Advances in the Development of PET Ligands Targeting Histone Deacetylases for the Assessment of Neurodegenerative Diseases

molecules

Review Advances in the Development of PET Ligands Targeting Histone Deacetylases for the Assessment of Neurodegenerative Diseases

Tetsuro Tago and Jun Toyohara * ID Research Team for Neuroimaging, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-3-3964-3241; Fax: +81-3-3964-1148

Received: 1 January 2018; Accepted: 29 January 2018; Published: 31 January 2018

Abstract: Epigenetic alterations of gene expression have emerged as a key factor in several neurodegenerative diseases. In particular, inhibitors targeting histone deacetylases (HDACs), which are enzymes responsible for deacetylation of histones and other proteins, show therapeutic effects in animal neurodegenerative disease models. However, the details of the interaction between changes in HDAC levels in the brain and disease progression remain unknown. In this review, we focus on recent advances in development of radioligands for HDAC imaging in the brain with positron emission tomography (PET). We summarize the results of radiosynthesis and biological evaluation of the HDAC ligands to identify their successful results and challenges. Since 2006, several small molecules that are radiolabeled with a radioisotope such as carbon-11 or ﬂuorine-18 have been developed and evaluated using various assays including in vitro HDAC binding assays and PET imaging in rodents and non-human primates. Although most compounds do not readily cross the blood-brain barrier, adamantane-conjugated radioligands tend to show good brain uptake. Until now, only one HDAC radioligand has been tested clinically in a brain PET study. Further PET imaging studies to clarify age-related and disease-related changes in HDACs in disease models and humans will increase our understanding of the roles of HDACs in neurodegenerative diseases.

Keywords: positron emission tomography; histone deacetylase; radioligand; imaging; neurodegenerative disease

1. Introduction Epigenetics is defined as mitotically and/or meiotically heritable changes in gene expression without alternations in the DNA sequence [1]. Several studies are actively examining the mechanisms of epigenetic regulation, including DNA methylation, histone modification, and RNA-based mechanisms [2]. In mammals, DNA methylation mainly involves the covalent addition of a methyl group at the 5-position of a cytosine followed by a guanine (50-CpG-30)[3]. This modification results in gene silencing by direct and/or indirect inhibition of transcription factor-DNA interactions. Mechanisms of DNA methylation are divided into two classes according to the corresponding DNA methyltransferase (DNMT): maintenance methylation carried out by DNMT1 and de novo methylation carried out by DNMT3a and DNMT3b [4,5]. Histone posttranslational modifications include acetylation, methylation, ubiquitination, and phosphorylation [6]. Acetylation of histones is one of the most highly studied processes and is regulated by opposing enzymes: histone acetyl transferases and histone deacetylases (HDACs) [7]. Acetylation of lysine residues by histone acetyl transferases neutralizes the positive charge of histones and consequently decreases the interaction of histones with the negatively charged phosphate group of DNA. Then, the chromatin structure becomes relaxed, allowing easier access of transcription factors to DNA. Conversely, deacetylation of

Molecules 2018, 23, 300; doi:10.3390/molecules23020300 www.mdpi.com/journal/molecules Molecules 2018, 23, 300 2 of 27 histones by HDACs induces gene silencing [8,9]. Histone methylation occurs mainly on lysine and arginine residues. These residues can be methylated multiple times (lysine: three times; arginine: twice), making the effects on gene regulation complex [1,7]. Diverse classes of RNA also regulate gene expression [10]. For example, small interfering RNAs directed to promoter regions result in transcriptional gene silencing through heterochromatin formation [11]. These epigenetic modifications have emerged as key factors in functions of the central nervous system (CNS) and in the development of common neurodegenerative diseases. So far, several studies into the role of epigenetics in CNS functions (e.g., learning and memory processes, fear memory formation, and drug addiction) have been reported [8,12–15]. Meanwhile, neurodegenerative diseases that may involve epigenetic alterations include Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease [2]. For example, significantly reduced levels of DNA methylation were observed in temporal neocortex neuronal nuclei in an AD monozygotic twin compared to his normal sibling [16]. Understanding the expression of enzymes associated with epigenetics and associated alterations in the human brain will help elucidate the pathological mechanisms of these neurodegenerative diseases and will accelerate development of therapeutic agents targeting epigenetics. The noninvasive imaging technique, positron emission tomography (PET), has received increasing attention in the past few decades, and now PET can be used to monitor various pathological changes in the living brain with a neurodegenerative disease [17,18]. This review summarizes the recent advances in development of PET ligands for imaging HDAC, as a representative example of an epigenetic enzyme, in the brain. With the aim of radiolabeling compounds with affinity for HDACs, various labeling methods using radioisotopes such as carbon-11 and fluorine-18 were investigated in previous studies. Unfortunately, even though more than 20 HDAC imaging radioligands have been developed since 2006, only one report describing imaging results in humans has been published. Summarizing the current advances and issues with HDAC PET ligands will help with future development of imaging techniques and will enable monitoring of the HDAC state in neurodegenerative diseases.

2. HDACs in the Brain In humans, 18 HDACs can be classified into five classes based on their characteristics and homology to yeast HDACs: class I (HDAC1, 2, 3, and 8), class IIa (HDAC4, 5, 7, and 9), class IIb (HDAC6 and 10), class III (sirtuins, SIRT1–7), and class IV (HDAC11) [9,19]. Generally, class III sirtuins, which are nicotinamide adenine dinucleotide-dependent deacetylases, are considered as separate from other HDAC classes, which have zinc-dependent catalytic activity. Class I HDACs mainly reside in the nucleus and are ubiquitously expressed. Class II HDACs shuttle between the nucleus and the cytoplasm and deacetylate non-histone proteins [20]. Compared to class I HDACs, class II HDACs show tissue specificity, and consequently, class II HDAC knockout mice tend to show local defects [21]. HDAC11, the only class IV HDAC, contains conserved residues in the catalytic core regions shared by both class I and II HDACs [22]. The distribution and expression of HDACs in normal and neurodegenerative brains of mammals, including humans, have been reported. Broide et al. conducted comprehensive gene expression mapping of the 11 HDAC isoforms in the rat brain using high-resolution in situ hybridization in 2007 [23]. The distribution of HDAC subtypes showed overlapping and distinct patterns, suggesting that HDACs play distinct physiological roles in the brain. Of the 11 isoforms, HDAC3, 4, 5, and 11 demonstrated the highest expression in the brain, particularly in the cortex. In addition, immunohistochemistry (IHC) showed that most HDACs are expressed primarily in neurons, whereas expression in other cell types is isoform-specific or limited in the rat brain. Regarding age-related changes in HDAC expression in rodent brain, global HDAC enzymatic activity in the hippocampus and frontal cortex of 18-month-old rats is higher than that in 3-month-old rats [24]. In disease models, concentrations of HDAC3 and 4 in whole brain hemispheres of 5xFAD mice, an AD model with amyloid-β deposition, are about 1.5 times higher than those in wild-type mice [25]. Expression of Molecules 2018, 23, 300 3 of 27 class I and II HDACs in the non-human primate (NHP) brain was reported by Yeh et al. in a study evaluating a radioligand for HDAC PET imaging [26]. In this report, they performed qualitative IHC analysis of the expression of 11 HDAC isoforms in brain regions including the nucleus accumbens, hippocampus, cortex, and cerebellum. In 2008, Lucio-Eterovic et al. assessed mRNA expression of HDACs in the human brain with quantitative real-time polymerase chain reaction as part of an investigation into HDAC expression levels in gliomas [27]. In normal brain tissue, expression of class I HDACs, HDAC6, and HDAC7 is relatively low, whereas HDAC4, 5, 9, 10, and 11 show higher expression. Anderson et al. determined HDAC expression in human brain with multiple reaction monitoring mass spectrometry [25]. Concentrations of HDAC1 + 2, 5, and 6 were reported as 1.10, 0.083, and 0.106 pmol/mg tissue protein, respectively, in the frontal cortex of aged controls. In 2016, Wey et al. assessed expression levels of HDAC1, 2, 3, and 6 in human brains diagnosed with no neuropathological abnormalities as part of a clinical study of an HDAC PET ligand [28]. The amounts of HDAC2 and 3 are significantly higher in the superior frontal gyrus relative to the corpus callosum. The expression levels of HDAC2, 3, and 6 are comparable in the superior frontal gyrus (0.12–0.16 pmol/mg total protein), whereas that of HDAC1 is obviously higher than others (1.7 pmol/mg total protein). In brains with neurodegenerative disease, Ding et al. reported that compared with aged controls, the expression of HDAC6 in the cerebral cortex and hippocampus of AD patients is increased by 52% and 91%, respectively [29]. In a study by Anderson et al. in 2015, the concentrations of HDAC1 + 2, 5, and 6 in the frontal cortex of AD patients were reported to be 0.7, 1.5, and 1.3 times those of aged controls [25]. Whitehouse et al. investigated the distribution and intensity of HDAC4, 5, and 6 in FTLD with IHC [30]. HDAC4 and 6 show higher immunoreactivity in the dentate gyrus in FTLD cases, especially in FTLD-tau Picks, compared with controls, and the difference in HDAC6 was more prominent. No changes were observed for HDAC5 between FTLD and controls.

3. HDAC Inhibitors In the last two decades, many types of inhibitors against zinc-dependent HDACs have been developed for treatment of cancer and neurodegenerative disease [21,31–33]. Typical small molecule HDAC inhibitors can be classified into four classes: hydroxamic acids, alkanoic acids, cyclic peptides, and ortho-aminoanilides [19]. Following the discovery of the class I and II HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA, also known as vorinostat) in 1996 [34], hydroxamic acid-based HDAC inhibitors compose the biggest compound library of the four classes. The chemical structure of HDAC inhibitors is generally composed of three motifs: a zinc-binding group that holds the zinc ion of HDAC, a cap group that interacts with the protein surface in the binding pocket, and a linker group that bridges the other two groups. For example, in SAHA, the zinc-binding, cap, and linker groups are N-phenylformamide, hydroxamic acid, and hexane groups, respectively (Figure1). SAHA is the first US Food and Drug Administration (FDA)-approved HDAC inhibitor for use in patients with cutaneous T-cell lymphoma. The anticancer effects of SAHA are thought to be caused by modulation of both gene expression and acetylation of proteins that regulate cell proliferation [35]. Besides SAHA, other HDAC inhibitors (e.g., panobinostat and belinostat) have been approved by the US FDA for the treatment of cutaneous T-cell lymphoma, peripheral T-cell lymphoma, or multiple myeloma [36,37]. HDAC inhibitors have shown promising results in preclinical studies using animal models of neurodegenerative diseases [33,38]. Ricobaraza et al. reported that sodium 4-phenylbutyrate treatment reverses spatial memory deficits in Tg2576 AD model mice without affecting β-amyloid levels [39]. They attributed this effect to both activation of gene transcription by histone deacetylation inhibition and normalization of tau hyperphosphorylation by glycogen synthase kinase 3β inhibition. Gardian et al. showed neuroprotective effects of phenylbutylate treatment in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease model mice [40]. Following phenylbutylate administration, depletion of dopamine in the striatum and reduction in Molecules 2018, 23, x 3 of 27

isoforms in brain regions including the nucleus accumbens, hippocampus, cortex, and cerebellum. In 2008, Lucio-Eterovic et al. assessed mRNA expression of HDACs in the human brain with quantitative real-time polymerase chain reaction as part of an investigation into HDAC expression levels in gliomas [27]. In normal brain tissue, expression of class I HDACs, HDAC6, and HDAC7 is relatively low, whereas HDAC4, 5, 9, 10, and 11 show higher expression. Anderson et al. determined HDAC expression in human brain with multiple reaction monitoring mass spectrometry [25]. Concentrations of HDAC1 + 2, 5, and 6 were reported as 1.10, 0.083, and 0.106 pmol/mg tissue protein, respectively, in the frontal cortex of aged controls. In 2016, Wey et al. assessed expression levels of HDAC1, 2, 3, and 6 in human brains diagnosed with no neuropathological abnormalities as part of a clinical study of an HDAC PET ligand [28]. The amounts of HDAC2 and 3 are significantly higher in the superior frontal gyrus relative to the corpus callosum. The expression levels of HDAC2, 3, and 6 are comparable in the superior frontal gyrus (0.12–0.16 pmol/mg total protein), whereas that of HDAC1 is obviously higher than others (1.7 pmol/mg total protein). In brains with neurodegenerative disease, Ding et al. reported that compared with aged controls, the expression of HDAC6 in the cerebral cortex and hippocampus of AD patients is increased by 52% and 91%, respectively [29]. In a study by Anderson et al. in 2015, the concentrations of HDAC1 + 2, 5, and 6 in the frontal cortex of AD patients were reported to be 0.7, 1.5, and 1.3 times those of aged controls [25]. Whitehouse et al. investigated the distribution and intensity of HDAC4, 5, and 6 in FTLD with IHC [30]. HDAC4 and 6 show higher immunoreactivity in the dentate gyrus in FTLD cases, especially in FTLD-tau Picks, compared with controls, and the difference in HDAC6 was more prominent. No changes were observed for HDAC5 between FTLD and controls.

3. HDAC Inhibitors In the last two decades, many types of inhibitors against zinc-dependent HDACs have been developed for treatment of cancer and neurodegenerative disease [21,31–33]. Typical small molecule HDAC inhibitors can be classified into four classes: hydroxamic acids, alkanoic acids, cyclic peptides, and ortho-aminoanilides [19]. Following the discovery of the class I and II HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA, also known as vorinostat) in 1996 [34], hydroxamic acid- based HDAC inhibitors compose the biggest compound library of the four classes. The chemical structure of HDAC inhibitors is generally composed of three motifs: a zinc-binding group that holds the zinc ion of HDAC, a cap group that interacts with the protein surface in the binding pocket, and a linker group that bridges the other two groups. For example, in SAHA, the zinc-binding, cap, and linker groups are N-phenylformamide, hydroxamic acid, and hexane groups, respectively (Figure 1). SAHA is the first US Food and Drug Administration (FDA)-approved HDAC inhibitor for use in Molecules 2018, 23, 300 4 of 27 patients with cutaneous T-cell lymphoma. The anticancer effects of SAHA are thought to be caused by modulation of both gene expression and acetylation of proteins that regulate cell proliferation [35]. tyrosineBesides SAHA, hydroxylase-positive other HDAC inhibitors neurons (e.g., in the panobino substantiastat nigra and arebelinostat) attenuated. have Inbeen R6/2 approved Huntington’s by the diseaseUS FDA model for the mice, treatment SAHA of treatment cutaneous improves T-cell lymp motorhoma, impairment peripheral in theT-cell mice lymphoma, as measured or multiple with the rotarodmyeloma performance [36,37]. test [41].

linker

zinc-binding cap

Figure 1. ChemicalChemical structure structure of of SAHA. SAHA. Three Three groups groups that that constitute constitute a typical a typical HDAC HDAC inhibitor inhibitor are areindicated. indicated.

