Novel Synthetic Strategies Enable the Efficient Development of Folate Conjugates for Cancer Radiotheranostics

Abstract

The folate receptor (FR) is an interesting target for radiotheranostics due to its overexpression in several tumor types. The progress in developing novel folate radioconjugates is, however, slow due to the synthetic challenges that folate chemistry presents. The goal of this study was, thus, to establish versatile solid-phase synthetic strategies for a convenient preparation of novel folate conjugates. Two approaches were established based on an orthogonal fluorenylmethyloxycarbonyl (Fmoc)-protection strategy to enable a modular buildup of an albumin-binding DOTA conjugate (known as OxFol-1) using folic acid (oxidized folate version) as a targeting agent. The main difference between the two approaches was the sequence of conjugating the single structural units. The approach that introduced the folate entity as the last unit appeared particularly useful for the preparation of conjugates based on 6R- or 6S-5-methyltetrahydrofolic acid (5-MTHF; a reduced folate version) as targeting entity. Three types of folate conjugates were synthesized either with a p-iodophenyl-based albumin binder (OxFol-1, 6R-RedFol-1, and 6S-RedFol-1) or without an albumin-binding entity (OxFol-14, 6R-RedFol-14, and 6S-RedFol-14). All six conjugates were obtained with high chemical purity (>98%) after 9–13 synthesis steps and a single final HPLC purification. Radiolabeling with lutetium-177 was feasible at high molar activity, and the resulting radioconjugates were stable over at least 24 h. Biodistribution and SPECT/CT imaging studies confirmed the favorable effect of an albumin-binding entity to increase the tumor uptake and reduce kidney retention of folate radioconjugates. The increased tumor-to-kidney ratios obtained with [177Lu]­Lu-6R-RedFol-1 and [177Lu]­Lu-6S-RedFol-1 as compared to [177Lu]­Lu-OxFol-1 indicated that 5-MTHF is the preferred FR-targeting agent for albumin-binding radioconjugates. This was, however, not the case for folate radioconjugates without an albumin binder. Thanks to the established synthesis strategy, the preparation of further folate radioconjugates will be facilitated, potentially enabling the optimization of the tissue distribution characteristics even more.

Design and Evaluation of Novel Albumin-Binding Folate Radioconjugates: Systematic Approach of Varying the Linker Entities

“… d The K D value of [ 177 Lu]­Lu-OxFol-1 was reprinted with permission from Deberle et al Bioconjug Chem 2021 , 32, 1617–1628. Copyright 2022 American Chemical Society; n.d. not determined. …”

“…Four novel folate radioconjugates with a hydrophobic or hydrophilic linker entity at two distinct positions of the molecule were synthesized and evaluated with the aim to better understand the interrelation between the structure of folate conjugates and their pharmacokinetics. The folate conjugates were obtained by employing the solid-phase chemistry technique as previously reported by Deberle et al 32 The folate conjugates were readily labeled with lutetium-177, and the resultant radioconjugates were all stable over 24 h in the presence of L-ascorbic acid, a commonly used radical scavenger for radiopharmaceutical formulations. 34,35 As expected, the incorporation of linker entities at different positions of the folate-based molecules did not affect the characteristics that are dependent on the folate entity, such as the FR-binding affinity or cell uptake and internalization into FR-positive cells.…”

“…An orthogonal protecting group strategy was applied to enable a modular build-up of the folate conjugates. OxFol-1 was synthesized as previously reported by Deberle et al OxFol-2 was prepared by introducing a d- Glu entity [N α -(9-Fmoc)- d -glutamic-acid-α- t -butyl ester (Fmoc- d -Glu-O t Bu)] between the lysine-based linker and the 4-( p -iodophenyl)­butanoate entity. The synthesis of OxFol-3 was performed using 4-(Fmoc-aminomethyl)­benzoic acid (Fmoc-AMBA-OH) instead of Fmoc- d -Glu-O t Bu.…”

“…Synthesis of Folate Conjugates. The folate conjugates were prepared as previously reported 32 using standard fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase synthesis techniques with a 2-chlorotrityl chloride resin as a solid support. An orthogonal protecting group strategy was applied to enable a modular build-up of the folate conjugates.…”

“…The FR-binding affinity of the folate radioconjugates (20 MBq/nmol) was determined using IGROV-1 tumor cells as previously reported 31 and compared to the K D value obtained for [ 177 Lu]Lu-OxFol-1. 32 The values were reported as the average ± SD obtained from n = 3−4 independent experiments, each performed in triplicate.…”