Abstract
1014
Objectives: Fibroblast activation protein-alpha (FAP), also known as seprase, is a marker of cancer-associated fibroblasts, a major regulator of the tumor microenvironment [1]. FAP plays a number of biological roles in cancer and is minimally expressed in normal tissue [2], rendering it a promising target for small molecule-based imaging and/or therapy in a range of cancers. Inhibitors containing a glycyl-2-cyanopyrrolidine [3,4] or a glycylpyrrolidine-2-boronic acid [5] core structure exhibit high (< 1 µM) affinity and selectively for FAP when conjugated to an aromatic or heteroaromatic ring. More recently, (4-quinolynoyl)-glycyl-2-cyanopyrrolidines have been derivatized with a DOTA moiety to enable radiolabeling with 68Ga [2,6] for imaging FAP using PET. Despite promising tissue distribution and contrast to background, the use of 68Ga currently limits the volume of patients that can be imaged with these ligands. Fluorine-18 is a particularly appealing isotope for PET imaging due to its physical properties and ease of scaling production. Our aim was to design and explore structure-activity relationships (SAR) of new fluorine-containing FAP inhibitors with a view towards developing an 18F-labeled radioligand for PET imaging of tumor stroma. Methods: An initial library of five candidates based on the 2-substituted glycylpyrrolidine pharmacophore was synthesized. Either 2-cyanopyrrolidine or pyrrolidine-2-ylboronic acid was conjugated to a glycine spacer, which, in turn, was linked via an amide bond to fluorobenzoyl, fluoropyridoyl, or iodobenzoyl substituents. The iodoquinoline derivative FAPI-01 [6] was prepared as a control. The affinity of the compounds for human FAP was determined by a competition binding assay against [131I]FAPI-01 in SW872 cells. Results: The IC50 of the ligands ranged from 30 nM to greater than 100 µM. The order of affinity was FAPI-01 > RPS-301, RPS-300 >> RPS-303, RPS-304 >> RPS-302. Two structure-activity relationships were evident. The FAP affinity of pyrrolidinyl-2-boronic acids was more than 100-fold higher than otherwise structurally-identical 2-cyanopyrrolidines. In addition, the introduction of N-containing aromatic heterocycles in place of aromatic carbacycles led to modest-to-high improvements in FAP affinity. Conclusions: The fluoronicotinamide analogue. RPS-301, with IC50 = 139 ± 45 nM, is a high affinity inhibitor of FAP. Facile preparation of the 18F-labeled derivative by nucleophilic aromatic substitution enhances the promise of this compound as a PET ligand for FAP. Efforts are underway to evaluate [18F]RPS-301 in a preclinical cancer model, and to prepare a library of structurally-related analogues of RPS-301 with improved affinity for FAP.