Customizable Porphyrin Platform Enables Folate Receptor PET Imaging Using Copper-64

Abstract

Folate receptors including folate receptor α (FRα) are overexpressed in up to 90% of ovarian cancers. Ovarian cancers overexpressing FRα often exhibit high degrees of drug resistance and poor outcomes. A porphyrin chassis has been developed that is readily customizable according to the desired targeting properties. Thus, compound O5 includes a free base porphyrin, two water-solubilizing groups that project above and below the macrocycle plane, and a folate targeting moiety. Compound O5 was synthesized (>95% purity) and exhibited aqueous solubility of at least 0.48 mM (1 mg/mL). Radiolabeling of O5 with 64Cu in HEPES buffer at 37 °C gave a molar activity of 1000 μCi/μg (88 MBq/nmol). [64Cu]Cu–O5 was stable in human serum for 24 h. Cell uptake studies showed 535 ± 12% bound/mg [64Cu]Cu–O5 in FRα-positive IGROV1 cells when incubated at 0.04 nM. Subcellular fractionation showed that most radioactivity was associated with the cytoplasmic (39.4 ± 2.7%) and chromatin-bound nuclear (53.0 ± 4.2%) fractions. In mice bearing IGROV1 xenografts, PET imaging studies showed clear tumor uptake of [64Cu]Cu–O5 from 1 to 24 h post injection with a low degree of liver uptake. The tumor standardized uptake value at 24 h post injection was 0.34 ± 0.16 versus 0.06 ± 0.07 in the blocking group. In summary, [64Cu]Cu–O5 was synthesized at high molar activity, was stable in serum, exhibited high binding to FRα-overexpressing cells with high nuclear translocation, and gave uptake that was clearly visible in mouse tumor xenografts.

Molecular designs with PEG groups for water-solubilization of sparsely substituted porphyrins

“…A facially encumbered porphyrin was prepared by a MacDonald-type condensation 27–31 (Scheme 2). Thus, condensation of 2,6-bis(prop-2-yn-1-yloxy)benzaldehyde ( 3 ) 32 and dipyrromethane ( 4 ) 33 was carried out upon catalysis by BF 3 ·O(Et) 2 followed by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and subsequent neutralization of the Lewis acid with triethylamine (TEA). In this manner, the trans -A 2 porphyrin 5,15-bis(2,6-bis(2-propynyloxy)phenyl)porphyrin ( 5 ) was obtained in 21% yield.…”

“…Zinc porphyrin Zn5 and mPEG 6 -azide were subjected to click reaction to give the corresponding tetra-pegylated zinc porphyrin Zn6 in 71% yield (Scheme 3). To explore the effect of the nature of the metalation state on aqueous solubility, and to gauge the ease of copper insertion for studies in radiochemistry with copper radionuclides, 32 zinc porphyrin Zn6 was demetalated with trifluoroacetic acid (TFA) at room temperature to give the free base porphyrin 6 in 96% yield. Free base porphyrin 6 was then treated overnight with Cu(OAc) 2 ·H 2 O at room temperature, affording the copper chelate Cu6 in quantitative yield.…”

“…A porphyrin scaffold ( Zn9 ) containing two propynyloxy groups and one TIPS-protected ethyne was recently prepared for successive elaboration with PEG and bioconjugatable groups. 32 Click chemistry with azido-PEG 6 -CO 2 t -Bu ( II ), sodium ascorbate, CuBr, and the ligand tris(hydroxypropyltriazolyl)methylamine (THPTA) followed by removal of the TIPS group with tetra- n -butylammonium fluoride (TBAF) afforded Zn10 32 in 70% yield (Scheme 4, left panel). Treatment of Zn10 with TFA in CH 2 Cl 2 for 2 h gave, in 94% yield, the free base porphyrin 11 for examination of aqueous solubility.…”

“…The porphyrin scaffold Zn9 also was treated with the short azido-PEG 2 -CO 2 t -Bu ( III ) via click chemistry to create porphyrin Zn12 in 73% yield. Removal of the TIPS group with TBAF gave in 93% yield porphyrin Zn13 , a homologue of zinc porphyrin Zn10 , 32 containing PEG 2 versus PEG 6 groups. Treatment with TFA in CH 2 Cl 2 gave the free base porphyrin 14 , a homologue of 11 , for studies of aqueous solubility (Scheme 4, right panel).…”

“…Despite the self-aggregation of 11 in the 20–200 μM range, a derivative thereof that contains a folic acid moiety tethered via a PEG 5 group (clicked onto the 4-ethynylphenyl moiety) did not show signs of self-aggregation across the concentration range of 4.8–480 μM in PBS at room temperature. 32 Such results indicate that incorporation of a polar motif with 11 substantially increases the aqueous solubility.…”