eukaryotic 
 cellular identity , growth , and homeostasis , extending 
 to the level of organs , systems , and whole organisms depends in part 
 on refined and adaptive regulatory control of gene expression .
nongenetic determinants of gene expression 
 variability  has highlighted the role of chromatin modifying 
 enzymes in integrating environmental cues into biological effects 
 via changes in transcriptional activity .
to date , much attention 
 in this regard has been paid to the histone 
 deacetylase ( hdac ) family of enzymes .
hdac enzymes are divided into four different classes based on sequence 
 homology , cellular localization , and phylogenic relationships to yeast 
 homologues : class i ( hdacs 1 , 2 , 3 , and 8) , class iia ( hdacs 4 , 5 , 
 7 , and 9 ) , class iib ( hdacs 6 , 10 ) , class iii ( sirtuins 17 ) , 
 and class iv ( hdac 11 ) . abnormal expression levels ( both increased 
 and decreased expression ) of hdac 1 , 2 , 3 , and 6 that have been correlated 
 with many diseases , including several forms of cancer , heart failure , 
 inflammatory diseases , and cognitive and psychiatric disorders ( table 1 ) . however
, our knowledge of normal hdac density 
 and alterations in hdacs with human disease remains extremely limited 
 and invasive methodologies used so far to investigate hdac distribution are not compatible with translational medicine .
therefore , our goal 
 was to develop a positron emission tomography ( pet ) imaging probe , 
 which can quantify the density of hdac 1 , 2 , 3 , and 6 in vivo . evaluating 
 the expression and distribution of epigenetic machinery in vivo will 
 improve our understanding of their relationship with disease and would 
 provide an opportunity to intervene with treatment .
the challenge for hdac research is developing a technique 
 for quantifying 
 the hdac density that can be used in living subjects .
the non - invasive 
 imaging technique , pet , is an excellent tool to visualize chromatin - modifying 
 enzymes in animals and human .
pet can provide molecular level insight 
 into protein density and interaction with drugs by quantifying the 
 distribution of a radiotracer with extraordinarily high sensitivity . 
 to date
there are no validated tools for assessing the densities of 
 hdac enzymes in human despite the importance of these enzymes and 
 their remarkably high expression .
efforts by others have resolved 
 non - invasive probes for imaging hdac enzyme density in vivo , however these tools have rapid binding kinetics and poor brain penetrating 
 ability or bind to a different subset of hdacs than the isoforms that 
 have been extensively implicated in diseases via study of patients 
 and preclinical animal models . herein , we report the development 
 of [ c](e)-3-(4-((((3r,5r,7r)-adamantan-1-ylmethyl)(methyl)amino)methyl)phenyl)-n - hydroxyacrylamide ( [ c]martinostat , [ c]6 ) ( figure 1 ) as a 
 molecule ready for translation to human pet imaging ( hdac 1 , 2 , 3 , 
 and 6 ) .
compound 6 is an adamantane - based hydroxamic 
 acid with selective binding to hdac 1 , 2 , 3 , and 6 with subnanomolar 
 potency and fast binding kinetics . using rodent and nonhuman primate 
 pet imaging , we demonstrate robust uptake and high specific binding 
 in brain , heart , spleen , kidney , and pancreas .
using this tool , measurement 
 of hdac density of target isoforms 1 , 2 , 3 , and 6 is possible in peripheral 
 organs and brain for the first time .
measuring hdac target density / occupancy 
 with the aformentioned non - invasive tool promises a major advance 
 to understand epigenetic mechanism in normal body tissues and diseases . 

self - blocking experiments have demonstrated the sensitivity of [ c]6 to resolve differences in hdac expression 
 in peripheral organs and brain .
further , blocking experiments with 
 the clinical approved hdac inhibitor , saha , underscore the utility 
 of this tool to evaluate hdac density and target engagement with different 
 therapeutics . taken together , our findings highlight the immediate 
 utility of [ c]6 as a preclinical pet tool 
 to investigate hdac expression in animal models .
furthermore , we outline 
 and discuss the optimal properties of [ c]6 as an hdac radiotracer that strongly support its development as 
 a clinical research tool .
we have developed a potent hdac imaging agent , termed [ c]6 , incorporating three key structural features to 
 create a versatile and translational probe for visualizing hdac expression 
 in vivo .
intravenous injection of trace amounts of [ c]6 ( nanogram scale ) in baboon and imaging by pet - mr demonstrates 
 quantifiable uptake in the brain and in diverse peripheral organs . 

this illustrates the potential for [ c]6 as 
 a broadly applicable tool in evaluating hdac density in humans .
hdac inhibitors 
 comprise diverse structural classes including hydroxamic 
 acids , natural cyclic peptides , and short - chain fatty acids as well 
 as aminoanilines and ketones .
most classes of hdac inhibitors contain 
 three main structural elements to their pharmacophore : ( i ) a capping 
 group , ( ii ) a linker domain , and ( iii ) a zinc - binding group , critical 
 for enzyme action .
modification of these structural elements has so 
 far revealed that the brain uptake of most hdac inhibitors is limited . to develop an hdac pet probe with broad disease applicability , we 
 wanted to integrate a chemical structure that would exhibit robust 
 exposure to both peripheral organs and the brain . in medicinal chemistry 
 efforts by others ,
the adamantyl group has been identified as a structural 
 subunit that confers cns distribution in several drugs classes including 
 antiviral drugs , cannabinoid receptor 
 modulators .
further , adamantane - based 
 hydroxamic acids were recently reported and showed strong evidence 
 of hdac inhibition . using these previous 
 studies to guide our synthesis efforts
, we designed compound 6 , a cinnamic acid - based hdac inhibitor containing an adamantyl 
 group as a  blood brain barrier ( bbb ) carrier ( figure 1 ) .
compound 6 was synthesized through 
 reductive amination , followed by conversion into a hydroxamic acid 
 in the presence of hydroxylamine and sodium hydroxide . the radiosynthesis 
 of [ c]6 was accomplished using its desmethyl 
 precursor in dmso with [ c]methyl iodide ( [ c]ch3i ) as outlined in scheme 1 .
reagents and conditions : ( a ) 
 nabh4 , meoh , overnight , rt , 75% ; ( b ) formaldehyde , acoh , 
 nabh4 , meoh , rt , overnight , 55% .
( c ) nh2oh ( aq ) , 
 1 m naoh , meoh / thf , 0 c to rt , 4 h , 42% for 6 , 40% 
 for 5 ; rcy of [ c]6 , 35% 
 ( non - decay corrected to trapped [ c]ch3i ) .
the in vitro inhibitory activities of 6 were measured 
 for each hdac isoform hdac1 through hdac9 ( table 2 ) using recombinant human enzymes , with ic50 values 
 for the fda approved hdac inhibitor drug , suberoylanilide hydroxamic 
 acid , vorinostat ( saha ) , measured in parallel as a reference ( to assess 
 the variability among different assays ) .
compound 6 showed 
 nanomolar ic50 values for class - i hdac isoforms ( hdac 13 , 
 0.3 , 2.0 , and 0.6 nm , respectively ) , and the class - iib hdac 6 ( 4.1 
 nm ) , with > 100-fold selectivity over each of the other hdac isoforms 
 assayed .
using [ c]6 in vitro , we measured 
 the partition coefficient ( log d ) at 2.03 and calculated 
 tpsa of 52.5 , which are in the preferred range for brain penetration , 
 and further detected plasma protein binding ( ppb ) , as measured in 
 blood from baboon ( 1.4% unbound ) and human ( 5.5% unbound ) , at levels 
 consistent with those for the clinical dopamine d2 receptor 
 radiotracer , [ c]raclopride ( figure s1 ) . in vitro ic50 ( nm ) 
 values using recombinant human enzymes for hdac subtypes 19 
 and specific substrates demonstrate that 6 is a selective 
 inhibitor of hdac 13 ( 0.32.0 nm ) with decreased potency 
 to inhibit hdac 6 ( 4.1 nm ) or other subtypes ( > 352 nm ) .
comparatively , 
 the hydroxamate hdac inhibitor saha exhibited lower affinity for hdac 
 targets 13 ( 3.011 nm ) .
response plots 
 in cultured primary mouse neuronal cells measuring relative h3k9ac 
 and h4k12ac levels revealed potent induction of histone acetylation 
 ( ec50 ) by 6 ( 100 nm ) compared to saha ( 19003400 
 nm ) . to test the selective 
 binding of 6 over other potential 
 protein targets , we screened the percentage inhibition of 6 ( 50 nm ) using radioligand displacement assay against 4 zinc - dependent 
 enzymes including matrix metalloproteinases and 80 additional non - hdac 
 or zn - dependent central nervous system ( cns ) targets .
we determined 
 minimal if any off - target binding , indicating high selectivity of 6 to the hdac family ( table s1 ) . 

