Development and Biological Evaluation of the First Highly Potent and Specific Benzamide-Based Radiotracer [18F]BA3 for Imaging of Histone Deacetylases 1 and 2 in Brain

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.


Introduction
Epigenetics describe inheritable and reversible regulation mechanisms of genes expression without altering the DNA sequence. It plays a pivotal role in gene expression by enzyme-mediated post-translational modifications (PTMs) of proteins, such as formylation, methylation, ubiquitination and acetylation. Two of the most studied modifications are the processes of deacetylation and acetylation of lysine side chains on histones, which are regulated by the opposing enzymes histone deacetylases (HDACs) and histone acetyltransferases (HATs). The HDAC family consists of 18 isoforms, which are subdivided into four classes. The enzymes of classes I, II and IV are zinc-dependent enzymes, whereas class III enzymes, also known as sirtuins, are NAD + -dependent (Table 1) [1][2][3]. The enzymatic deacetylation of lysine residues by HDACs leads to a positive charge on histones, and consequently modulates the chromatin structure by increasing the interaction of histones with the negatively charged phosphate groups of the DNA. The condensed chromatin structure restricts access of transcription factors to the DNA, and thus results in transcriptional repression. Conversely, the acetylation of histones by HATs is associated with a more open, and relaxed chromatin conformation and induced transcriptional activation. Over the past years, accumulating evidence showed that dysregulations of the histone deacetylation mechanism impact, among others, cell proliferation, apoptosis, cell differentiation and inflammation processes. These are associated with the progression of pathophysiological states, such as cancer, neurodegenerative disorders or memory impairment [2][3][4][5]. Furthermore, the upregulation of HDACs1/2 was discovered in several types of cancers, including gastric, colorectal, and prostate cancer as well as glioblastoma and several haematological malignancies. Both HDACs share a sequence similarity of around 93% and are usually co-expressed in several large co-repressor complexes, such as NuRD and MiDAC [6][7][8][9]. HDACs1/2 were also found to play a critical role in tumourigenesis and are inversely correlated with disease-free intervals and overall survival [4,[10][11][12][13][14][15]. Given their regulatory activity in multiple processes involved in cancer progression and resistance to radiotherapy and chemotherapy, the development of HDAC-targeted therapies using single agents or drug combinations is very attractive for tumour entities presenting a poor prognosis, and treatment resistance such as glioblastoma [16][17][18]. For these reasons, the use of HDAC inhibitors in the management of gliomas is studied in clinical trials using pan-inhibitors: valproic acid (NCT00302159), panobinostat (NCT00859222) or vorinostat (NCT01236560) as adjuvant therapy to radiotherapy and/or chemotherapy. However, the development of such a personalized therapy would benefit from a non-invasive imaging tool to help (1) assessing the drug targeting potential and (2) to gain more fundamental understanding of the HDAC1/2 density and role in healthy and pathogenic brain tissue, which is still not well understood. Table 1. Classification of HDACs and their distribution in human according to [2]. Therefore, we aim to develop a radiotracer for the non-invasive imaging of HDACs1/2 in the brain via positron emission tomography (PET). The evaluation of the expression and distribution patterns in vivo will help to understand the significance of these enzymes in neuronal processes and pathologies such as glioblastoma.