AlthoughHDAC inhibitors HDAC inhibitorshave shown are expectedpromising to results be promising in preclinical therapeutic studies tools using for neurodegenerative animal models of diseasesneurodegenerative as described diseases above, [33,38]. they may Ricobaraza have adverse et al. reported events [that42]. sodium HDAC inhibitors4-phenylbutyrate have pleiotropic treatment effects on the different types of cells in the CNS because they often inhibit multiple HDACs that deacetylate a wide variety of substrates, including histones, transcription factors, and cytoplasmic proteins [32]. For this reason, isoform-speciﬁc HDAC inhibitors are likely to broaden the application of HDAC-targeted therapy [19]. In addition, the in vivo target engagement studies of HDAC inhibitors using HDAC PET imaging techniques described in this review will help estimate the desired and undesired CNS effects of the drugs.

4. Radioligands for HDACs Radiosynthesis and preclinical characteristics of reported HDAC radioligands are summarized in the following sections. In this review, the radioligands were classiﬁed into four groups according to their structural properties: SAHA-based, adamantane-conjugated hydroxamic acid-based, other carboxylic acid- and hydroxamic acid-based, and ortho-aminoanilide-based ligands. Radioligands intended to image HDACs not only in the brain but also in tumors are included.

4.1. SAHA-Based Ligands In 2006, radiosynthesis of the first radiolabeled ligand for imaging of HDAC expression and activity was reported [43]. Aiming to evaluate the HDAC expression and to estimate the effectiveness of SAHA, Mukhopadhyay et al. developed an 18F-labeled SAHA analogue, 6-([18F]fluoroacetamido)-1- hexanoicanilide ([18F]FAHA, [18F]1) (Table1), which has an 18F-fluoroacetamido group as a substitute for a hydroxamic acid group. Although the fluoroacetamido group appeared to be a substrate for HDACs because of its similarity to acetyllysine, no characterization of its binding affinity and selectivity for HDACs was reported in the paper. Radiosynthesis of [18F]1 was achieved by 18F-fluorination with a bromide precursor (Scheme1). Using [ 18F]tetrabutylammonium fluoride ([18F]TBAF), reactions at 80 ◦C in several solvents such as acetonitrile (MeCN), tetrahydrofuran (THF), and dimethylsulfoxide (DMSO) for 20 min gave decay-corrected radiochemical yields of only 1.0–3.8% with an average of 1.8%, although non-radioactive fluorination with TBAF gave the desired product in >50% yield. The authors presumed that the low radiochemical yield was caused by loss of the product during a high-performance liquid chromatography (HPLC) purification step due to poor solubility of the product in the aqueous HPLC eluent. Meanwhile, reactions at 115 ◦C in MeCN for 25 min using [18F]KF/Kryptofix 2.2.2 gave [18F]1 in a decay-corrected radiochemical yield of 11 ± 1.7% after HPLC purification. The radiochemical purity and the molar activity following this method were >99% and >74 GBq/µmol, respectively, at the end of synthesis. Nishii et al. performed in vivo characterization of [18F]1 in rats using PET [44,45]. The uptake of [18F]1 in the rat brain increased rapidly after intravenous (i.v.) injection and reached 0.44% injected dose (ID)/g at 5 min post-injection (p.i.). Brain uptake Molecules 2018, 23, 300 5 of 27 was significantly decreased (p < 0.01; t-test) by pre-treatment with SAHA [50 mg/kg; intraperitoneal (i.p.) administration] an hour before a PET scan [44]. Tumor uptake of [18F]1 was also assessed using human breast carcinoma xenografts in rats, and as seen in the rat brain, the uptake was inhibited by pre-treatment with SAHA [45]. In 2009, Reid et al. reported a further biological study to evaluate the utility of [18F]1 for PET imaging of HDAC activity [46]. They anticipated that HDAC may cleave [18F]1 and generate a radiometabolite, [18F]fluoroacetate ([18F]FACE) (Figure2). [ 18F]FACE Moleculescrosses the2018 blood-brain,, 23,, xx barrier (BBB) and is observed as a radiometabolite of PET tracers containing5 of 27 a 2-[18F]fluoroethyl group [47–50]. In vivo generation of [18F]FACE could cause high background radioactivity and complicate interpretation of PET data [48]. [48]. Consequently, Consequently, they attempted to assess the in vivovivo biodistributionbiodistribution and metabolism of [[1818F]F]11 asas well well as as the biodistributionbiodistribution of independently synthesized [ 18F]FACEF]FACE [51]. [51]. In In baboons, baboons, almost almost all all [ 18F]F]11 waswaswas rapidlyrapidly metabolizedmetabolized withinwithin 55 minmin in in plasma, and regional differences inin brainbrain radioactivityradioactivity uptakeuptake andand kineticskinetics werewere observed.observed. InIn contrast,contrast, [18F]FACEF]FACE showed showed gradual gradual and and uniform uniform uptake uptake in the in thesame same baboon baboon brain, brain, and the and radioactivity the radioactivity peak peakwas much was much smaller smaller than than that thatof [ of18F] [181..F] SevenSeven1. Seven minmin min afterafter after administrationadministration administration ofof of [[18 [F]18F]1 1ininin rats,rats, rats, allall all ofof of the the radioactivity in the brain homogenateshomogenates waswas inin thethe formform ofof [[1818F]FACE.F]FACE. Radioactivity accumulation in the bone suggested some in vivovivo defluorinationdefluorination in rats.rats.rats. Altogether, Altogether, they they concluded that the rapid metabolism and ability of the radiometabolite to penetrate the BBB complicated PET image analysis and that furtherfurther studiesstudies aboutabout thethe interactioninteraction betweenbetween [[1818F]11 andand HDACs HDACs were needed.needed. Four Four years after this study, anan internationalinternational researchresearch groupgroup performedperformed anan [[1818F]1 PETPET study study in in rhesus macaques combining computed computed tomography tomography (CT) (CT) and and magnetic magnetic resonance resonance imaging imaging (MRI) (MRI) to quantify to quantify the rate the of HDAC-mediatedrate of HDAC-mediated accumulation accumulation of [18F]1 inin of thethe [18 F] brainbrain1 in usingusing the brain anan optimizeoptimize using and optimizedpharmacokinetics pharmacokinetics model [26]. Themodel pharmacokinetics [26]. The pharmacokinetics model involved model two involved blood plasma two blood input plasma functions input for functions [18F]1 andand for [ [18F]FACEF]1 and and[18F]FACE three tissue and three compartments. tissue compartments. In their study, In high their substrate study, high specificity substrate of specificity 1 forfor classclass ofIIaIIa1 HDACsHDACsfor class waswas IIa demonstratedHDACs was demonstrated with an in withvitro an assay,in vitro whichassay, whichwas consistent was consistent with with a preceding a preceding study study using trifluoroacetyl-lysinetrifluoroacetyl-lysine [52]. [52]. In In addition addition to to the multimodal imaging, they also assessed expression of individual HDACs and and the deacetylation status status of of histones in in the brain with IHC, andand confirmedconfirmed their heterogeneity inin differentdifferent brainbrain structuresstructures and and cell cell types. types. Quantitative Quantitative analysis analysis of of PET/CT/MRI PET/CT/MRI showed highhigh accumulation accumulation of [of18 F][181F]in1 the inin nucleus thethe nucleusnucleus accumbens accumbensaccumbens and cerebellum andand cerebellumcerebellum in which highinin whichwhich expression highhigh expressionlevels of class levels IIa of HDACs class IIa such HDACs as HDAC such 4as and HDAC 5 are 4 observed. and 5 are Theseobserved. results These indicated results that indicated [18F]1 thataccumulation [18F]1 accumulationaccumulation in macaque inin brains macaquemacaque depends brainsbrains on dependsdepends class IIa HDAConon classclass expression IIaIIa HDACHDAC and expressionexpression activity, andandand consequently, activity,activity, andand consequently,[18F]1 PET could [18F] be1 PETPET used couldcould for pharmacodynamics bebe usedused forfor pharmacodynamicspharmacodynamics studies of class studiesstudies IIa HDAC ofof classclass inhibitors IIaIIa HDACHDAC in inhibitorsinhibitors the brain. inRecently, the brain. the Recently, utility of the [18 utilityF]1 for of PET [18F] imaging1 forfor PETPET of imagingimaging lung cancer ofof lunglung was cancercancer evaluated waswas evaluatedevaluated using small usingusing animal smallsmall animalPET/CT PET/CT and a mouse and a mouse model [model53]. HDACs [53]. HDACs are also are expected also expected to be promising to be promising therapeutic therapeutic targets intargets lung incancer lung[ 54cancer]. A/J [54]. mice A/J treated mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific a tobacco- precarcinogen,specific precarcinogen, were used were as lungused canceras lung model cancer mice model [55]. miceIn vivo [55].PET In imagingvivo PET showed imaging significant showed significant[18F]1 uptake [18F] in1 lunguptakeuptake tumors inin lunglung with tumorstumors a >2.0 withwith tumor/nontumor aa >2.0>2.0 tumor/nontumortumor/nontumor ratio, which ratio,ratio, was whichwhich slightly waswas higher slightlyslightly than higherhigher those thanof [18 thoseF]fluorodeoxyglucose of [18F]fluorodeoxyglucose and [18F]nifene, and [18 anF]nifene,α4β2 nicotinic an α4β2 receptor nicotinic radioligand receptor radioligand [56]. [56].

[18F]KF/K.2.2.2/

K2CO3 MeCN, 115 °C, 25 min [18F]1 RCY: 11-15%

SchemeScheme 1.1. RadiosynthesisRadiosynthesis ofof [[1818F]F]11...

Figure 2.2. ChemicalChemical structure ofof [[1818F]FACE.F]FACE.

Molecules 2018, 23, 300 6 of 27

Table 1. Physiochemical properties of radiolabeled SAHA-based HDAC ligands.

Compound MW Log D RCY Target IC50 for HDACs In Vivo Properties in Rodents Reference Brain uptake: 0.44 and 0.40% ID/g at 5 and 60 min, respectively [18F]1 265.32 1.39 11–15% Class IIa – [26,43,44,46] (rat) Brain uptake: SUVs were around 1 in various brain regions during [18F]2 283.31 – 25% Class IIa – the 60-min scan [57] (rat) Brain uptake: SUVs were around 1 in various brain regions during [18F]3 301.30 – 22% Class IIa – the 60-min scan [57] (rat) HDAC1: 56 nM Brain uptake: 1.0, 0.7, and 0.4% ID/g at 30, 60, and 120 min, respectively HDAC5: 67 nM Deﬂuorination: 9.6, 11.1, and 13.4% ID/g bone uptake at 30, 60, [18F]4 309.37 1.01 19% Non-selective [58] HDAC6: 3 nM and 120 min, respectively HDAC7: 85 nM (tumor-bearing mouse) HDAC1: 9.0 nM HDAC2: 13 nM Brain uptake: Very limited in biodistribution study [18F]5 281.32 – 40% HDAC1–3/6 [59] HDAC3: 24 nM (mouse/tumor-bearing mouse) HDAC6: 50 nM [11C]6 278.34 0.5 23% – – – [60] [11C]7 247.33 1.7 1 48% – – – [60] 1 Clog P. MW: molecular weight; RCY: radiochemical yield (decay corrected); SUV: standardized uptake value; ID: injected dose. Molecules 2018, 23, 300 7 of 27

In 2015, two novel 18F-labeled successors to [18F]1 were reported [57]. Bonimi et al. developed 6- Molecules 2018, 23, x 7 of 27 (difluoroacetamido)-1-hexanoicanilide (DFAHA, [18F]2) and 6-(trifluoroacetamido)-1-hexanoicanilide 18 (TFAHA,In [ 2015,F]3) withtwo novel improved 18F-labeled selectivity successors and to substrate [18F]1 were efficiency reported [57]. for class Bonimi IIa et HDACs, al. developed which 6- have important(difluoroacetamido)-1-hexan roles in the brain developmentoicanilide (DFAHA, and function [18F]2) and (Table 6-(trifluoroacetamido)-1-hexanoicanilide1)[ 61]. These ligands were rationally designed(TFAHA, in accordance [18F]3) with withimproved previous selectivity reports and describing substrate ef theficiency substrate for class specificity IIa HDACs, of HDACswhich have[52 ,62]. Non-radiolabeledimportant roles2 inand the brain3 were development synthesized and function from 6-amino-1-hexanoicanilide (Table 1) [61]. These ligands were by reactions rationally with correspondingdesigned in di/trifluoroaceticaccordance with previous anhydride. reports Radiosynthesisdescribing the substrate of [18F] specif2 andicity [18 ofF] HDACs3 was achieved[52,62]. in a similarNon-radiolabeled manner as that 2 and of [183 F]were1 (Scheme synthesized2). For from [ 18 F]6-amino-1-hexanoicanilide2 radiosynthesis, a bromofluoro by reactions precursor with in corresponding di/trifluoroacetic anhydride. Radiosynthesis of [18F]2 and [18F]3 was achieved in a MeCN was added to dried [18F]KF/K.2.2.2 and then stirred at 100 ◦C for 20 min. HPLC purification similar manner as that of [18F]1 (Scheme 2). For [18F]2 radiosynthesis, a bromofluoro precursor in gave the desired product with a decay-corrected radiochemical yield of 25%, >95% radiochemical MeCN was added to dried [18F]KF/K.2.2.2 and then stirred at 100 °C for 20 min. HPLC purification 18 purity,gave and the molar desired activity product of 60–70with a GBq/decay-correctedµmol. For radiochemical [ F]3 radiosynthesis, yield of 25%, the >95% same radiochemical procedure except 18 ◦ F-fluorinationpurity, and withmolar additional activity of heating60–70 GBq/ at 110μmol.C For for [ 2518F] min3 radiosynthesis, was performed the tosame give procedure the desired except product with a18F-fluorination decay-corrected with radiochemical additional heating yield at of 110 22%, °C >95%for 25 radiochemicalmin was performed purity, to andgive molar the desired activity of 70–80product GBq/µ withmol. aIn decay-corrected vitro comparison radiochemical of substrate yiel affinityd of 22%, of 1>95%, 2, and radiochemical3 demonstrated purity, that and substitution molar of a fluorineactivity of atom 70–80 for GBq/ theμ hydrogenmol. In vitro atom comparison increased of k substratecat values affinity for class of 1 IIa, 2, HDACsand 3 demonstrated and decreased that vmax valuessubstitution for class Iof HDAC8. a fluorine In atom dynamic for the PET/CT hydrogen imaging, atom increased high accumulation kcat values for class of [18 IIaF] 2HDACsand [18 andF]3 was 18 observeddecreased in rat v brainmax values regions for class including I HDAC8. the cerebellum,In dynamic PET/CT nucleus imaging, accumbens, high andaccumulation hippocampus, of [ F] where2 and [18F]3 was observed in rat brain regions including the cerebellum, nucleus accumbens, and class IIa HDACs are highly expressed. The brain radioactivity of [18F]3 was higher than that of [18F]2, hippocampus, where class IIa HDACs are highly expressed. The brain radioactivity of [18F]3 was and it was significantly decreased by pre-treatment with SAHA (100 mg/kg; i.p. 30 min before [18F]3 higher than that of [18F]2, and it was significantly decreased by pre-treatment with SAHA (100 mg/kg; 18 i.v.). Thesei.p. 30 resultsmin before suggested [18F]3 i.v.). that These [ F] results3 is a moresuggested suitable that substrate-based[18F]3 is a more suitable radioligand substrate-based to image the expressionradioligand and activityto image of the class expression IIa HDACs and activity in the of brain. class IIa HDACs in the brain.

