Image-guided synthesis reveals potent blood-brain barrier permeable histone deacetylase inhibitors

Article abstract of DOI:10.1021/cn500021p

Recent studies have revealed that several histone deacetylase (HDAC) inhibitors, which are used to study/treat brain diseases, show low blood-brain barrier (BBB) penetration. In addition to low HDAC potency and selectivity observed, poor brain penetrance

Full text of DOI:10.1021/cn500021p

Research Article
pubs.acs.org/chemneuro
Image-Guided Synthesis Reveals Potent Blood-Brain Barrier
Permeable Histone Deacetylase Inhibitors
Young Jun Seo,†,‡ Yeona Kang,† Lisa Muench,§ Alicia Reid,∥ Shannon Caesar,† Logan Jean,†
Florence Wagner,⊥ Edward Holson,⊥ Stephen J. Haggarty,# Philipp Weiss,∇ Payton King,†
Pauline Carter,† Nora D. Volkow,§,◆ Joanna S. Fowler,†,¶ Jacob M. Hooker,*,†,○ and Sung Won Kim*,§,¶
†Biosciences Department, Brookhaven National Laboratory, Upton, New York 11973, United States
‡Department of Chemistry, Chonbuk National University, Jeonju, 561-756, South Korea
§Laboratory of Neuroimaging, National Institute on Alcohol Abuse and Alcoholism, Upton, New York 11973, United States
∥Physical, Environmental and Computer Sciences, Medgar Evers College, Brooklyn, New York 11225, United States
⊥Stanley Center for Psychiatric Research, Broad Institute of Massachusetts Institute of Technology and Harvard University,
Cambridge, Massachusetts 02142, United States
#Center for Human Genetic Research, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02142,
United States
∇Institut fur Organische Chemie, Johannes-Gutenberg Universitat Mainz, Duesbergweg 10-14, Mainz 55122, Germany
̈
̈
○Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital, Harvard Medical
School, Charlestown, Massachusetts 02129, United States
◆National Institute on Drug Abuse, National Institutes of Health, Bethesda, Maryland 20892, United States
¶Department of Chemistry, Stony Brook University, Stony Brook, New York 11794, United States
S
* Supporting Information
ABSTRACT: Recent studies have revealed that several
histone deacetylase (HDAC) inhibitors, which are used to
study/treat brain diseases, show low blood-brain barrier (BBB)
penetration. In addition to low HDAC potency and selectivity
observed, poor brain penetrance may account for the high
doses needed to achieve therapeutic efficacy. Here we report
the development and evaluation of highly potent and blood-
brain barrier permeable HDAC inhibitors for CNS applica-
tions based on an image-guided approach involving the parallel
synthesis and radiolabeling of a series of compounds based on the benzamide HDAC inhibitor, MS-275 as a template. BBB
penetration was optimized by rapid carbon-11 labeling and PET imaging in the baboon model and using the imaging derived
data on BBB penetration from each compound to feed back into the design process. A total of 17 compounds were evaluated,
revealing molecules with both high binding affinity and BBB permeability. A key element conferring BBB penetration in this
benzamide series was a basic benzylic amine. These derivatives exhibited 1−100 nM inhibitory activity against recombinant
human HDAC1 and HDAC2. Three of the carbon-11 labeled aminomethyl benzamide derivatives showed high BBB penetration
(∼0.015%ID/cc) and regional binding heterogeneity in the brain (high in thalamus and cerebellum). Taken together this
approach has afforded a strategy and a predictive model for developing highly potent and BBB permeable HDAC inhibitors for
CNS applications and for the discovery of novel candidate molecules for small molecule probes and drugs.
KEYWORDS: Histone deacetylase, positron emission tomography, blood-brain barrier permeability, benzamides
subsequent generation of cells thereby producing phenotypic
pigenetic regulation of gene expression via enzymatic
Emodification of DNA and histone proteins is implicated in diversity without changing DNA sequence.
development,1 inflammation,2 heart disease,3 cancer,4 and
neuropsychiatric disorders5,6 including depression, Alzheimer’s
disease, and substance use disorders. Two common and
influential epigenetic transformations are DNA methylation
and chromatin modifications, which provide a mechanism for
the transmission of environmentally cued information to
Histone acetyl transferase (HAT) and histone deacetylase
(HDAC) are histone modifying enzymes that regulate gene
expression by catalyzing the addition and removal of acetyl
Received: February 6, 2014
Revised: April 25, 2014
Published: April 29, 2014
© 2014 American Chemical Society
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Figure 1. Known HDAC inhibitor drugs (A) and the flowchart for image-guided systematic approach (B).