Class
In the last two decades, numerous HDAC inhibitors and radiotracers have been developed, but only a few were able to cross the blood-brain barrier (BBB) and exhibit suitable pharmacokinetics for brain imaging. HDAC inhibitors contain generally a zincbinding group (ZBG), a linker and a cap group, visualized in Figure 1 on the structures of published inhibitors. Over the years hydroxamic acids and benzamides have emerged as the most frequently used ZBGs. Starting from 4-bromo-2-nitroaniline, the Boc protection of the aromatic amino group was performed in two steps [48]. First of all, the amino group was treated with di-tert-butyl dicarbonate (Boc 2 O) and catalytic amounts of N,N-dimethyl-4-aminopyridine (DMAP) to afford the bis-Boc-protected intermediate followed by cleavage of one Boc group using trifluoroacetic acid (TFA) to give tert-butyl (4-bromo-2-nitrophenyl)carbamate (1) in a total yield of 95%. Under these conditions it was possible to achieve higher yields with this two-step synthesis compared with the reported yields [49][50][51][52][53] using a one-step synthesis. Subsequently, 1 was reacted with 2-/3-thienyl-, 2-/3-furanyl-or para-fluoro-phenylboronic acid in a Suzuki cross coupling reaction with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh 3 ) 4 ) in 1,4-dioxane/water (7:3, v/v) to provide the corresponding compounds 2a-e in very good yields (90-96%). In the first approach, the reduction in the aromatic nitro group was carried out under hydrogen atmosphere with palladium on carbon and afforded 3a-c in quantitative yields [49]. Using the same procedure for the 3-furan-containing compound 2e, the desired aniline product could not be obtained. The NMR analysis showed that both, the aromatic nitro group and the 3-furan, were entirely reduced to form the corresponding aniline derivative substituted with 3-tetrahydrofuran instead of 3-furan. Therefore, a reduction by sodium dithionite was carried out as a milder alternative, which gave the desired products 3d and 3e in good to moderate yields (83% and 40%, respectively) [54]. Subsequently, the amide couplings of 3a and 3b with 4-(Fmocaminomethyl)benzoic acid were performed in a straight forward synthesis under standard conditions using (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) as coupling reagent. After cleaving the Fmoc protecting group with a solution of 20% piperidine in N,N-dimethylformamide (DMF), the resulting amines were coupled with 2-fluoropropanoic acid under the same reaction conditions and led to the compounds 4a and 4b (23% and 40%, respectively, over three steps). To bypass the use of protecting groups and increase the yield of the amide coupling, we synthesized the 4-[(2-fluoropropanamido)methyl]-and 4-(2-fluoropropanamido)benzoic acids 7 and 8 by acylation of the amino group of 4-(aminomethyl)-(PAMBA) and 4-aminobenzoic acid (PABA) with 2-fluoropropanoyl chloride 6 in good yields (63% and 79%, respectively). With the compounds 7 and 8 we were then able to perform the final amide coupling in one instead of three reaction steps to synthesize the corresponding compounds 4a-e and 5a-e in better yields (57-95%). Finally, the Boc protecting group was removed with a solution of 30% TFA in dichloromethane (DCM) to give the final products BA1-BA10 after precipitation in moderate to good yields (15-91%).

Determination of the In Vitro Inhibitory Potency
All prepared compounds as well as the reference HDAC inhibitors vorinostat, entinostat and tacedinaline were tested regarding their inhibitory activity against the class I enzymes HDAC1-3. Additionally, HDAC6 was used as a control isoform to evaluate the selectivity towards class IIb HDACs. The IC 50 values are shown in Table 2.
The HDAC inhibitors containing (hetero)aryl substituted benzamides as ZBG showed, as expected, a high inhibitory activity against HDAC1 with IC 50 values ranging from 4.8 nM to 24.2 nM for BA1-BA10. In contrast, negligibly low inhibitions of HDAC3 and HDAC6 were observed. The screening also revealed that the compounds with the PAMBA linker (n = 1, BA1/BA3/BA5/BA7), compared with the PABA linker (n = 0, BA2/BA4/BA6/BA8), have a higher inhibitory potency towards HDAC1. The only exceptions are BA9 and BA10, which both bear a 3-furan substitution and exhibit no substantial differences in the IC 50 values against HDAC1. Compared with the approved hydroxamic acid-based HDAC inhibitor vorinostat and the benzamides tacedinaline and entinostat, the inhibitory potencies of the new compounds are significantly improved towards HDAC1. It is known that the use of a foot pocket-targeting (hetero)aromatic substituent at position 5 of the aminoanilide moiety leads to an increase in the specificity for HDAC1 and HDAC2 over HDAC3 [37][38][39][40][41]. This could be proven by the specificity ratios (HDAC1/HDAC3) determined for the synthesized compounds ranging from >74 (BA6) to 479 (BA1) compared with nearly no specificity of the reference compounds vorinostat, entinostat or tacedinaline, respectively. Out of the series, BA1 and BA3 showed the best inhibitory activities Pharmaceuticals 2022, 15, 324 6 of 30 against HDAC1. BA3 was selected for radiolabelling as the most active inhibitor of HDAC1 and HDAC2.

