Design, synthesis and biological evaluation of novel pyrrolo[2,3-d] pyrimidine as tumor-targeting agents with selectivity for tumor uptake by high affinity folate receptors over the reduced folate carrier

Tumor-targeted 6-substituted pyrrolo[2,3-d]pyrimidine benzoyl compounds based on 2 were isosterically modified at the 4-carbon bridge by replacing the vicinal (C11) carbon by heteroatoms N (4), O (5) or S (6), or with an N-substituted formyl (7), trifluoroacetyl (8) or acetyl (9). Replacement with sulfur (6) afforded the most potent KB tumor cell inhibitor, similar to 6-fold better than the parent 2. In addition, 6 retained tumor transport selectivity via folate receptor (FR) alpha and-beta over the ubiquitous reduced folate carrier (RFC). FR alpha-mediated cell inhibition for 6 was generally equivalent to 2, while the FR beta-mediated activity was improved by 16-fold over 2. N (4) and O (5) substitutions afforded similar tumor cell inhibitions as 2, with selectivity for FR alpha and-beta over RFC. The N-substituted analogs 7-9 also preserved transport selectivity for FR alpha and-beta over RFC. For FR alpha expressing CHO cells, potencies were in the order of 8 > 7 > 9. Whereas 8 and 9 showed similar results with FR beta-expressing CHO cells, 7 was similar to 16-fold more active than 2. By nucleoside rescue experiments, all the compounds inhibited de novo purine biosynthesis, likely at the step catalyzed by glycinamide ribonucleotide formyltransferase. Thus, heteroatom replacements of the CH2 in the bridge of 2 afford analogs with increased tumor cell inhibition that could provide advantages over 2, as well as tumor transport selectivity over clinically used antifolates including methotrexate and pemetrexed.