groups from the lysine groups of histone proteins, which in
association with DNA form chromatin. HDAC removes an
acetyl group of histone proteins (by hydrolysis of acetamido
group of ε-carbon of lysine residues), inducing a tight charge−
charge interaction between DNA and histone proteins, thereby
rendering the DNA inaccessible to transcription factor binding
and repressing gene expression. In general, histone acetylation
is associated with activation of gene expression exposing DNA
to transcription factors whereas deacetylation is linked to gene
repression by condensing the chromatin rendering DNA less
available for transcription.
There are four classes of HDAC (I, II, III, IV) and 11
isoforms. Class I HDACs (1, 2, 3, 8 subtype) are usually found
in the nucleus, while class II HDAC (4, 5, 7, 9 subtype) and
class IV (HDAC 11 subtype) occur in both cytoplasm and
nucleus. Except for Class III HDAC, all HDACs contain a Zn
ion in the substrate binding pocket where the hydrolysis of the
acetamide bond occurs. Class I HDAC inhibitors (HDACi)
have been developed as potential treatment of cancers.
Suberoylanilide hydroxamic acid (1, SAHA, Zolinza, Figure
1A) was approved as the first drug targeting pan HDAC for
cutaneous T-cell lymphoma treatment.7 In addition, SAHA has
been clinically tested for diverse types of cancers including
breast cancer8 and gliomas9 when used in combination with
conventional cancer drugs. Recently, a benzamide type of
HDACi, MS-275 (6, Entinostat, Figure 1A) has also been
under many clinical trials, notably, showing its clinical efficacy
for the treatment of estrogen-positive breast cancer.10
number of well-known HDAC inhibitors as potential templates
for the development of highly potent blood-brain barrier (BBB)
permeable HDAC inhibitors for CNS applications. In our initial
studies, we used carbon-11 labeled versions of the known
HDAC inhibitor drugs, butyric acid, valproic acid, and 4-
phenylbutyric acid, to measure their brain uptake and whole-
body pharmacokinetics.16 All of these drugs have CNS
applications, particularly valproic acid, which has been used
for decades to treat seizure disorders. Yet all of them have very
limited BBB permeability. Similarly, we found that the
benzamide HDACi, MS-275 (6), has low brain uptake when
administered intravenously to nonhuman primates,17 suggest-
ing its limitation as a therapeutic agent for CNS disorders.
Recently, Hanson et al.18 also demonstrated that the lack of
behavioral effects of SAHA is likely to be due to poor BBB
permeability, even though its therapeutic potential for CNS
applications was suggested by in vitro studies. Clearly, a
systematic approach to better predict BBB penetration of small
molecule probes and drugs for CNS therapeutics is needed,
including that for HDACi.19
Here we report a positron emission tomography (PET)
image-guided synthesis, radiolabeling, and evaluation of highly
potent and BBB permeable HDAC inhibitors targeting mainly
HDAC isoforms 120 and 221,22 due to their CNS disorder
relevance. We modeled our series on the HDAC inhibitor MS-
275 (6, Figure 1A). Although MS-275 is not BBB permeable, its
relatively low molecular weight (MW) and nonionic structure
at physiological pH provide the opportunity to vary different
parameters and the introduction of readily labelable functional
groups enabled the rapid assessment of brain uptake using PET.
In parallel, we assessed the in vitro potency for HDAC
inhibition of the compounds with the highest brain uptake.