[18F]KF/K.2.2.2 MeCN, 100 °C, 25 min [18F]2 RCY: 25%

[18F]KF/K.2.2.2 MeCN, 100 °C, 25 min [18F]3 RCY: 22%

SchemeScheme 2. 2.Radiosynthesis Radiosynthesis of [ 18F]F]22 andand [18 [F]183F]. 3.

In 2011, Zeglis et al. reported the synthesis and evaluation of a hydroxamic acid-based HDAC In 2011, Zeglis et al. reported the synthesis and evaluation of a hydroxamic acid-based HDAC radioligand, N1-(4-(2-fluoroethyl)phenyl)-N8-hydroxyoctanediamide (FESAHA, [18F]4) (Table 1) [58]. N1 N8 18 radioligand,Unlike [18F]1-(4-(2-fluoroethyl)phenyl)- that could be an HDAC substrate,-hydroxyoctanediamide [18F]4 was expected to (FESAHA,image the HDAC [ F]4 expression) (Table1)[ 58]. 18 18 Unlikeitself. [ F] [181F]that4 was could radiosynthesized be an HDAC from substrate, a tosylate [ F] 4precwasursor expected with a to one-pot, image thetwo-step HDAC reaction expression 18 itself.(Scheme [ F]4 3).was The radiosynthesized precursor was 18F-fluorinated from a tosylate by a reaction precursor with with[18F]TBAF a one-pot, in MeCN two-step at 90 °C reactionfor ◦ (Scheme20 min3). to The obtain precursor an 18F-intermediate, was 18F-fluorinated and then the by solvent a reaction was removed with [ 18byF]TBAF heating at in 90 MeCN °C under at a 90 C for 20slow min stream to obtain of argon. an 18F-intermediate, After the reaction and vial then wa thes cooled solvent to was0 °C, removeda solution by ofheating hydroxylamine at 90 ◦C in under a slowmethanol stream was of argon. added and After reacted the reaction for 30 min vial to wasconvert cooled the ethylester to 0 ◦C, group a solution into a of hydroxamic hydroxylamine acid in methanolgroup. was HPLC added purification and reacted and fo forrmulation 30 min togave convert the desired the ethylester product in group 19 ± into9% decay-corrected a hydroxamic acid group.radiochemical HPLC purification yield and and>99% formulation radiochemical gave purity. the The desired molar product activity inwas 19 about± 9% 2.3 decay-corrected GBq/μmol, which was relatively low for an 18F-labeled ligand. To 18F-radiolabel SAHA, they positioned a radiochemical yield and >99% radiochemical purity. The molar activity was about 2.3 GBq/µmol, fluoroethyl group at the para position of the aniline ring. In silico modeling suggested that the 18 18 whichmodified was relatively structure would low for bind an to theF-labeled active site ligand. of an HDAC-like To F-radiolabel protein [63].SAHA, Furthermore, they in positioned vitro a fluoroethylHDAC inhibition group atassays the paraconfirmed position its submicromolar of the aniline IC ring.50 values In silico ranging modeling from 3 nM suggested (HDAC6) that to the modified474 nM structure (HDAC9) would except bind 1.8 μ toM the for activeHDAC4. site Cell of anproliferation HDAC-like studies protein revealed [63]. identical Furthermore, cytostaticin vitro HDACeffects inhibition for 4 and assays SAHA confirmed against the its prostate submicromolar cancer cell lines, IC50 valuesLNCaP rangingand PC-3, from both 3of nM which (HDAC6) have to 474 nMincreased (HDAC9) HDAC except expression 1.8 µM [35,64]. for HDAC4. A biodistribution Cell proliferation study of [18F] studies4 was performed revealed identicalin mice bearing cytostatic effects for 4 and SAHA against the prostate cancer cell lines, LNCaP and PC-3, both of which have Molecules 2018, 23, 300 8 of 27

18 increasedMolecules HDAC 2018, 23 expression, x [35,64]. A biodistribution study of [ F]4 was performed in mice8 of bearing 27 LNCaP xenografts. Tumor uptake was 2.8 ± 0.3% ID/g at 30 min p.i. and decreased with time. BrainLNCaP uptake xenografts. was 1.0% ID/gTumor or uptake less over was the 2.8 course ± 0.3% ofID/g 120 at min 30 min p.i. Furthermore,p.i. and decreased significant with time. radioactivity Brain uptake was 1.0% ID/g or less over the course of 120 min p.i. Furthermore, significant radioactivity accumulationMolecules 2018 was, 23, x observed in the bone (9.6 ± 2.2% ID/g at 30 min p.i.), suggesting substantial8 of 27 accumulation was observed in the bone (9.6 ± 2.2% ID/g at 30 min p.i.), suggesting substantial in vivo in vivo defluorination of [18F]4, which was confirmed by small animal PET imaging. Altogether, LNCaPdefluorination xenografts. of [ 18TumorF]4, which uptake was was confirmed 2.8 ± 0.3% ID/gby small at 30 minanimal p.i. andPET decreasedimaging. withAltogether, time. Brain the 18 the radiolabelingradiolabelinguptake was 1.0% position position ID/g ofor of [ 18lessF] [ 4 F] overhas4 haslittle the little courseeffect effect on of its 120 onHDAC min its HDACp.i. inhibition Furthermore, inhibition properties, significant properties, but as aradioactivity PET but ligand, as a PET ligand,structuralaccumulation structural optimization optimizationwas observed is needed in is the needed due bone to (9.6 dueits metabolic± to2.2% its ID/g metabolic instability. at 30 min instability. p.i.), suggesting substantial in vivo defluorination of [18F]4, which was confirmed by small animal PET imaging. Altogether, the 18 radiolabeling position of [ F]4 has little effect on[18F]TBAF its HDAC inhibition properties, but as a PET ligand, structural optimization is needed due to its metabolicMeCN, instability. 90 °C, 20 min [18F]TBAF MeCN, 90 °C, NH2OH 20 min MeOH, 0 °C, 30 min [18F]4 NH2OH RCY: 19% MeOH, 0 °C, Scheme 3. Radiosynthesis of [1818F]4. 30Scheme min 3. Radiosynthesis of [ F]4. [18F]4 The same year, Hendricks et al. reported a close analogueRCY: 19% of SAHA, N-hydroxy-N’-(4-fluoro- 0 Thephenyl)octanediamide same year, Hendricks (p-fluoro et al.SAHA,Scheme reported [3.18 RadiosynthesisF]5 a), closefor characterization analogue of [18F]4. of SAHA, of SAHAN-hydroxy- as a cancerN -(4-fluoro-drug phenyl)octanediamide(Table 1) [59]. Non-radiolabeled (p-fluoro SAHA, p-fluoro [SAHA18F]5), was for synthesized characterization from 4-fluoroaniline of SAHA as by a following cancerdrug (Tablereported1)[The59]. strategiessame Non-radiolabeled year, [65]. Hendricks For radiosynthesisp -fluoroet al. reported SAHA of [18 F]a was5close, 1,4-dinitrobenzene synthesized analogue of fromSAHA, was 4-fluoroaniline usedN-hydroxy- as a startingN’-(4-fluoro- by reagent following reportedphenyl)octanediamide(Scheme strategies 4). The [ 65first]. Forintermediate, (p radiosynthesis-fluoro SAHA, [18F]1-fluoro-4-nitrobenzene, of[18F] [185F]), 5for, 1,4-dinitrobenzene characterization was obtained of was SAHA usedby microwaveas as a acancer starting heating drug reagent 18 (Scheme(Tableof dinitrobenzene4). 1) The [59]. first Non-radiolabeled intermediate, and [ F]KF/K [p218CO-fluoroF]3/K.2.2.21-fluoro-4-nitrobenzene, SAHA in wasDMSO synthesized at 120 °C wasfrom for obtained 54-fluoroaniline min. A bynitro microwave groupby following of the heating 18 reportedintermediate strategies was reduced [65].18 For by radiosynthesis NaBH4 and Pd/C of [ toF] 5obtain, 1,4-dinitrobenzene [18F]4-fluoroaniline,◦ was used and asthen a starting it was isolatedreagent of dinitrobenzene and [ F]KF/K2CO183/K.2.2.2 in DMSO at 120 C for 5 min. A nitro group of the (Schemeby solid 4).phase The extraction first intermediate, (SPE). Methyl [ F]1-fluoro-4-nitrobenzene, 8-chloro-8-oxooctanoate18 was was obtained added, by and microwave the mixture heating was intermediate was reduced by NaBH4 and Pd/C to obtain [ F]4-fluoroaniline, and then it was isolated ofstirred dinitrobenzene at room temperature and [18F]KF/K for 5 2min.CO3/K.2.2.2 Finally, in 50% DMSO hydroxylamine at 120 °C forand 5 1 min.M NaOH A nitro in methanol group of wasthe by solid phase extraction (SPE). Methyl 8-chloro-8-oxooctanoate18 was added, and the mixture was intermediateadded and reacted was reduced for 3 min by to NaBH convert4 and the Pd/C methyl to obtain ester group[ F]4-fluoroaniline, into a hydroxamic and then acid it group. was isolated HPLC stirredbypurification atsolid room phase temperaturegave extraction the desired (SPE). for product 5 Methyl min. Finally,with 8-chlo 39.5ro-8-oxooctanoate 50% ± 6.0% hydroxylamine decay-corrected was added, and radiochemical 1and M NaOHthe mixture yield in methanol wasand was addedstirred97.0 ± 4.7% at and room radiochemical reacted temperature for 3purity. min for 5 to 5min. has convert Finally,identical the 50% inhibitory methyl hydroxylamineester effects group as andSAHA into1 M against NaOH a hydroxamic HDAC1, in methanol 2, acid 3, wasand group. HPLCadded6. purificationThey and determined reacted gave for thethe 3 tissue min desired to distribution convert product the of methyl with[18F]5 39.5inester mice. ±group6.0% After into decay-correctedi.v. a injectionhydroxamic of the acid radiochemical radioligand, group. HPLC the yield and 97.0highestpurification± 4.7%radioactivity gave radiochemical the wasdesired observed purity.product in 5 thewithhas kidney identical39.5 ,± liver, 6.0% inhibitory anddecay-corrected blood, effects whereas radiochemical as brain SAHA uptake against yield was HDAC1, andthe 2, 3,lowest.97.0 and ± 6. 4.7% Bone They radiochemical uptake determined of radioactivity purity. the 5 tissue has wasidentical distribution not noticeable.inhibitory of effects [18TheyF]5 asnextin SAHA mice. assessed against After its HDAC1, i.v.accumulation injection 2, 3, and in of the 18 radioligand,tumors6. They determinedusing the highest a mouse the radioactivity tissuewith a distribution human was ovarian of observed [ F]cancer5 in mice. in xenograft the After kidney, i.v.and injection liver,small animal and of the blood, PET/CT.radioligand, whereas Tumor the brain highestaccumulation radioactivity of [18F] was5 was observed observed in inthe a kidney time-dependent, liver, and manner, blood, whereas and it decreasedbrain uptake by was23% theon uptake was the lowest. Bone uptake of radioactivity was not noticeable. They next assessed its lowest.pre-treatment Bone uptakewith SAHA. of radioactivity was not noticeable. They next assessed its accumulation in accumulationtumors using intumors a mouse using with a human mouse ovarian with a cancer human xenograft ovarian and cancer small xenograft animal PET/CT. and small Tumor animal 18 PET/CT. Tumor accumulation18 18 of [ F]5 was observed in a time-dependent manner,Methyl and it decreased accumulation of [ F]5[ wasF]KF/K observed2CO3/ in a time-dependentNaBH4, manner, and it decreased by 23% on by 23%pre-treatment on pre-treatment with SAHA. withK.2.2.2, SAHA. 10% Pd/C 8-chloro- 8-oxooctanoate DMSO, r.t., microwave, 10 min r.t., 18 Methyl [ F]KF/K120 °C,2CO3/ NaBH4, 5min K.2.2.2, 10% Pd/C 8-chloro- 5min 8-oxooctanoate DMSO, r.t., microwave, O 10 min r.t., H NH OH N 120 °C, 2 5min O 5min 1 M NaOH O r.t., 18F O 3 min H NH OH N 2 [18F]5 O 1 M NaOH RCY: 40% O r.t., 18F 3 min Scheme 4. Radiosynthesis of [18F]5. [18F]5 RCY: 40%

SchemeScheme 4. 4.Radiosynthesis Radiosynthesis of of [18 [18F]F]5. 5. Molecules 2018, 23, 300 9 of 27