the only potential off - target binding was the dopamine transporter 
 ( dat ) as implicated by the in vitro test . 6 ( 50 nm ) resulted in a 24% inhibition of dat ( where 
 50% inhibition is the minimum threshold to identify a potential binding 
 event ) .
therefore , to exclude a putative binding interaction of 6 to dat ( given that it is a relatively high dentistry brain 
 protein ) , we used the established radiotracer [ c]-cft 
 to evaluate dat density / occupancy in rats and observed no impact of 6 treatment ( 1 mg / kg , ip , 90 min prior ) ( figure s2 ) . to further establish that [ c]6 binding 
 was specific for hdac targets
we observed that specific binding of 
 [ c]6 was reduced by preincubation of sagittal 
 rat brain sections with either of the unlabeled hdac inhibitors : saha , 
 a hydroxamic acid - based hdac inhibitor ( selective for class i and 
 iib hdac isoforms ) , or ci-994 , a benzamide - based hdac inhibitor ( selective 
 for hdac 1 , 2 , and 3 isoforms ) ( figure s1c , d ) . to first test 
 [ c]6 as a radiotracer in vivo
, we conducted 
 pet imaging focused on rat brain . using pet - ct , we determined that 
 [ c]6 exhibited high bbb penetration and 
 sustained binding over the scanning time ( 60 min ) when administered 
 by intravenous bolus injection ( 0.91.1 mci per animal ) , as 
 shown in figure s3a . to confirm that [ c]6 was not effluxed from the brain by the xenobiotic 
 pump , p - glycoprotein ( p - gp ) , we measured radioligand uptake in brain 
 after treatment with the p - gp inhibitor , cyclosporin a ( csa ) , and 
 found no difference compared to control treatment ( figure s4a ) . additionally , [ c]6 binding 
 in the rat brain was disrupted by an iv challenge with unlabeled 6 20 min after tracer administration as evidenced by a rapid 
 decrease in the time 
this rapid loss 
 of radioactivity from brain regions provided confirmation of the reversibility 
 of [ c]6 binding to cns targets ( figure s4b ) . to determine the stability 
 of [ c]6 in brain , we measured the presence 
 of radiolabeled parent compound and polar metabolites in homogenized 
 rat brain tissue 30 min post - in vivo injection of [ c]6 . by eluting extracted and homogenized rat brain with solvents 
 containing fractional increases of acetonitrile in water
, we resolved 
 a pattern of radioactivity indicating intact parent compound in the 
 brain with limited degradation over the duration of a typical 3090 
 min pet scan ( figure s5a ) . a similar pattern 
 indicating unmetabolized [ c]6 was observed 
 in baboon plasma , 5 min post - injection of the tracer ( figure s5b ) .
to investigate the specificity 
 of [ c]6 , we performed pet imaging studies 
 in rats with a 5 min pretreatment of unlabeled 6 at different 
 doses ( 0.001 , 0.1 , 0.5 , and 2.0 mg / kg ) .
we found that [ c]6 binding in brain was blocked in a dose - dependent 
 manner with a stepwise reduction in the percent tracer uptake after 
 administration of unlabeled 6 ( figure 
 s3b ) . to test the sensitivity of [ c]6 against other hdac inhibitors
, we pretreated a rat with cn54 , a 
 highly brain penetrant hydroxamic acid hdac inhibitor as determined 
 by lc - ms - ms ( brain / plasma = 4.5 after 20 min administration of cn54 
 at 10 g / kg via iv ) , and saha , a hydroxamic acid hdac inhibitor 
 with limited brain penetrating ability . as expected , there was robust in vivo blockade of radioactivity 
 in brain with the treatment of cn54 , but not saha ( figure s3c ) .
next , we chose to utilize nonhuman primates to evaluate 
 the distribution 
 and specificity of [ c]6 . in baboons
, we 
 found that high brain uptake ( 5060 in vt , total volume of distribution ) of [ c]6 based on pet - mr focused on the head .
similar to the blocking results 
 we obtained in rats , injection of unlabeled 6 ( 0.5 and 
 1.0 mg / kg , iv ) dose - dependently reduced the radioactivity uptake ( measured 
 as vt ) in specific brain regions including 
 cerebellum , cortex , putamen , and caudate ( figure 2 ) .
the averaged bmax of whole 
 brain , 19.6  2.9 m of protein , was calculated from the 
 pet image on the basis of the established relationship between total 
 binding and dissociation rate and was 
 consistent with ex vivo protein quantification in rodents by two methods .
physiological effects ( i.e. , respiration rate , 
 blood pressure , heart rate , and end - tidal co2 ) were unchanged 
 at any administered dose of 6 or [ c]6 .
( a ) the total volume of distribution ( vt ) images from one representative animal show robust differences 
 in radiotracer uptake at baseline and after blocking ( 0.5 mg / kg iv , 
 10 min pretreatment ) ; ( b ) two independent baseline - blocking studies 
 were used to resolve quantitative vt data 
 which show that pretreatment with unlabeled 6 ( 0.5 or 
 1.0 mg / kg ) dose - dependently blocks tracer uptake in different baboon 
 brain regions .
wb : whole brain ; cb : cerebellum ; m1 : primary motor 
 cortex ; pu : putamen ; th : thalamus ; v1 : primary visual cortex ; ca : 
 caudate ; wm : white matter .
after [ c]6 intravenous bolus injection , radioactivity in arterial plasma 
 clears rapidly as expected and as depicted in figure s5c .
polar metabolites were observed in the plasma 
 ( 50% of the plasma radioactivity after 10 min ) ; however , radioactivity 
 in brain tissue of rodents at late time points ( 30 min post administration ) 
 was associated almost exclusively with the parent molecule .
these 
 data support that the pet images for the brain tissue largely represent 
 [ c]6 and not radiolabeled metabolites . for 
 quantitative analysis of the nhp pet imaging data ,
two - tissue 
 compartmental modeling was applied to analyze dynamic [ c]6 pet data , corrected for arterial input function 
 and parent compound metabolism to interpret results from intact and 
 perfused radiotracer .
based on the modeling results ( figure 2 ) , blocking with 0.5 and 1.0 mg / kg , vt decreased in all brain regions . based on our rodent 
 experiments and dose - equilibration for body mass , we assumed that 
 a 1.0 mg / kg dose would provide full saturation of hdac binding .
therefore , 
 5080% of the signal represented specific binding in the selected 
 brain regions suggesting that [ c]6 has sufficient 
 sensitivity to assess a wide range of cns diseases in which hdac may 
 be altered , such as alzheimer s disease and in mood disorders , and for evaluation of drug - hdac 
 engagement .
we further 
 evaluated the tracer uptake and blockade in peripheral organs in baboons . 

[ c]6 showed high uptake in major organs 
 including heart , kidney , pancreas , and spleen ( figure 3 ) .
the uptake in these organs was markedly decreased by pretreatment 
 with unlabeled 6 , indicating that the [ c]6 signal also represents hdac expression in peripheral organs . 
 to further confirm the sensitivity of [ c]6 to detect changes in hdac occupancy , we measured radiotracer uptake 
 in the baboon both before and after 75 min infusion of saha ( 5 mg / kg , 
 iv ) .
we found that radiotracer uptake in peripheral tissue was robustly 
 blocked by saha infusion , consistent with efficient distribution of 
 this hdac inhibitor blocking agent in nonbrain organs ( figure s6 ) .
axial views of summed 
 pet images ( 4080 min ) superimposed with mr images from the 
 same baboon following injection of radiotracer ( 4 mci / baboon ) . images 
 illustrate tracer uptake in organs of interest at baseline and after 
 pretreatment with unlabeled 6 ( 0.5 mg / kg ) .
robust blocking 
 was observed in organs of interest including : a , heart ; b , spleen ; 
 c , kidneys ; and d , pancreas .
activity curves ( baseline , 
 blue ; blocking , red ) demonstrate a high specific binding of [ c]6 in these peripheral organs as the percent 
 injected tracer dose per cm tissue is markedly reduced 
 by blocking .
epigenetic therapies that target regulatory mechanisms of the human 
 genome represent an emerging class of novel therapeutic agents .
the 
 importance of understanding aberrant enzyme expression of chromatin 
 modifying enzymes including hdacs is highlighted by the discovery 
 that protein coding mutations may play a role in human disease pathologies 
 linked to cancer in major organs , cardiac dysfunction , and cns disorders . 

the impact of these enzyme density changes is further implicated in 
 disease as recent large - scale genome - wide association studies ( gwas ) 
 have shown that gene regulatory regions key in many common human diseases 
 harbor an enrichment of single nucleotide polymorphisms ( snps ) .
this evidence of deregulated gene expression mechanism points to 
 a more general need to target epigenetic machinery that may be causally 
 involved in disease pathogenesis .
therefore , developing therapeutics 
 for many human diseases can be best accomplished by first understanding 
 the density and distribution of epigenetic machinery including hdac 
 enzymes in vivo .
development of specific pet radiotracers to 
 measure hdac density 
 or any other epigenetic component , with high uptake and major organs , 
 is challenging .
there are a number of 
 major factors ( summarized here a through d ) that determine the success 
 of a radiotracer candidate .
some factors can be predicted from in 
 vitro assays ( e.g. , binding affinity and selectivity ) , and those which 
 are more empirically determined ( e.g. , specific to nonspecific binding 
 ratio ) .
( a ) the binding affinity and selectivity of a radiotracer 
 for its target must be high enough to produce sufficient signal to 
 background for detection and selective for the target so that images 
 represent the biological target .
( b ) additionally , radiotracers must 
 exhibit specific binding , which is challenging to predict but can 
 be easily measured by presaturating the target with an unlabeled ( nonradioactive ) 
 form of the radiotracer thus resulting in decreased binding of radiotracer 
 to its target .
unlabeled compound binding is also expected to alter 
 the distribution and pharmacokinetics of the radioligand .
in general , small 
 molecular weight ( < 400 da ) molecules with relatively high lipophilicity 
 are required for a tracer to sufficiently penetrate the bbb .
predictive 
 models based on pet imaging data are emerging for hdac inhibitors 
 that suggest the total polar surface area ( tpsa ) needs to be < 65 .
( d ) with all of the above properties satisfied , 
 a tracer candidate can still be rendered unusable in vivo if it is 
 metabolized rapidly and those metabolites pervade the tissue regions 
 of interest .
each of the primary factors for a successful radiotracer 
 is met by [ c]6 and supported by our biochemical 
 and preclinical imaging data .
of the more than 100 compounds we assessed 
 through our tracer identification work , [ c]6 was unique . while we have observed some trends in molecular properties , 
 structure , and in vivo behavior , fully understanding the molecular 
 basis for [ c]6 success will require additional 
 structure  activity relationship determination and computational 
 modeling .
our data support [ c]6 as 
 a new tool to 
 visualize hdac density in the body and brain .
the robust and blockable 
 brain uptake with [ c]6 as a pet probe can 
 be used to quantify hdac density in brain disorders , such as neurodegenerative 
 diseases and mood disorders , and for demonstrating target engagement 
 of putative hdac inhibitor therapeutics early in compound development 
 pipeline .
we found stable probe uptake in diverse peripheral organs 
 in nonhuman primates , each with a distinct profile of hdac target 
 density and affinity as evidenced by time 
, we further 
 demonstrated that [ c]6 binding could be 
 prevented using a known , clinically applied hdac inhibitor drug ( figure s6 ) illustrating the direct utility of 
 this probe for understanding systemic impact of a cancer therapeutic . 