Though mostly investigated for the treatment of cancer, an
increasing numbers of studies are evaluating HDACi for central
nervous system (CNS) diseases, such as schizophrenia,
neurodegenerative disorders, seizures, depression, and addic-
tion.11−15 One potential advantage of HDAC drugs is the
potential for reversing abnormal cell transcription, rather than
treating downstream translational endophenotypes. The
potential therapeutic benefits that HDACi might confer in
CNS disorders stimulated us to investigate the brain uptake of a
RESULTS
■
Overview. The development of a BBB permeable HDAC
inhibitor is based on an iterative plan with four components: in
silico structural design and prediction, parallel synthesis and in
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Table 1. Inhibition Assay of HDAC1, HDAC2, and HDAC3 (IC50, nM)
a
Recombinant histone deacetylase (the value in parentheses was obtained after 3 h incubation).
vitro HDAC assay, radiolabeling, quantification, and in vivo
imaging (Figure 1B). Briefly, as a starting point, four known
highly potent HDAC drug candidates and their derivatives (7,
8, 19) including [11C]MS-275 (6)17 were labeled with C-11
and their BBB permeability was evaluated by in vivo PET
imaging. This first set of data was used to generate initial
guidelines for structural modification, compared with calculated
log BB (BB, ratio of brain to plasma concentration of drug in
steady state (Supporting Information (SI) Table 1).23 Then,
based on these results, the next set of compounds were
designed under the consideration of parallel synthesis, radio-
labeling, and in vitro assay. In general, three radiolabeled
compounds were designed at a time, creating one set for in vivo
evaluation and generating a quantitative structure−property
relationship (QSPR) model (SI). The accumulated set of
benzamides provided determinant physicochemical properties
to be modified for further optimization. We used baboon
(Papio Anubis) as it is expected to be similar to human24 and
allows more accurate blood analysis at multiple time points
than rodent.
Chemistry: Structural Design, Synthesis, and Radio-
chemistry. Despite the low BBB permeability of [11C]MS-275,
the core benzamide structure was chosen as a template for our
systematic approach varying polar surface area (PSA), charge,
molecular volume (MV) (SI Table 1), and lipophilicity.25
Structurally, substituent modification of benzamides was
confined at R1 of phenyl ring A and at R2 of phenyl ring B
(Figures 1B and 2) because both positions have been shown to
be critical for HDAC.26 Among various PET isotopes, carbon-
11 was chosen in that its 20.4 min half-life made multiple PET
studies possible in 1 day in the same animal. For facile and
rapid labeling with carbon-11, N-methylation was adopted
using [11C]methyl iodide27−29 or [11C]methyl triflate.30 In
some cases, N-acetylation was also performed with [11C]acetyl
chloride.31
Initially, we chose two known benzamides,32,33 CI-994 (7,
Tacedinaline) and 8, both of which have low molecular weight
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Figure 2. Parallel synthesis and radiolabeling of benzamides.
and sub micromolar HDAC inhibitory activity (Table 1).
Carbon-11 versions of these compounds were synthesized by
amidation using [11C]acetyl chloride (Figure 2B), which was
prepared from [11C]CO2. The spirobicyclic compound 19 was
also labeled with [11C]methyl iodide, since its nor-precursor 18
was one of the most potent HDAC1 inhibitors (IC50 = 8 nM)
among the known benzamides.26 Precursor and unlabeled
standards were synthesized by modification of known
procedures in multistep synthesis (SI). However, none of
these compounds proved to be BBB permeable in the baboon
brain PET imaging study.
The first QSPR model built with these compounds
(including [11C]MS-275) using the linear cross-correlation
matrix revealed that PSA was the main determinant property
(r2 = 0.39) for brain total distribution volume (VT, capacity of
the tissue to bind the drug, SI). A set of methylpiperazines
varying R1 was selected to reduce PSA in the second round. For
the rapid optimization of BBB permeability, we used the
parallel synthesis approach as shown in Figure 2.26 Boc-
protected phenylenediamines (2 and 3) were coupled with 4-
chloromethylbenzoyl choride (4) to provide various benza-
mides. We set R2 as a labeling position for N-methylation with
[11C]methyl iodide or [11C]methyl triflate, introducing a
piperazine ring and an N-mono or N-dimethyl amino group.
[11C]Methyl iodide or [11C]methyl triflate was used for N-
methylation of the piperazine group to give high radiochemical
yield (40−60%, decay-corrected) in N,N-dimethylformamide
(DMF) containing 1,2,2,6,6-pentamethylpiperidine (PMP)
within 1 h of radiosynthesis from the end of cyclotron
bombardment (Figure 2B). However, N-methylation of benzyl
amines, which were chosen as the third set of benzamides, did
not provide desired labeled product under these conditions
probably due to the reduced nucleophilicity. After exploring
many conditions with different combination of bases and
solvents, we found that using ammonia as a base gave a
moderate yield (10−30%) and sufficient labeled compound for
the in vivo imaging study.