In 2013, Seo et al. reported radiosynthesis and biological evaluation of two 11C-labeled SAHA-basedMolecules 2018 ligands, 23, x ([11C]6 and [11C]7) for HDAC imaging in the brain (Table1)[ 60]. [119 ofC] 276 was synthesized from a hydroxyl precursor by a two-step reaction of 11C-methylation and conversion of In 2013, Seo et al. reported radiosynthesis and biological evaluation of two 11C-labeled SAHA-11 a methylester group into a hydroxamic acid group (Scheme5). A mixture of the precursor, [ C]CH3I, based ligands ([11C]6 and [11C]7) for HDAC imaging in the brain (Table 1) [60]. [11C]6 was synthesized and sodium hydride in DMSO was heated to 70 ◦C for 5 min, and then hydroxylamine hydrochloride from a hydroxyl precursor by a two-step reaction of 11C-methylation and conversion of a methylester was added. The authors used potassium cyanide as a base in the second step instead of sodium group into a hydroxamic acid group (Scheme 5). A mixture of the precursor, [11C]CH3I, and sodium hydroxidehydride because in DMSO of was the heated slow reaction to 70 °C for rate 5 min, and by-productand then hydroxylamine formation [hydrochloride66]. HPLC purification was added. gave 11 the desiredThe authors product used in potassium 23 ± 3% decay-correctedcyanide as a base radiochemical in the second yieldstep instead (calculated of sodium from [hydroxideC]CH3I) and >99%because radiochemical of the slow purity. reaction The rate molar and activityby-product was formation 10.4 ± 1.9 [66]. GBq/ HPLCµmol purification at the time gave of the use. desired Synthesis 11 11 11 of [ productC]7, an in analogue 23 ± 3% ofdecay-corrected1, was achieved radiochemical using [2- yieldC]CH (calculated3COCl [67 from] (Scheme [ C]CH5).3I) Distillationand >99% of 11 radiochemical purity. The molar activity was 10.4 ± 1.9 GBq/μmol at the time of use. Synthesis of [2- C]CH3COCl into a mixture of the precursor and trimethylamine in THF resulted in the formation 11 11 of [11[C]C]7.7, HPLCan analogue purification of 1, was gave achieved the desired using product [2- C]CH in3COCl 48 ± 7%[67] decay-corrected(Scheme 5). Distillation radiochemical of [2-11C]CH3COCl into a mixture of the precursor and trimethylamine in THF resulted in the formation yield and >99% radiochemical purity. The molar activity was 6.0 ± 1.4 GBq/µmol at the time of of [11C]7. HPLC purification gave the desired product in 48 ± 7% decay-corrected radiochemical yield use. Using PET, organ uptake and clearance of [11C]6 and [11C]7 in baboons were assessed over and >99% radiochemical purity. The molar activity was 6.0 ± 1.4 GBq/μmol at the time of use. Using a 90-minPET, periodorgan uptake after i.v. and injection. clearance Arterialof [11C]6 bloodand [11C] plasma7 in baboons was also were collected, assessed andover thea 90-min fraction period of intact 11 radioligandafter i.v. wasinjection. measured Arterial with blood HPLC plasma at was different also collected, time points and the during fraction the of PETintact scan. radioligand [ C]6 wasshowed poormeasured brain uptake with HPLC in baboons, at different and time the points distribution during the pattern PET scan. was [11 homogeneousC]6 showed poor in brain different uptake brain regions.in baboons, Furthermore, and the no distribution difference pattern was observed was homo ingeneous time-activity in different curves brai inn peripheral regions. Furthermore, organs between the presenceno difference and was absence observed of SAHAin time-activity pre-treatment, curves in suggesting peripheral organs that no between specific the binding presence ofand [11 C]6 occursabsence in these of SAHA organs. pre-treatment, Contrary sugges to a predictionting that no by specific the authors, binding of brain [11C]6 uptake occurs in of these [11C] organs.7 was also 11 poorContrary even though to a prediction the chemical by the structureauthors, brain of [ 11uptakeC]7 closely of [ C]7 resembles was also poor that even of [though18F]1. the They chemical confirmed structure of [11C]7 closely resembles that of [18F]1. They confirmed that the unchanged [11C]7 fraction that the unchanged [11C]7 fraction in plasma at 10 min was higher than [18F]1 (10% versus 1%), in plasma at 10 min was higher than [18F]1 (10% versus 1%), and therefore, the difference was not due and therefore, the difference was not due to a metabolic difference. Thus, substitution of a hydrogen to a metabolic difference. Thus, substitution of a hydrogen atom for a fluorine atom affected brain atomuptake for a fluorine and substrate atom affectedspecificity brain for HDACs uptake more and substratethan expected. specificity In summary, for HDACs these radioligands more than expected. are In summary,not suitable these for radioligandsimaging HDACs are in not the suitable brain. for imaging HDACs in the brain.

11 i) [ C]CH3I, NaH, DMF, 70 °C, 5 min

ii) NH2OH·HCl, NaOH, KCN, MeOH/THF/water [11C]6 (1:1:0.4) RCY: 23%

11 [2- C]CH3COCl trimethylamine, THF [11C]7 RCY: 48%

SchemeScheme 5. 5.Radiosynthesis Radiosynthesis of [ 1111C]C]6 6andand [11 [C]117C]. 7.

4.2. Adamantane-Conjugated4.2. Adamantane-Conjugated Ligands Ligands In 2014, the first adamantane-conjugated HDAC imaging radioligand was reported by Hooker et al. In 2014, the first adamantane-conjugated HDAC imaging radioligand was reported by at Massachusetts General Hospital [68]. The adamantane “lipophilic bullet” is often conjugated to Hookerdrugs et to al. improve at Massachusetts their brain penetrance General [69–71], Hospital and [68adamantane-based]. The adamantane hydroxamates “lipophilic are highly bullet” is oftenpotent conjugated HDAC inhibitors to drugs [72] to. improveBased on prior their reports, brain penetranceWang et al. synthesized [69–71], and (E)-3-(4-((((3r,5r,7r)- adamantane-based hydroxamatesadamantan-1-ylmethyl)([ are highly potent11C]methyl)amino)methyl)phenyl)- HDAC inhibitors [72]. BasedN on-hydroxyacrylamide prior reports, Wang ([11C]Martinostat, et al. synthesized (E)-3-(4-((((3r,5r,7r)-adamantan-1-ylmethyl)([[11C]8) (Scheme 6) as a radioligand for quantification11C]methyl)amino)methyl)phenyl)- of the density of HDACs in theN-hydroxyacrylamide CNS and major ([11C]Martinostat,peripheral organs [11 C](Table8) (Scheme 2). The6 )radiosynthesis as a radioligand of [11 forC]8 quantificationwas achieved by of 11 theC-methylation density of HDACsof a in thedesmethylated CNS and major precursor. peripheral [11C]CH organs3I was trapped (Table2 ).in Thea solution radiosynthesis of the precursor of [ 11 inC] DMSO,8 was achievedand the by 11 solution was heated to 110 °C for 4 min. HPLC11 purification gave the desired product in 3–5% non- C-methylation of a desmethylated precursor. [ C]CH3I was trapped in a solution of the precursor in decay-corrected radiochemical yield (calculated from trapped [11C]CH3I) and ≥95% radiochemical DMSO, and the solution was heated to 110 ◦C for 4 min. HPLC purification gave the desired product purity with a molar activity of 37 ± 7.4 GBq/μmol. The log D value of [11C]8 was 2.03. In vitro in 3–5% non-decay-corrected radiochemical yield (calculated from trapped [11C]CH I) and ≥95% inhibitory activities of 8 against HDACs were measured using recombinant human enzymes.3 Low radiochemical purity with a molar activity of 37 ± 7.4 GBq/µmol. The log D value of [11C]8 was Molecules 2018, 23, 300 10 of 27

2.03. In vitro inhibitory activities of 8 against HDACs were measured using recombinant human enzymes. Low IC50 values were observed for HDAC1 (0.3 nM), 2 (2.0 nM), 3 (0.6 nM), and 6 (4.1 nM), moderate values were observed for HDAC4 (1970 nM) and 5 (352 nM), and high values were observed for HDAC7 (>20,000), 8 (>15,000), and 9 (>15,000). An in vitro radioligand displacement assay with four zinc-dependent enzymes and 80 additional non-HDAC and Zn-dependent CNS targets demonstrated that the dopamine transporter was a potential off-target binding site of [11C]8; however, binding of 2-β-carbomethoxy-3β-(4-fluorophenyl)-[N-11C-methyl]tropane, a dopamine transporter radioligand [73], in rat brains was not inhibited by 8. In vitro autoradiography (ARG) confirmed specific binding of [11C]8 in rat brain sections, and the binding was completely inhibited in the presence of excess SAHA. The authors assessed brain uptake of [11C]8 with PET imaging using both rats and baboons. In rat brains, radioactivity accumulation was observed after i.v. injection of [11C]8, and accumulation decreased following pre-treatment of unlabeled 8 in a dose-dependent manner. Furthermore, brain uptake of [11C]8 was not altered by pre-treatment with the P-glycoprotein (P-gp) inhibitor cyclosporine A (25 mg/kg, 30 min before administration of radioligand), suggesting that [11C]8 is not a substrate of P-gp. Metabolism of [11C]8 in rat brains was not noticeable. To analyze baboon PET data for [11C]8, a two-tissue compartmental model was used with a metabolite-corrected plasma time-activity curve. Plasma radioactivity was rapidly cleared after i.v. injection of [11C]8, although about 40% unchanged fraction still remained in plasma 30 min post-injection. As in rats, high brain uptake of [11C]8 was observed in baboons, and uptake decreased following pre-treatment with unlabeled 8 (Figure3). Following these promising results, the same group reported in vivo target engagement studies of a subset of HDAC inhibitors (5 hydroxamates, 4 ortho-aminoanilides, and 1 short-chain fatty acid) using [11C]8 PET imaging in the rat brain [74]. Small molecule HDAC inhibitors modulate CNSdisease-related behaviors in rodents [13,75]; however, direct evidence of target engagement in the brain has not been demonstrated. In this study, they found that i.v. pre-treatment with hydroxamates containing heterocyclic capping groups [76] 3–10 min before [11C]8 administration resulted in 20–40% blockage of [11C]8 binding in the brain. Meanwhile, i.p. pre-treatment with ortho-aminoanilides [75,77] resulted in limited blockage of [11C]8 binding. To test whether adamantane could improve brain penetrance of ortho-aminoanilides like hydroxamates, they synthesized an adamantane-conjugated ortho-aminoanilide named CN147. This novel compound showed 25% blockage of [11C]8 binding, suggesting that it enhanced HDAC engagement in the brain. Furthermore, an antidepressant-like effect of CN147 was confirmed with a rat behavioral test. These results suggested the utility of [11C]8 PET as a tool for investigating in vivo target engagement of HDAC inhibitors. Kinetic analysis with arterial plasma sampling of [11C]8 in different brain regions of the baboon was also reported [78]. Besides [11C]8, Strebl et al., from the same group at Massachusetts General Hospital, developed 18F-labeled adamantane- or cyclohexane-conjugated ligands in 2016 [79]. The three novel compounds have a similar structure to [11C]8 and a fluorine atom bound directly to a benzene ring ([18F]9–11) (Scheme7, Table2). [ 18F]9 and [18F]10 were synthesized using the same precursor, 3-(4-formyl-2-nitrostyryl)-5,5-dimethyl-1,4,2-dioxazole. A mixture of the precursor, Cs18F, and DMSO was heated to 110 ◦C for 5 min followed by transfer into another vial containing the corresponding amine and stirring at room temperature for 5 min. Then a saturated solution of sodium borohydride in isopropanol was added. After stirring at 60 ◦C for 10 min, the mixture was purified by semi-preparative HPLC. The isolated intermediate was passed through a C18 SPE cartridge and eluted with isopropanol. Camphor sulfonic acid was added, and the mixture was heated to 110 ◦C for 30 min. HPLC purification gave the desired products in 7% and 0.9% decay-corrected radiochemical yield for [18F]9 and [18F]10, respectively. [18F]11 was synthesized using methyl 4-formyl-3-nitrocinnamate as the precursor. 18 ◦ A mixture of the precursor, [ F]KF/K2CO3/K.2.2.2, and DMSO was heated to 110 C for 5 min. After SPE with a C18 cartridge, (1-adamantylmethyl)-N-methylamine was introduced. The mixture was reacted with NaB(CN)H3 and then purified with HPLC. Finally, a methyl ester group was converted to a hydroxamic acid group using hydroxylamine and sodium hydroxide. Molecules 2018, 23, 300 11 of 27

Table 2. Physiochemical properties of radiolabeled adamantane-conjugated HDAC ligands.

Compound MW Log D RCY Target IC50 for HDACs In Vivo Properties in Rodents Reference HDAC1: 0.3 nM Brain uptake: Around 0.5% ID/cc during the 60-min scan HDAC2: 2.0 nM [11C]8 353.49 2.03 3–5% 2 Class I/IIb Metabolism: Radiometabolites in the brain were limited [28,68,74,78] HDAC3: 0.6 nM (rat) HDAC6: 4.1 nM HDAC1: 1.6 nM Brain uptake: Rapid brain uptake and limited washout were observed HDAC2: 14 nM [18F]9 305.38 3.07 1 7% Class I/IIb Specific binding in the brain was confirmed in a blocking study [79] HDAC3: 0.5 nM (rat) HDAC6: 12 nM HDAC1: 0.8 nM Brain uptake: Rapid brain uptake and limited washout were observed HDAC2: 6.4 nM [18F]10 357.46 3.77 1 0.9% Class I/IIb Specific binding in the brain was confirmed in a blocking study [79] HDAC3: 0.5 nM (rat) HDAC6: 9.5 nM HDAC1: 0.8 nM Brain uptake: Rapid brain uptake and limited washout were observed HDAC2: 7.0 nM [18F]11 371.49 4.17 1 0.3% Class I/IIb Specific binding in the brain was confirmed in a blocking study [79] HDAC3: 0.8 nM (rat) HDAC6: 12 nM Brain uptake: Rapid brain uptake and limited washout were observed HDAC6: 60 nM [18F]12 345.45 – 8.1% 2 HDAC6 Specific binding in the brain was confirmed in a blocking study [80] Others: ≥1 µM (rat) 1 Clog P. 2 Non-decay corrected. MW: molecular weight; RCY: radiochemical yield (decay corrected). Molecules 2018, 23, 300 12 of 27 Molecules 2018, 23, x 12 of 27

11 [11C]CH3I [ C]CH3I DMSO, 110 °C, 4 min 11 4 min [11C]8 RCY: 3-5%*

1111 SchemeScheme 6.6. RadiosynthesisRadiosynthesis ofof [[11C]8.. * * Non-decay Non-decay corrected. corrected.

11 Figure 3. The total volume of distribution (VT) images from [11C]8 PET in baboon brains. Robust brain Figure 3. The total volume of distribution (VT) images from [11C]8 PET in baboon brains. Robust brain Figure 3. The total11 volume of distribution (VT) images from [ C]8 PET in baboon brains. Robust brain uptake (top) of [11C]8 was decreased by pre-treatment with unlabeled 8 (bottom). (Reproduced from uptake (top) of [11C]8 was decreased by pre-treatment with unlabeled 8 (bottom). (Reproduced from Wang et al. J Med Chem; published by The American Chemical Society, 2014 [68]). Wang etet al.al. JJ MedMed Chem;Chem; publishedpublished byby TheThe AmericanAmerican ChemicalChemical Society,Society, 20142014 [68[68]).]).