a recent report demonstrates that class i hdac inhibition by a prototypical 
 hdaci therapeutic , ms-275 , stimulates renal dysfunction .
dose - limiting toxicity may not be surprising 
 given the widespread expression and central role of hdacs in normal 
 biological function .
however , this underscores a mechanism - based liability 
 for hdac inhibitor leads proposed to date as cns disease therapeutics 
 where large mass dosing strategies are used to reach effective concentrations 
 of hdaci in brain .
importantly , the ability to simultaneously measure hdac target engagement 
 in peripheral organs and brain is a major development in not only 
 understanding cns disease mechanisms but also in prioritizing novel 
 hdac inhibitors with minimal systemic burden . in summary ,
[ c]6 binds to hdac subtypes 
 ( hdac 1 , 2 , 3 , and 6 ) with high selectivity and specificity and provides 
 a tool for quantitative imaging of hdac density in the brain and in 
 peripheral organs in vivo .
[ c]6 can be used 
 as a pet radioligand for studying hdacs in disorders where altered 
 regulation of hdacs is found .
the approach is also valuable for evaluation 
 of potential drugs in vivo , therapy response can be compared in the 
 same animal before drug treatment .
safety and toxicology studies have 
 been completed , and we are progressing [ c]6 forward for evaluation as the first hdac pet radiotracer and a novel 
 class of epigenetic imaging probes in humans .
all reagents and solvents 
 were of acs - grade purity or higher and used without further purification . 

nmr data were recorded on a varian 500 mhz magnet and were reported 
 in ppm units downfield from trimethylsilane .
analytical separation 
 was conducted on an agilent 1100 series hplc fitted with a diode - array 
 detector , quaternary pump , vacuum degasser , and autosampler .
mass 
 spectrometry data were recorded on an agilent 6310 ion trap mass spectrometer 
 ( esi source ) connected to an agilent 1200 series hplc with quaternary 
 pump , vacuum degasser , diode - array detector , and autosampler .
the 
 precursor and compound 6 were synthesized ( from sai life 
 sciences ltd . ) with > 98% purity , as determined by a high - pressure 
 liquid chromatography ( hplc ) system ( ymc triart c-18 ( 150 mm  
 4.6 mm  3 u ) mobile phase : a : 0.05% tfa in water / b : 0.05% tfa 
 in acetonitrile .
gradient : 15% b to 95% b in 
 8.0 min , hold until 9.5 min , 15% b in 13.0 min hold until 15.0 min . 

the quantitative nmr test were conducted by sai life sciences ltd . , 
 and 1,4-dimethoxybenzene was used as the reference .
[ c]co2 ( 1.2 ci ) was obtained via the n ( p , )c reaction on nitrogen with 2.5% 
 oxygen , with 11 mev protons ( siemens eclipse cyclotron ) , and trapped 
 on molecular sieves in a tracerlab fx - mei synthesizer ( general electric ) . 

[ c]ch4 was obtained by the reduction of [ c]co2 in the presence of ni / hydrogen at 350 c 
 and recirculated through an oven containing i2 to produce ch3i via a radical reaction .
all animal 
 studies were carried out at massachusetts general hospital ( phs assurance 
 of compliance no .
the subcommittee on research animal care 
 ( srac ) serves as the institutional animal care and use committee ( iacuc ) 
 for the massachusetts general hospital ( mgh ) .
pet - mr imaging was performed 
 in anesthetized ( ketamine , isoflurane ) 
 baboon ( papio anubis ) to minimize discomfort . 

highly trained animal technicians monitored animal safety throughout 
 all procedures , and veterinary staff were responsible for daily care . 
 all animals were socially housed in cages appropriate for the physical 
 and behavioral health of the individual animal .
animals were fed thrice 
 per diem , with additional nutritional supplements provided as prescribed 
 by the attending veterinarian .
audio , video , and tactile enrichment 
 was provided on a daily basis to promote psychological well - being . 

pet - ct imaging was performed in anesthetized ( isoflurane ) rats 
 ( sprague  dawley ) to minimize discomfort .
highly trained animal 
 technicians monitored animal safety throughout all procedures , and 
 veterinary staff were responsible for daily care .
all animals were 
 socially housed in cages appropriate for the physical and behavioral 
 health of the individual animal .
animals were given unlimited access 
 to food and water , with additional nutritional supplements provided 
 as prescribed by the attending veterinary staff .
animals were euthanized 
 at the end of the study using sodium pentobarbital ( 200 mg / kg , ip ) .
adamantan-1-ylmethanamine ( 1 ) ( 1 g , 6.0 mmol ) and ( e)-methyl 3-(4-formylphenyl)acrylate 
 ( 2 ) ( 1 g , 5.3 mmol ) was dissolved in meoh ( 30 ml ) , and 
 the mixture was stirred at room temperature for 2 h. sodium borohydride 
 ( 0.61 g , 16 mmol ) was then added , and the suspension was stirred overnight 
 at room temperature .
the white precipitate was filtered and dried 
 to obtain the product 3 ( 1.35 g , yield : 75%).h nmr ( 500 mhz , cdcl3 ) :  7.69 ( d , j = 16 hz , 1h ) , 7.48 ( d , j = 7 hz , 2h ) , 7.35 ( d , j = 7 hz , 2h ) , 6.43 ( d , j = 16 hz , 1h ) , 
 3.81 ( s , 2h ) , 3.80 ( s , 3h ) , 2.23 ( s , 3h ) , 1.96 ( s , 3h ) , 1.631.73 
 ( m , 6h ) , 1.53 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) : 167.55 , 144.78 , 143.81 , 132.87 , 128.35 ( 2c ) , 128.08 ( 2c ) , 117.10 , 
 62.15 , 54.28 , 51.65 , 40.85 ( 3c ) , 37.24 ( 3c ) , 33.49 , 28.48 ( 3c ) .
lc - ms 
 calculated for c22h29no2 expected 
 [ m ] : 339.2 ; found [ m + h ] : 340.3 .
to a solution 
 of 3 ( 0.5 g , 1.5 mmol ) in meoh / thf ( 5 ml/5 ml ) at 0 c 
 was added nh2oh ( 50% aq solution , 3 ml ) followed by 1 m 
 naoh ( 2 ml ) .
the mixture was stirred at 0 c for 2 h , warmed 
 to room temperature , and stirred for 2 h. acidification with 1 m hcl 
 to ph 78 ( ph paper ) resulted in product precipitation .
the 
 precipitate was filtered and dried to obtain 4 ( 0.2 g , 
 40% ) as white solid .
h nmr ( 500 mhz , cdcl3 ) : 
  7.69 ( d , j = 16 hz , 1h ) , 7.47 ( d , j = 7.5 hz , 2h ) , 7.39 ( d , j = 7.5 hz , 2h ) , 
 6.42 ( d , j = 16 hz , 1h ) , 3.80 ( s , 3h ) , 3.54 ( s , 2h ) , 
 2.19 ( s , 3h ) , 2.10 ( s , 2h ) , 1.95 ( s , 3h ) , 1.621.723 ( m , 6h ) , 
 1.53 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) : 164.92 , 
 141.31 , 139.69 , 133.75 , 128.61 ( 2c ) , 127.42 ( 2c ) , 116.88 , 71.09 , 61.08 , 
 53.31 , 40.41 ( 3c ) , 36.77 ( 3c ) , 32.84 , 28.48 ( 3c ) .
lc - ms calculated 
 for c21h28n2o2 expected 
 [ m ] : 340.5 ; found [ m + h ] : 341.3 .
quantitative nmr purity : 95.10% . to a solution of 3 ( 0.5 g , 1.5 mmol ) in meoh
( 30 ml ) 
 was added formaldehyde ( 33% aq solution , 2 ml ) followed by acetic 
 acid ( 0.1 ml ) .
the mixture was stirred at room temperature for 2 h. 
 sodium borohydride ( 0.61 g , 16 mmol ) was then added , and the suspension 
 was stirred overnight at room temperature .
the white precipitate was 
 filtered and purified by flash chromatography in hexanes : ethyl acetate 
 ( 4:1 ) to obtain the product 3 ( 0.29 g , yield : 55% ) as 
 white solid .
h nmr ( 500 mhz , meoh - d4 ) :  7.55 ( d , j = 16 hz , 1h ) , 7.52 
 ( d , j = 7.5 hz , 2h ) , 7.37 ( d , j = 
 7.5 hz , 2h ) , 6.47 ( d , j = 16 hz , 1h ) , 3.77 ( s , 2h ) , 
 2.21 ( s , 2h ) , 1.94 ( s , 3h ) , 1.661.76 ( m , 6h ) , 1.541.55 
 ( m , 6h ) ; c nmr ( 125 mhz , meoh - d4 ) : 164.93 , 141.32 , 139.71 , 133.76 , 128.62 ( 2c ) , 127.43 ( 2c ) , 
 116.8901 , 61.08 , 53.32 , 40.42 ( 3c ) , 36.78 ( 3c ) , 32.84 , 28.48 ( 3c ) . 