In Vitro HDAC Inhibition Assay. In vitro HDAC
inhibition assay data of benzamides are shown in Table 1.
The purpose of this evaluation is to compare the inhibitory
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potency between benzamides, based on the result of a well-
characterized hydroxamte inhibitor, SAHA (1) as a reference.
For HDAC1, inhibitory activity was measured after 1 and 3 h
incubation with each compound. Piperazinylmethyl substitu-
tion at R1 instead of an acetamido group of CI-994 decreases
potency 15-fold. Halogen substitution at R1 also decreased
potency. In contrast, introduction of a phenyl group at R1
dramatically increased inhibitory activity up to the nanomolar
level for HDAC1 and -2, but not -3, indicating cation−π
electron interaction between the phenyl group and guanidinium
cation of the arginine residue in the deep cavity inside the
HDAC binding pocket.34,35 Another notable point was that the
in vitro assay showed that compounds 15 and 16 displayed fast
and potent inhibitory activity similar to SAHA. These
properties are abolished with 4-fluoro substituent of the phenyl
ring at R2. Methylated spirobicyclic compound 19 also showed
similar potency to known nor-precursor compound 18.26 We
also found that a relatively simple amino compound, 20 was a
potent inhibitor for HDAC1 and -2. In order to reduce PSA
and molecular weight to improve BBB permeability, the
piperazinylmethyl group (compounds 9−17) was replaced
with simple aminomethyl substituent on R1 (compounds 21−
27), maintaining the C-11 labeling position. Similar to results
with the piperazine derivatives, potency was recovered by
adding an aromatic group at R1 resulting in a potency of 3 nM,
after 3 h incubation in vitro (Table 1).
PET Study: BBB Permeability and Binding Specificity.
PET studies were performed with 17 carbon-11 labeled
benzamides in the baboon brain. Arterial blood was sampled
over the time course of the study and selected samples were
analyzed by HPLC to measure the concentration of
unmetabolized parent benzamide. The % fraction of all intact
carbon-11 labeled benzamides in plasma was approximately 60
24% at 10 min after injection (Figure 3A). A series of
benzamides with N,N-di- or N-monomethylamino methyl
substituent on R2 showed significantly higher intact percent
(72 5%) (Figure 3A), compared with piperazinyl derivatives.
MS-275 (6) and CI-994 (7), which have been used for human
clinical trials, also showed relatively higher intact percent. A
methylated version of spirobicylic 19 showed a very low intact
fraction in the early time points and a low area under the curve
(AUC), which might be consistent with fast clearance of its
potent nor-compound, 18 in plasma in a prior report (Figure
3B).26
Figure 3C shows the brain uptake (% injected dose per cc (%
ID/cc)) of the selected carbon-11 labeled benzamides. The
carbon-11 labeled CI-994 as well as previously reported HDAC
inhibitor, 8,32 showed very low brain uptake, similar to
[11C]MS-275 (6). However, the whole brain uptake of 4-
methylpiperazinyl-1-methyl derivatives (10−14, 16, 17) was
4−8 times higher than [11C]MS-275 (6) at 15 min after
injection. Further improvement was shown in N,N-dimethyla-
minomethyl derivatives (23, 26−28) which showed peak brain
uptake up to 0.015%ID/cc, at 5 min post injection.
For highly potent benzamide HDAC inhibitors, the total
distribution volume (VT, as determined by a one-tissue
compartment model) in the brain and AUC ratio of brain
versus plasma (BAUC/PAUC) were calculated (Table 2) over the
90 min scan session. VT and BAUC/PAUC were 0−17 mL/cm3
and 0.1−7.5, respectively. While both parameters of MS-275
and CI-994 were minimal, N,N-dimethylamino compounds 26
Figure 3. Fraction of unchanged carbon-11 labeled benzamides (A),
area under the curves in baboon plasma over 60 m in after time of
injection (B), and time−activity curves of global brain uptake for the
selected benzamides (C).
Considering both in vitro binding potency and BBB
permeability, we chose compound 26 to evaluate binding
specificity to brain HDACs. In the baseline studies, the brain
and 28 showed very high VT and BAUC/PAUC
.