HPLC purification gave the desired product in 0.3% decay-corrected radiochemical yield. All HPLC purification gave the desired product in 0.3% decay-corrected50 radiochemical yield. All three three compounds showed high affinity for HDAC1, 2, 3, and 6 (IC50 values: <20 nM). Brain kinetics of compoundsthe radioligands showed was highassessed affinity with for PET HDAC1, imaging 2, in 3, ra andts and 6 (IC baboons.50 values: In <20 rats, nM). all radioligands Brain kinetics showed of the radioligandsrapid brain uptake was assessed after injection, with PET followed imaging by ina gradual rats and decrease baboons. in In radioactivity rats, all radioligands over the 2-h showed scan. rapidBy pre-treatment brain uptake with after unlabeled injection, 8, followed washout byrates a gradual of the radioligands decrease in radioactivityfrom the brain over were the increased, 2-h scan. Byand pre-treatment brain radioactivity with unlabeled decreased8, washoutto about rates50% of thepeak radioligands within 2 h. from Meanwhile, the brain PET were imaging increased, in andbaboons brain revealed radioactivity differences decreased between to the about radioligands 50% of peak in brain within kinetics. 2 h.Meanwhile, A brain standardized PET imaging uptake in 18 baboonsvalue (SUV) revealed from differences30 to 60 min between of [18F] the9, the radioligands cyclohexyl in compound, brain kinetics. was A only brain 0.57, standardized whereas those uptake of 18 18 18 9 value[18F]10 (SUV)and [18F] from11 reached 30 to 60 1.22 min and of 1.80, [ F] respectively,, the cyclohexyl suggeste compound,d that the wasadamantane only 0.57, group whereas enhanced those the of 18 18 18 11 [BBBF] 10permeability.and [ F]11 Onreached the other 1.22 andhand, 1.80, the respectively,brain uptake suggested of [18F]11 thatwas theslightly adamantane lower than group that enhanced of [11C]8 18 1811 thealthough BBB permeability. they are close Onanalogues. the other Furthermore, hand, the brainthe regional uptake SUV of [ ofF] 18F-labeledwas slightly ligands lower in baboon than thatbrains of 11 11 18 [wereC]8 comparedalthough to they that are of close[11C]8 analogues.. Furthermore, the regional SUV of F-labeled ligands in baboon brains were compared to that of [11C]8. 11 i) [18F]CsF, Although all threei) radioligands[18F]CsF, showed significant correlation with [ C]8, correlation between DMSO, i) HPLC 18 11 DMSO, 18 i) HPLC 11 [ F]11 and [ C]8 was110 the °C, highest.5 min Taken together, [ F]11ii) exhibitedSPE comparable properties to [ C]8, 110 °C, 5 min ii) SPE ii) RCH NH , iii) Camphor such as HDAC bindingii) RCH and2NH brain2, uptake, although furtheriii) Camphor optimization of radiosynthesis is needed. r.t., 5 min2 2 sulfonic acid, Very recently, Streblr.t., 5 etmin al. reported synthesis andsulfonic evaluation acid, of an 18F-labeled hydroxamic iii) NaBH4, iPrOH, iii) NaBH4, iPrOH, [18F]9: R = cyclohexane iPrOH,18 18 110 °C, 30 min [18F]9: R = cyclohexane acid-based radioligand,iPrOH, [ F]Bavarostat ([ F]12, Scheme1108),°C, with 30 min selectivityRCY: for 7% HDAC6 (Table2) [ 80]. 60 °C, 10 min RCY: 7% 60 °C, 10 min [18F]10: R = adamantane HDAC6 is receiving increased attention as a therapeutic target for the neurodegenerative[18F]10: R = adamantane diseases [81]. RCY: 0.9% [18F]12 has a similar structure as [18F]11 but without a vinyl group in theRCY: linker.0.9% Close approach i) [18F]KF, i) [18F]KF, K.2.2.2, DMSO, i) HPLC of a bulky moiety toK.2.2.2, the DMSO, zinc-binding group is one of the factorsi) HPLC that achieve the selectivity [82]. 110 °C, 5 min ii) SPE Radiolabeling of [18F]12110was °C, 5 achievedmin by a recently reported 18F-deoxyfluorinationii) SPE method [83–85]. First, ii) AdCH NHCH , iii) NH OH, ii) AdCH2NHCH3, iii) NH2OH, r.t., 52 min 3 NaOH,2 a mixture of 4-((((adamantan-1-yl)methyl)(methyl)amino)methyl)-3-hydroxybenzoate,r.t., 5 min NaOH, CpRu(cod)Cl, iii) NaB(CN)H3, MeOH/THF, ◦ iii) NaB(CN)H3, MeOH/THF, 18 N,N-bis(2,6-diisopropyl)phenyl-2-chloroimidazoliumiPrOH, chloride,r.t., and5 min. ethanol was heated[18F]11 to 85 C for iPrOH, r.t., 5 min. [ F]11 6 60 °C, 15 min RCY: 0.3% 30 min to form an η 60coordinated °C, 15 min ruthenium–phenol complex. The resulting solutionRCY: 0.3% was passed 18 through an F anion-bearing anion exchange cartridge, which18 was flushed with MeCN and DMSO. Scheme 7. Radiosynthesis of [18F]9–11. Molecules 2018, 23, x 12 of 27

11 [ C]CH3I DMSO, 110 °C, 4 min [11C]8 RCY: 3-5%*

Scheme 6. Radiosynthesis of [11C]8. * Non-decay corrected.

Molecules 2018, 23, 300 13 of 27

The elution was heated to 130 ◦C for 30 min, and then a hydroxylamine solution was added to convert a methylFigure ester group3. The total to volume a hydroxamic of distribution acid (V group.T) images HPLC from [11C] puriﬁcation8 PET in baboon gave brains. the Robust desired brain product in 8.1% non-decay-correcteduptake (top) of [11C]8 radiochemical was decreased by yield pre-tr witheatment a molar with unlabeled activity 8of (bottom 148 GBq/). (Reproducedµmol. Radiochemical from MoleculesWang 2018 et, 23al., xJ Med Chem; published by The American Chemical Society, 2014 [68]). 13 of 27 purity was not stated in the published article. Regarding selectivity, IC50 values of 12, determined µ µ 11 using recombinantAlthoughHPLC purification all enzymes, three radioligandsgave were the 0.06 desired showM product fored significant HDAC6 in 0.3% and correlation decay-corrected >1 M with for other [ C]radiochemical8 HDACs., correlation12 yield. betweenalso showedAll [18F]11 and [11C]8 was the highest. αTaken together, [18F]11 exhibited comparable properties to [11C]8, selectivethree deacetylation compounds showed inhibition high affinity of -tubulin, for HDAC1, a substrate 2, 3, and of 6 HDAC6,(IC50 values: in human<20 nM). induced Brain kinetics pluripotent of stemsuchthe cell-derived radioligands as HDAC neural binding was assessed progenitor and brain with uptake, cells. PET imagingIn although vitro inARG rafutsrther and using optimizationbaboons. rat brain In rats, sectionsof radiosynthesis all radioligands demonstrated is needed.showed speciﬁc 18 bindingrapid ofVery brain [18F] recently, 12uptake, and Streblafter the bindinginjection, et al. report wasfolloweded blocked synthesis by a bygradual and the evaluation HDAC6 decrease selectiveofin an radioactivity F-labeled inhibitor, overhydroxamic tubastatinthe 2-h scan.acid- A [82]. 18 18 BrainbasedBy kinetics pre-treatment radioligand, of [18F] with [12F]Bavarostatwas unlabeled assessed ([8, washoutF]12 with, Scheme PET rates 8), imaging of with the radioligandsselectivity in a baboon. for from HDAC6 Thethe (Tablebrain brain were 2) uptake [80]. increased, HDAC6 of [18 F]12 18 isand receiving brain radioactivityincreased attention decreased as a therapeutic to about 50%target of for peak the neurodegenerativewithin 2 h. Meanwhile, diseases PET [81]. imaging [ F]12 has in reached an SUV of 3 immediately18 after injection and gradually decreased over a 120-min period. ababoons similar revealedstructure differences as [ F]11 but between without the a radioligandsvinyl group in in the brain linker. kinetics. Close A approach brain standardized of a bulky moietyuptake Pre-treatmentto the zinc-binding with 1.0 group mg/kg is one unlabeled of the factors12 resulted that achieve in a substantialthe selectivity decrease [82]. Radiolabeling in baboon of brain [18F]12 uptake 18value (SUV) from 30 to 60 min of [18F]9, the cyclohexyl compound, was only 0.57, whereas those of of [ F]12. Preliminary analysis showed a18 good correlation between SUV and total distribution volume was[18F]10 achieved and [18F] 11by reached a recently 1.22 reportedand 1.80, respectively,F-deoxyfluorination suggested thatmethod the adamantane [83–85]. First, group a mixture enhanced of the 4- (V ). In summary, a promising HDAC6 PET radioligand with BBB permeability was successfully T ((((adamantan-1-yl)methyl)(methyl)amino)meBBB permeability. On the other hand, the brainthyl)-3-hydroxybenzoate, uptake of [18F]11 was slightly CpRu(cod)Cl, lower than thatN,N -bis(2,6-of [11C]8 developed,diisopropyl)phenyl-2-chloroimidazoalthough and they furtherare close biologicalanalogues. Furthermore, evaluationlium chloride, the and regional and optimization ethanol SUV of was 18F-labeled of heated radiosynthesis ligands to 85 °Cin baboonfor are 30 awaitedmin brains to for 6 a clinicalwereformstudy. comparedan η coordinated to that of ruthenium–phenol [11C]8. complex. The resulting solution was passed through an 18F anion-bearing anion exchange cartridge, which was flushed with MeCN and DMSO. The elution

was heated to 130 °C fori) [18 30F]CsF, min, and then a hydroxylamine solution was added to convert a methyl ester group to a hydroxamicDMSO, acid group. HPLC purificationi) HPLC gave the desired product in 8.1% non- 110 °C, 5 min ii) SPE decay-corrected radiochemical yield with a molar activity of 148 GBq/μmol. Radiochemical purity was ii) RCH2NH2, iii) Camphor not stated in the publishedr.t., 5 min article. Regarding selectivity,sulfonic acid,IC50 values of 12, determined using iii) NaBH , iPrOH, 4 18 recombinant enzymes, wereiPrOH, 0.06 μM for HDAC6 and >1 110μM°C, for 30 min other HDACs.[ F]9: R = cyclohexane 12 also showed selective 60 °C, 10 min RCY: 7% deacetylation inhibition of α-tubulin, a substrate of HDAC6, in human[18 inducedF]10: R = adamantane pluripotent stem cell- derived neural progenitor cells. In vitro ARG using rat brain sections demonstratedRCY: 0.9% specific binding 18 18 i) [ F]KF, of [ F]12, and the bindingK.2.2.2, DMSO, was blocked by the HDAC6 selectivei) HPLC inhibitor, tubastatin A [82]. Brain kinetics of [18F]12 was110 assessed °C, 5 min with PET imaging in a baboon. ii)The SPE brain uptake of [18F]12 reached an ii) AdCH2NHCH3, iii) NH2OH, SUV of 3 immediately r.t.,after 5 min injection and gradually decreased NaOH,over a 120-min period. Pre-treatment with 1.0 mg/kg unlabelediii) NaB(CN)H 12 resulted3, in a substantial decreaMeOH/THF,se in baboon brain uptake of [18F]12. iPrOH, r.t., 5 min. [18F]11 Preliminary analysis showed60 °C, 15 min a good correlation between SUV and total distributionRCY: 0.3%volume (VT). In

summary, a promising HDAC6 PET radioligand with BBB permeability was successfully developed, 1818 and further biological evaluationSchemeScheme and optimization 7. 7.Radiosynthesis Radiosynthesis of radiosynthesis of of [ [ F]F]9–911–11. are. awaited for a clinical study.

i) CpRu(cod)Cl, CIIM, EtOH, 85 °C, 30 min O ii) Anion exchange OH cartridge N H N iii) 18F anion, MeCN, DMSO, 18F 130 °C, 30 min [18F]12 iv) NH2OH, RCY: 8.1%* 5 M NaOH, THF, MeOH, r.t., 5 min

SchemeScheme 8. Radiosynthesis8. Radiosynthesis ofof [[1818F]F]1212. .* *Non-decay Non-decay corrected. corrected.

4.3. Other4.3. Other Carboxylic Carboxylic Acid- Acid- and and Hydroxamic Hydroxamic Acid-Based Acid-Based Ligands Pharmacokinetic evaluation of three 11C-labeled alkanoic acid-based HDAC inhibitors, n-butyric Pharmacokinetic evaluation of three 11C-labeled alkanoic acid-based HDAC inhibitors, n-butyric acid, 4-phenylbutylic acid, and valproic acid, was reported by Kim et al. in 2013 (Table 3) [86]. acid, 4-phenylbutylic acid, and valproic acid, was reported by Kim et al. in 2013 (Table3)[ 86]. Generally, HDAC inhibitory activities of these alkanoic acids (IC50 in the μM range) are weaker than Generally,those of HDAC hydroxamic inhibitory acid- activities or benzamide-based of these alkanoic inhibitors acids [87]. (IC Although50 in the µtheseM range) radioligands are weaker have than thosebeen of hydroxamic used to treat several acid- or diseases benzamide-based including CNS inhibitors diseases, information [87]. Although about thesetheir distribution radioligands and have beenpharmacokinetics used to treat several in the diseases brain was including limited. The CNS auth diseases,ors introduced information carbon-11 about at the their carbonyl distribution carbon and pharmacokineticsof carboxylic acid in theby reaction brain was of 11 limited.C-carbon Thedioxide authors and the introduced respectivecarbon-11 Grignard reagents at the carbonyl (Scheme 9). carbon of carboxylicBriefly, [11 acidC]CO by2 was reaction passed of through11C-carbon a THF dioxide solution and of the the precursor. respective The Grignard reaction was reagents quenched (Scheme by 9). water followed by sequential addition of 6 N hydrochloric acid and 6 N sodium hydroxide. For the Molecules 2018, 23, 300 14 of 27