Manual Radiosynthesis
For a direct radiofluorination of [ 18 F]BA3, the corresponding Boc-protected bromine precursor 9 was synthesized as shown in Scheme 1 in an overall yield of 34%. The radiolabelling process was investigated under thermal heating using 2-3 mg of precursor 9 and different (i) polar aprotic solvents, (ii) bases, (iii) different precursor to base ratios, (iv) temperatures, (v) reaction times and (vi) azeotropically dried 18  The reactions were carried out at 150 • C (DMF/DMSO) or 100 • C (MeCN) for up to 20 min. Aliquots of the reaction mixtures were taken every 5 min and analysed by radio-thin layer chromatography (radio-TLC) and/or radio-reversed phase high-performance liquid chromatography (radio-RP-HPLC) to determine the radiochemical yield (RCY) of the labelling process. In DMF or DMSO with K 2 CO 3 as base, the radiosynthesis of Boc-[ 18 F]BA3 was not possible. According to radio-RP-HPLC, under these reaction conditions a polar radioactive by-product was formed in high amounts (30% in DMF, 45% in DMSO). When using MeCN as a solvent, it was possible to obtain Boc-[ 18 F]BA3 in labelling yields between 1% and 2% (with KHCO 3 or K 2 CO 3 , respectively), but the same radioactive by-product (~10%) was formed as shown by radio-RP-HPLC. The use of K 2 C 2 O 4 instead of KHCO 3 /K 2 CO 3 and 18-crown-6 instead of K 2.2.2 did not result in an increase in the radiochemical yield of Boc-[ 18 F]BA3. Finally, the additional impact of microwave irradiation, which was investigated in DMSO, DMF and MeCN, respectively, did not improve the radiofluorination. For all syntheses, the (radio-)RP-HPLC chromatograms showed a decomposition of precursor 9 and Boc-[ 18 F]BA3 already after 5 min, as well as a large amount of unreacted [ 18 F]fluoride.
In general, these conditions are strongly basic, limiting the synthetic utility for basesensitive precursors since various side reactions, such as eliminations, can occur [56]. Assuming that the reaction conditions described above are not suitable for radiosynthesis of Boc-[ 18 F]BA3, the use of [ 18 F]tetra-n-butylammonium fluoride ([ 18 F]TBAF) was investigated as a milder reagent [57]. The impact of (i) different solvents, (ii) temperatures, and (iii) reaction times were studied. The radiofluorination of Boc-[ 18 F]BA3 was investigated first in MeCN at 100 • C achieving labelling yields up to 7% after 15 min, which were not increasing with further reaction time. Then, the influence of tert-butanol (tert-BuOH) as a solvent at 100 • C was investigated and a labelling yield of 5% was achieved after

Automated Radiosynthesis
An automated radiosynthesis of [ 18 F]BA3 was established based on the optimized reaction conditions using the radiosynthesis module TRACERlab FX2 N. The detailed configuration is shown in the Supplemental part ( Figure S1). Briefly, [ 18 F]fluoride (starting activities of 7-32 GBq) was trapped on a QMA cartridge and eluted into the reactor with TBAHCO 3 dissolved in MeCN/H 2 O and dried via azeotropic distillation. The nucleophilic substitution of the bromine precursor 9 (4 mg) was performed using [ 18 F]TBAF in MeCN at 100 • C for 15 min. The subsequent Boc deprotection was carried out by adding an aqueous 2 M HCl solution and stirring for 5 min at 80 • C. After neutralization with a 1 M solution of aqueous NaHCO 3 , the radiolabelled product [ 18 F]BA3 was isolated via semi-preparative RP-HPLC ( Figure 2) and purified by solid-phase extraction (SPE) on a C18 cartridge. Subsequent elution with ethanol (EtOH) and formulation in sterile 0.9% saline containing 10% of EtOH reproducibly provided [ 18 F]BA3 in a total synthesis time of approximately 85 min with RCYs of 0.9 ± 0.2% (EOB, n = 5; see Figure S2 for UVand radio-RP-HPLC-chromatograms of formulated [ 18 F]BA3). The identity of [ 18 F]BA3 was confirmed by analytical radio-and UV-RP-HPLC of the final product, co-injected with the reference compound BA3 (Figure 2). Finally, the radiotracer was obtained with a radiochemical purity of >96% and a molar activity in the range of 21-51 GBq/µmol (EOS, n = 5).

Lipophilicity and Radiochemical Stability of [ 18 F]BA3
The distribution coefficient (logD 7.4 ) was experimentally determined by the shake-flask method in the n-octanol/PBS system. The logD 7.4 value of 1.75 ± 0.16 (n = 8) for [ 18 F]BA3 suggests that a moderate passive diffusion through the BBB is most likely [58][59][60]. The radiotracer [ 18 F]BA3 was stable in n-octanol, as well as in the saline formulation containing 10% EtOH at room temperature for more than two hours (radiochemical purity > 99%).

