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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 radiosynthes

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 for cancer therapy. The aim of this work is the development of a novel ¹⁸F-labelled HDAC1/2-specific inhibitor with a benzamide-based zinc-binding group to visualize these enzymes in brain tumours by positron emission tomography (PET). BA3, exhibiting high inhibitory potency for HDAC1 (IC50 = 4.8 nM) and HDAC2 (IC50 = 39.9 nM), and specificity towards HDAC3 and HDAC6 (specificity ratios >230 and >2080, respectively), was selected for radiofluorination. The two-step one-pot radiosynthesis of [¹⁸F]BA3 was performed in a TRACERlab FX2 N radiosynthesizer by a nucleophilic aliphatic substitution reaction. 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). For the characterization of BA3, in vitro and in vivo experiments were carried out. The results of these pharmacological and pharmacokinetic studies indicate a suitable inhibitory potency of BA3, whereas the applicability for non-invasive imaging of HDAC1/2 by PET requires further optimization of the properties of this compound.

The data publication contain: 1) Radiosynthesis data (Scheme of the synthesis module; RP-HPLC chromatograms of formulated [18F]BA3; MLC chromatograms of in vivo metabolism studies) 2) Biological data (Baseline TAC of CD-1 mice brain, biodistribution after i.v. injection of [18F]BA3) 3) Analytical data (1H, 13C, 19F NMR spectra; LC-MS chromatograms for final products)

Ziel: Epigenetische Mechanismen wie die Methylierung und Acetylierung von Histonen regulieren die Genexpression auf Chromatin-Ebene. So beeinflusst der Grad der Acetylierung von Lysinresten der Histone die Zugänglichkeit der DNA und damit die Genexpression. HDACs sind in verschiedenen Tumorerkrankungen überexprimiert, woraus das Interesse an HDAC-Inhibitoren für die Therapie von Krebs resultiert. Das Ziel dieser Arbeit ist die Entwicklung neuer hochaffiner und selektiver fluortragender HDAC-Liganden, um HDAC1 und 2 in onkologischen Erkrankungen mittels PET darzustellen. Methodik: Basierend auf Tacedinalin wurden 10 fluorhaltige Derivate in bis zu 8 Synthesestufen hergestellt und ihre IC₅₀-Werte mittels eines biochemischen Enzymassays bestimmt. Von zwei Liganden mit hoher inhibitorischer Potenz und Selektivität für HDAC1 und 2 wurde HD70 ausgewählt und in einem Syntheseautomaten radiofluoriert. Zur biologischen Charakterisierung von [¹⁸F]HD70 wurden Untersuchungen in vitro und in vivo in der Maus durchgeführt. Ergebnisse: HD70 mit einem PAMBA-Linker (p-Aminomethylbenzoesäure) zeigt eine hohe inhibitorische Aktivität gegenüber HDAC1 (IC₅₀: 4,8 nM) und HDAC2 (IC₅₀: 39,9 nM). Die Radiosynthese von [¹⁸F]HD70 aus einem 2-Brompropionylpräkursor erfolgte automatisiert in zwei Stufen mit einer radiochemischen Ausbeute von 1 %. Die PET- und Metabolitenuntersuchungen in CD-1-Mäusen zeigten, dass der Radiotracer [¹⁸F]HD70 die Blut-Hirn-Schranke passiert (SUV5 min: 0,24). Allerdings betrug der Anteil an intaktem Tracer im Hirn nach 30 min nur 25 %. Schlussfolgerungen: Durch den hohen Anteil an hirngängigen Radiometaboliten wird [¹⁸F]HD70 für weitergehende Untersuchungen als ungeeignet eingestuft. Die erhaltenen Ergebnisse werden in das Design metabolisch stabilerer HDAC-Inhibitoren einfließen.

Epigenetics investigates heritable phenotype changes that do not involve alterations in the DNA sequence. The related phenomena play a key role in gene expression by enzyme-mediated post-translational modifications (PTMs) of proteins. One of the most relevant modifications is the process of deacetylation of the lysine side chains on histones, which are regulated by histone deacetylases (HDACs). The catalyzed deacetylation of lysine residues on histones modulates the chromatin and thus influences the gene expression and transcription. The class I histone deacetylases 1, 2 and 3 are overexpressed in several types of cancer, neurodegenerative diseases and inflammation. Inhibition of those zinc-dependent HDACs relaxes the chromatin structure and can result in transcriptional activation and anticancer effects, e.g. cell cycle arrest and induced differentiation. Consequently, radiolabeled HDAC inhibitors have emerged as a potential tool for the diagnostic imaging of tumors by positron emission tomography (PET). [1] The aim of this work is the development of novel highly affine and selective fluorine-containing derivatives of a class I selective HDAC inhibitor to obtain the corresponding 18F-labeled PET radiotracers with an ortho-aminoanilide as zinc-binding motif for targeting class I HDACs in tumors. Recently, we discovered a new highly affine HDAC 1 ligand LSH-A30 with an IC50 for HDAC 1 inhibition of 4.4 ± 0.1 nM. In this connection, the structure of LSH-A30 serves as lead for the development of a series of fluorinated reference compounds, which are currently synthesized. The binding affinities and selectivities towards the class I HDAC isoforms will be determined. Our strategy is mainly focused on the medicinal chemistry of fluorine-containing derivatives, which are suitable for direct and indirect nucleophilic radiofluorination. For the most promising compounds, precursors for radiolabeling will be synthesized, the evaluation of physicochemical properties, e.g. stabi

Epigenetics investigates heritable phenotype changes that do not involve alterations in the DNA sequence. The related phenomena play a key role in gene expression by enzyme-mediated post-translational modifications (PTMs) of proteins. One of the most relevant modifications is the process of deacetylation of the lysine side chains on histones, which are regulated by histone deacetylases (HDACs). The catalyzed deacetylation of lysine residues on histones modulates the chromatin and thus influences the gene expression and transcription. The class I histone deacetylases 1, 2 and 3 are overexpressed in several types of cancer, neurodegenerative diseases and inflammation. Inhibition of those zinc-dependent HDACs relaxes the chromatin structure and can result in transcriptional activation and anticancer effects, e.g. cell cycle arrest and induced differentiation. Consequently, radiolabeled HDAC inhibitors have emerged as a potential tool for the diagnostic imaging of tumors by positron emission tomography (PET). [1] The aim of this work is the development of novel highly affine and selective fluorine-containing derivatives of a class I selective HDAC inhibitor to obtain the corresponding 18F-labeled PET radiotracers with an ortho-aminoanilide as zinc-binding motif for targeting class I HDACs in tumors. Recently, we discovered a new highly affine HDAC 1 ligand LSH-A30 with an IC50 for HDAC 1 inhibition of 4.4 ± 0.1 nM. In this connection, the structure of LSH-A30 serves as lead for the development of a series of fluorinated reference compounds, which are currently synthesized. The binding affinities and selectivities towards the class I HDAC isoforms will be determined. Our strategy is mainly focused on the medicinal chemistry of fluorine-containing derivatives, which are suitable for direct and indirect nucleophilic radiofluorination. For the most promising compounds, precursors for radiolabeling will be synthesized, the evaluation of physicochemical properties, e.g. stabi