the docking studies.…”