In Vivo PET/MRI Studies of [ 18 F]BA3
The pharmacokinetics of [ 18 F]BA3 was investigated by dynamic PET scans on healthy CD-1 mice in order to evaluate the passage across the BBB, nonspecific binding and thus the expected background level. The time-activity curve (TAC) of the brain (peak standardized uptake value (SUV) at 0.8 min of~0.16) demonstrates a low brain uptake (see Figure 3 and Figure S4). Further radiotracer accumulation reaches a plateau at 15 min (~0.22 SUV) followed by a slow washout reaching a SUV of~0.16 at 60 min after i.v. administration. Notably, only 21% of the parent compound was found in the brain at 30 min p.i., suggesting that the major part of the measured activity originates from the nonspecific accumulation of radiometabolites. Although these first results do not support favourable imaging features of [ 18 F]BA3, the evaluation of its potential affinity to efflux drug transporter present at the BBB could help to predict the features of other derivatives of this class of compound. Thus, preadministration of cyclosporine A, an inhibitor of the P-glycoprotein (P-gp) efflux transporter, increased the initial uptake of [ 18 F]BA3 by about factor two (0.18 vs. 0.42 SUV) indicating that [ 18 F]BA3 is a substrate of P-gp.
Evaluation of the whole-body distribution derived from PET imaging revealed a low accumulation of tracer and/or metabolites in spleen, kidney, muscle, liver, and small intestines (initial uptake: SUV 5 min < 4), as well as a relatively constant activity in blood circulation (SUV 5 min to SUV 60 min : 1.47 vs. 1. 10). An elevated accumulation occurred in the gallbladder and the bladder, indicating both urinary and hepatobiliary excretion (Figures S5 and S6 and Table S1). In conclusion, although [ 18 F]BA3 presents a high in vitro inhibitory potency towards HDAC1 (IC 50 = 4.8 ± 0.6 nM) and HDAC2 (IC 50 = 39.9 ± 3.2 nM), and an excellent specificity of HDAC1 over HDAC3 and HDAC6 (specificity ratios >230 and >2080, respectively), its low passage across the BBB and the presence of radiometabolites in brain show the need for further ligand optimization.

In Vivo Metabolism of [ 18 F]BA3
The in vivo metabolism of [ 18 F]BA3 was investigated in the plasma, brain homogenate and urine obtained from mice at 30 min after injection into the tail vein. The radiometabolites and parent compound were analysed by (i) analytical radio-RP-HPLC and (ii) radiomicellar chromatography (radio-MLC) [61]. Prior to the analysis with radio-RP-HPLC, the plasma and brain samples were treated with MeCN/H 2 O (9:1, v/v) as extraction solvent. The recovery of activity in plasma and brain samples was 97% and 95%, respectively. Radio-RP-HPLC analysis revealed that the intact radiotracer accounted for about 5% and 21% (mean, n = 2) of the total activity in plasma and brain, respectively. Similar results were obtained with MLC (about 6% and 24% (mean, n = 2) in plasma and brain, respectively), where the samples are directly injected without previous protein precipitation (for corresponding MLC chromatograms see Figure S3). In urine intact radiotracer was not detected by neither radio-RP-HPLC nor radio-MLC. In the corresponding radio-chromatograms ( Figure 4) five radiometabolites ([ 18 F]M1-[ 18 F]M5) were detected in brain at 30 min p.i. In contrast, in plasma one single major radiometabolite, [ 18 F]M4, amounting to 74%, was found However, a conclusion, whether the radiometabolites in the brain originate from the plasma and pass the blood-brain barrier or are formed within the brain itself, was not possible, as only a single time point was investigated.
RP-HPLC, the plasma and brain samples were treated with MeCN/H2O (9:1, v/v) as extraction solvent. The recovery of activity in plasma and brain samples was 97% and 95%, respectively. Radio-RP-HPLC analysis revealed that the intact radiotracer accounted for about 5% and 21% (mean, n = 2) of the total activity in plasma and brain, respectively. Similar results were obtained with MLC (about 6% and 24% (mean, n = 2) in plasma and brain, respectively), where the samples are directly injected without previous protein precipitation (for corresponding MLC chromatograms see Figure S3). In urine intact radiotracer was not detected by neither radio-RP-HPLC nor radio-MLC. In the corresponding radio-chromatograms ( Figure 4) five radiometabolites ([ 18 F]M1-[ 18 F]M5) were detected in brain at 30 min p.i.. In contrast, in plasma one single major radiometabolite, [ 18 F]M4, amounting to 74%, was found However, a conclusion, whether the radiometabolites in the brain originate from the plasma and pass the blood-brain barrier or are formed within the brain itself, was not possible, as only a single time point was investigated.