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humans.40 Due to the short half-life of carbon-11, a serial study
with three different [11C]benzamides was readily achieved in
the same day using the same animal. We coupled this with a
QSPR model evaluating the utility of different molecular
descriptors for predicting BBB uptake.
Two measures of brain uptake relative to plasma were used
for the highly potent benzamide HDAC inhibitors: the total
tissue distribution volume (VT) and the area under the curve
(AUC) ratio of brain to plasma (BAUC/PAUC) (Table 2). VT
represents the capacity of the tissue to bind the drug, whereas
BAUC/PAUC represents the amount of drug in the brain over the
whole time course of the experiment relative to that in plasma
for the same time period. As expected, there was a strong linear
correlation between VT and BAUC/PAUC (R2 = 0.81, SI Figure
1). Measures of VT and BAUC/PAUC were highest for
compounds 26 and 28 in Table 2, whereas they were the
lowest for CI-994 (7) and MS-275 (6). On the other hand,
Table 2. Brain Total Distribution Volume and AUC Ratio of
Brain to Plasma for Selected [11C]Benzamides
a
b
compd
log D7.4
cLogP
PSA
VT (mL/cm3) BAUC/PAUC
6, MS-275
1.8
1.0
2.5
1.5
3.0
3.8
5.4
4.8
2.2
4.0
4.2
3.9
119.2
98.0
98.2
70.2
70.1
70.0
64.8
74.8
64.8
64.8
0
0.14
0.26
0.24
3.54
0.93
0.77
3.93
0.90
7.53
5.95
7, CI-994
0.41
0.22
8.02
4.81
1.35
5.51
2.13
11.96
17.81
8
16
19
20
23
25
26
28
2.2
4.7
2.1
a
b
Measured. Polar surface area (Å).
distribution of [11C]26 was heterogeneous, showing highest VT
in thalamus, cerebellum, and striatum. A repeated baseline
study of [11C]26 on the same day in the same animal showed
less than 10% of averaged variability of global brain uptake (n =
3). We examined the regional VT change of [11C]26 caused by
pretreatment with unlabeled compound 26 (1 mg/kg) or the
potent HDAC inhibitor, SAHA (1 mg/kg) (Table 3). The
degree of VT reduction ranged 8−24%, compared with the
baseline studies in various brain regions.
compound 19 has a higher VT (4.81 mL/cc) relative to BAUC
/
P
AUC. The higher than expected VT is most likely related to the
high log P and log D7.4, indicating that the higher lipophilicity
results in a slower brain clearance and therefore a higher VT.
Even though BAUC/PAUC for CI-994 is 0.256, which is similar to
the brain/plasma ratio (B/P) reported previously (0.43),41 we
point out that extrapolation of our result into steady-state B/P
would be limited because these data were obtained using bolus
IV administration with tracer-doses of the labeled compounds.
Considering chemical structure and based on our previous
results and predicted log BB (log(B/P)), the core benzamide
structure was selected and modified as a template for BBB
permeability optimization. We excluded charged and high
molecular weight derivatives. We found that the polar surface
area (PSA)42 is a critical property for brain uptake and it must
be not more than 65 for these types of benzamides (Table 2).
For example, the removal of one nitrogen atom by changing N-
methylpiperazine (16) to dimethylamino group (26) changed
the PSA from 70.19 to 64.75 and improved BBB permeability,
showing a 2-fold increase in BAUC/PAUC. Even more
dramatically, the slight difference between compound 25
(monomethyl) and 26 (dimethyl) resulted in a 7-fold increase
in BAUC/PAUC, which also could be explained by the number of
hydrogen bonding donors (HBD). Following this reasoning, we
suggest that the low brain permeability of CI-994 (7) may be
attributed to its high PSA (119.16) whereas both molecular
volume and PSA predicted the poor brain entry of MS-275. For
all previously reported hydroxamate-based compounds, it
became evident that their PSA (74−107) was not within the
appropriate range.
In in vitro binding assays, the potent hydroxamic acid
HDACi, SAHA, inhibits three isoforms of class I HDACs in the
low nM range without any apparent selectivity. In contrast,
benzamides showed selective inhibitory activity for HDAC1
and -2, which have recently been reported for their therapeutic
potential in CNS disorders.20−22,43 Interestingly, some of
piperazynyl derivatives (17) showed moderate HDAC2
selectivity. It also should be noted that most of the benzamides
reported here showed time-dependent inhibition, indicating
slower kinetics than SAHA. Chou et al. and others observed
slow kinetics but long residence time for other benzamides.44,45
However, we found that some of the piperazinyl compounds
(15, 16) demonstrated fast HDAC inhibitory activity (Table
1).