11 Briefly, [ C]CO2 was passed through a THF solution of the precursor. The reaction was quenched by waterMolecules followed 2018, 23 by, x sequential addition of 6 N hydrochloric acid and 6 N sodium hydroxide.14 of For 27 the synthesis of [11C]15, the reaction was performed with non-radioactive CO as a carrier with heating to Molecules 2018, 23, x 2 14 of 27 ◦ 11 45 Csynthesis for 2 min. of [ TheC]15 products, the reaction were was purified performed with HPLC.with non-radioactive Radiochemical CO yield2 as a (decaycarrier with corrected heating at the 11 endto ofsynthesis 45 cyclotron °C for of 2 [ 11min. bombardment)C]15 The, the products reaction and werewas molar performed purified activity with with wereHPLC. non-radioactive 31–50% Radiochemical and CO 7.4–372 as yield a carrier GBq/ (decay µwithmol corrected heating for [ at C] 13, 40–55%theto 45end and °C of for 7.4–30 cyclotron 2 min. GBq/ The bombardment) µproductsmol for were [11 andC] purified14 molar, and activity with 38–50% HPLC. were for Radiochemical31–50% [11C]15 and, respectively 7.4–37 yield GBq/ (decayμ (themol corrected molarfor [11C] activity at13 , 11 11 of [1140–55%theC]15 endwas andof cyclotron not7.4–30 mentioned). GBq/ bombardment)μmol for Radiochemical [ andC]14 molar, and activity38–50% purities were for of[ 31–50%C] all15 radioligands, respectively and 7.4–37 GBq/ were(theμ molarmol greater for activity [11 thanC]13 of, 98%. [11C]15 was not mentioned). Radiochemical11 purities of all radioligands11 were greater than 98%. Whole- Whole-body40–55% and pharmacokinetics 7.4–30 GBq/μmol of for these [ C]14 radioligands, and 38–50% was for [ determinedC]15, respectively withPET (the imagingmolar activity in baboons. of body[11C]15 pharmacokinetics was not mentioned). of these Radiochemical radioligands purities was determinedof all radioligands with PET were imaging greater inthan baboons. 98%. Whole- Of the Of the three radioligands, [11C]15 was the most stable in baboon plasma (>90% intact ligand over threebody radioligands,pharmacokinetics [11C] 15of thesewas the radioligands most stable was in determinedbaboon plas withma (>90% PET imaging intact ligand in baboons. over 90 Of min). the 90 min). PET images demonstrated very low uptake of these radioligands in the baboon brain of PETthree images radioligands, demonstrated [11C]15 wasvery the low most uptake stable of inthese baboon radioligands plasma (>90% in the intact babo ligandon brain over of 90less min). than 11 less than0.006%PET 0.006%images ID/cc, demonstrated ID/cc,presumably presumably due very to low thei due ruptake poor to their BBB of these poorpenetrance. radioligands BBB penetrance. Interestingly, in the Interestingly,babo [11C]on15 brain showed [of lessC] relatively15 thanshowed relativelyhigh0.006% heart high ID/cc, uptake heart presumably uptake consistent consistent due wi toth thei the withr cardiacpoor the BBB side cardiac penetrance. effects side of effects Interestingly,15 [88]. of These15 [ 88[ 11findingsC]].15 These showed obtained findings relatively by obtained the by theimaginghigh imaging heart studies uptake studies can consistent help can helpin understanding wi inth understanding the cardiac therapeutic side therapeutic effects profiles of 15 profiles [88].of the These inhibitors. of thefindings inhibitors. obtained by the imaging studies can help in understanding therapeutic profiles of the inhibitors. 11 i) [ C]CO2, 11THF, i) [ C]CO2, THF,r.t. ii) r.t.H2O, 6 M HCl, 11 ii) H2O, [ C]13 66 M M NaOH HCl, RCY:[11C] 31-50%13 6 M NaOH 11 i) [ C]CO2, RCY: 31-50% 11THF, i) [ C]CO2, THF,r.t.

ii) r.t.H2O, 6 M HCl, 11 ii) H2O, [ C]14 66 M M NaOH HCl, RCY:[11C] 40-55%14 6 M NaOH 11 RCY: 40-55% i) [ C]CO2, O 11THF, 11 i) [ C]CO2, OC OH 45 °C, 2 min THF, 11C OH 45 °C, 2 min ii) H2O, 11 6ii) M H HCl,2O, [ C]15 66 M M NaOH HCl, RCY:[11C] 38-50%15 6 M NaOH RCY: 38-50% Scheme 9. Radiosynthesis of [11C]13–15. SchemeScheme 9. 9.Radiosynthesis Radiosynthesis of of [11 [11C]C]1313–15–15. . In 2014, Wang et al. reported the synthesis and evaluation of radiolabeled hydroxamic acid- In 2014, Wang et al. reported the synthesis and evaluation of radiolabeled hydroxamic acid- basedIn 2014, ligands Wang for et HDAC al. reported imaging the [89]. synthesis Aiming andto investigate evaluation the of brain radiolabeled kinetics of hydroxamic hydroxamates, acid-based they based ligands for HDAC imaging [89]. Aiming to investigate the brain kinetics of hydroxamates, they ligandsselected for HDAC three known imaging HDAC [89]. Aiminginhibitors to and investigate modified the their brain structures kinetics offor hydroxamates, 11C-labeling (Scheme they selected 10, selected three known HDAC inhibitors and modified their structures for 11C-labeling (Scheme 10, Table 3) [90–92]. 11 threeTable known 3) HDAC[90–92]. inhibitors and modified their structures for C-labeling (Scheme 10, Table3) [ 90–92].

[11C]CH I 11 3 [ C]CH3I Cs2CO3, CsDMSO,2CO3, DMSO, 50 °C, 3 min [11C]16 50 °C, 3 min 11 RCY:[ C]16 9%* RCY: 9%*

11 [11 C]CH3I [ C]CH3I 1 M NaOH, 1 M NaOH, DMSO, DMSO, r.t., 3 min r.t., 3 min [11C]17 [11C]17 RCY:RCY: 3%* 3%*

11 [11C]CH3I [ C]CH3I 1 M NaOH, DMSO, r.t., 33 minmin 11 [11[ C]C]1818 RCY:RCY: 5%* 5%*

SchemeSchemeScheme 10. 10.Radiosynthesis RadiosynthesisRadiosynthesisof ofof [ [[111111C]C]1616–––181818.. *.* *Non-decayNon-decay Non-decay corrected. corrected. corrected. Molecules 2018, 23, 300 15 of 27

Table 3. Physiochemical properties of radiolabeled carboxylic acid- and hydroxamic acid-based HDAC ligands.

Compound MW Log D RCY Target IC50 for HDACs In Vivo Properties in Rodents Reference HDAC1: 16 µM HDAC2: 12 µM [11C]13 87.11 1.02 31–50% Class I –[86,87] HDAC3: 9 µM HDAC8: 15 µM HDAC1: 64 µM HDAC2: 65 µM [11C]14 149.18 −0.20 40–55% Class I –[86,87] HDAC3: 260 µM HDAC8: 93 µM HDAC1: 39 µM HDAC2: 62 µM [11C]15 143.21 0.26 38–50% Class I –[86,87] HDAC3: 161 µM HDAC8: 1103 µM HDAC1: 5 nM HDAC2: 32 nM Brain uptake: Around 0.1% ID/cc immediately after i.v. injection [11C]16 331.37 2.03 1 9% 3 HDAC1–3/6 [89] HDAC3: 3.4 nM (rat) HDAC6: 5 nM HDAC1: 0.2 nM HDAC2: 1.2 nM Brain uptake: Around 0.15% ID/cc immediately after i.v. injection [11C]17 348.43 2.66 1 3% 3 HDAC1–3/6 [89] HDAC3: 0.4 nM (rat) HDAC6: 1.6 nM Brain uptake: Around 0.2% ID/cc immediately after i.v. injection [11C]18 295.33 2.61 1 5% 3 HDAC8 HDAC8: 18 nM [89] (rat) Brain uptake: Radioactivity peaked in forebrain at 0.44 SUV and in HDAC1: 5.2 µM [11C]19 334.41 1.33 2 8% HDAC6 cerebellum at 0.48 SUV [93,94] HDAC6: 1.4 nM (rat) HDAC6: 4 nM [11C]20 334.41 1.33 2 16% HDAC6 –[82,95] Others: ≥1.3 µM Brain uptake: Biodistribution study was performed but brain uptake Class I, IIb, III: [64Cu]21 1009.02 – ≥98% Class I/IIb/III was not assessed [96] 94 nM (tumor-bearing mouse) 1 Log p. 2 clog D. 3 Non-decay corrected. MW: molecular weight; RCY: radiochemical yield (decay corrected). Molecules 2018, 23, 300 16 of 27 Molecules 2018, 23, x 16 of 27

11 11 Radiosynthesis of [11C]C]1616––1818 waswas achieved by a reaction of precursorsprecursors andand [[11C]CH3II in in DMSO 11 in the the presence presence of of base. base. Radiochemical Radiochemical yield yield (non-decay (non-decay corrected, calculated fromfrom trappedtrapped [[11C]CHC]CH3I)I) and molar activity werewere 9%9% andand 29.629.6± ± 7.47.4 GBq/GBq/µμmol forfor [[1111C]16, 3%3% andand 33.333.3 ±± 3.7 GBq/GBq/μµmolmol for [11C]C]1717, ,and and 5% 5% and and 25.9 25.9 ± 7.4± GBq/7.4 GBq/μmolµ formol [11 forC]18 [11, respectively.C]18, respectively. Chemical Chemical and radiochemical and radiochemical purities werepurities ≥95% were for all≥95% radioligands. for all radioligands. 16 and 17 showed16 and low17 ICshowed50 values low against IC50 classvalues I HDACs against (HDAC1–3) class I HDACs and HDAC6,(HDAC1–3) whereas and HDAC6, 18 exhibited whereas high 18HDAC8exhibited selectivity high HDAC8 with an selectivityIC50 value of with 18.3 an nM. IC50 Thevalue brain of 18.3kinetics nM. ofThe all brain three kinetics radioligands of all three was radioligands evaluated with was evaluatedPET imaging with in PET rodents imaging and in NHPs. rodents In and rats, NHPs. the radioligandsIn rats, the radioligands exhibited poor exhibited brain uptake poor brain with uptake less than with 0.25% less ID/cc than 0.25%over a ID/cc30-min over p.i. aperiod, 30-min and p.i. brainperiod, uptake and brainwas not uptake changed was by not pre-treatment changed by pre-treatmentwith 2 mg/kg unlabeled with 2 mg/kg compound. unlabeled Brain compound. uptake in aBrain baboon uptake was in also a baboon very limited. was also Altogether, very limited. these Altogether, imaging theseresults imaging indicated results that indicatedthese radioligands that these areradioligands not suitable are for not in suitable vivo HDAC for in imaging vivo HDAC in the imaging brain. in the brain. Lu et al. reported reported development development of of an an HDAC6-se HDAC6-selectivelective hydroxamic hydroxamic acid-based acid-based radioligand radioligand [93]. [93]. [11C]C]1919 isis a a 1111C-labeledC-labeled analogue analogue of of tubastatin tubastatin A, A, a a selective selective HDAC6 HDAC6 inhibitor inhibitor with with a a tetrahydro- tetrahydro-γγ-- carboline structure structure (IC (IC5050: 15: 15 nM nM for for HDAC6; HDAC6; 1640 1640 nM nM for forHDAC1) HDAC1) (Scheme (Scheme 11, Table 11, Table 3) [82,94].3)[ 82 11,94C-]. 11 MethylationC-Methylation at the at piperidine the piperidine nitrogen nitrogen atom was atom achieved was achieved using a desmethyl using a desmethyl precursor precursor and [11C]CH and3I. 11 11 [11C]CHC]CH33I I.was [ C]CH trapped3I was in a trappedsolution in of a the solution precursor of the and precursor KOH in and DMSO, KOH and in DMSO,the mixture and thewas mixture heated towas 80 heated °C for to4 min. 80 ◦C HPLC for 4 min.purification HPLC puriﬁcationgave the desired gave theproduct desired in 7.6% product radiochemical in 7.6% radiochemical yield from 11 [yield11C]CO from2 and [ >99%C]CO 2radiochemicaland >99% radiochemical purity. The molar purity. activity The molar wasactivity 96.2 GBq/ wasμmol. 96.2 IC GBq/50 valuesµmol. of IC 1650 werevalues 1.40 of 16 nMwere and 1.40 5180 nM nM and for 5180 HDAC6 nM for and HDAC6 HDAC1, and respectively HDAC1, respectively [94]. PET [imaging94]. PET with imaging a rhesus with monkeya rhesus demonstrated monkey demonstrated limited radioactivity limited radioactivity in the forebrain in the (SUV forebrain = 0.18) (SUV and cerebellum = 0.18) and (SUV cerebellum = 0.38). Furthermore,(SUV = 0.38). Furthermore,in rats pre-treated in rats with pre-treated a P-gp with inhibitor a P-gp 20 inhibitor min before 20 min radioligand before radioligand injection, injection, [11C]19 uptake[11C]19 inuptake the forebrain in the forebrain (SUV = 0.44) (SUV and = 0.44) cerebellum and cerebellum (SUV = 0.48) (SUV was = 0.48)also low. was alsoThe authors low. The assumed authors thisassumed low BBB this permeability low BBB permeability was due to was low due lipophilicity to low lipophilicity of the compound of the compound (cLogD = (cLog1.33).D = 1.33). Recently, thethe same same group group explored explored11C-labeling 11C-labeling of tubastatin of tubastatin A at the A carbonyl at the carboncarbonyl of hydroxamic carbon of hydroxamicacid using [acid11C]carbon using [11 monoxideC]carbon monoxide [95]. [11C]CO [95]. [11 hasC]CO the has potential the potential to radiolabel to radiolabel a wide a wide variety variety of ofcarbonyl-containing carbonyl-containing molecules molecules [97], [97], and and this labelingthis labeling method method could could be applied be applied to other to HDAC other ligandsHDAC ligandswith a hydroxamicwith a hydroxamic acid group. acid group. Initially, Initia thelly, authors the authors attempted attempted to synthesize to synthesize [11C] 20[11C]in20 a in one-step a one- steppalladium-mediated palladium-mediated reaction reaction using theusing iodinated the iodinated precursor, precursor, hydroxylamine, hydroxylamine, and [11C]CO; and however,[11C]CO; 11 11 however,the decay-corrected the decay-corrected radiochemical radiochemical yield of [ yieldC]20 ofwas [11 lessC]20than was 10%less (calculatedthan 10% (calculated from [ C]CO from2), [and11C]CO a carboxylic2), and a carboxylicacid derivative acid derivati was obtainedve was as obtained a major as product. a major Then product. they testedThen they a two-step tested a reaction two-step of reaction11C-methyl of 11 esterC-methyl formation ester followed formation by followed conversion by toconversion a hydroxamate. to a hydroxamate. The first step wasThe achievedfirst step withwas achieveda moderate with radiochemical a moderate yieldradiochemical of 18.5 ± yield6.2%, of but 18. the5 ± second6.2%, but step the with second hydroxylamine step with hydroxylamine hydrochloride hydrochloridedid not work. did Through not work. trial Through and error, trial theyand error, decided they to decided investigate to investigate the use the of ausep-nitrophenyl of a p-nitrophenyl ester, ester,with greaterwith greater susceptibility susceptibility toward toward aminolysis, aminolysis, as a labeledas a labeled intermediate intermediate (Scheme (Scheme 11). 11).