Expression of HDAC1 in F98 and U251-MG Cells
The expression of HDAC1 was evaluated by immunofluorescence microscopy of the glioblastoma cell lines F98 and U251-MG to check whether they are suitable for the assessment of the toxicity of HDAC inhibitors. As shown in Figure 5, both cell lines possess a HDAC1 nucleic localization, proved by the merged signals of the HDAC1 (green) enzyme and the nuclei (blue). Interestingly, the HDAC1 signal is exactly co-localized with the

Expression of HDAC1 in F98 and U251-MG Cells
The expression of HDAC1 was evaluated by immunofluorescence microscopy of the glioblastoma cell lines F98 and U251-MG to check whether they are suitable for the assessment of the toxicity of HDAC inhibitors. As shown in Figure 5, both cell lines possess a HDAC1 nucleic localization, proved by the merged signals of the HDAC1 (green) enzyme and the nuclei (blue). Interestingly, the HDAC1 signal is exactly co-localized with the nuclei in the F98 cells but additionally presents a slight cytoplasmic localization in U251-MG cells, as also previously found by Seto et al. [62].

The New HDAC Inhibitor BA3 Has Potential to Reduce the Proliferation of Cancer Cells
Inhibition of HDAC activity has been shown to hinder the proliferation of cancer cells. To assess the cytotoxic potential of BA3, the effect of this compound on the proliferation of the rat glioma F98 cells and the human glioblastoma U251-MG cells in comparison with the well-established HDAC inhibitors tacedinaline, vorinostat, and entinostat was investigated by an MTS assay (Table 3). Although solubility problems detected only after completion of the study (slight precipitation of BA3 at 50 µM in cell culture medium supplemented with 0.5% DMSO) preclude the direct comparison of the cytotoxic potency between the established HDAC inhibitors and BA3, the similar strongly diminished viability of F98 and U251-MG cells induced by BA3 at a concentration of even lower than 50 µM indicate nevertheless a high cytotoxic potential of BA3. Thus, although differently designed studies would be needed to determine proper cytotoxic IC 50 values and even though not relevant for imaging studies performed with radiotracers at low to sub-nanomolar concentrations, the presented data suggest, that the pharmacological potency of BA3 is at least comparable if not even higher than that of established and clinically tested HDAC inhibitors.

General
Chemicals were purchased from commercial suppliers in analytical grade and used without further purification. Solvents were dried before use, if required. Air-and moisturesensitive reactions were carried out under inert gas atmosphere using argon. The reaction monitoring was carried out by (radio-)TLC on pre-coated silica gel plates (Alugram ® Xtra SIL G/UV254; Polygram ® SIL G/UV254) purchased from Macherey-Nagel (Düren, Germany). The compounds were visualized with UV light at λ = 254 nm and 365 nm and/or by staining with aqueous KMnO 4 solution or ninhydrin solution.

Suzuki Coupling A1
An aqueous solution of K 2 CO 3 (2.6 eq.) was added to a solution of 1 (1 eq.) and the respective boronic acid (1.2 eq.) in 1,4-dioxane under argon atmosphere. After addition of tetrakis(triphenylphosphine)palladium(0) (0.05 eq.), the mixture was stirred under reflux until the complete consumption of 2. The 1,4-dioxane was evaporated; the crude product was dissolved in ethyl acetate (EA) and washed with water and brine. After drying over Na 2 SO 4 the solvent was evaporated and the product purified by flash chromatography.

Nitro Reduction with Hydrogen A2a
The nitro compound was dissolved in EA and diluted with MeOH (1:1, v/v). After flushing with argon, Pd/C (0.05 eq., 10% wt/wt) was added and the mixture was stirred under hydrogen atmosphere at room temperature for 12 h. The solvent was removed, the crude product was adsorbed on silica and purified by flash chromatography.

Nitro Reduction with Sodium Dithionite A2b
To a solution of nitro compound (1 eq.) in 1,4-dioxane/water (5:1, v/v), sodium dithionite (4 eq.) and sodium bicarbonate (10 eq.) were added. The heterogeneous mixture was stirred vigorously under reflux until the complete consumption of the nitro compound. The salts were filtered off and 1,4-dioxane evaporated. The organic compounds were transferred in a separation funnel with EA and H 2 O, extracted twice with EA, washed with brine, and dried over Na 2 SO 4 . After evaporation, the crude product was purified by flash chromatography.