Table 3. Difference of Total Distribution Volumes (VT) of
a
Test−Retest and Blocking Study (n = 3)
test/retest
pretreatment with SAHA pretreatment with 26
CB
12.6 ( 23.5)
−3.9 ( 14.6)
3.1 ( 19.8)
−2.5 ( 26.8)
−15.4 ( 11.1)
−8.9 ( 20.7)
2.5 ( 36.0)
−13.4 ( 18.4)
−12.9 ( 27.0)
−24.8 ( 5.7)
3.8 ( 48.5)
TH
TEMP
STR
−4.5 ( 21.6)
a
CB, cerebellum; TH, thalamus; TEMP, temporal cortex; STR,
striatum.
DISCUSSION
■
We report the development of BBB permeable, highly potent
HDAC inhibitors using PET in the nonhuman primate brain.
Previously, almost all HDAC inhibitors tested in vivo showed
lack of BBB permeability. Recently, similarly with two SAHA-
based hydroxamates,36,37 we and Shuiyu et al. also reported
minimal brain uptake of trichostatin A (TSA)-like hydrox-
amates38 and aryl hydroxamate derivative (KB631) in the
baboon brain,39 respectively. Brain efflux transporters may play
a critical role for these failures in that a hydroxamate drug,
SAHA is known to be a P-gp substrate in in vitro assay.18 In
contrast, the brain uptake of a benzamide, [11C]MS-275, was
not improved by pretreatment of P-gp substrate, verapamil.17
In order to develop a predictive approach for the
development of BBB permeable HDACi, we initiated a
systematic strategy (that allows rapid iterative feed-back to
structural modification from the initial design) using highly
sensitive image-guided quantification of BBB penetration in a
semi-high throughput manner. Carbon-11, a short-lived isotope
of carbon (half-life: 20.4 min) that decays by positron emission,
producing two highly energetic photons, can be detected
external to the body barrier using PET imaging. Carbon-11
labeled compounds and nonhuman primate PET imaging
provide the opportunity to assess the BBB penetration of drugs
noninvasively to facilitate their translation for applications in
We also point out that recent studies showed that
benzamides bind various HDAC-involving chromatin modify-
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ing complexes,46 indicating that the results of binding assays on
purified HDAC may not reflect in vivo HDAC activity.
Nonetheless, unlike SAHA, CI-994 was devoid of non-HDAC
target binding. Our findings suggest that the development of
hydroxamte and benzamide types radiotracers hold promise as
potential PET ligands for measuring brain HDACs in vivo.47
Based on its high brain uptake and the in vitro IC50 results,
compound 26 (IC50, 3 nM for HDAC1) was selected to
evaluate its in vivo binding specificity. While in vitro
autoradiography47 showed relatively high [3H]CI-994 binding
density in cerebellum in the rodent brain, VT reduction of [11C]
26 in the baboon cerebellum were similar to that in in thalamus
and striatum, all of which showed higher reduction than cortex.
There are no published reports of the regional density of
HDAC in the primate brain that might have helped us interpret
the regional differences we observed. The low displacement
signal might be due to the slow binding kinetics of benzamides
on HDAC as well as to nonspecific binding. However, since
there are no BBB permeable PET radiotracers available except
for [18]FAHA, compound [11C]26 would offer a good template
for further development of brain permeable HDAC radio-
tracers. Notably, while [18]FAHA, an HDAC substrate, binds
into mainly class II HDAC, benzamides are reversible HDAC I
inhibitors and therefore might be useful to measure class I
HDACs.
Study Limitations and Other Considerations. Some of
the synthesized benzamides could be substrates of efflux
transporters, and this would introduce another variable in our
studies. We reported that [11C]MS-27517 showed no
observable increase in brain uptake in the rodent after
verapamil pretreatment. In addition, BAUC/PAUC could be
affected by high brain nonspecific binding, reducing pharmaco-
logically active free fraction in the brain.48 BBB permeability
differences between species might result in different rates of
brain uptake of benzamides when translated into humans.