11 [ C]CH3I KOH, DMSO, 80 °C, 4 min [11C]19 RCY: 8%

11 [ C]CO NH2OH·HCl,

+ Pd2(dba)3, P1-t-Bu, Xantphos, DMSO, THF, ultrasound, 130 °C, 3 min r.t., 3 min [11C]20 RCY: 16%

SchemeScheme 11.11. RadiosynthesisRadiosynthesis ofof [[1111C]19 and [ 11C]C]2020. .

Using Pd2(dba)3 and Xantphos, [11C]CO insertion was successfully obtained with a radiochemical yield of 54.6 ± 8.0%. Furthermore, after investigation into the reaction condition, the second step with

Molecules 2018, 23, 300 17 of 27

11 Using Pd2(dba)3 and Xantphos, [ C]CO insertion was successfully obtained with a radiochemical yield of 54.6 ± 8.0%. Furthermore, after investigation into the reaction condition, the second step with a phosphazene base gave the desired product in 16.1 ± 5.6% decay-corrected radiochemical yield with 8.2 GBq/µmol molar activity. Aiming to evaluate the versatility of this labeling method, threeMolecules simple 2018 aryl, 23,iodides x (e.g., 1-chloro-4-iodobenzene or 4-iodoanisole) were assessed.17 However, of 27 although corresponding p-nitrophenyl esters were obtained in good radiochemical yields, the second a phosphazene base gave the desired product in 16.1 ± 5.6% decay-corrected radiochemical yield with step reaction gave carboxylic acid derivatives. Taken together, [11C]20 was labeled at the carbonyl 8.2 GBq/μmol molar activity. Aiming to evaluate the versatility of this labeling method, three simple carbon using [11C]CO, but application of the labeling method for the other hydroxamates required aryl iodides (e.g., 1-chloro-4-iodobenzene or 4-iodoanisole) were assessed. However, although furthercorresponding optimization. p-nitrophenyl esters were obtained in good radiochemical yields, the second step 11 18 Inreaction addition gave to carboxylicC- and acidF-labeled derivatives. HDAC Taken radioligands,together, [11C]20 radio-metal-labeled was labeled at the carbonyl ligands carbon have also beenusing developed. [11C]CO, In but 2013, application Meng et of al. the reported labeling method synthesis forand the other biological hydroxamates evaluation required of a 64 furtherCu-labeled hydroxamicoptimization. acid-based radioligand (Table3) [ 96]. In this study, a chelator, 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraaceticIn addition acid,to 11C- was and conjugated18F-labeled HDAC to an radioligands, HDAC inhibitor, radio-me CUDC-101tal-labeled [ligands98,99], have with also an been aliphatic linkerdeveloped. by Huisgen In 2013, cycloaddition. Meng et al. reported [64Cu] synthesis21 was and labeled biological following evaluation a reactionof a 64Cu-labeled of the hydroxamic precursor and 64 acid-based radioligand (Table 3) [96]. In this ◦study, a chelator, 1,4,7,10-tetraazacyclododecane- [ Cu]CuCl2 in ammonium acetate buffer at 60 C for 1 h (Scheme 12). HPLC puriﬁcation gave 1,4,7,10-tetraacetic acid, was conjugated to an HDAC inhibitor, CUDC-101 [98,99], with an aliphatic the desired product in ≥98% radiochemical yield and radiochemical purity. The molar activity linker by Huisgen cycloaddition. [64Cu]21 was labeled following a reaction of the precursor and was estimated to 2.4–2.9 GBq/µmol. The IC50 value of 21 against class I and II HDACs was [64Cu]CuCl2 in ammonium acetate buffer at 60 °C for 1 h (Scheme 12). HPLC purification gave the 94.47desired± 19.92 product nM. An in ≥in98% vitro radiochemicalbinding assayyield and using radiochemical MDA-MB-231, purity. aThe breast molar cancer activity cell was line estimated with high 64 HDACto 2.4–2.9 expression GBq/μmol. [100 The], demonstrated IC50 value of 21 against dose-dependent class I and II inhibitionHDACs was of 94.47 [ Cu]± 19.9221 nM.cell An uptake in vitro in the presencebinding of CUDC-101.assay using Subsequently,MDA-MB-231, a biodistribution breast cancer cell of [line64Cu] with21 inhigh mice HDAC bearing expression an MD-MBA-231 [100], xenograftdemonstrated was evaluated. dose-dependent PET imaging inhibition showed of tumor[64Cu]21 uptake cell uptake of radioactivity in the presence (1.20 ±of 0.21,CUDC-101.2.16 ± 0.08, and 2.36Subsequently,± 0.31% biodistribution ID/g at 2, 6, andof [64 24Cu] h21 p.i., in mice respectively), bearing an MD-MBA-231 and the uptake xenograft was reduced was evaluated. by half by coinjectionPET imaging of 20 mg/kgshowed tumor CUDC-101. uptake Theof radioactivity mice were (1.20 sacriﬁced ± 0.21, 2.16 immediately ± 0.08, and after2.36 ± the0.31% PET ID/g scan at and tissue2, radioactivity6, and 24 h p.i., respectively), was measured. and the The uptake biodistribution was reduced resultsby half by demonstrated coinjection of 20 tumor/muscle mg/kg CUDC- and 101. The mice were sacrificed immediately after the PET scan and tissue radioactivity was measured. tumor/blood uptake ratios of 9.61 ± 1.54 and 4.44 ± 0.88, respectively. The brain uptake is not The biodistribution results demonstrated tumor/muscle and tumor/blood uptake ratios of 9.61 ± 1.54 describedand 4.44 in the± 0.88, paper respectively. probably becauseThe brain radioligands uptake is not with described a bulky in motif the paper such asprobably metal chelatorsbecause are generallyradioligands not expected with a tobulky cross motif the such BBB. as These metal results chelators suggested are generally the feasibility not expected of HDACto cross imagingthe BBB. with radiometal-labeledThese results suggested ligands the in cancer.feasibility of HDAC imaging with radiometal-labeled ligands in cancer.

64 [ Cu]CuCl2

0.1 M NH4Ac (pH = 5.5), 60 °C, 1 h

[64Cu]21 RCY: ≥98% Scheme 12. Radiosynthesis of [64Cu]21. Scheme 12. Radiosynthesis of [64Cu]21. 4.4. Ortho-Aminoanilide-Based Ligands 4.4. Ortho-Aminoanilide-Based Ligands In 2010, Hooker et al. reported synthesis and evaluation of the first benzamide-based HDAC Inradioligand, 2010, Hooker [11C]MS-275 et al. reported([11C]22, Scheme synthesis 13), andand evaluationcharacterized of its the pharmacokinetics ﬁrst benzamide-based in the brain HDAC 11 11 radioligand,(Table 4) [[101].C]MS-275 22 (IC50: ~300 ([ C] nM22 ,for Scheme HDAC1; 13 ~8), andμM for characterized HDAC3 [102]) its is pharmacokineticsa potent, long-lasting, in brain the brain (Tableregion-selective4)[ 101]. 22 (IC HDAC50: ~300 inhibitor nM for th HDAC1;at shows a ~8 dose-dependentµM for HDAC3 increase [102 ])in ishistone a potent, 3 acetylation long-lasting, in the brain region-selectiverat brain [103]; HDAC however, inhibitor no thatdirect shows evidence a dose-dependent was obtained increasefor BBB inpermeability histone 3 acetylationof 22. They in the 11 11 11 rat brainsynthesized [103]; however, [ C]22 by no direct direct incorporation evidence was of [ obtainedC]CO2 into for the BBB carbamate permeability carbon of [104].22. They[ C]CO synthesized2 was 11 trapped in a mixture of 3-picolyl11 chloride hydrochloride, 4-(aminomethyl)-N11-(2-aminophenyl)- [ C]22 by direct incorporation of [ C]CO2 into the carbamate carbon [104]. [ C]CO2 was trapped benzamide dihydrochloride, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and DMF, which was then in a mixture of 3-picolyl chloride hydrochloride, 4-(aminomethyl)-N-(2-aminophenyl)-benzamide heated to 75 °C for 7 min. HPLC purification gave the desired product in 25% decay-corrected ◦ dihydrochloride,radiochemical 1,8-diazabicyclo[5.4.0]undec-7-ene yield and >98% radiochemical purity. (DBU),The molar and activity DMF, was which 100–229 was GBq/ thenμ heatedmol. Using to 75 C for 7 min.[11C]22 HPLC, they puriﬁcationperformed PET gave imaging the desired studies product in baboon in and 25% rat decay-corrected brains. In baboons, radiochemical no radioactivity yield and 11 >98%uptake radiochemical was observed purity. in the The brain molar after activity i.v. injection was 100–229 of [11C]22 GBq/. Pre-treatmentµmol. Using with [ theC] 22P-gp, they substrate, performed PET imagingverapamil studies (0.5 mg/kg, in baboon i.v., 5 min and before rat brains.[11C]22 injection), In baboons, had nono influenc radioactivitye on brain uptake pharmacokinetics was observed in the brainof [11C] after22, suggesting i.v. injection that of P-gp [11C] was22 .not Pre-treatment involved in [11 withC]22 exclusion the P-gp from substrate, the brain. verapamil Arterial (0.5plasma mg/kg, analysis demonstrated moderate metabolic stability of [11C]22; greater than 50% of radioactivity was derived from intact [11C]22 at 90 min p.i. A further PET study in rats also confirmed poor brain uptake

Molecules 2018, 23, 300 18 of 27 i.v., 5 min before [11C]22 injection), had no inﬂuence on brain pharmacokinetics of [11C]22, suggesting that P-gp was not involved in [11C]22 exclusion from the brain. Arterial plasma analysis demonstrated moderate metabolic stability of [11C]22; greater than 50% of radioactivity was derived from intact [11C]22 Moleculesat 90 min 2018 p.i., 23, x A further PET study in rats also conﬁrmed poor brain uptake of [11C]1822 of, and27 brain uptake was not altered by pre-treatment with unlabeled 19. These results suggested that MS-275 may of [11C]22, and brain uptake was not altered by pre-treatment with unlabeled 19. These results be not suitablesuggested for that the MS-275 treatment may be of not neurodegenerative suitable for the treatment diseases of neurodegenerative targeting HDACs, diseases but targeting it is also likely to haveHDACs, minimal but unwanted it is also likely CNS to effectshave minimal in treatment unwanted of CNS peripheral effects in cancer.treatment of peripheral cancer.

11 [ C]CO2 + DBU, DMF, 75 °C, 7 min. [11C]22 RCY: 25% Scheme 13. Radiosynthesis of [11C]22. Scheme 13. Radiosynthesis of [11C]22. Table 4. Physiochemical properties of radiolabeled benzamide-based HDAC ligands. Table 4. Physiochemical properties of radiolabeled benzamide-based HDAC ligands. Compound MW Log D RCY Target IC50 for HDACs In Vivo Properties in Rodents Reference Brain uptake: <0.10% ID/cm3 after 3 min Compound MW Log D RCY TargetHDAC1: IC50 for 60 HDACs nM Metabolism: In Vivo 80% Propertiesof radioactivity in Rodents in the Reference [11C]22 375.42 1.8 25% Class I [101,105] HDAC2: 153 nM Brainbrain uptake: was unchanged <0.10% ID/cm [11C]223 after 3 min HDAC1: 60 nM Metabolism: 80%(rat) of radioactivity in the [11C]22 375.42 1.8 25% Class I [101,105] HDAC1:HDAC2: 45 153 nM nM brain was unchanged [11C]22 [11C]23 268.30 1.0 10–15% Class I HDAC2: 31 nM – (rat) [105] HDAC3:HDAC1: 20 45nM nM [11C]23 268.30 1.0 10–15% Class I HDAC1:HDAC2: 10 31nM nM –[105] [11C]24 344.45 2.1 40% Class I – [105] HDAC2:HDAC3: 20 20nM nM HDAC1: 10 nM [11C]24 344.45 2.1MW: molecular 40% weight; Class I RCY: radiochemical yield (decay corrected).–[ 105] HDAC2: 20 nM Seo et al. conductedMW: molecular PET imaging-guided weight; RCY: radiochemical systematic yielddevelopment (decay corrected). of BBB-permeable HDAC inhibitors (Table 4) [105]. They repeated radiosynthesis, performed PET imaging in the baboon brain, Seoand et incorporated al. conducted the PETimaging imaging-guided data into the compound systematic design. development For this study, of several BBB-permeable 11C-labeled HDAC 11 11 11 inhibitorsbenzamides (Table4)[ were105 ].radiosynthesized They repeated using radiosynthesis, [ C]methyl iodide, performed [ C]methyl PET imagingtriflate, and in the[ C]acetyl baboon brain, chloride. Radiosynthesis of two representative benzamides, [11C]23 and [11C]24, is shown in Scheme and incorporated the imaging data into the compound design. For this study, several 11C-labeled 14. 23 is the known HDAC inhibitor, CI-994, with class I HDAC selectivity (dissociation constant: 11 11 11 benzamides0.055 μ wereM for radiosynthesizedHDAC1; 0.255 μM for using HDAC2; [ C]methyl0.024 μM for iodide, HDAC3 [ [106]).C]methyl For [11C] triflate,23 radiosynthesis, and [ C]acetyl 11 11 chloride.[1-11C]CH Radiosynthesis3COCl, prepared of twofrom representative [11C]CO2, was benzamides,added to a mixture [ C] 23of anda precursor, [ C]24 ,4- isN, shownN- in Schemedimethylaminopyridine, 14. 23 is the known and HDAC THF and inhibitor, heated to CI-994,60 °C for with5 min. classAfter HPLC I HDAC purification, selectivity the fraction (dissociation constant:containing 0.055 µ theM 11 forC-labeled HDAC1; intermediate 0.255 µ wasM for mixed HDAC2; with trifluoroacetic 0.024 µM acid, for HDAC3followed by [106 removal]). For of [11C]23 the solvent in11 vacuo. The desired product was obtained,11 and the decay-corrected radiochemical yield radiosynthesis, [1- C]CH3COCl, prepared from [ C]CO2, was added to a mixture of a precursor, and radiochemical purity were 10–15% and >95%, respectively. The molar activity was 200 MBq/μmol. 4-N,N-dimethylaminopyridine, and THF and heated to 60 ◦C for 5 min. After HPLC purification, For [11C]24 radiosynthesis, [11C]CH3OTf was trapped in the precursor containing DMSO, and the 11 the fractionmixture containing was heated the to 50 C-labeled°C for 3 min. intermediate HPLC purification was mixedgave the with desired trifluoroacetic product in 40% acid, decay- followed by removalcorrected of theradiochemical solvent in yield vacuo and. The99% desiredradiochemical product purity. was The obtained, molar activity and the was decay-corrected 185–666 radiochemicalGBq/μmol. yield IC50 and values radiochemical of 24 were 0.01 purity μM and were 0.02 10–15% μM against and HDAC1 >95%, respectively.and HDAC2, respectively. The molar activity Initially, the authors 11assessed the BBB permeability11 of some compounds including [11C]23, but brain was 200 MBq/µmol. For [ C]24 radiosynthesis, [ C]CH3OTf was trapped in the precursor containing uptake in baboon was very limited. After image-guided structural optimization, N,N-dimethylamino DMSO, and the mixture was heated to 50 ◦C for 3 min. HPLC purification gave the desired product derivatives such as [11C]24 demonstrated improved brain uptake (~0.015% ID/cc at 5 min p.i.). [11C]24 in 40% decay-correctedshowed the highest radiochemicalarea under the curve yield ratio and of 99% the brain radiochemical versus plasma purity. of 7.5. TheFurthermore, molar activity pre- was 185–666treatment GBq/µ mol.with unlabeled IC50 values 24 (1 mg/kg) of 24 wereresulted 0.01 in aµ decreaseM and in 0.02 VT inµ theM againstrange of 13–25% HDAC1 compared and HDAC2, respectively.with baseline Initially, in the the cerebellum, authors thalamus, assessed and the tempor BBBal permeability cortex. In this study, of some the polar compounds surface area including [11C]23,of but less brain than uptake65 appeared in baboon to be a wascritical very prop limited.erty for BBB After permeability, image-guided and a structural key element optimization, that N,N-dimethylaminoimproved brain derivatives uptake of the such benzamides as [11C] was24 demonstrateda benzylic amine. improved brain uptake (~0.015% ID/cc at 5 min p.i.). [11C]24 showed the highest area under the curve ratio of the brain versus plasma of 7.5. Furthermore, pre-treatment with unlabeled 24 (1 mg/kg) resulted in a decrease in VT in the range of 13–25% compared with baseline in the cerebellum, thalamus, and temporal cortex. In this study, the polar surface area of less than 65 appeared to be a critical property for BBB permeability, and a key element that improved brain uptake of the benzamides was a benzylic amine. Molecules 2018, 23, 300 19 of 27