Amide Coupling A3
To a solution of the carboxylic acid and N,N-diisopropylethylamine (DIPEA) in DMF, PyBOP was added in small portions and subsequently stirred for 30 min at room temperature. In the meantime, a solution of the respective amine in DMF was prepared and then added to the stirring solution. After stirring overnight most of the DMF was evaporated, the residue was dissolved in EA, washed with water and brine, and dried over Na 2 SO 4 . After adsorbing the crude product on silica, it was purified by flash chromatography.

Boc Cleavage A4
For the cleavage of the Boc group the corresponding compound (1 eq.) was dissolved in a 30% TFA (20 eq.) solution in DCM and stirred for 2-4 h. The solution was diluted with EA, washed extensively with a 5% aqueous solution of NaHCO 3 and dried over Na 2 SO 4 . The EA was removed until the product started to precipitate. Ice-cold heptane was added, and the product was filtered off and dried under reduced pressure.

Compounds
3.3.1. Synthesis of tert-butyl (4-bromo-2-nitrophenyl)carbamate (1) To a solution of 4-bromo-2-nitroaniline (10 g, 46 mmol, 1 eq.) in 150 mL THF, TEA (15.9 mL, 115 mmol, 2.5 eq.) and DMAP (1.1 g, 9.2 mmol, 0.2 eq.) were added successively. While stirring at room temperature, the first portion Boc 2 O (10 g, 46 mmol, 1 eq.) was added until it was completely dissolved. Then the second portion Boc 2 O (11.6 g, 53 mmol, 1.15 eq.) was added in smaller portions and the solution was stirred at room temperature for 4 h. The reaction volume was reduced to a third and an aqueous solution of citric acid (29 g, 151 mmol) was added. After extraction of the compound with EA, the organic layer was dried over Na 2 SO 4 and the solvent was evaporated to give a light-yellow solid. The crude product was dissolved in a solution of TFA (5.3 mL, 69 mmol, 1.5 eq.) in 140 mL DCM and stirred for 6 h. The reaction was monitored by TLC and after completion an aqueous solution of NaHCO 3 (6.7 g, 80 mmol) was added, the product was extracted twice with DCM, washed with brine, and dried over Na 2 SO 4 . It was purified by flash chromatography (silica, gradient PE/EA 20:1 → 18:1 → 16:1) to give 1 as yellow solid (13.93 g, 95%