lc - ms calculated for c21h28n2o2 expected [ m ] : 353.2 ; found [ m + h ] : 354.1 . to a solution 
 of 5 ( 0.5 g , 1.4 mmol ) in meoh / thf ( 3 ml/3 ml ) at 0 c 

was added nh2oh ( 50% aq solution , 3 ml ) followed by 1 m 
 naoh ( 2 ml ) .
the mixture was stirred at 0 c for 2 h , warmed 
 to room temperature , and stirred for 2 h. acidification with 1 m hcl 
 to ph 78 ( ph paper ) resulted in product precipitation .
the 
 precipitate was filtered and dried to obtain 6 ( 0.21 
 g , 42% ) as white solid .
h nmr ( 500 mhz , dmso - d6 ) :  7.49 ( d , j = 7.5 hz , 2h ) , 
 7.42 ( d , j = 16 hz , 1h ) , 7.33 ( d , j = 7.5 hz , 2h ) , 6.45 ( d , j = 16 hz , 1h ) , 3.48 ( s , 
 2h ) , 2.11 ( s , 3h ) , 2.06 ( s , 2h ) , 1.89 ( s , 3h ) , 1.551.65 ( m , 
 6h ) , 1.46 ( s , 6h ) ;  c nmr ( 125 mhz , dmso - d6 ) : 163.15 , 142.00 , 138.43 , 139.91 , 129.36 ( 2c ) , 
 127.77 ( 2c ) , 119.06 , 70.12 , 64.57 , 45.71 , 41.01 ( 3c ) , 37.21 ( 3c ) , 
 35.27 , 28.33 ( 3c ) .
lc - ms calculated for c22h30n2o2 expected [ m ] : 354.2 ; found [ m + h ] : 355.3 .
hdac inhibition assays : compounds were tested in a 12-point 
 dose curve with 3-fold serial dilution starting from 33.33 m . 

purified hdacs were incubated with 2 m ( the concentration is 
 kept the same for all the hdacs , below km of substrate ) carboxyfluorescein ( fam)-labeled acetylated or trifluoroacetylated 
 peptide substrate ( broad substrates a and b , respectively ) and test 
 compound for 60 min at room temperature , in hdac assay buffer that 
 contained 50 mm hepes ( ph 7.4 ) , 100 mm kcl , 0.01% bsa , and 0.001% 
 tween-20 . reactions were terminated by the addition of the known pan 
 hdac inhibitor lbh -589 ( panobinostat ) with a final concentration 
 of 1.5 m . substrate and product were separated electrophoretically , 
 and fluorescence intensity in the substrate and product peaks were 
 determined and analyzed by labchip ez reader .
the percent 
 inhibition was plotted against the compound concentration , and the 
 ic50 value was determined from the logistic dose 
mouse 
 primary neuronal cultures were generated in factory precoated poly - d - lysine 96-well plates ( bd biosciences no .
bd356692 ) treated 
 overnight with 75 l / well of laminin [ 0.05 mg / ml ] ( sigma no . 

e15 embryonic mouse forebrain was dissociated 
 into a single cell suspension by gentle trituration following trypsin / dnase 
 digestion ( trypsin : cellgro no .
12,500 cells per well were plated in 100 l neurobasal medium 
 ( gibco no . 21103 - 049 ) containing 2% b27 ( gibco no .
after 13 days , cultures were 
 treated with hdac inhibitors by pin transfer of compound ( 185 nl per 
 well ) using a cybi - well vario pinning robot ( cybio corp .
, germany ) 
 and subsequently incubated for 24 h at 37 c with 5% co2 and then fixed in 4% formaldehyde for 10 min . following two washes 
 with phosphate - buffered saline
, cells were permeabilized and blocked 
 with 0.1% triton x-100 and 2% bsa in pbs .
cells were stained with 
 an anti - ac - h4k12 antibody ( millipore , catalog no . 04119 ) and 
 alexa488-conjugated secondary antibody ( molecular probes ) .
cell nuclei 
 were identified by staining with hoechst 33342 ( invitrogen , h3570 ) . 

cell nuclei and histone acetylation signal intensity were detected 
 and measured using a laser - scanning microcytometer ( acumen ex3 , ttp 
 laptech ) .
acumen explorer software was used to identify a threshold 
 of histone acetylation signal intensity such that , without hdac inhibitor , 
 > 99.5% of cells had intensity levels below the threshold
. in the 
 presence 
 of hdac inhibitors , cells signal intensities above the threshold were 
 scored as  bright green cells  and expressed as a percentage 
 normalized to dmso controls .
ec50 values were determined 
 from curve fitting using graphpad prism v5 software ( graphpad software , 
 inc .
6 was submitted 
 to a panel of 80 binding assays for common transmembrane and soluble 
 receptors , ion channels , and monoamine transporters in the cns ( high - throughput 
 profile p-3 , cerep , france ) .
this concentration was > 10 the ic50 values 
 determined for hdac targets ( 0.34.1 nm , see table 2 , main text ) and in vast excess of the amount of 6 administered in pet imaging experiments . for example , injected 
 dose = 1 mci / rat ; specific activity ( see figure 
 s2 ) = 1 mci/1 nmole ; therefore , calculated dose = 1 nmole 6/ rat .
inhibition of control binding did not exceed 45% for 
 any target assayed , supporting that 6 predominantly binds 
 hdac targets .
[ c]ch3i was trapped in a tracerlab fx - m synthesizer 
 reactor ( general 
 electric ) preloaded with a solution of precursor ( 4 ) 
 ( 1.0 mg ) in dry dmso ( 300 l ) .
the solution was stirred at 100 
 c for 4 min , and water ( 1.2 ml ) was added .
the reaction mixture 
 was purified by reverse phase semipreparative hplc ( phenomenex luna 
 5u c8(2 ) , 250  10 mm , 5 m , 5.0 ml / min , 40% h2o + ammonium acetate ( 0.1 m)/ 60% ch3cn ) , and the desired 
 fraction was collected .
the final product was reformulated by loading 
 onto a solid - phase exchange ( spe ) c-18 cartridge , rinsing with 1 m 
 naoh aq ( 5 ml ) , eluting with etoh ( 1 ml ) , and diluting with 50 l 
 acetic acid in saline ( 0.9% , 9 ml ) .
the chemical and radiochemical 
 purity of the final product was tested by analytical hplc ( agilent 
 eclipse xdb - c18 , 150  4.6 mm ) .
the identity of the product was 
 confirmed by analytical hplc with additional co - injection of reference 
 standard .
the average time required for the synthesis from end of 
 cyclotron bombardment to end of synthesis was 35 min .
the average 
 radiochemical yield was 35% ( nondecay corrected to trapped 
 [ c]ch3i ) .
chemical and radiochemical purities 
 were 95% with a specific activity 1.0  0.2 ci/mol 
 ( eob ) .
specific radioactivity 
 ( sa ) of [ c]6 was calculated from an analytical 
 hplc sample of 100 l .
a calibration curve of known mass quantity 
 versus hplc peak area ( 254 nm ) was used to calculate the mass concentration 
 of the 100 l .
sa was determined using the mass concentration 
 value determined for the formulated [ c]6 solution , volume , and measured radioactivity .
the identity of the 
 mass peak assigned to [ c]6 was supported 
 by a second hplc injection , where [ c]6 was 
 mixed with nonradioactive compound 6 and yielded a single 
 peak .
an aliquot ( 50 
 l ) of the formulated radiotracer was added to a test tube containing 
 2.5 ml of octanol and 2.5 ml of phosphate buffer solution ( ph 7.4).the 
 test tube was mixed by vortex for 2 min and then centrifuged for 2 
 min to fully separate the aqueous and organic phase . a sample taken 
 from the octanol layer ( 0.1 ml ) and
an additional aliquot of the 
 octanol layer ( 2.0 ml ) was carefully transferred to a new test tube 
 containing 0.5 ml of octanol and 2.5 ml of phosphate buffer solution 
 ( ph 7.4 ) .
the previous procedure ( vortex mixing , centrifugation , sampling , 
 and transfer to the next test tube ) was repeated until six sets of 
 aliquot samples had been prepared .
the radioactivity of each sample 
 was measured in a well counter ( perkinelmer , waltham , ma).the log d of each set of samples was derived by the following equation : 
 log d = log ( decay - corrected radioactivity in octanol 
 sample  10/decay - corrected radioactivity in phosphate buffer 
 sample ) .
an aliquot of radiotracer 
 in saline ( 10 l ) was added to a sample of baboon or human plasma 
 ( 0.8 ml ) .
the mixture was gently mixed by repeated inversion and incubated 
 for 10 min at room temperature . following incubation a small sample 
 ( 20 l )
was removed to determine the total radioactivity in 
 the plasma sample ( at ; at = abound + aunbound ) .
an additional 0.2 ml of the plasma sample was 
 placed in the upper compartment of a centrifree tube ( amicon , inc .
the upper part of the 
 centrifree tube was discarded , and an aliquot ( 20 l ) from the 
 bottom part of the tube was removed to determine the amount of radioactivity 
 that passed through the membrane ( aunbound ) .
plasma protein binding was derived by the following equation : 
 % unbound = aunbound  100/at .
twenty m 
 thick sagittal 
 rat brain sections were cut using a 20 c cryostat , thaw - mounted 
 onto gelatin - coated slides , fixed in 4% paraformaldehyde containing 
 2% ethanol for 60 min at 4 c , and washed 10 min in ice - cold 
 10 mm tris - hcl , ph 7.4 .
sections were then incubated at room temperature 
 in a 50 ml bath containing either ( i ) 5% dmso as control ; ( ii ) unlabeled 6 ( 100 m ) or ( iii ) saha ( 100 m ) for 10 min ; 
 or ( iv ) ci-994 ( 100 m ) for 2 h to allow for slow hdac binding 
 kinetics known for benzamide compounds including ci-994 .
all incubation 
 baths contained 10 mm tris - hcl + 5% dmso , ph 7.4 .
[ c]6 was prepared as described , and 100 ci was added to 
 each bath . following 10 min incubation at room temperature
, sections 
 were washed 2  1 min in 10 mm tris - hcl ( ph 7.4 ) and carefully 
 wiped dry on absorbent towels .
sections were exposed for 1 h to a 
 multisensitive phosphorscreen and developed using a cyclone plus phosphorimager 
 ( both from perkinelmer ) . resulting parent image was evaluated using 
 imagej software ( nih ) with jet color lookup table ( lut ) with whole - image 
 intensity adjusted to enrich red color in control sections .
individual 
 images of sections were cropped using imagej with no additional adjustment 
 to color levels / thresholds .
male 
 sprague  dawley rats were utilized in pairs , anesthetized with 
 inhalational isoflurane ( forane ) at 3% in a carrier of 1.52 
 l / min medical oxygen , and maintained at 2% isoflurane for the duration 
 of the scan .
the rats were arranged head - to - head in a triumph trimodality 
 pet / ct / spect scanner ( gamma medica , northridge , ca ) .
rats were injected 
 standard references or vehicle via a lateral tail vein catheterization 
 at the start of pet acquisition .
dynamic pet acquisition lasted for 
 60 min and was followed by computed tomography ( ct ) for anatomic coregistration . 