Molecules 2018, 23, x 19 of 27

11 i) [1- C]CH3COCl, O THF, DMAP, N 60 °C, 5 min. H NHBoc ii) HPLC H2N iii) TFA [11C]23 RCY: 10-15%

11 [ C]CH3OTf DMSO, 50 °C, 3 min

[11C]24 RCY: 40% Scheme 14. Radiosynthesis of [11C]23 and [11C]24. Scheme 14. Radiosynthesis of [11C]23 and [11C]24. 4.5. First-in-Human PET Study 4.5. First-in-HumanFindings PET in HDAC Study radioligand studies with NHPs are summarized in Table 5. Unfortunately, almost all HDAC radioligands showed poor BBB penetrance in PET studies with NHPs, probably Findingsdue to in their HDAC low lipophilicity radioligand or metabolic studies instability. with NHPs are summarized in Table5. Unfortunately, almost all HDAC radioligands showed poor BBB penetrance in PET studies with NHPs, probably due to their low lipophilicity orTable metabolic 5. Brain PET instability. imaging studies of HDAC radioligands in NHPs. Compound Target Findings in PET Studies in NHPs Reference Table 5. Brain PETRadioactivity imaging accumulation studies of HDACwas brain radioligands region specific in rhesus NHPs. macaques (~0.03% ID/g) [18F]1 Class IIa By 30 min p.i., almost all [18F]1 was metabolized to [18F]FACE [26,46] Compound Target Findings in PET Studies in NHPs Reference Radioactivity in the brain was decreased by pre-treatment with RadioactivitySAHA accumulation in a dose-dependent was brain region manner speciﬁc in rhesus macaques (~0.03% ID/g) Brain uptake of both ligands was very low in baboons [18F]1 Class IIa By 30 min p.i., almost all [18F]1 was metabolized to [18F]FACE [26,46] [11C]6 (~0.004% ID/cc) – Radioactivity in the brain was decreased by pre-treatment with SAHA in [60] [11C]7 The unchanged fractiona dose-dependent of both ligands manner in baboon plasma was less Brain uptake of both ligandsthan was 20% very at 30 low min in baboonsp.i. (~0.004% ID/cc) [11C]6 – RegionalThe unchanged VT (90-min fraction scan) of both in the ligands baboon in baboonbrain ranged plasma from was less29.9 to [60] [11C]7 than 20%54.4 at mL/cm 30 min3 p.i. Parent fraction in plasma decreased gradually (50% at 30 min p.i. 3 [11C]8 Class I/IIbRegional VT (90-min scan) in the baboon brain ranged from 29.9 to 54.4 mL/cm [28,68,78] Parent fraction in plasmaand decreased40% at 60 gradually min p.i.) (50% at 30 min p.i. 11 [ C]8 Class I/IIb The mean VT in the brainand 40%decreased at 60 min byp.i.) 82.3 ± 5.5% with a 1-mg/kg [28,68,78] The mean VT in the brain decreasedblocking by dose 82.3 ± 5.5% with a 1-mg/kg blocking dose Whole brain SUV30–60 min of [18F]9, [18F]10, and [18F]11 in baboons 18 [18F]9 18 18 18 [ F]9 Whole brain SUVwere30–60 0.57, min of1.2, [ andF]9,[ 1.8,F] respectively10, and [ F]11 (2.3in baboonsfor [11C]8) were 0.57, 1.2, [18F]10[18F]10 Class I/IIbClass I/IIb and 1.8, respectively (2.3 for [11C]8) [79] [79] [18F]11 showed the highest correlation in regional brain distribution [18F]11[18F]11 [18F]11 showed the highest correlation in regional brain distribution with [11C]8 with [11C]8 ≈ ExcellentExcellent brain brain uptake uptake (SUV (SUV3 around ≈ 3 around 30 min 30 p.i.) min was p.i.) observed was observed in baboons in [18F]12 HDAC6 Nonspeciﬁc binding in the brain determined with 1 mg/kg unlabeled 12 was [80] baboons [18F]12 HDAC6 low (<1 SUV) [80] Nonspecific binding in the brain determined with 1 mg/kg [11C]13 unlabeled 12 was low (<1 SUV) [11C]14 Class I In the baboon brain, uptake of the three ligands was low (~0.006% ID/cc) [86] 11 [11C]13 [ C]15 In the baboon brain, uptake of the three ligands was low (~0.006% [11C]14 Class I [86] [11C]16 ID/cc) 11 HDAC1–3/6 Brain uptake in baboons was very low over the 80-min scan time [89] [11C]17[ C]15 [11C]16 [11C]18 HDAC8HDAC1–3/6 Brain Brain uptake inin baboonsbaboons was was very very low low over over the the 80-min 80-min scan scan time time [89] [89] [11C]17 [11C]18 HDAC8 BrainBrain uptake uptake in baboons in baboons was was very very low low over (<0.001% the 80-min ID/cc) scan time [89] over the 90-min scan time [11C]22 Class I [101] Approximately 60% of the plasma radioactivity was unchanged ligand at 40 min p.i.

11 11 Total VT values of [ C]23 and [ C]24 in the baboon brain were [11C]23 0.41 and 12 mL/cm3, respectively 11 Class I 11 [105] [ C]24 The degree of VT reduction of [ C]24 by unlabeled 24 or SAHA (1 mg/kg) ranged from 8–24% in various brain regions

Incidentally, in vivo P-gp blocking studies in rodents or NHPs were performed for some radioligands but showed no difference in brain uptake compared to baseline, suggesting that they are not P-gp substrates [68,93,101]. [18F]1, the first HDAC-targeting radioligand, showed a significant accumulation of radioactivity in the rhesus macaque brain, and substrate specificity analysis and Molecules 2018, 23, 300 20 of 27 immunohistochemical analysis for HDAC isoforms confirmed that these results reflect the level of class IIa HDACs in the brain [26]. However, quantitative analysis required consideration of the influence of brain uptake of a major radiometabolite, [18F]FACE. After this report in 2013, we could not find publications on clinical studies of [18F]1. Adamantane-conjugated hydroxamic acid-based HDAC radioligands tended to show preferable brain uptake in PET studies with NHPs, and especially, the study of kinetic analysis of [11C]8 is well advanced [68,79,80]. An in vivo blocking experiment using [11C]8 PET is also expected to be a tool to estimate brain penetrance of HDAC inhibitors for CNS diseases [74]. For 18F-labeled adamantane-conjugated ligands, further preclinical studies including metabolism analysis and brain kinetics analysis in NHPs are awaited. Following the promising preclinical studies, Wey and Gilbert et al. reported the first-in-human evaluation of [11C]8 PET in 2016 [28]. They conducted [11C]8 PET imaging on eight healthy volunteers (four females and four males; 28.6 ± 7.6 years old). [11C]8 showed rapid brain uptake after injection followed by a slight increase in brain radioactivity during 90 min p.i. Cortical SUV from 60 to 90 min was twice that of white matter, and consequently, white matter was selected as a reference region to determine SUV60–90 min ratios (SUVRs) (Figure4). The lowest SUVRs were observed in the hippocampus and amygdala (around 1.5), and the highest SUVRs were observed in the putamen and cerebellum (around 2). VT values determined using the metabolite-corrected arterial plasma as input fraction were stable beyond 50 min p.i., whereas information about the degree of unchanged radioligand in plasma was not described in the paper. The regional SUV60–90 min correlated well with the VT 11 values (r = 0.98, p < 0.0001); therefore, SUV60–90 min may be useful for quantification of [ C]8 in future studies without arterial blood sampling. Test-retest scans (3 h apart) in three subjects showed less than 3% variability in SUV60–90 min. Western blotting analysis of postmortem brain tissues, which were obtained independently from the imaging study, demonstrated that amounts of HDAC2 and 3 in the superior frontal gyrus (gray matter) were significantly higher than those in the corpus callosum (white matter), whereas differences in HDAC1 and 6 amounts were not significant between the superior frontal gyrus and corpus callosum. In further biochemical profiling using a thermal shift assay, [11C]8 was confirmed to selectively bind to HDAC1, 2, and 3. Although more research is needed to evaluate age-related and disease-related changes in HDAC expression, this study provided a critical foundation for quantification of epigenetic activity in the human brain. Molecules 2018, 23, x 21 of 27

Figure 4. [11C]8 PET images in a first-in-human study. (A) Brain SUV images averaged from 60 to Figure 4. [11C]8 PET90 min images p.i. of [ in11C] a8 first-in-human(174 MBq). PET images study. are overlaid (A) Brain on MRI; SUV (B) [11 imagesC]8 SUVR averagedimages of eight from 60 to 90 min p.i. 11 individual subjects. SUV60–90 min was normalized to white 11matter as a reference. (Reproduced with of [ C]8 (174 MBq).permission PET images from Wey are et al. overlaid Sci Transl on Med; MRI; The American (B)[ C] Association8 SUVR for images the Advancement of eight of individual subjects. Science, 2016 [28]). SUV60–90 min was normalized to white matter as a reference. (Reproduced with permission from Wey et al. Sci Transl Med;5. The Conclusions American Association for the Advancement of Science, 2016 [28]). Epigenetic abnormalities in the brain have been attracting more attention as therapeutic targets for neurodegenerative diseases. In this regard, non-invasive imaging techniques that can quantify the expression and/or activity of epigenetics-related enzymes in the living brain may deepen our understanding of the role of epigenetics in neurodegeneration and also help in development of new treatments; nevertheless, no clinically applicable technique currently exists. Although several types of epigenetic enzymes are expressed, development of PET imaging ligands for HDACs is particularly advanced at present. In this review, we summarized the radiosynthesis and preclinical characteristics of dozens of potential HDAC PET ligands developed in this decade to identify the achievements and challenges in the field. Brain uptake of just under 20 HDAC radioligands has already been assessed in NHPs with PET (Table 5); however, few of these radioligands readily enter the brain. Some radioligands showed low brain uptake in NHPs, although they had a preferable molecular weight and lipophilicity to cross the BBB by passive transport [107]; therefore, investigation into the background of the poor BBB permeability could help optimize radioligands for HDAC imaging in the brain. Besides the radioligands with binding affinity for multiple HDACs, the development of isoform-specific ligands is also awaited because disease-related changes in HDAC expression may be isoform specific. In this regard, evaluation of in vivo selectivity of radioligands (e.g., in vivo blocking assays using selective HDAC inhibitors and comparison of ex vivo ARG and immunohistochemical staining in rodent brain sections) is important in addition to in vitro HDAC inhibition assays. Moreover, PET imaging studies with disease model animals could reveal the time course of pathological changes and cognitive and behavioral manifestations by performing multiple longitudinal scans in the same subject. At the moment, the

Molecules 2018, 23, 300 21 of 27

5. Conclusions Epigenetic abnormalities in the brain have been attracting more attention as therapeutic targets for neurodegenerative diseases. In this regard, non-invasive imaging techniques that can quantify the expression and/or activity of epigenetics-related enzymes in the living brain may deepen our understanding of the role of epigenetics in neurodegeneration and also help in development of new treatments; nevertheless, no clinically applicable technique currently exists. Although several types of epigenetic enzymes are expressed, development of PET imaging ligands for HDACs is particularly advanced at present. In this review, we summarized the radiosynthesis and preclinical characteristics of dozens of potential HDAC PET ligands developed in this decade to identify the achievements and challenges in the field. Brain uptake of just under 20 HDAC radioligands has already been assessed in NHPs with PET (Table5); however, few of these radioligands readily enter the brain. Some radioligands showed low brain uptake in NHPs, although they had a preferable molecular weight and lipophilicity to cross the BBB by passive transport [107]; therefore, investigation into the background of the poor BBB permeability could help optimize radioligands for HDAC imaging in the brain. Besides the radioligands with binding affinity for multiple HDACs, the development of isoform-specific ligands is also awaited because disease-related changes in HDAC expression may be isoform specific. In this regard, evaluation of in vivo selectivity of radioligands (e.g., in vivo blocking assays using selective HDAC inhibitors and comparison of ex vivo ARG and immunohistochemical staining in rodent brain sections) is important in addition to in vitro HDAC inhibition assays. Moreover, PET imaging studies with disease model animals could reveal the time course of pathological changes and cognitive and behavioral manifestations by performing multiple longitudinal scans in the same subject. At the moment, the adamantane-conjugated radioligand, [11C]8, seems to be the most promising radioligand to image HDACs in the human brain. Clinical studies using [11C]8 to assess age-related and disease-related changes in brain HDACs could provide valuable information about the roles of HDACs in neurodegenerative diseases.

Acknowledgments: This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant No. 16H07486. Author Contributions: Tetsuro Tago and Jun Toyohara wrote and revised the manuscript. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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