Synthesis of 2-fluoropropanoyl chloride (6)
Sodium 2-fluoropropanoate (3 g, 26.3 mmol, 1 eq.) was poured in a two-necked flask, cooled down to 0 • C in an ice bath and stirred vigorously. Phosphorus pentachloride PCl 5 (6 g, 28.9 mmol, 1.1 eq.) was then added in small portions. After the complete addition of PCl 5 the mixture was stirred further at room temperature for 3 h. After distillation ( (7) To an ice-cold suspension of 6 (1 g, 9 mmol, 1.2 eq.) and 4-(aminomethyl)benzoic acid (1.14 g, 7.5 mmol, 1 eq.) in 13 mL chloroform, pyridine (730 µL, 9 mmol, 1.2 eq.) was added dropwise. After stirring at room temperature overnight, most of the chloroform was evaporated and the residue dissolved in an aqueous solution of Na 2 CO 3 (2.12 g, 20 mmol in 20 mL H 2 O). After washing twice with MTBE, the aqueous solution was acidified with 10 mL of a 6 M HCl solution, and the product started to precipitate. After stirring for a while, the solid was filtered off and dried under reduced pressure to give compound 7 (1.06 g, 63%) as white solid. To an ice-cold suspension of 6 (1 g, 9 mmol, 1.2 eq.) and 4-aminobenzoic acid (1.03 g, 7.5 mmol, 1.2 eq.) in 13 mL chloroform, pyridine (730 µL, 9 mmol, 1.2 eq.) was added dropwise. After stirring at room temperature overnight, most of the chloroform was evaporated and the residue dissolved in an aqueous solution of Na 2 CO 3 (2.12 g, 20 mmol in 20 mL H 2 O). After washing twice with MTBE, the aqueous solution was acidified with 10 mL of a 6 M HCl solution and the product started to precipitate. After stirring for a while, the solid was filtered off and dried under reduced pressure to give compound 8 (1.25 g, 79%) as white solid. To a solution of 4-(Fmoc-aminomethyl)benzoic acid (350 mg, 0.94 mmol, 1 eq.) and DIPEA (337 µL, 1.87 mmol, 2 eq.) in 7 mL DMF, PyBOP (536 mg, 1.03 mmol, 1.1 eq.) was added in small portions. After stirring the mixture for 30 min, a solution of 3b (355 mg, 1.22 mmol, 1.3 eq.) in 3 mL DMF was added and the solution was stirred at r.t. overnight. The amount of the DMF was reduced by evaporation, the residue was dissolved in EA, washed with water and brine and the organic phase was dried over Na 2 SO 4 . A filtration over a silica pad (silica, gradient PE/EA: 10:0 → 9:1 → 4:1) gave the crude Fmoc-protected compound (830 mg, 137%, mass balance) which was used without further analytics. The Fmoc-protected compound (830 mg, 0.94 mmol) was dissolved in 5 mL of a 20% piperidine solution in DMF and stirred at r.t. for 4 h. The amount of the DMF was reduced by evaporation, the residue was dissolved in EA, washed with water and brine and the organic phase dried over Na 2 SO 4 . After flash chromatography (silica, gradient DCM/MeOH: 100:0 → 99:1 → 95:5 → 90:10) the amine (240 mg, 60%) was used directly for the next reaction. To a solution of the amine (240 mg, 0.57 mmol, 1 eq.) and TEA (87 µL, 0.62 mmol, 1.1 eq.) in 4 mL DCM, 2-bromopropanoyl bromide (135 mg, 0.62 mmol, 1.1 eq.) was added. After stirring the mixture for 2.5 h, the solvent was evaporated and the crude product was purified by flash chromatography (silica, gradient PE/EA: 8:2 → 7:3 → 6:4 → 5:5) to give compound 9 (180 mg, 57%, 34% overall yield) as a white-beige solid. For labelling with [ 18 F]TBAF, the tetra-n-butylammonium hydrogen carbonate (TBAHCO 3 ) solution (150 µL, 0.075 M, ABX, Radeberg, Germany) was directly placed into the V-vial containing [ 18 F]fluoride and 1 mL of MeCN. The solution was azeotropically dried under vacuum and argon flow in the microwave cavity (Discover PETwave microwave CEM ® corporation, 50-60 • C, 75 W) for 8-10 min. Additional aliquots of MeCN (2; ×, 10mL), were added during the drying process and the final complex was dissolved in an appropriate volume of labelling solvent and used directly or divided in several portions. Thereafter, a solution containing the bromo precursor 9 (2-3 mg, 3.6-5.4 µmol) in 300 µL of an appropriate solvent was added, and 18 F-labelling was performed at different temperatures in dependence of the solvent used (80, 100 and 150 • C). The reaction was monitored in different time points (up to 20 min) via radio-thin layer chromatography (radio-TLC) and (radio-)reversed phase high-performance liquid chromatography ((radio)-RP-HPLC, see quality control).  Figure S1, entry 1, sorbent weight: 46 mg; preconditioned with 10 mL of an aqueous 0.5 M NaHCO 3 solution and 10 mL water, Waters, Milford, MA, USA) and eluted into the reactor with a solution of 150 µL of TBAHCO 3 (entry 2, 0.075 M ABX GmbH, Radeberg, Germany), 300 µL of H 2 O and 600 µL of MeCN. After azeotropic drying at 50 • C for 3 min, 2 mL MeCN (entry 3) were added and azeotropic drying was continued for further 3 min at 70 • C and 1 min at 40 • C. Thereafter, the nucleophilic aliphatic radiofluorination proceeded by adding the bromo precursor 9 (4 mg, 7.2 µmol) dissolved in 800 µL of MeCN (entry 4) and stirring the reaction mixture at 100 • C for 15 min. The reaction mixture was cooled to 40 • C and the Boc deprotection was carried out by adding 800 µL of a 2 M HCl aq solution (entry 5) and subsequent heating at 80 • C for another 5 min. After neutralization with a mixture of 1.6 mL of an aqueous 1 M NaHCO 3 solution and 1.8 mL of 100 mM aqueous phosphate buffer (pH = 6, entry 6), the solution was transferred into the injection vial (entry 7). [ 18 F]BA3 was isolated by semipreparative RP-HPLC (column: Reprosil-Pur C18-AQ, 250 × 10 mm, 10 µm; Dr. Maisch HPLC GmbH, Ammerbuch, Germany) with a solvent composition of 40% MeCN/20 mM NH 4 OAc aq at a flow rate of 4.0 mL/min (entry 8). The radiotracer [ 18 F]BA3 was collected in the HPLC collection vial containing 40 mL of H 2 O (entry 9), trapped on a Sep-Pak ® C18 light cartridge (entry 10, sorbent weight: 130 mg; preconditioned with 5 mL EtOH and 10 mL water, Waters, Milford, MA, USA) and followed by washing with 2 mL of H 2 O (entry 11) and elution of [ 18 F]BA3 with 1.3 mL EtOH (entry 12) in the product vial (entry 13). The ethanolic solution was transferred outside of the hotcell by remote control, the solvent was reduced manually in a stream of argon at 70 • C and the desired radiotracer was reconstituted in a saline solution (NaCl 0.9%) containing max. 10% EtOH (v/v). [ 18 F]BA3 was then ready for further biological characterization in a total synthesis time of about 85 min.
The molar activity was determined using analytical RP-HPLC with a Reprosil-Pur C18-AQ column (250 × 4.6 mm, 5 µm) and 38% MeCN/20 mM NH 4 OAc aq as eluent at a flow rate of 1.0 mL/min obtained at 254 nm.
The ammonium acetate concentrations stated as 20 mM NH 4 OAc aq correspond to the concentration in the aqueous component of an eluent mixture.