pet data were reconstructed using a 3d - mlem method resulting in a 
 full width at half - maximum ( fwhm ) resolution of 1 mm .
reconstructed 
 images were exported from the scanner in dicom format along with an 
 anatomic ct for rodent studies .
these files were imported to pmod 
 ( pmod technologies , ltd . ) and manually co - registered using six degrees 
 of freedom .
volumes 
 of interest ( vois ) 
 were drawn manually as spheres in brain regions guided by high - resolution 
 ct structural images and summed pet data , with a radius no < 1 mm 
 to minimize partial volume effects .
tacs were exported in terms of 
 decay corrected activity per unit volume at specified time points 
 with gradually increasing intervals .
papio anubis baboons , deprived of 
 food for 12 h prior to the study , were included 
 in the pet - mr scans .
atropine 0.05 mg / kg was used intramuscularly 
 to prevent excessive secretion ( 15 or 30 min of ketamine and xylazine ) . 

anesthesia was induced with intramuscular xylazine ( 0.52.0 
 mg / kg ) and ketamine ( 10 mg / kg ) .
after endotracheal intubation , v- 
 and a - lines were inserted , and anesthesia was maintained using isoflurane 
 ( 11.5% , 100% o2 or 50/50 o2/n2o , l l / min ) . during anesthesia , heart rate , respiration rate , blood 
 pressure , o2 saturation , and end tidal co2 were 
 monitored .
the baboon was catheterized antecubitally for radiotracer 
 injection , and a radial arterial line was placed for metabolite analysis .
pet - mr images were acquired in a biograph 
 mmr scanner ( siemens , munich , germany ) and pet compatible 8-channel 
 coil arrays for nonhuman primate brain imaging with a pet resolution 
 of 5 mm and field of view of 59.4 and 25.8 cm ( transaxial and axial , 
 respectively ) .
dynamic pet image acquisition was initiated followed 
 by administration of the radiotracer in a homogeneous solution of 
 10% ethanol and 90% isotonic saline .
an memprage sequence began after 
 30 min of the baseline scan for anatomic co - registration . to characterize 
 the specific binding of [ c]6 ,
a second imaging 
 experiment was carried out in which unlabeled 6 was co - administered 
 intravenously at the start of acquisition .
both scans were carried 
 out in the same animal on the same day , separated by 2.5 h. in both 
 scans , 45 mci of [ c]6 was administered 
 to the baboon .
dynamic data from the pet scans were recorded in list 
 mode and corrected for attenuation .
baboon data were reconstructed 
 using a 3d - osem method resulting in a fwhm resolution of 4 mm .
simultaneous pet - mr data were acquired 
 using a siemens biograph mmr system ( siemens medical solutions u.s.a . , 
 inc . ,
mr body imaging was performed with real - time respiratory 
 bellows - gating and using the body matrix coil and 
 the built - in spine coil as the receiving coil elements .
high - resolution 
 anatomical t1w axial , coronal , and sagittal dual - echo scan with the 
 following parameters : tr 115 ms , te1 1.23 ms , te2 2.46 ms , matrix 
 size 256  168 , fov 35 cm , phase fov 65.6% ) and 4 mm slice thickness 
 were obtained . additionally , in particular for the purpose of optimal 
 visualization of the pancreas , high - resolution fat - suppressed axial 
 t2w turbo spin echo ( tse ) data were obtained with the following parameters : 
 tr 5264 ms , te 98 ms , matrix size 488  235 , fov 18.0 cm , phase 
 fov 75% , fa 150 , and 3 mm slice thickness . for the purpose of 
 mr - based attenuation correction ( mrac ) of the pet data , default t1-weighted 
 ( t1w ) 2-point dixon 3d volumetric interpolated breath - hold examination 
 ( vibe ) scans were obtained .
pet data were obtained using a single - bed 
 acquisition with an axial field of view of 25.8 cm , transaxial field 
 of view of 59.4 cm , and axial and transaxial resolution of 4.8 
 and 4.7 mm , respectively .
pet data were acquired dynamically 
 for 80 min after administration of the radiotracer in a homogeneous 
 solution of 10% ethanol and 90% isotonic saline . to characterize the 
 specific binding of [ c]6 ,
a second imaging 
 experiment was carried out in which unlabeled 6 was co - administered 
 intravenously at the start of acquisition .
both scans were carried 
 out in the same animal on the same day , separated by 2.5 h. in both 
 scans , 45 mci of [ c]6 was administered 
 to the baboon . in order to evaluate the blocking of [ c]6 by saha , an separate dual - injection study of [ c]6 was performed . in that experiment
continuous 
 saha infusion ( 2 mg / ml at 0.16 ml / min ) was started after 45 min of 
 baseline scan with [ c]6 , and the infusion 
 was continued during the 80 min pet scan after a second injection 
 of [ c]6 . in all cases
pet data were recorded 
 in list mode , and reconstruction was performed using a 3d - osem method 
 resulting in a fwhm resolution of 4 mm .
attenuation correction was 
 performed using the aforementioned 2-point dixon 3d vibe mr - derived 
 attenuation maps .
data of each 80 min scan were reconstructed for 
 tac analysis using the following framing : 12  10 , 3  20 , 
 4  30 , and 15  300 s. for the dedicated self - blocking 
 and saha blocking experiments , additional static pet data were reconstructed 
 using data obtained between 40 and 80 min and 2045 min , respectively , 
 in order to visually display the effects of blocking in multiple organs .
pet data were motion - corrected , 
 spatially smoothed with a 2.5 mm fwhm gaussian filter , and registered 
 to the black baboon brain atlas using 
 jip tools optimized for nonhuman primate data processing ( www.nitrc.org/projects/jip ) .
image registration was carried out on high - resolution mprage mri 
 image using a 12 degree of freedom linear algorithm and a nonlinear 
 algorithm to the atlas brain .
kinetic modeling 
 was carried out in pmod ( pmod3.3 , pmod technologies ltd . ,
tacs were 
 exported from the whole brain , cerebellum , primary motor cortex , putamen , 
 thalamus , primary visual cortex , caudate , and white matter vois for 
 analysis . a two - tissue compartmental model
was used to estimate regional 
 volume of distribution ( vt ) with a metabolite - corrected 
 plasma tac .
voxel - wise vt maps were calculated 
 using a logan plot from the dynamic pet data .
the time until linearity of the plot was achieved and determined 
 for each scan .
in addition , regional k1/k2 and k3/k4 values were also obtained 
 from the baseline scans using 
 a two - tissue compartmental model .
it is known thatwith the assumption that kd = 0.4 m ( assumed from ex vivo hdac - complex kd measure for saha , which exhibits a similar ic50 in in 
 vitro activity - based assays we determined ) .
averaged bmax from the selected vois was estimated to be 19.6  
 2.9 m ( mean  sd ) .
arterial samples collected 
 during imaging from the baboon were centrifuged to obtain plasma , 
 which was then removed and placed in an automated gamma counter that 
 was calibrated to the pet scanner .
metabolite analysis was conducted 
 on a custom automated robot fitted with phenomenex spe strata - x 500 
 mg solid phase extraction cartridges that were primed with ethanol 
 ( 2 ml ) and deionized water ( 20 ml ) .
protein precipitation was achieved 
 by addition of plasma ( 300 l ) to acetonitrile ( 300 l ) , 
 which was centrifuged for 1 min to obtain protein - free plasma ( pfp ) . 