Though we focused on BBB permeability, some of the potent
compounds developed in this report including piperazinyl
derivatives could be good radioligand candidates for cancer
imaging and therapeutics for which high BBB permeability is
not necessary. Slow binding kinetics of some benzamides
observed by in vitro assay may limit the visualization of target
engagement in vivo over the short PET scan time.
and purified by reverse-phase HPLC (Column, Phenomenex Luna
C18(2), 250 mm × 10 mm; flow rate, 6 mL/min; eluent, ammonium
formate solution (0.1 M, water/acetonitrile = 25/75)). The desired
product (retention time, 40 min) was collected and concentrated using
a rotary evaporator under reduced pressure. The product was
reconstituted with sterile saline (4 mL) and filtered through a sterile
filter (Acrodisc 13 mm with 0.2 μm HT Tuffryn Membrane, Pall Co.,
Ann Arbor, MI) into a sterile, pyrogen-free vial. The total synthesis
time was 75 min from the end of bombardment. Radiochemical yield
was 40% (n = 16, decay corrected) and radiochemical purity was >99%
(specific activity = 5−18 Ci/μmol at the end of bombardment).
Radiochemical purity and identity were determined by analytical
HPLC (Column, Phenomenex Luna C18, 250 mm × 4.6 mm; flow
rate, 1.0 mL/min; eluent, ammonium formate solution (0.1 M, water/
acetonitrile = 32.5/67.5; retention time, 11 min)).
In Vitro HDAC Inhibition Assay. Purified HDACs were
incubated with 2 μM carboxyfluorescein (FAM)-labeled acetylated
or trifluoroacetylated peptide substrate (Broad Substrate 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 μM.
Substrate and product were separated electrophoretically, and
fluorescence intensity and substrate and product peaks were
determined and analyzed by Labchip EZ Reader. The reactions were
performed in duplicate for each sample. IC50 values were automatically
calculated by Origion8 using 4 Parameter Logistic Model. The percent
inhibition was plotted against the compound concentration, and the
IC50 value was determined from the logistic dose−response curve
fitting by Origin 8.0 software.49 Compounds were tested in duplicate
in a 12-point dose curve with 3-fold serial dilution starting from 33.33
μM.
PET Studies in the Baboon. Animal studies were approved by the
Brookhaven Institutional Animal Care and Use Committee. PET
studies were performed in 9 female baboons (Papio anubis, 13.5−21
kg). Briefly, anesthesia was induced by intramuscular injection of
ketamine hydrochloride (10 mg/kg) and then maintained with
isoflurane (Forane, 1−4%), nitrous oxide (1500 mL/min), and oxygen
(800 mL/min) during the PET scan. The brain was positioned in the
field of view of the PET scanner (Siemen’s high-resolution HR+).
Each labeled compound was injected into a radial arm vein and arterial
blood was sampled through the popliteal artery from the time of
injection and analyzed by HPLC. Each PET scan was performed in 3D
mode for 90 min, monitoring heart rate, respiration rate, and body
temperature. Arterial blood samples were collected at every 5 s for 2
min, then at 5, 10, 20, 30, 60, and 90 min after each radiotracer
injection. To evaluate reproducibility and specificity of compound
[11C]26, two injections in the same day in the same baboon were
carried out with or without pretreatment of compound 26 (bolus, 1
mg/kg, 20 min prior to radiotracer injection) or SAHA (1 mg/kg,
intravenous infusion for 30 min before the labeled compound).
In summary, an image-guided iterative synthesis approach
yielded new highly potent and BBB permeable benzamide type
HDAC inhibitors, one of which was assessed using PET.
Importantly, we determined the value of the molecular
descriptor PSA for predicting BBB permeability for this series.
Taken together, this approach has afforded a strategy and a
predictive model for developing highly potent and BBB
permeable HDAC inhibitors for CNS applications which
could also serve as a tool for the discovery of novel candidates
for small molecule probes and for drugs.
ASSOCIATED CONTENT
■
S
* Supporting Information
Information on chemical syntheses and radiosynthesis of
[11C]CI-994, QSPR model, and calculated values of molecular
descriptors for tested compounds are available. This material is
available free of charge via the Internet at http://pubs.acs.org.