Metabolite Analysis
The radiotracer [ 18 F]BA3 (~30 MBq in 200 µL isotonic saline) was administered via the tail vein in non-anesthetized female CD-1 mice (n = 2). At 30 min p.i., the animals were anesthetized by isoflurane inhalation and blood was sampled from the retro-orbital plexus. After cervical dislocation, urine was obtained and the brain was isolated. Plasma was obtained by centrifugation of the blood sample (8000× g, 2 min), and the brain was homogenized in~1 mL distilled water on ice using a glass/PTFE Potter Homogenizer (Potter S Homogenizer, B. Braun Melsungen AG, Melsungen, Germany).
Protein precipitation was performed by addition of an ice-cold MeCN/H 2 O mixture (9:1, v/v) in a ratio of 4:1 (v/v) of organic solvent to plasma or brain homogenate, respectively. The samples were vortexed for 3 min, equilibrated on ice for 3 min, and centrifuged for 10 min at 10,000 rpm. The precipitates were washed with 100 µL of the solvent mixture and subjected to the same procedure. The combined supernatants were concentrated at 70 • C under argon flow to a final volume of approximately 100 µL and analysed by analytical radio-RP-HPLC with a gradient system (see quality control section). The peak corresponding to the radiotracer in the radio-chromatogram was identified by co-injecting the sample with the reference compound BA3. To determine the percentage of activity in the supernatants compared with total activity, aliquots of each step as well as the precipitates were quantified by an automated gamma counter (1480 WIZARD, Perkin Elmer, Turku, Finland). For plasma and brain samples recoveries of 97% and 95%, respectively, of total activity were obtained.
The sodium dodecyl sulfate concentrations stated as 100 mM SDS aq correspond to the concentration in the aqueous component of an eluent mixture.
3.5.2. Cell Culture brain atlas template Ma-Benveniste-Mirrione-FDG. The activity data are expressed as mean SUV of the overall ROI.

Conclusions
A series of novel fluorinated benzamide-based HDAC inhibitors with high inhibition potency and specificity to HDAC1 and HDAC2 was synthesized. With IC 50 values in the low nanomolar range against HDAC1 (4.8 nM each) and HDAC2 (64.3 and 39.9 nM, respectively), the inhibitors BA1 and BA3 emerged as the most promising compounds of the series. BA3, the most potent inhibitor of HDAC1 and HDAC2 was subsequently selected for radiofluorination. After optimizing the radiolabelling conditions, a two-step one-pot automated radiosynthesis of [ 18 F]BA3 using the [ 18 F]TBAF-complex and the TRACERlab FX2 N radiosynthesizer was successfully established. [ 18 F]BA3 was finally obtained in a low radiochemical yield, but with a high radiochemical purity and good molar activities. The biological evaluation in mice revealed a low brain uptake along with a high amount of radiometabolites of [ 18 F]BA3 in the brain. These results led to the conclusion that the radiotracer [ 18 F]BA3 is not suitable for imaging HDAC1/2 in the brain. However, the pharmacological potency of BA3 in vitro is comparable to that of HDAC inhibitors possessing clinical efficacy in the treatment of cancer diseases, making BA3 a potential lead structure for the development of new HDAC inhibitors. Consequently, the scaffold of BA3 requires further modifications to optimize properties such as the metabolic stability and the brain penetrance.