300 l of pfp / acetonitrile solution was diluted into deionized 
 water ( 3 ml ) , loaded onto the c18 cartridge , and removed of polar 
 metabolites with 100% water .
next , a series of extractions were performed 
 using water and acetonitrile in quantities : 95:5 , 90:10 , 85:15 , 80:20 , 
 70:30 , 60:40 , 30:70 , and 100% acetonitrile at a volume of 4 ml .
a 
 control experiment was performed before metabolite analysis to determine 
 the retention of the parent compound by injection of a small amount 
 of [ c]6 onto a test series of extraction 
 cartridges .
each sample was counted in a wizard2 automatic gamma counter 
 to determine the presence of radiolabeled metabolites .
one rat under pentobarbital 
 anesthesia ( 50 mg / kg , ip ) was administered 0.538 mci of [ c]6 by tail vein injection in 0.7 ml and after 30 min , 
 sacrificed by pentobarbital overdose ( additional 150 mg / kg , ip ) , and 
 followed by rapid decapitation .
the brain was removed , sagitally hemisected , 
 and homogenized in 2 ml acetonitrile .
the resulting suspension was 
 centrifuged at 1500 rpm for 5 min , and 0.6 ml of clear supernatant 
 was vortexed with 3 ml water and applied to phenomenex spe strata - x 
 500 mg solid phase extraction cartridges , washed , and fractionally 
 eluted with water containing increasing proportions of acetonitrile 
 ( 100:0 , 95:5 , 90:10 , 85:15 , 80:20 , 70:30 , 60:40 , 30:70 , 0:100 ) .
each 
 sample was counted in a wizard2 automatic gamma counter to determine 
 the presence of radiolabeled metabolites .
adamantan-1-ylmethanamine ( 1 ) ( 1 g , 6.0 mmol ) and ( e)-methyl 3-(4-formylphenyl)acrylate 
 ( 2 ) ( 1 g , 5.3 mmol ) was dissolved in meoh ( 30 ml ) , and 
 the mixture was stirred at room temperature for 2 h. sodium borohydride 
 ( 0.61 g , 16 mmol ) was then added , and the suspension was stirred overnight 
 at room temperature .
the white precipitate was filtered and dried 
 to obtain the product 3 ( 1.35 g , yield : 75%).h nmr ( 500 mhz , cdcl3 ) :  7.69 ( d , j = 16 hz , 1h ) , 7.48 ( d , j = 7 hz , 2h ) , 7.35 ( d , j = 7 hz , 2h ) , 6.43 ( d , j = 16 hz , 1h ) , 
 3.81 ( s , 2h ) , 3.80 ( s , 3h ) , 2.23 ( s , 3h ) , 1.96 ( s , 3h ) , 1.631.73 
 ( m , 6h ) , 1.53 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) : 167.55 , 144.78 , 143.81 , 132.87 , 128.35 ( 2c ) , 128.08 ( 2c ) , 117.10 , 
 62.15 , 54.28 , 51.65 , 40.85 ( 3c ) , 37.24 ( 3c ) , 33.49 , 28.48 ( 3c ) .
lc - ms 
 calculated for c22h29no2 expected 
 [ m ] : 339.2 ; found [ m + h ] : 340.3 .
to a solution 
 of 3 ( 0.5 g , 1.5 mmol ) in meoh / thf ( 5 ml/5 ml ) at 0 c 
 was added nh2oh ( 50% aq solution , 3 ml ) followed by 1 m 
 naoh ( 2 ml ) .
the mixture was stirred at 0 c for 2 h , warmed 
 to room temperature , and stirred for 2 h. acidification with 1 m hcl 
 to ph 78 ( ph paper ) resulted in product precipitation .
the 
 precipitate was filtered and dried to obtain 4 ( 0.2 g , 
 40% ) as white solid .
h nmr ( 500 mhz , cdcl3 ) : 
  7.69 ( d , j = 16 hz , 1h ) , 7.47 ( d , j = 7.5 hz , 2h ) , 7.39 ( d , j = 7.5 hz , 2h ) , 
 6.42 ( d , j = 16 hz , 1h ) , 3.80 ( s , 3h ) , 3.54 ( s , 2h ) , 
 2.19 ( s , 3h ) , 2.10 ( s , 2h ) , 1.95 ( s , 3h ) , 1.621.723 ( m , 6h ) , 
 1.53 ( s , 6h ) ; c nmr ( 125 mhz , cdcl3 ) : 164.92 , 
 141.31 , 139.69 , 133.75 , 128.61 ( 2c ) , 127.42 ( 2c ) , 116.88 , 71.09 , 61.08 , 
 53.31 , 40.41 ( 3c ) , 36.77 ( 3c ) , 32.84 , 28.48 ( 3c ) .
lc - ms calculated 
 for c21h28n2o2 expected 
 [ m ] : 340.5 ; found [ m + h ] : 341.3 .
quantitative nmr purity : 95.10% . to a solution of 3 ( 0.5 g , 1.5 mmol ) in meoh
( 30 ml ) 
 was added formaldehyde ( 33% aq solution , 2 ml ) followed by acetic 
 acid ( 0.1 ml ) .
the mixture was stirred at room temperature for 2 h. 
 sodium borohydride ( 0.61 g , 16 mmol ) was then added , and the suspension 
 was stirred overnight at room temperature .
the white precipitate was 
 filtered and purified by flash chromatography in hexanes : ethyl acetate 
 ( 4:1 ) to obtain the product 3 ( 0.29 g , yield : 55% ) as 
 white solid .
h nmr ( 500 mhz , meoh - d4 ) :  7.55 ( d , j = 16 hz , 1h ) , 7.52 
 ( d , j = 7.5 hz , 2h ) , 7.37 ( d , j = 
 7.5 hz , 2h ) , 6.47 ( d , j = 16 hz , 1h ) , 3.77 ( s , 2h ) , 
 2.21 ( s , 2h ) , 1.94 ( s , 3h ) , 1.661.76 ( m , 6h ) , 1.541.55 
 ( m , 6h ) ; c nmr ( 125 mhz , meoh - d4 ) : 164.93 , 141.32 , 139.71 , 133.76 , 128.62 ( 2c ) , 127.43 ( 2c ) , 
 116.8901 , 61.08 , 53.32 , 40.42 ( 3c ) , 36.78 ( 3c ) , 32.84 , 28.48 ( 3c ) . 
 lc - ms calculated for c21h28n2o2 expected [ m ] : 353.2 ; found [ m + h ] : 354.1 . to a solution 
 of 5 ( 0.5 g , 1.4 mmol ) in meoh / thf ( 3 ml/3 ml ) at 0 c 
 was added nh2oh ( 50% aq solution , 3 ml ) followed by 1 m 
 naoh ( 2 ml ) .
the mixture was stirred at 0 c for 2 h , warmed 
 to room temperature , and stirred for 2 h. acidification with 1 m hcl 
 to ph 78 ( ph paper ) resulted in product precipitation .
the 
 precipitate was filtered and dried to obtain 6 ( 0.21 
 g , 42% ) as white solid .
h nmr ( 500 mhz , dmso - d6 ) :  7.49 ( d , j = 7.5 hz , 2h ) , 
 7.42 ( d , j = 16 hz , 1h ) , 7.33 ( d , j = 7.5 hz , 2h ) , 6.45 ( d , j = 16 hz , 1h ) , 3.48 ( s , 
 2h ) , 2.11 ( s , 3h ) , 2.06 ( s , 2h ) , 1.89 ( s , 3h ) , 1.551.65 ( m , 
 6h ) , 1.46 ( s , 6h ) ;  c nmr ( 125 mhz , dmso - d6 ) : 163.15 , 142.00 , 138.43 , 139.91 , 129.36 ( 2c ) , 
 127.77 ( 2c ) , 119.06 , 70.12 , 64.57 , 45.71 , 41.01 ( 3c ) , 37.21 ( 3c ) , 
 35.27 , 28.33 ( 3c ) .
lc - ms calculated for c22h30n2o2 expected [ m ] : 354.2 ; found [ m + h ] : 355.3 .
hdac inhibition assays : compounds were tested in a 12-point 
 dose curve with 3-fold serial dilution starting from 33.33 m . 

purified hdacs were incubated with 2 m ( the concentration is 
 kept the same for all the hdacs , below km of substrate ) carboxyfluorescein ( fam)-labeled acetylated or trifluoroacetylated 
 peptide substrate ( broad substrates a and b , respectively ) and test 
 compound for 60 min at room temperature , in hdac assay buffer that 
 contained 50 mm hepes ( ph 7.4 ) , 100 mm kcl , 0.01% bsa , and 0.001% 
 tween-20 . reactions were terminated by the addition of the known pan 
 hdac inhibitor lbh -589 ( panobinostat ) with a final concentration 
 of 1.5 m . substrate and product were separated electrophoretically , 
 and fluorescence intensity in the substrate and product peaks were 
 determined and analyzed by labchip ez reader .
the percent 
 inhibition was plotted against the compound concentration , and the 
 ic50 value was determined from the logistic dose 
mouse 
 primary neuronal cultures were generated in factory precoated poly - d - lysine 96-well plates ( bd biosciences no .
bd356692 ) treated 
 overnight with 75 l / well of laminin [ 0.05 mg / ml ] ( sigma no . 

