Synthesis of novel selective histone deacetylase inhibitors for the development of a suitable ¹⁸F-labelled radiotracer for the molecular imaging of HDAC1 in brain tumours

Objectives: Epigenetic mechanisms like methylation and acetylation of histones regulate the gene expression on the chromatin level. Thus, the degree of acetylation of lysine residues on histones influences the accessibility of DNA and furthermore the gene expression. Histone deacetylases (HDACs) are overexpressed in various tumour diseases, resulting in the interest in HDAC inhibitors (HDACi) for cancer therapy. The aim of this work is the development of a novel ¹⁸F-labelled HDAC1-selective inhibitor with an ortho-aminoanilide zinc-binding group (ZBG) to visualize this enzyme in brain tumours by positron emission tomography (PET).
Methods: Based on the selective HDAC1-3 inhibitors tacedinaline and entinostat, a series of fluorine-containing derivatives was synthesized and the IC₅₀ values were determined by an in-house biochemical enzyme assay. Out of several ligands with high inhibitory potency and selectivity for HDAC1, N-(2-amino-5-(thiophen-3-yl)phenyl)-4-((2-fluoropropanamido)methyl)benzamide (BA3, Figure 1A) was selected for radiofluorination. The two-step one-pot radiosynthesis of [¹⁸F]BA3 was performed by a nucleophilic aliphatic substitution reaction of the protected 2-bromopropionyl precursor 2 and subsequent deprotection. The process was successfully transferred to a TRACERlab FX2 N radiosynthesizer (Figure 1B). For the characterization of BA3, the in vitro stability in mouse and human liver microsomes and the cell toxicity in glioblastoma cell lines (U251-MG, F98) were assessed. In parallel, the in vivo metabolism of [¹⁸F]BA3 was investigated (mouse plasma and brain samples, 30 min p.i.) as well as PET studies in mice were carried out.
Results: BA3, containing a PAMBA linker (para-aminomethylbenzoic acid), shows a high inhibitory activity against HDAC1 and high selectivity towards HDAC3 and HDAC6 (see Table 1). The cell viability of U251-MG and F98 cells after incubation with 50 µM BA3 for 72h was only 64% and 36%, respectively. The automated radiosynthesis of [¹⁸F]BA3 resulted in a radiochemical yield of 1%, a radiochemical purity of > 96% and a molar activity between 21 and 51 GBq/µmol (n = 5, EOS). The PET studies in mice showed a low [¹⁸F]BA3 accumulation in the brain, suggesting a low blood-brain barrier penetration (SUV₅ₘᵢₙ: 0.24). Furthermore, the amount of intact radiotracer in the brain and plasma at 30 min p.i. was only 25% and 7%, respectively.
Conclusion: Due to the low blood-brain barrier penetration and the high amount of brain-penetrable radiometabolites, [¹⁸F]BA3 is classified as unsuitable for further PET-related investigations. The obtained results will be used in the design of metabolically more stable HDAC inhibitors.
Reference: [1] Krieger et al., J. Med. Chem. 2019, 62(24), 11260-11279.