METHODS
■
Radiosynthesis of [11C]26. [11C]carbon dioxide produced by the
14N(p,α)11C nuclear reaction on an EBCO cyclotron (TR-19, EBCO
Industry Ltd., Richmond, Canada) was converted into [11C]methyl
iodide using an automatic PETtrace MeI Microlab (GE Medical
Systems, Milwaukee, WI) apparatus, and then further converted to
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: hooker@nmr.mgh.harvard.edu (J.M.H.).
*E-mail: sunny.kim@nih.gov (S.W.K.)
Author Contributions
J.S.F., J.M.H., and S.W.K designed research; Y.J.S., L.M., A.R.,
S.C., F.W., E.H., P.K., P.C., S.J.H., P.W., S.W.K., and J.M.H.
[11C]methyl triflate by passage over a AgOTf furnace at 190
10
°C.30 [11C]Methyl triflate was transferred by argon flow to a V-shaped
reaction vessel (5 mL) containing compound 25 (1 mg) in DMSO
(300 μL) at room temperature. Upon completion of heating at 50 °C
for 3 min, the reaction mixture was diluted with HPLC eluent (1 mL)
594
dx.doi.org/10.1021/cn500021p | ACS Chem. Neurosci. 2014, 5, 588−596

ACS Chemical Neuroscience
Research Article
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Gilbert, M. R., Robins, H. I., Lieberman, F. S., Lassman, A. B.,
McGovern, R. M., Xu, J. H., Desideri, S., Ye, X. B., Ames, M. M.,
Espinoza-Delgado, I., Prados, M. D., and Wen, P. Y. (2012) Phase I
Study of Vorinostat in Combination with Temozolomide in Patients
with High-Grade Gliomas: North American Brain Tumor Consortium
Study 04−03. Clin. Cancer Res. 18, 6032−6039.
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Munster, P. N., Klein, P. M., Cruickshank, S., Miller, K. D., Lee, M. J.,
and Trepel, J. B. (2013) Randomized Phase II, Double-Blind, Placebo-
Controlled Study of Exemestane with or without Entinostat in
Postmenopausal Women with Locally Recurrent or Metastatic
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performed research; Y.K., J.L., J.M.H., and S.W.K. analyzed
data; J.L., N.D.V., J.S.F., J.M.H, and S.W.K. wrote the paper.
Funding
This work was supported by National Institutes of Health grant
1R01DA030321 (Y.J.S., J.M.H., S.W.K.), National Institute of
Alcohol Abuse and Alcoholism Intramural Program (S.W.K.,
L.M., N.D.V.), and the Deutscher Akademischer Austausch-
dienst (DAAD), Bonn, Germany (P.W.). This study was
carried out in part at Brookhaven National Laboratory under
contract DE-AC02-98CH10886 with the U.S. Department of
Energy and with infrastructure support from its Office of
Biological and Environmental Research.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank Michael Schueller for cyclotron operations and
Donald Warner for PET operations. We also thank Tiffany St.
Bernard, John Dobbs Jr., and Samuel Wilson supported by
BNL summer internship program for their efforts for synthesis
work. We used two computational chemistry software packages
available in the Center for Molecular Modeling (http://cmm.
cit.nih.gov) and the Helix Systems (http://helix.nih.gov) at the
National Institutes of Health, Bethesda, MD.
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ABBREVIATIONS
■
HDAC, histone deacetylase; BBB, blood-brain barrier; CNS,
central nervous system; PET, positron emission tomography;
HAT, histone acetyl transferase; HDACi, histone deacetylase
inhibitor; SAHA, suberoylanilide hydroxamic acid; MW,
molecular weight; QSPR, quantitative-property relationship;
MV, molecular volume; PSA, polar surface area; DMF, N,N-
dimethylformamide; PMP, 1,2,2,6,6-pentamethylpiperidine;
AUC, area under the curve; TSA, trichostatin A; VT, total
distribution volume
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Application of Histone Deacetylase Inhibitors for Central Nervous
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An Analysis of Recent Patenting Activity. Expert Opin. Ther. Pat. 17,
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C., Carter, P., King, P., Reid, A. E., Volkow, N. D., and Fowler, J. S.
(2013) Whole-Body Pharmacokinetics of Hdac Inhibitor Drugs,
Butyric Acid, Valproic Acid and 4-Phenylbutyric Acid Measured with
Carbon-11 Labeled Analogs by Pet. Nucl. Med. Biol. 40, 912−918.
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