e15 embryonic mouse forebrain was dissociated 
 into a single cell suspension by gentle trituration following trypsin / dnase 
 digestion ( trypsin : cellgro no .
12,500 cells per well were plated in 100 l neurobasal medium 
 ( gibco no .
17504 - 044 ) and 
 1% penicillin / streptomycin / glutamine ( cellgro no .
30 - 009-ci ) and cultured 
 at 37 c with 5% co2 . after 13 days
, cultures were 
 treated with hdac inhibitors by pin transfer of compound ( 185 nl per 
 well ) using a cybi - well vario pinning robot ( cybio corp .
, germany ) 
 and subsequently incubated for 24 h at 37 c with 5% co2 and then fixed in 4% formaldehyde for 10 min .
following two washes 
 with phosphate - buffered saline , cells were permeabilized and blocked 
 with 0.1% triton x-100 and 2% bsa in pbs .
cells were stained with 
 an anti - ac - h4k12 antibody ( millipore , catalog no . 04119 ) and 
 alexa488-conjugated secondary antibody ( molecular probes ) .
cell nuclei 
 were identified by staining with hoechst 33342 ( invitrogen , h3570 ) . 

cell nuclei and histone acetylation signal intensity were detected 
 and measured using a laser - scanning microcytometer ( acumen ex3 , ttp 
 laptech ) .
acumen explorer software was used to identify a threshold 
 of histone acetylation signal intensity such that , without hdac inhibitor , 
 > 99.5% of cells had intensity levels below the threshold . in the 
 presence 
 of hdac inhibitors ,
cells signal intensities above the threshold were 
 scored as  bright green cells  and expressed as a percentage 
 normalized to dmso controls .
ec50 values were determined 
 from curve fitting using graphpad prism v5 software ( graphpad software , 
 inc .
6 was submitted 
 to a panel of 80 binding assays for common transmembrane and soluble 
 receptors , ion channels , and monoamine transporters in the cns ( high - throughput 
 profile p-3 , cerep , france ) .
this concentration was > 10 the ic50 values 
 determined for hdac targets ( 0.34.1 nm , see table 2 , main text ) and in vast excess of the amount of 6 administered in pet imaging experiments .
for example , injected 
 dose = 1 mci / rat ; specific activity ( see figure 
 s2 ) = 1 mci/1 nmole ; therefore , calculated dose = 1 nmole 6/ rat .
inhibition of control binding did not exceed 45% for 
 any target assayed , supporting that 6 predominantly binds 
 hdac targets .
[ c]ch3i was trapped in a tracerlab fx - m synthesizer 
 reactor ( general 
 electric ) preloaded with a solution of precursor ( 4 ) 
 ( 1.0 mg ) in dry dmso ( 300 l ) .
the solution was stirred at 100 
 c for 4 min , and water ( 1.2 ml ) was added .
the reaction mixture 
 was purified by reverse phase semipreparative hplc ( phenomenex luna 
 5u c8(2 ) , 250  10 mm , 5 m , 5.0 ml / min , 40% h2o + ammonium acetate ( 0.1 m)/ 60% ch3cn ) , and the desired 
 fraction was collected .
the final product was reformulated by loading 
 onto a solid - phase exchange ( spe ) c-18 cartridge , rinsing with 1 m 
 naoh aq ( 5 ml ) , eluting with etoh ( 1 ml ) , and diluting with 50 l 
 acetic acid in saline ( 0.9% , 9 ml ) .
the chemical and radiochemical 
 purity of the final product was tested by analytical hplc ( agilent 
 eclipse xdb - c18 , 150  4.6 mm ) .
the identity of the product was 
 confirmed by analytical hplc with additional co - injection of reference 
 standard .
the average time required for the synthesis from end of 
 cyclotron bombardment to end of synthesis was 35 min .
the average 
 radiochemical yield was 35% ( nondecay corrected to trapped 
 [ c]ch3i ) .
chemical and radiochemical purities 
 were 95% with a specific activity 1.0  0.2 ci/mol 
 ( eob ) .
specific radioactivity 
 ( sa ) of [ c]6 was calculated from an analytical 
 hplc sample of 100 l .
a calibration curve of known mass quantity 
 versus hplc peak area ( 254 nm ) was used to calculate the mass concentration 
 of the 100 l .
sa was determined using the mass concentration 
 value determined for the formulated [ c]6 solution , volume , and measured radioactivity .
the identity of the 
 mass peak assigned to [ c]6 was supported 
 by a second hplc injection , where [ c]6 was 
 mixed with nonradioactive compound 6 and yielded a single 
 peak .
an aliquot ( 50 
 l ) of the formulated radiotracer was added to a test tube containing 
 2.5 ml of octanol and 2.5 ml of phosphate buffer solution ( ph 7.4).the 
 test tube was mixed by vortex for 2 min and then centrifuged for 2 
 min to fully separate the aqueous and organic phase . a sample taken 
 from the octanol layer ( 0.1 ml ) and
an additional aliquot of the 
 octanol layer ( 2.0 ml ) was carefully transferred to a new test tube 
 containing 0.5 ml of octanol and 2.5 ml of phosphate buffer solution 
 ( ph 7.4 ) .
the previous procedure ( vortex mixing , centrifugation , sampling , 
 and transfer to the next test tube ) was repeated until six sets of 
 aliquot samples had been prepared .
the radioactivity of each sample 
 was measured in a well counter ( perkinelmer , waltham , ma).the log d of each set of samples was derived by the following equation : 
 log d = log ( decay - corrected radioactivity in octanol 
 sample  10/decay - corrected radioactivity in phosphate buffer 
 sample ) .
an aliquot of radiotracer 
 in saline ( 10 l ) was added to a sample of baboon or human plasma 
 ( 0.8 ml ) .
the mixture was gently mixed by repeated inversion and incubated 
 for 10 min at room temperature . following incubation a small sample 
 ( 20 l )
was removed to determine the total radioactivity in 
 the plasma sample ( at ; at = abound + aunbound ) .
an additional 0.2 ml of the plasma sample was 
 placed in the upper compartment of a centrifree tube ( amicon , inc .
the upper part of the 
 centrifree tube was discarded , and an aliquot ( 20 l ) from the 
 bottom part of the tube was removed to determine the amount of radioactivity 
 that passed through the membrane ( aunbound ) .
plasma protein binding was derived by the following equation : 
 % unbound = aunbound  100/at .
twenty m 
 thick sagittal 
 rat brain sections were cut using a 20 c cryostat , thaw - mounted 
 onto gelatin - coated slides , fixed in 4% paraformaldehyde containing 
 2% ethanol for 60 min at 4 c , and washed 10 min in ice - cold 
 10 mm tris - hcl , ph 7.4 .
sections were then incubated at room temperature 
 in a 50 ml bath containing either ( i ) 5% dmso as control ; ( ii ) unlabeled 6 ( 100 m ) or ( iii ) saha ( 100 m ) for 10 min ; 
 or ( iv ) ci-994 ( 100 m ) for 2 h to allow for slow hdac binding 
 kinetics known for benzamide compounds including ci-994 .
all incubation 
 baths contained 10 mm tris - hcl + 5% dmso , ph 7.4 .
[ c]6 was prepared as described , and 100 ci was added to 
 each bath .
following 10 min incubation at room temperature , sections 
 were washed 2  1 min in 10 mm tris - hcl ( ph 7.4 ) and carefully 
 wiped dry on absorbent towels .
sections were exposed for 1 h to a 
 multisensitive phosphorscreen and developed using a cyclone plus phosphorimager 
 ( both from perkinelmer ) . resulting parent image was evaluated using 
 imagej software ( nih ) with jet color lookup table ( lut ) with whole - image 
 intensity adjusted to enrich red color in control sections .
individual 
 images of sections were cropped using imagej with no additional adjustment 
 to color levels / thresholds .
male 
 sprague  dawley rats were utilized in pairs , anesthetized with 
 inhalational isoflurane ( forane ) at 3% in a carrier of 1.52 
 l / min medical oxygen , and maintained at 2% isoflurane for the duration 
 of the scan .
the rats were arranged head - to - head in a triumph trimodality 
 pet / ct / spect scanner ( gamma medica , northridge , ca ) .
rats were injected 
 standard references or vehicle via a lateral tail vein catheterization 
 at the start of pet acquisition .
dynamic pet acquisition lasted for 
 60 min and was followed by computed tomography ( ct ) for anatomic coregistration . 

pet data were reconstructed using a 3d - mlem method resulting in a 
 full width at half - maximum ( fwhm ) resolution of 1 mm .
reconstructed 
 images were exported from the scanner in dicom format along with an 
 anatomic ct for rodent studies .
these files were imported to pmod 
 ( pmod technologies , ltd . ) and manually co - registered using six degrees 
 of freedom .
volumes 
 of interest ( vois ) 
 were drawn manually as spheres in brain regions guided by high - resolution 
 ct structural images and summed pet data , with a radius no < 1 mm 
 to minimize partial volume effects .
tacs were exported in terms of 
 decay corrected activity per unit volume at specified time points 
 with gradually increasing intervals .
papio anubis baboons , deprived of 
 food for 12 h prior to the study , were included 
 in the pet - mr scans .
atropine 0.05 mg / kg was used intramuscularly 
 to prevent excessive secretion ( 15 or 30 min of ketamine and xylazine ) . 

anesthesia was induced with intramuscular xylazine ( 0.52.0 
 mg / kg ) and ketamine ( 10 mg / kg ) .
after endotracheal intubation , v- 
 and a - lines were inserted , and anesthesia was maintained using isoflurane 
 ( 11.5% , 100% o2 or 50/50 o2/n2o , l l / min ) . during anesthesia , heart rate , respiration rate , blood 
 pressure , o2 saturation , and end tidal co2 were 
 monitored .
the baboon was catheterized antecubitally for radiotracer 
 injection , and a radial arterial line was placed for metabolite analysis .
pet - mr images were acquired in a biograph 
 mmr scanner ( siemens , munich , germany ) and pet compatible 8-channel 
 coil arrays for nonhuman primate brain imaging with a pet resolution 
 of 5 mm and field of view of 59.4 and 25.8 cm ( transaxial and axial , 
 respectively ) .
dynamic pet image acquisition was initiated followed 
 by administration of the radiotracer in a homogeneous solution of 
 10% ethanol and 90% isotonic saline .
an memprage sequence began after 
 30 min of the baseline scan for anatomic co - registration . to characterize 
 the specific binding of [ c]6 ,
a second imaging 
 experiment was carried out in which unlabeled 6 was co - administered 
 intravenously at the start of acquisition .
both scans were carried 
 out in the same animal on the same day , separated by 2.5 h. in both 
 scans , 45 mci of [ c]6 was administered 
 to the baboon .
dynamic data from the pet scans were recorded in list 
 mode and corrected for attenuation .
baboon data were reconstructed 
 using a 3d - osem method resulting in a fwhm resolution of 4 mm .
simultaneous pet - mr data were acquired 
 using a siemens biograph mmr system ( siemens medical solutions u.s.a . , 
 inc . ,
malvern , pa ) . mr body imaging was performed with real - time respiratory 
 bellows - gating and using the body matrix coil and 
 the built - in spine coil as the receiving coil elements .
high - resolution 
 anatomical t1w axial , coronal , and sagittal dual - echo scan with the 
 following parameters : tr 115 ms , te1 1.23 ms , te2 2.46 ms , matrix 
 size 256  168 , fov 35 cm , phase fov 65.6% ) and 4 mm slice thickness 
 were obtained .
additionally , in particular for the purpose of optimal 
 visualization of the pancreas , high - resolution fat - suppressed axial 
 t2w turbo spin echo ( tse ) data were obtained with the following parameters : 
 tr 5264 ms , te 98 ms , matrix size 488  235 , fov 18.0 cm , phase 
 fov 75% , fa 150 , and 3 mm slice thickness . for the purpose of 
 mr - based attenuation correction ( mrac ) of the pet data ,
default t1-weighted 
 ( t1w ) 2-point dixon 3d volumetric interpolated breath - hold examination 
 ( vibe ) scans were obtained .
pet data were obtained using a single - bed 
 acquisition with an axial field of view of 25.8 cm , transaxial field 
 of view of 59.4 cm , and axial and transaxial resolution of 4.8 
 and 4.7 mm , respectively .
pet data were acquired dynamically 
 for 80 min after administration of the radiotracer in a homogeneous 
 solution of 10% ethanol and 90% isotonic saline . to characterize the 
 specific binding of [ c]6 ,
a second imaging 
 experiment was carried out in which unlabeled 6 was co - administered 
 intravenously at the start of acquisition .
both scans were carried 
 out in the same animal on the same day , separated by 2.5 h. in both 
 scans , 45 mci of [ c]6 was administered 
 to the baboon . in order to evaluate the blocking of [ c]6 by saha , an separate dual - injection study of [ c]6 was performed . in that experiment
continuous 
 saha infusion ( 2 mg / ml at 0.16 ml / min ) was started after 45 min of 
 baseline scan with [ c]6 , and the infusion 
 was continued during the 80 min pet scan after a second injection 
 of [ c]6 . in all
cases pet data were recorded 
 in list mode , and reconstruction was performed using a 3d - osem method 
 resulting in a fwhm resolution of 4 mm .
attenuation correction was 
 performed using the aforementioned 2-point dixon 3d vibe mr - derived 
 attenuation maps .
data of each 80 min scan were reconstructed for 
 tac analysis using the following framing : 12  10 , 3  20 , 
 4  30 , and 15  300 s. for the dedicated self - blocking 
 and saha blocking experiments , additional static pet data were reconstructed 
 using data obtained between 40 and 80 min and 2045 min , respectively , 
 in order to visually display the effects of blocking in multiple organs .
pet data were motion - corrected , 
 spatially smoothed with a 2.5 mm fwhm gaussian filter , and registered 
 to the black baboon brain atlas using 
 jip tools optimized for nonhuman primate data processing ( www.nitrc.org/projects/jip ) .
image registration was carried out on high - resolution mprage mri 
 image using a 12 degree of freedom linear algorithm and a nonlinear 
 algorithm to the atlas brain .
kinetic modeling 
 was carried out in pmod ( pmod3.3 , pmod technologies ltd . ,
tacs were 
 exported from the whole brain , cerebellum , primary motor cortex , putamen , 
 thalamus , primary visual cortex , caudate , and white matter vois for 
 analysis .
a two - tissue compartmental model was used to estimate regional 
 volume of distribution ( vt ) with a metabolite - corrected 
 plasma tac .
voxel - wise vt maps were calculated 
 using a logan plot from the dynamic pet data .
the time until linearity of the plot was achieved and determined 
 for each scan .
in addition , regional k1/k2 and k3/k4 values were also obtained 
 from the baseline scans using 
 a two - tissue compartmental model .
it is known thatwith the assumption that kd = 0.4 m ( assumed from ex vivo hdac - complex kd measure for saha , which exhibits a similar ic50 in in 
 vitro activity - based assays we determined ) .
averaged bmax from the selected vois was estimated to be 19.6  
 2.9 m ( mean  sd ) .
arterial samples collected 
 during imaging from the baboon were centrifuged to obtain plasma , 
 which was then removed and placed in an automated gamma counter that 
 was calibrated to the pet scanner .
metabolite analysis was conducted 
 on a custom automated robot fitted with phenomenex spe strata - x 500 
 mg solid phase extraction cartridges that were primed with ethanol 
 ( 2 ml ) and deionized water ( 20 ml ) .
protein precipitation was achieved 
 by addition of plasma ( 300 l ) to acetonitrile ( 300 l ) , 
 which was centrifuged for 1 min to obtain protein - free plasma ( pfp ) . 

300 l of pfp / acetonitrile solution was diluted into deionized 
 water ( 3 ml ) , loaded onto the c18 cartridge , and removed of polar 
 metabolites with 100% water .
next , a series of extractions were performed 
 using water and acetonitrile in quantities : 95:5 , 90:10 , 85:15 , 80:20 , 
 70:30 , 60:40 , 30:70 , and 100% acetonitrile at a volume of 4 ml .
a 
 control experiment was performed before metabolite analysis to determine 
 the retention of the parent compound by injection of a small amount 
 of [ c]6 onto a test series of extraction 
 cartridges .
each sample was counted in a wizard2 automatic gamma counter 
 to determine the presence of radiolabeled metabolites .
one rat under pentobarbital 
 anesthesia ( 50 mg / kg , ip ) was administered 0.538 mci of [ c]6 by tail vein injection in 0.7 ml and after 30 min , 
 sacrificed by pentobarbital overdose ( additional 150 mg / kg , ip ) , and 
 followed by rapid decapitation .
the brain was removed , sagitally hemisected , 
 and homogenized in 2 ml acetonitrile .
the resulting suspension was 
 centrifuged at 1500 rpm for 5 min , and 0.6 ml of clear supernatant 
 was vortexed with 3 ml water and applied to phenomenex spe strata - x 
 500 mg solid phase extraction cartridges , washed , and fractionally 
 eluted with water containing increasing proportions of acetonitrile 
 ( 100:0 , 95:5 , 90:10 , 85:15 , 80:20 , 70:30 , 60:40 , 30:70 , 0:100 ) .
each 
 sample was counted in a wizard2 automatic gamma counter to determine 
 the presence of radiolabeled metabolites .
 epigenetic 
 enzymes are now targeted to treat the underlying gene 
 expression dysregulation that contribute to disease pathogenesis . 
 
 histone deacetylases ( hdacs ) have shown broad potential in treatments 
 against cancer and emerging data supports their targeting in the context 
 of cardiovascular disease and central nervous system dysfunction . 
 
 development of a molecular agent for non - invasive imaging to elucidate 
 the distribution and functional roles of hdacs in humans will accelerate 
 medical research and drug discovery in this domain . 
 herein , we describe 
 the synthesis and validation of an hdac imaging agent , [ 11c]6 . 
 our imaging results demonstrate that this probe 
 has high specificity , good selectivity , and appropriate kinetics and 
 distribution for imaging hdacs in the brain , heart , kidney , pancreas , 
 and spleen . 
 our findings support the translational potential for [ 11c]6 for human epigenetic imaging . 
Introduction
Results
Discussion
Experimental
Section
Chemical
Synthesis